JPH05399B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] The present invention provides an improved method for recovering daunorubicin hydrochloride from fermentation broth by utilizing a combination of liquid extraction and column chromatography. Regarding.

[Conventional technology]

Daunorubicin is an active substance against cancer and was the first anthracycline-type substance used in the clinic. It is formed by bacteria of the Streptomyces species. This component was independently isolated in the mid-1960s by researchers in Italy, France, and the Soviet Union [Belgian Patent No. 639897 and Belgian Patent No. 632391 and the journal “Antibiotiki” 11 , 763 (1966)] reference〕.

Each daunorubicin-producing strain creates a number of compounds that are chemically similar to daunorubicin during its fermentation. The various methods known to date for the separation and production of daunorubicin are therefore extremely complex and expensive. Pure substances are generally obtained by multi-step extractions carried out with different organic solvents or by a combination of extraction and adsorption methods (see van Marcel Dekker, New York, 1984).
The published book “Biotechnology of
"Industrial Antibiotics" pp. 569-594 and 1984
(See "Advances in Biotechnological Processes," Volume 3, pp. 141-161, edited by A. Mizurahi-Al van Wezel, published by Alan R. Liss. Inc., New York, 2003).

According to Belgian Patent No. 639897, the fermentation liquor is filtered in the presence of a filter aid. The crude daunorubicin, which is still a highly impure product, was separated separately from the mycelium mixed with the filter aid and the filtered fermentation liquor by multi-step extraction with n-butanol or chloroform. Ru. For purification of the crude product, a countercurrent extraction in n-butanol and phosphate buffer is carried out. The yield of this process is 21% based on the active substance content of the fermentation broth.

According to another extraction method developed next (Journal "Process Biochem." 14 , 6-11 (1979)), the fermented liquor is first mixed with n-butanol and then filtered. The phases of the filtrate are separated from each other and the butanol phase is concentrated by evaporation. The residue is mixed with n-hexane and then extracted with acidified water. The aqueous phase containing daunorubicin is washed with a mixture of acetone and toluol, and then the crude daunorubicin hydrochloride is precipitated out by addition of n-butanol. The crude product is recrystallized from a methanol/ethanol/chloroform mixture.

In the case of the extraction method combined with the adsorption method, the adsorbent is Amberlite IRC-50 (French Patent No. 1551195), a weakly acidic cation exchange resin, or a cation exchange resin and aluminum oxide (Belgian Patent No. 632391). issue) or cation exchange resin and silica gel [Magazine “Folia Microbiol.” 22 ,
275-285 (1977) was used.

[Problem that the invention seeks to solve]

All of the above-mentioned known processes have the common disadvantage that different organic solvents have to be used in significant amounts and that their yields are very low. Working with various organic solvents not only makes these methods extremely expensive, but also requires a series of measures in terms of health protection, environmental protection, etc., which makes them even more expensive. It becomes an expense.

Daunorubicin itself is known to be carcinogenic and cardiotoxic, thus requiring fundamental work protection measures. The more complex the method and the more steps required, the more difficult this becomes.

The aim of the present invention was to develop a method by which daunorubicin can be recovered from the fermentation broth and further purified in a simple and economical manner.

The present inventors incorporated a column chromatographic step into their separation method, using silica gel of appropriate particle size as the adsorbent and a chloroform/methanol mixture containing formic acid as the eluent. It has been found that the number of purification steps can be significantly reduced.

[Means for solving problems]

The method according to the invention will now be described in detail.

The optionally previously filtered culture solution is extracted with n-butanol under slightly alkaline conditions.
The extract is concentrated by evaporation in vacuo and the active substance is precipitated from the residue obtained in the form of the hydrochloride salt. The crude product thus obtained contains, in addition to daunorubicin, a number of unspecified anthracycle glycosides and aglucones. A simple single column chromatography yields pure daunorubicin hydrochloride from the highly impure crude product.
For this purpose, the crude product is dissolved in as little methanol as possible and the solution is charged to a column packed with silica gel, preferably with a particle size of 0.063-0.2 mm. In order to increase the sensitivity of the separation, it is advantageous to introduce the silica gel into the column in the form of a suspension prepared beforehand in chloroform. The chloroform used for this purpose must not contain methanol or other solvents. As an eluent, a liquid containing formic acid in a mixture of chloroform and methanol is used. For example, a mixture of chloroform:methanol:formic acid is used.
A mixture in the ratio 82:18:1 is used. The eluent is collected in fractions and these fractions are examined by thin layer chromatography. Each fraction containing daunorubicin hydrochloride is sharply separated from the other fractions. Each fraction containing the desired product is concentrated by evaporation.
The evaporated residue is recrystallized only once, and the precipitated daunorubicin hydrochloride is sufficiently pure for pharmacological purposes. The yield in terms of antibiotic content of fermentation liquid is 60-62%. Such a yield can never be obtained by known methods.

〔Example〕

Hereinafter, the present invention will be explained in more detail using some examples, but it is clearly stated that these are not intended to limit the present invention in any way.

Example 1 500 ml of fermentation broth (active substance content = 40 γ/ml) are added to 10 ml of 2% strength oxalic acid at room temperature. After stirring this fermented liquid for 45 minutes, 1% perlite for filtration was added and stirred for an additional 30 minutes. The mycelia are then separated by filtration. In order to recover the antibiotic from the mycelium, it is washed with 2% oxalic acid 100 for 30 minutes under stirring and then filtered.

The combined filtrates (530) are adjusted to a pH value of 8.0-8.4 with 40% strength caustic soda and then extracted twice with 200 g each of n-butanol. That n-
The butanol phase was washed with deionized water 100 °C, adjusted to pH 5.1−5.5 with 1N hydrochloric acid, and incubated at 45 °C in vacuo.
Concentrate by evaporation. Add 90 ml of deionized water to this evaporated concentrate (4.5). Then that
Adjust the pH value to 1.2−1.4 using 6N ethanolic hydrochloric acid.
Adjust to After the mixture has been evaporated to 3/4 of its volume at 45° C., crystallization is carried out at 5° C. for 24 hours. Crude daunorubicin hydrochloride (21 g, active substance content = 60%) was filtered and n
- Wash with butanol and dry in vacuo at 25°C. The mother liquor is again concentrated by evaporation to 3/4 of its volume and once again crystallized at 5°C. After 24 hours, a further 7 g of product (active substance content=58%) are separated and are washed and dried in the same manner as described above.

The combined crude product fractions (28 g) are purified by column chromatography. For this purpose, silica gel 5 with particle size of 0.063-0.2 mm was suspended in chloroform and column (diameter: height =
1:20). 124% of the above crude product
Dissolve in ml of methanol and charge to this column. A mixture of chloroform, methanol and formic acid adjusted in a ratio of 82:18:1 is then introduced into the column as an eluent (flow rate 800 ml/hr).
Collect 800 ml of each fraction and test by thin layer chromatography. The fractions containing daunorubicin and giving a unique mark are combined (12) and evaporated to 1/100 of its volume at 35° C. in vacuo. This concentrate is left at 5°C for 24 hours. The daunorubicin hydrochloride crystals are then filtered, washed with chloroform and dried in vacuo at 25°C. 12.5 g of product are obtained, which is a yield of 62% based on the total active substance content of the fermentation broth.

Elemental analysis: C ₂₇ H ₂₉ NO ₁₀ HCl (M=563.5) Calculated value (%): C57.50 H5.32 N2.48 Cl6.30 Actual value (%): C57.10 H5.40 N2.51 Cl6. 36 Melting point: 188-189°C (decomposed) Thin layer chromatography: 4:1:5 of n-butanol, acetic acid and water as eluent on Merck silicic acid gel 60 impregnated with 1% concentration of oxalic acid. using a mixture in the proportions of . Rf=0.4 UV-spectrum E1% 1cm (dissolved in methanol) 233nm 620 250nm 440 286nm 138 476nm 201 494nm 199 532nm 115 Example 2 A fermentation liquid of 500 was prepared at pH 8-8.4 (40% Extract with 400 g of n-butanol (adjust with a solution of caustic soda). Separate the phases from each other and wash the organic phase with half the volume of deionized water. The washed organic phase is treated in the same manner as described in Example 1. The product yield and each parameter are the same as in Example 1.

Claims (1)

[Claims] 1. Recovering daunorubicin hydrochloride from the fermentation broth by extraction with a solvent that is insoluble in water, evaporative concentration of the extract, precipitation of the crude product, and purification of the crude product by chromatographic methods. In this method, the chromatographic purification is carried out using an eluent consisting of a mixture of chloroform and methanol containing formic acid, and on a column containing silica gel as an adsorbent, to obtain a fraction containing daunorubicin hydrochloride. and optionally recrystallizing the product. 2. A process according to claim 1, wherein the eluent contains 0.2-2.0% by volume of formic acid and consists of chloroform and methanol in a ratio of 80:20 to 90:10.