JPH04235996A - Production of purified macrolide antibiotic - Google Patents

Abstract

PURPOSE:To produce a purified macrolide antibiotic having an aldehyde group. CONSTITUTION:A crude alkali metal hydrogensulfite adduct of macrolide antibiotic having an aldehyde group is treated with a strong alkali aqueous solution in an organic solvent capable of distributing the above-mentioned macrolide antibiotic into an organic layer. Thereby decomposition of the above-mentioned adduct and extraction of the macrolide antibiotic are simultaneously carried out to produce high-purity macrolide antibiotic in high yield.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing purified aldehyde group-containing macrolide antibiotics. More specifically, the present invention relates to a method for producing a purified aldehyde group-containing macrolide antibiotic, the main feature of which is to purify the crude sodium bisulfite adduct of the aldehyde group-containing macrolide antibiotic by decomposition and extraction.

0002

[Prior Art] Macrolide antibiotics exhibit antibacterial activity against a wide range of Gram-positive pathogenic bacteria such as Staphylococcus and mycoplasma, and are important as pharmaceuticals. As with the cultivation of general microorganisms, when producing macrolide antibiotics, in addition to the desired active ingredient, by-products that are structurally similar to the active ingredient are often simultaneously produced. Furthermore, macrolide antibiotics are often produced simultaneously with a variety of structurally similar active ingredients. Normally, in order to isolate and purify the target active ingredient from such a culture, it is necessary to consider the solubility and solubility differences in appropriate solvents, the precipitation properties and precipitation rates from solutions, and the adsorption affinities for various adsorbents. Isolation and purification methods that make use of differences in distribution between two types of liquid phases, etc., are appropriately selected, combined, and repeated, taking into consideration the target active ingredient, by-products, etc. It is common that

However, in order to produce and isolate and purify on an industrial scale, it is necessary to use an industrial isolation and purification method that uses as small-scale equipment as possible, has fewer steps, and can more simply and efficiently obtain the target effective substance with high purity. is desirable for improving productivity, and its probability is desired. Furthermore, when dealing with various macrolide antibiotics, it is desirable to establish more versatile industrial isolation and purification techniques.

[0004] Conventionally, as a method for separating and collecting multi-component basic macrolide antibiotics, there has been a known technique in which they are adsorbed onto a nonionic styrene resin and eluted with a mixed solvent of an organic solvent and an aqueous solution with a pH of 3 to 8. (Japanese Unexamined Patent Application Publication No. 48-76
No. 880).

[0005]

[Problem to be Solved by the Invention] However, this technique of separating and collecting individual active ingredients by adjusting the pH of the buffer solution not only requires an additional buffer solvent tank;
Buffer adjustment and pH control are also not easy. In addition, the YS-02930K-D substance shown by chemical formula 1 described in JP-A-61-268176, in which a multi-component basic macrolide antibiotic is simultaneously produced (
(hereinafter referred to as D substance) and YS-02930K-E substance (
Even when applied to YS-02930K group antibiotics such as YS-02930K group antibiotics (hereinafter referred to as Substance E), the purity remained at about 20-48%, and this method was by no means a method that could be used industrially.

[0006]

[Chemical formula 1]

[0007] The isolation and purification method described in JP-A No. 61-268176 is a laboratory production method, in which bacterial cells are removed by filtration, and the liquid is separated with ethyl acetate many times while the pH is adjusted. It is extracted and dried, developed using silica gel thin layer chromatography, the separated individual spots are scraped off, and further purified using silica gel column chromatography if necessary, but thin layer chromatography is not suitable for industrial production. Therefore, it was difficult to increase the purity to the desired level industrially even by extraction with ethyl acetate under pH adjustment.

[0008]

[Means for Solving the Problems] Based on the above state of the art, the present inventors aimed to establish an industrially advantageous isolation and purification method for macrolide antibiotics. Focusing on the aldehyde group that exists as We have discovered that it is possible to simultaneously extract and separate highly impurities, and to obtain the target macrolide antibiotic with high purity and high yield.

[0009] The present inventors also focused on the fact that many macrolide antibiotics have a dimethylamino group and are basic, and created the above adduct using an acidic cation exchange resin. For example, we found that not only can it be completely separated from related substances that do not contain aldehydes, but also that it is possible to efficiently improve the purity even when using inflow materials with many impurities.

Furthermore, the present inventors have demonstrated that related substances can be separated using a nonionic adsorption resin based on the difference in organic solvent concentration of a water-containing organic solvent without adjusting the pH with a buffer solution. We confirmed that by performing this separation prior to the step of creating the above adduct, it was possible to pre-separate most of the related substances containing aldehyde groups other than the target substance. .

The present invention was completed based on these findings, and one of its most distinctive features is the chemical method of converting an aldehyde group into an adduct with an alkali metal hydrogen sulfite and returning it with a strong alkali. The method can be used to purify macrolide antibiotics, and the yield and purity of macrolide antibiotics can be significantly increased using this method. Furthermore, the method of the present invention is applicable to the above YS-02
The effect was confirmed with 930K group macrolide antibiotics, but since it is a chemical method, it is versatile and can be used with all aldehyde group-containing macrolide antibiotics or aldehyde group-containing basic macrolide antibiotics. .

The present invention will be explained in detail below. As mentioned above, the macrolide antibiotic to be isolated and purified in the present invention may be any macrolide antibiotic containing an aldehyde group or an aldehyde group and a dimethylamino group. Not done. Such macrolide antibiotics include YS-02930K group, josamycin, leucomycin group, spiramycin group,
Typical examples include carbomycin group, silamycin group, tylosin, angoramycin, and derivatives thereof containing the above groups.

Each step of the present invention will be explained in detail below. For convenience of explanation, the process of adsorbing on a nonionic adsorption resin and eluting with a water-containing organic solvent is called the HP process, and the process of adsorbing on an acidic cation exchange resin and eluting the adduct with an aqueous solution of alkali metal bisulfite is called the HP process. The SK process and the process of extracting the target substance by treating the adduct with a strong alkaline aqueous solution in an organic solvent that can be separated and distributed are referred to as the extraction process.

(HP step) This step is a step in which the purified macrolide antibiotic is adsorbed onto a nonionic adsorption resin and separated into components containing the target macrolide antibiotic by the difference in concentration of the water-containing organic solvent. . As the nonionic adsorption resin used here, commercially available styrene adsorption resins can be used, and such commercial products include Diaion HP20 and HP2.
1. SP800 (both trade names: Mitsubishi Kasei) and Amberlite XAD-2 (trade names: Rohm & Haas) are advantageously used. The water-containing organic solvent may be one that can elute the target macrolide antibiotic by setting the organic solvent concentration. Such water-soluble organic solvents include ketones such as acetone,
Examples include alcohols such as methanol, ethanol, and isopropanol. Among them, acetone and isopropanol are preferably used. The concentration of the water-containing organic solvent must be set appropriately, taking into account the solubility of the target substance, which varies depending on the solvent and its concentration. For example, when the organic solvent is acetone for the YS-02930K group as a macrolide antibiotic, , approximately 20-35% v/v. Since acetone has good elubility, when using a solvent with poor elubility, the organic solvent concentration can be set higher, and when using a solvent with good elubility, it can be set to the same level or lower. Note that it is advantageous to use the crude macrolide antibiotic introduced into this HP step after removing bacterial cells from the fermentation broth by centrifugation or filtration using a conventional method. In addition, strong acids such as hydrochloric acid and sulfuric acid are added to this fermentation broth to improve bacterial cell aggregation and adsorption to resin.
It may be adjusted to about H1 to H3. According to this process of separation based on a set concentration difference of a water-containing organic solvent, related substances that are more hydrophobic than the target substance (including related substances with aldehyde groups are included) are not eluted due to the concentration difference, so separation is possible. Moreover, since salts are not adsorbed by this resin, they can be desalted, and since many impurities such as colored substances are hydrophobic, many of them remain without being eluted. Note that the elution is preferably carried out by appropriately selecting and controlling the conditions, taking into consideration the quality, yield, etc. of the eluate. For example, the temperature condition is about 20-30℃ and the elution rate SV (liquid hourly space velocity)
=0.5 to 2 is advantageous.

(SK step) As shown in chemical formula 2, in this step, a macrolide antibiotic having an aldehyde group and a dimethylamino group is adsorbed onto an acidic cation exchange resin, and then adsorbed with an aqueous solution of an alkali metal bisulfite salt. , is a step in which the adduct is eluted.

[0016]

[Case 2]

(In the formula, Resin is an acidic cation exchange resin having (Means the residue of a ride antibiotic.) As the acidic cation exchange resin, both a sulfonic acid type strongly acidic cation exchange resin and a carboxylic acid type weakly acidic cation exchange resin can be used. Strongly acidic cation exchange resins are advantageously used. Commercially available products can be used as such exchange resins, and such commercially available products include Diaion SK104 and SK104.
S, PK208, PK216 (all trade names: Mitsubishi Kasei), IR-118 (trade names: Rohm & Haas), and 50WX1, 50WX2, 50WX4 (trade names: Dow Chemical) can be used. Further, the alkali metal mentioned above may be either sodium or potassium, but sodium is advantageously used. Sodium bisulfite salt is usually commercially available as dimeric anhydrous sodium bisulfite (Na2S2O5), and when added to water it produces sodium bisulfite (N
aHSO3). During elution, the quality of the eluate,
The elution conditions for the aqueous solution of alkali metal bisulfite are selected and controlled as appropriate, taking into consideration the yield, etc. For example, the pH of the aqueous solution of alkali metal bisulfite is 3 to 9;
Preferably, the temperature is set to 4 to 8, and the temperature conditions during elution are set to 20 to 30 °C and the elution rate SV = about 0.5 to 2. Since the adduct is unstable, the eluate is cooled to 10 °C or less. It is preferable to do so. According to this process, related substances such as macrolide antibiotics that do not have aldehyde groups do not form adducts and remain uneluted, and the rest of the colored substances are not adsorbed or are not eluted even if adsorbed. Complete bleaching.

(Extraction step) In this step, as shown in Chemical formula 3, the alkali metal bisulfite adduct of a macrolide antibiotic having an aldehyde group is extracted with an organic compound that can be separated from water and from which the target antibiotic can be separated. This is a process in which a strong alkaline aqueous solution is added to a solvent and stirred to extract the target antibiotic.

[0019]

[Chemical formula 3]

(In the formula, M has the above-mentioned meaning, and A2 means a macrolide antibiotic residue excluding an aldehyde group.) Here, examples of organic solvents that can be separated and distributed include dichloromethane, dichloroethane, etc. Examples include halogenated hydrocarbon organic solvents, relatively hydrophobic alcohols such as n-hexyl alcohol and n-butanol, esters such as ethyl acetate, and ketones such as methyl isobutyl ketone. Dichloromethane at the point,
Halogenated hydrocarbon solvents such as dichloroquetane are advantageous. As the strong alkali aqueous solution, caustic alkalis such as sodium hydroxide and potassium hydroxide are preferably used. In this step, the macrolide antibiotic may be one that forms an adduct, and is not limited to a basic one; however, if it is treated continuously with the SK step, it will have a dimethylamino group. According to this process, the aldehyde group-containing macrolide antibiotic that is distributed in the organic layer is recovered, and the remaining hydrophilic impurities are completely removed. Related substances that are less hydrophobic than the target substance can also be made non-extractable by appropriately setting the type of organic solvent, the amount used, and the amount of caustic alkali, making it possible to remove them. The amount of organic solvent at this time is usually preferably set to 1/4 or more of the amount of eluate, but it is free to increase or decrease as appropriate, taking into consideration the degree of elubility. Also, in this extraction process, various distribution extraction conditions such as the pH of the strong alkaline aqueous solution are preferably selected and set appropriately and controlled in consideration of the quality and yield of the product obtained. The pH of the alkaline aqueous solution is set to 13 or more, preferably 14 or more.

Since the extract extracted in this way is strongly alkaline, it is neutralized or, if necessary, acidified, the same organic solvent is added again, the liquid is separated and extracted, and then concentrated and dried to achieve the desired purpose. Antibiotics can be obtained with high purity and high yield.

[0022] As an example of the present invention, YS-029
The purification method for 30K group antibiotics is listed, but since Substances D and E have similar physical and chemical properties, the only difference being the double bond and its epoxy form, they are extremely difficult to separate. . If it is desired to remove Substance E, since Substance E is unstable under acidic conditions, it can be removed like other coexisting epoxy substances by treating under acidic conditions before and after the above steps.

[0023]

Effects of the Invention According to the present invention, only the target aldehyde group-containing macrolide antibiotic can be produced from the raw material adduct with a substantially quantitative yield of at least 91%.
It is extremely useful as an innovative purification method for aldehyde group-containing macrolide antibiotics. Although it is known as an analytical method that the adduct is decomposed with a strong alkali and converted to the original aldehyde substance, this is the first time that it has been applied to macrolide antibiotics and that the decomposition and extraction were performed simultaneously. The ability to obtain purified macrolide antibiotics with high purity and high yield is completely unexpected in the field of antibiotic purification. Furthermore, by combining a process that uses this adduct with a HP process that uses only a difference in organic solvent concentration, the present invention solves the problem of purity that could not be achieved with conventional separation techniques that involve pH adjustment using a buffer solution. This is unexpected in that it was resolved all at once. Incidentally, the yield of the purified target macrolide antibiotic treated with the HP step, SK step, extraction step, and optionally the acid conversion step of the present invention is approximately 95 to 100%. The purity of the purified product reaches 90-95%, which is the value of 17 lots of 70 m3 produced in Example 2. From 64,000
The purity is about 13,000 times higher, and about 400 to 2,000 times higher than that of the filtrate solids (the value of 17 lots produced in 70 m3 in Example 2). Therefore, the present invention has achieved an industrially significant effect in that it has provided an innovative purification method for macrolide antibiotics. The above effects were confirmed as follows. 1. Yield and Purity in Extraction Step 80 mg of substance D is dissolved in 200 ml of sulfuric acid water at pH 3.5, and 100 mg of anhydrous sodium bisulfite is added to form an adduct. Add 200 ml of dichloromethane to this adduct-containing liquid.
and 12.5 ml of 25% aqueous sodium hydroxide solution to hydrolyze and extract the adduct. The extract was washed with 200 ml of water and concentrated to dryness, yielding 74 mg of Substance D. Yield = 91.9% Purity = 99.3%2. Recovery rate from SK process to acid conversion process A mixture containing substance D and substance E is added to the sample after the HP process of Example 3 to prepare solutions with concentrations of 223γ/ml and 154γ/ml, respectively. Add 250ml of this solution to Diaion SK-
10ml column packed with 104S (9φ x 16mm)
After adsorption and washing with water, 0.3 to 0.6% aqueous solution of anhydrous sodium sulfite was added for elution (the eluate was cooled to below 10°C), and then dichloromethane and 25% aqueous sodium hydroxide solution were used for elution. As a result of extraction, adjusting the pH to 2 using sulfuric acid, and acid conversion, the recovery rate of substance D was approximately 77.5 to 8.
It was 0.7%.

[0024]

[Example] The present invention will be explained in more detail with reference to Examples below. Note that the yield and purity in the text are based on actual production scale, and those skilled in the art will understand that the yield and purity are significant.

Example 1 2 m3 of culture solution obtained by the culture method described in the example of JP-A-61-268176 (Substance D = 218 g, Substance E = 19
0g), 2.2m3 of water, Radiolite Showa Kagaku Kogyo 1
Add 50 kg and stir. The pH is adjusted to 2.3 using sulfuric acid and then filtered. 5.2 m3 of the obtained filtrate was
21 columns (100L, φ28 x 162cm), S
After passing through the column at V=3 to adsorb the YS-02930K group antibiotics, the column was washed with 200 L of water to remove impurities. Next, using 400L of 25% acetone, SV = 1,
Elute at 20°C and collect 300L of main fraction. The obtained eluate was transferred to SK104S (10L, φ18×4
After adsorbing the YS-02930K group antibiotics, the column was washed with 20 L of water.
Emit acetone and other impurities. Next, using 100L of 0.4% anhydrous sodium bisulfite, SV = 0.5
, 20°C, and collect 90L of main fraction. 45L of dichloromethane and 5.6L of the obtained eluate
25% aqueous sodium hydroxide solution was added, and after stirring for 30 minutes, the dichloromethane layer was separated. Next, 15L of water was added to the dichloromethane layer, and the pH was adjusted to 2.0 using sulfuric acid.
After stirring for 30 minutes, the aqueous layer is separated. Add 7.5 L of dichloromethane to the obtained aqueous layer, and add 25 L of dichloromethane.
After adjusting the pH to 9.0 using % aqueous sodium hydroxide solution and stirring for 30 minutes, the dichloromethane layer was separated. After washing the dichloromethane layer with 0.3L of water,
When concentrated under reduced pressure at °C, substance D = 73%, substance E = 22%
200 g of white powder was obtained. Recovery rate: Substance D = 67% Purity: Substance D = 73% The purity of this substance has been previously treated with acid as described above.
This shows that if the substance is removed, substance D with a purity of 90% or more can be obtained. Furthermore, the physical and chemical properties of Substance D and Substance E were consistent with the specimens described in JP-A-61-268176.

Example 2 61 m3 of culture solution (D
Substance = 3.6kg, E substance = 3.0kg, purity 0.00
59%), add 120 m3 of water and 4200 kg of Radiolite, and stir. Filter to adjust pH to 2.0 using sulfuric acid. 180 m3 of the obtained filtrate (purity 0.18% as filtrate solids) was transferred to an HP21 column (10 m3, φ2.
1 x 2.9 m) at SV = 1 and YS-0293
After adsorbing the 0K group antibiotics, the column is washed with 20 m3 of water to flush out impurities. Next, elution is carried out using 32 m3 of 30% acetone water under the conditions of SV=1 and 20°C, and 30 m3 of the main fraction is separated. The obtained eluate was added to SK104.
After adsorbing the YS-02930K group antibiotics by passing the liquid through S (0.5 m3, φ0.8 x 1.0 m) at SV = 3,
．． Wash the column with 5 m3 of water to flush out acetone and other impurities. Next, elution is performed using 5.5 m3 of 0.4% anhydrous sodium bisulfite under the conditions of SV=0.5 and 20°C, and 5 m3 of the main fraction is collected. 2 to the obtained eluate
．． Add 5 m3 of dichloromethane and 0.31 m3 of 25% aqueous sodium hydroxide solution, stir for 30 minutes, and then separate the dichloromethane layer. Next, add 0.0% to the dichloromethane layer.
Add 8 m3 of water, adjust the pH to 2.0 using sulfuric acid, stir for 30 minutes, and then separate the aqueous layer. 0 in the resulting aqueous layer
．． Add 4 m3 of dichloromethane, adjust the pH to 9.3 using 25% aqueous sodium hydroxide solution, stir for 30 minutes, and then separate the dichloromethane layer. The dichloromethane layer was washed with 0.2 m3 of water and then concentrated under reduced pressure at 20 to 30°C to obtain 2 containing purity D substance = 72% and E substance = 24%.
．． 5 kg of white powder was obtained. The purity of this product was determined by pre-treatment with acid as described above in Example 1.
This shows that if the substance is removed, substance D with a purity of 90% or more can be obtained. Even at 72% purity, it is 12,200 times more effective from fermentation broth and 400 times more from solids, and at 90% purity it is 15,250 times more effective from broth and 500 times more from solids.

Claims (9)

[Claims] Claim 1: A hydrogen sulfite alkali metal adduct of a macrolide antibiotic having an aldehyde group is prepared in a strong alkaline aqueous solution in the presence of an organic solvent that can be separated and in which the macrolide antibiotic can be distributed into an organic layer. A method for producing a purified macrolide antibiotic, comprising: 2. The method according to claim 1, wherein the organic solvent is a halogenated hydrocarbon organic solvent. Claim 3: A crude macrolide antibiotic is adsorbed onto an acidic cation exchange resin, and an aqueous solution of an alkali metal bisulfite salt is added to obtain a sulfite compound containing only the macrolide antibiotic having an aldehyde group contained in the macrolide antibiotic. It is characterized by eluting the hydrogen alkali metal adduct and treating it with a strong alkaline aqueous solution in the presence of an organic solvent that is capable of separating the adduct and distributing the aldehyde group-containing macrolide antibiotic to an organic layer. A method for producing purified macrolide antibiotics. 4. The production method according to claim 3, wherein the acidic cation exchange resin is a sulfonic acid type cation exchange resin. 5. The production method according to claim 3 or 4, wherein the organic solvent is a halogenated hydrocarbon organic solvent. 6. A crude product containing the macrolide antibiotic is prepared by adsorbing the crude macrolide antibiotic onto a nonionic adsorption resin and adding a water-containing organic solvent that can elute the desired macrolide antibiotic. The eluted crude macrolide antibiotic is adsorbed onto an acidic cation exchange resin, and an aqueous solution of an alkali metal bisulfite salt is added to extract only the macrolide antibiotic containing an aldehyde group contained in the macrolide antibiotic. eluting the alkali metal hydrogen sulfite adduct, and treating the adduct with a strong alkaline aqueous solution in the presence of an organic solvent capable of separating the aldehyde group-containing macrolide into an organic layer. A method for producing purified macrolide antibiotics. 7. The production method according to claim 6, wherein the nonionic adsorption resin is a polystyrene adsorption resin. 8. The production method according to claim 6 or 7, wherein the acidic cation exchange resin is a sulfonic acid type cation exchange resin. 9. The production method according to claim 6, wherein the distributable organic solvent is a halogenated hydrocarbon organic solvent.