KR20010069097A - Process for preparation of β-lactam antibiotics by enzymatic acylation - Google Patents

Abstract

PURPOSE: The method for producing beta-lactam antibiotics using enzymatic acylation is provided, thereby the beta-lactam antibiotics can be simply and cheaply produced using enzymes. CONSTITUTION: The beta-lactam antibiotics represented by formula (3) is produced by acylation reacting a compound represented by formula (1) with a compound represented by formula (2) using enzyme penicillin amidase, in which, R1CO is diphenyl glycine or dipara hydroxyphenyl glycine, X is OR2 or NR3R4, wherein R2 is C1 to C7 alkyl or haloalkyl, and R3 and R4 are individually hydrogen or C1 to C3 alkyl, and the penicillin amidase is derived from E. coli, Acetobacter pasteurianum, Xanthomonas citri, Kluyveri citrophylla or Bacillus megaterium. The beta-lactam antibiotics represented by formula (5) is produced by acylation reacting a compound represented by formula (4) with a compound represented by formula (2) using enzyme penicillin amidase, in which Z is hydrogen, halogen, C1 to C3 alkyl, C2 to C4 alkenyl, nuclear philic group-substituted C1 to C3 alkyl; R1CO is diphenyl glycine or dipara hydroxyphenyl glycine; X is OR2 or NR3R4, wherein R2 is C1 to C7 alkyl or haloalkyl, and R3 and R4 are individually hydrogen or C1 to C3 alkyl.

Description

Process for preparation of β-lactam antibiotics by enzymatic acylation}

The present invention relates to a method for preparing beta-lactam antibiotics, and in particular, the conventional chemical acylation reaction in the production of beta-lactam antibiotics, such as ampicillin, amoxicillin, cephaclo, cephalexin, cephadroxyl, etc. It relates to a manufacturing method that can replace the misfire reaction.

Beta-lactam antibiotic preparations include beta, such as 6-aminophenicylanic acid (6-APA), 7-aminodesacetoxycephalosporinic acid (7-ADCA), or 7-aminocephalosporinic acid (7-ACA). It is made by condensation of an amino group of a lactam ring with an appropriate carboxyl group capable of causing an amidation reaction with these. For example, in the case of ampicillin, it is formed through the reaction of 6-amino peniclanic acid and di-phenylglycine derivative, and in the case of amoxicillin, it is formed by reaction of 6-aminophenicylanic acid and di-parahydroxyphenylglycine derivative.

However, to date, commercial beta-lactam antibiotic production processes are by chemical methods, which have some problems. For example, the formation of by-products by complex reactions such as the use of substrates with protected functional groups, the necessity of purification processes, the low temperature reaction below 0 ° C., and the use of organic solvents such as methylene chloride, etc. . In order to solve the drawbacks of this chemical process, a lot of research has been made on the preparation of beta-lactam antibiotics by acylating the beta-lactam ring and the carboxyl side chain using an enzyme.

However, in order to be a beta lactam synthesis process using economic enzymes, the beta lactam ring concentration, which is a substrate, needs to be raised to about 200 to 400 mM. In this case, the beta lactam antibiotic, which is a final product, is precipitated as a solid and shows high viscosity. A problem arises that the separation of the product is difficult.

According to U.S. Patent No. 5,525,483, there is an example in which a beta lactam antibiotic is prepared by an acylation reaction by enzyme even at a high concentration of about 400 mM, but there is a disadvantage in that the side chain is used in an amount of 4 equivalents or more with respect to the beta lactam ring. There is no mention of separation of enzymes from liquids, so commercial applicability is low. World Patent WO 9856945 reports that by using an excess of enzyme, the yield can be increased by 90% or more even when the side chain is used in an amount of 2 equivalents or less for the beta-lactam ring, but the amount of the enzyme used is several times higher than that of the substrate. As a matter of fact, there is a problem of inefficiency due to the high use of expensive enzymes and there is no mention of the enzyme separation process. According to World Patent WO 9602663A, the reaction process is semi-continuous for the separation of the final antibiotic to increase the separation efficiency and yield of the immobilized enzyme and the final reactant precipitated as a solid, but the minimum amount of commercially available immobilized enzyme is reported. The process is much more complicated than batch type, such as the use of an enzyme of 200 μm or more larger than 100 μm in diameter to increase the size of the sieve to facilitate separation of high-viscosity products and a raw material supply tank.

The present invention is to solve the problems of the prior art as described above, as a method different from the known production method, it is simple and easy to apply industrially to produce a beta lactam-based antibiotic in high purity, high yield The purpose.

In order to achieve the above object, the following manufacturing method is provided.

According to one aspect of the invention, the beta lactam-based compound of Formula 1 is reacted with a compound of Formula 2 to prepare a beta-lactam antibiotic of Formula 3.

(Wherein R ₁ CO is diphenylglycine or diparahydroxyphenylglycine, X is OR ₂ or NR ₃ R ₄ , R ₂ is an alkyl or haloalkyl group having 1 to 7 carbon atoms, and R ₃ , R ₄ is Each hydrogen or an alkyl group having 1 to 3 carbon atoms.)

According to another aspect of the present invention, the beta-lactam compound of Formula 4 is reacted with the compound of Formula 2 to prepare a beta-lactam antibiotic of Formula 5.

(Wherein Z is hydrogen, halogen, an alkyl group having 1 to 3 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an alkyl group having 1 to 3 carbon atoms substituted with a nucleophilic group, and R ₁ CO is diphenylglycine or diparahydroxyphenyl) Glycine, X is OR ₂ or NR ₃ R ₄ , R ₂ is an alkyl group or haloalkyl group having 1 to 7 carbon atoms, and R ₃ , R ₄ are each hydrogen or an alkyl group having 1 to 3 carbon atoms.)

In the present invention, it is necessary to use the side chains in excess of the beta-lactam ring in order to obtain high yield, which is a problem of the previous studies, and the problem of enzymatic reuse through separation of precipitated final reactants and expensive immobilized enzyme. A beta-lactam ring acylation process was developed. The process according to the present invention shows a reaction yield of 90% or more even when using a side chain of 1.4 to 4.0 equivalents, in particular 1.5 to 3.0 equivalents, and is easy to separate the final product from the enzyme, thereby reducing the loss in the separation process. to be.

The beta lactam ring of Formula 1 or 4 is a main chain having an amino group, and the amino group is acylated by the reaction of the present invention. Representative beta-lactam rings include 6-amino penicillic acid (6-APA), 7-aminodesacetoxycephalosporinic acid (7-ADCA), 7-aminocephalosporinic acid (7-ACA), 7-amino- 3-chlorocephalosporinic acid (7-ACCA) and the like. Such betaramtan rings are generally obtained through selective hydrolysis by enzymes such as penicillin, fermentation of cephalosporin (eg, penicillin V, penicillin G, cephalosporin C).

In the acylation reaction of the beta-lactam ring by an enzyme, the side chain carboxyl group is used for the reaction in the state substituted with various types of ester residues or amide residues derived from conventional carboxylic acids (Formula 2). Commonly used ester residues include alkyl esters of low carbon atoms, unsubstituted amides or substituted amide groups as amide residues.

The enzyme used in the present invention is an enzyme capable of catalyzing the acylation reaction of the beta lactam ring, and these enzymes have been known since about 1961. Microorganisms that produce such catalytic activity as penicillin amidase or penicillin acylase (EC3.5.1.11) -based enzymes include acetobacter, alkaligen, xanthomonas, mycoplana, protaminobacter, aeromonas ( German Patent Application No. 2,163,792) and, for enzymes derived from E. coli, are commercially available. In particular, enzymes of the immobilized form are advantageous to enable the reuse of enzymes. The enzymes derived from E. coli immobilized are as follows, and have been applied to the present invention. As enzymes immobilized on the polymer, PGA-450 (registered trademark) of Roche, Germany, and Super Enzyme (registered trademark) of Recordati, Italy, can be used, and can be reused as a crystalline enzyme. The form is applicable to Syntha CLEC-PA® from Altus Biologics, USA.

In the case of beta-lactam antibiotic synthesis reaction by enzyme, since the reaction is in aqueous solution, the proper concentration selection is very important because the final product, beta-lactam antibiotic, can be rehydrolyzed by the enzyme to obtain a certain yield or more. In general, since the final product has a lower solubility than the beta lactam ring used as the substrate, a higher yield can be reached by increasing the substrate concentration to reduce the rate of rehydrolysis by precipitation of the product as a solid, but the reaction solution viscosity is increased accordingly. This is accompanied by the difficulty of material transfer due to the effect of lowering the reaction yield rather than a certain concentration. In order to reach a reaction yield of 90% or more, the present inventors examined reaction conditions such as the amount of enzyme, substrate concentration, and equivalence ratio of the side chains to beta-lactam ring. The optimal concentration of beta lactam ring was 150-600mM, especially the highest yield at 200-400mM.

The optimal concentration of the side chains for the beta lactam ring was 1.2 to 4 equivalents, especially at about 1.5 to 3 equivalents.

In addition, there was a difference in the final yield according to the purity of the side chain used in the reaction, it was confirmed that more than 90% yield is possible only when the purity is at least 98%.

The amount of enzyme on the substrate was found to have no significant effect on the final yield but was correlated with the overall reaction time. However, when using a large amount of enzyme, it is shown that the proper enzyme activity is 500 ~ 4000 U / g 6-APA, especially 1000 ~ 2599 U / g 6-APA, because the reaction solution has a synergistic effect.

In the case of beta-lactam antibiotics, the solubility in aqueous solution is as low as a few grams per liter, depending on the substance. Under the general reaction conditions, in the case of the above substrate concentration, the reaction solution maintains the solid precipitated state from the beginning of the reaction, and when the yield is 90% or more, the final reaction solution forms a viscous solid solution. According to World Patent WO 9602663A, since an enzyme having a diameter of 200 to 500 µm is used, a sieve having a size of 180 µm is installed at the bottom of the reactor so that the final product smaller than the size of the sieve is passed, and the enzymes are not passed. The enzymes were separated and the enzymes were applied to the next reaction. When the present inventors applied this process, the amount of the product passing through the sieve was extremely small because the final reaction solution viscosity was high, and thus the yield of the final product actually obtained was about 60 to 70%. In addition, the size of the sieve to be installed in the reactor is about 80 ~ 90㎛ because the diameter of commercially used enzymes is 100㎛ or more, in this case it was confirmed that the separation difficulties due to the viscosity, etc. are further increased.

In the present invention, it is easy to separate the enzyme in the reaction solution by changing the pH of the final reaction solution to dissolve the reaction by-products precipitated in the reaction solution to lower the viscosity of the reaction solution. For example, the side chain used in the synthesis of amoxicillin is parahydroxyphenylglycine methyl ester, and most of the reactions at the end of the reaction are parahydroxyphenylglycine, a hydrolyzed by-product of the end product amoxicillin and the excess side chain used. . The amoxicillin production reaction is effective near pH 6, but since the solubility of amoxicillin and by-product parahydroxyphenylglycine is very low at this time, when the pH is raised to 7-8 using ammonia water, parahydroxyphenyl is a carboxylic acid. In the case of glycine, the solubility is much higher than that of amoxicillin, and as a result, the viscosity of the reaction solution is lowered, thereby facilitating separation between the enzyme and the produced solid. The reaction solution is then lowered to 4.5 to 5.5 again to maximize the precipitation of amoxicillin, and then filtered to obtain the final product. Before increasing the pH, 0.5 ~ 2 times the volume of the reaction solution, especially 0.8 ~ 1.2 times the volume of pure water was added to dilute the reaction solution, and it was found to be more effective.

In the process according to the invention, the reaction temperature is advantageously from 0 to 35 ° C, in particular from 20 to 30 ° C.

In addition, an appropriate pH varies depending on the type of substrate used, and generally 5 to 8 is appropriate, and in the case of amoxicillin synthesis, a pH of 5.5 to 6.4 is appropriate. It is also possible to keep the pH constant.

In the case of the proper reaction time, it depends on the amount of enzyme used and the activity, but in order to avoid high viscosity for the separation of enzyme after the reaction, it should be used in the amount of enzyme of 500 ~ 4000 U / g beta lactam ring, especially 1000 ~ 2500U / g beta lactam ring. In this case, according to the amount of enzyme used, the proper reaction time is about 3 to 8 hours, and since the product may be rehydrolyzed after reaching the highest yield, the reaction progress should be checked to determine the end point of the reaction. Purification of the final product is possible with recrystallization by changing pH, a method known in the art.

The following presents preferred examples and comparative examples to aid in understanding the invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Definition and analysis method

enzyme

Unit U of enzymatic activity corresponds to the amount of enzyme required to hydrolyze 1 μmol of penicillin G potassium salt for 1 minute (reaction conditions, 100 g · 1-1 penicillin G potassium salt, 0.05 M potassium phosphate buffer, pH 8.0, 28 ℃)

PGA-450 (Roche, Germany): 380 U / g

Super Enzyme (Recordari, Italy): 500 U / g

Syntha CLEC-PA (Altus Biologic, USA): 2100 U / g

HPLC analysis conditions

Column: Waters μBondapak C18 Column

Mobile phase: 0.7% (v / v) acetonitrile, 0.25M potassium phosphate buffer, pH5.0

Flow rate: 1 ml / min, wavelength: 230 nm

Example 1

Final yield by diparahydroxyphenylglycine methyl ester equivalents

Agitator and pH electrode are installed at the bottom, and 100M sieve is installed at the bottom of 1.5M, 20 ℃, 1.5L incubator and 6.aminophenic acid (6-APA) 43.2g (200mM) 1 L of oxyphenylglycine methyl ester (PHPGM), pure water, is added followed by immobilized penicillin amidase superenzyme 53600 U (Recordati, Italy). After 4 hours of reaction, the HPLC analysis results are shown in Table 1 below.

Amoxicillin Reaction Yield by Diparahydroxyphenylglycine Methyl Ester Equivalent PHPGM equivalent 1.5 2.0 3.0 4.0 Final yield (HPLC) 80% 93% 93% 90%

Example 2

Amoxicillin Production by Enzyme

Agitator and pH electrode were installed, and 43.2 g (200 mM) of 6-aminophenic silane acid (6-APA) in a 1.5 M, 20 ° C. incubator equipped with a 100 μm sieve at the bottom, and diparahydroxyphenylglycine methyl ester ( PHPGM) 72.4 g (2 equivalents to 6-APA), 1 L of pure water, followed by immobilized penicillin amidase superenzyme 53600 U (Recordati, Italy). The initial pH was 6.0 and it increased to about 6.8 as the reaction progressed and then dropped to 5.5 again. After 4 hours of reaction, the HPLC analysis showed that the amoxicillin yield was 92.6%. The reaction was stopped by raising the pH to 8.0 using 10% ammonia water. The reactor was opened to separate and recover the reaction solution from the enzyme, and the recovered reaction solution was filtered to obtain a solid product. The filtrate after filtering is put back into the reactor to wash off the solids remaining inside the reactor. Repeated three times, only the immobilized enzyme remained in the reactor, and the filtered solid product was re-dissolved in the final filtrate, and then the pH was adjusted to 24.8 sulfuric acid solution to 4.8. The reaction solution was filtered again to obtain 75 g of amoxicillin trihydrate (yield 90%).

Example 3

Amoxicillin Production by Enzyme

PGA-450 40700U (Roche, Germany) was used as the enzyme immobilized under the same reaction conditions as in Example 1, and the other reaction conditions were the same as in Example 1.

After 5 hours of reaction, 74 g of amoxicillin trihydrate was obtained (yield 80%).

Comparative Example 1

21.6 g (100 mM) of 6-aminophenicsilane acid (6-APA), 36.4 g of diparahydroxyphenylglycine methyl ester (PHPGM) (2 equivalents to 6-APA), pure water 1 L is added followed by immobilized penicillin amidase superenzyme 53600 U (Recordati, Italy). The initial pH was 6.0, and it increased to about 6.8 as the reaction progressed and then dropped to 5.5 again. After 2 hours of reaction, the highest yield was shown, after which the yield tended to decrease. HPLC analysis showed the highest amoxicillin yield of 62%. 21 g of amoxicillin trihydrate was obtained by the enzyme and reaction product separation method of Example 1 (final yield 50%).

Comparative Example 2

After the same reaction as in Example 2, after completion of the reaction without raising the pH with 10% ammonia water, the reaction solution and the enzyme are separated as it is. The reaction solution from which the enzyme has been separated is filtered to obtain a solid product, and the filtrate is added back to the reactor to wash off the remaining solid products. The same procedure as in Example 1 was repeated three times, but a large amount of solids were present in the reactor, and 53 g of amoxicillin trihydrate was obtained (yield 63%).

According to the method for producing beta-lactam antibiotics of the present invention, since a high concentration of the substrate is used, the reaction yield can be increased by 90% or more even when the side chain consumption is lowered, thereby satisfying the commercial productivity and the reaction yield at the same time, and also using the high concentration of the substrate. By facilitating the separation of high concentrations of solid products and enzymes, losses in the separation process can be minimized.

Claims (9)

A method for preparing a beta-lactam antibiotic of Formula 3 by acylating a compound of Formula 1 with a compound of Formula 2. (Wherein R ₁ CO is diphenylglycine or diparahydroxyphenylglycine, X is OR ₂ or NR ₃ R ₄ , R ₂ is an alkyl or haloalkyl group having 1 to 7 carbon atoms, and R _3, R ₄ are Each hydrogen or an alkyl group having 1 to 3 carbon atoms.) The method of claim 1, wherein the initial concentration of the compound of formula 1 is 150-600 mM, and the equivalent ratio of the compound of formula 2 is 1.2-4 times that of the compound of formula 1. The method of claim 2, wherein the initial concentration of the compound of formula 1 is 200-400 mM and the equivalent ratio of the compound of formula 2 is 1.5-3 times that of the compound of formula 1. The method of claim 1, further comprising, after completion of the acylation reaction, raising the pH of the reaction solution to 7.0 to 9.0, and then separating the compound of Formula 3 using a sieve having a size of 90 to 200 μm. A method for preparing a betalactam antibiotic of formula 5 by acylating a compound of formula 4 with a compound of formula 2. (Wherein Z is hydrogen, halogen, an alkyl group having 1 to 3 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an alkyl group having 1 to 3 carbon atoms substituted with a nucleophilic group, and R ₁ CO is diphenylglycine or diparahydroxyphenyl) Glycine, X is OR ₂ or NR ₃ R ₄ , R ₂ is an alkyl group or haloalkyl group having 1 to 7 carbon atoms, and R ₃ , R ₄ are each hydrogen or an alkyl group having 1 to 3 carbon atoms.) The method of claim 5, wherein the initial concentration of the compound of formula 4 is 150-600 mM, and the equivalent ratio of the compound of formula 2 is 1.2-4 times that of the compound of formula 4. The method of claim 5, further comprising, after completion of the acylation reaction, raising the pH of the reaction solution to 7.0 to 9.0, and then separating the compound of Formula 5 using a sieve having a size of 90 to 200 μm. The process according to claim 1 or 5, wherein the reaction temperature is 0 to 35 ° C. The method according to claim 1 or 5, wherein the enzyme is penicillin amidase derived from Escherichia coli, Acetobacter pasterianum, Xanthomonas citri, Clueberry citrophila or Bacillus megaterium.