DEP0010345MA - - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

Registration date: August 26, 1953 Advertised on February 2, 1956

GERMAN PATENT OFFICE

The invention relates to the production of pure oxytetracycline, known under the trademark "Terramycin", especially in the form of complex salts from its aqueous or others Solutions.

The raw oxytetracycline solution is prepared according to the method of the USA patent 2,516,080.

When obtaining oxytetracycline from the nutrient fluids, e.g. B. by the process of US Pat. No. 2,516,080, due to its solubility and that of its more or less insoluble, previously known compounds, considerable amounts are lost. The process of the present invention allows the production of pure oxytetracycline without incurring such losses. It has been found that some of the antibiotically active components from aqueous oxytetracycline raw solutions are temporarily deposited when alkali is added to a fermentation broth which contains 600 γ per ecm or more of oxytetracycline. This precipitate, however, dissolves again when the solution is made strongly alkaline, but it persists at a pH of around 8.5 to 9. However, some of the oxytetracycline remains in solution. It was also found that the precipitate consists of metal complex salts of oxytetracycline. and various divalent metals, such as calcium and

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Contains magnesium. These divalent metals are found as impurities in many crude aqueous oxytetracycline solutions present anyway, but seldom in such amounts that a satisfactory precipitate is obtained of the antibiotic for the purpose of extraction ensured wild. The addition of further quantities of selected however, divalent metal ions to these solutions cause fairly complete precipitation of the antibiotic and thus represents a valuable method for concentrating and cleaning Oxy tetracycline from its raw aqueous solutions.

It has now been found that effective 'oxy-

tetracycline in the form of a mixed complex compound with at least one of the metals barium, strontium or calcium together with at least one of the metals magnesium, zinc, beryllium, cadmium or mercury effectively from aqueous. Solutions at a _pH value of about 7 to 10 Removing

. Certain metals form precipitates, whereby most of the oxytetracycline is also precipitated from very dilute aqueous solutions, for example from fermentation solutions. These particularly effective metal pairs are calcium-magnesium, barium-magnesium and barium-zinc, whereby the second forms a corplex compound with oxytetracycline, which has the lowest solubility of the previously known. When using barium-magnesium salts, the oxytetracycline is completely removed from the nutrient solutions up to a concentration of about 60 γ per ecm a _pH value of only 5 starts and the precipitate appreciably dissolve only at a _pH value of over 10, it was found that it is more advantageous to operate at a _pH value between about 8 and. 9

The precipitated oxytetracycline salt is purer than that dissolved in the nutrient solutions. After that it is possible to further purify the precipitated oxytetracycline and bring it into a form in which it is therapeutic Use suitable. In addition, such a cleaning succeeds with only very little total loss ... effective substance. This treatment can too

- serve to remove oxytetracycline from residues or mother liquors from other manufacturing processes to win.

The quantitative ratio of the metal salts that have to be added in order to remove all of the oxytetracycline from a particular solution as far as possible depends on the oxytetracycline content and the type and percentage of the other dissolved substances. You need z. B. various acidic impurities, both organic and inorganic, which are present in fermentation solutions. can and form insoluble substances with the added metal compounds, first a certain amount of metal salt for precipitation before the oxytetracycline metal complex salt separates out completely. This is especially true for nutrient solutions that have been purified by sulfuric acid treatment, using considerable amounts of a metal salt, e.g. B. barium, are required to precipitate the acid as barium sulfate before oxytetracycline. begins to deposit. Through preliminary tests is. it is possible / to determine the exact amount of the metal salts to be used, which are required for the most complete possible precipitation. It is generally preferred to add a soluble barium salt and a soluble magnesium salt in slight excess. The ratio of the magnesium and barium salts used to one another is 1: 2 in some cases; however, it can change depending on the sulphate content and the other impurities present in the solution. A smaller amount of barium salt is required for solutions that have been purified with hydrochloric acid. The ratio of metal atoms to the Oxytetra- 'cyclin in the precipitates is not fixed but varies depending on the ratio of the O'xytetracyclinlösung added metal salts, and even after the metal ion content of this solution and the _pH value at which performed the precipitation will. Fermentation solutions often contain considerable amounts of metal ions, such as magnesium and calcium, which are involved in the formation of the oxytetrecycline metal complex salts according to the invention. The precipitate which is obtained by precipitation of an aqueous solution of pure oxytetracycline with a mixture of soluble barium and magnesium salts (1 ": 2) obtained at a _pH value of about 9, consists of 4 parts oxytetracycline, 2 parts magnesium and 1 Part of barium. However, this only applies to solutions of pure oxytetracycline.When using impure solutions, e.g. nutrient solutions, a different proportion of the metals must be maintained in order to achieve the most complete possible precipitation, and the precipitate often contains different amounts of oxytetracycline and the Metals.

The composition of the metal complex salts of oxytetracycline described can be changed by bringing the solid precipitate into contact with solutions of other metals. In this way you can z. B. change the composition of a precipitate consisting mainly of the calcium-magnesium complex salt of oxytetracycline so that it contains a considerable amount of barium after treatment with a solution of a soluble barium salt. As above. "Was emphasized, the barium-magnesium complex compound appears to be the least soluble of the complex compounds according to the invention. The barium-zinc salt has almost the same solubility, the calcium-magnesium salt is somewhat more soluble. If an oxytetracycline solution is used for the corresponding p _H value of a mixture of more than two water-soluble metal salts to, then first the least soluble metal complex salt separates.

The precipitated metal complex salt of the oxytetracycline is filtered off. The precipitation becomes ordinary washed with a small volume of water and then in an acid, preferably dilute mineral acid, especially in. dilute sulfuric acid, dissolved. When using sulfuric acid this will be insoluble filtered off barium sulfate washed with water. To get all of the oxytetracycline if possible To gain from this precipitate, it may be necessary to dilute the barium sulfate again with Treat acid. The acidic filtrate, the largest

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Contains part of the antibiotic, is then _{adjusted to a Pj 1} value of about 6 to 9, preferably about 7. In this _pH value, the anti-, biotikum separates as amphoteric compound from the solution from, but it may also substantial amounts of metal salts such. B. of calcium and magnesium.

In compliance with a slightly lower value p is _H, about 5, is the precipitation of oxytetracycline complex salt as a not quite complete. The oxytetracycline precipitate is again filtered off from the solution and washed with a small volume of water. Although this complex salt, which has a degree of purity of about 500 to 800 γ per mg, can already be used for certain therapeutic purposes, it is preferred to produce crystalline oxytetracycline or its salts for medical use from it. The precipitates are better dissolved in alkali than in acid in order to

■ 20 biotics can be obtained from it.

It was also found that the precipitates of this type contained considerable amounts of iron, chromium and May contain copper, which may have gotten into the solutions through metallic materials.

These metals, especially iron, interfere with the cleaning process. It has now been found that these metals can be obtained from the acid solutions of the precipitated . Metal complex salts of oxytetracycline by treatment with a soluble ferrocyanide such as Potassium ferrocyanide. The precipitate formed in this way is filtered off and the Excess of ferrocyanide by treating the solution with a soluble zinc, zirconium or precipitated another suitable metal salt. The end point of this setting can be with a solution of a copper salt. The clear filtrate is then mixed with a soluble alkali salt Precipitated antibiotic which is now essentially free of iron and other heavy metals, however contains certain amounts of calcium and magnesium. The product obtained in this way has a considerable lighter color and higher effectiveness than that treated without ferrocyanide, and the one with it incurred loss is very minor. Also in the manufacture of crystalline oxytetracycline or Oxytetracycline hydrochloride according to the method described exceeds the quality of the finished product by far that of the product treated without ferrocyanide. It has a pure, rather light yellow than brownish color, and its effectiveness is very high. In addition, this has no freeze-cyanide treatment obtained product has a tendency to darken considerably on standing. Furthermore, one obtains when using the ferrocyanide treatment according to the process according to the invention, a considerable amount better yield of crystalline salt.

The crystalline compounds can be prepared in various ways in known manner, e.g. B. by the method of U.S. Patent 2,516,080.

So the dried Rohoxytetracycline z. B. dissolved in methanol and concentrated enough Hydrochloric acid can be treated to dissolve the antibiotic. After treatment with decolorizing charcoal and filtration, the hydrochloride can be crystallized after adding more concentrated hydrochloric acid receive.

The present invention is illustrated in the following examples.

example 1

The following nutrient solution was prepared for oxytetracycline production:

Soybean meal 500 g

Glucose hydrate -... 500 g ⁷ ^

water-soluble extract from vinasse 25 g
Sodium chloride 250 g

Distilled water to make up to 50 1

The _pH value of the mixture was raised by the addition of sodium hydroxide to 7, then was added 50 g of calcium carbonate.

The nutrient solution was sterilized at 121 ^0, cooled, inoculated with Streptomyces rimosus and this fungus is cultured for 4 days at 28 ⁰ with constant shaking. By the addition of dilute sulfuric acid, then the solution was adjusted to a _pH value of 2.5 and subsequently nitrated. In the clear solution containing 44.9 million γ antibiotics, 120 g of barium chloride dihydrate and 48 g of magnesium chloride hexahydrate were dissolved with stirring. The resulting solution was then adjusted with sodium hydroxide to a _pH value of. 9 After standing for ten minutes, the precipitate formed was filtered off with the addition of a small amount of diatomaceous earth. It was found that the fermented nutrient solution and the washing liquids obtained contained a total of 1.92 million γ oxytetracycline. >

The precipitate was first suspended in water and then _{adjusted to a Pj 1} value of 1.2 with sulfuric acid.

The solution containing 36.8 million γ- oxytetracycline was filtered off from the insolubles. The oxytetracycline was precipitated by adjusting the solution with sodium hydroxide to a _pH value of. 7 The precipitate still contained about 34 million γ of the antibiotic and small amounts of calcium and magnesium. The filtrate of this precipitated product contained 2.3 million γ oxytetracycline.

Example 2

A Oxytetracyclinlösung prepared by the above method, the about 20 million γ of the antibiotic contained, however, had been clarified with hydrochloric acid at a _pH value of 2.5, was reacted with sodium hydroxide to a _pH value of. 9 Then 50 g of barium chloride dihydrate and 20 g of magnesium chloride hexahydrate were added, which heated in 200 ecm ;. Water had been dissolved. The mixture was ^{stirred for 1} Z ₂ hours, then 80 g of diatomaceous earth, known under the name "Supercel", were added and the precipitate was filtered off. The filtrate contained 0.8 million γ oxytetracycline. The precipitate was washed with one liter of water which dissolved 0.5 million γ . -

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Then the precipitate was triturated three times with 600 cc of water, the _pH value was each adjusted with sulfuric acid to 1.5 and filtering off the liquid from the solid. The combined filtrates contained 12,000 γ per ecm of the antibiotic. The combined filtrates was gradually added as much sodium hydroxide, until the _pH value reached 7, and precipitated Rohoxytetracyclin. This was filtered off and washed with 200 ecm of water. The filtrate and wash water contained about 2.3 million γ of the antibiotic. The precipitate was dried overnight in vacuo at 50 ^0, weighed 25 g, and the determination gave a content of 650 mg per γ of the antibiotic.

Example 3

A nutrient solution similar to that in Example 1, but containing the same amount by weight of an enzymatic casein extract instead of the vinasse, was fermented in a similar manner and with hydrochloric acid in one. p _H value of 2.5 cleaned. The clear solution was adjusted with sodium hydroxide to a _pH value of 9 and then several times with 10 volume percent n-butanol extracted; it was found that the antibiotic with metals such as calcium and magnesium was at least partially salted out. It is also possible to use the oxytetracycline as metal complex compounds with other metal salts, e.g. B. with barium-magnesium or barium-zinc salts to move out. The butanol extract was treated with 50 percent by volume 1η hydrochloric acid in water. The aqueous concentrate obtained contained 4600 γ per ecm of the antibiotic. 40 cc of this solution were contacted with dilute sodium hydroxide to a _pH value of 9.5; the resulting precipitate was centrifuged and separated from the clear liquid above. The clear liquid contained about 500 γ per cm of oxytetracycline; this means that about 90 ° / ₀ of the antibiotic had been precipitated., The precipitate was washed with water and dried. The investigation showed about 675 γ per mg. It was found that a sample of which contained about 35 ° / ₀ residue (Sulfate, were the same as quantities of calcium and magnesium).

As mentioned above, calcium and magnesium are used as complex salts of oxytetracycline the butanol layer.

Example 4

A nutrient solution according to Example 3, which contained 11.9 million γ of the antibiotic, was clarified with sulfuric acid and then treated with 175 g of barium, chloride dihydrate. Thereafter, sodium hydroxide was added sufficient to bring the p _H to value on. 9 After brief stirring, the solid which had precipitated out in the presence of a small amount of diatomaceous earth was filtered off and the precipitate was washed several times with small amounts of water. This precipitate consisted of a mixture of the barium-magnesium and calcium-magnesium complex salts of oxytetracycline. The filtrate still contained a total of 0.77 million γ oxytetracycline. The wet precipitate was suspended in the smallest possible amount of water and the _pH value _is brought to the suspension with sulfuric acid to 1.2. After the mixture was stirred well, the solids were filtered off and the residue was washed several times with water. The solution, which contained 9.8 million γ oxytetracycline, was treated slowly with stirring with sodium hydroxide solution reached until the _pH value. 7 The oxytetracycline precipitate was filtered off and washed with a small volume of water. The residue was sucked as dry as possible and then further dried in a vacuum desiccator at room temperature. The dried product weighed 12.4 g; the investigation showed 7007 per mg, a total of 8.68 million γ antibiotics. Of these, 7.7 million γ were suspended in 5 volumes of methanol and ι molar equivalent of 37% hydrochloric acid were slowly added. The solution was prepared using a small amount of diatomaceous earth, and some decolorizing carbon, known under the name of "Darco G-60", a second molar equivalent filtriertvDann the filtrate was 37 ^iJ / _o hydrochloric acid and a small amount of crystalline oxytetracycline hydrochloride was added as a vaccine. After _{stirring for 2} hours, the crystalline product was filtered off, washed with a small volume of methanol and dried. It weighed 5.10 g and the determination was 9007 per mg, which is almost the theoretical value for oxytetracycline hydrochloride.

Example 5 ■

A clarified fermentation solution according to Example 3, which contained 5.95 million of the antibiotic, was admixed with 75 g of barium chloride dihydrate with stirring. Thereafter, the solution with sodium hydroxide to a _pH value was adjusted to 9.5, added with 35 g of diatomaceous earth and filtered. The filter residue was washed with a total of 250 ecm of water. After drying under atmospheric pressure at 50 °, the substance was ground and suspended in 300 ecm of methanol. 4 ml of concentrated hydrochloric acid were added to the suspension; when diluted with ten times the volume of water it had a _pH value of about 3. The resulting suspension was filtered and the thus obtained solid substance in 150 cc of methanol was suspended again. Again, sufficient concentrated hydrochloric acid was added until the _pH value was 3, and then filtered off the insolubles. The combined methanol filtrates were concentrated to about 10 ecm and 25 ecm of a 1.5 η hydrochloric acid in methanol, which had been prepared by diluting 37% strength aqueous hydrochloric acid with methanol, was added. After inoculation with oxytetracycline hydrochloride crystals, the crystalline hydrochloride separated out after a short time and was filtered off and dried.

Example 6.

800 g of wet precipitate of the metal complex salt of oxytetracycline, which had been prepared according to the procedure of Example 1, were suspended in .'400 cc of water, and 5o ° / ₀ sulfuric acid with rapid stirring until the p _H a value of 1 , Reached 5. The oxytetracycline

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solution was obtained by filtering with a small amount of filtering agent. severed. The filter residue was washed with a small volume of water and then resuspended in a minimum volume of water. The _pH value was again adjusted to 1.5, then was filtered and washed with a small volume of water. The combined acidic filtrates had a total volume of about 1000 ecm and contained 20.5 million) / antibiotic.

The solution was added with stirring a io ° / ₀ solution of potassium ferrocyanide. After adding 11 ecm of this solution, a filtered sample showed no further precipitate when potassium ferrocyanide solution was added. A small amount of a filter agent was added to the suspension, and the precipitated. Filtered off impurities. The filter residue was washed with a small volume of water and the excess was removed from the filtrate by adding a little iron solution salt. Ferrocyanid checked. Since no

■ ao surplus was -jrhanden, the filtrate without Neueinstenüng the p _H -value was used for the next stage of the process.

The light amber-colored filtrate had a volume of 1140 ecm and contained 20 million γ oxytetra-

• 25 cyclin. Half of this filtrate was slowly adjusted by addition of a sodium hydroxide solution to a io% _pH value of 7, the precipitated oxytetracycline filtered off, gewasehen with 100 cc of water and then at 50 ⁰ dried in vacuo.

It weighed 12.5 g, the determination gave 740 γ per mg. When the acid solution was treated with decolorizing charcoal before the oxytetracycline was precipitated, the product obtained was a little lighter and more effective, but some substance was lost.

Crystalline oxytetracycline hydrochloride was obtained therefrom in the following manner: The dried Product was finely ground. 10 g of it were suspended in 40 ecm methanol and with 10 ecm one saturated solution of calcium chloride in methanol. The oxytetracycline gradually dissolved, and after treating with 0.5 g of decolorizing charcoal, the mixture was filtered. The filter residue was first with 10 ecm of a half-saturated solution of calcium chloride in methanol, then with 10 ecm Methanol, in which 1 ecm contains 37% hydrochloric acid were, and finally washed with 5 ecm of pure methanol. The methanol solution was stirred and slowly mixed with 6 ecm 37% hydrochloric acid.

Crystalline oxytetracycline hydrochloride was inoculated during the addition of the acid. When the crystallization had ended, the crystals were filtered off and washed with 10 ecm of 1,2 η-hydrochloric acid in methanol. After washing again with 10 ecm of anhydrous methanol, the product was dried for a short time at 50 °. The dried crystalline oxytetracycline hydrochloride weighed 6.64 g and the biochemical examination showed 900 γ per mg. It was bright yellow in gloss, contained only traces of metals, and was found to be useful for clinical purposes.

Contains a crude broth of oxytetracycline that enough suitable metal ions such as calcium and magnesium, needs only to the appropriate _pH value, preferably 8.5 to 9, is set to be "to skip a portion of the oxytetracycline. The amount of material which can be precipitated increases when concentrating the dilute Oxytetracyclinlösung before adjusting the p _H -value. However, this method is rarely so effective as the one in which the mixtures and proportions required divalent metal ions are additionally added.

Barium, strontium and calcium or or can be added as hydroxides, in order, if it is desired to simultaneously increase the _pH value.

Claims (1)

Patent claims: I. A process for recovering pure oxytetracycline and Oxytetracyclinkomplexsalzen from aqueous Oxytetracyclinrohlösungen, characterized in that the oxytetracycline from the crude solution, optionally substituted by an extraction at least about 600 cc per oxytetracycline γ containing solution with n-butanol at a basic _pH value and subsequent extraction of the n-butanol solution can be obtained with dilute aqueous acid, as a sparingly soluble metal complex salt with metal salts of barium, strontium and calcium, and at least one salt of magnesium, zinc, beryllium, cadmium and mercury at a _pH value of about 7 to 10 precipitates, sets the oxytetracycline free with an acid from the complex salts formed and, if necessary, subjects it to an additional purification with ferrocyanide. .2. Method according to claim 1, characterized in that that such solutions are used as raw solutions that have already been through the fermentation process Contain calcium and magnesium salts. 3. The method according to claim 1, characterized in that one uses a clarified crude solution, which was separated from the mycelium at a _pH value of 3.5 or less. Referred publications:
U.S. Patent No. 2,516,080. © 509 656/54 1.56