DE2041965C3 - Process for the microbiological 1 ^ -dehydration of corticosteroids - Google Patents

Description

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The dehydration of ring A in the 1,2-digit ring is still the most important microbiological steroid setting carried out on an industrial scale. The process was used to make androstadiene dione (US Pat. No. 2,844,513), prednisone (Swedish patent specification 2 12 999), prednisolone (German patent specification 12 87 075) and several substituted ones 1,2-dehydrated corticosteroids (e.g. U.S. Patents 29 38 834 and 30 37 912) used.

For the microbiological conversion, precisely because of their great importance, numerous processes which differ from each other essentially only in the bacterial strain used. So are For example, the following bacteria capable of dehydrating steroids in the 1,2 positions: Arthrobacter simplex, Corynebacterium Hoagii (US Patent 28 37 464), Bacterium cyclooxidans (US Patent 30 22 226), Bacterium mycoides (US Patent 30 37 913), Bacterium havaniensis (US Patent 30 37 914) and Alcaligenes faecalis (Hungarian patent specification 1 51 374).

A common. · Characteristic of the enumerated The method consists in growing the culture in the main fermentation plant and in the same plant the conversion of the substrate steroid is carried out simultaneously or subsequently, that is to say that the Nutrient medium after preparation with for dehydration, o tion capable bacteria is inoculated and simultaneously with it in the 1,2-position saturated steroid substrate added at the time of vaccination or at the time of bacterial growth or after bacterial growth.

In the case of Arthrobacter simplex and Corynebacte- _b 5 criterion is the dehydrogenation inducing steroid-1,2-dehydrogenase enzyme due to its inductive character originally in the bacterial cells not present (Hungarian patent specification 1 49 062), it is only in the presence of steroid on the The effect of a steroid additive is formed. The substrate steroid added in this way first causes the enzyme synthesis; After a corresponding amount of enzyme has been formed, the dehydration is then set in motion, the speed of which increases proportionally to the enzyme synthesis, step by step Dehydration has the highest steroid 1,2-dehydrogenase activity and thus the conversion takes place by far nldit with the optimal or maximum enzyme concentration

This disadvantage is compounded by the known fact that when steroid is added to the bacterial culture the other enzymes in the bacteria break down the steroid into compounds with lower carbon atoms and in addition, using the whole molecule as a carbon source (metabolizing) can (for example Z. Naturforsch. 18b, 988, 1963). The degradation process goes with great Speed of itself, which is why one of the most essential work steps in microbiological steroid 1,2-dehydration canceling the reaction within a few minutes at a specific point in time, if the dehydration is already complete, but the degradation is not yet essential. The rapid determination of the corresponding point in time and the rapid termination of the reaction are associated with many problems.

To reduce the steroid breakdown that occurs in the course of dehydration, see the following listed patents provided the use of various additives. These prevent essentially the metabolism of the bacteria or the function of those causing the degradation Enzymes and thus reduce the consumption of the steroid. Such additives are for example Alcohol or 0-phenylnaphthylamine (British Patent specification 9 08 976), antibiotics (US patent specification 30 10 876), sodium azide, monoiodoacetic acid and other inhibitors of enzyme function (Japanese Patent 25644/67).

Using these inhibitors increases the quantity of the 1,2-dehydrated product is always increased and thus it has been proven that without additives the steroid compounds are actually broken down by bacterial enzymes can. A disadvantage of the addition of substances which hinder cell function is that they cause them Present at the same time the synthesis of the steroid 1,2-dehydrogenase enzyme hinder or slow down its function and thus the implementation time of the substrate is increased several times. According to Japanese Patent 25 644/67 can For example, the dehydration of hydrocortisone compared to the otherwise possible 10 to 20 hours a very good yield can only be carried out within 48 to 60 hours.

In relation to these known processes, the Hungarian patent specification 1 49 062 is an essential one According to this, after establishing the inductive nature of the enzyme, the culture was first a smaller amount of inducer steroid was added. Thus, the synthesis of the 1,2-dehydrogenase enzyme induced and the substrate steroid to be converted is only the already active enzyme-containing culture added. For the induction of the steroid 1,2-dehydrogena-

seenzyms are the compounds of the androstan- or Prejnian series in equal measure. With the that Enzyme-containing culture will accelerate the dehydration of corticosteroids after induction and also happens faster. Thus it is made possible that the steroid to be converted is not part of the concentrated culture to add, but the implementation with a dilution of the culture in a ratio of 1:10 to 1:20 to undertake. The use of the diluted culture is advantageous because it enables cultivation on a large scale Extent eliminated as well as the steroid-splitting effect is reduced. This latter effect will partly achieved by reducing the number of bacteria causing the degradation in the reaction medium and partly because the culture medium, its remaining nutrients, primarily nitrogen-containing compounds, the utilization (des Substantially promote steroids as a carbon source, diluted is from the diluted and no additives containing culture, the dehydrated product can be extracted more easily and the resulting crystalline The product contains practically no foreign impurities.

The Hungarian patent specification 1 49 062 also throws in addition to the enumerated significant advantages unsolved problems. The dehydration can be carried out using the prepared according to that method diluted culture can only be carried out in relatively low concentrations. The product obtained does not always meet the quality requirements, as it is in a higher quantity than that in the pharmacopoeia Contains the prescribed 2% amount of unreacted substrate steroid saturated in the 1,2-position. This is in particular disadvantageous because the product steroid is separated by a single double bond distinguishing substrate afterwards only with great losses by means of a chemical process from Product steroid can be separated, which is why the product of corresponding quality only in quite low Yield is obtained.

The invention is directed to a method in which the advantages of carried out separately Steroid 1,2-dehydrogenase induction are utilized, at the same time, however, the disadvantages mentioned have been eliminated. According to the invention, in the induction phase by inducing secondary growth, an enzyme concentration in excess of the previous one by orders of magnitude (Enzyme level) achieved, which increases the substrate concentration in the dehydration phase enables, reduces the implementation time and a product of appropriate quality and with gives good yield.

The invention therefore relates to a process for the microbiological 1,2-dehydration of corticosteroids with Arthrobacter or Corynebacterium species using the enzyme induction that takes place after the start of cultivation and which precedes the dehydration, which is characterized in that the induction of the steroid-1 , 2-dehydrogenase by adding 0.02 to 0.2 mg / cm ^{3 of} a steroid of the _b0 androstane or pregnane series is carried out only after the bacteria have grown in the stationary phase of cultivation, with the culture at the same time being a 0.01 to 0.1% concentration resulting amount of a carbon and / or nitrogen source _{b5 is} added and maintained for the duration of the induction of secondary growth, whereupon the high activity culture obtained in this way immediately

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35 or after dilution thereof, a corticosteroid saturated in the 1,2-position is added all at once or in portions and the dehydration and isolation of the product obtained is carried out in a manner known per se.

In the course of induction, the steroid 1,2-dehydrogenase content of the culture is increased by one Order of magnitude achieved as follows

The inductor steroid is preferably in coarsely crystalline form or in the form of an aqueous one Suspension or in the form of crystals precipitated from an organic solution with a little water Culture added.

Advantageously, when the induction is started, the culture is or are Carbon and / or nitrogen source a mono- or Disaccharide, an amino acid or a natural substance containing such, in particular corn steep liquor, and / or the ammonium salt of an organic acid, in particular ammonium succinate, is added.

The cultivation of the microorganisms used for the reaction is carried out on a suitable known Culture medium, advantageously a glucose / amino acid culture medium, continued until growth pauses (the optical density reaches the maximum value), as one of the indispensable Components of the agar, the component serving as a carbon source sugar, organic acid or Amino acid or the component used as a nitrogen source, amino acid, ammonium or nitrate salt is exhausted The bacterial culture that has reached a stationary phase in this way becomes a secondary one Growth by adding such an amount of new carbon and / or nitrogen sources that consumed by the bacteria over the course of 2 to 5 hours. (Determination of the Nutrient according to: H. J. Conn: Manual of Microbiological Methods - Soc. At the. Bact. 1957). The selection the carbon and / or nitrogen source to be added depends on the lack of exhausted Nutrients. The carbon and / or nitrogen source is added in an aqueous solution, the Volume can be a maximum of 1/5 of the culture.

Simultaneously with the fresh carbon and / or nitrogen source, the inducer steroid is added to the culture, upon the action of which the steroid 1,2-dehydrogenase content of the culture reaches its maximum value in 2 to 5 hours of secondary growth. The inductor can be added in two ways. The solid steroid can be suspended in the aqueous solution of nutrients or the inducer can be dissolved in an organic solvent. In the latter case, the two solutions, namely the aqueous nutrient solution and the organic steroid solution, are combined with one another before the addition. In this case the inducer steroid is precipitated in the form of coarse crystals due to the small amount of water present, which is why the dissolution of the steroid in the culture is slowed down. Rapid degradation of the inductor is therefore not possible, so that a constant inductor concentration is ensured for the entire duration of the induction. During the induction, the culture is incubated at a temperature of 20 to 4O ⁰ C with aeration.

During induction, the steroid 1,2-dehydroRenase level increases of the culture measured according to the procedure of S i h and B e η e 11 (Biochim. Biophys. A. 56, 584, 1962). By the method according to the invention

the enzyme concentration is increased to a value of 150 to 400 units / cm ³ within 2 to 5 hours, while, in contrast, the amount of enzyme obtained according to the method of the Hungarian patent 1 49 062 is 25 to 40 units / cm ³ ; di.es means that the enzyme concentration increase is 3 to 1 times

The invention is based on the finding that the rate of induction of steroid 1,2-dehydrogenase and the level of the achievable enzyme concentration is the greatest when a culture is in the Growth phase is induced, that is, when food intake and metabolism of the Bacteria is active. In a medium lacking in nutrients in the stationary phase, itself is in In the presence of steroid, no significant amount of enzyme can be obtained

At the same time it was established by the applicant and also proved numerically that, while the active metabolic phase exerts an extremely beneficial effect on enzyme synthesis, on the other hand from the point of view of steroid 1 ^ dehydration is also disadvantageous, especially in this phase namely the most intense steroid degradation brought about by the culture and the disadvantageous ability to reduce the culture high, which is expressed in the reduction of the dehydrated steroid Difficulties have been eliminated by the method according to the invention, in that in the process according to the invention culture in the stationary phase only for the time of induction, specifically for this period of time secondary growth or a renewed active metabolism ensured and the actual reaction, namely the dehydration of the substrate steroid with one after the induction phase showing a certain lack of nutrient medium and are again in the stationary phase Culture is started.

The method according to the invention thus promotes enzyme synthesis to a considerable extent at the same time Eliminates dehydration in the presence of the nutrients that secure the growth of bacteria. A The result of this process is that when the substrate steroid is converted, the dehydration also occurs is extremely rapid in the diluted culture and is neither a steroid breakdown nor a steroid reduction can be proven. In a diluted culture, the hydrocortisone is in a concentration of 1.2 up to 1.6 g / l compared to a concentration of 0.8 to 1.0 g / l according to Hungarian patent specification 149 062 dehydrated and despite the increase in concentration, the dehydration process takes place instead of the usual 6 bis 10 hours in 3 to 5 hours. The product obtained corresponds to that prescribed in the pharmacopoeia Quality and the overall yield is up to 90%, based on the raw material used Hydrocortisone.

Another way of using the bacterial culture produced by the inventive method of high steroid-1,2-dehydrogenase activity sawn w is that which is not diluted by the induction in the stationary phase culture contained, but the same hydrocortisone above in a The amount described is added by 15 to 20 times the amount. At such a high substrate concentration, a dehydration was carried out by Japanese researchers (Swiss patent 3 64.? 60). However, this known method can only produce prednisolone with a high (5 to 10%) hydrocortisone content (E. Kondo, E. Masuo: J. Gea Appl. MicrobioL 7, 113, 1961). In order to reduce the remaining hydrocortisone content, the process according to the invention ensured the high dehydrogenase activity and low reduction capacity . In the induction carried out according to the method according to the invention, the enzyme is formed before the start of the reaction of the substrate and the desired rate of continuous feed can be calculated precisely from the enzyme activity achieved during induction or the reaction rate measured in the first hours of dehydration.

The high enzyme activity of the reaction culture is particularly important in those cases in which the Dehydration immediately with the fermentation broth of the previous microbiological conversion without Isolation of the product is carried out. Namely, in this case, many problems are eliminated, if the dehydrating culture brings about the implementation with good efficiency and appropriate speed and therefore it is sufficient to add 4 to 5% of the same amount to the steroid-containing broth to add.

The invention is explained in more detail with reference to the following examples, which are not to be interpreted as limiting.

example 1

There were 51 sterilized nutrient media with a pH of 6.8 to 7.0, containing 0.5% glucose, 0.3% Ammonium succinate, 0.1% yeast extract and 0.1% potassium dihydrogen phosphate inoculated with a suspension of Corynebacterium fascians The cultivation was under ventilation at 35 ° C and an oxygen supply of 80 millimoles / l / hour Stirring was continued for 14 hours until the ammonium succinate content was consumed of culturing 0.5 l of a sterile solution containing 0.8% ammonium succinate in which 0.5 g of hydrocortisone The incubation was continued in a similar manner for 5 hours during During this time the number of bacteria in the culture increased by about 20% and the activity of steroid 1 ^ dehydrogenase was as follows:

0 hour after the addition

1 hour after the addition

2 hours after the addition

4 hours after the addition

5 hours after the addition

6 hours after the addition

3.5 units / cm ³

17.0 finenesses / cm ³

40.0 units / cm ³

93.0 units / cm ³

107.0 units / cm ³

112.0 units / cm ³

If (according to Hungarian patent specification 1 49 062) a 2-hour induction was carried out in parallel after the same pre-cultivation with the addition of 0.1 mg / cm ^{3 of} hydrocortisone, the activity achieved was only 18 units / cm ³ .

Example 2

250 cm ³ nutrient medium containing 0.4% glucose, 0.4% peptone and 0.4% yeast extract were sterilized in a 750 cm ^{3 Erlenmeyer flask.} The culture medium was inoculated with a liquid culture of Arthrobacterium simplex and incubated at 35 ° C. on the shaking table until the determination of the bacterial count (optical density) indicated the beginning of the stationary phase. This occurred in the 19th to 20th hour of breeding; then 0.2 g of glucose were dissolved in 10 cm ^{3 of} water and 10 mg of prednisolone (inducer steroid) in 2 cm ^{3 of} methanol. The two solutions were combined and added to the culture and incubation continued for an additional 4 hours. After 4 hours of incubation, the activity of the culture was 180 units / cm ³ . The contents of the flask were ^{poured into 4750 cm 3 of} sterile water and 1.6 g / l, i.e. 8 g of hydrocortisone were dehydrated with the diluted active culture in such a way that the hydrocortisone was dissolved in methanol containing 10% calcium chloride in 4 portions every 2 hours Fermentation vessel was introduced. The incubation took place at 35 ^° C. and aeration with stirring to ensure an oxygen supply of 80 millimoles / l / hour. After 8 hours, the fermentation broth contained no hydrocortisone on the basis of the thin-layer chromatographic analysis. The broth was extracted 3 times with 0.3 volumes of ethyl acetate, the extract was drained and evaporated. The residual hydrocortisone content of the resulting crystalline prednisolone product (7.1 g) was 1%.

Prednisolone. The quality corresponded to that of the product of Example 2.

Example 3

200 liters of nutrient medium with a pH of 6.8 to 7.0 and containing 0.5% glucose, 0.1% asparagine and 0.2% corn steep liquor were sterilized. The fermentation vessel was inoculated with 101 of a culture of Arthrobacter simplex and the incubation was continued at 35 ° C and aeration ensuring an oxygen supply of 120 millimoles / l / hour with stirring. After 14 hours the acid content of the culture was 0.02%. Then 25 g of histidine were dissolved in 1000 cm ^{3 of} water and 25 g of hydrocortisone in 300 cm ^{3 of} methanol containing 10% calcium chloride. The two solutions were combined and added to the culture. The induction lasted 3 hours. After the sample taken in the third hour, the activity of the culture was 420 units / cm ³ , which corresponds to the conversion of 57 y / cm ^{3 of} hydrocortisone per minute

The culture containing steroid 1,2-dehydrogenase was diluted in a ratio of 1:20 with water to 40001. Then 3.2 kg of hydrocortisone were immediately added and after 2 hours 1.6 kg of hydrocortisone (a total of 4.8 kg = 1.2 g / l) were added The hydrocortisone could be used for a further hour with the aid of a thin-layer chromatographic examination can no longer be proven. The fennent broth was then extracted with 3x1 / 2 volumes of chloroform and the extract was cleared with charcoal and a filter aid and vacuumed to 201 concentrated. The product was crystallized, filtered and dried in the usual way. Yield: 4.2 kg

Example 4

The culture of Example 3 containing steroid 1,2-dehydrogenase was 20 g / l of hydrocortisone in the added in the following way:

There were 4 kg of finely ground or micronized hydrocortisone in 161 water in the 100 g of polyoxyethylene sorbitan monooleate (emulsifier) have already been dissolved, suspended; then 4 1 Methanol, in the presence of which the suspension is more suitable for feeding, added The suspension was passed through a homogenizer and then at a rate of 11.4 g hydrocortisone / minute added to the culture. The second and sixth hours of feeding were each 1 hour Paused to carry out the control analyzes. The one at 35 ° C and an oxygen supply of 80 Millimol / 1 / hour ensuring ventilation below Stirring continued reaction was followed by microscopic observation and after disappearance of the hydrocortisone granules (predominance of prednisolone needles) was a thin layer chromatographic method Control made. The fermentation was stopped when the amount of the remaining Hydrocortisone was below 2%. The duration of the reaction was 10 to 12 hours.

The prednisolone crystal mass was obtained from the fermentation juice through a separator, respectively Received separator. The prednisolone crystal mass was dissolved in 40 liters of a mixture of chloroform and methanol in a ratio of 1: 1 and the solution was made free of bacteria using a Seitz EK plate filtered, clarified with 100 g of activated charcoal, concentrated in vacuo and crystallized. The remaining hydrocortisone content the 3.45 kg of prednisolone obtained was 1.80 / o.

Example 5

To 61 of a 24 hour culture of the strain Curvularia prasadii grown on a medium containing 1.5% glucose and 2.5% corn steep liquor, 6 g of ^{Reichstein S compound dissolved in 60 cm 3 of} methanol containing 10% calcium chloride were added. The culture was incubated with stirring at 26 ° C. and an aeration ensuring an oxygen supply of 80 Miiiimoi / 'i / hour. Every 4 hours, a thin-layer chromatographic investigation was carried out paper chromatographic analysis a sample (I) was taken (Table I). ^{The culture was filtered, and 300 cm 3 of} a culture of Arthrobacter simplex (see Example 3) were added to the filtrate, which according to the paper chromatographic analysis contained predominantly hydrocortisone On the basis of the thin-layer chromatographic examination, a new sample (II) was taken after 4 hours for the paper-chromatographic analysis. The result of the analyzes is as follows:

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Table I.

investigation investigation No. I. No. Il Reichstein-S connection 12y / cm · ' - Hydrocortisone 608) '/ cm' - 11-ff-OH-Reichstein-S compound 5> '/ cnr' - 14-a-OH-Reichstein-S-compound 82> '/ cm' - I ^ -Dehydro-Reichstein-S-connection - 10 y / cm ³ Prednisolone - 560y / cm ' 20 _: /? - OH-Prednisolone - 16y / cm ³ 1,2-DehydiO-14-ir-C) ll-Reichstein-S compound - 75 y / crn ¹

The product was further recovered in the manner described in Example 3. Yield: 3.1 g of prednisolone.

Example 6

500 cm of sterile nutrient medium containing 0.3% glucose and 0.3% peptone were inoculated with a culture of Arthrobacter simplex. The bacteria were incubated on the shaker table at a temperature of 35 ° C and grown to the stationary phase (about 20 hours). Then the nutrients ensuring growth for the time of induction, namely 0.25 g of glucose and 0.15 g of glycocolla or glycine, were added. The induction was started by introducing a solution of 100 mg of 17-α-methyltestosterone in 10 cm ^{1 of} methanol. After induction carried out on the shaking table at 35 ° C. for 5 hours, the steroid 1,2-dehydrogenase activity of the culture was 220 units / cm ³ .

Then 500 cm ^{J of} the culture were ^{diluted with 4500 cm 1 of} water and, while stirring, a solution of 4 g of Reichstein S compound in 50 cm 'of methanol containing 10% CaC ^ was added. The dehydration was carried out with stirring and aeration and the reaction of the substrate, which took place within about 16 hours, was monitored by analysis by thin layer chromatography. During the soldering hour, a sample was taken for the paper chromatographic analysis, the result of which was as follows:

Table Il

Reichstein-S connection
1,2-dehydro-Reichstein-S compound
20- / K) xy-Reichstein-S connection

) '/ cm' fermentation juice

y / cm 'fermentation juice

30) '/ cm' fermentation juice

The fermentation broth was extracted with 3x (), 3 volumes of ethyl acetate, and then carried out in a known manner Isolation of the product gave 3.28 g of 1,2-dehydro-Reichstein-S compound.

Claims (1)

Claim: Process for the microbiological 1 ^ -Dehydration of corticosteroids with Arthrobacter or Corynebacterium anes using the enzyme induction that takes place after the start of the cultivation and that precedes the dehydration, characterized in that the induction of the steroid 1,2-dehydrogenase by adding ι ο from 0.02 to 0.2 mg / cm ^{3 of} a steroid of the androstane or pregnane series is carried out only after the growth of the bacteria in the stationary phase of the cultivation, at the same time giving the culture a 0.01 to 0.1% concentration Amount of a carbon and / or nitrogen source is added and secondary growth is maintained for the duration of the induction, whereupon a corticosteroid saturated in the 1,2-division is added all at once or in portions to the high activity culture thus obtained, either immediately or after dilution thereof, and carries out the dehydration and isolation of the product obtained in a manner known per se.