AU4756100A - 14beta-h-sterols, pharmaceutical compositions comprising them and use of these derivatives for the preparation of meiosis regulating medicaments - Google Patents

Description

WO 00/68245 PCT/EPOO/04092 1 14/-H-sterols, pharmaceutical compositions comprising them and use of these derivatives for the preparation of meiosis regulating medicaments 5 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 10 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 15 division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA synthesis. This division therefore results in the formation of the haploid germ cells with only 1c DNA. 20 The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the 25 female has a stock of oocytes which is drawn upon until the stock is exhausted. Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without 30 significant delay and produces 4 spermatozoa.

WO 00/68245 PCT/EPOO/04092 2 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: EppIg. J. J. et al Dev Biol 119 5 (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 10 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)]. 15 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, 20 WO97/00884, W098/28323 and W098/52965. 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. 25 The present invention relates to 14p-H-sterols of the general formula I: WO 00/68245 PCT/EPOO/04092 3 R22'

R

11 " H R22

R

3

R

8

R

1 5

R

3 HRR

R

4

R

4 ' wherein 5

R

3 designates a hydrogen atom or together with R 3 an additional bond,

R

3 designates a hydrogen atom or together with R 3 an additional bond,

R

4 designates a hydrogen atom or a methyl group,

R

4 ' designates a hydrogen atom or a methyl group, 10 R 7 designates a hydrogen atom or together with R' an additional bond,

R

8 designates a hydrogen atom or together with R 7 or together with R 9 an additional bond,

R

9 designates a hydrogen atom or together with R" or together with R 11 an additional bond, 15 R 15 designates a hydrogen atom, a hydroxy group, a halogen atom or together with R' 5 ' an oxo group or together with R 16 an additional bond or together with R 22 ' an oxygen bridge,

R

15 designates a hydrogen atom, an C1C linear or branched alkyl group, a C.-C1o aryl group or together with R" an oxo group, 20 R 1 6 designates a hydrogen atom, a hydroxy group, a halogen atom or together with R' 5 an additional bond or together with R 22 an additional bond,

R

22 designates a hydrogen atom, a C-C8 linear or branched optionally substituted alkyl or alkenyl group, an optionally substituted C6-C1O aryl group or together with R 16 an additional bond or together with R12 a C1C 25 linear or branched alkylidene group.

WO 00/68245 PCT/EPOO/04092 4

R

22 ' designates a hydrogen atom, a hydroxy group or together with R 1 5 an oxygen bridge or together with R 22 a CI-C8 linear or branched alkylidene group. 5 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,-C. alkyl designates an alkyl group having from one to eight carbon atoms: 10 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. R" together with R' is an 15 oxo group means that oxo (=O) is present in the 15 position and, consequently, there is no hydrogen atom in the 15 position. The term C-C aryl group designates a phenyl group, that is optionally substituted by halogen, (Cl

C

4 )alkoxy, hydroxy or (Cl-C 4 )alkyl groups. The term Cl-C, linear or branched alkylidene group stands for an alkyliden group having one to eight carbon atoms. 20 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. 25 It was surprisingly found that the l-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 14p-H-sterols, the meiosis activating effect of FF-MAS is diminished or completely extinguished. Meiosis-regulating substances described so far are either 14aX-H and A14 30 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 146l-H-sterols of this invention strongly antagonize the effect of the naturally WO 00/68245 PCT/EPOO/04092 5 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 % 5 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 isomeric forms and 10 mixtures thereof are within the scope of the invention (unless otherwise noted). Preferred compounds of formula I are such with an 3p-OH group and aA8 double bond. In addition, compounds of formula I are preferred, wherein R 15 designates a hydrogen atom or a hydroxy group. Other preferred compounds of formula I are such, wherein R 2 2 designates a C,-C linear or branched optionally substituted 15 alkyl group or together with R 22 a C,-Cs linear or branched alkylidene group. In another embodiment, the present invention relates to esters of compounds of the general formula 1. Such esters are formally derived by esterification of one or more hydroxylic groups of a compound of formula I with an acid which can for 20 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. 25 30 WO 00/68245 PCT/EPOO/04092 6 Especially preferred compounds of formula I of the present invention are the following: 4,4-dimethyl-5a, 1 4p-cholest-8-en-3p-ol 5 4,4-dimethyl-5a, 14p-cholest-8-ene-3p, 1 5p-diol 4,4-dimethyl-5a, 1 4p-cholest-8-ene-3p, 1 5ac-diol 4,4-dimethyl-5a, 1 4p-cholesta-8,15-dien-3p-ol 4,4-dimethyl-5a, 1 4p-cholesta-7,9(11),15-trien-3p-ol 4,4-dimethyl-5a, 1 4p-cholesta-8,15,23(E)-trien-3p-ol 10 4,4-dimethyl-5a, 1 4p-cholesta-8,15,24-trien-3p-ol 4,4-dimethyl-5a, 1 4p-cholesta-8,24-diene-3p, 1 5p-diol 4,4-dimethyl-5a, 14p-ergosta-8,22-dien-3p-ol 4,4-dimethyl-5a, 1 4p-ergosta-8,22-diene-3p, 1 5p-diol 4,4-dimethyl-5a, 1 4p-ergosta-8,22-diene-3p, 1 5a-diol 15 4,4-dimethyl-5a, 1 4p-ergosta-8,15,22-trien-3p-ol 4,4-dimethyl-24-nor-5ax, 1 4p-cholest-8-en-3p-ol 4,4-dimethyl-24-nor-5x, 1 4p-cholest-8-ene-3p, 1 5p-diol 4,4-dimethyl-24-nor-5a, 1 4p-cholest-8-ene-3p, 1 5a-diol 4,4-dimethyl-24-nor-5a, 1 4p-cholesta-8,15-dien-3p-ol 20 4,4-dimethyl-5a,14p-cholesta-8,15-dien-3p-ol hydrogen butanedioate 4,4-dimethyl-5a, 1 4p-ergosta-8,15,22-trien-3p-ol hydrogen butanedioate (20R)-4,4,20-trimethyl-1 6P,21 -cyclo-5a, 1 4p-pregn-8-ene-3p ,1 5a-diol (20R)-4,4,20-trimethyl-1 6P,21 -cyclo-5a, 1 4p-pregn-8-ene-3p, 1 5P-diol (20S)-20-hydroxymethyl-4,4-dimethyl-5a, 1 4-pregn-8-ene-3, 1 51-diol WO 00/68245 PCT/EPOO/04092 7 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 5 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 10 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: 15 The sterols that are used as starting materials can be synthesized according to literature procedures: 4,4-dimethyl-5a-cholesta-8,14-dien-33-ol [Biochem. J. 132 (1973), 439], 4,4 dimethyl-5a-ergosta-8,14,22-trien-3p-o [as benzoate: J. Org. Chem. 51 (1986), 20 4047], 5a-cholesta-8,14-dien-3p-ol [J. Am. Chem. Soc. 75 (1953), 4404], 5a ergosta-8,14,22-trien-3p-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. 25 The 14p-H-derivatives can be synthesized from A-8,14-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 WO 00/68245 PCT/EPOO/04092 8 deconjugated ketone 4 can be achieved by treatment with different acids or lewis acids like borontrifluorid [see: Chem. Pharm. Bull. 38 (1990), 1796]. scheme 1: ' H ii H HO BzO 2 H IH SRsRS 'H 11H 45H O H 0 BzO 3 BzO 5 The reactions can be carried out in the presence of different steroidal side chains (Rs) like cholesterol, ergosterol, sitosterol or stigmasterol side chain. If 15-ketones of general formula 4 are reduced with lithium aluminumhydride 3P,15p-diols of general formula 5 are obtained. The corresponding 15a-alcohols can be obtained as minor diastereomers of general formula 6 from the sodium 10 borohydride reduction. Subsequent deprotection of the 3-OH-group gives the 3P,,15a-diols of general formula 7 (scheme 2). scheme 2: WO 00/68245 PCT/EPOO/04092 9 - R - R "H "H H H OH BzO 4 HO 4 H5 -Rs Rs "IH "H H OH H bH HO BzO H BzO 6 7 The A8,15-diene of general formula 10 can be obtained by the following sequence. Sodium borohydride reduction of ketone 4 gives 150-alcohol 8 as the major diastereomer. Elimination to the A8,15-diene of general formula 9 can be 5 carried out with Martin's sulfurane. Deprotection gives the desired alcohols of general formula 10 (scheme 3). scheme 3: I-RS 4 Rs "H 'IH H 0 H OH BzO

-

BzO 4 H 8 Rs '-Rs "H IIH H H BzO 9 HO 10 WO 00/68245 PCT/EP00/04092 10 Derivatives which are saturated in position 15 can be obtained by the following route. 15P-Alcohols of the general formula 8 can be reacted with methanesulfonic acid chlorid. Mesylate 11 can then be reduced with lithium aluminiumhydride. 5 Compounds of general formula 12 are obtained (scheme 4). scheme 4: 4- Rs R "H "H H OH H OMs BzO - BzO H 8 H1 '-Rs 'IIH H HO HOH H 12 Side chain modifications can be performed via ozonolysis of the ergosterol side 10 chain in compound of formula 4 (with Rstero = 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. 15 WO 00/68245 PCT/EPOO/04092 11 scheme 5: Rsteroi "H "H OH HO HO BzO 4 BzO 13 Rstero = ergosterol side chain IIH OTs I H R22 H O Ho BzO BzO 1 H 14 H 15 These compounds can be transformed in the corresponding 3,15-diols (see scheme 2), A8,15-dienes (scheme 3) or 15 saturated compounds (scheme 4) as 5 described above. Other side chain modification like the 24-keto-, 24-hydroxy- and A24-compounds can be obtained by the following route. Ozonolysis of compound 4 and subsequent work up with triphenylphosphin gives aldehyde 16. This can be 10 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). 15 WO 00/68245 PCT/EPOO/04092 12 scheme 6: ' Rsteroi "H H H O H O BzO 4 BzO 1 HH 16 Rsterol ergosterol side chain 0OH IIH IIH BzOH O 4H O 1 BzO N17 BzO 1 H '0Co OH H 0H H9 OO H OH 2 Bz 9BzO 20 5 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). 10 WO 00/68245 PCT/EPOO/04092 13 scheme 7: HH BzOsH OH 20 H O 21 HH 22 H H IIH IH 4 H OH 231 24 HO =HO = H H 5 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 diisopropylamidle 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 aluminiumhyd ride to give diols26 10 and 27 (scheme 8).

WO 00/68245 PCT/EPOO/04092 14 scheme 8: H OTs 'IIH "H H O H O BzO BzO = H 14 H 25 H= H: H H H OH HOH HO HO H H 26 27 5 A further object of the present invention are pharmaceutical compositions comprising one or more compounds of the general formula I as active subs tances. The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disin 10 tegrating agents and other ingredients which are conventionally used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter. 15 Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with ex vivo and in vitro fertilization. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are men tioned in the list above. Further, a composition for transdermal administration of a 20 compound of this invention may be provided in the form of a patch and a WO 00/68245 PCT/EPOO/04092 15 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 5 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. 10 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 15 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 20 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 25 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. 30 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.

WO 00/68245 PCT/EPOO/04092 16 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 5 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, 10 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. 15 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 14B-hydrogen group in a sterol compound to increase the inhibitory activity of a meiosis 20 inhibiting substance. The present invention is further illustrated by the following examples: 25 30 WO 00/68245 PCT/EPOO/04092 17 5 Example 1: 4,4-dimethyl-5a,14p-ergosta-8,22-diene-3p,15p-diol a) 4,4-dimethyl-5a-ergosta-8,14,22-trien-3B-yl-benzoate 10 40 ml benzoylchloride were added in two portions to a solution of 30.9 g 4,4 dimethyl-5a-ergosta-8,22-dien-3p-oI 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-5a 15 ergosta-8,14,22-trien-3B-yl-benzoate (mp 146 0C). 1 H-NMR (CDCl 3 ): 5= 0,80 - 1,12 (8x CH 3 ); 4,75 (dd, J=16 Hz, 4 Hz, 1H, H-3a); 5,22 (m, 2H, H-22/23); 5,38 (s, broad, 1H, H-15); 7,46 (t, 2H); 7,57 (t, 1H); 8,07 (d, 2H) 20 b) 4,4-dimethyl-14,1 5a-epoxy-5a-ergosta-8,22-dien-3B-yl-benzoate A solution of 18 g 4,4-dimethyl-5a-ergosta-8,14,22-trien-3p-yl-benzoate in 60 ml dichloromethane was cooled to 10 *C. After addition of 3 g m-nitro 25 fluoroacetophenone and 10 ml saturated NaHCO 3 -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 WO 00/68245 PCT/EPOO/04092 18 and brine. Drying (MgSO 4 ) and removal of solvents in vacuo furnished crude epoxide (20 g), which was further reacted without purification. 5 c) 3B-benzoyloxy-4,4-dimethyl-5ca,14B-ergosta-8,22-dien-15-one 20 g of the crude epoxide were dissolved in 300 ml dioxane. The yellow 10 mechanically stirred solution was treated with 2.5 ml of boron triflouride-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 3B-benzoyloxy-4,4-dimethyl-5c,14B-ergosta 8,22-dien-15-one (mp 176 0C). 15 1 H-NMR (d5-pyridine): 5= 0,85 (2x d, J= 7 Hz, 6H, H-27/26); 0,91 (s, 3H, 4-p

CH

3 ); 0,93 (d, J= 7 Hz, 3H, H-28); 1,05 (s, 3H, H-18); 1,06 (s, 3H, 4-a-CH 3 ); 1,07 (s, 3H, H-19); 1,13 (d, J= 7 Hz, 3H, H-21); 4,89 (dd, J=12 Hz, 4 Hz, 1H, H-3X); 5,37 (m, 2H, H-22/23); 7,49 (t, 2H); 7,57 (t, 1H); 8,27 (d, 2H) 20 d) 4,4-dimethyl-5a, 14p-ergosta-8,22-diene-3p, 1 5p-diol 50 mg of LiAlH 4 were added to a stirred solution of 300 mg 3p-benzoyloxy-4,4 dimethyl-5ct,14p-ergosta-8,22-dien-15-one in 30 ml diethylether and the mixture 25 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-dimethyl-5a,14p-ergosta-8,22-diene-3p,15p diol. 30 1 H-NMR (CDCl 3 ): 5= 0,78 - 1,2 (8 x CH 3 ); 3,24 (dd, J=12 Hz, 4 Hz, 1H, H-3a); 3,7 (dd, broad, J=4 Hz, 4 Hz, 1H, H-15a); 5,24 (m, 2H, H-22/23) WO 00/68245 PCT/EPOO/04092 19 5 Example 2: 4,4-dimethyl-5a,14B-ergosta-8,22-diene-3B,15a-diol 10 a) 3B-benzoyloxy-4,4-dimethyl-5a,14B-ergosta-8,22-dien-15ax-ol A solution of 1 g 3B-benzoyloxy-4,4-dimethyl-5a,14B-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 15 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 3R-benzoyloxy-4,4-dimethyl 5a,14-ergosta-8,22-dien-15a-ol besides 770 mg of the corresponding 15p alcohol. 20 1 H-NMR (CDCI 3 ): 5= 0,8 - 1,12 (8 x CH 3 ); 4,07 (t, broad, J= 4 Hz, 1H, H 15 P); 4,76 (dd, J = 12 Hz, 4 Hz, 1 H, H-3a); 5,23 (m, 2H, H-22/23); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) 25 b) 4,4-dimethyl-5a, 14B-ergosta-8,22-diene-3, 1 5a-diol A solution of 40 mg 3B-benzoyloxy-4,4-dimethyl-5a,14B -ergosta-8,22-dien-15 a-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 30 25 mg 4,4-dimethyl-5a,14B1-ergosta-8,22-diene-3B,15a-diol were isolated as a white solid (mp 148 C).

WO 00/68245 PCT/EPOO/04092 20 1H-NMR (CDCI 3 ): 6 = 0,78 - 1,7 (8 x CH 3 ); 3,25 (dd, J = 11 Hz, 5 Hz, 1 H, H 3a); 4,04 (t, J= 4 Hz, 1H, H-15x); 5,22 (m, 2H, H-22/23); 5 Example 3: 4,4-dimethyl-5ax,14B-cholesta-8,15-dien-3-ol 10 a) 4,4-dimethyl-5ac-cholesta-8,14-dien-3B-yl-benzoate A solution of 1.0 g 4,4-dimethyl-5a-cholesta-8,14-dien-3B-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 15 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-5a-cholesta-8,14-dien-3R-yl-benzoate as a white solid 20 (mp 158 C*). 1 H-NMR (CDCl): 6= 0,83 (s, 3H, H-18); 0,87 + 0,88 (2x s, 3H, H-26/27); 0,95 (d, J = 6 Hz, 3H, H-21); 4,76 (dd, J = 11 Hz, 5 Hz, 1 H, H-3a); 5,38 (s, broad, 21, H-15); 7,45 (t, 2H); 7,57 (t, 1H); 8,06 (d, 2H) 25 b) 4,4-dimethyl-14,15a-epoxy-5a -cholest-8-en-3l-yl-benzoate To a well stirred suspension of 4,4-dimethyl-5a-cholesta-8,14-dien-3B-yl benzoate in 120 ml dichloromethane was added a solution of dimethyldioxirane 30 (130 ml, 0.09 - 0.11 M in acetone) at 0 C. After stirring for 30 minutes at 00C, the reaction mixture was concentrated in vacuo to dryness. The resulting crude epoxide was used without purificaton.

WO 00/68245 PCT/EPOO/04092 21 5 c) 3R-benzoyloxy-4,4-dimethyl-5a,14R-cholest-8-en-15-one A solution of 1 g of 4,4-dimethyl-14,15a-epoxy-5a-cholest-8-en-3B -yl-benzoate in 15 ml of dioxane was treated with 0.1 ml of boron triflouride-diethylether complex. The solution was stirred for 20 minutes at room temperature and then poured into 10 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 3R-benzoyloxy-4,4-dimethyl-5a,14B-cholest-8-en-15-one as a white solid (mp 153 -C). 15 1 H-NMR (CDCI 3 ): 6= 0,83 - 1,1 (7x CH 3 ); 2,31 (s, broad, 1H, H-14p); 4,76 (dd, J = 11 Hz, 5 Hz, 1 H, H-3a); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) d) 3B-benzoyloxy-4,4-dimethyl-5a,14R-cholest-8-en-15R-ol 20 A solution of 3R-benzoyloxy-4,4-dimethyl-5a,14B-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 0.1 N HCI and extracted with ethyl acetate. The organic layer was 25 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.

WO 00/68245 PCT/EPOO/04092 22 5 e) 4,4-dimethyl-5a,14B-cholesta-8,15-dien-3B-yl-benzoate A solution of 500 mg 3B-benzoyloxy-4,4-dimethyl-5a,14R-cholest-8-en-15B -o in 10 ml dichloromethane was cooled to 0 *C and 1.1 g Martin's sulfurane were 10 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-5a,14B-cholesta-8,15-dien-3R-yl benzoate as a waxy solid, which was used directly. 15 f) 4,4-dimethyl-5a, 14fB-cholesta-8,15-dien-3B-ol A solution of 315 mg 4,4-dimethyl-5ac,14B-cholesta-8,15-dien-3B-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 20 recrystallisation gave 135 mg 4,4-dimethyl-5a,14p-cholesta-8,15-dien-3R-ol as white needles (mp 138 *C). 1 H-NMR (CDCl 3 ): 6= 0,81 (s, 3H, H-18); 0,83 - 1,02 (6x CH 3 ); 2,49 (s, broad, 1H, H-14p); 3,24 (m, 1H, H-3a); 5,64 + 5,79 (2x m, 1H, H-15/16) 25 Example 4: 4,4-dimethyl-24-nor-5a,145-cholesta-8,15-dien-351-oI WO 00/68245 PCT/EPOO/04092 23 a) (20S)-3R-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5a, 14B-pregn-8-en-1 5 one and (20S)-3f5-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5a,14R-pregn-8 en-15BR-ol 5 A solution of 2.18 g 3B-benzoyloxy-4,4-dimethyl-5a,14R-ergosta-8,22-dien-15 one (example 1c) in 88 ml dichloromethane, 30 ml methanol and 1 ml pyridine was cooled to -70 *C. A ozone/oxygene-mixture (1:4) was passed through the solution for 12 min (controlled by TLC chromatography). Then 130 mg sodium borohydride were added and the stirred reaction mixture 10 was allowed to warm to 00C 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) 15 3R-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5ac,14B-pregn-8-en-15-one (mp 238.8 'C) and 330 mg of the the corresponding 15p-hydroxy-compound. 1 H-NMR (CDC3): 8= 0,9 - 1,1 (4x CH 3 ); 1,16 (d, J= 6 Hz, 3H, H-21); 2,33 (s, broad, 1H, H-14P); 3,42 (m, 1H, H-22a); 3,63 (m, 1H, H-22b); 4,75 (dd, J= 11 Hz, 20 4 Hz, 1H, H-3a); 7,45 (t, 2H); 7,57 (t, 1H); 8,05 (d, 2H) b) (20S)-35-benzoyloxy-4,4,20-trimethyl-21-toluolsulfonyloxy-5a,14B-pregn-8-en 15-one A solution of 385 mg (20S)-3R-benzoyloxy-20-hydroxymethyl-4,4-dimethyl 25 5a,1411-pregn-8-en-15-one in 5 ml pyridine was cooled to 00C. After addition of 190 mg p-toluenesulfonic chloride the reaction mixture was stirred over a period of 20 hours at 6 0C and then poured into brine and stirred for 20 min. The precipitate was collected, washed with ice-cold water and dried at 50 0C in vacuo. The crude product (500 mg) was used without further purification. 30 WO 00/68245 PCT/EP00/04092 24 c) 3fB-benzoyloxy-4,4-dimethyl-24-nor-5a,14B-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 0C to give a clear solution of Grignard reagent. After then the mixture 5 was cooled to -10 *C and 3.32 ml of a solution of Li 2 CuCl 4 (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 0C, whereupon a solution of 0.5 10 g (20S)-3B-benzoyloxy-4,4,20-trimethyl-21-toluolsulfonyloxy-5a,143-pregn-8-en 15-one in 10 ml tetrahydrofuran was added at 0 C. After 2 hours at 0 0C 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 15 over sodium sulfate and evaporated to dryness. The crude product purified by column chromatography to give 250 mg 3B-benzoyloxy-4,4-dimethyl-24-nor 5a,14R-cholest-8-en-15-one as a white foam. 1 H-NMR (CDC1 3 ): 6= 0,82 - 1,09 (7x CH 3 ); 2,31 (s, broad, 1H, H-143); 20 4,75 (dd, J = 11 Hz, 4 Hz, 1 H, H-3a); 7,44 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) d) 3R-benzoyloxy-4,4-dimethyl-24-nor-5a,14B-cholest-8-en-15B-ol 130 mg 3R-benzoyloxy-4,4-dimethyl-24-nor-5a,14B7-cholest-8-en-3-one were 25 treated with 100 mg sodium borohydrideas described in example 2a. Aqueous work-up yielded 130 mg 3B-benzoyloxy-4,4-dimethyl-24-nor-5a,14B-cholest-8 en-15l-ol as a white foam, which was used directly. e) 4,4-dimethyl-24-nor-5a,1 4B-cholesta-8,15-dien-3B-yl-benzoate WO 00/68245 PCT/EPOO/04092 25 400 mg of Martin's sulfurane were added in one portion to a solution of 100 mg 3f-benzoyloxy-4,4-dimethyl-24-nor-5a,1 4-cholest-8-en-1 5fl-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 5 dimethyl-24-nor-5a14B-cholesta-8,15-dien-3B-yl-benzoate as an oil, which was used without further purificatication. f) 4,4-dimethyl-24-nor-5a,14B-cholesta-8,15-dien-36-ol 10 100 mg 4,4-dimethyl-24-nor-5a,14R-cholesta-8,15-dien-3B-y 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-5a,14B-cholesta-8,15-dien-3R-ol (mp 104.3 -C). 15 1 H-NMR (CDCl 3 ): 6= 0,82 (s, 3H, H-18); 0,83 - 1,03 (6x CH 3 ); 2,49 (s, broad, 1H, H-14P); 3,24 (m, 1H, H-3a); 5,65 + 5,8 (m, 1 H, H-15 /16) Example 5: 4,4-dimethyl-24-nor-5a,14a-cholest-8-en-3E-ol 20 a) 1 5B-methanesulfonyloxy-4,4-dimethyl-24-nor-5a,14B-cholest-8-ene-3B-yl benzoate 40 mg of 3B-benzoyloxy-4,4-dimethyl-24-nor-5a,14R-cholest-8-en-15B-ol in 3 ml 25 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 156-methanesulfonyloxy-4,4-dimethyl-24-nor-5a,14B7 30 cholest-8-ene-3B-yl benzoate that was used directly.

WO 00/68245 PCT/EPOO/04092 26 b) 4,4-dimethyl-24-nor-5ac,14l-cholest-8-en-3B-o 44 mg 15R-methanesulfonyloxy-4,4-dimethyl-24-nor-5a,14B-cholest-8-ene-3B-yl benzoate were treated with 10 mg lithium aluminium hydride as described in 5 example 1d. The crude product was purified by chromatography to provide 32 mg 4,4-dimethyl-24-nor-5x, 14B-cholest-8-en-31-ol. 1 H-NMR (CDCI 3 ): 6= 0,81 (s, 3H, H-18); 0,82 - 1,03 (6x CH 3 ); 3,25 (dd, J= 11 Hz, 4 Hz, 1H, H-3c) 10 Example 6: 4,4-dimethyl-24-nor-5ax,145-cholest-8-ene-35,15B-diol 30 mg 3B-benzoyloxy-4,4-dimethyl-24-nor-5x,14B-cholest-8-en-15fB-ol were treated with 10 mg lithium aluminium hydride as described in example 1d. The 15 crude product was purified by column chromatography to give 23 mg 4,4 dimethyl-24-nor-5ax,14B-cholest-8-ene-3B,15B-diol as a white foam. 1 H-NMR (CDCl 3 ): 6= 0,82 (s, 3H, H-18); 0,84 - 1,04 (6x CH 3 ); 3,25 (d, broad,J= 12 Hz, 1H, H-3a); 3,7 (q, broad, J= 7 Hz, 1H, H-15a) 20 Example 7+8: (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene 3p,15p-diol and (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene 3p,15a-diol 25 a) (20R)-3p-benzoyloxy-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-en-15 one 1.10 g (20S)-3B-benzoyloxy-4,4,20-trimethyl-21-toluolsulfonyloxy-5c,14B-pregn 8-en-15-one were treated with 52 mmol phenylmagnesium bromide analoguously 30 to example 4c. After chromatography 560 mg (20R)-3p-benzoyloxy-4,4,20- WO 00/68245 PCT/EPOO/04092 27 trimethyl-16p,21-cyclo-5ca,14p-pregn-8-en-15-one were isolated beside other products, that result from tosylate substitution by the phenyl grignard reagent. 5 b) (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-3p,150-diol and (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-3p,15a-diol 460 mg (20R)-3p-benzoyloxy-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-en 10 15-one were treated with 50 mg lithium aluminium hydride as described in example 1d. The crude product was purified by column chromatography to give 40 mg (20R) 4,4,20-trimethyl-16p,21-cyclo-5x,14p-pregn-8-ene-3p,15p-diol and 30 mg (20R) 4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-3p,15a-diol as white solids. 15 (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-3p,15p-diol: 1 H-NMR (CDCl 3 ): 5= 0,73 (s, 3H, H-18); 0,82 (s, 3H, H-19); 1,02 (s, 6H, 4

CH

3 ); 1,09 (d, J = 8 Hz, 3H, H-21); 2,72 (m, 1H, H-16a); 3,25 (m, 1H, H 3c); 4,07 (q, J = 8Hz, 1H, H-15a) 20 (20R)-4,4,20-trimethyl-1 6P,21 -cyclo-5a, 14p-pregn-8-ene-3p, 1 5C-diol 1 H-NMR (CDCl 3 ): 6= 0,79 (s, 3H, H-18); 0,85 (s, 3H, H-19); 1,0 - 1,08 (3 * CH 3 ); 2,34 (d, J = 6 Hz, 1H, H-14P); 2,68 (m, 1H, H-16a); 3,25 (m, 1H, H-3ax); 4,07 (d, J = 6 Hz, 1H, H-15P) 25 Example 9: 4,4-dimethyl-5a,14B-cholesta-8,24-diene-35,15B-diol a) (20S)-3fB-benzoyloxy-4,4-dimethyl-1 5-oxo-5x,1 5R-pregn-8-ene-20 carbaldehyde 30 A solution of 2.2 g 3B-benzoyloxy-4,4-dimethyl-5a,14B -ergosta-8,22-dien-15-one in 87 ml dichloromethane and 53 ml methanol was cooled to -78 *C. A mixture of WO 00/68245 PCT/EPOO/04092 28 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)-3B-benzoyloxy-4,4-dimethyl-15-oxo 5 5a, 1 5B-pregn-8-ene-20-carbaldehyde (mp. 205 0C). 1 H-NMR (d5-pyridine): 6= 0,90 (s, 3H, H-19); 0,98 (s, 3H, H-18); 1,04 (s, 6H, 4-CH 3 ); 1,2 (d, J= 6 Hz, 3H, H-21); 2,41 (s, broad, 1H, H-14p); 4,83 (dd, J = 11 Hz, 4 Hz, 1 H, H-3a); 7,5 (t, 2H); 7,58 (t, 1 H); 8,27 (d, 2H); 9,82 10 (d, J=2 Hz, 1H, H-22) b) 3B-benzoyloxy-22-hydroxy-4,4-dimethyl-5ca,14f-cholest-8-ene-15,24-dione 25.8 ml of a n-butyllithium solution (1.6 M, hexane) were added to 6 ml 15 diisopropylamine in 48 ml tetrahydrofuran at -78 0C. 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 0C) solution of 6.0 g (20S)-3B-benzoyloxy-4,4-dimethyl-15-oxo-5a,14fB-pregn-8-en 20 20-carbaldehyde in 40 m tetrahydrofuran. The mixture was brought to 0 0C 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 25 column chromatography to give 6.26 g 3fB-benzoyloxy-22-hydroxy-4,4-dimethyl 5ac,14B-cholest-8-ene-15,24-dione as an oil. 1 H-NMR (CDC1 3 ): 8= 0,92 - 1,2 (7 x CH 3 ); 2,33 (s, broad, 1H, H-14P); 4,1 (m, 1 H, H-22); 4,74 (dd, J = 11 Hz, 4 Hz, 1 H, H-3a); 7,45 (t, 2H); 7,57 (t, 30 1H); 8,05 (d, 2H) c) 3R-benzoyloxy-4,4-dimethyl-5a,14B-cholesta-8,22-diene-15,24-dione WO 00/68245 PCT/EPOO/04092 29 A solution of 4.78 g 3B-benzoyloxy-22-hydroxy-4,4-dimethyl-5a,14B1-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 5 30 minutes at 0 0C. The solvents were removed in vacuo. Purification of the residue by column chromatography gave 4.18 g 3R-benzoyloxy-4,4-dimethyl 5a,14B-cholesta-8,22-diene-15,24-dione (mp 163 0C). 1 H-NMR (CDCl 3 ): 6= 0,9 - 1,2 (7 x CH 3 ); 2,31 (s, broad, 1H, H-14p); 4,75 10 (dd, J=11 Hz, 4 Hz, 1H, H-3a); 6,14 (d, J = 16 Hz, 1H, H-23); 6,71 (m, 1H, H-22); 7,45 (t, 2H); 7,57 (t, 1H); 8,05 (d, 2H) d) 3B-benzoyloxy-4,4-dimethyl-5a,14fB-cholest-8-ene-15,24-dione 15 500 mg palladium on charcoal (10%) were added to a solution of 4.2 g 3 benzoyloxy-4,4-dimethyl-5a, 14B-cholesta-8,22-diene-1 5,24-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 3B-benzoyloxy 4,4-dimethyl-5ax,145-cholest-8-ene-15,24-dione as a white crystalline residue (mp 20 129 C). 1 H-NMR (CDCl 3 ): 6= 0,92 - 1,16 (7 x CH 3 ); 2,33 (s, broad, 1H, H-14p); 4,74 (dd, J = 11 Hz, 4 Hz, 1 H, H-3aL); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) 25 e) 3B-benzoyloxy-4,4-dimethyl-5ac,14B-cholest-8-ene-15,24-diol and 3B-benzoyloxy-24-hydroxy-4,4-d imethyl-5cx, 14B-cholest-8-en- 1 5-one A solution of 4.2 g 3B-benzoyloxy-4,4-dimethyl-5a, 14B-cholest-8-ene-1 5,24-dione in 45 ml CH 2 Cl 2 was treated with 1.8 g tert-butylamine-borane complex. The 30 solution was refluxed for one hour. After cooling to 0 0C, 30 ml HCI (1 N, aqueous) were added. The mixture was stirred for one hour at 0 C. It was washed with HCI (1N, aqueous), water, saturated aqueous NaHCO 3 solution and brine. After drying WO 00/68245 PCT/EPOO/04092 30 over sodium sulfate, the solvents were removed in vacuo. Column chromatography gave 1.71 g 3B-benzoyloxy-4,4-dimethyl-5a,14B-cholest-8-ene 15,24-diol and 1.82 g 3B-benzoyloxy-24-hydroxy-4,4-dimethyl-5a.,14R-cholest-8 en-15-one as white foams. 5 3B-benzoyloxy-4,4-dimethyl-5a, 1 4Bl-cholest-8-ene-1 5,24-diol: 1 H-NMR (CDCl 3 ): S= 0,88 - 1,1 (7 x CH 3 ); 3,33 (m, 1H, H-24); 3,74 (m, 1 H, H-1 5); 4,75 (dd, J = 11 Hz, 4 Hz, 1 H, H-3a); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) 10 3B-benzoyloxy-24-hydroxy-4,4-dimethyl-5a, 14 B-cholest-8-en- 15-one: 1 H-NMR (CDCI 3 ): 6= 0,88 - 1,1 (7 x CH 3 ); 2,32 (s, broad, 1H, H-14P); 3,33 (m, 1 H, H-24); 4,74 (dd, J = 11 Hz, 4 Hz, 1 H, H-3ax); 7,45 (t, 2H); 7,57 (t, 1 H); 8,05 (d, 2H) 15 f) 3B-benzoyloxy-4,4-dimethyl-5a, 14B-cholesta-8,24-dien-15R-ol and 4,4-dimethyl-5a, 1 4B-cholesta-8,15,24-trien-3B-yl-benzoate 1.0 g 3B-benzoyloxy-4,4-dimethyl-5a,14R-cholest-8-en-15, 24-diol was treated 20 with 3.88 g of Martin's sulfurane as described in example 3e. Purification by column chromatography gave 305 mg 36-benzoyloxy-4,4-dimethyl-5a,14B cholesta-8,24-dien-15R-ol as a white foam and 870 mg 4,4-dimethyl-5aX,14R cholesta-8,15,24-trien-3R-yl-benzoate as a white solid. 25 3B-benzoyloxy-4,4-dimethyl-5a,14R-cholesta-8,24-dien-1 5B-ol: 1 H-NMR (d5-pyridine): S= 1,15 (s, 1H, H-18); 2,84 (m, 1H, H-7X); 4,1 (m, 1H, H-15); 4,93 (dd, J=11 Hz, 4 Hz, 1H, H-3a); 5,24 (t, J = 8 Hz, 1H, H 24); 7,45 (t, 2H); 7,57 (t, 1H); 8,25 (d, 2H) 30 4,4-dimethyl-5a,14B-cholesta-8,15,24-trien-3R-yl-benzoate WO 00/68245 PCT/EPOO/04092 31 1 H-NMR (CDCI 3 ): 6= 1,4 (s, 3H, H-19); 2,19 (m, 1H, H-17a); 2,51 (s, broad, 1H, H-14p); 4,75 (dd, J=11 Hz, 4 Hz, 1H, H-3ax); 5,1 (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, 1 H); 8,05 (d, 2H) 5 g) 4,4-dimethyl-5a,14B-cholesta-8,24-diene-3B,15R-diol 120 mg 3R-benzoyloxy-4,4-dimethyl-5a,14R-cholesta-8,24-dien-15B-ol were treated with 30 mg lithium aluminium hydride as described in example 1d. The 10 crude product was purified by flash column chromatography to give 80 mg 4,4 dimethyl-5a, 1 4B-cholesta-8,24-diene-3B, 1 5B-diol as a white foam. 1 H-NMR (CDCI 3 ): 5= 0,8 - 1,03 (5x CH 3 ); 1,6 + 1,68 (2x s, 3H, H-26/27); 3,24 (m, 1H, H-3ax); 3,70 (m, 1H, H-15a); 5,10 (t, J= 7 Hz, 1H, H-24) 15 Example 10: 4,4-dimethyl-5a,1 49-cholesta-8,15,24-trien-3B-ol 30 mg 4,4-dimethyl-5c,14B-cholesta-8,15,24-trien-3B-yl-benzoate (example 9f) were treated with 20 mg lithium aluminium hydride as described in example 1d. 20 The crude product was purified by column chromatography to give 20 mg 4,4 dimethyl-5a,14B-cholesta-8,15,24-trien-3B-ol as a white solid (mp 105 0C). 1 H-NMR (CDCl 3 ): 5= 0,81 (s, 3H, H-18); 0,94 - 1,02 (4x CH 3 ); 2,5 (s, broad, 1H, H-14p); 3,23 (m, 1H, H-3a); 5,11 (t, J = 9 Hz, 1H, H-24); 5,64 25 (m, 1H, H-15/16a); 5,79 (m, 1H, H-15/16b) Example 11: (20S)-20-hydroxymethyl-4,4-dimethyl-5a, 1 4B-pregn-8-ene 39,1511-diol 30 60 mg (20S)-20-hydroxymethyl-4,4-dimethyl-5a, 14B-pregn-8-ene-3B, 1 5B-diol were treated with 20 mg lithium aluminium hydride as described in example 1d.

WO 00/68245 PCT/EPOO/04092 32 The crude product was purified by column chromatography to give 42 mg (20S) 20-hydroxymethyl-4,4-dimethyl-5a,14R-pregn-8-ene-3B,15B-diol (mp 214 *C). 1 H-NMR (CDCl 3 ): 6= 0,82 (s, 3H, H-18); 0,93 - 1,04 (3x CH 3 );1,07 (d, J= 6 Hz, 3H, H-21); 3,23 (m, 1H, H-3c); 3,49 (m, 1H, H-22a); 3,68 (m, 1H, H 5 22b); 3,72 (m, 1H, H-1 5a) Example 12: Testing of meiosis-activating substances in the oocyte test 10 Animals Oocytes were obtained from immature female mice (C57Bl/6J x DBA/2J Fl hybrids, Bomholtgaard, Denmark) weighing 13 - 16 grams, that were kept under controlled lighting and temperature. The mice received an intra-peritoneal injection of 0.2 ml gonadotropins (Gonal F, Serono, Solna, Sweden , containing 15 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 20 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 a-minimum essential medium (ax-MEM without ribonucleosides, Gibco BRL, Cat.No. 22561) supplemented with 3 mM 25 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 IU/ml penicillin and 100 pg/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 30 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 WO 00/68245 PCT/EPOO/04092 33 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 % C02 in air. The culture time was 22 - 24 hours. 5 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 10 counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. The percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture. 15 Example 13: 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 20 oocytes (NO) were rinsed three times in Hx-medium. 4,4-Dimethylcholest-B, 14,24-trien-3Gl-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 pM FF-MAS in co-culture with the test compounds in different concentrations in 4-well multidishes (Nunclon, Denmark) 25 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 30 cultured in Hx-medium alone) was run simultaneously with the positive control.

WO 00/68245 PCT/EPOO/04092 34 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 5 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: 10 inhibition test compound (%) = 100% - [(GVB+PB (test compound) GVB+PB (negative control)) x 100 / GVB+PB(positive control) - GVB+PB (negative control)] % 15 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 20 and elicit the first so called polar body (PB).

WO 00/68245 PCT/EPOO/04092 35 Results: Table 1: Relative inhibition [%] of meiosis in naked mouse oocytes 5 Compounds Oocytes [n] Inhibition [%] GV GVB PB Control (Hx) 38 2 0 100 5pM FF-MAS 9 27 4 0 5pM FF-MAS + 10 pM 4,4- 28 11 1 66 dimethyl-5a,14p-cholesta-8,15 dien-3p-ol Control (Hx) 40 0 0 100 5pM FF-MAS 8 25 6 0 5pM FF-MAS + 10 pM 4,4- 37 0 0 100 dimethyl-5a,14p-ergosta-8,22 diene-3p,15p-diol Hx = Hypoxanthine GV = germinal vesicle GVB = germinal vesicle breakdown 10 PB = polar bodies n = number of 15

Claims (11)

1. Compounds of the general formula I R22' R11 IIH R22 R 1 9 I H R 1 6 HR15' R3'O R3R15 'R3 H R7 3R4 R4' R 5 wherein R 3 designates a hydrogen atom or together with R 3 an additional bond, R 3 ' designates a hydrogen atom or together with R 3 an additional bond, R 4 designates a hydrogen atom or a methyl group, 10 R 4 ' designates a hydrogen atom or a methyl group, R 7 designates a hydrogen atom or together with R" an additional bond, R 8 designates a hydrogen atom or together with R 7 or together with R 9 an additional bond, R 9 designates a hydrogen atom or together with R 8 or together with R" an 15 additional bond, R 15 designates a hydrogen atom, a hydroxy group, a halogen atom or together with R' an oxo group or together with R 1 6 an additional bond or together with R 22 an oxygen bridge, R 15 ' designates a hydrogen atom, an C-C8 linear or branched alkyl group, a 20 C6-Clo aryl group or together with R' 5 an oxo group, R' 6 designates a hydrogen atom, a hydroxy group, a halogen atom or together with R' 5 an additional bond or together with R 22 an additional bond, R 22 designates a hydrogen atom, a C1-C8 linear or branched optionally substituted alkyl or alkenyl group, an optionally substituted C-C, aryl WO 00/68245 PCT/EPOO/04092 37 group or together with R' 6 an additional bond or together with R 22 ' a C1C8 linear or branched alkylidene group. R 22 ' designates a hydrogen atom, a hydroxy group or together with R 15 an oxygen bridge or together with R 22 a Cl-C 8 linear or branched alkylidene 5 group. or esters thereof.

2. Compounds according to claim 1, wherein R 3 and R" designate hydrogen 10 atoms.

3. Compounds according to claim 1 or 2, wherein R 8 and R 9 designate together an additional bond. 15

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

5. Compounds according to any one of the claims 1 to 4, wherein R2 designates a C1C8 linear or branched optionally substituted alkyl group or 20 together with R2 a C1C8 linear or branched alkylidene group.

6. Compounds to any one of the claims 1 to 5 which are 4,4-dimethyl-5a,14p-cholest-8-en-3p-ol 4,4-dimethyl-5a,14-cholest-8-ene-3p,15p-diol 4,4-dimethyl-5a,14p-cholest-8-ene-3p,1 5a-diol 25 4,4-dimethyl-5a,14p-cholesta-8,15-dien-3p-ol 4,4-dimethyl-5a,14p-cholesta-7,9(11),15-trien-3p-ol 4,4-dimethyl-5a,14p-cholesta-8,15,23(E)-trien-3p-ol 4,4-dimethyl-5a, 1 4p-cholesta-8,15,24-trien-3p-ol WO 00/68245 PCT/EPOO/04092 38 4,4-dimethyl-5a, 14p-cholesta-8,24-diene-3p, 1 5p-diol 4,4-dimethyl-5a, 14p-ergosta-8,22-dien-3p-ol 4,4-dimethyl-5a, 1 4p-ergosta-8,22-diene-3p, 1 5p-diol 4,4-dimethyl-5a, 1 4p-ergosta-8,22-diene-3, 1 5a-diol 5 4,4-dimethyl-5a, 1 4p-ergosta-8,15,22-trien-3p-ol 4,4-dimethyl-24-nor-5a, 14p-cholest-8-en-3p-ol 4,4-dimethyl-24-nor-5a, 1 4p-cholest-8-ene-3p, 1 5p-diol 4,4-dimethyl-24-nor-5a, 1 4p-cholest-8-ene-3p, 1 5a-diol 4,4-dimethyl-24-nor-5a, 1 4p-cholesta-8,15-dien-3p-ol 10 4,4-dimethyl-5a,14p-cholesta-8,15-dien-3p-oI hydrogen butanedioate 4,4-dimethyl-5a,14p-ergosta-8,15,22-trien-33-o hydrogen butanedioate (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-3P,15a-diol (20R)-4,4,20-trimethyl-16p,21-cyclo-5a,14p-pregn-8-ene-30,15p-diol (20S)-20-hydroxymethyl-4,4-dimethyl-5a,14B-pregn-8-ene-3B,15&-diol 15

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. 20

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. 25 WO 00/68245 PCT/EPOO/04092 39

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. 5

11. Use of a 14B-hydrogen group in a sterol to increase the inhibitory activity of a meiosis inhibiting substance. 10