WO2000047604A1 - Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments - Google Patents

Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments Download PDF

Info

Publication number
WO2000047604A1
WO2000047604A1 PCT/EP2000/001074 EP0001074W WO0047604A1 WO 2000047604 A1 WO2000047604 A1 WO 2000047604A1 EP 0001074 W EP0001074 W EP 0001074W WO 0047604 A1 WO0047604 A1 WO 0047604A1
Authority
WO
WIPO (PCT)
Prior art keywords
cholesta
dimethyl
dien
designates
group
Prior art date
Application number
PCT/EP2000/001074
Other languages
French (fr)
Inventor
Thorsten Blume
Peter Esperling
Joachim Kuhnke
Christa Hegele-Hartung
Monika Lessl
Original Assignee
Schering Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Schering Aktiengesellschaft filed Critical Schering Aktiengesellschaft
Priority to BR0008065-9A priority Critical patent/BR0008065A/en
Priority to EP00910664A priority patent/EP1150993A1/en
Priority to AU32797/00A priority patent/AU3279700A/en
Priority to SK1138-2001A priority patent/SK11382001A3/en
Priority to JP2000598521A priority patent/JP2002536456A/en
Priority to IL14373500A priority patent/IL143735A0/en
Priority to CA002359687A priority patent/CA2359687A1/en
Priority to KR1020017009985A priority patent/KR20010101820A/en
Publication of WO2000047604A1 publication Critical patent/WO2000047604A1/en
Priority to NO20013901A priority patent/NO20013901D0/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0094—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 containing nitrile radicals, including thiocyanide radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

Definitions

  • the present invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions comprising them as active substances and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the unsaturated cholestane derivatives of the invention can be used for regulating meiosis.
  • Meiosis is the unique and ultimate event of germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1 n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA synthesis. This division therefore results in the formation of the haploid germ cells with only 1 c DNA.
  • the meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly.
  • All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty.
  • the female has a stock of oocytes which is drawn upon until the stock is exhausted.
  • Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell.
  • only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spermatozoa.
  • MAS meiosis-activating substance
  • MPS meiosis preventing substance
  • novel, stable compounds with interesting pharmacological properties are provided.
  • the compounds described here are useful for the regulation of meiosis in oocytes and in male germ cells.
  • novel compounds which are suitable substrates for the introduction of fluorescense markers are designed and synthesized for bioimaging purposes.
  • the present invention relates to unsaturated cholestane derivatives of the general formula I
  • R ⁇ designates a hydrogen atom, a C2-Cg-alkyl group, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR 1 group (with R 1 being a C-
  • R 2 designates a hydrogen atom, a C-j-Cs-alkyl group, a Cs-C ⁇ -alkenyl group, a C-i-C -hydroxyalkyl group, together with R 2' an optionally substituted benzylidene group, together with R 2 ' a hydroxymethylene group or together with R ⁇ an additional bond,
  • R 2' designates a hydrogen atom, together with R 2 an optionally substituted benzylidene group or together with R 2 a hydroxymethylen group
  • R3 designates a hydrogen atom or together with R ' an additional bond
  • R3' designates a hydrogen atom or together with R3 an additional bond
  • R4 designates a hydrogen atom or a methyl group
  • R4' designates a hydrogen atom or a methyl group
  • R8 designates together with R or with R14 an additional bond
  • R9 designates a hydrogen atom or together with R ⁇ an additional bond
  • R " l4 designates an ⁇ -hydrogen atom or together with R ⁇ an additional bond or together with R15 an additional bond
  • R15 designates a hydrogen atom or together with R ⁇ an additional bond
  • R ⁇ designates a hydrogen atom or together with R 2 ⁇ an additional bond
  • R ⁇ designates a hydrogen atom or together with R 2 ⁇ an additional bond
  • Preferred compounds of formula I are such wherein R ⁇ designates a hydrogen atom, a phenyl group or together with R 2 an additional bond.
  • Other preferred compounds are such wherein R 2 designates a C-j-Cs-alkyl group, an allyl group or an additional bond with R ⁇ .
  • Compounds wherein R 2 and R 2' designate an optionally substituted benzylidene group are also preferred.
  • esters of compounds of the general formula I are formally derived by estehfication of one or more hydroxylic groups of a compound of formula I with an acid which can for example be selected from the group of acids comprising succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulphonic acid, sulphamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.
  • an acid which can for example be selected from the group of acids comprising succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulphonic acid, sulphamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.
  • an alkyl group - when used alone or in combinations - may be a straight or branched alkyl group.
  • the expression C2-C5 alkyl designates an alkyl group having from two to six carbon atoms: preferred examples are ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and cyclohexyl more preferred ethyl.
  • C ⁇ -Cg alkyl designates an alkyl group having from one to eight carbon atoms; preferred examples are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl more preferred methyl, ethyl, propyl, isopropyl, butyl and tert-butyl, still more preferred methyl and ethyl.
  • Especially preferred compounds of formula I of the present invention are the following: 2 ⁇ -allyl-4,4-dimethyl-5 ⁇ -cholesta-8,14-dien-3 ⁇ -ol,
  • the compounds of the general formula I according to the invention can be synthesized analogously with the preparation of known compounds. Hence, synthesis of the compounds of formula I can follow the well established synthetic pathways described in the comprehensive sterol and steroid literature.
  • the following books can be used as the key source in the synthesis: L.F. Fieser & M. Fieser: Steroids: Reinhold Publishing Corporation, NY 1959; Rood ' s Chemistry of Carbon Compounds (editor: S. Coffrey): Elsevier Publishing Company, 1971 ; and especially Dictionary of Steroids (editors: R.A. Hill; D.N. Kirk; H.L.J. Makin and CM. Murphy): Chapman & Hall. The last one contains an extensive list of citations to the original papers covering the period up to 1990. All these books including the last mentioned citations are incorporated by reference.
  • the compounds of the present invention are synthesized according to the following general procedures:
  • sterols that are used as starting materials can be synthesized according to literature procedures:
  • the ⁇ -1 double bond can be introduced in an oxidation reaction with phenyl selenic anhydride as an oxidant in chlorobenzene [J. Chem Soc. Chem. Commun. 1978, 952] to give 4,4-dimethyl-5 ⁇ -cholesta-1 ,8,14-trien-3-one 3.
  • This reaction also works in the case of compounds without the 4,4-dimethyl group.
  • the new double bond can be selectively introduced as a ⁇ 1 -double bond.
  • the 3-keto group can then be reduced with sodium borohydride in the presence of cerium chloride [Luche reduction, for example: J. Am. Chem. Soc. 100 (1978), 2226] to give 4,4-dimethyl-5 ⁇ -cholesta-1 ,8,14-trien- 3 ⁇ -ol 4.
  • scheme 2 :
  • This amine can be used for further modification.
  • Substituents in position 2 can for example be introduced via aldol reactions and alkylations. If ketone 2 is treated with aromatic aldehydes in the presence of a base 2-benzylidene-substituted steroids of formula 10 are obtained. The aromatic ring can be substituted. Subsequent reduction with sodium borohydride in the presence of cerium chloride gives selectively the corresponding allylic 3 ⁇ - alcohols 11.
  • amine 12 is treated with hydroxysuccinimidyl ester derivatives of different carboxylic acids.
  • esters that contain fluorescence markers can be used (see example 11 in the experimental part).
  • Amides of formula 13 that contain the fluorescence markers can be used as molecular probes for bioimaging purposes.
  • Alkyl- and alkenyl substituents can be introduced in position 2 via deprotonation of ketone 2 and subsequent reaction of the enolate with alkyl- and alkenyl halides.
  • the 3-ketones of formula 14 can then be reduced with sodium borohydride to give two diastereomeric alcohols of formulae 15 and 16, which can be easily separated by column chromatography.
  • hydroxymethylsubstituents in position 2 can for example be introduced via a condensation reaction of a deprotonated 3-ketone with alkylformiates to give enol 17. This can be reduced with different reducing agents like sodium borohydride to give two diastereomeric alcohols 18 and 19.
  • scheme 7 :
  • compositions comprising one or more compounds of the general formula I as active substances.
  • the compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally used in the art.
  • solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter.
  • Liquid compositions include sterile solutions, suspensions and emulsions.
  • liquid compositions may be suitable for injection or for use in connection with ex vivo and in vitro fertilization.
  • the liquid compositions may contain other ingredients which are conventionally used in the art, some of which are men- tioned in the list above.
  • a composition for transdermal administration of a compound of this invention may be provided in the form of a patch and a composition for nasal administraton may be provided in the form of a nasal spray in liquid or powder form.
  • compositions of the invention are prepared by intimately bringing into association the active compound with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation.
  • the present invention relates to the use of the compounds of the general formula I for the preparation of a meiosis-regulating medicament.
  • the compounds of the present invention influence the meiosis in oocytes as well as in male germ cells.
  • the prospects of being able to influence the meiosis are several.
  • a compound of the general formula I can be used to stimulate the meiosis.
  • a compound of the general formula I can be used to stimulate the meiosis in humans.
  • the compounds of the general formula I are promising as new fertility-regulating agents without the usual side effects on the somatic cells which are known from the hitherto used hormonal contraceptives which are based on estrogens and/or gestagens.
  • the present invention concerns the use of the compounds of the general formula I for relieving infertility in females and males, particularly in mammals, more particularly in humans.
  • Meiosis-inducing substances of the general formula I can be used in the treatment of certain cases of infertility in females, including women, by administration thereof to females who, due to an insufficient own production of meiosis-activating substance, are unable to produce mature oocytes.
  • the compounds of the general formula I can also be used in artificial insemination procedures e.g. in in vitro fertilization or intracytoplasmic sperm injection.
  • in vitro fertilization is performed, better results can be achieved, when a compound of the invention is added to the medium in which the oocytes are cultured.
  • infertility in males, including men is caused by an insufficient own production of the meiosis-activating substance and thus a lack of mature sperm cells exists, administration of a compound of the invention may relieve the problem.
  • the compounds of the general formula I are useful as contraceptives in females and males, particularly in mammals, more particularly in humans.
  • a meiosis inducing substance can be administered so as to prematurely induce resumption of meiosis in oocytes while they are still in the growing follicle, before the ovulatory peak of gonadotropins occurs.
  • the resumption of the meiosis can, for example, be induced a week after the preceding menstruation has ceased.
  • the resulting overmature oocytes are then most likely not to be fertilized. The normal menstrual cycle is not likely to be affected.
  • contraception in females can also be achieved by administration of a compound of the invention which inhibits the meiosis, so that no mature oocytes are produced.
  • contraception in males can be achieved by administration of a compound of the invention which inhibits the meiosis, so that no mature sperm cells are produced.
  • the present invention relates to the use of the compounds of the general formula I as tool substances or as starting materials for the synthesis of tool substances for bioimaging purposes in order to clarify the mode of action of such substances.
  • meiosis-activating sterols that contain a fluorescence marker can be used to visualize the compartments of the germ cell where the active substances exert their biological function. This information may be helpful to clarify the mode of action of such substances.
  • the present invention relates to a method of regulating meiosis comprising administering to a subject in need of such a regulation an effective amount of one or more compounds of the general formula I.
  • the present invention relates to a method of regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vitro to a germ cell in need of such a regulation an effective amount of one or more compounds of the general formula I.
  • the germ cell may be an oocyte or a male germ cell.
  • compositions containing a compound of the invention may be any route which effectively transports the active compound to its site of action.
  • compositions which comprises at least one compound of the invention in connection with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for oral use, such compositions are preferably in the form of capsules or tablets.
  • the compounds of the invention When used as a contraceptive, the compounds of the invention will either have to be administered continuously or cyclically. When used as a contraceptive by females and not taken continuously, the timing of the administration relative to the ovulation will be important.
  • Example 4 1 ⁇ -cyano-4,4-dimethyl-5 ⁇ -cholesta-8,14-dien-3-one: 1.47 ml of a diethylaluminum cyanide solution (1 M in toluene) are added to a solution of 4,4-dimethyl-5 ⁇ -cholesta-1 ,8,14-trien-3-one in 5 ml tetrahydrofuran at 0 °C. The reaction mixture is allowed to warm to room temperature and stirred for one hour. After addition of 2.45 ml of a sodium hydroxide solution (1 M) at 0 °C the resulting mixture is diluted with water and extracted with diethylether.
  • a suspension of 64 mg 4-cyanobenzaldehyde and 27 mg potassium hydroxide in 2 ml ethanol is added dropwise to a suspension of 4,4-dimethyl-cholesta-8,14- dien-3-one in 4ml ethanol at room temperature.
  • the reaction mixture is stirred for 20 hours, diluted with water and extracted with dichloromethane. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered.
  • Example 8 (E)-N-[[4-[(3 ⁇ -hydroxy-4,4-dimethyl-5 ⁇ -cholesta-8, 14-dien-2- yliden)methyl]phenyl]methyl]octanamide: a) (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5 ⁇ -cholesta-8,14- dien-3 ⁇ -ol:
  • a solution of 200 mg 4,4-dimethyl-cholesta-8,14-dien-3-one in 3 ml THF is added dropwise to a freshly prepared lithium diisopropylamide solution (5.8 ml, 1 M) at - 70 °C.
  • the solution is stirred for one hour before 0.07 ml 3-iodo-propene are added.
  • the reaction mixture is poured into saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered.
  • 160 mg 2 ⁇ -allyl-4,4-dimethyl-5 ⁇ -cholesta-8,14-dien-3-one are treated with 55 mg sodium borohydride as described in example 5 to give 15 mg 2 ⁇ -allyl-4,4- dimethyl-5 ⁇ -cholesta-8,14-dien-3 ⁇ -ol and 80 mg 2 ⁇ -allyl-4,4-dimethyl-5 ⁇ - cholesta-8, 14-dien-3 ⁇ -ol.
  • Example 11 Testing of meiosis-activating substances in the oocyte test
  • mice were obtained from immature female mice (C57BI/6J x DBA/2J F1- hybrids, Bomholtgaard, Denmark) weighing 13 - 16 grams, that were kept under controlled lighting and temperature.
  • the mice received an intra-peritoneal injection of 0.2 ml gonadotropins (Gonal F, Serono, Solna, Sweden , containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU FSH) and 48 hours later the animals were killed by cervical dislocation.
  • gonadotropins Gonal F, Serono, Solna, Sweden , containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU FSH
  • the ovaries were dissected out and the oocytes were isolated in Hx-medium (see below) under a stereo microscope by manual rupture of the follicles using a pair of 27 gauge needles.
  • Spherical, naked oocytes (NO) displaying an intact germinal vesicle (GV) were placed in ⁇ -minimum essential medium ( ⁇ -MEM without ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg/ml Human Serum Albumin (HSA, State Serum Institute, Denmark), 0,23 mM pyrubate (Sigma, Cat. No.
  • Hx-medium 2 mM glutamine (Flow Cat. No. 16-801 ), 100 lU/ml penicillin and 100 ⁇ g/ml streptomycin (Flow, Cat No. 16-700).
  • This medium was designated Hx-medium.
  • the oocytes were rinsed three times in Hx-medium and cultured in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx- medium and 35 - 45 oocytes.
  • One control i.e. 35 - 45 oocytes cultured in Hx- medium with no addition of test compound was always run simultaneously with the test cultures, which were made with different concentrations of the compounds to be tested.
  • the cultures were performed at 37 °C and 100 % humidity with 5 % C0 2 in air.
  • the culture time was 22 - 24 hours.
  • the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment.
  • the percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture.
  • Example 12 Test of meiosis-inhibiting substances in the oocyte test
  • Germinal vesicle (GV) oocytes were obtained from immature FSH treated female mice using the same methods as described in Example 11 (see above). Naked oocytes (NO) were rinsed three times in Hx-medium. 4,4-Dimethyl-5 ⁇ -cholesta- 8,14,24-trien-3 ⁇ -ol (FF-MAS) has previously been shown to induce meiosis in NO in vitro (Byskov, A.G. et al. Nature 374 (1995) 559 - 562).
  • Hx-medium supplemented with 5 ⁇ M FF-MAS in co-culture with the test compounds in different concentrations in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx-medium and 35 - 45 oocytes.
  • One positive control i.e., 35 - 45 oocytes cultured in Hx-medium containing FF-MAS with no addition of test compound
  • one negative control 35 - 45 oocytes cultured in Hx-medium alone was run simultaneously with the positive control.
  • the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment.
  • the percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture.
  • Example 13 Testing of meiosis-activating substances in the ,in-vitro fertilisation' assay
  • NkO Naked oocytes
  • CEO cumulus enclosed oocytes
  • the oocytes (NkO and CEO) were pooled and cultured for approx. 20 hours in a modified ⁇ -MEM medium containing 3 mM hypoxanthine (Hx-medium) and 1 mg fetuin/ml culture medium.
  • Two oocyte-groups were used: (a) control oocytes, cultured in Hx-free medium (positive control group) and (b) oocytes, cultured in Hx-medium containing the test compound. After approx.
  • VVB germinal vesicle breakdown
  • GV germinal vesicle
  • GVB germinal vesicle breakdown
  • PB polar bodies
  • n number of oocytes
  • GVB germinal vesicle breakdown
  • PB polar bodies
  • n number of oocytes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Reproductive Health (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pregnancy & Childbirth (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Endocrinology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Steroid Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions comprising them as active substances and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the unsaturated cholestane derivatives of the invention can be used for regulating meiosis.

Description

Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
The present invention relates to pharmaceutically active unsaturated cholestane derivatives, to pharmaceutical compositions comprising them as active substances and to the use of these novel compounds for the preparation of medicaments. More particularly it has been found that the unsaturated cholestane derivatives of the invention can be used for regulating meiosis.
Meiosis is the unique and ultimate event of germ cells on which sexual reproduction is based. Meiosis comprises two meiotic divisions. During the first division, exchange between maternal and paternal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1 n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA synthesis. This division therefore results in the formation of the haploid germ cells with only 1 c DNA.
The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the female has a stock of oocytes which is drawn upon until the stock is exhausted. Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spermatozoa.
Only little is known about the mechanism which control the initiation of meiosis in the male and in the female, in the oocyte, new studies indicate that follicular purines, hypoxanthine or adenosine, could be responsible for meiotic arrest [Downs, S.M. et al. Dev Biol 82 (1985) 454-458: Epplg. J. J. et al Dev Biol 119 (1986) 313-321 ; and Downs, S.M. Mol Reprod Dev 35 (1993) 82-94]. The presence of a diffusible meiosis regulating substance was first described by Byskov et al. in a culture system of fetal mouse gonads [Byskov, A. G. et al. Dev Biol 52 (1976) 193-200]. A meiosis-activating substance (MAS) was secreted by the fetal mouse ovary in which meiosis was ongoing, and a meiosis preventing substance (MPS) was released from the morphologically differentiated testis with resting, non-meiotic germ cells. It was suggested that the relative concentrations of MAS and MPS regulated the beginning, arrest and resumption of meiosis in the male and in the female germ cells (Byskov, A.G. et al. in The Physiology of Reproduction [eds. Knobil. E. and Neill, J. D., Raven Press, New York (1994)]. Clearly, if meiosis can be regulated, reproduction can be controlled. A recent article [Byskov, A. G. et al. Nature 374 (1995), 559-562] describes the isolation from bull testes and from human follicular fluid of certain sterols that activate oocyte meiosis. Unfortunately, these sterols are rather labile and utilization of the interesting finding would thus be greatly facilitated if more stable meiosis- activating compounds were available.
Compounds being known to stimulate the meiosis and being different from the compounds claimed in the present patent application are described in WO 96/27658.
It is a purpose of the present invention to provide novel compounds for the treatment of infertility in females and males, particularly in humans via activation of meiosis.
It is a further purpose of the present invention to provide novel compounds useful as contraceptives in females and males, particularly in humans via inhibition of meiosis. According to the present invention there are provided novel, stable compounds with interesting pharmacological properties. In particular, the compounds described here are useful for the regulation of meiosis in oocytes and in male germ cells. It is a further purpose of the present invention to provide novel compounds which are suitable substrates for the introduction of fluorescense markers. The linked molecules are designed and synthesized for bioimaging purposes.
The present invention relates to unsaturated cholestane derivatives of the general formula I
Figure imgf000005_0001
wherein
R^ designates a hydrogen atom, a C2-Cg-alkyl group, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR1 group (with R1 being a C-|-C8-alkyl or an optionally substituted phenyl group) or together with R2 an additional bond,
R2 designates a hydrogen atom, a C-j-Cs-alkyl group, a Cs-Cρ-alkenyl group, a C-i-C -hydroxyalkyl group, together with R2' an optionally substituted benzylidene group, together with R2' a hydroxymethylene group or together with R^ an additional bond,
R2' designates a hydrogen atom, together with R2 an optionally substituted benzylidene group or together with R2 a hydroxymethylen group, R3 designates a hydrogen atom or together with R ' an additional bond, R3' designates a hydrogen atom or together with R3 an additional bond, R4 designates a hydrogen atom or a methyl group,
R4' designates a hydrogen atom or a methyl group,
R8 designates together with R or with R14 an additional bond,
R9 designates a hydrogen atom or together with R^ an additional bond
R"l4 designates an α-hydrogen atom or together with R^ an additional bond or together with R15 an additional bond
R15 designates a hydrogen atom or together with R^ an additional bond
R ^ designates a hydrogen atom or together with R2^ an additional bond
R ^ designates a hydrogen atom or together with R2^ an additional bond
or esters thereof,
with the proviso that compounds which are simultaneously unmodified in position 1 and 2 (R1 = R2 = R2'= H) are excluded.
The compounds of the general formula I have a number of chiral centres in the molecule and thus exist in several isomeric forms. All these isomeric forms and mixtures thereof are within the scope of the invention (unless otherwise noted).
Preferred compounds of formula I are such wherein R^ designates a hydrogen atom, a phenyl group or together with R2 an additional bond. Other preferred compounds are such wherein R2 designates a C-j-Cs-alkyl group, an allyl group or an additional bond with R^ . Compounds wherein R2 and R2' designate an optionally substituted benzylidene group are also preferred.
In another embodiment, the present invention relates to esters of compounds of the general formula I. Such esters are formally derived by estehfication of one or more hydroxylic groups of a compound of formula I with an acid which can for example be selected from the group of acids comprising succinic acid and other aliphatic dicarboxylic acids, nicotinic acid, isonicotinic acid, ethylcarbonic acid, phosphoric acid, sulphonic acid, sulphamic acid, benzoic acid, acetic acid, propionic acid and other aliphatic monocarboxylic acids.
As used in the present description and claims, an alkyl group - when used alone or in combinations - may be a straight or branched alkyl group. The expression C2-C5 alkyl designates an alkyl group having from two to six carbon atoms: preferred examples are ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and cyclohexyl more preferred ethyl. Similarly, the expression C^-Cg alkyl designates an alkyl group having from one to eight carbon atoms; preferred examples are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl and octyl more preferred methyl, ethyl, propyl, isopropyl, butyl and tert-butyl, still more preferred methyl and ethyl.
Especially preferred compounds of formula I of the present invention are the following: 2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
(E)-2-benzylidene-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
5α-cholesta-1 ,8,14-trien-3β-ol, 5α-cholesta-1 ,8,14-trien-3-one,
1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one,
4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3β-ol, 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3-one,
(E)-4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)- methyl]benzonitrile,
(E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2- ylidene)methyl]phenyl]methyl]octanamide, (E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)methyl]- phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3- yl)pentanamide,
2α-hydroxymethyl-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol,
2α-octyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol and (E)-4-[(3-oxo-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)methyl]benzonitrile.
The compounds of the general formula I according to the invention can be synthesized analogously with the preparation of known compounds. Hence, synthesis of the compounds of formula I can follow the well established synthetic pathways described in the comprehensive sterol and steroid literature. The following books can be used as the key source in the synthesis: L.F. Fieser & M. Fieser: Steroids: Reinhold Publishing Corporation, NY 1959; Rood's Chemistry of Carbon Compounds (editor: S. Coffrey): Elsevier Publishing Company, 1971 ; and especially Dictionary of Steroids (editors: R.A. Hill; D.N. Kirk; H.L.J. Makin and CM. Murphy): Chapman & Hall. The last one contains an extensive list of citations to the original papers covering the period up to 1990. All these books including the last mentioned citations are incorporated by reference.
Particularly, the compounds of the present invention are synthesized according to the following general procedures:
The sterols that are used as starting materials can be synthesized according to literature procedures:
4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol, 4,4-dimethyl-5α-cholest-8-en-3β-ol, 4,4-dimethyl-5α-cholest-8(14)-en-3β-ol [Biochem. J. 132 (1973), 439], 4,4- dimethyl-5α-cholesta-8,24-dien-3β-ol, 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β- ol [J. Am. Chem. Soc. 111 (1989), 278], 5α-cholesta-8,14-dien-3β-ol [J. Am. Chem. Soc. 75 (1953), 4404], 5α-cholest-8-en-3β-ol [J. Org. Chem. 46 (1981 ), 3421] and 5α-cholest-8(14)-en-3β-ol [Biochem. J. 144 (1974), 59].
In the following only the syntheses in the 4,4-dimethyl-Δ-8,14-series are described in detail. The derivatives in the Δ-8, Δ-8(14), Δ-8, 14,24 and Δ-8,24- series with and without the 4,4-dimethyl group can be synthesized from the corresponding starting materials in the same way.
The 3β-alcohols can be oxidized with different reagents to give the corresponding 3-ketones [for example: Tetrahedron Lett. 1967, 3699]. 4,4- dimethyl-5α-cholesta-8,14-dien-3β-ol 1 is treated with N-methyl-morpholine-N- oxide in the presence of tetrapropyl-ammonium-perruthenate to give 4,4- dimethyl-5α-cholesta-8,14-dien-3-one 2 [for example: Synthesis 1994, 639]. scheme 1 :
Figure imgf000010_0001
In the next step the Δ-1 double bond can be introduced in an oxidation reaction with phenyl selenic anhydride as an oxidant in chlorobenzene [J. Chem Soc. Chem. Commun. 1978, 952] to give 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3-one 3. This reaction also works in the case of compounds without the 4,4-dimethyl group. In these compounds the new double bond can be selectively introduced as a Δ1 -double bond. The 3-keto group can then be reduced with sodium borohydride in the presence of cerium chloride [Luche reduction, for example: J. Am. Chem. Soc. 100 (1978), 2226] to give 4,4-dimethyl-5α-cholesta-1 ,8,14-trien- 3β-ol 4.
Alkyl and aryl groups can be introduced in position 1 via cuprate additions to unsaturated ketones [Tetrahedron Lett. 35 (1994), 8591]. Cuprates which are formed from alkyl or aryl lithiums and cuprous iodide react with steroidal enones to give 1 -substituted derivatives. If for example enone 3 is treated with dialkylcuprates compounds of formula 5 (R1 = alkyl, aryl) are obtained. These ketones can then be reduced according to well known literature procedures to the two diastereome c alcohols 6 and 7 (R1 = alkyl, aryl), which are readily separable by column chromatography. scheme 2:
Figure imgf000011_0001
The introduction of a cyano group can be achieved in a similar way. Conjugate addition of cyanide to enone 3 gives the desired 1α-cyano-4,4-dimethyl-5α- cholesta-8,14-dien-3-one 5 (R1 = CN). Different reagents like diethylaluminum cyanide [J. Org. Chem. 59 (1994), 2766] and some alkali and earth alkali metals can be used in this reaction [Tetrahedron Lett. 28 (1987), 4189; Can. J. Chem. 59 (1981 ), 1641]. Cyanoketone 5 (R1 = CN) can also be reduced with standard reducing agents like sodium borohydride to give the two diastereomeric alcohols 6 and 7 (R1 = CN), which are readily separable by column chromatography.
If cyanoalcohol 6 (R1 = CN, synthesized as described above) is treated with lithium aluminumhydride 1α-aminomethyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β- ol 8 is obtained. This amine can be used for further modification. Amides of formula 9 (R1 = alkyl, aryl and fluorescence marker) can be synthesized from the amine by reaction with hydroxysuccinimidylester of various alkyl or aryl carboxylic acids. (For the use of hydroxysuccinimidylester of carboxylic acids that contain fluorescence marker see: Nonradioactive labeling and detection of biomolecules, Kessler C. ed., Springer Verlag, Berlin, 1992.) scheme 3:
Figure imgf000012_0001
Substituents in position 2 can for example be introduced via aldol reactions and alkylations. If ketone 2 is treated with aromatic aldehydes in the presence of a base 2-benzylidene-substituted steroids of formula 10 are obtained. The aromatic ring can be substituted. Subsequent reduction with sodium borohydride in the presence of cerium chloride gives selectively the corresponding allylic 3β- alcohols 11.
scheme 4:
Figure imgf000013_0001
Cyanobenzylidene-substituted compound 11 (R2 =4-CN) which can be synthesized as shown above can be used for further modification. The cyano group can be reduced with lithium aluminumhydride to the benzylic amine 12, which can be derivatized to corresponding amides of formula 13 (R2 = alkyl, aryl and fluorescence markers). For this purpose amine 12 is treated with hydroxysuccinimidyl ester derivatives of different carboxylic acids. For this reaction also commercially available esters that contain fluorescence markers can be used (see example 11 in the experimental part). Amides of formula 13 that contain the fluorescence markers can be used as molecular probes for bioimaging purposes.
scheme 5:
Figure imgf000014_0001
Alkyl- and alkenyl substituents can be introduced in position 2 via deprotonation of ketone 2 and subsequent reaction of the enolate with alkyl- and alkenyl halides. The 3-ketones of formula 14 can then be reduced with sodium borohydride to give two diastereomeric alcohols of formulae 15 and 16, which can be easily separated by column chromatography. scheme 6:
Figure imgf000014_0002
The hydroxymethylsubstituents in position 2 can for example be introduced via a condensation reaction of a deprotonated 3-ketone with alkylformiates to give enol 17. This can be reduced with different reducing agents like sodium borohydride to give two diastereomeric alcohols 18 and 19. scheme 7:
Figure imgf000015_0001
These alcohols can easily be separated with column chromatography. Longer chain hydroxyalkylsubstituents can for example be introduced via alkylation reactions as described above (see scheme 6).
A further object of the present invention are pharmaceutical compositions comprising one or more compounds of the general formula I as active substances. The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter. Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection or for use in connection with ex vivo and in vitro fertilization. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are men- tioned in the list above. Further, a composition for transdermal administration of a compound of this invention may be provided in the form of a patch and a composition for nasal administraton may be provided in the form of a nasal spray in liquid or powder form.
The dose of a compound of the invention to be used will be determined by a physician and will depend among several factors on the particular compound employed, on the route of administration and on the purpose of the use. In general, the compositions of the invention are prepared by intimately bringing into association the active compound with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation.
The present invention relates to the use of the compounds of the general formula I for the preparation of a meiosis-regulating medicament. The compounds of the present invention influence the meiosis in oocytes as well as in male germ cells. The prospects of being able to influence the meiosis are several. According to a preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate the meiosis. According to another preferred embodiment of the present invention, a compound of the general formula I can be used to stimulate the meiosis in humans. Thus, the compounds of the general formula I are promising as new fertility-regulating agents without the usual side effects on the somatic cells which are known from the hitherto used hormonal contraceptives which are based on estrogens and/or gestagens.
Accordingly, the present invention concerns the use of the compounds of the general formula I for relieving infertility in females and males, particularly in mammals, more particularly in humans. Meiosis-inducing substances of the general formula I can be used in the treatment of certain cases of infertility in females, including women, by administration thereof to females who, due to an insufficient own production of meiosis-activating substance, are unable to produce mature oocytes.
The compounds of the general formula I can also be used in artificial insemination procedures e.g. in in vitro fertilization or intracytoplasmic sperm injection. When in vitro fertilization is performed, better results can be achieved, when a compound of the invention is added to the medium in which the oocytes are cultured. When infertility in males, including men, is caused by an insufficient own production of the meiosis-activating substance and thus a lack of mature sperm cells exists, administration of a compound of the invention may relieve the problem.
In a further object of the present invention, the compounds of the general formula I are useful as contraceptives in females and males, particularly in mammals, more particularly in humans. For use as a contraceptive agent in females, a meiosis inducing substance can be administered so as to prematurely induce resumption of meiosis in oocytes while they are still in the growing follicle, before the ovulatory peak of gonadotropins occurs. In women, the resumption of the meiosis can, for example, be induced a week after the preceding menstruation has ceased. When ovulated, the resulting overmature oocytes are then most likely not to be fertilized. The normal menstrual cycle is not likely to be affected. In this connection it is important to notice, that the biosynthesis of progesterone in cultured human granulosa cells (somatic cells of the follicle) is not affected by the presence of a meiosis inducing substance whereas the estrogens and gestagens used in the hitherto used hormonal contraceptives do have an adverse effect on the biosynthesis of progesterone.
As an alternative to the method described above, contraception in females can also be achieved by administration of a compound of the invention which inhibits the meiosis, so that no mature oocytes are produced. Similarly, contraception in males can be achieved by administration of a compound of the invention which inhibits the meiosis, so that no mature sperm cells are produced.
In another aspect, the present invention relates to the use of the compounds of the general formula I as tool substances or as starting materials for the synthesis of tool substances for bioimaging purposes in order to clarify the mode of action of such substances. For example meiosis-activating sterols that contain a fluorescence marker can be used to visualize the compartments of the germ cell where the active substances exert their biological function. This information may be helpful to clarify the mode of action of such substances.
In a further aspect, the present invention relates to a method of regulating meiosis comprising administering to a subject in need of such a regulation an effective amount of one or more compounds of the general formula I.
In a still further aspect, the present invention relates to a method of regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vitro to a germ cell in need of such a regulation an effective amount of one or more compounds of the general formula I. The germ cell may be an oocyte or a male germ cell.
The route of administration of compositions containing a compound of the invention may be any route which effectively transports the active compound to its site of action.
Thus, when the compounds of this invention are to be administered to a mammal, they are conveniently provided in the form of a pharmaceutical composition which comprises at least one compound of the invention in connection with a pharmaceutically acceptable carrier. For oral use, such compositions are preferably in the form of capsules or tablets.
From the above it will be understood that administrative regimen called for will depend on the condition to be treated. Thus, when used in the treatment of infertility the administration may have to take place once only, or for a limited period, e.g. until pregnancy is achieved.
When used as a contraceptive, the compounds of the invention will either have to be administered continuously or cyclically. When used as a contraceptive by females and not taken continuously, the timing of the administration relative to the ovulation will be important.
The present invention is further illustrated by the following examples:
Example 1 : 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3-one
a) 4,4-dimethyl-5α-cholesta-8,14-dien-3-one:
To a solution of 6.40 g 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol and 4.03 g N- methyl-morpholin-N-oxide in 32 ml dichloromethane a few grains of molecular sieves are added and the mixture is stirred for five minutes. 408 mg tetrapropylammoniumperruthenate are added at room temperature and the resulting black reaction mixture is stirred for one hour. After filtration over celite the solvent is evaporated and the residue is chromatographed with a mixture of hexane and ethyl acetate to give 5.60 g 4,4-dimethyl-5α-cholesta-8,14-dien-3- one as a white solid.
1 H-NMR (CDCI3): δ= 0.83 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.07 (s, 3H); 1.12 (2x s, 6H); 2.55 (m, 2H); 5.41 (s, 1 H, H-15)
b) 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3-one:
To a solution of 2.00 g 4,4-dimethyl-5α-cholesta-8,14-dien-3-one in 30 ml chlorobenzene 1.75 g phenylselenic anhydride are added at room temperature. The reaction mixture is warmed to 100 °C for two hours. After cooling and evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 0.85 g 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3- one as an oil. 1 H-NMR (CDCI3): δ= 0.84 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.96 (d, J=7 Hz, 3H, H-21 ); 1.11 (s, 3H); 1.18 (s, 3H); 1.25 (s, 3H); 5.45 (s, 1 H, H-15); 5.95 (d, J=10 Hz, 1 H, H-2); 7.33 (d, J=10 Hz, 1 H, H-1)
Example 2: 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3β-ol
14 mg sodium borohydride are added to a suspension of 73 mg 4,4-dimethyl-5α- cholesta-1 ,8,14-trien-3-one and 67 mg cerium chloride heptahydrate in methanol at room temperature. The mixture is stirred for 4 hours, poured into water and extracted with diethylether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and diethylether to give 43 mg 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3β-ol as a white solid.
1 H-NMR (CDCI3): δ= 0.82 (s, 3H); 0.85 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H- 26/27); 0.96 (d, J=7 Hz, 3H, H-21 ); 1.02 (s, 3H); 1.10 (s, 3H); 3.89 (m, 1 H, H-3); 5.37 (s, 1 H, H-15); 5.50 (dd, J=10 Hz, 1 Hz, 1 H, H-1 ); 5.90 (d, J=10 Hz, 2 Hz, 1 H, H-2)
Example 3: 5α-cholesta-1 ,8,14-trien-3β-ol
a) 5α-cholesta-8,14-trien-3-one:
2.40 g 5α-cholesta-8,14-dien-3β-ol are treated with 1.11 g N-methyl-morpholin- N-oxide and 111 mg tetrapropylammoniumperruthenate in 13 ml dichloromethane as described in example 1a. After chromatography 1.81 g 5α- cholesta-8,14-dien-3-one are obtained as a white solid. 1 H-NMR (CDCI3): δ= 0.82 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21 ); 1.18 (s, 3H, H-19); 5.39 (s, 1 H, H-15)
b) 5α-cholesta-1 ,8,14-trien-3-one
1.81 g 5α-cholesta-8,14-dien-3-one are treated with 1.67 g phenylselenic anhydride in 29 ml chlorobenzene as described in example 1 b. After chromatography 0.53 g 5α-cholesta-1 ,8,14-trien-3-one are obtained as an oil.
1 H-NMR (CDCI3): δ= 0.84 (s, 3H, H-18); 0.86 (2x d, J=7 Hz, 6H, H-26/27); 0.96 (d, J=7 Hz, 3H, H-21 ); 1.20 (s, 3H, H-19); 5.43 (s, 1 H, H-15); 5.91 (d, J=10 Hz, 1 H, H-2); 7.42 (d, J=10 Hz, 1 H, H-1 )
c) 5α-cholesta-1 ,8,14-trien-3β-ol
150 mg 5α-cholesta-1 ,8,14-trien-3-one are treated with 15 mg sodium borohydride and 147 mg cerium chloride heptahydrate as described in example 2. After chromatography 47 mg 5α-cholesta-1 ,8,14-trien-3β-ol are obtained as a white solid.
1 H-NMR (CDCI3): δ= 0.82 (s, 3H, H-18); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.95 (d, J=7 Hz, 3H, H-21 ); 1.07 (s, 3H, H-19); 4.33 (m, 1 H, H-3); 5.37 (s, 1 H, H- 15); 5.56 (dd, J=10 Hz, 1 Hz, 1 H, H-1 ); 6.09 (d, J=10 Hz, 2 Hz, 1 H, H-2)
Example 4: 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one: 1.47 ml of a diethylaluminum cyanide solution (1 M in toluene) are added to a solution of 4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3-one in 5 ml tetrahydrofuran at 0 °C. The reaction mixture is allowed to warm to room temperature and stirred for one hour. After addition of 2.45 ml of a sodium hydroxide solution (1 M) at 0 °C the resulting mixture is diluted with water and extracted with diethylether. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 110 mg 1α- cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one as a white solid.
H-NMR (CDCI3): δ= 0.86 (2x d, J=7 Hz, 6H, H-26/27); 0.89 (s, 3H); 0.94 (d, J=7 Hz, 3H, H-21); 1.12 (s, 3H); 1.15 (s, 3H); 1.20 (s, 3H); 2.86 (m, 1H, H-2); 3.29 (dd, J=7 Hz, 5Hz, 1 H, H-1 ); 5.49 (ps, 1 H, H-15)
Example 5: 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol and 1α- cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol:
95 mg 1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3-one are treated with 33 mg sodium borohydride as described in example 5 to give 30 mg 1α-cyano-4,4- dimethyl-5α-cholesta-8,14-dien-3α-ol and 25 mg 1α-cyano-4,4-dimethyl-5α- cholesta-8,14-dien-3β-ol:
1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol:
1 H-NMR (CDCI3): δ= 0.83 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21 ); 1.09 (s, 3H); 1.17 (s, 3H); 3.08 (m, 1 H, H-1 ); 3.72 (bm, 1 H, H- 3); 5.43 (ps, 1 H, H-15)
1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol 1 H-NMR (CDCI3): δ= 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.91 (s, 3H); 0.93 (d, J=7 Hz, 3H, H-21 ); 1.08 (s, 3H); 1.15 (s, 3H); 1.28 (s, 3H); 2.92 (m, 1 H, H-1 ); 3.57 (ps, 1 H, H-3); 5.44 (ps, 1 H, H-15)
Example 6: (E)-4-[(3-oxo-4,4-dimethyl-5α-cholesta-8,14-dien-2- yliden)methyl]benzonitrile
A suspension of 64 mg 4-cyanobenzaldehyde and 27 mg potassium hydroxide in 2 ml ethanol is added dropwise to a suspension of 4,4-dimethyl-cholesta-8,14- dien-3-one in 4ml ethanol at room temperature. The reaction mixture is stirred for 20 hours, diluted with water and extracted with dichloromethane. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 168 mg (E)-4-[(3-oxo-4,4-dimethyl- 5α-cholesta-8,14-dien-2-yliden)methyl]benzonitrile as a white solid.
1 H-NMR (CDCI3): δ= 0.83 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21); 0.98 (s, 3H); 1.16 (s, 3H); 1.23 (s, 3H); 2.58 (d, J=17 Hz, 1H, H-1 ); 3.10 (d, J=17 Hz, 1 H, H-1 ); 5.47 (ps, 1 H, H-15); 7.42 (s, 1 H, H-2'); 7.53 (d, J=8 Hz, 2H, arom.); 7.68 (d, J=8 Hz, 2H, arom.)
Example 7: (E)-4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-yliden)- methyl]benzonitrile
156 mg (E)-4-[(3-oxo-4,4-dimethyl-5α-cholesta-8,14-dien-2-yliden)methyl]benzo- nitrile are treated wih 12 mg sodium borohydride in the presence of 111 mg cerium chloride heptahydrate as described in example 2 to give 93 mg (E)-4- [(3β-hydroxy-4,4-dimethyl-5α-cholesta-8, 14-dien-2-yliden)-methyl]benzonitrile as a white solid.
1 H-NMR (CDCI3): δ= 0.75 (s, 3H); 0.80 (s, 3H); 0.85 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.92 (d, J=7 Hz, 3H, H-21 ); 1.18 (s, 3H); 3.02 (d, J=17 Hz, 1 H, H-1 ); 3.94 (m, 1 H, H-3); 5.40 (ps, 1 H, H-15); 6.78 (s, 1 H, H-2'); 7.37 (d, J=8 Hz, 2H, arom.); 7.64 (d, J=8 Hz, 2H, arom.)
Example 8: (E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8, 14-dien-2- yliden)methyl]phenyl]methyl]octanamide: a) (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5α-cholesta-8,14- dien-3β-ol:
A suspension of 27 mg lithium aluminumhydride and 74 mg (E)-4-[(3β-hydroxy- 4,4-dimethyl-5α-cholesta-8,14-dien-2-yliden)-methyl]benzonitrile in 6 ml tetrahydro-furan is refluxed for 4 hours. After cooling the reaction mixture is worked up as described in example 3 b. Cristallization from ethylacetate gives 26 mg (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4-dimethyl-5α-cholesta-8, 14- dien-3β-ol as a white solid.
H-NMR (CDCI3): δ= 0.77 (s, 3H); 0.82 (s, 3H); 0.86 (2x d, J=7 Hz, 6H, H- 26/27); 0.89 (s, 3H); 0.92 (d, J=7 Hz, 3H, H-21 ); 1.18 (s, 3H); 3.15 (d, J=17 Hz, 1 H, H-1 ); 3.86 (s, 2H, Ar-CH2-N); 3.92 (ps, 1 H, H-3); 5.38 (ps, 1 H, H-15); 6.72 (s, 1 H, H-2'); 7.26 (m, 4H, arom.)
b) (E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2- yliden)methyl]phenyl]methyl]octanamide:
A solution of 8.6 mg N-hydroxysuccinimidyl caprylate in 1 ml dimethylforamide is added to a solution of 21 mg (E)-2-(4-aminomethyl-phenyl)-methylidene-4,4- dimethyl-5α-cholesta-8,14-dien-3β-ol in 4 ml dimethylformamide at room temperature. The reaction mixture is stirred for 20 hours, diluted with water and extracted with ethylacetate.
The organic layer is separated, washed with water, dried over anhydrous sodium sulphate and filtered. Evaporation of the solvent gives 21 mg (E)-N-[[4-[(3β- hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)methyl]phenyl]methyl]- octanamide as a white solid. 1H-NMR (CDCI3): δ= 0.77 (s, 3H); 0.82 (s, 3H); 0.86 (2x d, J=7 Hz, 6H, H- 26/27); 0.89 (s, 3H); 0.92 (d, J=7 Hz, 3H, H-21 ); 1.18 (s, 3H); 3.12 (d, J=17 Hz, 1 H, H-1 ); 3.92 (ps, 1 H, H-3); 4.44 (d, J=5 Hz, 2H, Ar-CH2-N); 5.37 (ps, 1 H, H- 15); 5.76 (t, J=5 Hz, 1 H, NH); 6.72 (s, 1 H, H-2'); 7.24 (m, 4H, arom.)
Example 9: (E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2- yliden)methyl]-phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacen-3-yl)pentanamide:
A solution of 5.0 mg 5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3- yl)pentanoic acid succinimidyl ester in 0.5 ml dimethylforamide is added to a solution of 8.9 mg (E)-2-(4-aminomethyl-phenyl)-methyiidene-4,4-dimethyl-5α- cholesta-8,14-dien-3β-ol in 2 ml dimethylformamide at room temperature. The reaction mixture is stirred for 44 hours, diluted with water and extracted with ethylacetate. The organic layer is separated, washed with water, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethyl acetate to give 6.5 mg (E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-yliden)- methyl]-phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacen-3-yl)pentanamide.
1 H-NMR (CDCI3): δ= 0.77 (s, 3H); 0.82 (s, 3H); 0.86 (2x d, J=7 Hz, 6H, H-
26/27); 0.89 (s, 3H); 0.92 (d, J=7 Hz, 3H, H-21 ); 1.19 (s, 3H); 2.25 (s, 3H, Ar- Me); 2.55 (s, 3H, Ar-Me); 3.12 (d, J=17 Hz, 1 H, H-1 ); 3.92 (ps, 1 H, H-3); 4.44 (d,
J=5 Hz, 2H, Ar-CH2-N); 5.37 (ps, 1 H, H-15); 5.87 (m, 1 H, NH); 6.09 (s, 1 H, arom.); 6.30 (d, J=5 Hz, 1 H, arom.); 6.70 (s, 1 H, H-2'); 6.90 (d, J=5 Hz, 1 H, arom.); 7.24 (m, 4H, arom.)
Example 10: 2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol
a) 2α-allyl-4,4-dimethyl-5α-cholesta-8, 14-dien-3-one:
A solution of 200 mg 4,4-dimethyl-cholesta-8,14-dien-3-one in 3 ml THF is added dropwise to a freshly prepared lithium diisopropylamide solution (5.8 ml, 1 M) at - 70 °C. The solution is stirred for one hour before 0.07 ml 3-iodo-propene are added. After being stirred for one additional hour at 0 °C the reaction mixture is poured into saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer is separated, washed with brine, dried over anhydrous sodium sulphate and filtered. After evaporation of the solvent the residue is chromatographed with a mixture of hexane and ethylacetate to give 160 mg 2α- allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3-one as a white solid.
1 H-NMR (CDCI3): δ= 0.82 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21 ); 1.10 (2x s, 6H); 1.30 (s, 3H); 2.60 (m, 1 H, allyl); 2.77 (m, 1 H, allyl); 5.05 (m, 2H, allyl); 5.38 (ps, 1 H, H-15); 5.80 (m, 1 H, allyl)
b) 2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol:
160 mg 2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3-one are treated with 55 mg sodium borohydride as described in example 5 to give 15 mg 2α-allyl-4,4- dimethyl-5α-cholesta-8,14-dien-3α-ol and 80 mg 2α-allyl-4,4-dimethyl-5α- cholesta-8, 14-dien-3β-ol.
2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol:
1 H-NMR (CDCI3): δ= 0.81 (s, 3H); 0.85 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H- 26/27); 0.94 (d, J=7 Hz, 3H, H-21 ); 1.03 (s, 3H); 1.05 (s, 3H); 2.50 (m, 1 H); 2.95 (d, J=11 Hz, 1 H, H-3); 5.06 (m, 2H, allyl); 5.37 (ps, 1 H, H-15); 5.89 (m, 1 H, allyl)
2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol:
1 H-NMR (CDCI3): δ= 0.82 (s, 3H); 0.87 (2x d, J=7 Hz, 6H, H-26/27); 0.90 (s, 3H); 0.94 (d, J=7 Hz, 3H, H-21); 1.00 (s, 3H); 1.05 (s, 3H); 3.36 (ps, 1 H, H-3); 5.05 (m, 2H, allyl); 5.35 (ps, 1 H, H-15); 5.85 (m, 1 H, allyl)
Example 11: Testing of meiosis-activating substances in the oocyte test
Animals Oocytes were obtained from immature female mice (C57BI/6J x DBA/2J F1- hybrids, Bomholtgaard, Denmark) weighing 13 - 16 grams, that were kept under controlled lighting and temperature. The mice received an intra-peritoneal injection of 0.2 ml gonadotropins (Gonal F, Serono, Solna, Sweden , containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU FSH) and 48 hours later the animals were killed by cervical dislocation.
Collection and culture of oocytes
The ovaries were dissected out and the oocytes were isolated in Hx-medium (see below) under a stereo microscope by manual rupture of the follicles using a pair of 27 gauge needles. Spherical, naked oocytes (NO) displaying an intact germinal vesicle (GV) were placed in α-minimum essential medium (α-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg/ml Human Serum Albumin (HSA, State Serum Institute, Denmark), 0,23 mM pyrubate (Sigma, Cat. No. S-8636), 2 mM glutamine (Flow Cat. No. 16-801 ), 100 lU/ml penicillin and 100 μg/ml streptomycin (Flow, Cat No. 16-700). This medium was designated Hx-medium. The oocytes were rinsed three times in Hx-medium and cultured in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx- medium and 35 - 45 oocytes. One control (i.e. 35 - 45 oocytes cultured in Hx- medium with no addition of test compound) was always run simultaneously with the test cultures, which were made with different concentrations of the compounds to be tested.
The cultures were performed at 37 °C and 100 % humidity with 5 % C02 in air. The culture time was 22 - 24 hours.
Examination of oocytes
By the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. The percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture.
Example 12: Test of meiosis-inhibiting substances in the oocyte test
Germinal vesicle (GV) oocytes were obtained from immature FSH treated female mice using the same methods as described in Example 11 (see above). Naked oocytes (NO) were rinsed three times in Hx-medium. 4,4-Dimethyl-5α-cholesta- 8,14,24-trien-3β-ol (FF-MAS) has previously been shown to induce meiosis in NO in vitro (Byskov, A.G. et al. Nature 374 (1995) 559 - 562). NO were cultured in Hx-medium supplemented with 5 μM FF-MAS in co-culture with the test compounds in different concentrations in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx-medium and 35 - 45 oocytes. One positive control (i.e., 35 - 45 oocytes cultured in Hx-medium containing FF-MAS with no addition of test compound) was always run simultaneously with the test cultures, which were supplemented with different concentrations of the compounds to be tested. In addition, one negative control (35 - 45 oocytes cultured in Hx-medium alone) was run simultaneously with the positive control.
Examination of oocytes
By the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. The percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture.
Example 13: Testing of meiosis-activating substances in the ,in-vitro fertilisation' assay
Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles of immature (C57BL/6xDBA 2) F1 mice (age 21-24 days), that had received 10 IU Pregnant Mare Serum Gonadotropin (FSH activity) i.p. 48 hours prior to collection.
The oocytes (NkO and CEO) were pooled and cultured for approx. 20 hours in a modified α-MEM medium containing 3 mM hypoxanthine (Hx-medium) and 1 mg fetuin/ml culture medium. Two oocyte-groups were used: (a) control oocytes, cultured in Hx-free medium (positive control group) and (b) oocytes, cultured in Hx-medium containing the test compound. After approx. 20 hours oocytes that exhibited germinal vesicle breakdown (GVB) were briefly washed in Hx-free medium and transferred to insemination dishes prepared in advance, which consisted of a motile sperm preparation from the cauda epididymis of male mice. The dishes were then incubated under defined gas conditions (5 % CO2) at 37°C in a modified α-MEM IVF-medium. Examination of the oocytes was carried out 20-22 hours after insemination, in order to check fertilisation and to record the number of 2-cell embryos. The percentage fertilisation (= fertilisation rate) was determined as number of oocytes that had cleaved into two-cell embryos, relative to the total number inseminated. Each IVF-experiment was performed with a total number of 50-200 GVB/PB-oocytes. A stimulation factor was calculated as the ratio between the fertilisation rate in the group containing the test compound and the control group.
Table 1 : Activation of meiosis in naked mouse oocytes
Figure imgf000031_0001
Hx = Hypoxanthine
GV = germinal vesicle
GVB = germinal vesicle breakdown
PB = polar bodies n = number of oocytes
Table 2: Relative inhibition of meiosis in naked mouse oocytes
Figure imgf000031_0002
Hx = Hypoxanthine GV = germinal vesicle GVB = germinal vesicle breakdown PB = polar bodies n = number of oocytes
Table 3: Relative inhibition of meiosis in naked mouse oocytes
Figure imgf000032_0001
Hx = Hypoxanthine GV = germinal vesicle
GVB = germinal vesicle breakdown PB = polar bodies n = number of oocytes

Claims

Claims
Compounds of the general formula I
Figure imgf000033_0001
wherein
R1 designates a hydrogen atom, a C2-Cø-alkyl group, an optionally substituted phenyl group, a cyano group, a CH2-NH-COR group ( (wwiitthh RR1 bbeeiinngg aa CC--||--CC88--aallkkyyll oorr aann ooppttiioonnaalllly substituted phenyl group) or together with R2 an additional bond,
R2 designates a hydrogen atom, a C^Cg-alkyl group, a C3-C6- alkenyl group, a C-|-Cg-hydroxyalkyl group, together with R2' an optionally substituted benzylidene group, together with R2' a hydroxymethylene group or together with R^ an additional bond,
R2' designates a hydrogen atom or together with R2 an optionally substituted benzylidene group or together with R2 a hydroxymethylene group,
R3 designates a hydrogen atom or together with R^' an additional bond, R3' designates a hydrogen atom or together with R3 an additional bond,
R4 designates a hydrogen atom or a methyl group,
R4' designates a hydrogen atom or a methyl group, R8 designates together with R? or with R^ an additional bond,
R9 designates a hydrogen atom or together with R^ an additional bond
R14 designates an α-hydrogen atom or together with R^ an additional bond or together with R^ an additional bond R15 designates a hydrogen atom or together with Rl4 an additional bond
R2^ designates a hydrogen atom or together with R2^ an additional bond
R25 designates a hydrogen atom or together with R2^ an additional bond
or esters thereof,
with the proviso that compounds which are simultaneously unmodified in position 1 and 2 (R1 = R2 = R2'= H) are excluded.
2. Compounds according to claim 1 , wherein R1 designates an hydrogen atom, a phenyl group or together with R2 an additional bond.
3. Compounds according to claim 1 or 2, wherein R2 designates a C-j-Cg-alkyl group, an allyl group or an additional bond with R^ .
4. Compounds according to any one of claims 1 or 2, wherein R2 and R2 designate an optionally substituted benzylidene group.
. Compounds according to any one of claims 1 to 4 which are
2α-allyl-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
(E)-2-benzylidene-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
5α-cholesta-1 ,8,14-trien-3β-ol, 5α-cholesta-1 ,8,14-then-3-one,
1α-cyano-4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol,
1 α-cyano-4,4-dimethyl-5α-cholesta-8, 14-dien-3-one,
4,4-dimethyl-5α-cholesta-1 ,8,14-trien-3β-ol,
4,4-dimethyl-5α-cholesta-1 ,8,14-then-3-one, (E)-4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8, 14-dien-2-ylidene)-methyl]- benzonitrile,
(E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)- methyl]phenyl]methyl]octanamide,
(E)-N-[[4-[(3β-hydroxy-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)- methyl]-phenyl]methyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacen-3-yl)pentanamide,
2α-hydroxymethyl-4,4-dimethyl-5α-cholesta-8,14-dien-3α-ol,
2α-octyl-4,4-dimethyl-5α-cholesta-8, 14-dien-3β-ol or
(E)-4-[(3-oxo-4,4-dimethyl-5α-cholesta-8,14-dien-2-ylidene)methyl]- benzonitrile.
6. Pharmaceutical compositions comprising one or more compounds of the general formula I according to any one of claims 1 to 5 as active substances.
7. Use of the compounds of the general formula I according to any one of claims 1 to 5 for the preparation of a meiosis regulating medicament.
8. Use according to claim 7 for the preparation of a medicament for the treatment of infertility in females or males, preferably in humans.
9. Use according to claim 7 for the preparation of a contraceptive medicament > for the treatment of females or males, preferably humans.
10. Use of the compounds of the general formula I according to any one of claims 1 to 5 to regulate the fertilisation rate in artificial insemination procedures.
11. Use of the compounds of the general formula I according to any one of claims 1 to 5 as tool substances or as starting materials for the synthesis of tool substances for bioimaging purposes in order to clarify the mode of action of such substances.
12. A method of regulating meiosis comprising administering to a subject in need of such a regulation an effective amount of one or more compounds of the general formula I according to any one of claims 1 to 5.
13. A method of regulating meiosis in a mammalian germ cell comprising administering ex vivo or in vitro to a germ cell in need of such a regulation an effective amount of one or more compounds of the general formula I according to any one of claims 1 to 5.
5 14. A method according to claim 13 wherein the germ cell is an oocyte or a male germ cell.
PCT/EP2000/001074 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments WO2000047604A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
BR0008065-9A BR0008065A (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of medicines that regulate meiosis
EP00910664A EP1150993A1 (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
AU32797/00A AU3279700A (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
SK1138-2001A SK11382001A3 (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
JP2000598521A JP2002536456A (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use in the production of meiosis regulators
IL14373500A IL143735A0 (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
CA002359687A CA2359687A1 (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
KR1020017009985A KR20010101820A (en) 1999-02-10 2000-02-09 Unsaturated Cholestane Derivatives and Their Use for the Preparation of Meiosis Regulating Medicaments
NO20013901A NO20013901D0 (en) 1999-02-10 2001-08-10 Unsaturated cholesterol derivatives and their use in the preparation of meiosis-regulating drugs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP99250040 1999-02-10
EP99250040.5 1999-02-10

Publications (1)

Publication Number Publication Date
WO2000047604A1 true WO2000047604A1 (en) 2000-08-17

Family

ID=8241140

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/001074 WO2000047604A1 (en) 1999-02-10 2000-02-09 Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments

Country Status (15)

Country Link
EP (1) EP1150993A1 (en)
JP (1) JP2002536456A (en)
KR (1) KR20010101820A (en)
CN (1) CN1368977A (en)
AU (1) AU3279700A (en)
BR (1) BR0008065A (en)
CA (1) CA2359687A1 (en)
CZ (1) CZ20012866A3 (en)
HU (1) HUP0200280A2 (en)
IL (1) IL143735A0 (en)
NO (1) NO20013901D0 (en)
PL (1) PL350557A1 (en)
SK (1) SK11382001A3 (en)
WO (1) WO2000047604A1 (en)
ZA (1) ZA200107387B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000235A1 (en) * 1994-06-23 1996-01-04 Novo Nordisk A/S Sterol derivatives used for regulation of meiosis
WO1996027658A1 (en) * 1995-03-06 1996-09-12 Novo Nordisk A/S Stimulation of meiosis
WO1997000884A1 (en) * 1995-06-23 1997-01-09 Novo Nordisk A/S Meiosis regulating compounds
WO1998028323A1 (en) * 1996-12-20 1998-07-02 Novo Nordisk A/S Meiosis regulating compounds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000235A1 (en) * 1994-06-23 1996-01-04 Novo Nordisk A/S Sterol derivatives used for regulation of meiosis
WO1996027658A1 (en) * 1995-03-06 1996-09-12 Novo Nordisk A/S Stimulation of meiosis
WO1997000884A1 (en) * 1995-06-23 1997-01-09 Novo Nordisk A/S Meiosis regulating compounds
WO1998028323A1 (en) * 1996-12-20 1998-07-02 Novo Nordisk A/S Meiosis regulating compounds

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HYLANDS, PETER J. ET AL: "Unsaturated steroids. Part 5. Synthesis of 4.alpha.-methylcholest- 8(9)-en-3.beta.-ol", J. CHEM. SOC., PERKIN TRANS. 1 (1977), (8), 817-19, XP002139138 *
L. HO-SHEN ET AL: "Synthesis and Biological Evaluation of a New Series of Sterols as Potential Hypocholesterolemic Agents", JOURNAL OF MEDICINAL CHEMISTRY., vol. 38, no. 2, 20 January 1995 (1995-01-20), AMERICAN CHEMICAL SOCIETY. WASHINGTON., US, pages 277 - 288, XP002139139, ISSN: 0022-2623 *
TAYLOR, FREDERICK R. ET AL: "Photoaffinity labeling of the oxysterol receptor", J. BIOL. CHEM. (1988), 263(5), 2264-9, XP002139137 *

Also Published As

Publication number Publication date
NO20013901L (en) 2001-08-10
CZ20012866A3 (en) 2002-01-16
IL143735A0 (en) 2002-04-21
CA2359687A1 (en) 2000-08-17
ZA200107387B (en) 2002-12-06
CN1368977A (en) 2002-09-11
BR0008065A (en) 2001-11-06
PL350557A1 (en) 2002-12-16
KR20010101820A (en) 2001-11-14
NO20013901D0 (en) 2001-08-10
AU3279700A (en) 2000-08-29
HUP0200280A2 (en) 2002-07-29
SK11382001A3 (en) 2002-01-07
EP1150993A1 (en) 2001-11-07
JP2002536456A (en) 2002-10-29

Similar Documents

Publication Publication Date Title
US6884796B2 (en) Meiosis regulating compounds
EP0857173B1 (en) Meiosis regulating compounds
AU746878B2 (en) Meiosis regulating compounds
US6645953B2 (en) Meiosis regulating compounds
EP1077992B1 (en) Use of meiosis regulating compounds
EP1150993A1 (en) Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
MXPA01007526A (en) Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments
US20020007079A1 (en) Meiosis regulating compounds
KR20020013541A (en) 14β-H-Sterols, Pharmaceutical Compositions Comprising Them and Use of These Derivatives for the Preparation of Meiosis Regulating Medicaments
EP0957108A1 (en) Unsaturated cholestane derivatives, pharmaceutical compositions comprising them and use of these derivatives for the preparation of meiosis regulating medicaments
EP1127890A1 (en) Steroid derivatives with an additional ring annulated to ring A and use of these derivatives for the preparation of meiosis regulating medicaments
CA2528904A1 (en) Thiomorpholino steroid compounds, the use thereof for the preparation of meiosis-regulating medicaments and method for the preparation thereof

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 00803662.4

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DK DM 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

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000910664

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 143735

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 2359687

Country of ref document: CA

Ref document number: 2359687

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/a/2001/007526

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2000 598521

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 32797/00

Country of ref document: AU

Ref document number: PV2001-2866

Country of ref document: CZ

WWE Wipo information: entry into national phase

Ref document number: 1020017009985

Country of ref document: KR

Ref document number: 11382001

Country of ref document: SK

WWE Wipo information: entry into national phase

Ref document number: 2001/07387

Country of ref document: ZA

Ref document number: 200107387

Country of ref document: ZA

WWP Wipo information: published in national office

Ref document number: 2000910664

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1020017009985

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 09913313

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: PV2001-2866

Country of ref document: CZ

WWW Wipo information: withdrawn in national office

Ref document number: 2000910664

Country of ref document: EP

WWR Wipo information: refused in national office

Ref document number: PV2001-2866

Country of ref document: CZ

WWW Wipo information: withdrawn in national office

Ref document number: 1020017009985

Country of ref document: KR