Patent GB-830921-A (GB)
Title: í¸-steroids and their preparation by a microbiological process
IPC: C12P33/00
Filed: 1956-03-21
Abstract: The invention comprises a process for the D 1-dehydrogenation of steroids by subjecting them to the dehydrogenating activity produced by Bacillus micro-organisms, especially Bacillus sphaericus.  The process, which is preferably applicable to D 4-3-keto-, D 5-3a -hydroxy, or D 5-3b -hydroxy-pregnenes, 3-keto-, 3a -hydroxy or 3b -hydroxypregnanes, any of which may contain e.g. 11-keto, 11b -hydroxy, 17-hydroxy, 20-keto, 21-hydroxy, 21-aldehydo, or 9a -halo substituents, and in which the 3- or 21-hydroxy groups may be acylated, e.g. with benzoyl or lower alkanoyl groups, is normally carried out (a) by adding the steroid (solid or in solution) under sterile conditions to a culture of the organism in a nutrient medium and agitating the mixture; (b) by agitating a buffered aqueous solution of the steroid with a suspension of the washed cell growth filtered from an established culture; or (c) by contacting the steroid with dehydrogenating enzyme preparations from the micro-organism.  Specified compounds obtained by the process include the following: D 1,4-pregnadiene - 3,20 - diones: the parent compound, 17a  - ol, 21 - ol, 17a ,21 - diol, 17a  - ol - 21 - al, 11 - keto, 11 - keto - 17a  - ol, 11 - keto - 21 - ol, 11 - keto - 17a ,21 - diol, 11 - keto - 17a  - ol - 21 - al, 11b  - ol, 11b  - ol - 21 - al, 11b ,21 - diol, 11b ,17a  - diol, 11b ,17a ,21 - triol, and the corresponding 9a -halo compounds, especially 9a -fluoro derivatives.  In examples: (1 and 8) hydrocortisone in dimethyl-formamide is agitated with a culture of B. sphaericus (A.T.C.C.-12488) in an aqueous nutrient medium at 28 DEG  C., the mixture extracted with ethyl acetate and the extracts subjected to paper chromatography, using formamide as the stationary phase and chloroform as the mobile phase, yielding hydrocortisone and D 1,4 - 3,20 - diketo - 11b ,17a ,21 - trihydroxypregnadiene; similarly (3) to (9), (12) and (13) the following compounds are dehydrogenated in the 1:2-position: cortisone, D 4 - 3,20 - diketo - 17a ,21 - dihydroxypregnene, 9a  - fluorohydrocortisone, D 4 - 3,20 - diketo - 11b ,21 - dihydroxypregnene, D 4 - 3,20 - diketo - 21 - hydroxypregnene, D 4 - 3,20 - diketo - 11b  - hydroxypregnene, D 4 - 3,20 - diketo - pregnene, D 4 - 3,20 - diketo - 11a ,17a ,21 - trihydroxypregnene, and D 4 - 3,17 - diketo-androstene; (10) D 4 - 3b - hydroxy - 20 - ketopregnene similarly yields D 1,4 - 3,20 - di - ketopregnadiene; (11) hydrocortisone is dehydrogenated using B. sphaericus A.T.C.C.-12488 on each of seven different media; (14) and (15) hydrocortisone is subjected to the action of B. sphaericus (A.T.C.C.-245) and B. sphaericus (A.T.C.C.-7054); (16) 9a -fluorohydrocortisone is dehydrogenated with B sphaericus (A.T.C.C.-7055); (17) the following steroids are dehydrogenated with B. sphaericus (A.T.C.C.-12634) and the D 1;4-steroids isolated by means of streak paper chromatography with various solvents: D 4-androstene-3,17-dione, D 5 - pregnen - 3b  - ol - 20 - one, D 4 - pregnene-17a ,21 - diol - 3,11,20 - trione, pregnane-17a ,21 - diol - 3,11,20 - trione 21 - acetate, pregnane-3b ,17a ,21 - triol - 20 - one 21 - acetate, pregname - 17a ,21 - diol - 3,20 - dione - 21 - acetate, D 4 - pregnene - 3b ,17a ,21 - triol - 11,20 - dione 21-acetate, and D 4-pregnene-3,20-dione and its 21 - ol, 17a ,21 - diol, 17a ,21 - diol 21 - acetate, 11b ,17a ,21 - triol, and 9a  - fluoro, 11b ,17a ,21 - triol derivatives.  Reference has been directed by the Comptroller to Specification 807,277.