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CN-122017051-A - Epigallouin hydrochloride intermediate K5 and separation and determination method of related impurities thereof

CN122017051ACN 122017051 ACN122017051 ACN 122017051ACN-122017051-A

Abstract

The invention belongs to the technical field of medicine analysis, and particularly relates to a separation and determination method for an epothilone hydrochloride intermediate K5 and related impurities thereof. The impurities comprise one or more of S-tertiary butyl sulfinamide, impurity K4a, impurity K4, benzyl chloride, impurity K3 and toluene. The method comprises the steps of using octadecylsilane chemically bonded silica as a filler, separating a mobile phase comprising a mobile phase A and a mobile phase B, wherein the mobile phase A and the mobile phase B are mixed solutions of phosphate buffer salt solution and acetonitrile, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase A is 50-70:50-30, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase B is 20-40:80-60, separating through linear gradient elution, detecting in a detector to obtain a chromatogram, and calculating the content of each impurity by an external standard method. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Inventors

  • CHEN WEN
  • WANG SHAOLI
  • LIU JIE
  • Xiang Hongfan
  • Hu jiangjun
  • HUANG SHIJIE

Assignees

  • 重庆华邦制药有限公司

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. A method for separating an epothilone hydrochloride intermediate K5 and impurities thereof based on high performance liquid chromatography, which is characterized in that the epothilone hydrochloride intermediate K5 and the impurities jointly form a composition, and the impurities comprise any one or more of S-tert-butylsulfinamide, impurities K4a, impurities K4, benzyl chloride, impurities K3 and toluene; The method comprises the steps of using octadecylsilane chemically bonded silica as a filler in a chromatographic column, wherein a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A and the mobile phase B are mixed solutions of phosphate buffer salt solution and acetonitrile, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase A is 50-70:50-30, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase B is 20-40:80-60, and separating a hydrochloride according to a pull ring element intermediate K5 and impurities thereof through linear gradient elution; each component in the composition has the following structural formula:
  2. 2. The method according to claim 1, wherein the phosphate buffer solution is dipotassium hydrogen phosphate in the mobile phase A, the volume ratio of the phosphate buffer solution to acetonitrile in the mobile phase A is 56-64:44-36, and the volume ratio of the phosphate buffer solution to acetonitrile in the mobile phase B is 29-31:71-69.
  3. 3. The method according to claim 1, characterized in that the procedure of the linear gradient elution is as follows: Time-minutes Mobile phase a- (parts by volume) Mobile phase B- (parts by volume) 0 90~100 10~0 3±0.5 90~100 10~0 20±0.5 10~0 90~100 45±0.5 10~0 90~100 46±0.5 90~100 10~0 55±0.5 90~100 10~0 。
  4. 4. The method according to claim 1, characterized in that the procedure of the linear gradient elution is as follows: Time-minutes Mobile phase a- (parts by volume) Mobile phase B- (parts by volume) 0 100 0 3 100 0 20 0 100 45 0 100 46 100 0 55 100 0 。
  5. 5. The method according to claim 1, wherein the flow rate is 0.8-1.2mL/min and the column temperature is 35-45 ℃.
  6. 6. The method for identifying the epothilone hydrochloride intermediate K5 and the impurities thereof is characterized by comprising the following steps: (1) Separating the composition by the method of any one of claims 1-5, and detecting with a detector having a detection wavelength of 220±10nm to obtain a chromatogram; (2) And comparing the chromatographic characteristics of the detection product with those of the reference product to determine whether the detection product contains the epothilone hydrochloride intermediate K5 and impurities thereof.
  7. 7. The method of claim 6, wherein the components of the composition are identified in ascending order of S-t-butylsulfinamide, impurity K4a, impurity K4, benzyl chloride, impurity K3, toluene, and epothilone hydrochloride intermediate K5.
  8. 8. The method according to claim 6, wherein the retention time is 3.1.+ -. 0.5, S-t-butylsulfinamide, impurity K4a, impurity K4, 16.9.+ -. 0.5, benzyl chloride, 17.5.+ -. 0.5, impurity K3, 18.0.+ -. 0.5, toluene, 20.9.+ -. 0.5, and epothilone hydrochloride intermediate K5 are determined.
  9. 9. The method for measuring the content of the epothilone hydrochloride intermediate K5 and the impurities thereof is characterized in that the method of any one of claims 6-8 is adopted to separate and identify the epothilone hydrochloride intermediate K5 and the impurities thereof, a chromatogram is obtained, and the content of each impurity is calculated by adopting an external standard method according to the obtained chromatogram.
  10. 10. The method of claim 9, wherein the sample formulation solvent is acetonitrile.

Description

Epigallouin hydrochloride intermediate K5 and separation and determination method of related impurities thereof Technical Field The invention belongs to the technical field of medicine analysis, and particularly relates to a separation and determination method for an epothilone hydrochloride intermediate K5 and related impurities thereof. Background The epothilone hydrochloride (ERAVACYCLINE) is the first fully synthesized tetracycline antibacterial drug. The composition has wide antibacterial spectrum, and is mainly used for treating complicated intraperitoneal infection (cIAI) adult patients caused by sensitive strains, including Escherichia coli, klebsiella pneumoniae, fulaudi citrate bacillus, enterobacter cloacae, klebsiella oxytoca, enterococcus faecium, staphylococcus aureus, streptococcus strainogenes, clostridium perfringens, bacteroides genus, and Paralopecis diradina. The original research company TETRAPHASE PHARMACEUTICALS, inc. Of epothilone hydrochloride discloses in document (Process Research and Development of an Enantiomerically Enriched Allyic Amine,One of the Key Intermediates for the Manufacture ofSynthetic Tetracyclines.Org.ProcessRes.Dev.2015,19,1784-1795) a process for the synthesis of the intermediate chiral enamine (document compound 3) which is specifically as follows: From this route, compound 3 and compound 2 were synthesized to obtain compound 1 (AB ring fragment), and compound 1 was further synthesized to obtain epothilone hydrochloride. One of the synthetic routes for compound 3 is as follows: It can be seen that compound 34 in the literature is a key intermediate in the synthesis of compound 3. The compound 34 has a Chinese cultural name of (S2S) -N- [ [3- (benzyloxy) -1,2-oxazol-5-yl ] methylene ] -2-methylpropane-2-sulfinamide, an English chemical name of (S2S) -N- [ [3- (Benzyloxy) -1,2-oxazol-5-yl ] methylidene ] -2-methylpropane-2-sulfinamide, a molecular formula of C 15H18N2O3 S, a molecular weight of 306.38 and a CAS number of 1575490-21-9. In the invention, the product is autonomously named as an epothilone hydrochloride intermediate K5, which is called as K5 for short. The quantitative detection and control of impurities in K5 are of great significance for the final synthesis of tetracycline compounds (such as epothilone hydrochloride, minocycline hydrochloride, tigecycline hydrochloride and the like). According to research, in the synthesis process of the epothilone hydrochloride intermediate K5, the impurities K3 and K4 are main synthesis raw materials, the impurity K4a is a main reaction byproduct, and the S-tert-butylsulfinamide, toluene and benzyl chloride are main reaction solvents and reagents. Wherein the impurity K4 has aldehyde group with a warning structure, benzyl chloride has halogenated alkane with a warning structure, and toluene is a 3-class carcinogen. The quality of the product can be controlled by detecting the impurities above in the epothilone hydrochloride intermediate K5, so that the subsequent high-quality preparation of the tetracycline compound AB ring segment is ensured. However, there is no report on the analysis method of S-tert-butylsulfinamide, impurity K4a, impurity K4, benzyl chloride, impurity K3 and/or toluene in epothilone hydrochloride intermediate K5. Disclosure of Invention Accordingly, one of the objectives of the present invention is to provide a method for separating epothilone hydrochloride intermediate K5 and impurities thereof based on high performance liquid chromatography, which can complete the separation of various substances in a short time. In order to achieve the above purpose, the technical scheme of the invention is as follows: A method for separating an epothilone hydrochloride intermediate K5 and impurities thereof based on high performance liquid chromatography, wherein the epothilone hydrochloride intermediate K5 and the impurities jointly form a composition, and the impurities comprise any one or more of S-tert-butylsulfinamide, impurities K4a, impurities K4, benzyl chloride, impurities K3 and toluene; The method comprises the steps of using octadecylsilane chemically bonded silica as a filler in a chromatographic column, wherein a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A and the mobile phase B are mixed solutions of phosphate buffer salt solution and acetonitrile, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase A is 50-70:50-30, the volume ratio of the phosphate buffer salt solution to the acetonitrile in the mobile phase B is 20-40:80-60, and separating a hydrochloride according to a pull ring element intermediate K5 and impurities thereof through linear gradient elution; each component in the composition has the following structural formula: the separated and detected product is used in the next production. The above impurities may be arranged and combined in various ways. For example, mode 1S-tert-butylsulfinamide and i