NL1013402C2 - Method for the preparation of a beta-lactam antibiotic. - Google Patents

Description

- 1 -

PROCESS FOR THE PREPARATION OF AN IS-LACTAM 5 ANTIBIOTIC

The invention relates to a method for the preparation of a β-lactam antibiotic by acylation of a β-lactam core using an acylating agent and in which a complexing agent is used.

Within the scope of the invention, an acylating agent is defined as an α-amino acid or a derivative thereof. Derivatives also include activated amino acids.

Well-known β-lactam antibiotics are cephalosporins and penicillins. The β-lactam antibiotics according to the invention contain at least three chiral centers.

Both cephalosporins and penicillins, as a rule, have at least three chiral centers in their structure. The β-lactam core contains at least two chiral centers, each of which usually has the L-configuration. The core coupled acylating agent contains at least 1 chiral center. One of the substituents at the chiral center in the acylating agent is an α-amino group. The α-amino group must be connected to the chiral center such that the residue of the acylating agent in the β-lactam antibiotic has the D-configuration.

It is usual in the acylation of 1013402 - 2 - to use a β-lactam core with the aid of an acylating agent, the acylating agent in the form of the D isomer, or substantially pure D isomer, so that immediately after the acylation the desired β-lactam antibiotic 5 is 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 substantially pure D isomer of the acylating agent is a drawback, because obtaining the D isomer from a (racemic) mixture of D and L isomers is an extra step in the preparation. In addition, part of the acylating agent is usually lost here.

The object of the invention is to provide a method which does not have the said drawback.

The invention is therefore characterized in that the acylating agent contains L-isomer and that an aldehyde is added during the preparation.

A further advantage of the method according to the invention is that the L-isomer of acylating agents can be used as such. It is conceivable that L isomer is available as a waste or by-product. The L isomer can now be used directly with the aid of the method according to the invention. With the method according to the invention, β-lactam antibiotics can be obtained by coupling a (racemic) mixture of D and L isomers or pure L isomers of the acylating agent to β-lactam nuclei. By adding an aldehyde and a complexing agent during the preparation, β-lactam 101 3402-3 antibiotics can be obtained in high yield.

After the β-lactam antibiotic complex has been selectively precipitated by the complexing agent, it can be separated from the reaction mixture, and the free β-lactam antibiotic can be recovered in high yield from the antibiotic complex, for example, by hydrolysis.

Acylation of a β-lactam core using an acylating agent can be carried out chemically or enzymatically. The enzymatic process is described, for example, in International Patent Application WO 9201061.

The chemical method is described, for example, in US-A-4,358,588 and EP-A-0 439 096 15 (preparations via Dane salt).

Suitable β-lactam nuclei are, for example, those nuclei represented by the general formula R, NHR1 V_L ^ \ OH (I) 20 where R0 is -H or -CH3; R3 is -H; Y is CH2, CHCH3, O, S25 \ and Z is / 101 34 02-4-XCH3. . or CH, ^ r2 ^ ^ r2 / ^ ch2 where R2 is -H, -Cl, -OH, -CH3, -CH2OH, -CHaCl, -5 CH2OC (O) CH3, CH2OC (0) NH2;

Suitable β-lactam nuclei are, for example, 7-Aminocephalosporic acid (7-ACA), 7-Amino-3'-desacetoxycephalosporic acid (7-ADCA), 7-Amino-3 '~ desacetylcephalosporic acid (7-ADAC), 7-Amino-3- chloro-10 3- (desacetoxymethyl) cephalosporic acid (7-ACCA), 7-

Amino-3-raethoxy-3- (desacetoxymethyl) cephalosporic acid, 7-Amino-3 - [(Z) -1-propenyl] -3- (desacetoxymethyl) cephalosporic acid (7-PACA), 7-Amino-3-vinyl-3 - (desacetoxymethyl) cephalosporic acid (7-AVCA), 7-Amino-3 '- (1,2,3-trizol-4-ylthio) -3'-desacetoxycephalosporic acid (7-TACA), 7-Amino-3'- (1-methyl-1H-tetrazol-5-ylthio) -3 '- desacetoxycephalosporic acid (7-TMCA), 7-Amino-3' - (5-methyl-1,3,4-thiadiazol-2-ylthio) -3 -20 desacetoxycephalosporic acid (7-TDA) and 7-Amino-3 '- (1-methylpyrrolidin-1-yl) -3- (desacetoxymethyl) cephalosporic acid, 7-Amino-3' - [(1- (2-dimethylamino)) ethyl) -lff-tetrazol-5-ylthio] -3'-desacetoxycephalosporic acid, 7-Amino-3'-methoxy-3-25 desacetoxycephalosporic acid, 7-Amino-3-methoxy-3- (desacetoxymethyl) cephalosporic acid, 7-Amino- 3 '- (pyridin-1-yl) -3' -desacetoxycephalosporic acid, 7-Amino-3 '- (2-furanylcarbonylthio) -3'- 101 3402 -5- desacetoxycephalosporic acid, 7-Amino-3'- ((1 2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-ylthio) -3'-desacetoxycephalosporic acid (7-ACT), 7-Am ino-3 '- (aminocarbonyloxy) -3'-desacetoxycephalosporic acid, 6-5 Aminopenicillic acid (6-APA) and (SR, 7R) -3-Chloro-7-amino-8-oxo-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid.

The β-lactam nuclei are often obtained by stripping fermentation products of their natural side chain.

As the acylating agent, all known acylating agents can be used, for example, dihydrophenylglycine, p-hydroxyphenylglycine (HPG), phenylglycine (PG) and derivatives thereof.

In one embodiment, the method according to the invention is characterized in that a mixture of D and L isomer containing more than 1% L isomer is used as an acylating agent.

In a preferred embodiment, the method according to the invention is characterized in that a racemic mixture is used as an acylating agent.

By reacting different acylating agents with different β-lactam nuclei, a multitude of β-lactam antibiotics can be obtained. Beta-25 lactam antibiotics that can be prepared by the method of the invention are, for example, Cefroxadine, Cefradine, Amoxicillin, Cefadroxil, Cefatrizine, Cefoperazone, Cefprozil, Ampicillin, Cefaclor, Cefalexin and Loracarbef.

The reaction conditions under which the 1013402-6 acylation reaction is carried out, for example pH and temperature, are different for chemical and enzymatic reactions, and are known to those skilled in the art.

In one embodiment, the acylation reaction is first performed, then the D, L antibiotic is isolated from the reaction mixture, and the aldehyde and complexing agent are added to a suspension or solution of the isolated D, L antibiotic.

In another embodiment of the method according to the invention, the method is carried out in one vessel. In a preferred embodiment, the method according to the invention is characterized in that the acylation reaction is first 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 can vary within wide limits, for instance between 2 and 10. Preferably the pH is between 4 and 9, in particular between 5 and 8.

The temperature of the reaction mixture at which the aldehyde is or is contacted with the reaction mixture is, for example, between -40 and 800C, preferably between -20 and 50 ° C, and in particular between 0 and 40 ° C.

Aromatic aldehydes are preferably used, for example benzaldehyde, salicylaldehyde or pyridoxal.

101 3402 - 7 -

The amount of aldehyde that can be used during the preparation can vary within wide limits, for instance between 0.01 and 1000 mol% relative to the amount of β-5 lactam core used. Preferably, the amount of aldehyde is between 1 and 10 mol% relative to the amount of β-lactam core used.

Any known complexing agent can be used as a complexing agent. The amount of complexing agent that can be used in the process of the invention can vary within wide limits, and is preferably at least 1 equivalent of complexing agent relative to the amount of β-lactam core used.

Suitable complexing agents are, for example, naphthalenes, quinolines, anthraquinone sulfonic acids and parabens. Examples of complexing agents are 1-naphthol, 2-naphthol, 2,6-dihydroxynaphthalene, anthraquinone-1,5-disulfonic acid.

For the purposes of the invention, yield is defined as the amount of β-lactam antibiotic complex obtained after isolation in mol% relative to the amount of β-lactam core used.

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

101 3402 - 8 -

EXAMPLES

Explanation of the abbreviations used: 6-APA: 6-Aminopenicillinic acid 5 7-ADCA: 7-Amino-3'-desacetoxycephalosporic acid D, L -PGA: D- and L-phenylglycine amide TMG: tetramethylguanidine DMF: dimethylformamide 10 is an immobilized

Escherichia coli penicillin acylase from E. coli ATCC 1105, as described in WO-A-97/04086. Immobilization was performed as described in EP-A-222462 using gelatin and chitosan as gelling agent and glutaraldehyde as crosslinker.

The final activity of the Escherichia coli penicillin acylase is determined by the amount of enzyme added to the activated beads and was 3 ASU / g dry weight with 1 20 ASU (Amoxicillin Synthesis Unit) defined as the amount of enzyme 1 g Amoxicillin per hour .3H20 generates from 6-APA and Dp-hydroxyphenylglycine methyl ester (at 20 ° C; 6.5% 6-APA and 6.5% Dp-hydroxyphenylglycine methyl ester).

25

Example I. Synthesis of Cefalexin

A solution A was prepared from 3.01 g (10 mmol) D, L-phenylglycine Dane salt and 15 ml of dichloromethane, 150 mg of n-methyl acetamide and 30 mg of γ-30 picoline. The temperature of the solution was lowered 101 3402-9 to about -40 ° C. Then 1.27 g (10.5 mmol) of pivaloyl chloride was added quickly. Stirred at a temperature between -20 and -30 ° C for 30 minutes. Then 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 over 30 minutes to solution A. HPLC was used to monitor the reaction. It was stirred for 1 hour at a temperature between -50 and -30 ° C, after which it was stirred for another 5 hours at a temperature between -30 and -20 ° C. A conversion of 81% was achieved.

Then 10 ml of H 2 O were added, and the mixture was acidified at -5 ° C with 37% aqueous HCl to pH 0.5. It was then stirred for 30 minutes. Then, the water and dichloromethane layer were separated and the dichloromethane layer was washed with 10 ml of water. The two layers of water were combined and neutralized to pH 7.0.

Then 850 mg of 1-naphthol was added and then 160 mg of pyridoxal. The resulting mixture was stirred at room temperature for 2 days.

The D-cephalexin complex was collected by filtration. The yield was 2.3 g (59%).

Example II. Synthesis of Cefadroxil

A solution A was prepared from 3.17 g (10 30 mmol) of L-p-hydroxyphenylglycine Dane salt and 15 ml of 1013402-10-dichloromethane, 2 ml of DMF and 30 mg of γ-picoline. The temperature of the solution was lowered to about -40 ° C. Then 1.27 g (10.5 mmol) of pivaloyl chloride was added quickly. Stirring was carried out at a temperature between -20 and -30 ° C for 30 minutes. Then 15 ml of dichloromethane was added, and the temperature was lowered to -50 ° C.

A solution B was prepared from 15 ml of dichloromethane and 1.71 g (8 mmol) of 7-ADCA to which 920 mg (8 mmol) of TMG was added at 10-5 ° C.

At a temperature below -50 ° C, solution B was then added dropwise over 30 minutes to solution A. HPLC was used to monitor the reaction. It was stirred for 1 hour at a temperature between -50 and -30 ° C, after which it was stirred for another 5 hours at a temperature between -30 and -20 ° C.

Then 10 ml of H 2 O was added, and the mixture was acidified at -5 ° C with an Hcl-20 solution (37% in water) to pH 1. Then stirred for 30 min. Then, the water and dichloromethane layer were separated and the dichloromethane layer was washed with 10 ml of water. The two layers of water were combined and neutralized to a pH of 7.0.

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

101 34 02 - 11 -

Example III. Synthesis of Cefalexin.

430 mg (2 mmol) 7-ADCA was dissolved in 10 ml of water at a pH between 7.5 and 8.0. 600 mg D, ΗΡΘΑ (4 mmol, in 10 ml water, pH 6) was added, keeping the pH above 7. 80 mg of pyridoxal was added. 1.6 g of Assemblase® (net wet weight) was added to the resulting reaction mixture.

160 mg of 1-naphthol was dissolved in 0.25 ml of methanol and added in portions over a period of 2.5 hours. The mixture was then stirred at room temperature overnight. The enzyme was then filtered off. The cephalexin complex was obtained by filtration. The yield was 780 mg (82%), with a purity of 82%.

15 1013402

Claims (12)

Method for the preparation of a β-lactam antibiotic by acylation of a β-lactam core 5 using an acylating agent and using a complexing agent, characterized in that the acylating agent contains L-isomer and that during the preparation a aldehyde is added. A method according to claim 1, characterized in that a mixture of D and L isomer containing more than 1% L isomer is used as an acylating agent. 3. A method according to any one of claims 1 or 2, characterized in that a racemic mixture is used as the acylating agent. 4. Process according to any one of claims 1-3, characterized in that the acylation reaction is first carried out, after which the aldehyde and the complexing agent are added to the reaction mixture. 5. Process according to any one of claims 1-4, characterized in that the pH (of the mixture?) At which the aldehyde is added is between 4 and 9. Process according to any one of claims 1 to 5, characterized in that the pH at which the aldehyde is added is between 5 and 8. 7. Process according to any one of claims 1-6, characterized in that the temperature at which the aldehyde is contacted or brought into contact with the 101 34 0 2 - 13 reaction mixture is between -20 and 50 ° C. Process according to claim 7, characterized in that the temperature at which the aldehyde is added is between 0 and 40 ° C. Process according to any one of claims 1 to 7, characterized in that an aromatic aldehyde is used. 10. Process according to claim 9, characterized in that the aldehyde used is benzaldehyde, salicylaldehyde or pyridoxal. 11. Process according to any one of claims 1-10, characterized in that the amount of aldehyde used during the preparation is between 1 and 10 mol% relative to the amount of β-lactam core used. A method according to any one of claims 1-11, characterized in that at least 1 equivalent of complexing agent relative to the amount of β-lactam core used is used. 20 1013402 REPORT ON NEW YEAR INVESTIGATION OF INTERNATIONAL TYPE OF IDENTIFICATION OF THE NATIONAL APPLICATION Characteristic of the applicant or of the authorized representative 9850 NL Dutch application no. Filing date 1013402 27 October 1999 Claimed priority date Applicant (Name) DSMN.V. Date of the request for an investigation of an international type Awarded by the International Research Authority (ISA) to a network of research of an international type. SN 33871 EN I. SUBJECT CLASSIFICATION (When using different classifications, please specify classification symbols) According to International Classification (IPC) lnt.CI.7: C07D501 / 06, C07D499 / 12 II. FIELDS OF TECHNIQUE EXAMINED _ Minimum documentation examined__ Classification system _ Classification symbols ____ lnt.CI.7: C07D C12P * Other documentation and minimum documentation insofar as such documents are included in the areas examined! H. NO RESEARCH AT ALL FOR CERTAIN CONCLUSIONS (comments on supplement sheet 'ir'- "' IV. LACK OF UNITY OF INVENTION (comments on supplement sheet)“ ACT / ISA / ZOf Ut 07. t970