DE1084719B - Process for the extraction of corticosteroid-like compounds from Gaerloesungen - Google Patents

Description

Process for the production of corticosteroid-like compounds from Fermentation solutions The invention relates to a process for the extraction of certain, Steroid compounds obtained by fermentation from fermentation solutions.

Over the past few years, many steroid compounds are using certain Microorganisms have been treated. For example, the oxidation of steroids, like converting the compound S into hydrocortisone, with a variety of different Microorganisms have been carried out. You also have hydrocortisone with different Microorganisms converted into prednisolone. There are also numerous related compounds, such as 14,15-oxidohydrocortisone, 14-oxyprednisolone and 14-oxyhydrocortisone, on a microbiological level Paths have been established.

There has now been a new process for obtaining compounds of the Above type found from these containing fermentation products. According to the new procedure the steroid is made from the fermentation solution with an organic one that is immiscible with water Solvent extracted in a known manner and the extract with a basic pH by extraction with a buffer substance, which is preferably a phosphate is cleaned. The solvent extract is then concentrated as much as it is for the crystallization of the product due to its solubility in the chosen one organic solvent is required. Buffer solution is added to the concentrate added and the mixture cooled and stirred to crystallize the product. This makes a crystalline product by a simpler process than before obtained from a high degree of purity.

The crystalline product can be obtained by recrystallization from a with Water-immiscible, halogenated solvents, preferably from chloroform, which contains a small amount of a lower alkanol of up to 3 carbon atoms, be cleaned further. By adding water to this mixture, the steroid becomes then failed. A product of very high purity is then obtained, its Quality is satisfactory for use in pharmaceutical Preparations are suitable.

The process according to the invention results in surprisingly high yields achieved. They generally exceed 70 per cent of the corresponding in steroid present in the fermentation solution. The fermentation products often contain more than one steroid. The present process now enables an excellent cleaning of undesirable Impurities with a very similar constitution.

If the procedure outlined above is modified, a steroid solution, by extracting a fermentation solution with a halogenated solvent of the following described type was obtained - before crystallization in the presence of aqueous Buffer solution -, with a small amount of a lower alkanol with up to treated to 3 carbon atoms. This treatment leads to the achievement of a product of high purity.

If the fermentation solution is treated with a ketone of the type described below extracted, so the concentrated ketone solution - before treatment with a Alkanol and crystallization in the presence of aqueous buffer solution - with a large Treated amount of a halogenated solvent of the type to be described. Generally, after treatment with the halogenated one, the ketone solution contains Solvent about 30 percent by volume ketone.

The fermentation solution is preferably extracted using a diatomaceous earth filter at a weakly acidic pH, d. H. at about 3 to about 6, filtered. Afterward the filtered product is extracted with a suitable solvent.

Used to carry out the method described above as the water-immiscible solvent for the first extraction of the steroid from the fermentation solution preferably methyl isobutyl ketone, methyl ethyl ketone, methylene chloride, Ethylene chloride or chloroform. The extraction carried out with these solvents can be discontinuous, i.e. H. by stirring a volume part with solvent one to five times the amount of fermentation solution followed by settling and separation of the phases. This process can be repeated several times to create a ensure complete separation. The extraction can but can also be carried out in packed columns, in which the filtered fermentation solution and the solvent can be passed in countercurrent. Preferred for this type of extraction are so-called Podbielniak extractors, as they have the best extraction effect is achieved: Is the solvent extract containing the steroid different from the aqueous Phase separated, then with an aqueous solution of a water-soluble buffer substance, such as a mixture of sodium phosphates, at a pH of about 7.5 to about 9.0, washed. The volume ratio of buffer substance to solvent extract can be about 1: 3 to about 2: 1. The buffer is in the form of a diluted aqueous solution used. A buffer can e.g. B. be produced in that a 1% aqueous solution of secondary sodium phosphate with so much more concentrated Sodium hydroxide solution is added so that a pH of 8 to 8.5 is reached. Other suitable buffer substances are mixtures of alkali metal carbonate and bicarbonate, Alkali metal acetate and acetic acid or alkali metal hydroxide combinations (depending on the desired pH value). Almost any water-soluble combination of a strong one Base and a weak acid adjusted to a suitable pH value, proves suitable for this purpose.

The extraction with a buffer can be discontinuous or in the countercurrent process described above take place. The purified steroid solvent extract is then preferably reduced to about -100 by distilling off the solvent in vacuo concentrated up to about 300 mg steroid per ccm solution. Depending on the particular to winning steroid compound, the most beneficial concentration varies somewhat. For prednisolone and hydrocortisone a concentration of about 250 mg / ccm is completely sufficient. If the concentration obtained is too high, the existing impurities can build up also precipitate during the crystallization of the desired compound.

After concentration, the solvent concentrate is about 1/2 to 2 volumes of a buffer of the type described above at a pH of about 7.5 to 9.0, preferably from 8 to 8.5. The resulting two-phase mixture is then cooled and stirred slowly for several days. Generally one applies a Temperature of 10 ° C or below. Temperatures well below 0 ° C out of the question. After several hours, i.e. H. after stirring for about 3 to 20 hours the crystalline product can be removed from the liquid two-phase system. this can be obtained by filtration, centrifugation, etc. The separated product is dried on it.

Although the product is obtained in high purity, it can, if so desired, be further purified by crystallization. For example, it can be dissolved in a mixture of one of the above halogenated solvents containing a small amount (5 to 35%) of a lower alkanol of up to 3 carbon atoms. Chloroform is preferably used as the solvent and methanol is used as the alkanol, there being about 30 to 100 mg of crystals per 1 cc of solvent. The recrystallized product can then be obtained by gradually adding water to the mixture, most advantageously using about 1/2 to 3 parts by volume of water per part by volume of solvent. The mixture is stirred and the recrystallized product separates out. It can be removed by filtration or centrifugation. If further cleaning is desired, the above process can be repeated. By concentrating the mother liquor, ie the filtrate or centrifugate from the purification process described above, further substances can be obtained which, however, may be very impure, so that they have to be returned for further purification.

The following examples serve to illustrate the process according to the invention: Example 1 The action of Mycobacterium smegmatis on hydrocortisone produced a fermentation solution which each contained 3.81 100 mg of 41-dehydrohydrocortisone. The fermentation solution also contained 41-dehydrohydrocortisone unconverted hydrocortisone and structurally related steroids. The fermentation solution was filtered through a diatomaceous earth filter at pH 4.0. Then it was extracted in a Podbielniak extractor with methyl isobutyl ketone, the ratio of solvent to fermentation solution being 1: 3. The methylsobutyl ketone extract was washed with 1 part by volume of 1% disodium phosphate, which had been adjusted to a pH of 8 using concentrated sodium hydroxide. The washed methyl isobutyl ketone solution of the steroid was concentrated by distillation to a solution which contained 250 mg of 41-dehydrohydrocortisone per cc. 1 part by volume of 1% disodium phosphate, the pH of which was adjusted to 8.5 with the aid of sodium hydroxide, was added to the solvent concentrate. The mixture was then cooled to 0 ° C. and slowly stirred for 10 hours. The crystalline product was filtered off and the crystals dried in vacuo with the application of some heat. The dried product was then dissolved in a mixture of 90 percent by volume of chloroform and 10 percent by volume of methanol, 50 mg of solids being used per ccm of solvent mixture. The purified product was crystallized by gradually adding the same volume of water with constant stirring. The 41-dehydrohydrocortisone was obtained with a purity of over 900 / in a yield of over 700 / .

EXAMPLE 2 A fermentation solution which, as described in US Pat. No. 2,658,023, had been prepared by cultivating Curvularia lunata in a fermentation medium and each containing 3.81 fermentation solution about 200 mg hydrocortisone in addition to other steroids, was at a pH Value of 4 filtered. The product was then extracted three times with 2 parts by volume of methyl isobutyl ketone each time. Then the combined methyl isobutyl ketone extracts were washed with a sodium phosphate buffer at pH 8 and the layers separated. The ketone solution was then concentrated to 250 mg of product per ccm of solution. 1 part by volume of 1% disodium phosphate adjusted to pH 8.0 with sodium hydroxide was added to the solvent concentrate. The mixture was then cooled to 5 ° C., stirred slowly for 15 hours and the precipitated crystalline product was recovered by filtration. The hydrocortisone was obtained with a purity of over 80% in a yield of over 70%.

Example 3 A fermentation solution; which had been produced by cultivating Nocardia opaca in a fermentation medium and each containing 3.81 fermentation solution about 200 mg prednisolone in addition to other steroids, was filtered at a pH of 4. The product was then extracted with chloroform in a full capacity Podbielniak extractor (model 6900), the ratio of solvent to fermentation solution being 1: 2. The chloroform solution was washed with a sodium bicarbonate buffer at pH 9.0. It was then concentrated to 250 mg of product per cc of solution. 1 part by volume of 1% sodium carbonate, the pH of which had been adjusted to 8.5 by means of sodium hydroxide, was added to the solvent concentrate. The mixture was then cooled to 0 ° C. and slowly stirred for 10 hours. The crystalline product was filtered and the crystals dried in vacuo with the application of some heat. The dried product was dissolved in a mixture of 90 percent by volume of ethylene chloride and 10 percent by volume of ethanol, 50 mg of solids being used per ccm of solvent mixture. The purified product was crystallized by gradually adding an equal volume of water with constant stirring. The crystalline deposited product was filtered and dried. Example 4 A fermentation solution each containing 3.81100 mg of prednisone was produced by the action of Micromonospora chalcea. The fermentation solution was filtered through a diatomaceous earth filter aid at a p.1 value of 4.0. It was then extracted in a Podbielniak Extractor (Model 6080) operating at full capacity on ethylene chloride with a 1.4: 1 ratio of solvent to fermentation solution. The ethylene chloride extract was washed with 1 part by volume of sodium acetate-sodium hydroxide buffer, the p], value of which was set to 7.5. The washed ethylene chloride solution of the steroid was concentrated by distillation to a solution which contained 250 mg of prednisone per cc. 1 part by volume of 1% disodium phosphate adjusted to pH 7.5 with sodium hydroxide was added to the solvent concentrate. The mixture was then cooled to 10 ° C. and stirred for 20 hours. The crystalline product was filtered off and the crystals dried in vacuo with the application of some heat. The dried product was then dissolved in a mixture of methylene chloride and alcohol and precipitated by the gradual addition of water with constant stirring. EXAMPLE 5 A fermentation solution which, as described in US Pat . No. 2658023, had been prepared by cultivating Curvularia lunata in a fermentation medium and each 3.81 fermentation solution contained about 200 mg hydrocortisone in addition to other steroids, was at a pH of 4 filtered. The product was then extracted in a Podbielniak extractor (model 7500) operating at full capacity with methyl ethyl ketone, the ratio of solvent to fermentation solution being 1: 3. The methyl ethyl ketone was then washed with sodium phosphate serving as a buffer at a pH of 8. The methyl ethyl ketone solution was concentrated to 250 mg of product per ccm of solution. Then 3 parts by volume of ethylene chloride and 1/6 parts by volume - based on the amount of ethylene chloride - added methanol. A 1% solution of sodium phosphate at a pH of 8.5 was then gradually added until the product had completely crystallized out. The mixture was stirred during the addition and for a few days thereafter. It was then filtered, the product washed with a small amount of mixed solvent and dried.

Example 6 A fermentation solution containing 100 mg of prednisone per 3; 81 was prepared by the action of Micromonospora chalcea and then filtered through a diatomaceous earth filter at a pH of 4.0. Extraction was then carried out in a Podbielniak extractor (model 6900) operating at full capacity with methylene chloride, the ratio of solvent to fermentation solution being 1.4: 1. The methylene chloride extract was washed with 1 part by volume of sodium acetate-sodium hydroxide buffer, the pH of which was adjusted to 7.5. The washed methylene chloride solution of the steroid was concentrated by distillation to a solution which contained 250 mg of prednisone per cc. 1 part by volume of 1% disodium phosphate adjusted to pH 7.5 with sodium hydroxide was added to the solvent concentrate. Then the mixture was cooled to 10 ° C. and stirred for 20 hours. The crystalline product was filtered and the crystals were dried in vacuo with the application of some heat. The dried product was then dissolved in a mixture of methylene chloride and alcohol and precipitated by the gradual addition of water with constant stirring.

Claims (3)

PATENT CLAIMS: 1. A method for obtaining corticosteroid-like compounds from fermentation solutions in which they were prepared, characterized in that the extract obtainable by filtering the fermentation solution from the mycelium and extracting this solution with a water-immiscible organic solvent at a pH of about 7.5 to about 9.0 is washed with an aqueous solution of a buffer, the buffer solution is deposited, the solvent extract washed with the buffer is concentrated, optionally again in a halogen-containing solvent, in particular methylene chloride, ethylene chloride or chloroform, and a smaller amount of one Treated lower alkanol with up to 3 carbon atoms or with a small amount of a lower alkanol with up to 3 carbon atoms, an aqueous solution of a buffer at a pH of about 7.5 to 9.0 was added to the concentrate or the solution of the steroid , the mixture cooled and stirred and the crystalline line steroid compound is separated and, if necessary, recrystallized. 2. The method according to claim 1, characterized in that for recrystallization a solution of the steroid in a mixed solvent of a lower alkanol with up to 3 carbon atoms and one water-immiscible, organic halogen-containing solvents, in particular methylene chloride, ethylene chloride or Chloroform, is mixed with water. 3. The method according to claim 1, characterized in that that the buffer solution is an aqueous one adjusted to a pg value of about 8.0 to 8.5 Solution of sodium phosphate and sodium hydroxide is used. Considered References: U.S. Patent No. 2,602,769.