阅读说明：本技术 一种加米霉素有关物质的分离、制备及纯化方法 (Method for separating, preparing and purifying gamithromycin related substances ) 是由 张许科 侯林 于 2018-06-28 设计创作，主要内容包括：本发明公开了一种加米霉素有关物质,其为具有式(I)所示结构式的化合物或其盐,<Image he="554" wi="406" file="DDA0001712405020000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该加米霉素有关物质能为加米霉素的质量监控提供保障。本发明还公开了加米霉素有关物质的制备及分析分离方法,本发明的制备方法工艺简单,能在产业上大规模应用。(The invention discloses a gamithromycin related substance which is a compound with a structural formula shown in a formula (I) or a salt thereof, the gamithromycin related substance can provide guarantee for quality monitoring of the gamithromycin. The invention also discloses a preparation and analysis separation method of the gamithromycin related substances, and the preparation method has simple process and can be applied to industry in large scale.)

1. A gamithromycin related substance, wherein the gamithromycin related substance is a compound shown as a structural formula (I) or a salt thereof

2. A process for preparing a substance relating to gaamicin according to claim 1, wherein the process is carried out by degradation of gaamicin; the degradation reaction comprises an acid degradation reaction, an alkali degradation reaction, a high-temperature degradation reaction, a high-humidity degradation reaction, an oxidation degradation reaction or an illumination degradation reaction; preferably, the degradation reaction is an oxidative degradation reaction.

3. The method for producing a gaamicin-related substance according to claim 2, wherein said oxidative degradation reaction comprises: and (3) carrying out reflux reaction on the gamithromycin and hydrogen peroxide in a non-polar solvent, quenching the hydrogen peroxide after the reflux reaction, carrying out layered separation on an organic phase, and drying the organic phase to obtain a coarse product of the related substances of the gamithromycin.

4. The process for producing a substance related to gamithromycin according to claim 3, wherein the nonpolar solvent is chloroform, the reflux reaction is carried out at a pH of 9 to 10, a temperature of 62 to 65 ℃ and a reflux reaction time of 0.5 to 4 hours; cooling to 0-5 deg.C in ice water bath, adding saturated sodium bisulfite solution at low temperature, quenching, and layering; drying the organic phase by vacuum evaporation at 30 ℃; preferably, the reflux reaction time is 1 h.

5. A purification method of a gamithromycin related substance, wherein the purification method comprises the steps of adding the crude gamithromycin related substance prepared by the preparation method of claim 3 into acetone, heating to 56-58 ℃ until the mixture is dissolved under reflux, filtering to remove insoluble substances, dripping water into the filtrate, cooling to 20 +/-5 ℃ after dripping is finished, stirring for 1-2 hours, and performing suction filtration, washing and drying to obtain the gamithromycin related substance.

6. The method for purifying a gaamicin-related substance according to claim 5, wherein the amount of acetone is 3-8 times the amount of the crude gaamicin-related substance; the amount of water is 20-50 times of the amount of the crude product of the gamithromycin related substance.

7. The purification process of a gaamicin-related substance according to claim 5, wherein the amount of acetone is 5-7 times the amount of the crude gaamicin-related substance, and the amount of water is 25-35 times the amount of the crude gaamicin-related substance.

8. An analytical separation method for a gazemycin-related substance according to claim 1, wherein said analytical separation method for a gazemycin-related substance is reversed-phase high performance liquid chromatography, and said analytical separation method comprises separating acetonitrile or a mixed acetonitrile/water solution of a gazemycin-related substance using a C18 preparative column and a mixed system of a 0.02% to 0.15% aqueous trifluoroacetic acid solution and acetonitrile as a mobile phase.

9. The analytical separation method for a gaamicin-related substance according to claim 8, wherein said elution flow rate is 10-50ml/min, the column temperature is 20-50 ℃; the sample injection volume is 0.1-0.5 ml, and the concentration of the related substances of the gamithromycin is 5-100 mg/ml;

when the elution procedure is 0min, the acetonitrile content is 5-25%; when the time is 10min, the acetonitrile content is 15-45%; when the time is 10.1min, the acetonitrile content is 70-100%; when the time is 12min, the acetonitrile content is 80-100%; when the time is 12.1min, the acetonitrile content is 55-5%;

preferably, the elution procedure is that when the time is 0min, the acetonitrile content is 10%; when the time is 10min, the acetonitrile content is 25 percent; when the time is 10.1min, the acetonitrile content is 75 percent; when the time is 12min, the acetonitrile content is 85 percent; at time 12.1min, the acetonitrile content was 15%.

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

Technical Field

The invention belongs to the technical field of chemical medicines, and particularly relates to a method for separating, preparing and purifying a gamithromycin related substance.

Background

Gamithromycin is a semi-synthetic macrolide antibiotic, and is a pentadecentricular macrolide antibiotic obtained by isomerizing, rearranging, reducing and propylating erythromycin A oxime (E type), compared with 15-membered semi-synthetic macrolide azithromycin and tulathromycin, the Gamithromycin undergoes an isomerizing step in the former synthesis to cause the position of N on the ring to be different after ring expansion. The gamithromycin structure contains two polar groups, belongs to diamine macrolide antibiotics and is greatly different from azalide and ketolide antibiotics.

Gamithromycin is marketed in European Union in 2007 and used for treating and preventing bovine respiratory diseases caused by sensitive bacteria such as Pasteurella haemolytica and Haemophilus somni, application to target animal pigs is increased in 2015, and the medicine is marketed in the United states in 2012. Pharmacological experiments and clinical practice results show that the drug effect of the gamithromycin is stronger than that of similar drugs, such as tylosin, tilmicosin and the like, which are widely used in the market, and the gamithromycin has wide application prospect in the breeding of cattle and pigs.

In order to ensure the safety of animals and foods of animal origin, strict quality control of animal-specific drugs for food animals is required, and structural identification of related substances and control of limited amounts of impurities are effective methods for ensuring the quality and safety of drugs, so that research on related substances in drugs is of considerable practical significance in industry. Meanwhile, analysis of related substances is also an important content of drug quality standards, and the analysis of related substances is considered as one of indexes of drug quality standards and is a key item for measuring the quality of drugs.

In the VICH (International coordination Committee for registration technical requirements of veterinary drugs) guideline, the impurity report limit of the veterinary drug special chemical drugs (excluding semisynthetic antibiotics) is 0.10%, the identification limit is 0.20%, and the control limit is 0.50%, and domestic veterinary drug management is carried out according to the guideline at present. Gamithromycin is a semisynthetic antibiotic, the impurity spectrum is complex and difficult to predict, the existing Gamithromycin literature reports a synthesis process of Gamithromycin, and the quality control of Gamithromycin, particularly the condition of related substances, is not reported.

The industry needs researches and methods for separating, preparing and identifying related substances of the gamithromycin.

Disclosure of Invention

In order to overcome the defects that the prior art lacks of gamithromycin related substances with definite structures, and cannot aim at the identification of the gamithromycin related substances and further cannot control the quality of the gamithromycin, the invention provides a gamithromycin related substance, a preparation method thereof and a separation and analysis method thereof.

The invention relates to a gamithromycin related substance, wherein the gamithromycin related substance is a compound with a structural formula shown in a formula (I) or a salt thereof,

the gamithromycin related substance provided by the invention is used as a necessity for quality control of the gamithromycin, and guarantees quality monitoring of large-scale production in the gamithromycin industry.

The invention also relates to a preparation method of the gamithromycin related substance, wherein the method is that the gamithromycin is prepared through degradation reaction; the degradation reaction comprises an acid degradation reaction, an alkali degradation reaction, a high-temperature degradation reaction, a high-humidity degradation reaction, an oxidation degradation reaction or an illumination degradation reaction.

In one embodiment of the present invention, in the method for producing a substance related to gamithromycin according to the present invention, the degradation reaction is an oxidative degradation reaction.

The invention can obtain the related substances of the gamithromycin and provide the structure thereof by a determined method, and the related substances of the gamithromycin prepared by the oxidative degradation reaction have definite structures and can be widely applied to the industry: thereby controlling the quality of the gamithromycin and providing a basis for the research of other unknown related substances of the gamithromycin.

In one embodiment of the present invention, in the method for preparing a gamithromycin-related substance according to the present invention, the oxidative degradation reaction includes: and (3) carrying out reflux reaction on the gamithromycin and hydrogen peroxide in a non-polar solvent, quenching the hydrogen peroxide after the reflux reaction, carrying out layered separation on an organic phase, and drying the organic phase to obtain a coarse product of the related substances of the gamithromycin.

The structure of the preparation method of the related substance is confirmed, and the structure correlation of the related substance and the gamithromycin is verified. The preparation method of the gamithromycin related substance has simple process and high yield, and can realize industrialized production.

As an embodiment of the invention, in the preparation method of the gamithromycin related substance, the nonpolar solvent is chloroform, the reflux reaction pH condition is 9-10, the temperature condition is 62-65 ℃, and the reflux reaction time is 0.5-4 h; cooling to 0-5 deg.C in ice water bath, adding saturated sodium bisulfite solution at low temperature, quenching, and layering; and drying the organic phase, namely organic phase, by vacuum evaporation, wherein the drying temperature is 30 ℃.

In a preferred embodiment of the present invention, in the method for preparing a substance related to gamithromycin according to the present invention, the reflux reaction time is 1 hour.

The invention also relates to a purification method of the gamithromycin related substances, wherein the purification method comprises the steps of adding the crude gamithromycin related substances prepared by the preparation method into acetone, heating to 56-58 ℃ until the mixture is dissolved by refluxing, filtering to remove insoluble substances, dripping water into the filtrate, cooling to 20 +/-5 ℃ after the dripping is finished, stirring for 1-2 hours, and carrying out suction filtration, washing and drying to obtain the gamithromycin related substances.

In one embodiment of the present invention, the amount of acetone in the purification method of a substance related to gamithromycin of the present invention is 3 to 8 times the amount of crude substance related to gamithromycin; the amount of water is 20-50 times of the amount of the crude product of the gamithromycin related substance.

In a preferred embodiment of the present invention, the amount of acetone is 5 to 7 times the amount of the crude gamithromycin-related substance, and the amount of water is 25 to 35 times the amount of the crude gamithromycin-related substance.

The invention also relates to an analytical separation method of the gamithromycin related substances, wherein the analytical separation method of the gamithromycin related substances is reverse phase high performance liquid chromatography, acetonitrile or acetonitrile/water mixed solution of the gamithromycin related substances is separated, a C18 preparation column is adopted, and a mobile phase is a mixed system of 0.02-0.15% trifluoroacetic acid aqueous solution and acetonitrile.

The analytical separation method of the gamithromycin related substances can be used for detecting and separating the gamithromycin related substances, and is efficient and accurate.

In the method for analyzing and separating the substance related to the gamithromycin, the elution flow rate is 10-50ml/min, the column temperature is 20-50 ℃, and the preparative chromatographic column is PhenomenexKintex; the sample introduction volume is 0.1-0.5 ml, and the concentration of related substances is 5-100 mg/ml; when the elution procedure is 0min, the acetonitrile content is 5-25%; when the time is 10min, the acetonitrile content is 15-45%; when the time is 10.1min, the acetonitrile content is 70-100%; when the time is 12min, the acetonitrile content is 80-100%; when the time is 12.1min, the acetonitrile content is 55-5%.

In a preferred embodiment of the present invention, in the method for analyzing and separating a substance related to gamithromycin according to the present invention, the elution procedure is performed for 0min, and the acetonitrile content is 10%; when the time is 10min, the acetonitrile content is 25 percent; when the time is 10.1min, the acetonitrile content is 75 percent; when the time is 12min, the acetonitrile content is 85 percent; at time 12.1min, the acetonitrile content was 15%.

The method for analyzing, separating, preparing and purifying the related substances lays a good foundation for controlling the medicine quality of the gamithromycin and researching unknown impurities.

The invention provides application of the gamithromycin related substance in impurity identification and analysis in quality control of gamithromycin.

Drawings

FIG. 1 shows the total ion flux of a crude product mass spectrum of gamithromycin;

FIG. 2 is a mass spectrum of an oxidation impurity of gamithromycin (3' -N-oxogamithromycin);

FIG. 3 is an HPLC chromatogram of degradation products of gamithromycin acid;

FIG. 4 is an HPLC chromatogram of a degradation product of gamithromycin base;

FIG. 5 is an HPLC chromatogram of an oxidative degradation product of gamithromycin;

FIG. 6 is an HPLC chromatogram of a high temperature degradation product of gamithromycin;

FIG. 7 is an HPLC chromatogram of a degradation product of gamithromycin by light;

FIG. 8 is an HPLC chromatogram of a blank control product of gamithromycin.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

Definition of

The invention relates to a gamithromycin related substance, which is introduced or degraded in a gamithromycin synthesis process. Related substance research is one of key projects in medicine quality research, and the content of related substances is a direct index reflecting the purity of medicines. The purity requirement of the medicine is based on the consideration of both safety and practical production conditions.

The Gamithromycin (Gamithromycin) is a novel semi-synthetic macrolide (Marolides) veterinary antibiotic, is one of representative drugs of second-generation macrolide antibiotics developed by French Merriya, and has antibacterial and bactericidal effects by inhibiting the synthesis of bacterial RNA-dependent protein. The gamithromycin has high antibacterial activity on Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and the like which cause bovine respiratory system diseases (BRD). It has wide antibacterial spectrum, high antibacterial activity, quick absorption, wide in vivo distribution, low in vivo residue, high safety, etc.

"non-polar solvent" refers to a class of solvents having a low dielectric constant, and non-polar solvents are not all non-polar, and some less polar solvents are classified as non-polar solvents, such as chloroform.

High Performance Liquid Chromatography (HPLC) is an important branch of Chromatography, in which Liquid is used as a mobile phase, a High-pressure infusion system is adopted, mobile phases such as single solvents with different polarities or mixed solvents and buffers with different proportions are pumped into a chromatographic column filled with a stationary phase, and after components in the column are separated, the mobile phases enter a detector for detection, so that analysis of a sample is realized.

The reversed-phase high performance liquid chromatography is a liquid chromatography system consisting of a non-polar fixed phase and a polar mobile phase, wherein the fixed phase is octadecyl bonded silica gel, and typical mobile phases are methanol and acetonitrile. Can be used for separating almost all organic matters which can be dissolved in polar or weak polar solvents. Reverse phase chromatography is suitable for separating non-polar, polar or ionic compounds, and most of the analytical tasks are performed by reverse phase chromatography.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.

In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.