CH369129A - Method for introducing a hydroxyl group into the 12-position of Reichstein's compound S. - Google Patents

Description

  

  Process for introducing a hydroxyl group into the 12-position of Reichstein's compound S It is already known that hydroxyl groups can be introduced as substituents in the most varied of positions in the molecule of steroids in a biochemical manner. However, an introduction to the 12-position in this way alone has not yet been described.

   All that is known is a simultaneous introduction of hydroxyl groups in the 12- and 15-position (compare Chem. Ber., Volume 90, 1957, page 2576 ff.) It has now been found that when Reichstein's compound S (4-Pregnen -17a, 21 diol-3,20-dione) with the fungus Coniothyrium hellebori, breed 078, from the class Fungi imperfecti and,

   of the Sphaeropsidales series, in addition to the hydrocortisone (4-pregne @ n-11ss, 17a-21-triol-3,20-dione), which is the main product in 50% yield, also 4-pregnen-12ss, 17a, 21-triol-3,20-dione is formed.



  This finding is all the more surprising as, according to the information in British patent specification number 749 414, when the same starting material was incubated with related fungi of the same class and series, only 11-epi-hydrocortisone was to be expected as a by-product.



  In the process according to the invention, the main product and by-product of the culture filtrate extracts are initially obtained as a crystalline mixture. Its complete separation by crystallization is difficult and lossy. If the mixture, which is not to be further separated, is acetylated under normal mild conditions, the monoacetates of both compounds are mainly formed, which are also difficult to separate.

       On the other hand, if the mixture is acetylated at a higher temperature, the 12ss, 21-diacetate of 4-pregnene-12ss, 17a-21-trio-3,20-dione is formed in addition to the 21-monoacetate of hydrocortisone. The separation of this compound by crystallization, countercurrent distribution or partition chromatography does not present any difficulties and succeeds practically quantitatively. The 4-pregnen-12ss, which can be obtained in this way,

  17a - 21-triol - 3,20-dione-12ss, 21-diacetate is a valuable starting product for syntheses in the steroid series, in particular for the production of pharmaceutically interesting compounds.



  <I> Example </I> 2.41 of a nutrient solution are prepared which contains the following components per liter: 50 g dextrose, 10 g organic malt, 2 g sodium nitrate, 1 g prim. Potassium phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulfate, 0.02 g iron sulfate.



  1.2 l each of the solution are placed in a 2 l laboratory fermenter and sterilized in an autoclave for 30 minutes at 120 and 1 atm. The fermenters consist of a glass vessel with a stainless steel lid; They are equipped with a turbine stirrer, an addition nozzle, an air inlet pipe and an air outlet.

   Each fermenter is inoculated with about 1 billion spores of Coniothyrium hellebori, breed 078, in the form of an aqueous suspension and then inoculated for 48 hours at 25 at 500 rpm. M. stirred. In the meantime, a solution of 1.5 kg dex trose, 30 g prime is in a steel fermenter with 50 l capacity. Potassium phosphate and 90 g sodium nitrate in 30 liters of tap water and sterilized for 40 minutes at 120 and 1 atm.

   The cultures from the two laboratory fermenters are removed sterile, mixed and 1.6 l of the mixture used for inoculating the 50-1 = Fermenfier.



  After stirring for 24 hours while passing through 1-2 m3 of air / hour (25), 5 g of Reichstein's compound S in 40 cm3 of sterile dimethylformamide are added.

   The same amount is added two more times, each with an interval of 24 hours. 24 hours after the last addition, the culture is removed from the fermenter, filtered and the culture filtrate is extracted four times with 41% methyl isobutyl ketone in a Westfalia centrifugal extractor.

   According to chromatographic analysis, the extract contains 7.9 g of hydrocortisone and 3.9 g of 4-pregnen-12ss, 17a, 21-triol-3,20-dione.



  The extract is evaporated to about 150 cm3 in a vacuum. On cooling, the first fraction crystallizes out, the filtrate is evaporated again and further crystal fractions are obtained by cooling, which are combined and dried.



  In this way, 11 g of a product are obtained which contain 66% hydrocortisone and 270 / a 4-pregnen-12ss, 17a, 21-triol-3,20-dione. The crude product is recrystallized twice from ethylene chloride, giving 7 g of chromatographically pure hydrocortisone with a melting point of 212-2.15.

   The mother liquors are evaporated and the residue is taken up in 10 cubic meters of pyridine, 5 cm3 of acetic anhydride are added and the mixture is heated to 50 for 8 hours.

   After the usual work-up, the reaction product is placed on a column which contains 20 g of Celite impregnated with ethylene glycol, that is to say a mixture of different pebbles known under this trade name. Elution with a mixture of 3 parts of cyclohexane and 1 part of methylene chloride gives 3.5 g of crystalline material.

   Recrystallization from a little aqueous methanol gives 3 g of 4-pregnene -12ss, 17a, 21-triol-3.20-dione-12ss, 21-di-acetate. F. 155-157; E238 17 000; [a] D = + 122 (chloro form).



  The elemental analysis confirms the composition of the compound. The mixed melting point with an authentic preparation (J. Chem. Soc. <I> 1955, </I> 870) does not give any depression. The JR spectra of both substances are practically identical.

 

Claims (1)

PATENT CLAIM Process for introducing a hydroxyl group into the 12-position of Reichstein's compound S, characterized in that Reichstein's compound S is exposed to the action of the enzymes of the fungus Coniothyrium hellebori from the Fungi imperfecti class and the Sphaeropsidales series in an aqueous medium Subjects the presence of oxygen and the mixture of the resulting hydroxylated steroids separates and separates. SUBCLAIM Method according to claim, characterized in that the separated mixture of the hydroxylated steroids, optionally after separation of the main amount of the hydirocortisone formed as the main product by crystallization from ethylene chloride, before further separation of an acetylation at elevated temperature subject and the resulting 4-pregnen-12ss, 17a-21-triol-3,20-dione-12ss, 21-diacetate is then separated off by means of partition chromatography from the hydrocortisone-21-monoacetate formed next to it.