BE549221A - - Google Patents

Description

       

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  The present invention relates to a biosynthetic process for the preparation of 11 / -liyfiroxy-stisroîdes of the series of the prút; nane (including prugnene and prégtz4diene), especially for the preparation ùe ± '- pregtladietm-1.1 / 3, 7,421-triol-3,2U-dione and its 21-esters, this process involving. microbiological oxidation of the corresponding 11-deoxy-steroid, .specially of 11-1,4-pregnadiene-17 c (, 21-diol-j, 2Ù-dione and its 21-esters.



  More particularly, the process according to the present invention consists in subjecting a LI -pr <g11a JG- ne-17 <, 21-diol-3,20-dione or a 21-ester of this compound to the action of 'an enzyme (or enzyme or system of enzymes) of a microorganism of the genus Coniotllyrium' (preferably, Coniothyriuta helleborine) in an aqueous medium, in the presence of oxygen, and to recover the fi 1,4-prégiiadièiie- 11/5, 17 d Y21-triol-3,20-dioiie or a 21-ester of this compound which formed.

   The action of the enzyme can be produced either by placing the steroid in an aerated culture of the microorganism in or on a suitable nutrient medium, or by bringing together in an aqueous medium ke
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 The presence of oxygen and the enzyme of non-proliferating cells of the microorganism, the first alternative being the preferred one.



   The steroids which can be used in the process according to the present invention include, inter alia, ¯ 1.4
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 Pregnadiene-17 ai, 1-diol-3, 20-d.ione and its 21-esters. Among the suitable esterifying acids, if a 21-ester is used, there may be mentioned organic carboxylic acids, in particular hydrocarbon carboxylic acids having

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 less than 10 carbon atoms, such as lower alkanoic acids (eg, acetic, propionic and enanthic acids),.,; lower alkanedioic acids (eg succinic acid), aromatic hydrocarbon acids (eg benzoic acid) and aralkanoic hydrocarbon acids (eg phenyl: -acetic acid and / 3 acid phenylpropionic).



   A suitable nutrient medium essentially comprises a source of nitrogenous factors and an assimilable source of carbon and energy. The latter can be a carbohydrate (such as sucrose, molasses, glucose maltose, starch or dextrin), a fatty acid, a fat and / or the steroid itself. Preferably, however, the medium comprises an assimilable source of carbon and energy in addition to the steroid.

   Among the fats that can be used for the purpose of the invention, there may be mentioned bacon oil, soybean oil, linseed oil, cottonseed oil, cottonseed oil, peanut, coconut oil, corn oil, castor oil, sesame oil, crude palm oil, mutton tallow, spermaceti oil, olive oil , tristearin, tripalmitin, triolein and trilaurine. Among the fatty acids which can be used for the purpose of the invention, there may be mentioned stearic acid, palmitic acid, oleic acid, linoleic acid and myristic acid.



   The source of nitrogenous factors can be organic (eg, soybean meal, corn maceration liquor, meat extract and / or distillers solubles) or synthetic (eg, simple synthetic organic and inorganic compounds. , such as ammonia salts, alkali nitrates, amino acids or urea).

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   An adequate supply of sterile air must be maintained during fermentation, for example by
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 customary methods of exposing a large surface area of the site to air or by using a submerged culture.



   The steroid can be added to the culture during the incubation period or included in the medium before sterilization or inoculation. The preferred (but not limited to) concentration of the steroid in the culture is about 0.01 to 0.105 The culture period or rather
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 . the time during which the steroid is subjected to the action of the enzyme can vary considerably, this time possibly being, for example, from about 6 to 98 hours (this range of time not being limiting).



   The 11/3 -hydroxy-steroid formed can be recovered from the culture, in which it is formed by extraction.
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 using an organic solvent (such as methyl isobutylketone, chloroform and methylene dichloride), followed by concentration of the extract and crystallization from an appropriate organic solvent.



   The following examples illustrate the invention.



     EXAMPLE 1.



   1.4
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 Li-prregnadiene-11! 3, 17 ci, 21-triol-), 20-dione. - (a) Fermentation - A fermentation medium of the following composition is prepared
Starch 20 gr syrup extracted from malted cereals 10 gr peptone 20 gr cerelose 44 gr
NaNO3 3 gr
KH2PO4 1 gr
KCl 0.5gr
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 1 "'1 ± S 4.120 0.5 gr FeSO 4' 7H20 0.0183 gr Water q.s. ad. 1 liter.



  The pH of the medium is adjusted to 7.0¯ 0.1 using
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 of a 2N solution of IdaUIi and 50 cc fractions of, ni-

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 place are distributed in Erlenmeyer flasks with a capacity of 250 cc. The vials are stoppered with a cotton swab and sterilized by heating in the autoclave for 30 minutes at 120 C.

   After cooling, each of the vials is inoolded with a 5% suspension of Coniothyrium he Lleborine spores, (which can be obtained in particular from the Kansas State Department of B tannic).
Middle School). / - The sporulated culture is obtained by growing pous. ser the microorganism on cracked corn for 2-3 weeks to allow maximum sporulation.



   The suspension is preferably prepared in 0.01 aqueous Duponal solutia. 2.5 cc of suspension, containing about 1 / 60th of the spores produced on 15g of maize are used to inoculate 5u cc from the first group of flasks to stir.



   The flasks are then mechanically shaken for 60 hours on a rotary stirrer at 280 revolutions per minute at 25 ° C. and a transfer of 6% by volume is then carried out. d in 40 flasks with a capacity of 250 cc containing 50 cc fractions of the following medium:
Glucose 40 gr
NaNO3 3 gr
KH2PO4 1 gr
Water q.s. ad. 1 litre
The inoculation is then carried out for 48 hours at 25 C, by mechanical agitation on a rotary stirrer at 280 revolutions per minute, after which 500 mgr of ¯ 1,4, pregnadiene-17 Ó, 21-diol-3,20 -dione are added to 20 cc of methanol. Incubation is continued for a further 48 hours.

   The contents of the vials are cooled, filtered through a Seitz pad and washed with about 10% water; The volume of filtrate and wash water is 1875 cc.

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  (b) IsolotJ \ 0nt of the IJ. 1,1'¯p, 'Ó;'; na <llLn} e-ll / .3, 1'1 d, 21-, ri.ol- (b): Lo7¯ertent of /, ¯ 1 ¯, ¯, .. ¯.¯ ... ¯ ¯ 3,20-dione.-
The culture filtrate thus obtained is extracted using 3 fractions of one liter of chloroform and: The so-
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 The remaining chloroform solution is evaporated in vacuo to dryness.

   The residue (about 238 is triturated
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 with chloroform and the resulting precipitate (about 140 mg). which is a mixture of the starting material and the desired hydroxylated product, is acetylated with 2 cc of anhydrous pyridine and 2 cc of acetic anhydride at room temperature for 18 hours.



  The mixture of acetates obtained by evaporation of the reagents is crystallized four times from 95% alcohol.
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  1,4¯Pregnadie 21-acetate is obtained! Fe -1y3 il / 0 (, 1-tr.o.-3, U-diot>, ef. F. About 237-239 C. Here J3 = + l12 (dioxeaie) i \ m 1, 9'lu, 3, U'7 lU, 5, 72 11) 5.81 r '6.16 u, 6.24 lt, 6.3U p. / this infra-red spectrum is identical to that of an authentic sample).



   By saponification of the aforementioned 21-acetate using potassium carbonate in methanol, one obtains
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 6 1,4¯Pregnadiene-1J3.7 c (, 21-trio7.-3, 'U-diotxe aut, hen- tic; m.p. about 239-240 C.



   The invention can be implemented in various other ways, without departing from the scope of the following claims.

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Claims (1)

CLAIMS.- 1.- Process for the production of a selected steroid EMI5.6 in the group comprising la to '' '-pregnadieno-11/5, 17 and 2l-triol-3,20-dion.e and its 21-esters, in which the process is subjected to a steroid selected from the group comprising la to lj4 ¯pregiiidir-% tie-l '/, 2.-diol-3, zU-.dione and se; r1-.esterr <Desc / Clms Page number 6> to the action of an enzyme of a microorganism of the genus Coniothyrium in an aqueous medium in the presence of oxygen, and the steroid formed is recovered. 2. - Method according to claim 1, in which the organism is Coniothyrium helleborine. 3. A process according to claim 2, in which the steroid is a 21-ester of ¯ 1,4-pregnadiene-17 Ó, 21-diol-3,20-dione with a hydrocarbon carboxylic acid having less than 10 atoms. of carbon. 4. A method according to claim 3, in which the acid is lower alkanoic acid. 5. A process according to claim 3, in which the acid is acetic acid. 6. - Process, in substance, as described above. 7. - ¯ 1,4-Pregnadiene-11/3, 17 Ó, 21-triol-3,20- dione and its 21-esters, when they are obtained by the process according to one or the other of previous claims.