HU176250B - Process for producing mikrobiologically 12-beta-hydroxycardenolide derivatives - Google Patents

Abstract

12beta-hydroxy-cardenolides of the secondary series are prepared by microbiological route from the corresponding 12-desoxy-cardenolides of the primary series by reacting the latter with submerged cultures of the strain Streptomyces purpurascens KA-26 (MNG 179), Str. Purpurascens KA-43 Purpurascens KA-82 (MNG 177), Str. Purpurascens KC-157 (MNG 176) or Str. Alboniger AB-318-ST (MNG 180), which are available under the indicated MNG numbers in the National Microorganism collection of the Hungarian National Institute of People's Health were deposited, fermented and the reacted product isolated from the fermentation liquid in a known per se.

Description

Stephen is okay. teacher 10%, Budapest

Method for microbiological production of 12β-hydroxycardenolide derivatives

The present invention relates to a novel process for the microbiological preparation of cardardolides of formula I wherein X is hydroxy, ¥ is hydroxy and R is hydrogen or IV or V.

It is known that some plant-derived cardardenolides, e.g. lanatoside C and digoxin, which can be used directly as a medicament; however, lanethoside A-acetyldigitoxin, digitoxin, only becomes therapeutically useful after prior chemical or microbiological conversion. The transformations are directed to the cleavage of the glucose and acetyl groups of the primary glucoside and, in particular, the hydroxylation of the 12-deoxyglucosides. Of these, hydrolysis is disclosed in German Patent Publication No. 29 32 577, which discloses a microbiological process for the preparation of digitoxin from lanatoside A using Streptomyces griseolus.

Japanese Patent Nos. 42,24,498 and 42,22611 to 4222614, respectively, show digoxin by hydroxylation of digoxin by strains of Streptomyces diastatochromogenes, Streptomyces owashiensis, Trichocladium asperum, Mortierella isabellina and Cirdnella muscae.

According to German Patent Publication No. 29 21 052, digoxin and acetyldigoxin can be prepared by digestion of Streptomyces praecox from digoxin or acetyldigitoxin.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a process which allows the conversion of primary 12-deoxycardololids 5 into single-series 12β-hydroxylic cardedrolides in a single fermentation step.

The present invention is based on the discovery that some of the 10 isolated fungal cultures of soil, namely strains KA-26, KA-43, KA-82, KC-157 and AB-318-ST, exhibit β-glucosidase activity in addition to 120-oxygenase activity. and deacetylase activity.

The most important diagnostic features of the strains were determined by comparing them with the description of the strains of the closely related species based on the tests detailed below.

1. The fiber of the mature aerial mycelium with the Tresner-Backus color wheel generally accepted in international practice [Tresner and Backus, Appl. Microbiol. 11, 335 (1963), Küster E .: Int Bull. Bact, Ma'am. Tax. 14

1 (1964) 1. The definition of color includes not only entering the correct color but also entering the correct color with a numeric code.

2. For determination of substrate mycelium and soluble pigment color, see the color chart prepared by Prauser in 1964 [H. Prauser: Z. Alig. Microbiol. 4, 95 (1964)] and Baumanns Farbtonkarte Atlas II. The definition of color includes the notation of color in the appropriate color group and the color numeric codes in Szabó & Martin 5 [l.M. Szabó, Márton M. Int. Bull. Bact. My wife. Tax. 14, 17 (1964)].

Determination of the indicator character of the substrate mycelium was the identification of the color change after a few drops of 0.05 N sodium hydroxide 10 and 0.05 N hydrochloric acid solution (after 15-20 minutes).

3. The solubility of the pigment was determined in a manner analogous to that of the substrate mycelium and indicator character.

4. The production of melanoid pigment was determined on standard M6 and M7 media.

Description of strain AB-318-ST

Morphology

Spore racks are Rectus flexible type, long, 25 straight, with more than 50 spores per spore chain. This morphology is observed on yeast extract malate extract agar, oatmeal agar, inorganic salt starch agar and glycerol-asparagine agar. Electron microscopic observations show that the spores have a smooth surface. The color of the aerial mycelium is white series (W) based on comparative studies with the Tresner-Backus color mixture. All of the above standard media can identify this color. Substrate mycelium on yeast extract malt agar: greyish-yellow 35 (Co 81), Yb, oatmeal agar: undetectable, inorganic salt-starch agar: yellow (Co37, Co 67) Y-b, glycerol-asparagine agar: yellow (Co 68) , Co70) Yb. The pigment of the substrate mycelium is not indicative. The former media space _{exopigment-40} mediators are not observed. The production of melanin-type pigment is not observed on peptone-iron agar and tyrosine agar: the melanoid pigment reaction is negative. It utilizes d-glucose, 1-arabinose, d-xylose, i-inositol, mannitol, d-fructose and raffinose, not 45 sells sucrose and rhamnose.

Comparison of key diagnostic features of strain AB-318-ST with standard description of Streptomyces alboniger strain (ISP 5043)

Table 1 summarizes.

Table 1

Name of the species Streptomyces AB-318-ST Streptomyces alboniger ISP 5043 55 Spore-bearing morphology RF RF 60 spore surface morphology smooth smooth 65

Continuation of Table 1

Name of the species Streptomyces AB-318-ST Streptomyces alboniger ISP 5043 Color of aerial mycelium W W Color of substrate mycelium Y b Y b Nature of substrate mycelium indicator - - Soluble pigment - - Nature of soluble pigment indicator - - - Melanoid M6 - - pigment M7 - - D-glucose 4- + L-arabinose + + D-xylose + different results 6Λ i-inositol 4 ~ + you mannit '03 vol + + 'G D-fructose different N CO results saccharose - - rhamnose - - raffinose + different

results

In the table, the notations are as follows:

RF = Rectus-Flexible Type Spore Holder W = White

Y-b = tan

M6 = peptone yeast extract iron agar

M7 = tyrosine agar

Based on the above data, strain AB-318-ST was determined as Streptomyces alboniger and deposited in the National Collection of Microorganisms under MNG 180.

The descriptions of KA-26, KA-43, KA-82 and KC-157 are summarized in Table 2.

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Table 2

Name of the species Streptomyces sp. KA-26 Streptomyces sp. KA-43 Streptomyces sp. KA-82 Streptomyces sp. KC-157 spore M2 S RA S RA morphology M3 S S S S M4 s S S s M5 RA RA S RA spore surface morphology prickly prickly prickly prickly aerial mycelium M2 R (7ca): R (7ca) R (7ca): R (7ca): color V (llca) V (llca) V (llca) M3 R (5ca) R (5ca) R (5ca) R (5ca) M4 R (7ca, 3ca) R (7ca, 3ca) R (7ca, 3ca) R (5ca, 7ca) M5 W (13ba) W (13ba) W (13ba) W (13ba) substrate Y — b + blue y b + Blue Y b + Blue Y b + Blue mycelium color o ed 4 * ed red «+ Ed substrate color change purple purple purple purple mycelium hydrochloric acid red red red red indicator effect nature color change purple purple purple purple sodium hydroxide blue blue violet blue effect Soluble M2 violet pale violet violet pigment violet M3 blue blue blue blue M4 violet violet violet violet M5 pale pale pale pale violet violet violet violet Soluble color change violet violet violet violet pigment hydrochloric acid orange orange orange orange indicator effect nature color change violet violet violet violet sodium hydroxide blue blue blue blue effect melanoid M6 + ♦ 4 4 pigment M7 - ~~ ' _{M is} D-glucose + 4 4 4 4 = L-arabinose 4 4 + 4 S is D-xylose 4 4 · 4 + i-inositol 4 + ♦ 4 D-mannitol 4 · 4 + 4 t-D-fructose + + 4 4 «2 sucrose 4 4- 4 4 rhamnose 4 4 4 4 raffinose 4- 4 4 4

In Table 2, the designations are as follows:

RA = Retinaculum - apertum spore holder

S = Spira Spore Holder 5

Y — b + blue red = tan + blue +> red

R (7ca) = red (light yellowish red)

V (llca) = purple (very light purple)

R (5ca) = red (light yellowish red) 10

R (3ca) = red (light orange yellow)

W (13ba) = i white (reddish white)

M2 = yeast extract malt extract agar

M3 = oatmeal agar

M4 = inorganic salt - starch agar

M5 = glycerol - asparagine agar

As can be seen from the data, there is complete taxonomic identity between the strains. Morphologically, the spore-holder is a Spira type, with occasional retinaculum apertum types only found on M2 and M5 media. Both the substrate mycelium and the soluble pigment are indicative,

Table 3 shows the standard description (ISP 5310) of strains KA-26, KA-43, KA-82, and KC-157 and Streptomyces purpurascens.

Table 3

Name of the species Streptomyces sp. KA-26, KA ^ 43, KA-82, KC-157 Streptomyces purpurascens ISP 5310 Spore-bearing morphology S. S Spore surface morphology prickly prickly Air mycelium color R (3ca, 5ca, 7ca) V (llca) R, W Substrate mycelium color Yb + blue Yb + blue red ++ Ed Substratal Color change: violet purple purple red nature of the indicator hydrochloric acid, sodium hydroxide purple blue violet from red effect Soluble pigment red: blue violet Soluble pigment as a substrate mycelium nature of the indicator indicator character Melanoid pigment M6 + + M7 - - , D-glucose + + «L-arabinose + + § D-xylose + + = i-inositol + mannitol + D-fructose + + <2 sucrose + rhamnose + + ⁰⁰ raffinose + +

(The notations are the same as in Tables 1 and 2

According to key diagnostic stamps, the strains were labeled with the genus Streptomyces purpurascens, and strains KA-26 of Streptomyces purpurascens were identified by MNG 179, strains of Streptomyces purpurascens KA-43, streptomyces purpurascens KA-43 and Strain 82 was deposited under MNG 177 and Streptomyces purpura-censed KC-157 under MNG 176.

Accordingly, the present invention provides a process for preparing a microbiological process for the preparation of cardiolides of Formula I wherein X is hydroxy, Y is hydrogen or hydroxy, R is IV or V, wherein X is hydroxy, uo250 hydrogen or hydroxy, Y is hydrogen, R is II, III, IV or V - cardardenolide Streptomyces purpurascens KA-26 (MNG 179) or Streptomyces purpurascens KA-43 (MNG 178) or Streptomyces purpurascens KA-82 ( MNG 177) or Streptomyces purpurascens KC-157 (MNG 176) or Streptomyces alboniger AB-318-ST (MNG 180) strain (deposited in National Collection of Microorganisms of National Institute of Public Health) and fermented from the product by self-fermentation and self-fermented product isolated in a known manner.

The most important aspect of practicing the method of the invention is to provide aseptic conditions at all times. Streptomyces strains are grown under conditions commonly used for the cultivation of ray fungi.

Strong growth is obtained using all sugars which are well utilized by the strains as carbon and energy sources, e.g. glucose, arabinose, xylose, mannitol, fructose, sucrose or raffinose. The nitrogen source used is hazelnut flour, corn jam, peptone, preferably soy flour, casein or hydrolyzate thereof, amino acids and inorganic ammonium salts.

The pH of the medium is adjusted to between 6 and 8, preferably to pH 7, and calcium carbonate can be used to increase its buffer capacity.

To prevent possible foaming, it is advisable to add vegetable oil, preferably palm or sunflower oil, or a synthetic antifoam agent to the medium prior to sterilization. The medium was sterilized for 30-60 minutes at 120 ° C for 1 min. overpressure.

Culturing and conversion is preferably carried out at 30-40 ° C, preferably 32 ° C, which is favorable for Streptomyces, but 32 ° C during growth and 37 ° C during transformation can be used. It is important to ensure proper air exchange throughout the process in the submerged culture by shaking the flasks or mixing the sterile air flowing through the fermentors properly.

Substrate conversion can be initiated by vigorously grown culture, preferably at a dry matter content of about 1%, but it is also possible to do so at the same time as culture.

For dosing, the substrate is introduced into the fermentation broth by dissolving in a water-miscible, non-inhibiting solvent, prior to filtration or heat sterilization. Preferably, substrate addition can be effected as described in U. S. Patent No. 2,034,353, which also allows for a significant increase in the substrate concentration of the fermentation broth by selecting the appropriate solvent. The progress of the conversion can be monitored by photometry of the color of the cardardenol content with dixantylurea after separation of the samples taken in the meantime.

Fermentation is terminated when the analytical test indicates the maximum growth of the desired product when the substrate is exhausted. The fermentation broth is then filtered and the resulting product is extracted from the cell mass or the fermentation broth with an organic solvent. The dry residue of the extract was purified by chromatography or recrystallization.

The main advantage of the process according to the invention is that, instead of two successive microbiological transformations, it enables the conversion of the primary 12-deoxycardenolides to the secondary 120-hydroxyl group in one single fermentation step. The loss of one fermentation step also saves the costly extraction-processing operation with 0 material losses. As a result, 12β-hydroxycardenolide can be prepared more easily and with much greater efficiency.

It is also of great importance that the process according to the invention does not require energy-intensive drying fermentation to aid in the conversion of the primary glycosides to the secondary glycosides.

This process allows the production of the most important digoxin, irrespective of the composition of the glycoside, due to the varying glycoside composition of the plant, the value of the total glucoside to lanososide C ratio.

A further advantage of the process according to the invention is that not only the lanatoside but also the purpurea-glucosides can be converted into a pharmaceutically useful product by using.

The process of the invention is illustrated by the following embodiments:

The qualitative and quantitative determination of the glycosides in the examples was carried out as follows:

The quality of the glycosides was determined by layer chromatography: Kieselgel HF ₂₅₄ (Merek, 5 Darmstadt, Federal Republic of Germany) was run on a 0.25 mm, 20 x 20 cm layer in a saturated steam Desaga bath, with chloroform-methanol 90: 10 as the primary glycosides. in the system (1), in the case of the secondary glycosides ethyl acetate - cyclohexane 0 absolute ethanol, in the system 55: 35: 10 (2), in each case developing the chromatogam three times.

The sample to be examined was applied to the layer in such a way that the volume of droplet contained 10-50 Mg of each glycoside. Standard reference glycosides were run alongside the samples to be identified for identification.

Chromatogram with anisaldehyde reagent and 0 A. Waldi chloramine reagent [Arch. Pharm. 292: 206 (1959)].

The anisaldehyde reagent (500 mL of 99.6% acetic acid, 3 mL of anisaldehyde and 4 mL of concentrated sulfuric acid) was uniformly sprayed until moistened and heated at 110 ° C for 10 minutes. In the chromatogram, each glycoside was represented by a characteristic color (Table 1.4).

In a chromatogram developed with Waldi reagent, glycosides under fluorescent UV light are typically fluorescent (E. Stahl, Dunnschicht-Chromatographie, Springer Verlag, 1967, 344).

Data for glycosides (Rf, Rg / Lanososide A and Digitoxin) and the colors given with the anisaldehyde reagent are given in Table 4.

Table 4

Ánizsaldehides

Primary glycosides Rf Rs (Lanatoside A) with reagent lanatoside A 0.26 1 gray-blue lanatoside A 0.13 0.5 blue purpurea A 0.09 0.35 gray-blue Secondary glycosides Rf (Digitoxin) digitoxin 0.61 0.43 1 gray-blue digoxin 0.7 blue 7 (3-hydroxy-digoxin 0.34 0.56 violet blue acetyl digitoxin 0.78 1.27 grayish blue acetyl digoxin 0.55 0.90 blue

The quantitative determination of the separated vékonyrétegkroma- ²⁰⁺ ográfiával glucosides based on specific dixantilkarbamiddal színreakcióján.

The chromatogram was pre-evaporated in iodine vapor, the spots were labeled according to standard glycosides, and the iodine was sublimated from the adsorbent in an air stream to completely whiten the adsorbent.

The labeled spots were scraped into stoppered test tubes and 4 ml of dixantylurea reagent [H. Pötter, Pharmazie 20, 737 (1965)].

After thoroughly shaking, the test tubes were placed in a 30 ° C water bath for 3 minutes. After cooling to room temperature, the agitated slurry was spun to sharpness. The color intensity of the clear supernatant was measured at 535 nm against a solution containing no glycosides. 35

The glucoside-free solution was prepared as described above by scrubbing the same area of the glycoside spot with the same area but without the glucoside free spot. 40

The concentration of each glycoside was calculated from the measured extinction by taking into account the respective dilution and volume applied, using the multiplication obtained from the standard concentration curve.

Example 1

A suspension of 50 cultures of potato dextrose slant agar on Streptomyces purpurascens KA-43 (MNG 178) for 3-4 weeks was prepared in 10 ml of sterile water. 1 ml of the suspension was inoculated into 100 ml PS medium sterilized in a 500 ml Erlenmeyer flask.

Composition of PS medium:

0.8% porosity

3.0% glucose pH 7.0 before sterilization. Sterilized at 120 ° C for 45 minutes. For the medium, glucose was separately sterilized in 50% solution for 30 minutes at 120 ° C.

Powdery soy preparation: A 10% suspension of extracted soybean meal sieved through a sieve with a 0.4 mm mesh sieve was hydrolysed with 0.4% pancreatin for 2 hours under heating with 1 atm of pressure and then cooled to 37 ° C with 0.4% pancreatin. with sodium hydroxide added between 7.5 and 8.0. The suspension was then centrifuged and the supernatant was spray dried. The product thus obtained is in the form of a powder containing 9% nitrogen.

The inoculated culture flask was shaken at 300 rpm for 2.5 days at 32 ° C on a flat-bed shaker and then treated with a solution of 50 mg of lanatoside A in sterile filtered in 0.5 ml of dioxane. After addition, the flask was shaken for another four days at 32 ° C and the culture was extracted with chloroform (2 x 50 mL). The glucoside content of the extract was determined to be 12 mg digoxin as described above.

Example 2

Proceeding as in Example 1, but adding 50 mg of acetyldithitoxin in 1 ml of dimethylsulfoxide instead of lanatoside A, 9 mg of digoxin was formed.

Example 3

Proceeding as in Example 1, but adding to the culture 50 mg of purpurea-glucoside A dissolved in 1 ml of tetrahydrofurfuric alcohol instead of lanatoside A, 8 mg of digoxin was formed.

Example 4

Proceeding as in Example 1, but adding 50 mg of digitoxin dissolved in 1 ml of dimethyl sulfoxide instead of lanatoside A, 8 mg of digoxin was formed. By definition, 15 mg of digitoxin remained unchanged.

Example 5

All procedures were carried out as described in Example 1, but the suspension in flask 1 was Streptomyces 65 purpurascens KA-26 (MNG 179), 2. lombiknál

Streptomyces purpurascens KA-82 (MNG 177), a

Flask 3 Streptomyces purpurascens KC-157 (MNG 176), Flask 4 Streptomyces alboniger

AB-316-ST (MNG 180). The glucoside content of the extracts is shown in Table 5.

Table 5

Serial number of flask A lanatoside Ac-digitoxin content Digitoxin in mg digoxin First 2.1 3.0 15.3 4.2 Second 1.8 5.5 12.4 7.3 Third 3.6 4.1 11.3 6.1 4th - 1.7 16.4 3.8

Example 6

Example 8

All procedures were carried out as described in Example 5 except that 50 mg of purpurea-glucoside A dissolved in 1 ml of tetrahydrofurfuryl alcohol were added to the cultures instead of lanatoside A. The glucoside content of the extracts is determined by

Table 6 illustrates this.

All were performed as described in Example 5, but cultures were treated with 1 ml of digitoxin dissolved in dimethylsulfoxide instead of lanatoside A. The glycoside content of the extracts is shown in Table 8.

Table 6

Serial number of flask Purpurea-Digitoxin Digoxin content in mg First 1.7 18.2 4 Second - 14.3 6.8 Third 2.8 12.7 7.2 4th 3.5 15.8 4.1

Table 8

Serial number of flask Digitoxin content Digoxin in mg 35 First 24.6 3.7 Second 21.5 4.9 Third 26.1 3.1 40 4th 28.6 2.5

Example 7

All were performed as described in Example 5, but 50 mg of acetidigitoxin dissolved in 1 ml of dimethyl sulfoxide was added to the cultures instead of lanatoside A. The glycoside content of the extracts is shown in Table 7. ⁵⁰

Example 9

All were carried out as in Example 5, but 50 mg of lanatoside C dissolved in 1 ml of dimethyl sulfoxide was added to the cultures instead of lanatoside A. The glucoside content of the extracts is shown in Table 9.

Table 7

Table 9

Serial number of flask AC-digitoxin Digitoxin content mg mg / b Digoxin m '55 Serial number of flask Lanatoside C content mg Digoxin content mg First 5.2 15.8 4.6 60 First 5.2 12.0 Second 4.5 14.3 6.4 Second 6.0 17.1 Third 6.1 15.5 4.8 Third 2.1 18.1 4th 7.3 16.0 3.7 65 4th 4.3 14.2

Example 10

In each case, the procedure of Example 1 was followed, but after addition, the fermentation was stopped after two days. The fermentation broth was extracted with chloroform. In addition to a small amount of untransformed lanososide A substrate, significant amounts of acetyldigitoxin, acetyldigoxin, digitoxin, and little digoxin were identified in the TLC.

Example 11

Streptomyces purpurascens strain KA-43 (MNG 178) was resuspended in 10 ml of sterile water from 15 grown cultures of 3-4 week potato-dextrose inclined agar. 1-1 ml of the suspension was inoculated into 100-100 ml PS medium sterilized in a 500 ml Erlenmeyer flask. The flasks were shaken at 32 ° C for 2 days, then inoculated with 4 flasks of 5 liters of PS medium containing 0.2% palm oil sterilized in a laboratory fermenter for 60 minutes at 120 ° C.

The inoculated fermenter was incubated in a 32 ° C water bath for 5 days at 5 L / min sterile air flow and 350 rpm at 25 rpm, and then added to a sterile filtered solution of 5 g of lanosozide A in 25 mL of dioxane. After another 5 days incubation, the; fermentation broth was filtered.

The cell-free filtrate was extracted twice with 1/3 volume of benzene. (The benzene extract contains the unmodified substrate, as well as digitoxin as an intermediate or part of the by-products. Thereafter, the fermentation broth was extracted three times with 1/3 volume of chloroform. The extract was dried over anhydrous sodium sulfate and filtered under vacuum at 50 ° C. It was evaporated to dryness at a temperature not higher than C. The resulting 2.1 g dry residue contained 40% digoxin, 14% 70-hydroxydigoxin and an additional 20% glycoside of unidentified structure Lanatoside A, acetyldigitoxin and acetyldigoxin were not .

The dry residue of the chloroform extract of the above composition was dissolved in a mixture of methanol (60 ml) and benzene (60 ml) and reacted with phenylboronic acid (400 mg) for 5 minutes. Water (60 mL) was added with constant stirring. After separation of the phases, the lower aqueous methanol layer was separated and extracted with 60 ml of benzene. After separation of the lower phase, the methanol was removed in vacuo at a temperature not higher than 50 ° C, the precipitated crystalline material was filtered off, washed with water (60 ml) and dried. 0.75 g of crystalline digoxin was obtained. [.Alpha.] D @ ₂₀ = + 13.6 DEG (c = 10%, pyridine).

Claims (1)

A process for the microbiological preparation of cardardenolides of formula I wherein X is hydroxy, Y is hydrogen or hydroxy, and R is IV or V, characterized in that a compound of formula I wherein X is hydrogen or hydroxy Y is hydrogen and R is a group of Formulas II, III, IV, or V - Cardardenolide Streptomyces purpurascens KA-43 (MNG 179) or Streptomyces purpurascens KA-82 (MNG 177) or Streptomyces Purpurascens KC-157 (MNG 176) or Streptomyces alboniger AB-318-ST (MNG 180) strain and isolate the transformed product or products from the fermentation broth in a manner known per se.