BE519106A - - Google Patents

Description

       

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  METHOD FOR THE MICROBIOLOGICAL OXIDATION OF STEROIDS.



   The invention relates to a process for the microbiological oxidation of steroids and relates to the preparation of certain oxidized derivatives of seroid, with particularly advantageous processes for obtaining these derivatives. These oxidized derivatives of steroids, in particular the llahydroxy derivatives of steroids from the group comprising 3-oxo-20-ce to-pregnenes and 3-oxy-20-ce to-pregnenes, are intermediates for the preparation of cortical hormones and / of other useful products.



   It has been established that steroids can be transformed into the oxidized derivatives (in particular the steroids of the non-'substituted in position 11 in the lla-hydroxy derivatives) by subjecting them to the action of enzymes of an oxidizing character. of aspergillus in the presence of oxygen. The action of the enzymes can be used either by reacting together, in an aqueous medium, the steroid, oxygen and the enzymes of the non-proliferating cells of aspergillus, or, preferably, by incorporating the steroid in. an aerated culture of aspergillus.



   The oxidizing species of aspergillus which can be used in the practice of the invention include, inter alia, Aspergillus niger, Aspergillus amstelodami, Aspergillus nidulans and aspergillus designated under the reference A.T.C.C. II, 267 (this number representing the identification given to the microorganism on deposit in the American Type Culture Collection, Washington, D. C.). Preferably, the tension of the Aspergillus niger used is that identified under the reference "Wisc. (Wisconsin) 72-2", or its equivalent, the derivation of this tension being described by Perlman, Kita and Peterson in Arch. of Biochemistry, volume II, p. 123 (1946).



    @
In general, the conditions for growing aspergillus for the purposes envisaged by the invention are (except as noted above)

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 passes for inclusion of the steroid to be oxidized) the same as those of the culture of microorganisms for the production of other metabolites, eg citric acid; thus aspergillus niger is developed on contact with an appropriate nutrient (in or on this agent) in the presence of oxygen (air). A suitable nutrient essentially comprises a source of nitrogenous factors and an assimilable source of carbon and energy.



  This agent can be a carbohydrate (such as sucrose, molasses, glucose, maltose, starch or dextrin), and / or the steroid itself. Preferably, however, the agent comprises an assimilable source of carbon and energy in addition to the steroid; and preferably also, this source is at least an essential part of a member of the group comprising (1) fatty acids having at least 14 carbon atoms and (2) fats. The use of this lipoid source of carbon and energy (in particular the use of a fatty oil) is advantageous because it enriches the utility of the steroid for the intended transformation.



   Among the fats which can be used for the purpose of the invention, mention may be made of: the oil which comes from bacon, soybean oil, linseed oil, cottonseed oil, peanut oil, 'coconut oil, grain oil, castor oil, sesame oil, crude palm oil, tallow from sheep fat, spermaceti oil, olive oil , tristearine, 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 the nitrogenous factors may be organic (e.g. soybean meal, liquor from a grain infusion, meat extract and / or soluble distillery products) or synthetic (e.g. consisting of simple compounds, organic and inorganics susceptible to synthesis, such as ammonium salts, alkali nitrogenates, amino acids or urea).



   Among the steroids which can be converted into the oxidized derivatives useful by the practice of the invention, there should be mentioned those of the series of 3-oxo (or oxy) -20-keto-pregnenes (or pregnanes) and of the series of 3-oxo (or oxy) -17-keto (or bydroxy) -androstenes (or andostrans), the series of 4-pregnene-3,20-diones (especially progesterone and 11-deoxy-17-hydroxycorticosterone, or l (acetate of these compounds) being preferred.

   These steroids are indicated by way of example by progesterone, 16a-hydro-
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 xyprogesterone, α-hydroxyprogesterone, 11-deoxy-17-hydroxycorticosterone (Reichstein's compound S), 11-deoxy-17-hydroxycorticosterone acetate, deoxycorticosterone, deoxycorticosterone acetate, pregnanolone, ¯ 5-pregnenolone., 3-succinyl- ¯ 5-pregnenolone,
 EMI2.2
 anârostenedione-3,17,16-dehydroprogesterone and testosterone. where an oxidizable hydroxy group is present in the steroid and its possible oxidation is not desired, this group can be transformed into an oxidation resistant group capable of reconversion to a hydroxyl group; for example an ester, an ether or a halogenated group.



   The examples which follow are only given to illustrate by way of example various embodiments of the invention (all the melting temperatures (MP) being in centigrade and all the specific rotations being in chloroform unless otherwise indicated. opposite).



     EXAMPLE I (a) Fermentation.-
A medium is prepared having the following composition: solids obtained from a liquor of an infusion of grains 3 g.



  Nh4H2PO4 3 g.



  CaCO3 2.5 g.

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 soybean oil 2.2 g. progesterone 0.20 g.



   Distilled water quantity to make one liter the pH of the medium is adjusted to 7.0 ¯ 0.1 (with sodium hydroxide solution); and 100 ml. portions of the medium are distributed in 500 ml. Erlenmeyer flasks, and the flasks are stoppered with cotton and are autoclaved for 30 minutes at 120. When the balloons are cold, each of them receives 5-10% of a plant seeding of Aspergillus niger (Wisc. 72-2; see Perlman, Kita and Peterson, Arch. Of Biochemistry, vol. II, p. 123, 1946) also DA Kita, Bachelor of Science thesis, University of Wisconsin, 1944), obtained as described below.

   The flasks are subjected to mechanical agitation for 72 hours in a chamber maintained at 25; then the contents of the flasks are cooled, adjusted to pH 4.0 ¯ 0.2 with sulfuric acid and filtered with suction through Seitz filter pads. (The plant seeding used is developed from cultures on strains (lyophilized flask or agar plants) for 24-72 hours (with or without successive periods of 24-72 hours) in a medium having the following composition: 15 g of solids from a liquor of an infusion of grains, 10 g of brown sugar, 6 g of NaNO3, 0.001 g.



  ZnSO4, 1.5 g. Anhydrous KH2PO4, 0.5 g. MgSO4.7H2O, 5g. CaCO3, 2 g. oil from bacon, and distilled water in sufficient quantity to make one liter, the medium being sterilized in the autoclave for 30 minutes at 120.



   (b) Extraction of oxidation products.



   1880 ml. of a culture filtrate obtained as described in a are exhausted with 4 1 liter parts of chloroform and the chloroform extract (combined) is filtered and evaporated to dryness in vacuo. The residue is taken up in 15 ml. 80% methanol (aqueous), and the residual solution is exhausted with 4 parts of 15 ml. hexane (which removes fatty impurities). The methanol solution is then concentrated to a small volume and is used up with 3 parts of 20 ml. of chloroform.



  The chloroform extract (combined) is dried over sodium sulfate and evaporated to dryness, leaving a residue (approx. 411 mg.) Which crystallizes on standing.



  (c) Separation of constituents I and II.



   The residue (containing components designated as I and II) is dissolved in hot acetone and then allowed to crystallize. Component I (a dihydroxyprogesterone) is sparingly soluble in acetone and therefore separates from solution, while component II (11a-hydroxyprogesterone) remains in the mother liquor. After repeating the crystallization four times by redissolving the crystallized residue in hot acetone and then letting it crystallize (collecting the mother liquor each time), component I is obtained substantially pure with a yield of about 8 mg. Upon recrystallization from 95% ethanol, component I has the following properties: M.P. about 250-2530 (by capillarity); Ó) 23 + 100 (c. 0.28); U.V., # alc.max. 236 m / u @ 15,000); . R.

   D 2.98 / u (0), z89 max. 6.04 = 15,000); I.R. Nujol 2.98 u (OH), 589 u (20-ketone), and 6.04 u (¯max. 4-3-ketone); analysis (calcd for C21H30O4; C, 72.80; H, 8.73) C. 72.66, and H. 8.75. These indications represent component I as being a dihydroxyprogesterone, probably 6,11a- = dihydroxyprogesterone (oxidation with chromic acid gives tetracetone and acetylation gives a diacetate, showing that the two hydroxy groups are se- condaires).



    (d) oxidation of component I for a tetracetone.



   Has a solution of 550 mg. dihydroxy-progesterone (I)

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 obtained cone described in c in 4 ml. of glacial acetic acid, 31.3 mg are added. chromic acid in 3 ml. acetic acid. After standing at room temperature - for 1/2 hour, 1 ml. alcohol is added and the solution is concentrated to dryness in vacuum. The residue is taken up in 2 ml. of water and 15 ml. of chloroform and the chloroform phase is washed with dilute sodium bicarbonate solution, then with water. After drying over sodium sulfate, the solvent is evaporated to dryness in vacuo, leaving a residue (weighing about 55.6 mg.), Which crystallizes on contact with acetone on contact.

   The crystallized residue is dissolved in 2 ml. of benzene and is adsorbed in an alumina column washed with sulfuric acid (lg.); then the column is eluted with benzene (10 fractions, making a total of 250 ml.), yielding about 40 mg. of light yellow crystals. After two recrystallizations from acetone-hexane, the product (a tetacetone) has the following properties: M.P. about 144 - 146; (Ó) 23 + 143 (c, 0.87); U.V., alc.max. 249 m / u (= Il.500); # 2% max. KOH in methanol 255 m / u (c 9,800), 370 m / u (# = 8,900); analysis (calculated for c21lH26o4: C 73.66; H 7.65) C. 73.77 and H, 7.66.

   The above absorption spectrum is characteristic of 3,6- ¯ 4-endiones, and one of the hydroxyl groups of component I is therefore in position -6. The second hydroxy group of component I is indicated as being in position 11a by the contributions to the molecular rotation made by this hydroxyl group in component I (¯ (M) D = -10), by the corresponding acetoxy group in the diacetate of 1 (-37) and by the keto group derived from the previous one by oxidation to give the above-mentioned tetracetone (+ 407). The contributions of lla-hydroxy, lla-acetoxy and 11-keto, when attached to progesterone, are (M) D - 16, -51 and +302, respectively.



  (d alternative) Acetylation of component I.



   18.3 mg. dihydroxyprogesterone (I) obtained as described in c is acetylated by treatment with 1 ml. of pyridine and 1 ml. acetic anhydride for 16 hours; acetylating agents are removed by evaporation in a vacuum; andthe residue (weighing about 24 mg.) is crystallized from an ether-ethyl-hexane mixture. The product (a diacetate of the formula C25H3406) has the following properties; M.P. about 154-155; (Ó) 23 + 80.6 (c., 0.92); Analysis (calculated: c, 69.74; and H, 7.96) C, 69.74 and H. 8.09.



  (e) Isolation of 11α-hydroxyprogesterone (component II)
All the mother liquor from the crystallization of component I (see section c) is combined and dissolved in 2 ml. of chloroform and 4 ml. benzene; and the solution is subjected to chromatography on a 6 g column. alumina washed with sulfuric acid. The column is eluted with 150 ml. of a mixture of 1 part chloroform and 2 parts benzene, and the solvents are evaporated in vacuo, yielding about 163 mg. of a crystalline fraction which is purified by crystallization from an acetone-ether mixture.

   The product, 11a-hydroxyprogesterone, has the following properties: M.P. about 166-167; (Ó) 22 +177.5 (c.1.22); U.V., # alc. 241 m / u (# -17,000); I.r., D max.



     # Nujol 2.91 / u (OH), 5.85 u (20-ketone) and 5.97 / u (4-3-ketone); analysis (calculated for C21H30O3: C. 76.33; H 9.16) C. 76.12 and H 9.28.



  The product is not identical to the known 11 ss -hydroxyprogesterone, which melts at 187 - 188; it forms a monoacetate; and it can be oxidized to give the known 11-oetoprogesterone.

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   (f) Acetylation of 11α-hydroxyprogesterone.



   19.2 mg. of lla-hydroxyprogesterone obtained as described in e are acetylated by treatment with 1 ml. of pyridine and 1 ml. acetic anhydride for 16 hours; acetylating agents are removed by evaporation in a vacuum; and the residue (weighing approx. 21.4 mg) is crystallized from acetone-hexane. The product, an acetate of the formula C23H3204, has the following properties: M.P. about 172-174; analysis (calculated: C.74.16; H. 8.66) C. 73.91, and H. 8.50 and (Ó (23 + 1550 (c.



  0.379).



   (f alternative) oxidation of 11a-hydroxyp [rogesterone to obtain 11-ketoprogesterone.



   8.5 mg. of chromic acid in 2.5 ml. of glacial acetic acid are added to a solution of 20.8 mg. 1α-hydroxyprogesterone contained as described in 2 ml. glacial acetic acid. After allowing to stand at room temperature for 1 hour, 1 ml. alcohol is added and the solution is evaporated to dryness in a vacuum. The residue is taken up in 2 ml. of water and 15 ml. of chloroform, and the chloroform phase is washed with dilute sodium bicarbonate solution, then with water. After drying over sodium sulfate, the solvent is removed in vacuo, leaving a residue (weighing about 20.4 mg) which spontaneously crystallizes.

   The product (11-ketoprogesterone), when recrystallized from an acetone-hexane mixture (or from ether) has the following properties: M.P. about 170-171.5 (capillarity); () 23 + 276 (c., 0.29), + 229 (c., 0.52 in acetone). Fuchs and Reichstein, Helv., 23, 684 (1940) reported that 11-ketoprogesterone melted at 173-175 (block) and (Ó) 14 + 238.4 (acetone) An example of 11-ketoprogesterone prepared
D from corticosterone by the method of Fuchs and Reichstein and melting at 170.5 - 172, had (Ó) 23 + 221 (c. 0.51 in acetone).



   D
When this sample is mixed with 11-ketoprogesterone obtained as described above, the melting point is about 169-172.5; and the infrared spectra of these 11-ketoprogesterone prepared under different conditions are identical in all respects.



   EXAMPLE 2
A medium having the following composition is prepared: 3 g. solids from a liquor of an infusion of grains; 2go of soybean oil; 3 g. NH4H2PO4; 0.15 g. 17-hydroxy-11-deoxycorticosterone acetate; and the quantity of distilled water to make 1 liter, the pH of the medium being adjusted to 6 with a 50 ml sodium hydroxide solution. of this medium are distributed in 250 ml. Erlenmeyer flasks and the flasks are sealed with cotton and are sterilized in the usual way (in an autoclave).

   When the balloons are cold, each is seeded with 2% of a plant seed of Aspergillus niger (Wisc. 72-2) which has grown on the same kind of medium but without the steroid for 72 hours. , and the flasks are kept at 25 on a mechanical stirrer for 48 hours. After incubation, the contents of the flasks are cooled and are used up three times with an equal volume of chloroform. The presence of an oxidized derivative of the steroid substrate in the chloroform extract is indicated by assay by the filter paper septum chromatographic method of Zaffaroni et al. (Science, vola III, p. 6, 1950). (toluene-propylene-glycol system), the oxidized derivative having a mobility equal or similar to that of hydrocortisone.



   EXAMPLE 3
Under the same fermentation conditions as those described in section a of Example 1, except that the prohesterone content of the medium is 0.5 g. per liter and that the seed organism

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 ment is Aspergillus type A.T.C.C. 11,267, and, using the same exhaustion, separation and isolation procedures as described in section b and following of Example 1, 6, 11a-dihydroxyprogesterone and 11a-hydroxyprogesterone are obtained in the same order of purity and yield.



   EXAMPLE (a) Fermentation.



   The same fermentation conditions as those described in the section of Example 1 are employed except that 11-deoxy-17-hydroxycorticosterone (Reichstein's compound S) is incorporated in a content of 0.50 g. per liter instead of progesterone.



   (b) Depletion and separation of oxidation products.



   3100 ml. of the culture filtrate obtained in a (based on a fermentation of 1.75 g. of the compound S) is exhausted with 5 parts of 1.5 liters of chloroform and the combined chloroform extract is filtered and is evaporated to at dryness in a vacuum, the temperature of the liquid which distills not exceeding 40. The semi-crystallized residue (weighing about 1.00 g.) Is suspended in 20 ml. of hot chloroform, and the crystalline precipitate (weighing about 424 mg) is filtered off.

   Upon recrystallization, the product obtained (¯ 4-pregnene-11a, 17a, 21-triol-3,20-dione) has the following properties: prismatic needles, M.P. about 216-219; (Ó) 24 + 117 (c 0.46 in absolute ethanol);
D alc.max. 242 m / u # = 15,000); analysis (calculated for C21H30O5; C, 69.50, and H. 8.34) C. 69.46 and H 8.390 (c) Isolation of another oxidation product.



   The combined chloroform mother liquor obtained in b is evaporated to dryness, and the residue is taken up in a small volume of acetone. By cooling in a refrigerator, a crystalline product is obtained; and two recrystallizations with the same solvent give a product having the following properties: M.P. about 248-250; (Ó) 24 +
D 97 (c. 0.50 in absolute ethanol); alc. 241 m / u (# D 16,000); max. analysis (calculated for C21HEO5; c. 69.58 and H. 8.34) C. 69.45 and H. 8.53.



  The product is isomeric with .6 r-pregnene-11a, 17a, 21-triol-3,20 = dione.



   (d) Acetylation of 4- = pregnene-11a, 17a, 21-triol-3,20-dione.



   14 mg. of ¯ 4-pregnene-11a, 17a, 21-triol-3,20-dione (see section b) are acetylated by treatment with 1 ml. of pyridine and 1 ml. acetic anhydride for 20 hours at room temperature. The acetylating agents are removed by evaporation in vacuo, the residue is dissolved in ether, and the ether is allowed to evaporate spontaneously at room temperature. Crystallization follows; and after recrystallization twice from an acetone-ether mixture, the monoacetate is obtained in the form of prisms having the following properties: M.P. about 205.5-208; (Ó)
22 + 117 (c. 0.84); analysis (calculated for C25H34O7: C.67.25 and H. 7.68)
C. 67.34 and H. 7.67.

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  (d alternative) Oxidation of ¯ 4-pregnene-11a, 17a, 21-triol-3,20-dione to obtain adrenosterone.



   A solution of 27.2 mg. of chromic acid in 5.5 ml. glacial acetic acid is added to a solution of 18.0 mg. of ¯ 4-pregnene-lla, 17a, 21-triol-3,20-dione (M.P. 216-219) in 2 ml. of ice acetic acid, the addition being carried out five times over an hour's interval.



  After allowing to stand for two more hours at room temperature, 1 ml. alcohol is added and the mixture is concentrated in vacuo to make a syrup. The residue is distributed between 3 ml. of water and 15 ml. chloroform; and the chloroform phase is separated, and is depleted with water, then with sodium bicarbonate solution and again with water. The chloroform is then evaporated, leaving a crystalline residue weighing about 13 mg; and, after two recrystallizations from alcohol, the product has the following properties: M.P. about 221-225; (¯ D 24 + 284 (c. 0.51); and analysis (calculated for C19H24O3: C. 75.97 and H. 8.05) C. 75.71 and H. 7.97.

   The product, adrenosterone, cannot be distinguished from an authentic sample of ¯ 4-androstene-3,11,17-trione (adrenosterone) prepared by oxidation of cortisone with chromic acid, regarding the melting point (no depression during mixing), the specific rotation and the infrared spectrum.



  (d alternative 2) Transformation of ¯ 4-pregnene-11a, 17a, 21-triol-3,20-dione to cortisone acetate.



   100 mg. of 4-pregnene-11a, 17a, 21-triol-3,20-dione (M. P.216-219) are dissolved in 0.5 ml. of pyridine which was dried completely over BaO, and 0.97 ml. of a solution of 159.6 mg. acetic anhydride in 5 ml. of dry pyridine are added at room temperature. The mixture is allowed to stand at room temperature for 12-16 hours, then the mixture is evaporated to dryness in vacuum; the residue is taken up in chloroform. The solution is made up with dilute hydrochloric acid, sodium bicarbonate solution and water; the chloroform is then removed in a vacuum.

   The amorphous residue (weighing about 109 mg.) Is dissolved in 2 ml. of glacial acetic acid and, while stirring, a solution of 22 mg. of Cr03 in 2 ml. of glacial acetic acid is added dropwise. After the reaction mixture has stood at room temperature for 1 hour, 1 ml. alcohol is added and the mixture is concentrated to form a syrup. The syrup is distributed between 3 ml. of water and 15 ml. of chloroform and the chloroform extract is washed with water (until free of chloroform), then with sodium bicarbonate solution and again with water. The chloroform solution is dried over sodium sulfate and evaporated to dryness in vacuo, leaving a crystalline residue weighing about 93 mg.



  This residue is recrystallized from acetone: the first product, weighing approximately 51 mg. melts at 237.8; and a second product of about 11 mg. is obtained. After recrystallization twice from acetone, the product (analytically pure cortisone acetate) has the following properties: M.P.



    243-244, the crystals turning to an opaque color at 85-90 and showing no depression in the melting point when mixed with an authentic sample of cortisone acetate melting at 242-244 and becoming opaque at 84-95; (Ó) 22 + 1680 (c 0.63 in acetone), + 204 (C 0.63) assay (calculated for C23H3006: C. 68.65 and H. 7.46) C. 68.67 and H. 7.54. The infrared spectrum of the product is identical to that of an authentic sample of cortisone acetate.



   EXAMPLE 5 (a) Fermentation.



   The same fermentation conditions as those described in

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 section a of Example 1 are used except that 11-deoxy-17-hydroxycorticosterone acetate is incorporated in a content of 0.50 g. per liter instead of progesterone.



  (b) Depletion of oxvdationo product
 EMI8.1
 The culture filtrate obtained in .a 'by fermentation of 1.9 g. 11-Deoxy-17-hydroxycorticosterone acetate is depleted with chloroform and the chloroform is removed from the extract as described in section b of Example 4. The residue, weighing about 420 mg., when it is removed. further processed as described in this section b provides approximately 156 mg. of crystals. After two recrystallizations with acetone, the product 4-pregnene IIa, 17a, 21-triol 3,2-dione melts at 215-218.

   (From the dried mycelium from this fermentation, ll-deoxy-17-hydroxycorticosterone acetate is recoverable by exhaustion with hexane to remove lipoid material, then with acetone, removing acetone in vacuum leaving a crystalline residue weighing about 1.35 g .; and recrystallizing the residue with acetone yielding about 930 mg of the pure compound melting at 234-2380).



   EXAMPLE 6 (a) Fermentation.



   The same fermentation conditions as those described in section.5!:. of Example 1 are used, except for the fact that deoxycorticosterone is incorporated in a content of 0.25 g. per liter instead of progesterone.
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  (b) Isolation of epicorticosterone.



     3.58 liters of culture filtrate obtained in .si (based on the fermentation of 1 g. Of deoxycorticosterone) is used up with 4 parts of 2 liters of chloroform, and the chloroform extracts are cooled and concentrated, providing approximately 1 , 01 g. of an amorphous solid product. This solid product is dissolved in 5 ml. of chloroform and 20 ml. of benzene and the solution is chromatographed on a column of 30 g. silica gel.

   Elution occurs as follows:
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<tb>
<tb> Eluat <SEP> Volume <SEP> Solids
<tb> fiai.) <SEP> (mg.)
<tb> Chloroform-benzene <SEP> (1: 4) <SEP> 300 <SEP> Traces
<tb> Chloroform-benzene <SEP> (1: 1) <SEP> 500 <SEP> Traces
<tb> Chloroform <SEP> 400 <SEP> Traces
<tb> Acetone-chloroform <SEP> (1: <SEP> 9) <SEP> 340 <SEP> Traces
<tb> Acetone-chloroform <SEP> (1: 4) <SEP> 850 <SEP> 670.2
<tb> Acetone-chloroform <SEP> (1: 1) <SEP> 525 <SEP> 103.2
<tb> Acetone <SEP> 250 <SEP> 19.5
<tb>
 
The solid products eluted with the acetone-chloroform mixture (1: 4) readily crystallize in the form of bars from acetone-ether mixtures, or in the form of prisms from ethyl acetate, having the following properties :
 EMI8.4
 M.P. 153-154;

   (al) 22.5 + 168 (c. 0.7); analysis (calculated for cZ1H3o 4;
D C. 72.80 and H. 80.73) C. 72.90 and H. 8.76. The product is ¯ 4-pregnene-

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 11a, 21-diol-3,20-dione, as prepared by the following transformation.



   (c) Conversion to dehydrocorticosterone acetate.



   One serving of 34.3 mg. of ¯ 4-pregnene-11a, 21-diol-3,20-dione obtained as described in section b is dissolved in 1.0 ml. of a solution of 114.0 mg. acetic anhydride in 10.0 ml. in dry pyridine. After allowing the solution to stand at room temperature for 12-16 hours, the solvents are removed by concentration in the vacuum; and the residue is dissolved in chloroform, washed with dilute hydrochloric acid, sodium bicarbonate solution and water, and dried over sodium sulfate. The solution is then concentrated to dryness and the crude acetate-21 is dissolved in 1.5 ml. of glacial acetic acid. 1.1 ml of a solution of 53.3 mgo of chromic acid in 5.0 ml. acetic acid is added.

   After 35 minutes, one ml. of methanol is added and the solution is concentrated to dryness in vacuum, then the dark green residue is taken up in chloroform and water, and the clear chloroform phase is washed with a solution of sodium bicarbonate and water and then dried over sodium sulfate. The chloroform is then evaporated, leaving about 36.9 mg. of a syrupy residue which begins to crystallize spontaneously. The product is recrystallized from a mixture of acetone and ether, yielding 18.6 mg of needles softening at 169 and melting at 172-177. Recrystallization with the same pair of solvents raises the melting point to 177.5 - 179.

   (An authentic sample of dehydrocorticosterone acetate melts at 178 - 179.5; there is no depression in the melting point of a mixture; and the infrared spectra of the two products are identical.) The product exhibits the following additional properties: (Ó) 22 + 239 (c. 0.5); analysis (calculated for C23H3005: C. 71.46 and H. 7.84) C. 71.54 and H. 7.99.



   EXAMPLE 7 (a) Fermentation
The same fermentation conditions as those described in section a of Example 1 are used, except that the 17α-hydroxy-progesterone is incorporated in a content of 0.25 g. per liter instead of progesterone.



    (b) Depletion and isolation of oxvdation products
The culture filtrate obtained in .Si (based on the fermentation of 1 g. Of 17α-hydroxyprogesterone) is stripped with chloroform as described in section b of Example 4. The residue, weighing about 514 mg ., is dissolved in chloroform and the solution is chromatographed on 15 g. silica. The chromatogram was eluted with 1300 ml of a mixture of 5% acetone in chloroform and the solvents were evaporated leaving about 245 mg. colorless crystals, which are recrystallized with an ethyl acetate-hexane mixture.

   The product, which is isomeric with 11a, 17a-dihydroxyprogesterone described below and is assumed to be 17a-methyl-D-homo- ¯ 4-androstene-11a, 17a-diol-3,20-dione (compound X) 'has the following properties: MP 261-262; (Ó) 3 + 46 (c. 0.74); analysis (calculated for C21H30O4: C. 72.80 and H. 8.73) C 72.92 and H. 8.840
Elution the chromatogram again with 550 ml. 10% acetone in chloroform and evaporation of the solvents provided about 150 mg. of a crystallized product.

   Upon recrystallization with an ethyl acetate-ether mixture, the product is obtained in the form of colorless plates having the following properties M.P. 219-221; (Ó) 22 + 87

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 (c. 0.57); and analysis (calculated for C21H30O4: C. 72.80 and H.8.73) C. 73.11 and H. 8.70. The product is 11a, 17a-dihydroxyprogesterone as prepared upon oxidation to form the known # 4-pregnene-17a-ol-3,11,20-trione.



    (c) Oxidation of compound X.



   24.3 mg. of compound X are oxidized with 15 mg. Chromic anhydride in glacial acetic acid, and the reaction mixture is taken up as described in the Example 6 section, and the product is crystallized with acetone-ether. The 11-ketone thus obtained in the form of colorless plaques has the following properties: M.P. 238-242; (Ó) 22 + 121 (co 0.48); and analysis (calculated for C21H2804 C, 73.23 and H.



    8.19) C. 73.33 and H. 8.34.



    (d) Oxidation of 11a, 17a-dihydroxyprogesterone.



   37.9 mg. of 11a, 17a-dihydrqxyprogesterone are oxidized with 20 mg. chromic anhydride as described above and the oxidation products are crystallized with an ethyl acetate-ether mixture in the form of colorless tetrahedrone having the following properties: -M.P. 232-235; (Ó) 22 + 186 (c. 0.33); and analysis (calculated for C21H28O4: C. 73.23 and H 8.19) C. 73.24 and H. 8.37. The melting point and specific rotation agree with the indications reported by Kritchevsky et al., J.A.C.S. 74, 483 (1952).



  (d alternative) Acetylation of 11a, 17a-dihydroxyprogesterone.



   22.1 mg. of 11a, 17a-dihydroxyprogesterone are acetylated by treatment with 0.5 ml. of pyridine and 0.5 ml. acetic anhydride at room temperature for 18 hours. The acetylating agents are removed by evaporation in a vacuum and the crystallized residue is recrystallized in an acetone-ether mixture, providing large prisms having the following properties: M.P. 205-208; (Ó) 24 + 65 (c 0.61); and analysis (calculated for C23H32O5: C.71.10 and H.8.30) C. 71.01 and H. 8.16.



   EXAMPLE 8 (to) Fermentation.



   The same fermentation conditions as described in the section of Example 1 are used, except that Aspargillus type A.T.C.C. 11.267 is used instead of aspergillus niger and 17α-hydroxyprogesterone is included, with a content of 0.25 g. per liter, instead of progesterone (b) Depletion of oxidation products.



   The culture filtrate obtained in a (based on the fermentation of 875 mg. Of 17α-hydroxyprogesterone) is depleted with chloroform as described in section II of Example 1 and the chloroform is removed. , leaving a residue of about 536 mg. The residue is dissolved in chloroform and the solution is chromatographed on 15 g. silica. Elution with 300 ml. of chloroform removes about 35 mg. unidentified by-products; another elution with 200 ml. of 5% acetone in chloroform removes about 20 mg. of the D-homo compound described in example
7;

   and elution with 1500 ml. of 10% acetone in chloroform eliminates about 410 mg. the primary product of 11a, 17a-dihydroxyprogesterone, melting at 215-219 and having (Ó) 24 + 87 (c. 0.52),
D

 <Desc / Clms Page number 11>

 
EXAMPLE 9 (a) Fermentation.



   The same fermentation conditions as those described in section a of Example 1 are used, except that 5pregnenolone is incorporated, with a content of 0.50 g. per liter, instead of progesterone.



  (b) Depletion and isolation of oxidation products.



   The culture filtrate obtained in a (based on the fermentation
 EMI11.1
 tion of 1 g. of A 5-pregnenolone) is depleted with chloroform - as described in section b of Example 1, yielding 169 mg. of a fat free steroid product. The product is dissolved in ml. of a mixture of chloroform and benzene in the proportions 1: 3, and the solution is chromatographed on 4 g. alumina washed with sulfuric acid. Elution of the chromatogram with 150 ml. of a chloroform-benzene mixture (1: 1) and evaporation of the solvents give about 40 mgo of lla-hydroxyprogesterone, and after recrystallization from an acetone-ether mixture, the product melts at 156-159 and has (Ó) 24 + 171 (c. 0.52).



   D
Elution the chromatogram again with 150 ml. of chloroform followed by evaporation of the chloroform to provide about 75 mg. of a product (probably 6, 11a-dihydroxyprogesterone) which, after recrystallization from acetone, melts at 247-250 and has # alc. 235 m / u (= max.



  14,000).



   Unoxidized ¯ 5-pregnenolone can be recovered by depleting the dry mycelium with chloroform, removing chloroform in a vacuum, keeping the residue in suspension in hexane, filtering
 EMI11.2
 crystals resulted "% (weighing about 654 mg.), and recrystallizing from acetone. The α1 -pregnenolone recovered melts at 186-190 and at (o:) D 2+ + 24 (tl, 4).



   EXAMPLE 10
 EMI11.3
 Using the method described in sections n and ¯b of Example 4, but with Aspergillus.type AoToCoCo 11.267 as the fermentation organism in place of Aspergillus niger, 582 mg is obtained. crystals insoluble in chloroform and having the following properties: M.P. 216-
 EMI11.4
 219; (0 <.) 23 + 117 (c. 1.00 in absolute ethanol); and analysis (calculated for Cz1Hp05: Co 69.58 and Ho 8.34) C. 69.88 and H. 8.090 The product, after recrystallization from acetone, shows in the infrared a spectrum identical to that of authentic L1 4-pregnene-11a, 17a, 21-triol-3,20-dione.



   EXAMPLE 11
Under the same conditions as those of Example 10, but by fermenting 11-deoxy-17-hydroxycorticosterone with a content of 1 g. per liter, we obtain, starting from 1 g., a yield of 467 mgo of
 EMI11.5
 -pregnene-lla, 17a, 2l-triol-3,20-dioneo
EXAMPLE 12 Under the same conditions as those of Example 10, but in
 EMI11.6
 using 11-deoxy-17-hydroxycorticosterone acetate $ a yield of about 204 mg is obtained. of 6.4-pregnene-l1a, 17a, 21-triol-3,20-dioneo

 <Desc / Clms Page number 12>

 
EXAMPLE 13
Under the same conditions as those of Example 12, -but by doubling the fermentation time, a yield of approximately 220 is obtained.
 EMI12.1
 mgo of d 4-pregnene-11a, 17a, 21-triol-3,20-dione.



   EXAMPLE 14
Under the same conditions as those of Example 4, but using a crude saponification mixture of 11-deoxy-17-hydroxycorticostone, approximately 589 mgo of ¯ 4-pregnene-11a, 17a, 21-triol is obtained. - 3,20-dione. The crude saponification mixture for addition to 2 liters of the medium is prepared by suspending 1.0 gb of 11-deoxy-17hydroxycorticosterone in a mixture of 47 ml. of methanol and a solution of 700 mgo of potassium bicarbonate in 13 ml. of water, then stirring the suspension for 18 hours at room temperature in a stoppered flask.



   EXAMPLE 15
Under the same conditions as those described in Example 4, but incorporating the steroid with a content of 0.25 gb per liter and using the microorganism aspergillus amstelodami instead of aspergillus niger, approximately 383 mg. of a steroidal product are obtained from
 EMI12.2
 0.5 gag of 11-deoxy 17-hydroxycorticosteronee This product is contained in 10 ml. of chloroform and the crystallized precipitate (weighing approximately 164 mg.) is recovered by filtration and is recrystallized from acetone. The product melts at 217-220 and its infrared spectrum is identical to that of
 EMI12.3
 Genuine 4¯pregnene-11a, 17a, 2l-triol-3,20-dione.



   The chloroform mother liquor is evaporated to dryness, is taken up in a little acetone and is cooled, and the resulting crystals (weighing about 33 mgo) are recrystallized from acetone. The product melts at 248.5 - 250 and its infrared spectrum is identical to that of the product described in section c of Example 4.



   EXAMPLE 16
Under the same conditions as those of Example 4, but using aspergillus nidulans instead of aspergillus niger, approximately 732 mg. of a steroid product are recovered from 1.0 g. of 11-deoxy-17-hydroxycorticosterone. From this steroid product, one obtains in-
 EMI12.4
 approximately 571 mg.

   de /), 4-pregnene-lla, 17a, 21-triol-3,20-dionl which, after recrystallization in acetone, melts at 216-219. and shows the correct infrared spectrum
Media other than those described in the preceding examples can be used for the purposes envisaged in the invention, the only requirement of course being that these media must be able to support the development or proliferation of aspergillus. especially in the presence of oxygen.

   Any citric acid obtained as a by-product (as in the case of the use of sucrose or molasses, for example as part or as all of the carbon and energy source), or any other valuable products obtained (for example amylases or proteinases, can be recovered according to the conventional methods for the recovery of these products starting from cultures of aspergillus.



   An adequate supply of oxygen (sterile) can be maintained during fermentation, which can be accomplished according to the conventional method of ensuring fermentations in the presence of oxygen, i.e. by exposing an oxygen. large area of the medium in the air or by immersing a ventilated culture. The incubation time can determine the degree of oxidation with

 <Desc / Clms Page number 13>

 certain steroid substrates and the incubation is of course stopped when the agent contains the highest content of the desired product, which is easy to determine for each kind of conditions.



   In the examples indicated above, the steroid substrate is incorporated into the fermentation agent before inoculation with aspergillus; and, in such a case, the steroid substrate is preferably dissolved in chloroform to facilitate handling (e.g. 1 ml of chloroform per 100 ml of the medium), the chloroform being removed from the medium during sterilization (i.e. i.e. before seeding).

   However, the steroid substrate can be added after seeding and even after substantial proliferation of aspergillus has occurred. Therefore the steroid substrate can be transformed into an oxidation product by bringing the steroid into common contact. steroid and air in an aqueous suspension of non-proliferating aspergillus cells or by bringing the steroid, air and aspergillus enzymes into common contact in an aqueous cell-free agent.

   Thus a three-day culture of aspergillus niger proliferated on the same medium as that used in the example but without progesterone, is subjected to centrifugation, is again suspended in distilled water, is subjected to a new centrifugation, is further suspended in distilled water; the suspension is placed in a vial; progesterone (for example) is added; flask is shaken at 25 for 24 hours; the suspension is filtered, and the lla-hydroxyprogesterone formed is recovered by depleting the filtrate with chloroform and by a new treatment such as that described in sections b and following of Example 1. Other expedients which are conventional in fermentation can be used for the practical implementation of the invention.

   This is how aspergillus can be grown on a medium best suited for its proliferation, the mycelium can be separated from the culture fluid after substantial proliferation, and the mycelium can be resuspended in a new medium containing the substrate. steroid (for example the medium described in Example 1). Also the aspergillus mycelium can be used repeatedly, i.e. the culture liquid containing the oxidized derivative of the steroid can be separated from the mycelium, the former being treated to recover said derivative, while the latter is resuspended in a further amount of the steroid substrate medium.



   The oxidized derivatives of the steroids obtained in the practical implementation of the invention are intermediates for the preparation of cortical hormones and / or other useful products. Thus, llahydroxyprogesterone is convertible into the known 11-keto-progesterone as described above, as well as cortisone acetate, for example.

   By way of illustration, 11a-hydroxyprogesterone can be catalytically reduced with palladium in ethyl acetate to form allopregnan-11a-ol-3,20-dione; the latter can be reduced with lithium aluminum hydride to the allopregnane-3,11a, 20-triol; the latter can be treated with an acylating agent (eg an acetylating agent) under mild conditions to obtain the 3-acyl derivative (eg allopregnane-3, 11a, 20-triol-3 acetate); the latter can be oxidized with chromic acid to form 3-acetoxy-allopregnane-11,20-dione; and the latter can be converted, by methods described in the art (for example by Rosenkranz, Pataki and Djerassi, J.A.C.S.



  73, 4055, 1951), in cortisone acetate. Such a conversion can, for example, be effected by treating 3-acetoxy-allopregnane-11,20-dione with acetic anhydride and p-toluenesulfonic acid to obtain.
 EMI13.1
 Remove the 11'20-dienol acetate from 3-acetoxy-allopregnane-11'20-d.ione and, without isolation, be treated with excess perbenzoic acid in chloroform, followed by saponification with a solution of 2N sodium hydroxide to obtain allopregnan-3,17a-diol-11,20-dione; the latter is brominated in chloroform to obtain 21-bromo-allopragnan-3,17a- diol-11,20-dione;

     the latter is treated with sodium iodide

 <Desc / Clms Page number 14>

 in aoetone, followed by potassium acetate, to provide allopregnane-3,17a-21-triol-11,20-dione-21 acetate (Reichstein's compound D); this acetate is oxidized with N-bromo-acetamide to provide al-lopregnane-3,11,20-trione-17a, 21-diol-21 acetate; this acetate is brominated and, without isolation, is treated with sodium iodide to obtain ¯ 4-2-iodo-pregnene-17a, 21-diol-3,11,20-trione-21 acetate ;

   and this acetate is in turn treated with zinc dust in dioxane and ethanol to provide cortisoneo acetate La ¯ 4-pregnene-11a, 17a, 21-triol- 3,20-dione is convertible into the known adrenosterone as described above, as well as into cortisone acetate for example, this acetate being the subject of alternative section d 2 of example 4. ¯ 4-pre- gnene -11a, 21-diol-3,20-dione is convertible to the known dehydrocorticosterone acetate as previously described. 11a, 17a-dihydroxyprogesterone is convertible to the known # 4-pregnene-17a-ol-3,11,20-trione as previously described.



   The invention can be carried out with variations without departing from the scope of this invention.


    

Claims (1)

ABSTRACT. The subject of the invention is: 1. A process for the conversion of a steroid into an oxidized derivative of this steroid, characterized by the following points, separately or in combination: The process comprises subjecting the steroid to the action of enzymes of an oxidizing type of Aspergillus in the presence of oxygen; 2 To introduce 1-oxygen at least at position-11a of an unsubstituted steroid at position 11, the steroid unsubstituted at position-11 is subjected to the action of enzymes of an oxidizing type of. aspergillus in the presence of oxygen; 3 To obtain a 11a-hydroxy derivative of a steroid from the group consisting of 3-oxo-20-keto-pregnenes and 3-oxy-20-keto-pregnenes, the method comprises subjecting an unsubstituted steroid to the position- 11 of the aforementioned group to the action of enzymes of an oxidizing type of Aspergillus in the presence of oxygen; When the steroid is subjected to the action of enzymes of an oxidizing type of Aspergillus in the presence of oxygen, this type of Aspergillus belongs to the group comprising Aspergillus niger Wise. 72-2, Aspergillus amstelodami, Aspergillus nidulans and Aspergillus type AoToC.C. He, 267; The steroid treated is progesterone; The steroid treated belongs to the group consisting of 17-hydroxy-11-deoxycorticosterone and esters thereof; 7 The steroid treated is deoxycorticosterone; 8 The steroid treated is 17α-hydroxyprogesterone; 9 The steroid treated is ¯ 5-pregnenolone; The steroid is incorporated into a fermentation agent capable of supporting the proliferation of an oxidizing type of Aspergillus, then this agent is inoculated with this type of aspergillus and subjected to incubation in the presence of. oxygen; <Desc / Clms Page number 15> EMI15.1 11 To separate 4-pregnene-11a, 17a, 21-triol-3,20-dione from the crude product of microbiological oxidation of 17-hydroxy-11-deoxy-corticosterone, the process consists of suspending this product of oxidation in a chlorinated hydrocarbon solvent, followed by recovering the product insoluble in said solvent; 12 For the separation provided for in paragraph 11, the solvent used belongs to the group comprising chloroform, ethylene dichloride and trichlorethylene. 2. The oxidized derivatives of the sterofids obtained by carrying out the process defined above are, for example: - One of the lla-hydroxy derivatives of 3-oxo-20-keto-pregnenes and of 3-oxy-20 -keto-pregnenes; - lla-hydroxyprogesterone; EMI15.2 - 0 4-pregnene-11a, 17a, 21-triol-3,20-dione; - A 4-pregnene-11a, 21-diol-3,20-dione; - 17a-methyl-D-homo- A 4-androstene-11a, 17a-diol-3,20-dione; - lla-17a-dihydroxyprogesterone.