CH646995A5 - METHOD FOR THE PRODUCTION OF 12BETA-HYDROXYCARDENOLIDE DERIVATIVES ON A MICROBIOLOGICAL WAY. - Google Patents

Description

The invention relates to a new process for the microbiological production of a 12β-hydroxyl group and optionally a 7ß-hydroxyl group-containing cardenolides of the formula I or mixtures thereof

R-O

wherein Y is a hydrogen atom or a hydroxyl group and R is a hydrogen atom or a group of the formula IV

0derV CH.

3 646995

mean.

It is known that some cardenolides of plant origin, e.g. the lanatoside-C and digoxin can be used directly as medicaments, while others, such as lanatoside-A, acetyldigitoxin and digitoxin, can only be brought into a therapeutically usable form by prior chemical or microbiological reaction. The purpose of these reactions is to split off the glucose or acetyl group of the primary glycosides, but in particular to hydroxylate the 12-deoxy-glycosides.

Such implementation methods are already known. A hydrolysis process is e.g. described in Hungarian Patent No. 175 601; According to this method, the lanatoside-A can be converted into digitoxin in a microbiological way, by using Streptomyces griseolus.

According to Japanese Patents 42 24 498 and 42 22 611 to 42 22 614, digitoxin can be converted into digoxin by hydroxylation using Streptomyces diastatochromogenes, 20 Streptomyces owasiensis, Trichocladium asperum, Mortierel-la isabellina and Circinelle muscae strains.

According to Hungarian Patent No. 175 474, digoxin and acetyl digoxin are prepared from digitoxin and acetyl digitoxin using a submerged culture of Streptomyces praecox.

The aim of the present invention was to provide a new process according to which cardenolides of the primary and secondary series can be converted into 12den-hydroxyl group-containing cardenolides of the secondary series in a single 30 fermentation step.

The invention is based on the knowledge that some radiation fungus cultures isolated from different soils, namely the strains KA-26, KA-43, KA-82, KC-157 and AB-318-ST, also ß in addition to the 12-oxygenase activity -Glycosidase and deacetylase activity.

The most important diagnostic features of these strains were determined - also in comparison with the description of typi-40 strains of closely related fungal species - on the basis of the studies described in detail below.

The description of the strain AB-318 ST morphology

The spore carriers are of the rectus flexibilis type, long, straight, with more than 50 spores per spore chain. These morphological properties can be observed on yeast extract-malt extract agar, oatmeal agar, glycerin-asparagine agar and on starch agar containing inorganic salts. According to electron microscopic observations, the spores have a smooth surface. According to comparative studies with the Tresner-Backus color wheel, the color of the aerial mycelia belongs to the "White" (W) series. This color 55 can be identified on all of the standard culture media mentioned above. The substrate mycelium shows the following colors on various nutrient media: yeast extract-malt extract agar: gray-yellow (Co 81): Yb, oatmeal agar: not determinable, glycerin-asparagine agar: yellow (Co 68; Co 70) Yb, starch -Agar with inorganic salts: yellow (Co 37; Co 67) 60 Yb. The pigment of the substrate mycelium is not an indicator. No exopigment formation can be observed on the nutrient media mentioned above. No pigment of the melanin type is formed on peptone iron agar and on tyrosine agar; the melanoid pigment reaction is negative. 65 The following carbohydrates were used well: d-glucose, 1-arabinose, d-xylose, i-inositol, mannitol, d-fructose and raffinose; Sucrose and rhamnose are not used.

646 995

4th

The most important diagnostic features of the AB-318-ST strain were summarized in Table 1 below and the corresponding features of a

Table 1

Streptomyces alboniger type strain (ISP 5043) compared.

Spore carrier morphology

Spore surface morphology

Color of the aerial mycelium

Color of the substrate mycelium

Indicator character of the

Substrate mycelium

Soluble pigment

Indicator character of the soluble pigment

Melanoid pigment M6

Melanoid pigment M7

Carbohydrate utilization:

d-glucose

1-arabinose d-xylose i-inositol mannitol d-fructose

Sucrose Rhamnose Raffinose

Streptomyces AB-318-ST

Streptomyces alboniger ISP 5043

RF smooth W Y-b

RF smooth W Y-b

+ + + + + +

+

+

deviating

Results +

H-

different results

+

different results

The symbols and abbreviations used in the tables above and in the following have the following meanings:

RF: Rectus flexibilis type spore carriers

RA: spore carrier of the retinaculum-aperture type

S: Spore carrier from Spira

M2: yeast extract-malt extract agar

M3: oatmeal agar

M4: Starch agar with inorganic salts

M5: glycerin-asparagine agar

M6: Peptone yeast extract iron agar

M7: Tyrosine agar

The data of the color series are given according to the names of the Tresner-Backus color determination [Tresner u. Backus: Appi. Microbiol. 11.335 (1963)); E Küster: Int. Bull. Bact, Nom. Tax. 14.1 (1964)].

40 On the basis of the above data, the strain AB-318-ST was determined as Streptomyces alboniger and on September 26, 1978 in the Hungarian National Collection of Medicai Bacteria, National Institute of Hygiene, Gyâli ut 2-6, H-1966 Budapest under no. MNG 180 deposited.

45 Table 2 below shows the characteristic properties of the strains Streptomyces sp. KA-26, Streptomyces sp. KA-43, Streptomyces sp. KA-82 and Streptomyces sp. KC-157 summarized.

Table 2

KA-26

KA-43

KA-82

KC-157

Spores

M2

S

RA

S

RA

carrier

M3

S

S

S

S

(Morpho

M4

S

S

S

S

logic)

M5

RA

RA

S

RA

Spores

surface

prickly prickly prickly prickly

Color of

M2

R (7ca):

R (7ca)

R (7ca):

R (7ca)

Aerial mycelium

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)

5

646 995

(Continued in Table 2)

KA-26

KA-43

KA-82

KC-157

Color of

Y-b + blue

Y-b + blue

Y-b + blue

Y-b + blue

Substrate mycelium

-red

- red

-red

- red

Indicator-

HCl from purple from purple from purple from purple

-Character

in red in red in red in red of the substrate

mycelium

NaOH

of purple of purple of purple of purple

in blue in blue in violet in blue

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

Indicator-

HCl from violet from violet from violet from violet

Character of

in orange in orange in orange soluble in orange

NaOH

from violet from violet from violet from violet

Pigments

in blue in blue in blue in blue

Melanoides

M6

+

+

+

+

pigment

M7

-

-

-

-

Utilization of d-glucose

+

+

+

+

from

1-arabinose

+

+

+

+

Carbon d-xylose

+

+

+

+

swell i-inositol

+

+

+

+

d-mannitol

+

+

+

+

d-fructose

+

+

+

+

Sucrose

+

+

+

+

Rhamnose

+

■ +

+

+

Raffinose

+

+

+

+

35

As can be seen from the above data, the strains described in Table 2 show complete taxonomic identity among themselves. The spore carriers belong to the “Spira” type morphologically, only on the M2 or M 5 culture media does the “Retinaculum-aperture” type occasionally appear. Both the substrate mycelium and the soluble pigment have an indicator character.

Table 3 below summarizes the standard descriptions of strains KA-28, KA-43, KA-82 and KC-157, and a strain of Streptomyces purpurascens type (ISP 5310).

40

Table 3

Spore carrier (morphology) Spore surface Color of the aerial mycelium

Streptomyces sp. KA-28, KA-43 KA-82, KC-157

S

Spiky R (3ca; 5ca; 7ca) V (1 Ica)

Streptomyces purpurascens ISP 5310

S

spiky R: W

Color of the substrate mycelium

Y-b + blue - red

Y-b + blue - red

Indicator character

HCl from purple to purple from purple to red

NaOH from red in violet from purple in blue

Soluble pigment Indicator character of the soluble pigment melanoides pigment

M6 M7

red: blue violet as with the substrate mycelium + +

646995

(Continued in Table 3)

d-glucose

+

+

1-arabinose

+

+

d-xylose

+

+

i-inositol

+

+

Mannitol

+

+

d-fructose

+

+

Sucrose

+

+

Rhamnose

+

+

Raffinose

+

+

The above strains were identified as Streptomyces purpurascens due to the agreement of the most important diagnostic features; the strain Streptomyces purpurascens KA-26 was registered under No. MNG179, the strain Streptomyces purpurascens KA-43 under No. MNG 178, the strain Streptomyces purpurascens KA-82 under No. MNG 177 and the strain Streptomyces purpurascens KC-157 under No. MNG 176 deposited on September 26, 1978 with the Hungarian National Collection of Medicai Bacteria, National Institute of Hygiene, Gyäli ut 2-6, H-1966 Budapest. Samples of the above microorganisms can be obtained from the institute mentioned.

The invention is thus a process for the preparation of cardenolides of the formula I defined above by microbiological means from cardenolide compounds of the formula IA

15

20th

R

25th

wherein X is hydrogen or a hydroxyl group and R1 is a hydrogen atom or a group of the formula II, III, IV 30 or V

646 995

(V)

mean, with the restriction that if X stands for a hydroxyl group, R1 can only mean a group of the formula II, and in the end product of the formula IR can only stand for a group of the formula IV or V, which is characterized in that Cardenolide compounds of the formula IA are fermented with a submerged culture of the strains Streptomyces purpurascens KA-26, Streptomyces purpurascens KA-43, Streptomyces purpurascens KA-82, Streptomyces purpurascens KC-157 or Streptomyces alboniger AB-318-ST and the resulting cardenolide or those obtained Cardenolides of formula I isolated from the cementation broth.

When practicing the method according to the invention, the most important condition is that one should work under aseptic conditions. The Streptomyces strains mentioned can also be grown under the conditions which are generally customary in the cultivation of radiation fungi.

Vigorous growth is achieved by using as the coal and energy source those sugars mentioned above which can be used well by these strains, e.g. Glucose, arabinose, xylose, mannitol, fructose, sucrose or raffinose. Hazelnut flour, corn steep liquor, peptone, furthermore advantageously soy flour, casein or hydrolysates thereof, amino acids or inorganic ammonium salts can be used as the nitrogen source.

The pH of the nutrient medium is adjusted between 6 and 8, advantageously to 7; Calcium carbonate can be added to increase the buffer capacity.

To avoid any foaming, a vegetable oil, advantageously palm oil or sunflower oil, or a synthetic antifoam agent is added to the nutrient medium, preferably before sterilization. The culture medium is sterilized at 120 ° C for 30 to 60 minutes.

The cultivation of the culture and the implementation are expediently carried out at the temperature which is favorable for the streptomycetes between 30 ° and 40 ° C., in particular at 32 ° C. but one can also use 32 ° C in the cultivation of the culture and 37 ° C in the implementation. It is important that the submerged culture should receive an appropriate supply of air during the entire process, which can be achieved by shaking the flask or by stirring the sterile air introduced into the fermenter.

The implementation of the substrate is expediently started with an already powerfully developed culture, for example at 1% dry matter content of the fermentation medium; however, the addition of the substrate can also start while the culture is growing.

The substrate is added to the fermentation medium in the form of a solution prepared with a water-miscible solvent which does not inhibit the reaction and which is sterilized by filtration or heating before the addition. When adding the substrate, the method described in Hungarian Patent No. 176 249 can advantageously be used; the concentration of the substrate in the fermentation medium can be increased considerably by an appropriate choice of the solvent to be used.

The progress of the implementation can be followed with the help of samples taken; after thin-layer chromatographic separation, the cardenolide content of the samples is determined by photometric measurement of the color obtained with dixanthylurea. The practical implementation of this method is also described in detail in the above-cited 30 Hungarian Patent No. 176 249 and also in Example 1 below.

The fermentation can be ended when the analytical test mentioned shows the complete consumption of the substrate and the maximum increase in the amount of the desired product. The fermentation liquid is then filtered off and the product obtained is extracted from the cell mass or from the fermentation liquid with an organic solvent. The dry residue of the extract can be purified by chromatography or by re-crystallization. (During the purification, the by-products formed can advantageously be separated off by using the method described in Hungarian Patent No. 181 880.)

The most important advantage of the process according to the invention is that the conversion of primary 12-deoxycardenolides into the corresponding secondary cardenolides containing a hydroxyl group in the 12β-position instead of the previously required two successive microbiological reactions in a single fermentation step can perform. The elimination of a fermentation step also means that there is no need for an expensive extraction and work-up step which causes material losses, so that the invention enables a much simpler and higher yield-producing method of 12β-hydroxy-cardenolides.

Another important advantage is that the energy-consuming drying fermentation during the processing of the drug - which is intended to facilitate the conversion of the primary Glyko-60 side into secondary glycosides - is also eliminated when using the method according to the invention.

By using the method according to the invention, the production of the digoxin which is most important from the therapeutic point of view can always be ensured regardless of the fluctuating glycoside composition of the drug (that is to say of the value of the ratio of total glycosides / lanato-sid-C).

646 995

As a further advantage, it should also be mentioned that not only the lanatosides but also the purpurea glycosides can be converted into therapeutically high-quality products with the method according to the invention.

The process according to the invention is illustrated in more detail by the examples below.

example 1

A suspension is prepared with 10 ml of sterile water from a 3 to 4 week old culture of the strain Streptomyces purpurascens KA-43 (MNG 178) grown on oblique potato dextrose agar. 100 ml «PS» medium is sterilized in a 500 ml Erlenmeyer flask. Composition of the PS culture medium:

0.8% soy powder

3.0% glucose pH before sterilization: 7.0

Sterilization for 45 minutes at 120 ° C. The glucose is sterilized separately in 50% aqueous solution, 30 minutes at 120 ° C. The soy powder is produced in the following way: From an extracted soybean sieve sieved through a sieve of 0.4 mm mesh size, a 10% suspension is prepared with tap water and this is firstly heated by heating at 1 atm for one hour, then cooled to 37 ° C hydrolyzed with 0.4% pancreatin for 2 hours, the pH being kept between 7.5 and 8.0 by the addition of sodium hydroxide solution. The suspension is then centrifuged and the supernatant liquid is evaporated to dryness with atomization. The product containing 9% nitrogen obtained in this way is the soy powder.

The sterilized nutrient medium prepared and sterilized in the above manner is inoculated with 1 ml of the microorganism suspension in the Erlenmeyer flask and shaken on a flat shaker with a 2.5 cm deflection and 300 rpm at 32 ° C. for two days. Then the solution of 50 mg of lanatoside-A in 0.5 ml of dioxane, which was previously sterilized by filtration, is added, the flask is then shaken for a further 4 days at 32 ° C., and then the fermentation liquid obtained in this way is mixed with 50 ml of chloroform extracted twice. After separation by thin layer chromatography, the glycoside content of the extract is determined in the following manner by photometric measurement of the color obtained with dixanthylurea:

Thin layer chromatographic separation: On a 0.25 mm thick layer of silica gel Merck H254 (E. Merck, Darmstadt, Germany), in a tub with a saturated vapor space; Solvent for primary glycosides chloroform-methanol 90:10, for secondary glycosides ethyl acetate-cyclo-hexane-Abs. Ethanol 55:35:10, with three runs each.

The samples to be examined are dripped onto the silica gel layer in such quantities that 10 to 50 [g] of the individual glycosides should be present in the volume applied.

After the three runs, the layer is carefully dried and the chromatogram developed in iodine vapor. The spots are marked according to the standard patterns and the iodine sublimed away in an air stream.

Color reaction: The marked stains are scraped off and placed in test tubes with cut glass stoppers. 3 ml of dixanthylurea reagent (see below) are added, the test tubes are closed, shaken thoroughly and kept in a boiling water bath for three minutes. The test tubes are then cooled in a cold water bath and, after shaking, centrifuged sharply. The color intensity of the clear supernatant liquid is measured at 535 nm in comparison with a glycoside-free solution.

The glycoside-free solution is made by scraping off one

10th

15

“Empty” (no glycoside-containing), at the same height as the glycoside in question, from the same area of the silica gel layer, produced in the manner described above.

5 The concentrations of the individual glycosides can be calculated from the measured extinction values, taking into account the possible dilution and the volume applied, with the help of the factor obtained from the standard concentration curve.

Dixanthylurea reagent: 10 mg of dixanthylurea are mixed with 50 ml of acetic acid and 1 ml of conc. Hydrochloric acid dissolved and the solution made up to 100 ml with acetic acid. The reagent is freshly prepared before the measurement.

The digoxin content of the extract obtained in the above manner, determined by the method described above, is 12 mg.

Example 2

20 The procedure is as in Example 1, with the difference that, instead of lanatoside A, 50 mg of acetyl digitoxin dissolved in 1 ml of dimethyl sulfoxide are added to the culture. The extract obtained in this way contains 9 mg digoxin.

25 Example 3

The procedure is as in Example 1, with the difference that, instead of lanatoside-A, 50 mg of purpureaglycoside-A dissolved in 1 ml of tetrahydrofurfuryl alcohol are added to the culture. The extract obtained in this way contains 8 mg

30 digoxin.

Example 4

The procedure is as in Example 1, with the difference that instead of lanatoside A 50 mg digitoxin in 1 ml di-

35 methyl sulfoxide dissolved in the culture are added. The extract obtained in this way contains 8 mg of digoxin, while 15 mg of digitoxin have remained unchanged.

Example 5

40 The procedure is as in Example 1, with the difference that the microorganism suspensions are produced from cultures of the following microorganism strains instead of Streptomyces purpurascens KA-43 (MNG 178):

Flask 1: Streptomyces purpurascens KA-26 (MNG

45 179),

Flask 2: Streptomyces purpurascens KA-82 (MNG 177),

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

see piston 4: Streptomyces alboniger AB-318-ST (MNG 180).

The amounts of glycosides found in the extracts obtained in this way are summarized in Table 4 below.

55

Table 4

piston

Lana-

Ac digit

Digitoxin

Digoxin

60

tosid-A

oxin (mg)

1

2.1

3.0

15.4

4.2

2nd

1.8

5.5

12.4

7.3

3rd

3.6

4.1

11.3

6.1

4th

-

1.7

16.4

3.8

65

Example 6

The procedure is as in Example 5, with the difference that instead of Lanatosid-A, the solution of 50 mg of purple

9

646 995

reaglycoside-A in 1 ml of tetrahydrofurfuryl alcohol is added to the cultures.

The amounts of glycosides found in the extracts obtained in this way are summarized in Table 5 below.

Table 5

piston

Purpurea glycoside

Digitoxin (mg)

Digoxin

1

1.7

18.2

4th

2nd

-

14.3

6.8

3rd

2.8

12.7

7.2

4th

3.5

15.8

4.1

Example 7

The procedure is as in Example 5, but with the difference that instead of lanatoside-A, the solution of 50 mg of acetyl digitoxin in 1 ml of dimethyl sulfoxide is added to the cultures. The amounts of glycosides obtained are summarized in Table 6 below.

Table 6

piston

Acetyl

Digitoxin

Digoxin

digitoxin

(mg)

1

5.2

15.8

4.6

2nd

4.5

14.3

6.4

3rd

6.1

15.5

4.8

4th

7.3

16.0

3.7

Example 8

The procedure is as in Example 5, but with the difference that instead of lanatoside A, the solution of 50 mg digitoxin in 1 ml dimethyl sulfoxide is added to the cultures. The amounts of glycosides so formed are summarized in Table 7 below.

Table 7

piston

Digitoxin

Digoxin

(mg)

IL

24.6

3.7

2nd

21.5

4.9

3rd

26.1

3.1

4th

28.6

2.5

Example 9

The procedure is as in Example 1, with the difference that the fermentation is ended 2 days after the addition of the substrate. The fermentation liquid is extracted with chloroform. The extract is chromatographed on a silica gel layer (silica gel HF254, Merck, Darmstadt; Dicek: _ 0.25 mm); Solvent: cyclohexane-ethyl acetate abs. Ethanol 35:55:10). The spots were developed with acetic acid anisaldehyde reagent. In addition to little unreacted lanatoside-A, significant amounts of acetyldigitoxin, acetyldigoxin and digitoxin as well as little digoxin were identified on the chromatogram obtained.

Example 10

A suspension with 10 ml of sterile water was prepared from a 3 to 4 week old culture of the strain Streptomyces purpurascens KA-43 (MNG 178) grown on oblique potato dextrose agar. 100 ml "PS" culture media are sterilized in 500 ml Erlenmeyer flasks and then inoculated with 1 ml of the microorganism suspension. The flasks are shaken at 32 ° C for two days.

In a laboratory fermentor, 5 liters of "PS" medium containing 0.2% palm oil are sterilized at 120 ° C for 60 minutes and, after cooling, inoculated with the combined contents of 4 flasks.

The contents of the fermenter kept in a water bath heated to 32 ° C. are incubated with aeration with 51 / min of sterile air and stirring at 350 rpm for one day and then the solution sterilized by filtration of 5 g of lanatoside-A in 25 ml of dioxane added. After another 5 days of incubation, the fermentation liquid is filtered off;

The cell-free filtrate is extracted twice with 1/3 vol. Benzene. The extract obtained with benzene contains the possibly unregulated substrate (Lanatosid-A) as well as the digitoxin formed as an intermediate and some of the by-products.

Subsequently, the fermentation liquid already extracted with benzene is extracted three times with each volume of chloroform, the extract is dried with anhydrous sodium sulfate, filtered and evaporated to dryness in vacuo at a maximum of 50 ° C. The residue obtained in this way contains 40% digoxin, 14% 7ß-hydroxy-digoxin and a further 20% other glycosides with an unidentified structure. Lanatosid-A, acetyldigitoxin and acetyldigoxin could not be detected in the residue.

This residue of the extract obtained with chloroform was dissolved in a mixture of 60 ml of methanol and 60 ml of benzene, the solution was mixed with 400 mg of phenylboric acid and left to stand for 5 minutes. The reaction mixture was then mixed with 60 ml of water with constant stirring. After the phases had been separated, the lower, aqueous rig-methanolic layer was separated and extracted with 60 ml of benzene. The lower layer was separated, the methanol removed in vacuo at a maximum of 50 ° C., the crystalline product which separated out was filtered off, washed with 60 ml of water and dried. In this way, 0.75 g

20th

obtained crystalline digoxin; [a] - ,, = +13.6 (c = 10%, in pyridine). 0

Example 11

The procedure according to Example 5 is followed, with the difference that, instead of lanatoside-A, 50 mg of lanatoside-C dissolved in 1 ml of dimethyl sulfoxide are added to the culture.

The glycosides found in the extracts obtained in this way are summarized in Table 8 below.

Table 8

piston

Lanatoside-C (mg)

Digoxin

1

5.2

12.0

2nd

6.0

17.1

3rd

2.1

18.1

4th

4.3

14.2

5

10th

15

20th

25th

30th

35

40

45

50

55

60

c

Claims (2)

646 995 PATENT CLAIMS 1. Process for the preparation of a 12β-hydroxyl group CH. O \ c = o or 35 wor * n X is hydrogen or a hydroxyl group and R1 is a hydrogen atom or a group of the formula II or III CH CH. (II) and (III) OH or of the above formula IV or V, with the restriction that if X represents a hydroxyl group, R1 can only mean one group of the formula II, by microbiological hydroxylation and / or deacetylation and / or glycose elimination, characterized in that one fermentes cardenolide compounds of formula IA with a submerged culture of the strains Streptomyces purpurascens KA-26, Streptomyces purpurascens KA-43, Streptomyces purpurascens KA-82, Streptomyces purpurascens K-157 or Streptomyces alboniger AB-318-ST and the resulting cardenolide or the cardenolides of the formula I obtained are isolated from the fermentation broth. 2. The method according to claim 1, characterized in that the cardenolide or cardenolides obtained is purified by chromatography or recrystallization.