NL1007828C2 - Complexes of beta-lactam antibiotics and 1-naphthol. - Google Patents

Description

- 1 - PN 9440

COMPLEXES OF β-LACTAM ANTIBIOTICS AND 1-NAFTOL

The invention relates to complexes of β-lactam antibiotics selected from cephradine and cefaclor, and 1-naphthol.

Complexes of β-lactam antibiotics and hydroxynaphthalenes are generally known from WO-A-93/12250, which explicitly describes the complexes of cephalexin and cefadroxil, and 2-naphthol.

The applicant has now found that the complexation of cephradine and cefaclor, which β-lactam antibiotics were shown to have a similar complexation behavior with 1-naphthol, is faster and more complete than with 2-naphthol, while the opposite is the case for other β-lactam antibiotics. .

The complexes according to the invention are particularly useful intermediates, for example in the enzymatic preparation of cephradine and cefaclor, in the recovery of the β-lactam antibiotics from reaction mixtures obtained after a chemical or enzymatic acylation reaction and in the purification of the β-lactam antibiotics . Cephradine is a β-lactam antibiotic which can be obtained by acylation of 7-aminodesacetoxycephalosporanoic acid (7-ADCA) with D-dihydrophenylglycine or a derivative thereof, for example an amide or, preferably lower (1-4 ° C) alkyl ester; cefaclor is a β-lactam antibiotic that can be obtained by acylation of 7-amino-3-chloro-cef-3-em-4-carboxylic acid with D-phenylglycine or a derivative thereof, preferably lower (1-4 C) alkyl ester , or an amide.

The complexes according to the invention can be prepared simply by contacting the β-lactam antibiotics with l-naphthol. The molar ratio of 1-naphthol to β-lactam antibiotic is preferably greater than 0.5 and in particular is between 0.5 and 2. The 1007828! The concentration of the β-lactam antibiotic is preferably chosen as high as possible, preferably greater than 0.01% by weight of β-lactam antibiotic in the reaction mixture. The temperature is not particularly critical and is, for example, between -10 and 100 ° C, preferably between -5 and 50 ° C. Nor is the pH at which the complexes are formed particularly critical; the residual concentration of the β-lactam antibiotic to be reached in solution after complexing with 1-naphthol appears to be virtually independent of the pH of the mixture in a wide range of pH values, for example from 1-10, in particular 2-9, more specifically 3-8. Consequently, that complex formation can be easily incorporated into various places in a process for the preparation of 3-lactam antibiotics, for example during an enzymatic acylation reaction, in the hydrolysis of protected 3-lactam antibiotics after a chemical acylation reaction, using protective groups, the purification of antibiotics and the isolation of β-lactam antibiotics from a reaction mixture obtained after the acylation reaction or from the mother liquor. Preferably a pH value between 2 and 9, in particular between 4 and 7, is chosen. The β-lactam antibiotic can be recovered from the complex in a manner well known to the person skilled in the art.

A particularly suitable application of the complexes according to the invention is in the enzymatic acylation of a 3-lactam core with an acylating agent, wherein at least part of the acylation reaction contains 1-naphthol in the reaction mixture. In a kinetically controlled coupling, hydrolysis of the acylating agent and the β-lactam antibiotic normally occurs during an enzymatic acylation reaction. Due to the presence of 1-naphthol in the reaction mixture and consequently the formation of the complexes according to 1007828! The invention provides a higher synthesis / hydrolysis ratio (S / H), the molar ratio of synthesis product (β-lactam antibiotic) to hydrolysis product, and less degradation of the β-lactam antibiotic takes place as a result of complexation. In addition, a higher reaction rate was realized, which limits the breakdown of the β-lactam antibiotic and the β-lactam nucleus.

The concentration at which the enzymatic acylation reaction is carried out is not particularly critical. For example, the concentration of the β-lactam core and of the acylating agent at the start of the acylation reaction is between 100 and 2,000 mM, preferably between 400 and 1,000 mM. Preferably, the β-lactam core and / or the acylating agent are supersaturated in the reaction mixture during at least part of the acylation reaction. This can be achieved, for example, by subjecting a mixture in which the / S-lactam core and / or the acylating agent are present concentrated to a pH increase or decrease or to a temperature decrease.

In principle, any enzyme suitable as a catalyst in the coupling reaction can be used as an enzyme in the acylation reaction. Such enzymes are, for example, the enzymes known under the general term penicillin amidase or penicillin acylase. Such enzymes are described, for example, in J.G. Shewale et. al. Process Biochemistry, August 1989 p. 146-154 and in J.G.

Shewale et. al. Process Biochemistry International,

June 1990, p. 97-103. Examples of suitable enzymes are enzymes derived from Acetobacter. in particular Acetobacter pasteurianum. Aeromonas, Alcaligenes, in particular Alcaliqenes faecalis.

Aphanocladium, Bacillus sp., In particular Bacillus megaterium, Cephalosporium. Escherichia, in particular Escherichia coli. Flavobacterium. Fusarium.

1007826-4 - in particular Fusarium oxvsporum and Fusarium solani, Kluwera, Mycoplana. Protaminobacter. Proteus. in particular Proteus rettgeri. Pseudomonas and Xanthomonas. in particular Xanthomonas citrii.

Preferably, an immobilized enzyme is used, since the enzyme can then be easily separated and reused.

For example, among the immobilized enzymes commercially available, the Escherichia coli enzyme from Boehringer Mannheim GmbH, commercially available under the name Enzygel®, the immobilized Penicillin-G acylase from Recordati and the immobilized Penicillin-G acylase from Pharma have proved particularly suitable. 15 Biotechnology Hanover. In addition, enzymes can also be used as a crystalline substance (CLEC's ™).

The temperature at which the enzymatic acylation reaction is carried out is not particularly critical and, due to the stability of the enzyme 20, is usually below 40 ° C, preferably between -5 and 35 ° C. The pH at which the enzymatic acylation reaction is carried out is usually between 5.5 and 9.5, preferably between 6.0 and 9.0.

Preferably, the reaction is stopped almost completely when the maximum conversion has been reached. A suitable embodiment to stop the reaction is to lower the pH, preferably to a value between 4.0 and 6.3, in particular between 4.5 and 5.7. Another suitable embodiment is to lower the temperature of the reaction mixture once the maximum conversion has been reached. A combination of both embodiments is also possible.

After the reaction has virtually stopped upon reaching maximum conversion, the reaction mixture is usually present in the form of a suspension containing multiple solids, for example, the antibiotic, D-phenylglycine and optionally immobilized enzyme 1007828-5. Due to the process economy, the immobilized enzyme is preferably recovered. This can conveniently be done, for example, by filtering the reaction mixture over a screen while stirring, preferably choosing the direction of rotation of the stirrer such that the suspension is pumped up at the center of the stirrer. Valuable components, for example the antibiotic and FG, can then be recovered, for example with the aid of a pH change.

Within the scope of the invention, a pH reduction can be effected, for example, by adding an acid. Suitable acids are, for example, mineral acids, in particular sulfuric, hydrochloric or nitric and carboxylic acids, for example acetic, oxalic and citric acid. For example, a pH increase can be accomplished by adding a base. Suitable bases are, for example, inorganic bases, in particular ammonia, caustic potash or caustic soda and organic bases, for example triethylamine and FGA. Ammonia is preferably used.

The enzymatic acylation reaction and the indicated measures, for example the preparation of the supersaturated mixtures, can be carried out in water. If desired, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30% by volume. Examples of organic solvents that can be used are alcohols with 1-7 carbon atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, especially ethylene glycol or a triol, especially glycerol.

The molar ratio of acylating agent to β-35 lactam core, i.e. the total amount of added acylating agent divided by the total amount of added β-lactam core expressed in moles, is 1007828-6 - less than 2.5. The molar ratio is preferably between 0.5 and 2.0, in particular between 0.7 and 1.8.

The enzymatic acylation reaction is preferably carried out as a batch process. If desired, it is also possible to carry out the reaction continuously.

The invention will be further elucidated by means of the examples without, however, being limited thereto.

10

Examples 7 -ACCA: 7 -amino- 3 -chloro-cef-3-em-4-carboxylic acid 7 -ADCA: 7 -aminodesacetoxycephalosporanoic acid 6-APA: 6-aminopenicillanic acid 15 CC1: cefaclor CEX: cephalexin FG: D-phenylglycine FGA: D-phenylglycine amide FGH: Dp-hydroxyphenylglycine 20 FGHM: Dp-hydroxyphenylglycine methyl ester

Assemblase ™ is an immobilized Escherichia coli penicillin acylase from E. coli ATCC 11105 as described in WO-A-97/04086. The immobilization has been 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 amounted to 3 ASU / g dry weight with 1 ASU (Amoxicillin Synthesis Unit) defined as the amount of enzyme containing 1 g Amoxicillin per hour. .3H20 generates from 6-APA and FGHM (at 20 ° C); 6.5% 6-APA and 6.5% FGHM).

1007828 - 7 -

Example I

Complexation of cephradine with 1-naphthol or 2-naphthol (comparative experiment) as complexing agent.

A basic solution was added dropwise to a (aqueous) solution of cephradine 5 with a concentration of 1.0 m% until the pH value of 6.3 was reached. An equimolar amount of 1-naphthol or 2-naphthol was then added at room temperature.

Samples were taken at various times during a stirring incubation. After filtration of these samples through a 0.45 µ filter, the concentration of the respective cephalosporin present in the filtrate was determined by HPLC. The 15 results are shown in Table 1.1.

Table 1.1

Complexation of cephradine with 1-naphthol or 2-naphthol as a complexing agent.

reaction time [cephradine] in filtrate (m%) (hours) 0 0.5 1.5 24 1-naphthol 1.0 0.65 0.05 0.03 25 2-naphthol * 1.0 0.80 0.65 0.65

* comparative experiment Example II

Example I was repeated for cefaclor instead of cephradine; now at a pH value of 7.0.

The results are shown in Table 1.2.

1007828 5 - 8 -Table 1.2

Complexation of cefaclor with 1-naphthol or 2-naphthol as complexing agent (pH 7.0, room temperature).

reaction time [cefaclor] in filtrate (m%) (hours) 0 0.5 1.5 24 1-naphthol 1.05 0.72 0.38 0.13 2-naphthol * 1.02 0.47 0.43 0 , 22 10 * comparative experiment

Comparative experiment A

Example I was repeated for cefadroxil in place of cephradine. The results are shown in Table 1.3.

Table 1.3 20 Complexation of cefadroxil with 1-naphthol or 2-naphthol as complexing agent.

reaction time [cefadroxil] in filtrate (m%) (h) 0 0.5 1.5 24 25 1-naphthol 1.0 0.98 0.80 0.73 2-naphthol 1.0 0.49 0.47 0 , 42

Example III

Complexation of cephradine with 1-naphthol as complexing agent at various pH values.

A (aqueous) cephradine solution with a (starting) concentration of 1.8% by mass was divided between 3 1007828-9 reaction vessels. The cephradine solution was adjusted to pH 4.5 in one of the reaction vessels using a dilute sulfuric acid solution. The pH value of the cephradine solution in the other reaction vessels was adjusted to pH 6.3 and pH 7.0 respectively with a dilute ammonia solution. An equimolar amount of 1-naphthol was then added at room temperature. Samples were taken at various times during a stirring incubation.

The concentration of cephradine present in filtrate samples is shown in Table 2.1.

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1007828 - 11 -

Example IV

To an aqueous solution (temperature 10 ° C) with 10 g FGA (454 mM) and 11.3 g 7-ACCA (328 mM) was added 3.4 g 1- or 2-naphthol (160 mM) at T = 10 ° C.

Before addition of Assemblase ™ (24 g), the pH was adjusted to 7.0 using 2N H 2 SO 4. Samples were taken at various times and analyzed by HPLC. The conversion (the number of moles of cefaclor formed relative to the number of moles of 7-10 ACCA with which the reaction started) is shown in Table 3.1.

Table 3.15 Enzymatic synthesis of cefaclor in the presence of 1-naphthol or 2-naphthol (10eC) conversion (%) time (min) 2 15 29 56 176 20 1-naphthol 4.8 16.6 34.8 59 1> 90 2-naphthol 0.9 9.7 19.1 40.2 84.6 1007828

Claims (9)

1. Complex of a jS-lactam antibiotic selected from cephradine and cefaclor and l-naphthol. A process for the preparation of a complex according to claim 1, wherein the corresponding i-lactam antibiotic is contacted with 1-naphthol. A process for the preparation of a complex according to claim 1, wherein the corresponding β-lactam antibiotic is prepared by acylation of the corresponding β-lactam core with a suitable acylating agent and wherein at least part of the acylation reaction 15-naphthol 15 is present. is in the reaction mixture. The method of claim 3, wherein the acylation is performed in the presence of an enzyme. The method of claim 3 or 4, wherein the complex is isolated and the β-lactam antibiotic is released from the complex. 6. A method for recovering a β-lactam antibiotic selected from cephradine and cefaclor, wherein a mixture containing the β-lactam antibiotic is contacted with 1-naphthol and the complex formed is recovered. The method of claim 6, wherein the β-lactam antibiotic is subsequently released from the complex. 8. Complexes and process for the preparation of complexes and 0-lactam antiobiotics therefrom as described and illustrated by the examples. 1007828 COOPERATION TREATY {PCI). REPORT ON NEWNESS RESEARCH OF INTERNATIONAL TYPE I0ENTIFIKAT1E OF OE NATIONAL APPLICATION Characteristic of "o · applicant ol of αβ gemacntigce 'j 9440NL j Dutch application no. Inawnngsdaajm 1007828 18 December 1997 - --- _ - ____ _____ ) | CHEMFERM vof | _ I Date of reconciliation for a turmoil * of mtemaaonal type Ooor Oe insanae for International Inazur (ISA) reconciled to a turmoil of mtemaoonaaJ type soegeKerxJ no. J SN 30489 EN I I. CLASSIFICATION OF THE SUBJECT (at eecassmg of verscnilienoe classifications. indicate all oasstficaoeymboien) According to oe inteinaoonaie dassilicaoe (IPC) Int.Cl.6: C 07 D 501/12, C 12 P 35/04 II. RESEARCHED FIELDS OF TECHNIQUE I_____ 1___ Minimum submission documentation _ 1 Classification System | _ClassificationSyrrccien __; _ j ί! - i Int. CL. 6: C 07 D, C 12 P ί I r I I i I i i i Ψ ... - - --------: -. _ - --------- - -! Onaerzocnte ancere coajmenaoe to ae miremum accurrenaoe feed as far as primordial people with a submarine geoeoen 13η eyes ί 1 | \; j I I I I i j IU. ; NO MCCEL1JK INQUIRY FOR CERTAIN CONCLUSIONS; ccner * inger. co supplement sheet ', IV._ LACK OF UNITY OF INVENTION (ccmerKircen cc supplemental',: sr— Dz ~: 5a, :: · 3: =