HUP0200280A2 - Unsaturated cholestane derivatives and pharmaceutical compositions containing the same - Google Patents

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Unsaturated cholestane derivatives, pharmaceutical compositions containing them, and the use of these derivatives in the preparation of meiosis-regulating drugs

The present invention relates to pharmaceutically active unsaturated cholestane derivatives and pharmaceutical compositions containing them as active ingredients, and to the use of these novel compounds in the preparation of medicaments. More specifically, it has been found that the unsaturated cholestane derivatives of the present invention can be used to regulate meiosis.

Meiosis is the unique and final event in germ cells, on which sexual reproduction is based. Meiosis consists of two meiotic divisions. During the first division, an exchange of maternal and paternal genes takes place, before which the chromosome pairs are divided into two daughter cells. These contain only half of the chromosomes (In) and 2c DNA. The second meiotic division takes place without DNA synthesis. Thus, this division results in haploid germ cells with only 1c DNA.

The meiotic events are similar in male and female germ cells, but the timing and differentiation processes that lead to oocytes and sperm cells are fundamentally different. All female germ cells enter prophase of the first meiotic division early in life, often before birth, but are arrested as oocytes in the later stages of prophase (dictyate state) until ovulation at puberty. Thus, women have a pool of oocytes from very early in life, which they then use until the pool is depleted. In women, meiosis does not occur completely until after fertilization, and results in only one egg and two abortive polar bodies per germ cell. Once initiated, meiosis in male cells proceeds without significant delay, resulting in 4 spermatozoa.

Little is known about the mechanism by which meiosis is initiated in both females and males. In the oocyte, recent studies suggest that purines, hypoxanthine, or adenosine in the follicle may be responsible for meiotic arrest [Downs, S.M. et al.: Dev. Biol. 82, 454-458 (1985); Epplg. J.J. et al.: Dev. Biol. 119, 313-321 (1986)]; Downs, S.M.: Mol. Repród. Dev. 35, 82-94 (1993)]. The diffusive meiotic regulator was first described by Byskov et al. in cultures prepared from mouse fetal gonads [Byskov, A.G. et al.: Dev. Biol. 52, 193-200 (1976)]. A meiosis-activating substance (MAS) is secreted by the mouse fetal ovary, in which meiosis is in progress, and a meiosis-inhibiting substance (MPS) is released from morphologically differentiated testes with resting, non-meiotic germ cells. The idea is that the relative concentrations of MAS and MPS regulate the initiation, arrest, and continuation of meiosis in male and female germ cells [Byskov, A.G. et al.: The Physiology of Reproduction, eds.: Knobil, E. and Neill, J.D., Raven Press, New York (1994)]. It is clear that if meiosis can be controlled, then reproduction can also be controlled. In a recent article [Byskov, A.G. et al. (1995) Nature 374:559-562) describe the isolation of certain sterols from bull testis and human follicular fluid that activate oocyte meiosis. Unfortunately, these sterols are very labile, and the use of this interesting result would be greatly facilitated if more stable meiosis-activating compounds were available.

Compounds known to stimulate meiosis and which differ from the compounds claimed in the present invention are disclosed in WO 96/27658.

The present invention provides novel compounds for the treatment of infertility in females and males, especially humans, through the activation of meiosis.

The present invention provides novel compounds useful as contraceptives in females and males, especially humans, through the inhibition of meiosis.

The present invention provides novel, stable compounds with interesting pharmacological properties. More specifically, the compounds described in the present invention are useful for regulating meiosis in oocytes and male germ cells.

It is a further object of the present invention to provide novel compounds that are suitable substrates for the delivery of fluorescent markers. The linked molecules are designed and produced for biological imaging purposes.

The present invention relates to unsaturated cholestane derivatives of the general formula I, in which

R ¹ represents a hydrogen atom, an alkyl group having 2-6 carbon atoms, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR ¹ ' group (wherein COR ¹ ' represents an alkyl group having 1-8 carbon atoms, or an optionally substituted phenyl group), or together with R ² they form a new chemical bond,

R ² represents a hydrogen atom, an alkyl group having 4-8 carbon atoms, an alkyl group having 3-6 carbon atoms, a hydroxyalkyl group having 1-6 carbon atoms, together with R ² ', an optionally substituted benzylidene group, together with R ² ', a hydroxymethylene group, or together with R ^x, a new chemical bond,

R ² ' is a hydrogen atom, together with R ² an optionally substituted benzylidene group, or together with R ² a hydroxymethylene group,

R ³ represents a hydrogen atom, or together with R ³ ', another chemical bond,

R ³ ' represents a hydrogen atom, or together with R ³ another chemical bond,

R ⁴ represents a hydrogen atom or a methyl group,

R ⁴ ' represents a hydrogen atom or a methyl group,

R ⁸ together with R ⁹ or R ¹⁴ represents a new chemical bond,

R ⁹ represents a hydrogen atom or, together with R ⁸ , a new chemical bond,

R ¹⁴ represents an α-hydrogen atom or together with R ⁸ a new chemical bond, or together with R ¹⁵ a new chemical bond,

R ¹⁵ represents a hydrogen atom, or together with R ¹⁴ forms a new chemical bond

R ²⁴ represents a hydrogen atom or together with R ²⁵ forms a new chemical bond

R ²⁵ represents a hydrogen atom or together with R ²⁴ a new chemical bond or esters thereof, with the proviso that compounds which are simultaneously unmodified in the 1- and 2-positions (R ^{1 =} R ² =R ² '=H) are excluded.

Compounds of formula I contain a number of chiral centers in the molecule and thus exist in a number of isomeric forms. All such isomeric forms and mixtures thereof are within the scope of the present invention (unless otherwise noted).

Preferred compounds of formula I are those in which R ¹ is hydrogen, phenyl, or together with R ² is a new chemical bond. Also preferred are compounds in which R ² is C 4 -C 8 alkyl, allyl, or together with R ^x is a new chemical bond. Also preferred are compounds in which R ² and R ² ' are optionally substituted benzylidene.

In another embodiment, the present invention provides esters of compounds of formula I. Such esters are formally derived from the esterification of one or more hydroxyl groups of a compound of formula I with an acid, which acid can be selected, for example, from the group consisting of succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarboxylic acid, phosphoric acid, sulfonic acid, sulfamic acid, benzoic acid, acetic acid, propionic acid, and other aliphatic monocarboxylic acids.

As used in this specification and the appended claims, an alkyl group, when used alone or in combination, may be straight or branched. The term C2-6 alkyl refers to an alkyl group containing 2-6 carbon atoms; preferred examples include ethyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and cyclohexyl, with ethyl being most preferred. Similarly, C1-8 alkyl refers to an alkyl group containing 1-8 carbon atoms; Preferred examples thereof are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl, of which methyl, ethyl, propyl, isopropyl, butyl and tert-butyl are preferred, and of which methyl and ethyl are more preferred.

Among the compounds of general formula I according to the present invention, the following are particularly preferred: 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol, (E)-2-benzylidene-4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol, • · ····· · ···· • · · · · · · *· ·

5a-cholesta-1,8,14-trien-3p-ol, 5oc-cholesta-1,8,14-ΰΐεη-3β-οη, la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-one, 4,4-dimethyl-5a-cholesta-1,8,14-trien-3p-ol, 4,4-dimethyl-5a-cholesta-1,8,14-trien-3p-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-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)methyl]phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacen-3-yl)pentanamide, 2oc-hydroxymethyl-4,4-dimethyl-5a-cholesta-8,14-dien-3a.-ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol and (E)-4-[(3-oxo-4,4-dimethyl-5a-cholesta-8,14-dien-2ylidene)methyl]benzonitrile

The compounds of formula I of the present invention can be synthesized by analogy with known compounds. Thus, the synthesis of compounds of formula I can follow the well-established synthetic biosynthetic pathways in the sterol and steroid literature. The following textbooks can be used as key sources for the synthesis: L.F. Fieser & M. Fieser: Steroids, Reihold Publishing Corporation, New York (1959); Rood's Chemistry of Carbon Compounds (ed.: S. Coffrey), Elsevier Publishing Company (1971); Dictionary of Steroids (ed.: R.A. Hill; D.N.

Kirk; H.L.J. Makin and G.M. Murphy), Chapman & Hall. The latter contains an extensive list of citations to the original publications, covering the period up to 1990. All books cited, including the last cited citations, are hereby incorporated by reference.

More specifically, the compounds of the present invention are synthesized according to the following general procedures.

The sterols used as starting materials can be synthesized using methods described in the literature:

4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol, 4,4-dimethyl-5acholesta-8-en-3β-ol, 4,4-dimethyl-5-α-cholesta-8(14)-en-3β-ol [Biochemical J. 132, 439 (1973)], 4,4-dimethyl-5-a-cholesta-8,24dien-3β-ol, 4,4-dimethyl-5-a-cholesta-8,14,24-ΐΓΪέη-3β-ο1 [Journal of the American Chemical Society 111, 278 (1989)], 5a-cholesta8,14-dien-3β-ol [Journal of the American Chemical Society 75, 4404 (1953)], 5a-cholesta-8-en-33-ol [J. Org. Chem. 46, 3421 (1981)], and 5-α-cholesta-8(14)-en-3β-ol [Biochemical J. 144, 59 (1974)].

In the following, only the synthesis of the 4,4-dimethyl-Δ-8,14-series is described in detail. The derivatives of the Δ-8, Δ-8(14), Δ-8,14,24 and Δ-8,24 series, with or without the 4,4-dimethyl group, can be synthesized from the corresponding starting materials in the same manner.

3β-Alcohols can be oxidized with various reagents to give the corresponding 3-ketones [Tetrahetron Letters 3699 (1967)]. In reaction scheme A, 4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol of formula II is treated with N-methylmorpholine-N-oxide in the presence of tetrapropylammonium perruthenate to give 4,4-dimethyl-5a-cholesta-8,14-dien-3-one of formula III [see, for example, Synthesis, 639 (1994)]. In the next step, the Δ-l double bond can be introduced by an oxidation reaction using selenic anhydride in chlorobenzene as the oxidizing agent [J. Chem. Soc. Chem. Comm., 952 (1978)], thus obtaining 4,4-dimethyl-5 a-cholesta-1,8,14-trien-3-one of formula IV. This reaction also occurs with compounds not containing the 4,4-dimethyl group. In these groups, the new double bond can be selectively introduced in the form of a Δ1 double bond. The 3-keto group can be reduced with sodium borohydride in the presence of cerium chloride [Luche reduction, see e.g.: Journal of the American Chemical Society 100, 2226 (1978)], thus obtaining 4,4-dimethyl-5 a-cholesta-1,8,14-trien-3 p-ol of formula V.

Alkyl and aryl groups can be introduced into the 1-position by cuprate addition to unsaturated ketones [Tetrahetron Letters 35, 8591 (1994)]. Cuprates formed from alkyl or aryl lithium and copper iodide react with steroid enones to give the 1-substituted derivatives. For example, if the enone of formula IV is treated with dialkyl cuprate compounds according to reaction scheme B, compounds of general formula VI are obtained (R ^{1 =} alkyl, aryl group). These ketones can then be reduced to the diastereomeric alcohols of general formula VII and VIII (R ^{1 =} alkyl, aryl group) by methods well known in the art, which can then be easily separated by column chromatography.

• · ··♦« t · · · · · ··· · · 4 · Μ ·

The introduction of the cyano group can be achieved in a similar manner. Addition of the cyanide conjugate to the enone IV yields the desired la-cyano-4,4-dimethyl-5a-cholesta8,14-dien-3-one VI (R ¹⁼ CN). Various reagents, such as diethylaluminum cyanide [J. Org. Chem. 59, 2766 (1994)], as well as some alkali and alkaline earth metals, can be used in this reaction [Tetrahetron Letters 28, 4189 (1987); Can. J. Chem. 59 1641 (1981)]. The cyanoketone VI (R ¹⁼ CN) can also be reduced with standard reducing agents, such as sodium borohydride, to give the two diastereomeric alcohols VII and VIII (R ¹⁼ CN), which can be easily separated by column chromatography.

According to reaction scheme AC, if the cyanoalcohol of general formula VII (R ^{1 =} CN, see synthesis above) is treated with lithium aluminum hydride, then 1αaminomethyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol of general formula IX is obtained. This amine can be used for further modification. Amides of general formula AX (R ^{1 =} alkyl, aryl group and fluorescent marker) can be synthesized from the amine by reacting with hydroxysuccinimidyl esters of various alkyl and aryl carboxylic acids. (For the use of hydroxysuccinimidyl esters of carboxylic acids containing fluorescent markers, see: Nonradioactive labeling and detection of biomolecules, ed.: Kessler, C., Springer Verlag, Berlin (1992)].

According to reaction scheme D, the substituents at position 2 can be introduced, for example, by aldol reactions and alkylations. When the ketone of formula III is treated with aromatic aldehydes in the presence of base

...· ·τ ·- ’v being present, then we obtain the 2-benzylidene-substituted steroids of general formula XI. The aromatic ring can be substituted. Reduction with sodium borohydride in the presence of cerium chloride selectively gives the corresponding allylic 3β-alcohol of general formula XII.

According to the reaction scheme E, the compound of general formula XII (R ² =4-CN), which can be synthesized as described above, can be used for further modifications. The cyano group can be reduced with lithium aluminum hydride to give the benzylamine of general formula XIII, from which the corresponding amides of general formula XIV can then be prepared (R ² =alkyl, aryl group and fluorescent marker). For this purpose, the amine of general formula XIII is treated with hydroxysuccinimidyl ester derivatives of various carboxylic acids. Commercial esters containing fluorescent markers can also be used in this reaction (see all. example in the experimental section). The amides of general formula XIV, which contain the fluorescent marker, can be used as molecular probes for biological imaging purposes.

According to Scheme F, alkyl and alkenyl substituents can be introduced at the 2-position by deprotonation of the ketone III and subsequent reaction of the enolate with alkyl and alkenyl halides. The 3-ketones XV can then be reduced with sodium borohydride to give the diastereomeric alcohols XVI and XVII, which can then be separated by column chromatography.

• ♦ ·«··· «*«>· ··* ··« 4 ·· ·

According to Scheme G, hydroxymethyl substituents at position 2 can be introduced, for example, by condensation of a deprotonated 3-ketone with alkyl formates to give the enol compound of formula XVIII. This can be reduced with various reducing agents, such as sodium borohydride, to give the diastereomeric alcohols of formula XIX and XX. These alcohols can be easily separated by column chromatography. Longer chain hydroxyalkyl substituents can be introduced, for example, by alkylation reactions as described above.

The present invention also provides pharmaceutical compositions comprising one or more compounds of formula I as active ingredients. The compositions may also contain pharmaceutically acceptable excipients well known in the art, such as carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting osmotic pressure, tablet disintegrating agents, and other additives conventionally used in the art. Solid carriers include, for example, magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, acacia, methylcellulose, sodium carboxymethylcellulose, low melting waxes, and cocoa butter.

Liquid formulations include sterile solutions, suspensions and emulsions. Such liquid formulations may be used for injection or in connection with ex vivo and in vivo fertilization. The liquid formulations may also contain other additives conventionally used in the art, some of which are mentioned in the above list. In addition, a formulation useful for transdermal administration of a compound of the present invention may be provided in the form of a patch, and a formulation useful for nasal administration may be provided in the form of a liquid or powder nasal spray.

The dosage of the compound of the present invention to be used will be determined by the attending physician and will depend, among other factors, on the compound being used, the route of administration, and the purpose of use. In general, the compositions of the present invention are prepared by very intimately mixing the active ingredient with liquid or solid auxiliary additives and, if necessary, bringing the product into the desired dosage form.

The present invention relates to the use of compounds of formula I in the preparation of meiosis-regulating reagents. The compounds of the present invention affect meiosis in oocytes and male germ cells. The ability to affect meiosis can have several advantages. According to a preferred embodiment of the present invention, the compound of formula I can be used to stimulate meiosis. According to another preferred embodiment of the present invention, the compound of formula I can be used to stimulate meiosis in humans. Thus, the compounds of formula I are promising new fertility-regulating agents, . ,·. ·Τ without exhibiting the usual side effects on somatic cells that are known for the hormonal contraceptives used to date, which are based on estrogens and/or gestagens.

Accordingly, the present invention relates to the use of compounds of formula I for the treatment of infertility in females and males, particularly in mammals, but especially in humans. The meiosis-inducing agents of the compound of formula I can be used to treat certain cases of infertility in females, including women, by administering these agents to women who are unable to produce mature oocytes due to their body not producing sufficient meiosis-activating agents.

The compounds of the general formula can also be used in artificial insemination procedures, such as in vitro fertilization or intracytoplasmic sperm injection. When in vitro fertilization is performed, better results are obtained if the compound of the present invention is added to the medium in which the oocytes are cultured.

If male infertility, including male infertility, is caused by insufficient production of meiosis-activating substance and therefore a lack of mature sperm cells, administration of the compound of the present invention may solve the problem.

It is also an object of the present invention that the compounds of the present invention are useful as contraceptive agents in males and females, especially mammals, but most notably in humans. For use as contraceptive agents in females, a meiosis inducing agent can be administered to prematurely induce the resumption of meiosis in oocytes while they are still in the growing follicles, before the ovulatory peak of gonadotropins occurs. In females, meiosis resumption can be induced one week after the previous menstruation has ceased. When ovulation occurs, the resulting over-mature oocytes are very likely not to be fertilized. This is unlikely to affect the normal menstrual cycle. In this context, it is worth noting that the biosynthesis of progesterone in human granulosa cells (somatic cells of the follicle) is not affected by the presence of the meiosis inducing agent, whereas estrogens, which have been used as hormonal contraceptives to date, and gestagens have a detrimental effect on the biosynthesis of progesterone.

In an alternative to the above method, contraception can be achieved in females by administering a compound of the present invention which inhibits meiosis and thereby produces mature oocytes. Similarly, contraception can be achieved in males by administering a compound of the present invention which inhibits meiosis and thus produces mature sperm cells.

In another aspect, the present invention provides the use of compounds of formula I as tool compounds or as starting materials in the synthesis of tool compounds in biological imaging methods, which methods are used to elucidate the mechanism of action of such compounds. For example, meiosis-activating sterols containing a fluorescent marker can be used to visualize the compartments of the germ cell in which the active substances exert their biological function. This information can be useful in elucidating the mechanism of action of such substances.

The present invention also provides a method for regulating meiosis, comprising administering to a subject in need of such regulation an effective amount of one or more of the compounds of formula I.

The present invention also provides a method for regulating meiosis in mammalian germ cells, which comprises administering to the germ cells in need of such regulation ex vivo or in vivo an effective amount of one or more of the compounds of formula I. The germ cell may be an oocyte or a male germ cell.

The route of administration of compositions containing a compound of the present invention may be any route of administration that effectively transports the active compound to its site of action.

Thus, when the compounds of the present invention are administered to a mammal, they can conveniently be provided in the form of a pharmaceutical composition comprising at least one of the compounds of the present invention together with a pharmaceutically acceptable carrier.

For oral administration, such compositions are preferably presented in capsule or tablet form.

From the above, it is clear that the amount of substance administered depends on the condition being treated. Thus, if it is used to treat infertility, the administration is sufficient if it is done only once, or only for a short period of time, i.e. until fertilization is achieved.

When used as a contraceptive agent, the compounds of the present invention can be administered either continuously or cyclically.

The present invention is illustrated by the following examples.

Example 1

4,4-dimethyl-5oc-cholesta-1,8,14-trien-3-one

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

A few grains of molecular sieves were added to a solution of 6.40 g of 4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol and 4.03 g of N-methylmorpholine-N-oxide in 32 ml of dichloromethane and the mixture was stirred for five minutes. 408 mg of tetrapropylammonium perruthenate was added at room temperature and the resulting black reaction mixture was stirred for one hour. The mixture was filtered through celite, the solvent was evaporated and the residue was chromatographed with hexane-ethyl acetate to give 5.60 g of 4,4-dimethyl-5a-cholesta-8,14-dien3-one as a white solid.

1H-NMR (CDCl 2 ): δ=0.83 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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).

b) 4,4-dimethyl-5a-cholesta-1,8,14-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 at room temperature was added 1.75 g of phenylselenic anhydride. The reaction mixture was heated at 100 °C for two hours. After cooling and evaporation of the solvent, the residue was chromatographed with hexane-ethyl acetate to give 0.85 g of 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one as an oil.

1 H-NMR (CDCl 2 ): δ = 0.84 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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-l)

Example 2

4,4-Dimethyl-5a-cholesta-1,8,14-trien-3β-ol mg sodium borohydride was added to 73 mg 4,4-dimethyl-5acholesta-1,8,14-trien-3-one and 67 mg cerium chloride heptahydrate in methanol at room temperature. The mixture was stirred at room temperature for 4 hours, poured into water, and extracted with diethyl ether. The organic phase was separated, washed with brine, dried over anhydrous sodium sulfate, and filtered. After evaporation of the solvent, the residue was chromatographed with hexane-diethyl ether to give 43 mg 4,4-dimethyl-5acholesta-1,8,14-trien-3β-ol as a white solid.

1H-NMR (CDCl 2 ): δ=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); 5.37 (s, 1H, H-15); 5.50 (dd, J=10 Hz, 1 Hz, H-l); 5.90 (d, J=10 Hz, 2 Hz, 1 Η, H-2).

Example 3

5a-cholesta-1,8,14-trien-3P-ol

a) 5a-cholest-8,14-trien-3-one

2.40 g of 5a-cholesta-8,14-dien-3p-ol were 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 1a. After chromatography, 1.81 g of 5a-cholesta-8,14-dien-3-one was obtained as a white solid.

1H-NMR (CDCl 2 ): δ=0.82 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.18 (s, 3H, H-19); 5.39 (s, 1H, H-15).

b) 5a-cholest-1,8,14-trien-3-one

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

Ή-NMR (CDCl ₃ ): δ=0.84 (s, 3H, Η-18); 0.86 (2x d, J=7 Hz, 6H, H26/27); 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, 1 H, H-2); 7.42 (d, J=10 Hz, 1H, H1)·

c) 5a-cholesta-1,8,14-trien-3β-ol

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

1H-NMR (CDCl ₃ ): δ=0.82 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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-15); 5.56 (dd, J=10Hz, 1Hz, 1H, H1); 6.09 (d, J=10 Hz, 2 Hz, 1H, H-2).

Example 4. la-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one

A solution of 1.47 diethylammonium cyanide (1 mol/l in toluene) was added to 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one (5 ml) in tetrahydrofuran at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for one hour. After addition of 2.45 ml of 1 mol/l sodium hydroxide solution at 0 °C, the mixture was diluted with water and extracted with diethyl ether. The organic phase was separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue was chromatographed with hexane-ethyl acetate to give 110 mg of 1-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one as a white solid.

1 H-NMR (CDCl ₃ ): δ=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); 1.20 (s, 3H); 2.86 (m, 1H, H-2); 3.29 (dd, J=7 Hz, 5 Hz, 1 H, H1); 5.49 (ps, 1H, H15).

Example 5. 1-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol and 1-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol mg of 1-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one was treated with 33 mg of sodium borohydride as described in Example 5 to give 30 mg of 1-cyano-4,4-dimethyl-5a-cholesta-8,14dien-3a-ol and 25 mg of 1-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol.

1α-Cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol 1R-NMR (CDCl 2 ): δ = 0.83 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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, 1H, H-15).

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

Ή-NMR (CDCl 3 ): δ=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, 1H, H-15).

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

Ή-NMR (CDCl 3 ): δ=0.83 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H26/27); 0.93 (d, J= 7Hz, 3H, H-21); 0.98 (s, 3H); 1.16 (s, 3H); 1.23 (s, 3H); 2.58 (d, J=17 Hz, 1H, H-1); 3.10 (d, J=17 Hz, 1H, H1); 5.47 (ps, 1H, H-15); 7.42 (s, 1H, H-2'); 7.53 (d, J=8 Hz, 2H, arom.); 7.68 (d, J=8 Hz, 2H, arom.)

Example 7 (E)-4-[(3-B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2ylidene)methyl]benzonitrile

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

1H-NMR (CDCl ₃ ): δ=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, H1); 3.94 (m, 1H, H-3); 5.40 (ps, 1H, H-15); 6.78 (s, 1H, H-2'); 7.37 (d, J=8 Hz, 2H, arom.); 7.64 (d, J=8 Hz, 2H, arom.).

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

a) A suspension of (E)-2-(4-aminomethylphenyl)methylidene-4,4-dimethyl-5acholesta-8,14-όΐέη-3β-ο1 mg lithium aluminum hydride and 74 mg (E)-4-[(3β-hydroxy4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)methyl]benzonitrile in 6 ml tetrahydrofuran was refluxed for 4 hours. After cooling, the reaction mixture was worked up as described in Example 3b. By crystallization from ethyl acetate, 26 mg (E)-2-(4-aminomethylphenyl)methylidene-4,4-di-

methyl-5a-cholesta-8,14-dien-3β-ol is obtained as a white solid.

Ή-NMR (CDCl 3 ): δ=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, H1); 3.86 (s, 2H, Ar-CH ₂ -N); 3.92 (ps, 1H, H-3); 5.38 (ps, 1H, H-15); 6.72 (s, 1H, H-2'); 7.26 (m, 4H, arom.).

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

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

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

1-NMR (CDCl 3 ): δ = 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, Ar-CH ₂ -N); 5.37 (ps, 1H, H-15); 5.76 (t, J=4 Hz, tH, NH); 6.72 (s, 1H, H-2'); 7.26 (m, 4H, arom.).

Example 9 (E)-N-[[4-[(33-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-sindacen-3-yl)pentanoic acid succinimidyl ester in 0.5 ml of dimethylformamide was added at room temperature to a solution of 8.9 mg of (E)-2-(4-aminomethylphenyl)methylidene-4,4-dimethyl-5a-cholesta-8,14dien-33-ol in 2 ml of dimethylformamide. The reaction mixture was stirred for 44 hours, diluted with water and extracted with ethyl acetate. The organic phase was separated, washed with water, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue was chromatographed with a hexane-ethyl acetate mixture to obtain 6.5 mg of (E)-N-[[4-[(33-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.

m-NMR (CDCl 2 ): δ=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-Me); 2.55 (s, 3H Ar-Me); 3.12 (d, J=17 Hz, 1H, H1); 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, arom.); 6.30 (d, J=5 Hz, IH, arom.); 6.70 (s, IH, H-2); 6.90 (d, J=5 Hz, IH, arom.); 7.24 (m, 4H, arom.).

Example 10

2a-allyl-4,4-dimethyl-5oc-cholesta-8,14-dien-30-ol a) 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-one

A solution of 200 mg of 4,4-dimethyl-5a-cholesta-8,14-diene-3β-one in 3 ml of tetrahydrofuran was added dropwise at 70 °C to 5.8 ml of freshly prepared 1 mol/l diisopropylamide. The solution was stirred for 1 hour before 0.07 ml of 3-iodopropene was added. The mixture was stirred for another hour at 0 °C, then poured into saturated ammonium chloride solution and extracted with ethyl acetate. The organic phase was separated, washed with brine, dried over anhydrous sodium sulfate and filtered. After evaporation of the solvent, the residue was chromatographed with hexane-ethyl acetate to give 160 mg of 2a-allyl-4,4-dimethyl-5α-cholesta-8,14-diene-3β-one as a white solid.

1H-NMR (CDCl ₃ ): δ=0.82 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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, allyl); 5.05 (m, 2H, allyl); 5.38 (ps, 1H, H-15); 5.80 (m, 1H, allyl).

b) 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3β-ol

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

2α-allyl-4,4-dimethyl-5cc-cholesta-8,14-dien-3β-ol 1H-NMR (CDCl ₃ ): δ=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, 1H, H-15); 5.89 (m, 1H, allyl).

2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol 1 H NMR (CDCl 3 ): δ = 0.82 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H26/27); 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, 1H, H-15); 5.85 (m, 1H, allyl).

Example 11

Testing for meiosis-activating substances in oocyte assay Animals

Oocytes were obtained from immature, 13-16 g female mice (C57BI/6J x DBA/2J FI hybrids, Bomholtgaard, Denmark) housed under controlled lighting and temperature. The mice were injected with 0.2 ml intraperitoneal gonadotropin (Gonal, F., Serono, Solna, Sweden, containing 20 IU FSH).

glaze, or alternatively Puregon, Organon, Swords, Ireland, which contains 20 IU FSH), and 48 hours later the animals are killed by cervical dislocation.

Oocyte collection and culture

The ovaries are excised and the oocytes are isolated under a stereomicroscope into Hx medium (see below), the follicles being ruptured with a pair of 27-gauge needles. Spherical, naked oocytes (NO), showing intact germinal vesicles (GV), were placed in a-minimal essential medium (oc-MEM, without ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mmol/L hypoxanthine (Sigma Cat. No. H-9377), 8 mg/mL human serum albumin (HAS, State Serum Institute, Denmark), 0.23 mmol/L pyruvate (Sigma, Cat. No. S-8636), 2 mmol/L glutamine (Flow Cat. No. 16-801), 100 IU/mL penicillin, and 100 pg/mL streptomycin (Flow Cat. No. 16-700). This medium is designated Hx medium.

Oocytes were rinsed three times with Hx medium and then cultured in four-well multiplates (Nunclon, Denmark), each well containing 0.4 ml of Hx medium and 35-45 oocytes. A control (i.e. 35-45 oocytes cultured in Hx medium and no test compound added) was always run in parallel with the test cultures, and different concentrations of test compound were added to the test cultures.

Cultivation is carried out at 37 °C, 100% humidity and 5% CO2. The cultivation period is 22-24 hours.

Examination of oocytes

Towards the end of the culture period, oocytes showing germinal vesicles (GV), germinal vesicle breakdown (GVB), or polar bodies (PB) are counted using a stereomicroscope or an inverted microscope equipped with a differential interference contrast device. The percentage of oocytes showing GVB relative to the total number of oocytes and the percentage of oocytes showing PB relative to the total number of oocytes in the test cultures are calculated and compared with the data from the control culture.

Example 12

Testing for meiosis inhibitors in the oocyte test

Germinal vesicle (GV) oocytes were obtained from immature, FSH-treated female mice as described in Example 11 (see above). The bare oocytes were rinsed three times with Hx medium. 4,4-dimethyl-5α-cholesta-8,14,24-ίηέη-3β-ο1Γ01 (FFMAS) has previously been shown to induce meiosis in NO in vitro [Byskov, A.G. et al.: Nature 374, 559-562 (1995)]. NO is cultured in Hx medium supplemented with 5 pmol/l FF-MAS, co-cultured with different concentrations of the test compounds, in four-well multiplates (Nunclon, Denmark), each containing 0.4 ml of Hx medium and 35-45 oocytes. A positive control (i.e. 35-45 oocytes, cultured in Hx medium containing FF-MAS, without the addition of the test compound) is always run in parallel with the test cultures, which is supplemented with different concentrations of the test compound. In addition, a negative control (35-45 oocytes, cultured in Hx medium only) is also run in parallel with the positive control.

Examination of oocytes

Towards the end of the culture period, oocytes showing germinal vesicles (GV), germinal vesicle breakdown (GVB), or polar bodies (PB) are counted using a stereomicroscope or an inverted microscope equipped with a differential interference contrast device. The percentage of oocytes showing GVB relative to the total number of oocytes and the percentage of oocytes showing PB relative to the total number of oocytes in the test cultures are calculated and compared with the data from the control culture.

Example 13

Testing for meiosis-activating substances in the “in vitro fertilization” assay

Naked oocytes (NkO) and cumulus-enclosed oocytes (CEO) are isolated from the follicles of immature (C57BL/6xDBA/2) (21-24 days old) FI mice that received 10 IU of Pregnant Mare Serum Gonadotropin (FSH activity) intraperitoneally 48 hours before collection.

Oocytes (NkO and CEO) are pooled and cultured for approximately 20 hours in modified a-MEM medium containing 3 mmol/l hypoxanthine and 1 mg fetuin per ml of culture. Two groups of oocytes are used: 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, oocytes showing germinal vesicle breakdown (GVB) are briefly washed in Hx-free medium and then transferred to pre-prepared fertilization plates containing a motile sperm preparation from the cauda epididymis of male mice. The plates are then cultured at 37°C under a defined gas composition (5% CO2) in modified a-MEM IVF medium. The oocytes are examined 20-22 minutes after fertilization to check for fertilization and to record the number of two-cell embryos. The percentage of fertilization (=fertilization rate) is defined as the number of oocytes that have split into two-cell embryos compared to the total number fertilized. Each IVF experiment is performed with a total of 50-200 GVB/PB oocytes. The stimulation factor is defined as the ratio of the fertilization rate in the test compound group to the control group.

Claims (14)

A compound of general formula I, in which R ¹ represents a hydrogen atom, an alkyl group having 2-6 carbon atoms, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR ¹ ' group (wherein COR ¹ ' represents an alkyl group having 1-8 carbon atoms, or an optionally substituted phenyl group), or together with R ² they form a new chemical bond, R ² represents a hydrogen atom, an alkyl group having 4-8 carbon atoms, an alkyl group having 3-6 carbon atoms, a hydroxyalkyl group having 1-6 carbon atoms, an optionally substituted benzylidene group together with R ² ', a hydroxymethylene group together with R ² ', or a new chemical bond together with R 2 , R ² represents a hydrogen atom, together with R ² an optionally substituted benzylidene group, or together with R ² a hydroxymethylene group, R ³ represents a hydrogen atom, or together with R ³ ', another chemical bond, R ³ ' represents a hydrogen atom, or together with R ³ another chemical bond, R ⁴ represents a hydrogen atom or a methyl group, R ⁴ ' represents a hydrogen atom or a methyl group, R ⁸ together with R ⁹ or R ¹⁴ represents a new chemical bond, R ⁹ represents a hydrogen atom or, together with R ⁸ , a new chemical bond, R ¹⁴ represents an α-hydrogen atom or together with R ⁸ a new chemical bond, or together with R ¹⁵ a new chemical bond, R ¹⁵ represents a hydrogen atom, or together with R ¹⁴ forms a new chemical bond R ²⁴ represents a hydrogen atom or together with R ²⁵ forms a new chemical bond R ²⁵ represents a hydrogen atom or together with R ²⁴ a new chemical bond or esters thereof, with the proviso that compounds which are in the 1- and 2-positions at the same time (unmodified R ¹⁼ R ² =R ² '=H) are excluded. 2. Compounds according to claim 1, wherein R ¹ is a hydrogen atom, a phenyl group, or together with R ² is a new chemical bond. 3. Compounds according to claim 1 or 2, wherein R ² is an alkyl group having 4-8 carbon atoms, an allyl group, or R 2 together with R 2 is a new chemical bond. 4. Compounds according to claim 1 or 2, wherein R2 and ^R2 ' are optionally substituted benzylidene groups. 5. A compound according to any one of claims 1-4, which may be: 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-33-ol, (E)-2-benzylidene-4,4-dimethyl-5a-cholesta-8,14-dien-3P-ol, 5a-cholesta-1,8,14-trien-3p-ol, 5a-cholesta-l,8,14-trien-33-one, lot-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, loc-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-one, 4,4-dimethyl-5a-cholesta-l,8,14-trien-3p-ol, 4,4-dimethyl-5a-cholesta-l,8,14-trien-3p-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-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)methyl]phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacen-3-yl)pentanamide, 2cc-hydroxymethyl-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-33-ol and (E)-4-[(3-oxo-4,4-dimethyl-5a-cholesta-8,14-dien-2ylidene)methyl]benzonitrile 6. Pharmaceutical compositions containing one or more of the compounds of general formula I according to any one of claims 1-5 as active ingredients. 7. Use of one or more of the compounds of general formula I according to any one of claims 1 to 5 as a medicament for regulating meiosis. 8. Use according to claim 7 for the preparation of a medicament for use in the treatment of infertility in females or males, preferably humans. 9. Use according to claim 7 for the preparation of a contraceptive medicament for use in the treatment of females or males, preferably humans. 10. Use of compounds of formula I according to any one of claims 1-5 for controlling the fertilization rate in artificial insemination procedures. 11. Use of compounds of general formula I according to any one of claims 1-5 as tools or as starting materials for the synthesis of compounds useful as tools or for biological imaging purposes, with the aim of elucidating the mechanism of action of such compounds. 12. A method for regulating meiosis, comprising administering to a subject in need of such regulation an effective amount of a compound of formula I according to any one of claims 1-5, ex vivo or in vivo. 13. A method for regulating meiosis in a mammalian germ cell, comprising administering to the germ cell in need of such regulation an effective amount of a compound of formula I according to any one of claims 1-5, ex vivo or in vivo. 14. The method of claim 13, wherein the germ cell is an oocyte or a male germ cell. Adam Jr. of Szentpéteri! patent attorney member of the SBG & K. Patent Attorneys Office r η,. , H-1062 Budapest, Andrássy út 113 ^r > Phone: 461-1000 Fax: 461-1099 THE