JP2728482B2 - Purification of macrolide antibiotics - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for purifying a macrolide antibiotic useful as an antibacterial agent.

2. Description of the Related Art Conventionally, as a method for purifying a macrolide antibiotic, an extraction method utilizing an amphipathic property that the compound is easily soluble in an organic solvent on the alkali side and easily soluble in water on the acidic side,
There are known an ion exchange method utilizing the basicity of amino sugars, and a method using a reversed-phase adsorbent utilizing the hydrophobicity of a macrolide skeleton.

In addition, when obtaining macrolide antibiotic products in the form of a free base, since they are generally poorly soluble in water, methylene chloride, methanol, acetone, Purification in the form of a solution in an organic solvent such as ethyl acetate is performed, and a method is known in which the solvent is distilled off or recrystallized to obtain crystals or powder of a free base, or water is added to reduce the solubility and precipitate. (For example, "Antibiotics" edited by Yusuke Sumiki, University of Tokyo Press 366
593, 926, and 1056 (1961)].

Problems to be Solved by the Invention However, these methods are disadvantageous in that the organic solvent used in the product remains and is not always preferable for use as a drug or a raw material of a drug, and a macrolide antibiotic containing no organic solvent A method for purifying the free base is desired.

Means for Solving the Problems According to the present invention, the macrolide antibiotic free base is easily dissolved in water at a low temperature, and the solubility in water is significantly affected by the temperature. A purification method for obtaining a free base free of an organic solvent without using an organic solvent is provided.

That is, the present invention relates to a method for purifying a macrolide antibiotic, wherein the compound is precipitated by raising the temperature of an aqueous solution of a free base of the macrolide antibiotic.

In carrying out the method of the present invention, the aqueous solution of the macrolide antibiotic free base is kept at 10 ° C or lower, preferably at 0 to 5 ° C.
When the temperature is increased from an arbitrary temperature, precipitation is usually observed at 10 to 50 ° C., depending on the rate of temperature increase.

Here, the macrolide antibiotics are basic macrolides to which amino sugars are bound, such as erythromycin and oleandomycin.
in), Carbomycin, Josamycin, Spiramycin, Tylosin, and Leucomyci
n) and the like.

An aqueous solution of a free base of a macrolide antibiotic used for purification may be any aqueous solution containing the antibiotic. For example, any of the reaction solution, fermentation solution, crudely purified solution, crudely purified solid, etc. containing the antibiotic can be used as it is or by removing the organic solvent as necessary, neutralizing, concentrating, etc., to obtain one of the free base of the antibiotic. It may be an aqueous solution containing up to 500 g / l.

In order to obtain the aqueous solution from various raw materials, for example, a fermentation solution containing the compound, which is roughly purified by the above-described extraction method, ion exchange method or reverse-phase adsorption, is prepared as follows, for example. .

When the crude fermentation broth is an aqueous solution containing an acid addition salt such as a mineral acid (eg, phosphoric acid, hydrochloric acid, sulfuric acid, etc.) salt or an organic acid (eg, succinic acid, lactobionic acid, propionic acid, etc.) salt After cooling the solution to 10 ° C. or lower, more preferably 0 to 5 ° C., sodium hydroxide,
It is prepared by neutralizing with an aqueous alkali solution such as potassium hydroxide or ammonia to generate a free base of the macroid.

When the crudely purified product is an organic solvent solution containing a free base, if it is a solvent that does not mix with water, add water and then add the same mineral acid or organic acid as described above to form an acid. An aqueous solution containing the compound transferred into the aqueous layer as an addition salt is obtained, and is prepared in the same manner as described above. If the organic solvent is a solvent that can be mixed with water, water is added to form an acid addition salt in the same manner, and then the organic solvent is distilled off by concentration.

Next, when the crudely purified product is a solid such as a crystal, a precipitate, or a powder, water is added, and if it is a free base, the obtained aqueous solution is prepared as it is if it is an acid addition salt, and the resulting aqueous solution is prepared as described above. I do.

Table 1 shows the results of studies on the solubility of free bases of typical macrolide antibiotics in water. It can be seen that the solubility of any of the macrolides increases as the temperature decreases, and that the solubility significantly increases particularly at 10 ° C. or lower.

By gradually increasing the temperature of the aqueous solution of the macrolide, the macrolide is precipitated as crystals or precipitates. If the heating rate is too fast, the precipitated crystals or precipitated particles become fine, so it is preferable that the heating rate be slow. Usually, the temperature is raised to about 10 ° C at a rate of 0.5 to 3 ° C / hour, and then 5 to 10 ° C. Temperature / hour. Crystals can also be enlarged by aging at the crystallization point (when the aqueous solution becomes cloudy) for 30 minutes to several hours.

The precipitated macrolide can be obtained as crystals or powder by ordinary operations such as centrifugation, filtration, washing, and drying.

Hereinafter, embodiments of the present invention will be described with reference to Examples and Comparative Examples.

Example 1, 100 l of spiramycin fermentation broth (containing 500 g of spiramycin) was centrifuged (5000 rpm) for 20 minutes, and then the supernatant liquid 85
l was collected. The supernatant is passed through 20 l of a cation exchange resin Amberlite 200 (NH ₄ ⁺ type) (manufactured by Rohm and Haas) at 20 l / h to adsorb spiramycin, and then with 1N aqueous ammonia. Spiramycin eluted.
This solution was adjusted to pH 7 with 2N sulfuric acid to obtain 40 l of a spiramycin sulfate solution (containing 400 g of spiramycin).

20 l of the above spiramycin sulfate solution was concentrated under reduced pressure to 0.5 l, cooled to 5 ° C, and pH was adjusted with 15N aqueous ammonia.
Adjusted 9.5. Thereafter, the temperature was gradually increased, and cloudiness started to be observed at about 10 ° C. After aging at this temperature for about 1 hour, the temperature was further gradually raised to 30 ° C. The obtained slurry was separated by a basket separator, and washed with 200 ml of water to remove contaminating ammonium sulfate. The solid matter remaining in the separator was vacuum-dried at 50 ° C. overnight to obtain 180 g of a dried matter.

Comparative Example 1. 20 l of the aqueous solution of spiramycin sulfate obtained in Example 1
After adding methylene chloride 1, the pH was adjusted to 9.5 with 10 N sodium hydroxide, and spiramycin was transferred to the methylene chloride layer. The solution was concentrated under reduced pressure to dryness,
After pulverization, vacuum drying was performed at 70 ° C. overnight to obtain 190 g of a dried product.

When the amount of the residual organic solvent was measured by gas chromatography for the dried spiramycin obtained from both Example 1 and Comparative Example 1, the dried product of Example 1 showed that
Although no organic solvent was detected, 100 ppm of methylene chloride was detected from the dried product of Comparative Example 1.

Example 2. Erythromycin fermentation broth 100 l (erythromycin 100 g
Was added, and the pH was adjusted to 10.5 with 10 N sodium hydroxide to transfer erythromycin into the methyl isobutyl ketone layer. After adding 10 l of water to this organic layer, the pH was adjusted to 6.5 with 2N hydrochloric acid,
Erythromycin was transferred to the aqueous layer to obtain 10 l of an erythromycin hydrochloride aqueous solution (containing 80 g of erythromycin).

5 l of the erythromycin hydrochloride aqueous solution was cooled to 5 ° C., adjusted to pH 9.0 with 10 N sodium hydroxide, and the temperature was gradually raised to 30 ° C. to precipitate crystals. The obtained slurry was passed through a nutsche, washed on the nutsche with 40 ml of water to remove contaminating sodium chloride, and dried under vacuum at 30 ° C overnight to obtain 30 g of a dried product.

Comparative Example 2. 5 l of erythromycin hydrochloride aqueous solution obtained in Example 2
After adding 250 ml of methylene chloride, the pH was adjusted to 9.0 with 10 N sodium hydroxide, and erythromycin was transferred to the methylene chloride layer. The solution was concentrated to dryness under reduced pressure, pulverized, and vacuum-dried at 50 ° C. overnight to obtain 37 g of a dried product.

When the amount of residual organic solvent was measured by gas chromatography for the dried erythromycin obtained from both Example 2 and Comparative Example 2, no organic solvent was detected from the dried product of Example 2; Example 2 300 ppm of methylene chloride was detected from the dried product.

Example 3 100 l of spiramycin fermentation broth (containing 500 g of spiramycin) was centrifuged (5000 rpm) for 20 minutes, and then the supernatant liquid 85
l was collected. The supernatant is passed through 20 l of an adsorbent resin HP-10 (manufactured by Mitsubishi Kasei) at 20 l / h to adsorb spiramycin, and then eluted with methanol. 20 l of methanol solution of spiramycin (containing 420 g of spiramycin) I got

90 l of water was added to 10 l of the above spiramycin methanol solution, adjusted to pH 7.0 with 2 N sulfuric acid, and then concentrated under reduced pressure to 1. After adding 9 l of water to the obtained concentrate, again,
Concentrated to 1. Further, 4 l of water was added to the concentrated solution, and the mixture was concentrated to 0.5 l to obtain 0.5 l of a spiramycin sulfate solution. The solution was cooled to 5 ° C. and adjusted to pH 9.5 with 10 N sodium hydroxide. Then, gradually raise the temperature to 10
Since the solution began to become cloudy at around 0 ° C, it was aged at this temperature for about 1 hour, and then gradually heated to 30 ° C. The obtained slurry was separated by a basket separator, and washed with 200 ml of water to remove contaminating sodium sulfate. The solid matter remaining in the separator was vacuum-dried at 50 ° C. overnight to obtain 190 g of a dried matter.

Comparative Example 3. 10 l of a methanol solution of spiramycin was concentrated under reduced pressure to dryness, pulverized, and vacuum dried at 70 ° C. overnight to obtain 200 g of a dried product.

When the residual organic solvent amount of the dried spiramycin obtained from both Example 3 and Comparative Example 3 was measured by gas chromatography, no organic solvent was detected from the dried product of Example 3; Example 3 From the dried product, 1000 ppm of methanol was detected.

Example 4 120 g of tylosin free base crude product powder was suspended in water 1 and dissolved at 5 ° C. Gradually raise the temperature of this solution to 8 ° C
Since turbidity began to be observed in the vicinity, the mixture was aged at this temperature for about 1 hour, and then gradually heated to 30 ° C. The obtained slurry was separated by a basket separator, and the solid matter remaining in the separator was vacuum-dried at 30 ° C. overnight to obtain 95 g of a dried product.

Comparative Example 4. 120 g of tylosin free base crude product powder was suspended in water 1 and adjusted to pH 7.0 with 2 N sulfuric acid to obtain an aqueous solution of tylosin sulfate. Add methylene chloride to this solution
400 ml was added, the pH was adjusted to 9.0 with 2N sodium hydroxide, and tylosin was transferred to the methylene chloride layer. The solution was concentrated to dryness under reduced pressure, pulverized, and vacuum-dried at 50 ° C. overnight to obtain 110 g of a dried product.

When the amount of the residual organic solvent was measured by gas chromatography for the tylosin dried product obtained from both Example 4 and Comparative Example 4, no organic solvent was detected from the dried product of Example 4; Example 4 300 ppm of methylene chloride was detected from the dried product.

Effects of the Invention The purification method of the present invention provides a free base of a macrolide antibiotic which does not contain an organic solvent.

Claims (5)

(57) [Claims] 1. A method for purifying a macrolide antibiotic, comprising heating the aqueous solution of a free base of the macrolide antibiotic to precipitate the compound. 2. The aqueous solution of a macrolide antibiotic free base according to claim 1, wherein the aqueous solution containing the acid addition salt of the compound is cooled to 10 ° C. or lower, and the solution is neutralized by adding an alkali solution. Purification method. 3. The purification method according to claim 1, wherein the temperature is raised from a temperature of 10 ° C. or lower. 4. The purification method according to claim 1, wherein the aqueous solution is an aqueous solution in which a macrolide antibiotic free base-containing substance is dissolved at 0 to 5 ° C. 5. The method according to claim 1, wherein the macrolide antibiotic is selected from erythromycin, oleandomycin, carbomycin, josamycin, spiramycin, tylosin and leucomycin.