DD290217A5 - PROCESS FOR PREPARING PREDNISOLONE-17-ACETATE - Google Patents

Abstract

The invention relates to a process for the microbial transformation of * to prednisolone 17-acetate. It is characterized by the fact that the starting materials which are esterified in the 17a-position and which may contain acylated hydroxyl groups in other positions, are hydroxylated with fungi, in particular with Cochliobulus lunatus 148, Absidia orchidis 409 and Absidia coerula 468 and by inhibition of the esterases the fungi in a fermentation run in the hydrogen ion concentration range of 5 to 7, the saponification reaction is inhibited.

Description

Field of application of the invention

The invention relates to the microbial transformation of 11-deoxy-1,4-diene-3-oxo-steroids of the pregnane series, which are esterified in the 17a position, to Prednlsolone 17-acetate. Prednisolone acetate is used as a medicine for the treatment of rheumatoid arthritis, asthma, dermatoses and other diseases.

The microbial hydroxylation of 11-deoxy-1,1-diene-S-oxo-steroids of the pregnane series esterified in the 17a-position to prednisolone-17-acetate is an economically and technically advantageous variant for obtaining this highly effective glucocorticoid.

Characteristic of the known state of the art

Of the numerous known microbial hydrolysis reactions on the steroid skeleton, 11β-hydroxylation is the most important factor. For the production of prednisolone or its esters, the following reaction steps are carried out industrially:

- 11 ß-hydroxylation of Reichstein's substance S (RSS) to hydrocortisone

- Isolation of hydrocortisone

- Microbial dehydration of hydrocortisone to prednisolone

- Isolation of the prednisolone.

The two biological stages with their separate isolation of fermentation products have a significant impact on the cost of the final products. It is known that the use of acetates as starting steroids makes the throughput more difficult and the effectiveness of the overall process is worsened. The microorganisms used, preferably species of Curvularia, Cunninghamella and Trichothecium, possess esterases, and the end product is a mixture of steroid alcohols and esters. Another chemical reaction step to achieve a uniform end product follows. 11β-hydroxylations of 1,4-dienoic steroids of the pregnane series are not known on an industrial scale. Such procedures are currently of academic interest only. The method known from DE-OS 2901 561 with the fungus Curvularia lunata allows only a throughput of 0.5 g Stero'd / I culture solution.

Object of the invention

The aim of the invention is, with suitable microorganisms, preferably fungi or their enzyme systems in conventional Fermentationsweise the batch culture or semicontinuous or continuous fermentation and / or immobilized cells of the microorganisms in the carrier-fixed state 17-acylated 11-deoxy-1 To transform 4-dienes of the pregnane series to the corresponding 17a-prednisolone acetate. In the microorganisms used, preferably Phycomycetes and Ascomycetes, it must be ensured that the contained in their Enzymbesteck next to hydroxylases catechases, which normally provide a mixture of prednisolone and 17 μ-prednisolone acetate, by the fermentation, especially when using the physiological state of the logarithmic growth phase be inhibited. This ensures that only 17a-prednisolone is present at the end of the fermentation, which is then particularly favorable to win from the culture solution. The method has to guarantee a high steroid throughput (1 to 2 g steroid / l culture solution), wherein in the 17-position acylated 11-deoxy-1,4-diene-3-oxo-steroids of pregnane series serve as starting substances, the number of by-products is low and their quantitative share does not disturb.

Explanation of the essence of the invention

The inventive experience for the production of α-prednisolone acetate is characterized in that 17-acylated 11-deoxy-1,4-diene-3-oxo-steroids of the pregnane series, which are esterified in the 17a position and which also acylated in other positions Hydroxyl groups can be transformed with fungi of the genera Cochliobulus and Absidia. Possess special suitability

Cochliobuluslunatur 148 Absidiaorchidis 409 Absidiacoerula 468. The esterases contained in the enzyme kit of the mushrooms mentioned are used by the fermentation guide when using the

optimal physiological state form of the mycelium for 11 ß-Hydrioylierung inhibited, so that only prednisolone acetate on

End of the transformation is isolated from the culture solution. It has surprisingly been found that the transformations for the introduction of an 11 ß-hydroxyl group in 17-acylated

11-dianoxy-1,4-diene-3-oxo-steroids of the pregnane series at the end of the logarithmic growth phase, shortly before the beginning of the

Carbohydrate limitation, can be made particularly advantageous. It has proven to be expedient to grow the mycelium in the form of the pellets in order to obtain optimal oxygen supply

to obtain short transformation times and high substrate throughputs. The mycelium can undergo repeated hydroxylation

aseptic conditions are used. φ

The phase of optimal hydroxylation is achieved when using the known analytical methods, a glucose Konzehtration

is determined from 0.02 g / l culture solution, the hydrogen ion concentration reaches a value of 5 to 7 and the biomass

Dry weight of 10 g / l culture solution. Hydroxylation is felt for longer times by lowering the pH by titration with carbohydrate solutions,

preferably glucose or sucrose, maintained at 0.02 to 0.04 g of glucose or sucrose per liter of culture solution and optionally adjusted with mineral acids such as sulfuric acid or hydrochloric acid to the pH range from 5 to 7 suitable for the hydroxylation reaction and the oxygenation by means of aeration and Stirring is brought to a degree of saturation of 40% to 50%. The throughput of starting steroid is limited by the oxygen supply.

The conditions indicated for the continuation of the hydroxylation reaction simultaneously inhibit the esterases whose

pH optimum is 7.5 to 8.5.

The starting materials are micronized or in a solvent such as acetone, dimethylformamide-water mixture

or lower alcohols, dissolved added. Suitable for continuous addition are methanolic CaCl ₂ -

Solutions in which the starting steroids are already dissolved at room temperature. The addition occurs during logarithmic growth. It begins just before the onset of carbohydrate limitation, in the Re (> '6 to 8 hours after inoculation. Fü »invention are by the choice of microorganisms, by the culture medium and by the fermentation Given conditions that give high yields of prednisolone 17-acetate. The inventive method is explained in more detail in the following embodiments: embodiments example 1 Preparation of 17-prednisolone acetate from 17-acetoxy-21-hydroxy-pregna-1,4-diene-3,20-dione (1-dehydro-RSS-17-acetate) with Cochliobulus lunatus

1.1. Incubation of i-DehydiO-RSS-17 acetate with Cochliobulus lunatus 148

Laboratory Fermentation:

2.5 L breast bottles with 500 ml of nutrient solution, which has a pH of 6.5 to 6.7 and the following composition

Corn steep liquor (100% dry weight) ad 1.0% glucose 2.5% NaNO ₃ 0.2% KH ₂ PO ₄ 0.1% _MgSO4 0.05% Aqua Dest. 1.01

are inoculated with 10% of a 22h culture solution of Cochliobulus lunatus. The submerged Vorzuchtstufe 22 hours at 26 ⁰ C ± 0.5 ⁰ C on a rotary shaker cultivated (240U / min; amplitude 4cm). 50ml culture suffice for inoculation of 500ml nutrient solution of the transformation step in 2.5-liter breast bottles The nutrient solution has the following composition:

Yeast extract 1.0%

Glucose 1,5%

KH ₂ PO ₄ 0.025%

MgSO ₄ 0.03%

Aqua Dest. ad 1.01

and must have a pH of 5.0.

The culture conditions are: temperature 29 ⁰ C ± 0.5 ⁰ C, aeration 240U / min, Amplitude 4cm. After 6 hours of growth, the nutrient solution is overgrown, the moist mycelium content is 7.5 to 10.5% (3000 rpm above

3 minutes). At this time, the first addition of 125 mg of 1-dehydro-RSS-17-acetate, dissolved in 1 ml Dimetnylformamid.

This process is repeated after 6 hours, so that in a batch of 500ml 250mg of 1-dehydro-RSS-17-acetate

are included.

The Transformationverlaut is followed by thin-layer chromatographic methods. He is terminated because no starting substance is more detectable. 18 hours after the last addition, the transformation is complete. The culture solution contains 58% prednisolone and 26% prednisolone 17-acetate.

1.2. Incubation of: Dehydro-RSS-17-acetate with Cochliobulus lunatus 148 in the 30 l Fermanter 20I nutrient solution of the following composition:

Corn steep liquor (based on dry weight) 3% sucrose 1% _MgSO4 0.15% KH ₂ PO ₄ 0.075% ZnSO ₄ 0.005% CaCl ₂ 0.1% polypropylene glycol 0.005% tap water

which has a pH of 5.2 are sterilized in a 30 l fermenter for 35 min at 123 ⁰ C. After cooling to 29 ⁰ C is inoculated with 21 seed medium from Steilbrustflaschen, whose nutrient solution, the composition, as in 1.1. described, vaccinated. After a growth phase of 6 hours (stirring 540 rpm, aeration: 11 l / l culture solution -min;

29 ° C ± 0.5 ° C; Oxygen saturation: 50%), a pH of 4.8 to 5.2 is reached, the glucose concentration is 1.2 g per liter of culture solution.

The addition of 30 g of 1-dehydro-RSS-17-acetate, dissolved in 11 methanolic CaCl ₂ solution, takes place continuously over a period of 10 hours. The transformation becomes, as in 1.1. The pH is maintained at pH 5.0 to 5.5 with 1 η H ₂ SO ₄ and the glucose concentration is adjusted by means of 50% sterile glucose solution at 0.2 g glucose / l culture solution. As a rule, if the fermentation regime is maintained, the starting material is converted 36 to 40 hours after the end of the addition.

After complete conversion, the experiment is worked up, the mycelium filtered off and washed with water. The culture filtrate combined with the washing liquid is exhaustively extracted with methylene chloride, the extract is concentrated in vacuo as far as possible and the concentrate is placed in the refrigerator overnight.

The precipitated crystals are filtered off, washed and dried in vacuo, then analyzed. The content of pure prednisolone 17-acetate is 81%, of which 56% are obtained as Erstkristallisat. The other 34% prednisolone 17-acetate can be obtained from the column chromatography of the mother liquor. Overall, 71% of the theoretically possible yield can be obtained.

Example 2

Preparation of 17-Predniso! Acetate from 17,21- (1'-ethoxyethylidene-dioxy) -pregna-1,4-diene-3,20-dione (1-dehydro-RSS-orthoester) with Cochliobulus lunatus 148

The conditions of growth and transformation correspond to those in Example 1 under 1.2.

After a growth period of 6 hours, 3g 1-dehydro-RSS-orthoesters are continuously added to the culture solution (31) over a period of 12 hours. The pH is adjusted to pH 5.0 to 5.5 after the pH drop at the beginning of growth.

From the 1-dehydro-RSS-orthoester 1-dehydro-RSS-17-acetate is formed under the chosen culture conditions, which is converted by the hydroxylases of the fungus to prednisolone-17-acetate.

From 3g 1-dehydro-RSS-orthoester 69% prednisolone-17-acetate is obtained.

Example 3

Preparation of 17-prednisolone acetate from 1-dehydro-RSS-17-acetate with Absidia orchidis 409 or Absidia coerula 468 21 nutrient solution of the following composition

Corn steep liquor (100% dw) 0.5% glucose 1.5% peptone 0.4% KH ₂ PO ₄ 0.05% _MgSO4 0.025% ZnSO ₄ 0.012% Antaphron (silicone oil) 0.005% Distilled water

which is adjusted to a pH of 5.8 to 6.2, are inoculated with 200 ml of culture solution of the fungus Absidia orchidis 409 (or Absidia coerula 468 in the same nutrient solution). After a growth phase of 30 hours (agitation: 630U / min; aeration: 11 air / l of culture solution · min; 40% oxygen saturation, temperature: 26 ⁰ C ± 0.5 "C) isteineMycelbildung from 10 to 12.5% biomass (3000 RPM, for 3 minutes) in the form of pellets, the pH of 4.5 to 5.0 and a glucose concentration of 0.3 to 0.4 g of glucose per liter of culture solution.

The addition of 3 g of 1-dehydrc-RSS-i7-acetate in 30 ml of methanol is carried out continuously over a period of 20 hours. The transformation is as in Example 1 under 1.1. describe, controlled. As a rule, if the fermentation regime is followed, the starting material is converted 48 to 60 hours after the end of the addition.

The workup is carried out as in Example 1 under 1.2. described. 65% prednisolone 17-acetate is obtained after vacuum distillation.

Example 4

Preparation of 17-Prednisolone Acetate from 1-dehydro-RSS-orthoester with Absidia orchidis 409 and / or Absidia coerula 468 2g 1-dehydro-RSS-orthoesters are added to the cultures of the fungi Absidia orchidis 409 and / or Absidia coerula 468 and are used as described in Example 3 described, so 63% prednisolone 17-acetate are obtained.

Claims (2)

1. A process for the preparation of prednisolone 17-acetate from 1 1-deoxy-1,4-diene-3-oxo-steroids, characterized in that the starting materials which are esterified in the 17-position and the acylated in other positions hydroxyl groups may be hydroxylated with mushrooms and the saponification reaction is inhibited by inhibiting the esterases of the fungi in a fermentation run in the hydrogen ion concentration range of 5 to 7. 2. A process for the preparation of prednisolone 17-acetate according to claim 1, characterized in that for microbial transformation, the fungi Cochliobulus lunatus 148
Absidiaorchidis 409 Absidiacoerula 468 be used.