HU192775B - Process for separating aminocyclitols, aminocyclitolaminogly-coside antibiotics and derivatives thereof - Google Patents

Abstract

Amino-cyclitols, amino-cyclitol-amino-glucoside antibiotics and their derivs. are sepd. form aq. soln. by binding a weakly acidic cation-exchange resins in their ammonium form, followed by chromatographic elution with aq. ammonium-hydroxide soln. contg. short chain alcohols and/or ketones and prepn. of the desired derivs. as salts. - The efficiency of the process is improved by this method, the capacity of the column and the productivity are increased. - A further advantage of the method is that the ratio of the starting components of raw genta-mycin can be changed and controlled at the desired value.

Description

The present invention relates to a process for the separation of aminocyclitols, aminocyclitol aminoglycoside antibiotics and their semisynthetic derivatives by ion exchange chromatography and preparation of their aqueous solutions, the binding of the components on the weakly acidic cation exchange resin and the desired .

It is known that in the preparation of aminocyclitol aminoglycoside antibiotics and their semi-synthetic derivatives, the target product must be separated from a number of other compounds of similar structure in order to achieve the desired purity. In the course of the biosynthesis of aminocyclitol aminoglycoside antibiotics, many aminoglycoside molecules with a structure similar to the target product are formed, and further the fermentation broth always contains biosynthetic intermediates. The quantity of these substances shall be kept below the limit prescribed in the final product. In many cases, however, it is also desirable to isolate these by-products in pure form, since no such by-product has a significant therapeutic effect (e.g., gentamicin B and gentamicin Bi). Biosynthetic intermediates isolated from fermentation broths (e.g., garamine) may serve as safeners for semisynthetic derivatives.

In the production of semisynthetic derivatives of aminocyclitol aminoglycoside antibiotics (due to the presence of several same functional groups), side reactions and thus the appearance of by-products are to be expected. Therefore, the production of aminocyclitol aminoglycoside antibiotics and their semisynthetic derivatives requires a suitably efficient, commercially available separation process.

In the vast majority of known processes, separation is carried out by column chromatography. It is well known that for column chromatography, the separation capacity of the column depends on the load on the column, i.e. the amount of material applied to the column.

The greater the amount of material mixture to be separated on the column, the greater the overlap and the yield will be degraded. From an economic point of view, it is advisable to work with the best possible load. However, too low a load is unfavorable in terms of raw and auxiliary material consumption and productivity.

The methods used in practice for separating aminocyclitol aminoglycoside antibiotics can be divided into two broad groups. In adsorption chromatography methods, the allophase is most often silica gel or cellulose powder and the mobile phase is a mixed solvent mixture, most often a chloroform-methanol-ammonium hydroxide system. Mixtures containing many components can be separated efficiently by such chromatographic techniques, but the load on the column is low. According to the data published so far, the capacity of silica gel separations is 5-20 g aminoglycoside base / lit.er stationary phase. A major disadvantage in the operational implementation of the method is the resistance of the columns on the operational scale. The chloroform-methanol-ammonium hydroxide mixture used as eluent is toxic and very aggressive and requires the use of a special structural material (Teflon).

Silica gel chromatographic separations are described, inter alia, in U.S. Patent Nos. 3,828,021, 3,651,042 and 4,085,208, Belgian Patent 818,431.

Another large group of methods use ion exchange resin as a stationary phase (Pirotta et al., Presentation at the Ion Exchange Symposium IV, 1980). In the case of highly basic anion exchangers, the eluent is deionized water and the weak acid cation exchanger is aqueous ammonium hydroxide. The disadvantage of ion-exchange chromatography methods is that they are not efficient enough in the separation, because of the overlaps, the desired product can be recovered only with lower efficiency or not isolated at all. The degree of separation depends strongly on the column load. so e.g. the crude gentamicin sulfate according to Hungarian Patent Application No. 168,778 can be isolated efficiently in addition to gentamicin X, gentamicin A, gentamicin B and Bi-isomycin Bi in a more or less pure state. However, according to the process described, the capacity of the column is only 25 g of base / liter of resin, which is barely better than that of silica gel processes. Raising the load of the resin column to 50-60 g of base / liter of resin results in primarily a decrease in the separation of minor amounts, with little change in the yield of the gentamicin C complex, e.g. with a load of 52 g of base / liter of resin according to Hungarian Patent Application No. 174,695, gentamicin B and bi are no longer separated, nor can gentamicin A and gentamicin X be separated.

For the preparation of semisynthetic aminocyclitol aminoglycoside derivatives in the ion-exchange chromatography methods described above, the loading capacity of the column is highly dependent on the reactions, their selectivity, the use of protecting groups and whether the final product or intermediate is prepared. In the chromatographic steps to ensure the purity of the final product, the column capacity is in most cases low (20-30 g of base / liter of resin). Such a procedure is described e.g. U.S. Patents 3,796,698 and 3,796,699; Hungarian Patents 172,446 and 175,575.

The aim is to develop a separation method that provides good separation even under high column load.

Surprisingly, our experiments have shown that the separation efficiency can be greatly improved and the yield increased by chromatography on a weakly acidic cation exchange resin with aqueous ammonium hydroxide solution containing 2-15% lower alcohol and / or ketone, preferably methanol and / or containing ethanol and / or acetone.

Thus, the present invention relates to the separation of aminocyclitols, aminocyclitol aminoglycoside antibiotics and their semisynthetic derivatives by ion-exchange chromatography by coupling the compounds to be separated on a weakly acidic cation exchange resin in the form of ammonium, followed by 2 to 15% and / or 0.05-0.5 N ammonium hydroxide solutions containing ethanol and / or acetone.

The process of the present invention allows the separation of aminocyclitols, aminocyclitol aminoglycoside antibiotics and their semi-synthetic derivatives by chromatography on a weakly acid cation exchange resin to improve the separation efficiency and at the same time increase the capacity of the column.

A further advantage of the process according to the invention is that when chromatographing crude gentamicin sulphate, the components of the gentamicin C complex can be separated more than when chromatographed with pure aqueous ammonium hydroxide solutions, so that the components of the gentamicin C complex have an unfavorable Gentamicin C sulphate can be obtained from raw gentamycin sulfate in a better yield, with a component composition according to the Pharmacopoeia. The process of the present invention enables the preparation of gentamicin C sulfate with a desired component composition over a wide range.

The following examples illustrate the ion-exchange chromatography of aminocyclitols, aminoglycoside antibiotics, and their semisynthetic derivatives by the process of the present invention, without limiting the scope of the invention.

Example 1 g of crude gentamicin sulfate, prepared according to Hungarian Patent Application No. 174,695, containing 540 U / mg gentamicin C base, 3% garamine sulfate, 5% gentamicin B-Bi sulfate, 1% gentamicin X sulfate. It contains 2% gentamicin A sulfate and 6% sisomycin sulfate - dissolved in 200 ml deionized water. The pH of the solution was adjusted to 7.0 with 5N ammonium hydroxide solution and the solution was clarified with 25 g of activated carbon. After filtration of the charcoal, the gentamicin content of the solution was bound to Amberlite CG-50 Type I resin in 750 ml of ammonium cycle. The resin column has a diameter of 3.6 cm and a height

74.5 cm. The linear rate of binding was 0.2 m / h, corresponding to a volumetric rate of 3.4 ml / min. After binding of the gentamicin antibiotics, the column was washed with deionized water at a linear speed of 0.2 m / h for 12 hours.

Then develop the chromatogram with 0.1 N ammonium hydroxide solution containing 2% methanol. The flow rate of the eluent is also 0.2 m / h. 500 ml fractions were collected. Elution was carried out with paper and thin layer chromatography (J. Antibiotics 28, 35, 1975) and high performance liquid chromatography.

(By HPLC). After the appearance of gentamicin Cz-2b, the ammonium hydroxide concentration in the eluent was raised to 0.3 n and the methanol content increased to 5%. Desirable substances are contained in the following fractions:

fractions number 23-27 gentamicin X 29-35 gentamicin A 37-46 gentamicin B 47-55 gentamicin Bi (some

contaminated with micin B)

59-70 garamin 73-93 sisomicin 97-180 gentamicin C complex

The appropriate fractions were combined and evaporated to give the concentrated concentrates as described in 168,778

E.g. The sulfuric acid formed is precipitated with methanol as described in Example 1, step 9 of the Hungarian patent. The amount of sulfate salts obtained is as follows:

1.6 g gentamicin B sulfate

1.7 g gentamicin Bi sulfate

0.5 g gentamicin X sulfate

1.2 g gentamicin A sulfate

2.1 g of garamine sulphate

4.1 g of schizomycin sulphate

65.9 g gentamicin C sulfate (component ratio:

gentamicin Cl 28.6%. gentamicin Cz-2b 49.2% ® gentamicin Cla 22.2%) '

Example 2

97-180 obtained according to the procedure of Example 1. The distribution of the individual gentamicin 3 components is as follows:

97-118 gentamicin Cz-2b

119-139 gentamicin C2-2b and gentamicin Cl

140-162 gentamicin C2-2b, gentamicin Ci and gentamicin Cu

163-171 gentamicin Ci and gentamicin Cla

172-180 gentamicin Cia

It can be seen that by combining or omitting appropriately selected fractions, the starting component ratio of crude gentamicin sulfate can be changed and adjusted to the desired value, e.g. 140-180. fractions were combined to give gentamicin C sulfate (54.0 g) having the following component ratio:

gentamicin Ci 31.0% gentamicin C2-2b 40.9% gentamicin Cia 28.1%

Example 3

100 ml of clarified crude sisomycin concentrate - prepared according to Hungarian Patents Nos. 179,145, 179,146, and 179,147, containing 40 g of the isomycin base corresponding to isomycin sulfate and 3 g of 4-demethylisomycin base and 1.5 g of 4-epi- contains demethylisomycin base - in 400 ml of ammonium cycle

Passed through Amberlite CG-50 Type I resin at 1.5 ml / min. After curing, the column was washed with deionized water for 12 hours at the same rate.

Chromatography was started with 0.1 N ammonium hydroxide solution containing 10% methanol. Speed of chromatography

1.5 ml / min. 200 ml fractions were collected and the antibiotic content was checked by paper and thin layer chromatography. After dissolution of the demethylisomycin epimers, the isomycin is dissolved in 0.2N ammonium hydroxide solution containing 10% methanol. The appropriate fractions were combined and reacted as described in Example 1 to give 2.5 g of 4'-epidemethyl-isomycin sulfate, 4.3 g of 4'-demethyl-isomycin sulfate and 32.0 g of isomycin sulfate.

Example 4

9 g of crude 2-deoxystreptamine sulfate obtained by hydrolysis of the gentamicin C complex with hydrochloric acid are dissolved in 25 ml of deionized water. The solution was adjusted to pH 7.0 with 5N ammonium hydroxide solution, and the solution was clarified with 1.5 g of activated carbon and filtered. The clarified solution was bound to 2-deoxystreptamine on '80 ml of Amberlite CG-50 ammonium ring. The linear velocity of the bond is 0.25 m / h. After curing, the column was washed with deionized water for 10 hours at a linear rate of 0.25 m / ό.

Chromatography was carried out with 0.1 N ammonium hydroxide solution containing 5% methanol. The chromatographic speed is the same. Fractions containing 100 ml were collected. The separation of the materials was monitored by thin layer chromatography (layer: silica gel, mobile phase: chloroform: methanol: c. Ammonium hydroxide: water 1: 4; 2: 1). The fractions containing 2-deoxystreptamine were combined and evaporated. The solution was concentrated to 25 ml with 5 N sulfuric acid

It is acidified to 4.5 and the solution is clarified with 1 g of activated carbon. From the clarified solution, 2-deoxystreptamine sulfate is precipitated with methanol. 6.2 g of 2-deoxystreptamine sulfate are obtained.

Example 5

As in Example 4, 9 g of crude 2-deoxystreptamine sulfate were chromatographed on 80 ml of Amberlite CG-50 ammonium ring resin. Elution was carried out with 0.1N ammonium hydroxide solution containing 8% ethanol. The fractions containing 2-deoxystreptamine were combined and performed as described in Example 4 to give 6.0 g of 2-deoxystreptamine sulfate.

Example 6

Similarly to Example 4, 2-deoxystreptamine sulfate was prepared using 0.1 N ammonium hydroxide in 5% acetone as eluent. 5.8 g of 2-deoxystreptamine sulfate are obtained.

Example 7

As in Example 4, 2-deoxystreptamine sulfate was prepared by eluting with 0.1N ammonium hydroxide solution containing 5% methanol and 5% acetone. 6.1 g of 2-deoxystreptamine sulfate are obtained.

Example 8

8 g of the reaction product obtained from the alkylation of isomycin (containing about 25% netilmicin sulfate and 40% unreacted isomycin sulfate by thin layer chromatography) are chromatographed on 200 ml of Amberlite CG-50 Ammonium Cycle. The reaction product (8 g) was dissolved in deionized water (25 mL) and bound to the resin at a rate of 1.5 mL / min. After setting, the column was washed with deionized water for 8 hours at the same rate. Chromatography speed as well

1.5 ml / min of 0.1 N ammonium hydroxide solution containing 10% methanol was eluted. Fractions (100 ml) were collected and monitored by TLC (stationary phase: silica gel, mobile phase: methylethylketone-ammonium hydroxide-ethanol 2: 1: 1). The appropriate fractions were combined and described in Example 1. Work-up gave 2 g of crude netilmicin sulfate and 3 g of isomycin sulfate.

Example 9

4 g of crude netilmycin sulphate obtained in Example 8 are chromatographed on 180 ml of Amberlite CG-50 ammonium ring resin. Dissolve in 0.1N ammonium hydroxide solution containing 1% methanol. The fractions containing 20 netilmicin were worked up as described in Example 1 to give 3.0 g of netilmicin sulfate.

Claims (6)

PATENT CLAIMS 1. A process for separating aminocyclitols, aminocyclytolaminoglycoside antibiotics and their semisynthetic derivatives by ion exchange chromatography and preparing them from their aqueous solutions, binding the components to a weakly acidic cation exchange resin in the form of ammonium and reacting with the desired solution of ammonium hydroxide. characterized in that the chromatography is carried out with ammonium hydroxide solution containing lower alcohols and / or ketones. 2. The process according to claim 1, wherein the lower alcohol is methanol, ethanol and the ketone is acetone. ~ 3. A process according to claim 1, wherein the concentration of the lower alcohol and / or ketone is from 2 to 15% by volume, preferably from 5 to 10% by volume. 4. A process according to claim 1, wherein the ammonium hydroxide solutions are present in a concentration of 0.05-0.5 n. 5. The process of claim 1, wherein the weakly acidic cation exchange resin is Amberlite CG-50. 6. A process according to claim 1 wherein the sulfate salt of the aminocyclitols, aminocyclitol aminoglycoside antibiotics and their semi-synthetic derivatives is prepared.