DE1070176B - Process for the production of new steroid compounds of the 1, 4 - Pregnadiene series - Google Patents

Description

^ o ^

FEDERAL REPUBLIC OF GERMANY

GERMAN M & m, PATENT OFFICE

kl. 12 ο 25/05

INTERNAT, ία. C 07 C EDITORIAL 1 070 176

A 25914 IVb / 12 ο

ANMEtDAY: OCTOBER 26, 1956

NOTICE THE REGISTRATION AND ISSUE OF THE EDITORIAL: DECEMBER 3, 1959

The invention relates to a process for the production of new steroid compounds of the 1,4-pregnadiene series.

Recently, a number of Pregnen and Pregnadiei series steroid compounds, e.g. Hydrocortisone and 1 (2) -dehydrocortisone have become important both as valuable therapeutics and as useful intermediates for the manufacture of other steroids useful for therapeutic purposes. The new 1,4-pregnadienes which can be produced by the process according to the invention are particularly suitable as anti-inflammatory or anti-inflammatory agents in the treatment of arthritis, asthma, burns, bursitis and the like in dermatology and in the treatment of collagen diseases . The compounds are used in combination with fillers, carriers, etc. in the form of tablets, powders, pills, etc., for use. In addition, they can be administered parenterally as a solution or as a suspension.

According to the invention, the new 1,4-pregnadienes are produced by adding a 4-pregnant compound to the general formula

CH ₂ OR

C = O

R '

Method of manufacture

new steroid compounds

the 1,4-pregnadia series

Applicant:

American Cyanamid Company,
New York, NY (V. St. A.)

Representative: Dr. F. Zumstein
and Dipl.-Chem. Dr. rer. nat. E. Assmanrt,
Patent Attorneys, Munich 2, Bräuhausstr. 4th

Claimed priority:
V. St. v. America March 30 and May 22, 1956

Robert Henry Blank, Seymour Bernstein,
William Shardlow Allen, Pearl River, NY,

Louis Israel Feldman, HiUcrest, NY,
and Robert Herman Lenhard, Ridgefield Park, NJ

(V. St. A.),
have been named as inventors

, x

in which X is hydrogen or halogen, Y is a methylene, oxymethylcn or carbonyl group, Z is an oxymethylene or alkanoyloxymethylene group with a lower alkanoyl radical, R 'is hydrogen or a hydroxyl group and R Means hydrogen or a lower alkanoyl group, in a known manner of the fermentative, enzymatic groups Exposure to organisms of the genus Nocardia or of the organism Corynebacterium simplex below. Those formed in the process according to the invention 1,4-Pregnadienes correspond to the general formula

CH ₂ OR

■ ί
C = O

O =

where the individual symbols are those given above Have meanings.

When carrying out the method according to the invention the following microorganisms can be used:

Mushrooms from Genus Nocardia (Bergey, Manual, 6th edition), e.g. coraUina ATCC999 and ATCC4273 (Lederle Culture No. 13), asteroides, ATCC 3308, keratolyticus, ATCC 12484, transvalensis, ATCC 12485, erythropolis, ATCC 4277, convoluta, ATCC 4275, gardneri, ATCC 9604,

leishmanii, ATCC 6855, opaca, ATCC 4276, blackwelü, ATCC 6846; caviae, ATCC 6848; cuniculi, ATCC 6864, globcrula, ATCC 9356; polychromogenes, ATCC 3409, sylvodifera, ATCC 7372; farcinica, ATCC 3318, sylvodifera, ATCC 4919 or other species of Nocardia as well

Corynebacterium simplex. The microorganism used in each case is aerobically in a suitable nutrient medium, containing a 4-pregin derivative. During the growth of the organism under favorable conditions are made from ring A of the steroid compound

Removed 2 hydrogen atoms, creating a double bond in the 1 (2) position. The exact one The process of this dehydration is not yet clear, but it is the result of the action of the Enzymes formed in the organism during growth.

909 687/421

A suitable nutrient medium contains a soluble source of carbon, nitrogen and mineral components. Sources of carbon include sugars such as glucose, sucrose, maltose, dextrose, X} * lose, and galactose; Alcohols e.g. B. glycerol or mannitol, corn starch, organic acids, e.g. B. citric acid, malic acid and acetic acid, and natural products containing many carbohydrates, e.g. B. corn steep liquor, soybean meal, cottonseed meal and many other available substances that have previously been used as carbon sources in fermentation experiments. Usually, any selection of the above can be used with good results for the medium.

Suitable nitrogen sources include some of the im substances mentioned above, e.g. B. Corn steeple, soybean meal, cottonseed meal and the like, and various other substances, e.g. B. meat extract, casein, yeast, enzymatically digested proteins, and breakdown products, including peptones, amino acids and many other available protein-like substances that have been found to be suitable for promoting fungal growth are to be expected. Inorganic nitrogen sources, which include, for example, urea, ammonium salts and nitrates, can be used as a source of in the medium assimilable nitrogen can be used; in the latter case, too, growth of the organism is preserved promoting nutrient fluids.

The minerals required for fermentation are usually supplied from the raw materials, most often used as carbon and nitrogen sources, or are present in the water used in the process. Usually, however, it is advisable to supplement the minerals normally present by adding further amounts in order to achieve maximum growth of the fungi of the genus Nocardia. The cations and anions that may be useful to add include sodium, potassium, calcium, magnesium, phosphate, sulfate, chloride, cobalt, manganese, and various others. The use of trace elements, e.g. B. boron, copper, cobalt, molybdenum and chromium is often useful.

The growth of the fungi of the genus Nocardia and \ 'on Corynebacterium simplex takes place under aerobic conditions. The ventilation of flasks, for example, can be achieved by shaking on a shaking machine with back and forth motion or on a rotary shaker and the ventilation of bottles or tanks by passing sterile air through the fermentation mixture. It is advisable to ^{pass the sterile air through in an amount of: t} / ₃ to 2 volume / volume of medium per minute. The movement in the bottles or fermentation tanks is effected with a mechanical stirrer. The fungus Nocardia grows at temperatures between 5 and 45 ⁰ C, but the method is preferably carried out at a temperature between 25 and 37 ° C. Corynebacterium simplex grows at temperatures between 10 and 45 ^° C., but temperatures between 25 and 38 ^° C. are preferably used in its cultivation.

For the preparation of vaccines, 1.0 ml of a washed vegetative cell suspension of an organism of the genus Nocardia or of Corynebacterium simplex is used to inoculate 100 ml of sterile trypticase soy broth in a 500 ml Erlenmeyer flask. . The trypticase soy broth contains 3 ⁰ Z _{0 of} a tryptic digestion product of soy proteins as a dehydrated powder (Baltimore Bacteriological Laboratories), 5% glycerol, 0.3% meat extract (Armor); Geroisch this is sterilized for 15 minutes at a temperature of 12O ⁰ C at about 1 atmosphere of steam pressure (15 poundi ,. steam pressure). After sterilization ^: the pH value is in the range from 7.4 to 7.7. The inoculated flask is incubated at 37 ° C for about 4 to 8 hours on a shaker. Such inocula can be used to inoculate larger batches of sterile medium in bottles, and these bottle cultures, after fermentation, can be used to inoculate large batches of the medium in fermentation tanks. At

ίο Place the trypticase soy broth described above other media can be used, as shown in the examples below.

The 4-position unsaturated compounds of the which can be used in the process according to the invention Pregnan series include, among others: 4-Pregnen-l 1 / 3.16 «, lTo ^ l-tctraol-S ^ O-dion, 4-Pregnen-9a, nuor-ll / ?, 16a, 17a, 21-tetraol-3,20-dione and 4-pregnen-16a, 17ct, 21-triol-3, ll, 20-trione and esters and similar derivatives of these compounds. When using the listed steroid substrates the free steroid compounds are formed during fermentation. The steroids are generally used introduced into the fermentation as a solution or in finely divided form. A preferred way of working consists of dissolving the steroid in ethanol or other water-miscible solvents and adding it to to add the desired process stage to the fermentation medium. Even if the steroid is at a such addition precipitates out of solution, it is distributed as a fine suspension throughout the medium and so on Easily available to the organism for oxidation. The amount of steroid added during fermentation can vary considerably, but is usually in the range of

Order of magnitude from ¹ Z ₁₀ to 1 g / liter of medium.

During fermentation it may be useful to add defoaming agents, e.g. B. silicone, glyceride oils and the like. To add. These compounds are added from time to time and in the amounts required in each case. When carrying out the method according to the invention, the 10 ml batches of inoculated medium are incubated in 100 ml Schiittel tubes usually for a period of about 16 to 40 hours at a temperature of about 32 ° C. At this point, a solution of 2 mg sterile substrate (4-Pregnenverbindung) introduced in 0.2 ml of ethanol and continue the fermentation at about 32 ⁰ C., the fermentation is carried so long that maximum conversion of the 4-Pregnens in the 1,4-pregnadiene is achieved. The time required for this can vary between 1 and 72 hours or more.

After the fermentation is complete, the desired 1,4-pregnadiene becomes as described below Method of operation, which is explained using the example of a fermentation with 10 ml, won. This way of working is generally applicable and for carrying out fermentations with the most varied of volumes suitable.

The contents of a fermentation tube are extracted 4 times with 4 volumes of methylene chloride each time. The four Extracts are combined, and the solution obtained is once with 2 ° / ^ 6γ aqueous sodium bicarbotate solution, which is saturated with sodium chloride, and then washed twice with saturated sodium chloride solution. The washed methylene chloride solution can be dried over anhydrous magnesium sulfate and filtered off.

The filtrate is concentrated to a small volume on the steam bath at atmospheric pressure, and the concentrate is transferred to a 10 ml graduated measuring vessel and made up with methylene chloride. This solution is used to determine steroid levels as follows described, used.

5 6

In the following examples, dehydration can be used in large-scale fermentations

Crude product or the crude products can be obtained from the compounds unsaturated in the 4-position

Fermentation liquid by simple solvent extraction Pregnenzeile with formation of the corresponding in 1- and

using a suitable steroid of the Pregnadic group which is not 4-position unsaturated with water miscible solvent, e.g. B. chlorinated low- 5 individual explained,

molecular hydrocarbons, alcohols, esters, ketones Example 1

etc., can be obtained. The further cleaning and _n " _o « «...., ο ™ j ·

Separation of the steroid compounds formed aas den M-Pregnadien-ga-fluor-ll ^ loa. ^ A ^ l-tetraol-S ^ O-dione

Extracts can be prepared by methods known per se. A test tube agar prepared with yeast extract

take place. In the separation and purification of an io from Nocardia sp. (ATCC 12483) was made more sterile with 7 ml

It is often necessary to chromatograph steroid mixtures. Washed saline solution, and the vegetative line obtained

The identification of the suspension described above was made to inoculate 100 ml more sterile

Extracts of the fermentation liquid containing steroid (as described above) trypticase soy broth. in one

bindings is used by paper strip chromatography 500 ml Erlenmeyer-Kolbeii. The flask was

performed. As a solvent system, water is incubated for 15 7 hours at 37 ° C. This inoculum was

Methanol — Benzene used by shaking it to inoculate sterile trypticase soy broth.

about 50 ° / ₀ water and 50 ° / ₀ methanol with benzene in medium used in 100 ml pouring tubes. 1 ml vaccine

a separatory funnel and separating the two fabric was used to inoculate 10 ml of a medium each time

forming layers is produced. A portion of 2 ° / ₀ molasses (Grandma), I ⁰ ₀ Z meat extract (Difco)

lower layer is used in an open dish on the bottom ao and 1 ° / ₀ glucose in a 100 ml shaker tube,

of a large glass cylinder. The top layer The tubes were incubated at 32 ° C for 40 hours. to

is the solvent phase and is used to fill the at this point each tube was a solution of

trough-shaped vessel used in the cylinder. 2 mg of 4-pregnen-9a-fluoi "-11 / ?, 16a, 17a, 21-tetraol-3,20-dione are prepared

Prepare a standard steroid solution by adding 10 mg in 0.2 ml of ethanol to each. The incubation was then

of the following steroid compounds in 10 ml of methylene 35 continued for a further 24 hours.

chloride dissolves: The 10 ml mash from one of the

4-Pregnen-II / ?, 17a, 21-triol-3,20-dione, bencn tubes were 4 times with 40 ml of methylene chloride

4-Pregnen-1 l / ?, 21-diol-3,20-dione, extracted. The extracts were combined and in vacuo

4-pregnen-17a, 21-diol-3,20-dione, evaporated to dryness. The residue was in 2 ml

4-Pregnen-lla, 17a, 21-triol-3,20-dione, 30 acetone, and a sample thereof was after

4-Pregnen-lljS, 16a, 17a, 21-tetraol-3,20-dione, paper strip method described above was analyzed.

4-Pregnen-ll / ?, 16a, 17a, 21-tetraol-3,20-dione-16,21- The chromatogram showed the presence of 1,4-Pre-

diacetate, gnadien-9a-fluoro-II / ?, 16u, 17a, 21-tetraol-3,20-dione.
4-Prcgnen-9a-fluoro-II / 5,16a, 17a, 21-tetraol-3,20-dione,

4-pregnene-9 <x-fluoro-ll _j Ö, L6A, 17a, 21-tetraol-3,20-dione 35 Example Z

16,21-diacetate. 1,4-Pregnadiene-II / ?, 16a, 17a, 21-tetraol-3,20-dione
(If appropriate, other steroid compounds can also be used for the standard solution.) A test tube manure obtained with potato dextrose was used for the standard solution Unknown compositions of the vegetative cell suspension obtained in this way were chromatographed for sedimentation. Exactly 0.025 ml of the standard inoculation of 10 ml portions of sterile, pn 7 steroid solution is used on the paper strip at the set medium # 13 in 100 ml shaker tubes starting line, approximately 10 cm from the top of the strip. The tubes were removed and applied for 40 hours at 32 ° C., which is placed in the trough-shaped vessel of a shaking device with to-and-fro movement and thereby shaken into the 45 contained therein and then immersed in each case with a solution of 2 mg of solvent phase. The strip is then developed 4-Pregnen-II ^, 16a, 17a, 21-tctraol-3,20-dione in 0.2 ml for 2 to 4 hours at about 37 ° C. In 70 ° / ₀ aqueous ethanol is added. The tubes were applied in the same way, 0.1 ml of the unknown solution, after 6, 24 or 72 hours of incubation after the addition of the composition, was applied to another strip and the substrate was harvested.

which was then placed in the same trough-shaped vessel and 50. Each of the fermentation mashes was 4 times with 40 ml each

extracted simultaneously with the strip containing the steroid standard methylene chloride. The individual extracts each

solution is developed. The trough-shaped mashes were combined and placed under reduced pressure

allows many stripes to be developed at the same time. evaporated to dryness. Each residue was in 2 ml

After properly carried out development of the pyridine added and with 0.5 ml of acetic anhydride

Paper strips are removed 55 from the device. These mixtures were left on for 10 to 15 minutes

and air dried at about 37 ° C. Thereafter, a steam funnel is heated and then in a vacuum

they evaporated to dryness with an alkaline solution of tetrazolium blue. Each residue was in 2 ml

sprayed, creating the places where the steroids dissolved acetone, and appropriate volume of the so obtained

become visible through their color development. Solutions were chromatographed. The Chromato-

The stained stripes were at least 60 grams indicated that the substrate was in 1,4-pregna-

Standard strips laid parallel and compared with this. dien-ll / ?, 16a, 17a, 21-tctraol-3,20-dione

The individual steroids can then be based on their location.

the stripes can be identified. B '13

The desired, 1- and 4-position unsaturated? I ^Sp / ~

Steroids are more polar than the corresponding ones in the 4-position 65 l, 4-pregnadiene-ll /?, 16o, l7a, 21-tetraol-3,20-dioii
unsaturated steroids. It should be noted that the desired 1,4-position unsaturated steroidal compounds agar prepared with potato starch, agar from Nocardia leishmanii (ATCC 6855), was washed with 5 ml of sterile saline, once isolated and identified. 1 ml portions of the standard steroid solution to improve the vegetative cell suspension obtained in this way could be used for the applied working method. 70 vaccinated from. 10 ml portions of sterile medium No. 13,

7 8

which had been adjusted to _{pH 7 was used.} Each incubated at 32 ° C and then with a solution of

these portions were in a 100 ml shaker tube. 2 mg substrate, 4-pregnen-ll /}, 16ct, 17a, 21-tetraol-3,20-

The tubes were mixed on a shaker with dione in 0.2 ml of 70 ° / ₀ aqueous ethanol.

Shaken reciprocating at 32 ° C for 40 hours; After a subsequent further incubation period of ¹ Z ₂ , at this point in time a solution of 2 mg 5 2, 7, 24 and 72 hours was harvested, respectively.

Substrate, 4-Pregnen-II / 5,16a, 17a, 21-tetraol-3,20-dione in each of the fermentation mashes was mixed 4 times with 40 ml each

0.2 ml of 70 ° / ₀ aqueous ethanol to each tube thylcnchlorid extracted. The extracts of each mash

given. After 6, 24 and 72 hours, the tubes were combined and concentrated under reduced pressure.

further incubation after adding the substrate. steams. Each of the residues thus obtained was in Each of the fermentation mashes was taken up 4 times with 40 ml io 2 ml pyridine and 0.5 ml acetic acid

Extracted methylene chloride. The anhydride added to each mash. These mixtures were 10 to IS Mi-

The extracts obtained were combined and heated on a steam funnel with trimmings and then im

reduced! Pressure evaporated to dryness. Each of these vacuum evaporated to dryness. Each of these return

Residue was taken up in 2 ml of pyridine and was dissolved in 2 ml of acetone, and appropriate mixed with 0.5 ml of acetic anhydride. The 15 volumes of the solutions obtained in this way were chromato-

Mixtures were steam graphed for 10 to 15 minutes. The chromatograms indicated that

funnel and then in a vacuum to dry the substrate in l ^ -Pregnadien-lljCMoa ^ a ^ l-tetraol-

evaporated. Any of the 3,20-dione thus obtained had been converted.

Residues were dissolved in 2 ml of acetone, and appropriate. .

Volumes of these solutions were chromatographed. 20 example 0

The chromatograms showed that the substrate was in 1,4-pregnadiene-II / 3,16a, 17a, 21-tetraol-3,20-dione

1,4-Pregnadien-ll / ?, 16a, 17a, 2) .- teraol-3,20-dione überge- A test tube agar prepared with yeast extract

had been leading. of Nocardia corallina (ATCC 999) was replaced with 7 ml

Example 4 ^ ^cr brine washed. "The suspension obtained

, .-r,, . ΛΑΛΑ, α ·, n <. ι ο ~,, ■ ² 5 became 100 ml of a trypticase soy broth

1.4-Pregnadien-ll / i.l6o, 17a.21-teti _a ol-3,2 (M ₁ on _{without glycerine in a} 100 ml Erknmcycr flask.

Put a test tube prepared with potato dextrose. The mixture was shaken at 37 ° C

Nocardia caviae agar slants (ATCC 6848) were incubated for 8 hours. Fifteen Erlenmeyer flasks that

Washed 5 ml of sterile saline solution. 1 ml portion of each 100 ml sterile medium No. 13 contained were

The vegetative cell suspension obtained was inoculated with 30 ml of this vaccine for inoculation. The pistons

of 10 ml portions of No. 13 sterile medium that were incubated with shaking at 32 ° C for 40 hours

pH 7 was used. Each of these proportions and each with a solution of 20 mg 4-Pregnen-II / J,

was in a 100 ml shaker tube. The tubes 16a, 17a, 21-tctraol-3,20-dione mixed in 2 ml of ethanol,

were then kept on a shaker at 32 ° C for 40 hours with shaking at 32 ° C during

shaken with back and forth movement and then incubated for _{35 8 * / 2 hours.} The contents of this flask were

combined with a solution of 2 mg of substrate, 4-pregnen-l 1 ß , and a mash with a pH of 7.3 was obtained.

16tt, 17a, 21-tetraol-3,20-dione, in0.2 ml 70 ° / ^ εηιwäßrigem The fermentation liquid combined after harvesting was

Ethanol added. The tubes were extracted after 6, 24 and in the usual manner, leaving 352 mg of crude crystals

72 more hours of incubation after the addition of the were obtained. By recrystallization from acetone-

Harvested substrate. 40 petroleum ether gave 145 mg of substance from F. - 216

Each of the fermentation mashes was mixed 4 times with 40 ml each to 218 "C. Recrystallization from acetone-petroleum ether - methylene chloride extracted. The extracts from each mash fermented the melting point to 229-231 ° C. The infrared was used combined and under reduced pressure to the spectrum was with that of an authentic sample Evaporated to dryness. Each of the residues was in by 1,4-Pregnadien-ll / ?, 16a, 17a, 21-tetraol-3,20-dioii 2 ml of pyridine taken up and identical to 0.5 ml of acetic acid 45.

anhydride added. These mixtures were 10 to 15 mi

grooves heated on a steam funnel and then in. . , ..,. _{Λ Λ} ~ _{Λ rt} », ...,, .--,.

Evaporated to dryness in vacuo. Each of the 1,4-pregnadiene-1,4, 16a, 17a, 2l-tetraol-3,20-dione thus obtained

Residues were then dissolved in 2 ml of acetone. Appropriate test tube volumes prepared with potato dextrose the solutions obtained were chromate agar slants from Nocardia globerula (ATCC 9356) graphed, and the chromatograms showed that this was washed with 5 ml of sterile saline, and proportions of Substrate in l ^ -Pregnadicn-ll ^ löa.Ua ^ l-tetraol-S ^ O- each 1 ml of the vegetative cell suspension obtained dion had been convicted. were used to inoculate 10 ml portions of sterile

dium no. 13, which was adjusted to pH 7, is used.

Example 5 _{55 each of the ante} j] _e was in a 100 ml shaker

1,4-Pregnadiene-ll, S, 16a, 17a, 2: l.-tetraol-3,20-dione _ΓΟηΓ . The pipes were on a reciprocating basis

A test tube shaking device obtained with potato dextrose is incubated at 32 ° C. for 40 hours and

Slant agar from Nocardia convoluta (ATCC 4275) was then each with a solution of 2 mg substrate, 4-Pre-

washed with 7 ml of sterile saline, and ImI of raising campaigns-ll ^, 16a, 17a, 21-tetraol-3,20-dione, in0,2ml70 _^0/0 sodium

The vegetative cell suspension obtained was mixed with 60 aqueous ethanol for inoculation. The pipes were after

of 10 ml of sterile medium No. 13 is used, which is added before the incubation is carried out for a further 6, 24 or 72 hours

the treatment in the autoclave has been set to pn 7, addition of the substrate has been gccrntet.

(pH value after treatment in the autoclave 6.75). Each of the fermentation mashes was 4 times with 40 ml Me-Das the 100 ml shaker tube used here was extracted onto ethylene chloride. The one obtained from each mash Shaker with reciprocating motion 65 tenen extracts were combined and mixed under vermin-16 Shaken at 32 ° C for hours. 1 ml portions of the dertem pressure evaporated to dryness. Everyone who has regressed Growth were stood for inoculation of five was taken up in 2 ml of pyridine and with Add 10 ml portions of sterile medium No. 13 to 100 ml 0.5 ml acetic anhydride. These mixtures Shaker tubes used. These tubes were placed on a steam funnel for 10 to 15 minutes heated with back and forth movement for 16 hours and then evaporated to dryness in vacuo.

9 10

The residues obtained in this way were converted into 1,4-pregnadiene-II / 3,16u, 17a, 21-tetraol-3,20-dione.

in each case 2 ml of acetone had been dissolved, and appropriate volumes of this had been carried out.

Solutions were chromatographed. The Chromate- At ό '111

grams showed that there '; Substrate in 1,4-pregnadiene _{n fl} iL · <, _{O (} ., „,.

11 / M6a, 17a, 21-tettaol-3,20-dione had been transferred. 5 ¹ > 4-pregnadien-9 "-fluoi-l / ?, 16a, 17", 21-tetraol ^ O-dione

A yeast extract are fermented slant \ ^r on

Example 8 Nocardia corallina (ATCC 999) became more sterile with 7 ml

1 / 1TJ j . η οι * i ι α washed on λ salt solution, and the suspension obtained

M-Pregnadien-ll / Uoa. ^ A ^ l-tetraol- ^ O-dione _{was za} f _m inoculated from 100 ml of medium No. 13 in

An agar slant of the culture prepared with yeast extract was used in a 500-nil Erlenmeyer flask. The Ge-Nocardia corallina (ATCC 999) was treated with 7 ml of sterile mixture was washed with shaking at 37 ° C for 7 ^l / ₂ hours salt solution, and incubated the resulting suspension. 1 ml portions of this culture were then used to inoculate 100 ml of No. 13 sterile medium to inoculate 10 ml portions of the sterile medium in a 500 ml Eiienmeyer flask. The No. 13 used in 100 ml fermentation tubes. The mixture was incubated with shaking for 7 ¹ Z hours at 37 "C. 15 inoculated tubes were incubated for 40 hours at 32" C. 1 ml portion of this culture was used for inoculation and then in each case with a solution of 2 mg 4-Pregncn of 10 ml portions of the sterile medium in 100 ml tubes 9a-fluorine-II / §, 16a, 17a, 21-tetraol-3, 20-dione used in 0.2 ml of atha. These tubes were added 40 hours at 32 NOJ ^Cl C. The tubes were incubated for a further 72 hours and then each incubated with a solution of 2 mg. The contents of a tube were then extracted 4 times with 4-Pregncn-II / 3.16 ^, 17α, 21-tctΓaol-3,20-dione-16,21-di- ao per 40 ml of methylene chloride. The combined acetate in 0.2 ml of ethanol was added. Then the extracts were evaporated to dryness in vacuo. Incubated for 24 hours. The contents of a tube (10 ml). The residue was taken up in 2 ml of pyridine and extracted 4 times with 40 ml of methylene chloride each time. The mixed with 0.5 ml of acetic anhydride. This mixture of combined extracts was evaporated to dryness under reduced pressure on a steam funnel for 10 to 15 minutes. The residue was evaporated to dryness with 25% and then in vacuo. A 2 ml pyridine was added and 0.5 ml acetic acid-suitable volume of the reverse anhydride dissolved in 2 ml acetone was added. This mixture was chromatographed on a stand. The funnel was heated by chromatography for 10 to 15 minutes and then considerable amounts of 1,4-Pregnadicn vacuum could be evaporated to dryness. A suitable volume of the residue dissolved in 2 ml of acetone was detected. It could thus be shown chromatographed. The Chrotnatogram showed that the l ^ -pregnadiene-ga-fluoro-llß.lou. ^ A ^ l-tedas substrate in 1,4-pregnadiene-II / 0,160,170,21-tetraol-traol-3,20-dione had been converted into larger 3,20-dione during fermentation. Had formed crowds.

Example 9 ₃₅ Example 12

1,4-pregnadiene-l 1 / ?, 16α, 17α, 21 -tetraol-S ^ -dione 1 ^ -Pß

The experiment described in Example 6 was carried out with 3,20-dione-16,21-diacetate
Nocardia sP (ATCC 12 483) in place of Nocardia 7 ml of sterile saline was repeated for washing one with corallina. A paper strip chromatogram of yeast extract prepared test tube slants from Node's product revealed l ^ -prgnadien-ll / Uoa.na ^ l-te-40 cardia corallina (ATCC 999) used, and the traol-3,20-dione obtained, which was obtained with the compound according to Example 6 The suspension obtained was used to inoculate 100 ml of sterile product which was identical. Medium No. 13 in a 500 ml Erlcnmeyer flask. The _. ^. _{1 (} .. Mixture was incubated for 8 hours at 37 ° C and two-. Example IU and thirty flasks with 500 ml capacity and each -ll ^ loa ^ a ^ l-tctraol-S ^ O-dione ₄₅ 100 ml sterile medium with 1 ml of this growth

A test tube inoculated with potato dextrose. These flasks were incubated at 32 ⁰ C 40 hours beschrägagar of Nocardia polychromogcnes (ATCC 3409) breeds and then in each case mg with a solution of 25 was washed with 5 ml of sterile saline, and 1 ml of 4-pregnene-9α-fmor-ll / 9, 16α, 17α, 21-tetraol-3,20-dione-16, portion of the vegetative cell suspension obtained was added to the 21-diacetate in 2.5 ml of ethanol. After inoculating 10 ml portions of pH 7 incubation for 53 hours, the contents of these flasks were combined and sterile medium No. 13, to which an equal volume of acetone was added in 100 ml shaking tubes. Which were found to be used. The tubes were filtered on a Mn solution through diatomaceous earth and the shaking device was evaporated at 32 ° C for 40 hours and acetone under reduced pressure. The shaken and then each with a solution aqueous phase was saturated with salt and extracted 5 times with 2 mg of substrate, 4-pregnene-llß, 16a, 17 «, 21-tetraol-55,500 ml of n-butanol. The butanol extract was 3,20-dione, ^{evaporated to dryness in 0.2 ml 70 0} Z ₀ IgCm aqueous ethanol under reduced pressure. After a further 6, 24 or.

The fermentation mashes were filtered 4 times with 40 ml of methylene 60 atomic earth each time. After evaporation of the acetone chloride extracted. The extracts of each mash were dissolved, the residue was dissolved in pyridine and combined with vinegar and acid anhydride evaporated to dryness under reduced pressure at room temperature overnight. Each residue was dissolved in 2 ml of pyridine. After addition of methanol, the solution was taken up under reduced pressure and evaporated to dryness with 0.5 ml of acetic anhydride. One replaces. These mixtures were kept on a steam funnel 65, 1.17 g of residue. Heated by partition chromatography for 10 to 15 minutes and then evaporated to dryness in vacuo for graphy using the solvent system. Each of the back thus obtained, 3 parts of ethyl acetate, 2 parts Pctroläther (Siedestände was dissolved in 2 ml of acetone, and proper range 90 to 10O ⁰ C), 3 parts of methanol, 2 parts of water volumes of these solutions were chromatographed. 0.50 g of the desired crude product, 1 ₍ 4-Pregna-Die chromatograms showed that the substrate in 70 dien-9a-fluoro-II / J, 16a, 17a, 21-tetrao1-16,21-diacetate

11 12

won. When recrystallizing the residue from strat in M-Pregnadien-II / J.lfi ^.

Acetone-petroleum ether gave 341 mg of substance which had been transferred.

with prior softening in the range of 153 to

Melted 233 ° C. The wide melting point range is similar

presumably due to solvation. 5. ,

1,4-Pregnadiene-llß, 16a, 17a, 21-tetraol-3,20-dione

Example 13 A test tube prepared with potato dextrose

. , τ, j _Λ ., η λc \ -τ ο, χ χ ι ο ^ r ₁ j slant agar of Nocardia sylvodifera (ATCC 4919)

1,4-prenadiene-llö, 16a, 17a, 21-tetraol-3,20-dione. r ι ± · ι c · τ ■, - ■, j α ±. · Ι

⁵ '''washed with 5 ml of a higher salt solution, and portions of

A are fermented with potato test tube io 1 ml of the obtained Vegetativzellsuspension were slant of Nocardia asteroides (ACCT 3308) was the adjusted to inoculate 10 ml portions to p _H 7 washed with 500 ml of sterile saline, and shares sterile medium no. 13, which were each in 100 ml of 1 ml each of the vegetative cell suspension obtained were shaking tubes were used. The tubes were placed in the inoculation of 10 ml portions of the pH, on a reciprocating shaker set at No. 13 sterile medium, shaken in 100-roll-15 32 "C for 40 hours and then shaken with a solution used. The tubes were of 2 mg substrate, 4-pregnene-ll / 3,16a, 17u, 21-tetraolauf dioii 3,20-a reciprocating shaker at / _o dissolved in 0.2 ml of aqueous 70 ° Ethanol was ^{shaken at 32 °} C. for 40 hours and then each set with one .. The tubes were incubated after a further 6, 24 or a solution of 2 mg of substrate, 4-Pregnen-II / S, 16a, 17a, 72 hours after the addition of the substrate 21-tetraol-3,20-dione in 0.2 ml of 70 ° / ₀ igern harvested aqueous 20th

Ethanol added. The tubes were after a further 6, 24 each of the fermentation mashes became 4roal with 40 ml each

or 72 hours of incubation after the addition of the substrate methylene chloride. The extracts of each mash harvested. were combined and prepared under reduced pressure

Each of the fermentation mashes was evaporated 4 times with 40 ml dryness each time. Each residue was extracted into 2 ml of methylene chloride. The combined extracts of pyridine were taken up and 0.5 ml of acetic anhydride from each mash were added under reduced pressure. These mixtures were evaporated on a datap dryer. Each of the residues obtained was funneled for 10 to 15 minutes and then taken up in vacuo in 2 ml of pyridine and evaporated to dryness with 0.5 ml. Acetic anhydride was added to each of these residues. These mixtures were dissolved in 2 ml of acetone and appropriate volumes of such a steam funnel heated for 10 to 15 minutes and then the resulting solutions were chromatographed. The: evaporated to dryness in vacuo. Each of the chromatograms thus showed that the substrate obtained in 1,4-pregna residues was dissolved in 2 ml of acetone, and diene-II / S, 16a, 17a, 21-tetraol-3,20-dione were transferred to appropriate volumes of these solutions chromate was.
graphed. The chromatograms showed that the sub- _R ■ ■ 1 ₁₆

strat in 1,4-pregnadiene-ll ^, 16a, 17o, 21-tetraol-3,20-dione 35. , _{B v (1Ä} 7- ° ₊₊ ,, ^ ,.

had been convicted. 1,4-Pregnadien-11 ^ 16 ", 17", 21-tetraol-3.20-dione

A yeast extract agar slant from Nocardia corallina Example 14 (ATCC 999) was washed with sterile saline.

l, 4-Pregnadien-l ^, 16a, 17a, 21-tetraol-3,20-dione Pj * obtained \ -egetative _Z cUsuspension was in a

^r 40 500 ml Erlenmeye.r flasks containing 100 ml of sterile medium

A No. 13 test tube prepared with potato dextrose was inserted. This flask was at

Slant agar of Nocardia cuniculi (ATCC 6864) was at 37 ⁰ C 7 ¹ Z ₂ hours on a reciprocating

Washed 7 ml of sterile saline solution. Leave 1 ml of the shaker obtained. 1 ml each of the formed

Vegetative cell suspension was used to inoculate 10 ml of growth was placed in five 100 ml shaker tubes

sterile medium No. 13, which before the treatment in 45 capacities with 10 ml of sterile medium

Autoclave was set to pn 7 (ρκ-value according to No. 13 introduced for inoculation. The tubes were opened

Treatment in the autoclave 6,75) and in a 100-inl- a reciprocating shaker

Schuttelrohx found used. The tube was incubated at 32 ° C for 40 hours and then each with one

a reciprocating shaker 16 hours solution of 2 mg 4-Pregnen-II / J, 16a, 17a, 21-tetraol-

the shaken at 32 ^{0 C.} Portions of each ImI des 50 3,20-dione-16,21-diacetate added in 0.2 ml of ethanol,

^{After a further 2, 7 χ} / ₂ , 24, 48 and 72 hours, respectively, growths formed were used to inoculate 5 species

Divide each 10 ml of sterile medium No. 13 into 100 ml- the tubes are harvested. The paper strip analysis of the

Shaking tubes used, which showed on a back and forth extracted fermentation mash that a considerable

giving shaking device for 16 hours at 32 ⁰ C loading amount of the starting material in 1,4-Pregnadien-Ilj9,16a,

hatched and then each had been transferred with a solution of 2 mg 55 17a, 21-tetraol-3,20-dione.
Substrate, 4-Pregnen-l Iß, 16a, 17a, 21 -tetraol- ^ O-dione, in

0.2 ml of 70 ° / ₀ aqueous ethanol were added. The example 17

After further incubation times of ¹ L, _A. - ^. ,. _Λ <"., "" _Nt j. iott> j-

Harvested 2,7,24 or 72 hours. . 1.4-Pregnadien-ll / ?, 16a, 17a, 21.tetr _a id-3,2 (Wion

Each of the fermentation mashes was mixed 4 times with 40 ml of 60 A reagent methylene chloride prepared with trypticase soy broth extracted. The extracts of each slant glass agar mash were washed with 5 ml of sterile water, were combined and reduced under reduced pressure to and the obtained cell suspension of Corynebacterium Evaporated to dryness. Each of the back simplex thus obtained was used to inoculate 100 ml of sterile Stand was taken up in 2 ml of pyridine and mixed with trypticase soy broth medium in a 500 ml alder 0.5 ml of acetic anhydride are added. These mixtures used 65 Meyer flasks. This mixture was at were incubated on a steam funnel for 10 to 15 minutes at 37 ° C for 8 hours with shaking. Five-and-a-half heats and then evaporated to dryness in vaciium. twenty Erlenmcycr flasks with 500 ml capacity each this residue was dissolved in 2 ml of acetone, each containing 100 ml of sterile trypticase soy broth and suitable volumes of these solutions were chroma medium without glycerine contained, were each with 1 ml des tographed. The chromatograms showed that the sub-70 inoculated 8 hour old inoculum. These pistons were

13 14

incubated at 32 ^C C for 40 hours and then each with an Example 19

Solution of 40 mg 4-pregnen-110,16a, 17a, 21-tetraol-

3,20-dione in 40 ml of ethanol was added. The fermentation was l, 4-Pregnadieii-U / ?, 16a, 17a, 21-tetraol-

continued for a further 8 hours at 32 ° C. The contents of all 3,20-dione-16,21-diacetate

Twenty-five flasks were combined using a 5 A mixture of 100 mg of 1,4-pregnadiene-llic?
Mash received from pn 8.1. 21-tetraol-3,20-dione, 10 ml pyridine and 2 ml acetic acid

The combined mash obtained after harvesting became anhydride and was left to stand overnight at room temperature once with 3 liters of methylene chloride and 3 times with 2 liters of methylene chloride each time. The solution was then extracted under methylene chloride. The combined extracts were evaporated to dryness under the pressure. The solid Rücklmal washed with saturated saline solution and stood under io, while crystallizing from ethyl acetate, evaporated to dryness under reduced pressure. One ester petroleum ether (boiling range 90 to 100 ° C) 105 mg received 509 mg of an oily residue, which in 1.5 ml of the (86 ° / ₀ ) M-Prcgnadien-U / J.loa. ^ Ct ^ l-tetraol- S ^ O-dione stationary phase of the system: 3 parts of ethyl acetate 16,21-diacetate with a temperature of 147 to 150 ° C. Recrystallize ester, 2 parts of petroleum ether (boiling point 90 to 100 ° C), from the same pair of solvents increased the 3 parts Dissolved methanol, 2 pebbles of water and mixed with 3 g of 15 melting point to 161 to 163 ° C. Infrared spectrum diatomaceous eggs. The so impregnated ν * £ - 3458, 1758, 1668, 1632, 1612 (shoulder) 1234, diatomaceous earth was then on the upper end of a 1060 cm "¹; ultraviolet spectrum λ ^" - 242 ηιμ glass column with the dimensions 1.5 times 35 a to ge (ε = 14 200).

attaches the 25 g with 12.5 ml of the stationary phase Example? 0

of the above-mentioned system of impregnated diatoms-20

earth develOps. The desired. Connection was then 1,4-Pregnadien-9a_nuor-ll / ?, 16 «, 17a, 21-tet: raol-

with the mobile phase of the above system 16,21-diacetate

eluted, yielding 207 mg of a crude solid. A test tube slant of Coryncbacteriuin

obtained during crystallization on acetone - simplex was washed with 5 ml of sterile saline solution, petroleum ether (boiling range 60 to 70 ° C) 56 mg of a 2.5 and the resulting spore suspension became 100 ml of substance with a temperature of 195 to 200 " C (Kofler block) sterile trypticase soy broth medium in a 500 ml dose. Recrystallize from the same solution Erlenmeyer flask. The mixture was incubated at medium pairs, the melting point increased to 202 to 32 ° C for 8 hours, 1 ml each this culture was 205 ° C (BLOCK); mp = 229 to 231 ° C (capillary). Ultra- for inoculating 10 flasks, each 100 ml sterile violet spectrum X ™ '"~ 241 ηιμ [ε - 14 500). 3 »Trypticase soy butter medium was used. This

ten flasks were left with shaking at 32 ° C for 16 hours

Infrared spectrum ν ^ = 3412, 1718, 1667, 1622, 1612 and then each incubated with a solution of 20 mg (shoulder) 1098, 1072 cm- ¹ 4-Pregnen-9a-fluor-II / 3.16a, 17a, 21- tetraol-3,20-dione-

16,21-diacetate in 2 ml of ethanol was added. Flask Analysis: C ₂₁ H ₂₈ O (376.44) 35 contents were combined and this combined solution Calculated C 67 00 H 7 SO ⁰ '· was found several times with a large volume of methylene .'. '.'. C (n, m, H 7 ^ 62 / "'chloride extracted, washed with saturated brine

and evaporated under reduced pressure. The return

The physical and chemical properties stood was dissolved in methanol, treated with activated carbon were the same as those of 1,4-pregnadiene-l 10,16a. ⁴ ° T ^{d by Ώ} * ^Χο ™ * ™ ^ ^ftltriert · After another one-17a 21-tetraol-3 20-dione evaporate to obtain 277 mg of a Ols, the overnight

was acctylated. The chromatograms showed about

equal amounts of substrate and a more polar Example 18 product (1,4-Pregnadiene-9a-fluoro-ll _j Ö, l6a, 17a, 2l-tetraol-

1.4-Pregnadien-H / i, 16a, 17a, 21-tetraol-3.20-dione ⁴⁵ 3,20-dione-16,21-diacetate) and very small amounts of

two less polar products. Through distribution

The mashes combined after harvesting were a like chromatography of 0.25 g of the residue

Described in Example 17 fermentation carried out less polar products and separated the substrate

was filled with 7 parts of 2 1 methylene chloride each (diatomaceous earth as a column filling; system: 2 parts

hiert, and the combined extracts were 50 ethyl acetate, 3 parts of petroleum ether, boiling range

evaporated to dryness under reduced pressure. 90 to 100 ° C., 3 parts of methanol, 2 parts of water were obtained. The GE-

1.585 g of a crude semi-solid, the chromate- wanted most polar product remained on the

was graphed. The column of the desired compound was removed and it was eluted with 500 ml of methanol. The at

After combining and evaporating the methanol, the holding fractions yielded a residue that remained

Evaporation to dryness 601 mg of a crystalline residue of 90 mg was redistributed on diatomaceous earth

stands. Crystallization from acetone-petroleum ether gave (system: 3 parts of ethyl acetate, 2 parts of petroleum

278 mg substance with a temperature of - 229 to 231 ° C. Recrystalline ether, boiling range 90 to 100 ° C, 3 parts of methanol,

sizing from the same pair of solvents increased the 2 parts of water), and the desired product

Melting point at 231 to 232 ^C C [a] g = - + 77 ° (methanol) holding fraction, which due to the UV absorption

Ultraviolet spectrum: A ^ "= 241 to 242n ^ (e ^ 14800). 60 spectrum was determined, was under reduced

Lifra red spectrum v ™ = 3436, 1715, 1664, 1621, 1603, pressure evaporated. 18 mg of solid substance were obtained.

11.29, 1063 cm ■ *. Crystallization from acetone — petroleum ether gave

13 mg of colorless needles of l ^ -prcgnadien ^ a-nuor-

Analysis: L ₂₁ M ₂₈ Uf, (o / 6.44) llp.loa.lva ^ l-tetraol-S ^ O-dione-lö ^ l-diacetate vom

Calculated C 67.00, H 7.50 "/ · 65 F. = 150 to 21O ⁰ C (Kofler block) obviously under

found C bö.SZ ,, Ji 7.27 ° / ° Loss of solvent at 150 ° C. By recrystallization from acetone-petroleum ether, the melting point was

The product was unchanged with a maintenance point according to Example 17. Ultraviolet spectrum: λ tL '"*"''"'

tenen sample of M-Prcgnadien-ll / Uoci. ^ a ^ l-tetraol- = 239 ηιμ (e = 15200). Spectrum in sulfuric acid

3,20-dione identical. 70 X Jw = 261, 308, 387 ηιμ. The 2,4-bis-dinitrophenyl

hydrazone of this compound was prepared and its ultraviolet absorption spectrum was determined: λ , '"!! /' ^! - 258, 300 (inflection), 312 (inflection) and 400nui.

Example 2 \

1,4-Pregnadiene-9 a-fluoro-11 ß \ 6 a, 17 a, 21-tctraol 3,20-dione

A solution of 100 mg 1,1-Pregnädien-i'tt-fluorII / M6a, 17a, 21-tetraol-3,20-dione-16,21-diacetate in 10 ml of methanol the mixture was cooled to 0 ° C. and, after nitrogen had been passed through, with a solution of 35 mg of potassium hydroxide added in 2 ml of methanol. After standing for 1 hour at room temperature, the solution was with Glacial acetic acid neutralized and evaporated to a white solid under nitrogen. After adding water and cooling, the product was filtered off and washed with water. 52 mg of 1,4-pregnadiene-9a-fluoro-II ^, 16a, 17a, 21-tetraol-3,20-dione were obtained from the F. = 246 to 249 ° C.! By 3 times recrystallization from acetone - Petroleum ether gave 29 mg of the tetraol with a temperature of 260 to 262.5 ° C.

Analysis: C ₂₁ H ₂₇ O ₆ F

Calculated
found .

C 63.94, H 6.90, F 4.82%;
C 64.19, H 7.17, F 4.90%.

Claims (2)

PATENT CLAIMS; 1. A process for the preparation of steroid compounds of the 1,4-pregnadiene series, characterized in that one
general formula a 4-pregnen connection of the CH ₂ OR R ' ,. • ^J <. in which X is hydrogen or halogen, Y is a methylene, oxymethylene or carbonyl group, Z is an oxymethylene or alkanoyloxymethylene group with lower alkanoyJrest, R 'hydrogen or a Hydroxyl group and R denotes hydrogen or a lower alkanoyl group, in a known manner the fermentative, enzymatic action of organisms of the genus Nocardia or the organism Subjects to Corynebacterium simplex. 2. The method according to claim 1, characterized in that that one of the following species is used as an organism of the genus Nocardia: corallina, asteroides, keratolyticus, transvalensis, erythropolis, convoluta, gardneri, leishmanii, opaca, blackwelii, caviae, cuniculi, globerula, polychromogenes, sylvodifera, farcinica or sj'lvodifera. Considered publications:
Journ. Amer. Chem. Soc, Vol. 77, 1955, p. 4184. 909 687/421 11:59