CS209853B2 - Method of making the semisynthetic cephalosporine - Google Patents

Abstract

1480850 Enzymatic hydrolysis on synthesis of cephalosporins SNAMPROGETTI SpA 16 Dec 1974 [20 Dec 1973] 54336/74 Heading C2C Hydrolysis of a cephalosporin produced by increasing the size of the thia ring of penicillin G and/or the synthesis of a semi-synthetic cephalosporin from a 7-aminocephalosporanic acid whose thia ring has been produced by increasing the size of the thia ring of penicillin G, is effected by contacting the said cephalosporin or 7-aminocephalosporanic acid in a phosphate buffer at a pH at which said hydrolysis or synthesis takes place with bacterial cells of a strain of Escherichia coli or with penicillin acylase extracted from said cells and purified, a said synthesis being carried out in the presence of a molar excess of an acylating agent. Preferably cephalosporin G is hydrolysed to yield 7 - aminodesacetoxycephalosporanic acid. Alternatively a synthesis comprising reacting 7 - aminodesacetoxycephalosporanic acid or (7 - ADCA) 7 - aminocephalosporanic acid (7 - ACA) with a compound of the formula in which X<SP>1</SP> is H or a group inert during said synthesis, X<SP>11</SP> is an alkoxy or -NH 2 group and X<SP>111</SP> is H or a -NH 2 group, may be effected in the presence of the cells or enzyme and phosphate buffer. For example, 7-ADCA may be reacted with D(-)phenyl glycine methyl ester HCl(PGM) to obtain cephalexin, 7-ACA may be reacted with PGM to obtain cephaloglycine, or 7-ACA may be reacted with phydroxyphenylglycine ethyl ester to form 7#[21 - amino - 2 - (4 - hydroxyphenyl)acetamido] - 3 - hydroxymethyl(acetate)ceph- 3-em-4-carboxylic acid.

Description

(54) A method for producing semi-synthetic cephalosporinia

The invention relates to a process for the preparation of a semisynthetic cephalosporin of the formula

CH 2 CO-NH

O

CfyOCOCH

COOH hydrolysis. of a derivative resulting from the chemical expansion of the penicillin G ring, followed by the use of the hydrolysis product as a starting compound for its synthetic conversion to the desired semisynthetic cef and lo-sporin.

The importance that semi-synthetic cephalosporin derivatives have gained in recent years is well known. They are widely used in pharmacology to replace antibiotics resistant to the effects of β-lactamase (cephalosporinase). In addition, they are sufficiently stable in an acidic environment, are well tolerated and have a broad spectrum of antibiotic activity.

The current known state of their production consists of the following chemical stages:

(a) penicillin G ring widening,

(b) hydrolysis of the product obtained;

c) isolation of the starting material (7-desoxyacetylcephalosporanic acid, 7-ADCA),

d) synthesis of new compounds from this starting material.

In the deoxyacetyl derivatives obtained by the above process, i.e. in the methyl radical at the 3-position, the methyl group can be oxidized to the corresponding hydroxyacetyl derivatives whose structure may be related to that of natural cephalosporin (cephalosporin C).

It has now been found that some steps of these processes, used in the preparation of semisynthetic cephalosporins, can be carried out with bacterial cells or cell extracts, bringing substantial improvements, saving the starting material, reaction under milder conditions and with good yields. The cells are cultured by a conventional fermentation procedure; cellular extracts can be readily obtained by mechanically damaging the cells, thereby releasing the desired enzyme, which is partially purified.

Enzymatic processes can replace the following chemical steps:

(a) hydrolysis of the product obtained by chemical ring expansion of the benzylpnicnicillin

b) chemical acylation of the obtained 7-amino acid resulting in the final product.

Thus, the present invention provides a process for the preparation of a semi-synthetic cephalosporin of formula

COOH ^{1 3} by hydrolysis of the corresponding derivative formed by chemical expansion of the penicillin G ring and subsequent use of the hydrolysis product - as a - starting compound for its synthetic conversion to the desired semisynthetic cephalosporin, the principle of which is: by the action of penicillin acylase from Escherichia co-li, whereupon the resulting compound of formula I

the reaction with a compound of formula II is reported.

^^ - CHCCOOH (ID

The method of the invention can be carried out by hydrolyzing a penicillin G derivative by contacting it with bacterial cells of Escherichia coli or with Penicillin acylase extracted and purified from Escherichia-coli cells.

In the process according to the invention, cephalosporin C is preferably used as a penicillin G derivative.

The hydrolysis - is preferably carried out at a pH in the range of - 6 - to 9 and at a temperature in the range of 0 to 50 ° C.

The process according to the invention is preferably carried out by reacting the compounds I and II at a pH in the range of 3 to 8, with a molar excess of the acylating agent.

The hydrolytic reaction with the cellular and enzymatic extracts takes place at pH 6.0 to 9.0, preferably at pH 8 to 8.5. The temperature -can vary -from 0 to 50 ^Q C, most preferably C. The synthetic reaction is carried out in the presence of a cellular or enzymatic extract, preferably at a pH of 3.0 to 8.0, preferably at a pH of 7.0.

Performing the synthesis requires - the presence of a molar excess of acylating agent to shift the equilibrium; - the molar ratio between the reagent and acylačmm cephalosporanic acid can be varied ^Me zi 1 to '5, preferably - 2.5. Since the enzyme catalyzes the hydrolysis of the ester or amide group of the acylating agent -and-amide bond of the product, it is desirable to interrupt the reaction rapidly when the maximum concentration has been reached. If cells were used, this can be done by removing them from the reaction medium by centrifugation if an enzymatic extract was used by lowering the pH to a value close to 2.0.

The following examples illustrate the process according to the invention without limiting it.

Example 1

Es < 96 &gt; and Ghia Coli.ATCC 9637 was fermented in submerged aerobic culture. The composition of the culture medium, expressed in g / l, was as follows: meat extract 10, peptone 10, N.aCl 5, phenylacetic acid 2, the pH was adjusted to 7 and the temperature maintained at 24 ° C. The fermentation was discontinued after 20 hours. 1 liter of this culture contained an average amount of E. Coli biomass - capable of hydrolyzing 40 millimoles of penicillin G in 1 hour at 37 ° C. pH 7.8 (40,000 units).

To the biomass obtained from 2 L of culture (80,000 units), separated by centrifugation and washed with a buffer (potassium phosphate) at pH 7.8, was added with stirring 0.5 L of a 6% w / v solution, 7- (2'-phenylacetamido) ^{-3-methylcellulose!} 4-carboxylic acid (cephalosporin G) (90 mmol) in 0.1 M phosphate buffer solution at pH 7.8 and 37 ° C, - the pH of the solution was maintained by the continuous addition of sodium hydroxide solution under control of a constant pH device. After 2.5 hours, when the stoichiometric amount of NaOH was consumed, the reaction mixture was centrifuged, the filtrate was cooled to -4 ° C, 209853 no 200 ml of butyl acetate was added and the pH of the solution was adjusted to pH 2.0 with 6N HCl. The pH of the separated aqueous phase was adjusted to 4.6 with 5N NaOH and diluted with an equal amount of ethyl alcohol.

The precipitate thus obtained was collected, washed with water / ethanol (50:50, v / v) and dried. 18.7 g (87 mmol) of 7-amino-3-methylceph-3-em-4-carboxylic acid 7-aminosacetoxycephalosporanic acid (7-ADCA) with a 95% content were obtained.

Example 2

Escherichia Coli was fermented as in Example 1. The biomass obtained from 3 L culture medium was suspended in Tris-HCl buffer 0.01M at pH 8.5 and homogenized by double recycling in an M-anton-Gaulm homogenizer at 700 kg / / cm ² . Hexadecylpyridinium chloride was then added to a concentration of 0.4% and the pH adjusted to 4.5. The temperature was maintained at 37 ° C. After three hours, the insoluble material was centrifuged. Penicillin acylase was recovered from the clear solution by precipitation with ammonium sulfate (65% saturation).

The resulting precipitate was suspended in 0.1 M phosphate buffer pH 7.8 and dialyzed against 20 volumes of the same buffer. 30 g (90 mmol) of cephalosporin G were added to the 500 ml of dialyzed product thus obtained and the pH of the reaction mixture was maintained at pH 7.8 by means of a pH stat. After 2.5 hours after complete hydrolysis of cephalosporin G, 18.1 g of 7-ADCA was isolated, as in Example 1, with a conversion yield of 93%.

Example 3 g (23.3 mmol) of 7-ADCA and 10 g (50 mmol) of D- (-) - phenylglycine methyl ester hydrochloride (PGM) were suspended in phosphate buffer pH 7.0 at 25 ° C and the volume was adjusted to 500 ml. Biomass obtained from 2 L of E. coli culture obtained as in Example 1 was added to the solution thus obtained with stirring. The formation of cephalexin was followed by spectrophotometric method (Analyst 92, 247, 1967). After 1 hour the reaction was discontinued, the cells separated by centrifugation. 550 ml of the solution contained 8 mg / ml cephalexin (4.4 g), corresponding to 54% k-onversion.

Example 4 g (23.3 mmol) of 7-ADCA and 10 g (50 mmol) of PGM were dissolved in 500 ml of 0.1 M phosphate buffer pH 7.0 containing the amount of penicillin acylase obtained from a 3 L culture as this is described in Example 2.

After 1 hour at 25 ° C, the solution contained a volume of 555 ml of 7.8 mg / ml cefalexin, corresponding to a 52% 7-ADCA conversion.

Example 1 -ad 5 g (23.2 mmol) of 7-ADCA and 10 g (51 mmol) of D - (-) - p -hydroxyphenylglycine ethyl ester (p-OH PGE) were dissolved in 1.5 L, 1 M phosphate buffer pH 7.0 at 25 ° C. Biomass from 2 L of E. coli culture obtained according to Example 1 was added to the solution, which was maintained at pH 7.0. After 1 hour, the reaction was stopped. The solution contained 2 mg / ml of 7beta- [2 amino-amino-2‘- (4-hydroxyphenyl) asamido] -3-methylceph-3-em-4-carboxylic acid corresponding to a 35% 7-ADCA conversion.

Example 6 g (23.3 mmol) of 7-ADCA and 10 g [51 mmol] of p-OH PGE were dissolved in 1500 ml of 0.1 M phosphate buffer at pH 7.0 at 25 ° C containing penicillin acylase, obtained from 3 L of culture as in Example 2. After 1 hour, 1.8 mg / ml of beta- [2'-amino-2'- (4-hydroxyphenyl) acetamido] -3-methylcephene- was found in the solution. 3-em-4-carboxylic acid, which corresponds to a 32% 7-ADCA conversion.

Example 7 g (18.36 mmol) of 7-aminocephalosporanic acid 1-ACA and 10 g (50 mmol) of PGM were dissolved in 1500 ml of 0.1 M phosphate buffer pH 7.0 at 25 ° C. Biomass obtained from 2 liters of E. Coli culture medium was added to the solution with stirring. After 1 hour, the solution contained 3 mg / ml of 7- (D-alpha-aminophenylacetamido / cephalosporanic acid) corresponding to a 60% 7-ACK conversion.

Example 8 g (18.36 mmol) of 7-ACK and 10 g (50 mmol) of PGM were dissolved in 1500 ml phosphate buffer containing penicillin acylase extracted from 3 L culture, maintaining the pH of the solution at 7.0 by continuous addition of NaOH, temperature at 25 ° C. After one hour, the solution contained 3.1 mg / ml cephaloglycine, which corresponds to a 62% 7-ACK conversion.

Example 9 g (18.36 mmol) of 7-ACK and 9.75 g (50 mmol of p-hydroxyphenylglycine ethyl ester) were dissolved in 1500 ml of 0.1 M phosphate buffer pH 7.0 at 25 ° C. with stirring, the btomase obtained from

1 cultures of E. Coli. Pc · 1 hour, the solution contained 1.7 mg / ml acid 7Ье1а- [2 -ат1по ^4-2- (4-hydroxyphenyl 1) acetamido] -3-hydroxymethyl-1 (vinegar) ceph-3-em-4-carboxylic acid, corresponding to a 33% 7-ACK conversion.

Example 10 obtained from 3 L of E. coli culture. At the end of the reaction, a 30% conversion was recorded

Preparation procedure as in Example 7-ACK.

9, biomass replaced by enzymatic extract-

Claims (8)

SUBJECT OF THE INVENTION A process for the preparation of a semisynthetic cephalosporin of the formula CK-OCOCH. COOH ^hydrolyzates corresponding derivative formed by chemical ring expansion of penicillin G followed using the product of hydrolysis of the starting compounds for a synthetic conversion into the desired semi-synthetic cephalosporin, characterized in that the hydrolysis of a derivative of penicillin G takes place by the action of penicillin acylase from Escherichia coli, after which the resulting reacting a compound of formula I with a compound of formula II. θ-сн ^ оон lil) 2. The method of claim 1, wherein the hydrolysis of the penicillin G derivative is carried out by contacting the bacterial cells with Escherichia coli. 3. A method according to claim 1, wherein the hydrolysis of the penicillin G derivative is carried out by contacting it with penicillin acylase extracted and purified from Escherichia coli cells. 4. The method of claim 1, wherein the penicillin G derivative is cephalosporin C. 5. The process of claim 1, wherein the hydrolysis is carried out at a pH in the range of 6 to 9. 6. The process of claim 1, wherein the hydrolysis is carried out at a temperature in the range of 0 to 50 ° C. 7. The process of claim 1 wherein the reaction is carried out at a pH in the range of 3 to 8. 8. The process of claim 1 wherein the reaction is carried out in the presence of a molar excess of the acylating agent.