AU3279700A - Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments - Google Patents

Description

WO 00/47604 PCT/EP0O/01074 1 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments 5 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 10 medicaments. More particularly it has been found that the unsaturated cholestane derivatives of the invention can be used for regulating meiosis. Meiosis is the unique and ultimate event of germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first 15 division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA synthesis. This division therefore results in the formation of the haploid germ cells with only 1 c DNA. 20 The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the 25 female has a stock of oocytes which is drawn upon until the stock is exhausted. Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without 30 significant delay and produces 4 spermatozoa. Only little is known about the mechanism which control the initiation of meiosis in the male and in the female. In the oocyte, new studies indicate that follicular WO 00/47604 PCT/EPOO/01074 2 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 a diffusible meiosis regulating substance was first described by 5 Byskov et al. in a culture system of fetal mouse gonads [Byskov, A. G. et al. Dev Biol 52 (1976) 193-200]. A meiosis-activating substance (MAS) was secreted by the fetal mouse ovary in which meiosis was ongoing, and a meiosis preventing substance (MPS) was released from the morphologically differentiated testis with resting, non-meiotic germ cells. It was suggested that the relative concentrations 10 of MAS and MPS regulated the beginning, arrest and resumption of meiosis in the male and in the female germ cells (Byskov, A.G. et al. in The Physiology of Reproduction [eds. Knobil. E. and Neill, J. D., Raven Press, New York (1994)]. Clearly, if meiosis can be regulated, reproduction can be controlled. A recent article [Byskov, A. G. et al. Nature 374 (1995), 559-562] describes the isolation 15 from bull testes and from human follicular fluid of certain sterols that activate oocyte meiosis. Unfortunately, these sterols are rather labile and utilization of the interesting finding would thus be greatly facilitated if more stable meiosis activating compounds were available. Compounds being known to stimulate the meiosis and being different from the 20 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 for the treatment of infertility in females and males, particularly in humans via activation 25 of meiosis. It is a further purpose of the present invention to provide novel compounds useful as contraceptives in females and males, particularly in humans via inhibition of meiosis. 30 According to the present invention there are provided novel, stable compounds with interesting pharmacological properties. In particular, the compounds described here are useful for the regulation of meiosis in oocytes and in male germ cells.

WO 00/47604 PCT/EPOO/01074 3 It is a further purpose of the present invention to provide novel compounds which are suitable substrates for the introduction of fluorescense markers. The linked molecules are designed and synthesized for bioimaging purposes. 5 The present invention relates to unsaturated cholestane derivatives of the general formula I R24 R25 IR 101 R1 H 9 R' R14' R3'T R8 R14 R15 H R3 R4 R4' wherein 10 R1 designates a hydrogen atom, a C2-C6-alkyl group, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR group (with Rs being a C-C8-alkyl or an optionally substituted phenyl group) or together with R2 an additional bond, 15 R2 designates a hydrogen atom, a C4-C8-alkyl group, a C3-C6-alkenyl group, a C1-C6-hydroxyalkyl group, together with R 2 ' an optionally substituted benzylidene group, together with R 2 ' a hydroxymethylene group or together with R1 an additional bond, 20 R2' designates a hydrogen atom, together with R2 an optionally substituted benzylidene group or together with R2 a hydroxymethylen group,

R

3 designates a hydrogen atom or together with R 3 ' an additional bond, 25 R 3 ' designates a hydrogen atom or together with R 3 an additional bond, WO 00/47604 PCT/EPO0/01074 4

R

4 designates 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 designates a hydrogen atom or together with R 8 an additional bond 5 R 1 4 designates an a-hydrogen atom or together with R 8 an additional bond or together with R 15 an additional bond

R

15 designates a hydrogen atom or together with R 14 an additional bond

R

24 designates a hydrogen atom or together with R 25 an additional bond

R

25 designates a hydrogen atom or together with R 24 an additional bond 10 or esters thereof, with the proviso that compounds which are simultaneously unmodified in position 1 and 2 (R 1 = R2 = R 2 '= H) are excluded. 15 The compounds of the general formula I have a number of chiral centres in the molecule and thus exist in several isomeric forms. All these isomeric forms and mixtures thereof are within the scope of the invention (unless otherwise noted). 20 Preferred compounds of formula I are such wherein R 1 designates a hydrogen atom, a phenyl group or together with R 2 an additional bond. Other preferred compounds are such wherein R 2 designates a C 4

-C

8 -alkyl group, an allyl group or an additional bond with R 1 . Compounds wherein R 2 and R 2 ' designate an optionally 25 substituted benzylidene group are also preferred. In another embodiment, the present invention relates to esters of compounds of the general formula 1. Such esters are formally derived by esterification of one or more hydroxylic groups of a compound of formula I with an acid which can for 30 example be selected from the group of acids comprising succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, WO 00/47604 PCT/EP0O/01074 5 phosphoric acid, sulphonic acid, sulphamic 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 5 or in combinations - may be a straight or branched alkyl group. The expression C2-C6 alkyl designates an alkyl group having from two to six carbon atoms: preferred examples are ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and cyclohexyl more preferred ethyl. Similarly, the expression C1-C8 alkyl designates an alkyl group having from one to eight carbon atoms; preferred examples are 10 methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl more preferred methyl, ethyl, propyl, isopropyl, butyl and tert-butyl, still more preferred methyl and ethyl. 15 20 25 Especially preferred compounds of formula I of the present invention are the following: 30 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, (E)-2-benzylidene-4,4-dimethyl-5ax-cholesta-8,14-dien-3p-ol, 5a-cholesta-1,8,14-trien-3p-ol, WO 00/47604 PCT/EPOO/01074 6 5a-cholesta-1,8,14-trien-3-one, 1 a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, 1 a-cyano-4,4-dimethyl-5ax-cholesta-8,14-dien-3-one, 4,4-dimethyl-5ca-cholesta-1,8,14-trien-3p-ol, 5 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one, (E)-4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl]benzonitrile, (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5ac-cholesta-8,14-dien-2 ylidene)methyl]phenyl]methyl]octanamide, 10 (E)-N-[[4-[(3p-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, 2c-hydroxymethyl-4,4-dimethyl-5ax-cholesta-8,14-dien-3a-ol, 2c-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-oI and 15 (E)-4-[(3-oxo-4,4-dimethyl-5c-cholesta-8,14-dien-2-ylidene)methyl]benzonitrile. 20 The compounds of the general formula I according to the invention can be synthesized analogously with the preparation of known compounds. Hence, synthesis of the compounds of formula I can follow the well established synthetic pathways described in the comprehensive sterol and steroid literature. The 25 following books can be used as the key source in the synthesis: L.F. Fieser & M. Fieser: Steroids: Reinhold Publishing Corporation, NY 1959; Rood's Chemistry of Carbon Compounds (editor: S. Coffrey): Elsevier Publishing Company, 1971; and especially Dictionary of Steroids (editors: R.A. Hill; D.N. Kirk; H.L.J. Makin WO 00/47604 PCT/EP00/01074 7 and G.M. Murphy): Chapman & Hall. The last one contains an extensive list of citations to the original papers covering the period up to 1990. All these books including the last mentioned citations are incorporated by reference. Particularly, the compounds of the present invention are synthesized according 5 to the following general procedures: The sterols that are used as starting materials can be synthesized according to 10 literature procedures: 4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, 4,4-dimethyl-5u-cholest-8-en-3p-ol, 4,4-dimethyl-5a-cholest-8(14)-en-3p-o [Biochem. J. 132 (1973), 439], 4,4 dimethyl-5a-cholesta-8,24-dien-3p-ol, 4,4-dimethyl-5a-cholesta-8,14,24-trien-3p ol [J. Am. Chem. Soc. 111 (1989), 278], 5a-cholesta-8,14-dien-3p-ol [J. Am. 15 Chem. Soc. 75 (1953), 4404], 5a-cholest-8-en-3p-ol [J. Org. Chem. 46 (1981), 3421] and 5ax-cholest-8(14)-en-3p-ol [Biochem. J. 144 (1974), 59]. In the following only the syntheses in the 4,4-dimethyl-A-8,14-series are described in detail. The derivatives in the A-8, A-8(14), A-8,14,24 and A-8,24 20 series with and without the 4,4-dimethyl group can be synthesized from the corresponding starting materials in the same way. The 3p-alcohols can be oxidized with different reagents to give the corresponding 3-ketones [for example: Tetrahedron Lett. 1967, 3699]. 4,4 dimethyl-5a-cholesta-8,14-dien-3p-ol 1 is treated with N-methyl-morpholine-N 25 oxide in the presence of tetrapropyl-ammonium-perruthenate to give 4,4 dimethyl-5a-cholesta-8,14-dien-3-one 2 [for example: Synthesis 1994, 639].

WO 00/47604 PCT/EPOO/01074 8 scheme 1: 11H 11H HO 10 2 H0 H 11 H > 00 3 HO*4 In the next step the A-1 double bond can be introduced in an oxidation reaction 5 with phenyl selenic anhydride as an oxidant in chlorobenzene [J. Chem Soc. Chem. Commun. 1978, 952] to give 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 Al-double bond. The 3-keto group can then be reduced with sodium 10 borohydride in the presence of cerium chloride [Luche reduction, for example: J. Am. Chem. Soc. 100 (1978), 2226] to give 4,4-dimethyl-5a-cholesta-1,8,14-trien 3p-ol 4. 15 Alkyl and aryl groups can be introduced in position 1 via cuprate additions to unsaturated ketones [Tetrahedron Lett. 35 (1994), 8591]. Cuprates which are formed from alkyl or aryl lithiums and cuprous iodide react with steroidal enones to give 1-substituted derivatives. If for example enone 3 is treated with 20 dialkylcuprates compounds of formula 5 (R 1 = alkyl, aryl) are obtained. These WO 00/47604 PCT/EP0O/01074 9 ketones can then be reduced according to well known literature procedures to the two diastereomeric alcohols 6 and 7 (R' = alkyl, aryl), which are readily separable by column chromatography. scheme 2: 11H R1 11H 5 The introduction of a cyano group can be achieved in a similar way. Conjugate addition of cyanide to enone 3 gives the desired 1at-cyano-4,4-dimethyl-5aL cholesta-8,14-dien-3-one 5 (R 1 = ON). Different reagents like diethylaluminum cyanide [J. Org. Chem. 59 (1994), 2766] and some alkali and earth alkali metals 10 can be used in this reaction [Tetrahedron Lett. 28 (1987), 4189; Can. J. Chem. 59 (1981), 1641]. Cyanoketone 5 (R 1 = ON) can also be reduced with standard reducing agents like sodium borohydride to give the two diastereomeric alcohols 6 and 7 (R 1 = ON), which are readily separable by column chromatography. 15 If cyanoalcohol 6 (R 1 = ON, synthesized as described above) is treated with lithium aluminumhydride 1ac-aminomethyl-4,4-dimethyl-5aL-cholesta-8, 14-dien-3p3 ol 8 is obtained. This amine can be used for further modification. Amides of formula 9 (R 1 = alkyl, aryl and fluorescence marker) can be synthesized from the 20 amine by reaction with hydroxysuccinimidylester of various alkyl or aryl carboxylic WO 00/47604 PCT/EP00/01074 10 acids. (For the use of hydroxysuccinimidylester of carboxylic acids that contain fluorescence marker see: Nonradioactive labeling and detection of biomolecules, Kessler C. ed., Springer Verlag, Berlin, 1992.) scheme 3: R1 11H H2N1- 11H HO 6 (Rl=CN) HO 8 4 R H HO 9 5 Substituents in position 2 can for example be introduced via aldol reactions and alkylations. If ketone 2 is treated with aromatic aldehydes in the presence of a base 2-benzylidene-substituted steroids of formula 10 are obtained. The aromatic ring can be substituted. Subsequent reduction with sodium borohydride 10 in the presence of cerium chloride gives selectively the corresponding allylic 3p alcohols 11.

WO 00/47604 PCT/EPOO/01074 11 scheme 4: R2" HH 1H R2" IH HO 11 Cyanobenzylidene-substituted compound 11 (R2" =4-CN) which can be synthesized as shown above can be used for further modification. The cyano group can be reduced with lithium aluminumhydride to the benzylic amine 12, 5 which can be derivatized to corresponding amides of formula 13 (R 2 = alkyl, aryl and fluorescence markers). For this purpose amine 12 is treated with hydroxysuccinimidyl ester derivatives of different carboxylic acids. For this reaction also commercially available esters that contain fluorescence markers can be used (see example 11 in the experimental part). Amides of formula 13 10 that contain the fluorescence markers can be used as molecular probes for bioimaging purposes. 15 WO 00/47604 PCT/EPOO/01074 12 scheme 5: R2" NH2 IH 'I H HO 11 (R2"=4-CN) HO12 N YR2 Y H Alkyl- and alkenyl substituents can be introduced in position 2 via deprotonation of 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 5 borohydride to give two diastereomeric alcohols of formulae 15 and 16, which can be easily separated by column chromatography. scheme 6: HI H I',J R2,1 0 2 01 H HH H0015 HO" 16 WO 00/47604 PCT/EP00/01074 13 The hydroxymethylsubstituents in position 2 can for example be introduced via a condensation reaction of a deprotonated 3-ketone with alkylformiates to give enol 17. This can be reduced with different reducing agents like sodium borohydride to give two diastereomeric alcohols 18 and 19. 5 scheme 7: "H I H HO O 2 0 H H 17H HO HO HO 18 HO 19 These alcohols can easily be separated with column chromatography. Longer chain hydroxyalkylsubstituents can for example be introduced via alkylation reactions as described above (see scheme 6). 10 A further object of the present invention are pharmaceutical compositions comprising one or more compounds of the general formula I as active subs tances. The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, 15 preservatives, buffers, agents for adjusting the osmotic pressure, tablet disin tegrating 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 carboxymethyl cellulose, low melting waxes and cacao butter. 20 WO 00/47604 PCT/EPOO/01074 14 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 men 5 tioned in the list above. Further, a composition for transdermal administration of a compound of this invention may be provided in the form of a patch and a composition for nasal administraton may be provided in the form of a nasal spray in liquid or powder form. 10 The dose of a compound of the invention to be used will be determined by a physician and will depend among several factors on the particular compound employed, on the route of administration and on 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 15 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 meiosis-regulating medicament. The compounds of the present invention influence the meiosis in oocytes as well as in male germ cells. 20 The prospects of being able to influence the meiosis are several. According to a preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate the meiosis. According to another preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate the meiosis in humans. Thus, the compounds of the general 25 formula I are promising as new fertility-regulating agents without the usual side effects on the somatic cells which are known from the hitherto used hormonal contraceptives which are based on estrogens and/or gestagens. Accordingly, the present invention concerns the use of the compounds of the 30 general formula I for relieving infertility in females and males, particularly in mammals, more particularly in humans. Meiosis-inducing substances of the general formula I can be used in the treatment of certain cases of infertility in females, including women, by administration thereof to females who, due to an insufficient WO 00/47604 PCT/EPO0/01074 15 own 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 e.g. in in vitro fertilization or intracytoplasmic sperm injection. When in 5 vitro fertilization is performed, better results can be achieved, when a compound of the invention is added to the medium in which the oocytes are cultured. When infertility in males, including men, is caused by an insufficient own production of the meiosis-activating substance and thus a lack of mature sperm cells exists, administration of a compound of the invention may relieve the problem. 10 In a further object 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 so as to prematurely 15 induce resumption of meiosis in oocytes while they are still in the growing follicle, before the ovulatory peak of gonadotropins occurs. In women, the resumption of the meiosis can, for example, be induced a week after the preceding menstruation has ceased. When ovulated, the resulting overmature oocytes are then most likely not to be fertilized. The normal menstrual cycle is not likely to be 20 affected. In this connection it is important to notice, 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 whereas the estrogens and gestagens used in the hitherto used hormonal contraceptives do have an adverse effect on the biosynthesis of progesterone. 25 As an alternative to the method described above, contraception in females can also be achieved by administration of a compound of the invention which inhibits the meiosis, so that no mature oocytes are produced. Similarly, contraception in males can be achieved by administration of a compound of the invention which 30 inhibits the meiosis, so that no mature sperm cells are 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 bioimaging purposes in order to clarify the mode of action of such WO 00/47604 PCT/EPOO/01074 16 substances. For example meiosis-activating sterols that contain a fluorescence marker can be used to visualize the compartments of the germ cell where the active substances exert their biological function. This information may be helpful to clarify the mode of action of such substances. 5 In a further aspect, the present invention relates to a method of regulating meiosis comprising administering to a subject in need of such a regulation an effective amount of one or more compounds of the general formula 1. 10 In a still further aspect, the present invention relates to a method of regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vitro to a germ cell in need of such a regulation an effective amount of one or more compounds of the general formula 1. The germ cell may be an oocyte or a male 15 germ cell. The route of administration of compositions containing a compound of the invention may be any route which effectively transports the active compound to its site of action. Thus, when the compounds of this invention are to be administered to a 20 mammal, they are conveniently provided in the form of a pharmaceutical composition which comprises at least one compound of the invention in connection with a pharmaceutically acceptable carrier. For oral use, such compositions are preferably in the form of capsules or tablets. 25 From the above it will be understood that administrative regimen called for will depend on the condition to be treated. Thus, when used in the treatment of infertility the administration may have to take place once only, or for a limited period, e.g. until pregnancy is achieved. 30 When used as a contraceptive, the compounds of the invention will either have to be administered continuously or cyclically. When used as a contraceptive by WO 00/47604 PCT/EPOO/01074 17 females and not taken continuously, the timing of the administration relative to the ovulation will be important. 5 The present invention is further illustrated by the following examples: Example 1: 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one 10 a) 4,4-dimethyl-5ca-cholesta-8,14-dien-3-one: To a solution of 6.40 g 4,4-dimethyl-5ca-cholesta-8,14-dien-3p-ol and 4.03 g N methyl-morpholin-N-oxide in 32 ml dichloromethane a few grains of molecular sieves are added and the mixture is stirred for five minutes. 408 mg 15 tetrapropylammoniumperruthenate are added at room temperature and the resulting black reaction mixture is stirred for one hour. After filtration over celite the solvent is evaporated and the residue is chromatographed with a mixture of hexane and ethyl acetate to give 5.60 g 4,4-dimethyl-5a.-cholesta-8,14-dien-3 one as a white solid. 20 1 H-NMR (CDCI 3 ): 6= 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, 1H, H-15) 25 b) 4,4-dimethyl-5c-cholesta-1,8,14-trien-3-one: To a solution of 2.00 g 4,4-dimethyl-5a-cholesta-8,14-dien-3-one in 30 ml chlorobenzene 1.75 g phenylselenic anhydride are added at room temperature. The reaction mixture is warmed to 100 *C for two hours. After cooling and 30 evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 0.85 g 4,4-dimethyl-5a-cholesta-1,8,14-trien-3 one as an oil.

WO 00/47604 PCT/EPOO/01074 18 1 H-NMR (CDCl 3 ): 8= 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); 1.18 (s, 3H); 1.25 (s, 3H); 5.45 (s, 1H, H-15); 5.95 (d, J=10 Hz, 1H, H-2); 7.33 (d, J=10 Hz, 1H, H-1) 5 Example 2: 4,4-dimethyl-5ca-cholesta-1,8,14-trien-3p-ol 14 mg sodium borohydride are added to a suspension of 73 mg 4,4-dimethyl-5a 10 cholesta-1,8,14-trien-3-one and 67 mg cerium chloride heptahydrate in methanol at room temperature. The mixture is stirred for 4 hours, poured into water and extracted with diethylether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and 15 diethylether to give 43 mg 4,4-dimethyl-5a-cholesta-1,8,14-trien-3p-ol as a white solid. 1 H-NMR (CDCl 3 ): 6= 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, 1H, H-3); 20 5.37 (s, 1H, H-15); 5.50 (dd, J=10 Hz, 1 Hz, 1H, H-1); 5.90 (d, J=10 Hz, 2 Hz, 1H, H-2) Example 3: 5a-cholesta-1,8,14-trien-3p-ol 25 a) 5a-cholesta-8,14-trien-3-one: 2.40 g 5a-cholesta-8,14-dien-3p-ol are treated with 1.11 g N-methyl-morpholin N-oxide and 111 mg tetrapropylammoniumperruthenate in 13 ml 30 dichloromethane as described in example la. After chromatography 1.81 g 5a cholesta-8,14-dien-3-one are obtained as a white solid.

WO 00/47604 PCT/EPOO/01074 19 1 H-NMR (CDCl 3 ): 6= 0.82 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.18 (s, 3H, H-19); 5.39 (s, 1H, H-15) 5 b) 5ax-cholesta-1,8,14-trien-3-one 1.81 g 5a-cholesta-8,14-dien-3-one are treated with 1.67 g phenylselenic anhydride in 29 ml chlorobenzene as described in example 1b. After 10 chromatography 0.53 g 5a-cholesta-1,8,14-trien-3-one are obtained as an oil. 1 H-NMR (CDC13): 6= 0.84 (s, 3H, 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, 1H, H-15); 5.91 (d, J=10 Hz, 1H, H-2); 7.42 (d, J=10 Hz, 1H, H-1) 15 c) 5a-cholesta-1,8,14-trien-3p-ol 150 mg 5c-cholesta-1,8,14-trien-3-one are treated with 15 mg sodium borohydride and 147 mg cerium chloride heptahydrate as described in example 20 2. After chromatography 47 mg 5a-cholesta-1,8,14-trien-3p-ol are obtained as a white solid. 1 H-NMR (CDCl 3 ): 6= 0.82 (s, 3H, 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, 1H, H-3); 5.37 (s, 1H, H 25 15); 5.56 (dd, J=10 Hz, 1 Hz, 1H, H-1); 6.09 (d, J=10 Hz, 2 Hz, 1H, H-2) 30 Example 4: 1 a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one: WO 00/47604 PCT/EP0O/01074 20 1.47 ml of a diethylaluminum cyanide solution (1 M in toluene) are added to a solution of 4,4-dimethyl-5ax-cholesta-1,8,14-trien-3-one in 5 ml tetrahydrofuran at 0 0C. The reaction mixture is allowed to warm to room temperature and stirred 5 for one hour. After addition of 2.45 ml of a sodium hydroxide solution (1 M) at 0 0C the resulting mixture is diluted with water and extracted with diethylether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 110 mg 1 a 10 cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one as a white solid. 1 H-NMR (CDC13): 6= 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, 1H, H-2); 3.29 (dd, J=7 Hz, 5Hz, 1H, H-1); 5.49 (ps, 1 H, H-15) 15 Example 5: 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol and 1 a cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol: 95 mg 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one are treated with 33 20 mg sodium borohydride as described in example 5 to give 30 mg 1ac-cyano-4,4 dimethyl-5a-cholesta-8,14-dien-3a-ol and 25 mg 1a-cyano-4,4-dimethyl-5a cholesta-8,14-dien-3p-ol: 1 a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol: 25 1 H-NMR (CDC1 3 ): 6= 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, 1H, H-1); 3.72 (bm, 1H, H 3); 5.43 (ps, 1 H, H-15) 30 1 a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol: WO 00/47604 PCT/EP00/01074 21 1 H-NMR (CDCl 3 ): 8= 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, 1H, H-1); 3.57 (ps, 1H, H-3); 5.44 (ps, 1 H, H-15) 5 Example 6: (E)-4-[(3-oxo-4,4-dimethyl-5ac-cholesta-8,14-dien-2 yliden)methyl]benzonitrile A suspension of 64 mg 4-cyanobenzaldehyde and 27 mg potassium hydroxide in 2 ml ethanol is added dropwise to a suspension of 4,4-dimethyl-cholesta-8,14 10 dien-3-one in 4ml 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 sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 168 mg (E)-4-[(3-oxo-4,4-dimethyl 15 5ax-cholesta-8,14-dien-2-yliden)methyl]benzonitrile as a white solid. 1 H-NMR (CDC1 3 ): 6= 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); 1.23 (s, 3H); 2.58 (d, J=17 Hz, 1H, H-1); 3.10 (d, J=17 Hz, 1H, H-1); 5.47 (ps, 1 H, H-15); 7.42 (s, 1 H, H-2'); 7.53 20 (d, J=8 Hz, 2H, arom.); 7.68 (d, J=8 Hz, 2H, arom.) Example 7: (E)-4-[(3p-hydroxy-4,4-dimethyl-5c-cholesta-8,14-dien-2-yliden) methyl]benzonitrile 25 156 mg (E)-4-[(3-oxo-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)methyl]benzo nitrile are treated wih 12 mg sodium borohydride in the presence of 111 mg cerium chloride heptahydrate as described in example 2 to give 93 mg (E)-4 [(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)-methyl]benzonitrile as a white solid. 30 1 H-NMR (CDC1 3 ): 6= 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, 1H, WO 00/47604 PCT/EP00/01074 22 H-1); 3.94 (m, 1H, H-3); 5.40 (ps, 1 H, H-15); 6.78 (s, 1 H, H-2'); 7.37 (d, J=8 Hz, 2H, arom.); 7.64 (d, J=8 Hz, 2H, arom.) Example 8: (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2 5 yliden)methyl]phenyl]methyl]octanamide: a) (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5a-cholesta-8,14 dien-3p-ol: A suspension of 27 mg lithium aluminumhydride and 74 mg (E)-4-[(3p-hydroxy 4,4-dimethyl-5ax-cholesta-8,14-dien-2-yliden)-methyl]benzonitrile in 6 ml 10 tetrahydro-furan is refluxed for 4 hours. After cooling the reaction mixture is worked up as described in example 3 b. Cristallization from ethylacetate gives 26 mg (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5X-cholesta-8,14 dien-3p-ol as a white solid. 15 1 H-NMR (CDCl 3 ): 6= 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, 1H, H-1); 3.86 (s, 2H, Ar-CH 2 -N); 3.92 (ps, 1H, H-3); 5.38 (ps, 1 H, H-15); 6.72 (s, 1 H, H-2'); 7.26 (m, 4H, arom.) 20 b) (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2 yliden)methyl]phenyl]methyl]octanamide: A solution of 8.6 mg N-hydroxysuccinimidyl caprylate in 1 ml dimethylforamide is added to a solution of 21 mg (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4 dimethyl-5o-cholesta-8,14-dien-3p-o in 4 ml dimethylformamide at room 25 temperature. The reaction mixture is stirred for 20 hours, diluted with water and extracted with ethylacetate. The organic layer is separated, washed with water, dried over anhydrous sodium sulphate and filtered. Evaporation of the solvent gives 21 mg (E)-N-[[4-[(3p hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)methyl]phenyli]methyl] 30 octanamide as a white solid.

WO 00/47604 PCT/EPOO/01074 23 1 H-NMR (CDC1 3 ): 8= 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, 1H, H-1); 3.92 (ps, 1H, H-3); 4.44 (d, J=5 Hz, 2H, Ar-CH 2 -N); 5.37 (ps, 1 H, H 5 15); 5.76 (t, J=5 Hz, 1H, NH); 6.72 (s, 1 H, H-2'); 7.24 (m, 4H, arom.) Example 9: (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5ax-cholesta-8,14-dien-2 yliden)methyl]-phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s 10 indacen-3-yl)pentanamide: A solution of 5.0 mg 5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3 yl)pentanoic acid succinimidyl ester in 0.5 ml dimethylforamide is added to a solution of 8.9 mg (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5a 15 cholesta-8,14-dien-33-ol in 2 ml dimethylformamide at room temperature. The reaction mixture is stirred for 44 hours, diluted with water and extracted with ethylacetate. The organic layer is separated, washed with water, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 20 6.5 mg (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden) methyl]-phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s indacen-3-yi)pentanamide. 25 1 H-NMR (CDCl 3 ): 6= 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); 1.19 (s, 3H); 2.25 (s, 3H, Ar 30 Me); 2.55 (s, 3H, Ar-Me); 3.12 (d, J=17 Hz, 1H, H-1); 3.92 (ps, 1H, H-3); 4.44 (d, J=5 Hz, 2H, Ar-CH 2 -N); 5.37 (ps, 1H, H-15); 5.87 (m, 1H, NH); 6.09 (s, 1H, WO 00/47604 PCTIEP00/01074 24 arom.); 6.30 (d, J=5 Hz, 1H, arom.); 6.70 (s, 1 H, H-2'); 6.90 (d, J=5 Hz, 1H, arom.); 7.24 (m, 4H, arom.) Example 10: 2a-ally-4,4-dimethyl-5c-cholesta-8,14-dien-3p-ol 5 a) 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-one: A solution of 200 mg 4,4-dimethyl-cholesta-8,14-dien-3-one in 3 ml THF is added dropwise to a freshly prepared lithium diisopropylamide solution (5.8 ml, 1 M) at 10 70 0C. The solution is stirred for one hour before 0.07 ml 3-iodo-propene are added. After being stirred for one additional hour at 0 0C the reaction mixture is poured into saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is 15 chromatographed with a mixture of hexane and ethylacetate to give 160 mg 2

U.

allyl-4,4-dimethyl-5ax-cholesta-8,14-dien-3-one as a white solid. 1 H-NMR (CDC1 3 ): 6= 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, 1H, allyl); 2.77 (m, 1H, 20 allyl); 5.05 (m, 2H, allyl); 5.38 (ps, 1 H, H-15); 5.80 (m, 1H, allyl) 25 b) 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol: 160 mg 2a-allyl-4,4-dimethyl-5ax-cholesta-8,14-dien-3-one are treated with 55 mg sodium borohydride as described in example 5 to give 15 mg 2a-allyl-4,4- WO 00/47604 PCT/EPO0/01074 25 dimethyl-5ac-cholesta-8,14-dien-3a-oI and 80 mg 2a-allyl-4,4-dimethyl-5a cholesta-8,14-dien-3p-ol. 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol: 5 1 H-NMR (CDCI 3 ): 6= 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, 1H); 2.95 (d, J=11 Hz, 1H, H-3); 5.06 (m, 2H, allyl); 5.37 (ps, 1 H, H-15); 5.89 (m, 1H, allyl) 10 2a-allyI-4,4-dimethyl-5c-cholesta-8,14-dien-3ax-ol: 1 H-NMR (CDCl 3 ): 6= 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, 1H, H-3); 5.05 (m, 2H, allyl); 5.35 (ps, 1 H, H-15); 5.85 (m, 1H, allyl) 15 Example 11: Testing of meiosis-activating substances in the oocyte test Animals 20 Oocytes were obtained from immature female mice (C57Bl/6J x DBA/2J Fl hybrids, Bomholtgaard, Denmark) weighing 13 - 16 grams, that were kept under controlled lighting and temperature. The mice received an intra-peritoneal injection of 0.2 ml gonadotropins (Gonal F, Serono, Solna, Sweden , containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU 25 FSH) and 48 hours later the animals were killed by cervical dislocation. Collection and culture of oocytes The ovaries were dissected out and the oocytes were isolated in Hx-medium 30 (see below) under a stereo microscope by manual rupture of the follicles using a pair of 27 gauge needles. Spherical, naked oocytes (NO) displaying an intact germinal vesicle (GV) were placed in a-minimum essential medium (a-MEM WO 00/47604 PCT/EPOO/01074 26 without ribonucleosides, Gibco BRL, Cat.No. 22561) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg/mI Human Serum Albumin (HSA, State Serum Institute, Denmark), 0,23 mM pyrubate (Sigma, Cat. No. S-8636), 2 mM glutamine (Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 pg/ml 5 streptomycin (Flow, Cat No. 16-700). This medium was designated Hx-medium. The oocytes were rinsed three times in Hx-medium and cultured in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx medium and 35 - 45 oocytes. One control (i.e. 35 - 45 oocytes cultured in Hx medium with no addition of test compound) was always run simultaneously with 10 the test cultures, which were made with different concentrations of the compounds to be tested. The cultures were performed at 37 0C and 100 % humidity with 5 % C02 in air. The culture time was 22 - 24 hours. 15 Examination of oocytes By the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential 20 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 was calculated in the test cultures and compared to the control culture. Example 12: Test of meiosis-inhibiting substances in the oocyte test 25 Germinal vesicle (GV) oocytes were obtained from immature FSH treated female mice using the same methods as described in Example 11 (see above). Naked oocytes (NO) were rinsed three times in Hx-medium. 4,4-Dimethyl-5aX-cholesta 8,14,24-trien-3B-ol (FF-MAS) has previously been shown to induce meiosis in 30 NO in vitro (Byskov, A.G. et aL Nature 374 (1995) 559 - 562). NO were cultured in Hx-medium supplemented with 5 pM FF-MAS in co-culture with the test compounds in different concentrations in 4-well multidishes (Nunclon, Denmark) in which each well contained 0.4 ml of Hx-medium and 35 - 45 oocytes. One WO 00/47604 PCT/EPOO/01074 27 positive control (i.e., 35 - 45 oocytes cultured in Hx-medium containing FF-MAS with no addition of test compound) was always run simultaneously with the test cultures, which were supplemented with different concentrations of the compounds to be tested. In addition, one negative control (35 - 45 oocytes 5 cultured in Hx-medium alone) was run simultaneously with the positive control. Examination of oocytes By the end of the culture period, the number of oocytes with germinal vesicle 10 (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo 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 was calculated in the test cultures and compared to the control culture. 15 Example 13: Testing of meiosis-activating substances in the ,in-vitro fertilisation' assay Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from 20 follicles of immature (C57BL/6xDBA/2) F1 mice (age 21-24 days), that had received 10 IU Pregnant Mare Serum Gonadotropin (FSH activity) i.p. 48 hours prior to collection. The oocytes (NkO and CEO) were pooled and cultured for approx. 20 hours in a modified a-MEM medium containing 3 mM hypoxanthine (Hx-medium) and 1 mg 25 fetuin/ml culture medium. Two oocyte-groups were used: (a) control oocytes, cultured in Hx-free medium (positive control group) and (b) oocytes, cultured in Hx-medium containing the test compound. After approx. 20 hours oocytes that exhibited germinal vesicle breakdown (GVB) were briefly washed in Hx-free medium and transferred to insemination dishes prepared in advance, which 30 consisted of a motile sperm preparation from the cauda epididymis of male mice. The dishes were then incubated under defined gas conditions (5 % C02) at 37 0 C in a modified a-MEM IVF-medium. Examination of the oocytes was carried out 20-22 hours after insemination, in order to check fertilisation and to record the WO 00/47604 PCT/EP0O/01074 28 number of 2-cell embryos. The percentage fertilisation (= fertilisation rate) was determined as number of oocytes that had cleaved into two-cell embryos, relative to the total number inseminated. Each IVF-experiment was performed with a total number of 50-200 GVB/PB-oocytes. A stimulation factor was calculated as the 5 ratio between the fertilisation rate in the group containing the test compound and the control group.

WO 00/47604 PCT/EPOO/01074 29 Table 1: Activation of meiosis in naked mouse oocytes Compounds Oocytes [n] Activation [%] GV GVB PB GVB+PB Control (Hx) 39 0 1 2.6 10 pM FF-MAS 5 29 4 86.8 10 pM 4,4-dimethyl-5ax-cholesta- 9 18 5 71.9 1,8,14-trien-3p-ol 5 Hx = Hypoxanthine GV = germinal vesicle GVB = germinal vesicle breakdown PB = polar bodies n = number of oocytes 10 Table 2: Relative inhibition of meiosis in naked mouse oocytes Compounds Oocytes [n] Inhibition[%] GV GVB PB Control (Hx) 34 2 0 100 5 pM FF-MAS 6 28 0 0 5 pM FF-MAS + 40 pM 1c-cyano- 38 0 0 107 4,4-dimethyl-5a-cholesta-8,14 dien-3-one 15 Hx = Hypoxanthine GV = germinal vesicle GVB = germinal vesicle breakdown WO 00/47604 PCT/EPOO/01074 30 PB = polar bodies n = number of oocytes 5 Table 3: Relative inhibition of meiosis in naked mouse oocytes Compounds Oocytes [n] Inhibition[%] GV GVB PB Control (Hx) 34 2 0 100 5 pM FF-MAS 6 28 0 0 5 pM FF-MAS + 10 pM (E)-N-[[4- 25 13 0 63 [(3p-hydroxy-4,4-dimethyl-5a cholesta-8,14-dien-2-yliden) methyl]phenyl]methyl]octanamide Hx = Hypoxanthine 10 GV = germinal vesicle GVB = germinal vesicle breakdown PB = polar bodies n = number of oocytes 15 20 25

Claims (10)

1. 2. Compounds according to claim 1, wherein R 1 designates an hydrogen atom, a phenyl group or together with R 2 an additional bond. 25
2. 3. Compounds according to claim 1 or 2, wherein R 2 designates a C 4 -C 8 -alkyl group, an allyl group or an additional bond with R 1 .
3. 4. Compounds according to any one of claims 1 or 2, wherein R 2 and R 2 30 designate an optionally substituted benzylidene group. WO 00/47604 PCT/EPOO/01074 33
4. 5. Compounds according to any one of claims 1 to 4 which are 2a-allyl-4,4-dimethyl-5c-cholesta-8,14-dien-3p-ol, (E)-2-benzylidene-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, 5a-cholesta-1,8,14-trien-3p-ol, 5 5a-cholesta-1,8,14-trien-3-one, 1 a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one, 4,4-dimethyl-5a-cholesta-1,8,14-trien-3p-ol, 4,4-dimethyl-5x-cholesta-1,8,14-trien-3-one, 10 (E)-4-[(3p-hydroxy-4,4-dimethyl-5c-cholesta-8,14-dien-2-ylidene)-methyl] benzonitrile, (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) methyl]phenyl]methyl]octanamide, (E)-N-[[4-[(3p-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene) 15 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-5ac-cholesta-8,14-dien-3a-ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-oI or (E)-4-[(3-oxo-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)methyl] 20 benzonitrile.
5. 6. Pharmaceutical compositions comprising one or more compounds of the general formula I according to any one of claims 1 to 5 as active substances. 25 7. Use of the compounds of the general formula I according to any one of claims 1 to 5 for the preparation of a meiosis regulating medicament. WO 00/47604 PCT/EPOO/01074 34
6. 8. Use according to claim 7 for the preparation of a medicament for the treatment of infertility in females or males, preferably in humans.
7. 9. Use according to claim 7 for the preparation of a contraceptive medicament 5 for the treatment of females or males, preferably humans.
8. 10. Use of the compounds of the general formula I according to any one of claims 1 to 5 to regulate the fertilisation rate in artificial insemination procedures. 10
9. 11. Use of the compounds of the general formula I according to any one of claims 1 to 5 as tool substances or as starting materials for the synthesis of tool substances for bioimaging purposes in order to clarify the mode of action of such substances. 15
10. 12. A method of regulating meiosis comprising administering to a subject in need of such a regulation an effective amount of one or more compounds of the general formula I according to any one of claims 1 to 5. 20 13. A method of regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vitro to a germ cell in need of such a regulation an effective amount of one or more compounds of the general formula I accor ding to any one of claims 1 to 5. 25 14. A method according to claim 13 wherein the germ cell is an oocyte or a male germ cell.