JPH0369918B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 12α-hydroxypregna-4-en-3-one-20-carbaldehyde and a method for producing the same. 12α-Hydroxypregnath-4-en-3-one-20-carbaldehyde provided by the present invention is a new compound that has not been described in known literature, and is typified by prednisone and prednisolone, which have excellent anti-inflammatory effects. It is useful as an intermediate for producing 12α-hydroxypregna-4-ene-3,20-dione, which is a raw material for the synthesis of corticoid steroids. Traditionally, prednisone has been manufactured by using deoxycholic acid as the starting material and going through more than 20 steps [Co-authored by L.F. Fieser and M. Fieser; Steroids (published by Reinhold, Inc.;
(1959), pp. 634-647] is known.
This method uses expensive reagents and requires a long process, so it is not necessarily suitable for industrial implementation. The present inventors conducted intensive studies on useful intermediates for producing prednisone, and found that 12α
-Hydroxypregna-4-en-3-one-20
- Carbaldehyde is easily obtained by reacting specific bacteria belonging to the genus Pseudomonas with deoxycholic acid and/or its salt, and 12α-hydroxypregna-4-ene-3 is a raw material for the synthesis of prednisone. , 20-dione can be easily derived, leading to the present invention. or (In the above formula, R represents a hydrogen atom, an alkali metal or an alkaline earth metal, and R ¹ represents an alkyl group.) According to the present invention, 12α-hydroxyprecipitates using deoxycholic acid and/or a salt thereof as a substrate. Guna
12α-Hydroxypregnane-4-
En-3-one-20 carbaldehyde can be obtained with high selectivity and high yield. The above bacteria belonging to the genus Pseudomonas include Pseudomonas putida D4014−A357
(Pseudomonas putidaD4014-A357) strain (Feikoken Article No. 206). This strain is Pseudomonas putida D4014 collected from soil.
(Pseudomonas putidaD4014) strain (Feikoken Joyori No. 205) was subjected to mutation treatment. Pseudomonas putida obtained by the present inventors
D4014 strain and Pseudomonas putida D4014−
The following table lists the mycological properties of the A357 strain.

【table】

[Table] Based on the mycological properties shown in the table above, Pseudomonas putida D4014 strain and Pseudomonas putida D4014-A357 strain were identified.
Pseudomonas putida strain D4014 is a bacillus, has polar flagella, has negative Gram staining, positive oxidase and catalase reactions, and is aerobic. Based on the physiological properties such as the O-F test result being oxidative, bacteria belonging to the genus Pseudomonas are classified according to the Verges Manual of Determinative Bacteriology, 7th and 8th editions. It was identified that Furthermore, Pseudomonas putida strain D4014 has a fluorescent color in its culture,
It was identified as a bacterium belonging to the Pseudomonas genus Putida species because it does not liquefy gelatin, grows at 37°C, and produces arginine dihydrolase. Furthermore, it is generally believed that a mutant strain belongs to the same species as its parent strain, and it was determined that the Pseudomonas putida strain D4014-A357 belongs to the species Putida of the genus Pseudomonas. 12α-Hydroxypregna-4- according to the invention
En-3-one-20-carbaldehyde is produced using 12α-hydroxypregn-4-en-3-en-3-one using deoxycholic acid and/or its salt as a substrate.
The method is carried out by culturing bacteria belonging to the genus Pseudomonas that produce 1-20-carbaldehyde in a medium containing deoxycholic acid and/or its salt. Specific examples of the salts of deoxycholic acid include salts of deoxycholic acid with alkali metals such as sodium and potassium, and salts with alkaline earth metals such as calcium and magnesium. The concentration of deoxycholic acid and/or its salt may normally be in the range of about 1 to 200 g/, but depending on the yield of 12α-hydroxypregn-4-en-3-one-20-carbaldehyde produced, culture conditions, From an economical point of view such as operability, the amount is preferably in the range of about 2 to 50 g/. The culture method is basically the same as that used for aerobic culture of general microorganisms, but usually a shaking culture method using a liquid medium or an aerated agitation culture method is used. The medium contains a nutrient source that can be assimilated and utilized by bacteria belonging to the genus Pseudomonas that produces 12α-hydroxypregn-4-en-3-one-20-carbaldehyde using the above deoxycholic acid and/or its salt as a substrate. It is fine as long as it is done. As the carbon source, deoxycholic acid and/or its salt may be used as the sole carbon source, or glucose, glycerin, peptone, meat extract, yeast extract, etc. may be used in combination with deoxycholic acid and/or its salt. Examples of nitrogen sources include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate,
Inorganic nitrogen sources, such as potassium nitrate, or organic nitrogen sources, such as polypeptone, peptone, meat extract, are used. In addition, inorganic salts such as dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate are added. Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually carried out at 25 to 35°C for 10 hours to 7 days. 12α-Hydroxypregn-4-en-3-one-20-carbaldehyde accumulated in the culture solution in this way has significantly lower solubility in water than the substrate deoxycholic acid or its salt. Usually, it precipitates out in the culture solution. this
To separate and collect 12α-hydroxypregn-4-en-3-one-20-carbaldehyde, the precipitated 12α-hydroxypregna-4-en-3-one-20-carbaldehyde is decanted. The precipitated 12α-hydroxypregnane-4-ene is separated by centrifugation at a rotation speed that does not precipitate the floating bacteria, or by centrifugation at a rotation speed that does not precipitate the floating bacteria. The method used is to precipitate -3-one-20-carbaldehyde and then separate 12α-hydroxypregn-4-en-3-one-20-carbaldehyde by the above-mentioned decantation. A culture filtrate obtained by separating and removing bacterial cells and other insoluble components contained in a culture solution from which precipitated 12α-hydroxypregn-4-en-3-one-20-carbaldehyde has been removed by filtration or centrifugation. Or, after making the culture filtrate or supernatant alkaline by adding an alkali such as sodium hydroxide, potassium hydroxide, or calcium hydroxide to the supernatant, an organic solvent that dissolves the aldehyde and phase separates from water, e.g. An extraction operation is performed using ethyl acetate, chloroform, a mixture of chloroform and methanol, etc., the resulting extract is collected, and the solvent is distilled off from it to remove the 12α- dissolved in the culture solution. Hydroxypregna-4-en-3-one-20-carbaldehyde can be recovered. This extraction operation using an organic solvent can be performed not only on the culture filtrate or supernatant, but also on the culture solution itself. The precipitate or extract obtained by the above method contains 12α-hydroxypregn-4-ene-3-
Besides 1-20-carbaldehyde, it contains almost no residual substrate deoxycholic acid and/or its salts and by-products, and it is easy to obtain high-purity 12α-hydroxypregnathic acid by recrystallization from an aqueous methanol solution, for example. -4-en-3-one-20-carbaldehyde can be obtained. 12α-Hydroxypregn-4-en-3-one-20-carbaldehyde obtained by the present invention is obtained by (Method-1) reacting it with piperidine in the presence of a dehydrating agent such as potassium carbonate or barium oxide. 12α-hydroxy-22-(N-piperidyl)
Bisnor-4,20(22)-colladien-3-one and then ozone oxidation of this product yielded 12α-hydroxypregna-4-ene-3,20
-Can be induced into diones. Also, according to (Method 2), 12α-hydroxypregnath-4-en-3-one-20-carbaldehyde and the general formula () R ¹ COOH … () (wherein R ¹ represents an alkyl group) 12α-acyloxypregn-4-en-3-one represented by the general formula () can be obtained by reacting a carboxylic acid represented by the formula () or a reactive derivative thereof, such as an acid halide, an acid anhydride, etc., by a conventional method. -20-carbaldehyde is obtained. As a typical reaction example, the reaction between 12α-hydroxypregn-4-en-3-one-20-carbaldehyde and the chloride of a carboxylic acid represented by the general formula () is a reaction with a tertiary chloride such as triethylamine or pyridine. Performed in the presence of an amine. This reaction is preferably carried out in a solvent, methylene chloride,
Chloroform or these together with benzene, toluene,
A mixed solvent with ethyl acetate or the like is preferably used. This reaction is usually carried out at room temperature, but if necessary, it can be carried out with heating up to about 60°C. After the reaction, the reaction mixture is washed with dilute hydrochloric acid, sodium bicarbonate, water, etc., and then dried, and then the low boiling point substances are distilled off to obtain 12α represented by the general formula ().
-Acyloxypregna-4-en-3-one-
A crude product of 20-carbaldehyde is obtained. This crude product can be used as it is in the next reaction. By reacting 12α-acyloxypregn-4-en-3-one-20-carbaldehyde represented by the general formula () with a secondary amine such as piperidine, pyrrolidine, morpholine, etc. Enamines are produced. The secondary amine is used in an equimolar to twice molar amount relative to 12α-acyloxypregn-4-en-3-one-20-carbaldehyde represented by the general formula ().
Water produced as a by-product during the reaction is removed from the reaction system under heating under reflux using a solvent that is azeotropic with water, such as benzene or triene. Although this reaction does not particularly require a catalyst, it can also be carried out in the presence of a catalyst such as p-toluenesulfonic acid. After the reaction, by distilling off low-boiling substances from the reaction mixture under reduced pressure,
A crude enamine product represented by the general formula () is obtained. This crude product can be used as it is in the next reaction. By oxidizing the enamine represented by the general formula () using ozone oxidation or chromic anhydride, pyridinium chlorochromate, sodium dichromate, etc.,
3,20-dione can be obtained. Note that the oxidation reaction using chromic anhydride is usually carried out in a pyridine solvent. In this case, a mixture of chromic anhydride and pyridine is gradually added to a pyridine solution in which the enamine represented by the general formula () is dissolved, or the enamine represented by the general formula () is added to the mixture of chromic anhydride and pyridine. The reaction is carried out by gradually adding a pyridine solution in which . This oxidation reaction is carried out under ice cooling or at room temperature. After the reaction,
Dilute the reaction mixture with benzene, toluene, etc.
After removing solid matter from this by filtration, dilute hydrochloric acid water is added to the filtrate, followed by extraction with benzene, toluene, etc., and by distilling off low-boiling substances from the extract, 12α-acyloxy A crude product of pregna-4-ene-3,20-dione is obtained. This crude product is purified by silica gel column chromatography or recrystallization as necessary to obtain a highly pure 12α-acyloxypregn-4-ene-3,20 represented by the general formula (). - Diones can be obtained. 12α-hydroxypregna-4-ene-3,20-dione is obtained by subjecting 12α-acyloxypregna-4-ene-3,20-dione represented by the general formula () to a conventional hydrolysis reaction. It will be done. For example, this hydrolysis reaction can be performed using methanol,
It is carried out in a solvent such as ethanol in the presence of potassium hydroxide, sodium hydroxide, etc. at room temperature to the reflux temperature of the solvent. After the reaction, the reaction mixture is concentrated under reduced pressure, diluted with benzene, toluene, etc., washed with water, diluted hydrochloric acid, etc., dried, and then the lower boiling point substances are distilled off to obtain 12α.
A crude product of -hydroxypregna-4-ene-3,20-dione is obtained. This crude product can be purified, for example, by recrystallization from ethyl acetate. 12α-Hydroxypregna-4-ene-3,20
-dione can be derived into prednisone, for example, by the following method. [In the above formula, Me represents a methyl group and Ac represents an acetyl group. ] That is, 12α-hydroxypregna-4-ene-3,20-dione is oxidized with chromium trioxide, and the oxidation product is further oxidized with selenium dioxide in the presence of potassium hydroxide to produce pregnane.
4,9(11)-diene-3,12,20-trione is obtained. This pregna-4,9(11)-diene-3,12,
20-trione is hydrogenated in the presence of platinum to give 12α-hydroxypregna-4,9(11)-diene-3,20-dione, and this product is then treated with methanol under acidic conditions. 12α-Methoxypregna-4,9(11)-diene-3,20-dione was obtained, and this product was treated with hydrogen chloride to produce 12-chloropregna-4,9(11)-diene-
3,20-dione is obtained. By treating this 12-chloropregna-4,9(11)-diene-3,20-dione with sodium hydrogen carbonate, pregna-4,
11-diene-3,20-dione was obtained, and this product was treated with bromine to produce 11,12-dibromopregna-4.
-ene-3,20-dione, which was then hydrolyzed in the presence of silver chromate and chromium trioxide to produce 12-bromopregn-4-ene-3,
11,20-trione is obtained, and this product is treated with metallic zinc to form pregna-4-ene-3,11,20-trione. This pregna-4-en-3,
Treatment of 11,20-trione with acetic anhydride yields 20-acetoxy-4,17(20)-diene-3,11
- Obtain dione. By reacting this product with perterephthalic acid, 20-acetoxy-17α,
20α-epoxypregna-4-ene-3,11-dione is obtained, which is hydrolyzed to obtain 17α-hydroxypregna-4-ene-3,11,20-trione, and then this product is treated with iodine. Let it work 17α
-hydroxy-21-indopregna-4-ene-
We get 3,11,20-trion. This 17α-hydroxy-21-indopregna-4-ene-3,11,
Treatment of 20-trione with potassium acetate in acetone yields 17α-hydroxy-21-acetoxypregna-4-ene-3,11,20-trione. Next, a microorganism capable of forming a double bond at the 1-position of this 17α-hydroxy-21-acetoxypregnath-4-ene-3,11,20-trione, such as the easily available Arthrobacter simplex IAM1660, is then added. (Arthrobacter simplex
IAM1660) 21-acetoxy-prednisone is obtained by reacting the bacteria using a conventional fermentation method, and prednisone can be obtained by hydrolyzing this. Also, 12α-hydroxypregna-4-ene-
3,20-dione can be derived into hydrocortisone and further prednisolone via the known compound pregna-4,11(12)-diene-3,20-dione by the following method. (In the above formula, R ² represents an alkyl group.) That is, by subjecting 12α-hydroxypregna-4-ene-3,20-dione to a normal sulfonation reaction, a compound represented by the general formula () is obtained. A sulfonate is obtained. For example, in this sulfonation reaction, 12α-hydroxypregn-4-ene-3,20-dione is dissolved in pyridine, picoline, or a mixed solvent of these and benzene, toluene, etc., and this solution is added to the general formula () R ² SO ₂ Cl ...() (In the formula, R ² represents an alkyl group.) The sulfonic acid chloride represented by: Add molar amount, at room temperature or as needed, approx.
It is carried out under heating up to 80°C. After the reaction, the reaction mixture is poured into diluted hydrochloric acid, etc., extracted with benzene, etc., the extract is washed with diluted hydrochloric acid, sodium bicarbonate, water, etc., dried, and the low boiling point substances are distilled off to obtain the general formula. A crude product of the sulfonate shown in parentheses is obtained. A highly pure sulfonate represented by the general formula () can be obtained by recrystallizing this crude product from, for example, ethyl acetate. The sulfonate represented by the general formula () is induced into the known compound pregna-4,11(12)-diene-3,20-dione by subjecting it to a desulfonic acid reaction. This desulfonic acid reaction is typically performed using potassium acetate, lithium chloride,
It is carried out in the presence of a reaction aid (a compound capable of scavenging sulfonic acid) such as collidine and potassium t-butoxide. The amount of the reaction aid to be used is equimolar to 20 times the molar amount of the sulfonate represented by the general formula (). This reaction is preferably carried out in a solvent such as hexamethylphosphortriamide or N,N-dimethylformamide, and is usually carried out by heating at a temperature of about 80 to 140°C. Pregna-4,11(12)-diene-3,20-dione is induced into hydrocortisone and further into prednisolone by a conventional method. The present invention will be explained in more detail below using Examples and Reference Examples. Reference example: Method for obtaining Pseudomonas putida D4014-A357 strain Medium 1 (composition: deoxycholic acid 0.5%, sodium hydroxide 0.05%, peptone 0.5%, yeast extract
Pseudomonas putida grown on slants of 0.5% sodium chloride, 0.5% sodium chloride and 1.5% agar).
A loopful of D4014 strain was prepared in advance in a test tube in medium 2 (composition: 2% deoxycholic acid, 0.2% sodium hydroxide, 0.2% ammonium nitrate, 0.1% potassium dihydrogen phosphate, 0.6 potassium dihydrogen phosphate).
%, magnesium sulfate heptahydrate 0.02%, and yeast extract 0.02%) and cultured with shaking at 30°C for 14 to 15 hours. Culture medium 3 (composition: deoxycholic acid 0.5
%, sodium hydroxide 0.05%, glucose 0.1%,
Ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1
%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02% and yeast extract 0.02%).
ml and cultured at 30°C for 8 to 9 hours. Then,
The cells in the logarithmic growth phase were collected by aseptic filtration using a 0.45 μ membrane filter, washed with 20 ml of 0.1 M phosphate buffer (PH: 7.0), and washed with 25 ml of the same buffer.
ml. Mutation treatment was carried out by adding N-methyl-N'-nitro-N-nitrosoguanidine to the mixture at a final concentration of 50 μg/ml and allowing it to stand for 3 to 4 minutes. The cells subjected to mutation treatment were collected by filtration using a 0.45μ membrane filter, washed with 20ml of 0.1M phosphate buffer (PH: 7.0), and then suspended in 20ml of the same buffer. The obtained bacterial suspension was diluted with sterile physiological saline and added to medium 4.
(Composition: Deoxycholic acid 0.5%, sodium hydroxide 0.05%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02
% and agar 1.5%) on an agar plate so that 500 to 1000 colonies appear, and then incubated at 30°C.
The cells were cultured for 3 to 4 days. After isolating the tiny colonies among the colonies that appeared on the slant of medium 1,
Culture medium 5 (composition: deoxycholic acid 0.2%, sodium hydroxide) prepared in advance in a test tube
0.02%, glucose 0.1%, ammonium nitrate 0.2
%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%
and yeast extract 0.02%) and inoculated at 30℃.
It was cultured with shaking for 24 hours. The products in each culture solution obtained were assayed by thin layer chromatography, and the target 12α-hydroxypregnane-4-
We found a strain that selectively accumulates en-3-one-20-carbaldehyde and named it Pseudomonas putida D4014-A357. Example 1 Pseudomonas putida D4014-A357 strain (Feikoken Joyori No. 206) was cultured by the following method. Deoxycholic acid 0.2g, glucose 0.1g,
Ammonium nitrate 0.2g, potassium dihydrogen phosphate 0.1
g, dipotassium hydrogen phosphate 0.6 g, magnesium sulfate heptahydrate 0.02 g, and yeast extract 0.02 g, add 50 ml of tap water, add 1N sodium hydroxide to this solution to adjust the pH to 8.4, Further water was added to bring the volume to 100 ml, and this was used as a culture medium. Pour this medium into a 500ml Sakaguchi flask and heat at 120℃ for 15 minutes.
Steam sterilization was performed for 1 minute. Add 10 ml of the inoculum grown in advance in a test tube shaker for one day in the same medium as the above medium to the above 500 ml Sakaguchi flask,
Culture was carried out with shaking at 30°C for 2 days. After culturing, the precipitate and bacterial cells produced during the culture were collected using a centrifuge, washed with water, and then 50 ml of methanol was added thereto. After sufficiently dissolving the precipitate in methanol, the solution was centrifuged again to obtain a methanol solution of the supernatant. By distilling off methanol using a rotary evaporator, 65 mg of 12α-hydroxypregna-4-en-3-one-20-carbaldehyde was obtained. Take a portion of the obtained 12α-hydroxypregn-4-en-3-one-20-carbaldehyde, add methanol to it to make a 4% solution, and apply 25μ of this solution to a Microbondapak C-18 column. The mixture was injected into a high performance liquid chromatography system (model HLC-GPC-244, manufactured by Waters, USA). As the mobile phase, a 30/70 volume ratio mixture of water/methanol adjusted to pH 4.0 was flowed at a flow rate of 1 ml/min.
Detection was performed using the refractive index method. The area ratio of each peak in the obtained liquid chromatograph was measured using an integrator (Shimadzu Chromatopack C-R1A, manufactured by Shimadzu Corporation).
The purity of the above 12α-hydroxypregna-4-en-3-one-20-carbaldehyde was determined to be 98% from this area ratio. 12α-hydroxypregna-4 obtained above
-En-3-one-20-carbaldehyde was confirmed by the following method. Melting point: 179-181℃ Mass spectrum m/Z: 344 [M] ⁺ 326 [M-H ₂ O] ⁺ 316 [M-CO] ⁺ Also, from m/Z = 124, the presence of 3-keto-4-ene is confirmed. NMR spectrum (90MHz) δ ^CDCl3 _HMS : 0.72 (3H, s) 18-CH ₃ 1.10 (3H, d) 21-CH ₃ 1.15 (3H, s) 19-CH ₃ 3.95 (1H, s) 12β-H 5.70 ( 1H, s) 4-H 9.40 (1H, d) 22-CHO.

Claims (1)

[Claims] 1 12α-Hydroxypregna-4-ene-3-
on-20-carbaldehyde. 2 12α-Hydroxypregn-4-ene-3- using deoxycholic acid and/or its salt as a substrate
12α-Hydroxypregn-4-ene-3, characterized in that bacteria belonging to the genus Pseudomonas that produce 1-20-carbaldehyde are cultured in a medium containing deoxycholic acid and/or its salts.
A method for producing -one-20-carbaldehyde.