Bisacodine genotoxic impurity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine synthesis, and discloses a bisacodine genotoxic impurity and a preparation method and application thereof.The bisacodine genotoxic impurity is specifically bisacodine nitric oxide, and the preparation method comprises the following steps that bisacodine serves as a raw material and is oxidized through an oxidizing agent; extracting with ethyl acetate, collecting an ethyl acetate layer, and concentrating under reduced pressure. The invention successfully confirms the structure of bisacodine nitrogen oxide, the provided preparation method has the characteristics of simple operation, common and easily available raw materials, mild reaction conditions, no high-temperature and high-pressure step, thorough conversion of reaction raw materials, high yield and the like, and the obtained product has high purity, can be used as a reference substance for quality control of bisacodine raw materials and preparations, and has wide application prospects. And a guarantee is provided for safe medication of the bisacodine.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and in particular relates to a bisacodyl genotoxic impurity and a preparation method and application thereof. Background Art

[0002] Bisacodyl is an organic compound with the chemical formula C22H19NO4 and its Chinese name is 4,4'-(2-pyridylmethylene) diphenol diacetate. Bisacodyl is a stimulant laxative originally developed by Boehringer Ingelheim. It mainly acts on the large intestine and is clinically used for acute and chronic constipation. Suppositories can be used for colonoscopy, intestinal X-ray examination or intestinal cleaning before abdominal surgery.

[0003] Some impurities contained in drugs may have biological activity. If the content exceeds a certain standard, it may cause side effects and threaten the patient's life, which is directly related to the safety of clinical drug use. Impurities can interact with drugs and affect the effectiveness of drugs. Impurities in chemically synthesized drug raw materials and preparations, especially genotoxic impurities, affect the safety of drugs. In terms of the selection of drug dosage forms, formulation of prescriptions and process decisions, impurities have a great impact on the analysis methods and storage research of products, and are the focus of innovative drug quality research and safety research.

[0004] In order to ensure the quality of drugs and prevent drug safety, it is necessary to study the related impurities of bisacodyl. After consulting foreign pharmacopoeias, it was found that the United States Pharmacopoeia, the Japanese Pharmacopoeia, and the European Pharmacopoeia included inspection items for related substances of bisacodyl APIs. The United States Pharmacopoeia clearly defines the structures of the six impurities of bisacodyl, which are named 4,4′-(pyridin-2-ylmethylene)diphenol (impurity A), 2,4′-(pyridin-2-ylmethylene)diphenol (impurity B), 4-[(4-hydroxyphenyl)(pyridin-2-yl)methyl]phenyl acetate (impurity C), impurity 1 (0.85), 2-[(4-acetoxyphenyl)(pyridin-2-yl)methyl]phenyl acetate (impurity E), and impurity 2 (2.6), and control them as known impurities. The standards for related substances of bisacodyl APIs included in the European Pharmacopoeia are basically consistent with those of the United States Pharmacopoeia. The only difference is that the European Pharmacopoeia does not specify known impurity 1 (0.85), the impurity numbers are not completely consistent, and the limits of individual impurities are also different. The limits of impurity A and impurity B in the United States Pharmacopoeia are ≤0.15%, and the limits of impurity A and impurity B in the European Pharmacopoeia are ≤0.1%. The Japanese Pharmacopoeia's related substance inspection items are spot plates, and there is no clear impurity limit. The United States Pharmacopoeia and the European Pharmacopoeia also have settings for chromatographic conditions. The quality standards for bisacodyl suppositories and enteric-coated tablets in my country were first published in Part II of the 1990 edition of the "Chinese Pharmacopoeia". The current standards are published on pages 84 and 85 of Part II of the 2020 edition of the "Chinese Pharmacopoeia". However, the related substance inspection items in the quality standards for bisacodyl suppositories and enteric-coated tablets are thin layer chromatography, which is the same as the Japanese Pharmacopoeia, which makes the product have certain quality risks. Moreover, for the 6 known impurities in the United States Pharmacopoeia and the European Pharmacopoeia, there are currently basically no reports on related preparations in domestic and foreign literature.

[0005] However, according to the synthesis route and reaction mechanism of bisacodyl, the raw material 2-pyridinecarboxaldehyde may contain 2-pyridinecarboxaldehyde nitrogen oxide, which will participate in the reaction of the subsequent steps together with 2-pyridinecarboxaldehyde, thereby resulting in the bisacodyl product containing the structural formula Figure 1 The impurity Bisacodyl N-oxide (Chinese name is 2-(bis(4-acetoxyphenyl)methyl)pyridine 1-oxide) is shown. Since the impurity contains the pyridine N-oxide structure, that is, it contains the genotoxic impurity warning structure, it is determined to be a genotoxic impurity. It can be seen that using this impurity as a reference substance is of great significance for the quality control research and detection of bisacodyl raw materials and preparations, but there is currently no relevant report on this Bisacodyl N-oxide. Summary of the invention

[0006] In view of this, the present invention aims to develop a method for preparing the above-mentioned bisacodyl genotoxic impurity, to provide a reference substance for the quality control of bisacodyl raw materials and preparations, and to ensure the safety of medication.

[0007] The technical solution of the present invention is as follows: The first aspect of the present invention provides a method for preparing a bisacodyl genotoxic impurity, wherein the bisacodyl genotoxic impurity is specifically a structural formula as shown in Figure 1 The bisacodyl nitrogen oxide shown in the invention comprises the following steps: S1. dissolving bisacodyl in an organic solvent, and adding an oxidant to react; wherein the organic solvent is one or more of methanol, ethanol, isopropanol, and acetone, and the oxidant is hydrogen peroxide and / or m-chloroperbenzoic acid; S2. After the reaction in step S1 is completed, water and ethyl acetate are added to carry out liquid-liquid extraction, and the ethyl acetate layer is collected, washed, and concentrated under reduced pressure to obtain the product.

[0008] Preferably, in the above preparation method, the mass volume ratio of bisacodyl to the organic solvent is 1:(2-10), and the molar ratio of bisacodyl to the oxidant is 1:(1.2-10); wherein the molar ratio of bisacodyl to the oxidant is more preferably 1:(1.5-5).

[0009] Preferably, in the above preparation method, the oxidant is meta-chloroperbenzoic acid.

[0010] Preferably, in the above preparation method, the reaction temperature of step S1 is 0-80° C., and the reaction time is 1-20 h.

[0011] Preferably, in the above preparation method, during extraction, the amount of water added is 5-10 times the volume of the organic solvent, and the amount of ethyl acetate added is 15-20 times the volume of the organic solvent.

[0012] Preferably, in the above preparation method, the temperature of the reduced pressure concentration in step S2 is 35-50°C.

[0013] Preferably, in the above preparation method, the following steps are also included: S3. Separate and purify the product obtained in step S2 by silica gel column chromatography, and obtain high-purity bisacodyl genotoxic impurity (white powder solid) after the eluate is concentrated under reduced pressure and dried; more preferably, the temperature for reduced pressure concentration is 35-50°C.

[0014] Based on the prepared bisacodyl genotoxic impurities, the second aspect of the present invention provides the use of bisacodyl genotoxic impurities in the quality control of bisacodyl raw materials and preparations. The bisacodyl genotoxic impurities prepared by the method of the present invention have high purity and can be used as a reference substance for the quality control of bisacodyl raw materials and preparations.

[0015] Preferably, in the above application, the bisacodyl genotoxic impurities in the bisacodyl drug substance or preparation are qualitatively or quantitatively detected based on HPLC and under the following chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel (Waterssymmetry C18 (250×4.6 mm, 5 μm) or an equivalent chromatographic column); the mobile phase was an aqueous solution of ammonium formate (1.58 g / L, pH adjusted to 5.0 with formic acid) as mobile phase A, and acetonitrile solution as mobile phase B; the running time was 45 min; the flow rate was 1.5 mL / min; the column temperature was 30°C; the injection volume was 20 μL; and the wavelength was 265 nm.

[0016] More preferably, isocratic elution is performed in HPLC, and mobile phase A:mobile phase B=55:45 (v:v).

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention identifies for the first time the structure of a new bisacodyl impurity, namely bisacodyl nitrogen oxide, and develops a method for synthesizing the impurity in a simple and economical manner. The method of the present invention uses bisacodyl as a raw material, obtains the impurity pure product by adding an oxidant for oxidation synthesis, ethyl acetate extraction, silica gel column chromatography purification and the like, and the purity is greater than 98% and the yield is as high as 95%. The method also has the advantages of simple process, common and easily available materials, little environmental pollution, safety and environmental protection, etc.

[0018] The bisacodyl nitrogen oxide prepared by the invention has the characteristic of high purity, meets the requirements of being a reference substance for qualitative and quantitative analysis and detection of bisacodyl preparations, and the quality standard of bisacodyl preparations can be improved through the research and detection of the impurities, thus providing a guarantee for the safe use of bisacodyl. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the structural formula of bisacodyl nitrogen oxide in the present invention; Figure 2 The technical roadmap for preparing bisacodyl nitrogen oxide of the present invention; Figure 3 1H-NMR diagram of bisacodyl nitrogen oxide prepared by the present invention; Figure 4 This is a full scan of the mass spectrum of the bisacodyl nitrogen oxide prepared by the present invention; Figure 5 The HPLC purity chromatogram of the bisacodyl nitrogen oxide prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0021] In view of the fact that the domestic pharmacopoeia does not control the related substances of bisacodyl suppositories and enteric-coated tablets, and considering the safety of clinical medication, the inventors have previously studied the related substances of bisacodyl suppositories and enteric-coated tablets and found that according to the synthesis route and reaction mechanism of bisacodyl, the bisacodyl raw material is very likely to contain the structural formula Figure 1 The bisacodyl nitrogen oxide impurity shown in the figure. This genotoxic impurity affects the safety of the drug. In terms of the selection of drug dosage forms, formulation of prescriptions and process decisions, the impurity has a great impact on the analysis method and storage research of the product. In order to better control the quality of bisacodyl, it is necessary to prepare a pure product of bisacodyl nitrogen oxide and confirm its structure. Therefore, the present invention has developed a method for synthesizing the above-mentioned genotoxic impurity, and its synthesis route is as follows Figure 2 As shown, the specific steps include: (1) dissolving bisacodyl in an organic solvent and adding an oxidant to react; wherein the organic solvent is one or more of methanol, ethanol, isopropanol, and acetone, and the oxidant is hydrogen peroxide and / or m-chloroperbenzoic acid; (2) After the reaction in step (1) is completed, water and ethyl acetate are added for liquid-liquid extraction, the extraction is performed 1-2 times, the ethyl acetate layers are combined, washed with water 2-4 times, and concentrated under reduced pressure to obtain a crude product of bisacodyl genotoxic impurities; (3) Separating and purifying the genotoxic impurity of bisacodyl by silica gel column chromatography and drying to obtain a pure product.

[0022] The structure of the product synthesized by the method of the present invention was confirmed by using a variety of techniques. Figure 1 As shown, the results of nuclear magnetic resonance detection are as follows Figure 3 The mass spectrometry results are shown in Figure 4 shown.

[0023] In the method of the present invention, the reaction conditions are mild and there is no high temperature and high pressure step. It should be noted that the type, equivalent and reaction temperature of the oxidant are key factors in synthesizing the genotoxic impurity of the present invention. The inventors have almost completely converted bisacodyl into bisacodyl nitrogen oxide by controlling the above factors.

[0024] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0025] Example 1 This example provides a method for preparing bisacodyl genotoxic impurities, comprising the following steps: Weigh 0.60 g of bisacodyl raw material, add 3 mL (5 V) of methanol to dissolve, add 0.5 mL (3 eq) of 30% hydrogen peroxide solution, and react at 30°C for 3 h; add 15 mL of water and 45 mL of ethyl acetate, liquid-liquid extraction for 3 min, collect the ethyl acetate layer, wash with water 3 times, and check that the ethyl acetate layer does not turn blue when detected by starch potassium iodide paper to ensure that hydrogen peroxide is completely removed. Collect the ethyl acetate layer, concentrate under reduced pressure at 40°C to obtain the crude bisacodyl nitrogen oxide impurity; separate and purify by silica gel column chromatography, concentrate the eluate under reduced pressure at 40°C and dry, and obtain 0.60 g of white powder solid (i.e., bisacodyl nitrogen oxide) with a yield of 95%.

[0026] Example 2 This example provides a method for preparing bisacodyl genotoxic impurities, comprising the following steps: Weigh 0.60 g of bisacodyl raw material, add 6 mL (10 V) of ethanol to dissolve it, add 0.8 mL (5 eq) of 30% hydrogen peroxide solution, and react at 50°C for 2 h; add 30 mL of water and 90 mL of ethyl acetate, liquid-liquid extraction for 3 min, collect the ethyl acetate layer, wash it with water 3 times, and check that the ethyl acetate layer does not turn blue when detected by starch potassium iodide paper to ensure that the hydrogen peroxide is completely removed. Collect the ethyl acetate layer, concentrate it under reduced pressure at 45°C to obtain the crude bisacodyl nitrogen oxide impurity; separate and purify it by silica gel column chromatography, concentrate the eluate under reduced pressure at 45°C and dry it to obtain 0.61 g of white powder solid (i.e., bisacodyl nitrogen oxide) with a yield of 97%.

[0027] Example 3 This example provides a method for preparing bisacodyl genotoxic impurities, comprising the following steps: Weigh 0.60 g of bisacodyl raw material, add 3 mL (5 V) of isopropanol to dissolve it, add 0.86 g (3 eq) of m-chloroperbenzoic acid, and react at 70°C for 1 h; add 15 mL of water and 45 mL of ethyl acetate, perform liquid-liquid extraction for 3 min, collect the ethyl acetate layer, wash it twice with water, collect the ethyl acetate layer, and concentrate it under reduced pressure at 50°C to obtain the crude bisacodyl nitrogen oxide impurity; separate and purify it by silica gel column chromatography, concentrate the eluate under reduced pressure at 50°C and dry it to obtain 0.60 g of white powder solid (i.e., bisacodyl nitrogen oxide) with a yield of 95%.

[0028] Example 4 This example provides a method for preparing bisacodyl genotoxic impurities, comprising the following steps: Weigh 0.60 g of bisacodyl raw material, add 6 mL of acetone (10 V) to dissolve it, add 1.43 g (5 eq) of m-chloroperbenzoic acid, and react at 40°C for 5 h; add 30 mL of water and 90 mL of ethyl acetate, perform liquid-liquid extraction for 3 min, collect the ethyl acetate layer, wash it twice with water, collect the ethyl acetate layer, and concentrate it under reduced pressure at 40°C to obtain the crude product of bisacodyl nitrogen oxide impurity, which is separated and purified by silica gel column chromatography, and the eluate is concentrated under reduced pressure at 40°C and dried to obtain 0.61 g of white powder solid (i.e., bisacodyl nitrogen oxide) with a yield of 97%.

[0029] Example 5 This example provides a method for detecting bisacodyl genotoxic impurities based on HPLC, and the chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel (Waterssymmetry C18 (250×4.6 mm, 5μm)); Mobile phase: 1.58 g / L ammonium formate aqueous solution (adjusted to pH 5.0 with formic acid) as mobile phase A, acetonitrile solution as mobile phase B; isocratic elution, mobile phase A:mobile phase B=55:45; running time, 45 min; flow rate, 1.5 mL / min; detection wavelength, 265 nm; injection volume, 20 μL; column temperature, 30°C.

[0030] Based on the detection method in Example 5, the purity analysis of the bisacodyl genotoxic impurities prepared in Examples 1-4 was performed, and the specific operation was as follows: 25 mg (precisely weighed) of the bisacodyl genotoxic impurities were weighed and placed in a 25 mL volumetric flask, dissolved and diluted to the scale with a diluent (acetonitrile: water = 35:5), and shaken well; 20 μL was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded until 45 min, and the HPLC purity of the bisacodyl genotoxic impurities was calculated by the peak area normalization method. The results are shown in Tables 1 and Figure 3 As shown, Figure 3 Specifically, the chromatogram of the genotoxic impurities of bisacodyl obtained in Example 1 is as follows: Table 1 Purity of genotoxic impurities of bisacodyl obtained in various examples

[0031] The raw materials listed in the present invention, as well as the upper and lower limits and interval values ​​of the raw materials of the present invention, and the upper and lower limits and interval values ​​of the process parameters can all realize the present invention, and the embodiments are not listed one by one here.

[0032] In summary, the method for preparing bisacodyl genotoxic impurities provided by the present invention has the advantages of complete reaction conversion and high yield. The obtained bisacodyl genotoxic impurities have high purity and can be used as a reference substance for quality control of bisacodyl preparations, which is of great significance to the safe use of bisacodyl.

[0033] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for preparing bisacodyl genotoxic impurities, characterized in that: The structural formula of the bisacodyl genotoxic impurity is as follows: ； The preparation method comprises the following steps: S1. dissolving bisacodyl in an organic solvent, and adding an oxidant to react; the organic solvent is one or more of methanol, ethanol, isopropanol, and acetone, and the oxidant is hydrogen peroxide and / or m-chloroperbenzoic acid; S2. After the reaction in step S1 is completed, water and ethyl acetate are added to carry out liquid-liquid extraction, and the ethyl acetate layer is collected, washed, and concentrated under reduced pressure to obtain the product.

2. The preparation method according to claim 1, characterized in that: Also includes: S3, separating and purifying the product obtained in step S2 by silica gel column chromatography.

3. The preparation method according to claim 1, characterized in that: The mass volume ratio of the bisacodyl to the organic solvent is 1:(2-10), and the molar ratio of the bisacodyl to the oxidant is 1:(1.2-10).

4. The preparation method according to claim 1, characterized in that The reaction temperature in step S1 is 0-80° C., and the reaction time is 1-20 h.

5. The preparation method according to claim 1, characterized in that: In step S2, the amount of water added is 5-10 times the volume of the organic solvent, and the amount of ethyl acetate added is 15-20 times the volume of the organic solvent.

6. The preparation method according to claim 1, characterized in that: The temperature of the reduced pressure concentration in step S2 is 35-50°C.

7. A genotoxic impurity of bisacodyl prepared according to any one of claims 1 to 6.

8. Use of the bisacodyl genotoxic impurity as claimed in claim 7 in the quality control of bisacodyl raw materials or preparations.

9. The use according to claim 8, characterized in that: Based on HPLC, the qualitative or quantitative detection of bisacodyl genotoxic impurities is performed under the following chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel; the mobile phase was ammonium formate aqueous solution as mobile phase A and acetonitrile solution as mobile phase B; the running time was 45 min; the flow rate was 1.5 mL / min; the column temperature was 30°C; the injection volume was 20 μL; and the wavelength was 265 nm.

10. The use according to claim 9, characterized in that: Isocratic elution was performed in HPLC with mobile phase A:mobile phase B=55:45.

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