FI61517B - FAR OIL FRAMSTAELLNING AV 6-D - (-) ALPHA-AMINO-ALPHA- (P-HYDROXYPHENYLACETAMIDO) PENICILLAN SYRIC - Google Patents

Description

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(71) Bristol-Myers Company, 3U5 Park Avenue, New York, N.Y. 10022, USA (72) Daniel Bouzard, Franconville, Abraham Weber, Paris, France-Frankfurt (FR) (7U) Oy Kolster Ab (5U) Method 6- / D - (-) - Ct-amino-Oi. - (for the preparation of p-hydroxyphenylacetamido / penicillanic acid - Forfarande för framställning av 6- / D- (-) - C * -amino- 0 (, - (p-hydroxyphenylacetamido) 7penicillan) The present invention relates to a new process for the preparation of 6-D - (-) - c <-amino- <β- (p-hydroxyphenylacetamido) penicillanic acid, its hydrate or a pharmaceutically acceptable salt thereof.

The process according to the invention is characterized in that an aqueous solution of 6-D- (-) - c * -amino-cX- (p-acetoxyphenylacetamido) penicillanic acid is treated with an esterase, namely citrus esterase, wheat bran or wheat germ, at a pH of about 5.0-7, 5, optionally in the presence of a buffer, wherein the esterase concentration is about 5-10 mg / ml of the total solution, and the product is isolated by methods known per se, after which, if desired, the products in free acid or hydrate form are converted into pharmaceutically acceptable salts.

2,61517

Of particular commercial interest is a process for the preparation of 6-D - (-) - Of-amino-O- (p-hydroxyphenylacetamido) penicillanic acid, hydrate or a pharmaceutically acceptable salt, wherein 6-D - (-) - OC-amino-CV - <p- acetoxyphenylacetamido) an aqueous solution of penicillanic acid is treated with a commercially available esterase, namely coarse wheat bran, either at pH 5.5 to 6.0 or in the presence of buffer at pH 7.0, with a concentration of wheat bran of 10 mg / ml of total solution volume, and the product separated by methods, and if desired, the product in the form of the free acid or hydrate is converted into a pharmaceutically acceptable salt.

The preparation of the starting material is described in parent application 751596.

6-D - (-) - Of-amino-Of- (p-hydroxyphenylacetamido) penicillanic acid (amoxicillin) prepared by this method is known to be an effective antibacterial agent and useful in the treatment of infectious diseases caused by gram-negative or positive bacteria in animals , in poultry and also in humans.

The following examples illustrate the method of the invention.

Example 1

The following solutions were prepared: 0.5 mg / ml of 6-D- (-) - Of-amino-O- (p-acetoxyphenylacetamido) -penicillanic acid (p-acetoxyampicillin) in normal saline.

0.5 mg / ml p-acetoxyampicillin in citrus esterase solution (esterase concentration equal to that of wheat bran esterase solution, 10 mg / ml) diluted 10-fold with 0.1 M potassium phosphate buffer to adjust the pH to 7.0, and 0.5 mg / ml p-acetoxyampicillin in a solution containing 10 mg / ml crude wheat bran (Shiloh Farms, Inc., Sulfur Springs, Arkansas, USA) in 0.1 M potassium phosphate buffer.

All these solutions were stirred at 37 ° C, and sampled for chromatography at 0, 2, 4, 8, and 24 hours.

5 μΐ samples were pipetted onto Whatman No. 1 paper strips 12.7 mm wide, dried and developed in a solvent mixture containing 80 parts of butyl acetate, 15 parts of n-butanol, 40 parts of acetic acid and 24 parts of part of the water. The samples were then bioautographed on plates inoculated with Bacillus subtilis at pH 6.0.

These biochromatograms showed that p-acetoxyampicillin is stable in normal saline, but is rapidly hydrolyzed to the p-hydroxy form by both citrus esterase and bran esterase.

Example 2

The following reaction mixtures were prepared; they were mixed at 28 ° C and sampled at 0, 1.5, 1, 2, 3, 4, and 6 hours as shown in Example 1.

1. 25 mg of non-fat bran (wheat bran obtained from Shiloh, treated with acetone and dried), 4.5 ml of 0.1 M potassium phosphate buffer, pH 6.0 and 0.5 ml of a solution containing 5 mg / ml 6-D - (-) - Q? -Amino-Qi-j (p-acetoxyphenylacetamido) penicillanic acid (p-acetoxyampicillin) in the same buffer.

2. 25 mg of non-fat bran, 4.5 ml of 0.1 M potassium phosphate buffer, pH 7.0, and 0.5 ml of a solution containing 5 mg / ml p-acetoxyampicillin in the same buffer.

3. 25 mg of non-fat bran, 4.5 ml of 0.1 M potassium phosphate buffer, pH 7.5, and 0.5 ml of a solution containing 5 mg / ml p-acetoxyampicillin in the same buffer.

4. 50 mg of non-fat bran, 4.5 ml of 0.1 M potassium phosphate buffer, pH 6.0 and 0.5 ml of a solution containing p-acetoxy-ampicillin in the same buffer.

5. 50 mg of non-fat bran, 4.5 ml of 0.1 M potassium phosphate buffer, pH 7.0 and 0.5 ml of a solution containing 5 mg / ml acetoxyampicillin in the same buffer.

6. 50 mg non-fat, bran, 4.5 ml 0.1 M potassium phosphate buffer, pH 7.5 and 0.5 ml solution containing 5 mg / ml p-acetoxyampicillin in the same buffer.

The results of the biochromatograms are given in Table IV.

4 6151 7

Table IV

% conversion to p-hydroxyampicillin

Reaction time (hours)

Reaction n; o 0_1 / 2 1 2 3 / 4_6 1 12 23 32 64 100 91 95% 2 11 34 45 68 120 75 77% 3 11 32 39 45 68 75 98% 4 9 32 55 59 80 80 77% 5 14 34 64 109 131 104 104% 6 16 66 66 91 98 116 86%

Optimal results are thus obtained using reaction mixture No. 5; then the total conversion to p-hydroxyampicillin is complete in 2 hours at a bran concentration of 10 mg / ml and a pH of 7.0.

Example 3

The following reaction mixture was prepared containing 50 mg of nonfat bran (Shiloh), 2.5 mg of 6-D - (-) - (β-amino-cA- (p-acetoxyphenylacetamido) penicillanic acid (p-acetoxyampicillin) and 5, 0 ml of water The mixture was stirred at about 28 [deg.] C. and chromatographed every 1, 2 and 3 hours.

The results of the biochromatograms showed that 100% conversion from p-acetoxyampicillin to p-hydroxyampicillin occurred in 3 hours. The pH of the reaction mixture remained constant (about 5.7) despite the absence of buffer.

Example 4

The following ingredients were combined: 20 g of non-fat bran (Shiloh), 1.0 g of 6-D- (-) - o (-amino-? - (p-acetoxyphenylacetamido) penicillanic acid (p-acetoxyampicillin) and 2 l of 0.01- m of potassium phosphate buffer solution, pH 7.0, The resulting mixture was stirred at 28 ° C and sampled every hour as in Example A.

From the biochromatograms it was found that 100% conversion was achieved in 3 hours. The mixture was then centrifuged. The supernatant was collected, the pH was adjusted to 4.0 with hydrochloric acid, and lyophilized. The lyophilate was further examined as described above to find approximately 900 mg of p-hydroxyampicillin, 6151 7 5

The lyophilate, 6.0 g, was mixed with 20 ml of water. To the resulting mixture was added 6N hydrochloric acid to gradually lower the pH to 2.0. Stirring was continued for a further 15 minutes, followed by filtration of the solid. The filtrate was treated with 1.0 g of decolorizing carbon, the carbon was filtered off, and the clear filtrate was adjusted to pH 4.5. Crystallization was initiated by scraping and allowed to continue for 4 hours. The crystals were collected on a filter, washed with water and acetone, and dried to give 180 mg of 6-D - (-) - 5-amino-C * - (p-hydroxyphenylacetamido) penicillanic acid trihydrate, Decomposition temperature 201 ° C,

Elemental analysis for C 18 H 28 N 2 O 2 S% theoretical% found C 45.82 45.87 H 6.01 5.71 N 10.02 10.48 K.F, H 2 O 12.89 13.68

Biological information In vivo activity

Mean effective doses (CDcn) in mice of various 50 pathogenic microorganisms were determined for 6- [D - (-) - <= f-amino- (i- (4-hydroxyphenyl) acetamido] penicillanic acid. CD50 data are given below in mg / kg.

Organism Route of administration CD50 (m9 / kg) 1 1 1 '«' —— Il m '1'« y ·· n ~ .i Tl »"> ......— - 1 —1

Staphylococcus aureus Smith intramuscularly 0.2 orally 0.9

Salmonella enteritidis intramuscularly 5.4 orally 3.5-4.0

Klebsiella pneumoniae intramuscularly 7 orally 7

Oral Absorption Assays were performed by oral administration of 6- / 5 - (-) - β-amino-β- (4-hydroxyphenyl) acetamido / penicillanic acid to mice. In the experiment, four mice were orally administered 30 mg / kg of compound. The mean blood levels were as follows:

Time (hours) Concentration (μg / ml) 0.5 8.1 1.0 4.0 2.0 2.0 1.40 3.5 1.1

Claims (2)

6 6151 7 A process for the preparation of 6-D- (-) - <X-amino- (X- (p-hydroxyphenylacetamido) penicillanic acid, its hydrate or a pharmaceutically acceptable salt thereof, characterized in that 6-D- (-) - An aqueous solution of ε-amino- (β- (p-acetoxyphenylacetamido) penicillanic acid is treated with an esterase, namely citrus esterase, wheat bran or wheat germ, at a pH of about 5.0 to 7.5, optionally in the presence of a buffer, the esterase concentration being about 5-10 mg / ml of the total solution, and the product is isolated by methods known per se, after which, if desired, the product in the form of the free acid or hydrate is converted into a pharmaceutically acceptable salt. Process according to Claim 1, characterized in that an aqueous solution of 6-D - (-) - ίλ-amino-cX- (p-acetoxyphenylacetamido) penicillanic acid is treated with a commercially available esterase, namely raw wheat bran, at a pH of 5.5 to 6.0. or alternatively in the presence of a buffer at pH 7.0, the bran concentration being 10 mg / ml of the total solution, and the product is isolated by methods known per se, after which, if desired, the product in free acid or hydrate form is converted into the corresponding pharmaceutically acceptable salt.