DE1207552B - Process for cleaning amphoteric or basic antibiotics - Google Patents

Description

Methods for Purifying Amphoteric or Basic Antibiotics The invention refers to the purification of amphoteric and basic antibiotics.

Used in the manufacture of amphoteric and basic antibiotics an aqueous culture medium in which the antibiotic in question is dissolved, a subjected to combined preparation or regeneration and cleaning process. If an extraction process is used here, the mycelium of the organism that produced the antibiotic is usually first separated from the culture medium. The antibiotic is then treated with an organic solvent That extracts as much as possible of the antibiotic in the organic pH Phase is dissolved, the volume of the organic phase being significantly less than that of the culture medium. The organic phase obtained in this way contains the amphoteric or basic antibiotic in concentrated form. It also becomes an essential Part of the impurities contained in the culture medium do not enter the organic Phase transferred so that the antibiotic is both concentrated and a first Has been subjected to purification. The antibiotic is then reassigned with an aqueous one Phase brought together at a pH value at which the antibiotic in the aqueous Phase has a much higher solubility than in the organic solvent. Since in the first extraction stage the volume of the extractant, i.e. the aqueous Phase, much lower than that of the organic phase can be kept, results an even more concentrated solution of the antibiotic. Be at the same time an amount of the impurities contained in the organic phase in this phase held back. This gives a concentrated aqueous solution of the antibiotic, of which the majority of the impurities originally present in the culture medium was separated.

The extraction process described above can be repeated for so long until a solution is obtained in which the antibiotic is in sufficient in a pure and concentrated form, the same applies to later crystallization or (other) processing crystallized out or precipitated in a pure state to become.

So-called carrier substances were used in the aforementioned extraction processes used, the transition of the antibiotic in question from the aqueous to the promote organic phase. In the production of chlorotetracycline and tetracycline were surface-active substances of the general formula R - 0n - SOZOH in the R is a hydrophobic organic radical and n is 1 or 0, or quaternary ammonium compounds used as carrier substances and the organic solvent with which the Chlorotetrazyclin or tetracycline to be extracted from the culture medium is added.

As for the use of quaternary ammonium compounds as carriers is concerned, an extraction method has been proposed in which the culture medium, that the chlorotetracycline and / or tetracycline together with the mycelium of the organism, that produced these antibiotics is not contained immediately with one with water miscible polar organic solvent is extracted after the quaternary Ammonium compound was added, both the pH of the culture medium as also regulates the concentration of the calcium and magnesium ions it contains or was monitored. The extraction had to be carried out discontinuously, as it has been found necessary in practice, the first extraction to extract the resulting filter cake a second time.

In the production of chlorotetrazycline and its derivatives as well in the production of streptomycin, sulfates or sulfuric acid esters became the above given general formula used as carrier substances. When using this However, the carrier substances is the extraction of the aqueous Culture medium not complete, since fluctuating amounts of antibiotic remain in the aqueous phase. In addition, the extraction of the Chlorotetrazyclins with an organic Solvent carried out and then the organic phase by evaporation concentrated. Thereafter, an acid salt of Chlorotetrazyclins after adding Hydrochloric acid crystallized from the concentrated organic phase. It's tough, to carry out these work stages continuously. It also results from recovery the carrier substance for the purpose of further use difficulties.

Starting from a process for cleaning amphoteric or basic Antibiotics when extracting the same from a fermentation solution in the presence one or more carriers by means of one with little or no water Immiscible organic solvent has now been found that, surprisingly good results can be obtained when the extraction is carried out with a mono- or diester orthophosphoric acid or monoester of phosphorous acid or a salt thereof Ester is carried out as a carrier substance, the organic radical being the ester is hydrophobic to such an extent that the amount of ester used in the organic Solvent is completely soluble. During the extraction, the mycelium may still be present, but also have already been removed beforehand.

For the purposes of the invention, they must be used as carrier substances organic esters meet certain conditions.

First, it is important that the ester has at least one dissociable contain (removable) hydrogen or metal atom in their molecule; to have to so be acidic esters or salts of acidic esters. In the case of the organic esters of orthophosphoric acid are therefore only the monoesters and diesters or their salts suitable for the purposes of the invention while as an ester of phosphorous acid only their monoesters or their salts come into consideration.

Second, the organic forming part of the ester molecule must be Radical have such a hydrophobic character that the hydrophilic behavior of the (phosphoric or phosphorous) acidic component of the ester molecule is sufficiently suppressed is so that the esters are soluble in the organic solvent in which the antibiotic from the aqueous phase, in which it is in solution, for the purpose of purification and later extraction is to be transferred. It has been found that this is satisfactory Degree can be achieved if the organic radical has at least 4 carbon atoms contains.

In the process according to the invention, this will still be the impurities containing antibiotic into a hardly or not at all miscible with water organic solvent transferred in the presence of a carrier substance, wherein a Feature of the invention is that as a carrier substance for the antibiotic one or more organic esters of phosphoric or phosphorous acid are used are, these esters in their molecule at least one dissociable hydrogen or Contain metal atom and the organic radical or the radicals of the ester one have sufficient hydrophobic character so that the esters in the organic mentioned Solvents are soluble.

Orthophosphoric acid diesters suitable for the purposes of the invention have the general formula in which R1 and R2 represent an aliphatic group which contains at least 4 and up to 10 carbon atoms. Examples are the dibutyl, dipentyl, dihexyl, diheptyl, dioctyl (e.g. di-2-ethylhexyl) and dinonyl orthophosphoric acid esters. However, it is not necessary that R1 and R2 be the same aliphatic groups.

If RI and R2 are a lauryl group, the ester is not complete soluble in the organic solvent and can function as a good carrier for the antibiotics don't meet. The same applies to the diphenyl and dibenzyl orthophosphoric acid esters.

In the case of the monoester of phosphoric or phosphorous acid can the organic radical R1, which forms part of the ester, is an aliphatic group with more. than 10 carbon atoms, for example a stearyl or lauryl group, or an aromatic group such as a phenyl or benzyl group.

The corresponding esters of pyrophosphoric acid can also be used be used.

Generally it can be done on hand with small samples Tests are carried out to determine whether there is a specific phosphoric or phosphorous acid ester suitable for the purposes of the invention.

The amount of the present invention used as a carrier for the antibiotics Ester depends on the particular antibiotic, its concentration and the amount the impurities present in the antibiotic. To get good results, may be the ester in an amount of about at least 1 percent by weight, based on the organic solvent used. With increasing concentration of the antibiotic, larger amounts of ester are also required. For economic For reasons, however, it is not advisable to use the ester in amounts of more than 15 to 20 Percentage by weight, based on the organic solvent, to be used.

The different amphoteric and basic antibiotics require different ones Amounts of ester in order to achieve an optimal cleaning effect. In connection with tetracycline, the ester is preferably used in an amount of 2 to 5 percent by weight, based on the organic solvent used, while an amount of 1.5 up to 2 percent by weight for polymyxin and 5 to 10 percent by weight for streptomycin is preferred.

The esters mentioned are amphoteric or basic among the various Antibiotics are not equally effective. While the di- (2-ethylhexyl) -orthophosphoric acid and the dinonyl orthophosphoric acid in both tetracycline and streptomycin as well Polymyxin is an effective carrier substance is dibutylorthophosphoric acid only suitable for tetracycline as a carrier.

In the process according to the invention, any organic solvent, that with water at all is not or only slightly miscible is used and used as a solvent for the antibiotic in question. Such Solvents are well known. There are, for example, butyl acetate, chloroform, Called amyl alcohol and methyl isobutyl ketone.

When extracting the said antibiotics using a or more of the aforementioned esters as carriers are high yields of antibiotic obtained, which is due to the fact that the carrier substance with regard to the antibiotic, the distribution ratio in favor of the organic The phase in which the carrier substance is contained is changed. As a result, it becomes a high Percentage of the antibiotic contained in the culture medium in the organic Phase transferred.

According to one embodiment of the invention, this is in an aqueous Antibiotic dissolved in the organic medium along with the impurities Solvent containing one or more of the esters mentioned, transferred. This working method can be used in connection with all of the antibiotics examined and can be carried out with particular advantage on an industrial scale.

In another embodiment of the invention, in context with the purification of tetracycline, the contaminated tetracycline from an aqueous Medium precipitated in the presence of one or more of the esters mentioned. After that the resulting precipitate is extracted with an organic solvent. With this embodiment, slightly higher yields than with the first mentioned Embodiment can be obtained, but it is only for batch work suitable and not so easy to use.

After the cleaning process according to the invention has been carried out, the antibiotics are further processed in a manner known per se.

It has already been suggested, a basic antibiotic treat the streptomycin type with dicresyl phosphate, lecithin and (caprylic) SNa5Pß02o, to make salt-like compounds from the antibiotic and the mentioned phosphates. These compounds are said to be therapeutically effective. With the well-known Process used phosphates can, however, the process according to the invention do not carry out successfully, since these phosphates are not those required according to the invention Satisfy conditions. This is referred to in the example? reproduced results of comparative experiments with a phosphoric acid ester according to the invention and such Referenced phosphoric acid esters, which meet the requirements of the invention not meet.

The following examples first illustrate the cleaning effect that can be achieved according to the invention on crude and contaminated solutions of tetracycline, polymyxin B and streptomycin. Example 1 After the mycelium had been filtered off, 300 cc of the fermentation liquid from a tetracycline fermentation were successively three times with 30 cc of a solution of 2% di- (2-ethylhexyl) orthophosphoric acid and 0.5% tris-n-butyl phosphate in Butyl acetate extracted. The pH was kept at 6.30 by adding sodium hydroxide. The aqueous residue contained less than 0.5% of the tetracycline activity contained in the starting solution. The organic phases were combined and extracted twice with 15 cc of water which had a pH of 1.20, which was adjusted by adding concentrated hydrochloric acid to the water before the extraction was carried out. The volume of the two extracts was 30 ccm, which means a concentration of ten times that of the starting liquid. The extracts contained more than 95% of the tetracycline activity based on the starting liquid, whereas the organic phase had less than 5% of the activity. Example 2 321 of a filtered culture liquid from a polymyxin B fermentation, which contained 50-106 units of polymyxin B, were mixed with 6.41 and then with 3.21 of a solution of 10 /, di- (2-ethylhexyl) orthophosphoric acid in Butyl acetate extracted at pH 7.0. The aqueous residue containing from 1.5 to 101 units of polymyxin B (3 ° / o) and was discarded. The organic phase (8.81) contained 50-106 units (100 ° / B) and was extracted first with 600 cc of water and then with 200 cc of water with a pH of 2.0, which was achieved by adding concentrated hydrochloric acid to each the water content has been ensured. There were 880 ml of a concentrated aqueous solution obtained in the 49,3 - templates 1011 units of polymyxin B (99 ° / o), and an organic residue, which from 0.1 to 106 units (less than 1 ° / o) containing . The residue was used in the next extraction of the culture liquid. Example 3 400 ml of a filtered streptomycin fermentation liquid which contained 4850 units of streptomycin per cubic centimeter was successively three times with 50 cc of a solution of 5 % di (2-ethylhexyl) orthophosphoric acid and 0.5% trisn-butyl phosphate extracted in chloroform at pH 6.30. The aqueous residue contained 167 units per cubic centimeter (3.5 %), whereas the chloroform extracts (35 cc with 21400 units per cubic centimeter, 48 cc with 15 100 units per cubic centimeter and 43 cc with 5000 units per cubic centimeter) 860 /, the Contained streptomycin activity. The organic phases were extracted twice with 20 cc of water each time at a pH of 2.0, which was adjusted by adding concentrated hydrochloric acid to each of the water components. The chloroform residue contained less than 100 units of streptomycin per cubic centimeter, whereas the aqueous phases contained 79 % of the streptomycin activity, based on the starting liquid; the degree of purity was more than 50%. Example 4 100 cc of a contaminated solution of tetracycline containing 4700 units per cubic centimeter was extracted three times with 25 cc each time of a mixture of 100 % di-n-butyl phosphate in chloroform. This contained an extract of 62 cc with 420 mg tetracycline (89%), whereas the aqueous residue contained 690 units per cubic centimeter (15%). Example s After the mycelium had been filtered off from a tetracycline fermentation culture fluid, 250 ml of the filtrate were mixed three times with 30 cc of a mixture of 90 cc of butyl acetate and 10 cc of "Lensodel A" (manufactured by Shell, England), which contains sodium dinonyl phosphate as the active component contains, extracted, the pH of the filtrate being adjusted to 5.5 by adding sodium hydroxide. The aqueous residue contained less than 50 units of tetracycline activity per cubic centimeter. Most of the tetracycline activity was found in the organic phase, from which the tetracycline can be converted into an aqueous phase in the manner described in Example 1. EXAMPLE 6 50 g of kieselguhr and 50 g of di (2-ethylhexyl) orthophosphoric acid were added to 51 of a filtered tetracycline culture liquid with vigorous stirring, while sodium hydroxide was added to adjust the pH to 8.5. The mixture was filtered and the precipitate washed out with water. The filtrate, which had 30 % of the original activity, was discarded. The precipitate was dispersed in 800 cc of butyl acetate and filtered, washed with 50 cc of butyl acetate and redispersed in 250 cc of butyl acetate, filtered and washed again with 50 cc of butyl acetate. A total of 1270 cc of butyl acetate extract with 92% of the original activity was obtained.

The tetracycline can be converted from the butyl acetate extract into an aqueous phase, as described in Example l. Example ? In the comparative experiments with the esters given in the table below, the procedure was as follows: 40 cc of streptomycin culture fluid with a streptomycin activity of 5150 units per cubic centimeter were extracted with 20 cc of chloroform which contained 5% ester. Two parallel extractions were carried out, namely one at a pH of 8 and the other at a pH of 2. The streptomycin activity of the chloroform phase was then determined and the percentage of streptomycin activity transferred to the chloroform phase was calculated. The results are shown in the table below: Streptomycin proportion of the pH activity in the chloroform phase Ester chloroform phase passed over Units of streptomycin g 2 per milliliter in ° / a Di- (2-ethylhexyl) orthophosphoric acid. . . . ... . . . . . . X 3890 37.8 X <100 <1 Tri-n-butyl phosphate ............................ X <100 <1 '@ X <l00 <1 Tricresyl phosphate .............................. @ X <100 <1 i X <100 <1 Di-p-cresyl phosphate. . . . . . . . . . . . . . . . . . . . . . . . . . . . X <100 <1 X 82 0.8

Claims (4)

Claims: 1. Process for cleaning amphoteric or basic Antibiotics when extracting the same one from fermentation broth in the presence one or more carriers by means of one with little or no water immiscible organic solvent, characterized in that the extraction with a mono- or diester of orthophosphoric acid or monoester of phosphorous Acid or a salt of these esters is carried out as a carrier substance, the organic radical of the ester is hydrophobic to such an extent that the Amount of ester is completely soluble in the organic solvent. 2. Procedure according to claim 1, characterized in that the organic radical of the ester is a is an aliphatic hydrocarbon group. 3. The method according to claim 1, characterized in that the ester has the general formula where R1 and R- 'are identical or different aliphatic groups, preferably aliphatic hydrocarbon groups, which contain at least 4 and up to 10 carbon atoms. 4. Method according to one or more of the preceding claims, characterized in that for cleaning tetracycline the contaminated antibiotic in the presence of one or more of the foregoing Claims characterized ester precipitated from an aqueous medium and the obtained Precipitate is extracted with an organic solvent. Considered Publications: German Patent Specifications No. 859198, 835 324.