DE1593502A1 - Method of making steroids - Google Patents

Description

Method of making steroids

The present invention relates to a process for the preparation of 14a-hydroxysteroids of the general

lOrmel

OH

lower

wherein R-,, Rp, R ^ are hydrogen or a preferably acyl radical mean and which according to the invention all 4 - by variation the steric arrangement on the possible carbon atoms denoted by # - should include diastereomers both in the isolated state and in any proportion of the individual diastereomers.

The new process products are characterized by their high effectiveness as insect metamorphosis hormones. Besides they show profound influences on cell metabolism

009829 / t, 4_82 BAD ORIGINAL

- 2 - P. 934 / April 7, 1966 . _: .

in other living beings, especially in warm-blooded animals. Effects on the central nervous system are also observed. This results in a multiple technical usability, for example as pharmaceuticals in human and veterinary medicine or as a pesticide in crop protection. Show the known corresponding 2o-deoxy compound (ecdysone) the new process products, such as the Calliphora test using the example of Δ -5β-cholesten-2β, 3β, 14a, 2o, 22,25-hexaol-6-one (2o-hydroxy-ecdysone), consider.

Like ecdysone, so is naturally occurring 2o-i-hydroxy ecdysone from z. B. insects - e.g. B. from silkworm pupae Bombyx mori in a kenge of 47 mg / 2.8 t - can be isolated. Around To be able to use highly effective products in practice, their synthetic production is of technical importance.

The new connections are obtained by going into connections the general formula

OH

OH

according to methods of steroid chemistry known per se, be it purely chemically, in particular through the action of selenium Acid, be it by biochemical means, especially by exposure of such microorganisms or those formed by them

009829/1482

BATH ORIGINAL ~ ³ ~

SCHERING AG. . - 3 - P. 934 / April 7, 1966

Enzymes known to be capable of 14a-hydroxylation are, such as B. Enzymes from strains of the genus Curvularia or Heliocostyium sov / ie Absidia regnieri, the lAa-permanent Introduces hydroxyl group are desired either to start from sterically uniform starting materials or that according to the invention Process primarily obtained diastereomer mixture in itself known way z. B. by fractional distillation, chromatography or countercurrent distribution afterwards into its components to separate. After the introduction of the I4a-hydroxyl groups - and zv; ar before or after the separation of the diastereomers - can gevünsehtenfalis the 2- and / or 3- and / or 22-rowed ones Hydroxyl groups are usually acylated verier.

In particular, these hydroxyl groups are esterified Acetic acid, propionic acid, butyric acid or their reactive ones Derivatives in question.

_{The from t} rar.gs:r.äteriul, not previously described in the literature, for the production of which iir. Barge the present. Registration ochutz: not desired, iziain after per se known riethoden of the steroid ohe ~ «ie, z. . 3. from 2i 3: - Liacetc: -: yA ^'-:: -l pregnene-ü, 2c-dicn (prepared from 2o-A "thylendic:';yi; .i rre.rr <.an- 3a-oi-co: i: analogous to the working methods described in Tetrahedral Letters 1; 6ό , 1337} according to the following reaction schemes:

009829/1482

- Δ -

BAD ORIGfNAL

RINGAG

P. 934 / April 7, 1966 '

-ep / η ere

Aco

CH

009829 / U82

BATH ORIGINAL

SCHERINGAG -5- P. 934 / 7.4.196 *

example 1

300 mg of 20- (l ^{l, l} 4-dihydroxy-4- ^{l-methyl-pentyl)} -A ⁷ -5ß-pregnene-2ß.3ß.20-triol-6-one (prepared from 2ß-r3ß-diacetoxy-A ⁷ -5ßpregnen-6.20-dione by vinyl grignardation at Cp ₀ , ozone cleavage to 20-aldehyde, grignardation with 2-methyl-butyn-2-oltetrahydropyranyl ether, hydrogenation of the triple bond and hydrolysis) are dissolved in 15 ml of dioxane and treated with 300 mg of selenium dioxide for 70 minutes heated to 80 ° C. The solvent is removed in vacuo and the residue is purified by preparative layer chromatography (silica gel PF ₂ 54 ¹ ^ ⁰ K ^AG * development with chloroform / methanol). 20 mg of Δ'-50-ChOIeSten-2β.j5β are obtained. 140-20.22.25-HeXaOl-O-On, which melt after recrystallization from tetrahydrofuran / ethyl acetate at 237.5 to 239.5 ° C.

UV: ^ ₂ k2 = 12,400 (methanol)

Example 2

200 ecm of a medium consisting of 4.5 % glucose, 2 % peptone and 0.5 % itcorn steep liquor are sterilized in a shake flask. Absidia regnieri is then inoculated and shaken at 28 ° C. for 2 days 90 mg of 20- (1 ', 4'-dihydroxy-4'-methyl-pentyl) -A ^ -5ß-pregnen-2ß, 3ß-20-triol-6-one in 5 ml of 2.5 % methanol are added to this culture After the fermentation, which can be determined by thin-layer chromatography, the cell mass is filtered off and extracted with acetone and ethyl acetate, after which all the extracts are combined. The extracts are washed with sodium hydrogen carbonate solution and water, dried over sodium sulfate and to dryness

009829 / U82

BATH ORIGINAL -6-

SCHERING AG -6- p.934 / 7.4.1966

concentrated in vacuo. After purification by preparative thin-layer chromatography, the 20- (l *, 4 ^l -dihydroxy-4 ^l -methylpentylM -5ß-pregnen-2ß, 3ß * l ^ ^a -20-tetrol-6-one; P. 236 bis 240 ° C.

UV: ^ ₂₄₂ = 12,400 (methanol)

" ¹

IR: (KBr) ^ _0H 3440 cm

1650 cm " ¹ 16IO cm" ¹

KMR: (Proton resonance _{spectrograph if} Varian HP 100 ", perdeuterated pyridine, reference substance tetramethylsilane)

C-18 methyl protons ^c 1.19 C-19 methyl protons δ 1.05 C-21 methyl protons δ 1.55

C ₂ C and C ₆ methyl δ 1.35 ^{3 protons}

C ₇ vinyl proton δ 6.20

Example 3

A nutrient solution consisting of

5 % sucrose

1 # beet sugar molasses 0.2 % NaNO,

0.1 % KH ₂

0.05 $ KCl

MgSO ₄
0.001 ^ PeSO ₄

0.5 % corn steep water (known under the trade name

"Cornsteep Liquor) from pH ·

009829/1482 ORIGINAL BATHROOM

SCHERING AG -7- P «934 / 7.4.1966

is sterilized by heating to 120 ° C for 90 minutes and, after cooling, with a spore suspension of Curvularia lunata inoculated, which was obtained by washing away a 7-day maize culture with physiological saline.

After 2 days of propagation at 25 ° C with stirring and aeration, 280 ml of the culture produced are removed under sterile conditions and placed in a fermenter with 4.7 liters of a nutrient solution of 5 % sucrose, 1 % beet sugar molasses, 0.2 % NaNO-, , 0.1 % KH ₂ PO ^ transferred. After cultivation with stirring and aeration for 24 hours, 1.13 g of 20- (1 ^f , V-dihydroxy-V-methyl-pentyl) -A'-5β-pregnen-2fl, 3β, 20-triol-6-one in 50 ml of ethanol added. After the fermentation has ended, the procedure described in Example 2 is continued. Isolating the 20- (l ^l, 4'-dihydroxy-4 ^l methyl-pentyl) -A -5ß-pregnene-2ß, j5ß, L4A, 20-tetrol-6-one. M.p. 237.5 to 239 ° C.

009829 / U 8 2

Claims (7)

^ HERING AG P. 934 / April 7, 1966. Patent claims 1. Process for the preparation of 14oc-hydroxysteroids of general formula OH -L OH H 0 before R-,, R _? , R _{7 is} hydrogen or a preferably lower acyl radical ", characterized in that compounds of the general formula * £. OH HO either according to known methods of steroid chemistry a) purely chemically, in particular by exposure of selenous acid or 009829/1482 ORIGINAL BATHROOM SCHERING AG P. 934 / April 7, 1966 b) on biochemical V / ege through the action of such microorganisms or or enzymes formed by them, which are known to be capable of 14α-hydroxylation, such as. B. the enzymes from strains of the genus Curvularia or Heliocostylum as well as Absidia regnieri, which introduces the 14a hydroxyl group, and, if desired, subsequently esterifying the 2- and / or 3- and / or 22-hydroxyl group. 2. The method according to claim 1, characterized in that one starts from diastereomer mixtures and the primary 14a-hydroxylation products subsequently separates into their components according to methods known per se. 3. The method according to claim 1, characterized in that one starts from a uniform diastereomer. 4. The process products according to claim 1 as a mixture of diastereomers and as isolated diastereomers. 5 · Compounds of the formula R ₂ O OH 009829 / U82 IHERINGAG - & - P. 934 / 7- April 1966 where R-,, R _{? t mean} R, hydrogen or a preferably lower acyl radical, if produced synthetically. 6. A ⁷ -5β-Cholesten-2β, 3β, 14a, 2o, 22,25-hexanol-6-oii, prepared synthetically. BATH ORIGINAL