DD143927A5 - PROCESS FOR PREPARING 12BETA HYDROXY CARDENOLIDE COMPOUNDS - Google Patents

Description

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Process for the preparation of iso-hydroxy-cardenolide compounds

Field of application of the invention; I _- . ·

The invention relates to a process for the microbiological production of cardenolide compounds of the general formula I in which X is hydrogen or a hydroxyl group and R is hydrogen or a group of the general formula III and in the latter formula R is hydrogen or an acetyl group. -.

Characteristic "of the known technical. Lösung.ent

It is known that the commercially available Hersmittel lanatoside C, acetyldigoxin and digoxin weraen obtained from the leaves of the plant Digitalis lanata ₀ The leaves of this plant contain a mixture of the glycosides lanatoside A,

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-B, -C and -D ₀ However, under the action of the hydrolyzing enzymes present, a cleavage of the part of the glycoside and of the acetyl group may also occur, whereby, in addition to the mentioned primary glycosides, the four corresponding secondary glycosides digitoxin, gitoxin, Digoxin and biginatine are present in the drug * The ratio of the primary to the secondary glycosides varies depending on the breeding conditions of the plant, but this is not advantageous from the point of view of the technology of obtaining the glycosides. In this case the Lanatosemia remains with the Recovery of the usable as drugs * in the 12ß-position a hydroxyl group-containing glycosides as useless by-product back «

For the hydroxylation of the Aglykons of 12-deoxy-Herzglykoßiden different microorganisms can be used according to the literature. Thus, for example, Fusarium lini (HeIv. Shim * Acta, £ 1, 297 / I960 /), Gibberella fujikuroii and Melicostylum piriforme (Nature 184 "» 4 & 9/1959 /), Gibberella saubinetti (Chem. Pharm. Bull. 8, 530 / I960 /), Oaloneetria decora and Nigrospora sphaerica (Abstr ₀ Symposium on the Chem. Of Digitalis Cardiac Glycosides, 114 / I960 /), but the hydroxylation of the therapeutically applicable cardiac glycoside digitoxin has been the concern of only one Japanese research group, which has successfully used the microorganism strains Streptorayces owashiensis, Streptomyces diastatochromogenes, Mortierella isabellina, Trichocladium asperam and Circinella muscae for the production of digoxin (Agr _o Biol. Chern, 2 ^., 783/1965 /).

Aim of the invention:

The aim of the present invention was the elaboration of a new, simple and large-scale economical

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Applicable method for the preparation of therapeutically valuable cardiac glycosides containing a hydroxyl group in the 12ß-position *

Invention··*

The invention has been made possible by the ability to isolate and grow such a Streptomyces strain from a forest soil which has the ability to introduce a 12β-hydroxyl group into the aglycone portion of 12-desoxy-derived glycosides in the course of aerobic fermentation. Taxonomic the new strain is to be considered as Streptomyces praecox for its properties, which are described in detail below (see Waksmant The Actinomycetes, IPo1, 1961); the strain was in the Hungarian National Collection of Microorganisms under He. MG 127 deposited ·

The new strain shows the following characteristics *

Morphology * The spores originating from the hyphae of the aerial mycelia show monopodial branching, the 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-edged , ellipsoidal ¹ · her size is 0.95 * 1.00 χ 1.10 - 1.28 / U ·

Sucrose-Hitrat agar colonies show only scattered growth, aerial mycelium is-kremfarbig that Hährbodenmyzelien have a bright cinnamon color \ ~ no Löo royal pigment is produced ·

To Tyrosine Agar? no. Growth <> '· ..-. . ·· ... The strain selects at nutrient agar with mediocre speed * There are no aerial mycelia developing, the

ground mycelia are colorless, with a waxy surface; ee no soluble pigments are formed

At Glycose asparagine agar, the strain shows very rapid growth, the aerial mycelia have dusty surface and spider tissue like shape; both the aerial mycelia and the medullary myceliums are drappfarbig; there is no soluble pigment.

On broth agar the colonies grow fast; there is no aerial mycelium, the nutrient-cell mycelia form a very thin layer and are slimy; no soluble pigment diffuses into the agar.

Potato-glucose agar: fast growth; the aerial mycelia are initially white, later cinnamon-colored and finally drappo. The nutrient-cell mycelia are drappy or cinnamon-colored; there is no soluble pigment «,

Sabouraud Glucose Agars the strain grows with mediocre. Velocity, aerial mycelia are slow and sparse with white color; the nutrient-cell mycelia are creamy; no soluble pigment «

Löffler ¹ shear curdled whey brothi good-Y / sturgeon, no aerial mycelia, the nutrient-cell mycelia look like bacteria colonies and have waxy surface; no soluble pigment is produced, the proteolytic effect is minimal «

Cellulose as sole carbon source; good growth. Gelatinous positive

Potato cut: fast growth '} cinnamon-colored aerial mycelia, no soluble pigment »

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Blood ~ Agarj gray-brown colonies with a waxy surface, 1 mm in diameter; after one week incubation at 26 ⁰ G no sporulation, weak beta-hemolysis »

Assimilation of Kohlens.toffquellens

D-glucose + ' Melesitose weak D-Galaktöse ⁺ . soluble starch + sucrose D-XyIose ++ maltose ++ L-arabinose + lactose ++ Ii-Ehamnose - raffinose galactitol , - sorbose - H-mannitol + cellobiose , -f inositol trehalose salicin Θ melibiose - ώ-methyl -D-glucoside weak

@ in the nutrient agar well-diffusing brown pigment + or ++ $ good bzwo very good assimilation.

The object of the present invention is therefore a new process for the preparation of cardenolide compounds of the above-defined general formula I on a microbiological route which is characterized in that cardiac glycosides of the general formula II in which R and X are as in the general formula I. haberij in aqueous MeäLuni with a submerged, aerated and stirred culture of the strain Streptomyces (MjUG-127 * deposited in the Hungarian National Collection of Microorganisms) in contact and then separates the resulting, biologically active, hydroxylated product from the reaction mixture ·

In the practice of the method according to the invention, the strain Streptomyces praecox or Vari-

of which are known to be cultivated using growth media suitable for cultivating streptococci. Thus, glycose, maltose, galactose, lactose, starch or dextrin may advantageously be used as sources of energy. The source of nitrogen may be organic substances of natural origin, such as casein hydrolyzate, soybean meal, peanut meal, Corn steep liquor, peptone or yeast extract, and also synthetic products such as amino acids, urea or ammonium salts. The mahrbodenbestandteile kö'n-. It is expedient to add to the soil also inorganic salts, especially phosphates, potassium, calcium, magnesium and / or iron salts. The amounts and proportions of the carbon source and the nitrogen source in the nutrient medium can be between wide limits (about 0.5 % to 4 %) vary. The decrease in the pH of the fermentation liquid can be prevented by adding calcium carbonate • to the nutrient medium »

The fermentation can be between 20 ⁰ C and 4O ⁰ C, but especially at low for streptomycetes temperature of 28 - are carried out 30 ⁰ C · The cultivation takes place in shaken Erlenmeyer flasks or in equipped with stirrer fermentors · The air flow can by vigorous shaking of the flask or, in the case of submerged fermentation in fermentors, by blowing air into the fermentation medium. The hydroxylating activity of the microorganisms can be increased by repeated inoculation of the culture onto fresh nutrient medium ₀

The substrate to be hydroxylated may be added to the already fully developed culture or during the phase of intensive growth; However, the same result is achieved even if you add the substrate to the culture medium before the beginning of the growth, since the growth of the culture by the presence of cardiac glycosides

It is also possible to work by separating the grown culture from the nutrient medium by filtration and suspending it in a buffer solution and then adding the substrate to this suspension. However, the oxygen supply of the reaction mixture and the appropriate temperature must always be maintained to be secured ₀ "

As substrate any 12-deoxy-Herzgl: ykoside of the general formula IX, or the corresponding AgIucone itself, used as "^ -, acetyl-digitoxin, digitoxin or digitoxigenin ₀ Me Me as a substrate to be used compound is appropriate in one with water miscible organic solvent, z _o B. dissolved in ethanol or methanol, and fed in this form to the fermentation medium; but the reaction can also be carried out with good results, if 'one' suspends the finely powdered compound in water and attaches it in this form to the fermentation medium.

The product of the fermentation process obtained in the 12ß-position containing a hydroxyl group of the general formula I can be obtained by extraction with a Y / asser immiscible solvent, preferably with chloroform, bichloromethane or ethyl acetate from the fermentation liquid after concentration of the thus obtained organic solution and after chromatography of the residue, the crude crude product can be purified by recrystallization.

The most important distribution of the invention is that it enabled the economical production of therapeutically effective cardiac glycosides. When lanatoside C is obtained from the plant Digitalis lanats, large quantities of the therapeutically worthless lanatoside A are always retained during the production of digoxin the therapeutically worthless Digitoxin obtained as a byproduct

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becomes; By applying the method according to the invention, therapeutically valuable cardiac glycosides can be obtained from these worthless by-products

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The process of the invention is further illustrated by the following examples

Example 1 . , , , "

The strain Streptomyces praecox MNG 127 is on

1.0% glucose

0.2 % casamine

0.1 % meat extract

0.01 % Lanatoside A and

1.7 % agar

Cultivated culture medium containing sputum containing 5 ml of distilled water from Schrägagar is used as a vaccine of the submerged culture.

100 ml of nutrient solution S ^U 'containing 2% Sojamehrl, 3% glucose and 0.5% Oalsiumöarbonat, are inoculated with 1 ml of the above suspension "Before seeding, the soil, its pH is 7, at 120 G ⁰ Sterilized for 25 minutes. Cultivation of the Streptomycetes is carried out at 28 ⁰ O, on a shaker with 2.5 cm rash, 300 U / min · After 2 days incubation, the culture with the solution of 50 mg digitoxin in 2 ml of methanol and the culture at 37 ° 0 further shaken. The progression of the biochemical reaction is monitored by TLC analysis of the chloroform extract from daily samples of the medium. Kieselgel HF ₂₅ , plates (E _c Merck, Darmstadt, Germany) are used for this purpose; Eluant: 35j55j1O Mixture of cyclohexanes ethyl acetate and abs _c ethanol, -The stains of the reaction product

visualized by the use of acetic anisaldehyde solution at 105 ° C. In the most favorable time point, on the basis of the thin-layer chromatographic analysis, about 90 hours after the addition of digitoxin, the fermentation is stopped; From the 4 liter fermentation liquid obtained from 40 shaken flasks, the settling constituents are separated by centrifuging, extracted with 500 ml of methanol and the methanol solution evaporated to dryness. The supernatant liquid obtained during the centrifugation is extracted three times with 3 liters of chloroform each time and the extracts are combined , IIJ this combined chloroform solution and the dry KENE residue of the above methanol extract is solved "the solution over anhydrous sodium sulfate, dried and evaporated in vacuo at 40 ⁰ C» The oily residue is taken up in 10 ml Iji mixture of chloroform and methanol and 20 g of silica gel (quality suitable for column chromatography). The silica gel carrying the crude product is introduced into a column of 70 cm height and 4 cm diameter. Chromatography is started on a dry support and carried out in an ascending direction. Initially, a 35 * 55 * 2.5 mixture of cyclohexanes, ethyl acetate and abs. Ethanol, which is then modified to reach 1100 ml eluate volume linearly to 35 l 55:10 modified vyird. 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 remaining unchanged appears in the. Fractions between 960 ml and 1070 ml, then from the fractions between 1070 and 1150 ml the main conversion product digoxin and from the following fractions the likewise formed 7B-hydroxydigoxin are obtained.,

The digoxin-containing fractions are kept for 2 days

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-20 ⁰ C held; during this time, the crude digoxin (330 mg) precipitates in the form of acicular crystals. The mother liquor is evaporated in vacuo, the oily residue dissolved in 5 ml of chloroform and the solution is added dropwise with cyclohexane until the onset of turbidity · The turbidity is resolved by the addition of a few drops of methanol, the mixture cooled to -20 ⁰ C and to crystallize In two days, 140 mg of digoxin precipitate in crystalline form. The two crude products are combined and recrystallized from hot 80% methanol. On this plant 450 mg of crystalline digoxin are obtained; F. ι 245 - 250 ⁰ C; 10% (+ 1, pyridine). IR (KBr): 3400, 3510, 3090, 1720, 1620, 1400, 1270, 860, - 820 cm " ¹ .

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

Peak values of the mass spectrum: 390 (M ⁺ ), 372 (30 %) , • 354 (70 μg), 262 (42 #), 219 (40 %), 201 (100 #), 147 (40 %) _t 111 (41 %).

UV (in cc · H ₂ SO ₄ ): λ 'max .: 228, 317, 388 mn »BMR" (in dimethyl sulfoxide): <f "0.7 s (18-Me), 0.9 s - (19 -Me), 3, .85 (3 ~ H), - 4.05s (14-0H), "4,2 d (3-OH), 4,6 d (12-0H), 4,9 s (21-H), 5.8s (22-H) ppm.

UV (in CCoHpSO 4): λ _{n v} t -228, 317 and 388 mn. £ τ * max.

From the evaporation residue of 7ß-hydroxy-digoxin-containing fractions are obtained after recrystallization from 80% methanol 175 mg of crystalline product; M.p .: 210-217 ⁰ C; £ luj ² £ «+ 16.1 ° (c - 1, pyridine)»

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The 7β-hydroxy-digoxygenin produced from the resulting crystalline product by acid hydrolysis shows the following spectral data ι

Peak values of the mass spectrumi 403 (M ⁺ , 3 %) $ 281

(36 SS), 278 (30th SS), 163, 145 (43 SS). IR CKBr) ί 3570, 3370, -2910, 1710, 1600, 1430, 1310, 860,

- 825 cm "" ¹

WATCH (in dimethyl sulfoxide) χ «Γ« 0.7 s (18-Me), 0.9 s

- (19-Me), 3.9 (3-H), 4.25 d (3-OH), 4.65 df (12-0H), 4.9 s (21-H), 5 * 3 s (14-0H), 5 * 5 d (7-OH), 5.8 s * (22-H) ppm. , - -

UV (in cc »HpSOJ Λ _moT * 23o, 380 and 460 nnu £ * r max»

Example 2

The growth of the strain Streptomyces praecox (MMG-127) and the microbiological reaction of the added substrate are carried out below the ratios described in Example 1. "To the grown culture as substrate to each 100 ml of the culture 20 mg pi -acetyldigitoxin in 1 ml of ethanol The fermentation is continued for 4 days, then the product obtained is extracted in the manner described in Example 1 and purified by chromatography, with the difference that the solvent gradient is adjusted so that the initial volume ratio 35 * 55s2,5 of the mixture of cyclohexane, ethyl acetate and absolute ethanol until the flow of IOOOO ml of Bluat should be diluted to 35-55ί8; in the further course of EIuierens then this relationship is maintained. The fractions collected between 15 ml and 1770 ml eluate voices are pooled and evaporated; the residue is crystallized from 80% methanol; The resulting crystalline product can be characterized as having a thin-layer chromatographic properties as a c / y-acetyldigoxin

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The product is further purified by preparative thin-layer chromatography. The analytically pure pi- acetyldigoxin obtained in this way melts at 224-229 ° C .; PdjJ ² ^ «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

From a 5 to 6 weeks old culture of the strain Streptomyces praecox MNG 127 grown from a slant agar grown on potato extract and glucose, a suspension is made with 10 ml sterile distilled water. 100 ml are sterilized with 500 ml Erlenmeyer flasks per 1 ml of this suspension PS nutrient solution (containing 0.8 % dusty soy extract and 3 % glucose). The nutrient solution, the pH of which was before the Strilisieren 7.0, was sterilized prior to inoculation for 45 minutes at 120 ⁰ C; sterilization of the glucose to be added to the nutrient solution separately, in 50% solution, at 120 ^° C. for 30 minutes.

The dusty soy extract will be on the. prepared as follows: extracted soy flour is sieved through a sieve with O ₅ 4 mm mesh size and suspended in tap water. The suspension containing 10 % flour is heated under 1 atm for one hour, then cooled to 37 ⁰ C and hydrolyzed with 0.4 % pancreatin for 2 hours with constant stirring, the pH of the reaction mixture with added as needed sodium hydroxide during the hydrolysis is maintained between 7.5 and 8.0 ", after completion of the hydrolysis, the mixture is centrifuged and the supernatant liquid is dried under atomization."

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13 - £ ]., JU $

The nitrogen content of the powdered soy extract obtained in this way is 9 % o

The incubation of the Streptomyces strain in the inoculated flask is carried out at 32 ^° C. on a shaking table with 2.5 μm rash and 300 rpm. With the 400 ml shake culture fully grown under such circumstances in 24 hours, in a glass laboratory fermentor with 10 l Volume 5 1 Inoculated PS nutrient medium · In addition to the soy extract and glucose, 0.2 % palm oil is added to this Sail dsm medium to prevent foaming. The fermentation is continued at 32 ⁰ C, under aeration with 5 l / min sterile air and with stirring at 350 revolutions / min one day, then the culture temperature is raised to 37 ° 0, and it is sterilized by filtration solution of 2J5 After 2 days of uncubation, the perforating liquid is filtered off and the filtrate is extracted twice with 1/3 volume of benzene and then three times with 1/3 volume of chloroform. The chloroform extract becomes 750 mg Digoxin and 440 mg Tβ-hydroxydigoxin were obtained.

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HQ- (/ OH

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Claims (2)

- 14 - 2 1 Invention claim: 1 »Process for the preparation of cardenolide compounds
of the general formula I, wherein X is hydrogen or a hydroxyl group and R is hydrogen or a group of
general formula III, and in the latter formula R
Is hydrogen or an acetyl group, characterized in that oardenolide compounds of the general formula II in which 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 praecox MTG 127, deposited. in the Hungarian National Collection of Microorganisms, and then separating the resulting biologically active hydroxylated product from the reaction mixture 2. The method according to item 1, characterized by ,, that Digitoxin used as a cardenolide compound of the general formula II Process according to item 1, characterized in that d / acetyldigitoxin was used as the cardenolide compound of general formula II