MXPA00003769A - PROCESS FOR THE PREPARATION OF A&bgr;-LACTAM ANTIBIOTIC - Google Patents

Abstract

Process for the preparation of a&bgr;-lactam antibiotic in which a&bgr;-lactam nucleus is subjected to an enzymatic acylation reaction with the aid of an acylation agent at a molar ratio of acylation agent/&bgr;-lactam nucleus of less than 2.5, with the acylation agent and/or the&bgr;-lactam nucleus being supersaturated in the reaction mixture during at least part of the acylation reaction. In the process, a concentrated slurry or solution, for instance, of the&bgr;-lactam nucleus and/or the acylation agent with a different pH or a higher temperature than the pH or temperature at which the acylation reaction is carried out is added to the reaction mixture during the acylation reaction. Both the&bgr;-lactam nucleus and the acylation agent may be supersaturated in the reaction mixture.

Description

PROCEDURE FOR THE PREPARATION OF AN ANTIBIOTICS OF ß- LACTAMA DESCRIPTIVE MEMORY The present invention relates to a process for the preparation of a β-lactam antibiotic, in which a β-lactam nucleus is subjected to an enzymatic acylation reaction with the aid of an acylating agent at a molar ratio of acylation / ß-lactam core, less than 2.5. A similar procedure is described, for example, in WO-A-96/23897. The yield of the β-lactam antibiotic that will be obtained in the enzymatic acylation reaction according to the prior art by amount of ß-lactam nucleus used and by amount of acylating agent used, is generally relatively low, since the β-lactam nuclei and / or β-lactam antibiotics are often relatively unstable, while the reaction time is relatively long due to the usually low solubility of the reactants. In addition, at the aforementioned relatively low ratio of acylating agent: β-lactam nucleus, only a relatively low yield of β-lactam antibiotic can be achieved by the amount of ß-lactam nucleus used. The invention provides a method in which in an enzymatic acylation reaction a shorter reaction time is achieved and a higher yield of β-lactam antibiotic is achieved by amount of ß-lactam nucleus and / or by amount of acylating agent used, the ratio of the amount of acylating agent being relatively low: amount of β-lactam core used. This is achieved according to the invention because the acylating agent and / or the β-lactam nucleus are supersaturated in the reaction mixture during at least part of the acylation reaction. The applicant has found that it is possible to achieve a high degree of supersaturation of the β-lactam nucleus and / or the acylating agent in the reaction mixture and that, especially surprisingly, the supersaturation can be kept stable for hours. This allows the concentration of the β-lactam nucleus and / or the dissolved acylating agent to be markedly increased, so that the reaction proceeds more rapidly with less degradation of the β-lactam antibiotic and / or reagents, and also with a greater yield of β-lactam antibiotic by amount of ß-lactam nucleus used and / or by amount of acylating agent used. This also results in a higher production capacity. In addition, it is known from the literature, for example from the document WO-A-92/01061, that a high yield of β-lactam antibiotic can be obtained by amount of β-lactam nucleus, applying a high molecular relationship between the acylating agent and the β-lactam nucleus. However, a disadvantage of applying a high molecular ratio between the acylating agent and the β-lactam nucleus is that large amounts of acylating agent are lost as a result of hydrolysis of the acylating agent (and possibly the β-antibiotic). lactam). Accordingly, a low synthesis / hydrolysis ratio (S / H) is obtained, the molar ratio between the synthesis product (β-lactam antibiotic) and the hydrolysis product. Furthermore, it has been found that the preparation of the β-lactam antibiotic is often hindered by a relatively large amount of hydrolyzed acylating agent relative to the β-lactam antibiotic which is present in the reaction mixture obtained after the acylation reaction enzymatic, as a result of which a smaller amount of ß-lactam antibiotic can be isolated. For the purposes of the present invention, the yield of β-lactam antibiotic per amount of reagent (β-lactam nucleus or acylating agent) that will be achieved in the acylation reaction means the (molar) amount of the β-antibiotic -lactam formed in the acylation reaction by quantity (molar) of the reagent used. For the purposes of the present invention, the solubility of a compound in a mixture means the dissolved concentration of the compound in the presence of all other components of the mixture, expressed in mmoles / liter or mass percent. Solubility is measured by dissolving the compound at constant temperature and pH and in the presence of all components of the mixture. In this way, the solubility can be calculated from the amount of the dissolved compound upon reaching equilibrium (saturated solution). For the purposes of the present invention, a compound is supersaturated in a mixture when the dissolved concentration of that compound in the mixture is greater than the solubility. The supersaturation factor means the ratio between the two solubilities mentioned above (supersaturated divided by saturated). The supersaturation factor that will be achieved and the time during which the supersaturation is maintained depend on a number of factors such as the nature and concentration of the compound, the nature and concentrations of the other components in the mixture, the pH and temperature. The supersaturation factor that will be obtained depends largely on the compound involved, and is preferably greater than 2, more particularly greater than 5. The concentration of the dissolved β-lactam nucleus is expressed as the amount of the β-lactam nucleus dissolved in moles per kilogram of the liquid reaction mixture; the total concentration of the dissolved and undissolved ß-lactam nucleus is expressed as the amount of the ß-lactam nucleus in moles per kilogram of the total reaction mixture; The total reaction mixture may contain, in addition to the solution, a plurality of solids, for example β-lactam nucleus, β-lactam antibiotic, acylating agent (hydrolyzate) and immobilized enzyme. Similar definitions are applicable for the acylating agent and the β-lactam antibiotic.

A mixture in which the ß-lactam nucleus or the acylating agent, respectively, is supersaturated, can be obtained by means of, for example, a change in pH. For this purpose, if necessary, a concentrated mixture can first be prepared as a suspension or solution by dissolving the β-lactam nucleus or the acylating agent, respectively, present in solid form with the aid, for example, of an increase in the pH or a decrease in pH, or a decrease in pH, respectively. It is preferred that the β-lactam nucleus and / or the acylating agent be dissolved in the mixture obtained. However, it is also possible that a portion of the β-lactam nucleus and / or the acylating agent is still present in solid form. Subsequently, this suspension or solution can be subjected to a decrease in pH or an increase in pH, or an increase in pH, respectively. In this way, a solution or suspension is obtained in which the ß-lactam nucleus or the acylating agent is supersaturated. Any solid β-lactam nucleus present can be dissolved, for example, by lowering the pH until a pH of less than 3, preferably less than 2, in particular less than 1; or by increasing the pH to a pH greater than 6, preferably greater than 7, in particular greater than 8. In practice, the final pH is preferably chosen so that the ß-lactam nucleus barely enters completely in solution, so to obtain a solution as concentrated as possible. In practice, the concentrated solution will usually have a concentration of the β-lactam core of at least 5% by weight. Usually, the final pH will be less than 10 and greater than O. Subsequently, a supersaturated solution of a solution can be obtained, whose pH may or may not have been decreased or increased, increasing or decreasing the pH to a value between, for example, 3.0 and 9.0, preferably between 4.0 and 8.5, in particular between 4.5 and 8.0. Any solid acylating agent present can be dissolved, for example, by lowering the pH until a value of less than 8.0, preferably less than 6.5, in particular less than 5.0; preferably, the final pH is chosen, so that the acylating agent is hardly completely dissolved, and in this way a solution as concentrated as possible is obtained. In practice, the concentrated solution will have a concentration of the acylating agent of at least 5% by weight. The final pH will usually be greater than 1. A supersaturated mixture with, for example, the acylating agent, can be obtained from a mixture, preferably a solution, whose pH could optionally have been reduced, increasing the pH to a higher value of , for example, 4.5, preferably greater than 5.5, in particular greater than 6.0. Another way to obtain a mixture in which the β-lactam nucleus and / or the acylation agent is supersaturated is, for example, by a temperature decrease, optionally after some solid β-lactam nucleus and / or agent of present solid acylation has been first dissolved at least partially, by an increase in temperature or a change in pH. The solution can be effected, for example, by an increase in temperature until, for example (virtually), all the solid matter is in solution, for example, up to a temperature higher than 15 ° C, preferably higher than 20 ° C. , in particular higher than 25 ° C. Subsequently, a supersaturated solution or suspension can be obtained from the mixture obtained by lowering the temperature to a temperature below 20 ° C, preferably below 15 ° C, in particular below 10 ° C. The supersaturated mixture is preferably prepared by changing the pH in view of the fact that a higher supersaturation factor can then be reached. In practice, a change in pH and a decrease in temperature will usually be applied simultaneously in the preparation of a supersaturated solution. In a suitable embodiment of the method according to the invention, a mixture is first prepared in which the ß-lactam nucleus and / or the acylating agent are supersaturated, after which the acylation reaction is initiated, for example, by adding enzyme (immobilized). The concentration of the reactants will decrease during the acylation reaction, so that the supersaturation will have passed around the term of the acylation reaction. In another suitable embodiment, the acylation reaction is initiated with a portion of the β-lactam core and / or the acylating agent, which may or may not be supersaturated, after which the supersaturation is maintained or carried out by adding to the reaction mixture, for example by titration, a concentrated mixture of the β-lactam nucleus and / or the acylating agent with a different pH or a higher temperature, than the pH or the temperature at which the acylation reaction is carried out. performed. In practice, the β-lactam nucleus and the acylating agent may be present in a supersaturated condition during the acylation reaction, for example, by dosing in the acylation reactor a suspension or concentrated solution of the β-lactam nucleus (with a high pH or optionally with a low pH), and at the same time a suspension or concentrated solution of the acylating agent (with a low pH). By doing so, if desired, the pH can be kept constant during the acylation reaction, for example, by titration. The process according to the invention can be used very adequately in the preparation of cefaclor. Cefaclor exhibits reduced stability at high pH (>; 6.5), while the solubility of the corresponding β-lactam nucleus (corresponding 7-amino-3-chloro-cef-3-em-4-carboxylic acid; 7-ACCA) is low at said pH values (approximately 6.0-6.5) ) to which the degradation of cefaclor is still relatively low. As a result, the yield of cefaclor was low at relatively high and relatively low pH, so that a technically and commercially attractive procedure was not possible. The yield of cefaclor by amount of 7-ACCA used and by amount of acylating agent used could be markedly improved by the use, for example, of naphthol as a complexing agent, however, the use of such toxic auxiliary materials in the preparation of antibiotics is disadvantageous because they need to be completely removed, which involves additional procedural steps, with a noticeably negative effect on the economics of the procedure. Surprisingly, it has been found that the process according to the invention allows an exceptionally high supersaturation factor (> 10) to be achieved, so that the acylation reaction can be carried out at a relatively low pH ( 6.0-6.5) while, however, the concentration of the β-lactam nucleus is sufficiently high. In this way, it is now possible to prepare cefaclor in high yields by enzymatic acylation without the use of auxiliary materials, so that a technically and commercially attractive process can be achieved. Another application of the process according to the invention is the preparation, for example, of ampicillin by acylation of 6-aminopenicillanic acid (6-APA) with the aid of D-phenylglycine amide (FGA). Since 6-APA and ampicillin degrade relatively rapidly at high concentration and at high pH, it is important to make the acylation reaction proceed as quickly as possible at as low a pH as possible. By increasing, for example, the pH of a mixture of 6-APA and FGA to a value between 7.0 and 8.0, and then decreasing it immediately to a pH between 6.0 and 6.5 and then initiating the enzymatic reaction immediately, it has been possible to achieve, in a short period, a high yield of ampicillin by amount of 6-APA used and by amount of acylating agent used, a relatively high concentration of 6-APA being present in the solution only for a short time, and the degradation of Ampicillin at a relatively low pH value. At the same time, the hydrolysis of FGA in D-phenyl glycine (FG) is restricted. The process according to the invention can also be applied with advantage in the enzymatic preparation of cephalexin by the acylation of 7-aminodesacetoxycephalosporanic acid (7-ADCA) with the aid of D-phenyl glycine amide (FGA). Usually, the acylation reaction is carried out at a relatively high pH value, for example between 7.5 and 8.5, and is assisted by (undesirable) hydrolysis of FGA in D-phenyl glycine (FG). The reaction can be accelerated, and the hydrolysis of the acylating agent can be limited, by applying the process according to the invention, for example, by first raising the pH of a mixture of FGA and 7-ADCA to, for example, a pH between 8.0 and 9.0, and then acidifying the mixture again to a pH between 6.5 and 8.5. Another example of the method according to the invention is its application in the enzymatic preparation of amoxicillin from 6-APA and Dp-hydroxyphenyl glycine methyl ester (FGHM), and in the preparation of cefadroxil from 7-ADCA and FGHM. The solubility of FGHM is relatively low, and decreases as the pH increases, while the solubility of 6-APA and 7-ADCA is also relatively low and decreases as the pH decreases. It has been found that by subjecting a mixture of, for example, 6-APA or 7-ADCA and FGHM to a pH decrease to a value at which the FGHM is virtually completely dissolved (for example, a pH between 5 and 6), and then at an increase in the pH to a value between, for example, 6 and 7.5, a supersaturation of the FGHM can be achieved by a factor of 3 to 5. In this way, the acylation reaction can be carried out at a slightly higher pH, with a higher concentration of the dissolved β-lactam nucleus, as long as the concentration of the acylating agent is also relatively high. Another embodiment is to dissolve 7-ADCA at a relatively high concentration with a base at a pH between, for example, 8 and 9, and then add a concentrated acid solution of FGHM, in which process the pH decreases. In this modality, the supersaturation of 7-ADCA and FGHM can be achieved at the same time. Enzymatic reactions can be started after all the reagents have been added. It is also possible to add (a portion of) the solution (or suspension) of FGHM or concentrated 7-ADCA during the reaction. Another example of the process according to the invention is its application in the enzymatic preparation of cefazolin from tetrazole-1-acetic acid and as a core of β-lactam 7-aminocephalosporanic acid (7-ACA) or 7-amino-3 acid - (5-methyl-1, 3,4-thiadizolo-2-yl-thiomethyl) -3-cef-em-4-carboxylic acid (7-ACA-MMTD). Since the acylating agent does not contain an a-amino group, a so-called thermodynamically controlled coupling reaction with the acid can be carried out. The optimum pH for said thermodynamically controlled coupling reaction is preferably between 4.0 and 6.5. At this pH, the solubility of the β-lactam nucleus is relatively low. It appears that the conversion in the coupling reaction can be markedly increased by adding a concentrated solution of the β-lactam core with a relatively high pH (e.g., between 7.0 and 9.0) to the enzyme reaction mixture, while the pH of the mixture of reaction is maintained at a value between 4.0 and 6.5, for example, by titration with an acid. The process according to the invention can be conveniently applied in the preparation of β-lactam antibiotics, for example cephalexin, ampicillin, cefaclor, amoxicillin, cephradine, cefadroxil, cefotaxime, cefazolin, cefprozil, loracarbef and cephaloglycine. Any ß-lactam nucleus can be used in principle, in particular a ß-lactam nucleus with the general formula (1): wherein R 0 represents H or an alkoxy group having from 1 to 3 carbon atoms; Y represents CH2, O, S or an oxidized form of sulfur; and Z represents: wherein Ri represents, for example, H, OH, halogen, an alkoxy group having from 1 to 5 carbon atoms, an alkyl group having from 1 to 5 carbon atoms, a cycloalkyl group having 4 to 8 carbon atoms, an aryl or heteroaryl group having from 6 to 10 carbon atoms, in which the groups may or may not be substituted with, for example, an alkyl, aryl, carboxy or an alkoxy group having 1 to 8 carbon atoms; and wherein the carboxylic acid group can be an ester group, if so desired. Suitable examples of β-lactam nuclei which can be used in the process according to the invention are derivatives of penicillin, for example 6-aminopenicillanic acid (6-APA) and cephalosporic acid derivatives for example, 7-aminocephalosporanic acid with or without a substituent at site 3, for example 7-aminocephalosporanic acid (7-ACA), 7-aminodesacetoxycephalosporanic acid (7-ADCA), 7-amino-3-chloro-cef-em-4-carboxylic acid (7-) ACCA), 7-amino-3- (1-propenyl) -cef-3-em-4-carboxylic acid (7-PACA), 7-amino-3- (5-methyl-1, 3,4-thiadiazole acid -2-yl-thiomethyl) cef-3-em-4-carboxylic acid (7-ACA-MMTD) and 7-amino-3-chloro-8-oxo-1-azabicyclo [4.2.0] oct-2-en -2-carboxylic acid. In the acylation (enzymatic) reaction, the acylating agent can be, for example, a phenyl glycine in activated form, preferably an amide (primary, secondary or tertiary), or salt thereof, or a lower alkyl ester (from 1 to 4 carbon atoms), for example, a methyl ester; Suitable phenyl glycines are, for example, substituted and unsubstituted phenyl glycines, in particular phenyl glycine, p-hydroxyphenyl glycine and dihydrophenyl glycine. In addition, a-substituted acetic acid derivatives and the corresponding amides and esters can be applied, for example phenyl acetic acid, phenoxy acetic acid, tetrazole-1-acetic acid, mandelic acid or thienyl acetic acid. In principle, any enzyme that is suitable as a catalyst in the coupling reaction can be used as the enzyme. Said enzymes include the enzymes referred to collectively as 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, 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, Proteus, in particular Proteus rettgari, Pseudomonas and Xanthomonas, in particular Xanthomonas citrii. Preferably, an immobilized enzyme is used, since in that case, the enzyme can be easily isolated and reused. Particularly suitable enzymes among the immobilized enzymes that are commercially available are, for example, the Escherichia coli enzyme from Boehringer Mannheim GmbH, which is commercially available under the name Enzygel®, immobilized penicillin-G acylase from Recordati and penicillin- G acylase immobilized from Pharma Biotechnology Hannover. In addition, enzymes in crystalline form (CLEC's ™) can also be used. The temperature at which the enzymatic acylation reaction is usually carried out is less than 40 ° C, preferably between -5 and 35 ° C. The pH at which the enzymatic acylation reaction is usually carried out is between 3.0 and 9.5, preferably between 4.0 and 9.0. The optimum pH for a kinetically controlled coupling reaction is relatively high, for example between 4.5 and 9.0, preferably between 5.5 and 8.5, in particular between 6.0 and 8.0. The optimum pH of a thermodynamically controlled coupling reaction is generally lower and is, for example, between 3.0 and 7.0, preferably between 4.0 and 6.5. The reaction is preferably stopped almost completely when the maximum conversion has virtually been achieved. A suitable mode for stopping 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 reduce the temperature of the reaction mixture, after achieving the maximum conversion. A combination of the two modalities is also possible. After the reaction has stopped at maximum conversion, the reaction mixture is usually present in the form of a suspension comprising a plurality of solids, for example the antibiotic, D-phenyl glycine and, possibly, the immobilized enzyme. Preferably, the immobilized enzyme is recovered when there is an interest in the economics of the process. This can be suitably achieved, for example, by filtering the reaction mixture in a sieve, while stirring, the direction of rotation of the agitator being chosen, so that the suspension is pumped upwards to the center of the agitator. Subsequently, valuable components such as the antibiotic and FG can be recovered by, for example, a change in pH. For the methods of the invention, a change in pH can occur by adding an acid. Suitable acids are, for example, mineral acids, in particular sulfuric acid, hydrochloric acid or nitric acid, and carboxylic acids, for example acetic acid, oxalic acid and citric acid. An increase in pH can occur, for example, by adding a base. Suitable bases are, for example, inorganic bases, in particular ammonium hydroxide, potassium hydroxide or sodium hydroxide, and organic bases, for example triethylamine and FGA. Preferably, ammonium hydroxide is used. The enzymatic acylation reaction and the measures mentioned, for example, the preparation of the supersaturated mixtures, can be carried out in the presence of 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 suitable organic solvents are alcohols having from 1 to 7 carbon atoms, for example a monoalcohol, in particular methanol or ethanol; a diol, in particular ethylene glycol, or a triol, in particular glycerol. The molar ratio of acylating agent: β-lactam nucleus, ie, the total amount of acylating agent added, divided by the total amount of the added β-lactam nucleus, expressed in moles, is less than 2.5. It is preferred that the molar ratio be between 0.5 and 2.0, in particular between 0.7 and 1.8. The enzymatic acylation reaction is preferably carried out as an intermittent process. If desired, the reaction can also be carried out continuously. The invention will be further elucidated by means of the following examples, without however being restricted thereto.

Abbreviations 7-ACCA: 7-amino-3-chloro-cef-3-em-4-carboxylic acid 7-ADCA: 7-aminodesacetoxycephalosporanic acid 6-APA: 6-amino-penicillanic acid AMPI: ampicillin CCI: cefaclor CEX: cephalexin FG: D-phenylglycine FGA: D-pheniglycinamide FGH: Dp-hydroxyphenylglycine FGHM: Dp-hydroxyphenylglycine methyl ester Assemblase ™ is a penicillin acylase from Escherichia coli immobilized from E. coli ATCC 1105 as described in WO-A-97/04086. Immobilization was performed as set forth in EP-A-222462, gelatin and chitosan being used as gelling agents and glutaraldehyde as the entanglement agent. The final activity of the penicillin acylase of Escherichia coli by the amount of enzyme added to the activated blood cells and amounted to 3 ASU / g of dry weight, 1 ASU (amoxicillin synthesis unit) being defined as the amount of enzyme capable of producing one gram of amoxicillin.3H20 from of 6-APA and FGHM per hour (at 20 ° C, 6.5% of 6-APA and 6.5% of FGHM).

COMPARATIVE EXAMPLE 1 Synthesis of cefaclor (7-ACCA not oversaturated) An enzyme reactor (1.5 I, diameter 11 cm), equipped with a sieve bottom with 175 μm mesh, of 100 g of wet Assemblase ™ net was filled. A preparation reactor (1.2 I) of 75.4 g of FGA was filled (0.500 mol), 4.0 g of sodium bisulfite, 700 g of water, 6.6 g of H2SO4 at 4N and 72.1 g of 7-ACCA (0.300 mol). This mixture was stirred at T = 10 ° C; the pH was 7.5. Subsequently, the mixture was transferred to the enzyme reactor with the aid of 118 ml of water (T = 10 ° C). The stirrer was connected in the enzyme reactor at t = 0. The temperature was maintained at 10 ° C. The pH was maintained at 7.5 by titration with H2SO4 at 4N. At t = 48 minutes, the enzyme reactor contained approximately: 215 mmoles of CCI (conversion = 72%) 70 mmoles of 7-ACCA 175 mmoles of FGA 95 mmoles of FG A t = 81 minutes, the enzyme reactor contained approximately: 212 mmoles of ICC (conversion = 71%) 61 mmoles of 7-ACCA 83 mmoles of FGA 182 mmoles of FG After that, the amount of CCI decreased. The concentrations (C) in mmoles / kg of FGA in solution (FGAS), total FGA (FGAt) are shown in figure 1. 7-ACCA in solution (ACCAS), total 7-ACCA (ACCAt) and total CCL (CCLt) during the reaction, as a function of time (t) in minutes (min.).

EXAMPLE I Synthesis of cefaclor (7-ACCA supersaturated) An enzyme reactor (1.5 I, diameter 11 cm) was filled, equipped with a sieve bottom with 175 μm mesh, of 300 g of net Assemblase ™ wet. A preparation reactor (1.2 I) of 86.6 g of 7-ACCA (0.360 mol), 67.8 g of FGA (0.450 mol), 4.0 g of sodium bisulfite and 402 g of water was filled. This mixture was stirred for 5 minutes at T = 10 ° C; the pH was 7.4. The pH was adjusted to 8.0, while stirring, with the aid of 16.8 g of concentrated ammonium hydroxide. Subsequently, the pH was decreased from 8.0 to 6.4 by metering 73.8 ml of H2SO4 at 4N in 20 minutes. Then, at t = 0, the mixture of the preparation reactor was transferred to the enzyme reactor with the aid of 140 ml of water (T = 10 ° C). The stirrer was connected in the enzyme reactor at t = 0; T = ° C. The pH was maintained at 6.4 by titration with H2SO4 at 4N. After 7 hours, 66.0 ml of acid had been added. The reactor now contained: 300 mmoles of ICC (conversion = 83%) 55 mmoles of 7-ACCA 100 mmoles of FGA 45 mmoles of FG The concentrations (C) in mmoles / kg of FGA in solution are shown in Figure 2 (FGAS) ), Total FGA (FGAt). 7-ACCA in solution (ACCAS), total 7-ACCA (ACCAt) and total CCL (CCLt) during the reaction, as a function of time (t) in minutes (min.).

EXAMPLE II Synthesis of ceclafor (7-ACCA supersaturated and 7 -ACC A being introduced metered during the enzymatic reaction An enzyme reactor (1.5 I, diameter 11 cm) was filled, equipped with a sieve bottom with 175 μm mesh, of 150 g of wet Assemblase ™ net. A preparation reactor (1.2 I) of 48.1 g of 7-ACCA (0.200 mol), 75.4 g of FGA (0.500 mol), 4.0 g of sodium bisulfite and 175 g of water was filled. This mixture was stirred for 5 minutes at T = 10 ° C; the pH was 7.43. The pH was adjusted to 9.0, while stirring, with the help of 15.5 g of concentrated ammonium hydroxide. Subsequently, the pH was lowered from 9.0 to 6.4 by metering 80.6 ml of H2SO4 at 6N in 45 minutes. Then, at t = 0, the mixture of the preparation reactor was transferred to the enzyme reactor with the aid of 40 g of water (T = 10 ° C). The stirrer was connected in the enzyme reactor at t = 0. The temperature was maintained at T = 10 ° C. 244 g were metered in (0.200 mol) of 7-ACCA solution at constant speed in 109 minutes. The solution had been prepared recently by suspending 48.1 g of 7-ACCA (0.200 mol) in 183 g of water at T = 3 ° C and raising the pH to 8.2 with the help of 13.2 g of concentrated NH3, procedure in which all the 7-ACCA was dissolved. From t = 0 onwards, the pH of the enzyme reactor was maintained at 6.4 by titration with H2SO4 at 6N. At t = 350 minutes, the enzyme reactor contained: 350 mmoles of CCI (conversion = 88%) 45 mmoles of 7-ACCA 80 mmoles of FGA 65 mmoles of FG A t = 400 minutes, the amount of ceclafor was maximum and the pH was decreased to 5.0 by adding H2SO4 to 6N. The enzyme reactor now contained: 370 mmoles of CCI (conversion = 92%) 25 mmoles of 7-ACCA 40 mmoles of FGA 85 mmoles of FG The concentrations (C) in mmoles / kg of FGA in solution are shown in figure 2 (FGAS), total FGA (FGAt). 7-ACCA in solution (ACCAS), total 7-ACCA (ACCAt) and total CCL (CCLt) during the reaction, as a function of time (t) in minutes (min.).

COMPARATIVE EXPERIMENT B Synthesis of ampicillin (6-APA not oversaturated) First, a solution of FGA.1 / 2-H2SO4 was prepared. 301.6 g of FGA (2.00 mol) were suspended in 650 of water at T = 5 ° C. 102.1 g of 96% H2SO (1.00 mol) were added dropwise while stirring, the temperature being maintained at T < 25 ° C by means of cooling. Then, the enzymatic condensation was carried out. An enzyme reactor (1.5 I, diameter 11 cm), equipped with sieve bottom with 175 μm mesh, was filled with 300 g of wet Assemblase ™ net. A preparation reactor (1.2 I) of 131.6 g of 6-APA (0.600 mol), 30.2 g of FGA (0.200 mol) and 400 ml of water (T = 10 ° C) was filled. This mixture was stirred for 15 minutes at T = 10 ° C and subsequently, at t = 0 it was transferred to the enzyme reactor with the aid of 100 ml of water (T = 10 ° C). The stirrer was connected in the enzyme reactor at t = 0. 423.7 g (0.800 mol) were added to the solution at a constant speed in 283 minutes, maintaining the temperature at 10 C. The pH was 6.3. From t = 328 minutes onwards, the pH was maintained at 6.3 by titration with H2SO4 at 6N. At t = 540 minutes, the amount of ampicillin was maximum and the pH decreased to 5.6 by adding H2SO4 to 6N. The enzyme reactor now contained: 575 mmoles of AMPI (= 96% relative to the amount of 6-APA used) 15 mmoles of 6-APA 50 mmoles of FGA 365 mmoles of FG The concentrations are shown in Figure 4 (C ) in mmoles / kg of FGA in solution (FGAS), total FGA (FGAt), APA session in solution (APAS), total 6-APA (APAt) and total Ampi (Ampit), which occur during the reaction, depending on the time (t) in minutes (min.).

EXAMPLE III Synthesis of ampicillin (supersaturated 6-APA) An enzyme reactor (1.5 I, diameter 11 cm), equipped with sieve bottom with 175 μm mesh, of 300 g of wet Assemblase ™ net was filled. A preparation reactor (1.2 I) of 550 ml of water was filled (T = 10 * C), 138.8 g (0.920 mol) of FGA and 131.6 g (0.600 mol) of 6-APA. After 5 minutes at 10 ° C, a clear solution with a pH value of 7.4 was obtained. Subsequently, the pH was adjusted to 6.5 with 96% H2SO (approximately 16 g) and after stirring for 5 minutes at 10 ° C the solution was transferred to the enzyme reactor at t = 0 with the aid of 100 ml of water (T = 10 ° C). At t = 0, the temperature was 10 ° C and the pH = 6.3. During the enzyme reaction, the pH was maintained at 6.3 by titration with H2SO4 at 6N. At t = 300 minutes, the amount of AMPI was maximal and the pH was decreased to 5.6 by adding H2SO4 to 6N. The enzyme reactor now contained: 575 mml of AMPI (= 96% relative to the amount of 6-APA used) 15 mmole of 6-APA 40 mmole of FGA 295 mmole of FG The concentrations are shown in Figure 4 ) in mmoles / kg of FGA in solution (FGAS), total FGA (FGAt), 6-APA in solution (APAS), total APA assignment 6-APA (APAt) and total Ampi (Ampit), which occur during the reaction, as a function of time (t) in minutes (min.).

COMPARATIVE EXAMPLE C Synthesis of cephalaccin (7-ADCA not oversaturated) An enzyme reactor (1.5 I, diameter 11 cm) was filled, equipped with sieve bottom with 175 μm mesh, of 75 g of wet Assemblase ™ net. A preparation reactor (1.2 I) of 668 g of water was filled (T = 4 ° C), 4.0 g of sodium bisulfite, 130.3 g of 7-ADCA (0.600 mol) and 75.4 g of FGA (0.500 mol). 7.8 g of concentrated ammonium hydroxide were added, whereupon the suspension was stirred for 15 minutes at T = 4 ° C. The pH was 7.8. Subsequently, at t = 0, the suspension was transferred to the enzyme reactor with the aid of 50 ml of water (T = 4 ° C). The agitator was connected to the enzyme reactor at t = 0. The temperature was maintained at T = 4 ° C at all times. All 7-ADCA had dissolved after approximately 75 minutes; after that, a clear solution was present, apart from the solid Assemblase ™. After 210 minutes, the pH had risen to 8.6. The reactor now contained: 393 mmoles of CEX (conversion = 66%; S / H = 6.1 200 mmoles of 7-ADCA 64 mmoles of FG 33 mmoles of FGA.They are shown in Figure 6 S / H (in mmoles of CEX / mmoles of FG) depending on the conversion (in%).

EXAMPLE IV Synthesis of sephalexin (supersaturated 7-ADCA).

An enzyme reactor (1.5 I, diameter 11 cm) was filled, equipped with sieve bottom with 175 μm mesh, of 75 g of wet Assemblase ™ net. A reactor for preparation (1.2 I) of 600 ml of water (T = 4 ° C), 4.0 g of sodium bisulfite, 130.3 g of 7-ADCA (0.600 mol) and 75.4 g of FGA (0.600 mol) was filled. The suspension was stirred for 15 minutes at T = 4 ° C. The pH of 7.6. The pH was adjusted to 8.6, with stirring, using 43.8 g of concentrated ammonium hydroxide. The stirring was carried out for 5 minutes T = 4 ° C. Subsequently, 21.2 g of concentrated H2SO4 was added. The clear solution was stirred for 15 minutes at T = 4 ° C. The pH was 7.4. Subsequently, T = 0, the suspension was transferred to the enzyme reactor with the aid of 50 ml of water (T = 4 ° C). The stirrer was connected to the enzyme reactor at t = 0. The temperature was maintained at T = 4 ° C at all times. At t = 90, the pH had risen to 8.05. 5.5 g of concentrated H2SO4 were added, causing the pH to decrease to 7.75. A clear solution was present, apart from the solid Assemblase ™, all the time in the enzyme reaction. After 340 minutes, the pH had felt at 8.5. the reactor now contained: 445 mmoles of CEX (conversion = 74%; S / H = 7.2 146 mmoles of 7-ADCA 62 mmoles of FG 80 mmoles of FGA.They are shown in figure 6 S / H (mmoles of CEX / mmoles of FG) depending on the conversion (in%).

EXAMPLE V Synthesis of cephalexin (supersaturated 7-ADCA).

An enzyme reactor (1.5 I, diameter 11 cm) was filled, equipped with sieve bottom with 175 μm mesh, of 75 g of wet Assemblase ™ net. A preparation reactor (1.2 I) of 500 ml of water (T = 4 ° C), 4.0 g of sodium bisulfite, 162.9 g of 7-ADCA (0.750 mol) and 113.1 g of FGA (0.750 mol) was filled. The suspension was stirred for 15 minutes at T = 4 ° C. The pH was 7.8. the suspension is stirred for 15 minutes T = 4 ° C. The pH of 7.6 The pH was adjusted to 8.7, with stirring, using 55.6 g of concentrated ammonium hydroxide. Stirring was carried out for 5 minutes at T = 4 ° C. Subsequently, 9.6 g of concentrated H2SO was added. The clear solution was stirred for 15 minutes at T = 4 ° C. The pH was 8.0. Subsequently, at t = 0, the suspension was transferred to the enzyme reactor with the aid of 50 ml of water (T = 4 ° C). The stirrer was connected to the enzyme reactor at t = 0. The temperature was maintained at T = 4 ° C at all times. After 60 minutes, the pH had risen to 8.3. The pH was now maintained at 8.3 by titration with concentrated H2SO. At t = 300 minutes, a total of 23.4 g of concentrated H2SO4 had been titrated. At this point, the titration was stopped; t = 450 minutes, the pH had risen to 8.7. A clear, solid Assemblase ™ solution was present, all the time in the enzyme reaction. At t = 450 minutes, the reactor now contained 545 mmoles of CEX (conversion = 73%, S / H = 8.5 195 mmoles of 7-ADCA 64 mmoles of FG 129 mmoles of FGA, shown in Figure 6 S / H ( mmoles of CEX / mmoles of FG) depending on the conversion (in%).

COMPARATIVE EXPERIMENT D Synthesis of cefradixil (7-ADCA NI FGHM OVERHEAD An enzyme reactor (1.5 I, diameter 11 cm), equipped with sieve bottom with 175 μm mesh, of 300 g of wet Assemblase ™ net was filled. A preparation reactor (1.21) of 63.7 ml of 7-ADCA (0.293 mol), 107.1 g of FGHM (0.590 mol), 4.0 g of sodium bisulfite and 440 g of water was filled. This mixture was stirred for 5 minutes at T = 10 C maintaining the pH at 7.0 with the aid 7.5 g of concentrated ammonium hydroxide. At t = 0, the suspension of the reactor for preparation was transferred to the enzyme reactor with the aid of 50 ml of water (T = 10 ° C). The stirrer was connected in the enzyme reactor at t = 0. The temperature T = 10 ° C was maintained; the pH remained constant at 7.0 (without titration).

After 380 minutes, the enzyme reactor contained: 68 mmoles of Cefadroxil (conversion = 23%; S / H = 0.67) 222 mmoles of 7-ADCA 420 mmoles of FGHM 101 mmoles of FGH The concentrations are shown in Figure 7 C) in mmoles / kg. of total cefadroxil (cefadroxilt), total FGH (FGHt), total FGHM (FGHMt), FGHM in solution (FGHMS), total 7-ADCA (7-ADCAt) and 7-ADCA in solution (7-ADCAs), which occur during the reaction, as a function of time (t) in minutes (min. ).

EXAMPLE VI Synthesis of cefadroxil (both FGHM and 7 -ADCA oversaturated) An enzyme reactor (1.5 I, diameter 11 cm), equipped with sieve bottom with 175 μm mesh, of 270 g of net Assemblase ™ was filled. A preparation reactor (1.2 I) of 97.7 ml of 7-ADCA was filled (0.448 mol), 4.0 g of sodium bisulfite and 250 g of water (T = 10 ° C). The suspension was stirred for 5 minutes at T = 10 ° C. Subsequently, the pH was adjusted to 8.1 with the aid of 40.3 g of NH 3, a procedure in which a clear solution was produced (T = 10 ° C). A second reactor for the preparation of 98.5 g of FGHM (0.538 mol), 60 g of water (T = 10 ° C) and 105.1 g of H2S04 solution at 6N was filled. This solution was adjusted to T = 10 ° C; pH 2.3. The 7-ADCA solution was transferred from the first preparation reactor to the enzyme reactor with the aid of 20 ml of water (T = 10 ° C). The stirrer was connected in the enzyme reactor. From t = 0, the FGHM solution of the second reactor of preparation to the reactor of enzymes at a constant speed in 120 minutes was metered in. The temperature was maintained at T = 10 ° C. After 30 minutes, the pH had decreased from 8.1 to 6.8. Then, the pH was maintained at 6.8 by titration with concentrated ammonium hydroxide. At t = 120 minutes, 8.3 g of concentrated ammonium hydroxide had been added. The degree was stopped; the pH continued to be 6.8. After 420 minutes, the enzyme reactor contained: 390 mmoles of Cefadroxil (conversion = 87%, S / H = 4.0) 50 mmoles of 7-ADCA 44 mmoles of FGHM 97 mmoles of FGH The concentrations are shown in Figure 8 C) in mmoles / kg. of total cefadroxil (cefadroxilt), total FGH (FGHt), total FGHM (FGHMt), FGHM in solution (FGHMS), total 7-ADCA (7-ADCAt) and 7-ADCA in solution (7-ADCAs), which occurs during the reaction, as a function of time (t) in minutes (min.).

Claims (16)

NOVELTY OF THE INVENTION CLAIMS

1. - Process for the preparation of a β-lactam antibiotic in which the β-lactam nucleus is subjected to an enzymatic acylation reaction with the aid of an acylating agent at a molar ratio of acylating agent / β-lactam nucleus of less than 2.5, characterized in that the acylating agent and / or the β-lactam nucleus is supersaturated in the reaction mixture for at least part of the acylation reaction.

2. Method according to claim 1, further characterized in that the acylating agent and / or the β-lactam nucleus is supersaturated in the reaction mixture at the beginning of the acylation reaction.

3. Method according to claim 1 or 2, further characterized in that a suspension or concentrated solution of the β-lactam nucleus and / or the acylating agent is added, with a different pH or a temperature higher than the pH or the temperature at which the acylation reaction is carried out, to the reaction mixture during the acylation reaction.

4. Method of compliance of any of claims 1-3, further characterized in that the ß-lactam nucleus is supersaturated in the reaction mixture.

5. - Process according to claim 4, further characterized in that the mixture in which the ß-lactam nucleus is dissolved is subjected to a decrease in pH or an increase in pH until a pH between 3.0 and 9.0 is reached.

6. Method according to claim 5, further characterized in that the decrease in pH or increase in pH is effected until a pH of between 4.0 and 8.5 is reached.

7. Process according to any of claims 1-6, further characterized in that the acylating agent is supersaturated in the reaction mixture.

8. Method according to claim 7, further characterized in that a mixture in which the acylating agent is dissolved at an increase in pH is subjected.

9. Method according to claim 8, further characterized in that the pH is increased to a pH higher than 5.5.

10. Method according to claim 9, further characterized in that the pH is increased to a pH higher than 6.

11. Method according to any of claims 4-10, further characterized in that a mixture containing dissolved ß-lactam nucleus and / or acylating agent at a temperature decrease.

12. Method according to claim 11, further characterized in that the temperature is lowered at a temperature below 15 ° C.

13. Method according to claim 12, further characterized in that the temperature is lowered at a temperature below 10 ° C.

14. Process according to any of claims 7-13, further characterized in that the methyl ester of p-hydroxyphenylglycine is used as the acylating agent.

15. Process according to any of claims 1-13, further characterized in that an amide is used as an acylating agent.

16. Process according to any of claims 1-15, further characterized in that 7-aminodesacetoxycephalosporanic acid (7-ADCA), 7-amino-3-chloro-cef-3-em-4-carboxylic acid (7) is used. -ACCA), 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3- (1-propenyl) -cef-3-em-4-carboxylic acid (7-PACA ), 7-amino-3- (5-methyl-1, 3,4-thiadiazol-2-yl-thiomethyl) -cef-3-em-4-carboxylic acid (7-ACA-MMTD) or 7-amino acid -3-chloro-8-oxo-1-azabicyclo [4.2.0] oct-2-en-2-carboxylic acid, as the ß-lactam nucleus.