JP2728482C - - 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, methods for purifying macrolide antibiotics include an extraction method using 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, and an amino sugar. There are known an ion exchange method utilizing the basicity of the above, and a method using a reversed-phase adsorbent utilizing the hydrophobicity of a macrolide skeleton. In addition, when a macrolide antibiotic product is obtained in the form of a free base, since it is generally insoluble in water, the final step after the above-described purification step involves:
Purify in the form of a solution in an organic solvent such as methylene chloride, methanol, acetone, ethyl acetate, etc., and remove the solvent or recrystallize to obtain crystals or powder of the free base, or add water to reduce the solubility and precipitate. (For example, see "Antibiotics", edited by Yusuke Sumiki, Tokyo University Press, p. 366, p. 593, p. 926, p. 1056, etc. (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 from 10 ° C. or lower. In carrying out the method of the present invention, the temperature of the aqueous solution of the macrolide antibiotic free base is raised from an arbitrary temperature of 10 ° C. or lower, preferably from 0 to 5 ° C .; Precipitation is seen. Here, the macrolide antibiotics are basic macrolides to which amino sugars are bound, such as erythromycin, oleandmycin (Ole
andomycin), carbomycin, josamycin, spiramycin, tylosin, leucomycin, 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 bases 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 is roughly purified by the above-described extraction method, ion exchange method, or reverse phase adsorption method, and is prepared, for example, as follows. . 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 The solution is kept at 10 ° C. or lower, more preferably 0 to
After cooling to 5 ° C., it is prepared by neutralizing with an aqueous alkali solution such as sodium hydroxide, potassium hydroxide or ammonia to generate a free base of the macrolide. 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. Obtaining an aqueous solution containing the compound transferred into the aqueous layer as an addition salt,
Hereinafter, it is prepared in the same manner as described above. If the organic solvent is a solvent that mixes 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. The resulting aqueous solution is prepared in the same manner as described above. Next, when the crude product is a solid such as a crystal, a precipitate, or a powder, water is added. 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. When the temperature of the aqueous solution of the macrolide is gradually increased, the macrolide is precipitated as crystals or precipitates. If the heating rate is too fast, the precipitated crystals or precipitated particles become finer, so the slower rate is preferred, usually 0.5 to
The temperature is raised to about 10 ° C. at a rate of 3 ° C./hour and then at a rate of 5-10 ° C./hour. The crystals can also be made larger 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. Embodiment 1 FIG. After 100 l of spiramycin fermentation broth (containing 500 g of spiramycin) was centrifuged (5000 rpm) for 20 minutes, 85 l of the supernatant was collected. 20 l of this supernatant was added to 20 l of a cation exchange resin Amberlite 200 (NH ₄ + type) (manufactured by Rohm and Haas).
After sporamycin was adsorbed in some cases, spiramycin was eluted with 1 N aqueous ammonia. 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). After concentrating 20 l of the above spiramycin sulfate aqueous solution to 0.5 l under reduced pressure,
After cooling to 5 ° C., the pH was adjusted to 9.5 with 15N ammonia water. Thereafter, the temperature was gradually increased, and the solution began to become cloudy at around 10 ° C. After aging at this temperature for about 1 hour, the temperature was further gradually increased to 30 ° C. The resulting slurry was separated by a basket separator and washed with 200 l 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 1 l of methylene chloride was added to 20 l of the aqueous solution of spiramycin sulfate obtained in Example 1, and the pH was adjusted to 9.5 with 10 N sodium hydroxide to transfer spiramycin to the methylene chloride layer. This solution was concentrated under reduced pressure to dryness, pulverized,
Vacuum drying was performed at 70 ° C. overnight to obtain 190 g of a dried product. Regarding the dried spiramycin obtained from both Example 1 and Comparative Example 1,
When the amount of the residual organic solvent was measured by gas chromatography, no organic solvent was detected from the dried product of Example 1, but 100 ppm of methylene chloride was detected from the dried product of Comparative Example 1. Embodiment 2. FIG. 30 l of methyl isobutyl ketone was added to 100 l of erythromycin fermentation broth (containing 100 g of erythromycin), the pH was adjusted to 10.5 with 10 N sodium hydroxide, and erythromycin was transferred to the methyl isobutyl ketone layer. After 10 l of water was added to the organic layer, the pH was adjusted to 6.5 with 2N hydrochloric acid, erythromycin was transferred to the aqueous layer, and 10 l of erythromycin hydrochloride aqueous solution (erythromycin 8
0 g). Five liters of the erythromycin hydrochloride aqueous solution was cooled to 5 ° C., adjusted to pH 9.0 with 10N sodium hydroxide, and the temperature was gradually raised to 30 ° C. to precipitate crystals. The obtained slurry was filtered with Nutsche, washed on Nutsche with 40 ml of water to remove contaminating sodium chloride, and dried in vacuo at 30 ° C. overnight to obtain 34 g of a dried product. Comparative Example 2. To 5 l of the erythromycin hydrochloride aqueous solution obtained in Example 2, 250 ml of methylene chloride was added, and the pH was adjusted to 9.0 with 10 N sodium hydroxide to transfer erythromycin to the methylene chloride layer. The solution was concentrated under reduced pressure to dryness,
After the pulverization, vacuum drying was performed 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. Embodiment 3 FIG. After 100 l of the spiramycin fermentation broth (containing 500 g of spiramycin) was centrifuged (5000 rpm) for 20 minutes, 85 l of the supernatant was collected. This supernatant is adsorbed resin H
Spiramycin was adsorbed on 20 liters of P-10 (manufactured by Mitsubishi Kasei Co., Ltd.) at a rate of 20 liters / hour and eluted with methanol to obtain 20 liters of a spiramycin methanol solution (containing 420 g of spiramycin). 90 l of water was added to 10 l of the above spiramycin methanol solution, the pH was adjusted to 7.0 with 2N sulfuric acid, and the mixture was concentrated under reduced pressure to 1 l. After adding 9 l of water to the obtained concentrate, it was again concentrated to 1 l. Further, 4 liters of water was added to the concentrated solution, and 0.5
Concentration to 1 l gave 0.5 l of spiramycin sulfate solution. The solution was cooled to 5 ° C. and adjusted to pH 9.5 with 10 N sodium hydroxide. Thereafter, the temperature was gradually increased, and the solution began to become cloudy at around 10 ° C. After aging at this temperature for about 1 hour, the temperature was further gradually increased to 30 ° C. The resulting slurry is separated by a basket separator,
Washing was carried out using 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 to dryness under reduced pressure, pulverized, and vacuum dried at 70 ° C. overnight to obtain 200 g of a dried product. Regarding the dried spiramycin obtained from both Example 3 and Comparative Example 3,
When the amount of the residual organic solvent was measured by gas chromatography, no organic solvent was detected from the dried product of Example 3;
00 ppm was detected. Embodiment 4. FIG. 120 g of crude powder of tylosin free base was suspended in 1 l of water and dissolved at 5 ° C. The temperature of the solution was gradually increased, and the solution began to become turbid at about 8 ° C.
After aging for a period of time, the temperature was further gradually raised 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 1 l of water and adjusted to pH 7.0 with 2 N sulfuric acid to obtain an aqueous solution of tylosin sulfate. To this solution was added 400 ml of methylene chloride, the pH was adjusted to 9.0 with 2N sodium hydroxide, and tylosin was transferred to the methylene tallolide layer. This solution was concentrated to dryness under reduced pressure, crushed, 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 with respect to the dried tylosin 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 (1)

Claims: 1. Purification of a macrolide antibiotic, wherein the compound is precipitated by raising the temperature of an aqueous solution of a free base of the macrolide antibiotic from 10 ° C or lower. Law. 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 an alkali solution is added to neutralize the aqueous solution. Purification method. 3. The purification method according to claim 1, wherein the temperature is raised from a temperature of less than 10 ° C. 4. The purification method according to claim 1, wherein the aqueous solution is an aqueous solution in which a substance containing a free base of a macrolide antibiotic 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.