PROCESS FOR THE PREPARATION OF A beta -LACTAM ANTIBIOTIC

Abstract: The invention relates to a process for the preparation of a beta -lactam antibiotic by enzymatic acylation of a beta -lactam core by means of an acylation agent, with the molar ratio between the beta -lactam core and the acylation agent being between 0.5:1 and 2:1, the concentration of inorganic salts in the reaction mixture being lower than 1000:n mM, n representing the valency of the anion, and the sum of the concentrations of the beta -lactam antibiotic and the beta -lactam core being between 200 and 800 mM. This process offers the advantage that the enzyme can be re-used many times without much loss of activity of the enzyme. This makes it possible to obtain a commercially attractive process for the preparation of beta -lactam antibiotics via enzymatic acylation of beta -lactam cores.

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 enzymatic acylation of a β-lactam core by means of an acylation agent. Such a process is described for instance in WO- A-9201061. In the process described in that publication a large excess of acylating agent relative to the β-lactam core is applied. The pH is brought to a certain, relatively low level and kept constant. If in such a process the enzyme is re-used a number of times it appears that the enzyme activity declines relatively strongly. In practice, however, it appears that in order to get a commercially attractive process, the enzyme to be applied should be suitable to be re-used many times (for instance more than 50 times, in particular more than 100 times) without much loss of activity. The object of the invention is to obtain a process for the enzymatic acylation of a β-lactam core whereby said drawback is eliminated. This is achieved according to the invention in a process for the preparation of a β-lactam antibiotic by enzymatic acylation of a β-lactam core by means of an acylation agent with the molar ratio between the β-lactam core and the acylation agent being between 0.5:1 and 2:1, the concentration of inorganic salts in the reaction mixture being lower than 1000:n mM, n representing the valency of the anion, and the sum of the concentrations of the β-lactam antibiotic and the β-lactam core being between 200 and 800 mM. - 2 - The fact is that the applicant has found that the enzyme activity in successive cycles depends on the concentration of the inorganic salts in the reaction mixture during the enzymatic acylation reaction, as well as on the sum of the concentrations of the β-lactam antibiotic and the β-lactam core. Suitable examples of β-lactam cores that can be used in the process according to the invention are penicillic acid derivatives, for instance 6- aminopenicillic acid (6-APA), and cephalosporanic acid derivatives, for instance 7-aminocephalosporanic acid (7- ACA), 7-aminodesacetoxy-cephalosporanic acid (7-ADCA) and 7-amino-3-chlorocephalosporanic (7-ACCA) . Suitable acylation agents that can be used in the process according to the invention are for instance α- amino acid derivatives, in particular amides and esters of phenyl glycine, p-hydroxyphenyl glycine and dihydrophenyl glycine. In principle any enzyme can be used that is suitable as catalyst in the coupling reaction. Such enzymes are for instance the enzymes that are known under the general designations 'penicillin amidase' and 'penicillin acylase'. Examples of suitable enzymes are enzymes derived from Acetobacter, Aeromonas, Alcaliαenes, Aphanocladium, Bacillus SD. , Cephalosporium. Escherichia. Flavobacterium, Kluvvera, Mvcoplana, Protaminobacter, Pseudomonas and Xanthomonas, in particular Acetobacter pasteurianum, Alcaliαenes faecalis, Bacillus meσaterium. Escherichia coli and Xanthomonas citrii. Preferably an immobilized enzyme is used, since the enzyme can be easily re-used then. Immobilized enzymes are known as such and are commercially available. Highly suitable enzymes have appeared to be the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name 'Enzygel*', the immobilized Penicillin-G acylase from Recordati, the immobilized Penicilline-G acylase from Pharma Biotechnology Hannover, and an Escheria coli penicilline acylase isolated as described in WO-A-92/12782 and immobilised as described in EP-A-222462. The enzymatic acylation reaction is mostly carried out at a temperature between -5 and 35°C, in particular between 5 and 35°C preferably between 0 and 28°C, most preferably between 15 and 28°C. The pH at which the acylation reaction is carried out is mostly between 6 and 8.5. The optimum pH depends on, among other things, the antibiotic, since the stability and the solubility of the β-lactam antibiotic as well as the β-lactam core depend on the pH. If a phenyl glycine derivative is used as acylation agent the pH is preferably between 6.2 and 8.5, most preferably between 7 and 8; if a p-hydroxyphenyl glycine derivative is used as acylation agent the pH is preferably between 6 and 7.5, most preferably between 6 and 7. Besides, the enzyme activity is also pH-related. In practice the acylation reaction is mostly carried out in water. Optionally, the reaction mixture may also contain an organic solvent or a mixture of organic solvents, preferably less than 30 vol.%. Examples of organic solvents that can be used are alcohols with 1-7 carbon atoms, for instance a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol or a triol, in particular glycerol. The concentration of the β-lactam antibiotic and the concentration of the β-lactam core are chosen such that the sum of the two concentrations is between 200 and 800 mM. Preferably the concentrations are chosen such that the sum of the two concentrations is between 300 and 700, most preferably between 300 and 600 mM. The molar ratio between the acylating agent and the β-lactam core is between 0.5:1 and 2:1, preferably between 0.7:1 and 1.3:1, most preferably between 0.8:1 and 1.2:1. The molar ratio between the acylating agent and the β-lactam core is preferably chosen such that the pH of the reaction mixture throughout the process does not exceed the above-mentioned limits, without titration with acid. The fact is that the applicant has found that if in such processes the pH is kept constant by titration with a strong acid, the impairment of the enzyme stability is aggravated by the occurrence of so-called 'hot spots' (low-pH spots) as a result of the direct addition of acids to the reaction mixture. This effect occurs in addition to the effect which is caused by the increase of the salt concentration as a result of the titration. It has been found that these two effects reinforce each other. Since ammonia is released in the acylation reaction if amides are used as acylation agent, inorganic salts will accumulate in the reaction mixture in the presence of anions, for instance Cl", S0 4 2 ", P0 4 3 ", N0 3 ". It has been found that if the concentration of inorganic salts becomes too high, for instance higher than 1000:n mM, where n is the valency of the anion, the enzyme activity declines strongly after several cycles. The concentration of inorganic salts in the process according to the invention is therefore kept below 1000:n mM, preferably below 700:n mM, with n as defined above, which implies that for chlorides and nitrates n is 1 and for sulphates it is 2. In order to keep the concentration of inorganic salts low, it is preferred to ensure that the amount of inorganic salts formed during the enzymatic acylation reaction is restricted to a minimum. In the preparation of the known β-lactam antibiotics, such as for instance cephalexin, amoxicillin, ampicillin, cephaclor, cephradin, cephadroxyl and cephotaxim, frequently used acylation agents are phenyl glycine and p-hydroxyphenyl glycine derivatives, for instance their amides or esters. In the known processes these are often used in the form of the salt of a strong acid, for instance phenyl glycine amide.HC1, phenyl glycine amide.*$H 2 S0 4 , phenyl glycine methyl ester.HC1 or dihydrophenyl glycine methyl ester.HC1. In the process according to the invention the free amide or the free ester is preferably used in order to prevent the formation of additional inorganic salts as a result of neutralization of the salts of the acylation agents. Free amides in solid form are particularly suitable for this purpose. It is known that the free amide of D-phenyl glycine (D-PGA) is difficult to recover in solid form. It has been found now that free D-phenyl glycine amide can be obtained simply by neutralization with a base, preferably ammonia, of an aqueous solution of D-phenyl glycine amide in the form of a salt of a strong acid, and recovery, after cooling, of the free D-phenyl glycine amide in solid form. The concentration of the sulphuric acid salt of D- phenyl glycine amide preferably is between 0.5 M and 4 M, in particular between 1 and 3 M. The temperature after cooling is mostly between -5 and 25°C, preferably between 0 and 15°C. The β-lactam antibiotic formed can be recovered in a known manner from the reaction mixture, for instance by means of complexation or crystallization. The invention will be further elucidated now by means of the following examples, without however being restricted thereto. General description of the enzyme recycling experiments In each of the following examples, I to X, enzyme recycling experiments are described, i.e. in one experiment a certain amount of enzyme is subjected several times to identical reaction conditions. This was achieved by performing the experiments in a double-walled, stirred reaction vessel, the bottom of which consisted of a G-3 glass filter. At the end of each run the reaction mixture was removed by filtration with suction; the enzyme remaining behind was washed and then used directly in the following run. In each of the examples I to X immobilized Pen-G acylase from Recordati (Milan) was used. This enzyme is commercially available in a mixture of water and glycerol ('wet enzyme'); before each application it was washed three times with water. The temperature in each of these experiments was 25°C. In the title and the description of each example the concentrations of the acylation agent and the β-lactam core in the starting mixture (or 'feed') are stated, as well as in what form the acylation agent is applied (as a salt of an acid or in free form), the development of the pH in the reaction and whether titration takes place and if so, with what acid. If titration has been effected, this means that the pH has been kept constant at the stated final value. In each example a standard reaction time for the entire series was used. After each run the concentration of β-lactam antibiotic in the filtrate after filtration and washing was determined. The development of these concentrations is shown in the corresponding graph; the derived direction coefficient of the curve is taken as a measure of the decline of the enzyme activity in the experiment concerned. In order to obtain good insight into the decline of the enzyme activity, the standard reaction time chosen for each example was so short that already in the first run no optimum quantity of β-lactam antibiotic was produced. Example I Enzyme recycling experiment with 600 mM of D-PGA.%H 2 S0 4 and 400 mM of 7-ADCA, pH 7.5 → 7.8, and titration with 12N H 2 S0 4 . 20 g of wet enzyme was washed with water and introduced into the reaction vessel. To this was added a feed consisting of 61.0 g of D-PGA.*jH 2 S0 4 , 43.7 g of 7-ADCA (purity 98.3 mass ), 1.8 g of D-PG, 375 ml of water and 27.5 ml of 25 mass% aqueous NH 4 OH (pH of the feed = 7.5). The reaction mixture was stirred at 25°C. While this was done the pH increased to 7.8, after which it was kept constant at 7.8 by means of titration with 12N H 2 S0 4 . After two hours the mixture was suction filtered. Then the enzyme was washed twice with water; the washing waters were also filtered and added to the first filtrate. The remaining clear solution was weighed, after which the cephalexin content was determined by means of HPLC. Results: see graph 1 for the quantity of cephalexin after each run. The estimated average decline of the enzyme activity is about 5.5% per run. Example II Enzyme recycling experiment with 600 mM of D-PGA.>$H 2 S0 4 an( 3 400 mM of 7-ADCA, pH 7.5 -♦ 7.8, and titration with 2N H 2 S0 4 . Carried out analogously to example I, with 20 g of wet enzyme and a feed consisting of 61.0 g of D- PGA.J $ H 2 S0 4 , 43.7 g of 7-ADCA, 1.8 g of D-PG, 375 ml of water and 27.5 ml of 25 mass% aqueous NH 4 OH. Initial pH = 7.5, final pH = 7.8, kept constant by means of titration with 2N H 2 S0 4 . Reaction time: 2 hours. Results: see graph 1. Estimated average decline of the enzyme activity: 3.4% per run. Example III Enzyme recycling experiment with 600 mM of D-PGA and 400 mM of 7-ADCA, pH 7.5 -♦ 7.8, and titration with 12N H 2 S0 4 . Carried out analogously to example I, with 20 g of wet enzyme and a feed consisting of 45.0 g of free D- PGA, 43.7 g of 7-ADCA, 1.8 g of D-PG, 413 ml of water and 5 ml of 25 mass% aqueous NH 4 0H. Initial pH - 7.6, final pH = 7.8, kept constant by means of titration with 12N H 2 S0 4 . Reaction time: 2 hours. Results: see graph 1. Estimated average decline of the enzyme activity: 2.4% per run. Example IV Enzyme recycling experiment with 500 mM of D-PGA and 500 mM of 7-ADCA, pH 7.4 → 7.55, in the presence of 380 mM of (NH 4 ) 2 S0 4 . Carried out analogously to example I, with 25 g of wet enzyme and a feed consisting of 37.5 g of free D- PGA, 54.6 g of 7-ADCA, 25 g of (NH 4 ) 2 S0 4 and 375 ml of water. No titration. Initial pH = 7.4, final pH = 7.55. Reaction time 75 minutes. Results: see graph 2. Estimated decline of the enzyme activity: 0.5% per run. Example V Enzyme recycling experiment with 600 mM of D-PGA.> $ H 2 S0 4 and 400 mM of 7-ADCA, pH 7.2 -♦ 7.5, in the presence of 100 mM of (NH 4 ) 2 S0 4 . Carried out analogously to example I, with 25 g of wet enzyme, 60.0 g of D-PGA.JjH 2 S0 4 , 43.8 g of 7-ADCA, 6.6 g of (NH 4 ) 2 S0 4 , 14.2 g of 25 mass% aqueous NH 4 0H and 390 ml of water. No titration. Initial pH = 7.2, final pH = 7.5. Reaction time 75 minutes. Results: see graph 2. Estimated decline of the enzyme activity: 0.9% per run. Example VI Enzyme recycling experiment with 500 mM of D-PGA and 500 mM of 7-ADCA, pH 7.3 → 7.5, in the presence of 500 mM of NH 4 C1. Carried out analogously to example I, with 25 g of wet enzyme and a feed consisting of 37.5 g of free D- PGA, 54.6 g of 7-ADCA, 13.4 g of NH 4 C1 and 375 ml of water. No titration. Initial pH = 7.3, final pH = 7.5. Reaction time 75 minutes. Results: see graph 2. Estimated decline of the enzyme activity: 0.6% per run. Example VII Enzyme recycling experiment with 500 mM of D-PGA and 500 mM of 7-ADCA, pH 7.3 → 7.4, in the presence of 1000 mM of NH 4 C1. Carried out analogously to example I, with 25 g of wet enzyme and a feed consisting of 37.5 g of free D- PGA, 54.6 g of 7-ADCA, 26.8 g of NH 4 C1 and 375 ml of water. No titration. Initial pH = 7.3, final pH = 7.5. Reaction time 75 minutes. Results: see graph 2. Estimated decline of the enzyme activity: 1.3% per run. Example VIII Enzyme recycling experiment with 300 mM of D-PGA and 300 mM of 7-ADCA, pH 7.2 → 7.8. Carried out analogously to example I, with 50 g of wet enzyme and a feed consisting of 45.0 g of free D- PGA, 65.0 g of 7-ADCA, 1.8 g of D-PG and 900 ml of water. No ammonia used and no titration. Initial pH = 7.2, final pH = 7.8. Reaction time 75 minutes. Results: see graph 3. Estimated decline of the enzyme activity: < 0.05% per run. Example IX Enzyme recycling experiment with 400 mM of D-PGA and 400 mM Of 7-ADCA, pH 7.3 ■ * 7.8. Carried out analogously to example I, with 25 g of wet enzyme and a feed consisting of 30.0 g of free D- PGA, 43.7 g of 7-ADCA and 425 ml of water. No ammonia used and no titration. Initial pH = 7.3, final pH = 7.8. Reaction time 75 minutes. Results: see graph 3. Estimated decline of the enzyme activity: 0.1% per run. Example X Enzyme recycling experiment with 500 mM of D-PGA and 500 mM of 7-ADCA, pH 7.4 → 7.8. Carried out analogously to example I, with 25 g of wet enzyme and a feed consisting of 37.5 g of free D- PGA, 54.6 g of 7-ADCA and 375 ml of water. No ammonia used and no titration. Initial pH = 7.4, final pH = 7.8. Reaction time 75 minutes. Results: see graph 3. Estimated decline of the enzyme activity: 0.2% per run. Conclusions drawn from the enzyme recycling experiments From graph 2 and from a comparison of graph 2 with graph 3 it appears that the enzyme declines if inorganic salts are present. This effect increases with increasing salt load, as appears from a comparison of examples VI and VII. From graph 1 it also appears that the level of the salt load has an effect on the enzyme activity, which is seen from a comparison of examples I and III, in which D-PGA.JjH 2 S0 4 and D-PGA, respectively, is started from (in the first case a higher salt load is created). Further, a hot-spot effect occurs as a result of titration with a strong acid, which appears from a comparison of examples I and II, where a difference between titration with 12N and 2N H 2 S0 , respectively, is seen, and from a comparison of graph 1 with graph 2, based on examples without titration. The effect of the substrate concentrations appears from graph 3, which shows that the enzyme stability declines at a high substrate concentration. If high substrate concentrations are applied (as in example X), which is desirable in practice, it appears that the decline of the enzyme activity can be minimized by carrying out the enzyme reaction without any inorganic salts. From graph 3 it appears that the decline of the enzyme activity is by far the least if there are not any inorganic salts left, so if the free form of the acylation agent is used and without titration. The decline of the enzyme stability is very small then and the enzyme can be recycled many times (» 50 times), also at high substrate concentrations, as appears from example X. Example XI Preparation of D-phenyl glycine amide from D-phenyl glycine amide.JH 2 S0 4 600 g of D-PGA.J$H 2 S0 4 was suspended in 750 ml of water at 25°C. With stirring, 260 ml of 25 mass% NH 4 OH was added, the temperature being kept at ≤ 35°C by cooling. The suspension was cooled to 5°C. The crystals that formed were filtered off, re-washed with 4x200 ml of water of 5°C and dried to constant weight. Yield: 421 g (93%). Characterization of the D-phenyl glvcine amide obtained: D-PGA was analyzed by means of HPLC: content of D-PGA: 100 ± 0.5 mass% content of D-PG: < 0.01 mass% e.e. D (enantiomeric excess of the D-enantiomer) = 99.7% In addition, the content was determined by means of titration: content of D-PGA: 99.5 mass% Content of S0 4 2 " = 0.14 mass% Melting point = 134-135°C The specific rotation was determined in methanol and water: α D 25 = -115.8° (c = 1, methanol) *o J 25 - = -97.8° (c = 1, water) X H NMR (D 2 0): δ 7.40 (s, 5H) , 4.52 (s, 1H) 13 C NMR (D 2 0): δ 179.1, 140.4, 129.6, 128.8, 127.3, 58.9

Claims: C L A I M S 1. Process for the preparation of a β-lactam antibiotic by enzymatic acylation of a β-lactam core by means of an acylation agent with the molar ratio between the β-lactam core and the acylation agent being between 0.5:1 and 2:1, the concentration of inorganic salts in the reaction mixture being lower than 1000:n mM, n representing the valency of the anion, and the sum of the concentrations of the β-lactam antibiotic and the β-lactam core being between 200 and 800 mM. 2. Process according to claim 1, wherein a free amino acid amide or a free amino acid ester is used as acylation agent. 3. Process according to claim 1 or 2, characterized in that the molar ratio between the β-lactam core and the acylation agent is between 0.7:1 and 1.3:1. 4. Process according to any one of claims 1-3, with the temperature being between 5 and 35°C. 5. Process according to claim 4, with the temperature being between 15 and 28°C. 6. Process according to any one of claims 1-5, with avoidance of direct addition of an acid to the reaction mixture. 7. Process according to any one of claims 1-6, with the total concentration of β-lactam antibiotic and β- lactam core being between 300 and 600 mM. 8. Process according to any one of claims 1-7, with application of an immobilized enzyme. 9. Process according to any one of claims 1-8, wherein after the acylation reaction the enzyme is separated out of the reaction mixture and subsequently re-used in a following acylation reaction. 10. Process according to claim 9, wherein the enzyme is re-used more than 50 times in an acylation reaction. 11. Process according to any one of claims 1-10, wherein a free amino acid amide is used as acylation agent. 12. Process according to claim 11, wherein the acylation agent used is solid D-phenyl glycine amide, prepared by using a base to neutralize D-phenyl glycine amide in the form of a salt of a strong acid with a concentration between 0.5 and 4 M, and recovering, after cooling to a temperature between -5 and 25°C» the free D-phenyl glycine amide in solid form.

Inventors: BOESTEN WILHELMUS HUBERTUS JOS, SMEETS JOHANNA CHRISTINA MARIA, VAN DOOREN THEODORUS JOHANNES, BOESTEN, WILHELMUS, HUBERTUS, JOSEPH, SMEETS, JOHANNA, CHRISTINA, MARIA, VAN DOOREN, THEODORUS, JOHANNES, GODFRIED, MARIA
Applicants: BOESTEN WILHELMUS H J, CHEMFERM VOF, DOOREN THEODORUS JOHANNES GODF, SMEETS JOHANNA CHRISTINA MARIA, BOESTEN, WILHELMUS, HUBERTUS, JOSEPH, CHEMFERM V.O.F., SMEETS, JOHANNA, CHRISTINA, MARIA, VAN DOOREN, THEODORUS, JOHANNES, GODFRIED, MARIA
Application year: 1996
Main classification: C12P35
IPC codes: C07D501, C12P35