DE1022230B - Process for the production of cycloserine and its water-insoluble metal salts - Google Patents

Description

Process for obtaining cycloserine and its water-insoluble ones Metal salts The invention relates to a process for the pure preparation of cycloserine and its water-insoluble metal salts from a nutrient solution in which it passes Microorganisms is formed.

Cycloserine has a wide spectrum of activity and is effective against both gram-negative and gram-positive bacteria, including mycobacteria. It is formed by the microorganism species Streptomyces orchidaceaus, is an amphoteric polypeptide that has weakly acidic and weakly basic groups, is easily soluble in water, sparingly soluble in glycols, isopropyl alcohol, methanol, ethanol and acetone and in hexane, benzene, chloroform, ether , Petroleum ether, dioxane, butanol (1), ethyl acetate and ethylene dichloride are insoluble. It melts at about 153 'with decomposition and with 1 N hydrochloric acid at 140' hydrolysed.

For the production of cycloserine, the microorganism Streptomyces orchidaceaus is grown in a nutrient solution which contains a suitable protein and carbohydrate source, the nutrient solution being supplied with air and the temperature being kept between about 20 and 30 ' .

In itself, the use of ion exchangers in antibiotics already known for the purification of the raw aqueous solutions. It is also known to be water-insoluble Prepare salts of the antibiotics, decompose them and then the antibiotic amorphous or crystallized.

It has now become a pure preparation process for cycloserine and its water-insoluble metal salts from impure aqueous solutions obtained by filtration of the fermentation liquid containing the cycloserine, found that conveniently economical and effective the production of cycloserine in good yields in amorphous or crystalline form.

The new process for the pure preparation of cycloserine and its water-insoluble metal salts consists in the fact that one cycloserine from either his impure aqueous solution obtained by filtering the fermentation liquid containing the cycloserine was obtained, or from the known per se after the adsorption of the raw cycloserine Way to a basic anion exchange resin and washing out with a suitable one aqueous salt or acid solution obtained by adding a water-soluble, inorganic metal salt which, together with cycloserine, is a metal salt that is insoluble in water forms, as insoluble metal salt falls, this from the supernatant liquid separates, the water-insoluble cycloserine salt is slurried with water, the metal ion of the insoluble cycloserine salt falls as a water-insoluble salt, which after separation of the inorganic precipitate remaining purified aqueous cycloserine solution either freeze-dried in vacuo or the cycloserine with an excess of water-soluble, organic precipitant precipitates in crystalline form. Very special has been in the present Case the production of the silver salt as extremely practical and economical proved, as will be explained in more detail below.

In practicing the invention, the impure aqueous solution becomes of cycloserine, e.g. B. the fermentation liquid in which the cycloserine was formed is, first filtered or otherwise clarified; preferably one Solution first filtered and then decolorized with active col-de, or else adsorbed the cycloserine from the aqueous solution to a strongly basic anion exchange resin in the hydroxide form. Suitable strongly basic anion exchange resins used will are known in large numbers, e.g. B. under the Trade names bel #: annten "Dowex" - and "Arnberlite" -. '# Ustanscherharze.

After the cycloserine has been adsorbed by the strongly basic anion exchange resin it is washed out of the resin with an aqueous solution of a salt or an acid, which have an anion and are able to displace the Cvcloserin from the resin. Suitable Eluting agents are water-soluble sulfates, chlorides, acetates, formates, phosphates, Citrates, oxalates, sulfuric acid, hydrochloric acid, acetic acid, formic acid, phosphoric acid, Citric acid and oxalic acid. The process of adsorbing cycloserine to the Anion exchange resin is known per se; a suitable method is to to pass the aqueous solution of cycloserine through a column containing the resin, until most of the cycloserine is adsorbed by the resin. This is made possible by the The cycloserine appears at the bottom of the column, which is then larger Quantities found in the effluent from the column. Washing out the cycloserine Your resin with the eluent is continued until the cycloserine is complete is washed out, what by its reduced amounts and finally by its complete absence in the process is indicated.

Then, if necessary, the Eltiat is decolorized with activated charcoal and the cycloserine is precipitated from the aqueous solution as an insoluble metal salt. So a water-soluble metal salt is added to the aqueous solution of cycloserine, which forms an insoluble salt with cycloserine and thus precipitates. Before the metal salt is added to the cycloserine solution, its pH is adjusted to about 5.5 to 9.0 and, after the metal salt has been added, it is brought back to a p.1 value in this range. A p11 value of 6.5 is preferred. At a pH value below 5.5 very little insoluble metal salt of cycloserine is formed, and at a pn value above 9.0 excessive amounts of metal hydroxide are formed when the water-soluble metal salt is added to the aqueous solution of cycloserine. Forming metals, the insoluble salts with cycloserine, silver, are 'copper, mercury, zinc, lead, aluminum and cobalt. Suitable water-soluble salts are accordingly silver nitrate, cupric chloride, copper sulfate, mercuric acetate, zinc nitrate, zinc sulfate, lead acetate, lead nitrate, aluminum chloride, cobalt nitrate, cobalt chloride and cobalt sulfate. The cycloserine is preferably precipitated with silver nitrate or a water-soluble copper salt.

One can of course choose the insoluble metal salts of cycloserine precipitate each of its aqueous solutions. It was found that the cycloserine as insoluble metal salt from a clarified nutrient solution in which it is formed, can be precipitated without the need to attach the cycloserine to an anion exchange resin to adsorb and wash it out of this. According to this procedure, the nutrient solution, in which the cycloserine was formed, filtered and clarified with activated charcoal, whereby a partially purified aqueous solution of cycloserine is obtained from which it can be precipitated as an insoluble metal salt.

After the precipitation of the cycloserine and the separation of the insoluble one Metal salt from the solution above becomes the insoluble metal salt in water slurried and mixed with a precipitant for the metal ion, whereby the Cycloserine made free goes into solution in water. If z. B. the cycloserine as If the silver salt has been precipitated, the silver salt of cycloserine can be found in water Slurry and add hydrochloric acid. The silver precipitates as super chloride, and that Cycloserine goes into solution as a free acid. If enough hydrochloric acid is used, the cycloserine can be converted into the hydrochloride, while the silver as Silver chloride is precipitated. Hydrogen sulfide can be used in the same way the silver precipitates as silver sulfide and the cycloserine in the MTasser goes into solution. But you can also use a mixture of hydrochloric acid and hydrogen sulphide use to completely remove the silver. If the insoluble caper of the Cycloserine is suspended in water and hydrogen sulfide is added, falls the copper turns out as copper sulfide, and the cycloserine goes into solution in the water. In Similarly, mercury can be precipitated as a sulfide; the others too Metals can be removed in a similar way.

After removing the metal ion that leads to the formation of the -insoluble Metal salt of cycloserine was used, the aqueous solution of cycloserine is optionally decolorized and then subjected to gel drying in vacuo, wherein it is obtained in amorphous form of very high purity. For most uses of cycloserine, the amorphous form is suitable.

The cycloserine can, however, also be obtained in crystalline form from the aqueous solution. It is obtained in crystallized form to its purified aqueous solution by adding a water-miscible solvent in which it is only slightly or not at all soluble. Suitable water-miscible solvents which can be used to crystallize cycloserine from aqueous solution are acetone, methanol and dioxane. In particular, the silver salt of cycloserine is prepared by the process of the invention by adding silver nitrate either to an impure aqueous cycloserine solution clarified by the addition of clarifying agents and / or filtration or to that after adsorption of the crude cycloserine in a manner known per se on a basic one Anion exchange resin and washing with a suitable aqueous salt or seed solution are obtained at a pff value between 5.5 and 9.0 and the precipitated silver cycloserine is filtered off, washed with acetone and dried in vacuo.

Usually Cvcloserin is not very stable if it is not in pure form is in an anhydrous form. As a silver salt, however, it is well permanent, unlimited Can be stored for a long time and can be converted into pure crystalline cycloserine as required will.

The following examples illustrate the invention. Example 1 of -1.8 units) e1,29COylcINosäelirrisntojefflcösumngwumridteeninfielmtriCe.rethaulntd passed through a column which contained 101 strongly basic anion exchange resin in the OH form and is known under the trade name "Dowex-2", at a flow rate of 1200 ccm per minute . The exchange resin was then washed with water and the cycloserine from the resin with 0.2 N sulfuric acid at a rate of 1200 cc per minute eluted, the eluate was collected in fractions eighteen to 2. 1 The pI value of the eluate fraction was then adjusted to a pH of about 7.0 , this was treated with 1 percent by weight of decolorizing charcoal and the mixture was filtered. Then 0.5 mg of silver nitrate per unit of active cycloserine were added to the decolorized eluate and the pI value was adjusted to 6.5 with sodium hydroxide. The crystallized silver salt of cycloserine separated out from the eluate; it was filtered off, washed with acetone and dried in vacuo at a temperature of 65.5 '. The following table gives the results for nine of the eluate fractions mentioned above. Eluate Ehlat units decolorized filtrate starch units added 9 silver salt units Volume volume AgNo, No. per Cem per Ccm per cein per Ccm 9 9 per cem 10 2260 15 2400 12 22.6 12.8 1.92 11 2280 27-21 24.0 2-1.4 1.23 12 2180 34 21 340 31 35.0 33.9 1.80 13 2020 47 2120 - 43.4 29.7 2.12 14 2220 55 2400 - 53.3 38.2 2.5 15 2160 32 2465 28 36.7 29, -1 2.4 16 2130 25 2370 20 28.8 24.4 2.0 17 2050 17 2220 13 22.6 21.8 1.18 18 1960 11 2100 8 12.7 10.4 0 10 g of crystallized silver salt from eluate 14 were mixed with 60 cc of water and then with 89 cc of 0.5165 N hydrochloric acid. The silver chloride was precipitated from the solution and separated off by filtration. The remaining solution was subjected to freeze-drying in vacuo, giving 4.4 g of solid amorphous cycloserine with 5.15 units per mg.

b) 1 g of the amorphous cycloserine obtained according to the information under a) was dissolved in 7.5 cc of water and 5 cc of acetone were added to this solution. Crystalline Cvcloserin precipitated out of the solution. A further 5 cc of acetone was then added to bring about complete crystallization. The crystals were filtered off and washed with acetone to obtain 0.6177 g of air-dry crystals of 4.32 units per mg and containing 140% moisture. After removing the moisture by drying, the crystallized Cvcloserin contained 5.02 units per mg. Example 2 102 cc. Nutrient solution containing 5 units per cc of cycloserine was stirred with 4% activated charcoal and then filtered. By adding 170 mg of silver nitrate, the silver salt of cycloserine was precipitated in the form of small white crystals. The crystals were filtered off and dried, whereby 160 mg of the crystallized silver salt of cycloserine were obtained. This was then suspended in 10 cc of water and treated with 1.47 cc of 0.505 N hydrochloric acid. The superchloride precipitated out of the solution and was centrifuged off. The remaining solution was treated with activated charcoal as in Example 1 and then subjected to freeze-drying in vacuo, whereby the Cvcloserin hydrochloride with 4.85 units per mg was obtained.

EXAMPLE 3 100 cc of aqueous cycloserine solution containing 88 units of cycloserine per cc was admixed with 4.89 g of copper sulfate, and the pH of the mixture was adjusted to 6.7 with sodium hydroxide. The precipitated copper salt of cycloserine was filtered off, washed with acetone and dried, whereby 3.92 g of the cycloserine copper of 1.88 units per mg were obtained. 1 g of the copper salt was slurried in 20 cc of water and hydrogen sulfide was bubbled through the slurry for 5 minutes. The insoluble copper sulfide precipitated out of the solution and was filtered off. The filtrate was subjected to freeze-drying in vacuo, whereby 0.457 g of cycloserine having 3.3 units per mg was obtained.

Claims (3)

PATF, NTCLAIMS-. 1. A process for the production of cycloserine and its water-insoluble metal salts, characterized in that cycloserine is either obtained from its impure aqueous solution obtained by filtering the fermentation liquid containing the cycloserine, or from the fermentation liquor containing the cycloserine after adsorption in a manner known per se a basic anion exchange resin and washing with a suitable aqueous salt or acid solution obtained by adding a water-soluble, inorganic metal salt, which forms a metal salt insoluble in water with cycloserine, as an insoluble metal salt with a pr value of 5, 5 to 9 falls, this is separated from the supernatant liquid, the water-insoluble cycloserine salt is slurried with water, the metal ion of the insoluble Cvcloserine salt falls as a water-insoluble salt, the purified aqueous cycloserine solution remaining after the separation of the inorganic precipitate is either freeze-dried in vacuo or there s Cycloserine precipitates in crystalline form with an excess of water-soluble, organic precipitant. 2. The method according to claim 1, characterized in that the cycloserine is precipitated with silver nitrate or a water-soluble copper salt. 3. The method according to claim 1 and 2, characterized in that the cycloserine or copper is broken down with hydrochloric acid or hydrogen sulfide or mixtures thereof. Contemplated publications: USA. Patents No. 2 541420, 2479832, 2667441;. French Patent Nos. 952 376, 986 277, 1017 468; British Patent No. 653 088.