阅读说明：本技术 一种头孢米诺7β-甲氧基立体异构体的分离纯化方法 (Separation and purification method of cefminox 7 beta-methoxy stereoisomer ) 是由 王辉 高全喜 于 2020-03-26 设计创作，主要内容包括：本发明属于药物技术领域,具体涉及一种头孢米诺7β-甲氧基立体异构体的分离纯化方法。该分离纯化方法包括步骤：1)以7-氨基-7-甲氧基-3-[(1-甲基-1H-四唑-5-基)硫甲基]-3-头孢烯-4-羧酸二苯甲酯为原料合成头孢米诺溶液,利用旋转蒸发仪除去大部分溶剂,得到含有头孢米诺7β-甲氧基立体异构体的粘稠状液体；2)用制备液相色谱进行分离纯化,根据色谱图收集头孢米诺7β-甲氧基立体异构体对应的洗脱流分,进行预冻后,放入真空冻干机中进行冻干,即得目标产物。本发明通过制备液相色谱分离纯化,经冷冻干燥得到白色粉末状头孢米诺异构体纯品。本发明得到的头孢米诺7β-甲氧基异构体质量好、纯度高,液相纯度在98%以上。(The invention belongs to the technical field of medicines, and particularly relates to a separation and purification method of cefminox 7 beta-methoxyl stereoisomer. The separation and purification method comprises the following steps: 1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid diphenylmethyl ester as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer; 2) separating and purifying by using a preparative liquid chromatography, collecting an elution fraction corresponding to the cefminox 7 beta-methoxy stereoisomer according to a chromatogram, pre-freezing, and freeze-drying in a vacuum freeze dryer to obtain the target product. The invention obtains a white powdery cefminox isomer pure product by separation and purification of preparative liquid chromatography and freeze drying. The cefminox 7 beta-methoxyl isomer obtained by the method has good quality and high purity, and the liquid phase purity is more than 98%.)

1. A separation and purification method of cefminox 7 beta-methoxyl stereoisomer is characterized in that: the separation and purification method comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparative liquid chromatogram, wherein a chromatographic column is a stationary phase of octadecylsilane chemically bonded silica, a mobile phase is a 4-8% acetonitrile water solution, isocratic elution is carried out, the flow rate is 10-20 mL/min, the wavelength is 190-400 nm, the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer is prepared into a solution with the mass concentration of 5-15 mg/mL by using the mobile phase, 5-15 mL of the solution is injected into the preparative liquid chromatogram for separation and purification, collecting elution fractions corresponding to the cefminox 7 beta-methoxy stereoisomer according to the chromatogram, pre-freezing, and (3) putting the mixture into a vacuum freeze dryer for freeze drying to obtain the target impurity cefminox 7 beta-methoxyl stereoisomer.

2. The method for separating and purifying cefminox 7 beta-methoxy stereoisomer of claim 1, which comprises the following steps: the mobile phase of 4-8% acetonitrile water solution, with the mass percentage content of 0.05% acetic acid and ammonia water to adjust the pH value to 5.5.

3. The method for separating and purifying cefminox 7 beta-methoxy stereoisomer of claim 1, which comprises the following steps: the separation and purification method comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separation of the preparative liquid phase: separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparation solution, wherein a chromatographic column is fixed phase octadecylsilane chemically bonded silica, a mobile phase is 6% acetonitrile, isocratic elution is carried out, the flow rate is 15mL/min, the wavelength is 254nm, the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer is prepared into a solution with the mass concentration of 10mg/mL by using a mobile phase, 10mL of the solution is injected into a preparation liquid phase for separation and purification, and an effluent liquid of a target peak is collected;

3) separating and purifying all the viscous liquid obtained in the step 1) according to the method in the step 2), and combining effluent liquid containing target peaks to obtain a solution;

4) pre-freezing the solution obtained in the step 3) at-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze-drying to obtain the target product cefminox 7 beta-methoxyl stereoisomer.

4. The method for separating and purifying cefminox 7 beta-methoxy stereoisomer of claim 1, which comprises the following steps: the separation and purification method comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separation of the preparative liquid phase: separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparation solution, fixing a chromatographic column on octadecylsilane chemically bonded silica, carrying out isocratic elution by using a mobile phase of 4% acetonitrile, carrying out flow rate of 20mL/min and wavelength of 254nm, matching the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer into a solution with the mass concentration of 10mg/mL by using the mobile phase, injecting 10mL of the solution into the preparation liquid phase for separation and purification, and collecting an effluent liquid of a target peak;

3) separating and purifying all the viscous liquid obtained in the step 1) according to the method in the step 2), and combining effluent liquid containing target peaks to obtain a solution;

4) and (4) pre-freezing the solution obtained in the step (3) at the temperature of-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze drying to obtain the target product.

5. The method for separating and purifying cefminox 7 beta-methoxy stereoisomer of claim 1, which comprises the following steps: the separation and purification method comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separating and purifying the isomer sample by using a preparation solution, wherein a chromatographic column is a stationary phase of octadecylsilane chemically bonded silica, a mobile phase is 8% acetonitrile water, isocratic elution is performed, the flow rate is 10mL/min, the wavelength is 254nm, the viscous liquid in the step 1) is matched into a solution of 10mg/mL by using a mobile phase, 10mL of the solution is injected into the preparation liquid phase for separation and purification, and an effluent liquid of a target peak is collected;

3) separating and purifying all the viscous liquid obtained in the step 1) according to the method in the step 2), and combining effluent liquid containing target peaks to obtain a solution;

4) pre-freezing the solution obtained in the step 3) at-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze drying to obtain a target product.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a separation and purification method of cefminox 7 beta-methoxyl stereoisomer.

Background

Cefminox is a cephamycin that entered the market in china in the last 90 th century. As a broad-spectrum antibacterial drug, the compound has broad-spectrum antibacterial action on anaerobic bacteria such as aerobic gram-negative bacteria, gram-positive bacteria, bacteroides fragilis and the like, is stable to broad-spectrum beta lactamase and extended-spectrum beta lactamase generated by bacteria, and is mainly used for septicemia, tonsillitis, tracheitis, pneumonia, pulmonary suppuration, pyelonephritis, cystitis, cholecystitis, peritonitis, pelvic inflammation, adnexitis, endometritis, abdominal postoperative infection and the like caused by sensitive bacteria.

Because the different process routes of the synthesis of cefminox (the structure is shown in figure 1) can generate 7 beta-methoxy cefminox acid (the structure is shown in figure 2) which is an impurity different from the original medicine, the impurity is cefminox 7 beta-methoxy stereoisomer, and a reference substance of the impurity must be identified and provided according to the requirement of the registration report of the imitation drugs, so the 7 beta-methoxy stereoisomer is prepared by liquid phase separation and purification according to the requirements of the registration and the process research of the imitation drugs.

The invention takes 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazole-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid diphenylmethyl ester as a raw material to synthesize a cefminox solution, and the reference literature Tetrahedron Lett, 1982, 23, (29): 2977-2980.

Disclosure of Invention

The invention aims to provide a separation and purification method of cefminox 7 beta-methoxy stereoisomer, which is based on cefminox reaction liquid synthesized by taking 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazole-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material and prepares cefminox 7 beta-methoxy stereoisomer by liquid phase purification.

A method for separating and purifying cefminox isomer impurities comprises the following steps:

a separation and purification method of cefminox 7 beta-methoxy stereoisomer comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparative liquid chromatogram, wherein a chromatographic column is a stationary phase of octadecylsilane chemically bonded silica, a mobile phase is 4-8% of acetonitrile water, isocratic elution is carried out, the flow rate is 10-20 mL/min, the wavelength is 190-400 nm, the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer is prepared into a solution with the mass concentration of 5-15 mg/mL by using the mobile phase, 5-15 mL of the solution is injected into the preparative liquid chromatogram for separation and purification, collecting elution fractions corresponding to the cefminox 7 beta-methoxy stereoisomer according to the chromatogram, pre-freezing, and (3) putting the mixture into a vacuum freeze dryer for freeze drying to obtain the target impurity cefminox 7 beta-methoxyl stereoisomer.

Preferably, acetonitrile water in 4-8% of the mobile phase is adjusted to pH5.5 by using 0.05% by mass of acetic acid water and ammonia water.

Preferably, the separation and purification method of the cefminox 7 beta-methoxy stereoisomer comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separation of the preparative liquid phase: separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparation solution, wherein a chromatographic column is fixed phase octadecylsilane chemically bonded silica, a mobile phase is 6% acetonitrile, isocratic elution is carried out, the flow rate is 15mL/min, the wavelength is 254nm, the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer is prepared into a solution with the mass concentration of 10mg/mL by using a mobile phase, 10mL of the solution is injected into a preparation liquid phase for separation and purification, and an effluent liquid of a target peak is collected;

3) completely separating and purifying the viscous liquid obtained in the step 1) according to the method of the step 2), and combining effluent liquid of all target peaks to obtain a solution;

4) pre-freezing the solution obtained in the step 3) at-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze-drying to obtain the target impurity cefminox 7 beta-methoxyl stereoisomer.

Preferably, the separation and purification method of cefminox 7 beta-methoxy stereoisomer comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separation of the preparative liquid phase: separating and purifying the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer prepared in the step 1) by using a preparation solution, fixing a chromatographic column on octadecylsilane chemically bonded silica, carrying out isocratic elution by using a mobile phase of 4% acetonitrile, carrying out flow rate of 20mL/min and wavelength of 254nm, matching the viscous liquid containing the cefminox 7 beta-methoxy stereoisomer into a solution with the mass concentration of 10mg/mL by using the mobile phase, injecting 10mL of the solution into the preparation liquid phase for separation and purification, and collecting an effluent liquid of a target peak;

3) completely separating and purifying the viscous liquid obtained in the step 1) according to the method of the step 2), and combining effluent liquid of all target peaks to obtain a solution;

4) and (4) pre-freezing the solution obtained in the step (3) at the temperature of-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze drying to obtain the target impurity.

Preferably, the separation and purification method of cefminox 7 beta-methoxy stereoisomer comprises the following steps:

1) synthesizing a cefminox solution by using 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-diphenylmethyl carboxylate as a raw material, and removing most of a solvent by using a rotary evaporator to obtain a viscous liquid containing a cefminox 7 beta-methoxy stereoisomer;

2) separating and purifying the isomer sample by using a preparation solution, wherein a chromatographic column is a stationary phase of octadecylsilane chemically bonded silica, a mobile phase is 8% acetonitrile water, isocratic elution is performed, the flow rate is 10mL/min, the wavelength is 254nm, the viscous liquid in the step 1) is matched into a solution of 10mg/mL by using a mobile phase, 10mL of the solution is injected into the preparation liquid phase for separation and purification, and an effluent liquid of a target peak is collected;

3) completely separating and purifying the viscous liquid obtained in the step 1) according to the method of the step 2), and combining effluent liquid of all target peaks to obtain a solution;

4) pre-freezing the solution obtained in the step 3) at the temperature of-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze drying to obtain the target impurity.

The technical advantages of the invention are as follows:

the invention takes 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazole-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid diphenylmethyl ester as the raw material to synthesize cefminox reaction liquid, and obtains a cefminox isomer pure product by liquid chromatography separation and purification, and freeze drying. The method has simple steps and saved working procedures, can quickly and simply obtain the impurity reference substance, does not need dangerous and highly toxic chemical reagents, and is very environment-friendly. The cefminox pure product obtained by the method has good quality and high purity, and the liquid phase purity is more than 98%.

Drawings

FIG. 1 shows the structural formula of cefminox.

FIG. 2 shows the structural formula of 7 beta-methoxy cefminox acid.

FIG. 3 is an analytical chromatogram of a viscous liquid containing 7 β -methoxy cefminox acid.

FIG. 4 is a chromatogram of 7 β -methoxy cefminox acid preparation.

FIG. 5 is an analytical chromatogram of 7 β -methoxycefminox acid obtained in example 1.

FIG. 6 is an analytical chromatogram of 7 β -methoxycefminox acid obtained in example 2.

FIG. 7 is an analytical chromatogram of 7 β -methoxycefminox acid obtained in example 3.

FIG. 8 is the hydrogen spectrum of the target pure compound 7 beta-methoxy cefminox acid.

FIG. 9 is a carbon spectrum diagram of the obtained target pure compound 7 beta-methoxy cefminox acid.

FIG. 10 is the mass spectrum of the obtained target pure compound 7 beta-methoxy cefminox acid.

Detailed Description

The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.

Examples 1

First, experimental material

Preparing a liquid phase: HT7100A

Analyzing a liquid phase: waters2996/2695

Preparing a chromatographic column: RPS-C18-50um (50 um, 15 x 460 mm)

Analyzing a chromatographic column: HT-ODS-P (5um, 4.6x 250 mm)

Experimental materials: 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid benzhydryl ester.

Second, test methods and results

1) 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid diphenylmethyl ester is used as a raw material to synthesize a solution of cefminox, most of solvent is removed by using a rotary evaporator to be viscous, and viscous liquid containing cefminox 7 beta-methoxy stereoisomer is obtained, an analytical chromatogram of the viscous liquid is shown in figure 3, and the viscous liquid can be seen to contain 7 beta-methoxy cefminox acid.

2) Separation of the preparative liquid phase: separating and purifying the sample containing 7 beta-methoxy cefminox acid by using the preparation solution, carrying out isocratic elution by using a chromatographic column with RPS-C18-50um (50 um, 15 x 460 mm) and a mobile phase with 6% acetonitrile water (0.05% acetic acid water and ammonia water for adjusting the pH value to be 5.5), matching the flow rate of the viscous liquid in the step 1) into a solution with the wavelength of 254nm by using the mobile phase, injecting 10mL of the viscous liquid into the preparation solution for separation and purification, collecting the effluent of a target peak, and obtaining a preparation chromatogram shown in figure 4.

3) Separating and purifying the viscous liquid obtained in the step 1) according to the method of the step 2), and combining the effluent liquid of all target peaks.

4) Pre-freezing the solution obtained in the step 3) at-20 ℃, and directly putting the solution into a vacuum freeze dryer for freeze-drying to obtain the target impurity substance, wherein the yield is about 61.3%, and the HPLC normalized purity reaches 98.79% (see figure 5).

EXAMPLES example 2

First, experimental material

Preparing a liquid phase: HT7100A

Analyzing a liquid phase: waters2996/2695

Preparing a chromatographic column: RPS-C18-50um (50 um, 15 x 460 mm)

Analyzing a chromatographic column: HT-ODS-P (5um, 4.6x 250 mm)

Experimental materials: 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid benzhydryl ester.

Second, test methods and results

1) In the same manner as in example 1, a solution of cefminox was synthesized from 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid benzhydryl ester, and most of the solvent was removed by a rotary evaporator to a viscous state, thereby obtaining a viscous liquid containing cefminox 7 β -methoxy stereoisomer.

2) Separating and purifying a sample of cefminox 7 beta-methoxy stereoisomer by using preparative liquid chromatography, wherein a chromatographic column is RPS-C18-50um (50 um, 15 x 460 mm), a mobile phase is 4% acetonitrile water (0.05% acetic acid water, ammonia water for adjusting pH to 5.5), isocratic elution is carried out, the flow rate is 20mL/min, the wavelength is 254nm, the viscous liquid in the step 1) is prepared into a solution of 10mg/mL by using the mobile phase, 10mL of the solution is injected into the preparative liquid phase for separation and purification, and effluent of a target peak is collected.

3) Separating and purifying the viscous liquid obtained in the step 1) according to the method of the step 2), and combining the effluent liquid of all target peaks.

4) And (3) pre-freezing the solution obtained in the step (3) at-20 ℃, directly putting the solution into a vacuum freeze dryer for freeze drying to obtain the target impurity substance, wherein the yield is about 59.8%, and the HPLC normalized purity reaches 98.77%, which is shown in figure 6.

EXAMPLE 3

First, experimental material

Preparing a liquid phase: HT7100A

Analyzing a liquid phase: waters2996/2695

Preparing a chromatographic column: RPS-C18-50um (50 um, 15 x 460 mm)

Analyzing a chromatographic column: HT-ODS-P (5um, 4.6x 250 mm)

Experimental materials: 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid benzhydryl ester.

Second, test methods and results

1) 1) in the same manner as in example 1, 7-amino-7-methoxy-3- [ (1-methyl-1H-tetrazol-5-yl) thiomethyl ] -3-cephem-4-carboxylic acid benzhydryl ester was used as a raw material to synthesize a solution of cefminox, and most of the solvent was removed by a rotary evaporator to a viscous state, thereby obtaining a viscous liquid containing a cefminox 7 β -methoxy stereoisomer.

2) Separating and purifying a sample of cefminox 7 beta-methoxy stereoisomer by using preparative liquid chromatography, wherein a chromatographic column is RPS-C18-50um (50 um, 15 x 460 mm), a mobile phase is 8% acetonitrile water (0.05% acetic acid water, ammonia water for adjusting pH to 5.5), isocratic elution is carried out, the flow rate is 10ml/min, the wavelength is 254nm, the viscous liquid in the step 1 is prepared into a solution of 10mg/ml by using the mobile phase, 10ml of the solution is injected into the preparative liquid phase for separation and purification, and effluent of a target peak is collected.

3) The viscous liquid obtained in step 1 is completely separated and purified according to the method of step 2, and the effluent liquid of all target peaks is combined.

4) And (3) pre-freezing the solution obtained in the step (3) at the temperature of-20 ℃, directly putting the solution into a vacuum freeze dryer for freeze drying to obtain the target impurity substance, wherein the yield is about 60.1%, and the HPLC normalized purity reaches 97.97%, which is shown in figure 7.

The obtained pure compound is determined to be the target 7 beta-methoxy cefminox acid through structure identification, and the structure identification data is as follows:

¹H-NMR（400Mz，D₂O）:δ5.15(s，1H，CH)，4.32-4.29（dd，1H，CHN），4.06（s，3H，NH），4.00-3.98（dd，2H，CH ₂），3.78-3.47（dd，2H，CH ₂），3.54（s，3H，CH ₃），3.52（d，2H，CH ₂），3.24-3.15（dq，2H，CH ₂). See fig. 8.

¹³C-NMR（400Mz，D₂O): δ 173.1, 172.4, 167.2, 159.7, 153.4, 130.5, 119.1, 92.3, 63.2, 53.5, 53.0, 36.3, 34.9, 34.1, 32.9, 26.4. See fig. 9.

ESI-MS (m/z): 558.03[ M + K ] +. See fig. 10.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.