ITMI960033A1 - PROCEDURE FOR THE ENZYMATIC SYNTHESIS OF B-LACTAM ANTIBIOTICS IN THE PRESENCE OF AN ENZYME INHIBITOR - Google Patents

Abstract

A process for the production of penicillins and cephalosporins by reaction between a lactam nucleus and an amide. The reaction is catalyzed by a penicillin acylase enzyme and occurs in the presence of enzyme inhibitors.

Description

Description of an industrial invention in the name:

The present invention relates to an improved enzymatic process for obtaining penicillins and cephalosporins of formula (I) and (II)

where X is S or CH.2, R is an optionally substituted 6-membered hydrocarbon ring and R1 is a hydrogen atom, a halogen atom, a methyl group, a methoxyl group, a C1-C4 alkenyl group or a methylene bonded to an organic radical by means of an oxygen, sulfur or nitrogen atom.

US-A-3816253 describes a process for obtaining penicillins or cephalosporins by reacting an a-substituted α-amino acid with a compound deriving from 7-aminocephalosporanic or 7-aminodesacetoxycephalosporanic acid in the presence of an active microorganism or enzyme , in aqueous solution, at a temperature between 5 ° C and 50 ° C, preferably between 20 ° C and 40 ° C. It has been found that, by operating as described in the previously mentioned USP, the yield of the desired final product is strongly decreased due to concomitant parallel reactions with the formation of by-products which are difficult to separate from the reaction mixture. To eliminate or at least reduce the aforementioned drawbacks, EP-A-0473008 proposes a method for producing penicillins and cephalosporins of formula (I) or (II). According to said method, the 6-amino penicillanic acid or 7-amino cephalosporanic acid is reacted with an α-amino acid or substituted with the formula R-CH (NH2) -COOH or with a reactive derivative of the same, in the presence of an immobilized penicillin acylase, carrying out the reaction in an aqueous medium at a temperature between -5 ° C and 20eC, preferably at about 4 ° C. The reaction conditions are clearly provided in EP-A-0473008 which also reports, in detail, 25 embodiments with very good yields (90% and more) of the desired final product. In this regard, it should be noted that a process is industrially feasible only when the yields are sufficiently high (about 90%) and the final product is easily purifiable. ; EP-A-0473008 mentions in generic terms the α-substituted α-amino acids of the formula R-CH (NH2) ~ COOH or their reactive derivatives which can be used as starting substances. However, the only amino acid specifically exemplified is only the methyl ester of D-phenylglycine. The present applicants have carried out many and accurate experimental tests following the procedure described in the examples reported in EP-A-0473008: the results obtained, however, were all discouraging. ; It has in fact been verified that, if in the reaction medium the molar ratio of the aforementioned amino acid is less than 4 moles per 1 mol of 6-amino penicillanic or 7-amino cephalosporanic acid used, the yield of the desired final product is very low (lower about 60%) and, as such, it is not industrially acceptable. Good yields, industrially acceptable, are instead obtained only if the number of moles of the D-phenylglycine methyl ester in reaction is in a ratio between 4 and 6 with respect to 1 mol of 6 - amino-penicillanic or 7-aminocephalosporanic acid used. In this case, however, not only an unacceptable increase in costs occurs (the price of D-phenylglycine methyl ester is very high), but also by-products that are difficult or completely unavailable to eliminate from the desired final penicillins or cephalosporins of the reaction. Attempts have been made using different esters of D-pheniglycine or different amino acids: all these attempts have been unsuccessful. Equal negative results were obtained using numerous reactive amino acid derivatives. ; The German patent application DOS 2214444 reports an enzymatic synthesis of cephalosporins (in particular of cephalexin) by reaction of 7-ADCA with phenylglycine amide in the presence of the enzyme penicillin acylase: the yields obtained are very low. ; PCT / DK91 / 00188 (W092 / 01061) and PCT / DK92 / 00388 (WO93 / 12250) report methods for the preparation of β-lactam antibiotics by means of enzymatic acylation, according to which an amide of formula ;; ;; it is reacted with 6-amino-penicillanic or 7-amino-cephalosporanic acid in the presence of an immobilized penicillin acylase enzyme. ; These methods prove to be feasible: however, it has been found that an appreciable amount of amide (III) is hydrolyzed by the enzyme itself, thus increasing the number of impurities of the desired final products: even a fair amount of the final product is hydrolyzed by the same enzyme. ; Since the first applications of Penicillin Acylase (E.C. 3.5..1.11) it is known that this enzyme is inhibited by the presence of phenylacetic acid, phenoxyacetic acid and / or other inhibitors, in the presence of which the enzyme loses its activity and catalysis tends This is a problem when addressing the acylation of β-lactam nuclei which may contain traces of phenylacetic acid or phenoxyacetic acid or other inhibitors. ; This problem has been addressed by many by looking for enzymes that do not show this type of inhibition (ref. D.D.Y. Ryu et al., Biotechnology and Bioengineering 1985, Voi XXVII pp. 953-960. A.M. Blinkowsky et al., Enzyme Microbial Technology 1993, Vol 15 , pages 965-973): enzymes have been selected (often called Ampicillin Acylase or Amidase, Cephalosporin Acylase or Amidase, enzyme synthesizing Cephalessin etc.) coming from various microbial sources (Acetobacter spp., Xantomonas spp.). However, none of these enzymes are commercially available nor are they prepared on a large scale as is necessary for industrial uses; this approach is therefore disadvantaged by the fact that it requires considerable investments in research to develop a specific catalyst for the synthesis of β-lactam antibiotics. ; Another way to overcome the inhibition of the enzyme is to eliminate the aforementioned inhibitors before carrying out enzymatic acylation, for example by extraction with organic solvent as described in PCT / DK91 / 00188. The main disadvantage of this method consists precisely in the use of organic solvents; since the enzymatic acylations of the β-lactam nuclei have been introduced expressly to eliminate the use of said solvents and therefore obtain processes with high environmental compatibility, it follows that even this approach has not so far led to satisfactory results. ; The applicants have surprisingly discovered, while carrying out specific studies on the reactions catalyzed by Penicillin Acylase, that the aforementioned inhibition can be circumvented, thus obtaining the synthesis of βlactam antibiotics in the presence of even significant quantities of the inhibitors mentioned above. In some cases it is also possible to exploit the inhibition of the enzyme in terms useful for the synthesis, as described below, by introducing certain quantities of an inhibitor not initially present or present in small quantities. As a consequence, the present invention relates to an improved enzymatic process for producing penicillins and cephalosporins of formula (I) and (II) respectively as specified above in which a 6-amino-penicillanic or 7-amino cephalosporanic acid of formula (IV ) or (V) ;; ;; in which X and R1 have the meaning mentioned above, it is reacted at a temperature between -50C and 35'C, in the presence of a Penicillin Acylase enzyme in free or immobilized form, with an amide of formula (III) ;;; ;;; where R is as specified above, R2 and R3 are each independently a hydrogen atom or a linear branched alkyl group of 1 to 3 carbon atoms, their salts, in a molar ratio of 1 to 6 moles of amide (III) per mole of acid (IV) or (V), characterized in that; in the reaction mixture there is an enzymatic inhibitor of formula (VI): ;; where:; R4 is a linear or branched C1-C8 alkyl group, a substituted or unsubstituted aromatic ring, a halogen, a proton, Y can be absent or, if present, is 0, S CH2, phenyl or halogen; ; R5 is a linear or branched C1-C4 carboxyl or ester or a -CONH2 group; ; R6 is H, OH OCHO, CH3; ; this inhibitor being present in concentrations from 0.0001 to 0.5 molar. ; In particular, said inhibitor (VI) is present in a concentration ranging from 0.0005 to 0.2 molar and is chosen from phenylacetic acid, phenoxyacetic acid, mandelic acid or C1-C9 esters and starches ( linear or branched) of these acids. The invention also relates to pharmaceutical compositions comprising penicillins or cephalosporins prepared by means of the process described in the present invention, as well as to pharmaceutically acceptable diluents or carriers. The pharmaceutical compositions containing the penicillins or cephalosporins are commonly prepared according to conventional methods and are administered in suitable pharmaceutical form. With reference to formulas (I) and (II), R can be for example a phenyl, cyclohexadienyl, cyclohexenyl group not substituted or substituted with a hydroxyl, halogen, alkyl, alkoxy, carboxyl, nitro or amino group. ; Ri can be a hydrogen atom, a halogen atom, a methyl or inethylene group bonded to an organic group and in particular to an alkoxy, alkoxycarbonyl group or a five- or six-membered heterocyclic group containing from 1 to 4 heteroatoms selected from 0, S and N bonded to the inethylene group by means of an atom of 0, S, N and optionally substituted with one or more groups selected from a hydroxyl, halogen, alkyl, alkoxy, carbonyl, carboxy, cyano, amino and the like. The terms alkyl and alkoxy used herein refer to groups having from 1 to 6 carbon atoms, preferably from 1 to 4 carbon atoms. With reference to the α-amino acids whose starches have formula (III), D-phenylglycine, D-p-hydroxyphenylglycine and D-1,4-cyclohexadien-1-yl-glycine can be mentioned as examples. ; Suitable salts of the amide are those with an inorganic acid such as hydrochloric, hydrofluoric, hydrobromic, sulfuric or nitric acid, or with an organic acid, for example acetic, formic or maleic acid. ; The following acids are of particular interest among the compounds of formula (IV) and (V):; 6-amino-penicillanic (6-APA), 7-aminodesacetoxycephalosporanic (7-ADCA), 7-aminocephalosporanic, 7-amino-3-chloro-cephalosporan, 7-amino-3-methoxycephalosporan, 7-aminophenacetylhexacetoxycephalosporan, 7-aminophenosiacethyldhexacetoxycephalosporan, 7-amino-3-chloro-carbacefem, penicillin G and penicillin V. penicillin G or V and 7-aminophenacetyl-desacetoseicefalosporanic acid are commonly used as raw materials for the preparation of 6-APA and 7-ADCA respectively, in turn used to produce Ampicillin and Amoxicillin, Cefalexin and Cefadroxil: up to now these intermediates (6APA and 7-ADCA) had to be purified from phenylacetic or phenoxyacetic acid in order to use them in enzymatic acylation reactions, as described in PCT / DK91 / 00188. The possibility of carrying out the synthesis of these compounds even in the presence of inhibitors therefore entails the advantage of carrying out the synthesis without the need for purification of the intermediates, with a considerable saving of work and avoiding the use of organic solvents. The enzyme inhibitor can be present in the reaction mixture from the beginning of the synthesis or it can be added at any time during the course of the reaction; said inhibitor can also be added at the end of the enzymatic reaction to facilitate the isolation of the product and the subsequent treatments in general. ; The enzyme used as a catalyst in this process is a Penicillin Amidohydrolase classified E.C. 3.5.1.11 (otherwise also called penicillin acylase, penicillin hydrolase, ampicillin acylase etc.) coming from any microbiological source and in particular from Xantomonas, Pseudomonas, Arthrobacter, Kluyvera, Acetobacter, Escherichia, Bacillus, Acromonas strains. Particularly preferred are enzymes deriving from natural or engineered strains of Escherichia coli: some of these are commercially available in large quantities and also in immobilized form. ; The enzyme can be used in free form (i.e. soluble) or it can be immobilized on a solid matrix: among the latter the supports expressly dedicated to the immobilization of biomolecules such as synthetic epoxy or azlattonic resins are particularly preferred. ; EXAMPLE 1; Synthesis of Cephalexin by enzymatic acylation of 7-ADCA with D (-) phenylglycinamide; 9 g (42 mmol) of 7-aminodesacetoxycephalosporanic acid and 15.7 g (100 mmol) of D (-) phenylglycinamide are dissolved in water (final volume 300 ml). The solution was cooled to about 4 ° C and brought to pH = 6.8, then transferred to a refrigerated reactor connected to a control system (hereinafter called pH-stat) which maintains the pH constant by automatically adding sulfuric acid. 4 N.; To the above solution, 5400 IU of penicillin amidohydrolase enzyme were added in immobilized form (on azlatonic or epoxy resin), incubating at constant 4 * C and pH = 6.8.

After about 75 minutes the enzyme was separated from the solution by filtration; about 4 g of b-naphthol dissolved in dilute soda were added to the solution. 16.6 g of product were recovered in the form of the cephalexin b-naphthol complex, with a molar yield with respect to the starting nucleus of 74%. From the complex with b-naphthol the form of Cephalexin monohydrate with good quality characteristics was then obtained (with known procedures).

EXAMPLE 2

Synthesis of Cephalexin in the presence of phenylacetic acid

17 g (0.077 mmol) of crude 7-amino-deacetoxycephalosporanic acid containing 1.18% of phenylacetic acid and 1.63% of 7-aminophenacetyl-desacetoxycephalosporanic acid as impurities and 42.68 g (0.28 mmol) of D (-) -phenylglycinamide are dissolved in water obtaining a final volume of 600 ml.

The pH-stat is adjusted to pH = 7.6, the thermostat at 2 ° C, 7560 IU of immobilized enzyme are used and then the reaction is carried out as described in example 1. After 5 hours and 30 minutes the reaction is stopped filtering the penicillin Acylase and the product is precipitated with 6 g of b-naphthol obtaining a molar yield of 87.5%. Cephalexin monohydrate is then obtained as already described in Example 1.

EXAMPLE 3

Synthesis of cephalexin from 7-aminophenacetyl-desacetoxycephalosporanic acid and D (-) phenylglycinamide

16 g (0.048 moles) of 7-aminophenacetylhexacetoxycephalosporanic acid were dissolved in water by adding diluted NaOH as needed to reach pH = 8.0 then the solution was heated to 28 ° C and diluted with water as required to 320 ml and then transferred in a suitable reactor, connected to a pH-stat system and immersed in a thermostatic bath.

About 14000 IU of penicillin acylase enzyme in immobilized form were added to this solution (solution A) and therefore the pH (by adding NaOH 2 N) and temperature conditions were kept constant.

Separately, an aqueous solution of D (-) pheniglycinamide was prepared at a concentration of about 180 g / 1 (solution B).

After about two hours of reaction, about 190 ml of solution B were added to solution A, correcting the pH and temperature if necessary; the pH was kept constant by adding 4 N sulfuric acid.

After seven hours the enzyme was removed by vacuum filtration; Cephalexin was recovered from the resulting solution by precipitation of the Cephalexin bnaphtholate complex in a quantity corresponding to a molar yield of 80.8%. Cephalessin monohydrate having a satisfactory quality for marketing was obtained from the Cefalexin bnaphtolate complex by means of known procedures.

EXAMPLE 4

Use of inhibitors in the enzymatic acylation of 7-amino-3-chlorocephalosporanic acid

The reaction is carried out as in Example 1, but using 9.85 g (0.042 mmoles) of 7-amino-3-chlorocephalosporanic acid. At the end of the reaction, 1.5 g of phenylacetic acid dissolved in dilute NaOH are added, then the Cefaclor bnaftolato is precipitated as already described in Examples 1-3 with regard to Cephalexin.

The molar yield from 7-amino-3-chlorocephalosporanic acid was 70%. Cefaclor monohydrate is obtained by decomposition (with known techniques) of the Cefaclor b-naphtholate complex.

EXAMPLE 5

Enzymatic acylation of 6-aminopenicillanic acid in the presence of inhibitors

By means of known processes, 6-aminopenicillanic acid (hereinafter referred to as 6-APA) is obtained from Penicillin G by enzymatic hydrolysis, isolating it from the aqueous solution by precipitation with acids: the product thus obtained has phenylacetic acid impurities.

20.8 g of crude wet 6-APA having 80% titre (0.077 moles) are dissolved in water and mixed with D - (-) phenylglycinamide as described in Example 2, obtaining a solution containing about 0.7 g / 1 of phenylacetic acid. 6670 units of penicillin acylase are added and the reaction is carried out analogously to that described in Example 2: a conversion of 6-APA of 82.4% is obtained.

EXAMPLE 6

Hydrolytic inhibition of penicillin G acylase during the synthesis of cefalexin. in the presence of phenylacetic acid.

13 g (60.75 mmol) of 7-aminodesacetoxycephalosporanic acid (7-ADCA), 46.5 g (230.84 mmol) of D-phenylglycine methyl ester. HCl and 140 mg (1.03 mmol) of phenylacetic acid were dissolved in Ha0 for one total volume of 1000 ml.

The acylation was performed with 17.5 g (3000 I.U.) of penicillin G acylase under the following conditions: the initial pH was 7.25 at a temperature of 3 ° C. After approximately 3 hours, 93% of 7-ADCA was transformed into cefalexin.

After 93% conversion of 7-ADCA to cefalexin, the molar ratio of synthetic cefalexin production to hydrolytic formation of D-phenylglycine was 2.15 compared to 1.44 obtained without inhibitor.

Claims (5)

CLAIMS 1. A process for producing a penicillin or cephalosporin of formula (I) or (II) where X is S or CH2, R is an optionally substituted 6-membered hydrocarbon ring and R1 is a hydrogen atom, a halogen atom, a methyl group, a methoxyl group, a C1-C4 alkenyl group or a bonded methylene group to an organic radical by means of an oxygen, sulfur or nitrogen atom, wherein: a. 6-amino-penicillanic or 7-aminocephalosporanic acid of formula (IV) or (V) in which X and R1 are as defined above, it is reacted in an aqueous medium at a temperature between -5 ° C and 35 ° C in the presence of an immobilized penicillin acylase enzyme or in soluble form with an amide of formula (III) in which R is as defined above, R2 and Ra are each independently a hydrogen atom or a linear or branched alkyl group of 1 to 3 carbon atoms or their salts, in a molar ratio of 1 to 6 moles of said amide (III) per 1 mole of acid (IV) or (V), characterized in that an enzymatic inhibitor of formula (VI) is added to the reaction medium: where is it: R4 is selected from a group consisting of a linear or branched C1-C5 alkyl, a substituted or non-substituted aromatic ring, a halogen, a proton; Y can be absent or, if present, is O, S, CH2, phenyl or halogen; R5 is selected from the group consisting of a carboxyl, a linear or branched C1-C4 ester or a -CONH2 group; R6 is selected from the group consisting of H, OH, OCHO, CH3, this inhibitor being present in concentrations from 0.0001 to 0.5 molar. 2. A process according to claim 1, characterized in that the concentration of said inhibitor is preferably from 0.0005 to 0.2 molar. 3. A process according to claims 1 and 2, characterized in that said inhibitor (VI) is selected from the group consisting of phenylacetic acid, phenoxyacetic acid, mandelic acid or the esters and starches C1-C8 (linear or branched) of these acids. 4. A process according to claims 1 and 2 characterized in that said amide (III) is selected from the group consisting of D-pheniglycine amide or a salt thereof with an organic or inorganic acid. 5. Pharmaceutical composition containing a penicillin or cephalosporin obtained according to the processes of claims 1 to 4.