WO2001030783A1

WO2001030783A1 - PROCESS FOR THE PREPARATION OF A β-LACTAM ANTIBIOTIC

Info

Publication number: WO2001030783A1
Application number: PCT/NL2000/000636
Authority: WO
Inventors: Jie Zhu; Binne Zwanenburg; Gerardus Johannes Kemperman
Original assignee: Dsm N.V.
Priority date: 1999-10-27
Filing date: 2000-09-08
Publication date: 2001-05-03
Also published as: AU7459400A; NL1013402C2

Abstract

Process for the preparation of a β-lactam antibiotic by acylation of a β-lactam nucleus with the aid of an acylating agent, use being made of a complexing agent, and the acylating agent containing L-isomer and an aldehyde being added during the preparation. This process makes it possible to use a racemic mixture as acylating agent in the preparation of a β-lactam antibiotic.

Description

PROCESS FOR THE PREPARATION OF A β-LACTAM ANTIBIOTIC

The invention relates to a process for the preparation of a β-lactam antibiotic by acylation of a β-lactam nucleus with the aid of an acylating agent, this process involving the use of a complexing agent.

In the framework of the invention an acylating agent is defined as an α-amino acid or a derivative thereof. Derivatives are also understood to mean activated amino acids. Preferred activated amino acids are esters or amides of α-amino acids.

Known β-lactam antibiotics are cephalosporins and penicillins. The β-lactam antibiotics according to the invention contain at least three chiral centres . Both cephalosporins and penicillins as a rule have at least three chiral centres in their structure. The β-lactam nucleus contains at least two chiral centres, which as a rule each have the L- configuration. The acylating agent coupled to the nucleus contains at least 1 chiral centre. One of the substituents at the chiral centre in the acylating agent is an α-amino group. Preferred antibiotics are those in which the α-amino group in the acylating agent is connected to the chiral centre in such a manner that the residue of the acylating agent in the β-lactam antibiotic has the D-configuration.

Customarily, in the acylation of a β-lactam nucleus with the aid of an acylating agent, the acylating agent is used in the form of the D-isomer, or virtually pure D-isomer, so that after the acylation the desired β-lactam antibiotic is immediately obtained. As a rule, the enantiomeric excess (e.e.) of the acylating agent is greater than 95%, usually greater than 99%. The use of an optically pure or virtually pure D-isomer of the acylating agent is a drawback because the recovery of the D-isomer from a mixture of D- and L-isomers is an extra step in the preparation. Moreover, usually part of the acylating agent is lost in this step.

It is the aim of the invention to provide a process that does not have said drawback. To this end, the invention is characterized in that the acylating agent contains L-isomer and in that an aldehyde is present during the preparation of the β-lactam antibiotic. If the aldehyde is not present at the start of the preparation, it may be added during the preparation.

A further advantage of the process according to the invention is that the L-isomer of acylating agents can be used as such. It is conceivable for the L-isomer to be available as waste product or by-product. With the process according to the invention the L-isomer can now be used directly.

With the process according to the invention β-lactam antibiotics can be obtained by coupling a mixture of D- and L-isomers, which mixture may be a racemic mixture, or pure L-isomers of the acylating agent to β-lactam nuclei. By the presence of an aldehyde and a complexing agent during the preparation, β-lactam antibiotics can be obtained in a high yield.

When a complexing agent is present, a antibiotic complex is formed by the complexing agent and the D-β-lactam antibiotic or the L-β-lactam antibiotic, after which the complex precipitates. In the framework of this text a D-β-lactam antibiotic means a β-lactam antibiotic in which the residue of the acylating agent in the antibiotic contains an α-amino group which is connected to the chiral centre of the residue in such a manner that the residue has the D- configuration. Preferably, a complex is selectively formed between the complexing agent and antibiotics comprising a nucleus having two chiral centres having the L-configuration and comprising a residue of the acylating agent that contains an α-amino group which is connected to the chiral centre of the residue in such a manner that the residue has the D-configuration.

After the D- or L-antibiotic-complex has selectively been precipitated, it can be separated from the reaction mixture and the free β-lactam antibiotic can be recovered in a high yield from the antibiotic complex, for instance by hydrolysis.

Acylation of a β-lactam nucleus by means of an acylating agent can be carried out chemically or enzymatically. The enzymatic process is for instance described in International patent application WO 9201061.

The chemical process is for instance described in US-A-4, 358,588 and EP-A-0 439 096. Suitable β-lactam nuclei are for instance the nuclei represented by the general formula

(I) where

Ro is -H or -OCH₃; Ri is -H; Y is CH₂, CHCH₃, 0, S

\ and Z is

where R₂ is H, Cl, OH, CH₃ , CH₂OH, CH₂C1, CH₂OC(0)CH₃,

CH₂OC(0)NH₂;

Examples of suitable β-lactam nuclei are 7- aminocephalosporanic acid (7-ACA) , 7-amino-3'- desacetoxycephalosporanic acid (7-ADCA), 7-amino-3'- desacetylcephalosporanic acid (7-ADAC) , 7-amino-3- chloro-3- (desacetoxymethyl) cephalosporanic acid (7-

ACCA) , 7-amino-3-methoxy-3- (desacetoxymethyl) cephalosporanic acid, 7-amino-3- [ (Z) 1-propenyl] -3- (desacetoxymethyl) cephalosporanic acid

(7-PACA) , 7-amino-3-vinyl-3-

(desacetoxymethyl) cephalosporanic acid (7-AVCA) , 7- amino-3 '-(1,2, 3-trizol-4-ylthio) -3 ' - desacetoxycefalosporanic acid (7-TACA), 7-amino-3 ' - (1- methyl-lH-tetrazol-5-ylthio) -3 ' - desacetoxycephalosporanic acid (7-TMCA) , 7-amino-3 ' - (5- methyl-1, 3 , 4-thiadiazol-2-ylthio) -3 ' - desacetoxycephalosporanic acid (7-TDA) and 7-amino-3'- (1-methylpyrrolidin-l-yl) -3-

(desacetoxymethyl) cephalosporanic acid, 7-amino-3 ' - [ (1-

(2-dimethylamino) ethyl) -lH-tetrazol-5-ylthio] -3 ' - desacetoxycephalosporanic acid, 7-amino-3 ' -methoxy-3- desacetoxycephalosporanic acid, 7-amino-3-methoxy-3- (desacetoxymethyl) cephalosporanic acid, 7-amino-3'-

(pyridin-1-yl) -3 ' -desacetoxycephalosporanic acid, 7- amino-3 ' - (2-furanylcarbonylthio) -3 ' - desacetoxycephalosporanic acid, 7-amino-3 ' - ( (1 , 2 , 5, 6- tetrahydro-2-methyl-5, 6-dioxo-l, 2 , 4-triazin-3-ylthio) - 3 ' -desacetoxycephalosporanic acid (7-ACT) , 7-amino-3'-

(aminocarbonyloxy) -3 ' -desacetoxycephalosporanic acid,

6-aminopenicillanic acid (6-APA) and ( 6R, 1R) -3-chloro-

7-amino-8-oxo-l-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid. The β-lactam nuclei are mostly obtained by removing the natural side chains from fermentation products .

As acylating agent use can be made of all known acylating agents, for instance dihydrophenylglycine, p-hydroxyphenylglycine (HPG) , phenylglycine (PG) and derivatives thereof.

In an embodiment the process according to the invention is characterized in that a mixture of D- and L-isomers containing more than 1 mole% L-isomer, relative to the total number of moles of D- and L- isomer, is used as acylating agent. In a preferred embodiment the process according to the invention is characterized in that a racemic mixture is used as acylating agent.

By reacting various acylating agents with various β-lactam nuclei, a plurality of β-lactam antibiotics can be obtained. Examples of β-lactam antibiotics that can be prepared using the process according to the invention are cefroxadin, cefradine, amoxicillin, cefadroxil, cefatrizine, cefoperazone, cefprozil, ampicillin, cefaclor, cefalexin and loracarbef.

The reaction conditions under which the acylation reaction is carried out, for instance pH and temperature, are different for chemical and enzymatic reactions and are known to one skilled in the art. In an embodiment first the acylation reaction is carried out and subsequently the D,L- antibiotic is isolated from the reaction mixture, after which the aldehyde and complexing agent are added to a suspension or solution of the isolated D, -antibiotic . With D, -antibiotic is meant the antibiotic obtained after the acylation reaction according to the invention. In another embodiment of the process according to the invention the process is carried out in a single vessel. In a preferred embodiment the process according to the invention is characterized in that first the acylation reaction is carried out, after which the aldehyde and the complexing agent are added to the reaction mixture. It does not matter in which order the aldehyde and the complexing agent are added. The pH at which the aldehyde can be added to the reaction mixture may vary within broad limits, and for instance lies between 2 and 10. Preferably, the pH of the mixture is between 4 and 9, in particular between 5 and 8.

The temperature of the reaction mixture at which the aldehyde is or has been contacted with the reaction mixture for instance lies between -40 and

80°C, preferably between -20 and 50°C and in particular between 0 and 40°C.

Preferably, use is made of aromatic aldehydes, for instance benzaldehyde, salicylic aldehyde or pyridoxal.

The amount of aldehyde that can be used during the preparation may vary between broad limits and for instance lies between 0.01 and 1000 mol% relative to the amount of β-lactam nucleus applied. Preferably, the amount of aldehyde lies between 1 and 10 mol% relative to the amount of β-lactam nucleus applied.

As complexing agent use can be made of any known complexing agent. The amount of complexing agent that can be used in the process according to the invention may vary between broad limits and is preferably at least 1 equivalent of complexing agent relative to the amount of β-lactam nucleus applied.

Examples of suitable complexing agents are naphthalenes, quinolines, anthraquinone sulphonic acids and parabenes . Examples of complexing agents are 1- naphthol, 2-naphthol, 2 , 6-dihydroxynaphthalene and anthraquinone-1, 5-disulphonic acid.

In the framework of the invention yield is defined as the amount of β-lactam antibiotic complex that is obtained, in mol%, upon isolation relative to the amount of β-lactam nucleus applied.

The invention will now be elucidated on the basis of a few examples, without being restricted thereto .

Examples

The abbreviations used stand for: 6-APA; 6-aminopenicillanic acid

7-ADCA: 7-amino-3 ' -desacetoxycephalosporanic acid D,L-PGA: D- and L-phenylglycinamide TMG: tetramethylguanidine DMF : dimethylformamide

Asse blase® is an immobilized Escherichia coli penicillin acylase from E. coli ATCC 1105, as described in WO-A-97/04086. The immobilization was carried out as described in EP-A-222462, using gelatine and chitosan as gelling agents and glutaric aldehyde as crosslinker .

The final activity of the Escherichia coli penicillin amylase is determined by the amount of enzyme that is added to the activated spheres and amounted to 3 ASU/g dry weight, 1 ASU (amoxicillin Synthesis Unit) being defined as the amount of enzyme generating 1 g of Amoxicillin.3H₂0 per hour from 6-APA and D-p-hydroxyphenylglycine methylester (at 20°C; 6.5 mass% 6-APA and 6.5 mass% D-p-hydroxyphenylglycine methylester, the mass percentage being calculated relative to the mass of the reaction mixture) . Example I Synthesis of cefalexin

A solution A was prepared from 3.01 g (10 mmol) D, L-phenylglycine Dane salt and 15 ml dichloromethane, 150 mg n-methylacetamide and 30 mg γ- picoline. The temperature of the solution was lowered to about -40°C. Then 1.27 g (10.5 mmol) pivaloyl chloride was rapidly added. After stirring for 30 minutes at a temperature between -20 and -30°C the temperature was lowered to -50°C.

A solution B was prepared from 10 ml dichloromethane and 1.71 g (8 mmol) 7-ADCA to which 920 mg (8 mmol) TMG was added at -5°C.

At a temperature below -50°C solution B was then added dropwise in 30 minutes to solution A. HPLC was used to monitor the reaction. Stirring took place for 1 hour at between -50°C and -30°C, followed by another 5 hours' stirring at a temperature between -30 and -20°C. A conversion of 81% was achieved. Subsequently, 10 ml H₂0 was added and the mixture was acidified at -5°C using 37% aqueous HCl until the pH was 0.5. This was followed by stirring for 30 minutes, after which the water and dichloromethane layers were separated. The dichloromethane layer was washed with 10 ml water. The two water layers were combined and neutralized to pH 7.0.

Subsequently, 850 mg 1-naphthol was added and then 160 mg pyridoxal . The resulting mixture was stirred for 2 days at room temperature. The D-cefalexin complex was recovered by means of filtration. The yield was 2.3 g (59%) . Example II Synthesis of cefadroxil

A solution A was prepared from 3.17 g (10 mmol) L-p-hydroxyphenylglycine Dane salt and 15 ml dichloromethane, 2 ml DMF and 30 mg γ-picoline. The temperature of the solution was lowered to approx. - 40°C. Then 1.27 g (10.5 mmol) pivaloyl chloride was rapidly added. After stirring for 30 minutes at a temperature between -20 and -30°C, 15 ml dichloromethane was added and the temperature was lowered to -50°C.

A solution B was prepared from 15 ml dichloromethane and 1.71 g (8 mmol) 7-ADCA to which 920 mg (8 mmol) TMG was added at -5°C.

At a temperature below -50°C solution B was then added dropwise in 30 minutes to solution A. HPLC was used to monitor the reaction. Stirring took place for 1 hour at between -50°C and -30°C, followed by another 5 hours' stirring at a temperature between -30 and -20°C. Subsequently, 10 ml H₂0 was added and the mixture was acidified at -5°C using an HC1 solution (37% in water) until the pH was 1. Stirring took place for 30 minutes, after which the water and dichloromethane layers were separated and the dichloromethane layer was washed with 10 ml water. The two water layers were combined and neutralized to pH 7.0.

Subsequently, 640 mg 2,7-naphthol was added and then 160 mg pyridoxal . The resulting mixture was stirred for 24 hours. The D-cefadroxil complex was recovered by means of filtration. The yield was 2.41 g (58%) . Example III Synthesis of cefalexin

420 mg (2 mmol) 7-ADCA was dissolved in 10 ml water at a pH between 7.5 and 8.0. 600 mg D,L-PGA (4 mmol, in 10 ml water, pH 6) was added, while the pH was kept above 7. 80 mg Pyridoxal was added. To the reaction mixture obtained 1.6 g Assemblase® (net wet weight) was added.

160 mg 1-Naphthol was dissolved in 0.25 ml methanol and added in portions over a 2.5-hour period. The mixture was then stirred overnight at room temperature. Then the enzyme was removed by filtration. The cefalin complex was obtained by filtration. The yield was 780 mg (82%), with a purity of 82%.

Claims

1. Process for the preparation of a β-lactam antibiotic by acylation of a β-lactam nucleus with the aid of an acylating agent and involving the use of a complexing agent, this process being characterized in that the acylating agent contains L-isomer and in that an aldehyde is present during the preparation.

2. Process according to claim 1, characterized in that the aldehyde is added during the preparation .

3. Process according to claim 1 or 2 , characterized in that a mixture of D- and L-isomers containing more than 1 mole% L-isomer is used as acylating agent .

4. Process according to any one of claims 1-3, characterized in that a racemic mixture is used as acylating agent .

5. Process according to any one of claims 2-4, characterized in that first the acylating reaction is carried out, following which the aldehyde and the complexing agent are added to the reaction mixture.

6. Process according to any one of claims 2-5, characterized in that the pH at which the aldehyde is added lies between 4 and 9.

7. Process according to any one of claims 2-6, characterized in that the pH at which the aldehyde is added lies between 5 and 8.

8. Process according to any one of claims 1-6, characterized in that the temperature at which the aldehyde has been or is contacted with the reaction mixture lies between -20 and 50°C.

9. Process according to claim 8, characterized in that the temperature at which the aldehyde is added lies between 0 and 40°C.

10. Process according to any one of claims 1-9, characterized in that use is made of an aromatic aldehyde.

11. Process according to claim 10, characterized in that as the aldehyde use is made of benzaldehyde, salicylic aldehyde or pyridoxal.

12. Process according to any one of claims 1-11, characterized in that the amount of aldehyde used during the preparation lies between 1 and 10 mol% relative to the amount of β-lactam nucleus used.

13. Process according to any one of claims 1-11, characterized in that use is made of at least 1 equivalent of complexing agent relative to the amount of β-lactam nucleus used.