阅读说明：本技术 一种加米霉素有关物质及其合成与分离方法 (Gamithromycin related substance and synthesis and separation method thereof ) 是由 陈强 许留群 杨申永 李建国 王玲 于 2020-04-30 设计创作，主要内容包括：本发明涉及一种加米霉素有关物质,属于化学合成领域,其特征在于,所述加米霉素有关物质为式Ⅰ所示结构的化合物或其盐,其合成方法是以去甲基阿奇霉素为反应底物,以低级醇为溶剂,将反应底物和溶剂置于高压反应釜中,加入丙醛和钯炭,在氢气保护条件下,保持氢气压力,控制反应温度,搅拌反应；该产品是加米霉素质量控制的必需品,本发明的合成分离方法制得的该物质能达到克级,为加米霉素的质量控制及未知杂质的研究奠定基础。(The invention relates to a gamithromycin related substance, which belongs to the field of chemical synthesis and is characterized in that the gamithromycin related substance is a compound with a structure shown in a formula I or a salt thereof, the synthesis method is characterized in that demethyl azithromycin is used as a reaction substrate, lower alcohol is used as a solvent, the reaction substrate and the solvent are placed in a high-pressure reaction kettle, propionaldehyde and palladium carbon are added, under the condition of hydrogen protection, hydrogen pressure is kept, the reaction temperature is controlled, and the stirring reaction is carried out; the product is a necessity for controlling the quality of the gamithromycin, and the substance prepared by the synthetic separation method can reach gram level, thereby laying a foundation for the quality control of the gamithromycin and the research of unknown impurities.)

1. A gamithromycin related substance is characterized in that the gamithromycin related substance is a compound with a structure shown as a formula I or a salt thereof,

2. the method for synthesizing the gamithromycin related substance as claimed in claim 1, wherein demethyl azithromycin is used as a reaction substrate, lower alcohol is used as a solvent, the reaction substrate and the solvent are placed in a high-pressure reaction kettle, propionaldehyde and palladium carbon are added, under the condition of hydrogen protection, the hydrogen pressure is maintained, the reaction temperature is controlled, and the stirring reaction is carried out;

after the reaction is finished, cooling to room temperature, replacing gas in the reaction kettle with nitrogen, filtering, washing, decompressing and evaporating the solvent, adding dichloromethane and water, adjusting the pH value to 9-11 with sodium hydroxide aqueous solution, and layering; the organic phase is washed by water, and the solvent is evaporated to dryness to obtain a crude product of the N-propyl azithromycin.

3. The method of claim 2, wherein the weight of the propanal is 1-5 times that of the demethylated azithromycin, and the weight of the palladium on carbon is 0.5-1 times that of the demethylated azithromycin.

4. The method for synthesizing a substance related to gamithromycin of claim 2, wherein the lower alcohol is at least one of methanol, ethanol and isopropanol, the hydrogen pressure is 0.1-0.6Mpa, the reaction temperature is 20-50 ℃, and the reaction time is 4-10 h.

5. A method for separating a substance related to gamithromycin according to claim 1, comprising the steps of: dissolving the N-propyl azithromycin crude product by using a small amount of solvent A, transferring the solution to a silica gel column for adsorption, and dissolving the N-propyl azithromycin crude product by using a solvent A: solvent B: eluting with mixed solvent of ammonia water, tracking and detecting by TLC, collecting eluent containing N-propyl azithromycin, and evaporating the solvent to dryness under reduced pressure to obtain the N-propyl azithromycin.

6. The method according to claim 5, wherein the solvent A is at least one selected from the group consisting of ethyl acetate, n-heptane and dichloromethane, the solvent B is at least one selected from the group consisting of methanol, ethanol and acetone, and the concentration of ammonia water is 20-35%.

7. The method for separating a substance related to gamithromycin according to claim 5, wherein the ratio of the solvent A: solvent B: the volume ratio of ammonia water is 5-20: 1: 0.1-0.5.

8. The method for separating a substance related to gamithromycin according to claim 1, wherein the liquid chromatography comprises the steps of: dissolving the N-propyl azithromycin crude product by using a dissolving phase, injecting a liquid phase, performing isocratic elution by using a mobile phase, collecting eluent containing the target substance, and freeze-drying.

9. The method for separating a substance related to gamithromycin of claim 8, wherein the chromatographic column is a C18 preparative column, the column temperature is 20-40 ℃, the sample injection concentration is 50-100mg/mL, the dissolved phase is 30-70% acetonitrile in water, and the mobile phase is acetonitrile: water: ammonia water is 100:30-70: 0.1-0.6.

10. Use of the gamithromycin-related substance of claim 1 as an impurity identification assay in quality control of gamithromycin.

Technical Field

The invention relates to the field of chemical synthesis, in particular to a gamithromycin related substance and a synthesis and separation method thereof.

Background

The gamithromycin is a second generation macrolide aza 7 alpha antibiotic, and has a structure shown as a formula II.

The gamithromycin is white to off-white, is insoluble in water, is stable at high temperature and is not sensitive to light. An antibiotic injection ZACTRAN (EMEA CVMP,2008) for animals developed by French Meiria CO Limited company is approved by European Union for use in 9 months 2007, and the European Union drug administration authenticates the structure and the efficacy of the antibiotic injection ZACTRAN, can be used for using animals, has the advantages of quick absorption, low in-vivo residue, wide in-vivo distribution, safety, high efficiency and the like, and has no adverse reaction report temporarily. Gamithromycin is an azamacrolide with a single propyl substitution at the 7 alpha position, and this particular structure is beneficial to rapid absorption under physiological pH conditions and long-term action in the target tissue lung. Macrolide antibiotics exert bacteriostatic and bactericidal effects by interfering with the synthesis of bacterial proteins. Macrolide antibiotics inhibit the combination of bacterial protein synthesis 50S ribosome subunits and prevent peptide chain extension, thereby achieving the effects of bacteriostasis and sterilization. The bacteriostatic mechanism of gamithromycin is the same as that of macrolides. The gamithromycin mainly acts on bovine respiratory diseases caused by main pathogenic bacteria such as haemolytic mannheimia, pasteurella multocida, histophilus somni and the like.

In order to ensure the safety of animal medicines, the quality of the animal professional medicines needs to be strictly controlled, the measurement of medicine content, the structural identification of unknown impurities and the limitation of impurities are effective methods for controlling the medicine quality, and the analysis of impurities is important content of the medicine quality standard. Gamithromycin is a semi-synthetic macrolide antibiotic, and the impurity spectrum is complex and difficult to predict. In the prior art, relevant patent publications describe that the N group on the sugar ring on the 11-position carbon is easy to oxidize by using gamithromycin as a substrate under the oxidizing condition, and gamithromycin oxide is generated through degradation reaction; in addition, there is a patent publication that gazemycin or a precursor of gazemycin is used as a reaction substrate, and that gazemycin is unstable under acidic conditions, and a sugar ring on the 13-position carbon is easily dropped to produce descladinose and gazemycin.

Based on the complex and difficult prediction of the gamithromycin hybrid spectrum, the structure confirmation of related substances generated in the synthesis process of the gamithromycin and a corresponding synthesis and separation method are urgently needed in the industry.

Disclosure of Invention

The invention aims to solve the problem that the prior art is lack of structure confirmation of related substances generated in the synthesis process of the gamithromycin and the corresponding synthesis and separation methods and the like, and provides a gamithromycin related substance and a synthesis and separation method thereof.

Gamithromycin is a semi-synthetic macrolide antibiotic, and is a fifteen-membered macrolide antibiotic obtained by isomerizing, rearranging, reducing and propylating E-erythromycin A oxime. The isomerization of the E-erythromycin A oxime cannot convert the E-erythromycin A oxime into the Z-erythromycin A oxime in a percent, and the E-erythromycin A oxime is continuously increased in the process of placing the Z-erythromycin A oxime, so that the Z-erythromycin A oxime necessarily contains the E-erythromycin A oxime. The E-erythromycin A oxime generates demethyl azithromycin through rearrangement reduction, and finally generates the N-propyl azithromycin through propyl. Therefore, the N-propyl azithromycin is an important process impurity in related substances of the gamithromycin and is a necessity for researching the quality of the gamithromycin.

The technical scheme for solving the technical problems is as follows: a gamithromycin related substance is characterized in that the gamithromycin related substance is a compound with a structure shown as a formula I or a salt thereof,

the synthesis method of the gamithromycin related substances is characterized in that demethyl azithromycin is taken as a reaction substrate, lower alcohol is taken as a solvent, the reaction substrate and the solvent are placed in a high-pressure reaction kettle, propionaldehyde and palladium carbon are added, the hydrogen pressure is kept under the condition of hydrogen protection, the reaction temperature is controlled, and the stirring reaction is carried out, wherein the specific reaction formula is as follows:

after the reaction is finished, cooling to room temperature, replacing gas in the reaction kettle with nitrogen, filtering, washing, decompressing and evaporating the solvent, adding dichloromethane and water, adjusting the pH value to 9-11 with sodium hydroxide aqueous solution, and layering; the organic phase is washed by water, and the solvent is evaporated to dryness to obtain a crude product of the N-propyl azithromycin.

The weight of the propionaldehyde is 1-5 times of that of the demethyl azithromycin, the weight of the palladium carbon is 0.5-1 time of that of the demethyl azithromycin, and the weight of the lower alcohol is 5-15 times of that of the demethyl azithromycin;

the lower alcohol is at least one of methanol, ethanol and isopropanol, the hydrogen pressure range is 0.1-0.6Mpa, the reaction temperature is 20-50 ℃, and the reaction time is 4-10 h.

And a method for separating the gamithromycin related substances, which is characterized in that the conventional silica gel column separation method comprises the following steps: dissolving the N-propyl azithromycin crude product by using a small amount of solvent A, transferring the solution to a silica gel column for adsorption, and dissolving the N-propyl azithromycin crude product by using a solvent A: solvent B: eluting with mixed solvent of ammonia water, tracking and detecting by TLC, collecting eluent containing N-propyl azithromycin, and evaporating the solvent to dryness under reduced pressure to obtain the N-propyl azithromycin.

Wherein the solvent A is at least one of ethyl acetate, n-heptane and dichloromethane, the solvent B is at least one of methanol, ethanol and acetone, and the concentration of the ammonia water is 20-35%; the solvent A: solvent B: the volume ratio of ammonia water is 5-20: 1: 0.1-0.5;

and another method for separating a substance related to gamithromycin as described above, characterized in that the liquid chromatography comprises the steps of: dissolving the N-propyl azithromycin crude product by using a dissolving phase, injecting a liquid phase, performing isocratic elution by using a mobile phase, collecting eluent containing the target substance, and freeze-drying.

Wherein the chromatographic column adopts a C18 preparative column, the column temperature is 20-40 ℃, and the sample injection concentration is 50-100 mg/mL; the dissolved phase is 30-70% acetonitrile in water, and the mobile phase is acetonitrile: water: ammonia water is mixed system with the volume ratio of 100:30-70: 0.1-0.6.

The invention has the beneficial effects that:

1. the N-propyl azithromycin prepared by the synthesis and separation method overcomes the defects of lack of structure confirmation of related substances generated in the synthesis process of the gamithromycin, corresponding synthesis and separation methods and the like in the prior art, and the obtained product can reach gram level, meets the market demand and lays a foundation for the quality control of the gamithromycin and the research of unknown impurities;

2. the separation method creatively adopts a solvent A: solvent B: the mixed eluent of ammonia water, the three components have synergistic effect, and the product purity is greatly improved.

Drawings

FIG. 1 is an HPLC chromatogram of a crude N-propyl azithromycin of the present invention;

FIG. 2 is an HPLC profile of N-propyl azithromycin of the invention;

FIG. 3 is a mass spectrum of N-propyl azithromycin of the invention;

FIG. 4 is a drawing showing the preparation of N-propylazithromycin of the invention¹H NMR spectrum;

fig. 5 is a partially enlarged view of fig. 4.

Detailed Description