DE2050982C3 - - Google Patents

Description

1-aminocyclohexylpenicillin is a broad-spectrum penicillin, that against the most diverse gram-negative and gram-positive pathogenic germs works.

This penicillin is resistant to numerous bacterial pathogens that cause infections in humans, more active in vivo than in vitro. In this respect it differs from α-aminobenzylpenicillin, the

35 is also a broad-spectrum penicillin, but is inferior to 1-aminocyclohexylpenicillin in terms of in vivo activity.

l-aminocyclohexylpenicillin has so far been used by Acylation of 6-aminopenicillanic acid, hereinafter referred to as 6-APS, with l-aminocyclohexylcarboxylic acid or a reactive derivative of this compound

The object of the invention was to provide a new process for the preparation of l-aminocyclohexylpenicillin microbiological way to create. This object is achieved by the invention

The invention thus relates to the subject matter characterized in the claim.

The penicillin G or V used according to the process or the 6-aminopenicillanic acid are used as alkali or Alkaline earth metal salt, in particular as sodium, potassium or calcium salt, used 6-aminopenicillanic acid can also be in the form of a salt, an acid, e.g. B. as hydrochloride, hydrobromide, hydroiodide or hydrogen phosphate, can be used.

The 1-aminocyclohexylcarboxylic acid used in the process of the invention can be used in the form of the free acid. Preferably, the amide, an alkyl ester with 1 to 4 carbon atoms in Alkyl radical and alkali metal salts of 1-aminocyclohexylcarboxylic acid, in particular a salt of an acid of these derivatives is used, particularly preferred the methyl or ethyl esters. However, the sodium or potassium salts of the acid can also be used will. As salts of an acid, salts can be used which differ from mineral acids, such as hydrochloric acid, Derive hydrobromic acid, hydriodic acid, phosphoric acid or sulfuric acid. The hydrochloride is preferred. The following reaction equations illustrate the process of the invention.

(a) Using 6-APS as a substrate:

COOH

+ H ₂ NCC

O = C N-C-COOH

NH,

CO-NH-C-C

NH,

1-aminocyclo- 6-aminopenicillanic acid (6-APS) hexyl carboxylic acid

O = C N-C-COOH

1-Aminocyclohexylpenicillin

(b) Using penicillin G as a substrate:

COOH

y \

NH,

HH
CH ₇ -CO-NH-CC

O = C N-C-COOH

l-aminocyclo-penicillin G.

hexyl carboxylic acid

CH ₇ -COOH +

HH
CO-NH-C-C

O = C N —C — COOH

Phenylacetic acid

1-Aminocyclohexylpenicillin

The microorganisms used in the process of the invention have a very high formation activity of 1-aminocyclohexylpenicillin from o-APS or Penicillin G or V and l-aminocyclohexylcarboxylic acid and their amide or alkyl esters with 1 to 4 carbon atoms in the alkyl radical.

To obtain the enzyme or enzyme system used in the method of the invention, the The aforementioned strains of microorganisms are bred to produce the desired enzyme or enzyme system forms within the cell body and preferably also within the culture fluid. The cultivation time is preferably adjusted so that the enzyme can be formed and enriched extracellularly in the manner described below.

As a growing medium for the process The microorganisms used are culture media that, in addition to a carbon or energy source, such as glucose, cane sugar, starch, molasses, sorbitol or other natural substances, also an organic one Contain nitrogen source. For example, peptone, meat extract, yeast extract or corn steep liquor can be used be used. The medium further contains suitable amounts of a nitrogen source such as Urea, ammonia, ammonium sulfate or ammonium chloride, or a natural source of nitrogen, such as corn steep liquor, peptone, meat extract or yeast extract. The nutrient medium preferably contains nor inorganic salts such as phosphates, e.g. B. primary and secondary potassium phosphate, magnesium salts, such as magnesium sulfate, metal ions such as iron, sodium, potassium, manganese, zinc or calcium ions, as well as anions such as chloride and nitrate ions. If necessary, additional nutrients are added added, which are necessary for the growth of the microorganisms.

The cultivation is carried out at temperatures of 20 to 50 ^° C. and a pH value in the range from about 5.0 to 9.0 under aerobic conditions, e.g. B. carried out by shaking culture or submerged culture with aeration and stirring. The cultivation period is usually 1 to 7 days. In this way, the enzyme used for synthesis is formed in the cultured cell bodies, and it is usually also present in the culture fluid.

The enzyme source used in the method of the invention can be the culture fluid itself, the Cell bodies, the culture fluid freed from cell bodies or the free enzyme that z. B. by Salting out with ammonium sulfate, dialysis, precipitation with acetone or ethanol or chromatography can be cleaned. When using the cell bodies, these are preferably in a suspension or used in acetone dried state. When using the culture fluid, the substrate added to the culture liquid and subjected to the reaction after adjusting the pH to below specified range is set

The enzymatic conversion takes place in a reaction liquid carried out, which contains the two substrates and a sufficient amount of the enzyme. The reaction medium can be the culture liquid itself to which the substrates have been added. The A buffer solution is preferably added to the reaction medium in order to keep the reaction within the optimum to be carried out in the pH range. The implementation

is possible in a relatively wide pH range from 4.5 to 7.5. The reaction temperature is at about 30 to 38 C. The reaction time is 1 to 24 hours.

After the reaction has ended, the t-aminocyclohexylpenicillin is isolated by known methods, z. B. by countercurrent distribution, precipitation by means of an organic solvent or by Adjustment of the pH value to the isoelectric point. The product can also be used for cleaning Ion exchangers or by column chromatography.

The examples illustrate the invention.

example 1

Kluyvera citrophila ATCC 21 285 is used as the microorganism. The sowing medium contains 1% peptone, 1% meat extract, 0.5% yeast extract and 0.3% sodium chloride. A platinum loop of the seeding microorganism is inoculated into 20 ml of the nutrient medium in a 250 ml fiber-filling Erlenmeyer flask and cultured at 30 ° C. for 24 hours with shaking. Then 2 ml of the seed culture liquid are inoculated into 20 ml of a nutrient medium in a 250 ml Erlenmeyer flask. This nutrient medium contains 0.5% peptone, 0.5% yeast extract, 0.5% sodium L-glutamate and 0.3% sodium chloride and 0.2% phenylacetic acid. The pH of the nutrient medium before sterilization is adjusted to 7.3 with 5N sodium hydroxide solution. The culture is grown with shaking at 30 ^° C. for 48 hours. The cell bodies are then separated from the culture fluid by centrifugation and washed twice with 0.9% strength aqueous sodium chloride solution. The cell bodies are then suspended in a 1/30 molar phosphoric acid buffer of pH 6.8 in a concentration of 10 mg dry cells / ml. The suspension is mixed with 3 mg / ml of the potassium salt of penicillin G and 5 mg / ml of l-aminocyclohexylcarboxamide

f, ₅ hydrochloride added. The reaction is then carried out at 35 ° C. for 4 hours. Thereafter, 2.5 mg / ml 1-aminocyclohexylpenicillin have formed in the solution.

Example 2

Streptomyces phaeochromoeenes ATCC 21 289 or Nocardia globerula ATCC 21 292 are used. The main fermentation medium is a Medium used containing 3% soluble starch, 2% soybean meal, 0.5% yeast extract and 0.1% calcium carbonate contains. The pH of this medium before sterilization is 7.3. The breeding conditions are the same as in Example 1. On the fourth breeding day the culture broth with 2.5 mg / ml penicillin G (when using Streptomyces phaeochromogenes) or Penicillin V (when using Nocardia globerula) and 10 mg / ml of 1-aminecyclohexylcarboxylic acid ethyl ester sulfate offset. The pH of the fermentation mash is adjusted to 6.8 and fermentation is continued. During fermentation the pH of the fermentation mash is adjusted to 6.8 at hourly intervals with hydrochloric acid or sodium hydroxide solution set. 6 hours after adding the substrates, Streptomyces phaeochromogenes 1.12 mg / ml or when using Nocardia globerula 1.85 mg / m) 1-aminocyclohexylpenicillin educated.

The superiority of the method according to the invention is evident from the following comparative tests emerged:

Attempt 1

The manufacture of l-aminocyclohexylpenicillin was carried out according to Example 14 of DT-OS 18 00 698, instead of 0.8 Mo! Dimethylaniline dihydrochloride however, 1.6 moles of dimethylaniline monohydrochloride were used used because the dihydrochloride was not accessible. The following results were obtained:

Yield 16.8g

Conversion rate of 6-APS 49.3%

Product purity 97%

Attempt 2

The preparation of l-aminocyclohexylpenicillin was carried out with the strains listed below according to Example 1, but without the addition of phenylacetic acid and using 1.5 mg / ml 6-APS and 5 mg / ml 1-aminocyclohexylcarboxylic acid methyl ester hydrochloride repeated. The following results were obtained:

tribe Educated yield Urnwind purity Amount of lung of product rate of Per 6-APS dukts (mg ml) (B) (%) (%) K. citrophila 2.13 30.5 56.5 98.5 ATCC 21 285 N. globerula 2.07 26.0 54.6 99 ATCC 21 292 S. phaeochromo- 2.05 25.7 54.0 98 genes ATCC 21 289

The product was isolated as follows:
After separating the mycelium, about 2 liters of the culture liquid are passed through a column which is filled with a strongly acidic potassium resin exchange.

} o it is filled in the soda form. and the one before Use was buffered with 0.2 molar citrate buffer of pH 2.0. That on the resin exchanger adsorbed product is then with 0.2 molar citrate buffer of pH 4.0 and then

Eluted for 3s with 0.2 molar citrate buffer of pH 7.0. The fractions which contain the l-aminocyclohexylpenicillin are combined, adsorbed on 1.5 liters of activated carbon and then eluted with 70% aqueous methanol. The eluate is evaporated ^{at 40 ° C. under reduced pressure.} When acetone is slowly added to the concentrate, the product separates out in crystalline form.

Claims (1)

Claim: Process for the preparation of f-aminocyclohexylpenicillin, thereby characterized in that 6-aminopenicillanic acid, its alkali or Alkaline earth metal salt or its salt with an acid or penicillin G, penicillin V or their alkali or Alkaline earth metal salt with 1-Aininocyclohexyl- ι ο carboxylic acid or its amide or alkyl ester with 1 to 4 carbon atoms in the alkyl radical or Salt of an acid in the presence of a 1-aminocyclohexylpenicillin-forming enzyme, which in the aerobic cultivation of Kluyvera citrophila ATCC 21 285, Nocardia globerula ATCC 21 292 or Streptomyces phaeochromogenes ATCC 21 289 in a standard nutrient medium at temperatures from 20 to 50 ° C and at a pH value of 5 to 9, at temperatures from 30 to 38 ° C and at: o brings a pH of 4.5 to 7.5 to the implementation.