CZ20012866A3 - Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments - Google Patents

Abstract

The present invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions comprising them as active substances and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the unsaturated cholestane derivatives of the invention can be used for regulating meiosis.

Description

Technical field

The invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions containing them as active ingredients, and to the use of these novel compounds for the preparation of medicaments. In particular, it has been found that unsaturated cholestane derivatives can be used to control meiosis.

BACKGROUND OF THE INVENTION

Meiosis is the specific and major germ cell manifestation on which sexual reproduction is based. Meioza includes two meiotic divisions. During the first meiotic division, there is an exchange between mother and father genes before the chromosome pairs are separated into two daughter cells. These daughter cells contain only half (1n) chromosomes and 2c DNA. The second meiotic division proceeds without DNA synthesis. This division therefore results in the formation of haploid germ cells with only 1c DNA.

Meiotic processes occur similarly in both male and female germ cells, but the timing and differentiation processes that lead to ova and spermatozoids vary significantly. All female germ cells enter the prophase of the first meiotic division at an early stage of life, often before birth, but all are later stopped in the prophase as oocytes (dictate) until ovulation after puberty. In this way, the female has an oocyte supply at an early stage of life, which is pumped until the supply is depleted. Meioza in females is completed after fertilization and results in only one egg and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells undergo meiosis starting from puberty and constitute a stem cell population throughout life. Upon initiation, meiosis occurs in male cells without significant delay and produces 4 spermatozoids.

Very little is known about the mechanism that regulates the initiation of meiosis in male and female. Concerning oocytes, some recent studies have reported that follicular purines, hypoxanthine or adenosine may be responsible for meiotic arrest [Downs, S.M. et al., Dev Biol 82 (1985) 454-458; Epplg.J.J. et al. Dev Biol 119 (1986) 313-321; and Downs, S.M. Mol Reprod Dev 35 (1993) 82-94]. The presence of diffusible meiosis regulating agents was first described by Byskov et al. in the culture of fetal mouse gonads [Byskov, A.G. et al. Dev Biol 52 (1976) 193-200]. Certain meiosis-activating agent (MAS) was secreted by the fetal mouse ovary in which meiosis occurred, while another meiosis-inhibiting agent (MPS) was released from morphologically differentiated testes with resting non-meiotic germ cells. Relative concentrations of MAS and MPS have been reported to regulate the onset, arrest, and relaunch of meiosis in male and female germ cells (Byskov, AG et al., The Psysiology of Reproduction [ed. Knobil E. and Neil, JD, raven Press , New York (1994)] It is therefore understood that if meiosis can be controlled, then it can also be controlled

reproduction. In a recent article [Byskov A.G. et al., Nature 374 (1995), 559-562] describes the isolation of certain sterols from bovine testes and from human follicular fluid, said sterols reported to activate oocyte meiosis. Unfortunately, these sterols are somewhat labile and the use of this interesting finding would be greatly facilitated if more stable compounds capable of activating meiosis were available.

Compounds known to stimulate meiosis other than those claimed in this patent application are described in WO 96/27658.

Thus, it is an object of the invention to provide novel compounds for treating female and male infertility, particularly in humans, by activating meiosis.

It is a further object of the invention to provide novel compounds useful in male and female, especially human, as meiosis inhibition contraceptives.

SUMMARY OF THE INVENTION

Within the scope of the invention, new, stable compounds with interesting pharmacological properties are provided. The compounds described herein are particularly useful for controlling meiosis in oocytes and male germ cells.

Yet another object of the invention is to provide novel compounds which are suitable substrates for introducing fluorescent labels. Bound molecules are designed and synthesized for biosimaging purposes.

The object of the invention

are unsaturated cholestane derivatives of formula I

in which

R ¹ represents a hydrogen atom; C 2 -C 6 alkyl; optionally substituted phenyl; cyano; a C 1 -C 4 -NH-COR ¹ group in which R ¹ represents an alkyl group having 1 to 8 carbon atoms or an optionally substituted phenyl group; or together with R @ ² means an additional bond;

'

R is hydrogen; C 4 -C 8 alkyl; (C 3 -C 6) alkenyl; a (C 1 -C 6) hydroxyalkyl group; together with R ^{2 an} optionally substituted benzylidene group; Together with 2 '

R is a hydroxymethylene group; or together with R additional bond; ,

R is hydrogen; together with R an optionally substituted benzylidene group; or together with R ² a hydroxymethylene group;

R is hydrogen; or together with R additional bond;

* 3

R is hydrogen; or together with R additional bond;

• · ·

• · · · · · · · · · · · ·

represents a hydrogen atom; or methyl; represents a hydrogen atom; or methyl;

914 represents together with R or R an additional bond;

R is hydrogen; or together with R additional bond;

148

R is α-hydrogen; or together with R additional bond; or together with R additional bond;

R ( ¹⁵⁾ is hydrogen; or together with R ¹⁴ an additional bond;

25

E represents a hydrogen atom; or together with R additional bond; and

24

R is hydrogen; or together with R additional bond;

or esters thereof, except that said compounds do not include compounds that are not simultaneously modified at the 1 and 2 positions, i.e., when R = R = R = H.

The compounds of formula I have a number of chiral centers per molecule and therefore exist in several isomeric forms. All such isomeric forms and mixtures thereof are included within the scope of the invention, unless expressly stated otherwise.

Preferred compounds of the invention are those compounds of formula (I) wherein R ¹ is hydrogen or phenyl, or together with R ¹ is an additional bond. Further preferred compounds of the invention are those compounds of formula I wherein R is C 4 -C 8 alkyl or allyl or forms an additional bond with R ¹ . Also preferred are compounds of formula I wherein R and R are optionally substituted benzylidene.

In another embodiment, the invention relates to esters of the compounds of formula I. Such esters are formally derived by esterifying one or more hydroxyl groups of a compound of formula I with an acid, which may for example be selected from succinic acid and other aliphatic dicarboxylic acids. nicotinic acid, isonicotinic acid, ethanecarboxylic acid, phosphoric acid, sulfonic acid, sulfamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.

The term alkyl, both when used alone or when used in combination, is to be understood as an alkyl group with direct examples of alkyl atoms being isopropyl, cyclohexyl ethyl, pentyl or branched chain, groups containing 2 to 6 propyl, hexyl more preferably, butyl, wherein

Preferred carbon, tert-butyl and ethyl are ethyl. Preferred examples of the alkyl group having 1 to 8 carbon atoms are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl, with methyl, ethyl being more preferred , propyl, isopropyl, butyl and tert-butyl, and even more preferred are methyl and ethyl.

Particularly preferred compounds of the invention are compounds of formula I selected from the group consisting of:

2alpha-allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol, (E) -2-benzylidene-4,4-dimethyl-5alpha-cholesta-8,14-diene-3beta- ol, * »

5alpha-cholesta-1,8,14-trien-3beta-ol,

5alpha-cholesta-1,8,14-trien-3-one, 1alpha-cyano-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol, 1alpha-cyano-4,4-dimethyl- 5alpha-cholesta-8,14-3-one,

4,4, dimethyl-5alpha-cholesta-1,8,14-trien-3beta-ol,

4,4-dimethyl-5alpha-cholesta-1,8,14-trien-3-one, (E) -4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-diene- 2-ylidene) methyl] benzonitrile, (E) -N - [[4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide, (E) - N - [[4- [3beta-Hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4'-dimethyl) - difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacen-3-yl) pentanamide,

2alpha-hydroxymethyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3alpha-ol,

2alpha-octyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol and (E) -4 - [(3-oxo-4,4-dimethyl-5alpha-cholesta-8,14- dien-2-ylidene) methyl] benzonitrile.

The compounds of the formula I according to the invention can be synthesized in a manner analogous to that for the synthesis of structurally similar compounds. The synthesis of compounds of formula (I) may thus proceed according to the well-known reaction scheme described in the literature for sterols and steroids. The following literature can be used as a key source for the synthesis of compounds of Formula I of the invention: L.F. Fieser & M. Fieser: Steroids: Reinhold Publishing Corporation, NY 1959; Ross's Chemistry of Carbon Compounds (published by S. Cooffrey): Elsevier Publishing Company, 1971; and in particular Disctionary of Steroids

99 99 • • · • 9 • · • · • 9 99 • · Φ · · • 9 9 9 · 9 • · • 9 9 · 9 > 9 9 · • 9 > 9 9

(published by R.A.Hill; D.N.Kirk; H.L.J.Makin and G.M.Murphy): Chapman & Hal. The latter publication contains an extensive list of references to original publications covering the period up to 1990.

In particular, the compounds of formula I are synthesized by the following general procedures.

Sterols used as starting materials can be synthesized according to the procedures described in the following references:

4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol,

4,4-dimethyl-5alpha-cholest-8-en-3beta-ol a

4,4-dimethyl-5alpha-cholest-8 (14) en-3beta-ol [Biochem. J. 132 (439);

4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol a

4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol [J.Am.Chem.Soc.111 (1989278);

5alpha-cholesta-8,14-dien-3beta-ol [J.Am.Chem.Soc.75 (1953)]

4404];

5alpha-cholest-8-en-3beta-ol [J. Org.Chem. 46 (1981) 3421] and

5alpha-cholest-8 (14) -en-3beta-ol [Biochem. 144 (1974) 59].

In the following part of the description they are described in detail only

4.4-dimethyl-A-8,14-series. The derivatives in Δ-8, A-8 (14), Δ-8,14,24 and A-8,24-series, with or without 4,4-dimethyl group, can be synthesized in the same manner using the corresponding starting materials.

3beta-Alcohols can be oxidized using various reagents to give the corresponding 3-ketones [e.g., Tetrahedron Lett.1967, 3699].

4,4-Dimethyl-5alpha-cholesta-8,14-dien-3beta-ol is given in • 4 4 • 4

treatment with N-methylmorpholine-N-oxide in the presence of tetrapropylammonium perruthenate to give 4,4-dimethyl-5α-cholesta-8,14-dien-3-one 2 [for example: Synthesis 1994, 639].

Reaction Scheme 1:

In the next step, a vaz-1 double bond can be introduced by an oxidation reaction with phenylselenic anhydride, used as an oxidizing agent, in chlorobenzene [J.Chem.Soc.Chem.Commun.197 8, 952] to form

4,4-dimethyl-5alpha-cholesta-1,8,14-trien-3-one 3. This reaction also takes place for compounds without

4.4-dimethyl groups. In these compounds, a new double bond can be selectively introduced as a double bond Δ1. The keto group can then be reduced with sodium borohydride in the presence of cerium chloride [for example, by the mechanism of the Luche reaction: J. Am. Chem. Soc. 100 (1978), 2278] to give 4,4-di. -5alpha-cholesta-1,8,14-trien-3beta-ol 4.

An alkyl and aryl group can be introduced at the 1-position by adding cuprates to unsaturated ketones [Tetrahedron Lett.35 (1994), 8591]. Copperates, which are formed from alkyl or aryl lithium and cuprous iodide, react with steroid enones to form 1-substituted derivatives. If, for example, enone 3 is reacted with dialkyl copper (II), then compounds of formula 5 (R ¹ = alkyl, aryl) are obtained. These ketones can then be reduced by methods well known in the literature to two diastereomeric alcohols 6 and 7 (R ¹ = alkyl, aryl), which can be easily separated by column chromatography.

Reaction Scheme 2:

The introduction of the cyano group can be carried out in the same manner. Simultaneous addition of cyanide to enone 3 gives the desired 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one 5 (R ¹ = CN). Various reagents such as diethylaluminium cyanide [J. Org.Chem.59 (1994), 2766] and certain alkali and alkaline earth metals may be used in this reaction.

[Tetrahedron Lett. 28 (1987) 4189; Can.J.Chem. 59 (1981), 1641]. Cyanoketone 5 (R ¹ = CN) can be reduced using standard reducing agents such as sodium borohydride to give two diastereomeric alcohols 6 and 7 (R ¹ = CN), which can be easily separated by column chromatography.

When cyanoalcohol 6 (R ¹ = CN, synthesized as described above) is reacted with lithium aluminum hydride, then 1α-aminomethyl-4,4-dimethyl-5α-cholesta-8,14-dien-3-beta-ol 8 is obtained. This amine can be used to further modify the molecule. Amides of formula 9 (R ¹ = alkyl, aryl and fluorescent label) can be synthesized with said amine by reaction with hydroxysuccinimidyl esters of various alkyl or arylcarboxylic acids (using hydroxysuccinimidyl carboxylic acid ester containing a fluorescent label, see: C., Springer Verlag, Berlin, 1992).

For example, the substituents at the 2-position can be introduced by aldol reactions and alkylations. If ketone 2 is reacted with aromatic aldehydes in the presence of a base, then

to give 2-benzylidene-substituted steroids of formula 10. The aromatic ring may be substituted. Subsequent reduction using sodium borohydride in the presence of cerium chloride affords selectively the corresponding ally-3beta-alcohols 11.

Reaction Scheme 4

Cyanobenzylidene-substituted compound 11 (R ² =

4-CN) _r which can be synthesized in the above manner can be used for further modification. The cyano group can be reduced using lithium aluminum hydride to benzylamine 12, which can be derivatized to the corresponding amides of formula 13 (R ² = alkyl, aryl and fluorescent label). To this end, the amine 12 is reacted with hydroxysuccinimidyl ester derivatives of various carboxylic acids. Commercially available esters containing a fluorescent label can also be used for this reaction (see Example 11 of the Example section). Amides of Formula 13 that contain fluorescent markers can be used as molecular probes for biosimaging.

9 purposes.

Reaction Scheme 5:

The alkyl and alkenyl substituents can be introduced at the 2-position by the deprotonation mechanism of ketone 2 and subsequent reaction of the enolate with the alkyl and alkenyl halides. The 3-ketones of formula 14 can then be reduced with sodium borohydride to give two diastereomeric alcohols 15 and 16, which can be easily separated by column chromatography.

♦ · · · ·

Reaction scheme 6

For example, the hydroxymethyl substituents introduced at the 2-position may be deprotonated by the condensation reaction

Of 3-ketone with alkyl formates to give. This enol can be reduced using various reducing agents, for example using sodium borohydride, to form two diastereomeric alcohols 18 and 19.

Reaction Scheme 7:

• ·

• · ♦ · · ·

These alcohols can be easily separated by column chromatography. For example, longer chain hydroxyalkyl substituents may be introduced by the above alkylation reactions (see Reaction Scheme 6).

A further object of the invention is to provide pharmaceutical compositions comprising one or more compounds of formula (I) as an active ingredient. The compositions may further comprise well-known pharmaceutically acceptable excipients such as carriers, diluents, absorption enhancers, preservatives, buffers, osmotic pressure adjusting agents, tablet disintegrating agents, and other additives commonly used in the pharmaceutical industry. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting waxes, and cocoa butter.

Liquid compositions include sterile solutions, suspensions, and emulsions. Such liquid compositions may be suitable for use in connection with ex vivo and in vivo fertilization. These liquid compositions may contain other additives commonly used in the art, some of which have already been mentioned above. Further, a composition for transdermal administration of a compound of the invention in the form of a patch and a composition for nasal administration in the form of a nasal spray in liquid or powder form may be provided.

The dose of the compound of the invention will be determined by the attending physician depending on a number of factors, including in particular the specific type of compound used, the mode of administration and the method of use. In general, the compositions of the invention are prepared by thoroughly mixing the active compound with liquid or solid excipients and optionally shaping the resulting mixture into the desired formulation.

• · · · · · · · · · · · · · · · · · · · ·

The invention relates to the use of the compounds of formula I for the preparation of a medicament for controlling meiosis. The compounds of the invention affect meiosis in both oocytes and male germ cells. There are several mechanisms capable of influencing meiosis. In a preferred embodiment of the invention, the compound of formula I may be used to stimulate meiosis. In another embodiment of the invention, the compound of formula I may be used to stimulate meiosis in humans. The compounds of formula (I) are promising novel fertility-regulating factors that do not have the usual somatic cell side effects of the hitherto known hormonal contraceptives which are based on estrogens and / or gestagens.

Accordingly, the invention relates to the use of the compounds of formula I for treating infertility in females and males, particularly humans. The meiosis-inducing compounds of Formula I can be used in the treatment of certain types of infertility in females, including females, by administering these compounds to females which, due to their inadequate production of the meiosis-activating agent, are not producing mature oocytes.

The compounds of formula I may be used in artificial insemination processes, for example, in vitro fertilization or intracytoplasmic spermatoid injection. If fertilization is performed in vitro, better results can be obtained when the compound of the invention is added to the oocyte culture medium.

If infertility in males, including males, is due to insufficient intrinsic production of the meiosis-activating agent and thus lack of mature spermatoid cells, administration of a compound of the invention may help to overcome this deficiency.

• · · * · ·

According to a further aspect of the invention, the compounds of the formula I are useful as contraceptives in females and males, in particular in mammals and in particular in humans. When used as a contraceptive in females, the meiosis-inducing agent may be administered to prematurely induce the recurrence of meiosis in oocytes that are still in the growth follicle before the gonadotrophin ovulation peak occurs. In women, for example, re-initiation of meiosis may be induced a week after the end of previous menstruation. In the case of ovulation, the resulting overripe oocytes will most likely not be fertilized. The normal menstrual cycle is unlikely to be affected. In this context, it is important to note that biosynthesis of progesterone in cultured human granulosa cells (somatic cells affected by the presence of estrogen-inducing agent and progestogen used contraceptives have in this effect on progesterone biosynthesis).

follicle) is not meiosis, while as a horminal case undesirable

In an alternative form of the method described above, contraception in women can be achieved by administering a compound of the invention that inhibits meiosis, thereby producing immature oocytes. Similarly, contraception in men can be achieved by administering a compound of the invention that inhibits meiosis, thereby producing immature sperm.

The invention also relates to the use of the compounds of the formula I according to the invention as research substances or as starting materials for the synthesis of research substances for biosimaging purposes, in order to clarify the mode of action of such substances. For example, meiosis-activating sterols that contain a fluorescent marker can be used to visualize the germ cell regions in which the active agents exert their biological function. This information • · · · * ♦

may be helpful in elucidating the mechanism of action of these substances.

The invention also relates to a method for controlling meiosis, comprising administering to a subject in need thereof an effective amount of one or more compounds of Formula I.

The invention also relates to a method for controlling meiosis in mammalian germ cells comprising administering to said germ cell in need of such regulation an effective amount of one or more compounds of Formula I ex vivo or in vitro. The germ cell may be an oocyte or a male germ cell.

The route of administration of the compositions comprising the compound of the invention may be any route of administration that effectively delivers the active compounds to their site of action.

When the compounds of the invention are to be administered to a mammal, the compounds are suitably provided in the form of a pharmaceutical composition comprising at least one compound of the invention in combination with a pharmaceutically acceptable carrier. When administered orally, such compositions preferably take the form of capsules or tablets.

It will be apparent from the above that the route of administration selected will depend upon the condition being treated. In the case of infertility treatment, administration may be performed only once or for a limited time, limited, for example, by pregnancy.

When used as a contraceptive, the compounds of the invention will be administered continuously or cyclically. If the compounds of the invention are to be used as contraceptives in females and are not 9 9 99 99

When administered continuously, then it will be important to adjust the time of administration in relation to the ovulation.

In the following, the invention will be explained in more detail with reference to the following examples, which are not to be construed as limiting the scope of the invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

4,4-Dimethyl-5alpha-cholesta-1,8,14-trien-3-one

(a) 4,4-dimethyl-5α-cholesta-8,14-dien-3-one

To a solution of 6.40 g of 4,4-dimethyl-5α-cholesta-8,14-dien-3-beta-ol and 4.03 g of N-methylmorpholine-N-oxide in 32 ml of dichloromethane was added several grains of molecular sieves and the mixture was Stir for five minutes. At ambient temperature, 408 mg of tetrapropylammonium perruthenate are added and the resulting black reaction mixture is stirred for one hour. After filtration through celite, the solvent was evaporated and the residue was chromatographed using hexane / ethyl acetate to give 5.60 g of 4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one in white solid product.

¹ H-NMR (CDC1 _3):

δ = 0.83 (s, 3H, H-18); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.94 (d, J = 7Hz, 3H, H-21); 1.07 (s, 3H);

1.12 (2x s, 6H); 2.55 (m, 2 H);

5.41 (s, 1H, H-15).

• · ··· ·

b) 4,4-Dimethyl-5alpha-cholesta-1,8,14-trien-3-one

To a solution of 2.00 g of 4,4-dimethyl-5α-cholesta-8,14-dien-3-one in 30 ml of chlorobenzene was added 1.75 g of phenylselenic anhydride at room temperature. The reaction mixture was heated at 100 ° C for two hours. After cooling and evaporation of the solvent, the evaporation residue is chromatographed, eluting with hexane / ethyl acetate, to give 0.85 g.

4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one as an oil.

^* H-NMR (CDC1 _3):

δ - 0.84 (s, 3H, H-18); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.96 (d, J = 7Hz, 3H, H-21); 1.1 (s, 3H);

1.18 (s, 3H); 1.25 (s, 3H);

5.45 (s, 1H, H-15); 5.95 (d, J = 10 Hz, 1H, H-2);

7.33 (d, J = 10 Hz, 1H, H-1).

Example 2

4,4-Dimethyl-5alpha-cholesta-1,8,14-trien-3beta-ol

To a suspension of 73 mg

4,4-dimethyl-5alpha-cholesta-1,8,14-trien-3-one and 67 mg of cerium chloride in methanol are added at room temperature to 14 mg of sodium borohydride. The resulting mixture was stirred for 4 hours, then poured into water and extracted with diethyl ether. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered. After evaporation of the solvent, the evaporation residue is chromatographed with hexane / diethyl ether to give 43 mg of 4,4-dimethyl-5α-1H-cholesta-1,8,14-trien-3-beta-ol as a white solid. product.

·· ····

Η-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.82 (s, 3H); 0.85 (s. 3H);

0.87 (2x d, J = 7Hz, 6H, H-26/27); 0.96 (d, J = 7Hz, 3H, H-21);

1.02 (s, 3H);

3.89 (m, 1H, H-3);

1.10 (s, 3H);

5.37 (s, 1H, H-15);

5.50 (dd, J = 10Hz, 1Hz, 1H, H-1); 5.90 (d, J = 10 Hz, 2 Hz, 1H, H-2)

Example 3

5alpha-Cholesta-1,8,14-trien-3beta-ol

(a) 5alpha-Cholesta-8,14-trien-3-one

2.40 g of 5α-cholesta-8,14-dien-3-beta-ol are mixed with

1.11 g of N-methylmorpholine-N-oxide and 111 mg of tetrapropylammonium perruthenate in 13 ml of dichloromethane as described in Example 1a. It is obtained after chromatography

1.81 g of 5alpha-cholesta-8,14-dien-3-one as a white solid.

^χ Η-ΝηΚ1βέτηί magnetic resonance spectrum (CDC1 ₃ ):

Δ = 0.82 (s, 3H, H-18); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.94 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H, H-19 each);

5.39 (s, 1H, H-15).

b) 5alpha-1,8,14-trien-3-one

1.81 g of 5α-cholesta-8,14-dien-3-one were reacted with 1.67 g of phenylselenic anhydride in 29 ml of chlorobenzene as described in Example 1b. After chromatography, 0.53 g of 5alpha-cholesta-1,8,14-trie-3-one is obtained as an oil.

¹ H-NMR (CDC1 _3):

· «« ·························

9 9 9 9 99 9 9 ·

9 9 9 9 9

9 9 9 9 9 9 9 9 99 9 δ = Ο, 84 (s, 3Η, Η-18);

0.96 (d, J = 7Hz, 3H, H-21);

5.43 (s, 1H, J-15);

7.42.

0.86 (2χ d, J = 7Hz, 6Η, Η-26/27);

1.20 (s, 3H, H-19);

5.91 (d, J = 10 Hz, 1H, H-2);

c) 5alpha-Cholesta-1,8,14-trien-3beta-ol

150 mg of 5α-cholesta-1,8,14-trien-3-one is reacted with 15 mg of sodium borohydride and 147 mg of cerium chloride heptahydrate. as described in Example 2. After chromatography 47 mg of 5α-cholesta-1,8,14-trien-3-beta-ol was obtained as a white solid.

and

Η-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.82 (s, 3H, H-18); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.95 (d, J = 7Hz, 3H, H-21); 1.07 (s, 3H, H-19);

4.33 (m, 1H, H-3); 5.37 (s, 1H, H-15);

5.56 (dd, J = 10Hz, Hz, 1H, H-1); 6.09 (d, J = 10 Hz, 2 Hz, 1H, H-2)

EXAMPLE 4 [alpha] 1alpha-Cyano-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one

To a solution of 4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one in 5 mL of tetrahydrofuran at 0 ° C was added 1.47 mL of diethylaluminium cyanide solution (1M in toluene). The reaction mixture is then allowed to warm to ambient temperature and stirred at this temperature for one hour. After adding

2.45 ml of 1M sodium hydroxide solution at 0 ° C, the resulting mixture was diluted with water and extracted with diethyl ether. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered. After evaporation of the solvent, the evaporation residue is chromatographed with hexane-ethyl acetate to give 110 mg of .alpha.-cyano-4,4-dime. · · · · · · ·

9 ♦ ···· · 9 9 9 9 9 9 9 · 99 1 99 1 99 1

ethyl-5alpha-cholesta-8,14-dien-3-one as a white solid.

¹ H-NMR (CDC1 _3):

δ = 0.86 (2x d, J = 7Hz, 6H, H-26/27); 0.89 (s. 3H);

0.94 (d, J = 7Hz, 3H, H-21); 1.12 (s, 3H);

1.20 (s, 3H); 2.86 (m, 1H, H-2);

3.29 (dd, J = 7Hz, 5Hz, 1H, H-1); 5.49 (ps, 1H, H-15).

EXAMPLE 5 [alpha] 1alpha-Cyano-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol and 1alpha-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3alpha-ol

To 95 mg of .alpha.-cyano-4,4-dimethyl-5.alpha.-cholesta-8,14-diene-3-one is added 33 mg of sodium borohydride as described in Example 5 to give 30 mg of .alpha.-cyano-4, 4-dimethyl-5alpha-cholesta-8,14-dien-3alpha-ol and 25 mg of 1alpha-cyano-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol.

¹ H-Cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-beta-ol: ¹ H-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.83 (s, 3H); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.94 (d, J = 7Hz, 3H, H-21); 1.09 (s, 3H);

1.17 (s. 3H); 3.08 (m, 1H, H-1);

3.72 (bm, 1H, H-3); 5.43 (ps, 1H, H-15);

.alpha.-cyano-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-3.alpha.-ol: ¹ H-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.87 (2x d, J = 7 Hz, 6H) H-26/27); 0.91 (s, 3H);

0.93 (d, J = 7Hz, 3H, H-21); 0.08 (s, 3H);

1.15 (s, 3H); 2.92 (m, 1H, H-1);

3.57 (ps, 1H, H-3); 5.44 (ps, 1H, H-15).

Example 6 φ φφφφ φφφ φφ »»φφφ φ

ΦΦΦ φφφφ φ »φφφφφφφφφφφφφ

Φ ΦΦΦ ΦΦΦ Φ φ φ ΦΦΦ ΦΦΦ (Ε) -4 - [(3--χο-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile

To a suspension of 4,4-dimethylcholesta-8,14-dien-3-one in 4 mL of ethanol was added a suspension of 64 mg at room temperature.

Of 4-cyanobenzaldehyde and 27 mg of potassium hydroxide. The reaction mixture was stirred for 20 hours, diluted with water and extracted with dichloromethane. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered. After evaporation of the solvent, the residue is chromatographed using hexane / ethyl acetate to give 168 mg of (E) -4 - [(3-oxo-4,4-dimethyl-5alpha-cholesta-8,14-diene). 2-ylidene) methyl] benzonitrile as a white solid. ¹ H-NMR (CDC1 _3): δ = 0.83 (s, 3H); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.93 (d, J = 7Hz, 3H, H-21); 0.93 (d, J = 7Hz, 3H, H-21);

0.98 (s, 3H); 1.16 (s, 3H);

1.23 (s. 3H); 2.58 (d, J = 17 Hz, 1H, H-1);

3.10 (d, J = 17 Hz, 1H, H-1); 5.47 (ps, 1H, H-15);

7.42 (s, 1H, H-2 '); 7.53 (d, J = 8Hz, 2H, arom.);

7.68 (d, J = 8Hz, 2H, arom.).

Example 7 (E) -4 - [(3beta-Hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile

156 mg of (E) -4 - [(3-oxo-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile was mixed with 12 mg of sodium borohydride in the presence of 111 mg of chloride heptahydrate cerium as described in Example 2 to give 93 mg of (E) -4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl ] benzonitrile as a white solid.

* · It's the ·· • »^ a Ti-NMR (CDC1 _3): δ = 0.75 (s, 3H); 0.80 (s, 3H)

0.87 (2x d, J = 7Hz, 6H, H-26/27); 0.92 (d, J 17 Hz, 3H, H-21); 1.18 (s, 3H); 3.02 (d, J = 17 Hz, 1H, H-1);

3.94 (m, 1H, H-3); 5.40 (ps, 1H, H-15);

6.78 (s, 1H, H-2 '); 7.37 (d, J = 8Hz, 2H, arom.);

7.64 (d, J = 8 Hz, 2H, arom.).

Example 8 (E) -N - [[4 - [(3beta-Hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide

a) (E) -2- (4-Aminomethylphenyl) methylidene-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol

A suspension of 27 mg of lithium aluminum hydride and 74 mg of (E) -4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile in 6 ml of tetrahydrofuran is heated to a temperature of under reflux for 4 hours. After cooling, the reaction mixture was worked up as described in Example 3b. Crystallization from ethyl acetate gave 26 mg of (E) -2- (4-aminomethylphenyl) methylidene-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-beta-ol as a white solid.

¹ H-NMR (CDC1 _3): δ = 0.77 (s, 3H); 0.82 (s, 3H);

0.86 (2x d, J = 7Hz, 6H, H-26/27); 0.89 (s. 3H);

0.92 (d, J = 7Hz, 3H, H-21);

3.15 (d, J = 17 Hz, 1H, H-1);

3.92 (ps, 1H, H-3);

6.72 (s, 1H, H-2 ');

1.18 (s. 3H);

3.86 (s, 2H, _Ar-CH2 -N);

5.38 (ps, 1H, H-15);

7.26 (m, 4H, arom.).

** w ·· «· * · e · 100 *

W · · · · ··· *

9 9 9 9 9 9 9

9 9 9 9 9

9 99 99 »4 4 · ·

b) (E) -N - [[4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide

To a solution of 21 mg of (E) -2- (4-aminomethylphenyl) methylidene-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-beta-ol in 4 ml was added a solution of 8.6 mg of N at room temperature. -hydroxysuccinimidyl caprylate in 1 ml dimethylformaldehyde. The reaction mixture was stirred for 20 hours, diluted with water and extracted with ethyl acetate.

The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and filtered. Evaporation of the solvent gave 21 mg of (E) -N - [[4 - [(3-beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] phenyl] methyl ] octanamide as a white solid.

¹ H-NMR (CDC1 _3): δ = 0.77 (s, 3H); 0.82 (s, 3H)

0.86 (2x d, J = 7Hz, 6H, H-26/27); 0.89 (s. 3H);

0.92 (d, J = 7Hz, 3H, H-21);

3.12 (d, J = 17 Hz, 1H, H-1);

4.44 (d, J = 5 Hz, 2H, _Ar-CH2 N);

5.76 (t, J = 5Ηζ, Ηζ, ΝΗ);

1.18 (s. 3H);

3.92 (ps, 1H, H-3);

5.37 (ps, 1H, H-15);

6.72 (s, 1H, H-2);

7.24 (m, 4H, arom.).

Example 6 (E) - N - [[4 - [(3beta-Hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4, (-, -)) 4-Difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacen-3-yl) pentamide

To a solution of 8.9 mg of (E) -2- (4-aminomethylphenyl) methylidene-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-beta-ol in 2 ml of dimethylformamide is added a solution of 5 at room temperature, 0 mg

5- (4,4-difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacen-3-yl) pentanoic acid as succinyl ester in 0.5 ml

dimethylformamide. The reaction mixture was stirred for 44 hours, then diluted with water and extracted with ethyl acetate. The organic layer was separated, washed with water, dried over anhydrous sodium sulfate, and filtered. After evaporation of the solvent, the residue is chromatographed using hexane / ethyl acetate to give 6.5 mg of (E) -N - [[4 - [(3beta-hydroxy-4,4-dimethyl-5beta-cholesta)]. -8,14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4-difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacene-3- yl) pentamide.

¹ H-NMR (CDC1 _3):

δ = 0.77 (s, 3H); 0.82 (s, 3H);

0.86 (2x d, J = 7Hz, 6H, H-26/27); 0.89 (s. 3H);

0.92 (d, J = 7Hz, 3H, H-21); 1.19 (s, 3H);

2.25 (s, 3H, Ar-Me); 2.55 (s, 3H, Ar-Me);

3.12 (d, J = 17 Hz, 1H, H-1); 3.92 (ps, 1H, H-3);

4.44 (d, J = 5 Hz, 2H, _Ar-CH2 -N); 5.37 (ps, 1H, H-15);

5.87 (m, 1H, NH); 6.09 (s, 1H, arom.);

6.30 (d, J = 5 Hz, 1H, arom.); 6.70 (s, 1H, H-2 ');

6.90 (d, J = 5Hz, 1H, arom.); 7.24 (m, 4H, arom.).

Example 10

a) 2alpha-Allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one

To a freshly prepared solution of lithium diisopropylamide (5.8 mL, 1M) at 70 ° C was added a solution of 200 mg of 4,4-dimethylcholesta-8,14-dien-3-one in 3 mL of tetrahydrofuran. The solution obtained is stirred for one hour and then 0.07 ml of 3-propene is added. After stirring for an additional hour at 0 ° C, the reaction mixture was poured into saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered.

··· ·

After evaporation, the residue is chromatographed using hexane / ethyl acetate to give 160 mg of 2alpha-allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one as a white solid. .

¹ H-NMR (CDC1 _3):

δ = 0.82 (s, 3H); 0.87 (2x d, J = 7Hz, 6H, H-26/27);

0.93 (d, J = 7Hz, 3H, H-21); 1.10 (2x s, 6H);

1.30 (s, 3H); 2.60 (m, 1H, allyl);

2.77 (m, 1H, allyl); 5.05 (m, 2H, allyl);

5.38 (ps, 1H, H-15); 5.80 (m, 1H, allyl).

b) 2alpha-Allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol

160 mg of 2alpha-Allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one are mixed with 55mg of sodium borohydride as described in Example 5 to give 15mg of 2alpha-allyl-4,4 dimethyl-5alpha-cholesta-8,14-diene-3alpha-ol and 80 mg of 2alpha-allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol.

2alpha-Allyl-4,4-dimethyl-5alpha-cholesta-8,14-3beta-ol: ¹ H-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.81 (s, 3H); 0.85 (s. 3H)

0.87 (2x d, J = 7Hz, 6H, H-26/27); 0.94 (d, J = 7Hz, 3H, H-21);

1.03 (s, 3H); 1.0 (s, 3H);

2.50 (m, 1H); 2.95 (d, J = 11 Hz, 1H, H-3);

5.06 (m, 2H, allyl); 5.37 (ps, 1H, H-15);

5.89 (m, 1H, allyl);

2alpha-allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3alpha-ol: ¹ H-Nuclear Magnetic Resonance Spectrum (CDCl ₃ ): δ = 0.82 (s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27);

0.90 (s, 3H); 0.94 (d, J = 7Hz, 3H, H-21);

• ·

1.05 (s. 3H);

5.05 (m, 2H, allyl);

5.85 (m, 1H, allyl).

1.00 (s, 3H);

3.36 (ps, 1H, H-3);

5.35 (ps, 1H, H-15);

Example 11

Testing of meiosis activating agents in the oocyte test

Animals

Oocytes were obtained from juvenile female mice (C57BI / 6J x DBA / 2J Fl-hybrids, Bomholtgaard, Denmark) with a body weight of 13 to 16 grams, which were kept under controlled temperature and light conditions. Mice were injected intraperitoneally with 0.2 ml of gonadotropins (Gonal F, Derono, Salna, Sweden containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU FSH) and after 48 hours the animals were sacrificed by cervical dislocation.

Isolation and cultivation of oocytes

After ovaries were cut off, the oocytes were isolated in Hx medium (see below) under a stereomicroscope by manually rupturing the follicles using a pair of 27 gauge needles. Spherical exposed oocytes (NO) having intact embryonic bladder (GV) were stored in alpha-minimal essential medium (α-MEM) ribinucleoside-free, Gibco BRL, Cat # 22561) enriched with 3 mM hypoxanthine (Sigma, Cat # H-9377), 8 mg / ml Human Serum Albumin (HSA, Stata Serum Institute, Denmark), 0.23 nM pyrubate (Sigma, Cat. No. S-8636), 2 mM glutamine (Flow, Cat. No. 16-801), 100 IU / ml penicillin and 100 gg / ml streptomycin (Flow, Cat. No. 16-700). This environment is known as the Hx environment. Oocytes were rinsed three times with Hx medium and cultured on 4-well multi-plates (Nuncion, plates containing 0.4 concurrently with test control cultured compounds), at different concentrations of test compounds.

Denmark), with ml of Hx medium and 35 to 45 oocytes.

cultivations (in which in Hx medium each well was performed each one was 35, without the addition of oocytes tested using

The cultures were carried out at 37 ° C and 100% humidity of air containing 5% carbon dioxide. The cultivation time was 22 to 24 hours.

Examination of oocytes

At the end of the culture period, the numbers of oocytes with germinal bladder (GV) or with damaged germinal bladder (GVB) or polar oocytes (PB) were counted using a stereomicroscope or inverted imaging microscope equipped with a differential interference contrast control device. In the cultures tested, the percentage of oocytes with GVB based on total oocyte counts and the percentage of oocytes with PB based on total oocyte counts were compared to the results obtained for control culture.

Example 12

Testing of Meiosis Inhibitors in the Oocyte Test

Germ bladder (GV) oocytes were obtained from juvenile FSH-treated female mice using the procedures used in Example 11 (see above). Exposed oocytes (NO) were flushed three times in Hx medium. U 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-olu

³¹ (FF-MAS) has previously been shown that induces meiosis in exposed oocytes in vitro (Byskov AG et al. Nature 374 (1995) 559-562. NO oocytes were cultured in Hx medium enriched with 5 μΜ FF-MAS as co-cultures with test compounds used at different concentrations in 4 wells multipot plates (Nunclon, Denmark), each well containing 0.4 ml of Hx medium and 35-45 oocytes, and one positive control culture (35-45 oocytes cultured in Hx medium containing FF-MAS without in test cultures, test compounds were used at various concentrations, and in addition, one negative control culture (35-45 oocytes, which were cultured in Hx medium alone) was performed. .

Examination of oocytes

At the end of the culture period, the numbers of oocytes with germinal bladder (GV) or with damaged germinal bladder (GVB) or polar oocytes (PB) were counted using a stereomicroscope or inverted imaging microscope equipped with a differential interference contrast control device. In the cultures tested, the percentage of oocytes with GVB based on total oocyte counts and the percentage of oocytes with PB based on total oocyte counts were compared to the results obtained for control culture.

Example 13

In vitro fertilization testing of meiosis activating agents

Exposed oocytes (NkO) and cumulated oocytes (CEO) were isolated from juvenile FI follicles (C57BL / 6xDBA / 2) (21-24 days old) given 48 hours before oocyte isolation with 10 IU of Pregnant Mara. Serum Gonadotropin (FSH activity). Oocytes (NkO and CEO) were collected and cultured for about 20 hours in a modified α-MEM medium containing 3 mM hypoxanthine (Hx medium) and 1 mg fetuin per ml culture medium. Two groups of oocytes were used: a) control oocytes cultured in Hx-free medium (positive control group) and b) oocytes cultured in Hx medium containing test compound. After about 20 hours, the germinal bladder (GVB) oocytes were briefly washed with Xx-free medium and transferred to preformed insemination dishes containing a male cauda epididymis motile spermatoid product. The plates were then incubated under defined gaseous medium conditions (5% CO ₂ ) at 37 ° C in a modified α-MEM IVF medium. Oocyte examinations were performed 20-22 hours after insemination to control fertilization and count

2-cell embryos. The percentage of fertilization (= degree of fertilization) was determined as the number of oocytes that had been split into two-cell embryos based on the total number of inseminated oocytes. Each IVF experiment was performed with a total of 50-200 GVB / PB-oocytes. The stimulation factor was calculated as the ratio between the degree of fertilization in the test compound containing group and the degree of fertilization in the control group.

• ···· * ·· ·. <

· · ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** _and »» »

Table 1

Activation of meiosis in oocytes of nude mice

Compounds GV Oocytes [n] GVB PB Activation [ %] GVB + PB Control (Hx) 39 0 1 2.6 10 μΜ FF-MAS 5 29 4 86, 8

μΜ 4,4-dimethyl-5α-cholesta-1,8,14-triene-3-ο1 9

5

71, 9

Hx = hypoxanthine

GV = Germ Bladder

GVB = broken embryonic bladder

PB = polar body n = oocyte count • · «·

Table 2

Relative inhibition of meiosis in oocytes of nude mice

Compounds GV Oocytes [n] GVB PB Inhibition [%] Control (Hx) 34 2 0 100 ALIGN! 5 μΜ FF-MAS 6 28 0 0

ΜΜ FF-MAS + 40 ΜΜ la-cyano-4,4-dimethyl-5α-cholesta-8,14dien-3-one

0 0

107

Hx = hypoxanthin GV = embryonic bladder GVB = broken embryonic bladder PB = polar body n = oocyte count

· T 4 4 4 4 4 9 9 9 9 9 9 9 9 9 9 9 9 9

Table 3

Relative inhibition of meiosis in oocytes of nude mice

Compounds GV Oocytes [n] GVB PB Inhibition [%] Control (Hx) 34 2 0 100 ALIGN! 5 μΜ FF-MAS 6 2 8 0 0

μΜ FF-MAS + μΜ (10 μΜ (E) -N - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide 25

0

Hx = hypoxanthin GV = embryonic bladder GVB = broken embryonic bladder PB = polar body n - oocyte count

• · · ·

• f (/ όύθϋ1 ~)

Claims (13)

PATENTOVÉ Claims Compounds of the general formula I in which R is hydrogen; an alkyl group having 2 to 6 carbon atoms; optionally substituted phenyl; cyano; a C ¹ -NH-COR ¹ group in which R ¹ represents an alkyl group having 1 to 8 carbon atoms or an optionally substituted phenyl group; or together with R represents an additional bond; R is hydrogen; C 4 -C 8 alkyl; (C 3 -C 6) alkenyl; a (C 1 -C 6) hydroxyalkyl group; together with R ^{2 an} optionally substituted benzylidene group; together with R ² a hydroxymethylene group; or together with R ¹ an additional bond; R ² is hydrogen; together with R ^{2 an} optionally substituted benzylidene group; or together with R ² a hydroxymethylene group; • ·· R ³ means atom hydrogen; or together with 3 ' R additional a bond; R ³ ' means atom hydrogen; or together with 3 R additional a bond; R ⁴ means atom hydrogen; or methyl a group; R ⁴ means atom hydrogen; or methyl a group; R ⁸ means together with R ⁹ or 14 with R additional bond; R ⁹ means atom hydrogen; or together with G R additional a bond; 14 8 R is α-hydrogen; or together with R additional R ¹⁵ a bond; or í together '15 s R additional a bond; means atom hydrogen; or together with R ¹⁴ additional a bond; E ²⁴ means atom hydrogen; or together with R ²⁵ additional a bond; and R ²⁵ means atom hydrogen; or together with R ²⁴ additional a bond; or their esters, with the exception that the scope of the said compounds belong compounds which are simultaneously unmodified in position 1 and 2, i.e. when R ¹ - R ² = R ² = H. Compounds according to claim 1, in which R ¹ represents a hydrogen atom, a phenyl group or together with 2 R additional bond. Compounds according to claim 1 or 2, in which R represents an alkyl group having 4 to 8 carbon atoms, an allyl group or an additional bond with Compounds according to either of Claims 1 or 2 of the general formula (s): - - - - - - - - - Formula I in which R ² and R ² are an optionally substituted benzylidene group. Compounds according to any one of claims 1 to 4 which are: 2alpha-allyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol, (E) -2-benzylidene-4,4-dimethyl-5alpha-cholesta-8,14-diene-3beta- ol, 5alpha-cholesta-1,8,14-trien-3beta-ol, 5alpha-cholesta-1,8,14-trien-3-one, 1alpha-cyano-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol, 1alpha-cyano-4,4-dimethyl- 5alpha-cholesta-8,14-3-one, 4,4, dimethyl-5alpha-cholesta-1,8,14-trien-3beta-ol, 4,4-dimethyl-5alpha-cholesta-1,8,14-trien-3-one, (E) -4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-diene- 2-ylidene) methyl] benzonitrile, (E) -N - [[4 - [(3beta-hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide, (E) - N - [[4- [3beta-Hydroxy-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4-) - difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacen-3-yl) pentanamide, 2alpha-hydroxymethyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3alpha-ol, 2alpha-octyl-4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol; and (E) -4 - [(3-Oxo-4,4-dimethyl-5alpha-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile. Pharmaceutical compositions comprising one or more compounds of the formula I as claimed in any one of claims 1 to 5 as active substances. Ί. Use of a compound of formula I according to any one of claims 1 to 5 for the preparation of a medicament for controlling meiosis. Use according to claim 7 for the preparation of a medicament for treating infertility in female or male, preferably human. Use according to claim 7 for the preparation of a contraceptive medicament for the treatment of females or males, preferably humans. Use of compounds of formula I according to any one of claims 1 to 5 for controlling the degree of fertilization in artificial insemination processes. Use of compounds of the formula I according to any one of claims 1 to 5 as research substances or as starting materials for the synthesis of research substances for biosimaging purposes in order to elucidate the mechanism of action of such substances. 12. A method for controlling meiosis comprising administering to a subject in need thereof an effective amount of one or more compounds of Formula I according to any one of claims 1 to 5. 13. A method for controlling meiosis in mammalian germ cells, comprising administering to a germ cell in need of such regulation an effective amount of one or more compounds of formula I according to any one of claims 1 to 5, ex vivo or in vitro. The method of claim 13, wherein the germ cell is an oocyte or a male germ cell.