CS254050B1 - Preparation method of acetyldigoxine - Google Patents

Abstract

The solution relates to a process for the preparation of acetyldigoxin by placing it in a liquid medium containing the carbon and nitrogen source inoculate Penicillium vermiculatum Dang. CCM F-276 or Aspergillus niger CCM F- -330, then 10-60 mg of lanatoside is added C per 100 ml of cathode and allowed to cultivate at 26 to 30 ° C for 8 to 24 hours. The filtrate the fermented soil is extracted with chlorinated hydrocarbons, preferably chloroform, the extract is concentrated and obtained solid the product is purified by recrystallization from the mixture solvent chloroform: benzene. The filtrate also the acid esters can be extracted acetic acid, preferably ethyl acetate.

Description

. The present invention relates to a process for the preparation of acetyldigoxin by the selective microbial deglucosidation of lanatoside C using the microorganisms Penicillium vermiculatum Dang. CCM F-276 or Aspergillus niger CCM F-330.

The present invention provides a process for the preparation of acetyldigoxin, which comprises adding to the nutrient medium containing a source of carbon, organic and inorganic nitrogen inoculated with a culture of P. vermiculatum, lanatoside C (100-500 mg. L ^-1 ) dissolved in polar a solvent, preferably dimethylformamide, and cultured for 8-24 hours at 26-30 degrees Celsius with stirring and aeration. After transformation, the filtrate of the soil containing acetyldigoxin is extracted with chloroform, the extract obtained is dried, dried under reduced pressure, and the crude product is recrystallized from a solvent mixture of benzene: ethanol (3: 1). Other chlorinated hydrocarbons having a carbon number of 1-5, preferably dichloromethane or acetic acid esters, preferably ethyl acetate, can also be used to extract the substance from the filtrate.

Acetyldigoxin is a white crystalline substance with the following values measured: mp 203-206 ° C (decomposition), for the general formula C 43 H 66 O 15 823.0, calculated: C 62.75 washes. H 8.08%, found C 62.59%, H 7.96%. IC (KBr) in cm ⁴ : 3410, 2966, 2943, 1741, 1720, 1631, 1457, 1347. The crystalline material provided by thin-layer chromatography on Silufol in benzene: ethanol:: water (30: 10: 0.3] one spot with Rf values of 0.41 and ethyl acetate:: methanol: water (79: 19: 11) with an Rf value of 0.85, aglycone was detected after hydrolysis of the obtained substance with sulfuric acid. on Silufole ^R in benzene: ethyl acetate (1: 1) gave one spot with Rf 0.28 and in ethyl acetate also one spot with Rf 0.11, which were identical to those obtained by the same procedure from the acetyldigoxin standard.

Both the P. vermiculatum and A. niger strains used in the experiments transformed the added lanatoside C very selectively, with almost no accompanying metabolites, almost quantitative in 8-24 hours.

The following examples show methods for transforming lanatoside C to acetyldigoxin.

Example 1

Prepare a soil of the following composition in 25 500-ml flasks: (g. L ^-1 ) glucose 20, yeast extract 5, soya flour 5, NaCl 5, KH2PO4 5, tap water 1 liter, pH 6,5-7, 0th The pod was sterilized for 15 minutes at 120 ° C and, after cooling, inoculated with 15 ml of a vegetative inoculum of Penicillium vermiculatum. Cultivation at 28 ° C on a 220 rpm rotary shaker. . min ^-1 . After 24 hours of culture, 30 mg of lanatoside C dissolved in 0.3 ml of dimethylformamide was added to each flask. A total of 720 mg of lanatoside C was used in the experiment. The mycelium of the microorganism used was collected by filtration to obtain the filtrate (1.900 mL, pH)

6.1), was extracted three times in succession with chloroform (2: 1 ratio), with constant stirring for 15-20 minutes, at room temperature. The extracts obtained were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. After concentration and cooling to + 4 ° C, acetyldigoxin precipitated, which was collected by filtration and washed with diethyl ether. After addition of diethyl ether, which was obtained after washing the first crop of crude product to the mother liquors, an additional portion of acetyldigoxin precipitated. The crude products obtained were combined and recrystallized from chloroform: benzene (1: 3). A total of 420 mg of pure acetyldigoxin was obtained, representing a yield of 62% relative to the substrate used.

Example 2

The soil of the same composition as in Experiment 1 was used in the experiment. After cultivation for 24 hours, 60 mg of lanatoside C per 100 ml of soil were added to the soil. Transformation was complete at 24 hours. Clear filtrate (1,800 ml, pH

6.2) was extracted three times in succession with ethyl acetate (2: 1). The combined extracts after drying over anhydrous sodium sulfate were concentrated and, upon cooling to +4 ° C, acetyldigoxin precipitated. A double volume of diethyl ether was added to the mother liquors to give an additional portion of acetyldigoxin. The crude products were recrystallized from ethyl acetate: benzene (3: 1). In total, 510 mg of pure acetyldigoxin was obtained, which is 63.7% yield based on the substrate used.

Example 3

The procedure for biotransformation of lanatoside C was the same as in Example 1, but the acetyldigoxin formed was extracted with dichloromethane. The yield with respect to the substrate used was 57.2%.

Example 4

The biotransformation procedure was the same as in Example 1, but the Aspergillus niger strain was used as the microorganism.

CCM F-330 The yield of acetyldigoxin relative to the substrate used was 48.7%.

SUBJECT

Process for preparing acetyldigoxin, characterized in that a culture of Penicillium vermiculatum Dang is inoculated into a liquid medium containing a source of carbon, organic and inorganic nitrogen. CCM F-276 or Aspergillus niger CCM F-330, add 10 to 60 mg of lanatoside C dissolved in a polar solvent per 100 ml of soil, culturing at 26 to 30 ° C with stirring and aeration for 12 to 24 h , the filtrate is extracted with organic solvents, the extract is concentrated after drying and the residue is crystallized.

2. The method of claim 1, characterized by

Claims (4)

INVENTION in that dimethylformamide is used as the polar solvent for dissolving lanatoside C. 3. The method of claim 1, wherein the filtrate after cultivation is extracted with chlorinated hydrocarbons having a carbon number of 1 to 4, preferably chloroform, or esters of acetic acid with alcohols having a carbon number of 1 to 3, preferably ethyl acetate. 4. The process of claim 1 wherein the residue after evaporation of the extender is crystallized from chloroform-benzene or ethyl acetate-benzene.