TECHNICAL FIELD

This invention relates to a an improved process for preparation of β-Lactam derivatives by enzymatic acviation of ^• che parent amino jS-lactam with an acylating agent.

BACKGROUND ART

Today, semisynthetic β-lactam derivatives such as a-picillin, amoxicillin, cefaclor, cephalexin, cephadrcxil and cephaloglycin are, in an industrial scale, prepared by che-ical methods, for example by reacting an amino /J-lactam such as 6-aminopenicillanic acid, usually having its carboxyl group protected, with an activated side chain derivative, followed by the removal of the protecting group by hydrolys- is. For example, ampicillin (6-D-α—aminophenylscetamido- peni illanic acid) can be prepared by reacting 6-A?.-., having a suitable protected carboxyl group, with D-pher.ylgiycine acid chloride, followed by removal of the protecting group by hydrolysis. These reactions typically involve cosrly steps such as sub zero degree Celcius conditions and organic solvents like ethylene chloride and silylation reagents.

Within the last years, there has been an increasing amount of publications concerning the possibility cf enzym¬ atic preparation of penicillins and cephalosporins by react- ion cf an acylating agent, be it in acid form or in activated forr. (for example, amide or ester) and the parent amir.o β- lactam (for example, 6-APA or 7-ADCA) . For example, enzym¬ atic production of ampicillin from 6-APA and a D-phenyl- gly ine derivative (such as a lower alkyl ester) is known

froπ West German patent application having publication No. 2,163,792, Austrian Patent No. 243,986, Dutch patent application No. 70-09138, West German patent application having publication No. 2,621,618, European patent application having publication No. 339,751 and PCT patent application having publication No. 92/01061.

The enzymatic acylation of an amino /3-lactam with an acylating agent may be performed in the presence of a suitable amidase or acylase whereby the desired /3-lactam derivative is formed. The parent amino /3-lactam and the β- lacta derivative have the same 3-lactam nucleus. In the β- lactam derivative endproduct the 6-NH2 side chain of the peneπ parent /3-lactam or the -NH ₂ side chain of the cephem parent /3-lactam are acylated.

This reaction can be illustrated by the following reaction scheme I:

Reaction scheme I

Acylating agent + amino 3-lactam - /3-lactam derivative

If this process is performed batchwise, it has been discovered that at the beginning of the reaction, an increasing amount of /3-lactam derivative is formed whereas, after a certain period of time, the amount of 5-lactam derivative present in the reaction mixture is decreasing. The decomposition of the _/ 3-lactam derivative formed may be due to hydrolysis thereof whereby amino /3-lactam and the acid form of the acylating agent is formed. In addition, the enzyme present may decompose the starting acylating agent. Consequently, at least two sorts of decomposition nay take place during such a reaction.

One object of this invention is to furnish a process whereby the molar ratio between the amount of /3-lactam derivative

which can be recovered from the reaction mixture and the acid form' of the acylating agent formed during the reaction is increased, compared with this ratio for the known processes.

BRIEF STATEMENT OF THIS INVENTION

It has now, surprisingly, been found that improved process conditions are obtained if the enzymatic acylation of the amino /3-lactam is performed at constantly high concentrations of both the parent amino /3-lactam and the corresponding acylating agent. Preferably the concentrations of the parent amino /3-lactam and of the corresponding acylating agent are higher than 70%, preferably higher than 85% of the lowest value of both the saturated concentration of the parent amino /3-lactam and of the saturated concentration of the corresponding acylating agent.

The advantages of this invention are, inter alia, as follows:

1) An improved molar ratio between the amount of β- lactam derivative which can be recovered from the reaction mixture and the amount of the acid form of the acylating agent, compared with this ratio for the known processes, is obtained. This improvement has direct impact on the economy of the process due to the reduced consumption of acylating agent versus /3-lactam derivative which can be recovered from the reaction mixture. This improvement also has an indirect impact on the economy of the process due to the lower amount of the acid form of the acylating agent which is to be removed from the desired /3-lactam derivative.

2) An increased yield of the 3-lactam derivative is obtained.

3) An increase in the productivity of the /3-lactam derivative is obtained. This productivity is defined as the

amount of _/ 3-lactam derivative which can be recovered per time unit per volume unit of the reaction mixture.

DETAILED DESCRIPTION OF THIS INVENTION

Herein, the terms (parent) amino /3-lactam and acylating agent have been used for the two starting materials. The term β- lactam derivative has been used for the desired end product.

As -entioned above, two undesired decomposition reactions take place during the process in question and these reactions have been illustrated in the following reaction scheme II which also shows the parent reaction from reaction scheme I:

Reaction scheme II:

^v l

Acylating agent + amino /3-lactam - /3-lactam derivative

I v ₂ I v ₃

Acid form of the Acid form of the acylating agent acylating agent

+ amino /3-lactam

In reaction scheme II, v- _j _ is the reaction rate for the reaction: acylating agent + amino /3-lactam - /3-lactam derivative, v ₂ is the reaction rate for the reaction: acylating agent - acid form of the acylating agent, and v ₃ is the reaction rate for the reaction: /3-lactam derivative → acid form of the acylating agent + amino /3-lactam.

One way of performing the process at constantly high concentrations of reactants according to this invention is to maintain the rate of formation of the desired 3-lactam

derivative in the reaction mixture (v^ within a certain range for a certain period of time. Preferably, the rate of formation of the desired /3-lactam derivative does, during a period of not less than 5 hours, preferably not less than 10 hours, not deviate more than ± 50 %, more preferred not more than ± 20 %, most preferred not more than ± 10 %, from the average rate of formation of the desired /3-lactam derivative during the same period of time.

To take an example, if the average rate cf format- ion cf amoxicillin during a period of 20 hours is 2.5 μmoles/ minute/ml reaction mixture, then the rate of formation of amoxicillin during this period of time should preferably be within the range from 1.3 to 3.9 μmoles/minute/ l.

Another way of performing the process at constantly high concentrations of reactants according to this invention is to maintain the net rate of formation of the desired 3-lactam derivative in the reaction mixture (v± minus v ₃ ) within a certain range for a certain period of time. Preferably, the net rate of formation of the desired /3-lactam derivative does, during a period of not less than 5 hours, preferably not less than 10 hours, not deviate more than ± 50 %, more preferred not more than z 20 %, most preferred not -ore than z 10 %, from the average net rate of formation of the desired /3-lactam derivative during the same period of time.

A still further way of performing the process at constantly high concentrations of reactants according to this invention is to maintain the ratio between, on one hand, the net rate of formation of the desired /3-lactam derivative in the reaction mixture (v- _j _ - v ₃ ) and, on the other hand, the total rate of formation of the acid form of the acylating agent in the reaction mixture (v ₂ + v ₃ ) within a certain range for a certain period of time. Hence, this ratio is (v ₁ - v ₃ )/(v ₂ + v ₃ ) . Preferably, the ratio between the net rate of formation of the desired /3-lactam derivative and the rate of formation of the acid form of the acylating agent does, during a period

of not less than 5 hours, preferably not less than 10 hours, not deviate more than ± 50 %, more preferred not more than ± 20 %, most preferred not more than ± 10 %, from the average ratio between the net rate of formation of the desired β- lacta derivative and the rate of formation of the acid form of the acylating agent during the same period of time.

The 3-lactam derivative formed may precipitate during the reaction and, also, the acid form of the acylating agent such as D-phenylglycine and D-p-hydroxyphenylglycine may precipitate. Hence, in some cases the reaction mixture will be a slurry throughout the reaction.

During the process of this invention, it is preferred that the total content of the desired 3-lactam derivative, both in dissolved and optionally in precipitated form, in the reaction mixture is currently kept below a certain concentration. One object of doing so is to try tc prevent that the viscosity of the reaction mixture becomes too high. Consequently, during the performance of the process at constantly high concentrations of reactants cf this invention, it is preferred that the total content of the desired 3-lactam derivative, both in dissolved and optionally in precipitated form, does not exceed 350 mM/liter reaction mixture and preferably does not exceed 250 mM/liter reaction mixture.

The process according to this invention may be performed in a reactor which is equipped with a device for stirring or mixing and with means for maintaining the reaction temperature within a certain range. Also, it is recommended that this reactor be connected with an equipment maintaining the pH value within a certain range.

During the continuous performance of the process, the amino /3-lactam and the acylating agent may ze added continuously or semicontinuously so as to maintain the desired concentrations thereof in the reaction mixture. The

resulting 3-lactam derivative and the acid form of the acylating agent may be removed continuously or semicontinuously from the reactor, for example, by removal, from the reactor, of a part of the reaction mixture from which the resulting /3-lactam derivative and, possibly, the acid form of the acylating agent is removed, for example, by centrifugation or filtration. The mother liquor from this centrifugation or filtration may be returned to the reactor or may be used to dissolve the amino 3-lactam or the acylating agent.

Herein, the semicontinuous performance of an action means that the action in question is performed several times within a certain period of time, for example, during an hour or during a day and night.

The (parent) amino 0-lactam has a free amino group which is acylated by the reaction according to this invention. The amino /3-lactam may conveniently be β-APA, 7-ADCA, 7-ACA or 7- ACCC.

The amino 3-lactam, for example 6-APA or 7-ADCA, used in the process of this invention may be obtained by enzymatic hydrolysis of the fermented penicillins or cephalosporins, (for example penicillin V, penicillin G or cephalosporin C) or their ring enlarged analogues (for example V-DCA and G-DCA) or derivatives thereof followed by removal of the hydrolysis by-product, if desired (phenoxyacetic acid etc.). Advantageously, the crude solution can be used directly without further purification or dilution.

The acylating agent may be in an activated form. Preferably, the acylating agent is an amide or an ester. The acylating agent may be a derivative of D-phenylglycine, D-JD- hydroxyphenylglycine, D-2,5-dihydrophenylglycine or mandelic acid, such as a lower alkyl ester (methyl, ethyl, n-propyl or isopropyl ester) or an amide which is unsubstituted or substituted in the -CONH ₂ group. The acylating agent may be

used in the form of a salt, for example, the hydrochloride salt or the H ₂ S0 ₄ salt. The acylating agent may be added in an active form or the active form may be formed in situ.

The solubility of the acylating agent such as the D-phenylglycine or D-p_-hydroxyphenylglycine derivative will vary with the identity of the derivative and with the composition of the reaction medium. In an aqueous system as used in the examples, the solubility of the hydrochloride salt of D-phenylglycine amide is typically approximately 450 mM. However, the solubility is very dependent on the salt components in the solution, as well as on the pH value and the temperature of the solution. As a further example, the solubility of the sulphate form of D-phenylglycin amide is about 3.3 M within a pH range from 2.5 to 6.5.

As appears from the above explanation, the acylating agent and the /3-lactam derivative may be decomposed forming, inter alia, the acid form of the acylating agent. The acid form of the acylating agent is the parent carboxylic acid corresponding to the acylating agent in question.

Exar.ples of _/ 3-lactam derivatives that may be prepared by the process of this invention are ampicillin, amoxicillin, cefaclor, cephalexin, cephadroxil, cephradine, epicillin and cefamandol.

The enzyme to be used in the process of this invention may be any enzyme catalyzing the reaction in question. Such enzymes have been known since around 1961. Enzymes to be used are, for example, termed penicillin amidase or penicillin acylase and classified as E.C. 3.5.1.11. A number of nicrobial enzymes are known to have this activity, derived from for example Acetobacter, Alcaliσenes. Xanthomonas. Mvcoclana. Protaminobacter, Aeromonas (West German patent application having publication No. 2,163,792) Pseudomonas (Austrian Patent No. 243986) , Flavobacterium (Dutch patent application No. 70-09138) , Aphanocladium, Cephalosporiu (West German

patent application having publication No. 2,621,618), Aceto- bacter pasteurianu , Alcaliαenes faecalis. Bacillus meσaterium. Xanthomonas citrii (European patent application having publication No. 339,751), Kluwera citrophila fAσr.3iol.Chem. 37 (1973), 2797 - 2804) and Escherichia coli (West German patent application having publication No. 2,930,794). The Escherichia coli enzyme is commercially available. The enzyme also may be a so-called anpicillin hydrclase, acylase or amidase. In this connection, reference is, inter alia, made to Hakko to Koσvo 38 (1980) , 216 et seσ. , the content of which is incorporated by reference.

It is preferred to use the enzyme in a rauseable form, for example, in entrapped or immobilized form. Immobilization may be done by any known method. Immobilized Escherichia coli enzyme is commercially available from Boehringer Mannheim GmbH, Germany, under the trade name Enzycel ^® , and Recordati under the trade name Super Enzyme ^® . Preferably, as enzyme Penicillin G acylase, immobilized on a carrier consisting of a gelling polycarbohydrate and a polymer containing free amino groups, is applied.

The process of this invention is generally carried cut in a system containing water. If desired, an organic solvent may be added.

Generally, the reaction temperature of the process of this invention may vary between 0 and 35"C, especially between 10 and 20°C. Temperatures in the range from 20 to 30°C may be preferred for convenient operation.

The suitable pH value depends, inter alia, on the type of the enzyme used. Using Escherichia coli enzyme, the pH value is typically in the range from 5 to 8, preferably in the range from 6.1 to 7. For the preparation of amoxicillin, a pH value in the range from 5.5 to 6.4 is preferred. Control of the pH value may be used.

Suitable reaction times are above 5 hours. The reaction time may be days or weeks.

Suitable enzyme concentrations may be from 1 to 100 U/ml (1 U = one unit of enzyme activity, see below) .

Before the process is started, the reactor may be filled with the components in the desired amounts. Using the process according to this invention, an extraordinary high molar ratio between the amount of 3-lactam derivative which can be recovered and the total amount of acid form of the acylating agent can be obtained. These high ratios are obtained using the teachings of this invention and properly selecting the concentration of the acylating agent, the ratio between the concentration of acylating agent and the starting amino 3-lactam, the pH value and the enzyme. Thus, a ratio of 2.4 was obtained in Example 1 beiow using the process according to the present invention. In a compara- tive batch process, vide Example 1 below, a molar ratio of only 1.4 was obtained. In addition, the yields of isolated product obtained in this example were 90 and 85 %, respectively.

Purification of the product can be achieved by methods known per se, for example by crystallisation.

Herein, the following abbreviations have been used: 7-ACA is 7-ar.inocephalosporanic acid, 7-ACCC is 7-amino-3-chloro-3- cephem-4-carboxylate, 7-ADCA is 7-aminodesacetoxycephalo- spcranic acid, 6-APA is 6-aminopenicillanic acid, D-HPG is D- p-hydroxyphenylglycine, D-PG is D-phenylglycine, D-FGA is D- pher.ylglycine amide, D-PGM is D-phenylglycine methyl ester, D-HFGM is D-p_-hydroxyphenylglycine methyl ester, G-DCA is 7- pher.ylacetamidodesacetoxycephalosporanic acid, D-HPGA is D-p_- hydroxyphenylglycine amide, and V-DCA is 7-phenoxyacetamido- desacetoxycephalosporanic acid.

Any novel feature or combination of features described herein is r.aterial to this invention.

This invention is further illustrated by the following examples which, however, are not to be construed as limiting.

DEFINITIONS and METHODS of ANALYSIS

Enzvme:

As enzyme Penicillin G acylase (26250 U, size 200- 500 urn) isolated according to WO application 012782 immobilized on a carrier consisting of a gelling poly- carbohydrate and a polymer containing free amino groups according to European patent 22642, is applied.

As definition of penicillin G acylase activity the following is used: one unit (U) corresponds to the amount of enzyme that hydrolyses per minute 1 μmole penicillin G under standard conditions (100 g.l" ^"1 Penicillin G potassium salt, 0.05 M potassium phosphate buffer, pH value 8.0, 28"C) .

HPLC analysis of ampicillin and cephalexin:

Column: RP LC-18, (250 x 4.6 mm; 5μm)

Eluent A: 25 mM phosphate buffer, pH value 6.5

Eluent B: acetonitrile

Gradient: Time, minutes eluent B, %

0 → 10 1 - 20

10 → 20 20

Flow: 1 ml/min. Detection: 215 nm.

Retention times in minutes: 4.1 (D-PG) ; 6.3 (7-ADCA) ; 8.1 (6- APA) ; 9.1 (D-PGA) ; 13.4 (Cephalexin) ; 13.9 (Ampicillin) ; 18 (D-PGM) .

HPLC analysis of amoxicillin:

Column: Chromspher C18, 5 μm (100 x 3.0 mm)

Solvent: 25 % acetonitrile in 12 mM phosphate buffer + 0.2% sodiumdodecylsulphate, pH value: 2.6. Flow: 1 ml/min. UV- detection at 214 nm.

Retention times in minutes: 1.9 (D-p_-hydroxyphenylglycine) ; 7.3 (D-HPGM) ; 3.1 (D-HPGA) ? 3.4 (6-APA) ; 4.8 (Amoxicillin) .

EXAMPLE 1 ENZYMATIC PREPARATION OF AMOXICILLIN FROM D-HPGM AND 6-APA

The equipment for this experiment consisted of (see Jig. 1) a thermostated reactor having a volume of 1.5 liters, equipped with a three-bladed impeller and a sieve with slots 180 μm (open area about 32%) . The reactor was connected to an autotitrator system using 4 M sulphuric acid as titrand. A valve was positioned at the outlet of the reactor. The outlet of the valve was connected via a pump to a basket centrifuge equipped with a polypropylene bag having a density of 1-5 μm. The outlet from the centrifuge was connected via a pump to a feed tank equipped with a stirrer and a glass sinter bottom. The outlet from the feed tank was connected via a pur.p to the reactor.

A mixture consisting D-HPGM (36.2 g, 200 mmol) and 6-APA (43.2 g, 200 mmol) in 800 ml water was added to the reactor with the bottom valve closed. The stirring was started. Immo- bilized Penicillin G acylase (26250 U, size 200-500 μm) made up to 200 ml was added to the reactor. The pH value was maintained at 6.1. The reaction temperature was about 20 °C. Under these conditions, the reaction mixture was almost saturated with D-HPGM and 6-APA. Then, the bottom valve was opened allowing the reaction mixture from the reactor to enter the centrifuge. Thereafter, the mother liquor from the centrifuge was pumped into the feed tank wherein D-HPGM

(21.7 g, 120 mmol) and 6-APA (20.0 g, 92.5 mmol) were loaded. The total volume of the suspension in the feed tank was kept at about 75 ml. A flow of about 100 ml/min was maintained in the system. The concentration of reaction components in the reactor, in the reactor outlet, in the centrifuge outlet, in the feed tank and in the feed tank outlet were monitored by analytical HPLC.

As the reaction proceeded, the amoxicillin and D-HPG formed started to precipitate out of solution. The crystals were separated from the immobilized enzyme particles by the bottom sieve in the reactor and the crystal suspension was led to the centrifuge where the crystals were separated from the mother liquor. When the mother liquor, which was now undersaturated with respect to D-HPGM and 6-APA, passed through the feed tank, some of the solid D-HPGA and 6-APA present dissolved such that the outlet from the feed tank contained saturated D-HPGM and 6-APA. When the total concentrations of D-HPGM and 6-APA came down to about 225 mM HPGM and 225 mM 6-APA, more solid substrate was added to the feed tank. At intervals the crystals in the centrifuge were washed with water, the washing liquid was added to the reactor. The amount of water used was sufficient to keep the volume in the reactor at its starting level. After washing the crystals in the centrifuge, the centrifuge was e-ptied.

After about 12 hours, the dosing of D-HPGM and β-APA was stopped. After 14 hours, the 6-APA concentration reached 20 mM and the reaction was stopped. The amounts of reaction components are given in Table 1, below.

Table 1. substrate crystal cake in solution added at the end at the end

D-HPGM 606.5 g 2.0 g 10.9 g (3351 mmol) (11 mmol) (60 naol)

6-APA 523.4 g 9.0 g 4.3 g (2423 mmol) (42 mmol) (20 mmol)

Amoxicillin. 0 909.6 g 8.4 g 3H ₂ 0 (2171 mmol) (20 mmol)

D-HPG 0 123.7 g 30.0 g (741 mmol) (180 mmol)

Based on the above results, the following yield can be calcu¬ lated: amoxicillin yield (isolated product, based on 6-APA added) : 90%.

Amoxicillin productivity: 156 mmol/hour/liter.

Hereinafter, the molar ratior between the amount of β-latam derivative which is formed and the amount of the acid form of the acylating agent formed has been designated R. In this example R = 2.4.

A comparative experiment was performed at batchwise conditions and the reaction was stopped at the moment where the optimum yield of amoxicillin was obtained (2 hours) . The reaction temperature was about 20 °C, the pH value was about 6.1 and 26250 units of the enzyme, mentioned in this example above, was used. The total volume of the reaction mixture was 1 liter. The reaction was performed in the reactor mentioned above. After 2 hours the bottom valve was opened, and the crystals were separated from the immobilized enzyme particles by the bottom sieve in the reactor. The crystal suspension

was filtered. The amounts of reaction components are given in Table 2, below.

Table 2 substrate crystal cake in solution added at the end at the end

D-HPGM 90.5 g 0.1 g 34.2 g (500 mmol) (0.6 mmol) (189 mmol)

6-APA 43.2 g 0.7 g 3.0 g (200 mmol) (3 mmol) (14 mmol)

Amoxicillin. 0 70.8 g 4.2 g 3H ₂ 0 (169 mmol) (10 mmol)

D-HPG 0 0.3 g 21.7 g (2 mmol) (130 mmol)

Amoxicillin yield (isolated product, based on 6-APA added) 85%.

Amoxicillin productivity: 90 mmol/hour/liter.

R = 1.4.

EXAMPLE 2 ENZYMATIC PREPARATION OF AMOXYCILLIN FROM D-HPGA AND 6-APA

The equipment for this experiment is described in example 1,

A mixture consisting of D-HPGA (20.0 g, 120 mmol) and 6-APA in 800 ml water, which was adjusted at a pH value of 6.0 by adding 4 M ammonium hydroxide, was added to the reactor with the bottom valve closed. The stirring was started. Immobilized Penicillin G acylase (26250 U, size 200-500 μm) made up to 200 ml was added to the reactor. The pH value was

maintained at 6.0. The reaction temperature was about 20 ^* C. Under these conditions the reaction mixture was almost saturated with D-HPGA and 6-APA. Then the bottom valve was opened allowing the reaction mixture from the reactor to enter the centrifuge. Thereafter, the mother liquor from the centrifuge was pumped into the feed tank wherein D-HFGA (20.0 g, 120 mmol) and 6-APA (20.0 g, 92.5 mmol) were loaded. The total volume of the suspension in the feed tank was kept at about 75 ml. A flow of about 100 ml/min was maintained in the system. The concentration of reaction components in the reactor, in the reactor outlet, in the centrifuge outlet, in the feed tank, and in the feed tank outlet were monitored by analytical HPLC.

The crystals formed in the reactor were separated from the immobilized enzyme particles by the bottom sieve in the reac¬ tor. The crystal suspension was led to the centrifuge where the crystals were separated from the mother liquor. The mother liquor was passed through the feed tank. When the total concentrations in the feed tank came down to about 150 mM HPGA and 225 mM 6-APA, more solid was added to the feed tank. At intervals the crystals in the centrifuge were washed with water, the washing liquid was added to the reactor. The amount of water used was sufficient to keep the volume in the reactor at its starting level. After washing the crystals in the centrifuge, the centrifuge was emptied.

After about 12 hours, the dosing of 6-APA to the feed tank was stopped while the D-HPGA dosing was maintained in order to keep D-HPGA at saturation. After 14 hours, the 6-APA concentration reached 20 mM and the reaction was stopped. The amounts of reaction components are given in Table 3, below.

Table 3 .

substrate crystal at in solution added the end at the end

D-HPGA 483.9 g 1.3 g 22.4 g (2915 mmol) (8 mmol) (135 mmol)

6-APA 443.0 g 8.0 g 4.3 g (2051 mmol) (37 mmol) (20 mmol)

Amoxicillin. 0 758.4 g 8.4 g 3H ₂ 0 (1810 mmol) (20 mmol)

D-HPG 0 103.2 g 29.7 g (618 mmol) (178 mmol)

Amoxicillin yield (isolated product, based on 6-APA added) 88%.

Amoxicillin productivity: 131 mmol/hour/liter.

R = 2.3