DD290216A5 - PROCESS FOR THE PREPARATION OF PREDNISOLONE - Google Patents

Abstract

The invention relates to a process for the preparation of prednisolone from 1-dehydro-11-deoxy-steroids. It is characterized in that the starting materials which are esterified in the 17-position and which may contain acylated hydroxyl groups in other positions, are hydroxylated and saponified with fungi of the genera Cochliobulus lunatus 148, Absidia orchidis 409 and Absidia coerula 468, the fermentation after completed hydroxylation in a hydrogen ion concentration range of 7 to 9 is carried out.

Description

Cochliobulus lunatus 148

Absidiaorchidis409

Absidiscoerula468

be used.

3. Process for the preparation before. Prednisolone according to Claims 1 and 2, characterized in that the fermentation is carried out after completion of the hydroxylation, preferably in a hydrogen ion concentration range of 7.4 to 8.4.

Field of application of the invention

Subject of the invention is the microbieile transformation of 11-deoxy-1,4-dienes steranes of the pregnane series to the corresponding 11 ß-hydroxy derivatives. Steroids with the functional 11β-hydroxyl group serve as drugs for the treatment of rheumatoid arthritis, asthma, dermatoses and other diseases. Only microbial hydroxylation to introduce an OH group into the 11-position of the pregnane skeleton allows the economical production of glucocorticoids.

Characteristic of the known technical solutions

Of the steroid transformations known since 1952 on the pregnane backbone, 11β-hydroxylation still has the greatest importance. Suitable species of microorganisms from the literature are the species Cunninghamella

Curvularia

Trichothecium and

Absidia known.

Thus, for example, DE-PS1618599,2803660 and 2803661 describe the selective 11β-hydroxylation of steroids.

It was possible to obtain high yields of 11β-hydroxylation products by consideration of the substrate specificity of the competent microorganisms by modification or substitution of the starting material (about 88% by weight). Cuivularia species were used as microorganisms which form undesired by-products (7.14-hydroxylated compounds according to US Pat. No. 2,783,255) with a yield of less than 40% under conventional conditions of culture culture and in Reichstein's substance S as starting steroid.

Object of the invention

The aim of the invention is to overcome with selected microorganisms in continuous or semi-continuous Fermentationsführung dio disadvantages of the described technical solutions in the 11 ß-hydroxylation of steroids of the pregnane series, such fungi are to be used, which are fast growing, the logarithmic in a few hours Complete the growth phase and form the mycelial form of the pellets in the stationary phase with sugar limitation. In this case, the fungi used in their Enzymbesteck next to hydroxylases have esterases that cause the saponification of the steroids of the pregnane series at the set culture conditions, so that a subsequent chemical saponification can be omitted. The fungi have to guarantee a high steroid throughput with the use of 11-desoxy-1,4-dieno-steroids, wherein predominantly 17-substituted steroids are to serve as starting substances, the number and amount of the by-products are low and their occurrence in the Isolation should not disturb.

-2- 290 216 Explanation of the nature of the invention

With the invention, the problem is solved, with selected microorganisms 11-deoxy-1,4-dien-steroids to transform economically to prednisolone.

The process according to the invention for the preparation of prednisolone is characterized in that 11-deoxy-1,4-diene steroids which are esterified in the 17-position and which may contain acylated hydroxyl groups in other positions are hydroxylated and saponified with fungi, the fermentation after completed hydroxylation in a hydrogen ion concentration range of 7 to 9 is performed.

It has surprisingly been found that especially the species of the genera

Cochlibulus lunatus 148 Absidia orchidis 409 and Absidia coerula468

capable of introducing a hydroxyl group into the 11 ß-position, when the above-mentioned compounds serve as starting material. The fungi found to be suitable also possess esterases, so that the esters, preferably acetates of the 11-deoxy-1,4-diene steroids, can serve as the starting substance.

It was found that the transformation to introduce a 11 ß-hydroxyl group in the stationary growth phase at a hydrogen ion concentration of pH 4.8 to 5.3, the subsequent saponification at pH 7 to 9, preferably 7.4 to 8.4 below strictly aerobic conditions must be made (pO ₂ saturation must not fall below 30%). The phase of optimal 11 (3-hydroxylation is achieved when using the known analytical methods

- no carbohydrates

- no organic and inorganic nitrogen

is more evidence and the hydrogen ion concentration has reached a pH of 4.8 to 5.3. The pH is maintained at pH 4.8 to 5.3 by titration with acids, preferably sulfuric acid. The hydroxylation reaction is strictly oxygen-requiring, the throughput of starting steroid is limited by the supply of oxygen.

The starting substances are micronised or added in solvents such as acetone, dimethylformamide-water mixture, ethanol or methanolic calcium chloride solution. On the one hand, it can be added during the resting stage of the submerged culture, on the other hand it is possible to separate the biomass from the culture solution at the optimal mycelium stage and to resuspend the pellets in an isotonic medium.

For the invention, conditions are given by the choice of the microorganism, a fungus of the genera Absidia and Cochliobulus, the nutrient medium and the fermentation guide, which allow high yields of 11 ß-hydroxylated compounds. Complete transformations of the 11-deoxy-i, 4-dien-steroids of the pregnane series were achieved.

Throughputs of 0.5 g to 1.5 g starting substance / l culture solution are possible.

The method according to the invention is explained in the following exemplary embodiments:

embodiments

example 1

Preparation of prednisolone from 17-acetoxy-21-hydroxy-pregna-1,4-diene-3,20-dione with Cochliobulus lunatus 101 nutrient solution of the following composition

glucose 1% Corn steep liquor 2% KH ₂ PO ₄ 0.0025% MgSO ₄ .7H ₂ O 0.02% Sunflower oil 0.005% AntaphronR 0.005% Distilled water PH value 5.0 to 5 ,:

are sterilized for 35 min at 120 ^° C. in a 15 l laboratory fermenter. At 30 ⁰ C is inoculated with 11 seed medium which has grown on the same medium for 46 hours at 20 ⁰ C in culture bottles on a rotary shaker (240 U / min).

After a growth phase of 8 to 14 hours (stirring 620U / min, aeration 11 air / l culture solution, temperature 29 ⁰ C ± 0.5 ⁰ C) is a mycelial formation of 15 to 17.5% biomass (3000 U / min, 3min long) and a pH of 4.8 to 5.2 in the culture solution.

The addition of 10 g of 1-dehydro-RSS-17-acetate, dissolved in 500 ml of methanolic CaCl ₂ solution, takes place from the

8. Fermentation hour continuously over a period of 12 hours. As a rule, when the fermentation regime is followed, the transformation is completed 24 to 36 hours after the addition is complete.

The transformation process is monitored by thin-layer chromatographic methods. It is finished when no starting substance is detectable.

After complete conversion, the experiment is worked up:

After filtering off the mycetium, the culture filtrate is extracted three times with 0.8% by volume of i-methylene chloride. The combined organic extracts are concentrated in vacuo and the resulting oil is dissolved in 300 ml of ethyl acetate and separated from insoluble constituents. The organic extract is washed three times with 150 ml of 0.1 N NaOH, then with water until neutral

washed, dried over sodium sulfate 3t and concentrated to incipient crystallization. After 18 hours at 5 ° C, the

crystallized product separated and dried. This gives 7.3 of a crystal mixture which contains in addition to small impurities substantially prednisolone.

The crystal mixture is dissolved in 600 ml of methanol at room temperature. After saturation with nitrogen, 520 mg of KOH are added, and it is stirred for 40 min at room temperature. The resulting clear solution is neutralized with 1N acetic acid and stirred with 4 g of activated charcoal. After filtering and concentration of the reaction solution is recrystallized from 6 ml of chloroform. The product obtained consists of 6.5 g of prednisolone in a yield of 70%.

Example 2

Preparation of prednisolone from 17a, 21- (1'-ethoxy-ethylidene-dioxy) -pregna-1,4-diene-3,20-dione (17a, 21-orthoester) with Cochliobulus lunatus

A nutrient solution of the following composition, contained in a 2.5 l Erlenmeyer flask

glucose 1% Corn steep liquor 2% MgSO ₄ -7H ₂ O 0.002% KH ₂ PO ₄ 0.0075% Aqua Dest. pH 5.0 to 5 ,:

is inoculated with 10% of a 22 hour old culture solution, which was obtained as follows: From a spore Iyophil preserve of the strain Cochliobulus lunatus by addition of physiological saline a suspension and with 0.5ml of which a submerse propagation passage in 500ml Erlenmeyer Piston created. The nutrient solution has the following composition:

glucose 2.5% Corn steep liquor 0.5% peptone 0.25% NaNO ₃ 0.12% KH ₂ PO ₄ 0.01% MgSO ₄ .7H ₂ O 0.002% Aqua dcst. pH 6.5 to 6

The first submerged Vorzuchtstufe of the strain is described Cochliobulus lunatus 64 to 68 hours at 26 ⁰ C ± 0.5 ⁰ C on a rotary shaker, as in Example 1, cultured. 10ml of the first pre-breeding stage are used to inoculate the second propagation passage (250ml nutrient solution in the 2.5L Erlenmeyer flask). The nutrient solution has the following composition:

glucose 1.5% 22 to 24 hours. Corn steep liquor 1% KH ₂ PO ₄ 0.025% MgSO ₄ -7H ₂ O 0.02% Aqua Dest. pH 6.5 to 6.7. jcuiiiyuiiyun öiiiu. temperature 26 ° C ± 0.5 ° C Ventilation: Rotary shaker (240 rpm, amplitude 4 cm) Kulturzeit

For the following culture conditions of the transformation stage:

Temperature 29 ⁰ C ± 0.5 ⁰ C

ventilation

Rotary shaker (240 rpm, amplitude 4cm)

is 8 to 10 hours after inoculation achieved the glucose limitation and a pH of 4.5 to 4.8 given. At this time, the I.Zugabe of 125 mg 17a, 21-orthoester, dissolved in 2.5 ml of methanolic CaCl ₂ solution under aseptic conditions. This process is repeated three more times, so that in a batch of 500 ml 500 mg 17a, 21-orthoester are included. After 24 to 30 hours, the hydroxylation step b is sent, and the fermentation process is continued for a further 16 to 20 hours to give a hydrogen ion concentration of pH 7.5 to 8.5 at which complete saponification of the prednisolone acetate to prednisolone occurs

 After complete conversion, the experiment is worked up as follows:

The mycelium is removed from the fermentation solution and the filtrate is exhaustively extracted with 3% by volume of ethyl acetate. The combined extracts are concentrated under reduced pressure to the oil and dissolved in methanol to remove insoluble matter. The filtered methanol solution is concentrated to dryness, dissolved in 17 ml of chloroform and washed three times with 5 ml of a saturated sodium acetate solution. After drying with sodium sulfate, the chloroform solution is mixed with 10 ml of isopropanol and concentrated in vacuo until incipient crystallization. After standing for 24 hours at 5 ⁰ C, the crystal slurry is filtered off with suction, washed with ice-cold isopropanol and dried. Yield: 382.6 mg Prednisolone (88%)

Example 3

Preparation of prednisolone from 17,21-diacetoxy-pregna-1 / -diene-3,20-dione d-dehydro-RSS-IV ^ I-diacetate) with Cochliobulus lunatus

According to Example 2, 500 mg of 1-dehydro-RSS-17,21-diacetate are added in portions to a 10 hour culture of Co'hliobulus lunatus. This approach is worked up as follows:

The culture solution is filtered and the filtrate is extracted three times with 2.5%, 10% butyl acetate. The combined extracts are concentrated under reduced pressure to give the oil and dissolved in 22 ml of chloroform to which 4% by volume of methanol has been added. The solution is passed through a Kieselgelsäuie to separate the by-products. When concentrated, 290.2 mg of prednisolone are obtained from the main fraction as white crystals (yield 69%).

Example 4

Preparation of prednisolone from 17,21-dihydroxy-pregna-1,4-diene-3,20-dione (1-dehydro-RSS) with Cochliobulus lunatus According to Example 2, a 10 hour old culture of Cochliobulus lunatus 125mg 1-dehydro-RSS added. After 72 hours 52% prednisolone is formed by thin-layer chromatography. Two more polar products than prednisolone are present at 18%, in addition to 17% of the original substance.

Example 5

Preparation of prednisolone from 17a, 21- (1'-ethoxy-ethylidene-dioxy) -pregna-1,4-diene-3,20-dione (17a, 21-orthoester) with Absidia orchidis 409

2.5 liter breast bottles with 500ml nutrient solution of the following composition:

glucose 1.5% Corn steep liquor (dry weight) 0.3% Peptone for bakt. purposes 0.2% _MgSO4 0.025% KH ₂ PO ₄ 0.025% Aqua dist 11 pH 5.0 to 5.2

were inoculated with 10% of a 22 hour old culture solution, which was obtained as follows: From a spore Iyophil preserve of the strain Absidia orchidis is prepared by addition of physiological saline suspension and with 0.5ml of which is a submerse propagation passage in 500ml Erlenmoyer Inoculated. The nutrient solution has the following composition:

glucose 0.5% Corn steep liquor (dry weight) 1.5% peptone 0.1% Distilled water on 11 pH 5.2 to 5.4.

The submerged preemergence stage is cultivated on a rotary shaker for 22 hours at 25 ° C. (240 rpm, amplitude 4 cm).

The culture conditions of the transformation stage correspond to those of the pre-breeding stage. After 34 to 36 hours of growth, the mycelium has developed in the form of pellets. The moist mycelium content is 15 to 17.5% (3000rpm, over 3min). At this time, the I.Zugabe in the form of 125 mg 17a, 21-orthoester is dissolved in 2.5 ml of ammoniacalmethanol solution. This process ^v: j repeated three more times. 24 to 30 hours after the last addition, the hydroxylation is complete and the fermentation conditions for the biological saponification are set: aeration: 1.51 l / l broth, pH 7.5 to 8.5, temperature 28 ° C. The saponification process lasts 18 to 22 hours. The resulting free prednisolone is isolated and recovered as described in Example 2.

Examples

Preparation of prednisolone from 17a, 21-orthoester with Absidia coerula 468

If, according to example 5, Absidia coerula 468 is used instead of Absidia orchidis dor fungus, prednisolone is obtained under the same conditions.

Example 7

Preparation of prednisolone from 17a-monoacetate and 17a, 21-diacetate of 1-dehydro-RSS with Absidia orchidis 409 and Absidia coerula 468

If the 17a-monoacetate or the 17a, 21-diacetate of 1-dehydro-RSS are used as starting material for the fungi Absidia orchidis 409 and Absidia coerula 468 and the procedure is as described in example 5, prednisolone is in a formation mode of 75 to 85 % receive.

Claims (2)

1. A process for the preparation of prednisolone from i-dehydro-11-deoxy-steroids, characterized in that the starting materials which are esterified in the 17-position and which may contain acylated in other positions hydroxyl groups are hydroxylated and saponified with fungi, wherein the fermentation is carried out after completion of the hydroxylation in a hydrogen ion concentration range of 7 to 9 and then the prednisolone is isolated in a known manner. 2. A process for the preparation of prednisolone according to item 1, characterized in that for microbial transformation, the fungi