The use of compounds of sterol to obtain drugs that regulate meiosis, and how the regulation of meiosis

 

(57) Abstract:

The invention relates to biology, can be used in medicine and concerns the stimulation of meiosis of germ cells in mammals. To do this, as stimulant use sterols, which are intermediates in the biosynthesis of cholesterol, as well as new, structurally they are related synthetic sterols. The method allows to increase the efficiency of stimulation of meiosis. 2 S. and 36 C.p. f-crystals, 6 PL.

The technical field to which the invention relates

The present invention relates to the field of organic chemistry, medicine and veterinary medicine, in particular to some derived Sterol and to their use as medicines. In particular, it was discovered that certain derivatives of Sterol can be used for the regulation of meiosis.

Art

Meiosis is the very important event in germ cells, which is based on sexual reproduction. Meiosis consists of two meiotic division. During the first division are exchanged between the maternal and paternal genes, before pairs of chromosomes will be divided into two daughter cells. They have only half the number (1n) chromosomes and 2C DNA. Plotnik germ cells only with 1C DNA.

Meiotic events similar in male and female germ cells, but the temporary program and the processes of differentiation, leading to the formation of eggs and sperm are totally different. All female gametes are profeso the first meiotic division in early life, often before birth, but are delayed in development in the form of oocytes at a later stage of prophesy (dictyate state) until ovulation after puberty. Thus in early life, the female has a stock of oocytes, which is consumed until it is exhausted. Meiosis in females does not end until the fertilization and results in only one Mature egg and two underdeveloped polar bodies on reproductive cell. In contrast, only some of the male germ cells enter meiosis since puberty and go from ancestral populations of germ cells throughout life. Once begun, the meiosis of male cells continues without substantial delay and gives 4 sperm.

Only little is known about the mechanisms that control the occurrence of meiosis in males and females. New research shows that responsible for stopping meiosis can the Downs, S. M. Mol. Reprod. Dev. 35 (1993) 82-94. ) Diffundere meiosis-regulating substances was originally described By Byskov et al. in connection with the cultural system of the fetal gonads of mice (Byskov, A. G. et al. Dev. Biol. 52 (1976) 193-200). Ovary fetal mouse, which took place in meiosis, was secreted meiosis-inducing substance (MIS), and morphologically differentiated testis with the rest of nematicheskii gametes were allocated meiosis-preventing substance (MPS). It was hypothesized that the relative concentrations of MIS and MPS regulated start, stop and resumption of meiosis in male and female germ cells (Byskov, A. G. et al. in The discrimination of Reproduction, (eds. Knobil, E. and Neill, J. D. Raven Preess, New York (1994)). Obviously, if meiosis can be adjusted, it is possible to control reproduction. Unfortunately it is still impossible to identify the meiosis-inducing substance.

The invention

It has been unexpectedly discovered that certain sterols, known as intermediates in the biosynthesis of cholesterol, and some new, structurally related synthetic sterols, can be used for the regulation of meiosis.

Thus, the present invention relates to a compound of General formula (I)


config C1-C6alkyl which may be substituted with halogen or hydroxy, or R1and R together with the carbon atom to which they are attached, form a cyclopentane or cyclohexane ring,

R3and R4together form an additional bond between the carbon atoms to which they are attached, and in this case, R5represents hydrogen and R6and R7or are hydrogen or together form an additional bond between the carbon atoms to which they are attached, or

R5and R6together form an additional bond between the carbon atoms to which they are attached, and in this case, R3represents hydrogen and R6and R7or are hydrogen or together form an additional bond between the carbon atoms to which they are attached, or

R4and R6together form an additional bond between the carbon atoms to which they are attached, and in this case, R3, R5and R7all represent hydrogen,

R8and R9represent hydrogen or together form an additional bond between the carbon atoms to which they are connected is alphagroup, or R10represents a group which, together with the rest of the molecule forms a simple ether for use as a medicine.

The present invention relates to new compounds of General formula (I).

In the present context the expression "regulation of meiosis" is understood as meaning that connections can be used for stimulation of meiosis in vitro, in vivo and ex vivo.

Thus, more specifically, the present invention concerns the use of compounds of the above General formula (I) for the regulation of meiosis.

The present invention also relates to a method of regulation of meiosis in sexual cell of a mammal, comprising the treatment of the reproductive cells in need of such treatment an effective amount of compounds of the above General formula (I).

Information confirming the possibility of carrying out the invention

It was found that the meiosis-inducing substance, extracted both from bovine testes, and from human follicular fluid, can cause the resumption of meiosis in cultured mouse oocytes (ocity test), as well as to stimulate meiosis in male germ cells cultured and various mammals, including men, and found in Mature follicles of the ovary mammalian species, including women. From examples 1 and 2 shows that the meiosis-inducing substance identified in bovine testes, is 4,4-dimethylpiperidin, and the meiosis-inducing substance identified in human follicular fluid, -4,4-dimethyl-5-cholesta-8,14,24-triene-3-ol.

The existence of the meiosis-inducing substances has been known for some time. But so far nothing is known about the identification of a meiosis-inducing substance or substances. To the authors ' knowledge the present invention, still other compounds of General formula (I) did not find practical application in medicine. In particular, compounds of General formula (I) still have not been used as medicines for the regulation of meiosis.

There are several types of effects on meiosis. In accordance with a preferred embodiment of the present invention compounds of General formula (I) used for stimulation of meiosis. In accordance with another preferred variant of the present invention compounds of General formula (I) used for stimulation of meiosis in humans. Thus, the compounds of formula (I) is very Persian is key, which are known from experience to use this time of hormonal contraceptives, made on the basis of extrarenal and/or gestagen. When used as contraceptives in females meiosis-inducing substance can be introduced to cause premature resumption of meiosis in oocytes when they are still in the growing follicle, to the peak of gonadotropins. The woman resumption of meiosis may, for example, be due one week after the termination of the previous period. Being ovwelrowudy received overripe oocytes most likely will not be fertilized.

Normal menstrual cycle, apparently, will not be realized. In this regard, it is important to note that the presence of the meiosis-inducing substance does not affect the biosynthesis of progesterone in cultured granular cells (somatic cells of the follicle), whereas the estrogen and the progestogen used in the previously used hormonal contraceptives have an adverse effect on the biosynthesis of progesterone.

In accordance with another aspect of the present invention meiosis-inducing substance of General formula (I) can be used to treat some svinnogo generating MIS are unable to produce Mature oocytes. In addition, when performing in vitro fertilization the best results are achieved when added to a medium containing the oocytes, meiosis-inducing substance of General formula (I).

In addition, infertility in males (including men), due to lack of own production meiosis-inducing substance, the introduction of the meiosis-inducing substance of General formula (I) may alleviate the problem.

The introduction of compositions containing a compound of the formula (I) may be carried out in any way, effectively ensuring the transport of active compounds to the site of its action.

So, if necessary, the introduction of the mammal the compounds of the present invention can easily produce in the form of pharmaceutical compositions containing at least one compound of General formula (I) in combination with a pharmaceutically acceptable carrier. For oral administration, such compositions are made in the form of capsules or tablets.

From the above it is clear that the required scheme is the introduction of the composition depends on the state that you want to cure. Thus, in the treatment of infertility introduction can be only once or for a limited period, nab the General formula (I) take or continuously, either periodically. When a woman accepts it as a contraceptive is not continuous, then it is important to observe the agreement in time relative to the menstrual cycle.

Pharmaceutical compositions can include carriers, excipients, enhancers suction, preservatives, buffers, substances for regulation of osmotic pressure, substances to improve raspadaemosti tablets and other components that are traditionally used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragakant, methylcellulose, sodium carboxymethyl cellulose, nikopensius wax and cocoa butter.

Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with fertilization In vitro and ex vivo. The liquid composition may contain other components conventionally used in the art, some of which are mentioned in the above enumeration.

Further, the composition for percutaneous introduction of the compounds of the present invention can be manufactured in the form of a patch, and competitivene of the present invention determined by the treating physician and depends among other things, on the specific compound, the route of administration and from the target application.

Preferred compounds of formula (I) are the following:

The cholesterol-7-EN-3-ol,

4-Metalholic-7-EN-3-ol,

4-Atilhan-7-EN-3-ol,

4,4-Dimethylpent-7-EN-3-ol,

4-Methyl-4-atilhan-7-EN-3-ol,

4-Ethyl-4-metalholic-7-EN-3-ol,

4,4-Diestelhorst-7-EN-3-ol,

4-Profilelist-7-EN-3-ol,

4-Bootyholes-7-EN-3-ol,

4-Isobutyryl-7-EN-3-ol,

4,4-Tetramethylene-7-EN-3-ol,

4,4-Pentamethylene-7-EN-3-ol,

The cholesterol-8-EN-3-ol,

4-Metalholic-8-EN-3-ol,

4-Atilhan-8-EN-3-ol,

4,4-Dimethylpent-8-EN-3-ol,

4-Methyl-4-atilhan-8-EN-3-ol,

4-Ethyl-4-metalholic-8-EN-3-ol,

4,4-Diestelhorst-8-EN-3-ol,

4-Profilelist-8-EN-3-ol,

4-Bootyholes-8-EN-3-ol,

4-Isobutyryl-8-EN-3-ol,

4,4-Tetramethylene-8-EN-3-ol,

4,4-Pentamethylene-8-EN-3-ol,

The cholesterol-8(14)-EN-3-ol,

4-Metalholic-8(14)-EN-3-ol,

4-Atilhan-8(14)-EN-3-ol,

4,4-Dimethylpent-8(14)-EN-3-ol,

4-Methyl-4-atilhan-8(14)-EN-3-ol,

4-Ethyl-4-metalholic-8(14)-EN-3-ol,

4,4-Diestelhorst-8(14)-EN-3-ol,

4-Profilelist-8(14)-EN-3-ol,

4-Bootyholes-8(14)-EN-3-ol,

4 And is of alesta-8,14-Dien-3-ol,

4-Methylfolate-8,14-Dien-3-ol,

4-Atelierista-8,14-Dien-3-ol,

4,4-Dimethylpent-8,14-Dien-3-ol,

4-Methyl-4-atelierista-8,14-Dien-3-ol,

4-Ethyl-4-methylfolate-8,14-Dien-3-ol,

4,4-Diestelhorst-8,14-Dien-3-ol,

4-Propylgallate-8,14-Dien-3-ol,

4-Butyrolactam-8,14-Dien-3-ol,

4-Isobutyrate-8,14-Dien-3-ol,

4,4-Tetramethylaniline-8,14-Dien-3-ol,

4,4-Pentamethylenebis-8,14-Dien-3-ol,

Cholesta-8,24-Dien-3-ol,

4-Methylfolate-8,24-Dien-3-ol,

4-Atelierista-8,24-Dien-3-ol,

4,4-Dimethylpent-8,24-Dien-3-ol,

4-Methyl-4-atelierista-8,24-Dien-3-ol,

4-Ethyl-4-methylfolate-8,24-Dien-3-ol,

4,4-Diestelhorst-8,24-Dien-3-ol,

4-Propylgallate-8,24-Dien-3-ol,

4-Butyrolactam-8,24-Dien-3-ol,

4-Isobutyrate-8,24-Dien-3-ol,

4,4-Tetramethylaniline-8,24-Dien-3-ol,

4,4-Pentamethylenebis-8,24-Dien-3-ol,

Cholesta-8,14,24-triene-3-ol,

4-Methylfolate-8,14,24-triene-3-ol,

4-Atelierista-8,14,24-triene-3-ol,

4,4-Dimethylpent-8,14,24-triene-3-ol,

4-Methyl-4-atelierista-8,14,24-triene-3-ol,

4-Ethyl-4-methylfolate-8,14,24-triene-3-ol

4,4-Diestelhorst-8,14,24-triene-3-ol,

4-Propylgallate-8,14,24-triene-3-ol,

4-Butyrolactam-8,14,24-triene-3-ol,

4-Isobutyrate-8,14,24-triene-3-ol,

4,4-Tet is s.

Preferred esters of the formula (I) are those in which R10represents an acyl group of the carboxylic acid, which may be branched or unbranched, or cyclic and may contain optionally substituted by an amino group and/or 1 or 2 oxygen atom in addition to the carbonyl oxygen of the ester group that binds R10with Sterol skeleton. When R10represents an acyl group, it preferably contains from 1 to 20 carbon atoms, more preferably from 1 to 12 carbon atoms, even more preferably from 1 to 10 carbon atoms and most preferably from 1 to 7 carbon atoms. Acid, which produced the radical R10may be a dicarboxylic acid. Examples R10are acetyl, benzoyl, pivaloyl, butyryl, nicotinoyl, isonicotinoyl, hemisuccinate, hemilateral, Hemamali, hemipterous, butylcarbamoyl, phenylcarbamoyl, butoxycarbonyl, tert-butoxycarbonyl, etoxycarbonyl, 4-dimethylaminomethylphenol, 4-Diethylaminoethanol, 4-dipropyleneglycol, 4-(morpholinomethyl)-benzoyl, 4-(4-methyl-1-piperazinylmethyl)-benzoyl, 3-dimethylaminomethylphenol, 3-Diethylaminoethanol, CAE (1) means an ester or salt sulfate) or phosphono (in this case, (1) means an ester or salt of the phosphate).

Preferred ethers of the formula (I) are those in which R10represents methyl, methoxymethyl, benzyl or pivaloyloxymethyl group.

Compounds of the present invention, naturally occurring, can be obtained from natural sources by known methods. In accordance with another way they can how and structurally related synthetic sterols of the present invention, be obtained by synthesis by known methods.

Further, the present invention is illustrated by several examples, which should not be considered as limiting the amount of protection. The characteristics disclosed in the description and in the following examples, can serve both individually and in any combination, material for realizing the invention in its various forms.

Information confirming the possibility of carrying out the invention

EXAMPLE 1

Isolation, purification and identification of a meiosis-inducing substance (MIS) from bovine testes

Testes six-year-old bull (Danish Land race) were removed immediately after slaughter of the animal. Removing the protein shell fabric testes were placed on dry ice and kept at -80oC. Frozen t). Dried tissue was extracted with 400 ml n-heptane (Lichrosolv. Merck 4390, Germany) under nitrogen with stirring for 24 h at 20oC. the Suspension was filtered, and the solid material was extracted as described above. The combined organic phases were evaporated to dryness on a rotary evaporator at room temperature, resulting in 981 mg of extracted material. This material was dissolved in n-heptane and distributed in 15 tubes, of which evaporated n-heptane. The tubes were stored under nitrogen at 4oWith in the dark.

The extracts were purified by three-stage HPLC (HPLC):

In the first stage, the contents of one test tube was dissolved in 50 μl of 50% (volume/volume) tetrahydrofuran (THF) in water and applied on the column for HPLC with reversed phase (Lichro Spher 100 RP-8, muted, 5 μm, h mm EXT. diameter, Merck). Was suirable at 40oC using a linear gradient of 50%-100% THF for 15 min (flow rate: 1 ml/min). Collected 18 fractions of 1 ml and determined them to MIS-activity.

In the second stage of the fractions obtained in the first stage, and with activity in oocytes test, was dissolved in 50-100 ál of 70% THF and was applied on the column, similar to what was used in the first stage. Suirable ml and determined them to MIS-activity.

In the third stage, the fraction obtained in the second stage, and with activity in oocytes test, was dissolved in 100 μl of a mixture of n-heptane:2-propanol (98: 2, vol/vol) and were applied to prepreparation column for HPLC (Chromspher 5 μm Si, 25010 mm EXT. diameter, Chrompack). Was suirable at room temperature using a mobile phase consisting of a mixture of n-heptane: 2-propanol, 98:2 (volume/volume) (flow rate: 5 ml/min). Collected 5 fractions of 2.5 ml and determined them to MIS-activity.

At all stages of the elution was monitored by UV-detectice at 220 nm.

The material that passed through the above procedure three-stage purification, was used to study the molecular structure of active compounds by spectrometry nuclear magnetic resonance (NMR) spectroscopy and by mass spectrometry.

To obtain the NMR spectrum approximately 1 mg of purified material was dissolved in 0.6 ml of deuterochloroform. Split the NMR spectrum of the proton13-1H-NMR-spectrum (with increasing resolution or without) and 2D TOCSY spectrum was recorded on a NMR spectrometer Brurer AMX2 400, equipped with a broadband inverse 5 mm head gradient coil. Chemical shifts13C-NMR in M. D. (()) for the selected MIS are given in table 1 al. Magn. Res. Chem. 27 (1989) 1012).

Mass spectrometry was performed using device YG Trio 1000 for LC and MS interface L1NC for beam particles and software LAB-BASE 2.1 (Fisons Instrumtnts) and HPLC system containing a column (Chrompack), Chromspher Si, 3 μm, 1004,6 mm HPLC was performed at room temperature using a mixture of n-heptane:2-propanol, 98:2 (volume/volume) as the mobile phase (flow rate: 0.6 ml/min). Sample input MIS was dissolved in n-heptane. The mass spectrometer operated in the electron impact. The results are given in table 2, where the relative heights of the peaks for the selected product are compared with the data for 4,4-dimethylpiperidine from reference 1. Link 2 "+" means that the corresponding peak reported in this study; "-" under the link 1 or 2 means that the corresponding peak in these studies does not say.

On the basis of the13C-NMR spectrum and the molecular weight of 412 determined by mass spectroscopy (MS), it was hypothesized that the structure of MIS, isolated from bovine testes, represents 4,4-dimethyl-5-cholesta-8,24-Dien-3-ol, also identified as 4,4-dimethylpiperidin (DMZ). The chemical shifts of the individual carbon atoms MIS-active material with the third stage of purification by HPLC cf is abrudaime proton chemical shifts, constant interaction and CBT-correlation definitively confirm that the selected compound is 4,4-dimethylpiperidino.

EXAMPLE 2

Isolation, purification and identification of a meiosis-inducing substance (MIS) from human follicular fluid

Follicular fluid of a person (PP) were obtained from follicles allocated to obtain oocytes for the treatment of infertility by in vitro fertilization. Liquid liofilizirovanny and was extracted with n-heptane extract was purified as described in example 1. The connection from the active peak had a molecular ion with m/z=410 and the mass spectrum showed that the chemical structure of the molecule FF-MIS - 4,4-dimethyl-5-cholesta-8,14,24-triene-3-ol.

Methods: Mass spectrometry was performed using device YG Trio 1000, for LC and MS LINC interface for beam particles and software LAB-BASE 2.1 (Fisons Instruments) connected to the system premiani HPLC, containing column (Chrompack), ChromSper Si, 3 μm, h mm C. D. and a mixture of n-heptane: 2-propanol: methanol: ammonia (68:30:2:0,2) as mobile phase (flow rate: 0.5 ml/min). Sample input MIS was dissolved in n-heptane. The mass spectrometer operated in the electron impact. The results are shown in table 3.

EXAMPLE 3

Getting 4-m is RA YPG and were grown for 3 days at 30oWith temperature-controlled incubator. The shoals were added 5 ml of sterile environment of the UE and yeast colonies suspended in a liquid medium by shaking the tubes on a vortex mixer. Then the cell suspension was collected 5 ml sterile syringe and was made in the initial flask with the two tabs on the bottom. The flask contained 200 ml of medium ZYM. The flask was fixed on a rocking chair, and incubated for 24 h at 30oC at 250 rpm/min was Added to the flask, 0.4 ml sterile filtered solution of amphotericin b and incubated for another 25 hours the Yeast cells were collected by centrifugation (Beckman JG, 5oC, 10 min, 4000 rpm) and washed once with water. Cell suspension was placed in a small plastic container and kept at -18oWith up to the final extraction of sterols.

The above-mentioned culture medium and the solution of amphotericin b had the following composition:

Agar PG

Yeast extract, 4 g

KN2RHO41 g

Mo5SO47H2O - 0.5 g

Glucose - 15g

Agar - 20 g

Deionized water 1000 ml

the pH was brought to 5.8 before autoclaving at 121oWith 20 minutes

Wednesday UE

Yeast extract, 10 grams

Deionized water 1000 ml

Autoclave odna water 1000 ml

the pH was brought to 6.5 and 6.6 before autoclaving at 121oC, 20 min, glucose was added separately after autoclaving) 60 g

A solution of amphotericin b

1 mg Fungizone (freeze-dried cake of 50 mg of amphotericin b, 41 mg desoxycholate sodium and 20.2 mg of sodium phosphate production F. "Squibb") was dissolved in 1 ml deionized water.

Stage

Cultured cells from the stage And suspended in 10 ml of water was added 10 ml of 40% KOH in methanol. The mixture was heated under reflux for 4 hours, left overnight at room temperature and then was extracted twice with 20 ml n-heptane. The combined extracts washed with 10% sodium chloride solution and then with water to neutral reaction (five times) and dried. After evaporation of solvent received 40 mg of the crude sterols.

Stage

The crude sterols from the stage In was dissolved in 1 ml of a mixture of n-heptane:2-propanol (98:2) and which on a vortex mixer, and centrifuged at 5000 rpm for 10 min and then subjected to HPLC:

Column: Lichvosorb DIOL 10 μm, 2504 mm max. diameter (Merck)

Eluent: a mixture of n-heptane:2-propanol (98:2)

Flow rate: 1.1 ml/min, 20oWITH

Detection: UV at 220 nm

The data of the mass spectrum shown in table 4, is identical to the data for 4-methylinosine registered in the library of the National Bureau of standards.

EXAMPLE 4

Getting 4,4-dimethylpent-8,14-Dien-3-ol

This compound was obtained as described by Schroepfen et al.: Chemistry and Physics of Lipids 47 (1988) 187, and had the same physical constants as described in the literature.

EXAMPLE 5

Getting 4,4-dimethylpent-8-EN-3-ol

Stage AND

2,48 g 4,4-dimethylpent-8,14-Dien-3-ol was dissolved at 0oWith in 20 ml of pyridine (example 4). To the resulting solution was added 1.7 g of benzoyl chloride and the mixture was stirred at room temperature overnight. After evaporation to dryness was added 25 ml of toluene and after standard water treatment, evaporation and trituration with acetone obtained 2.3 g (74%) of crystalline benzoate.

1H-NMR spectrum (CDCl3, ) showed characteristic signals at: 8,1 (d, 2H), 7,55 (t, 1H), and 7.4 (t, 2H), 5,4 (s, broad, 1H), 4.2V (DD, 1H).

Stage

2,04 g of 3-benzoyloxy-4,4-dimethylpent-8,14-diene was dissolved in 50 ml of THF (stage A) and added dropwise atoWith 360 ml of 1 M solution and thereto is added dropwise 140 ml of water, and then 360 ml of 10% aqueous sodium hydroxide and 378 ml of 30% hydrogen peroxide. After stirring for 90 minutes, to the mixture was added 100 ml of diethyl ether and the aqueous phase was twice extracted with diethyl ether. The combined organic phases are washed twice with a solution of sodium bisulfite and then water. After evaporation the product was purified by chromatography on SiO2(2% diethyl ether in toluene) and as a result got to 0.62 g of 3-benzoyloxy-4,4-dimethylpent-8-EN-15-ol.

MS (molecular ion): 534,4.

1H-NMR spectrum (CDCl3, ) showed characteristic signals at: 8,0 (d, 2H), 7.5 (t, 1H), and 7.4 (t, 2H), and 4.75 (m, 1H), 4,1 (m, 1H).

Stage

of 0.54 g of 3-benzoyloxy-4,4-dimethylpent-8-EN-15-ol was dissolved at 0oWith 2.7 ml of pyridine and the resulting solution was carefully added 33 mg of dimethylaminopyridine and 287 mg of phenylchloropyruvate. The mixture was stirred for 20 hours at room temperature. After adding 25 ml of diethyl ether and the mixture was washed 6 times with a saturated solution of copper sulfate, water, twice 10% sodium hydroxide, water and brine and evaporated to obtain 0.66 g of the crude 3-benzoyl-4,4-dimethylpent-8-EN-15-phenylthiocarbamide, further processed by a solution of the P>oC for 20 minutes and repeat the same processing. After evaporation the mixture was subjected to rough purification by chromatography on SiO2(heptane:methylene chloride = 70: 30) and as a result received 150 mg of crude 3-benzoyloxy-4,4-dimethylpent-8-ene mixed with the appropriate 8,14-diene (stage A).

Stage D

150 mg of the mixture obtained in stage C, was dissolved in 2 ml of methylene chloride, cooled to 0owas added dropwise 0.7 ml of diisobutylaluminium and after 15 minutes was carefully added 0.15 ml of water. Then add 25 ml of diethyl ether and the organic phase was washed with a saturated solution of tartrate of potassium-sodium twice with water and brine and evaporated to obtain 130 mg of the mixture, which was subjected to chromatography on a mixture of AgNO3/SiO2(obtained as described in Chem. & Phys. of Lipids63 (1992) 115) with elution with toluene. Crystallization from a mixture of ether-methanol gave 49 mg of the target compound. Melting point: 154-155oC.

MS (molecular ion): 414,4.

13C-NMR spectrum (CDCl3, 100,6 MHz) showed characteristic signals at: 78,49 (C3); 127,49 (C8), 135,35 (C9).

EXAMPLE 6

Obtain 3-acetoxy-4,4-dimethylpent-8,14-Dien

7.5 ml Piri is 88) 187) and the solution was stirred at 23oWith all night. The mixture was evaporated in vacuum, was desirerable twice with toluene and purified by flash chromatography on SiO2(toluene). The first 300 ml of the eluate was evaporated and the product was led from diethyl ether to obtain 140 mg of 3-acetoxy-8,14-dimethylxanthene.

Melting point: 120-125oC (with decomposition).

MS (molecular ion): 454,4.

1H-NMR spectrum (CDCl3, ) showed characteristic signals at: 5,4 (s, broad, 1H), 4,5 (DD, 3H), 2,0 (s, 3H).

EXAMPLE 7

Getting cholesta-8,14-Dien-3-ol

In a mixture of 2.7 ml of benzene, 19 ml of ethanol and 2.7 ml of concentrated chloroethanol acid was dissolved 770 mg dehydrocholesterol and the solution was heated at the boiling point under reflux for 3 hours. The mixture was cooled in an ice bath, resulting in the first portion (110 mg) crystals. Evaporation of the filtrate to dryness and crystallization from a mixture of ether/methanol gave the second portion (220 mg) crystals, which were combined with the first portion and chromatographically on a mixture of AgNO3/SiO2(obtained as described in example 5, step D) with elution with 2.5% acetone in toluene, resulting in 94 mg of pure cholesta-8,14-Dien-3-ol.

Temperature p is exerted characteristic signals at: to 5.35 (s, broad, 1H), 3,6 (m, 1H).

13C-NMR spectrum (CDCl3and 50.3 MHz) showed characteristic signals at: 70,99 (C3), 117,42 (C15), 123,1 (C8), 140,8 (C9), 151,1 (C14).

EXAMPLE 8

Getting 4,4-tetramethylaniline-8,14-Dien-3-ol

Stage AND

1,15 g dehydrocholesterol was dissolved in 15 ml of 2-butanol, was added 0.34 g of isopropoxide aluminum and the mixture was heated at the boiling point under reflux for 75 minutes. The mixture was cooled in an ice bath, was added 15 ml of 2 N chloroethanol acid, are of the same phase and the organic phase is washed twice with 7.5 ml of 2 N chloroethanol acid. The aqueous phase was extracted with toluene, the combined organic phases are washed with water and brine, dried and evaporated to obtain 1.18 g of crude cholesta 5,7-Dien-3-one as a viscous oil.

1H-NMR spectrum showed characteristic signals at: 5,8 (s, 1H), and 5.2 (m, 1H), 3,2 (d, 1H), and 2.7 (DD, 1H).

Stage

of 0.67 g of tert-butoxide potassium was dissolved in 16 ml of tert-butanol at 45oWith added 0,57 g cholesta-5,7-Dien-3-one and the mixture was stirred for 10 minutes. Added to 0.47 g of 1,4-diiodobutane and the mixture was stirred for 30 minutes. The solvent evaporated, the residue was again dissolved in toluene is Oh and the combined aqueous phase was extracted once with toluene. The combined toluene extracts were dried and evaporated to obtain 0.45 g of foamed material, which after crystallization from a mixture of diethyl ether-methanol gave 0.35 g of crystalline 4,4-tetramethylene-5,7-Dien-3-one.

MS (molecular ion): 436,4.

1H-NMR spectrum (CDCl3, ) showed characteristic signals at: of 5.75 (d, 1H), 5,5 (m, 1H).

Stage

130 mg diAlH4suspended in 6 ml of THF and the suspension was added 1.97 g of 4,4-tetramethylaniline-5,7-Dien-3-one, dissolved in 40 ml of THF is added dropwise within 30 minutes. 15 minutes after the end of the add was still some unreacted starting material (TLC) and therefore was added 65 mg LiAlH4. After stirring for 30 minutes the reaction was over, and then dropwise added to 0.9 ml of water in 5 ml of THF. After 30 minutes of stirring was added excess amount of magnesium sulfate and the mixture was stirred for another 30 minutes, filtered and evaporated to dryness. The residue was dissolved in 25 ml of diethyl ether and 25 ml of methanol and ether was carefully removed under vacuum. The mixture was stirred over night, filtered and received a 1.75 g of crystalline 4,4-tetramethylaniline-5,7-Dien-3-ol.

MS (molecules is).

Stage D

770 mg of the compound obtained in stage C, was dissolved in a mixture of 2.38 ml of benzene, and 17.5 ml of ethanol and of 2.38 ml of concentrated chloroethanol acid, and the solution was heated under reflux for 16 hours, and evaporated in vacuum. The residue was again dissolved in 5 ml of toluene, filtered and chromatographically on medium pressure column with AgNO3/SiO2; (heptane:toluene = 10: 90), receiving 35 mg of 4,4-tetramethylaniline-8,14-Dien-3-ol.

MS (molecular ion): 438,4.

1H-NMR spectrum (CDCl3that of the product showed characteristic signals at: 5,35 (s, broad, 1H), 3,3 (DD, 1H).

13C-NMR spectrum (CDCl3, 100,6 MHz) showed characteristic signals at: 79,0 (C3), 117,4 (C15), 122,9 (C8), 141,3 (C9), 151,1 (C14).

EXAMPLE 9

Getting 4,4-dimethylpent-8(14)-EN-3-ol

580 mg of 4,4-dimethylpent-8-EN-3-ol was dissolved in 20 ml of diethyl ether and 20 ml of acetic acid. Added 60 mg of 10% Pd/C catalyst and the mixture was stirred overnight under hydrogen at 3.5 ATM. Remove the catalyst and the filtrate was concentrated to 10 ml, which resulted crystallization. Added 10 ml of methanol and after 16 hours collecting the crystals. The cross is(9)and 8(14)-isomers.

The mixture was again dissolved in 10 ml of diethyl ether and 10 ml of acetic acid. Added 75 mg of 5% Pt/C catalyst and the mixture was treated overnight with hydrogen at atmospheric pressure. Removing the catalyst, the solvent evaporated and the crystalline residue is triturated with 5 ml of methanol, resulting in 190 mg of pure 4,4-dimethylpent-8(14)-EN-3-ol.

MS (molecular ion): 414,4.

13C-NMR-spectrum (l3, 100,6 MHz) showed characteristic signals at: 79,24 (C3), 126,11 (C8), 142,20 (C14).

EXAMPLE 10

Ocity test to determine the meiosis-inducing activity of compounds

Animals

Immature female mice (B6D 2-F1, line C57B1/2J) were kept under controlled lighting (14 h light, 10 h dark) and temperature with food and water at will. When the animals reached the weight 13-16 g (which corresponds to the age of 20-22 days old, they did one injection (intraperitoneally) human menopausal gonadotropin (Humegon, Organon, netherlands), containing approximately 20 IU of FSH (follicle stimulating hormone) and 20 IU of LH (luteinizing hormone) (Ziebe, S. et al. Hum. Reprod. 8 (1993) 385-88). After 8 hours the animals were killed by cervical (neck) offset.

Collection and multiwired for collection and cultivation consisted of minimal eagle medium (Flow, USA) containing 4 mm gipoksantina, 3 mg/ml bovine serum albumin, 0.23 mm sodium pyruvate, 2 mm glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all Sigma, USA). This environment is called the NC environment. The same medium, but without gipoksantina used as the control environment.

Antral ovarian follicles were punctured with a needle under No 27 preprofile a magnifying glass. Were taken prisoners in the world (the totality of membranes) cells (CEO) of the same size and washed them three times with fresh NC environment.

Oocytes are released from cells of the world, i.e., naked oocytes (DO) was obtained by careful bandwidth SEO through a pipette with a hole of very small diameter. SEO and DO were cultured in 4-hole plates (Nunclon, Denmark), containing 0.5 ml NC-environment, except the control, which were cultured in the control medium. Each hole contained 35-50 oocytes. Culture medium contained different concentrations of the tested compounds, as indicated in table 5.

The cups were incubated at 37oC and 100% humidity in an atmosphere of 5% POPs within 24 hours.

Analysis of oocytes

At the end of the cultivation period were counting the number of oocytes with germinal vesicle (GV), with the destruction of the germs is but-contrast equipment. Calculate the percentage of GVBD oocytes with the total number of oocytes and the percentage of oocytes with PB from GVBD. Results for DO and SEO calculated as units of activity MIS, are given in table 5. One unit activity of the MIS is defined as

< / BR>
and the number of activity units MIS calculated as

< / BR>
EXAMPLE 11

Gonadal test to determine the meiosis-inducing activity of compounds

Gonadal test was performed essentially as described Byskov. A. G. et al. Mol. Reprod. Dev. 34 (1993) 47-52. The results are shown in table 6, were evaluated semi-quantitatively as described Westergaard, L. et al. Fertil. Steril. 41 (1984) 377-84.

EXAMPLE 12

Getting mono (5-cholesta-8,14-diene)-3-succinate

0.50 g of 5-cholesta-8,14-Dien-3-ol was dissolved in 10 ml of THF, and then added 0.39 g of succinic anhydride and 16 mg of 4-dimethylaminopyridine. The solution was heated with reverse holodilnik overnight and then was evaporated to dryness. The residue is suspended in 10 ml of water, the precipitate was filtered, washed with water and dried, resulting in of 0.48 g of the target compound, which was further purify by dissolving in a mixture of an aqueous solution of sodium bicarbonate and ethanol, adding chloroethanol acid to pH 2 and then to concentrate the solution to obtain a precipitate.l3that of the product showed characteristic signals at: are 5.36 (s, 1H), and 4.75 (m, 1H), to 2.67 (m, 2H), AND 2.6 (M, 2H)

13C-NMR spectrum (CDCl3, 100,6 MHz) showed characteristic signals at: 73,4, 117,1, 122,7, 140,0, 150,5, 171,2, 177,2.

EXAMPLE 13

Obtaining 3-ethoxycarbonyl-5-cholesta-8,14-Dien

0.50 g of 5-cholesta-8,14-Dien-3-ol was dissolved in a mixture of 5 ml of toluene and 5 ml of pyridine cooled in an ice bath. Was added within 5 min to 2.3 ml ethylchloride dissolved in 5 ml of toluene. After 30 minutes the ice bath was removed and continued stirring for 20 hours at room temperature, and then 2 hours at 60oC. the Reaction mixture was evaporated to dryness in vacuo and the residue triturated with 10 ml of ethanol, resulting in worn: 0.505 g of the target compound, which can be further purified by recrystallization from ethanol.

Melting point: 101-106oWITH

MS (molecular ion): 456,3

1H-NMR-spectrum (l3that of the product showed characteristic signals at: and 5.30 (s, 1H), 4,50 (m, 1H), 4,12 (kV, 2H), 1,24 (t, 3H).

13C-NMR spectrum (CDCl3, 100,6 MHz) showed characteristic signals at: 62,6, 116,6, 122,2, 139,4, 150,0, 153,6.

EXAMPLE 14

Obtaining 3-fosforico-4,4-dimethyl-5-cholesta-8,14-Dien

Melting point: 183-185oWITH

1H-NMR spectrum (CDCl3+ 2 drops CD3OD) of the product showed characteristic signals at: are 5.36 (s, 1H), with 3.89 (m, 1H).

13C-NMR spectrum (CDCl3+ 2 drops of CO3D, 100,6 MHz) of the product showed characteristic signals at: 85,1, 116,9, 122,3, 140,9, 150,5,

EXAMPLE 15

Obtaining 3-isonicotinoyl-5-cholesta-8,14-Dien

0.50 g of 5-cholesta-8,14-Dien-3-ol was dissolved in 5 ml of pyridine, and then added to 1.16 g isonicotinohydrazide. The suspension was heated under reflux during the night is washed with water and dried, receiving 0.97 g of the crude product, which was recrystallized from a mixture of acetone-water to obtain 0.40 g of the target compound. Melting point: 129-131oWITH

1H-NMR-spectrum (l3that of the product showed characteristic signals at: 8,77 (d, 2H), to 7.84 (d, 2H), 5,39 (s, 1H), 4,49 (m, 1H).

13C-NMR spectrum (CDCl3and 50.3 MHz) showed characteristic signals at: 75,0, 117,7, 122,8, 123,3, 138,0, 140,3, 150,5, 150,9, 164,6.

1. Means for stimulation of meiosis, representing sterols General formula (I)

< / BR>
where R1and R2independently selected from hydrogen, unbranched or branched C1-C6the alkyl, optionally substituted with halogen or hydroxy, or R1and R2together with the carbon atom to which they are attached, form a cyclopentane or cyclohexane ring;

R3and R4together form an additional bond between the carbon atoms to which they are attached, and in this case, the5represents hydrogen and R6and R7or are hydrogen or together form an additional double bond between the carbon atoms to which they are attached, or

R5and R4the UP> represents hydrogen and R6and R7or are hydrogen or together form an additional bond between the carbon atoms to which they are attached, or

R6and R4together form an additional bond between the carbon atoms to which they are attached, and in this case, the3, R5and R7represent hydrogen;

R8and R9represent hydrogen or together form an additional bond between the carbon atoms to which they are attached;

R10represents hydrogen or acyl group, sulfonyloxy group or postonline group, or a group which together with the rest of the molecule forms a simple ether.

2. Means under item 1, in which R1represents hydrogen or methyl.

3. Means under item 1, in which R1choose from ethyl and unbranched and branched C3-C6the alkyl.

4. Means under item 1, in which R1represents an unbranched or branched hydroxyalkyl group having up to six carbon atoms.

5. Means under item 1, in which R1represents an unbranched or branched --hydroxy is yet an unbranched or branched alkyl group, substituted with halogen.

7. Means under item 1, in which R1represents trifluoromethyl.

8. Means under item 1, in which R2represents hydrogen or methyl.

9. Means under item 1, in which R2choose from ethyl or unbranched and branched C3-C6the alkyl.

10. Means under item 1, in which R2represents an unbranched or branched hydroxyalkyl group having up to six carbon atoms.

11. Means under item 1, in which R2represents an unbranched or branched hydroxyalkyl group having up to six carbon atoms.

12. Means under item 1, in which R2represents an unbranched or branched alkyl group substituted with halogen.

13. Means under item 1, in which R2represents trifluoromethyl.

14. Means under item 1, in which R1and R2together with the carbon atom to which they are attached, form a cyclopentane ring.

15. Means under item 1, in which R1and R2together with the carbon atom to which they are attached, form a cyclohexane ring.

16. Means on p. is connected, and R5represents hydrogen.

17. Means under item 1, in which R5and R4together form an additional bond between the carbon atoms to which they are attached, and R3represents hydrogen.

18. Means under item 1, in which R6and R4together form an additional bond between the carbon atoms to which they are attached, and R3, R5and R7represent hydrogen.

19. Means under item 1, in which R6and R7represent hydrogen.

20. Means under item 1, in which R6and R7together form an additional bond between the carbon atoms to which they are attached.

21. Means under item 1, in which R8and R9represent hydrogen.

22. Means under item 1, in which R8and R9together form an additional bond between the carbon atoms to which they are attached.

23. Means under item 1, in which R1represents hydrogen.

24. Means under item 1, in which R10represents an acyl group which is derived from acid having 1-20 carbon atoms.

25. Means on p. 24, in which R10represents and is Oila, hemiglutarate, butylcarbamoyl, phenylcarbamoyl, butoxycarbonyl, tert-butoxycarbonyl and ethoxycarbonyl.

26. Means under item 1, in which R10represents alkyl, Uralkaliy, alkyloxyalkyl or alkanoyloxy group containing up to 10 carbon atoms, preferably up to 8 carbon atoms, which together with the rest of the molecule forms a simple ether.

27. Means on p. 26, in which R10represents methoxymethyl or juliolemelin group.

28. Means under item 1, in which R10represents sulfonyloxy group.

29. Means under item 1, in which R10represents postonline group.

30. Means under item 1, representing 4,4-dimethyl-5-cholesta-8,14,24-triene-3-ol.

31. Means under item 1, representing 4,4-dimethyl-5-cholesta-8,24-Dien-3-ol.

32. The method of stimulation of meiosis in sexual cell of a mammal, comprising the treatment of the reproductive cells an effective amount according to any one of paragraphs. 1-31.

33. The method according to p. 32, which stimulates meiosis tool is a 4,4-dimethyl-5-cholesta-8,14,24-triene-3-ol.

34. The method according to p. 32, kotorom of PP. 32-34, in which the sexual cell process tool according to any one of paragraphs. 1-31 through the introduction of specified funds to the mammal containing the specified sexual cell.

36. The method according to any of paragraphs. 32-35, in which sexual cell is an oocyte.

37. The method according to p. 36, in which the oocyte is treated with a tool according to any one of paragraphs. 1-31 ex vivo.

38. The method according to any of paragraphs. 32-35, in which sex cell is the male reproductive cell.

Priority points:

23.06.1994 - PP. 1-31;

09.03.1995 - PP. 32-38.

 

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EFFECT: higher efficiency of therapy.

3 ex

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