DE1000380B - Process for the separation of aldosterone or its diacetate from adrenal extracts - Google Patents

Description

It has already been proposed (Experientia, Vol. 9, ^IO -53> p. 333) that aldosterone, which is a highly effective, chemically uniform new compound, should be obtained from the adrenal glands and presented in pure form. The production of this new hormone, however, meets with great difficulties because of the low yield.

It has now been found that the yield of the above-mentioned active ingredient can be increased by at least dan Can increase ten to twenty fold when first treating shredded adrenal glands with oxygen and then separating the active ingredient in free form or as a functional derivative.

The crushed adrenal glands are used, for. B. those from cattle or pigs and homogenized or cut them. The homogenization is preferably carried out in the cold in the presence of suitable buffer solutions and near constant p _H. It is advantageous to add 11-deoxycorticosterone to the comminuted adrenal glands, such as the homogenates.

The enzyme systems that are important for the reaction can be modified by adding suitable substances and pausing appropriate environmental conditions can be maintained in a functional manner. Suitable additives are in particular those of substances known as substrates of the cyclophorase system, e.g. B. the acids of the citric acid cycle or related compounds, e.g. B, citric acid, aconitic acid, isocitric acid, Oxalsuccinic acid, a-ketoglutaric acid, Succinic acid, fumaric acid, malic acid, pyruvic acid, oxaloacetic acid, but also malonic acid, Glutaric acid, adipic acid, glutamic acid, aspartic acid, asparagine. Alanine, glycocolla, serine, and more Ascorbic acid, lactic acid, dioxy-tartaric acid, proline, tyrosine, tryptophan or mixtures thereof. Additions of adenosine triphosphoric acid, adenosine diphosphoric acid, Triphosphopyridine nucleotide and diphosphopyridine nucleotide, nicotinic acid amide, adenine, adenosine and Adenylic acid.

To maintain appropriate environmental conditions, an aqueous agent is used to which components of physiological solutions such as carbohydrates, inorganic and / or organic salts, for example sodium phosphate, alkali metal chlorides or magnesium sulfate, are advantageously added. With the added salts in particular the p _H and the ionic strength should be kept the reaction solution during the reaction in the most favorable range. The reaction is preferably carried out at a molar 0.01 p _H = 6.5 to 9 and a salt concentration of 0.5 to. Accordingly, a physiological liquid such. B.

Process for the separation of aldosterone or its diacetate
from adrenal extracts

Applicant:
CIBA Aktiengesellschaft, Basel (Switzerland)

Representative: Dipl.-Ing. E. Splanemann, patent attorney, Hamburg 36, Neuer Wall 10

Claimed priority:
Switzerland from November 6, 1953 and August 30, 1954

ao Dr. Albert Wettstein, Dr. Friedrich Werner Kahnt, Basel, and Dr. Robert Neher, Binningen (Switzerland),
have been named as inventors

Plasma, use. In this case, a preservative, for example penicillin, is advantageous fed.

For treatment with oxygen, in addition to pure oxygen, oxygen-containing gas mixtures, like air, pass over or through the reaction mixture. In addition, the latter can also be shaken or be stirred.

The active ingredient is separated off from the reaction mixture, depending on the starting materials used and reactants, respectively, according to the procedures described in Experientia, Vol. 9, 1953, p. 333 and HeIv. Chin. Act., Vol. 37, 1954, p. 1163, detailed methods.

In an analogous manner, the active ingredient can also be used in the form of its functional derivatives, eg. B. as diacetate, furthermore by partial acylation or partial hydrolysis as a monoester, furthermore as an ether or Carbonyl derivatives.

The following examples explain the process according to the invention.

example 1

243 g freshly slaughtered beef adrenal glands are ^{homogenized for 5 minutes with 550 cm 8 of} an ice-cold aqueous solution in a homogenizer. The solution used contains 3.63 g sodium chloride, 1.86 g potassium chloride, 3.56 g secondary sodium phosphate, 0.98 g magnesium sulfate, 18 g per liter

3 4

Glucose, 6.4 g sodium fumarate, 3 g niootimic acid amide, an increase in the yield by about ten

and is on p _? 7.50 set. During the homofold compared to the extraction of 243 g untreated

Genizing is achieved by adding 1 η sodium hydroxide solution to the deleterious adrenal glands.

prevents the p _H is below 7.3. The emulsion To separate the active ingredient, the ether-chloroform extracts (26.3 mg) combined with a stirrer and gas inlet tube are emptied from the vessel and, with stirring and one-five approaches, a preparative paper chromatography is passed through of oxygen for 3 1/2 hours Leave 30 ⁰ . chromatography in the system of propylene glycol-toluene-The p _H is with a glass electrode during subjected (24 hours). The monitored by ultraviolet photocopy reaction between p _H 7.3 and 7.5 at the same specific band of aldosterone, as held vorbleibend. The reaction mixture is then described in 10 upright position, cut out, eluted and given extra 5 ^{liters of cold acetone and mixed with 250 cm 3 of} acetone each time. The extract (2.1 mg) is left to rinse twice after drying. The solution, left to stand for 16 hours in the cold in a high vacuum from acetone-ether and a trace, is stirred occasionally and water crystallizes. This gives 0.9 mg pure and then suction filtered. The residue is washed with aldosterone from double ^{melting point 104 to 112 0} ml with 500 cm ^{3 of} acetone and the combined acetone 15 and 153 to 158 ^{0 are} evaporated. Further amounts can be obtained from the clay solutions in a water jet vacuum at 40 to 50 ⁰ mother liquor.
Bath temperature to about 200 cm ³ .

The remaining aqueous fat suspension extra- Example 2
one hosed four times with 50 cm ⁸ petroleum ether (b.p. = 50

up to 70 ⁰ ) and then three times with 100 cm »fresh ao 225 g freshly slaughtered beef adrenal glands are added

distilled ethylene chloride, occasionally on- 550 cm ^{8 of} an ice-cold aqueous solution that is im

Emerging emulsions or suspensions through centri- liter 3.63 g sodium chloride, 1.86 g potassium chloride,

fugues are broken. After distilling off the 3.56 g of secondary sodium phosphate, 0.98 g of magnetic

dried ethylene chloride solutions remain a re-siumsulfat, 18 g glucose, 6.4 g sodium fumarate, 3 g

stood at 191.1 mg. This extract is in methylene 95 nicotinic acid amide, 1 g adenosine triphosphoric acid Na-

is dissolved chloride and contains an adsorption trium, and adjusted to p _H 7.50, in a

subjected to silica gel. 5 g of silica gel are homogenized in a homogenizer for 5 minutes. While

Methylene chloride is suspended and in a suitable homogenizing is done by adding 1N Na-

Column filled. First, the above hydroxide obtained is added prevents the p _H is below 7.3. the

Extract solution and then eluted on top of each other - the emulsion is poured into a stirrer and gas inlet

then emptied a vessel provided with methylene chloride ¹ (three fractions), chlorine tube and stirring

form (five fractions), chloroform-acetone 9: 1 and introducing oxygen 3 ¹ / * hours at 30 ⁰

(four fractions), 1: 1 (four fractions) and acetone left. The p _H is selected with a glass electrode.

(two fractions) - (10cm ⁸ per fraction). The one rend of the reaction followed and between 7.3 and 7.5

The individual fractions are kept constant on paper chromatography. The reaction mixture is

graphically for their adrenal cortex content in 5.5 l of cold acetone and each with 250 cm ³

steroids approved. All fractions were rinsed twice with cortisone and acetone. The 16 hours in the

17-oxycorticosterone (12 to 16) are combined. Solution left in the cold is occasionally turned around.

(24.5 mg) and stirred on a wide cellulose sheet and then suction filtered. The residue will

(known under the trade name "Whatman No. ι") 40 ^{washed with 500 cm 8} acetone; the United

with the system methanol-water-ethyl acetate-toluene acetone solutions are in a water jet vacuum at

(5: 5: 1: 9) chromatographed again, whereupon in the 40 to 45 ° bath temperature to about 200 cm ³

Sheet chromatogram each steamed in ultraviolet.

absorbing bands by ultraviolet photocopy. Further work-up is carried out as shown in Example 1. The one described for the 17-Oxycorti- 45. Degreased the Äthylenchloridextrakt (736.7 mg) costeron corresponding band which is also the aldosterone again by containing it in 40 cm ^3, is cut, with 20 ⁰ / pc alcohol methanol dissolves 95% ethanol and 40 cm ³ of petroleum ether and thoroughly eluted and the eluate, after concentration in this solution, was successively ^{mixed with 2.2, 5> 4 and} vacuum to three quarters of its original volume of 6.8 cm ^{3 of} water, ten times with the same volume of water after each water meter In addition, the petroleum ether solution which separates out from the purified mixture of ether-chloroform (3: 1) is separated and discarded.

extracted. After drying and evaporation, the defatted extract (188 mg) is applied to 4 g of silica gel (known as 500 γ in the propylene glycol-toluene system for 24 hours under the trade name) as described in the extract (4.6 mg) obtained in vacuo (4.6 mg) "Silicagel") chromatographed on paper, whereby the aldosterone 55 phiert are detectable (a total of 38.7 mg) are acetylated with reducing power to silver diammine or acetic anhydride in pyridine at 20 ⁰ and prepa-3, 3 - dianisole - to -4, 4 '- (3, 5 -diphenyl-tetrazolium- relative to paper with the system formamide Benchlorid at a defined point of the Chromate- ⁶ ° zol-cyclohexane (1: 1).? chromatographed at 40 ⁰ program (i _F value, which is the relative papierchromato- the acetate of the new aldosterone entsprec current graphical migration speed; see e.g. B. Bande (capable of about 11 -Dehydrocorticosteron-Journ. Of Biol. Chem., Vol. 177, S. in; about like acetate) can be detected analogously to the cortisone described in Example 1 under these conditions). Way localized, eluted and extracted. The ultraviolet absorption and reducing power of the extract (3.6 mg) are chromatographed again on probably standard substances such as 17-oxycorticosterone paper in the formamide-benzene systems and the new aldosterone is combined with one-formamide-benzene-cyclohexane (1: 1), whereupon the other is compared and from this the amount of the latter is determined semi-quantitatively with the indicators mentioned in Example 1. There are therefore 208 bis of the aldosterone acetate determined. In this way ge-240 γ of the new aldosterone has been formed, which is 70 depends on obtaining 220 γ diacetate, correspondingly

5 6

about an increase in the yield by the ten-toluene (5: 5: 1: 9) chromatographed again, whereupon the individual in the ultra-work-up of 225 g of untreated adrenal glands were times higher than that carried out in the sheet chromatogram. violet absorbing bands by ultraviolet. . photocopy can be made visible. The band corresponding to 17-Oxy-B e 1 s ρ 1 e 1 3 ₅ corticosteron, which also contains the

250 g of freshly slaughtered beef adrenal glands are contained with aldosterone, are cut out, with 20% 600 cm ^{3 of} an ice-cold aqueous solution, the addition of which is thoroughly eluted and the eluate, after being used in Example 1, is reduced to three quarters in vacuo Originally, siert in a homogenizer for 5 minutes homogenous volume ten times with the same volume. After 1 minute of homogenization, 10% of a purified mixture of ether-chloroform is added to 0.5 g of sodium adeno-triphosphate; (3: 1) extracted. After drying and then homogenizing for another 5 minutes. By evaporation in vacuo the obtained extract (6.1 mg) adding 1 η sodium hydroxide solution is prevented that the γ are 250 in the system of propylene glycol toluene p _H falls below 7.3. The emulsion is papierchromatographiert in the in the 24 hours, with the Examples 1 and manner described in detail 2 separates 15 aldosterone of 17-Oxy corticosterone and * treated by 3 ¹ / h at 30 ⁰ with oxygen and its absorbance in the ultraviolet at 240 πιμ reaction mixture in a corresponding manner of extraction and subjected to tion by its reducing power. Silver diamine or 3, 3-dianisole-bis-4, 4 '- (3, 5-di-

251.4 mg of ethylene chloride extract are obtained, which is chromatographed on phenyl) tetrazoliium chloride at a defined point 5 g of silica (known under the trade name 20 of the chromatogram (R _P - value roughly like cortisone "silica gel"). The fractions under these conditions) can be proven. Ultra-10 to 16 contain the corticosteroids (total violet absorption and reducing power both 21.2 mg). The elution and extraction of the 17-oxy- from standard substances such as 17-oxycorticosterone as the corticosterone band of the leaf chromatogram also from the new aldosterone are compared with each other (system methanol-water-ethyl acetate-toluene) and the amount of the latter gives half of 4.1 mg, which determines 405 γ of the new aldosterone de-quantitatively. In 250 γ extract, 33 to 36 γ of the new aldosterone can be retained, corresponding to an increase in the yield in this way, about twenty times as much as in the extraction, 805 to 880 γ per 250 g of adrenal glands, which is one of 250 g of untreated adrenal glands. Increase in yield by about thirty-five. . 30 to forty times that of the extraction of 250 g of Example 4 corresponds to untreated adrenal glands.

250 g freshly slaughtered beef adrenal glands are used to separate the active substance.

550 cm ^{3 of} an ice-cold aqueous solution, which is part of the extract (5.85 mg) of a preparative

Liters 3.63 g sodium chloride, 1.86 g potassium chloride, paper chromatography in the system propylene glycol-

Subjected to 3.56 g of secondary sodium phosphate, 0.98 g of Magne-35 toluene (24 hours). The ultra-

sium sulfate, 18 g glucose, 6.4 g sodium fumarate, violet photocopy certain band of the Aldoste-

i g contains sodium adenosine triphosphate and rons is cut out as described above,

is set to p _H 7.50, eluted in a homogenizer and extracted. The extract (1.2 mg) is left

Homogenized for 5 minutes. During the homogenization after drying in a high vacuum from acetone-ether

sierens is crystallized by adding 1 η sodium hydroxide solution and a trace of water. You get so

prevents that the p _H is below 7.3. For this pulp 0.55 mg of pure aldosterone from the double melting point

0.125 g of ιi-deoxycorticosterone are also given. ^I0 4 to 112 ⁰ and 153 to 158 ⁰ . More quantities

This emulsion is poured into a stirrer and gas inlet can be obtained from the mother liquor,
pipe provided vessel emptied and under. .

Stirring and introduction of oxygen 3 ¹ / "45 hours Example

leave at 30 ^0. The p _H is with a glass electrode battle 250 g fresh bovine adrenal be with

monitored during the reaction and between p _H 7.3 550 cm ³ of an ice cold aqueous solution which

and 7.5 kept constant. The reaction mixture liter 3.63 g sodium chloride, 1.86 g potassium chloride,

is then poured into 5 l of cold acetone and mixed with 3.56 g of secondary sodium phosphate, 0.98 g of magnetic

250 cm ^{3 of} acetone rinsed twice. The 16 hours 50 sulfate, 18 g glucose, 6.4 g sodium fumarate,

Solution left in the cold is occasionally 3 g of nicotinic acid amide, 1 g of adenosine triphosphoric acid

lent stirred and then sucked off. Contains the residue of sodium and is adjusted to p _H 7.50, in

it is washed with 500 cm ^{3 of} acetone and evaporated, the homogenized in a homogenizer for 5 minutes,

agreed acetone solutions in a water jet vacuum at. During homogenization, by adding

40 to 50 ⁰ bath temperature to about 200 cm ³ . 55 of 1 η sodium hydroxide solution prevents the p _H at 7.3

The remaining aqueous fat suspension sinks. To this slurry are still 0.125 g ii-Desoxyhiert to four times with 50 cm ³ of petroleum ether (bp. = Added 50 corticosterone. This emulsion is in a with up to 70 ⁰⁾ and then three times with 100 cm ^{3 of} fresh stirrer and gas inlet tube provided vessel umdestilliertem ethylene chloride wherein up occasionally emptied and left under stirring and passing in Sauer Treading emulsions or suspensions by centrifugal material 60 3 ¹ / * hours at 30 ^0th The p _H will be broken with fugues. After distilling off of a glass electrode during the reaction followed dried Äthylenchloridlösungen remains a back and kept constant between 7.3 and p _H 7.5, was 75.3 mg. This defatted extract is then added to 4 g of silica and ^{rinsed twice with 250 cm s of} acetone each time (known under the trade name "Silicagel") 65 in 5 l of cold acetone as described in Example 1. The 16 hours left in the cold is chromatographed. The solution in fractions 11 to 16 is occasionally stirred and then detectable adrenal cortical steroids are sucked off. The residue is washed with 500 cm ³ (14.7 mg) and acetone on a wide cellulose sheet and the combined acetone solutions are evaporated in a water jet vacuum (known under the trade name "Whatman No. ι") at a bath temperature of 40 to 45 ° using the methanol system —Water — ethyl acetate— 70 to about 200 cm ³ .

The remaining aqueous fat suspension is extracted four times with 50 cm ⁸ petroleum ether (b.p. - = 50 to 70 ⁰ ) and then three times with 100 cm ⁸ freshly distilled ethylene chloride, occasional emulsions or suspensions being broken by centrifugation. After the dried ethylene chloride solutions have been distilled off, a residue of 161 mg remains. This defatted extract is, as described in Example 1, chromutographed on 7 g of silica (known under the trade name "Silicagel"). The adrenal cortical steroids detectable in fractions 1 to 16 are combined (21.3) and placed on a broad cellulose sheet (known under the trade name "Whatman No. 1") using the system methanol - water - ethyl acetate - toluene (5: 5: 1: 9) chromatographed again, whereupon the individual bands absorbing ultraviolet in the sheet chromatogram are made visible by ultraviolet photocopy. The band corresponding to the 17-Oxycorticosteron, which also contains the aldosterone is excised, ehiiert thoroughly with 20 ° / pc alcohol and methanol da.s eluate, after concentration in vacuo to three-quarters of its original volume ten times with the same volume of a purified mixture of ether - Chloroform (3: 1) extracted. From the extract (5.4 mg) obtained after drying and evaporation in vacuo, 250 γ in the propylene glycol-toluene system are paper chromatographed for 24 hours, the aldosterone separating from 17-oxycorticosterone and its absorption capacity in the ultraviolet at 240 ΐημ and its reducing capacity compared to silver diamine or 3, 3-dianisole-bis-4, 4 '- (3, 5-diphenyl) tetrazolium chloride at a defined point on the chromatogram (Rp value roughly like cortisone under these conditions). Ultraviolet absorption and reducing power both of standard substances such as 17-oxycorticosterone and of aldosterone are compared with one another and the amount of the latter is determined semi-quantitatively from this. 250 y extract 27 to 29 γ aldosterone can thus prove or 580-625 γ per 250 g adrenal glands, corresponding to an increase of the yield by about twenty-five to thirty times with respect to the extraction of 250 grams untreated adrenal glands.

Claims (6)

PATENT CLAIMS: 1. Process for the separation of aldosterone ι »or its diacetate from adrenal extracts, characterized in that comminuted adrenal glands are first treated with oxygen and then separates the aldosterone in a manner known per se as a free compound or as a diacetate. 2. The method according to claim 1, characterized in that that one in the cold in the presence of suitable buffer solutions homogenized cattle or Pig adrenal glands used. »0 3. The method according to claim 1 and 2, characterized characterized in that the enzyme systems important for the reaction are added by adding suitable Maintains functional materials and compliance with appropriate environmental conditions. »5 4. The method according to claim 1 to 3, characterized characterized in that as substances which the enzyme systems get functional, the as Substrates of the cyclophorase system known substances, e.g. B. the acids of the citric acid cycle or compounds related to this last-mentioned, in particular fumaric acid, adenosine triphosphoric acid and nicotinic acid amide is used. 5. The method according to claim 1 to 4, characterized in that the reaction mixture u-deoxycorticosterone adds. 6. The method according to claim 1 to 4, characterized in that zwisehen the oxygen treatment in the presence of components of physiological solutions, especially at a _pH value 6.5 and 9, performs.