DE1161276B - Process for the preparation of derivatives of Cephalosporin C. - Google Patents

Description

Process for the preparation of derivatives of cephalosporin C The invention relates to a process for the preparation of derivatives of cephalosporin C.

For cephalosporin C is the structure has been proposed.

There has also been proposed a method of cleaving the @ -aminoadipyl side chain from cephalosporin C, and the product thus obtained has the structure and is referred to as 7-aminocephalosporanic acid. In addition, the preparation of N-acyl derivatives of 7-aminocephalosporanic acid other than the compound formulated first has also been proposed.

The invention relates to a process for the preparation of derivatives of cephalosporin C of the general formula where R represents a hydrogen atom or an N-acyl group, and its salts, which is characterized in that one is made from a compound of the general formula in which R has the above meaning, or from a soluble salt of such a compound the O-acetyl group with a citrus acetyl esterase at a temperature between about 20 and 40 ° C and at a pH between about 5.5 and 8.0 removed enzymatically and the process product, optionally after reaction with a suitable sodium, potassium or ammonium salt, isolated in the usual way. The acid addition salts can also be prepared by reacting the compound of the general formula in which R is a hydrogen atom with an acid.

In the process according to the invention, preference is given to using a compound of the general formula II as starting material, where R is an optionally substituted benzoyl or @ -aminoadipinyl radical or a group of the general formula represents in which R 1 and R 2 are hydrogen atoms, alkyl, phenyl, alkyloxy or optionally substituted phenoxy groups and these groups can be identical or different. If R1 and Rz are alkyl or alkoxy groups, these can contain 1 to 10 carbon atoms, in particular 1 to 6 carbon atoms. Preferred substituents for the phenyl radicals are nitro, alkyl, alkoxy groups and chlorine atoms. In the case of the alkoxy groups, methoxy and ethoxy groups are particularly suitable.

Particularly important compounds falling under the general formulas are the corresponding phenylacetyl, phenoxyacetyl, n-propionyl-a-phenoxypropionyl, Isobutyryl, hexanoyl, p-nitrophenylacetyl and p-nitrophenoxyacetyl and 2,6-dimethoxybenzoyl derivatives.

Citrus acetylesterase and its recovery have been described by J a n s e n, J a n g and M a c D o n e 11 in »Archive. Biochem., Vol. 15, 1947, pp. 415. The best deacetylation results are obtained when maintaining a pH value between 6.0 and 7.0 and a temperature between 25 and 37 ° C.

The respective process product can pass through from the reaction medium known separation process z. B. by chromatography and ion exchange be separated.

Desacetylcephalosporin C is a particularly important one according to the invention established connection. It is preferably made using the sodium salt of cephalosporin C as the starting material and can be made from the reaction mixture according to The preparation can be separated by applying the mixture to a column of an anion exchange resin there, which is in the form of its salt with a weak acid, and the desacetylcephalosporin C eluted with a solution containing the anion of a weak acid. solutions of volatile buffers, such as ammonium acetate or pyridine acetate, with a pH value of about 5, are suitable for this purpose. The deacetylcephalosporin C in the eluate can be detected in different ways depending on the eluant used be e.g. B. by the UV absorption spectrum of the product, its reaction with Ninhydrin and its antibacterial effectiveness. Is the eluent a solution? a volatile buffer, the product can be obtained as a solid, by evaporating the appropriate fractions of the eluate. A suitable salt of the product, e.g. B. the sodium salt, can then in pure form by crystallization can be obtained.

Desacetylcephalosporin C can be characterized by the following properties 1. It shows no C-methyl in the Kuhn-Roth determination.

2. The UV absorption spectrum of its sodium salt at a concentration of 0.05 mg / ml in aqueous solution shows a maximum at 261 mla. (as shown in Fig. 1).

3. The infrared spectrum of its sodium salt (in liquid paraffin) is in F i g. 2 reproduced.

4. The activity of its sodium salt is approximately 2.3 units per milligram versus staph. aureus and 1.8 units per milligram against Salmonella typhi (cf. Cephalosprorin C sodium salt, whose activity is 8 to 10 units each Milligrams to both organisms).

5. Chromatograph on »Whatlnan no. 1 «paper (applied in the form of the sodium salt and with descending solvent), it shows the following R-Glycol values: Solvent R Glycol (A) n-butanolic acetic acid, water (4: 1: 4 volume). . . . . . . . . . . 0.57 (B) propan-l-ol! Water (7: 3 volume). ... . .... .. 0.60 In solvent B, it is separated from cephalosporin C (R-glycoll = 0.77) and from cephalosporin C, (R-glycocoll = 0.98).

6. It is subjected to electrophoresis on paper (14 V / cm 3.3 Hours) in pyridine acetate buffer at pH 4.5, it migrates 7.9 cm towards the anode (cephalosporin C migrates 7.5 cm in Towards the anode and cephalosporin C, 1.9 cm towards the cathode).

If the electrophoresis is carried out in 10 percent by volume acetic acid, so it migrates 1.2 cm towards the cathode (cephalosporin C migrates 0.8 cm towards the cathode, and cephalosporin C <migrates 4.6 cm towards on the cathode).

7. Dissolve it in 0.1 N hydrochloric acid, where it quickly becomes into the cephalosporin C ,. convicted. This conversion can easily be demonstrated by making a Sample of the reaction solution in 10 volts percent acetic acid on paper by electrophoresis subject. The newly formed cephalosporin C migrates 4.6 cm towards the Cathode and can be detected by its antibacterial activity.

B. If it is allowed to react with certain acid chlorides, such as phenylacetyl chloride, it forms N-acyl derivatives with an increased activity towards Staph. aureus. The acylation can, as previously suggested, be carried out on paper. These N-acyl derivatives have the structure where R4 represents an N-acyl group.

9. If it is titrated electrometrically in water at 20 ° C, it shows Groups with pKa values of approximately <2.5, 3.0 and 9.6. If you titrate a solution, which has been kept at a pH of 1.9 for 2 hours, back with alkali, so it turns out that about 28% of the one acidic group has disappeared (as a result of the lactone formation in the formation of the cephalosporin Co). If you titrate a solution, which was held at pH 11.5 for 30 minutes, back with acid, so results found that partial hydrolysis occurred with the formation of a new acid group Has. This is presumably the result of the opening of the l-lactam ring. According to the invention Compounds prepared can be used as intermediates for the synthesis of antibiotic effective cephalosporins can be used.

The following examples illustrate the invention (for the process according to Example 3, a) protection is not sought) Example 1 52 mg sodium salt des Cephalosporin C is dissolved in a solution of citrus acetylesterase. The solution is heated to 30 ° C and the pH value quickly by adding 0.1 N sodium hydroxide solution set to 6.5. The temperature is kept at 30 ° C and the pH through Addition of 0.1 N sodium hydroxide solution kept in the range from 6.2 to 6.8. The rate of addition of the alkali necessary to maintain the pH value drops rapidly during the first 15 minutes, and the pH remains after 40 minutes without any further addition of alkali and thus indicates the end of the reaction. The solution will be cooled to room temperature and placed on a column (20-0.9 cm) of the under the trade name "Amberlite Xe-58" known ion exchanger in acetate form (0.12 to 0.074 mm) applied, previously with an ammonium acetate solution, 0.2 molar of ammonium ion, has been equilibrated at pH 5.0. Is used for elution a solution of ammonium acetate buffer with the same concentration is used, collecting a 3 ml fraction every 35 minutes. The parliamentary groups are examined by measuring their absorbance at 260 m # t; the greatest absorption will be between the 16th and 26th parliamentary groups determined. The fractions 17 to 24 are in a few millimeters Water combined and freeze-dried twice to get as much ammonium acetate as possible to remove. The residue contains the ammonium salt of deacetylcephalosporin C. The absorbance at 260 mV indicates a yield of 70010. Example 2 601 mg of the sodium salt of Cephalosporin C are dissolved in 3 ml of water and made into a Solution of citrus acetylesterase (5 ml) is added, previously adjusted to pH 6.5 will. A 0.25 N sodium hydroxide solution is added periodically to bring the pH between 6.2 and 6.9 to keep; the solution is kept at 30 ° C. by means of a water jacket. After 95 minutes, the necessary rate of addition of alkali becomes quite low; a further 5 ml of the enzyme solution are added. After 200 minutes the pH is up practically constant without the addition of alkali. The solution is made with acetic acid on a pH 5.0 acidified, cooled to room temperature and transferred to a column (21 - 2.0 cm) of the ion exchanger known under the trade name »Amberlite Xe-58« given in acetate form (0.12-0.074 mm). The elution is carried out with a pyridine acetate solution, 0.3 molar of acetate ion, carried out at pH 5.0. Fractions of 4.5 ml are all Collected for 14 minutes and checked by drawing on paper and staining with ninhydrin. As soon as this shows a maximum, take 0.1 ml samples and leave them with you react with a ninhydrin solution (M o r e and S t e i n, »Journal of Biological Chemistry «, Vol. 176, 1948, p.367) and determines the color density. The greatest absorption lies between fraction 45 and 77. Fractions 50 to 62 are combined and freeze dried. The freeze-dried powder is in a minimum of water dissolved, precipitated with acetone (about 50 ml) and under acetone to a fine powder ground and washed with another amount (approximately 50 ml) of anhydrous acetone. This powder is dissolved in 3 ml of water and the pH is adjusted by adding 0.1 N sodium hydroxide solution set to 7.9. More water is added (10 ml) and the solution freeze dried. The freeze-dried product, Desacetylcephalosporin C Sodium is crystallized from aqueous ethanol. A yield of 440 mg (80%) is obtained. Example 3 a) The outer peels of 80 oranges are removed and weighed (1334 g). The grated shells are washed with acidic sand (100g) and sodium chloride (20g) mixed and pressed through a filter cloth. The juice obtained is made with sodium oxalate saturated and filtered through a filter paper ("Whatman" No. 42). The extract (310 ml) is mixed with an equal volume of saturated ammonium sulfate solution and the precipitate formed was recovered by centrifugation (2000 rpm) for 15 minutes. The precipitate is resuspended in 0.1 molar sodium oxalate solution (60 ml) and dialyzed against an 0.1 molar sodium oxalate solution at 5 ° C. overnight. b) 1.8 g of the sodium salt of Cephalosporin C is dissolved in 9 ml of water and with 15 ml of the treated according to a) citrus acetylesterase, which was previously adjusted to pH 6.5 will. The mixture is stirred and the pH is adjusted by adding 0.1 N sodium hydroxide solution held between 6.2 and 6.9. If the rate of addition slows down, then more citrus enzyme (15 ml at pH 6.5) was added and the mixture was warmed to 30 ° C. The reaction is usually ended after 33.7 ml of 0.1 N sodium hydroxide solution have been added are (theoretically 34.0 ml). The bioautograph only shows one active spot accordingly the deacetylcephalosporin C.

The bulk of the reaction mixture is adjusted to pH 5.0 with 2N acetic acid and placed on a 21-2.5 cm column of the ion exchanger known under the trade name "Amberlite Xe-58", which is buffered to pH 4.0 . The column is then developed with 0.3 molar acetic acid that has been buffered to pH 5.0 with pyridine. 5 ml fractions are collected from the column and 0.1 ml samples are tested with ninhydrin according to the method of M oore and S tein (op. Cit.). The greatest activity lies between fraction 66 and 101. These fractions are collected and lyophilized. The solid is taken up in 10 ml of water and precipitated with 200 ml of acetone. The oil is finally solidified by mild evaporation in vacuo. This solid is obtained by centrifugation (1500 rpm) for 15 minutes and ground under 50 ml of anhydrous acetone. The solid is filtered, dissolved in 10 ml of water and brought to pH 7.9 with 0.1 N sodium hydroxide solution. The resulting solution of the sodium salt of desacetylcephalosporin C is lyophilized and the yield is 526 mg. Crystallization from aqueous ethanol yields 342 mg of a product which only gives one place on the bioautograph corresponding to DesacetylcephalosporinC. Activity of desacetylcephalosporin C o / 0 Organism desacetyl cephalosporin C B. subtilis C 289. . . . . . . . . . . . . 58.0 Staph.aureus C 864 ............. 23.0 V. cholerae C 833. . . . . . . . . . . . . j 22.9 Example 4 52 µg of sodium 7-phenylacetamidocephalosporanic acid are dissolved in 0.2 ml of water. 0.1 ml of this solution is drawn off and 0.1 ml of a solution of citrus acetyl esterase, buffered to pH 7.4 with molar phosphate solution, is added and the solution is incubated at 30 ° C. for 1 hour. 10; d samples of this solution and 5 µl samples of the original solution are then placed on an electrophoresis sheet. A sample of Cephalosporin C is used as a marker and the leaf is dried at room temperature. The sheet is then sprayed with molar pyridine acetate solution (pH 7.0), dried almost at room temperature and hung in a pyridine atmosphere (vapor in equilibrium with molar pyridine acetate solution of pH 7.0) at 37 ° C. for 18 hours. The sheet is hung in a container for 1 hour, with marks of the original solution; the solution is incubated with enzyme and then cephalosporin C is applied; the sheet is then electrophoresed for 3 hours in 0.05 molar pyridine acetate solution at pH 4.5 under a voltage of 14 Vlcm. The electrophorogram is hung in a container for 1 hour and then on agar floors using a bioautograph, with Staph. aureus vaccinated, tested. The results are summarized in the table below. The stretches traversed in the direction of the cathode are marked with a minus sign and the stretches traversed in the direction of the anode with a plus sign. Wandered through Connection route (cm) * Cephalosporin G. . . . . . . . . . . . . . . -6.6 Cephalosporin G with pyridine treated ....... . . . . . . . . . . . . . -1.4 Cephalosporin G with enzyme treated .................... +7.4 Cephalosporin G with enzyme, then treated with pyridine ... ... ... -, - 7.1 Cephalosporin C. . . . . . . . . . . . . . . . -6.6 Cephalosporin C with pyridine treated . . . . . . . . . . . . . . . . . . . . -1.3 * Cephalosporin G means 7-phenylacetamidoceplialosporan- acid. 7-Phenylacetamidocephalosporanic acid is converted by the action of citrus acetylesterase into a substance which does not give a derivative of the CA type when treated with pyridine under the conditions used, whereby the formation of the deacetyl compound is confirmed. The new substance migrates similarly to 7-phenylacetamidocephalosporanic acid and has a high antistaphylococcal activity, but not as high as 7-phenylacetamidocephalosporanic acid itself. Example s 1 mg of 7-aminocephalosporanic acid is dissolved in 0.1 ml of a 0.1 molar phosphate buffer solution of pH 7.0 . A sample of this solution (10 µl) is mixed with 10 µl of water and the solution obtained is used as a comparison solution.

The remaining 90 μl of the 7-aminocephalosporan solution are mixed with 90 u1 citrus acetylesterase solution (adjusted to pH 7.0) mixed.

The mixture and the comparison solution are kept at 30 ° C for 1.5 hours held. 5-VJ samples of both the mixture and the control solution are applied a »Whatman no. 1 ″ paper is applied and the paper lightly covered with molar pyridine acetate sprayed (pH 7.0), hung in an atmosphere of pyridine acetate overnight and finally air dried. Another 5-, al-samples of both the mixture as also the comparison solution are applied to a paper and overnight in the Laboratory air kept. Both the pyridine-treated and the untreated spots are then electrophoresis on the papers (14 V jcm) 2.5 hours with a Subjected to buffer at pH 4.5. The ring connections are then on the paper using Phenylacetyl chloride, as described above, implemented. The stains of active phenylacetylated material are finally determined by bioautography with staph. aureus (Oxford strain) as a test organism.

The following results are obtained: 1. 7-Aminocephalosporanic acid gives large active spots migrating 3.4 cm towards the anode.

2. The reaction product of 7-aminocephalosporanic acid with acetylesterase provides slightly smaller spots that migrate 3.6 cm towards the anode.

3. The reaction product of 7-aminocephalosporanic acid with pyridine provides a broad active spot due to the formation of the cephalosporin CA core (Pyridine) migrating 3 cm towards the cathode.

4. The reaction product of 7-aminocephalosporanic acid with a) acetylesterase and b) pyridine shows only a trace of product directed towards the cathode wanders. The 7-aminocephalosporanic acid turns into an active compound by acting converted by acetylesterase, which is similar to 7-aminocephalosporanic acid itself electrophoresis at pH 4.5 migrates to a derivative, but not with pyridine of the CA type responds. This shows the production of desacetyl-7-aminocephalosporanic acid at.

Claims (3)

Claims: 1. Process for the preparation of derivatives of cephalosporin C of the general formula in which R represents a hydrogen atom or an N-acyl group, and their salts, characterized in that a compound of the general formula wherein R has the above meaning, or from a soluble salt of such a compound the O-acetyl group with a citrus acetyl esterase at a temperature between about 20 and 40 ° C and at a pH between about 5.5 and 8.0 enzymatically removed and the process product, optionally after reaction with a suitable sodium, potassium or ammonium salt or, if R is a hydrogen atom, isolated with an acid in the customary manner. 2. The method according to claim 1, characterized in that a compound of the general formula II is used as the starting material, where R is a β-aminoadipinyl radical or a group of the general formula represents in which R 1 and R 2 are hydrogen atoms, alkyl, phenyl, alkoxy or optionally substituted phenoxy groups and these groups can be identical or different. 3. The method according to claim 1 or 2, characterized in that one performs the enzyme treatment at a pH of 6.0 to 7.0.