U.S. patent application number 10/705398 was filed with the patent office on 2004-08-05 for steroid derivatives.
This patent application is currently assigned to Arch Development Corporation, an Illinois corporation. Invention is credited to Liao, Shutsung, Song, Ching.
Application Number | 20040152681 10/705398 |
Document ID | / |
Family ID | 26829742 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152681 |
Kind Code |
A1 |
Liao, Shutsung ; et
al. |
August 5, 2004 |
Steroid derivatives
Abstract
Steroid derivatives of this invention interact with nuclear
liver X receptor (LXR) and ubiquitous receptor (UR) and can be used
to treat a variety of LXR- or UR-mediated disorders.
Inventors: |
Liao, Shutsung; (Chicago,
IL) ; Song, Ching; (Chicago, IL) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Arch Development Corporation, an
Illinois corporation
|
Family ID: |
26829742 |
Appl. No.: |
10/705398 |
Filed: |
November 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10705398 |
Nov 10, 2003 |
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09560236 |
Apr 28, 2000 |
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6645955 |
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60131728 |
Apr 30, 1999 |
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60191864 |
Mar 24, 2000 |
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Current U.S.
Class: |
514/169 ;
514/319; 514/408; 514/517; 514/529; 514/559; 514/623 |
Current CPC
Class: |
A61K 31/585 20130101;
A61P 1/16 20180101; A61P 43/00 20180101; C07J 41/00 20130101; A61P
7/06 20180101; A61K 31/575 20130101; C07J 9/00 20130101; A61K
31/565 20130101; A61P 35/02 20180101; C07J 3/00 20130101; A61P 3/10
20180101; A61P 25/28 20180101; A61P 35/00 20180101; C07J 17/00
20130101; A61P 29/00 20180101; A61K 31/57 20130101; A61P 9/10
20180101 |
Class at
Publication: |
514/169 ;
514/319; 514/408; 514/517; 514/559; 514/529; 514/623 |
International
Class: |
A61K 031/56; A61K
031/445; A61K 031/40; A61K 031/255; A61K 031/215; A61K 031/165 |
Goverment Interests
[0002] This invention was made with Government support under NIH CA
58073 from National Institute of Health. The Government may have
certain rights in this invention.
Claims
What is claimed is:
1. A compound of the following formula: 12wherein R.sup.3 is
hydrogen, amino, carboxyl, oxo, halo, sulfonic acid, --O-sulfonic
acid, or alkyl that is optionally inserted with --NH--,
--N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--,
--CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--, or
--N(alkyl)-CO--, and further optionally substituted with hydroxy,
halo, amino, carboxyl, sulfonic acid, or --O-sulfonic acid; each of
R.sup.1, R.sup.2, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid,
--O-sulfonic acid, or alkyl that is optionally inserted with
--NH--, --N(alkyl)-, --O--, --S--, --S--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, or --N(alkyl)-CO--, and further optionally substituted
with hydroxy, halo, amino, carboxyl, sulfonic acid, or --O-sulfonic
acid; each of R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and
R.sup.14, independently, is hydrogen, alkyl, haloalkyl,
hydroxyalkyl, alkoxy, hydroxy, or amino; R is --X--Y-Z, in which X
is a bond, or alkyl or alkenyl, optionally inserted with --NH--,
--N(alkyl)-, --O--, or --S--, and further optionally forming a
cyclic moiety with R.sup.16 and the 2 ring carbon atoms to which
R.sup.16 and R.sup.17 are bonded; Y is --CO--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, --N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl,
and is optionally substituted with hydroxy, alkoxy, amino, halo,
sulfonic acid, --O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being a side chain of an amino acid, and B being
hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each of
R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or alkyl;
and n is 0, 1, or 2; provided that when Z is substituted with
carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z
contains at least one double bond, and that when Y is a bond,
either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl;
or a salt thereof.
2. The compound of claim 1, wherein n is 0.
3. The compound of claim 1, wherein R.sup.3 is amino, carboxyl,
halo, sulfonic acid, --O-sulfonic acid, or alkyl; R.sup.6 is
hydroxy, amino, carboxyl, halo, sulfonic acid, --O-sulfonic acid,
or alkyl; and each of R.sup.3 and R.sup.6, independently, is in the
.alpha.-configuration.
4. The compound of claim 1, wherein R.sup.5 is hydrogen and is in
the .beta.-configuration.
5. The compound of claim 1, wherein R.sup.3 is oxo; each of
R.sup.1, R.sup.2, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, oxo, halo, sulfonic acid, --O-sulfonic acid, or
alkyl.
6. The compound of claim 5, wherein each of R.sup.1, R.sup.2,
R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.15,
R.sup.16, and R.sup.17', independently, is hydrogen, hydroxy, or
oxo; and each of R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and
R.sup.14, independently, is hydrogen or hydroxy; or a salt
thereof.
7. The compound of claim 6, wherein X is a bond or alkyl.
8. The compound of claim 7, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
9. The compound of claim 1, wherein X is a bond or alkyl.
10. The compound of claim 9, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --N.sup.aR.sup.b or --COOR.sup.c
11. The compound of claim 6, wherein Y is --CO--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--NH--,
--NH--CO--, or a bond.
12. The compound of claim 11, wherein Z is alkyl, alkenyl, aryl,
heteroaryl, aralkyl, or heteroaralkyl, and is optionally
substituted with hydroxy, alkoxy, halo, sulfonic acid, carboxyl,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl; or is
--CH(A)-B.
13. The compound of claim 1, wherein Z is alkyl or aryl, each of
which being optionally substituted with hydroxy; or is --CH(A)-B
with A being an amino acid side chain having an aromatic moiety,
and B being --NR.sup.aR.sup.b, or --COOR.sup.c.
14. The compound of claim 1, wherein R.sup.17 contains a straight
chain having 6-20 chain atoms.
15. The compound of claim 14, wherein R.sup.17 contains a straight
chain having 8-16 chain atoms.
16. The compound of claim 1, wherein X is
--CH(CH.sub.3)--CH.sub.2--, Y is a bond, and Z is
--CH.sub.2--CH.dbd.C(R')(CH.sub.3) with R' being hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio.
17. The compound of claim 1, wherein said compound is: 1314
18. A compound of the following formula: 15wherein each of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid,
--O-sulfonic acid, or alkyl that is optionally inserted with
--NH--, --N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, or --N(alkyl)-CO--, and further optionally substituted
with hydroxy, halo, amino, carboxyl, sulfonic acid, or --O-sulfonic
acid; each of R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and
R.sup.14, independently, is hydrogen, alkyl, haloalkyl,
hydroxyalkyl, alkoxy, hydroxy, or amino; R.sup.17 is --X--Y-Z, in
which X is a bond, or alkyl or alkenyl, optionally inserted with
--NH--, --N(alkyl)-, --O--, or --S--, and further optionally
forming a cyclic moiety with R.sup.16 and the 2 ring carbon atoms
to which R.sup.16 and R.sup.17 are bonded; Y is --CO--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, --N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl,
and is optionally substituted with hydroxy, alkoxy, amino, halo,
sulfonic acid, --O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being an amino acid side chain containing an
aromatic moiety, and B being hydrogen, --NR.sup.aR.sup.b, or
--COOR.sup.c wherein each of R.sup.a, R.sup.b, and R.sup.c,
independently, is hydrogen or alkyl; and n is 0, 1, or 2; provided
that when Z is substituted with carboxyl or alkyloxycarbonyl, Y is
a bond and either X or Z contains at least one double bond, and
that when Y is a bond, either X is --NH-alkyl-, --NH-alkenyl-,
--N(alkyl)-alkyl-, --N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-,
--S-alkyl-, or --S-alkenyl-; or Z is substituted with halo,
sulfonic acid, --O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl,
or is alkenyl; or a salt thereof.
19. The compound of claim 18, wherein n is 0.
20. The compound of claim 18, wherein each of R.sup.3 and R.sup.6,
independently, is hydroxy, amino, carboxyl, halo, sulfonic acid,
--O-sulfonic acid, or alkyl, and is in the
.alpha.-configuration.
21. The compound of claim 18, wherein R.sup.5 is hydrogen and is in
the .beta.-configuration.
22. The compound of claim 18, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, oxo, halo, sulfonic acid, --O-sulfonic acid, or alkyl.
23. The compound of claim 22, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, or oxo; and each of R.sup.5, R.sup.8, R.sup.9, R.sup.10,
R.sup.13, and R.sup.14, independently, is hydrogen or hydroxy.
24. The compound of claim 23, wherein X is a bond or alkyl.
25. The compound of claim 24, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
26. The compound of claim 18, wherein X is a bond or alkyl.
27. The compound of claim 26, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
28. The compound of claim 18, wherein Y is --CO--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--NH--,
--NH--CO--, or a bond.
29. The compound of claim 28, wherein Z is alkyl, alkenyl, aryl,
heteroaryl, aralkyl, or heteroaralkyl, and is optionally
substituted with hydroxy, alkoxy, halo, sulfonic acid, carboxyl,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl; or is
--CH(A)-B.
30. The compound of claim 18, wherein Z is alkyl or aryl, each of
which being optionally substituted with hydroxy; or is --CH(A)-B
with A being an amino acid side chain having an aromatic moiety,
and B being --NR.sup.aR.sup.b, or --COOR.sup.c.
31. The compound of claim 18, wherein R.sup.17 contains a straight
chain having 6-20 chain atoms.
32. The compound of claim 31, wherein R.sup.17 contains a straight
chain having 8-16 chain atoms.
33. The compound of claim 18, wherein X is
--CH(CH.sub.3)--CH.sub.2--, Y is a bond, and Z is
--CH.sub.2--CH.dbd.C(R')(CH.sub.3) with R' being hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio.
34. The compound of claim 18, wherein said compound is: 161718
35. A compound of the following formula: 19wherein each of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid,
--O-sulfonic acid, or alkyl optionally inserted with --NH--,
--N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--,
--CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--, or
--N(alkyl)-CO--, and further optionally substituted with hydroxy,
halo, amino, carboxyl, sulfonic acid, or --O-sulfonic acid; each of
R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and R.sup.14,
independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy,
hydroxy, or amino; R.sup.17 is --X--Y-Z, in which X is a bond, or
alkyl or alkenyl, optionally inserted with --NH--, --N(alkyl)-,
--O--, or --S--, and further optionally forming a cyclic moiety
with R.sup.16 and the 2 ring carbon atoms to which R.sup.16 and
R.sup.17 are bonded; Y is --CO--, --SO--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--,
--N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally
substituted with hydroxy, alkoxy, amino, halo, sulfonic acid,
--O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being a side chain of an amino acid, and B being
hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each of
R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or alkyl;
and n is 0, 1, or 2; provided that when Z is substituted with
carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z
contains at least one double bond, and that when Y is a bond,
either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl;
and further provided that at least one of R.sup.3 and R.sup.4,
R.sup.4 and R.sup.5, R.sup.5 and R.sup.6, R.sup.7 and R.sup.8,
R.sup.12 and R.sup.13, and R.sup.15 and R.sup.16, independently, is
deleted to form a double bond; or a salt thereof.
36. The compound of claim 35, wherein at least one of R.sup.3 and
R.sup.4, R.sup.4 and R.sup.5, R.sup.12 and R.sup.13, and R.sup.15
and R.sup.16, independently, is deleted to form a double bond.
37. The compound of claim 35, wherein n is 0.
38. The compound of claim 35, wherein R.sup.3 is hydroxy, amino,
carboxyl, halo, sulfonic acid, --O-sulfonic acid, or alkyl, and is
in the .alpha.-configuration.
39. The compound of claim 35, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, oxo, halo, sulfonic acid, --O-sulfonic acid, or alkyl.
40. The compound of claim 39, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, or oxo; and each of R.sup.5, R.sup.8, R.sup.9, R.sup.10,
R.sup.13, and R.sup.14, independently, is hydrogen or hydroxy.
41. The compound of claim 40, wherein X is a bond or alkyl.
42. The compound of claim 41, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
43. The compound of claim 35, wherein X is a bond or alkyl.
44. The compound of claim 35, wherein Y is --CO--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--NH--,
--NH--CO--, or a bond.
45. The compound of claim 35, wherein Z is alkyl or aryl, each of
which being optionally substituted with hydroxy; or is --CH(A)-B
with A being an amino acid side chain having an aromatic moiety,
and B being --N.sup.aR.sup.b, or --COOR.sup.c.
46. The compound of claim 35, wherein R.sup.17 contains a straight
chain having 6-20 chain atoms.
47. The compound of claim 46, wherein R.sup.17 contains a straight
chain having 8-16 chain atoms.
48. The compound of claim 35, wherein X is
--CH(CH.sub.3)--CH.sub.2--, Y is a bond, and Z is
--CH.sub.2--CH.dbd.C(R')(CH.sub.3) with R' being hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio.
49. The compound of claim 35, wherein Z is alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl.
50. The compound of claim 49, wherein n is 0.
51. The compound of claim 49, wherein R.sup.3 is hydroxy, amino,
carboxyl, halo, sulfonic acid, --O-sulfonic acid, or alkyl, and is
in the .alpha.-configuration.
52. The compound of claim 49, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, oxo, halo, sulfonic acid, --O-sulfonic acid, or alkyl.
53. The compound of claim 52, wherein each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, or oxo; and each of R.sup.5, R.sup.8, R.sup.9, R.sup.10,
R.sup.13 and R.sup.14, independently, is hydrogen or hydroxy.
54. The compound of claim 53, wherein X is a bond or alkyl.
55. The compound of claim 54, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
56. The compound of claim 49, wherein X is a bond or alkyl.
57. The compound of claim 56, wherein Y is --C(.dbd.O)--NH-- or
--NH--C(.dbd.O)--; and Z is --CH(A)-B with A being a side chain of
Tyr or Phe, and B being --NR.sup.aR.sup.b or --COOR.sup.c
58. The compound of claim 49, wherein Y is --CO--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--NH--,
--NH--CO--, or a bond.
59. The compound of claim 49, wherein R.sup.17 contains a straight
chain having 6-20 chain atoms.
60. The compound of claim 59, wherein R.sup.17 contains a straight
chain having 8-16 chain atoms.
61. The compound of claim 49, wherein X is
--CH(CH.sub.3)--CH.sub.2--, Y is a bond, and Z is
--CH.sub.2--CH.dbd.C(R')(CH.sub.3) with R' being hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio.
62. The compound of claim 49, wherein said compound is: 202122
63. A pharmaceutical composition for treating a UR- or a
LXR-mediated disorder, said composition comprising a
pharmaceutically acceptable carrier and an effective amount of a
compound of the following formula: 23wherein R.sup.3 is hydrogen,
amino, carboxyl, oxo, halo, sulfonic acid, --O-sulfonic acid, or
alkyl that is optionally inserted with --NH--, --N(alkyl)-, --O--,
--S--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--, --CO--O--, --O--CO--,
--CO--NH--, --CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and
further optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid; each of R.sup.1, R.sup.2,
R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.15,
R.sup.16, and R.sup.17', independently, is hydrogen, hydroxy,
amino, carboxyl, oxo, halo, sulfonic acid, --O-sulfonic acid, or
alkyl that is optionally inserted with --NH--, --N(alkyl)-, --O--,
--S--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--, --CO--O--, --O--CO--,
--CO--NH--, --CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and
further optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid; each of R.sup.5, R.sup.8,
R.sup.9, R.sup.10, R.sup.13, and R.sup.14, independently, is
hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or
amino; R.sup.17 is --X--Y-Z, in which X is a bond, or alkyl or
alkenyl, optionally inserted with --NH--, --N(alkyl)-, --O--, or
--S--, and further optionally forming a cyclic moiety with R.sup.16
and the 2 ring carbon atoms to which R.sup.16 and R.sup.17 are
bonded; Y is --CO--, --SO--, --SO.sub.2--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--O--,
--O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--,
--N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally
substituted with hydroxy, alkoxy, amino, halo, sulfonic acid,
--O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being a side chain of an amino acid, and B being
hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each of
R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or alkyl;
and n is 0, 1, or 2; provided that when Z is substituted with
carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z
contains at least one double bond, and that when Y is a bond,
either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl;
or a salt thereof.
64. The composition of claim 63, wherein said compound is: 2425
65. A pharmaceutical composition for treating a UR- or a
LXR-mediated disorder, said composition comprising a
pharmaceutically acceptable carrier and an effective amount of a
compound of the following formula: 26wherein each of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid,
--O-sulfonic acid, or alkyl that is optionally inserted with
--NH--, --N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, or --N(alkyl)-CO--, and further optionally substituted
with hydroxy, halo, amino, carboxyl, sulfonic acid, or --O-sulfonic
acid; each of R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and
R.sup.14, independently, is hydrogen, alkyl, haloalkyl,
hydroxyalkyl, alkoxy, hydroxy, or amino; R.sup.17 is --X--Y-Z, in
which X is a bond, or alkyl or alkenyl, optionally inserted with
--NH--, --N(alkyl)-, --O--, or --S--, and further optionally
forming a cyclic moiety with R 6 and the 2 ring carbon atoms to
which R.sup.16 and R.sup.17 are bonded; Y is --CO--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, --N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl,
and is substituted with hydroxy, alkoxy, amino, halo, sulfonic
acid, --O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being an amino acid side chain containing an
aromatic moiety, and B being hydrogen, --NR.sup.aR.sup.b, or
--COOR.sup.c wherein each of R.sup.a, R.sup.b, and R.sup.c,
independently, is hydrogen or alkyl; and n is 0, 1, or 2; provided
that when Z is substituted with carboxyl or alkyloxycarbonyl, Y is
a bond and either X or Z contains at least one double bond, and
that when Y is a bond, either X is --NH-alkyl-, --NH-alkenyl-,
--N(alkyl)-alkyl-, --N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-,
--S-alkyl-, or --S-alkenyl-; or Z is substituted with halo,
sulfonic acid, --O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl,
or is alkenyl; or a salt thereof.
66. The composition of claim 65, wherein said compound is:
272829
67. A pharmaceutical composition for treating a UR- or a
LXR-mediated disorder, said composition comprising a
pharmaceutically acceptable carrier and an effective amount of a
compound of the following formula: 30wherein each of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11,
R.sup.12, R.sup.15, R.sup.16, and R.sup.17', independently, is
hydrogen, hydroxy, amino, carboxyl, oxo, halo, sulfonic acid,
--O-sulfonic acid, or alkyl optionally inserted with --NH--,
--N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--,
--CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--, or
--N(alkyl)-CO--, and further optionally substituted with hydroxy,
halo, amino, carboxyl, sulfonic acid, or --O-sulfonic acid; each of
R.sup.5, R.sup.8, R.sup.9, R.sup.10, R.sup.13, and R.sup.14,
independently, is hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy,
hydroxy, or amino; R.sup.17 is --X--Y-Z, in which X is a bond, or
alkyl or alkenyl, optionally inserted with --NH--, --N(alkyl)-,
--O--, or --S--, and further optionally forming a cyclic moiety
with R.sup.16 and the 2 ring carbon atoms to which R.sup.16 and
R.sup.17 are bonded; Y is --CO--, --SO--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--,
--N(alkyl)-CO--, or a bond; and Z is hydrogen, alkyl, alkenyl,
alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl,
and is optionally substituted with hydroxy, alkoxy, amino, halo,
sulfonic acid, --O-sulfonic acid, carboxyl, oxo, alkyloxycarbonyl,
alkylcarbonyloxy, alkylaminocarbonyl, alkylcarbonylamino,
alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or alkylthio; or is
--CH(A)-B with A being a side chain of an amino acid, and B being
hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each of
R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or alkyl;
and n is 0, 1, or 2; provided that when Z is substituted with
carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z
contains at least one double bond, and that when Y is a bond,
either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl;
and further provided that at least one of R.sup.3 and R.sup.4,
R.sup.4 and R.sup.5, R.sup.5 and R.sup.6, R.sup.7 and R.sup.8,
R.sup.12 and R.sup.13, and R.sup.15 and R.sup.16, independently, is
deleted to form a double bond; or a salt thereof.
68. The composition of claim 67, wherein said compound is: 313233
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 USC .sctn. 119(e), this application claims
the benefit of prior U.S. provisional application 60/131,728, filed
Apr. 30, 1999; and 60/191,864, filed Mar. 24, 2000.
BACKGROUND
[0003] Nuclear receptors are a family of transcription factors
modulated by small hydrophobic signaling molecules, like steroids,
thyroid hormone, free fatty acids, vitamin D and retinoids. Nuclear
receptors are important pharmacological targets for drug
intervention in disease management. For example, Tamoxifen, an
estrogen antagonist, interacts with estrogen receptor to deliver
its therapeutic effects on breast cancer; RU486, an antagonist of
progesterone receptor, is used for termination of pregnancies and
menopause-related disorders; and Dexamethasone interacts with
glucocorticoid receptor to suppress immune system function and is
useful for treating inflammatory diseases such as asthma.
[0004] Nuclear receptors have three independent domains I, II and
III. Domains I and III modulate transcriptional activities by
interacting with other factors of the transcription complex; Domain
II involves in DNA-binding; and Domain III is the ligand-binding
domain. Domain II is the most conserved region within the nuclear
receptor family, with a unique feature of four pairs of cysteine
chelated with two zinc atoms which form a "zinc finger" structure.
The three domains of nuclear receptors are functionally
interchangeable between different members. For example, the
androgen receptor DNA-binding domain can be fused to the
ligand-binding domain of estrogen receptor and the resulting AR-ER
chimeric receptor can modulate androgen-responsive genes by binding
to estrogen.
[0005] Amino acid sequence homology of the DNA-binding domain
between members of nuclear receptor family allows identification of
new members of this family through low stringency nucleotide-probe
screening. Human genome project also facilitates identification of
new genes coding for new nuclear receptors. At present, a few
dozens of nuclear receptors have been identified and sequenced, but
their ligands have yet to be identified. Recently, a novel nuclear
receptor was cloned through degenerate oligonucleotide screening
from human and rat cells and was named ubiquitous nuclear receptor
("UR"), because of its ubiquitous expression pattern in the body.
UR has been found to form heterodimers with RXR receptors and binds
to double-stranded DNA with the sequence motif: AGGTCANNNNAGGTCA
(SEQ ID NO: 1) ("DR4"). Promoters containing DR4 can be activated
by UR and RXR heterodimer in cultured cells.
[0006] LXRa, another new member of the nuclear receptor family has
been cloned recently. Amino acid sequence analysis revealed that it
shares over 80% homology with UR in the DNA- and ligand-binding
domain. The expression of LXRa mRNA is limited to liver and a few
other tissues. LXRa has been identified as a transcriptional
activator of the cholesterol 7.alpha.-hydroxylase gene and plays an
important role in cholesterol catabolism.
[0007] Recently other nuclear proteins interacting with nuclear
receptors have been identified through yeast two-hybrid screening
techniques, among which are co-activators and co-repressors of
nuclear receptors, e.g., SRC1, 2, 3, and Grip1. These proteins
interact with nuclear receptors in a ligand-dependent manner. This
property is useful to set up biochemical assays for ligand-receptor
interaction.
[0008] Steroid derivatives described in this invention are found to
modulate the transcriptional activities via binding to UR or LXRa,
and thus can be used to treat disorders mediated by such receptors
such as atherosclerosis.
SUMMARY
[0009] An aspect of this invention relates to steroid derivatives
of formula (I): 1
[0010] R.sup.3 is hydrogen, amino, carboxyl, oxo, halo, sulfonic
acid, --O-sulfonic acid, or alkyl that is optionally inserted with
--NH--, --N(alkyl)-, --O--, --S--, --SO--, --SO.sub.2--,
--O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--,
--CO--, --CO--O--, --O--CO--, --CO--NH--, --CO--N(alkyl)-,
--NH--CO--, or --N(alkyl)-CO--, and further optionally substituted
with hydroxy, halo, amino, carboxyl, sulfonic acid, or --O-sulfonic
acid. Each of R.sup.1, R.sup.2, R.sup.4, R.sup.4', R.sup.6,
R.sup.7, R.sup.11, R.sup.12, R.sup.15, R.sup.16, and R.sup.17',
independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo,
sulfonic acid, --O-sulfonic acid, or alkyl that is optionally
inserted with --NH--, --N(alkyl)-, --O--, --S--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and further
optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid. Each of R.sup.5, R.sup.8,
R.sup.9, R.sup.10, R.sup.13, and R.sup.14, independently, is
hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or
amino. R.sup.17 is --X--Y-Z. X is a bond, or alkyl or alkenyl,
optionally inserted with --NH--, --N(alkyl)-, --O--, or --S--, and
further optionally forming a cyclic moiety with R.sup.16 and the 2
ring carbon atoms to which R.sup.16 and R.sup.17 are bonded. Y is
--CO--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, --N(alkyl)-CO--, or a bond. Z is
alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl, and is optionally substituted with hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio; or is --CH(A)-B. A being a side chain of an amino acid,
and B is hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each
of R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or
alkyl. n is 0, 1, or 2. Note that when Z is substituted with
carboxyl or alkyloxycarbonyl, Y is a bond and either X or Z
contains at least one double bond, and that when Y is a bond,
either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is
alkenyl.
[0011] Another aspect of this invention relates to steroid
derivatives having the formula (I) as depicted above. Each of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7,
R.sup.11, R.sup.12, R.sup.15, R.sup.16, and R.sup.17',
independently, is hydrogen, hydroxy, amino, carboxyl, oxo, halo,
sulfonic acid, --O-sulfonic acid, or alkyl that is optionally
inserted with --NH--, --N(alkyl)-, --O--, --S--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and further
optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid. Each of R.sup.5, R.sup.8,
R.sup.9, R.sup.10, R.sup.13, and R.sup.14, independently, is
hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or
amino. R.sup.17 is --X--Y-Z. X is a bond, or alkyl or alkenyl,
optionally inserted with --NH--, --N(alkyl)-, --O--, or --S--, and
further optionally forming a cyclic moiety with R.sup.16 and the 2
ring carbon atoms to which R.sup.16 and R.sup.17 are bonded. Y is
--CO--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, --N(alkyl)-CO--, or a bond. Z is
alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl, and is optionally substituted with hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio; or is --CH(A)-B. A is an amino acid side chain
containing an aromatic moiety, and B is hydrogen,
--NR.sup.aR.sup.b, or --COOR.sup.c wherein each of R.sup.a,
R.sup.b, and R.sup.c, independently, is hydrogen or alkyl. n is 0,
1, or 2. Note that when Z is substituted with carboxyl or
alkyloxycarbonyl, Y is a bond and either X or Z contains at least
one double bond, and that when Y is a bond, either X is
--NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-, --N(alkyl)-alkenyl-,
--O-alkyl-, --O-alkenyl-, --S-alkyl-, or --S-alkenyl-; or Z is
substituted with halo, sulfonic acid, --O-sulfonic acid,
alkylsulfinyl, or alkylsulfonyl, or is alkenyl.
[0012] A further aspect of this invention relates to steroid
derivatives of formula (1), supra. Each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.4', R.sup.6, R.sup.7, R.sup.11, R.sup.12,
R.sup.15, R.sup.16, and R.sup.17', independently, is hydrogen,
hydroxy, amino, carboxyl, oxo, halo, sulfonic acid, --O-sulfonic
acid, or alkyl optionally inserted with --NH--, --N(alkyl)-, --O--,
--S--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--, --CO--O--, --O--CO--,
--CO--NH--, --CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and
further optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid. Each of R.sup.5, R.sup.8,
R.sup.9, R.sup.10, R.sup.13, and R.sup.14, independently, is
hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or
amino. R.sup.17 is --X--Y-Z. X is a bond, or alkyl or alkenyl,
optionally inserted with --NH--, --N(alkyl)-, --O--, or --S--, and
further optionally forming a cyclic moiety with R.sup.16 and the 2
ring carbon atoms to which R.sup.16 and R.sup.17 are bonded. Y is
--CO--, --SO--, --SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--,
--O--SO.sub.3--, --SO.sub.3--O--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, --N(alkyl)-CO--, or a bond. Z is
alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl, and is optionally substituted with hydroxy, alkoxy,
amino, halo, sulfonic acid, --O-sulfonic acid, carboxyl, oxo,
alkyloxycarbonyl, alkylcarbonyloxy, alkylaminocarbonyl,
alkylcarbonylamino, alkylcarbonyl, alkylsulfinyl, alkylsulfonyl, or
alkylthio; or is --CH(A)-B. A is a side chain of an amino acid, and
B is hydrogen, --NR.sup.aR.sup.b, or --COOR.sup.c wherein each of
R.sup.a, R.sup.b, and R.sup.c, independently, is hydrogen or alkyl.
n is 0, 1, or 2. Note that when Z is substituted with carboxyl or
alkyloxycarbonyl, Y is a bond and either X or Z contains at least
one double bond, and that when Y is a bond, either X is
--NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-, --N(alkyl)-alkenyl-,
--O-alkyl-, --O-alkenyl-, --S-alkyl-, or --S-alkenyl-; or Z is
substituted with halo, sulfonic acid, --O-sulfonic acid,
alkylsulfinyl, or alkylsulfonyl, or is alkenyl; and that at least
one of R.sup.3 and R.sup.4, R.sup.4 and R.sup.5, R.sup.5 and
R.sup.6, R.sup.7 and R.sup.8, R.sup.12 and R.sup.13, and R.sup.15
and R.sup.16, independently, is deleted to form a double bond. One
subset of the just-described steroid derivatives encompasses
compounds which are featured by the presence of at least one double
bond in the rings, which are formed by deleting one or more of the
following pairs of substituents: R.sup.3 and R.sup.4, R.sup.4 and
R.sup.5, R.sup.12 and R.sup.13, and R.sup.15 and R.sup.16. Another
subset encompasses compounds which are featured by that Z is
alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,
heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl,
and optionally substituted with hydroxy, alkoxy, amino, or halo; or
is --CH(A)-B. A and B are as described above.
[0013] Note that X and Z optionally join together to form a cyclic
moiety. For example, if both X and Z are alkyl, and Y is
--C(.dbd.O)--O--, a lactone results from joining X and Z.
[0014] A salt of the steroid derivative of this invention is also
within the scope of this invention and can be formed, for example,
between the steroid of this invention having a carboxylate and a
cationic counterion such as an alkali metal cation, e.g., a sodium
ion or a potassium ion; or an ammonium cation that can be
substituted with organic groups, e.g., a tetramethylammonium ion or
a diisopropyl-ethylammonium ion. A salt of this invention can also
form between the steroid derivative of this invention having a
protonated amino group and an anionic counterion, e.g., a sulfate
ion, a nitrate ion, a phosphate ion, or an acetate ion.
[0015] Set forth below are some examples of steroid derivatives of
this invention: 23456789
[0016] As used herein, the term "alkyl" in this disclosure denotes
a straight or branched hydrocarbon chain containing 1-8 carbon
atoms. Some examples of an alkyl group are methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl,
octyl, or 2-methylpentyl. By the term "cycloalkyl" is meant a
cyclic hydrocarbon chain that has 3-8 carbon atoms. The cycloalkyl
groups herein described may also contain fused rings. Fused rings
are rings that share a common carbon-carbon bond. Examples of
cycloalkyl groups include, but not limited to, cyclopropyl,
cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl.
[0017] The term "alkenyl" refers to a straight or branched
hydrocarbon chain containing 2-8 carbon atoms and characterized in
having one or more double bonds. Examples of a typical alkenyl
include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl
and 3-octenyl groups. The term "cycloalkenyl" is meant a cyclic
hydrocarbon chain containing 3-8 carbon atoms and having at least
one or more double bonds. Similar to the definition of cycloalkyl
groups above, cycloalkenyl groups may also contain fused rings.
Some examples of cycloalkenyl groups are cyclopentenyl,
cyclohexenyl, cycloheptenyl, norbornylenyl, and cyclooctenyl
groups.
[0018] The term "alkynyl" refers to a straight or branched
hydrocarbon chain containing 2-8 carbon atoms and characterized in
having one or more triple bonds. Some examples of a typical alkynyl
are ethynyl, 2-propynyl, and 3-methylbutynyl.
[0019] The terms "heterocycloalkyl" and "heterocycloalkenyl" refer
to cycloalkyl and cycloalkenyl groups which contain one or more
heteroatoms, such as, nitrogen, oxygen, or sulfur. Typical
heterocycloalkyl and heterocycloalkenyl groups include
tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,
pyrrolinyl and pyrrolidinyl.
[0020] "Aryl" represents an aromatic moiety which contains 6-12
carbon atoms and can contain fused rings. A fused ring is an
aromatic group which contains at least two aryl rings sharing a
common carbon-carbon bond. Typical examples of aryl include phenyl
and naphthyl.
[0021] "Heteroaryl" groups in this disclosure are aromatic groups
containing 5 to 12 ring atoms, in which one or more of these ring
atoms are heteroatoms as defined above. Some examples of heteroaryl
groups are pyridyl, pyrazinyl, furyl, pyrrolyl, thienyl, thiazolyl,
benzimidazolyl, and imidazolyl.
[0022] The positions of substituents on each of the cyclic groups
described herein may be at any available position, unless specified
otherwise. For example, a methyl substituent on a benzene ring can
be attached at the ortho, meta, or para position.
[0023] The term "alkoxy" is defined as the moiety "--O-alkyl." Some
examples are methoxy, ethoxy, propoxy, isopropoxy, and t-butoxy.
"Halo" represents a halogen atom, such as, fluoro, chloro, bromo,
or iodo. By the terms "haloalkyl" and "hydroxyalkyl" are meant
alkyl groups which are respectively substituted with one or more
halogen atoms and one or more hydroxy groups. The nitrogen atom in
an amino or amido group present in a steroid derivative of this
invention can be mono- or di-substituted with an alkyl, a
cycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl.
[0024] For convenience, a divalent moiety is named herein as if it
were a monovalent moiety. For example, "alkyl," such as CH.sub.3,
which is assigned to X, actually stands for "alkylene," such as
--CH.sub.2--. As recognized by a skilled person in the art, steroid
derivatives described herein contain stereocenters. Both the
racemic mixtures of isomers and the optically pure isomers are
within the scope of this invention.
[0025] Yet another aspect of this invention relates to a
pharmaceutical composition for treating a UR- or LXRa-mediated
disorder which contains a pharmaceutically acceptable carrier and
an effective amount of one or more of the steroid derivatives
described above. The use of such a steroid derivative or a salt
thereof for the manufacture of a medicament for treating the
above-mentioned disorders is also within the scope of this
invention.
[0026] A still further aspect of this invention relates to a
pharmacological composition for treating cancer, including solid
tumors and leukemia, and immune dysfunction. The pharmacological
composition contains a pharmaceutically acceptable carrier and an
effective amount of one or more of a steroid derivative of formula
(I), supra. Each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.4',
R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.15, R.sup.16, and
R.sup.17', independently, is hydrogen, hydroxy, amino, carboxyl,
oxo, halo, sulfonic acid, --O-sulfonic acid, or alkyl that is
optionally inserted with --NH--, --N(alkyl)-, --O--, --S--, --SO--,
--SO.sub.2--, --O--SO.sub.2--, --SO.sub.2--O--, --O--SO.sub.3--,
--SO.sub.3--O--, --CO--, --CO--O--, --O--CO--, --CO--NH--,
--CO--N(alkyl)-, --NH--CO--, or --N(alkyl)-CO--, and further
optionally substituted with hydroxy, halo, amino, carboxyl,
sulfonic acid, or --O-sulfonic acid. Each of R.sup.5, R.sup.8,
R.sup.9, R.sup.10, R.sup.13, and R.sup.14, independently, is
hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxy, or
amino. R.sup.17 is --X--Y-Z, in which X is a bond, or alkyl or
alkenyl, optionally inserted with --NH--, --N(alkyl)-, --O--, or
--S--, and further optionally forming a cyclic moiety with R.sup.16
and the 2 ring carbon atoms to which R.sup.16 and R.sup.17 are
bonded; Y is --CO--, --SO--, --SO.sub.2--, --O--SO.sub.2--,
--SO.sub.2--O--, --O--SO.sub.3--, --SO.sub.3--O--, --CO--O--,
--O--CO--, --CO--NH--, --CO--N(alkyl)-, --NH--CO--,
--N(alkyl)-CO--, or a bond; and Z is alkyl, alkenyl, alkynyl,
cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl,
aryl, heteroaryl, aralkyl, or heteroaralkyl, and is optionally
substituted with hydroxy, alkoxy, amino, halo, sulfonic acid,
--O-sulfonic acid, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy,
alkylaminocarbonyl, alkylcarbonylamino, alkylcarbonyl,
alkylsulfinyl, alkylsulfonyl, or alkylthio; or is --CH(A)-B with A
being a side chain of an amino acid, and B being hydrogen,
--NR.sup.aR.sup.b, or --COOR.sup.c wherein each of R.sup.a,
R.sup.b, and R.sup.c, independently, is hydrogen or alkyl; and n is
0, 1, or 2. When Z is substituted with carboxyl, Y is a bond and
either X or Z contains at least one double bond, and when Y is a
bond, either X is --NH-alkyl-, --NH-alkenyl-, --N(alkyl)-alkyl-,
--N(alkyl)-alkenyl-, --O-alkyl-, --O-alkenyl-, --S-alkyl-, or
--S-alkenyl-; or Z is substituted with halo, sulfonic acid,
--O-sulfonic acid, alkylsulfinyl, or alkylsulfonyl, or is alkenyl.
The use of a just-described steroid derivative or a salt thereof
for the manufacture of a medicament for treating the
above-mentioned disorders is also within the scope of this
invention.
[0027] Still another aspect of the present invention relates to a
method of treating a UR- or LXRa-mediated disorder by administering
to a patient in need thereof an effective amount of one of the
pharmaceutical compositions described above. Some examples of UR-
or LXRa-mediated disorders are: liver cirrhosis, gallstone disease,
hyperlipoproteinemias, Alzheimer's disease, anemia, chronic
inflammatory diseases (e.g., rheumatoid arthritis), metabolic
disorders (e.g., diabetes), and cancers which are associated with
UR expression, e.g., breast cancer, colon cancer, prostate cancer,
and leukemia. Patients with other disorders such as atherosclerosis
and liver cholestasis can also be treated with one of the
pharmaceutical compositions described above.
[0028] Other features or advantages of the present invention will
be apparent from the following detailed description of several
embodiments, and also from the appending claims.
DETAILED DESCRIPTION
[0029] A steroid derivative of this invention can be prepared by
forming an amide bond between a steroid having a C17
carboxyl-containing substituent and an amino-containing compound or
between a steroid having a C17 amino-containing substituent and a
carboxyl-containing compound. Similarly, an ester bond can be
formed between a steroid with a C17 carboxyl-containing substituent
and a hydroxyl-containing compound, or between a steroid with a C17
hydroxyl-containing substituent and a carboxyl-containing compound.
Some examples of a steroid that can be used as a starting material
are cholic acid (e.g., ursodeoxycholic acid, hyocholic acid, and
hyodeoxycholic acid), androstan-17-carboxylic acid (e.g.,
androstan-3-oxo-17-carboxylic acid and d5-androsten-3-ol-17-carbox-
ylic acid) and pregnan-20-ol (e.g., d5-pregnen-3,17-diol or
pregnan-17-ol-3-one). Synthesis of these steroids can be found in
the literature, e.g., Roda A. et al., F. Lipid Res. vol. 35, pages
2268-2279 (1994) and Roda A. et al., Dig. Dis. Sci. vol. 34, pages
24S-35S (1987). Some examples of compounds that can be used to
couple to a steroid to form a steroid derivative of this invention
are aniline, glycine, phenylalanine, or benzoic acid. Examples of a
coupling reagent that can be used in the amide- or ester-forming
reaction include 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide
(EDC), dicyclohexyl-carbodiimide (DCC), N-hydroxybenzo-triazole
(HOBt), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate (HBTU), or
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (PyBOP). The amide- or ester-forming reaction
can take place in any solvents that are suitable with the starting
materials and reagents. Note that if the reaction takes place in an
aqueous solvent, e.g., a buffered solution (or in combination with
other miscible organic solvents such as alcohol), isolation of the
steroid product for in vitro or in vivo screening assays is not
necessary, as the product is already in suitable assaying
conditions, i.e., in an aqueous buffered medium. Protection of
functional groups, e.g., hydroxyl or keto, on the steroids is not
needed. See, e.g., Example 1 below. Due to the simplicity of the
reaction, it can be easily automated. Isolation and quantification
of the product can be done by thin-layer chromatography, high
pressure liquid chromatography, gas chromatography, capillary
electrophoresis, or other analytical and preparative procedures.
Trifluoromethyl- and taurine-conjugated steroid derivatives can be
prepared according to methods described in Li, S. et al., Chem.
Phys. Lipids 99:33-71 (1999) and Kurosawa, T. et al., Steroids,
60:439-444 (1995), respectively. As to the preparation of
3.beta.-hydroxy-5-cholesten-25(R)-26-carboxylic acid derivatives,
see Kim, H. et al., J. Lipid Res. 30:247 (1989) and Varma, R. K. et
al., J. Org. Chem. 40:3680 (1975). Steroid derivatives having a
side chain that contains a double bond, e.g., between C24 and C25,
can be prepared according to the following scheme: 1011
[0030] 3-beta-t-butyldimethylsilyloxy-delta[5]-cholen-24-al and
3-alpha,6-alpha-di(t-butyldimethylsilyloxy)5-beta-cholan-24-al were
prepared using NaBH4 and pyridinium chlorochromate according to
methods described in Somanathan et al., Steroids 43:651-655 (1984).
Ethyl-3-beta-t-butyldimethylsilyloxy-delta[5,24]-cholestenoate and
ethyl-3a,6a-di(t-butyldimethylsilyloxy)-delta[24]-cholestanoate
were then prepared via Wittig-Homer reaction using triethyl
2-phosphono-propionate and a suitable base according to methods
described in Lund et al., Arterioscler. Thromb. Vasc. Biol.
16:208-212 (1996). After the t-butyldimethylsilyloxyl groups were
removed, ethyl ester groups were hydrolyzed under alkaline
conditions.
[0031] As mentioned above, a pharmaceutical composition containing
a steroid derivative or a salt of this invention in an effective
amount can be used to treat UR- or LXRa-mediated disorders. Also
within the scope of this invention is a method of treating a UR- or
LXRa-mediated disorder such as astherosclerosis by administering to
a patient such a composition. An effective amount is defined as the
amount of the derivative which, upon administration to a patient in
need, confers a therapeutic effect on the treated patient. The
effective amount to be administered to a patient is typically based
on body surface area, patient weight, and patient condition. The
interrelationship of dosages for patients (based on milligrams per
meter squared of body surface) is described by Freireich et al.,
Cancer Chemother. Rep. 1966, 50, 219. Body surface area may be
approximately determined from height and weight of the patient.
See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New
York, 1970, 537. An effective amount of a compound of this
invention used to practice the invention can range from about 1
mg/kg to about 2 g/kg, e.g., from about 1 mg/kg to about 1 g/kg, or
from about 1 mg/kg to about 500 mg/kg. Effective doses will also
vary, as recognized by those skilled in the art, dependant on route
of administration, excipient usage, and the possibility of co-usage
with other therapeutic treatments.
[0032] The pharmaceutical composition may be administered via the
parenteral route, including subcutaneously, intraperitoneally,
intramuscularly and intravenously. Examples of parenteral dosage
forms include aqueous solutions of the active agent, in a isotonic
saline, 5% glucose or other well-known pharmaceutically acceptable
excipient. Solubilizing agents such as cyclodextrins, or other
solubilizing agents well-known to those familiar with the art, can
be utilized as pharmaceutical excipients for delivery of the
therapeutic compounds.
[0033] The steroid derivatives of this invention can also be
formulated into dosage forms for other routes of administration
utilizing well-known methods. They can be formulated, for example,
in dosage forms for oral administration in a gel seal, a syrup, a
capsule, or a tablet. Capsules may comprise any well-known
pharmaceutically acceptable material such as gelatin or cellulose
derivatives. Tablets may be formulated in accordance with the
conventional procedure by compressing mixtures of the compound of
this invention and a solid carrier, and a lubricant. Examples of
solid carriers include starch and sugar bentonite. The steroid
derivatives of this invention can also be administered in a form of
a hard shell tablet or a capsule containing a binder (e.g., lactose
or mannitol) and a conventional filler.
[0034] The level of interaction between the UR or LXRa protein and
a steroid derivative of this invention can be preliminarily
evaluated using various assays as described below:
[0035] Protease protection assay is a simple assay for measuring
the level of interaction between a test steroid and the UR or LXRa
protein. This assay can be done by using a .sup.35S-Met
radiolabeled rat UR or human LXRa protein. The radiolabeled protein
is then incubated with the steroid of this invention and digested
with a protease, e.g., trypsin. A control experiment is done by
incubating UR receptor with a protease but without the steroid.
Protein fragments from both assays are electrophoresed on a
polyacrylamide gel. The fragments from each of the assays can be
visualized by exposing the gel to X-ray films and compared
side-by-side. A test steroid, if binds to the UR or LXRa protein,
will protect the receptor from being digested by the protease. As a
result, reactions that result in binding between the steroid and UR
will lead to fewer bands of low molecular weights than those that
do not result in binding between the two molecules.
[0036] The co-activator binding assay employs a fusion protein
formed between a glutathione S-transferase (GST) and a co-activator
of UR, e.g., Grip1. The GST moiety of the fusion protein binds to a
glutathione-coated solid support, thereby immobilizing the fusion
protein. UR and a steroid of this invention are then incubated with
the immobilized fusion protein. Subsequently, any bound UR is
released and collected from the solid support. The proteins are
then electrophoresed on a polyacrylamide gel and visualized by
exposing the gel to X-ray films. If the steroid interacts with UR,
less UR will bind to the fusion protein, and a lighter band would
therefore result on the gel. By monitoring the intensity of the
band of the bound UR, one can estimate the binding of the steroid
to UR.
[0037] Yeast two-hybrid binding assay is a sensitive assay for
identifying UR modulating compounds by monitoring transcriptional
activation. General descriptions of these assay can be found in,
e.g., Chien C. T. et al., Proc. Natl. Acad. Sci. USA, vol. 88,
9578-9582 (1991); Fields, S. et al., Nature, vol. 340, 245-247
(1989); and Green, M. B. et al., Curr. Biol., vol. 2, 403-405
(1992). In this screening method, a steroid of this invention that
modulates the interaction of UR or LXRa with its natural ligand
will have an effect on the transcriptional activation of a reporter
gene. In a specific assay, two plasmids are introduced into a yeast
cell. One expresses a fusion protein having a GAL4 DNA binding
domain and a UR natural ligand, and the other expresses a fusion
protein containing a UR ligand binding domain and a GAL4 activation
domain. If the steroid interacts with UR and disrupts the binding
of UR to its natural ligand, the activity of the reporting gene
(Gal4) will be altered. The changes in reporter activities (i.e.,
.beta.-galactosidase activities) can be measured with a commercial
luminescence kit.
[0038] Mammalian cell transfection can also be used to screen
steroid derivatives that affect the interaction between the UR
protein and a steroid of this invention. A rat UR or human LXR gene
and a human RXRa gene are cloned into a mammalian expression vector
(e.g., pSG5 from Strategene) and overexpressed. A heterologous
promoter is formed by inserting four tandem repeats of a hormone
response element DR4 into the vector upstream to a c-fos promoter
sequence, which is followed by a sequence encoding luciferase. The
entire construct is named DR4-fos-luc. DR4-fos-luc is then
co-transfected with pSG5/rUR or CMV/hLXR and pSG5/hRXRa into
mammalian cells, e.g., COS-1 cells. An ethanol solution containing
a steroid of this invention is then added to the transfected cells.
The steroid, if interacts to the UR or LXRa protein, affects the
level at which the luciferase gene is activated. The cells are then
lysed and assayed for luciferase activity with a commercial assay
kit and a luminometer. A high intensity of luminescence indicates
that the steroid is a potent UR or LXR agonist.
[0039] Another chimeric receptor that can be used in this assay is
constructed by fusing oligonucleotides encoding the ligand-binding
domain of rat UR to a human AR gene lacking ligand-binding site
coding region. For this chimeric receptor, a reporter gene
ARE-fos-luc is constructed by inserting three tandem repeats of
Androgen Response Element (ARE) into the vector upstream to a c-fos
promoter which is followed by a luciferase reporter gene. After
adding a steroid of this invention to the medium of the transfected
cells, the steroid can interact with UR and affect the level of
activation of ARE-fos-luc in cultured cells. The level of
luminescence activity thus indicates the level of UR modulation by
the steroid.
[0040] Yet another assay involves expressing rUR gene in PC-3 cells
by retroviral infection. See Underwood et al., J. Biol. Chem., vol.
273, pages 4266-4274 (1998). The transfected cells are then seeded
in media containing delipidated serum and then treated with a
solution containing a steroid of this invention. The PC-3 cells are
later washed with phosphate buffered saline (PBS) and treated with
100 mg/ml amphotericin B in DMEM media without serum at 37EC.
Amphotericin B functions to kill cells containing cholesterol in
the cell membrane. The cells are then fixed in 10% TCA and stained
with Sulforhodamine B after more washing. Viable cells are stained
and can then be assessed using a colorimetric assay. The amount of
dye is directly proportional to number of surviving cells on the
culture plates. From comparing the number of viable cells between
assays with and without a steroid, one can estimate the effect the
steroid has on the de novo synthesis of cholesterol.
[0041] A still further assay makes use of nitrogen monoxide (NO) as
an indicator of the level of inflammation. Cells from a murine
macrophage cell line RAW264.7 are incubated with a steroid of this
invention for 24 hours. The macrophages are then activated by
adding lipopolysaccharide (LPS) and gamma-interferon. The NO
production of activated macrophages can be monitored indirectly by
quantifying NO.sub.2 in the media according to Green L. et al.,
Anal. Biochem., vol. 126, 131-138 (1982). The reduced amount of NO
in comparison to that of a control experiment in which no steroid
is used indicates that the steroid used in the assay has inhibitory
effect on inflammation.
[0042] Using the same murine macrophage cell line RAW264.7,
constitutive expression of rat UR and human RXRa gene by retroviral
systems transforms these cells into foam-cell-like morphology and
integrated into clumps while increasing cell sizes and undergo
apoptosis. Foam cells originated from macrophages are the major
components in pathological plaque which is usually found on the
inner wall of blood vessels in patients suffering from
atherosclerosis. Steroid derivatives of this invention which
modulate UR can suppress the progression of macrophage-foam cell
transformation at different stages, and can be used in the
treatment or prevention of atherosclerosis. See Kellner-Weibel et
al., Arterioscler. Thromb. Vasc. Biol., vol. 18, pages 423-431
(1998).
[0043] Yet another assay measures the effect of a steroid of this
invention has on the level of adipocyte differentiation on
fibroblasts. Specifically, the level of adipocyte differentiation
in murine fibroblasts 3T3-L1 containing rat UR gene at
sub-confluent conditions is measured. Constitutive expression of
rat UR gene in murine fibroblasts 3T3-L1 can be done by using
retroviral systems. Full-length rat UR cDNA are inserted into
retroviral expression vector MV7. Infected 3T3-L1 cells that are
G418-resistant are treated with insulin, dexamethacine, and
1-methyl-3-isobutylxanthine (MIX) to induce adipocyte
differentiation. A control experiment can be done by inserting
human UR cDNA into MV7 in the antisense orientation. Cells infected
with hUR-antisense constructs and parent 3T3-L1 cells are also
treated with the same insulin cocktail under same cell density.
Cells infected with rUR are shown to accumulate more Red oil O
positive lipid drops than parent cells, while cells infected with
hUR antisense are shown to have less Red oil O positive lipid
drops. Thus, the finding shows that the expression of UR in
fibroblasts plays a role in adipocyte differentiation.
[0044] Without further elaboration, it is believed that one skilled
in the art can, based on the description herein, utilize the
present invention to its fullest extent. The following specific
examples, which described syntheses, screenings, and biological
testings of various compounds of this invention, are therefore, to
be construed as merely illustrative, and not limitative of the
remainder of the disclosure in any way whatsoever. All publications
recited herein, including patents, are hereby incorporated by
reference in their entirety.
[0045] Preparation of Phenylalanine Conjugated-Steroid
Derivatives
[0046] To a stirred solution of L-(or D-) phenylalanine ester
hydrochloride (2 mmol) in dry DMF (10 mL) was added triethylamine
(2 mmol) and the mixture was stirred at room temperature for 10
minutes. Bile acid (1 mmol) and
1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide (2 mmol) were then
added and the suspension was stirred at room temperature overnight.
The reaction mixture was diluted with water and ethyl acetate. The
organic layer was separated and the water layer was extracted with
ethyl acetate again. The combined organic layer was then washed
with 1N HCl, water, 1N NaOH and water, and dried (MgSO.sub.4). The
solvent was removed under reduced pressure to afford the steroid
derivatives which were then analyzed by Thin Layer Chromatography,
High Pressure Liquid Chromatography, and/or proton-NMR.
[0047] Preparation of
Ethyl-3-alpha,6-alpha-dihydroxy-delta[24]-5-beta-cho-
lestanoate
[0048]
Ethyl-3-alpha,6-alpha-dihydroxy-delta[24]-5-beta-cholestanoate was
prepared according to methods described above. .sup.1H NMR: 0.63
(C18); 0.90 (C19); 1.29 (C21); 1.88 (C26); 3.61 (C3); 4.04 (C6);
4.22 (C28); 5.88 (C24).
[0049] Preparation of
3-alpha,6-alpha-dihydroxy-delta[24]-5-beta-cholestan- -27-oic
Acid
[0050] 3-Alpha,6-alpha-dihydroxy-delta[24]-5-beta-cholestan-27-oic
acid was prepared according to methods described above. .sup.1H
NMR: 0.63 (C18); 0.90 (C19); 1.29 (C21); 1.88 (C26); 3.61 (C3);
4.04 (C6); 4.22 (C28); 6.85 (C24).
[0051] Preparation of
Ethyl-3-beta-hydroxy-delta[5,24]-cholestenoate
[0052]
Ethyl-3-alpha,6-alpha-dihydroxy-delta[24]-5-beta-cholestanoate was
prepared according to methods described above. .sup.1H NMR: 0.68
(C18); 0.95, 1.00 (C19, C21); 1.83 (C26); 3.50 (C3); 4.19 (C28);
5.34 (C5); 6.74 (C24); .sup.13C NMR: 72.0 (C3); 121.9 (C5); 143.3
(C6); 168.8 (C27); 127.8, 141.2, 144.0 (C24, C25).
[0053] Preparation of 3-beta-hydroxy-delta[5,24]-cholesten-27-oic
Acid
[0054] 3-Alpha,6-alpha-dihydroxy-delta[24]-5-beta-cholestan-27-oic
acid was prepared according to methods described above. .sup.1H
NMR: 0.68 (C18); 0.95, 1.00 (C19, C21); 1.83 (C26); 3.50 (C3); 4.19
(C28); 5.34 (C5); 6.79 (C24).
[0055] Yeast Two-Hybrid Binding Assay
[0056] A commercial yeast two-hybrid kit from Stratagene,
HybriZAP-2.1.TM., was used to construct primary screening system.
Four pairs of degenerated oligonucleotides were annealed, digested
with EcoRI and SalI, and purified. The sequences of the four pairs
of oligonucleotides are listed as follows (N represents A, G, T or
C):
1 WB1: 5'-GTA TCG CCG GAA TTC NNN TTG NNN (SEQ ID NO: 2) NNN TTG
TTG NNN NNN TAA GTC GAC TCT AGA GCC-3' WB2: 5'-GGC TCT AGA GTC GAC
TTA NNN NNN (SEQ ID NO: 3) CAA CAA NNN NNN CAA NNN GAA TTC CGG CGA
TAC-3' LS1: 5'-GTA TCG CCG GAA TTC ATC TTG CAC (SEQ ID NO: 4) AGA
TTG TTG CAA GAA TAA GTC GAC TCT AGA GCC-3' LS2: 5'-GGC TCT AGA GTC
GAC TTA TTC TTG (SEQ ID NO: 5) CAA CAA TCT GTG CAA GAT GAA TTC CGG
CGA TAC-3' WD1: 5'-GTA TCG CCG GAA TTC NNN TTG NNN (SEQ ID NO: 6)
NNN TGG TTG TTG NNN NNN TAA GTC GAC TCT AGA GCC-3' WD2: 5'-GGC TCT
AGA GTC GAC TTA NNN NNN (SEQ ID NO: 7) CAA CAA CCA NNN NNN CAA NNN
GAA TTC CGG CGA TAC-3'
[0057] The purified fragments were cloned into the yeast vector
pBD-GAL4 (Strategene) of the same restriction sites. The resulting
plasmid pCAM/BDs expressed a fusion protein with a GAL4 DNA-binding
domain (amino acid 1-147 of Gal4) and a polypeptide of ten amino
acid in length with a LXXLL (SEQ ID NO: 8) or LXXWLL (SEQ ID NO: 9)
motif. UR ligand binding domain (amino acids 141 to 443 of rUR) was
generated with PCR and inserted into another yeast vector
pAD-GAL4-2.1 (Strategene) with cloning site EcoRI and XhoI. The
resulting plasmid, p2.1/rURLB, expressed a fusion protein
containing a Gal4 transcription activation domain (amino acids
761-881 of Gal4) and a rUR ligand binding domain.
[0058] Plasmids pCAM/BDs and p2.1/rURLB were co-transformed into an
appropriate yeast strain by using lithium acetate and polyethylene
glycol. The yeast was then grown on selective medium until the
formed yeast colonies reached 2 mm. Colonies were picked and grown
in selective medium for 15 hours at 30.degree. C. and
.beta.-galactosidase activities were measured with a commercial
luminescence kit.
[0059] Mammalian Cell Transfection Assay (1)
[0060] Rat UR and human RXRa gene were cloned into a mammalian
expression vector pSG5 (Strategene) by transfection with calcium
phosphate and overexpressed in cultured cells. A heterogeneous
promoter was constructed by inserting into the vector four tandem
repeats of DR4 with sequence 5'-TTC AGG TCA CAG GAG GTC AGA GAG
CT-3' (SEQ ID NO: 10) upstream to a c-fos promoter sequence
(-56-+109) which was followed by a sequence encoding luciferase.
The entire construct was named DR4-fos-luc. DR4-fos-luc was then
co-transfected with pSG5/rUR and pSG5/hRXRa into COS-1 cells. 16-24
hours after transfection, a steroid derivative in ethanol was added
to the medium until the maximum final concentration is 2 .mu.M. The
final concentration of solvent ethanol is 0.2%. After 24-48 hours,
cells that were treated with the steroid were lysed and assayed for
luciferase activity with a commercial assay kit and a
luminometer.
[0061] A wide variety of compounds of this invention were tested
and found to modulate transactivation activity of UR or LXRa. For
example, steroid (1) (see page 5, supra), unexpectedly increased
the luciferase activity by 15-fold in comparison to absence of
steroid only for UR but not LXRa; steroid (2) unexpectedly
increased the luciferase activity by 60-fold in comparison to
absence of steroid only for LXRa but not UR; steroid (3), (5) or
(10) can activate both UR or LXRa; steroid (7), (8), or (9) can
antagonize UR or LXRa transactivation acitvity.
[0062] Mammalian Cell Transfection Assay (2)
[0063] In a similar fashion to the experiment described above,
another chimeric receptor was constructed by fusing
oligonucleotides encoding the ligand-binding domain of rat UR (141
to 443 amino acid residues) to a human AR gene lacking
ligand-binding coding region (human AR 1 to 623 amino acid
residues) and overexpressed in cultured cells. For this chimeric
receptor, a reporter gene ARE-fos-luc was constructed by inserting
into the vector three tandem repeats of Androgen Response Element
(ARE) with a sequence 5'-TCG AGT CTG GTA CAG GGT GTT CTT TTG-3'
(SEQ ID NO: 11) upstream to a c-fos promoter sequence (-56-+109)
which was followed by a sequence encoding luciferase.
[0064] Various steroid derivatives of this invention were found to
modulate UR transactivation activity on DR4-fos-luc expression in
the cultured cells. For example, steroid derivative (6) (see page
6, supra) unexpectedly increased the luciferase activity by 5-fold
in comparison to the steroid starting material.
[0065] Mammalian Cell Transfection Assay (3)
[0066] Human embryonic kidney 293 cells were seeded into 48-well
culture plates at 10.sup.5 cells per well in DMEM supplemented with
10% fetal bovine serum. After 24 hours, cells were transfected by a
calcium phosphate coprecipitation method with 250 ng of the
pGL3/UREluc reporter gene which consists of three copies of
AGGTCAagccAGGTCA fused to nucleotides -56 to +109 of the human
c-fos promoter in front of the firefly luciferase gene in the
plasmid basic pGL3 (Promega), 40 ng pSG5/RXRa, 40 ng pSG5/rUR or
CMX/hLXR, 10 ng pSG5/hGrip1, 0.4 ng CMV/R-luc (transfection
normalization reporter, Promega) and 250 ng carrier DNA per well.
Alternatively, 500 ng of the pGL2/7aluc reporter gene which
consists of a single copy of nucleotides -101 to -49 of the rat
7a-hydroxylase gene fused to the SV40 promoter in front of the
firefly luciferase gene in the plasmid basic pGL2 (Promega) was
used instead of pGL3/UREluc. This reporter does not have response
elements for COUP-TFII or HNF4. In some experiments, 500 ng of the
human 7.alpha.-hydroxylase gene reporter, PH/hCYP7A-135, which
consists of a single copy of nucleotides -135 to +24 of the human
CYP7A gene fused to the firefly luciferase gene in the plamid basic
pGL3 (Promega), was used instead of pGL2/7aluc. After another 12-24
hours, cells were washed with PBS and refed with DMEM supplemented
with 4% delipidated fetal bovine serum. Steroid derivatives
dissolved in ethanol were added in duplicate to the medium so that
the final concentration of alcohol was 0.2%. After 24-48 hours,
cells were harvested and luciferase activity was measured with a
commercial kit (Promega Dual luciferase II) on a Monolight
luminometer (Beckton Dickenson). Both LXR and UR form heterodimers
with RXR for gene transactivation. The ligand for RXR, 9-cis
retinoic acid, is known to activate the LXR/RXR heterodimer but
addition of 9-cis retinoic acid to transactivation assays did not
change the potency of either .DELTA..sup.5 or 6.alpha.-hydroxy
steroids for activation of LXR or UR (data not shown). The
expression of endogenous LXR and UR (and TR which also binds to a
DR4 response element) were apparently low since reporter activation
was low in the absence of added expression vectors for LXR or UR.
Reporter activation was also low when the DR4 response-element was
replaced with a glucocorticoid receptor response element. Each
experiment was repeated as least twice to demonstrate
reproducability. Relative light units were about 2.times.10.sup.7
for pGL3/UREluc, 1.times.10.sup.6 for pGL2/7aluc, 5.times.10.sup.4
for PH/hCYP7A-135 and 5.times.10.sup.5 for CMV/R-luc. Purity of
synthesized steroid derivatives was verified by thin layer
chromatography and structures were confirmed using proton and
C.sup.13 magnetic resonance spectrometry.
3-Oxo-6.alpha.-hydroxy-5.beta.-- cholanoic acid methyl ester,
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid methyl ester,
and 3.alpha.,6.alpha.,7.alpha.-trihydroxy-5.beta.-chol- anoic acid
methyl ester were found to be as potent as
3.beta.-hydroxy-.DELTA..sup.5-cholanoic acid methyl ester as
activators for LXR, with ED.sub.50's of about 150 nM. Loss of
activity was seen when the 6.alpha.-hydroxy group was changed to a
6.beta. configuration. In contrast to activity with LXR,
3.beta.-hydroxy-.DELTA..sup.5-cholanoic acid methyl ester
(ED.sub.50 of 130 nM) was more active than
3-oxo-6.alpha.-hydroxy-cholanoic acid methyl ester (ED.sub.50 of
550 nM) and 3.alpha.,6.alpha.-dihydroxy-cholanoic acid methyl ester
(ED.sub.50 of 500 nM) for UR activation.
[0067] Using the same assay, ED.sub.50's of 6.alpha.-hydroxylated
steroids with 24-keto side chains include free and conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid and
3.alpha.,6.alpha.,7.alpha.-trihydroxy-5.beta.-cholanoic acid were
determined. These steroid derivatives were found to be more
selective activators of LXR than UR.
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid activated LXR
with an ED.sub.50 of 17 mM for the free acid and 3 mM for its
taurine conjugate. Free and taurine-conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acids activated UR
with ED.sub.50 of 55 mM and 11 mM, values three to four times
higher than those for LXR. Cholanoic acid derivatives containing
trifluoromethyl moiety were also found to be selective activators
of LXR.
[0068] The ability of taurine-conjugated
3.alpha.,6.alpha.-dihydroxy-5.bet- a.-cholanoic acid to activate
LXR using the natural response element derived from the rat
7a-hydroxylase promoter was also investigated. It was found that
taurine-conjugated 3.alpha.,6.alpha.-dihydroxy-5.beta.-cho- lanoic
acid activated LXR but not UR using this reporter gene, with an
ED.sub.50 of 10 mM. To investigate if LXR can activate human CYP7A
gene transcription, a chimeric reporter plasmid, in which the
nucleotides -135 to +24 of the human CYP7A promoter were fused to
the luciferase gene, was used in a co-transfection assay in human
embryonic kidney 293 cells along with LXR, RXR and Grip1 expression
plasmids. It was found that LXR can activate reporter gene
expression in the presence of taurine-conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid.
Taurine-conjugated 3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanoic
acid, on the other hand, suppressed reporter gene expression.
Another compound, 3.beta.-hydroxy-5-cholesten-25(R)-26-carboxylic
acid activated LXR with an ED.sub.50 of 300 nM and UR with an
ED.sub.50 of over 2 .mu.M. Its taurine-conjugated counterpart was
also found to be able to transactivate both LXR and UR. On the
other hand, many of its related metabolites were found to be
inactive on either receptors.
[0069] Protease Protection Assay
[0070] Rat UR protein radio-labeled with .sup.35S-Met is produced
with a commercial kit in an in vitro system. The radio-labeled
protein is incubated with steroid derivatives with final
concentration of up to 1 mM for 2 hours on ice, and digested with
trypsin for 30 minutes at 37.degree. C. for 20 minutes. The
protected fragments were separated from free .sup.35S-Met by
polyacrylamide electrophoresis and visualized by exposing dried
gels to X-ray films.
[0071] The patterns of the X-ray film indicate that steroid
derivatives of this invention bind to and protect UR from being
digested by trypsin. Some examples of such a steroid derivative
include 5.beta.-androstan-3a, 17b-diol,
5.beta.-androstan-3a-ol-16-one, .DELTA..sup.5-Pregnen-3b-ol-20--
one, 5a-androstan-3-one, 5.alpha.-androstan-17-ol-3-one,
5a-androstan-3b-ol-17-carboxylic acid, 5a-pregnan-3,20-dione, and
.DELTA..sup.5-androsten-3b,17b-diol.
[0072] Incubation of UR with increasing concentrations of trypsin
in the absence of 3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic
acid methyl ester leads to extensive digestion of the receptor. In
contrast, when UR was incubated with 5 mM
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid methyl ester,
two protease-resistant fragments of 35 and 26 kDa were observed. A
similar protected pattern was observed with taurine-conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid.
[0073] Co-activator Binding Assay
[0074] A fusion protein formed between glutathione S-transferase
and Grip1 (termed GST-Grip1) was expressed in E. Coli. The bacteria
was lysed by sonication in the presence of detergent NP40 0.1% and
Tween-20 0.5%. Soluble GST-Grip1 was separated from insoluble
debris by centrifugation at 50,000 G at 4.degree. C. for 30
minutes. The soluble fusion protein was then immobilized to
glutathione-agarose. Radiolabeled rat UR protein was incubated with
GST-Grip1 coated glutathione-agarose in the presence of a test
compound of this invention for 2 hours at 22.degree. C. under
agitation. UR that did not bind to the agarose was washed away.
Bound UR was eluted with solution containing SDS and
.beta.-mercaptoethanol and separated from free .sup.35S-Met with
polyacrylamide electrophoresis, and finally visualized by exposure
the dried gel to X-ray films. Diosgenin was shown to be capable of
promoting UR and Grip1 protein interaction in this assay.
[0075] Another fusion protein GST-rUR was expressed in E. Coli
strain BL21 using the expression plasmid pGEX using a method
similar to that as described above. Transfected cells were lysed by
one cycle of freeze-thaw and sonication. Supernatant, prepared by
centrifugation at 45,000 G for 1 hour, was incubated with
glutathione-agarose for 10 min at 4.degree. C. The agarose was
washed with binding buffer (20 mM Hepes, pH 7.5, 10 mM EDTA, 10 mM
Na.sub.2MoO.sub.4, 1 mM .beta.-mercaptoethanol, 1 mM DTT, 0.5 mM
PMSF, 2 ug/ml aprotinin). Human Grip1 was produced by in vitro
translation using a rabbit reticulocyte lysate and labeled with
[.sup.35S]-methionine. [.sup.5]-Grip1 in reticulate lysate (2 ml)
was added to GST-UR bound to agarose beads in 100 ul binding
buffer. Test chemicals in ethanol were added to the mixture and the
slurry was shaken at room temperature for 30 min. The agarose beads
were then washed three times with binding buffer. Bound protein was
eluted with SDS-PAGE loading buffer and separated on a 8% SDS-PAGE
gel. Gels were dried and subjected to autoradiography. Radioactive
Grip1 was measured with a STORM phosphoimager (Molecular
Dynamics).
[0076] Both 3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid
methyl ester and 22R-hydroxy cholesterol promoted interaction of
Grip1 with GST-UR and taurine-conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholano- ic acid promoted
interaction of Grip1 with GST-LXR. Taurine-conjugated
3.alpha.-hydroxy-5.beta.-cholanoic acid, taurine-conjugated
3.alpha.-hydroxy-5.beta.-cholanoic acid, and taurine-conjugated
3.alpha.,7.alpha.-dihydroxy-5.beta.-cholanoic acid all failed to
enhance coactivator-receptor interaction under the same
conditions.
[0077] Using the same conditions,
3.beta.-hydroxy-5-cholesten-25(R)-26-car- boxylic acid was found to
bind to and form complexes with LXR and nuclear receptor
co-activator Grip 1, indicating that this acid bound to LXR and
induced a conformation change that favored co-activator binding. In
a dose response analysis,
3.beta.-hydroxy-5-cholesten-25(R)-26-carboxylic acid increased the
amount of [.sup.35S]-Grip1 bound to LXR with an EC.sub.50 value of
300 nM, which correlates with the cell-based transfection assay.
These data showed that 3.beta.-hydroxy-5-cholesten-25-
(R)-26-carboxylic acid is a LXR agonist.
[0078] Inhibition of De Novo
[0079] Cholesterol Synthesis in Cultured Cells
[0080] On day 1, PC-3 cells stably expressing rUR gene by
retroviral infection were seeded in media containing delipidated
serum. On day 2, cells were treated with an ethanol solution
containing a test compound at maximum concentration of 2 .mu.M. On
day 3, cells were washed with PBS and treated with 100 mg/ml
amphotericin B in Dulbecco's Modified Eagle Medium (DMEM) without
serum at 37.quadrature.C. 4 hours later, cells were then washed and
treated with solution containing 80% water and 20% DMEM for 30
minutes. Surviving cells were assessed using a colorimetric assay.
Cells were fixed in 10% trichloroacetic acid (TCA) and stained with
sulforhodamine B. The amount of dye is linear to number of fixed
cells on the culture plates. Cells with cholesterol in the cell
membrane were killed by amphotericin B treatment.
[0081] Compounds of this invention were found to inhibit
cholesterol synthesis of the cell to various extent.
[0082] Measuring the Level of Inflammation
[0083] In Cells by Monitoring the Amount of NO.sub.2
[0084] Murine macrophage cell line RAW264.7 were incubated with a
test compound at maximum final concentration of 2 .mu.M for 24
hours. The macrophages were then activated by adding
lipopolysaccharide (100 ng/mL) and .gamma.-interferon (100
units/mL). The nitrogen monoxide (NO) production of activated
macrophages was measured indirectly by quantifying nitrogen dioxide
(NO.sub.2) in the media according to Green L. et al., Anal.
Biochem. 126, 131-138 (1982). Compounds of this invention were
found to inhibit cholesterol synthesis of the cell to various
extent.
[0085] Macrophage-Foam Cell Transformation
[0086] Constitutive expression of rat UR and human RXRa gene by
retroviral systems in RAW264.7 transformed these cells into
foam-cell-like morphology and integrated into clamps while
increasing cell sizes and undergoing apoptosis. Foam cells
originated from macrophages are the major components in
pathological plaques formed on the inner wall of blood vessels
which are a characteristic feature in atherosclerosis. Compounds of
this invention were shown to be able to suppress the progression of
macrophage-foam cell transformation at different stages, and thus
can be used in the treatment or prevention of atherosclerosis.
[0087] Adipocyte Differentiation
[0088] Constitutive expression of rat UR gene in murine fibroblasts
3T3-L1 was done by using retroviral systems. Full-length rat UR
cDNA was inserted into retroviral expression vector Mv7. Infected
3T3-L1 cells that are G418-resistant were treated with 5 .mu.g/ml
insulin, 250 nM dexamethacine, and 0.5 mM
1-methyl-3-isobutylxanthine (MIX) to induce adipocyte
differentiation. A control experiment was done by inserting human
UR cDNA into MV7 in the antisense orientation. Cells infected with
hUR-antisense constructs and parent 3T3-L1 cells were also treated
with the same insulin cocktail under same cell density. Cells
infected with rUR were shown to accumulate more Red oil O positive
lipid drops than parent cells, while cells infected with hUR
antisense were shown to have less Red oil O positive lipid
drops.
[0089] Erythrocyte Differentiation
[0090] Constitutive expression of rat UR gene in murine NN10,
IW32.1 or IW201 was done by using retroviral systems. Full-length
rat UR cDNA was inserted into retroviral expression vector MV7.
Infected cells that were G418-resistant were cultured upto 5 days
to induce erythrocyte differentiation. A control experiment was
done by using parent MV7 vector. NN10, IW32.1 or IW201 cells
infected with parent MV7 construct were also treated with G418 in
parallel under same cell density. More cells infected with rUR were
shown to accumulate hemoglobin protein (stained with benzidine)
than parent or control cells. When IW32.1/rUR cells were cultured
on fibronectin-coated plates, some cells differentiated into mature
enucleated reticulocytes.
[0091] Animal Studies
[0092] Male Sprague-Dawley rats that were 50 days old were fed a
regular chow diet and tap water ad libitum for 1 week during
acclimatization, and then randomly divided into groups that were
given different dietary treatments. Both control and treatment
groups were initially fed ad libitum a cholesterol-enriched diet,
which was prepared by adding 2% cholesterol and 1%
3.alpha.,7.alpha.,12.alpha.-trihydroxy-5.beta.-cholano- ic acid to
the regular chow diet. The treatment group received the same diet
supplemented with 0.03% test steroid derivative. Rats were fasted
overnight before determining body and liver weight and drawing
blood from the tail vein for serum total cholesterol measurements.
Total cholesterol was determined enzymatically with a diagnostic
kit (Sigma, St. Louis, Mo.) on day 0 and 7. Average food
consumption was 20-25 g/rat/day and average feces production was 9
g/rat/day. There was no statistical difference between control and
treatment groups for food consumption and feces production. The
dose for test steroid derivative in the treatment group was 40-50
mg/kg/day. Rats on high cholesterol/bile acid diet and treated with
a trifluoromethyl conjugated 3.alpha.,6.alpha.-dihydroxy-5.b-
eta.-cholanoic acid had a 20% drop (p<0.05) in the serum total
cholesterol compared with the level in untreated animals (Table 1).
Food consumption, body and liver weight were similar in the control
and treatment groups. In another experiement, rats were made
hypercholesterolemic with a high cholesterol/cholic acid diet and
then treatment with the same trifluoromethyl conjugated
3.alpha.,6.alpha.-dihydroxy-5.beta.-cholanoic acid again lowered
the serum total cholesterol by 20% compared with untreated
animals.
[0093] Other Embodiments
[0094] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. For example, the moiety
A can be a side chain of an amino acid which is structurally
similar to a naturally occurring amino acid described above. One
specific example of A is a side chain of phenylglycine. Thus, other
embodiments are also within the claims.
* * * * *