CH335488A - Process for the manufacture of a 14-oxy-steroid - Google Patents

Description

  

  Additional patent to the main patent No. 329194 Process for the production of a 14-oxy-steroid The invention relates to the production of a new steroid, the 14a, 17a, 21-trioxy-4-pregnen-3,20-dione from 17a, 21- Dioxy-4-pregnen-3,20-dione or its 21-acyl ester by biochemical oxidation with enzymes produced by a fungus of the genus Helicostyhtm, which belongs to the order l @ Iucorales, e.g. B.

         Helicostylum piriforme. The isolated enzymes of the fungi can be used. The preferred method, however, consists in conducting the oxidation under fermentation conditions using a viable fungus in the presence of a nutrient solution and then separating off the oxidized steroid, as will be explained in detail in the following example.



  The 7.4a, 17a, 21-trioxy-4-pregneii-3,20-dione, which shows pharmacological effects, can be mixed with (", chromium oxide to 14a-oxy-4-androstene-3,17-dione with a melting point j-on 259 to 263 C are oxidized.



       Example. A nutrient medium consisting of 20 g of lactic acid (edamine, enzymatic lactic albumin extract), 3 g of corn steep liquor and 50 g of technical dextrose was diluted in one liter of tap water, and to a 1), 1 value of 4 , 3 to 4.5 brought, manufactured ..

   12 liters of the sterilized culture medium were inoculated with Helieostyliim piriforme spores (ATCC No. 8992). and incubated for 24 hours at a temperature of 28 C with aeration and stirring, so that the oxygen uptake is 6.3 to 7 millimoles per liter and hour of Na2803 according to the method of Cooper, Fernstrom and Miller, Ind. Eng. Chem. 36, 504 (1944).

   To this medium, which contained a 24 hour old culture of Helicostylum piriforme, 2 g of the compound S from Reichstein [17a, 21-Dioxy-4-pregnen-3,20-dione] in a minimum of ethanol were added, to obtain a suspension of the steroid in the culture. After a further incubation period of 24 hours under the same temperature and ventilation conditions, the wort and mycelium were extracted.

   The mycelium was filtered off and washed twice in a volume of acetone each approximately corresponding to the amount of mycelium and then finitely extracted twice with a volume of methylene chloride approximately corresponding to the amount of mycelium. The acetone and methylene chloride extracts, including the solvent, were added to the wort filtrate.

    The mixed extracts and the spice filtrates were gradually extracted with two volumes of methylene chloride corresponding to half their volume and then finitely two volumes of methylene chloride corresponding to one quarter of their volume.

   The combined methylene chloride tracts were washed with two parts corresponding to one-tenth of their volume of a 2% strength aqueous sodium bicarbonate solution and then with two parts of water corresponding to one-tenth of their volume.

   After drying the methylene chloride extracts with about 3-5 g of anhydrous sodium sulfate per liter of solvent and filtering them off, the solvent was removed by distillation. The residue was dissolved in a minimum of 12 ethylene chloride, filtered and the solvent evaporated. The resulting Rohkri stalle were dried and then five times with. washed 5 em3 ether per gram of raw crystals. There was a residue of 4.354 g.

   The residue was dissolved in 10 cubic meters of ethylene dichloride and chromatographed over 350 g of Florisil, a synthetic magnesium silicate, using 550 cm3 portions of weight-bearing solvent fractions according to the table.
EMI0002.0031
  
    <I> table </I>
<tb> Helicostylum <SEP> piriforme
<tb> Fraction <SEP> Solvent <SEP> Yield <SEP> in <SEP> mg
<tb> 1. <SEP> Ä <SEP> ethylene dichloride <SEP> 479.5
<tb> 2 <SEP> ethylene dichloride <SEP> 403.0
<tb> 3 <SEP> ethylene dichloride-aeetone <SEP> 15:

  1 <SEP> 815.5
<tb> 4 <SEP> <SEP> <B> 15: 1 </B> <SEP> 36.5
<tb> 5 <SEP> <SEP> 12: 1 <SEP> 36.0
<tb> 6 <SEP> <SEP> 12: 1. <SEP> 50.0
<tb> 7 <SEP> <SEP> 12: 1 <SEP> 109.5
<tb> 8 <SEP> <SEP> <B> 10: 1 </B> <SEP> 159.0
<tb> 9 <SEP> <SEP> <B> 10: 1 </B> <SEP> 148.0
<tb> 10 <SEP> <SEP> <B> 8: 1 </B> <SEP> 145.0
<tb> 11 <SEP> <SEP> <B> 8: 1 </B> <SEP> 104.5
<tb> 12 <SEP> <SEP> <B> 8: 1 </B> <SEP> 98.0
<tb> 13 <SEP> <SEP> <B> 5: </B> <SEP> 1. <SEP> 170.0
<tb> 14 <SEP> <SEP> <B> 5: 1- </B> <SEP> 123.0
<tb> 15 <SEP> <SEP>: 1 <SEP> 123.5
<tb> 16 <SEP> <SEP> <I> 3: 1 </I> <SEP> 1.27.5
<tb> 17 <SEP> <SEP> <B> 3: 1 </B> <SEP> 200.0
<tb> 1.8 <SEP> <SEP> 1. <SEP>: 1 <SEP> 456.0
<tb> 19 <SEP> <SEP> 1 <SEP>:

  1 <SEP> 356.0
<tb> 20 <SEP> acetone <SEP> 226.5 Including fractions 13-16, freed from the solvent, were recrystallized from 10 cm3 of ether by slowly evaporating the ether at room temperature. The crystallization produced 228.6 mg of crystals with a melting point of 218 to 2261 ° C. Two further recrystallizations, each obtained from 10 em3 of ether-acetone in a ratio of 1: 1, gave crystals with a melting point of 232 to 235 C.

   Recrystallization of a sample of this substance, 57 mg, from 5 cm3 of methanol gave 14a, 17a, 21-trioxy-4-pregnene-3,20-dione with a melting point of 234 to 237a C, [a] D + 155 (c = 1.13 in methanol) with a uniform infrared spectrum. Analysis: / o calculated for C2111300.5: C = 69.58 1I = 8.34 o / 0 found: C. = 69.42 11 = 8.40 The use of a 21-ethyl ester, e.g. B.

    of acetate gives the same results.

 

Claims (1)

PATENT CLAIM Process for the production of 14a, 17a, 21-trioxy-4-pregnen-3,20-dione, characterized in that 17a, 21-dioxy-4-pregnene-3,20-dione or its 21-acyl ester is one bio-chemical oxidation with enzymes which are produced by a fungus of the genus Helieostylum. SUBCLAIMS 1. Method according to patent claim, characterized in that the fungus used is Helicostylum piriforme. 2. The method according to claim, characterized in that the oxidation takes place under fermentation conditions and using a viable fungus in the presence of a nutrient medium for the fungus.