WO2000068245A1

WO2000068245A1 - 14β-H-STEROLS, PHARMACEUTICAL COMPOSITIONS COMPRISING THEM AND USE OF THESE DERIVATIVES FOR THE PREPARATION OF MEIOSIS REGULATING MEDICAMENTS

Info

Publication number: WO2000068245A1
Application number: PCT/EP2000/004092
Authority: WO
Inventors: Peter Esperling; Joachim Kuhnke; Henry Laurent; Thorsten Blume; Christa Hegele-Hartung; Monika Lessl
Original assignee: Schering Aktiengesellschaft
Priority date: 1999-05-10
Filing date: 2000-05-05
Publication date: 2000-11-16
Also published as: SK16212001A3; CZ20013981A3; HUP0201098A2; AU4756100A; BR0010449A; PL352029A1; JP2002544136A; CN1350543A; EP1177205A1; CA2373442A1; EE200100592A; IL145373A0; NO20015488D0; NO20015488L; BG106093A; ZA200110073B; KR20020013541A; EA200101157A1

Abstract

The present invention relates to pharmaceutically active 14β-H-sterols, to pharmaceutical compositions comprising them and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the 14β-H-sterols of the invention can be used for regulating meiosis.

Description

14β-H-sterols, pharmaceutical compositions comprising them and use of these derivatives for the preparation of meiosis regulating medicaments

The present invention relates to pharmaceutically active sterols, to pharmaceutical compositions comprising them as active substances and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the sterols 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 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 (1 n) 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 1c DNA.

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 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 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 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 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 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 from bull testes and from human follicular fluid of certain sterols (T-MAS and FF- MAS) that activate oocyte meiosis.

Compounds being known to regulate the meiosis are described in WO 96/27658, WO97/00884, WO98/28323 and WO98/52965.

It is purpose of the present invention to provide novel compounds useful as contraceptives in females and males, particularly in humans via inhibition of meiosis.

The present invention relates to 14β-H-sterols of the general formula

wherein

R³ designates a hydrogen atom or together with R^3' an additional bond, R^3' designates a hydrogen atom or together with R³ an additional bond, R⁴ designates a hydrogen atom or a methyl group, R^4' designates a hydrogen atom or a methyl group, R⁷ designates a hydrogen atom or together with R⁸ an additional bond,

R⁸ designates a hydrogen atom or together with R⁷ or together with R⁹ an additional bond, R⁹ designates a hydrogen atom or together with R⁸ or together with R¹ an additional bond, R¹⁵ designates a hydrogen atom, a hydroxy group, a halogen atom or together with R^15' an oxo group or together with R¹⁶ an additional bond or together with R^22' an oxygen bridge, R^15' designates a hydrogen atom, an

linear or branched alkyl group, a C₆-C₁₀ aryl group or together with R¹⁵ an oxo group, R¹⁶ designates a hydrogen atom, a hydroxy group, a halogen atom or together with R¹⁵ an additional bond or together with R²² an additional bond, R²² designates a hydrogen atom, a

linear or branched optionally substituted alkyl or alkenyl group, an optionally substituted C₆-C₁₀ aryl group or together with R ^S an additional bond or together with R^22' a

linear or branched alkylidene group. R²²' designates a hydrogen atom, a hydroxy group or together with R¹⁵ an oxygen bridge or together with R²² a

linear or branched alkylidene group.

or esters thereof.

As used in the present description and claims, an alkyl group - when used alone or in combinations - may be a straight or branched alkyl group. The expression C.-Cg alkyl designates an alkyl group having from one to eight carbon atoms: preferred examples are methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, tert-butyl, pentyl, iso-pentyl, hexyl and cyclohexyl. The term alkenyl group refers to an unsaturated alkyl group. Preferred examples are vinyl, allyl, isopropenyl and prenyl. The term halogen means fluoro, chloro, bromo or iodide. As used in the present description and claims, a statement that e.g. R¹⁵ together with R^15' is an oxo group means that oxo (=0) is present in the 15 position and, consequently, there is no hydrogen atom in the 15 position. The term C₆-C₁₀ aryl group designates a phenyl group, that is optionally substituted by halogen, (C C₄)alkoxy, hydroxy or (C₁-C₄)alkyl groups. The term C,-C₈ linear or branched alkylidene group stands for an alkyliden group having one to eight carbon atoms. This group is connected via a double bond to carbon 22 of the steroid. Preferred examples are methylene, ethylidene, propylidene, isopropylidene, butylidene, iso- butyliden, pentylidene, iso-pentylidene, neo-pentylidene and cyclohexylidene.

It was surprisingly found that the β-configuration of the hydrogen at position 14 (numbering of the C atoms is according to the IUPAC nomenclature) is important for the meiosis-inhibiting activity. In the presence of the 14β-H-sterols, the meiosis activating effect of FF-MAS is diminished or completely extinguished. Meiosis-regulating substances described so far are either 14α-H and Δ14- cholestane derivatives [Byskov, A. G. et al. Nature 374 (1995), 559-562] and show either a meiosis-activating activity or a low inhibitory activity. In contrast, the 14β-H-sterols of this invention strongly antagonize the effect of the naturally occuring FF-MAS and are thus inhibitors of meiosis. These findings make the described compounds especially interesting for contraception. Preferred compounds of formula I are those which inhibit the germinal vesicle breakdown by at least 20 %, preferably at least 40 % , especially preferred at least 60 % when tested in an oocyte test as described in example 13 and which do not activate meiosis in an oocyte test as described in example 12.

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 isome c forms and mixtures thereof are within the scope of the invention (unless otherwise noted). Preferred compounds of formula I are such with an 3β-OH group and aΔ8 double bond. In addition, compounds of formula I are preferred, wherein R¹⁵ designates a hydrogen atom or a hydroxy group. Other preferred compounds of formula I are such, wherein R²² designates a C.-C₈ linear or branched optionally substituted alkyl group or together with R^22' a

linear or branched alkylidene group.

In another embodiment, the present invention relates to esters of compounds of the general formula I. 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 example be selected from the group of acids comprising succinic acid, glutaric acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulphonic acid, sulphamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.

Especially preferred compounds of formula I of the present invention are the following:

4,4-dimethyl-5 J4β-cholest-8-en-3β-ol 4,4-dimethyl-5αJ4β-cholest-8-ene-3βJ5β-diol

4,4-dimethyl-5 J 4β-cholest-8-ene-3βJ 5α-diol

4,4-dimethyl-5α, 14β-cholesta-8J 5-dien-3β-ol

4,4-dimethyl-5αJ4β-cholesta-7,9(11 )J5Jrien-3β-ol

4,4-dimethyl-5α, 14β-cholesta-8J 5,23(E)-trien-3β-ol 4,4-dimethyl-5α, 14β-cholesta-8J 5,24Jrien-3β-ol

4,4-dimethyl-5 J 4β-cholesta-8,24-dieπe-3βJ 5β-diol

4,4-dimethyl-5αJ4β-ergosta-8,22-dien-3β-ol

4,4-dimethyl-5αJ 4β-ergosta-8,22-diene-3βJ 5β-diol

4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3βJ 5α-diol 4,4-dimethyl-5α, 14β-ergosta-8J 5,22-trien-3β-ol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-3β-ol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-ene-3βJ 5β-diol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-ene-3βJ 5α-diol

4,4-dimethyl-24-nor-5 J4β-cholesta-8J5-dien-3β-ol 4,4-dimethyl-5αJ4β-cholesta-8J 5-dien-3β-ol hydrogen butanedioate

4,4-dimethyl-5αJ4β-ergosta-8J5,22-trien-3β-ol hydrogen butanedioate

(20R)-4,4,20-trimethyl-16β,2Ncyclo-5α, 14β-pregn-8-ene-3βJ 5α-diol

(20R)-4,4,20-trimethyl-16β,21-cyclo-5αJ4β-pregn-8-ene-3βJ 5β-diol

(20S)-20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-ene-3β, 15^β-diol 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 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 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.

Particularly, the compounds of the present invention are synthesized according to the following general procedures:

The sterols that are used as starting materials can be synthesized according to literature procedures:

4,4-dimethyl-5α-cholesta-8J4-dien-3β-ol [Biochem. J. 132 (1973), 439], 4,4- dimethyl-5α-ergosta-8J4,22-trien-3β-ol [as benzoate: J. Org. Chem. 51 (1986), 4047], 5α-cholesta-8J4-dien-3β-ol [J. Am. Chem. Soc. 75 (1953), 4404], 5α- ergosta-8J4,22Jrien-3β-ol [J. Org. Chem. 53 (1988), 1563].

In the following only the compounds with 4,4-dimethyl group are described in detail. Compounds that are unsubstituted in position 4 are obtained by analogous routes. The 14β-H-dehvatives can be synthesized from Δ-8J4-diene-systems 1 via protection of the alcohol, epoxydation and subsequent opening of the epoxide (scheme 1). The 3-alcohol can be protected as a benzoate. Epoxidation can be achieved with reagents like dimethyldioxirane, hydrogenperoxide in the presence of different catalysts, m-CPBA and other peracids. The rearrangement to the deconjugated ketone 4 can be achieved by treatment with different acids or lewis acids like borontrifluond [see: Chem. Pharm. Bull. 38 (1990), 1796] scheme 1 :

The reactions can be carried out in the presence of different steroidal side chains (R_s) like cholesterol, ergosterol, sitosterol or stigmasterol side chain

If 15-ketones of general formula 4 are reduced with lithium aluminumhydπde 3βJ5β-dιols of general formula 5 are obtained. The corresponding 15α-alcohols can be obtained as minor diastereomers of general formula 6 from the sodium borohydπde reduction. Subsequent deprotection of the 3-OH-group gives the 3βJ 5α-diols of general formula 7 (scheme 2) scheme 2:

The Δ8J5-diene of general formula 10 can be obtained by the following sequence. Sodium borohydride reduction of ketone 4 gives 15β-alcohol 8 as the major diastereomer. Elimination to the Δ8J5-diene of general formula 9 can be carried out with Martin^'s sulfurane. Deprotection gives the desired alcohols of general formula 10 (scheme 3). scheme 3:

Derivatives which are saturated in position 15 can be obtained by the following route. 15β-Alcohols of the general formula e can be reacted with methanesulfonic acid chlorid. Mesylate 11 can then be reduced with lithium aluminiumhydride. Compounds of general formula 12 are obtained (scheme 4). scheme 4:

Side chain modifications can be performed via ozonolysis of the ergosterol side chain in compound of formula 4 (with R_stero, = ergosterol side chain). After reductive work up with sodium borohydride, the 22-alcohol 13 can be transformed to a leaving group like a tosylate (scheme 5). This tosylate 14 can be coupled with different branched or linear alkyl, alkenyl or aryl grignard reagents under copper catalysis to give compounds of general formula 15. scheme 5:

R sterol

R_stero, = ergosterol side chain

These compounds can be transfomed in the corresponding 3,15-diols (see scheme 2), Δ8J 5-dienes (scheme 3) or 15 saturated compounds (scheme 4) as described above.

Other side chain modification like the 24-keto-, 24-hydroxy- and Δ24-compounds can be obtained by the following route. Ozonolysis of compound 4 and subsequent work up with triphenylphosphin gives aldehyde 16. This can be coupled in an aldol reaction with isopropyl-methyl-ketone to give compound 17 as a mixture of diastereomers. Elimination with Martin^'s sulfurane, hydrogenation and subsequent reduction gives diol 20 as a mixture of diastereomers (scheme 6). scheme 6:

R_steroi = ergosterol side chain

Diols 20 can be eliminated to to the following sterols. Treatment with Martin^'s sulfurane gives a mono- and a bis-eliminated product. Subsequent cleavage of the benzoate can easily be achieved by reduction to give diol 23 and triene 24 respectively (scheme 7). scheme 7:

Compounds with an additional bond between C16 and C22 can be obtained by the following route. Tosylate 14 can be treated with a base like lithium diisopropylamide or different grignard compounds to deprotonate the ketone at position 16. The enolate alkylates the 22-tosytate intramolecularly to give pentacyclus 25. This can be treated with lithium aluminiumhydride to give diols26 and 27 (scheme 8). scheme 8

A further object of the present invention are pharmaceutical compositions comprising one or more compounds of the general formula I as active substances The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents aosorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally used in the art Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter

Liquid compositions include sterile solutions, suspensions and emulsions Such liquid compositions may be suitable for injection or for use in connection with ex vivo and in vitro fertilization The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are mentioned in the 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

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 and then, if necessary, shaping the product into the desired formulation

Usually, not more than 1000 mg, preferably not more than 100 mg, and in some preferred instances not more than 10 mg of a compound of formula I is to be administered to mammals, e g to humans, per day

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 The compounds of the general 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

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 regulating substance whereas the estrogens and gestagens used in the hitherto used hormonal contraceptives do have an adverse effect on the biosynthesis of progesterone

Contraception in females can 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 inhibits the meiosis, so that no mature sperm cells are produced 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 I.

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 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.

When used as a contraceptive, the compounds of the invention will either have to be administered continuously or cyclically.

In a further aspect, the present invention relates to the use of a 14β-hydrogen group in a sterol compound to increase the inhibitory activity of a meiosis inhibiting substance.

The present invention is further illustrated by the following examples:

Example 1 : 4,4-dimethyl-5α,14β-ergosta-8,22-diene-3β,15β-diol

a) 4,4-dimethyl-5α-ergosta-8,14,22-trien-3β-yl-benzoate

40 ml benzoylchloride were added in two portions to a solution of 30.9 g 4,4- dimethyl-5α-ergosta-8,22-dien-3β-ol in 154 ml of pyridine at 60°C. The reaction was stirred for 1 hour at the same temperature and then poured into ice-water. The precipitate was collected, washed with ice-cold ethanol, recrystallized from dichloromethane/methanol and dried for 12 h to give 32 g 4,4-dimethyl-5α- ergosta-8J4,22-trien-3β-yl-benzoate (mp 146 °C).

¹ H-NMR (CDCI_g): δ = 0,80 - 1 ,1 2 (8x CH₃); 4,75 (dd, j = 1 6 Hz, 4 Hz, 1 H, H-3α); 5,22 (m, 2H, H-22/23); 5,38 (s, broad, 1 H, H-1 5); 7,46 (t, 2H); 7,57 (t, 1 H); 8,07 (d, 2H)

b) 4,4-dimethyl-14J 5 -epoxy-5α-ergosta-8,22-dien-3β-yl-benzoate

A solution of 18 g 4,4-dimethyl-5α-ergosta-8J4,22-trien-3β-yl-benzoate in 60 ml dichloromethane was cooled to 10 °C. After addition of 3 g m-nitro- fluoroacetophenone and 10 ml saturated NaHCO₃-solution the reaction was stirred for 15 min. Then 12 ml of hydrogen peroxide (aqueous 30 %) were added and the solution stirred at 10 °C for 20 h. 20 mL of saturated sodiumthiosulfate- solution were added and the mixture was stirred for 20 minutes, diluted with dichloromethane, washed with sodium hydroxide solution (aqueous 5%), water and brine. Drying (MgSOJ and removal of solvents in vacuo furnished crude epoxide (20 g), which was further reacted without purification

c) 3^β-benzoyloxy-4,4-dimethyl-5αJ4β-ergosta-8,22-dιen-15-one

20 g of the crude epoxide were dissolved in 300 ml dioxane The yellow mechanically stirred solution was treated with 2 5 ml of boron tnflounde-etherate for 30 minutes and then poured into ice-water The precipitate was collected, washed with ice-cold water and dried The crude material was purified by chromatography to give 11 55 g 3β-benzoyloxy-4,4-dιmethyl-5αJ4β-ergosta- 8,22-dιen-15-one (mp 176 °C)

H-NMR (d5-pyrιdine): δ= 0,85 (2x d, J= 7 Hz, 6H, H-27/26); 0,91 (s, 3H, 4-β- CH₃); 0,93 (d, j= 7 Hz, 3H, H-28); 1 ,05 (s, 3H, H-18); 1 ,06 (s, 3H, 4-α-CH₃); 1 ,07 (s, 3H, H-19); 1 ,13 (d, j= 7 Hz, 3H, H-21 ), 4,89 (dd, j=12 Hz, 4 Hz, 1 H, H-3α); 5,37 (m, 2H, H-22/23), 7,49 (t, 2H), 7,57 (t, 1 H); 8,27 (d, 2H)

d) 4,4-dιmethyl-5α,14β-ergosta-8,22-dιene-3β,15β-dιol

50 mg of LiAIH₄ were added to a stirred solution of 300 mg 3β-benzoyloxy-4,4- dιmethyl-5αJ4β-ergosta-8,22-dιen-15-one in 30 ml diethylether and the mixture was stirred for 30 minutes at room temperature. Then 1 ml of saturated ammonium chloride solution was added After 10 minutes the solution was filtered and the solvent removed in vacuo The residue was separated by chromatography to give 100 mg 4,4-dιmethyl-5αJ4β-ergosta-8,22-dιene-3βJ5β- diol.

¹ H-NMR (CDCI3): δ = 0,78 - 1 ,2 (8 x CH₃); 3,24 (dd, J = 1 2 Hz, 4 Hz, 1 H, H-3α); 3,7 (dd, broad, J = 4 Hz, 4 Hz, 1 H, H-1 5α), 5,24 (m, 2H, H-22/23)

Example 2: 4,4-dimethyl-5α,14^β-ergosta-8,22-diene-3β,15α-diol

a) 3^β-benzoyloxy-4,4-dimethyl-5αJ4β-ergosta-8,22-dien-15α-ol

A solution of 1 g 3β-benzoyloxy-4,4-dimethyl-5α,14β-ergosta-8,22-dien-15-one in 75 ml tetrahydrofuran and 25 ml methanol was treated with 750 mg of sodium borohydride. After stirring at room temperature for 30 minutes, 1 ml of saturated aqueous ammonium chloride solution was added and the mixture was stirred for 15 minutes at room temperature. Filtration, removal of solvent in vacuo and purification by chromatography gave 60 mg of 3β-benzoyloxy-4,4-dimethyl- 5 J4β-ergosta-8,22-dien-15α-ol besides 770 mg of the corresponding 15β- alcohol.

¹ H-NMR (CDCI₃): δ = 0,8 - 1 , 1 2 (8 x CH₃); 4,07 (t, broad, J = 4 Hz, 1 H, H- 1 5β); 4,76 (dd, J = 1 2 Hz, 4 Hz, 1 H, H-3α); 5,23 (m, 2H, H-22/23); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

b) 4,4-dimethyl-5αJ4β-ergosta-8,22-diene-3βJ 5α-diol

A solution of 40 mg 3β-benzoyloxy-4,4-dimethyl-5αJ4β-ergosta-8,22-dien-15α-ol in 10 ml diethylether was treated with 10 mg lithium aluminium hydride for 30 min at room temperature as described in example 1d. After column chromatography 25 mg 4,4-dimethyl-5αJ4β-ergosta-8,22-diene-3βJ 5α-diol were isolated as a white solid (mp 148 °C). ¹ H-NMR (CDCI₃): δ = 0,78 - 1 ,7 (8 x CH₃); 3,25 (dd, J = 1 1 Hz, 5 Hz, 1 H, H- 3α); 4,04 (t, J = 4 Hz, 1 H, H-1 5α); 5,22 (m, 2H, H-22/23);

Example 3: 4,4-dimethyl-5αJ4β-cholesta-8J5-dien-3β-ol

a) 4,4-dimethyl-5α-cholesta-8J4-dien-3β-yl-benzoate

A solution of 1.0 g 4,4-dimethyl-5α-cholesta-8J4-dien-3β-ol in 5 ml pyridine was treated with 1.5 ml of benzoylchloride. After stirring for 3 hours at room temperature the solution was poured into water and the aqueous layer was extracted with ethyl acetate. The organic layers were combined and washed with 0.1 N aqueous HCI, saturated aqueous sodium bicarbonate solution and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give an oil that was purified by column chromatography to give 900 mg 4,4-dimethyl-5α-cholesta-8J4-dien-3β-yl-benzoate as a white solid (mp 158 C°).

¹ H-NMR (CDCI₃): δ = 0,83 (s, 3H, H-1 8); 0,87 + 0,88 (2x s, 3H, H-26/27); 0,95 (d, J = 6 Hz, 3H, H-21 ); 4,76 (dd, j = 1 1 Hz, 5 Hz, 1 H, H-3α); 5,38 (s, broad, 21 , H-1 5); 7,45 (t, 2H); 7,57 (t, 1 H); 8,06 (d, 2H)

b) 4,4-dimethyl-14J5α-epoxy-5α -cholest-8-en-3β-yl-benzoate

To a well stirred suspension of 4,4-dimethyl-5α-choiesta-8J4-dien-3β-yl- benzoate in 120 ml dichloromethane was added a solution of dimethyldioxirane (130 ml, 0.09 - 0.1 1 M in acetone) at 0 °C. After stirring for 30 minutes at 0°C, the reaction mixture was concentrated in vacuo to dryness. The resulting crude epoxide was used without purificatoπ.

c) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15-one

A solution of 1 g of 4,4-dimethyl-14J 5α-epoxy-5 -cholest-8-en-3β-yl-benzoate in 15 ml of dioxane was treated with 0J ml of boron triflouride-diethylether complex. The solution was stirred for 20 minutes at room temperature and then poured into saturated aqueous sodium bicarbonate solution. The aqueous layer was extraced with ethyl acetate, washed with water and brine, dried over sodium sulfate and concentrated under reduced pressure. Column chromatography gave 440 mg of 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15-one as a white solid (mp 153 °C).

¹ H-NMR (CDCI₃): δ = 0,83 - 1 , 1 (7x CH₃); 2,31 (s, broad, 1 H, H-14β); 4,76 (dd, J = 1 1 Hz, 5 Hz, 1 H, H-3α); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

d) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15β-ol

A solution of 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15-one (440 mg, 0.83 mmol) in dry tetrahydrofuran (15 ml) and methanol (1 ml) was treated at room temperature with sodium borohydride (125 mg). After 2 hours the solution was poured into 0J N HCI and extracted with ethyl acetate. The organic layer was washed water and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give a white foam, which was used without further purification.

e) 4,4-dimethyl-5α, 14β-cholesta-8, 15-dien-3β-yl-benzoate

A solution of 500 mg 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15β-ol in 10 ml dichloromethane was cooled to 0 °C and 1.1 g Martin^'s sulfurane were added in one portion. The reaction was stirred for 1 hour at room temperature. The solvents were removed in vacuo. Purification of the residue by chromatography furnished 315 mg 4,4-dimethyl-5αJ4β-cholesta-8J 5-dien-3β-yl- benzoate as a waxy solid, which was used directly.

f) 4,4-dimethyl-5αJ4β-cholesta-8J5-dien-3β-ol

A solution of 315 mg 4,4-dimethyl-5αJ4β-cholesta-8J5-dien-3β-yl-benzoate in dry diethylether was treated with 35 mg of lithium aluminium hydride as described in example 1d. Column chromatography of the crude product and subsequent recrystallisation gave 135 mg 4,4-dimethyl-5αJ4β-cholesta-8J 5-dien-3β-ol as white needles (mp 138 °C).

¹ H-NMR (CDCI₃): δ = 0,81 (s, 3H, H-18); 0,83 - 1 ,02 (6x CH₃); 2,49 (s, broad, 1 H, H-14β); 3,24 (m, 1 H, H-3α); 5,64 + 5,79 (2x m, 1 H, H-1 5/1 6)

Example 4: 4,4-dimethyl-24-nor-5α,14β-cholesta-8,15-dien-3β-ol a) (20S)-3β-benzoyloxy-20-hydroxymethyl-4,4-dιmethyl-5α, 14β-pregn-8-en-15- one and (20S)-3β-benzoyloxy-20-hydroxymethyl-4,4-dιmethyl-5α, 14β-pregn-8-

A solution of 2 18 g 3β-benzoyloxy-4,4-dιmethyl-5αJ4β-ergosta-8,22-dιen-15- one (example 1c) in 88 ml dichloromethane, 30 ml methanol and 1 ml pyndine was cooled to -70 °C A ozone/oxygene-mixture (1 4) was passed through the solution for 12 mm (controlled by TLC chromatography)

Then 130 mg sodium borohydride were added and the stirred reaction mixture was allowed to warm to 0°C over a 2 hour period and poured into saturated aqueous ammonium chloride solution The solution was extracted with ethyl acetate and the combined extracts were further washed with water and brine and then dried over sodium sulfate The solution was filtered and evaporated in vacuo The crude product was purified by chromatography to yield 1 28 g (20S)- 3β-benzoyloxy-20-hydroxymethyl-4,4-dιmethyl-5α, 14β-pregn-8-en-15-one (mp 238 8 °C) and 330 mg of the the corresponding 15β-hydroxy-compound

¹ H-NMR (CDCI₃) δ= 0,9 - 1 ,1 (4x CH₃), 1 ,16 (d, J= 6 Hz, 3H, H-21 ), 2,33 (s, broad, 1 H, H-14β), 3,42 (m, 1 H, H-22a), 3,63 (m, 1 H, H-22b), 4,75 (dd, j= 1 1 Hz, 4 Hz, 1 H, H-3α), 7,45 (t, 2H), 7,57 (t, 1 H), 8,05 (d, 2H)

b) (20S)-3β-benzoyloxy-4,4,20-tπmethyl-21-toluolsulfonyloxy-5αJ4β-pregn-8-en- 15-one

A solution of 385 mg (20S)-3β-benzoyloxy-20-hydroxymethyl-4,4-dιmethyl- 5αJ4β-pregn-8-en-15-one in 5 ml pyndine was cooled to 0°C After addition of 190 mg p-toluenesulfonic chloride the reaction mixture was stirred over a period of 20 hours at 6 °C and then poured into brine and stirred for 20 mm The precipitate was collected, washed with ice-cold water and dried at 50 °C in vacuo The crude product (500 mg) was used without further purification c) 3^β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-15-one

3.42 g 2-methyl propylbromide were slowly added to stirred suspension of magnesium (powder 1.6 g) in 25 ml anhydrous tetrahydrofuran over a period of 30 min at 50 °C to give a clear solution of Grignard reagent. After then the mixture was cooled to -10 °C and 3.32 ml of a solution of Li₂CuCI₄ (prepared by mixing 99 mg cupric chloride, 86 mg lithium chloride and 10 ml tetrahydrofuran) were added dropwise.

The reaction mixture was stirred for 1 hour at -30 °C, whereupon a solution of 0.5 g (20S)-3β-benzoyloxy-4,4,20-trimethyl-21 -toiuolsulfonyloxy-5α, 14β-pregn-8-en- 15-one in 10 ml tetrahydrofuran was added at 0 °C. After 2 hours at 0 °C the mixture was stirred at room temperature overnight. Ethyl acetate and saturated ammonium chloride solution were added and the solution was stirred for 15 minutes. The organic extracts were washed with 1N HCI, water and brine, dried over sodium sulfate and evaporated to dryness. The crude product purified by column chromatography to give 250 mg 3β-benzoyloxy-4,4-dimethyl-24-nor- 5 J4β-cholest-8-en-15-one as a white foam.

¹ H-NMR (CDCI₃): δ = 0,82 - 1 ,09 (7x CH₃); 2,31 (s, broad, 1 H, H-14β); 4,75 (dd, j = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

d) 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ 4β-cholest-8-en-15β-ol

130 mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-3-one were treated with 100 mg sodium borohydride as described in example 2a. Aqueous work-up yielded 130 mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4^β-cholest-8- en-15β-ol as a white foam, which was used directly.

e) 4,4-dimethyl-24-nor-5αJ4β-cholesta-8J 5-dien-3β-yl-benzoate 400 mg of Martin^'s sulfurane were added in one portion to a solution of 100 mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-15β-ol in 10 ml dichloromethane. The mixture is stirred at room temperature for 18 hours. Evaporation under reduced pressure and chromatography gave 100 mg 4,4- dimethyl-24-nor-5α14β-cholesta-8J5-dien-3β-yl-benzoate as an oil, which was used without further purificatication.

f) 4,4-dimethyl-24-nor-5αJ4β-cholesta-8J 5-dien-3β-ol 100 mg 4,4-dimethyl-24-nor-5αJ4β-cholesta-8J5-dien-3β-yl benzoate were treated with 20 mg lithium aluminium hydride as described in example 1d. Column chromatography of the crude product and recrystallization (hexane/ethyl acetate) gave 36 mg 4,4-dimethyl-24-nor-5αJ4β-cholesta-8J5-dien-3β-ol (mp 104.3 °C).

¹ H-NMR (CDCI₃): δ = 0,82 (s, 3H, H-1 8); 0,83 - 1 ,03 (6x CH₃); 2,49 (s, broad, 1 H, H-14β); 3,24 (m, 1 H, H-3α); 5,65 + 5,8 (m, 1 H, H-1 5/1 6)

Example 5: 4₎4-dimethyl-24-nor-5α,14β-cholest-8-en-3β-ol

a) 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-ene-3β-yl benzoate

40 mg of 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-15β-ol in 3 ml pyridine were treated with 0.2 ml of methanesulfonic chloride at 0 °C. After stirring at room temperature for 2 hours the reaction mixture was diluted with water and extracted with ethyl acetate. The extracts were combined and washed with 1 N HCI, water and brine, dried over sodium sulfate and filtered. Removal of the solvents gave 40 mg 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5 J4β- cholest-8-ene-3β-yl benzoate that was used directly. b) 4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β-ol

44 mg 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5αJ4^β-cholest-8-ene-3β-yl benzoate were treated with 10 mg lithium aluminium hydride as described in example 1d. The crude product was purified by chromatography to provide 32 mg 4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-3β-ol.

¹ H-NMR (CDCI₃): δ = 0,81 (s, 3H, H-18); 0,82 - 1 ,03 (6x CH₃); 3,25 (dd, j = 1 1 Hz, 4 Hz, 1 H, H-3α)

Example 6 : 4,4-dimethyl-24-nor-5α,14β-cholest-8-ene-3β,15β-diol

30 mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-15β-ol were treated with 10 mg lithium aluminium hydride as described in example 1d. The crude product was purified by column chromatography to give 23 mg 4,4- dimethyl-24-nor-5αJ4β-cholest-8-ene-3β,15β-diol as a white foam.

¹ H-NMR (CDCI₃): δ = 0,82 (s, 3H, H-18); 0,84 - 1 ,04 (6x CH₃); 3,25 (d, broad,J = 1 2 Hz, 1 H, H-3α); 3,7 (q, broad, j = 7 Hz, 1 H, HJ 5α)

Example 7+8: (20R)-4,4,20-trimethyl-16β,21-cyclo-5α,14β-pregn-8-ene- 3βJ5β-diol and (20R)-4,4,20-trimethyl-16β,21-cyclo-5α,14β-pregn-8-ene- 3β,15α-diol

a) (20R)-3β-benzoyloxy-4,4,20-trimethyl-16β,21-cyclo-5α, 14β-pregn-8-en-15- one

1 J0 g (20S)-3β-benzoyloxy-4,4,20-trimethyl-21-toluolsulfonyloxy-5αJ4β-pregn-

8-en-15-one were treated with 52 mmol phenylmagnesium bromide analoguously to example 4c. After chromatography 560 mg (20R)-3β-benzoyloxy-4,4,20- tπmethyl-16β,21-cyclo-5αJ4β-pregn-8-en-15-one were isolated beside other products, that result from tosylate substitution by the phenyl gπgnard reagent

b) (20R)-4,4,20-tπmethyl-16β,2Ncyclo-5αJ4β-pregn-8-ene-3βJ 5β-dιol and (20R)-4,4,20Jrιmethyl-16β,21 -cyclo-5α, 14β-pregn-8-ene-3βJ 5α-dιol

460 mg (20R)-3β-benzoyloxy-4,4,20-trιmethyl-16β,21-cyclo-5αJ4β-pregn-8-en- 15-one were treated with 50 mg lithium aluminium hydride as described in example 1 d

The crude product was purified by column chromatography to give 40 mg (20R)- 4,4,20Jrιmethyl-16β,21 -cyclo-5αJ4β-pregn-8-ene-3βJ5β-dιol and 30 mg (20R)- 4,4,20-tnmethyl-16β,21-cyclo-5αJ4β-pregn-8-ene-3βJ 5α-dιol as white solids

(20R)-4,4,20-trιmethyl-16β,21-cydo-5αJ4β-pregn-8-ene-3βJ 5β-dιol ¹ H-NMR (CDCI₃): δ = 0,73 (s, 3H, H-18); 0,82 (s, 3H, H-1 9), 1 ,02 (s, 6H, 4- CH3); 1 ,09 (d, J = 8 Hz, 3H, H-21 ); 2,72 (m, 1 H, H-1 6α); 3,25 (m, 1 H, H- 3α); 4,07 (q, j = 8Hz, 1 H, H-1 5α)

(20R)-4,4,20-trιmethyl-16β,21 -cyclo-5α, 14β-pregn-8-ene-3β, 15α-dιol ¹ H-NMR (CDCI3): δ = 0,79 (s, 3H, H-1 8), 0,85 (s, 3H, H-1 9); 1 ,0 - 1 ,08 (3 * CH3), 2,34 (d, J = 6 Hz, 1 H, H-14β), 2,68 (m, 1 H, H-1 6α), 3,25 (m, 1 H, H-3α); 4,07 (d, J = 6 Hz, 1 H, H-1 5β)

Example 9: 4,4-dimethyl-5α,14β-cholesta-8,24-diene-3β,15β-diol

a) (20S)-3β-benzoyloxy-4,4-dιmethyl-15-oxo-5αJ 5β-pregn-8-ene-20- carbaldehyde

A solution of 2.2 g 3β-benzoyloxy-4,4-dιmethyl-5αJ4β-ergosta-8,22-dιen-15-one in 87 ml dichloromethane and 53 ml methanol was cooled to -78 °C A mixture of ozone/oxygen (1 :4) was passed into the solution for 16 min. 2.62 g triphenylphosphine were added and the solution was brought to room temperature. The solvents were removed in vacuo and the residue was purified by chromatography to give 1.26 g (20S)-3β-benzoyloxy-4,4-dimethyi-15-oxo- 5αJ 5β-pregn-8-ene-20-carbaldehyde (mp. 205 °C).

¹ H-NMR (d5-pyridine): δ = 0,90 (s, 3H, H-1 9); 0,98 (s, 3H, H-1 8); 1 ,04 (s, 6H, 4-CH₃); 1 ,2 (d, J = 6 Hz, 3H, H-21 ); 2,41 (s, broad, 1 H, H-14β); 4,83 (dd, J = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,5 (t, 2H); 7,58 (t, 1 H); 8,27 (d, 2H); 9,82 (d, J = 2 Hz, 1 H, H-22)

b) 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-dione

25.8 ml of a n-butyllithium solution (1.6 M, hexane) were added to 6 ml diisopropylamine in 48 ml tetrahydrofuran at -78 °C. The mixture is stirred for 20 minutes. A solution of 4.5 ml 3-methyl-2-butanone in 10 ml tetrahydrofuran was added and the mixture was stirred at -78 °C for 15 min. The resulting kinetic enolate was transferred via a cannula under nitrogen into a cooled (-78 °C) solution of 6.0 g (20S)-3β-benzoyloxy-4,4-dimethyl-15-oxo-5 J4β-pregn-8-en- 20-carbaldehyde in 40 m tetrahydrofuran. The mixture was brought to 0 °C over a one hour period and then poured into saturated aqueous ammonium chloride solution. The mixture was extracted with ethyl acetate, and the combined organic extracts were further washed with water and brine and then dried over sodium sulfate. Removal of solvents in vacuo gave a residue which was separated by column chromatography to give 6.26 g 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-

5αJ4β-cholest-8-ene-15,24-dione as an oil.

¹ H-NMR (CDCI3): δ = 0,92 - 1 ,2 (7 x CH3); 2,33 (s, broad, 1 H, H-14β); 4, 1 (m, 1 H, H-22); 4,74 (dd, j = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

c) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholesta-8,22-diene-15,24-dione A solution of 4.78 g 3^β-benzoyloxy-22-hydroxy-4,4-dimethyl-5αJ4β-cholest-8- ene-15,24-dione in 60 ml dichloromethane was cooled to 0 °C and 9.31 g Martin^'s sulfurane were added in one portion. The reaction mixture was stirred for 30 minutes at 0 °C. The solvents were removed in vacuo. Purification of the residue by column chromatography gave 4.18 g 3β-benzoyloxy-4,4-dimethyl- 5αJ4β-cholesta-8,22-diene-15,24-dione (mp 163 °C).

¹ H-NMR (CDCI₃): δ = 0,9 - 1 ,2 (7 x CH3); 2,31 (s, broad, 1 H, H-14β); 4,75 (dd, J = 1 1 Hz, 4 Hz, 1 H, H-3α); 6J 4 (d, J = 1 6 Hz, 1 H, H-23); 6,71 (m, 1 H, H-22); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

d) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-dione

500 mg palladium on charcoal (10%) were added to a solution of 4.2 g 3β- benzoyloxy-4,4-dimethyl-5 J4β-cholesta-8,22-diene-15,24-dione in 60 ml ethyl acetate. The mixture was stirred under a hydrogen atmosphere for 3 hours. The mixture was filtered. Removal of the solvent in vacuo gave 4.22 g 3β-benzoyloxy- 4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-dione as a white crystalline residue (mp 129 °C).

¹ H-NMR (CDCI3): δ = 0,92 - 1 , 1 6 (7 x CH3); 2,33 (s, broad, 1 H, H-14β); 4,74 (dd, J = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

e) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-diol and 3β-benzoyloxy-24-hydroxy-4,4-dimethyl-5αJ4β-cholest-8-en-15-one

A solution of 4.2 g 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-dione in 45 ml CH₂CI₂ was treated with 1.8 g tert-butylamine-borane complex. The solution was refluxed for one hour. After cooling to 0 °C, 30 ml HCI (1 N, aqueous) were added. The mixture was stirred for one hour at 0 °C. It was washed with HCI (1 N, aqueous), water, saturated aqueous NaHCO₃ solution and brine. After drying over sodium sulfate, the solvents were removed in vacuo. Column chromatography gave 1.71 g 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-ene- 15,24-diol and 1.82 g 3β-benzoyloxy-24-hydroxy-4,4-dimethyl-5αJ4β-cholest-8- en-15-one as white foams.

3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-ene-15,24-diol: ¹ H-NMR (CDCI₃): δ = 0,88 - 1 ,1 (7 x CH3); 3,33 (m, 1 H, H-24); 3,74 (m, 1 H, H-1 5); 4,75 (dd, J = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

3β-benzoyloxy-24-hydroxy-4,4-dimethyl-5αJ4β-cholest-8-en-15-one: ¹ H-NMR (CDCI3): δ = 0,88 - 1 , 1 (7 x CH3); 2,32 (s, broad, 1 H, H-14β); 3,33 (m, 1 H, H-24); 4,74 (dd, j = 1 1 Hz, 4 Hz, 1 H, H-3α); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H)

f) 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholesta-8,24-dien-15β-ol and 4,4-dimethyl-5αJ 4β-cholesta-8J 5,24-trien-3β-yl-benzoate

1.0 g 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholest-8-en-15,24-diol was treated with 3.88 g of Martin^'s sulfurane as described in example 3e. Purification by column chromatography gave 305 mg 3β-benzoyloxy-4,4-dimethyl-5αJ4β- cholesta-8,24-dien-15β-ol as a white foam and 870 mg 4,4-dimethyl-5αJ4β- cholesta-8J5,24-trien-3β-yl-benzoate as a white solid.

3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholesta-8,24-dien-15β-ol:

¹ H-NMR (d5-pyridine): δ = 1 J 5 (s, 1 H, H-1 8); 2,84 (m, 1 H, H-7α); 4, 1 (m, 1 H, H-1 5); 4,93 (dd, j = 1 1 Hz, 4 Hz, 1 H, H-3α); 5,24 (t, J = 8 Hz, 1 H, H- 24); 7,45 (t, 2H); 7,57 (t, 1 H); 8,25 (d, 2H)

4,4-dimethyl-5αJ4β-cholesta-8J 5,24Jrien-3β-yl-benzoate ¹H-NMR (CDCI3): δ= 1,4 (s, 3H, H-19); 2J9 (m, 1H, H-17α); 2,51 (s, broad, 1H, H-14β); 4,75 (dd, j = 11 Hz, 4 Hz, 1H, H-3α); 5J (t, J = 8 Hz, 1H, H-24); 5,65 (m, 1H, H-16); 5,79 (m, 1H, H-15); 7,45 (t, 2H); 7,57 (t, 1H); 8,05 (d, 2H)

g) 4,4-dimethyl-5αJ4β-cholesta-8,24-diene-3βJ5β-diol

120 mg 3β-benzoyloxy-4,4-dimethyl-5αJ4β-cholesta-8,24-dien-15β-ol were treated with 30 mg lithium aluminium hydride as described in example 1d. The crude product was purified by flash column chromatography to give 80 mg 4,4- dimethyl-5α,14β-cholesta-8,24-diene-3βJ5β-diol as a white foam.

¹H-NMR (CDCI3): δ= 0,8 - 1,03 (5x CH₃); 1,6 + 1,68 (2x s, 3H, H-26/27); 3,24 (m, 1H, H-3α); 3,70 (m, 1H, H-15α); 5J0 (t, J- 7 Hz, 1H, H-24)

Example 10: 4,4-dimethyl-5α,14β-cholesta-8,15,24-trien-3β-ol

30 mg 4,4-dimethyl-5αJ4β-cholesta-8J5,24-trien-3β-yl-benzoate (example 9f) were treated with 20 mg lithium aluminium hydride as described in example 1d. The crude product was purified by column chromatography to give 20 mg 4,4- dimethyl-5αJ4β-cholesta-8J5,24-trien-3β-ol as a white solid (mp 105 °C).

¹H-NMR (CDCI3): δ= 0,81 (s, 3H, H-18); 0,94 - 1,02 (4x CH3); 2,5 (s, broad, 1H, H-14β); 3,23 (m, 1H, H-3α); 5,11 (t, J = 9 Hz, 1H, H-24); 5,64 (m, 1H, H-15/16a); 5,79 (m, 1H, H-15/16b)

Example 11: (20S)-20-hydroxymethyl-4,4-dimethyl-5α,14β-pregn-8-ene-

60 mg (20S)-20-hydroxymethyl-4,4-dimethyl-5αJ4β-pregn-8-ene-3βJ5β-diol were treated with 20 mg lithium aluminium hydride as described in example 1d. The crude product was purified by column chromatography to give 42 mg (20S)- 20-hydroxymethyl-4,4-dimethyl-5αJ4^β-pregn-8-ene-3βJ5β-diol (mp 214 °C).

¹ H-NMR (CDCI₃): δ = 0,82 (s, 3H, H-1 8); 0,93 - 1 ,04 (3x CH₃); 1 ,07 (d, j = 6 Hz, 3H, H-21 ); 3,23 (m, 1 H, H-3α); 3,49 (m, 1 H, H-22a); 3,68 (m, 1 H, H- 22b); 3,72 (m, 1 H, H-1 5α)

Example 12: Testing of meiosis-activating substances in the oocyte test

Animals

Oocytes were obtained from immature female mice (C57BI/6J x DBA/2J F1- 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 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 (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 α-minimum essential medium (α-MEM without ribonucleosides, Gibco BRL, Cat.No. 22561 ) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg/ml 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 lU/ml penicillin and 100 μg/ml 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 the test cultures, which were made with different concentrations of the compounds to be tested.

The cultures were performed at 37 °C and 100 % humidity with 5 % CO₂ in air. The culture time was 22 - 24 hours.

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 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 13: Test of meiosis-inhibiting substances in the oocyte test

Germinal vesicle (GV) oocytes were obtained from immature FSH treated female mice using the same methods as described in Example 1 (see above). Naked oocytes (NO) were rinsed three times in Hx-medium. 4,4-Dimethylcholest-B, 14,24-trien-3β-ol (FF-MAS) has previously been shown to induce meiosis in NO invitro (Byskov, A.G. et al. Nature 374 (1995) 559 - 562). NO were cultured in Hx-medium supplemented with 5 μM 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 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 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 (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 + PB per total number of oocytes were calculated in the test cultures and in the control (positive and negative) culture groups. The relative inhibition of the test compound was calculated by the following formula:

inhibition test compound (%) = 100% - [(GVB+PB (test compound) - GVB+PB (negative control)) x 100 / GVB+PB(positive control) - GVB+PB (negative control)] %

Oocytes arrested in meiosis are characterised by an intact nucleus with a prominent nucleolus, known as germinal vesicle (GV). Upon reinitiation of meiosis the nucleolus and the nuclear envelope disappear and this is characterised by a breakdown of the GV, which than is called germinal vesicle breakdown (GVB). Some hours later the oocyte complete a reductional division and elicit the first so called polar body (PB).

Results:

Table 1 : Relative inhibition [%] of meiosis in naked mouse oocytes

Hx = Hypoxanthine

GV = germinal vesicle

GVB = germinal vesicle breakdown

PB = polar bodies n = number of

Claims

1. Compounds of the general formula I

wherein

R³ designates a hydrogen atom or together with R^3' an additional bond, R^3' designates a hydrogen atom or together with R³ an additional bond, R⁴ designates a hydrogen atom or a methyl group, R^4' designates a hydrogen atom or a methyl group,

R⁷ designates a hydrogen atom or together with R⁸ an additional bond,

R⁸ designates a hydrogen atom or together with R⁷ or together with R⁹ an additional bond, R⁹ designates a hydrogen atom or together with R⁸ or together with R¹¹ an additional bond,

R¹⁵ designates a hydrogen atom, a hydroxy group, a halogen atom or together with R^15' an oxo group or together with R¹⁶ an additional bond or together with R^22' an oxygen bridge, R^15' designates a hydrogen atom, an C_rC₈ linear or branched alkyl group, a C₆-C₁₀ aryl group or together with R¹⁵ an oxo group,

R¹⁶ designates a hydrogen atom, a hydroxy group, a halogen atom or together with R¹⁵ an additional bond or together with R²² an additional bond, R²² designates a hydrogen atom, a linear or branched optionally substituted alkyl or alkenyl group, an optionally substituted C₆-C₁₀ aryl group or together with R¹⁶ an additional bond or together with R^22' a C C₈ linear or branched alkylidene group.

R²²' designates a hydrogen atom, a hydroxy group or together with R¹⁵ an oxygen bridge or together with R²² a

linear or branched alkylidene group.

or esters thereof.

2. Compounds according to claim 1 , wherein R³ and R^3' designate hydrogen atoms.

3. Compounds according to claim 1 or 2, wherein R⁸ and R⁹ designate together an additional bond.

4. Compounds according to any one of the claims 1 to 3, wherein R¹⁵ designates a hydrogen atom or a hydroxy group.

5. Compounds according to any one of the claims 1 to 4, wherein R²² designates a C C₈ linear or branched optionally substituted alkyl group or together with R^22' a C^C-e linear or branched alkylidene group.

6. Compounds to any one of the claims 1 to 5 which are 4,4-dimethyl-5αJ4β-cholest-8-en-3β-ol 4,4-dimethyl-5αJ4β-cholest-8-ene-3βJ5β-diol 4,4-dimethyl-5αJ 4β-cholest-8-ene-3βJ 5α-diol 4,4-dimethyl-5αJ4β-cholesta-8J5-dien-3β-ol

4₎4-dimethyl-5αJ4β-cholesta-7,9(11)J5Jrien-3β-ol 4,4-dimethyl-5αJ 4β-cholesta-8J 5,23(E)-trien-3β-ol 4,4-dimethyl-5αJ4β-cholesta-8,15,24-trien-3β-ol 4,4-dimethyl-5αJ4β-cholesta-8,24-diene-3βJ5β-diol

4,4-dimethyl-5αJ4β-ergosta-8,22-dien-3β-ol

4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3βJ 5β-diol

4,4-dimethyl-5αJ4β-ergosta-8,22-diene-3βJ5α-diol 4,4-dimethyl-5αJ4β-ergosta-8J5,22-trien-3β-ol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-en-3β-ol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-ene-3βJ5β-diol

4,4-dimethyl-24-nor-5αJ4β-cholest-8-ene-3βJ5α-diol

4,4-dimethyl-24-nor-5αJ4β-cholesta-8J5-dien-3β-ol 4,4-dimethyl-5αJ4β-cholesta-8J5-dien-3β-ol hydrogen butanedioate

4,4-dimethyl-5αJ4β-ergosta-8J5,22-trien-3β-ol hydrogen butanedioate

(20R)-4,4,20-trimethyl-16β,21-cyclo-5αJ4β-pregn-8-ene-3βJ 5α-diol

(20R)-4,4,20-trimethyl-16β,21 -cyclo-5α, 14β-pregn-8-ene-3βJ 5β-diol

(20S)-20-hydroxymethyl-4,4-dimethyl-5αJ4β-pregn-8-ene-3βJ 5β-diol

7. Pharmaceutical compositions comprising one or more compounds of the general formula I according to any one of the claims 1 to 6 together with a pharmaceutically acceptable excipient.

8. Use of the compounds of the general formula I according to any one of the claims 1 to 6 for the preparation of a meiosis regulating medicament.

9. Use according to claim 8 for the preparation of a contraceptive medicament for the treatment of females or males, preferably humans.

10. 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 6.

11. Use of a 14β-hydrogen group in a sterol to increase the inhibitory activity of a meiosis inhibiting substance.