3-methylene-steroid derivatives, pharmaceutical composition, method for treatment

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

 

This invention relates to new 3-meilensteine derived, providing a therapeutic effect, as well as to receive pharmaceutical products, including 3-alkylidene steroid.

Steroids with methylene substituents in position 3 of the steroid skeleton and 1-2 or 4-5 double bonds are known and reported as useful for therapeutic applications (BE 696235 and BE 654772, respectively). We offer medical purpose includes anabolic, estrogenic and gestagenna action. None of these compounds was used for this purpose, either as drugs in other areas, despite the fact that the treatment of many diseases that are currently available drugs still remains unsatisfactory. It should be noted that diseases of the immune system such as rheumatoid arthritis and autoimmune diseases, need better drugs. Sometimes for the treatment of these diseases apply corticosteroid means, however, necessary improvements on the effectiveness and on the number or severity of side effects.

This invention provides 3-meilensteine derivatives and their prodrugs, these steroid derivatives have the General formula 1

in which R1represents H or, together with R3forms β-epoxide, or R1absent in the presence of 5-10 or 4-5 double bond;

R2is (C1-C5) alkyl or CF3;

R3is βN, βCH3or together with R1forms β-epoxide, or R3absent in the presence of 5-10 double bond;

R4represents H, lower alkyl;

Y represents [H, H] [IT, N], =O, [HE lower alkyl], [IT, (C2-C5) alkenyl], [IT, (C2-C5) quinil] or (C1-C6) alkylidene, with the specified alkyl, alkenyl, quinil and alkyliden optional galogenidov; or =NOR5in which R5represents H, a lower alkyl.

Dotted lines represent an optional double bond.

Preferred compounds in accordance with this invention are such compounds in which R4represents N and Y represents [IT, N], =O, [HE lower alkyl], [IT, (C2-C5) alkenyl], [IT, (C2-C5) quinil] or (C1-C6) alkylidene, with the specified alkyl, alkenyl, quinil and alkyliden can be optional galogenirovannyie.

In this description, the terms have the following meanings.

Lower alkyl means a branched or unbranched alkyl group having preferably 1-atomov carbon such as hexyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl and methyl. Most preferred are alkyl groups having 1-3 carbon atom.

(C1-C5)alkyl means a branched or unbranched alkyl group having 1-5 carbon atoms, e.g. methyl, ethyl, isopropyl, butyl, sec-butyl, tert-butyl, etc. Preferred are alkyl groups having 1-3 carbon atom.

(C2-C5)alkenyl means a branched or unbranched alkenylphenol group having from 2 to 5 carbon atoms, such as ethynyl, 2-butenyl etc. are Preferred alkeneamine group having 2-3 carbon atoms.

(C2-C5) quinil means a branched or unbranched group having 2-5 carbon atoms, such as ethinyl, PROPYNYL, etc. are Preferred alkyline group having 2-3 carbon atoms.

(C1-C5)alkyliden means a branched or unbranched Gildenlow group having 1-5 carbon atoms, such as metanil, butylidene etc. are Preferred alkylidene group having 2-3 carbon atoms.

Halogen means fluorine, chlorine, bromine and iodine.

Prodrugs are compounds that are intended for the formation of compounds in accordance with this invention in the body of the recipient with C is poured treatment. In General, such prodrugs can represent complex or simple esters of 17-hydroxyl functional group.

The preferred implementation of the present invention relates to the above described connection with 5-10 double bond. Most preferred is the compound (7α, 17α)-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol. In this invention, the following compounds are excluded: (7α, 17β)-7α-methyl-3-methylene-4-estren-17-ol, (7α, 17α)-7-methyl-3-methylene-19-norpregna-4-EN-17-ol, (7α,17α)-7-methyl-3-methylene-19,21-dynorphin-4-EN-17-ol, (7α)-17-keto-7-methyl-3-methylene-4-estren, (7α, 17α)-methyl-3-methylene-19-norpregna-4-EN-20-in-17-ol, 17β-hydroxy-7α-methyl-3-methylene-17α-propen-2-yl-4-estren and 17β-hydroxy-7α-methyl-3-methylene-17α-butene-2-yl-4-estren. The above exceptions are associated with the descriptions presented in the BE 696235.

Epoxy compounds (R1and R3together form β-O-) in accordance with this invention can be obtained by oxidation of compounds having the formula 2, in which the symbols have the above meanings, with metallocarboxypeptidase acid

3-Methylene group in the compound in accordance with the present invention can be obtained by implementation of the Wittig reaction (Wittig) with application b is Amida methyltriphenylphosphonium and tert-butoxide potassium with the corresponding 3-keto-steroid precursors, characterized by the formula 3, in which Y2has the same meanings as given above Y, except for a =O, and the remaining symbols have the above values.

17-Alkylidene compounds in accordance with this invention can be obtained by implementation of the Wittig reaction derived 3-Catala 17-ketopropane according to the formula 4, in which the symbols have the above meanings, with bromide alkyldiphenylamine with subsequent hydrolysis of the 3-Catala. Compounds in which Y=H, and H is in position 17 (formula 2), in accordance with this invention can be obtained by reduction of wolf-Kishner (Wolff-Kishner) 17-keto remainder of the compound 3-Catala formula 4, in which the symbols have the above meanings. Derivatives of 17-oxime in accordance with this invention can be obtained by condensation of the 17-keto function (formula 4) with hydroxylamine derivatives. 16-Alkyl compound in accordance with this invention can be obtained by alkylation provisions 16 17 catasetinae (Y=O, formula 4).

The above reagents for the conversion of the parent compounds and their interactions with compounds known in this area, but still they were not applied to the connection group formed by the DL is producing compounds in accordance with this invention. Source materials can be obtained by methods described in the literature. Especially close to publication is Van Vliet et al. The Reel. Trav. Chim. Pays-Bas; EN; 105; 4; 1986; 111-115 included in this description by reference. It should be noted that it describes the possible receipt of a 17-alkyl-Z-ketopropionic in accordance with the formula 3, in which Y2is [HE lower alkyl], and the other symbols have the above meanings, by catalytic hydrogenation, for example, with the use of PtO2/H2the compounds in accordance with formula 3, in which Y2is [HE, alkenyl] or [HE quinil], and the other symbols have the above values.

Derivatives in accordance with formula 5 can be obtained by purification from a mixture of compounds formed after the restoration of Birch (Birch) derived 4-EN-3-one according to formula 6, as in the formula 5 and formula 6, the symbols have the above values.

Derived 4-EN-3-one according to formula 6, in which R3is N, and the other symbols have the above meanings, can be obtained from compounds in accordance with formula 7 by the isomerization of double bonds (in accordance with the method described by van Vliet et al., 1986).

Derived 4-EN-3-one according to F. what rmulas 6, in which R3is methyl, and the other symbols have the above meanings, can be obtained in accordance with methods described in the publication Grunwell et al.; Steroids, Vol.27, pages 759-771, 1976, cited here as a reference.

Further methods for producing compounds in accordance with formula 7 as a starting material described by van Vliet et al., 1986.

The use of compounds defined by formula 1, is intended for immunomodulation in mammals. Connection in accordance with this invention can be, in particular, used to treat arthritis, such as rheumatoid arthritis (RA)and autoimmune diseases (AIDs), such as Sjogren syndrome and systemic lupus erythematosus (SLE). It can also be used for disease prevention. Moreover, the connection in accordance with this invention can be used to produce pharmaceutical preparations containing a compound in accordance with this invention as the active ingredient.

The present invention also relates to a pharmaceutical composition comprising a steroid compound in accordance with this invention, mixed with one or more pharmaceutically acceptable auxiliary substances, for example, described in the standard reference, Gennaro et al., Remmington''s Pharmaceutical Sciences (18thed., Mack publising Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture). A mixture of one or more steroid compounds in accordance with this invention and one or more pharmaceutically acceptable excipients may be compressed into solid dosage forms such as pills, tablets, or processed to produce capsules or suppositories. When using a pharmaceutically suitable liquids these compounds can also be used as a drug for injection in the form of a solution, suspension, emulsion or aerosol, for example, a nasal spray. To get dosage forms, e.g. tablets, used known additives such as fillers, dyes, polymeric binder, etc. In General, can be used in any pharmaceutically acceptable additive, not affecting the function of the active compounds. Steroid compounds in accordance with this invention can also be incorporated into the implant, the pad, gel and any other products with slow release.

Suitable carrier materials that can be entered compositions include lactose, starch, cellulose derivatives, etc. or mixtures thereof, used in appropriate quantity.

Another aspect of the invention relates to the use of steroid compounds in accordance with this invention for receipt of the medicinal product, designed to modulate the immune system of a mammal. More specifically, the invention relates to the use of steroid compounds in accordance with this invention for obtaining a medicinal product intended for the treatment of arthritis, such as rheumatoid arthritis (RA)and autoimmune diseases (AIDs), such as Sjogren syndrome and systemic lupus erythematosus (SLE). The medicines can also be used for the prophylaxis of these diseases. Drug preferably attach the appropriate form, in particular, for treatment of individuals meeting the requirements of health authorities in different countries. The use of 3-meilensteine derivatives, such as (7α, 17β)-7α-methyl-3-methylene-4-estren-17-ol, (7α, 17α)-7-methyl-3-methylene-19-norpregna-4-EN-17-ol, (7α-17α)-7-methyl-3-methylene-19,21-dynorphin-4-EN-17-ol, (7α)-17-keto-7-methyl-3-methylene-4-estren, (7α,17α)-7-methyl-3-methylene-19-norpregna-4-EN-20-in-17-ol, 17β-hydroxy-7α-methyl-3-methylene-17α-propen-2-Il-4-estren and 17β-hydroxy-7α-methyl-3-methylene-17α- butene-2-yl-4-estren, included in the scope of the present invention.

Further, this invention relates to the treatment of arthritis, such as rheumatoid arthritis (RA)and autoimmune diseases (AIDs), such as the syndrome of Serena and systemic lupus erythematosus (LE), includes introduction to the patient the above compounds (in the form of a suitable pharmaceutical dosage forms).

The dose of these steroids is in the range from 0.001 to 100 mg per administration to a patient in need of treatment. Therefore, the dosage unit for the practical application of the compounds in accordance with the present invention may contain an amount of active ingredient in the range of 0.001 to 100 mg

Immunomodulating properties of compounds in accordance with this invention can be demonstrated and used in the following methods.

In the implementation of allergic reactions of the delayed type (DTH) effect of compounds on the development of immune reactions can be traced on the example of mice (please see example 16). Briefly, the compound is administered daily, and then animals are subjected to immunization with antigen in Freund. After 7 days the animals stimulated locally by local injection of antigen (mainly in the foot), measuring subsequently formed local tumor in response to the introduction of the antigen. The degree of this swelling is associated with the development of the immune response against the provoking antigen. The inhibitory activity of the compounds in accordance with the present invention on the development of this tumor can be established by comparing the treated groups with the unity and placebo. As a comparison can be used introduction glucocorticoids.

The action of the compounds in accordance with this invention is also examined in mice with arthritis.

According to this method, mice are subjected to immunization with collagen type II, mainly from bovine cartilage, in Freund. Three weeks later introduce an auxiliary agent for swelling (booster) with the same antigen. Approximately 7-10 days after the booster reaction can be observed swelling of the joints (especially on the back and front feet). This swelling increases rapidly, causing redness, inflammation and pathological changes in normal function. X-ray examination reveals a distortion of the normal shape of the joints. Histological examination of these joints reveals severe inflammation, leading to distortion of the normal shape of the joints. The degree of observed changes can be classified; moreover, the degree of histological changes can also be classified.

Next, the action of compounds in accordance with this invention can be investigated on the example does not obese mice with diabetes (NOD).

According to such a method in mice spontaneously and gradually develop autoimmune disease with symptoms that resemble the symptoms of insulin-dependent diabetes (IDDM) man and Sjogren syndrome. Syndrome SEG the s is characterized by the development of infiltrates in the salivary and lacrimal glands. Do not obese mice gradual development of infiltrates characterized especially salivary gland. Connection in accordance with this invention depending on the dose inhibits the development of infiltrates in the submandibular gland.

Insulin-dependent diabetes mellitus is characterized by the development of infiltrates in the pancreas, resulting in the destruction of the islets of Langerhans produce insulin. After the gradual destruction of all such islets insulin is no longer produced, which leads to the development of IDDM.

The following examples illustrate the invention.

Example 1

Obtaining (7α, 17α)-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol

Bromide methyltriphenylphosphonium (26,4 g, 73,9 mmol.) add to the mix a solution of tert-butoxide potassium (7.9 g, of 70.4 mmol) in 130 ml of dry THF under nitrogen atmosphere. The yellow suspension is stirred for 45 minutes at room temperature. To the suspension quickly (within approximately 30 seconds) add a solution of 20 g, 64 mmol. tibolona in 200 ml of dry THF, which leads to a small exothermic reaction (from 20 to 33°). The yellow reaction mixture is stirred for 30 minutes at room temperature, poured into ice water (700 ml) and extracted with ethyl acetate (700 ml). The organic layer was washed with water (300 ml), dried (Na2SO4) and evaporated dash is, getting 41.8 g of a yellow oil. In the result column chromatography using toluene/ethyl acetate 95/5 (about./vol.%) as eluent get colorless oil, containing approximately 90% (7α, 17α)-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol and 10% isomeric Δ4derived. As a result of recrystallization (3 times) using heptane/petroleum ether as solvent receive specified in the header connection (6,44 g, yield 32%) as a white solid, TPL of 91.6°C.

Example 2

Obtaining (7α, 17β)-7-methyl-3-methylenethf-5(10)-EN-17-ol

Bromide methyltriphenylphosphonium (4,21 g of 11.8 mmol) add to the mix a solution of tert-butoxide potassium (1.26 g, and 11.2 mmol.) in 20 ml of dry THF under nitrogen atmosphere. The yellow suspension is stirred for 45 minutes at room temperature. To the suspension quickly add a solution of (7α, 17β)-7-methyl-3-keto-variety-5(10)-EN-17-ol (3.4 g, to 11.8 mmol) in 25 ml of dry THF. The yellow reaction mixture is stirred for 30 minutes at room temperature, poured into ice water and extracted with ethyl acetate. The organic layer was washed with water, dried (Na2SO4) and evaporated to dryness, obtaining 5.3g specified in the title compound as a yellow oil. In the results column chromatography using toluene/ethyl acetate (vol./vol.%) as eluent floor is anxious 2.3 g of colorless oil. As a result of crystallization using heptane/petroleum ether get (7α, 17β)-7-methyl-3-methylenethf-5(10)-EN-17-ol (1.47 g, 44%) as a white solid, TPL 111,8°C.

Example 3

Receive (5β, 7α, 17α)-5,10-epoxy-7-methyl-3-methylene-19-norpregna-20-in-17-ol

Connection (7α, 17α)-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol (390 mg, of 1.26 mmol), obtained in accordance with example 1 is stirred in 15 ml of dry dichloromethane under nitrogen atmosphere. The solution is cooled to 0°and add meta-chloroperoxybenzoic acid (1,26 mmol, 310 mg of 70% mcpba). The reaction mixture is stirred for 2 hours at 0°C, washed with saturated sodium thiosulfate solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, dried (Na2SO4) and evaporated to dryness, receiving 450 mg of crude (5β, 7α, 17α)-5,10-epoxy-7-methyl-3-methylene-19-norpregna-20-in-17-ol. In the result column chromatography using heptane/ethyl acetate 95/5 (about./vol.%) as eluent get mentioned in the title compound (150 mg, 37%) as a foam.

Example 4

Receive (5β, 7α, 17β)-5,10-epoxy-7-methyl-3-methyltetra-17-ol

Connection (7α, 17β)-7-methyl-3-methylenethf-5-(10)-EN-17-ol (350 mg, 1,22 mmol), obtained in accordance with example 2 are stirred in 15 ml of dry dichloromethane in the atmosphere and the PTA. The solution is cooled to 0°and then add meta-chloroperoxybenzoic acid (mcpba) (1,22 mmol, 301 mg of 70% mcpba). The reaction mixture is stirred for 75 minutes at 0°C, washed with saturated sodium thiosulfate solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, dried (Na2SO4) and evaporated to dryness, obtaining 370 mg of crude (5β, 7α, 17β)-5,10-epoxy-7-methyl-3-methyltetra-17-ol. In the result column chromatography using heptane/ethyl acetate 95/5 (about./vol.%) as eluent a cleaner specified in the title compound (230 mg, 63%). As a result of crystallization using heptane/ethyl acetate 95/5 (about./vol.%) get cleaned specified in the title compound (120 mg, 33%) as a white solid, TPL 125°C.

Example 5

Receive (5α, 7α, 17α)-7-methyl-3-methylene-19-norpregna-20-ene-17-ol

Bromide methyltriphenylphosphonium (688 mg, of 1.93 mmol) add to the mix a solution of tert-butoxide potassium (196 mg, about 1.75 mmol) in 2 ml of dry THF under nitrogen atmosphere. The yellow suspension is stirred for 45 minutes at room temperature. To the suspension quickly add a solution of (5α, 7α, 17α)-7-methyl-3-keto-19-norpregna-20-ene-17-ol (220 mg, 0.7 mmol.) in 3 ml of dry THF. The yellow reaction mixture is stirred for 2.5 hours at room temperature, you is more in ice-cold water and extracted with ethyl acetate. The organic layer was washed with water, dried (Na2SO4) and evaporated to dryness, receiving 500 mg of oil. In the result column chromatography using toluene/ethyl acetate 95/5 (about./vol.%) as eluent receive 200 mg of crude (5α,7α, 17α)-7-methyl-3-methylene-19-norpregna-20-ene-17-ol. As a result of crystallization from heptane get cleaned specified in the title compound (120 mg, 55%)as a white solid, TPL 105°C.

Example 6

Receive (5α, 7α, 17α)-7-methyl-3-methylene-19-norpregna-20-in-17-ol

Bromide methyltriphenylphosphonium (1.85 g, 5.18 mmol) add to the mix a solution of tert-butoxide potassium (527 mg, 4.7 mmol) in 4 ml of dry THF under nitrogen atmosphere. The yellow suspension is stirred for 45 minutes at room temperature. To the suspension is added a solution of (5α, 7α, 17α)-7-methyl-3-keto-19-norpregna-20-in-17-ol (590 mg, 1.88 mmol.) in 8 ml of dry THF. The yellow reaction mixture is stirred for 2.5 hours at room temperature, poured into ice water and extracted with ethyl acetate. The organic layer was washed with water, dried (Na2SO4) and evaporated to dryness, obtaining 1.8 g of oil. In the result column chromatography using toluene/ethyl acetate 95/5 (about./vol.%) as eluent obtain 590 mg of crude (5α, 7α, 17α)-7-methyl-3-methylene-19-norpregna-20-in-17-ol. The result Krista the implementation of heptane get cleared specified in the title compound (290 mg, 50%), in the form of a white solid, TPL 97°C.

Example 7

Receive (5β, 7α,17α)-7-methyl-3-methylene-19-norpregna-17-ol

Bromide methyltriphenylphosphonium (5,4 g, 15.1 mmol) add to the mix a solution of tert-butoxide potassium (1,62 g, 14.4 mmol) in 27 ml of dry THF under nitrogen atmosphere. The yellow suspension is stirred for 40 minutes at room temperature. To the suspension quickly add a solution of (5β, 7α, 17α)-7-methyl-3-keto-19-norpregna-17-ol (4,56 g, 14.4 mmol) in 50 ml of dry THF. The yellow reaction mixture is stirred for 30 minutes at room temperature, poured into ice water and extracted with ethyl acetate. The organic layer was washed with water and brine, dried (Na2SO4) and evaporated to dryness, obtaining of 8.2 g of oil. In the result column chromatography using toluene/ethyl acetate 95/5 (about./vol.%) as eluent get (5β, 7α, 17α)-7-methyl-3-methylene-19-norpregna-17-ol (3,65 g, 81%)that solidified upon storage, TPL 104°C.

Example 8

Obtaining (7α)-7-methyl-3,17-dimethylene-5(10)-ene

Bromide methyltriphenylphosphonium (2.2 g, 6 mmol) add to the mix a solution of tert-butoxide potassium (680 mg, 6 mmol) in 25 ml dry THF under nitrogen atmosphere. The yellow suspension is stirred for 30 minutes at room temperature. To the suspension rapidly approx. 30 seconds) add a solution of (7α)-7-methyl-3-keto-17-methylenethf-5(10)-ene (850 mg, 3 mmol.) in 25 ml of dry THF, which leads to a small isothermal reaction. The reaction mixture is stirred for 30 minutes at room temperature, poured into ice water (100 ml) and extracted with ethyl acetate (100 ml). The organic layer was washed with water (50 ml), dried (Na2SO4) and evaporated to dryness, obtaining (7α)-7-methyl-3,17-dimethylene-5(10)-ene (680 ml, 80%) as a colourless oil, [a]D+115° (0,185 in ethanol).

Example 9

Obtaining (7α, 16α, 17β)-7,16-dimethyl-3-methylene-17-(1-PROPYNYL)variety-5-(10)-EN-17-ol

The solution (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-she (1.5 g, 4.5 mmol.) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU, 1.2 ml, 9.9 mmol) in 30 ml of diet. THF is added in approx. 8 minutes to mix the solution letibit(trimethylsilyl)amide (LiHMDS, 4,96 ml, 1 M in THF) in 15 ml of distilled THF at -40°C in nitrogen atmosphere. The reaction mixture is stirred for 45 minutes at -40°C. Add a solution iodomethane (730 μl, 11.7 mmol) in 1 ml ditch, stirred for 1 hour, and the temperature give the opportunity to rise to 0°C. Add water and a saturated solution of ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness. In the column of chromatogr is the philosophy using heptane/ethyl acetate 95/5 (about./vol.%) as eluent get (7α ,16α)-3,3-dimethoxy-7,16-dimethylester-5(10)-EN-17-one (990 ml, yield 65%).

Propyne (approx. 4 g, 100 mmol) bubbled through a solution of n-BuLi (9 ml, 1.6 M in hexane) in 19 ml of dry THF at -70°receiving an exothermic reaction (from -70 to -30°). The solution (7α,16α)-3,3-dimethoxy-7,16-dimethylester-5(10)-EN-17-she (990 mg, of 2.86 mmol) in 10 ml of dry THF added dropwise within about 5 minutes in the white suspension in a nitrogen atmosphere. The reaction mixture is stirred for 45 minutes and the temperature give the opportunity to rise slowly to room temperature. Add water and a saturated solution of ammonium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness, obtaining (7α,16α,17β)-3,3-dimethoxy-7,16-dimethyl-17-(1-PROPYNYL)variety-5(10)-EN-17-ol. A solution of oxalic acid (72 mg, or 0.57 mmol.) in 6 ml of water are added to a solution (7α,16α,17β)-3,3-dimethoxy-7,16-dimethyl-17-(1-PROPYNYL)variety-5(10)-EN-17-ol (2,86 mmol.) in 25 ml of ethanol and the reaction mixture is stirred for 45 minutes at room temperature. Add a saturated solution of sodium bicarbonate, and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness. In the result column chromatography using heptane/ethyl acetate 9/1 (about./vol.%) as eluent get (7α, 16α, 17&x003B2; )-7,16-dimethyl-17-hydroxy-17-(1-PROPYNYL)variety-5(10)-EN-3-one (940 mg, yield 97%).

Bromide methyltriphenylphosphonium (1.7 g, 4.7 mmol) add to a suspension of tert-butoxide potassium (470 mg, 4.2 mmol,) in 31 ml of dry toluene under nitrogen atmosphere. The reaction mixture is stirred for 45 minutes at a temperature of education phlegmy. The reaction mixture is cooled to 4°and add a solution of (7α,16α,17β)-7,16-dimethyl-17-hydroxy-17-(1-PROPYNYL)variety-5(10)-EN-3-one (940 mg, was 2.76 mmol) in 39 ml of dry toluene for approx. 10 minutes. The reaction mixture is stirred for 20 minutes at 4°add dilute sodium chloride and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness. In the result column chromatography using heptane/ethyl acetate 95/5 (about./vol.%) as eluent get (7α,16α,17β)-7,16-dimethyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-EN-17-ol (630 mg, yield 67%) as oil. Product lyophilizate of dioxane. [α]D+83° (from 0.15 in ethanol).

Example 10

Synthesis of (7α, 16α, 17β)-16-ethyl-7-methyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-EN-17-ol

This connection receive in accordance with the above-described method, using as starting materials (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-one and ethyliodide. [α]D+54° (from 0.13 in ethanol).

Example 11

Synthesis of (7#x003B1; on 17β)-7-methyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-EN-17-ol

This connection receive in accordance with the above-described method, using as a starting material (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-one. [α]D+80° (from 0.14 in ethanol).

Example 12

Synthesis of (7α, 16β, 17β)-16-ethyl-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol

This connection receive in accordance with the above-described synthesis, using as a starting material (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-one. [α)D+94° (0,10 in ethanol).

Example 13

Obtaining (7α)-3-methylene-7-methylestr-5(10)-ene

Potassium hydroxide (1.5 g, 27 mmol) and hydrazine monohydrate (3 ml, 62 mmol) are added to the suspension (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-she (1 g, 3 mmol.) in 17 ml of diethylene glycol at room temperature in a nitrogen atmosphere. The reaction mixture is heated for 1 hour at 130°and then for 1 hour and 15 min at 230°C. For the Department of water and excess hydrazine is used to trap Dean-stark. The reaction mixture is cooled to room temperature, add water and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness, obtaining a dry product (7α)-3,3-dimethoxy-7-methylestr-5(10)-ene, which in turn (7α,16α, 17β)-7,16-dimethyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-e the-17-ol in accordance with the above-described method, so pl. 57°C.

Example 14

Synthesis of (7α)-3-methylene-7-methylestr-5(10)-EN-17-one O-methyloxime

(7α)-3,3-Dimethoxy-7-methylestr-5(10)-EN-17-one (100 mg, 0.3 mmol) added to a solution of methoxylamine. HCl (100 mg, 1.2 mmol) and sodium acetate (123 mg, 1.5 mmol) in 6 ml of Meon. The reaction mixture is stirred for 3 days at room temperature. Add water and a saturated solution of sodium bicarbonate, and extracted with ethyl acetate. The organic layer was washed with brine, dried (Na2SO4) and evaporated to dryness give crude (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-one O-methyloxime, which in turn (7α)-3-methylene-7-methylestr-5(10)-EN-17-one O-methyloxime in accordance with the above-described method. [α]D+149° (0.11 in ethanol).

Example 15

Synthesis of (7α)-3-methylene-7-methylestr-5(10)-EN-17-one of oxime

This oxime is obtained from (7α)-3,3-dimethoxy-7-methylestr-5(10)-EN-17-she and hydroxylamine. HCl in accordance with the above-described method, TPL 149°C.

Example 16

Allergic reaction of the delayed type (DTH)

In the beginning of the experiment using a group of eight female mice of Balb/c mice (Harlan, Zeist, The Netherlands) at the age of about 8 weeks. Prior to the experiment used mice are placed in Macrolon cages under normal conditions and will have acclimatised in 3-5 days. Groups receive either a connection or a placebo.

Their vesvese is up in the 1st and last day of the experiment. Animals do subcutaneous (sc) injection once daily in the back of the neck or administered orally through a feeding tube, starting from the 1st till the 11th day, of the studied compound at a concentration factor of 1,2; 3, 4, 6 to 12 mg/kg of Volume injection rate of 0.1 ml filler for injection, consisting of a saline solution containing 0.5% gelatin and 5% mannitol. In each experiment include the group receiving placebo (filler), and the group receiving 4 mg/kg of dexamethasone as a comparative compound.

On day 2, animals are subjected to immunization with 0.1 ml of a stable suspension of 3.75 Lf antigen Tetanus Toxoid (TT from RIVM, Zeist, The Netherlands) in 1 mg/ml bromide of dimethyldioctadecylammonium (DDA), the resulting phase separation in 2 places on the chest, intradermal.

On the 9th day of the animals stimulate on ventral side, right (R) hand with the application of 0.05 ml of a solution containing 50 Lf TT and 1 mg Al(Oh)3on Jr. In the bottom of the left (L) legs (control) was injected only filler with Al(Oh)3. After 24 and 48 hours the thickness of the sole of the left and right legs (the parameters L and R, respectively) is measured using a densitometer with calipers in mm and determine the percentage of antigen-specific swelling of the soles of the feet in accordance with the following formula: [(R-L)/L]×100%.

Results
ConnectionThe number of connections, input SC. mg/kg, resulting in a 50% decrease in DTH response compared to placebo
Example 11,9-3,4
Example 220
Example 311
Example 4>20
Example 510
Example 76

Example 17

Not obese mice with diabetes (NOD)

Use NOD mice grown on site (pairs for reproduction original purchase in Hattori, Boston, USA, 58-generation or purchased in Bomholtgard (Denmark), at the age of 6-7 weeks.

For evaluating the effect of compounds on the spontaneous development of sialadenitis characterized by infiltration of leukocytes submandibular gland, use only mice-females.

Groups of 6-8 mice-females contain under normal conditions and once a day injected them subcutaneously or orally) with 8 - to 14 - or 20 weeks of age 0.1 ml of the compound (at a concentration of 1,2; 4 or 12 mg/kg/day) or placebo (filler), or nothing type. Starting from the 12th week, they make weekly tests for the presence of glucose in urine (Diabur-test 5000, Boehringer Mannheim).

On the 8th (pre-introduction), 14 or 20 weeks, after 6 or 12 weeks of injection, respectively, animals kill the ml ether anesthesia; the bodies are removed, cut the fat and weighed organs. The submandibular gland is fixed in sublimate-formalin for 18-24 hours and transferred to 70% alcohol. The tissue sections are placed in paraffin and stained with application NOT in accordance with customary methods. To assess the tissue section on the infiltration carried out by two independent researchers under the microscope in accordance with customary methods. Such a study may serve to demonstrate the activity of the compounds in accordance with this invention.

Example 18

Collagen arthritis

Female mice of the DBA-1J/BOM acquire in Bomholtgard, Denmark. All animals contain under normal conditions and will have acclimatised for at least 7 days.

At the age of 10-12 weeks all animals subjected to immunization intradermally at the base of the tail with 100 μg of an emulsion of purified bovine collagen type II [S] (2 mg/ml in 0.05 acetic acid), emulsified in an equal volume of complete adjuvant's adjuvant (CFA containing 4 mg/ml MT H37Ra, Difco Laboratories, Detroit, USA). On day 21 after immunization doing intraperitoneal booster injection of 100 μg C, dissolved in saline solution. The introduction is carried out once a day, starting from the 1st day (the day before immunization) to autopsy at the 44th day.

Groups comprising from 9 to 10 animals injected or investigational compound (1.5 mg/kg/day up to 22 days,and then - only 3 times a week), or cyclosporin a (initially 100 mg/kg/day and 4-day - 20 mg/kg)or dexamethasone (2 mg/kg/day)or filler (5% Mulgofen (EL 719, GAF) in saline solution). Also include are not receiving drugs control group.

Mice are weighed every week, while the activity of clinical arthritis (visual manifestation of arthritis in peripheral joints) determines (in accordance with conventional ways) the same observer, with no information about the input preparations, starting with the 19th day, every 2-3 days until the autopsy on the 44th day.

Clinical arthritis assess on a scale of 0-2 on the paw and expressed as the cumulative arthritic number on the mouse with the maximum magnitude of the component 8. At the end of the experiment the knee and ankle joints are isolated and subjected to x-ray analysis as an indicator of bone destruction followed by immediate fixation in 4% formaldehyde for histology.

X-rays carefully examined under a stereo microscope, assessing the destruction of the bones of the joints on a scale of 0-5 in accordance with conventional methods, since the intact joints to their complete destruction. Such a study may serve to demonstrate the activity of the compounds in accordance with this invention.

That is person 1

ALLERGIC REACTION of the DELAYED TYPE (DTH)
ExampleThe number of connections required for the formation of swelling (statistically significant), mg/kg
Example 13-12
Example 212
Example 312
Example 512
Example 612
Example 712
Example 96-24
Example 1012
 
ExampleSwelling, %
Placebo77.1
Example 16.09
Example 469.1
Example 533
Example 620.5
 
ExampleSwelling, %
Placebo56
Example 117.9
Example 852.6
Example 933.5
Example 1030.6
Example 1128.9
Example 1238.6
Example 1335.3
Example 1434.3
Example 15 43.5

1. 3-Meilensteine derivative having the General formula 1

Formula 1

in which

R1represents H or, together with R3forms β-epoxide, or R1absent in the presence of 5-10 double bond;

R2is (C1-C5)alkyl;

R3is βH βCH3or together with R1forms β-epoxide, or R3absent in the presence of 5-10 double bond;

R4represents H, lower alkyl;

Y represents [H, H] [IT, N], [IT, (C2-C5) alkenyl], [IT, (C2-C5)quinil] or (C1-C6) alkylidene; or =NOR5in which R5represents H, lower alkyl;

dotted lines represent an optional double bond;

provided that the 3-meilensteine derived is not one of the following compounds: (7α, 17β)-7α-methyl-3-methylene-4-estren-17-ol, (7α, 17α)-7-methyl-3-methylene-19-norpregna-4-EN-17-ol, (7α, 17α)-7-methyl-3-methylene-19,21-dynorphin-4-EN-17-ol, (7α)-17-keto-7-methyl-3-methylene-4-estren, (7α, 17α)-7-methyl-3-methylene-19-norpregna-4-EN-20-in-17-ol, 17β-hydroxy-7α-methyl-3-methylene-17α-(2-propenyl)-4-estren and 17β-hydroxy-7α-methyl-3-methylene-17α-(2 methyl-propenyl)-4-estren.

2. 3-Meilensteine derivative according to claim 1, characterized in that the 3-meilensteine derivative has 5-10 double bond, a R1and R3no.

3. 3-Meilensteine derivative selected from the group consisting of:

(7α, 17α)-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol;

(7α,17β)-7-methyl-3-methylenes-5(10)-EN-17-ol

(5β, 7α, 17α)-5,10-epoxy-7-methyl-3-methylene-19-norpregna-20-in-17-ol

(5β, 7α, 17β)-5,10-epoxy-7-methyl-3-methyltetra-17-ol

(5α, 7α, 17α)-7-methyl-3-methylene-19-norpregna-20-ene-17-ol

(5α, 7α, 17α)-7-methyl-3-methylene-19-norpregna-20-in-17-ol

(5β, 7α, 17α)-7-methyl-3-methylene-19-norpregna-17-ol

(7α)-7-methyl-3,17-dimethylene-5(10)-ene

(7α, 16α, 17β)-7,16-dimethyl-3-methylene-17-(1-PROPYNYL)variety-5-(10)-EN-17-ol

(7α, 16α, 17β)-16-ethyl-7-methyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-EN-17-ol

(7α, 17β)-7-methyl-3-methylene-17-(1-PROPYNYL)variety-5(10)-EN-17-ol

(7α, 16β, 17β)-16-ethyl-7-methyl-3-methylene-19-norpregna-5(10)-ene-20-in-17-ol

(7α)-3-methylene-7-methylestr-5(10)-ene

(7α)-3-methylene-7-methylestr-5(10)-EN-17-one O-methyloxime

(7α)-3-methylene-7-methylestr-5(10)-EN-17-one of oxime

4. 3-Meilensteine derivative (7α, 17α)-7-methyl-3-methylene-19-norpregna-(10)-ene-20-in-17-ol.

5. 3-Meilensteine derivative according to any one of claims 1 to 4 as a medicinal product.

6. 3-Meilensteine derivative according to any one of claims 1 to 4, to obtain a medicinal product intended for the treatment and/or prevention of arthritis and/or autoimmune diseases.

7. Pharmaceutical composition for treatment and/or prevention of arthritis and/or autoimmune diseases, including 3-meilensteine derivative according to claims 1 to 3, or its pharmaceutically acceptable salt, or MES in a mixture with a pharmaceutically acceptable auxiliary substance.

8. The method of treatment and/or prevention of arthritis and/or autoimmune diseases by introducing 3-meilensteine derivative according to claim 6 to a patient in need of such treatment or prevention.



 

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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: chemical technology, natural materials, medicine, pharmacy.

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EFFECT: improved preparing method.

1 cl, 4 ex

FIELD: organic chemistry, steroids, pharmacy.

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EFFECT: valuable properties of compounds and composition.

4 cl, 3 sch, 1 tbl, 8 ex

FIELD: organic chemistry, steroids, pharmacy.

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11 cl, 1 tbl, 9 ex

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