ZA200107387B - 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 PDFInfo
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- ZA200107387B ZA200107387B ZA200107387A ZA200107387A ZA200107387B ZA 200107387 B ZA200107387 B ZA 200107387B ZA 200107387 A ZA200107387 A ZA 200107387A ZA 200107387 A ZA200107387 A ZA 200107387A ZA 200107387 B ZA200107387 B ZA 200107387B
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- ZA
- South Africa
- Prior art keywords
- cholesta
- dimethyl
- dien
- designates
- compounds
- Prior art date
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- 230000021121 meiosis Effects 0.000 title claims description 35
- 230000001105 regulatory effect Effects 0.000 title claims description 13
- 238000002360 preparation method Methods 0.000 title claims description 9
- 239000003814 drug Substances 0.000 title claims description 7
- 150000001837 cholestane derivatives Chemical class 0.000 title description 5
- 150000001875 compounds Chemical class 0.000 claims description 75
- 210000000287 oocyte Anatomy 0.000 claims description 55
- 239000000203 mixture Substances 0.000 claims description 28
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 24
- 239000000126 substance Substances 0.000 claims description 23
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzenecarbonitrile Natural products N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 16
- -1 hydroxymethylene group Chemical group 0.000 claims description 15
- 210000004602 germ cell Anatomy 0.000 claims description 11
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 239000003433 contraceptive agent Substances 0.000 claims description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 7
- 125000000649 benzylidene group Chemical group [H]C(=[*])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 6
- 208000000509 infertility Diseases 0.000 claims description 6
- 230000036512 infertility Effects 0.000 claims description 6
- 231100000535 infertility Toxicity 0.000 claims description 6
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- 230000009471 action Effects 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 4
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- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 239000007858 starting material Substances 0.000 claims description 4
- 230000002254 contraceptive effect Effects 0.000 claims description 3
- IPWFJLQDVFKJDU-UHFFFAOYSA-N pentanamide Chemical compound CCCCC(N)=O IPWFJLQDVFKJDU-UHFFFAOYSA-N 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 30
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 21
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 20
- 239000002609 medium Substances 0.000 description 18
- 210000004508 polar body Anatomy 0.000 description 17
- 238000012360 testing method Methods 0.000 description 15
- 229910052739 hydrogen Inorganic materials 0.000 description 14
- 125000000217 alkyl group Chemical group 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 12
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- 229940093499 ethyl acetate Drugs 0.000 description 10
- 235000019439 ethyl acetate Nutrition 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- 239000012279 sodium borohydride Substances 0.000 description 9
- 229910000033 sodium borohydride Inorganic materials 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 8
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- JXUJDVNCKDZIKF-SRSKCPMFSA-N (5r,10s,13r,17r)-4,4,10,13-tetramethyl-17-[(2r)-6-methylheptan-2-yl]-6,7,11,12,16,17-hexahydro-5h-cyclopenta[a]phenanthren-3-one Chemical compound C([C@@]12C)=CC(=O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCCC(C)C)CC=C21 JXUJDVNCKDZIKF-SRSKCPMFSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 6
- 125000003118 aryl group Chemical group 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 6
- 239000012044 organic layer Substances 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 description 6
- 235000011152 sodium sulphate Nutrition 0.000 description 6
- LFQXEZVYNCBVDO-PBJLWWPKSA-N 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@@H](CCC=C(C)C)C)CC=C21 LFQXEZVYNCBVDO-PBJLWWPKSA-N 0.000 description 5
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- 238000007792 addition Methods 0.000 description 5
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- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 4
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- 239000007758 minimum essential medium Substances 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
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- QXFOKPYCBXATOI-SRSKCPMFSA-N (5r,10s,13r,17r)-4,4,10,13-tetramethyl-17-[(2r)-6-methylheptan-2-yl]-2,5,6,7,11,12,16,17-octahydro-1h-cyclopenta[a]phenanthren-3-one Chemical compound C([C@@]12C)CC(=O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCCC(C)C)CC=C21 QXFOKPYCBXATOI-SRSKCPMFSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- KPZSTOVTJYRDIO-UHFFFAOYSA-K trichlorocerium;heptahydrate Chemical compound O.O.O.O.O.O.O.Cl[Ce](Cl)Cl KPZSTOVTJYRDIO-UHFFFAOYSA-K 0.000 description 3
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- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
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- 238000010521 absorption reaction Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- 230000002411 adverse Effects 0.000 description 1
- 238000005575 aldol reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- HFEHLDPGIKPNKL-UHFFFAOYSA-N allyl iodide Chemical compound ICC=C HFEHLDPGIKPNKL-UHFFFAOYSA-N 0.000 description 1
- 125000000746 allylic group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000003934 aromatic aldehydes Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 125000001743 benzylic group Chemical group 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
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- 239000002775 capsule Substances 0.000 description 1
- 150000001722 carbon compounds Chemical class 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000023752 cell cycle switching, mitotic to meiotic cell cycle Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- LSXDOTMGLUJQCM-UHFFFAOYSA-M copper(i) iodide Chemical compound I[Cu] LSXDOTMGLUJQCM-UHFFFAOYSA-M 0.000 description 1
- GTBRTGPZZALPNS-MXHVRSFHSA-N cyanoketone Chemical compound C1C=C2C(C)(C)C(=O)[C@H](C#N)C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@](C)(O)[C@@]1(C)CC2 GTBRTGPZZALPNS-MXHVRSFHSA-N 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 230000005595 deprotonation Effects 0.000 description 1
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- 235000019425 dextrin Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 150000002085 enols Chemical class 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 102000013361 fetuin Human genes 0.000 description 1
- 108060002885 fetuin Proteins 0.000 description 1
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- 108010006578 follitropin alfa Proteins 0.000 description 1
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- 238000009472 formulation Methods 0.000 description 1
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- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
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- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
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- 239000003068 molecular probe Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
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- 235000001968 nicotinic acid Nutrition 0.000 description 1
- WWZKQHOCKIZLMA-UHFFFAOYSA-M octanoate Chemical compound CCCCCCCC([O-])=O WWZKQHOCKIZLMA-UHFFFAOYSA-M 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
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- 239000001814 pectin Substances 0.000 description 1
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- 229920001277 pectin Polymers 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
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- 230000000284 resting effect Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
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- 229960005322 streptomycin Drugs 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical compound NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical compound OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 description 1
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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
Description
-_—
Unsaturated cholestane derivatives and their use for the preparation of meiosis regulating medicaments ee
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 patemal genes take place before the pairs of chromosomes are separated into the two daughter cells. These contain only half the number (1n) of chromosomes and 2c DNA. The second meiotic division proceeds without a DNA synthesis. This division therefore results in the : formation of the haploid germ celis with only 1c DNA.
The meiotic events are similar in the male and female germ cells, but the time schedule and the differentiation processes which lead to ova and to spermatozoa differ profoundly. All female germ cells enter the prophase of the first meiotic division early in life, often before birth, but all are arrested as oocytes later in the prophase (dictyate state) until ovulation after puberty. Thus, from early life the female has a stock of oocytes which is drawn upon until the stock is exhausted.
Meiosis in females is not completed until after fertilization, and results in only one ovum and two abortive polar bodies per germ cell. In contrast, only some of the male germ cells enter meiosis from puberty and leave a stem population of germ cells throughout life. Once initiated, meiosis in the male cell proceeds without significant delay and produces 4 spermatozoa.
Only little is known about the mechanism which control the initiation of meiosis in the male and in the female. In the oocyte, new studies indicate that follicular
Tey 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
S 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 feds. 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.
Itis 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.
. < Co oo 3 itis 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
R24 ~, R25 00
Ra Zz
R14 R11
R30 = Rs
Ras
R4 R#
I wherein : R1 designates a hydrogen atom, a Co-Cg-alkyl group, an optionally substituted phenyl group, a cyano group, a CHo-NH-COR" group (with
R" being a C4-Cg-alkyl or an optionally substituted phenyl group) or together with R2 an additional bond,
R2 designates a hydrogen atom, a C4-Cg-alkyl group, a C3-Cg-alkenyl group, a Cq-Cg-hydroxyalkyl group, together with RZ" an optionally substituted benzylidene group, together with RZ’ a hydroxymethylene group or together with R1 an additional bond,
RZ’ designates a hydrogen atom, together with RZ an optionally substituted benzylidene group or together with RZ a hydroxymethylen group,
R3 designates a hydrogen atom or together with R3' an additional bond,
R3' designates a hydrogen atom or together with R3 an additional bond,
* oo Co ~ 4
R4 designates a hydrogen atom or a methyl group, r4 designates a hydrogen atom or a methyl group,
R8 designates together with RS or with R14 an additional bond,
RY designates a hydrogen atom or together with R8 an additional bond
R14 designates an a-hydrogen atom or together with R8 an additional bond or together with R15 an additional bond
R15 designates a hydrogen atom or together with R14 an additional bond
R24 designates a hydrogen atom or together with R25 an additional bond
R25 designates a hydrogen atom or together with R24 an additional bond or esters thereof, with the proviso that compounds which are simultaneously unmodified in position 1 and 2 (R! = RZ = R2'= H) are excluded.
The compounds of the general formula | 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 | are such wherein R1 designates a hydrogen atom, a phenyl group or together with R2 an additional bond. Other preferred compounds are such wherein R2 designates a C4-Cg-alkyl group, an allyl group or an additional bond with R1. Compounds wherein R2 and RZ’ designate an optionally substituted benzylidene group are also preferred. in another embodiment, the present invention relates to esters of compounds of the general formula |. Such esters are formally derived by esterification of one or more hydroxylic groups of a compound of formula | 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,
» < Lo Co 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 5 or in combinations - may be a straight or branched alkyl group. The expression
Co-Cg alkyl designates an alkyl group having from two to six carbon atoms: preferred examples are ethyl, propyl, isopropyl, buty!, tert-butyl, pentyl, hexyl and cyclohexyl more preferred ethyl. Similarly, the expression C4-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 | of the present invention are the following: 2ca-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3B-ol, (E)-2-benzylidene-4,4-dimethyl-5a-cholesta-8, 14-dien-3p-ol, 5a-cholesta-1,8,14-trien-33-ol,
€ . . i . y 6
Sa-cholesta-1,8,14-trien-3-one, 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one, 4,4-dimethyl-5a-cholesta-1,8,14-trien-3p-al, 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one, (E)-4-{(3B-hydroxy-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene)- methyi)benzonitrile, (E)-N-[[4-[(3B-hydroxy-4,4-dimethyl-5a-~-cholesta-8,14-dien-2- ylidene)methyl]phenyllmethyl]octanamide, (E)-N-[{4-[(3B-hydroxy-4,4-dimethyl-5a-cholesta-8, 1 4-dien-2-ylidene)methyl}- phenyllmethyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3- yl)pentanamide, 20-hydroxymethyl-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p-o! and (E)-4-[(3-ox0-4,4-dimethyl-5a-cholesta-8, 1 4-dien-2-ylidene)methyl]benzonitrile.
The compounds of the general formula | according to the invention can be synthesized analogously with the preparation of known compounds. Hence, synthesis of the compounds of formula | 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
» ‘ . . 7 and G.M. Murphy): Chapman & Hall. The last one contains an extensive list of citations to the original papers covering the period up to 1990. 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-5a-cholesta-8,14-dien-3p-ol, 4,4-dimethyl-Sa-cholest-8-en-3p-ol, 4,4-dimethyl-Sa-cholest-8(14)-en-3B-ol [Biochem. J. 132 (1973), 439), 4.4- dimethyl-5a.-cholesta-8,24-dien-3B-ol, 4,4-dimethyl-5a-cholesta-8,14,24-trien-3p- ol [J. Am. Chem. Soc. 111 (1989), 278], S5a-cholesta-8,14-dien-3B-ol [J. Am.
Chem. Soc. 75 (1953), 4404], Sa-cholest-8-en-3B-ol [J. Org. Chem. 46 (1981), 3421] and 5a-cholest-8(14)-en-3B-ol [Biochem. J. 144 (1974), 59].
In the following only the syntheses in the 4 4-dimethyl-A-8,14-series are described in detail. The derivatives in the A-8, A-8(14), A-8,14,24 and A-8,24- series with and without the 4,4-dimethyl group can be synthesized from the corresponding starting materials in the same way.
The 3B-alcohols can be oxidized with different reagents to give the corresponding 3-ketones [for example: Tetrahedron Lett. 1967, 3699]. 4 4- dimethyl-5a-cholesta-8,14-dien-3B3-0l 1 is treated with N-methyl-morpholine-N- oxide in the presence of tetrapropyl-ammonium-perruthenate to give 4,4- dimethyl-5a-cholesta-8,14-dien-3-one 2 [for example: Synthesis 1994, 639].
[
Lo. oo v 8 scheme 1:
WH 1H —_— 2 o) = 2
HO v 1 H f nH nH —— —_— HO 4 fo) 3
In the next step the A-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-5a-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 Al-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-5a-cholesta-1,8,14-trien- 3p3-0l 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 ary! 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 (R' = alkyl, aryl) are obtained. These
« .o oo ketones can then be reduced according to well known literature procedures to the two diastereomeric alcohols 6 and 7 (R' = alkyl, aryl), which are readily separable by column chromatography. scheme 2: "nH R! Ul; ——— A
E) 3 - 5 o} V (0) ;
Rr] "WH rR "WH
RE suns = + + 6 w = 7
HO : HO a ’ 5
The introduction of a cyano group can be achieved in a similar way. Conjugate addition of cyanide to enone 3 gives the desired 1a-cyano-4,4-dimethyl-5a- cholesta-8,14-dien-3-one 5 (R' = 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 (R' = CN) can also be reduced with standard reducing agents like sodium borohydride to give the two diastereomeric alcohols 6 and 7 (R' = CN), which are readily separable by column chromatography.
If cyanoalcohol 6 (R' = CN, synthesized as described above) is treated with lithium aluminumhydride 1o-aminomethyl-4,4-dimethyl-5a-cholesta-8,14-dien-3p- ol 8 is obtained. This amine can be used for further modification. Amides of formula 9 (R" = alkyl, aryl and fluorescence marker) can be synthesized from the amine by reaction with hydroxysuccinimidylester of various alkyl or aryl carboxylic
«
Co. , 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:
Rl 1H HN WH ———
HO a 6 (R'=CN) HO 0 8
RUA HH
—_—— 0 _A ’ = 9
HO WY
5 .
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 10 in the presence of cerium chloride gives selectively the corresponding allylic 38- alcohols 11.
‘ Lo oo scheme 4: ) rR2" 1H (7 g 1H — ID © A 2 0 v 10 @ S nH 0S
HO ht 1
Cyanobenzylidene-substituted compound 11 (R®=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 (R*" = alkyl, ary! 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.
< oo oo . 12 scheme 5:
NH r2" 2 (7) C) nH ® 0H
SUSS 9% (I 0S = 11 (R2'=4-CN =
HO A ( ) HO ; 12
No
Oo 2, nH _ 0 09 - 13
HO ht
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: iH iH
R2 = ’s = 2 = 0 a © ni 14
WH nH
RZ R2 —— +
H = YN:
Oo w 15 HO H 16
. A . . .
The hydroxymethyisubstituents 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: dls) WH —_— HO” o = 2 oO A 17 1H nH
HO" HO" —_— +
HO H 18 HO A 19
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 | as active subs- tances. The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disin- tegrating agents and other ingredients which are conventionally used in the art.
Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter.
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 ; 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 | can be used to stimulate the meiosis. According to another preferred embodiment of the present invention, a compound of the general formula | can be used to stimulate the meiosis in humans. Thus, the compounds of the general formula | 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 | for relieving infertility in females and males, particularly in mammals, more particularly in humans. Meiosis-inducing substances of the general formula | 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
J
< M . . . . 15 own production of meiosis-activating substance, are unable to produce mature oocytes.
The compounds of the general formula | 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 | 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 | 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 |.
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
. Lo. A. 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-5a-cholesta-1,8,14-trien-3-one a) 4,4-dimethyl-5a-cholesta-8,14-dien-3-cne:
To a solution of 6.40 g 4,4-dimethyl-5a-cholesta-8,14-dien-3B-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-5a-cholesta-8,14-dien-3- one as a white solid.
TH-NMR (CDCl3): &= 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. 1H, H-15) b) 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one:
To a solution of 2.00 g 4.4-dimethyl-5a-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-5a-cholesta-1,8, 14-trien-3- one as an oil.
TH-NMR (CDCl3): 8= 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, 1H,
H-15), 5.95 (d, J=10 Hz, 1H, H-2); 7.33 (d, J=10 Hz, 1H, H-1)
Example 2: 4 4-dimethyl-5a-cholesta-1 ,8,14-trien-33-0! 14 mg sodium borohydride are added to a suspension of 73 mg 4,4-dimethy!-5a- 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-5a-cholesta-1,8,14-trien-3B-ol as a white solid.
TH-NMR (CDCi3): 3= 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, 1H, H-3); 5.37 (s, 1H, H-15); 5.50 (dd, J=10 Hz, 1 Hz, 1H, H-1); 5.90 (d, J=10 Hz, 2 Hz, 1H, H-2)
Example 3: 5a-cholesta-1,8,14-trien-33-ol a) 5a-cholesta-8,14-trien-3-one: 2.40 g 5a-cholesta-8,14-dien-3p3-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 5a- cholesta-8,14-dien-3-one are obtained as a white solid.
J
TH-NMR (CDCl): 8= 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, 1H, H-15) b) Sa-cholesta-1,8,14-trien-3-one 1.81 g Sa-cholesta-8,14-dien-3-one are treated with 1.67 g phenylselenic anhydride in 29 ml chlorobenzene as described in example 1b. After chromatography 0.53 g 5a-cholesta-1,8,14-trien-3-one are obtained as an oil.
TH-NMR (CDCI3): 8= 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, 1H, H-15); 5.91 (d, J=10
Hz, 1H, H-2); 7.42 (d, J=10 Hz, 1H, H-1) c) 5a-cholesta-1,8,14-trien-3B-ol : 150 mg Sa-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 Sa-cholesta-1,8,14-trien-3p-ol are obtained as a white solid.
TH-NMR (CDClg): 8= 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, 1H, H-3); 5.37 (s, 1H, H- 15); 5.56 (dd, J=10 Hz, 1 Hz, 1H, H-1); 6.09 (d, J=10 Hz, 2 Hz, 1H, H-2)
Example 4: 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3-one:
N
1.47 mi of a diethylaluminum cyanide solution (1 M in toluene) are added to a solution of 4,4-dimethyl-5a-cholesta-1,8,14-trien-3-one in 5 mi 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 1a- cyano-4,4-dimethyl-5a-choiesta-8,14-dien-3-one as a white solid.
TH-NMR (CDCl3): 8= 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, 1H, H-1); 5.49 (ps, 1 H, H-15)
Example 5: 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-33-ol and 1a- cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol; 95 mg 1a-cyano-4,4-dimethyl-Sa-cholesta-8,14-dien-3-one are treated with 33 mg sodium borohydride as described in example 5 to give 30 mg 1a-cyano-4,4- dimethyl-5a-cholesta-8,14-dien-3a-ofl and 25 mg to-cyano-4,4-dimethyl-5a- cholesta-8,14-dien-3p-ol: 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol:
TH-NMR (CDCl3). &= 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, 1H, H-1); 3.72 (bm, 1H, H- 3); 5.43 (ps, 1 H, H-15) 1a-cyano-4,4-dimethyl-5a-cholesta-8,14-dien-3a-ol :
LJ
TH-NMR (CDCl3): 8= 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 (mM, 1H, H-1); 3.57 (ps, 1H, H-3); 5.44 (ps, 1 H, H-15)
Example 6: (E)-4-[(3-ox0-4,4-dimethyl-5a-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-ox0-4,4-dimethyl-
Sa-cholesta-8,14-dien-2-yliden)methyf]lbenzonitrile as a white solid.
TH-NMR (CDCI3): 8= 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, 1H, 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-[(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)- methyl]benzonitrile 156 mg (E)-4-[(3-0x0-4,4-dimethyl-5a-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- [(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)-methyl]lbenzonitrile as a white solid.
TH-NMR (CDCl3): 8= 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, 1H,
»
H-1); 3.94 (m, 1H, H-3); 540 (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-[(3pB-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2- yliden)methyllphenyl)methyljoctanamide: a) (E)-2-(4-aminomethyi-phenyl)}-methylidene-4,4-dimethyl-5a-cholesta-8,14- dien-3p3-ol:
A suspension of 27 mg lithium aluminumhydride and 74 mg (E)-4-[(3B-hydroxy- 4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)-methyllbenzonitrile 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-phenyt)}-methylidene-4,4-dimethyl-5a-cholesta-8,14- dien-3p-ol as a white solid.
TH-NMR (CDCl3): 8= 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, 1H, H-1); 3.86 (s, 2H, Ar-CH-N); 3.92 (ps, 1H, H-3); 5.38 (ps, 1 H, H-15); 6.72 (s. 1H, H-2'); 7.26 (m, 4H, arom.)
Db) (E)-N-[[4-{(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2- yliden)methyllphenyllmethyl]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-5a-cholesta-8,14-dien-3B-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-[(3B- hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)methyl)phenyijmethyl}- octanamide as a white solid.
[J . Lo oo 23
TH-NMR (CDCl3). &= 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, 1H, H-1); 3.92 (ps, 1H, H-3); 4.44 (d, J=5 Hz, 2H, Ar-CH-N); 5.37 (ps, 1 H, H- 15). 56.76 (t. J=5 Hz, 1H, NH); 6.72 (s, 1 H, H-2'); 7.24 (m, 4H, arom.)
Example 9: (E)-N-[[4-[(3p-hydroxy-4,4-dimethyi-5a-cholesta-8,14-dien-2- yliden)methyl]-phenyllmethyl]-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)-methylidene-4,4-dimethyl-5a- cholesta-8,14-dien-3p-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-[(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-yliden)- methyl]-phenyilmethyl}-5-(4,4-diflucro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacen-3-yl)pentanamide.
TH-NMR (CDCig): 6= 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, 1H, H-1); 3.92 (ps, 1H, H-3); 4.44 (d,
J=5 Hz, 2H, Ar-CH2-N); 5.37 (ps, 1H, H-15); 5.87 (m, 1H, NH); 6.09 (s, 1H,
» arom.); 6.30 (d, J=5 Hz, 1H, arom.); 6.70 (s, 1 H, H-2'), 6.90 (d, J=5 Hz, 1H, arom.); 7.24 (m, 4H, arom.)
Example 10: 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-ol a) 2a-allyl-4,4-dimethyl-5a-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 (6.8 mi, 1M) 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 2a- allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-one as a white solid.
TH-NMR (CDCl3): 8= 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, 1H, allyl); 2.77 (m, 1H, allyl); 5.05 (m, 2H, allyl); 5.38 (ps, 1 H, H-15); 5.80 (m, 1H, ally!) b) 2a-allyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3p-ol: 160 mg 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3-one are treated with 55 mg sodium borohydride as described in example 5 to give 15 mg 2a-allyl-4,4-
* dimethyl-Sa-cholesta-8,14-dien-3a-0f and 80 mg 2a-aliyl-4 4-dimethyl-5a- cholesta-8,14-dien-3p-ol. 20-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3B-ol:
TH-NMR (CDCI3): &= 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, 1H); 2.95 (d, J=11 Hz, 1H, H-3); 5.06 (m, 2H, allyl); 5.37 (ps, 1 H, H-15); 5.89 (m, 1H, allyl) 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-3a-0l:
TH-NMR (CDCl3). 8= 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, 1H, H-3); 5.05 (m, 2H, allyl); 5.35 (ps, 1 H, H-15); 5.85 (m, 1H, allyl) ] 15
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 1U
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 o-minimum essential medium (o-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 IU/ml penicillin and 100 ug/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 % CO, 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-5a-cholesta- 8,14,24-trien-3R-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 pM FF-MAS in co-culture with the test compounds in different concentrations in 4-well multidishes (Nunclon, Denmark) in which each well contained 0.4 ml of Hx-medium and 35 — 45 oocytes. One
. < positive control (i.e., 35 — 45 oocytes cultured in Hx-medium containing FF-MAS with no addition of test compound) was always run simultaneously with the test cultures, which were supplemented with different concentrations of the compounds to be tested. In addition, one negative control (35 — 45 oocytes cultured in Hx-medium alone) was run simultaneously with the positive control.
Examination of oocytes
By the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. The percentage of oocytes with GVB 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 (CEQ) 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 c~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 % CO,) at 37°C in a modified a—~MEM IVF-medium. Examination of the oocytes was carried out 20-22 hours after insemination, in order to check fertilisation and to record the
F 3 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
Compounds Oocytes [n] Activation [%]
GV GVB | PB GVB + PB 110 uM 4 4-dimethyl-5a-cholesta- 18 5 71.9 1,8,14-trien-3p-ol
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
Compounds Occytes [n] Inhibition[%)]
GV GVB PB
5 uM FF-MAS + 40 uM 1a-cyano- 38 107 4,4-dimethyl-5a-cholesta-8,14- dien-3-one
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
Compounds Oocytes [n] Inhibition[%]
GV GvB PB ! 5 uM FF-MAS + 10 uM (E)-N-[(4- 25 13 0 63 [(3B-hydroxy-4,4-dimethyl-5c- : cholesta-8, 14-dien-2-yliden)- i methyl]phenyl)methyljoctanamide :
Hx = Hypoxanthine
GV = germinal vesicle
GVB = germinal vesicle breakdown
PB = polar bodies n = number of oocytes
Claims (21)
1. Compounds of the general formula R24 “, R25 R1 : | 1H R% Z R14 RR R30 Rs Rs H R4 R4 I wherein R1 designates a hydrogen atom, a C»-Cg-alkyl group, an optionally substituted phenyl group, a cyano group, a CHo-NH-COR" group (with RY being a C4-Cg-alkyl or an optionally substituted phenyl group) or together with R2 an additional bond, 16 R2 designates a hydrogen atom, a C4-Cg-alkyl group, a C3-Cg- alkenyl group, a C4-Cg-hydroxyalkyl group, together with RZ an : optionally substituted benzylidene group, together with R2' a hydroxymethylene group or together with R1 an additional bond, RZ designates a hydrogen atom or together with R2 an optionally substituted benzylidene group or together with RZ a hydroxy- methylene group, R3 designates a hydrogen atom or together with R3' 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 R9 or with R14 an additional bond, RS designates a hydrogen atom or together with R8 an additional bond R14 designates an a-hydrogen atom or together with R8 an additional bond or together with R15 an additional bond R15 designates a hydrogen atom or together with R14 an additional bond R24 designates a hydrogen atom or together with R25 an additional bond R25 designates a hydrogen atom or together with R24 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 RZ designates a C4-Cg-alkyl group, an allyl group or an additional bond with R1.
4, Compounds according to any one of claims 1 or 2, wherein R2 and R2' designate an optionally substituted benzylidene group.
5. Compounds according to any one of claims 1 to 4 which are 2a-allyl-4,4-dimethyl-5a-cholesta-8,14-dien-33-0l, (E)-2-benzylidene-4,4-dimethyl-5a-cholesta-8,14-dien-3p-ol, Sa-cholesta-1,8,14-trien-33-ol, Sa-cholesta-1,8,14-trien-3-one, 1a-cyano-4,4-dimethyl-5a-cholesta-8, 14-dien-3p-ol, 1a-cyano-4,4-dimethyl-Sa-cholesta-8, 14-dien-3-one, 4 ,4-dimethyl-5a-cholesta-1,8,14-trien-3p-ol, 4 4-dimethyl-5o-cholesta-1,8,14-trien-3-one, (E)-4-[(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)-methyl}- benzonitrile, (E)-N-[[4-[(3B-hydroxy-4,4-dimethyl-5a-cholesta-8,14-dien-2-ylidene)- methyilphenyljmethylJoctanamide, (E)-N-[[4-[(3B-hydroxy-4 4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene)- methyl}-phenyljmethyl]-5-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacen-3-yl)pentanamide, 2a-hydroxymethyl-4,4-dimethyl-5a-cholesta-8, 14-dien-3a-~ol, 2a-octyl-4,4-dimethyl-5a-cholesta-8,14-dien-3f3-ol or (E)-4-[(3-0x0-4,4-dimethyl-5a-cholesta-8, 14-dien-2-ylidene)methyl]- benzonitrile.
6. Pharmaceutical compositions comprising one or more compounds of the general formula | according to any one of claims 1 to 5 as active substances.
7. Use of the compounds of the general formula | according to any one of claims 1 to 5 for the preparation of a meiosis regulating medicament.
i} 34 PCT/EPO0O/01074
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 compounds of the general formula | 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 | 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 | 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 | according to any one of claims 1 to 5.
14. A method according to claim 13 wherein the germ cell is an oocyte or a male germ cell.
15. A substance or composition for use in a method of regulating meiosis, said substance or composition comprising one or more compounds of the general formula | according to any one of claims 1 to 5, and said method comprising administering to a subject in need of such a regulation an effective amount of said substance or composition. AMENDED SHEET
} 3b PCT/EPOO/01074
16. A compound according to claim 1, substantially as herein described and illustrated.
17. A composition according to claim 6, substantially as herein described and illustrated.
18. Use according to any one of claims 7-11, substantially as herein described and illustrated.
18. A method according to any one of claims 12-14, substantially as herein described and illustrated.
20. A substance or composition for use in a method of treatment according to claim 15, substantially as herein described and illustrated.
21. A new compound, new composition, new use of a compound as claimed in any one of claims 1 to 5, new non-therapeutic method of treatment, new ex vivo or in vitro method of regulating meiosis, or a substance or composition for a new use in a method of treatment, substantially as herein described. AMENDED SHEET
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP99250040 | 1999-02-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ZA200107387B true ZA200107387B (en) | 2002-12-06 |
Family
ID=8241140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ZA200107387A ZA200107387B (en) | 1999-02-10 | 2001-09-06 | 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) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5716777A (en) * | 1994-06-23 | 1998-02-10 | Novo Nordisk A/S | Regulation of meiosis using sterols |
| PL322106A1 (en) * | 1995-03-06 | 1998-01-05 | Novo Nordisk As | Meiosis stimulation |
| HUP9900454A3 (en) * | 1995-06-23 | 1999-11-29 | Novo Nordisk As | Meiosis regulating compounds and their use |
| IL130249A0 (en) * | 1996-12-20 | 2000-06-01 | Novo Nordisk As | Meiosis regulating compounds |
-
2000
- 2000-02-09 JP JP2000598521A patent/JP2002536456A/en active Pending
- 2000-02-09 CN CN00803662A patent/CN1368977A/en active Pending
- 2000-02-09 CZ CZ20012866A patent/CZ20012866A3/en unknown
- 2000-02-09 PL PL00350557A patent/PL350557A1/en not_active Application Discontinuation
- 2000-02-09 IL IL14373500A patent/IL143735A0/en unknown
- 2000-02-09 SK SK1138-2001A patent/SK11382001A3/en unknown
- 2000-02-09 WO PCT/EP2000/001074 patent/WO2000047604A1/en not_active Application Discontinuation
- 2000-02-09 CA CA002359687A patent/CA2359687A1/en not_active Abandoned
- 2000-02-09 HU HU0200280A patent/HUP0200280A2/en unknown
- 2000-02-09 KR KR1020017009985A patent/KR20010101820A/en not_active Application Discontinuation
- 2000-02-09 AU AU32797/00A patent/AU3279700A/en not_active Abandoned
- 2000-02-09 EP EP00910664A patent/EP1150993A1/en not_active Withdrawn
- 2000-02-09 BR BR0008065-9A patent/BR0008065A/en not_active Application Discontinuation
-
2001
- 2001-08-10 NO NO20013901A patent/NO20013901D0/en not_active Application Discontinuation
- 2001-09-06 ZA ZA200107387A patent/ZA200107387B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CZ20012866A3 (en) | 2002-01-16 |
| NO20013901L (en) | 2001-08-10 |
| KR20010101820A (en) | 2001-11-14 |
| CA2359687A1 (en) | 2000-08-17 |
| CN1368977A (en) | 2002-09-11 |
| IL143735A0 (en) | 2002-04-21 |
| AU3279700A (en) | 2000-08-29 |
| JP2002536456A (en) | 2002-10-29 |
| NO20013901D0 (en) | 2001-08-10 |
| BR0008065A (en) | 2001-11-06 |
| PL350557A1 (en) | 2002-12-16 |
| HUP0200280A2 (en) | 2002-07-29 |
| EP1150993A1 (en) | 2001-11-07 |
| SK11382001A3 (en) | 2002-01-07 |
| WO2000047604A1 (en) | 2000-08-17 |
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