Method of producing 6-methyleneandrost-4-ene-3,17-dione from androst-4-ene-3,17-dione, method of producing 6-methyleneandrost-1,4-diene-3,17-dione (exemestane) using obtained 6-methyleneandrost-4-ene-3,17-dione
SUBSTANCE: invention relates to improved methods of producing 6-methyleneandrost-4-ene-3,17-dione and 6-methyleneandrost-1,4-diene-3,17-dione (MNN exemestane) using the obtained 6-methyleneandrost-4-ene-3,17-dione. 6-methyleneandrost-4-ene-3,17-dione is obtained using a method involving preliminary enolisation of Δ4-3-ketofunction of androst-4-ene-3,17-dione with formation of 3-alkoxyandrost-3,5-dien-17-one, subsequent three-component condensation with a secondary amine and formaldehyde in the medium of a polar protonic solvent, deamination of the N,N-disubstituted amino group to form a 6-methylene group in the medium of an aprotic solvent. 6-methyleneandrost-1,4-diene-3,17-dione is obtained through 1,2-dehydrogenation of 6-methyleneandrost-4-ene-3,17-dione via microbiological transformation in a medium containing up to 40% water-miscible aprotic solvent using Nocardioides simplex cells VKM As-2033D.
EFFECT: high output and selectivity under mild conditions for carrying out the process.
11 cl, 4 ex, 1 tbl
The present invention relates to the field of organic synthesis and biotechnology, specifically for the production of steroidal compounds 6-medienanstalt-1,4-diene-3,17-dione and can be used in chemical, microbiological and pharmaceutical industries.
6-Medienanstalt-1,4-diene-3,17-dione (6-methylene-ADD, INNS exemestane) is a high - performance modern inactivator aromatase, known under the trade name "Aromasin" (Manufacturer Pharmacia Italia, Italy), which is widely used in the treatment of malignant tumors of the breast [US 4808616, 1989; D.Giudici, G.Ornati, G.Briatico, F.Buzzetti, P.Lombardi and E. di Salle Journal of Steroid Biochemistry, Volume 30, Issues 1-6, 1988, Pages 391-394]. Clinical trials have shown high efficacy and safety of exemestane in the treatment of postmenopausal women (natural or induced), suffering from metastatic breast cancer progressing on therapy with antiestrogens, non-steroidal aromatase inhibitors or progestins [Mbotenana "aromatase Inhibitors in the treatment of disseminated breast cancer patients in menopause". Materials of the IV all-Russian annual Oncology conference. Moscow, November 21-23, 2000].
Known methods for producing exemestane 1,2-dehydrogenation of 6-medienanstalt-4-ene-3,17-dione (6-methylene-AD).
Known methods 1,2-degeri the Finance 6-methylene-AD using chemical dehydrating agents. Thus, known methods for producing 6-medienanstalt-1,4-diene-3,17-dione by dehydrogenation of 6-methylene-AD using 2,3-dichloro-5,6-dicyan-1,4-benzoquinone (DDQ) boiling of the reaction mixture in dioxane for 15 h (output 50,3%) [GB 2177700, 1987, example 1] or in an inert solvent for 8 h in the presence of aromatic carboxylic acids (output 50,7%) [CN 1453288, 2003].
Known methods for producing 6-medienanstalt-1,4-diene-3,17-dione by dehydrogenation of 6-methylene-AD using selenium dioxide (boiling in the environment tert-butyl alcohol for 30 h, the output 40,2%) [GB 2177700, 1987, example 2] or using a 2-iodoxybenzoic acid [CN 1491957, 2004].
Also known process exemestane reaction dehydrogenation of 6-methylene-HELL in the 1,2-position in the presence of quinone, cilleruelo agent and an acid having a pKa in aqueous solution ≤1 [WO 2009/077454, 2009, example 1]. At the same time as the quinone use 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), as cilleruelo agent using bis(trimethylsilyl)triptorelin, as a strong acid - triftormetilfullerenov acid; technical product Obtained with a yield of 81.8% of the product is recrystallized, however, the yield of pure product is not specified. Thus, a method of obtaining 6-medienanstalt-1,4-diene-3,17-dione by reaction of 6-methylene-HELL with chloranil in a convenient solvent, as a cat who was used dimethyl sulfoxide (DMSO) or N-methylpyrrolidine (N) [WO 2009093262, 2009]. From 100 g of 6-methylene-AD (example 3) receive only 55 g exemestane 97.3% of the content.
A method of obtaining 6-medienanstalt-1,4-diene-3,17-dione, in which the dehydrogenation of 6-methylene-HELL is bromirovanii 6-methylene-HELL bromine in an organic solvent at a temperature of from 0 to 10°C with the formation of tribrom connection, dibromononane tribrom-coupling reaction with iodide of an alkali metal in an organic ketone solvent with the formation of 2-bromo derivatives of 6-methylene-HELL and dehydrobrominated 2-bromo derivatives of 6-methylene-AD by reaction with a basic agent in a polar solvent at a temperature of 120°C for 2.5 h, the total output 40,13%) [US 4990635, 1991, examples 2-4] (figure 1).
Figure 1. Synthesis of exemestane of 6-methylene-AD chemical methods
The General lack of chemical methods 1,2-dehydrogenation is the use of expensive and toxic reagents, harsh conditions of the reactions, and in most cases, the necessity of using column chromatography for purification, and as a consequence low output exemestane.
You know the introduction of the 1,2-double bond in the steroid molecule using methods microbiological transformation.
The implementation of the multienzyme complex processes into a single process stage in mild conditions, against the sustained fashion high outputs the obtained target product, the possibility of inhibiting side reactions determine the prospects of using microorganisms as biocatalysts reaction of 1,2-dehydrogenation, in particular, to obtain exemestane.
As biocatalysts process 1,2-dehydrogenation use culture with 3-ketosteroid-Δ1-dehydrogenase (EU 184.108.40.206) genera: Arthrobacter, Alternaria, Alcaligenes, Calonectria, Nauchnye, Corynebacterium, Bacillus, Nocardia, Streptomyces, Bacterium, Mycobacterium, Fusarium, Cylindrocarpon, Pseudomonas, Protaminobacter, Septomyxa, Didymella, etc. [Charney W., Herzog H. Microbial transformation of Steroids. Academic Press. Inc. New York. 1967, pp.236-261]. Most often the process of 1,2-dehydrogenation of steroid compounds of the number pregnane and androstane are strains of the species Arthrobacter simplex.
The bioconversion carried out using the culture fluid of native cells (60-85% humidity) [US 4749649, 1988], the dried cells obtained by treatment with acetone, dried in vacuum or in air when heated [US 4704358, 1987], in conditions of lyophilization and use a cell-free extracts obtained by ultrasound destruction or destroyed by passing the frozen cells through a Spinneret under pressure [EP 0350488, 1993]. In order to eliminate additional processing steps, preferably using native cells.
The main problem of obtaining 1,2-dehydrogenated derivatives of a number of androstane, in particular exames the Ana, microbiological method is the low efficiency of the process due to the low solubility of the substrate in the aquatic environment, the need for these conditions, the use of small concentrations to create the conditions of its availability to the cells of the microorganisms and to achieve the most complete conversion to product.
To achieve in an environment of higher concentration of steroid substrates and process efficiency 1,2-dehydrogenation used organic solvents immiscible with water in an amount necessary for dissolving the source substrate.
So, there is a method of transformation of 1,2-saturated steroid 1.2-dehydrothermal, which includes the interaction of 1,2-saturated steroid with Arthrobacter simplex or Bacterium cyclooxydans in the presence of exogenous carrier electrons, water-immiscible aromatic hydrocarbon solvent, where the oxygen concentration is maintained at a minimum concentration of oxygen required for oxidation, or where the reaction is carried out below the flash temperature of the mixture provided that the 1,2-saturated steroid has a solubility in water-immiscible aromatic hydrocarbon solvent, more than 5 g/l [US 4684610, 1987]. The process is carried out in a two-phase system, using water-immiscible organic solvents: ensol, toluene, xylene.
Also known is a method of obtaining 1,2-degidro-3-ketosteroid, which is that the 1,2-saturated 3-ketosteroid subjected to interaction with Arthrobacter simplex or Bacterium cyclooxydans in the presence of exogenous carrier electrons and water-immiscible solvents, including aromatic hydrocarbons [GB 2131811, 1984]. According to this method (examples 1 and 2) the transformation process carried out by cells of A. simplex in the presence of menadione in the environment K2HPO4buffer solution (pH 7.5)containing 10% toluene in 4 days.
The disadvantage of these methods is the need to create and maintain strict minimum allowable concentration of dissolved oxygen in the environment to avoid the flash vapor gas mixture and at the same time to provide optimum conditions for oxidation of the substrate. The implementation of these methods requires special technological equipment, which effectively precludes their practical implementation in the conditions of industrial production. However, known methods receive androsta-1,4-diene-3,17-dione (ADD) from androst-4-ene-3,17-dione (AD), androsta-1,4,9(11)-triene-3,17-dione (Δ9(11)-ADD) from androsta-4,9(11)-diene-3,17-dione (Δ9(11)-AD), and other 1,2-dihydroprogesterone number of androstane, and they are not described receiving exemestane (6-methylene-ADD) of 6-methylene-HELL.
It is known that exogenous electron acceptors - finishingtouches (FMS), menadione (2-methyl-1,4-naphthoquinone), 1,4-naphthoquinone, menadione bisulphite is used not only to promote the process of 1,2-dehydrogenation of steroid compounds, but also for the inhibition of side reactions restoration of nature and the destructive activity of the cells of microorganisms. The formation of by-products, destruction of the target product reduces the yield of the main product.
In the processes of microbiological transformation of steroid compounds, along with the use of organic solvents immiscible with water, use miscible with water and organic solvents, such as dimethylsulfoxide, dimethylformamide, methanol, ethanol, acetone, etc.
Describes the use of dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) as miscible with water and organic solvents used in the processes of microbiological 1,2-dehydrogenation.
For example, the known method for producing 1,2-degidro-Δ4-3-ketosteroid, which is that the corresponding 1,2-saturated Δ4-3-ketosteroid subjected to interaction with air-dried or heat cells of Arthrobacter simplex containing from 1 to 10% moisture, where the cells are dried in the absence of organic solvent [US 4524134, 1985]. According to SP is soba (example 2) suspension of micronized substrate in DMF contribute in phosphate buffer solution (pH 7.5), containing a suspension of dried cells of A. simplex was ATSS 6946 (10 g/l) and menadione (86 mg/l). The concentration of DMF in the environment is 2% v/v, and 1,2-dehydrogenation is carried out with the load of the substrate of 2.5 g/l within 24 h at a temperature of 31aboutC. However, in this way get ADD from HELL, Δ9(11)-ADD of Δ9(11)-HELL, and other 1,2-dihydroprogesterone number of androstane and pregnane, and it is not described receiving exemestane (6-methylene-ADD) of 6-methylene-HELL.
The relatively low concentration of substrate characterizes this method of producing 1,2-dehydrofreezing as ineffective.
Also there is a method of transformation of 1,2-saturated 3-keto steroids through 1.2-degidro-3-ketosteroids, which is that the 1,2-saturated 3-ketosteroids is subjected to interaction with the drug, with steroid-1,2-dehydrogenase activity of Arthrobacter simplex or Bacterium cyclooxydans, in the presence of exogenous carrier electrons and one or more additional scavenger of toxic oxygen selected from the group consisting of catalase, superoxide dismutase or platinum [US 4749649, 1988]. According to this method steroid substrate can be made in the form of powder, aqueous paste, or in the form of a solution (or suspension) in miscible with water, an organic solvent, such as DMF, DMSO, ethanol, methanol, acetone, in an amount not more than 5% of konechno the medium volume. The method is illustrated by example 1, the description of which, however, does not specify which of the above types making of the substrate used (load substrate 3.5 g/l). In bioconversions environment, even in the presence of menadione buildup Δ1-dehydrogenation takes place only at 50%.
Thus, the application of miscible with water and organic solvents at low concentrations up to 5% v/v not significantly increase the solubility of the substrate in the aquatic environment and its availability to the cells of the microorganism and does not allow to increase the efficiency of the process.
Consider the way [US 4749649, 1988] proposes the use of exogenous enzyme catalase. Adding catalase in an amount of 10 mg/l (equivalent to 16000 IU/l) contributes to the active transformation of the substrate and increases its load to 10 g/l (example 1). For 22 hours, the degree of conversion of the substrate is 83%for 46 hours - 95%. In addition, in the presence of catalase and of menadione is complete suppression of destructive activity.
However, the appreciation of the technology due to the need to add to the environment of the absorber (enzyme catalase, or superoxide dismutase, or platinum)that eliminates the inhibitory effect of singlet oxygen on the activity of dehydrogenase, defines the main disadvantage of this method also in a known manner get ADD from HELL, Δ9(11)-ADD of Δ9(11)-HELL, and other 1,2-dihydroprogesterone number of androstane, and it is not described receiving exemestane (6-methylene-ADD) of 6-methylene-HELL.
It is known that to increase the solubility of steroid substrates in the aquatic environment also use α, β, or γ-cyclodextrins, and derivatives of β-cyclodextrin (β) methyl-β and hydroxypropyl-β at a concentration of 1-50 g/l [Szejtli J. The use of cyclodextrins in biotechnological operations. Ed.D.Duchene, Published in France by Editions de Sante. 1991, 625 p]. In particular, there is a method for producing 1,2-dehydrofreezing Delta-3-ketosteroids by transforming Delta-3-ketosteroids using microorganism Arthrobacter globiformis 193 in the presence of cyclodextrin, characterized in that the cyclodextrin used is a water-soluble chemically modified derivatives of β-cyclodextrin [EN 2156302, 2000]. According to this method, the use of methyl-β allows the 1,2-dehydrogenation of the substrate at a load of 20 g/L. When the weight ratio of methyl-β/substrate 7/1 full conversion of the substrate is completed within 6 hours. The content of 1,2-negidrirovannogo product in the culture fluid is 95%. However, the practical significance of this way of 1,2-dehydrogenation largely limited to the use of large quantities of expensive methyl-β. Additionally, in a known manner get ADD from HELL (example 12) and it is not described receiving exemestane (6-methylene-ADD) of 6-methylene-HELL.
Along with the use of cyclodextrins to enhance the concentration and availability for microorganisms steroid substrates using synthetic polymers of the class of N-vinylamide - polyvinylpyrrolidone (PVP), polyvinylcaprolactam (PVA) and polyvinyl alcohol (PVA) [V AV, Andryushina VA, Arinbasarova, A., Pashkin I.I., Savinova T.S., Stetsenko T.S., Voishvillo N.E. Biotechnology, 2008, No.1, p.46-50].
A known method of producing dehydrolinalool steroids, including microbiological transformation of the corresponding Δ4-3-ketosteroids in the presence of a polymer material, followed by separation from the reaction medium of the target product, characterized in that the polymer material used Homo - or copolymer of N-vinylcaprolactam with molm 104-106or copolymers of N-vinylcaprolactam with vinyl alcohol, vinyl acetate or vinylethylene with the contents thereof to 46%, 45% and 15%, respectively [EN 2042687, 27.08.1995]. However, the known method declared in the General form and the invention is not illustrated examples, it is not described receiving exemestane of 6-methylene-HELL.
Intermediate compound in the synthesis of exemestane of androst-4-ene-3,17-dione (AD) is 6-methylene-HELL.
Known methods for producing 6-methylene-HELL from HELL.
In particular, a method of obtaining a 6-methylene-3-oxo-Δ4-Stero the Dov, which includes the processing of 3-Talavera 6-hydroxydutasteride acidic reagent capable of regenerating Δ4-3-ketosteroids from their 3-telefonov and simultaneously to recover 6-hydroxymethylene group in the 6-methylene [US 3112305, 1963]. According to this method, a 6-methylene-HELL is obtained from 3-methyl Talavera 6-hydroxymethyl-HELL dehydration 90% formic acid, which can be obtained from the HELL by the following sequence of reactions: obtain 3-methyl Talavera HELL, its interaction with the reagent of Vilsmeier [US 3114750, 1963], hydrogenation of the obtained 6-formyl derivative [US 3095411, 1963] (outputs not specified) (figure 2).
Figure 2. Synthesis of 6-methylene-HELL from HELL using the reaction of Vilsmeier
Also known is a method of obtaining a 6-methylene-Δ4-3-ketosteroid some of the androstane or pregnane, including reaction of the corresponding Δ4-3-ketosteroid with a derivative of formaldehyde in an inert solvent in the presence of a strong acidic condensing agent [US 4322349, 1982; Annen, Klaus; Hofmeister, Helmut; Laurent, Henry; Wiechert, Rudolf: Synthesis, 1982, No. 1, 34-40] (method of direct γ-methyltyrosine). As a derivative of formaldehyde can be used trioxane or dialkylated formaldehyde (methyl, ethyl or isopropyl), and as a condensing agent - pentoxide phospho what and or phosphorus oxychloride. For example, according to this method, the interaction HELL with diethylacetal formaldehyde is carried out in the presence of phosphorylchloride and sodium acetate in an inert solvent boiling within 5.5 hours, the product is distilled chromatographytandem on silica gel (figure 3).
Figure 3. Synthesis of 6-methylene-HELL from HELL by direct γ-methyltyrosine [US 4322349, 1982]
However, despite a much shorter path to the target product, this method does not allow to obtain an output of 6-methylene-HELL from HELL higher than 68%.
A method of obtaining 6-N,N-disubstituted aminomethylphosphonic steroids [GB 1280569, 1972] and the method of obtaining 6-meilensteine of 6-N,N-disubstituted aminomethylphosphonic steroids [GB 1280570, 1972]. The way to obtain 6-N,N-disubstituted aminomethylphosphonic steroids [GB 1280569, 1972] is that mixed secondary amine, formaldehyde and steroid compound containing in the core of the 3-alkoxy-3,5-diene group, the reaction is carried out in an environment of tetrahydrofuran, and as a secondary amine is used, among others, N-methylaniline. The way to obtain 6-meilensteine [GB 1280570, 1972] is that 6-(N-methyl-N-phenyliminomethyl)-derived steroids obtained in a manner analogous to [GB 1280569, 1972], mixed with a strong acid. According to this method, 6-(N-methyl-N-phenyliminomethyl)-steroide is derived dissolved in 6 N hydrochloric acid, the solution was incubated for 3 h at room temperature, the precipitate is filtered off. However, there are ways to get 6-(N-methyl-N-phenyliminomethyl)-derivative Δ4-3-catasetinae series pregnane and 6-(N-methyl-N-phenyliminomethyl)-derivative of 17β-hydroxy-17α-methylandrosta-4,9(11)-Dien-3-one, and their 6-methylene analogues, and they do not describe how to get 6-(N-methyl-N-phenyliminomethyl)-derived from AD and 6-methylene-HELL.
The closest entity to the proposed method of obtaining 6-methylene-HELL from HELL is a method of obtaining exemestane and its derivatives by the reaction of 3-Talavera HELL or its derivatives with 30-40% aqueous solution of formaldehyde equivalent and 1-2 kaleidotrope amine salt in an organic solvent at a temperature of from 30 to 50°C To produce 6-methylene-HELL or its derivatives described Along and Lombardi [US 4990635, 1991]. The method is illustrated by an example (example 1), namely, that in the synthesis of 6-methylene-HELL from HELL last process triethylorthoformate in the environment of tetrahydrofuran containing ethanol (13,3%), in the presence of p-toluenesulfonic acid at a temperature of 40°C for 2 h, then added N-methylaniline and 40% aqueous formaldehyde solution and after exposure for 2 h at 40°C. the reaction mass is treated with concentrated hydrochloric acid at room temperature. The product is crystallized of rasba the population of the reaction mixture with water. 20 g of starting compound obtain 14.8 g of 6-methylene-HELL with a molar yield of 71%. (Figure 4).
Figure 4. Synthesis of 6-methylene-HELL from HELL using the reaction of manniche [US 4990635, 1991].
A similar method of obtaining 6-methylene-HELL from HELL used in patents [CN 1415624, 2003; CN 1491957, 2004], while the product obtained with the yield of 68.6%.
The disadvantages described in US 4990635 method are:
- long duration of phase enolizatsii (2 h at 40°C);
- fractional flow of catalyst p-toluenesulfonic acid in the process enolizatsii;
- the use of large amounts of hydrochloric acid during deamination;
- low yield of 6-methylene-HELL, despite the fact that the process is carried out without isolation of intermediates.
It is known that the reaction mechanism of manniche (acid catalysis) is the initial formation of a highly reactive intermediate particles - iminium cation formed from an amine and carbonyl components, which stabilised acid. High reactivity iminium salts imposes some restrictions on the choice of solvent for the reaction of aminomethylpyridine. Usually in the synthesis of non-steroidal bases of manniche prefer to use a highly polar solvents (for salt was in the solution), unable to chemical wsimages the view with iminium salt: acetonitrile, dimethyl sulfoxide, dimethylformamide, etc.
In addition, the choice of solvent for the reaction of manniche should take into account the peculiarities of the steroid substrate. To ensure regioselectively of aminomethylpyridine on atom6and to avoid adverse effects on the atom2located in the α-position to the 3-ketogroup, conduct preliminary enolization Δ4-3-closetime. From the solubility and stability of the latter in the process of aminomethylpyridine depends on the efficiency of the reaction. Therefore, the choice of solvent environment is essential.
In the known methods of obtaining 6-metalinguistic steroids number pregnane and androstane through the formation of 6-(N-methyl-N-phenyliminomethyl)-intermediate using the reaction of manniche in the solvent used tetrahydrofuran [GB 1280569, 1972; US 4990635, 1991; CN 1415624, 2003; CN 1491957, 2004]. Thus, in the patent [GB 1280569, 1972] it is noted that alcohols (methanol, ethanol, propanol, isopropanol, t-butanol) though can be used as solvents in the process of aminomethylpyridine, however, polar aprotic solvents, such as tetrahydrofuran, dioxane, 1,2-dimethoxyethane more effective. In addition, the use of aliphatic alcohols as solvents in the reaction of manniche with non-steroidal compounds of the structure is considered to be undesirable because the spirits of the venture is able to react with formaldehyde and a secondary amine with the formation of the product O-aminomethylpyridine.
However, in the patent [GB 1280569, 1972] does not describe how to obtain 6-(N-methyl-N-phenyliminomethyl)-derived HELL. In addition, information about the study of the effect of solvent environment on the process parameters 6-aminomethylpyridine steroids available in the literature is missing.
It is also known that the splitting of manniche (including steroids) may be ambiguous. In addition to the target product of the deamination hectometre-compound (A) can be formed product reversible process desamination (C)representing the source compound in the synthesis (figure 5).
Figure 5. Directions of cleavage steroid grounds manniche.
Moreover, the selectivity of the process of deamination significantly depends on the conditions of its carrying out. Despite the fact that the optimal way of splitting due to C-N phenylsilane of aminomethylation (R1=Ph) with the formation of 6-methylenephosphonic is handling the concentrated mineral acid [Mannich bases, Maurilio Tramontini, Luigi Angiolini //CRC Press. Inc. 1994. P 78-80], the nature of the solvent medium and temperature are also important. Ensuring regioselectively splitting of N,N-disubstituted 6-aminomethyl-derived HELL is of great importance in deciding the b efficiency and industrial applicability of this method. However, information about the impact of the last two factors on the reaction selectivity deamination 6-aminomethylphosphonic steroids available in the literature is virtually nonexistent. In the patent [GB 1280570, 1972] there is no information about the quality of the products obtained deamination 6-(N-methyl-N-phenyliminomethyl)-steroids. In addition, not described the splitting of 6-(N-methyl-N-phenyliminomethyl)-derived HELL with the formation of 6-methylene-HELL.
In the literature there is no information about the study of the feasibility of combining sequential processes enolizatsii, aminomethylpyridine and deamination and holding them without isolation of intermediates.
The closest entity to the proposed method of obtaining exemestane of 6-methylene-HELL is a method of obtaining 6-medienanstalt-1,4-diene-3,17-dione, which consists in contacting 6-medienanstalt-4-ene-3,17-dione with Δ1-dehydrating enzymes A. Simplex in the presence of water-immiscible organic solvent and exogenous electron acceptor [WO 0104342, 2001]. According to the method for carrying out the process of the 1,2-dehydrogenation of 6-methylene-AD using whole cells or cell-free extract of Arthrobacter simplex ATCC 6946, with the use of whole cells is preferable. The process is carried out in a two-phase system using immiscible with the ode organic solvent, selected from the group of toluene, xylene, benzene, heptane, methylene chloride, n-octanol, or mixtures of these solvents, in the presence of chemical exogenous electron acceptor (4% of menadione in relation to the weight of the substrate) and the enzyme catalase. Although the authors claimed that the substrate 6-medienanstalt-4-ene-3,17-dione is present in the environment at concentrations from 10 to 125 g/l (p and 19 formula), the example And illustrating the method, the concentration of the primary substrate is of 55.6 g/L. Thus, for the process 6-medienanstalt-4-ene-3,17-dione (50 g) and menadione (2.0 g) is mixed with toluene (800 ml). The aqueous phase (pH 8.7-8.9), prepared as a mixture of catalase, discalificata, cellular concentrate A. Simplex (50 ml) and water (50 ml), add to intensively mix the mixture in toluene. The process is carried out at 30aboutC, the reaction mixture is blown adjustable mixture of air and nitrogen and air (0.1% SCHF) and nitrogen (0.3% SCHF). If necessary during the reaction type cells A. Simplex to complete the process. Moreover, the number of added advanced cell concentrate is not specified. After the process is complete, the organic phase is separated, the aqueous phase is extracted with toluene. After filtration and clarification toluene solution is concentrated and to the residue add a 2.5-fold volume number octane crystalline product is filtered off.
Under the headed the remainder described in WO 0104342 process are:
- use of the enzyme catalase in the amount of 0.03% by weight of the substrate, which increases the technological process and limits its industrial application;
- the use of large amounts of flammable organic solvents in the transformation stage, which requires a special fermentation equipment and the special rules of fire safety;
- the use of toxic organic compounds poses a threat to the health professionals involved in the production. Used aromatic hydrocarbons (benzene, toluene, xylene) are potent poisons that affect the function of blood in the body. So, for example, toluene causes cyanosis, hypoxia, Central nervous system, as well as toluene substance abuse, which has carcinogenic effects.
- the need to solve environmental problems;
the difficulty of carrying out the process in terms of the necessary regulation of the composition of the gas mixture supplied into the reaction environment;
- lack of information about the selectivity of the transformation process and the efficiency of the allocation process of the crystalline product, i.e. on the degree of conversion of the substrate into the product and the yield of crystalline product (indicated only that the number of nontransgenic source substrate composition is employed, 0,1%);
- lack of information about the quality of the received exemestane;
- the absence of the sample, confirming the applicability of the method when the load of the substrate to 125 g/L.
The technical task of one of the inventions claimed the group is, therefore, greater efficiency, maintainability and safety of the microbiological 1,2-dehydrogenation and getting 6-medienanstalt-1,4-diene-3,17-dione 6-medienanstalt-4-ene-3,17-dione by the way, devoid of these shortcomings.
Technical problem in the claimed group of inventions is to increase the yield of the target products by reducing the probability of occurrence of adverse reactions during the formation of 3-Talavera HELL, at the stage of introduction of N,N-disubstituted aminomethyl substituent in position With6molecules of Androstenedione, almost complete elimination of by-products formation at the stage of deamination and education 6-methylene group and stage of 1,2-dehydrogenation and getting 6-medienanstalt-4-ene-3,17-dione and 6-medienanstalt-1,4-diene-3,17-dione ways, devoid of the above disadvantages.
The technical problem is solved by a method of obtaining 6-medienanstalt-4-ene-3,17-dione of the formula (I)
from Androstenedione formula (II)
including pre-enolization Δ4 -3-closetime with the formation of 3-Talavera Androstenedione formula (III),
three-component condensation with formaldehyde and a secondary amine under conditions of acid catalysis with the formation of N,N-disubstituted 6-aminomethylpropanol Androstenedione formula (IV)
deamination in a medium containing a strong mineral acid, characterized in that as the medium for the reaction of aminomethylpyridine use proton polar solvents, and as a medium for the reaction deamination use aprotic solvent or mixture of aprotic solvents. Proton solvents as the medium for the reaction of 6-aminomethylpyridine steroid substrate range of androstane earlier in this process has not been applied. Aprotic solvents of the stated group of solvents or mixtures thereof as a medium for the reaction deamination of N,N-disubstituted 6-aminomethylphosphonic steroids number of androstane previously in similar processes were not applied.
Another invention claimed is the method of obtaining 6-medienanstalt-1,4-diene-3,17-dione, 1,2-dehydrogenation using as the starting material 6-medienanstalt-4-ene-3,17-dione, obtained by the above described method according to izobreteny is, microbiological method transformation using bacterial cells Nocardioides simplex VKM AC-D and subsequent selection of the target product from the culture fluid, wherein the transformation is carried out in an aqueous medium containing up to 40% miscible with water and organic solvent. The use of large concentrations miscible with water and organic solvent in the environment for the process of 1,2-dehydrogenation of steroid substrate is not obvious and previously large concentration miscible with water, organic solvent such processes were not applied.
The technical problem in obtaining 6-medienanstalt-1,4-diene-3,17-dione using as a source (intermediate compound) obtained 6-medienanstalt-4-ene-3,17-dione is achieved by obtaining 6-medienanstalt-1,4-diene-3,17-dione of formula (V)
from 6-medienanstalt-4-ene-3,17-dione of the formula (I),
microbiological method 1,2-dehydrogenation in a medium containing exogenous electron acceptor and miscible with water aprotic polar solvent, using an initial substrate concentrations from 5 to 100 g/l and the selection of the target product from the culture fluid.
Thus, the essence of the claimed group of inventions, to the which includes obtaining 6-medienanstalt-4-ene-3,17-dione and next 6-medienanstalt-1,4-diene-3,17-dione with its use is that Androstenedione is first subjected enolizatsii Δ4-3-closetime, then a three-component condensation with formaldehyde and a secondary amine under conditions of acid catalysis with the formation of N,N-disubstituted 6-aminomethylpropanol of Androstenedione in the environment of the proton polar solvent, then the deamination with the formation of 6-methylene derivative HELL in the environment aprotic solvent (or mixture of aprotic solvents) with the subsequent conduct of microbiological reactions of 1,2-dehydrogenation in an aqueous medium containing up to 40% polar aprotic solvent, miscible with water.
With the aim of increasing output and simplify enlever HELL are aminomethylpropanol position With6the reaction manniche in the environment of the proton polar solvent and then regioselective cleavage of Mannich bases before the formation of the 6-methylene group in the environment aprotic solvent (or mixture of aprotic solvents).
This three-component condensation-type reaction of manniche spend with the release of the product from the reaction medium, and the deamination reaction is carried out in optimal conditions.
In addition, as the proton polar solvent used aliphatic alcohols from the group of methanol, ethanol, propanol, isopropanol, etc.
In addition, as APRO the traditional organic solvent used dealkylation (for example, acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g. benzene, toluene), chlorinated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane), and mixtures thereof in an effective ratio.
In addition, as a secondary amine using N-methylaniline, and the formaldehyde used in the form of 37-40% aqueous solution.
In addition, follow-1,2-dehydrogenation order to obtain 6-medienanstalt-1,4-diene-3,17-dione is performed with the use of bacterial cells Nocardioides simplex VKM AC-D.
In addition, as a polar aprotic organic solvent in the conduct of the 1,2-dehydrogenation using dimethylformamide or dimethylsulfoxide, preferably dimethyl sulfoxide. The concentration of DMF in the aquatic environment is not less than 8% vol., as the concentration of DMSO in the aquatic environment is 8-40%.
In addition, the concentration of the starting substrate while conducting 1,2-dehydrogenation is from 5 to 100 g/l, preferably 100 g/l
In addition, as an exogenous electron acceptor use mutation in an effective amount.
The advantages of the proposed method are as follows:
- No need to use multiple crystallization of the main product or chromatography was carried out with the aim of cleaning it.
- High efficiency of the process enolizatsii. In the course of being received for Talavera HELL is 94,4%.
- High selectivity and facilitate reaction of aminomethylpyridine. Exit at the stage of obtaining N,N-disubstituted 6-aminomethylpropanol HELL quantitative;
The high selectivity of the process of deamination. The output stage is 85-88%;
- Total output 6-medienanstalt-4-ene-3,17-dione from Androstenedione is 83%.
The method according to the present invention allows the process 1,2-dehydrogenation:
- under high load of the substrate to 100 g/l in soft conditions;
without the use of flammable organic solvents;
without additional steroid compounds, ensuring stabilization 1,2-dehydrogenase activity;
the method is environmentally safe, as well as mixing with the water solvent serves DMSO - high-boiling liquid with low toxicity and a flash point of 89°C (closed Cup), used in medicine in the form of aqueous solutions as a local drug anti-inflammatory and analgesic actions (trade name Dimexide);
the method eliminates the use of the enzyme catalase and any other scavengers of singlet oxygen;
the method can be implemented on standard manufacturing equipment.
Getting 6-medienanstalt-4-ene-3,17-dione of the formula is (I) and 6-medienanstalt-1,4-diene-3,17-dione of formula (V) are carried out according to the scheme, figure 6.
Figure 6. Synthesis of exemestane from HELL through a 6-methylene-AD by the claimed method
Below cited as an example of a detailed description of the invention.
Androstenedione formula (II)
put enolizatsii Δ4-3-closetime (emitting product enolizatsii (III), for example by the action of a catalytic amount of sulfosalicylic acid. Obtained access to 94.4% of methyl 3-enlever AD (III) is subjected to interaction with the reagent manniche formed in situ under the conditions of acid catalysis (p-TAC) from formaldehyde and a secondary amine (N-methylaniline), in the environment of the proton polar organic solvent to form N,N-disubstituted 6-aminomethylpropanol (IV), which after separation from the reaction mass is subjected to regioselective the deamination under the conditions of catalysis with a strong mineral acid (sulfuric or hydrochloric) in aprotic solvent (or mixture of aprotic solvents) with the formation of 6-methylene derivative (I). Obtained with the yield up to 88% 6-medienanstalt-4-ene-3,17-dione (I) next turn 6-medienanstalt-1,4-diene-3,17-dione (V) the introduction of the 1,2-double bond method microbiological dehydrogenation involving cells of the microorganisms, about adusa 3-ketosteroid-Δ 1-dehydrogenase activity (preferably cells of bacteria Nocardioides simplex VKM AC-D), and the load of the source substrate is from 5 to 100 g/l, and the length of fermentation - from 24 to 52 hours and the process is carried out in an aqueous medium containing exogenous electron acceptor and miscible with water aprotic polar solvent is DMF or DMSO, preferably DMSO).
Using miscible with water and organic solvents on the stage of the 1,2-dehydrogenation provides translation of steroid substrate - 6-methylene-HELL, or the greater part thereof in soluble form and process at high (50-100 g/l) loads substrate greatly reduces the effect of cocrystallization substate and product and formation of mixed crystals. In the literature there are no data on the use miscible with water and organic solvents used in high concentrations (20-40% V/V).
Analysis of the products of microbial transformation carried out by thin layer chromatography (TLC) and HPLC.
Determination of the contents of the products by HPLC performed on the chromatograph Agilent 1100/1200 (Agilent, Germany) with precolonial Symmetry C18, 5 μm, of 3.9×20 mm and column Symmetry C18, 5 μm, a 4.6×250 mm (Waters, Ireland); in the system with mobile phase (v/v): 60% acetonitrile, 10% isopropanol, in 0.01% acetic acid and water to 100%, at a flow rate of 1 m is/min, 50°C, and detection by absorption at 250 nm; typical retention times: 6-methylene-HELL of 4.9 min and 6-methylene-ADD (exemestane) of 3.2 minutes
Selection 6-medienanstalt-1,4-diene-3,17-dione (V) of the culture liquid can be carried out by extraction of water-immiscible solvent or extraction of steroid pasta biomass in any suitable solvent, after preliminary separation from the aqueous phase. To extract steroid can also be applied, for example, sorption method of extraction, as well as any other effective method. The selectivity of the formation of 6-medienanstalt-1,4-diene-3,17-dione (V) at the stage of microbiological 1,2-dehydrogenation is 95-98%. The yield of crystalline 6-medienanstalt-1,4-diene-3,17-dione (V) at the stage of microbiological 1,2-dehydrogenation after isolation and purification is 85-87%.
The overall yield of 6-medienanstalt-1,4-diene-3,17-dione (V) from Androstenedione is from 70 to 72%.
The claimed group of inventions is illustrated by the following examples without limiting it.
Education 3-Talavera HELL, make use of a minimal number of necessary trialkylamine in the environment corresponding absolute alcohol in the presence of an acidic catalyst. As the acidic catalyst can be used in a strong organic acid, n is an example of p-toluensulfonate or sulfosalicylic acid.
N,N-Disubstituted 6-aminomethylpropanol Androstenedione formula (IV) are obtained in aqueous aliphatic alcohol with a water content of from 5 to 10%.
As a secondary amine using N-methylaniline, and the formaldehyde is used in the form of a 37% aqueous solution.
As proton solvent at the stage of aminomethylpyridine in the implementation of the present invention, the method of obtaining 6-medienanstalt-4-ene-3,17-dione can be used, for example, aliphatic alcohols (methanol, ethanol, propanol, isopropanol and others).
As the aprotic solvent in carrying out the invention is a method of producing 6-medienanstalt-4-ene-3,17-dione under deamination can be used, for example, dealkylation (for example, acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g. benzene, toluene), chlorinated hydrocarbons (e.g. dichloromethane, chloroform, dichloroethane), and mixtures thereof in an effective ratio.
As the acidic catalyst under deamination can be used mineral acid (preferably sulfuric or hydrochloric acid).
Microbiological 1,2-dehydrogenation in the implementation of the present invention, the method of obtaining 6-medienanstalt-1,4-diene-3,17-dione can be implemented resting (washed) cells of bacteria of the genus Nocardioides, specifically Nocardioides simplex VKM AC-D.
As a component of the environment during implementation of the invention is a method of producing 6-medienanstalt-1,4-ene-3,17-dione can be used miscible with water and organic polar aprotic solvents such as DMF or DMSO, preferably DMSO.
The method is illustrated by examples.
Example 1. Obtaining 3-methoxyindole-3,5-Dien-17-she (3-enlever HELL, III).
A suspension of 100 g AD (I) in the mixture for 85.6 ml triethylorthoformate and 130 ml of anhydrous methanol is stirred for 15 minutes, then slowly add 120 ml of a solution of 0.2 g of anhydrous sulfosalicylic acid in methanol. The reaction mass is maintained at 20-25°C for 1 hour and 15 minutes. After aging the reaction mass is then cooled to 10-15°C, slowly add 75 ml of dry acetone and incubated for 1 hour at the same temperature. Then add 0.8 ml of triethylamine to pH 7-8, the reaction mass is cooled to 0-2°C and add 225 ml of water. After aging the precipitate is filtered off, washed on the filter with water and 0.25% solution of triethylamine in methanol. Get 99 g 3-Talavera AD (III) with the release of 94,4%. TPL=171-172°C (lit. 171-173°C [RU 2163606, 2001]). NMR1N, δ, ppm: 0.90 c (3 H, 18-CH3), 0.98 c (3 H, 19-CH3), 1.02-2.52 m (17 H), 3.56 (3 H, och3), 5.13 d, J=1.2 Hz, 1 H, H4), 5.22-5.25 m (1 N, N6).
Example 2. Getting 6ξ-(N-methyl-N-phenyliminomethyl)-androst-4-ene-3,17-dione a Mixture of 6α and 6β isomers, IV).
To a suspension of 30 g of 3-Talavera AD (III) in 150 ml of methanol add to 12.9 ml of N-methylaniline and was 7.45 ml of 37% aqueous formaldehyde solution, and then slowly add a solution of 0.75 g of p-TJC in 10 ml of methanol. The suspension is incubated for 15-30 min at room temperature, then 1 h at a temperature of 30-40°C. to dissolve the suspension. After the reaction solution was poured in a 10-fold volumetric amount of water containing 0.1 g of NaOH. The precipitate is filtered off, washed with water until pH~7. Get 40,42 g of compound IV (a mixture of 6α and 6β isomers).
Found: 80.16%; N 8.92%; N, 3.71%, C27H35NO2. Calculated: 79.96%; N, 8.70%, N 3.45%
6α-isomer: NMR1N, δ, ppm: 0.92 (3H, 18-CH3), 1.31 (3H, 19-CH3), 2.98 (3H, 21-CH3-N), 3.25 m (1H, CH2-N), 3.77 m (1H, CH2-N), 5.84 (1H, H4), 6.50-7.25 m (5H, Ph);13C, δ, ppm: 199.02, 170.44, 148.58, 129.17, 126.53, 116.49, 112.14, 56.97, 55.33, 54.10, 52.88, 50.50, 47.24, 42.52, 39.99, 39.07, at 38.15, 37.24, 35.51, 34.65, at 33.85, 31.04, 30.34, 21.24, 20.13, 18.21, 13.55. 6β-isomer: NMR1N, δ, ppm: of 0.87 (3H, 18-CH3), to 1.21 (3H, 19-CH3), 2.97 (3H, CH3-N), 5,74 (1H, H4).
Example 3. Getting 6-medienanstalt-4-ene-3,17-dione (6-methylene-HELL, I).
To a suspension of 40 g of a mixture of 6α and 6β isomers of compound IV in 200 ml of acetone add to 28.5 ml of 32% hydrochloric acid. The mass is maintained at room temperature for 1 h and poured into 10-fold volume of water. The precipitate is filtered off, washed with water until pH~7 Obtain 26.7 g of 6-methylene-AD (I) with the release of 90.7 percent.
After recrystallization from aqueous ethanol gain of 25.9 g of compound I with the release of 88%.
MP.=160-163°C (lit. 159-162°C [CN 1415624, 2003]).
NMR1N, δ, ppm: 0.86 (3H, 18-CH3), 1.07 (3H, 19-CH3), 5,00 d (J=44.3 Hz, 2H, 6-C=CH2), 5.88 (1H, H4).
To a suspension of 13.5 g of a mixture of 6α and 6β isomers of compound IV in a mixture of 100 ml of toluene and 50 ml of methyl ethyl ketone is added 15 ml of 60% aqueous sulfuric acid. The mass is maintained at room temperature for 1 h, separated acidic layer and extracted twice with 20 ml of toluene. A solution of the product in toluene was washed with 60% sulfuric acid solution and water, brightening process coal. The solvent is evaporated to dryness, the residue is crystallized from aqueous ethanol. Obtain 8.5 g of 6-methylene-AD (I) exit for 85.6%.
Example 4. Getting 6-medienanstalt-1,4-diene-3,17-dione (V)
In use, the strain Nocardioides simplex VKM AC-D, which is supported on agar medium of the following composition (g/l): glucose - 10, soy peptone - 4, yeast extract - 4, MgSO4- 0.5, KH2PO4- 2, K2HPO44 agar - 20, distilled water, pH 7.0-7.2. The cultivation is carried out in the next 3-4 days at 30aboutC.
The cultivation of the 1st inoculum
Washing the culture with the surface of the agar medium in 5 ml of saline solution to make a 750 ml Erlenmeyer flask with 50 ml of medium (g/l): glucose - 15, the soy peptone - 6, yeast extract - 6, MgSO4- 0.5, KH2PO4- 2, K2HPO4- 4, and cultured at 28 to 30aboutFor 24-26 hours.
The cultivation of transforming culture.
5 ml of the culture fluid at 24-26 hours growth 1 inoculum make 100 ml of medium of the same composition and spend growing in similar conditions for 15 hours, then make the inducer of the synthesis of steroid-Δ1-dehydrogenase - acetate cortisone in the form of an ethanol solution (20 mg in 1 ml ethanol). The induction of the synthesis of Δ1-dehydrogenase is carried out in the next 24 to 26 hours. Cells of microorganisms precipitated by centrifugation at 5000 g for 30 min and used for process 1-dehydrogenation or stored at -18aboutWith over a month to use.
Transformation of 6-methylene-HELL
The transformation of 6-methylene-AD (I) is carried out in flasks of 250 ml containing 25 ml of 0.02 M Na-phosphate buffer solution, pH 8.0. Consistently contribute 125 mg of substrate in powder form and add 0.2 g of biomass (dry weight biomass).
The bioconversion is carried out in aerated conditions at 30aboutC on a rotary shaker at 200 rpm Process stop for 72 hours cultivation at achievement of contents 6-methylene-ADD - 91-93 (±3%).
Analysis of the products of the conversion carried out by TLC and HPLC.
The way assests the Ute for option 1, but the reaction medium presents 0.02 M Na-phosphate buffer solution, pH 8.0 (23 ml) and DMSO (2 ml) with menadione.
The method is carried out as per option 2, but the reaction medium presents 0.02 M Na-phosphate buffer solution, pH 8.0 (23 ml) and DMF (2 ml).
The methods provide for option 2, but with a range of concentrations of the substrate from 10.0 to 100.0 g/l (table).
The culture fluid is extracted with ethyl acetate, the extract washed with water, dried over anhydrous MgSO4, lighten activated charcoal and evaporated to dryness in a vacuum. The residue is triturated with petroleum ether, the precipitate is filtered off. Get 6-medienanstalt-1,4-diene-3,17-dione with the release of 85-87%.
MP.=183-185°C (lit. 188-191°C [US 4904650, 1990]). NMR1N, δ, ppm: 0.93 (3H, 18-CH3), 1.16 (3H, 19-CH3), 5.00 d (J=24.7 Hz, 2H, 6-C=CH2), 6.16 d, J=1.9 Hz, 1H, H4), 6.24 DD, J=1.9 Hz, J=10.1 Hz, 1H, H2), 7.07 d (J=10.2 Hz, 1H, H1).
|Option No.||Volume environment, ml||Biomass, g/l (dry weight biomass)||The load of the substrate, g/l||Way to make||Concentration of the solvent, %||The duration of the process, watch the||The content of the product in the environment %|
1. The way to obtain 6-medienanstalt-4-ene-3,17-dione of the formula (I)
from Androstenedione formula (II)
including pre-enolization Δ4-3-closetime with the formation of 3-Talavera Androstenedione formula (III)
three-component condensation with formaldehyde and a secondary amine under conditions of acid catalysis with the formation of N,N-disubstituted 6-aminomethylpropanol Androstenedione formula (IV)
deamination in a medium containing a strong mineral acid, characterized in that as the medium for the reaction of aminomethylpyridine use proton polar solvents, and as a medium for the reaction deamination use aprotic solvent or mixture of aprotic solvents.
2. The method according to claim 1, characterized in that as the proton polar solvent used aliphatic alcohols from the group of methanol, ethanol, propanol, isopropanol, etc.
3. The method according to claim 1, characterized in that as the aprotic organic solvent used dealkylation (for example acetone, methyl ethyl ketone), aromatic hydrocarbons (e.g. benzene, toluene), chlorinated hydrocarbons (for example dichloromethane, chloroform, dichloroethane), and mixtures thereof in an effective ratio.
4. The method according to claim 1, characterized in that as a secondary amine using N-methylaniline, and the formaldehyde used in the form of 37-40%aqueous solution.
5. The way to obtain 6-medienanstalt-1,4-diene-3,17-dione of formula (V)
from 6-stands the androst-4-ene-3,17-dione of the formula (I)
by microbiological 1,2-dehydrogenation in an aqueous medium containing exogenous electron acceptor and miscible with water, the solvent, using the cells of the microorganism with 3-ketosteroid-Δ1-dehydrogenase activity, characterized in that as component of the environment of the use of aprotic polar solvent at a concentration of up to 40%vol.
6. The method according to claim 5, characterized in that the microorganism with 3-ketosteroid-Δ1-dehydrogenase activity using Nocardioides simplex BKM Ac-D.
7. The method according to claim 5, characterized in that as the polar aprotic organic solvent used is dimethylformamide or dimethylsulfoxide, preferably dimethyl sulfoxide.
8. The method according to claim 5, characterized in that as an exogenous electron acceptor use menadione in an effective amount.
9. The method according to claim 5, characterized in that the concentration of the primary substrate is from 5 to 100 g/l, preferably 100 g/l
10. The method according to claim 7, characterized in that the concentration of DMF in the aquatic environment is not less than 8 vol.%.
11. The method according to claim 7, characterized in that the concentration of DMSO in the aquatic environment is 8-40%.
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed invention relates to process of crystallisation, obtaining and isolation of novel crystalline form of fusidic acid, to application of said processes in production of pharmaceutical composition or medication, and to application of said form of crystalline fusidic acid in treatment of bacterial infections.
EFFECT: obtaining pharmaceutical composition for treatment of bacterial infections.
11 cl, 10 ex, 1 tbl, 10 dwg
SUBSTANCE: invention enables synthesis of 3-methoxy-2-fluoro-18ethyl-8α-gona-1,3,5(10)-trienes, having osteoprotector and hypocholesteremic activity.
EFFECT: invention has osteoprotector and hypocholesteremic activity and can be used in medicine for hormonal replacement therapy.
1 cl, 3 ex, 1 tbl, 3 dwg
SUBSTANCE: claimed invention relates to application of steroid compounds, preferably 17 α-ethinyl derivatives of androstane.
EFFECT: insuring treatment of inflammatory or autoimmune disease in subject and/or infection prevention.
7 cl, 12 ex, 18 dwg
SUBSTANCE: invention provides administration of triterpene glycosides of holothurian Cucumaria okhotensis chosen from a group consisting of Frondoside A1, Ochotoside B1, Ochotoside A1-1, Ochotoside A2-1 or Cucumarioside A2-5 or their mixtures, as an agent stimulating cell-mediated immunity in mammals, as well as for preparing a pharmaceutical composition stimulating cell-mediated immunity in mammals.
EFFECT: extended range of the agents stimulating cell-mediated immune reaction in mammals.
4 dwg, 1 tbl, 2 cl
SUBSTANCE: invention refers to a method of Δ4-3-ketosteroids 11 β-hydroxylation by a biomass Curvularia lunata strain mycelium, RNCIM No. F-988. For the transformation, Curvularia lunata strain mycelium, RNCIM No. F-988 not older than 30 h and washed of nutrient medium is used. Mycelium is taken in such amount that the relation of the biomass to the transformed steroid makes 1.5-2.5:1. The transformation is performed in a buffer solution, and a steroid substratum is added as a microcrystal suspension, or as a water-soluble methyl - β-cyclodextrine complex with steroid related thereto as 1:1-0.6:1 (mol/mol). The yield of 11β-hydroxyderivatives is 50-80 %.
EFFECT: offered invention allows for higher selectivity of 11β-hydroxylation process, concentration of the transformed steroid substratum up to 20 g/l and reduced reaction period to 24-50 h.
1 tbl, 10 ex, 2 cl
FIELD: medicine, pharmaceutics.
SUBSTANCE: there are described compounds of the following structure or their salts: where A, B, R2, R4, R6, R7, R10, R16, R17α, R17β, Z, Y, X have the values specified in the description. Some of these compounds exhibiting tissue-specific antiandrogen activity and tissue-specific androgen activity can be applied for treating or reducing risk of the diseases associated with androgen stimulation loss.
EFFECT: preparation of the compounds which reducing probability of the androgen-dependent diseases, such as prostate cancer, benign prostate hyperplasia, polycystic ovary syndrome, acne, hirsutism, seborrhoea, etc.
41 cl, 176 ex, 4 tbl
SUBSTANCE: there are described compounds of formula in a free form or in the form of salt where R1 and R2 have values specified in the description of the application which are used for treating inflammatory conditions, first of all inflammatory or obstructive respiratory tract diseases. Besides the application describes the pharmaceutical compositions containing said compounds, and methods for preparing said compounds.
EFFECT: compounds exhibits improved efficiency.
5 cl, 8 ex
SUBSTANCE: described is a 15β-substituted oestradio derivative having selective oestrogenic activity. The preferred compound is 7α-ethyl-15β-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,17β-diol.
EFFECT: obtaining compounds which can be used in treating or preventing diseases or physiological conditions related to oestrogen receptors.
11 cl, 4 ex, 2 tbl
SUBSTANCE: invention relates to new 3.15-substituted estrone derivatives that are inhibitors of 17β-hydroxysteroid-dehydrogenase type 1 (17β-HSD1), to their salts, pharmaceutical compositions, containing specified compounds and to methods of such compound producing. Besides the invention refers to application in medicine of specified new 3,15-substituted estrone derivatives firstly to their application for treatment or prevention of steroid-dependent diseases or disorder such as steroid-dependent diseases or disorders treatment of which requires inhibition of 17β-hydroxysteroid dehydrogenase type 1 and/or reducing of endogenous 17β-estradiol concentration. The invention also relates to general application of selective inhibitors of 17β-hydroxysteroid-dehydrogenase type 1 that do not bind with estrogen receptor or display antipathic affinity to estrogen receptor.
EFFECT: possibility of application for treatment and prevention of benign gynaecologic diseases.
49 cl, 836 ex, 42 tbl
SUBSTANCE: invention refers to synthesis of [18F]fluororganic compounds ensured by reaction of [18F]fluoride and relevant halogenide or sulphonate with alcoholic vehicle of formula 1 where R1, R2 and R3 represent hydrogen atom or C1-C18 alkyl.
EFFECT: possibility for mild process with low reaction time and high yield.
21 cl, 2 tbl, 27 ex
FIELD: organic chemistry, steroids, pharmacy.
SUBSTANCE: invention relates to a new type of selective estrogens comprising steroid structure of the general formula (I) with nonaromatic ring A and free of bound hydroxyl group at carbon atom 3 wherein R1 means hydrogen atom (H), (C1-C3)-alkyl or (C2-C3)-acyl; R2 means hydrogen atom (H), α-(C1-C4)-alkyl, α-(C2-C4)-alkenyl or α-(C2-C4)-alkynyl; R3 means hydrogen atom (H) or (C1-C4)-alkyl at position 16 of steroid structure; R4 means ethynyl; R5 means hydrogen atom (H), (C1-C3)-alkyl or (C2-C3)-acyl; R6 means (C1-C5)-alkyl, (C2-C5)-alkenyl, (C2-C5)-alkynyl being each of that is substituted optionally with chlorine or fluorine atom; dotted line means the optional double bond. Compounds of the formula (I) elicit the selective affinity to ERα-receptors.
EFFECT: valuable properties of compounds and composition.
4 cl, 3 sch, 1 tbl, 8 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention relates to a new compound of the general formula (2) and a method for its preparing wherein R1 represents hydrogen atom or salt-forming metal; R2 represent a direct or branched (C1-C7)-halogenalkyl group; m represents a whole number from 2 to 14; n represents a whole number from 2 to 7; A represents a group taken among the following formulae: (3) , (4) ,
(17) , (18) , (19) , (20) , (23) , (25) and (26) wherein R3 in formula (6) represents a direct or branched group (C1-C5)-alkyl group; R8 in formulae (18) and (20) represents a direct or branched (C1-C5)-alkyl group, a direct or branched (C2-C5)-alkenyl group or a direct or branched (C2-C5)-alkynyl group; in formula (23) each R21, R22, R23 and R24 represents independently hydrogen atom, a direct or branched (C1-C5)-alkyl group, a direct or branched (C1-C7)-halogenalkyl group, halogen atom or acyl group; in formulae (25) and (26) X represents halogen atom; or enantiomers of compound, or hydrates, or pharmaceutically acceptable salts of compound, or its enantiomers. Also, invention relates to a pharmaceutical composition containing indicated compound as an active component and to a therapeutic agent used against breast cancer based on thereof.
EFFECT: valuable medicinal properties of compounds.
10 cl, 2 tbl, 39 ex
FIELD: organic chemistry, steroids, medicine, pharmacy.
SUBSTANCE: invention relates to 3-methylene-steroid derivative of the general formula (1):
wherein R1 means hydrogen atom (H), or in common with R3 it forms β-epoxide; or R1 is absent in the presence of 5-10-double bond; R2 means (C1-C5)-alkyl; R3 means βH, βCH3 or in common with R1 it forms β-epoxide; either R3 is absent in the presence of 5-10-double bond; R4 means hydrogen atom, lower alkyl; Y represents [H, H], [OH, H], [OH, (C2-C5)-alkenyl], [OH, (C2-C5)-alkynyl] or (C1-C6)-alkylidene, or =NOR5 wherein R5 means hydrogen atom (H), lower alkyl; dotted lines represent optional double bond. Compound can relate also to its prodrug used for treatment of arthritis and/or autoimmune diseases.
EFFECT: valuable medicinal properties of compounds, improved method for treatment.
38 cl, 1 tbl, 18 ex
FIELD: organic chemistry, medicine, pharmacy.
SUBSTANCE: invention represents new derivatives of 17,20-dihydrofusidic acid of the formula (Ia)
wherein Q1 and Q2 are similar or different and mean -CO-, -CHOH-, -CHRO- wherein R means (C1-C4)-alkyl; Q3 means -CH2-; Y means hydrogen atom (H); A means -O- or -S-; R1 means (C1-C4)-alkyl, (C2-C4)-olefin, (C1-C6)-acyl, (C3-C7)-cycloalkylcarbonyl, benzoyl. These derivatives are used in pharmaceutical compositions for treatment of infectious diseases, in particular, in composition for topical applying for treatment of infectious diseases of skin and eyes.
EFFECT: valuable medicinal properties of compounds.
22 cl, 7 tbl, 41 ex
FIELD: organic chemistry, steroids, chemical technology.
SUBSTANCE: invention describes a method for synthesis of 7-substituted steroid compounds of the general formula (I):
wherein R1 means hydrogen atom (H) or -COR2 group wherein R2 means (C1-C6)-alkyl, (C1-C6)-alkoxy-group; Z1 means -CH2- or wherein R3 is in α-configuration; R3 means H or -COR2; Z2 means -CH-, or Z1 and Z2 mean in common a double bond; Q means ,,,,,,; Y means -CN, -CH2-CH=CH2 or -CHR4C(O)Ar, -CHR4C(O)-(C1-C6)-alkyl, -CHR4C(O)XAr or -CHR4C(O)X-(C1-C6)-alkyl wherein R4 means -O-(C1-C6)-alkyl or aryl X means oxygen (O) or sulfur (S) atom that are intermediate compounds used in synthesis of eplerenon.
EFFECT: improved method of synthesis.
7 cl, 1 tbl, 2 dwg, 20 ex
FIELD: organic chemistry, steroids, medicine, pharmacy.
SUBSTANCE: invention describes novel halogen- and pseudohalogen-substituted 17-methylene-4-azasteroids of the general formula (I) wherein each R20 and R20a means independently fluorine, chlorine, bromine atom, (C1-C4)-alkyl, hydrogen atom (H), cyano-group; R4 and R10 mean hydrogen atom or methyl group; both R1 and R2 represent hydrogen atom and form an additional bond. Compounds are inhibitors of 5α-reductase and can be used in treatment of diseases caused by the enhanced blood and tissue testosterone and dihydrotestosterone level.
EFFECT: valuable medicinal and biochemical properties of compounds.
9 cl, 5 dwg, 1 tbl, 10 ex
FIELD: organic chemistry, steroids, medicine.
SUBSTANCE: invention relates to steroid compounds of the general formula (I) given in the invention description wherein R1 means oxygen atom (O); R2 and R3 mean independently hydrogen atom (H), CH3, C2H5, and at least radical among R2 and R3 means CH3 and C2H5; R4 means H. Compounds are useful in treatment associated with androgens, such as androgen deficiency and contraception in males and females.
EFFECT: valuable medicinal properties of compounds.
5 cl, 1 tbl, 7 ex
FIELD: organic chemistry, steroids, medicine, pharmacy.
SUBSTANCE: invention relates to compositions containing 16α-bromo-3β-hydroxy-5α-androstane-17-one semihydrate and one or more excipients wherein the composition contains less 3% of water. Compositions are useful in preparing improved pharmaceutical compositions. Invention describes methods for discontinuous dosing steroid compounds, such as analogs of 16α-bromo-3β-hydroxy-5α-androstane-17-one and compositions useful in such dosing regimens. Also, invention describes compositions and methods for inhibition of pathogenic viral replication, improving symptoms associated with disorders in immune response and modulation of immune response in a patient by using indicated compounds and their analogs. Also, invention describes methods for their preparing and using these immunomodulatory compositions.
EFFECT: improved preparing method, valuable medicinal properties of compounds and pharmaceutical compositions.
63 cl, 3 tbl, 13 sch, 13 dwg, 37 ex
FIELD: organic chemistry, steroids, chemical technology.
SUBSTANCE: invention relates to novel effective methods for synthesis of 9,11-epoxy-17α-hydroxy-3-oxopregn-4-ene-7α,21-dicarboxylic acid, γ-lactone, methyl ester (eplerenone). Also, invention describes novel intermediate compounds of the general formula (I): wherein R1 means hydrogen atom (H), -COR4 wherein R4 means (C1-C6)-alkyl, (C1-C6)-alkoxy-group; R3 means (C1-C)-alkyl; Z1 means compound of the formula wherein -O-COR4 is at α-position; Z2 means -CH-, or Z1 and Z2 form in common a double bond; Q means compounds of formulas .
EFFECT: improved methods of synthesis.
28 cl, 3 sch, 17 ex
FIELD: organic chemistry, pharmaceuticals.
SUBSTANCE: invention relates to improved method for production of 4,17(20)-E-pregnadiene-3,16-dione (E-guggulsterone) of formula III and 4,17(20)-Z-pregnadiene-3,16-dione (Z-guggulsterone) of formula IV including oxidation of compound of formula II , wherein C-OH or =O; ----- is optional double bond with pyridinium chlorochromate, pyridinium dichromate etc to produce 4,17(20)-E-pregnadiene-3,16-dione of formula III followed by conversion thereof by photochemical, thermochemical reaction or reaction in presence of acidic catalyst. Compounds of formulae III and IV effectively decrease increased low density lipoprotein levels and high cholesterol levels.
EFFECT: improved method for production of 4,17(20)-Z-pregnadiene-3,16-dione.
8 cl, 46 ex, 9 dwg