NO118033B - - Google Patents

Description

Process for the production of new corticosteroids.

The present invention relates to a process for the production of 11-oxy-generate-1,4-pregnadiene-17a,21-diol-3,20-

diones and esters thereof.

It has been shown that the new connections

is in possession of very valuable therapeutic

tical properties, especially 1-dehydro-cor-

tison and 1-dehydro-cortisol (1-dehydro-hydro-cortisone) and their 21-esters, or can be used as intermediate products in the production of

position of compounds with therapeutic properties. Some of the most important new 1,4-pregnadiene derivatives are given in Formula Scheme I. The formula shows:

I. Pregnancy skeleton with numbering

of C atoms

II. 1-dehydro-cortisone and its 21-esters

III. 1-dehydro-cortisol and its 21-esters

IV. 9a-halo-l-dehydro-cortisone and

its 21-esters

V. 9a-halo-l-dehydro-cortisol and

its 21 esters

WE. General formulas for compounds II

to V

VII. 11-epi-l-dehydro-cortisol and its 21-

ester

The procedure for producing them

new 11-oxygenated-1,4-pregnadiene-17,21-diol-3,20-diones and esters of such ex-

is characterized by the fact that a compound of the pregnane series which is unsaturated at C-5 and in-

holds a keto-, hydroxy- or esterified hydroxy group in the 3-position, and a keto-

or hydroxy group in the 20-position is exposed to the action of a microorganism that dehydrogenates in the 1(2)-position without pre-

carbon skeleton of other pregnane compounds,

with the exception of microorganisms of the genus Fusarum, that there are thus

held 3,20-diketo-1,4-pregnadiene com-

if necessary, in a manner known per se, an 11-, 17a- and/or 21-hydroxy group and, if desired, a 9a-halogen atom are introduced, and that in the thus obtained 11-oxygenated-1,4- pregnadien-17a,21-

diol-3,20-dione, if desired, one or more hydroxy groups are esterified, one or more esterified hydroxy groups are hydrolyzed and/

or an 11-hydroxy group is oxidized to an 11-keto group, in a manner known per se.

In the method according to the invention

then cultures of dehydrating microorganisms with the special characteristic

cause them to introduce a double bond between

lom the carbon atoms and the 1- and 2-positions without simultaneous degradation of the pregnane's carbon atom skeleton such as a cleavage of the side chain at the carbon atom in the 17-position or opening of the D-ring. The dehydrating microorganisms are also capable of hydro-

lighter ester groups, generally in the 3- and 21-positions, and with different rates

names or different completeness out-

oxidation of hydroxyl groups is then also carried out

per in the 3 and 20 positions.

The dehydrating microorganisms vir-

works best on substrates (i.e. steroid compounds

the part to be changed) which, in addition to dob-

the belt bond at the carbon atom in the 5-position contains either a hydroxyl group which is relatively stable towards this organism, i.e. not quickly oxidised to keto-oxygen, or an ester of such a hydroxyl group and which is hydrolysed by cultivation of microorganisms. For this reason, compounds such as progesterone are not satisfactory as starting materials, and the same applies to pregnenolone (5-pregnen-3-ol-20-one) as this compound's hydroxyl group in the 3-position is quickly oxidized to ketone oxygen during the formation of progesterone.

The hydroxyl group in the 11-position can be introduced into compounds which do not have substituents on the carbon atom in the 11-position using a culture of lla-hydroxylating or lip-hydroxylating microorganisms. Such introduction of a hydroxyl group in the 11-position can be carried out not only with 4-pregnane or 5-pregnane compounds but also with 1,4-pregnadienes, 1,4,16-pregnatrienes and 4,5-pregnadienes. As compounds with a hydroxyl group in the lla position generally have little or no physiological activity, the lla hydroxyl group is preferably oxidized by a mild oxidizing agent such as chromic acid in pyridine at room temperature and preferably after acylation of the 21-hydroxyl group when such a group is present.

Synthesis of • compounds corresponding to the formulas II to V also includes the introduction of a hydroxyl group in the 17a position, when such a group is not present in the starting material. Such hydroxylation in the 17a position can be carried out either chemically by starting from a 16-dehydropregnan compound, e.g. by forming the corresponding 16,17-epoxide with subsequent hydrolysis, or by starting from compounds that are saturated in the D-ring and introducing the hydroxyl group in the 17a position using cultures of known 17a-hydroxylating microorganisms.

The hydroxyl group in the 21-position can be introduced into the 21-methyl group in a known manner by purely chemical means, but it is preferred to do this with the help of a culture of 21-hydroxylating microorganisms.

As mentioned above, the invention also includes the production of certain esters of compounds corresponding to the formulas II to V. It has been found that these esters increase the duration of the compounds' effect, so that these can be given at relatively long intervals, especially as far as introduction by injection is concerned.

The 9a-halogen group can be present in the 11-keto- or 11|3-hydroxy starting material or it can be introduced by means of

various chemical processes during any stage of the preparation of compounds corresponding to the formulas II to V in which a lla- or ll(3-hydroxyl group is present. In both cases a 9a-halo-lip-hydroxy compound is obtained which can is oxidized to the 9a-halo-II-keto form.

The new 1,4-pregnadiene derivatives have been found in clinical tests to have an extremely improved physiological effect in comparison with the monounsaturated compounds (cortisone, hydrocortisone and 21-esters of these compounds). Thus, said compounds show potentials that are several times greater than the potential of corresponding known hormones, such as e.g. cortisone and hydrocortisone in the treatment of acute rheumatism. Moreover, the unwanted side effects of the known hormones only show themselves to a lesser extent when using these new compounds, and the intensity of these side effects is further reduced due to the fact that the new compounds can be used in much smaller doses than the corresponding monounsaturated known compounds. Both the starting doses and the doses to maintain the treatment in the treatment of acute rheumatism can be greatly reduced by the use of the new compounds compared to the doses of cortisone and hydrocortisone required. Even with the reduced doses, the new compounds do not require the simultaneous use of codeine or other analgesic agents to relieve pain, whereas such analgesic doses have often been used together with cortisone and cortisol.

The therapeutically active dienes produced according to the present invention are preferably given by mouth in the form of tablets containing a complete daily dose, e.g. 50 mg, or doses of which 50 mg is a multiple, e.g. 25 or 20 mg or only 10 mg, mixed with a solid pharmacological carrier containing one or more of the usual ingredients such as starch, sugar, gums, clays and the like. However, the compounds can also be given by intravenous or intramuscular injection, dissolved or suspended in suitable non-toxic liquid carriers. The compounds can also be given in solid form by subcutaneous implantation or in the form of suppositories in which they are dissolved or suspended in fatty or waxy carriers

which melts at temperatures around

body temperature. The connections as well

as their 9a-halogen derivatives can also be given locally in the form of ointments or creams in which they are dissolved in such ointments or

cream bases of known composition.

The introduction of the second double bond in various intermediates with a single double bond and which are known to have therapeutic activity has been found to improve the activity of such compounds as well. Examples of such compounds are corticosterone and 11-dehydrocorticosterone.

The various reactions that are briefly discussed in the preceding section will be explained in more detail below.

A. Introduction of a 1,2-double bond by means of a culture of a dehydrating microorganism.

An essential feature of the invention is the microbiological introduction of a 1,2-double bond in a pregnene derivative which has a double bond at the 5-carbon atom by incubating or pre-fermenting the steroid used as starting material with a culture medium containing a dehydrating microorganism which does not at the same time deconstructs or splits the pregnane molecule. Suitable microorganisms of this type belong to the family Corynebactriaceae of which the species Corynebacterium simplex (American Type Culture Collection 6946) and Corynebacterium hoagii (A.T.C.C. 7005) have given very satisfactory results. The first of these species is a soil bacterium, while the other is found in the human throat (where it apparently produces no pathological condition) and in some cases as a contaminant in cultures exposed to the atmosphere. However, the real or original habitat of the species is not known.

The starting materials can have hydroxyl groups, keto groups, halogen atoms and ester groups in different positions in the core or on the side chain. Thus, hydroxyl groups can be present in one or more of the 3-, 11-, 17-, 20- and 21-positions, while keto groups can occupy one or more of the 3,11- and 20-positions, and halogen atoms which fluorine or bromine atoms may be attached to the carbon atom in the 9-position, or to other locations in the core or side chain. The presence of a free hydroxyl group appears to promote the chemical transformation. Many different ester groups and preferably esters of acids which are generally used in the synthesis of steroid hormones for therapeutic use and especially of the lower alkanoic acids such as the acetates, can be found in one or more of the 3-, 11-, 17-, 20- and 21- the positions. The special character of the ester is not decisive in the method according to the invention, and other esters of both organic and inorganic acids can be used, such as e.g. cyclopentyl and cyclohexyl acetates, propionates and butyrates. Likewise, phosphates, polyphosphates and sulfates can be used, the only condition being that the esters are not toxic to the microorganism. The hydroxylated products from the process can, if desired, be transferred to the corresponding esters using known methods, e.g. in its lower alkane esters and especially acetic acid esters. The hydroxyl groups in the 3- and 11-positions can be the α-epimers or the β-epimers. As will be described in more detail below, in particular the ester in the 21-position can be the ester of an acid which acts in the direction of prolonging the duration of the activity of the therapeutic compounds.

When using dehydration microorganisms according to the method according to the invention, one can transfer e.g. 4-pregnane-17a,21-diol-3,11-20-trione (cortisone) in 1,4-pregnadiene-17a,21-diol-3,11-20-trione (1-dehydro-cortisone), 4-pregnane -lip, 17a-21-triol-3,20-dione (hydrocortisone or cortisol) in 1,4-pregnadien-lip,17a,21-triol-3,20-dione (1-dehydro-cortisol),

4- pregnene-17a,21-diol-3,20-dione (Reichstein's compound S) in 1,4-pregnadiene-17a,21-diol-3,20-dione and 1,4-pregnadiene-17a,20p,21- triol-3-one,

5-pregnene-3p,20-diol, i 1,4-pregnadiene-3,20-dione,

4-pregnane-lip-21-diol-3,20-dione (corticosterone) in 1,4-pregnadiene-lip,21-diol-3,20-dione,

4-pregnen-21-ol-3,20-dione (desoxycorticosterone) in 1,4-pregnadien-21-ol-3,20-dione, 4- pregnen-21-ol-3,11,20-trione (Kendall's compound A) in 1,4-pregnadien-21-ol-3,11-20-trione, 9a-fluoro-4-pregnen-lip,17a,21-triol-3,20-dione (fluorocompound F) in 9a-fluoro- 1,4-pregnadiene-lip-17a,21-triol-3,20-dione, 5-pregnene-3p,17a,21-triol-20-one-3,21-di-acetate i 1,4-pregnadiene-17a ,21-diol-3,20-dione and 4-pregnene-17nt,21-diol-3,20-dione-21-acetate,

4- pregnen-20-ol-3-one in 1,4-pregnadien-3,20-dione, 5- pregnen-3,17a,20,21-tetrol-ll-one and its 3- and/or 21- ester in 1,4-pregnadiene-17α,21-diol-3,11-20-trione and its 21-ester, 5-pregnene-3,11a,17u,20-21-pentol and its 3-and/or 21- ester in 1,4-pregnadiene-lla,17a,-21-triol-3,20-dione and its 21-ester.

Instead of the 3-keto starting materials, the corresponding 3-hydroxy compounds and their 3-esters, such as e.g. 5-pregnene-3,11[3,17u,21-tetrol-20-one and 5-pregnene-3,17a,21-triol-11,20-dione and their 3-acetates or 3,21-diacetates to produce the same 3-ketodiene end products, the 3-ester group being hydrolysed and the 3-hydroxyl group subsequently oxidized to keto-oxygen. Ester groups in the 11- and 17-positions are generally not hydrolyzed, at least not to any appreciable extent, while ester groups in the 21-position may or may not be hydrolyzed depending on the reaction conditions. Thus, when the starting material is a 3,21-diester, the reaction product may be a 3-keto-21-ester compound or a 3-keto-21-hydroxy compound. Together with the 3-hydroxyl group, a 20-hydroxyl group is often oxidized to a keto group. It will thus be seen that the dehydrating microorganisms used in the method according to the present invention are selective with regard to the oxidation step, as this is practically completely limited to the 3- and 20-positions, while the hydrolysis can be limited to the 3-ester groups.

The method according to the invention can also be used to treat the 11a-hydroxy epimers of the above-mentioned lip-hydroxy starting materials, such as e.g. 4-pregnene-lla,17a,21-triol-3,20-dione, 4-pregnene-lla,17a,20,21-tetrol-3-one, 5-pregnene-3,lla,17a,21-tetrol- 20-one, 5-pregnen-3,lla,-17a,20,21-pentol and their mono- and polyesters, such as the 3-acetates, 3,21-diacetates and 3,17a,21-triacetates. These starting materials give 1,4-pregnadiene-11a,17a,21-triol-3,20-dione or an ester of this compound. The 11-epimers of the 1,4-diene of 1-dehydrocortisol and its esters can be transferred to the 1,4-diene of cortisone and its ester by oxidation of the 11a-hydroxyl group in a known manner as with the theoretical amount of chromic acid with or without the presence of pyridine or acetic acid at room temperature or lower temperature (5 to 15°C), preferably after esterification of the 21-hydroxyl group if this is free. These 11-hydroxy starting materials can be produced relatively easily with a high yield, as is previously known, and are therefore advantageous starting materials for the production of 1-dehydrocortisone and 1-dehydrocortisol.

The method according to the invention can also be used on 9a-fluoro, chloro and bromosteroids and then gives the corresponding substituted reaction products. The various reactions can be represented by equations 1, 2 and 3 on attached sheet 2. These equations show the formation of the desired end products and also intermediate products that can be transferred into them.

In equation 1, X denotes

(a or p) Y denotes -CO CH2OH and Z' denotes OH or H. In equation 2, W denotes H, F, Cl or Br, X denotes H2, =0 or

Y denotes -CO.CH2OH, -CO CH2-

OOCR' or -CHOH.CH2OH, Z' denotes OH or H, and R' denotes a lower alkyl radical. In equation 3, R denotes a lower alkanoyl group, Y denotes -CO.CH.OH, -CHOH. CH,„ -CO.CH2OOCR' or CHOH.CHgOH, Z' denotes H or OH and R' denotes a lower alkyl radical.

In order to achieve satisfactory growth of dehydrating microorganisms such as Corynebacterium simplex and C hoagii for the method according to the present invention, a suitable nutrient substrate is prepared containing carbohydrates, organic nitrogen, auxiliary factors ("cofactors") and inorganic salts. It is possible to skip the carbohydrates without severely damaging the organism's growth. After cultivation of the micro-organisms in the nutritious substrate, the cell mass can be collected by centrifuging the culture, decanting the upper liquid layer and suspending the cell mass in salt solution. A suitable volume of the cell suspension is then inoculated into a nutritious substrate which is advantageous for supporting the growth of the microorganism. As a nutritious substrate, you can use the yeast extract ("Difco"), casein hydrolyzate (N-Z-amine) (Type B Sheffield), corn casting liquid, aqueous extracts of soybean meal, lactalbumin hydrolyzate (Edamine-Sheffield Enzymatic), fish extracts and the like.

The steroid compound is in the form of a solid or dissolved or suspended in ethanol, acetone or other solvent which is miscible with water and is not toxic to the organism added to the cultured microorganism in the nutrient substrate under sterile conditions. The culture is then shaken, aerated or simultaneously aerated and stirred to promote the microorganism's growth and the steroid's biochemical reaction. The steroid can be added to the nutrient substrate and then inoculated with bacteria or the cultured microorganism in the nutrient substrate can be added to the steroid. In certain cases, which depend on the conditions in the reaction medium, it may be desirable to achieve optimal growth of the microorganism before the steroid is added. Alternatively, enzyme preparations obtained in a known manner from cultures of microorganisms can be used for carrying out the method.

It is advantageous to have inorganic salts present, so that a pH value of between 6.8 and 7.2 is maintained in the reaction medium. However, the use of organic salts as buffers in the reaction mixture can also be omitted. If inorganic salts are not added, the pH rises from an initial value of 6.8 to 7.7-8. However, this increase will still allow the formation of the desired steroid end products. The optimum temperature for the cultivation of the microorganism is 37°C, but the temperature can vary between 25 and 37°C and even between 20 and 40°C. The reaction time can vary from 3 hours to 48 hours. The amount of time used to carry out the reaction depends on the steroid compound being treated. Any solvent which is miscible with water and is not toxic to the microorganism can be used to dissolve or suspend the steroid. Ethanol and acetone are preferably used in such proportions that the final concentration of these solvents in the reaction mixture is no higher than 7 percent and down to traces. Due to evaporation, the final concentration of the organic solvent can be practically equal to 0.

After the completion of the oxidation or dehydration process which may be accompanied by partial or complete hydrolysis when mono- or polyesters are used, the reaction products may be recovered from the mixture by extraction with a suitable solvent immiscible with water, by filtration, by adsorption on a suitable adsorbent or by other operations which are generally used in the art. In extraction, chlorinated lower hydrocarbons, ketones and alcohols are advantageous extractants. Among the special extractants of these groups that can be used are chloroform, methylene chloride, trichloroethane, ethylene dichloride, butanol and diethyl ketone. It is preferred to use extraction as a method for isolating the steroid products. After extraction, the products are then isolated by concentrating the extracts to a small volume or to dryness. Cleansing

of the residues can then be carried out in individual cases

recrystallizations from suitable solvents or mixtures of solvents such as acetone, methylene chloride, ethanol, mixtures of acetone and hexane, mixtures of methylene chloride and hexane, etc. The desired diene is then obtained in very good yield and in a very pure state.

The chemical reactions that can be carried out by subjecting the various pregnenes to the influence of a culture of dehydrating microorganisms are thus of many different kinds, and can take place individually, or two or more such reactions can take place simultaneously or one after the other.

The different reactions seem to be

unaffected by other substituents in the core of the steroids or in their side chain.

It will be obvious from the foregoing that it is not of essential importance that the starting material has its double bond in the 4-position of the A-ring, but the double bond can thus be in the 5-position. The starting material can also have more than one double bond. Thus, 4,6-pregnadienes can be dehydrogenated and reformed in this way in the manner described above, so that 1,4,6-pregnatrienes are obtained, which can easily be reduced in a known manner to 1,4-pregnadienes. As an example, it is mentioned that 4,6-pregnadiene-17a,21-diol-3,11-20-trione-21-acetate is transferred to 1,4,6-pregnatriene-17a,-21-diol-3,11,20- the triene or its 21-ester. The corresponding 11- or 11(3-hydroxy compounds can be transferred in a similar way to 1,4,6-trienes. The trienes can be reduced to 1-dehydrocortisone or to 1-dehydrocortisol or similar compounds using known methods. The starting material can also have a double bond' in the 16-position or in the 9(11)-position, which will be explained below.

The fish extracts discussed above are commercially available as herring and menhaden extracts and various mixtures of such extracts subjected to enzymatic hydrolysis. This material can be added directly to the culture medium to provide nutrients. When using fish extracts (with 50% solids) which have not been subjected to enzymatic hydrolysis, such extracts should be diluted with water and treated with steam for about 10 minutes at 90°C and then filtered, preferably using Filter-Cel .

A further example of a dehydrating microorganism which has been found to carry out the reaction described above is Bacillus sphaericus (American Type Culture Collection 7055) which is also capable of introducing a double bond into the A ring without simultaneously causing degradation of the side chain or cleavage of the D ring. The resulting steroids with an unsaturated A-ring are highly stable in cultures of the aforementioned microorganism, and it is not necessary to use special precautions to prevent destruction of the desired end product. The incubation time can be as long as 96 hours and the yields are high, often almost quantitative.

As is the case with the other dehydration microorganisms mentioned above, instead of a growing culture of B. sphaericus, enzymes or enzyme extracts secreted from such cultures can be used.

In general, B. sphaericus will carry out the same chemical designs in the above-mentioned pregnene compounds as the Corynebacterium organisms, except that the former organism does not so easily and in some cases does not hydrolyze the 3-ester group. It is therefore recommended with this organism to use 3-keto- and 3-hydroxy-pregnenes and not the corresponding 3-esters. The steroid substrates may contain double bonds in the core in addition to the double bonds at C-4 or C-5, and such further double bonds may be saturated with halogen or hydrogen halides, or by forming adducts of dienophilic compounds such as maleic acid and maleic anhydride. Except that 3-esters are not used, the different starting materials will be transferred to the same end products of B. sphaericus as of Corynebacterium simplex and hoagii.

The culture method for B. sphaericus and the method of inoculation of the nutrient medium, the nature of this medium and the way in which the steroid substrate is brought into contact with the growing culture of the microorganism may be the same as with the Corynebacterium species, except that the incubation time as already indicated may be longer . It is generally from about 24 to 96 hours. In all cases, the steroid-containing culture is stirred and aerated.

In addition to the nutrient media already mentioned, protopeptones can also be used. The microorganism's growth is preferably initiated at a pH value of around 6.6 to 7.2. The optimum temperature for growing B. sphaericus is around 28°C, but the temperature can vary between 19 and 32°C.

The products from the biochemical reaction are preferably recovered by extraction with the above-mentioned solvents and in the above-mentioned manner. However, any other suitable known chemical method may also be used.

Instead of the dehydration microorganisms mentioned above, their mutants can also be used.

B. Combination of the microbiological 1, 2- dehydration with 11- and 21- hydroxylation and 11- oxidation.

The dehydrogenation of the A-ring by means of the dehydrogenating microorganisms described above can be combined with other microbiological reactions that are individually known, and with a chemical oxidation step (when 1-dehydrocortisone is to be produced), so that 17a-hydroxy-progesterone (4-pregn -17a-ol-3,20-dione) and 5-pregnen-3,17a-diol-20-one are transferred to 1-dehydro-cortisone or 1-dehydro-cortisol. The operations can be carried out in different orders, with the exception that the chemical oxidation step must follow the microbiological process which introduces a hydroxyl group in the 11-position. The microbiological processes are particularly of three types as follows: 1) Introduction of an A' unsaturation

(ie a double bond in the 1,2 position)

with simultaneous oxidation of a 3-hydroxyl group when such is present by the action of dehydrogenation microorganisms as described above, whereby a 1,4-dien-3-keto system is obtained. 2) Introduction of a lia- or an 11(3-hydroxyl group by the action of an 11-hydroxylating microorganism of the type Rhizopus nigricans and Curvularia lunata, and 3) introduction of a 21-hydroxyl group by the action of a microorganism of the species Ophiobolus.

The order of these three microbiological functions can be varied, and can e.g. be 1), 2), 3) or 2), 1), 3) or 1), 3), 2) or 3), 1), 2).

If 1-dehydro-cortisone is to be produced, the intermediate product is oxidized at any stage after the introduction of the 11-hydroxyl group, e.g. with chromic acid in pyridine, preferably at room temperature or lower temperature, whereby the 11-hydroxyl group is transferred to ketone oxygen. However, when the 11-hydroxyl group has the |3 configuration and it is desired to obtain the 1-dehydro derivative of hydrocortisone, the oxidation step is omitted.

The introduction of an 11a-hydroxyl group as indicated above is preferably effected by the action of a culture (or of the separated enzymatic material) of Rhisopus nigricans in the manner described in U.S. Pat. patent no. 2,602,769, while the introduction of an 11(3-hydroxyl group is preferably carried out with a culture (or the separated enzymatic material from a culture) of Curvularia lunata, as described in U.S. patent no. 2,658,023. However, where other lla-hydroxylating organisms such as Aspergillus niger, Rhizopus arrhizus and other (3-hydroxylating organisms such as Cunninghamella blakesleeana or the like) are also used.

The 21-hydroxyl group is introduced into the steroid molecule by means of a hydroxylating organism of the species Ophiobolus, preferably O. herbotrichus in the manner described

written by Meystre and colleagues in Helvetica Chim. Acta 37, 1948 (1954).

The dehydrogenation of the steroid compound's A ring to introduce an A' double bond is carried out with a dehydrogenating organism, e.g. of those stated above. This dehydrogenation step can be applied to the starting material or to any step in the finished process.

The different orders of reactions stated above appear from the equations below. The steps where included

1,4-pregnadienes, illustrate features of

the method according to the invention. The other steps concern the production of starting materials.

In the first order (method A) 17ct-hydroxyprogesterone (Ic) or 5-pregnen-3,17a-diol-20-one (I'c) is easily transferred by the action of C. simplex or C. hoagii to 1,4 -pregnadiene-17α-ol-3,20-dione (lic).

Hydroxylation of He with Rhizopus nigricans (in the manner described in U.S. Patent No. 2,602,769) gives compound III. On the other hand, hydroxylation of He with Curvularia lunata (following the method described in U.S. Patent No. 2,658,023) gives compound IVc.

Other lla-hydroxylating organisms such as Aspergillus niger, Rhizopus arrhizus, etc. are equally effective in transferring He to Ille, while other lla-hydroxylating organisms such as Cunninghamella blakesleeana can be used to transfer He to IVc. Ille or IVc can be oxidized to Vc by the action of a mild oxidizing agent such as chromic acid in pyridine. Hydroxylation of the compounds Ille, IVc or Vc at the 21-position is carried out with e.g. Ophiobolus herbotrichus by which one obtains. respectively the compounds Vila, Illa, or Ha (see sheet 1). Both compound VIIa and compound IIa (preferably in the form of their 21-acetates or other lower alkanoyl-21-esters) can be converted to compound Ha by mild oxidation with chromic acid in pyridine. The ester group can then be saponified, e.g. by refluxing with alcoholic potassium bicarbonate.

In the second of the indicated sequences of reactions (method B), the sequence of steps 1 and 2 is reversed, which results in the formation of three new intermediates, namely the compounds VIc, VIIc and VIIIc. However, the individual steps are the same and the final products are likewise the same. C. Combination of the microbiological 1, 2-dehydrogenation with an 11- and 17a-hydroxylation.

The preferred end products, namely 1-dehydrocortisone and 1-dehydrocortisol and their esters can also be obtained from desoxylcorti-costerone (Id below) (4-pregnen-21-ol-3,20-dione) and from 5-pregnen-3,21 -diol-20-one and their esters by a combination of the microbiological 1,2-dehydrogenation with an 11- and 17a-hydroxylation.

The microbiological reactions are of three types as follows: 1) introduction of an A<1->unsaturation

(ie a double bond in the 1,2 position)

with simultaneous oxidation of a 3-hydroxyl group when such is present, or hydrolysis of a 3-ester with subsequent oxidation of the resulting 3-hydroxyl group by the action of a dehydrating microorganism. 2) Introduction of a lin- or a 11(3-hydroxyl group by the action of an organism of the type Rhizopus nigricans or of Curvularia lunata, and 3) introduction of a 17a-hydroxyl group by the action of a culture of a hydroxylating microorganism of the species Trichothecium.

The sequence of these three microbiological reactions can be varied as desired and can be 1), 2), 3) or 2), 1), 3) or 1), 3), 2) or 3), 1), 2). If 1-dehydrocortisone or its esters are to be prepared, the intermediate is oxidized, preferably in the form of its 21-ester at any stage after the introduction of the 11-hydroxyl group, preferably with chromium trioxide at lower temperatures, whereby the 11-hydroxyl group is transferred to the ketooxy gen. However, when the 11-hydroxyl group has the (3) configuration and it is desired to obtain the A<1 >derivative of hydrocortisone, the oxidation step is omitted.

The 17a-hydroxyl group is introduced by means of a hydroxylating organism selected from the species Trichothecium and preferably T. roseum in the manner and with the apparatus described by Meystre et al., Helvetica Chim. Acta 37, 1548 (1954).

The dehydration of the A-ring may be accompanied by a partial or complete hydrolysis when using mono- or polyesters and when this dehydration is complete, the reaction products may be recovered from the mixture by extraction with a suitable solvent immiscible with water, by filtration, by adsorption on a suitable absorbent or by any other method commonly used in the art. Suitable solvents for the extraction are indicated above.

The ester groups may be the same as indicated above. The end products can, if they contain free 21-hydroxyl groups, be transferred to the corresponding esters using known methods, e.g. to their lower alkane esters and especially acetic acid esters.

The different sequences in which the steps mentioned above can be carried out are shown in the diagram below. The steps involving 1,4-pregnadienes illustrate features of the method according to the invention. The other steps concern the production of starting materials.

In this scheme, the compounds Uld, IVd, Vid, Vlld, Illa and Vila are shown as free 21-alcohols for the sake of clarity, but in practice it is preferred to strengthen the 21-hydroxyl group, e.g. with a lower alkanoylating agent prior to the oxidation to compounds Vd, VHId and Ha, the ester group being hydrolysed at any following stage. With regard to the compounds Vid and Vlld, hydrolysis can be carried out together with the introduction of the A'-double bond' by means of Corynebacteria or other dehydrating bacterial cultures.

In the sequence indicated first in the scheme (method C), desoxycorticosterone (Id) is easily transferred by the action of C. simplex to 1,4-pregnadien-21-ol-3,20-dione (Ild), which is also formed by similar treatment of 5-pregnen-3,21-diol-20-one (I'd). Hydroxylation of Ild with Rhizopus nigricans gives Wool. On the other hand, hydroxylation of Ild with Curvularia lunata gives the compound IVd. All these hydroxylation processes can be easily carried out.

As indicated above, other 11a-hydroxylating organisms, such as Aspergillus niger, Rhizopus arrhizus, etc., are equally effective in converting compound Ild to ■■ compound Wool, while other ll(3-hydroxylating organisms such as Cunninghamella Blakesleeana can be used to convert compound Ild to compound IVd . Compound Uld or IVd, or preferably these compounds' 21-acetates or other lower alkanoyl esters can be oxidized to compound Vd or its corresponding ester by the action of a mild oxidizing agent such as chromic acid in pyridine. Hydroxylation of Uld, IVd or Vd at 17- is carried out with T .roseum and gives respectively Vila, Illa and Ila. Both the compounds Vila and Illa (or preferably the 21-acetates of these compounds) can be transferred to compound Ha by mild oxidation with chromic acid in pyridine.

In the second sequence indicated on the form (method D), the starting material (compound Id) is first 11-hydroxylated and then dehydrated in the A ring, whereby three new products Vid, Vlld and VIHd are obtained. However, the final products remain the same and so do the methods used. D. Use of a 16, 17-unsaturated pregnene derivative as starting material.

With the help of the present invention, it is also possible to transfer the relatively cheap and easily available 16-dehydropregnenolone (le) and its esters, and likewise 16-dehydroprogesterone (Ille) to the highly active cortical hormone substances 1,4-pregnadien-ll|3, 17<x,21-triol-3,20-dione (1-dehydrocortisol) and 1,4-pregnadiene-17a,21-diol, 3,11,20-trione (1-dehydrocortisone) and their esters, such as e.g. their lower alkanoyl esters by a relatively small number of chemical and biochemical operations. These operations include: a) Transfer of the 3-hydroxy-AG system to the 3-keto-A4 system (using

16- dehydro-pregnenolone),

b) Introduction of a 17«-hydroxyl group above the 16,17-epoxide. c) Introduction of a 21-hydroxyl group by the action of a microorganism of the species Ophiobolus, e.g. O. herbotrichus. d) Introduction of an lln- or ll[3-hydroxyl group by the action of a suitable microorganism as described above, and e) introduction of the Ai double bond by means of Corynebacterium simplex, or dehydrating microorganisms under similar action, as described above.

Only the introduction of the 1,2-double bond and those of the conversions mentioned here which are carried out after the introduction of this double bond come under the method according to the invention.

The oxidation of the 3-hydroxyl group of the 16-dehydropregnenolone (le) can be carried out by means of Oppenauer's reaction or by careful oxidation with chromic acid at room temperature or lower temperature, but it is preferred to carry out this oxidation simultaneously with the introduction of the A'-double bond by treating a culture of a dehydrating microorganism or with an enzyme extract of such a culture, as described in more detail above. However, such a treatment should follow the introduction of a hydroxyl group in one or more of the 11-, 17- and 21-positions, as otherwise the yields will be poor. If desired, esters such as lower alkanoyl esters of 16-dehydropregnenolone can be used as starting material for the microbiological reaction. The introduction of the 17a-hydroxyl group can be carried out by treatment with a peracid such as perphthalic acid and perbenzoic acid, whereby the intermediate product 16,17-epoxide is obtained. This intermediate product is then preferably exposed to the action of hydrogen iodide in a mixture with acetic acid and acetic anhydride, whereby the 16-iodo-17a-hydroxy compound is obtained. The iodine atom is then removed by heating the compound dissolved in a lower aliphatic alcohol such as ethanol, and containing a small amount of an organic acid such as acetic acid and in the presence of Rahney nickel catalyst. The heating thus takes place during reflux cooling. In this reaction, the hydrogen adsorbed on the catalyst will replace the iodine group.

The 21-hydroxyl group is introduced in the manner already described by exposing the 21-methyl compound to a culture of a member of the species Ophiobolus. The introduction of the lla- or ll|3-hydroxyl group is likewise carried out biochemically as described above in connection with other substances. It has been found that the 11(3-hydroxyl group can be introduced in the desired positions, even when treated with 1,16- and 1,4-pregnadienes, 1,4,16-pregnatrienes and 1,4-pregnadienes. Likewise, the 11a-hydroxyl group can is introduced without saturation of any of the double bonds.

The introduction of the double bond is carried out as described above, preferably after hydroxylation. Cultures of dehydrating microorganisms are able to introduce an A1 double bond even though the steroid molecule already contains a double bond at the carbon atom in the 16-position in addition to the double bond at the carbon atom in the 5-position. As is the case with the other starting materials listed above, the culture is capable not only of introducing an A1 double bond, but also of oxidizing a 3-hydroxyl group to a keto group with concomitant displacement of a 5,6-double bond to a 4,5- the position, and under certain conditions the culture can also effect oxidation of a 20-hydroxyl group to a 20-keto group.

When 1-dehydrocortisone is desired as the end product, it is preferred to introduce a 11a-hydroxyl group rather than a 1113-hydroxyl group as the 1113-hydroxyl group is generally easier to introduce and can subsequently be oxidized to a 11-keto group.

The intermediates have the chemical constitution shown by formulas VIII and IX (formula scheme II). In these formulas

OH

denotes X H„ O or < (a or (3),

H

while R denotes H, OH or OAcyl, the acyl group in each case being the acyl group of an organic carboxylic acid, but preferably of a lower alkanoic acid, such as formic acid, acetic acid, propionic acid, butyric acid, valerian acid or caproic acid. However, other acids such as substituted alkanoic acids and aromatic acids, such as e.g. cyclohexylacetic acid, cyclopentylpropionic acid and benzene acid, not excluded.

Compounds in which X denotes hydr-

oxyl and R an acyl group, is prepared by esterifying 16,17-oxido-1,4-pregnadiene-1,21-diol-3,20-dione so that the 21-ester is obtained before the oxidation of the 11-hydroxyl group. The oxidation is then opened with HJ or HBr so that the 16,17-halohydrin is formed. The halogen is then removed with a Raney nickel catalyst or with hydrogen in the presence of a catalyst consisting of palladium on charcoal.

As already mentioned, the different steps in the method can be carried out in different sequences. Some of these sequences are indicated in the following, where the steps that give pregnanes as a product relate to the production of starting materials by the method according to the invention, while the steps that give or convert 1,4-pregnadienes illustrate the actual method according to the invention:

Most of these steps are completely independent of each other so that the sequence of operations can be changed in many ways in accordance with what is appropriate in each case for the achievement of good results.

E. 11-hydroxylation and subsequent 11-oxidation of 1,4-pregnadiene derivatives.

It turned out that cultures of microorganisms or enzymes separated from these which have the known property of being able to introduce a hydroxyl group in the 11-position (in the a or (3) position) on saturated pregnanes or monounsaturated pregnanes are able to introduce such an 11- hydroxyl group also in 1,4-pregnadienes which are unsubstituted in the 11-position, and that the conjugated double bond system in the A-position is not modified by such microorganisms.

1,4-pregnadiene-17a,21-diol-3,20-dione

and its 21-esters (If below) can thus, despite the presence of two unsaturated bonds in the A-ring, be transferred to the corresponding 11-hydroxyl group by treatment with various known 11-hydroxylating microorganisms. Thus, for-

bond If is either lla-hydrolyzed by the action of one of the various known llcc-hydroxylating organisms with Rhizopus arrhizus, Rhizopus delmar, Rhizopus nigricans, 1 Rhizopus reflexus, Aspergillus niger, Phycomyces blakesleeanus, so that one obtains Hf, or it can be lip-hydrolyzed by action of Cunninghamella blakesleeana, Curvularia lunata or one of the other known 11(5-hydroxylating organisms so that one obtains Illa or Illb. For products from the lla-hydroxylation, compound Hf, in the form of its 21-esters, preferably the acetate, is then oxidized by the action of chromic acid dissolved in pyridine, or by means of other oxidizing agents with equivalent potential to 1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-acetate (Hb), which can be easily hydrolyzed to The 21-OH compound (1-dehydrocortisone, II a). The product of lip hydrolysis is the 21-ester of 1-dehydrocortisone (compound III b) which can be oxidized to the 21-acetate of 1-dehydrocortisone with subsequent hydrolysis of 21 -ester.

The reactions that take place in this procedure can be shown by the following form:

If compound If is in the form of the free 21-alcohol, after the 11-hydroxylation it is temporarily transferred to the 21-ester before it is oxidized. As before, the acyl radical is preferably acetic acid radicals, but can also be a radical of other lower fatty acids such as formic acid, propionic acid, butyric acid, valeric acid or succinic acid, or of aromatic acids such as benzoic acid, salicylic acid, phthalic acid and veratric acid (3,4-dimethoxy-benzoic acid ). As regards dibasic acids, the ester is preferably in the form of a half-ester. If desired, the acyl group can be hydrolyzed, e.g. by heating with alcoholic potassium bicarbonate.

G. Introduction of halogen in 9 a-position one.

It is not necessary for the preparation of compounds with formulas IV and V to use starting materials with the halogen atom (fluorine, chlorine or bromine) in the 9a position. This substituent can be introduced into the steroid molecule after the dehydration of the A ring. The substitution is carried out by starting from the II (a or (3)-hydroxy-1,4-pregnadiene compound and transferring it to the 1,4,9(11)-pregnatriene, which can then be treated in various ways to introduce the desired halogen atom in the 9a-position and at the same time to introduce a lip-hydroxyl group. The 11-hydroxyl group can then be oxidized under favorable conditions so that it is replaced by keto-oxygen (eg to 9a-halo-1-dehydrocortisone).

As starting material, it is preferred to use 1,4-pregnadiene-lip,17a,21-triol-3,20-dione-(III) or the corresponding 11a-hydroxyl compound (VII) or 21-ester of these compounds. The starting material is first esterified (III b) in the 21-position (if there is not already a 21-ester) by treatment with an acid anhydride in pyridine, or by using an acid chloride so that you get e.g. a lower alkanoyl ester such as the 21-acetate or 21-propionate or any other suitable ester such as 21-cyclopentylpropionate, 21-enanthate or the like. Dehydration to achieve the introduction of the 9,11-double bond is carried out in the case of compound III b, by the action of phosphorus oxychloride in pyridine. However, it is preferred to form the 1,4,9 (11)-triene by treating the 21-ester of the lla-hydroxy compound (VII b) with p-toluenesulfonyl chloride in pyridine, whereby the corresponding lla-tolylate (llct-p -toluenesulfonate) (I j). When treating the latter compound with a basic substance such as sodium acetate in acetic acid (or with potassium acetate, lithium propionate or the like), the removal of the p-toluenesulfonic acid is carried out while introducing the 9(11) double bond. Instead of the p-toluenesulfonate, the corresponding methanesulfonate, benzenesulfonate or other similar sulfonic acid esters can be produced there as an intermediate product. These reactions are shown in the following form:

The constitutional formula for 1,4,9(11)-pregnatrienes, of which compound II j is an example, is shown in formula XV in formula scheme II. In this formula, R denotes hydrogen or acyl, preferably lower alkanoyl.

The step (II j) is now treated with a compound that gives bromine ions, e.g. N-bromoacetamide or N-bromosuccinimide in perchloric acid or other strong acid such as acetic acid. This gives the 9cc-bromo derivative (III j). If it is desired to substitute fluorine or chlorine in the 9ct position, the 9a-bromo compound (the bromohydrin III j) is dehalogenated by the action of a weak base such as potassium acetate or sodium acetate in a relatively neutral solvent such as acetone or alcohol. This gives the epoxy IV j. This is exposed to the action of anhydrous hydrogen fluoride, whereupon the epoxidation is opened and a 9a-fluorine atom is introduced together with a ll|3-hydroxyl group. You then get compound V (formula I), where Z in this case denotes fluorine. By using anhydrous hydrogen chloride, the analogous 9a-chloro compound is formed. This can then be hydrolysed to cleave off the 21-acyl group, e.g. with the aid of aqueous acid, whereby the 21-alcohol is obtained.

The 9a-fluorine compound can be subjected to mild oxidation, e.g. with chromic acid in pyridine at room temperature whereby

The ll(3-hydroxyl group is replaced by keto-oxygen. You then get compound IV b which can be hydrolyzed with acid to give 9a-fluoro-1,4-pregnadiene-17a,21-diol-3,11,-20-trione .

It is preferred to use 1,4-pregnadiene-11a,17a,21-triol-3,20-dione or the corresponding lip-hydroxyl compound as starting material. This new 11a-hydroxyl compound is prepared in the manner described above. It is important that before the treatment with p-toluenesulfonyl chloride, the 21-hydroxyl group is selectively protected before this group is esterified with a sulfonic acid derivative. It has been found that if one does not protect the 21-hydroxyl group from the action of sulfonyl halides such as p-toluenesulfonyl chloride, one obtains mixtures which cannot be rectified. Furthermore, it is preferred for the esterification in the 21-position to use a relatively mild acylating agent such as the anhydrides described above, whereby real selectivity is achieved and the 11-hydroxyl group remains unesterified.

H. Preparation of esters with increased duration of action.

The invention also includes the production of compounds with the formulas II to V in the form of 21-esters which are not as quickly converted by metabolism in the body as the free 21-alcohol compounds, so that the physiological effect of the compounds is maintained for longer periods of time and the frequency of the injections can thereby be significantly reduced.

The acids whose esters have been found to have longer periods of action of cyclohexanecarboxylic acid, 4-methylcyclohexanecarboxylic acid, 3-ethylcyclohexylacetic acid, cyclohexylpropionic acid, cyclopentylpropionic acid, phenylacetic acid, trimethylacetic acid, tertiary butylacetic acid, butoxybutyric acid, ethoxycaproic acid, methylthiovaleric acid , isopropylthioacetic acid, phenylthioacetic acid, caproic acid, isobutyric acid, enanthic acid, isocaproic acid, cyclohexylcaproic acid, undecylenic acid, 2-ethylbutyric acid, methylbenzoic acid and ethoxybenzoic acid.

It has been found that acids from the groups of aryloxyalkanoic acids and the pyroslimic acids in particular give a significantly longer duration of hormone activity, and thus enable a more effective, convenient and generally beneficial therapy than what can be achieved with the corresponding original hormones. Typical of beneficial acids of this group are phenoxyacetic acid, p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 5-chlorofuronic acid, 5-methylfuronic acid, 5-bromofuronic acid, 4-bromophenoxyacetic acid, 4-methylphenoxy- acetic acid, 4-methoxyphenoxyacetic acid, 4-tertiary-butyl-phenoxyacetic acid, 5-tertiary-butylfuronic acid, furonic acid and phenoxypropionic acid.

The esters of the above-mentioned acids have been found to increase the duration of activity not only of 1-dehydrocortisone, 1-dehydro-cortisol, dehydro-9a-halo-cortisone and 1-dehydro-9a-halo-cortisol (in which the halo-gen atom is a chlorine atom or a bromine atom), but also of the physiologically important intermediates such as 1-dehydrocorticosterone, 1-dehydro-11-desoxycorticosterone, 1-dehydro-11-desoxycortisone, 1-dehydro-17-desoxycortisone and 1-dehydro-aldosterone .

As examples of the increased duration of activity, it has been found that injection of 0.25 mg of 1-dehydrocortisone or 1-dehydrocortisol or their acetates in adrenalectomized mice causes eosinopendia from two to four days. A similar injection of 1-dehydrocortisone-21-(2',4'-dichlorophenoxyacetate) lasts 12 to 20 days, that of 1-dehydrocortisone's 21-(5'-bromofuroate) lasts 10 to 18 days, that its 21-furoate of 1-dehydrocortisol lasts 10 to 16 days and that of its 21-(4'-tert-butyl-phenoxyacetate) lasts 14 to 20 days.

In another attempt, an in-

injection of 1-dehydrocortisone 21-(5'-chloro-furoate) in rats reduced the weight of the bracken for a period that was from 5 to 8 times longer than that caused by the free hormone, while 1-dehydrocortisol 21-(4'-tertiary -butylphenoxyacetate) caused shape changes ("involution") from 4 to 6 times longer than the free hormone. These samples are standard indications of glycocorticoid (ie anti-arthritic) activity. Likewise, the mineralocorticoid activity of the hormones is improved by esterification with these acids. Thus, 1-dehydro-11-desoxycorticosterone-21-(4'-chlorophenoxy-acetate) maintains the life of adrenalectomized mice 5 to 7 times longer than the free hormone, and 1-dehydro-aldosterone-21-furoate maintains the life of such animals 6 to 10 times longer than the corresponding hormone.

In human therapy, the same protection of the hormone from destruction is observed with a simultaneous extension of the duration of the level of the therapeutic activity. Thus, one or two weekly injections of l-dehydrocortisone-21-furoate or l-dehydrocortisol-21-(4'-tertiary-butyl-phenoxyacetate) replace seven daily doses of the free hormone. Likewise, injections of 50—100 mg of 1-dehydrocortisol-21-(5'-tertiary-butylfuroate or 1-dehydrocortisone-21-(4'-chlorophenoxyacetate) have an activity lasting 7 to 10 days, while the activity of the free hormone in equivalent doses only lasts one day.

Particular examples of 21-esters of 9α-halogen derivatives of 1-dehydrocortisone and 1-dehydrocortisol are 21-(2'-furoate), 21-[ (5'-tertiary-butyl)-2'-furoate) ], 21-phen -oxyacetate and 21-(2',4',5'-trichlorophenoxyacetate).

The esters can be given in the manner usual for cortical hormones. Thus, they can be mixed with inert material to produce tablets for oral use or to be held under the tongue. Another common form of application is ointments or liquid preparations for local use. The most effective way to give the remedy is to inject it

intramuscularly, subcutaneously or between

the joints (inter-articular) in the form of solutions or suspensions in oils or aqueous media.

The esters can be produced in several ways. The simplest is esterification of the corresponding free hormones, e.g. by treating the hydroxysteroid with the corresponding acid anhydride or chloride, possibly in the presence of a base, or by treating the hydroxysteroid with the free acid in the presence of an acid catalyst under dewatering conditions.

For illustrative purposes, the following examples provide a detailed description of various embodiments of the invention.

Example 1.

Conversion of cortisone to 1-^ e-hydrocortisone.

27 portions of 100 ml each are taken from a solution of 30 g of yeast extract ("Difco") in 3.0 liters of tap water, containing 13.2 g of potassium dihydrogen phosphate and 26.4 g of disodium hydrogen phosphate (the -H value of the solution equal 6.9). These portions are placed in 300 ml Erlenmeyer flasks and sterilized by autoclaving for 15 minutes at a steam pressure of 1.05 kg/cm2

(120°C). After autoclaving and cooling the substrate, 1 ml of a suspension of Corynebacterium simplex (A.T.C.C. 6946) is placed in each flask. The flask is then shaken on a shaking table at 220 rpm. my. and 28°C for 24 hours.

150 mg of Kendal's Compound E are placed in each of the 27 Erlenmeyer flasks. The flasks with their contents are then sterilized for 15 minutes at a steam pressure of 1.05 kg/j ems (120°C). Then add 5.0 ml of ethanol to each bottle. The 24-hour-old bacterial culture is then transferred aseptically, and the resulting suspensions are shaken on a shaking table at 220 rpm. min for 48 hours at 28°C. The final pH value is 7.2.

The contents of all flasks are combined and extracted with the entire 9.0 liters of chloroform in three equal portions. The combined extracts are then concentrated to a residue which is recrystallized from a mixture of acetone and hexane. 1.1 g of 1-dehydrocortisone-(1,4-pregnadiene-17a,21-diol-3.11:20-trione) is obtained with m.p. 210—215°C (decomposition). After several recrystallizations, the substance's melting point rose to 230-232°C (decomposition).

[a] -Ds = + 175.3 (dioxane), e2.!8<1>5.400 (methanol).

Found on analysis:

C 70.38%, H 7.67% Calculated for C.MH2(10:-: C 70.37%, H 7.51% 21-acetylation of 1-dehydrocortisone. A solution of 0 .5 g of 1-dehydro-cortisone in 5 ml of anhydrous pyridine is added to 3 ml of acetic anhydride. The reaction mixture is allowed to stand overnight at room temperature, after which it is diluted with ice and water. The resulting precipitate is filtered from the mixture and recrystallized from a mixture of acetone and hexane. 0.35 g of l-dehydro-cortisone-21-acetate is obtained with m.p. 227 —228°C (decomposition). After several recrystallizations from a mixture of acetone and hexane, this substance melts at 233-236°C (decomposition).

Example 2.

Transfer of cortisol to 1- dehydro-cortisol. 27 portions of 150 mg of cortisol (4-pregnen-lip,17rt,21-triol-3,20-dione) each in its Erlenmeyer flask, are treated as described in Example 1. The pH value at the end of the shaking period is 7.0 .

The contents of all flasks are combined and extracted with 9.0 liters of chloroform in three equal portions. The combined extracts are then concentrated to a residue weighing 3.75 g. The residue's m.p. is 227-232°C. 2.75 g of this raw material is slurried with 50 ml of acetone and the suspension is cooled. By filtering the suspension, 1.35 g of 1-dehydro-cortisol (1,4-pregnadiene-llfS,17(z,21-triol-3,20-dione)) with m.p. 237-239°C (decomposition ).A further amount of product can be recovered from the mother liquor. Upon recrystallization from acetone, the substance's m.p. rose to 239-241°C (decomposition),

[a]2ns = + 107° (dioxane), e24:j 14.600 (methanol).

Found on analysis:

C 70.24 per cent, H 8.13 per cent.

Calculated for C21H2R0-:

C 69.97 per cent, H 7.83 per cent.

21-acylation of 1-dehydrocortisol.

A solution of 0.85 g of 1-dehydrocortisol in 5 ml of pyridine is added to 3 ml of acetic anhydride. The reaction mixture was allowed to stand overnight at room temperature, after which it was diluted with ice water. The resulting precipitate is filtered from the mixture and recrystallized from a mixture of acetone and hexane. 0.45 g of 1,4-pregnadiene-116,17d;21-triol-3,20-dione-21-acetate (Cl-dehydro-cortisol-21-acetate) is obtained with m.p. 235-239°C. During recrystallization, the substance's m.p. rose. to 237-239°C.

[a] <2>Ds = + 116°C (dioxane), e2.1:, 15,000 (methanol).

Found on analysis:

C 68.62 percent, H 7.78 percent.

Calculated for C^ R^ O^.

C 68.73 per cent, H 7.51 per cent.

Example 3.

Conversion of Reichstein's compound S to 11-hydroxy-l-dehydro-Reichctein's compound S

100 ml of a 1.0% yeast extract concentrate Containing 9.0 ml 0.2 M

KH2PO, and 9.0 ml of 0.2 M Na2HP04 are sterilized as above and inoculated with a 1.0% suspension of Corynebacterium simplex (A.T.C.C. 6946) from a 24 hour culture. The newly inoculated culture is incubated and shaken (shaking table) for 20 hours at 28°C. After the incubation, the culture is aseptically transferred to a sterile 300 ml Erlenmeyer flask containing 150.0 mg of sterile Reichctein's compound S-(4-pregnene-17a,21-diol-3,20-di-one)- in 5.0 ml of ethanol or acetone. The pH value of the reaction mixture is 7.0. The bacterial culture containing the steroid and the solvent is incubated and shaken for a period of 48 hours at 28°C. The final pH value of the reaction mixture is 7.2-7.4. The culture is then thoroughly extracted with chloroform. The extracts are combined and evaporated to dryness on a water bath. The crude extract weighs 196.0 mg.

The entire quantity of the crude extract is slurried with methanol and 80 mg of a crystalline solid with m.p. 246

-250°C. After two crystallizations from acetone, m.p. 246—249°C, cleavage, [a] V

= + 76° (CHCl 1 ), <e>243 15,000 (C 2 H 5 OH). Calculated for C21H28<6>4<:>

C 73.22 percent, H 8.19 percent.

Found:

C 73.56 per cent, H 8.40 per cent.

By means of the infrared spectrum and chemical analysis, it was established that the product is 1,4-pregnadiene-17a,21-diol-3,20-dione. This is then hydrolyzed in the 11-position to the desired end product.

21- acetylation of 11- hydroxy- l- dehydro-Reichstein- compound- S.

A solution of 0.25 g of 11-hydroxy-1-dehydro-Reichstein's compound-S in 2 ml of pyridine was added to 1 ml of acetic anhydride. The reaction mixture was allowed to stand at room temperature overnight, after which it was diluted with ice and water. The resulting precipitate was filtered off and recrystallized from a mixture of methylene chloride and hexane, whereby 0.20 g of 1,4-pregnadiene-11,17a,21-triol-3,20-dione-21-acetate was obtained.

Example 4.

Transfer of Reichstein's compound-S to 11-hydroxyl-l-dehydro-Reichstein's compound-S.

100 ml of a medium consisting of 1% fish extract ("fish solubles"), 0.1% yeast extract, 9.0 ml 0.2 M KH2PO4 and 9.0 ml 0.2 M Na2HP04 are sterilized as described above and inoculated with a suspension of Corynebacterium simplex from a 24 hour culture. The newly inoculated culture

incubated and shaken (shaking table) for 20 hours at 28°C. After incubation, the culture is aseptically transferred to another sterile 300 ml Erlenmeyer flask containing 150 mg of sterile Reichstein's Compound-S in 5.0 ml of ethanol. The reaction mixture's pH value is 6.9. The bacterial culture containing steroid and solvent is incubated and shaken for 48 hours at 28°C. The final pH value

is 7.3. The culture is extracted with 2 liters of chloroform in five equal portions, the extracts are combined and the mixture obtained is concentrated on a steam bath.

The crude residue is taken up in methylene chloride and chromatographed over Florisil. 30 g of 1,4-pregnadien-17a,21-diol-3,20-dione (as well as 90 g of 1,4-pregnadien-17a,20,21-triol-3-one) are isolated from the chromatographic starting material (15 mg), which after introduction of an 11-hydroxy group gives the desired product.

Instead of the ethanol, other water-miscible organic solvents that are not toxic to the microorganism can be used, such as e.g. acetone, mixtures of ethanol and acetone and the like.

Example 5.

II - hydroxy- l- dehydro- Reichstein's compound- S.

100 ml of culture containing 0.1% yeast extract concentrate, 9.0 ml of 0.2 M KH2-P04 and 9.0 ml of 0.2 M Na2HP04 is inoculated with 1 ml of a 24 hour culture of Corynebacterium simplex. The flask is incubated at 28°C for 24 hours.

150 ml sterile 5-pregnen-ip,17a,21-triol-20-one-3,21-diacetate in 50 ml acetone is inoculated into a 300 ml Erlenmeyer flask with the 24-hour culture and incubated for 48 hours at 28 to 30°C .

The products are extracted with chloroform, the chloroform extracts are concentrated to a small volume and chromatographed on Florisil. : The sequence of elution is first unreacted starting material (75 mg), then Reichstein's compound S-21-acetate (15 mg) and finally . 1-dehydro-Reichstein's compound-S (30 mg). All products were identified by comparing their spectra in infrared with such spectra for authentic samples. In a known manner, a hydroxy group is then introduced in the 11-position, whereby the desired product is obtained.

By using the same reaction medium and the same organism as in example 5, as well as the same quantity ratio of steroid, 4-pregnene-lip,21-diol-3,20-dione (corticosterone) is transferred to crystalline 1,4-pregnadiene lip,21-diol-3,20-dione (1-dehydro-corticosterone). Likewise, 4-pregnen-21-ol-3,20-dione (desoxycorticosterone) gives 1,4-pregnadien-21-ol-3,20-dione as a crystalline solid, while 4-pregnen-21-ol-3.11, 20-trione (11-dehydrocorticosterone) is converted to crystalline 1,4-pregnadiene-21-01-3,11,20-trione. The necessary hydroxy groups are then introduced in the 11- and/or 17-position.

With regard to the acetylation of the dienes described in examples 1, 2 and 3, it will be obvious that other esters of the diene compounds can be prepared in a similar way, e.g. by reaction with the anhydride or chloride of the acid in a known manner, so that one obtains the formates, propionates, butyrates and valerates and likewise the esters of other non-toxic acids such as the benzoates as well as the neutral and acidic esters of polybasic acids, such as succinic acid, maleic acid , malic acid, citric acid, tartaric acid, phthalic acid and hexahydrophthalic acid. In the production of acidic esters, the metal salts can be formed in the usual way by reaction with the hydroxide, carbonate or bicarbonate of the metal, such as e.g. an alkali metal or an alkaline earth metal.

Instead of first forming the 1,4-diene of cortisone and hydrocortisone and then esterifying the products, the corresponding esters of cortisone and hydrocortisone and of their intermediates can be subjected to the process according to the present invention and brought to give the dienes of the esters. Thus, in example 1, cortisone can be replaced by its 21-ester or by its 17a,21-diester or by 5-pregnene-3,17a,21-triol-11,20-di-one-21-acetate or 17a,21- diacetate or 3,17a,21-triacetate or other esters, while in example 2 cortisol can be replaced by its 21-acetate, or 17a,21-diacetate or HS.17a,21-triacetate, or by 5-pregnene-3.11|3 ,17a,21-tetrol-20-one-3,21-diacetate,

3,17α,21-triacetate or 3,lip,17α,21-tetraacetate. The latter group of compounds can be replaced by the corresponding 11a-hydroxyepimers, so that one obtains 11-epi-1-dehydro-cortisol and its 21-esters and 17<x,21-

diesters. The polyesters can in any case be mixed esters such as 3-propionate-21-acetate. For example, cortisol can be transferred to 1-dehydro-cortisol (example 2) or cortisol-21-acetate can be transferred to 1-dehydro-cortisol or its 21-acetate. From 1.0 g of cortisol-21-acetate, 0.22 g of 1-dehydro-cortisol (Illa) with m.p. 239-241°C (decomposition) is obtained.

When it is desired to avoid saponification in position 21, the same conditions as stated above are used with the exception that the ambient temperature for the cultivation and the reaction step of the process is raised to 36° C. The product is isolated in the usual way. From 1.0 g of cortisol-21-acetate, you get 0.13 g of 1-dehydro-cortisol-21-acetate (IHb) with m.p. 237—239° C (decomposition).

Similarly, by proceeding as indicated in example 1, 1.0 g of cortisone-21-acetate can be transferred to 1-dehydro-cortisone, of which 0.17 g is isolated with m.p. 230-232°C. If the ambient temperature during cultivation and the reaction step of the process is raised to 36°C, 1.0 g of cortisone acetate, 0.11 g of 1-dehydro-cortisone-21-acetate with m.p. 230— 233°C (decomposition).

Example 6.

Transfer of cortisol to 1- dehydro-cortisol.

10 portions each of 100 ml, placed in a 300 ml Erlenmeyer flask and sterilized by autoclaving for 15 minutes with 1.05 kg/cm2 steam pressure (120°C). After the autoclave treatment and cooling of the culture, 1 ml of a suspension of Corynebacterium hoagii (American Type Culture Collection 7005) is placed in each Erlenmeyer flask. The flasks are then shaken on a shaking table at 220 rpm. minute and 28°C for 16<1>4 hours.

In each of the 10 Erlenmeyer flasks, under aseptic conditions, 50 mg of cortisol, dissolved in 1 ml of 90% methanol, is added. The flasks are again placed on the shaking table and incubated for 7 hours. The pH value at the end of the shaking period is 6.82.

The contents of all flasks are combined and extracted with a total of 3 liters of chloroform in three equal portions. The combined extracts are concentrated to a residue of 425 mg. Crystallization of the residue from acetone yields 248 g of 1-dehydrocortisol.

Example 7.

Transfer of cortisol to 1- dehydro-cortisol.

Ten portions of 100 ml each are taken from a solution of 0.5 g "Basa-min Busch" (Anheuser Busch) in 1 liter of tap water, placed in Erlenmeyer flasks and sterilized by treatment for 15 minutes at 1.05 kg/ cms vapor pressure (120°C). After autoclaving and cooling the culture, 1 ml of a suspension of Corynebacterium simplex is placed in each flask. The flasks are then shaken on a shaking table at 220 rpm. minute and at 28°C for 24 hours.

In each of the ten Erlenmeyer flasks, 50 g of cortisol, dissolved in 0.8 ml of absolute methanol, is added under aseptic conditions. The flasks are placed again on the shaking table and incubated for a further 4-7 hours. The pH value at the end of the shaking period is 7.2-7.6.

The contents of all flasks are combined and extracted with a total of 3 liters of chloroform in 3 equal portions. The combined extracts are concentrated to a residue of 490 mg. Crystallization of the residue from acetone yields 403 mg of 1-dehydrocortisol. Sm.p. 238

—240°C (decomposition), [a] y = + 105°

(dioxane) e.M;, 14,500 methanol.

By using cortisone instead of cortisol in this example, and increasing the reaction time to 6-12 hours, crude 1-dehydro-cortisone is obtained with a yield of 85 per cent.

This example (7) shows that the yield is increased by reducing the concentration of the starting material.

The other 4-pregnene compounds described above can likewise be treated as indicated in Example 7. As in all other examples indicated above, the 21-esters can be used as starting material and likewise the analogous 5-pregnene-3- hydroxy compounds.

Example 8.

Transfer of 6-dehydrocortisone-21-acetate to 1-dehydrocortisone.

0.85 g of 4,6-pregnadiene-17α,21-diol-3,11,20-trione-21-acetate (6-dehydro-cortisone-21-acetate), obtained on it in J. Biol, Chem. 197, 261 (1952) described manner, is transferred to 1,4,6-pregnatriene-17a,21-diol-3,11,20-trione

in the manner described above by the action of enzymes of a culture of Corynebacterium simplex. You get a dividend of around 18 per cent. The step obtained has

adreno-cortical hormone properties. It can be partially hydrogenated to the corresponding 1,4-d<i>ene by reduction under relatively mild conditions, e.g. by means of a zinc, acetic or ascorbic acid in the manner described in the above-referenced publication.

In the manner described, other 1.4.6-pregnatrienes can be prepared from starting materials with a system of conjugated double bonds distributed in the A and B rings. Such pregnatrienes can be partially hydrolysed in a manner similar to that mentioned above, so that the corresponding 1,4-pregnadienes are obtained. The substituent in the 3-position can also be a hydroxyl group and in that case the conjugated double bonds will generally run between the 5,6- and 7,8-carbon atoms. The substituent in the 11-position may be an α- or (3-hydroxyl group or may be absent, and the group in the 21-position may be methyl instead of hydroxymethyl or acyloxymethyl. Thus,

6-dehydro-cortisol and 5,7-pregnadien-3.11(3,17n.21-tetrol-20-one under the influence of

the cultures described above are transferred to 1,6-bis-dehydro-cortisol, while 4,6-pregnadiene-17a,21-diol-3,20-dione is transferred to 1,4,6-pregnatriene-17a,21-diol- 3.20-dione, with subsequent introduction of an 11-hydroxy group. In each case, the 21-ester can

is used and this can be hydrolysed or not hydrolysed during the method according to

the invention. By partial hydration as described above, the corresponding 1,4-pregnadienes are obtained.

Example 9.

Transfer of 4-pregnene-lln, 17n, 21-triol-3, 20-dione to 11-epi-l-dehydro-cortisol.

The reaction is carried out exactly as described in connection with the transfer of cortisol to the corresponding diene, and the product is isolated by extraction with chloroform and crystallized from a mixture of acetone and hexane. From 1.0 g of 11-epi-cortisol, 0.25 g of 11-epi-1-dehydro-cortisol is isolated as a crystalline solid, m.p. 245—246°C (decomposition).

Acetylation of 11-epi-1-dehydro-cortisol (1.0 g) is carried out by dissolving the compound in 15 ml of anhydrous pyridine with subsequent addition of 0.3 g of acetic anhydride. The reaction mixture is allowed to stand overnight at room temperature and then poured into ice water. The precipitate thus obtained is separated by filtration and recrystallized from a mixture of acetone and hexane, whereby crystalline 11-epi-1-dehydro-cortisol-21-acetate is obtained.

Example 10.

1, 4- pregnadiene- 9a- fluorine- 17' a, 21 -

diol-3,11,20-trione.

In a 300 ml Erlenmeyer flask, place 100 ml of 0.1% yeast extract ("Difco") containing 9.0 ml of 0.2 M calcium dihydrogen phosphate and 9.0 ml of 0.2 M disodium hydrogen phosphate. The flask and its contents are sterilized by autoclaving for 15 minutes at 120°C and 1 ml of a 1% suspension of Corynebacterium simplex is added to the sterile medium. The flask and its contents are incubated at 28°C for 24 hours.

In another 300 ml Erlenmeyer flask, place 2 ml of ethanol and 25 mg of 9a-fluoro-11-dehydro-corticosterone. After 24 hours of cultivation, the culture is transferred under aseptic conditions to the flask containing the steroid, after which the mixture is incubated at 28°C and shaken for 10 hours. At the end of this time, the reaction mixture is thoroughly extracted with chloroform and the chloroform extracts are concentrated to a residue. The residue is crystallized from a mixture of acetone and hexane, which gives 4 mg of 9a-fluoro-1,4-pregnadien-21-ol-3,11,20-trione, into which a 17a-hydroxy group is then introduced in a known manner.

The 21-acetate of the compound according to this example is prepared by allowing 0.3 g of acetic anhydride to react with a solution of 1 g of the 21-ol in 20 ml of anhydrous pyridine at room temperature overnight with subsequent dilution with ice water.

In a similar way, the method according to this example gives (10) 9a-fluoro-1,4-pregnadiene-1ip,17a,2l-triol-3,20-dione with a yield of 5 mg from 25 mg of unsaturated starting material with a double bond. The 21-acetate is obtained by the usual acetylation.

By using a 1% suspension of Corynebacterium hoagii instead of C. simplex according to example 10 and otherwise the same conditions, 25 mg of 9a-chloro-corticosterone is transferred to 9a-chloro-1,4-pregnadien-lip,21- diol-3,20-dione with a yield of 7 mg. This product can be transferred to the 21-propionate by reaction with propionic anhydride. With the same culture (C. hoagii) 9α-bromo-corticosterone-21-acetate is transferred to 9α-bromo-1,4-pregnadiene-11(3,21-diol-3,20-dione.

By proceeding as indicated in Example 10, but using an incubation period of 48 hours at 28-30°C, 9a-fluorocortisol is converted to 9a-fluoro-1-dehydrocortisol. Using the same procedure, 9a-fluorine, 9a-chloro and 9a-bromo-cortisone are transferred to 9a-fluorine, 9a-chloro and 9a-bromo-1-dehydro-cortisone, respectively. 9a-chloro and 9a-bromo-cortisol are transferred in the same way to 9a-chloro- and 9a-bromo-1-dehydrocortisol.

The 17-deoxy compounds can be transferred to the corresponding 17a-hydroxy compounds

(9a-halogen derivatives of 1-dehydro-cortisol and 1-dehydro-cortisone) by the action of a culture of Trichothecium reseum as described above.

Example 11..

1, 4-pregnadien-ll, 17a, 21-triol-3, 20-dione.''

Bacillus sphaericus (A.T.C.C. 70 55) is incubated in a nutrient agar consisting of Bacto beef extract 3 g, Bacto peptone 5 g, sodium chloride 8 g, agar 15 g and tap water 1 liter for 24 hours at 28°C.

100 ml of a sterile nutrient medium consisting of Bacto beef extract 3 g, Bacto-peptone 5 g, filled to 1 liter with

tap water in a 300 ml flask is added to a small portion of the incubated culture and the culture mixture is further incubated for 24 hours at 28°C on a shaker under aerobic conditions. The culture thus obtained is used as a 1 percent inoculation medium.

To each of the ten flasks containing 100 ml of sterile nutrient medium, 1 ml of this inoculation medium is added. The contents of the flasks are stirred by shaking them on a rotating shaker for 8 hours at 28°C with 240 strokes per minute. minute. After this cultivation period, 25 mg of Reichstein's compound-S in 0.5 ml of methanol is added to each flask under aseptic conditions. The flasks are shaken again and incubated for a further 24 hours. The final pH value is 7.8.

The contents of the flasks are then combined and extracted three times with 2 liters of chloroform in each extraction. The combined chloroform extracts are evaporated to dryness, whereby 310 g of crude product is obtained. This steroid crude product is purified by chromatography in the system described by G.M. Shull: Abstracts of Papers of the 126th Meeting of the American Chemical Society, December 12-17, 1954, page 9a, Paper No. 24. The chromatographic determination shows quantitative conversion of the starting material to the 1,4-diene .

Recrystallization of the crude product from acetone yields 225 mg of 1-dehydro-Reichstein's compound-S, (m.p. 246-248° C), into which an 11-hydroxy group is then introduced in a known manner.

Example 12.

Production of 1-dehydrocortisone.

A. 1,4-pregnadiene-17a-ol-3,20-dione (Ile).

150.0 mg of sterile 4-pregnene-17ct-ol-3,20-dione (17a-hydroxyprogesterone) (Ic) in 5.0 ml of ethanol is dehydrated using the culture described in example 3. The crude extract obtained weighs 178 mg. This crude product is recrystallized from a mixture of acetone and hexane, whereupon crystals of pure 1,4-pregnadien-17a-ol-3,20-dione (lic) are obtained.

B. 1, 4- pregnadien-lla, 17 a- diol- 3, 20-

dion (Ille).

The transfer of this compound is carried out using a culture of Rhizopus nigricans in the manner described in detail in J. Am. Chem. Soc. 74, 5933 (1952): 6 liters of a 24 hour culture of Rhizopus nigricans is added to 1 g of compound lic in 200 ml of ethanol. After a 48-hour transfer period in the apparatus and medium described in the aforementioned publication, the culture is extracted with methylene chloride, the solvent is evaporated from the extract and the crude, crystalline residue is recrystallized from a mixture of acetone and hexane, whereby crystalline 1,4-pregnadiene-lla,17a-diol-3,20-di-one (Ille). C. 1, 4- pregnadiene- 17a-ol- 3, 11, 20- trione (Vc).

A solution of 3.0 g of compound III in 30 ml of pyridine is added slowly to a suspension of 1.5 g of chromic acid in 15 ml of pyridine (Poos et al., J. Am. Chem. Soc. 75, 422 (1953) and the the resulting mixture is stirred overnight at room temperature. To the reaction mixture is then added 4.5 g of sodium sulfite in 45 mL of water and stirring is continued overnight at room temperature. To the reaction mixture is then added 4.5 g of sodium sulfite in 45 mL of water and stirring is continued for 2 hours. The reaction mixture is poured in 600 ml of water and the resulting solution is extracted with methylene chloride. The extracts are washed with dilute sulfuric acid, aqueous sodium carbonate solution and water until neutral and dried over magnesium sulfate. By evaporation of the dried solution and recrystallization of the residue from a mixture of acetone and hexane crystalline 1,4-pregnadien-17o-ol-3,11,20-trione (Vc) is obtained.

D. 1-dehydro-cortisone (Ila).

4 liters of 1% yeast extract ("Difco") are sterilized in a shaking flask and inoculated with a strain of Ophiobolus herbotrichus. The culture is incubated for three days with shaking at 27°C. The contents of the shaking flask are then added under aseptic conditions, 1.0 g of compound Vc in 25 ml of sterile

acetone and the shaking is continued for another 3 days at 27°C. The mycelium is separated from the reaction mixture and both the mycelium and it

aqueous filtrate is thoroughly extracted with three portions of chloroform. The extracts are combined, washed with water, dried and concentrated. The concentrated solution is chromatographed over "Florisil" (30 g)) and the column

washed with methylene chloride. By elution with 0.5% and 1.0% methanol in methylene chloride, 1-dehydrocortisone is removed from the column and recrystallized from a mixture of acetone and hexane.

Example 13.

Production of 1-dehydrocortisone.

A. Preparation of 1, 4-pregnadiene-llfi,-17a-diol-3, 20-dione (IVc) from lic.

A culture of Curvularia lunata (QM 120h) is grown in flasks containing the medium described in Example 13 (see also U.S. Patent No. 2,658,023). 100 ml of this culture is added under sterile conditions to 2 liters of an aqueous medium containing the following substances:

The mixture is adjusted to a pH value of 7 with sulfuric acid and 0.25 percent calcium carbonate is added, after which the mixture is sterilized. The inoculated medium is aerated at a rate of about half to one volume part of air per volume part resolution per minute at 27-28°C for 24 hours. During this time, the mixture is stirred with a stirrer that rotates at around 1,700 revolutions per minute. minute. 0.5 g of 1,4-pregnadien-17a-ol-3,20-dione (lic) is dissolved in 20 ml of 95% ethanol. The solution is added to the culture mixture under sterile conditions. The reaction was then allowed to continue for a further 24 hours under exactly the conditions described above.

The culture solution is extracted with three portions of methylene chloride. The combined extracts are dried over magnesium sulfate and concentrated to a small volume. The concentrated solution is fed to a "Florisil" column (30 g), which is prepared with hexane and the column is eluted with hexane containing ether in proportions varying from 1 per cent to 99 per cent. The desired product is collected from the eluates with the greatest possible polarity (25 wt. ether 99 wt. ether) and crystallized from acetone and hexane, whereby crystalline 1,4-pregnadiene-llp,17a-diol-3,20-dione (IVc) is obtained.

B. Preparation of Compound Vc from Compound IVc.

This reaction is carried out exactly as the transfer of compound Ille to compound Vc, except that compound IVc is used as starting material instead of compound Ille. The product is then transferred to 1-dehydrocortisone (compound IIa) in the manner described in example 12.

Example 14.

Preparation of ll-epi-l-dehydro-^ortisol (Vila) from Ule.

The same procedure is used as for the transfer of compound Vc to compound Ila, with the exception that compound Ille is used as the steroid substrate and that the product compound Vila is isolated by chromatography over "Florisil" in the eluates consisting of 1% and 2% methanol in methylene chloride . Crystallization of compound Vila from acetone gives a crystalline product with m.p. 243-245°C.

Using the same procedure used to transfer compound Vc to Ha, compound IVc is transferred to compound Illa. By recrystallization of compound Illa from acetone, crystals with m.p. 238-240°C.

Oxidation of compounds IIa or VIIa, preferably as the 21-acetates or lower alkanoyl esters (IIIb and VIIb), is carried out by the method described for transferring compound IIIa to Vc. The 21-acetate group is hydrated with potassium bicarbonate in aqueous methanol, whereby compound Ila is obtained.

Transfer of compound Ic to 4-pregnene-11a,17a-diol-3,20-dione (compound

VIc) (Method B) is carried out by the same procedure used in the transfer of compound 11c to Ille (Example 12B).

In a similar way, 4-preg-

nen-ll(3,17a-diol-3,20-dione (VIIc) from Ic, as IVc is prepared from He. Compounds VIc, VIIc are both oxidized to 4-pregnen-17a-ol-3,11,20-trione (VIIIc) by the method used to transfer compounds Ille to IVc to compound Vc.

The other reactions according to method B follow the procedure in method A. Thus, compound VIc is transferred to Ille and compound VIIIc is transferred to Vc, while compound VIIc is transferred to IVc in the same way as Ic is transferred to He, i.e. by treatment with a culture or an enzymatic extract of a culture of Corynebacterium simplex or hoagii. From this point the reaction is the same as in method A.

Example 15.

Production of 1-dehydrocortisone (Ila).

A. 1, 4- pregnadiene- 21-ol- 3, 20-dione ( Fire).

150.0 mg sterile 4-pregnen-21-ol-3,20-dione (Id) in 5.0 ml ethanol is subjected to cultivation in the same medium, with the same microorganism in the same way as stated in example 12 A. One obtains a crude extract weighing 137 mg. This crude product is recrystallized from a mixture of acetone and hexane, whereby crystalline 1,4-pregnadien-21-ol-3,20-dione (Ild) is obtained.

B. 1,4-pregnadien-lla,21-diol-3,20-dione (II Id).

1.0 g of compound Ild in 200 ml of ethanol is treated with a culture of Rhizopus nigricans in the manner described in example 12 B. The crude crystalline residue is recrystallized from a mixture of acetone and hexane, whereby crystalline 1,4-pregnadien-lla,21-diol-3,20-dione (Uld) is obtained. C. 1,4-pregnadien-21-ol-3,11,20-trione (Vd).

A solution of 3.0 g of compound Uld in 30 ml of pyridine is oxidized in the manner described in example 12 C. By evaporation of the dried methylene chloride extract and crystallization of the residue from a mixture of acetone and hexane, crystalline 1,4-pregnadien-21-ol-3,11,20-trione (Vd) is obtained.

D. 1-dehydro-cortisone (Ila).

2 liters of Czapek-Dox medium are sterilized in a shaking flask and inoculated with a strain of Trichothecium roseum. After 3 days of shaking at 27°C, 500 mg of 1,4-pregnadien-21-ol-3,11,20-trione (Vd) in 15 ml of acetone is added to the culture under aseptic conditions. The shaking is continued for 60 hours at 27°C. The mycelium is separated and both the mycelium and the aqueous filtrate are thoroughly extracted with methylene chloride. The combined extracts are washed well with water, dried and concentrated. The concentrated solution is chromatographed over "Florisil" and 1-dehydro-cortisone (Ila) is eluted in the fractions with 0.5% and 1% methanol in methylene chloride after washing the column with methylene chloride. By recrystallization of the aforementioned fractions from acetone, crystals of compound IIa with m.p. 226—229°C (decomposition).

Example 16.

Production of 1-dehydrocortisone (Ila).

A. l, 4- pregnadien- ll$, 21- diol- 3, 20- dione ( IVd).

A culture of Curvularia lunata (identified by the "United States Quartermaster Corps", Philadelphia) is grown in flasks containing the medium indicated in Example 1, namely the medium according to U.S. patent no. 2 658 023. The culture is used exactly as described in example 13A for the treatment of 1,4-pregnadien-21-ol-3,20-dione (Ild), likewise dissolved in 20 ml of 95% ethanol. From the eluates with the highest polarity (25% ether > 99% ether) 1,4-pregnadiene-11p,21-diol-3,20-dione (IVd) is obtained, which is recrystallized from a mixture of acetone and hexane.

B. Preparation of 1,4-pregnadien-21-ol-3,11,20-trione (Vd).

This reaction is carried out exactly like the transfer of compound Uld to Vd except that compound IVd is used as starting material instead of compound Uld.

Compound Uld is transferred to 1,4-pregnadiene-lla,17a,21-triol-3,20-dione (Vila)

by the same procedure used to transfer compound Vd to IIa. The product is isolated by chromatography over "Florisil" in the eluates consisting of 1% and 2% methanol in methylene chloride. By recrystallization of compound Vila from acetone, the product is obtained in crystalline form with m.p. 243-245°C.

Likewise, the method used to transfer compound IVd to Illa is the same as that used to transfer compound Vd to Ha, and the product Illa is isolated by chromatography over "Florisil" in the eluates consisting of 1% and 2% methanol in methylene chloride. By recrystallization of compound Illa from acetone, the compound is obtained in crystalline form with m.p. 238-240°C.

Oxidation of compounds Illa or Vila, preferably as the 21-acetates obtained by allowing the 21-alcohols to react at room temperature with an equivalent of acetic anhydride is carried out by the method described for the transfer of compound Uld to Vd. The 21-acetate group is hydrolysed by heating with potassium bicarbonate in aqueous methanol, whereby compound Ila is obtained.

The transfer of 4-pregnene-lln,21-diol-3,20-dione (Vid), 4-pregnene-llj3,21-diol-3,20-dione (Vlld) and 4-pregnene-21-ol-3; 11,20-trione (VUId) to compounds Uld, IVd and Vd respectively, is carried out in the same way as the transfer of compound Id to Ild.

Similarly, for the transfer of compound Id to the intermediate products Vid and Vlld, the method described above is used for the transfer of compound Ild to Uld, respectively of compound Ild to IVd. The compounds Vid and Vlld are oxidized to compound VUId by oxidation under mild conditions, as described above.

As indicated above, instead of desoxycorticosterone and its esters, 5-pregnen-3,21-diol-20-one and its 21-esters can be used as the acetate and other lower alkanoyl esters as starting material. During the treatment with a culture of or an isolated enzyme of the dehydration microorganism, such as e.g. C. simplex or C. hoagii, in the manner already described, the 3-hydroxyl group is oxidized to ketone oxygen, while the 5,6-double bond moves to the 4,5-position. Hereby, 5-pregnen-3,21-diol-20-one and its 21-esters are transferred to compound IIa and its 21-ester.

You will generally get a better yield under less critical conditions

the oxidation of lla-OH and ll(3-OH compounds to the corresponding 11-keto compounds, when the 21-hydroxyl group is first esterified in the methods described in examples 15C and 16B, possibly with subsequent hydrolysis. Methods that are suitable for the partial esterification of the 11,21-diols and for the hydrolysis of the 11-keto-21-esters obtained by oxidation, have already been described.

The 21-acetate of compound Vd can be obtained by oxidation of the 11-hydroxyl group in the

the bonds Uld and IVd 21-acetates with chromic acid in pyridine as already described in connection with compounds related to these. The compound's Vd 21 ester is easily hydrolyzed by heating under reflux with potassium carbonate in aqueous methanol.

Compounds Vid and Vlld can be converted to their 21-acetates in the same way as compounds Uld and IVd. When oxidizing the compounds Vid and Vlld 21-acetate to the compound VUId acetate, the same procedure is used as for the oxidation of the compound Uld 21-acetate to the compound Vd 21-acetate.

In one of the schematic representations in the foregoing, the transfer of 16-dehydropregnenolone (le) and 16-dehydroprogesterone (Ille) to 1-dehydro-cortisone (Ila) is shown by means of the 16,17-epoxides and following different pathways. These pathways also include addition of an 11"- or lip-hydroxyl group, addition of a 21-hydroxyl group, the introduction of a 17cc-hydroxyl group by opening the 16,17-epoxidation, the introduction of the 1,2-double bond and oxidation of 11 -hydroxyl group of the ketone oxygen. The sequence of these reactions can vary with certain limitations (thus, for example, the oxidation step must follow the introduction of the 11-hydroxyl group), but it is preferred to carry out the treatment with the dehydrating microorganism after the introduction of a hydroxyl group at least in one of 11,17 - and the 21-positions, or after the formation of the 16,17-epoxide.

Example 17.

Production of 1-dehydrocortisone.

A. Preparation of the starting material used 4-pregnen-lla, 17a, 21-triol-3, 20-dione (IXe).

1 g of 16-dehydroprogesterone (Ille) dissolved in 25 ml of chloroform is allowed to react at room temperature for about 15 hours with the equivalent amount of perbenzoic acid dissolved in chloroform. Water is then added to the reaction mixture, the organic layer is washed with sodium sulphite solution, then with sodium bicarbonate solution, then with water and then dried over sodium sulphate. The chloroform is evaporated and the residue is crystallized from a mixture of acetone and hexane, whereby 16,17-epoxy-4-pregnene-3,20-dione (IVe) is obtained.

A suspension of 1 g of the 16,17-epoxide in 10 ml of glacial acetic acid is mixed with a solution of 1.1 g of 47% aqueous hydrogen iodide, 10 ml of acetic acid and 4.1 g of acetic anhydride. The temperature is kept at around 15 to 20° C during the entire mixing operation and for half an hour afterwards. The mixture is then poured into 5 volumes of water, the precipitate that precipitates is filtered off, washed with water and dried. The crude product is 16-iodo-4-pregnene-17a-ol-3,20-dione. This is dissolved in 100 ml of ethanol and 1 ml of acetic acid and heated under reflux for about 6 hours with 2.5 g of Raney nickel catalyst. The catalyst is then removed by filtration and the filtrate is concentrated. When water is added to this concentrated filtrate, a precipitate consisting of 4-pregnen-17a-ol-3,20-dione (17a-hydroxy-progesterone, Vlle) is obtained. 4 liters of 1% yeast extract ("Difco") are sterilized in a shaking flask and inoculated with a strain of Ophiobolus herbotrichus. The culture is incubated for 3 days with shaking at 27°C, after which 1.0 g of 17'-hydroxyprogesterone in 25 ml of acetone (sterile) is added under aseptic conditions to the contents of the shaking flask and the shaking is continued for a further 3 days at 27°C. The mycelium is separated from the reaction mixture and both this and the aqueous filtrate are thoroughly extracted with three portions of chloroform. The extracts are combined, washed with water, dried, concentrated and chromatographed on Florisil. The material obtained by elution, e.g. with 0.5% to 1.5% methanol in methylene chloride, is crystallized from a mixture of acetone and hexane, whereby 4-pregnene-17a,21-dibl-3,20-dione is obtained (Ville).

A solution of 1.0 g of compound Ville in 150 ml of ethanol is added to one liter of a 24 hour culture of Rhizopus nigricans. After a reaction period of 48 hours, the mixture is extracted with methylene chloride and the extract is evaporated to dryness. The crude product is crystallized from acetone and hexane, whereby 4-pregnen-lla,-17a,21-triol-3,20-dione (IXe) is obtained.

B. 1-dehydrocortisone.

Compound IXe is exposed to a culture of a dehydrating microorganism such as Corynebacterium simplex. After incubation for about 48 hours at 28°C, 1,4-pregnadiene-11a,17a,21-triol-3,20-dione (Xe, Vila) is obtained. This compound is thus oxidized as described above to 1-dehydro-cortisone (Ila).

By using a culture of Curvularia lunata (QM 120h), thus as described in Example 13A instead of a culture of Rhizopus nigracans, the compound 1,4-pregnadiene-11|3,17a,-21-triol is obtained from compound IXe -3,20-dione (1-dehydro-cortisol, Illa) which can also be oxidized to 1-dehydro-cortisone.

Example 18.

1-dehydrocortisone.

16-dehydroprogenolone is transferred to the 16,-17-epoxide by treatment with perphthalic acid. The 16,17-epoxy-5-pregnen-3-ol-20-one (Ile) is then converted to the 11a-hydroxy derivative (compound Xle) by treatment with a culture of Rhizopus nigricans. Compound Xle is subjected to the action of a culture of Ophiobolus herbotrichus, whereby 16,17-epoxy-5-pregnen-3,lla,-21-triol-20-one (Xlle) is obtained. The epoxidation is then opened by treatment with hydroiodic acid as described above and 5-pregnen-3,lla,17a,21-tetrol-20-one (XHIe) is obtained. By exposing the latter compound to the influence of a culture of a dehydrating microorganism, 11-epi-1-dehydro-cortisol (Xe) is obtained, which can then be oxidized to 1-dehydro-cortisone.

If desired, 16-dehydropregnenolone can first be treated with a culture of Ophiobolus herbotrichus, whereby 5,16-pregnadien-3,21-diol-20-one is obtained which is then acetylated to the diacetate in a known manner. When treated with a dehydrating microorganism, the diacetate is transferred to 1,4,16-pregnatrien-21-ol-3,20-dione. This compound is transferred to the 16,17-epoxide by treatment with perphthalic acid, after which the epoxidation is opened by treatment with hydroiodic acid, after which it is heated under reflux with a Raney nickel catalyst. This gives 1,4-pregnadiene-17a,21-diol-3,20-dione. This compound is converted to 1-dehydro-cortisol by exposure to a culture of Curvularia lunata.

Instead of the peracids such as peracetic acid, perbenzoic acid and perphthalic acid, hydrogen peroxide together with an alkali metal base, preferably sodium hydroxide or] potassium hydroxide, can be used to form the oxidation. The mixture may contain an alcohol such as methanol and tertiary butanol to support the dissolution of the steroid compound, but the presence of an alcohol is generally just as absolutely necessary.

Additional starting materials are 5,16-pregnadien-3,11-diol-20-one and its 3-esters, 5,16-pregnadien-3,21-diol-20-one and its 3- and 21-esters, 5, 16-pregnadien-3,11,21-triol-20-one and its 3- and 21-esters and 4,16-pregnadien-21-ol-3,11,20-trione and its 21-esters. These can be exposed to the action of a culture of a dehydrating microorganism and to the action of a peracid or hydrogen peroxide. The steps can be carried out in any sequence.

Example 19.

Production of 1-dehydrocortisone.

A. 11-epi-l-dehydro-cortisol (Vila).

Using the method described in J. Am. Chem. Soc. 74, 5933 (1952) is added to 6 liters of a 24 hour culture of Rhizopus nigricans 1.0 g of 1,4-pregnadiene-17a,21-diol-3,20-dione (which can be prepared as described in examples 3 and 5 or by known chemical agents) i

200 ml of ethanol. After a 48-hour reaction period in the apparatus and medium described in the above-mentioned article, the extraction is carried out with methylene chloride and the crude, crystalline, solid residue is washed three times with 5 ml portions of ice-cold chloroform. The yield of compound Vila is 0.9 g. The product is recrystallized from a mixture of acetone and hexane.

B. 1-dehydro-cortisone (Ila).

To a solution of 0.4 g of compound Vila in 10 ml of anhydrous pyridine is added 0.11 g of acetic anhydride. The reaction mixture is allowed to stand overnight at room temperature, and then a mixture of 0.15 g of chromium trioxide in 15 ml of pyridine is added (the chromium trioxide-pyridine reagent is prepared as indicated by Poos et al., J. Am. Chem. Soc. 75, 422 (1953)). The resulting mixture is allowed to stand at room temperature overnight and then poured into 5 parts by volume of water. The product thus obtained is extracted with methylene chloride and the extracts are washed with water, dilute sulfuric acid and again with water. The methylene chloride solution is dried over magnesium sulfate and concentrated in vacuo. The crystallization of the 21-acetate in compound Ha is carried out from a mixture of acetone and hexane, whereby 0.21 g of crystalline solid substance (compound Hb) is obtained.

To a solution of 0.12 g of the 21-acetate of compound Ha in 3.3 ml of chemically pure methanol is added 0.0315 g of potassium bicarbonate in 0.5 ml of water. The mixture is heated under reflux for 5 minutes and cooled rapidly. It is diluted with water, neutralized with dilute hydrochloric acid and extracted with methylene chloride. The extracts are dried and concentrated. The residue is recrystallized from a mixture of acetone and hexane, whereby 0.051 g of 1-dehydro-cortisone (compound IIa) is obtained.

Example 20.

A. 1-dehydro-cortisol (Illa).

1,4-pregnadiene-17a,21-diol-3,20-dione

(0.5) is treated with a culture of Curvularia lunata (QM 120H) which is prepared in the following way. Curvularia lunata is grown for 2 days in a shaking flask at 28°C in a medium consisting of malt, sucrose and salts. 100 ml of the resulting culture is used to inoculate 2000 ml of the same medium in a culture apparatus equipped with a submerged aerator. The inoculated medium is stirred at 1,700 revolutions at a rate of 0.5 parts by volume of air per volume part medium per minute for 22 hours at 28°C in a water bath. 50 g of moist mycelium obtained by filtering the resulting culture is added to 2000 ml of water and the above steroid compound in a similar culture apparatus. The suspension of mycelium and steroid compound is stirred in the manner described above for 22 hours and then extracted with chloroform. The chloroform extracts are dried, concentrated to a residue which is chromatographed on Florosil. The Florosil column is prepared with hexane, the mixture containing the steroid is added dissolved in methanol chloride and a mixture of methylene chloride and methanol. Unreacted starting material is eluted with 0.5% methanol in methylene chloride and the desired compound Illa is eluted with 1-2% methanol in methylene chloride. From 0.5 g of 1-dehydro-Reichstein-compound-S, 0.17 g of compound Illa is obtained as a crystalline solid with m.p. 237-239°C.

B. 1-dehydro-cortisone (Ila).

Compound Illa is transferred to compound Ha using the same procedure used to transfer compound Vila to compound Ha. From 0.400 g of compound Illa, 0.205 g of compound Ila is obtained.

Example 21.

Preparation of 9a-fluoro-l-dehydro-cortisone.

A. 1,4,9(II)-pregnatriene-17a,21-diol-3,20-dione-21-acetate (Ilj).

Option 1.

1,4-pregnadiene-llfS,17a,21-triol-3,20-dione (Illa) is acetylated with acetic acid in pyridine, and to 1.22 g of the obtained ester dissolved in pyridine and cooled in ice is added 0.30 ml phosphorus oxychloride. The reaction mixture is allowed to stand overnight at room temperature.

return. At the end of this time, the solution is concentrated in vacuo at 25°C to a volume of 5 ml and 50 ml of water is added. The oily substance that separates out is taken up in ethyl acetate and the extracts are washed once with water, then with 1 N hydrochloric acid until they are free of pyridine, again with water and then with 5% sodium bicarbonate solution. The ethyl acetate solution is then dried and evaporated in vacuo. The residue is triturated with ether, whereby 1,4,9 (11)-pregnatriene-17a,21-diol-3,20-dione-21-acetate crystallizes.

Option 2.

11-epi-l-dehydro-cortisol (Vila) is 21-acetylated. A solution of 1.0 g of the acetate in 15 ml of anhydrous pyridine is added at 0°C with 1.0 g of p-toluenesulfonyl chloride. The reaction mixture is allowed to warm to room temperature and stand overnight. It is then diluted with ice water and the precipitate of 11a-p-toluene-sulfonate-21-acetate (compound Ij) which is thereby obtained is filtered from the solution.

A solution of 1.0 g of compound Ij in 25 ml of acetic acid containing 2.0 g of sodium acetate is heated under reflux for 45 minutes. The solution obtained is evaporated in vacuo and the residue is leached with methylene chloride. The methylene chloride extract is washed free of acetic acid with water and dried over anhydrous magnesium sulphate. The anhydrous solution is evaporated and the residue triturated with ether. The product, 1,4,9(11)-pregnatriene-17a,21-diol-3,20-dione-21-acetate (IIj), is recrystallized from a mixture of acetone and hexane.

B. 9 a- fluoro- 1- dehydro-cortisol.

To a suspension of 2.0 g of compound Ilj in a finely divided state in 200 ml of purified dioxane and 20 ml of water is added 1.0 g of N-bromoacetamide and 10 ml of N aqueous perchloric acid. The mixture is stirred from time to time until the suspended solid has completely dissolved. After 1 hour, aqueous sodium sulphite is added to cleave the excess of N-bromoacetamide. The solution obtained is then thoroughly extracted with methylene chloride and the extracts are washed free of acid with water. The solution is dried over magnesium sulfate, evaporated in vacuo and the residue recrystallized from acetone, whereby crystalline 9a-bromo-1,4-pregnadiene-11(3,17a,21-triol-3,20-dione-21-acetate is obtained.

A solution of 1.0 g of the 9a-bromo compound in 100 ml of methanol containing 2 g of anhydrous potassium acetate is heated under reflux for 1 hour and then evaporated to give a residue. The solid residue is thoroughly extracted with methylene chloride and the extracts are washed well with water. The extracts are dried, evaporated and recrystallized from a mixture of acetone and hexane, whereby crystalline 9,11-oxido-1,4-pregnadiene-17a,21-diol-3,20-dione-21-acetate is obtained.

A solution of 1.0 g of the oxydo compound in 50 ml of alcohol-free chloroform is saturated with anhydrous hydrogen fluoride at 0°C. After 5 hours at this temperature, the excess hydrogen fluoride and the solvent are removed in vacuo, likewise at 0°C. The residue is recrystallized from a mixture of acetone and hexane, whereby crystalline 9a-fluoro-1-dehydro-cortisol-21-acetate is obtained. This substance is hydrolysed in a mixture of methanol and chloroform with aqueous hydrochloric acid at 20-25°C over a period of about 48 hours, whereby the free 21-ol (9a-fluoro-1-dehydro-cortisol) is obtained, which is recrystallized from a mixture of acetone and hexane.

9α-Fluoro-1-dehydro-cortisol-21-acetate

can be oxidized to 21-acetate of 9a-fluoro-1-dehydro-cortisone by adding dropwise a solution of 162 mg of the former compound in 10 ml of glacial acetic acid to a solution of 30 mg of chromium trioxide in 0.5 ml of water and 2 ml of glacial acetic acid. After 6 hours, the solution, which has a green colour, is diluted with 2 ml of methanol and evaporated in vacuo. The residue is extracted thoroughly with methylene chloride and the extracts are dried over anhydrous magnesium sulphate. When the anhydrous solution is evaporated, crystalline 9a-fluoro-1-dehydro-cortisone-21-acetate is obtained, which can be easily hydrolyzed to the free 21-ol (compound IV where Z = F).

Example 22.

9a-chloro-l-dehydrocortisol.

A solution of 1.0 g of 9(11)-oxydo-1,4-pregnadiene-17a,21-diol-3,20-dione-21-acetate in 50 ml of alcohol-free chloroform is saturated with anhydrous hydrogen chloride at 0° C. After 5 hours at this temperature, the excess hydrogen chloride and the solvent are removed in vacuum at 0°C. The residue is recrystallized from a mixture of acetone and hexane, whereby crystalline 9a-chloro-1-dehydro-cortisone-21-acetate is obtained. This compound is then hydrolysed, whereby the free 21-ol compound is obtained.

By oxidizing the acetate in the manner described in the preceding example, the 21-acetate of 9a-chloro-1-dehydro-cortisone is obtained, which can be hydrolyzed to the free 21-ol. Example 23. 1-dehydro-cortisol-21-(5'-tert.butyl-furoate). 1 g of 1-dehydrocortisol dissolved in 10 ml of pyridine is cooled by stirring in an ice bath and treated with 0.6 g of 5-tert.butylfuroyl chloride. After stirring overnight, the mixture is poured into dilute sulfuric acid and the resulting solid is extracted with ether. The crude product is recrystallized from a mixture of benzene and methanol, whereby the pure product is obtained which melts on cleavage below about 237-239°C.

Example 24.

9a- fluoro- 1 - dehydro- cortisone- 21 -

(2'-furoate).

1.0 g of 9a-fluoro-1-dehydro-cortisone is dissolved in 20 nil of dry pyridine and the resulting solution is cooled to 0°C. It is then added dropwise and with stirring 0.45 g of 2-furoyl chloride. The reaction mixture is allowed to warm slowly to room temperature and is stirred overnight. The mixture is then poured into 200 ml of cold 10% aqueous sulfuric acid. The precipitate that is thereby filtered off, washed with water, then with an aqueous bicarbonate solution and finally again with water. By recrystallization from a mixture of methanol and water, the 21-(2'-furoate) ester is obtained in crystalline form.

In a similar way, the 21-(2'-furoate) ester of 9α-fluoro-1-dehydrocortisol and the 9α-chloro analogues of both compounds can be obtained. By using 5-tert-butyl-2-furoyl chloride, phenoxyacetyl chloride and 2,4,5-tri-chlorophenoxyacetyl chloride, 21-[(5'-tert-butyl)-2' is obtained in a similar manner -furoate], 21-phenoxyacetate and 21-(2',4',5'-trichlorophenoxy-acetate of the 9a-fluoro and 9a-chloro derivatives of 1-dehydro-cortisol and of 1-dehydro-cortisone.

By allowing the corresponding free 21-alcohols to react with the acid chloride, the following further esters are produced: 1,4-pregnadien-21-ol-3,20-dione-21- (5'-bromofuroate),

1,4-pregnadiene-11(3,17a,21-triol-3,20-dione-21-furoate, m.p. 240-242°C (under decomposition),

1,4-pregnadiene-lip,21-diol-3,20-dione-21-(4'-methylphenoxyacetate),

1,4-pregnadiene-lip,17a,21-triol-3,20-dione-21-(5'-chlorofuroate),

1,4-pregnadiene-lip,17αi21-triol-3,20-dione-21-(4'-tert-butylphenoxyacetate), m.p. 203—206°C (under decomposition),

1,4-pregnadien-21-ol-3,20-dione-21-phenoxyacetate,

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-(4'-chlorophenoxyacetate), m.p. 180—

182°C,

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-(4'-methoxyphenoxyacetate), m.p. 130—

135°C (during loss of solvent),

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-(5'-bromofuroate),

1,4-pregnadiene-11(5,17a,21-triol-3,20-dione-21-phenoxyacetate, m.p. 196-199°C,

1,4-pregnadiene-lip,17,21-triol-3,20-dione-21-(4'-bromophenoxyacetate),

1,4-pregnadiene-17a,21-triol-3,11,20-trione-21-furoate, in two forms, m.p. 233-235°C

and 251-254°C,

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-(4'-tert-butylphenoxyacetate), m.p.

130—135°C (during loss of solvent),

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-phenoxyacetate, m.p. 205—207°C and

1,4-pregnadiene-17a,21-diol-3,11,20-trione-21-(5'-tert-butyl furoate) m.p. 241—-243°C.

These esters can be administered on the

various ways indicated above.

Claims (3)

1. Method for the production of corticosteroids with improved activity and consisting of 11-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-diones or esters thereof, characterized in that a compound of the pregnane series which is unsaturated at C-5 and contains a keto-, hydr oxy- or esterified hydroxy group in the 3-position, and a keto- or hydroxy group in the 20-position are exposed to the action of a microorganism that dehydrogenates in the 1-(2)-position without changing the carbon skeleton of pregnane compounds, with the exception of microorganisms of the genus Fusarium, that an 11-, 17a- and/or 21-hydroxy group and, if desired, a 9a -halogen atom, and that in the thus obtained 11-oxygenated-1,4-pregnadiene-17a,21-diol-3,20-dione, if desired, one or more hydroxy groups are esterified, one or more esterified hydroxy groups are hydrolyzed and/ or an 11-hydroxy group is oxidized to an 11-keto group, in a manner known per se. 2. Method according to claim 1, characterized in that an organism of the family Corynebacteri-aceae, such as of the species C. simplex or C. is used as the dehydrogenating microorganism. hoagie. 3. Process according to claim 1 or 2, characterized in that cortisone, hydrocortisone, 9a-fluoro-hydro-cortisone or an ester of these compounds are used as starting material.