CZ20013981A3 - 14beta-H-sterols, their use as medicaments and pharmaceutical preparations in which the sterols are comprised - Google Patents

Abstract

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

Description

143-H-jterols, their use as medicaments and pharmaceutical compositions containing them

Technical field

The present invention relates to pharmaceutically active sterols, pharmaceutical compositions containing such sterols as active ingredients, and the use of these novel compounds for the preparation of medicaments. In particular, it has been found that the sterols of the present invention can be used to control meiosis.

BACKGROUND OF THE INVENTION

Meiosis is a unique and basic process of germ cells on which sexual reproduction is based. Meiosis involves two meiotic divisions. During the first meiotic division, the exchange between the maternal and paternal genes takes place before the chomosome pairs are divided into two daughter cells. These contain only half the number of chromosomes, i.e. 1n, and 2c DNA. The second meiotic division proceeds without DNA synthesis. Therefore, this division results in an array of haploid germ cells containing only 1c DNA.

Meiotic processes are similar in male and female germ cells, but the time course and differentiation processes that result in egg and sperm vary widely. In all female germ cells, the prophase of the first meiotic division ends early in life, often before birth, but all are later suspended as oocytes in the prophase, i.e., the meiosis is at rest, i.e., in the dictyotene stage, until ovulation after puberty. Thus, from the beginning of life, the female has a supply of oocytes that are released until the supply is depleted. Meiosis in females is not fully completed until post-fertilization, resulting in only one egg and two abortive polar bodies per germ cell. In contrast, only a few males after puberty 4 4

Germ cells terminate meiosis and leave the germ cell population throughout life. As soon as meiosis begins in male cells, it proceeds without noticeable delay and results in 4 spermatozoa.

little is known about the mechanism that regulates the onset of meiosis in males and females. New studies have shown that in oocytes, follicular purines, such as hypoxanthine or adenosine, may be responsible for suspending meiosis. e.g., Downs, S.M. et al., Dev Biol 82, 1985, pp. 454-458, Epplg, J. J. et al., Dev Biol 119, 1986, pp. 313-321, and Downs, S. Μ. Mol Reprod Dev 35, 1993, pp. 82-94. The presence of a diffusible meiosis regulating agent was first described by Byskov et al. in the fetal mouse gonadal culture system in Byskov, AG et al., Dev Biol 52, 1976, pp. 193-200. The meiosis-activating substance (MAS) was secreted by the fetal murine ovary in which meiosis continued and the meiosis precursors (MPS) were it released from a morphologically differentiated testicle with resting germ cells in which the meiosis did not occur. This indicated that relative concentrations of MAS and MPS regulate the onset, pause, and resumption of meiosis in female and male germ cells. For example, Byskov, A.G. et al., The Physiology of Reproduction, (edited by Knobil, E. and Neill, J. D.), Raven Press, New York, 1994. If meiosis can be controlled, of course reproduction can also be controlled. Recent articles, such as Byskov, A.G. et al., Nature 374, 1995, pp. 559-562, describe the isolation of certain sterols, T-MAS and FF-MAS, that activate meiosis in oocyte, bull testicles and human follicular fluid.

Compounds known to control meiosis are described in WO 96/27658, WO 97/00884, WO 98/28323 and WO 98/52965.

It is an object of the present invention to provide novel compounds useful as contraceptives and in females and males, particularly humans, which act by inhibiting meiosis.

SUMMARY OF THE INVENTION

The present invention relates to 14β-H-sterols of formula I,

(I) wherein R ³ or together with R ³ '

R

R ⁴

R ⁷

R ^a

R ^s

15 'means hydrogen atom forms another bond, means hydrogen atom forms another bond, represents hydrogen atom or methyl group, represents hydrogen atom or methyl group, represents hydrogen atom or together with a substituent forms another bond, represents hydrogen atom or together with R ⁷ or together with R ⁹ forms a further bond, represents a hydrogen atom or together with R ⁸ , or or together with R ¹¹ forms an additional bond, represents a hydrogen atom, a hydroxy group, a halogen atom, or together with a substituent R ¹⁵ forms an oxo group, or together with R ¹⁶ forms another bond, or together with R ²² 'forms an oxygen bridge, means a hydrogen atom, a branched or unbranched alkyl group having 1 to 8 carbon atoms, or together with a substituent R ³ · ·

4 ·

R 'is C 6 -C 10 aryl, or together with R ¹⁵ forms oxo, represents hydrogen, hydroxy, halogen, or together with R ¹⁵ forms another bond, or together with R ²² forms another bond, a hydrogen atom, an optionally substituted, branched or unbranched, alkyl or alkenyl group having 1 to 8 carbon atoms, an optionally substituted aryl group having 6 to 10 carbon atoms, or together with R ¹⁶ forms

R '

22 'further bond, or together with the substituent R a branched or unbranched alkylidene containing 1 to 8 carbon atoms, represents a hydrogen atom, a hydroxy group, or

22 'together with the substituent R forms together with the substituent unbranched alkylidene carbon atoms, or esters thereof.

group together or an oxygen bridge,

v. /

They form a branched or group containing from 1 to 8

The term "alkyl, used alone or in combination," means a straight or branched alkyl group in the present specification and claims. The term "C 1 -C 8 alkyl" refers to an alkyl group containing from one to eight carbon atoms, preferred examples of which are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl and cyclohexyl. The term "alkenyl" refers to an unsaturated alkyl group. Preferred examples are vinyl, allyl, isopropenyl and prenyl. The term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. For example, "R ^15, together with R ¹⁵ ', forms the oxo group used in the present specification and claims, meaning that the oxo group, i.e., the O = group, is present at the 15-position, and therefore there is no atom at the 15-position. hydrogen. The term "C 6 -C 10 aryl" refers to a phenyl group optionally substituted by halogen, C 1 -C 4 alkoxy, hydroxy or C 1 -C 4 alkyl. The term "straight or branched alkylidene group having 1 to 8 carbon atoms" means an alkylidene group having one to eight carbon atoms. This group is bonded at the 22-position through a double bond to the steroid carbon atom. Preferred examples are methylene, ethylidene, propylidene, isopropylidene, butylidene, isobutylidene, pentylidene, isopentylidene, neopentylidene and cyclohexylidene.

Surprisingly, it has been found that the β-configuration of the hydrogen atom at position 14 is important for meiosis-inhibiting activity (this is the numbering of carbon atoms according to the IUPAC nomenclature). In the presence of 14β-H-sterols, the meiosis-inhibiting effect of FF-MAS is impaired or completely suppressed. The meiosis regulating agents hitherto described are 14a-H and 1414-cholestane derivatives (Byskov, AG et al., Nature 374, 1995, pp. 559 to 562), showing either meiosis-activating activity or low inhibitory activity. The H-sterols of the present invention strongly antagonize the effect of natural FF-MAS and are thus meiosis inhibitors.These findings have made the disclosed compounds of particular interest for contraception Preferred compounds of Formula I are those that inhibit germinal disintegration by at least 20%, preferably 40 %, refined by 60% when tested with an oocyte assay as described in Example 13 and which did not activate meiosis in the oocyte assay described in Example 12.

• · • ·

The compounds of formula I have several chiral centers in the molecule and therefore occur in several isomeric forms. All such isomeric forms and mixtures thereof are within the scope of the present invention unless otherwise indicated. Preferred compounds of formula (I) are those having a 3β-hydroxy group and a δδ-double bond. Further preferred are compounds of formula I wherein R ¹⁵ is hydrogen or hydroxy. Further preferred compounds of formula I are those wherein R ²² is a straight or branched, optionally substituted, C 1 -C 8 alkyl group or, together with R ²² ', forms a straight or branched C 1 -C 8 alkylidene group atoms.

In another embodiment, the present invention relates to esters of compounds of formula I. Such esters are formally derived by esterifying one or more hydroxyl groups of a compound of formula I with an acid, which may be selected from the group consisting of succinic acid, glutaric acid and other aliphatic dicarboxylic acids. nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulfonic acid, amidosulfonic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.

Particularly preferred compounds of formula I of the present invention are as follows:

4,4-dimethyl-5α, 14β-cholest-8-en-3β-ol,

4,4-dimethyl-5α, 14β-cholest-8-en-3β, 15β-α1,

4,4-dimethyl-5α, 14β-cholest-8-en-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-3β-ol,

4,4-dimethyl-5α, 14β-cholesta-8,15,23 (E) -trien-3β-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-diol,

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

4,4-dimethyl-5α, 14β-ergosta-8,15,22-trien-3β-ol,

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

4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β, 15β-α1,

4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β, 15α-diol,

4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β-ol,

4,4-dimethyl-5α, 14β-cholesta-8,15-dien-3β-ol hydrogenbutanedioate,

4,4-dimethyl-5α, 14β-ergosta-8,15,22-triene-3β-ol hydrogenbutanedioate, (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8- en-3β, 15α-diol, (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-3β, 15β-diol, (20R) -20-hydroxymethyl- 4,4-dimethyl-5α, 14β-pregn-8-en-3β, 15β-diol,

The compounds of formula (I) of the present invention can be synthesized in a manner similar to the preparation of known compounds. Therefore, the syntheses of the compounds of formula (I) may follow the well established synthetic routes described in the extensive sterol and steroid literature. The following literature can be used as an aid in synthesis:

L. Fieser & M. Fieser: Steroids, Reinhold Publishing

Corporation, NY 1959

Year with Chemistry of Carbon Compounds (editor S. Coffrey):

Elsevier Publishing Company, 1971, and in particular

Dictionary of Steroids (editors by R. A. Hill, D. N. Kirk,

H. L. J. Makin and G. M. Murphy): Chapman &

The latter contains a comprehensive list of citations to original articles covering the period up to 1990.

»· · · ·

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

Sterols used as starting materials can be synthesized by methods described in the literature:

4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol (Biochem. J. 132,

1973, p. 439)

4,4-dimethyl-5α-ergosta-8,14,22-trien-3β-ol (as benzoate:

J. Org. Chem. 51, 1986, p. 4047)

5α-cholesta-8,14-dien-3β-ol (<J. Am. Chem. Soc. 75, 1953, p. 4404)

5α-ergosta-8,14,22-ηη-3β-ο1 (J. Org. Chem. 53, 1988, p.

1563).

In the following, only the compounds containing

4,4-dimethyl group. Compounds which are not substituted at the 4-position can be obtained in a similar manner.

benzoate. such as in the presence of peroxyacids.

formula 4 can be

14P-H-derivatives can be synthesized from Δ-8,14-pulp systems of Formula 1 through alcohol protection, epoxidation and subsequent epoxide opening (see Scheme 1). 3-Alcohol Can Be Protected As Epoxidation can be achieved with reagents similar to, for example, dimethyldioxiran, hydrogen peroxide, various catalysts, m-CPBA and other.

Lewis acids such as boron trifluoride (see Chem. Pharm. Bull. 38, 1990, p. 1796).

Scheme 1

BzO

Tt tttttttt tt «tt tttttt

The reactions can be carried out in the presence of various steroid side chains, i.e. R _s , such as, for example, cholesterol, ergosterol, sitosterol or stigmasterol side chains.

When the 15-ketones of formula 4 are reduced with lithium aluminum hydride, 3β, 15β-γίο1γ of formula 5 are obtained. The corresponding 15α-alcohols can be obtained as less common diastereomers of formula 6 by reduction with sodium borohydride. Subsequent cleavage of the hydroxy protecting group at the 3-position results in the 3β, 15α-diols of formula 7 (see Scheme 2).

Scheme 2

The following general formula (4) can be obtained by the general formula (8), i.

to 88,15-diene

8,15-diene of general formula 10 according to the procedure. Reduction of the sodium borohydride ketone results in a 15β-alcohol more common diastereomer. The elimination of formula 9 protecting formula 10 can be accomplished with Martin's sulphate. Deleting a group leads (see scheme 3).

the desired alcohols

Scheme 3

Rc

Derivatives which are saturated at the 15-position can be obtained by the following route. The 15β-alcohols of formula 8 can be reacted with methanesulfonic acid chloride. The mesylate of formula 11 can then be reduced with lithium aluminum hydride. The result is compounds of formula 12 (see Scheme 4).

Scheme 4

BzO

BzO

9 9

9

Modification of the side chains of the ozonolysis of the ergosterol side formula 4, wherein R _s teroi is an ergosterol side chain. After reduction with sodium borohydride, the hydroxy group at the 22-position of the 22-alcohol of formula 13 can be converted to a leaving group such as tosylate (see Scheme 5). The tosylate 14 can be reacted with a variety of branched or straight chain alkyl, alkenyl, or aryl Grignard reagents to catalyze the copper, resulting in compounds of Formula 15.

can be accomplished by the chain of compound i

Scheme 5

These compounds can be converted to the corresponding 3,15-diols (see Scheme 2), 88,15-dienes (see Scheme 3) or saturated compounds (see Scheme 4) as described above.

4 ·

4444

4 4 • 4 4 44 • 4 4

444

Other side chain modifications, such as the 24-keto, 24-hydroxy and A24 compounds, can be obtained by the following route. Ozonolysis of the compound of formula 4 and subsequent treatment with triphenylphosphine results in an aldehyde of formula 16. This can be subjected to an aldol reaction with an isopropyl methyl ketone to give a compound of formula 17 as a mixture of diastereomers. Elimination with Martin's sulphate, hydrogenation and subsequent reduction affords the diol of formula 20 as a mixture of diastereomers (see Scheme 6).

Scheme 6

BzO

^H OH ·· ♦♦♦ ·

The diols of formula 20 can be subjected to elimination resulting in the following sterols. Treatment with Martin's sulphate yields the mono- and bis-eliminated product. Subsequent cleavage of the benzoate can readily be achieved by reduction, resulting in a diol of formula 23 and a triene of formula 24, respectively (see Scheme 7).

Scheme 7 · «φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ · * »· ···

Compounds with an additional bond between the carbon atom at the 16-position and the carbon atom at the 22-position can be obtained by the following route. The tosylate 14 can be reacted with a base, such as lithium diisopropylamide or various Grignard compounds, to deprotonate the ketone at the 16-position. The enolate is intramolecularly alkylating the 22-tosylate to form the pentacyclic ring 25. This can be reacted with lithium aluminum hydride,

99 «· · 9999

99999 9 9 99 9

9 «« · 9 9 · 9 · 9

9 9 9 999

999 999 999 99 999 resulting in diols of formula 26 and formula 27 (see Scheme 8).

Scheme 8

The present invention further relates to pharmaceutical compositions containing one or more compounds of formula I as active ingredient. The compositions may further comprise pharmaceutically acceptable excipients well known in the art, such as carriers, diluents, absorption enhancers, preservatives, buffers, osmotic pressure control agents, tablet disrupting agents, and other additives commonly used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting waxes, and cocoa butter.

Liquid compositions include sterile solutions, suspensions, and emulsions. Such liquid compositions may be suitable for injection or use in connection with ex vivo and in vitro fertilization. Liquid compositions may contain components that are commonly used in the art, some of which are mentioned in tt tt tt tttttttt tttttt tttt tttt tttttttt · tt tt tttttt · ^z · t · ttt · t · «• tt enumeration above. Further, the composition for transdermal administration of the compounds of the present invention may be provided in the form of a patch, and the composition for nasal administration may be provided in the form of a nasal spray, in liquid or powder form.

The particular dose of the compound of the invention to be used will be determined by the attending physician, which dose will depend on a variety of factors, for example, the particular compound employed, the mode of administration and the intended use. In general, the compositions of the invention are prepared by intimately mixing the active compound with liquid or solid excipients and then, if necessary, shaping them into the desired form.

Usually, no more than 1000 mg, preferably no more than 100 mg, and in some preferred cases no more than 10 mg, of a compound of Formula I are administered to mammals, e.g.

in Compound oocytes

The present invention relates to the use of compounds of formula I for the preparation of a medicament for controlling meiosis. The compounds of the present invention affect meiosis as well as in male germ cells, Formula I appears to be promising new fertility regulating agents without the usual somatic cell side effects known to date of estrogen-based and / or estrogen-based hormonal contraceptives. gestagenech.

In this context, it is important to note that progesterone biosynthesis in human granulosa cell culture, i.e. somatic follicle cells, is not affected by the presence of a meiosis regulating agent, whereas estrogens and gestagens contained in hitherto used hormonal contraceptives have an adverse effect on progesterone biosynthesis. .

• 4 9 9 4 3 4 5 6 7 8 9

4 4 4 4 9 9 4 4 • 44 4

445 445 44 4 4 «4

Contraception in females can be achieved by administering a compound of the invention that blocks meiosis so that no mature oocytes are formed. Similarly, male contraception can be achieved by administering a compound of the invention that blocks meiosis so that no mature sperm is formed.

In another embodiment, the present invention relates to a method for controlling meiosis, comprising administering to a patient in need thereof an effective amount of one or more compounds of Formula I.

The route of administration of compositions comprising a compound of the invention may be any route that provides effective transport of the active compound to its site of action.

Therefore, when administered to a mammal, the compounds of the present invention are generally provided in the form of a pharmaceutical composition comprising at least a compound of the invention together with a pharmaceutically acceptable carrier. Such compositions for oral administration are preferably in the form of capsules or tablets.

The compounds of the invention should be administered either continuously or cyclically when used as contraceptives.

In another embodiment, the present invention relates to the use of a 14β-hydrogen group in a sterol compound to enhance the inhibitory activity of a meiosis inhibiting agent.

The present invention is further illustrated by the following examples.

• • • • • · * • • · • • ·· • • • • • * • • • • • • • t ··· a · a ··· »4

DETAILED DESCRIPTION OF THE INVENTION

Example 1

4,4-dimethyl-5α, 14β-ergosta-8,22-ßίβη-3β, 15β- <ϋο1

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

To a solution of 30.9 g of 4,4-dimethyl-5α-ergosta-8,22-β-β-3β-η1 in 154 ml of pyridine was added at 60 ° C 40 ml of benzoyl chloride in two portions. The reaction mixture was stirred at the same temperature for 1 hour and then poured into ice water. The precipitate was collected, washed with ice-cold ethanol, recrystallized from a mixture of dichloromethane and methanol, and then dried for 12 hours. 32 g were obtained

4,4-dimethyl-5α-er-gosta-8,14,22-η-3-β-1-β-α, m.p. 146 ° C.

^* H-NMR (CDCl₃): 8 = 0.80 to 1.12 (8x _CH3); 4.75 (dd, J = 16Hz, 4Hz, 1H, H-3a); 5.22 (m, 2H, H-22/23); 5.38 (broad s, 1H, H-15); 7.46 (t, 2 H); 7.57 (t, 1H); 8.07 (d, 2H)

(b) 4,4-dimethyl-14,15a-epoxy-5a-ergosta-8,22-β-β-β-1-β-benzoate

A solution of 18 g of 4,4-dimethyl-5α-ergosta-8,14,22-trien-3β-yl-benzoate in 60 ml of dichloromethane was cooled to 10 ° C. After addition of 3 g of m-nitrofluoroacetophenone and 10 ml of saturated sodium bicarbonate solution, the reaction mixture was stirred for 15 minutes. Then 12 ml of 30% aqueous hydrogen peroxide solution (30% aqueous solution) was added and the solution was stirred for 20 hours at 10 ° C. Then, 20 ml of saturated sodium thiosulfate solution was added and the mixture was stirred for 20 minutes, diluted with dichloromethane, washed with 5% aqueous sodium hydroxide solution, water and brine. As a result of drying with magnesium sulfate and elimination

·· ·· <* · • ·· * • ·· • • ·· • ·· • · • · • · • • « • • • • • • • · ·· ··· ··· ·· «·

The solvent in vacuo is 20 g of crude epoxide which was further reacted without being purified.

c) 3β-Benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15-one

20 g of crude epoxide was dissolved in 300 ml of dioxane. The yellow, mechanically stirred solution was treated with 2.5 ml of boron trifluoride etherate for 30 minutes and then poured into ice water. The precipitate was then collected, washed with ice-cold water and dried. The crude material was purified by chromatography to give 11.55 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-β-β 3, 3- 8,22-dien-15-one, m.p. 176 ° C.

¹ H-NMR (d 5 -pyridine): δ = 0.85 (2x d, J = 7 Hz, 6H, H-27/26); 0.91 (s, 3H, 4-β-ΟΗ _3); 0.93 (d, J = 7Hz, 3H, H-28); 1.05 (s, 3H, H-18); 1.06 (s, 3H, 4- _c -CH3); 1.07 (s, 3H, H-19 each); 1.13 (d, J = 7Hz, 3H, H-21); 4.89 (dd, J = 12Hz, 4Hz, 1H, H-3a); 5.37 (m, 2H, H-22/23); 7.49 (t, 2 H); 7.57 (t, 1 H); 8.27 (d, 2H).

d) 4,4-dimethyl-5α, 14β-β9θ3β3-8,22-dlen-3β, 15β-βlen1

50 mg of LiAlH ₄ was added to a stirred solution of 300 mg of 3β-εηζογ1οχγ-4,4-dimethyl-5α, 14β-βτς 5ί3-8,22-dien-15-one in 30 ml diethyl ether, and the mixture was then 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 was removed in vacuo. The residue was separated by chromatography to give 100 mg

4,4-dimethyl-5α, 14β-β-α3β3-8,22β-β-3β, 15β-α1υ.

¹ H-NMR (CDCl₃): δ = 0.78 to 1.2 (8x CH _3); 3.24 (dd, J = 12Hz, 4Hz, 1H, H-3a); 3.7 (broad dd, J = 4Hz, 4Hz, 1H, H-15a); 5.24 (m, 2H, H-22/23)

Example 2

4,4-dimethyl-5α, 14β-ergosta-8,22-βίβη-3β, 15α-diol

(a) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15α-ol

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

'H-NMR (CDCl₃): O = 0.8 to 1.12 (8x _CH3); 4.07 (broad t, J = 4 Hz, 1H, [beta] -15β); 4.76 (dd, J = 12Hz, 4Hz, 1H, H-3a); 5.23 (m, 2H, H-22/23); 7.44 (t, 2 H); 7.57 (t, 1H); 8.05 (d, 2H).

b) 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-diol

A solution of 40 mg of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15α-ol in 10 mL of diethyl ether was treated with 10 mg of lithium aluminum hydride for 30 minutes at room temperature as described in Example 1d. After column chromatography, 25 mg of 4,4-dimethyl-5α, 14β-ergosta-8,22-diene-3β, 15α-diol was isolated as a white solid, m.p. 148 ° C.

¹ H-NMR (CDCl₃): δ = 0.78 to 1.7 (8x CH _3); 3.25 (dd, J = 11Hz, 5Hz, 1H, H-3a); 4.04 (t, J = 4 Hz, 1H, H-15a); 5.22 (m, 2H, H-22/23);

Example 3

4,4-dimethyl-5α, 14β-cholesta-8,15- <θθη-3β-ο1

(a) 4,4-dimethyl-5α-cholesta-8,14-dien-3β-yl-benzoate

A solution of 1.0 g of 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in 5 mL of pyridine was treated with 1.5 mL of benzoyl chloride. After stirring at room temperature for 3 hours, the solution was poured into water and the aqueous layer was extracted with ethyl acetate. The organic layers were combined and washed with 0.1 M aqueous hydrochloric acid, saturated sodium bicarbonate solution 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 900 mg of 4,4-dimethyl-5α-cholesta-8,14-di-en-3β- 158 ° C.

^* H-NMR (CDCl₃): δ = 0.83 (s, 3H, H-18); 0.87 + 0.88 (2x s; 3H, H-26/27); 0.95 (d, J = 6Hz, 3H, H-21); 4.76 (dd,

J = 11 Hz, 5 Hz, 1H, H-3c); 5.38 (broad s, 21, H-15); 7.45 (t, 2 H); 7.57 (t, 1H); 8.06 (d, 2 H)

b) 4,4-dimethyl-14,15a-epoxy-5α-cholest-8-en-3β-yl benzoate

To a well-stirred suspension of 4,4-dimethyl-5α-cholesta-8,14-dien-3β-yl-benzoate in 120 mL of dichloromethane was added 130 mL of a 0.09-0.11 M solution of dimethyldioxirane in acetone at 0 ° C. After stirring for 30 minutes at 0 ° C, the reaction mixture was concentrated in vacuo to dryness. The resulting crude epoxide was used without being purified.

• · · · · · · · · · · · · · · · · · ·

c) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15-one

A solution of 1 g of 4,4-dimethyl-14,15a-epoxy-5α-cholest-8-en-3β-yl-benzoate in 15 mL of dioxane was treated with 0.1 mL of boron trifluoride diethyl ether complex. The solution was stirred for 20 minutes at room temperature 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. Column chromatography gave 440 mg of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15-one as a white solid, m.p. 153 ° C.

¹ H-NMR (CDCl₃): δ = 0.83 - 1.1 (7x _CH3); 2.31 (broad s, 1H, [beta] -14 [beta]); 4.76 (dd, J = 11Hz, 5Hz, 1H, H-3a); 7.44 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

d) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15β-ol

A solution of 440 mg, 0.83 mmol of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15-one in 15 mL of dry tetrahydrofuran and 1 mL of ethanol was treated with 125 mg of sodium borohydride at room temperature. After 2 hours, the solution was poured into 0.1M hydrochloric acid 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 being purified.

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

A solution of 500 mg of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-en-15β-ol in 10 mL of dichloromethane was cooled to 0 ° C, and then 1.1 g of Martin's sulfurate was added in one portion. The reaction mixture was stirred for 1 hour at room temperature. The solvents were removed in vacuo. The residue was purified

by chromatography to give 315 mg of 4,4-dimethyl-5α, 14β-cholesta-8,15-dien-3β-yl-benzoate as a waxy solid which was used directly.

f) 4,4-dimethyl-5α, 14β-cholesta-8,15-dien-3β-ol

A solution of 315 mg of 4,4-dimethyl-5α, 14β-cholesta-8,15-dien-3β-yl-benzoate in dry diethyl ether was treated with 35 mg of lithium aluminum hydride as described in Example 1d. Column chromatography of the crude product followed by recrystallization resulted in 135 mg of 4,4-dimethyl-5α, 14β-cholesta-8,15-dien-33-ol as white needles, m.p. 138 ° C.

@ ¹ H-NMR (CDCl3): .delta. = 0.81 (s, 3H, H-18); 0.83 - 1.02 (6x CH ₃ ); 2.49 (broad s, 1H, [beta] -14 [beta]); 3.24 (m, 1H, H-3a);

5.64 + 5.79 (2x m, 1H, H-15-16)

Example 4

4,4-dimethyl-24-nor-5α, 14β-ο1ιο1β3ΕΒ-8,15-β-β-ο1

(a) (20S) -3β-5-β-γ-1-γ-20-1-α-1,4-dimethyl-5α, 14β-pregn-8-en-15-one and (20S) -3β-benzoyloxy-20-hydroxymethyl-4; 4-dimethyl-5α, 14β-pregn-8-en-15β-one

A solution of 2.18 g of 3β-β-γ-γ-γ-4,4-dimethyl-5α, 14β-β-β-α3-8,22-dien-15-one (see Example 1c) in 88 ml of dichloromethane, 30 ml of methanol and 1 ml of pyridine was cooled to -70 ° C.

The solution was passed through a 1: 4 mixture of ozone / oxygen for 12 minutes under control by thin-layer chromatography.

Then 130 mg of sodium borohydride was added and the stirred reaction mixture was warmed to 0 ° C for 2 hours and then 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 was purified by chromatography to give 1.28 g of (20 S) -3β-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-en-15-one, m.p. 8 ° C and 330 mg of the corresponding 15β-hydroxy compound.

@ ¹ H-NMR (CDCl3): .delta. = 0.9 - 1, 1 (4x ch ₃ ); 1.16 (d J = 6Hz, 3H, H-21); 2.33 (broad s, OH, Η-14β) ; 3.42 (m, ÍH H-22a); 3.63 (m, 1H, H-22b); 4.75 (dd, J = 11 Hz, 4 Hz, ÍH

H-3a); 7.45 (t, 2 H); 7.57 (t, 1H); 8.05 (d, 2H).

(b) (20S) -3β-Benzoyloxy-4,4,20-trimethyl-21-toluenesulphonyloxy-5α, 14β-η-8-η-15-η

A solution of 385 mg of (20S) -3β-benzoyloxy-20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-en-15-one in 5 mL of pyridine was cooled to 0 ° C. After addition of 190 mg of p-toluenesulfonyl chloride, the reaction mixture was stirred for 20 hours at 6 ° C 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 resulting crude product was used without further purification.

c) 3β-Benzoyloxy-4,4-dimethyl-24-nor-5α, 4-cholest-8-en-15-one

To a stirred suspension of 1.6 magnesia powder in 25 ml of anhydrous tetrahydrofuran was slowly added 3.42 g of 2-methylpropyl bromide over 30 minutes at 50 ° C. The result was a clear solution of Grignard reagents. Then the mixture was cooled to -10 ° C and 3.32 ml of LiClCl 4 solution prepared by mixing 99 mg of copper chloride, 86 mg of lithium chloride and 10 ml of tetrahydrofuran was added dropwise.

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

¹ H-NMR (CDCl₃): δ = 0.82 to 1.09 (7x _CH3); 2.31 (broad s, 1H, [beta] -14 [beta]); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3a); 7.44 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

d) 3β-β-γ-γ-γ-4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-15β-ol

130 mg of 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8en-3-one was treated with 100 mg of sodium borohydride as described in Example 2a. After water treatment, 130 mg of 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-δ-εη-δδ-οΐη was obtained as a white foam which was used directly.

e) 4,4 ίππθθ] ^ 1-24-ηο ^ 5α, 14β-ο1ιο1θ3ί3-8,15 ^ββ-3β ^ 1 ^ θηζοέί

400 mg of Martin sulphate was added in one portion to a solution of 100 mg of 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-15β-ο1υ in 10 mL of dichloromethane. The mixture was stirred for 18 hours at room temperature. Evaporation under reduced pressure and chromatography gave 100 mg of 4,4-dimethyl-24-ηοΓ-5α, 14β-ηή 1β3β 3-8,15-dien-3β-yl-benzoate as an oil which was used without purification.

• · Φ 1 φ φ φφφφ • φ

f) 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β-ol

100 mg of 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β-yl benzoate were treated with 20 mg of lithium aluminum hydride as described in Example 1d. Column chromatography of the crude product and recrystallization with hexane / ethyl acetate gave 36 mg of 4,4-dimethyl-24-nor-5α, 14β-cholesta-8,15-dien-3β-ol, m.p. 104.3 ° C.

^* H-NMR (CDCl₃): δ = 0.82 (s, 3H, H-18); 0.83 to 1.03 (6x _CH3); 2.49 (broad s, 1H, [beta] -14 [beta]); 3.24 (m, 1H, H-3a); 5.65 + 5.8 (m, 1H, H-15-16)

Example 5

4,4-dimethyl-24-nor-5α, 14β-σΚο1β3ύ-8-βη-3β-ο1

a) 15β-β-β-3-η 3-η-η-1-ω-4,4-dimethyl-24-nor-5a, 14β-οΗο1β3ΐ-8-ηη-3β-1-β-1 mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5a, 14β -cholest-8-βη-15β-ο1 in 3 ml pyridine was treated with 0.2 ml methanesulfonyl chloride at 0 ° C. After stirring for 2 hours at room temperature, the reaction mixture was diluted with water and extracted with ethyl acetate. The extracts were combined and washed with 1M hydrochloric acid, water and brine, dried over sodium sulfate and filtered. Removal of the solvents resulted in 40 mg of 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β-yl-benzoate, which was used directly.

(b) 4,4-dimethyl-24-nor-5a, 14β-η1β3ΐ-8-η 1 mg 15β-methanesulfonyloxy-4,4-dimethyl-24-nor-5α, 14β-σ-1β3ΐ-8-ηη -3β-ν1-βηθοέ1 was treated with 10 mg of lithium aluminum hydride as described in Example 1d.

4444 »♦ · 4 4 4 · 4

The crude product was purified by chromatography to give 32 mg of 4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3P-ol.

^* H-NMR (CDCl₃): δ = 0.81 (s, 3H, H-18); 0.82 to 1.03 (6x _CH3); 3.25 (dd, J = 11Hz, 4Hz, 1H, H-3a)

Example 6

4,4-dimethyl-24-nor-5α, 14β-cholest-8-en-3β, 15β-diol mg 3β-benzoyloxy-4,4-dimethyl-24-nor-5α, 14β-cholest-8-en- The 15β-ol was treated with 10 mg of lithium aluminum hydride as described in Example 1d. The crude product was purified by column chromatography to give 23 mg of 4,4-dimethyl-24-nor-5α, 14β-cholest-8-ene-3β, 15β-diol as a white foam.

@ ¹ H-NMR (CDCl3): .delta. = 0.82 (s, 3H, H-18); 0.84 - 1.04 (6x CH ₃ ); 3.25 (broad d, J = 12 Hz, 1H, H-3a); 3.7 (broad q, J = 7Hz, 1H, H-15a)

Examples 7 + 8 (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-33,15β-diol and (20R) -4,4,20-trimethyl- 16β, 21-cyclo-5α, 14β-pregn-8-en-3β, 15α-diol

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

1.10 g of (20S) -3β-benzoyloxy-4,4,20-trimethyl-21-toluenesulfonyloxy-5α, 14β-pregn-8-en-15-one was treated with 52 mmol of phenylmagnesium bromide, analogous to Example 4c. 560 mg of (20R) -3β-benzoyloxy-4,4,20-β-piperidin-16β, 21-cyclo-5α, 14β-pregn-8-en-15-one was isolated after chromatography

9 ···· ♦ 9 ·· • 9 9 99 · · · · ·

9 9 *

999 ··· other products resulting from the substitution of tosylate with phenyl Grignard reagent.

(b) (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-3β, 15β-diol and (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-3β, 15α-diol

460 mg of (20 R) -3β-benzoyloxy-4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-15-one was treated with 50 mg of lithium aluminum hydride as described in Example 1d . The crude product was purified by column chromatography to give 40 mg of (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-3β, 15β-diol and 30 mg ( 20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-ene-33,15α-diol as a white solid.

(20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-β-η-8-βη-3β, 15β-diol:

@ ¹ H-NMR (CDCl3): .delta. = 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-16a); 3.25 (m, 1H, H-3a); 4.07 (q, J = 8Hz, lH,

H-15a) (20R) -4,4,20- ππ 1-1-1ββ, 21-cyclo-5a, 14β-β-η-8-ηη-3β, 15α-diol:

@ ¹ H-NMR (CDCl3): .delta. = 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, [beta] -14β); 2.68 (m, 1H, H-16a); 3.25 (m, 1H, H-3a); 4.07 (d, J = 6Hz, 1H,

Η-15β)

Example 9

4,4-dimethyl-5a, 14β- <2ΐιο1β3Ε3-8,24-ωίθη-3β, δδβ ^ ίοΐ

a) (20S) -3β-Benzoyloxy-4,4-tetrahydro-4-carboxylic acid

4 9 9 9

4 · 99

A solution of 2.2 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-ergosta-8,22-dien-15-one in 87 mL of dichloromethane and 53 mL of methanol was cooled to -78 ° C. The solution was purged with a 1: 4 ozone / oxygen mixture for 16 minutes. 2.62 g of 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 yield 1,26 (20S) -3P-benzoyloxy-4,4-dimethyl-15-oxo-5α, 14β-pregn-8-ene-20-carbaldehyde with mp 205 ° C.

¹ H-NMR (d 5 -pyridine): δ = 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 (broad s, 1H, [beta] -14 [beta]); 4.83 (dd, J = 11Hz, 4Hz, 1H, H-3a); 7.5 (t,

1H); 7.58 (t, IH); 8.27 (d, 2 H); 9.82 (d, J = 2Hz, 1 H, H-22)

(b) 3β-Benzoyloxy-22-hydroxy-4,4-dimethyl-5α, 14β-cholest-8-ene;

15.24-dione

25.8 mL of a solution of n-butyllithium (1.6 M, hexane) was added to 6 mL of dilsopropylamine in 48 mL of tetrahydrofuran at -78 ° C. The mixture was stirred for 20 minutes. A 4.5 ml solution was added

Of 3-methyl-2-butanone in 10 mL of tetrahydrofuran and the mixture was stirred at -78 ° C for 15 minutes. The resulting kinetic enolate was transferred via cannula under nitrogen to a -78 ° C cooled solution of 6.0 g of (20S) -3β-benzoyloxy-4,4-dimethyl-15-oxo-5α, 14β-pregn-8-en-20. -carbaldehyde in 40 ml of tetrahydrofuran. The mixture was brought to 0 ° C over one hour and then poured into a 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 the solvents in vacuo gave a residue which was separated by column chromatography to give 6.26 g of 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24- Dion in the form of oil.

·· ·· · ^X H-NMR (CDCl₃): δ = 0.92 to 1.2 (7 x _CH3); 2.33 (broad s, 1H, [beta] -14 [beta]); 4.1 (m, 1H, H-22); 4.74 (dd, J = 11Hz,

Hz, 1H, H-3a); 7.45 (t, 2 H); 7.57 (t, 2 H); 8.05 (d, 2H) 9 9 9 9 9 »» »»

9

9 9,999 9 9 9

c) 3β-ηθζογ1οχγ-4,4-dimethyl-5α, 14β-cholesta-8,22-diene-15,24-dione

A solution of 4.78 g of 3β-benzoyloxy-22-hydroxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-dione in 60 mL of dichloromethane was cooled to 0 ° C and then added at once. , 31 g Martin's uranium sulphate. The reaction mixture was stirred for 30 minutes at 0 ° C. The solvents were then removed in vacuo. Purification of the residue by column chromatography gave 4.18 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,22-diene-15,24-dione, m.p. 163 ° C.

^* H-NMR (CDCl₃): δ = 0.9 to 1.2 (7 x _CH3); 2.31 (broad s, 1H, [beta] -14 [beta]); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3a);

6.14 (d, J = 16Hz, 1H, H-23); 6.71 (m, 1H, H-22); 7.45 (t,

2H); 7.57 (t, 1 H); 8.05 (d, 2H).

d) 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-dione

500 mg of palladium on charcoal (10%) was added to a solution of 4.2 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,22-diene-15,24-dione in 60 mL of ethyl acetate. The mixture was stirred under a hydrogen atmosphere for 3 hours. The mixture was then filtered. Removal of the solvent in vacuo resulted in 4.22 g of 3β-η-η-η 4,4 -α-α-β-η 1-5α, 14β-ο-ω-1β-β-8-η-15,24-dione as a white crystalline residue, m.p. 129 ° C.

¹ H-NMR (CDCl₃): δ = 0.92 to 1.16 (7 x _CH3); 2.33 (broad s, 1H, [beta] -14 [beta]); 4.74 (dd, J = 11Hz, 4Hz, 1H, H-3a);

7.45 (t, 2 H); 7.57 (t, 1 H); 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

A solution of 4.2 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-dione in 45 mL of CH ₂ Cl ₂ was treated with 1.8 g of tert-butylamine-borane complex . The solution was heated to reflux for one hour. After cooling to 0 ° C, 30 ml of a 1M aqueous hydrochloric acid solution was added. The mixture was stirred for 1 hour at 0 ° C. It was then washed with 1M aqueous hydrochloric acid, water, saturated aqueous sodium bicarbonate solution and brine. After drying over sodium sulfate, the solvents were removed in vacuo. Column chromatography resulted in 1.71 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-diol and 1.82 g of 3β-benzoyloxy-24-hydroxy-4,4- dimethyl-5α, 14β-cho-lest-8-en-15-one as a white foam.

3β-Benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-diol:

^* H-NMR (CDCl₃): δ = 0.88 - 1.1 (7 x _CH3); 3.33 (m, 1H, H-24); 3.74 (m, 1 H, H-15); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3a); 7.45 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

3ss-benzoyloxy-24-hydroxy-4,4-dimethyl-5a, 14ss-cholest-8-en-15-one ^X H-NMR (CDCl₃): δ = 0.88 - 1.1 (7 x CH ₃ ); 2.32 (broad s, 1H, [beta] -14β); 3.33 (m, 1H, H-24); 4.74 (dd, J = 11Hz, 4Hz, 1H, H-3a); 7.45 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

(f) 3β-Benzoyloxy-4,4'-piperazin-1-yl] -5-5α, 14β-οΗο1β3β3-8,24-dien-15β-ol and 4,4-dimethyl-5α, 14β-cholesta-8,15,24-triene - 3β-yl-benzoate

1.0 g of 3β-benzoyloxy-4,4-dimethyl-5α, 14β-cholest-8-ene-15,24-diol was treated with 3.88 g of Martin's sulfurate as described in Example 3e. Column chromatography purification resulted in 305 mg of 3β-benzoyloxy-4,4-di-3-methyl-5α, 14β-cholesta-8,24-dien-15β-ol as a white foam and 870 mg of 4,4-dimethyl -5α, 14β-cholesta-8,15,24-trien-3β-yl-benzoate as a white solid.

3β-Benzoyloxy-4,4-dimethyl-5α, 14β-cholesta-8,24-diene-15β-α: 1 H-NMR (d 5 -pyridine): δ = 1.15 (s, 1H, H-18 ); 2.84 (m, 1H, H-7a); 4.1 (m, 1H, H-15); 4.93 (dd, J = 11Hz, 4Hz, 1H, H-3a); 5.24 (t, J = 8Hz, 1H, H-24); 7.45 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-yl-benzoate:

^* H-NMR (CDCl₃): δ = 1.4 (s, 3H, H-19); 2.19 (m, 1 H,

H-17a); 2.51 (broad s, 1H, [beta] -14β); 4.75 (dd, J = 11Hz, 4Hz, 1H, H-3a); 5.1 (t, J = 8Hz, 1H, H-24); 5.65 (m, 1H, H-16); 5.79 (m, 1H, H-15); 7.45 (t, 2 H); 7.57 (t, 1 H); 8.05 (d, 2H).

(g) 4,4-dimethyl-5α, 14β-cholesta-8,24-diene-3β, 15β-α1

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

'H-NMR (CDCl₃): δ = 0.8 to 1.03 (5x CH _3); 1.6 + 1.68 (2x s; 3H, H-26/27); 3.24 (m, 1H, H-3a); 3.70 (m, 1H, H-15a); 5.10 (t, J = 7Hz, lH, H-24)

Example 10

4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-ol mg 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-yl-benzoate, see example 9f was treated with 20 mg of lithium aluminum hydride as described in Example 1d. Crude • 4 · «·· * 4

The 444 product was purified by column chromatography to give 20 mg of 4,4-dimethyl-5α, 14β-cholesta-8,15,24-trien-3β-ol as a white solid, m.p.

105 ° C.

^* H-NMR (CDCl₃): δ = 0.81 (s, 3H, H-18); 0.94 - 1.02 (4x CH ₃ ); 2.5 (broad s, 1H, Η-14β); 3.23 (m, 1H, H-3a); 5.11 (t, J = 9Hz, 1H, H-24); 5.64 (m, 1H, H-15 / 16a); 5.79 (m, 1H, H-15-16b)

Example 11 (20S) -20-hydroxymethyl-4,4-dimethyl-5α, 14β-β-η-8-βη-3β, 15β-diol mg (20S) -20-hydroxymethyl-4,4-dimethyl-5α, 14β- pregn-8-εη-3β, 15β-β-1η was treated with 20 mg of lithium aluminum hydride as described in Example 1d. The crude product was purified by column chromatography to give 42 mg of (20S) -20-hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-ene-3β, 15β-diol, m.p. 214 ° C.

^* H-NMR (CDCl₃): δ = 0.82 (s, 3H, H-18); 0.93 - 1.04 (3x CH ₃ ); 1.07 (d, J = 6Hz, 3H, H-21); 3.23 (m, 1H, H-3a); 3.49 (m, 1H, H-22a); 3.68 (m, 1H, H-22b); 3.72 (m, 1 H, H-15a)

Example 12

Testing of meiosis activating agents in oocyte test

Animals

Oocytes were obtained from juvenile female mice (Fl-hybrids C57BI / 6J x DBA / 2J, Bomholtgaard, Denmark), weighing 13-16 grams, which were kept under controlled temperature and light. Mice were injected intraperitoneally with 0.2 ml gonadotropins (Gonal F,

Serono, Salna, Sweden, containing 20 mes. j. FSH, alternatively Puregon, Organon, Swords, Ireland, containing 20 mes. 48 animals were sacrificed by cervical dislocation.

Collection and cultivation of oocytes

After ovaries were harvested, oocytes were isolated in Hx-medium (see below) under a stereomicroscope using manual follicle rupture using a pair of 27-gauge needles. Spherical, naked oocytes (NO) having intact embryonic bladder (GV) were placed in alpha-minimal essential medium ( Ribonucleoside-free MEM, Gibco BRL, Cat # 22561) enriched with 3 mmol hypoxanthine (Sigma, Cat # H-9377), 8 mg / ml human serum albumin (HSA, State Serum Institute, Denmark), 0, 23 mmol of pyrubate (Sigma, cat. No. S-8636), 2 mmol of glutamine (Flow, cat. No. 16-801), 100 IU / ml penicillin and 100 µg / ml streptomycin (Flow, cat. No. 16-700). This medium was designated as Hxmedium. Oocytes were rinsed three times in Hx-medium and cultured on 4-well multiplots (Nuncion, Denmark), each containing 0.4 ml Hx-medium and 35-45 oocytes. In parallel to the test cultures, which were performed with different concentrations of test compounds, one control culture was performed, i.e. 35 to 45 oocytes were cultured in Hx-medium without addition of the test compound.

Cultivations were carried out at 37 ° C and 100% humidity of air containing 5% carbon dioxide. The cultivation time ranged from 22 to 24 hours.

Examination of oocytes

At the end of the culture period, a number of contrast-contrast-contrast-contrast controls were detected using a stereomicroscope or inverted-image microscope.

9

9

9,999 ·

9

9 9 9 · 9 99

999 999 999 99 999 Oocytes with germinal bladder (GV) or breakdown of germinal bladder (GVB) and oocytes with polar body (PB). In the cultures tested, the percentage of oocytes with GVB relative to total oocytes and the percentage of oocytes with PB relative to total oocytes were calculated and compared to control cultures.

Example 13

Testing of Meiosis Inhibitors in Oocyte Test

Germ bladder oocytes (GV) were obtained from juvenile FSH-treated female mice using the same procedures described in Example 12 (see above). Naked oocytes (NO) were flushed three times in Hx-medium. 4,4-Dimethylcholest-B, 14,24-trien-3p-ol (FF-MAS) has previously been shown to induce meiosis in naked oocytes in vitro (Byskov, AG et al. Nature 374, 1995, p. 559 to 562). Naked oocytes were cultured in Hx-medium supplemented with 5 μπιοί FF-MAS in culture concurrently with test compounds at various concentrations in 4-well multiplots (Nunclon, Denmark), each well containing 0.4 ml Hx-medium and 35-45 oocytes . In parallel to the test cultures, which were carried out with different concentrations of the test compounds, one positive control culture was performed, ie. 35 to 45 oocytes were cultured in Hx-medium containing FF-MAS without addition of test compound. In parallel to the positive control culture, one negative control culture was performed, i.e., 35 to 45 oocytes were cultured in Hx-medium alone.

Examination of oocytes

At the end of the cultivation period, the number of oocytes with germinal bladder (GV) or germinal disintegration was detected by means of a stereomicroscope or inverted imaging microscope equipped with a differential interference contrast control device.

4

444 ·

444 4 • 4 * · 4 · 4 4 4 * 4

4 4 4 4

444 444 44 »» · bladder (GVB) and polar body oocytes (PB). In the tested and control cultures, ie positive and negative, the percentage of oocytes with GVB + PB was calculated relative to the total number of oocytes. The relative inhibition of test compounds was calculated using the following formula:

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

Oocytes suspended during meiosis are characterized by conspicuous (GV). After the envelope disappears with intact nucleus bladder nucleus and nuclear disintegration of embryonic bladder, embryonic bladder (GVB). completes the reduction division and the body (PB).

and are known as the relaunch of meiosis and this is characterized as a breakdown A few hours later, oocytes form the first so-called polar

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Results

Table 1

Relative inhibition (%) of meiosis in nude mouse oocytes

Compounds Number of oocytes Inhibition GV GVB PB (%) Control culture (Hx) 38 2 0 100 ALIGN! 5μπιο1 FF-MAS 9 27 Mar: 4 0 ομπιοί · Εξ-MÁS + 10. μπιοί 4,4-Diinethyl-5-t-4β-cholesta-8,15-dier-3β-ol 28 11 - BM Control culture (Hx) 40 0 0 100 ALIGN! 5μπιοί FF-MAS 8 25 6 0 5μπιο1 ··, FF-MAS. +, 10-μπιοί '4,4-methylethyl- (η 1, 4β-chloro-β-21,2-e' - -β, ' ¹ β- ·'; i. · '. · 1 M · - M BBBMI

Hx hypoxanthin

GV Bladder

GVB disintegration of embryonic bladder PB polar body

9999 9 9 9 9 9

9 9 9 99 9 9 999 999

9 ··

999 99 999

Claims (9)

PATENT CLAIMS 14β-H-sterols of general formula I, in which R ³ is hydrogen or together with R ^{3 forms} an additional bond, R ³ 'represents a hydrogen atom or together with R ³ forms another bond, R ⁴ represents hydrogen or methyl, R ⁴ 'represents a hydrogen atom or a methyl group, R ⁷ represents a hydrogen atom or together with R ⁸ forms another bond, R ⁸ represents a hydrogen atom or together with R ⁷ , or together with R ⁹ , forms another bond, R ⁹ represents a hydrogen atom or together with R ⁸ , or together with R ¹¹ , forms a further bond, R ¹⁵ represents a hydrogen atom, a hydroxy group, a halogen atom, or together with the substituent R ¹⁵ forms an oxo group, or together with the substituent R ¹⁶ forms another bond, or together with the substituent R ²² 'forms an oxygen bridge, R ¹⁵ 'represents a hydrogen atom, a branched or unbranched alkyl group having 1 to 8 carbon atoms, 9 A AAAA A AAA A A A A A 9 99 999 AAA a (C 6 -C 10) aryl group, or together with R ¹⁵ forms an oxo group, R represents a hydrogen atom, a hydroxyl group, a halogen atom or together with R ¹⁵ forms a further bond, or together with R ²² forms a further bond, R ²² represents a hydrogen atom, an optionally substituted, branched or unbranched, alkyl or alkenyl group having 1 to 8 carbon atoms, an optionally substituted aryl group having 6 to 10 carbon atoms, or together with R ¹⁶ forms another bond, or together with a substituent R ²² 'forms a branched or unbranched alkylidene group containing 1 to 8 carbon atoms, R ²² 'represents a hydrogen atom, a hydroxy group, or together with R ¹⁵ forms an oxygen bridge, or together with R ²² forms a branched or unbranched alkylidene group having 1 to 8 carbon atoms, or esters thereof. 14β-H-sterols according to claim 1, of the general formula I, in which R ³ and R ³ 'are hydrogen. The 14β-H-sterols according to claim 1 or 2, wherein R ⁸ and R ⁹ together represent a further bond. The 14β-H-sterols of any one of claims 1 to 3, wherein R ¹⁵ is hydrogen or hydroxy. The 14β-H-sterols according to any one of claims 1 to 4, wherein R ²² is an optionally substituted, straight or branched alkyl group containing 1 to 4. · ♦ · · <· <<<<<< C 8 -C 8, or together with R ²² ', represents a straight or branched alkylidene group containing 1 to 8 carbon atoms. The 14β-H-sterols according to any one of claims 1 to 5, which are 4,4-dimethyl-5α, 14β-cholest-8-en-3β-ol, 4,4-dimethyl-5α, 14β-cholest-8-en-3β, 15β-α1, 4,4-dimethyl-5α, 14β-ούο1θ3ύ-8-ηη-3β, 15α-diol, 4,4-dimethyl-5α, 14β-ούο1θ3ΐ3-8,15-dien-3p-ol, 4,4-dimethyl-5α, 14β-cholesta- 7.9 (11), 15-chloro-3β-ο1, 4,4-dimethyl-5a, 14β-ούο1θ3-3-8,15,23 (Ε) - β-3β-ο1, 4,4-dimethyl-5a, 14β-σύο1θ3ύ3-8,15, 24-β-β-β-ο1, 4,4-dimethyl-5α, 14β-ούο1θ3ύ3-8,24-ωίθη-3β, 15β-ωί1, 4,4-dimethyl-5α, 14β-βΓςο3ύ3-8,22-ßίβη-3β-ο1, 4,4-dimethyl-5α, 14β-βΓςο3β3-8,22-β-3β, 15β-β1, 4,4-dimethyl-5a, β-β-β-ε-η-δ, 22-β-β-β-15β, 15α-diol, 4,4-dimethyl-5a, 14β-βΓςο3-3-8,15,22-β-3β-ο1, 4,4-dimethyl-24-nor-5α, 14β-ο1το1θ3ί-8-ηη-3β-ο1, 4,4-dimethyl-24-nor-5α, 14β-η1ο 1θ3ύ-8-ηη-3β, 15β-ωί1, 4,4-dimethyl-24-nor-5α, 14β-ούοΐΘ3ΐΘ-8-ηη-3β, 15α-diol, 4,4-dimethyl-24-nor-5α, 14β-ούο1θ3ύ3-8,15-ßίβη-3β-ο1, 4,4-Dimethyl-5a, 14β-ούο1β3ύ3-8,15-dien-3β-ol hydrocene enbutanedioate, 4,4-dimethyl-5α, 14β-β-α3,3-8,15,22-trien-3β-ol hydrogenbutanedioate, (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-β-β-ol 8-ηη-3β, 15α-diol, (20R) -4,4,20-trimethyl-16β, 21-cyclo-5α, 14β-pregn-8-en-3β, 15β-α1, (20R) -20- hydroxymethyl-4,4-dimethyl-5α, 14β-pregn-8-ene-3β, 15β-diol, A pharmaceutical composition comprising one or more 14β-H-sterols of formula I according to any one of claims 1 to 6, together with a pharmaceutically acceptable excipient. Use of 14β-H-sterols of formula I according to any one of claims 1 to 6 for the preparation of a medicament for controlling meiosis. 9. Use according to Claim 8 for preparation contraceptive medicines k healing female or female male, preferably people. 10. Method regulation of meiosis ..... .mu.Ci s e comprising administering to a patient in need of such regulation an effective amount of one or more 14β-H-sterols of formula I according to any one of claims 1 to 6. Use of a 14β-hydrogen substituent on sterol to enhance the inhibitory activity of a meiosis inhibiting agent.