20-aminosilane steroids or their acid additive salts, method of production thereof, pharmaceutical composition, intermediate products

 

(57) Abstract:

Describes new compounds of formula I in which R1and R2-same or different alkyl, C1-C12, R3-L - methyl group, n = 2, R4- alkyl, C1-C12, R5is a hydrogen atom, acyl group1-C12wavy lines indicate that asymmetric centers 17 and 20 may independently of one another have the absolute configuration R or S, or an acid additive salt. The compounds of formula I have a strong affinity to the Sigma receptor and the activity against the tide of calcium in sperm. Also describes the method of production thereof, pharmaceutical composition, the intermediate product. 4 C. and 4 h.p. f-crystals, 3 PL.

The present invention relates to new steroid containing in position 20 aminosilane circuit, method of their production, to their use as medicaments, containing their pharmaceutical compositions and to intermediate compounds for the synthesis of steroids.

The object of the present invention are the compounds of formula (I):

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in which R1and R2identical or different, represent an alkyl radical comprising from 1 to 12 atoms with which they are associated, rich heterocycle with 5 or 6 atoms, which may contain another heteroatom, selected from among oxygen, nitrogen and sulfur, R3the position represents an alkyl radical comprising from 1 to 8 carbon atoms, n has an integer value from 2 to 15, R4represents an alkyl radical comprising from 1 to 12 carbon atoms, R5represents a hydrogen atom, acyl group containing not more than 12 carbon atoms, or an alkyl radical containing not more than 12 carbon atoms and the wavy lines indicate that asymmetric centers 17 and 20 may independently of one another have the absolute configuration R or S, as well as their acid additive salt.

When R1, R2, R4and R5represent an alkyl group containing from 1 to 12 carbon atoms, it can go from such radicals as methyl, ethyl, sawn, ISO-propyl, boutigny, isobutylene, tert-boutigny, n - pentelenyi, n-sexily, 2-methylpentyl, 2,3-dimethylbutyl, n-Gately, 2-methylhexane, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentane, n-octillery, 2,2-dimethylhexane, 3,3-dimethylhexane, 3-methyl - 3-ethylpentane, monilinia, 2,4-dimethylheptyl or nUB> and R2are aracelio group containing from 7 to 15 carbon atoms, it is, first of all, benzyl or fenetylline group.

When R1and R2form together with the nitrogen atom to which they are bound, a saturated a heterocycle with 5 or 6 atoms, which may include another heteroatom, selected from among oxygen, nitrogen and sulphur, it is, first of all, about such groups as piperidino, Martinova, thiomorpholine, pieperazinove or pyrolidine.

When R3represents an alkyl group containing from 1 to 8 carbon atoms, we can talk about radical, such as methyl, ethyl, sawn, ISO-propyl, boutigny, isobutylene, tert-boutigny, n-pentelenyi, n-sexily, 2 - methylpentyl, 2,3-dimethylbutyl, n-Gately, 2 - methylhexane, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentane. Preference is given to the methyl radical.

Under acyl group containing not more than 12 carbon atoms, understand in the first place, group, selected from acetyl, propionyl, butyryl, benzoyl, Valerie, hexanoyl, acryloyl and crotonoyl. You can also call and formyl group.

This invention has been implemented with such acids, as hydrochloric, nitric, sulfuric, phosphoric, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, Glyoxylic, aspartic, hydrogen bromide, alkanesulfonyl, such as methanesulfonate or econsultation, arylsulfonate, such as benzosulfimide or paratoluenesulfonyl and arylcarbamoyl. Preference is given to salts with hydrochloric acid.

The object of the present invention are, in particular, compounds of General formula (I) defined above, in which n is equal to 2, as well as their acid additive salt.

The object of the present invention are, in particular, the compounds of the formula defined above, meets the General formula I':

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in which R1and R2have the same meaning as above, and their acid additive salt.

The object of the present invention are, in particular, the following compounds of General formula (I):

- (20R) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5(10)- triene-3-ol,

- (20S) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3, 5(10)-triene-3-ol,

- (20R) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5(10)- triene-3-ol,

- (20 CLASS="ptx2">

The object of the present invention is, in particular, the following compound of formula (I):

(20S) (8,9,13,14,17) 20(((dimethylamino)ethyl)amino)19-norpregna-1,3,5 (10)-triene-3-ol,

and its salts with acids.

The object of the present invention is also a method of obtaining compounds of formula (I) defined above, characterized in that the compound of formula (II):

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in which R3has the meaning specified above, subject if necessary to effect Alliluyeva or alkylating substances to obtain the compounds of formula (IIA):

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in which R3has the meaning specified above, a R5' has the same meaning as R5above, except hydrogen; a compound of formula (II) or (IIA) exposed to tianyoude substances to obtain the compounds of formula (III):

< / BR>
in which R3and R5have the meanings indicated above, and in which the wavy line indicates that the product has the form of the pure stereoisomers (17-OH,17-CN) or (17 CN,17-OH) or form a mixture, which is subjected to dehydration reaction to obtain compounds of formula (IV):

< / BR>
in which R3and R5have the meanings indicated above, which is subjected to reaction boschet, Deputy CN is in position 17 or 17, or in the form of a mixture of 17 and 17, and R3and R5have the meanings specified above,

which is exposed to ORGANOMETALLIC reagent derived radical R4specified above, and then the impact of the means of acid hydrolysis to obtain the compounds of formula (VI):

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in which R3, R4and R5have the meanings specified above and the wavy line means that the Deputy COR4is in position 17 or 17, or in the form of a mixture of 17 and 17,

which is exposed to the salt of the hydroxylamine to obtain the compounds of formula (VII):

< / BR>
in which R3, R4and R5have the meanings specified above and the wavy line means that the Deputy C(R4)=N-OH is in position 17 or 17, or in the form of a mixture of 17 and 17, and the oxime is in the position of SYN, anti, or in the form of a mixture of SYN and anti,

which is subjected to reduction reaction of the oxime to obtain the compounds of formula (VIII):

< / BR>
in which the wavy line means that the Deputy NH2is in position 20R or 20S, or in the form of a mixture of 20R and 20S, and in which R3, R4and R5have the same meaning as above,

to the which X represents a halogen atom, R1and R2such as defined above, n' is equal to n-1 and n are defined as above, and then, perhaps, selective hydrolysis in position 3 dialling compounds, formed in the intermediate stage, to obtain the compounds of formula (IX):

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in which the wavy line, R1, R2, R3, R4, R5and n' have the meanings indicated above,

which is subjected to reduction reaction of the keto-amide group, and then, if desired and if necessary, subjected to one or more of the following reactions, in any order:

- acylation at position 3,

- alkylation at position 3,

the saponification Allexinno group in position 3,

- a selection of different stereoisomers,

the salt formation by impact of salts of organic or inorganic acids.

Allermuir matter is, first of all, carboxylic acid derivative, for example, chloride or anhydride in the presence of a base, such as pyridine.

Possible alkylation is performed by conventional methods. Used alkylating reagent, which is preferably an alkyl halide, such as iodide of alkyl or alkyl sulfate.

Ciallella preferably in a lower alcohol, such as methyl, in the presence of acetic acid.

The dehydration reaction can be carried out using a dehydrating reagent such as phosphorus oxychloride in pyridine.

The reduction of a double bond 16-17 can be carried out either by catalytic hydrogenation, while hydrogenation reagent is hydrogen in the presence of a catalyst such as palladium on charcoal, or rhodium reagent, such as a reagent Wilkinson, either by exposure to sodium borohydride in ethanol, or the influence of magnesium in methanol.

The specified recovery or stereospetsifichno and allows you to get the Deputy CN in position 17 or in position 17 or estereotipo. Thus obtained mixture of stereoisomers (17+17), which, if necessary, produce by conventional methods such as crystallization or chromatography.

ORGANOMETALLIC reagents derived from radical R4represent the usual reagents, i.e. organolithium (R4-Li), magnetogenesis (R4-Mg-X) radicals, while X is a halogen, selected from among Cl, Br and I. Preference is given to Br.

Reaction of acid hydrolysis, following the reaction with meta is Aliz is performed under normal conditions of imine hydrolysis in an acidic medium, such as hydrochloric acid, oxalic acid or acetic acid.

The formation of the oxime of formula (VII) is produced primarily by the impact of hydroxylamine hydrochloride in the presence of a base, such as pyridine, sodium hydroxide or sodium carbonate.

The reduction product of the formula (VII) can be produced by various methods, such as catalytic hydrogenation using as a hydrogenation reagent hydrogen in the presence of catalysts, such as palladium on coal or platinum dioxide by exposure to zinc in acetic acid environment of the sodium in alcohol, such as ethanol or n - propanol, or by adding DIBORANE in diglyme.

The specified recovery or stereospetsifichno and allows you to get the Deputy NH2in position 20 or position 20S or estereotipo. Thus obtained mixture of stereoisomers 20R + 20S, which, if necessary, produce by conventional methods such as crystallization or chromatography.

Condensation of compounds of formula X-CO-(CH2)n'-NR1R2in which X is a halogen atom, selected from among Cl, Br and I, and n', R1and R2such as described above with soedinenii, such as dimethylformamide (DMF). The reaction is carried out mainly among the triethylamine/dimethylformamide (tea/DMF).

Selective hydrolysis of O-acyl compounds which can be formed in the intermediate stage, is performed under normal conditions with the help of an agent, which may be alkaline base, such as caustic soda or sodium hydroxide in a lower alcohol such as methanol or ethanol.

The restoration of the keto-group of the amide compounds of formula (IX) is produced, for example, using a metal hydride, such as alumalite lithium (AlLiH4) in a polar aprotic solvent such as tetrahydrofuran (THF) or ether, or with alkaline borhydride, such as sodium borohydride (NaBH4in the presence of acids, such as acetic acid.

If desired and if necessary, the reaction of acylation or alkylation of the group-OH at position 3 are produced by the methods described above.

The saponification reaction is performed, if desired and if necessary, in the presence of, preferably, alkaline base, such as caustic soda or caustic potash, tert-butyl potassium or acetylenic lithium in ethylenamine. The saponification reaction is performed preferably in a lower alcohol which, if necessary, conventional methods of crystallization or chromatography.

The acid of the salt formation is performed under normal conditions. The operation is performed mainly using hydrochloric acid, for example in ether solution.

When exposed tianyoude substances, leading to the product of formula (III) ORGANOMETALLIC reagent, leading to the product of formula (VI) or a salt of hydroxylamine, leading to the obtaining of the product of formula (VII), it is possible to obtain a product of formula (III), (VI) or (VII), in which alloctype gidrolizirovanny.

This invention extends to a method such as described above, which product of formula (III), (VI) or (VII), in which the specialised acyloxy group optionally subjected to re-acylation.

The products of formula (V), (VI), (VII), (VIII) and (IX) can be obtained, if necessary, in the form of mixtures of stereoisomers. These products are subjected, if desired or if necessary, the allocation of these stereoisomers.

Thus, the invention extends to a method such as described above, which can be allocated to different stereoisomers obtained in prinia, have:

1) a strong affinity to Sigma receptors (see pharmacological tests);

2) activity against the tide of calcium in sperm.

The test results show that some of the products anchored on Sigma receptors, stimulate the flow of calcium into the sperm, while others have a debilitating effect on the tide of calcium stimulated or not stimulated by progesterone molecule, which according to the data associated with the Sigma receptor.

These properties due to their use in therapy. Therefore, in accordance with the present invention the compounds defined by the above formula (I) and their salts with acceptable from a pharmaceutical standpoint acids, can be used as medicines.

As a drug primarily used products corresponding to the formula (I') above, as well as their additive salts with acceptable from a pharmaceutical standpoint acids.

More specifically, in the quality of medicines use the following compounds of General formula (I):

-(20R) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5(10)triene - 3-ol,

-(20S) (8,9,13,14,17) 20-(((dim is(10)triene - 3-ol,

-(20S) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5(10)triene - 3-ol,

as well as their additive salts with acceptable from a pharmaceutical standpoint acids.

Medicines, according to the present invention, include, in particular, the following compound of General formula (I):

-(20S) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5(10)triene-3-ol,

and its salts with acceptable from a pharmaceutical standpoint acids.

The compounds of formula (I) having agonistic activity, stimulate the flow of calcium into the sperm. Medicines, according to the present invention can be used in the treatment of certain forms of sterility, the characteristic feature of which is the lack of fertilizing capacity of spermatozoa.

The compounds of formula (I), which has antagonistic activity, have a debilitating effect on the flow of calcium into the sperm. Medicines, according to the present invention can potentially be used to control acrosome reaction and, consequently, affect the fertilizing ability of sperm. Thus, they are the exact tools.

They can also be used in the veterinary field as a means of contraception for males Pets (dogs, cats, and so on) or to limit the breeding of all kinds of pests, including rodents and pigeons.

Commonly used doses depend on the disease to be treated, but also on the method of use of a medication. They can be, for example, from 10 to 1000 mg per day for adults by ingestion.

The present invention also concerns pharmaceutical compositions comprising as an active beginning at least one of the above compounds of formula (I) in an effective amount.

The compounds of formula (I) applied orally, parenterally or locally, for example, percutaneous, in particular for women, or in the veterinary field by injection, including subcutaneous. They can be as simple or draeven of tablets, capsules, granules, suppositories, injectables, balls of medicinal substance, in particular vaginal balls, ointments, creams, gels, microspheres, implants, patches that receive conventional methods.

Active principle (active start) is introduced into snooze, amidon, magnesium stearate, cocoa butter, aqueous or anhydrous binder, fats of animal or vegetable origin, derived paraffin, glycols, various moisturizing, dispersion or emulsion agents, preservatives.

The object of the present invention are as new industrial products that can be used, in particular, when implementing the method according to the present invention, the products of formula (IIA), (III), (IV), (V), (VI), (VII), (VIII) and (IX) excluding products of the formula (IIA), in which R5' is an alkyl group containing not more than 12 carbon atoms.

The product of formula (II) can be obtained by the methods described in the following developments:

- J. H. HUTCHINSON and other Tetrahedron Letters 1985 26(15), pp. 1819-1822,

- L. L. SMITH and others, J. Am. Chem. Soc. 1966, page 3120-3128.

The following example illustrates the invention, however, limiting it.

Example 1: (20S) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5 (10)-triene-3-ol

Stage a: 8,9,13,14 3 atomic charges-östra-1,3,5(10)-triene-17-one

In a suspension of 33.3 g antipodal estrone (the receipt of which is described in the document: J. N. HUTCHINSON and other Tetrahedron Letters 1985 26(15), pp. 1819-1822, in 67 ml of pyridine type of 33.5 ml of acetic anhydr the solution shaken for 18 hours at a temperature of 182oC, after which he poured into a mixture of ice water (660 ml) and hydrochloric acid 22oB (76 ml). After crystallization suspension is left at rest for 1 h, filtered, washed with water and dried. Thus, the gain of 38.7 g of the desired crude product which is purified by means of hot and cold recrystallization from 83 ml of absolute alcohol, followed by treatment of the activated carbon L2S, filtration and drying. The result was 32.7 g of the desired product (tpl.128oC).

Stage B: (8,9,13,14,17) -3-(atomic charges)17-hydroxy-östra-1,3,5(10)-triene - 17-carbonitrile

In an environment of inert gas into a solution of 32.7 g astronautica obtained at stage a, 654 ml of methanol and 167 ml of acetic acid is injected to 91.6 g of potassium cyanide and shaken for 16 h at room temperature. Then in suspension add 330 ml of a mixture of ice/water. After observing significant crystallization mixture is introduced into 3 l of ice water, filtered and washed with water. Raw not the product is again dissolved in 1.2 l of ethyl acetate. The organic phase is washed, dried, filtered and concentrated to crystallization. After cooling at a temperature of -10oC for 1 h perform the filtering, washing and drying. The result is train-17 - carbonitrile

At the boiling point under reflux for 4 h heat of 27.2 g of the product obtained at stage B, in 82 ml of pyridine and 25 ml of phosphorus oxychloride. Then cooled to 20oC and poured into 450 ml of crushed ice. After observing the exothermic precipitation add sulfuric acid, diluted to 1/5, to obtain a pH close to 1. Then extracted using ethyl acetate, washed with water and then with sodium bicarbonate solution, dried, filtered and evaporated to dryness under reduced pressure. The residue is again dissolved in 60 ml of ethanol and incubated with shaking for 1 h at a temperature of 0oC, filtered and dried. The result of 17.7 g of the desired product (tpl.120oC).

Stage G: (8,9,13,14,17) -3-hydroxy-östra-1,3,5(10)-triene-17-carbonitrile

In a nitrogen atmosphere in a suspension of 17.7 g of the product obtained at stage b, 354 ml of ethyl acetate and cent to 8.85 g of 10% palladium hydroxide on coal injected for 14 min 1,425 l of hydrogen and shaken for 30 minutes Then filtered, evaporated to dryness under reduced pressure, re-dissolved dry extract in 90 ml of ethanol, shaken for 1 h at a temperature of -10oC, centrifuged and dried. The result 15,35 g ECOMOG the 19-norpregna-1,3,5(10)-triene-20-he

In an environment of inert gas in a mixture of 46 g of magnesium shavings in 307 ml of benzene and 307 ml of ether is added at the boiling point under reflux for 1 h 121 ml of methyl iodide. Then heated at boiling temperature under reflux for 30 min and injected prepared just before use the solution 15,35 g of the product obtained in stage G, 154 ml of benzene and 154 ml of ether. Then shaken at boiling point for 93 hours stop Boiling, slowly pour in suspension in a mixture of water and ice and add 340 ml of acetic acid (pH = 4). After concentration, perform centrifugation, washing and drying. So get to 13.7 g of crude product which is purified in 840 ml of acetone and 0.6 g of activated charcoal 3SA, filtered, concentrated to about 5., shaken for 1 h at a temperature of -10oC, centrifuged and dried. The result is 12.2 g of the desired product (tPL248oC; ()D= of 156.6oC (C = 0,5% CHCl3)).

Stage E: (8,9,13,14,17) 20 hydroxyimino-19-norpregna-1,3,5(10)-triene-3-ol

In a nitrogen atmosphere in a solution of 10 g of the product obtained in stage D, in 100 ml of pyridine added 4.5 g of hydroxylamine hydrochloride and heated at a temperature of 80-85oC for 1 h 30 min, then up to aniu and recrystallized from 120 ml of ethanol at the boiling point under reflux. Then add 75 ml of demineralized water and see significant crystallization of the product. Then shaken for 30 min at a temperature of 0oC, centrifuged and dried. The result of 9.45 desired product (tPL234oC).

Stage G: (20S) (8,9,13,14,17) 20-amino-19-norpregna-1,3,5(10)-triene-3-ol

In suspension 2,94 g of platinum dioxide in 304 ml acetic acid is added to 7.35 g of the product obtained in stage E, 550 ml (+368 ml to rinse) acetic acid and produce a hydrogenation with total absorbed amount 1075 ml of hydrogen for 6 h 30 min

Obtaining hydrochloride

After filtering, perform concentration under reduced pressure to obtain a dry extract, which is taken in an acidic environment, consisting of a mixture 4,15 ml of hydrochloric acid 53.5 ml of ethanol and 1.2 ml of water. Then, the resulting solution was added 92 ml of ether, shaken for 1 h at a temperature of 0oC, centrifuged and dried. The crude hydrochloride is subjected to recrystallization by dissolving in boiling in 50 ml of ethanol with 0.5% hydrochloric acid, shaken for 1 h at temperatures from 0 to +5oC and centrifuged.

Getting the Foundation of the odes at the boiling point under reflux, slowly add at an elevated temperature of 140 ml of demineralized water, watching crystallization, shaken for 1 h at a temperature of from 0 to 5oC, centrifuged and dried. So get of 3.77 g of crude base, which is purified by dissolving in boiling temperature in 120 ml of ethanol, concentrated under normal pressure and in an atmosphere of nitrogen to a volume of 40 ml, shaken for 1 h at a temperature of from 0 to 5oC, centrifuged and dried. The result is 3,285 g of the desired product (tPL235oC).

Step 3: (20S) (8,9,13,14,17) 2-dimethylamino-N-(3-hydroxy-19-norpregna-1,3,5 (10)-triene-20-yl)ndimethylacetamide

In the atmosphere of inert gas into a solution of the product obtained at stage W, 91,5 ml of dimethylformamide, 28.4 ml of triethylamine, obtained at a temperature of +80oC and cooled to +5oC, quickly add 12 g of the hydrochloride salt of N, N'-dimethylglycine, shaken for 3 h and poured into a saturated solution of 370 ml of acid sodium carbonate in 550 ml of a mixture of ice and water. Then shaken for 1 h and extracted three times with 100 ml dichloromethane, washed with water and then with sodium bicarbonate solution and aqueous salt solution. Organic solutions coeditor absorb in the atmosphere of inert gas in 37 ml of methanol and 11 ml of 5 N. caustic soda, shaken for 1 h to dissolve, slowly add 220 ml of demineralized water, bubbled carbon dioxide (pH 8), add 14.7 ml of triethylamine, shaken for 15 min, extracted using 250 ml, and then 100 ml of dichloromethane, washed 5 times with 100 ml of water, dried organic solution is treated with activated carbon 3SA, filtered, concentrated under reduced pressure to obtain dry extract (oily concentration), which is subjected to recrystallization by double treatment with ethanol. Thus get x 6.15 g of the desired crude product, which was dissolved at the boiling temperature of 80 ml of ethanol, treated with activated charcoal L2S, filtered, concentrated at normal pressure to a volume of 40 ml, see crystallization, add 10 ml of water, shaken for 1 h at a temperature of from 0 to 5oC, centrifuged and dried. The result 3.03 g of the desired product (tPL226oC).

Phase I: (20S) (8,9,13,14,17) 20-(((dimethylamino)ethyl)amino)19-norpregna-1,3,5 (10)-triene-3-ol

In the atmosphere of inert gas suspended in 1,845 g of lithium aluminum hydride and 5.25 g of aluminium chloride in 131 ml of tetrahydrofuran was added when t is. the donkey cooling to a temperature of from 0 to 5oC add 20 ml of ethyl acetate and then 100 ml of saturated solution of sodium chloride. Then the suspension is filtered and consistently absorb a mixture of water/6 N. HCl (80 ml/50 ml), filtered, absorbed with a mixture of 60 ml of 60% ethanol/8 ml triethylamine and filtered.

In the solution, water is added, see deposition, is extracted using dichloromethane, washed, dried, treated with activated charcoal L2S and concentrate under reduced pressure to obtain 1.9 g of dry extract. Then the specified dry extract was dissolved at the boiling temperature of 60 ml of ethyl acetate and 4 drops of triethylamine for 15 min, then concentrated under reduced pressure to a final volume of 30 ml, cooled for 1 h at a temperature of from 0 to 5oC, centrifuged and dried. The result of 1.03 g of the target product (tPL177oC; ()D= -80,6oC (C = 0,5% EtOH)).

The chemical composition (C24H38ON2: 370,56)

Calculated, %: C 77,78; H 10,34; N 7,56

Found,%: C 77,9; H 10,3; N 7,4

Biological tests

The test report

The preparation of the sperm of a man

Human sperm get from healthy donors. Mobile spermatocele environment BWW, includes: NaCl 166 mmol, KCl 5 mm, CaCl21.3 mmol, KH2PO41.2 mmol, Mg3SO41.2 mmol, glucose 5.5 mmol, lactic acid sodium 21 mmol, sodium pyruvate 0.25 mmol, NaHCO325 mmol, HEPES 20 mmol and 0.8% HSA (410 mOsm/l), pH of 7.4 at room temperature.

Measurement of intracellular calcium

Mobile spermatozoa incubated for at least 2 h in medium BWW/HSA. They are then incubated (at a concentration of 5-10 106/ml) with Fura-AM (final concentration of 2 µmol) at a temperature of 37oC for 45 minutes After washing by centrifuging in centrifusion box 600 g for 10 min in an environment BWW without HSA sperm again suspension at a concentration of 4106/ml. Fluorescent signal measured at a temperature of 37oC using spectrofluorometry when exciting the wave of 340 and 380 nm (PTIM 2001-Kontron) or 340, 360 and 380 nm (Hitachi F-2000 - Braun Century Science Tec. ). Fluorescence emission is recorded at 505 nm. Progesterone or test the products dissolved in absolute ethanol, is added to the incubation medium to a final concentration of 0.1% ethanol. If you need to determine the antagonistic effect of progesterone, the product is added to the medium for 2 min before injection of progesterone. In Konz; the ATEM sperm permeabilities using 0.05% of Triton X-100, and then add 10 mmol EGTA (pH 9.5) is to measure the minimum fluorescence signal. These values allow us to calculate the intracellular concentration of calcium ([Ca2+]i) according to the method described by Grynkiewicz and others (Grunkiewicz G., Poenie, M. and Tsien, R. Y. (1985) J. Biol. Chem. 260, pp. 3440-3450). The obtained values of the concentration of intracellular calcium are expressed relative to a baseline level, arbitrarily taken as 1.

The Sigma receptor: a measure of the relative affinity of communication

The relative affinity of the bond is determined on the brain drugs and semenkovich membranes of the rat.

Preparation of membranes

Used male rats Sprague Dawle weighing about 200 g, coming from Iffa Credo. Animals are exposed to death by decapitation. Then have them cut out the brain and testes, which are homogenized at a temperature of 4oC 10 to 25 volumes of buffer solution of Tris-HCl 50 mmol (pH of 7.7) using the device Ultraturrax. Then the homogenates centrifuged (30,000 g) for 15 min at a temperature of 4oC, after which the precipitate washed three times by suspending (in the same buffer solution), centrif the Incubation

Used marker of Sigma receptors is3H PPP (propyl-3-(3-hydroxyphenyl)piperidine) NEN, having a specific activity 3404 GBq/mmol.

Membrane again suspension in a buffer solution of Tris-HCl 50 mm (pH 8.0) to obtain a protein concentration of about 0.6 mg/ml for testes and 1 mg/ml for the brain. Homogeneous aliquots are subjected to incubation at a temperature of 25oC for 90 min (total volume 0.5 ml) with 3 nmol3H PPP in the presence of increasing concentrations of the control product (haloperidol) or of the products tested. At the end of the incubation 3H PPP, associated with membranes, separated from the free3H PPP speed by filtration using filters Whatman GF/C, pre-treated 0.05% polyethylenimine. Then the precipitate washed twice with 5 ml of buffer solution of Tris-HCl. The calculation of the radioactivity produced after adding 20 ml scintillation fluid equalit (Baker).

Measure of the relative affinity of communication (OCC)

Draw two curves: the percentage associated titiraupenga token 100B/BO depending on the logarithm of the concentration of cold control product or depending on the logarithm of concentration is 100(BO/BO + Bmin/BO)/2, that is,

I50=100(1+Bmin/BO)/2 = 50(1 + Bmin/BO),

where:

- BO: concentration associated titiraupenga marker in the absence of any cool product.

- B: concentration associated titiraupenga marker in the presence of concentrations X cold product;

- Bmin: concentration associated titiraupenga marker in the presence of a significant excess of cold control product (5000 nmol).

The intersection of the line I50and curves allow us to calculate the concentration of cold control product (CH) and cold test product (CX) which reduce by 50% the specific communication titiraupenga token with the receptor. The relative affinity of communication (OCC) of the product being tested is determined using the equation:

OCC = 100(CH)/(CX)

OCC haloperidol is taken arbitrarily set to 100.

Pharmacological tests

1. The relative affinity of communication (OCC) Sigma-receptor (see tab. 1).

2. Measurement of intracellular calcium

The effect of progesterone with a concentration of 10-5mol after 2 minutes pre-treatment product of U with different dosage from 10-8mol to 10-5mol [Ca2+]i Mean SEM n = 3 (see tab. 2).

The antagonistic effect of the product of example 10-6M on the effect of prog. 10-5M Mean SEM n = 8 (see table. 3).

These results are expressed relative to a baseline level, arbitrarily taken as 1.

The effect on intracellular calcium sperm of a man

Progesterone concentration of 10-5mol causes a transient increase in [Ca2+]i followed the second phase, or [Ca2+]i slightly above the baseline level.

As for the product of example (10-5mol), he fully antagonistic effect of progesterone when it is added to the environment for two minutes before last.

The relative affinity of communication (OCC) Sigma-receptor

This product, like progesterone, is able to move3H PPP. OCC calculated on the membranes of rats brain, have been calculated on the testes; the results obtained are given in tables 1-3.

Difference found between OCC on the brain and testes, may be explained by different distribution of different types of plots of Sigma receptor in these two organs.

Thus, such products can inhibit acrosomal reaction (the most important stage of fertilization) if t is

1. 20-Aminosilane steroids of General formula I

< / BR>
in which R1and R2the same or different alkyl, C1- C12, R3- -methyl group;

n = 2;

R4- alkyl, C1- C12;

R5is a hydrogen atom, acyl group C1- C12;

wavy lines indicate that asymmetric centers 17 and 20 may independently of one another have the absolute configuration R or S,

or their acid additive salt.

2. The compounds of formula I on p. 1 conforming to General formula I'

< / BR>
where R1and R2have the values listed in paragraph 1,

or their acid additive salt.

3. The compounds of formula I under item 1, with the following names: 20(R)(8,9,13,14,17)20-[(/dimethylamine/ethyl)amino] 19-norpregna-1,3,5(10)-triene-3-ol, 20(S)(8,9,13,14,17)20-[(/dimethylamine/ethyl)amino] 19-norpregna-1,3,5(10)-triene-3-ol, 20(R)(8,9,13,14,17)20-[(/dimethylamine/ethyl)amino]19-norpregna-1,3,5(10)-triene-3-ol, 20(S)(8,9,13,14,17)20-[(/dimethylamine/ethyl)amino] 19-norpregna-1,3,5(10)-triene-3-ol, or an acid additive salt.

4. The compound of formula I under item 1, with the following title: 20(S)(8,9,13,14,17)20-[(/dimethylamine/ethyl)amino] 19-norpregna-1,3,5(10)-triene-3-ol or its acid salt additive.

5. The method of obtaining soudnitsina, specified in paragraph 1, subject if necessary to effect Alliluyeva substances to obtain compounds of General formula IIA< / BR>
< / BR>
where R3have the above significance, and R'5has the same meaning as R5in paragraph 1, except for values of R5hydrogen, the compound of General formula II or IIAexpose tianyoude substances to obtain compounds of General formula III

< / BR>
where R3and R'5have the above values, and the wavy line means that the product has the form of pure stereoisomers (17-OH,17-CN) or the form of a mixture, which is then subjected to dehydration reaction to obtain compounds of General formula IV

< / BR>
where R3and R5have the above values, with subsequent restoration 16 - 17 double bond to obtain the compounds of General formula V

< / BR>
where the wavy line indicates that the Deputy CN is in position 17 or 17 or in the form of a mixture of 17 and 17, and R3and R5have the above values, which expose ORGANOMETALLIC reagent derived from radical R4as indicated in paragraph 1, and then the exposure means to acid hydrolysis to obtain a product of General formula VI


4is in position 17 or 17 or in the form of a mixture of 17 and 17, which is then exposed to a salt of hydroxylamine to obtain compounds of General formula VII

< / BR>
where R3, R4and R5have the above values and the wavy line means that the Deputy C(R4) = N - OH is in position 17 or 17 or in the form of a mixture of 17 and 17, and the oxime is in the position of SYN, anti, or in the form of a mixture of SYN and anti, which is subjected to reduction reaction of the oxime to obtain compounds of General formula VIII

< / BR>
where R3, R4and R5have the above values, and the wavy line means that the Deputy NH2is in position 20R 20S or or in the form of a mixture of 20R and 20S with its subsequent interaction with the acyl halide of General formula

X-CO-(CH2)n'-NR1R2,

where X is a halogen atom, R1and R2have the values listed in paragraph 1, n' = 1,

and then probably spend the selective hydrolysis in position 3 dialling compounds obtained in the intermediate stage, to obtain compounds of General formula IX

< / BR>
where R1, R2, R3, R4n and R5have the above values, which is then subjected to reaction reset is koleobrazovaniya by salt of organic acid.

6. The compounds of formula I on PP.1 to 5, with affinity to the Sigma receptor and/or activity against the tide of calcium in sperm.

7. Pharmaceutical composition having affinity for Sigma receptors and/or activity against the tide of calcium in sperm, including as applicable the beginning of at least one of the compounds of formula I on PP.1 to 5 and a pharmaceutically acceptable base.

8. The products of formula IIA, III, IV, V, VI, VII, VIII and IX, as defined in paragraph 6 of the claims, except for products of formula IIAin which R'5is an alkyl group containing not more than 12 carbon atoms, as new industrial products.

 

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