SU1547711A3 - Method of dawnorubicin-hydrochloride - Google Patents

Abstract

This invention relates to the microbiological industry, in particular the production of antibiotics. The purpose of the invention is to increase the yield of the target product and simplify the method. The fermentation broth, obtained by growing the producer culture STREPTOMYCES SPP., Is extracted with a water-immiscible solvent. The extract obtained is evaporated and the desired product is precipitated. Purification is carried out by chromatography on silica gel and eluted with a mixture of chloroform and methanol, taken in a ratio of 80:20 - 90:10, with the addition of 0.2-2% by volume of formic acid. The yield of the target product is 59.7-62% of the content of the antibiotic in the fermentation broth.

Description

one

(21). 4203039 / 30-13

(22) 07/31/87

(46) 02.28.90. Bul № 8

(71) Biogal Gyögyatryd p (HU)

(72) Janos Balint, Juanna Emri, Jozsef Fazekas, Attila Jakab and György Toth (HU)

(53) 615.779.93 (088.8)

(56) Felia Microbiology, 22, 1977,

p. 275-285. i

(54) METHOD FOR OBTAINING DAUNORUBICIN HYDROCHLORIDE

(57) The invention relates to the microbiological industry, in particular to the production of antibiotics.

The purpose of the invention is to increase the yield, the desired product and simplify the process. The fermentation broth, obtained by growing the culture-produce-; cent of Stroptomyces spp., extracted with a water-immiscible solvent. The extract obtained is evaporated and the desired product is precipitated. Purification is carried out by chromatography on silica gel and eluted with a mixture of chloroform and methanol, taken in a ratio of 80:20 - 90:10 with the addition of 0.2-2% by volume of formic acid. The yield of the target product is 59.7-62% of the content of the antibiotic in the fermentation broth.

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The invention relates to the microbiological industry, in particular to the production of antibiotics.

The purpose of the invention is to increase the yield of the target product and simplify the method.

Example 1. To 500 l of enzyme broth (active substance content 40 u / ml) at room temperature add 10 l of 2% oxalic acid. The fermentation broth is stirred for 45 minutes, then mixed with 1% filter pearlite and again mixed for 30 minutes. Then the mycelium is filtered. To obtain an antibiotic from a mycelium, it is leached with 100 liters of 2% oxalic acid for 30 minutes with stirring, and then filtered.

Purified filtrates (530 l) using 40% sodium hydroxide

adjusted to pH 8.0-8.4, and then extracted twice with 200 l of n-butanol. The n-butanol phase is washed with 100 l of ion-free water, the pH is adjusted to 5.1-5.5 with hydrochloric acid and evaporated in vacuo at 45 ° C. 90 ml of water free of ions is added to the concentrate from evaporation (4.5 L). , 3 then using 6 n ethanolic hydrochloric acid, the pH is adjusted to 1.2-1.4, the mixture is evaporated at 45 ° C to 3/4 of its volume, and then crystallized at 5 ° C for 24 hours. Raw daunorubium cing hydrochloride (21 g, active ingredient 60%) is filtered off, washed with n-butanol and dried in vacuum at 25 ° C. The mother liquor is again evaporated to 3/4 of its volume and again crystallized at 5 ° C until crystals form. After 24 h the remaining 7 g

SP 4b

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the product (active ingredient 58%) is separated, washed in the manner described above and dried.

The purified fractions of the crude product (28 g) are purified by the method of column chromatography. For this, 5 liters of silica gel with a particle size of 0.06-0.2 mm are suspended in chloroform and placed in a column (height D-JVJ height 1:20). The raw product is dissolved in 125 ml of methanol and placed in a column. Then, a mixture of chloroform, methanol and formic acid (speed 800 ml / h) prepared in a ratio of 81: 18: 1 is fed as an elu / ent to the column. The fractions are trapped at 800 ml and examined by thin layer chromatography. Daunorubicin containing fractions, showing only the spot, are purified (12 L) and evaporated in vacuum at 35 ° C per 1/100 of the volume. The concentrate is isolated at 5 ° C for 24 hours. Then the daunorubicin-hydrochloride crystals are filtered, washed with chloroform and dried in vacuum at 25 ° C. 12.5 g of product are obtained, which, in terms of the total content of the active substance of the enzyme broth, is 62% of the yield.

EXAMPLE 2: 500 l of enzyme broth is extracted at a pH of 8-8.4 (adjusted with 50% sodium alkali), 400 l of n-butanol without first filtering off the mycelium. The phases are separated from each other, and the organic phase is washed with half the volume of water without ions. The washed organic phase is treated as in example 1. The yield and parameters are similar to example 1.

PRI me R 3. 500 l of fermentation broth, which is obtained by applying a strain of Streptomyces sp. 69 (content of biologically active substance 43 μg / ml), treated according to Example 1. 22.5 g of red daunomycin-hydrochloride are obtained with a content of biologically active substance of 59%. From the masterbatch

solution, you can get an additional 7.5 g of 59% crude product.

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The combined crude product (30 g) as described in Example 1 was purified on silica gel using column chromatography. A mixture of chloroform, methanol and formic acid (2%) in a volume ratio of 80: 20: 2 is used as an eluent. The yield is 13.0 g (60.5% of the antibiotic from the content in the fermentation broth).

PRI me R 4. 500 l of fermentation broth, as in example 3, is treated as described in example 1. 20.0 g of crude daunomycin hydrochloride are obtained (the content of the biologically active substance is 60%) and an additional 8 g is obtained from the mother liquor (the content of the active substance is 58%). The combined amount of 28 g of the crude product is purified as described in example 1 using column chromatography. The eluting agent is a mixture of chloroform, methanol and formic acid (0.2%) in a ratio of 90:10: 0.2. The output of 12.8 g (59.7% of contain neither in fermentation broth).

The use of the method allows to simplify the technology and increase the yield of the target product (according to the prototype, a yield of 0.5 g, which is 33% of the content in the fermentation broth).

Claims (1)

Invention Formula The method of producing daunorubicin hydrochloride by extracting the fermentation broth of the producer-culture Streptomyces sp. a water-immiscible solvent, evaporation of the extract followed by precipitation of the target product and purification by chromatography on silica gel and elution with organic solvents, characterized in that, in order to increase the yield of the target product and simplify the process, the elution is carried out with a mixture of chloroform and methanol , taken in a ratio of 80-90: 10-20, in the presence of 0.2-2% by volume of formic acid.