RU2205224C2 - Method for preparing androst-4-en-3,17-dione or their derivatives from sterol of vegetable and animal origin - Google Patents

Abstract

FIELD: organic chemistry, steroids, biotechnology, microbiology. SUBSTANCE: invention relates for preparing steroids and proposes method for preparing androst-4-en-3,17-dione of the general formula:

where R₁ is hydrogen atom or C₂H₅; R₂ is hydrogen atom or CO-(CH₂)₂-COOX (where X means group NH₄, Na or K) or their derivatives from sterol of vegetable and animal origin using the strain Mycobacterium smegmatis VKPMAc-1552 resistant to streptomycin. Method ensures to transform indicated sterols in the loading 10-30 g/l to form androst-4-en-3,17-dione with yield up to 66.2% of theoretical value without taking into account the recovered parent substrate. Isolation of product of sterol transformation is carried out by sorption method. Modified copolymers of ethylstyrene and divinylbenzene are used as sorbents. Indicated sorbents can be used in cycles sorption-desorption many times. Sorbent is added to medium for transformation in the process of fermentation or after its completion. EFFECT: increased yield, enhanced quality of product, decreased labor intensity of process. 3 cl, 1 tbl

Description

 The invention relates to the field of biotechnology, and in particular to a method for producing androst-4-en-3,17-dione from sterols of plant and animal origin and their derivatives, and can be used in the pharmaceutical, microbiological and chemical industries.

 Androst-4-en-3,17-dione (hereinafter AD) is a valuable steroid compound that can be used as a key intermediate in the synthesis of steroid drugs from plant and animal sterols (sitosterol or cholesterol), which are the cheapest and most affordable source of steroid raw materials.

 Steroid drugs are widely used in medicine, animal husbandry and veterinary medicine as hormonal anti-inflammatory, anti-allergic, diuretic, contraceptive, anabolic and other drugs [1].

 Microbiological transformations of cholesterol and sitosterol to 3,17-diketosteroids (in particular, in blood pressure) are widely studied and documented. The ability of various bacterial strains of the genera Arthrobacter, Brevibacterium, Corynebacterium, Pseudonocardia and Mycobacterium to transform sterols with the formation of blood pressure is known. However, most of the known methods for producing blood pressure from sterols are based on the use of bacteria of the genus Mycobacterium.

 A known method of producing blood pressure by microbiological transformation of sterols with strain Mycobacterium fortuitum [2]. When implementing this method, the transformation of sterol is carried out at a load of 30 g / l for 14 days. According to this method, in addition to the target blood pressure, the product of its deeper oxidation, androsta-1,4-diene-3,17-dione (hereinafter abbreviated as ADD) accumulates in the culture fluid during the transformation. The amount of ADD is very significant: the ratio of AD / ADD is 8: 1. Separation of this mixture is a difficult task. In addition, the disadvantage of this method of obtaining blood pressure is the long duration of the process (14 days), which increases the risk of the appearance of extraneous microflora.

 A known method of producing blood pressure using a strain of Mycobacterium vaccae [3]. This strain is resistant to streptomycin and rifampicin; which makes it possible to use an antibiotic if it is necessary to suppress the growth of extraneous microorganisms that can otherwise modify the target transformation product. However, a disadvantage of the method based on the use of this strain is the transformation at low loads of the initial steroid substrate (1 g / l).

 The closest to the proposed technical essence and the achieved effect is a method of producing blood pressure from cholesterol at a load of 0.3 g / l using strain Mycobacterium smegmatis SG-99 [4]. The low specificity of this method with respect to blood pressure and the low load of the initial substrate are its significant drawback: blood pressure is formed in a mixture with ADD in a ratio of 1: 4. ADD is disposed of in the second stage of fermentation, which is carried out under anaerobic conditions for 2 days. During this period, ADD is completely restored to blood pressure. Thus, firstly, there is a complication of the technology for producing blood pressure, and secondly, under anaerobic conditions, in addition to the recovery of blood pressure, undesired recovery of the 17-keto group of fermentation products with the formation of side compounds can also occur.

 The specified method involves the selection of the target product of the transformation of blood pressure by extraction from the culture fluid with large volumes of water-immiscible organic solvents. Moreover, the extract, in addition to the target blood pressure and by-products, contains an untransformed initial sterol. The presence of impurities of the initial substrate in the technical product, as well as the presence of side compounds make it difficult to isolate blood pressure in pure form. An additional cleaning operation is applied. After evaporation of the solvent, the target product is separated from other steroids by crystallization. HELL output not specified.

 The problem to which the present invention is directed, is to intensify the process of obtaining blood pressure by microbiological transformation of sterols.

The technical result that can be obtained by carrying out the invention is as follows:
- increase the load of the initial substrate and the degree of its conversion;
- increase the yield and quality of the target product;
- reducing the number of technological operations and the complexity of the process;
- reduction in the amount of organic solvent used to isolate the target product;
- improving the environmental safety of the process.

где R₁ - H или С₂Н₅; R₂ - H или СО(СН₂)₂СООХ (X - Na, К или NH₄-группа).The essence of the invention lies in the fact that according to the method for producing blood pressure by microbiological transformation from sterols of plant and animal origin or their derivatives, the strain Mycobacterium smegmatis VKPM-Ac-1552 is used as a transformer microorganism, and compounds of the general formula are used as sterols and their derivatives:

where R ₁ is H or C ₂ H ₅ ; R ₂ is H or CO (CH ₂ ) ₂ COOX (X is Na, K or NH ₄ group).

 Extraction of the transformation product from the reaction medium is carried out using a nonionic sorbent.

 Microbiological transformation is carried out using the new mutant strain Mycobacterium smegmatis VKPM Ac-1552, which carries a chromosomal marker of resistance to streptomycin and obtained as a result of laboratory selection. The specified strain is able to selectively split the side chain of sterols at a load of 10-30 g / l with the formation of blood pressure without ADD impurities and transform in the presence of an antibiotic without significantly reducing the yield of blood pressure and lengthening the fermentation time. The following examples show that the steroid-transforming activity of the culture of Mycobacterium smegmatis VKPM Ac-1552 does not decrease when switching from fermentation in flasks to transformations in fermenters.

 The advantage of using the above modified sterols is a significant increase in the specific activity of biomass (Table).

 In addition, the modification of the sterol molecule leads to a significant change in the hydrophilic properties of the initial substrate, which allows its use in transformation without prior micronization. The substrate is introduced in the form of a colloidal solution without the use of detergents, solubilizing additives and any other complexing compounds that contribute to the change in the solubility of hydrophobic sterol or to increase the stability of its suspension. This eliminates the complex energy and labor-intensive operation of grinding the substrate. The transformation is carried out in aqueous solutions of a carbon source, for example carbohydrate (preferably glucose, fructose or galactose), or other sugars, as well as alcohols (preferably glycerol) in the absence of other components of the medium.

Also a distinctive feature of this method is the extraction of the target product from the reaction medium by sorption using a nonionic polar or slightly polar macroporous sorbent in an effective amount. As sorbents, modified copolymers of ethyl styrene and divinylbenzene can be used, characterized by a large specific surface area (500-1000 m ² / g), pore volume of more than 1 cm ¹ / g, high mechanical strength and stability, for example, polyurethane, wofatite, levatite. The above sorbents can be used repeatedly in sorption-desorption cycles.

 The introduction of the sorbent into the fermentation medium can be carried out in a different period of transformation: simultaneously with the initial substrate, during the process, or after its completion. Due to the selective adsorption ability of the sorbent, the transformation in the presence of the sorbent allows you to remove the target product from the reaction medium as it forms and thereby prevent the product from inhibiting cell mass growth. In addition, due to the selective adsorption capacity of the sorbent, the method eliminates the time-consuming operation of separating the target product from the residual starting sterol and thereby simplifies the isolation step.

 Information on the use of chemically modified sterol derivatives of the general formula 1 as initial substrates in the processes of obtaining 3.17-ketoandrostanes (in particular, AD) by microbiological transformation as a strain of the transformer Mycobacterium smegmatis VKPM Ac-1552, as well as information on the possibility of transformation sterols to blood pressure in the presence of a sorbent are absent in the literature.

 The HPLC method was used to control the accumulation of blood pressure in the culture fluid, as well as to assess the content of the main substance in the transformation product. Sorption and desorption processes were monitored by TLC analysis.

 The method is illustrated by examples.

Example 1
Mycobacterium smegmatis VKPM Ac-1552 is grown for 7-10 days on a solid medium of the following composition (g / l): glucose - 10, soy flour - 3.0, citric acid - 2.2, urea - 0.5, ammonium chloride - 1.0, monosubstituted potassium phosphate -0.5, magnesium sulfate - 0.5, calcium carbonate - 1.5, ferrous sulfate - 0.05, agar-agar - 2.5, pH 7.0 - 7.5 . The biomass is transferred to a liquid medium of the same composition (without agar), in which the culture is grown for 48-70 hours under aerobic conditions on a shaker at a temperature of 30 ^o C (culture medium is poured into 100 ml conical flasks with a volume of 750 ml). The resulting seed in an amount of 10 vol. % is transferred to the medium for transformation of the following composition (g / l): glucose - 10, soy flour - 10, citric acid - 2.2, urea - 0.5, ammonium phosphate disubstituted - 1.5, magnesium sulfate - 0.5 , calcium carbonate - 1.5, iron sulfate - 0.05, cholesterol with a particle size of 3-10 microns - 10, sorbital - 3.2, pH 7.2 -7.4. The transformation medium is poured into 50 ml into 500 ml Erlenmeyer flasks. The transformation is carried out under aerobic conditions on a rocking chair at a temperature of 30 ^o C for 90-100 hours

The culture fluid containing blood pressure and unreacted sterol is acidified to pH 2-3 and mixed with porolas in the amount of 1/7 part of the weight of the original substrate. The suspension is maintained at a temperature of 20-25 ^o C until the completion of sorption. The sorbent is separated, the adsorbed BP is eluted with acetone. The solution is clarified with activated carbon. The solvent is evaporated, the residue is triturated with water, the precipitate is filtered off, washed with water. Get a white powder with a BP content of 97%, so pl. 169-171 ^o C, yield 66.2% (of theory).

 The culture fluid is centrifuged after separation of the foam. The biomass is treated with acetone. The resulting solution was clarified with activated carbon, the solvent was evaporated. The residue is triturated with water, the precipitate is filtered off. A white powder is obtained containing, according to GLC, 94% cholesterol suitable for reuse. The yield of blood pressure in terms of the sterol that has reacted is 71%.

Example 2
Inoculum obtained analogously to example 1 (1st inoculum), contribute to 500 ml of fresh nutrient medium of the same composition as for the 1st inoculum, incubated on a shaker in the conditions of example 1 for 24 hours. Receive the 2nd inoculum, which is transferred to a 10 L fermenter filled with 5 L of the transformation medium of the composition shown in Example 1, but with the addition of an antifoam. The transformation is carried out for 96-100 hours at 30 ^o C, stirring 700 rpm and air supply with an intensity of up to 0.6 l / l of medium / min. The product is isolated analogously to example 1. Get a technical product with a yield of blood pressure of 60.7% (of theory.) With a basic substance content of 95%, so pl. 167-170 ^o C.

Example 3
The transformation is carried out analogously to example 1, but with the replacement of cholesterol with β-sitosterol. Isolation is carried out analogously to example 1. Get the product with a yield of 54.4% (of theory.) With a basic substance content of 96%, so pl. 169-171 ^o C. In addition, unreacted sitosterol is isolated. The yield of blood pressure in terms of the sterol that has reacted is 85%.

Example 4
The transformation of β-sitosterol at a load of 10 g / l is carried out in a fermenter as in Example 2. The product is isolated as in Example 1. A technical product is obtained with a yield of 51.4% (of theory) with a basic substance content of 94%, so pl. 167-170 ^o C. The yield of blood pressure in terms of the reacted sitosterol is 82%.

Example 5
Seeds obtained according to example 1, contribute in an amount of 10 vol. % into the transformation medium of the following composition (g / l): glycerin - 20, starch - 5.0, yeast extract - 0.5, urea - 0.4, potassium phosphate monosubstituted - 0.7, ammonium chloride - 0.7, magnesium sulfate - 0.5, ferric chloride - 0.05, cholesterol - 20 (in the form of particles 3-10 microns), sorbital - 6.5, pH 7.0-7.2. The medium is poured into 50 ml into 750 ml conical flasks. Fermentation is carried out under the conditions of Example 1 for 230 hours. During this period, according to the HPLC analysis, 10 g / L of BP accumulates in the culture fluid. According to HPLC, there is no ADD impurity in the transformation products, and the present impurity in an amount of 4% consists of Δ ^1.4 - and Δ ⁴ -22-alcohols.
The product is isolated analogously to example 1. Wofatit EP 60 is used as the sorbent. A white powder is obtained with a 96% HELL content, so pl. 169-171 ^o C, yield 58.5% (of theory). Given the returned sterol, the yield of blood pressure is 66.2%.

Example 6
The transformation of cholesterol at a load of 30 g / l is carried out under the conditions of example 1 for 330 hours. The product is isolated with a yield of 52.6% (of theory) and a basic substance content of 97%.

Example 7
The transformation of cholesterol at a load of 30 g / l is carried out under the conditions of example 1 for 6 days, after which streptomycin is added to the culture fluid and fermentation is continued for 8 days. The selection of the product is carried out analogously to example 1. Get the product with a yield of 49.5% (of theory.) With a basic substance content of 96%.

Example 8
The seed and transformation medium are prepared analogously to example 1 (10 g / l of cholesterol), fermentation is carried out for 24 hours also under the conditions of example 1, then separately sterilized porolas is added to the culture fluid in an amount of 20% of the volume of medium. Fermentation is continued under the same conditions for another 3 days. The sorbent is separated from the culture fluid and treated analogously to example 1. Receive blood pressure with a yield of 58.4% (from theory.).

Example 9
The non-converted cholesterol returned after the transformations described in examples 1, 2, 5-8, is transformed at a load of 10 g / l under the conditions of example 1 for 96 hours. Receive blood pressure in 53.1% yield, which is isolated according to example 1 using Lewatit OS 1062.

Example 10
A gel of the ammonium salt of cholesterol hemisuccinate (cholesterol concentration 20 g / l) is mixed with an aqueous solution of glycerol in a ratio of 1: 1 (the content of the carbon source is 1%), the pH of the resulting solution is 7.5-7.8.

The solution is dispensed in 50 ml conical flasks with a volume of 500 ml and sterilized for 20 min at 110 ^° C. The seed prepared according to Example 1 is introduced into sterile solutions in an amount of 20 vol% and transformation is carried out on a rocking chair under the conditions of Example 1. The formation of blood pressure begins after a day of fermentation, during which the pH is maintained above 7.0. The selection of blood pressure is carried out analogously to example 1.

Example 11
The seed and transformation medium are prepared analogously to example 1 (10 g / l of sitosterol). Into a flask with a capacity of 0.75 L make 20 ml of seed 50 ml of transformation medium (the number of components based on the volume of the medium is 100 ml), 10 ml of a 10% suspension of sitosterol and 20 ml of porolas. The transformation is carried out for 96 hours. The allocation of blood pressure is carried out analogously to example 1 using Wofatit EP 62 as a sorbent. Blood pressure is obtained with a basic substance content of 90% and a yield of 66.7% (of theory). Taking into account the content of the main substance in the initial substrate (90%), the blood pressure yield is 74%.

Sources of information
1. Mashkovsky M.D. "Medicines", M., "Medicine", 1993, v. 1, S. 690-710.

 2. Pat. USA 4 345 033, Cl. 435-55.

 3. Doc. Bolg. AN, vol. 46, p. 123-126, 1993

 4. Pat. GDR 137 661, C 07 J 1/00.

Claims (3)

1. The method of producing androst-4-ene-3,17-dione by microbiological transformation of sterols of plant and animal origin or their derivatives, characterized in that the strain Mycobacterium smegmatis VKPM As-1552 is used as a transformer microorganism, as sterols and their derivatives use compounds of the general formula

where R ₁ is H or C ₂ H ₅ ;
R ₂ is H or CO (CH ₂ ) ₂ COOX (X is a group of NH ₄ , Na or K).  2. The method according to p. 1, characterized in that the extraction of the transformation product from the reaction medium is carried out using a nonionic sorbent.  3. The method according to p. 1, characterized in that as the sorbent use modified copolymers of ethyl styrene and divinylbenzene.

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