SK11382001A3 - 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 a specific and major germ cell manifestation on which sexual reproduction is based. Meiosis involves two meiotic divisions. During the first meiotic division, maternal and paternal genes are exchanged 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 generation of haploid germ cells with only 1c DNA.

Meiotic events 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 embryonic cells enter the prophase of the first meiotic division in the early period of life, often before birth, but all are later arrested in the prophase as oocytes (dictate) until ovulation after puberty. In this way, the female has an oocyte supply at an early stage of her life, which is pumped until that supply is depleted. Meiosis in females is completed only after fertilization and results in only one egg and two abortive polar bodies. one germ cell. In contrast, only some of the male germ cells undergo meiosis starting from puberty and constitute the stem cell population throughout life. After initiation, meiosis proceeds 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. With regard to oocytes, some recent studies report that follicular purines, hypoxanthine or adenosine could 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 a culture of fetal mouse gonads

[Byskov, A.G. and al. Dev Biol (1976) 193-200]. some meiosis-activating substance (LAG) was secreted fetal mice ovary, v which proceeded meiosis, while another meiosis-inhibiting substance (MPS) was released from morphologically

differentiated testes with key non-meiotic germ cells. The 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, A.G. et al., The

Psysiology of Reproduction [publ. Knobil E. and Neil, J.D., raven

Press, New York (1994)]. It is therefore clear that if meiosis can be controlled, then reproduction can also be controlled. In a recent article [Byskov A.G. et al., Nature 374 (1995), 559-562] describes the isolation of certain sterols from bull testicles and from follicular fluid, wherein said sterols allegedly are oocyte meiosis.

Unfortunately, these sterols are relatively humane to activate labile, and the use of this in the event that an interesting finding would be available more readily facilitated significantly by the compounds capable of activating meiosis.

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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 the treatment of 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-based contraceptives.

SUMMARY OF THE INVENTION

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

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 of formula I are unsaturated cholestane derivatives ,, 5 ²⁴ . with R25 R1 LjR ⁹ I // (I) R3 fC R ⁴ R ⁴ ' p ruR ¹⁵

in which

R ¹ represents a hydrogen atom; (C 2 -C 6) -alkyl; optionally substituted phenyl;

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cyano; a CH 2 -NH-COR ¹ group wherein R ¹ represents an alkyl group having 1 to 8 carbon atoms or an optionally substituted phenyl group; or together with ^R2 represents an additional bond;

R ² is H; (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 H; together with ^R2 an optionally substituted benzylidene; or together with R ² a hydroxymethylene group;

R ³ means a hydrogen atom; or common with R ³ additional a bond; R ³ ' means a hydrogen atom; or common with R ³ additional a bond; R ⁴ means a hydrogen atom; or methyl group; R ⁴ ' means a hydrogen atom; or methyl group; R ⁸ means together with R ⁹ or with R ¹⁴ additional bond; R ⁹ means a hydrogen atom; or common with R ⁸ additional vä zbu;

R ¹⁴ is α-hydrogen; or together with R ^{3 an} additional bond; or together with R ^{15 an} additional bond;

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

E ²⁴ represents a hydrogen atom; or together with R ^{25 an} additional bond; and

R ²⁵ represents a hydrogen atom; or together with R ^{24 an} additional bond; or their esters, except that the compounds do not include those which are not simultaneously modified

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at positions 1 and 2, i.e., where R ¹ = R ² = R ² = 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 ^{2 is an} additional bond. Other preferred compounds of the invention are those compounds of formula I wherein R ² is C 4 -C 8 alkyl or allyl or forms an additional bond with R ¹ . Also preferred compounds of the invention are compounds of formula I, wherein R ² and R ² represent an optionally substituted benzylidene.

In another embodiment, the invention relates to esters of 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 and when used in combination, refers to a straight or branched chain alkyl group. Preferred examples of C 2 -C 6 alkyl are ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and cyclohexyl

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

2a-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol, (E) -2-benzylidene-4,4-dimethyl-5α-cholesta-8,14-diene-3β- ol,

5a-cholesta-l, 8,14-trien-3.beta.-ol,

5α-cholesta-1,8,14-trien-3-one, 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol, 1α-cyano-4,4-dimethyl- 5a-cholesta-8.14-3-one,

4,4-dimethyl-5α-cholesta-1,8,14-trien-3β-ol,

4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one, (E) -4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-diene-2-) (E) -N - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide, (E) - N 7 - [[4 - [(3β-Hydroxy-4,4-dimethyl-5α-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,

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

2a-octyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol; and

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(E) -4 - [(3-Oxo-4,4-dimethyl-5α-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 the compounds of the formula I can thus take place on the basis of 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;

Rood's Chemistry of Carbon Compounds (published by S. Cooffrey): Elsevier Publishing Company, 1971; and in particular Disctionary of Steroids (ed. R. A. Hill; D. N. Kirk; H. L. J. Makin and G. M. Murphy): Chapman & The latter 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-5α-cholesta-8,14-dien-3β-ol,

4,4-Dimethyl-5α-cholest-8-en-3β-ol a

4,4-dimethyl-5α-cholest-8 (14) εη-3β-ο1 [Biochem. J.132 (439)];

4,4-Dimethyl-5α-cholesta-8,24-dien-3β-ol a

4,4, dimethyl-5α-cholesta-8,14,24-trien-3β-ol [J.Am.Chem.Soc.111 (1989), 278)];

5α-cholesta-8,14-dien-33-ol [J.Am.Chem.Soc.75 (1953) 4404];

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

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5α-cholest-8 (14) -βη-3β-ο1 [Biochem. J.144 (1974) 59].

Only 4,4-dimethyl-Δ-β, 14-series are described in detail in the following. The derivatives in Δ-8, Δ-8 (14), Δ-8,14,24 and á-8,24-series with or without 4,4-dimethyl group can be synthesized in the same manner using the corresponding starting materials.

3β-Alcohols can be oxidized using various reagents to give the corresponding 3-ketones [e.g., Tetrahedron Lett. 1967, 3699]. 4,4-Dimethyl-5α-cholesta-8,14-dien-33-ol is reacted with N-methylmorpholine-N-oxide in the presence of tetrapropylammonium perruthenate to give 4,4-dimethyl-5α-cholesta-8,14- dien-3one 2 [e.g.: Synthesis 1994, 639].

Reaction Scheme 1:

In the next step, a väz-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. 1978, 952] to give 4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one 3. This reaction also takes place for compounds without

4,4-dimethyl group. In these compounds, a new double bond can be selectively introduced as a double bond Δ1. Keto group up

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it can then be reduced with sodium borohydride in the presence of cerium chloride [for example by the Luche reaction mechanism: J. Am. Chem. Soc. 100 (1978), 2278] to give 4,4-dimethyl-5α-cholesta-1,8,14-triene- 3p-ol 4.

The alkyl and aryl groups 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 copper (I) iodide, react with steroid enones to form 1-substituted derivatives. For example, if enone 3 is reacted with dialkyl cuprates, 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 1-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one 5 (R ¹ = CN). Various reagents such as diethylaluminum10 cyanide [J. Org. Chem. 59 (1994), 2766] and certain alkali and alkaline earth metals [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 the alcohols borohydride and 7 (R ¹ sodium, to give the two diastereomers can be readily separated by column chromatography.

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

Reaction scheme 3

For example, the substituents at the 2-position can be introduced by aldol reactions and alkylations. If the ketone 2 reacts with the aroma, the ketone 2 is reacted with the aroma. · · Et k k k In the presence of a base, 2-benzylidene-substituted steroids of formula 10 are obtained. The aromatic ring may be substituted. Subsequent reduction using sodium borohydride in the presence of cerium chloride provides selectively the corresponding allyl-3β-alcohols 11.

Reaction scheme 4

The cyanobenzylidene substituted compound 11 (R ² = 4-CN), which can be synthesized as described above, 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, 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 which contain fluorescent markers can be used as molecular probes for biosimaging purposes.

Reaction scheme ·· • · • ·

Alkyl and by the mechanism of alkyl reduction of alcohols alkenyl substituents can be introduced by deprotonation of ketone 2 followed by alkenyl halides. 3-Ketones of the formula sodium borohydride to give two and 16, which can be chromatographed.

Reaction scheme 6 at position 2 by reaction of the enolate can then be easily separated by diastereomeric columnar.

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

3-ketone with alkyl13 formates to form enol 17. This enol can be reduced

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using various reducing agents, for example using sodium borohydride, to form two diastereomeric alcohols 18 and 19.

Reaction scheme 7

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

A further object of the invention is to provide pharmaceutical compositions comprising one or more compounds of formula I as active ingredient. These 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 such as magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low waxes

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melting point 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 before. 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 mode of use. In general, the compositions of the invention are prepared by intimately 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 can 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 which do not have the usual side effects on somatic cells, which occur in the hitherto known hormonal contraceptives which are based on estrogens and / or gestagens.

Accordingly, the invention relates to the use of compounds of general interest

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of formula I for the treatment of infertility in females and males, in particular in mammals, and in particular in humans. The compounds of formula I indicating meiosis may be used to treat some types of infertility in females, including females, by administering these compounds to females which are unable to produce mature oocytes due to their inadequate production of the meiosis activating agent.

Compounds of formula (I), artificial insemination, intracytoplasmic perform fertilization, for example, in spermatoid in vitro, may be used in in vitro processes or by fertilization by injection. In the case of, in vitro, better results can then be obtained if the environment in which the compound of the invention is cultivated with oocytes.

was added

If infertility in males, including males, is due to insufficient intrinsic production of the meiosis-activating agent and thus lack of mature sperm 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 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 meiosis relaunch in oocytes that are still in the growth follicle before the gonadotrophin ovulatory peak occurs. In women, for example, relapsing 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 progesterone biosynthesis in cultured human granulosa cells (somatic follicle cells) is not affected by the presence of a meiosis-inducing substance, while estrogens and gestagens used as hormonal agents

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contraceptives in this case have an adverse effect on the progesterone biosynthesis.

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, male contraception 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 areas of the germ cell in which the active agents perform their biological function. This information may be helpful in explaining 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 of 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 compositions comprising a compound of the invention may be any route of administration that effectively delivers effective

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compounds at the site of their action.

When the compounds of the invention are to be administered to a mammal, the compounds are conveniently 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.

From the foregoing, it will be appreciated that the mode 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 used as a contraceptive in females and are not administered continuously, then it will be important to adjust the time of administration relative to ovulation.

In the following, the invention will be further elucidated by means of specific examples thereof, the examples being illustrative only and not in any way limiting the scope of the invention, which is clearly defined by the definition of the claims and the description.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

4,4-dimethyl-5a-cholesta-l, 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-30-ol

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and 4.03 g of N-methylmorpholine-N-oxide in 32 ml of dichloromethane are added several grains of molecular sieves and the mixture is stirred for 5 minutes. 408 mg of tetrapropylammonium perruthenate are added at ambient temperature 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-5α-cholesta-8,14-dien-3-one as a white solid. solid product.

^X H-Nuclear Magnetic Resonance Spectrum (CDCl3):

5 = 0, 83 (with, 3H, H-18); 0.87 (2x d, J = 7Hz, 6H, H-26/27); 0 94 (D, J 7 Hz, 3H, H-21; 1, 07 (with, 3H); 1 12 (2x WITH, 6H); 2.55 (M, 2H); 5 41 (with, 1H, H-15).

b) 4,4-Dimethyl-5α-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 ambient temperature. The reaction mixture was heated at 100 ° C for 2 hours. After cooling and evaporation of the solvent, the evaporation residue is chromatographed using hexane / ethyl acetate to give 0.85 g of 4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one as oil.

^ Nuclear Magnetic Resonance Spectrum (CDCl3):

δ = 0.84 (with, 3H, H-18); 0 87 (2x d, J = 7Hz, 6H, H-26/27); 0, 96 (D, J = 7 Hz, 3H, H-21; 1 11 (with, 3H); 1, 18 (with, 3H); 1 25 (WITH, 3H); 5, 45 (with, 1H, H-15); 5 95 (D, J = 10Hz, 1H, H-2); 7.33 (D, J = 10Hz, 1 H, H — 1).

Example 2

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

14 mg of sodium borohydride are added to a suspension of 73 mg of 4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one and 67 mg of cerium chloride in methanol at ambient temperature. 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 residue is chromatographed using hexane / diethyl ether to give 43 mg of 4,4-dimethyl-5α-cholesta-1,8,14-trien-3β-ol as a white solid.

^ TLC analysis (CDC1 _3):

0, 82 (with, 3H); 0, 85 (with, 3H); 0, 87 (2x d, J = 7Hz, 6H, H-26/27); 0, 96 (D, J = 7Hz, 3H H-21); 1, 02 (with, 3H); 1, 10 (with, 3H); 3, 89 (M, 1H, H-3); 5, 37 (with, 1H, H-15); 5, 50 (Dd, . J = 10 Hz 1Hz, 1H, H-1); 5, 90 (D ,. J lCHz, 2 Hz, H, H-2).

Example 3

5a-cholesta-l, 8,14-trien-3p-ol

(a) 5α-Cholesta-8,14-trien-3-one

2.40 g of 5α-cholesta-3,14-trien-3β-ol are mixed with 1.11 g of N-methylmorpholine-N-oxide and 11 mg of tetrapropylammonium perruthenate in 13 ml of dichloromethane as described in Example 1a. After chromatography, 1.81 g of 5α-cholesta-8,14-dl-3-one is obtained as a white solid.

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

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,

1 H, H-15).

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

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

^ TLC analysis (CDC1 _3):

δ = 0.84 (with, 3H, H-18); 0, 86 (2x d, J = 7Hz, 6H, H-26/27); 0, 96 (D, J = 7Hz, 3H, H-21; 1.20 (with, 3H, H-19); 5.43 (with, 1H, H-15); 5.91 (D, J = 10Hz, 1H, H-2); 7, 42 (D, J = 10Hz, 1 H, H-1).

c) 5α-Cholesta-1,8,14-trien-3β-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. 47 g of 5α-cholesta are obtained after chromatography. 1-8,14-trien-3P-ol as a white solid. ¹ H-NMR (CDC1 _3):

δ = 0.82 (with, 3H, H-18); 0, 87 (2x d, J = 7Hz, 6H, H-26/27); 0, 95 (D, J = 7Hz, 3H, H-21; 1, 07 (with, 3H, H-19); 4, 33 (M, 1H, H-3); 5, 37 (with, 1H, H-15); 5.56 (Dd, J = 10 Hz, 1H, H-1); 6, 09 (D, J = 10Hz, 2 Hz, 1H, H-2).

Example 4 1a-Cyano-4,4-dimethyl-5α-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 diethyl aluminum cyanide solution (1M in toluene). The reaction mixture was then allowed to warm to ambient temperature and stirred at this temperature for one hour. After addition of 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 residue was chromatographed using hexane / ethyl acetate to give 110 mg of 1-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one as a white solid.

^{'; H} NMR (CDCl3):

5 = 0, 86 (2x d, J = 7Hz, 6H, H-26/27); 0, 89 (with, 3H); 0 94 (D, J = 7Hz, 3H, H-21; 1, 12 (with, 3H); 1 20 (with, 3H); 2.86 (M, 1H, H-2); 3 29 (Dd, J = 7Hz, 5Hz, 1H, H-1); 5, 49 (Ps. 1 H, H-15).

EXAMPLE 5 1α-Cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol and 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol

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

la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol:

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

δ = 0.83 (with, 3H); 0, 87 0, 94 (D, J 7 Hz, 3H, H-21; 1.09 1.17 (with, 3H); 3.08 3, 72 (Bm, 1H, H-3); 5, 43

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

(s, 3H);

(m, 1H, H-1);

(ps, 1 H, H-15).

la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-ol:

^ TLC analysis (CDC1 _3):

Δ = 0.87 (2x d, J = 7Hz, 6H, H-26/27); 0, 91 (with, 3H); 0, 93 (D, J = 7Hz, 3H, H-21; 0, 08 (with, 3H); 1, 15 (with, 3H); 2, 92 (M, 1H, H-1); 3, 57 (Ps. 1H, H-3); 5, 44 (Ps. 1 H, H-15).

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

Example 6 (E) -4 - [(3-Oxo-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 at room temperature was added a suspension of 64 mg of 4-cyanobenzaidehyde 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 eluting with hexane / ethyl acetate to give 168 mg of (E) -4 - [(3-oxo-4,4-dimethyl-5α-cholesta-8,14-diene). 2-ylidene) methyl] benzonitrile as a white solid.

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

0, 83 (with, 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 (with, 3H); 1 16 (with, 3H); 1, 23 (with, 3H); 2 58 (D, J = 17 Hz, 1 H, H-1); 3, 10 (D, J = 17 Hz, 1H, H-1); 5 47 (PS, . 1H, H-15); 7.42 (with, 1 H, H-2 '); 7 53 (D, J = 8 Hz, 2H, arom.); 7, 68 (D, J = 8 Hz, 2H, and: rom.).

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

156 mg of (E) -4 - [(3-oxo-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile was mixed with 12 mg of sodium borohydride in the presence of 11 mg of chloride heptahydrate cerium as described in Example 2 to give 93 mg of (E) -4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile as a white solid.

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

• · « • ·· ·· · • · · ·· · · • • • · • · · • · · • • • ··· · • · · · • • • · • · · • • ·· ·· ··· ·· · · · ·

0.75 (with, 3H); 0, 80 (with, 3H); 0, 87 (2x d, J = 7 Hz, 6H, H-26/27); 0, 92 (D, J 7 Hz, 3H, H-21); 1, 18 (WITH, 3H); 3, 02 (D, J 17 Hz, 1H, H-1); 3, 94 (M, 1H, H-3); 5, 40 (Ps. 1H, H-15); 6, 78 (with, 1H, H-2 '); 7.37 (D, J 8 Hz, 2H, arom.); 7, 64 (D, J = 8Hz, 2H, arom.).

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

a) (E) -2- (4-Aminomethylphenyl) methylidene-4,4-dimethyl-5α-cholesta-8,14-diene-3β-ol

A suspension of 27 mg of lithium aluminum hydride and 74 mg of (E) -4 - [(3β-hydroxy-4,4-dimethyl-5α-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-5α-cholesta-8,14-dien-33-ol as a white solid.

H-NMR NMR spectrum (CDCl3): δ = 0.77 (with, 3H); 0, 82 (with, 3H); 0, 86 (2x d, J = 7 Hz, 6H, H-26/27); 0.89 (with, 3H); 0, 92 (D, J = 7Hz, 3H, H-21); 1, 18 (with, 3H); 3.15 (D, J = 17 Hz, 1 H, H-1); 3, 86 (WITH, 2H, Ar-CH-N); 3, 92 (ps (1H, H-3); 5, 38 (Ps. 1H, H-15); 6.72 (with, 1H, H-2 '); 7.26 (M, 4H, flavorings.).

b) (E) -N - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-diene-224)]

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

ylidene) methyl] phenyl] methyl] octanamide

To a solution of 21 mg of (E) -2- (4-aminomethylphenyl) methylidene-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in 4 ml was added a solution of 8.6 mg of N at ambient 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. After evaporation of the solvent, 21 mg of (E) -7 - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide is obtained in the form of white solid product.

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

0, 77 (with, 3H); 0 82 (with, 3H); 0.86 (2x d, J = 7 Hz, oH, H-26/27); 0 89 (with, 3H); 0, 92 (D, J = 7Hz, 3H, H-21; 1 18 (with, 3H); 3.12 (D, J = 17Hz (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, 76 (T, J = 5 Hz 1H, NH); 6. 72 (WITH, 1H, H-2 '); 7.24 (M, 4 H, arom •) ·

Example 9 (E) -N - [[4 - [(3β-Hydroxy-4,4-dimethyl-5α-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-5α-cholesta-8,14-dien-3- [3-ol in 2 ml of dimethylformamide was added a solution at ambient temperature. 5.0 mg of 5- (4,4-difluoro-5,7-dimethyl-4-boron-3α, 4α-diaza-s-indacen-3-yl) pentanoic acid as the succinate ester in 0.5 mL of 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 evaporation residue is chromatographed using hexane / ethyl acetate to give 6.5 mg of (E) -ΛΖ - [[4 - [(3β-hydroxy-4,4-ωίιηθίγ1-5β-οΗο1θ5ί3)]. -8,14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4-difluoro-5,7-dimethyl-4-bra-3a, 4a-diaza-s-indacen-3-yl) ) pentanamide.

^ TLC analysis (CDC1 _3):

0 77 (with, 3H); 0, 82 (with, 3H) F 0 86 (2x d, J 7 = Hz, 6H, H-26/27); 0, 89 (with, 3H) r 0 92 (D, J 7 Hz, 3H, H-21; 1.19 (with, 3H) T 2 25 (with, 3H, ar- Me); 2.55 (with, 3H, Ar-Me); 3 12 (D, J 17 Hz , 1H , H-l); 3, 92 (Ps. 1H H-3); 4. 44 (D, J 5 Hz, 2H, Ar-CH ₂ -N); 5, 37 (Ps. 1H H-15); 5 87 (M, 1H, NH) s 6, 09 (with, 1H, arom.); 30 (D, J 5 Hz, 1H, arom.); 6.70 (with, 1H, H-2 '); 6. 90 (D, J 5 Hz, 1H, arom.); 7.24 (M, 4H, flavorings.).

Example 10

a) 2α-Allyl-4,4-dimethyl-5α-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 was stirred for one hour, then 0.07 mL of 3-iodopropene was added. The mixture was stirred for an additional hour at 0 ° C, then 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 of the solvent, the residue is chromatographed using hexane / ethyl acetate as the eluent to give 160 mg of 2o-allyl-4,4-dimethyl. * • ··· · ······ · · · ······················

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

¹ H-Nuclear Magnetic Resonance Spectrum (CDCl3):

δ = 0.82 (with, 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 (with, 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) 2a-Allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol

160 mg of 2a-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3-one is mixed with 55 mg of sodium borohydride as described in Example 5 to give 15 mg of 2a-allyl-4,4 dimethyl-5α-cholesta-8,14-dien-3α-ol and 80 mg of 2a-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol.

2oí-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol:

^ TLC analysis (CDC1 _3):

0, 81 (with, 3H) T 0, 85 (s, 3H); 0, 87 (2x d. J = 7Hz, 6H, H-26/27); 0, 94 (d, J = 7Hz, 3H, H-21); 1, 03 (with, 3H) F 1.05 (s, 3H); 2, 50 (M, 1H) / 2, 95 (d, J = 11 Hz, 1 H, H-3); 5, 06 (M, 2H, allyl); 5, 37 (ps, 1 H, H-15); 5, 89 (M, 1H, allyl).

2-Allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-ol:

H-NMR (CDC1 _3):

δ = 0.82 (with, 3H); 0 87 (2x d, J = 7Hz, 6H, H-26/27); 0, 90 (with, 3H); 0 94 (D, J = 7Hz, 3H, H-21); 1, 00 (with, 3H); 1 05 (with, 3H); 3, 36 (Ps. 1H, H-3); 5 05 (M, 2H, allyl); 5, 35 (Ps. 1H, H-15); 5 85 (M, 1H, allyl).

• ··············

Example 11

Testing of meiosis activating agents in the oocyte test.

The 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 gonadotropins (Gonal F, Derono, Solna, 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 the ovaries were cut off, the oocytes were isolated in Hx medium (see below) under a stereomicroscope by manually breaking the follicles using a pair of 27 gauge needles. Spherical exposed oocytes (NO) having intact germ bladder (GV) were stored in α-minimal essential medium (α) -MEM without ribinucleosides, Gibco BRL, cat.no. 22561) enriched with 3 mM hypoxanthine (Sigma, cat.no. H-9377), 8 mg / ml Human Serum Albumin (HSA, State Serum)

Institute, Denmark), 0.23 nM pyrubate (Sigma, Cat. No.-8636), 2 mM glutamine (Flow, Cat. No. 16-801), 100 IU / ml penicillin and 100 µg / ml streptomycin (Flow , Cat. No. 16-700). This environment is referred to as the Hx environment. Oocytes were washed three times with Hx medium and cultured on 4-well multiplates (Nuncion, Denmark), each well containing 0.4 ml Hx medium and 35-45 oocytes. One control culture (in which 35-45 oocytes were cultured in Hx medium without addition of the test culture) was performed in parallel to the test cultures.

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

cultures were performed using different concentrations of test compounds.

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

Investigation of oocytes

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

Example 12

Testing of Meiosis Inhibitors in an Oocyte Test

Germ bladder oocytes (GV) were obtained from juvenile FSH-treated female mice using the procedures used in Example 11 (see above). The exposed oocytes (NO) were flushed three times in Hx. 4,4-Dimethyl-5α-cholesta-8,14,24-triene-3β-ol (FF-MAS) was previously shown to induce meiosis in exposed oocytes in vitro (Byskov AG et al. Nature 374 (1995) NO oocytes were cultured in Hx medium enriched with 5 μΜ FF-MAS as co-cultures with test compounds used at various concentrations in 4-well multiplates.

• ·· • ·· · · • · · ·· · · • • · · • · · • · · • • • ··· · • · · • · • «· • · · • • ·· ·· ··· ·· · · · ·

(Nunclon, Denmark), each well containing 0.4 ml Hx medium and 35-45 oocytes. One positive control culture (35-45 oocytes cultured in Hx medium containing FF-MAS without addition of test compound) was performed simultaneously with the test cultures; in test cultures, test compounds were used at various concentrations. In addition, one negative control culture (35-45 oocytes cultivated in Hx medium alone) was performed.

Investigation of oocytes

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

Example 13

Testing of meiosis-activating substances in the 'in vitro fertilization' test

Exposed oocytes (NkO) and cumulated oocytes (CEO) were isolated from juvenile FI follicles (C57BL / 6xDBA / 2) (21-24 days of age) given 48 hours prior to oocyte isolation with 10 IU Pregnant Mare Serum Gonadotropin (activity) FSH). Oocytes (NkO and CEO) were collected and cultured for 20 hours in modified form

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

α-ΜΕΜ medium containing 3 mM hypoxanthine (medium Hx) and 1 mg fetuin per ml of culture medium. Two groups of oocytes were used: a) control oocytes cultured in Hx-free environment (positive control group) and b) oocytes cultured in Hx environment containing the test compound. After about 20 hours, the bladder-disrupted oocytes (GVB) were briefly washed with Xx-free media and transferred to preformed insemination plates containing a male spermatozoon motile sperm. 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 record the number of 2-cell embryos. Percent fertilization rate (= 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.

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

Table 1: Activation of meiosis in exposed mouse oocytes

compounds Oocytes [n] Activation [%] GVB + PB GV GVB PB Control (Hx) 39 0 1 x 2.6 10 μ FF-MAS 5 29 4 86, 8 10 μΜ 4,4-Dimethyl-5α-cholesta-1,8,14-βίβη-3β-ο1 9 18 5 71, 9

Hx = hypoxanthine GV = germinal oocytes GVB = oocytes with broken fetal bladder PB = oocytes with a polar body n = oocyte count

Table 2: Relative inhibition of meiosis in exposed mouse oocytes

compounds oocytes [N] PB Inhibition [%] GV GVB Control (Hx) 34 2 0 100 5 μΜ FF-MAS 6 28 0 0 5 μΜ FF-MAS + 40 μΜ la-cyano-4,4-dimethyl-5a-cholesta-8,14- dien-3-one 38 0 0 107

Hx =

GV

Hypoxanthine oocytes with germ bladder

• · · • ·· · · • · · ·· · · • • · · • · · • · · • • • Hl · • · · • · • • · • · · • • ·· ·· ··· ·· · · · ·

GVB = oocytes with broken fetal bladder

PB = polar body oocytes n = oocyte count

Table 3: Relative inhibition of meiosis in exposed mouse oocytes

compounds oocytes [N] PB Inhibition [%] GV GVB Control (Hx) 34 2 0 100 5 μ FF-MAS 6 28 0 0 5 μΜ FF-MAS + 10 μΜ (E} -N - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide 25 13 0 63

Hx = Hypoxanthine

GV = oocytes with germinal vesicles

GVB = oocytes with broken fetal bladder

PB = polar body oocytes number of oocytes

Claims (14)

Compounds of general formula I (I) wherein: R ¹ represents a hydrogen atom; (C 2 -C 6) -alkyl; optionally substituted phenyl; cyano; a CH 2 -NH-COR ¹ group wherein R ¹ represents an alkyl group having 1 to 8 carbon atoms or an optionally substituted phenyl group; or together with ^R2 represents an additional bond; R ² is H; (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 H; together with R 'an optionally substituted benzylidene group; or together with R ² a hydroxymethylene group; R ³ represents a hydrogen atom; or together with R ^{3 an} additional bond; R ³ represents a hydrogen atom; or together with R ^{3 an} additional bond; R ⁴ is H; or methyl; R ⁴ represents a hydrogen atom; or methyl; R ³ together with R ⁹ or R ^{14 represents an} additional bond; ·························· R ⁹ represents a hydrogen atom; or together with R ^{8 an} additional bond; R ¹⁴ is α-hydrogen; or together with R ^{8 an} additional bond; or together with R ^{15 an} additional bond; R ( ¹⁵⁾ is hydrogen; or together with R ^{14 an} additional bond; E ²⁴ is hydrogen; or together with R ^{2 an} additional bond; and R ²⁵ represents a hydrogen atom; or together with R ^{24 an} additional bond; or esters thereof, with the exception that that the scope of the compounds does not include compounds which are also modified at positions 1 and 2, i.e., where 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 R ^{2, an} additional bond. Compounds according to claim 1 or 2, in which R ² represents an alkyl group having 4 to 8 carbon atoms, an allyl group or an additional bond with R ¹ . Compounds according to either of Claims 1 and 2, in which R ² and R ² represent an optionally substituted benzylidene group. Compounds according to any one of claims 1 to 4, which are 2a-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol, (E) -2-benzylidene-4,4-dimethyl-5α-cholesta-8,14-diene-3β- ol, 5a-cholesta-l, 8,14-trien-3.beta.-ol, 5a-cholesta-l, 8,14-trien-3-one, • ·· • ·· · · • · · ·· · · • • · · • · · • · · • · • ··· · • · · • · • • · • · · • • ·· ·· ··· ·· · · • · 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol, 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-3-one, 4,4, dimethyl-5α-cholesta-1,8,14-trien-3β-ol, 4,4-dimethyl-5α-cholesta-1,8,14-trien-3-one, (E) -4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile, (E) -N - [[4 - [(3β-hydroxy-4,4) (E) -N - [[4 - [(3β-hydroxy-4,4-dimethyl-5α-cholesta) -dimethyl-5α-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide -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 . 2-hydroxymethyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-ol, 2a-octyl-4,4-dimethyl-5α-cholesta-8,14-dien-38-ol or (E) -4 - [(3-oxo-4,4-dimethyl-5α-cholesta-8,14- dien-2-ylidene) methyl] benzonitrile. Pharmaceutical compositions, characterized in that they contain one or more compounds of the formula I according to any one of claims 1 to 5 as active substances. Use of compounds 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 the treatment of infertility in female or male, preferably human. Use according to claim 7 for the preparation of a contraceptive for the treatment of females or males, preferably humans. Use of the compounds of the general formula I according to • ·· • ·· ·· · M · · ·· · · • · · · • · · • · · • · · • ··· · • · · · • · · • · • · · • · · ··· ♦ · ··· ·· ·· ·· 4. The method of claim 1, wherein the fertilization rate of artificial insemination processes is controlled. * Use of compounds of 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. 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.