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Title:
COMPLEXES OF ß-LACTAM ANTIBIOTICS AND 1-NAPHTHOL
Document Type and Number:
WIPO Patent Application WO/1999/031109
Kind Code:
A1
Abstract:
The invention relates to complexes of cephradine and cefaclor and 1-naphthol. It has been found that 1-naphthol shows better complexing behaviour then for example 2-naphthol. The invention also relates to a process for the preparation of such complexes, the corresponding ß-lactam antibiotic being prepared through acylation of the corresponding ß-lactam nucleus with a suitable acylating agent and 1-naphthol being present in the reaction mixture during at least part of the acylation reaction. The acylation is preferably carried out in the presence of an enzyme. The ß-lactam antibiotic can subsequently be released from the complex in a known manner.

Inventors:
KEMPERMAN GERJAN (NL)
DE GELDER RENE (NL)
RAEMAKERS-FRANKEN PETRONELLA C (NL)
Application Number:
PCT/NL1998/000714
Publication Date:
June 24, 1999
Filing Date:
December 14, 1998
Export Citation:
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Assignee:
DSM NV (NL)
KEMPERMAN GERJAN (NL)
GELDER RENE DE (NL)
RAEMAKERS FRANKEN PETRONELLA C (NL)
International Classes:
C07D501/00; C07D501/12; C12P35/04; (IPC1-7): C07D501/12; C12P35/04
Domestic Patent References:
WO1993012250A11993-06-24
Foreign References:
US4003896A1977-01-18
Other References:
CHEMICAL ABSTRACTS, vol. 84, no. 21, 24 May 1976, Columbus, Ohio, US; abstract no. 150644, KODAMA T. ET AL.: "Purification of cephalosporins" XP002059554
Attorney, Agent or Firm:
Jacobs, Monique Sophie Nicole (Octrooibureau DSM P.O. Box 9 MA Geleen, NL)
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Claims:
C L A I M S
1. Complex of a ßlactam antibiotic chosen from the group comprising cephradine and cefaclor and 1 naphtol.
2. Process for the preparation of a complex according to Claim 1, in which the corresponding ßlactam antibiotic is brought into contact with 1 naphtol.
3. Process for the preparation of a complex according to Claim 1, in which the corresponding ßlactam antibiotic is prepared through acylation of the corresponding ßlactam nucleus with a suitable acylating agent and in which 1naphtol is present in the rection mixture during at least part of the acylation rection.
4. Process according to Claim 3, in which the acylation is carried out in the presence of an enzyme.
5. Process ac : ording to Claim 3 or Claim 4, in which the complex is isolated and the ßlactam antibiotic is released from the complex.
6. Process for the recovery of a ßlactam antibiotic chosen from the group comprising cephradine and cefaclor, a mixture containing the ßlactam antibiotic being brought into contact with 1 naphtol and the complex formed being recovered.
7. Process according to Claim 6, the ßlactam antibiotic subsequently being released from the complex.
Description:
COMPLEXES OF ß-LACTAM ANTIBIOTICS AND 1-NAPHTOL The invention relates to complexes of ß- lactam antibiotics chosen from the group comprising cephradine and cefaclor, and 1-naphthol.

Complexes of ß-lactam antibiotics and hydroxynaphthalenes are known in general terms from WO- A-93/12250, which explicitly describes the complexes of cephalexine and cephadroxyl, and 2-naphthol.

The applicant has now found that the formation of a complex of cephradine and cefaclor, which ß-lactam antibiotics proved to show comparable complexing behaviour, proceeds faster and more completely with 1-naphtol than with 2-naphthol, whereas the opposite holds for other ß-lactam antibiotics.

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

or a derivative thereof, preferably a lower (1-4 C) alkyl ester, or an amide.

The complexes according to the invention can be prepared in a simple manner by bringing the ß- lactam antibiotic into contact with 1-naphthol. The molar ratio of the 1-naphtol and the ß-lactam antibiotic is preferably greater than 0.5 and is in particular between 0.5 and 2. The concentration of the ß-lactam antibiotic is preferably chosen to be as high as possible, preferably greater than 0.01 wt. % ß-lactam antibiotic in the rection mixture. The temperature applied is not particularly critical and is for example between-10 and 100°C, preferably between-5 and 50°C.

The pH at which the complexes are formed is not particularly critical either; the residual concentration of the ß-lactam antibiotic in solution to be obtained after complexing with 1-naphtol proves to be virtually independent of the mixture's pH in a wide range of pH values, for example between 1 and 10, in particular 2 and 9, more in particular 3 and 8. That complex formation can consequently be incorporated in a simple manner at various points in a process for the preparation of ß-lactam antibiotics, for example during an enzymatic acylation rection, in the hydrolysis of protected ß-lactam antibiotics after a chemical acylation rection in which use is made of protecting groups, in the purification of antibiotics or in the .,, j isolation of ß-lactam antibiotics from a rection mixture obtained after the acylation rection or from the mother liquor. Preferably a pH value of between 2 and 9, in particular between 4 and 7, is chosen. The ß- lactam antibiotic can be recovered from the complex in

a manner that is generally known to those skilled in the art.

A particularly suitable application of the complexes according to the invention is in the enzymatic acylation of a ß-lactam nucleus with an acylating agent, 1-naphtol being present in the rection mixture during at least part of the acylation rection. With a kinetically controlled coupling, hydrolysis of the acylating agent and the ß-lactam antibiotic usually occurs during an enzymatic acylation rection. Owing to the presence of 1-naphtol in the rection mixture, and consequently the formation of the complexes according to the invention, a higher synthesis/hydrolysis ratio (S/H), the molar ratio of the synthesis product (ß-lactam antibiotic) and the hydrolysis product, is obtained and less decomposition of the ß-lactam antibiotic occurs as a result of the complexing. In addition, a higher rection rate was realised, as a result of which the decomposition of the ß-lactam antibiotic and the ß-lactam nucleus is restricted.

The concentration at which the enzymatic acylation rection is carried out is not particularly critical. The concentration of the ß-lactam nucleus and of the acylating agent at the beginning of the acylation rection is for example between 100 and 2,000 mM, preferably between 400 and 1,000 mM. Preferably the ß-lactam nucleus and/or the acylating agent are during at least part of the acylation rection present in the rection mixture in a supersaturated form. This can for example be realised by subjecting a mixture in which the ß-lactam nucleus and/or the acylating agent are

present in a concentrated form to an increase or reduction in pH or to a reduction in temperature.

As the enzyme in the acylation rection any enzyme can in principle be used that is suitable for use as a catalyst in the coupling rection. Such enzymes are for example the enzymes known under the general name of penicillin amidase or penicillin acylase. Such enzymes are for example described in J. G.

Shewale et al., Process Biochemistry, August 1989, pp.

146-154, and in J. G. Shewale et al., Process Biochemistry International, June 1990, pp. 97-103.

Examples of suitable enzymes are enzymes derived from Acetobacter, in particular Acetobacter pasteurianum, Aeromonas, Alcaligenes, in particular Alcaligenes faecalis, Aphanocladium, Bacillus sp., in particular Bacillus megaterium, Cephalosporium, Escherichia, in particular Escherichia coli, Flavobacterium, Fusarium, in particular Fusarium oxysporum and Fusarium solani, Kluyvera, Mycoplana, Protaminobacter, Protes, in particular Proteus rettgeri, Pseudomonas and Xanthomonas, in particular Xanthomonas citrii.

Preferably use is made of an immobilised enzyme, because the enzyme can then be separated and reused in a simple manner. of the commercially available immobilised enzymes, the Escherichia coli enzyme of Boehringer Mannheim GmbH that is commercially available under the name of Enzygel@, the immobilised Penicillin-G acylase of Recordai and the immobilised Penicillin-G acylase of Pharma Biotechnology Hannover for example have proved to be very suitable. Enzymes can also be used as a crystalline substance (CLECsTM).

The temperature at which the enzymatic acylation rection is carried out is not particularly critical and is, on account of the enzyme's stability, usually lower than 40°C, preferably between-5 and 35°C.

The pH at which the enzymatic acylation rection is carried out is usually between 5.5 and 9.5, preferably between 6.0 and 9.0.

Preferably the rection is almost completely stopped as soon as almost the maximum degree of conversion has been reached. A suitable mode of stopping the rection is lowering the pH, preferably to a value of between 4.0 and 6.3, in particular between 4.5 and 5.7. Another suitable mode is lowering the temperature of the rection mixture as soon as the maximum degree of conversion has been reached. A combination of the two modes is also possible.

After the rection has been virtually stopped when the maximum degree of conversion has been reached, the rection mixture is usually present in the form of a suspension containing several solid substances, for example the antibiotic and D-phenylglycine, while immobilised enzyme may also be present. The immobilised enzyme is preferably recovered, in view of process economics. This can for example be carried out in a suitable manner by filtering the rection mixture through a sieve, with stirring, the stirrer's direction of rotation preferably being chosen so,, 5hat the suspension is pumped upwards at the centre of the stirrer. Valable components, for example the antibiotic and PG, can subsequently be recovered, for example with the aid of a change in pH.

A reduction in pH can in the context of the invention for example be effected by adding an acid.

Suitable acids are for example mineral acids, in particular sulphuric acid, hydrochloric acid or nitric acid, and carboxylic acids, for example acetic acid, oxalic acid or citric acid. An increase in pH can for example be effected by adding a base. Suitable bases are for example inorganic bases, in particular ammonia, potassium hydroxide or sodium hydroxide, and organic bases, for example triethylamine and D-phenylglycine amide. Preferably ammonia is used.

The enzymatic acylation rection and the indicated mesures, for example the preparation of the supersaturated mixtures, can be carried out in water.

The rection mixture may optionally also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol. %. Examples of organic solvents that can be uzed are alcools with 1-7 C atoms, for example a monoal. cohol, in particular methanol or ethanol; a diol, in particular ethylene glycol, or a triol, in particular glycerol.

The molar ratio of the acylating agent and the ß-lactam nucleus, i. e. the total amount of acylating agent supplie divided by the total amount of ß-lactam nucleus supplie expressed in moles, is smaller than 2.5. Preferably the molar ratio is between 0.5 and 2.0, in particular between 0.7 and 1.8.

The enzymatic acylation rection is preferably carried out as a batch process. It is optionally also possible to carry out the rection continuously.

The invention will be further elucidated with reference to the examples without however being limited thereby.

Examples 7-ACCA: 7-amino-3-chloro-ceph-3-em-4-carboxylic acid 7-ADCA: 7-aminodesacetoxycephalosporanic acid 6-APA: 6-aminopenicillanic acid CCl: cefaclor CEX: cephalexine PG: D-phenylglycine PGA: D-phenylglycine amide HPG: D-p-hydroxyphenylglycine HPGM: D-p-hydroxyphenylglycine methyl ester Assemblase is an immobilised Escherichia coli peniC411in acylase from E. coli ATCC 11105, as described in WO-A-97/04086. The immobilisation was carried out as described in EP-A-222462, using gelatine and chitosan as the gelling agents and glutaraldehyde as a crosslinker.

The ultimate activity of the Escherichia coli penicillin acylase is determined by the amount of enzyme added to the activated spheres and was 3 ASU/g of dry weight, 1 ASU (Amoxicillin Synthesis Unit) being defined as the amount of enzyme that generates 1 g of Amoxicillin. 3H20 per hour from 6-APA and HPGM (at 20°C) ; 6.5% 6-APA and 6.5% HPGM).

Exam, ple I Complexing of cephradine using 1-naphtol or 2-naphtol (comparative experiment) as the complexing agent.

A basic solution was added, drop by drop, to a (n aqueous) solution of cephradine having a concentration of 1.0 m. % until a pH of 6.3 was obtained. Next, an equimolar amount of 1-naphtol or 2- naphtol was added at room temperature.

Samples were taken at different moments during a stirring incubation. After these samples had been filtered through a 0.45 p filter, the concentration of the cephalosporine in question present in the filtrate was determined with the aid of HPLC.

The results are presented in Table 1.1.

Table 1.1 Complexing of cephradine using 1-naphtol or 2-naphtol as the complexing agent. rection [cephradine] in filtrate (m. t) time (hours) 0 0.5 1.5 24 1-naphtol 1.0 0.65 0.05 0.03 2-naphthol* 1.0 0.80 0.65 0.65 * comparative expriment

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

The results are presented in Table 1.2.

Table 1.2 Complexing of cefaclor using 1-naphtol or 2-naphtol as a complexing agent (pH 7.0, room temperature). rection [cefaclor] in filtrate (m. %) time (hours) 0 0.5 1.5 24 1-naphtol 1.05 0.72 0.38 0.13 2-naphthol* 1.02 0.47 0. 43 0.22 l

* comparative expriment AComparativeExperiment Example I was repeated for cefadroxil instead of cephradine. The results are presented in Table 1.3.

Table 1.3 Complexing of cefadroxyl using 1-naphtol or 2-naphtol as the complexing agent. reactionreaction[cefadroxyl] in filtrate (m.%) time (hours) 0 0.5 1.5 24 1-naphthol 1.0 0.98 0.80 0.73 2-naphthol 1.0 0.49 0.42 Example III Complexing of cephradine using 1-naphtol as the complexing agent at various pH values.

A (n aqueous) cephradine solution having a (n initial) concentration of 1.8 percent by mass was divided between 3 rection vesses. With the aid of a diluted sulphuric acid solution the cephradine solution in one of the rection vessels was brought to a pH of 4.5. The pH values of the cephradine solutions in the other rection vessels were brought to 6.3 and 7.0 respectively, with a diluted ammonia solution. Next, an equimolar amount of 1-naphtol was added at room temperature. Samples were taken at various moments during a stirring incubation.

The concentrations of cephradine present in filtrate samples are indicated in Table 2.1.

Table 2.1<BR> Complexing of cephradine using 1-naphthol as a complexing agent at various pH reaction [cephradine] in filtrate (m.%) time (hours) 0 0.5 1.5 24 pH 4.5 6.3 7.0 4.5 6.3 7.0 4.5 6.3 7.0 4.5 6.3 7.0 1- 1.8 1.8 1.8 1.64 1.26 1.22 0.84 0.88 1.13 0.79 0.78 1.0 naphthol

Example IV To an aqueous solution (temperature 10°C) containing 10 g of PGA (454 mM) and 11.3 g of 7-ACCA (328 mM) was added 3.4 g of 1-or 2-naphtol (160 mM) at T = 10°C.

Before AssemblaseTM (24 g) was added, the pH was brought to 7.0 with the aid of 2N H2SO4. Samples were taken at various moments and analyse with the aid of HPLC. The degree of conversion (the number of moles of cefaclor formed relative to the number of moles of 7-ACCA with which the rection began) is shown in Table 3.1.

Table 3. 1 Enzymatic synthesis of cefaclor in the presence of 1- naphtol or 2-naphtol (10°C) Degree of conversion (%) 2152956176Time(min.) 1-naphthol1-naphthol4.8 16.6 34.8 59.1 >90 2-naphthol 19.140.284.69.7