RU2512076C1 - Method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C¹⁷ with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.

EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.

11 cl, 1 tbl, 1 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, specifically relates to the production of the steroid compound androsta-4,9 (11) -diene-3,17-dione from phytosterol and can be used in the biotechnological and chemical-pharmaceutical industries for the production of steroid preparations.

BACKGROUND

Androsta-4,9 (11) -diene-3,17-dione (Δ ^{9 (11)} -AD, 9 (11) -dehydroandrostenedione, CAS 1035-69-4) is a key compound in the synthesis of corticosteroid preparations from sterols, for example such as hydrocortisone, prednisone, as well as their 9-halogen derivatives (e.g., Cortinef, 9-fluoroprednisolone, etc.), and preparations also having substituents at C ¹⁶ , in particular alkyl groups and hydroxyl groups (e.g. , triamcinolone, dexamethasone, betamethasone, mometasone, etc.) [V. vanRheenen, KP Shepard, J. Org. Chem ., 1979, 44 (9), 1582-1584]. In addition, Δ ^{9 (11)} -AD can be used as a starting substrate in the synthesis of the androgenic preparation of fluorooxymesterone (11β-hydroxy-9α-fluoro-17α-methyltestosterone, CAS 76-43-7) [CN 102040639, 2011] used for the treatment of, for example, seborrhea [MXNL05000077, 2007], breast cancer [WO 0040230, 2000].

Methods for producing Δ ^{9 (11)} -AD are known.

Thus, a method for the complete synthesis of Δ ^{9 (11)} -AD is described based on (1S, 7aS) -1- t -butoxy-7a-methyl-5,6,7,7a-tetrahydroindan-5-one [U. Eder, G. Sauer, G. Haffer, J. Ruppert, R. Wiechert, A. Furst, and W. Meier. Helvetica Chimica Acta , 1976, 59 (4), 999-1005]. The disadvantage of this method is multi-stage, low product yield (27.8%), the use of chromatographic purification of intermediates and product, the use of expensive reagents, as well as reagents, the use of which in industrial production is a serious danger.

In addition, methods are known for producing Δ ^{9 (11)} -AD by dehydration of steroid alcohols, specifically derivatives of androst-4-en-3,17-dione (AD), containing a hydroxyl group at the C ⁹ or C ¹¹ atom (in 11α- or 11β- configuration). It should be noted that in multistage syntheses of steroid preparations by modification of the 17-keto group of hydroxyl-containing derivatives of AD, the hydroxyl group is dehydrated first to form Δ ^{9 (11)} -AD, in which the 9.11 double bond is more stable under the conditions of subsequent reactions than the hydroxyl group at C ¹¹ . So, for example, in the synthesis of fluorooxymesterone at the first stage, Δ ^{9 (11)} -AD is obtained by dehydration of 11α-hydroxyandrost-4-en-3,17-dione [CN 102040639, 2011].

Known methods for producing Δ ^{9 (11)} -AD by dehydration of 11β-hydroxyandrost-4-en-3,17-dione: under conditions of ketalization [Q. Zhao, Zh. Li. Synthetic Communications , 1993, 23 (10), 1473-1478], by the action of N, N′-thionyl diimidazole in tetrahydrofuran [S. Solyom, K. Szilágyi, L. Toldy. Journal für Praktische Chemie , 1988, 330 (2), 309-312] or SO ₂ in pyridine [GB1081307, 1967, Example 2]. However, the yield of Δ ^{9 (11)} -AD does not exceed 45-50% in the first two and 77.3% in the latter embodiment.

In the synthesis of a corticosteroid chain based on the 17-keto group of 9α-hydroxyandrost-4-en-3,17-dione (9-OH-AD), the dehydration of the 9α-hydroxy group is also usually carried out in the first stage [J.G. Reid, T. Debiak-Krook,Tetrahedron lett., 1990, 31 (26), 3669-3672], since the resulting Δ^{9 (11)}-AD is more stable in further synthesis. This allows us to avoid possible rearrangements involving the tertiary hydroxyl group at C⁹, especially rearrangements in ring A [L. Chinn, R. Dodson,J. Org. Chem., 1959, 24 (6), 879].

9-OH-AD is an intermediate in the synthesis of Δ ^{9 (11)} -AD from natural sterols (cholesterol or phytosterol). The word "phytosterol" means sterols of plant origin (β-sitosterol, campesterol, stigmasterol, etc.) or mixtures thereof.

9-OH-AD is obtained from phytosterol by microbiological transformation using the methods and cultures of microorganisms described in the literature.

9-OH-AD can be obtained by a two-stage biotechnological process, including the step of transforming sterols into AD and the step of 9α-hydroxylation of the isolated AD using known methods ([C. Perez, A. Falero, LH Duc, et al. , J. Ind. Microbiol. Biotechnol., 2006, 33 (8) , 719-723; N.V. Rodina, M.A. Molchanova,

NOT. Voishvillo, V.A. Andryushina, T.S. Stytsenko , Prikl . biochem . microbiol. , http://elibrary.ru/issues.asp?id=7955&jyear=2008&selid=439695 , http://elibrary.ru/issues.asp?id=7955&volume=44&selid=439695 (1), 56-62] and [ GB862701, 1961; US3065146, 1962; US4397947, 1983; B. Angelova, P. Fernandes, A. Cruz, HM Pinheiro, S. Mutafova and JMS Cabra , Enzym. Microb. Tech., 2005, 37 (7), 718-722; N.V. Homeland, V.A. Andryushina, T.S. Stytsenko, T.P. Turova, R.V. Baslerov, A.N. Panteleeva, N.E. Voishvillo , App . biochem . microbiol. , http://elibrary.ru/issues.asp?id=7955&jyear=2008&selid=439695 , http://elibrary.ru/issues.asp?id=7955&volume=44&selid=439695 (4), 439-445; RU2351645, 2009] respectively).

where R = C ₂ H ₅ - β-sitosterol; R = CH ₃ - campesterol; R = C ₂ H ₅ and ∆ ^{22 (23)} - stigmasterol)

Also known is a one-stage two-stage microbiological process consisting in the sequential use of a sterol-transforming strain and a 9α-hydroxylating culture without isolation of the intermediate-formed side chain degradation product, androstenedione (AD), from the culture fluid [B.A. Andryushina, N.V. Homeland, T.S. Stytsenko, Lew Dook Hee,

A.V. Druzhinin, V.V. Yaderets, N.E. Voishvillo, Prikl . biochem . microbiol . , 2011, 47 (3), 297-301].

Known single-stage methods of microbiological transformation of phytosterol in 9-OH-HELL [M .G. Wovcha, F. J. Antosz, J. C. Knight, L. A. Kominek, and

T. R. Pyke,Biochim. Biophs. Acta1978, 539, 308; US4035236, 1977; US4175006, 1979; US4397946, 1983; DD298278, 1992; DD298279, 1992; RU2077590, 1997; M.V. Donova, S.A. Gulevskaya, D.V., Dovbnya, I.F. Puntus,Appl. Microbiol. Biotechnol.2005, 67 (5), 671-678; M.V. Donova, D.V., Dovbnya, G.V. Sukhodolskaya, S.M. Khomutov V.M. Nikolayeva, I. Kwon, K. Han,J. Chem. Technol. Biotechnol.,2005, 80, 55-60, etc.].

It is known that the effectiveness of the microbiological method for producing 9-OH-AD by sterol transformation also depends on the efficiency of the method of extracting 9-OH-AD from the culture fluid. Typically, the degree of recovery of the transformation product is from 70 to 90%.

For example, a method is known [US 4035236, 1977], according to which transformation of phytosterol into 9-OH-AD is carried out by bacterial cellsMycobacterium fortuitum NRRL B-8119 with a loading of the initial substrate of 10 g / l. The cells of the culture after transformation are separated, extracted with a solvent (miscible or immiscible with water). The cell-free culture fluid is extracted with a water-immiscible organic solvent, for example methylene chloride (Example 2). The extract was filtered through kieselguhr and evaporated to dryness in vacuo. The residue is dissolved in a mixture of methanol and chloroform, concentrated in a stream of nitrogen in a steam bath until crystallization begins. After cooling the residue to room temperature, the precipitate of non-transformed sterol is filtered off (for example, sitosterol, example 2). From the supernatant, after evaporation of the solvent, a 9-OH-AD pond is obtained, in which traces of the impurity of the 17-hydroxy derivative are present. An additional amount of 9-OH-HELL can be obtained from the mother liquor by further processing.

Also known is a method of producing 9-OH-AD from phytosterol [GB 2197869, 1988] with a substrate load of 20 g / l, according to which bacterial cells are separated after fermentation, the product is washed off with methanol from the cells. An additional amount of 9-OH-AD is recovered from the supernatant by extraction after separation of the products of incomplete oxidation of the sterol side chain. Purification of 9-OH-HELL is carried out by silica gel column chromatography. From 15.1 g of 9-OH-AD crudes, only 5.7 g of purified product is obtained.

A known method for the selective isolation, purification and separation of monohydroxylated 3,17-diketosteroid compounds from a solution obtained by the microbiological method containing a mixture of steroid compounds by extraction, purification and selective crystallization [WO 2008032131, 2008]. Example 4 describes the isolation of 9-OH-AD obtained by biotransformation of β-sitosterol cultureMycobacterium fortuitum NRRL B-8119 with a load of the starting substrate of 30 g / l under the conditions of the patent [US 40635236, 1977]. Moreover, according to HPLC analysis, the content of 9-OH-AD in the culture fluid at the end of the transformation is 12 g / l: 1.5 g / l of this amount is in the liquid phase, and the rest is on the surface of the filtered biomass. After separate extraction of the product from biomass and extraction from the supernatant, subsequent purification and crystallization, 9-OH-AD was obtained with a basic substance content of 95.6% and a yield of 92%, based on the content in the culture liquid (degree of extraction, thus, 88 %).

A known method for the preparation and isolation of 9-OH-AD [DD 298279, 1992], which consists in conducting microbiological transformation of sterols with Mycobacterium vaccae ZIMET 11053 culture with a load of 10 g / l in the presence of an adsorption polymer (divinylbenzene-ethyl styrene copolymer).

In addition, a similar method has been described for the isolation of 9-OH-AD from the culture fluid obtained by transformation of sitosterol by Mycobacterium sp. 207 with a substrate load of 5 g / l in the presence of a synthetic resin, which is a modified copolymer of ethyl styrene and divinylbenzene (Porolas)

[E.A. Borman, Yu.A. Redikyultsev, K.A. Koschenko, A.M. Turuta, A.V. Kamernitsky, Prikl. Biochem. Microbiol., 1992, 28 (4), 551-56]. The method involves separating the resin with the adsorbed product, washing, eluting with 9-OH-AD by ethyl acetate, evaporation and crystallization. The output at the isolation stage is about 70%.

Since fermentation takes place in the presence of an adsorbent, the main drawback of these methods for producing 9-OH-AD from sterols is the need to use special equipment to prevent the destruction of the resin with stirring and to ensure effective mass transfer between the polymer resin and the steroid product. This limits the scaling and practical use of such processes.

From the foregoing, it is obvious that the methods for extracting 9-OH-AD from the fermentation medium are complex, multi-stage and cannot do without significant losses of the basic substance.

Methods for producing Δ ^{9 (11)} -AD by dehydration of 9-OH-AD are known. A feature of the dehydration of 9-OH-AD is the possibility of the formation of the undesirable isomer of androst-4.8 (9) -diene-3.17-dione (Δ ^{8 (9)} -AD).

Moreover, the isomeric Δ ^{9 (11)} - and Δ ^{8 (9)} -olefins have the same value of R _f on TLC and cannot be separated by crystallization. The determination of the impurity of the Δ ^{8 (9)} isomer can be carried out, for example, spectroscopically ( ¹ H NMR, ¹³ C NMR) [EP 0253415, 1988; EP 0294911, 1988] or chromatographic (HPLC [EP 0253415, 1988; EP 0294911, 1988], GLC [US 4102907, 1978]).

Mineral and organic acids, anhydrides and acid chlorides of mineral and organic acids, as well as other reagents, are used as a dehydrating agent.

The dehydration reaction of 9-OH-AD with the formation of Δ ^{9 (11)} -AD can be carried out either in the medium of a dehydrating agent without using a solvent, or in an environment of an organic solvent.

The combination of the essential features of the claimed invention is to conduct the dehydration reaction of 9-OH-AD with the formation of Δ ^{9 (11)} -AD in an inert aprotic organic solvent, in particular in an aromatic hydrocarbon (e.g. benzene, toluene) or chlorine-containing hydrocarbon (e.g. dichloromethane, dichloroethane, chloroform), using a mineral acid as a dehydrating agent, including water containing, in particular, phosphoric or perchloric acid.

Known methods for the dehydration of 9-OH-HELL with the formation of Δ ^{9 (11)} -HELL in an organic solvent. It is known that chlorine-containing hydrocarbons, aromatic hydrocarbons, cyclic ethers, and organic bases can be used as the medium.

A known method of dehydration of 9-OH-HELL in a cyclic ether medium, in particular tetrahydrofuran (THF).

Thus, the dehydration of 9-OH-AD by the action of N, N is known.^one-thionyl-diimidazole formed from thionyl chloride and imidazole in THF medium [S. Solyom, K. Szilágyi, L. Toldy,Journal für Praktische Chemie1988, 330 (2), 309-312]. The product is obtained in 93% yield. However, the reaction is non-selective: a mixture of Δ isomers is formed^{9 (11)}-AD and Δ^{8 (9)}-AD in the ratio of 69:31. Moreover, their ratio, as noted by the authors, is not affected by changes in either the temperature regime or the nature of the solvent.

Known methods for the dehydration of 9-OH-HELL with the formation of Δ ^{9 (11)} -HELL in an organic base.

In the article [CG Bergstrom, RT Nicholson, RM Dodson, J. Org. Chem ., 1963, 28 (10), 2633-2640] describes a method for producing Δ ^{9 (11)} -AD from 9-OH-AD by the action of a reagent formed from HF and pyridine containing 30% pyridine. However, under these conditions, the formation of an equilibrium mixture containing 9α-fluoro-androstenedione (9α-fluoro-AD) occurs.

Another known method for producing Δ^{9 (11)}-AD - treatment of 9-hydroxysteroids, including 9-OH-AD, with an equimolar amount of thionyl chloride in an organic base medium (eg, pyridine and triethylamine) [US 3065146, 1962]. However, only the possibility of obtaining Δ^{9 (11)}-AD in general form (example 7) without indicating the yield and quality of the product. The use of these conditions, as noted in [V. VanRheenen, K. P. Shephard,J. Org. Chem., 1979, 44 (9), 1582-1584], does not allow cleavage of the hydroxyl group at 9-OH-AD is regioselective: a mixture of isomeric products Δ^{9 (11)}-AD and ∆^{8 (9)}-HELL. So, in the patent [US 4102907, 1978] describes the receipt of Δ^{9 (11)}-AD from 9-OH-AD by the action of thionyl chloride in pyridine medium (0-5 ° C, 2 min). It is shown that under these conditions the formation of a mixture Δ^{9 (11)}-HELL and ∆^{8 (9)}-AD with a content of 43.4-55.5% and 43.7-56%, respectively, according to GLC analysis [Preparation 1 - 3]. An unsatisfactory result gives the use of a mixture of bromine and SO for dehydration₂ in the environment of pyridine [US 4102907, 1978. Preparation 4 - 5]. The product is a mixture of ∆^{9 (11)}-HELL and ∆^{8 (9)}-AD with a content of 50-64.4% and 43.7-50%, respectively, according to GLC analysis.

A known method of producing Δ ^{9 (11)} -AD [US 4102907 , 1978, claim 25 of the formula, Example 1] by two-stage synthesis, including 1) sulfinylation of 9α-hydroxy-androstenedione by phenylsulfinyl chloride to form 9-phenyl sulfinate 9α-hydroxyandrostenedione and 2) desulfinylation 9-phenylsulfinate of 9α-hydroxyandrostenedione by heating to 40 ° C in the presence of silica gel and p-methyl-phenylsulfonic acid (p-toluenesulfonic acid, p-TSC).

The production of 9-OH-AD sulfate ester is carried out in pyridine medium (10-20 ° C), subsequent desulfinylation in chloroform medium at boiling point in the presence of silica gel and p-TSC. The maximum achievable yield of ∆ ^{9 (11)} -AD with a melting point of 191 to 201 ° C does not exceed 91.78%, and the content of the side ∆ ^{8 (9)} -AD is not indicated.

A known method of producing Δ ^{9 (11)} -AD [US 3005834, 1961, claim 6 of the formula], which includes contacting 9-OH-AD, 11α-hydroxy-AD and 11β-hydroxy-AD in anhydrous conditions in the presence of pyridine with 1 ) N-bromoacetamide and 2) anhydrous sulfur dioxide, with sulfur dioxide being added until the hydroxyl group is completely oxidized to form Δ ^{9 (11)} -AD. However, despite the mention of 9-OH-AD as the starting compound in the dehydration reaction with the formation of Δ ^{9 (11)} -AD, there is no example of its use. Therefore, it is impossible to evaluate the effectiveness of this method as applied to 9-OH-AD.

Known methods for the dehydration of 9-OH-HELL in the environment of aromatic hydrocarbons.

Thus, a method is described for using p-toluenesulfonic acid in dry benzene to dehydrate 9-OH-AD [CG Bergstrom and RMDodson, Chem.and Ind ., 1961, 1530]. However, instead of the expected Δ ^{9 (11)} -AD, the authors obtained a mixture containing 9,10-secoandrost-4-ene-3,9,17-trion (A) and 9α-hydroxy-4-methylestra-4-en-1 , 17-dione (B).

A known method of producing 9 (11) -dehydroandrostanes by dehydration of the corresponding 9α-hydroxyandrostane, characterized in that the dehydration is carried out in the presence of aromatic sulfonic acid and silica gel [EP 0253415, 1988]. The reaction is carried out in an aromatic hydrocarbon medium at the boiling point of a steroid solution, while p-TSC or naphthalenesulfonic acid can be used as aromatic sulfonic acid. The method is characterized in that a sulfonic acid adsorbate (for example, p-TSC monohydrate adsorbate) on chromatographic quality silica gel is preliminarily obtained. Significant disadvantages of this method is the use of a significant amount of silica gel (more than 10 times the weight of the starting substrate) and low product yield: the achieved maximum yield of Δ ^{9 (11)} -AD does not exceed 65.8%.

A method for producing 9 (11) -dehydrosteroids of the androstane series and driven by dehydration of the corresponding 9α-hydroxysteroid is described in the patent [EP0294911, 1988]. The method is characterized in that the dehydration is carried out in an inert organic solvent in the presence of a Lewis acid selected from the group consisting of iron chloride, boron trifluoride or their complexes, antimony pentachloride, titanium tetrachloride and mixtures of these compounds with silica gel. So, example 1 describes the receipt of ∆^{9 (11)}-AD from 9-OH-AD by the specified method in anhydrous benzene at a boiling point by the action of a reagent formed from iron chloride and silica gel. The product does not contain impurities ∆^{8 (9)}-HELL. The disadvantages of this method are the use of chromatographic purification method and low yield Δ^{9 (11)}-AD (from 0.4 g of 9-OH-AD, the authors obtained 0.16 g of ∆^{9 (11)}-AD, which corresponds to a molar yield of 42.6%). Example 3 describes the dehydration reaction of 9-OH-HELL in a methylene chloride medium at room temperature by the action of antimony pentachloride. The main disadvantages of this option are the use of a toxic reagent, an extremely low yield ∆^{9 (11)}-AD (from 604 mg of 9-OH-AD, 139 mg Δ^{9 (11)}-AD, which corresponds to a molar yield of 24.5%). The best result was obtained using boron trifluoride complexes in benzene (boron trifluoride etherate in example 4 at boiling point, a complex of boron trifluoride with methanol at room temperature in example 5, and a complex of boron trifluoride with acetic acid at boiling point in example 6). From 3.02 g of 9-OH-HELL (Example 4), 2.3 g of Δ^{9 (11)}-AD, which corresponds to a molar yield of 81%. In this case, the melting point obtained Δ^{9 (11)}-AD is 204-205.5 ° C, which confirms the purity of the product. Output ∆^{9 (11)}-AD in example 5 was 92.4%, but the content of the basic substance was only 89.5%. Output ∆^{9 (11)}-AD in example 6 was 97.5%. The authors noted that the presence of ∆^{8 (9)}-isomer was not detected (according to HPLC). A common disadvantage of the options described in examples 4-6, is the use of a hazardous reagent boron trifluoride, which has extremely high corrosiveness and toxicity.

A known method of obtaining Δ^{9 (11)}-AD by dehydration of 9-OH-AD with chlorosulfonic acid in a medium of chlorine-containing hydrocarbon, particularly methylene chloride [US 4127596, 1978, Examples 1-8]. The disadvantage of this method is that chlorosulfonic acid, which must utilize the water generated by the dehydration of the 9-hydroxy group, thereby catalyzing the process, also reacts with water, which may be present in the organic solvent. In addition, to prevent the destruction of 9-OH-AD or Δ^{9 (11)}-AD reaction is carried out at low temperatures (specifically, at a temperature of from minus 20 to 5 ° C). Output ∆^{9 (11)}-AD is 85% (in example 1) and ~ 95% (in examples 2-7). When carrying out the reaction at a temperature of 20-27 ° C, the output ∆^{9 (11)}-AD drops to 84.4% (example 8).

One of the essential features of the present invention is the use of a mineral acid as a dehydrating agent, including water, more specifically, phosphoric or perchloric acid.

A known method of obtaining Δ^{9 (11)}-steroids of a number of androstane, including ∆^{9 (11)}-AD by the reaction of 9α-hydroxysteroid (in particular, 9-OH-AD) with non-aromatic oxygen-containing acid with pK_a ≤1.0 [US4127596, 1978]. Moreover, the acid is selected from the group: chlorosulfonic, sulfuric, phosphoric, methanesulfonic, perchloric and trifluoroacetic. The reaction is carried out in an oxygen-containing acid medium. So, for example, when using aqueous sulfuric acid (a mixture of 3 parts of water and 7 parts of 70% sulfuric acid, which corresponds to ~ 55%), Δ^{9 (11)}-AD with a yield of 97.4%. However, the duration of the reaction is not indicated, while the product contains up to 1% of the side ∆^{8 (9)}-HELL. Examples 12-15 describe the use of methanesulfonic acid (MSC). It is shown that the highest yield ∆^{9 (11)}-AD is obtained by carrying out the reaction without adding water (example 13, yield 95.6%, reaction time 80 min at 25 ° C). Carrying out the reaction in a medium of a mixture of MSC and water with increasing water content increases the duration of the process and decreases the yield ∆^{9 (11)}-AD, and there is a need for a reaction at a higher temperature. So, with a volume ratio of MSC and water 4: 1, the duration of the process at 31-39 ° C is 5 hours, the output ∆^{9 (11)}-AD - 87.2%, mp 198.5-201 ° C (example 12); when the volume ratio of MSC and water is 1: 1, the duration of the process at a temperature of 100 to 60 ° C is 2 hours, the output of the crud ∆^{9 (11)}-HELL with so pl. 170-175 ° C is 75.9% (example 15), and with a volume ratio of MSC and water 2: 3, the duration of the process at 68 ° C is 6 hours, and the output Δ^{9 (11)}-HELL with so pl. 186-195 ° C is only 55.7% (example 14). Thus, the disadvantage of using MSCs is the long process time and a significant decrease in yield and quality ∆^{9 (11)}-AD in the presence of water and with an increase in the temperature of the process above room temperature.

Also known is a method of producing Δ ^{9 (11)} -AD from 9-OH-AD by dehydration with polyphosphoric acid [HUT36138, 1985]. The reaction is carried out in a medium of polyphosphoric acid for 2 hours and 20 minutes at a temperature of 40 ° C, using ~ 3-fold volume of acid per 1 part by weight of 9-OH-HELL. At the end of the reaction, the reaction mass is diluted with water, the precipitate is filtered off. The product was obtained with a yield of 98.46% and so pl. 199-201ºС.

A common drawback of dehydration methods in the environment of a dehydrating agent, especially in the presence of water, is the use of a large excess of a dehydrating agent, a long reaction time, and in most cases the need for column chromatography to purify the product, and, as a result, either a low yield or poor quality.

The closest analogue (prototype) according to one of the essential features of the claimed invention, which is the nature of the dehydrating agent used, namely the use of mineral acids, including those containing water, more specifically the use of phosphoric acid or perchloric acid, is a method for producing Δ^{9 (11)}-steroids of a number of androstane, including ∆^{9 (11)}-AD by the reaction of 9α-hydroxysteroid (in particular, 9-OH-AD) with a non-aromatic oxygen-containing acid with pK_a ≤1.0 [US 4127596, 1978, Examples 10-11 and Example 16, respectively (reprinted as USRE 33364, 1990].

An example of a prototype [US 4127596, 1978] describes the receipt of Δ^{9 (11)}-AD by the reaction of 9-OH-AD with 85% phosphoric acid, and phosphoric acid simultaneously serves as both a dehydrating agent and a reaction medium. 12.2 moles of 85% phosphoric acid were used per 1 mol of 9-OH-AD. The reaction mass is stirred at a temperature of 45-55 ° C for 24 hours. Then it is diluted with water three times, the precipitate is filtered off. Get ∆^{9 (11)}-AD with a molar yield of 95.1%. ∆ content^{9 (11)}-AD in the sample is not indicated. However, the melting point of the obtained product is only 193-195 ° C, which is an indicator of the low content of the basic substance and is probably associated with prolonged heating of the reaction mass. For specialists working in the field of synthesis of steroid compounds, it is obvious that the melting temperature is an important characteristic of the purity of steroid compounds. It is known that the melting point ∆^{9 (11)}-AD, practically free of impurities, is in the range from 203 to 206 ° C (for example, 204-205.5 ° C [EP0294911, 1988]). In addition, another significant drawback of the described method is the long duration of the process. Significant dilution of the reaction mass with phosphoric acid (up to 73 times the initial steroid) allows reducing the reaction time from 24 hours to 7.5 hours at a temperature of 35-48 ° С and obtain ∆^{9 (11)}-AD with a yield of 96.2% [US 4127596, 1978, example 10]. Although the melting point of the product rises to 197-201 ° C, GLC analysis shows the presence of 1% side ∆^{8 (9)}-HELL. The disadvantage of this variant of the method is the use of a large amount of dehydrating agent and a long process time.

Example 16 of the prototype [US 4127596, 1978] describes the receipt of Δ^{9 (11)}-AD by the reaction of 9-OH-AD using dilute perchloric acid, with perchloric acid simultaneously serving as both a dehydrating agent and a reaction medium. The reaction is carried out in an environment of ~ 28% aqueous perchloric acid (the medium is a mixture of 5 ml of water and 2 ml of 70% perchloric acid), and 23.2 moles of perchloric acid are used per 1 mol of 9-OH-AD. The method is extremely inefficient, since in addition to a long process time (66 h) at a temperature of 54 ° С, the molar yield ∆^{9 (11)}-AD is only 69.6%.

Common disadvantages of the methods for producing Δ ^{9 (11)} -AD using mineral acids, including those containing water, used as a solvent medium, in particular 85% phosphoric acid and 28% perchloric acid, are:

• A large amount of mineral acid (up to 73 times the weight of the original steroid);

• Long reaction time (from 7.5 hours to 24 hours for 86% phosphoric acid and 66 hours for 28% perchloric acid);

• Low product yield (69.6% when using perchloric acid).

Thus, the analysis of 9-OH-AD dehydration methods known from available literature shows that when carrying out the reaction in an organic solvent, in particular in an aromatic hydrocarbon or chlorine-containing hydrocarbon, the use of mineral acids, including those containing water, is unknown. The reaction is described by organic acids or their derivatives, anhydrides or acid chlorides of mineral acids, and the presence of an anhydrous medium is a condition for the reaction to provide a positive result. As follows from the prior art, the use of mineral acids, including those containing water, to produce Δ ^{9 (11)} -AD is possible and known in the processes of dehydration of 9-OH-AD without the use of an organic solvent, however, these methods have significant drawbacks and the results obtained cannot be considered satisfactory.

Available literature does not contain information on the dehydration reaction of 9-OH-AD with the formation of Δ ^{9 (11)} -AD using mineral acids, including those containing water, as a dehydrating agent in an aprotic organic solvent.

In addition, the available literature also lacks information on the intensification of the dehydration process by removing excess water during the dehydration reaction, for example, by azeotropic distillation.

In addition, there is no information available in available literature on the possibility of obtaining Δ ^{9 (11)} -AD from phytosterol through the formation of 9-OH-AD without isolating the latter. Known methods, for example, described above [US 4035236, 1977; GB 2197869, 1988], provide for the transformation of phytosterol with the release of crystalline 9-OH-HELL. Other known methods for the preparation of Δ ^{9 (11)} -AD include the introduction of crystalline 9-OH-AD into the dehydration reaction.

In addition, there is no information in available literature on the possibility of carrying out the dehydration reaction of 9-OH-AD with the formation of Δ ^{9 (11)} -AD in the extract of the culture fluid containing 9-OH-AD and obtained as a result of microbiological transformation of sterols.

SUMMARY OF THE INVENTION

The aim of the present invention is to simplify and reduce the cost of technology for producing Δ ^{9 (11)} -AD from phytosterol.

The essence of the invention is the combination of two stages of the synthesis of Δ ^{9 (11)} -AD from phytosterol - the biotechnological stage, namely the microbiological transformation of phytosterol with the formation of 9-OH-HELL, and the chemical stage of dehydration of 9-OH-HELL - in one production process, carried out without isolation of the intermediate 9-OH-HELL from the clarified culture fluid extract.

A similar technical solution is not known from the prior art. Implemented by us for the first time.

The technical result of the present invention is provided by a combination of essential features, which are:

• Conducting dehydration of 9-OH-HELL obtained from phytosterol by microbiological transformation in an extract of the culture fluid from which the bacterial cells of the transforming strain were previously removed;

• Use of ∆ ^{9 (11)} -AD for the extraction of organic solvents, namely aromatic hydrocarbons or organochlorine hydrocarbons;

• Use as a dehydrating agent of mineral acids, including those containing water, more specifically, phosphoric acid or perchloric acid;

• Removing water from the reaction mass during the reaction, for example, by azeotropic distillation.

The proposed technical solution is new (not known from the prior art), does not explicitly follow from the prior art, is industrially applicable.

The technical result of the proposed technical solutions are:

• Optimization and simplification of the method of producing ∆ ^{9 (11)} -AD from phytosterol by combining the two stages of synthesis of ∆ ^{9 (11)} -AD from phytosterol - the biotechnological stage, namely the microbiological transformation of phytosterol with the formation of 9-OH-AD, and the chemical stage dehydration of 9-OH-AD - in one production process carried out without isolation of the intermediate 9-OH-AD from the extract of clarified culture fluid;

• Reducing the number of technological operations at the stage of the biotechnological process of obtaining 9-OH-HELL from phytosterol, namely, the exclusion of the following operations from the technological process: evaporation of the extract, crystallization of 9-OH-HELL, filtration of technical sediment 9-OH-HELL, drying of technical 9- OH-AD, purification of technical 9-OH-AD (includes crystallization / recrystallization, filtration, drying);

• The complete exclusion of losses of 9-OH-AD in the operations of its isolation described above (from 10 to 30% of the main substance), including mechanical losses and losses of the main substance in the mother liquor;

• Conducting a dehydration reaction in the extract without loss of efficiency compared with the dehydration of crystalline 9-OH-AD in an organic solvent;

• Reduction of the duration of the dehydration reaction of 9-OH-AD, reduction of amount of a dehydrating agent and thereby improving the yield and quality of Δ ^{9 (11)} -AD,

• Reducing the duration of the production process and thereby energy costs;

• Reduction of material costs.

The technical problem to be solved by the claimed invention is aimed at simplifying and increasing the efficiency of the process of obtaining Δ ^{9 (11)} -AD from phytosterol through the formation of intermediate 9-OH-HELL due to the complete elimination of losses of 9-OH-HELL, increasing the yield of the target product ∆ ^{9 (11)} -AD from phytosterol by almost completely eliminating the formation of a by-product of ∆ ^{8 (9)} -AD at the stage of 9-OH-AD dehydration and obtaining ∆ ^{9 (11)} -AD from phytosterol in a way devoid of the above disadvantages.

The implementation of two complex processes in one technological stage determines the prospects of using the method according to the invention to obtain Δ ^{9 (11)} -AD from phytosterol.

The technical problem of obtaining androsta-4,9 (11) -diene-3,17-dione from phytosterol using 9α-hydroxyandrost-4-en-3,17-dione obtained by microbiological transformation as an intermediate is solved by the method of producing androsta 4.9 (11) -diene-3.17-dione of the formula (I)

from phytosterol of the general formula (II),

where R = C ₂ H ₅ - β-sitosterol; R = CH ₃ - campesterol; R = C ₂ H ₅ and ∆ ^{22 (23)} - stigmasterol, R = CH ₃ and ∆ ^{22 (23)} - brassicasterin,

including microbiological oxidative elimination of the side chain with the formation of 9α-hydroxyandrost-4-en-3,17-dione of the formula (III),

separation of biomass, extraction of 9α-hydroxyandrost-4-en-3,17-dione (III) from the clarified culture fluid and subsequent dehydration of the 9α-hydroxy group with a dehydrating agent, characterized in that the 9α-hydroxy group is dehydrated in the culture fluid extract, and mineral acids, including those containing water, are used as the dehydrating agent, and the removal of excess water can be carried out during the dehydration reaction. The dehydration reaction of 9-OH-HELL obtained by microbiological transformation from phytosterol, without isolation from the extract of the culture fluid, has not been previously described in literature.

Thus, the essence of the claimed invention, which includes obtaining 9-OH-AD from phytosterol and then Δ ^{9 (11)} -AD using it, lies in the fact that phytosterol is first subjected to microbiological transformation with a culture of the genus Mycobacterium with the formation of 9-OH- HELL, then 9-OH-HELL is extracted from a pre-clarified culture fluid with an aprotic organic solvent, followed by a dehydration reaction in the obtained extract, for which mineral acid, including water, is added to the extract When in use, excess water can be removed during the reaction, e.g., azeotropic distillation.

In this case, the microbiological transformation of phytosterol in order to obtain 9-OH-AD is carried out using bacteria cells of Mycobacterium sp. VKM Ac-1817D.

In addition, an aromatic hydrocarbon (e.g., benzene or toluene) or a chlorine-containing hydrocarbon (e.g., dichloromethane, or chloroform, or dichloroethane) is used as an aprotic organic solvent.

In addition, the concentration of phytosterol during microbiological transformation is from 4 to 20 g / l.

The advantages of the proposed method are as follows:

• No need to extract from the extract, crystallization and purification of 9-OH-HELL; the absence of losses of 9-OH-AD occurring in these operations;

• High regioselectivity of the dehydration reaction. The degree of conversion of 9-OH-AD to Δ ^{9 (11)} -AD 98-100% (according to HPLC analysis); the recovery rate of Δ ^{9 (11)} -AD 96-98%.

• The total yield of ∆ ^{9 (11)} -AD from phytosterol reaches 71%.

The method of the present invention allows the process of dehydration of 9-OH-HELL:

• with a minimum amount of dehydrating agent, namely from 1.6 to 9 mol per 1 mol of 9-OH-AD, in the extract of the culture fluid;

• without the use of hazardous and expensive dehydrating agents, such as anhydrides and acid chlorides of mineral and organic acids, boron trifluoride complexes;

• without the use of expensive anhydrous organic solvents;

• The method can be implemented on standard processing equipment.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining androsta-4,9 (11) -diene-3,17-dione of the formula (I) from the phytosterol of the formula (II) is carried out according to the scheme shown in Figure 1.

Figure 1 - Synthesis of Δ ^{9 (11)} -AD (I) from phytosterol (II) through 9-OH-AD (III) of the claimed method, where R = C ₂ H ₅ - β-sitosterol; R = CH ₃ - campesterol; R = C ₂ H ₅ and ∆ ^{22 (23)} - stigmasterol, R = CH ₃ and ∆ ^{22 (23)} - brassicasterin

The following is an example of a detailed description of the invention.

Phytosterol of the general formula (II),

where R = C ₂ H ₅ - β-sitosterol; R = CH ₃ - campesterol; R = C ₂ H ₅ and ∆ ^{22 (23)} - stigmasterol, R = CH ₃ and ∆ ^{22 (23)} - brassicasterin,

subjected to microbiological oxidative elimination of the side chain at the C ¹⁷ atom with the simultaneous formation of a Δ ⁴ -3-ketosystem and 9α-hydroxylation of a culture of bacterial cells of the genus Mycobacterium (preferably bacteria cells Mycobacterium sp. VKM Ac-1817D), while the load of the initial substrate is from 4 to 20 g / l, and the duration of the fermentation is from 72 to 144 hours; the process is carried out in an aqueous medium with a selectivity of conversion to 9α-hydroxyandrost-4-en-3,17-dione (III) up to 72%, after which the cells of the transforming strain are separated. 9α-Hydroxyandrost-4-ene-3,17-dione (III) is extracted from the obtained clarified culture fluid containing practically no phytosterol with an organic solvent (aromatic hydrocarbon or chlorine-containing hydrocarbon) and without isolation from the extract is reacted with a dehydrating agent to form androst -4.9 (11) -diene-3,17-dione (I), for which mineral acid is added to the resulting extract.

Analysis of the products of microbiological transformation is carried out by thin layer chromatography (TLC) and HPLC. Determination of the content of products by HPLC was carried out on an Agilent 1200 chromatograph (USA) with a Symmetry C18 pre-column, 5 μm, 3.9x20 mm and a Symmetry C18 column, 5 μm, 4.6x250 mm (Waters, USA) in a mobile phase system (%) : acetonitrile - 52, acetic acid - 0.01 and water -47.99; at a flow rate of 1 ml / min, a temperature of 50 ° C, with peak detection by optical absorption at 240 nm; with the calculation of concentrations according to the external standard method. Typical retention times: for 9-OH-HELL 4.27 min and for Δ ^{9 (11)} HELL 8.25 min.

Isolation of 9-OH-AD (III) from the clarified culture fluid can be carried out by extraction with a water-immiscible solvent after preliminary separation of the biomass from the aqueous phase. For steroid extraction, for example, a sorption extraction method, as well as any other effective method, can also be applied. The selectivity of the formation of 9α-hydroxyandrost-4-en-3,17-dione (III) at the stage of microbiological oxidation of phytosterol (II) is 68-72%. The selectivity of the formation of Δ ^{9 (11)} -AD (I) at the stage of dehydration is 98-100%. The degree of extraction of crystalline Δ ^{9 (11)} -AD from the crud is 96-98%.

The total yield of Δ ^{9 (11)} -AD (I) from phytosterol reaches 71%.

The claimed invention is illustrated by the following examples, not limiting it.

For transformation, soybean phytosterol with the content of transformable sterols from 90 to 100% can be used. When carrying out the invention, a method for producing androsta-4,9 (11) -diene-3,17-dione in the example described below, phytosterol was used with a content of transformable sterols of 95.47%, including (%): β-sitosterol - 42, 39; campesterol - 23.48; stigmasterol - 26.08; brassicasterin - 3.52.

Microbiological oxidation of phytosterol in the implementation of the invention, a method for producing androsta-4,9 (11) -diene-3,17-dione can be carried out by bacteria cells of the genus Mycobacterium , specifically Mycobacterium sp. VKM Ac-1817D.

As an organic solvent in the implementation of the invention, a method for producing androst-4,9 (11) -diene-3,17-dione in the operation of extraction of 9-OH-AD and subsequent dehydration, an aromatic hydrocarbon (for example, benzene or toluene) can be used or a chlorine-containing hydrocarbon (e.g. dichloromethane or chloroform or dichloroethane).

Mineral acids, including those containing water, for example, perchloric acid with a water content of up to 43%, or phosphoric acid with a water content of up to 15%, or phosphoric acid with a content of 9% OH, can be used as a dehydrating agent for the dehydration of 9-OH-AD. up to 50% pyrophosphoric acid.

The intensification of the dehydration process in the implementation of the invention, the method of producing androst-4,9 (11) -diene-3,17-dione by removing excess water during the dehydration reaction can be carried out, for example, by azeotropic distillation or by carrying out the reaction in the presence of an effective amount of pyrophosphoric acid , which, reacting with water, turns into phosphoric acid under the conditions of the reaction.

Table 1 presents the results of the dehydration of 9α-hydroxyandrost-4-en-3,17-dione with the formation of Δ ^{9 (11)} -AD in an extract of clarified culture fluid under the conditions of the present method and the methods described in the literature.

Examples

Example 1 Preparation of androsta-4,9 (11) -diene-3,17-dione (I)

A) Growing inoculum

Bacteria Mycobacterium sp. VKM Ac-1817D is cultivated in a shaker-thermostat for 25 hours at a temperature of 30 ° C and a shaking frequency of 220 rpm in rocking flasks with a capacity of 750 ml containing 100 ml of a liquid nutrient medium of the following composition (g / l): dry yeast extract - 10; (NH ₃ ) ₂ HPO ₄ - 1.5; KH ₂ PO ₄ - 0.5; K ₂ HPO ₄ x 3H ₂ O - 0.5; glycerin - 10; polyethylene glycol sorbitol monooleate - 3.0; MgSO ₄ x 7H ₂ O 0.2; FeSO ₄ x 7H ₂ O - 0.005; ZnSO ₄ x 7H ₂ O - 0.002; distilled water - up to 1 l, pH 6.8 - 7.2.

B) Transformation

The resulting seed is introduced into the medium for transformation in an amount of 10% (by volume). Microbiological transformation of phytosterol is carried out in a shaker-thermostat for 72 hours at a temperature of 30 ° C and a shaking frequency of 220 rpm in rocking flasks with a capacity of 750 ml containing 100 ml of medium for transformation of the following composition (g / l): KH ₂ PO ₄ - 0.8; K ₂ HPO ₄ x 3H ₂ O - 4.2; (NH ₃ ) ₂ SO ₄ - 3.0; glycerin - 5.0; urea - 0.13; MgSO ₄ x 7H ₂ O 0.2; FeSO ₄ x 7H ₂ O - 0.01; ZnSO ₄ x 7H ₂ O - 0.002; polyethylene glycol sorbitol monooleate - 1.0; phytosterol (calculated on transformable sterols) - 4.0; distilled water - up to 1 l, pH 6.8 - 7.2. After sterilization of the medium, phytosterol is homogenized by ultrasound (42 kHz, 100 W) for 2 minutes.

C) Preparation of the extract

At the end of the transformation, the culture fluid is centrifuged at

10,000 g for 40 minutes at a temperature of 20-25 ° C. A clarified culture fluid (supernatant) containing 9-OH-AD (transformation selectivity 71.48%) is obtained.

 9-OH-AD is extracted from the supernatant with an organic solvent. An extract is obtained containing 9-OH-HELL (hereinafter the extract).

D) Conducting a dehydration reaction

Option 1. The extract is placed in a flask equipped with a reflux condenser. Mineral acid is added to the extract. The reaction mass is heated to boiling and maintained with stirring until the end of the reaction. Then the mixture was cooled to room temperature and a 5% aqueous NaCl solution was added. The organic layer was separated, the aqueous layer was extracted with the same solvent. The combined organic phases are washed with 5% NaCl until neutral and water, then dried with Na₂SO_four and evaporated to dryness in vacuo. Get crud containing ∆^{9 (11)}-AD and not containing 9-OH-AD according to HPLC analysis.

Option 2 (with azeotropic distillation of water). The extract is placed in a flask equipped with a Dean-Stark nozzle and reflux condenser. Then, mineral acid is added to the extract. The reaction mass is heated to boiling and maintained with stirring until the end of the reaction, collecting the released water in the nozzle. Then the mixture was cooled to room temperature. Processing the reaction mass is carried out as described in the previous embodiment.

Selectivity dehydration reaction is evaluated by HPLC analysis of the initial extract (determination of 9-OH-AD) and Crude (content of Δ ^{9 (11)} -AD).

After isolation from the crud, Δ ^{9 (11)} -AD is obtained with a melting point of 203.5 to 206.5ºC and a degree of recovery of 96-98%.

¹ H NMR Spectrum ( δ , ppm; CDCl ₃ ): 5.75 (d, J = 1.8 Hz, 1H, 4-C H ); 5.55 (m, 1H, 11-C H ); 1.35 (s, 3H, 19-C H ₃ ); 0.87 (s, 3H, 18-C H ₃ ).

¹³ C NMR spectrum ( δ , ppm; CDCl ₃ ): 221.0 (17- C (O)); 199.0 (3- C (O)); 168.9 (5- C ); 145.1 (9- C ); 124.2 (4- C H); 118.1 (11- C H); 48.0; 45.8; 41.1 36.8; 36.2; 34.2; 33.8; 33.4; 32.6; 31.1; 26.2; 22.6; 13.9 (19-C H ₃ ).

Table 1

Way Solvent Dehydrating agent (mol / mol 9-OH-AD) Temperature, reaction time The selectivity of the formation of ∆ ^{9 (11)} -AD,% (HPLC) Exit
∆ ^{9 (11)} -HELL,%
(degree of extraction,%) Note According to the method of US 4127596 - 85% H ₃ PO ₄
12,2 45-55 ° C,
24 h  - 95.1 Crystalline 9-OH-HELL According to the method of US 4127596 - H ₃ PO ₄ 85%
73v / 1w 35-48 ° C,
7.5 h  -  96.2 Crystalline 9-OH-HELL The inventive method Chloroform
85% H ₃ PO ₄
1,6 Boiling,
1 h 25 min  ~ 100 - Extract The inventive method Benzene
85% H ₃ PO ₄
1,6 Boiling,
1 h 10 min ~ 100 (97.8) Extract The inventive method Toluene
85% H ₃ PO ₄
1,6 Boiling,
30 minutes 98.4
- Extract The inventive method Dichloroethane 85% H ₃ PO ₄
8.9 Boiling
(78 ° C), 30 min ~ 100 (96.86) Extract The inventive method Toluene
85% H ₃ PO ₄
1,6 Boiling,
5 minutes ~ 100 - Extract. Carrying out the reaction with azeotropic distillation of water The inventive method CH ₂ Cl ₂ 85% H ₃ PO ₄
1,6 Boiling, 2 hours  99 (96.1)
Extract. Carrying out the reaction with azeotropic distillation of water The inventive method Toluene 46.5% H ₄ P ₂ O ₇ / H ₃ PO ₄ Boiling,
40 min 98.52 - Extract The inventive method Chloroform
46.5% H ₄ P ₂ O ₇ / H ₃ PO ₄
5,4 Boiling,
1 h 40 min
99.48 - Extract By the way
HUT36138 - (H) _{n + 2} ^. (P) _n ^. (O) _{3n + 1}
3v / 1w 40 ° C
2 h 20 min - 98.46 Crystalline 9-OH-HELL The inventive method Benzene
57% HClO ₄
1.83 Boiling,
10 min ~ 100 (96.0) Extract. Carrying out the reaction with azeotropic distillation of water According to the method of US 4127596 - 28% HClO ₄
23,2 54 ° C
66 h - 69.6 Crystalline 9-OH-HELL

Claims (11)

1. The method of producing androsta-4,9 (11) -diene-3,17-dione of the formula (I)

from phytosterol of the general formula (II),

where R = C ₂ H ₅ - β-sitosterol; R = CH ₃ - campesterol; R = C ₂ H ₅ and ∆ ^{22 (23)} - stigmasterol; R = CH ₃ and Δ ^{22 (23)} - brassicasterin,
including microbiological oxidative elimination of the side chain at the C ¹⁷ atom with the formation of 9α-hydroxyandrost-4-en-3,17-dione of the formula (III),

separation of biomass, extraction of 9α-hydroxyandrost-4-en-3,17-dione (III) from the clarified culture fluid and subsequent dehydration of the 9α-hydroxy group with a dehydrating agent, characterized in that compound (III) is extracted from the clarified culture fluid with an aprotic organic a solvent selected from aromatic hydrocarbons or organochlorine hydrocarbons, followed by the dehydration reaction of the 9α-hydroxy group of compound (III) in the obtained extract, using as a dehydrating agent dissolved mineral acid, including water-containing and selected from the group comprising orthophosphoric, pyrophosphoric and perchloric acid, and the removal of excess water is performed during the dehydration reaction. 2. The method according to claim 1, characterized in that for the transformation using soybean phytosterol with the content of transformable sterols from 90 to 100%. 3. The method according to claim 1, characterized in that the microbiological transformation of phytosterol is carried out by the bacteria Mycobacterium sp. VKM Ac-1817D. 4. The method according to claim 1, characterized in that benzene or toluene is used as an aromatic hydrocarbon. 5. The method according to claim 1, characterized in that dichloromethane or chloroform or dichloroethane is used as the chlorine-containing hydrocarbon. 6. The method according to claim 1, characterized in that the removal of excess water can be carried out in the process of dehydration by azeotropic distillation. 7. The method according to claim 1, characterized in that the removal of excess water can be carried out during the dehydration reaction in the presence of an effective amount of pyrophosphoric acid. 8. The method according to claim 6, characterized in that they use mineral acid in an amount of from 1 to 10 mol per 1 mol of 9α-hydroxyandrost-4-en-3,17-dione, preferably from 2 to 5 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. 9. The method according to claim 6, characterized in that 85% orthophosphoric acid is used as a mineral acid. 10. The method according to claim 6, characterized in that 57% perchloric acid is used as a mineral acid. 11. The method according to claim 1, characterized in that as the dehydrating agent using phosphoric acid containing up to 50% pyrophosphoric acid.