DE1071080B - Process for the production of 3-keto-zlM-steroids, 3-keto-44-steroids or mixtures thereof - Google Patents

Description

GERMAN

njoO.

P 19645 IVb / 12 ο

REGISTRATION DAY: NOVEMBER 13, 1957

NOTICE THE REGISTRATION AND ISSUE OF THE EDITORIAL:

DECEMBER 17, 1959

The invention relates to a process for the dehydration of the core of saturated 3-keto-steroid compounds by microorganisms of the Micromonospora genus, in particular the Micromonospora chalcea species, or by oxidizing enzymes produced by these microorganisms.

Either the 4-position double bond alone or, depending on the conditions used, such as reaction time, etc., a 1-position and 4-position double bond can be introduced into the core. This conversion is of so great importance because many Δ ^ -steroids, such as zJ ^l ' ⁴ -pregnadiene-ll / S, 17a, 21-triol-3,20-dione (prednisolone), Zl'' ⁴ -pregnadiene- 17a, 21-diol-3,11,20-trione (prednisone), 14-hydroxy-prednisolone or 14-hydroxy-prednisone, are very valuable for the treatment of certain diseases of the human body, particularly rheumatoid arthritis.

There are many easy-to-prepare, core-saturated steroids that can be used as relatively cheap starting materials for the synthesis of these compounds, but no viable method for introducing the required double bonds into the Α-ring was previously known. Such a method for introducing A ¹ - and Δ ″ double bonds into the Α ring is therefore of great importance for the production of certain pharmaceutical preparations.

It has already been described that a double bond at the 1-position can be introduced ^{into certain 3-keto-J 4 compounds by organisms other than the organisms used in this invention.} Very many organisms have been tested for their ability to carry out this conversion, but very many have been found to be unusable. It has been found that organisms of the Micromonosporachalcea species are able to carry out this reaction with high yield.

Further advantages of the organisms of this Micromonospora chalcea species are that the product obtained is relatively free of by-products and therefore easy to clean. Since cleaning is expensive, this freedom from by-products is a very great asset. Another advantage of the Micromonospora chalcea lies in the fact that the organism grows relatively quickly and is easily grown on cheap nutrient media can be.

The procedure is carried out by dissolving the selected steroidal compound in aqueous solution or Suspension with either an actively growing Micromonospora chalcea culture or with removed from the growing culture and suspended in a suitable medium Micromonospora chalcea cells or with extracts of oxidizing enzymes from the Micromonospora chalcea culture brings in touch.

A variety of core saturated 3-keto steroid procedures for the production

-steroids ^4, - from ^3-keto-1 J

3-keto-zl ⁴ steroids or mixtures thereof

Applicant:

Chas. Pfizer & Co., Inc.,
Brooklyn, NY (V. St. A.)

Representative:

Dr. W. Beil and A. Hoeppener, lawyers,
Frankfurt / M.-Höchst, Antoniterstr. 36

Claimed priority:
V, St. v. America February 12, 1957

Gilbert Malcolm Shull, Huntington Station, N.Y.

(V. St. A.)
has been named as the inventor

Compounds can be used as starting materials for the reaction according to the invention. This includes:

Androstan-S-on.Androstan-S.lT-dione.Bisnorcholan-S-on, Pregnan-3-one, Pregnan-3,20-dione, 4-dihydrocorticosterone, 17a-Oxyandrostan-3-one, Östran-3-one, 4-Dihydrodcsoxycorticosteroti, Alloprcgnan-3-one, allopregnan-3,20-djon, 17a-oxypregnan-3-one and -3,20-dione, 11/9-oxypregnan-3-one and -3,20-dione, 20-oxy-pregnan-3,20, 14a-oxypregnan-3-one and -3,20-dione, pregnan-and allopregnan-3,11-dione and -3,11,20-trione, ll / S.lfa ^ l-trioxy-prcgnan-3,20-dione, 17a, 21-dioxypregnane-3, ll, 20-trione, 11 / 8,21-dioxypregnane-3,20-dione, 11 / ?, 14a, 17a, 21-tetraoxypregnane-3,20-dione, 21-Oxvpregnan-3,20-dione.

The method according to the invention is particularly good at essentially saturated 3-keto steroid reactants feasible with 18 to 21 atoms in the carbon skeleton. The products of this implementation can through an accurate comparison of paper chromatograms of those made with known steroid compounds Products are determined. This procedure has been tried and performed on a variety of compounds known to produce reliable results.

The micrononospora chalcea organism is described in " Actionomycetes and their Antibiotics" on p. 127 by Waksman and Lcchevalier, 1953, Williams and WiI-kens, Baltimore. The one in the catalog of the

909 689/574

American culture collection as Micromonospora special magnesium and a smaller amount of Ade-Art No. 10 026 listed organism was contained by this nosine triphosphate.

public cultural collection related and than to the After implementation one leads the most expediently Identified as belonging to Micrononospora chalcea species. A paper chromatography. The one after the previous one Culture of an organ isolated from the nutrient medium - 5 method described below nism, which is classi- fied as the Micromonospora chalcea species, can be obtained by extraction with various, with water was fected, became miscible, organic solvents from the aqueous in the American culture Collection, Washington, D. C, and received the solution to be isolated. Are particularly suitable for this ATCC number 12 452. Both the Micromonospora lower halogenated hydrocarbons such as chloroform, chalcea ATCC 12 452 as well as the Micromonospora species io After the extraction, the solvents can be abde no. 10 026 correspond to the stilled on page 127 of the aforementioned and the solid product can thus be isolated. This recognized text by Waksman and Lechevalier substance can be recrystallized from organic given description of the Micromonospora chalcea solvents or by chromatography, e.g. B. on Art. The reactions according to the invention can be carried out either on alumina columns or on other suitable, solid further purified with an organism isolated from the ground as absorbent substances also with that of the American culture collection. The related ones proved to be particularly advantageous Organism. Turning a silica gel-ethanol column with

The dehydration process according to the invention can mix methylene chloride and 2 to 5 percent by volume

run in different ways. Ethanol (95%) as a developer. Method of manufacture

one can use a carbohydrate, an organic nitrogen- ao of products of this kind are in the specialist literature

source, containing mineral salts and trace elements. For some uses it is not

Culture media with inclined culture media from Micromonospora necessary to separate the products; the raw mix can

inoculate chalcea, cultivate it for 2 to 3 days and use it as such. Proved in some cases

then use it as a vaccine for larger tanks, as appropriate to acylate the raw products and

which are stirred and aerated. The 25 to be dehydrated with the resulting esters, which are somewhat more stable,

Steroid then becomes the breeding ground for at least one to work.

Hydrogen acceptor, a divalent metal, in particular the various Δ ^{1> 4} and Zl ⁴ products are suitable

Magnesium, and should contain buffer substances, after as intermediates for the synthesis of other valuable

adjusting the p _H -value just in solid form to-full compounds. The dehydrated products that

set. 30 in the 1 (2) position together with the 3-keto group

The dehydration of steroids generally and in the 4 (5) position requires unsaturation to be separated Hours to several days. The most favorable time holdings are, for example, for an and temperature conditions can be easily experi- mented particularly suitable. Through this, oestrone-mental determine. derivatives formed.

Instead of cultivating the microorganism in advance, 35 Except for the above-mentioned advantageous options If the steroid compound is also sterilized, many of the breeding grounds are cultivated by this conversion add directly and then with microfabricated compounds because of their biological active monospora Inoculating the chalcea, it is very important. Is z. B. ll / ?, 17a,

The dehydration can also be carried out with a growing 21-trioxypregnene-3,20-dione with Micromonospora chalcea

Culture removed cells are carried out, which are treated in 40 according to the new process, so that predni-

A solute can be suspended in relation to nasolone, which is very useful and in the treatment of

trium fumarate or another hydrogen acceptor of rheumatoid arthritis is superior to hydrocortisone.

as well as magnesium sulfate 0.01 molar and with regard to Will 17a, 21-Dioxypregnan-3, ll, 20-trione with Micromono-

Sodium citrate is 0.03 molar. The spora chalcea turns out to be treated, so it arises as prednisone

simultaneous presence of a certain amount of Ade- 45 known compound, which is found in the treatment of

nosine triphosphate, e.g. B. 0.125%, as seltr advantageous. Rheumatoid arthritis also found to be very beneficial

The medium mentioned is shown before the addition of the centrifuge.

fused, washed cells with z. B. Citric Acid It has also been found that other 3-keto

adjusted to a _pH value of about. 6 Δ ^ -steroids have great effectiveness than adrenal glands

The cells of about 100 ecm of the stirred and cortical hormones possess and are in favor of the same therapy

aerated Micromonospora chalcea cultures result in how hydrocortisone are suitable. Many naturally occurring

Suspending in about 20 ecm of the said solution is good steroids as well as from the naturally occurring easily

Results. This quantitative ratio can, however, have strong steroids, although they are essentially saturated

can be varied. The steroid compound is based on a ver 3-keto structure, but no 3-keto-4 ¹⁺ compound

A ratio of around 25 to around 200 mg per 100 mg of fertilization is readily available as raw materials. thats why

Settlement mixture used. the present process, which involves the dehydration of a

Another method is used for the deny or both A-ring bonds, those with the 3-ketodration the oxy group of the saturated steroid nucleus produced by Micromonospora chalcea is linked, in acting enzymes used. These can be a single stage in a technically large scale a number of processes from the cells of the rods made possible, of very large scale chosen organism to be isolated, e.g. B. mechanical importance.

by autolysis, by rapidly successive processes The following examples explain the invention

freezing and thawing, ultrasonic treatment procedures,

or leave to stand in water-miscible solution - Example 1

agents, especially acetone. 65

For dehydration, the enzymes are put into the same series of Fernbach flasks, each 500 ecm

Media given as they were contained in the dehydration with the following nutrient medium:

separated cells were used, i.e. H. in media containing enzymatic casein hydrolyzate (known

a hydrogen acceptor such as Furaarat, a buffer under the trade name N-Z-Amin B;

substance, in some cases a divalent metal, especially 70 Sheffield Farms casein hydrolyzate) 10 g

Dextrose hydrate 10 g

Yeast extract 5 g

Calcium carbonate Ig

and tap water to make up to 11 was provided. The flasks were sealed with cotton, sterilized and then with a suspension of the Spores and plant growth from a sloping bottom culture of Micromonospora chalcea ATCC 10 026 was obtained, vaccinated. The flasks were shaken for 4 days at 28 ° C, with each flask 50 mg II / 9.17a, 21-trioxypregnane-3,20-dione in the form of a sterile solution in acetone was added. Shaking continued for 3 days. Then each became a flask extracted three times with the same amount of chloroform each time. The combined chloroform extracts were placed on a silica gel chromatography column, which was eluted with a mixture of methyl chloride and ethanol. This is how prednisolone was obtained.

Example 2

30th

The procedure was as in Example 1, only this time the organism used was Micromonospora chalcea (ATCC No. 12,452). It returned to prednisolone.

Example 3

The procedure of Examples 1 and 2 was repeated, using 17a, 21-dioxypregnane-3, ll, 20-trione as starting material was used. In each case, prednisone was obtained.

Example 4

The procedure of Example 1 was repeated using 4-dihydro-deoxycorticosterone as the starting material. After one day of the three-day conversion period, a sample was removed and tested. Deoxycorticosterone was obtained in a mixture with some / d ^l -dehydro-deoxycorticosterone. An examination of the product carried out at the end of the three-day reaction showed that it consisted largely of Δ M-hydro-deoxycorticosterone with traces of deoxycorticosterone.

In all cases the yields obtained were between 25 and 35%.

Example p

A series of Fernbach flasks, each containing 500 ecm of the following nutrient medium:

Enzymatic casein hydrolyzate, known
under the trade name NZ-AminB (Sheffield Farms Casein Hydrolyzate) 10 g

Dextrose hydrate 10 g

Yeast extract 5 g

Calcium carbonate Ig

and tap water to make up to 11 was provided. The flasks were closed with cotton wool, sterilized and then with a suspension of the spores and vegetative growth from a slant culture

SS inoculated from Micromonospora chalcea (ATCC 1.0 026). The flasks were shaken at 28 ° C. for 4 days, after which a sterile solution of 50 mg androstan-3-one in acetone was added to each flask. Then it was shaken for another 3 days. Each flask was then extracted three times with an equal volume of chloroform. The combined chloroform extracts were applied to a silica gel column which was eluted with mixtures of methyl chloride and ethanol. J ¹ ' ⁴ -androstadien-3-one was obtained in a yield of 25%.

Example 6

A series of Fernbach flasks, each 500 ecm of the following nutrient medium:

Enzymatic casein hydrolyzate, known
under the trade name NZ-AminB (Sheffield Farms Casein Hydrolyzate) 10 g

Dextrose hydrate 10 g

Yeast extract 5 g

Calcium carbonate Ig

and tap water to make up to 1 liter was provided. The flasks were closed with cotton wool, sterilized and then inoculated with a suspension of the spores and vegetative growth from a slant culture of Micromonospora chalcea (ATCC 10 206). The flasks were shaken at 28 ° C. for 4 days, after which a sterile solution of 50 mg androstane-3,17-dione in acetone was added to each flask. Then it was shaken for another 3 days. Each flask was then extracted three times with an equal volume of chloroform. The combined chloroform extracts were applied to a silica gel column which was eluted with mixtures of methylene chloride and ethanol. Δ ^li4 -androstadiene-3,17-dione was obtained in a yield of 30%.

Claims (4)

Patent claims: 1. A process for the preparation of 3-keto-Zl ¹ ^ ⁴ -steroids, 3-keto-z] ⁴ -steroids or mixtures thereof, characterized in that a 3-keto-steroid saturated in the core is mixed in a known manner with the oxidizing Enzymes of an organism of the Micromonospora chalcca species are dehydrated. 2. The method according to claim 1, characterized in that the starting compounds in the core uses saturated 3-keto steroids with 18 to 21 atoms in the carbon skeleton. 3. The method according to claim 1 and 2, characterized in that the starting compound is 3-ketoandrostane, a 3-ketopregnane or 3-ketoallopregnan, a bisnorcholane, II / 9,17a, 21-trioxypregnane-3,20-dione, ^ a ^ l-Dioxypregnan-S.ll ^ O-trione, 11 ß, 2i -Dioxypregnan ^ O-dione, 11 ß, 14ct, l 7ct, 21-Tetraoxypregnan-3,20-dione or 21-Oxypregnan-3,20- dion used. 4. The method according to claim 1 to 3, characterized in that the microorganism Micromonospora chalcea ATCC 10026 or 12 452 used. Q 909 689/574 12:59