MXPA01007526A - 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

- i - UNSATURATED COLESTANATE DERIVATIVES AND THEIR USE FOR THE PREPARATION OF REGULATORY MEDICINES OF THE MEIOSIS DESCRIPTION OF THE INVENTION The present invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions comprising them, to 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 to regulate meiosis. Meiosis is the unique and final phenomenon of the germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, an exchange is made between the maternal and paternal genes before the pairs of chromosomes in two daughter cells are separated. These contain only half the number (ln) of chromosomes and 2c DNA. The second meiotic division is carried out without DNA synthesis. This division therefore results in the formation of aploid germ cells with only lc of DNA. The meiotic phenomena are similar in male and female germ cells, but the time protocol and differentiation processes which lead to ovules and sperm differ profoundly. All female germ cells enter prophase of the first meiotic division early in life, often before birth, but all are suppressed as oocytes later in the prophase (dictiato state) until ovulation after puberty. Thus, from early life, the female has a fixed amount of oocytes which are extracted until this amount is exhausted. Meiosis in females is not completed until after fertilization and results only in one egg and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a pluripotent population of germ cells during life. Once initiated, meiosis in the male cells is carried out without significant delay and produces 4 spermatozoa. Only little is known about the mechanism which controls the onset of meiosis in males and females. In the oocyte, new studies indicate that follicular purines, hypoxanthine or adenosine, may be responsible for meiotic suppression [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 a diffusible substance that regulates meiosis was described for the first time by Byskov et al. , in a fetal mouse adrenal culture system [Byskov, A. G. eü al. Dev Biol 52 (1976) 193-200]. A meiosis activating substance (MAS) is secreted by the mouse fetal ovary in which meiosis occurs, and a substance that prevents meiosis (MPS) of morphologically differentiated testes is released with resting non-meiotic germ cells. It is suggested that the relative concentrations of MAS and MPS regulated at the beginning, suppress the resumption of meiosis in male and female germ cells (Byskov, AG et al., In The Physiology of Reproduction [eds Knobil, E. and Neill, JD, Raven Press New York (1994).] Clearly, if meiosis can be regulated, reproduction can be controlled A recent article [Byskov, AG et al., Na ture 374 (1995), 559-562] describes the isolation of certain sterols of testes of bull and human follicular fluid, which activate the meiosis of the oocytes Unfortunately, these sterols are labile and the use of the interest found would be facilitated if more stable meiosis activating compounds were available. meiosis and are different from the compounds claimed in the present patent application are described in WO 96/27658 It is a purpose of the present invention to provide novel compounds dosos for the treatment of infertility in women and men, particularly in humans via the activation of meiosis.

A further purpose of the present invention is to provide novel compounds useful as contraceptives in women and men, particularly in humans via inhibition of meiosis. In accordance with the present invention, novel stable compounds with interesting pharmacological properties are provided. In particular, the compounds described herein are useful for the regulation of meiosis in oocytes and in male germ cells. A further purpose of the present invention is to provide novel compounds which are suitable substrates for the introduction of fluorescent labels. The linked molecules are designed and synthesized for bioimaging purposes. The present invention relates to unsaturated cholestane derivatives of the general formula I wherein R1 designates a hydrogen atom, an alkyl group of 2 to 6 carbon atoms, an optionally substituted phenyl group, a cyano group, a CH ^ -NH-COR1 'group (wherein R1' is an alkyl of 1 to 8 carbon atoms or an optionally substituted phenyl group) or together with R2 , an additional bond, R 'designates a hydrogen atom, an alkyl group of 4 to 8 carbon atoms, an alkenyl group of 3 to 6 carbon atoms, a hydroxyalkyl group of 1 to 6 carbon atoms, together with R2 'a benzylidene group optionally substituted together with R2' a hydroxymethylene group or together with R1 a bond additional, R2 denotes a hydrogen atom, together with R2 an optionally substituted benzylidene group or together with R2 a hydroxymethylene group, R designates a hydrogen atom or together with R3 'an additional bond, RJ designates a hydrogen atom or together with R3 a bond additionally, R 4 denotes a hydrogen atom or a methyl group, R 4 'designates a hydrogen atom or a methyl group, / R 8 designates together with R 9 or with R 14 an additional bond, R 9 denotes a hydrogen atom or together with R 8 a bond further, R1 designates an a-hydrogen atom or together with R8 an additional bond or together with R15 an additional bond, R 15 designates a hydrogen atom or together with R 14 an additional bond R 2- designates a hydrogen atom or together with R25 an additional bond, R2 designates a hydrogen atom or together with R24 an additional bond or esters thereof, with the condition that compounds are excluded which are simultaneously not modified in positions 1 and 2 (R1 = R2 = R2 '= H). The compounds of the general formula I have several chiral centers in the molecule and therefore exist in various isomeric forms. All of these isomeric forms and mixtures thereof are within the scope of the invention (e- less than otherwise indicated). Preferred compounds of formula I are those in which R 1 denotes a hydrogen atom, a phenyl group or together with R 2, an additional bond. Other preferred compounds are those in which R 2 denotes an alkyl group of 4 to 8 carbon atoms, an allyl group or an additional bond with R 1. Also preferred are compounds wherein R2 and R2 'designate a substituted benzylidene group.

In another embodiment, the present invention relates to esters of compounds of general formula I. Such esters are formally derived by esterification of one or more hydroxyl groups of a compound of formula I with an acid which can be selected, for example, from the group of acids comprising succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulfonic acid, sulfamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids. As used in the present description and claims, an alkyl group-when used alone or in combinations-can be a linear or branched alkyl group. The term "alkyl of 2 to 6 carbon atoms" denotes an alkyl group having 2 to 6 carbon atoms: preferred examples are ethyl, propyl, isopropyl, butyl, tertbutyl, pentyl, hexyl and cyclohexyl. More preferably ethyl. Similarly, the term "alkyl of 1 to 8 carbon atoms" designates an alkyl group having from 1 to 8 carbon atoms; preferred examples are methyl, ethyl, propyl, isopropyl, butyl, tertbutyl, pentyl, hexyl and octyl, more preferably methyl, ethyl, propyl, isopropyl, butyl and tertbutyl, and most preferably methyl and ethyl. The especially preferred compounds of formula I of the present invention are the following: 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-diene-3ß-ol, (E) -2-benzylidene-4, 4 -dimethyl-5-cholesterol-8, 14-diene-3ß-ol, 5a-cholesta-1, 8, 14-trien-3ß-ol, 5a-cholesta-1, 8, 14-trien-3-one, -cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol, la-cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3-one, 4.4 -dimethyl-5a-cholesta-l, 8, 14-trien-3ß-ol, 4, 4 -dimethyl-5a-cholesta-l, 8, 14. trien-3 -one, (E) -4- [(3ß -hydroxy-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) -methyl] benzonitrile, (E) -N- [[4- [(3β-hydroxy-4, 4-dimethyl- 5a-cholesta-8, 14-dien-2-ylidene) methyl] phenyl] octanamide, (E) -N- [[4- [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8, 14 -dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-yl) pentanamide, 2a -hydroxymethyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3a-ol, 2a-octyl-4, 4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol and (E) -4- [(3 -oxo-4, 4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile. The compounds of the general formula I according to the invention can be synthesized analogously to the preparation of known compounds. Therefore, the synthesis of the compounds of formula I can follow the well-established synthetic routes described in the comprehensive literature of sterol and steroids. The following books can be used as a key source in the synthesis: L.F. Fieser & M. Fieser: Steroids: Reinhold Publishing Corporation, NY 1959; Rood's Chemistry of Carbon Compounds (ed.: S. Coffrey): Elsevier Publishing Company, 1971; and especially Dictionary of Steroids (editors: R.A. Hill; D.N. Kirk; H.L.J. Makin and G.M. Murphy): Chapman & Hall. The latter contains an extensive list of citations of the original documents covering a period up to 1990. All of these books, including the citations mentioned at the end, are incorporated by reference. Particularly, in the compounds of the present invention they are synthesized according to the following general procedures. The sterols that are used as starting materials can be synthesized according to procedures in the literature: 4,4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol, 4, 4-dimethyl-5a-cholest-8 -in-3ß-ol, 4, 4-dimethyl-5a-cholest-8 (14) -en-3ß-ol [Biochem. J. 132 (1973), 439], 4, 4-dimethyl-5a-cholesta-8, 24-dien-3β-ol, 4,4-dimethyl-5a-cholesta-8, 14, 24-trien-3β -ol [J. Am. Chem. Soc. 111 (1989), 278], 5a-cholesta-8, 14-dien-3β-ol [J ". Am. Chem. Soc. 75 (1953), 4404], 5a-cholest-8 -in-3 ß-ol [J. Org. Chem. 46 (1981), 3421] and 5a-cholest-8 (14) -in-3ß-ol [Biochem. J. 144 (1974), 59]. the following is described in detail only the synthesis in the series 4, 4-dimethyl -? - 8, 14. Derivatives in the series? -8,? -8 (14),? -8,14,24 and? - 8.24 with and without the 4,4-dimethyl group, can be synthesized from the corresponding starting materials in the same manner The 3β-alcohols can be oxidized with different reagents to provide the corresponding 3-ketones [for example: Tetrahedron Lett. 1967, 3699.] 4,4-Dimethyl-5a-cholesta-8,14-dien-3β-ol 1 is treated with N-methyl-morpholin-N-oxide in the presence of tetraprommonium perruthenate to provide 4, 4- dimethyl-5a-cholesta-8, 14-dien-3-one 2 [for example, Synthesis 1994, 639].

Reaction Scheme 1: In the next step, the double bond? -l can be introduced into an oxidation reaction with phenylsenic anhydride as an oxidant in chlorobenzene [J. Chem. Soc. Chem. Commun. 1978, 952] to provide 4,4-dimethyl-5a-cholesta-1, 8, 14-trien-3 -one 3. This reaction also works in the case of compounds without the 4, 4-dimethyl group. In these compounds, the new double bond can be selectively introduced as a double bond? L. The 3-keto group can then be reduced with sodium borohydride in the presence of cerium chloride [Luche reduction, for example: J. Am. Chem. Soc. 100 (1978), 2226] to provide 4, 4-dimethyl- 5a-cholesta-l, 8, 14-trien-3ß-ol 4. The alkyl and aryl groups can be introduced in position 1 via additions of cuprate to unsaturated ketones [Tetrahedron Lett. 35 (1994), 8591]. Cuprates which are formed from alkyl and aryllithium and cuprous iodide react with steroidal enones to provide 1-substituted derivatives. If, for example, enone 3 is treated with dialkylcuprate compounds of formula 5 (R 1 = alkyl, aryl) are obtained. These ketones can then be reduced according to procedures well known in the literature to two diastereomeric alcohols 6 and 7 (R 1 = alkyl, aryl) which are easily separable by column chromatography.

Reaction Scheme 2: The introduction of a cyano group can be obtained in a similar way. The conjugate addition of cyanide to enone 3 gives the desired lacyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one (R 1 = CN). Different reactants such as diethylaluminum cyanide [J. Org. Chem. 59 (1994), 2766] and some alkaline and alkaline earth metals [Tetrahedron Lett. 28 (1987), 4189; Can. J. Chem. 59 (1981), 1641]. Cyanoketone 5 (R1 = CN) can also be reduced with conventional or standard reducing agents such as sodium borohydride, to provide the two diastereomeric alcohols 6 and 7 (R1 = CN), which can be easily separated by column chromatography. If the cyanoalcohol 6 (R1 = CN, synthesized as described above) is treated with lithium aluminum hydride, la-aminomethyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3β-ol 8. This amine can be used for additional modification The amides of formula 9 (R1 '= alkyl, aryl and fluorescence label) can be synthesized from the amine by reaction with hydroxysuccinimidylester of various alkyl or arylcarboxylic acids (for the use of hydroxysuccimidylester of carboxylic acids containing fluorescence label see : Nonradioactive labeling and detection of biomoleculess, Kessler C. ed., Springer Verlag, Berlin, 1992).

Esq-uema of Reaction 3: The substituents at position 2 can be introduced, for example via aldol reactions and alkylations. If the ketone 2 is treated with aromatic aldehydes in the presence of a base, 2-benzylidene-substituted steroids of formula are obtained . The aromatic ring may be substituted. The subsequent reduction with sodium borohydride in the presence of chloride -erium selectively provides the corresponding 3-allylic alcohols 11.

Reaction Scheme 4: Compound 11 substituted with cyanobenzylidene (R2"= 4-CN) which can be synthesized as shown above, can be used for further modification.The cyano group can be reduced with lithium aluminum hydride to the benzylic amine. , which can be derivatized to the corresponding amides of formula 13 (R2"'= alkyl, aryl and fluorescence labels). For this purpose, amine 12 is treated with hydroxysuccinimidylester derivatives of different carboxylic acids. For this reaction, commercially available esters containing fluorescence labels can also be used (see example 11 in the experimental part). The amides of formula 13 containing the fluorescence labels can be used as molecular probes for purposes of image bioformation.

Reaction Scheme 5 Alkyl and alkenyl substituents can be introduced in the 2-position via deprotonation of the ketone 2 and subsequent reaction of the enolate with alkyl and alkenyl halides. The 3-ketones of formula 14 can then be reduced with sodium borohydride to provide two diastereomeric alcohols of formulas 15 and 16, which can be easily separated by column chromatography.

Reaction Scheme 6 The hydroxymethyl substituents at the 2-position can be introduced, for example, via a condensation reaction of a deprotonated 3-ketone with alkyl formates to provide enol 17. This can be reduced with different reducing agents such as sodium borohydride to provide two diastereomeric alcohols 18 and 19.

Reaction Scheme 7 These alcohols can be separated. easily with column chromatography. Longer chain hydroxyalkyl substituents can be introduced, for example, via alkylation reactions as described above (see reaction scheme 6). A further objective of the present invention are pharmaceutical compositions comprising one or more compounds of the general formula I as active substances. The compositions may further comprise pharmaceutically acceptable excipients well known in the art as carriers, diluents, absorption improvers, preservatives, buffers, osmotic pressure adjusting agents, tablet disintegrating agents and other ingredients which are conventionally used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethylcellulose, waxes with low melting point and cocoa butter. Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with ex vivo and in vitro fertilization. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are mentioned in the above list. In addition, a composition for transdermal administration of a compound of this invention can be provided in the form of a patch and a composition for nasal administration in the form of a nasal spray in liquid or powdered form can be provided. The dose of a compound of the invention to be used will be determined by the physician and will depend, among several factors, of the particular compound used, the route of administration and the purpose of the use. In general, the compositions of the invention are prepared by intimately bringing into association the active compound with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation. The present invention relates to the use of the compounds of the general formula I for the preparation of a medicament regulating meiosis. The compounds of the present invention influence meiosis in oocytes as well as in male germ cells. There are several prospects capable of influencing meiosis. According to a preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate meiosis. According to another preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate meiosis in humans. Therefore, the compounds of the general formula I are promising as new fertility regulating agents, without the usual collateral effects on somatic cells which are known from the hormonal contraceptives used up to now which are based on estrogen or gestagens or both. Accordingly, the present invention relates to the use of the compounds of the general formula I to alleviate infertility in females and males, particularly in mammals, and more particularly in humans. The meiosis inducing substances of the general formula I can be used in the treatment of certain cases of infertility in females, including the female, by administering it to females who, due to a pro insufficient production of meiosis activating substance , are unable to produce mature oocytes. The compounds of the general formula I can also be used in artificial insemination procedures, for example in in vitro fertilization or intracytoplasmic sperm injection. When in vitro fertilization is performed, better results can be obtained when a compound of the invention is added to the medium in which the oocytes are grown. When infertility in males, including man, is caused by an insufficient and self-produced meiosis activating substance and therefore there is a lack of mature sperm cells, administration of a compound of the invention can solve the problem. In a further objective of the present invention, the compounds of the general formula I are useful as contraceptives in females and males, particularly in mammals, more particularly in humans. For use as a contraceptive agent in females, a meiosis-inducing substance can be administered in a manner that prematurely induces the generation of meiosis again in oocytes while still in the growing follicle, before the gonadotropin ovulatory peak occurs. . In women, resolution of meiosis can be induced, for example, one week after the previous menstruation has ceased. When they ovulate, the resulting oocyte oocytes are more likely not to be fertilized. The normal menstrual cycle is not likely to be affected. In this regard, it is important to note that the biosynthesis of progesterone in cultured human granulosa cells (somatic cells of the follicle) is not affected by the presence of a meiosis-inducing substance while the estrogens and progestins used in the hormonal contraceptives used so far have an adverse effect on progesterone biosynthesis. As an alternative to the method described above, contraception in females can also be carried out by administering a compound of the invention which inhibits meiosis, so that mature oocytes are not produced. Sarly, male contraception can be obtained by administration of a compound of the invention which inhibits meiosis, so that mature sperm cells are not produced. In another aspect, the present invention relates to the use of the compounds of the general formula I as tool substances or as starting materials for the synthesis of tool substances for purposes of image bioforming in order to clarify the mode of action of such substances. For example, sterols that activate meiosis can be used which contain a fluorescence marker to visualize the components of the germ cells where the active substances exert their biological function. This information can be useful to clarify the mode of action of such substances. In a further aspect, the present invention relates to a method for regulating meiosis which comprises administering to a subject in need of such regulation an effective amount of one or more compounds of the general formula I. In a further aspect, the present invention relates to a method for regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vi tro to a germ cell in need of such regulation, an effective amount of one or more compounds of the general formula I. The germ cell can be an oocyte or a male germ cell. The route of administration of the compositions containing a compound of the invention can be any route which effectively transports the active compound to its site of action. Therefore, when the compounds of this invention are to be administered to a mammal, they are conveniently provided in the form of a pharmaceutical composition which comprises at least one compound of the invention together with a pharmaceutically acceptable carrier. For oral use, such compositions are preferably in the form of capsules or tablets. From the above, it will be understood that the administration regime that is required will depend on the condition in question. Thus, when used in the treatment of infertility, the administration can be carried out only once, or for a limited period, for example until pregnancy is achieved. When used as a contraceptive, the compounds of the invention can be administered continuously or cyclically. When used as a contraceptive by females and not taken continuously, it will be important to synchronize administration in relation to ovulation. The present invention is further illustrated by the following examples: Example 1: 4, 4-dimethyl-5a-cholesta-1, 8, 14-trien-3-one a) 4, 4-dimethyl-5a-cholesta-8, 14-dien-3-one: To a solution of 6.40 g of 4,4-dimethyl-5a-cholesta-8,14-diene-3ß-ol and 4.03 g of N-methylmorpholine-N-oxide in 32 ml of dichloromethane are added some grains of molecular sieves and The mixture is stirred for 5 minutes. 408 mg of tetrapropylammonium perruthenate are added at room temperature and the resulting black reaction mixture is stirred for 1 hour. After filtration over Celite, the solvent is evaporated and the residue is subjected to chromatography with a mixture of hexane and ethyl acetate to provide 5.60 g of, 4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol as a white solid.

XH NMR (CDC13): d = 0.83 (s, 3H, H-18); 0.87 (2x, d, J = 7 Hz, 6H, H-26/27); 0.94 (d, J 7 Hz, 3H, H-21); 1.07 (s, 3H); 1.12 (2x s, 6H); 2.55 (m, 2H); 5.41 (s, ÍH, H-15). b) 4, 4-dimethyl-5a-cholesta-l, 8, 14 -trien-3 -one: To a solution of 2.00 g of 4,4-dimethyl-5a-cholesta-8,14-dien-3-one in 30 ml of chlorobenzene, 1.75 g of phenylsenic anhydride are added at room temperature. The reaction mixture is heated at 100 ° C for 2 hours. After cooling and evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and ethyl acetate to give 0.85 g of 4,4-dimethyl-5a-cholesta-1, 8, 14-trien-3-one: as an oil.

H-NMR (CDCl 3): d = 0.84 (s, 3H, H-18); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.96 (d, J = 7 Hz, 3H, H-21); 1.11 (s, 3H); 1.8 (s, 3H); 1.25 (s, 3H); 5.45 (s, ÍH, H-15); 5.95 (d, J = 10 Hz, 1H, H-2); 7.33 (d, J = 10 Hz, ÍH, H-1). Example 2: 4, 4 -dimethyl-5a-cholesta-l, 8, 14-trien-3ß-ol 14 mg of sodium borohydride is added to a suspension of 73 mg of 4,4-dimethyl-5a-cholesta-1, 8, 14-trien-3 -one and 67 mg of cerium chloride heptahydrate, in methanol at room temperature . The mixture is stirred for 4 hours, poured into water and extracted with diethyl ether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and diethylether to provide 43 mg of 4, -dimethyl-5a-cholesta-1,8,8-trien-3β-ol as a white solid.

X H NMR (CDC13): d = 0.82 (s, 3H); 0.85 (s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.96 (d, J = 7 Hz, 3H, H-21); 1.02 (s, 3H); 1.10 (s, 3H); 3.89 (m, ÍH, H-3); 5.37 (s, 1H, H-5); 5.50 (dd, J = 10 Hz, 1 Hz, ÍH, H-1); 5.90 (d, J = 10 Hz, 2 Hz, ÍH, H-2).

Example 3: 5a-cholesta-l, 8, 14-trien-3 -one: 2.40 of 5a-cholesta-8, 14-diene-3ß-ol are treated with 1.11 g of N-methylmorpholine-N-oxide and 111 mg of tetrapropylammonium perruthenate in 13 ml of dichloromethane, as described in Example la. After chromatography, 1.81 g of 5a-cholesta-8, 14-dien-3 -one is obtained as a white solid.

X H NMR (CDCl 3): d = 0.82 (s, 3 H, H-18); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.94 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H, H-19); 5.39 (s, ÍH, H-15) b) 5a-cholesta-l, 8, 14 -trien-3 -one 1.81 g of 5a-cholesta-8, 14-dien-3-one are treated with 1.67 g of phenylsenic anhydride in 29 ml of chlorobenzene, as described in example Ib. After chromatography, 0.53 g of 5a-cholesta-1, 8, 14-trien-3-one is obtained as an oil.

X H NMR (CDCl 3): d = 0.84 (s, 3 H, H-18); 0.86 (2x d, J = 7 Hz, 6H, H-26/27); 0.96 (d, J = 7 Hz, 3H, H-21); 1.20 (s, 3H, H-19); 5.43 (s, ÍH, H-15); 5.91 (d, J = 10 Hz, ÍH, H-2); 7.42 (d, J = 10 Hz, ÍH, H-l) c) 5a-cholesta-l, 8, 14-trien-3ß-ol 150 mg of 5a-cholesta-1, 8, 14-trien-3-one are treated with 15 mg of sodium borohydride and 147 mg of cerium heptahydrate chloride, as described in example 2. After chromatography, 47 mg of 5a-cholesta-1, 8, 14-trien-3ß-ol as a white solid.

X H NMR (CDCl 3): d = 082 (s, 3 H, H-18); 0.87 (2x, d, J = 7 Hz, 6H, H-26/27); 0.95 (d, J = 7 Hz, 3H, H-21); 1.07 (s, 3H, H-19); 4.33 (m, ÍH, H-3); 5.37 (s, ÍH, H-15); 5.56 (dd, J = 10 Hz, 1 Hz, ÍH, H-1); 6.09 (d, J = 10 Hz, 2 Hz, ÍH, H-2) Example 4 la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one: 1.47 ml of a solution of diethylaluminum cyanide (1 M in toluene) are added to a solution of 4,4-dimethyl-5a-cholesta-1,8,8-trien-3 -one in 5 ml of tetrahydrofuran at 0 °. C. The reaction mixture is allowed to warm to room temperature and is stirred for 1 hour. After the addition of 2.45 ml of a 1 M solution of sodium hydroxide at 0 ° C, the resulting mixture is diluted with water and extracted with diethyl ether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and ethyl acetate to provide 110 mg of lacyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one as a white solid.

X H NMR (CDCl 3): d = 0.86 (2x d, J = 7 Hz, 6H, H-26/27); 0.89 (s, 3H); 0.94 (d, J = 7 Hz, 3H, H-21); 1.12 (s, 3H); 1.-15 (s, 3H); 1.20 (s, 3H); 2.86 (m, ÍH, H-2); 3.29 (dd, J = 7 Hz, 5 Hz, ÍH, H-1); 5.49 (ps, 1 H, H-15). Example 5: la-cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol and la-cyano-4, 4-dimethyl-5a -cholesterol-8, 14-dien-3a-ol: 95 mg of la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one are treated with 33 mg of sodium borohydride as described in example 5 to provide 30 mg of la-cyano -4,4-dimethyl-5a-cholesta-8, 14-diene-3a-ol and 25 mg of la-cyano-4,4-dimethyl-5a-cholesta-8,14-diene-3ß-ol: l-cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol: NMR (CDC13): d = 0.83 (s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.94 (d, J = 7 Hz, 3H, H-21); 1.09 (s, 3H); 1.17 (s, 3H); 3.08 (m, ÍH, H-1); 3.72 (broad m, ÍH, H-3); 5.43 (ps, 1 H, H-15). la-cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3a-ol: X H NMR (CDCl 3): d = 0.87 (2x, d, J = 7 Hz, 6H, H-26/27); 0.91 (s, 3H); 0.93 (d, J = 7 Hz, 3H, H-21); 1.08 (s, 3H); 1.15 (s, 3H, 1.28 (s, 3H) 2.92 (m, ÍH, H-l), 3.57 (ps, ÍH, H-3), 5.44 (ps, 1 H, H-15).

Example 6; (E) -4- [(3 -oxo-4, 4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) methyl] benzonitrile A suspension of 64 mg of 4-cyanobenzaldehyde and 27 mg of potassium hydroxide in 2 ml of ethanol is added dropwise to a suspension of 4,4-dimethyl-cholesta-8,14-dien-3-one in 4 ml of Ethanol at room temperature. The reaction mixture is stirred for 20 hours, diluted with water and extracted with dichloromethane. The organic layer is separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and ethyl acetate to provide 168 mg of (E) -4 - [(3-oxo-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) methyl] benzonitrile as a white solid.

X H NMR (CDC13): d = 0.83 (s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.93 (d, J = 7 Hz, 3H, H-21); 0.98 (s, 3H); 1.16 (s, 3H); 2.58 (d, J = 17 Hz, ÍH, H-1); 3.10 (d, J = 17 Hz, ÍH, H-1); 5.47 (ps, 1 H, H-15); 7.42 (s, 1 H, H-2 '); 7.53 (d, J = 8 Hz, 2H, aromatic); 7.68 (d, J = 8 Hz, 2H, aromatic).

Example 7: (E) -4- [(3β-hydroxy-4, 4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) methyl] benzonitrile 156 mg of (E) -4- [(3-oxo-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile are treated with 12 mg of sodium borohydride in the presence of 111 mg of cerium chloride heptahydrate as described in example 2, to provide 93 mg of (E) -4- [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8, 14-dien-2- ylidene) methyl] benzonitrile as a white solid.

XH NMR (CDC13): d = 0.75 (s, 3H); 0.80 (s, 3H); 0.85 s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.92 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H); 3.02 (d, J = 17 Hz, ÍH, H-1); 3.94 (m, ÍH, H-3); 5.40 (ps, 1 H, H-15); 6.78 (s, 1 H, H-2 '); 7.37 (d, J = 8 Hz, 2H, aromatic); 7.64 (d, J = 8 Hz, 2H, aromatic).

Example 8; (E) -N- [[4 [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) methyl] phenyl] octanamide: a) (E) -2- (4-aminomethyl-phenyl) -methylidene-4,4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol: A suspension of 27 mg of lithium aluminum hydride and 74 mg of (E) -4- [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] benzonitrile in 6 ml of tetrahydrofuran it is refluxed for 4 hours. After cooling, the reaction mixture is worked up as described in example 3b. Crystallization from ethyl acetate affords 26 mg of (E) -2- (4-aminomethyl-phenyl) -methylidene-4,4-dimethyl-5a-cholesta-8,14-diene-3ß-ol as a solid White.

X H NMR (CDC13): d = 0.77 (s, 3H); 0.82 (s, 3H); 0.86 (2x d, J = 7 Hz, 6H, H-26/27); 0.89 (s, 3H); 0.92 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H); 3.15 (d, J = 17 Hz, ÍH, H-1); 3.86 (s, 2H, Ar-CH2-N); 3.92 (ps, ÍH, H-3); 5.38 (ps, 1 H, H-15); 6.72 (s, 1 H, H-2 '); 7.26 (m, 4H, aromatic) b) (E) -N- [[4- [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide: A solution of 8.6 mg of N-hydroxysuccinimidyl caprylate in 1 ml of dimethylformamide is added to a solution of 21 mg of (E) -2- (4-aminomethyl-phenyl) -methylidene-4,4-dimethyl-5a-cholesta -8, 14-dien-3ß-ol in 4 ml of dimethylformamide at room temperature. The reaction mixture is stirred for 20 hours, diluted with water and extracted with ethyl acetate. The organic layer is separated, washed with water, dried over anhydrous sodium sulfate and filtered. Evaporation of the solvent affords 21 mg of (E) -N- [[4- [(3β-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] phenyl] methyl] octanamide as a white solid.

X H NMR (CDCl 3): d = 0.77 (s, 3H); 0.82 (s, 3H); 0.86 (2x d, J = - 7 Hz, 6H, H-26/27); 0.89 (s, 3H); 0.92 (d, J = 7 Hz, 3H, H-21); 1.18 (s, 3H); 3.12 (d, J = 17 Hz, ÍH, H-1); 3.92 (ps, ÍH, H-3); 4.44 (d, J = 5 Hz, 2H, Ar-CH2-N); 5.37 (ps, 1 H, H-15); 5.76 (t, J = 5 Hz, ÍH, NH); 6.72 (s, 1 H, H-2 '); 7.24 (m, 4H, aromatic).

Example 9; (E) -N- [[4- [(3β-Hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl] -phenyl] methyl] -5- (4,4) -difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-yl) pentanamide A solution of 5.0 mg of 5- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3-yl) pentanoic acid succinimidyl ester in 0.5 ml of dimethylformamide is add to a solution of 8.9 mg of (e) -2- (4-aminomethyl-phenyl) -methylidene-4,4-dimethyl-5a-cholesta-8,14-diene-3ß-ol in 2 ml of dimethylformamide at room temperature ambient. The reaction mixture is stirred for 44 hours. It is diluted with water and extracted with ethyl acetate. The organic layer is separated, washed with water, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and hexyl acetate to provide 6.5 mg of (E) -N - [[4- [(3β-hydroxy-4,4-dimethyl- 5a-cholesta-8, 14-dien-2-ylidene) methyl] -phenyl] methyl] -5- (4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacen -3-yl) pentanamide.

XH NMR (CDC13): d = 0.77 (s, 3H), 0.82 (s, 3H); 0.86 (2x d, J = 7 Hz, 6H, H-26/27); 0.89 (s, 3H); 0.92 (d, J = 7 Hz, H-21); 1.19 (s, 3H); 2.25 (s, 3H, Ar-Me); 2.55 (s, 3H, Ar-Me); 3.12 (d, J = 17 Hz, ÍH, H-1); 3.92 (ps, 1H, H-3); 4.44 (d, J = 5 Hz, 2H, Ar-CH2-N); 5.37 (ps, ÍH, H-15); 5.87 (m, ÍH, NH); 6.09 (s, ÍH, aromatic); 6.30 (d, J = 5 Hz, ÍH, aromatic); 6.70 (s, 1 H, H-2 '); 6.90 (d, J = 5 Hz, 1H, aromatic); 7.24 (m, 4H, aromatic).

Example 10: 2a-Allyl-4,4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol a) 2a-allyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3-one: A solution of 200 mg of 4,4-dimethyl-cholesta-8,14-dien-3-one in 3 ml of THF is added dropwise to a freshly prepared solution of lithium diisopropylamide (5.8 ml, IM) at -70. ° C. The solution is stirred for 1 hour before 0.07 ml of 3-iodopropene is added. After stirring for an additional 1 hour at 0 ° C, the reaction mixture is poured into a saturated solution of ammonium chloride and extracted with ethyl acetate. The organic layer is separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue is subjected to chromatography with a mixture of hexane and ethyl acetate to give 160 mg of 2-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-one as a white solid.

XH NMR (CDC13): d = 0.82 (s, 3H); 0.87 (2x d, J = 7 Hz, 6H, H-26/27); 0.93 (d, J = 7 Hz, 3H, H-21); 1.10 (2x s, 6H); 1.30 (s, 3H); 2.60 (m, ÍH, allyl); 2.77 (m, ÍH, allyl); 5.05 (m, 2H, allyl); 5.38 (ps, 1 H, H-15); 5.80 (m, ÍH, alil). b) 2a-allyl-4, 4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol: 160 mg of 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-one are treated with 55 mg of sodium borohydride as described in example 5 to provide 15 mg of 2a-allyl 4,4-dimethyl-5a-cholesta-8, 14-diene-3a-ol and 80 mg of 2a-allyl-4,4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol. 2a-allyl-4, 4-dimethyl-5a-cholesta-8, 14-diene-3ß-ol: NMR K (CDCl 3): d = 0.81 (s, 3H); 0.85 (s, 3H); 0.87 (2x, d, J = 7 Hz, 6H, H-26/27); 0.94 (d, J = 7 Hz, 3H, H-21); 1.03 (s, 3H); 1.05 (s, 3H); 2.50 (m, ÍH); 2.95 (d, J = 11 Hz, 1H, H-3); 5.06 (m, 2H, allyl); 5.37 (ps, 1 H, H-15); 5.89 (m, ÍH, alil). 2a-allyl-4, 4-dimethyl-5a-cholesta-8, 14-diene-3a-ol: X H NMR (CDCl 3): d = 0.82 (s, 3H); 0.87 (2x, D, J = 7 Hz, 6H, H-26/27); 0.90 (s, 3H); 0.94 (d, J = 7 Hz, 3H, H-21); 1.00 (s, 3H); 1.05 (s, 3H); 3.36 (ps, ÍH, H-3); 5.05 (m, 2H, allyl); 5.35 (ps, 1 H, H-15); 5.85 (m, ÍH, alil).

Example 11: Test of substances that activate meiosis in oocyte tests.

Animals Oocytes are obtained from immature female rats (C57BI / 6J x DBA / 2J hybrids Fl, Bomholtgaard, Denmark) weighing 13-16 grams, and kept under controlled lighting and temperatures. Mice receive an intraperitoneal injection of 0.2 ml of gonadotropins (Godal F. Serono, Solna, Sweden, containing 20 IU of FSH, or alternatively Puregon, Organon, Swords, Ireland, containing 20 IU of FSH) and 48 hours then the animals are sacrificed by cervical dislocation.

Collection and Cultivation of Oocytes The ovaries are removed by dissection and the oocytes are isolated in Hx medium (see below) under a stereoscopic microscope by manual rupture of the follicles using a pair of 27 gauge needles. The spherical bare oocytes (NO) show an intact germ cell vesicle ( GV) are placed in a minimal essential medium (a-MEM) without ribonucleosides, Gibco BRL, cat. No. 22561) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377). human serum albumin 8 mg / ml (HSA, State Serum Institute, Denmark), 0.23 mM pyruvate (Sigma, Cat. No. S-8636), 2 mM g-glutamine (Flow, Cat. No. 16-801), 100 IU / ml of penicline and 100 μg / ml of streptomycin (Flow, Cat. No. 16-700). This medium is called Hx medium. The oocytes are rinsed three times with Hx medium and cultured in 4-well, multi-well containers (Nuncion, Denmark) in which each well contains 0.4 ml of Hx medium and 35-45 oocytes. Control is always run (ie, 35-45 oocytes cultured in Hx medium without addition of the test compound) simultaneously with the test cultures, which are made with different concentrations of the compounds to be tested. The cultures are carried out at 37 ° C and 100% humidity with C02 5% in air. The culture time is 22-24 hours.

Oocyte examination At the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle rupture (GVB) and those with polar bodies (PB) are counted using a stereoscopic microscope or an inverted microscope with differential interference contrast equipment . The percentage of oocytes with GVB per total number of oocytes, and the percentage of oocytes with PB per total number of oocytes is calculated in the test cultures and compared with the control culture.

Example 12: Test of substances that inhibit meiosis in the oocyte test Germ cell vesicle (GV) oocytes are obtained from female mice treated with immature FSH using the same methods as those described in Example 11 (see above). Naked oocytes are rinsed (NO) three times with Hx medium. It has previously been shown that 4,4-dimethyl-5a-cholesta-8, 14, 24-trien-3ß-ol (FF-MAS) induces meiosis in NO in vi tro (Byskov, A.G. et al. Nature 374 (1995) 559-562). They are grown NO in Hx medium supplemented with 5 μM FFR-MAS in coculture with the test compounds at different concentrations in multiple 4-well containers (Nunclon, Denmark) in which each well contains 0.4 ml of Hx medium and 35-45 oocytes . A positive control (ie, 35-45 oocytes cultured in Hx medium containing FF-MAS without addition of the test compound) is always run simultaneously with the test cultures, which is supplemented with different concentrations of the compounds that are they are going to try. In addition, a negative control (35-45 oocytes cultured in Hx medium alone) is run simultaneously with the positive control.

Oocyte estimation At the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle rupture (GVB) and those with polar body (PB) are counted using a stereoscopic microscope or an inverted microscope with differential interference contrast equipment . The percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes is calculated in the test cultures and compared with the control culture.

Example 13: Testing of substances that activate meiosis in the in vitro fertilization test Naked oocytes (NkO) and oocytes enclosed in clusters (CEO) are isolated from immature mouse follicles Fl (C57BL / 6xDBA / 2) (age 21-24 days) who have received 10 IU pregnant Pregnant Mare gonadotropin (FSH activity) i.p. 48 hours before collection. . The oocytes (NkO and CEO) are accumulated and cultured for about 20 hours in a modified α-MEM medium containing 3 mM hypoxanthine (Hx medium) and 1 mg fetuin / ml culture medium. Two groups of oocytes are tested: (a) control oocytes, cultured in Hx-free medium (positive control group) and (b) oocytes cultured in Hx medium containing the test compound. After approximately 20 hours, the oocytes showing germinal vesicle rupture (GVB) are briefly washed in liquid Hx medium and transferred to insemination vessels prepared in advance, which consist of a mobile sperm preparation of the caudal epidimid. of male mice. The containers are then incubated under defined gas conditions (C02 5%) at 37 ° C in an IVF medium modified with a-MEM. The examination of the oocytes is carried out 20-22 hours after the insemination, in order to verify the fertilization and register the number of embryos with two cells. The percentage of fertilization (= fertilization rate) is determined, such as the number of oocytes that have been separated in embryos of two cells, in relation to the total number inseminated. Each IVF experiment is performed with a total number of 50-200 GVB / PV oocytes. A stimulation factor is calculated as the ratio between the fertilization rate and the group containing the test compound and the control group.

Table 1: Activation of meiosis in nude mouse oocytes Hx = hypoxanthine GV = germinal vesicle GVB = rupture of the germinal vesicle PB = polar bodies n = number of oocytes Table 2: Relative inhibition of meiosis in nude mouse oocytes Hx = hypoxanthine GV = germinal vesicle GVB = rupture of the germinal vesicle PB = polar bodies n = number of oocytes Table 3: Relative inhibition of meiosis in nude mouse oocytes Hx = hypoxanthine GV = germinal vesicle GVB = rupture of the germinal vesicle PB = polar bodies n = number of oocytes

Claims (14)

1. CLAIMS Compounds of the general formula I wherein R1 designates a hydrogen atom, an alkyl group of 2 to 6 carbon atoms, an optionally substituted phenyl group, a cyano group, a CHj-NH-COR1 'group (wherein R1' is an alkyl of 1 to 8 carbon atoms or an optionally substituted phenyl group) or together with R 2, an additional bond, R 2 designates a hydrogen atom, an alkyl group of 4 to 8 carbon atoms, an alkenyl group of 3 to 6 carbon atoms, a group hydroxyalkyl of 1 to 6 carbon atoms, together with R 2 a benzylidene group optionally substituted together with R 2 'a hydroxymethylene group or together with R 1 an additional bond, R denotes a hydrogen atom, together with R 2 an optionally substituted benzylidene group or together with R 2 a hydroxymethylene group, R designates a hydrogen atom or together with R 3 'an additional bond, designates a hydrogen atom or together with R 3 an additional bond R4 designates a hydrogen atom or a methyl group, R4 denotes a hydrogen atom or a methyl group, R8 designates together with R9 or with R14 an additional bond, R9 designates a hydrogen atom or together with R8 an additional bond, R1 designates an a-hydrogen atom or together with R8 an additional bond or together with R15 an additional bond, R1 designates a hydrogen atom or together with R14 an additional bond R denotes a hydrogen atom or together with R2S an additional bond, R 25 designates a hydrogen atom or together with R24 an additional bond or esters thereof with the proviso that the compounds are excluded which are simultaneously not modified in positions 1 and 2 (R1 = R2 = R2 '= H).
2. 2. Compounds as described in the claim 1, wherein R 1 designates a hydrogen atom, a phenyl group or together with R 2, an additional bond.
3. 3. Compounds as described in claim 1 or 2, wherein R 2 denotes an alkyl group of 4 to 8 carbon atoms, an allyl group or an additional bond with R 1.
4. 4. Compounds as described in claim 1 or 2, wherein R2 and R2 'designate an optionally substituted benzylidene group.
5. 5. Compounds as described in any of claims 4, which are: 2a-allyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol, (E) -2-benzylidene-4 , 4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol, 5-cholesta-l, 8, 14 -trien-3ß-ol, 5a-cholesta-1, 8, 14 -trien-3- ona, la-cyano-4,4-dimethyl-5a-cholesta-8, 14-dien-3ß-ol, la-cyano-4, 4-dimethyl-5a-cholesta-8, 14-dien-3-one, 4,4-dimethyl-5a-cholesta-l, 8, 14-trien-3-ß-ol, 4,4-dimethyl-5a-cholesta-l, 8, 14. trien-3 -one, (E) -4 - [(3-β-hydroxy-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) -methyl] benzonitrile, (E) -N- [[4- [(3β-hydroxy)] 4, 4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene) methyl] phenyl] octanamide, (E) -N- [[4- [(3β-hydroxy-4,4-dimethyl-5a- cholesta-8, 14-dien-2-ylidene) methyl] phenyl] methyl] -5- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacen-3 il) pentanamide, 2a-hydroxymethyl-4,4-dimethyl-5a-cholesta-8, 14-diene-3a-ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3β-ol and (E) -4- [(3 -oxo-4,4-dimethyl-5a-cholesta-8, 14 -dien-2-ylidene) methyl] benzonitrile.
6. 6. Pharmaceutical compositions comprising one or more compounds of the general formula I, as described in any of claims 1 to 5, as active substances.
7. 7. Use of the compounds of the general formula I, as described in any of claims 1 to 5, for the preparation of a medicament regulating meiosis.
8. 8. Use as described in claim 7 for the preparation of a medicament for the treatment of infertility in females or males, preferably in humans.
9. 9. Use as described in claim 7 for the preparation of a contraceptive medicament for the treatment of females or males, preferably humans.
10. 10. Use of the compounds of the general formula I, as described in any of claims 1 to 5, to regulate the rate of fertilization in artificial insemination procedures.
11. 11. Use of the compounds of the general formula I, as described in any of claims 1 to 5, as tool substances or as starting materials for the synthesis of tool substances for purposes of image bioforming in order to clarify the mode of action of such substances.
12. 12. A method for regulating meiosis, comprising administering to a subject in need of such regulation an effective amount of one or more compounds of the general formula I, according to any of claims 1 to 5.
13. 13. A method for regulating meiosis in a mammalian germ cell, comprising administering ex vivo or in vi tro to a germ cell in need of such regulation, an effective amount of one or more compounds of the general formula I as described in any of claims 1 to 5.
14. 14. A method according to claim 13, wherein the germ cell is an oocyte or a male germ cell.