JP2002544136A - 14β-H-sterols, pharmaceutical compositions comprising them, and the use of their derivatives for the modulation of meiotic regulatory medicaments - Google Patents

Abstract

  (57) [Summary] The present invention relates to pharmaceutically active 14β-H-steros, pharmaceutical compositions comprising them and the use of these compounds for the preparation of medicaments. More specifically, it has been found that the 14β-H-sterols of the invention can be used to regulate meiosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutically active sterols, pharmaceutical compositions comprising them as active substance, and the use of these novel compounds for the preparation of medicaments. More specifically, it has been found that the sterols of the present invention can be used for the regulation of meiosis. Meiosis is a unique and ultimate phenomenon of germ cells in which sexual reproduction is formed. Meiosis comprises two meiosis. During the first division, an exchange occurs between the maternal and paternal genes, after which the chromosome pair is separated into two daughter cells. They contain only half (1n) of the chromosomes and 2c DNA. The second meiosis is DN
A Proceed without synthesis. Thus, this division results in the formation of haploid germ cells with only 1c DNA.

[0002] Meiotic events are similar in male germ cells, but the time schedule and differentiation process leading to eggs and sperm are very different. All female germ cells enter the first phase of meiosis early in life, often before life extension, but all in the early phase (dictyate state) until ovulation after puberty. Blocked as an oocyte. Thus, from an early life, females have their egg stock withdrawn based on their depletion. Meiosis in females is not completed until after fertilization and results in only one egg and two defective polar cells per germ cell. In contrast, some of the male germ cells enter meiosis from puberty and release a germ cell stem population throughout life. Upon initiation, meiosis in male cells proceeds without significant delay and produces four spermatozoa.

[0003] To a small extent, mechanisms that regulate the onset of meiosis in males and females are known. New studies on oocytes have shown that follicular purine, hypoxanthine or adenosine can be responsible for meiotic arrest [Downs, SM
Biol. 82 (1985) 454-458; Epplg. JJ et al. Dev. Biol. 119 (19
86) 313-321; and Downs, SM Mo. Reprod. Dev. 35 (1993) 82-94]. The presence of diffusible meiotic regulators was first identified in culture systems of exterminating mouse gonads by By
skov et al. [Byskov, AG et al. Dev. Biol. 52 (1976) 193-2
00].

[0004] Meiotic activators (MAS) are secreted by the ovaries of fetal mice undergoing meiosis, and meiotic inhibitors (MPS) are morphologically differentiated with quiescent non-meiotic germ cells. The testis was released. It has been proposed that relative concentrations of MAS and MPS regulate the onset, arrest and resumption of meiosis in male and female germ cells [Byskov, AG et al. The Physiology of Reproduction (eds. Knobil. E
and Neill. JD, Raven Press, New York (1994))]. Obviously, if meiosis can be regulated, reproduction can be controlled. Recent literature [Byskov. AG et al. Na
ture 374 (1995), 559-562] describe the isolation of certain sterols (T-MAS and FF-MAS) that activate oocyte meiosis from bull testis and human follicular fluid.

[0005] Compounds known to regulate meiosis are described in WO 96/27658, WO 97/00884
No. WO98 / 28323 and WO98 / 52965. It is an object of the present invention to provide new compounds useful as contraceptives in females and males, especially humans, through the inhibition of meiosis. The present invention has the following general formula I:

[0006]

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Wherein R ³ represents an additional bond together with a hydrogen atom or R ^{3 ′} , R ^{3 ′} represents an additional bond together with a hydrogen atom or R ³ , and R ⁴ represents hydrogen represents an atom or a methyl group, R ^{4 'represents} a hydrogen atom or a methyl group, R ⁷ is a hydrogen atom, or together with R ^8, represents an additional bond, R ⁸ is hydrogen atom, or R ⁷ or R with ⁹ represents an additional bond, R ⁹ is a hydrogen atom, or together with R ⁸ or R ^11, represents an additional bond, R ¹⁵ is a hydrogen atom, a hydroxy group, a halogen atom, or together with R ^{15 ',} oxo Represents an oxygen bridge, together with a group, or R ^16, with an additional bond, or R ^{22 ′} ,

R ^{15 ′} represents a hydrogen atom, a C ₁ -C ₈ linear or branched alkyl group, a C ₆ -C ₁₀ aryl group, or an oxo group together with R ¹⁵ , R ¹⁶ represents a hydrogen atom, Represents an additional bond with a hydroxy group, a halogen atom, or R ¹⁵ or R ²² , wherein R ²² is a hydrogen atom, a C ₁ -C ₈ linear or branched optionally substituted alkyl or alkenyl group, optionally optionally substituted C ₆ -C even though ₁₀ aryl group, or together with R ^16, an additional bond, or together with R ^{22 ',} represents a C ₁ -C ₈ linear or branched alkylidene radical, R ^{22 ′} represents a hydrogen atom, a hydroxy group, or an oxygen bridge together with R ¹⁵ , or a C ₁ -C ₈ linear or branched alkylidene group together with R ²² ], or 14β-H-sterol represented by Regarding the ester.

As used herein, the term alkyl, when used alone or in combination, can be a straight or branched chain alkyl group. The expression C ₁ -C ₈ alkyl denotes an alkyl group having 1 to 8 carbon atoms; preferred examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, 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 herein, for example, the meaning that R ¹⁵ together with R ^{15 ′} is an oxo group means that oxo (OO) is at position 15 and thus a hydrogen atom at position 15 Does not exist. The term C ₆ -C ₁₀ aryl group refers to halogen,
Represents a phenyl group optionally substituted by a (C ₁ -C ₄ ) alkoxy, hydroxy or (C ₁ -C ₄ ) alkyl group. The term is C ₁ -C ₈ straight or branched chain alkylidene group, represents an alkylidene group having 1 to 8 carbon atoms. This group is attached to carbon 22 of the steroid through a double bond. Preferred examples are methylene,
Ethylidene, propylidene, isopropylidene, butylidene, pentylidene, isopropylidene, neopentylidene and cyclohexylidene.

It has surprisingly been found that the β-form of hydrogen at position 14 (numbering of C atoms follows the IUPAC nomenclature) is important for meiotic inhibitory activity. 14β-
In the presence of H-sterols, meiosis, which activates the effects of FF-MAS, is reduced or completely abolished. The meiotic regulators described so far are 14α-
H and Δ14-cholestane derivatives [Byskov. AG et al. Nature 374 (1995), 559-
562] and exhibit either meiotic activating or low inhibitory activity.

[0012] In contrast, the 14β-H-sterols of the present invention strongly potentiate and inhibit the effects of naturally occurring FF-MAS, and are therefore inhibitors of meiosis. These findings make the compounds described have particular interest for contraception. Preferred compounds of the formula I, when tested in the oocyte test, have a germinal vesicle degradation of at least 20%, preferably at least 40%, in particular at least 60%, as described in Example 13.
%, And those compounds that do not activate meiosis in the oocyte test, as described in Example 12.

The compounds of general formula I have a number of chiral centers in the molecule and are therefore
And exists in several isomeric forms. All their isomeric forms and their mixtures
Is within the scope of the present invention (unless otherwise stated). Preferred compounds of formula I
The compound has a 3β-OH group and a Δ8 double bond. Further, the formula I [where R ^Fifteen Represents a hydrogen atom or a hydroxy group]. Formula I [Formula
Medium, R^{twenty two}Is C₁−C₈A linear or branched optionally substituted alkyl group of^{twenty two '} With C₁−C₈Represents a linear or branched alkylidene group of the formula
Are preferred.

In another aspect, the invention relates to esters of the compounds of general formula I. Such esters formally include, for example, succinic acid, glutaric acid, and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethyl carboxylic acid, phosphoric acid,
By esterification of one or more hydroxyl groups of a compound of formula I with an acid selected from the group of acids comprising sulfonic, sulfamic, benzoic, acetic, propionic and other aliphatic monocarboxylic acids. Be guided.

Particularly preferred compounds of the formula I according to the invention are: 4,4-dimethyl-5α, 14β-cholest-8-en-3β-ol, 4,4-dimethyl-5α, 14β- Cholest-8-ene-3β, 15β-diol, 4,4-dimethyl-5α, 14β-cholest-8-ene-3β, 15α-diol, 4,4-dimethyl-5α, 14β-cholesta-8,15-diol Diene-3β-ol, 4,4-dimethyl-5α, 14β-cholesta-7,9 (11), 15-trien-3
β-ol, 4,4-dimethyl-5α, 14β-cholesta-8,15,23 (E) -triene-3
β-ol, 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-ol,

4,4-dimethyl-5α, 14β-cholesta-8,24-diene-3β, 15β-diol, 4,4-dimethyl-5α, 14β-ergosta-8,22-dien-3β-ol, , 4-Dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15β-
Diols, 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-
Diols, 4,4-dimethyl-5α, 14β-ergosta-8,15,22-triene-3β-
All, 4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β-ol, 4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3β, 15β
-Diols,

4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3β, 15α
-Diol, 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-diene-3β
-Ol, 4,4-dimethyl-5α, 14β-cholesta-8,15-diene-3β-ol hydrogen butanediate, 4,4-dimethyl-5α, 14β-ergosta-8,15,22-triene-3β −
(20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15α-diol, (20R) -4,4,20-diol Trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15β-diol, (20S) -20-hydroxymethyl-4,4-dimethyl-5,14β-pregn-
8-ene-3β, 15β-diol.

The compounds of the general formula I according to the invention can be synthesized analogously to the preparation of known compounds. Thus, the synthesis of compounds of formula I can follow well-established synthetic routes described in the comprehensive sterol and steroid literature. The following references can be used as key sources in synthesis: LF Fieser & M. Fieser: Steroids: Reinhold P
ublishing Corporation, NY 1959; Rood's Chemistry of Carbon Compounds (e
ditor: S. Coffrey): Elsevier Publishing Company, 1971; and especially Diction
ary od Steroids (editors: RA Hill; DN Kirk; HLJ Makin and G.
M. Murphy): Chapman & Hall. The last document contains an extensive list of references to the first document covering the period up to 1990.

In particular, the compounds of the present invention are synthesized according to the following general methods. The following sterols used as starting materials can be synthesized according to the following literature method: 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol [Bi
ochem. J. 132 (1973), 439], 4,4-dimethyl-5α-ergosta-8,14
, 22-trien-3β-ol [as benzoate: J. Org. Chem. 51 (1986)
4047], 5α-Cholesta-8,14-dien-3β-ol [J. Am. Chem. Soc
75 (1953) 4404], 5α-ergosta-8,14,22-trien-3β-ol [J. Org. Chem. 53 (1988) 1563].

In the following, only compounds having a 4,4-dimethyl group will be described in detail. Compounds that are not substituted at the 5-position are obtained by a similar route. 14β-H-derivatives can be synthesized from the Δ-8,14-diene-system through protection of the alcohol, epoxidation and subsequent opening of the epoxide (Scheme 1).
3-Alcohols can be protected as benzoates. Epoxidation can be achieved with reagents such as dimethyldioxirane, hydrogen peroxide in the presence of various catalysts, m-CPBA and other peracids. Transfer to the unconjugated ketone 4 can be achieved by treatment with various acids or Lewis acids, such as boron trifluoride [Chem. Pharm. B
ull. 38 (1990), 1796].

Scheme 1 :

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The reaction can be carried out in the presence of a different steroid chain (R _s ), for example cholesterol, ergosterol, sitosterol or stigmasterol side chain field. When the 15-ketone of general formula 4 is reduced by lithium aluminum hydride,
3β, 15β-diol of the general formula 5 is obtained. The corresponding 15α-alcohol is obtained as a minor diastereomer of general formula 6 from sodium borohydride reduction. Subsequent deprotection of the 3-OH group gives the 3β, 15α-diol of general formula 7 (Scheme 2).

Scheme 2 :

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The Δ8,15-diene of the general formula 10 is obtained in the following order. Sodium borohydride reduction of ketone 4 gives 15β-alcohol 8 as a minor diastereomer. The elimination of general formula 9 to Δ8,15-diene can be carried out by Martin sulfuran. Deprotection gives the desired alcohol of general formula 10 (Scheme 3
).

Scheme 3 :

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The derivative saturated at the 15 position can be obtained by the following route. The 15β-alcohol of general formula 8 can be reacted with methanesulfonic acid chloride. Next, the mesylate 11 can be reduced by lithium aluminum hydride. A compound of general formula 12 is obtained (Scheme 4).

Scheme 4 :

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Side chain modification can be performed through ozonolysis of the ergosterol side chain in compounds of formula 4 (R _sterol = ergosterol side chain). After reduction with sodium borohydride, the 22-alcohol 13 can be rearranged to a leaving group such as tosylate (Scheme 5). This tosylate 14 is coupled with different branched linear alkyl, alkenyl or aryl Grignard reagents under copper catalysis to give compounds of general formula 15.

Scheme 5 :

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The compounds are prepared as described above with their corresponding 3,15-diols (see Scheme 2), Δ8,15-dienes (Scheme 3) or 15 saturated compounds (see Scheme 2).
In scheme 4), it can be rearranged. Other side chain modifications such as 24-keto-, 24-hydroxy- and Δ24-compounds can be obtained by the following route. Ozonolysis of compound 4 and subsequent work with triphenylphosphine gives aldehyde 16. This is linked to an aldol reaction with isopropyl-methyl-ketone to give compound 17 as a mixture of diastereomers. Elimination, hydrogenation and subsequent reduction with Martin's sulfuran gives diol 20 as a mixture of diastereomers (Scheme 6).

Scheme 6 :

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The diol 20 can be eliminated to the next sterol. Treatment with Martin's sulfuran gives a one- or two-eliminated product. Subsequent decomposition of the benzoate can be easily achieved by reduction, yielding the diol 23 and triene 24, respectively (Scheme 7).

Scheme 7 :

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Compounds having an additional bond between C16 and C22 are obtained by the following route. Tosylate 14 can be treated with a base such as lithium diisopropylamide or various Grignard compounds, and the ketone at position 16 can be deprotonated. The enolate intraalkylates 22-tosylate to give the pentacyclic 25. This can be treated with lithium aluminum hydride to give diols 26 and 27 (Scheme 8):

Scheme 8 :

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A further object of the invention is a pharmaceutical composition comprising one or more compounds of the general formula I as active substances. The composition may further comprise pharmaceutically acceptable excipients well known in the art, such as carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting osmotic pressure, tablet disintegration. Peptizers and other ingredients commonly used in the art may be included. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, kratose, sugar, talc,
Gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax and cocoa butter.

[0038] Liquid compositions include sterile solutions, suspensions, and emulsions. Such liquid compositions are suitable for injection or for use with ex vivo and in vitro fertilization. The liquid composition can include other components conventionally used in the art, some of which have been mentioned above. Additionally, compositions for transdermal administration of a compound of the present invention may be supplied in the form of a patch, and compositions for intranasal administration may be provided in the form of an intranasal spray in liquid or powder form. obtain.

The dosage of the compound of the present invention used will be determined by a physician, and will depend on the particular compound used, the route of administration and the intended purpose. In general, the compounds of the invention associate the active ingredient with the liquid or solid auxiliary ingredient intimately, and then
If necessary, it is prepared by shaping the product into the desired formulation. In general, no more than 1000 mg, preferably no more than 100 mg, and preferably no more than 10 mg of a compound of formula I should be administered to a mammal, for example a human, per day.

The present invention relates to the use of a compound of general formula I for the preparation of a meiotic-modulating agent. The compounds of the present invention affect meiosis in oocytes and male germ cells. The compounds of the general formula I are promising as novel fertility-modulating agents based on estrogens and / or gestagens without the usual side effects on somatic cells known from the hormonal contraceptives used hitherto. . In this regard, the biosynthesis of progesterone in cultured human granulosa cells (follicular somatic cells) is not affected by the presence of meiotic regulators, whereas estrogen and estrogens used in hormonal contraceptives that have been used so far. It is important to note that gestagen adversely affects progesterone biosynthesis.

Contraception in women can be achieved by administration of a compound of the present invention that inhibits meiosis and consequently does not produce mature oocytes. Similarly, contraception in men can be achieved by administration of a compound of the present invention, which inhibits meiosis and consequently does not produce mature sperm cells. In a further aspect, the present invention relates to a method of modulating meiosis comprising administering an effective amount of one or more compounds of general formula I to a subject in need of meiosis modulation. The route of administration of a composition comprising a compound of the present invention can be any route that effectively delivers the active compound to its site of action.

Thus, when the compounds of the present invention are administered to a mammal, they are conveniently supplied in a pharmaceutical composition comprising at least one compound of the present invention, together with a pharmaceutically acceptable carrier. You. For oral use, such compositions are preferably in the form of capsules or tablets. When used as a contraceptive, the compounds of the invention should be administered continuously or periodically. In a further aspect, the present invention relates to the use of a 14β-hydrogen group in a sterol compound for increasing the inhibitory activity of a meiotic inhibitor. The invention is further illustrated by the following example.

[0042] Example Example 1: 4,4-dimethyl-5.alpha, 14.beta. ergosta -8,22- diene -3β, 15β- diol: a) 4,4-Dimethyl -5α- ergosta -8,14,22- Trien -3β-yl -benzoate : 40 ml of benzoyl chloride in 4,154 dimethyl-5α in 154 ml of pyridine at 60 ° C.
Ergosta-8,22-dien-3β-ol 30.9 was added in two portions to the solution. The reaction was stirred at the same temperature for 1 hour and then poured into ice water.

The precipitate is collected, washed with ice-cold ethanol, recrystallized from dichloromethane / methanol and dried for 12 hours, 4,4-dimethyl-5α-ergosta-8,14,22-triene- 32 g of 3β-yl-benzoate (mp = 146 ° C.) were obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.80-1.12 (8 × CH ₃ ); 4.75 (dd, J = 16 Hz, 4 Hz, 1H, H
-3α); 5.22 (m, 2H, H-22 / 23); 5.38 (s, wide, 1H, H-15); 7.46 (t, 2H); 7.
57 (t, 1H); 8.07 (d, 2H).

[0044] b) 4,4-Dimethyl -14,15α- epoxy -5α- ergosta -8,22- diene emissions -3β- yl - benzoate: dichloromethane 60 ml, 4,4-dimethyl -5α- ergosta -8, 14, 22−
A solution of 18 g of trien-3β-yl-benzoate was cooled to 10 ° C. After addition of 3 g of m-nitrofluoroacetophenone and 10 ml of saturated sodium hydrogencarbonate solution,
The reaction was stirred for 15 minutes. Then, 12 ml of hydrogen peroxide (30%) were added and the solution was stirred at 10 ° C. for 20 hours. 20 ml of a saturated sodium thiosulphate solution were added and the mixture was stirred for 20 minutes, diluted with dichloromethane and washed with sodium hydroxide solution (5% aqueous solution), water and brine. Drying (magnesium sulfate) and removal of the solvent under vacuum provided the crude epoxide (20 g), which was reacted further without purification.

C) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22 -dien-15-one : Crude epoxy 20 was dissolved in 300 ml of dioxane. The mechanically stirred yellow solution is treated with 2.5 ml of boron trifluoride-etherate for 30 minutes and then
Ice-poured into water. The precipitate was collected, washed with ice-cold water and dried. The crude material is purified by chromatography and 3β-benzoyloxy-
11.55 g of 4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15-one
(mp = 176 ° C.).

¹ H-NMR (d5-pyridine): δ = 0.85 (2 × d, J = 7 Hz, 6H, H-27 / 26); 0.91 (s,
3H, 4-β-CH ₃ ); 0.93 (d, J = 7Hz, 3H, H-28); 1.05 (s, 3H, H-18); 1.6 (s,
3H, 4-α-CH ₃ ); 1.07 (s, 3H, H-19); 1.13 (d, J = 7Hz, 3H, H-21); 4.89 (dd,
J = 13Hz, 4Hz, 1H, 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-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 5β
-Diol : 50 mg of LiAlH ₄ in 30 ml of diethyl ether in 3β-benzoyloxy-4,4
-Dimethyl-5α, 14β-ergosta-8,22-dien-15-one was added to a stirred solution of 300 mg and the mixture was stirred at room temperature for 30 minutes. Next, 1 ml of a saturated ammonium chloride solution was added. After 10 minutes, the solution was filtered and the solvent was removed under vacuum. The residue was separated by chromatography to give 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15β-diol. ¹ H-NMR (CDCl ₃ ): δ = 0.78-1.2 (8 × CH ₃ ); 3.24 (dd, J = 12 Hz, 4 Hz, 1 H, H-
3α); 3.7 (dd, wide, J = 4Hz 4Hz, 1H, H, 15α); 5.24 (m, 2H), H-22 / 23).

Example 2 : 4,4-Dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-diol : a) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8 , 22-Dien-15α-ol : 3β-benzoyloxy-4 in 75 ml of tetrahydrofuran and 25 m of methanol
A solution of 1 g of 1,4-dimethyl-5α, 14β-ergosta-8,22-dien-15-one was treated with 750 mg of sodium borohydride. After stirring at room temperature for 30 minutes, 1 ml of a saturated aqueous ammonium chloride solution was added and the mixture was stirred at room temperature for 15 minutes.

By filtration, removal of the solvent under vacuum and purification by chromatography, 3β
60 mg of benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15α-ol and 770 mg of the corresponding 15β-alcohol were obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.8-1.12 (8 × CH ₃ ); 4.07 (t, wide, J = 4 Hz, 1H, H
-15β); 4.76 (dd, J = 12Hz, 4Hz, 1H, H-3α); 5.23 (m, 2H, H-22 / 23); 7.44
(t, 2H); 7.57 (t, 1H); 8.05 (d, 2H).

B) 4,4-Dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α -diol : 3β-benzoyloxy-4,4-dimethyl-5α in 10 ml of diethyl ether
A solution of 40 ml of 14,14-ergosta-8,22-dien-15α-ol was treated with 10 mg of lithium aluminum hydride at room temperature for 30 minutes as described in Example 1d. After column chromatography, 25 mg of 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-diol was added to a white solid (mp = 148
C). ¹ H-NMR (CDCl ₃ ): δ = 0.78-1.7 (8 × CH ₃ ); 3.25 (dd, J = 11 Hz, 5 Hz, 1 H, H-
3α); 4.04 (t, J = 4 Hz, 1H, H-15α); 5.22 (m, 2H, H-22 / 23).

[0051] Example 3: 4,4-dimethyl-5.alpha, 14.beta. cholesta -8,15- diene -3β- O Lumpur: a) 4,4-Dimethyl -5α- cholesta-8,14-diene -3β- yl - benzoate: in pyridine 5 ml, 4,4-dimethyl -5α- cholesta-8,14-diene -3β
-A solution of 1.0 g of all was treated with 1.5 ml of benzoyl chloride. 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 0.1N aqueous HCl, saturated aqueous sodium bicarbonate and brine.

The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give an oil, which was purified by column chromatography to give 4,4-dimethyl-5α
-Cholesta-8,14-dien-3 [beta] -yl-benzoate 900mg was obtained as a white solid (mp = 158 [deg.] C). ¹ H-NMR (CDCl ₃ ): δ = 0.83 (s, 3H, H-18); 0.87 + 0.88 (2 × s, 3H, H-26 / 2)
7); 0.95 (d, J = 6Hz, 3H, H-21); 4.76 (dd, J = 11Hz, 5Hz, 1H, H-3α); 5.38 (
s, wide, 21, H-15); 7.45 (t, 2H); 7.57 (t, 1H); 8.06 (d, 2H).

B) 4,4-Dimethyl-14,15α-epoxy-5α-cholest-8-ene-3β -yl-benzoate : 4,4-dimethyl-5α-cholesta-8,14-diene in 120 ml of dichloromethane. To a well-stirred suspension of -3β-yl-benzoate, a solution of dimethyldioxirane (130 ml; 0.09-0.11 M in acetone) was added at 0 ° C. 30 at 0 ° C
After stirring for minutes, the reaction mixture was concentrated to dryness under vacuum. The resulting crude epoxide was used without purification.

C) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8- en-15-one : 4,4-dimethyl-14,15α-epoxy-5α-cholest- in 15 ml of dioxane. A solution of 1 g of 8-en-3β-yl-benzoate was treated with 0.1 ml of boron trifluoride-diethyl ether complex. The solution was stirred at room temperature for 20 minutes and then poured into a saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate, washed with water and brine, dried over sodium sulfate and concentrated under reduced pressure. By column chromatography, 3β-benzoyloxy-4
440 mg of 4,4-dimethyl-5α, 14β-cholest-8-en-15-one were obtained as a white solid (mp = 153 ° C.). ¹ H-NMR (CDCl ₃ ): δ = 0.83-1.1 (7 × CH ₃ ); 2.31 (s, wide, 1H, H-14β);
4.7 (dd, J = 11Hz, 5Hz, 1H, H-3α); 7.44 (t, 2H); 7.57 (t, 1H); 8.05 (d,
2H).

D) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8- en-15β-ol : 3β-benzoyloxy-4,4-in tetrahydrofuran (15 ml) and methanol (1 ml). Dimethyl-5α, 14β-cholest-8-en-15-one (440 mg
, 0.83 mmol) was treated with sodium borohydride (125 mg) at room temperature.
After 2 hours, the solution was poured into 0.1N HCl and extracted with ethyl acetate. The organic layer was washed with 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 : 3β-benzoyloxy-4,4-dimethyl-5α in 10 ml of dichloromethane
A solution of 500 mg of 14β-cholest-8-en-15β-ol was cooled to 0 ° C. and 1.1 g of Martin's sulfane was added in one portion. The reaction was stirred at room temperature for 1 hour. The solvent was removed under vacuum. Purification of the residue by chromatography gave 4
315 mg of 2,4-dimethyl-5α, 14β-cholester 8,15-dien-3β-yl-benzoate were obtained as a waxy solid which was used directly.

F) 4,4-Dimethyl-5α, 14β-cholesta-8,15-dien-3β-ol : 4,4-dimethyl-5α, 14β-cholesta-8,15 in anhydrous diethyl ether
A solution of 315 mg of -dien-3β-yl-benzoate was treated with 35 mg of lithium aluminum hydride as described in Example 1d. Column chromatography of the crude product and subsequent recrystallization gave white needles (mp = 138 ° C.)
4,4-dimethyl-5α, 14β-cholesta-8,15-dien-3β-ol 135m
g was obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.81 (s, 3H, H-18); 0.83-1.02 (6 × CH ₃ ); 2.49 (s
, Wide, 1H, H-14β); 3.24 (m, 1H, H-3α); 5.64 + 5.79 (2 x m, 1H, H-15 / 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-Jim
Chill-5α, 14β-pregn-8-en-15-one and (20S) -3β -benzo
Iloxy-20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn- 8-en-15β-ol : 3β-benzoyloxy-4,4-dimethyl-5α, in 88 ml of dichloromethane, 30 ml of methanol and 1 ml of pyridine. A solution of 2.18 g of 14β-ergosta-8,22-dien-15-one (Example 1c) was cooled to -70 ° C. Ozone / oxygen mixture (1: 4)
Was passed through the solution for 12 minutes (adjusted by TLC chromatography).

Next, 130 mg of sodium borohydride were added and the stirred reaction mixture was warmed to 0 ° C. for 2 hours and poured into a 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 is purified by chromatography,
1.28 g of (20S) -3β-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-en-15-one (mp = 238.8 ° C.) and 330 mg
Of the corresponding 15β-hydroxy-compound. ¹ H-NMR (CDCl ₃ ): δ = 0.9-1.1 (4 × CH ₃ ); 1.16 (d, J = 6 Hz, 3H, H-21); 2.3
3 (s, wide, 1H, H-14β); 3.42 (m, 1H, H-22a); 3.63 (m, 1H, H-22b); 4.75
(dd, J = 11Hz, 4Hz, 1H, H-3α); 7.45 (t, 2H); 7.57 (t, 1H); 8.05 (d, 2H)
.

B) (20S) -3β-benzoyloxy-4,4,20-trimethyl-21-toluo
Ylsulfonyloxy-5α, 14β-pregn-8-en-15-one : (20S) -3β -benzoyloxy-20-hydroxymethyl- in 5 ml of pyridine
A solution of 385 mg of 4,4-dimethyl-5α, 14β-pregn-8-en-15-one was added to 0%.
Cooled to ° C. After the addition of 190 mg of p-toluenesulfonic acid chloride, the reaction mixture was stirred at 6 ° C. for 20 hours and then poured into brine and stirred for 20 minutes. The precipitate was collected, washed with ice-cold water and dried at 50 ° C. under vacuum. The crude product (500 mg) was used without further purification.

[0061] c) 3.beta .- benzoyloxy-4,4-dimethyl-24-yl-5.alpha, 14.beta. Kore strike-8-en-15-one: 2-methylpropyl bromide 3.42 g, in anhydrous tetrahydrofuran 25 ml, magnesium (1.6 g of powder) was added slowly at 50 ° C. over 30 minutes to the stirred suspension to give a clear solution of Grignard reagent. Then the mixture was cooled to -10 ° C and 3.32 ml of a solution of Li ₂ Cul ₄ (prepared by mixing 99 mg of cupric chloride, 86 mg of lithium chloride and 10 ml of tetrahydrofuran) were added dropwise.

The reaction mixture was stirred at −30 ° C. for 1 hour and added thereto in 10 ml of tetrahydrofuran.
A solution of 0.5 g of (20S) -3β-benzoyloxy-4,4,20-trimethyl-21-toluolsulfonyloxy-5α, 14β-pregn-8-en-15-one 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 extract was washed with 1N HCl, water and brine, dried over sodium sulfate and evaporated to dryness. The crude product is purified by column chromatography to give 3β-benzoyloxy-4,4-dimethyl- as a white foam.
250 mg of 24-nor-5α, 14β-cholest-8-en-15-one were obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.2-1.09 (7 × CH ₃ ); 2.31 (s, wide, 1H, H-14β);
4.75 (dd, J = 11Hz, 4Hz, 1H, H-3α); 7.44 (t, 2H), 7.57 (t, 1H); 8.05 (d,
2H).

[0063] d) 3.beta .- benzoyloxy-4,4-dimethyl-24-nor-5.alpha, 14.beta. Kore strike-8-en -15β- ol: 3.beta .- benzoyloxy-4,4-dimethyl-24-nor - 130 mg of 5α, 14β-cholest-8-en-3-one was treated with 100 mg of sodium borohydride as described in Example 2a. By aqueous work, 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-15 was obtained as a white foam.
130 mg of β-ol were obtained, which was used directly.

E) 4,4-Dimethyl-24-nor-5α, 14β-cholesta-8,15-dien - 3β -yl-benzoate : 400 mg of Martin's sulfuran are added to 3β-benzoyloxy-4, It was added all at once to a solution of 100 mg of 4-dimethyl-24-nor-5α, 14β-cholest-8-en-15β-ol. The mixture was stirred at room temperature for 18 hours. Evaporation under reduced pressure and chromatography gave 100 mg of 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β-yl-benzoate as an oil, which was further purified Used without purification.

F) 4,4-Dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β -ol : 4,4-dimethyl-24-nor-5α, 14β-cholesta-8, 15-diene-3β
100 mg of -yl-benzoate were treated with 20 mg of lithium aluminum hydride as described in Example 1d. Column chromatography of the crude product and recrystallization (hexane / ethyl acetate) gave 4,4-dimethyl-24-nor-5α, 1
36 mg of 4β-cholesta-8,15-dien-3β-ol (mp = 104.3 ° C.) was obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.82 (s, 3H, H-18); 0.83-1.03 (6 × CH ₃ ); 2.49 (s,
3.24 (m, 1H, H-3α); 5.65 + 5.8 (m, 1H, H-15 / 16).

Example 5 : 4,4-Dimethyl-24-nor-5α, 14β-cholest-8-en-3β -ol : a) 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5α, 14β -cholest-8-en-3β-yl-benzoate : 3β-benzoyloxy-4,4-dimethyl-24-nor-5 in 3 ml of pyridine.
40 mg of α, 14β-cholest-8-en-15β-ol was treated at 0 ° C. with 0.2 ml of methanesulfone sanchloride. After stirring at room temperature for 2 hours, the reaction mixture was diluted with water and extracted with ethyl acetate. Combine the extracts and add 1N H
Washed with C1, water and brine, dried over sodium sulfate and filtered. Removal of the solvent gave 15β-methanesulfonyloxy-4,4-dimethyl-24.
40 mg of -nor-5α, 14β-cholest-8-en-3β-yl-benzoate were obtained, which was used directly.

[0067] b) 4,4-dimethyl-24-nor-5.alpha, 14.beta. cholest-8-en -3β- O Lumpur: 15Beta- methanesulfonyloxy-4,4-dimethyl-24-nor--15α, 14β −
44 mg of cholest-8-en-3β-yl-benzoate were treated with 10 mg of lithium aluminum hydride as described in Example 1d. The crude product was purified by column chromatography to obtain 32 mg of 4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β-ol. ¹ H-NMR (CDCl ₃ ): δ = 0.81 (s, 3H, H-18); 0.82-1.03 (6 × CH ₃ ); 3.25 (dd,
J = 11Hz, 4Hz, 1H, H-3α).

Example 6 4,4-Dimethyl-24-nor-5α, 14β-cholest-8-ene-3β , 15β-diol : 4,4-dimethyl-24-nor-5α, 14β-cholest-8- Ene-3β-ol: 30 mg of 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-15β-ol were prepared as described in Example 1d by lithium aluminum hydride. Treated with 10 mg. The crude product is purified by column chromatography and 4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3
23 mg of β, 15β-diol was obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.82 (s, 3H, H-18); 0.84-1.04 (6 × CH ₃ ); 3.25 (d,
Wide (J = 12Hz, 1H, H-3α); 3.7 (q, wide, J = 7Hz, 1H, H-15α).

Example 7 + 8 : (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β -pregun-8-ene-3β, 15β-diol and (20R) -4,4 20-bird
Methyl -16β, 21- cycloalkyl-5.alpha, 14.beta. Puregun 8-ene-3.beta, 15 [alpha] di-ol: a) (20R) -3β- benzoyloxy -4,4,20- trimethyl -16β, 21-
Cyclo-5α, 14β-pregn-8-en-15-one : (20S) -3β-benzoyloxy-4,4,20-trimethyl-toluolsulfonyloxy-5α, 14β-pregn-8-en-15- 1.10g on Example 4c
And treated with 52 mmol of phenylmagnesium bromide. After the chromatographic treatment, 560 mg of (20R) -3β-benzoyloxy-4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-15-one was converted to tosylate with a phenyl Grignard reagent. Isolated, in addition to other products resulting from the substitution.

B) (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pre
Gun-8-ene-3β, 15β-diol and (20R) -4,4,20-trimethyl
-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15α-diol : (20S) -3β-benzoyloxy-4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-diol 460 mg of pregn-8-en-15-one were treated with 50 mg of lithium aluminum hydride as described in Example 1d. The crude product is purified by column chromatography and (20R) -4,4,20-trimethyl-16β
, 21-Cyclo-5α, 14β-pregn-8-ene-3β, 15β-diol 40 mg and (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene- 30 mg of 3β, 15α-diol was obtained as a white solid.

(20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15β-diol: ¹ H-NMR (CDCl ₃ ): δ = 0.73 ( s, 3H, H-18); 0.82 (s, 3H, H-19); 1.02 (s,
_{6H, 4- CH 3); 1.09} (d, J = 8Hz, 3H, H-21); 2.72 (m, 1H, H-16α); 3.25 (m, 1H
, H-3α); 4.07 (q, J = 8 Hz, 1H, H-15α).

(20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15α-diol: ¹ H-NMR (CDCl ₃ ): δ = 0.79 ( s, 3H, H-18); 0.85 (s, 3H, H-19); 1.0-1.08
(3-CH ₃ ); 2.34 (d, J = 6Hz, 1H, H-14β); 2.68 (m, 1H, H-16α); 3.25 (m, 1H,
H-3α); 4.07 (q, J = 6 Hz, 1H, H-15β).

Example 9 : 4,4-Dimethyl-5α, 14β-cholesta-8,24-diene-3β, 15β -diol : a) (20S) -3β -benzoyloxy-4,4-dimethyl-15- Oxo-5α
, 15β-Pregn-8-en-20-carbaldehyde : 3β-benzoyloxy-4,4 in 87 ml of dichloromethane and 53 ml of methanol.
A solution of 2.2 g of -dimethyl-5α, 14β-ergosta-8,22-dien-15-one was cooled to -70 ° C. An ozone / oxygen mixture (1: 4) was passed through the solution for 16 minutes. 2.62 g of triphenylphosphine were added and the solution was brought to room temperature. The solvent is removed under vacuum and the residue is purified by chromatography, (20
S) -3β-Benzoyloxy-4,4-dimethyl-15-oxo-5α, 15β-
1.26 g of pregun-8-ene-20-carbaldehyde (mp = 205 ° C.) was obtained.

¹ H-NMR (d5-pyridine): δ = 0.85 (2 × d, J = 7 Hz, 6H, H-27 / 26); 0.91 (s,
3H, 4-β-CH ₃ ); 0.90 (s, 3H, H-19); 0.98 (s, 3H, H-18); 1.04 (s, 6H, 4-
CH ₃ ); 1.2 (d, J = 6Hz, 3H, H-21); 2.41 (s, wide, 1H, H-14α); 4.83 (dd, J =
11Hz, 4Hz, 1H, H-3α); 7.5 (t, 2H); 7.58 (t, 1H); 8.27 (d, 2H); 9.82 (d
, J = 2Hz, 1H, H-22).

B) 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-5α, 14β -cholest-8-en-15,24-dione : 25.8 ml of n-butyllithium solution (1.6 M, hexane) was added. Was added to 6 ml of diisopropylamine in 48 ml of tetrahydrofuran at -78 ° C. The mixture for 20 minutes
Stirred. A solution of 4.5 ml of 3-methyl-2-butanone in 10 ml of tetrahydrofuran was added and the mixture was stirred at -78 ° C for 15 minutes. The resulting kinetic enolate is then passed through a cannula under nitrogen in 40 ml of tetrahydrofuran (
20S) -3β-benzoyloxy-4,4-dimethyl-15-oxo-5α, 14β
-Transferred to a cooled (-78 ° C) solution of 6.0 g of pregn-8-en-20-carbaldehyde. The mixture was brought to 0 ° C. for 1 hour and poured into saturated aqueous ammonium chloride.

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.
After removal of the solvent under vacuum, the residue was separated by column chromatography,
6.26 g of 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-5α, 14β-cholest-8-en-15,24-dione were obtained as an oil. ¹ H-NMR (CDCl ₃ ): δ = 0.92-1.2 (7 × CH ₃ ); 2.33 (s, wide, 1H, H-14β);
4.1 (m, 1H, H-22); 4.74 (dd, J = 11Hz,, 4Hz, 1H, H-3β); 7.45 (t, 2H); 8.05 (d
, 1H); 4.07 (q, 2H).

C) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta- 8,22-diene-15,24-dione : 3β-benzoyloxy-22-hydroxy-4,4 in 60 ml of dichloromethane. −
A solution of 4.78 g of dimethyl-5α, 14β-cholest-8-ene-15,24-dione was added to 0.
C. and 9.31 g of Martin's sulfuran was added in one portion. The reaction mixture was stirred at 0 ° C. for 30 minutes. The solvent was removed under vacuum. The residue was purified by column chromatography to give 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,22-diene-15,24-dione (mp = 163 ° C.) 4.1
8 g were obtained. ¹ H-NMR (CDCl ₃ ): δ = 0.9-1.2 (7 × CH ₃ ); 2.31 (s, wide, 1H, H-14β); 4.
75 (dd, J = 11Hz, 4Hz, 1H, H-3α); 6.14 (d, J = 11Hz, 1H, H-23); 6.71 (m, 1H,
H-22); 7.45 (t, 2H); 7.57 (t, 1H); 8.05 (d, 2H).

D) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8- ene-15,24-dione : On charcoal, 500 mg of palladium (10%) is added to 3β- Benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,22-diene-15,24-
Dione was added to a solution of 4.2 g. The mixture was stirred under a hydrogen atmosphere for 3 hours. The mixture was filtered. After removal of the solvent under vacuum, 4.22 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15,24-dione was obtained as a white crystalline residue. ¹ H-NMR (CDCl ₃ ): δ = 0.9-1.16 (7 × CH ₃ ); 2.33 (s, wide, 1H, H-14β);
4.74 (dd, J = 11Hz, 4Hz, 1H, H-3α); 7.45 (t, 2H); 7.57 (t, 1H); 8.05 (d, 2H)
.

E) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8- ene-15,24-diol and 3β-benzoyloxy-24-hydroxy-4,4 -dimethyl-5α, 14β -Cholest -8-en-15-one : 3β-benzoyloxy-4,4-dimethyl-5α, 14 in 45 ml of methyl chloride.
A solution of 4.2 g of β-cholest-8-ene-15,24-dione was added to tert-butylamine-
Treated with 1.8 g of borane complex. The solution was refluxed for 1 hour. After cooling to 0 ° C., 30 ml of HCl (1N, aqueous) were added. The mixture was stirred at 0 ° C. for 1 hour. It was washed with HCl (1N, aqueous), water, saturated aqueous sodium bicarbonate and brine. After drying over sodium sulfate, the solvent was removed in vacuo.
By column chromatography, as a white foam, 3β-benzoyloxy-24-hydroxy-4,4-dimethyl-5α, 14β-cholest-8-ene- was obtained.
1.71 g of 15-one was obtained.

3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-diol: ¹ H-NMR (CDCl ₃ ): δ = 0.88-1.1 (7 × CH ₃ ) ; 3.33 (m, 1H, H-24); 3.74 (m, 1
H, H-15); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3α); 7.45 (t, 2H); 7.57 (t, 1H); 8
.05 (d, 2H). 3β-benzoyloxy-24-hydroxy-4,4-dimethyl-5α, 14β-
Cholest-8-en-15-one: ¹ H-NMR (CDCl ₃ ): δ = 0.88-1.1 (7 × CH ₃ ); 2.32 (s, wide, 1H, H-14β);
33 (m, 1H, H-22); 4.74 (dd, J = 11Hz, 4Hz, 1H, H-3α); 7.45 (t, 2H); 7.57 (t
, 1H); 8.05 (d, 2H).

F) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta -8,24- dien-15β-ol and 4,4-dimethyl-5α, 14β-cholesta- 8,15,24- Trien -15β-yl-benzoate : 1.0 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15,24-diol was added to Martin's sulfran 3.88 as described in Example 3e. g. By column chromatography, 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8 was obtained as a white foam.
, 24-Diene-15β-ol (305 mg) and 4,4-dimethyl- as a white solid
5α, 14β-cholesta-8,15,24-trien-15β-yl-benzoate 870 m
g was obtained.

3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,24
-Dien-15β-ol: ¹ H-NMR (d5-pyridine): δ = 1.15 (s, 1H, H-18); 2.84 (m, 1H, H-7α); 4
.1 (m, 1H, H-1); 4.93 (dd, J = 11Hz, 4Hz, 1H, H-3α); 5.24 (t, J = 8Hz, 1H, H-
24); 7.45 (t, 2H); 7.57 (t, 1H); 8.25 (d, 2H). 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-15β-yl-benzoate: ¹ H-NMR (d5-pyridine): δ = 1.4 (s, 3H, H-19); 2.19 (m, 1H, H-17α); 2.
51 (s, wide, 1H, H-14β); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3α); 5.1 (t, J = 8Hz
, 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-5α, 14β-cholesta-8,24-diene-3β, 15β- diol : 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,24
120 mg of -dien-15β-ol were treated with 30 mg of lithium aluminum hydride as described in Example 1d. The crude product was subjected to flash column chromatography to obtain 80 mg of 4,4-dimethyl-5α, 14β-cholesta-8,24-diene-3β, 15β-diol as a white foam. ¹ H-NMR (CDCl ₃ ): δ = 0.8-1.01 (5 × CH ₃ ); 1.6 + 1.68 (2 × s, 3H, H-26 / 27);
3.24 (m, 1H, H-3α); 3.70 (m, 1H, H-15α); 5.10 (t, J = 7 Hz, 1H, H-24).

Example 10 : 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β -ol : 4,4-dimethyl-5α, 14β-cholesta-8,15,24-triene 30 mg of -15β-yl-benzoate (Example 9f) were treated with 20 mg of lithium aluminum hydride as described in Example 1d. The crude product was purified by column chromatography to give 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-ol (mp = 105 ° C.) as a white solid. . ¹ H-NMR (CDCl ₃ ): δ = 0.81 (s, 3H, H-18); 0.94-1.02 (4 × CH ₃ ); 2.5 (s, wide, 1H, H-14β); 3.23 (m, 1H) , H-3α); 5.11 (t, J = 9Hz, 1H, H-24); 5.64 (m, 1
H, H-15 / 16a); 5.79 (m, 1H, H-15 / 16b).

Example 11 : (20S) -20-hydroxymethyl-4,4-dimethyl-5α, 14β-prop
Regun-8-ene-3β, 15β-diol : 60 mg of (20S) -20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-8-ene-3β, 15β-diol are described in Example 1d. Thus, it was treated with 20 mg of lithium aluminum hydride. The crude product is purified by column chromatography and treated with (20S) -20-hydroxymethyl-4,4-dimethyl-
42mg of 5α, 14β-pregn-8-ene-3β, 15β-diol (mp = 214 ° C)
I got ¹ H-NMR (CDCl ₃ ): δ = 0.82 (s, 3H, H-18); 0.93-1.04 (3 × CH ₃ ); 1.07 (d, J
= 6Hz, 3H, H-21); 3.23 (m, 1H, H-3α); 3.49 (m, 1H, H-22a); 3.68 (m, 1H, H-2
2b); 3.72 (m, 1H, H-15α).

Example 12 : Testing of meiosis-activators in the oocyte test : Animals : 13-16 g immature female mice (C) maintained under controlled light and temperature
Oocytes were obtained from 57Bl / 6J × DBA / 2J F1-hybrid, Bomholtgaard, Denmark). Mice were treated with 0.2 ml of gonadotropin (containing 20 IU of FSH, Gonal F, Serono,
Solna, Sweden, on the other hand, received an intraperitoneal injection of Puregon, Organon, Swords) containing 20 IU of FSU, and 48 hours later the animals were killed by cervical dislocation.

Oocyte Harvesting and Culture : The ovaries are dissected and the oocytes are excised by manual cleavage of vesicles using a pair of 27 gauge needles under a stereomicroscope under Hx-medium ( See below). Naked spherical oocytes (NO) exhibiting intact germinal vesicles (GV) were purified from 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg / ml human serum albumin (HAS, State Serum).
Institute, Denmark), 0.23 mM pyruvate (Sigma. Cat. No. S-8636), 2 mM
Glutamine (Flow Cat. No. 16-801), 100 IU / ml penicillin and 100 μg / ml
Α-MEM essential medium (α-MEM without ribonucleoside, Gibco BRL, Cat. No. 22561) supplemented with Streptomycin (Flow, Cat. No. 16-700).

This medium was named Hx-medium. Oocytes were rinsed three times with Hx-medium and cultured in 4-well multi-dishes (Nuncion, Denmark), where each well contained 0.4 ml of Hx-medium and 35-45 cells. Oocytes included. One control (i.e., 35-45 cultured in Hx-medium without the addition of test compound)
Oocytes) were always tested at the same time as test cultures performed with different concentrations of the compound to be tested. Cultures were performed at 37 ° C. and 100% humidity in air under 5% CO ₂ . The culture time is
22-24 hours.

Testing of oocytes : At the end of the culture period, the number of oocytes with germinal vesicles (GV) or germinal vesicle breakdown (GVB) and those with polar bodies (PB) were determined Counting was performed using a stereo microscope or an inverted microscope equipped with a differential interference contrast device. GV per total number of oocytes
The percentage of oocytes with B and the percentage of oocytes with PB per total number of oocytes were calculated in the test culture and compared to the control culture.

Example 13 : Testing of meiosis-inhibitors in the oocyte test : Germinal vesicle (GV) oocytes were isolated using the same method as described in Example 1 (see above). Obtained from adult FSH-treated female mice. Naked oocytes (NO) were converted to Hz-
Rinse several times with medium. 4,4-Dimethylcholest-B, 14,24-trien-3β-ol (FF-MAS) has previously been shown to induce meiosis in NO in vitro (Byskov, AG et al. Nature 374). (1995) 559-562)
.

NO was cultured in 4-well multi-dish (Nunclon, Denmark) with test compound at different concentrations in Hx-medium supplemented with 5 μM FF-Mas, where the individual wells were , 0.4 ml of Hx-medium and 35-45 oocytes. One positive control (i.e., 35-45 oocytes cultured in Hx-medium with FF-MAS without the addition of test compound) was treated with various concentrations of the compound to be tested. It was always tested at the same time as the supplemented test culture. In addition, one negative control (35-45 oocytes cultured in Hx-medium only) was tested simultaneously with the positive control.

Testing of oocytes : At the end of the culture period, the number of oocytes with germinal vesicles (GV) or germinal vesicle breakdown (GVB) and those with polar bodies (PB) was determined. Counting was performed using a stereo microscope or an inverted microscope equipped with a differential interference contrast device. GV per total number of oocytes
The percentage of oocytes with B + PB was calculated in test and 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)) × 100 / GVB + PB (positive control) −GVB + PB (negative control)]%. Oocytes arrested in meiosis are characterized by intact nuclei with protruding nuclei, known as germinal vesicles (GV). Upon meiotic reinitiation, the nucleus and nuclear envelope are extinguished, which is characterized by a breakdown of GV called germinal vesicle breakdown (GVB). After several hours, the oocyte has completed meiosis and triggers the first so-called polar body (PB). result:

[0094]

[Table 1]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA , ZW (72) Inventor Blume, Torsten Germany, D-13467 Berlin, Kurhausstrasse 18 (72) Inventor Hegele-Hartung, Krista Germany, D-45470 Mülheim / Luer, Boerenbeck 101 (72) Inventor Wrestle, Monica Germany, Day 13467 Berlin, Bilhelmstrasse 9F term (reference) 4C086 AA01 AA02 AA03 DA08 MA01 MA02 MA03 MA04 MA05 NA14 ZA81 ZA86 ZB21 ZC54 4C09 1 AA01 BB10 BB20 CC01 DD01 EE04 EE05 FF02 FF06 GG01 HH01 JJ03 LL04 LL09 MM03 NN01 PA02 PA05 PB04 PB05 QQ01 QQ05

Claims (11)

[Claims] 1. The following general formula I: [Wherein, R ³ represents an additional bond together with a hydrogen atom or R ^{3 ′} , R ^{3 ′} represents an additional bond together with a hydrogen atom or R ³ , and R ⁴ represents a hydrogen atom or methyl. Represents a group, R ^{4 ′} represents a hydrogen atom or a methyl group, R ⁷ represents an additional bond together with a hydrogen atom or R ⁸ , R ⁸ represents a hydrogen atom, or together with R ⁷ or R ⁹ , Represents an additional bond, R ⁹ represents a hydrogen atom, or together with R ⁸ or R ¹¹ , represents an additional bond, R ¹⁵ represents a hydrogen atom, a hydroxy group, a halogen atom, or an oxo group together with R ^{15 ′} , or with R ^16, an additional bond, or R ^{22 'together} represent an oxygen bridge, R ^15' represents a hydrogen atom, C ₁ -C ₈ linear or branched alkyl group, C ₆ -C ₁₀ aryl group, or Together with R ¹⁵ represents an oxo group; R ¹⁶ represents a hydrogen atom, a hydroxy group, a halogen atom, or an additional bond together with R ¹⁵ or R ²² , R ²² is a hydrogen atom, C ₁ -C ₈ linear or branched optionally optionally substituted alkyl or alkenyl group, optionally substituted on C ₆ -C ₁₀ aryl group, or with R ^16, an additional bond, or R ^{22 'together} represent a C ₁ -C ₈ linear or branched chain alkylidene group, R ^22' represents a hydrogen atom, hydroxy group, or together with R ^15, an oxygen bridge, or with R ^22, C ₁ -C ₈ linear or compounds represented by the representative of the branched chain alkylidene group, or an ester. 2. The compound according to claim 1, wherein R ³ and R ^{3 ′} represent a hydrogen atom. 3. The method of claim 1, wherein R ⁸ and R ⁹ together represent an additional bond.
A compound as described. 4. The compound according to claim 1, wherein R ¹⁵ represents a hydrogen atom or a hydroxy group. Wherein R ²² is, C ₁ -C ₈ linear or optionally substituted which may be an alkyl group branched, or together with R ^{22 ',} C ₁ -C ₈ straight or branched chain 5. The compound according to claim 1, which represents an alkylidene group. 6. 4,4-dimethyl-5α, 14β-cholest-8-ene-3β
-Ol, 4,4-dimethyl-5α, 14β-cholest-8-ene-3β, 15β-diol, 4,4-dimethyl-5α, 14β-cholest-8-ene-3β, 15α-diol, 4,4 -Dimethyl-5α, 14β-cholesta-8,15-dien-3β-ol, 4,4-dimethyl-5α, 14β-cholesta-7,9 (11), 15-triene-3
β-ol, 4,4-dimethyl-5α, 14β-cholesta-8,15,23 (E) -triene-3
β-ol, 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-ol, 4,4-dimethyl-5α, 14β-cholesta-8,24-diene-3β, 15β- Diol, 4,4-dimethyl-5α, 14β-ergosta-8,22-dien-3β-ol, 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15β-
Diols, 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-
Diols, 4,4-dimethyl-5α, 14β-ergosta-8,15,22-triene-3β-
All, 4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β-ol, 4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3β, 15β
-Diol, 4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3β, 15α
-Diol, 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-diene-3β
-Ol, 4,4-dimethyl-5α, 14β-cholesta-8,15-diene-3β-ol hydrogen butanediate, 4,4-dimethyl-5α, 14β-ergosta-8,15,22-triene-3β −
(20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15α-diol, (20R) -4,4,20-diol Trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15β-diol, (20S) -20-hydroxymethyl-4,4-dimethyl-5,14β-pregn-
The compound according to any one of claims 1 to 5, which is 8-ene-3β, 15β-diol. 7. A pharmaceutical composition comprising one or more compounds of the general formula I according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient. 8. Use of a compound of general formula I according to any one of claims 1 to 6 for the preparation of a meiosis regulating agent. 9. Use according to claim 8, for the preparation of a contraceptive medicament for the treatment of women or men, preferably humans. 10. A method of regulating meiosis, comprising administering an effective amount of one or more compounds of general formula I according to any one of claims 1 to 6 to a subject in need of such regulation. A method comprising: 11. Use of a 14β-hydrogen group in a sterol for increasing the inhibitory activity of a meiosis inhibitor.