DE1693041C - Oxazoline Steroid and Process for Their Manufacture - Google Patents

Description

The invention relates to sieroid oxa-olines of the formula CII ₁ R

co _vl

C -

wherein R is either hydrogen, the hydroxyl or aceloxy group and R 'is hydrogen or the methyl radical means.

The new compounds have a high level of anti-inflammatory and hormone-like agents Degree of pharm ological efficacy.

The inventive method for manufacturing The new stcroid-oxazoline consists in taking a steroid-oxazoline of the formula

CH, R CO

I /

t I

C R '

in which R and R 'have the meaning given above, by fermentation with Curvularia lunata NRRL 2380 b / w. Cunninghamclla blaekesleeana NRRL8688A or Absidia orchidis ATCC ^- 6647 enzymatically hydroxylieri

To carry out this process, the microorganisms mentioned are cultured and omitted aerobic conditions in a suitable nutrient medium to a steroid oxazoline of the above Act formula. Due to the oxidizing effect of the enzymes formed by the microorganisms is here in the 11-position of the steroid oxazoline introduced a hydroxyl group.

A suitable nutrient medium must be a carbon dioxide and contain a nitrogen source and mineral base materials. Usable carbon sources are Starch, dextrose. Sucrose. Galactose. Xylose. Glycerin and other carbohydrates and polyvalent ones Alcohols, as well as other natural sources of carbohydrates, like corn steep water. Cottonseed meal. Some of the useful sources of slicksloff include abovementioned substances, such as. B. Corn soaking water, cottonseed breath. other substances, such as beef extract, Mcfc, peptones, amino acids, generally enzymatically hydrolyzed proteins and other proteinaceous substances. Inorganic nitrogen sources, too, such as urea, nitrates, ammonium salts and the like are usable.

The required minerals are either in the same substances listed above, if in raw State used, or present in the water used to make the medium. If the medium is to receive an additive, a suitable amount of inorganic substances, such as phosphate, Sulphate, chloride, alkali and alkaline earth metals. Iron, manganese, and cobalt are added to the medium with cations and anions.

The steroid to be subjected to I! -Hydroxylation can be given before or after the Add the medium with the culture of the microorganism.

The fermentation is then continued at a temperature between about 26 and 32 C for 12 to 12 hours. After the fermentation is finished if necessary, ablillricrt the medium from the mycelium. the 11-hydroxy lated steroid with a water-insoluble organic solvent in which the Steroid dissolves, extracted and then the organic extract in a vacuum to dryness or / u evaporated to a small volume. In the latter case, the addition of one forms with the for Extraction solvent used:; ischhareii Solvent in which the steroid is insoluble, a precipitate for concentrated steroid solution of the product. This can then be chromatographically or by another suitable method like recrystallization. getting cleaned.

According to a further procedure, after good mycelial growth has been achieved in a medium Part of the mycelium separated from the listed composition and placed in a phosphate buffer solution transferred to a pH between 7.0 and 7.5 where it comes into contact with the steroid to be converted is brought. The steroid is then extracted from the liquid as described above isolated.

Compared with other known ones, the same Sleroids possessing pharmacological properties, such as hydrocortisone. show the invention Compounds have strong anti-inflammatory properties. The results of this effectiveness that were evaluated in the case of granules in rats, can be seen from TaOeIIe I. The one in 10% Gunimi Arabic dissolved or slurried products were orally administered to male Wistar rats weighing about 120 to 150 g.

Table I.

connection of the example

3
Hydrocortisone

Doms

in ηιμ kg

by «» -,

Number of Γ Ii rf

Ahn.ihiue des (lunulonis in ".

3 ^l ) .6
34

1H.4
■ »5

It was found that the connection of the Example 3 also shows a gluconcogenic activity exhibited. The tests were carried out according to

fco von Olsen et al. \\\ Endocrinol 35 (1944). P. 40, described method was carried out on surreclomized male Wistar rats. If one compares the results of the compound according to the invention with the result obtained with hydrocortisone under the same experimental conditions, one finds that the former is more effective. The following table II summarizes the results found in both cases.

I 693 041

Γίΐhelle II

ti Cs Hl ¹ J. (M

Hydrocoriison

Control attempts

in: Hiii Ιιιμ π; 11

500
2000

500
2000

Zillll OlT KiIlL-Il

10
10
10
10
10

ι S.li.-lny
prn HKl μ
UlT K '.) (ilUL'll- | l!
I iL-w kill
; || U 1.14 12.84 t 2.34 3 3.7 ¹ yr I. 0. ¹ Y 7 7.04 -fc 1.43 05/20 (- 0.46 1.18 fc

The following examples illustrate the invention:

Example I.

Pregn-4-e »-11 //. 21-dinl-3.20-dione- [17u.J6.i-dl-2-methyloxazoline-21-acetal

Several 500 ml conical colons. each of which 100 ml of the following nutrient medium contained:

Dextrose 10.0 g

Soybean meal 5.0 g

Peptone 5.0 g

Busumin Bush 3.0 g

KH ₁ PO, 5.0 g

NaCl 5.0 g

Silicone oil 0.1 ml

filled up with distilled water / u I 1 (whereby the pH value before sterilization η., ί NaC) H to pH 68 is discontinued), with a 4-day-old Schrüg culture of Curvularia lunata NRRL 2380 inoculated after incubation for 24 hours at 28 C on a rotating shaker (250IJ min.) good my / eh growth is achieved.

4 l of a medium of the specified composition are in a pre-fermentation vessel with 5 volume percent (V V) of the culture obtained above and inoculated under the following conditions incubated: 1.5 1 air per liter of medium per minute (1.5 1 1 min). Stirring speed 8 (K) U Min. Temperature 28 C. Time 24 hours

4 l of the same medium described above are added to a new fermentation vessel 2 (X) ml of the fermentation produced above Inoculated nutrient broth is carried out under the same conditions as in the previous step. After 24 hours, the Separated mycelium by filtration

The isolated, moist mycelium »weight about 3 (KIg) is in 4 l of a phosphate buffer M 15 at a Slurried pH 7.3 in a fermentation vessel and the slurry for 10 minutes long with a speed ν, οη 8 (K) Ü min. touched. The air is fed in at a rate of 1.51 per liter of liquid per minute and the temperature set to 28 C.

Prcgn-4-cn-2l-ol-3,20-dione- [l7-i.l6. (- d] -2'-meth>'_; -oxazoline-21-acetal is used in an amount of 0.5% ( Oew./Vol.) Of the medium, dissolved in 40 ml of acetone, added to the medium and the mass exposed to the above-mentioned stirring, air supply and temperature conditions for 24 hours, at which point the reaction is stopped by adding chloroform.

The mixture is nitrated, the mycelium several times Washed chloroform and extracted the filate with chloroform. The united, received at home washing C'hlornformanieile and extracts become smaller in a vacuum. Volume evaporated. Then petroleum ether is added to form a precipitate. The crude product is through Countercurrent flow with a mixture of methanol : Water: chloroform: ligroin in ratio

cleaned to 2: 1: 2: 1. The wheezing is 0.5 g of Pregn-4-ene-11 / i, 21-diol-3.20-dione -117 ". I (vi-d] -2'-met hyloxazoMn, which forms 21-acetate with lissigsäureanhydrid in pyridine Melting point 224 to 225 ° C, [, <]: + 108 (c = 0.5, ClICI ₁ ).

if. The identity of the Vet bond obtained is established by exposing it to the action of the enzyme produced during the fermentation of Corynebaclerium simplex SCH 2141 under the conditions generally used when a double bond is introduced into the I (2) colony of the steroid nucleus will. This process gives the well-known Pregna-1.4-diene-11,. '. 21 -diol-3.20-dione- [17 ". 16.id] -2'-methy lo \ azolin-2! -acetate. Melting point 256 to 257 C / _x 241 to 243 (CH ₁ OHl

The starting compound necessary for carrying out the process described in this example is produced by the following process steps. A mixture of 5 g of Pregn-5-cn-3,; - ol-20-one- [i7.i.l6.i-d] -2'-niethyloxazoline. 40 ml of tetrahydrofuran. 40 ml of methanol. 7.5 g calcium oxide and 0.11g of azo-bis-isobutyronitrile is vigorously Tubes gradually added a solution of 5 g of iodine in 25 ml of tetrahydrofuran and 13 ml of methanol. The mixture is then filtered and the filtrate is evaporated to dryness in vacuo the residue dissolved in 80 ml of methylene chloride. the organic solution is first made with sodium sulfate and then washed with water and then evaporated to dryness in vacuo This residue (the 21-iodine derivative of steroid si is dissolved in 40 ml of acetone and one to 50 to 60 ° C heated solution of 28 ml of acetic acid. 14 ml of acetone and 50 ml of triethylamine are then added the mixture was refluxed for 90 minutes

The acetone is distilled off, and then the mixture 350 ml of warm water are gradually added. After cooling, the precipitate formed is separated off and washed The yield entrusts 4.8 g of pregn-5-en-3 / ;. 2l-diol-20-one [17.i.l6.i-d] -2-methhvlo \ azoline-21-acetate. Melting point 232 to 234 C. [<«]: 20.4 (c - 0.5. T HCI,)

By known processes is applied to the recovered steroid using Aluminiumisopropylal and cyclohexanone in toluene a Oppenaucr oxidation performed from 35 g of the starting pregn-5-ene, 30 g (yield 86 ¹ O) pregn-4-ene-21 - Oil 3.20-dione - [I7 ". | 6., - d] - 2 - melhyloxazohn-21-acetal obtained. Melting point 204-206 C. [><]': +86 (<= 0.5. CHCl,). El ^ 401.8. >. """ 240 μ

fio (CH ₁ OlI).

Example 2

Pregn-4-en-11 f / -ol-3,20-dione- [17 «.16.i-d] -oxazoline

The preparation is carried out exactly as described in Example I, with Prcgn-4cn-3,20-dione- [17.i, 16ii-d] -oxazoline as the starting compound and C'unninghamella blackcsleeana NRRL 8688A as a formulation organism is used.

The melting point of the Siemid oxa / oline obtained is I ¹ JS to 2 (M) C.

Example 3

l'regn-4-en-11 / ί.21-diol-3.2 () -dione- [I Tu. 16n-d] -2-melhyloxazoline

The production takes place exactly as described in Example I, the starting compound being Pregn-4-en-2l-ol-3,20-dione- [17u, 16ii-d] -2'-methyIoxazo | in and Absidia orchidis ATCC 6647 as a fermented organism is used,

The melting point of the obtained sleroid oxazoliiis is 235 C.

Claims (1)

Patent claims:
I. Sieroid oxazolines of the general formula C-R ' 25th where R is hydrogen, the hydroxyl or acctoxy group and R 'is hydrogen or methyl.
2. Prcgn-4-cn- 11 //. 2I - diol - 3.20 - dione - [17, j. ! 6 «-d] -2-methyloxazoline. λ method / ur production of a Sicntid-Oxazolins tier aligemeiiv-Ίι I-'orniel CILK
CO 110- C R ' in which R is hydrogen, the hydroxyl or aceloxy group and R 'represents hydrogen or the methyl radical, characterized in that one Oxazoline steroid of the general formula CH ₁ R CO C R ' in which R and R 'have the meaning given, by fermentation with Curvularia lunata NRRL 2380 or Cunninghamella blackesleeana NRRL 8688 A or Absidia orchidis ATCC 6647 enzymatically hydror.ylicrt.