RU2532902C1 - Method of obtaining 11beta, 17 alpha, 21-trihydroxy-16alpha-methyl-9alpha-fluoropregna-1,4-diene-3,20-dione (dexamethazone) from phytosterol - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of obtaining dexamethasone from phytosterols (β-sitosterol, campesterol, stigmasterol, brassicasterol) by a method, including a sequence of microbiological and chemical reactions, namely: microbiological oxidative elimination of a side chain of phytosterol with the formation of 9α-hydroxyandrost-4-en-3,17-dione by culture Mycobacterium sp.VKM Ac-1817D; processing 9α-hydroxyandrost-4-en-3,17-dione with mineral acid in a medium of aprotic organic solvent with the formation of 9(11)-double bond; 17-hydrocyanation of androst-4,9(11)-diene-3,17-dione; protection of 3-ketogroup of 17α-hydroxy-17β-cyanandrost-4,9(11)-dien-3-one by the formation of cyclic ketal, protection of 17α-hydroxygroup by the formation of 1-alkoxyethyl ether, alkylation of 17-CN-group with further removal of protective groups, C21-acetoxylation of the obtained 17α-hydroxy-20-ketopregn with the application of a method of direct C21-iodination; etherification of 17α-hydroxygroup of 21-acetoxy-17α-hydropregn-4,9(11)-dien-3,20-dione and elimination of 17α-acetoxygroup, microbiological 1,2-dehydration of 21-acetoxypregn-4,9(11),16-triene-3,20-dione with a culture of Nocardioides simplex VKM Ac-2033D with the formation of 21-acetoxypregn 1,4,9(11),16-tetraene-3,20-dione, reaction of the catalytic 1,4-connection of the Grignard reagent by Δ16-20-ketosystem with the formation of magnesiumhaloidderivative of 21-acetoxy-20-hydroxy-16a-methylpregn-1,4,9(11),17(20)-tetraen -3-one; introduction of 17α-hydroperoxygroup by autooxidation of the latter; reduction of 17α-hydroperoxygroup in 17α-hydroxyl; 9,11-bromohydroxylation of 21-acetoxy-17α-hydroxyl-16α-methylpregn-1,4,9(11)-triene-3,20-dione and further 9β(11)-epoxidation with the simultaneous hydrolysis of the acetoxygroup at C21; disclosure of 9β(11)-epoxy ring of 17α,21-dihydroxy-16α-methyl-9β(11)epoxypregn-1,4-diene-3,20-dione by the action of hydrofluoric acid.

EFFECT: method makes it possible to obtain dexamethasone with the 23 percent output.

24 cl, 3 tbl, 16 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, specifically relates to the production of the steroid compound 11β, 17α, 21-trihydroxy-16α-methyl-9α-fluoropregna-1,4-diene-3,20-dione (dexamethasone) from phytosterol and can be used for the production of substances of dexamethasone and its derivatives.

BACKGROUND

11β, 17α, 21-Trihydroxy-16α-methyl-9α-fluorpregna-1,4-diene-3,20-dione (dexamethasone, 9α-fluoro-16α-methylprednisolone, CAS 50-02-2) is a glucocorticosteroid drug strong anti-inflammatory and anti-allergic effect [M.D. Mashkovsky. Medicines In two volumes. Ed. 13th. Kharkov: Torsing, 1997, vol. 2. p.35]. In addition, dexamethasone can be used as a starting substrate in the synthesis of its derivatives, for example, dexamethasone disodium phosphate 21 (dexamethasone disodium phosphate, CAS 2392-39-4), used for intravenous and intramuscular administration, as well as in ophthalmology for the treatment of allergic conjunctivitis , keratitis, reduction of inflammatory manifestations after eye operations and injuries.

To date, there are no published data on the scheme for the synthesis of dexamethasone from phytosterol per se. However, there are reports of a synthesis of its various intermediates. The search and study of various approaches to the synthesis of dexamethasone has been conducted by leading pharmaceutical companies in the world over the past 50 years and is determined by the variability of sources of steroid raw materials and the search for possible synthesis intermediates common to dexamethasone and several other steroid preparations.

The most promising source of steroid raw materials are sterols. Sterols are structural precursors in the production of steroid hormones (gestagens, androgens, estrogens, corticoids, etc.). Phytosterol - a plant-based sterol - can be obtained from waste wood or soybeans. The word "phytosterol" means sterols of plant origin (β-sitosterol, campesterol, stigmasterol, etc.) or mixtures thereof. In recent years, the world has developed effective methods for the oxidative degradation of the side chain of sterols with the formation of androst-4-en-3,17-dione (AD), which may be accompanied by its simultaneous or sequential functionalization with the formation of 1,2-dehydro- or 9α-hydroxy derivatives of blood pressure.

9α-Hydroxyandrost-4-en-3,17-dione (9-OH-AD) is an ideal intermediate in the synthesis of corticosteroids from sterols (for example, hydrocortisone, prednisolone, dexamethasone, triamcinolone, 6α-methylprednisolone, etc.). This is especially important, since it allows to exclude the stage of microbiological 11-hydroxylation from the technological process.

The synthesis of dexamethasone from phytosterol through 9-OH-HELL is a multi-stage sequence of transformations, including the following stages of functionalization:

- dehydration of the 9α-hydroxy group with the formation of the Δ9 (11) bond;

- the introduction of 17α-hydroxy-17β-pregnan chain;

- the introduction of a hydroxyl group at the 21-position of 17α-hydroxy-C20-ketopregnan with the formation of a corticosteroid (dioxiacetone) chain;

- dehydration of the 17α-hydroxy group with the formation of Δ16-3,20-diketopregnan;

- the introduction of a 1,2-double bond (chemical or microbiological method);

- the introduction of a 16α-methyl-17α-hydroxy fragment at the ∆16 bond;

- introduction of the 9α-fluoro-11β-hydroxy fragment through the Δ9 (11) bond.

As follows from the literature, these steps can be carried out in any order. The choice of the sequence of stages and methods of synthesis is a complex task, the solution of which determines success in creating a cost-effective production technology.

Methods of oxidative degradation of the side chain of phytosterol with the formation of Δ4-3,17-diketoandrostanes, including the formation of 9-OH-AD, are known. 9-OH-AD is obtained from phytosterol by microbiological transformation using the methods and cultures of microorganisms described in the literature [Prikl. biochem. microbiol., 2011, 47 (3), 297-301; Biochim. Biophs. Acta, 1978, 539, 308; US4035236, 1977; US4175006, 1979; US4397946, 1983; DD298278, 1992; DD298279, 1992; RU2077590, 1997; Appl. Microbiol. Biotechnol., 2005, 67 (5), 671-678; J. Chem. Technol. Biotechnol., 2005, 80, 55-60, etc.].

Dehydration of the 9α-hydroxy group to form the Δ9 (11) bond

In the synthesis of the corticosteroid chain based on the 17-keto group 9-OH-AD, the dehydration of the 9α-hydroxy group is usually carried out at the first stage, since the resulting androsta-4,9 (11) -diene-3,17-dione (Δ9 (11) -AD ) is more stable in further synthesis (for example, in the synthesis of hydrocortisone acetate from sterol [Tetrahedron Lett., 1990, 31 (26), 3669-3672]). This avoids possible undesirable reactions involving the tertiary hydroxyl group at C9 in the next stages of the synthesis. In addition, the presence of a 9.11-double bond is a necessary condition for the further introduction of a halogen to position 9α in the synthesis of 9-halogenated corticosteroids, including dexamethasone. It is also advisable to carry out dehydration in the absence of hydroxyl groups in other positions. Thus, Δ9 (11) -AD is a key compound in the synthesis of corticosteroids from natural sterols (cholesterol or phytosterol).

Methods for producing Δ9 (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 (Δ8 (9) -AD).

In this case, the isomeric Δ9 (11) - and Δ8 (9) - olefins have the same Rf value on TLC and cannot be separated by crystallization. The determination of the impurity of the Δ8 (9) isomer can be carried out, for example, spectroscopically (1 H NMR, 13 C NMR) [EP0253415, 1988; EP0294911, 1988] or chromatographic (HPLC [EP0253415, 1988; EP0294911, 1988], GLC [US4102907, 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 Δ9 (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.

A combination of the essential features of the claimed invention in terms of carrying out the dehydration of the 9α-hydroxy group with the formation of the Δ9 (11) bond is the dehydration reaction of 9-OH-AD with the formation of the Δ9 (11) -AD in an inert aprotic organic solvent, specifically, in 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, specifically ofosfornoy, sulfuric or perchloric acid.

Known methods for the dehydration of 9-OH-HELL with the formation of Δ9 (11) -HELL in an organic solvent. It is known that chlorinated hydrocarbons, aromatic hydrocarbons, cyclic ethers, and organic bases can be used as the medium. However, N, N1-thionyl-diimidazole formed from thionyl chloride and imidazole (in tetrahydrofuran medium) was used as a dehydrating agent [Journal für Praktische Chemie, 1988, 330 (2), 309-312]; or p-toluenesulfonic acid (p-TSC, in benzene) [Chem.and Ind., 1961, 1530]; or an aromatic sulfonic acid adsorbate on silica gel (in an aromatic hydrocarbon medium) [EP0253415, 1988]; or Lewis acids - iron chloride, boron trifluoride or their complexes, antimony pentachloride, titanium tetrachloride - and mixtures of these compounds with silica gel (in an inert organic solvent) [EP0294911, 1988]; or chlorosulfonic acid (in the environment of methylene chloride) [US4127596, 1978, examples 1-8].

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, sulfuric or perchloric acid.

A known method of producing Δ9 (11) -steroids of the androstane series, including Δ9 (11) -AD, by the reaction of 9α-hydroxysteroid (in particular, 9-OH-AD) with non-aromatic oxygen-containing acid with pKa ≤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.

Also known is a method of producing Δ9 (11) -AD from 9-OH-AD by dehydration with polyphosphoric acid [HUT36138, 1985].

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 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 unsatisfactory quality.

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 Δ9 (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.

Thus, in the available literature there is no information about the dehydration reaction of 9-OH-AD with the formation of Δ9 (11) -AD using mineral acids, including those containing water, as a dehydrating agent in an aprotic organic solvent.

In addition, in available literature there is no information on the intensification of the dehydration process by removing excess water during the dehydration reaction, for example, by azeotropic distillation.

In addition, the available literature does not contain information on the possibility of obtaining Δ9 (11) -AD from phytosterol through the formation of 9-OH-AD without isolating the latter from the culture fluid extract obtained as a result of microbiological transformation.

Introduction 17α-hydroxy-17β-pregnan chain

Hydrocyanation

One of the well-known methods for the synthesis of 17α-hydroxy-17β-acetyl steroids is the cyanohydrin method, which consists in the interaction of 17-ketosteroids with hydrogen cyanide and the subsequent alkylation of the obtained 17α-hydroxy-17β-nitrile. The chemical synthesis of pregnanes from 17-ketoandrostanes using cyanohydrin synthesis is known and described in the literature [Pharmaceutical Chemistry Journal, 1987, 21, (4), 225-300]. The cyanide anion donor can serve as salts of hydrocyanic acid [DD281394, 1990], and acetone cyanohydrin [J. Am. Chem. Soc., 1953, 75 (3), 650-653; RU2156255, 2000], as well as mixtures thereof [US4500461, 1985; US3043833, 1962; US4548748, 1985] and other cyanide-containing organic compounds, for example diethylaluminum cyanide [US3496169, 1970]. The reaction with cyanide-containing compounds is carried out in an alkaline medium, in addition to alkali metal hydroxides, triethylamine can be used as an alkaline agent [GB1109626, 1968].

The cyanohydrin variant of constructing the side chain of corticosteroids from AD, 9α-OH-AD and other 17-ketosteroids has several advantages: the selectivity of the hydrocyanation reaction for the 17-keto group [Organic Reactions, 1977, 25, p. 255]. However, hydrocyanation of 17-ketosteroids proceeds with the formation of an equilibrium mixture of epimeric 17β- and 17α-carbonitriles. Depending on the conditions of the hydrocyanation process, one or another isomer may predominate in the mixture.

It should be noted that this method has received the greatest development and application in the synthesis of 17-cyanohydrin AD. There is a method of introducing a pregnan side chain in blood pressure using acetone cyanohydrin as a cyanating agent [DD147669, 1979, example 2].

The use of acetone cyanohydrin alone as an agent for hydrocyanation of Δ9 (11) -AD, although claimed in patents [RU2156255, 2000; US4500461, 1985], but not supported by examples. The cyanohydrin method of constructing a pregnan chain based on AD is described by Japanese authors [Bull. Chem. Soc. Jpn., 1985, 58 (3), 978-980]. However, hydrocyanation of Δ9 (11) -AD with acetone cyanohydrin under the conditions described by the authors proceeds with the formation of an extremely undesirable 17α-cyan-17β-hydroxy epimer, while the reaction in the presence of a 9-hydroxy group under the same conditions gives the extremely desirable 17β-cyan -17α-hydroxy epimer [Bull. Chem. Soc. Jpn., 1985, 58 (3), 978-980]. The 17β-cyan-17α-hydroxy-Δ9 (11) -epimer can be obtained by subsequent isomerization of the initially formed 17α-cyan-17β-hydroxy-isomer by the action of, for example, sodium cyanide or triethylammonium hydrocyanide in a lower alcohol environment [JPS5762298, 1982; JPS5762299, 1982].

Thus, we can conclude that the technical result obtained by the example of blood pressure in the patents [RU2156255, 2000 and RU2156256, 2000] may not be obvious when using Δ9 (11) -AD as the starting compound in the synthesis of 17β-cyan 17α-hydroxy derivative.

The authors of the patent [DD281394, 1990] managed to obtain 17β-cyan-17α-hydroxy-epimer with a yield of 93% based on Δ9 (11) -AD in an aqueous alcohol containing acetone using sodium cyanide as a cyanating agent and the method epimerization, the essence of which consisted in the gradual precipitation of the 17β-cyan epimer by changing the concentration of water. A similar method for the preparation of ∆9 (11) -AD cyanohydrin using potassium cyanide is described in the patent [US4548748, 1985], and a mixture of potassium cyanide and acetone cyanohydrin in the patent [US4500461, 1985, example 2].

Thus, we did not find information on the use of acetone cyanohydrin alone for the stereoselective hydrocyanation of Δ9 (11) -AD.

3-keto protection

To prevent possible alkylation of the 3-keto group, it is protected. The protection of the 3-keto group can be carried out by the formation of enol ether (methyl or ethyl) [J. Am. Chem. Soc., 1953, 75 (3), 650-653; J. Org. Chem., 1961, 26 (10), 3925-3928; US3516991, 1970; US4500461, 1985, EP263569, 1988; US4921638, 1990], cyclic ketal [US3516991, 1970; US4500461, 1985; http://www.springerlink.com/content/106464/?p=077e6ed5e8a848f38bd231d4c7dbdf27&pi=0, 1984, 20 (3), 302-304; JPS5762296, 1982; JPS5762298, 1982; JPS5762299, 1982; JPS5762300, 1982; Tetrahedron Letters, 1990, 31 (26), 3669-3672] or enamine [US3629298, 1971]. Ethylene-ketal protection has found the greatest application and is most often mentioned in literary sources. So, in the works [US4500461, 1985; JPS5762296, 1982; 1987, 21 (4), 225-300; EP263569, 1988], 3-keto group protection was used by the formation of a five-membered 1,3-dioxolane ring by reaction with ethylene glycol. However, the synthesis of 3.3-ethylenedioxy-17α-hydroxy-17β-cyanoanodrost-5.9 (11) -diene describes the introduction of ethylene ketal protection before the hydrocyanation stage [JPS5762296, 1982], and not after the hydrocyanation stage, as proposed in the present method.

The method of hydrocyanation of 17-ketone after protecting the 3-keto group of ∆9 (11) -AD by forming 3-methyl ester in the synthesis of hydrocortisone from 9-OH-AD is described in [Tetrahedron Lett., 1990, 31 (26), 3669-3672] .

Thus, a description of an example of the protection of carbonyl at the C3 atom by the formation, for example, of ethylene ketal after the preparation of the 17α-hydroxy-17β-cyano derivative from Δ9 (11) -AD (cyanohydrin Δ9 (11) -AD) in literature was not found by us .

The use of 2,2-dimethyl-1,3-propanediol (neopentyl glycol) for protecting the 3-keto group of the AD molecule to form the 1,3-dioxane cycle was first reported in patents [RU2156255, 2000; RU2156256, 2000]. However, despite the fact that the general claims claimed the applicability of this protection for the alkylation of Δ9 (11) -AD cyanohydrin, there are no examples of the preparation and use of the Δ9 (11) -AD 1,3-dioxane ketal cyanhydrin.

Both in the case of the introduction of dioxane, and in the formation of dioxolane protection, trialkyl orthoformates (triethyl and trimethylorthofrmiate) are used [RU2156256, 2000] or dimethylformamide diethylacetal. This allows you to shift the equilibrium in the ketalization reaction due to the chemical binding of water formed during the reaction. As the catalyst, p-TSC or sulfosalicylic acid is used. For ketalization of 17α-hydroxy-17β-cyanandrost-4-en-3-one, the classical method of boiling with ethylene glycol in benzene in the presence of a catalytic amount of p-TSC with azeotropic distillation of the resulting water can be applied [Chemistry of Natural Compounds, 1984, 20 (3 ), 302-304]. However, it is less effective than the one described above.

Protection of the 17α-hydroxy group

It is known that under alkylation conditions, the cyanohydrin group can be eliminated with the regeneration of the carbonyl group with the subsequent formation of its alkylation products. The content of the latter can reach significant amounts, which leads to loss of yield and contamination of the target compound. To prevent these undesirable processes, resort to the protection of the 17α-hydroxy group by the formation of ether. Alkyl vinyl esters (ethyl vinyl [US4500461. 1985; US4548748, 1985] or butyl vinyl [JP57062296, 1982]) are used as the esterifying agent. Also known methods of protection by the formation of 17-tetrahydropyranyl ether [J. Am. Chem. Soc., 1959, 81 (21), 5725-5727; EP263569, 1988] by reaction with 2,3-dihydropyran under conditions of acid catalysis (p-TSC or phosphorus oxychloride) or trimethylsilyl ether by reaction with trimethylchlorosilane [Tetrahedron Lett. 1971, 2005-2008]. However, during the formation of the 17-tetrahydropyranyl ether, partial isomerization of the 17β-cyan compound with an increase in the impurity content of the undesirable 17α-cyan-17β-tetrahydropyranyloxy isomer is observed. The trimethylsilyl protective group is also undesirable, since it is bulky and exhibits a shielding effect in the subsequent stage of alkylation of the nitrile group. This necessitates the use of harsh alkylation conditions (prolonged boiling in THF or the use of high-boiling solvents, for example, anisole) and is accompanied by a side reaction on the 1,3-dioxolane ring with the formation of its cleavage product on the C-O bond 3ξ-methyl-3ξ- (hydroxyethoxy) -pregnan in high yield (in anisole, for example, quantitative) [Chemistry of Natural Compounds, 1984, 20 (3), 302-304; J. Chem. Soc., Perkin Trans. I, 1993, (7), 783-793]. Therefore, in our opinion, protection of the 17-hydroxy group of 17β-carbonitrile by the formation of 17α- (1-alkoxy) ethyl ester is most acceptable.

Introduction of a 20-keto-pregnane side chain by alkylation of a carbonitrile group

Further conversion of the 17β-cyan-17α-hydroxy compound to 20-ketopregnans is carried out by reacting the corresponding cyanohydrins with protected 3-keto and 17α-hydroxy groups with methyl lithium [EP263569, 1988; Pharmaceutical Chemistry Journal, 1987, 21 (4), 297-300; Bull. Chem. Soc. Jpn., 1985, 58 (3), 978-980] or methyl magnesium bromide [Chemistry of Natural Compounds, 1984, 20 (3), 302-304].

The closest in essence to the claimed method in terms of constructing the pregnane side chain based on 17-ketoandrostanes using the cyanohydrin method, cyclic protection of the 3-keto group and protection of the 17α-hydroxyl group by the formation of 17α- (1-alkoxy) ethyl ester is the method described in the patent [RU2156255, 2000].

However, the method in the patent formula [RU2156255, 2000] is claimed in general terms. Examples confirmed the receipt of 17α-hydroxyprogesterone from AD. The use of Δ9 (11) -AD as the starting compound is not confirmed by examples. ∆9 (11) -AD is not a homologue of AD. As is known to a person skilled in the art, and as mentioned above, a 9.11-double bond is a functional reaction-specific group, that is, an additional reaction center, therefore, the technical result obtained by the example of AD cannot be obvious when using Δ 9 (11) -AD as the starting compound.

The synthesis sequence of 17α-hydroxypregn-4,9 (11) -diene-3,20-dione (Δ9 (11) -HPG) from Δ9 (11) -AD in accordance with the present invention is not described in available literature.

Introduction of a hydroxyl group at the 21-position of 17α-hydroxy-C20-ketopregnan with the formation of a corticosteroid (dioxiacetone) chain

It is known that in the synthesis of corticosteroids from 17α-hydroxypregnans, the preliminary stage, as a rule, is the halogenation of the 21-position with the formation of the 21-halogen-17α-hydroxypregnane intermediate, which is then subjected to the substitution reaction for the acetoxy group with alkali metal acetate.

A known method of producing 21-acetate of the substance “S” from 17α-hydroxyprogesterone with a previously protected 3-keto group by the formation of an N-morpholinyl derivative, which consists in the fact that 20-ketopregnan is α-halogenated at position C21 by introducing a bromine atom in the presence of HCl and then the resulting mixture of 21-bromo and 21-chloro derivatives is treated with potassium acetate in the presence of a small amount of sodium iodide in acetone [Chem. farm.zh. 1990, 24 (11), 55-57].

The synthesis of 21-acetoxy-17α-hydroxypregn-4,9 (11) -diene-3,20-dione (21-acetoxy-Δ9 (11) -PGH) can also be carried out from the 21-bromo derivative Δ9 (11) - GPG [US4041055, 1977] or a 21-chloro derivative [US4342702.1982; US4357279, 1982] by sequentially replacing a bromine or chlorine atom first with an iodine atom by a reaction with potassium iodide, and then with an acetoxy group by reacting 21-iodine-Δ9 (11) -GPG with potassium acetate in acetone [US4041055, 1977] or a mixture acetone and dichloromethane [US4342702.1982].

However, the transition from Δ9 (11) -GPG to the 21-bromo or 21-chloro derivative requires protection of the Δ4-3-ketosystem, for which the formation of methyl or ethyl 3,5-dienol ethers, pyrrolidine or morpholine enamines, or ketal protection. Otherwise, the formation of a by-product of α-halogenation (for example, bromination) is observed at the C2 atom and, with a greater excess of bromine, the halogenation product at position C6.

Последующая реакция замещения атома иода на ацетоксигруппу может быть проведена с применением как ацетата калия, так и ацетата натрия. Кроме того, может быть использован ацетат триэтиламина в среде ацетона [CN101125877, 2008] с общим выходом на стадиях иодирования и ацетоксилирования до 88% [Bull. Chem. Soc. Jpn., 1985, 58, 1081-1082].

The subsequent reaction of substitution of the iodine atom for the acetoxy group can be carried out using both potassium acetate and sodium acetate. In addition, triethylamine acetate in acetone can be used [CN101125877, 2008] with a total yield of up to 88% at the stages of iodination and acetoxylation [Bull. Chem. Soc. Jpn., 1985, 58, 1081-1082].

A method of 21-acetoxylation of pregnanes using the direct iodination method without protection of the Δ4-3 keto group is also well known. This method was first proposed by Hogg et al. In 1955 [J. Am. Chem. Soc., 1955, 77 (16), 4436-4438] to obtain cortexolone 21-acetate and hydrocortisone acetate from 17-hydroxyprogesterone, according to which the C21-methyl group of the steroid molecule reacts with iodine in the presence of a base and the 21-mono-iodo derivative is formed in treatment with an alkali metal acylate transforms into the corresponding 21-acyloxysteroid.

Direct iodination has been most widely used in the production of cortexolone acetate from 17α-hydroxyprogesterone [J. Am. Chem. Soc., 1958, 80 (1), 250; http://www.springerlink.com/content/106490/?p=97444ae762a94bcfab26466d7f1502ed&pi=0, 1990, 24 (11), 839-842; http://www.springerlink.com/content/0091-150x/ "\ o" Link to the Journal of this Article 1969, http://www.springerlink.com/content/0091-150x/3/12/ " \ o "Link to the Issue of this Article), 709-712; SU151331, 1966; RU2156256, 2000]. In the patent [GB882388, 1961], this process is described on 6-methyl substituted 17α-hydroxyprogesterone, in the patent [US5565588, 1996] on 9.17α-dihydroxyprogesterone.

The closest in essence to the claimed method in terms of the sequence of construction of the dioxia-acetone side chain based on 17-ketoandrostanes, namely, using the cyanohydrin method, cyclic protection of the 3-keto group, protection of the 17α-hydroxyl group by formation of 17α- (1-alkoxy) ethyl ether, alkylation nitrile group and 21-acetoxylation using the direct iodination method, is the method described in the patent [RU2156256, 2000].

The patent [RU2156256, 2000] claims the use of the direct iodination method in the synthesis of corticosteroids with the formation of a 21-mono-iodo derivative and subsequent acetoxylation under the conditions of L. G. Gatsenko and V.N. Petrova [Chem. farm. Well, 1972, 10, 27]. However, the method in the patent formula [RU2156256, 2000] is claimed in general terms. Examples confirmed the receipt of 21-acetoxy-17α-hydroxyprogesterone from AD. Examples of the preparation of 21-iodo-Δ9 (11) -HPG and further 21-acetoxy-Δ9 (11) -HPG are absent.

In literature there is no information on the receipt of 21-iodine-Δ9 (11) -GPG by direct iodination of Δ9 (11) -GPG.

The sequence of synthesis of 21-acetoxy-Δ9 (11) -GPG from Δ9 (11) -AD through the formation of 21-iodo-Δ9 (11) -GPG of the present invention is not described in available literature sources.

Dehydration of the 17α-hydroxy group to form Δ16-3,20-diketopregnan

In order to transfer from 17α-hydroxy-20-ketopregnan to a compound with the Δ16 bond necessary for introducing the 17α-hydroxy-16α-methyl fragment, the 17α-hydroxy group is dehydrated.

Dehydration of the 17α-hydroxy group to the corresponding 16.17-dehydroduct may be carried out, for example, with a mixture of phosphorus oxychloride and pyridine.

A known method of introducing Δ16-bonds in the molecule of 21-acetoxy-Δ9 (11) -GPG [US3493563, 1970]. The method consists in treating the 17-hydroxy compound with thionyl chloride in pyridine and provides preliminary protection of the 3-keto group with ketalization, and the 20-keto group with the formation of a methoxyimine derivative.

However, the stepwise yields of intermediates are low.

In 1970, Salse, Hazen and Schoenewaldt reported that heating 17α-acyloxy derivatives of 20-oxosteroids at a temperature of 100-105 ° C in potassium acetate-containing dimethylformamide for 7 hours leads to elimination of the acyloxy function with the formation of 16-dehydro-20-oxosteroids [J. Org. Chem., 1970, 35, p 1681; US 3631076, 1971]. An important condition for the progress of this reaction is the presence of an ester group at the C21 atom, which was established as a result of a study of the influence of the nature of the substituent at C21 on the elimination of the 17α-acyloxy group in 17α-acyloxy-20-oxosteroids by Solo and Suto in 1980 [J. Org. Chem., 1980, 45, 2012].

In this case, the product yield in the presence of 21-acetoxy group did not exceed 62%. In the presence of the 21-hydroxy group, an acyl migration of the acetoxy group from position C17 to position C21 occurred, and in the case of 21-chloro-substituted, the elimination reaction did not proceed at all.

The esterification of the spatially hindered tertiary 17α-hydroxy group is carried out by acylation of mainly 21-acetates of 20-oxopregnans with a mixture of carboxylic acid and carboxylic anhydride in the presence of acid catalysts at a temperature of 18-35ºС. As catalysts, p-TSC, 55-70% perchloric acid, sulfuric acid, etc. can be used [SU668288, 1994; GB1375770, 1974]. The main adverse reaction of 17-acylation of Δ4-3-keto compounds is enolation at the C3 atom, which proceeds under the same conditions of acid catalysis. C3-Acylate can then be hydrolyzed to regenerate the Δ4-3 ketostructure. However, in the case of corticoids - compounds with a dioxia-acetone side chain at C17 - a new problem arises in the hydrolysis of 3-acylate: hydrolysis of the 21-acetoxy group, acyl migration of the ester group from position 17 to the C21 atom, and its further hydrolysis with the formation of 17α, 21-dihydroxy compounds.

In addition, the 17-acetylation reaction can be carried out under basic catalysis [US3678082, 1972] with dimethylaminopyridine in the Ac2O / triethylamine system. However, there are no examples of the use of this method for 17-acetylation of corticoids in the patent.

The esterification of sterically hindered hydroxyl groups without affecting the Δ4-3-keto group is possible by carrying out the reaction in Ac2O medium in the presence of CaO at boiling point [JP53009755, 1978]. In addition to CaO, the authors used Al2O3, ZnO, as well as acetates of magnesium, calcium, manganese, zinc or barium. However, the preparation of 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione by this method is absent in the formula and description.

The patent [DE2055221, 1972] describes the acylation of 17α-hydroxypregnans in high yield using anhydride or acyl chloride in a mixture with anhydrous SnCl4 in nitromethane. Carboxylic acid halides or anhydrides can also be used in the presence of a phosphate catalyst (for example, H3PO4 or its mono- or dialkyl ethers), for example Ac2O / H3PO4. However, the description of the preparation of 17α, 21-diacetoxypregna-4,9 (11) -diene-3,20-dione is absent in this patent.

A known method of acetylation of the 17α-hydroxy group of the molecule 21-acetoxy-Δ9 (11) -GPG [US4154748, 1979, example 1], according to which the formation of 17α, 21-acetoxypren-4,9 (11) -diene-3,20-dione carried out by the action of acetic anhydride under conditions of catalysis with phosphoric acid derivatives. It was noted that in the catalysis of 85% phosphoric acid (Ac2O / AcOH), the product was obtained with a yield of 97.5% and trace impurities of the starting compound and 3-enol acetate.

The closest in essence the method of acetylation used in the present invention is the method [GB1097164, 1967]. The acylation of the 17α-hydroxy group in the presence of the 21-acetoxy group is carried out by the mixed anhydride method, which consists in treating the initial steroid with a mixture of trifluoroacetic anhydride and acid, the preparation of the ester of which is the aim of the reaction, at a temperature of 80 ° C for 1 h [GB1097164, 1967]. However, the preparation of 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione by this method is absent in the formula and description. Moreover, the use of the mixed anhydride method for acetylation of the 17α-hydroxy group in 21-acetoxy-17α-hydroxy-pregna-4,9 (11) -diene-3,20-dione was not found in available literature.

A known method [US3631076, 1971], according to which 17,21-diacetate can be converted to the Δ16-derivative by reaction with potassium acetate at a temperature of 100 ° C for 7 hours in a medium of dimethylformamide. However, an example of the preparation of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione is not described in this patent and product yields are not indicated.

A more effective method for eliminating the 17α-acetoxy group is described in the patent [SU 819119, 1981], which consists in treating the initial steroid with potassium acetate in dimethyl sulfoxide and the process is carried out at a temperature of 80-85 ° C. However, the preparation of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione by this method is absent in the formula and description.

The introduction of a 1,2-double bond microbiological method

Microbiological methods for carrying out the process of 1,2-dehydrogenation of steroids, including steroids of the pregnane series, are known.

As biocatalysts of the 1,2-dehydrogenation process, cultures with 3-ketosteroid-Δ1-dehydrogenase (3-KSD) (EC 1.3.99.4) genera are used: Arthrobacter, Alternaria, Alcaligenes, Calonectria, Ophiobolus, Corynebacterium, Bacillus, Nocardia, Streomy , Bacterium, Mycobacterium, Fusarium, Cylindrocarpon, Pseudomonas, Protaminobacter, Septomyxa, Didymella, Rhodococcus et al. [Charney W., Herzog H. Microbial transformation of Steroids. Academic Press. Inc. New York 1967, p. 236-261; J. Basic. Microbiol. 1990, 30 (6), 415-423; J. Biochem., 1999, 126, 662-667]. Most often, strains of the species Arthrobacter simplex are used to carry out the 1,2-dehydrogenation process of steroid compounds of a number of pregnan and androstane.

A feature of the 1,2-dehydrogenation process of acetylated derivatives of 21-hydroxy-Δ4-3-ketosteroids by microorganisms is the support of the 1,2-dehydrogenation reaction by deacetylation (hydrolysis of the ester group) [Steroids, 2003, 68 (5), 415-421]. This is a significant drawback of the known methods, since it becomes necessary to carry out the acetylation reaction of the hydroxyl group at C21.

The closest in essence to the proposed method in terms of the introduction of 1,2-double bonds is the method described in the patent [US4524134, 1985, example 4]. The authors use the growing Arthrobacter simplex culture for 1,2-dehydrogenation of 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione with a steroid load of 8 g / l in phosphate buffer medium (pH 7.5) in the presence of menadione, and the steroid substrate is introduced into the medium for transformation in a solution of dimethylformamide, the concentration of which in the medium is 2 vol.%. However, a 1,2-dehydroproduct was obtained only with a yield of 18.1%, while the content of the basic substance was only 59.1%. The product also contains 40.9% of the non-transformed substrate and a significant amount of other undesirable steroid compounds. The authors note that these disadvantages are associated with the use of a growing culture and can be avoided if dried cells are used under the same transformation conditions. A method for producing 1,2-dehydro-Δ4-3-ketosteroids, comprising treating the corresponding 1-saturated Δ4-3-ketosteroid with dried Arthrobacter simplex cells containing 1 to 10% moisture, the cells being dried in the absence of any organic solvent, is described in the same example [US 4524134, 1985, example 4]. Moreover, the 1,2-dehydro-Δ4-3-ketosteroid or 21-monoester is selected from the group including 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (claim 2 of the formula ) According to this method, the process of 1,2-dehydrogenation of 21-acetoxypregna-4.9 (11), 16-triene-3.20-dione is carried out in phosphate buffer (pH 7.5) with dried cells (10 g / l) with a steroid load 8 g / l in the presence of menadione, and the steroid substrate is introduced into the medium for transformation in a solution of dimethylformamide, the concentration of which in the medium is 2 vol.%. The incubation duration is 24 hours at a temperature of 31 ° C. However, as noted in Example 3, as a result of the bioconversion of 21-acetylated steroids, mixtures of 21-acetoxy-Δ1,4-compounds and their 21-hydroxy analogs are obtained. So, the yield of a mixture of products is 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (21-tetraene acetate) and 21-hydroxypregna-1,4,9 (11), 16- tetraen-3,20-dione (tetraene) —with the use of dried cells, it was 79.6%, the content of the main substance having a Δ1,4 structure, 97.8%, the content of the residual substrate was 2.1%.

Thus, the main disadvantage of this method is the need for preliminary drying of the cells to a moisture content of 1 to 10% in the absence of an organic solvent. As described in example 2 of the patent [US 4524134, 1985], the culture cells after incubation are subjected to centrifugation and then dried in vacuum at 55 ° C for 24 hours. After that, the dried cell material is placed in an airtight container and stored at 50 ° C. use in bioconversion. The process of drying cells is complex and energy intensive. In addition, despite the high yield of the transformation product using dried cells (1.25 g of cells per 1 g of steroid substrate), it contains a mixture of tetraene 21-acetate and its 21-deacylated analogue. Thus, an analysis of the materials presented in the patent indicates that the 1,2-dehydrogenation process of the 21-acetylated derivatives of Δ4-3-keto-steroids of the series was disrupted, both using dried cells and a growing culture, is not selective. 1,2-Dehydrogenation is accompanied by deacetylation. In the analyzed information material there is no data on the quantitative yield of 1,2-dehydrogenated acetylated target product. The degree of hydrolysis and the ratio of dehydrated 21-acetylated and dehydrogenated 21-deacetylated products are not indicated, which are significant disadvantages of this method.

Closest to the claimed method in terms of the introduction of a 1,2-double bond according to the technical result is a method for producing a 1,2-dehydro-3-ketosteroid, which consists in the fact that the 1,2-saturated 3-ketosteroid is reacted with A. simplex or Bacterium cyclooxydans in the presence of an exogenous electron carrier and a water-immiscible solvent, including aromatic hydrocarbons [GB2131811, 1984]. According to this method (example 1), the transformation process of 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione with a load of 8 g / l is carried out by A. simplex cells in the presence of 1% weight. menadione in a phosphate buffered saline (pH 7.5) containing 10 vol.% toluene, for 25 hours at a temperature of 28 ± 1 ° C. The toluene phase contains 94.3% of tetraene 21-acetate, 4.2% of tetraene and 1.5% of the original 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione. However, the description does not contain information on the result of the isolation of the product and, accordingly, on its yield and quality.

Also known is a method of converting 1,2-saturated 3-ketosteroids to 1,2-dehydro-3-ketosteroids, which consists in the fact that 1,2-saturated 3-ketosteroids are reacted with an agent having a steroid-1,2-dehydrogenase activity of A. simplex or B. cyclooxydans, in the presence of an exogenous electron carrier and one or more additional toxic oxygen scavengers selected from the group consisting of catalase, superoxide dismutase or platinum [US4749649, 1988]. Moreover, the 1,2-saturated 3-ketosteroid is a 1,2-saturated 3-ketopregnan (claim 9) selected from the group including 21-acetoxypregna-4,9 (11), 16-trien-3,20 Dion. According to this method, the process is carried out in a buffer water system having a molarity from 0.01 M to 2.0 M and containing a water-immiscible solvent (toluene, xylene or benzene) and a preparation having steroid-1-dehydrogenase activity, which may be cultural liquid, moist paste of cells with a moisture content of 60% to 85% or dry cell mass with a moisture content of 1 to 30%. The method under consideration [US4749649, 1988] also proposes the use of an exogenous enzyme, catalase. However, the method is illustrated by a single example of transformation of a compound of the pregnane series — hydrocortisone (Example 3) —in a phosphate buffer medium (50 mM) using dry cells and a substrate load of 0.5 g / L. Moreover, after 23 h of incubation in the presence of catalase, the conversion of hydrocortisone to prednisolone is 69%, and without catalase, it is 27% lower. The cost of technology, due to the need to add an absorber (a catalase enzyme, or superoxide dismutase, or platinum) to the medium, eliminating the inhibitory effect of singlet oxygen on dehydrogenase activity, determines the main disadvantage of this method, which limits the application of this method on an industrial scale. In addition, despite the mention of the possibility of 1,2-dehydrogenation of 21-acetoxy-4,9 (11), 16-triene-3,20-dione in a known manner, there is no description of the preparation of 21-tetraene 21-acetate.

Thus, the common disadvantages of the methods for producing tetraene 21-acetate by microbiological 1,2-dehydrogenation of its Δ4-3-ketoprecursor in a two-phase medium using water-immiscible organic solvents are:

• low concentration of substrate (up to 8 g / l), long duration of the transformation process (at least 24 hours) and lack of information on the method of isolation, as well as on the yield and quality of the isolated product;

• the use of large volumes of flammable organic solvents at the transformation stage, which requires special fermentation equipment and compliance with special fire safety rules; the requirement for the use of special technological equipment virtually eliminates the practical implementation of these methods in industrial production;

• the complexity of the process under the necessary control of the composition of the gas mixture supplied to the reaction medium: the need to create and strictly maintain the minimum permissible concentration of dissolved oxygen in the medium in order to avoid an outburst of vapor of the gas-air mixture and at the same time provide optimal conditions for the oxidation of the substrate;

Another close in essence to the proposed method in terms of the introduction of a 1,2-double bond is a method involving the transformation of 21-acetoxypren-4,9 (11), 16-trien-3,20-dione [J. Steroid Biochem. Mol. Biol., 2003, 87 (4-5), 434-439]. Bioconversion is carried out without the addition of methyl-β-cyclodextrin (MCD) in a medium of 0.01 M sodium phosphate buffer solution (pH 7.4-8.1) at a temperature of 30 ° C. The initial substrate (load 1.0 g / l) is added as micronized powder. During the conversion of the substrate by native cells with a biomass content of 330 mg / L, active deacetylation is observed, followed by subsequent 1,2-dehydrogenation. After 20 hours of transformation, an accumulation of 70% of the deacetylated 1,2-dehydrogenated tetraene product takes place, 30% is the content of the deacetylated substrate during the complete conversion of the loaded substrate.

The use of WDC can significantly increase the efficiency of this process [J. Steroid Biochem. Mol. Biol., 2003, 87 (4-5), 434-439]. During substrate conversion (1.0 g / l) by native cells with a biomass content of 330 mg / l and a steroid: MCD molar ratio of 1: 2, in the presence of PMS (0.4 mg / ml), the maximum accumulation of tetraene 21-acetate is observed after 1 h incubation and is 75%. A subsequent decrease in its content to zero correlates with the accumulation of a deacetylated 1,2-dehydrogenated product - tetraene - reaching 90% in 5 hours. The content of the deacetylated substrate is 21-hydroxypregn-4.9 (11), 16-trien-3 , 20-dione - does not exceed 10% (the substrate is completely transformed after 5 hours of incubation). To reduce the contribution of deacetylation in the initial hours of conversion, a low biomass of 33 mg / ml is used; while the conversion of the substrate (1.0 g / l) with a molar ratio of steroid: MCD equal to 1: 2, in the presence of PMS (0.4 mg / ml) is completed in 5.5 hours with a content of 92% of tetraene 21-acetate and less than 5-7% of its deacetylated analogue.

The main disadvantage of this process is the low concentration of the initial substrate (1 g / l).

The introduction of 16α-methyl-17α-hydroxy fragment at ∆16 bonds

To date, there is little information in the literature on the synthesis of 21-acetoxy-17α-hydroxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione (1,4,9-triene) from 21 tetraene acetate. There is literature data on the functionalization of the positions of C16 and C17 in 21-acetoxy compounds with a cross-conjugated system in ring A. Moreover, the introduction of the Δ9 (11) bond is usually carried out last.

Thus, a known method for producing 1,4,9-triene [US3284477, 1966]. However, the synthesis is carried out from 21-acetate of 16α-methylprednisolone by dehydration of the 11β-hydroxyl group by the action of methanesulfonyl chloride in pyridine medium.

1,4,9-Triene is a key intermediate in the synthesis of corticosteroids from 9-OH-AD having a methyl group in the α configuration at the C16 atom and a hydroxyl group at the C17 atom, for example, dexamethasone, icomethasone (9-chloro analog of dexamethasone), flumethasone (6α-fluoro-dexamethasone), parametasone (6α-fluoro-16α-methylprednisolone), mometasone (11,17-dihydroxy-9,21-dichloro-16-methylpregna-1,4-diene-3,20-dione) and etc.

The introduction of 17α-hydroxy and 16α-methyl substituents in -Δ16-20-ketopregnan is carried out, as a rule, by a two-step process, initially carrying out 16α-alkylation. The introduction of the 16α-methyl group to the Δ16-20-keto fragment of the steroid molecule is usually carried out by the 1,4-addition reaction of methylmagnesium halide in the presence of copper compounds.

The process of 16α-alkylation of tetraene 21-acetate is known to produce 21-hydroxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione, followed by 21-acetylation [CA1029659, 1978].

However, from 100 g of tetraene 21-acetate, the authors obtain, using chromatographic purification and recrystallization from ethyl acetate, only 22.7 g of the 16α-methyl derivative.

The 17α-hydroxy group can be introduced into the 16α-methyl-Δ4-3,20-diketo compound by microbiological transformation with a culture of Dactylium dendroides [GB891594, 1962]. However, in literature there is no information about the possibility of microbiological 17-hydroxylation of 16α-methyl-Δ1,4,9 (11) -3,20-diketo compounds.

It is known that 17α-hydroxylation of 20-ketopregnans can be carried out, for example, by direct oxidation according to Barton [GB963287, 1964] or by the method of Gallagher and Krichevsky [US2562030, 1951]. These methods are based on the idea of oxidizing the Δ17 (20) -20-hydroxy derivative obtained by preliminary enolization of the keto group at the C20 atom. In the first case, the oxidation is carried out with oxygen in the presence of potassium tert-butylate, in the second - by the action of peracid. However, the enolacetylation of the 20-keto group in 16α-methylsteroids is difficult. The reaction proceeds slowly and with a low yield.

Known methods for producing 1,4,9-triene from tetraene 21-acetate using the modified Gallagher and Krichevsky method, which consists in processing 16α-methyl-Δ17 (20) -20-enolate with an acylating agent [EP0165037, 1985, examples 1 and 2; US4929395, 1990, examples 1 and 2]. However, the yield of 1,4,9-triene is not indicated in both patents, and it is impossible to evaluate the effectiveness of the method.

A known method for producing 1,4,9-triene from tetraene 21-acetate [US4530795, 1985, example 2], including the formation of enolsulfinate, capable of undergoing sulfenate-sulfoxide rearrangement. The latter occurs spontaneously at room temperature with the formation of sulfenate ether, the decomposition of which with a slight excess of thiophil (methylxanthate or sodium thiocyanate) leads to 17α-hydroxy-16α-methyl compound (in particular, to 1,4,9-triene).

This method eliminates the difficulties in the enolacetylation of 16α-methyl-20-ketopregnans that arise during 17α-hydroxylation by the Gallagher and Krichevsky method, and, as a result, leads to an increase in the yield of 17α-hydroxy-16α-methyl compounds (from 3.66 g of 21-acetate tetraene receive 2.35 g of 1,4,9-triene, which corresponds to a molar yield of 59%). However, this method has significant disadvantages: multi-stage, the use of expensive and dangerous reagents, purification of reaction products using column chromatography.

Known methods for introducing 17α-hydroxy-16α-methyl fragment into Δ16-20-ketosteroids by reduction of 17α-hydroperoxy products obtained by molecular oxidation of molecular oxygen by the intermediate 16α-methyl-Δ17 (20) -20-magnesium halides [Lieb. Ann. Chem. 1971, B. 752 (1). S. 78-85; DE1921396, 1970; GB2001990, 1979]. It is known that intermediate 16α-methyl-Δ17 (20) -magnesium enolates are very reactive and easily oxidized by atmospheric oxygen to form 17α-hydroperoxy-20-oxo compounds. So, Kerb and Wichert [Lieb. Ann. Chem. 1971, B. 752 (1). S. 78-85; DE1921396, 1970] showed that the 17α-hydroperoxy group can be easily introduced simultaneously with the 16α-methyl group in the 18-methyl-Δ16-20-ketosteroids of the Δ5-series or estran series. The yield of 17α-hydroperoxy-16α-methyl compound was 40 to 65%.

Восстановление 17α-гидропероксигруппы в 17α-гидроксильную может быть проведено известными методами, например, действием цинковой пыли в среде уксусной кислоты [DE1921396, 1970; DE2407967,1974; GB1377975, 1974; GB2001990, 1979] или каталитическим гидрированием над палладием на угле [DE1921396, 1970; US2562030, 1951; GB898093, 1962; GB2001990, 1979]. 17α-Гидропероксигруппа может быть восстановлена в 17α-гидроксильную триметилфосфитом [GB1196984, 1970] или триэтилфосфитом [Comptes Rendus, 1964, 258(23), 5669-5671; Tetrahedron, 1971, 27(10), 1909-1915]. В связи с неустойчивостью 17α-гидропероксисоединений обычно проводят их восстановление в 17α-гидроксианалоги без выделения из реакционной массы.

The reduction of the 17α-hydroperoxy group to the 17α-hydroxyl group can be carried out by known methods, for example, by the action of zinc dust in an acetic acid medium [DE1921396, 1970; DE2407967.1974; GB1377975, 1974; GB2001990, 1979] or by catalytic hydrogenation over palladium on carbon [DE1921396, 1970; US2562030, 1951; GB898093, 1962; GB2001990, 1979]. The 17α-hydroperoxy group can be reduced to 17α-hydroxyl trimethylphosphite [GB1196984, 1970] or triethylphosphite [Comptes Rendus, 1964, 258 (23), 5669-5671; Tetrahedron, 1971, 27 (10), 1909-1915]. Due to the instability of 17α-hydroperoxy compounds, they are usually reduced to 17α-hydroxy analogs without isolation from the reaction mixture.

A known method of producing derivatives of 16α-methylprednisolone, including dexamethasone [GB2001990, 1979], using the method of autooxidation of 16α-methyl-Δ17 (20) -enolate and reduction of 17α-hydroperoxy to 17α-hydroxy group. However, the yield of dexamethasone obtained by these authors does not exceed 26% by weight, which indirectly indicates a low content of 17α-hydroperoxy compound in the technical product. In addition, the description of obtaining 1,4,9-triene from 21-tetraene acetate in a known manner is missing.

The closest in essence to the claimed method in terms of introducing a 16α-methyl-17α-hydroxy fragment through Δ16 bonds are methods for producing 16α-methylated steroids [RU2125575, 1999] and [RU2127278, 1999], where the function of the ketone in position 3 can be protected in the form of ketal, thioketal, hemithioketal or enol ether. The preparation of 1,4,9-triene from tetraene 21-acetate is described in Example 3 of the patent [RU2125575, 1999]. This method includes the methylation of a 16-unsaturated steroid in the presence of a copper catalyst, hydrolysis of the obtained metal enolate to form the corresponding 16α-methyl-Δ17 (20) -enol, its oxidation with molecular oxygen in the presence of a reducing agent and an agent for the formation of an inorganic copper element and obtaining the target compound. Cu (OAc) 2 monohydrate (2.73% w / 5 mol%) is used as a catalyst, the 1,4-addition reaction is carried out in tetrahydrofuran medium by the action of a 3 M solution of methyl magnesium chloride in ether at a temperature of minus 20 ° С for 1 h. At the end of the alkylation, a 13.6% aqueous solution of potassium hydrogen phosphate is added to the reaction mixture at a temperature of 0 ° C, and then dimethyl sulfide in the presence of air is added at a temperature of 15 ° C. Air was bubbled into the mixture for 18 hours, while condensing the distilled dimethyl sulfide.

The reaction mass is extracted with ethyl acetate. The product is purified by silica gel chromatography. However, the yield of 1,4,9-triene is extremely low: from 0.733 g of tetraene 21-acetate, the authors obtained only 0.16 g of 1,4,9-triene (which corresponds to a molar yield of 20%).

The same company claimed a similar method for producing 1,4,9-triene from tetraene 21-acetate [RU2127278, 1999, example 2], in which the hydrolysis of metal enolate and autooxidation is carried out with a mixture of phosphoric acid and sodium hydroxide in the presence of hydrogen peroxide, and instead dimethyl sulfide as a reducing agent use a suspension of peracid in THF, prepared from phthalic acid and hydrogen peroxide. Moreover, from 0.733 g of tetraene 21-acetate, the authors obtain 0.4 g of 1,4,9-triene (which corresponds to a molar yield of 50%). In addition to the low yield of the target compound, a significant disadvantage of this method is the use of dangerous acids in industrial production.

Thus, a common drawback of the known methods for introducing the 16α-methyl-17α-hydroxy fragment at the Δ16 bond is the low yield of the target product.

Introduction of the 9α-fluoro-11β-hydroxy fragment through the Δ9 (11) bond

The introduction of the 9α-fluoro and 11β-hydroxy moieties into Δ9 (11) steroids is usually carried out by a three-step process, initially carrying out hypohalogenation (e.g., hypobromation) of the Δ9 (11) bond, dehydrohalogenation with the formation of 9.11β-epoxide, and then the opening of the epoxide with hydrofluoric acid.

In the hypobromination reaction, dibromantin is used as a reagent. In addition to dibromantin, N-bromosuccinimide or N-bromoacetamide can be used as a donor of hypobromic acid [US3211758, 1965].

The closure in 9,11β-epoxide can be carried out by treating the 9α-bromo derivative with potassium acetate in acetone [US3211758, 1965].

By reacting with HF or with a reagent containing HF, 9,11β-Epoxide is converted to an 9α-fluoro-11β-hydroxy compound under anhydrous or aqueous conditions. A complex of urea and hydrogen fluoride [US3211758, 1965], a solution of anhydrous hydrogen fluoride in tetrahydrofuran [US3700702, 1972, US3780177, 1973] can be used as an HF-containing reagent. In addition, the synthesis of fluorohydrin can be carried out directly from the Δ9 (11) compound, bypassing the stages of hypobromation and epoxidation. However, in this case, the formation of a significant amount of side compounds occurs, which complicates the purification of dexamethasone.

SUMMARY OF THE INVENTION

The aim of the present invention is to simplify and reduce the cost of technology for producing dexamethasone from phytosterol.

The essence of the invention is the optimal combination of biotechnological and chemical stages of the synthesis of 11beta, 17alpha, 21-trihydroxy-16alpha-methyl-9alpha-fluoropregna-1,4-diene-3,20-dione (dexamethasone) from phytosterol. The synthesis of dexamethasone from phytosterol is a sequence of transformations that includes the following stages of functionalization:

- microbiological elimination of the side chain of phytosterol with the formation of 9-OH-AD by the culture of bacterial cells of Mycobacterium sp. VKM Ac-1817D;

- dehydration of the 9α-hydroxy group with the formation of the Δ9 (11) bond;

- the introduction of 17α-hydroxy-17β-pregnan chain;

- the introduction of a hydroxyl group at the 21-position of 17α-hydroxy-C20-ketopregnan with the formation of a corticosteroid (dioxiacetone) chain;

- acetylation of the 17α-hydroxy group;

- cleavage of the 17α-acetoxy group with the formation of Δ16-3,20-diketopregnan;

- microbiological 1,2-dehydrogenation using cells of the microorganism Nocardioides simplex VKM Ac-2033D;

- the introduction of a 16α-methyl-17α-hydroxy fragment at the ∆16 bond;

- the introduction of a 9α-fluoro-11β-hydroxy fragment through the Δ9 (11) bond.

The technical result of the present invention is to increase the overall yield of the target product, which is ensured by the claimed sequence of biotechnological and chemical reactions and a combination of essential features, which are:

Conducting dehydration of 9-OH-HELL obtained from phytosterol by microbiological transformation, the action of a mineral acid in an aprotic organic solvent medium previously isolated in the form of a crystalline powder or in an extract medium of a culture fluid from which bacterial cells of a transform strain were previously removed; while for the extraction of 9-OH-AD use organic solvents, namely aromatic hydrocarbons or organochlorine hydrocarbons.

Use as a dehydrating agent of mineral acids, including those containing water, more specifically, the use of phosphoric acid, or sulfuric acid, or perchloric acid; removal of water from the reaction mass during the reaction, for example, by azeotropic distillation.

Construction of a corticosteroid (dioxiacetone) chain by phasing in stereoselective hydrocyanation reactions, protecting a ketone at the C3 atom by forming a cyclic ketal, protecting a 17α-hydroxy group by forming a 17α- (1-alkoxy) ethyl ester, alkylating a nitrile group, introducing a hydroxyl group into the 21-position of 17α-hydroxy -C20-ketopregnan using the direct C21-iodination method.

Acetylation of the 17α-hydroxy group by the mixed anhydride method and the elimination of the 17α-acetoxy group to form Δ16-3,20-diketopregnan.

The introduction of a 1,2-double bond by the microbiological method in an aqueous medium using cells of the microorganism Nocardioides simplex VKM Ac-2033D, using a polar solvent miscible with water, in a concentration of up to 8% vol., In a buffer solution with a pH from 5.0 up to 5.5 at a temperature of 25 ° C for 6 to 14 hours

The introduction of the 16α-methyl-17α-hydroxy fragment via the Δ16 bond by the copper-catalyzed 1,4-addition of Grignard reagent followed by the autooxidation of the 16α-methyl-Δ17 (20) -enolate and the reduction of the 17α-hydroperoxy group to 17α-hydroxyl.

The introduction of the 9α-fluoro-11β-hydroxy fragment by the Δ9 (11) linkage by a sequence including hypobromation, dehydrobromination and hydrofluorination of the formed 9β, 11-epoxide.

The overall yield of pharmacopoeial quality dexamethasone from phytosterol reaches 23%.

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 problem of obtaining dexamethasone from phytosterol is solved by the method of obtaining 11β, 17α, 21-trihydroxy-16α-methyl-9α-fluorpregna-1,4-diene-3,20-dione (dexamethasone) of the formula (I)

from phytosterol of the general formula (II)

where R = C2H5 is β-sitosterol; R = CH3 is campesterol; R = C2H5 and Δ22 (23) - stigmasterol; R = CH3 and Δ22 (23) - brassicasterin,

including microbiological oxidative elimination of the side chain of phytosterol at the C17 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, treatment of 9α-hydroxyandrost-4-en-3,17-dione with a dehydrating agent in an aprotic solvent or in an extract of the culture liquid with the formation of androsta-4,9 (11) -diene-3,17-dione of the formula (IV)

17-hydrocyanation of androsta-4,9 (11) -diene-3,17-dione by interaction with a cyanating agent in a proton solvent under basic catalysis with the formation of 17α-hydroxy-17β-cyanandrosta-4,9 (11) -diene- 3-one formulas (V)

protection of the 3-keto group of 17α-hydroxy-17β-cyanandrost-4,9 (11) -diene-3-one by ketalization to form a cyclic ketal of the general formula (VI)

protection of the 17α-hydroxy group by the action of alkoxyethylene with the formation of a 17α-esterified cyclic ketal derivative of the general formula (VII)

where R '= CH (CH3) OC2H5 or CH (CH3) OC4H9;

alkylation of the carbonitrile group of the 17α-esterified cyclic ketal derivative of the general formula (VII) with an alkylating agent in an aprotic solvent and removal (deprotection) of the protective groups to form 17α-hydroxypregn-4,9 (11) -diene-3,20-dione of the formula (VIII )

C21-iodination of 17α-hydroxypregn-4,9 (11) -diene-3,20-dione with the formation of 17α-hydroxy-21-iodpregn-4,9 (11) -diene-3,20-dione of the formula (IX)

C21-acetoxylation of 17α-hydroxy-21-iodine-4,9 (11) -diene-3,20-dione by replacing the iodine atom on the acetoxy group by the action of alkali metal acetate with the formation of 21-acetoxy-17α-hydroxy-pregna-4.9 (11) -diene-3,20-dione of the formula (X)

the esterification of the 17α-hydroxy group of 21-acetoxy-17α-hydroxypregn-4,9 (11) -diene-3,20-dione with an acetylating agent to form 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione formulas (XI)

elimination of the 17α-acetoxy group of 17α, 21-diacetoxypregna-4,9 (11) -diene-3,20-dione with the formation of 21-acetoxypregna-4,9 (11), 16-triene-3,20-dione of the formula (XII)

microbiological 1,2-dehydrogenation of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione with the formation of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione formulas (XIII)

the catalytic 1,4-addition reaction of methylmagnesium halide according to the Δ16-20 keto system of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione in an aprotic solvent (or mixture of solvents) with the formation of a 21-magnesium halide derivative acetoxy-20-hydroxy-16α-methylpregna-1,4,9 (11), 17 (20) -tetraen-3-one of the formula (XIV)

autooxidation of the magnesium halide derivative 21-acetoxy-20-hydroxy-16α-methylpregna-1,4,9 (11), 17 (20) -tetraen-3-one with the formation of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4 9 (11) -trien-3,20-dione of the formula (XV)

the reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione to the 17α-hydroxyl group with a reducing agent to form 21-acetoxy-17α-hydroxy-16α methylpregna-1,4,9 (11) -trien-3,20-dione of the formula (XVI)

the reaction of 9,11-bromohydroxylation of 21-acetoxy-17α-hydroxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione by the action of hypobromic acid with the formation of 21-acetoxy-9α-bromo-11β, 17 -dihydroxy-16α-methylpregn-1,4-diene-3,20-dione of the formula (XVII)

9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregn-1,4-diene-3,20-dione and simultaneous removal of the ester protecting group at C21 to form 17α, 21-dihydroxy -16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione of the formula (XVIII)

and the reaction of opening the 9β (11) -epoxide ring of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione with hydrofluoric acid.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining 11β, 17α, 21-trihydroxy-16α-methyl-9α-fluorpregna-1,4-diene-3,20-dione (dexamethasone) of formula (I) from phytosterol of formula (II) is carried out according to the scheme shown in Figure 1.

Figure 1 - Synthesis of dexamethasone (I) from phytosterol (II) by the claimed method, where: R = C2H5 - β-sitosterol; R = CH3 is campesterol; R = C2H5 and Δ22 (23) - stigmasterol, R = CH3 and Δ22 (23) - brassicasterin; R '= CH (CH3) OS2H5 or CH (CH3) OS4H9

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

Phytosterol of the general formula (II),

where R = C2H5 is β-sitosterol; R = CH3 is campesterol; R = C2H5 and Δ22 (23) - stigmasterol, R = CH3 and Δ22 (23) - brassicasterin,

subjected to microbiological oxidative elimination of the side chain at the C17 atom with the simultaneous formation of the Δ4-3-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 into 9-OH-HELL (III) up to 72%, after which the cells of the transforming strain are separated. 9-OH-AD (III) is extracted from the obtained clarified culture fluid, practically free of phytosterol, with an organic solvent (aromatic hydrocarbon or chlorine-containing hydrocarbon). The obtained 9-OH-AD (III) with isolation in the form of a crystalline powder or without isolation from the extract is reacted with a dehydrating agent to form Δ9 (11) -AD (IV). For this, mineral acid is added to the extract or to a solution of crystalline 9-OH-AD (III) in an aprotic organic solvent. At the same time, 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 selectivity of the formation of 9-OH-AD (III) at the stage of microbiological oxidation of phytosterol (II) is 68-72%. The selectivity of Δ9 (11) -AD (IV) formation at the dehydration stage is 98-100%. The degree of extraction of crystalline Δ9 (11) -AD from the crud is 96-98%. The total yield of Δ9 (11) -AD from phytosterol reaches 71%. Next, Δ9 (11) -AD is reacted with acetone cyanohydrin in the presence of sodium hydroxide in aqueous methanol, whereby Δ9 (11) -AD (V) cyanohydrin is obtained in a yield of more than 94%. After that, the 3-keto group of cyanohydrin Δ9 (11) -AD (V) is protected by the formation of a cyclic ketal (five-membered or six-membered) by reaction with ethylene glycol or neopentyl glycol in the presence of an acid catalyst and a water binding agent, for example triethylorthoformate. Then, the 17α-hydroxy group is protected by the formation of 17α- (1-alkoxy) ethyl ether, after which the nitrile group of compound VII is subjected to alkylation by the action of methyl lithium or methyl magnesium halide. Acid hydrolysis of the protective groups gives Δ9 (11) -GPG (VIII) with a yield of 89 to 92.4%, depending on the nature of the protective groups. The obtained Δ9 (11) -GPG (VIII) is converted to 21-acetoxy-Δ9 (11) -GPG (X) by direct C21-iodination followed by substitution of the iodine atom for the acetoxy group by the action of potassium acetate, and the substitution reaction can be carried out without isolation 21 iodine-Δ9 (11) -HPG (IX). The 17α-hydroxy group of 21-acetoxy-Δ9 (11) -HPG (X) is acetylated by the mixed anhydrides method in methylene chloride using an acetylating mixture consisting of acetic acid, trifluoroacetic anhydride and a catalytic amount of p-TSC containing the minimum amount of trifluoroacetic anhydride which allows to completely eliminate the formation of an undesirable product of enolacetylation of the carbonyl group at the C3 atom. Compound XI obtained in 92.54% yield is then treated with dimethyl sulfoxide in potassium acetate to give 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione (XII), which is subjected to microbiological 1,2-dehydrogenation with the participation of N. simplex VKM Ac-2033D cells with 3-ketosteroid-∆1-dehydrogenase activity, the load of the initial substrate being from 10 to 20 g / l, and the duration of fermentation is from 6 to 14 hours and the process is carried out at a temperature 25 ° C in a buffer solution at pH ~ 5, containing up to 8 vol.% Polar solvent, mixing water, (preferably dimethyl sulfoxide or ethanol), methyl-β-cyclodextrin in an amount of not more than 2 moles per 1 mol of the initial substrate and an exogenous electron acceptor - menadione - in an effective amount. Isolation of tetraene (XIII) 21-acetate from the culture liquid is carried out by extraction with a water-immiscible solvent or extraction of the steroid from biomass paste with any suitable solvent after preliminary separation from the aqueous phase. The selectivity of the formation of tetraene (XIII) 21-acetate from 21-acetoxypregn-4.9 (11), 16-trien-3.20-dione (XII) is 92-95%. The yield of crystalline tetraene (XIII) 21-acetate after isolation is 83.5-84%. After that, a catalytic 1,4-addition reaction of methylmagnesium bromide is carried out on the Δ16-20-ketosystem of tetraene 21-acetate (XIII) in an aprotic solvent (or solvent mixture) to form a magnesium halide derivative 21-acetoxy-20-hydroxy-16α-methylpregna-1, 4.9 (11), 17 (20) -tetraen-3-one (XIV), which is hydrolyzed by the action of an aqueous solution of ammonium chloride in the presence of atmospheric air. The 17α-hydroperoxy group of the 21-acetoxy-17α-hydroperoxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione (XV) obtained by spontaneous oxidation is reduced to 17α-hydroxyl to form 1,4,9 -triene (XVI), which is carried out by the action of potassium iodide in a medium of a mixture of aromatic hydrocarbon and acetone, and this sequence can be carried out without isolation of 21-acetoxy-17α-hydroperoxy-16α-methylpregn-1,4,9 (11) -triene- 3,20-dione (XV). Then 1,4,9-triene (XVI) is subjected to bromo-hydroxylation by the action of hypobromic acid in an aprotic polar solvent (for example, tetrahydrofuran or dialkyl ketone) containing an effective amount of water to form hypobromic acid in situ, using dibromantin as a source of hypobromic acid (1,3-dibromo-5,5-dimethylhydantoin). Subsequent 9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione (XVII) and removal of the ester protecting group at C21 is carried out simultaneously in aqueous a medium containing a mixture of an aprotic polar solvent (e.g. tetrahydrofuran or dialkyl ketone) and a proton solvent (e.g., aliphatic alcohol), an alkaline agent (e.g., alkali metal hydroxide or carbonate). In this case, the sequence of reactions of bromohydroxylation of 1,4,9-triene (XVI) and 9β (11) -epoxidation of compound XVII with simultaneous removal of the ester protecting group at C21 can be carried out without isolating compound XVII from the reaction mixture. The opening reaction of the 9β (11) -epoxide ring of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione (XVIII) is carried out in a medium of 70% aqueous hydrofluoric acid, which reagent. Dexamethasone (I) is obtained with a yield of 90.91% and a basic substance content of 91.1% (HPLC).

After purification, pharmacopeia-grade dexamethasone with a basic substance content of 97.8% (HPLC) is obtained. The purification yield is 81.2%. The total yield of dexamethasone from phytosterol is 22.8%.

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

For the transformation of phytosterol, soybean phytosterol with the content of transformable sterols from 90 to 100% can be used. When carrying out the invention, a method for producing 9-OH-AD (III) 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 of producing dexamethasone from phytosterol can be carried out by bacteria cells of the genus Mycobacterium, specifically Mycobacterium sp. VKM Ac-1817D.

Aromatic hydrocarbons (for example, benzene or toluene) or chlorine-containing can be used as an organic solvent in the implementation of the invention method in the extraction operation of 9-OH-AD (III) and for obtaining Δ9 (11) -AD (IV) in a subsequent dehydration reaction hydrocarbons (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 sulfuric acid with a water content of up to 30%, or phosphoric acid, can be used as a dehydrating agent in the dehydration stage of 9-OH-AD (III) with a water content of up to 15%, as well as phosphoric acid with a content of 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 dehydration of crystalline 9-OH-HELL with the formation of Δ9 (11) -AD, as well as the results of dehydration in the extract of clarified culture fluid, under the conditions of the present method and the methods described in the literature.

Hydrocyanation of Δ9 (11) -AD (IV) is carried out by reaction with acetone cyanohydrin as a hydrocyanating agent, and a mineral base (for example, alkali metal hydroxide) is used as the main catalyst. The reaction is carried out in aqueous methanol.

The protection of the 3-keto group of 17α-hydroxy-17β-cyanandrost-4,9 (11) -dien-3-one (V) is carried out by ketalization by reaction with 1,2-diol (e.g. ethylene glycol) to form dioxolane ketal or by reaction with 1, 3 diols (e.g. nonpentyl glycol) to form a hexacyclic (1,3-dioxane) ketal.

The protection of the 17α-hydroxy group of the cyclic ketal of general formula (VI) is carried out by esterification by the action of alkoxyethylene (for example, ethoxyethylene or butoxyethylene).

The alkylation of the carbonitrile group of the 17α-esterified cyclic ketal derivative of the general formula (VII) is carried out using methyl lithium or methyl magnesium halides (e.g. methyl magnesium bromide or methyl magnesium chloride) as an alkylating agent

C21-Iodination of Δ9 (11) -GPG (VIII) is carried out in a medium of a mixture of methanol and dichloromethane by the action of molecular iodine in the presence of calcium oxide and calcium chloride, and to carry out the reaction of subsequent replacement of the iodine atom by the acetoxy group in 21-iodine-Δ9 (11) - GPG (IX) aprotic polar organic solvents (e.g., dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, acetonitrile), as well as dialkyl ketones (e.g. acetone) can be used. In addition, the sequence of processes of C21-iodination Δ9 (11) -HPG (VIII) and C21-acetoxylation of 21-iodo-Δ9 (11) -GPG (IX) can be carried out without isolation of 21-iodine-Δ9 (11) -GPG ( IX) from the reaction solution.

The esterification of the 17α-hydroxy group of 21-acetoxy-Δ9 (11) -HPG (X) is carried out with an acetylating agent, which can be used as an anhydride, or acetic acid chloride, or a mixture of acetic acid and trifluoroacetic anhydride in an effective ratio.

Elimination of the 17α-acetoxy group of 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione (XI) is carried out by the action of an alkali metal acetate in an aprotic organic solvent (for example, dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, etc. )

1,2-Dehydrogenation of 21-acetoxypregna-4.9 (11), 16-triene-3.20-dione of the formula (XII) is carried out microbiologically using cells of the microorganism Nocardioides simplex VKM Ac-2033D having 3-ketosteroid-1- dehydrogenase activity. The main parameters of the microbiological 1,2-dehydrogenation process of 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione are shown in Tables 2 and 3. A substrate with a mass fraction of 21-acetoxypregna-4.9 ( 11), 16-triene-3,20-dione from 80% to 95%. The load of the initial substrate is given in terms of the content of the basic substance.

The catalytic 1,4-addition reaction of methylmagnesium bromide by Δ16-20-ketosystem of tetraene 21-acetate (XIII) is carried out at a temperature of minus 70 to minus 10 ºС, and copper (I) chloride is used as a catalyst.

The autooxidation of the magnesium halide derivative of 21-acetoxy-20-hydroxy-16α-methylpregna-1,4,9 (11), 17 (20) -tetraen-3-one (XIV) is carried out at a temperature of minus 10 to plus 10 ºС with atmospheric molecular oxygen in a heterogeneous medium consisting of an aprotic solvent miscible with water (e.g. tetrahydrofuran, acetonitrile, or a mixture thereof) and a non-miscible aprotic solvent (aromatic, e.g. benzene, toluene) in an effective ratio.

The reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione of the formula (XV) to the 17α-hydroxyl group is carried out by the action of a reducing agent, which is used as alkali metal iodide (sodium or potassium) in an effective amount. The reaction is carried out in a dialkyl ketone (e.g. acetone, methyl ethyl ketone) or in an aromatic hydrocarbon (e.g. benzene, toluene) in the presence of a dialkyl ketone (e.g. acetone) in an effective amount.

In addition, a reaction sequence including a catalytic 1,4-addition of methylmagnesium halide (e.g., methylmagnesium bromide or methylmagnesium chloride) according to the Δ16-20-ketosystem of tetraene 21-acetate (XIII), autooxidation of the magnesium halide derivative 21-acetoxy-20-hydroxy-16α-methylpregn , 4.9 (11), 17 (20) -tetraen-3-one (XIV) and reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -trien-3 , 20-dione (XV) into a 17α-hydroxyl group, can be carried out without isolation of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dio and (XV).

The reaction of bromohydroxylation of 1,4,9-triene (XVI) by the action of hypobromic acid is carried out in an aprotic polar solvent (e.g. tetrahydrofuran or dialkyl ketone) containing an effective amount of water to form hypobromic acid in situ, the source of hypobromic acid being selected from the group containing 1,3-dibromo-5,5-dimethylhydantoin, N-bromosuccinimide, N-bromoacetamide and the like.

9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione (XVII) and removal of the ester protecting group at C21 is carried out simultaneously in an aqueous medium containing a mixture of an aprotic polar solvent (e.g. tetrahydrofuran or dialkyl ketone) and a protonic solvent (e.g., an aliphatic alcohol selected from the group: methyl, ethyl, isopropyl) in an effective amount, by the action of an alkaline agent (e.g., alkali metal hydroxide or carbonate). In addition, the sequence of reactions of bromohydroxylation of 1,4,9-triene (XVI) and 9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17-dihydroxy-16α-methylpregna-1,4-diene-3,20 -dione (XVII) with simultaneous removal of the ester protecting group at C21 can be carried out without isolation of 21-acetoxy-9α-bromo-11β, 17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione (XVII) from the reaction mixture.

Examples

Example 1 Preparation of 9α-hydroxyandrost-4-en-3,17-dione (9-OH-HELL, III)

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; (NH3) 2HPO4 1.5; KH2PO4 0.5; K2HPO4 x 3H2O 0.5; glycerin 10; polyethylene glycol sorbitol monooleate 3.0; MgSO4 x 7H2O 0.2; FeSO4 x 7H2O 0.005; ZnSO4 x 7H2O 0.002; distilled water 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). The microbiological transformation of phytosterol is carried out in a thermostat shaker 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): KH2PO4 0.8 ; K2HPO4 x 3H2O 4.2; (NH3) 2SO4 3.0; glycerol 5.0; urea 0.13; MgSO4 x 7H2O 0.2; FeSO4 x 7H2O 0.01; ZnSO4 x 7H2O 0.002; polyethylene glycol sorbitol monooleate 1.0; phytosterol (calculated on transformable sterols) 4.0; distilled water 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) the allocation of 9-OH-HELL

The solvent was evaporated in vacuo until steroid crystallization began, the concentrate was diluted with n-hexane in a ratio of 1: 1 by volume, stirred, cooled to 4 ° C and incubated for 2-3 hours. The resulting crystals were separated by filtration and dried. Two recrystallizations from ethanol are carried out, the mother liquors are combined, evaporated under vacuum until crystallization begins, the concentrate is cooled to 4 ° C and incubated for 4-6 hours. The crystals formed are separated by filtration, dried and recrystallized from ethanol. All crystals obtained are combined, crushed and dried under vacuum. Get 9-OH-HELL with a degree of extraction of 82%, the content of the main substance ≥ 97%, the impurity content of HELL <0.5% and a melting point of 217-220 ° C (lit. 219-221 ° C [US4237220, 1980, example 2 ]).

Example 2 Preparation of androsta-4,9 (11) -diene-3,17-dione (Δ9 (11) -AD, IV)

The description of some variants of Example 2 for the preparation of Δ9 (11) -AD (IV) is given. The parameters and results of other variants of example 2 are shown in table 1.

Option 1. The extract obtained according to p. B) of example 1, 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 a 5% NaCl solution until neutral and water, then dried with Na2SO4 and evaporated to dryness in vacuo. Get a crude containing Δ9 (11) -AD and not containing 9-OH-AD according to HPLC analysis.

Option 2 (with azeotropic distillation of water). The extract obtained according to item B) of example 1, is placed in a flask equipped with a Dean-Stark nozzle and a 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.

The selectivity of the dehydration reaction is evaluated by HPLC analysis of the initial extract (determination of the content of 9-OH-AD) and crud (content of Δ9 (11) -AD).

After isolation from the crud, Δ9 (11) -AD is obtained with a recovery of 96-98%. The melting point is from 203.5 to 206.5ºC (lit. 201-204.5ºC [US4127596, 1978, example 7].

[α] D20 + 217 to +221 º (c = 1.0, CHCl3) (lit. +220 º (CHCl3) [US4127596, 1978, example 6]).

¹ H NMR Spectrum (δ, ppm; CDCl3): 5.75 (d, J = 1.8 Hz, 1H, 4-CH); 5.55 (m, 1H, 11-CH); 1.35 (s, 3H, 19-CH3); 0.87 (s, 3H, 18-CH3).

¹³ C NMR Spectrum (δ, ppm; CDCl3): 221.0 (17-C (O)); 199.0 (3-C (O)); 168.9 (5-C); 145.1 (9-C); 124.2 (4-CH); 118.1 (11-CH); 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-CH3).

Option 3. To a suspension of 5.0 g of 9-OH-AD (III) in 60 ml of dry 1,2-dichloroethane, 10 ml of 85% phosphoric acid are added with stirring. The reaction mixture is heated to boiling, while complete dissolution of the starting material is observed. The reaction mass is boiled for 30 minutes, then cooled to room temperature and 30 ml of 5% aqueous NaCl solution are added. Stirred for 3-5 minutes. The organic layer was separated, the aqueous layer was extracted with 1,2-dichloroethane. The combined organic extracts are washed with a 5% aqueous NaCl solution until neutral, treated with clarifying charcoal.

The solvent was evaporated at atmospheric pressure to a small volume, after which a 5% aqueous NaCl solution was added and distillation continued. The resulting suspension was cooled to room temperature, the precipitate was filtered off, washed on the filter with water and dried to constant weight. 4.46 g of Δ9 (11) -AD (IV) are obtained in a yield of 95.15%.

T. pl. 198-200ºC.

Option 4. To a solution of 115 mg of 9-OH-AD (III) in 1.5 ml of absolute chloroform, add 0.2 ml of 80% H2SO4. The mixture is kept at boiling for 1.5 hours, then diluted with water and transferred to a separatory funnel. The aqueous layer was separated, the organic layer was washed with water to pH ~ 7. The solvent was evaporated in vacuo to stop the run, the residue was crystallized from ethyl acetate. 97.34 mg of compound IV is obtained in a yield of 90%. Mp 198-202ºС.

Example 3 Preparation of 17α-hydroxy-17β-cyanoandrost-4.9 (11) -dien-3-one (V)

To a suspension of 50 g of Δ9 (11) -AD (IV) in 140 ml of methanol in a stream of nitrogen at room temperature, 45 ml of acetone cyanohydrin and 7 ml of 2.68 N. caustic soda solution in water. The suspension is heated to a temperature of 35 ° C and incubated for 50-60 minutes at the same temperature. Then 30 ml of distilled water are slowly added, the reaction mass is cooled to room temperature and incubated for 19-21 hours. After that, the reaction suspension is slowly diluted with 130 ml of water. The precipitate is filtered off, washed first with a mixture of water and methanol, then with water to pH ~ 7. Obtain 43.84 g of compound V.

Mp 225-229 ° C (lit. 211-220 ° C [DD281394, 1990, example 1]);

[α] D20 +115 º (c = 1.0, CHCl3) (lit. +118 º (c = 1.0, CHCl3) [US4500461, 1985]).

7.81 g of compound V with a melting point of 7.81 g are further recovered from the mother liquor 214-218 ° C. The impurity content of the isomeric 17β-hydroxy-17α-cyanandrost-4.9 (11) -dien-3-one is less than 1%.

A total of 51.65 g is obtained with a yield of 94.2%.

1H NMR spectrum (CDCl3) δ: 5.75 (m, 1H, 4-H), 5.57 (m, 1H, 11-H), 1.35 (s, 3H, 19-CH3), 0.94 (s, 3H, 18-CH3 )

13C NMR spectrum (CDCl3) δ: 199.7 (C-3), 169.8 (C-5), 144.3 (C-9), 124.0 (C-4), 120.7 (CN), 118.2 (C-11), 77.2 ( S-17), 47.8, 45.3, 41.0, 38.5, 38.0, 34.2, 33.8, 32.8, 32.2 31.0, 26.7, 24.7 (19-СН3), 16.1 (18-СН3).

Example 4 Preparation of 3.3- (2,2-dimethylpropylene) -dioxio-17α-hydroxy-17β-cyanoandrosta-5.9 (11) -diene (VIa)

To a suspension of 20 g of 17α-hydroxy-17β-cyanoandrost-4,9 (11) -dien-3-one (V) in 40 ml of dry methylene chloride in a stream of nitrogen, 20 g of 2,2-dimethylpropane-1,3- diol and 20 ml of triethylorthoformate. The reaction mass is cooled to 0-2 ° C, 2 g of anhydrous p-TSC are added and stirred for 7-8 hours. Then 2 ml of triethylamine and 160 ml of water are added. The suspension is stirred for 30 minutes, the precipitate is filtered off, washed with water to pH 7, and dried to constant weight.

Obtain 21.8 g of the first crystallizate of compound VIa.

3.47 g of a second crystallizate of compound VIa are additionally recovered from the mother liquor.

A total of 25.27 g of compound VIa was obtained in 99% yield.

Mp analytical sample 253-255ºС (with decomposition).

1H NMR Spectrum (δ, ppm; CDCl3): 5.51 (m, 1H, 11-CH); 5.40 (m, 1H, 6-CH); 3.50 (m, 4H, 1'-OCH2); 1.18 (s, 3H, 19-CH3); 0.98 (s, 3H, 18-CH3); 0.91 (d, J = 3.4 Hz, 6H, 3'-CH3)

13C NMR spectrum (δ, ppm; CDCl3): 145.7 (9-C); 138.0 (5-C); 121.7 (CN); 120.8 (6-CH); 116.0 (11-C); 98.6 (3-CH); 70.5; 70.0; 48.6; 46.3; 44.0; 39.0; 38.9; 35.1; 33.0; 32.7; 31.3; 30.4; 28.1; 27.6; 25.1; 23.0; 22.9; 16.1 (19-CH3).

Found: C, 75.40%; H, 8.68%; N, 3.51%. C25H35NO3. Calculated C 75.53%, H 8.87%, N 3.52%.

 Example 5 Preparation of 3.3-ethylenedioxy-17α-hydroxy-17β-cyanoanodrost-5.9 (11) -diene (VIb)

To a solution of 0.165 g of anhydrous p-TSC in 9.6 ml of ethylene glycol, 96 ml of dichloromethane, 9.5 g of 17α-hydroxy-17β-cyanoandrost-4.9 (11) -diene-3-one (V) and 4.7 were added ml of trimethylorthoformate. The reaction solution was stirred for 2 hours at room temperature. Then, the solvent was distilled off from the reaction mass at atmospheric pressure to half the initial volume, the residue was cooled to room temperature and 0.25 ml of dry triethylamine was added.

The reaction solution was transferred to a separatory funnel and washed with water. The solvent was evaporated to dryness. 10 ml of methanol was added to the residue, triturated, the resulting suspension was cooled to 0 ° C and held for at least 6 hours at the same temperature. The precipitate is filtered off, washed with 1.6 ml of methanol, dried to constant weight. 10.04 g of compound VIb are obtained in a yield of 92.5%.

Mp 201-203 ° C (lit. 202-205 ° C [Bull.Chem.Soc.Jpn. 1985, 58 (3), 978-980, compound 10a]).

1H NMR Spectrum (δ, ppm; CDCl3): 5.54 (m, 1H, 11-CH); 5.40 (m, 1H, 6-CH); 3.95 (m, 4H, OCH2-OCH2); 1.20 (s, 3H, 19-CH3); 0.91 (s, 3H, 18-CH3).

Example 6 Preparation of 3,3- (2,2-dimethylpropylene) -dioxo-17α- (1-ethoxyethoxy) -17β-cyanoadrost-5.9 (11) -diene (VII)

To a mixture of 1 g of 3,3 (2,2-dimethylpropylene) -dioxio-17α-hydroxy-17β-cyanoandrost-5.9 (11) -diene (VIa) in 10 ml of absolute THF, cooled to 0 ° С, add 1 ml of ethoxyethylene and 100 mg of anhydrous p-TSC. Stirred for 1.5 hours. Upon completion of the reaction, 0.1 ml of triethylamine was added and held for 10 minutes. Then the reaction mass is poured into 100 ml of water. The precipitate formed is filtered off, washed on the filter with water until neutral and dried to constant weight.

Obtain 0.92 g of compound VII with a yield of 78% (as a mixture of two diastereomers in a 1: 1 ratio).

1H NMR Spectrum (δ, ppm; CDCl3): 5.52 (m, 1H, 11-CH); 5.41 (m, 1H, 6-CH); 5.10, 5.00 (q, J = 5.3 Hz, 1 H, O2CH); 3.73-3.36 (m, 6H, 1'-OCH2, OCH2); 1.35, 1.31 (d, J = 5.3 Hz, 3H, CH3CH); 1.22, 1.18 (t, J = 7.1 Hz, 3H, CH3CH2O); 1.19 (s, 3H, 19-CH3); 0.97 (s, 3H, 18-CH3); 0.92 (m, 6H, 3'-CH3).

Example 7 Preparation of 17α-hydroxy-pregna-4,9 (11) -diene-3,20-dione (VIII)

Option 1. To a solution of 90.65 g of 3,3- (2,2-dimethylpropylene) -dioxo-17α-hydroxy-17β-cyanoandrosta-5.9 (11) -diene (VIa) in 907 ml of absolute diethyl ether, cooled to 0-2 ° C, in a stream of argon add 90.7 ml of ethoxyethylene and 9.07 g of anhydrous p-TSC. The reaction mixture is stirred for 4 hours at a temperature of 0-2 ° C.

Then the reaction mass is cooled to a temperature of minus 25 ° C, 9 ml of triethylamine are added and stirred for 5 minutes. Then, 384 ml of a 1.6 M solution of methyl lithium in diethyl ether are added dropwise and cooling is stopped. Warm the reaction mass to room temperature and incubate for 30 minutes. A mixture of 49.4 ml of methanol, 2.7 ml of water and 2.7 ml of concentrated HCl is then added. After 5 min, the reaction mass was diluted with 181 ml of methanol and 181 ml of ~ 20-21% diluted HCl was added. It is stirred for 18 hours. The ether is distilled off at atmospheric pressure, gradually adding 1.36 L of distilled water to the reaction mass. The suspension in water is cooled to a temperature of 0-2 ° C and incubated for 1 hour. The precipitate is filtered off, washed with water until neutral and dried to constant weight. Obtain 79 g of technical product, which is treated with clarifying charcoal in dichloromethane, crystallized from acetone.

Obtain 57.33 g of the first crystallizate of compound VIII with so pl. 207-211 ° C (lit. mp 206-211 ° C [US 4041055, 1977, example 58].

An additional 9.47 g of the second crystallizate of compound VIII with a melting point of 89 ml are additionally recovered from the mother liquor 207-209 ° C.

A total of 66.8 g of compound VIII was obtained in a yield of 89.2%.

1H NMR Spectrum (δ, ppm; CDCl3): 5.73 (d, J = 1.5 Hz, 1H, 4-CH); 5.53 (m, 1H, 11-CH); 2.27 (s, 3H, 21-CH3); 1.33 (s, 3H, 19-CH3); 0.68 (s, 3H, 18-CH3).

Option 2. In the conditions of option 1 from 10 g of 3.3-ethylenedioxy-17α-hydroxy-17β-cyanandrost-5.9 (11) -diene (VI) receive 8.54 g of compound VIII with a yield of 92.4%.

 Mp 206-209 ° C.

Option 3. To the reaction mass obtained under the conditions of option 1 from 10 g of 3.3-ethylenedioxy-17α-hydroxy-17β-cyanandrost-5.9 (11) -diene (VIb) and 11 ml of butoxyethylene in 40 ml of toluene, at a temperature of minus 5 ° C. add 120 ml of a solution of CH3MgCl in toluene, obtained from 44 ml of a solution of CH3MgCl in THF (Merck) by distillation of THF. The reaction mass is stirred for 1.5 hours, then 40 ml of a 10% aqueous solution of NH4Cl and 8 ml of concentrated HCl are added. After 20 minutes, the reaction solution was transferred to a separatory funnel, the organic layer was separated, and the aqueous was extracted with toluene. The steroid solution in toluene is washed with water to pH 7 and evaporated to dryness in vacuo. 100 ml of methanol was added to the residue. The suspension is heated to boiling, add 1 ml of concentrated HCl and incubated for 10-15 minutes. Then, 22 ml of a 12.5% aqueous solution of sodium acetate are added to the reaction mixture, cooled to room temperature, and the solvent is evaporated in vacuo. The resulting suspension is diluted with 100 ml of water, the precipitate is filtered off, washed with water. The precipitate is dried to constant weight and recrystallized from methanol.

After purification, 6.25 g of compound VIII is obtained with a melting point of 210-212 ° C.

Example 8 Preparation of 17α-hydroxy-21-iodopregn-4,9 (11) -diene-3,20-dione (IX)

To a solution of 30 g of 17α-hydroxypregn-4,9 (11) -diene-3,20-dione (VIII) in a mixture of 225 ml of dichloromethane and 75 ml of methanol add 22.7 g of calcium oxide, 1.5 ml of distilled water and a solution 30 g of iodine and 5.1 g of calcium chloride in 75 ml of methanol. The reaction mass is stirred at room temperature for 1 h and 300 ml of water are added. Stirred for 5 minutes. The inorganic precipitate is filtered off, washed successively with a mixture of methylene chloride and methanol (4: 1) and dichloromethane. The aqueous layer was separated and extracted with methylene chloride. The combined extract was evaporated in vacuo to stop the run.

The residue was triturated with methanol, and the precipitate was filtered off. 41.5 g of compound IX are obtained in quantitative yield.

Mp 148-150 ° C (lit. 142-149 ° C [US3009935, 1954, example 2]).

1H NMR Spectrum (δ, ppm; CDCl3): 5.74 (d, J = 1.5 Hz, 1H, 4-CH); 5.53 (m, 1H, 11-CH); 4.20 (d, Jobs = 11.1 Hz (AB system), 1H, 21-CH2I); 3.98 (d, Jobs = 11.1 Hz (AB system), 1H, 21-CH2I); 1.32 (s, 3H, 19-CH3); 0.65 (s, 3H, 18-CH3).

Example 9 Preparation of 21-acetoxy-17α-hydroxy-pregn-4,9 (11) -diene-3,20-dione (X)

Option 1. To a solution of 41.5 g of 17α-hydroxy-21-iodpregn-4,9 (11) -diene-3,20-dione (IX) in 420 ml of acetone in an inert gas stream add 41.5 g of calcined potassium acetate . The suspension is boiled for 2.5 hours, then cooled to room temperature and poured into 4.3 L of water. Stirred for 30 minutes. The precipitate is filtered off, washed on the filter with water, dried to constant weight. Obtain 32.5 g of compound X with a yield of 92%.

Mp 225-230 ° C. (lit. 230-232 ° C [US 4041055, 1977, example 66])

1H NMR Spectrum (δ, ppm; CDCl3): 5.73 (s, 1H, 4-CH); 5.53 (m, 1H, 11-CH); 5.02 (d, Jobs = 17.4 Hz (AB system), 1H, 21-CH2 O); 4.83 (d, Jobs = 17.4 Hz (AB system), 1H, 21-CH2 O); 2.16 (s, 3H, 21-OS (O) CH3); 1.32 (s, 3H, 19-CH3); 0.64 (s, 3H, 18-CH3).

Option 2. To a solution of 15 g of 17α-hydroxy-21-iodopregn-4,9 (11) -diene-3,20-dione (IX) in 150 ml of acetonitrile in an inert gas stream was added 21 g of calcined potassium acetate. The suspension is boiled for 2.5 hours, then cooled to room temperature and poured into 1.5 liters of water. Stirred for 30 minutes. The precipitate is filtered off, washed on the filter with water, dried to constant weight. 12.41 g of compound X are obtained with a yield of 97.2% and a content of related compounds of not more than 5%.

Mp 219 - 220 ° C.

Option 3. To the residue after evaporation of the solvent containing 17α-hydroxy-21-iodpregn-4,9 (11) -diene-3,20-dione (IX) and representing a thick yellow oil obtained in example 8 from 66.8 g of 17α -hydroxypregna-4,9 (11) -diene-3,20-dione (VIII), add 1 l of acetone and 84 g of anhydrous potassium acetate. The reaction mass is heated to a temperature of 60-65 ° C and maintained for 2.5 hours. At the end of the reaction, the reaction mass is cooled to room temperature and poured into 10 l of water. The precipitate is filtered off, washed with water. 78 g of compound X are obtained in a yield of 99.24%, counting on VIII.

After purification, 66.9 g of compound X is obtained with a yield of 85.8% for purification and a content of related compounds of not more than 2%.

Mp 229.1 - 229.8 ° C

Option 4. To the residue after distillation of the solvent containing 17α-hydroxy-21-iodpregna-4,9 (11) -diene-3,20-dione (IX) obtained in example 8 from 33.1 g of 17α-hydroxypregna-4 , 9 (11) -diene-3,20-dione (VIII), add 330 ml of dimethylformamide and 48.3 g of anhydrous potassium acetate. The reaction mass is heated to a temperature of 60-65 ° C, incubated for 2 hours, then cooled to a temperature of 0 ° C. After holding for 3 hours, the precipitate is filtered off, washed with dimethylformamide and water.

34.2 g of compound X are obtained with a yield of 87.8%, counting on VIII. The content of related compounds is 2.5%, the content of DMF is 5%.

Mp 207-209 ° C.

Example 10 Preparation of 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione (XI)

To an acetylating mixture consisting of 29.8 g of anhydrous p-TSC, 0.34 L of glacial acetic acid and 0.125 L of trifluoroacetic anhydride, 1.34 L of methylene chloride add 66.9 g of 21-acetoxy-17α-hydroxy-pregna-4. 9 (11) -diene-3,20-dione (X). The reaction mass is stirred at room temperature for 2.5 hours, then poured into 3.24 L of a 5% aqueous ammonia solution. It is kept for 15 min at a temperature of 10-15 ° C, after which the organic layer is separated, the aqueous layer is extracted with methylene chloride. The combined extracts are washed with water until neutral. The solvent was evaporated at atmospheric pressure. The residue is crystallized from water. The precipitate is filtered off, washed with water, dried to constant weight. 68.6 g of compound XI are obtained with a yield of 92.54%.

Mp 220 ° C with decomposition (lit. 215-220 ° C [Bulletin of the USSR Academy of Sciences, Chemical Series, 1991, 40 (12), 2875-2878]).

1H NMR Spectrum (δ, ppm; CDCl3): 5.75 (s, 1H, 4-CH); 5.55 (m, 1H, 11-CH); 4.88 (d, Jobs = 16.7 Hz (AB system), 1H, 21-CH2 O); 4.65 (d, Jobs = 16.7 Hz (AB system), 1H, 21-CH2 O); 2.16 (s, 3H, 21-OS (O) CH3); 2.08 (s, 3H, 17-OS (O) CH3); 1.33 (s, 3H, 19-CH3); 0.70 (s, 3H, 18-CH3).

Example 11 Preparation of 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione (XII)

The reaction suspension, consisting of 68.6 g of 21.17-diacetoxypregn-4.9 (11) -diene-3.20-dione (XI), 34.3 g of calcined potassium acetate in 686 ml of absolute dimethyl sulfoxide, is heated in an inert stream gas to 80 ° C and incubated for 2 hours, after which the reaction mixture was cooled and slowly poured into 6.9 L of water. Stirred for 30 minutes. The precipitate is filtered off, washed with water and dried to constant weight. 58.87 g of compound XII are obtained in a yield of 99.8%.

T. pl. 114-117 ° C. According to TLC and PMR analysis, there is no impurity of compound XI. The amount of extraneous (related) steroids is less than 5%.

After cleaning, mp 128-130.5 ° C (lit. 128-129 ° C from ether [US4290963, 1981, example 15].

1H NMR Spectrum (δ, ppm; CDCl3): 6.76 (m, 1H, 16-CH); 5.73 (br s, 1H, 4-CH); 5.53 (m, 1H, 11-CH); 5.02 (d, Jobs = 16.1 Hz (AB system), 1H, 21-CH2 O); 4.90 (d, Jobs = 16.1 Hz (AB system), 1H, 21-CH2 O); 2.16 (s, 3H, 21-OS (O) CH3); 1.33 (s, 3H, 19-CH3); 0.86 (s, 3H, 18-CH3).

Example 12 Preparation of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (XIII)

In the work using the strain Nocardioides simplex VKM Ac-2033D, which is supported on a mown agarized corn-glucose medium of the following composition (g / l): corn extract 10.0; glucose 10.0; tap water (up to 1 liter), pH 7.0-7.2. Cultivation is carried out for 3-5 days at a temperature of 30 ° C. The culture grown on a mown agar medium is used to obtain the first inoculum or stored for 1-2 months in a refrigerator at 4 ° C.

A) Growing the 1st inoculum

The washing of N. simplex culture from the surface of the agar medium is carried out with sterile tap water; in this case, when diluted 10 times, the optical density of the resulting cell suspension should be 0.35-0.40 (cell length 0.5 cm, wavelength 536 nm, Specord (Germany)). The resulting cell suspension (2-3 ml) was added to 750 ml Erlenmeyer flasks containing 50 ml of medium (g / l): glucose 10, soy peptone 4, yeast extract 4, MgSO4 0.5, KH2PO4 2, K2HPO4 4, pH 7.0-7.2. Incubated on a shaker (220 rpm) at a temperature of 30 ° C for 42-48 hours.

B) Growing the 2nd inoculum

5 vol.% Of the grown first inoculum was added to fresh medium of the same composition and incubated for 24-26 hours under the same conditions.

C) Growing a transforming culture

To obtain a culture with 1 (2) dehydrogenase activity, N. simplex cells were grown in an ANKUM-2M fermenter (Russia) with a working volume of 5 l under periodic culture conditions with recharge. Inoculum (2nd inoculum) is added in an amount of 5 vol.% To the medium of the following composition (g / l): yeast extract 10, soy peptone 20, glucose 20, MgSO4 0.5, KH2PO4 2, K2HPO4 4, pH 7, 0. The glucose-free medium is sterilized in a flask at an overpressure of 1 atm for 30 minutes. A glucose solution (30 g in 150 ml of water) is sterilized separately (0.5 atm, 30 min). After 6-8 hours of cultivation, cortisone acetate (in the form of a 1% alcohol solution) is introduced to induce the synthesis of 3-ketosteroid-1-dehydrogenase. The mode of cultivation of the culture in the fermenter: temperature 29 ° C; pH 6.5-7.5; agitator shaft rotation speed from 350 to 800 rpm; initial air flow from 0.25 to 4 l / min. Duration of cultivation 36-38 hours

The resulting culture fluid with cells is used immediately or frozen and stored at a temperature of minus 18 ° C for subsequent use. The concentration of cells in the culture fluid (dry weight) is 13.4 g / L.

Microorganism cells are pelleted by centrifugation at 5000 x g for 30 minutes and used to carry out the 1,2-dehydrogenation process or stored at
minus 18 ° C for a month before use.

Incubation is carried out under aerated conditions on a rotary shaker at a temperature of 25 ° C

Steroids are extracted with ethyl acetate. The main process parameters and the content of steroid compounds in the extract in comparison with known analogues are presented in table 2. Yield and quality parameters of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (XIII) (according to HPLC) obtained after isolation without further purification in comparison with the known analogue, are presented in table 3.

Mp 173-175 ° C (lit. 172-174 ° C [US2864834, 1958, example 1]).

1 H NMR Spectrum (δ, ppm; CDCl 3): 7.21 (d, J = 10.1 Hz, 1H, 1-CH); 6.76 (m, 1H, 16-CH); 6.28 (dd, J = 10.1 Hz, J = 1.8 Hz, 1H, 2-CH); 6.06 (t, J = 1.8 Hz, 1H, 4-CH); 5.55 (m, 1H, 11-CH); 5.03 (d, Jobs = 16.1 Hz (AB system), 1H, 21-CH2 O); 4.89 (d, Jobs = 16.1 Hz (AB system), 1H, 21-CH2 O); 2.17 (s, 3H, 21-OS (O) CH3); 1.42 (s, 3H, 19-CH3); 0.91 (s, 3H, 18-CH3).

Option 1. Bioconversion of 21-acetoxy-4,9 (11), 16-trien-3,20-dione (XII) is carried out in a medium containing a solution of statistically methylated β-cyclodextrin (MCD) in 0.05 M Na-acetate buffer (pH 5), with a concentration of 10.0 g / l. A steroid substrate (0.5 g in a 100% calculation of the basic substance) is added to 46.25 ml of medium in the form of a solution in dimethyl sulfoxide (DMSO), which also contains 20 mg of menadione (4% by weight of the substrate), while the molar ratio of MCD: substrate is 1.5: 1, and the concentration of DMSO is 4% vol., counting on the final volume of the transformation medium (50 ml). Then add 3.75 ml of culture fluid, the weight ratio of biomass: substrate is 1:10. Incubation is carried out for 6 hours under aerated conditions (220 rpm). The results (according to TLC analysis) are presented in table 2.

Option 2. Bioconversion of 21-acetoxy-4,9 (11), 16-trien-3,20-dione (XII) at a concentration of 20.0 g / l (1.0 g of steroid substrate in 100% of the calculation of the basic substance) is carried out as described in option 1, but using 38.34 ml of MCD solution in a 0.02 M NaH2PO4 solution (acidified to pH 5.0) and 10.66 ml of culture fluid, the molar ratio of MCD: substrate is 2: 1, the weight ratio of biomass: the substrate is 1: 7. The steroid substrate and menadione are introduced into a solution of ethanol. Incubation is carried out for 14 hours. The results (according to TLC analysis) are presented in table 2.

Option 3. Bioconversion of 21-acetoxy-4,9 (11), 16-trien-3,20-dione (XII) at a concentration of 20.0 g / l (1 g of steroid substrate in 100% calculation of the basic substance) is carried out as described in option 2, but using 38.34 ml of MCD solution in 0.05 M Na-acetate buffer (acidified to pH 5.0) and 10.66 ml of culture fluid. Moreover, the molar ratio of WDC: substrate is 2: 1, and the weight ratio of biomass: substrate is 1: 7. Incubation is carried out for 9 hours. The results (according to HPLC analysis) are presented in table 2.

Option 4. Transformation of 21-acetoxypregn-4.9 (11), 16-triene-3.20-dione (XII) at a concentration of 20.0 g / l (24 g of steroid substrate in 100% calculation of the basic substance) in the ANKUM fermenter -2M with a working volume of 1.2 L is carried out under the conditions of option 3, but using 805.5 ml of MCD solution in 0.05 M Na-acetate buffer (acidified to pH 5.0) and 298.5 ml of culture fluid. The molar ratio of WDC: substrate is 2: 1, and the weight ratio of biomass: substrate is 1: 6. Incubation is carried out for 14 hours at a stirrer shaft speed of 800 rpm, the level of dissolved oxygen is maintained above 30% of the saturation level. The results (according to HPLC analysis) are presented in table 2.

Option 5. Bioconversion of 21-acetoxy-4,9 (11), 16-triene-3,20-dione (XII) at a concentration of 20.0 g / l (1 g of steroid substrate in 100% calculation of the basic substance) is carried out as described in option 2, but use a solution of MCD in 0.02 M Na-citrate buffer (pH 5.0) and 670 mg of wet biomass paste stored frozen at minus 18 ° C for 1 month (corresponding to 142.8 mg of dry biomass weight ) In this case, the molar ratio of MCD: substrate is 2: 1, the weight ratio of biomass: substrate is 1: 7, and the ethanol concentration is 8% based on the final volume of the transformation medium (50 ml). Incubation is carried out for 11 hours. Results (by TLC analysis) are presented in table 2.

Option 6. Bioconversion of 21-acetoxy-4,9 (11), 16-trien-3,20-dione (XII) at a concentration of 20.0 g / l (1 g of steroid substrate in 100% calculation of the basic substance) is carried out as described in option 5, but use a solution of MCD in a 0.02 M solution of NaH2PO4 (acidified to pH 5.0). Incubation is carried out for 14 hours. The results (according to TLC analysis) are presented in table 2.

Option 7. Bioconversion of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione (XII) at a concentration of 20.0 g / l (1 g of steroid substrate in 100% calculation of the basic substance) is carried out as described in embodiment 5, but using a solution of MCD in 0.05 M Na-acetate buffer (pH 5.0). Incubation is carried out for 9 hours. The results (according to TLC analysis) are presented in table 2.

Example 13 Preparation of 21-acetoxy-17α-hydroxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione (XVI)

To a solution of 1.05 g of CuCl in 633 ml of acetonitrile, 45.48 g of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (XIII), 180 ml of tetrahydrofuran and 690 ml of toluene are added. The reaction mass is cooled to a temperature of minus 40 ° C and 304 ml of a 1.4 M solution of CH3MgBr in a mixture of toluene and THF (3: 1 by volume) are added. Then the reaction mass is maintained at a temperature of minus 40ºС for 15 minutes. At the end of the reaction, the reaction mass is slowly poured into 2.28 L of a 20% aqueous solution of NH4Cl at a temperature not exceeding 5 ° C with vigorous stirring. After additional exposure for 30 min at a temperature of 0-5 ° C, the reaction mass containing 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione (XV) is transferred to separatory funnel. The organic layer was separated, washed with 20% aqueous NH4Cl and water.

To the resulting solution of compound XV in toluene in an inert gas stream was added 544 ml of acetone and 15.8 g of potassium iodide. The suspension is stirred at room temperature for 1 hour. Then add a 10% aqueous solution of sodium sulfite to discolor the reaction mass and 20 ml of water. After this, the reaction mass containing 21-acetoxy-17α-hydroxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione (XVI) is transferred to a separatory funnel. The organic layer was separated, washed with 10% aqueous sodium sulfite and water. The solvent was evaporated in vacuo until the cutoff ceased, the toluene residue was distilled off with methanol. A solution of compound XVI in methanol was poured into a 10-fold amount of water. The precipitate is filtered off, washed with water, dried to constant weight.

46.7 g of compound are obtained in a molar yield of 94.4%, based on compound XIII. T. pl. 188-192ºС. After crystallization from diethyl ether, 35.63 g of compound XVI is obtained with a yield of 76.3% in purification.

T. pl. 207-208.5 ° C (lit. 206-211 ° C [US3947409, 1976, example 1]).

1H NMR Spectrum (δ, ppm; CDCl3): 7.17 (d, J = 10.1 Hz, 1H, 1-CH); 6.26 (dd, J = 10.1 Hz, J = 1.8 Hz, 1H, 2-CH); 6.05 (t, J = 1.8 Hz, 1H, 4-CH); 5.53 (m, 1H, 11-CH); 4.96 (d, Jobs = 17.5 Hz (AB system), 1H, 21-CH2 O); 4.79 (d, Jobs = 17.5 Hz (AB system), 1H, 21-CH2 O); 2.16 (s, 3H, 21-OS (O) CH3); 1.38 (s, 3H, 19-CH3); 0.91 (d, J = 7.1 Hz, 3H, 16-CH3); 0.73 (s, 3H, 18-CH3).

Example 14 Preparation of 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione (XVII)

To a solution of 50 g of 21-acetoxy-17α-hydroxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione (XVI) in 350 ml of acetone, cooled to a temperature of 10 ° C, add 100 ml distilled water and 54 ml of a ~ 6.5% aqueous solution of HClO4. Then, 25.24 g of dibromantin are added to the reaction mass in portions over 25-30 minutes. The reaction mass is incubated for 45 minutes, add a 10% solution of sodium sulfite in water until discoloration.

Then 560 ml of water are added to the reaction mass. The precipitate is filtered off, washed with water. 78 g of a wet paste of compound XVII are obtained, which, without drying, is used to obtain compound XVIII.

1H NMR spectrum of a vacuum dried sample (δ, ppm; DMSO-d6): 7.33 (d, J = 10.1 Hz, 1H, 1-CH); 6.22 (dd, J = 10.1 Hz, J = 1.8 Hz, 1H, 2-CH); 5.95 (br s, 1H, 4-CH); 5.57 (d, J = 5.4 Hz, 1H, 11-OH); 5.22 (s, 1H, 17-OH); 5.03 (d, Jobs = 17.6 Hz (AB system), 1H, 21-CH2 O); 4.78 (d, Jobs = 17.6 Hz (AB system), 1H, 21-CH2 O); 4.57 (br.s, 1 H, 11-CH); 2.08 (s, 3H, 21-OS (O) CH3); 1.63 (s, 3H, 19-CH3); 0.88 (s, 3H, 18-CH3); 0.79 (d, J = 7.3 Hz, 3H, 16-CH3).

Example 15 Preparation of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione (XVIII)

Option 1. To the reaction mass obtained in example 14 after decolorization with 10% sodium sulfite solution, add 300 ml of methanol. The reaction mass is cooled to a temperature of 0 ° C, 200 ml of a 7.5% aqueous NaOH solution are added and maintained at the same temperature for 40 minutes. Then the reaction mass is neutralized with acetic acid. The solvent was evaporated to a small volume, 500 ml of water was added to the residue. The precipitate is filtered off, washed with water, dried to constant weight. 42.51 g of compound XVIII are obtained in a yield of 90.97%, counting on compound XVII, the impurity content is less than 1% in total.

Mp 257-259 ° C (lit. 238-239.5 ° C [US4990612, 1991, example 8]).

1H NMR Spectrum (δ, ppm; CDCl3): 6.58 (d, J = 10.1 Hz, 1H, 1-CH); 6.18 (dd, J = 10.1 Hz, J = 1.8 Hz, 1H, 2-CH); 6.14 (br s, 1H, 4-CH); 4.60 (dd, Jobs = 20.0 Hz (AV system), J = 4.5 Hz, 1H, 21-CH2 O); 4.22 (dd, Jobs = 20.0 Hz (AV system), J = 4.5 Hz, 1H, 21-CH2 O); 3.21 (br.s, 1 H, 11-CH); 3.06 (t, J = 4.5 Hz, 21-OH); 1.42 (s, 3H, 19-CH3); 0.92 (s, 3H, 18-CH3); 0.89 (d, J = 7.2 Hz, 3H, 16-CH3).

Option 2. 7 g of a wet paste 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregn-1,4-diene-3,20-dione XVII obtained in example 14 of 4.45 g of compound XVI, dissolved in 56 ml of methanol and 2.57 g of K2CO3 are added in an inert gas stream. The reaction mass is kept at room temperature for 2 hours. At the end of the reaction, the reaction mass is neutralized with acetic acid and 24 ml of water are added. The precipitate is filtered off, washed with water. Obtain 3.6 g of compound XVIII with a yield of 86.5%.

Example 16 Preparation of 11β, 17α, 21-trihydroxy-16α-methyl-9α-fluoropregna-1,4-diene-3,20-dione (dexamethasone, I)

To 213 ml of 70% aqueous hydrofluoric acid at a temperature not exceeding minus 20 ° C, 42.51 g of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxyprena-1,4-diene-3,20- are added in uniform portions. dion (xviii). The reaction mass is stirred for 1.5 hours at the same temperature. At the end of the reaction, the reaction mass is poured into 4.68 L of a ~ 2% aqueous solution of ammonia at a temperature not exceeding 10 ° C. The suspension is maintained at a temperature of 0-2 ° C for 2 hours. The precipitate is filtered off, washed with water, and dried to constant weight. 40.71 g of dexamethasone (I) are obtained with a yield of 90.91% and a basic substance content of 91.1% (HPLC).

After purification, dexamethasone is obtained with a basic substance content of 97.8% (HPLC). The purification yield is 81.2%.

Mp 264-265 ° C (lit. 263 ° C with decomposition [US3947409, 1976, example 7]).

1 H NMR Spectrum (δ, ppm; DMSO-d6): 7.28 (d, J = 10.1 Hz, 1H, 1-CH); 6.21 (dd, J = 10.1 Hz, J = 1.8 Hz, 1H, 2-CH); 5.99 (br s, 1H, 4-CH); 5.26 (br s, 1H, 11-OH); 4.93 (s, 1H, 17-OH); 4.65 (t, J = 5.9 Hz, 1H, 21-OH); 4.49 (dd, Jobs = 19.2 Hz (AV system), J = 5.9 Hz, 1H, 21-CH2 O); 4.13 (m, 1H, 11-CH);); 4.07 (dd, Jobs = 19.2 Hz (AV system), J = 5.9 Hz, 1H, 21-CH2 O); 1.47 (s, 3H, 19-CH3); 0.85 (s, 3H, 18-CH3); 0.77 (d, J = 7.3 Hz, 3H, 16-CH3)

Table 1.

The main parameters of the process of obtaining androsta-4,9 (11) -diene-3,17-dione (IV) by the claimed method and known analogues

table 2

The main parameters of the microbiological process of 1,2-dehydrogenation of 21-acetoxypregn-4,9 (11), 16-triene-3,20-dione (XII) by the claimed method and known analogues

Table 3

The yield and quality parameters of the selected 21-acetoxypregn-1,4,9 (11), 16-tetraen-3,20-dione (XIII) according to the claimed method and the known analogue

Example
No. Yield of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (XIII),
% Mass fraction of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione (XIII),% Mass fraction of impurities,% 21-hydroxy-1,4,9 (11), 16-tetraen-21-ol-3,20-dione 21-acetoxy-pregna-4.9 (11), 16-triene-3.20-dione 21-hydroxy-4.9 (11), 16-triene-3.20-dione The inventive method (example 3)
Growing culture 83.5 95.6 3.77 0.59 0.02 99.37 (total) The inventive method (example 4)
Growing culture 83.8 96.03 3.11 0.82 0.02 99.14 (total) US 4524134
(example 4)
Growing culture 18.1 59.1 (total) 40.9 - US 4524134
(example 4)
dry cells 79.6 97.8 (total) 2.1 -

Claims (24)

1. The method of obtaining 11β, 17α, 21-trihydroxy-16α-methyl-9α-fluoropregna-1,4-diene-3,20-dione (dexamethasone) of the formula (I)

from phytosterol of the general formula (II)

where R = C₂H₅ - β-sitosterol; R = CH₃ - campesterol; R = C₂H₅ and ∆^{22 (23)}- stigmasterol; R = CH₃ and ∆^{22 (23)}- brassicasterin,
including microbiological oxidative elimination of the side chain of phytosterol at atom C¹⁷ with the formation of 9α-hydroxyandrost-4-en-3,17-dione of the formula (III)

treatment of 9α-hydroxyandrost-4-en-3,17-dione with a dehydrating agent in an aprotic solvent to form androst-4,9 (11) -diene-3,17-dione of the formula (IV)

17-hydrocyanation of androsta-4,9 (11) -diene-3,17-dione by interaction with a cyanating agent in a proton solvent under basic catalysis with the formation of 17α-hydroxy-17β-cyanandrosta-4,9 (11) -diene- 3-one formulas (V)

protection of the 3-keto group of 17α-hydroxy-17β-cyanandrost-4,9 (11) -diene-3-one by ketalization to form a cyclic ketal of the general formula (VI)

protection of the 17α-hydroxy group by the action of alkoxyethylene with the formation of a 17α-esterified cyclic ketal derivative of the general formula (VII)

where R '= CH (CH₃) OC₂H₅or CH (CH₃) OC_fourH₉;
alkylation of the carbonitrile group of the 17α-esterified cyclic ketal derivative of the general formula (VII) with an alkylating agent in an aprotic solvent and removal (deprotection) of the protective groups to form 17α-hydroxypregn-4,9 (11) -diene-3,20-dione of the formula (VIII )

FROM²¹iodination of 17α-hydroxypregn-4,9 (11) -diene-3,20-dione with the formation of 17α-hydroxy-21-iodpregn-4,9 (11) -diene-3,20-dione of the formula (IX)

FROM²¹-acetoxylation of 17α-hydroxy-21-iodopregn-4,9 (11) -diene-3,20-dione by replacing the iodine atom on the acetoxy group by the action of alkali metal acetate with the formation of 21-acetoxy-17α-hydroxypregna-4.9 (11) - diene-3,20-dione of the formula (X)

the esterification of the 17α-hydroxy group of 21-acetoxy-17α-hydroxypregn-4,9 (11) -diene-3,20-dione with an acetylating agent to form 17α, 21-diacetoxypregn-4,9 (11) -diene-3,20-dione formulas (XI)

elimination of the 17α-acetoxy group of 17α, 21-diacetoxypregna-4,9 (11) -diene-3,20-dione with the formation of 21-acetoxypregna-4,9 (11), 16-triene-3,20-dione of the formula (XII)

microbiological 1,2-dehydrogenation of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione with the formation of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione formulas (XIII)

catalytic 1,4-addition of methylmagnesium halide according to Δ¹⁶-20-keto system of 21-acetoxypregna-1,4,9 (11), 16-tetraen-3,20-dione in an aprotic solvent (or solvent mixture) to form a magnesium halide derivative of 21-acetoxy-20-hydroxy-16α-methylpregna- 1,4,9 (11), 17 (20) -tetraen-3-one of the formula (XIV)

autooxidation of the magnesium halide derivative 21-acetoxy-20-hydroxy-16α-methylpregna-1,4,9 (11), 17 (20) -tetraen-3-one with the formation of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4 9 (11) -trien-3,20-dione of the formula (XV)

the reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione to the 17α-hydroxyl group with a reducing agent to form 21-acetoxy-17α-hydroxy-16α methylpregna-1,4,9 (11) -trien-3,20-dione of the formula (XVI)

the reaction of 9,11-bromohydroxylation of 21-acetoxy-17α-hydroxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione by the action of hypobromic acid with the formation of 21-acetoxy-9α-bromo-11β, 17α -dihydroxy-16α-methylpregn-1,4-diene-3,20-dione of the formula (XVII)

9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione and simultaneous removal of the ester protecting group at C²¹with the formation of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione of the formula (XVIII)

and the reaction of opening the 9β (11) -epoxide ring of 17α, 21-dihydroxy-16α-methyl-9β (11) -epoxypregna-1,4-diene-3,20-dione with hydrofluoric acid. 2. The method according to claim 1, characterized in that the microbiological transformation of the phytosterol of the formula (II) is carried out by the cells of the bacteria Mycobacterium sp. VKM Ac-1817D. 3. The method according to claim 1, characterized in that the dehydration of the 9α-hydroxy group of the 9α-hydroxyandrost-4-en-3,17-dione of the formula (III) is carried out in an aprotic organic solvent (chlorine-containing, for example dichloromethane, chloroform, dichloroethane, or aromatic, for example benzene, toluene) by the action of a dehydrating agent. 4. The method according to claim 1, characterized in that mineral acids (for example, phosphoric, pyrophosphoric, sulfuric, perchloric) are used as the dehydrating agent, including those containing water, moreover, the removal of excess water can be carried out during the dehydration reaction. 5. The method according to claim 1, characterized in that the dehydration of the 9α-hydroxy group of 9α-hydroxyandrost-4-en-3,17-dione of the formula (III) can be carried out in an extract of clarified culture fluid after separation of the biomass after the end of the microbiological process of oxidative elimination of the lateral phytosterol chains of the formula (II), and mineral acids, including those containing water, were used as the dehydrating agent, and the removal of excess water can be carried out during the dehydration reaction. 6. The method according to claim 1, characterized in that the hydrocyanation of androst-4,9 (11) -diene-3,17-dione of the formula (IV) is carried out by reaction with acetone cyanohydrin as a hydrocyanating agent, and a mineral base is used as the main catalyst (e.g. alkali metal hydroxide). 7. The method according to claim 1, characterized in that the protection of the 3-keto group of 17α-hydroxy-17β-cyanandrost-4,9 (11) -diene-3-one of the formula (V) by ketalization is carried out using 1,2-diols with the formation of dioxolane ketal or with the help of 1.3 diols, including 2,2-disubstituted, with the formation of hexacyclic (1,3-dioxane) ketal. 8. The method according to claim 1, characterized in that for the preliminary protection of the 17α-hydroxy group of the cyclic ketal of general formula (VI), it is esterified with alkoxyethylene (for example, ethoxyethylene or butoxyethylene). 9. The method according to claim 1, characterized in that for the alkylation of the carbonitrile group of the 17α-esterified cyclic ketal derivative of the general formula (VII), methyl lithium or methyl magnesium halides (for example methyl magnesium bromide or methyl magnesium chloride) are used as the alkylating agent. 10. The method according to claim 1, characterized in that as a medium for carrying out the reaction of substitution of an iodine atom for an acetoxy group in 17α-hydroxy-21-iodine-4,9 (11) -diene-3,20-dione of the formula (IX) aprotic polar organic solvents (e.g., dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, acetonitrile) and dialkyl ketones (e.g. acetone) can be used. 11. The method according to claim 1, characterized in that the sequence of processes of C ²¹ iodination of 17α-hydroxypregn-4,9 (11) -diene-3,20-dione of formula (VIII) and C ²¹ -acetoxylation of 17α-hydroxy-21 iodine-4,9 (11) -diene-3,20-dione of the formula (IX) by replacing the iodine atom with an acetoxy group can be carried out without isolation of 17α-hydroxy-21-iodine-4,9 (11) -diene-3, 20-dione of formula (IX) from the reaction solution. 12. The method according to claim 1, characterized in that the esterification of the 17α-hydroxy group of 21-acetoxy-17α-hydroxypregn-4,9 (11) -diene-3,20-dione of the formula (X) is carried out with an acetylating agent, which can an anhydride or acetic acid chloride or a mixture of acetic acid and trifluoroacetic anhydride in an effective ratio may be used. 13. The method according to claim 1, characterized in that the elimination of the 17α-acetoxy group 17α, 21-diacetoxypren-4,9 (11) -diene-3,20-dione (XI) is carried out by the action of alkali metal acetate in an aprotic organic solvent ( for example, dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide and the like). 14. The method according to claim 1, characterized in that the 1,2-dehydrogenation of 21-acetoxypregna-4.9 (11), 16-triene-3,20-dione of the formula (XII) is carried out microbiologically using cells of the microorganism Nocardioides simplex VKM Ac-2033D possessing 3-ketosteroid-1-dehydrogenase activity. 15. The method according to 14, characterized in that the microbiological transformation is carried out in an aqueous medium containing an exogenous electron acceptor menadione in an effective amount and methyl β-cyclodextrin in an amount of not more than 2 moles per 1 mol of the starting substrate, using a polar solvent, miscible with water, at a concentration of the specified solvent in an aqueous medium from 4 to 8 vol.%, in a buffer solution with a pH from 5.0 to 5.5, preferably 5.1 ± 0.1 at a temperature of 25 ° C for from 6 to 32 hours (optimally from 6 to 14 hours), while menadione contribute into the medium for transformation simultaneously with the steroid substrate in the form of a solution in a polar solvent. 16. The method according to claim 1, characterized in that the catalytic 1,4-addition reaction of methylmagnesium halide (for example, methylmagnesium bromide or methylmagnesium chloride) according to Δ ¹⁶ -20-keto system 21-acetoxypregna-1,4,9 (11), 16 -tetraen-3,20-dione of the formula (XIII) is carried out at a temperature from minus 70 to minus 10 ° C, and copper (I) chloride is used as a catalyst. 17. The method according to claim 1, characterized in that the autooxidation of the magnesium halide derivative of 21-acetoxy-20-hydroxy-16α-methylpregn-1,4,9 (11), 17 (20) -tetraen-3-one of the formula (XIV ) is carried out with atmospheric molecular oxygen in a heterogeneous medium consisting of an aprotic solvent miscible with water (e.g. tetrahydrofuran, acetonitrile, or a mixture thereof) and an aprotic solvent miscible with water (aromatic, e.g. benzene, toluene) in an effective ratio. 18. The method according to 17, characterized in that the autooxidation of the magnesium halide derivative of 21-acetoxy-20-hydroxy-16α-methylpregna-1,4,9 (11), 17 (20) -tetraen-3-one of the formula (XIV ) is carried out at a temperature from minus 10 to plus 10 ºС. 19. The method according to claim 1, characterized in that the reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -triene-
3,20-dione of the formula (XV) in the 17α-hydroxyl group is carried out by the action of a reducing agent, which is used as an alkali metal iodide (sodium or potassium) in an effective amount. 20. The method according to claim 1, characterized in that the reduction of the 17α-hydroperoxy group of 21-acetoxy-17α-hydroperoxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione of the formula (XV) to 17α -hydroxyl group is carried out in a dialkyl ketone (e.g. acetone, methyl ethyl ketone) or in an aromatic hydrocarbon (e.g. benzene, toluene) in the presence of dialkyl ketone (e.g. acetone) in an effective amount. 21. The method according to claim 1, characterized in that the reaction sequence comprising a catalytic 1,4-addition of methylmagnesium halide according to the Δ ¹⁶ -20-keto system 21-acetoxyprena-1,4,9 (11), 16-tetraen-3,20 -dione of the formula (XIII), autooxidation of the magnesium halide derivative of 21-acetoxy-20-hydroxy-16α-methylpregn-1,4,9 (11), 17 (20) -tetraen-3-one of the formula (XIV) and reduction of the 17α-hydroperoxy group 21 -acetoxy-17α-hydroperoxy-16α-methylpregna-1,4,9 (11) -trien-3,20-dione of formula (XV) into a 17α-hydroxyl group, can be carried out without isolation of 21-acetoxy-17α-hydroperoxy- 16α-methylpregna-1,4,9 (11) -trien-3,20-dione of the formula (XV). 22. The method according to claim 1, characterized in that the bromohydroxylation reaction of 21-acetoxy-17α-hydroxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione of the formula (XVI) is carried out by the action of hypobromic acid in an aprotic polar solvent medium (for example, tetrahydrofuran or dialkyl ketone) containing an effective amount of water to form hypobromic acid in situ, the source of hypobromic acid being selected from the group consisting of 1,3-dibromo-5,5-dimethylhydantoin, N-bromosuccinimide N-bromoacetamide and the like 23. The method according to claim 1, characterized in that 9β (11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione of the formula (XVII ) and the removal of the ester protecting group at C ^{21 is} carried out simultaneously in an aqueous medium containing a mixture of an aprotic polar solvent (for example, tetrahydrofuran or dialkyl ketone) and a protonic solvent (for example, an aliphatic alcohol selected from the group: methyl, ethyl, isopropyl) in an effective amount, the action of an alkaline agent (e.g. alkali hydroxide or carbonate wow metal). 24. The method according to claim 1, characterized in that the sequence of reactions of bromohydroxylation of 21-acetoxy-17α-hydroxy-16α-methylpregn-1,4,9 (11) -trien-3,20-dione of the formula (XVI) and 9β ( 11) -epoxidation of 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione of the formula (XVII) with simultaneous removal of the ester protecting group at C ²¹ can be carried out without isolating 21-acetoxy-9α-bromo-11β, 17α-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione of formula (XVII) from the reaction mixture.