U.S. patent application number 11/014153 was filed with the patent office on 2005-05-12 for meiosis regulating compounds.
This patent application is currently assigned to Novo Nordisk A/S. Invention is credited to Blume, Thorsten, Breinholt, Jens, Faarup, Peter, Gronvald, Frederick Christian, Murray, Anthony.
Application Number | 20050101573 11/014153 |
Document ID | / |
Family ID | 27512801 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050101573 |
Kind Code |
A1 |
Faarup, Peter ; et
al. |
May 12, 2005 |
Meiosis regulating compounds
Abstract
Certain novel sterol derivatives can be used for regulating the
meiosis in oocytes and in male germ cells.
Inventors: |
Faarup, Peter; (Vaerlose,
DK) ; Gronvald, Frederick Christian; (Vedbaek,
DK) ; Blume, Thorsten; (Berlin, DE) ; Murray,
Anthony; (Hellerup, DK) ; Breinholt, Jens;
(Soborg, DK) |
Correspondence
Address: |
NOVO NORDISK, INC.
PATENT DEPARTMENT
100 COLLEGE ROAD WEST
PRINCETON
NJ
08540
US
|
Assignee: |
Novo Nordisk A/S
Bagsvaerd
DK
Schering AG
Berlin
DE
|
Family ID: |
27512801 |
Appl. No.: |
11/014153 |
Filed: |
December 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11014153 |
Dec 16, 2004 |
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10162843 |
Jun 4, 2002 |
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10162843 |
Jun 4, 2002 |
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09760237 |
Jan 12, 2001 |
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6407086 |
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09760237 |
Jan 12, 2001 |
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09332235 |
Jun 14, 1999 |
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60086306 |
May 21, 1998 |
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60092983 |
Jul 16, 1998 |
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Current U.S.
Class: |
514/129 ;
514/169; 552/506; 552/518 |
Current CPC
Class: |
C07J 53/004 20130101;
C07J 9/00 20130101; C07J 41/0094 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/129 ;
514/169; 552/506; 552/518 |
International
Class: |
A61K 031/66; A61K
031/56; C07J 001/00; C07J 041/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 1999 |
WO |
PCT/DK99/00263 |
Jun 19, 1998 |
DK |
PA 1998 00811 |
May 14, 1998 |
DK |
PA 1998 00657 |
May 14, 1998 |
EP |
EPO 98250166.0 |
May 14, 1998 |
EP |
98250166.0 |
Claims
1. Novel compounds of the general formula I: 8wherein R.sup.1 is
hydrogen, halogen, methyl, hydroxy or oxo; R.sup.2 is selected from
the group comprising hydrogen, hydroxy, C.sub.1-C.sub.3 alkyl,
vinyl, C.sub.1-C.sub.3 alkoxy and halogen, or R.sup.2 designates,
together with R.sup.3, an additional bond between the carbon atoms
at which R.sup.2 and R.sup.3 are placed; R.sup.3 is selected from
the group comprising hydrogen, optionally substituted alkoxy,
acyloxy, sulphonyloxy, phosphonyloxy, halogen, lower alkyl or a
perfluoro(lower alkyl) group; or R.sup.3 designates, together with
R.sup.2 an additional bond between the two carbon atoms at which
R.sup.2 and R.sup.3 are placed; or R.sup.3 designates, together
with R'.sup.3, oxo or a group of the general formula
.dbd.NOR.sup.38 wherein R.sup.38 is hydrogen or lower alkyl;
R'.sup.3 designates hydrogen or hydroxy; with the proviso that
R.sup.3 and R'.sup.3 are not, simultaneously, hydrogen; R.sup.4 and
R'.sup.4, which are different or identical with the proviso that
they are not both hydroxy, are selected from the group comprising
hydrogen, halogen, hydroxy and C.sub.1-C.sub.6 alkyl which may be
substituted by halogen, hydroxy or cyano, or wherein R.sup.4 and
R'.sup.4 together designate methylene or oxo or, together with the
carbon atom to which they are bound, form a cyclopropane ring, a
cyclopentane ring, or a cyclohexane ring; or R.sup.4 designates,
together with R.sup.'4 and R.sup.5, a methano bridge between the
carbon atoms in 4 and 5 position or an additional bond between the
carbon atoms in 4 and 5 position; R.sup.5 is hydrogen, halogen,
hydroxy, lower alkyl, cyano, hydroxymethyl, a carbaldehyde, an
oxime derived from a carbaldehyde, a carboxylic acid, a primary or
secondary amide derived from a carboxylic acid, an ester with a
C.sub.1-C.sub.6-alcohol group; or R.sup.5 designates, together with
R.sup.6, an additional bond between the carbon atoms at which
R.sup.5 and R.sup.6 are placed; R.sup.6 is hydrogen, hydroxy,
halogen or oxo, or R.sup.6 designates, together with R.sup.5 or
R.sup.7, an additional bond between the carbon atoms at which
R.sup.6 and R.sup.5 or R.sup.7 are placed; R.sup.7 is selected from
the group comprising hydrogen, hydroxy, lower alkoxy, acyloxy,
halogen and lower alkyl; or R.sup.7 designates, together with
R.sup.6 or R.sup.8, an additional bond between the carbon atoms at
which R.sup.7 and R.sup.6 or R.sup.8 are placed; R'.sup.7 is
hydrogen, or, if R.sup.7 is lower alkyl, R'.sup.7 is hydrogen or
hydroxy; or R.sup.7 designates, together with R'.sup.7, methylene,
oxo or a group of the general formula .dbd.NOR.sup.36, wherein
R.sup.36 is hydrogen or lower alkyl; R.sup.8 is hydrogen, hydroxy
or halogen, or R.sup.8 designates, together with R.sup.7, R.sup.9
or R.sup.14, an additional bond between the carbon atoms at which
R.sup.8 and R.sup.7, R.sup.9 or R.sup.14 are placed; R.sup.9 is
hydrogen, hydroxy or halogen, or R.sup.9 designates, together with
R.sup.8 or R.sup.11, an additional bond between the carbon atoms at
which R.sup.9 and R.sup.8 or R.sup.11 are placed; R.sup.11 is
selected from the group comprising hydrogen, hydroxy, lower alkoxy,
acyloxy, halogen and lower alkyl, or R.sup.11 designates, together
with R.sup.9 or R.sup.12, an additional bond between the carbon
atoms at which R.sup.11 and R.sup.9 or R.sup.12 are placed;
R'.sup.11 is hydrogen or, if R.sup.11 is lower alkyl, R'.sup.11 is
hydrogen or hydroxy, or R.sup.11 designates, together with
R'.sup.11, methylene, oxo or a group of the general formula
.dbd.NOR.sup.37, wherein R.sup.37 is hydrogen or lower alkyl;
R.sup.12 is selected from the group comprising hydrogen, halogen,
C.sub.1-C.sub.4 alkyl, methylene, hydroxy, lower alkoxy, acyloxy,
oxo and a group of the general formula .dbd.NOR.sup.33 wherein
R.sup.33 is hydrogen or C.sub.1-C.sub.3 alkyl, or R.sup.12
designates, together with R.sup.11, an additional bond between the
carbon atoms at which R.sup.11 and R.sup.12 are placed; R.sup.14 is
hydrogen or hydroxy, or R.sup.14 designates, together with
R.sup.15, an additional bond between the carbon atoms at which
R.sup.14 and R.sup.15 are placed; R.sup.15 is selected from the
group comprising hydrogen, halogen, lower alkyl, methylene,
hydroxy, lower alkoxy, oxo and a group of the general formula
.dbd.NOR.sup.32 wherein R.sup.32 is hydrogen or C.sub.1-C.sub.3
alkyl, or R.sup.15 designates, together with R.sup.14 an additional
bond between the carbon atoms at which R.sup.15 and R.sup.14 are
placed; R.sup.16 is selected from the group comprising hydrogen,
halogen, C.sub.1-C.sub.3 alkyl, methylene, hydroxy, lower alkoxy,
oxo and a group of the general formula .dbd.NOR.sup.34 wherein
R.sup.34 is hydrogen or lower alkyl, or R.sup.16 designates,
together with R.sup.17, an additional bond between the carbon atoms
at which R.sup.16 and R.sup.17 are placed; R.sup.17 is hydrogen or
hydroxy, or R.sup.17 designates, together with R.sup.16, an
additional bond between the carbon atoms at which R.sup.17 and
R.sup.16 are placed; R.sup.20 is selected from the group comprising
hydrogen, lower alkyl and hydroxymethyl, or R.sup.20 and R'.sup.20
together designate methylene or oxo; R'.sup.20 is hydrogen,
halogen, lower alkyl or hydroxy; R'.sup.22 is hydrogen, hydroxy or
oxo; R.sup.22 represents phenyl optionally substituted by one or
more of the following groups which substituents may be different or
identical: hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a
group of the general formula --COOR.sup.39, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; benzyl optionally substituted by one or more of
the following groups which substituents may be different or
identical: hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a
group of the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; cyclohexyl optionally substituted by one or more
of the following groups which substituents may be different or
identical: hydroxy, alkoxy, halogen, amino, cyano, carboxy, a group
of the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; cyclohexylalkyl optionally substituted by one or
more of the following groups which substituents may be different or
identical: hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a
group of the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; alkyl optionally substituted by one or more of the
following groups which substituents may be different or identical:
hydroxy, alkoxy, halogen, amino, cyano, carboxy, a group of the
general formula --COOR.sup.39, oxo, N-acylamino, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; or alkenyl optionally substituted by one or more
of the following groups which substituents may be different or
identical: hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a
group of the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; and R.sup.39 represents lower alkyl or aralkyl,
and additionally, when R.sup.1, R.sup.2, R'.sup.4, R.sup.5,
R.sup.11, R.sup.12, R.sup.15, R.sup.16, R'.sup.20 and R'.sup.22 are
each hydrogen, R.sup.3 is hydrogen, lower alkyl or perfluoro(lower
alkyl), R'.sup.3 is hydroxy, or R.sup.3 designates, together with
R'.sup.3, oxo, R.sup.4 is hydrogen or together with R.sup.5 a
methano bridge or together with R.sup.5 an additional bond, R.sup.5
is lower alkyl, cyano, hydroxymethyl, a carbaldehyde, an oxime
derived from a carbaldehyde, a carboxylic acid, a primary or
secondary amide derived from a carboxylic acid, an ester with a
C.sub.1-C.sub.6-alcohol group or together with R.sup.4 a methano
bridge or together with R.sup.4 an additional bond, R.sup.7 is
together with R.sup.8 an additional bond or hydrogen, if R.sup.8
and R.sup.9 or R.sup.8 and R.sup.14 stand together for an
additional bond, R.sup.8 is together with R.sup.7 or with R.sup.9
or with R.sup.14 an additional bond, R.sup.9 is together with
R.sup.8 an additional bond or a hydrogen atom, if R.sup.7 and
R.sup.8 or R.sup.9 and R.sup.14 stand together for an additional
bond, R.sup.14 is together with R.sup.8 an additional bond or a
hydrogen atom, if R.sup.7 and R.sup.8 or R.sup.8 and R.sup.9 stand
together for an additional bond, R.sup.17 is hydrogen in the alpha
position, R.sup.19 is methyl in the beta position, and R.sup.20 is
methyl in the alpha position, then R.sup.22 is 3-methylbutyl; with
the proviso that the following compounds are disclaimed:
cholesta-4,7-dien-3-one; cholesta-4,8-dien-3-one;
cholesta-4,8(14)-dien-3-one; cholesta-4,7-dien-3.alpha.-ol;
cholesta-4,7-dien-3.beta.-ol; 5-cyano-5,-cholest-7-en-3-one;
5-cyano-5.beta.-cholest-7-en-3.beta.-ol;
5-methyl-5,-cholest-7-en-3-one;
5-methyl-50-cholest-7-en-3.beta.-ol;
5-methyl-50-cholest-7-en-3.beta.-ol;
3.beta.,7.alpha.-dihydroxycholest-5-- ene;
3.beta.,7.beta.-dihydroxycholest-5-ene;
3.beta.-hydroxycholest-5-en-7- -one; 3.beta.-hydroxycholest-7-one;
7.alpha.-hydroxycholest-4-en-3-one; cholest-3,6-dione;
3.beta.-hydroxycholest-6-one; 3.beta.,6.beta.-dihydrox-
ycholestane; cholest-4-en-3,6-dione;
3.beta.,5.alpha.,6.beta.-trihydroxych- olestane;
3.beta.,5.alpha.-dihydroxycholestane-; 3.beta.,4.beta.-dihydroxy-
cholest-5-ene; cholest-2-en-6one; cholest-4,6-dien-3-one;
cholest-4,7-dien-3-one; cholest-3,5-dien-7-one;
19-nor-21-methylpregna-4,- 9-dien-17.alpha.-hydroxy-3,20-dione;
19-nor-21-methylpregna-4,9-dien-17.al- pha.-acetoxy-3,20-dione;
19-nor-21,21-dimethylpregna-4,9-diene-17.alpha.-h-
ydroxy-3,20-dione;
177.alpha.-21,21-dimethyl-19-nor-pregna-4,9-dien-3,20-d- ione;
3.alpha.-acetoxy-24-nor-cholan-23-one;
3.alpha.-hydroxy-26,27-di-nor-
-23-trans-5.beta.-cholest-23-en-25-carboxylic acid methyl ester;
3.alpha.-hydroxy-26,27-di-nor-5.beta.-cholesta-25-carboxylic acid
methyl ester; 3-keto-26,27-di-nor-5.beta.-cholesta-25-carboxylic
acid methyl ester;
3-keto-4-bromo-26,27-di-nor-5.beta.-cholesta-25-carboxylic acid
methyl ester; 3-keto-26,27-di-nor-cholest-4-en-25-carboxylic acid
methyl ester;
3.beta.-acetoxy-26,27-di-nor-cholesta-3,5-dien-25-carboxylic acid
methyl ester;
3.beta.-hydroxy-26,27-di-nor-cholest-5-en-25-carboxylic acid methyl
ester; 3-keto-26,27-di-nor-cholest-4,6-dien-25-carboxylic acid
methyl ester;
3.beta.-hydroxy-26,27-di-nor-cholesta-3,5,7-trien-25-c- arboxylic
acid methyl ester; and 3.beta.-hydroxy-26,27-di-nor-cholesta-5,7-
-dien-25-carboxylic acid methyl ester;
3.beta.,22-diacetoxy-cholesta-5-en-- 25-ol;
3.beta.,22-diacetoxy-25-fluorocholesta-5-ene;
22-hydroxycholesta-5-en-25-fluoro-3.beta.-hemisuccinate;
3.beta.,22-diacetoxy-25-dichlorocholesta-5-ene;
3.beta.,22-dihydroxy-25-c- hlorocholesta-5-ene;
22-hydroxy-25-chlorocholesta-5-en-3.beta.-hemisuccina- te;
3.beta.,22-di-hydroxy-25-bromocholesta-5-ene;
3.beta.,22-dihydroxy-25-- fluorocholesta-5-ene; cholenic acid;
3.beta.-acetoxycholesta-5-en-25-des-d- imethyl-24-one;
3,24-diacetoxycholesta-25-des-dimethyl-5,23-diene;
3.beta.-acetoxycholesta-25-des-methyl-5-en-24-difluoro-25-one;
3.beta.-acetoxy-24-di-fluorocholesta-5,7-dien-25-ol;
3.beta.-hydroxycholest-5-en-24-one;
3.beta.-acetoxycholest-5-en-24-one; 5.beta.-cholest-24-one;
5.beta.-cholestan-24.alpha.-homo-24-one;
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholest-24-one;
3.alpha.,6.alpha.-diacetoxy-5,-cholest-24-one;
3.alpha.,6.alpha.-diacetox-
y-5.beta.-cholest-24.alpha.-homo-24-one;
3.alpha.,6.alpha.-dihydroxy-5.bet-
a.-cholest-24.alpha.,2413-bis-homo-24-one;
3.alpha.-hydroxy-53-cholest-24-- one; 3.alpha.-acetoxy
5.beta.-cholesta-24-one; 3.alpha.-benzoyloxy-5.beta.-
-cholesta-24-one;
3.alpha.-ethyloxycarbonyloxy-5.beta.-cholesta-24-one;
3.alpha.-hydroxy-5.beta.-cholestan-24.alpha.-homo-24-one;
3.alpha.-hydroxy-24.alpha.,24.beta.-bis-homo-5.beta.-cholestane;
3.beta.-hydroxycholesta-5,7-dien-24-one;
3.beta.-acetoxycholesta-5,7-dien- -24-one;
1.alpha.,3.beta.-dihydroxycholesta-5,7-diene-24-one;
1.alpha.,3.beta.-diacetoxycholesta-5,7-diene-24-one;
chenodeoxycholic acid; ursodeoxycholic acid; trimebutyn salt of
chenodeoxycholic acid; trimebutyn salt of ursodeoxycholic acid;
3.beta.,25-dihydroxycholest-5-en- -24-one;
3.beta.-acetoxy-cholest-5-en-24-one; 3.beta.-acetoxy-25-hydroxych-
olest-5-en-24-one; 3.beta.,25-dihydroxycholest-5-en-24-one;
3.beta.-hydroxycholest-5-en-24-one;
3.beta.-hydroxy-25-hydroperoxycholest- -5-en-24-one;
3.beta.,24,25-trihydroxycholest-5-ene;
1.alpha.,3.beta.-dihydroxycholest-5-en-24-one;
1.alpha.,3.beta.,25-trihyd- roxy-cholest-5-en-24-one;
1.alpha.,3.beta.,24,25-tetrahydroxycholest-5-en-- 24-one;
3.alpha.-acetoxy-7.alpha.-bromo-cholest-5-ene;
3.alpha.-acetoxycholesta-5,7-diene;
3.alpha.-acetoxy-25-hydroxycholesta-5- ,7-diene;
313,25-dihydroxy-26,27-hexafluorocholest-5-ene;
1.alpha.,3.beta.,25-trihydroxy-26,27-hexafluoro-cholest-5-ene;
3.alpha.,7.alpha.,12.alpha.,24R,26,27-hexahydroxycholestane;
3.alpha.-hydroxy-7-cholanic acid;
3.alpha.,7.alpha.-dihydroxycholanic acid;
3.alpha.,7.beta.-dihydroxycholanic acid; and lithium
3.alpha.,7.beta.-dihydroxycholanate; cholesta-1,4,6-trien-3-one;
cholest-5-en-1.alpha.,3.beta.-diol;
1.alpha.-hydroxycholesta-4,6-diene-3-- one;
1.alpha.,3.beta.-dihydroxycholest-5-ene;
25-hydroxycholesta-1,4,6-tri- ene-3-one;
1.alpha.,3.beta.-25-trihydroxycholest-5-ene;
3.alpha.,71-dihydroxydeoxycholic acid;
3.alpha.,7.alpha.-dihydroxydeoxych- olic acid; 7-ketodeoxycholic
acid; 3.alpha.,7.beta.-dihydroxydeoxycholic acid; cholic acid;
7-ketocholic acid; 3.alpha.,7.beta.,12.alpha.-cholic acid;
12-ketocholic acid; cholic acid; 3.alpha.-hydroxy-7-ketocholic
acid; 3.alpha.,7.alpha.-diacetoxycholic acid;
3.alpha.,7.alpha.-diacetoxy- -12-ketocholic acid;
3.alpha.,7.alpha.-dihydroxydeoxycholic acid; 7-ketodeoxycholic
acid; 3.alpha.,7.beta.-dihydroxydeoxycholic acid;
3.alpha.,7.beta.-dihydroxy-12-ketocholic acid; cholic acid; cholic
acid methyl ester; 3-acetylcholic acid methyl ester;
3-(2-propenyl)-cholic acid methyl ester; 3-(3-hydroxypropyl)cholic
acid-3-methyl ester; desoxycholic acid; 12-ketodesoxycholic acid;
3.beta.-acetyloxy-12-keto-de- soxycholic acid;
3.beta.-(hydroxyethyloxy)-cholic acid methyl ester;
3.beta.-(hydroxypropyloxy)cholic acid methyl ester;
3.beta.-(hydroxybutyl-oxy)cholic acid methyl ester;
3.beta.-(hydroxypentyloxy)cholic acid methyl ester;
3.beta.-(hydroxy-hexyloxy)cholic acid methyl ester;
3.beta.-(hydroxydecanoyloxy)cholic acid methyl ester;
3.beta.-(2-hydroxyethyloxyethyloxy)cholic acid methyl ester;
3.beta.-(2-hydroxypropyloxy)cholic acid methyl ester;
3.beta.-(hydroxyethyloxy)desoxycholic acid methyl ester;
3.beta.-(hydroxypropyloxy)desoxycholic acid methyl ester;
3.beta.-(hydroxypentyloxy)desoxycholic acid methyl ester;
30-(hydroxy-decyloxy)desoxycholic acid methyl ester;
3.beta.-(2-hydroxyethyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(3-hydroxypropyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(5-hydroxy-pentyloxy)chenodesoxycholic acid methyl ester;
30-(10-hydroxydecyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(2-hydroxyethyloxy)litocholic acid methyl ester;
3.beta.-(3-hydroxypropyl-oxy)litocholic acid methyl ester;
3.beta.-(5-hydroxypenthyloxy)lithocholic acid methyl ester;
3.beta.-(10-hydroxydecayloxy)lithocholic acid methyl ester;
3.beta.-(benzyloxyethyloxy)cholic acid methyl ester;
3.beta.-(benzyloxyethyloxy)cholic acid tert.butyl ester;
3.beta.-(2-hydroxyethyloxy)cholic acid tert.butyl ester;
3.beta.-(2-hydroxyethyloxy)-7.alpha.,12.alpha.-diacetyloxycholic
acid methyl ester;
3.beta.-(propionyloxy)-7.alpha.,12.alpha.-diacetyloxy-24-ca-
rboxylic acid methyl ester; chenodeoxycholic acid; sitosterol;
3.alpha.-hydroxycholestane; 3.beta.-hydroxycholestane;
25-fluorocholest-5-en-3.beta.,22-diol;
25-chlorocholest-5-en-3.beta.,22-d- iol;
22-hydroxy-25-fluorocholest-5-en-3.beta.-hemisuccinate;
22-hydroxy-25-chlorocholest-5-en-3.beta.-hemisuccinate;
cholesta-5-en-3.beta.,22,25-triol;
(3.beta.,5.alpha.,20R)-4,4,20-trimethy-
l-21-phenylpregna-8,14-dien-3-ol;
(3.beta.,5.alpha.,20R)-4,4,20-trimethyl--
21-(3-methyl-phenyl)pregna-8,14-dien-3-ol; and
(3.beta.,5.alpha.,20R)-4,4--
dimethyl-23-phenyl-24-norchola-8,14-dien-3-ol; and esters, salts,
active metabolites and prodrugs thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/162,843, filed Jun. 4, 2002, which is a continuation of Ser. No.
09/760,237, now U.S. Pat. No. 6,407,086, which is a continuation of
U.S. Ser. No. 09/332,235, filed on Jun. 14, 1999, now abandoned,
which is a continuation of PCT/DK99/00263 filed on May 11, 1999 and
claims priority under 35 U.S.C. 119 of Danish application no. PA
1998 00657 filed on May 13, 1998, Danish application no. PA 1998
00811 filed on Jun. 19, 1998, European application no. 98250166.0
filed on May 14, 1998, U.S. provisional application No. 60/086,306
filed on May 21, 1998, and U.S. provisional application No.
60/092,983 filed on Jul. 16, 1998, the contents of which are fully
incorporated herein by reference.
FIELD OF THIS INVENTION
[0002] The present invention relates to certain pharmacologically
active compounds, to pharmaceutical compositions containing certain
compounds as active substance and to their use as medicaments. More
particularly, it has been found that the derivatives described
herein can be used for regulating the meiosis.
BACKGROUND OF THIS INVENTION
[0003] 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 1c DNA.
[0004] 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 fertilisation, 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.
[0005] Only little is known about the mechanisms which control the
initiation of meiosis in the male and in the female. In the oocyte,
new studies indicate that follicular purines, hypoxanthine or
adenosine, could be responsible for meiotic arrest (Downs, S. M. et
al. Dev. Biol. 82 (1985) 454-458; Eppig, J. J. et al. Dev. Biol.
119 (1986) 3.beta.-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. in 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. in 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 utilisation of the interesting
finding would thus be greatly facilitated if more stable meiosis
activating compounds were available.
[0006] In Aust. J. Chem. 35 (1982), 629-640, Horn et al. deals with
compounds possibly having biological activity (insect moulting
hormones). Examples of compounds specifically mentioned therein are
5-cyano-5.beta.-cholest-7-en-3-one;
5-cyano-5.beta.-cholest-7-en-3.beta.-- ol;
5-methyl-5.beta.-cholest-7-en-3-one;
5-methyl-5.beta.-cholest-7-en-3.a- lpha.-ol; and
5-methyl-5.beta.-cholest-7-en-3.beta.-ol.
[0007] In Bull. Soc. Chim. Fr. (1971), 2037-2047, Levisalles et
al., cholesta-4,8(14)-dien-3-one is described as an
intermediate.
[0008] In Just. Lieb. Ann. Chem. 542 (1939), 218-224, Windaus et
al. mentions cholesta-4,7-dien-3-one;
cholesta-4,7-dien-3.alpha.-ol; and cholesta-4,7-dien-3.beta.-ol as
intermediates.
[0009] In Pharm. Bull. 1 (1953), 224-227, Arima mentions
cholesta-4,8-dien-3-one as intermediate.
[0010] In Lipids 13 (1978), 704 et seq., Kandutsch et al. describes
some cholestane derivatives which may be potent inhibitors of
sterol synthesis. Compounds specifically mentioned therein are, in
FIG. 1, 3.beta.,7.alpha.-dihydroxycholest-5-ene;
3.beta.,7.beta.-dihydroxycholest- -5-ene;
3.beta.-hydroxycholest-5-en-7-one; 3.beta.-hydroxycholest-7-one;
7.alpha.-hydroxycholest-4-en-3-one; in FIG. 2 (compounds 1-5),
cholest-3,6-dione; 3.beta.-hydroxycholest-6-one;
3.beta.,6.beta.-dihydrox- ycholestane; cholest-4-en-3,6-dione;
3.beta.,5.alpha.,6.beta.-trihydroxych- olestane; in FIG. 3,
3.beta.,5.alpha.-dihydroxycholestane;
3.beta.,4.beta.-dihydroxycholest-5-ene; and, in FIG. 4 (compounds
1, 3, 4 and 5), cholest-2-en-6-one; cholest-4,6-dien-3-one;
cholest-4,7-dien-3-one; cholest-3,5-dien-7-one.
[0011] Danish patent application with publication number 130,992,
deals with compounds possibly having progestomimetic properties.
Examples of compounds specifically mentioned therein are
19-nor-21-methylpregna-4,9-d- ien-17.alpha.-hydroxy-3,20-dione;
19-nor-21-methyl-pregna-4,9-dien-17.alph- a.-acetoxy-3,20-dione;
19-nor-21,21-dimethylpregna-4,9-diene-17.alpha.-hyd-
roxy-3,20-dione; and
17.alpha.-21,21-dimethyl-19-nor-pregna-4,9-dien-3,20-- dione.
[0012] In the Danish patent application with publication number
136,909, 3.alpha.-acetoxy-24-nor-cholan-23-one;
3.alpha.-hydroxy-26,27-di-nor-23-t-
rans-5.beta.-cholest-23-en-25-carboxylic acid methyl ester;
3.alpha.-hydroxy-26,27-di-nor-5.beta.-cholesta-25-carboxylic acid
methyl ester; 3-keto-26,27-di-nor-5.beta.-cholesta-25-carboxylic
acid methyl ester;
3-keto-4-bromo-26,27-di-nor-5.beta.-cholesta-25-carboxylic acid
methyl ester; 3-keto-26,27-di-nor-cholest-4-en-25-carboxylic acid
methyl ester;
3.beta.-acetoxy-26,27-di-nor-cholesta-3,5-dien-25-carboxylic acid
methyl ester;
3.beta.-hydroxy-26,27-di-nor-cholest-5-en-25-carboxylic acid methyl
ester; 3-keto-26,27-di-nor-cholest-4,6-dien-25-carboxylic acid
methyl ester;
3.beta.-hydroxy-26,27-di-nor-cholesta-3,5,7-trien-25-c- arboxylic
acid methyl ester; and 3.beta.-hydroxy-26,27-di-nor-cholesta-5,7-
-dien-25-carboxylic acid methyl ester are mentioned as
intermediates.
[0013] Danish patent application with publication number 146,390,
deals with compounds possibly having pharmacological properties,
e.g. an inhibiting action on the production of serum cholesterol.
Examples of compounds specifically mentioned therein are
3.beta.,22-di-acetoxycholest- a-5-en-25-ol;
3.beta.,22-diacetoxy-25-fluorocholesta-5-ene;
22-hydroxycholesta-5-en-25-fluoro-3.beta.-hemisuccinate;
3.beta.,22-diacetoxy-25-dichlorocholesta-5-ene;
3.beta.,22-di-hydroxy-25-- chlorocholesta-5-ene;
22-hydroxy-25-chlorocholesta-5-en-3.beta.-hemisuccin- ate;
3.beta.,22-dihydroxy-25-bromocholesta-5-ene; and
3.beta.,22-dihydroxy-25-fluorocholesta-5-ene.
[0014] In the Danish patent applications with publication numbers
156,726 and 156,644, cholenic acid;
3.beta.-acetoxycholesta-5-en-25-des-dimethyl-- 24-one;
3,24-diacetoxycholesta-25-des-dimethyl-5,23-diene;
3.beta.-acetoxycholesta-25-des-methyl-5-en-24-difluoro-25-one; and
3.beta.-acetoxy-24-difluorocholesta-5,7-dien-25-ol are mentioned as
intermediates.
[0015] In the Danish patent application with publication number
158,790,31-hydroxy-cholest-5-en-24-one;
3.beta.-acetoxycholest-5-en-24-on- e; 5.beta.-cholest-24-one;
5.beta.-cholestan-24.alpha.-homo-24-one;
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholest-24-one;
3.alpha.,6.alpha.-diacetoxy-5.beta.-cholest-24-one;
3.alpha.,6.alpha.-diacetoxy-5.beta.-cholest-24.alpha.-homo-24-one;
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholest-24.alpha.,24.beta.-bis-homo-2-
4-one; 3.alpha.-hydroxy-5.beta.-cholest-24-one; 3.alpha.-acetoxy
5.beta.-cholesta-24-one;
3.alpha.-benzoyl-oxy-5.beta.-cholesta-24-one;
3.alpha.-ethyloxycarbonyloxy-5.beta.-cholesta-24-one;
3.alpha.-hydroxy-5.beta.-cholestan-24.alpha.-homo-24-one;
3.alpha.-hydroxy-24.alpha.,24.beta.-bis-homo-5.beta.-cholestane;
3.beta.-hydroxy-cholesta-5,7-dien-24-one;
3.beta.-acetoxycholesta-5,7-die- n-24-one;
1.alpha.,3.beta.-dihydroxycholesta-5,7-diene-24-one; and
1.alpha.,3.beta.-diacetoxycholesta-5,7-diene-24-one are mentioned
as intermediates.
[0016] Danish patent application with publication number 159,456,
deals with compounds possibly having utility in the treatment of
gall diskinese. Examples of compounds specifically mentioned
therein are chenodeoxycholic acid; ursodeoxycholic acid; trimebutyn
salt of chenodeoxycholic acid; and trimebutyn salt of
ursodeoxycholic acid.
[0017] In the Danish patent application with publication number
162,648, 3.beta.,25-dihydroxy-cholest-5-en-24-one;
3.beta.-acetoxycholest-5-en-24-- one;
3.beta.-acetoxy-25-hydroxycholest-5-en-24-one;
3.beta.,25-dihydroxycholest-5-en-24-one;
3.beta.-hydroxycholest-5-en-24-o- ne;
3.beta.-hydroxy-25-hydroperoxycholest-5-en-24-one;
3.beta.,24,25-trihydroxycholest-5-ene;
1.alpha.,3.beta.-dihydroxy-cholest- -5-en-24-one;
1.alpha.,3.beta.,25-trihydroxycholest-5-en-24-one;
1.alpha.,3.beta.,24,25-tetrahydroxy-cholest-5-en-24-one are
mentioned as intermediates.
[0018] In the Danish patent application with publication number
165,410, 3.alpha.-acetoxy-7.alpha.-bromocholest-5-ene;
3.alpha.-acetoxycholesta-5,- 7-diene; and
3.alpha.-acetoxy-25-hydroxycholesta-5,7-diene are mentioned as
intermediates.
[0019] In the Danish patent application with publication number
165,695, 3.beta.,25-dihydroxy-26,27-hexafluorocholest-5-ene; and
1.alpha.,3.beta.,25-trihydroxy-26,27-hexafluorocholest-5-ene are
mentioned as intermediates for the preparation of vitamin D
analogues.
[0020] Danish patent application with publication number 167,220
deals with compounds possibly having utility for the treatment of
liver disorders. An examples of a compound specifically mentioned
therein is
3.alpha.,7.alpha.,12.alpha.,24R,26,27-hexahydroxycholestane.
[0021] In U.S. Pat. No. 4,425,274, the compounds
3.alpha.-hydroxy-7-cholan- ic acid;
3.alpha.,7.alpha.-dihydroxycholanic acid; 3.alpha.,7.beta.-dihydr-
oxycholanic acid; and lithium 3.alpha.,7.beta.-dihydroxycholanate
are described as intgermediates.
[0022] In the Norwegian patent application with publication number
144,264, cholesta-1,4,-6-trien-3-one;
cholest-5-en-1.alpha.,3.beta.-diol;
1.alpha.-hydroxycholesta-4,6-diene-3-one;
1.alpha.,3.beta.-di-hydroxychol- est-5-ene;
25-hydroxycholesta-1,4,6-triene-3-one; and
1.alpha.,3.beta.-25-trihydroxy-cholest-5-ene are mentioned as
intermediates for the preparation of 1.alpha.-hydroxy steroids of
the cholestane serie.
[0023] Norwegian patent application with publication number
158,423, deals with compounds possibly having utility in the
treatment of biliary dyskinesis. An example of a compound
specifically mentioned therein is
3.alpha.,7.beta.-dihydroxydeoxycholic acid.
[0024] In the Norwegian patent application with publication number
162,562, 3.alpha.,7.alpha.-di-hydroxydeoxycholic acid;
7-ketodeoxycholic acid; 3.alpha.,7.beta.-dihydroxydeoxycholic acid;
cholic acid; 7-ketocholic acid; 3.alpha.,7.beta.,12.alpha.-cholic
acid; and 12-ketocholic acid are mentioned as being intermediates
in the preparation of ursodeoxycholanic acid.
[0025] In the Norwegian patent application with publication number
162,665, cholic acid; 3.alpha.-hydroxy-7-ketocholic acid;
3.alpha.,7.alpha.-diacetoxycholic acid;
3.alpha.,7.alpha.-diacetoxy-12-ke- tocholic acid;
3.alpha.,7.alpha.-dihydroxydeoxycholic acid; 7-ketodeoxycholic
acid; 3.alpha.,7.beta.-dihydroxydeoxycholic acid; and
3.alpha.,7.beta.-dihydroxy-12-ketocholic acid are mentioned as
being intermediates in the preparation of ursodeoxycholanic
acid.
[0026] In the Norwegian patent application with publication number
303,450, cholic acid; cholic acid methyl ester; 3-acetylcholic acid
methyl ester; 3-(2-propenyl)cholic acid methyl ester;
3-(3-hydroxypropyl)cholic acid-3-methyl ester; desoxycholic acid;
12-keto-desoxycholic acid; 3.beta.-acetyloxy-12-keto-desoxycholic
acid; 3.beta.-(hydroxyethyloxy)cholic acid methyl ester;
3.beta.-(hydroxypropyloxy)cholic acid methyl ester;
3.beta.-(hydroxybutyloxy)cholic acid methyl ester;
3.beta.-(hydroxypentyloxy)cholic acid methyl ester;
3.beta.-(hydroxyhexyloxy)cholic acid methyl ester;
3.beta.-(hydroxydecanoyloxy)cholic acid methyl ester;
3.beta.-(2-hydroxyethyloxyethyloxy)cholic acid methyl ester;
3.beta.-(2-hydroxypropyloxy)cholic acid methyl ester;
3.beta.-(hydroxyethyloxy)desoxycholic acid methyl ester;
3.beta.-(hydroxypropyloxy)desoxycholic acid methyl ester;
3.beta.-(hydroxy-pentyloxy)desoxycholic acid methyl ester;
3.beta.-(hydroxydecyloxy)desoxycholic acid methyl ester;
3.beta.-(2-hydroxyethyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(3-hydroxypropyloxy)-chenodesoxycholic acid methyl ester;
3.beta.-(5-hydroxypentyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(10-hydroxydecyloxy)chenodesoxycholic acid methyl ester;
3.beta.-(2-hydroxy-ethyloxy)litocholic acid methyl ester;
3.beta.-(3-hydroxypropyloxy)litocholic acid methyl ester;
3.beta.-(5-hydroxypenthyloxy)lithocholic acid methyl ester;
3.beta.-(10-hydroxydecayloxy)lithocholic acid methyl ester;
3.beta.-(benzyloxyethyloxy)cholic acid methyl ester;
3.beta.-(benzyloxyethyloxy)cholic acid tert.butyl ester;
3.beta.-(2-hydroxyethyloxy)cholic acid tert.butyl ester;
3.beta.-(2-hydroxyethyl-oxy)-7.alpha.,12.alpha.-diacetyloxycholic
acid methyl ester; and
3.beta.-(propionyloxy)-7.alpha.,12.alpha.-diacetyloxy-2-
4-carboxylic acid methyl ester are mentioned as intermediates.
[0027] In the Swedish patent application with publication number
385,905, chenodeoxycholic acid is mentioned to have utility for the
treatment of cholelithiasis and cholic acid is mentioned as an
intermediate for the preparation thereof.
[0028] Swedish patent application with publication number 402,462
mentions sitosterol which may have medical application, e.g. for
the prevention or reduction of absorption of cholesterol in the
small intestine, and campesterol is mentioned to lower the effect
of sitosterol.
[0029] In the Swedish patent application with publication number
413,247, 3.alpha.-hydroxy-cholestane and 3.beta.-hydroxycholestane
are mentioned to have antiinflammatoric properties and slightly
side effects.
[0030] Swedish patent application with publication number 430,508
deals with compounds possibly having pharmacological properties,
i.e. inhibition of HMG-CoA reductase and inhibition of the
formation of serum cholesterol. Examples of compounds specifically
mentioned therein are 25-fluorocholest-5-en-3.beta.,22-diol;
25-chlorocholest-5-en-3.beta.,22-d- iol;
22-hydroxy-25-fluorocholest-5-en-3.beta.-hemisuccinate;
22-hydroxy-25-chlorocholest-5-en-3.beta.-hemisuccinate; and
cholesta-5-en-3.beta.,22,25-triol.
[0031] Compounds being known to stimulate the meiosis and being
different from the compounds claimed in the present patent
application are described in International patent applications
having Nos. WO 96/00235, 96/27658 and 97/00884 (Novo Nordisk A/S)
and 98/55498. In International patent application having No. WO
98/52965, filed on 11 May 1998 and published on 26 Nov. 1998, it is
stated that certain 20-aralkyl-5.alpha.-pregnan derivatives can be
used in the preparation of a medicament for the control of
fertility and some of the specific compounds mentioned therein are
(3.beta.,5.alpha.,20R)-4,4,20-trimethyl-2-
1-phenylpregna-8,14-dien-3-ol (example 1);
(3.beta.,5.alpha.,20R)-4,4,20-t-
rimethyl-21-(3-methylphenyl)pregna-8,14-dien-3-ol (example 2A); and
(3.beta.,5.alpha.,20R)-4,4-dimethyl-23-phenyl-24-norchola-8,14-dien-3-ol
(example 7A).
[0032] The compounds described herein have advantages compared with
the known compounds.
SUMMARY OF THIS INVENTION
[0033] A main purpose of this invention is to furnish compounds
which can be used to regulate meiosis.
[0034] One purpose of the present invention is to provide compounds
and methods useful for relieving infertility in females and males,
particularly in mammals, more particularly in humans.
[0035] In a further object, the present invention concerns the use
of the compounds of the general formula Ic (stated in the claims,
below) for relieving infertility in females and males, particularly
in mammals, more particularly in humans.
[0036] 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.
[0037] In another embodiment, the invention relates to esters,
salts, active metabolites and prodrugs of compound of the general
formula Ia.
[0038] In still another preferred embodiment, the present invention
relates to compounds of the general formula Ib (stated in the
claims below) or esters, salts, active metabolites or prodrugs
thereof as a medicament.
[0039] In a further preferred embodiment, this invention relates to
compounds of the general formula Ic (stated in the claims below) or
esters, salts, active metabolites or prodrugs thereof in the
manufacture of a medicament for use in the regulation of
meiosis.
[0040] In a further preferred aspect, the present invention relates
to the use of a compound of formula Ib/Ic or an ester, salt, active
metabolite or prodrug thereof as a medicament, in particular as a
medicament for use in the regulation of meiosis. The compound may
be used neat or in the form of a liquid or solid composition
containing auxiliary ingredients conventionally used in the
art.
[0041] In the present context, the expression "regulating the
meiosis" is used to indicate that certain of the compounds of
formula Ia, Ib or Ic can be used for stimulating the meiosis in
vitro, in vivo, or ex vivo. Thus, the compounds of formula Ia, Ib
or Ic which may be agonists of a naturally occurring meiosis
activating substance, can be used in the treatment of infertility
which is due to insufficient stimulation of meiosis in females and
in males. Other compounds of formula Ia, Ib or Ic, which may be
antagonists of a naturally occurring meiosis activating substance,
can be used for regulating the meiosis, preferably in vivo, in a
way which makes them suited as contraceptives. In this case the
"regulation" means partial or total inhibition.
[0042] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the regulation of the
meiosis of an oocyte, in particular a mammalian oocyte, more
particularly a human oocyte.
[0043] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the stimulation of the
meiosis of an oocyte, in particular a mammalian oocyte, more
particularly a human oocyte.
[0044] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the inhibition of the
meiosis of an oocyte, in particular a mammalian oocyte, more
particularly a human oocyte.
[0045] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the regulation of the
meiosis of a male germ cell, in particular a mammalian male germ
cell, more particularly a human male germ cell.
[0046] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the stimulation of the
meiosis of a male germ cell, in particular a mammalian male germ
cell, more particularly a human male germ cell.
[0047] In a still further preferred aspect, the present invention
relates to the use of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof in the inhibition of the
meiosis of a male germ cell, in particular a mammalian male germ
cell, more particularly a human male germ cell.
[0048] In a yet still further preferred aspect, the present
invention relates to a method of regulating the meiosis in a
mammalian germ cell which method comprises administering an
effective amount of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof to a germ cell in need of such
a treatment.
[0049] In a still further aspect, the present invention relates to
a method of regulating the meiosis in a mammalian germ cell wherein
a compound of formula Ic or an ester, salt, active metabolite or
prodrug thereof is administered to the germ cell by administering
the compound to a mammal hosting said cell.
[0050] In a still further aspect, the present invention relates to
a method wherein the germ cell the meiosis of which is to be
regulated by means of a compound of formula Ic or an ester, salt,
active metabolite or prodrug thereof is an oocyte.
[0051] In a still further aspect, the present invention relates to
a method of regulating the meiosis in an oocyte wherein a compound
of formula Ic or an ester, salt, active metabolite or prodrug
thereof is administered to the oocyte ex vivo.
[0052] In a still further aspect, the present invention relates to
a method of regulating the meiosis of a male germ cell by
administering a compound of formula Ic or an ester, salt, active
metabolite or prodrug thereof to the cell.
[0053] In a still further aspect, the present invention relates to
a method whereby mature male germ cells are produced by
administering in vivo or in vitro a compound of formula Ic or an
ester, salt, active metabolite or prodrug thereof to testicular
tissue containing immature cells.
[0054] In a still further aspect, the present invention relates to
compounds of formula Ia, Ib and Ic having improved stability.
[0055] According to the present invention there are provided novel
compounds of formula Ia (stated in claim 1, below) with interesting
pharmacological properties. The compounds described herein are
useful for regulating the meiosis in oocytes and in male germ
cells.
DETAILED DESCRIPTION OF THIS INVENTION
[0056] It has, surprisingly, been found that compounds having a
side chain (R.sup.22) which is different from the cholesterol and
lanosterol side chains or compounds having certain specifically
elected substituents in the ring system, have superior
properties.
[0057] Preferred compounds of formula Ia, Ib and Ic are such having
a double bond.
[0058] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is hydrogen.
[0059] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is halogen.
[0060] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is methyl.
[0061] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is hydroxy.
[0062] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is oxo.
[0063] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2, together with R.sup.3, designates an additional
bond between the carbon atoms at which R.sup.2 and R.sup.3 are
placed.
[0064] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2 is hydrogen.
[0065] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2 is hydroxy.
[0066] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R is C.sub.1-C.sub.3 alkyl.
[0067] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2 is C.sub.1-C.sub.3 alkoxy.
[0068] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2 is halogen.
[0069] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is hydrogen.
[0070] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is hydroxy.
[0071] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is alkoxy, aralkyloxy, alkoxyalkoxy or
alkanoyloxyalkyl, each group comprising a total of up to 10 carbon
atoms, preferably up to 8 carbon atoms.
[0072] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is C.sub.1-C.sub.4 alkoxy.
[0073] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is methoxy.
[0074] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is ethoxy.
[0075] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is CH.sub.3OCH.sub.2O--.
[0076] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is pivaloyloxymethoxy.
[0077] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is an acyloxy group derived from an acid having
from 1 to 20 carbon atoms.
[0078] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is an acyloxy group selected from the group
comprising acetoxy, benzoyloxy, pivaloyloxy, butyryloxy,
nicotinoyloxy, isonicotinoyloxy, hemi succinoyloxy, hemi
glutaroyloxy, butylcarbamoyloxy, phenylcarbamoyloxy,
butoxycarbonyloxy, tert-butoxycarbonyloxy and
ethoxycarbonyloxy.
[0079] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is sulphonyloxy.
[0080] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is phosphonyloxy.
[0081] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 together with R'.sup.3 is oxo.
[0082] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is the group .dbd.NOH.
[0083] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is a group of the general formula .dbd.NOR.sup.38,
wherein R.sup.38 is C.sub.1-C.sub.3 alkyl.
[0084] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is halogen.
[0085] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is hydroxy and C.sub.1-C.sub.4 alkyl bound to the
same carbon atom of the sterol skeleton.
[0086] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.3 is perfluoro-(lower alkyl), preferably
perfluoro(lower alkyl) having 1 through 6, preferably 1 through 3,
carbon atoms in the alkyl group, more preferred
trifluoromethyl.
[0087] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4 are both hydrogen.
[0088] Other preferred compounds of formula Ia, Ib and Ic are such
wherein one of R.sup.4 and R'.sup.4 is hydrogen while the other is
methyl.
[0089] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4 are both methyl.
[0090] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 is branched or unbranched C.sub.1-C.sub.6 alkyl,
optionally substituted by halogen, hydroxy or cyano.
[0091] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.4 is branched or unbranched C.sub.1-C.sub.6 alkyl,
optionally substituted by halogen, hydroxy or cyano.
[0092] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 is hydroxy and R.sup.4 is selected from the group
comprising hydrogen and branched or unbranched C.sub.1-C.sub.6
alkyl which may be substituted by halogen, hydroxy or cyano.
[0093] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.4 is hydroxy and R.sup.4 is selected from the group
comprising hydrogen and branched or unbranched C.sub.1-C.sub.6
alkyl which may be substituted by halogen, hydroxy or cyano.
[0094] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4 together designate methylene.
[0095] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4, together with the carbon atom to
which they are bound, form a cyclopropane ring.
[0096] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4, together with the carbon atom to
which they are bound, form a cyclopentane ring.
[0097] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.4 and R'.sup.4, together with the carbon atom to
which they are bound, form a cyclohexane ring.
[0098] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is hydrogen.
[0099] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is halogen.
[0100] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is hydroxy.
[0101] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is alkyl with 1 through 3 carbon atoms, preferably
methyl, cyano or hydroxymethyl, or R.sup.5 is, together with
R.sup.4, a methano bridge or R.sup.5 is, together with R.sup.4, an
additional bond.
[0102] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is a primary or secondary amide derived from a
carboxylic acid.
[0103] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is an ester with a C.sub.1-C.sub.6-alcohol
group.
[0104] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.6 is hydrogen.
[0105] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.6 is halogen.
[0106] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.6 is oxo.
[0107] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.6 is hydroxy.
[0108] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.6, together with R.sup.5, designates an additional
bond between the carbon atoms at which R.sup.5 and R.sup.6 are
placed.
[0109] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is hydrogen.
[0110] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is methylene.
[0111] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is hydroxy.
[0112] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is methoxy or acetoxy.
[0113] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is halogen.
[0114] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is oxo.
[0115] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is the group .dbd.NOH.
[0116] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 is a group of the general formula .dbd.NOR.sup.36,
wherein R.sup.36 is C.sub.1-C.sub.3 alkyl.
[0117] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7 simultaneously is hydroxy and C.sub.1-C.sub.4 alkyl
both being bound to the same carbon atom of the sterol skeleton,
i.e. in the 7 position.
[0118] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7, together with R.sup.9, designates an additional
bond between the carbon atoms at which R.sup.7 and R.sup.6 are
placed.
[0119] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.7, together with R.sup.8, designates an additional
bond between the carbon atoms at which R.sup.7 and R.sup.8 are
placed.
[0120] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.8, together with R.sup.9, designates an additional
bond between the carbon atoms at which R.sup.8 and R.sup.9 are
placed.
[0121] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.8 is hydrogen.
[0122] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.8 is halogen.
[0123] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.8 is hydroxy.
[0124] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.9 is hydrogen.
[0125] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.9 is halogen.
[0126] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.9 is hydroxy.
[0127] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is hydrogen.
[0128] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is methylene.
[0129] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is hydroxy.
[0130] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is halogen.
[0131] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is methoxy or acetoxy.
[0132] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is oxo.
[0133] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is the group .dbd.NOH.
[0134] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 is a group of the general formula .dbd.NOR.sup.37,
wherein R.sup.37 is C.sub.1-C.sub.3 alkyl.
[0135] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11 simultaneously is hydroxy and C.sub.1-C.sub.4
alkyl both being bound to the same carbon atom of the sterol
skeleton, i.e. in the 11 position.
[0136] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11, together with R.sup.9, designates an additional
bond between the carbon atoms at which R.sup.11 and R.sup.9 are
placed.
[0137] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.11, together with R.sup.12, designates an additional
bond between the carbon atoms at which R.sup.11 and R.sup.12 are
placed.
[0138] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is hydrogen.
[0139] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is halogen.
[0140] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is C.sub.1-C.sub.4 alkyl.
[0141] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is methylene.
[0142] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is hydroxy.
[0143] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is methoxy or acetoxy.
[0144] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is oxo.
[0145] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is the group .dbd.NOH.
[0146] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.12 is a group of the general formula .dbd.NOR.sup.33,
wherein R.sup.33 is C.sub.1-C.sub.3 alkyl.
[0147] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.14 is hydrogen.
[0148] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.14 is hydroxy.
[0149] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.14, together with R.sup.8, designates an additional
bond between the carbon atoms at which R.sup.14 and R.sup.8 are
placed.
[0150] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is hydrogen.
[0151] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is halogen.
[0152] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is C.sub.1-C.sub.4 alkyl.
[0153] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is methylene.
[0154] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is hydroxy.
[0155] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is methoxy.
[0156] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is oxo.
[0157] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is the group .dbd.NOH.
[0158] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15 is a group of the general formula .dbd.NOR.sup.32,
wherein R.sup.32 is C.sub.1-C.sub.3 alkyl.
[0159] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.15, together with R.sup.14, designates an additional
bond between the carbon atoms at which R.sup.15 and R.sup.14
.alpha.re placed.
[0160] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is hydrogen.
[0161] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is halogen.
[0162] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is C.sub.1-C.sub.3 alkyl.
[0163] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is methylene.
[0164] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is hydroxy.
[0165] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is methoxy.
[0166] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is oxo.
[0167] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is the group .dbd.NOH.
[0168] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 is a group of the general formula .dbd.NOR.sup.34,
wherein R.sup.34 is C.sub.1-C.sub.3 alkyl.
[0169] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.16 together with R.sup.17, designates an additional
bond between the carbon atoms at which R.sup.16 and R.sup.17 are
placed.
[0170] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.17 is hydrogen.
[0171] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.17 is hydroxy.
[0172] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.17 is in the .alpha. position.
[0173] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.20 is hydrogen.
[0174] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.20 is hydroxymethyl.
[0175] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.20 is C.sub.1-C.sub.4 alkyl.
[0176] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.20 together with R'.sup.20 designates methylene.
[0177] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.20 together with R'.sup.20 designates oxo.
[0178] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.20 is hydrogen.
[0179] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.20 is halogen.
[0180] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.20 is methyl.
[0181] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.20 is hydroxy.
[0182] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.22 is cyclohexyl optionally substituted by one or
more of the following groups which substituents may be different or
identical: hydroxy, alkoxy, halogen, amino, cyano, carboxy, a group
of the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio;
[0183] cyclohexylalkyl optionally substituted by one or more of the
following groups which substituents may be different or identical:
hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a group of
the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio;
[0184] alkyl optionally substituted by one or more of the following
groups which substituents may be different or identical: hydroxy,
alkoxy, halogen, amino, cyano, carboxy, a group of the general
formula --COOR.sup.39, oxo, N-acylamino, N-alkylamino or
N,N-dialkylamino wherein the N-alkylamino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio; or
[0185] alkenyl optionally substituted by one or more of the
following groups which substituents may be different or identical:
hydroxy, lower alkoxy, halogen, amino, cyano, carboxy, a group of
the general formula --COOR.sup.39, oxo, N-alkylamino or
N,N-dialkylamino wherein the N-alkyl-amino or N,N-dialkylamino
substituent optionally is substituted by carboxy, lower alkoxy or
lower alkylthio;
[0186] and R.sup.39 represents lower alkyl or aralkyl, e.g.
benzyl.
[0187] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.22 is phenyl, cyclohexyl, benzyl, o-tolyl, m-tolyl,
p-tolyl, but-3-enyl, 3-methylbut-3-enyl, 2-methylpropyl,
2-oxo-2-ethoxyethyl, 2-oxo-2-(N,N-dimethylamino)ethyl,
carboxymethyl, 3-hydroxymethylbutyl, 2-cyanoethyl,
cyclohexylmethyl, 3-chloro-3-methylbutyl,
2-(N,N-dimethylamino)-2-cyanoethyl, 2-chloroethyl, 2-iodoethyl,
ethyl, 2-phenylethyl, 2-methoxyethyl, 2-benzyloxyethyl or
2-acetoxy-ethyl.
[0188] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R'.sup.22 is hydrogen.
[0189] Other preferred compounds of formula Ia, Ib and Ic are such
wherein the side chain in the 17 position (i.e.
--C(R.sup.20)(R.sup.'20)--CH(R.su- p.22)(R.sup.'22)) is in the
.beta. position.
[0190] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.2 together with R.sup.3 is an additional double bond
and R.sup.'3 is hydrogen.
[0191] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1 is hydrogen, halogen, methyl, hydroxy or oxo;
R.sup.2 is selected from the group comprising hydrogen, hydroxy,
C.sub.1-C.sub.3 alkyl, vinyl, C.sub.1-C.sub.3 alkoxy and halogen,
or R.sup.2 designates, together with R.sup.3, an additional bond
between the carbon atoms at which R.sup.2 and R.sup.3 are placed;
R.sup.3 is selected from the group comprising hydrogen, hydroxy,
optionally substituted alkoxy, acyloxy, sulphonyloxy,
phosphonyloxy, oxo, halogen, C.sub.1-C.sub.4 alkyl and a group of
the general formula .dbd.NOR.sup.38 wherein R.sup.38 is hydrogen or
C.sub.1-C.sub.3 alkyl, or R.sup.3 designates, together with
R.sup.2, an additional bond between the carbon atoms at which
R.sup.2 and R.sup.3 are placed; wherein R.sup.4 and R.sup.4', which
are different or identical with the proviso that they are not both
hydroxy, are selected from the group comprising hydrogen, halogen,
hydroxy and branched or unbranched C.sub.1-C.sub.6 alkyl which may
be substituted by halogen, hydroxy or cyano, or wherein R.sup.4 and
R.sup.4' together designate methylene or oxo or, together with the
carbon atom to which they are bound, form a cyclopropane ring, a
cyclopentane ring, or a cyclohexane ring; R.sup.5 is hydrogen,
halogen or hydroxy, or R.sup.5 designates, together with R.sup.6,
an additional bond between the carbon atoms at which R.sup.5 and
R.sup.6 are placed; R.sup.6 is hydrogen, hydroxy, halogen or oxo,
or R.sup.6 designates, together with R.sup.5 or R.sup.7, an
additional bond between the carbon atoms at which R.sup.6 and
R.sup.5 or R.sup.7 are placed; R.sup.7 is selected from the group
comprising hydrogen, methylene, hydroxy, methoxy, acetoxy, oxo,
halogen, C.sub.1-C.sub.4 alkyl and a group of the general formula
.dbd.NOR.sup.36 wherein R.sup.36 is hydrogen or C.sub.1-C.sub.3
alkyl, or R.sup.7 designates, together with R.sup.6 or R.sup.8, an
additional bond between the carbon atoms at which R.sup.7 and
R.sup.5 or R.sup.8 are placed; R.sup.8 is hydrogen, hydroxy or
halogen, or R.sup.8 designates, together with R.sup.7, R.sup.9 or
R.sup.14, an additional bond between the carbon atoms at which
R.sup.8 and R.sup.7, R.sup.9 or R.sup.14 are placed; R.sup.9 is
hydrogen, hydroxy or halogen, or R.sup.9 designates, together with
R.sup.8 or R.sup.11, an additional bond between the carbon atoms at
which R.sup.9 and R.sup.8 or R.sup.11 are placed; R.sup.11 is
selected from the group comprising hydrogen, methylene, hydroxy,
methoxy, acetoxy, oxo, halogen, C.sub.1-C.sub.4 alkyl and a group
of the general formula .dbd.NOR.sup.37 wherein R.sup.37 is hydrogen
or C.sub.1-C.sub.3 alkyl, or R.sup.11 designates, together with
R.sup.9 or R.sup.12, an additional bond between the carbon atoms at
which R.sup.11 and R.sup.9 or R.sup.12 are placed; R.sup.12 is
selected from the group comprising hydrogen, halogen,
C.sub.1-C.sub.4 alkyl, methylene, hydroxy, methoxy, acetoxy, oxo
and a group of the general formula .dbd.NOR.sup.33 wherein R.sup.33
is hydrogen or C.sub.1-C.sub.3 alkyl, or R.sup.12 designates,
together with R.sup.11, an additional bond between the carbon atoms
at which R.sup.11 and R.sup.12 are placed; R.sup.14 is hydrogen or
hydroxy, or R.sup.14 designates, together with R.sup.15, an
additional bond between the carbon atoms at which R.sup.14 and
R.sup.15 are placed; R.sup.15 is selected from the group comprising
hydrogen, halogen, C.sub.1-C.sub.4 alkyl, methylene, hydroxy,
methoxy, oxo and a group of the general formula .dbd.NOR.sup.32
wherein R.sup.32 is hydrogen or C.sub.1-C.sub.3 alkyl, or R.sup.15
designates, together with R.sup.14 an additional bond between the
carbon atoms at which R.sup.15 and R.sup.14 are placed; R.sup.16 is
selected from the group comprising hydrogen, halogen,
C.sub.1-C.sub.3 alkyl, methylene, hydroxy, methoxy, oxo and a group
of the general formula .dbd.NOR.sup.34 wherein R.sup.34 is hydrogen
or C.sub.1-C.sub.3 alkyl, or R.sup.16 designates, together with
R.sup.17, an additional bond between the carbon atoms at which
R.sup.16 and R.sup.17 are placed; R.sup.17 is hydrogen or hydroxy,
or R.sup.17 designates, together with R.sup.16, an additional bond
between the carbon atoms at which R.sup.17 and R.sup.16 are placed;
R.sup.20 is selected from the group comprising hydrogen,
C.sub.1-C.sub.4 alkyl and hydroxymethyl, or R.sup.20 and R.sup.20'
together designate methylene or oxo; R.sup.20' is hydrogen,
halogen, alkyl or hydroxy, R.sup.22' is hydrogen, hydroxy or oxo;
R.sup.22 represents phenyl optionally substituted by one or more of
the following groups which substituents may be different or
identical: hydroxy, alkoxy, halogen (chloro, bromo or iodo), amino,
N-alkylamino, N,N-dialkylamino, cyano, carboxy or oxo; benzyl
optionally substituted by one or more of the following groups which
substituents may be different or identical: hydroxy, alkoxy,
halogen (chloro, bromo or iodo), amino, N-alkyl-amino,
N,N-dialkylamino, cyano, carboxy or oxo; cyclohexyl optionally
substituted by one or more of the following groups which
substituents may be different or identical: hydroxy, alkoxy,
halogen (chloro, bromo or iodo), amino, N-alkylamino,
N,N-dialkylamino, cyano, carboxy or oxo; cyclohexylalkyl optionally
substituted by one or more of the following groups which
substituents may be different or identical: hydroxy, alkoxy,
halogen (chloro, bromo or iodo), amino, N-alkylamino,
N,N-dialkylamino, cyano, carboxy or oxo; alkyl optionally
substituted by one or more of the following groups which
substituents may be different or identical: hydroxy, alkoxy,
halogen (chloro, bromo or iodo), amino, N-alkylamino,
N,N-dialkyl-amino, cyano, carboxy or oxo; alkenyl optionally
substituted by one or more of the following groups which
substituents may be different or identical: hydroxy, alkoxy,
halogen (chloro, bromo or iodo), amino, N-alkylamino,
N,N-dialkylamino, cyano, carboxy or oxo; and esters thereof.
[0192] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.1, R.sup.2, R'.sup.4, R.sup.5, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, R'.sup.20 and R'.sup.22 is each hydrogen,
R.sup.3 is hydrogen, lower alkyl or perfluoro(lower alkyl),
R'.sup.3 is hydroxy, or R.sup.3 designates, together with R'.sup.3,
oxo, R.sup.4 is hydrogen or together with R.sup.5 a methano bridge
or together with R.sup.5 an additional bond, R.sup.5 is lower
alkyl, cyano, hydroxymethyl, a carbaldehyde, an oxime derived from
a carbaldehyde, a carboxylic acid, a primary or secondary amide
derived from a carboxylic acid, an ester with a
C.sub.1-C.sub.6-alcohol group or together with R.sup.4 a methano
bridge or together with R.sup.4 an additional bond, R.sup.7 is
together with R.sup.8 an additional bond or hydrogen, if R.sup.8
and R.sup.9 or R.sup.8 and R.sup.14 stand together for an
additional bond, R.sup.8 is together with R.sup.7 or with R.sup.9
or with R.sup.14 an additional bond, R.sup.9 is together with
R.sup.8 an additional bond or a hydrogen atom, if R.sup.7 and
R.sup.8 or R.sup.8 and R.sup.14 stand together for an additional
bond, R.sup.14 is, together with R.sup.8, an additional bond or a
hydrogen atom, if R.sup.7 and R.sup.8 or R.sup.8 and R.sup.9 stand
together for an additional bond, R.sup.17 is hydrogen in the alpha
position, R.sup.19 is methyl in the beta position, R.sup.20 is
methyl in the alpha position, and R.sup.22 is 3-methylbutyl; and
ester, salt, active metabolite or prodrugs thereof.
[0193] Other preferred compounds of formula Ia, Ib and Ic are such
wherein R.sup.5 is a C.sub.1-C.sub.3-alkyl group, preferably a
methyl group, a cyano group, a hydroxymethyl group or together with
R.sup.4 a methano bridge or together with R.sup.4 an additional
bond.
[0194] It is to be understood that the above preferred substituents
can be combined in any way with each other.
[0195] Examples of interesting and preferred compounds of the
general formula Ia, Ib and Ic are as follows:
[0196]
(20R)-20-Methyl-21-phenyl-5.alpha.-pregna-8,14-dien-3.beta.-ol;
(20R)-20-methyl-21-(3-methylphenyl)-5.alpha.-pregna-8,14-dien-3.beta.-ol;
(20R)-20-methyl-21-(3-hydroxyphenyl)-5.alpha.-pregna-8,14-dien-3.beta.-ol-
;
(20R)-20-methyl-21-(cyclopentyl)-5.alpha.-pregna-8,14-dien-3.beta.-ol;
24-nor-cholest-8,14-dien-3.beta.-ol;
(20R)-20-methyl-21-(cyclohexyl)-5.al-
pha.-pregna-8,14-dien-3.beta.-ol;
(20R)-20-methyl-22-phenyl-5.alpha.-pregn- a-8,14-dien-3.beta.-ol;
23,24-dinor-cholest-8,14-dien-3.beta.-ol; (20R)-20-methyl-21
cyclobutyl)-5.alpha.-pregna-8,14-dien-3.beta.-ol;
4,4-dimethyl-17.beta.-((1R)-methyl-3-methyl-2-butenyl)androsta-8,14-dien--
3-.beta.-ol;
(20R)-20-methyl-23-dimethylamino-5.alpha.-pregna-8,14-dien-3.-
beta.-ol; 3.beta.-hydroxy-5.alpha.-cyanochol-8-en-24-oic
acid-N,N-dimethyl amide; 5.beta.-methychol-8-en-3-on-24-oic
acid-N,N-dimethyl amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.,14.beta.-chola-8,15-dien-24-oic
acid-N,N-dimethyl amide;
3.beta.-hydroxy-5.alpha.-cyanocholest-8-en-24-on- e;
5.beta.-methylchol-8-en-3,24-dione;
3.beta.-hydroxy-4,4-dimethyl-5.alph-
a.,14.beta.-cholesta-8,15-dien-24-one;
3.beta.-hydroxy-5.alpha.-cyanochol-- 8-en-24-oic acid cyclohexyl
ester; 5.beta.-methylchol-8-en-3-on-24-oic acid cyclohexyl ester;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.,14.beta.-cho-
la-8,15-dien-24-oic acid cyclohexyl ester;
3.beta.-hydroxy-5.alpha.-chola-- 8,14-dien-24-oic
acid-N-(4-methylpiperazinyl)amide;
3.beta.-hydroxychola-5,7-dien-24-oic
acid-N-(4-methylpiperazinyl)amide;
3.beta.-hydroxy-5.alpha.-cyanochol-8-en-24-oic
acid-N-(4-methylpiperaziny- l)amide;
5.beta.-methylchol-8-en-3-on-24-oic acid-N-(4-methylpiperazinyl)a-
mide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.,14.beta.-chola-8,15-dien-24-oi-
c acid-N-(4-methylpiperazinyl)amide;
(20R)-20-methyl-21-phenyl-5.alpha.-pr- egna-5,7-dien-3.beta.-ol;
(20R)-20-methyl-21-(3-methylphenyl)-5.alpha.-pre-
gna-5,7-dien-3.beta.-ol;
(20R)-20-methyl-21-(3-hydroxyphenyl)-5.alpha.-pre-
gna-5,7-dien-3.beta.-ol;
(20R)-20-methyl-21-(cyclopentyl)-5.alpha.-pregna--
5,7-dien-3.beta.-ol; 24-nor-cholest-5,7-dien-3.beta.-ol;
(20R)-20-methyl-21-(cyclohexyl)-5.alpha.-pregna-5,7-dien-3.beta.-ol;
(20R)-20-methyl-22-phenyl-5.alpha.-pregna-5,7-dien-3.beta.-ol;
23,24-dinor-cholest-5,7-dien-3.beta.-ol;
(20R)-20-methyl-21-(cyclobutyl)--
5.alpha.-pregna-5,7-dien-3.beta.-ol;
4,4-dimethyl-17.beta.-((1R)-methyl-3--
methyl-2-butenyl)androsta-5,7-dien-3.beta.-ol;
(20R)-20-methyl-23-dimethyl-
amino-5.alpha.-pregna-5,7-dien-3.beta.-ol;
cholesta-5,7-dien-25-chloro-3.b- eta.-ol;
cholesta-5,7-dien-26-chloro-3-.beta.-ol; cholesta-5,7-dien-26-ol;
nor-24-cholesta-8,11-dien-3.beta.-ol; cholesta-4,8-dien-3.beta.-ol;
cholesta-4,8-dien-3.alpha.-ol; cholesta-4,8(14)-dien-3.beta.-ol;
cholesta-4,8(14)-dien-3.alpha.-ol;
5-cyano-5.alpha.-cholest-7-en-3.alpha.- -ol;
5-cyano-5.alpha.-cholest-7-en-3.beta.-ol;
5-cyano-5.beta.-cholest-7-e- n-3.alpha.-ol;
5-cyano-5.alpha.-cholest-8-en-3.alpha.-ol;
5-cyano-5.alpha.-cholest-8-en-3.beta.-ol;
5-cyano-5.beta.-cholest-8-en-3.- alpha.-ol;
5-cyano-5.beta.-cholest-8-en-3.beta.-ol;
5-cyano-5.alpha.-cholest-8(14)-en-3.alpha.-ol;
5-cyano-5.alpha.-cholest-8- (14)-en-3.beta.-ol;
5-cyano-5.beta.-cholest-8(14)-en-3.alpha.-ol;
5-cyano-5.beta.-cholest-8(14)-en-3.beta.-ol;
3',4.alpha.-dihydrocycloprop-
a[4,5]-5.beta.-cholest-7-en-3.beta.-ol;
3',4.beta.-dihydrocyclopropa[4,5]--
5.alpha.-cholest-7-en-3.alpha.-ol;
3',4.alpha.-dihydrocyclopropa[4,5]-5.be-
ta.-cholest-8-en-3.beta.-ol;
3',4.beta.-dihydrocyclopropa[4,5]-5.alpha.-ch-
olest-8-en-3.alpha.-ol;
3',4.alpha.-dihydrocyclopropa[4,5]-5.beta.-cholest-
-8(14)-en-3.beta.-ol;
3',4.beta.-dihydrocyclopropa[4,5]-5.alpha.-cholest-8-
(14)-en-3.alpha.-ol;
5-(hydroxymethyl)-5.alpha.-cholest-7-en-3.beta.-ol;
5-(hydroxymethyl)-5.beta.-cholest-7-en-3.alpha.-ol;
5-(hydroxymethyl)-5.alpha.-cholest-8-en-3.beta.-ol;
5-(hydroxymethyl)-5.beta.-cholest-8-en-3.alpha.-ol;
5-(hydroxymethyl)-5.alpha.-cholest-8(14)-en-3.beta.-ol;
5-(hydroxymethyl)-5.beta.-cholest-8(14)en-3.alpha.-ol;
5-methyl-5.beta.-cholest-8-en-3-one;
5-methyl-5.beta.-cholest-8-en-3.beta- .-ol;
5-methyl-5.beta.-cholest-8-en-3.alpha.-ol;
5-methyl-5.beta.-cholest-- 8(14)-en-3-one;
5-methyl-5.beta.-cholest-8(14)-en-3.beta.-ol;
5-methyl-5.beta.-cholest-8(14)-en-3.alpha.-ol;
3.alpha.-(trifluoromethyl)- cholesta-4,7-dien-3.beta.-ol;
3.beta.-(trifluoromethyl)cholesta-4,7-dien-3- .alpha.-ol;
3.alpha.-(tri-fluoromethyl)cholesta-4,8-dien-3.beta.-ol;
3.beta.-(trifluoromethyl)cholesta-4,8-dien-3.alpha.-ol;
3.alpha.-(tri-fluoromethyl)cholesta-4,8(14)-dien-3.beta.-ol;
3.beta.-(trifluoromethyl)cholesta-4,8(14)-dien-3.alpha.-ol;
5-methyl-24-nor-5.beta.-cholest-8(14)-en-3-one;
(20R)-5,20-dimethyl-21-ph- enyl-5.beta.-pregn-8(14)-en-3-one;
(20R)-21-cyclohexyl-5,20-dimethyl-5.bet- a.-pregn-8(14)-en-3-one;
5-methyl-24-nor-5.beta.-cholesta-8(14),23-dien-3-- one;
4,4-dimethyl-24-benzoylamido-5.alpha.-chola-8,14-dien-3.beta.-ol;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic acid
N-phenylalanine amide;
mono(3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8- ,14-dien)-24
succinate; 3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-d-
ien-24-oic acid (1-methyl-4-hydroxypiperidinyl)ester;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(norleucine)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,1- 4-dien-24-oic
acid-N-(arginine)amide; 3.beta.-hydroxy-4,4-dimethyl-5.alpha-
.-chola-8,14-dien-24-oic acid-N-(glutamic acid)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(leucine)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-d- ien-24-oic acid
methyl ester; 3.beta.-hydroxy-4,4-dimethylchola-5,7-dien-2- 4-oic
acid methyl ester;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-- dien-24-oic acid
ethyl ester; 3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola--
8,14-dien-24-oic acid;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-di- en-24-oic acid
cyclohexyl ester; 3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cho-
lesta-8,14-dien-24-one;
3.beta.-hydroxy-4,4,24-trimethyl-5.alpha.-chola-8,- 14-dien-24-one;
3.beta.-hydroxy-4,4-dimethyl-24-phenyl-5.alpha.-chola-8,14-
-dien-24-one;
3.beta.-hydroxy-4,4-di-methyl-24-(3-pentyl)-5.alpha.-chola-8-
,14-dien-24-one;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-- oic
acid-N-phenyl amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-- dien-24-oic acid
amide; 4,4-dimethyl-24-phenylamino-5.alpha.-chola-8,14-di-
en-3.beta.-ol;
4,4-dimethyl-24-amino-5.alpha.-chola-8,14-dien-3.beta.-ol;
4,4-dimethyl-5.alpha.-chola-8,14-dien-3.beta.,24-diol;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-aldehyde;
4,4-dimethyl-17.beta.-((1R)-methyl-4-methyl-3-pentenyl)-androsta-8,14-die-
n-3.beta.-ol;
4,4-dimethyl-5.alpha.-cholesta-14,16,24-triene-3.beta.-ol;
4,4-dimethyl-17.beta.-((1R)-methyl-3-methyl-2-butenyl)androsta-8,14-dien--
3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(4-methylphenyl)-5.alpha.-pregna-8,1-
4-dien-3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(2-methylphenyl)-5.alpha.-pre-
gna-8,14-dien-3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(cyclohexyl)-5.alpha.--
pregna-8,14-dien-3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(3-hydroxyphenyl)-5-
.alpha.-pregna-8,14-dien-3.beta.-ol;
(20R)-4,4,20-trimethyl-22-(cyclohexyl-
)-5.alpha.-pregna-8,14-dien-3.beta.-ol;
24-nor-4,4-dimethyl-5.alpha.-chole- st-8,14-dien-3.beta.-ol;
27-nor-4,4-dimethyl-5.alpha.-cholest-8,14,25-trie- n-3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(cyclobutyl)-5.alpha.-pregna-8,14--
dien-3.beta.-ol;
(20R)-4,4,20-trimethyl-21-(cyclopentyl)-5.alpha.-pregna-8-
,14-dien-3.beta.-ol;
25-chloro-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-3.- beta.-ol;
4,4-dimethyl-24-(N,N-dimethylamino)-24-cyano-5.alpha.-cholesta-8-
,14-dien-3.beta.-ol; 4,4-dimethylcholest-8,14,25-trien-3.beta.-ol;
4,4-dimethyl-17.beta.-((1R)-methyl-4-iodobutyl)androsta-8,14-dien-3.beta.-
-ol;
4,4-dimethyl-17.beta.-((1R)-methylbutyl)androsta-8,14-dien-3.beta.-ol-
;
4,4-dimethyl-17.beta.-((1R)-methyl-4-cyanobutyl)androsta-8,14-dien-3.bet-
a.-ol;
4,4-dimethyl-17.beta.-((1R)-methyl-4-cyanobutyl)androsta-8,14-dien--
3.beta.-ol;
27-nor-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-die-
n-26-oic acid benzyl ester;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,- 14-dien-24-oic
acid-N-(methionine methyl ester)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(methionine)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,1- 4-dien-24-oic
acid-N-(4-methylpiperazinyl)amide; 3.beta.-hydroxy-4,4-dimet-
hyl-5.alpha.-chola-8,14-dien-24-oic acid-N-tert-butylamide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(isonipecotic acid ethyl ester)amide;
3.beta.-hydroxy-4,4-dimethyl- -5.alpha.-chola-8,14-dien-24-oic
acid-N-(isonipecotic acid)amide;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(phenylalanine methyl ester)amide;
3.beta.-hydroxy-4,4-dimethylcho- la-5,7-dien-24-oic acid;
3.beta.-hydroxy-4,4-dimethylchola-5,7-dien-24-oic acid-N-dimethyl
amide; 4,4-dimethyl-24-acetamido-5.alpha.-chola-8,14-dien-
-3.beta.-ol;
4,4-dimethyl-24-acetoxy-5.alpha.-chola-8,14-dien-3.beta.-ol;
4,4-dimethyl-24-methoxy-5.alpha.-chola-8,14-dien-3.beta.-ol;
4,4-dimethyl-24-benzyloxy-5.alpha.-chola-8,14-dien-3.beta.-ol;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic acid
benzyl ester;
26,27-diethyl-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14--
dien-26,27-dioate;
3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-die-
n-26,27-dioic acid; and
27-nor-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-chole-
sta-8,14-dien-26-oic acid.
[0197] Preferred compounds of formula Ia, Ib and Ic are such which
when tested by the method described below for agonistic properties
(example 71) shows a relative activity of at least 50, preferably
at least 80, or when tested by the method described below for
antagonistic properties (example 72) shows a IC.sub.50 value below
10, preferably below 2.
[0198] Examples of other preferred compounds are such not being
active at the oestrogen receptor, and preferably compounds not
being active at other hormone receptors.
[0199] Further preferred embodiments are mentioned in the appended
claims.
[0200] As used in the present description and claims, a lower alkyl
group--when used alone or in combinations--may be a straight or
branched alkyl group. Preferably, said alkyl group contains not
more than 6 carbon atoms. Examples of preferred alkyl groups are
methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl and
hexyl, more preferred methyl, ethyl, propyl, isopropyl, butyl and
tert-butyl, still more preferred methyl and ethyl. In a preferred
embodiment of this invention, the alkyl group contains not more
than 4 carbon atoms, preferably not more than 3 carbon atoms.
[0201] As used in the present description and claims, alkoxy
designates a straight or branched alkoxy group, preferably
containing not more than 6 carbon atoms, preferably not more than 4
carbon atoms, most preferred not more than 3 carbon atoms. Examples
of preferred alkoxy groups are methoxy, ethoxy and propoxy, more
preferred methoxy and ethoxy.
[0202] As used in the present description and claims, N-alkylamino
is an alkyl group connected to an amino group. Preferably, said
alkyl group is a lower alkyl group as defined above. Preferred
N-alkylamino groups are methylamino and ethylamino.
[0203] As used in the present description and claims,
N,N-alkylamino is two alkyl group which are the same or different
and which are connected to an amino group. Preferably, said alkyl
group are lower alkyl groups as defined above. Preferred
N,N-alkylamino groups are N,N-dimethylamino, N,N-diethylamino and
N-methyl-N-ethylamino.
[0204] As used in the present description and claims, the
expression alkenyl designates a straight or branched alkenyl group
preferably containing not more than 6 carbon atoms, preferably not
more than 3 carbon atoms.
[0205] As used in the present description and claims, the
expression halogen preferably designates chloro, bromo or iodide.
Another group of preferred halogens are fluoro and chloro,
preferably fluoro.
[0206] As used in the present description and claims, the
expression C.sub.3-C.sub.6 cycloalkyl designates a cycloalkyl group
containing 3 through 6 carbon atoms in the ring. Preferred examples
are cyclopropyl and cyclopentyl.
[0207] As used in the present description and claims, the
expression acyloxy designates a monovalent substituent comprising
an optionally substituted C.sub.1-6-alkyl or phenyl group linked
through a carbonyloxy group; such as e.g. acetoxy, propionyloxy,
butyryloxy, isobutyryloxy, pivaloyloxy, valeryloxy, benzoyl and the
like. Optionally said acyloxy group may contain a hydroxy and/or
carboxy group.
[0208] As used in the present description and claims, a statement
that, e.g., R.sup.1 is oxo means that oxo (.dbd.O) is present in
the 1 position and, consequently, there is no hydrogen atom in the
1 position. Analogous considerations apply for similar situations.
In other instances, two symbols together may represent oxo, e.g.,
R.sup.3 and R'.sup.3.
[0209] As used in the present description and claims, a statement
that, e.g., R.sup.12 is methylene means that methylene
(.dbd.CH.sub.2) is present in the 12 position and, consequently,
there is no hydrogen atom in this position. Analogous
considerations apply for similar situations. In other instances,
two symbols together may represent methylene, e.g., R.sup.4 and
R'.sup.4.
[0210] As used in the present description and claims, a statement
that alkoxy is optionally substituted (R.sup.3) means that the
alkoxy group is substituted with a convenient substituent such as
hydroxy or carboxy.
[0211] As used in the present description and claims, the
expressions "a primary or secondary amide derived from a carboxylic
acid" used for the substituent R.sup.5 is a group of the general
formula --CONHR.sup.40 wherein R.sup.40 is hydrogen or lower
alkyl.
[0212] As used in the present description and claims, the
expressions "an ester with a C.sub.1-C.sub.6-alcohol group" used
for the substituent R.sup.5 is a group of the general formula
--COOR.sup.41 wherein R.sup.41 is lower alkyl or aralkyl.
[0213] As used in the present description and claims, the
expression arakyl designates alkyl substituted by an aryl group,
e.g. benzyl.
[0214] Salts of compounds of formula Ia, Ib and Ic are preferably
pharmaceutically acceptable salts, especially acid-addition salts,
including salts of organic acids and mineral acids. Examples of
such salts include salts of organic acids such as formic acid,
fumaric acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, succinic acid, malic acid,
tartaric acid, citric acid, benzoic acid, salicylic acid and the
like. Suitable inorganic acid-addition salts include salts of
hydrochloric, hydrobromic, sulphuric and phosphoric acids and the
like. Further examples of pharmaceutically acceptable inorganic or
organic acid addition salts include the pharmaceutically acceptable
salts listed in Journal of Pharmaceutical Science, 66 (1977), 2 et
seq.
[0215] Esters of compounds of formula Ia, Ib and Ic are formally
derived by esterification of one or more hydroxylic groups of a
compound of formula Ia, Ib or Ic, respectively, 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.
[0216] A "metabolite" of a compound of formula Ia, Ib or Ic is an
active derivative of a compound of formula Ia, Ib or Ic which is
produced when the compound of formula Ia, Ib or Ic is metabolised.
Metabolites of compounds of formula Ia, Ib or Ic can be identified
either by administration of a compound of formula Ia, Ib or Ic to a
host and an analysis of blood samples from the host, or by
incubation of a compound of formula Ia, Ib or Ic with hepatic cells
in vitro and analysis of the incubant.
[0217] A "prodrug" is a compound that either is converted into a
compound of formula Ia, Ib or Ic in vivo or which has the same
active metabolites as a compound of formula Ia, Ib or Ic.
[0218] The compounds of formula Ia, Ib or Ic have a number of
chiral centres in the molecule and thus exists in several isomeric
forms. All these isomeric forms and mixtures thereof are within the
scope of the invention.
[0219] The compounds of the general formula Ia, Ib and Ic can be
prepared analogously with the preparation of known compounds.
Hence, synthesis of the compounds of formula Ia, Ib and Ic can
followed 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, N.Y. 1959;
Rood's Chemistry of Carbon Compounds (editor: S. Coffrey): Elsevier
Publishing Company, 1971; J. Fried and J. A. Edwards: Organic
Reactions in Steroid Chemistry, Vol. 1 and 11, Van Nostrand
Reinhold Company, New York, 1972; and especially Dictionary of
Steriods (editors: R. A. Hill; D. N. Kirk; H. L. J. Makin and G. M.
Murphy): Chapmann & 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. In addition, information in all the
above publications (including patent specifications) dealing with
preparation of compounds similar with compounds of formula Ia, Ib
and Ic is incorporated by reference.
[0220] Particularly, the compounds of the present invention may be
synthesised according to the following general procedures:
[0221] Cholesta-5,8-dien-3-ol 1, which is synthesised as described
in the literature [J. Lip. Res. 37, 1529, (1996)], can be oxidised
in an Oppenauer reaction to give cholesta-4,8-dien-3-one 2 (scheme
1). In this reaction, the sterol is treated with a ketone like
acetone, quinone or cyclohexanone in the presence of aluminum
isopropoxide or aluminum tert-butoxide [e.g. J. Chem. Soc. Perkin I
2667 (1994)]. The sterol can also be oxidised with pyridinium
dichromate [vide Synth. Commun. 20 (1990), 1167]. The same
oxidation reaction can be carried out with
cholesta-5,8(14)-dien-3.beta.-ol, which is also synthesised as
described in the literature [J. Lip. Res. 37 (1996), 1529,] to give
cholesta-5,8(14)-dien-3-one. For this ketone, a laborious synthesis
is described in the literature [Bull. Soc. Chim. Fr. 2037, (1971)].
Cholesta-4,7-dien-3-one is synthesised according to literature
procedures [Liebigs Ann. Chem. 542 (1939), 218,] (in scheme 1, the
series with the .DELTA..sup.8-double bond are shown as an example,
analogous reactions have to be performed in the .DELTA..sup.7- and
.DELTA..sup.8(14)-series).
[0222] In the following, only the syntheses in the
.DELTA..sup.8-series are described. The derivatives in the
.DELTA..sup.7 and .DELTA..sup.8(14)-series can be synthesised by a
skilled artisan from the corresponding starting materials in the
same way. 1
[0223] Enone 2 can be treated with different
C.sub.1-C.sub.6-Grignard reagents to give two diastereomeric
alcohols 3 and 4 (with R.sup.3.dbd.C.sub.1-C.sub.6-alkyl), which
are easily separated by column chromatography [e.g. J. Med. Chem.
40 (1997), 61].
[0224] Cholesta-4,8-dien-3-one 2 can be reduced according to well
known literature procedures. Lithium aluminum hydride, sodium
borohydride and diisobutylaluminum hydride are especially useful
[e.g. Liebigs Ann. Chem. 542 (1939), 218]. Two diastereomeric
alcohols of the formulae 3 and 4 (with R.sup.3=hydrogen) can be
obtained and readily separated by column chromatography.
[0225] For the introduction of perfluoroalkyl substituents in
position 3, cholesta-4,8-dien-3-one 2 can be treated with
perfluoroalkyltrialkylsilan- es in the presence of fluoride sources
like tetrabutyl-ammonium fluoride or caesium flouride. The
trifluoromethyl group is preferentially introduced with reagents
like trimethylsilyltrifluoromethane or
triethylsilyltrifluoromethane [J. Org. Chem. 56 (1991), 984; J.
Org. Chem. 54 (1989), 2873].
[0226] Again two diastereomeric alcohols of the formulae 3 and 4
(with R.sup.3=perfluoroalkyl, preferentially: trifluoromethyl) can
be obtained and readily separated by column chromatography.
[0227] The cyanoketones 5 and 6 are available starting from
cholesta-4,8-dien-3-one 2 via a conjugate addition of cyanide
(scheme 2). Different reagents like diethylaluminum cyanide [J.
Org. Chem. 59 (1994), 2766] and some alkali and earth alkali metal
cyanides [Tetrahedron Lett. 28 (1987), 4189; Can. J. Chem. 59
(1981), 1641] can be used in this reaction. 2
[0228] The readily separated cyanoketones 5 and 6 can be reduced
according to well known literature procedures. Lithium aluminum
hydride, sodium borohydride and diisobutyl-aluminum hydride are
used preferentially [e.g. Aust. J. Chem. 35 (1982), 629]. In the
reduction reactions, two diastereomeric alcohols 7 and 8
(R.sup.3.dbd.H), respectively, 9 and 10 (R.sup.3.dbd.H) are
obtained.
[0229] The cyanoketones of the formulae 5 and 6 can also be treated
with Grignard reagents [e.g. Chem. Pharm. Bull. 9 (1961), 854] to
give two diastereomeric tertiary alcohols 7 and 8 or 9 and 10 (with
R.sup.3.dbd.C.sub.1-C.sub.6-alkyl), respectively.
[0230] The hydroxymethyl derivatives of the formulae 12 and 14 can
be synthesised in two step sequences from the cyanoalcohols 8 and
9, respectively (scheme 3). First the cyano group can be reduced
with an electrophilic reducing agent like diisobutylaluminum
hydride to give the corresponding imines, which are hydrolysed in
situ to the carbaldehydes 11 and 13. In the second step, the
carbaldehydes can be further reduced with well known reducing
agents like lithium aluminum hydride, sodium borohydride or
diisobutylaluminum hydride to the desired hydroxymethyl derivatives
[e.g. J. Med. Chem. 39 (1996), 5092]. 3
[0231] The methano derivatives of the formulae 15 and 16 can be
synthesised form the allylic alcohols 3 and 4, respectively (scheme
4). Different variations of the Simmons-Smith reaction can be
employed. As reagents, diiodomethane in the presence of
zinc/copper-couple [e.g. J. Org. Chem. 31 (1966), 3869; J. Med.
Chem. 39 (1996), 4218] as well as chloroiodomethane in the presence
of a diethylzinc solution [J. Am. Chem. Soc. 108 (1986), 6343] can
be used in this cyclopropanation reaction. 4
[0232] 5.beta.-Methylcholest-8-en-3-one 17 can be synthesised from
cholesta-4,8-dien-3-one 2 via a conjugate addition reaction (scheme
5). The methyl group is introduced either with lithium
dimethylcuprate [e.g. Aust. J. Chem. 35 (1982), 629] or with
methyl-Grignard compounds or trimethylaluminum in the presence of
nickel catalysts [e.g. Tetrahedron Lett. 35 (1994), 6075; Synthesis
3 (1995), 317]. 5
[0233] Subsequent reduction of 5.beta.-methylketone 17 is achieved
with different well known reducing agents like lithium aluminum
hydride, sodium borohydride and diisobutylaluminum hydride [e.g.
Aust. J. Chem. 35 (1982), 6291. Two diastereomeric alcohols 18 and
19 (R.sup.3=hydrogen) can be obtained and readily separated by
column chromatography.
[0234] If 5.beta.-methylketone 17 is treated with Grignard
reagents, two diastereomeric tertiary alcohols (18 and 19; with
R.sup.3.dbd.C.sub.1-C.s- ub.6-alkyl) are obtained, which are easily
separated by column chromatography.
[0235] Analogues that combine a 5-cyano-substituent with different
steroidal sidechains can be synthesized by the following general
route (see scheme 6): Starting from lichesterol, which can be
synthesized as described in the literature [J. Chem. Soc. Perkin
Trans. 1 (1981), 2125], an enone can be generated by an oppenauer
oxidation [e.g. J. Chem. Soc. Perkin Trans. 1 (1994), 2667]. A
cyano group can be introduced by conjugate addition [J. Org. Chem.
59 (1994), 2766]. After protection of the 3-ketone as an ketal, the
side chain can be cleaved by ozonolysis and subsequent reductive
work up [Synthesis 3 (1990), 193]. The 22-alcohol can be
transformed to the corresponding tosylate to generate the
appropriate leaving group for the copper catalyzed addition of
grignard reagents. By this addition, different alkyl- and aryl side
chains can be intoduced [Chem. Pharm. Bull. 28 (1980), 606]. After
deprotection of the ketone, the latter can be reduced to the
corresponding .alpha.- and .beta.-alcohols by standard methods.
67
[0236] The compounds of the present invention will influence the
meiosis in oocytes as well as in male germ cells.
[0237] The existence of a meiosis inducing substance in nature has
been known for some time. However, until recently the identity of
the meiosis inducing substance or substances was unknown.
[0238] The prospects of being able to influence the meiosis are
several. According to a preferred embodiment of the present
invention, a compound of formula Ia, Ib or Ic or an ester, salt,
active metabolite or prodrug thereof can be used to stimulate the
meiosis. According to another preferred embodiment of the present
invention, a compound of formula Ia, Ib or Ic or an ester, salt,
active metabolite or prodrug thereof can be used to stimulate the
meiosis in humans. Thus, the compounds of formula Ia, Ib or Ic and
ester, salt, active metabolite or prodrugs thereof are promising as
new fertility regulating agents without the usual side effect on
the somatic cells which are known from the hitherto used hormonal
contraceptives which are based on estrogens and/or gestagens.
[0239] 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 fertilised. 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.
[0240] According to another aspect of this invention, a meiosis
inducing substance of formula Ia, Ib or Ic or an ester, salt,
active metabolite or prodrug thereof 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. Also, when in vitro fertilisation is performed,
better results can be achieved, when a compound of formula Ia, Ib
or Ic or an ester, salt, active metabolite or prodrug thereof is
added to the medium in which the oocytes are cultured.
[0241] 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, administration of a compound of
formula Ia, Ib or Ic or an ester, salt, active metabolite or
prodrug thereof may relieve the problem.
[0242] As an alternative to the method described above,
contraception in females can also be achieved by administration of
a compound of formula Ia, Ib or Ic or an ester, salt, active
metabolite or prodrug thereof which inhibits the meiosis, so that
no mature oocytes are produced. Similarly, contraception in males
can be achieved by administration of a compound of formula Ia, Ib
or Ic or an ester, salt, active metabolite or prodrug thereof which
inhibits the meiosis, so that no mature sperm cells are
produced.
[0243] The route of administration of compositions containing a
compound of formula Ia, Ib or Ic or an ester, salt, active
metabolite or prodrug thereof may be any route which effectively
transports the active compound to its site of action.
[0244] 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 formula Ia, Ib or Ic or an ester, salt, active
metabolite or prodrug thereof in connection with a pharmaceutically
acceptable carrier. For oral use, such compositions are preferably
in the form of capsules or tablets.
[0245] 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 formula Ia, Ib or Ic or ester, salt, active metabolite
or prodrugs thereof 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.
[0246] Pharmaceutical Compositions
[0247] Pharmaceutical compositions comprising a compound of formula
Ia, Ib or Ic or an ester, salt, active metabolite or prodrug
thereof may further comprise 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, methyl cellulose, sodium
carboxymethyl cellulose, low melting waxes and cocoa butter.
[0248] 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
fertilisation. The liquid compositions may contain other
ingredients which are conventionally used in the art, some of which
are mentioned in the list above.
[0249] 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 administration may be provided in the
form of a nasal spray in liquid or powder form.
[0250] The dose of a compound of formula Ia, Ib or Ic to be used
will be determined by a physician and will depend, inter alia, 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.
[0251] Usually, not more than 1000 mg, preferably not more than 100
mg, and in some preferred instances not more than 10 mg, of a
compound of formula Ia, Ib or Ic is to be administered to mammals,
e.g. to man, per day.
[0252] None of the compounds of formula Ia, Ib and Ic have shown to
be toxic when administered to man in an amount of 1000 mg per
day.
[0253] The compounds of formula Ia, Ib or Ic thereof can be
synthesised by methods known per se.
[0254] The present invention is further illustrated by the
following examples which, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, in any
combination thereof, be material for realising the invention in
diverse forms thereof.
EXAMPLE 1
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic Acid
Methyl Ester
[0255] 4,4-Dimethyl-3-oxochol-5-en-24-oic acid methyl ester (19.8
g) (G. Aranda et al., Tetrahedron 43 (1987), 4147) is reduced with
sodium borohydride (9.9 g) in 1900 ml methanol at room temperature.
The solution is stirred for 20 hours. After aqueous work-up,
3.beta.-hydroxy-4,4-dimet- hylchol-5-en-24-oic acid methyl ester
(19.2 g) is isolated. Melting point: 136-138.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.=5.55 (1H, m); 3.67 (3H, s); 3.23
(1H, m).
[0256] 3.beta.-Hydroxy-4,4-dimethylchol-5-en-24-oic acid methyl
ester (19.1 g) is suspended in 390 ml of N,N-dimethylformamide
(hereinafter designated DMF) and 39 g of imidazol and 34.6 g of
tert-butyldimethylsilyl choride is added. The reaction mixture is
stirred at 70.degree. C. for 20 hours. After aqueous work-up, a
crude product 23.9 g is isolated. Crystallization from
tetrahydrofurane/methanol (hereinafter tetrahydrofurane and
methanol are designated THF and MeOH, respectively) yields
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-chol- -5-en-24-oic
acid methyl ester (19.55 g). Melting point: 144-146.degree. C.
.sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.52 (1H, m); 3.66 (3H,
s); 3.18 (1H, m); 0.89 (9H, s); 0.02 (6H, m).
[0257]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethylchol-5-en-24-oic
acid methyl ester (9.75 g) is dissolved in a warm mixture of 122 ml
of benzene and 475 ml of n-hexane,
1,3-dibromo-5,5-dimethylhydantoin (3.95 g) is added and the mixture
is refluxed for 15 minutes. After cooling (fast) and evaporating to
dryness under reduced pressure, 475 ml of o-xylene and 21.7 ml of
quinaldine is added and the mixture is refluxed for 1 hour. After
aqueous work-up and trituration with methanol
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethylchola-5,7-dien-24-oic
acid methyl ester (8.22 g) is isolated. Melting point:
116-124.degree. C. .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.88
(1H, m); 5.52 (1H, m); 3.65 (3H, s); 3.32 (1H, m); 0.89 (9H, s);
0.04 (6H, m).
[0258]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethylchola-5,7-dien-24-oi-
c acid methyl ester (1.0 g) is suspended in a mixture of 25 ml
methanol and 3.4 ml of concentrated hydrogen chloride acid and
refluxed overnight. After evaporation to dryness under reduced
pressure the remanens is triturated with methanol and thereafter
recrystallized with methanol/water to give the title compound (0.47
g). Melting point: 156-157.degree. C. .sup.1H-NMR (CDCl.sub.3, 400
MHz): .delta.=5.36 (1H, s); 3.67 (3H, s); 3.23 (1H, m). MS
(abbreviation for mass spectroscopy): Calculated: 414.6. Found
414.3.
EXAMPLE 2
3.beta.-Hydroxy-4,4-dimethylchola-5,7-dien-24-oic Acid Methyl
Ester
[0259]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethylchola-5,7-dien-24-oi-
c acid methyl ester (0.50 g) is dissolved in 5 ml of dry THF and
0.48 g of tetra-butylammoniumfluoride hydrate and 0.4 g of
pulverised molecular sieve is added and the mixture is stirred for
4 days. After column chromatography and crystallization from
methanol/water and methanol, the title compound (195 mg) is
isolated. Melting point: 124-128.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.=5.91 (1H, m); 5.53 (1H, m); 3.67
(3H, s). 3.4 (1H, m). MS: Calculated: 414.6. Found 414.3.
EXAMPLE 3
30-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic Acid Ethyl
Ester
[0260]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethylchola-5,7-dien-24-oi-
c acid methyl ester (4.0 g) is suspended in a mixture of 60 ml of
96% ethanol, 10.1 ml of benzene and 10.1 ml of concentrated
hydrogen chloride acid and refluxed for 3.5 hours. After aqueous
work-up and column chromatography followed by crystallization from
ethanol the title compound (1.37 g) is isolated. Melting point:
122-124.degree. C. .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35
(1H, s); 4.13 (2H, q); 3.25 (1H, m). 1.25 (3H, t). MS: Calculated:
428.7. Found 428.3.
EXAMPLE 4
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid
[0261] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid ethyl ester (1.9 g) is suspended in a mixture of 190 ml of 96%
ethanol and 63 ml of 1M sodiumhydroxide. The mixture is stirred for
3.5 h at room temperature. After aqueous work-up and
crystallization from ethanol/water, the title compound (1.48 g) is
isolated. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.36 (1H, s);
3.22 (1H, m); 1.03 (3H, s). 1.01 (3H, s); 0.97 (3H, d); 0.83 (3H,
s); 0.81 (3H, s). MS: Calculated: 400.6. Found 400.3.
EXAMPLE 5
30-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic Acid
Cyclohexyl Ester
[0262] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid (0.2 g) is suspended in 10 ml of cyclohexanol, 0.1 ml of boron
trifluoride diethyl etherate is added and the mixture is stirred at
55-60.degree. C. for 20 hours. After evaporation to dryness under
reduced pressure, the remanens is purified by column chromatography
and crystallized from methanol to give the title compound (49 mg).
Melting point: 130-132.degree. C. .sup.1H-NMR (CDCl.sub.3):
.delta.=5.35 (1H, s); 4.75 (1H, m); 3.24 (1H, m). MS: Calculated:
482.8. Found 482.4.
EXAMPLE 6
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-24-one
[0263] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid methyl ester (18.2 g) is suspended in 400 ml of DMF and 14.8 g
of imidazol and 13.0 g of t-butyldimethylsilylchloride is added.
The reaction is stirred at 65.degree. C. for 20 hours. After
aqueous work-up and crystallization from ether/methanol,
3.beta.-tert-butyldimethylsilylo-
xy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic acid methyl ester
(21 g) is obtained. Melting point: 124-125.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.=5.34 (1H, s); 3.68 (3H, s); 3.2 (1H,
m); 0.9 (9H, s); 0.04 (6H, m).
[0264]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid methyl ester (12 g) is dissolved in a mixture of
500 ml THF and 400 ml ethanol. 60 ml 1M sodium hydroxide is added
and the mixture is stirred for 20 hours. After aqueous work-up and
crystallization from ethanol/water,
3.beta.-tert-butyldimethylsilyloxy-4,-
4-dimethyl-5.alpha.-chola-8,14-dien-24-oic acid (11.26 g) is
obtained. Melting point: 181-184.degree. C. .sup.1H-NMR
(CDCl.sub.3, 300 MHz): .delta.=5.34 (1H, s); 3.19 (1H, m); 0.9 (9H,
s); 0.04 (6H, m).
[0265]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (5.0 g) is dissolved in 250 ml of dry
dichloromethane. After cooling to -15.degree. C., 2.14 ml of
N-methylmorpholine and 1.27 ml isobutylchloroformate is added and
the mixture is stirred at -15.degree. C. for 20 minutes, whereupon
0.98 g of N,O-dimethylhydroxy/amine, HCl is added and the mixture
is stirred overnight and the temperature is slowly elevated to room
temperature. After aqueous work-up,
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.-
alpha.-chola-8,14-dien-24-oic acid-N-methoxy-N-methyl amide (5.37
g) is obtained. Melting point: 134-135.degree. C. .sup.1H-NMR
(CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s); 3.69 (3H, s); 3.2 (1H,
m); 3.18 (3H, s); 0.9 (9H, s); 0.04 (6H, m).
[0266]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid-N-methoxy-N-methyl amide (0.57 g) is dissolved in
10 ml of dry THF and added to a solution of 10 mmol
isopropylmagnesium bromide in 10 ml THF, whereupon it was cooled on
an ice-bath. The mixture is stirred for some h and left overnight
at 5.degree. C. After aqueous work-up, column chromatography and
crystallization from methanol,
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-cholesta-8,14-di-
en-24-one (238 mg) is obtained, Melting point: 120-122.degree. C.
.sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s); 3.2 (1H,
m); 2.61 (1H, m); 0.9 (9H, s); 0.04 (6H, m).
[0267]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-cholesta-8-
,14-dien-24-one (0.1 g) is dissolved in 5 ml of ethanol, 0.2 ml of
6N hydrogen chloride is added and the mixture is stirred for 2
days. After aqueous work-up, column chromatography and
crystallization from ethanol/-water, the title compound (64 mg) is
obtained. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.35 (1H, s);
3.24 (1H, m); 2.62 (1H, m). MS: Calculated: 426.7. Found 426.3.
EXAMPLE 7
3.beta.-Hydroxy-4,4,24-trimethyl-5.alpha.-chola-8,14-dien-24-one
[0268]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24 oic acid-N-methoxy-N-methyl amide (0.57 g) is reacted with
methylmagnesiumbromide and hydrolysed with ethanol/HCl following
the procedure outlined in example 6 to give the title compound
(0.20 g). Melting point: 183-185.degree. C. .sup.1H-NMR
(CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s); 3.24 (1H, m); 2.17
(3H, s). MS: Calculated: 398.6. Found 398.3.
EXAMPLE 8
3.beta.-Hydroxy-4,4-dimethyl-24-phenyl-5.alpha.-chola-8,14-dien-24-one
[0269]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24 oic acid-N-methoxy-N-methyl amide (0.57 g) is reacted with
phenylmagnesiumbromide and hydrolysed with ethanol/HCl following
the procedure outlined in example 6 to give the title compound
(0.31 g). Melting point: 196-199.degree. C. .sup.1H-NMR
(CDCl.sub.3, 300 MHz): .delta.=8.0-7.4 (5H, m); 5.35 (1H, s); 3.24
(1H, m); 3.0 (2H, m). MS: Calculated: 460.7. Found 460.3.
EXAMPLE 9
3.beta.-Hydroxy-4,4-dimethyl-24-(3-pentyl)-5.alpha.-chola-8,14-dien-24-one
[0270]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24 oic acid-N-methoxy-N-methyl amide (0.57 g) is reacted with
3-pentylmagnesiumbromide and hydrolysed with ethanol/HCl following
the procedure outlined in example 6 to give the title compound (13
mg). .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s); 3.23
(1H, m). MS: Calculated: 454.7. Found: 454.3.
EXAMPLE 10
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-phenyl Amide
[0271]
30-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-
-24 oic acid (0.50 g) is dissolved in 15 ml of dry dichloromethane.
After cooling to -15.degree. C., 0.188 ml of N-methyl-morpholine
and 0.153 ml of isobutylchloroformiate is added and the mixture is
stirred at -15.degree. C. for 20 minutes, whereupon 0.44 ml of
aniline is added. The mixture is stirred overnight and the
temperature is slowly elevated to room temperature. After aqueous
work-up and crystallization from methanol,
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola--
8,14-dien-24-oic acid-N-phenyl amide (0.431 g) is obtained.
.sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.53 (2H,d); 7.34
(2H,t); 7.17 (1H,s); 7.12 (1H,t); 5.35 (1H,s); 3.21 (1H,m); 0.9
(9H,s); 0.04 (6H,m).
[0272]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid-N-phenyl amide (50 mg) is dissolved in 5 ml of
ethanol, 0.2 ml of 6N hydrogen chloride is added and the mixture is
stirred at room temperature overnight. After aqueous work-up and
crystallization from methanol the title compound is obtained. (36
mg). H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.53 (2H, d); 7.33 (2H,
t); 7.18 (1H, s); 7.12 (1H, t); 5.36 (1H, s); 3.26 (1H, m). MS:
Calculated: 475.7. Found: 475.4.
EXAMPLE 11
30-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic Acid
Amide
[0273]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24 oic acid (1.0 g) is reacted with ammonia and hydrolysed
with ethanol/HCl following the procedure outlined in example 10 to
give the title compound (147 mg). Melting point: 233-235.degree. C.
.sup.1H-NMR (CDCl.sub.3, 400 MHz)): .delta.=5.36 (1H, s); 5.45-5.2
(2H, broad d); 3.25 (1H, m). MS: Calculated: 399.6. Found:
399.3.
EXAMPLE 12
4,4-Dimethyl-24-phenylamino-5.alpha.-chola-8,14-dien-3.beta.-ol
[0274]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24 oic acid (0.15 g) is reacted with aniline following the
procedure outlined in example 10 and reduced with lithium aluminium
hydride (0.15 g) in THF at room temperature. Aqueous work-up and
crystallization from methanol gives
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-24-phenyla-
mino-5.alpha.-chola-8,14-dien (106 mg). .sup.1H-NMR (CDCl.sub.3,
300 MHz)): .delta.=7.17 (2H, t); 6.69 (1H, t); 6.6 (2H, d); 5.35
(1H, s); 3.6 (1H, s); 3.2 (1H, m); 3.09 (2H, m); 0.9 (9H, s); 0.04
(6H, m).
[0275]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-24-phenylamino-5.al-
pha.-chola-8,14-dien (100 mg) is hydrolysed with ethanol/HCl at
50.degree. C. Aqueous work-up and crystallization from ethanol
gives the title compound (53 mg). Melting point: 178-180.degree. C.
.sup.1H-NMR (CDCl.sub.3, 300 MHz)): .delta.=7.19 (2H, t); 6.7 (1H,
t); 6.63 (2H, d); 5.36 (1H, s); 3.6 (1H, s); 3.26 (1H, m); 3.1 (2H,
m). MS: Calculated: 461.7. Found: 461.3.
EXAMPLE 13
4,4-Dimethyl-24-amino-5.alpha.-chola-8,14-dien-3.beta.-ol
[0276] The compound is synthesised following the procedure outlined
in example 12. .sup.1H-NMR (DMSO-d.sub.6, D.sub.2O+HCl):
.delta.=5.3 (1H, s); 3.03 (1H, m); 2.77 (2H, m). MS: Calculated:
385.6. Found: 385.3.
EXAMPLE 14
4,4-Dimethyl-5.alpha.-chola-8,14-dien-3.beta.,24-diol
[0277] 4,4-Dimethyl-3-oxochol-5-en-24-oic acid methyl ester (6.0 g)
(G. Aranda et al., Tetrahedron 43 (1987), 4147) is reduced with
lithium aluminium hydride (3.3 g) in THF (600 ml). After aqueous
work-up and crystallization from diethyl ether,
4,4-dimethyl-5-ene-3.beta.,24-diol (5,21 g) is obtained. Melting
point: 201-202.degree. C. .sup.1H-NMR (CDCl.sub.3, 400 MHz):
.delta.=5.55 (1H, m); 3.61 (2H, m); 3.23 (1H, m). MS: Calculated:
388.6. Found: 388.4.
[0278] A mixture of 4,4-dimethylchol-5-ene-3.beta.,24-diol (57 g),
imidazole (125 g) and tert-butyldimethylsilyl chloride (110.5 g) in
DMF is stirred at 70.degree. C. for 20 hours. After aqueous work-up
and crystallization from methanol,
3.beta.,24-bis(tert-butyldimethylsilyloxy)- -4,4-dimethylchol-5-ene
(87.7 g) is obtained. Melting point: 161-162.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.=5.53 (1H, m); 3.58 (2H, m); 3.21
(1H, m); 0.9 (18H, m); 0.03 (12H, m).
[0279]
3.beta.,24-Bis(tert-butyldimethylsilyloxy)-4,4-dimethylchol-5-ene
(44 g) is dissolved in a warm mixture of hexane (2 l) and benzene
(540 ml). 1,3-Dibromo-5,5-dimethylhydantoin (15.32 g) is added and
the mixture is refluxed for 20 min and then cooled rapidly to room
temperature, and the insoluble material is removed by filtration.
The filtrate is concentrated under reduced pressure and o-xylene (2
l) and quinaldine (84 ml) is added. The mixture is refluxed for 1
hour. After aqueous work-up and trituration with methanol,
3.beta.,24-bis(tert-butyldimethylsilyloxy)-
-4,4-dimethylchola-5,7-diene (39 g) is isolated. Melting point:
98-106.degree. C. .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.9
(1H, m); 5.54 (1H, m); 3.58 (2H, m); 3.35 (1H, m); 0.91 (18H, s);
0.05 (12H, m).
[0280] A mixture of
3.beta.,24-bis(tert-butyldimethylsilyloxy)-4,4-dimethy-
lchola-5,7-diene (18.8 g) in 99.9% ethanol (375 ml), benzene (55
ml) and concentrated hydrochloric acid (55 ml) is heated to reflux
for 4 hours. After standing overnight at room temperature, the
reaction mixture is concentrated under reduced pressure.
Crystallization of the remanens from ethanol/water gives 8.44 g of
the title compound. Melting point: 203-208.degree. C. .sup.1H-NMR
(CDCl.sub.3, 400 MHz): .delta.=5.36 (1H, s); 3.62 (2H, m); 3.23
(1H, m).
EXAMPLE 15
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-aldehyde
[0281] 4,4-Dimethyl-5.alpha.-chola-8,14-dien-3.beta.,24-diol (0.21
g) is dissolved in benzene (21 ml).
Tris(tri-phenylphosphine)ruthenium(II) chloride (1.04 g) is added
and the mixture is stirred at room temperature for 20 hours.
Purification by column chromatography gives the title compound (129
mg). .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=9.79 (1H, s); 5.36
(1H, s); 1.05 (3H, s),1.01 (3H, s); 0.95 (3H, d); 0.82 (3H, s);
0.80 (3H, s).
EXAMPLE 16
4,4-Dimethyl-17.beta.-((1R)-methyl-4-methyl-3-pentenyl)androsta-8,14-dien--
3.beta.-ol
[0282] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.35 (1H, s);
5.18 (1H, t); 3.24 (1H, dd),1.7 (3H, s); 1.6 (3H, s).
EXAMPLE 17
4,4-Dimethyl-5.alpha.-cholesta-14,16,24-triene-3.beta.-ol
[0283] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.98 (1H, m); 5.8
(1H, m); 5.12 (1H, t), 3.24 (1H, m); 1.7 (3H, s); 1.58 (3H, s).
EXAMPLE 18
4,4-Dimethyl-17.beta.-((1R)-methyl-3-methyl-2-butenyl)androsta-8,14-dien-3-
.beta.-ol
[0284] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.35 (1H, s);
4.93 (1H, d); 3.24 (1H, m), 1.69 (3H, s); 1.65 (3H, s).
EXAMPLE 19
(20R)-4,4,20-Trimethyl-21-phenyl-5.alpha.-pregna-8,14-dien-3.beta.-ol
[0285] 19 A: According to Steroids 26 (1975), p. 339-357, the
4,4-dimethylstigmasterole has been synthesised.
[0286] 19 B: Ozonolysis of compound 19A at -70.degree. C. by slowly
introduction of O.sub.3, and following reduction at -70.degree. C.
by sodium-bis-(2-methoxyethoxy) aluminium hydride and further
reduction at a temperature about -30.degree. C. with lithium
aluminium hydride gave afforded
23-nor-4,4-di-methyl-5.alpha.-cholest-5-en-3.beta.,22-diol, which
was diacetylated in pyridine with acetic acid anhydride. (1B).
[0287] 19 C: Compound 19B was isomerised by reflux in ethanol/6M
hydrochloric acid giving
23-nor-4,4-dimethyl-5.alpha.-cholest-8,14-dien-3-
.beta.,22-diol.
[0288] 19 D: Compound 19C was selectively C-22 tosylated by
treatment of p-toluene sulfonyl chloride in pyridine by standing
over night in room temperature. The compound was purified by colom
chromathography and crystalised.
[0289] NMR: H ppm: 0.78 s CH.sub.3; 0.82 s CH.sub.3; 0.98 d
CH.sub.3; 1.02 s CH.sub.3; 2.46 s CH.sub.3-aromat; 3.22 m H 3 a;
5.38 s H15; 7.33 d 2H; 7,78 d 2H.
[0290] This intermediate, 19D, is used in several of the following
examples.
[0291] 19 E: Compound 19D was reacted with phenylmagnesium bromide
catalysed by Li.sub.2CuCl.sub.4 giving the title compound. (Chem.
Pharm. Bull. 28 (1980), p. 606-611; Masuo Monsaki et al.).
[0292] NMR: H ppm: 0.82 d CH.sub.3; 0.83 s CH.sub.3; 1.02 s
CH.sub.3; 1.03 s CH.sub.3; 2,55 m 1H; 2,91 dd 1H; 3.25 m 3 a H; 5.4
s 1H (15); 7.17 m 2H; 7.28 m 3H.
EXAMPLE 20
(20R)-4,4,20-Trimethyl-21-(3-methylphenyl)-5.alpha.-pregna-8,14-dien-3.bet-
a.-ol
[0293] According to example 19 (compound 19 D) was reacted with
3-methylphenyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the
title compound.
[0294] NMR: H ppm: 0.80 d CH.sub.3; 0.82 2s 2CH.sub.3; 1.0 2s
2CH.sub.3; 2.33 s CH.sub.3-aromat; 3.27 m H3 a 5.40 s 1H; 6.98 m
3H; 7.17 1H.
EXAMPLE 21
(20R)-4,4,20-Trimethyl-21-(4-methylphenyl)-5.alpha.-pregna-8,14-dien-3.bet-
a.-ol
[0295] According to example 19 (compound 19 D) was reacted with
4-methylphenyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the
title compound.
[0296] NMR: H ppm: 0.84 s, 2d 3 CH.sub.3; 1.04 2s 2CH.sub.3; 2.33 s
CH.sub.3-aromat; 3.25 m H3 a; 5.40 s 1H; 7.05 m 4H.
EXAMPLE 22
(20R)-4,4,20-Trimethyl-21-(2-methylphenyl)-5.alpha.-pregna-8,14-dien-3.bet-
a.-ol
[0297] According to example 19 (compound 19 D) was reacted with
2-methylphenyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the
title compound. NMR: H ppm: 0.84 s+2d, 3 CH.sub.3; 1.04 2s
2CH.sub.3; 2.32 s CH.sub.3-aromat; 3.25 m H3 a; 5.40 s 1H; 7.10 m
4H.
EXAMPLE 23
(20R)-4,4,20-Trimethyl-21-(cyclohexyl)-5.alpha.-pregna-8,14-dien-3.beta.-o-
l
[0298] According to example 19 (compound 19D) was reacted with
cyclohexyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the
title compound.
[0299] NMR: H: mmp: 0.81 s CH.sub.3; 0.82 s CH.sub.3; 0.90 d
CH.sub.3; 1.03 s CH.sub.3; 104 s CH.sub.3; 3.24 s H3 a; 5.37 s
H;
[0300] MS: 424.4.
EXAMPLE 24
(20R)-4,4,20-Trimethyl-22-(phenyl)-5.alpha.-pregna-8,14-dien-3.beta.-ol
[0301] According to example 19 (compound 19 D) was reacted with
tolyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the title
compound.
[0302] NMR: H; ppm: 0.80 s CH.sub.3; 0.82 s CH.sub.3; 1.02 s
CH.sub.3; 1.04 s CH.sub.3; 1.06 d CH.sub.3; 3.25 m H3 a; 5.35 s H;
7.18 m 3H; 7.27 m 2H.
EXAMPLE 25
(20R)-4,4,20-Trimethyl-21-(3-hydroxyphenyl)-5.alpha.-pregna-8,14-dien-3.be-
ta.-ol
[0303] According to example 19 (compound 19 D) was reacted with
3-trimethylsilyloxyphenyl magnesium bromide and Li.sub.2CuCl.sub.4
giving the title compound.
[0304] NMR: H; ppm: 0.80 d CH.sub.3; 0.83 s CH.sub.3; 0.84 s
CH.sub.3; 1.01 s CH.sub.3; 1.03 s CH.sub.3; 3.26 m H3 a; 5.40 s H;
6.66 m 2H; 6.76 m H; 7.16 m H.
[0305] MS: 434.3.
EXAMPLE 26
(20R)-4,4,20-Trimethyl-22-(cyclohexyl)-5.alpha.-pregna-8,14-dien-3.beta.-o-
l
[0306] According to example 19 (compound 19 D) was reacted with
cyclohexylmethyl magnesium bromide and CuLi.sub.2Cl.sub.4 giving
the title compound.
[0307] NMR: H ppm: 0.80 s CH.sub.3; 0.83 s CH.sub.3; 0.91 d
CH.sub.3; 1.01 s CH.sub.3; 1.03 S CH.sub.3; 3.24 m H3 a; 5.35 s
H.
[0308] MS: 483.3.
EXAMPLE 27
24-Nor-4,4-dimethyl-5.alpha.-cholest-8,14-dien-3.beta.-ol
[0309] According to example 19 (compound 19 D) was reacted with
isobutyl magnesium bromide and CuLi.sub.2Cl.sub.4 giving the title
compound. NMR: H; ppm: 0.80 s CH3; 0.84 s CH3; 0.86 d CH3; 0.88 d
CH.sub.3; 0.91 d CH.sub.3; 1.02 s CH.sub.3; 1.04 s CH.sub.3; 3.25 m
H3 a; 5.35 s H.
EXAMPLE 28
27-Nor-4,4-dimethyl-5.alpha.-cholesta-8,14,25-trien-3.beta.-ol
[0310] According to example 19 (compound 19 D) was reacted with
cyclopropylmethyl magnesium bromide and Li.sub.2CuCl.sub.4 giving
the title compound.
[0311] NMR: H; ppm: =0.82 s CH.sub.3; 0.84 S CH.sub.3; 0.95 d
CH.sub.3; 1.02 s CH.sub.3; 1.04 s CH.sub.3; 3.24H3 a; 4.97 dd 2H;
5.37 s 1H; 5.82 m 1H.
EXAMPLE 29
(20R)-4,4,20-Trimethyl-21-(cyclobutyl)-5.alpha.-pregna-8,14-dien-3.beta.-o-
l
[0312] In the reaction of example 28 there was further isolated a
compound which was identified as the title compound. NMR: H; ppm;
0.80 s CH.sub.3; 0.84 s CH.sub.3; 0.89 d CH.sub.3; 1.01 s CH.sub.3;
1.03 s CH.sub.3; 3.25 m H3 a; 5.35 s 1H.
EXAMPLE 30
(20R)-4,4,20-Trimethyl-21-(cyclopentyl)-5.alpha.-pregna-8,14-dien-3.beta.--
ol
[0313] According to example 19 (compound 19 D) was reacted with
cyclopentyl magnesium bromide and Li.sub.2CuCl.sub.4 giving the
title compound.
[0314] NMR: H; ppm: 0.80 s CH.sub.3; 0.82 s CH.sub.3; 0.94 d
CH.sub.3, 1.01 s CH.sub.3; 1.03 s CH.sub.3; 3.25 m H3 a; 5.35 s
H.
EXAMPLE 31
25-Chloro-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-3.beta.-ol
[0315] A mixture of
3.beta.-(tert-butyldimethylsilyloxy)-4,4-dimethylchole-
sta-5,7,25-triene (50 mg), benzene (5 mL), ethanol (5 mL) and
concentrated HCl (2 mL) was heated under reflux for 4 hours. The
reaction was concentrated to half volume under reduced pressure and
water (5 mL) was added. Extraction of the aqueous phase with
dichloromethane and concentration under reduced pressure gave a
residue, which was purified by flash chromatography.
Recrystallisation from ethyl acetate:hexane gave the title compound
as a white solid (35 mg).
[0316] Melting point 131-133.degree. C.
[0317] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.78 (s), 0.80 (s, 3H), 0.92 (d), 0.99
(s), 1.02 (s), 1.52 (s), 1.55 (s), 3.22 (dd), 5.33.
[0318] The mass spectrum showed characteristic peaks at: 446.3
(M.sup.+).
EXAMPLE 32
4,4-Dimethyl-24-(N,N-dimethylamino)-24-cyano-5.alpha.-cholesta-8,14-dien-3-
.beta.-ol
[0319] To a mixture of
3-.beta.-hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-d-
ien-24-aldehyde (10 mg. 0.03 mmol), dimethylamine hydrochloride (18
mg), sodium acetate (16 mg) and molecular seives (100 mg) in
methanol (2 mL) was added sodium cyanoborohydride (8 mg) and the
reaction stirred for 1.5 hours. Dilute HCl (0.2 mL) was added,
followed by sodium bicarbonate solution (0.3 mL) and the seives
were removed by filtration. Concentration of the remaining solution
gave a residue which was purified by flash chromatography to give
the title compound (5 mg).
[0320] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.80 (s, 3H), 0.82 (s, 3H), 0.95 (d),
1.01 (s, 3H), 1.02 (s, 3H), 2.30 (s, 6H), 3.20 (dd, 1H), 3.39-3.49
(m, 1H), 5.32 (s, 1H). The .sup.13C-NMR spectrum (CDCl.sub.3, d)
showed characteristic signals at: 151.3, 142.3, 123.2, 117.5,
117.4, 79.1. The mass spectrum showed characteristic peaks at:
438.4 (M+).
EXAMPLE 33
4,4-Dimethylcholest-8,14,25-trien-3.beta.-ol
[0321] Step 1
[0322] A solution of
(25R)-4,4-dimethyl-5.alpha.-cholesta-8,14-diene-3.bet- a.,26-diol
(69 mg, 0.16 mmol), toluene sulphonyl chloride (45 mg, 2.4 mmol)
and pyridine 1.5 ml was stirred for 6 h at ice bath temperature and
3 h at room temperature. Concentration under reduced pressure and
purification by flash chromatography gave
(25R)-(26-tosyloxy)-4,4-dimethy-
l-5.alpha.-cholesta-8,14-diene-3.beta.-ol (61 mg).
[0323] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.75 (s), 0.82 (s), 0.84 (s), 0.86 (s),
1.00 (s), 1.03 (s), 1.20 (s), 2.42 (s, 3H), 3.20-3.30 (m, 1H),
3.62-3.80 (m, 2H), 5.33 (s, 1H), 7.32 (d, 2H), 7.75 (d, 2H).
[0324] Step 2
[0325]
(25R)-(26-Tosyloxy)4,4-dimethyl-5.alpha.-cholesta-8,14-diene-3.beta-
.-ol (61 mg), sodium iodide (150 mg and dimethylformamide (2 mL)
was heated at 60.degree. C. for 4 hours. Water was added and the
aqueous layer extracted with dichloromethane, concentration under
reduced pressure gave
(25R)-26-iodo-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-3.be-
ta.-ol, which was disssolved in chloroform (5 mL).
1,8-Diazabicyclo(5.4.0)- undec-7-ene was added and the solution was
heated to reflux for 5 h, concentrated under reduced pressure and
purified by flash chromatography. Recrystallisation from methanol
gave the title compound (5 mg).
[0326] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.80 (s), 0.82 (s), 0.93 (d), 1.00 (s),
1.02 (s), 1.51 (s), 1.71 (s), 3.20-3.30 (m, 1H), 4.62 (d, 2H), 5.33
(s, 1H).
EXAMPLE 34
4,4-Dimethyl-17.beta.-((1R)-methyl-4-chlorobutyl)androsta-8,14-dien-3.beta-
.-ol
[0327] Step 1
[0328] A mixture of
4,4-dimethyl-5.alpha.-chola-8,14-diene-3.beta.-24-diol (900 mg, 23
mmol), tosyl chloride (470 mg, 24 mmol) in pyridine (20 mL) was
stirred 6 h at ice bath temperature, poured into water and
extracted with diethyl ether. Drying over magnesium sulphate,
concentration under reduced pressure and purification by flash
chromatography gave
24-toluene-sulphonyloxy-4,4-dimethyl-5.alpha.-chola-8,14-diene-3-ol
(1.1 g).
[0329] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.79 (s, 3H), 0.83 (s, 3H), 0.90 (d,
3H), 1.01 (s, 3H), 1.02 (s, 3H), 2.42 (s, 3H), 3.21 (dd, 1H), 3.52
(m, 2H), 4.01 (t, 2H), 5.33 (s, 1H), 7.35 (d, 2H), 7.80 (d,
2H).
[0330] Step 2
[0331] A mixture of
24-toluenesulphonyloxy-4,4-dimethyl-5.alpha.-chola-8,1-
4-diene-3-ol (45 mg, 0.8 mmol) and lithium chloride (0.6 mmol) in
dimethylformamide (1.5 mL) under nitrogen was heated at 60.degree.
C. for 2 hours. The reaction was then poured into water and
extracted with diethyl ether. Drying over magnesium sulphate,
concentration under reduced pressure and purification by flash
chromatography followed by recrystallised from methanol gave the
title compound, 12 mg.
[0332] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.80 (s, 3H), 0.82 (s, 3H), 0.97 (d,
3H), 1.02 (s, 3H), 1.04 (s, 3H), 3.25 (dd, 1H), 3.52 (m, 2H), 5.36
(s, 1H). The mass spectrum showed characteristic peaks at: 404.2
(M.sup.+).
EXAMPLE 35
4,4-Dimethyl-17.beta.-((1R)-methyl-4-iodobutyl)androsta-8,14-dien-3.beta.--
ol
[0333] A mixture of
24-toluenesulphonyloxy-4,4-dimethyl-5.alpha.-chola-8,1-
4-diene-3-ol (100 mg, 0.18 mmol) and sodium iodide (0.9 mmol) in
dimethylformamide (2 mL) under nitrogen was heated at 60.degree. C.
for 3 hours. The reaction was then poured into water and extracted
with diethyl ether. Drying over magnesium sulphate, concentration
under reduced pressure and purification by flash chromatography
followed by recrystallised from methanol gave the title compound
(40 mg).
[0334] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.80 (s, 3H), 0.82 (s, 3H), 0.95 (d,
3H), 1.01 (s, 3H), 1.02 (s, 3H), 3.10-3.31 (m, 2H), 5.35 (s, 1H).
The mass spectrum showed characteristic peaks at: 496.1 (M+).
EXAMPLE 36
4,4-Dimethyl-17.beta.-((1R)-methylbutyl)androsta-8,14-dien-3.beta.-ol
[0335] To a solution of
24-toluenesulphonyloxy-4,4-dimethyl-5.alpha.-chola-
-8,14-diene-3-ol (100 mg, 0.18 mmol) in diethyl ether (25 mL) was
added lithium aluminium hydride (40 mg, 1.1 mmol) and the reaction
was stirred 16 hours at room temperature. The reaction was then
poured into water and extracted with diethyl ether. Drying over
magnesium sulphate, concentration under reduced pressure and
purification by flash chromatography gave the title compound (41
mg).
[0336] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.80 (s, 3H), 0.82 (s, 3H), 0.87 (t, 3H)
0.95 (d, 3H), 1.00 (s, 3H), 1.02 (s, 3H), 3.20-3.31 (m, 1H), 5.35
(m, 1H). The .sup.13C-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 79.1, 117.8 (C-15), 123.2 (C-14), 142.1
(C-9), 151.4 (C-8). The mass spectrum showed characteristic peaks
at: 370.3 (M.sup.+).
EXAMPLE 37
4,4-Dimethyl-17.beta.-((1R)-methyl-4-cyanobutyl)androsta-8,14-dien-3.beta.-
-ol
[0337] A mixture of
24-toluenesulphonylloxy-4,4-dimethyl-5.alpha.-chola-8,-
14-diene-3-ol (500 mg, 0.5 mmol) and sodium cyanide (90 mg, 2 mmol)
in dimethylsulphoxide (2 mL) under nitrogen was heated at
140.degree. C. for 2.5 hours. The reaction was then poured into
ammonium chloride solution and extracted with dichloromethane.
Drying over magnesium sulphate, concentration under reduced
pressure and purification by flash chromatography gave the title
compound (240 mg).
[0338] The .sup.1H-NMR spectrum (CDCl.sub.3, d) showed
characteristic signals at: 0.81 (s, 3H), 0.83 (s, 3H), 0.97 (d,
3H), 1.00 (s, 3H), 1.02 (s, 3H), 3.18-3.31 (m, 1H), 5.35 (m, 1H).
The mass spectrum showed characteristic peaks at: 395.3
(M.sup.+).
EXAMPLE 38
27-Nor-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-26-oic
Acid Benzyl Ester
[0339] The compound is synthesised following the procedures
outlined in example 52, below.
[0340] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.35 (5H, m);
5.34 (1H, s); 5.11 (2H, s); 3.23 (1H, m).
EXAMPLE 39
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-(methionine Methyl Ester)amide
[0341] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid methyl ester (18.0 g) is suspended in 300 ml of DMF and 14.8 g
of imidazol, and 13 g of tert-butyldimethylsilylchloride is added.
The reaction mixture is stirred at 70.degree. C. for 20 hours.
After aqueous work-up, and crystallisation from diethyl
ether/methanol, 21.0 g of
30-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oi-
c acid methyl ester is isolated. Melting point: 124-125.degree.
C.
[0342] .sup.1H-NMR(CDCl.sub.3, 400 MHz): .delta.=5.34 (1H, s); 3.67
(3H, s); 3.19 (1H, m); 0.9 (9H, s); 0.03 (6H, m).
[0343]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid methyl ester (3.6 g) is dissolved in a mixture of
150 ml of THF, 120 ml of ethanol and 18 ml of 1M sodium hydroxide.
The mixture is stirred for 20 hours at room temperature and 2 hours
at 50.degree. C. After aqueous work-up and crystallisation from
ethanol/water, 2.48 g of
30-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oi-
c acid is isolated.
[0344] .sup.1H-NMR(CDCl.sub.3, 300 MHz): .delta.=5.33 (1H, s); 3.19
(1H, m); 0.9 (9H, s); 0.03 (6H, m).
[0345]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (0.5 g) is dissolved in 10 ml of dry
dichloromethane and 0.213 ml of N-methylmorpholine. After cooling
to -15.degree. C. 0.132 ml of isobutylchloroformate is added and
the mixture is stirred at -15.degree. C. for 20 minutes, whereupon
0.232 g of L-methioninemethyl ester, hydrochloride is added and the
mixture is stirred overnight and the temperature is slowly elevated
to room temperature. After aqueous work-up,
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8-
,14-dien-24-oic acid-N-(methionine methyl ester)amide (0.60 g) is
obtained.
[0346] .sup.1H-NMR(CDCl.sub.3, 300 MHz): .delta.=6.12 (1H, d); 5.34
(1H, s); 4.73 (1H, m); 3.76 (3H, s); 3.2 (1H, m); 2.1 (3H, s); 9.0
(9H, s); 0.03 (6H, m).
[0347]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid-N-(methionine methyl ester)amide (0.20 g) is
suspended in 20 ml methanol, and 0.1 ml 6M hydrogen chloride is
added and the mixture is stirred at 50.degree. C. for 1.5 hours and
at room temperature for 20 hours. After crystallisation by adding
of water, the title compound (165 mg) is obtained. Melting point:
138-141.degree. C.
[0348] .sup.1H-NMR(CDCl.sub.3, 300 MHz): .delta.=6.12 (1H, d); 5.35
(1H, s); 4.73 (1H, m); 3.76 (3H, s); 3.23 (1H, m); 2.1 (3H, s). MS:
Calculated: 545.8. Found 545.3.
EXAMPLE 40
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-(methionine)amide
[0349] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(methionine methyl ester)amide (75 mg) is saponified
overnight in a mixture of 10 ml of methanol, 5 ml of THF, 1 ml of
water, and 0.6 ml of 1M sodium hydroxide. After aqueous work-up and
crystallisation from methanol, the title compound (56 mg) is
obtained.
[0350] .sup.1H-NMR(CDCl.sub.3, 300 MHz): .delta.=6.35 (1H, d); 5.35
(1H, s); 4.7 (1H, m); 3.25 (1H, m); 2.12 (3H, s). MS: Calculated:
531.8. Found 531.8.
EXAMPLE 41
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(4-methylpiperazinyl)amide
[0351]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (0.40 g) is reacted with N-methylpiperazine and
hydrolysed with HCl/ethanol following the procedure outlined in
example 39 to give the title compound (80 mg). Melting point:
189-191.degree. C.
[0352] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s);
3.63 (2H, m); 3.48 (2H, m); 3.24 (1H, m); 2.31 (3H, s). MS:
Calculated: 482.8. Found 482.3.
EXAMPLE 42
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-tert-butylamide
[0353]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (0.50 g) is reacted with tert-butylamine and
hydrolysed with HCl/ethanol following the procedure outlined in
example 39 to give the title compound (204 mg). Melting point:
171-176.degree. C.
[0354] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s);
5.21 (1H, s); 3.24 (1H, m); 1.36 (9H, s). MS: Calculated: 455.7.
Found 455.4.
EXAMPLE 43
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-(isonipecotic Acid Ethyl ester)amide
[0355]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (0.50 g) is reacted with ethylisonipecotate and
hydrolysed with HCl/ethanol following the procedure outlined in
example 39 to give the title compound (85 mg). Melting point:
116-119.degree. C.
[0356] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.35 (1H, s);
4.43 (1H, m); 4.15 (2H, q); 3.82 (1H, m); 3.25 (1H, m); 3.11 (1H,
m); 2.8 (1H, m); 1.28 (3H, t). MS: Calculated: 539.8. Found
539.4.
EXAMPLE 44
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-(isonipecotic Acid)amide
[0357] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid-N-(isonipecotic acid ethyl ester)amide (36 mg) is saponified
overnight in a mixture of 3 ml of ethanol and 0.2 ml of 1M
sodiumhydroxide. After aqueous work-up and crystallisation from
ethanol/water, the title compound (10 mg) is obtained. Melting
point: 228-231.degree. C.
[0358] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.36 (1H, s);
4.45 (1H, m); 3.84 (1H, m); 3.27 (1H, m); 3.16 (1H, m); 2.86 (1H,
m). MS: Calculated: 497.7. Found 497.6.
EXAMPLE 45
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
Acid-N-(phenylalanine methyl ester)amide
[0359]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (0.40 g) is reacted with phenylalanine methyl
ester and hydrolysed with HCl/methanol following the procedures
outlined in example 39 to give the title compound (86 mg). Melting
point: 158-160.degree. C.
[0360] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.27 (3H, m);
7.09 (2H, m); 5.86 (1H, d); 5.35 (1H, s); 4.89 (1H, m); 3.73 (3H,
s); 3.25 (1H, m); 3.13 (2H, m). MS: Calculated: 561.8. Found
561.5.
EXAMPLE 46
3.beta.-Hydroxy-4,4-dimethylchola-5,7-dien-24-oic Acid
[0361] 3.beta.-Hydroxy-4,4-dimethylchola-5,7-dien-24-oic acid
methyl ester (120 mg) is saponified in a mixture of 15 ml of
methanol, 15 ml of THF and 0.7 ml of 1M sodium hydroxide at
50.degree. C. After aqueous work-up and crystallisation from
methanol, the title compound (76 mg) is isolated. Melting point:
210-213.degree. C.
[0362] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.92 (1H, d);
5.55 (1H, m); 3.38 (1H, m). MS: Calculated: 400.6. Found 400.2.
EXAMPLE 47
3.beta.-Hydroxy-4,4-dimethylchola-5,7-dien-24-oic Acid-N-dimethyl
Amide
[0363]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethylchola-5,7-dien-24-oi-
c acid methyl ester (3.5 g) is saponified and reacted with
dimethylamine following the procedure outlined in example 39. The
tert-butyldimethylsilyl protecting group is split of by treatment
with tetra-butylammonium-fluoride hydrate according to the
procedure outlined in example 2 to give the title compound.
[0364] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.92 (1H, d);
5.54 (1H, m); 3.4 (1H, m); 3.03 (3H, s); 2.96 (3H, s). MS:
Calculated: 427.7. Found 427.4.
EXAMPLE 48
4,4-Dimethyl-24-acetamido-5.alpha.-chola-8,14-dien-3.beta.-ol
[0365]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid (5.0 g) is reacted with ammonia following the
procedure outlined in example 39 and reduced with lithium aluminium
hydride (3.0 g) in THF at room temperature. Aqueous work-up and
crystallisation from methanol gives 2.6 g of
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-2-
4-amino-5.alpha.-chola-8,14-diene.
[0366] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.34 (1H, s); 3.2
(1H, m); 3.0 (2H, broad s); 2.7 (1H, m); 0.9 (9H, s); 0.03 (6H, m).
MS: Calculated: 499.9. Found 499.4.
[0367]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-24-amino-5.alpha.-c-
hola-8,14-diene (0.50 g) is acetylated in a mixture of 20 ml of
pyridine and 7 ml of acetic anhydride and hydrolysed with
HCl/ethanol. After aqueous work-up and crystallisation from
ethanol/water, the title compound (0.24 g) is isolated. Melting
point: 219-221.degree. C.
[0368] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.43 (1H, s);
5.35 (1H, s); 3.23 (3H, m); 1.98 (3H, s). MS: Calculated: 427.7.
Found 427.4.
EXAMPLE 49
4,4-Dimethyl-24-acetoxy-5.alpha.-chola-8,14-dien-3.beta.-ol
[0369]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-oic acid methyl ester (5.29 g) is reduced with lithium
aluminium hydride (1.75 g) in 300 ml of THF at room temperature.
After aqueous work-up and crystallisation from ethanol/water, 4.58
g of
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-dien--
24-ol is isolated.
[0370] .sup.1H-NMR (CDCl.sub.3, 400 MHz): .delta.=5.35 (1H, s);
3.65 (2H, m); 3.2 (1H, m); 0.9 (9H, s); 0.03 (6H, m).
[0371]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-ol (150 mg) is acetylated in a mixture of 2 ml of pyridine
and 1 ml of acetic anhydride. The tert-butyldimethylsilyl
protecting group is split of by treatment with tetra-butylammonium
fluoride hydrate according to the procedure outlined in example 2.
After column chromatography and crystallisation from acetone/water,
the title compound (36 mg) is obtained. .sup.1H-NMR (CDCl.sub.3,
300 MHz): .delta.=5.35 (1H, s); 4.05 (2H, m); 3.23 (1H, m); 2.04
(3H, s). MS: Calculated: 428.7. Found 428.3.
EXAMPLE 50
4,4-Dimethyl-24-methoxy-5.alpha.-chola-8,14-dien-3.beta.-ol
[0372]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-ol (100 mg) is methylated with sodium hydride 60% (16 mg)
and methyl iodine (0.125 ml) in 1 ml of DMF. The protecting
tert-butyldimethylsilyl group is split of by treatment with
HCl/ethanol. After aqueous work-up and crystallisation from
methanol/water, the title compound (5 mg) is isolated.
[0373] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s);
3.36 (2H, m); 3.32 (3H, s); 3.23 (1H, m). MS: Calculated: 400.7.
Found 400.3.
EXAMPLE 51
4,4-Dimethyl-24-benzyloxy-5.alpha.-chola-8,14-dien-3.beta.-ol
[0374] The compound is synthesised following the procedure in
example 50. Melting point: 114-115.degree. C.
[0375] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=7.38-7.23 (5H,
m); 5.35 (1H, s); 4.51 (2H, s); 3.45 (2H, m), 3.24 (1H, m). MS:
Calculated: 476.7. Found: 476.3.
EXAMPLE 52
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic Acid
Benzyl Ester
[0376] 3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-chola-8,14-dien-24-oic
acid (100 mg) is suspended in 5 ml of dry DMF. 811 mg of cesium
carbonate and 0.29 ml of benzyl chloride is added and the mixture
is stirred at 50.degree. C. overnight. After aqueous work-up,
column chromatography and crystallisation from acetone/water 55 mg
of the title compound is obtained. Melting point: 118-119.degree.
C.
[0377] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=7.35 (5H, m);
5.35 (1H, s); 5.12 (2H, s); 3.23 (1H, m). MS: Calculated: 490.7.
Found: 490.3.
EXAMPLE 53
26,27-Diethyl-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-26,-
27-dioate
[0378]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-chola-8,14-
-dien-24-ol (4.0 g) is dissolved in 80 ml of dry pyridine at
0.degree. C. 3.04 g of p-toluenesulphonyl chloride is added and the
mixture is stirred at 5.degree. C. for 20 hours and at room
temperature for 5 hours. After aqueous work-up and crystallisation
from methanol, 3.01 g of
3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-24-p-toluenesulphonyloxy--
5.alpha.-chola-8,14-diene is obtained.
[0379] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=7.79 (2H, d);
7.34 (2H, d); 5.33 (1H, s); 4.02 (2H, m); 3.19 (1H, m); 2.43 (3H,
s); 0.9 (9H, s); 0.03 (6H, m).
[0380]
3.beta.-tert-Butyldimethylsilyloxy-4,4-dimethyl-24-p-toluenesulphon-
yloxy-5.alpha.-chola-8,14-diene (0.50 g) is added to a mixture of
0.58 ml of diethyl malonate and 137 mg of sodium hydride (60%) in
10 ml of dry THF at -70.degree. C. Stirring at -70.degree. C. for
15 minutes, whereupon elevating of the temperature and reflux for 9
hours. After aqueous work-up and crystallisation from
ethanol/water, 0.432 g of
26,27-diethyl-3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alpha.-ch-
olesta-8,14-dien-26,27-dioate is obtained.
[0381] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.33 (1H, s);
4.21 (4H, q); 3.32 (1H, t); 3.2 (1H, m); 0.9 (9H, s); 0.03 (6H, m).
MS: Calculated: 643.0. Found: 642.4.
[0382]
26,27-Diethyl-3.beta.-tert-butyldimethylsilyloxy-4,4-dimethyl-5.alp-
ha.-cholesta-8,14-dien-26,27-dioate (0.41 g) is dissolved in 40 ml
of ethanol and 0.5 ml of 6M hydrogen chloride. Stirring at room
temperature for 48 h and 40.degree. C. for 1 hour. The product is
crystallised by adding of water and recrystallised from ethanol to
give 141 mg of the title compound. Melting point: 105-106.degree.
C.
[0383] .sup.1H-NMR (CDCl.sub.3, 300 MHz): .delta.=5.35 (1H, s); 4.2
(4H, q); 3.33 (1H, t); 3.25 (1H, m); 1.25 (6H, t). MS: Calculated:
528.8. Found: 528.4.
EXAMPLE 54
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-26,27-dioic
Acid
[0384]
26,27-Diethyl-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-d-
ien-26,27-dioate (95 mg) is saponified in a mixture of 5 ml of 96%
ethanol, 5 ml of THF and 5 ml of 1M sodium hydroxide at room
temperature. After aqueous work-up, the title compound is isolated
(60 mg). Melting point: 188-190.degree. C.
[0385] .sup.1H-NMR (DMSO-d.sub.6, 400 MHz): .delta.=12.63 (2H, s);
5.3 (1H, s); 4.37 (1H, s); 3.2 (1H, t); 3.03 (1H, m). MS:
Calculated: 472.7. Found: 473.2 [M+H].sup.+.
EXAMPLE 55
27-Nor-3.beta.-hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-26-oic
Acid
[0386]
3.beta.-Hydroxy-4,4-dimethyl-5.alpha.-cholesta-8,14-dien-26,27-dioi-
c acid (535 mg) is suspended in 30 ml of o-xylene and refluxed
overnight. After evaporation of o-xylene, the title compound is
isolated by crystallisation from methanol/ether. Yield: 338 mg.
[0387] .sup.1H-NMR (DMSO-d.sub.6, 400 MHz): .delta.=11.95 (1H, s);
5.28 (1H, s); 4.35 (1H, m); 3.0 (1H, m). MS: Calculated: 428.7.
Found: 428.2.
EXAMPLE 56
Synthesis of the Intermediate cholesta-4,8-dien-3-one
[0388] A solution of 2.20 g cholesta-5,8-dien-3.beta.-ol in 27 ml
toluene and 6 ml cyclohexanone is refluxed for 10 minutes in a
Dean-Stark apparatus. 0.57 g aluminum isopropoxide are added and
the reaction mixture is refluxed for 30 minutes. After cooling and
addition of sulfuric acid (2 N), the resulting mixture is extracted
with ethyl acetate. The organic layer is separated, washed with
saturated sodium bicarbonate solution and 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 1.53 g cholesta-4,8-dien-3-one as a white
solid.
[0389] .sup.1H-NMR (CDCl.sub.3): .delta.=0.68 (s, 3H, H-18); 0.87
(2.alpha.d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.35
(s, 3H, H-19); 5.77 (s, 1H, H-4).
[0390] Cholesta-4,8(14)-dien-3-one is synthesised from
cholesta-5,8(14)-dien-3.beta.-ol in the same way.
Cholesta-4,7-dien-3-one is synthesised according to literature
procedures [Just. Liebigs Ann. Chem. 542 (1939), 218,].
EXAMPLE 57
Scheme 1
Cholesta-4,8-dien-3.alpha.-ol and cholesta-4,8-dien-3.beta.-ol
[0391] To a solution of 520 mg cholesta-4,8-dien-3-one in 45 ml
tetrahydrofuran 1.70 ml of a L-Selectride solution (1 N, in
tetrahydofuran) are added dropwise at -75.degree. C. The reaction
mixture is warmed to room temperature within 4 hours, poured into
hydrochloric acid (1 N) 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 ethyl
acetate to give 286 mg cholesta-4,8-dien-3.beta.-ol and 20 mg
cholesta-4,8-dien-3.alpha.-ol as white solids.
Cholesta-4,8-dien-3.alpha.-ol
[0392] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.86
(2.times.d, J=7 Hz, 6H, H-26/27); 0.92 (d, J=7 Hz, 3H, H-21); 1.11
(s, 3H, H-19); 4.04 (m, 1H, H-3); 5.47 (d, H=5 Hz, 1H, H-4).
Cholesta-4,8-dien-3.beta.-ol
[0393] .sup.1H-NMR (CDCl.sub.3): .delta.=0.64 (s, 3H, H-18); 0.86
(2.alpha.d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21); 1.23
(s, 3H, H-19); 2.47 (m, 1H); 4.19 (m, 1H, H-3); 5.32 (s, 1H,
H-4).
[0394] Cholesta-4,7-dien-3.alpha.-ol and
cholesta-4,7-dien-3.beta.-ol are synthesised according to
literature procedures [Just. Liebigs. Ann. Chem. 542 (1939),
218].
EXAMPLE 58
Scheme 2
a) 5-Cyano-5.beta.-cholest-8-en-3-one and
5-cyano-5.alpha.-cholest-8-en-3-- one
[0395] 30 ml of a diethylaluminum cyanide solution (1 N, in
toluene) are added to a solution of 3.82 g cholesta-4,8-dien-3-one
in 60 ml tetrahydrofuran at 0.degree. C. The reaction mixture is
warmed to room temperature and stirred for 4 hours. 20 ml of a
sodium hydroxide solution (1 N) are added before the mixture is
extracted with diethyl ether. The organic layer is separated, 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 1.46 g 5-cyano-5.beta.-cholest-8-en-3-one
and 1.76 g 5-cyano-5.alpha.-cholest-8-- en-3-one as pale yellow
crystals.
5-Cyano-5.beta.-cholest-8-en-3-one
[0396] .sup.1H-NMR (CDCl.sub.3): .delta.=0.69 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.95 (d, J=7 Hz, 3H, H-21); 1.43
(s, 3H, H-19); 2.63 (m, 1H).
5-Cyano-5.alpha.-cholest-8-en-3-one
[0397] .sup.1H-NMR (CDCl.sub.3): .delta.=0.64 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.95 (d, J=7 Hz, 3H, H-21); 1.37
(s, 3H, H-19); 2.52 (m, 2H).
[0398] 5-Cyano-5,-cholest-7-en-3-one [which is known from the
literature: Aust. J. Chem. 35 (1982), 629],
5-cyano-5.alpha.-cholest-7-en-3-one,
5-cyano-50-cholest-8(14)-en-3-one and
5-cyano-5.alpha.-cholest-8(14)-en-3- -one are synthesised from the
corresponding starting materials in the same way.
b) 5-Cyano-5.alpha.-cholest-8-en-3.beta.-ol and
5-cyano-5.alpha.-cholest-8- -en-3.alpha.-ol
[0399] 327 mg sodium borohydride are added to a solution of 1.76 g
5-cyano-5.alpha.-cholest-8-en-3-one in 200 ml ethanol at room
temperature. The reaction mixture is stirred for 4 hours. After
addition of hydrochloric acid (1 N) the resulting mixture is
extracted with dichloromethane. The organic layer is separated,
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 0.36 mg
5-cyano-5.alpha.-cholest-8-en-3.beta.-ol and 1.00 g
5-cyano-5.alpha.-cholest-8-en-3.alpha.-ol as white solids.
5-Cyano-5.alpha.-cholest-8-en-3.beta.-ol
[0400] .sup.1H-NMR (CDCl.sub.3): .delta.=0.59 (s, 3H, H-18); 0.86
(2.times.d, J=7 Hz, 6H, H-26/27); 0.92 (d, J=7 Hz, 3H, H-21); 1.08
(s, 3H, H-19); 4.12 (bm, 1H, H-3).
5-Cyano-5.alpha.-cholest-8-en-3.alpha.-ol
[0401] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.89
(2.times.d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.06
(s, 3H, H-19); 4.11 (bm, 1H, H-3).
[0402] 5-Cyano-5.alpha.-cholest-7-en-3.beta.-ol,
5-cyano-5.alpha.-cholest-- 7-en-3.alpha.-ol,
5-cyano-5.beta.-cholest-7-en-3.alpha.-ol,
5-cyano-5.beta.-cholest-8-en-3.beta.-ol,
5-cyano-5.beta.-cholest-8-en-3.a- lpha.-ol,
5-cyano-5.alpha.-cholest-8(14)-en-3.beta.-ol,
5-cyano-5.alpha.-cholest-8(14)-en-3.alpha.-ol,
5-cyano-5.beta.-cholest-8(- 14)-en-3.beta.-ol and
5-cyano-5.beta.-cholest-8(14)-en-3.alpha.-ol are synthesised in the
same way.
EXAMPLE 59
Scheme 3
a) 3.beta.-Hydroxy-5.alpha.-cholest-8-ene-5-carbaldehyde
[0403] 4.66 ml of a diisobutylaluminum hydride solution (1.2 N, in
toluene) are added to a solution of 230 mg
5-cyano-5.alpha.-cholest-8-en-- 3.beta.-ol in 18 ml toluene at
-10.degree. C. The reaction mixture is stirred for 3 hours before
3.6 ml sulfuric acid (1 N) are added. After refluxing for one hour
the resulting mixture is cooled to room temperature, diluted with
water and extracted with dichloromethane. The organic layer is
separated, dried over anhydrous sodium sulphate and filtered. After
evaporation of the solvent 220 mg 3.beta.-hydroxy-5.alpha-
.-cholest-8-ene-5-carbaldehyde are obtained as a white solid.
[0404] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.86
(2.times.d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7 Hz, 3H, H-21); 1.17
(s, 3H, H-19); 2.23 (m, 2H); 3.61 (m, 1H, H-3); 9.86 (s, 1H,
5-CHO)
b) 5-(Hydroxymethyl)-5.alpha.-cholest-8-en-3.beta.-ol
[0405] A suspension of 56 mg lithiumaluminum hydride in 5 ml
tetrahydrofuran is added to a solution of 200 mg
3.beta.-hydroxy-5.alpha.- -cholest-8-en-5-carbaldehyde in 20 ml
tetrahydrofuran at room temperature. After being heated to
50.degree. C. for two hours the reaction mixture is cooled to room
temperature. 0.06 ml water, 0.06 ml of a sodium hydroxide solution
(1 N) and 0.18 ml water are added subsequently. The resulting
suspension is stirred for 15 minutes and filtered over anhydrous
sodium sulphate. After evaporation of the solvent the residue is
crystallized from ethylacetate to give 80 mg
5-(hydroxymethyl)-5.alpha.-cholest-8-en-3- .beta.-ol as a white
solid.
[0406] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21); 1.16
(s, 3H, H-19); 3.58 (s, 2H, 5-CH.sub.2OH); 3.97 (m, 1H, H-3).
[0407] 5-(Hydroxymethyl)-5.alpha.-cholest-7-en-3.beta.-ol,
5-(hydroxymethyl)-5.beta.-cholest-8-en-3.alpha.-ol and
5-(hydroxymethyl)-5.alpha.-cholest-8(14)-en-3.beta.-ol are
synthesised in the same way.
EXAMPLE 60
Scheme 4
3',4.alpha.-Dihydrocyclopropa[4,5]-5.beta.-cholest-8-en-3.beta.-ol
[0408] 300 mg zinc dust and 0.03 ml glacial acetic acid are added
to a solution 5.4 mg cupric acetate in 1.2 ml dimetoxyethane at
room temperature. The mixture is stirred for 30 minutes before 0.01
ml triethylamine are added. After 5 minutes a solution of 100 mg
cholesta-4,8-dien-3b-ol in 0.4 ml dimethoxyethane is added. Then
0.24 ml diiodomethane are added at such a rate that the reaction
temperature does not rise above 40.degree. C. The reaction mixture
is stirred for further 6 hours. After addition of saturated
ammonium chloride solution the resulting mixture is 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 ethyl acetate to give 32 mg
3',4.alpha.-dihydrocyclopropa[4,5]-5.beta.-cholest-8-en-3.beta.-ol
as a white solid.
[0409] .sup.1H-NMR (CDCl.sub.3): .delta.=0.25 (dd, J=9 Hz, 5 Hz,
1H, 4,5-CH.sub.2); 0.63 (s, 3H, H-18); 0.76 (dd, J=5 Hz, 5 Hz, 1H,
4,5-CH.sub.2); 0.87 (2.times.d, J=7 Hz, 6H, H-26/27); 0.94 (d, J=7
Hz, 3H, H-21); 1.11 (s, 3H, H-19); 4.32 (m, 1H, H-3).
EXAMPLE 61
Scheme 5
a) 5-Methyl-5.beta.-cholest-8-en-3-one
[0410] 0.85 ml of a methyllithium solution (1.6 N, in diethyl
ether) are added to a suspension of 130 mg cuprous iodide in
diethyl ether at 0.degree. C. The resulting mixture is stirred for
one hour before a solution of 130 mg cholesta-4,8-dien-3-one in 1
ml diethyl ether is added. After being stirred for 30 minutes 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 ethyl acetate to give
80 mg 5-methyl-5.beta.-cholest-8-en-3-one as a white solid.
[0411] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.86
(2.times.d, J=7 Hz, 6H, H-26/27); 0.90 (s, 3H, 5-CH.sub.3); 0.93
(d, J=7 Hz, 3H, H-21); 1.06 (s, 3H, H-19); 2.42 (m, 1H).
b) 5-Methyl-5.beta.-cholest-8-en-3.alpha.-ol and
5-methyl-5.beta.-cholest-- 8-en-3.beta.-ol
[0412] 0.31 ml of a K-Selectride solution (1 N, in tetrahydrofuran)
are added to a solution of 63 mg
5-methyl-5.beta.-cholest-8-en-3-one in 4.4 ml tetrahydrofuran at
-65.degree. C. After being stirred for 2 hours the reaction mixture
is allowed to warm to room temperature, 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 dichloromethane and
acetone to give 26 mg 5-methyl-50-cholest-8-en-3.alpha.-ol and 21
mg 5-methyl-50-cholest-8-en-30-ol as white solids.
5-Methyl-5.beta.-cholest-8-en-3.alpha.-ol
[0413] .sup.1H-NMR (CDCl.sub.3): .delta.=0.59 (s, 3H, H-18); 0.85
(2.times.d, J=7 Hz, 6H, H-26/27); 0.86 (s, 3H); 0.90 (s, 3H); 0.92
(d, J=7 Hz, 3H, H-21); 3.82 (m, 1H, H-3).
5-Methyl-5.beta.-cholest-8-en-3.beta.-ol
[0414] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.89 (s, 3H, 5-CH.sub.3); 0.93
(d, J=7 Hz, 3H, H-21); 0.99 (s, 3H, H-19); 3.87 (m, 1H, H-3)
5-Methyl-5.beta.-cholest-8(14)-en-3-one,
5-methyl-5B-cholest-8(14)-en-3.b- eta.-ol and
5-methyl-5.beta.-cholest-8(14)-en-3.alpha.-ol are synthesised in
the same way.
EXAMPLE 62
Scheme 1
3.beta.-(Trifluoromethyl)cholesta-4,8-dien-3.alpha.-ol and
3.alpha.-(trifluoromethyl)cholesta-4,8-dien-3.beta.-ol
[0415] 0.2 ml triethylsilyltrifluoromethane and 328 mg
tetrabutylammonium fluoride trihydrate are added to a solution of
200 mg cholesta-4,8-dien-3-one in 10 ml tetrahydrofuran at room
temperature. After being stirred for 2 hours the reaction mixture
is diluted with ethyl acetate, washed with water and 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 45 mg
3.beta.-(trifluoromethyl)cholesta-4,8-dien-3.alpha.-ol as a
colourless oil and 170 mg
3.alpha.-(trifluormethyl)cholesta-4,8-dien-3.beta.-ol as a white
solid.
3.beta.-(Trifluoromethyl)cholesta-4,8-dien-3.alpha.-ol
[0416] .sup.1H-NMR (CDCl.sub.3): .delta.=0.65 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21); 1.18
(s, 3H, H-19); 2.41 (m, 1H,); 5.39 (s, 1H, H-4).
3.alpha.-(Trifluoromethyl)cholesta-4,8-dien-3.beta.-ol
[0417] .sup.1H-NMR (CDCl.sub.3): .delta.=0.64 (s, 3H, H-18); 0.87
(2.times.d, J=7 Hz, 6H, H-26/27); 0.93 (d, J=7 Hz, 3H, H-21); 1.25
(s, 3H, H-19); 2.43 (m, 1H,); 5.25 (s, 1H, H-4).
EXAMPLES 63+64
(20R)-5-Cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
and
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
a) Ergosta-4,8,22-trien-3-one
[0418] 1.90 g ergosta-5,8,22-trien-30-ol (lichesterol) were treated
with 0.50 g aluminum-tris-iso-propylate as described in example 56.
Column chromatography gave 1.54 g ergosta-4,8,22-trien-3-one as a
white solid.
[0419] .sup.1H-NMR (CDCl.sub.3): .delta.=0.69 (s, 3H, H-18);
0.82-1.05 (4.times.d, 4.times.Me); 1.36 (s, 3H, H-19); 5.22 (m, 2H,
H-22/23); 5.77 (s, 1H, H-4).
b) 5-Cyano-5.alpha.-ergosta-8,22-dien-3-one
[0420] 1.54 g ergosta-4,8,22-trien-3-one were treated with 11.73 ml
diethylaluminum cyanide solu-tion (1 N, toluene) as described in
example 58. After aqueous work up and chromatography 0.90 g
5-cyano-5.alpha.-ergosta-8,22-dien-3-one were isolated as a white
solid (beside the corresponding 5.beta.-cyano-compound).
[0421] .sup.1H-NMR (CDCl.sub.3): .delta.=0.66 (s, 3H, H-18);
0.82-1.05 (4.times.d, 4.times.Me); 1.28 (s, 3H, H-19); 5.21 (m, 2H,
H-22/23).
c)
5-Cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-ergosta-8,22-diene
[0422] A mixture of 900 mg
5-cyano-5.alpha.-ergosta-8,22-dien-3-one, 0.97 ml ethylene glycol,
25 mg p-toluenesulfonic acid in 20 ml toluene was refluxed at a
dean-stark-trap for 2 hours. After cooling, the reaction mixture
was poured into saturated sodium bicarbonate solution, extracted
with ethyl acetate and washed with water. The combined organic
extracts were dried and evaporated to give 900 mg
5-cyano-3-(spiro-2',5'-dioxa-cyc-
lopentyl)-5.alpha.-ergosta-8,22-diene as a white solid.
[0423] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18);
0.82-1.05 (4.times.d, 4.times.Me); 1.11 (s, 3H, H-19); 3.90-4.15
(m, 4H, 3-ketal); 5.20 (m, 2H, H-22/23).
d)
(20R)-5-Cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pregn-8-ene-20-
-methanol
[0424] 450 mg
5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-ergosta-8-
,22-diene were treated with ozone as described in example 19b.
After reductive work up, 172 mg
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-
-5.alpha.-pregn-8-ene-20-methanol were isolated as a white
solid.
[0425] .sup.1H-NMR (CDCl.sub.3): .delta.=0.65 (s, 3H, H-18); 1.06
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 3.35 (m, 1H, H-22); 3.66
(m, 1H, H-22); 3.90-4.12 (m, 4H, 3-ketal).
e)
(20R)-5-Cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pregn-8-ene-20-
-methanol-4-methylbenzene Sulfonate
[0426] 1.13 g (20R)-5-cyano-5.alpha.-pregn-8-ene-20-methanol was
treated with 869 mg p-toluenesulfonyl chloride as described in
example 19 d. After aqueous work up and column chromatography, 1.19
g
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pregn-8-ene-20-m-
ethanol-4-methylbenzene sulfonate were isolated.
[0427] .sup.1H-NMR (CDCl.sub.3): .delta.=0.59 (s, 3H, H-18); 1.00
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 3.70-4.12 .mu.m, 4H
(3-ketal)+2H(H-22)].
f)
(20R)-5-Cyano-21-cyclohexyl-20-methyl-3-(spiro-2',5'-dioxa-cyclopentyl)-
-5.alpha.-pregn-8-ene
[0428] 258 mg
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pre-
gn-8-ene-20-methanol-4-methylbenzene sulfonate were treated with
cyclohexyl magnesium bromide analoguously to example 19e. After
column chromatography, 147 mg
(20R)-5-cyano-21-cyclohexyl-20-methyl-3-(spiro-2',-
5'-dioxa-cyclopentyl)-5.alpha.-pregn-8-ene were isolated as a pale
yellow solid.
[0429] .sup.1H-NMR (CDCl.sub.3): .delta.=0.62 (s, 3H, H-18); 0.90
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 3.90-4.12 (m, 4H,
3-ketal).
g)
(20R)-5-Cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3-one
[0430] A mixture of 128 mg
(20R)-5-cyano-21-cyclohexyl-20-methyl-3-(spiro--
2',5'-dioxa-cyclopentyl)-5.alpha.-pregn-8-ene, 31 mg amberlyste 15
and 8 ml acetone was stirred at room temperature for 20 hours.
After filtration and evaporation of the solvent 94 mg
(20R)-5-cyano-21-cyclohexyl-20-methy- l-5.alpha.-pregn-8-en-3-one
were isolated.
[0431] .sup.1H-NMR (CDCl.sub.3): .delta.=0.67 (s, 3H, H-18); 0.90
(d, J=7 Hz, 3H, H-21); 1.27 (s, 3H, H-19); 2.55 (pseudo-s, 2H).
h)
(20R)-5-Cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
and
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
[0432] 90 mg
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3-o- ne
were treated with 33 mg sodium borohydride as described in example
58b. After column chromatography, 15 mg
(20R)-5-cyano-21-cyclohexyl-20-methyl--
5.alpha.-pregn-8-en-3.beta.-ol and 25 mg
(20R)-5-cyano-21-cyclohexyl-20-me-
thyl-5.alpha.-pregn-8-en-3.alpha.-ol were isolated.
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
[0433] .sup.1H-NMR (CDCl.sub.3): .delta.=0.63 (s, 3H, H-18); 0.93
(d, J=7 Hz, 3H, H-21); 1.11 (s, 3H, H-19); 4.12 (broad-m, 1H,
H-3).
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
[0434] .sup.1H-NMR (CDCl.sub.3): .delta.=0.62 (s, 3H, H-18); 0.92
(d, J=7 Hz, 3H, H-21); 1.06 (s, 3H, H-19); 4.12 (narrow-m, 1H,
H-3).
EXAMPLES 65+66
(20R)-5-Cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
and
(20R)-5-cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
a)
(20R)-5-Cyano-21-phenyl-20-methyl-3spiro-2',5'-dioxa-cyclopentyl)-5.alp-
ha.-pregn-8-ene
[0435] 400 mg
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pre-
gn-8-ene-20-methanol-4-methylbenzene sulfonate (example 63e) were
treated with phenyl magnesium bromide analoguously to example 63f.
After column chromatography, 190 mg
(20R)-5-cyano-21-phenyl-20-methyl-3-(spiro-2',5'-d-
ioxa-cyclopentyl)-5.alpha.-pregn-8-ene were isolated as a white
solid.
[0436] .sup.1H-NMR (CDCl.sub.3): .delta.=0.65 (s, 3H, H-18); 0.84
(d, J=7 Hz, 3H, H-21); 1.12 (s, 3H, H-19); 2.90 (dd, J=12 hz, J=3
Hz, 1H, H-21); 3.90-4.12 (m, 4H, 3-ketal); 7.12-7.30 (m, 5H,
ph).
b) (20R)-5-Cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3-one
[0437] 180 mg
(20R)-5-cyano-21-phenyl-20-methyl-3-(spiro-2',5'-dioxa-cyclo-
pentyl)-5.alpha.-pregn-8-ene were treated with amberlyste 15
.alpha.s described in example 63g. After filtration and evaporation
of the solvent, 164 mg
(20R)-5-cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3-o- ne were
iso-lated.
[0438] .sup.1H-NMR (CDCl.sub.3): .delta.=0.68 (s, 3H, H-18); 0.84
(d, J=7 Hz, 3H, H-21); 1.27 (s, 3H, H-19); 2.55 (pseudo-s, 2H);
2.90 (dd, J=12 hz, J=3 Hz, 1H, H-21); 7.12-7.30 (m, 5H, ph).
c) (20R)-5-Cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
and
(20R)-5-cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
[0439] 152 mg
(20R)-5-cyano-21-cyclohexyl-20-methyl-5.alpha.-pregn-8-en-3-- one
were treated with 50 mg sodium borohydride as described in example
58b. After column chromatography 36 mg
(20R)-5-cyano-21-phenyl-20-methyl-- 5.alpha.-pregn-8-en-3.beta.-ol
and 46 mg (20R)-5-cyano-21-phenyl-20-methyl-
-5.alpha.-pregn-8-en-3.alpha.-ol were isolated.
(20R)-5-Cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.beta.-ol
[0440] .sup.1H-NMR (CDCl.sub.3): .delta.=0.67 (s, 3H, H-18); 0.85
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 2.90 (dd, J=12 hz, J=3
Hz, 1H, H-21); 4.15 (broad-m, 1H, H-3); 7.12-7.30 (m, 5H, ph).
(20R)-5-Cyano-21-phenyl-20-methyl-5.alpha.-pregn-8-en-3.alpha.-ol
[0441] .sup.1H-NMR (CDCl.sub.3): .delta.=0.65 (s, 3H, H-18); 0.84
(d, J=7 Hz, 3H, H-21); 1.05 (s, 3H, H-19); 2.90 (dd, J=12 hz, J=3
Hz, 1H, H-21); 4.12 (narrow-m, 1H, H-3); 7.12-7.30 (m, 5H, ph)
EXAMPLES 67+68
5-Cyano-24-nor-5.alpha.-cholest-8-en-3.beta.-ol and
5-cyano-24-nor-5.alpha.-cholest-8-en-3.alpha.-ol.
a)
5-Cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-24-nor-5.alpha.-cholest-8-ene
[0442] 400 mg
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pre-
gn-8-ene-20-methanol-4-methylbenzene sulfonate (example 63e) were
treated with isobutyl magnesium bromide analoguously to example
63f. After column chromatography 206 mg
5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-24-nor-5.-
alpha.-cholest-8-ene were isolated as a white solid.
[0443] .sup.1H-NMR (CDCl.sub.3): .delta.=0.62 (s, 3H, H-18);
0.85-0.95 (3.times.d, J=7 Hz); 1.10 (s, 3H, H-19); 3.88-4.10 (m,
4H, 3-ketal).
b) 5-Cyano-24-nor-5.alpha.-cholest-8-en-3-one
[0444] 188 mg
5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-24-nor-5.alpha.-ch-
olest-8-ene were treated with amberlyste 15 .alpha.s described in
example 63g. After filtration and evaporation of the solvent, 175
mg 5-cyano-24-nor-5.alpha.-cholest-8-en-3-one were isolated.
[0445] .sup.1H-NMR (CDCl.sub.3): .delta.=0.65 (s, 3H, H-18);
0.85-0.95 (3.times.d, J=7 Hz); 1.27 (s, 3H, H-19); 2.55 (pseudo-s,
2H).
c) 5-Cyano-24-nor-5.alpha.-cholest-8-en-3.beta.-ol and
5-cyano-24-nor-5.alpha.-cholest-8-en-3.alpha.-ol
[0446] 173 mg 5-cyano-24-nor-5.alpha.-cholest-8-en-3-one were
treated with 67 mg sodium borohydride as described in example 58b.
After column chromatography, 35 mg
5-cyano-24-nor-5.alpha.-cholest-8-en-30-ol and 64 mg
5-cyano-24-nor-5.alpha.-cholest-8-en-3.alpha.-ol were isolated.
5-Cyano-24-nor-5.alpha.-cholest-8-en-3.beta.-ol
[0447] .sup.1H-NMR (CDCl.sub.3): .delta.=0.60 (s, 3H, H-18);
0.85-0.95 (3.times.d, J=7 Hz); 1.10 (s, 3H, H-19); 4.13 (broad-m,
1H, H-3).
5-Cyano-24-nor-5.alpha.-cholest-8-en-3.alpha.-ol
[0448] .sup.1H-NMR (CDCl.sub.3): .delta.=0.62 (s, 3H, H-18);
0.85-0.95 (3.times.d, J=7 Hz); 1.05 (s, 3H, H-19); 4.10 (broad-m,
1H, H-3).
EXAMPLES 69+70
5-Cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.beta.-ol and
5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.alpha.-ol
a)
5-Cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-24-nor-5.alpha.-cholesta-8,23-
-diene
[0449] 400 mg
(20R)-5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-5.alpha.-pre-
gn-8-ene-20-methanol-4-methylbenzene sulfonate (example 63e) were
treated with the grignard reagent from 1-bromo-2-methylpropene
analoguously to example 63f. After column chromatography, 206 mg
5-cyano-3-(spiro-2',5'-d-
ioxa-cyclopentyl)-24-nor-5.alpha.-cholesta-8,23-diene were isolated
as a white solid.
[0450] .sup.1H-NMR (CDCl.sub.3): .delta.=0.62 (s, 3H, H-18); 0.92
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 1.59, 1.70 (2.times.s,
2.times.3H); 3.90-4.10 (m, 4H, 3-ketal); 5.12 (t, J=7 Hz; 1H,
H-23).
b) 5-Cyano-24-nor-5.alpha.-cholesta-8,23-dien-3-one
[0451] 190 mg
5-cyano-3-(spiro-2',5'-dioxa-cyclopentyl)-24-nor-5.alpha.-ch-
olesta-8,23-diene were treated with amberlyste 15 as described in
example 63g. After filtration and evaporation of the sol-vent, 170
mg 5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3-one were
isolated.
[0452] .sup.1H-NMR (CDCl.sub.3): .delta.=0.60 (s, 3H, H-18); 0.90
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 1.61, 1.72 (2.times.s,
2.times.3H); 2.55 (pseudo-s, 2H); 5.12 (t, J=7 Hz; 1H, H-23).
c) 5-Cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.beta.-ol and
5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.alpha.-ol
[0453] 170 mg 5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3-one were
treated with 67 mg sodium borohydride as described in example 58b.
After column chromatography and HPLC for purification, 8 mg
5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.beta.-ol and 19 mg
5-cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.alpha.-ol were
isolated.
5-Cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.beta.-ol
[0454] .sup.1H-NMR (CDCl.sub.3): .delta.=0.60 (s, 3H, H-18); 0.90
(d, J=7 Hz, 3H, H-21); 1.10 (s, 3H, H-19); 1.59, 1.72 (2.times.s,
2.times.3H); 4.13 (broad-m, 1H, H-3); 5.12 (t, J=7 Hz; 1H,
H-23).
5-Cyano-24-nor-5.alpha.-cholesta-8,23-dien-3.alpha.-ol
[0455] .sup.1H-NMR (CDCl.sub.3): .delta.=0.61 (s, 3H, H-18); 0.90
(d, J=7 Hz, 3H, H-21); 1.05 (s, 3H, H-19); 1.59, 1.72 (2.times.s,
2.times.3H); 4.11 (narrow-m, 1H, H-3); 5.12 (t, J=7 Hz; 1H,
H-23).
EXAMPLE 71
[0456] An agonistic oocyte assay can be performed as follows:
[0457] Oocytes were obtained from immature female mice
(C57BL/6J.times.DBA/2J F1, Bomholtgaard, Denmark) weighing 13-16
grams, that were kept under controlled temperature (20-22.degree.
C.), light (lights on 06.00-18.00) and relative humidity (50-70%).
The mice received an intra-peritoneal injection of 0.2 ml
gonadotropins (Gonal-F, Serono) containing 20 IU FSH and 48 hours
later the animals were killed by cervical dislocation.
[0458] 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 oocytes displaying an intact germinal vesicle
(hereinafter designated GV) were divided in cumulus enclosed
oocytes (hereinafter designated CEO) and naked oocytes (hereinafter
designated NO) and placed in a-minimum essential medium
(.alpha.-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561)
supplemented with 3 mg/ml bovine serum albumin (BSA, Sigma Cat. No.
A-7030), 5 mg/ml human serum albumin (HSA, Statens Seruminstitute,
Denmark), 0.23 mM pyruvate (Sigma, Cat. No S-8636), 2 mM glutamine
(Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 t.sub.g/ml
streptomycin (Flow, Cat No. 16-700). This medium was supplemented
with 3 mM hypoxanthine (Sigma Cat. No. H-9377) and designated
Hx-medium.
[0459] The oocytes were rinsed three times in Hx-medium and oocytes
of uniform size were divided into groups of CEO and NO. CEO and NO
were cultured in 4-well multidishes (Nunclon, Denmark) in which
each well contained 0.4 ml of Hx-medium. One control well (i.e.,
35-45 oocytes cultured in identical medium with no addition of test
compound) was always cultured simultaneously with 3 test wells
(35-45 oocytes per well supplemented with test compound).
[0460] The oocytes were cultured in a humidified atmosphere of 5%
CO.sub.2 in air for 24 hours at 37.degree. C. By the end of the
culture period, the number of oocytes with germinal vesicle
(hereinafter designated GV), germinal vesicle breakdown
(hereinafter designated GVB) and polar bodies (hereinafter
designated PB), respectively, were counted using a stereomicroscope
(Wildt, Leica MZ 12). The percentage GVB, defined as percentage of
oocytes undergoing GVB per total number of oocytes in that well,
was calculated as: % GVB=(number of GVB+number of PB/total number
oocytes).times.100.
[0461] The % PB was defined as percentage of oocytes displaying one
extruded polar body per total number of oocytes in that well.
[0462] The effect of the tested compounds has been indexed against
control level and
4,4-dimethyl-5.alpha.-cholesta-8,14,24-trien-3.beta.-ol
(hereinafter designated FF-MAS) where controls and FF-MAS are
indexed to an effect of 0 and 100, respectively. The relative
effect of the tested compound is calculated as follows:
Relative effect=((test GVB %-control GVB %)/(FF-MAS GVB %-control
GVB %)).times.100.
[0463] Results
1TABLE 1 The mean percentage GVB, the mean percentage PB and mean
Relative Effect of compounds after culture of naked oocytes (NO) in
vitro for 24 hours. Concentration; mean mean mean mikroM % GVB % PB
Relative Effect CONTROL 0 11.9 5.5 0 FF-MAS 10 86.1 28.5 100
Example 4 10 10.5 3.5 -10 Example 6 10 88 11 110 Example 10 10 10 3
-10 Example 21 10 82 29.5 98 Example 23 10 73.5 21.5 98 Example 33
10 70 14.5 105
EXAMPLE 72
[0464] An antagonistic oocyte assay can be performed as
follows:
[0465] Animals
[0466] Oocytes were obtained from immature female mice
(C57BI/6J.times.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.
[0467] Test of Meiosis-Inhibiting Substances in the Oocyte Test
[0468] 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 .alpha.-minimum essential medium (.alpha.-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, Statens Seruminstitut, Denmark), 0.23 mM pyrubate
(Sigma, Cat. No. S-8636), 2 mM glutamine (Flow Cat. No. 16-801),
100 IU/ml penicillin and 100 .mu.g/ml streptomycin (Flow, Cat No.
16-700). This medium was designated Hx-medium.
[0469] Naked oocytes (NO) were rinsed three times in Hx-medium.
4,4-Dimethyl-5.alpha.-cholesta-8,14,24-trien-3.beta.-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 .mu.M 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 the
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.
[0470] Examination of Oocytes
[0471] 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+PB per total number
of oocytes were calculated in the test cultures and in the control
(positive and negative) culture groups. The relative inhibition of
the test compound was calculated by the following formula:
Inhibition of test compound (in percentage)=100-[(GVB.sub.test
compound-GVB.sub.negative control).times.100/(GVB.sub.positive
control-GVB.sub.negative control)].
[0472] In case of a dose response curve, an IC.sub.50 (dose, which
lead to a 50% inhibition) was calculated.
* * * * *