CN113365973B

CN113365973B - Phenoxyacetic acid derivative and method for preparing penicillin V salt by using same through enzymatic method

Info

Publication number: CN113365973B
Application number: CN202080011415.2A
Authority: CN
Inventors: 许岗; 张娇; 曾红宇; 黄斌; 宋子博
Original assignee: Hunan Flag Biotechnology Co ltd
Current assignee: Hunan Flag Biotechnology Co ltd
Priority date: 2019-01-30
Filing date: 2020-01-15
Publication date: 2024-04-19
Anticipated expiration: 2040-01-15
Also published as: CN109628541A; CN113365973A; WO2020156176A1; CN109628541B

Abstract

The application belongs to the technical field of antibiotic medicines, and relates to a phenoxyacetic acid derivative and a method for preparing penicillin V salt by using the same through an enzymatic method. The derivative is phenoxyacetic glyceride. The method comprises the following steps: (1) Reacting 6-APA with said derivative in an aqueous solution under the catalysis of penicillin acylase; (2) After the reaction is completed, penicillin V obtained by acidification reaction is separated to obtain penicillin V salt after salinization. The method for preparing penicillin V salt by using the phenoxyacetic acid derivative and the enzymatic method can prepare penicillin V salt by using the phenoxyacetic acid derivative with better enzymatic method.

Description

Phenoxyacetic acid derivative and method for preparing penicillin V salt by using same through enzymatic method

Cross Reference to Related Applications

The present application claims priority from chinese patent application number 201910089112.1 filed on 1/30 2019, the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The application belongs to the technical field of antibiotic medicines, and relates to a phenoxyacetic acid derivative and a method for preparing penicillin V salt by using the same through an enzymatic method.

Background

Penicillin V salts, such as penicillin V potassium and penicillin V sodium, are clinically common antibiotic drugs, with penicillin V potassium being used more widely. Penicillin V potassium beta-lactamase antibiotics have an antibacterial spectrum similar to penicillin G, can destroy bacterial cell walls, have bactericidal effect, are the first choice drugs for treating gram positive bacteria and partial negative bacteria infection, and have been widely applied to the clinic for treating abscess, suppurative meningitis, pneumonia, gonorrhea and other diseases. Compared with penicillin G potassium/sodium, penicillin V potassium is more stable to acid, is not easy to damage by gastric acid, is better to be absorbed orally, is particularly suitable for children, and is more effective to infection caused by drug-resistant staphylococcus aureus because of slower damage by beta-lactamase.

Penicillin V potassium tablets were first applied to the clinic in 1952, and were approved by the FDA in the united states in 9 of 1957, and received in the united states pharmacopeia, british pharmacopeia, japan drug administration, etc. At present, penicillin V potassium tablets are produced in the United states, canada, england, germany, japan and other countries, and are an oral antibiotic with wide clinical application in the world.

Penicillin has a high probability of anaphylactic reaction, and common anaphylactic reactions comprise rash, urticaria, dermatitis, fever, angioneurotic edema, asthma, anaphylactic shock and the like, wherein the anaphylactic shock is the most serious and even can cause death. In order to prevent anaphylactic reaction, especially serious anaphylactic reaction, the penicillin injection needs to be subjected to skin sensitivity test before use, and a skin test negative medicament can be used for patients, and if the skin test is positive, the skin test is forbidden; the penicillin drugs orally taken abroad are skin-free.

In recent years, the demand for oral penicillin V preparations is greatly increased, but the quality of penicillin V salt bulk pharmaceutical products still has a great problem. Therefore, the production of penicillin V salt bulk drug with high quality level (low impurity content, especially low polymer impurity content) is of great significance. Penicillin V potassium raw materials and preparations may contain synthesis precursors, process byproducts and various degradation products thereof; in addition, penicillin V potassium may also polymerize itself, producing a high molecular weight polymer. The largest sources of process byproducts, degradation products and polymer impurities are the production process, and the longer the production process, the greater the probability of impurity production. Allergic reactions caused by beta-lactam antibiotics have been examined for many years and have been shown to be associated with the presence of high molecular weight polymers in pharmaceuticals. The existing method for producing penicillin V potassium has the defects of long fermentation period and long production procedure, so that various process byproducts, degradation products and polymer impurities are high in content.

Penicillin V salt is not prepared by a biological enzyme method at present, and is not prepared by reversely catalyzing 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid derivatives by penicillin acylase.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

The primary object of the present application is to provide phenoxyacetic acid derivatives, in order to be able to use their better enzymatic processes for the preparation of penicillin V salts.

To achieve this object, in a basic embodiment, the present application provides phenoxyacetic acid derivatives, said derivatives being phenoxyacetic acid glycerides (including glyceryl monophenoxyacetate, glyceryl diphenoxyacetate, glyceryl triphenoxyacetate).

In an alternative embodiment, the present application provides phenoxyacetic acid derivatives wherein the derivative is glycerol monophenoxyacetate.

In an alternative embodiment, the instant application provides a phenoxyacetic acid derivative wherein the derivative has the structure shown in formula (I):

A second object of the present application is to provide a process for enzymatic preparation of penicillin V salt using the aforementioned phenoxyacetic acid derivatives, so as to enable better enzymatic preparation of penicillin V salt.

To achieve this object, in a basic embodiment, the present application provides a process for preparing penicillin V salt by the aforementioned phenoxyacetic acid derivative enzymatic method, said process comprising the steps of:

step (1): reacting 6-APA with said derivative in an aqueous solution under the catalysis of penicillin acylase;

step (2): after the reaction is completed, penicillin V obtained by acidification reaction is separated to obtain penicillin V salt after salinization.

In an alternative embodiment, the present application provides a method for preparing penicillin V salt by using the phenoxyacetic acid derivative enzyme method, wherein in the step (1), the penicillin acylase is an immobilized penicillin acylase.

In an alternative embodiment, the present application provides a method for preparing penicillin V salt by using the phenoxyacetic acid derivative enzyme method, wherein in the step (1), the penicillin acylase is a mutant of natural penicillin acylase, and has an amino acid sequence shown in SEQ ID No. 1.

In an alternative embodiment, the present application provides a process for preparing penicillin V salt by using the aforementioned phenoxyacetic acid derivative enzyme method, wherein in step (1),

The molar ratio of the 6-APA to the derivative is 1:1.1-1.5, wherein the weight ratio of the 6-APA to the penicillin acylase is 1:2-4,

The reaction temperature is 5-40 ℃, the reaction pH is 4.0-8.0, and the reaction time is 1-2h.

In a further alternative embodiment, the present application provides a process for preparing penicillin V salt by using the aforementioned phenoxyacetic acid derivative enzyme method, wherein in step (1), the reaction temperature is 10-30 ℃, the reaction pH is 5.0-6.0, and the reaction time is 1-2 hours.

In an alternative embodiment, the present application provides a method for preparing penicillin V salt by using the phenoxyacetic acid derivative enzyme method, wherein in the step (2), the acidification is to adjust the pH of the solution to 2.5-3.5 by using acid, and the crystallization is carried out for 10-30 minutes.

In an alternative embodiment, the application provides a method for preparing penicillin V salt by using the phenoxyacetic acid derivative enzyme method, wherein in the step (2), the salinization is that the pH value of a solution is firstly regulated to 5.5-7.5 by using carbonate, then a water carrying agent (one or more of ethanol, isopropanol, n-butanol and isobutanol) is added into the solution, and the solution is evaporated and concentrated until penicillin V salt crystals are separated out.

In an alternative embodiment, the present application provides a process for the enzymatic preparation of penicillin V salts using the aforementioned phenoxyacetic acid derivatives, wherein in step (2) the penicillin acylase may also be separated from the solution prior to the acidification.

The embodiment of the application has the beneficial effects that:

1) The application discovers that the latest reported penicillin G acylase mutant and penicillin V acylase mutant have good catalytic activity on the reaction between 6-APA and phenoxyacetic acid derivatives for the first time, and can realize the efficient synthesis of penicillin V so as to further obtain high-purity penicillin V salt.

2) The penicillin V salt has short production period, and the whole process of penicillin V salt synthesis, purification and crystallization does not exceed 24 hours.

3) The penicillin V salt production process is simple to operate, and only one-step synthesis and two-step crystallization steps are needed.

4) The synthesis process of the penicillin V salt is green and pollution-free, and does not need to use butyl acetate and the like for extraction and purification.

5) The penicillin V salt synthesis of the application takes 6-APA and phenoxyacetic acid derivatives as raw materials, the phenoxyacetic acid derivatives have good water solubility and low synthesis cost, can react with 6-APA in solution under the catalysis of penicillin acylase catalyst to generate penicillin V, and has the advantages of small required penicillin acylase catalyst amount, repeated utilization of penicillin acylase and low cost.

6) The penicillin V salt prepared by the application has less impurities, good product quality, high product purity and high yield, is far higher than the penicillin V salt produced by the existing fermentation method, is higher than the quality standard of similar foreign raw medicines, has the 6-APA conversion rate of 99.5 percent and the liquid phase purity of more than 99.7 percent, is not detected by 4-hydroxyphenoxymethyl penicillin (impurity D), has the polymer impurity lower than 0.01 percent, and can change the current situation that the prior penicillin V medicine in China has to be subjected to skin test.

7) The byproduct generated in the whole process of penicillin V salt synthesis is glycerin, which is harmless to enzymes and has no pollution to the environment.

Drawings

FIG. 1 is an NMR spectrum of glycerol monophenoxyacetate prepared in example 1.

Detailed Description

Specific embodiments of the present application are further described below with reference to examples and drawings.

Example 1: preparation of glyceryl monophenoxyacetate of the structure shown in formula (I)

Step (1): weighing 30.0g of phenoxyacetic acid, adding 270.0g of glycerol, heating to 60 ℃ to enable the phenoxyacetic acid to be fully dissolved, pouring the mixture into a closed reactor, adding 30.0g of immobilized lipase, uniformly mixing the mixture, keeping the vacuum degree of a reaction system below-0.1 MPa, keeping the reaction temperature at 50 ℃, reacting for 360 minutes, and separating the immobilized enzyme from a reaction solution by a screen to obtain a reaction product.

Step (2): adding 200ml of water into the reaction product obtained in the step (1), regulating the pH value to 7.0 by using 10% (m/v) sodium carbonate solution, adsorbing by using macroporous adsorption resin, eluting by using 30% (v/v) ethanol solution, collecting the solution with the purity of the compound I higher than 98.0%, carrying out reduced pressure distillation at 50 ℃ to the minimum volume, and collecting the concentrated solution.

The reaction product obtained in the step (1) and the concentrated solution obtained in the step (2) are respectively sampled for HPLC analysis, and the analysis conditions are as follows:

Chromatographic column: diamosil C18 (2) 5 μm 4.6X1250 mm

Mobile phase: acetonitrile: 0.1% (m/v) aqueous acetic acid ammonia solution=60:40

Column temperature: 25 DEG C

Detection wavelength: 220nm

Total flow rate: 1.0ml/min

Sample injection amount: 5 μl

Preparing a test solution: taking 30-35mg of sample to be measured in a 10ml volumetric flask, dissolving with mobile phase, and fixing the volume to scale.

Sampling the concentrated solution obtained in the step (2) for ESI mass spectrometry and NMR analysis (ESI condition: thermo Finnigan mass spectrometer, model LCQ ADVANTAGE, solvent methanol; NMR condition: bruce Nuclear magnetic resonance spectrometer, model AVANCE III HD 400MHz, solvent deuterated chloroform), the obtained HPLC analysis results are shown in Table 1 below, the obtained mass spectrometry detection result is 474.69[2M+Na ], and the obtained NMR spectrum is shown in FIG. 1 (¹H-NMR(CDCl₃,400MHz)：δ7.293(1H,d,H-2);δ7.274(1H,d,H-6);δ6.908(1H,S,H-3);δ6.888(1H,S,H-5);δ7.002(1H,t,H-4);δ4.699(2H,m,H-7);δ4.269(2H,m,H-9);δ3.918(1H,t,H-10);δ3.656(2H,m,H-11);δ3.319(1H,S,OH-10);δ2.927(1H,S,OH-11)).

TABLE 1 HPLC analysis results

Wherein, the structure of the compound I is as follows:

The structure of the compound II is as follows:

The structure of the compound III is as follows:

example 2: enzymatic method for preparing penicillin V salt

25.0G of 6-APA is taken, 400mL of pure water is added, 3mol/L of ammonia water solution is added dropwise to fully dissolve the 6-APA, phenoxyacetic acid derivatives are added and uniformly mixed, then immobilized penicillin acylase with an amino acid sequence shown as SEQ ID NO.1 (namely, mutant penicillin G acylase SPGA-4 with an amino acid sequence shown as SEQ ID NO.3 in China patent application CN201510736957.7, the preparation method of the mutant penicillin G acylase SPGA-4 is shown as example 1-3 of China patent application CN 201510736957.7) is added dropwise, and the reaction pH is regulated by adding 3mol/L of ammonia water solution. The reaction is carried out at 20 ℃, the HPLC detection is carried out by sampling at regular time, and the detection conditions are as follows:

chromatographic column: diamosil C18,5 μm, 4.6X1250 mm

Mobile phase a phase: phosphate buffer pH3.5 (0.5 moL/L potassium dihydrogen phosphate was adjusted to pH 3.50 with phosphoric acid) -methanol-water (10:30:60)

Mobile phase B phase: phosphate buffer pH3.5 (0.5 moL/L potassium dihydrogen phosphate was adjusted to pH 3.50 with phosphoric acid) -methanol-water (10:55:35)

Column temperature: 25 DEG C

Detection wavelength: 268nm

Total flow rate: 1.0mL/min

Sample injection amount: 20 μl.

Gradient elution was performed as follows:

Time (min)	Mobile phase a (%)	Mobile phase B (%)
			0	40	60
27	40	60
			47	0	100
62	0	100
			65	40	60

After the reaction is completed, separating the immobilized enzyme from the reaction solution by using a screen, regulating the pH value of the reaction solution to 2.8 by using 6mol/L hydrochloric acid, growing the crystal for 20 minutes, and filtering to obtain the penicillin V acid filter cake. The filter cake was transferred to 4-fold weight of pure water, the pH of the mixed solution was adjusted to 6.3 with 10% (m/v) potassium carbonate solution, and n-butanol was then added to 63% (v/v) n-butanol. Concentrating under reduced pressure until penicillin V salt crystals are separated out, filtering, washing and drying the crystals to obtain penicillin V potassium crystal powder.

The above different operating conditions and the detection results are shown in tables 2 and 3 below.

TABLE 2 different operating conditions

TABLE 3 detection results corresponding to different operation conditions

Other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present application without departing from the spirit and scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The foregoing examples or implementations are merely illustrative of embodiments of the present application, and the present application may be embodied in other specific forms or with other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The scope of the application should be indicated by the appended claims, and any changes that are equivalent to the intent and scope of the claims are intended to be encompassed within the scope of the application.

Claims

1. A phenoxyacetic acid derivative having a structure represented by the following formula (I):

2. a process for preparing penicillin V salt by using the phenoxyacetic acid derivative enzyme method of claim 1, comprising the steps of:

3. The method according to claim 2, wherein the penicillin acylase in step (1) is an immobilized penicillin acylase.

4. The method according to claim 2, wherein the penicillin acylase of step (1) is a mutant of a natural penicillin acylase having the amino acid sequence shown in SEQ ID No. 1.

5. The method of claim 2, wherein the molar ratio of 6-APA to the derivative in step (1) is 1:1.1-1.5, wherein the weight ratio of the 6-APA to the penicillin acylase is 1:2-4.

6. The process according to claim 5, wherein the reaction temperature in step (1) is 5-40 ℃, the reaction pH is 4.0-8.0, and the reaction time is 1-2h.

7. The method of claim 2, wherein the acidification in step (2) is carried out by adjusting the pH of the solution to 2.5-3.5 with acid, and growing the crystals for 10-30 minutes.

8. The method according to claim 2, wherein the salinization in step (2) is performed by adjusting the pH of the solution to 5.5-7.5 with carbonate, adding a water-carrying agent into the solution, and evaporating and concentrating until penicillin V salt crystals are separated out.

9. The method of claim 2, wherein in step (2), the penicillin acylase is further separated from the solution prior to the acidification.