BE540478A - - Google Patents

Description

       

  



  PREPARATION OF PREGNADIENES.



  International Convention, *, Patent applications of the United States of America filed under the numbers, dates and by the applicants indicated below of which 1 the applicant is the beneficiary: 449,257 11. 8. 1954 Arthur NOBILE 460.508 5.10.1954 Hershel L.HERZOG and David H. GOULD 464.159 22.10.1954 Arthur NOBILE 466.207 .1, Il, 1954 Eugene P.OLIVETO and David H. GOULD 481.271 il? 1. 1955 Hershel L.HERZOG 481.279 11. 1.1955 Arthur NOBILE 482. 890 19.1.1955 Hershel L.HERZOG 483.167 20. 1.1955 'Hershel L.HERZOG and David H.

   GOULD 484,302 26. 1. 1955 Eugene P.OLIVETO and Hershel L.HERZOG 484,588 27. 1. 1955 Hershel L.HERZOG and Eugene P.OLIVETO 485,829. 2. 2. 1955 Eugene P.OLIVETO and Hershel L.HERZOG 486.037 3. 2. 1955 David H. GOULD and Hershel L.HERZOG 489.282 18. 2.1955 Eugene P.OLIVETO and Hershel L.HERZOG 505.542 2.5.1955 Hershel L.HERZOG 506.085 4. 5.1955 William CHARNEY and David SUITER 513.902 7. 6.

   1955 Arthur NOBILE

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The present invention relates to the synthesis of 1-dehydrocortisone (1,4-pregnadiene-17 [alpha], 21-diol-3,11,20-trione) from 1-dehydrocortisol (1,4-pregnadiene-11ss, 17 [alpha], 21-triol-3,20-dione), their 9 0 (-halo-derivatives and their esters, and to intermediates convertible into such dienes to the novel compounds thus produced and to the compositions pharmaceuticals containing the therapeutically active compounds.



   The invention relates mainly to the preparation of the 10,13-dimethylsteroids-1,4-dienes mentioned above, which can be considered as derivatives of the hydrocarbon pregnane and, to facilitate the understanding of the same. The invention shows in formula I of sheet 1 of the appended formulas the conventional numbering of the carbons of the pregnane backbone. Usually the CH3 group attached to co-ones 10 and 13 is omitted, and only the vertical lines are shown.



  The letters A, B, C, D are used conventionally to identify the four nuclei.



   The main end products of the invention are represented by the formulas II (1-dehydrocortisone and its 21-esters) III (1-dehydrocortisol and its 21-esters), IV (9 [alpha] -halogeno-1-dehydrocortisone and their 21-esters) and V (9 [alpha] -halo-1-dehy- drocortisol and their 21-esters), all of these formulas being combined in composite formula VI. The formula.VII represents 11-epi-1-dehydrocortisol and its 21-esters, that is to say 1,4-pregnadiene-11 [alpha], 17 [alpha], 21-triol-3,20 -dione and its 21-esters, these compounds being valuable intermediates for the preparation of 1-dehydrocortisone (II).

   According to the present invention, a wide variety of starting compounds can be employed for the preparation of compounds II to V, and during the processing of the various starting compounds, which will be described in detail hereinafter, one obtains various new intermediate compounds, a number of which have

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 even of value from a therapeutic point of view.



   When carrying out the synthesis according to the invention of the 1,4-pregnadienes mentioned above, a saturated or an unsaturated pregnane having an oxygen function at positions 3 and 20, but lacking one or more of the following characteristics: (a) the # 1 double bond, (b) the # 4 double bond, (c) the X group, (d) the OH group at 17 [alpha], (e) the OR group at 21 and (f) the halogen group at 90 (,. is subjected to one or more treatments by which the missing characteristic (s) are introduced into the molecule.



   In the implementation of the invention, a second double bond is introduced on a carbon of ring A, this same ring A already having a double bond attached to one of the carbon atoms. Thus, in the practice of the invention, there is introduced into the starting compound a: double bond in position 1,2, with or without one or more of the following modifications of this compound which may be necessary to produce any of Compounds II to V, performed in any suitable order:

   (1) introduction of a 4 or 5 double bond (the # 5 double bond finally migrating to position 4.5), (2) introduction of an oxygen function in 11, (3) introduction of an oxygen function in 17 [alpha], (4) introduction of an oxygen function at 21, (5) introduction of a halogen at 9 [alpha].



   The ketone group at position 3 is not stated as a separate characteristic to be introduced into the starting compound because in a new process according to the invention for introducing the 1,2 double bond, a hydroxyl or ester group is simultaneously there. replaced by ketone oxygen.



   The starting unsaturated compounds may have a double bond attached to one or more of positions 4, 6, 9 (11) and

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 16. In a modification of the present process, the starting compounds may in fact already have the two double bonds in ring A, which are characteristic of the end products represented by formulas II to V.



   Various routes can be used for the synthesis of compounds II to V depending on the nature of the starting compound, all of these routes having in common the characteristic of employing methods to introduce one or more of the characteristics (a) to (f ) listed above, and providing end products in the form of 1,4-pregnadienes having the substituents at positions 3, 11, 17, 20 and 21, indicated by structural formulas II to V, and with or without the halogen group at 9.



   The operations by which the necessary changes in the starting compound are effected may be microbiological, or all chemical, or a combination of microbiological and chemical processes, depending on the character of the starting compound. or according to preference in the cases. in particular where certain conversions can be carried out conveniently both with the aid of certain microorganisms and with purely chemical reagents.



   While a number of the reactions described, both chemical and biochemical, are widely known in connection with the preparation of other compounds, one of the novel modifications effected by the present process involves the use of cultures of dehydrogenating microorganisms having the specific property of introducing a double bond between the 1,2-carbons without simultaneous degradation of the carbon skeleton of pregnane, such as for example the splitting of the side chain at carbon 17 or the opening of the D ring. Dehydrogenating microorganisms are also able to hydrolyze ester groups, usually at the 3 and 21 positions, and with different rates or degrees of completion they also effect the oxida-

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 tion of hydroxyl groups at 3 and 20.

   These microorganisms are extremely effective at introducing # 1 unsaturation, particularly in compounds having a double bond attached to the 4,5 or 5,6 carbons.



   Dehydrogenating microorganisms work best on substrates (i.e., the steroid compound to be modified) that have a. double bond attached to carbon 5 and which contain either a hydroxyl group which is relatively stable to the organism, i.e. which is not rapidly oxidized to ketone oxygen, or an ester of such a hydroxyl group which is hydrolyzed by the culture of the microorganism. For this reason, compounds such as progesterone are not satisfactory starting compounds, and this is also true with respect to pregnenolone (5-pregnen-3-ol-20-one) since its hydroxyl group in 3 is rapidly oxidized to ketone oxygen, giving progesterone.



   The 11-hydroxyl group can be introduced into compounds lacking a substituent on the 11-carbon atom by the use of a culture of 11-hydroxylating [alpha] or 11ss-hydroxylating microorganisms. This 11-hydroxyation can take place not only on 4-pregnene or 5-pregnene compounds, but also on 1,4-pregnadienes, 1,4,16-pregnadienes and 4,6-pregnadienes, such as 11 [alpha] -hydroxy compounds usually have little or no physiological activity, 11 [alpha] hydroxy is preferably oxidized by a mild oxidizing agent, for example chromic acid in pyridine at room temperature or lower,

   and preferably after acylation of hydroxyl 21 when the latter group exists.



   The synthesis according to the invention of compounds II to V also comprises the introduction of a 17 [alpha] hydroxyl group when this group does not exist in the starting compound. This hydroxyla-

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   yours. in 17 [alpha] can be done either chemically, starting from a 16-dehydropregnane compound, for example by forming the corresponding epoxide 16, 17 and then hydrolyzed, or starting from compounds which are saturated in the D ring and in introducing the 17 [alpha] hydroxyl group using cultures of known 17 [alpha] hydroxylating microorganisms.



     - .. As regards the 21-hydroxyl group, although this hydroxyl can be introduced into the methyl group 21 in a known manner by purely chemical means, it is preferred to obtain this result by means of a culture of a microorganism. hydroxylating at 21.



   As already indicated, it is also possible to introduce the unsaturation into ring A by purely chemical means, for example bromination followed by dehydrobromination. In this process, the starting compound is a saturated pregnane and the two double bonds are introduced into ring A by dehydrohalogenation of the 2,4-dibromo compound.



   However, it is also possible to form only a single bond in ring A of an already unsaturated pregnant compound once, by purely chemical means. This can be done by substituting an oxygen function at position 2 of cortisone and hydrocortisone and their esters. The oxygen function is preferably an ester group which is finally replaced by a 2-hydroxyl. The 2-hydroxy-3-keto-4-pregnene compound thus obtained is then dehydrated, for example with an acid, to remove the 2-hydroxyl group together with a hydrogen atom in position 1, to produce the corresponding compound 3 -keto-1,4-pregnadiene.



   Another route within the scope of the present invention for forming the 3-keto-1,4-pregnadiene structure consists of starting from the once unsaturated 1-pregnene analogous to cortisone, hydrocortisone and their esters. Cn first forms the 1,2-epoxide of

  <Desc / Clms Page number 9>

 starting compound, after which the oxide ring is opened to form
 EMI9.1
 deny the saturated halohydrin (1-hydroxy-2-halo-3-keto-presnane) After removal of the halogen and acylation of the hydroxyl 1, the saturated compound is halogenized in position 4 and it is <1-lijreti oiialogenizes to form 1-acyloy-3-ccto --- prenne. By cleavage of the 1-acyloxy group, the double bond is introduced in position 1,2, forming the compound 1,4-presnadiene.



   The invention also contemplates the preparation of certain esters of the compounds of formulas II to V which have been shown to increase the potency of the compounds, and thus require less frequent administration and particularly injection.
 EMI9.2
 



  The 9ce-halo group may exist in the starting compound 11-keto or .1 3 -hydroxy, or it may be introduced by various chemical methods at any stage of preparation. compounds II 'to (V in those which hydroxyl group II \ or II is present, in each case obtaining a 9 "\ - halo-; cmo-11 -hydroxy compound which can be oxidized to the 9, -haloz form; cno- 11-keto.



   By clinical trials' it was found that compounds 1-
 EMI9.3
 c. hydro-cortisone, 1-dehydrocortiool and their 21-esters have an extraordinarily improved physiological action compared to the corresponding one-time unsaturated compounds (cortisone, hydrocortisone and their 21-esters respectively).



  Thus, they exhibit several times the power of the corresponding known hormones, such as cortisone and hydrocortisone,
 EMI9.4
 in the treatment of rYiuratoide arthritis.



  In addition, the undesirable side reactions of known hormones are manifested only to a lesser extent in the use of the new compounds; the intensity of these reactions
 EMI9.5
 condaires jet further reduced dava: 1tacc by the fact naked: 1. ' we can c; .j rent .. "f] f3 C105es much more bazzfiz ae ;;: 0 calves coi-yo> Jés j>:;. cmp¯va.'¯. ¯: i .i =; r .. .¯ ¯ :: .... ¯.¯¯¯; c :: - 2. ""; J: Î 011. And:

   ;?, ne c i 11

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 times unsaturated. Both the initial and maintenance doses in the treatment of rheumatoid arthritis can be greatly reduced by the use of the new compounds, compared to the doses required with cortisone and hydrocortisone. Even with reduced doses, the new compounds do not require the simultaneous use of codeine or other analgesics for pain relief, whereas these analgesics were frequently used in conjunction with cortisone and the compound.



   The therapeutically active dienes of the present invention are preferably administered orally in the form of tablets containing a full daily dose, for example 50 mg or a sub-multiple thereof, i.e. 25 or 20. mg, or even 10 mg, in admixture with a solid pharmaceutical carrier containing one or more of the usual components such as starch, sugar, gums, clays, etc.

   However, they can also be administered by intravenous and intramuscular injection, in solution or in suspension in a non-toxic liquid vehicle. <appropriate; or they can be administered in the solid state by subcutaneous implantation, or in the form of suppositories dissolved or suspended in a fatty or waxy vehicle melting at approximately body temperature. These compounds, as well as their 90 {-halogenated derivatives, can also be administered topically in the form of an ointment or cream, in solution in an ointment or cream base of known composition.



   It has been found that the introduction of the second double bond into various one-time unsaturated intermediates known to have therapeutic activity also enhances the activity of these compounds. Examples of the latter are corticosterone and 11-dehydrocorticosterone.



   We. will now explain in great detail the various convergions that have been briefly alluded to

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 EMI11.1
 previously.



  A. Introduction of a double bond using a culture of a deShrdrOβllc ^ 11t microorganism.



  It has been found that we can efficiently and
 EMI11.2
 inexpensive chemical modification of pregnant compounds, and. especially the dehydrogenation of ring A of pregnenes already having a double bond attached to a carbon of ring A, with or without one or more of the operations of oxidation, reduction and ester hydrolysis, by incubating or fermenting the ester. starting steroid with a culture medium containing a dehydrogenating microorganism which does not degrade or split the molecule at the same time. Appropriate microorganisms
 EMI11.3
 of this genus belong. to the Coyebactriaceae family,

   parsnip which 'species CÉ9 nebacter1uln simplex (American ope Culture Collection 6946) t'COr7118actèriwn, boagii (A.T.0.0.



    7005) have given very satisfactory results. The first of these is a bacterium,,; , while the second one meets
 EMI11.4
 be in the ', rore' of man (in which apparently he does not p ro d -Li r) ao a pathological state) and by: Cois as a contaminant of- cultures e, -poo, fl, the. to 1, "atinosphere although its real or original habitat is not known.



     The starting compounds can have hydroxyl, keto, halogen, and ester groups in various ring or side chain positions; thus, hydroxyl groups may exist
 EMI11.5
 ter in one or more of the positions'>, 11,17,20 and 21; ketone groups can occupy one or more of the 3,11 and 20 positions, while halogen, for example fluorine or bromine, can be attached to carbon 5 or other points of the ring or chain lateral. The presence of a hydroxy group
 EMI11.6
 'the free serble promote chemical transformation.

   A wide variety of ester groups, and preferably the acid esters usually employed in the synthesis of steroids and in the

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 the preparation of steroid hormones for therapeutic use, particularly lower alkanoic acids such as acetate, can be located at one or more of positions 3, 11,17,20 and 21. The specific character of ester n is not critical to the present process, and others can be employed. very esters, both organic acids and mineral acids, for example cyclopentyl- and cyclohexyl-acetates propionates and butyrates, as well as phosphates, polyphosphates and sulfates; what is only necessary is that these esters are not toxic to the microorganism.

   If desired, the hydroxy products of the present process can be converted into their corresponding esters by known methods, for example their lower alkanoic esters and more particularly their acetic esters. Hydroxyl groups at
 EMI12.1
 positions 3 and 11 may be the "0 epimers (or (3. As will be disclosed more fully below, the ester, particularly in position 21, may be the ester of an acid which acts to to prolong the duration of the activity of therapeutic compounds.



   By using a dehydrogenating microorganism according to the invention, for example 4-pregnene- can be converted.
 EMI12.2
 17c, 21-diol-3,11,20-trione (cortisone, compound E of Kendall) in 1,4-pregnadiene-17t, 2l-diol-3,11,20-trione (1-dehydrocortisone), la 4 -Pregnene-11 (3,17c>, /, 21-triol-3,20-dione (hydrocortisone or cortisol, compound F from Kendall) in 1,4-pregnadiene-11 1A, 170 (, 21-triol-3, 20-dione (1-dehydrocortisol), 4-pregn.ene-17o (, 21-diol-3,20-dione (Reichstein's compound S) to 1,4-pregnadiene-17o (, 21-diol-3, 20-dione and 1,4-pre; n.adiene-17o, 203, 21-triol-3-one, 5-pregn.ene-3'3,20-diol to 1,4-pregnadiene-3.20 -dione, the 4-pr: leads-11;

  3,21-diol-3,20-dione (corticosterone) in 1,4-pregnadiene-11 3,21-diol-j, 2C-dione, 4-pregnene-21-ol-3,20-dione (deoxycorticosterone ) 1,4-prenadiene-21-ol-3,20-dione

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 EMI13.1
 the L .-, re; zWne-21-ol->, 11, 20-trione (composed A from Kendall) in 1, - ,: é, xladiùzle-21-O1 ->, 11,20-trione, the 9 a -iluoro-4-pifleginne- Il? J, 17 '; z, 21-triol-3,20-dione (fluoro-compound 1') in 9 C \ -1'luo1'o- 1,.-y- renadin-11 3.17x. , 21-triol-, 20-dione, 5-pregnene-5, 17-n {, 21-triol-20-one, 1,4-pr.; Nadiene-17.) '', 2l-diol-3,20-dione and 21-acetate of 4-pregnene-17> m, l-diol-3, 2 (,, 1 dione, 4-preznane-2C-ol-5-one in 1, 4-Pregnadiene 33,20-dione, 5-hrenene-â, 17a, 20,21-tetrol-11-one and its 3 and / or 21 esters in 1, ° -pre;

  nodine - 17-, 21-diol-3,11,20-trione and its 21-esters, and 5-prenne-3,11 x, 17, 20,21-pentol and its esters 3 and% or 21 in 1,4-pree; nadiene-llrx, 17 -, x 21-triol-3, 20-dione and its 21-ester.



   Instead of the 3-ketone starting compounds, one can use
 EMI13.2
 er the corresponding 3-llydroxylated compounds and their: 5-e. ' ¯.rs, such as 5-prenene-3, 11, 3, 17 ^. (, 21-tetrol-20-one and 5-pregn-3.17 x, 21-triol-11,20- dione and their 3-acetates or 3,21-diacetates, to produce the same 3-c6to-diene end compounds, the 3-ester group being hydrolyzed and the 3-hydroxyl group then oxidized to ketone oxygen. positions 11 and 17 are generally not hydrolyzed, at least to a significant extent, while an ester group at position 21 may or may not be hydrolyzed, depending on the reaction conditions. Thus, when the starting compound is a 3, 21- diester, the reaction product may be a compound 3-keto-21-
 EMI13.3
 ester, or a j-keto-21-hydroxy compound.

   Along with 3-hydroxyl, a 20-hydroxyl will frequently be oxidized to a ketone group. It will thus be seen that the dehydrogenating organisms employed in the present invention are selective with regard to the oxidation stage, the latter being limited in practice.
 EMI13.4
 coupling at positions 3 and 20, while hydrolysis may be limited to 3-ester groups. 25
The method is also applicable to the treatment of
 EMI13.5
 ear;.:

  eres 11-. -hydroxy of the starting compounds 11 3 -hydroxy mcn-

  <Desc / Clms Page number 14>

 
 EMI14.1
 yours above, for example 4-prenene-11 x, 17 x, 21-triol-3,20-dione, 4-prenene-11, I x, 20, 21-tetrol-3-one , 5- pregnne-3-11 <, 17, x, 21-tetrol-20-one, 5-prenene-3,11 x, 1Ïa 20,21-pentol, and their mono- and polyesters, for example 3-acetates, 3,21-diacetates and 3,17,21-triacetates, these starting compounds providing 1,4-prenadien-11 x, 17 (, 21-triol-3,20-dione or an ester thereof.

   The 1,4-diene 11-epimers of compound F (-1-dehydrocortisol) and its esters can be converted to cortisone 1,4-diene and its esters by
 EMI14.2
 oxidation of the 11-hydroxyl group in a known manner by exempt. with. the theoretical amount of chromic acid, with or without pyridine or acetic acid, at or below room temperature (5 to 15 0), preferably after esterification of the hydroxyl 21 if the latter is free.

   These starting compounds
 EMI14.3
 110 (-hydroxylated) are relatively easily prepared in a high yield, which is well known in the art, and therefore constitute advantageous compounds.
 EMI14.4
 for the preparation of 1-dehydrocortisone and 1, -dehydrocortisol,
Further transformations that can be accomplished with dehydrogenating microorganisms are the conversion of
 EMI14.5
 5-prenene-5,17,21-diacetate in a mixture of 1,4-pre; nadiZne-17 ', 21-diol-3,20-dione and 21- 4-prenene-17x, 21-diol-3,20-dione acetate.

   By modifying the fermentation medium, compound S can be made to provide not only 1,1-prenadiene-17x, 21-diol-3,20-dione, but also the slow 1 , 4-1renadiene-17 - 20 3, 21-triol-5-one, i.e. the reduction takes place on the 20-keto group. Like âcja 1.22C11.Ç'tl <., microorganisms can also carry out the oxidation (iqun secondary hydroxyl group 20, as in the conversion of 5-o: r: Wne-S, L.; - Viol to 1,4-pr:; nadiene-, 20-dione The process of the invention is also applicable to 9 \ -

  <Desc / Clms Page number 15>

 
 EMI15.1
 fluoro-, chloro- and bromo-steroids and will provide the corresponding substituted reaction products.

   Various transformations can be. represented by equations (1), (2) and (3) on sheet 1, which show the formation of the desired end products and also the intermediates which can be converted into these end products.
 EMI15.2
 



  'Q .2' "In equation (1), X is, H2, - 0, OR <OH (a or i3), is -C0. CH20H and Z 'is NH or E., In equation (2), YI is H, F, Cl or Br, X is H2e = 0 OUNOHP Y is -CO.CHOH, -CO.CH, 200CR' or - CI-IOII ,. OH 29He Z 'is OH or H and R' is a lower alkyl radical. In equation (3), R is lower aikaiicyl group, Y is, -CO: CH20H, -CIiOHI.CH ,, -CO.CH200CR '. or CHOHI CH20H,, Z 'is H or', 01-1 and R 'is a radical, lower alkyl.



  In order to achieve the desired growth of the dehydrogenation microorganism, for example, O oryneb rium simplex and O. hdagii, for the process of the present invention, a suitable nutrient medium is prepared containing a. oarbohydrate, organic aote, cofactors and mineral gels. It is possible to omit the carbohydrate use without! to completely impede the growth of the organism. After culturing the microorganism in the nutrient medium, the cell mass can be harvested by wringing out the nutrient broth, decanting the supernatant liquid and suspending the cell mass in saline.

   An appropriate volume of the cell suspension is then seeded into a nutrient medium desired to assist the growth of the microorganism. The nutrient medium used can be a yeast extract (Difco), a casein hydrolyzate
 EMI15.3
 (11-Îi-Ainine) (Type B Sheffield), a corn maceration liquor, an aqueous extract of soybean oil meal, a lactalbumin hydrolyzate (Edamine-Sheffield Enzymatic), soluble extracts of fish, etc. .
 EMI15.4
 The compound stu'roivle in solid form or in solution or

  <Desc / Clms Page number 16>

 ion in ethanol, acetone or other solvent Any water miscible which is non-toxic to the organism is added to the cultured microorganism in the culture medium under sterile conditions.

     This culture is then agitated, aerated, or alternatively ventilated and agitated simultaneously, in order to enhance the growth of the microorganism and the biochemical conversion of the steroid substrate. The steroid can be added to the culture medium and then inoculated with the bacteria, or the microorganism grown in the culture medium can be added to the steroid. In some cases, depending on the conditions of the reaction medium, it may be desirable to achieve optimum growth of the microorganism before addition of the steroid. Or, for the implementation of the process, it is also possible to use enzyme preparations obtained in a known manner from cultures of microorganisms.



   Inorganic salts are desirable to maintain a pH level in the reaction medium between 6.8 and 7.2. However, the use of inorganic salts to buffer the reaction mixture can be omitted. The omission of mineral salts causes the pH to rise from an initial value of 6.8 to about 7.7-8. This, however, still allows the formation of the desired end steroid products. The optimum temperature for the growth of the microorganism is 37 C, but temperatures can vary between 25 and 37 C, and even between 20 and 40 C. The reaction time can vary from as little as 3 hours to as little as 3 hours. time as big as 48 hours. The time that will be used will depend on the steroid to be converted.

   Any water miscible solvent which is non-toxic to the body can be used to dissolve or suspend the steroid. The use of ethanol or acetone is preferred in amounts such that the final concentration of these solvents in the reaction mixture does not exceed approximately 7µ1, and these solvents

  <Desc / Clms Page number 17>

   can exist only in trace amounts; as a result of evaporation, the final concentration of the organic solvent may even be practically zero.



   After completion of the oxidation or dehydrogenation process which may be accompanied by partial or total hydrolysis when using mono- or poly-esters, the reaction products can be recovered from the mixture. by extractio with an appropriate solvent insoluble in water, by filtration, by adsorption on a suitable adsorbent or by any other methods commonly used in this field. For extraction, chlorinated lower hydrocarbons, ketones and alcohols are useful. These compounds include chloroform, methylene chloride, trichloroethane, ethylene dichloride, butanol, diethyl ketone and other compounds. We prefer to employ extraction as a method of isolating steroidal products.

   After extraction, the products can be isolated by concentrating the extracts to low volume or to dryness. Residues can then be purified by simple recrystallizations from a suitable solvent or solvent mixture, for example acetone, methylene chloride, ethanol, acetone-hexane, methylene chloride. - hexane, etc., which provides the desired diene in excellent yield and in a very high state of purity.



   The chemical transformations which can be carried out by subjecting the various pregnens to the action of a culture of dehydrogenating microorganisms are thus of widely different kinds; they can occur in isolation, or two or more of these transformations can occur simultaneously or successively. The various reactions appear to be unaffected by other substituents in the steroid ring or in the side chain.



   From the above it is evident that it is not essential

  <Desc / Clms Page number 18>

 that the starting compound has its double bond in position 4 of ring A; therefore, the double bond can be in position 5.



  The starting compound can also contain more than one double bond. Thus, 4,6-pregnadienes can be dehydrogenated and otherwise processed as described above to produce 1,4,6-pregnadienes, the latter easily being reduced in known manner to 1,4-pregnadienes. For exemple
 EMI18.1
 4,6-Pregnadiene-17,21-diol-3,11,20-trione 21-acetate can be converted to 1,4,6-pregnatriene-17 r, 21-diol-3,11,20-trione or its 21-ester. The corresponding 110 (or 11ss - hydroxy compounds will likewise be converted to 1,4,6-trienes. The trienes can be reduced by known methods to 1-dehydrocortisone or 1-dehydrocortisol or the like.

   The starting compound can also have a double bond at position 16, or at position 9 (11), as explained below.



   The soluble extracts of fish referred to above can be obtained presently in commerce in the form of an extract of herring, menhaden, and mixture.
 EMI18.2
 various of these which have been subjected to enzymatic hydrolysis. This material can be added directly to the culture broth to provide nutrient material. When soluble fish extracts (50% solids content) are available and these have not been subjected to enzymatic hydrolysis, such extracts should be diluted with water and treated with water. steam for about 10 minutes at 90 ° C, then filtered, preferably using Filter-Cel.



   Another example of a dehydrogenating microorganism which has been shown to accomplish the reactions described above is the Bacillus
 EMI18.3
 sehaerieus var. fusiformis (American Type Culture Collection 7055) which likewise is capable of introducing a double bond into the A ring without simultaneously causing the degradation of the side chain or the cleavage of the D ring.

  <Desc / Clms Page number 19>

 
 EMI19.1
 - 1-1b unsaturated in nucleus A are very stable in a culture of this microorganism, and it is not necessary to take special precautions in order to prevent destruction of the desired end product. The incubation time can be as high as 96 hours and the yields are very high,

   these frequently being almost quantitative.
 EMI19.2
 



  As with the other dehydrogenating microorganisms mentioned above, it is possible to use instead of a growing culture of B. s haericus var. fusiformis separate enzymes or enzyme extract from this culture. The starting pregnenes can likewise be very varied, but it is preferable to employ a starting compound having either a C-4 or a C-5 double bond, an example being given by a 3-keto. 4- or 3-hydroxy-5-pregnene.
 EMI19.3
 



  In general, B-spha ricus var. fusiformis will perform the same chemical transformation in the pregnene compounds mentioned above as the Corynebacteritui organisms, except that it does not hydrolyze as easily, and in some cases not at all, a 3-ester group, and this is why we recommend that with this gold-
 EMI19.4
 canisse is used 3-keto and -hydroxy-prégni: nes, and not the corresponding 3-esters. Steroid substrates can contain nuclear double bonds in addition to those at C-4 and C-5, and these additional double bonds can be saturated with halogen or a halogenated hydracid, or by form-
 EMI19.5
 dicnophilic addition compounds such as maleic acid and human anhydride.

   With the exception that the 3-ester; are not used, the various starting compounds will be converted into the same end products by B. s haericus var. fusiformis and: .Lïsâ1 by Oorynebacterium simplex and 1101, Fr. 7 ...



  The cultivation node of 3. sphaericus var. fusiformis and the method of inoculation of the nutrient medium, the nature of the nutrient medium, the:., ni.r e to contact the steroldc substrate

  <Desc / Clms Page number 20>

 
 EMI20.1
 , 1.-C = 1 culture grown. rici% oorj¯. ;;. ni-cme "po; 1, r <#xrl be the.J i..0un cu 'with Jori species. = ne -1, cfezsiigg, sc.uf c = ui, co - .l-ù ja indicates, the incubation period can be 311tü 1C 'I,' I'¯3, é'va, usually about 24 to 96 hours. In all cases, the culture containing the steroid is subjected to agitation and aeration.



   In addition to backgrounds. nutrients revealed above, we can
 EMI20.2
 also er-deploy protopeptones. The growth of microoranism is preferably initiated at a pil of about 11.6 to. 7.2; the optimum temperature for the growth of B. u s phaerious var. fusi-rormis is around 28 C, but the temperature can vary between 19 C and 32 C.



   The products of the biochemical reaction are preferably recovered by extraction with the solvents and following the procedure described above, but any other suitable method known to chemists can be used.
 EMI20.3
 



  Obviously, instead of the dehydrogenating microorganisms described above, their mutants can be employed which have similar dehydrogenating activity without exerting a destructive action on the pregnane carbon skeleton.



   We will now describe more complicated syntheses, starting from simpler compounds, to prepare compounds II to V, and also intermediate compounds which are converted or are capable of being converted into these compounds, however employing a dehydrogenating microorganism for- the introduction of a double bond in ring A.
 EMI20.4
 



  B. Binding of 1-ac: li; rdrOGGrtisone and 1-deh, ¯hydrocortisol from, 17'Q, igrdroxy + -proiJterone and 5- rêrn: ne-> 17 '- diol-20-cne.



  According to a modification of the present process, it has been found that one has to combine the dehydrogenation of the ring A, by means of; .iicroorj; anisms àési; j = droj6nai; ts described above, with

  <Desc / Clms Page number 21>

 other microbiological conversions, which are widely known individually, and with a chemical oxidation process step (when it comes to producing 1-dehydrocortisone), to convert 17 [alpha] -hydroxy- progesterone (4-pregnene -17 [alpha] -ol-3,20-dione) and 5-pregnene-3,17 [alpha] -diol-20-one in the much more valuable # 1-dehydro derivatives of cortisone and hydrocortisone, these operations being capable of being used in different sequences,

   except that the operational phase of chemical oxidation is consecutive to the biological process which introduces a hydroxyl group in position 11. Microbiological processes are in principle of three types; These include the following: (1) introduction of a # 1 unsaturation (i.e. of a double bond in the .1,2 position) with secondary oxidation of a 3-hydroxyl, when it exists. , by the action of a dehydrogenating microorganism such as those described above, to produce a 1,4-diene-3-ketone system;

   (2) intruduction of an 11 [alpha] or 11ss 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 member belonging to the genus Ophiobolus.



   The sequences of these three microbiological transformations can vary; can be, for example: (1), (2), (3); (2), (1), (3); (1), (3), (2); or (3), (1), (2).



   If the production of 1-dehydrocortisone is contemplated, then at any point after the introduction of the 11-hydroxy group the intermediate compound is oxidized, for example with chronic acid in pyridine, preferably at room temperature or below, to convert 11-hydroxyl to ketone oxygen.

   However, in case the 11-

  <Desc / Clms Page number 22>

 
 EMI22.1
 hydroxyl has the '3 configuration, and we desire 0 -etij.r 1-dehydro-Jerivated from hydrocortisone, we oriet phaso Oßsti;.' i O¯i..f. tr
 EMI22.2
 oxidation.
 EMI22.3
 The introduction of a 11 ydrofle, retort .; indicates more
 EMI22.4
 high, is preferably done by the action of a culture (or
 EMI22.5
 separate enzymatic material) from Rhizopus nif, ricams, as described in the US patent to Lurray et al. ,?
 EMI22.6
 2,602,769, July 8, 1952; while the introduction of a
 EMI22.7
 group 11 1i is preferably made with a culture (or the enzyme material separated from a culture) of Curvularia gi "alta ,, co: = 1.ie described in the US patent of Shull et al., If '2,658.02
 EMI22.8
 of 3 November 1953.

   However, it is also possible to use other
 EMI22.9
 very llc-hydroxylalit organisms such as 11-isuergillus itir; r, lc:
 EMI22.10
 
 EMI22.11
 Rhizopus arrhizus; and it is also possible to use other cr.anis- .. / /. my fr -hydroxy.lant ,, s such as Çunninghamella blahesleeana, etc.



  The hydroxyl is introduced into the steroid molecule by an organism of the genus, 0 hiobolus, preferably the.
 EMI22.12
 
 EMI22.13
 0. herbotriclius' as described by Meystre et al. , lIelvetica Chim. Acta 37, 1548 '(.1954-).



  The dehydrogenation of the, 41ojra-u-, A. Of the steroid compound to introduce a l, l double bond is effected by a de-
 EMI22.14
 hydrogenating agent, for example one of those described above. This dehydrogenation operating phase can be applied to the compound.
 EMI22.15
 landing or at any intermediate point in the
 EMI22.16
 complete process.



  'The various sequences indicated above can be represented by the following equations:
 EMI22.17
 Here is :
 EMI22.18
 17-hydroxy 1, - pren.dene- 1,; .- pr: n: Līnc¯ rro: 38teroe lloe, 17, diol-5, 2c-d-ione llx, l7a;, 1-; iio1- ::, ¯ - Ic 1. ' 1 1 LIT - C t "### dione VIIa /
 EMI22.19
 
 <tb>
 <tb>
 
 EMI22.20
 1, 4 -p r é gnad iene - 1 ü.¯ = ^ vi'li: C .. 'E i l! ¯- 7, a'¯ ¯¯z't!) ¯i., -. fiat-ȯ-3, C1-cio? '? e 7-Q - :, 11,' - trio? 7x ---- l¯51 - w, 'I- 7c "" will sort The 5-p' esnene -. 1,4-recndie- 1,, 4-presnaBîène- 3, 7'-C ICZ-Î-C5'L¯ ¯l,! 7x - C.1.01-f, '..- ir? T .; ¯ J? . 1 C, 2.Z-Y '¯u' .- ¯ Z 'c l'î, 1 - "3,20-a10ne

  <Desc / Clms Page number 23>

 
 EMI23.1
 'ro..IJ] .:

   
 EMI23.2
 i.-preyene-11x-17x - diol-3, 20-dione IIIc -VIIa, 1 VIc v 4-preguene-17X-
 EMI23.3
 
 <tb> 01-3,11,20-trione <SEP> @ <SEP> Vc <SEP> @ <SEP> IIa
 <tb>
 <tb> VIIIc <SEP> @
 <tb>
 
 EMI23.4
 \ ° -renene-113.17x-
 EMI23.5
 
 <tb> diol-3 <SEP>, <SEP> 20-dione <SEP> - <SEP> # <SEP> IVc <SEP> - <SEP> ---- <SEP> IIIa
 <tb> VIIc
 <tb>
 
In the first sequence (lane A), 17 [alpha] -hydroxyprogesterone (Ic), or 5-pregnene-3,17-diol-20-one (l'c) is easily
 EMI23.6
 transformed by the action of C. simplex or C. hoaall to 1,4-pregnadiene-17-01-3,20-dione (IIc). Hydroxylation of IIe by Rhizopus nigricans (as described in Aan patent 2,602,769) provides compound IIIc.

   On the other hand, the hydroxylation of IIc with Curvularia lunata (following the process
 EMI23.7
 described in US Patent UO '2,658,023) gives the compound IVO.



  Other 11-hydroxylating organisms such as Aspergillus, Rhizopus arrhizus, etc., are also effective for the conversion of IIc to IIIc, while other 11-hydroxylating organisms, such as Cunninhamella Blakesleeana can be used for the conversion. conversion from IIc to IVc. Either IIIc or IVe can be oxidized to Vc by the action of a mild oxidizing agent such as the pyridine-chromic acid reagent. The hydroxylation of IIIc, IVc or Vc in position 21 is carried out, for example, with Ophiobolus herbotrichus, giving respectively VIIa, IIIa or IIa (see sheet 1).

   VIIa and IIIa (preferably in the form of their
 EMI23.8
 21-acetates or other 21-lower alkanoic esters) are both convertible to IIa by controlled oxidation with the pyridine-chronic acid reagent. The ester group can subsequently be saponified, for example by heating under reflux with alcoholic potassium bicarbonate.



   In the second sequence (channel B), the order of the phases operates

  <Desc / Clms Page number 24>

 ratories (1) and (2) (listed above) is reversed, resulting in the production of three new intermediate compounds VIc, VIIc and'Ville. However, the individual process phases are the same and the end products are also the same.



   As can easily be seen from the equations above, compounds IIIc, IVe and Vc are produced by both sequences; these compounds constitute important intermediates for the preparation of # 1 -dehydro-derivatives of cortisone and hydrocortisone. They are encompassed by the formula X (sheet 2) where X is = 0, / 3 -OH, H or - \ -OH, H.



  C. Conversion of deoxycorticosterone and 5-pregnene-
3,21-diol-20-one and their esters in 1-dehydrocortisone t 1-dehydrocortisol.



   Preferred end products, namely 1-dehydrocortisone and 1-dehydrocortisol, as well as their esters, can also be obtained from deoxycorticosterone (Id, below) (4-pregnene-21- ol-3,20-dione) and 5-pregnene-3,21-diol-20-one, and their esters, by a combination of some of the operating phases described above, with the operating phase of introducing 17 [alpha ] -hydroxyl by means of a 17 [alpha] -hydroxylating microorganism, for example one selected from the genus Trichothecium, the process also comprising the process step of oxidizing an 11 [alpha] - or a 113 -hydroxyl, introduced during the process, when 1-dehydrocortisone is the desired end product.



   The various process steps can be employed in any sequence except that the chemical oxidation process phase follows the microbiological process which introduces 11-hydroxyl.



   There are three types of microbiological transformations; they are the following:

  <Desc / Clms Page number 25>

 (1) introduction of a # 1 unsaturation (i.e. a double bond in the 1,2 position) with simultaneous oxidation of a 3-hydroxyl, when it exists, or hydrolysis of a 3-ester followed by the oxidation of the resulting 3-hydroxyl by the action of a dehydrogenating microorganism.



   (2) introduction of an 11 [alpha] - or 11ss -hydroxyl group by the action of an organism of the Rhizopus nigricans type or
Curvularia lunata; and (3) introduction of a 17 [alpha] -hydroxyl group by the action of a culture of a hydroxy microorganism selected from the genus Trichothecium.



   The sequences of these three microbiological transformations can vary at will and can be: (1), (2), (3): (2), (1), (3); or (1), (3), (2); or. (3), (1), (2). If 1-dehydrocortisone or its esters are to be produced, then at any point after the introduction of the 11-hydroxyl group, the intermediate compound is oxidized, preferably in the form of its 21-ester, for example with chromium trioxide at low temperatures to convert 11-hydroxyl to ketonic oxygen. However, in the case where the 11-hydroxyl has the ss configuration, and it is desired to obtain the # 1 -hydrocortisone derivative, the operational oxidation phase is omitted.



   The 17 [alpha] -hydroxyl group is attached by a hydro-xylating organism selected from the genus Trichothecium, and preferably T. roseum, in the manner and with the equipment described by Meystre et al., Helvetica Chim. Acta, 37 1548 (1954).



   After completion of the dehydrogenation of ring A, which may be accompanied by partial or total hydrolysis when mono- or poly-esters are employed, the reaction products can be recovered from the mixture by extraction with a. suitable solvent immiscible with water, by filtration, by

  <Desc / Clms Page number 26>

 adsorption onto a suitable adsorbent or by any of the other methods in common use in this field, suitable solvents being disclosed above.



   The ester groups can be the same as those which have been disclosed previously. If the end products contain free 21-hydroxyl, they can be converted to their corresponding esters by known methods, for example their
 EMI26.1
 lower aikanoic esters and particularly their acetic esters.



   The different sequences according to which this modification of the present process can be carried out are explained by the following diagram: ROUTE C:
 EMI26.2
 4-Presnene-21-1,4-prenadien-e-1,4-pregnadiene-0l-3, 20-dione III, 21-.tiol-II .. <, 17d, 21-triol-. ' 3, 20-dione 3, 20-dione / 'IIId 1 11 1,4 ± pregnadiene-84-pregndiene% -21 = ¯ 1,4-pregnadbene-1, -pregnadiene-1,4-pregndiene-21- 1, 4-prognadene-21-0l-3, 20-dione-01-3, 11 20-trione ', -' 17a, 21-diol-3, 11, 20-. 1 y,; 1 ... , IIa 5-prégi7.ene-3, 21- 1, 4-préna.ièn¯e- 1,4-pregnadiene-diol-2Qone 1,3,21-diol-3,20-ll (3,, 171, 21triol-
 EMI26.3
 
 <tb> I'd <SEP>, <SEP> dione. <SEP> ' <SEP> 3,20-dione
 <tb> IVd <SEP>. <SEP> IIIa
 <tb> TRACK <SEP> D <SEP>:

    <SEP>
 <tb>
 
 EMI26.4
 '4-Pregnene-.Il, 21-diol-3,20-aione. - IIId - - VIIa VId J 4 - prëgnëne-21- ol-3,11, Ç0-trione bzz¯¯- Vd - IIa
 EMI26.5
 
 <tb> Id <SEP> VIIId
 <tb>
 
 EMI26.6
 q 4-Pregnene-11.3,21- \ ¯diol-3, 20-dione - - .. IVd -, .-. IIIa
 EMI26.7
 
 <tb> #VIId
 <tb>
 
In the above diagrams, for the sake of simplicity, compounds IIId, IVd, VId have been shown; VIId, IIIa and VIIa as free 21-alcohols, but in actual practice it is preferred to esterify the 21-OH group, for example with a lower alkanylating agent, prior to oxidation to compounds Vd.

  <Desc / Clms Page number 27>

 
VIIId and IIa, the ester group being hydrolyzed at any time thereafter.

   In the case of compounds VId and VIId, the hydrolysis can be carried out simultaneously with the introduction of the # 1 double bond by Corynebacterium or other dehydrogenating culture.



   In the first sequence (path C), deoxycorticosterone (Id) is easily transformed by the action of C. simplex into 1,4-pregnadiene-21-ol-3,20-dione (IId), which is also formed by similar processing of 5-pregnene-3-21-diol-20-one (l'd).



  Hydroxylation of IId by Rhizopus nigricans provides IIId.



  On the other hand, hydroxylation of IId with Curvularia lunata provides IVd. Either of these hydroxylating processes can be performed with ease.



   As indicated above, other 11 [alpha] -hydroxy- ing organisms, such as Aspergillus niger, Rhizopus arrhizus, etc., are also effective in converting IId to IIId, while one can employ other 11ss-hydroxylating organisms, such as Cunninghamella blakesleeaa., to convert IId to IVd. IIId or IVd, or preferably their 21-acetates or other lower alkanoyl esters, can be oxidized to Vd or its corresponding ester by the action of a mild oxidizing agent such as the pyridine-chromic acid reagent. The hydroxylation of IIId, IVd or Vd at position '17 is carried out with T. roseum, giving respectively VIIa, IIIa or IIa.

   VIIa and also IIIa (or preferably their 21-acetates) can be converted to IIa by mild oxidation with the pyridine-chromic acid reagent.



   In the second sequence (route D), the starting compound (Id) is first hydroxylated at 11 and then dehydrogenated in ring A, thus resulting in the production of three new intermediate compounds VId, VIId and VIIId. However, the end products remain the same and so do the processes employed.

  <Desc / Clms Page number 28>

 
 EMI28.1
 



  B. rent and see: terw r c1G "',, wlrCi ..L. 1-QP. y; Î droG'or lsot'3.C'. Pt l, ', dur0"' Àā¯ # éôzj ll; .- àro #; rc; <ester¯one in 1-dehydro cortisone and 1-J.hydro- C'.G'i't.L ^ C? 1.



  According to the invention, it is also possible to convert it. 16 -: -: 'llj-ù.r0: 9r (::; nJ: .lolone (Ie) and its esters, relatively sparingly.- cCÜeU8G and readily available, as is 16-dehydroprogesterone (IIIe) , in the cortical hormonal substances 1 = active Js 1, -I - réxiadine-11, 17 x, 21-triol -, G- 6ione (l-dehydrocortisol) and 1, .- hré; 2adi4ne-12 21 -diol-5, 11, 20-trione (1-dehydrocortisone) and their esters, such as their lower aikanoyed esters, by a relatively small number
 EMI28.2
 chemical and biochemical operations during which
 EMI28.3
 obtains new intermediates.

   These operations are (a) conversion of the 3-hydroxy-a5 system to the 5-Cto- system (in the case of 16 = oeéhyàro-pregnenolone), (b) introduction of 17 -hydroxyl via 16,17- epoxy,
 EMI28.4
 (c) introduction of a 21-hydroxyl group by the action of a
 EMI28.5
 member belonging to the genus 0'lziobolus, for example
 EMI28.6
 
 EMI28.7
 0. lierbotriclluis, (d) introduction of an II ,,,, - or II / '' 3-hydroxyl group by
 EMI28.8
 the action of a suitable microorganism, as described above, and
 EMI28.9
 (e) introduction of the Ol double bond by means of Corynebacterium simplex or dehydro-cenellate microorsanisms operating in the same way, as described above. e r: lI1l e nt.



  The oxidation of the 5-hydroxyl group of IS-dei2; 'droprc: t ± fr? No- lone (le) can be accomplished by the Cppcnauer reaction or by careful oxidation with chronic acid at room temperature. or one below this, but it is preferred to carry out this o :: - ,: 1.ation simultaneously with the introduction of the double ii = ison à by treatment with a culture of a microorganimilc

  <Desc / Clms Page number 29>

 dehydrogenating or with an enzymatic extract of such a culture, as more fully described would precede,

       but this treatment must follow the addition of a hydroxyl group at one or more of the 11, 17 and 21 positions, otherwise the yields are poor. If desired, esters, for example lower alkanoyl esters of 16-dehydropregenolone can be used as the starting material for this microbiological conversion. The introduction of the 17 [alpha] -hydroxyl group can be done by treatment with a peracid, for example perphthalic and perbenzoic acids, obtaining the intermediate 16,17-epoxide.

   The latter is then preferably subjected to the action of iodidic acid mixed with acetic acid and acetic anhydride, obtaining the compound 16-iodo-17 [alpha] -hydroxylated. The iodine atom is then removed by boiling under reflux the co @ o- se dissolved in a lower aliphatic alcohol such as ethanol and containing a small amount of an organic acid such as acetic acid, in presence of a Raney nickel catalyst. In this reaction, the hydrogen adsorbed under the catalyst acts to replace the iodo group with hydrogen.



   The 21-hydroxyl group is introduced, in the manner already described, by subjecting the 21-methyl compound to the action of a culture of a member belonging to the genus Ophiobolus, while the mode of introduction of the 11 [alpha ] or 11ss -hydroxyl is likewise performed biochemically, as described in connection with other compounds above. It has been found that the 11ss -hydroxyl group can be introduced at the desired positions even in the case of 1,16- and 1,4-pregnadienes and 1,4,16-pregnatrienes and, in the case of 1,4-Pregnadienes, also the 11 [alpha] -hydro-xyl group, without saturating any of the double bonds.



   The operating phase of dehydrogenation is carried out, in which a double bond is introduced in position 1 of the pregnant compound, by subjecting the starting compound, preferably,

  <Desc / Clms Page number 30>

 rence after hydroxylation conne stated above, to the action of a. dehydrogenating organism as described above.

   Cultures of dehydrogenating microorganisms are able to introduce a 'double bond, although the steroid molecule already contains a C16 double bond and also an O 5-attached double bond. As with the other starting compounds previously described, the culture is capable of introducing not only a # 1 double bond, but also of oxidizing a 3-hydroxyl group to a ketone group with simultaneous migration of a 5,6 double bond in the 4.5 position; under certain conditions, the culture is also capable of effecting the oxidation of a 20-hydroxyl to a 20-ketone group.

   In the event that the compound with which one operates has an ester group in positions 3 or 21, this ester group will be hydrolyzed, the hydrolysis of the C21 ester group generally being favored by a pH of 6.8 to @. , 1 and a temperature of about 26 to 29 C.



   When the final product is to be 1-dehydrocortisone, it is preferred to introduce a 110 (-hydroxyl group rather than an 11, -3-hydroxyl group, since the 110 {-hydroxyl group can in general be introduced more easily. and can then subsequently be oxidized to an 11-ketone group.In the event that an 11,3-hydroxyl is introduced into the steroid molecule, this group can be kept unchanged in the final product to give the # 1 -dehydro-derivative of hydrocortisone, which is likewise very active.



   The intermediate compounds included in the present invention for the preparation of 61-derivatives of cortisone, hydrocortisone and their esters, and prepared by the modification of the present process which is just described, have the structures indicated. in formulas VIII and IX (sheet 2).

   In these formulas, is H2, OH ([alpha] or), while R re-reformules, X is H2, O, or OH ([alpha] or ss), while R represents H, CH or 0 acyl, the acyl group in each case being

  <Desc / Clms Page number 31>

 colui .. An organic carboxylic acid, preferably from a lower alkanoic acid such as formic, acetic, propionic, butyric, valeric or caproic acids, although other acids such as alkanoic acids are not excluded. substitutes and aromatics such as cyclohex-yl-acetic, cyclopentyl-propionic and benzoic acids.



   Cn prepares the compounds in which X represents a hydroxyl and R an acyl group by esterifying 16,17-oxydo-1,4-pregnadiene-11,21-diol-3,20-dione to obtain the 21-ester pra - addition to the oxidation of 11-hydroxyl, after which the oxido ring is opened with III or IIBr to form the 16,17-halohydrin; the halogen is then removed with Raney nickel or with hydrogen in the presence of a palladium on charcoal catalyst.

   The hydrolysis of the 21-ester group can be carried out at any point after the oxidation ...,
As already indicated, the different operating phases involved in this form of the present process can be carried out according to variable sequences, a certain number of which is given in the following diagram:

  <Desc / Clms Page number 32>

 
 EMI32.1
 
 <tb> Xe
 <tb>
 
 EMI32.2
 5-prÉncne-5, 11 (c or 3), I :: e 17 :, 21-t c: t rol-2 C-one
 EMI32.3
 
 <tb> XIIIth <SEP> t
 <tb>
 
 EMI32.4
 15, 17-epox;

   -5-prenene- 16,17-epoxy-4- 3, II (, or 3), 21- triol¯ - preJl1ene-II (c (or / 3) 2C-one, 2l-diol-3,20-dionc ZIIe XVe 16, 17-époxT-5-pré Jnne- 16, 17-époXY-4- 3, 11 (:, or - ') -diol-20-one - - --- - z pre; nene-11 ( a or / 3) life, 01-3,20-dione -del # j-dro- xve 16-dbhydro- r3rc :: rnolonē 16, 17-epoxy-5- 16,17-epoxy-. 17a Wydro: y - ire - -pr6gnene-3-01- 4-pregnene- - --- progesterone
 EMI32.5
 
 <tb> 20-one <SEP> # <SEP> 3,20-dione
 <tb> IIe <SEP> IVth <SEP> # <SEP> Vile <SEP>! <SEP>
 <tb>
 
 EMI32.6
 16 - dehydro '16,17-epoxy- progesterone, 4-pregnadiene-
 EMI32.7
 
 <tb> 3 <SEP>, <SEP> 20-dione <SEP>
 <tb>
 <tb> 3rd <SEP> VIe
 <tb>
 
 EMI32.8
 16,17-epoxy-1,4-1,4-prer;

  nn.adiene- prcgnadiene-21-¯¯ 17x, 21-diol-3, 20- ol-3, 20-dione dione) 1-dehydro- 1, 4-pr6nadiene-4-pregnene-4-pregnene-cortisone 11 ( '' <or b), 17x, 11 (x or 3), 17, 17x, 21-diol- IIa - 21-triol-j, 20-diône 21-triol-3, 20- ¯ ---- 3, 20- dione
 EMI32.9
 
 <tb> dione
 <tb> Xe <SEP> IXth <SEP> 8th
 <tb>
 
Most of these operating phases are completely independent of each other so that the sequence of operations can be varied in various ways giving a good result. For example, the compound Ie can first be converted to 16,17-epoxy-pregnenolone (IIe), and then to 16,17-
 EMI32.10
 epoxy-progesterone (IVe), or it can be converted to 16-dehydro-progesterone (IIIe), then to IVe.

   Any of these known intermediates (IIe, IIIe, IVe) can be hydroxylated at position 11 (either x or 3) and position 21, and dehydrogenated to have the 3-keto system # 1,4 -diene; these three sequences can be carried out in any order, except that the hydro-xylation or epoxidation preferably precedes the treatment.
 EMI32.11
 with the .1Ícroorcanis :: the dehydrogenator. Compound IIIe, containing

  <Desc / Clms Page number 33>

 a # 16 double bond, is converted to 16,17-epoxide (IVe) by the usual methods (either with peracids or with alkaline hydrogen peroxide).

   Compound IVe is then treated with an acid to open the oxido ring by cleavage in order to form 17 [alpha] - hydroxy-progesterone (VIIe). The latter is then treated with a culture of a 21-hydroxylating microorganism to have VIIIe; the latter can then be hydroxylated to form compound IXe. This last compound is then treated with a culture of
 EMI33.1
 dehydrogenating microorganism to have the compound, 1,4-pregnadiene (Xe) which is then oxidized to 1-del> ydrocortisone (IIa).
 EMI33.2
 



  The compound Xe can also be produced by first hydroxylating the 16,17-epoxy compound IIe at 11, obtaining XIe which is then hydroxylated at 21%. give compound 12th. We ..
 EMI33.3
 then opens the epoxy ring of the latter compound for '!: rr' '' odui- 're the' group 1701 -hydroxyl.e, 'as indicated in' compound XIII, which can be chemically oxidized to convert the 3rhydroxyl to ketonic oxygen, obtaining IXe, which is "aior J, ldehydrogenated 1 in, Xe ;;> where one can immediately treat the compound, 'XzIIe with the dehydrogenating microorganism for a6irldirect Xe.



  As further indicated in the diagram, it is possible to hydroxylate compound IVe (XIVe) 1 at 11 and then hydroxylate it at 21 to have compound XVe. The latter is then treated to open the epoxy ring 16,17 to give compound IXe which is then dehydrogenated in Xe. The diagram shows various, other trans- formations which will be easily understood from the, des-
 EMI33.4
 previous description.



     The point in the process where the 16,17-epoxy ring is opened, for example with hydriodic acid (HI) to give
 EMI33.5
 the 170th-hydroxyl group, or the point at which the 11-hydroxyl group is oxidized to 11-ketone, is irrelevant, provided that during these reactions all hydroxyls present at positions 3 and 21 are protected by ester groups which may be in-

  <Desc / Clms Page number 34>

 raised thereafter.
 EMI34.1
 E. Conversion of 1,4-zr; nadiene-17a -21-diol ->! 2G-diome and its esters to 1-dehydrocortisone and 1-deyhydrocortisol and their esters.



   An example of the use of a starting compound which already has the
 EMI34.2
 3-keto-1,4-pregnadiene structure for the preparation of 1-deliy-drocortisone and 1-dehydrocortisol and their esters is 1,4-pregnadiene-17cx, 21-diol-3,20-dione (the 1- dehydro-derivative of the compound S of Reichstein) which can be prepared from the compound S either chemically (see American patent of Djerassi et al. No. 2,579,479 of December 25, 1951) or by treatment with a dehydrogenating microorganism as previously described. .



   It has been found that cultures of microorganisms, or their separate enzymes, having the known property of introducing a hydroxyl group at the 11-position of saturated or single-unsaturated pregnans, either at the 11x'or 11ss position, are capable of Introducing such an 11-hydroxyl group also into 1,4-pregnadienes which are not substituted in this position, and the conjugated double bond system in ring A is not modified by these microorganisms. From which we can easily prepare 1-dehydrocortisone and 1-dehydro-
 EMI34.3
 cortisol when the pl-dehydro-derivative of the S compound is available.



   According to the present modification of the process, it has been found
 EMI34.4
 that 1,4-pregnadien-17rx, 21-diol-3,20-dione and its 21-esters (If, below) can be converted, despite the presence of the double unsaturation in the A ring , in compound 11-hydro-
 EMI34.5
 xyl corresponding by treatment with various known 11-hydroxylating microorganisms.

   So the compound If can be either 11th <-hydrox ;;. r1é by the action of a toes various organisms 110 <'- hy- al'c, :: y1: mtu ct - :. iui-, for example iliiii-oni = x 1, rriiizu =, le: u17¯G (if11.9 it'1 GL: u, ï: ü! 1Ízoyms ùel ;; ar, the 211izGT, us Z'G = ¯1G = lis, the. li..3: .C.'r '¯ll¯f

  <Desc / Clms Page number 35>

 
 EMI35.1
 ± Lµ.> I :, 1-;? L17'oO o'7C ':) 0 bla081oeanus., To give LU, or ii can be 11 -il;,. Rō = lc by the action of 1.i ! lr; l1.8 ± l.ell.a 1Jlale :: toean8 ". of S. JurvLÜc lU11ata or one of the other ll o - hydroxylating organisms known to give IIIa or IIIb.

   The product of the 11 -h3rdroxirlation, IIi, under the decoration of its 21-ester, preferably the acetate, is then oxidized by the action of chromic acid in pyridic solution, or by other oxidizing agents
 EMI35.2
 mild of equivalent power, in 21-acetate of 1,4-preZiiadiùne-17, 21-diol-3,11,20-trione (IIb), which can be easily hydrolyzed to compound 21-OH (1-dehydrocortisone) (IIa). The product of II O -hydrolysis is the 21-ester of 1-dehydrocortisone (IIIb) which can be oxidized to 21-acetate of 1-dehydrocortisone and after that comes the hydrolysis of the 21-ester.



   The reactions involved in this process are shown in the following diagram:
 EMI35.3
 1,4-pre; nadiene-17n (, 21-diol 1,4-prc: nadiene-11, 17.a (, 21-
 EMI35.4
 
 <tb> 3,20-dione <SEP> or <SEP> 21-ester <SEP> # <SEP> triol-3,20-dione <SEP> or <SEP> 21-ester
 <tb> I-f <SEP>. <SEP> i <SEP> IIf
 <tb>
 
 EMI35.5
 l, 4-pr; nadi: born-11 ,,, 17x, 21- l, 4-prnadine-17X, 21-triol-3,20-dione or 21-cster diol-3,11,20 - brione
 EMI35.6
 
 <tb> or <SEP> 21-ester <SEP> ' <SEP>
 <tb> IIIa <SEP> or <SEP> IIIb <SEP> IIa <SEP> or <SEP> IIb
 <tb>
 
If If is in the state of free 21-alcohol, then after 11-hydroxylation it is intermediately converted to 21-ester before oxidation.

   The acyl radical, as before, is preferably that of acetic acid, but it can also be that of other lower fatty acids such as the acids.
 EMI35.7
 formic, propionic, butyric, valeric or succinic or aronatic acids such as benzoic, salicylic, phthalic and veratric acids. In the case of dibasic acids, the es- is preferably in the form of the semi-ester. If necessary, the acyl group can be hydrolyzed, for example by heating with alcoholic potassium bicarbonate.

  <Desc / Clms Page number 36>

 



  F. Introduction of unsaturation into nucleus A by chemical means.



   It is apparent from the foregoing description that dehydrogenating microorganisms can effect either single ring unsaturation or double unsaturation, as in the case of 5-pregnen-3-ol compounds (or their esters, which are hydrolyzed. to free 3-OH compounds), and in this case by oxidation of the 3-OH group to a ketone group, the 5,6 double bond is caused to migrate to the 4,5 position. However, one or both double bonds of ring A can also be introduced by purely chemical means.



   For example, it has been found that the 21-esters of pregnan-17 [alpha],
21-diol-3,11,20-trione (Ig, below) as well as the 21-ester and the 11,21-diesters of pregnan-11 ([alpha] or ss), 17 [alpha], 21 -triol-
3,20-dione (IIg, compound 11ss-OH) can be halogenated in positions 2 and 4 by treatment with a halogen having an atomic weight greater than that of fluorine, to produce the 2,4-dihalides in satisfactory yield. correspondents. Esterification, particularly of group 11 (3-hydroxyl, improves yield. The halogenation at both positions can be done in one or two stages of the process and /, in the latter case, halogens can be employed. different types such as chlorine and bromine.



   The dihalide is then treated with a dehydrohalogenation agent to introduce two-double bonds in ring A at positions 1,2 and 4,5, obtaining IIb, 'IIIb, (21-ester or 11ss,
21-diester) or VIIb (sheet 1). If desired, then the resulting diene ester can be hydrolyzed in known manner with an acid or a base to produce the primary alcohol of the dienes (IIa, IIIa or VIIa).



   It is preferred to employ bromine as the halogenating agent and this modification of the invention will therefore be described later in connection with the use of this halogen. Through

  <Desc / Clms Page number 37>

 
 EMI37.1
 reaction with brone, the 2,4-dibromides of the 21-esters of pret; nane-17 x, 21-diol-3,11,20-trione and pregnant-. 1,17, 21-t-riol are obtained. -3.2C-dione. It is preferred to carry out the dehydrobromination by boiling under reflux with collidine. after which the 1,4-diene 21-ester is separated by chromatography or otherwise. Instead of collidine, other high boiling organic bases such as auinaldine and dimethylaniline can be employed.
 EMI37.2
 



  -this method is applicable both to the allo-pregnan series and to the normal series. Thus, the dibromination of 21-acetate of allo-pre; nane-17-, 21-diol ->, 11,20-trione and of 21-acetate of allo-prégnane-11 3,17c, 21-triol- 3,20-dione provides the corresponding bromides (IIIg and III'g) which, on dehydrobromuration, give the 21-acetates of dienes II and III, respectively.



   You can also prepare dienes in the same way
 EMI37.3
 of llà-hydroxylated steroids. The dibromination of the 21-acetate of pre t; nane-110! , 17 C, 21-triol-, 2 C-dione. gives the corresponding 2,4-dibromide (IVg) which, by dehydrobromuration, gives the 21-acetate of 1,1q-pre; nadiene-11,17 (, 21-triol-3, 20-dione (VIIb). Oxidation of VIIb (R = acetyl) gives IIb in good yield.



    Hydrolysis of IIb gives IIa.



   The reactions involved in this chemical process are evidenced by the following equations:
 EMI37.4
 
 <tb> (1) <SEP> 21-ester <SEP> of <SEP> 21-ester <SEP> of
 <tb>
 
 EMI37.5
 pr nane-17-, 1, 4-prenadi: ne 21-diol-5, 11,20- 2 2,4-dibromo -17p (, 21-diol- IIa trione compound 3, 11,20-trione
 EMI37.6
 
 <tb> Ig <SEP> IIb
 <tb>
 <tb> (2) <SEP> 21-ester <SEP> of <SEP> 21-ester <SEP> of <SEP> 1,4-preg-
 <tb>
 
 EMI37.7
 prgn..ne-11 3, '2 4- <ii LJromo nadiÈ; ne-llj. 3, 17 et, IIIeI 17,1-triol- r2 2,4-dibroDo 21-triol- IIIa 5, 2C-dione corxnose 3,20-worthy
 EMI37.8
 
 <tb> They <SEP> IIIb
 <tb>
 
 EMI37.9
 1-e; ter due 2, <% - to. he;

   ro;, io- C'11.10 - '.' g ', rîi: .2lG -'- ¯¯¯¯ IIb 7¯'î x, 21- iol->, 11, G-
 EMI37.10
 
 <tb> trione
 <tb>
 <tb> IIIg
 <tb>
 

  <Desc / Clms Page number 38>

 
 EMI38.1
 
 <tb> (4) <SEP> 21-ester <SEP> dice
 <tb>
 
 EMI38.2
 ; ::, 4-dibrol.l0-, llo-ir: Zaxx- ¯¯¯¯¯ IIIb 113,17,21-trioTI
 EMI38.3
 
 <tb> 3,20-dione
 <tb> III'g '
 <tb>
 <tb> (5) <SEP> 21-ester <SEP> of <SEP> 21-ester <SEP> of
 <tb>
 
 EMI38.4
 2,4-dibromo 1,4-prégn.diene ç prÉ; Jüane-11C, l1x, 17oc, 21-triol- 3. IIb IIa
 EMI38.5
 
 <tb> 17,21-triol- <SEP> 3,20-dione
 <tb>
 <tb> 3,20-dione
 <tb>
 <tb> IVg <SEP> VIIb
 <tb>
 
The new intermediates obtained by this chemical process are represented by the formula XI (sheet 2) in
 EMI38.6
 where X is = 0, t / OH or '.01, and R is H or a lower alloying radical.

   Pregnane nucleus can have either normal or allo configuration.
 EMI38.7
 



  The bromuratiort of the starting compound, which is pref8rcn,:; r. a 21-ester, is made in acetic acid or other substantially anhydrous organic solvent which is inert to bromine.



  It is not necessary to purify said bromide thus formed before the reaction with the dehydobromiding agent, which, as has already been said, is preferably a high boiling organic base, the collidine being in. generally the one that suits best. If desired, the resulting doubly unsaturated ester can be hydrolyzed with an alkali metal base, for example alkali metal carbonates, bicarbonates and hydroxides, in known manner in a suitable neutral solvent such as than methyl and ethyl alcohols.



   The 11 [alpha] and 11ss hydroxyls can be oxidized to keto oxygen either before or after hydrolysis. Although a variety of oxidizing agents can be used, chromic acid has been found to be an effective and inexpensive agent for this purpose.



   We showed more. high that we can introduce the double
 EMI38.8
 1,2 bond in 4-pregnene-3-ones having a hydroxyl group elsewhere in the molecule, by treatment with a desJio microorganism, drojé = 1ar; t. In accordance with a new feature of

  <Desc / Clms Page number 39>

 
 EMI39.1
 the 'ol'c, n., c: iaE.' invention, such a 1,2 double bond can also be chemically introduced into unsaturated pregnenes;

   in addition, the 4,5 double bond can also be introduced by purely chemical reactions into 3-keto-pregnens in which the double bond is located in position 1,2.,
In order to carry out the introduction of the 1,2 double bond in 4-pregnene-3-ones, it is possible to start from 6-bromo-4-pregnene-
 EMI39.2
 17 x, 21-diol-3,11,20-trione available (Kendall et al., J.



  Diol. Chem., 197, 261 (1952)) and 6-brorao-4-pregnene-11,3,17 -> (, 21-triol-3,20-diol 11-formate 21-acetate, ie (resulting from the Kendall oromuration of 4-prene-11 3,17 (, 21-triol-3,20-dione) 11-formate 21-acetate which is treated with an alkali metal salt of a lower alkro10ic acid, preferably sodium acetate or potassium acetate. In this way the respective 2-alkanoates are prepared. Mild hydrolysis with aqueous or alcoholic acids or bases can be employed to convert the 2-alkanoates. Dehydration by eliminating 2-hydroxyl simultaneously with hydrogen from carbon atom 1 produces the introduction of the 1,2 double bond.

   Participating reactions are shown in the following sets of equations:
 EMI39.3
 
 <tb> (1) <SEP> 21-ester <SEP> of <SEP> 2,21-ester <SEP> of
 <tb>
 
 EMI39.4
 6-bromo-4-pregnene-4-pregnene-2,17 ,, \ /, 17-n, 21-diol-3elle2O- 21-triol-3,11,20- ¯¯¯¯¯
 EMI39.5
 
 <tb> trione <SEP> triol
 <tb> Ih <SEP> IIh
 <tb>
 
 EMI39.6
 ° -pre; nene-2, 17c, 21-triol-3,11,20-trione IIa
 EMI39.7
 
 <tb> IIIh <SEP> @
 <tb>
 
 EMI39.8
 (2) 11-forKiia-be 21- 11-formate 21- allnanoate of 6-bromo-4-pregnene-, 4-pregnene-2,11'3, 113,17,21-triol-17a, 21- ttrol- ###### 5,20-dione 3,20-dione I iTh Vh 4-: d ^: nne-2, 113, 17x, Î-tc: trol- 5, 2C-dione. ¯¯¯¯¯¯ IIIa screw ######

  <Desc / Clms Page number 40>

 
 EMI40.1
 -bas structural formulas IIIh and VIh are indicated in XII and XIII in sheet 2.

   In these formulas, R is hydrogen or a lower alkanoic acid radical. In the
 EMI40.2
 Formula XIII, ie group 11 3 -OTl can be replaced by 08.



   Instead of dehydrating compound IIIh or VIh, the 2,21-diesters can be subjected to the action of the dehydrating agent.



  Thus, the 2,21-diacetate of compound IIIh can be treated with hydrobromic acid in acetic acid to have the
 EMI40.3
 1-Dehydrocortisone 21-acetate.



  The 1,2 double bond can also be introduced by the
 EMI40.4
 route of a hydroxy or acyloxy substituent on carbon 1, for example the 21-esters of cdrtisone and hydrocortisone. The structure of these compounds is shown in XIV (sheet 2); they can be prepared from by the sequence sul @@ ante
 EMI40.5
 of reactions, X representing = 0 or Ii- 'H', and Y representing OH or Oacyl:
 EMI40.6
 2l = ester of '1>) 21-extlr of 1-pregncne-llX-' tl, 2 "-epo, xy-llX-. ' 17Jj2i-diol-1 ′ 1 pregnan-17, '21 - ...



  '>', 20.-diQ.e 1, diolT, 20-dionë 11. .1 <'). i, j 1111 '' 21-estt'e te ,,, 21-ester of pretuan, e-2-bromo-préghane- 'llX-l, 17,21-triol-11X-.l 17t, '21 - triol- \, 20-d.ane ¯¯¯¯¯¯¯ 3, 20-d; ione, -, '; , 1111 '' - 'e, IVi
 EMI40.7
 
 <tb> 1,21-diester <SEP> of <SEP> 1,21-diester <SEP> of
 <tb>
 <tb>
 <tb>
 <tb>
 <tb>
 <tb> pregnane-llX- <SEP> 4-bromo-pregnan
 <tb>
 
 EMI40.8
 1,17,21-triol -.1X-1,17 x, 21- 3,20-dione ¯¯¯¯¯¯¯ triol-3,20-dione ¯¯¯¯¯¯¯
 EMI40.9
 
 <tb> Vi <SEP> VIi
 <tb>
 <tb> 1,21-diester <SEP> of
 <tb>
 
 EMI40.10
 4-pret1nellle-llX-
 EMI40.11
 
 <tb> 1.17x, 21-triol-
 <tb>
 <tb>
 <tb>
 <tb> 3,20-dione <SEP> # <SEP> II <SEP> or <SEP> III
 <tb>
 <tb>
 <tb> VIIi
 <tb>
 
The ester groups in the above equations are preferably those of lower alkanoic radicals.

   For example,
 EMI40.12
 in the conversion of 1-preGene-17,2l-diol-3,11 21-acetate,

  <Desc / Clms Page number 41>

 
 EMI41.1
 1 # ± -.1; r.ioiie (Mattox et vendait, J.13.G., 1 ,. ', 287 (1951)) in 4- p: cn' xze-1, '1 lx, 21-triol -3,11,20-throne and 1,2l-diacetate corresponding, the raw material (Ii) is epoxidized by means of a (agents tt 'Elol>,' ,. TtI.G t10x1 well known such as perbelizolque acid, nonoperphthalic acid, peracetic acid, etc. Cn is preferably used in this operation perbenzoic acid
 EMI41.2
 or Dono acid: perphthalate in an inert medium such as chloroform or methylene chloride.

   The product of the epoxidation (IIi) is then treated with an anhydrous halogenated hydracid such as hydrobromic acid in chloroform, hydriodic acid in glacial acetic acid, etc. The resulting halohydrin (IIIi), preferably iodohydrin, but also bromhydrin, is then dehalogenated with Raney nickel in acetic acid, or with hydrogen and a hydrogen catalyst.
 EMI41.3
 palladium on calcium carbonate, in. Pregnan-1,17 ', 21-triü1-3,11,20-trione (IVi). The compound IVi is acylated with a lower alkanoic acid anhydride or chloride such as acetic anhydride, in pyridic solution, to obtain Vi.

   Alternatively, the compound Vi can be prepared by reversing the order of the last gentle steps. Bromination and dehydrobromination
 EMI41.4
 de Vi provides the desired 1,21-diacetate of 4-prenne-1,17,21-triol-5,11,20-trione (VIIi). The bromination is preferably carried out with bromine in the presence of sodium acetate to control the acidity. Dehydrobromuration is the most suitable
 EMI41.5
 nobly using zemi-carbaziàe. The resulting semi-carbon is converted to VIIi by the action of pyruvic acid.



   Analogously, IVi can be converted to 21-acetate
 EMI41.6
 of.-prc'nene-1,1'7x, 21-triol-3, 11, 20-trione (VIIIi) by omitting the operational phase of acylation. Brorination in the presence of sodium acetate followed by the same dehydrobromination process effects the desired transformation.



   The corresponding 113 -hydroxy compounds of

  <Desc / Clms Page number 42>

 
 EMI42.1
 la ', ê \ 1 \ e Starting from 21-acetate of I-pre.sni:.: ne-J¯lf3' i c (.



  - .¯w. vi-, 0-c.¯: clL3.



  On:, ë: 1.: J .; ¯; .ai:;: r. the soups VIIi, VIIIi and the compounds 1 ¯ / .3-hydr, xyl É, c.:::"::E.re::.1tÚ; with an acid or a base such as a.lu-mic, triton B (-. Dro -; ,,. A from tetr ethy 1-arr¯roniuu), 'etc, for :: éP21l "Gl' le 1 -s .¯scuC = ¯, '' '¯ obtaining the substances powerfully anti -arthritics II and III (sheet 1).



  G. j1J = -l; .s: ti tutl dēJo 'halogen? :: ne in position S 3 ..



   It is not necessary for the preparation of compounds of formulas IV and V that the starting compounds contain the atom
 EMI42.2
 of ¯aloGt: ne (fluorine, chlorine or bromine) in position 9. This substituent can be introduced into the steroid molecule after the
 EMI42.3
 dehydrogenation of nucleus A.

   The substitution is carried out starting from the compound 1 ('X' or (3) -hydroxy-1,4-pregnadiene and by converting to 1,4,9 (II) -pregnatri, Y, l.é. which can then be treated in various ways to introduce the desired halogen in the 9 [alpha] position and at the same time an 11/3-hydroxyl. The 11-hydroxyl can then be oxidized in a mild manner to replace it by
 EMI42.4
 ketone oxygen to form Se / -halo-1-dehydro-cortisone.



   It is preferred to use as a starting material either 1,4-
 EMI42.5
 prcrnadiene-111, 17 ot, 21-triol-3,2G-dione (III) or the corresponding 11 -11clrozcy compound (VII) or their 21-esters. The starting compound is esterified (IIIb) first in position 21 (if it is not already not a 21-ester) by treatment with an anhydride
 EMI42.6
 c'ae.iae in pyndine or using acid chloride, so as to produce by e: '3: -: 1ple a lower e: ter ¯llcanoyl such as 21 - ,, ket # fe or propionate , or any other suitable ester such as lc; -lo '' 3üJJ: rlr: ro :: - 'iol [', te, Ll-oenanthylate, etc.



  The dehydration to effect the introduction of the double bond Si, 11 this does cā: o :: ent in the case of C01-: lpOSb 'IIIb in:; "zÜr ..: a' :: 't act -the 0¯TC¯: lOrWrP of r; Oc :: lore danc of pyridinc.

  <Desc / Clms Page number 43>

 
 EMI43.1
 



  However, it is necessary to force the 1,., 9 (11) -trin by treating the 21-ester of llg-li> -1roJz3r-conposed (VIIb) with p- tO111È11v chloride - Sti.1fo22Vlg in de pyridine to produce the corresponding 11'x - tosylate (11-p-toluene-sulfonate) (Ij). By treating the latter compound with a basic compound such as sodium acetate in acetic acid (or with potassium acetate, lithium propionate, etc.), the eli - mination of p-toluenesulfonic acid with the introduction of the double bond 9 (11). Instead of p-toluenesulphonate, the corresponding methanesulphonate, benzene sulphonate or other similar sulphonic esters can be produced as an intermediate.

   These reactions are shown in the following diagram:
 EMI43.2
 
 <tb> anhydride <SEP> 21-acetate <SEP> of <SEP> 1.4.9
 <tb>
 
 EMI43.3
 aeë-binue pool (II) -pregnatriene- 1-dehydro- ace-Lque 21-acetate 3 l7rX21-diol - 3,2C- cortisol pyridine pyridin dione IIIa, pyx idine IIIb, IIj 1 ,, chloride 1,, 4- preena. p-tou- '11-p- diene1D <', 21-acetate o1: l1c: "sulfotoluene 17c (, 21-triol. Nyl .. .sulfonate' cl1 their 3,20..dione i," 21-ace'ate a0 II, V'II VIIb , ij 1 CH'CQOÈ3 3
 EMI43.4
 The. structure of 1,4,9 (II) -prenatrienes, of which compound IIj is an example shown in XV (sheet 2) where, R, is H or acyl, preferably lower alkanoyl.



   The triene (IIj) is then treated with a compound which provides a bromium ion, such as N-bromoacetamide or N-bromosuccinimide in perchloric acid or other strong acid such as
 EMI43.5
 acid: sUl; furic. The 9x -bromo-derivative 111j) is thus obtained. If it is desired to replace fluorine or chlorine in position 9- \, the 9 [alpha] -bromine-compound (bromhydrin IIIj) is dehalogenized by the action of a weak base such as acetate. potassium or sodium in a relatively neutral solvent such as acetone or alcohol, thereby producing epoxide IVj.

   The latter is subjected to the action of anhydrous hydrofluoric acid, following which

  <Desc / Clms Page number 44>

 the epoxy ring is open and a fluorine atom 9 [alpha] is introduced
 EMI44.1
 simultaneously produced with a 11-3V-droxyl group, obtaining the compound V (Z = F). Using anhydrous hydrochloric acid, the 9; -chlorinated analog is formed. The latter can then be hydrolyzed to separate the 21-acyl group and produce the 21-alcohol, for example by means of an aqueous acid.



   The 9 [alpha] -fluorinated compound can be subjected to a mild oxidation, for example with chromic acid. In pyridine at room temperature to replace the 11 (3-hydroxyl with an oxygen ketone, to have the compound IVb which can then be hydrolyzed with an acid to produce 9 [alpha] -fluoro-1,4-
 EMI44.2
 Pregnadiene-17r /, 21-diol-3,11,20-trione.



  It is preferable to use 1, ° -pregnad.ene-117 ° C, '21-triol-5', 20-dione rather than 1,3-hydroxy-compound 1 <., weighing as the starting material, this new 11-hydroxy-compound being obtained as described above. It is important, before treatment with p-toluenesulfonyl chloride, to selectively protect the 21-hydrbxyl before the 11 [alpha] - hydroxyl group is esterified with a sulfonic derivative. It has been found that failure to protect the 21-hydroxyl group against the action of a sulfonyl halide, for example p-toluene-sulfonyl chloride, results in non-rectifiable mixtures.
 EMI44.3
 



  Further, when esterifying the 21-position, it is preferred to employ a relatively mild acylating agent such as the anhydrides described above, and thus true selectivity is obtained and the 11-hydroxyl remains unesterified.



  H. Preparation of esters having an increased duration of activity.



   The invention also contemplates the preparation of compounds of formula II to V in the form of 21-esters which are not metabolized as rapidly in the body as the free 21-alcoholic compounds, if, although the physiological activity of the compounds is. maintained for a longer period of time and the

  <Desc / Clms Page number 45>

 injection rate is therefore considerably reduced.



   The acids whose esters have been found to have a longer duration of activity are cyclohexane-carboxylic acid, 4-ethylcyclohexane-carboxylic acid, 3-ethyl-cyclohexylacetic acid, cyclohexylpropionic acid, cyclopentylpropionic acid, phenylacetic acid, trimethylacetic acid, t.-butylacetic acid, butoxybutyric acid, aci-
 EMI45.1
 of ethoxycaproic acid, methylthibvaleric acid, isopropylthioacetic acid, phenylthioacetic acid, caproic acid, isobutyric acid, enanthylic acid, isocaprylic acid, cyclohexylcaproic acid, undecylenic acid
 EMI45.2
 2-Cthylbutyric acid, toluic acid and ethoxybeiizoic acid.



   In particular, it has been found that acids belonging to the aryloxyalkanoic and furoic acid groups give an exceptionally long duration of hormonal activity, thus providing a more effective, convenient and useful therapy than that obtainable. with related hormones.

   Typically interesting acids belonging to these groups are: phenoxyacetic acid, p-chlorophenoxyacetic acid,
 EMI45.3
 2, ° -dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 5-chlorofuric acid, 5-methylfuroic acid, 5-bromoi'uroic acid, 4-bromophenoxyacetic acid , 4-methylphenoxyacetic acid, 4-methoxyphenoxyacetic acid,
 EMI45.4
 4-t.-Butyl-phenoxyacetic acid, 5-t.-Butylfuroic acid, furoic acid and phenoxypropionic acid.



   It has been found that the esters of the acids mentioned above increase the duration of activity of not only 1-dehydrocortisone, 1-dehydrocortisol, dehydro-9-halo-cortisone.
 EMI45.5
 and 1-dellydro-g ,: -haloeeno-cortisol (the haloel atom: not being chlorine or bromine) but also. of physi0lo {; iq, ueJüent active compounds such as 1-delydrocorticostérone, 1- de- l: rîro-11-tlso:; rcortico "t; rane, 1-deydro-11-deoxycortisone,

  <Desc / Clms Page number 46>

 
 EMI46.1
 --ceilyti'v - s' -c'¯so because i.lSGi2e and 1-.ClrCl1'O-2¯ûOStÉrOlle.



   As examples of the increased duration of activity, it was found that in mice having undergone adrenalectonia, an injection
 EMI46.2
 0, L5 n, s of 1-dehydrocortisone or 1-dehydrocortisol or their acetates causes a lowering of the normal level of eosinophils
 EMI46.3
 wearing 2 to 4 days. A similar injection of 21- (2 ', 4'-diclilorophenoxy-acetate) 1-dehydrocortisone lasts 12 to 20 days. Its 21- (5'-bromofuroate) lasts 10 to 18 days, 1-de-hydroccrtisol 21-furoate lasts 10 to 16 days and its 21- (4'-t.-butylphenoxy-acetate) lasts 14 to 20 days .



   In another trial, an injection of 21- (5'-chlorofuroate)
 EMI46.4
 of 1-dehydrocortisone in rats caused the weight of the thymus gland to decrease for a period 5 to 8 times longer than in the case of the free hormone, while the 21- 4'-
 EMI46.5
 1-Dehydroçortisol t-butylphenoxyacetate causes thymus uvolution to be 4 to 6 times greater than in the case of the free hormone. These tests are indications, standards of glucocorticoid activity (ie anti-arthritic). Likewise, the mineralocorticoid activity of hormones is enhanced by ester-
 EMI46.6
 rification with these acids.

   Thus the 21- (4'-chlorophenoxyacetate) of 1-dehydro-11-deoxycorticosteronerhaint the life of mice having undergone adrenalectomy for a time 5 to 7 times greater compared to the free hormone, and the 21-furoate of 1- dehydroaldosterone maintains a life 6 to 10 times longer than in the case of the corresponding hormone.



   In human therapy, the same protection of the hormone against destruction is observed, with concomitant extension of the duration of the therapeutic level of activity. For example, one or two injections per week of 1-dehydrocortisone 21-furoate or
 EMI46.7
 21 - (.'- t. = Eutylplenoxyacetate) of 1-dehydrocortisol replace 7 daily medications with the free hormone. Similarly, injections of 50-100 mg of 21- (5'-t.-butylfuroate) of 1-dehydro-

  <Desc / Clms Page number 47>

 
 EMI47.1
 cortisol or 1-dehydroeortisone 21- (41-ohlorophenoxyacetate) have an activity of 7 to 10 days, compared to an efficacy of 1 day for the free hormone.



   Specific examples of the 21-esters of 9 [alpha] -halogen-derived 1-dehydrocortisone and 1-dehydrocortisol are 21-
 EMI47.2
 (2'-furoate), 21 - (51-t.-butyl) -21-furoate, 21-phenoxy-acetate and 21- (2 ', 4', 5'-trichlorophenoxyacetate).



   The esters can be administered as is usual with cortical hormones. Thus, it can be made into tablet form compositions with an inert material for oral or sublingual use. Another commonly used form is an ointment or lotion for topical use. In general the most effective mode of administration is by injection of solutions or suspensions in oil or aqueous media.
 EMI47.3
 intramuscularly, subcutaneously or intraarticularly ,.



   The esters can be prepared in various ways, the simplest being the esterification of the corresponding free hormones.
 EMI47.4
 tes, for example by treating the hydroxysteroid with the corresponding acid anhydride or chloride, in the presence or absence of a base, or by treating the hydroxysteroid with the free acid in the presence of an acid catalyst under dehydrating conditions.



   Esters can also be prepared by esterifying intermediates in the preparation of hormones, using these esters rather than acetate as the pro- moieties.
 EMI47.5
 tectors. Thus, in the preparation of 1-dehydrocortisone and 1-dehydrocortisol by the chemical processes described above, the 21-ester of one of the deposit and reserve producing acids mentioned above can be formed intermediately, ev, nt introduction of 1 ", alo-'ne i ti (usually bromine) in position 2 and -F, this process is conveniently carried out by reacting the;" '1-ùr0L10-cO :: ll, OS8 from uteroid with metal salt

  <Desc / Clms Page number 48>

 
 EMI48.1
 J. t: ..- :. ci ": - 3. 4 '..' ..: on.tr3 this in 1 '# iqu-ztà, oii consesuta Û.e, 11.8 1.G' .. C 4îC :. She:

   it is understood that the COG! JOf: é 'D <'-; 11 ±: "lC de Géjxatf can have the configuration 110 1' :: 113. La or hello:
 EMI48.2
 21-brcno- 21-ester de 21-estor àe prt: [: l18.ne- pl'8 ± 113.ne-l1X- 1, -i - 4s1 4 'v-ic "llsi-17 -ol- 17 , 21-cli01- ïl¯?: - ¯Z: .-: 5, 20-dione: 5, 20-dione?,; .- ctira: a- 17x,.-C: .ial- àéi% ivé 3 , 20-dione Cl8ll ve,. '- (,. lone
In this equation, X represents O or H, OH, while the ester is that of any of the acids described above which increase the activity time of the diene.



   In the following examples, detailed methods for carrying out the various forms are presented by way of explanation: the carrying out of the invention, and the dehy-
 EMI48.3
 droenation of the nucleus' A using microorganisms.



  T'ye "" lJ2le 1 conversion of compound 1. into a 1, ° -r r ¯no, dine-lnc '21-diol-3,11,20-trione.



   From a solution of 30 g of yeast extract (Difco) in 3.0 liters of tap water containing 13.2 g of phos-
 EMI48.4
 plmte monopotassic acid and μ6.4 g. of disodium acid phosphate (pU of solution: 6.9), 27 portions of 100 cm3 each are taken, and they are placed in 300 cm3 Erlenmeyer flasks. and sterilized in an autoclave for 15 minutes with a steam pressure of 6.795 kg (120 ° C.).

   After autoclaving and cooling the broth, 1 cm3 of a suspension of
 EMI48.5
 arS-neb; .cterilua simplex (A.T.C.C. 6946) in each flask, an then shakes the flasks on a shaking table at 220 revolutions per minute, at 28 C for 24 hours.



   In each of the 27 Erlenmeyer flasks are placed 150 mg of Mendall's compound E. The vials and their contents are then sterilized for 15 minutes at a water vapor pressure of.
 EMI48.6
 . , 79 1 ;; -; (l2Qo :;). To each vial is then added 5.0 cr: 1J of etrha-Rol. C'n t :: ans ± era. :: l1B1Ü ts asepti :: ue ::: r;: summer bacterial culture

  <Desc / Clms Page number 49>

 
 EMI49.1
 '- vs; : ..., hours and the resulting suspensions are added over Ü, 1Ùa seCOUSS-3i.- at 220 revolutions per minute, at 20 ° C. for .C hours. The final pH is 7.2.



   The contents of all vials are combined and extracted with a total of 9.0 liters of chloroform in three rack portions. The combined extracts are then concentrated to a residue which is crystallized from acetone-hexane. We obtain
 EMI49.2
 1.1 g of 1,4-prenadiene-17cX, 21-diol-3,11,20-trione, m.p. 210-215 0 (dec.).



  Several additional recrystallizations raise the mp to 230-232 C (dec.);
 EMI49.3
 LQ (¯75 + 175.3 (dioxane); C :::: 238.15.400 (methanol).



  Anal .: hold. for C21Ii2G05. This 70.37; H, 7.31. Found: Ce 70.3C; II, 7.67.



  21-acetlation of 1,4-reatiZne-17C. (, 21-diol-3,1120 - l, lione.



  Has a solution of 0.5; 1,4-Pregnadiene-17o (, 21-diol-j, 11,20-trione in 5 cm3 of anhydrous pyridine is added 3 c3 of acetic anhydride. The reaction mixture is left to stand overnight at room temperature, then diluted with ice and water The resulting precipitate is filtered off and recrystallized from acetone-hexane.
 EMI49.4
 0.35 g 1,1-prenadiene-17,21-diol-3,11,2C-21-acetate, m.p. 227-228 C, (dec.). After several recrystallizations from acetone-hexane, it melts at 233-236 C (dec.).
 EMI49.5
 



  : 0 :: - :: 611] 116 "2. Conversion of colipos6 P to 1,4-iJresnadiene-11 / 3,17ex ', 21-triol-3,20-dione.



   As described in Example 1, 150 mg of compound are treated
 EMI49.6
 Kendall's F in each of z7 vials i; rlenmeer. The fold at the end of the shaking period is 7.0.



   Combine the contents of all the vials and extract with a total of 9. C liters of chloroform in three portions

  <Desc / Clms Page number 50>

 
 EMI50.1
 es 422 then concentrates the CO :: J7..il :: extracts; to 1 residue which p2.se 5.75. The ±, h ". Of the residue, is 22 (-252 V. from, the, 2,75, c: of this r: atiÈ: re raw by triturating with 50 CE13 (1 '", - c--: to "1G and cooling, recovered by filtration 1, # prß22C.C11.G11.e-11j, 17,21.-triol-5,2C-Ct10i2e, P.:;". 237- 239 8 (tlt ': c.). Additional product can be recovered from the mother liquor. Recrystallization from acetone
 EMI50.2
 raised the p. p. at 239-2410C (dec.).



     [[alpha]] D25 + 107 (dioxane); # 243 14,600 (methanol).



  Anal .: calc. for C21H28O5: C, 69.97, H, 7.83. Found: C, 70.24, H, 8.13.
 EMI50.3
 



  21-acetylation of 1, 4-jrr '; xladiene-11 3.17 21-tri01- 3.20-therefore.



  To a solution of G, 35 µ of 1,4-prenadiene-11 3,17 :, ¯¯ triol-3,20-dione in 5 cm3 of pyridine, 3 cm3 of acetic anhydride is added. The reaction mixture is allowed to stand! room temperature overnight, then diluted with
 EMI50.4
 1. "Ice-water. The. Resulting precipitate is separated from the mixture by the. . filtratio'n and recrystallp-pe from acetone-hexane.



  We recover G, Y4; 5. 1,4-preg.adiene-5.1r, 17t, 21-triol-3,20-dione, .P. 5-23g C. On recrystallization, the P.P. amounts to 4t 2.3 7-2 59%. 25 +: q.6 ',' (dioxane) ;, E 245 - - ...



  15,000 (methanol). Anal ..: calc. for G 23 I 30 G. G G,, 68.63, Ile 7.51 Found: C, 68.62, H, 7.78.



    Example 3.
 EMI50.5
 



  Conversion of compound S to 14-rË, nadiene-17C 21-diol-3,20-dione.



   As previously, 100 cm3 of a 1.0% yeast extract concentrate, comprising 9.0 cm3 of 0.2 I.I KH2PO4, are sterilized.
 EMI50.6
 and, G cc; 5 of 0.2 h ITa2FIFG4 and inoculated with a 1.0% suspension of Corynebacterium simplex (A.2.0.C. 6946) from a 24 hour broth culture. Cn incubate the culture

  <Desc / Clms Page number 51>

 
 EMI51.1
 1101Jve}, '1, e: lIlc;: lt C: "; C.1O: 1C ..] C and stirred (table li i.iC3CCll, .l3pC; 110n- Û 2r 12 -' 20 hours a 21 = J. After incubation, we i: 1.'i:;, n 1: 7 'C3: ,,: c <31. "Ticll10- ment lc. Culture in broth in a sterile sterile sterile E.'1C1-- Gtil1 sodium hydroxide flask, containing 150.0 µl i16 'cci-.1-, t) os J. (-? rice: .i2e-17,21-diol-, C-dioil) in 5.02 / 11.30 etanol or acetone.

   The pi-I of the reaction medium is 7.0.



  The bacterial culture containing the steroid and the solvent is
 EMI51.2
 i-: ise to incubate and stirred for a period of 48 hours: 28-IC. The final fold of the reaction mixture is 7.2-7.4. The culture is then extracted thoroughly with chloroform. The extracts are combined and concentrated to dryness on the steam bath. The raw extract weighs 196.0 mg.



   The total crude extract is triturated with methanol and 80 mg of crystalline solid are obtained, m.p. 245-250 C. After two
 EMI51.3
 recrystallizations from acetone, B.l '.. ext from 2t.-24 C, oec., L c' ¯75 + 76 (Cacl - 3) 9 = 245 15. penny (C2II5CII).



  Hold. for C21H28O4: C, 73.22, II, 8.19. Found: C, 73.56, II, 8.40. Infrared spectral analysis and chemical analysis
 EMI51.4
 establish that the product is 1,4-prenadine-17X, 21-diol-j, 20- (liÓne.



  21-r.LCUt lation of 1 4 r; nadine-1.7t 21-diol-5,2C-dione.



  To a solution of 0.25 g of 1,4-preL; nadine-17,21-diol-3,20-diode in 2 cm3 of pyridine is added 1 cm3 of acutic anhydride. The reacted mixture is allowed to stand at ordinary temperature overnight, then diluted with ice and water. The resulting precipitate is filtered off and
 EMI51.5
 recz'i; .v :: llise from methylene-hexane chloride, obtaining C, 20 5 of 21-acetate of 1,1 - pr.nadicne-l7qy21-c.iol-5,20-dione, 1 '.?. 6.5-L2C C.



  ± xen, the 4.



  '? 2.0: ± rE: .. i0l'l 11u 3 o ::' o s C in 1 - nr ne, dzene-17 2: .J-dio1- 3, 2'0- lic- = <; and 1 ...?. '. 1. ,, - n.w-1 i A'21-triol3 one.

  <Desc / Clms Page number 52>

 
 EMI52.1
 . "'I' t41 -: r m
100 cm3 of medium are sterilized as described above. consisting of 1% soluble fish products, 0.1% ex-
 EMI52.2
 line of yeast, 9.0 cm3 of 0.2 LI I (H2PO4 and 9.0 cm3 C, 2% Ia2: 04 and inoculated with a suspension of Corynebacterium simplex from 24 hour broth culture. incubate the newly seeded culture and shake (shaking table) for 20 hours at 28 C. After incubation, the broth culture is aseptically transferred to a second sterile 300 cm3 rlenmeyer flask, containing 150 mg of compound. S sterile in 5.0 cc of ethanol The pH of the reaction mixture is 6.9.

   The bacterial culture containing the steroid and the solvent is incubated and stirred for 48 hours at 28 ° C. The final pH is 7.3. The culture is extracted with 2 liters of chloroform in 5 equal portions, the extracts are combined, and the whole is concentrated on a steam bath.



   The crude residue is taken up in methylene chloride and chromatographed on Florisil. We isolate from the chromatogram
 EMI52.3
 starting material (15 mg), 1,4-prcg'nadiene-17X, 21-diol-3,20-dione (30 mg) and 1,4-pregnadiene-171X ', 20,21 - triol-3-one (90 mg) which, recrystallized from acetone-hexa-, melts at 195-196 C, [[alpha]] D25 + 33 (methanol). Anal .: hold. for C21H30O4: C, 72.80, H, 8.73. Found: 0.72.79, H, 9.08.



   Instead of ethanol, other water-soluble organic solvents which are not toxic to the microorganism can be used, for example acetone, mixtures of ethanol and acetone, etc.



    Example 5.



  Reaction of 5-pregnene-3 3,20-diol.



   100 cm3 of 0.1% yeast extract concentrate comprising
 EMI52.4
 C-1 1.0 cm3 of 0.2LI I # I2P04 and 9.0 cm3 of 0.2il Ha211P04 are autoclaved in a 300 cm3 Erlenmeyer flask. After an autoclave for 15 minutes at 6.795 kg (120 C), the mixture is allowed to cool.

  <Desc / Clms Page number 53>

 flask at room temperature. We then inoculate the vial
 EMI53.1
 with a suspension of Coryneoacterium simplex. The seeded flask is incubated and shaken (shaking table) for 24 hours at 28 C.



   A second 300 cm3 Erlenmeyer flask is sterilized. eon-
 EMI53.2
 .holding 150 mg of 5-pregnene-3A, 20-diol, in an autoclave for 15 minutes at 6.795 kg (120 ° C). To this flask is then added 5.0 cc of acetone or ethanol to dissolve the steroid.
 EMI53.3
 The 24 hour Corynebacterium siEJ21ex culture was transferred aseptically to the steroid vial and the reaction mixture was stirred (on a shaking table) for
36 hours at 28 ° C. At the end of the transformation period, the pH is 7.1-7.2.



   The reaction mixture was then extracted thoroughly with chloroform, the chloroform extracts were combined and the resulting solution concentrated to a residue (0.20 g). The crude extract is crystallized from ether in the form of elongated prisms melting at 135-138 ° C. Two crystallizations from methylene chloride-hexane afford 0.06 g of.
 EMI53.4
 1,4-pre; nadiene-3,20-dione, PF 152-153 0, -c (5 + 122 (CIIC13) 'é 245 15,000 (C2II50H). The infrared spectrum indicates the presence of a L1'4 system -clien-3-one, another carbonyl (6 member ring or side chain) and the complete absence of hydroxyl, example 6.
 EMI53.5
 ls = .- x:; nadi: ne-17 '(, 21-d.iol-3,20-dione.



  100 em3 of culture broth containing a concentration of yeast extract of 0.1; #, 9.0 cm3 of 0.2i, 1 KII2P04 and 9.0 cm3 of 0.2â ITa2I # O 4 ', are inoculated with 1 cm3 of a z hour culture broth of Cor7nebactet'iur.l simplex. The: Gc.con 21 is incubated at 28 C for 24 hours.



  150 cm3 of 3.21-

  <Desc / Clms Page number 54>

 
 EMI54.1
 dJacea.t de 5'-p'eg6.r'e-3/3, le /, lt.'ic-20-ùu nL. & ri.le dauu 5C c -: l "'actono, it: : i: has an iole i 12i1: 16 '; i ¯.3 300 ci.15, ei a iaand incubate for 41 hours at a temperature of 25 to .3o'J; J.



  The products are extracted with C1210'0 orr .., cLi1c. <a.tr?:; chloroform extracts to low volume and ohrouatography on Florisil. The order of elution is first and foremost unreacted starting material (75 mg), then 21-acetate of compound S (15 mag) and finally 1,4-precgradiune-17 W, 21-diol-3,20-dione (30 mg). All products are identified by comparison of their infrared spectrum with those of genuine samples.



   By using the same reaction medium and the same organism as in Example 6, and with the same proportion of substrate
 EMI54.2
 steroid, 4-pregnene-11, 2.-diol-3,20-dior- (cortico; ster, one) is converted into 1,4-pregnadiene-11, -3,21-diol-3,20: - crystalline dici.e. Likewise, 4-pregnene-21-01-3,20-dione '(de-soxycorticosterone)' provides 1,4-pr4gnadiene-21-ol-5,20-dione as a solid. crystalline, while 4-pregnadiene-2.-01-3,11,20-trione (11-dehydrooortiéo'sterone) is transformed into'1,4 # pregnadiene-21-01-3,11,20-trio 'not crystalline.



  As regards the acetylation of the dienes described in Examples 1, 2 and 3, it is evident that it is possible to prepare likewise other esters of the diene compounds, for example by reaction with anhydride of the acid or with its chloride in a known manner, for example formates, propionates, butyrates and valerate, as well as esters of other non-toxic acids such as benzoates, and also neutral esters and acids of polybasic acids such as succinic, maleic, malic, citric, tartaric, phthalic and hexahydrophthalic acids.

   In the case of acid esters, the metal salts can be formed in the usual way by reaction with the hydroxide, carbonate or bicarbonate of the metal, for example

  <Desc / Clms Page number 55>

 example of alkali and alkaline earth metals.



   Instead of forming the 1,4-dienes of cortisone and hydrocortisone and then esterifying the products, the corresponding esters of cortisone and hydrocortisone and their intermediate compounds can be subjected to the process of the present invention and made in so that they provide the dienes of the esters.



  Thus, in Example 1, the compound E can be replaced by its
 EMI55.1
 21-ester or by its 17G, 21-diester, or by 21-acetate, 17, 21-diacetate, 3,17C, 21-triacetate or other esters of 5-prégnc- ne-3,17, 21-triol- 11,20-dione, while in Example 2, compound F can be replaced by its 21-acetate, 17r, 21-diacetate or 11 ', 17oC. , 21-triacetate, or by 3,21-diacetate, 3,17, 21-triacetate or 3, 11, 17 cc, 21-ôetracetate of 5-pregnene-3, 11 J, 17, 21-tetrol-20 -one. The last group of d-compounds can be replaced by the corresponding 11'c-hydroxy-epimers, to have the 11-epimers of the diene of compound F and its 21-esters and
 EMI55.2
 17c, 21-diesters. In all cases, the polyesters can be mixed esters such as 3-propionate-21 = acetate.



   For example, by treatment as described for converting compound F to the corresponding diene (Example 2), the acetate of compound F can be converted to 1,4-diene or its 21-acetate, and the compound is isolated. produced by extraction with chloroform and crystallization from acetone. From 1.0 g of 21-acetate of compound F, 0.22 g of 1-dehydrocortisol is obtained
 EMI55.3
 (IIIa) m.p. 239-2415 C (dec.).



   When it is desired to suppress the deacetylation reaction, the same conditions as above are used, except that the ambient temperature for the growth and reaction phases of the process is raised to 36 C. The product is isolated in the usual manner. .

   From 1.0 g of 21-acetate of compound F, 0.13 -, 1-dehydrocortisol 21-acetate (IIIb), P.F.
 EMI55.4
 257-251 1 (d ')

  <Desc / Clms Page number 56>

 
 EMI56.1
 : J, :: ".: 308, following the procedure of Exeuldle 1, Cte 21-acetate of CO 1 -sone is converted to 1-delxydrocortisone, from which 0.17, r .. 230-252 J If we raise the open temperature for the growth and reaction phases of the process to 36 C, then from 1.0 g of cortisone acetate we get
 EMI56.2
 holds C, 11 g of 1-dehydrocortisone 21-acetate, P.P. 2: r0-2> d (dec.).



  * Example 7.
 EMI56.3
 Conversion of commode F into 1-dehvdrocortisol.



   From a solution of 1 g of yeast extract (Difco) in 1.0 liter of tap water containing 4.4 g of monopotassium acid phosphate and 8.8 g of disodium acid phosphate (pH of the solution: 6.9), 10 portions of 100 cm3 are taken
 EMI56.4
 each which is placed in 5F cm3 Brienmeyer flasks and sterilized in an autoclave for 15 minutes under a pressure of 6.795 kg of water vapor (120 C). After autoclaving and cooling the broth, 1 cm3 of a suspension is placed in each flask.
 EMI56.5
 pension of aorynebacteriilm hoai {(pmerican Type Culture Collec- -Lion 7005). The vials were then stirred on a shaking table at 220 rpm, 28 ° C for 16 1/2 hours.



   In each of the 10 Erlenmeyer flasks are aseptically added 50 mg of Kendall's compound F dissolved in 1 cm3 of
 EMI56.6
 i.utlxanol at 90;:. Replace the vials on the shaker and incubate for 7 hours. The pII at the end of the shaking period is 6.82.



   The contents of all the vials were 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 provides 248 mg
 EMI56.7
 of 1, =; - rregua.èae-11, 17-, 21-triol-3,20-dione.



  Ex.E1¯Û- :.



  EXemflg 8 C0r1..I ± J ':: icl1 .li: cO:.:' '\ Ooé' 2 in 1 - .. IÉI2-Wroco-rtisol.

  <Desc / Clms Page number 57>

 
 EMI57.1
 



  From a solution of t), 5 g of Basamin 8uech (ànheuser-, Busch) in 1 liter of tap water, 10 portions are taken !? of 100 cm3 each, they are placed in Erlenmeyer flasks of
300 cm3 and sterilized by autoclaving for 15 minutes under a steam pressure of 6.795 kg (120 ° C). After autoclaving and cooling the broth, 1 cc of a suspension of Corynebacterium simplex is added to each flask. The vials are then shaken on a shaking table at 220 revolutions per minute at 28 ° C. for 24 hours.



   To each of the 10 Erlenmeyer flasks are aseptically added 50 mg of Kendall's compound F dissolved in 0.8 cm 3 of absolute methanol. The vials are returned to the shaker and incubated for a further 4-7 hours. The pH at the end of the shaking period is 7.2-7.6.



   The contents of all the vials were combined and the whole was extracted with a total of 3 liters of chloroform in three equal portions: The combined extracts were concentrated to a residue of 490 mg. Crystallization of the residue from acetone furnace
 EMI57.2
 nit 403 mg of 14-prégradiene-11,170 (, 2l-triol-3,20-dione, p.r.28-24o c (dec.), 25 + 103o (dioxane) 243 14-500 (methanol).



   By using the compound instead of compound F in this example and increasing the conversion time from 6 to 12 hours crude dehydrocortisone is obtained with a yield of 85%.



   Example 8 shows that the yield is increased with a reduction in the concentration of the starting compound.



   The other 4-pregnene compounds disclosed above can likewise be treated according to the methods of Example 8. As with the other examples mentioned above, the 21-esters can be used as starting compounds as well as the compounds.
 EMI57.3
 5-Pregnene-3-hydroxy analogs.

  <Desc / Clms Page number 58>

 



  Example 9.
 EMI58.1
 



  Caw ": -, - ¯cW1 i '! U. Cü¯: = 10..yt: 3iYjL - elr.'drocoytijone.



  Cn ... and incubate the r.acillus¯ser: rcu j = sg. ïil = t¯C1Y'I97i: (A.'2.0.0. 7055) on a nutrient agar consisting of Lacto-beef extract, 3 2acfo-pepto = 1e, 5 g, sodium chloride, 8 2, garl 15 water dispensing, 1 liter, for 24 hours 28 C.
 EMI58.2
 z1 100 of a sterile nutrient broth (compound el'e =: trii-1 Dacto-beef, 3 g, Bacto-peptone, 5 ii and complete to 1 liter with tap water) in a 300 cm3 flask , a spoonful of the incubated culture is added and the culture medium is reincubated for 24 hours at 28 ° C. on a shaking machine under aerobic conditions. The culture broth thus obtained is used as a 1% inoculum. .



   In each of 10 flasks containing 100 cm3 of broth
 EMI58.3
 sterile nutrient is added 1 cm3 of iloculum. 1 μl was stirred on a rotary shaker under aerobic conditions for 8 hours at 28 ° C. and 240 layers per minute. After this period of growth, aseptically added to each vial a solution of 25 mg of cortisone in 0.5 cm3 of methanol; we asylum again and incubate for an additional 24 hours.



  The final pH is close to 7.8. The contents of the vials are removed, extracted with chloroform and treated in the usual manner, obtaining 1-dehydrocortisone.



   Instead of compound E, the material of
 EMI58.4
 start of 5 - prégncne-3, 17 oc; 21-txi: .l-11, 20-dione, of the 5- pre. 'Ilene-3,17? , 20,21-tetrol-11-one 0- their 3-acetates or other esters which are not toxic to the microorganism o @
 EMI58.5
 which do not inhibit its enz, -ne; of mr. 2, instead of compound P 1, 5-prenene-3,11317a (, 21-tetrol-20-one, 5-preYnGne-3,11.3, 17 (, 20,21-pentol o- 3-Acetates or Other Esters As indicated above, the 20-hydroxy group will be oxidized to a ketone group.

  <Desc / Clms Page number 59>

 



  Example 10.



   Conversion of compound P to 1-dehydrocortisol.



   A growth medium is prepared from 30 8 of soluble condensed fish products (treated with enzymes),
4.49 g of KH2PO4 and 8.83 g of Na2HPO4.7 H2O, the whole being diluted to 1 liter with tap water. The pH of this medium is close to 6.8. In each of 10 flasks of 300 cm3 we place
100 cm3 of the growth medium and the vials and their contents are autoclaved for 15 minutes under a steam pressure of 6.795 kg. To each cooled flask is then added 1 cm3 of the inoculum described in Example 9 and the growth is allowed to continue for 12 hours. To each vial is then added aseptically 25 mg of hydrocortisone and the aerobic incubation is continued for an additional 24 hours.

   The fermentation mixture is then treated as described above for the recovery of 1-dehydrocortisol.



   The methods of Examples 9 and 10 can be modified by employing as the nutrient medium a 1% yeast extract (Dif-co) in tap water. 'Example 11.



  Conversion of 4,6-Pregnadiene-17 [alpha], 21-diol-3,11,20-trione 21-acetate to 1-dehydrocortisone.



   0.85 g of 4,6-pregnadiene-17 [alpha], 21-diol-3,11,20-trione (also known as 6-dehydrocortisone acetate) obtained as described in J. Biol. Chem. 197, 261 (1952), is converted to 1,4,6-pregnatriene 17 [alpha], 21-diol-3,11,20-trione in the manner described above by the action of enzymes from a culture of Corynebacterium simplex, with a yield close to 18%.

   This new trine exhibits the properties of adrenocortical hormones, but it can be partially hydrogenated to the corresponding 1,4-diene by reduction under relatively mild conditions, for example by means of zinc, acetic acid and ascorbic acid

  <Desc / Clms Page number 60>

   bique, as described in the cited publication.



   We can prepare, as we have just described, others
 EMI60.1
 1,4,6-pr.é, j; n; trienes from starting compounds having a system of conjugated double bonds distributed in the rings A and B, and it is likewise possible to partially hydrogenate these prégna-
 EMI60.2
 triùnes to have the corresponding 1,4-pr.égnadiéneà. Likewise the substituent at position 3 can be a hydroxyl group, and in this case the conjugated double bonds will generally be attached to carbons 5,6 and 7,8.

   The substituent at position 11 may be [alpha] - or / 3-hydroxyl or it may be entirely lacking; the group at position 21 may be methyl instead
 EMI60.3
 hydromethyl or acyloxymethyl. Thus, 4,6-prenc, diene-I1,17oC, 21-triol-3,20-dione and 5,7-pre-no, diene-3, 11, 170 (, 21-tetrol- 20-one by the action of the cultures previously revealed in l, 4., 6-prenatriene-11, 170 (, 21-tz:., Ï 3,20-dione, while ,, that we can convert the 4 , 6-Pregnadiene-LL, 21-diol-3, 20-dion in 1,1 °, 6-prenatriene-17 r, 21-diol-3, 2U-dione. In each case, the 21-ester, which may or may not be hydrolyzed in the process.
 EMI60.4
 tielle as described above, .on will obtain the corresponding 1,4-prégnadiènex.



    Example 12.
 EMI60.5
 



  Conversion of 4-rene-11 17 21-triol-3 20-dione to -1 ° -renadiene-11qi 17. 2l-triol> 20-dione.



   The reaction is carried out exactly as described for the conversion of compound F to the corresponding diene and the product is isolated by extraction with chloroform and crystallization at
 EMI60.6
 from acetone-hexane. Starting from 1.0 g of 11-epi-cornposé F, one isolates zg of 1,4-pre; naoiene-11, 17 a ', 21-triol-3, 2G-; ione in the state of melting crystalline solid at 245-246 G (dec.).



  From a cc: t;, rlation of the diene of 11-epi-corpus P (1.0 g) is done by dissolving in 15 cm3 of anhydrous pyridine, followed by the addition

  <Desc / Clms Page number 61>

 
 EMI61.1
 , :: L -: ': iOl1 Ci :) C, 3 g of acetic anhydride. The reaction mixture is left to stand at ordinary temperature overnight at 2 ... then poured into a mixture of ice and water. The resulting precipitate is filtered off and recrystallized as a crystalline solid (21-acetate) from acetone-hexane.



  Example 13.
 EMI61.2
 



  14-i; r adiene-9, luoro-21-0l-311: 20-trione.



  In a 300 cm3 3rlelTIûeyer flask is added 100 cm3 of 0.1% yeast extract (Difco) containing 9.0 cm3 of 0.2M monopotassium acid phosphate and 9.0 cm3 of 0.2M disodium acid phosphate. The vial and its contents are sterilized in an autoclave for 15 minutes at 120 C and added to the sterile medium.
 EMI61.3
 1 cm3 of an oel suspension of OorYA., Eb. @ :. 9terium siin-olex. The vial and its contents are incubated at 28 ° C. for 24 hours.



   In a second Erlenmeyer flask of 300 cm3 we add
 EMI61.4
 2 em3 of ethanol and 25 ny of 9 no-: Cluoro-11-dehydrocortieosterone.



  The 24 hour old culture is transferred aseptically to the vial containing the steroid and the mixture is incubated at 28 ° C and stirred for 10 hours. At the end of this time the reaction mixture is extracted carefully with chloroform and the chloroform extracts are concentrated to a residue. This is crystallized from acetone-hexane,
 EMI61.5
 obtaining 4 mg of: -luoro-1,4-pregnadiene-21-ol-3, 11, 20-trione.



   The 21-acetate of the compound of this example is prepared by reacting 0.3 g of acetic anhydride with a solution of 1 g of 21-ol in 20 cm 3 of anhydrous pyridine at room temperature overnight. after which it is diluted with water and ice.



   Likewise, by the process of Example 13,
 EMI61.6
 '-i';. t-oro-1, -r-prnaditxe-11,, 21-diol-3,20-dione with a yield of 5 mg from 25 mg of starting compound a single

  <Desc / Clms Page number 62>

 
 EMI62.1
 L 21P'Jk't -. 'S.-W ï ::. f Ci-1 obtains the 21-acetate by the usual procedure G 'x; cG-tylatici. ln employing an a leo suspension of Li0rz'l2Cb? .GterI.LLT11 110: '- t- instead of 0. sinle = of Example 15, the other conditions remaining the same, 25 mg of 01., - are converted. -chlorocorticostL- rone in 9 -chloro-1,4-prenadiene-11 3, 21-diol-3,20-aione in a yield of 7 mg. The product can be converted to 21-propionate by reaction with propionic anhydride.

   With the same
 EMI62.2
 culture (2.lioa ii), the 9C-bromocorticosterone 21-acetate is converted to 9o (-bromo-1,4-prenadiene-113, 21-diol-3,20-dione.



   Following the method of Example 13, but using an incubation period of 48 hours at a temperature of 28 to 30 C
 EMI62.3
 the 9 -fluoro'-derivative compound F is converted to the corresponding 1, ° -diene. With the same process, we convert the 9 [alpha] -fluoro,
 EMI62.4
 Compound 9 n-Chloro and 9c-bromo-deified (cortisone) respectively in 9 Of.-loro, 9 -chlorc and 9 -bromine-derivatives of 1-dehydocortisane.

   The 9r'-èlil kro .ét; 9r-bromo = derivatives 'of compound F sonf, in the same way' CoMiertïs ': respect, vèment en '91%' - chloro and 9 K àbfgnio-4ér $ véx of 1-Délly , r'ocorti * sol., ', On. can convert the 17 - deoxß-domposed to the corresponding 17-hydroxy-compounds (9n (-hâlo, en6-derivatives of 1-dehydrocortisone and 1-dehydrbcortisone) by the action of Trichothecium culture roseum, as described below.
 EMI62.5
 



  As a result, other 1,4-pre; adienes produced using dehydrogenating mieroorcaiiism can be converted by the methods described in more detail below to the desired high potency end products.



    Example 14.
 EMI62.6
 1,4-prenadine-17 '21-diol-5,20-dione.



   1 cm3 of a culture broth, prepared as an inoculum (1%) according to Example 9, is added to each of 10 flasks containing

  <Desc / Clms Page number 63>

 
 EMI63.1
 l ::: 1.1lt lCC cc-i3 of sterile nutrient broth as described in this example. The vials were stirred on a rotary sealer for 8 hours at 26 ° C at 240 strokes per minute. After this period of growth, aseptically-: slowly a solution of 25 mg of Reich's compound S is added to each vial.
 EMI63.2
 stein in It <, 5 cents of methanol, then stirred and subjected to incubation for 24 hours additional. The final pH is 7.8.



   The contents of the vials are then combined and extracted 3 times with 2 liters of chloroform by extraction. The combined chloroform extracts were evaporated to dryness to give 310 mg of crude product. The crude steroid is purified by chroma-
 EMI63.3
 toraphy on the chromatographic system described by G.l ;. o 11ull, from the publications of the 126th Congress of the American Chemical Society, December 12-17, 1954, page 9a, publication No. 24.
 EMI63.4
 Chromatography examination shows a quantitative conversion of starting material to diene when an authentic sample of S-dicne is used as a control.



   Or, the crude product is recrystallized from aceto.
 EMI63.5
 ne, which gives 225 mg of 1,1-prLnadine-170, 21-diol-3, 1-dione, 1 '. . a.G-2.3 C. Example 15.



  1-ciiy¯di oeor-Lisol.



   From a solution of 1% yeast extract (Difco) in tap water, 10 portions of 100 cm3 each are taken, placed in flasks of 300 cm3 and sterilized by autoclave for 15 minutes under steam pressure
 EMI63.6
 of water at J, 795 .'ti ;. Where autoclaving and cooling of the oil, one places, t .. ± '. 118 c: the 1 cc flask; 5 of a shaken culture of 8 aci. Nlmerica var. iUS: ij'Ol: 1, tE, cultivated in broth of culture COJ.J.:.:c described from - 1s example 14. - Cnra.i te then the flasks; J1.: r a tal :,1; shaking for 12 hours at 2G C, due to

  <Desc / Clms Page number 64>

 
 EMI64.1
 9-40 runs pa.1 '.-Lil5t1tC.
 EMI64.2
 



  In 0 :: "1 (.!." L ".. 'flask we add s..s-3, -' dCJ. Welcome a solution of 25 '' '' '0' of" "'-1'1 --'- '/ FC 5 c ---' -the nethanol and the flask and its contents are stirred and incubated for an additional 6 hours. The combination of the contents of all the vials and e: ctrac- tiozi with cLIoroforj-ie conae described in Example 14, on G, 't, a :: l3t 3C4 ## z of crude diene. Cn carried out the purification by L' :, :(; :;. 'iGtL' .. llÜ: 30: i0l1 starting from aèGt'one with the use of decolorizing charcoal, and thus 230 mg (1, ° -prenadiene-11, 17, '1-triol-3 , 20-ûione, melting at 2..0-21 C.



   Using a total of 250 mg of 21-acetate of compound F instead of the compound F itself gives 220 mg of 21-
 EMI64.3
 1,4-prenadiene-11,3,11,21-triol-3,2C-dione acetate, melting at 237-239 C after recrystallization from acetone-hexane.
 EMI64.4
 Example 16. 1-Hydrocortisone.



   A growth medium is prepared from 30 g of soluble condensed fish products (treated with enzymes),
 EMI64.5
 4, L% 9 of K "12po 4 and 8.83 g of ÎFalIP0.71120e which is diluted to 1 liter with tap water (the pH of this medium is close to 6.8). Each of 10 flasks of 300 cm3 is introduced 100 cc of growth medium and the flasks and their contents are autoclaved for 15 minutes under a steam pressure of 6.795 kg. Each flask is inoculated with 1 cm3.
 EMI64.6
 of Dacillus sehaeriatis var. fusifornis coe in Example 15 and allowed to grow for 12 hours.

   To each vial is then added aseptically a sclution of 50 mg of compound E in
 EMI64.7
 1 cm3 of r = é-t1; anol, then stirring and incubation are continued for an additional 24 hours.



   The contents of the vials are combined and extracted with

  <Desc / Clms Page number 65>

   chloroform as described in Example 14, obtaining 630 mg of crude di.ne of compound E. Purification is carried out by recrystallization from acetone in the presence of decolorizing charcoal, thus obtaining 420 mg of 1-dehydrocortisone, melting point 233-235 vs.



   Likewise, according to the method of Example 16, corticosterone is converted to 1,4-pregnadiene-11ss, 21-diol-3,20-dione. Using the same reaction medium and the same organism described in Example 14, 9 [alpha] -fluoro-4-pregnene-11ss, 17 [alpha], 21-triol-3,20-dione is converted to 1 Corresponding 4-pregnadiene which is obtained as a crystalline solid.



   With the culture described in Example 16, 5-pregnene-30,20-diol is converted to 1,4-pregnadiene-3,20-dione, mp 152-153 C after extraction with chloroform, evaporation and re. - crystallization of the residue from ether.



   Likewise, deoxycorticosterone can be converted to 1,4-pregnadiene-21-ol-3,20-dione with the culture described in Example 14.



   As can be seen from the foregoing, the dehydrogenating microorganisms carry out the introduction of a double bond in position 4,5 with oxidation of 3-hydroxyl of 3-hydroxy-5-pregnenes with simultaneous dehydrogenation at the 1,2 carbons; although cultures of dehydrogenating microorganisms can be made to act on 3-hydroxy- or 3-keto-1-pregnenes, it is preferred to employ starting compounds having a double bond attached to carbon 5.



   The following Examples 17 to 19 illustrate various combinations of the process phase of introducing a 1,2 double bond into the steroid molecule using the dehydrogenating microorganisms described above, combined with other methods. microbiology, thereby introducing various substituents into the molecule to convert various known starting compounds into therapeutically active pregnadienes with production of

  <Desc / Clms Page number 66>

 
 EMI66.1
 intermediate compounds which themselves are therapeutically active or can be converted to 1-dehydrocortisone or 1-de-hydrocortisol, and their esters.



   Example 17.
 EMI66.2
 



  A. 1,4-prefMr.adiene-17G -01-3,20-dione (IIC).



  Using the culture described in Example 3, 150.0 mg of sterile 4-pregnene-17 (-1? 1-3,20-dione (17o (-progesterone) (le) are dehydrogenized in 5.0. ethanol The crude extract weighs 178 mg The crude product is crystallized from acetone-hexane to give pure crystals of 1,4-pregnadiene.
 EMI66.3
 17G -01-3,20-dione (IIc).



  B. 1,4-prex.adiene-I10. 17Ç -diol-3, 20-dione (IIc). ,
This conversion is carried out using a culture of Rhizdpus nigricans as described in detail in J. Am.
 EMI66.4
 



  Chem ISOC., 74, 5933 (7.952) É A 6 liters of a broth. ' of 24 hours of Rhizopus ni0ri 'is added 1.0. g of IIc in .1 .. 1 1. l ', 1,200 cc13 of etha11, ol,. After ur., 3, -pdi-iodine' 'de.ra'ns'fo'rmotion of 48 hours in the apparatus and the medium described in. In this publication, the culture is extracted with methylene chloride, the solvent is evaporated off and the crude crystalline residue is recrystallized.
 EMI66.5
 starting from acetone-hexane to give crystalline * 1 ', 4-pregadiene-11a, 117 C -diol-3, 20-dione (IIIc). '' C. 1 4- re waditne-17 A-ol-3 11 20-trione (Vc) ..



   Slowly add a solution of 3.0 g of IIIc in
30 cm3 of pyridine to a pasty product consisting of 1.5 g of chrpmic acid in 15 cm3 of pyridine (Poos et al., J. Am. Chéri.



   Soc., 75, 422 (1953)) and the resulting mixture is stirred overnight at room temperature. To the reaction mixture is then added 4.5 g of sodium sulfite in 45 cm 3 of water and stirring is continued for 2 hours. The reaction mixture is poured into dry cm 3 of water and the resulting solution is extracted with methylene chloride. The extracts are washed until new.

  <Desc / Clms Page number 67>

 Treated with dilute sulfuric acid, aqueous sodium carbonate and water, and dried over magnesium sulfate. The concentration of the dried solution and the crystallization of
 EMI67.1
 residue from acetone-hexane gives crystalline, 4-pregnadiene-17 -ol-321le2O-trione (Vc).



  D. 1-dehydrocortisone (IIa).



   4 liters of 1% yeast extract (Difco) are sterilized in a shaking flask and inoculated with a strain of Ophiobolus herbotrichus. The culture was incubated for 3 days with shaking at 27 ° C. Then 1.0 g of Vc in 25 cm3 of acetone (sterile) was added aseptically to the shaking flask and stirring was continued for 3 more. days at 27 ° C. The mycelium is separated from the reaction medium and the mycelium and the aqueous filtrate are thoroughly extracted with 3 portions of chloroform.



  The extracts are combined, washed with water, then dried and concentrated. The concentrated solution is chromatographed on Floricil (30 g) and the polonne is washed with methylene chloride: Elution with 0.5% and 1.0% methanol in methylene chloride removes 1-dehydrocortisone. which is re-crystallized from acetone-hexane.



    Example 18.
 EMI67.2
 



  A. Preparation of 1,4-Pregnadiene-11/3, 17 af -diol-3,20-dione (IVc) from IIc.



   A culture of Curvularia lunata (QM 120h) is grown in flasks containing the same medium as described in Example 17 (see also US Pat. No. 2,658,023).



  100 cm 3 of this inoculum is added under sterile conditions to 2 liters of an aqueous medium containing. what follows :
 EMI67.3
 
 <tb> sucrose <SEP> 1
 <tb>
 <tb> Difco <SEP> tryptone <SEP> 1
 <tb>
 <tb> nitrate <SEP> of <SEP> sodium <SEP> 0.2
 <tb>
 <tb> phosphate <SEP> acid <SEP> dipotassium <SEP> 0.1
 <tb>
 <tb> sulfate <SEP> of <SEP> magnesium <SEP> heptahydrate <SEP> 0.05
 <tb>
 <tb> chloride <SEP> of <SEP> potassium <SEP> 0.05
 <tb>
 <tb> sulfate <SEP> ferrous <SEP> heptahydrate <SEP> 0.001
 <tb>
 

  <Desc / Clms Page number 68>

 
This mixture is adjusted to pH 7 with sulfuric acid and 0.25 is added; of calcium carbonate before sterilization of the mixture.

   The inoculated medium is aerated at a rate of about 1/2 to 1 volume of air per volume of solution per minute at 27-280 for 24 hours. During this period, the mixture is stirred with a stirrer rotating at about 1700 revolutions per minute.
 EMI68.1
 minute. 1/2 g of 1,4-pregnadiene-17D (-0l-, 20-dione (IIc) is dissolved in 20 cm3 of 95% ethanol The solution is added to the fermentation medium under sterile conditions. then the reaction for a further 24 hours, exactly under the same conditions as those described above.



   The fermentation mixture was extracted thoroughly with 3 portions of methylene chloride and the combined extracts dried over magnesium sulfate, then concentrated to low volume. The concentrated solution was added to a Lorisil column (30g) prepared with hexane and the column was stripped with hexane containing ether in amounts ranging from 1% to 99%.



  The desired product is collected from the more polar eluates (25% ether # 99% ether) and crystallized from acetone-hexane, obtaining 1,4-pregnadiene. -
 EMI68.2
 II / 13,17 -diol-> ', 20-dione crystalline (IVe).



  B. Preparation of Vc from IVe.



   This reaction is carried out exactly like the conversion from IIIc to Vc, except that IVe is used instead of IIIc.



  The product is then converted to IIa as described above.



  Example 19.
 EMI68.3
 



  Preparation of 1 4 - nrérnadiene-11 .t 17 c 21-triol-3 20-ûione (VIla) from IIIC.



   The same procedure as that used to convert Vc to IIa is used, except that the steroid substrate is IIIc and the product, VIIa, is isolated by chromatography on Plorioil in

  <Desc / Clms Page number 69>

 eluates 1 and 2% methanol in methylene chloride. The recrystallization of Vils. from acetone gives a crystalline product melting at 243-245 C.



   By the same process as that used to convert Vc to IIa, we convert IVe to IIIa. Recrystallization of IIIa from acetone gives crystals melting at 238-240 C.



   The oxidation of IIIa or VIIa, preferably in the form of 21-acetates or other lower alkanoyl esters (IIIb and VIIb) is carried out by the method described for converting IIIc to Vc.



  The 21-acetate rump is hydrolyzed with potassium bicarbonate in aqueous methanol to recover IIa.
 EMI69.1
 



  The conversion of Ic to 4-pregnene-llc, 17-diol-3,20-dione (VIc) (route B) is carried out by the same process as that which is used in the conversion of compound IIc to compound IIIc (Example 17 B).
 EMI69.2
 



  Likewise, 4-pre; n; .ne-11,170 (-diol-, U-dione (VIIC) is prepared from Ic in the same way as IVc from IIc.



  Compounds VIc and VIIc are both oxidized to 4-pre-17n (-ol-3,11,2 (7-trione (Ville) by the method employed for the conversion of compounds IIIc and IVc to compound Vc.



   The rossant of the transformations following route B also follow the procedures of route A. Thus, coin- posed VIe is converted to IIIc and compound VIIIc to Vc, while compound VIIc is converted to IVc (the same way that Ic is conver-
 EMI69.3
 ti in IIe, cloo4u - ie by trc ,, itoi-hereit with an auwatinu culture or extract of ue culture ûe GorTnebacl; erium simplex or boagii.

   From this point, the transformations are the same as in lane A In the specific application to the conversion of deoxy-
 EMI69.4
 coiwicN-téßozve (--regnne - '? 1-0l-, W-ûioz.e), from the 5-pre, <; nino-, 1- iol-20-o: ze and do theirc esters in L!,. 1¯Ô, Gh: rrlrocorti8ollo and 11¯àëh "yèl'occ.: ct.LOOl (4e-àéhydi'Goo. .: po = 1é il) and their esters, we

  <Desc / Clms Page number 70>

 
 EMI70.1
 p. : r-li <.jU'3i 'the process of the invention such i Lt' i1 'E i4d: Lt1? C: c.â.i' .... f3 loz e :: e'los 2C and Ll which follow.



  10 LC. l¯? iiadinej-21-3, C: LO (IId).



  150.0 rii-- of sterile 4 - prcgnbne-21-ol-3,20-dione (here) are fermented in 5.0 cm3 of ethanol, in the same medium, with the same microorganism and the lead. Way as in the example
 EMI70.2
 r
 EMI70.3
 17A, obtaining a crude extract weighing 157 mg. The crude product is tallized from acetone-hexane, obtaining crystalline 1,4-presnadiene-21-ol-3,20-dione (IId).



  B. 1,4-rc; nadine-11 ", 2lcliol-> 20-. Dione (IIId).



  1.0 g of IId in 200 cc of ethanol is treated with a culture of Rhizopus ni, 7, ricalis as described in Example 17I3. The crude crystalline residue is recrystallized from ketone-hexane to give. 1, 4-pr ,, é '; nadine-11 ((, 21-d.cl -
 EMI70.4
 Crystalline 3,20-dione (IIId).
 EMI70.5
 



  VS . lré, diixa.e-21-ol-3, 11, 20-triox.e (Vcl ± ..



  A solution of 3.0 g of IIId in 30 cm 3 of pyridine is subjected to the oxidation, in the manner described in Example 170.



  Evaporation of the dried extract of methylene chloride and crystallization. of the residue will start from acetone-hexane to produce crystalline 1,1q-prenadine-21-ol-, 11,20-trione (Vd).



  D. 1-d6l ± trocor "lisoTie 'IIa).



  Cn sterilizes liters of Czapek-Dox medium in a vial. a; ite and inoculated with a strain of Trichothecium roseum.



  After 3 days of agitation. 2; 0, 5CO isg of 1,1-argnudi: ne-21-ol-3,11,20-trione (Vd) in acetone was added aseptically to the culture. Stirring is continued for 60 hours at 27 ° C. The mycelium is separated off and the mycelium and the aqueous filtrate are extracted thoroughly with metric chloride. The combined extract is washed suitably with water, dried and concentrated. The concentrated solution is chromatographed on

  <Desc / Clms Page number 71>

 
 EMI71.1
 1 <'iorizil and the 1-dehj, rdrocor% isone (IIa) in the 0.5% and 1% fractions of methanol in methylene chloride, following washing the column with " methylene chloride.



  Recrystallization of the indicated fractions from acetone afforded crystals of IIa, m.p. 226-229 C (dec.).



  Example 21.
 EMI71.2
 



  A. 1 4-d; n.adiene-113, 21-diol-3 "20-dione (IVd).



   A culture of Curvularia lunata (QM 120h) is grown in flasks containing the same medium as described in Example 1 of the US patent to Gilbert M. Shull et al. , N 2,658,023 and is employed exactly as described in Example 18A for the conversion of 1,4-pregnadiene-21-ol-3,20-dione (IId) likewise in solution in 20 cm3. ethanol
 EMI71.3
 at 95%. From the more polar eluates (25 ether # # 99) (ether), we obtain 1,4-pre; n .dine-11 2l-diol-), O-dione (IVd) as 'one recrystallizes from acetone-hexane. l3. Preparation of 14-nadiene-21-ol-5 ll 20-trione Vd.



   This reaction is performed exactly like the -Lion transformation from IIId to Vd, except that IVd is used in place of IIId.
 EMI71.4
 



  Compound IIId is converted to 1,4-pregnadibne 110 (, 170 (, 21-txiol-, 2-dione (VIIa) by the same method as that used to convert Vd to IIa. The product is isolated by chromium-. Matography on Florisil in methanol eluates 1 and 2 in methylene chloride Recrystallization of VIIa from acetone provides the product in crystalline form,
 EMI71.5
 P. I. '-. 5 C.



  De, llêJ.1c, the method employed to convert IVd to hydrocortisone (III) is that which is used to convert Vû on IT4, and the product IIIa is isolated by ccro.Liatot, ra-ohie on chlorisone. in eluates at 1 and z (methanol in iatii chloride;. rl: ne. LL, recrystallization of IIIA from acetone gives L; 1; ciic product: c. llim melting at 238-, c. 8.

  <Desc / Clms Page number 72>

 
 EMI72.1
 i 'oY, .- c.', ii10II of IIa or VIIa, preferably 11 et-u- of Ll-aciitate which is obtained by reacting 1-a, leool ;: 1a.



    Ordinary temperature with one equivalent of acetic anhydride is carried out according to the method described for converting IIId to Vd.



  The 21-acetate group is hydrolyzed by heating with potassium bicarbonate in aqueous methanol to recover IIa.
 EMI72.2
 



  The transformation of 4-prenene-11, 21-diol-, 2G-c.iox.e (VId), 4-pre; n.ene-11J, 21-diol-3,2G-dione (VIId) and 4-prcnene-21-ol ->, 1L, 20-trione (VIIId) to IIId, IVd and Vd, respectively, is carried out in the same manner as the conversion of Id to IId.



   Analogously, the conversion of Id to the intermediate compounds VId and VIId follows the methods described above for the conversion of IId to IIId and IId e respectively.
 EMI72.3
 IVd. Zes cômp ss VId and VIId are oxidized to compound VIIId dar .. ci.e; mild oxidizing conditions such as those described above.



   As indicated above, the place of deoxycorticosterone and its esters,! Can be used as starting compounds
 EMI72.4
 J 5-prenene-, '2l-diol-20-one. and its 21-esters such as acetate and other lower aikanoyne esters) since during the treatment with a culture or an enzyme separated from the
 EMI72.5
 dehydrogenating microorganism such as C. simplex or C.- lioaii as already described, 3-hydroxyl is oxidized to ketone oxygen while the 5,6 double bond migrates into position
 EMI72.6
 4.5. In this way, 5-prenene 3,21-diol-20-one and sea bzz1-esters are converted into IIa and its 21-esters.



   In general, better yields are obtained in oxy-
 EMI72.7
 dation of compounds llr-, -0- ':! and 11,, O -eJ to the corresponding 11-ketoniquos compounds and with less critical conditions if the 21- is first esterified in the methods described in Examples 20C and 21B, with subsequent hydrolysis if it is

  <Desc / Clms Page number 73>

 longed for. Processes suitable for the partial esterification of 11-21-diols and the hydrolysis of 11-keto-21-esters obtained by oxidation have already been described.



   The 21-acetate of Vd can be obtained by oxidation of the 11-hydroxyl of the 21-acetates of IIId and IVd with chromic acid in pyridine, as already described in connection with related compounds. The 21-ester of Vd is readily hydrolyzed by boiling under reflux with potassium carbonate in aqueous methanol.



   Compounds VId and VIId can be converted to their 21-acetaes in the same manner as IIId and IVd. The oxidation of 21-acetate from VId and 21-acetate from VIId to 21-acetate from VIIId is carried out according to the process of oxidation of 21-acetate from IIId to 21-acetate from Vd. The hydrolysis of 21 -acetate of VIIId en Ville *, in case this hydrolysis has not been carried out or has not been completed by the microorganism employed to introduce the 1,2 double bond, for example when using higher fermentation temperatures high, is carried out by the hydrolysis process of 21-acetate from Vd.



   In one of the preceding diagrams, we show the conversion of 16-dehydropregnenolone (Ie) and 16-dehydroprogesterons (IIIe) into 1-dehydrocortisone (IIa) by the 16,17-epoxide route and by various routes: These routes also include the addition of an 11 (0 ('or (3) -hydroxyl, the addition of a 21-hydroxyl, the introduction of a 17 [alpha] -hydroxyl by opening the ring 16, 17- epoxide, the introduction of the 1, 2 double bond and the oxidation of 11-hydroxyl to ketone oxygen.

   Although the order of these reactions can vary between certain limits (for example the oxidation phase must follow the introduction of 11-hydroxyl), it is preferred to carry out the treatment with the dehydrogenic microorzanism after the introduction of 'a hydroxylated group at least in one of the 11, 17 and 21 positions, or after the formation of the 16, 17-

  <Desc / Clms Page number 74>

 
 EMI74.1
 epoxyJc. Cn gives in examples 22 and 1J'JCéi, .f,: i explains what concerns the conduct of the different orders; ii conversions on 4,16- and 6,15 prénc-dienes. people 22.



  1-del? Tdro c ortisone.



  1 g of 16-dehydroprogesterone (IIIe), in solution of 25 cm 3 of chloroform, is reacted 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 sulfite solution, then with sodium bicarbonate solution, water, then dried over sodium sulfite. The chloroform is evaporated; and the residue is crystallized from a mixture of acetone and hexane, which '' friend
 EMI74.2
 l, 17-epoxy-4.-re; nene>, ca.-dione (iVe) ...



  A suspènsàon, of 1 g is mixed. of, 16.17. epoxide in 10 cm 3 of acetic acid glasiai 'with a solution of 1.1 g of aqueous iodlydric acid at 47%! 10Jcm3 'of acetic acid and 4.1 g of acetic anhydride. Maintain.the.temperature, at about 15-
 EMI74.3
 20 ° C, stop the addition, continue for 1/2 hour. The mixture is poured into 5 volumes of water, the precipitate is filtered; we wash it with water and dry it. The gross product,
 EMI74.4
 lÉ-iodo'-ÀLpréànéne-17 <Lol-3, ç0-dio> ie, 'is dissolved in 100 cm3 of ethanol and 1 cm3 of acetic acid, then it is boiled under reflux for about 6 hours with 2.5 g of catalyst. Raney nickel.

   The catalyst is then removed by filtration, the filtrate is concentrated and, by addition of water, the product is obtained.
 EMI74.5
 a precipitate of 4-pre, ene-17! c -0l-3,20-dione (17o (-hydroly-progesterone, VII).



   4 liters of a 1% yeast extract (Difco) are sterilized
 EMI74.6
 in a shaking flask and inoculated with a strain of Onhiobo1us herbotrichus. 01-1 incubate the cultur3 r.e =; éa.; A% 3 days, with

  <Desc / Clms Page number 75>

 
 EMI75.1
 agitation at "7"; 1.0 g of 17% hydroxy-proesterone in 25 cm3 of acetone (sterile) is then added aseptically to the shaking flask, followed by continuing. stir for an additional 3 days at 27 ° C. The mycelium is separated from the reaction mixture and the mycelium and aqueous filtrate are thoroughly extracted with 3 portions of chloroform. The extracts are combined, washed with water, dried, concentrated and chromatographed on Florisil.

   The material obtained on elution, for example with 0.5% to 1.5% methanol in methylene chloride, is crystallized.
 EMI75.2
 from acetone-hexane to give 4-pregnene-17o (-2l-diol-3,20-dione (VIIIe).



   A solution of 1.0 g of Ville in 150 cc of ethanol is added to 1 liter of a 24 hour culture of Rhizopus niricana. After a 48 hour transformation period, the mixture is treated with methylene chloride and the organic extract is evaporated to dryness. The crude product is crystallized at
 EMI75.3
 Starting with acetone-hexane to have 4-pregnene-11c /, 170,21-triol-3,20-dione (IXe).



   Compound IXe is then subjected to the action of a culture of a dehydrogenating microorganism such as Corynebacterium simplex. After incubation for about 48 hours at 28 C, we obtain
 EMI75.4
 l, ° -pre; nadi: ne-110 (, 17, (, 21-triol-3,20-dione '(Xe, VIIa). This compound is then oxidized as described above to 1-dehydrocortisone (IIa).



   By employing a culture of Curvularia lunata (QM 120h) as described in Example 18 A in place of a culture of
 EMI75.5
 R1Jisopns nigricans, we obtain 2 .. from IXe the compound 1,4-pregnacliè; ie-113, lit, 21-triol ->, 2G-dione (1-dehydrocortisol) (IIIu) which can likewise be oxidized in 1 -delydrocortisone.



  Exenpie 2]>.



  1-, 11-yû.roccrti; onc. converts 15-.éll ;, rdropré ;; n; nolone to 15,17-epoxide

  <Desc / Clms Page number 76>

 
 EMI76.1
 ;, 'a1' .t = ii-te:, ient with perplitalic acid. On C01LVj: it alet'Si la l, 17 - :. ß, ox.r-5-prex2ene - ol-20-ox2e (IIe) in derivative 11-ilytro- :::: '1- (:: le ) by treatment with a culture of Rl1izOiJUS nir: ricans.



  Cn then subjects the compound Ale to the action of a culture of Ophio-001us herbotrichus to produce 16,17-epoxyr-5-pregJ1ene-5, 110 (, 21-triol-2v-one (XIIe). opens the epoxy ring by treatment with hydriodic acid, as described above, to give 5-Preb-nene-3, 11c 170 /, 21-tetrol-20-one (XIIIe). the latter compound to the action of a culture of a,
 EMI76.2
 uehydrogenating microorganism, 1,4-pregnadiene-110,17 A /, 21-triol-3, 20-dione is obtained which is then oxidized to 1-dehydrocortisone.



   If desired, you can first process the 16-dehydro-
 EMI76.3
 Pregnexiolonated with a culture of 0 "¯hiobolus herbotrichus to have 5,16-pregnadiene-3,21-diol-20-one which is then added in a known manner to obtain the diacetate. By treatment with a dehydrogenating microorganism, the
 EMI76.4
 diacetate in 1,1 °, 16-pregnatriene-21-ol-3,20-dione. The latter is then converted to 16,17-epoxide by treatment with perphthalic acid, after which the epoxy ring is opened with hydriodic acid, followed by boiling under reflux with a nickel catalyst. Raney, which gives 1,4-prégnadiè-
 EMI76.5
 ne-17, 21-diol-j, 20-tlione.

   The latter can then be converted into 1-dehydrocortisol by the action of a culture of Curvularia lunata.



   Instead of peracids, such as peracetic, perbenzoic and perphthalic acids, for the formation of the oxidation ring, hydrogen peroxide can be used together with an alkali metal base, preferably sodium hydroxide. or potassium. This mixture may contain some amount of an alcohol, such as methanol and t.-butanol, to aid in the dissolution of the steroid compound, but this alcohol in general is not.

  <Desc / Clms Page number 77>

 
 EMI77.1
 'bsa7.umn't héce5Sare.



  D'3L'uti'as composes; starting ..30ilt la., 1> - "'' ?. 'GxlC.lL7 .- ,, J .. ^ dj-ol-20-oe and its 3-est = a, 1:, 1-.s 'txlwdine-3,21-zxo1-C- one and its 3- and 2i-e;:' ta: ';, la 5,1ü-rc :, viacliçx.e-, 11,1-txW o1- 20- ce and 3v 3- and 21-esters and la. r: -. 32 '', ilc.C) .1: .21e-1-O1-.7, -a-, 20-tl'i01lG and these Si -esters, which can be subjected to an alum cult ::. icTOOr (; ' <. ::.: nisLl8 dehydrosinating to form the conjugated µ-keto-1,4-to-iine system, and 5. the action of peracic1e or hydrogen peroxide to force the cycle 16,17- oxide, 'in that order or in the other order.
 EMI77.2
 



  17..i; jC'3i..171 of a compound already having the structure 1,4 - diene- Í-oetoniClU8 which can be converted to 1-dehydrocortisone and 1-dehydrocortisol is 1-dbhydro-dbrive of compound S. The necessary transformations can be carried out according to Examples 24 and 25.
 EMI77.3
 



  : fxr7, the.



  J¯ 4 'n1'c: nadién, e-110 (17 r, 21-t.riol-3 2U-dione VIIa).



    According to the process described in 'J. Am. Chem. Soc. , 74., 5933
 EMI77.4
 (1c> 2) is added to 6 liters of a 24 hour culture of I3hi- .gr ', t} -: s. : g, = h.LrJcans 1.0 of 1, .- prenadi: ne-llo (, 21-dxol-3,20-dione (which can be prepared as described in Examples 3 and 6 below) above or according to known chemical methods) in 200 cm3 of ethanol After a transformation period of 48 hours in the apparatus and the medium described in the aforementioned article, the extraction is carried out with methylene chloride and washed the crude crystalline solid residue three times with
 EMI77.5
 5 cc portions of: cold lace-eh10roforme. The yield in coxipo <-é 'iIIa es.t de e, 9.

   The product is recrystallized from acetone-hexane.



  (B) Z.- '.' 'C''¯:':. 'R CCO1''lSOxie (I 1 a).



  A solution of 0.4 a of 7IIc. 0.11 g of acetic anhydride is added to 100m3 of anhydrous pyridine. Cn let rest

  <Desc / Clms Page number 78>

 ser the mixture Lthe reaction at room temperature working overnight, then it is added to a mixture of 0.15 g of chromium trioxide in 15 cm3 of pyridine (the chromium trioxide-pyridine reagent is prepared according to the method of 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 volumes of water. The product is extracted with methylene chloride and the extracts washed with water, dilute sulfuric acid and again with water. The methylene chloride solution is dried over magnesium sulfate and concentrated in vacuo. Crystallization of the 21-acetate of IIa takes place from acetone-hexane to give 0.21 g of crystalline solid (IIb).



   To a solution of 0.12 g of IIa 21-acetate in 5-cm3 of chemically pure methanol is added 0.0315 g of potassium bicarbonate in 0.5 cm3 of water. The mixture is boiled under reflux for 3 minutes and cooled rapidly. Then it is diluted with water, neutralized with dilute hydrochloric acid and extracted with methylene chloride. The extracts are dried and concentrated. The residue is crystallized from acetone-hexane to give 0.051 g of 1-dehydrocortisone (IIa).



  Example 25.



  (A) 1,4-Pregnadiene-11ss, 17 [alpha], 21-triol-3,20-dione (IIIa).



   1,4-Pregnadiene-17 [alpha], 21-diol-3,20-dione (0.5 g) is treated with a culture of Curvularia lunata (QM 120h) which is prepared in the following manner. The curvularia lunata is grown for 2 days in flasks stirred at 28 ° C. on a malt-sugar-salts medium. 100 cc of the resulting culture is used to inoculate 2000 cc of the same medium contained in a fermentation apparatus equipped with a submerged aerator. The inoculated mixture is stirred at 1700 rpm and acre at a rate of 0.5 volume of air per volume of Medium per minute for 22

  <Desc / Clms Page number 79>

 hours in a double boiler at 28 C.

   50 g of a wet mycelium, obtained by filtration of the resulting broth, is added to 2000 cm3 of water, and to the steroid mentioned above in a similar fermenting apparatus. After the mycelium-steroid suspension is stirred as described above for 22 hours, it is extracted with chloroform. The chloroform extracts are dried, concentrated to a residue and chromatographed on Florisil. The Florisil column is prepared with hexane, the steroid mixture is added in a methylene chloride solution, and the elution is carried out with methylene chloride and methylene chloride-methanol mixtures.

   Unreacted starting material was eluted in 0.5% methanol methylene chloride and the desired product IIIa eluted in 1-2% methanol methylene chloride. From
 EMI79.1
 0.5z; 1-delzydro-compound S is obtained 0.17 g of IIIa in the form of a crystalline solid melting at 237-239 C.
 EMI79.2
 (B) 1-Dehydrocortisone (IIa).



   Compound IIIa is converted to compound IIa by the same method as that used to convert compound VIIa to compound IIa. From 0.400 g of IIIa, 0.205 g of IIa is obtained.



   In the following Examples 26 and 27, a chemical process for the introduction of the 1,2 double bond is described.



  Example 26.
 EMI79.3
 



  1-dehydrocortisone (IIa).



  A solution of 1 g of allopregnane-170 (, 21-diol-3,11,20-trione in 15 cm3 of acetic acid is bromined by adding 0.80 g of bromine in 10 cm3 of acetic acid. Addition of water precipitates 2,4-dibromo-allopreg- 21-acetate.
 EMI79.4
 nane-17c, 21-diol-3,11,20-trione.



   Without purification, the dibromide (1.5 g) is boiled under reflux in 50 cm3 of S-collidine for one hour. We add

  <Desc / Clms Page number 80>

   chloroform and water, the organic extract is washed with dilute sulfuric acid, then with water, then dried and evaporated. The crude product is chromatographed on Florisil
 EMI80.1
 to have 1,4-Pregnadiene-1,21-dol-3,1,20-trione 21-acetate (Ib, R = acetyl), m.p. 232-235 C (dec.).



   Similarly, by bromination of 21-acetate
 EMI80.2
 pregnane-17o, 2l-diol-3, ll, 20-trione with two equivalents of bromine and dehydrobromuration by means of collidine, we obtain
 EMI80.3
 1-d.hydrocortisone 21-acetate.



  By bromination of aliopregnane-11 / 3,17c /, 21-triol-3,, 20-dione at positions 2 and 4 by treatment with bromine followed by dehydrobronfuration with collidine ., 1-dehydro'cortisol 21-acetate is obtained.



  Likewise, dibromidation of Pregnane-11-acetate-11, -1? P, 21-triol = 3,20-dione with 2-moles of bromine, followed by dehydrobromination with collidine, gives the 1-dehydrocortisol acetate. L. Alloprene21-acetate, 21-d.iol-, li, 20-triohe ,. employed in this modification of the present process, can be prepared by hydrogenating 2.00 g of cortisone acetate in 200 cm3 of ethyl acetate with hydrogen at normal pressure and at room temperature, using 0.20 g of 5% palladium charcoal as a catalyst. The reaction is allowed to continue overnight, the catalyst is then removed by filtration and the filtrate is evaporated under reduced pressure.



  The allopregnane compound is recrystallized from acetone.
 EMI80.4
 



  Likewise, allopregnane-II 21-acetate (3 '17,21-triol-3,20-dione can be obtained by hydrogenation of 5.0 g of 21-acetate of compound F in 750 cm3 of compound F. ethyl acetate with hydrogen in the presence of 0.5 g of 5% palladium charcoal as a catalyst.



   The 21-acetate obtained from IIa can be hydrolyzed, as can

  <Desc / Clms Page number 81>

 that of IIIa, by boiling under reflux with potassium bicarbonate in aqueous methanol, in order to obtain the free alcohol. example 27.
 EMI81.1
 



  14-renadiene-11 A 21-acetate 17,2l-tr.ol-3 20-dioaae ..



  A solution of 210 g of Pregnane 110 21-acetate (, 17 A, 21-triol-3, 20-dione in 25- cm3 of acetic acid is bromined in position 2 and 4 by adding 1.60 g of bromine. in 15 cm3 of acetic acid, the 21-ao-
 EMI81.2
 crude 2,4-dibromopregnane-11,17 D (, 21-triol-3,20-dione.



   Dehydrobromination with 100 om3 of collidine for 1 hour gives, after chromatography, 1,4-preg- 21-acetate.
 EMI81.3
 . naene-11 1,17i, 21-triol-5, 20-aion (VIIb).



   Hydrolysis with potassium bicarbonate in aqueous methanol for 10 minutes under reflux gives 1,4-Pregna-.
 EMI81.4
 diene-11 p (, 17o (, 21-triol-3,20-dion (VIIa).



  Oxidation of the acetate with, Cr07., N-bromoacetamide or N-bromosuccinimide gives IIb.



   Examples 28-32 illustrate methods of chemically introducing the 1,2-double bond.



  Example 28.
 EMI81.5
 



  A. 4-Pregnene-, 2,21-diacetate, 17O {, 2l-triol-3,11,20-trione (IIh).



   A solution of 2.0 g of 6-bromo-4-pregnene 21-acetate
 EMI81.6
 -17 (, 21-diol-3,11,20-trione in 100 cm3 of glacial acetic acid is heated under reflux in a nitrogen atmosphere with 10 g of anhydrous potassium acetate for 4 hours. The reaction mixture is mixed with water and the products are extracted with ethyl acetate, the extracts are dried, concentrated in vacuo and the residue taken up in a small volume of methylene chloride. chromatographs the resulting solution on '.'lori. ,, il (magnesium sillicate) and the solid,

  <Desc / Clms Page number 82>

 
 EMI82.1
 which elutes with 50% ether-hexane, is recreated several times from acetone-hexane to give 4-pregnene-2,17t 2,21-diacetate , 21-triol-3,11,2C-trione,? .F.



  ,, - 10-215 0,) Max alcohol 238 (= 14,700).



  B. ° --prenene-2, 17 D (, 21-triol-3,, 11, 20-trione (IIIn).



   To a solution boiling under reflux of 1.0 g of IIh in 100 cm3 of chemically pure methanol, in a nitrogen atmosphere, 0.45 g of potassium bicarbonate dissolved in 10 cm3 of water is added. The mixture is boiled under reflux for 3 to 5 minutes and the pH at 6.8-7.0 is then added by the addition of acetic acid. The resulting mixture is then concentrated in vacuo to a solid residue, the residue is thoroughly stripped with ethyl acetate, and the extracts are concentrated. The residues are then crystallized from acetone-hexane, this
 EMI82.2
 which gives 4-pregnene-2,170 (, 21-triol-3,11,20-trione or * - talline.



  Example 29.
 EMI82.3
 A. -bromo-4 11-formate 21-acetate = prenene-11/3 L17 0.21-triol-3,20-dione (IVh).



   1.6 g of 11-formate are added to 160 em3 of chlorobenzene
 EMI82.4
 4-Pregnene-11 ', 170 (, 21-triol-3,20-dione 21-acetate and the mixture is heated until complete dissolution of the steroid.



  Then 180 cc of dry carbon tetrachloride is added and the mixture is boiled to remove the water. Then 4.2 cc of a 10-fold solution of pyridine in carbon tetrachloride and 0.79 g of N-bromosuccinimide are added, carbon dioxide is bubbled through the mixture, the mixture is illuminated with a lamp. 50 watt frosted, placed in contact with the flask, and the reaction mixture is quickly brought to the boil. After 10 to 20 minutes of heating, the N-bromo-succinimide has completely reacted; the solution is cooled, washed with water and concentrated in vacuo. We can do

  <Desc / Clms Page number 83>

 
 EMI83.1
 c.,>: i- # 1; ll; 1:> ei 'the residue from chloride of 1 = a.i c, ui gives crystalline compound IV'tl.



  L. 1: Cornaiate Z 21-di2ctate de.-Rine 2 11 17r 21-zral 2C-dione.



  The reaction is carried out exactly as described in Example 2 ('1 @, except that IVh is the starting steroid. The above-mentioned product is chromatographed on Florisil, the crystalline fractions are combined. (50 '. Ether-hexane and 100 from utlior) and recrystallized from acetone-hexane.



  C. 4-pregnene-2, ll to, 171>, 21-tetrol-3,20-dione (VIh) e A solution of 0.5 g of the triester in 50 cm3 of
 EMI83.2
 chemically pure mcthanol with a solution of 0.165 g of sodium hydroxide in 5 cm3 of water, in an argon atmosphere. The reaction mixture is allowed to stand at room temperature overnight and then neutralized with ac acid. - tick. The solution is concentrated in vacuo and the residue is extracted thoroughly with ethyl acetate. The extracts are concentrated and the residue crystallized from acetone-hexane, which
 EMI83.3
 which gives crystalline -prenene-2,11,17pC,, 21-tetroi-3,20-dione (VIh).



  Example 30.
 EMI83.4
 



  A. 21-acetate e. 1,2-oxido rermane-17 nc 21-diol-> 11 20-trioll (Ili).



  Has a solution of 0.4 gade 21-aoctate of 1-pregnene-17 m,.



  'C -'l-diol-3,11,20-triol (Ii) in 100 cm3 of chloroform at 5 ° C 0.14 g of perbenzoyl acid in 25 c3 of chloroform is added The resulting mixture is allowed to stand at 5 ° C. C overnight, then washed with dilute aqueous sodium bicarbonate. We
 EMI83.5
 : ¯..the chloroform solution is then concentrated under vacuum. The residue crystallizes from aoctone-hexane to give crystalline compound IIi.



  21-zc State ge. 2 -br omo p r é nan e -1.17. 2J. triol-3, ll, 2p - tri on (IIIi).

  <Desc / Clms Page number 84>

 



   To a solution of 0.42 g of IIi in 100 cm3 of chloroform at 5 ° C., 0.08 g of anhydrous hydrobromic acid dissolved in 100 cm3 of chloroform is added. The mixture is allowed to stand at 5 ° C. for one hour, then concentrated in vacuo. The residue is crystallized from methylene chloride-hexane to give crystallized compound IIIi.
 EMI84.1
 



  0. 21-nreaetate; n.ane-1,17x ', 21-triol-3,11,20-trione (IVi).



   A solution of 1 g of IIIi in 100 cm 3 of methanol with 10 g of 10% palladium on calcium carbonate catalyst is stirred in a hydrogen atmosphere at atmospheric pressure. When one equivalent of hydrogen has been absorbed (which requires several hours of stirring), the catalyst is filtered off from solution and the filtrate is concentrated in vacuo to a solid residue (IVi).
 EMI84.2
 



  TI. Prenane-1,21-diacetate-1,17oCt2l-triol-3,11i20-trion =; Ti).



   The crude product IVi is dissolved in a mixture of 0.5 cm3 of acetic anhydride and 5 cm3 of pyridine. The reaction is allowed to continue overnight, then the mixture is diluted with an ice-water mixture. The resulting precipitate is filtered off and recrystallized from methylene chloride-hexane, obtaining
 EMI84.3
 crystalline Pregnane-1,21-diacetate-1,17 ', 21-triol-3,11,20-trione (Vi).



  E. 4-brominated 1,21-diacetate; nane-1,1? oe, 21-triol-3,1120-trione (VIi).



   To a solution of 0.46 g of Vi in 50 cm3 of glacial acetic acid is added 0.5 cm3 of 0.28H hydrobromic acid in acetic acid. a solution containing 0.16 g of bromine, 0.08 g of sodium acetate and 15 cm3 of glacial acetic acid is then added dropwise thereto, with good stirring, at a rate such that each drop has the opportunity to react before the other is added. When the addition is complete, the reaction mixture is diluted with 5 volumes of water and collected by

  <Desc / Clms Page number 85>

 
 EMI85.1
 w- um 'v .l'iî. "; 1., ats. <> i. la -30., iiiosLl VIi precipitated.



  >, gg = (jg @ -ggt -i è ¯ 4 -ni, µ # gµ n e - 1 ¯Jg ±, ¯gg% = @ g1 ¯ µ¯āgl;, 10 -trio / i & -dim.



  To a solution of 0.54 g (the VIi in 5 cm3 cl '-, here -'L 00 that glacial is added, in a carbon dioxide atmosphere, a solution containing C, 25 g of semi-hydrochloride. carba-
 EMI85.2
 side, 180 mg of anhydrous sodium acetate, 10 cm3 of water and 10 ei.15 of glacial acetic acid. The mixture is stirred for 10 minutes and then 20 cm3 of 1N sodium acetate in glacial acetic acid are added thereto. Stirring is continued for another 10 minutes, 2 cm 3 of pyruvic acid are added and the mixture is boiled under reflux for 10 minutes.

   The cooled solution is diluted with water and extracted with methylene chloride. On.wash in water the extracts until there is no
 EMI85.3
 more acid and the solution is dried over inagi sulfate. ... Lum.



  Concentration of the solution dries up the addition of hexane to initiate crystallization of VIIi. Pres-11a21e-1,17,21-triol-21-acetate can be brominated and deahydrobro-mured. 3, f.1,20-tr.oile ,, r the process described in e; cemle 30 'obtaining, dil .2l = aétate.-Pré.ène-l, 17n (, example 50'DE and F, obtaining, 21-acetate of 4-prepiene-1,17 Crystalline, 21-triol-3,11,2 O-irione. ggw goc 31 ..



  Conversion of 2lOacetate 1-pre; qxn) nemll 1, 17 ±, 21, -triol-5, 20- dione to 4-renene-1,11 21-diacetate 2, 17c, 21-tetrol- 3,20-dione.



   By the same series of reactions as that which is revealed in Examples 30 A to 30 F, and substantially in the
 EMI85.4
 .iCiic-under conditions, the 21-acetate of 1-precnene-11 d, 17 0, 21-tr iol-3, 2C-dione is converted by the same operating steps and via the 21-acetate of 1,2-oxydopré , nane-11, 170 (, 21-triol-3,20-dione, 21-cc t, te.; 2-bromopregnan-1,11, 17 0 !, 21-tetrol->, 20- -Loi -ic, Pregnane-1,11 A, 21-acetate, 17, K, 21-.tGtrol-3, 2E- rt: l O11G, l, 21, -d 1.CC tate de rremn el, 11, l7 (, 21-tetxol-, 4-

  <Desc / Clms Page number 86>

 
 EMI86.1
 dione and -brcmppxenane-1,113,101,21-diacetate, lô (, 21té-Gr ol-3, 20-dione, -pregnene 1,21-diacetate, ll / 3, 17 D !, 21- tetrol-3,20-dione.



   It is also possible to bromide and dehydrobromide 21-acetate
 EMI86.2
 of prenar¯e-1,11,17,21-tetrol-3,20-dione by the methods described above to give the 21-acetate of 4-pregnene-1,113, 17c, 21-tetrol-3,20- dione.



  Z:;: ermle 32.



  The 1-hydroxyyl group is combined with alumina.



  A. A solution of 0.1 g of 4-Pregnene 21-acetate-1,113, 17 is passed through; 21- tetrol-3, 20-dione, in 100 em3 key chloroform, on an activated alumina column (30 g passing through a sieve
 EMI86.3
 at 0.1 ° 9-0.07 ° mm mesh opening) and the column is then washed with methanol. The combined eluates are concentrated: @e residue, which crystallizes from acetone, is identical to all
 EMI86.4
 aspects of 1,1 ° -pren.adiene-11/3, 17 a (, 21-triol-3,20-dione (1-dehydrocortisol 21-acetate).



    B. A solution of 0.1 g of
 EMI86.5
 21-acetate of 4-pregnne-1,17cl, 21-triol-3,11,20-trione, in 100 cm3 of chloroform, on an activated alumina column (30 g passing through a sieve of 0.149-0.074 mm d ' mesh opening) and then the column is washed with methanol. Concentrate the combined eluates and crystallize the residue from aceto.
 EMI86.6
 born. The product is 1,1 ° -Pregradiene-17r, 21-diol-3,11,0-trione 21-acetate (1-dehydrocortisone 21-acetate).



   In Examples 33 to 35 are described methods for introducing the 9 \ -halogen group into 1,4-pregnadiene-2-ol compounds.
 EMI86.7
 



  Ex.e ': l1 "\ l 33.



  '.-¯'1.-cetwCe ôe 1,., (11) -: rnC.trin-17 of, 21-diol-3,20-dione (II.



  Or acetyl of 1,; - rrenadine-11 ,, 17, 21-triol-3,20-dione (IIIa) with acetic acid in pyridine, and to

  <Desc / Clms Page number 87>

   1.22 g of this ester dissolved in pyridine and cooled
 EMI87.1
 ; :; # ix 'of lc, ülace C, 51 - C ci? 3 of phosphorus oxychloride is added, then the reaction mixture is left to stand at room temperature overnight. At the end of this period, we con-
 EMI87.2
 Center the solution under vacuum at 25 ° C to a voluria of 5 cc: i3, and 50 cm3 of water is added.

   The oil which separates is taken up in ethyl acetate and the extracts are washed once with water, with 1N hydrochloric acid until the pyridine has disappeared, again with water. 'water and then with a 5% sodium bicarbonate solution. The ethyl acetate solution is then dried and concentrated in vacuo. The residue is triturated with ether, which causes crystallization of the 21-acetate 1,4,9
 EMI87.3
 (11) - meadow, natriéne-17 (, 21-dio: -, 2U-dione.



  Example, 34- '9r-fltioro-1,4-pr'f- "adip-113 l', 21-triol-2U-dione.



  ..> Acetylated in .. 21 du, l, 4-prfnadiene; llDC, 7! Q0l, 2 - tr: iol-3, 20-diohe (VIIa) ;, to a solution of 1, 0 of acetate in 15 increments of anhydrous pyridine at Q to which 1.0 g of p-toluene chloride is added. 0 let the de-reaction return to
 EMI87.4
 temperature and left to stand overnight. It is then diluted with ice water and the resulting precipitate of 21-acetate is removed by filtration from the solution.
 EMI87.5
 11 c --tolttene-su.onate (T j).



   A solution of 1.0% in 25 cc of acetic acid containing 2.0 g of sodium acetate is boiled under reflux for 45 minutes. The resulting solution is concentrated in vacuo and the residue is stripped with methylene chloride. The methylene chloride extract is washed with water to remove acetic acid and dried over anhydrous magnesium sulfate. The dried solution is concentrated and the residue is triturated with ether. A fact crystallize the product (IIj)
 EMI87.6
 from c'c ^ :. CÉ: -t0: lG'-128i: 2.'¯2e.

  <Desc / Clms Page number 88>

 
 EMI88.1
 



  At U11e ".p'-ioL '-le 2.0 g of II j ìne: .. in% <.i.rà.xé in 200 cm3 of .l; j, cri - .. to purified and z0 e # = 15 a '; :: C. "1.0 g of Y-hi.J = ¯ is added oa> é'ia:.; i; 1.ec "t eiiJ 1. , '1' L \ ft.Jl-'t :): -oae - ": t \:.: L:; ¯: 3 ot 1C 01: i ...) C'.C'.1W. '. . Aqueous chloric acid 1 1; '. The.-éīan $ L do' vca: îī'E, -. is stirred until the solid in i ::: li.; J:, C : 2. :: '1i (11 3; .. L'W ce., Tii, i?>;. # O # 1 "u in solution. After one hour, we e..r.02¯' :. : aqueous sodium sulphite yOLl. # destroy excess 1: 1- 1) rCU02ctiùe. VS The resulting solution is then extracted thoroughly with methylene chloride and the extracts washed with water to remove the residue.

   After setting over raa.ize: sâ¯wn sulfate, the solution is concentrated in vacuo and the solution is crystallized.
 EMI88.2
 r 1.1, '¯ from acetone, which gives 9-bromo-1,4-pr 21-acetate; =' zadien-11, 17 ', 21-triol-, 2G-dione crystallizes.



   .



  A solution is boiled under reflux for one hour.
 EMI88.3
 1.0 g of the 9 'compound aror.ié in 100 cm3 of methanol contG "' <.: 1t 2 g of anhydrous potassium acetate, .. then concentrated to a residue. The solid is thoroughly stripped with methylene chloride and the extracts washed well with water. After drying, the extracts are concentrated and crystallized from acetone-hexane to give 9,11- 21-acetate.
 EMI88.4
 oxyldo-1,4-iJi, Ôcnadiene-17 li, 21-diol-3, 20-d, ione crystallizes.

   A solution of 1.0 g of the oxido-o-compound, in 50 cm3 of alcohol-free chloroform, is saturated with anhydrous hydrofluoric acid at 0 C. After 5 hours at this temperature, the mixture is removed under vacuum at 0 C excess hydrofluoric acid and the sol-
 EMI88.5
 is worth and the residue is crystallized from rI I8.CtoIle-lc: ': [:. nc: to have the 21-acetate of 9-fluoro-1-dehydroccrtisol crystallized. The latter is then hydrolyzed in a methanol-chloroform solution with aqueous hydrochloric acid at 2G-: 5 C, over a period of about 40 hours, to obtain the free 21-ol (9 -fl-uoro-1 -àé] i.yàroc-1rtisol) in which tJrÜ:; - ;: 11Ü .. ::: did not start from tsn-hexane.



  We can oxidize 21-acta Le in 21-aoé: ta ';;; of 9 Ql -s :: - I ;;. L o-

  <Desc / Clms Page number 89>

 
 EMI89.1
 1-dchydrocortisone by adding dropwise to an above 162 mg of the first, in 10 cm3 of glacial acetic acid, a solution of 30 mg of chromium trioxide in 0.5 cm3 of water and 2 cm3 of acid glacial acetic. After 6 hours, the green solution is diluted with 2 cm3 of methanol and concentrated in vacuo.



  The residue is thoroughly stripped with methylene chloride and the extracts are dried over anhydrous magnesium sulfate. The con-. Centering the dried solution affords crystalline 9 @ - fluoro-1-dehydrocortisone 21-acetate which can be readily hydrolyzed to the free 21-ol (IV, Z = F).



  Example 35.
 EMI89.2
 



  9 -chloro-1-renadien-III13 17oC 21-triol-20-dione.



  Saturated a solution of 1.0 g of 21-acetate of 9 (11) - oxydo-lp4-pi6gnadiene-17, x ', 21-diol-3,20-di-onc, in 50 cm3 of free chloroform. alcohol, with anhydrous hydrochloric acid at 0 ° C. After 5 hours at this temperature, the excess hydrochloric acid and the solvent are removed under vacuum at 0 ° C.
 EMI89.3
 and the residue is crystallized from acetone-hexane to have crystallized 9 -ohloro-1-dehydrocortiBol 21-acetate. The latter is then hydrolyzed to obtain the 21-ol-free compound.



   By oxidation of the acetate in the manner described in the previous example, 9 [alpha] -chloro-1-dehydrocortisone 21-acetate is obtained which can then be hydrolyzed to the free 21-ol.



   The ester of Example 33 can be hydrolyzed by boiling under reflux a mixture of 1.0 g of the ester in 10 cm3 of methanol under nitrogen and adding 0.22 g of sodium bicarbonate in 1 cm3 of nitrogen. 'water. Boiling is continued under reflux for 10 minutes, then the reaction mixture is neutralized with acetic acid. The solvents are removed in vacuo, the residue is exhausted with methylene chloride and the free 21-alcohol is crystallized by the addition of hexane.

  <Desc / Clms Page number 90>

 to the concentrated solution of methylene chloride. The following examples include the preparation of long acting esters of 1,4-pregnadienes and show how these ester groups can be used to protect 21-hydroxyl during the synthesis of pregnadienes.



  Example 36.
 EMI90.1
 



  14-renadiene-11 21- (5'-t-butylfuroate) 17. 21-triol-3,20-dione.



  1 g of 1,4-pregnadiene-113,17,21-triol-3,20-dione is dissolved in 10 cm3 of pyridine, refrigerated with stirring in an ice bath and treated with 0.6 g of chloride. 5-t-butylfuroyl. After stirring for one night, pour the med-
 EMI90.2
 Mix 4 years de'l'acid sulfuri% ue, diluted and the solid is extracted with ether. The crude product is crystallized from a benzene-methanol mixture to have the product pure.
 EMI90.3
 with decomposition at about = 237-23 C.



  Example'37. 1 1 # 'A. 21- 2' 'q-' -dzchloro hénox s, tae de ré ane-3 17 C 21- 'trio l-11, 20-dio'ne.



  100 g of pregnan-3 y, 17cC -dio '.- 1Z, .20-dion.e are dissolved in 1 liter of chloroform containing 1% ethanol, the mixture is cooled to 10 ° C. and treated with 12. g of gaseous hydrobromic acid. After refrigeration to -20 ° C, and remaining below -1010, the mixture is treated with a solution of 48 g of bromine in 650 cc of chloroform for a period of 3 hours.



  The reaction mixture is then concentrated to 250 cm3, under vacuum and below 25 C. To the solution is added 800 cm3 of acetone, f
 EMI90.4
 then 200 g of 2 ″ 4-diciloronhér2oxyacétote potassium (obtained by evaporation under vacuum of a methanolic solution of 2,4-dichloroplidiio., -.- acutioue acid neutralized with potassium carbonate) The mixture is treated with 200 cm3 of water, mixed well and boiled under reflux for 16 hours.
 EMI90.5
 removes chloro the solvaiit by entrainment with water vapor after

  <Desc / Clms Page number 91>

 
 EMI91.1
 addition Ciw Vv Ci: l :) Co. t eci.L <., and we heat 10 ..clC.l, t. '. pcjidant 30: i.: il1uto: J 2 1CC "" '. The product is collected by filtration and it is raised 5 times in hot water.

   It is crystallized from aqueous methanol to obtain the 21- (2 ', 41-dichloropliélio .-- lac, - tate) of pregnane-3: C, 17, x, 21-triol-11, 2O-diOxle pure .



  B. 21- (2, ° -dichloropheno-ette) çlFé.E2} Fle - = \ - 7 "X, 21-dJo- 3,11,20-trione.



     The wet crude residue of Example 37A is dissolved in 1170 cm3 of acetone and 100 cm3 of water. The solution is cooled to -5 ° C and the pH is adjusted to 2.4 with hydrochloric acid.
 EMI91.2
 611 (0.2-1.0 cm3). In the dark, 89.8 C of it-uromosuccinimide is added and the mixture is maintained at -5 C for one hour.



  To the red solution is added sufficient sodium sulphite solution (123 g of sodium sulphite in G70 cm3 of water) to bring the pH to 4.5-5.0 and the mixture is brought to the boiling point. afraid of water until the temperature reaches 100 0 for an hour. The product is separated by filtration and washed 3 times with hot water and then dried. We purify the
 EMI91.3
 21- (2 ', q .'- dichlorophenoxyacetate) of pre; nane-17o, 21-diol-3,11, 20-trione by crystallization from an acetone-methanol mixture.
 EMI91.4
 



  C. 21- (2 'L °' -dic: hloroohc: xzo = Lj% o.céto.te) of 2, ° -dibromopréFfnane- 17 x 21-ol-3 11 20- = trione.



   Ten grams of the product of Example 37B are dissolved in 250 cm3 of glacial acetic acid, stirred and drop.
 EMI91.5
 a solution of F, 1'5, j of bromine in 50 cru.3 of acid. acetic as quickly as the color dissipates (about 30 minutes). Addition of the crude dibromide to the precipitate, which is separated by filtration. Crystallization from aceto
 EMI91.6
 IIG-'1thnll01 gives 21- (2 ', .'- dichloroplxc, no: r¯yactate) of 2,4- dilror.iolz .. vs: l: a-17 oC, 21- :? Pure iol-3, 11, 2C-trione.



  21- (2'¯, 4 r 1 - ,;} ¯-: Pl'é'1 (lj; .. è: ¯n¯-17c (.J .. t ¯-, 'i. ¯, = Licï : loroi "¯.4xlO ra'clCE; ty.te) de 1.-nzna clner 17U,, l- ,:: Lol - :: L.1l., t1.: la-tri one,

  <Desc / Clms Page number 92>

 
 EMI92.1
 ,,: 1, ¯1¯¯: 3 t t 5 G of the product of example '; 7;:; L, 11 tj 5 c-: t3 de, ..;. L, - :. 1 \ 'l1'.0 and we keep it at:' '> - 1 t .. v for 1 hour.



  , l1: r:.: itG the mixture is steamed with water to remove the quinaldine and the ester is extracted with ether. The solution is washed with dilute sulfuric acid and water, dried.
 EMI92.2
 and evaporated to a residue. Chlorination from benzone on activated magnesium silicate gives pure 21- (2t, 4t-dichlorophenoxyacetate) 1,4-Pregnadiene-17 .x,: '1-diol-, 11, 20- pure trione. , which is eluted with benzene; it has a rotation of about ([alpha]) D + 116 (dioxane). example 38.
 EMI92.3
 



  A. Pregnane-11 3 17 (21-triol-3,20-dione 21- (4'-chlorophenoacetate).



  In the same way as in Example 36, 2 g of
 EMI92.4
 prenan-11; , 17 (, 21-triol-3, 20-dione with 1.4 g of chloru: =: of 4-chlorophenoxyacetyl. The crude product is dissolved in methanol and concentrated to obtain 21- (4'-chlorophenoxy -
 EMI92.5
 acetate) crystalline pregnane-11,17x, 21-triol-5, 20-dione.



  B. 21- (4'-chlorophunoxyacetate) 1,4-pregnadiene-11,317: X 21-triol -, 2C-dione.



  1 g of the product of Example 38A is brominated as in
 EMI92.6
 example bzz1 with 0 f 62 r of bromine in acetic acid. The precipitated crude dibromide is dried with water and dissolved in 10 cm 3 of toluene and 5 cm 3 of 2,4, C-collidine. The mixture is boiled under reflux for 16 hours and entrained with water vapor to remove the solvents. We process the product
 EMI92.7
 crude and cl.ro1 # atoci'apiiie conne in Example 37D and 11gluant bellZéni'lue is crystallized from methanol to have the 21- (e'-chloro: -inoîrc, etate) 1,4-prenadiene -11, 17 C, 21-trio1-5, L0-dione, PB 184-186 0.



  Ex: item 39.



  A. h 1 n. no <1¯ 'allopré <; nz-iie-11/17 <, 21- -j; Y-i ol ¯ = 2Q- i oü.

  <Desc / Clms Page number 93>

 
 EMI93.1
 



  We 8, 'W ¯1':.: 1.1C 1, "{le ±) -g, to; =; r: Eiie-11 / $, 17 çl, 21-.ti, iol-µ, 11-. l5ic> n- # designed in the 3G people with 7, C {'; 4-chlo- l'v ",; :: 0: ..:' :. o:; :: rac, t: Tle. in crystallizes the crude product from Ct 't: .8, y: ol a S'5: to have the 21- (4'-chlorophenoxyacetate) of, -'c: ßrio-11, 17,,, 2l- pure triol-3,20-dione. Z1 ot 5 g of UGt ester suspended in 150 µl of ethyl acetate and 1 g of charcoal to 5) 1 of palladium is added. We gi t: 1 :) nl8.fl:; e with hydrogen until one equivalent (231 ci, µ at 25 C) is absorbed and the absorption stops. We filter 1C: .l <ÙC.llL ': G and the solution is concentrated to 25 Jm3. The product which crystallizes out is removed by filtration and dried.

   It can be further crystallized from acetone to have the 21- (1% '- chlorophenoxyaJeta'te) of allopregnalia-11, 17,, 1-triol-3,20-dione. It is also possible to prepare r e; tcr by hydrolysis of 21-acetate of allopregnane-11 J, 17: \, 21- -t; riol ->, a0-d: ione and re-esterification. 10 g of 21-acctate are dissolved in 200 cm3 of methanol and treated with a solution of 2.95 f.S of potassium bicarbonate in 25 µm of water.



  \. 'n a. ('; i tG le 1111c..nr; c for 24 hours; 3 and nitrogen is bubbled slowly through it. then acetic acid is added until neutral, then add 5C C111) of water and 1 concentrate the mixture f101 \ f1 vit'So to 125 e #: 13. The precipitate is collected, washed with ub water or crystallized ii from dilute methanol for; ii <: 1: 1 a1¯to1'êglzîc-113.17fl (, 21-triol-5, Ei-dione. Jn esterifies 5 g, 10 this {c..cJl1: 1'0 CC.:ltlG in the 'G: i'.8..1ple 56 "voc ::;, 5 (from e llLJ J': tr0 do.; -Oh1 oro 3H:;. 0 :: TC" oé t;: rle. Ion crystallized the product at p2l.:J..".L 'èe ii: = a: Lalm: -. itl: .d, 1101 to have the 2l- (4'-cliloro- j'> linked ::,> ;.: j; -; ¯, 1 e1; r .ii) of allogrégle.ne-1l, lÎ'p (, 21-triol-3,0-dione.



  "., - ('1 h" l,.' .. .. ¯, j) ja - 4L, 'egna.d. :: 1' '"] l 1" / - / 21- L 1. O u. J r ... V -â.f-! : '::.



  .... 11 covers 2 g. 1 l .rcè, bed of Example 39A B, '/ oc 25 cm3 of glacial acetiv acid, on 8gi - :: ;; ')' on .1¯i? IrYQCal5li 'drop drop

  <Desc / Clms Page number 94>

 
 EMI94.1
 1, 25: L bromine in 5 CDU of acetic acid. After the color has disappeared, water is added, the crude dibromide is filtered off and dried.



  * Cn suspends 1 g dudibromide in 5 cm3 of diethylaniline and is heated to 95-100 0 on a bath. steam with agitation for 6 hours. The mixture is then poured into dilute sulfuric acid and extracted with methylene chloride.



  The solution is dried and evaporated, and the residue is crystallized three times from methanol-acetone to obtain
 EMI94.2
 1,4-Pregnadien-113, 17-0 21- (4'-ch16rophenoxyac "state) <21-triol-3,20-dione, m.p. 184-186 C, which is the same product as in Example 38B.



  Example 40 ..
 EMI94.3
 



  79 -fluoro-14- '21- (2'-furoate), dienene-17o (21-diol-: 1, 20-trione.



  Dissolve 1.0 g of 9 -f lu: oro-1, 4-pregnadiene-17 aC, 21-diol-3,11,20-trione in 20 cm3 of anhydrous pyridine and cool the resulting solution. at 0 C .. 0.45 g of 2-furoyl chloride is added dropwise to the stirred solution. The reaction mixture is allowed to warm slowly to room temperature and stirred overnight; The mixture is then poured into 200 cc of cold 10% aqueous sulfuric acid. The resulting precipitate is filtered and washed successively with water, aqueous sodium bicarbonate and water. Recrystallization
 EMI94.4
 from methanol-water provides the'21- (2'-furoate), as a crystalline substance.



   Analogously one can obtain 21- (2'-furoate) of
 EMI94.5
 9 ci ...;.: Cluoro-1,4-prée; nadiene-11 (3, 17, 21-triol-3,20-d3.one and 9 i. -Cl: 10ro-analogues of these two compounds By employing 5-t-butyl-2-furoyl chloride, phenoxyacetyl chloride and 2,4,5-trichlorophenoxyacetyl chloride, 21- is obtained.



  (5'-t-butrl) -2'fztrozte%, 21-phenoxyacetate and 21- (2 ',

  <Desc / Clms Page number 95>

 
 EMI95.1
 ', 5'-U; ricnloroyW ¯loxTacetate) of the 9 x -rluor o- and 3 \ -chloro- derivatives of 1-dehydrocortisol and 1-dehydrocortisone.



   By reacting the corresponding free 21-alcohol with the chloride of the acid, the following additional esters were obtained:
 EMI95.2
 1,4-Pregnadiene-21- (5'-bromofuroate) 1,4-Pregnadiene-21-ol-5,20-dione, 1,4-Pregnadiene-11 3,17 x 2l-furoate, 21-triol-3,20-dioxie , melting at 240-242 C (with decomposition),
 EMI95.3
 21- (4'-methylphenoxyacetate) 1,4-Pregnadiene-11 3,21-diol-3,20-dione, 21 - (5'-chlorofuroate) 1,4-prenadine-11 J, 17,> ,, 21-triol-3,20-dione, 21- (41-t-butylphenoxyacetate) 1,4-pregnadiene-11, 17, 21-triol-3,20-dione, mp 203-206 C (dec .) 1,4-Pregnadibile-21-phenoxyacetate-21-ol-3,20-dioil, 21- (4'-chlorophenoxyacetate) .e 1,4-pregnadibile-17c, 21-diol- 3,11,20- trione, the .F! , ± 3U-3. ± 32 C, 21- (4'-rnethoxyphenoxyaceta, 1,4'Pregnadiene-17 x, 21-dio3 - 3,11,20-trione, P.

   .r1.35C (loss of solvate), 21- (5'-bromofuroate from lpC; nadii.e-17, 21-diol-3,11,20-trione, 21-phenoxyacetate from l, prenadienll, 17 x, 21 -triol-3,2U- dione, PF 19Gwl99'Cf 21- (4'-bromophenoxyac; ate) 1,4-pre; nadine-113,17 (, 21-triol-3,20-dione, 21-furoate 1,4-p.enadine-17., 21-diol-3,11,20-trione in the forms, 233-35'C and 251-254 C, 21- (4'-t-butylphenoxyacÓtate) of l, q - prenadiene-17 ', 21-diol- 3,11,20-trione, Pi 130-135 0 (loss of solvate), 21-pheno: 1,4-pre- cyacetate, adiene-17 -> , 21-diol-3,11,20- vrione P, F 205-207 C, and 21- (5'-t-butylfuroate) 1,4-hrenadine-17, 21-diol-3,11,20- trione I, F. 241-243 C.



   These esters are administered according to the various methods described above.



   As will be understood from the foregoing, the sequence of operations for preparing a desired product from a given starting compound will be chosen in accordance with the principles of the invention taking into account the possible effect of groups or configurations. interfering with the subsequent operating phases of the process, and the necessary presence or the preliminary introduction of a group in which a particular operating phase

  <Desc / Clms Page number 96>

   process culture operates.



   By the term "oxygen function" as used herein is meant ketone oxygen and groups convertible therein.



    CLAIMS.
 EMI97.1
 ------------------------------ 1.- Process for preparing a compound of formula:
 EMI97.2
 wherein X is 0 or (H, OH), R is H or acyl and W is H, F, 01 or Br, characterized in that a saturated pregnan or an unsaturated pregnan lacking one or more several of the following: (a) the 1,2 strong bond, (b) the 4,5 double bond, (c) the X group, (d) the 17 [alpha] -OH group, (e) the group 21-OR, and (f) group 9 [alpha] -fluoro, chloro or bromo, is subjected to one or more treatments by means of which the absent characteristic or characteristics are introduced into the molecule.



   2. - Process according to claim 1, characterized in that the starting compound comprises an oxygen function in positions 3 and 20;
3. - Process according to claim 2, characterized in that the oxygen function is a ketone oxygen, a hydroxyl group or an ester group.



   4.- Method according to claims 1 to 3, characterized in that the starting compound is subjected to at least one of the following operating phases in any order: (a) introduction of a 1,2 double bond, (b) introduction of a double bond attached to carbon 5.



   (c) introduction of an 11-oxygen function, (d) introduction of a 17 [alpha] -oxygen function, (e) introduction of a 21-oxygen function, (f) introduction of a 9-fluoro group , chloro or bromo,

  <Desc / Clms Page number 98>

 these operating phases comprising in any case the introduction of the 1,2 double bond.



   5. - Process according to claims 1, 2 and 3, characterized in that the starting compound has a double bond attached in at least one, but not in more than three of the positions 1, 4, 6, 9. (11) and 16.



   6. - Process according to claims 1 to 5, characterized in that the starting compound has a double bond attached to a carbon of ring A.



   7. - Process according to claims 1 to 6, characterized in that the starting compound comprises a hydroxyl or ester group at least in one of positions 11, 17, 20 and 21.



   8. - Process according to claims 1 to 7, characterized in that a double bond is introduced into the ring A treatment of the starting compound with a culture of a dehydrogenating microorganism which does not simultaneously degrade the molecule. , or with the enzymatic material separated from this culture.



     9. A process according to claims 1 to 7, characterized in that the introduction of a double bond. 1, '2 is carried out by treatment with a culture of a dehydrogenating microorganism which does not simultaneously degrade the molecule. , or with the enzymatic material separated from this culture.



   10. A method according to claims 8 and 9, characterized in that the microorganism belongs to the Cornne-bactriaceae family.



   II.- Method according to claim 10, characterized in that the microorganism belongs to the genus Corynebacterium.



   12. A method according to claim 11, characterized in that the microorganism is Corynebacterium simplex.



   13. - Process according to claim 11, characterized in that the microorganism is Corynebacterium hoagii.



   14.- A method according to claims 8 or 9, characterized

  <Desc / Clms Page number 99>

 in that the microorganism is Bacillus sphaericus var. fusiformis.



   15. - Process according to claims 1 to 14, characterized in that the starting compound has an 11-hydroxyl group.



   16. - Process according to claims 1 to 14, characterized in that the 11-hydroxyl group is microbiologically introduced.



   17. - Process according to claims 1 to 14, characterized in that the 11/3-hydroxyl group is introduced chemically.



   18. - Process according to claims 1 to 17, characterized in that the 21-hydroxyl group is introduced microbiologically.



   19. A method according to claims 1 to 18, characterized in that the 17 [alpha] -hydroxyl group is introduced microbiologically.



   20. - Process according to claims 1 to 18, characterized in that the 174 / -hydroxyl group is introduced chemically.



   21. - Process according to claims 1 to 20, characterized in that the 9Y-halo group is introduced into a 1,4-pregnadiene.



   22. - Process according to claims 1 to 14 and 18 to 20, characterized in that the 9, -halogeno and 11ss-hydroxyl groups are introduced into a 1,4-pregnadiene.



   23. A process according to claims 15 to 17 and 22, characterized in that the 11-hydroxyl is subsequently oxidized to ketone oxygen.



   24. - Process according to claims 1 to 7, characterized in that the 1,2 double bond is introduced chemically.



   25. - Process according to claim 24, characterized in that an oxygen function is introduced in position 1 and in this

  <Desc / Clms Page number 100>

 that it is separated later.



   26. - Process according to claim 24, characterized in that an oxygen function is introduced in position 2 and in that it is subsequently separated.



   27. - Process according to claims 1 to 7, characterized in that the two double bonds in the ring A are introduced chemically.



   28. - Process according to claim 27, characterized in that the ring A of the starting compound is saturated and in that it is brominated at positions 2 and 4, after which the compound is dehydrobromided.



   29. - Process according to claim 21, characterized in that the atom 9 ,, - ,, '- halogen is introduced into a 1,4,9 (11) -prégna- triene.



   30. - Process according to claims 1 to 14, 16, 17 and
 EMI100.1
 21 to 24, characterized in that 4-pregnene-17oC, 21-diol-3,20-dione or its ester is hydroxylated in position 11 and in that it is dehydrogenated to produce a double bond between the carbons. 1,2 bones in any order.



   31.- Process according to claims 1 to 14 and 16 to 24, characterized in that the progesterone is hydroxylated in position 11, in position 17 [alpha] and in position 21, and in that it is dehydrogenated. into ring A to introduce a 1,2 double bond after hydroxylation.



   32. - Process according to claims 1 to 14, 18 to 21 and 24 to 28, characterized in that an 11-hydroxyl is introduced in
 EMI100.2
 1,4-Pregnadiene-1 ?, 21-dioh-3,20-dione or its ester and, if desired, the 11-hydroxyl is oxidized to ketonic oxygen or the ester group is hydrolyzed.



   33. - Process which can be used in the preparation of pregnadienes according to claims 1 to 4 and 7, characterized by the operating phase which consists in subjecting a compound

  <Desc / Clms Page number 101>

 pregnene, having a double bond attached to carbon 5 and a hydroxyl group at a position other than the 3 position, to the action of a culture of a dehydrogenating microorganism which does not simultaneously degrade the molecule or enzymatic material separated from this culture, with a view to introducing a double bond into ring A, and in that the pregnadiene thus formed is separated.



   34.- Process according to claim 33, characterized in that the starting compound is 5-pregnene-3,20-diol.



     3. A method according to claims 1 to 24, characterized in that the starting compound is a 3-keto-4-pregnene or a 3-hydroxy-5-pregnene, or an ester thereof.



   36. - Process according to claims 8 to 14, character
 EMI101.1
 in that the starting compound is 5-pregnene-3.17 A ',. ¯ ,. triol-11,20-dione, 5-pregnene-3,17, ', 20,21-tetrol-11-one, 5-pregnene-3,11, (or), 17 0,21-tetrol -20-one, 5-pregnene-3,11 (eX or (3), 17, 20,21-pentol, 4-pregnene-170 (.



    21-diol-3,20-dione or an ester of these compounds.



   37. - Process which can be used in the preparation of the compounds defined in claim 1, characterized by the
 EMI101.2
 operative phase which consists in subjecting 4-pregnene-11, 17c (, 21-triol-3,20-dione, 4-pregnene-17oC, 21-diol-3,20-dione, corticosterone, deoxycorticosterone, 5-pregnene-3, 20-diol-17ol-hydroxy-progesterone, 4-pregnene-11 / 3,21-diol-3,20-dione, 4-pregnene-21 -ol-3,20-dione, 4-pregnene-21-ol-3,11,20-trione, 4-pregnene-llp (, 17, 20,21-tetrol-3-one or 5-Pregnene-3,11c. (, 17ç (, 21-tetrol-20-one with the action of a dehydrogenating microorganism as defined in claims 8 to 14.



   38. - Process according to claims 9 to 14, characterized in that the starting compound is cortisone, hydrocortisone or an ester thereof.

  <Desc / Clms Page number 102>

 



   39. - Process according to claims 1 to 24, characterized in that the starting compound is a pregnadiene having a double bond attached to each of the carbons 4 and 6 and in that it comprises the operating phase of partial hydrogenation of the
 EMI102.1
 1,4,6-Pregna.triene formed to saturate the double bond attached to carbon 6.



   40. - Process according to claims 1 to 20, characterized in that the starting compound contains a 9 [alpha] -fluoro, chloro or bromo group.



   41.- The method of claim 40, characterized in that the 9 [alpha] -halogen group is a fluoro group.



   42.- Method according to claim 40, characterized in that
 EMI102.2
 that the starting compound is, 9 a-ha, logeno-4-pregnene-21-01-3,11; 20-trione, 9c'-halo-4-pregnene-1? r, 21 -d '^. 3,11,20-trione, 9-haloàeno-4-p'regnene-il (3. ,, 21.-d'iol-3 and 20-dione of 9-halo-4-pregnene-. 13,, 1c ;, 21-triol-3,20-dione.



   43.- Process according to claims 1 to 14 and 16 to 18, characterized in that the starting compound is 17 [alpha] -hydro-
 EMI102.3
 ZY-progeoterone or 5-pregnene-3,1? -Diol-20-one.



     44 .-. Process according to claims 1 to 14, 16 'and 19, characterized in that the starting compound is deoxycorticosterone or 5-pregnene-3,21-diol-20-one or a of their esters, the treatments being carried out in any order.



   45. - Method according to claim 20, characterized in that
 EMI102.4
 that a 1,4,16-pregnatrtene is reacted with a peracid and with hydrogen peroxide to form a 16,17-oxydo-derivative which is then hydrolyzed to introduce a 17-hydroxyl group.



   46. - Process according to claim 45, characterized in that the triene is formed by dehydrogenation of a pregnadiene having a double bond attached to carbon 5 and a second double bond in position 16,17, the oxido group being formed

  <Desc / Clms Page number 103>

 prior to dehydrogenation if there is no hydroxyl present in a position other than position 3.



   47.- Process according to claim 45, characterized in that the starting compound contains a hydroxyl or aoyloxy group in the 21 position or a hydroxyl group in the 11 position.



   48. - Process according to claims 1 to 13, 16, 18 'and
20, characterized in that a pregnadiene having a double bond attached to carbon 5 and a second double bond at position 16, 17 is first oxygenated, and subsequently dehydrogenated in ring A.



   49. - Process according to claim 48, characterized in that the starting compound is 16-dehydropregnenolone or its ester or 16-dehydroprogesterone.



   50. - Process according to claims 1 to 13, 16 and
 EMI103.1
 characterized in that 1a5,16-pregnadiene-3,2.- diol-20-one, or a diester thereof, is converted to the epoxide 16,17, in any order, and in that it is hydrolyzed to introduce
 EMI103.2
 a -17 (x'-hydroxyl.



   51.- Process according to claims 1 to 13 and 18, characterized in that 16-dehydropregnenolone is subjected to the action of a culture of a 21-hydroxylating microorganism, and subsequently to the action of a dehydrogenating mioroorganism to pro-
 EMI103.3
 reduce 1,4,16-prégnatriéne-21-01-5,20-àione.



   52. - Process according to claim 27, characterized in that normal- or allo-pregnan-2,4-dibromo- is reacted
 EMI103.4
 11X-17c, 21-diol-3,20-dione, or its 21-ester, with dehydrobromidating agent to introduce two double bonds into ring A, X being 0 or H, OH ([alpha] or ss).



   53.- Process according to claim 52, characterized in that
 EMI103.5
 Pregnan.e-17 01, 21-diol-3,1., 20-acetate is reacted with bromine to form 2,4-dibromide, in that the dibromide is di-dehydrobromide to '' produce 21-acetate

  <Desc / Clms Page number 104>

   1,4-Pregnadiene-17 [alpha], 21-diol-3,11,20-trione and, if desired, in that the ester group is hydrolyzed.



   54. - Process according to claim 27, characterized in that the 21-acetate of pregnan-11 [alpha], 17 [alpha], 21-triol-3,20-dione is reacted with bromine to form 2 , 4-dibromide, in that the dibromide is di-dehydrobromideed to produce the corresponding 1,4-pregnadiene-compound, and in that the 11 [alpha] -hydroxy is oxidized to a ketone group.



   55. - The method of claim 27, characterized in that reacting 21-acetate of normal- or allo-pregnan-11ss, 17 [alpha], 21-triol-3,20-dione with bromine to form 2,4-dibromide, and in that the dibromide is di-dehydrobromide to form the corresponding 1,4-pregnadiene compound.



   56. - Method according to claim 26, characterized. e converting a 3-keto-1-pregnene into the corresponding 1,2-oxido compound, hydrolyzing the latter with a halogenated hydracid to produce a 1-hydroxy-2-halo-3-keto pregnance, in that the halogen is separated, in that the 1-hydroxy group is replaced by an acyloxy group, in that halogen is introduced in position 4, in that one submits the compound undergoes dehydrohalogenation to produce a 4,5 double bond and in that the 1-acyloxy group is subsequently separated to introduce a 1,2 double bond.



   57. - Process according to claim 56, characterized in that the starting compound is a 1-pregnene-11X-17 [alpha], 21-diol- 3, 20-dione and its 21-esters, X being '0 or H, OH ([alpha] or ss), and the halogenating agent being bromine, while the halogenated hydracid is hydrobromic acid.



   58. - Method according to claim 25, characterized in that heating a 3-keto-6-bromo-l-pregnene with an alkali metal salt of a lower alkanoic acid to form a 2-alkano-yloxy-3 -keto-1-pregnene, and in that the

  <Desc / Clms Page number 105>

 acyl group in position 2 to introduce a 1,2 double bond.



     59.- Process according to claim 58, characterized in that the starting compound is a 1-pregnene-6-bromo-11X-17 [alpha], 21-diol-3,20-dione, X being 0, H, OH, or H, OR, R being lower alkanoyl.



   60. - Process according to claim 58, characterized in that the starting compound is 21-acetate of 6-bromo-4-pregnene-17 [alpha], 21-diol-3,11,20-trione, and in what salt is that of acetic acid.



     @ 1.- A method according to claim 58, characterized in that the starting compound is 11-formate-21-acetate 6-brom-4-pregnene-11 3,17, 21-triol-3,20 -dione, and in that the salt is that of acetic acid.



   62. - Process according to claims 21 and 22, characterized in that a 21-ester of 1-dehydrocortisol is dehydrated to introduce a 9 (11) double bond, and in that the triene is then reacted with a reagent for introducing a 9 x -bromo group and an 11 3 -hydroxyl group.



   63. - Process according to claim 62, characterized in that the product is heated with a metal salt of a lower fatty acid to introduce a 9 (11) -oxydo group, and in that the oxydo group is opened with hydrofluoric acid to produce 9 [alpha] -fluoor-11ss-hydroxy compound.



   64. - A method according to claim 62, characterized in that the dehydrating agent is phosphorus oxyehloride.



   65. - Process according to claims 21 and 22, charac- terized in that a 21-ester of 1,4-pregnadiene-11 [alpha], 17 [alpha], 21-triol-3,20-dione is treated. with a sulfonating agent to produce the 11 [alpha] -sulfonate, in that the diester is then heated with a mild base to separate the sulfonic acid group and introduce a 9 (II) double bond, and then in that we treat the sort-

  <Desc / Clms Page number 106>

 ene with a reagent to introduce a 9-bromo group and an II (3-hydroxyl.



   66. - Process according to claims 1 to 65, characterized in that acetylated any free 21-hydroxyl in the product.



   67.- Process according to claims 1 to 65, characterized in that a free 21-hydroxyl is esterified in the initial, intermediate or final compounds with an aryloxy-alkanoylation agent or a furoylation agent, or a product alkyl-, alkoxy- or halo-substituted thereof.



   68. - Process according to claim 67, characterized in that the ester formed 'is 21- (2'-furoate).



   69. - Process according to claim 67, characterized in that the ester formed is 231- [5 '- (t-butyl) -2'-fuorate].



   70. - Process according to claim 67, characterized in that the ester formed is 21- (2 ', 4', 5'-trichloro) -phenoxyacetate.



   71. - Process according to claims 1 to 66, characterized in that an 11 -or 11ss-hydroxyl group is oxidized with a mild oxidizing agent in ketone oxygen.



   72. - Process according to claims 1 to 20 and 23 to 28, characterized in that the 1-dehydrocortisone is esterified with 5-t-butyl-furoyl chloride.



   73. - Process according to claims 1 to 20 and 23 to 28, characterized in that 1-dehydrocortisol is esterified with 5-t-butyl-furoyl chloride.



   74. - The new processes described in the present patent.



   75.- Compound of general formula:
 EMI106.1
 

  <Desc / Clms Page number 107>

 wherein X is 0 or H, ss -OH, R is H or acyl, and W is H, F, Cl or Br.



   76. - 1-Dehydrocortisone.



   77. - 1-dehydrocortisol.



   78. - 21-esters of 1-dehydrocortisone and 1-dehydrocortisol.



   79.- 1-Dehydrocortisone and 1-Dehydrocortisol 21-acetate.
 EMI107.1
 



  80.- 1,4-Pregnadiene-11, 170 (, 21-trial-3,20-dione and its
21-esters.



  81.- 9 [alpha] -fluoro, chloro- and bromo-derivatives of 1-dehydrocortisone and 1-dehydrocortisol.
 EMI107.2
 



  82.- 9-Fluoro-1-dehydrocortisone.



  83.- 9 --fluoro-1-dehydrocortisol.



  84. Esters of 1-dehydro-cortical hormones with aryloxy-alkanoic lines and furoic acid and their alkyl-, alkoxy- and halo-substituted products.
 EMI107.3
 



  85.- Esters of aryloxyacetic, haloaryloxyacetic, alkoxy-aryloxyacetic and alkylaryloqyaoetic acids of cortical 1-dehydrohormones.



   86.- Esters of furoic, halofuric and alkylfurol-
 EMI107.4
 ques of 1-dehydro-cortical hormones.



  87. - Compounds according to claim 84, characterized in
 EMI107.5
 that the cortical hormones are 1-dehydrocortigone, l-dehydrocortisol, 1-dehydx-oo'icottérQne, l-dehydro-11-deoxy-corticosterone; 1-dehyàro-11-deoxy-oortisone, 1- dehydro-17-deoxy-prtisore, 1-de4ydro-aldo-sterone, 1- dehydro-9 -halo.ocxt, spue and 1-dehydro- - halo-cort3.sol in which the halogen atom is fluorine, chlorine or bromine.



   88. - 21- (4t-t-butylphenoxyacetate), 21- (4'-chlorophenoxy-
 EMI107.6
 acetate), 21- (2 ', 4'-dichlorophenoxyacetate), 21- (2', 4 ', 5'-tx "i-chlorophenoxyacetate), 21- (2'-furoate), 21- (5'-chlorofuroate ), 2l- (5'-bromofuroate) and 21-L- (5'-t-butyl) -2t-furoatē7des com-

  <Desc / Clms Page number 108>

 of claims 76 to 88.
 EMI108.1
 



  89.- 21- / 5 '- (t-butyl) -2'-furoatē7 of 9 -fluoro-1-dehydro-cortisol.



  90.- 21 - / - 5- (t-butyl) -2-furoate% 9g-fluoro-1-dehydro-cortisone.



   91. - 21- (2 ', 4', 5'-trichlorophenoxyacetate) 9 -fluoro-1-dehydrocortisone.



   92.- 9 [alpha] -fluoro-1-dehydrocortisol 21- (2 ', 4', 5'-trichlorphenoxyacetate).
 EMI108.2
 



  Q3 9p-fluoro-1-dehydrocortisol 21- (2'-furoate).



  94.- 1,4,16-prregnatriene-3,20-diones.



  95.- 1,4,16-Pregnatriene-21-ol-5,20-diones and their lower alkanoyl esters.



  96.- 1,4,16-Pregnatriene-11X-21R-3,20-diones, in 1-X is 0, H, 0H (d or / 3) or H, acyloxy (R or / 3), and R is H, OH or acyloxy.



   97. - 1,4,16-Pregnatriene-3,11,20-triones and their 21-ol-derivatives.
 EMI108.3
 



  98.- 1,4,16-Pregnatriene-11,21-diol-3,20-diones and their lower alkanoyl esters.



   99. - 16,17-oxydo-1,4-pregnadiene-11X-21R-3,20-diones in which X is H2, 0, or H, OH ([alpha] or ss), and R is H, OH or lower alkanoyloxy.



   100. - 16,17-oxydo-1,4-pregnadiene-3,20-diones.
 EMI108.4
 



  101.- 16,17-oxydo-1,4-pregnadiene-3,11,20-trionea.



  102.- 16,17-oxydo-114-pregnadiene-21-ol-3,20-diones and their lower alkanoyl esters.



  103.- 16917-oxydo-1,4-pregnadiene-21-ol-3,11,20-triones and their lower alkanoyl esters.



  104.- 16,17-oxydo-1,4-pregnadiene-11,21-diol-3,20-diones and their lower alkanoyl esters.



  105.- 16,17-oxydo-1,4-pregnadiene-11-ol-3,20-diones and their

  <Desc / Clms Page number 109>

 
 EMI109.1
 C: 1te: r3 lower a1kanoylés.
 EMI109.2
 106.- 2-hydroxy- and 2-alkanoyloxy (lower) -derivatives of
 EMI109.3
 4-Pregnene-III-17F, 21-ol-3,20-diones and their lower aika-walnut 21-esters, X being 0 or H, OH (a! Or).



  107.- 4-Pregnene-2,17,21-diacetate, 2l-triol-3,11,20-
 EMI109.4
 trione.
 EMI109.5
 108.- 4-Pregnene-2.11 il-formate-2,21-α-acetate ..!, 17 ,,
 EMI109.6
 21-tetrol-3,20-dione.
 EMI109.7
 



  109.- 1-hydroxy- and 1-alkanoyloxy (inférietir) -derivatives of 4- pre-rene-11X-17vC, 21-diol-3,20-diones and their alkanoyl esters
 EMI109.8
 lower, X being 0 or H, OH.
 EMI109.9
 110.- 4-Pregnene-1-acetate and 1,21-diacetate-1,17,21-
 EMI109.10
 triol-311,20-trione.
 EMI109.11
 111- 4-Pregnene-1,11 1-acetate and 1,21-diacetate. ,. 7 \, 21-tetrol-3,20-dione. , I 112.- 1,4,9 (11) -pregnatrièri-17 ', 21-.diol-, 20-d.one and its 21-lower alkanoyl esters
 EMI109.12
 
 EMI109.13
 113., - 1,4-Pregnadiene-11 (, z 7 diol-3,20'-dione.



  . It 114.-1,4-Pregnad3.ene-11.1o (-diol-3.2o, -diane.
 EMI109.14
 
 EMI109.15
 



  115.- 1,4-Pregnad3: ene-7-0.-3.11, OTtrione.



  116.-, 4-Pregnadiene-1X-17, 20,21-t, # iol-3-ones and their 21-lower alkanoyl esters, where. X is N2t 0, HPOH (, e or / 3).



  11'l.- 1,4-prégns, s..llX-21i-, 20-diones, in which X is H2 '0 or X, 0H (tyJàù (j), zur being H, OH or an a1kanoy10xy
 EMI109.16
 inferior.
 EMI109.17
 118.- 1.4 - pxeg.n-1? , 20,21-triol-3-one and its esters
 EMI109.18
 lower alkanoyls,
 EMI109.19
 119.- 1,4-Pregnadiene-3,20-dione. 120.- 1,4-Pregnadiene-11 (3,21-diol-3,20-dinone and its esters
 EMI109.20
 lower alkanoyls.
 EMI109.21
 121.- 1,4-Pregn, adiene-21-ol-3,20-diome and its alkano- esters
 EMI109.22
 lower yles.

  <Desc / Clms Page number 110>

 
 EMI110.1
 



  122.- 1,4-Pregnadiene-21-ol-3911920-trione and its lower alkanoyl esters.



  123.- 9 -halogen-1,4-pregnadiene-11X-21-ol-3,20-diones, the halogen being F, Cl or Br, and X being 0 or H, ss -OH, and their 21- lower alkanoyl esters.
 EMI110.2
 



  124.- 9c (, - fluoro-1,4-pregnadiene-11X-21-ol-3,20-diones, X being 0 or H, / 3 -OH, and their lower alkanoyl esters.



  125.- 21-normal-2,4-dibromoprene-1 acetate? , 21-diol-3,11,20-trione.
 EMI110.3
 126.- 21-normal-2,4-dibromopregnane-11,17 d, 21-triol-3,20-dione acetate.



   127. Pharmaceutical preparations comprising 1-dehydrocortisone or an ester thereof, in admixture with a pharmaceutical carrier.



   128. - Pharmaceutical preparations comprising 1-dehydro-cortisol or its esters, in admixture with a pharmaceutical carrier.



   129. - Pharmaceutical preparations according to claims 127 and 128, characterized in that the pharmaceutical carrier is a solid and in that the preparation is in the form of tablets.



   130. - Pharmaceutical preparations according to claims 127 and 128, characterized in that the pharmaceutical carrier consists of a base of wax, fatty substance or cream.



   131. Pharmaceutical preparations according to any one of claims 127 and 128, characterized in that the pharmaceutical carrier is a non-toxic liquid.



   132.- New products described in this patent.


    

Claims (1)

<Desc / Clms Page number 1> EMI1.1 <Desc / Clms Page number 2> EMI2.1 2) The same claim, lines 3/4, should read: EMI2.2 "2-alkanoyloxy-3-keto -4-pregnene" instead of: "2-alkanoyloxy-3-keto-1-pregnene"; 3) In claim 59, it should read: "4 - pregnne -6-bromo ..,." Instead of: 'Tl.-pregnne-6-bromo ..., ";