CH642978A5 - METHOD FOR PRODUCING 12-BETA HYDROXYCARDENOLIDE COMPOUNDS. - Google Patents

Description

The invention relates to a new process for the microbiological production of cardenolide compounds of the formula I.

OH.

CH

CH

OH

R — 0

H

wherein X is hydrogen or a hydroxyl group and R is hydrogen or a group of the general formula III

CH

OH

OH

HO

(III)

,1

and in the latter formula R1 is hydrogen or an acetyl group.

It is known that the heart products Lanatosid-C, acetyldigoxin and digoxin, which are commercially available, are obtained from the leaves of the Digitalis lanata plant. The leaves of this plant contain a mixture of the glycosides

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Lanatoside-A, -B, -C and -D. However, under the action of the hydrolyzing enzymes present, the glycose part and the acetyl group can also be split off, which means that in addition to the primary glycosides mentioned, the four corresponding secondary glycosides digitoxin, gitoxin, digoxin and diginatin are also present in the leaves of the Digitalis Lanata. The quantitative ratio of the primary to the secondary glycosides varies depending on the breeding conditions of the plant, but this is not at all advantageous from the point of view of the technology of obtaining the glycosides. The lanatoside-A remains as an unusable by-product when the glycosides which can be used as medicaments and contain a hydroxyl group in the 12β-position are obtained.

According to the literature, various microorganisms can be used to hydroxylate the Àglycone of 12-deoxy cardiac glycosides. For this purpose, e.g. Fusarium lini (Helv. Chim. Acta. 41,297 [I960]), Gibberella fujikuroii and Helicostylum piriforme (Nature 184,469 [1959]), Gibberella saubinetti (Chem. Pharm. Bull. 8, 530 [I960], Calonectria decora and Nigrospora sphaerica ( Abstr. Symposium on the Chem. Of Digitalis Cardiac Glyco-sides, 114 [I960]). However, only a Japanese research group has dealt with the hydroxylation of the therapeutically applicable cardiac glycoside digitoxin, which strains the microorganisms Streptomyces owashiensis, Strep-tomyces diastatochromogenes, Mortierella hasabellina, Tri-chocladium asperum and Circinella muscae successfully used for the production of digoxin (Agr. Biol. Chem. 29, 783 [1965]).

The aim of the present invention was to develop a new, simple and commercially usable process for the production of therapeutically valuable cardiac glycosides containing a hydroxyl group in the 12β-position.

The invention was made possible by the fact that it was possible to isolate and grow such a streptomycese strain from a forest floor which has the ability to introduce a 12β-hydroxyl group into the aglycone part of 12-deoxy cardiac glycosides in the course of an aerobic fermentation . The new strain is taxonomically to be regarded as Streptomyces praecox due to its properties described in detail below (cf. Waksman: The Actinomycetes, Vol. 2,1961); the strain was deposited on June 4, 1974 in the National Microorganism Strain Collection of the Hungarian National Institute for Health Care, Gyâli ut 2-6, Budapest, under number MNG 127.

The new strain shows the following characteristics:

Morphology: the spore carriers originating from the hyphae of the aerial mycelia show monopodial branches, the individual branches are short, about 50% tendril-shaped, in 50% they consist of loose or compact spirals showing 2 to 3 loops. The spores are smooth-walled, ellipsoidal; their size is 0.95-1.00x1.10-1.28 p ..

In saccarose nitrate agar, the colonies show only scattered growth, the aerial mycelium is cream-colored, the nutrient mycelia have a light cinnamon color; no soluble pigment is formed.

On tyrosine agar: no growth.

The strain of nutrient agar grows at a moderate rate. No aerial mycelia develop, the nutrient medium mycelia are colorless, with a waxy surface; there is no soluble pigment.

The strain shows very rapid growth on glucose-asparagine agar; the aerial mycelia have a dusty surface and a spider-like shape; both the aerial mycelia and the culture medium mycelia are drapy; there is no soluble pigment.

The colonies on broth agar grow rapidly; there is no aerial mycelium, the nutrient mycelia form a very thin layer and are slimy; no soluble pigment diffuses into the agar.

Potato-glucose agar: rapid growth; the aerial mycelia are initially white, later cinnamon and finally drape. The nutrient medium mycelia are draped or cinnamon-colored; there is no soluble pigment.

Sabouraud glucose agar: the strain grows at a moderate rate, aerial mycelia form only slowly and sparsely with a white color; the nutrient medium mycelia are cream-colored; no soluble pigment.

Löffler's curdled whey culture medium: good growth, no aerial mycelia, the culture medium mycelia look like bacterial colonies and have a waxy surface; there is no soluble pigment, the proteolytic effect is minimal.

Cellulose as the only carbon source: good growth.

Gelatin liquefaction: positive.

Potato cut: rapid growth; cinnamon-colored aerial mycelia, no soluble pigment.

Blood agar: gray-brown colonies with a waxy surface, 1 mm in diameter; after one week incubation at 26 ° C no spore formation, weak beta hemolysis.

Assimilation of carbon sources:

D-glucose

+

Melesitosis weak

D-galactose

+

soluble starch

+

Sucrose

+

D-xylose

+ +

Maltose

4- +

L-arabinose

+

lactose

+ +

L-rhamnose

-

Raffinose

-

Galactite

-

Sorbose

-

N-mannitol

+

Cellobiose

+

Inositol

+ +

Trehalose

+ +

Salicin

©

Melibiosis

-

a-Methyl-D-glucoside weak

© well diffusing brown pigment + or ++ in nutrient agar: good or very good assimilation.

The object of the present invention is thus a new process for the preparation of cardenolide compounds of the general formula I defined above by microbiological means, which is characterized in that cardiac glycosides of the general formula II

CH

OH

0

(II)

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wherein R and X have the meanings given in the general formula I, in an aqueous medium with a submerged, aerated and stirred culture of the strain Streptomyces paraecox (MNG 127, deposited in the Hungarian National Collection of Microorganisms) and then the resulting, biologically active, hydroxylated product from the reaction mixture.

In the practical implementation of the method according to the invention, the strain Streptomyces praecox or variants thereof can be grown using nutrient medium components known to be suitable for growing Streptomycetes. For example, glucose, maltose, galactose, lactose, starch or dextrin can advantageously be used as energy sources. Organic substances of natural origin, such as casein hydrolyzate, soy flour, peanut flour, corn steep liquor, peptone or yeast extract, and also synthetic products such as amino acids, urea or ammonium salts can be used as the nitrogen source. The nutrient medium components can be used in various combinations. It is advisable to add inorganic salts, especially phosphates, potassium, calcium, magnesium and / or iron salts, to the nutrient medium. The amounts and the ratio of the carbon source and the nitrogen source in the nutrient medium can vary between wide limits (about 0.5% to 4%). The drop in the pH of the fermentation liquid can be prevented by adding calcium carbonate to the culture medium.

The fermentation can be carried out between 20 ° C and 40 ° C, but especially at the temperature of 28-30 ° C, which is favorable for streptomycetes. Cultivation takes place in shaken Erlenmeyer flasks or in fermenters equipped with stirrers. The air can be supplied by vigorously shaking the flask or, in the case of submerged fermentation in fermenters, by blowing air into the fermentation medium. The hydroxylating activity of the microorganisms can be increased by repulping the culture on fresh nutrient media.

The substrate to be hydroxylated can be added to the already fully developed culture or during the phase of intensive growth; the same result is achieved even if the substrate is added to the nutrient medium before growth begins, since the growth of the culture is not influenced by the presence of cardiac glycosides. It is also possible to work in such a way that the adult culture is separated from the culture medium by filtration and suspended in a buffer solution and then the substrate is added to this suspension. However, the oxygen supply to the reaction mixture and the suitable temperature must always be ensured.

Any 12-deoxy cardiac glycosides of the general formula II or the corresponding aglucones themselves, that is to say a-acetyl-digitoxin, digitoxin or digitoxigenin, can be used as the substrate. The compound to be used as a substrate is conveniently in a water-miscible organic solvent, e.g. dissolved in ethanol or methanol and added to the fermentation medium in this form; however, the reaction can also be carried out with good results if the finely powdered compound is suspended in water and added to the fermentation medium in this form.

The compound of the general formula I obtained as the product of the fermentation process and containing a hydroxyl group in the 12β position can be obtained from the fermentation liquid by extraction with a water-immiscible solvent, advantageously with chloroform, dichloromethane or ethyl acetate. After this

Concentration of the organic solution thus obtained and after chromatography of the residue, the crude product obtained can be purified by recrystallization.

The most important advantage of the invention is that it enables the economical production of therapeutically active cardiac glycosides. When lanatoside C is obtained from the Digitalis lanata plant, large amounts of the therapeutically worthless lanatoside A always remain, while in the production of digoxin the digitoxin, which is also therapeutically worthless, is obtained as a by-product; By using the method according to the invention, therapeutically valuable cardiac glycosides can be obtained from these worthless by-products.

The practical implementation of the method according to the invention is illustrated in more detail by the examples below.

example 1

The strain Streptomyces praecox MNG 127 is on a

Bred 1.0% glucose 0.2% casamin 0.1% meat extract 0.01% lanatoside A and 1.7% agar medium. A spore wash made with 5 ml of distilled water from Schrägagar is used as the vaccine of the submerged culture.

100 ml nutrient solution "S", which contains 2% soy flour, 3% glucose and 0.5% calcium carbonate, is inoculated with 1 ml of the above suspension. Before inoculation, the culture medium, the pH of which is 7, is sterilized at 120 ° C for 25 minutes. The streptomycetes are grown at 28 ° C., on a shaking table with a 2.5 cm deflection, 300 rpm. After 2 days of incubation, the culture is mixed with the solution of 50 mg digitoxin in 2 ml methanol and the culture is further shaken at 37 ° C. The progress of the biochemical reaction is monitored by thin-layer chromatography analysis of the chloroform extract from samples of the medium taken daily. For this purpose, silica gel HF254 plates (E. Merck, Darmstadt, FRG) are used; Mobile solvent: 35: 55: 10 mixture of cyclohexane, ethyl acetate and abs. Ethanol. The spots of the reaction product are made visible by using acetic acid anisaldehyde solution at 105 ° C. At the point in time which appears to be the most favorable from the point of view of digoxin formation based on the thin-layer chromatographic analysis, about 90 hours after the addition of digitoxin, the fermentation is ended; the constituents which separate out are separated from the 4 liters of fermentation liquid obtained from 40 shaken flasks by centrifugation, extracted with 500 ml of methanol and the methanol solution is evaporated to dryness. The supernatant liquid obtained during centrifugation is extracted three times with 3 liters of chloroform and the extracts are combined. The dry residue of the above methanol extract is also dissolved in this combined chloroform solution, the solution is dried over anhydrous sodium sulfate and evaporated in vacuo at 40.degree. The oily residue is taken up in 10 ml of a 1: 1 mixture of chloroform and methanol and dried on 20 g of silica gel (quality suitable for column chromatography). The silica gel containing the crude product is introduced into a column 70 cm high and 4 cm in diameter; Chromatography is started with a dry carrier and in an ascending direction

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carried out. Initially, a 35: 55: 2.5 mixture of cyclohexane, ethyl acetate and abs. Ethanol is used, which is then linearly modified to 35:55:10 until 1100 ml of elution volume is reached. Then the ratio of the solvents remains unchanged during the further elution, but the initial flow rate of 30 ml / h is reduced to 20 ml / h. The digitoxin which remains unchanged appears in the fractions between 960 ml and 1070 ml, then the main reaction product digoxin is obtained from the fractions between 1070 and 11150 ml and the 7β-hydroxydioxin also formed from the subsequent fractions.

The fractions containing digoxin are kept at -20 ° C for 2 days; during this time the crude digoxin (330 mg) is excreted in the form of acicular crystals. The mother liquor is evaporated in vacuo, the oily residue is dissolved in 5 ml of chloroform and cyclohexane is added dropwise to the solution until it begins to become cloudy. The turbidity is dissolved by adding a few drops of methanol, the mixture is cooled to -20 ° C. and left to crystallize. In two days, 140 mg of digoxin are excreted in crystalline form. The two crude products are combined and recrystallized from hot 80% methanol. In this way 450 mg of crystalline digoxin are obtained; F .: 245-250 ° C; Md = + 10.8 ° (c = 1, pyrides).

IR (KBr): 3400.3510.3090, 1720.1620.1400, 1270, 860,

820- '.

The genin produced from the crystalline product obtained by acid hydrolysis shows the following spectral data:

Mass spectrum peaks: 390 (M +), 372 (30%), 354 (70%), 262 (42%), 219 (40%), 201 (100%), 147 (40%), 111 (41%) .

UV (in cc.H2SO4): W: 228.317.388 nm.

NMR (in dimethyl sulfoxide): 8 = 0.7 s (18-Me), 0.9 s (19-Me), 3.85 (3-H, 4.05 s (14-OH), 4.2 d (3-OH), 4.6 d (12-OH), 4.9 d (21 -H, 5.8 s (22-H) ppm.

UV (in cc.H2SO4): Àmax .: 228.317 and 388 nm.

After recrystallization from 80% methanol, 175 mg of crystalline product are obtained from the evaporation residue of the fractions containing 7β-hydroxy-digoxin;

F .: 210-217 ° C; [afe0 = + 16.1 ° (c = 1, pyridine).

The 7ß-hydroxy-digoxygenin prepared from the crystalline product obtained by acid hydrolysis shows the following spectral data:

Mass spectrum peaks: 403 (M +, 3%), 281 (36%), 278 (30%), 163.145 (43%).

IR (KBr): 3570.3370, 2910, 1710, 1600, 1430, 1310, 860, 825 cm-1.

NMR (in dimethyl sulfoxide): 5 = 0.7 s (18-Me), 0.9 s (19-Me), 3.9 (3-H), 4.25 d (3-OH), 4.65 d (12-OH), 4.9 s (21-H), 5.3 s (14-OH), 5.5 d (7-OH), 5.8 s (22-H) ppm.

UV (in cc.H2SO4) Imax .: 230.380 and 460 nm.

Example 2

The cultivation of the strain Streptomyces praecox (MNG-127) and the microbiological conversion of the added substrate are carried out under the conditions described in Example 1. To the adult culture, 20 mg of a-acetyldigitoxin dissolved in 1 ml of ethanol are added as substrate to every 100 ml of the culture. The fermentation is continued for 4 days, then the product obtained is extracted and purified by chromatography in the manner described in Example 1, with the difference that the solvent gradient is adjusted such that the initial volume ratio 35: 55: 2.5 of the mixture of Cyclohexane, ethyl acetate and abs. Ethanol should be diluted to 35: 55: 8 until 1600 ml of eluate has drained away; this ratio is then maintained in the further course of the elution. The fractions collected between 1550 ml and 1770 ml of eluate volume are combined and evaporated; the residue is crystallized from 80% methanol; The kirstalline product obtained can be identified as a-acetyldigoxin due to its thin-layer chromatographic properties. The product is further purified by preparative thin layer chromatography. The analytically pure a-acetyldigoxin obtained in this way melts at 224-229 ° C .;

[a] o = 18.2 ° (c = 1, pyridine).

The genin obtained after hydrolysis of this product shows the same physico-chemical data as the digoxygenin obtained according to Example 1.

Example 3

A suspension of 10 ml of sterile distilled water is prepared from a 5 to 6 week old culture of the Streptomyces praecox MBG 127 strain grown on inclined agar containing potato extract and glucose. 1 ml of this suspension is used to inoculate 100 ml of sterilized PS nutrient solution (containing 0.8% dusty soy extract and 3% glucose) in 500 ml Erlenmeyer flasks. The nutrient solution, the pH of which was 7.0 before sterilization, was sterilized at 120 ° C for 45 minutes before inoculation; the glucose to be added to the nutrient solution was sterilized separately, in 50% strength solution, at 120 ° C. for 30 minutes.

The dust-like soy extract is produced in the following way: extracted soy flour is sieved through a sieve with a 0.4 mm mesh size and suspended in tap water. The suspension containing 10% flour is heated for 1 hour under a pressure of 1 bar, then cooled to 37 ° C. and hydrolyzed with 0.4% pancreatin for 2 hours with constant stirring, the pH of the reaction mixture being added with sodium hydroxide as required maintained between 7.5 and 8.0 during hydrolysis. After the hydrolysis has ended, the mixture is centrifuged and the supernatant liquid is dried with atomization. The nitrogen content of the powdery soy extract obtained in this way is 9%.

The Treptomyces strain is incubated in the inoculated flask at 32 ° C on a shaking table with a 2.5 cm deflection and 3000 rpm. With the 400 ml shake culture fully grown in such circumstances in 24 hours, 51 HP nutrient medium is inoculated in a glass laboratory sensor with a volume of 1-1. In addition to the soy extract and glucose, 0.2% palm oil is added to the nutrient medium to prevent foaming. The fermentation is continued for one day at 32 ° C., with aeration with 51 / min of sterile air and with stirring at 350 rpm, then the temperature of the culture is raised to 37 ° C. and the solution of 2 sterilized by filtration is 5 g of digitoxin in 100 ml of methanol are added. After 2 days of incubation, the fermentation liquid is filtered off and the filtrate is extracted twice with x / i vol. Benzene and then three times with V3 vol. Chloro-from. 750 mg of digoxin and 440 mg of 7β-hydroxydigoxin are obtained from the chloroform extract.

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Claims (3)

642 978 PATENT CLAIMS 1. Process for the preparation of cardenolide compounds of the formula I O OH CH CH OH R — 0 H wherein X is hydrogen or a hydroxyl group and R is hydrogen or a group of the formula III CH OH OH HO (III) and in the latter formula R1 is hydrogen or an acetyl group, characterized in that cardenolidine compounds of the formula II 0 CH CH OH O H wherein R and X are those given in general formula I. Meanings in an aqueous medium with a submerged, aerated and stirred culture of the strain Strep-tomyces praecos MNG 127, deposited in the Hungarian National Collection of Microorganisms, and then the resulting, biologically active, hydroxylated product separates the reaction mixture. 2. The method according to claim 1, characterized in that digitoxin is used as the cardenolide compound of the general formula II. 3. The method according to claim 1, characterized in that a-acetyl digitoxin is used as the cardenolide compound of the general formula II.