DE1468178A1 - Process for the isolation of the antibiotic fusidic acid from fermentation broths by extraction - Google Patents

Description

DB. ING.F.WTJESTHOFF 8 MUNICH θ

ΒΙΡΙ »ING. G. PUI <S schwbiobkstbassb 2

DR. E. ν. PECHMANN m. * Ro »as Οβ 51 FKOTBCTFATBJfT MßlfClIKlf

1A-26 253 1468178

B JB ₁ aohreibung to the patent application

L0VEMS IEMISKB FABHIK, VED. A. KOHGSTiSD, 11, Ballerup Byvej, Ballerup, Denmark

concerning

Process for the isolation of the antibiotic puaidic acid also used . a _URC h extraction.

The invention relates to a method aur isolation of the new antibiotic pusidina acid, the also called antibiotic ZH-6, ouch, the culture fluids containing aea in good yield and in a degree of purity which is satisfactory for therapeutic purposes.

Daa antibiotic is produced by placing a microorganism in a controlled environment grows in a suitable culture medium. The well-known Pilse Oephaloaporium lamellaeoola and Tuaidium ooccineum Fuck K. Tubaki produce this

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Substance under suitable cooling conditions in, for industrial production, quantities satisfactory. The fermentation process itself falls but not within the scope of the invention.

The antibiotic fusidic acid is a weak one Acids, salts with various inorganic and organic bases and also solvates, these are molecules » compounds with certain solvents.

Me electrometric titration of the antibiotic in 80% aqueous ethanol provides one pK-rfert, which has a pK -Y / ert of about 5 »3 in Water corresponds. The titration also shows an equivalent valence weight of the substance of 516.

Studies of the chemical structure and analytical data for this substance suggest based on the formula CU-jH ^ gOg. The connection seems a Cyelopentenopolyhydrophenanthrene Hingsystein, that by two hydroxyl groups, one aoetoxy group and four methyl groups is substituted and in the 17-position via a double bond with the c < Carbon atom linked by 5-methyl-4,5-heptenic acid is to be included.

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U68178 The structure of the antibiotic fusidic acid

has not yet been finally clarified, however • vird cuijenoiomen that the connection below Formula (I) in which the wavy lines show that the configuration at the point in question Carbon atom is un ^ ev / iss and the broken lines that the relevant nest is in <X position »

J-CO ₂ H

CH ₂ -OH ₂ -GH β C:

CH, CH,

0OC.CH,

(D

The antibiotic fusidic acid is sparingly soluble in water and hexane and soluble in ethanol, acetone, methyl butyl ketone, amyl acetate, chloroform and
similar solvents.

As mentioned, the antibiotic can have molecular compounds, so-called solvates, with some organic solvents, especially benzene and methanol,

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form and theee solvates are well crystallizing Links. Therefore one can see the formation of solvates during the extraction and purification of the Operating the connection · The crystalline alkali salts dee The antibiotic fusidic acid are easily soluble in tfaeti he and can advantageously be used as ingredients

en
pharmaceutical preparation / can be used. The salts of the antibiotic fusidic acid with organic amines and especially those salts whose amine component does not contain any hydrophilic groups, for example hydroxyl groups, are sparingly soluble or insoluble in water, but easily soluble in certain organic solvents. Because of this, the antibiotic fuuidic acid can be made from a small amount of the antibiotic

containing aqueous phase in the form of one of the above mentioned amine salts by extraction of the aqueous

Phase can be isolated with an organic solvent suitable for the salt in question.

In bacteriological tests, the antibiotic fusidic acid was found to be effective against various pathogenic microorganisms, in particular against otaphylococcue aureus _t geisserla gonorrhoea » » including penioillin-resistant strains thereof,

in
Meisseria mengitidea » Myoobacterium tuberoulosle ^ and the streptomycin-, ißonikotinsäurehydracid- and p-aiüino-salicylic acid-resistant strains of these organisms and against Gorynebaoterium diphteriae.

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1468178 The isolation method according to the invention is based

on the acidic properties of the antibiotic and its «solubility properties and described above those of other salts. By adding an o amine in free form odeipin the form of one of its salts or an acid in amounts such that substantial amounts of the antibiotic present in the culture medium Pusidic acid reacts with these, turns the antibiotic into one, in organic solvents soluble, brought into form · The antibiotic is then placed in the wean with a vein that is immiscible with water immiscible, organic solvents, e.g. a ester, a ketone or a chlorinated hydrocarbon, extracted and then isolated from the extractant, optionally in the form of one of its üalts with a pharmaceutically acceptable base or in the form of a solvate of the compound.

During extraction on an industrial scale, methyliaobutyl ketone, amylolite and butyl acetate were found particularly suitable for extraction.

Preferably, the extraction is carried out after removing the solids from the culture medium, e.g. Microorganisms or the nutrient components used, by filtration or centrifugation.

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If the antibiotic has been extracted from the cooling medium in the form of the free acid, then the organic phase can be concentrated to a small volume, from which the antibiotic is excreted when cooling or by adding a component that reduces the solubility of the antibiotic. The extract can also be evaporated to dryness and the residue can then be recrystallized from a suitable solvent or solvent mixture, preferably from a solvent with which the antibiotic forms a well-crystallizing solvate, for example benzene or methanol.

Furthermore, by extracting the organic phase with an aqueous solution or suspension of a, containing the desired cation, alkalis effective otoffes the antibiotic can be isolated in the form of one of its water-soluble iialze.

If the antibiotic is extracted after acidification of the culture medium, then preferably acidification to a pH./ert of 3 to 6, for example from 5 to 6, since this creates favorable conditions for a successful extraction of the fermentation liquid, after that? Removal of the mycelium.

If the antibiotic is extracted from the culture medium in the form of one of its amine salts, then the

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pH preferably drops to 7.0 or above.

For this type of execution dee according to the invention Process one prefers strongly bulky amines, the a higher, non-substituted, hydrocarbon group contained in the molecule and in particular quaternary amines, e.g. acetyl-pyridinium chloride.

Also the one containing the unin salt of the ioitibiotic, In the organic phase, the compound can be more advantageous in the form of one of its water-soluble salts iaoliert ./erden by mixing the organic phase with an aqueous alkaline solution containing the desired cation Solution extracted.

The antibiotic can also be isolated according to other known methods, e.g. by double conversion of the amine salts8 in the extraction medium with another, in this soluble or sparingly soluble salt, which contains a cation with which the antibiotic becomes a ^ in This extra-extraction medium forms poorly soluble, sale-resistant.

Furthermore, the extraction medium containing the auine salt of the antibiotic can be treated with an aqueous solution of a strong acid in order to remove the amine component from the extraction medium, whereby an organic phase with the antibiotic in the form of the free acid can be obtained from this acid

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the antibiotic, as above »can be isolated.

In a suitable embodiment according to the invention, the extraction can be carried out as a multistage process be carried out by first mixing the antibiotic with an organic solvent in a countercurrent and then the organic solvent with an aqueous solution or suspension of an alkaline active substance extracted "whereupon the resulting aqueous" solution is acidified and that Antibiotic with a, with barrels not mismatchable, Solvent extracted

For the countercurrent extraction, which can expediently be carried out in a Podbielniak extraction device, butyl acetate or methyl isobutyl ketone, for example, can be used as extraction agents. From the organic obtained in this way Lüsung the antibiotic with an aqueous or an aqueous suspension latriuahydroxydlösung ron Xalsiumhydroxyd can be shaken out, and NaOH, the separation and acidification of the aqueous phase finally extracted with methyl isobutyl ketone this example, or benzene.

The inventive method is through the following examples are explained in more detail

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Example 1i

Sine PermentEfciona liquid with a Activity corresponding to a content of 70 mg of the antibiotic tuaidic acid per liter was taken from the myoel freed by filtration, the pH in 700 l of the Piltrata medium · a 25 # strength HgSO ^ solution adjusted to 3.3 and in a Podbielniak Extraktionsvorrlchtung in countercurrent with 230 1 Butyl acetate extracted. The ao obtained butyl acetate phase was mixed with 77 l of water to which a 10 # NaOH solution was added to a pH of 10.0 was »extracted and the phases separated. The pH-tfert the aqueous phase was now adjusted to 3.2 by adding a 25% HgSO, solution and the Solution extracted with 40 1 methyliaobutyl ketone. The phases were separated, the organic phase treated with 40 g of activated charcoal and in vacuo at a Boiling temperature of 25 ° C evaporated to dryness, The The evaporation residue was dissolved in 500 ml of benzene and the solution was kept in the Üiaachrank over Haoht Here, the benzene solvate of the antibiotic pusidina acid crystallized filtered off and recrystallized from benzene ^ whereby one 12.0 g of the pure substance, P 189.0 to 189.5 ° ^, received.

By drying the dolvata at 50 ° C / 0.01 mm Hg

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bia sur Gewiohtskonatan «became a benzene-free product, P 191 to 192 ⁰ O, ΠΚ7Ζ2 « 9 ° (c »1 Ji,

D chloroform), obtained

example 2 \

15 OOCjtL fermentation broth with a content of 3.75 kg of the antibiotic fusidic acid was adjusted to a pH of 6.0 and in a PodbielniaJc extraction device extracted with 3000 l of methyl isobutyl ketone. The extract contained 3.4 kg of the antibiotic fusidic acid. Eb was made with 600 liters of aqueous sodium hydroxide with a pH value extracted from 11.5. Immediately after the extraction, the aqueous phase was brought to a pH of 3.3 and Concentrated to a volume of 150 l in vacuo. These contained 3.25 kg of the antibiotic pusidic acid.

40 l of benzene were added and, while stirring, the pH of the aqueous phase was adjusted to 5.0 by adding hydrochloric acid. Stirring was continued for 4 hours and then the precipitate consisting of the benzene solvate of the antibiotic fuaidic acid was filtered off, washed with ./asser and benzene and dried. The crude product was 2.8 kg. Us was dissolved in 12 l of methylene chloride, filtered and evaporated to dryness in vacuo. The residue was dissolved in 3.2 l v / poor methanol

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dissolved and ^{cooled to 0} ° C., a methanol solvate crystallizing out, which was filtered off, washed 3 times with 300 ml of ice-cold methanol each time and ge. was dried to give 2.48 kg of colorless methanol solvate.

This product was suspended in a mixture of 750 ml of methanol and 1750 ml of acetone, a few drops of phenolphthalein were added and then, while stirring, 33% aqueous sodium hydroxide until Appearance of a pale reddish color added · The solution was replaced by Dicalite, a diatomaceous earth existing filter medium, filtered, mixed with 5 1 methyl butyl ketone and reduced in vacuo to approximately 3 1 concentrated * Crystallized during the Sinengen · the sodium salt · This was filtered off, washed with acetone and then with ether and dried » 2.39 kg of the pure sodium salt of the antibiotic fusidic acid were obtained.

Example 3t

10 1 clarified fermentation broth (with a Content of about 700 mg of the antibiotic fueidic acid 1) each was adjusted to a pH of 7-0.

I.

90 mg of acetylpyridinium chloride were added

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joined and dl · ventilation 2 aal old j · 500 al Methylobutyl ketone extracted.

laoh dtr extraction were in the aqueous * » Phase less than 35 ag / 1 Antibiotilnui raeldlneättre found.

SI · Tereinlgten Methylobutylketon-lxt »» kt · became old Waeeer gtwaeohen and then «la Ubereehul a saturated aqueous Kaliuaaietmt-Leenag and stirring added.

I "oh one hour at skin temperature, the raw Kalluaeale de Antibiotilcuaa fusltlaetmi ¹ " old Aieton was blown and dried.

Sa · received rope was for dl · Hereiellvag SalsMi dee Antibiotikuae Fu «ldlaeäure la pheraa- ■ eutlMA pure QtulitKt well suited.

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Claims (1)

-13- 1A-26 253 Procedure for isolating the antibiotic fusidic acid on a culture medium containing it, ₉ by using an amine or one of its acids or an acid in close proximity so that substantial amounts of the antibiotic fusidic acid present in the culture are in organic solvents and the antibiotics "" with a water-immiscible or largely non-miscible organic solvent, in particular a "set" of a ketone or a "chlorinated carbon dioxide, whereupon" the antibiotic is isolated from the extraction agent, possibly in the form of one of his Salse with a phareaneutiaoh sulässlge Baee or in the form of one of his Solrates 2. Method according to Claim 1, because there is no problem with the culture medium, yorsugswelse acidified to a pH value τοη 3 - 6, + and the antibiotics «« it a «immiscible with water or world-wide non-miscible organic solvents, In particular an Xster, a ketone or a « coal Chloxraraastoff , extracted, whereupon the antibiotic is isolated from the extract, optionally in the form of one of its salts or solvates with an organic solvent BATH -H- U * 6 »3 3 · The method according to claim 1, characterized in that the culture medium is on a pH of 7.0 or above one divides strong amine in such amounts that one A substantial amount of the amine salts * of the antibiotic is produced and the salt with one with water does not Miscible or largely immiscible organic solvents, especially a Bster, a ketone or a chlorinated hydrocarbon »extracted, whereupon the consensus antibiotic from the Extract isolated, possibly in the form of one of my salts with pharmaceutically acceptable bases or in the form of one of its solvates with an organic solvent 4. The method according to claims 1 to 3 »thereby characterized in that the extraction is carried out as a 4-leg extraction. 5. The method according to claims 1 to 4, characterized in that one with «barrels immiscible phase containing the antibiotic pusidic acid extracted with aqueous alkali and from the resulting aqueous solution of a salt of the antibiotic isolates the salt or the Antibiotic precipitates by acidifying the solution. -15- 1A-26 253 6. The method of claim 5 NaOH "dadttreh g k β β α η -seiohnet, ^DAFL> ba an aqueous solution • inea Salm dta antibiotic fusldlnsäure susetst Bensol In solohsB amounts of DAB which is converted in the solution Torhandent antibiotic in its Beniol-SolTat" Before the acid is acidified, the benmol solute of the antibiotic precipitates, this is separated off and, if necessary, converted into the antibiotic "Fusidinsäar" or a salt thereof. 7 · Procedure according to claims 5 or 6 »daduroh It is known that the isolated crude antibiotic yusidic acid is obtained from a solvent, which for the formation of solrates is old Antibiotic capable is recrystallized, whereupon the extracted solrat de antibiotic is isolated and, if necessary, into the antibiotic which transfers a saIs of the same. 909807/1054