U.S. patent application number 10/305603 was filed with the patent office on 2004-04-29 for inhibitors of type 5 and type 3 17beta-hydroxysteroid dehydrogenase and methods for their use.
This patent application is currently assigned to Endorecherche, Inc.. Invention is credited to Belanger, Alain, Gauthier, Sylvain, Labrie, Fernand, Luu-The, Van, Merand, Yves, Poirier, Donald, Provencher, Louis, Singh, Shankar M..
Application Number | 20040082556 10/305603 |
Document ID | / |
Family ID | 26759352 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082556 |
Kind Code |
A1 |
Labrie, Fernand ; et
al. |
April 29, 2004 |
Inhibitors of type 5 and type 3 17beta-hydroxysteroid dehydrogenase
and methods for their use
Abstract
Novel methods of medical treatment and/or inhibition of
development of diseases are disclosed for diseases that are
sensitive to androgenic or estrogenic activity. The treatments
utilize inhibitors of type 5 and/or type 3 17.beta.-hydroxysteroid
dehydrogenase. Novel inhibitors of type 5 17.beta.-hydroxysteroid
dehydrogenase are also disclosed, as are novel inhibitors of type 3
17.beta.-hydroxysteroid dehydrogenase.
Inventors: |
Labrie, Fernand;
(Sainte-foy, CA) ; Belanger, Alain; ( Cap-Rougc,
CA) ; Gauthier, Sylvain;
(Saint-Augustin-de-Desmaures, CA) ; Luu-The, Van;
(Charny, CA) ; Merand, Yves; (Sainte-foy, CA)
; Poirier, Donald; (L' Ancienne-Lorette, CA) ;
Provencher, Louis; (Ste-Julie, CA) ; Singh, Shankar
M.; (Sainte-Foy, CA) |
Correspondence
Address: |
OSTROLENK FABER GERB & SOFFEN
1180 AVENUE OF THE AMERICAS
NEW YORK
NY
100368403
|
Assignee: |
Endorecherche, Inc.
|
Family ID: |
26759352 |
Appl. No.: |
10/305603 |
Filed: |
November 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10305603 |
Nov 26, 2002 |
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09265807 |
Mar 10, 1999 |
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6541463 |
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60077510 |
Mar 11, 1998 |
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60095623 |
Aug 7, 1998 |
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Current U.S.
Class: |
514/178 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/566 20130101; A61P 17/00 20180101; A61P 15/00 20180101;
C07J 21/00 20130101; A61K 31/585 20130101; A61K 45/06 20130101;
A61P 17/08 20180101; A61P 5/28 20180101; A61P 13/08 20180101; A61P
5/24 20180101; A61P 17/14 20180101; A61K 31/5685 20130101; A61P
43/00 20180101; A61P 17/10 20180101; A61K 31/566 20130101; A61K
2300/00 20130101; A61K 31/5685 20130101; A61K 2300/00 20130101;
A61K 31/585 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/178 |
International
Class: |
A61K 031/57 |
Claims
What is claimed is:
1. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 115wherein the dotted
line is optional pi bond; wherein A is selected from the group
consisting of straight or branched C.sub.1-C.sub.4 alkyl,
--OR.sup.c (R.sup.c being a C.sub.1-C.sub.8 alkyl radical), and
--N(R.sup.d)R.sup.e (R.sup.d and R.sup.e being independently
hydrogen or C.sub.1-C.sub.8 alkyl or aryl), and unsaturated analogs
of any of the foregoing definitions for substituent A; wherein
R.sup.1 is selected from the group consisting of hydrogen and
methyl and ethyl; wherein R.sup.6 is selected from the group
consisting of hydrogen, and halogen, and C.sub.1-C.sub.8 alkyl;
wherein R.sup.a is selected from the group consisting of straight
or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and R.sup.b is selected from the group consisting of
hydrogen, substituted or unsubstituted phenyl, C.sub.2-C.sub.10
acyl, C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl,
and halogen.
2. The method of claim 1 wherein the optional pi bond at 6 is
present.
3. The method of claim 1 wherein R.sup.6 is methyl.
4. The method of Cairn 1 wherein R.sup.a is C.sub.1-C.sub.6
alkylene.
5. The method of claim 1 where A is either methyl or
--N(R.sup.d)R.sup.e.
6. The method of claim 1 where A is --N(R.sup.d)R.sup.e.
7. The method of claim 6 wherein R.sup.d is methyl.
8. The method of claim 6 wherein R.sup.e is C.sub.1-C.sub.6 alkyl
or C.sub.7-C.sub.12 phenyl alkyl.
9. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 116wherein the dotted
line is an optional pi bond; wherein R.sup.16.beta. is selected
from the group consisting of hydrogen, C.sub.1-C.sub.8 alkyl,
fluoro, chloro, C.sub.1-C.sub.8 haloalkyl, a moiety which together
with R.sup.16.alpha. is C.sub.4-C.sub.7 spirocycloalkyl,
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16
being C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.; wherein R.sup.16.alpha. is
selected from the group consisting of hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 haloalkyl, a moiety which together with
R.sup.16.beta. forms C.sub.4-C.sub.7 spirocycloalkyl,
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16
being C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoings; wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl; wherein R.sup.19 is either --H
or --CH.sup.3; and wherein R.sup.6 is selected from the group
consisting of --H, --CH.sub.3, and halo; provided that
R.sup.16.beta., R.sup.16.alpha., and R.sup.15.alpha. are not
simultaneously hydrogen.
10. The method of claim 9 wherein R.sup.16.alpha. is a larger
substituent than R.sup.16.beta..
11. The method of claim 9 wherein R.sup.6 is hydrogen.
12. The method of claim 9 wherein the optional pi bond at position
1 is not present.
13. The method of claim 9 wherein R.sup.16.alpha. is a
C.sub.3-C.sub.5 alkyl chain.
14. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 117wherein the dotted
line is optional pi bond wherein X is C.sub.1-C.sub.3 alkyl;
wherein Y is hydrogen or an acyloxy moiety; wherein R.sup.6 is --H
or --CH.sub.3; wherein R.sup.16 is --H or halo; wherein R.sup.1 is
--H or --CH.sub.3.
15. The method of claim 14 wherein R.sup.6 is methyl.
16. The method of claim 14 wherein the optional pi bond at position
1 is present.
17. The method of claim 14 wherein Y is a C.sub.3-C.sub.6
fluoroacyloxy.
18. The method of claim 14 wherein X is methyl.
19. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 118wherein n is an
integer from 1-2; wherein the dotted lines are optional double
bonds; wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl; wherein Z is selected from the group
consisting of --H and (C.sub.1-C.sub.3)alkyl; wherein R.sup.3 is
selected from the group consisting of hydrogen, acyl, carboxyl,
alkoxycarbonyl, substituted or unsubstituted carboxamide, cyano,
alkoxy, alkoxyalkoxy, alkythioalkoxy, acyloxy; hydroxy, halo,
--O--SO.sub.2R.sup.a (R.sup.a being selected from the group
consisting of C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and
a moiety which, together with R.sub.2, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom; wherein
R.sup.2 is selected from the group consisting of hydrogen, amido,
acyloxy, carboxyl, carboxamide, alkoxycarbonyl, cyano, halo, nitro,
C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a moiety which, together
with R.sub.3, is a 5-6 member ring containing at least one oxygen
and one nitrogen atom; wherein R.sup.4 is hydrogen or halo; wherein
R.sup.6 is selected from the group consisting of hydrogen and oxo;
wherein R.sup.9 is --H or --OH provided that X, Y, and Z are not
all hydrogen when R.sup.3 is methoxy.
20. The method of claim 19 wherein R.sup.3 is alkoxyalkoxy.
21. The method of claim 19 wherein R.sup.3 is carboxyl or
alkoxyl;
22. The method of claim 19 wherein at least one of X, Y or Z is
methyl.
23. The method of claim 19 wherein R.sup.3 is carboxamide.
24. The method of claim 19 wherein both X and Y are methyl.
25. The method of claim 19 wherein n=1 or 2.
26. The method of claim 19 wherein R.sup.6 is oxo.
27. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 119wherein the dotted
lines are optional pi bonds; wherein n=1 or 2; and wherein a is
either --H or --CH.sub.3, wherein b and c are independently
hydrogen or methyl; wherein Z is oxygen or sulfur.
28. The pharmaceutical composition of claim 27 wherein n=1.
29. The method of claim 27 wherein at least one of b or c is
methyl.
30. The method of claim 27 wherein both b and c are methyl.
31. The method of claim 27 wherein Z is oxygen.
32. A method of inhibiting the activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase selected from the group consisting of:
120121122123124
33. The method of claim 32 wherein an inhibitor of
17.beta.-hydroxysteroid dehydrogenase selected from the group
consisting of: 125is administered.
34. The method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase selected from the group consisting of: 126127and
compounds having the molecular structure: 128wherein the dotted
lines are optional pi bonds; wherein A is selected from the group
consisting of straight or branched C.sub.1-C.sub.4 alkyl,
--OR.sup.c (R.sup.c being a C.sub.1-C.sub.4 alkyl radical), and
--N(R.sup.d)R.sup.e (R.sup.d and R.sup.e being independently
hydrogen or C.sub.1-C.sub.8 alkyl or aryl), and unsaturated analogs
of any of the foregoing definitions for substituent A; wherein
R.sup.1 is selected from the group consisting of hydrogens, and
methyl; wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-C.sub.8 alkyl; wherein R.sub.16 is
selected from the group consisting of H,H and CH.sub.2; wherein
R.sup.a is selected from the group consisting of straight or
branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7 cycloalkylene;
and R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen.
35. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 129wherein the dotted
line is an optional pi bond; wherein R.sup.16.beta. is selected
from the group consisting of hydrogen, C.sub.1-C.sub.8 alkyl
fluoro, chloro, C.sub.1-C.sub.8 haloalkyl, a moiety which together
with R.sup.16.alpha. forms C.sub.4-C.sub.7 spirocycloalkyl,
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16
being C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.; wherein R.sup.16.alpha. is
selected from the group consisting of hydrogen, C.sub.1-C.sub.8
alkyl C.sub.1-C.sub.8 haloalkyl, a moiety which together with
R.sup.16.beta. forms C.sub.4-C.sub.7 spirocycloalkyl
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16
being C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing; wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl; wherein R.sup.19 is either --H
or --CH.sub.3; and wherein R.sup.6 is selected from the group
consisting of --H, --CH.sub.3, and halo; provided that
R.sup.16.beta., R.sup.16.alpha., and R.sup.15.alpha. are not
simultaneously hydrogen.
36. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 130wherein n=1 or 2;
wherein the dotted lines are optional pi bonds; wherein X and Y are
independently selected from the group consisting of --H,
(C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3) alkenyl, and
methoxycarbonyl; wherein Z is selected from the group consisting of
--H and (C.sub.1-C.sub.3)alkyl; wherein R.sup.3 is selected from
the group consisting of acyl, carboxyl, alkoxycarbonyl, substituted
or unsubstituted carboxamide, cyano, alkoxy, alkoxyalkoxy,
alkythioalkoxy, acyloxy; hydroxy, halo, --O--SO.sub.2R.sup.a
(R.sup.a being selected from the group consisting of
C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and a moiety
which, together with R.sub.2, is a 5-6 member ring containing at
least one oxygen and one nitrogen atom; wherein R.sup.2 is selected
from the group consisting of hydrogen, amido, acyloxy, carboxyl,
carboxmide, alkoxycarbonyl, cyano, halo, nitro, C.sub.1-C.sub.8
alkyl and CF.sub.3 and a moiety which, together with R.sub.3, is a
5-6 member ring containing at least one oxygen and one nitrogen
atom; wherein R.sup.4 is hydrogen or halo; wherein R.sup.6 is
selected from the group consisting of hydrogen and oxo; wherein
R.sup.9 is --H or --OH; provided that X, Y and Z are not all
hydrogen.
37. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 131wherein the dotted
lines are optional pi bonds; wherein n=1 or 2; and wherein a is
either --H or --CH; wherein b and c are independently hydrogen or
methyl; wherein Z is oxygen or sulfur.
38. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 132wherein the dotted
line is an optional pi bond; wherein A is selected from the group
consisting of straight or branched C.sub.1-C.sub.4 alkyl, OR.sup.c
(R.sup.c being a C.sub.1-C.sub.8 alkyl radical), and
--N(R.sup.d)R.sup.e (R.sup.d and R.sup.e being independently
hydrogen or C.sub.1-C.sub.8 alkyl or aryl), and unsaturated analogs
of any of the foregoing definitions for substituent A; wherein
R.sup.1 is selected from the group consisting of hydrogen and
methyl; wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-C.sub.8 alkyl; wherein R.sup.a is
selected from the group consisting of straight or branched
C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7 cycloalkylene; and
R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen; provided that wherein A is methyl, R.sup.a and R.sup.b
together have at least two carbon atoms and R.sup.1 is methyl.
39. A method of inhibiting activity of type 5
17.beta.-hydroxysteroid dehydrogenase comprising administering to a
patient in need of such treatment a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 133wherein the dotted
lines are optional pi bonds wherein X is C.sub.1-C.sub.3 alkyl;
wherein Y is hydrogen or an acyloxy moiety; wherein R.sup.6 is --H
or --CH.sub.3; wherein R.sup.16.alpha. is --H or halo; wherein
R.sup.1 is --H or --CH.sub.3.
40. A pharmaceutical composition comprising a pharmaceutically
acceptable excipient, diluent or carrier and a therapeutically
acceptable amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 134wherein the dotted
lines are optional pi bonds; wherein A is selected from the group
consisting of straight or branched C.sub.1-C.sub.4 alkyl,
--OR.sup.c (R.sup.c being a C.sub.1-C.sub.8 alkyl radical), and
--N(R.sup.d)R.sup.e (R.sup.d and R.sup.e being independently
hydrogen or C.sub.1-C.sub.8 alkyl or aryl), and unsaturated analogs
of any of the foregoing definitions for substituent A; wherein
R.sup.1 is selected from the group consisting of hydrogen and
methyl; wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-C.sub.8 alkyl; wherein R.sup.a is
selected from the group consisting of straight or branched
C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7 cycloalkylene; and
R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen; provided that when A is methyl, R.sup.a and R.sup.b
together have at least two carbon atoms, and R.sup.1 is methyl.
41. The pharmaceutical composition of claim 40 wherein the optional
pi bond at 6 is present.
42. The method of claim 40 wherein R.sup.6 is methyl.
43. The pharmaceutical composition of claim 40 wherein R.sup.a is
C.sub.1-C.sub.6 alkylene.
44. The pharmaceutical composition of claim 40 where A is either
methyl or --N(R.sup.d)R.sup.e.
45. The pharmaceutical composition of claim 40 where A is
--N(R.sup.d)R.sup.e.
46. The pharmaceutical composition of claim 45 wherein R.sup.d is
methyl.
47 The pharmaceutical composition of claim 45 wherein R.sup.e is
C.sub.1-C.sub.6 alkyl or phenyl C.sub.1-C.sub.6 alkyl.
48. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 135wherein the dotted
line is an optional pi bond; wherein R.sup.16.beta. is selected
from the group consisting of hydrogen, C.sub.1-C.sub.8 alkyl,
fluoro, chloro, C.sub.1-C.sub.8 haloalkyl, a moiety which together
with R.sup.16.alpha. is C.sub.4-C.sub.7 spirocycloalkyl,
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16
being C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.; wherein R.sup.16.alpha. is
selected from the group consisting of hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 haloalkyl, a moiety which together with
R.sup.16.beta. forms C.sub.4-C.sub.7 spirocycloalkyl,
C.sub.4-C.sub.7 halospirocycloalkyl, or .dbd.--R(R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoings; wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl; wherein R.sup.19 is either --H
or --CH.sub.3; and wherein R.sup.6 is selected from the group
consisting of --H, --CH.sub.3, and halo; provided that
R.sup.16.beta., R.sup.16.alpha., and R.sup.15.alpha. are not
simultaneously hydrogen.
49. The pharmaceutical composition of claim 48 wherein
R.sup.16.alpha. is a large substituent than R.sup.16.beta..
50. The pharmaceutical composition of claim 48 wherein R.sup.6 is
hydrogen.
51. The pharmaceutical composition of claim 48 wherein the optional
pi bond at position 1 is not present.
52. The pharmaceutical composition of claim 48 wherein
R.sup.16.alpha. is a C.sub.3-C.sub.5 alkyl chain.
53. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 136wherein the dotted
line is an optional pi bond wherein X is C.sub.1-C.sub.3 alkyl;
wherein Y is hydrogen or an acyloxy moiety; wherein R.sup.6 is --H
or --CH.sub.3; wherein R.sup.16 is --H or halo; wherein R.sup.1 is
--H or CH.sub.3.
54. The pharmaceutical composition of claim 53 wherein R.sup.6 is
methyl.
55. The pharmaceutical composition of claim 53 wherein the optional
pi bond at position 1 is present.
56. The pharmaceutical composition of claim 53 wherein Y is a
C.sub.3-C.sub.6 fluoroacyloxy.
57. The pharmaceutical composition of claim 53 wherein X is
methyl.
58. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 137wherein n is an
integer from 1-2; wherein the dotted lines are optional pi bonds;
wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl; wherein Z is selected from the group
consisting of --H and (C.sub.1-C.sub.3)alkyl; wherein R.sup.3 is
selected from the group consisting of hydrogen acyl, carboxyl,
alkoxycarbonyl, substituted or unsubstituted carboxamide, cyano,
alkoxy, alkoxyalkoxy, alkythioalkoxy, acyloxy; hydroxy, halo,
--O--SO.sub.2R.sup.a (R.sup.a being selected from the group
consisting of C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and
a moiet which, together with R.sub.2, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom; wherein
R.sup.2 is selected from the group consisting of hydrogen, amido,
acyloxy, carboxyl, carboxamide, alkoxycarbonyl, cyano, halo, nitro,
C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a moiety which, together
with R.sub.3, is a 5-6 member ring containing at least one oxygen
and one nitrogen atom; wherein R.sup.4 is hydrogen or halo; wherein
R.sup.6 is selected from the group consisting of hydrogen and oxo;
wherein R.sup.9 is --H or --OH; provided that X, Y, and Z are not
all hydrogen.
59. The pharmaceutical composition of claim 58 wherein R.sup.3 is
alkoxyalkoxy.
60. The pharmaceutical composition of claim 58 wherein R.sup.3 is
carboxyl or alkoxyl;
61. The pharmaceutical composition of claim 58 wherein at least one
of X, Y or Z is methyl.
62. The pharmaceutical composition of claim 58 wherein both X and Y
are methyl.
63. The pharmaceutical composition of claim 58 wherein n=1 or
2.
64. The pharmaceutical composition of claim 58 wherein R.sup.6 is
oxo.
65. The pharmaceutical composition of claim 58 wherein R.sup.3 is
carboxamide.
66. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 138wherein the dotted
lines are optional pi bonds; wherein n=1 or 2; and wherein a is
either --H or --CH.sub.3; wherein b and c are independently
hydrogen or methyl; wherein Z is oxygen or sulfur.
67. The pharmaceutical composition of claim 66 wherein n=1.
68. The pharmaceutical composition of claim 66 wherein at least one
of b or c is methyl.
69. The pharmaceutical composition of claim 66 wherein both b and c
are methyl.
70. The pharmaceutical composition of claim 66 wherein Z is
oxygen.
71. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having a molecular structure selected from the group
consisting of: 139140141142143
72. The pharmaceutical composition of claim 71 wherein said
inhibitor of 17.beta.-hydroxysteroid dehydrogenase is selected from
the group consisting of: 144
73. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 145wherein the dotted
line is optional pi bond; wherein A is selected from the group
consisting of straight or branched C.sub.1-C.sub.4 alkyl,
--OR.sup.c (R.sup.c being a C.sub.1-C.sub.8 alkyl radical), and
--N(R.sup.d)R.sup.e (R.sup.d and R.sup.e being independently
hydrogen or C.sub.1-C.sub.8 alkyl or aryl), and unsaturated analogs
of any of the foregoing definitions for substituent A; wherein
R.sup.1 is selected from the group consisting of hydrogen and
methyl; wherein R.sup.6 is selected from the group consisting
halogen and C.sub.1-C.sub.8 alkyl; wherein R.sup.a is selected from
the group consisting of straight or branched C.sub.1-C.sub.8
alkylene, C.sub.3-C.sub.7 cycloalkylene; and R.sup.b is selected
from the group consisting of hydrogen, substituted or unsubstituted
phenyl, C.sub.2-C.sub.10 acyl, C.sub.2-C.sub.10 acyloxy,
C.sub.2-C.sub.10 alkoxycarbonyl, and halogen; provided that when A
is methyl, R.sup.1 is methyl.
74. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 146wherein the dotted
lines are optional pi bonds wherein X is C.sub.1-C.sub.3 alkyl;
wherein Y is hydrogen or an acyloxy moiety; wherein R.sup.6 is --H
or wherein R.sup.16 is --H or halo; wherein R.sup.1 is --H or
--CH.sub.3.
75. An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 147wherein the dotted line is
optional pi bond; wherein A is selected from the group consisting
of straight or branched C.sub.1-C.sub.4 alkyl, --OR.sup.c (R.sup.c
being a C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (Rd
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A; wherein R.sup.1 is selected from the
group consisting of hydrogen and methyl; wherein R.sup.6 is
selected from the group consisting of halogen and C.sub.1-C.sub.8
alkyl; wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene,
C.sub.3-C.sub.7cycloalkylene; and R.sup.b is selected from the
group consisting of hydrogen, substituted or unsubstituted phenyl,
C.sub.2-C.sub.10 acyl, C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10
alkoxycarbonyl, and halogen; provided that when A is methyl,
R.sup.a and R.sup.b together have at least two carbon atoms, and
R.sup.1 is methyl.
76. The inhibitor of claim 75 wherein the optional pi bond at 6 is
present.
77. The inhibitor of claim 75 wherein R.sup.6 is methyl.
78. The inhibitor of claim 75 wherein R.sup.a is C.sub.1-C.sub.6
alkylene.
79. The inhibitor of claim 75 where A is either methyl or
--N(R.sup.d)R.sup.e.
80. The inhibitor of claim 75 where A is --N(R.sup.d)R.sup.e.
81. The inhibitor of claim 80 wherein R.sup.d is methyl.
82. The inhibitor of claim 80 wherein R.sup.e is C.sub.1-C.sub.6
alkyl or phenyl C.sub.1-C.sub.6 alkyl.
83. An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 148wherein the dotted line is an
optional pi bond; wherein R.sup.16.beta. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl, fluoro, chloro,
C.sub.1-C.sub.8 haloalkyl, a moiety which together with
R.sup.16.alpha. is C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions for R.sup.16.beta.; wherein R.sup.16.alpha.
is selected from the group consisting of hydrogen, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 haloalkyl, a moiety which together with
R.sup.16.beta. is C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R(R'.sup.16 being C.sub.1-C.sub.3
alkyl) and unsaturated analogs of any of the foregoings; wherein
R.sup.15.alpha. is selected from the group consisting of hydrogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkenyl and C.sub.1-C.sub.4
alkynyl; wherein R.sup.19 is either --H or --CH.sub.3; and wherein
R.sup.6 is selected from the group consisting of --H, --CH.sub.3,
and halo; provided that R.sup.16.beta., R.sup.16.alpha., and
R.sup.15.alpha. are not simultaneously hydrogen.
84. The inhibitor of claim 83 wherein R.sup.16.alpha. is a larger
substituent than R.sup.16.beta..
85. The inhibitor of claim 83 wherein R.sup.6 is hydrogen.
86. The inhibitor of claim 83 wherein the optional pi bond at
position 1 is not present.
87. The inhibitor of claim 83 wherein R.sup.16l is a
C.sub.3-C.sub.5 alkyl.
88. An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 149wherein the dotted line is
optional pi bond wherein X is C.sub.1-C.sub.3 alkyl; wherein Y is
hydrogen or an acyloxy moiety; wherein R.sup.6 is --H or
--CH.sub.3; wherein R.sup.16 is --H or halo; wherein R.sup.1 is --H
or CH.sub.3.
89. The inhibitor of claim 88 wherein R.sup.6 is methyl.
90. The inhibitor of claim 88 wherein the optional pi bond at
position 1 is present.
91. The inhibitor of claim 88 wherein Y is a C.sub.3-C.sub.6
fluoroacyloxy.
92. The inhibitor of claim 88 wherein X is methyl.
93. An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 150wherein n is an integer from
1-2; wherein the dotted lines are optional pi bonds; wherein X and
Y are independently selected from the group consisting of --H,
(C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3) alkenyl, and
methoxycarbonyl; wherein Z is selected from the group consisting of
--H and (C.sub.1-C.sub.3)alkyl; wherein R.sup.3 is selected from
the group consisting of hydrogen, acyl, carboxyl, alkoxycarbonyl,
substituted or unsubstituted carboxamide, cyano, alkoxy,
alkoxyalkoxy, alkythioalkoxy, acyloxy; hydroxy, halo,
--O--SO.sub.2R.sup.a (R.sup.a being selected from the group
consisting of C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and
a moiety which, together with R.sub.2, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom; wherein
R.sup.2 is selected from the group consisting of hydrogen, amido,
acyloxy, carboxyl, carboxamide, alkoxycarbonyl, cyano, halo, nitro,
C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a moiety which, together
with R.sub.3, is a 5-6 member ring containing at least one oxygen
and one nitrogen atom; wherein R.sup.4 is hydrogen or halo; wherein
R.sup.6 is selected from the group consisting of hydrogen and oxo;
wherein R.sup.9 is --H or H; provided that X, Y, and Z are not all
hydrogen.
94. The inhibitor of claim 93 wherein R.sup.3 is alkoxyalkoxy.
95. The inhibitor of claim 93 wherein R.sup.3 is carboxyl
unsubstituted carboxamide or alkoxyl;
96. The inhibitor of claim 93 wherein at least one of X, Y or Z is
methyl.
97. The inhibitor of claim 93 wherein both X and Y are methyl.
98. The inhibitor of claim 93 wherein n=1.
99. The inhibitor of claim 93 wherein R.sup.6 is oxo.
100. The inhibitor of claim 93 wherein R.sup.3 is carboxamide.
101. An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 151wherein the dotted lines are
optional pi bonds; wherein n=1 or 2; and wherein a is either --H or
wherein b and c are independently hydrogen or methyl; wherein Z is
oxygen or sulfur.
102. The inhibitor of claim 101 wherein n=1.
103. The inhibitor of claim 101 wherein at least one of b or c is
methyl.
104. The inhibitor of claim 101 wherein both b and c are
methyl.
105. The inhibitor of claim 101 wherein Z is oxygen.
106 An inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
having a molecular structure selected from the group consisting of:
152153154155156
107. The inhibitor of claim 106 wherein said inhibitor of
17.beta.-hydroxysteroid dehydrogenase selected from the group
consisting of: 157
108. A method of inhibiting type 3 17.beta.-hydroxysteroid
dehydrogenase comprising administering to a patient in need of such
treatment a therapeutically effective amount of an inhibitor of
type 3 17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 158wherein R is selected from the group consisting of
alkoxy, alkylthio, alkoxyalkoxy, alkoxyalkylthio, alkylthioalkoxy,
and alkylthioalkylthio, or 159wherein n is an integer from 1 to
4.
109. The method of claim 108 wherein said inhibitor is selected
from the group consisting of: 160161
110. A pharmaceutical composition comprising a pharmaceutically
acceptable diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 162wherein R is
selected from the group consisting of alkoxy, alkylthio,
alkoxyalkoxy, alkoxyalkylthio, alkylthioalkoxy, and
alkylthioalkylthio, or 163wherein n is an integer from 1 to 4.
111. The pharmaceutical composition of claim 110 wherein said
inhibitor is selected from the group consisting of: 164165
112 An inhibitor of type 3 17.beta.-hydroxysteroid dehydrogenase
having the molecular structure: 166wherein R is selected from the
group consisting of alkoxyethoxy, alkoxyalkylthio, alkylthioalkoxy,
and alkylthioalkylthio, or 167wherein n is an integer from 1 to
4.
113 The inhibitor of claim 112 wherein said inhibitor is selected
from the group consisting of: 168169
114. A method of treating, or reducing the risk of developing,
prostate cancer, comprising administering to a patient in need of
such treatment or reduction a therapeutically effective amount of
an inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase, and
an amount of an LHRH agonist or antagonist effective to reduce
testicular secretion of sex steroids.
115. The method of claim 114, further comprising administering a
therapeutically effective amount of an antiandrogen.
116. The method of claim 115, further comprising administering a
therapeutically effective amount of a 5.alpha.-reductase
inhibitor.
117. The method of claim 114, further comprising administering a
therapeutically effective amount of a 5.alpha.-reductase
inhibitor.
118. A method of treating, or reducing the risk of developing
prostate cancer, comprising administering to a patient in need of
such treatment or reduction a therapeutically effective amount of
an inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase and of
an inhibitor of type 3 17.beta.-hydroxysteroid dehydrogenase.
119. The method of claim 118 further comprising a therapeutically
effective amount of a 5.alpha.-reductase inhibitor.
120. The method of claim 118 further comprising a therapeutically
effective amount of an LHRH agonist or antagonist.
121. The method of claim 118 further comprising a therapeutically
effective amount of an antiandrogen.
122. The method of claim 118 further comprising a therapeutically
effective amount of an LHRH agonist or antagonist and an
antiandrogen.
123. The method of claim 118 further comprising a therapeutically
effective amount of an LHRH agonist or antagonist, an antiandrogen,
and a 5.alpha.-reductase inhibitor.
124. The method of claim 118 further comprising a therapeutically
effective amount of an antiandrogen and a 5.alpha.-reductase
inhibitor.
125. The method of claim 118 further comprising a therapeutically
effective amount of an LHRH agonist (or antagonist) and a
5.alpha.-reductase inhibitor.
126. A method of treating, or reducing the risk of developing,
benign prostatic hyperplasia comprising administering to a patient
in need of such treatment or reduction, a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase.
127. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of an agent
selected from the group consisting of an antiestrogen or an
aromatase inhibitor.
128. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of an
antiandrogen.
129. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of an antiandrogen
and a 5.alpha.-reductase inhibitor.
130. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of a
5.alpha.-reductase inhibitor.
131. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of a
5.alpha.-reductase inhibitor and an antiestrogen or an aromatase
inhibitor.
132. The method of claim 126, further comprising administering to
said patient a therapeutically effective amount of a
5.alpha.-reductase inhibitor, an antiandrogen, an antiestrogen or
an aromatase inhibitor.
133. A method of treating or reducing the risk of developing, acne,
seborrhea, hirsutism or androgenic alopecia, comprising
administering to a patient in need of such treatment or reduction,
a therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase.
134. The method of claim 133, further comprising administering to
said patient at therapeutically effective amount of an
antiandrogen.
135. The method of claim 133, further comprising administering to
said patient at therapeutically effective amount of a
5.alpha.-reductase inhibitor.
136. The method of claim 133, further comprising administering to
said patient at therapeutically effective amount of a
5.alpha.-reductase inhibitor and an antiandrogen.
137. A method of treating, or reducing the risk of developing,
polycystic ovarian syndrome comprising administering to a patient
in need of such treatment or reduction a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase.
138. The method of claim 137, further comprising administering a
therapeutically effective amount of an inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase.
139. A method of treating, or reducing the risk of developing,
polycystic ovarian syndrome comprising administering to a patient
in need of such treatment or reduction, a therapeutically effective
amount of an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase.
140. The method of claim 137 further comprising administering a
therapeutically effective amount of an antiandrogen.
141. A method of treating, or reducing the risk of developing,
polycystic ovarian syndrome comprising administering to a patient
in need of such treatment or reduction a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase, an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase, and an antiandrogen.
142. The method of claim 139 further comprising administering a
therapeutically effective amount of an antiandrogen.
143. A method of treating, or reducing the risk of developing,
breast cancer comprising administering to a patient in need of such
treatment or reduction, a therapeutically effective amount of an
inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase and a
therapeutically effective amount of an antiestrogen.
144. The method of claim 143 further comprising administering a
therapeutically effective amount of an LHRH agonist or
antagonist.
145. The method of claim 143, further comprising administering a
therapeutically effective amount of an androgenic compound.
146. The method of claim 145, further comprising administering an
amount of an LHRH agonist effective to reduce ovarian secretion of
sex steroids.
147. A method of treating, or reducing the risk of developing,
endometriosis or leiomyoma comprising administering to a patient in
need of such treatment or reduction, a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase.
148. The method of claim 147, further comprising administering to
said patient a therapeutically effective amount of an antiestrogen
or of an inhibitor of aromatase.
149. The method of claim 147, further comprising administering to
said patient an amount of an LHRH agonist (or antagonist) effective
to reduce ovarian secretion of sex steroids.
150. The method of claim 149, further comprising administering a
therapeutically effective amount of an antiestrogen or of an
aromatase inhibitor.
151. A method of inhibiting testicular hormonal secretions
comprising administering to a patient in need of such inhibition an
amount of an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase effective to reduce testicular production of sex
steroids.
152. The method of claim 151 further comprising administering a
therapeutically effective amount of an antiandrogen.
153. The method of claim 152 further comprising administering a
therapeutically effective amount of a 5.alpha.-reductase
inhibitor.
154. The method of claim 151, further comprising administering an
LHRH agonist.
155. The method of claim 154, further comprising administering an
antiandrogen.
156. The method of claim 154, further comprising administering an
antiandrogen and a 5.alpha.-reductase inhibitor.
157. The method of claim 151, further comprising administering an
LHRH antagonist.
158. The method of claim 157, further comprising administering an
antiandrogen.
159. The method of claim 157, further comprising administering an
antiandrogen and a 5.times.-reductase inhibitor.
160. A method of treating precocious puberty comprising
administering to a male or female patient in need of such treatment
a therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase.
161. The method of claim 160 comprising administering to a male
patient a therapeutically effective amount of an LHRH agonist or
antagonist.
162. The method of claim 160 comprising administering to a male or
female patient a therapeutically effective amount of an
antiandrogen.
163. The method of claim 160 comprising administering to a male
patient a therapeutically effective amount of an antiandrogen and
an LHRH agonist or antagonist.
164. The method of claim 160 comprising administering to a male
patient a therapeutically effective amount of an inhibitor of type
3 17.beta.-hydroxysteroid dehydrogenase.
165. A method of treating precocious puberty comprising
administering to a male or female patient in need of such treatment
a therapeutically effective amount of an inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase.
166. The method of claim 165 further comprising administering to a
male patient a therapeutically effective amount of an LHRH agonist
or antagonist.
167. The method of claim 165 further comprising administering to a
male or female patient a therapeutically effective amount of an
antiandrogen.
168. The method of claim 165 further comprising administering to a
male patient a therapeutically effective amount of an antiandrogen
and an LHRH agonist or antagonist.
169. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one antiandrogen.
170. A pharmaceutical composition of claim 169 wherein said
composition further comprising a therapeutically effective amount
of a 5.alpha.-reductase inhibitor.
171. A pharmaceutical composition of claim 169 wherein said
composition further comprising a therapeutically effective amount
of at least one antiestrogen or one aromatase inhibitor.
172. A pharmaceutical composition of claim 170 wherein said
composition further comprising a therapeutically effective amount
of at least one antiestrogen or one aromatase inhibitor.
173. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase
174. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one antiandrogen.
175. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one 5.alpha.-reductase inhibitor.
176. A pharmaceutical composition of claim 174 wherein said
composition further comprising a therapeutically effective amount
of at least one 5.alpha.-reductase inhibitor.
177. A pharmaceutical composition of claim 173 wherein said
composition further comprising a therapeutically effective amount
of at least one antiandrogen.
178. A pharmaceutical composition of claim 173 wherein said
composition further comprising a therapeutically effective amount
of at least one 5.alpha.-reductase inhibitor.
179. A pharmaceutical composition of claim 177 wherein said
composition further comprising a therapeutically effective amount
of at least one 5.alpha.-reductase inhibitor.
180. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one 5.alpha.-reductase inhibitor.
181. A pharmaceutical composition of claim 180 wherein said
composition further comprising a therapeutically effective amount
of at least one antiestrogen or one aromatase inhibitor.
182. A pharmaceutical composition comprising: a) a pharmaceutically
acceptable excipient, diluent or carrier; b) a therapeutically
effective amount of at least one inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase; and c) a therapeutically
effective amount of at least one antiestrogen or one aromatase
inhibitor.
183. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one antiandrogen.
184. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one inhibitor of type 3 17.beta.-hydroxysteroid dehydrogenase
185. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one antiandrogen.
186. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one 5.alpha.-reductase inhibitor.
187. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one 5.times.-reductase inhibitor.
188. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutically effective amount
of at least one inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase and a therapeutically effective amount of at least
one antiestrogen or one aromatase inhibitor.
189. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutic effective amount of at
least one inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
and an amount of at least one LHRH agonist or antagonist.
190. A kit having a plurality of containers wherein contents of
each container differ in whole or in part from content of another
container, said kit comprising a therapeutic effective amount of at
least one inhibitor of type 3 17.beta.-hydroxysteroid dehydrogenase
and an amount of at least one LHRH agonist or antagonist.
191. A method of treating, or reducing the risk of developing,
prostate cancer, comprising administering to a patient in need of
such treatment or reduction a therapeutically effective amount of
an inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase, and
an amount of an LHRH agonist or antagonist effective to reduce
testicular secretion of sex steroids wherein the inhibitor of type
5 17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
192. A method of treating, or reducing the risk of developing,
benign prostatic hyperplasia comprising administering to a patient
in need of such treatment or reduction, a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase wherein the inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
193. A method of treating or reducing the risk of developing, acne,
seborrhea, hirsutism or androgenic alopecia, comprising
administering to a patient in need of such treatment or reduction,
a therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase wherein the inhibitor of type
5 17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
194. A method of treating, or reducing the risk of developing,
polycystic ovarian syndrome comprising administering to a patient
in need of such treatment or reduction a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase wherein the inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
195. A method of treating, or reducing the risk of developing,
breast cancer comprising administering to a patient in need of such
treatment or reduction, a therapeutically effective amount of an
inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase and a
therapeutically effective amount of an antiestrogen wherein the
inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase is not
medroxyprogesterone acetate, megestrol acetate, chlormadinone
acetate, 1-dehydromegestrol acetate, melengestrol acetate,
nomegestrol acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
196. A method of treating, or reducing the risk of developing,
endometriosis or leiomyoma comprising administering to a patient in
need of such treatment or reduction, a therapeutically effective
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase wherein the inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
197. A method of treating precocious puberty comprising
administering to a male or female patient in need of such treatment
a therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase.
198. The method of claim 1 wherein the inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
199. The method of claim 38 wherein the inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase is not medroxyprogesterone
acetate, megestrol acetate, chlormadinone acetate,
1-dehydromegestrol acetate, melengestrol acetate, nomegestrol
acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
200. The pharmaceutical composition of claim 40 wherein the
inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase is not
medroxyprogesterone acetate, megestrol acetate, chlormadinone
acetate, 1-dehydromegestrol acetate, melengestrol acetate,
nomegestrol acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
201. The pharmaceutical composition of claim 73 wherein the
inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase is not
medroxyprogesterone acetate, megestrol acetate, chlormadinone
acetate, 1-dehydromegestrol acetate, melengestrol acetate,
nomegestrol acetate, 1-dehydromelengestrol acetate, and cyproterone
acetate.
202. The method of claim 139 wherein the inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase is not androsterone.
203. The method of claim 151 wherein the inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase is not androsterone.
204. The method of claim 165 wherein the inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase is not androsterone.
Description
RELATED APPLICATION
[0001] This patent application is a regular, non-provisional
application claiming priority of provisional application Serial No.
60/077,510, filed Mar. 11, 1998, the entire specification of which
is incorporated by reference as though fully set forth herein. This
application also claims priority of U.S. Provisional Application
Serial No. 60/095,623, filed Aug. 7, 1998, the entire disclosure of
which is incorporated by reference as though fully set forth
herein.
FIELD OF THE INVENTION
[0002] The present invention relates to inhibitors of enzymes
involved in the biosynthesis of sex steroids from natural
precursors and to their use in the treatment of sex steroid
dependent diseases. In particular, inhibitors are disclosed which
suppress the activity of type 3 and type 5 17.beta.-hydroxysteroid
dehydrogenase, thus diminishing the production of androgens
catalyzed by these two enzymes. Pharmaceutical use of the
inhibitors may reduce the natural production of androgens such as
testosterone and dihydrotestosterone, and thereby beneficially
treat diseases whose onset or progress is aided by androgenic
activity. Because androgens formed by reactions catalyzed by type 3
or type 5 enzyme are precursors to estrogens, the invention also
has applicability to diseases whose onset or progress is aided by
estrogenic activity.
BACKGROUND OF THE RELATED ART
[0003] Many androgen-sensitive diseases, i.e. diseases whose onset
or progress is aided by androgenic activity, are known. They
include but are not limited to prostate cancer, benign prostatic
hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia,
precocious puberty, adrenal hyperplasia, and polycystic ovarian
syndrome. Estrogen sensitive diseases, i.e. diseases whose onset or
progress is aided by estrogenic activity are also known. They
include but are not limited to breast cancer, endometriosis,
leiomyoma, and precocious puberty.
[0004] Precocious puberty is usually associated with an excess of
androgen secretion, usually of adrenal origin. The current
treatments include blockade of adrenal secretion by glucocorticoids
with its associated side effects. Blockade of type 5 17.beta.-HSD
would be an advantage, thus desirably reducing the dose or avoiding
the use of glucocorticoids. Another treatment is the use of LHRH
agonists to cause medical castration. A better controlled
inhibition of androgen formation could be achieved with type 5 and
3 17.beta.-HSD inhibitors.
[0005] Polycystic ovarian syndrome is associated with an excess of
androgen secretion by the ovaries. LHRH agonists are used among
other, as treatment, to cause medical castration. The use of an
inhibitor of 17.beta.-HSD would be advantageous.
[0006] Estrogen sensitive diseases may vary in their responses to
androgens. They may, for example, respond favorably, unfavorably or
not at all to androgens. Likewise, androgen-sensitive diseases may
respond differently to estrogens. Thus, the treatment of sex
steroid sensitive diseases may involve increasing or decreasing
androgenic activity depending on whether the disease in question
responds favorably or unfavorably to androgenic activity. Treatment
may also involve increasing or decreasing estrogenic activity
depending on whether the disease in question responds favorably or
unfavorably to estrogenic activity. Breast cancer, for example, is
known to respond favorably to androgenic activity and negatively to
estrogenic activity. Benign prostatic hyperplasia is believed to
respond negatively to both androgenic and estrogenic activity.
[0007] Androgenic and estrogenic activity may be suppressed by
administering androgen receptor antagonists ("antiandrogens") or
estrogen receptor antagonists ("antiestrogens"), respectively. See
e.g. WO 94/26767 and WO 96/26201. Androgenic and estrogenic
activity may also be reduced by suppressing androgen or estrogen
biosynthesis using inhibitors of enzymes that catalyze one or more
steps of such biosynthesis or by suppressing ovarian or testicular
secretions by known methods. See e.g. WO 90/10462, WO 91/00731, WO
91/00733, and WO 86/01105. Type 5 17.beta.-hydroxysteroid
dehydrogenase is described in WO 97/11162.
[0008] Effective inhibitors of type 5 17.beta.-hydroxysteroid
dehydrogenase enzyme are provided by the present invention. The
prior art is not believed to have provided compounds sufficiently
effective at simultaneously (1) inhibiting type 5 or type 3
17.beta.-hydroxysteroid dehydrogenase while (2) desirably failing
to substantially inhibit other 17.beta.-hydroxysteroid
dehydrogenases or other catalysts of sex steroid degradation.
Medroxyprogesterone acetate, megestrol acetate, and chlormadinone
acetate which the prior art as used as pharmaceutical agents for
other purposes are not believed to have been disclosed as
inhibitors of type 5 17.beta.-hydroxysteroid dehydrogenase,
although applicants' research has now shown them to inhibit that
type 5 enzyme.
SUMMARY OF THE INVENTION
[0009] It is accordingly an object of the present invention to more
selectively and effectively inhibit type 3 and/or type 5
17.beta.-hydroxysteroid dehydrogenase while preferably avoiding
inhibition of other 17.beta.-hydroxysteroid dehydrogenases, type 1
or 2 3.alpha.-hydroxysteroid dehydrogenases, or other androgen
degradation enzymes.
[0010] It is another object to provide novel inhibitors of types 3
and 5 17.beta.-hydroxysteroid dehydrogenase and pharmaceutical
compositions thereof.
[0011] It is another object to provide treatment and prevention
regimens for androgen and estrogen sensitive diseases which
regimens include inhibiting activity of type 3 or type 5
17.beta.-hydroxysteroid dehydrogenase.
[0012] In one embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 1
[0013] wherein the dotted line is optional pi bond;
[0014] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, --OR.sup.c (R.sup.c being a
C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0015] wherein R.sup.1 is selected from the group consisting of
hydrogen and methyl;
[0016] wherein R.sup.6 is selected from the group consisting of
hydrogen, and halogen, and C.sub.1-C.sub.8 alkyl;
[0017] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and
[0018] R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen;
[0019] provided that when A is methyl, R.sup.a and R.sup.b together
have at least two carbon atoms, and R.sup.1 is methyl.
[0020] It is preferred that the optional pi bond at 6 is present
that R.sup.6 is methyl, that R.sup.a is C.sub.1-C.sub.6 alkylene or
that A is either methyl or --N(R.sup.d)R.sup.e.
[0021] It is also preferred when wherein A is --N(R.sup.d)R.sup.e
that R.sup.d is methyl or that R.sup.e is C.sub.1-C.sub.6 alkyl or
C.sub.7-C.sub.12 phenyl alkyl.
[0022] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 2
[0023] wherein the dotted line is an optional pi bond;
[0024] wherein R.sup.16.beta. is selected from the group consisting
of hydrogen, fluoro, chloro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16 is C.sub.4-C.sub.7
spirocycloalkyl, C.sub.4-C.sub.7 halospirocycloalkyl, or
.dbd.--R'.sup.16 (R'.sup.16 being C.sub.1-C.sub.3 alkyl) and
unsaturated analogs of any of the foregoing definitions of
R.sup.16.beta.;
[0025] wherein R.sup.16.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.beta. forms
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing.;
[0026] wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl;
[0027] wherein R.sup.19 is either --H or --CH.sub.3; and
[0028] wherein R.sup.6 is selected from the group consisting of
--H, CH.sub.3, and halo;
[0029] Provided that R.sup.16.beta., R.sup.16.alpha., and
R.sup.15.alpha. are not simultaneously hydrogen.
[0030] It is preferred that R.sup.16.alpha. is a larger substituent
than R.sup.16.beta., that R.sup.6 is hydrogen, that the optional pi
bond at position 1 is not present or that R.sup.16.alpha. is a
C.sub.3-C.sub.5 alkyl chain.
[0031] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 3
[0032] wherein the dotted line is optional pi bond
[0033] wherein X is C.sub.1-C.sub.3 alkyl;
[0034] wherein Y is hydrogen or an acyloxy moiety;
[0035] wherein R.sup.6 is --H or --CH.sub.3;
[0036] wherein R.sup.16 is --H or halo;
[0037] wherein R.sup.1 is --H or --CH.sub.3.
[0038] It is preferred that R.sup.6 is methyl that the optional pi
bond at position 1 is present, that Y is a C.sub.3-C.sub.6
fluoroacyloxy or that X is methyl.
[0039] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 4
[0040] wherein n is an integer from 1-2;
[0041] wherein the dotted lines are optional double bonds;
[0042] wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl;
[0043] wherein Z is selected from the group consisting of --H and
(C.sub.1-C.sub.3)alkyl;
[0044] wherein R.sup.3 is selected from the group consisting of
hydrogen, acyl, carboxyl, alkoxycarbonyl, substituted or
unsubstituted carboxamide, cyano, alkoxy, alkoxyalkoxy,
alkythioalkoxy, acyloxy; hydroxy, halo, --O--SO.sub.2R.sup.a
(R.sup.a being selected from the group consisting of
C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and a moiety
which, together with R.sub.2, is a 5-6 member ring containing at
least one oxygen and one nitrogen atom;
[0045] wherein R.sup.2 is selected from the group consisting of
hydrogen, amido, acyloxy, carboxyl, carboxamide, alkoxycarbonyl,
cyano, halo, nitro, C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a
moiety which, together with R.sup.3, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom;
[0046] wherein R.sup.4 is hydrogen or halo;
[0047] wherein R.sup.6 is selected from the group consisting of
hydrogen and oxo;
[0048] wherein R.sup.9 is --H or --OH;
[0049] provided that X, Y, and Z are not all hydrogens when R.sup.3
is methoxy.
[0050] It is preferred that R.sup.3 is alkoxyalkoxy, carboxamide,
carboxyl or alkoxyl, that at least one of X, Y or Z is methyl, that
both X and Y are methyl, that n is 1 or that R.sup.6 is oxo.
[0051] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 5
[0052] wherein the dotted lines are optional pi bonds;
[0053] wherein n=1 or 2; and
[0054] wherein a is either --H or --CH.sub.3,
[0055] wherein b and c are independently hydrogen or methyl;
[0056] wherein Z is oxygen or sulfur.
[0057] It is preferred that n is 1, that at least one of b or c is
methyl, both b and c are methyl or that Z is oxygen.
[0058] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase selected from the group
consisting of: 678910
[0059] It is preferred that the type 5 inhibitor is selected from
the group consisting of: 11
[0060] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase selected from the group
consisting of: 1213
[0061] wherein the dotted lines are optional pi bonds;
[0062] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, OR.sup.c (R.sup.c being a
C.sub.1-C.sub.4 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0063] wherein R.sup.1 is selected from the group consisting of
hydrogens, and methyl;
[0064] wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-C.sub.8 alkyl;
[0065] wherein R.sub.16 is selected from the group consisting of
H,H and CH.sub.2;
[0066] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and R.sup.b is selected from the group consisting of
hydrogen, substituted or unsubstituted phenyl, C.sub.2-C.sub.10
acyl, C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl,
and halogen.
[0067] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 14
[0068] wherein the dotted line are optional pi bonds;
[0069] wherein R.sup.16.beta. is selected from the group consisting
of hydrogen, fluoro, chloro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.alpha. forms
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.;
[0070] wherein R.sup.16.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.beta. forms
C.sub.4-C.sub.7 spirocycloalkyl C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing;
[0071] wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl;
[0072] wherein R.sup.19 is either --H or --CH.sub.3; and
[0073] wherein R.sup.6 is selected from the group consisting of
--H, --CH.sub.3, and halo;
[0074] provided that R.sup.16.beta., R.sup.16.alpha., and
R.sup.15.alpha. are not simultaneously hydrogen.
[0075] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 15
[0076] wherein n=1 or 2;
[0077] wherein the dotted lines are optional pi bonds;
[0078] wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl;
[0079] wherein Z is selected from the group consisting of --H and
(C.sub.1-C.sub.3)alkyl;
[0080] wherein R.sup.3 is selected from the group consisting of
acyl, carboxyl, alkoxycarbonyl, substituted or unsubstituted
carboxamide, cyano, alkoxy, alkoxyalkoxy, alkythioalkoxy, acyloxy;
hydroxy, halo, --O--SO.sub.2R.sup.a (R.sup.a being selected from
the group consisting of C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10
aryl), and a moiety which, together with R.sub.2, is a 5-6 member
ring containing at least one oxygen and one nitrogen atom;
[0081] wherein R.sup.2 is selected from the group consisting of
amido, acyloxy, carboxyl, carboxamide, alkoxycarbonyl, cyano, halo,
nitro, C.sub.1-C.sub.8 alkyl and CF.sub.3 and a moiety which,
together with R.sub.3, is a 5-6 member ring containing at least one
oxygen and one nitrogen atom;
[0082] wherein R.sup.4 is hydrogen or halo;
[0083] wherein R.sup.6 is selected from the group consisting of
hydrogen and oxo;
[0084] wherein R.sup.9 is --H or OH;
[0085] provided that X, Y, and Z are not hydrogens when R.sup.3 is
methoxy.
[0086] It is preferred that R.sup.3 is alkoxyalkoxy, carboxamide,
carboxyl or alkoxyl, that at least one of X, Y or Z is methyl, that
both X and Y are methyl, that n is 1 or that R.sup.6 is oxo.
[0087] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 16
[0088] wherein the dotted lines are optional pi bonds;
[0089] wherein n=1 or 2; and
[0090] wherein a is either --H or CH.sub.3;
[0091] wherein b and c are independently hydrogen or methyl;
[0092] wherein Z is oxygen or sulfur.
[0093] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 17
[0094] wherein the dotted line is an optional pi bond;
[0095] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, --OR.sup.c (R.sup.c being a
C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0096] wherein R.sup.1 is selected from the group consisting of
hydrogen, methyl, and ethyl;
[0097] wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-C.sub.8 alkyl;
[0098] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and
[0099] R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen;
[0100] provided that when A is methyl, R.sup.1 is methyl.
[0101] In another embodiment, the invention provides a method of
inhibiting activity of type 5 17.beta.-hydroxysteroid dehydrogenase
comprising administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 5
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 18
[0102] wherein the dotted lines are optional pi bonds
[0103] wherein X is C.sub.1-C.sub.3 alkyl;
[0104] wherein Y is hydrogen or an acyloxy moiety;
[0105] wherein R.sup.6 is --H or --CH.sub.3;
[0106] wherein R.sup.16 is --H or halo;
[0107] wherein R.sup.1 is --H or --CH.sub.3.
[0108] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 19
[0109] wherein the dotted lines are optional pi bonds;
[0110] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, --OR.sup.c (R.sup.c being a
C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0111] wherein R.sup.1 is selected from the group consisting of
hydrogen and methyl;
[0112] wherein R.sup.6 is selected from the group consisting of
hydrogen, halogen, and C.sub.1-Q alkyl;
[0113] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and
[0114] R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen;
[0115] provided that when A is methyl, R.sup.a and R.sup.b together
have at least two carbon atoms, and R.sup.1 is methyl.
[0116] It is preferred that the optional pi bond at 6 is present,
that R.sup.6 is methyl, that R.sup.a is C.sub.1-C.sub.6 alkylene or
that A is either methyl or --N(R.sup.d)R.sup.e.
[0117] It is also preferred when A is --N(R.sup.d)R.sup.e, that
R.sup.d is methyl or that R.sup.e is C.sub.1-C.sub.6 alkyl or
phenyl C.sub.1-C.sub.6 alkyl.
[0118] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 20
[0119] wherein the dotted line is an optional pi bond;
[0120] wherein R.sup.16.beta. is selected from the group consisting
of hydrogen, fluoro, chloro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.alpha. is
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.;
[0121] wherein R.sup.16.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.beta. forms
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoings;
[0122] wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C.sub.1-C.sub.4 alkynyl;
[0123] wherein R.sup.19 is either --H or --CH.sub.3; and
[0124] wherein R.sup.6 is selected from the group consisting of
--H, --CH.sub.3, and halo;
[0125] provided that R.sup.16.beta., R.sup.16.alpha., and
R.sup.15.alpha. are not simultaneously hydrogen.
[0126] It is preferred that R.sup.16.alpha. is a larger substituent
than R.sup.16.beta. that R.sup.6 is hydrogen, that the optional pi
bond at position 1 is not present or that R.sup.16.alpha. is a
C.sub.3-C.sub.5 alkyl chain.
[0127] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 21
[0128] wherein the dotted line is an optional pi bond
[0129] wherein X is C.sub.1-3 alkyl;
[0130] wherein Y is hydrogen or an acyloxy moiety;
[0131] wherein R.sup.6 is --H or --CH.sub.3;
[0132] wherein R.sup.16 is --H or halo;
[0133] wherein R.sup.1 is --H or --CH.sub.3.
[0134] It is preferred that R.sup.6 is methyl, that the optional pi
bond at position 1 is present, that Y is a C.sub.3-C.sub.6
fluoroacyloxy or that X is methyl.
[0135] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 22
[0136] wherein n is an integer from 1-2;
[0137] wherein the dotted lines are optional pi bonds;
[0138] wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl;
[0139] wherein Z is selected from the group consisting of --H and
(C.sub.1-C.sub.3)alkyl;
[0140] wherein R.sup.3 is selected from the group consisting of
hydrogen, acyl, carboxyl, alkoxycarbonyl, substituted or
unsubstituted carboxamide, cyano, alkoxy, alkoxyalkoxy,
alkythioalkoxy, acyloxy; hydroxy, halo, --O--SO.sub.2R.sup.a
(R.sup.a being selected from the group consisting of
C.sub.1-C.sub.6 alkyl and C.sub.6-C.sub.10 aryl), and a moiety
which, together with R.sub.2, is a 5-6 member ring containing at
least one oxygen and one nitrogen atom;
[0141] wherein R.sup.2 is selected from the group consisting of
hydrogen, amido, acyloxy, carboxyl, carboxamide, alkoxycarbonyl,
cyano, halo, nitro, C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a
moiety which, together with R.sub.3, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom;
[0142] wherein R.sup.4 is hydrogen or halo;
[0143] wherein R.sup.6 is selected from the group consisting of
hydrogen and oxo;
[0144] wherein R.sup.9 is --H or --OH;
[0145] provided that X, Y, and Z are not all hydrogen when R.sup.3
is methoxy.
[0146] It is preferred that R.sup.3 is alkoxyalkoxy, carboxamide,
carboxyl or alkoxyl, that at least one of X, Y or Z is methyl, that
both X and Y are methyl, that n is 1 or that R.sup.6 is oxo.
[0147] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 23
[0148] wherein the dotted lines are optional pi bonds;
[0149] wherein n=1 or 2; and
[0150] wherein a is either --H or --CH.sub.3;
[0151] wherein b and c are independently hydrogen or methyl;
[0152] wherein Z is oxygen or sulfur.
[0153] It is preferred that n is 1, that at least one of b or c is
methyl, that both b and c are methyl or that Z is oxygen.
[0154] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having a molecular structure selected from the group
consisting of: 2425262728
[0155] It is preferred that the type 5 inhibitor is selected from
the group consisting of: 29
[0156] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 30
[0157] wherein the dotted line is an optional pi bond;
[0158] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, --OR.sup.c (R.sup.c being a
C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0159] wherein R.sup.1 is selected from the group consisting of
hydrogen and methyl;
[0160] wherein R.sup.6 is selected from the group consisting of
hydrogen, and halogen, and C.sub.1-Cg alkyl;
[0161] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and
[0162] R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen;
[0163] provided that when A is methyl, R.sup.1 is methyl or
ethyl.
[0164] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 31
[0165] wherein the dotted lines are optional pi bonds
[0166] wherein X is C.sub.1-C.sub.3 alkyl;
[0167] wherein Y is hydrogen or an acyloxy moiety;
[0168] wherein R.sup.6 is --H or --CH.sub.3;
[0169] wherein R.sup.16 is --H or halo;
[0170] wherein R.sup.1 is --H or CH.sub.3.
[0171] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 32
[0172] wherein the dotted line is optional pi bond;
[0173] wherein A is selected from the group consisting of straight
or branched C.sub.1-C.sub.4 alkyl, R.sup.c (R.sup.c being a
C.sub.1-C.sub.8 alkyl radical), and --N(R.sup.d)R.sup.e (R.sup.d
and R.sup.e being independently hydrogen or C.sub.1-C.sub.8 alkyl
or aryl), and unsaturated analogs of any of the foregoing
definitions for substituent A;
[0174] wherein R.sup.1 is selected from the group consisting of
hydrogen and methyl;
[0175] wherein R.sup.6 is selected from the group consisting of
hydrogen, and halogen, and C.sub.1-C.sub.8 alkyl;
[0176] wherein R.sup.a is selected from the group consisting of
straight or branched C.sub.1-C.sub.8 alkylene, C.sub.3-C.sub.7
cycloalkylene; and
[0177] R.sup.b is selected from the group consisting of hydrogen,
substituted or unsubstituted phenyl, C.sub.2-C.sub.10 acyl,
C.sub.2-C.sub.10 acyloxy, C.sub.2-C.sub.10 alkoxycarbonyl, and
halogen;
[0178] provided that when A is methyl, R.sup.a and R.sup.b together
have at least two carbon atoms, and R.sup.1 is methyl.
[0179] It is preferred that the optional pi bond at 6 is present,
that R.sup.6 is methyl; or that R.sup.a is C.sub.1-C.sub.6
alkylene.
[0180] It is preferred that A is either methyl or
--N(R.sup.d)R.sup.e.
[0181] It is also preferred when A is N(R.sup.d)R.sup.e, that
R.sup.d is methyl or that R.sup.e is C.sub.1-C.sub.6 alkyl or
phenyl C.sub.1-C.sub.6 alkyl.
[0182] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 33
[0183] wherein the dotted line is an optional pi bond;
[0184] wherein R.sup.16.beta. is selected from the group consisting
of hydrogen, fluoro, chloro, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl, a moiety which together with R.sup.16.alpha. is
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoing definitions of R.sup.16.beta.;
[0185] wherein R.sup.16.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
haloalkyl and a moiety which together with R.sup.16.beta. forms
C.sub.4-C.sub.7 spirocycloalkyl, C.sub.4-C.sub.7
halospirocycloalkyl, or .dbd.--R'.sup.16 (R'.sup.16 being
C.sub.1-C.sub.3 alkyl) and unsaturated analogs of any of the
foregoings;
[0186] wherein R.sup.15.alpha. is selected from the group
consisting of hydrogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkenyl and C alkynyl;
[0187] wherein R.sup.19 is either --H or --CH.sub.3; and
[0188] wherein R.sup.6 is selected from the group consisting of
--H, --CH.sub.3, and halo;
[0189] provided that R.sup.16.beta., R.sup.16.alpha., and
R.sup.15.alpha. are not simultaneously hydrogen.
[0190] It is preferred that R.sup.16.alpha. is a larger substituent
than R.sup.16.beta., that R.sup.6 is hydrogen, that the optional pi
bond at position 1 is not present or that R.sup.16.alpha. is a
C.sub.3-C.sub.5 alkyl chain.
[0191] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 34
[0192] wherein the dotted line is optional pi bond
[0193] wherein X is C.sub.1-C.sub.3 alkyl;
[0194] wherein Y is hydrogen or an acyloxy moiety;
[0195] wherein R.sup.6 is --H or --CH.sub.3;
[0196] wherein R.sup.16 is --H or halo;
[0197] wherein R.sup.1 is --H or --CH.sub.3.
[0198] It is preferred that R.sup.6 is methyl that the optional pi
bond at position 1 is present, that Y is a C.sub.3-C.sub.6
fluoroacyloxy or that X is methyl.
[0199] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 35
[0200] wherein n is an integer from 1-2;
[0201] wherein the dotted lines are optional pi bonds;
[0202] wherein X and Y are independently selected from the group
consisting of --H, (C.sub.1-C.sub.3)alkyl, (C.sub.2-C.sub.3)
alkenyl, and methoxycarbonyl;
[0203] wherein Z is selected from the group consisting of --H and
(C.sub.1-C.sub.3)alkyl;
[0204] wherein R.sup.3 is selected from the group consisting of
hydrogen, acyl, carboxyl, alkoxycarbonyl, substituted or
unsubstituted carboxamide, cyano, alkoxy, alkoxyalkoxy,
alkythioalkoxy, acyloxy; hydroxy, halo, O--SO.sub.2R.sup.a (R.sup.a
being selected from the group consisting of C.sub.1-C.sub.6 alkyl
and C.sub.6-C.sub.10 aryl), and a moiety which, together with
R.sub.2, is a 5-6 member ring containing at least one oxygen and
one nitrogen atom;
[0205] wherein R.sup.2 is selected from the group consisting of
hydrogen, amido, acyloxy, carboxyl, carboxamide, alkoxycarbonyl,
cyano, halo, nitro, C.sub.1-C.sub.8 alkyl, and CF.sub.3 and a
moiety which, together with R.sub.3, is a 5-6 member ring
containing at least one oxygen and one nitrogen atom;
[0206] wherein R.sup.4 is hydrogen or halo;
[0207] wherein R.sup.6 is selected from the group consisting of
hydrogen and oxo;
[0208] wherein R.sup.9 is --H or --OH;
[0209] provided that X, Y, and Z are not hydrogen when R.sup.3 is
methoxy.
[0210] It is preferred that R.sup.3 is alkoxyalkoxy; carboxamide,
carboxyl or alkoxyl, that at least one of X, Y or Z is methyl, that
X and Y are methyl, that n is 1 or R.sup.6 is oxo.
[0211] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 36
[0212] wherein the dotted lines are optional pi bonds;
[0213] wherein n=1 or 2; and
[0214] wherein a is either --H or --CH.sub.3;
[0215] wherein b and c are independently hydrogen or methyl;
[0216] wherein Z is oxygen or sulfur.
[0217] It is preferred that n is 1, that at least one of b or c is
methyl, that both b and c are methyl or that Z is oxygen.
[0218] In another embodiment, the invention provides an inhibitor
of type 5 17.beta.-hydroxysteroid dehydrogenase having a molecular
structure selected from the group consisting of: 3738394041
[0219] It is preferred that the type 5 inhibitor is selected from
the group consisting of: 42
[0220] In another embodiment, the invention provides a method of
inhibiting type 3 17.beta.-hydroxysteroid dehydrogenase comprising
administering to a patient in need of such treatment a
therapeutically effective amount of an inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase having the molecular
structure: 43
[0221] wherein R is selected from the group consisting of alkoxy,
alkylthio, alkoxyalkoxy, alkoxyalkylthio, alkylthioalkoxy, and
alkylthioalkylthio, or 44
[0222] wherein n is an integer from 1 to 4.
[0223] It is preferred that the said type 3 inhibitor is selected
from the group consisting of: 4546
[0224] In another embodiment, the invention provides a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient, diluent or carrier and a therapeutically acceptable
amount of an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase having the molecular structure: 47
[0225] wherein R is selected from the group consisting of alkoxy,
alkylthio, alkoxyalkoxy, alkoxyalkylthio, alkylthioalkoxy, and
alkylthioalkylthio, or 48
[0226] wherein n is an integer from 1 to 4.
[0227] It is preferred that the said type 3 inhibitor is selected
from the group consisting of: 4950
[0228] In another embodiment, the invention provides an inhibitor
of type 3 17.beta.-hydroxysteroid dehydrogenase having the
molecular structure: 51
[0229] wherein R is selected from the group consisting of
alkoxyethoxy, alkoxyalkylthio, alkylthioalkoxy, and
alkylthioalkylthio, or 52
[0230] wherein n is an integer from 1 to 4.
[0231] It is preferred that the said type 3 inhibitor is selected
from the group consisting of: 5354
[0232] A patient in need of treatment or reducing the risk of onset
of a given disease is one who has either been diagnosed with such
disease or one who is particularly susceptible to acquiring such
disease, for example, one at higher risk of acquiring the disease
than the general population.
[0233] Except where otherwise stated, the preferred dosage of the
active compounds of the invention is identical for both therapeutic
and prophylactic purposes. The dosage for each active component
discussed herein is the same regardless of the disease being
treated (or prevented).
[0234] As used in the methods of medical treatment or methods of
reduction of risk of onset of disease herein, an "inhibitor of type
5 or 3 17.beta.-hydroxysteroid dehydrogenase" means a compound
whose IC.sub.50 of inhibition for the enzyme in question (computed
in the same manner as described in connection with Table 1 herein)
is no higher than 500 nM. It is preferred that such inhibitor be no
higher than 20 nM, most preferably lower than 10 nM. In some
embodiments of the type 5 or type 3 inhibitor, it is also preferred
that IC.sub.50 of undesirable inhibition of type 2
17.beta.-hydroxysteroid dehydrogenase be more than 5 times that for
inhibition of type 5, preferably more than 10 times, and most
preferably more than 25 times. In some embodiments, it is preferred
that the percentage of inhibition of the binding of [.sup.3H]R1881
on the androgen receptor (as described in D--Androgen Receptor (AR)
assays, supra), by the inhibitor of type 5 or 3
17.beta.-hydroxysteroid dehydrogenase at the concentration of
10.sup.-6 M be less than 30% most preferably less than 20%.
[0235] Where two or more different active agents are discussed as
part of a combination therapy herein (e.g. an enzyme inhibitor and
an antiandrogen), a plurality of different compounds are
administered rather than a single compound having multiple
activities.
[0236] Except where otherwise noted or where apparent from context,
dosages herein refer to weight of active compounds unaffected by
pharmaceutical excipients, diluents, carriers or other ingredients,
although such additional ingredients are desirably included, as
shown in the examples herein. Any dosage form (capsule, tablet,
injection or the like) commonly used in the pharmaceutical
industry, is appropriate for use herein, and the terms
"ingredient", "diluent" or "carrier" include such non active
ingredients as are typically, included together with active
ingredients, used in such dosage form in the industry.
BRIEF DESCRIPTION OF THE DRAWINGS
[0237] FIGS. 1a to 1d show autoradiographs of .sup.3H-labelled type
5 17.beta.-HSD antisense and sense riboprobes (35 base pairs)
hybridized in situ to human benign prostatic hyperplasia (BPH)
tissue.
[0238] FIG. 1.sup.a shows semithin Epon section (0.7 .mu.m-thick)
hybridized with the antisense probe. Epithelial cells lining the
tube-alveoli as well as some of the surrounding stromal cells are
labelled. In the epithelium, the dashed line approximately
indicates the boundary between the basal and the luminal cells. The
basal cells are intensively labelled in comparison with the luminal
cells where only a few grains are seen. (X 1000)
[0239] FIG. 1.sup.b shows similar area from the same prostate
hybridized with the sense probe as a control. Only scattered silver
grains can be detected. (X 1000)
[0240] FIG. 1.sup.c shows the antisense probe generates strong
radioautographic signals in the wall of blood vessels (arrows). (X
600)
[0241] FIG. 1.sup.d shows similar blood vessels hybridized with the
sense probe as a control. No significant labeling could be seen. (X
600)
[0242] FIG. 2 shows the specificity of the antiserum used in the
immunostaining of type 5 17.beta.-HSD. Transfected human kidney
cells (293) with human type 5 17.beta.-HSD, types 1 and 3
3.alpha.-HSD and types 1 and 2 5.alpha.-reductase, were used for
immunoblot analysis. The results indicate that the antiserum
specifically reacts with type 5 17.beta.-HSD. Types 1 and 3
3.alpha.-HSD which share 84 and 86% amino acid identity with type 5
17.beta.-HSD, respectively, and types 1 and 2 5.alpha.-reductase,
two other androgen-synthesizing enzymes present at high
concentration in prostatic tissue were used as controls.
[0243] FIGS. 3a to 3e show paraffin sections of normal human
prostate and cultured epithelial cells (PrEC 5500-1) immunostained
with antibody to type 5 17.beta.-HSD (X 800).
[0244] FIG. 3a shows the staining reaction is observed in basal
cells (arrows) and in most of the stromal cells situated below. The
luminal cells of the epithelium (above the basal cells) are not
reactive.
[0245] FIG. 3b shows all epithelial cells are immunoreactive.
[0246] FIG. 3c shows the basal cells are not very well seen (thick
arrows) but they are labelled as well as some luminal cells (thin
arrows) of the tube-alveoli.
[0247] FIG. 3d shows an immunostained paraffin section of normal
human prostate epithelial cells (PrEC 5500-1). The staining
reaction can be seen in the cytoplasm of most of the cells.
[0248] FIG. 3e shows the same cultured cells as in FIG. 3d, but the
antiserum was incubated with an excess of antigen. No staining
reaction can be seen.
[0249] FIGS. 4a to g show paraffin sections of BPH tissue
immunostained with antibody to type 5 17.beta.-HSD.
[0250] FIG. 4a shows all of the basal cells show a strong positive
staining reaction (arrowheads). Note the absence of reaction in the
luminal cells (L) while the fibroblasts of the stroma are stained
(arrows) (X 500).
[0251] FIG. 4b shows some of the luminal cells are immunoreactive
(arrows) as well as basal cells (arrowheads) (X500).
[0252] FIG. 4c shows a section consecutive to that shown in FIG.
4a. Immunoabsorption of the antiserum with an excess of antigen
completely prevents immunostaining (X500).
[0253] FIG. 4d shows, in the stroma, smooth muscle cells (arrows)
are not labeled while the surrounding fibroblasts are stained
(X800).
[0254] FIG. 4e shows the positive staining in the wall of a large
vein (V). The endothelial cells of small blood vessels are
well-labeled (arrows) (X 800).
[0255] FIG. 4.sup.f shows the low magnification of an artery
showing the labelling of endothelial cells (arrows) and of
fibroblasts of the tunica adventitia (arrowheads) while smooth
muscle cells of tunica media (m) are weakly labeled (X 200).
[0256] FIG. 4g shows the high magnification of an artery which
clearly demonstrates the labelling in the cytoplasm of endothelial
cells (arrows) as well as of fibroblasts (arrow heads) while smooth
muscle cells of the tunica media (m) are not labeled (X 800).
[0257] FIGS. 5a to e show paraffin sections of BPH tissue
immunostained with antibodies for type 5 17.beta.-HSD (a),
3.beta.-HSD (b) and androgen receptor (c, d and e) (X800).
[0258] FIGS. 5a and b show consecutive sections immunostained for
type 5 17.beta.-HSD (5a) and 3.beta.-HSD (5b). Although the
reaction for 3.beta.-HSD is somewhat weaker than that obtained for
type 5 17.beta.-HSD, the distribution of the two enzymes in basal
(arrowheads) and luminal cells (L) is similar.
[0259] FIG. 5c shows an androgen receptor immunoreactivity was
found to be exclusively localized in the nuclei. In the epithelium,
the reaction can be seen in the majority of the luminal cells (L)
nuclei, but not in the nuclei of most basal cells (arrowheads).
[0260] FIG. 5d shows, in the fibromuscular stroma, most of the
nuclei are labeled. Some smooth muscle cell nuclei are labeled
(arrows) while others show not detectable reaction
(arrowheads).
[0261] FIG. 5e shows the wall of the blood vessels shows labeled
and unlabeled nuclei. Some of the nuclei of the endothelial cells
lining the lumen of an arteriole are labeled (arrows), while others
show no staining (arrowheads).
[0262] FIG. 6 shows human skin hybridized with type 5 17.beta.-HSD
cRNA probe.
[0263] FIG. 7 shows monkey ovary hybridized with type 5
17.beta.-HSD.
[0264] FIG. 8 is a schematic diagram showing the biosynthetic
pathway of active androgens in human prostatic tissue.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0265] There are several types of 17.beta.-hydroxysteroid
dehydrogenases. Of the various types of 17.beta.-hydroxysteroid
dehydrogenases, type 1 catalyzes conversion of estrone to
estradiol. Types 2 and 4 catalyze the reverse reaction. Type 1 also
catalyzes conversion of DHEA to 5-diol while type 4 catalyzes the
reverse reaction. Types 3 and 5 catalyze conversion of 4 dione to
testosterone while type 2 catalyzes the reverse reaction. Types 3
and 5, the primary concern of the present application, catalyze in
vivo the biosynthesis of androgenic compounds, such as the
biosynthesis of testosterone from androst-4-ene-dione ("4-dione")
or the conversion of androstanedione (A-dione) to DHT. The reaction
catalyzed by type 3 or type 5 17.beta.-hydroxysteroid dehydrogenase
is a reversible reaction, and the reverse reaction can be catalyzed
by type 2 17.beta.-hydroxysteroid dehydrogenase, whose activity is
preferably not inhibited. It is also desirable to avoid inhibition,
for example, of type 4 17.beta.-hydroxysteroid dehydrogenase which
desirably (for androgen sensitive diseases) converts a different
testosterone precursor to another compound.
[0266] For purposes of treating diseases which respond unfavorably
to androgenic activity, it is desirable to inhibit androgen
formation without hindering androgen degradation. Thus, in one
aspect, the present invention endeavors to reduce the level of
androgens by selectively inhibiting the type 3 and type 5
17.beta.-hydroxysteroid dehydrogenases while desirably allowing the
type 2 and type 4 enzymes to remain free of inhibition. Thus, the
equilibrium of the reversible androgen (or androgen precursor)
synthesis reactions is beneficially affected by inhibiting the
forward androgen synthesis reactions while permitting any androgens
or precursors that are synthesized (either locally or systemically)
to be reconverted to their precursors by action of the type 2
and/or type 4 17.beta.-hydroxysteroid dehydrogenases.
[0267] The type 5 enzyme is better inhibited by certain compounds
discussed in more detail infra, while the type 3 enzyme is better
inhibited by other compounds (also discussed infra). It is
therefore possible to provide best inhibition of androgen synthesis
by a combination therapy which includes administering both a type 5
inhibitor and a type 3 inhibitor.
[0268] Type 5 enzyme tends to predominate in liver, adrenal glands,
prostate, and the peripheral tissues whereas type 3 enzyme tends to
predominate in the testes. In the prior art, testicular androgens
have been suppressed by either surgical or chemical castration,
frequently by administration of an LHRH agonist. The present
invention provides inhibitors or type 3 17.beta.-hydroxysteroid
dehydrogenase which, like the LHRH agonist of the prior art, reduce
testicular androgen production. Thus, in some embodiments of the
invention, the type 3 inhibitors of the invention may be used to
inhibit testicular androgen secretion, either alone or in
combination with prior art chemical or surgical castration. In some
embodiments, antiandrogen and/or 5.alpha.-reductase inhibitor is
also provided. In some embodiments, the inhibitors of the type 3
enzyme are used in addition to chemical castration by LHRH agonist.
An undesirable initial upward spike of androgenic production and
secretion by the testes occurs during the very early portion of
treatment with LHRH agonist, prior to the beginning of the
beneficial effect of androgen reduction caused by LHRH agonist.
Using type 3 17.beta.-hydroxysteroid dehydrogenase inhibitor in
conjunction with LHRH agonist is expected to reduce that initial
undesirable androgen elevation.
[0269] Both androgen-related and estrogen-related diseases may be
treated with the 17.beta.-hydroxysteroid dehydrogenase inhibitors
of the present invention. Androgen-sensitive diseases are those
whose onset or progress is aided by androgen activation of androgen
receptors, and should respond favorably to treatment with the
present invention because of the reduction of androgen biosynthesis
that is achieved thereby. Estrogen-sensitive diseases (diseases
whose onset or progress is aided by activation of the estrogen
receptor) should also benefit because many androgens whose
biosynthesis is suppressed by the present invention are precursors
to estrogens, and the present invention may therefore reduce
estrogen biosynthesis as well. Androgen-sensitive diseases include
but are not limited to prostatic cancer, benign prostatic
hyperplasia, acne, seborrhea, hirsutism, androgenic alopecia, and
polycystic ovarian syndrome. Estrogen-sensitive diseases include
but are not limited to breast cancer, endometrial cancer,
endometriosis, and endometrial leiomyoma.
[0270] For the treatment of androgen-sensitive diseases, it is of
course preferred that the inhibitors of 17.beta.-hydroxysteroid
dehydrogenase that are administered (whether type 5 or type 3 or
both) do not possess inherent androgenic activity. However, breast
cancer (and some other estrogen-sensitive diseases, e.g. ovarian
cancer, uterine cancer, and endometrial cancer) respond favorably
to androgens. Therefore, a compound which inhibits type 5 or type 3
17.beta.-hydroxysteroid dehydrogenase, and which is also
androgenic, can be especially useful for the treatment of breast
cancer and other diseases which respond negatively to estrogen and
positively to androgen.
[0271] Type 5 and/or type 3 inhibitors of 17.beta.-hydroxysteroid
dehydrogenase may, in accordance with the invention, be utilized as
part of a combination therapy with other strategies (listed below)
which, attack androgen- or estrogen-sensitive diseases through
other mechanisms, thus providing synergistic combinations. These
combination therapies include in addition to type 3 or type 5
inhibitors of 17.beta.-hydroxysteroid dehydrogenase one or more of
the following strategies:
[0272] Strategy 1: Suppression of testicular or ovarian hormonal
secretion by chemical or surgical castration. This is useful in
treatment of diseases which respond adversely to androgen or
estrogen, respectively. When surgical or chemical castration is
utilized, chemical castration is preferred utilizing either an
LHRH-agonist, an LHRH antagonist or inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase (as discussed herein).
Suitable LHRH agonists are reported in U.S. Pat. No. 4,659,695, but
any LHRH agonist showing the ability to induce chemical castration
can be used since they all act through the same mechanisms as
described (Labrie et al., J. Androl. 1: 209-228, 1980). Dosages are
known in the art. Some suitable LHRH antagonists are reported in
U.S. Pat. No. 4,666,885 but any LHRH antagonist is acceptable, if
used according to the recommendation of the manufacturer.
[0273] Strategy 2: Utilizing androgen or estrogen receptor
antagonists ("antiandrogens" or "antiestrogens") to prevent
activation of androgen or estrogen receptors by androgens or
estrogens. Strategy 2 is useful against diseases that respond
adversely to androgenic or estrogenic activity, respectively.
Antiandrogens, and dosages therefor, are known in the art (e.g.
Flutamide (N-[4-nitro-3-(trifluoromethyl)phenyl)]-2-methyl
propanamide) at a dosage of 250 mg, 2 or 3 times a day, Nilutamide
at a dosage of 150 mg/day, Casodex at a dosage of 50 to 500 mg/day
or EM-250 of the following structure: 55
[0274] Synthesized and used as reported in International
Publication WO94/26767.
[0275] When antiestrogens are used in accordance with the
invention, either alone or as part of one of the combination
therapies described herein, the attending clinician should, at
least initially, use the dosages recommended by the manufacturer.
However, the attending clinician should monitor individual patient
response and metabolism and adjust patient dosage accordingly.
Indeed, that will be true of all of the strategies discussed
herein. One preferred antiestrogen is EM-800 reported in
PCT/CA96/00097 (WO 96/26201) The molecular structure of EM-800 is:
56
[0276] Other suitable antiestrogens include but are not limited to
Tamoxifen ((Z)-2-[4
(1,2-diphenyl-1-butenyl)]-N,N-dimethylethanamine) and ICI 182780
(available from Zeneca, UK), Toremifene (available from
Orion-Farmos Pharmaceuticla, Finland), Droloxifene (Pfizer Inc.,
USA), Raloxifene (Eli Lilly and Co., USA), LY 335563 and LY 353381
(Eli Lilly and Co., USA), Iodoxifene (SmithKline Beecham, USA),
Levormeloxifene (Novo Nordisk, A/S, Denmark). Any antiestrogen used
as required for efficacy, as recommended by the manufacturer, can
be used. Appropriate dosages are known in the art. Any other
antiestrogen commercially available can be used according to the
invention.
[0277] Strategy 3: Suppression of sex steroid biosynthesis by
inhibition of 3.beta.-hydroxysteroid dehydrogenase [e.g. Trilostane
(2.alpha.-cyano-4.alpha.,5.alpha.-epoxy-17.beta.-hydroxyandrostan-3-one),
Sterling-Winthrop Research Institute, Renslaer, New York, USA] by
administering such inhibitor at a dosage of 1 to 500 mg/day).
Desirable serum levels of 3.beta.-hydroxysteroid dehydrogenase
inhibitor range from 5 ng/ml to 500 ng/ml, preferably 10 ng/ml to
100 ng/ml. Strategy 3 is useful against both (1) diseases that
respond adversely to androgenic activity and (2) some diseases that
respond adversely to estrogenic activity.
[0278] Strategy 4: Suppression of conversion of the androgen
testosterone to the more potent androgen dihydrotestosterone (DHT)
by inhibiting the activity of testosterone 5.alpha.-reductase (e.g.
by administering Proscar, available from Merck Sharp and Dohme
Canada, at the recommended dosage). Any other potent
5.alpha.-reductase inhibitor can be used. The dosage used can be 2
to 20 mg daily orally. The dosage should be the one recommended by
the manufacturer. Strategy 4 is useful against diseases that
respond adversely to androgenic activity.
[0279] Strategy 5: Utilizing an aromatase inhibitor to reduce
estrogen production. Strategy 5 is useful against diseases that
respond adversely to estrogenic activity or estrogen
receptor-mediated exacerbation of the type of androgen-sensitive
diseases that are also estrogen-sensitive diseases (e.g. benign
prostatic hyperplasia). When aromatase inhibitors are used in
accordance with the invention, either alone or as part of one of
the combination therapies described herein, the attending clinician
should initially use the dosage recommended by the manufacturer.
When administered orally, the dosage which is usually effective to
provide the desired serum levels is between 1.0 mg and 20 mg of
active ingredient per day per 50 kg of body weight. For example,
Arimidex (Zeneca) is taken at the oral dose of 1 mg daily. However,
the attending clinician should monitor individual patient response
and metabolism and adjust patient dosage accordingly. Aromatase
inhibitors include those set forth in U.S. Pat. No. 5,227,375.
Aromatase inhibition may also be achieved, for example, by
administering Arimidex (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-
-1,3-phenylene bis (2-methylpropiononitrile)) available from
Zeneca, UK, at a dosage of 1 mg/day. Any other aromatase inhibitor
can be used according to the recommendations of the
manufacturer.
[0280] Strategy 6: Administering an androgenic compound. Strategy 6
is useful against a disease which responds favorably to androgenic
activity. Preferred androgens include but are not limited to
medroxyprogesterone acetate, and megestrol acetate at a dosage of
5-200 mg/day. Desirable low dose and sustained release formulations
of such androgens are described in U.S. Pat. No. 5,434,146.
[0281] Any of the above strategies useful against diseases that
respond negatively to estrogenic activity may also be used to help
alleviate estrogenic side effects that may result from treatment of
purely androgen-sensitive diseases in accordance with the
invention. For example, many androgen precursors are also estrogen
precursors. Strategies which suppress formation of certain
androgens may increase levels of precursors thereto which are also
estrogen precursors thus undesirably increasing estrogen formation.
For example, administering inhibitors of testosterone
5.alpha.-reductase to suppress conversion of testosterone to
dihydrotestosterone may increase the conversion of testosterone to
estradiol. In that case, strategy 5 may desirably reduce that
unwanted estrogen production Strategy 2 (estrogen antagonist
aspect) and strategy 3 may also be helpful in that regard.
[0282] In general, for both androgen-sensitive diseases and
estrogen-sensitive diseases, simultaneous treatment with inhibitors
of sex steroid biosynthesis (inhibitors of enzymes which catalyze
one or more steps of estrogen or androgen biosynthesis), and with
estrogen receptor antagonists and/or androgen receptor antagonists,
are believed to have additive rather than redundant effect because
they are acting in a beneficial manner by a different mechanism.
Likewise, the activity of two different enzyme inhibitors (enzymes
which catalyze one or more different steps of sex steroid
biosynthesis) are believed to provide additive effect, especially
where the inhibitors affect more than one synthetic pathway. Such
an approach permits to achieve a more complete effect.
[0283] Different sex steroid-dependent diseases respond differently
to both androgen receptor activation and estrogen receptor
activation. For example, breast cancer responds unfavorably to
estrogen receptor activation, but favorably to androgen receptor
activation. On the other hand, benign prostatic hyperplasia
responds unfavorably to activation of either the estrogen or
androgen receptor. The type 5 and/or type 3 inhibitors of the
invention may be used in any combination with any of the strategies
1-6 above whose effect (increasing or decreasing androgenic or
estrogenic activity) is consistent with desirable effect on the
disease in question. With that in mind, set forth below are a list
of representative diseases which may be treated, or risk of which
may be reduced, in accordance with the present invention. Beneath
each disease, is several preferred combination therapies for
treatment, or risk reduction, of that particular disease. However,
these combinations may be supplemented using one or more of the six
strategies listed above, limited only by whether a particular
disease responds favorably or adversely to estrogenic activity or
to androgenic activity.
[0284] A) Prostate cancer (responds adversely to androgenic
activity, favorably to estrogenic activity)
[0285] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH-agonist (or antagonist)
[0286] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase.
[0287] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist (or antagonist)
[0288] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH-agonist (or antagonist)+antiandrogen
[0289] 5. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0290] 6. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist (or antagonist)+antiandrogen
[0291] 7. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+5.alpha.-reductase inhibitor+LHRH
agonist (or antagonist)
[0292] 8. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist+5.alpha.-reductase inhibitor
[0293] 9. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+5.alpha.-reductase inhibitor
[0294] 10. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+5.alpha.-reductase inhibitor
[0295] 11. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist (or
antagonist)+antiandrogen+5.alpha.-reductas- e inhibitor
[0296] B) Benign prostatic hyperplasia (responds adversely to both
androgenic activity and estrogenic activity)
[0297] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0298] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen or aromatase inhibitor
[0299] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0300] 4. inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+5.alpha.-reductase
inhibitor+antiestrogen or aromatase inhibitor
[0301] 5. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+5.alpha.-reductase inhibitor
[0302] 6. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+5.alpha.-reductase inhibitor
[0303] 7. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+5.alpha.-reductase inhibitor+antiestrogen or
aromatase inhibitor
[0304] C) Acne, seborrhea, hirsutism, androgenic alopecia (responds
adversely to androgenic activity)
[0305] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0306] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0307] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+5.alpha.-reductase inhibitor
[0308] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+5.alpha.-reductase inhibitor+antiandrogen
[0309] D) Polycystic ovarian syndrome (responds adversely to
androgenic stimulation)
[0310] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0311] 2. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0312] 3. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase
[0313] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0314] 5. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0315] 6. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0316] E) Breast cancer (responds favorably to androgenic activity,
adversely to estrogenic activity)
[0317] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen
[0318] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen+androgenic compound
[0319] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen+LHRH agonist (or antagonist)
[0320] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen+LHRH-agonist (or antagonist)+androgenic
compound
[0321] F) Endometriosis, leiomyoma (responds adversely to
estrogenic activity)
[0322] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0323] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiestrogen or inhibitor of aromatase
[0324] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH-agonist (or antagonist)
[0325] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH-agonist (or antagonist)+antiestrogen or
aromatase inhibitor
[0326] G) Precocious puberty (male and female) (responding
adversely to androgenic activity)
[0327] 1. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase
[0328] 2. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist (or antagonist) (male only)
[0329] 3. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0330] 4. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+LHRH agonist (or antagonist) (male
only)
[0331] 5. Inhibitor of type 5 17.beta.-hydroxysteroid
dehydrogenase+inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase (male only)
[0332] 6. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase
[0333] 7. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+LHRH agonist (or antagonist) (male only)
[0334] 8. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen
[0335] 9. Inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase+antiandrogen+LHRH agonist (or antagonist) (male
only)
[0336] When type 5 17.beta.-hydroxysteroid inhibitors are used in
accordance with the invention, either alone or as part of one of
the combination therapies described herein, the attending clinician
desirably will target patient serum concentration of the type 5
inhibitor between 0.5 ng/ml and 100 ng/ml, preferably between 1
ng/ml and 20 ng/ml, and most preferably between 1 ng/ml and 10
ng/ml. Serum concentration may be measured by LC/MS. When
administered orally, the dosage which is usually effective to
provide the desired serum levels is between 1.0 mg and 1,000 mg of
active ingredient per day per 50 kg of body weight, preferably
between 10 mg and 500 mg and most preferably between 10 mg and 100
mg. However, dosage should vary with the bioavailability of the
chosen inhibitor and with individual patient response. For example,
when EM-1394, or EM-1404 are chosen, oral dosage is preferably
between 5 mg and 500 mg per day per 50 kg body weight, more
preferably between 10 mg/day and 300 mg/day, for example between 20
mg/day and 100 mg/day. The attending clinician should monitor
individual patient response and metabolism and adjust patient
dosage accordingly. When administered by injection, a lesser dosage
is usually appropriate, e.g. 10 mg to 100 mg per day per 50 kg of
body weight.
[0337] When type 3 17.beta.-hydroxysteroid inhibitors are used in
accordance with the invention, either alone or as part of one of
the combination therapies described herein, the attending clinician
desirably will target patient serum concentration of the type 3
inhibitor between 0.5 ng/ml and 100 ng/ml, preferably between 1
ng/ml and 20 ng/ml and most preferably between 1 ng/ml and 10
ng/ml. When administered orally, the dosage is preferably between
1.0 mg and 1,000 mg of active ingredient per day per 50 kg of body
weight, preferably between 5 mg and 500 mg and most preferably
between 10 mg and 100 mg. However, the attending clinician should
monitor individual patient response and metabolism and adjust
patient dosage accordingly.
[0338] All of the active ingredients used in any of the therapies
discussed herein may be formulated in pharmaceutical compositions
which include one or more of the other active ingredients.
Alternatively, they may each be administered separately but
sufficiently simultaneous in time so that a patient eventually has
elevated blood levels or otherwise enjoys the benefits of each of
the active ingredients (or strategies) simultaneously. In some
preferred embodiments of the invention, for example one or more
active ingredients are to be formulated in a single pharmaceutical
composition. In other embodiments of the invention, a kit is
provided which includes at least two separate containers wherein,
the contents of at least one container differs in whole or in part
from the contents of at least one other container with respect to
active ingredients contained therein. Two or more different
containers are used in these combination therapies of the
invention. Combination therapies discussed herein also include use
of one active ingredient of the combination in the manufacture of a
medicament for the treatment (or prevention) of the disease in
question where the treatment or prevention further includes the
other active ingredient(s) or strategy of the combination. Some
embodiments of the methods of treating or preventing disease
discussed herein, utilize the type 5 and/or type 3 inhibitors of
17.beta.-hydroxysteroid dehydrogenase discussed herein (i.e. the
molecular structures discussed herein).
[0339] It is believed that, in some situations, the desirability of
using an inhibitor of type 5 17.beta.-hydroxysteroid dehydrogenase
implies the desirability of also using an inhibitor of type 3
17.beta.-hydroxysteroid dehydrogenase and vice versa, depending
upon the expression level of both types in implicated tissues. Type
3 17.beta.-hydroxysteroid dehydrogenase is mainly expressed in the
testis while type 5 17.beta.-hydroxysteroid dehydrogenase is mainly
expressed in liver, adrenal glands, prostate, ovary, skin, mammary
gland, testis, and in a series of peripheral tissues. LHRH agonists
and LHRH antagonists may be used interchangeably to suppress either
testicular or ovarian hormonal secretions by known techniques,
except where preferences are otherwise stated herein.
[0340] In other embodiment, pharmaceutical composition for
combination therapy may contain one type 5 17.beta.-hydroxysteroid
dehydrogenase inhibitor and at least one of other active
ingredients selected from the group consisting of type 3
17.beta.-hydroxysteroid dehydrogenase inhibitor, antiestrogen,
antiandrogen, aromatase inhibitor and 5.alpha.-reductase
inhibitor.
[0341] In another embodiment, pharmaceutical composition for
combination therapy contains one type 3 17.beta.-hydroxysteroid
dehydrogenase inhibitor and at least one of the other active
ingredients selected from the group consisting of type 5
17.beta.-hydroxysteroid dehydrogenase inhibitor, antiestrogen,
antiandrogen, aromatase inhibitor and 5.alpha.-reductase
inhibitor.
[0342] In another embodiment, kits for combination therapy may
contain in two or more containers, one type 5
17.beta.-hydroxysteroid dehydrogenase inhibitors and at least one
of other active ingredients selected from the group consisting of
type 3 17.beta.-hydroxysteroid dehydrogenase inhibitor,
antiestrogen, antiandrogen, aromatase inhibitor, 5.alpha.-reductase
inhibitor, and an LHRH agonist or antagonist.
[0343] In another embodiment, kits for combination therapy may
contain in two or more containers one type 3
17.beta.-hydroxysteroid dehydrogenase inhibitors and at least one
of other active ingredients selected from the group consisting of
type 5 17.beta.-hydroxysteroid dehydrogenase inhibitor,
antiestrogen, antiandrogen, aromatase inhibitor, 5.alpha.-reductase
inhibitor, and an LHRH agonist or antagonist.
[0344] It is desired that activation of glucocorticoid receptors be
minimized when administering the active ingredients of the
invention. In preferred embodiments, active ingredients have no
more effect on glucocorticoid receptors than is provided by
megestrol acetate.
[0345] Type 5 17.beta.-HSD, 3.beta.-HSD and AR in human prostate.
In order to obtain precise information on the cellular distribution
of type 5 17.beta.-HSD and gain a better knowledge of the role of
this enzyme in the human prostate, we performed in situ
hybridization and immunocytochemical localization studies in human
hyperplastic prostatic tissue (BPH). Normal human prostate tissue
and the epithelial prostate cell line (PrEC) were also investigated
by immunostaining. In the same series of experiments, the
immunocytochemical localization of 3.beta.-HSD was examined to
compare the distribution of the two enzymes which are both involved
in the biosynthesis of androgens from DHEA. In order to determine
the site(s) of action of the locally produced androgens, we have
also identified the immunocytochemical localization of the androgen
receptor.
[0346] Materials and Methods
[0347] Tissue preparation. Adult prostatic tissue was obtained from
12 patients with symptomatic benign prostatic hyperplasia (BPH)
undergoing transurethral prostatectomy. The specimens were fixed by
immersion in 2% glutaraldehyde, 4% formaldehyde and 3% dextran in
0.05 M phosphate buffer (pH 7.4). After 4 h, the specimens were
processed and embedded in paraffin or frozen at -70.degree. C. Four
paraffin blocks of normal human prostate, fixed in 4% formaldehyde
(age of patients between 37 and 73) were kindly provided by Dr.
Bernard Tetu, Dep. of Pathology, Hotel-Dieu de Qubec.
[0348] Cultured cells. Normal prostate epithelial cells PrEC 5500-1
were cultured in PrEGM medium (Clonetics) and harvested after the
third passage using a rubber policeman. The cells were then fixed
in 2% glutaraldehyde, 4% formaldehyde and 3% dextran in 0.05M
phosphate buffer for 20 min and centrifuged at 700 rpm for 5 min.
After removing the supernatant, 2% agarose in 0.05 M phosphate
buffer was added to the pellet at 55.degree. C. (the volume of
agarose was twice the volume of the pellet).
[0349] After mixing the cells with agarose, the pellet was
solidified at 4.degree. C. and immersed in the same fixative for
two hours, then washed, processed and paraffin embedded.
[0350] In situ hybridization. Two different procedures were used
for in situ hybridization of BPH tissue. In the first one, 10 .mu.m
sections were cut from frozen tissue with a cryostat and processed.
The second procedure will be described in detail elsewhere (El-Alfy
et al, unpublished data). In brief, thick paraffin sections (20
.mu.m) were cut and the unmounted sections were deparaffinized in
toluene. The sections were subsequently rehydrated, postfixed in 2%
glutaraldehyde, 4% formaldehyde and 3% dextran in 0.05M phosphate
buffer and washed in the same buffer containing 7.5% glycine.
Hybridization of the floating sections was performed overnight at
40.degree. C. with a .sup.3H-UTP riboprobe. Following
hybridization, they were postfixed in osmium tetroxide,
flat-embedded in Epon and cut at 0.7 .mu.m with an ultramicrotome.
Both frozen (10 .mu.m) and semithin (0.7 .mu.m) sections were
coated with liquid photographic emulsion (Kodak NTB-2) and
processed after 14 (semithin sections) or 28 days (frozen sections)
of exposure.
[0351] Sense and antisense riboprobes were generated by in vitro
transcription from the p-Bluescript phagemid containing a cDNA
insert of 35 nucleotides of the human type 5 17.beta.-HSD.
[.sup.35S]- and [.sup.3H]-UTP riboprobes were used for
hybridization with the frozen and floating deparaffinized sections,
respectively.
[0352] Immunocytochemistry. Twelve paraffin-embedded BPH samples,
four normal prostate specimens and PrEC cells in paraffin blocks
were serially cut at 4 .mu.m. Sections were incubated overnight at
4.degree. C. with the human type 5 17.beta.-HSD antiserum diluted
at 1:1000 in Tris-saline, pH 7.6. The sections were then washed and
incubated at room temperature for 4 h with peroxidase-labelled goat
anti-rabbit .gamma.-globulin (Hyclone, Logon, Utah) diluted 1:500.
Endogenous peroxidase activity was eliminated by preincubation with
3% H.sub.2O.sub.2 for 30 min and peroxidase was then revealed
during incubation with 10 mg of 3,3-diaminobenzidine in 100 ml of
Tris-saline buffer containing 0.03% H.sub.2O.sub.2. The intensity
of staining was controlled under the microscope. The sections were
then counterstained with hematoxylin. On other sections,
immunostaining was performed using a commercial kit (Vectastain ABC
Kit; Vector Laboratories, Burlingame, Calif.) and diaminobenzidine
was used as the chromogen to visualize the biotin
streptavidin-peroxidase complex. A microwave retrieval technique
was applied for the androgen receptor staining Control experiments
were performed on adjacent sections by substituting non-immunized
rabbit serum (1:1000). In the case of type 5 17.beta.-HSD antiserum
(diluted 1:1000), immunoabsorption with an excess (10.sup.-6 M) of
the synthetic peptide used to raise the antibodies was also
performed. The number of stained cells (type 5 17.beta.-HSD) and
nuclei (AR) were counted from colored photographs and their number
presented in Table 1. The intensity of staining was compared and
evaluated between the different stained cell types of the prostate
on the same section. Similarly, the density of silver grains was
compared between the labeled cells on the same section. The
intensity of immunostaining and in situ hybridization reaction was
presented in Table 2. Paraffin sections of cultured cells were
immunostained using type 5 17.beta.-HSD antiserum as mentioned
above and the number of immunostained cells presented as a
percentage of stained cells.
[0353] Antibody Preparation
[0354] Type 5 17.beta.-HSD. The type 5 17.beta.-HSD peptide
sequence N-GLDRNLHYFNSDSFASHPNYPYS located at amino acid position
297 to 320 of the human type 5 17.beta.-HSD was synthesized by "Le
Service de Squence de Peptides de l'Est du Qubec" (SSPEQ) (CHUL
Research Center, Qubec, Canada) and purified by HPLC. New Zealand
rabbits (2.5 Kg) received a subcutaneous injection of 100 .mu.g of
the peptide solubilized in 1 ml phosphate saline buffer containing
50% of complete Freund's adjuvant. The animals received at
one-month intervals two successive booster injections with 50 .mu.g
of the peptide in 1 ml of incomplete Freund's adjuvant. Two weeks
after the last injection, the rabbits were killed and the blood
collected. The antiserum was obtained by decantation and separation
by centrifugation, then affinity purified and stored at 80.degree.
C.
[0355] Specificity of the antiserum was examined by immunoblot
analysis. In brief, human embryonal kidney cells (293) were
transfected with CMV-neo vectors expressing human type 5
17.beta.-HSD, types 1 and 3 3.alpha.-HSD and types 1 and 2
5.alpha.-reductase, respectively. Stable transfectants were
selected by their resistance to 10.sup.-7M G-418. Positive clones
were confirmed by their ability to efficiently transform the
appropriate substrate (Luu-The, et al. unpublished data). Forty
micrograms of protein of the homogenate of each cell line were
electrophoresed on a 5-15% sodium dodecyl sulfate
(SDS)-polyacrylamide gel, before being transferred to the
nitrocellulose filter using a Bio-Rad apparatus for 4 h at 60V. The
blot was treated 3 times with 5% fat-free milk in PBS containing
0.1% Nonidet P-40 for 30 min. The antiserum developed against the
type 5 17.beta.-HSD peptide was diluted to {fraction (1/1000)} and
the blot was then incubated at 4.degree. C. for 18 h in the diluted
antiserum. The blot was then washed three times with PBS containing
5% fat-free milk and 0.1% Nonidet P40. After incubation with
horseradish peroxidase-conjugated anti-rabbit IgG in solution for 2
h, the membrane was washed and bound antibodies were detected with
ECL detection reagents (Amersham) and finally the membrane was
exposed to Hyperfilm.
[0356] 3.beta.-HSD. The antiserum used for immunocytochemical
studies was raised by immunizing rabbits with purified human
placental 3.beta.-HSD. This antiserum has been widely used to
localize the enzyme in tissues of several species including the
human.
[0357] Androgen Receptor (AR). AR rabbit antiserum was generated
against a synthetic peptide corresponding to the first 20 amino
acid residues of the N-terminal domain of the human and rat AR. The
antiserum was purified by immunoprecipitation and did not show any
crossreactivity with estrogen or progesterone receptors. This
antiserum was kindly provided by Dr. Tho H. van der Kwast, Dept. of
Pathology, Erasmus University Rotterdam, The Netherlands.
[0358] Results
[0359] Type 5 17.beta.-HSD
[0360] In situ hybridization. In the prostate specimens hybridized
with the [3H]-labeled type 5 17.beta.-HSD probe, all basal cells
are intensively labeled while the luminal secretory cells are
poorly labeled (FIG. 1a). In the fibromuscular stroma, none or only
few silver grains are located over smooth muscle cells, while the
fibroblasts dispersed throughout the stroma or in association with
the wall of blood vessels are well labeled (FIG. 1c).
Interestingly, the endothelial cells lining blood vessels are
strongly labeled. The smooth muscle cells and fibroblasts of the
tunica media and adventitia show variable labeling intensity. When
the hybridization was performed using .sup.3H-labelled sense
riboprobe as control, only a few scattered silver grains were
detected over the epithelium (FIG. 1b) and blood vessels (FIG. 1d).
The results obtained using [.sup.35S]-UTP riboprobes (not shown)
were found to be similar to the above mentioned results.
[0361] Immunostaining
[0362] Type 5 17.beta.-HSD Distribution. As illustrated in FIG. 2,
immunoblot analysis indicates that the antiserum specifically
reacts with type 5 17.beta.-HSD. In fact, no crossreactivity was
detected, either with types 1 or 3 3 .alpha.-HSD which share 84%
and 86% identity with type 5 17.beta.-HSD, respectively, or with
types 1 and 2 5.alpha.-reductase, two enzymes which are abundant in
prostatic tissue
[0363] When immunostained paraffin sections of BPH specimens and
normal prostatic tissues were examined, similar results were found
(FIGS. 3a-c, 4a,b). Some variation in the distribution and
intensity of immunostaining was observed between the twelve BPH
specimens examined. A similar degree of variation was found between
the normal prostate specimens, and the overall pattern was similar
between normal and BPH prostatic tissues. The variation in staining
of the epithelium lining the tube-alveoli was observed not only
between the different specimens but also between the tube-alveoli
of the same specimens. A constant finding was the positive reaction
detected in the stromal fibroblasts while the smooth muscle cells
were not stained (FIGS. 3a-c, FIGS. 4a, b, d). Strong staining was
consistently found in the basal cells of the epithelium (FIGS. 3a,
4a, 5a). In contrast, the luminal secretory cells exhibited highly
variable and usually low immunoreactivity. In fact, in most alveoli
(about 85%), no luminal cells were labelled (FIGS. 3a, 4a) while
about 10% of alveoli contained a low detectable but positive
reaction of all luminal cells (FIG. 3b), the basal cells being
always strongly labelled. In some alveoli (about 5%), labeling of
few luminal cells and all basal cells (Table 1) was found (FIGS.
3c, 4b).
[0364] When the antiserum was immunoabsorbed with the antigen or
when non-immunized rabbit serum was used, no staining could be
detected (FIG. 4c). The endothelial cells of small (FIG. 4e) and
large (FIGS. 4e-g) blood vessels were strongly immunoreactive. The
staining reaction was variable in the smooth muscle cells of the
tunica media, while fibroblasts of the tunica adventitia were
intensively stained. Veins appeared strongly labelled because of
the large number of fibroblasts in their walls (FIG. 4e). In
arteries, the tunica media was lightly stained (FIGS. 4f, g), while
the tunica adventitia was well stained (FIG. 4f).
[0365] When the paraffin sections of cultured epithelial cells were
examined after immunostaining using type 5 17.beta.-HSD antibody,
58% of these cells were found to be positively stained (FIG.
3d).
[0366] 3.beta.-HSD Distribution. The results obtained following
immunostaining with antibody to 3.beta.-HSD were found to be very
similar to those generated with the type 5 17.beta.-HSD antiserum
(FIGS. 5a, b). Although the staining reaction was generally weaker
for 3.beta.-HSD, the cellular distribution of the enzyme
corresponds very well to that described above for type 5
17.beta.-HSD. In the glandular epithelium of the prostate, all the
basal cells were generally labelled while, in the luminal cells,
the staining was variable, being intense in some cells and weak or
absent in most others. In the stroma, the staining was restricted
to the cytoplasm of fibroblasts. As observed for type 5
17.beta.-HSD, specific immunolabelling was found in the endothelial
cells and fibroblasts of blood vessel walls including arteries,
veins and capillaries. In all the 3.beta.-HSD-containing cells, the
staining was restricted to the cytoplasm, no significant nuclear
staining being detected.
[0367] Androgen receptor distribution. The AR appears exclusively
localized in the nuclei of prostate cells in all the specimens
examined. In the epithelium, immunostaining is detected in almost
all the nuclei of the luminal cells, while most of the basal cell
nuclei do not exhibit positive staining (FIG. 5c and Table 1). In
the stroma, the majority of nuclei of the fibromuscular cells are
labelled, but unstained nuclei of smooth muscle cells are also
observed (FIG. 5d and Table 1). In the blood vessels, several
nuclei of the endothelial cells lining the lumen are positive, but
some display no reaction (FIG. 5e). In the tunica media of the
arteries, most of the nuclei of the smooth muscle cells are stained
while some remain negative (not shown). Comparable results were
obtained for the nuclei of fibrocytes of the tunica adventitia.
[0368] Discussion
[0369] In the present study, we have used two complementary
approaches, namely in situ hybridization (using BPH specimens) and
immunocytochemistry (using BPH, normal prostate tissues and
cultured epithelial cells), to identify the cells which express
type 5 17.beta.-HSD in the human prostate. This enzyme was found
mainly in the basal cells of the tube-alveoli, the fibroblasts of
the stroma and blood vessels as well as in the endothelial cells of
the blood vessels (Table 1, 2). This double approach permits to
identify not only type 5 17.beta.-HSD mRNA but also the enzyme
itself. The present data are in agreement with results from this
laboratory, which indicated the presence of androgenic 17.beta.-HSD
activity in human and rhesus monkey prostates.
[0370] The stratified epithelium lining the tube-alveoli is divided
into two layers, namely the basal layer made of low cuboidal cells
and a layer of columnar secretory cells (luminal cells). It is
generally believed that prostatic stem cells are located in the
basal cell layer. As revealed by both in sift hybridization and
immunocytochemistry, the basal cells are expressing type 5
17.beta.-HSD at a much higher level than the luminal cells. In
fact, while many luminal cells exhibited some detectable
hybridization signal, they have shown a high degree of variation
and usually low level of immunostaining (FIGS. 3c, 4b). On the
other hand, the majority of alveoli contained only strongly labeled
basal cells (FIGS. 3a, 4a). However, in a few alveoli, staining was
detectable either in some luminal cells (FIGS. 3c, 4b) or in all of
them (FIG. 3b). This variable staining in luminal cells might be
explained by variations in the biosynthetic activity among alveoli
or among different luminal cells in the same alveolus. It is quite
possible that the low level of the protein in an unknown proportion
of luminal cells cannot be detected by immunocytochemistry. It is
noteworthy to mention that very similar results were obtained with
the antibody against 3.beta.-HSD.
[0371] The cultured epithelial cells PrEC 5500-1 have shown
approximately the same pattern of expression of type 5 17.beta.-HSD
as the epithelial cells of BPH and normal prostate tissues. We
assumed that the cultured epithelial cells are a mixture of basal
and luminal cells. Therefore, it is not surprising to find that
only 58% of these cells expressed the enzyme.
[0372] It has been reported that types 1 and 2 5.alpha.-reductases
are produced by both epithelial and stromal cells in the prostate.
Using immunocytochemistry, it has been shown that staining for type
2 5.alpha.-reductase could be seen in both basal and luminal
epithelial cells. On the basis of studies performed with human
prostatic in vitro models, it has been suggested that the basal
cells exert a stem cell role. On the other hand, in vivo studies
performed in the rat prostate during maturation have established
that both basal and secretory luminal cells are self-replicating
cell types. The presence of type 5 17.beta.-HSD, 3.beta.-HSD and
5.alpha.-reductase isoenzymes in the basal cells suggests that this
cell type is actively involved in androgen production and cannot be
considered as being only a precursor of the luminal secretory
cells.
[0373] Using cells transfected with the cDNAs of different types of
17.beta.-HSD, Luu-The et al have shown that types 1 and 3
17.beta.-HSD catalyze the reduction of estrone to estradiol and
4-androstenedione to testosterone, respectively. They have also
shown that these enzymes are substrate and orientation selective.
In fact, type 3 and 5 17.beta.-HSDs have the same selective
function, but type 3 was detected only in the testis and was not
found in the human prostate. Therefore, in the prostate, the
reduction of 4-Dione to testosterone is probably due to type 5
17.beta.-HSD. Since type 5 17.beta.-HSD and 3.beta.-HSD are both
highly expressed in basal cells while the androgen receptor is
mainly present in luminal cells (FIG. 5c and Table 1), it is
tempting to suggest that testosterone synthesized in the basal
cells reaches the luminal cells in a paracrine fashion to be
ultimately transformed into DHT in the luminal cells where the
androgenic action is exerted and AR is highly expressed. DHT made
in the luminal cells by the action of 5.alpha.-reductase would then
exert its action in the luminal cells themselves, thus meeting the
definition of intracrine activity. The involvement of two cell
types in the biosynthesis of steroids has already been shown to
occur in the ovary. In fact, in the ovary, C19 steroids
(androstenedione and testosterone) synthesized by theca interna
cells are transferred to granulosa cells where they are aromatized
into estrogens. The present data suggest the possibility of a
similar two-cell mechanism of androgen formation in the human
prostate: testosterone is first synthesized in the basal cells
before diffusing into the luminal cells where transformation into
DHT occurs.
[0374] In the present study, the fibroblasts present in the stroma
as well as those associated with blood vessels are shown to contain
type 5 17.beta.-HSD mRNA as well as the immunoreactive type 5
17.beta.-HSD and 3.beta.-HSD enzymes. The two types of
5.alpha.-reductase have also been detected in this cell type by
various techniques. The role of the steroidogenic enzymes in
fibroblasts remains to be established but since androgen receptors
are present in the nuclei of most stromal cells (Table 1), it is
likely that DHT could act in the fibroblasts themselves (intracrine
action) to modulate the activity of these cells.
[0375] Previous study has shown that in normal prostate, basal
cells contained the mRNA for AR but lacked an immunodetectable
receptor while in the luminal cells both mRNA and immunodetectable
receptor were present. These authors have also stated that AR
localization in BPH was identical to that observed in normal
prostate. Similarly, it has been found that the nuclei of the
luminal and the majority of stromal cells were positive to androgen
receptor antibody in hyperplastic as well as normal prostatic
glands. It has also been found that primary, as well as metastatic,
prostate carcinomas show nuclear staining for AR and that the
proportion and the intensity of immunostained human prostate tumor
cells decreased in the more aggressive tumors. The basal cells also
express nuclear AR in normal and hyperplastic tissue. However, the
receptor was most frequently expressed at lower levels in the basal
cells when compared with the staining intensity detected in
secretory luminal cells. It has been found that AR immunostaining
was localized to the nuclei of luminal cells but was weak or absent
in basal cells and of variable intensity in the stromal cells. In
the present study, while 94% of luminal cells expressed nuclear AR,
only 37% of basal cells were stained (Table 1) and their staining
intensity was lower than that of luminal cells (Table 2). The
majority (66%) of fibro-muscular stromal cells also expressed AR.
The findings of the present study are thus in agreement with
previous studies performed in human, rat and mouse tissue. Since
the stroma/epithelium cell ratio is higher in the hyperplastic
prostate, it can be hypothesized that androgens synthesized
intracellularly by fibroblasts can influence the production of
collagen and elastic fibers in the stroma.
[0376] An unexpected finding was the localization of type 5
17.beta.-HSD and 3.beta.-HSD in blood vessel walls, including the
endothelial cells. This observation, however, correlates well with
recent findings from this laboratory indicating the presence of
types 1 and 2 5.alpha.-reductase mRNA in blood vessel walls in
human prostate and skin. Recently, we have also observed that
immunoreactive type 5 17.beta.-HSD is present in the blood vessel
walls in other tissues such as skin, breast, uterus and ovary. The
role of the steroidogenic intracrine enzymes in these vascular
structures is unknown.
[0377] Previously, it has been shown that androgen receptors were
present in vascular smooth muscle and endothelial cells of human
skin. It has been found that nuclear androgen receptors were
present in the muscular layer of almost all arteries within the rat
testis. It has been suggested that testicular blood vessels could
be a target organ for androgens and may mediate some of the effects
of androgens on testicular microcirculation. Furthermore, in the
developing human prostate, AR was positive in the nuclei of
vascular smooth muscle and endothelial cells. Since androgen
receptors are present in the endothelial cells, smooth muscle cells
and fibroblasts of blood vessels (Table 1), it may be speculated
that locally biosynthesized androgens are exerting a paracrine
and/or intracrine action in blood vessels. It is also possible that
these androgens are, up to an unknown extent, released into the
blood circulation to reach some target tissues, although their
global impact is likely to be minimal. Interestingly, it has been
shown that in the rat prostate, testosterone could induce a rapid
response of the vasculature which precedes growth of the glandular
epithelium. It might well be that cells of the blood vessels are
stimulated by locally made androgens to produce paracrine growth
factors, which could promote the growth of the secretory
epithelium. Further studies are required to elucidate the role of
the steroids synthesized by cells of the blood vessel walls. The
present data clearly indicate new mechanisms of androgen formation,
which may play an important role, not only in normal human prostate
physiology, but also in the pathogenesis of benign prostatic
hyperplasia and possibly prostate cancer.
1TABLE 1 Percentage of immunostained cells of BPH and normal human
prostate tissues. Type 5 Androgen 17.beta.-HSD receptor (%) (%)
Epithelium Basal cells 100 37 Luminal cells 22(*) 94 Fibro-muscular
Fibrocytes 100 66(o) stromal cells Smooth muscle cells 0 Blood
vessels Endothelial cells of 100 75 tunica intima Smooth muscle
cells 35(.cndot.) 82 of tunica media Fibrocytes 100 56 of tunica
adventitia (*)This percentage represents the number of stained
luminal cells only in about 5% of the alveoli, as shown in FIGS. 3c
and 4b. The vast majority of alveoli (about 85%) did not show
stained luminal cells while all luminal cells were stained in about
10% of them (FIG. 3b). (o)The number represents the percentage of
stained nuclei of fibrocytes and smooth muscle cells together.
(.cndot.)The staining intensity is low in the stained smooth muscle
cells of tunica media (as seen in FIG. 4f) as compared with other
stained cells.
[0378]
2TABLE 2 Intensity of the in situ hybridization and immunostaining
reactions in the different cell types of BPH and normal human
prostatic tissue. In situ Immunostaining hybridization Type 5 Type
5 3.beta.- 17.beta.- Androgen 17.beta.-HSD HSD HSD receptor
Epithelium Basal cells +++ +++ +++ +/- Luminal cells + +/- +/-
+++/- Fibro- Fibrocytes +++ +++ +++ +++/- muscular Smooth - - -
+++/- stromal cells muscle cells Blood Endothelial +++ +++ +++
+++/- vessels cells of tunica intima Smooth +/- +/- +/- +++/-
muscle cells of tunica media Fibrocytes +/- ++ ++ +++/- of tunica
adventitia The presence of silver grains or positive immunostaining
reaction is indicated by (+), graded from 1 to 3. The number of (+)
thus corresponding to the intensity of the reaction and takes into
account the percentage of labelled cells. The absence of reaction
is indicated by (-). The possibility of being positively or
negatively labeled is indicated by (+/-).
[0379] Human skin hybridized with type 5 17.beta.-HSD cRNA probe.
In a preliminary experiment of in situ hybridization, human skin
has been hybridized with type 5 17.beta.-HSD antisense and sense
riboprobes the results show that the epidermis (except stratum
corneum) (FIGS. 6a,b), the wall of blood vessels (FIGS. 6c,d), hair
follicles as well as most of the fibrocytes of the dermis are
labeled.
[0380] When paraffin sections immunostained with a specific
antibody to type 5 17.beta.-HSD were examined, the obtained results
were in agreement with the in situ hybridization results. In the
epidermis, few cells of the stratum granulosum were found to be
heavily immunostained than all the other stained cells.
[0381] Localization of type 5 17.beta.-HSD in human mammary gland.
Immunostaining of tissue sections from several normal human mammary
gland show that the epithelial cells lining the ducts and the
alveoli are not stained while the connective tissue surrounding
cells are stained. In the only one examined case of mammary gland
tumor, the tumor cells themselves were not labeled but the
epithelial cells lining the ducts were found to be heavily labeled
(FIGS. 7a, b).
[0382] Localization of tope 5 17.beta.-HSD in monkey ovary. In a
preliminary study of in situ hybridization, one of the growing
follicles was examined. Theca cells, granulosa cells and the oocyte
were found to be labeled.
Preferred Inhibitors of Type 5 17.beta.-Hydroxysteroid
Dehydrogenase
[0383] Set forth in the tables below are lists of compounds which
we have found to be useful as inhibitors of type 5
17.beta.-hydroxysteroid dehydrogenase. The tables also include in
many instances further tests of a particular compound on other
important parameters such as androgenic and antiandrogenic activity
and the effect of a compound on androgen receptors,
androgen-sensitive cells, and other effects more fully explained
below. In each of tables 1-5 and 1'-5' below that do not include a
"prime" (') in its table number, details of molecular structure of
preferred inhibitors are set forth. The corresponding table with a
"prime" (') in its table number shows certain information about the
functional efficacy of each tested compound. The numbers in the
column headings correspond to a description at the end of all of
the tables regarding what information is reported in each column
and how it is determined. Entries left blank are not yet
determined.
3TABLE 1 57 Name Example A R.sup.1 =CH.sub.3 R.sup.6 R.sub.a
R.sub.b .DELTA.1 .DELTA.6 EM-996 1 CH.sub.3 NO CH.sub.3
--C.sub.5H.sub.10 Br NO YES EM-1029 1 CH.sub.3 " CH.sub.3
--C.sub.4H.sub.8 Cl " YES EM-950 1 CH.sub.3 " CH.sub.3
cycloC.sub.5H.sub.8 H " YES CS-245 2 CH.sub.3 YES CH.sub.3
--C.sub.3H.sub.6 H " YES EM-1003 1 CH.sub.3 NO CH.sub.3
--C.sub.3H.sub.6 Br " YES EM-1291 1 CH.sub.3 " CH.sub.3 --CH.sub.2
.PHI.OCH.sub.3 (p) YES YES EM-1280 1 CH.sub.3 " CH.sub.3
--C.sub.2H.sub.4 H YES YES CS-251 1 CH.sub.3 " CH.sub.3
--(CH.sub.2).sub.3 COCH.sub.3 NO YES CS-243 2 CH.sub.3 YES CH.sub.3
--C.sub.2H.sub.4 H " YES EM-1195-CS 1 CH.sub.3 NO CH.sub.3
--C.sub.4H.sub.8 F " YES EM-928 1 CH.sub.3 " CH.sub.3 --C
(CH.sub.3).sub.2 H " YES CS-241 1 CH.sub.3 " CH.sub.3
--C.sub.3H.sub.6 Cl " YES EM-1182-CS 1 CH.sub.3 " CH.sub.3
--C.sub.5H.sub.10 F " YES EM-1123-CS 1 CH.sub.3 " CH.sub.3
--C.sub.4H.sub.8 Br " YES EM-1171 1 CH.sub.3 " CH.sub.3
--C.sub.5H.sub.10 Cl " YES EM-949 1 CH.sub.3 " CH.sub.3 --CH.sub.2
.PHI. " YES EM-978 1 CH.sub.3 " CH.sub.3 cycloC.sub.6H.sub.10 H "
YES EM-979 1 CH.sub.3 " CH.sub.3 tBu H " YES EM-1044 1 CH.sub.3 "
CH.sub.3 CH.sub.2 .PHI.Cl(p) " YES CS-209 2 CH.sub.3 YES CH.sub.3
--isoC.sub.3H.sub.6 H " YES CS-256 1 CH.sub.3 NO CH.sub.3
--CH.sub.2 .PHI.F " YES EM-1022 2 CH.sub.3 YES CH.sub.3
--cycloC.sub.5H.sub.8 H " YES EM-1049 1 CH.sub.3 No CH.sub.3
--CH.sub.2 .PHI.OCH.sub.3(p) " YES EM-1107 1 CH.sub.3 No CH.sub.3
--CH.sub.2 .PHI.OCOterC.sub.4H.sub.9(p) " YES EM-952 2 CH.sub.3 YES
CH.sub.3 --CH.sub.2 H " YES Megestrol Acetate nil CH.sub.3 NO
CH.sub.3 --CH.sub.2 H " YES EM-995 2 CH.sub.3 YES CH.sub.3
--C.sub.5H.sub.10 H " YES EM-994 2 CH.sub.3 YES CH.sub.3
--C.sub.4H.sub.8 H " YES CS-220 2 CH.sub.3 YES CH.sub.3 -tBu H "
YES CS-206 2 C.sub.4H.sub.9 NO CH.sub.3 --CH.sub.2 H " YES EM-1023
2 C.sub.2H.sub.5 NO CH.sub.3 --CH.sub.2 H " YES EM-1059 2 CH.sub.3
YES CH.sub.3 CH.sub.2 .PHI. " YES EM-1159 9 N--(CH.sub.3).sub.2 NO
CH.sub.3 --CH.sub.2 H " YES EM-1181-CS 9
--N(CH.sub.3)C.sub.2H.sub.5 NO CH.sub.3 --(CH.sub.2).sub.3 Cl " YES
EM-1079 9 --N(CH.sub.3)C.sub.2H.sub.5 NO CH.sub.3 --CH.sub.2 H "
YES EM-1165 9 --N(CH.sub.3)C.sub.4H.sub.9 NO CH.sub.3 --CH.sub.2 H
" YES EM-1158 9 --N(CH.sub.3).PHI. NO CH.sub.3 --CH.sub.2 H " YES
EM-1230-CS 9 --N(CH.sub.3)C.sub.2H.sub.5 NO CH.sub.3
--C(CH.sub.3).sub.2 H " YES EM-1264 9 --N(CH.sub.3)CH.sub.3 NO
CH.sub.3 --C.sub.2H.sub.4 H " YES EM-1268 9 --N(CH.sub.3)CH.sub.3
NO CH.sub.3 --C.sub.3H.sub.6 H " YES EM-1315 9
--N(CH.sub.3)CH.sub.2.PHI. NO CH.sub.3 --C.sub.3H.sub.6 H " YES
EM-1316 9 --N(CH.sub.3)CH.sub.2.PHI. NO CH.sub.3 --C.sub.2H.sub.4 H
" YES EM-1326-CS9 9 --N(CH.sub.3)C.sub.3H.sub.7 NO CH.sub.3
--C.sub.2H.sub.4 H " YES EM-1327 9 --N(CH.sub.3)C.sub.3H.sub.7 NO
CH.sub.3 --C.sub.2H.sub.4 H " YES EM-1317 9
--N(CH.sub.3)C.sub.3H.sub.5 NO CH.sub.3 --C.sub.2H.sub.4 H " YES
EM-1318 9 --N(CH.sub.3)C.sub.3H.sub.5 NO CH.sub.3 --C.sub.3H.sub.6
H " YES EM-1321-CS 9 --N(CH.sub.3)CH(CH.sub.3).sub.2 NO CH.sub.3
--C.sub.2H.sub.4 H " YES EM-1322-CS 9 --N(CH.sub.3)CH.sub.2CH(CH.s-
ub.3).sub.2 NO CH.sub.3 --C.sub.2H.sub.4 H " YES EM-1323-CS 9
--N(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2 NO CH.sub.3
--C.sub.3H.sub.6 H " YES EM-1271-CS 9 --N(CH.sub.3)CH.sub.3 NO
CH.sub.3 --C(CH.sub.3).sub.2 H " YES EM-1010 11 --OCH.sub.3 NO
CH.sub.3 --CH.sub.2 H " YES EM-1106 10 CH.sub.2.PHI. NO CH.sub.3
--CH.sub.2 H " YES EM-923 1 CH.sub.3 NO CH.sub.3 C.sub.4H.sub.8 H "
YES EM-948 1 CH.sub.3 NO CH.sub.3 --(CH.sub.2).sub.2cyclo
C.sub.5H.sub.8 H " YES EM-1007 4 CH.sub.3 NO CH.sub.3
C.sub.3H.sub.6CO.sub.2CH.sub.2 H " YES EM-1081 10 C.sub.3H.sub.7 NO
CH.sub.3 --CH.sub.2 H " YES EM-917 1 CH.sub.3 NO CH.sub.3
--C.sub.5H.sub.10 H NO YES EM-918 1 CH.sub.3 NO CH.sub.3
--C.sub.2H.sub.4 H NO YES EM-920 1 CH.sub.3 NO CH.sub.3
C.sub.3H.sub.6 H NO YES EM-946 1 CH.sub.3 NO CH.sub.3
C.sub.2H.sub.4 .phi. NO YES EM-1018 2 CH.sub.3 YES CH.sub.3
C.sub.4H.sub.10 H NO YES EM-1048 1 CH.sub.3 NO CH.sub.3
C.sub.7H.sub.14 Br NO YES EM-1075 Not CH.sub.3 NO CH.sub.3
C.sub.3H.sub.6 CO.phi. NO YES reported EM-1081 10 C.sub.3H.sub.7 NO
YES CH.sub.2 H NO YES EM-1103 Not CH.sub.3 NO NO CH.sub.2 H NO YES
reported EM-1127 1 CH.sub.3 NO YES C.sub.5H.sub.10 H NO YES EM-1188
2 CH.sub.3 YES YES CH.sub.2 .phi.F(o) NO YES EM-1175 10
--C(CH.sub.3)C.sub.2H.sub.5 NO YES CH.sub.2 H NO YES EM-1141 10
C(CH.sub.3).sub.3 NO YES CH.sub.2 H NO YES EM-1209 10
C.sub.4H.sub.9 NO YES CH.sub.2 H NO YES EM-1204 2 CH.sub.3 YES YES
CH.sub.2 .phi.F(p) NO YES EM-1213 2 CH.sub.3 YES YES CH.sub.2
.phi.OCH.sub.3(p) NO YES EM-1216 2 CH.sub.3 YES YES CH.sub.2
.phi.Br(p) NO YES EM-1217 2 CH.sub.3 YES YES CH.sub.2 .phi.Cl(p) NO
YES EM-1224 2 CH.sub.3 YES YES CH.sub.2 .phi.OC.sub.2H.sub.5(p) NO
YES EM-1231 1 CH.sub.3 NO YES -CH(CH.sub.3)-- .phi. NO YES EM-1240
2 CH.sub.3 YES YES CH.sub.2 .phi.CH.sub.3(o) NO YES EM-1241 2
CH.sub.3 YES YES CH.sub.2 .phi.CH.sub.3(p) NO YES EM-1242 2
CH.sub.3 YES YES CH.sub.2 .phi.CH.sub.3(m) NO YES EM-1243 2
CH.sub.3 YES YES CH.sub.2 .phi.CF.sub.3(p) NO YES EM-1246 2
CH.sub.3 NO YES CH.sub.2 .phi.CH.sub.3(p) NO YES EM-1253 1 CH.sub.3
NO YES --C(C.sub.2H.sub.2)-- .phi. NO YES EM-1263-CS 2 CH.sub.3 YES
YES CH.sub.2 .phi.NO.sub.2(p) NO YES EM-1219-CS 2 CH.sub.3 YES YES
--C(C.sub.2H.sub.2)-- .phi. NO YES EM-1282-CS 1 CH.sub.3 NO YES
--C(CH.sub.3).sub.2 .phi. NO YES EM-1290 2 CH.sub.3 YES YES
CH.sub.2 .phi.-di-(OCH.sub.3)(m) NO YES EM-1292-CS 2 CH.sub.3 YES
YES CH.sub.2 .phi.OH(p) NO YES EM-1297 1 CH.sub.3 NO YES CH.sub.2
.phi.OH(p) NO YES EM-1312 1 CH.sub.3 NO YES CH.sub.2
.phi.CH.sub.3(o) NO YES EM-1339-CS 1 CH.sub.3 NO YES
--CH(CH.sub.3)-- .phi.OCH.sub.3(p) NO YES EM-1340 1 CH.sub.3 NO YES
CH.sub.2 .phi.F(o) NO YES EM-1343 1 CH.sub.3 NO YES CH.sub.2
.phi.OCH.sub.3(o) NO YES EM-910 1 CH.sub.3 NO YES CH.sub.2 H YES
YES EM-991 1 CH.sub.3 YES YES CH.sub.2 H YES YES EM-1294 1 CH.sub.3
NO YES CH.sub.2 .phi. YES YES EM-1308 2 CH.sub.3 NO YES
--C(CH.sub.3).sub.2-- H YES YES EM-1309 1 CH.sub.3 NO YES
C.sub.3H.sub.6 H YES YES EM-1319-CS 1 CH.sub.3 NO YES
--C(CH.sub.3).sub.2CH.sub.2-- H YES YES
[0384]
4TABLE 1' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Inhibi- Inhibi-
tion tion Type 1 Inhibi- Inhibi- Inhibi- Inhibi- Type V 17B- tion
tion tion tion Oral 17B- HSD Type 2 Type 3 Type 1 Type 3 Androgenic
Bio- HSD Activity 17B-HSD 17B-HSD 3a-HSD 3a-HSD and availa-
IC.sub.50 IC.sub.50 Activity Activity Activity Activity Anti- %
Inhibition bility (nM) (nM) IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50
androgenic % Inhibtion % Inhibition Gluco- % Inhibition AUC (% Inh.
Reversi- (% Inh. (nM) (nM) (nM) (nM) Shionogi Androgen Progesterone
corticoid Estrogen 0-7 h At bility at (% Inh. (% Inh. (% Inh. (%
Inh. DHT ZR-75-1 Activity Receptor Receptor Receptor Receptor (ng/
(3.10.sup.-7 (%) of 3.10.sup.-4 at at at at Basal E.sup.-7 E.sup.-6
Basal E.sub.2 *(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5) *(E.sup.-7
E.sup.-5) *(E.sup.-7 E.sup.-5) Name mL .multidot. h) 3.10.sup.-6)
control) M) 3.10.sup.-4 M) 3.10.sup.-4 M) 3.10.sup.-4 M)
3.10.sup.-4 M) E.sup.-7 E.sup.-6 [IC.sub.50 (nM)] E.sup.-8 E.sup.-6
3E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8
E.sup.-6 E.sup.-8 E.sup.-6 EM-996 ND 1.8 .+-. 0.1 -11 -32 -78 -126
0 -8 -35 -53 0 1 9 88 0 67 0 0 [158 .+-. 59] EM-1029 10 .+-. 2.5 3
[13] -24 -15 -123 -146 0 -10 -62 -64 1 1 5 80 8 98 0 0 [9.3] EM-950
ND 2.7 .+-. 0.39 -23 -25 -79 -110 -17 0 -39 -46 13 4 2 58 18 95 0 0
[44 .+-. 5] CS-245 18 .+-. 5 3.2 .+-. 0.1 -28 -34 -106 -109 ND ND 5
71 0 26 0 43 0 2 [12] EM-1003 16 .+-. 5 3.5 .+-. 0.3 -17 -15 -83
-101 -9 -13 -67 -73 0 67 9 85 2 79 0 0 [35 .+-. 6] EM-1291 94 .+-.
23 7 .+-. 1 27 [67] -8 -44 -13 -48 ND ND 0 0 0 33 1 69 0 0 EM-1280
18 .+-. 6.6 3 .+-. 0.5 31 [59] 0 0 -94 -111 ND ND 0 44 0 39 9 93 0
0 [23] CS-251 ND 11 .+-. 1 5 -11 -22 -109 -23 0 -60 -47 0 3 0 12 0
57 1 1 CS-243 11 .+-. 2.5 3.5 .+-. 0.1 -13 -23 -102 -129 0 0 -60
-57 0 33 0 27 0 29 1 0 [11] EM-1195 31 .+-. 5.5 4 .+-. 0.5 [40 ]
-27 -34 -105 -110 -11 18 -38 -40 1 62 0 70 3 90 0 0 CS EM-928 ND
3.7 .+-. 0.3 [27] 0 -13 -60 -107 0 -11 -50 -84 4* 72* 11* 90* 21*
99* 0* 0* [67 .+-. 6] CS-241 ND 4.1 .+-. 0.6 5 23 -91 -93 ND ND 10
91 0 61 9 96 1 3 [10] EM-1182 ND 4.4 .+-. 0.4 [50] -14 -16 -94 -109
ND ND 0 51 18 78 5 90 0 0 [18 5] EM-1173- 4.1 .+-. 1.4 4.6 .+-. 1.1
[28] -17 -54 -70 -121 197 196 0 0 0 28 6 76 5 89 0 0 CS [33]
EM-1171 4 .+-. 1.3 5.3 .+-. 0.3 [14] -6 -30 -82 -111 98 182 0 -29 1
36 5 76 8 90 0 0 [61] EM-949 ND 5.5 .+-. 1.1 [64] -19 -13 -45 -102
-8 0 -23 -17 2* 41* 37* 97* 11* 94* 0* 0* [193 .+-. 41] EM-978 ND
6.8 .+-. 0.4 [39] -31 -48 -79 -132 -13 -13 -35 -48 7 17 0 48 14 93
0 0 [93 .+-. 10] EM-979 ND 7.2 .+-. 0.7 [20] -15 -41 -39 -117 -12 0
-26 -40 6 7 1 41 7 88 0 0 EM-1044 53 .+-. 23 5.1 4 -27 -21 -87 0 19
0 -24 0 0 0 74 0 81 0 0 CS-209 ND 10.5 .+-. 2.7 [62] 13 -30 -67
-122 0 0 -44 -48 2 8 2 38 0 29 0 0 [82] CS-256 33 .+-. 7 5 .+-. 1
-10 -24 -17 -89 ND ND 4 5 2 75 2 51 8 0 EM-1022 ND 5.4 .+-. 0.6
[62] -12 -35 -68 -155 0 20 -30 -40 0 9 0 25 0 44 0 0 [128] EM-1049
83 .+-. 16 9 .+-. 1 10 -33 -12 66 0 0 -52 -71 0 1 0 63 0 62 0 0
EM-1107 ND 17 .+-. 3 [64] -9 -6 3 -34 ND ND 4 3 0 48 1 64 0 0
EM-952 ND 17.4 .+-. 5.1 -19 -4 -88 -104 0 0 -17 -13 0 20 4 50 1 14
0 3 [41 .+-. 4] Megestrol 92 .+-. 17 17.4 .+-. 2.8 ND [10 .+-. 1.8]
ND -70 -81 9* 92* 36* 97* 17* 98* 01* 08* Acetate 5 39 3 77 0 82 0
0 EM-995 ND 21.3 .+-. 6.7 -4 -21 -51 -121 -29 -9 -12 -19 0 10 5 72
0 27 0 0 EM-994 ND 31 .+-. 5.1 [42] -10 -27 -82 -122 0 -8 -10 -35 0
19 6 64 0 33 0 3 [75 .+-. 10] CS-220 ND 31 .+-. 3.3 [32] 0 -31 15
-106 0 11 -25 -11 0 0 0 20 0 23 2 0 [674] CS-206 ND 40 .+-. 8.5
[80] -18 -40 -75 -118 0 0 -12 -40 5 15 0 29 0 45 1 0 EM-1023 ND 50
.+-. 7 [20] -11 -14 -108 -133 14 0 61 66 0 43 0 44 0 85 0 0 [40]
EM-1059 ND (70 92) -7 -12 -34 -81 ND ND 0 3 0 41 0 23 1 0 EM-923 10
.+-. 2 (94 96) [31] 10 5 -73 -94 0 -12 60 -105 0 3 0 41 0 23 1 0
EM-948 ND (91 93) [49] -26 -19 -49 -105 0 0 -29 -36 ND ND ND ND
EM-1007 16.4 (81 86) 0 -31 -14 -76 -8 0 -41 -52 0 0 0 0 0 0 0 0
EM-1159 452 .+-. 170 14 .+-. 2 15 -14 1 -71 ND ND 1 1 2 4 8 59 0 1
EM-1181- 130 .+-. 35 5 .+-. 1 [45] -6 -12 -89 -104 ND ND 0 27 ND 2
70 7 0 CS [15.1] EM-1079 252 .+-. 61 17 .+-. 1 [28] -15 -23 -7 -71
ND ND 0 0 0 0 3 45 0 0 EM-1165 24 .+-. 3.6 2.5 .+-. 5 5 -17 -14
-102 ND ND 0 1 0 7 0 26 0 0 [232] EM-1158 ND 25 .+-. 5 0 0 -16 -82
ND ND 1 2 0 6 0 53 EM-1230- 82 .+-. 15 8 .+-. 1 [65] -9 -30 0 -66
ND ND 1 0 0 0 1 45 3 0 CS EM-1264 257 .+-. 81 6 .+-. 1 [51] -25 -56
-25 -108 ND ND 0 0 0 5 7 80 3 0 [535] EM-1268 58 .+-. 10 5.8 [80]
-25 -45 -86 -113 ND ND 0 12 1 7 14 90 1 6 [101] EM-1315 ND 6.8 [88]
-32 -62 -66 -121 ND ND 1 13 0 14 18 66 0 0 EM-1316 23 .+-. 5 11.6
.+-. 3 -23 -64 -32 -118 ND ND 0 1 0 13 3 84 0 0 EM-1326- ND 6 .+-.
1 [87] -28 -56 -47 -116 ND ND 0 0 0 4 3 47 0 0 CS EM-1327 ND 6 .+-.
1 [75] -41 -59 -101 -119 ND ND 0 19 0 7 12 40 0 0 EM-1317 ND 8 .+-.
1 [81] -20 -56 -32 -113 ND ND 0 0 0 0 1 64 0 0 EM-1318 ND 7 .+-. 1
[80] -30 -53 -85 -121 ND ND 0 15 0 11 5 68 0 0 EM-1321- ND 7 .+-. 2
[84] 0 0 -25 -99 ND ND 0 1 0 5 15 29 0 0 CS EM-1322- ND 4 .+-. 1
[83] -38 -40 -53 -112 ND ND 0 2 0 3 0 45 0 0 CS EM-1323- ND 5 .+-.
1 [79] -33 -44 -92 -116 ND ND 1 16 0 11 9 30 0 1 CS EM-1271- 181
.+-. 28 4.1 [73] -25 -42 0 -68 ND ND 0 0 0 0 9 82 0 0 CS EM-1010 ND
31 .+-. 3.4 -26 -25 -83 -109 0 0 -50 -60 0 28 0 47 0 45 0 0 [58
.+-. 9] EM-1106 29 .+-. 1.6 (83 88) [62] -19 -33 -65 -108 ND ND 1
23 1 24 1 50 1 0 [55] EM-917 -4 -19 -69 -109 EM-918 43 11 -50 -97
EM-920 88 78 -26 -50 EM-946 -25 -7 -39 -82 EM-1018 (58 82) -9 -36
-34 -142 EM-1048 (51 84) -4 -30 -18 -97 EM-1075 (40 90) 10 -26 -10
-82 EM-1081 (61 82) -13 -23 -94 -108 EM-1103 (59 86) -5 -40 -104
EM-1127 (71 91) 85 92 EM-1188 (58 88) -18 -32 -24 -61 EM-1175 (32
86) -30 -47 -35 -106 EM-1141 (25 79) 0 -5 -8 -75 EM-1209 (95 94)
-35 -40 -53 -105 EM-1204 (33 88) 3 -24 -22 -60 EM-1213 (35 83) -9
-32 -22 -51 EM-1216 (33 77) -14 -36 -22 -63 EM-1217 (36 81) -5 -36
-21 -59 EM-1224 (2 22) -6 -28 -18 -55 EM-1231 (91 96) -28 -36 -58
-112 EM-1240 (47 89) -10 -33 -8 -56 EM-1241 (4 53) -6 -41 -3 -56
EM-1242 (19 78) -6 -414 -12 -63 EM-1243 (2 16) -14 -52 -28 -89
EM-1246 (74 95) -17 -34 -25 -97 EM-1253 (39 85) -25 -63 -41 -122
EM- (7 22) -13 -60 -32 -115 1263-CS EM- (10 43) -32 -52 -52 --
1279-CS EM- (91 94) -32 -59 -39 -114 1282-CS EM-1290 (9 3) -25 -10
-48 -62 EM- (45 87) -14 -31 -19 -53 1292-CS EM-1297 (93 95) -27 -49
-17 -65 EM-1312 (93 96) -7 -37 -28 -100 EM- (69 85) 0 -44 -22 -97
1339-CS EM-1340 (81 82) -5 -21 -25 -102 EM-1343 (70 87) 7 -20 -1
-68 EM-910 EM-991 (44 87) EM-1294 (93 94) EM-1308 EM-1309 (95 96)
79 72 -63 -84 EM- (94 95) 1319-CS
[0385]
5TABLE 2 58 Name Example .DELTA..sup.1 R.sup.6 R.sup.15.alpha.
R.sup.16.alpha. R.sup.16.crclbar. R.sup.19 EM-1183 8 NO H H
C.sub.3H.sub.7 F CH.sub.3 EM-1134 8 NO H H
CH.sub.2-isoC.sub.3H.sub.7 CH.sub.3 CH.sub.3 EM-1211 5 NO H H H
CH.sub.2-isoC.sub.3H.sub.7 CH.sub.3 EM-1061 6 NO H H 16-spiro
C.sub.5H.sub.8 H EM-1273-CS 5 NO H H CH.sub.2-isoC.sub.3H.sub.8 H
CH.sub.3 EM-1097 5 NO H H C.sub.3H.sub.7 H CH.sub.3 EM-1082 6 NO H
H 16-spiro C.sub.6H.sub.10 H CS-195 6 NO H H 16-spiro
C.sub.5H.sub.8 CH.sub.3 EM-1042 8 NO H H C.sub.3H.sub.7 CH.sub.3
CH.sub.3 EM-1077 6 NO H H 16-spiro C.sub.5H.sub.8 CH.sub.3
EM-1151-CS 8 NO H H 59 60 CH.sub.3 EM-922 6 NO H H
16-spiroC.sub.6H.sub.10 CH.sub.3 CS-204 6 YES H H
16-spiroC.sub.6H.sub.10 CH.sub.3 EM-1261 12 NO H
CH.sub.2CH.dbd.CH.sub.2 H H CH.sub.3 EM-1227 13 NO H H
.dbd.CH--C.sub.2H.sub.5 CH.sub.3 EM-1299 14 NO H H spiro bicyclo
[3.1.0] hexane CH.sub.3 SA-208-59 15 NO H --C.sub.3H.sub.6-- H
CH.sub.3 CS-239 Not NO H H CH.sub.3 C.sub.3H.sub.7 CH.sub.3
reported EM-1039 Not YES H H 16-spiro C.sub.5H.sub.8 CH.sub.3
reported EM-1057 6 NO CH3 H 16-spiro C.sub.5H.sub.8 CH.sub.3
EM-1135 8 NO H H C.sub.3H.sub.6Cl CH.sub.3 CH.sub.3 EM-1155-CS 8 NO
H H C.sub.3H.sub.6Br CH.sub.3 CH.sub.3 EM-1192 8 NO H H
C.sub.5H.sub.11 CH.sub.3 CH.sub.3 EM-1245 12 NO H C.sub.3H.sub.7 H
H CH.sub.3 EM-1287 8 NO H H C.sub.4H.sub.9 F CH.sub.3 EM-1260 12 NO
H C.sub.3H.sub.7 H H CH.sub.3 EM-1262 8 NO H H C.sub.3H.sub.5 F
CH.sub.3 EM-1272-CS Not NO H H 16-spiro C.sub.5H.sub.8
(CH.sub.3).sub.2 CH.sub.3 reported EM-1313 8 NO H H C.sub.4H.sub.7
H CH.sub.3 EM-1314 8 NO H H C.sub.3H.sub.5 H CH.sub.3 EM-1349-CS 8
NO H H C.sub.2H.sub.3F.sub.2 CH.sub.3 CH.sub.3 EM-1353 13 NO H H
.dbd.CHCH(CH.sub.3).sub.2 CH.sub.3 EM-1359 8 NO H H C.sub.3H.sub.5
Cl CH.sub.3 EM-1361 12 NO H C.sub.4H.sub.7 H H CH.sub.3
[0386]
6TABLE 2' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Inhibi- Inhibi-
tion tion Type 1 Inhibi- Inhibi- Inhibi- Inhibi- Type V 17B- tion
tion tion tion Oral 17B- HSD Type 2 Type 3 Type 1 Type 3 Androgenic
Bio- HSD Activity 17B-HSD 17B-HSD 3a-HSD 3a-HSD and availa-
IC.sub.50 IC.sub.50 Activity Activity Activity Activity Anti- %
Inhibition bility (nM) (nM) IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50
androgenic % Inhibtion % Inhibition Gluco- % Inhibition AUC (% Inh.
Reversi- (% Inh. (nM) (nM) (nM) (nM) Shionogi Androgen Progesterone
corticoid Estrogen 0-7 h At bility at (% Inh. (% Inh. (% Inh. (%
Inh. DHT ZR-75-1 Activity Receptor Receptor Receptor Receptor (ng/
(3.10.sup.-7 (%) of 3.10.sup.-4 at at at at Basal E.sup.-7 E.sup.-6
Basal E.sub.2 *(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5) *(E.sup.-7
E.sup.-5) *(E.sup.-7 E.sup.-5) Name mL .multidot. h) 3.10.sup.-6)
control) M) 3.10.sup.-4 M) 3.10.sup.-4 M) 3.10.sup.-4 M)
3.10.sup.-4 M) E.sup.-7 E.sup.-6 [IC.sub.50 (nM)] E.sup.-8 E.sup.-6
3E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8
E.sup.-6 E.sup.-8 E.sup.-6 EM-1183 ND 4.6 .+-. 4 [34] 0 -17 0 -51
ND ND 0 1 9 45 0 12 0 0 EM-1134 ND 20 .+-. 4 0 -13 -36 -85 ND ND 1
7 0 13 0 2 0 1 [471] EM-1211 ND (87 95) [46] 0 -20 -7 -18 ND ND 1 3
0 2 1 0 0 0 EM-1061 ND (85 95) 14 -6 0 -10 -10 0 0 -20 0 0 0 0 0 0
0 1 EM-1273 85 .+-. 26 9 40 [31] 12 -36 9 -43 0 0 0 -30 0 0 0 0 0 1
0 0 -CS EM-1097 122 .+-. 5.7 10 45 [64] [53] [82] 18 -12 -3 -71 -8
10 0 0 0 5 2 0 0 0 0 1 EM-1082 ND 36 .+-. 11 [32] 4 -20 0 0 ND ND 0
0 1 2 0 0 0 0 CS-195 ND 19.4 .+-. 2 [09] 0 -18 -4 -31 -4 0 0 -15 0
0 0 2 0 2 0 0 EM-1042 ND 35 .+-. 5 0 -22 -14 -85 0 10 -12 -12 -11 0
0 0 0 0 0 EM-1077 ND 35 .+-. 4 [32] 0 -24 0 -34 ND ND 0 1 0 0 4 3 0
3 EM-1151 ND (76 95) 0 -25 -13 -48 ND ND 0 3 0 14 0 1 8 9 EM-992 ND
66 .+-. 9 [27] 22 48 0 24 0 0 0 0 ND ND ND ND CS-204 ND 94 [23] 0
-17 -25 -13 0 0 0 -25 0 5 0 2 0 0 0 0 EM-1261 ND (92 96) [61] 0 -31
-11 -123 ND ND 0 0 0 0 0 1 0 0 EM-1277 ND (82 97) [49] 9 -16 11 -22
ND ND 0 0 0 0 2 1 0 0 EM-1299 ND (73 99) [49] 5 10 0 -27 ND ND 1 3
0 0 0 0 3 3 SA- ND (72 88) ND ND ND ND ND ND ND ND 208-59 EM-925
(46 81) [13] 11 -4 15 -7 EM-1057 (57 90) 16 -2 -20 -64 EM-1135 (67
95) 7 -17 -29 -90 EM- (49 90) 0 -12 -7 -93 1155-CS EM-1192 (73 91)
-15 -17 -48 -98 CS-239 (20 62) 20 0 11 -19 EM-1257 (96 97) 0 -37 -8
-49 EM-1260 (8 57) -3 -27 -10 -43 EM-1262 (93 97) -1 -29 4 -47 EM-
(66 93) -7 -26 -6 -13 1272-CS EM-1313 -7 -40 2 -51 EM-1314 -2 -41 6
-53 EM- (61 93) -6 -35 -21 -71 1349-CS EM-1353 (66 95) 3 -5 -17 -22
EM-1359 (86 93) 2 -19 7 -52 EM-1361 (69 92) 0 -22 -22 -39 EM-1039
(64 82) [3] 8 -5 25 8
[0387]
7 61 Name Example .DELTA. R.sup.1 R.sup.6 R.sup.16a Y EM-1201-CS 16
.sup.6.DELTA. H CH.sub.3 H OCOC.sub.4H.sub.8Cl EM-1202 16
.sup.6.DELTA. H CH.sub.3 H OCOC.sub.5H.sub.10F EM-1196-CS 16
.sup.6.DELTA. H CH.sub.3 H OCOC.sub.4H.sub.8F EM-1078 16
.sup.6.DELTA. H CH.sub.3 H OCO(CH.sub.2).sub.2CH.sub.3 EM-1172-CS
16 .sup.6.DELTA. H CH.sub.3 H OCOC.sub.5H.sub.10Cl CS-237 16
.sup.1.DELTA.,.sup.6.DELTA. H CH.sub.3 H OAc EM-1091 16
.sup.6.DELTA. H CH.sub.3 H OCOC.sub.2H.sub.5 CS-259 16
.sup.6.DELTA. H CH.sub.3 H OCO(CH.sub.2).sub.5Br CS-260 16
.sup.6.DELTA. H CH.sub.3 H OCO(CH.sub.2).sub.4CH.sub.3 EM-1205-CS
16 .sup.6.DELTA. H CH.sub.3 Br H EM-1154-CS 16 .sup.6.DELTA.
.alpha.-CH.sub.3 CH.sub.3 H OCOcycloC.sub.5H.sub.9 EM-1143-CS 16
.sup.6.DELTA. .alpha.-CH.sub.3 CH.sub.3 H OCOCH(CH.sub.3).sub.2
EM-1098-CS 16 .sup.6.DELTA. H CH.sub.3 H OCOCH.sub.2.PHI.
EM-1108-CS 16 .sup.6.DELTA. H CH.sub.3 H
OCO(CH.sub.2).sub.3CH.sub.3 EM-1146 16 .sup.6.DELTA. H CH.sub.3 H
OCOCH.sub.2.PHI.OCOC(CH.sub.3).sub.3 CS-240 16 .sup.6.DELTA.
.alpha.-CH.sub.3 CH.sub.3 H OAc EM-1142-CS 16 .sup.6.DELTA.
.alpha.-CH.sub.3 CH.sub.3 H OCOC.sub.5H.sub.11 CS-1Q98 16
.sup.6.DELTA. H CH.sub.3 H OAc EM-1117-CS Not .sup.6.DELTA.
.alpha.-CH.sub.3 CH.sub.3 H OCOC.sub.6H.sub.11 reported EM-1121-CS
Not .sup.6.DELTA. .alpha.-CH.sub.3 CH.sub.3 H OCOC.sub.4H.sub.9
reported EM-1418-CS Not .sup.6.DELTA. H CH.sub.3 H
OCOCH.sub.2.phi.OMe(o) reported EM-1293 Not .sup.1,6.DELTA. H
CH.sub.3 H OCO-iso-C.sub.3H.sub.7 reported EM-1144-CS Not
.sup.6.DELTA. .alpha.-CH.sub.3 CH.sub.3 H OCO-CH.sub.2.phi.
reported EM-1295-CS Not .sup.1,6.DELTA. H CH.sub.3 H
OCOC.sub.2H.sub.5 reported EM-1296 Not .sup.1,6.DELTA. H CH.sub.3 H
OCOC.sub.3H.sub.7 reported
[0388]
8TABLE 3' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Inhibi- Inhibi-
tion tion Type 1 Inhibi- Inhibi- Inhibi- Inhibi- Type V 17B- tion
tion tion tion Oral 17B- HSD Type 2 Type 3 Type 1 Type 3 Androgenic
Bio- HSD Activity 17B-HSD 17B-HSD 3a-HSD 3a-HSD and % Inhibition
availa- IC.sub.50 IC.sub.50 Activity Activity Activity Activity
Anti- % Inhibtion % Inhibition Gluco- % Inhibition bility (nM) (nM)
IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 androgenic Androgen
Progesterone corticoid Estrogen AUC (% Inh. Reversi- (% Inh. (nM)
(nM) (nM) (nM) Shionogi 0-7 h At bility at (% Inh. (% Inh. (% Inh.
(% Inh. DHT ZR-75-1 Activity Receptor Receptor Receptor Receptor
(ng/ (3.10.sup.-7 (%) of 3.10.sup.-4 at at at at Basal E.sup.-7
E.sup.-6 Basal E.sub.2 *(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5)
*(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5) Name mL .multidot. h)
3.10.sup.-6) control) M) 3.10.sup.-4 M) 3.10.sup.-4 M) 3.10.sup.-4
M) 3.10.sup.-4 M) E.sup.-7 E.sup.-6 [IC.sub.50 (nM)] E.sup.-8
E.sup.-6 3E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6
E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 EM-1201- ND 26 [24] -29 -36 -99
-110 ND ND 7 45 8 73 2 90 0 0 CS EM-1202 ND 34 [21] -28 -37 -88
-109 ND ND 4 30 7 64 10 86 0 0 EM-1196- ND 6.5 .+-. 0.5 32 [48] -30
-32 -98 -109 ND ND 0 37 1 44 7 79 0 0 CS EM-1078 ND 3.8 .+-. 0.6
[15] 5 45 -79 -72 ND ND 7 83 0 53 20 94 5 0 EM-1172- ND 6.4 .+-.
1.1 [13] -12 -29 -84 -114 ND ND 0 22 5 73 10 87 0 0 CS CS-237 ND 9
.+-. 1 -22 -29 -51 -106 ND ND 0 11 0 0 7 69 0 0 [95] EM-1091 ND 11
.+-. 1 [38] -25 -21 -88 -105 ND ND 1 34 2 47 13 78 2 0 [15] CS-259
ND 4 .+-. 0.4 [63] -25 -25 -18 -108 ND ND 2 12 3 68 0 17 2 0 [40]
CS-260 ND 3 .+-. 0.3 [66] -29 -31 -80 -109 ND ND 4 21 7 74 2 72 1 0
[30] EM-1205- ND 29.5 [45] -15 -38 -16 -107 ND ND 6 15 1 9 1 19 0 0
CS EM-1154- ND 24.2 0 -22 -23 -107 ND ND 1 5 2 35 0 30 10 13 CS
[184] EM-1143- ND 38.6 -13 -17 -19 -95 ND ND 0 2 0 24 0 9 0 0 CS
[300] EM-1098- ND 14.6 [56] 0 -17 -13 -100 0 0 -34 -4 1 3 0 66 0 24
0 0 CS EM-1108- ND 8.9 [70] -28 -14 -92 -104 ND ND 4 42 0 55 4 73 0
0 CS [19] EM-1146 ND 29.7 13 -11 9 -19 ND ND 0 0 1 24 4 30 0 0
CS-240 ND 28.5 .+-. 2.7 -12 -32 -41 -106 ND ND 1 9 0 9 0 13 0 1
[151] EM-1142- ND (78 95) -15 -32 -10 -102 ND ND 0 6 10 74 0 19 3 3
CS CS-198 ND 38.5 .+-. [13] -27 -33 -84 -128 -10 -12 -23 -4 4 23 0
44 1 73 0 0 10.7 [82 .+-. 23] EM-1117- ND (72 95) [75] -39 -3 -93
CS EM-1121- ND (82 94) [83] -8 -23 -53 -109 CS EM-1144- (36 89) 0
21 -13 -55 CS EM-1418- ND [69] -7 -34 -18 -71 CS EM-1293 ND (94 94)
[67] -37 -53 -38 -111 EM-1295- (93 95) [46] -48 -50 -81 -112 CS
EM-1296 (94 95) [27] -41 -39 -102 -111
[0389]
9TABLE 4 62 Name Example R.sup.2 R.sup.3 R.sup.4 R.sup.6 R.sup.9 X
Y Z EM-1404 28 H --CONH.sub.2 H H H CH.sub.3 CH.sub.3 H EM-1403 28
H --CON(CH.sub.3).sub.2 H H H CH.sub.3 CH.sub.3 H EM-1401 28 H
--COOH H H H CH.sub.3 CH.sub.3 H EM-1394 31 H --OCH.sub.3 H .dbd.O
H CH.sub.3 CH.sub.3 H EM-1424-CS 29 CONH.sub.2
--O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1413-CS
29 CON(CH.sub.3).sub.2 --O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3
CH.sub.3 H EM-1402-CS 29 COOCH.sub.3 --O(CH.sub.2).sub.2OCH.sub.3 H
H H CH.sub.3 CH.sub.3 H EM-1396 30 CN --O(CH.sub.2).sub.2OCH.sub-
.3 H H H CH.sub.3 CH.sub.3 H EM-1393-CS 24 F
--O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1131 19
NO.sub.2 --OH H H H H CH.sub.3 H EM-1125-CS 19 NO.sub.2 --OH H H H
CH.sub.3 CH.sub.3 H EM-1408-CS 31 H --O(CH.sub.2).sub.2OCH.sub.3 H
.dbd.O H CH.sub.3 CH.sub.3 H EM-1407-CS 32 --CH.dbd.N--O-- H H H
CH.sub.3 CH.sub.3 H EM-1126 19 NO.sub.2 --OH H H H CH.sub.3 H H
EM-1118 19 NO.sub.2 --O(CH.sub.2).sub.2OCH.sub.3 H H H H H H
EM-1124 19 NO.sub.2 --OH H H H H H H CS-224 18 H --H H H H CH.sub.3
CH.sub.3 H EM-1157-CS 26 H --F H H H CH.sub.3 CH.sub.3 H EM-1365-CS
27 H --OSO.sub.2CH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1364-CS 27 H
--OSO.sub.2C.sub.2H.sub.5 H H H CH.sub.3 CH.sub.3 H EM-1392-CS 23
Cl --OSO.sub.2CH.sub.3 Cl H H CH.sub.3 CH.sub.3 H EM-1391-CS 23 Cl
--OH Cl H H CH.sub.3 CH.sub.3 H EM-1371-CS 22 Cl
--O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1368-CS
21 H --O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1405
29 COOH --O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H
EM-1386 32 H --OCH.sub.3 H .dbd.O OH CH.sub.3 CH.sub.3 H EM-1388 28
H --COOCH H H H CH.sub.3 CH.sub.3 H EM-1370 21 H --OCH.sub.3 H H H
CH.sub.3 CH.sub.3 H EM-1369 21 H --OC.sub.2H.sub.5 H H H CH.sub.3
CH.sub.3 H EM-1389-CS 21 H --O(CH.sub.2).sub.2N(CH.sub.3).sub.2 H H
H CH.sub.3 CH.sub.3 H EM-1412-CS 33 CH.sub.3 --OCH.sub.3 H H H
CH.sub.3 CH.sub.3 H EM-1390-CS 21 H
--O(CH.sub.2).sub.2cycloN--C.sub.5H.sub.10 H H H CH.sub.3 CH.sub.3
H EM-1385-CS 23 Br --OCH.sub.3 Br H H CH.sub.3 CH.sub.3 H
EM-1366-CS 27 H --OSO.sub.2C.sub.6H.sub.5 H H H CH.sub.3 CH.sub.3 H
EM-1025 28 H --H H H H CH.sub.3 H H EM-1170-CS 26 H --F H H H H H H
CS-242 25 H CH.sub.3SC.sub.2H.sub.4O-- H H H H H H EM-919 Not H
HO-- H H H H H H reported EM-916 18 H --H H H H H H H PB-132-140 35
H HO-- H H H H nil nil PB-132-146 35 H HO-- H H H H COOCH.sub.3 H
PB-132-152 35 H HO-- H H H H CH.sub.2CHCH.sub.2 H PB-132-142 35 H
HO-- H H H H H CH.sub.3 EM-1438 33 CF.sub.3
--O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H EM-1382-CS
23 Br HO-- Br H H CH.sub.3 CH.sub.3 H EM-1372 Not H
--O(CH.sub.2).sub.3CH.sub.3 H H H CH.sub.3 CH.sub.3 H reported
EM-1373 Not H --O(CH.sub.2).sub.2OC.sub.2H.sub.5 H H H CH.sub.3
CH.sub.3 H reported EM-1398 Not H .phi.CH.sub.2OCO-- H H H CH.sub.3
CH.sub.3 H reported EM-1409 Not CF.sub.3 --OCH.sub.3 H H H CH.sub.3
CH.sub.3 H reported EM-1406-CS Not --CON(CH.sub.3).sub.2
--O(CH.sub.2).sub.2OCH.sub.3 H .dbd.O H CH.sub.3 CH.sub.3 H
reported EM-1413-CS Not --CON(CH.sub.3).sub.2
--O(CH.sub.2).sub.2OCH.sub.3 H H H CH.sub.3 CH.sub.3 H reported
EM-1416 Not H --OH H H H C.sub.2H.sub.5 C.sub.2H.sub.5 H reported
EM-1419 Not H --OH H H H C.sub.2H.sub.5 C.sub.2H.sub.5 H reported
EM-01607-C Not H --OH H H H CH.sub.3 CH.sub.3 H reported EM-1608-D
Not H --OH H .dbd.O H CH.sub.3 CH.sub.3 H reported EM-01645 Not
--CN --OH H H H CH.sub.3 CH.sub.3 H reported EM-01646 Not H --CN H
H H CH.sub.3 CH.sub.3 H reported EM-01660 Not --CONH.sub.2 --OH H H
H CH.sub.3 CH.sub.3 H reported EM-01661-B Not --CON(CH.sub.3).sub.2
--OH H H H CH.sub.3 CH.sub.3 H reported EM-01662 Not
--CO.sub.2CH.sub.3 --OH H H H CH.sub.3 CH.sub.3 H reported
EM-01667C Not --Cl --OH H H H CH.sub.3 CH.sub.3 H reported EM-01668
Not --CO.sub.2H --OH H H H CH.sub.3 CH.sub.3 H reported EM-1692 Not
--I --OH H H H CH.sub.3 CH.sub.3 H reported EM-1695 Not H --CN H H
H H H H reported EM-01807-B Not --CN --OCH.sub.3 H H H CH.sub.3
CH.sub.3 H reported EM-01905-B Not --CF.sub.3 --OH H H H CH.sub.3
CH.sub.3 H reported EM-2016-D Not --CN --OC.sub.2H.sub.5 H H H
CH.sub.3 CH.sub.3 H reported
[0390]
10 TABLE 4' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Inhibi- Inhibi-
tion tion Type 1 Type 2 Inhibi- Inhibi- Inhibi- Inhibition 17B-
17B- tion tion tion Androgenic Type V Re- HSD HSD Type 3 Type 1
Type 3 and % Inhibition 17B-HSD versi Activity Activity 17B-HSD
3a-HSD 3a-HSD Anti- IC.sub.50 -bili IC.sub.50 IC.sub.50 Activity
Activity Activity androgenic (nM) ty (nM) (nM) IC.sub.50 IC.sub.50
IC.sub.50 Activity % Inhibtion % Inhibition Gluco- % Inhibition
Oral Bio- (% Inh. (%) (% Inh. (% Inh. (nM) (nM) (nM) Shionogi
Androgen Progesterone corticoid Estrogen availability At of at at
(% Inh. (% Inh. (% Inh. DHT ZR-75-1 Activity Receptor Receptor
Receptor Receptor AUC 0-7 h (3.10.sup.-7 con- 3.10.sup.-4
3.10.sup.-4 at at at Basal E.sup.-7 E.sup.-6 Basal E.sub.2
*(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5)
*(E.sup.-7 E.sup.-5) Name (ng/mL .multidot. h) 3.10.sup.-6) trol)
M) M) 3.104 M) 3.10.sup.-4 M) 3.10.sup.-4 M) E.sup.-7 E.sup.-6
[IC.sub.50 (nM)] E.sup.-8 E.sup.-6 3E.sup.-8 E.sup.-6 E.sup.-8
E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6
EM-1404 3842 .+-. 197 3.2 .+-. 1.5 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 0 0 [.about.1000] 0 0 0 0 0 0 0 0 0 0 0 0
EM-1403 7.2 .+-. 1.6 19 0 0 -54 ND ND 0 0 0 0 0 0 0 0 EM-1401 1629
.+-. 181 4 .+-. 1.5 31 >>>10.sup.4 [76]
>>>10.sup.4 [76] >>>10.sup.4 2 0 [>1000] ND ND
0 0 0 0 4 2 5 2 EM-1394 1764 .+-. 69 9 23 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 4 -12 0 -23 ND ND 0 0 2 4 3 2 4 8 EM-1424 368
.+-. 70 9.5 17 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4 0
-17 -22 -58 0 0 -11 -2 2 2 1 0 0 0 3 3 EM-1413-CS ND 17 37
>>>10.sup.4 0 0 -17 -44 ND ND EM-1402-CS 214 .+-. 36 .+-.
12 25 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4 0 -9
-13 -87 0 -10 0 -61 0 0 2 4 4 7 1 0 (EM-1405 576 .+-. 93) EM-1396
196 .+-. 53 13 28 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4 0
-15 -16 -29 0 0 +12 -1 0 2 0 0 0 1 3 2 EM-1393-CS ND 16 38
>>>10.sup.4 88 -10 27 -104 ND ND EM-1131 124 .+-. 27 3
[70] 0 0 -15 -47 7 15 0 0 2 1 2 10 0 0 5 3 EM-1125-CS 95 .+-. 6.5 2
3 -10 -30 -13 10 34 0 0 2 1 5 7 0 1 2 2 EM-1408-CS ND 5.5 .+-. 1
[66] 0 -12 -19 -94 ND ND 0 0 0 0 0 0 0 6 EM-1407-CS ND 12 .+-. 3
[68] 0 -23 -16 -96 ND ND 0 0 0 3 0 0 2 0 EM-1126 ND 6 .+-. 1 [59] 0
-8 0 -49 0 -89 0 0 2 10 5 14 0 0 1 0 EM-1118 138 .+-. 10 4 [97] 15
-20 0 -25 0 0 12 15 1 0 0 0 0 0 0 0 EM-1124 5.1 .+-. 1 (8 h) 3 .+-.
0.2 0 -11 0 -65 0 12 0 0 1 7 1 20 1 1 0 0 CS-224 18 .+-. 3.5 2.9
.+-. 0.4 17 0 23 -11 16 128 13 19 0 0 2 5 1 5 0 2 10.7 EM-1157-CS
40 .+-. 7 4 28 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4 16
-12 0 -19 ND ND 0 0 2 5 1 0 0 0 10 EM-1365-CS ND 12 49
>>>10.sup.4 >>>10.sup.4 >>>10.sup.4
>>>10.sup.4 >>>10.sup.4 4 -28 -17 -53 -17 0 -17
-48 0 0 2 2 0 5 2 4 EM-1364-CS ND 17 [73] 0 -16 -11 -59 0 +36 -12
-37 0 0 0 0 0 3 0 5 EM-1392-CS 305 .+-. 49 19 38
>>>10.sup.4 7 -35 0 -75 ND ND 0 0 2 2 0 8 0 0 EM-1391-CS
28 .+-. 3.4 19 49 >>>10.sup.4 0 -13 0 -19 ND ND 0 0 1 1 0
2 0 0 EM-1371-CS 64 .+-. 21 19 40 >>>10.sup.4 58 92 0 -76
0 +137 0 0 0 1 0 3 1 0 2 1 20 EM-1368-CS ND 21 [73] 8 -22 0 -44 0
+15 0 -28 0 0 0 3 3 3 0 0 EM-1405 ND 23 53 >>>10.sup.4 0 0
0 0 ND ND 0 0 0 0 0 0 0 0 EM-1386 5595 .+-. 789 25 54
>>>10.sup.4 5 -17 0 -27 0 -12 0 -16 0 0 0 0 3 0 3 0
EM-1388 136 .+-. 33 25 52 >>>10.sup.4 -18 -23 0 -84 ND ND
0 0 2 3 2 0 0 0 EM-1370 ND 41 [33] 12 -11 0 -28 0 +24 0 0 3 2 0 0 3
0 0 2 EM-1369 ND 46 [62] 0 0 0 -24 0 +84 +18 -1 0 1 1 3 1 2 2 0
EM-1389-CS 169 .+-. 2 25 40 >>>10.sup.4 0 41 -94 -103 ND
ND 0 0 3 0 0 3 0 0 EM-1412-CS ND 54 71 >>>10.sup.4 0 -11 0
-60 ND ND 0 0 0 0 0 2 0 0 EM-1390-CS 401 .+-. 5 48 50
>>>10.sup.4 -18 -13 -36 -119 ND ND 0 0 0 2 2 0 0 0
EM-1385-CS 301 .+-. 36 51 60 >>>10.sup.4 -11 -28 0 -61 ND
ND 0 0 2 2 0 5 2 4 EM-1366-CS ND 54 [62] 0 0 -13 -67 0 0 +19 -4 6 0
0 0 1 3 6 3 EM-1025 ND 6.1 .+-. 0.7 [64] 5 0 -14 -25 20 201 0 0 0 0
0 4 0 0 1 4 4.3 .+-. 0.7 EM-1170-CS ND 7 .+-. 1 [84] 12 -27 15 -38
ND ND 0 1 1 24 4 3 0 0 CS-242 0 6.6 .+-. 0.9 0 -32 9 -66 ND ND 0 0
0 0 0 0 0 0 EM-919 4.8 .+-. .8 10 .+-. 1.4 [86] +73 -17 +38 -95 0
-20 -18 -23 0* 6* 8* 68* 0* 33* 07* 90* [609 .+-. 59] EM-916 ND
10.3 .+-. 0.8 [88] +17 -1 +2 -46 0 0 0 -17 0* 12* 4* 70* 0* 13* 0*
13* [2100] PB-132-140 ND 49 .+-. 5 30 -18 16 0 15 71 0 -28 ND ND ND
ND PB-132-146 ND 72 .+-. 3 [75] 29 0 14 -40 0 91 0 -36 ND ND ND ND
PB-132-152 ND 107 .+-. 9 [50] 39 0 14 -19 39 90 -11 -44 ND ND ND ND
PB-132-142 ND 19 .+-. 1.5 [69] 25 -17 0 -13 38 94 0 -35 ND ND ND ND
EM-1438 ND (90.6 [72] 10 -14 0 -53 ND ND 0 10 0 0 1 0 4 0 95.2)
EM-1382-CS ND (74 86) [70] 0 0 0 0 ND ND 0 0 2 0 2 4 0 3 EM-1372 12
.+-. 7 (39 81) [65] -13 -35 -13 -35 EM-1373 32 .+-. 7 (77 88) [69]
-5 -31 -4 -42 EM-1398 ND (70 89 [64] 13 -10 0 -37 EM-1409 36 .+-.
13 171 .+-. 70 [4] 5 -23 -5 -105 EM-1406-CS ND (84 94) [65] -3 11
-18 -13 EM-1413-CS ND [69] -1 -24 -18 -44 EM-1416 ND [76] 28 -4 26
-41 EM-1419 ND [74] 16 -29 2 -64 EM-01607-D ND EM-01608-D ND 3.6
.+-. 0.5 [58] EM-01645 52 .+-. 3 4.5 .+-. 0.3 [38] -5 35 -6 -10
EM-01646 ND [42] -4 -42 8 -74 EM-01660 ND 5.6 .+-. 0.4 [59] -4 -31
-20 -51 EM-01661-B ND 19.2 .+-. 1 [55] 0 -13 3 -15 EM-01662 ND 35
.+-. 5 [70] 0 -27 -11 -51 EM-01667-C 152 .+-. 18 [83] 6 -3 9 -42
EM-01668 ND (81 90) [63] -3 26 11 2 EM-1692 ND (88 95) [25] 8 -12 3
-78 EM-1695 ND (94 97) [85] 6 -18 -27 -98 EM-01807-B 449 .+-. 115
[39] 4 -12 -7 -12 EM-1808-B 388 .+-. 71 [0] 2 -30 -14 -58
EM-01905-B ND [66] 0 -16 -5 -24 EM-2016-D ND
[0391]
11TABLE 5 63 Name Example X R Unsaturation n b c EM-1122-CS 17 O
CH.sub.3 1.DELTA. 1 H H CS-254 17 O H nil 2 H H EM-980 17 O
CH.sub.3 nil 1 H H CS-201 17 O H nil 1 H H EM-1086 17 S H nil 1 H H
EM-1266-CS Not O H .sup.1.DELTA. 1 CH.sub.3 CH.sub.3 reported
EM-1267-CS Not O H nil 1 CH.sub.3 CH.sub.3 reported EM-1265 Not O
CH.sub.3 .sup.1.DELTA. 1 CH.sub.3 CH.sub.3 reported EM-1269 Not O
CH.sub.3 nil 1 CH.sub.3 CH.sub.3 reported
[0392]
12 TABLE 5' 1 2 3 4 5 6 7 8 9 10 Inhibi- Inhibi- tion tion Type 1
Inhibi- Inhibi- Inhibi- Inhibi- Type V 17B- tion tion tion tion
Oral 17B- HSD Type 2 Type 3 Type 1 Type 3 Androgenic Bio- HSD
Activity 17B-HSD 17B-HSD 3a-HSD 3a-HSD and availa- IC.sub.50
IC.sub.50 Activity Activity Activity Activity Anti- bility (nM)
(nM) IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 androgenic AUC (% Inh.
Reversi- (% Inh. (nM) (nM) (nM) (nM) Shionogi 0-7 h At bility at (%
Inh. (% Inh. (% Inh. (% Inh. DHT (ng/ (3.10.sup.-7 (%) of
3.10.sup.-4 at at at at Basal E.sup.-7 E.sup.-6 Name mL .multidot.
h) 3.10.sup.-6) control) M) 3.10.sup.-4 M) 3.10.sup.-4 M)
3.10.sup.-4 M) 3.10.sup.-4 M) E.sup.-7 E.sup.-6 [IC.sub.50 (nM)]
EM-1122- ND (80 95) [90] 0 -11 -14 -74 CS [447] CS-254 ND 20 .+-. 2
0 7 -5 -7 EM-980 ND 27.8 .+-. 10 [68] 0 -34 9 58 CS-201 ND 31.4
.+-. 5.4 [70] -26 -37 -23 7 CS-205 ND 83 .+-. 18 [31] -26 -37 -74
-112 EM-1266- ND 26 [81] 0 -22 -12 -10 CS EM-1267- ND 29 [43] 0 -24
-12 -25 CS EM-1265 ND (83 95) [77] 0 0 -11 -80 EM-1269 ND (74 93)
[57] 0 0 -10 -59 11 12 13 14 15 16 % Inhibition % Inhibtion %
Inhibition Gluco- % Inhibition Androgen Progesterone corticoid
Estrogen ZR-75-1 Activity Receptor Receptor Receptor Receptor Basal
E.sub.2 *(E.sup.-7 E.sup.-5) *(E.sup.-7 E.sup.-5) *(E.sup.-7
E.sup.-5) *(E.sup.-7 E.sup.-5) Name E.sup.-8 E.sup.-6 3E.sup.-8
E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6 E.sup.-8 E.sup.-6
E.sup.-8 E.sup.-6 EM-1122- ND ND 1 8 0 3 0 0 2 0 CS CS-254 ND ND 0
0 0 0 3 0 2 1 EM-980 0 0 0 -19 3 5 1 0 0 0 0 0 CS-201 0 0 15 0 0 0
0 0 0 0 0 0 CS-205 0 0 0 25 1 15 0 7 0 0 2 2 EM-1266- ND ND 0 0 1 0
0 0 3 6 CS EM-1267- ND ND 0 0 0 3 3 1 3 4 CS CM-1265 EM-1269
[0393] Legends to Tables
[0394] In column 1, the oral bioavailability of preferred type 5
inhibitors, expressed in ng/mL.h, was determined as described below
in "Other Tests, A--In Vivo Assays of Bioavailability of Human Type
5 17.beta.-HSD Inhibitors". Higher number are desirable. ND means
that a determination was not done.
[0395] In column 2, the inhibition of human type 5
17.beta.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition (IC.sub.50 in nM) is
reported (centered numbers). The manner in which IC.sub.50 was
determined is described in "Efficacy of the Preferred Inhibitors".
Lower numbers for IC.sub.50 are desirable. When IC.sub.50 was not
determined, the percentage of inhibition is reported in parentheses
at 3.10.sup.-7M (left number) and 3.10.sup.-6M (right number).
[0396] In column 3, the reversibility of the inhibition of type 5
17.beta.-hydroxysteroid dehydrogenase activity expressed in
percentage of control is reported. The manner in which the
reversibility is. determined is described in "Efficacy of Preferred
Inhibitors: V. Reversibility of Human Type 5 17.beta.-HSD
Inhibitory Activity". Lower number is desirable. Blank means that a
determination was not done.
[0397] In column 4, the inhibition of human type 1
17.beta.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition of enzymatic activity
(IC.sub.50 in nM) is reported. The manner in which IC.sub.50 was
determined is described in "Efficacy of Preferred Inhibitors: VI.
Enzymatic assay for types 1, 2, and 3 17.beta.-HSD and types 1 and
3 3.alpha.-HSD". Higher numbers of IC.sub.50 are desirable. Blank
means that a determination was not done. The % of inhibition at the
concentration of 3.10.sup.-6M determined in preliminary screening
test is reported in square braquets.
[0398] In column 5, the inhibition of human type 2
17.beta.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition of enzymatic activity
(IC.sub.50 in nM) is reported. The manner in which IC.sub.50 was
determined is described in "Efficacy of Preferred Inhibitors: VI.
Enzymatic assay for types 1, 2, and 3 17.beta.-HSD and types 1 and
3 3.alpha.-HSD". Higher numbers of IC.sub.50 are desirable. Blank
means that a determination was not done. The % of inhibition at the
concentration of 3.10.sup.-6M determined in preliminary screening
test is reported in square braquets.
[0399] In column 6, the inhibition of human type 3
17.beta.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition of enzymatic activity
(IC.sub.50 in nM) is reported. The manner in which IC.sub.50 was
determined is described in "Efficacy of Preferred Inhibitors: VI.
Enzymatic assay for types 1, 2, and 3 17.beta.-HSD and types 1 and
3 3.alpha.-HSD". Lower numbers of IC.sub.50 are desirable. Blank
means that a determination was not done. The % of inhibition at the
concentration of 3.10.sup.-6M determined in preliminary screening
test is reported in square braquets.
[0400] In column 7, the inhibition of human type 1
3.alpha.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition of enzymatic activity
(IC.sub.50 in nM) is reported. The manner in which IC.sub.50 was
determined is described in "Efficacy of Preferred Inhibitors: VI.
Enzymatic assay for types 1, 2, and 3 17.beta.-HSD and types 1 and
3 3.alpha.-HSD". Higher numbers of IC.sub.50 are desirable. Blank
means that a determination was not done. The % of inhibition at the
concentration of 3.10.sup.-6M determined in preliminary screening
test is reported in square braquets.
[0401] In column 8, the inhibition of human type 3
3.alpha.-hydroxysteroid dehydrogenase activity expressed by the
concentration which produce 50% of inhibition of enzymatic activity
(IC.sub.50 in nM) is reported. The manner in which IC.sub.50 was
determined is described in "Efficacy of Preferred Inhibitors: VI.
Enzymatic assay for types 1, 2, and 3 17.beta.-HSD and types 1 and
3 3.alpha.-HSD". Higher numbers of IC.sub.50 are desirable. Blank
means that a determination was not done. The % of inhibition at the
concentration of 3.10.sup.-6M determined in preliminary screening
test is reported in square braquets.
[0402] In column 9, the androgenic activity of preferred type 5
inhibitors expressed as the percentage of stimulation of
proliferation of Shionogi cells at concentrations of 10.sup.-7 M
(left number) and 10.sup.-6 M (right number) of inhibitor. The
manner in which the stimulation is determined is described in
"Other Tests; B--Androgenic/Antiandrogenic Activity". Lower numbers
are desirable. ND means that a determination was not done.
[0403] In column 10, the antiandrogenic activity of preferred type
5 inhibitors expressed by the concentration which produce 50% of
inhibition (IC.sub.50 in nM) of DHT-induced proliferation of
Shionogi cells is reported (bracketed centered numbers). The
percentage of inhibition of DHT-induced proliferation of Shionogi
cells at concentrations of 10.sup.-7M (left number) and 10.sup.-6M
(right number) of inhibitor is also reported. For example, EM-1403
in Table 4', column 10, the number -54 means that at a
concentration of .sup.10-6 M, the stimulation of DHT-induced
proliferation of Shionogi cells was 54% reduced. The manner in
which the inhibition is determined is described in "Other Tests;
B--Androgenic/Antiandrogenic Activity". Lower numbers are
desirable. ND means that a determination was not done.
[0404] In column 11, the estrogenic activity of preferred type 5
inhibitors expressed as the percentage of stimulation of the
proliferation of ZR-75-1 cells at concentrations of 10.sup.-7M
(left number) and 10.sup.-6M (right number) of inhibitor. The
manner in which the stimulation is determined is described in
"Other Tests; C--Estrogenic/Antiestrogenic activity" Lower numbers
are desirable. ND means that a determination was not done.
[0405] In column 12, the antiestrogenic activity of preferred type
5 inhibitors expressed as percentage of inhibition of E2-induced
proliferation of ZR-75-1 cells at a concentrations of 10-7N (left
number) and 10.sup.-6 (right number) of inhibitor is reported. For
example, EM-1402-CS in Table 4', column 12, the number -61 means
that at a concentration of 10M, the stimulation of E2-induced
proliferation of ZR-75-1 cells was 61% reduced. The manner in which
the inhibition is determined is described in "Other Tests;
C--Estrogenic/Antiestrogenic Activity". Lower numbers are
desirable. ND means that a determination was not done.
[0406] In column 13, the binding on androgen receptor expressed as
percentage of inhibition of the binding of [.sup.3H]R1881 at the
concentration of 10.sup.-8M (stared number at 10.sup.-7 M) (left
number) and 10.sup.-6M (stared number at 10.sup.-5 M) (right
number) of inhibitor is reported. The manner in which the
percentage of inhibition is determined is described in "Other
Tests; D--Androgen Receptor (AR) Assays". Lower numbers are
desirable.
[0407] In column 14, the binding on progesterone receptor expressed
as percentage of inhibition of the binding of [.sup.3H]R5020 at the
concentration of 10.sup.-8M (stared number at 10.sup.-7 M) (left
number) and 10M (stared number at 10.sup.-5 M) (right number) of
inhibitor is reported. The manner in which the percentage of
inhibition is determined is described in "Other Tests;
E--Progesterone Receptor Assay". Lower numbers are desirable.
[0408] In column 15, the binding on glucocorticoid receptor
expressed as percentage of inhibition of the binding of
[6,7-.sup.3H*(N)]-dexamethason- e at the concentration of
10.sup.-8M (stared number at 10.sup.-7 M) Deft number) and
10.sup.-6M (stared number at 10.sup.-5 M) (right number) of
inhibitor is reported. The manner in which the percentage of
inhibition is determined is described in "Other Tests;
F--Glucocorticoid Receptor Assay".
[0409] In column 16, the binding on estrogen receptor expressed as
percentage of inhibition of the binding of [.sup.3H]E.sub.2 at the
concentration of 10.sup.-8M (stared number 10.sup.-7) (left number)
and 10.sup.-6M (stared number 10.sup.-5) (right number) of
inhibition is reported. The manner in which the percentage of
inhibition is determined is described in "Other Tests; G--Estrogen
Receptor (ER) Assay".
Efficacy of the Preferred Inhibitors
[0410] A The Preferred Inhibitors of the Invention are Tested for
Their Type 5 17.beta.-HSD Inhibitory Activity by the Following
Method
[0411] I) Cloning of Type 5 1710-HSD cDNA
[0412] Using a oligoprimer pair
(5'-GGA-AAT-CGT-GAC-AGG-GAA-TGG-ATT-CCA-AA- C-AG-3',
5'-GGA-ATT-CTT-TAT-TGT-ATT-TCT-GGC-CTA-TGG-AGT-GAG-3') derived from
human aldoketoreductase (Qin et al., J. Steroid Biochem. Molec.
Biol. 46: 673-679, 1993) and polymerase chain reaction (PCR), we
have amplified from a human placental .lambda.gt11 cDNA library
(Clontech Laboratories Inc., Palo Alto, Calif.) a cDNA fragment
that was used as probe to screen again a human placental
.lambda.gt11 cDNA to get the full length cDNA clone. The amplified
cDNA fragment was purified on agarose gel and labeled with
[.alpha..sup.32P]dCTP (Amersham Corp.) using the random primer
labeling kit from Pharmacia Inc. PCR amplification reaction medium
contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.1% gelatin, 1 mM
MgCl.sub.2, 250 .mu.M dNTP, 0.25 .mu.M oligonucleotide primers, and
200 ng of DNA prepared from human placental .lambda.gt11 cDNA
library. The aqueous phase was overlaid with 75 .mu.L of mineral
oil to avoid evaporation. The mixture was heated at 98.degree. C.
for 5 min, following the temperature reduction to 72.degree. C., 1
unit of Taq DNA polymerase was added. Using a DNA Thermal Cycler
(Perkin-Elmer Corp.), 30 cycles of amplification were carried out
using a step program (94.degree. C., 1 min; 60.degree. C., 1 min;
and 72.degree. C., 1 min)/cycle.
[0413] Approximately 5.times.10.sup.5 recombinant phage plaques
were screened with 1.5.times.10.sup.6 cpm/ml of the probe.
Prehybridization was performed for 4 h at 42.degree. C. in 50%
formamide, 5.times. Denhardt's solution (1.times. Denhardt's being
0.02% polyvinyl pyrrolidone, 0.02 Ficoll, 0.02 bovine serum
albumin), 5.times.SSPE (1.times.SSPE being 0.18M NaCl, 10 mM
NaH.sub.2PO.sub.4, pH 7.4, 1 mM EDTA), 0.1% SDS, and 100 .mu.g/ml
yeast tRNA. The probe was denatured by heating at 100.degree. C.
for 5 min, before being added to the hybridization solution at
42.degree. C. overnight. Filters were then washed twice at room
temperature in 2.times.SSC (1.times.SSC being 0.015 M NaCl, 0.15M
sodium citrate, pH 7.0), 0.1% SDS, and twice in 0.1.times.SSC, 0.1%
SDS at 60.degree. C. Positive recombinant plaques were purified by
replating twice and grown in liquid culture. Phage DNA were
isolated by centrifugation for 90 min at 105,000.times.g, and DNA
was then isolated by phenol extraction and precipitation with
isopropanol (Karen et al. 1987; In: Current Protocols in Molecular
Biology (F. M. Ausubel, R. Brent, E. Kingston, D. D. Moore, T. G.
Seidman, T. A. Smith and K. Struhl, eds), Wiley & Sons, New
York, pp. 1.13.1-1.13.6).
[0414] II) Construction of Expression Vector, and Nucleotide
Sequence Determination
[0415] The phage DNA were digested with EcoRI restriction enzymes
and the resulting cDNA fragments were inserted into the EcoRI site
downstream to the cytomegalovirus (CMV) promoter of the pCMIV
vector (kindly provided by Dr. Matthew, Cold Spring Harbor, N.Y.,
USA). Recombinant pCMV plasmids were amplified in Escherichia Coli
DH5.alpha. competent cells, and were isolated by alkaline lysis
procedure (Maniatis et al., In: Molecular cloning: a laboratory
manual, Cold Spring Harbor Laboratory., 1982). Sequencing of
double-stranded plasmid DNA was performed according to the dideoxy
chain termination method (Sanger et al., PNAS 74: 5453-5467, 1977)
using a T7 DNA polymerase sequencing kit (Pharmacia LKB
Biotechnology). To avoid errors, all sequences were determined by
sequencing both strands of the DNA. Oligonucleotide primers were
synthesized in our laboratory with a 394 DNA/RNA synthesizer
(Applied Biosystem).
[0416] The pCMV vector contain 576 nucleotides of the pCMV promoter
(followed by 432 bp of small t intron (fragment 4713-4570) and poly
adenylation signal (2825-2536)of SV40, followed by 1989 bp of the
Pvu II(628)-AatII (2617) fragment from pUC 19 vector (New England
Biolabs) that contains an E. Coli origin of replication and an
ampicillin resistance gene for propagation in E. Coli.
[0417] III) Cells Overexpressing 17.beta.-HSDs in Transformed
Embryonal Kidney (293) Cells
[0418] A. Transient Transfection
[0419] Transfection was performed by the calcium phosphate
procedure (Kingston et al., In: Current Protocols in Molecular
Biology (Ausubel et al., eds), pp. 9.1.1-9.1.9, John Wiley &
Sons, Inc., New York, 1987) using 1 to 10 .mu.g of recombinant
plasmid DNA per 10.sup.6 cells. The total amount of DNA is keep at
10 .mu.g of plasmid DNA per 10.sup.6 by completing with pCMV
plasmid without insert. The cells were initially plated at 10.sup.4
cells/cm.sup.2 in Falcon culture flasks and grown in Dulbecco's
modified Eagle's medium containing 10% (vol/vol) fetal bovine serum
(Hyclone, Logan, Utah) under a humidified atmosphere of
air/CO.sub.2 (95/5%) at 37.degree. C. supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 IU penicillin/ml, and 100
.mu.g streptomycin sulfate/ml.
[0420] B. Stable Transfection
[0421] Cells were cultured in 6-wells falcon flasks to
approximately 3 105 cells/well in DMBM. Five 4 g of pCMV-neo
plasmid that expresses type 5 17.beta.-HSD and Neomycin resistant
gene, were transfected into 293 cells using lipofectin transfection
kit (Life Technologies, Burlinton, ON). pCMV-neo vector were
constructed by inserting 2685 bp of a BamHI-BamHI DNA fragment that
expresses a neomicin resistance gene isolated from the pMAMneo
vector (Clontech Laboratories, Palo Alto, Calif.) at the BamH1 site
of the pCMV vector. After 6 h incubation at 37.degree. C., the
transfection medium was removed and 2 ml of DMEM were added. Cells
were further cultured for 48 h then transferred into a 10 cm petri
dishes and cultured in DMEM containing 700 .mu.g/ml of G-418 (Life
Technologies, Burlinton, ON), in order to inhibit the growth of
non-transfected cells. Medium containing G-418 was changed every
two days until resistant colonies were observed. Resistant clones
that incorporated Neomycin resistant gene were tested for
17.beta.-HSD activity. The most actif clone was cultured and keep
frozen at -70.degree. C. for further inhibition study.
[0422] I) Assay of Enzymatic Activity
[0423] Determination of activity was performed as described
(Luu-The et al., Mol. Endocrionol. 4: 268-275, 1990, Lachance et
al., J. Biol. Chem. 265: 20469-20475, 1990; Luu-The et al., DNA
& Cell Biol. 14: 511-518, 1995). Briefly, 0.1 .mu.M of the
indicated .sup.14C-labeled substrate (Dupont Inc. (Canada), namely,
DHEA, 4-androstene-3,17-dione (.DELTA.4), in absence or presence of
increasing concentration of preferred inhibitor of the invention,
was added to freshly changed culture medium in a 6-well culture
plate. After incubation for 1 h, the steroids were extracted twice
with 2 ml of ether. The organic phase were pooled and evaporated to
dryness. The steroids were solubilized in 50 .mu.l of
dichloromethane, applied to Silica gel 60 thin layer chromatography
(TLC) plate (Merck, Darmstad, Germany) then separated by migration
in the toluene-acetone (4:1) solvent system. Substrates and
metabolites were identified by comparison with reference steroids
and revealed by autoradiography and quantitated using the
Phosphoimager System (Molecular Dynamics, Sunnyval, Calif.).
Transfection could be also performed with HeLa, SW-13, 293, COS-1
cells, the preferred cell line is 293 cells.
[0424] V. Reversibility of Human Type 5 17.beta.-HSD Inhibitory
Activity
[0425] The reversibility assay was performed as described above for
the standard type 5 17.beta.-HSD enzymatic assay, except that
before adding the .sup.14C-labeled substrate, the cells were
preincubated with 0.3 .mu.M of the indicated inhibitor for 1 h, (or
the indicated time interval), followed by two washes with phosphate
saline buffer (PBS).
[0426] VI. Enzymatic Assay for Types 1, 2, and 3 17.beta.-HSD and
Types 1 and 3 3.alpha.-HSD
[0427] Enzyme sources. 293 cells transiently transfected with
expression vectors encoding types 1, 2 and 3 17.beta.-HSD (Luu-The
et al., J. Steroid Biochem. Molec. Biol., 55: 581-587, 1995), types
1 and 2 5.alpha.-reductase (Luu-The et al., J. Invest. Dermatol.,
102: 221-226, 1994) and types 1 and 3 3.alpha.-HSD (Dufort et al.
Biochem. Biophys. Res. Commun. 228: 474479, 1996), using the
calcium phosphate procedure (Kingston et al., In: Current Protocols
in Molecular Biology. Edited by E. M. Ausbel, R. Brent, R. E.
Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhi. John
Wiley & Sons, New York, pp. 9.1.1-9.1.9, 1991; Luu-The et al.,
J. Invest. Dermatol., 102: 221-226, 1994). For assays using cell
subfractions, cells were sonicated in 50 mM sodium phosphate buffer
(pH 7.4), containing 20% glycerol and 1 mM EDTA and centrifuged at
10 000.times.g for 30 min before centrifugation for 100 000.times.g
for 1 h to separate the mitochondrial and microsomal fractions,
respectively. The cytosol fractions (100 000.times.g supernatant)
was used to determine type 1 activity while the microsomal fraction
(pellet at 100 000.times.g) was used for measurement of types 2 and
3 17.beta.-HSD activities.
[0428] Incubation. The enzymatic reaction was carried out at
37.degree. C. in 1 ml of 50 mM sodium phosphate buffer, pH 7.4,
containing 20% glycerol, 1 mM EDTA, and 2 mM cofactors (NADPH or
NAD.sup.+) for 1 h in the presence of 0.1 .mu.M .sup.14C-labeled
substrate: estrone and 4-dione for types 1 and 2 17.beta.-HSD,
respectively, testosterone for type 2 17.beta.-HSD as well as types
1 and 2 5.alpha.-reductases, DHT for types 1 and 3 3.alpha.-HSD
activities, and the indicated concentration of inhibitor.
[0429] Extraction of metabolites, migration on TLC and
quantification were as described above for the assay of type 5
17.beta.-HSD.
[0430] Other Tests
[0431] A--In vivo Assays of Bioavailability of Human Type 5
17.beta.-HSD Inhibitors
[0432] 1) Principle
[0433] The assays of the bioavailability of 17.beta.-HSD Type 5
inhibitors were performed in male Sprague Dawley rats by measuring
the plasma concentrations of the compounds after single oral
administration of the compounds. The measurements at various time
intervals were for values greater than or equal to 1.0 ng/mL and
less than or equal to 50 ng/mL.
[0434] a) Animals and Treatment
[0435] Male Sprague-Dawley rats [Crl:CD(SD)Br] weighing 275-350 g
were obtained from Charles-River Canada Inc. and housed 2 per cage
during the acclimation period and individually during the study
period. The animals were maintained under a regimen of 12 hours
light: 12 hours dark (lights on at 08:00). Animals received
certified Rodent feed (Lab Diet #5002, pellets) and tap water ad
libitum. Rats were fasted (access to water only) starting on the
evening prior to dosing.
[0436] Each compound to be tested was administered to three animals
as a suspension in 0.4% methylcellulose by oral gavage at a dose of
0.5 mg/rat (1.0 ml/rat). Four to eight new compounds were tested
each day and one group of animals received megestrol acetate (MGA)
under the same conditions on each dosing day as a reference. One
blood sample of .about.0.7 ml was collected from the jugular vein
of rats under Isoflurane-induced anesthesia at 1, 2, 3, 4, and 7
hours post-gavage. Blood samples were immediately transferred into
a refrigerated 0.75 ml Microtainer containing EDTA and kept in an
ice-water bath until centrifugation at 3000 rpm for 10 minutes.
Plasma separation was performed rapidly (less than 50 minutes)
after blood collection. One aliquot of 0.25 ml of plasma was then
transferred into a borosilicate tube (13.times.100) and was rapidly
frozen on dry-ice. Plasma samples were kept at -80.degree. C. until
measurement of plasma concentration of the inhibitor(s) by
LCMS/MS.
[0437] 2) LCMS Measurements
[0438] a) Apparatus
[0439] 1. Vacuum manifold
[0440] 2. Turbo Vap LV evaporator
[0441] 3. Mass spectrometer API III or API-300 (PE/Sciex) with
associated peripherals
[0442] 4. Automatic Injector
[0443] 5. HPLC pump
[0444] 6. Infusion pump
[0445] 7. Calibrated pipets
[0446] b) Reagents and Solutions
[0447] 1. Methanol, HFLC grade
[0448] 2. Water, Ultrapure (Super Q)
[0449] 3. Ethanol, reagent grade
[0450] 4. N-butyl chloride, HPLC grade
[0451] 5. Acetone, HPLC grade
[0452] 6. Male rat plasma (EDTA)
[0453] 7. 17.beta.-HSD type 5 inhibitors in reference standard
ethanol solution approximately 100 .mu.g/mL
[0454] 8. EM 248 Internal Standard reference standard (solution of
50 ng/mL)
[0455] 9. Mass calibrator solution Polypropylene Glycol
(PE/Sciex)
[0456] c) Mass Spectrometer Conditions
[0457] Detector: Mass spectrometer API-300 (PE/Sciex)
[0458] Interface: Turbo Ion spray inlet (split 1/5)
[0459] Auxiliary flow: 4.5L/minute (nitrogen)
[0460] Nebulizer Flow: 11
[0461] Curtain Gas Flow: 11
[0462] Probe Temperature: 460.degree. C.
[0463] Pressure: Approximately 3.times.10.sup.-5 Torr
[0464] CAD gas thickness: 3
[0465] Count Control: 1
[0466] Mobile Phase: Gradient of Methanol with 1 mm Ammonium
formate and Water with 1 mm Ammonium formate
[0467] Flow Rate: 1 mL/minute
[0468] d) Mass Spectrometer Analysis Parameters for EM-1118
[0469] Dwell time: 150 msec
[0470] Pause time: 30 msec
[0471] Duration: 4 minute
[0472] MRM mode for
[0473] EM-1118 analysis: 444.2 and 398.3
[0474] Injection: 10 .mu.L
[0475] Data handling: "API Standard Software" update version.
[0476] e) Preparation of Standard Solutions
[0477] Stock solutions for each type 5 inhibitor were prepared in
methanol and, when not in use, the methanol solutions were stored
at -20.degree. C. Calibration curve standard solutions for each
compound were prepared in male rat plasma as illustrated in Table
1.
[0478] A solution of internal standard in methanol containing
EM-248 at 50 ng/mL, was prepared from stock standard solutions of
EM-248 stored at -20.degree. C.
13 Concentration of inhibitor 17.beta.-HSD Volume of solution
Volume of plasma Std 50 ng/mL 90 .mu.l of 1 .mu.g/mL 1.71 mL Std 20
ng/mL 0.8 mL of 50 ng/mL 1.2 mL Std 10 ng/mL 0.9 mL of 20 ng/mL 0.9
mL Std 5 ng/mL 0.8 mL of 10 ng/mL 0.8 mL Std 2 ng/mL 0.6 mL of 5
ng/mL 0.9 mL Std 1 ng/mL 0.5 mL of 2 ng/mL 0.5 mL Std 0 N/A 0.5 mL
Blank N/A 0.5 mL
[0479] f) Extraction Procedure for Type 5 Inhibitors From Rat
Plasma
[0480] Aliquots of rat plasma (0.250 mL) were transferred to
13.times.100 mm borosilicate tubes. Water (1.0 mL) and internal
standard solution (0.1 mL) were added to each sample and vortexed
for 2 min. A mixture of N-butyl chloride and acetone (v:v, 7:3) (3
mL) was added to each sample and vortexed for 2 min. This step was
repeated and the combined organic phases were evaporated to dryness
under nitrogen in a Turbo Vap evaporator at 35.degree. C. The
residue was reconstituted with 1 mL of methanol and evaporated in a
Turbo Vap evaporator at 35.degree. C. The final extract was
reconstituted into 0.1 mL of methanol/water (v:v, 75:25) and then
transferred into a conical vial for injection into the mass
spectrometer.
[0481] g) Assay
[0482] The assay procedure was performed by analyzing, in
duplicate, rat plasma samples spiked at six different Type 5
inhibitor concentrations (1, 2, 5, 10, 20 and 50 ng/mL). As
example, the results of EM-1118 are presented in FIG. 1. The lower
limit of quantitation (LOQ) was established at 1.0 ng/mL. Values
lower than 1.0 ng/mL were expressed as below limit of
quantification (BLQ).
[0483] h) Linearity
[0484] The assay procedures for EM-1118 were found to be linear
over the 1.0 to 50 ng/mL range. Weighted (1/X) linear regression
analysis gave a correlation (r.sup.2) of 0.991.
[0485] i) Calculation of AUC Values
[0486] For all compounds studied, the area under the plasma
concentration versus time curve (AUC) from time 0 to 7 hours
post-dosing was determined. AUC.sub.0-7 values were calculated by
the linear trapezoidal method (model-independent) for each rat and
data were expressed as mean AUC.sub.0-7.+-.SEM (n=3).
[0487] B--Androgenic/Antiandrogenic Activity
[0488] Androgenic/antiandrogenic activity of some preferred
compounds has been measured using the Shionogi mouse mammary
carcinoma cells.
[0489] Materials
[0490] Minimal essential culture medium (MEM), non-essential amino
acids, and fetal calf serum were purchased from Flow Laboratories.
In order to remove endogenous steroids, serum was incubated
overnight at 4.degree. C. with 1% activated charcoal (Norit A,
Fisher) and 0.1% Dextran T-70 (Pharmacia). A 2-h supplementary
adsorption was performed at 25.degree. C. in order to further
remove protein-bound steroids. Serum was also inactivated by a
20-min incubation at 56.degree. C. 5.alpha.-dihydrotestosterone
(DHT) was obtained from Steraloids. The antiandrogen
hydroxyflutamide (OH-FLU) was kindly supplied by Drs. T. L.
Nagabuschan and R. Neri (Schering Corporation, Kenilworth,
U.S.A.).
[0491] Cell Dispersion, Culture and Cloning
[0492] Shionogi male mice bearing androgen-sensitive mammary tumors
were obtained from Drs. Keishi Matsumoto, Osaka, Japan, and Yvonne
Lefebvre, Ottawa, Canada. For primary culture, tumors were excised
and washed in ice-cold sterile 25 mM Hepes buffer (137 mM NaCl; 5
mM KCl; 0.7 mM Na.sub.2HPO.sub.4; 10 mM glucose, pH 7.2). After
mincing with scissors, the tumor minces were digested for 2 h at
37.degree. C. in Hepes buffer containing 3.8 mg/ml collagenase
(Clostridium, Boehringer), 1.5 mg/ml hyaluronidase II (Sigma), and
3% bovine serum albumin fraction V (Schwartz-Mann). Dispersed cells
were collected by centrifugation (500.times.g for 10 min), washed
twice by suspension in minimal essential medium (MEM) containing 5%
dextran-coated charcoal-treated fetal calf serum (DCC-FCS), 1%
non-essential amino acids, 10 IU/ml penicillin, 50 .mu.g/ml
streptomycin, and 100 nM dihydrotestosterone (DHT)
(Steraloids).
[0493] Cells were plated in the same medium at a density of 75 000
cells/ml in 75 cm.sup.2 flasks under an atmosphere of 5% carbon
dioxide in air at 37.degree. C. The medium was changed weekly. The
steroids and antisteroids were dissolved in ethanol and kept in
stock solutions chosen to yield final ethanol concentrations less
than 0.01.degree.% in the culture medium. Such a concentration of
ethanol does not affect cell growth.
[0494] Cells were subcultured at near-confidence by gentle
digestion in a solution of 0.1% pancreatin (Flow Laboratories) in
Hepes buffer containing 3 mM ethylenediaminetetraacetic acid (EDTA)
(pH 7.2). Cells were pelleted by centrifugation, resuspended in
culture medium, counted in a Coulter counter, and replated as
described above. Soft agar cloning was performed as described
(Stanley et al., Cell 10: 35-44, 1977) in the presence of 100 nM
DHT.
[0495] Measurement of Cell Growth and Sensitivity to Steroids and
Antisteroids
[0496] Cells were plated in 24-well plates at a density of 20 000
cells/well. The indicated increasing concentrations of agents were
added to triplicate dishes, and cells were grown for 10-12 days
with changes of medium every 3-4 days. Cell number was measured by
direct counting in a Coulter counter.
[0497] Calculations and Statistical Analysis
[0498] ED.sub.50 values of action of DHT and glucocorticoids were
calculated according to a least-square regression as described
(Rodbard, Endocrinology 94: 1427-1431, 1974). Statistical
significance was calculated according to a multiple-range test
(Kramer, Biometrics 12: 307-310, 1956).
[0499] C--Estrogenic/Antiestrogenic Activity
[0500] Estrogenic/antiestrogenic activity of some preferred
compounds has been measured using the ZR-71-1 human breast cancer
cell line as described in more detail below.
[0501] Maintenance of Stock Cultures
[0502] ZR-75-1 cells (83.sup.rd passage) were obtained from the
American Type Culture Collection (Rockville, Md.) and routinely
cultured in phenol red free RPMI 1640 supplemented with 1 nM
estradiol (E.sub.2), 2 mM L glutamine, 1 mM sodium pyruvate, 15 mM
N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid, 100 IU
penicillin/ml, 100 .mu.g streptomycin/ml, and 10% (v/v) fetal
bovine serum (Hyclone, Logan, Utah) under a humidified atmosphere
of 95% air, 5% CO.sub.2, at 37.degree. C. All media and medium
supplments were purchased from Sigma. Cells were subcultured weekly
by treatment with a pancreatic solution containing EDTA (0.2 g/L).
The cell cultures used for the experiments herein described were
between passages 89 and 94.
[0503] Measurements of Cell Proliferation
[0504] Cells in their logarithmic growth phase were harvested,
briefly centrifuged, and resuspended in RPMI 1640. Cells were then
plated in triplicate in LIMBRO 24-well plastic culture plates (2
cm.sup.2/well). Since plating density influences the effect of
hormones on ZR-75-1 cell growth, cells were plated at a density of
1.times.10.sup.4 cells/well. After 72 h, medium was replaced with
fresh medium containing the inhibitor at the concentration of
3.10.sup.-7 and 10.sup.-6 M in absence or presence of 0.1 M
estradiol (E.sub.2). Control cultures received the ethanol vehicle
only. Cells were then allowed to grow at 37.degree. C. for 10 days
with medium changes (of identical composition) every 2 days. In
absence of inhibitors, in 0.1M estradiol (E.sub.2)-containing
medium, ZR-75-1 cells have doubling time of about 48 h.
[0505] After E.sub.2 and/or antiestrogen treatment, cells were
harvested by addition of 0.5 ml of a pancreatin solution (Sigma)
for 5-10 min at 37.degree. C. before addition of 0.5 ml of RPMI
1640 containing 5% dextran coated charcoal-free bovine serum in
order to block enzymatic action. Cell number (0.10 ml aliquot) was
determined by measurement of DNA content as previously described
(Simard et al., Endocrinology 126: 3223-3231, 1990).
[0506] D--Androgen Receptor (AR) Assays
[0507] Tissue Preparation
[0508] Male Sprague-Dawley rats (Crl: CD(SD)Br) weighing 200-300 g
were obtained from Charles-River Inc. (St-Constant, Qubec, Canada).
The rats were gonadectomized under general anesthesia (Isoflurane)
and killed by cervical dislocation 24 hours later. The ventral
prostates were rapidly removed, dissected free from adhering tissue
and frozen on dry-ice. Prostates were kept at -80.degree. C. until
assay.
[0509] All subsequent steps were performed at 0-4.degree. C.
Prostates were homogenized in 5 vol (wt/vol) of buffer A (25 mM
Tris-HCI, 1.5 mM EDTA disodium salt, 10 mM
.alpha.-monothioglycerol, 10% glycerol, and 10 mM sodium molybdate,
pH 7.4), using a Polytron PT-10 homogenizer (Brinkman Instruments,
Canada) at a setting of 5 for three periods of 10 sec, with
intervals of 10 sec for cooling. The homogenate was then
centrifuged at 105,000.times.g for 60 min in a Beckman L5-65
ultracentrifuge (Fullerton, Calif.). The protein concentration of
the cytosol fraction was measured according to the method of
Bradford (Anal. Biochem. 72: 248-254, 1976), using bovine serum
albumin as standard.
[0510] Androgen Receptor Assay
[0511] Androgen binding was measured using the hydroxylapatite
assay (HAP). In brief, the radioactive steroid [.sup.3H]R1881
solubilized in ethanol was diluted into buffer A. Aliquots of
prostate cytosol preparation (0.1 ml) were then incubated with 8 nM
[.sup.3H]R1881 (0.1 ml, 200,000 cpm) in the presence or absence of
the indicated concentrations of unlabeled compounds (0.1 ml,
prepared in buffer A containing 10% ethanol) for 16-18 h at
0-4.degree. C. Triamcinolone acetonide (150 nM) was added in order
to mask progesterone receptors. Unbound steroids were separated by
incubation for 40 min at 0-4.degree. C. with 0.3 ml HAP prepared in
buffer P (50 mM, Tris-HCl, 10 mM KH.sub.2PO.sub.4, pH 7.4) as
follows: 10 g HAP were washed with buffer P until the supernatant
reached a pH of 7.4 and then following centrifugation and
decantation of the supernatant, 37.5 ml of buffer P were added.
After incubation with HAP and 10 minutes of centrifugation at
1,000.times.g, the pellet was washed 3 times with 1 ml buffer P.
Thereafter, the radioactivity was extracted from the pellet by
incubation at room temperature for 60 minutes with 1 ml EtOH. After
centrifugation, the supernatant was decanted into a scintillation
vial and the pellet was extracted again with ethanol. Thereafter,
10 ml Formula-989 scintillation liquid was added to pooled
supernatant and the radioactivity was measured in a Beckman
counter.
[0512] Calculations
[0513] The results were reported as the percentage of inhibition of
the binding of [.sup.3H]R1881 at the concentrations of 10.sup.-8
and 10.sup.-6 M of the inhibitor.
[0514] E--Progesterone Receptor Assay
[0515] Chemicals
[0516] [17.alpha.-methyl-3H]-promegestone (R5020) (84 Ci/mmol) and
the corresponding unlabeled compound were purchased from New
England Nuclear (Lachine, Qubec, Canada). All other chemicals were
of analytical grade. Stock solutions of the unlabeled steroids were
kept at 4.degree. C. in ethanol. The desired steroid solutions were
then prepared by appropriate dilution in buffer B (10 mM Tris-HCl,
1.5 mM EDTA, 10 mM .alpha.-monothioglycerol, pH 7.4) containing 30%
ethanol.
[0517] Tissue Preparation
[0518] Female Sprague-Dawley rats weighing 200-300 g were obtained
from Charles-River Inc. (St-Constant, Qubec, Canada). The rats were
gonadectomized under general anesthesia (Isoflurane) and killed by
cervical dislocation 24 hours later. The uteri are rapidly removed,
dissected free from adhering tissue and frozen on dry-ice. Tissues
were kept at -80.degree. C. until use.
[0519] Cytosol Preparation
[0520] All procedures were performed at 4.degree. C. Tissues were
pulverized frozen in dry ice with a Thermovac pulverizer. The
samples were then homogenized in 10 vol (w/v) of buffer A (25 mM
Tris-HCl, 1.5 mM EDTA, 10 mM .alpha.-monothioglycerol, 10%
glycerol, 10 mM sodium molybdate, pH 7.4) using a Polytron FT-10
homogenizer (Brinkmann Instruments, Canada) at a setting of 5 for
two periods of 10 sec, with intervals of 10 sec for cooling. The
homogenate was then centrifuged at 105,000.times.g for 90 min. The
supernatant was used immediately for assay.
[0521] Binding Assays
[0522] Progesterone binding was measured using the dextran-coated
charcoal adsorption technique. Incubations were performed at
0-4.degree. C. for 16-18 h using 100 .mu.l of cytosol, 100 .mu.l of
[.sup.3H]-R5020 (5 nM final, which contained 1,000 nM of
dexamethasone in order to mask the glucocorticoid receptors) and
100 .mu.l of unlabeled compounds at the indicated concentrations.
Each concentration was done in triplicate. Assay was ended with 300
.mu.l of DCC (1% Norit A and 0.1% Dextran T-70 in Buffer B). After
10 min of incubation, tubes were centrifuged at 2,000.times.g for
10 min. and decanted in vials with 6 ml of BCS liquid scintillation
(New England Nuclear, Dupont). The radioactivity was measured in a
Beckman counter at a counting efficiency of 35%.
[0523] Calculations
[0524] The results were reported as the percentage of the
inhibition of the binding of [.sup.3H]R5020 at the concentrations
of 10.sup.-8 and 10.sup.-6M of the inhibitors.
[0525] F--Glucocorticoid Receptor Assay
[0526] Chemicals
[0527] [6,7-.sup.3H(N)]-Dexamethasone (39 Ci/mmol) was purchased
from New England Nuclear (Lachine, Qubec, Canada) while unlabeled
dexametasone was obtained from Steraloids (Wilton, N.H.). All other
chemicals were of analytical grade.
[0528] Stock solutions of the unlabeled steroids were kept at
4.degree. C. in ethanol. The desired steroid solutions were then
prepared by appropriate dilution in buffer B (10 mM Tris-HCl, 1.5
mM EDTA, 10 mM .alpha.-monothioglycerol, pH 7.4) containing 30%
ethanol.
[0529] Tissue Preparation
[0530] Male Sprague-Dawley rats weighing 200-300 g were obtained
from Charles-River Inc. (St-Constant, Qubec, Canada). The rats were
killed by cervical dislocation and the liver were rapidly removed,
dissected free from adhering tissue and frozen on dry-ice. Tissues
were kept at -80.degree. C. until use.
[0531] Cytosol Preparation
[0532] All procedures were performed at 4.degree. C. Tissues were
eminced and homogenized in 10 vol (w/v) of buffer A (25 mM
Tris-HCl, 1.5 mM EDTA, 10 mM .alpha.-monothioglycerol, 10%
glycerol, 10 mM sodium molybdate, pH 7.4) using a Polytron FT-10
homogenizer (Brinkmann Instruments, Canada) at a setting of 5 for
two periods of 10 sec, with intervals of 10 sec for cooling. The
homogenate was then centrifuged at 105,000.times.g for 90 min. The
supernatant was used immediately for assay.
[0533] Binding Assays
[0534] Glucocorticoid binding was measured using the dextran-coated
charcoal adsorption technique. Incubations were performed at
0-4.degree. C. for 16-18 h using 100 .mu.l of cytosol, 100 .mu.l of
[.sup.3H]-Dexamethasone (5 nM final) and 100 .mu.l of unlabeled
compounds at the indicated concentrations. Each concentration was
done in triplicate. Assay was ended with 300 .mu.l of DCC (2.5%
Norit A and 0.25% Dextran T-70 in Buffer B). After 10 min of
incubation, tubes were centrifuged at 2,000.times.g for 10 min. and
decanted in vials with 6 ml of BCS liquid scintillation (New
England Nuclear, Dupont). The radioactivity was measured in a
Beckman counter at a counting efficiency of 35%.
[0535] Calculations
[0536] The results were reported as the percentage of the
inhibition of the binding of [.sup.3H]-dexamethasone at the
concentrations of 10.sup.-8 and 10.sup.-6M of the inhibitor.
[0537] G--Estrogen Receptor (ER) Assay
[0538] Tissue Preparation
[0539] Female Sprague-Dawley rats (Crl: CD(SD)Br) weighing 200-300
g were obtained from Charles-River Inc. (St-Constant, Qubec,
Canada). The rats were gonadectomized under general anesthesia
(Isoflurane) and killed by cervical dislocation 24 hours later. The
uteri were rapidly removed, dissected free from adhering tissue and
frozen on dry-ice. Uteri were kept at -80.degree. C. until
assay.
[0540] All subsequent steps were performed at 0-4.degree. C. Uteri
were homogenized in 10 vol (wt/vol) of buffer A (25 mM Tris-HCl,
1.5 mM EDTA disodium salt, 10 mM .alpha.-monothioglycerol, 10%
glycerol, and 10 mM sodium molybdate, pH 7.4), using a Polytron
PT-10 homogenizer (Brinkman Instruments, Canada) at a setting of 5
for three periods of 10 sec, with intervals of 10 sec for cooling.
The homogenate was then centrifuged at 105,000.times.g for 60 min
in a Beckman L5-65 ultracentrifuge (Fullerton, Calif.). The protein
concentration of the cytosol fraction was measured according to the
method of Bradford (Anal. Biochem. 72: 248-254, 1976), using bovine
serum albumin as standard.
[0541] Estrogen binding was measured using the dextran-coated
charcoal adsorption technique as described previously (Asselin et
al., Endocrinology, 101: 666-671, 1977; Asselin and Labrie, J.
Steroid Biochem., 9: 1079-1082, 1978). Briefly, [.sup.3H]E.sub.2
solubilized in ethanol were diluted into buffer A. Aliquots of
uterine cytosol preparation (0.1 ml) were incubated with 5 nM
[.sup.3H]E.sub.2 (.about.200,000 cpm, 0.1 ml) in the presence or
absence of the indicated concentrations of unlabeled compounds (0.1
ml, prepared in buffer A containing 10% ethanol) for 3 h at room
temperature. Unbound steroids were then separated by incubation for
15 min at 0-4.degree. C. with 0.3 ml 0.5% Norit-A and 0.05% Dextran
T-70 in buffer B (1.5 mM EDTA disodium salt, 10 mM
monothioglycerol, and 10 mM Tris-HCl, pH 7.4) and centrifuged at
3,000.times.g for 15 min. Aliquots of the supernatant (0.3 ml) were
removed for radioactivity measurement. After the addition of 10 ml
Formula-989 scintillation liquid (New England Nuclear-DuPont), the
radioactivity was measured in a Beckman counter at a counting
efficiency of 62%.
[0542] Calculations
[0543] The results were reported as the percentage of inhibition of
the binding of E.sub.2 at the concentrations of 10.sup.-8 and
10.sup.-6M of the inhibitor.
[0544] Primary criteria in selecting preferred inhibitors include
bioavailability, desirable inhibition of type 5
17.beta.-hydroxysteroid dehydrogenase, extent of undesirable
inhibition on type 2 17.beta.-hydroxysteroid dehydrogenase and
androgenicity. It is believed that the methyl groups in 5' position
in EM 1394 and analogous compounds promote selectivity of type 5
inhibition (versus undesirable type 2 inhibition). Of the compounds
in the foregoing tables, the most preferred and their molecular
structures are set forth below: 6465666768
Preferred Inhibitors of Type 3 17.beta.-Hydroxysteroid
Dehydrogenase
[0545] Natural androsterone provides inhibition of type 3
17.beta.-hydroxysteroid dehydrogenase with a IC.sub.50=200 nM. In
vitro testing has shown the following compounds to also be capable
of inhibiting the type 3-catalyzed conversion of 4-dione to
testosterone:
14TABLE 6 69 % of Inhibition of the Type 3 17.beta.-HSD Activity *
Name R 3.10.sup.-7 M 3.10.sup.-6 M EM-1071
--O(CH.sub.2).sub.2OC.sub.2H.sub.5 73 91 EM-1065
--S(CH.sub.2).sub.2SCH.sub.3 63 94 EM-1066 --OCH.sub.2SCH.sub.3 63
93 EM-1064 --SCH.sub.2OCH.sub.3 62 92 EM-1074
--O(CH.sub.2).sub.3CH.sub.3 62 92 EM-1073 --O(CH.sub.2).sub.2OCH.s-
ub.3 51 87 EM-1070 --OCH.sub.2OCH.sub.3 57 90 * The inhibition of
human type 3 17.beta.-hydroxysteroid dehydrogenase activity,
expressed as the percentage of inhibition of enzymatic activity at
3.10.sup.-7 M (left column) and 3.10.sup.-6 M (right column), is
reported. The manner in which the percentage of inhibition was
obtained is described in "Efficacy of types 1, 2, and 3
17.beta.-HSD and types 1 and 3 3.alpha.-HSD supra". Higher numbers
are desirable.
[0546] or
15TABLE 7 70 Inhibition of the Type 3 17.beta.-HSD Activity* Name n
IC.sub.50 (nM) CS-213 1 31 .+-. 4 EM-1324-CS 2 31 .+-. 4 *The
inhibition of human type 3 17.beta.-hydroxysteroid dehydrogenase
activity, expressed by the concentration which produce 50% of
inhibition of enzymatic activity (IC.sub.50 is nM), is reported.
The manner in which IC.sub.50 was determined is described in
"Efficacy of types 1, 2, and 3 17.beta.-HSD and types 1 and 3
3.alpha.-HSD". Lower numbers of IC.sub.50 are desirable.
EXAMPLES OF SYNTHESIS OF PREFERRED INHIBITORS
[0547] The IR spectra herein were taken on a Perkin-Elmer 1600
Series FT-IR spectrophotometer. Proton NMR spectra were recorded on
a Brucker AC-F 300 instrument. The following abbreviations have
been used: s, singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quadruplet; and m, multiplet. The chemical shifts (6)
were referenced to chloroform (7.26 ppm for .sup.1H and 77.00 ppm
for .sup.13C) and were expressed in ppm. Optical rotations were
measured at room temperature on a Jasco DIP 360 polarimeter. Mass
spectra (MS) were obtained on a V. G. Micromass 16F machine.
Thin-layer chromatography (TLC) was performed on 0.25 mm Kieselgel
60F254 plates (E. Merck, Darmstadt, FRG). For flash chromatography,
Merck-Kieselgel 60 (230400 mesh A.S.T.M.) was used. Unless
otherwise noted, starting material and reactant were obtained
commercially and were used as such or purified by standard means.
All solvents and reactants purified and dried were stored under
argon. Anhydrous reactions were performed under an inert
atmosphere, the set-up assembled and cooled under argon. Organic
solutions were dried over magnesium sulfate, evaporated on a
rotatory evaporator and under reduced pressure. Starting materials
and reagents were available from Aldrich Chemical Company, Inc.
(Milwaukee, Wis.)
[0548] List of Abbreviations
[0549] DHP 3,4-dihydro-2H-pyran
[0550] EDTA Ethylenediaminetetraacetic acid
[0551] HPLC High pressure liquid chromatography
[0552] PTSA p-toluenesulfonic acid
[0553] THF Tetrahydrofuran
[0554] THP Tetrahydropyranyl
[0555] TMS Tetramethylsilyl
Example 1
Synthesis of 6-methyl
4,6-pregnadien/1,4,6-pregnatrien-17-ol-3,20-dione
Alkanoates/Benzoates
[0556] These syntheses are described in Schemes 1 and 2. 71 72
Example 1A
[0557] 6-methyl-1,4,6-Pregnatrien-17.alpha.-ol-3,20-dione acetate
(2; EM-910):
[0558] Megestrol acetate (2.5 g; 6.7 mmol) and DDQ (5.07 g; 22
mmol) in dioxane (30 mL) were refluxed for 2 h. Solvent was removed
and the mixture in EtOAc was washed with saturated NaHCO.sub.3
solution (3 times). Solvent was dried (MgSO.sub.4) and evaporated
to give the product. Purification on silica gel column
(hexanes/acetone) gave the trienone (2.1 g) in 90% yield; IR (KBr,
cm.sup.-1) 2953, 2895, 1729, 1709, 1658, 1646, 1610, 1365, 1264,
1247, 887. .sup.1H NMR (CDCl.sub.3) .delta. 0.69 (s, 3H, H-C18),
1.13 (s, 3H, H-C19), 1.87 (s, 3H, 6-CH3), 2.0 (s, 3H) 2.02 (s, 3H),
2.89-2.98 (m, 1H), 5.79 (s, 1H), 6.22 (s, 1H), 6.20-6.24 (m, 1H,
2-H), 7.04 (d, 1H, 1-H, J=10 Hz); .sup.13C NMR (CDCl.sub.3) .delta.
203.7, 186.4, 170.6, 163.2, 153.4, 134.5, 131.8, 127.7, 121.6,
96.2, 49.1, 47.9, 47.1, 41.1, 37.7, 31.1 30.3, 26.4, 23.2, 21.5,
20.5 19.2, 14.4.
Example 1B
[0559] 6-methyl-4,6-Pregnadien-17.alpha.-ol-3,20-dione (3):
Megestrol acetate (11.2 g) was dissolved in 200 mL of boiling MeOH
and to this, was added 2N NaOH (20 mL). The mixture was refluxed
for 5 h. Approx. 100 mL of MeOH was removed, and water (50 mL) was
added and the suspension was cooled to 0.degree. C. to give a
solid, which was filtered and washed with water (9.6 g, 96%); IR
(KBr, cm.sup.-1) 3493, 2942, 2767, 1702, 1658, 1646, 1623, 1575.
.sup.1H NMR (CDCl.sub.3) .delta. 0.75 (s, 3H, H-C18), 1.06 (s, 3H,
H-C19), 1.81 (s, 3H, 6-CH.sub.3), 2.25 (s, 3H), 2.96 (s, 1H), 5.83
(s, 1H), 5.95 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 211.3,
200.0, 164.4, 138.5, 131.1, 121.0, 89.5, 50.3, 48.7, 47.9, 36.9,
36.1, 34.0 33.5, 30.2, 27.7, 23.3, 20.1, 19.8 16.3, 15.2.
Example 1C
[0560] 6-methyl-4,6Pregnadien-17.alpha.-ol-3,20-dione
aklanoates/benzoates (4): Esterification was conducted by following
the method A or B, C, D, E.
[0561] Condition A; Using Trifluoromethanesulfonic Anhydride: To a
concentrated solution of the organic acid (1.1 eq.) in
CH.sub.2Cl.sub.2 (1 mol/L), was added trifluoromethanesulfonic
anhydride (1.2 eq.) at room temperature. After 15 min, the mixed
anhydride was added to a solution of megestrol 3 in
CH.sub.2Cl.sub.2 (1 mol/L) at 0.degree. C. The mixture was stirred
for 1 h at 0.degree. C. follwed by 1 h at room temperature.
Saturated solution of NaHCO.sub.3 was added and the aq. layer was
extracted with CH.sub.2Cl.sub.2 (3 times). The combined organic
layer was dried and evaporated to give the dark brown oil, which
was purified on a silica gel column using hexanes/acetone as an
eluent to give pure product (yields range from 40 to 80%)
[0562] Condition B; Using Trifluoroacetic Anhydride: Acid (5.61
mmol) was added to trifluoroacetic anhydride (5.33 mmol) in
CH.sub.2Cl.sub.2 (5 mL) at room temperature and the solution was
stirred for 3 h. The resulting solution was transferred to compound
3 (0.56 mmol) in CH.sub.2Cl.sub.2 (5 mL) at room temperature and
the reaction was followed by TLC. The reaction mixture was diluted
with CH.sub.2Cl.sub.2 (50 mL) and the organic solution was washed
with saturated NaHCO.sub.3 solution and then with saturated NaCl
solution, dried (MgSO.sub.4). Evaporation of solvents gave the
crude product which was purified by column chromatography using
hexanes/acetone as an eluent to give the pure product.
[0563] Condition C; Using Acid chloride or Acid anhydride and
p-Toluenesulfonic acid: To a solution of megestrol 3 in
CH.sub.2Cl.sub.2 was added p-toluenesulfonic acid (1 eq.) followed
by acid chloride/acid anhydride (2 to 3 eq.). The reaction mixture
was stirred for 8 to 10 h depending upon the alkyl chains. The
mixture was poured in ice water and extracted with
CH.sub.2Cl.sub.2. The organic layer was washed with saturated
NaHCO.sub.3, dried, and solvents removed to give the product which
was purified by column chromatography using hexanes/acetone as an
eluent.
[0564] Condition D; Using Acid chloride or Acid anhydride and
phosphoric acid: To a solution of megestrol 3 in CH.sub.2Cl, was
added 85% aq phosphoric acid (1 eq.) followed by acid chloride/acid
anhydride (2 to 3 eq.). The reaction mixture was stirred for 8 to
10 h depending upon the alkyl chains. The mixture was poured in ice
water and extracted with CH.sub.2Cl.sub.2. The organic layer was
washed with saturated NaHCO.sub.3, dried, and solvents removed to
give the product which was purified by column chromatography using
hexanes/acetone as an eluent.
[0565] Condition E; Using Acid anhydride and Scandium
Trifluoromethanesulfonate: To compound 3 (1.12 mmol), was added
propionic acid (47 mmol) and scandium trifluoromethanesulfonate (1
mol %; 5 mg) under argon and left for 3 h while stirring. The
mixture was diluted with EtOAc and washed twice with saturated
NaHCO.sub.3 solution. Organic solvents were evaporated to give the
product which was purified by column chromatography using
hexanes/EtOAc as a solvent system to give pure product.
Example 1D
[0566] The followings are non-limiting examples of physicochemical
characteristics of inhibitors of Example 1.
[0567] EM-917 (4; R=n-C.sub.5H.sub.11); Yield, 74%; IR (NaCl,
cm.sup.-1) 2950, 2870, 1731, 1661, 1626, 1580, 1458, 1352, 1249,
1159, 1110; .sup.1H NMR (CDCl.sub.3) .delta. 0.69 (s, 3H, H-C18),
0.86 (t, 3H, H-C6', J=7 Hz), 1.07 (s, 3H, H-C19), 1.82 (s, 3H,
6-CH.sub.3), 2.02 (s, 3H, H-C21), 2.31 (t, 2H, H-C2', J=7 Hz),
2.44-2.56 (m, 2H), 2.92-3.01 (m, 1H), 5.85 (s, 1H), 5.94 (s, 1H);
.sup.13C NMR (CDCl.sub.3) .delta. 203.9, 199.8, 173.3, 164.0,
137.9, 131.4, 121.3, 96.2, 50.3, 49.1, 47.6, 37.1, 36.1, 34.4,
34.1, 33.6, 31.3, 31.2, 30.4, 26.4, 24.5, 23.3, 22.3, 20.3, 19.9,
16.4, 14.3, 13.9.
[0568] EM-923 (4; R=n-C.sub.4H.sub.9); Yield, 82%; IR (NaCl,
cm.sup.-1) 2951, 2872, 1731, 1660, 1626, 1580, 1458, 1387, 1351,
1263, 1160, 1110; .sup.1H NMR (CDCl.sub.3) .delta. 0.71 (s, 3H,
H-C18), 0.90 (t, 3H, H-C5', J=7 Hz), 1.08 (s, 3H, H-C19), 1.84 (s,
3H, 6-CH.sub.3), 2.03 (s, 3H, H-C21), 2.20-2.27 (m, 1H), 2.38 (t,
2H, H-C2', J=7 Hz), 2.40-2.59 (m, 2H), 2.94-3.03 (m, 1H), 5.86 (s,
1H), 5.95 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.9, 199.8,
173.3, 164.0, 137.9, 131.4, 121.2, 96.1, 50.3, 49.1, 47.5, 37.1,
36.1, 34.2, 34.1, 33.6, 31.1, 30.4, 26.8, 26.4, 23.3, 22.2, 20.3,
16.4, 14.3, 13.6.
[0569] EM-928 (4; R=CH.sub.3).sub.2); Yield, 58%; IR (NaCl,
cm.sup.-1) 2947, 2874, 1731, 1660, 1626, 1580, 1469, 1387, 1351,
1272, 1215, 1197, 1154, 1111, 1084, 1060; .sup.1H NMR (CDCl.sub.3)
.delta. 0.71 (s, 3H, H-C18), 1.08 (s, 3H, H-C19), 1.18 (t, 6H,
2'-CH.sub.3, J=7 Hz), 1.83 (s, 3H, 6-CH3), 2.01 (s, 3H, H-C21),
2.45-2.60 (m, 3H), 2.93-3.02 (m, 1H,), 5.86 (s, 1H), 5.94 (s, 1H);
.sup.13C NMR (CDCl.sub.3) .delta. 203.8, 199.8, 176.2, 164.0,
137.9, 131.4, 121.3, 96.0, 50.3, 49.2, 47.7, 37.1, 36.1, 34.2,
34.1, 33.6, 31.2, 30.4, 26.3, 23.3, 20.3, 19.9, 18.8, 18.6, 16.4,
14.4.
[0570] EM-948 (4; R=CH.sub.2Cypent); Yield, 68%; IR (NaCl,
cm.sup.-1) 2947, 2867, 1731, 1681, 1626, 1579, 1455, 1352, 1262,
1160; .sup.1H NMR (CDCl.sub.3) .delta. 0.66 (s, 3H, H-C18), 1.00
(s, 3H, H-C19), 1.79 (s, 3H, 6-C.sub.3), 1.98 (s, 3H, H-C21), 2.29
(t, 2H, H-C2', J=8 Hz), 2.38-2.54 (m, 2H), 2.89-2.97 (m, 1H), 5.84
(s, 1H), 5.92 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.9,
199.8, 173.4, 164.0, 137.9, 131.4, 121.3, 96.1, 50.3, 49.1, 47.5,
39.6, 37.1, 36.1, 34.1, 33.7, 33.6, 32.4, 32.3, 31.1, 30.8, 30.4,
26.4, 25.1, 23.3, 20.3, 19.9, 15.3, 14.3.
[0571] EM-949 (4; R=CH.sub.2Ph); Yield, 47%; IR (NaCl, cm.sup.-1)
2946, 2870, 1730, 1658, 1625, 1579, 1454, 1351, 1264, 1141; .sup.1H
NMR (CDCl.sub.3) .delta. 0.67 (s, 3H, H-C18), 1.07 (s, 3H, H-C19),
1.86 (s, 3H, 6-CH3), 1.90 (s, 3H, H-C21), 2.00-2.04 (m, 1H),
2.17-2.19 (m, 1H), 2.43-2.64 (m, 2H), 2.90-2.98 (m, 1H), 3.65 (s,
2H, H-C2'), 5.90 (s, 2H), 7.22-7.33 (m, 5H, Ar); .sup.13C NMR
(CDCl.sub.3) .delta. 203.6, 199.9, 170.8, 164.0, 137.9, 133.4,
131.4, 129.3, 128.7, 127.3, 121.3, 96.8, 50.4, 48.9, 47.7, 41.9,
37.1, 36.1, 34.2, 33.5, 31.0, 30.2, 26.2, 23.2, 20.3, 19.9, 16.4,
14.3.
[0572] EM-950 (4; R=Cypent); Yield, 49%; IR (NaCl, cm.sup.-1);
2948, 2869, 1726, 1660, 1625, 1578, 1475, 1351, 1271, 1193, 1155,
1110; .sup.1H NMR (CDCl.sub.3) .delta. 0.72 (s, 3H, H-C18), 1.09
(s, 3H, H-C19), 1.85 (s, 3H, 6-CH.sub.3), 2.03 (s, 3H, H-C21),
2.21-2.24 (m, 1H), 2.41-2.63 (m, 2H), 2.72-2.79 (m, 1H, H-C2'),
2.94-3.03 (m, 1H), 5.88 (s, 1H), 5.95 (S, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 203.9, 199.8, 175.9, 164.0, 138.0, 131.4,
121.3, 96.0, 50.3, 49.2, 47.6, 44.0, 37.1, 36.1, 34.1, 33.6, 31.2,
30.5, 29.7, 29.6, 26.3, 25.7, 25.6, 23.4, 20.3, 19.9, 16.4,
14.3.
[0573] EM-978 (4; R=Cyhex); Yield, 48%; IR (NaCl, cm.sup.-1) 2935,
2857, 1726, 1660, 1625, 1579, 1449, 1351, 1316, 1247, 1159; .sup.1H
NR (CDCl.sub.3) .delta. 0.71 (s, 3H, H-C18), 1.09 (s. 3H, H-C19),
1.85 (s, 3H, 6-CH.sub.3), 2.01 (s, 3H, H-C21), 2.24-2.37 (m, 2H),
2.42-2.64 (m, 2H), 2.94-3.03 (m, 1H), 5.88 (s, 1H), 5.95 (s, 1H);
.sup.13C NMR (CDCl.sub.3) .delta. 203.8, 199.8, 175.3, 164.0,
138.0, 131.4, 121.3, 95.9, 50.3, 49.2, 47.6, 43.2, 37.1, 36.1,
34.0, 33.6, 31.3, 30.5, 28.8, 28.7, 26.3, 25.6, 25.3, 23.4, 20.3,
19.9, 16.4, 14.4.
[0574] EM-979 (4; R=t-Bu); Yield, 82%; IR (NaCl, cm.sup.-1) 2949,
2872, 1724, 1660, 1626, 1580, 1152; .sup.1H NMR (CDCl.sub.3)
.delta. 0.71 (s, 3H, H-C18), 1.09 (s, 3H, H-C19), 1.22 (s, 9H,
2'-CH.sub.3), 1.84 (s, 3H, 6-CH.sub.3), 2.01 (s, 3H, H-C21),
2.21-2.25 (m, 1H), 2.42-2.62 (m, 2H), 2.95-3.04 (m, 1H), 5.87 (s,
1H), 5.94 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.7, 199.8,
177.5, 164.0, 137.9, 131.4, 121.3, 95.9, 50.4, 49.4, 47.8, 39.0,
37.1, 36.1, 34.0, 33.6, 31.4, 30.4, 26.9, 26.2, 23.3, 20.3, 19.9,
16.4, 14.3.
[0575] EM-996 (4; R=(CH.sub.2).sub.5Br); Yield, 43%; IR (NaCl,
cm.sup.-1) 2946, 2868, 1731, 1660, 1626, 1579, 1457, 1387, 1352,
1260, 1194, 1123, 1110; .sup.1H NMR (CDCl.sub.3) .delta. 0.70 (S,
3H, H-C18), 1.07 (s, 3H, H-C19), 1.83 (s, 3H, 6-CH.sub.3), 2.03 (s,
3H, H-C21), 2.35 (t, 2H, H-C2, p=7 Hz), 2.93-3.02 (m, 1H), 3.37 (t,
2H, H-C6', J=7 Hz), 5.85 (s, 1H), 5.94 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 203.8, 199.8, 172.9, 164.0, 137.8, 131.4,
121.2, 96.2, 50.2, 49.0, 47.5, 37.0, 36.0, 34.1, 34.0, 33.5, 33.3,
32.2, 31.1, 30.4, 27.6, 26.5, 23.9, 23.2, 20.2, 19.8, 16.3,
14.3.
[0576] EM-1003 (4; R=(CH.sub.2).sub.3Br); Yield, 13%; IR (NaCl,
cm.sup.-1) 2947, 1731, 1659, 1625, 1579, 1444, 1124; .sup.1H NMR
(CDCl.sub.3) .delta. 0.74 (s, 3H, H-C18), 1.01 (s, 3H, H-C19), 1.85
(s, 3H, 6-CH.sub.3), 2.07 (s, 3H, H-C21), 2.55 (t, 2H, H-C2', J=5
Hz), 2.96-3.05 (m, 1H), 3.47 (t, 2H, H-C6', J=7 Hz), 5.89 (s, 1H),
5.96 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.6, 199.7,
172.0, 163.9, 137.7, 131.3, 121.2, 96.4, 50.1, 48.9, 47.5, 37.0,
36.0, 33.9, 33.5, 32.5, 32.4, 31.0, 30.4, 27.2, 26.5, 23.2, 20.2,
19.8, 16.3, 14.2.
[0577] EM-1029 (4; R=(CH.sub.2).sub.4Cl); Yield, 30%; IR (NaCl,
cm.sup.-1) 2947, 2871, 1730, 1659, 1625, 1579, 1458, 1445, 1353,
1271, 1059; .sup.1H NMR (CDCl.sub.3) .delta. 0.67 (s, 3H, H-C18),
1.04 (s, 3H, H-C19), 1.79 (s, 3H, 6-CH.sub.3), 2.00 (s, 3H, H-C21),
2.16-2.23 (m, 1H), 2.34 (t, 2H, H-C2', J=7 Hz), 2.89-2.98 (m, 1H),
3.49 (t, 2H, H-C5', J=7 Hz), 5.81 (s, 1H), 5.91 (s, 1H); .sup.13C
NMR (CDCl.sub.3) .delta. 203.6, 199.6, 172.5, 163.9, 137.8, 131.2,
121.0, 96.2, 50.1, 48.9, 47.4, 44.2, 36.9, 35.9, 33.9, 33.4, 31.6,
30.9, 30.3, 26.4, 23.1, 21.9, 20.1, 19.7, 16.2, 14.1.
[0578] EM-1044 (4; R=CH.sub.2PhCl(p)); Yield, 38%; IR (KBr,
cm.sup.-1) 2948, 1734, 1656, 1625, 1578, 1492, 1459, 1352, 1262,
1154, 1089, 1017; .sup.1H NMR (CDCl.sub.3) .delta. 0.67 (s, 3H,
H-C18), 1.07 (s, 3H, H-C19), 1.86 (s, 3H, 6-CH.sub.3), 1.92 (s, 3H,
H-C21), 2.15-2.22 (m, 1H), 2.41-2.67 (m, 2H), 2.88-2.96 (m, 1H),
3.61 (s, 2H, H-C2'), 5.89 (s, 2H), 7.18 (d, 2H, J=8.3 Hz), 7.27 (d,
2H, J=8.3 Hz); .sup.13C NMR (CDCl.sub.3) .delta. 203.6, 199.9,
170.3, 164.0, 137.7, 133.4, 131.8, 131.5, 130.7, 128.8, 121.3,
96.9, 50.4, 48.9, 47.8, 41.1, 37.1, 36.1, 34.1, 33.6, 31.0, 30.3,
26.4, 23.1, 20.3, 19.9, 16.4, 14.3.
[0579] EM-1049 (4; R=CH.sub.2PhOMe(p)); Yield, 40%; IR (KBr,
cm.sup.-1) 2947, 2871, 1729, 1657, 1625, 1578, 1513, 1459, 1352,
1302, 1249, 1151, 1035; 1H N NMR (CDCl.sub.3) .delta. 0.64 (s, 3H,
H-C18), 1.05 (s, 3H, H-C19), 1.83 (s, 3H, 6-CH.sub.3), 1.87 (s, 3H,
H-C21), 1.96-2.08 (m, 1H), 2.13-2.20 (m, 1H), 2.39-2.62 (m, 2H),
2.85-2.93 (m, 1H), 3.56 (s, 2H, H-C2'), 3.72 (s, 3H, p-OCH.sub.3),
5.86 (s, 1H), 5.89 (s, 1H), 6.83 (d, 2H, J=8.5 Hz), 7.18 (d, 2H,
J=8.5 Hz); .sup.13C NMR (CDCl.sub.3) .delta. 203.8, 199.9, 171.1,
164.1, 158.9, 137.9, 131.4, 130.3, 125.4, 121.3, 114.0, 96.7, 55.3,
50.3, 48.9, 47.7, 40.9, 37.1, 36.1, 34.1, 33.6, 31.0, 30.2, 26.2,
23.2, 20.3, 19.9, 16.4, 14.3.
[0580] EM-1107 (4; R=CH.sub.2PhOCO-t-Bu(p)); Yield, 38%; IR (NaCl,
cm.sup.-1) 2972, 2872, 1731, 1659, 1625, 1579, 1508, 1263, 1202,
1118; .sup.1H NMR (CDCl.sub.3) .delta. 0.67 (s, 3H, H-C18), 1.07
(s, 3H, H-C19), 1.33 (s, 9H, tert-Butyl), 1.85 (s, 3H, 6-CH.sub.3),
1.91 (s, 3H, H-C21), 3.64 (s, 2H, H-C21), 5.88 (s, 1H), 5.93 (s,
1H), 6.97-7.0 (m, 2H), 7.25-7.28 (m, 2H); .sup.13C NMR (CDCl.sub.3)
.delta. 203.7, 200.0, 177.0, 170.6, 164.1, 150.4, 138.0, 131.4,
130.6, 130.2, 121.8, 121.3, 96.9, 50.3, 49.0, 47.7, 41.2, 39.1,
37.1, 36.1, 34.0, 33.6, 30.9, 30.2, 27.1, 26.3, 23.2, 20.2, 19.9,
16.4, 14.3.
[0581] CS-251 (4; R=(CH.sub.2).sub.3COCH.sub.3); Yield, 26%; IR
(NaCl, cm.sup.-1) 2949, 2873, 1732, 1660, 1626, 1580, 1463, 1356,
1258, 1060; .sup.1H NMR (CDCl.sub.3) .delta. 0.63 (s, 3H, H-C18),
1.00 (s, 3H, H-C19), 1.77 (s, 3H, 6-CH.sub.3), 1.96 (s, 3H, H-C6'),
2.05 (s, 3H, H-C21), 2.29 (t, 2H, J=7 Hz), 2.43 (t, 2H, J=7 Hz),
5.77 (s, 1H), 5.87 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta.
207.5, 203.6, 199.6, 172.5, 163.8, 137.7, 131.1, 120.9, 96.0, 50.0,
48.8, 47.4, 42.0, 36.8, 35.8, 33.8, 33.4, 33.1, 30.8, 30.2, 29.7,
26.3, 23.1, 20.0, 19.6, 18.4, 16.1, 14.1.
[0582] CS-256 (4; R=CH.sub.2PhF(p)); Yield, 32%; IR (KBr,
cm.sup.-1) 2948, 2872, 1734, 1657, 1625, 1578, 1510, 1458, 1352,
1264, 1224, 1152; .sup.1H NMR (CDCl.sub.3) .delta. 0.65 (s, 3H,
H-C18), 1.05 (s, 3H, H-C19),1.84 (s, 3H, 6-CH.sub.3), 1.88 (s, 3H,
H-C21),1.96-2.02 (m, 1H), 2.13-2.21 (m, 1H), 2.38-2.49 (m, 2H),
2.87-2.95 (m, 1H), 3.59 (s, 2H, H-C2'), 5.86 (s, 1H), 5.89 (s, 1H),
6.93-6.99 (m, 2H), 7.18-7.82 (m, 2H); .sup.13C NMR (CDCl.sub.3)
.delta. 203.6, 199.9, 170.6, 164.0, 163.7, 160.5, 137.8, 131.4,
130.9, 130.8, 129.12, 129.07, 121.3, 115.7, 115.4, 96.9, 50.4,
48.9, 47.7, 40.9, 37.0, 36.1, 34.1, 33.6, 31.0, 30.2, 26.3, 23.1,
20.3, 19.9, 16.4, 14.3.
Example 2
Synthesis of
1.alpha.-alkyl-6-methyl-4,6-pregnadien-17.alpha.-ol-3,20-dion- e
alkanoates/benzoates
[0583] These syntheses are described in Scheme 2
Example 2A
[0584]
1.alpha.-alkyl-6-methyl-4,6-Pregnadien-17.alpha.-ol-3,20-dione
acetate (5): The following example is a representative: To trienone
2 (500 mg; 1.33 mmol) in THF (30 mL), were added CuBr (12 mg; 0.08
mmol), trimethylaluminum (2.6 mL; 5.2 mmol) and
chlorotrimethylsilane (0.659 mL; 5.2 mmol) under argon. The
reaction mixture was stirred for 1 h. Exess of reagents was
destroyed by the slow addition of MeOH at 0.degree. C. The mixture
was extracted with CH.sub.2Cl.sub.2, dried and solvents removed.
Purication on silica gel column using hexanes/EtOAc as an eluent
gave the 1.alpha.-methyl compound, EM-952 (466 mg) in 90% yield; IR
(KBr, cm.sup.-7) 2972, 2892, 1734, 1715, 1661, 1625, 1368, 1259;
.sup.1H NMR (CDCl.sub.3) .delta. 0.66 (s, 3H, 18-CH.sub.3), 0.93
(d, 3H, 1-CH3, J=7 Hz), 1.12 (s, 3H, 19-CH.sub.3), 1.88 (s, 3H,
6-CH3), 1.99 (s, 3H), 2.05 (s, 3H), 2.78-2.97 (m, 2H), 5.81 (s,
1H), 5.89 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.6, 199.2,
170.5, 160.3, 137.8, 131.8, 120.5, 96.3, 49.0, 47.3, 446, 41.1,
39.2, 36.8, 35.6, 30.99, 30.2, 26.2, 23.2, 21.5, 19.7, 19.6, 18.9,
14.8, 14.2.
Example 2B
[0585]
1.alpha.-alkyl-6-methyl-4,6-Pregnadien-17.alpha.-ol-3,20-dione (6):
The following example is a representative: To
1.alpha.-Methyldienone 5 (517 mg; 1.3 mmol) in MeOH (30 mL), was
added aq NaOH (52 mg in 1 mL of water and the mixture was refluxed
for 1 h. The mixture was diluted with brine and extracted with
CH.sub.2Cl.sub.2 (3.times.50 mL), dried, and solvents were removed
under reduced pressure to the 17.alpha.-hydroxy compound 6 (430 mg)
in 93% yield; IR (KBr, cm.sup.-1) 3445, 2972, 2892, 1703, 1647,
1622, 1576, 1259; .sup.1H NMR (CDCl.sub.3) .delta. 0.75 (s, 3H,
H-C18), 0.91 (d, 3H, 1-CH.sub.3, J--7 Hz), 1.14 (s, 3H, H-C19),
1.81 (s, 3H, 6-CH3), 2.25 (s, 3H COCH.sub.3), 2.69-2.88 (m, 2H),
3.0 (s, 1H), 5.83 (s, 1H), 5.93 (s, 1H); .sup.13C NMR (CDCl.sub.3)
.delta. 211.3, 199.6, 160.9, 138.5, 131.8, 120.5, 89.6, 48.9, 48.2,
44.8, 42.1, 39.2, 36.8 35.7, 33.5, 30.2, 27.7, 23.3, 19.8, 19.6,
18.9, 15.3, 14.8.
Example 2C
[0586]
1.alpha.-alkyl-6-methyl-4,6-Pregnadien-17.alpha.-ol-3,20-dione
aklanoates/benzoates (7):
[0587] Esterification was conducted by following the method A or B,
C, D, E of Example 1.
Example 2D
[0588] The following is a non-limiting example of physicochemical
characteristics of inhibitors of Example 2.
[0589] EM-1022 (7; R=Cypent); Yield 54%; IR (CDCl.sub.3) 1733,
1717, 1652, 1623, 1576 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.
0.69 (s, 3H), 0.95 (d, 3H, J=7 Hz), 1.15 (s, 3H), 1.82 (s, 3H),
2.00 (s, 3H), 2.71-3.00 (m, 3H), 5.84 (s, 1H), 5.91 (s, 1H); 2 (s,
3H), .sup.13C NMR (CDCl.sub.3) .delta. 203.8, 199.3, 175.9, 160.4,
137.9, 131.9, 120.6, 95.9, 49.3, 47.6, 44.7, 43.7, 42, 39.2, 36.9,
35.7, 31.2, 30.4, 29.7, 29.5, 26.2, 25.7, 25.6, 23.3, 19.9, 19.7,
19, 14.8, 14.3.
[0590] EM-1059 (7; R=CH.sub.2Ph); Yield 74%; IR (CDCl.sub.3) 1731,
1651, 1576 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.59 (s,
3H), 0.92 (d, 3H, J=7 Hz), 1.08 (s, 3H), 1.79 (s, 3H), 1.84 (s,
3H), 2.85 (m, 2H), 3.59 (s, 2H), 5.81 (s, 2H) 7.19 (m, 5H);
.sup.13C NMR (CDCl.sub.3) .delta. 203.6, 199.3, 170.7, 160.5,
137.9, 133.4, 131.9, 129.3, 128.6, 127.2, 120.6, 96.7, 49.1, 47.8,
44.7, 42.1, 41.8, 39.2, 39.9, 35.9, 35.8, 30.8, 30.1, 26.1, 23.1,
19.9, 19.7, 19, 14.9, 14.2.
[0591] CS-209 (7; R=CH(CH.sub.3).sub.2); Yield 33%; IR (CDCl.sub.3)
1731, 1659, 1624, 1577 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.
0.72 (s, 3H), 0.98 (d, 3H, J=7 Hz), 1.18 (s, 3H), 1.18 (d, 3H, J=7
Hz), 1.20 (d, 3H, J=7 Hz), 1.85 (s, 3H), 2.03 (s, 3H), 2.56-2.66
(m, 1H), 2.85-3.05 (m, 2H), 5.88 (s, 1H,) 5.94 (s, 1H); .sup.13C
NMR (CDCl.sub.3) .delta. 203.7, 199.4, 176.3, 160.4, 137.9, 132,
120.6, 95.9, 49.3, 47.7, 44.8, 42, 39.2, 36.7, 35.7, 34, 31.2,
30.4, 26.2, 23.3, 19.9, 19.7, 19, 18.7, 18.6, 14.9, 14.3.
[0592] CS-243 (7; R=CH.sub.2CH.sub.3); Yield 67%; IR (CDCl.sub.3)
1732, 1716, 1659, 1625, 1577 cm.sup.-1; .sup.1H NMR (CDCl.sub.3)
.delta. 0.68 (s, 3H), 0.94 (d, 3H, J=7 Hz), 1.12 (t, 3H, J=5.8 Hz),
1.14 (s, 3H), 1.81 (s, 3H), 2 (s, 3H), 2.80-2.90 (m, 2H), 5.83 (s,
1H,) 5.91 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 203.8, 199.4,
173.9, 160.5, 137.9, 132, 120.7, 96, 49.1, 47.5, 44.6, 41.9, 39.1,
36.9, 35.6, 31, 30.2, 27.7, 26.2, 23.2, 19.8, 19.6, 18.9, 14.8,
14.2, 8.9.
[0593] CS-245 (7; R=(CH.sub.2).sub.2CH.sub.3); Yield 70%; IR
(CDCl.sub.3) 1731, 1715, 1660, 1625, 1577 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.67 (s, 3H), 0.91 (t, 3H, J=7 Hz), 0.94 (d,
3H, J=7 Hz), 1.14 (s, 3H), 1.81 (s, 3H), 1.99 (s, 3H), 2.29 (t, 2H,
J=7 Hz), 280-2.90 (m, 2H), 5.82 (s, 1H,) 5.9 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 203.7, 199.3, 173.1, 160.4, 137.9, 131.9,
120.6, 96, 49.1, 47.5, 44.6, 42, 39.2, 36.9, 36.2, 35.7, 31, 30.3,
262, 23.2, 19.8, 19.6, 18.9, 18.2, 14.8, 14.2, 13.6.
Example 3
[0594] 6-methyl-4,6-Pregnadien-17.alpha.-ol-3,20-dione
4-carbomethoxybutyrate (EM-1007): To a solution of megestrol 3 (400
mg; 1.17 mmol) in CH.sub.2Cl.sub.2, was added p-toluenesulfonic
acid (90 mg; 0.47 mmol).) followed by glutaryl chloride (2.1 g;
12.5 mmol) at 20.degree. C. After addition, the reaction mixture
was stirred for 7.5 h. The mixture was cooled to 0.degree. C. and
quenched with exess of MeOH. After 10 min, the mixture was poured
in brine and extraced with CH.sub.2Cl.sub.2. The organic layer was
washed with saturated NaHCO.sub.3, dried, and solvents removed to
give the product which was purified by column chromatography using
hexanes/acetone: 80/20 as an eluent to give EM-1007 (161 mg) in 30%
yield; IR (NaCl, cm.sup.-1) 2949, 2873, 1732, 1860, 1626, 1580,
1443, 1363, 1260, 1198, 1144; .sup.1H NMR (CDCl.sub.3) .delta. 0.72
(s, 3H, H-C18), 1.09 (s, 3H, H-C19), 1.85 (s, 3H, 6-CH.sub.3), 1.96
(t, 2H, H-C4', J=7 Hz), 2.06 (s, 3H, H-C21), 2.21-2.27 (m, 1H),
2.39 (t, 2H, H-C2', J=7 Hz), 2.95-3.04 (m, 1H), 3.67 (s, 3H,
5'-OCH.sub.3), 5.88 (s, 1H), 5.95 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 203.6, 199.6, 173.0, 172.3, 163.9, 137.8,
131.2, 121.0, 96.2, 51.4, 50.0, 48.8, 47.4, 36.9, 35.9, 33.8, 33.4,
33.2, 32.7, 30.9, 30.2, 26.3, 23.1, 20.1, 19.7, 16.2, 14.1.
Example 4
[0595] 6methyl-17.alpha.-pentyloxy-4,6Pregnadien-3,20-dione
(EM-1127): Powered potassium hydroxide (263 mg; 4.7 mmol) was added
to DMSO (4 mL) under argon. After 5 min, megestrol (3) (400 mg;
1.17 mmol) was added followed by 1-iodopentane (305 .mu.l; 2.3
mmol). The reactiom mixture was stirred for 1 h and water (20 mL)
was added, and extracted with CH.sub.2Cl.sub.2 (3.times.20 ml). The
organic layer was washed with brine (20 mL), dried, and solvents
removed to give the crude product which was purified on silica gel
column using hexanes/EtOAc as eluent to give pure product (33 mg)
in 7% yield: IR (NaCl, cm.sup.-1) 2932, 2868, 1706, 1662, 1625,
1579; .sup.1H NMR (CDCl.sub.3) .delta. 0.63 (s, 3H, H-C18), 0.88
(t, 3H, J=6.9 Hz), 1.06 (s, 3H, H-C19), 1.82 (s, 3H, 6-CH3), 2.11
(s, 3H, COCH.sub.3), 2.42-2.56 (m, 3H), 2.91 (app. q, 1H), 3.34
(app. q, 1H), 5.85 (s, 1H), 5.95 (s, 1H); .sup.13C NMR (CDCl.sub.3)
.delta. 211.2, 200.1, 164.5, 138.7, 131.0, 121.0, 95.9, 64.4, 50.1,
48.5, 48.1, 37.2, 36.1, 33.9, 33.6, 30.7, 29.9, 28.3, 26.5, 23.5,
23.1, 22.5, 20.4, 19.8, 16.3, 14.7, 14.0.
Example 5
16.alpha.-substituted 4,6-androstadien-3,17-dione Derivatives
[0596] These syntheses are described in Scheme 3. 73
Example 5A
[0597] General method for 16a-substituted
4,6-androstadien-3,17-dione (13) 16.alpha.-substituted
5-androsten-3,17-dione 3-ethyleneketal (11) Under argon atmosphere,
a solution of 5-androsten-3,17-dione 3-ethyleneketal 10 in
anhydrous THF (3.3% W/V) was cooled at 0.degree. C. and treated
with a 1.0 M solution of lithium bis(trimethylsilyl)amide in THF
(1.0 equiv) and HMPA (2.0 equiv). The solution was stirred 20 min
at room temperature, cooled at -78.degree. C., and treated with 1.2
equiv of alkyl iodide. The reaction mixture was allowed to reach
room temperature and stirred overnight (for R=isobutyl). For
smaller R group (e.g.: R=Pr), the reaction was allowed to reach
-40.degree. C. and stirred 3 h. The reaction was quenched with
saturated ammonium chloride and diluted with ethyl acetate. The
organic phase was washed with brine, dried over magnesium sulfate,
filtered, and evaporated. The crude mixture was purified by flash
chromatography (hexanes to hexanes-acetone 19-1) to provide
compound 11 (e.g., R=isobutyl, 8%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.88 (d, J=6.3 Hz, 3H), 0.92 (s, 3H), 0.92 (d,
J=6.1 Hz, 3H), 1.05 (s, 3H), 1.08-1.84 (m, 17H), 2.11 (m, 2H), 2.46
(m, 1H), 2.58 (m, 1H), 3.95 (m, 4H), 5.37 (m, 1H).
[0598] 16.alpha.-substituted 4-androsten-3,17-dione (12). Under
argon atmosphere, a solution of compound 11 and p-toluenesulfonic
acid (0.11 equiv) in acetone (1.0% W/V) was refluxed for 2.5 h. The
crude mixture was warmed to room temperature, diluted with
distilled water, and evaporated (acetone). The aqueous phase was
extracted with dichloromethane. The organic phase was washed two
times with saturated sodium bicarbonate and brine, dried over
magnesium sulfate, filtered, and evaporated. The crude mixture was
purified by flash chromatography (hexanes-acetone 49-1 to
hexanes-acetone 19-1) to provide compound 12 (e.g., R=isobutyl,
81%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (d, J=6.5 Hz,
3H), 0.92 (d, J=6.5 Hz, 3H), 0.96 (s, 3H), 1.21 (s, 3H), 0.98-2.02
(m, 16H), 2.38 (m, 5H), 5.75 (s, 1H).
[0599] 16.alpha.-substituted 4,6-androstadien-3,17-dione (13).
Under argon atmosphere, a solution of compound 12 and chloranil
(4.8 equiv) in anhydrous tert-butanol (2.6% W/V) was refluxed for 3
h. The reaction mixture was cooled at room temperature and the
suspension was filtered to remove excess of chloranil and then
evaporated. The crude mixture was diluted with dichloromethane and
the obtained solution was washed 3 times with distilled water, 4
times with 5% sodium hydroxide, and 4 times with distilled water.
The organic phase was dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by 2 flash
chromatographies (hexanes to hexanes-acetone 9-1; and hexanes-ethyl
acetate 97-3 to hexanes-ethyl acetate 17-3) to provide compound 13
(e.g., EM-1273C, R=isobutyl, 29%): IR (CHCl.sub.3) 3010, 2957,
2871, 1733, 1656, 1619, 1467 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.89 (d, J=6.5 Hz, 3H), 0.93 (d, J=6.4 Hz, 3H),
1.01 (s, 3H, 1.14 (s, 3H), 1.05-2.05 (m, 13H), 2.34-2.58 (m, 4H),
5.70 (s, 1H), 6.18 (m, 2H; .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 14.45, 16.32, 19.92, 21.35, 23.44, 26.57, 27.45, 31.52,
33.87, 36.13, 36.97, 40.02, 43.06, 46.56, 48.88, 50.86, 124.16,
128.76, 138.37, 162.91, 199.30, 221.43.
Example 6
16-Symmetrical-disubstituted 4,6-androstadien-3,17-dione and
4,6-estradien-3,17-dione Derivatives
[0600] 74
Example 6A
[0601] General method for 16-symmetrical-disubstituted
4,6-androstadien-3,17-dione and 4,6-estradien-3,17-dione (17)
[0602] 16-symmetrical-disubstituted 5-androsten-3,17-dione
3-ethyleneketal (15). Under argon atmosphere, a solution of
5-androsten-3,17-dione 3-ethyleneketal (14) in anhydrous THF (2.4%
W/V) was treated with sodium hydride in 60% dispersion in mineral
oil (10 equiv) and alkyl dihalide (1.5 equiv) or alkyl halide (10
equiv) and refluxed for 20 h. The reaction mixture was cooled at
room temperature and quenched with ethanol, and then the solvents
were evaporated. The crude mixture was diluted with dichloromethane
and washed with saturated ammonium chloride and brine. The organic
phase was dried over magnesium sulfate, filtered, and evaporated.
The crude compound 16 (e.g. R.sup.1=CH.sub.3,
R.sup.2=--(CH.sub.2).sub.4--) was directly used for the next step:
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (s, m, 3H), 1.05 (s,
3H), 1.0-2.2 (m, 24H), 2.58 (m, 1H), 3.95 (m, 4H), 5.37 (m, 1H).
When alkyl halide was used, compounds 15 were purified by flash
chromatography.
[0603] 16-symmetrical-disubstituted 4androsten-3,17-dione (16). The
same procedure for compound 12 was used, starting from compound 15.
The crude mixture was purified by flash chromatography
(hexanes-acetone 19-1 to hexanes-acetone 4-1) to provide compound
16 (e.g. EM-844, R.sup.1=CH.sub.3, R.sup.2=--(CH.sub.2).sub.4--,
74%). A sample was recrystallized from hexanes-acetone 4-1: IR
(KBr) 2950, 2925, 2858, 1730, 1674, 1617, 1448 cm.sup.-1; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 0.89 (s, 3H), 1.18 (s, 3H),
0.93-2.04 (m, 19H), 2.26-2.44 (m, 4H), 5.70 (s, 1H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 14.03, 17.36, 20.31, 25.37, 25.93,
30.93, 31.89, 32.58, 33.89, 34.71, 35.65, 38.18, 38.66, 39.21,
39.93, 48.00, 48.54, 53.94, 56.15, 124.06, 170.39, 199.22,
225.41.
[0604] 16-symmetrical-disubstituted 4,6-androstadien-3,17-dione
(17). A solution of compound 16 in acetic anhydride (12.4 equiv)
and pyridine (1.1 equiv) was treated with acetyl chloride (13.0
equiv), refluxed for 3 h, cooled at room temperature, and stirred
overnight. The reaction mixture was evaporated and coevaporated 2
times with ethanol and 1 time with toluene. The crude dienol
acetate was dissolved in wet DMF (97%). The solution was treated
with N-bromosuccinimide (1.03 equiv), stirred for 45 min, treated
with lithium carbonate (2.4 equiv) and lithium bromide (1.02
equiv), and heated at 100.degree. C. for 3 h. The reaction mixture
was cooled at room temperature, treated with saturated ammonium
chloride, and extracted 3 times with ethyl acetate. The combined
organic phase was dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes to hexanes-acetone 19-1) and recrystallization
(hexanes-acetone 49-1) to provide compound 17 (e.g. CS-195,
R.sup.1=CH.sub.3, R.sup.2=--(CH.sub.2).sub.4--, 61%): IR
(CHCl.sub.3) 2947, 2872, 1731, 1657, 1619 cm.sup.-1; .sup.1H RMN
(300 MHz, CDCl.sub.3) .delta. 0.98 (s, 3H), 1.14 (s, 3H), 1.2-2.05
(m, 18H), 2.35-2.65 (m, 3H), 5.70 (s, 1H), 6.17 (m, 2H); .sup.13C
RMN (75 MHz, CDCl.sub.3) .delta. 14.04, 16.33, 20.00, 25.38, 25.93,
31.86, 33.87, 36.17, 36.65, 37.73, 39.20, 39.98, 46.57, 48.84,
50.91, 56.24, 124.12, 128.70, 138.68, 163.00, 199.32, 224.69.
Example 6B
[0605] 16-symmetrical-disubstituted 1,4,6-androstatrien-3,17-dione
(18). Under argon atmosphere, a solution of compound 17 and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.7 equiv) in toluene
(4.3% W/V) was refluxed for 5 h. The reaction mixture was cooled at
room temperature, diluted with ethyl acetate, and washed with
saturated sodium carbonate and brine. The organic phase was dried
over magnesium sulfate, filtered, and evaporated. The crude mixture
was purified by flash chromatography (hexanes to hexanes-acetone
19-1) and triturated in hexanes to provide compound 18 (e.g.
EM-1039, R.sup.1=CH, R.sup.2=--(CH.sub.2).sub.4--, 52%): IR
(CHCl.sub.3) 2946, 1732, 16.52, 1603 cm.sup.-1; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 1.01 (s, 3H), 1.23 (s, 3H), 1.29-2.04 (m,
16H), 2.45 (m, 1H), 6.03 (m, 2H), 6.29 (m, 2H), 7.06 (d, J=10.1 Hz,
1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.14, 20.76, 21.22,
25.37, 25.93, 31.84, 37.20, 37.70, 39.18, 39.99, 41.18, 46.75,
48.40, 48.58, 56.22, 124.28, 128.34, 128, 49, 135.99, 152.41,
161.78, 186.13, 224.32.
Example 6C
[0606] 1.alpha.-methyl-16-symmetrical-disubstituted
4,6-androstadien-3,17-dione (19). Under argon atmosphere, a
solution of compound 18 and cuprous bromide (0.01 equiv) in
anhydrous THF (15% W/V) was treated with a 2.0 M solution of
trimethylaluminum in toluene (1.1 equiv) and stirred 1 h at room
temperature. The reaction mixture was hydrolyzed with water and the
solid filtered off and washed. The crude mixture was purified by
flash chromatography (hexanes-acetone 19-1) to provide compound 19
(e.g. EM-1077, R.sup.1=CH.sub.3, R.sup.2=--(CH.sub.2).sub.4--,
55%): IR (CHCl.sub.3) 3014, 2948, 2873, 1731, 1651, 1618, 1253
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.95 (d, J=6.8
Hz, 3H), 0.99 (s, 3H), 1.23 (s, 3H), 1.25-2.05 (m, 16H), 2.19-2.38
(m, 3H), 2.85 and 2.91 (dd, J=5.4 and 17.8 Hz, 1H), 5.71 (s, 1H),
6.17 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.10,
14.85, 19.02, 19.47, 25.40, 25.96, 31.84, 35.82, 36.63, 37.77,
39.23, 39.32, 40.02, 42.38, 45.47, 46.79, 49.00, 56.99, 123.70,
129.54, 138.41, 159.77, 198.95, 224.71.
Example 6D
[0607] 16-symmetrical-disubstituted
4-androsten-3,17-dione-6-methylene (22) A solution of
16-symmetrical-disubstituted 4-androsten-3,17-dione (16), sodium
acetate (4.4 equiv), diethoxymethane (89 equiv), and phosphorus
oxychloride (15 equiv) in chloroform (3.3% W/V) was refluxed for 1
h. The reaction mixture was cooled at rt, quenched slowly with
saturated sodium carbonate, and diluted in chloroform. The organic
phase was washed with distilled water and brine. The crude mixture
was purified by flash chromatography (hexanes-ethyl acetate 9-1 to
hexanes-ethyl acetate 4-1) and recrystallization (acetone-hexanes
1-19) to provide compound XI (e.g. EM-921, R.sup.1=CH.sub.3,
R.sup.2=--(CH.sub.2).sub.5--, 59%): IR (KBr) 2931, 2857, 1726,
1673, 1596, 1444, 1267, 1227 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.91 (s, 3H), 1.11 (s, 3H), 1.14-2.08 (m, 22H),
2.35-2.50 (m, 2H), 2.58 and 2.26 (dd, J=3.3 and 12.9 Hz, 1H), 4.98
(d, J=1.9 Hz, 1H), 3.09 (d, J=2.0 Hz, 1H), 5.92 (s, 1H); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 14.29, 17.10, 20.24, 22.40, 22.55,
25.42, 31.87, 31.92, 33.00, 33.79, 34.77, 35.10, 36.65, 39.02,
39.18, 48.25, 48.56, 50.47, 52.80, 114.44, 121.91, 145.54, 168.43,
199.61, 223.84.
Example 6E
[0608] 16-symmetrical-disubstituted
4,6-androstadien-3,17-dione-6-methyl(2- 3) A solution of
16-symmetrical-disubstituted 4-androsten-3,17-dione-6-met- hylene
(22) in benzene (0.67% W/V) was treated with acetic anhydride (15
equiv) and a solution of p-toluenesulfonic acid (5 equiv) in
benzene (8.4% W/V), and refluxed for 3.5 h. The reaction mixture
was cooled at rt, quenched with distilled water, and diluted with
benzene. The organic phase was washed 1 time with distilled water,
2 times with 5% sodium bicarbonate, and 4 times with brine. The
organic phase was dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 9-1 to hexanes-ethyl acetate 4-1) and
recrystallization (acetone-hexanes 1-19) to provide compound 23
(e.g. EM-925, R.sup.1=CH.sub.3, R.sup.2=--(CH.sub.2).sub.5--, 60%);
IR (KBr) 2926, 2858, 1726, 1662, 1629, 1582, 1444, 1268 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.97 (s, 3H), 1.11 (s,
3H), 1.20-2.62 (m, 26H), 5.89 (s, 1H), 6.06 (s, 1H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 14.31, 16.39, 19.89, 19.98, 22.46,
22.58, 25.40, 31.81, 32.01, 32.53, 33.61, 34.02, 36.22, 36.63,
46.13, 49.27, 50.59, 51.13, 121.45, 131.85, 136.56, 163.88, 199.82,
223.45.
Example 7
[0609] 16 symmetrical disubstituted 1,4androstadien-3,17-dione
(21). The same procedure for compound 18 was used, starting from
compound 16. The reflux period was 20 h instead of 5 h. The crude
mixture was purified by 2 flash chromatographies (hexanes to
hexanes-acetone 9-1) to provide compound 21 (e.g. EM 1123,
R.sup.1=CH.sub.3, R.sup.2=--(CH.sub.2).sub.4--- , 74%). A sample
was recrystallized in acetone-hexanes (1-49): IR (CHCl.sub.3) 3014,
2946, 2871, 1730, 1661, 1622, 1603 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.96 (s, 3H), 1.26 (s, 3H), 1.14-2.05 (m, 19H),
2.35-2.60 (m, 2H), 6.09 (s, 1H), 6.23 and 6.26 (dd, J=2.0 and 10.2
Hz), 7.05 (d, J=10.2 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 14.16, 18.72, 214, 25.38, 25.94, 31.81, 32.50, 32.59,
34.71, 38.37, 39.23, 39.98, 43.49, 48.16, 5245, 56.13, 124.13,
127.72, 155.34, 168.37, 186.22, 225.19.
Example 8
16-asymmetrical-disubstituted 6methyl-4,6-androstadien-3,17-dione
(27)
[0610] These syntheses are described in Scheme 5. 75
Example 8A
[0611] General Method for 16-asymmetrical-disubstituted
4,6-androstadien-3,17-dione (27)
[0612] 16-monosubstituted 5-androsten-3,17-dione 3-ethyleneketal
(24). Under argon atmosphere, a solution of 5-androsten-3,17-dione
3-ethyleneketal (10) in anhydrous Tim (4.6% W/V) was cooled at
-20.degree. C., treated with a 1.0 M solution of lithium
bis(trimethylsilyl)amide in THF (1.0 equiv), warmed to room
temperature for 15 min, cooled at -78.degree. C., and treated with
alkyl iodide (1.05 equiv). The reaction mixture was warmed to room
temperature, stirred for 2 h, quenched with saturated ammonium
chloride, and evaporated (THF). The aqueous phase was extracted
with dichloromethane. The combined organic phase was washed with
brine, dried over magnesium sulfate, filtered, and evaporated. The
crude mixture was purified by flash chromatography (hexanes-acetone
99-1 to hexanes-acetone 19-1) to provide compound 24 (e.g.
R.sup.1=CH.sub.3, 7:3 16.alpha./.beta. ratio, 69%): .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.84 (s, 0.9H), 0.91 (s, 2.1H), 1.04
(s, 3H), 1.09 (d, J=6.5 Hz, 2.1H), 1.20 (d, J-7.0 Hz, 0.9H),
1.25-1.86 (m, 13H), 2.05-2.16 (m, 3H), 2.56 (m, 2H), 3.95 (m, 4H),
5.36 (m, 1H).
[0613] 16-asymmetrical-disubstituted 5-androsten-3,17-dione
3-ethyleneketal (25). Under argon atmosphere, a solution of
compound 24 in anhydrous THF (4.0% W/V) was cooled at -40.degree.
C., treated with 0.5 M solution of potassium
bis(trimethylsilyl)amide in toluene (1.2 equiv), warmed to room
temperature for 15 min, cooled at -78.degree. C., and treated with
1.5 equiv of alkyl iodide. The reaction mixture was allowed to
reach room temperature, refluxed for 2 h, and cooled at room
temperature. The reaction mixture was quenched with saturated
ammonium chloride, evaporated (THF), and extracted with
dichloromethane. The combined organic phase was washed with brine,
dried over magnesium sulfate, filtered, and evaporated. The crude
mixture was purified by flash chromatography (hexanes-acetone 99-1
to hexanes-acetone 19-1) to provide compound 25 (e.g.
R.sup.1=CH.sub.3, R.sup.2=isobutyl, 56%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.85 (d, J=6.6 Hz, 3H), 0.90 (d, J=6.6 Hz, 3H),
0.93 (s, 3H), 1.05 (s, 3H), 1.15 (s, 3H), 1.27-2.16 (m, 19H), 2.56
(m, 1H), 3.95 (m, 4H), 5.37 (m, 1H).
[0614] 16-asymmetrical-disubstituted 4androsten-3,17-dione (26).
The same procedure for compound 12 was used, starting from compound
25. The crude mixture was purified by flash chromatography
(hexanes-acetone 49-1 to hexanes-acetone 19-1) to provide compound
26 (e.g. EM-1133, R.sup.1=CH.sub.3, R.sup.2=isobutyl, 96%): IR
(KBr) 2951, 2869, 1734, 1676, 1616, 1454 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.86 (d, J=6.6 Hz, 3H), 0.90 (d,
J=6.6 Hz, 3H), 0.96 (s, 3F), 1.17 (s, 3H), 1.21 (s, 3H), 1.02-2.07
(m, 16H), 2.31-2.49 (m, 4H), 5.75 (s, 1H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 15.06, 17.30, 20.13, 24.64, 24.88, 25.15,
25.96, 30.85, 32.05, 32.55, 33.89, 34.65, 35.12, 35.61, 38.66,
46.27, 47.74, 48.21, 48.90, 54.02, 124.06, 170.40, 199.31,
224.70.
[0615] 16-asymmetrical-disubstituted 4,6-androstadien-3,17-dione
(27). The same procedure for compound 13 was used, starting from
compound 26. The crude mixture was purified by flash chromatography
(hexanes-acetone 49-1 to hexanes-acetone 19-1) to provide compound
27 (e.g. EM-1134, R.sup.1=CH.sub.3, R.sup.2=isobutyl, 57%): IR
(KBr) 2954, 2870, 1735, 1665, 1618, 1584, 1466 cm.sup.-1; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 0.88 (d, J=6.7 Hz, 3H), 0.91 (d,
J=6.6 Hz, 3H), 1.01 (s, 3H), 1.14 (s, 3H), 1.20 (s, 3H), 1.22-2.65
(m, 16H), 5.71 (s, 1H), 6.18 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 15.10, 16.29, 19.82, 24.56, 24.89, 25.15,
25.97, 31.99, 33.78, 33.87, 34.80, 36.15, 36.59, 45.77, 46.27,
48.98, 50.92, 124.07, 128.77, 138.70, 163.05, 199.34, 224.32.
Example 8B
[0616] 16.alpha.-alkylhalide-16.beta.-alkyl
4,6-androstadien-3,17-dione (27b). Under argon atmosphere, a
solution of 16.alpha.-hydroxyalkyl-16.be- ta.-alkyl
4,6-androstadien-3,17-dione (27a) and triphenylphosphine (1.2
equiv) in dichloromethane (10% W/V) was cooled at 0.degree. C. and
treated with N-chlorosuccinimide (or N-bromosuccinimide) (1.2
equiv). The reaction mixture was warmed to room temperature for 30
min and diluted with dichloromethane. The organic phase was washed
with brine, dried over magnesium sulfate, filtered, and evaporated.
The crude mixture was purified by 2 flash chromatographies
(hexanes-ethyl acetate 9-1 to hexanes-ethyl acetate 7-3; and
dichloromethane to dichloromethane-ethyl acetate 9-1) to provide
compound 27b (e.g. EM-1135, R.sup.1=CH.sup.3,
R.sup.2=(CH.sub.2).sub.3Cl, 51%): IR (CHCl.sub.3) 3010, 2946, 2873,
1733, 1657, 1619 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.02 (s, 3H), 1.14 (s, 3H), 1.21 (s, 3H), 1.26-2.11 (m,
14H), 2.35-2.65 (m, 3H), 3.49 (m, 2H), 5.71 (s, 1H), 6.17 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.91, 16.32, 19.80,
25.16, 27.97, 31.78, 33.80, 33.89, 34.83, 35.25, 36.16, 36.60,
45.19, 46.01, 48.10, 49.36, 50.89, 124.21, 128.89, 138.43, 162.92,
199.40, 223.36.
Example 9
17.alpha.-acyloxy-6-methyl-4,6-androstadien-3-one-17.beta.-carboxamide
(34)
[0617] These syntheses are described in Scheme 6. 7677
Example 9A
[0618] General method for
17.alpha.-acyloxy-6-methyl-4,6-androstadien-3-on-
e-17.beta.-carboxamide (34)
[0619] 4androsten-17.alpha.-ol-3-one-17.beta.-carboxylic acid
methyl ester (29). A solution of 4pregnen-17,21-diol-3,20-dione
(28) (6.93 g, 0.0200 mol) in methanol (500 mL) was treated with a
0.075 M periodic acid (300 mL, 0.0225 mol) and 2.5 M sulfuric acid
(70 mL, 0.175 mol) and stirred 3 h. The reaction mixture was
evaporated (methanol) and filtered. The crude mixture was dried to
afford 4androsten-17.alpha.-ol-3-one-17.beta.-carbox- ylic acid
(6.50 g). A solution of the carboxylic acid (4.56 g, 0.0137 mol)
and 6 drops of concentrated sulfuric acid in methanol (250 mL) was
refluxed for 30 h. The reaction mixture was cooled to room
temperature, evaporated, diluted with ethyl acetate,
dichloromethane, and water, and treated with sodium bicarbonate.
The mixture was extracted 3 times with ethyl acetate. The combined
organic phase was washed with brine, dried over magnesium sulfate,
filtered, and evaporated. The crude mixture was purified by flash
chromatography (hexanes to hexanes-ethyl acetate 3-2) to provide
the methyl ester 29 (4.12 g, 87%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.72 (s, 3H), 1.18 (s, 3H), 0.94-1.89 (m, 13H),
2.01 (m, 1H), 2.36 (m, 4H), 2.65 (m, 1H), 2.85 (s, 1H), 3.75 (s,
3H), 5.72 (s, 1H).
Example 9B
[0620]
6-methylene-4-androsten-17.alpha.-ol-3-one-17.beta.-carboxylic acid
methyl ester (30). A suspension of sodium acetate (6.65 g, 0.081
mol), diethoxymethane (201 mL, 1.62 mol) and phosphorus oxychloride
(25 mL, 0.27 mol) in chloroform (250 mL) was refluxed for 1 h,
treated with a solution of compound 29 (6.02 g, 0.018 mol) in
chloroform (50 mL), and refluxed for 2 h. The reaction mixture was
cooled at room temperature, diluted with water, and extracted with
dichloromethane. The combined organic phase was stirred in the
presence of sodium carbonate for 1 h, decanted, washed with water
and brine, dried over magnesium sulfate, filtered, and evaporated.
The crude mixture was purified by flash chromatography (hexanes to
hexanes-ethyl acetate 1-1) to provide the methyl ester 30 (3.60 g,
56%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.71 (s, 3H), 1.08
(s, 3H), 1.13-2.08 (m, 13H), 2.45 (m, 3H), 2.67 (m, 1H), 2.85 (m,
1H), 3.76 and 3.77 (2s, 2.2 and 0.8H), 4.93 (d, J=1.9 Hz, 1H), 5.05
(d, J=1.6 Hz, 1H), 5.90 (s, 1H).
Example 9C
[0621]
17.alpha.-acetoxy-6-methyl-4,6-androstadien-3-one-17.beta.-carboxyl-
ic acid methyl ester (31). The same procedure for compound 23 was
used, starting from compound 30. The crude mixture was purified by
flash chromatography (hexanes to hexanes-ethyl acetate 7-3) to
provide compound 31 (EM-1010, 75%): IR (KBr) 2967, 2871, 1742,
1668, 1628, 1584, 1458, 1366, 1260, 1245 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.77 (s, 3H), 1.09 (s, 3H), 1.23-2.02
(m, 11H), 1.84 (broad s, 3H), 2.04 (s, 3H), 2.24 (m, 1H), 2.42-2.65
(m, 2H), 2.98 (m, 1H), 3.71 (s, 3H), 5.87 (s, 1H), 5.96 (s, 1H);
.sup.13C NMR (75 MHz, CDCl.sub.3) 14.43, 16.38, 19.86, 20.15,
21.05, 23.36, 30.35, 31.90, 33.61, 34.08, 36.12, 37.23, 48.01,
48.40, 50.36, 52.08, 90.58, 121.22, 131.37, 138.02, 164.12, 170.25,
170.41, 199.88.
Example 9D
[0622]
6-methyl-4,6-androstadien-17.alpha.-ol-3-one-17.beta.-carboxylic
acid (32). A solution of compound 31 (3.00 g, 7.50 mmol) in
methanol (225 mL) and 10% potassium carbonate (62 mL, 0.045 mol)
was refluxed for 21 h. The crude mixture was cooled at room
temperature, evaporated (methanol), acidified with 10% hydrochloric
acid, and diluted with water. The precipitate was filtered, washed
with water, and dried. The filtrate was extracted with
dichloromethane. The organic phase was washed 3 times with brine,
dried over magnesium sulfate, filtered, and evaporated. A solution
of the combined solids in methanol-dichloromethane 1-1 (300 mL) was
treated with 10% hydrochloric acid (60 mL), stirred for 3 h, and
evaporated (methanol and dichloromethane). The aqueous phase was
extracted 3 times with dichloromethane. The combined organic phase
was washed 4 times with brine, dried over magnesium sulfate,
filtered, and evaporated to provide carboxylic acid 32 (2.05 g,
80%): .sup.1H NMR (CDCl.sub.3, CD.sub.3OD) .delta. 0.68 (s, 3F),
0.95 (s, 3H), 1.05-1.88 (m, 13H), 1.68 (s, 3H), 2.09 (m, 1H), 2.55
(m, 1H), 5.69 (s, 1H), 5.89 (s, 1H).
Example 9E
[0623]
6-methyl-4,6-androstadien-17.alpha.-ol-3-one-17.beta.-carboxamide
(33). A solution of compound 32 and 1,3-bis(trimethylsilyl)urea
(1.5 equiv) in dichloromethane-pyridine 8-1 (5.7% W/V) was refluxed
for 21 h. The reaction mixture was cooled at room temperature,
filtered, evaporated (dichloromethane), dissolved in
dichloromethane, filtered, and evaporated. A solution of the
silylated .alpha.-hydroxycarboxylic acid in
chloroform-dimethylformamide 99-1 (5.1% W/V) was cooled at
0.degree. C., treated with oxalyl chloride (1.2 equiv), stirred for
1 h, and warmed to room temperature for 30 min. The reaction
mixture was cooled at 0.degree. C., treated with pyridine (4
equiv), and amine (3 equiv), stirred for 1 h, and warmed to room
temperature for 3 h. The reaction mixture was diluted with water
and extracted with ethyl acetate. The combined organic phase was
washed with 10% hydrochloric acid and brine, dried over magnesium
sulfate, filtered, and evaporated. A solution of the crude mixture
in methanol-acetone 1-1 (10 mL) was treated with 10% hydrochloric
acid (2 mL) and stirred for 3 h. The reaction mixture was
evaporated and extracted 3 times with ethyl acetate. The combined,
organic phase was washed 2 times with brine, dried over magnesium
sulfate, filtered, and evaporated. The crude mixture was purified
by flash chromatography (hexanes to hexanes-ethyl acetate 2-1) to
provide compound 33 (e.g. R.sup.1=Me, R.sup.2=Et, 41%): .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.83 (broad s, 3H), 1.09 (s, 3H),
1.13 (t, J=7.0 Hz, 3H), 1.22-2.23 (m, 12H), 1.84 (s, 3H), 2.45-2.63
(m, 3H), 3.08 (m, 5H), 3.80 (m, 1H), 5.86 (s, 1H), 5.98 (s,
1H).
Example 9F
[0624]
17.alpha.-acyloxy-6-methyl-4,6-androstadien-3-one-17.beta.-carboxam-
ide (34). A solution of compound 33 and p-toluenesulfonic acid
(0.14 equiv) in acid chloride (4.7% W/V) was stirred 10 h at room
temperature. The reaction mixture was quenched with methanol and
water, stirred for 30 min, and extracted with ethyl acetate. The
combined organic phase was washed with saturated sodium bicarbonate
and brine, dried over magnesium sulfate, filtered, and evaporated.
The crude mixture was purified by 2 flash chromatographies (hexanes
to hexanes-ethyl acetate 3-2 and dichloromethane to
dichloromethane-acetone 49-1) to provide compound 34 (e.g.
EM-1181-CS, R.sup.1=Me, R.sup.2=Et, R.sup.3=(CH.sub.2).sub.3Cl,
50%): IR (KBr) 2944, 2871, 1738, 1659, 1640, 1631, 1580 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.84 (s, 3H), 1.10 (m,
6H), 1.25-2.25 (m, 14H), 1.85 (s, 3H), 2.40-2.63 (m, 4H), 2.89 and
2.99 (2s, 0.7 and 2.3H), 3.43 (m, 3H), 3.62 (t, J=6.1 Hz, 2H), 5.88
(s, 1H), 5.98 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) 611.49,
14.70, 16.32, 19.85, 20.54, 22.91, 27.12, 30.99, 32.93, 33.57,
33.99, 34.10, 35.44, 36.02, 37.28, 43.87, 45.18, 48.51, 48.91,
50.04, 94.76, 121.15, 131.28, 138.10, 164.07, 167.31, 170.78,
199.87.
Example 10
17.beta.-acyl-17.alpha.-acyloxy-6-methyl-4,6-androstadien-3-one
[0625] These syntheses are described in Scheme 7. 7879
[0626] General method for 17.beta.-acyl-17.alpha.-acyloxy-6-methyl
4,6-androstadien-3-one (40)
Example 10A
[0627] 5-androsten-17.alpha.-ol-3-one-17.beta.-carboxylic acid
3-ethyleneketal methyl ester (35). A solution of
4androsten-17.alpha.-ol-- 3-one-17.beta.-carboxylic acid methyl
ester (29) (5.16 g, 0.0154 mol) and ethylene glycol (2.87 g, 0.0463
mol) in benzene (500 mL) was refluxed with a Dean-Stark apparatus
for 1 h, treated with p-toluenesulfonic acid (100 mg, 0.53 mmol),
and refluxed for 6.5 h. The reaction mixture was cooled to room
temperature, diluted with ethyl acetate, washed with saturated
sodium bicarbonate, and 2 times with water, dried over magnesium
sulfate, filtered, and evaporated. The crude mixture was purified
by flash chromatography (hexanes to hexanes-ethyl acetate 7-3) to
provide the ketal 35 (5.12 g, 85%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.70 (s, 3H), 1.03 (s, 3H), 1.08-1.82 (m, 15H),
2.01 (m, 1H), 2.10 and 2.15 (dd, J=2.6 and 14.2 Hz, 1H), 2.61 (m,
2H), 2.78 (s, 1H), 3.75 (s, 3H), 3.94 (m, 4H), 5.35 (m, 1H).
Example 10B
[0628]
17.alpha.-(2'-tetrahydro-2'H-pyranyloxy)-5-androsten-3-one-17.beta.-
-carboxylic acid 3-ethyleneketal methyl ester (36). A solution of
compound 35 (4.94 g, 0.0126 mol) in 3,4dihydro-2H-pyran (100 mL)
was cooled at 0.degree. C., treated with p-toluenesulfonic acid
(300 mg, 1.6 mmol), and stirred for 5 h. The reaction mixture was
diluted with ethyl acetate, washed with saturated sodium
bicarbonate and 2 times with water, dried over magnesium sulfate,
filtered, and evaporated. The crude mixture was purified by flash
chromatography (hexanes to hexanes-ethyl acetate 21-4) to
quantitatively provide the diketal 36 (6.10 g): .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.66 (s, 1.1H), 0.67 (s, 1.9H), 1.01 (s
3H), 1.09-2.15 (m, 22H), 2.47 (m, 3H), 3.37 (m, 0.7H), 3.51 (m,
1.3H), 3.65 (s, 1.7H), 3.69 (s, 1.3H), 3.93 (m, 4), 4.56 (m, 0.6H),
4.77 (m, 0.4H), 5.34 (m, 1H).
Example 10C
[0629]
17.alpha.-(2'-tetrahydro-2'H-pyranyloxy)-5-androsten-3-one-17.beta.-
-carboxylic acid 3-ethyleneketal (37). Compound 36 (3.73 g, 0.00786
mol) was treated with 1 N potassium tert-butoxide in dimethyl
sulfoxide (118 mL). The reaction mixture was stirred 4 h at room
temperature, diluted with water, acidified to pH 2, and extracted 3
times with ethyl acetate. The combined organic phase was washed
with brine and water, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes to ethyl acetate) to provide the carboxylic acid 37 (2.50
g, 69%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.76 (s, 2.0H),
0.80 (s, 1.0H), 0.82-2.15 (m, 22H), 1.03 (s, 3H), 2.38-2.58 (m,
3H), 3.4-3.6 (2m, 2H), 3.95 (m, 4H), 4.68 (m, 1H) 5.35 (m, 1H).
Example 10D
[0630] 17.beta.-acyl-4-androsten-17.alpha.-ol-3-one (38). Under
argon atmosphere, a solution of compound 37in dry benzene (3.0%
W/V) was cooled at 0.degree. C., treated with an organolithium
solution (7.0 equiv), and stirred 20 h at room temperature. The
reaction mixture was cooled to 0.degree. C., quenched with
saturated ammonium chloride, diluted with water, and extracted 3
times with ethyl acetate. The combined organic phase was washed
with brine, dried over magnesium sulfate, filtered, and evaporated.
The crude mixture was dissolved in methanol-acetone 1-1 (2% W/V),
treated with 2.5 M hydrochloric acid, and refluxed for 1 h. The
reaction mixture was cooled at room temperature, evaporated
(solvents), diluted in water, and extracted 3 times with ethyl
acetate. The combined organic phase was washed with brine and
water, dried over magnesium sulfate, filtered, and evaporated. The
crude mixture was purified by flash chromatography (hexanes to
hexanes-ethyl acetate 3-1) to provide compound 38 (e.g. R.sup.1=Bn,
42%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.74 (s, 3H),
0.96-1.88 (m, 13H), 1.17 (s, 3H), 2.02 (m, 1H), 2.35 (m, 4H), 2.76
(m, 2H), 3.77 (d, J=16.5 Hz, 1H), 4.07 (d, J=16.4 Hz, 1H), 5.72 (s,
1H), 7.16-7.33 (m, 5H).
Example 10E
[0631] 17.beta.-acyl-6-methylene-4-androsten-17.alpha.-ol-3-one
(39). A solution of compound 38, paraformaldehyde (5.4 equiv), and
N-methylanilinium trifluoacetate (4.8 equiv) in THF (2.9% W/V) was
refluxed for 2.5 h. The reaction mixture was cooled at room
temperature, treated with 2.5 M hydrochloric acid, stirred for 15
min, and extracted 3 times with ethyl acetate. The combined organic
phase was washed with water, saturated sodium bicarbonate, and
brine, dried over magnesium sulfate, filtered, and evaporated. The
crude mixture was purified by flash chromatography (hexanes to
hexanes-ethyl acetate 4-1) to provide compound 39 (e.g. R.sup.1=Bn,
33%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.75 (s, 3H), 1.09
(s, 3H), 1.15-2.08 (m, 13H), 2.45 (m, 3H), 2.78 (m, 2H), 3.78 (d,
J=16.3 Hz, 1H), 4.07 (d, J=16.3 Hz, 1H), 4.94 (s, 1H), 5.06 (s,
1H), 5.91 (s, 1H), 7.17-7.34 (m, 5H).
Example 10F
[0632]
17.beta.-acyl-17.alpha.-acyloxy-6methyl-4,6-androstadien-3-one
(40). The same procedure for compound 23 was used, starting from
compound 39. The crude mixture was purified by 2 flash
chromatographies (dichloromethane to dichloromethane-acetone 99-1
and hexanes to hexanes-ethyl acetate 17-3) to provide compound 40
(e.g. EM-1106, R.sup.1=Bn, R.sup.2.dbd.CH, 45%): IR (KBr) 2943,
2857, 1736, 1718, 1656, 1628, 1579, 1266, 1236; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.72 (s, 3H), 1.09 (s, 3H), 1.26-2.25 (m,
12H), 1.86 (s, 3H), 2.14 (s, 3H), 2.43-2.64 (m, 2H), 3.06 (m, 1H),
3.68 (m, 2H), 5.89 (s, 1H), 5.97 (s, 1H), 7.16-7.33 (m, 5H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.52, 16.39, 19.94,
20.35, 21.35, 23.30, 30.76, 31.21, 33.64, 34.07, 36.12, 37.11,
45.02, 47.91, 49.06, 50.23, 96.42, 121.29, 126.85, 128.32, 129.94,
131.49, 133.88, 137.93, 164.08, 170.76, 199.97, 203.41.
Example 11
17.alpha.acyloxy-6-methyl-4,6-androstadien-3-one-17.beta.-carboxyester
[0633] These syntheses are described in Scheme 8. 80
Example 11A
[0634] 4-androsten-17.alpha.-ol-3-one-17.beta.-carboxyester (41).
The same procedure for compound 29 was used and provide compound 41
(e.g., compound 41, R.sup.1=CH.sub.3).
Example 11B
[0635]
6-methylene-4-androsten-17.alpha.-ol-3-one-17.beta.-carboxester
(42). The same procedure for compound 30 was used and provide
compound 42 (e.g. compound 42, R.sup.1=CH.sub.3).
Example 11C
[0636]
17.alpha.-acyl-6-methyl-4,6-androstadien-17.alpha.-ol-3-one-17.beta-
.-carboxester (43). The same procedure for compound 31 was used and
provide compound 43 (e.g. compound 43,
R.sup.1,R.sup.2=CH.sub.3).
Example 12
15.alpha.-substituted 4,6-androstadien-3,17-dione derivatives
[0637] These synthesis are described in Scheme 9. 81
Example 12A
[0638] 5,15-androstadien-3,17-dione 3-ethyleneketal (44). Under
argon atmosphere, a solution of 5-androsten-3,17-dione
3-ethyleneketal 10 (4.95 g, 0.0150 mol) in anhydrous THF (150 mL)
was cooled at 0.degree. C. and treated with 1.0 M solution of
lithium bis(trimethylsilyl)amide in THF (18 mL, 0.018 mol). The
solution was stirred 20 min at room temperature, cooled at
-78.degree. C., and treated with chlorotrimethylsilane (2.24 mL,
0.0176 mol). The reaction was allowed to warm to room temperature,
then evaporated. The residue was diluted with dichloromethane,
washed two times with water and brine, dried over magnesium
sulfate, filtered, and evaporated. A solution of the crude
silylenol ether in a dichloromethane-acetonitrile 2-5 (140 mL) was
treated with palladium acetate (4.04 g, 0.0180 mol) and refluxed
for 30 min. The reaction mixture was cooled to room temperature and
evaporated. The black residue was purified by flash chromatography
(hexanes-ethyl acetate 4-1) to provide the enone 44 (3.49 g, 71%):
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.085 (s, 3H), 1.09 (s,
3H), 1.21-1.93 (m, 11H), 2.15 (dd, J=2.7 and J=14.1 Hz, 1H),
2.26-2.34 (m, 2H), 2.38 (d, J=14.1 Hz, 1H), 3.95 (m, 4H), 5.40 (d,
J=5.1 Hz, 1H), 6.04 (dd, J=3.2 and J=6 Hz, 1H), 7.50 (d, J=5.8 Hz,
1H).
Example 12B
[0639] 17.alpha.-substituted allyl
5,15-androstadien-17.beta.-ol-3-one 3-ethyleneketal 1 (45). Under
argon atmosphere, a solution of compound 44 in anhydrous THF (10%
W/V) was cooled at 0.degree. C., treated with a solution of
substituted allylmagnesium bromide or chloride in THF or ether (3
equiv) and stirred 3 h. The reaction mixture was quenched with
saturated ammonium chloride and diluted with ethyl acetate. The
organic phase was washed with brine, dried over magnesium sulfate,
filtered, and evaporated. The crude residue was purified by flash
chromatography (hexanes-ethyl acetate 9-1 to hexanes-ethyl acetate
4-1) to provide compound 45 (e.g., R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.5=H, 70%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
0.92 (s, 3H), 1.06 (s, 3H), 1.11-1.81 (m, 11H), 1.95 (d, J=9.3 Hz,
1H), 2.10-2.21 (m, 3H), 2.30-2.36 (m, 1H), 2.57 (d, J=5 Hz, 1H),
3.95 (m, 4H), 5.10-5.16 (m, 2H), 5.36 (broad d, J=5 Hz, 1H), 5.61
(dd, J=3.1 and 5.7 Hz, 1H), 5.83 (d, J=5.8 Hz, 1H), 5.84-5.94 (m,
1H).
Example 12C
[0640] 15.alpha.-substituted allyl-5-androsten-3,17-dione
3-ethyleneketal (46). Under argon atmosphere, a solution of
compound 45 in dry THF (10%) W/V) was cooled at 0.degree. C.,
treated with potassium hydride in 35% dispersion in mineral oil (2
equiv), and stirred 30 min. Then, the resulting suspension was
treated with 18-crown-6 ether (1.5 equiv), allowed to reach room
temperature, and stirred 2 h. The black mixture was quenched with
saturated ammonium chloride then diluted with ethyl acetate. The
organic phase was washed with brine, dried over magnesium sulfate,
filtered, and concentrated. The oily residue was purified by flash
chromatography (hexanes-ethyl acetate 4-1) to provide compound 46
(e.g., R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5=H, 71%): .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 0.94 (s, 3H), 1.06 (s, 3H),
1.1-1.5 (m, 5H), 1.5-2.0 (m, 11H), 2.14 (d, J=14.1 Hz, 1H),
2.19-2.29 (m, 2H), 2.62 (dd, J=8.3 and 19.3 Hz, 2H), 3.95 (m, 4H),
4.66 (d, J=16 Hz, 2H), 5.35 (s, 2H).
Example 12D
[0641] 15.alpha.-substituted allyl 4-androsten-3,17-dione (47). The
same procedure for compound 12 was used, starting from compound 46.
The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 9-1 to hexanes-ethyl acetate 4-1) to provide
compound 47 (e.g., EM-1284, R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5=H, 85%): IR (CHCl.sub.3) 3020, 2954, 1733, 1664, 1617,
1275, 1230 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
0.97 (s, 3H), 1.21 (s, 3H), 0.99-1.60 (m, 4H), 1.60-2.05 (m, 10H),
2.05-2.50 (m, 4H), 2.65 (dd, J=8.7 and 16.2 Hz, 2H), 5.03 (m, 2H),
5.67-5.73 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 15.30,
17.62, 20.26, 31.09, 32.40, 32.57, 33.91, 35.79, 35.88, 36.59,
38.73, 40.17, 42.42, 49.94, 53.91, 54.36, 116.78, 123.72, 135.93,
169.93, 199.15, 219.08.
Example 13E
[0642] 15.alpha.-substituted allyl 4,6-androstadien-3,17-dione
(48). The same procedure for compound 13 was used, starting from
compound 47. The crude residue was purified by 2 flash
chromatographies (hexanes-acetone 9-1 and hexanes-ethyl acetate
17-3) to provide compound 48 (e.g., EM-1261, R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5=H, 61%): IR (CHCl.sub.3) 3013, 2943,
2865, 1734, 1655, 1619, 129 cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. 1.02 (s, 3H), 1.12 (s, 3H), 1.30-1.55 (m, 4H),
1.64-2.20 (m, 6H), 2.30-2.80 (m, 6H), 5.03-5.08 (m, 2H), 5.65-5.85
(m, 2H), 6.17 (dd, J=2.7 and 10.0 Hz, 1H), 6.42 (dd, J=1.8 and 10.0
Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 15.41, 16.31,
19.79, 31.12, 33.85, 33.95, 35.29, 36.03, 38.40, 39.96, 42.25,
50.57, 52.61, 117.31, 123.97, 128.29, 135.19, 139.01, 162.35,
199.12, 218.20.
Example 13
(E)-16monosubstituted methylene 4,6-androstadien-3,17-dione
[0643] These syntheses are described in Scheme 10. 82
Example 13A
[0644] 16-monosubstituted hydroxymethyl 5-androsten-3,17-dione
3-ethyleneketal (49). Under argon atmosphere, a solution of
5-androsten-3,17-dione 3-ethyleneketal (10) in anhydrous THF (3.3%
W/V) was cooled at 0.degree. C. and treated with a 1.0 M solution
of lithium bis(trimethylsilyl)amide in THF (1.0 equiv) and HMPA
(2.0 equiv). The solution was stirred 20 min at room temperature,
cooled at -78.degree. C., and treated with 1.2 equiv of aldehyde.
The reaction mixture was allowed to reach room temperature and
stirred over 3 h. The reaction was quenched with saturated ammonium
chloride and diluted with ethyl acetate. The organic phase was
washed with brine, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was used to carry out the next step
without further purification (e.g., R.sup.2=isopropyl, quantitative
yield): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.89-0.99 (m,
9H), 1.01-2.00 (m, 14H), 2.2-2.6 (m, 6H), 3.5-4.0 (m, 5H), 5.72 (m,
2H).
Example 13B
[0645] (E)-16-monosubstituted methylene 4androsten-3,17-dione (50).
The same procedure for compound 12 was used, starting from compound
49. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 97-3 to hexanes-ethyl acetate 17-3) to
provide compound 50 (e.g., R.sup.2=isopropyl, 19%): .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 0.88 (s, 3H), 0.99 (d, J=6.6 Hz, 3H),
1.00 (d, J=6.6 Hz, 3H), 1.18 (s, 3H), 1.1-1.5 (m, 5H), 1.6-1.8 (m,
3H), 1.8-2.1 (m, 4H), 2.2-2.6 (m, 6H), 5.70 (s, 1H), 6.38 (m,
1H).
Example 13C
[0646] (E)-16-monosubstituted methylene 4,6androstadien-3,17-dione
(51). The same procedure for compound 13 was used, starting from
compound 50. The crude mixture was purified by 2 flash
chromatographies (hexanes-ethyl acetate 19-1 to hexanes-ethyl
acetate 7-3) to provide compound 51 (e.g., EM-1353,
R.sup.2=isopropyl, 60%): IR (CHCl.sub.3) 2961, 2868, 1722, 1656,
1619, 1465, 1374, 1269, 1209, 914, 733 cm.sup.-1; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.98 (s, 3H), 1.05 (d, J=6.4 Hz, 3H), 1.07
(d, J=5.9 Hz, 3H), 1.16 (s, 3H), 1.25-1.55 (m, 4H), 1.71-1.80 (m,
2H), 1.94-2.05 (m, 2H), 2.18-2.28 (m, 1), 2.40-2.65 (m, 4H), 2.74
and 2.79 (dd, J=6.5 and 15.2 Hz, 1H), 5.71 (s, 1H), 6.19 (s, 2H),
6.49 (m, 21); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.26,
16.29, 19.97, 21.79, 25.74, 29.19, 31.21, 33.73, 33.83, 36.13,
36.56, 46.39, 48.50, 50.74, 124.13, 128.76, 133.68, 138.44, 144.27,
162.88, 199.25, 208.08.
Example 14
4,6androstadien-3,17-dione-16-spirobicyclo [3'.1'.0'] hexane
[0647] These syntheses are described in Scheme 11. 83
Example 14A
[0648] 5-androsten-3,17-dione-16-spirobicyclo[3'.1'.0']-hexane
3-ethyleneketal (52). Under argon atmosphere, a solution of
5-androsten-3,17-dione 3-ethyleneketal (10) (3.96 g, 0.0120 mot),
potassium tertbutylate (0.70 g, 0.059 mol) and
4-pentynyltriphenylphospho- nium iodide (2,90 g, 0.0635 mol) in dry
toluene (3% W/V) was heated at 100.degree. C. for 1 h. The reaction
mixture was cooled at room temperature, poured into ice-water and
extracted several times with ethyl acetate. The combined organic
phase was washed with water and brine, dried over magnesium
sulfate, filtered, and evaporated. The obtained solid was purified
by flash chromatography (hexanes-ethyl acetate 9-1 to hexanes-ethyl
acetate 4-1) to provide spirobicycloketal 52 (0.95 g, 20%): .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 0.94 (s, 3H), 1.06 (s, 3H),
1.15-2.04 (m, 22H), 2.16 (dd, J=2.7 and J=14.1 Hz, 1H), 2.57 (m,
2H), 3.96 (m, 4H), 5.38 (s, 1H).
Example 14B
[0649] 4androsten-3,17-dione-16-spirobicyclo[3'.1'.0']-hexane (53).
The same procedure for compound 12 was used, starting from compound
52. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 9-1) to provide spirobicycloenone 53 (50%):
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.95 (s, 3H), 1.18 (s,
3H), 1.02-2.10 (m, 22 h), 2.25-2.45 (m, 3H), 5.8 (s, 1H).
Example 14C
[0650] 4,6-androstadien-3,17-dione-16-spirobicyclo[3'.1'.0']-hexane
(EM-1299). The same procedure for compound 13 was used, starting
from compound 53. The crude mixture was purified by flash
chromatography (hexanes-ethyl acetate 9-1 to hexanesethyl acetate
41) to provide spirobicyclodienone EM-1299 (51%): IR (CHCl.sub.3)
3008, 2943, 2878, 1730, 1643, 1611 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 1.01 (s, 3H), 1.14 (s, 3H), 1.15-1.80 (m, 13H),
1.84-2.15 (m, 5H), 2.38-2.54 (m, 3H), 5.70 (s, 1H), 6.14-6.24 (m,
2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.51, 16.32, 19.92,
23.12, 23.24, 25.77, 31.21, 33.83, 35.35, 36.16, 36.28, 47.54,
48.58, 50.87, 124.12, 128.70, 138.65, 163.01, 199.31, 218.66.
Example 15
15.alpha., 16.alpha., (E-ring)4,6-androstadien-3,7-dione
derivatives
[0651] These syntheses are described in Scheme 12. 8485
Example 15A
[0652]
15.alpha.-[1-(3-hydroxypropyl)]-5-androsten-17.beta.-ol-3-one
3-ethyleneketal (54). Under argon atmosphere, a solution of
compound 46 (1.20 g, 3.24 mmol) in dry THF (6 mL) was cooled at
0.degree. C. and treated with a 0.5 M solution of 9-BBN in THF (20
mL, 10 mmol). The reaction mixture was allowed to warm to room
temperature and stirred overnight. The reaction mixture was cooled
at 0.degree. C. and treated with few drops of methanol, 3 N sodium
hydroxide (5 mL) and 30% hydrogen peroxide (5 mL). The reaction
mixture was vigorously stirred 2 h and neutralized with 10%
hydrochloric acid. After evaporating of the solvent, the aqueous
phase was extracted with dichloromethane. The organic phase was
washed with brine, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(dichloromethane-ethyl acetate 9-1 to dichloromethane-ethyl acetate
4-1) to provide the diol 54 (0.97 g, 77%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 0.78 (s, 3H), 1.04 (s, 3H), 1.07-1.89 (m, 19H),
2.11 (dd, J=2.6 and 14 Hz, 2H), 2.55 (d, J=14.1 Hz, 1H), 3.60 (m,
3H), 3.92 (m, 4H), 5.32 (s, 1H).
Example 15B
[0653]
15.alpha.-[1-(3-tosyloxypropyl)]-5-androsten-17.beta.-ol-3-one
3-ethyleneketal (55). Under argon atmosphere, a solution of
compound 54 (558 mg, 1.43 mmol) in dry pyridine (5 mL) was cooled
at 0.degree. C., treated with p-toluenesulfonyl chloride (328 mg,
1.72 mmol), and stirred 5 h. The reaction mixture was diluted with
ethyl acetate and washed with 10% hydrochloric acid and brine. The
organic phase was dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 9-1) to give the hydroxytosylate 55 (389 mg,
50%): .sup.1H NMR (300 MHz, CDCl.sub.3) 0.74 (s, 3H), 1.04 (s, 3H),
1.06-1.76 (m, 19H), 2.12 (dd, J.apprxeq.2 and 15 Hz, 2H), 2.45 (s,
3H), 2.55 (d, J=15 Hz, 1H), 3.58 (t, J.apprxeq.8 Hz, 1H), 3.92-4.12
(m, 4H), 5.29 (s, 1H), 7.34 (d, J'8.4 Hz, 2H), 7.75 (d, J=8.2 Hz,
2H).
Example 15C
[0654] 15.alpha.-[1-(3-tosyloxypropyl)]-5-androsten-3,17-dione
3-ethyleneketal (56). Under argon atmosphere, a solution of
compound 55 (258 mg, 0.474 mmol) in dry dichloromethane (10 mL) was
treated with 4-methylmorpholine N-oxide (82 mg, 0.70 mmol) and
molecular sieves 4 .ANG., stirred 30 min, treated with a catalytic
amount of tetrapropylammonium perruthenate (5 mg) and stirred 2 h.
Filtrating and evaporating of the reaction mixture afforded a black
residue which was purified by flash chromatography (hexanes-ethyl
acetate 7-3) to provide the ketotosylate 56 (129 mg, 50%): .sup.1H
NMR (300 MHz, CDCl.sub.3) a 0.74 (s, 3H), 1.05 (s 3H), 1.08-2.16
(m, 20H), 2.45 (s, 3H), 2.54-2.66 (m, 2H), 3.90-4.00 (m, 41),4.03
(t, J=6.4 Hz, 2H), 5.30 (s, 1H), 7.35 (d, J=8.4 Hz, 2H), 7.79 (d,
J=8.2 Hz, 2H).
Example 15D
[0655] 15.alpha.,16.alpha.-(1,3-propylene)-5-androsten-3,17-dione
3-ethyleneketal (57). Under argon atmosphere, a solution of
compound 56 in dry THF (10 mL) was cooled at 0.degree. C. and
treated with 1 M solution of lithium bis(trimethylsilyl)amide in
THF (0.50 mL, 0.50 mmol) and HMPA (57.5 .mu.L, 0.330 mmol). The
reaction mixture was stirred 10 min, quenched with saturated
ammonium chloride, evaporated (THF) and extracted with
dichloromethane. The organic phase was washed with brine, dried
over magnesium sulfate, filtered, and evaporated. The crude mixture
was purified by flash chromatography (hexanes-ethyl acetate 4-1 to
hexanes-ethyl acetate 7-3) to provide the pentacyclic ketone 57 (96
mg, 78%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.95 (t, J=10.4
Hz, 1H), 1.02 (s, 3H), 1.07 (s, 3H), 1.13-1.88 (m, 17H), 2.12 (dd,
J=2.6 and 14.2 Hz, 2H), 2.55 (m, 2H), 2.99 (m, 1H), 3.95 (m, 4H),
5.3 (s, 1H).
Example 15E
[0656] 15.alpha.,16.alpha.-(1,3-propylene)-4androsten-3,17-dione
(58). The same procedure for compound 12 was used, starting from
compound 57. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 4-1) to provide pentacyclic enone 58
(.about.100%): .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 0.85 (t,
J=10.6 Hz, 1H), 0.94-1.02 (m, 1H), 1.05 (s, 3H), 1.22 (s, 3H),
1.24-1.87 (m, 14), 2.06 (m, 2H), 2.31-2.48 (m, 3H), 2.58 (m, 1H),
3.01 (m, 1H), 5.30 (s, 1H).
Example 15F
[0657]
15.alpha.,16.alpha.-(1,3-propylene)4,6-androstadien-3,17-dione
(59). The same procedure for compound 13 was used, starting from
compound 58. The crude mixture was purified by 2 flash
chromatographies (hexanes-acetone 9-1 and hexanesthyl acetate 97-3
to hexanes-ethyl acetate 17-3) to provide the pentacyclic dienone
59 (60%). The .sup.1H NMR spectrum shows the presence of
pentacyclic enone 58 in 10%: .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.04 (t, J=.apprxeq.10 Hz, 1), 1.09 (s, 3H), 1.13 (s, 3H),
1.22-1.86 (m, 12), 1.99 (m, 1H), 2.16-2.60 (m, 3), 2.72 (q, J=8.2
Hz, 1H), 3.05 (m, 1H), 5.70 (s, 1H), 6.18 (dd, J=9.8 and 2.5 Hz,
1H), 6.32 (d, J=10 Hz, 1H).
Example 16
6,17.alpha.,.beta.-dimethyl/1.alpha.,6,17a.beta.-trimethyl-D-Homo-4,6-andr-
ostadien/1,4,6-androstatrien-17a.alpha.-ol-3,17-dione Alkanoate
[0658] These syntheses are described in Scheme 13. 86
Example 16A
[0659]
6,17a.beta.-dimethyl-D-Homo-4,6-androstadien-17a.alpha.-ol-3,17-dio-
ne (60): The following example is a representative: Megestrol (44.1
g; 129 mmol) was dissolved in THF (550 mL). Sodium hydride (10.2 g;
3.2 eq.) was added portion-wise at 0.degree. C. After addition, the
mixture was stirred for 9 h at room temperature and cooled to
0.degree. C., and water (300 mL) was added slowely. Approximately
THF (300 mL) was evaporated under vacuum and another 300 mL of
water was added. The suspension was cooled to 0.degree. C. Solid
was filtered and washed with water. Solid, thus obtained was
triturated with boiling MeOH (150 mL) and cooled to room
temperature. This gave pure product which was filtered and washed
with MeOH, and dried (36.4 g; 82% yield).
Example 16B
[0660]
6,17a.beta.-dimethyl-D-Homo-1,4,6-androstatrien-17a.alpha.-ol-3,17--
dione (61): Compound 60 (5 g; 14.7 mmol) and DDQ (10 g; 3 eq) in
dioxane (60 mL) were refluxed for 3 h. Solvent was removed and the
mixture in EtOAc was washed with saturated NaHCO.sub.3 solution (3
times). Solvent was dried (MgSO.sub.4) and evaporated to give the
product. Purification on silica gel column (hexanes/acetone) gave
the trienone 61 (3.7 g) in 76% yield.
Example 16C
[0661]
1.alpha.,6,17a.beta.-trimethyl-D-Homo-4,6-androstadien-17a.alpha.-o-
l-3,17-dione (63): This compound was prepared by following the
methods, described above.
Example 16D
[0662]
6,17a.beta.-dimethyl/1.alpha.,6,17a.beta.-trimethyl-D-Homo-4,6-andr-
ostadien-17.alpha.-ol-3,17-dione alkanoate (64): The alcohols were
esterified by using the conditions A, B, C, D or E.
Example 16E
[0663] The followings are non-limiting examples of physicochemical
characteristics of inhibitors of Example 16.
[0664] EM-1078 (64, R'=H, R=(CH.sub.2).sub.2CH); Yield, 66%; IR
(KBr, cm.sup.-1) 2948, 2871, 1735, 1660, 1624, 1577, 1458, 1375,
1317, 1269, 1188, 1099; .sup.1H NMR (CDCl.sub.3) .delta. 0.80 (s,
3H, H-C18), 0.96 (t, 3H, H-C4', J=7 Hz), 1.06 (s, 3H, H-C19), 1.34
(s, 3H, 17.alpha.-CH3), 1.87 (s, 3H, 6-CH3), 2.01-2.06 (m, 2H),
2.32 (t, 2H, H-C2', J=7 Hz), 5.89 (s, 1H), 6.13 (s, 1H); .sup.13C
NMR (CDCl.sub.3) .delta. 208.0, 200.0, 172.5, 163.7, 136.5, 131.8,
121.0, 87.6, 49.5, 45.4, 40.4, 37.3, 36.3, 36.1, 33.7, 33.4, 31.8,
26.1, 20.2, 19.8, 18.3, 16.2, 13.6, 13.0.
[0665] EM-1091 (64, R'=H, R=CH.sub.2CH.sub.3); Yield, 53%; IR (KBr,
cm.sup.-1) 2944, 1734, 1664, 1625, 1581, 1444, 1352, 1273, 1194,
1098; .sup.1H NMR (CDCl.sub.3) .delta. 0.77 (s, 3H, H-C18), 1.01
(s, 3H, H-C19), 1.12 (t, 3H, H-C3', J=7 Hz), 1.30 (s, 3H,
17.alpha.-CH3), 1.83 (s, 3H, 6-CH3), 2.34 (q, 2H, H-C2', J=7 Hz),
5.84 (s, 1H), 6.10 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta.
207.8, 199.8, 173.2, 163.5, 136.4, 131.9, 121.4, 121.2, 87.7, 49.7,
45.5, 40.6, 37.4, 36.2, 33.9, 33.6, 31.9, 27.9, 26.1, 20.2, 19.9,
16.3, 13.7, 13.1, 9.0.
[0666] EM-1098 (64, R'=H, R=CH.sub.2Ph); Yield, 37%; IR (KBr,
cm.sup.-1) 2946, 2869, 1735, 1657, 1624, 1578, 1456, 1268, 1127,
1098; .sup.1H NMR (CDCl.sub.3) .delta. 0.72 (s, 3H, H-C18), 1.01
(s, 3H, H-C19), 1.30 (s, 3H, 17a-CH.sub.3), 1.90 (s, 3H,
6-CH.sub.3), 3.65 (s, 2H, H-C2'), 5.93 (s, 1H), 6.00 (s, 1H), 7.26
(s, 5H, Aromatics); .sup.13C NMR (CDCl.sub.3) .delta. 207.5, 199.9,
169.7, 165.6, 136.5, 131.6, 129.3, 128.6, 127.2, 121.0, 88.2, 49.6,
45.5, 42.0, 39.9, 37.2, 37.1, 36.1, 33.9, 33.5, 31.8, 25.8, 20.1,
20.2, 19.9, 19.2, 13.6, 14.0.
[0667] EM-1146 (64, R'=H, R=CH.sub.2PhOCO-t-Bu(p)); Yield, 14%; IR
(KBr, cm.sup.-1) 2955, 2871, 1736, 1660, 1624, 1582, 1508, 1458,
1393, 1270, 1202, 1166, 1117; .sup.1H NMR (CDCl.sub.3) .delta. 0.69
(s, 3H, H-C18), 1.05 (s, 3H, H-C19), 1.31 (s, 3H, 17.alpha.-CH,),
1.34 (s, 9H, t-Butyl), 1.88 (s, 3H, 6-CH3), 3.64 (s, 2H, H-C2'),
5.90 (s, 1H), 6.10 (s, 1H), 6.97 (d, 2H, J=8.3 Hz), 7.28 (d, 2H,
J=8.3 Hz); .sup.13C NMR (CDCl.sub.3) .delta. 207.7, 199.9, 177.0,
169.7, 163.6, 1150.5, 136.7, 131.7, 130.5, 130.4, 121.9, 121.2,
88.4, 49.8, 45.6, 41.4, 40.2, 39.1, 37.4, 37.2, 36.2, 33.8, 33.6,
31.8, 27.1, 25.9, 20.2, 19.9, 16.3, 13.7, 13.1.
[0668] CS-259 (64, R'=H, R=(CH.sub.2).sub.5Br); Yield, 32%; IR
(KBr, cm.sup.-1) 2943, 2869, 1734, 1660, 1624, 1579, 1458, 1375,
1270, 1194, 1099; .sup.1H NMR (CDCl.sub.3) .delta. 0.81 (s, 3H,
H-C18), 1.06 (s, 3H, H-C19), 1.34 (s, 3H, 17.alpha.-CH3), 1.87 (s,
3H, 6-CH.sub.3), 1.89-2.07 (m, 1H), 2.38 (t, 2H, H-C2', J=7 Hz),
3.40 (t, 2H, H-C6', J=7 Hz), 5.89 (s, 1H), 6.13 (s, 1H); .sup.13C
NMR (CDCl.sub.3) .delta. 207.8, 199.8, 172.2, 163.5, 136.4, 131.8,
121.0, 87.8, 49.5, 45.3, 40.4, 37.3, 36.1, 34.1, 33.7, 33.4, 32.1,
31.8, 27.5, 26.0, 23.8, 20.2, 19.8, 16.1, 13.6, 13.0.
[0669] CS-260 (64, R'=H, R=(CH.sub.2).sub.4CH.sub.3); Yield, 41%;
IR (KBr, cm.sup.-1) 2953, 2870, 1735, 1660, 1624, 1579, 1458, 1375,
1317, 1270, 1098; .sup.1H NMR (CDCl.sub.3) .delta. 0.79 (s, 3H,
H-C18), 0.87 (t, 3H, H-C6'), 1.06 (s, 3H, H-C19), 1.32 (s, 3H,
17.alpha.-CH3), 1.86 (s, 3H, 6-CH.sub.3), 2.00-2.06 (m, 2H), 2.33
(t, 2H, H-C2', J=7 Hz), 5.88 (s, 1H), 6.12 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 208.0, 199.9, 172.7, 163.6, 136.5, 131.9,
121.2, 87.7, 49.7, 45.5, 40.5, 37.4, 36.2, 34.4, 33.8, 33.5, 31.9,
31.3, 26.1, 24.5, 22.3, 20.2, 19.9, 16.2, 13.9, 13.7, 13.1.
[0670] CS-237 (64, R.sup.1=H, .quadrature.1, R=CH.sub.3); Yield
67%; IR (CDCl.sub.3) 1735, 1652, 1609, 1582 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.79 (s, 3H), 1.1 (s, 3H), 1.29 (s, 3H), 1.89
(s, 3H), 2.02 (s, 3H), 2.41-2.50 (m, 1H), 5.96 (s, 1H) 6.17 (s,
1H), 6.24 (d, 1H, J--10 Hz), 7.06 (d, 1H, J=10 Hz); .sup.13C NMR
(CDCl.sub.3) .delta. 207.6, 186.3, 169.9, 162.7, 153.1, 132.7,
132.3, 127.8, 121.5, 87.7, 77.4, 77, 76.6, 47.2, 45, 41.1, 40.7,
38, 37.2, 31.7, 26.1, 21.3, 21.1, 20.2, 19.5, 13.6, 13.
[0671] CS-240 (64, R=CH.sub.3); Yield 70%; IR (CDCl.sub.3) 1737,
1651, 1622, 1578 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.78
(s, 3H), 0.96 (d, 3H, J=7 Hz), 1.12 (s, 3H), 1.31 (s, 3H), 1.84 (s,
3H), 2.01 (s, 3H), 2.80-2.90 (dd, 1H, J=5.1, 17.5 Hz), 5.85 (s, 1H)
6.1 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 207.8, 199.4, 169.9,
159.9, 136.5, 132.5, 120.6, 87.9, 45.5, 44, 42, 40.6, 39.3, 37.4,
37.3, 35.5, 31.8, 26, 21.2, 20.2, 19.3, 18.8, 14.8, 13.7, 13.
[0672] EM-1117 (64, R=Cyhex); Yield 38%; IR (CDCl.sub.3) 1731,
1703, 1658, 1619, 1578 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.
0.80 (s, 3H), 0.99 (d, 3H, J=7 Hz), 1.14 (s, 3H), 1.31 (s, 3H),
1.87 (s, 3H), 5.89 (s, 1H,) 6.1 (s, 1H); .sup.13C NMR (CDCl.sub.3)
.delta. 208.1, 199.7, 174.7, 160.2, 136.7, 132.5, 120.6, 87.4,
45.7, 44.2, 43, 42.8, 42.1, 40.9, 39.4, 37.4, 35.6, 32, 29.7, 28.9,
28.8, 28.7, 26.2, 25.6, 25.3, 25.2, 20.3, 19.4, 18.9, 14.9, 13.8,
13.2.
[0673] EM-1121 (64, R=(CH.sub.2).sub.3CH.sub.3); Yield 70%; IR
(CDCl.sub.3) 1735, 1657, 1622, 1578 cm.sup.-4; .sup.1H NMR
(CDCl.sub.3) .delta. 0.77 (s, 3H), 0.85 (t, 3H, J=7 Hz), 0.94 (d,
3H, J=7 Hz), 0.87 (s, 3H), 1.11 (s, 3H), 1.83 (s, 3H), 2.33 (t, 2H,
J=7.4 Hz), 2.47 (m, 1H), 2.80-2.88 (dd, 1H, 5.3, 17.8 Hz), 5.84 (s,
1H,) 6.09 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 207.9, 199.4,
172.7, 160, 136.5, 132.4, 120.5, 87.6, 45.5, 44, 42, 40.7, 39.2,
37.4, 37.3, 35.5, 34, 31.9, 26.7, 26, 22.1, 20.2, 19.3, 18.8, 14.8,
13.7, 13.6, 13.
[0674] EM-1142 (64, R=(CH.sub.2).sub.4CH.sub.3); Yield 39%; IR
(CDCl.sub.3) 1735, 1657, 1621, 1578 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.80 (s, 3H), 0.87 (t, 3H, J=7 Hz), 0.98 (d,
3H, J=7 Hz), 1.15 (s, 3H), 1.33 (s, 3H), 1.87 (s, 3H), 2.84-2.91
(dd, 1H, J=5.1, 17.6 Hz), 5.89 (s, 1H,) 6.11 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 208.10, 199.6, 172.8, 160.1, 136.6, 132.5,
120.6, 87.7, 45.6, 44.2, 42.1, 40.8, 39.4, 37.5, 37.6, 34.4, 33.8,
32, 31.2, 26.1, 24.4, 22.3, 20.3, 19.4, 18.9, 14.9, 13.9, 13.2.
[0675] EM-1143 (64, R=CH(CH.sub.3).sub.2); Yield 34%; IR
(CDCl.sub.3) 1733, 1657, 1622, 1578 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.80 (s, 3H), 0.96 (d, 3H, J=7 Hz), 1.14 (s,
3H), 1.17 (d, 3H, J=7 Hz), 1.18 (d, 3H, J=7 Hz), 1.31 (s, 3H), 1.86
(s, 3H), 2.84-2.92 (dd, 1H, J=5.1, 17.6 Hz), 5.87 (s, 1H,) 6.1 (s,
1H); .sup.13C NMR (CDCl.sub.3) .delta. 207.8, 199.5, 175.8, 160,
136.5, 132.6, 120.7, 87.5, 61.9, 45.8, 44.2, 42.1, 40.9, 39.4,
37.4, 35.6, 34.1, 32, 26.2, 20.3, 19.4, 18.9, 18.7, 14.9, 14.1,
13.8, 13.1.
[0676] EM-1144 (64, R=CH2Ph); Yield 34%; IR (CDCl.sub.3) 1734,
1654, 1623, 1578 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.72
(s, 3H), 0.99 (d, 3H, J=7 Hz), 1.06 (s, 3H), 1.29 (s, 3H), 1.89 (s,
3H), 2.83-2.90 (dd, 1H, J=5, 17.4 Hz), 3.66 (s, 2H), 5.91 (s, 1H,)
5.96 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 207.6, 199.5,
169.7, 160, 136.6, 133.6, 132.2, 129.4, 128.7, 127.2, 120.5, 88.1,
45.7, 44, 42, 40.1, 39.2, 37.2, 37.1, 35.6, 31.8, 25.8, 20.2, 19.3,
18.8, 14.9, 13.7, 13.
[0677] EM-1154 (64, R=Cypent); Yield 30%; IR (CDCl.sub.3) 1731,
1657, 1622, 1578 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.79
(s, 3H), 0.97 (d, 3H, J=7 Hz), 1.14 (s, 3H), 1.31 (s, 3H), 1.86 (s,
3H), 2.79-2.84 (m, 2H), 5.87 (s, 1H,) 6.1 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 208.1, 199.5, 175.5, 160, 136.6, 132.6, 120.7,
87.5, 45.7, 44.2, 43.8, 42.1, 40.9, 39.4, 37.4, 37.3, 35.6, 32,
29.9, 29.6, 26.2, 25.8, 25.7, 20.3, 19.4, 18.9, 14.9, 13.8,
13.1.
Example 17
Synthesis of 4-aza-androstan-3-one-spiro .gamma./.delta.-lactone
Derivatives
[0678] These syntheses are described in Scheme 14. 87
Example 17A
[0679] General Procedure for 66a and 66c:
[0680] A flask was charged with HC.ident.C(CH.sub.2).sub.nOTHP (n=2
or 3) (41.5 mmol) and dry THF (300 mL) under argon, and cooled to
-50.degree. C. in a cold bath. To this solution, was added n-BuLi
(1.6 M, 41.4 mmol) via a syringe slowly, and the solution was
allowed to warm up to 0.degree. C. over a course of 2 h. Then, the
solution was cooled to -78.degree. C., and the ketone 65a (10.4
mmol) was added as a solid. The reaction mixture was stirred and
allowed to warm up to room temperature over a course of 3 h, and
stirred for additional 2 h at room temperature. Saturated aqueous
NH.sub.4Cl solution (50 mL) was added. The resulting two phase was
seperated, and water phase was extracted with CH.sub.2Cl.sub.2
(2.times.100 mL). The combined solvents were removed and the
residue was extracted with CH.sub.2Cl.sub.2 (2.times.150 mL).
Solution was washed with brine and dried over MgSO.sub.4. Solvent
was removed on a rotary evaporator. The purification of the residue
by flash column gave the product as a white solid.
[0681]
17.beta.-Hydroxy-17.alpha.-{4'-(2"-tetrahydro-2"H-pyranyloxy)butyn--
1'-yl}aza-5.alpha.-androstan-3-one (66a): Yield, 73%; IR (KBr,
cm.sup.-1) 3520-3210 (br), 2906, 2838, 1642; .sup.1H NMR
(CDCl.sub.3) .delta. 0.81 (s, 3H, 18-CH.sub.3), 0.88 (s, 3H,
19-CH.sub.3), 2.52 (dd, 2H, J=4.8, 11.2 Hz), 2.61 (t, 2H, J=7.7
Hz), 3.01 (dd, 1 h, J=3.2, 12.2 Hz), 3.48-3.56 (m, 2H), 3.78-3.86
(m, 2H), 4.64 (t, 1H, J=3.1 Hz), 6.14 (br, s, 1H); .sup.13C NMR
(CDCl.sub.3) 172.4, 98.6, 84.6, 83.1, 79.6, 65.8, 62.0, 60.7, 50.9,
49.9, 35.7, 35.6, 33.3, 32.6, 30.5, 29.6, 29.2, 29.0, 28.5, 27.2,
25.4, 22.9, 20.8, 20.3, 19.3, 12.8, 11.3.
[0682]
17.beta.-Hydroxy-17.alpha.-{5'-(2"-tetrahydro-2"H-pyranyloxy)pentyn-
-1'-yl}aza-5.alpha.-androstan-3-one (66c): Yield, 70%; IR (KBr,
cm.sup.-1) 3242, 3136, 2906, 2842, 1642; .sup.1H NMR (CDCl.sub.3)
.delta. 0.78 (s, 3H, 18-CH3), 0.86 (s, 3H, 19-CH.sub.3), 2.28-2.58
(m, 3H), 3.0 (dd, 1H, J=3.4, 12.3 Hz), 3.39-3.47 (m, ml), 3.74-3.84
(m, 2H), 4.55 (br s, 1H), 6.44 (s, 1H); .sup.13C NMR (CDCl.sub.3,
ppm) .delta. 172.3, 98.7, 85.3, 84.0, 79.5, 65.8, 62.1, 60.7, 50.9,
49.8, 46.9, 38.9, 35.6, 33.3, 32.5, 30.5, 29.3, 29.0 28.9, 28.5,
27.1, 25.3, 22.9, 20.8, 19.4, 15.6, 12.8, 11.3.
Example 17B
[0683]
17.beta.-Hydroxy-17.alpha.-{4'-(2"-tetrahydro-2"H-pyranyloxy)butyn--
1-yl}-4-methyl-4-aza-5.alpha.-androstan-3-one (66b): A flask was
charged with HC.ident.C(CH.sub.2).sub.2OTHP (3.05 g, 19.8 mmol) and
anhydrous THF (200 mL) under argon, and cooled to -50.degree. C. in
a cold bath. To this solution was added n-BuLi (1.6 M, 19.8 mol)
and the solution was allowed to warm up to 0.degree. C. over a
course of 2 h. Then, the solution was cooled to -78.degree. C., and
ketone 65b in dry THF (100 mL) was added via a cannula. The
reaction mixture was stirred under argon and allowed to warm up to
room temperature over a course of 3 h, and stirred for additional 2
h at room temperature. Saturated aqueous NH.sub.4Cl solution (50
mL) was added. The resulting two phase was seperated, and water
phase was extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The
combined solvents were removed and the residue was extracted with
CH.sub.2Cl.sub.2 (2.times.150 mL). Solution was washed with brine
and dried over MgSO.sub.4. Solvent was removed on a rotary
evaporator. The purification of the residue by a flash column gave
the product as a white solid (3.18 g, 7.12 mmol, 72%); IR (KBr,
cm.sup.-1) 3530-3130 (br), 2920, 2850, 1620; .sup.1H NMR
(CDCl.sub.3) .delta. 0.84 (s, 3H, 18-CH.sub.3), 0.90 (s,
19-CH.sub.3), 2.54 (t, 2H, J=7.1 Hz), 2.93 (s, 3H), 3.54 (dd, 1 h,
J=3.3, 12.9 Hz), 3.51-3.59 (m, 2H), 3.78-3.89 (m, 2H), 4.11-4.64
(m, 1H); .sup.13C NMR (CDCl.sub.3, ppm) .delta. 172.1, 98.2, 84.1,
83.1, 79.4, 65.9, 65.2, 62.1, 60.9, 60.7, 53.8, 50.7, 49.8, 46.5,
38.3, 35.6, 35.4, 33.1, 31.3, 29.4, 28.3, 27.5, 26.8, 25.7, 21.4,
20.9, 20.6, 12.7, 11.4.
Example 17C
[0684] General procedure for 67a-67c: Compound 66 (5 mmol) was
dissolved in methanol (100 mL) and amberlyst-15 (0.4 g) was added.
The raction mixture was stirred for 2 to 3 h (monitored by TLC) at
room temperature and filtered. Solvent was removed on a rotary
evaporator, and purification of the residue by flash column gave
product as a white solid.
[0685]
17.beta.-Hydroxy-17.alpha.-{4'-hydroxybutyn-1'-yl}-4aza-5.alpha.-an-
drostan-3-one (67a): Yield, 87%; IR (KBr, cm.sup.-1) 3490-3108
(br), 2910, 2832, 1636; .sup.1H NMR (CDCl.sub.3) .delta. 0.80 (s,
3H, 18-CH.sub.3), 0.86 (s, 3H, 19-CH.sub.3), 2.45 (t, 2H, J=6.9
Hz), 3.02 (dd, 1H, J=3.4, 12.1 Hz), 3.67 (t, 2H, J=6.8 Hz), 6.23
(br, s, 1H); .sup.13C NMR (CDCl.sub.3, ppm) .delta. 172.6, 85.6,
82.7, 79.3, 60.8, 60.6, 50.8, 49.7, 49.2, 46.8, 38.8, 35.6, 33.1,
32.6, 29.0, 28.4, 27.0, 23.1, 22.9, 20.7, 12.8, 11.2.
[0686]
17.beta.-Hydroxy-17.alpha.-{4'-hydroxybutyn-1'-yl}-4-methyl-4-aza-5-
.alpha.-androstan-3-one (67b): Yield, 91%; IR (KBr, cm.sup.-1)
3540-3120, 2904, 2836, 1624; .sup.1H NMR (CDCl.sub.3) .delta. 0.83
(s, 3H, 18-CH.sub.3), 0.89 (s, 3H, 19-CH.sub.3), 2.43-2.51 (m, 4H,
2.92 (s, 3H), 3.35 (dd, 1H, J=3.4, 12.5 Hz), 3.69 (t, 2H, J=6.2
Hz); .sup.13C NMR (CDCl.sub.3, ppm) .delta. 171.1, 85.6, 82.8,
79.4, 65.7, 60.9, 60.7, 51.6, 49.9, 46.9, 38.9, 36.4, 34.9, 32.8,
29.7, 29.6, 29.2, 29.0, 25.2, 23.2, 22.9, 20.8, 12.9, 12.4
[0687]
17.beta.-Hydroxy-17.alpha.-{5'-hydroxypentyn-1'-yl}-4-aza-5.alpha.--
androstan-3-one (67c): Yield, 84%; IR (KBr, cm.sup.-1) 3530-3082,
2902, 2842, 1618; .sup.1H NMR (CDCl.sub.3) .delta. 0.83 (s, 3H,
18-CH.sub.3), 0.89 (s, 3H, 19-CH.sub.3), 2.24 (t, 2H, J=6.9 Hz),
2.29 (dd, 2H, J=4.7, 5.5 Hz), 2.95 (dd, 1H, J=3.8, 12.6 Hz), 3.59
(t, 2H, J=6.3 Hz), 6.26 (br, s 1H); .sup.13C NMR (CDCl.sub.3, ppm)
.delta. 173.0, 84.9, 83.9, 79.2, 60.7, 60.4, 50.7, 49.7, 46.7,
38.7, 35.4, 32.9, 31.1, 28.9, 28.8, 28.1, 26.7, 22.8, 20.6, 15.1,
12.7, 11.1.
Example 17D
[0688] General procedure for 68a-68c: A flask was charged with
compound 67 (5 mmol), 10 mmol % of Pd/C, CH.sub.3OH (30 mL), ethyl
acetate (120 mL) and stir bar, and then the compound was
hydrogenated using a balloon. The reaction mixture was stirred at
room temperature for 3 h and filtered. Solvent was removed on a
rotary evaporator, and purification of the residue by flash column
gave product as a white solid.
[0689]
17.beta.-Hydroxy-17.alpha.-{4'-hydroxybutan-1'-yl}-4-aza-5.alpha.-a-
ndrostan-3-one (68a): Yield, 65%; .sup.1H NMR (CDCl.sub.3) .delta.
0.87 (s, 3H, 18-CH.sub.3), 0.91 (s, 3H, 19-CH.sub.3), 2.39-2.44 (m,
2H), 3.04 (dd, 1H, J=4.2, 11.9 Hz), 3.67 (t, 2H, J=5.8 Hz), 5.93
(br, s 1H).
[0690]
17.beta.-Hydroxy-17.alpha.-{4'-hydroxybutan-1'-yl}-methyl-4-aza-5.a-
lpha.-androstan-3-one (68b): Yield, 81%; .sup.1H NMR (CDCl.sub.3)
.delta. 0.87 (s, 3H, 18-CH.sub.3), 0.89 (s, 3H, 19-CH.sub.3),
2.41-2.46 (m, 2H), 2.92 (s, 3H), 3.02 (dd, 1H, J=3.5, 12.5 Hz),
3.68-7.26 (br, s, 2H).
[0691]
17.beta.-Hydroxy-17.alpha.-{5'-hydroxypentan-1'-yl}-4-aza-5.alpha.--
androstan-3-one (68c): Yield, 76%; IR (thin film, cm.sup.-1) 1663
(s); .sup.1H NMR (CDCl.sub.3) .delta. 0.87 (s, 3H, 18-CH.sub.3),
0.92 (s, 3H, 19-CH.sub.3), 2.41-2.46 (m, 2H), 3.08 (dd, 1H, J=4.8,
11.5 Hz), 3.62 (br, s, 2H), 5.48 (br, s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 172.3, 83.2, 62.8, 60.8, 51.2, 50.1, 46.6,
36.6, 35.9, 35.8, 34.3, 33.4, 32.7, 31.4, 29.4, 28.6, 27.3, 26.6,
23.6, 23.3, 20.9, 14.5, 11.4.
Example 17E
[0692] General procedure for 69a and 69b: Alcohol 68 (5 mmol) was
dissolved in 150 mL of acetone and the solution was cooled to
0.degree. C. in an ice bath. Jones' reagent (0.5 M, 12.5 mmol) was
added dropwise. After addition, the mixture was stirred for 30 min
at 0.degree. C. Then 100 mL of water was added and producted was
extracted with CH.sub.2Cl.sub.2 (3.times.150 mL). The combined
organic layer was washed with saturated aqueous NaHCO.sub.3
solution, water and brine, and then dried over MgSO.sub.4. Solvents
were removed on a rotary evaporator and the purification of the
residue by flash column gave product as a white solid.
[0693]
4aza-5a-androstan-3-one-17(R)-spiro-2'-(6'-oxo)tetrahydropyran)
(69a): Yield, 58%; IR (thin film, cm.sup.-1) 1725 (s), 1663 (s).
.sup.1H NMR (CDCl.sub.3) .delta. 0.92 (s, 3H, 18-CH3), 0.98 (s, 3H,
19-CH.sub.3), 5.53 (s, 1H, br, NH); .sup.13C NMR (CDCl.sub.3)
.delta. 172.2, 171.9, 93.0, 59.5, 51.0, 49.3, 47.1, 35.7, 35.4,
33.8, 33.4, 31.7, 29.3, 29.2, 28.5, 27.8, 27.1, 23.6, 20.6, 15.8,
14.4, 11.3. Anal. Calcd for C.sub.22H.sub.33NO.sub.3: C 73.50; H
9.25, N 3.90. Found: C 73.23; H 9.29, N 3.78.
[0694]
4-methyl-4-aza-5.alpha.-androstan-3-one-17(R)-spiro-2'-(6'-oxo)tetr-
ahydropyran) (69b): Yield, 64%; IR (thin film, cm.sup.-1) 1727 (s),
1641 (s); .sup.1H NMR (CDCl.sub.3) .delta. 0.89 (s, 3H,
18-CH.sub.3), 0.97 (s, 3H, 19-CH.sub.3), 2.91 (s, 3H, NMe); 13C NMR
(CDCl.sub.3, ppm) .delta. 171.9, 170.6, 93.0, 65.6, 51.7, 49.3,
47.0, 36.4, 34.7, 33.9, 32.9, 31.8, 29.9, 29.4, 29.0, 27.8, 25.2,
23.6, 20.6, 15.8, 14.4, 12.3. Anal. Calcd for
C.sub.23H.sub.35NO.sub.3: C 73.96; H 9.44, N 3.75. Found: C 73.85;
H 9.59, N 3.50.
Example 17F
[0695] General procedure for 71a and 71b: A flask was charged with
69 (1.11 mmol), DDQ (1.11 mmol) and anhydrous dioxane (8 mL).
[0696] Bis(trimethylsilyl) trifluoroacetamide (BSTFA) (4.56 mmol)
was added via a syringe. The reaction mixture was stirred at room
temperature under argon for 18 h, and then refluxed for 8 h. The
mixture was poured into CH.sub.2Cl.sub.2 (80 mL), and the solution
was washed with saturated NaHCO.sub.3 (2.times.50 mL) and Brine.
The organic layer was dried over MgSO.sub.4. Solvents were removed
on a rotary evaporator and the residue purified by a flash column
to give product as a white solid.
[0697]
4-aza-5.alpha.-androst-1-en-3-one-17(R)-spiro-2'-(6'-oxo)tetrahydro-
pyran (71a): Yield, 52%; IR (thin film, cm.sup.-1) 1715 (s), 1674
(s), 1599 (m). .sup.1H NMRS (CDCl.sub.3) .delta. 0.95 (s, 3H,
18-CH.sub.3), 0.96 (s, 3H, 19-CH.sub.3), 5.76 (d, 1H, J=10.0 Hz,
CH.dbd.CH), 6.67 (br, s, 1H, NH), 6.76 (d, 1H, J=10.0 Hz,
CH.dbd.CH); .sup.13C NMR (CDCl.sub.3) .delta. 172.0, 166.9, 151.0,
123.0, 92.9, 59.6, 49.3, 47.4, 47.2, 39.3, 35.6, 33.8, 31.7, 29.4,
29.1, 27.8, 25.6, 23.7, 20.7, 15.7, 14.5, 11.9.
[0698]
4-methyl-4-aza-5.alpha.-androst-1-en-3-one-17(R)-spiro-2'-(6'-oxo)t-
etrahydropyran (71b): Yield, 25%; IR (thin film, cm.sup.-1) 1725
(s), 1661 (s), 1604 (m); .sup.1H NMR (CDCl.sub.3) .delta. 0.92 (s,
3H, 18-CH.sub.3), 0.98 (s, 3H, 19-CH.sub.3), 2.93 (s, 3H, NMe),
5.83 (d, 1H, J=9.9 Hz, CH.dbd.CH), 6.67 (d, 1H, J=9.9 Hz,
CH.dbd.CH); .sup.13C NMR (CDCl.sub.3) .delta. 171.9, 165.5, 148.5,
123.2, 92.9, 63.7, 49.2, 47.6, 47.1, 39.5, 35.0, 33.9, 31.7, 29.6,
29.4, 27.8, 27.6, 24.3, 23.6, 20.7, 15.7, 14.5, 12.1.
Example 17G
[0699]
17.beta.-Hydroxy-17.alpha.-{4'-carboxybutan-1'-yl}-4-methyl-4-aza-5-
.alpha.-androstan-3-one (70): Alcohol 68c (1.82 g, 5.00 mmol) was
dissolved in 100 mL of acetone and the solution was cooled to
0.degree. C. in an ice bath. Jones' reagent (0.5 M, 25.0 mL, 12.5
mmol) was added dropwise. Then saturated aqueous NaHCO.sub.3 (150
mL) and ethyl acetate (100 mL) were added and the mixture was
stirred vigorously for overnight. Two phases were separated and the
water phase was acidified using 1 M HCl. The acidic solution was
extracted with CH.sub.2Cl.sub.2 (3.times.120 mL). The combined
organic layer were washed with water and brine, and then dried over
MgSO.sub.4. Removal of solvents gave the product as a white solid
(1.25 g, 3.20 mmol, 64%); IR (thin film, cm.sup.-1) 1704 (s), 1632
(s); 1H NMR (pyridine-d.sub.5) .delta. 1.22 (s, 3H,
18-CH.sub.3),1.45 (s, 3H, 19-CH.sub.3), 8.41 (s, 1H, NH); .sup.13C
NMR (pyridine-d.sub.5) .delta. 176.1, 171.4, 82.6, 60.9, 51.5,
50.5, 47.2, 37.5, 36.2, 35.9, 35.2, 34.5, 34.0, 32.1, 29.9, 29.4,
27.5, 26.8, 24.2, 21.3, 15.4, 11.4.
Example 17H
[0700] Preparation of the lactone 72: To acid 70 (100 mg, 0.258
mol) in 20 mL of CH.sub.2Cl.sub.2 was added oxalyl chloride (1.2
eq, 42 mL) at 0.degree. C. under argon, and the reaction mixture
was stirred for 1 h. After 1 h, no more acid was detected on the
TLC. Then, pyridine (4 eq, 50 mL) was added and the mixture was
stirred for 48 h at room temperature. The product was extracted
with ethyl acetate, dried over MgSO.sub.2 and purified by a flash
column using CH.sub.2Cl.sub.2/MeOH (gradient, 2 to 8%) as an eluent
to give the lactone 72 as a white solid (43 mg, 45%); IR (thin
film, cm.sup.4-) 1714 (s), 1660 (s); .sup.1H NMR (CDCl.sub.3, ppm)
.delta. 0.88 (s, 3H, 18-CH.sub.3), 0.96 (s, 3H, 19-CH.sub.3), 6.57
(s, 1H, NH); .sup.13C NMR (CDCl.sub.3, ppm) .delta. 175.6, 172.8,
60.7, 51.2, 48.8, 36.1, 35.6, 33.6, 33.3, 30.9, 30.6, 29.7, 28.5,
27.1, 24.2, 24.1, 23.1, 22.6, 20.6, 15.3, 14.1, 11.4.
Example 18
Syntheses of
Unsubstituted-A-ring-1,3,5(10)-estratrien-17-spiro-.delta.-la-
ctone Derivatives
[0701] These syntheses are described in Scheme 15. 88
Example 18
Syntheses of
Unsubstituted-A-ring-1,3,5(10)-estratrien-17-spiro-.delta.-la-
ctone Derivatives
[0702] These syntheses are described in Scheme 15. 89
Example 18A
[0703] Synthesis of 3-deoxygenated-estrone (74). At 0.degree. C.,
1.0 g (3.7 mmol) of estrone (73) was dissolved in 50 mL of dry
pyridine and 1.24 mL (7.4 mmol) of trifluoroacetic anhydride
(Tf.sub.2O) was added slowly to the solution. After 1 h, the crude
solution was poured into a cold aqueous solution of CuSO.sub.4 (1M)
and the organic phase washed with the same solution until the blue
color disappear. Next, the solution was extracted with EtOAc and
the organic phase washed with water, and dried over MgSO.sub.4.
After evaporation of the solvent, the crude estrone-triflate was
dissolved in 50 mL of dry DMF. To the resulting mixture, we added
1.54 mL (11 mmol) of Et.sub.3N, 0.41 mL (11 mmol) of HCOOH, 0.143
mg (0.55 mmol) of PPM, and 30.8 mg (0.14 mmol) of Pd(OAc).sub.2.
After 3 h at room temperature, the solution was quenched with
aqueous HCl (5%) and extracted with CH.sub.2Cl.sub.2. The organic
phase was washed with water, dried over MgSO.sub.4, and solvents
evaporated to dryness. The crude compound was purified by flash
chromatography with hexanes/EtOAc (9:1) as eluent to give 615 mg
(65% two steps) of 1,3,5(10)-estratrien-17-one (74). White solid;
IR .nu. (film): 1738 (C.dbd.O); .sup.1H NMR (CDCl.sub.3) .delta.
0.94 (s, 3H, 18-CH.sub.3), 2.94 (m, 2H, 6-CH.sub.2), 7.16 (m, 3H,
1-CH, 2-CH, 3-CH), 7.32 (d, J=5.9 Hz, 1H, 4-CH); .sup.13C NMR
(CDCl.sub.3) .delta. 13.76 (C-18), 21.51 (C-15), 25.61 (C-11),
26.42 (C-7), 29.29 (C-6), 31.56 (C-12), 35.77 (C-16), 38.04 (C-8),
44.40 (C-9), 47.88 (C-13), 50.49 (C-14), 125.22 (C-3), 125.73 (C-2
and C-1), 128.98 (C4), 136.37 (C-5), 139.62 (C-10), 220.63
(C-17).
Example 18B
[0704] Synthesis of alcohol 75. To a solution of
tetrahydro-2-(butynyloxy)- -2-H-pyran (0.94 mL, 5.9 mmol) in 30 mL
of dry THF was added at 0)C, 3.53 mL of n-BuLi 1.6 M (5.7 mmol),
and the mixture was stirred for 40 min. A solution of
3-deoxygenated-estrone (74) (500 mg, 1.96 mmol) in 10 mL of THF was
then added dropwise at -78.degree. C. and the mixture stirred for
11 h. After this time, a solution of aqueous NaHCO.sub.3 (5%) was
added and aqueous phase was extracted with EtOAc. Organic layer was
washed with brine, and dried over MgSO.sub.4. After evaporation of
solvent, the crude compound was purified by flash chromatography
with hexane/EtOAc (9:1) as eluent to give 566 mg (68%) of
17.beta.-hydroxy-17.alpha.-[4'-[(tetrahydr- o-2"H-pyranyl)oxy]
butynyl]-1,3,5(10)-estratrien (75). Colorless oil; IR .nu. (film):
3438 (OH, alcohol), 2233 very weak (C.ident.C); .sup.1H NMR
(CDCl.sub.3) .delta. 0.88 (s, 3H, 18-CH.sub.3), 2.57 (t, J=7.0 Hz,
2H, C.ident.CCH.sub.2), 2.87 (m, 2H, 6-CH.sub.2), 3.55 and 3.86
(2m, 4H, CH.sub.2O of side chain and CH.sub.2O of THP), 4.68
(s.sub.app, 1H, CH of THP), 7.13 (m, 3H, 1-CH, 2-CH, 3-CH), 7.31
(d, J=6.7 Hz, 1H, 4-CH); .sup.13C NMR (CDCl.sub.3) .delta. 12.72
(C-18), 19.21 (C4" of THP), 20.30 (C-3'), 22.76 (C-15), 25.37 (C-5"
of THP), 26.18 (C-11), 27.13 (C-7), 29.49 (C-6), 30.48 (C-3" of
THP), 32.87 (C-12), 38.98 (C-16), 39.14 (C-8), 43.07 (C-9), 47.07
(C-13), 49.53 (C-14), 61.95 (C-2" of THP), 65.78 (C-4'), 79.81
(C-17), 83.07 (C-2'), 84.68 (C-1'), 98.58 (C-1" of THP), 125.25
(C-3), 125.47 (C-2), 125.51 (C-1), 128.91 (C4), 136.62 (C-5),
140.22 (C-10).
Example 18C
[0705] Synthesis of lactone 77
[0706] a) Reduction of triple bond (75->78a). To a solution of
compound 75 (650 mg, 1.6 mmol) in EtOAc was added 40 mg of
palladium on activated charcoal (10%), and the mixture was stirred
at room temperature overnight under an hydrogen atmosphere. After
this time, it was filtered through celite, washed with EtOAc, and
evaporated to dryness to give the compound 78a as a white foam.
[0707] b) Hydrolysis of THP group (78a->78b). The crude alcohol
78a was dissolved in 60 mL of methanol and 20 mg of P-TSA was
added. After 2 h at room temperature, water was added to the
mixture, the methanol was evaporated, and the resulting mixture
extracted with EtOAc. The organic phase was washed with water and
dried over MgSO.sub.4. Then the solvent was evaporated to dryness
to give 455 mg of crude diol 78b.
[0708] c) Jones' oxidation with lactonization (78b->77) The
crude diol 78b (450 mg) was dissolved in 30 mL of acetone and 0.9
mL of Jones' reagent (2.7M) was added dropwise at 0.degree. C.
After the addition was completed, the mixture was stirred at room
temperature for 2 h. Then 2 mL of isopropyl alcohol was added and
the resulting green solution was evaporated to dryness. The solid
was dissolved in water and EtOAc, and the mixture extracted with
EtOAc. Organic layer was washed with brine and dried over
MgSO.sub.4. After evaporation of solvent, the crude compound was
purified by flash chromatography with hexanes/EtOAc (8:2) as eluent
to give 392 mg (65%, three steps) of
1,3,5(10)-estratrien-17(R)-spiro-2'-- (6'-oxo)tetrahydropyran (77).
White solid; IR .nu. (KBr): 1732 (C.dbd.O, lactone); .sup.1H NMR
(CDCl.sub.3) .delta. 1.03 (s, 3H, 18-CH.sub.3), 2.88 (m, 2H,
6-CH.sub.2), 7.12 (m, 3H, 1-CH, 2-CH, 3-CH), 7.29 (d, J=5.8 Hz, 1H,
4-CH); .sup.13C NMR (CDCl.sub.3) .delta. 14.03 (C-18), 15.59
(C-2'), 23.22 (C-15), 25.54 (C-11), 27.13 (C-1'), 27.63 (C-7),
29.17 (C-6 and C-3'), 31.69 (C-12), 33.67 (C-16), 38.56 (C-8),
43.83 (C-9), 46.92 (C-13), 48.64 (C-14), 92.93 (C-17), 124.92
(C-3), 125.39 (C-1 and C-2), 128.70 (C-1), 136.18 (C-10), 139.56
(C-5), 171.70 (C-4'); EI-HRMS: calcd for C.sub.22H.sub.28O.sub.2
324.20892, found 324.20702.
Example 18D
[0709] Synthesis of lactones 76a and 76b. A mixture of 127 .mu.L
(1.0 mmol) of diisopropylamine, 0.5 mL (0.80 mmol) of n-BuLi (1.6M)
and 5 mL of dry THF was stirred at 0.degree. C. for 30 min. The
solution was cooled at -78.degree. C., and 75 mg (0.233 mmol) of
lactone 77 in 10 mL of dry THF was added dropwise. After 1 h, 90
.mu.L of CH.sub.3I was added and the mixture stirred overnight and
let warm to room temperature. Then, the solution was quenched with
water and extracted with EtOAc. The organic phase was washed with
water and dried over MgSO.sub.4. After evaporation of the solvent,
the crude compound was purified by flash chromatography with
hexanes/EtOAc (95:5) as eluent to give 28 mg (36%, two steps) of
mono-methylated lactone 76a and 33 mg (40%, two steps) of
dimethylated lactone 76b.
[0710]
1,3,5(10)-estratrien-17(R)-spiro-2'-(5'-methyl-6'-oxo)tetrahydropyr-
an (76a). [major isomer only]. White solid; IR .nu. (film): 1734
(C.dbd.O, lactone); .sup.1H NMR (CDCl.sub.3) .delta. 1.02 (s, 3H,
18-CH.sub.3), 1.25 (d, J=6.8 Hz, 3H, CHCH.sub.3), 2.56 (m, 1H,
CHCH3), 2.87 (m, 2H, 6-CH.sub.2), 7.12 (m, 3H, 1-CH, 2-CH, 3-CH),
7.29 (d, J=6.1 Hz, 1H, 4-CH); .sup.13C NMR (CDCl.sub.3) .delta.
14.34 (C-18), 17.26 (CHCH.sub.3), 23.52 (C-15), 24.44 (C-2'), 26.78
(C-11), 27.28 (C-1'), 27.40 (C-7), 29.41 (C-6), 32.01 (C-12), 33.51
(C-3'), 34.00 (C-16), 38.84 (C-8), 44.14 (C-9), 47.21 (C-13), 48.76
(C-14), 92.75 (C-17), 125.22 (C-3), 125.56 (C-1 and C-2), 128.97
(C4), 135.51 (C-10), 139.87 (C-5), 175.84 (C4').
[0711]
1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetrahyd-
ropyran (76b). White solid; IR .nu. (film): 1724 (C.dbd.O,
lactone); .sup.1H NMR (CDCl.sub.3) .delta. 1.02 (s, 3H,
18-CH.sub.3), 1.28 (s, 6H, 2.times.CH.sub.3), 2.88 (m, 2H,
6-CH.sub.2), 7.13 (m, 3H, 1-CH, 2-CH, 3CH), 7.30 (d, J=6.2 Hz, 1H,
4-CH); .sup.13C NMR (CDCl.sub.3) .delta. 14.41 (C-18), 23.29
(C-15), 25.59 (C-1'), 25.84 (C-11), 27.39 (C-7), 27.54 and 27.76
(2.times.CH.sub.3), 29.42 (C-6), 31.51 (C-12), 32.02 (C-16), 34.82
(C-2'), 37.79 (C-3'), 38.87 (C-8), 44.15 (C-9), 47.25 (C-13), 48.84
(C-14), 93.55 (C-17), 125.21 (C-3), 125.52 (C-2), 125.56 (C-1),
128.99 (C4), 135.52 (C-10), 139.87 (C-5), 177.82 (C4').
Example 19
Synthesis of 2-nitro-1,3,5(10)-estratrien-17-spiro-.delta.-lactone
Derivatives
[0712] These syntheses are described in Scheme 16. 90
Example 19A
[0713] 3-hydroxy-2-nitro-1,3,5(10)-estratrien-17-one (79a). The
titled compound was prepared as described by Stubenrauch and
Knuppen[2]. The procedure is described below.
[0714] Estrone (37, 18.004 g, 66.6 mmol) was dissolved in boiling
acetic acid (540 mL) and allowed to cool down to 50.degree. C. The
nitrating mixture was prepared from 70% nitric acid (4.5 mL, 70
mmol), water (10 mL) and a few crystal of sodium nitrite, warmed up
to 50.degree. C. and added dropwise to the solution of estrone with
stirring. After stirring overnight at room temperature, the yellow
precipitate was filtered by suction and recrystallized from 92%
aqueous acetic acid. 4-nitro derivative (6.800 g, 32%) was thus
obtained as a pale yellow solid. IR (.nu.) 3227 (OH), 2931, 2864,
1723 (C.dbd.O), 1626, 1584; 1523, 1458, 1404, 1374, 1295, 1264,
1245, 1211, 1169, 1085, 1062, 1027, 954, 930, 908, 881, 823, 796,
719, 654, 588, 556, 530, 494 cm.sup.-1; .sup.1H NMR
(Pyridine-d.sub.5) .delta. 0.85 (3H, s, 18'-CH.sub.3), 2.85 (2H, d,
6'-CH.sub.2), 5.00 (1H, s, OH), 7.11 (1H, d, J=8.7 Hz, 2'-CH), 7.26
(1H, d, J=8.7 Hz, 1'-CH); .sup.13C NMR (Pyridine-d.sub.5) .delta.
13.8 (C-18), 21.6 (C-15), 24.4 (C-11), 25.7 (C-7), 26.2 (C-12),
32.0 (C-6), 35.9 (C-16), 37.7 (C-8), 44.0 (C-14), 47.9 (C-13); 50.1
(C-9), 115.4 (C-2), 128.4 (C-1), 129.0 (C-10), 131.8 (C-5), 148.4
(C-3), 219.2 (C-17).
[0715] The reaction filtrate from above was evaporated under
reduced pressure and the residue was recrystallized from
EtOH/H.sub.2O 8.5:1.5. A brown solid (7.854 g) was obtained which
was further purified by flash chromatography on SiO.sub.2 column
(EtOAc/hexanes, gradient 8-20%) to give pure compound 79a (6.284 g,
30%) as a yellow solid. IR (n): 3300 (OH), 2933, 2864, 1737
(C.dbd.O), 1630, 1562, 1522, 1480, 1431, 1372, 1311, 1252, 1216,
1146, 1084, 1054, 1035, 1008, 905, 832, 762, 722, 662, 600, 520
cm.sup.-1. .sup.1H NMR (Pyridine-d.sub.5) .delta. 0.85 (3H, s,
18'-CH.sub.3), 2.76 (2H, d, 6'-CH.sub.2), 4.99 (1H, s, OH), 6.98
(1H, s, 4'-CH), 7.96 (1H, s, 1'-CH). .sup.13C NMR
(Pyridine-d.sub.5) .delta. 13.8 (C-18), 21.7 (C-15), 25.8 (C-11),
26.1 (C-7), 29.6 (C-12), 31.9 (C-6), 35.9 (C-16), 37.8 (C-8), 43.5
(C-14), 47.9 (C-13), 50.3 (C-9), 119.8 (C4), 122.2 (C-1), 132.8
(C-10), 147.8 (C-2), 152.6 (C-3), 219.1 (C-17).
Example 19B
[0716]
3-(tert-butyldimethylsilyloxy)-2-nitro-1,3,5(10)-estratrien-17-one
(79b). A solution of 2-nitro-estrone (79a, 1.118 g, 3.55 mmole),
imidazole (0.670 g, 9.84 mmole) and TBDMSCl (0.781 g, 5.18 mmole)
in dry DMF (50 mL) was stirred under Ar(g) overnight. The mixture
was then poured onto ice/water (80) mL. The white precipitate was
filtered, washed with water and then dried in vacuo to give (79b)
as a yellowish powder (1.447 g, 95%).
[a].sup.25.sub.D+123.9.degree. (c 1.03, CHCl.sub.3); IR (NaCl)
2933, 2860, 1736 (s, C.dbd.O), 1617, 1561, 1518, 1492, 1408, 1351,
1291, 1256, 1054, 909, 832, 790, 697 cm.sup.-1; .sup.1H NMR .delta.
0.24 (6H, s, Si(CH.sub.3).sub.2), 0.92 (3H, s, 18-CH.sub.3), 1.01
(9H, s, SiC(CH.sub.3).sub.3), 1.40-1.78 (6H, m), 1.90-2.35 (5H, m).
2.37-2.60 (2H, m), 2.90 (2H, m, 6-CH.sub.2), 6.67 (1H, s, 4-CH),
7.76 (1H, s, 1-CH); .sup.13C NMR .delta. 220.2, 147.2, 143.8,
139.6, 133.3, 122.6, 122.1, 50.3, 47.9, 43.6, 37.8, 35.8, 31.3,
29.4, 26.1, 25.7, 25.6, 21.5, 18.2, 13.8, 4.4.
Example 19C
[0717]
3-(tert-butyldimethylsilyloxy)-17.beta.-hydroxy-2-nitro-17.alpha.-(-
4'-(2"-tetrahydro-2"H-pyranyloxy)-butynyl)-1,3,5(10)-estratriene
(80). To a stirred solution of tetrahydro-2-(butynyloxy)-2H-pyran
(1.71 mL, 10.91 mmole) in dry THF (75 mL) under Ar (g) at
-35.degree. C. was dropwise added (MeLi 1.4M in ether 7.80 mL,
10.92 mmole). The solution was stirred for 45 min. after which was
added at -35.degree. C. a solution of ketone 79b (1.294 g, 3.01
mmole) in dry THF (20 mL). After 75 min. ice (20 g) and saturated
aqueous NaHCO.sub.3 (70 mL) were added to the reaction mixture and
the aqueous phase was extracted with EtOAc. The combined organic
layers were washed with brine, dried with magnesium sulfate,
filtered and then concentrated in vacuo. The crude yellow oil was
purified on SiO.sub.2 (40 g, 2:8 EtOAc/hexanes) to give compound 80
as a yellow foam (1.617 g, 92%). [.alpha.].sub.D.sup.25
-57.5.degree. (c 0.72, CHCl.sub.3); IR (NaCl) 3423 (broad, OH),
2936, 2870, 2366, 1654, 1630, 1578, 1560, 1527, 1481, 1458, 1438,
1313, 1268, 1121, 1080, 1032, 899, 869, 761, 669 cm.sup.-1; .sup.1H
NMR .delta. 0.23 (6H, s, Si(CH.sub.3).sub.2), 0.87 (3H, s,
18-CH.sub.3), 1.00 (9H, s, SiC(CH.sub.3).sub.3), 1.20-2.35 (20H,
m), 2.55 (2H, t, J=6.9 Hz, CCCH.sub.2), 2.84 (2H, m, 6-CH.sub.2),
3.55 (2H, m, CH.sub.2O of chain), 3.85 (2H, m, CH.sub.2O of THP),
4.65 (1H, m, CH of THP), 6.65 (1H, s, 4-CH), 7.76 (1H, s, 1-CH);
.sup.13C NMR .delta. 147.0, 144.2, 139.5, 133.9, 122.6, 122.0,
98.8, 84.5, 83.4, 79.8, 65.8, 62.2, 49.4, 47.1, 43.2, 38.9, 32.6,
30.6, 29.6, 26.7, 26.2, 25.6, 25.4, 22.8, 20.4, 19.4, 18.2, 12.7,
-4.4.
Example 19D
[0718]
3-(tert-butyldimethylsilyloxy)-17.beta.-hydroxy-2-nitro-17.alpha.-(-
4'-(2"-tetrahydro-2"H-pyranyloxy)-butyl)-1,3,5(10)-estratriene
(81). A solution of compound 80 (2.00 g, 3.42 mmol) and 5%
Pd/CaCO.sub.3 (400 mg) in dry MeOH (400 mL) was stirred under
H.sub.2 (g) atmosphere (balloon) for 1 h. The mixture was then
filtered through celite and the filtrate rotary evaporated. The
residue was purified on silica gel (2:8 EtOAc/hexanes) to give
compound 81 as a white foamy solid (1.483 g, 74%).
[.alpha.].sub.D.sup.25+1.3.degree. (c 0.90, CHCl.sub.3); IR (NaCl)
3458 (broad, OH), 2935, 2860, 1616, 1563, 1518 (NO.sub.2), 1491,
1408, 1348 (NO.sub.2), 1291, 1256, 1119, 1070, 1023, 925, 893, 832,
784, 672 cm.sup.-1; .sup.1H NMR 6 0.23 (6H, s, Si(CH).sub.2), 0.91
(3H, s, 18-CH3), 1.00 (9H, S, SiC(CH.sub.3).sub.3), 1.20-2.38 (26H,
m), (2.85 (2H, m, 6-CH.sub.2), 3.48 (2H, m, CH.sub.2O of chain),
3.83 (2H, m, CH.sub.2O of THP), 4.59 (1H, m, CH of THP), 6.65 (1H,
s, 4-CH), 7.75 (1H, s, 1-CH); .sup.13C NMR 146.9, 144.1, 139.4,
134.0, 122.4, 121.9, 98.9, 83.2, 67.6, 67.6, 62.4, 62.4, 49.3,
46.6, 36.3, 34.1, 31.3, 30.7, 30.3, 29.5, 26.9, 26.0, 25.5, 25.4,
23.3, 20.4, 19.6, 18.1, 14.3, -4.4.
Example 19E
[0719]
17.alpha.-(4'-hydroxybutyl)-3,17.beta.-dihydroxy-2-nitro-1,3,5(10)--
estratriene (82). A solution of compound 81 (300 mg, 0.510 mmol) in
5% HCl in MeOH (10 mL) was stirred at room temperature and under
argon atmosphere for 12 h. The reaction mixture was then poured
into NaHCO.sub.3/ice and MeOH was evaporated under reduced
pressure. The aqueous phase was extracted with EtOAc and the
combined organic layers were washed with brine, dried over
MgSO.sub.4, filtered and evaporated to dryness. This gave a crude
yellow foam (198 mg, 100%). Purification by flash chromatography
(column loaded with CH.sub.2Cl.sub.2 and then eluted with
EtOAc/CH.sub.2Cl.sub.2 2:8, 4:6, 1:1, 6:4, 9:1) gave compound 82 as
a yellow solid (127.0 mg, 64%). Rf 0.21 (8:2 EtOAc/Hexanes); M.p.
184-6.degree. C.; [.alpha.].sup.26.sub.D+58.8.degree. (c. 1.00,
CHCl); R (.nu.) 3335, 2934, 2865, 1735, 1719, 1654, 1630, 1576,
1522, 1479, 1434, 1373, 1305, 1266, 1169, 1112, 1067, 1033, 1000,
896, 874, 762, 659 cm.sup.-1; .sup.1H NMR .delta. 0.90 (3H, s),
3.69 (2H, d, J=5.7 Hz), 6.84 (1H, s), 7.98 (1H, s), 10.42 (1H, s);
.sup.13C NMR .delta. 14.3, 19.8, 23.3, 26.1, 26.8, 29.8, 31.2,
33.3, 34.3, 36.1, 39.0, 43.2, 46.5, 49.4, 61.7, 62.8, 83.4, 118.8,
121.4, 131.6, 133.7, 149.2, 152.8.
Example 19F
[0720]
2-Nitro-1,3,5(10)-estraetriene-3-ol-17(R)-spiro-2'-(6'-oxo)tetrahyd-
ropyran (EM-1124). To a stirred solution of compound 82 (128 mg,
0.33 mmole) in dry acetone (25 mL) at 0.degree. C. was slowly added
a first 1.1 equivalent of Jones' reagent (1.25 M, 0.29 mL, 0.80
mmol). The orange solution was then stirred for 0.5 h then a second
equivalent was added. The dark solution was stirred for a further
0.5 h then quenched with isopropanol (green precipitate formed).
the mixture was stirred for 10 min. then filtered through celite
and the filtrate rotary evaporated. The residue was taken in EtOAc
then washed with aq. sat. NaHCO.sub.3, H.sub.2O, brine, dried
(MgSO.sub.4), filtered, rotovaped. The crude solid was purified by
flash chromatography on SiO.sub.2 (3:7 EtOAc/Hexanes) to give
EM-1124 (108 mg, 85%) as a yellow solid. M.p. 213.degree. C.;
[.alpha.].sup.25.sub.D+90.0.degree. (c 0.70, CHCl.sub.3); IR (NaCl)
3198, 2934, 2876, 2245, 1720 (s, C.dbd.O, lactone), 1630, 1577,
1522, 1480, 1434, 1378, 1314, 1267, 1234, 1199, 1169, 1151, 1120,
1070, 1036, 1024, 992, 914, 851, 759, 732, 662, 585 cm.sup.-1;
.sup.1H NMR .delta. 1.02 (3H, s, 18-CH3), 1.20-2.23 (16H, m),
2.25-2.65 (3H, m), 2.90 (2H, m, 6-CH.sub.2), 6.85 (1H, s, 4-CH),
7.97 (1H, s, 1-CH), 10.41 (1H, s, OH phenol); .sup.13C NMR 5 171.9,
152.8, 148.9, 133.3, 131.7, 121.5, 118.9, 93.0, 48.8, 47.1, 43.1,
38.4, 33.9, 31.6, 29.7, 29.4, 27.9, 26.8, 25.8, 23.4, 15.8,
14.2.
Example 19G
[0721]
2-Nitro-1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5'-methyl-6'-oxo)-
tetrahydropyran (EM-1126, EM-1131). LDA was prepared as follows: To
a stirred solution of diisopropylamine (92 .mu.L, 71 mg, 0.70 mmol)
in dry TH (5 mL) at -78.degree. C. under Ar(g) was added n-BuLi
(1.2 M/Hexane, 580 .mu.L, 0.68 mmol) and the solution was then
stirred at 0.degree. C. for 25 min. then cooled down to -78.degree.
C. A solution of EM-1124 (66 mg, 0.17 mmol) in dry THF (5 mL) was
added and the resulting dark orange solution was then stirred for
30 min. Dry HMPA (2 mL) was added and after 15 min., MeI (107
.mu.L, 243 mg, 1.71 mmol). The solution was then stirred for a
further 4 h. The reaction was quenched with aqueous saturated Cl
and extracted with EtOAc. The organic phase was washed with 1 M
aqueous CuSO.sub.4 (4.times.), 1120, aqueous 1M Na.sub.2SO.sub.3,
brine, dried (MgSO.sub.4), filtered then rotary evaporated to give
a crude solid (103 mg). Purification by flash chromatography on
SiO.sub.2 (1:9-->2:8 EtOAc/Hexanes) provided first EM-1126 (11
mg, 16%) closely followed by EM-1131 (34 mg, 34%) both as yellow
solids. EM-1126: M.p. 2046.degree. C.;
[.alpha.].sup.25.sub.D+73.4.degree. (c 1.67, CDCl.sub.3); IR .nu.
3422? (b, r, OH), 2937, 2874, 1725 (vs, CO), 1630, 1577, 1525,
1479, 1458, 1432, 1378, 1311, 1269, 1249, 1205, 1188, 1150, 1118,
1088, 1071, 1007, 990, 934, 896, 760, 731, 668, 585, 495 cm.sup.-1;
.sup.1H NMR .delta. 1.03 (311, s), 1.30 (3H), d, J=7.1 Hz),
1.31-1.77 (10H, m), 1.89-2.03 (5H, m), 2.15 (1H, td, J=7.1 Hz,
J'=5.0 Hz), 2.30-2.50 (2H, m), 2.90 (211, dd, J=8.3 Hz, J'=4.9 Hz),
6.85, (1H, s), 7.98 (1H, s), 10.43 (1H, s, OH); .sup.13C NMR
.delta. 174.8, 152.9, 149.0, 133.4, 131.7, 121.5, 118.9, 93.4,
48.7, 47.1, 43.1, 38.5, 36.2, 34.6, 31.6, 29.7, 28.6, 26.9, 25.9,
25.2, 23.4, 17.4, 14.4.
[0722] EM-1131 (5'-epimer of EM-1126, real configuration not
determined): M.p. 206-8.degree. C.;
[.alpha.].sup.25.sub.D+62.6.degree. (c 0.68, CDCl.sub.3); IR .nu.
3422 (br, OH), 3192, 2934, 2876, 2858, 2824, 1721 (vs, CO), 1631,
1578, 1522, 1482, 1458, 1436, 1377, 1314, 1271, 1237, 1204, 1173,
1120, 1103, 1082, 1051, 1019, 1002, 933, 901, 877, 860, 759, 663,
638, 600, 495 cm.sup.-1; .sup.1H NMR .delta. 1.01 (3H, s), 1.24
(3H, d, J=7.0 Hz), 1.31-1.80 (10H, m), 1.89-2.20 (6H, m), 2.35 (1H,
br s), 2.55 (1H, sextuplet, J=7.5 Hz), 2.89 (2H, t, J=5.2 Hz), 6.84
(1H, s), 7.96 (1H, s), 10.41 (H, S, OH); .sup.13C N .delta. 175.8,
152.8, 148.9, 133.3, 131.6, 121.4, 118.8, 92.5, 77.4, 77.0, 76.6,
48.5, 47.0, 43.0, 38.4, 33.8, 33.4, 31.6, 29.7, 27.16, 26.7, 25.8,
24.3, 23.6, 17.2, 14.3.
Example 19H
[0723]
2-Nitro-1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-dimethyl-6'-
-oxo)tetrahydropyran (EM-1125). LDA was prepared as follows: To a
stirred solution of diisopropylamine (206 .mu.L, 159 mg, 1.57 mmol)
in dry THF (12 mL) at -78.degree. C. under Ar(g) was added n-BuLi
(1.2 M/Hexane, 1,28 mL, 1.53 mmol) and the solution was then
stirred at 0.degree. C. for 20 min. then cooled down to -78.degree.
C. A solution of a mixture of EM-1126 and EM-1131 (153 mg, 0.38
mmol) in dry THE (10 mL) was added and the resulting dark orange
solution was then stirred for 20 min. Dry HMPA (4.7 mL) was added
and after 15 min., MeI (238 .mu.L, 544 mg, 3.83 mmol). The solution
was then stirred for 5 min. then was warmed up to -30.degree. C.
and stirred for a further 1 h. The reaction was quenched with
aqueous saturated NH.sub.4Cl and extracted with EtOAc. The organic
phase was washed with brine (6.times.), aqueous 1M
Na.sub.2SO.sub.3, brine, dried (MgSO.sub.4), filtered then rotary
evaporated to give a crude liquid. Purification by flash
chromatography on SiO.sub.2 (1:9-->2:8 EtOAc/Hexanes) provided
EM-1125 (82 mg, 52%) as a yellow solid. M.p. 195-7.degree. C.;
[.alpha.].sup.25.sub.D+72.8.degree. (c 1.61, CDCl.sub.3); IR .nu.
3421 (br, OH), 3194, 2954, 2927, 2873, 1718 (vs, CO), 1631, 1578,
1523, 1476, 1458, 1438, 1386, 1312, 1298, 1271, 1204, 1151, 1118,
1059, 1032, 1016, 931, 898, 872, 855, 758, 663, 595 cm.sup.-1;
.sup.1H NMR .delta. 1.02 (3H, s), 1.28 (6H, s), 1.32-1.77 (10H, m),
1.85-2.15 (6H, m), 2.36 (1H, br s), 2.89 (2H, dd, J=8.2 Hz, J'=4.9
Hz), 6.85 (1H, s), 7.97 (1H, s), 10.42 (H, s, OH); .sup.13C NMR
.delta. 177.7, 152.8, 149.0, 133.3, 131.7, 121.5, 118.9, 93.4,
48.6, 47.1, 43.1, 38.5, 37.8, 34.7, 31.6, 31.5, 29.7, 27.7 (4),
27.6 (8), 26.7, 25.9, 25.5, 23.3, 14.4.
Example 191
[0724]
2-Nitro-3-methylthioethyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(6-
'-oxo) tetrahydropyran (EM-1118). To a solution of EM-1124 (40 mg,
0.117 mmol) in anhydrous acetonitrile (20 mL) at room temperature
under Ar(g) was added K.sub.2CO.sub.3 (16 mg, 0.117 mmol) and
chloroethylmethyl sulfide (39 mg, 35 mL, 0.350 mmol). The solution
was refluxing for 44 h. The acetonitrile solution was evaporated to
dryness and ethyl acetate (40 mL) was then added. The organic phase
was washed with water, brine and dried with MgSO.sub.4 to gave
crude product (49 mg). Purification by flash chromatography on
silica gel (SiO.sub.2, 3 g) using ethyl acetate/hexanes (2:8) as
eluent gave EM-1118 (35 mg, 70%). IR (v) 2921, 1727, 1608, 1576,
1499, 1466, 1438, 1382, 1348, 1330, 1310, 1280, 1236, 1187, 1159,
1100, 1067, 1036, 992, 931, 914, 860, 818, 731 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) .delta. 1.01 (3H, s), 1.25-2.57 (19H, m), 2.20
(3H, s) 2.85 (4H, t, J=6.88 Hz), 4.12 (2H, t, J=6.74 Hz), 6.62 (1H,
d, J=2.49 Hz), 6.71 (1H, dd, J1=2.58 Hz J2=5.89 Hz), 7.19 (1H, d,
J=8.54 Hz); .sup.13C NMR(CDCl.sub.3) 514.8, 15.9, 16.2, 23.5, 26.1,
27.5, 28.0, 29.5, 29.7, 32.0, 33.1, 34.0, 39.1, 43.7, 47.3, 48.9,
67.4, 93.3, 112.1, 114.6, 126.3, 132.6, 137.9, 156.5, 172.1.
Example 20
Synthesis of
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-
-6'-oxo) tetrahydropyran (89)
[0725] This synthesis described in Scheme 17 9192
[0726] 3-t-butyldimethylsilyloxy-1,3,5(10)-Estratrien-17-one (83).
The ether was prepared from estrone (37) following the method
described Pelletier et al. (Steroids 59: 536-547, 1994).
[0727]
3-t-butyldimethylsilyloxy-17.beta.-hydroxy-17.alpha.-{4'-(2"-tetrah-
ydro-2"H-pyranyl)butyn-1'-yl}-1,3,5(10)-estratriene (84). To a
solution of HC.ident.C(CH.sub.2).sub.2OTHP (18.3 mL, 117 mmol) in
dry THF (600 mL) at 0.degree. C., was added dropwise n-butyllithium
(43.7 mL, 109 mmol) and the mixture was stirred for 90 min. The
mixture was cooled to -78.degree. C. and a solution of
TBDMS-estrone 37 (15 g, 39 mmol) in THF (500 mL) was added
dropwise. Then, the reaction mixture was allowed to come to room
temperature and left stirring for a period of 15 h. Solvents were
evaporated to the half volume and 200 mL of water was added. The
mixture was extracted with EtOAc (3.times.200 mL), the organic
layer was washed with brine, dried (MgSO.sub.4) and evaporated to
dryness. The residue was purified over silica gel column
chromatography with hexanes/EtOAc (9/1) as an eluent to furnish
15.1 g (72%) of the product; IR (NaCl cm.sup.-1) 3432, 2934, 2858,
1607, 1495, 1287, 1256, 1033, 958, 839; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.12 (d, 1H, J=8.4 Hz), 6.62 (dd, 1H, J=24, 8.4
Hz), 6.54 (d, 1H, J=2.2 Hz), 4.66 (br.s., 1H), 3.89-3.79 (m, 2H),
3.56-3.50 (m, 2H), 2.79 (br.s., 2H), 2.56 (t, 2H, J=7.0 Hz),
2.35-2.17 (m, 3H), 2.07-1.23 (m, 17H), 0.98 (s, 9H), 0.87 (s, 3H,
18-Me), 0.19 (s, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
153.3, 137.8, 133.0, 126.1, 119.9, 117.1, 98.7, 84.7, 83.2, 80.0,
65.8, 62.1, 49.5, 47.2, 43.7, 39.4, 39.0, 32.9, 30.6, 29.7, 27.3,
26.4, 25.7, 25.4, 22.8, 20.3, 19.3, 18.1, 12.8, 4.4.
[0728] 3-t-butyldimethylsilyloxy
17.beta.-Hydroxy-17.alpha.-{4'-(2"-tetrah-
ydro-2"H-pyranyl)butan-1'-yl}-1,3,5(10)-estratriene (85). 5%
Palladium on activated carbon (1.5 g, 10% wt) was added to a
solution of the alkyne 84 (15.1 g, 28 mmol) in EtOAc (500 mL) at
room temperature. The flask was purged with H.sub.2 three times
(vacuum followed by H.sub.2) and left stirring under 1 atm pressure
of H.sub.2. The reaction was followed by TLC. After a period of 3
h, the mixture was filtered over a plug of celite and the solvent
was removed under reduced pressure. The crude product was used in
the next step without further purification; IR (NaCl, cm.sup.-1)
3474, 2935, 2858, 1607, 1570, 1496, 1471, 1286, 1257, 1156, 1137,
1119, 1033, 954, 839, 780; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.12 (d, 1H, J=8.4 Hz), 6.62 (dd, 1H, J=2.1, 8.4 Hz), 6.55
(s, 1H), 4.59 (br.s., 1H), 3.92-3.73 (m, 2H), 3.55-3.38 (m, 2H),
2.82-2.77 (m, 2H), 2.30-1.33 (m, 26H), 0.97 (s, 9H), 0.90 (s, 3H,
18-Me), 0.18 (s, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
153.27, 137.81, 133.08, 126.02, 119.87, 117.06, (98.90, 98.84),
83.38, 67.61, 62.33, 49.50, 46.67, 43.81, 39.58, 36.35, 34.28,
31.60, 30.75, 30.36, 29.62, 27.51, 26.26, 25.67, 25.47, 23.37,
20.45, 19.66, 18.12, 14.35, -4.43.
[0729] 3-t-butyldimethylsilyloxy
17.beta.-hydroxy-17.alpha.-(4'-hydroxybut-
an-1'-yl)-1,3,5(10)-estratriene (86). To a solution of the THP
ether 85 (15.1 g, 28 mmol) in MeOH (400 mL), was added
p-toluenesulfonic acid monohydrate (150 mg, 0.8 mmol) and the
reaction was stirred over a period of 5 h. A saturated solution of
NaHCO.sub.3 (100 mL) was added and volume of solvent was reduced to
half on a rotary evaporator. The mixture was extracted with
CH.sub.2Cl.sub.2, the organic phase was washed with brine, dried
(MgSO.sub.4) and evaporated to dryness. The crude product was used
in the next step without pufification; IR (NaCl, cm.sup.-1) 3356,
2931, 2858, 1608, 1496, 1471, 1286, 1256, 954, 839, 780; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.12 (d, 1H, J=8.5 Hz), 6.61 (dd,
1H, J=2.5, 8.5 Hz), 6.55 (s, 1H), 3.69 (br.d, 2H, J=5.2 Hz),
2.82-2.78 (m, 2H), 2.35-2.26 (m, 1H), 2.20-1.94 (m, 2H), 1.90-1.81
(m, 1H), 1.62-1.22 (m, 17H), 0.98 (s, 9H), 0.90 (s, 3H, 18-Me),
0.19 (s, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 153.27,
137.81, 133.05, 126.02, 119.89, 117.09, 83.59, 62.56, 49.50, 46.69,
48.81, 39.58, 35.98, 34.32, 33.20, 31.61, 29.62, 27.51, 26.26,
25.68, 23.37, 19.74, 18.14, 14.36, -4.40.
[0730]
3-t-butyldimethylsilyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(6'-o-
xo) tetrahydropyran (87). To a solution of the diol 86 (12.5 g, 27
mmol) in acetone (500 mL) at 0.degree. C., was added dropwise of a
2.7 M solution of Jone's reagent (15.1 mL, 41 mmol). The reaction
was stirred for 30 min. 2-Propanol (100 mL) was added, followed by
of a saturated solution of NaHCO.sub.3 (200 mL). The volume of
solvents was reduced to half by evaporation and the mixture was
extracted with EtOAc. The organic phase was washed with brine,
dried (MgSO.sub.4) and concentrated under reduced pressure. The
residue was purified a silica gel column chromatography with
hexanes/acetone (6/1) to afford 8.6 g of the lactone. (68% yield
for 3 steps); IR (NaCl, cm.sup.-1): 2960, 2930, 2857, 1732, 1607,
1496, 1284, 1264, 1244, 1037, 958, 840; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.11 (d, 1H, J=8.4 Hz), 6.61 (dd, 1H, J=2.3,
8.4 Hz), 6.56 (s, 1H), 2.85-2.79 (m, 2H), 2.58-2.39 (m, 2H),
2.38-2.25 (m, 1H), 2.21-2.10 (m, 1H), 2.03-1.27 (m, 15H), 1.02 (s,
3H, 18-Me), 0.97 (s, 9H), 0.18 (s, 6H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 172.00, 153.36, 137.63, 132.62, 126.02, 119.92,
117.19, 93.25, 48.88, 47.26, 43.68, 39.05, 33.98, 31.96, 29.50,
29.48, 27.94, 27.46, 25.98, 25.67, 23.48, 18.12, 15.87, 14.30,
-4.43.
[0731]
3-t-butyldimethylsilyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5'-5-
'-dimethyl-6'-oxo) tetrahydropyran(88). In a dry 1L flask under
argon, was dissolved the lactone 87 (8.6 g, 19 mmol) in dry THF
(300 mL), and cooled to 0.degree. C. A 1M solution of LiHMDS (47.3
mL, 47.3 mmol) was added dropwise. The mixture was stirred 15 min
at 0.degree. C. and cooled to -78.degree. C. and then methyl iodide
(5.9 mL, 79 mmol) was added. The reaction was stirred 1 h at this
temperature and then allowed to warm to room temperature over a
period of 2 h. A saturated solution of NH.sub.4Cl (200 mL) was
added and the mixture was extracted with EtOAc. The organic layer
was washed with a saturated solution of Na.sub.2S.sub.2O.sub.3,
brine, dried (MgSO.sub.4) and concentrated under reduced pressure.
The residue was purified by column chromatography with
hexanes/acetone (5/1) as an eluent to afford 7.4 g (81%) of the
dimethyl compound; IR (NaCl, cm.sup.-) 2954, 2930, 2858, 1725,
1496, 1287, 1258, 1150, 1137, 956, 840; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.11 (d, 1H, J=8.5 Hz), 6.62 (dd, 1H, J=2.4,
8.5 Hz), 6.55 (d, 1H, J=2.1 Hz), 2.81-2.78 (m, 2H), 2.36-2.28 (m,
1H), 2.20-1.38 (m, 16H), 1.28 (s, 3H), 1.27 (s, 3H), 1.02 (s, 3H,
18-Me), 0.97 (s, 9H), 0.18 (s, 6H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 177.79, 153.33, 137.62, 132.62, 125.99, 119.90,
117.14, 93.66, 48.67, 47.24, 43.65, 39.06, 37.74, 34.79, 31.96,
31.56, 29.50, 27.73, 27.61, 27.42, 26.01, 25.65, 25.55, 23.26,
18.11, 14.42, 4.43.
[0732]
1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)
tetrahydropyran (89): To a solution of the silyl ether 88 (7.1 g,
14.7 mmol) in THF (300 mL) at 0.degree. C., was added dropwise a 1M
solution of TBAF (17.6 mL, 17.6 mmol) and the reaction was stirred
for 15 min. Ice water (200 mL) was added to precipitate the
compound. The flask was placed on a retary evaporator to reduce the
volume of THF, and then placed on an ice bath. The precipitate was
collected by filtration, washed with cold water and dried in an
oven (30.degree. C.) over a period of 24 h to furnish 5.4 g (100%)
of the 3-OH compound; IR (NaCl, cm.sup.-1): 3357, 2932, 2871, 1695,
1287, 1158; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.14 (d, 1H,
J=8.4 Hz), 6.63 (dd, 1H, J=2.6, 8.4 Hz), 6.55 (d, 1H, J=2.6 Hz),
4.62 (br.s, 1H, OH), 2.81-2.79 (m, 2H), 2.38-2.29 (m, 1H),
2.20-1.81 (m, 5H), 1.76-1.31 (m, 11H), 1.29 (s, 3H), 1.28 (s, 3H),
1.01 (s, 3H, 18-Me); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
178.06, 153.52, 138.08, 132.19, 126.42, 115.26, 112.74, 93.80,
48.69, 47.29, 43.65, 39.14, 37.81, 34.84, 31.98, 31.61, 29.53,
27.76, 27.64, 27.39, 26.12, 25.59, 23.29, 14.43.
Example 21
Synthesis of
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-
-6'-oxo)tetrahydropyran-3-derivatives
[0733] These syntheses are described in Scheme 18. 93
Example 21A
[0734] Synthesis of
3-ethoxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-di-
methyl-6'-oxo)-tetrahydropyran (EM-1369). To a solution of lactone
89 (50 mg, 0.136 mmol) in anhydrous THE (10 ml) and under argon was
added K.sub.2CO.sub.3 (26 mg, 0.190 mmol), 18-crown-6 (14 mg, 0.054
mmol) and bromoethane (303 .mu.l, 4.08 mmol). The solution was
refluxed for 24 h. Then water (10 ml) was added and the product was
extracted with ethyl acetate (2.times.20 ml), washed with brine,
and dried with MgSO.sub.4. After evaporation of solvent, the crude
product was purified by flash chromatography on silica gel using
ethyl acetate/hexanes (1:9) as eluent to give the pure EM-1369.
White solid; 59% yield; IR (film NaCl) .nu. 2930, 2872, 1732, 1608,
1573, 1500, 1477, 1455, 1385, 1309, 1236, 1149, 1137, 1114, 1051,
1017; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.02 (s, 3H,
18-CH.sub.3), 1.28 (2s, 6H, 2.times.CH3), 1.30 to 2.36 (m, 17H),
1.39 (t, J=7.0 Hz, 3H, CH.sub.3CH.sub.2O), 2.84 (m, 2H, 6-CH2),
4.00 (q, J=7.0 Hz, 2H, CH.sub.3CH.sub.2O), 6.62 (d, J=2.5 Hz, 1H,
4-CH), 6.70 (dd, J.sub.1=2.5 Hz and J.sub.2=8.5 Hz, 1H, 2-CH), 7.19
(d, J=8.5 Hz, 1H, 1-CH); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
14.44, 14.91, 23.30, 25.61, 26.11, 27.48, 27.66, 27.77, 29.73,
31.63, 32.01, 34.85, 37.80, 39.17, 43.69, 47.30, 48.71, 63.31,
93.70, 112.05, 114.50, 126.21, 132.10, 137.76, 156.88, 177.85; HRMS
calculated for C.sub.26H.sub.37O.sub.3 (M.sup.++H): 397.27426,
found: 397.27520.
Example 21B
[0735] Typical procedure for the synthesis of EM-1368-CS,
EM-1389-CS, and EM-1390-CS. To a solution of lactone 89 (0.20 mmol)
in anhydrous acetonitrile (20 ml) and under argon was added
K.sub.2CO.sub.3 (0.20 mmol). The appropriate electrophile (16.0
mmol) (chloroethylmethylether, 2-dimethylaminoethylchloride.HCl,
and 1-(2-chloro-ethyl)-piperidine-HCl). The solution was refluxed
for 56 h. The acetonitrile was evaporated to dryness and ethyl
acetate (40 ml) was then added. The organic phase was washed with
water, brine, and dried with MgSO.sub.4 to gave the crude products
which were purified by flash chromatography on silica gel using, as
eluent, ethyl acetate/hexanes (1:9) for EM-1368CS and
CH.sub.2Cl.sub.2/Et.sub.3N (95.5:0.5) for EM-1389CS and
EM-1390CS.
[0736]
3-Methoxyethoxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-
-6'-oxo)-tetrahydropyran (EM-1368-CS). White solid; 74% yield; IR
(film NaCl) .nu. 2930, 2873, 1722, 1609, 1574, 1499, 1455, 1385,
1309, 1237, 1201, 1135, 1064, 1032, 1017; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 1.02 (s, 3H, 18-CH.sub.3), 1.28 (2s, 6H,
2.times.CH.sub.3), 1.30 to 2.35 (m, 17H), 2.85 (m, 2H, 6-CH.sub.2),
3.44 (s, 3H, OCH.sub.3), 3.73 (t, J=4.7 Hz, 2H,
CH.sub.3OCH.sub.2CH.sub.2O), 4.09 (t, J=4.7 Hz, 2H,
CH.sub.3OCH.sub.2CH.sub.2O), 6.66 (d, J=2.5 Hz, 1H, 4-CH), 6.73
(dd, J.sub.1=2.5 Hz and J.sub.2=8.6 Hz, 1H, 2-CH), 7.19 (d, J=8.6
Hz, 1H, 1-CH); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.44,
23.29, 25.61, 26.09, 27.45, 27.66, 27.76, 29.71, 31.62, 31.99,
34.85, 37.79, 39.14, 43.67, 47.29, 48.70, 59.16, 67.20, 71.12,
93.70, 112.16, 114.67, 126.21, 132.49, 137.78, 156.72, 177.85; HRMS
calculated for C.sub.27H.sub.39O.sub.4 (M.sup.++H): 427.28482,
found: 427.28690.
[0737]
3-(N,N-dimethylaminoethyl)-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',-
5'-dimethyl-6'-oxo)-tetrahydropyran (EM-1389-CS). White solid; 40%
yield; IR (film NaCl) .nu. 2936, 2871, 2819, 2770, 1723, 1609,
1575, 1499, 1456, 1385, 1309, 1290, 1256, 1238, 1202, 1149, 1032;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.01 (s, 3H,
18-CH.sub.3), 1.28 (2s, 6H, 2.times.CH.sub.3), 1.30 to 2.20 (m,
17H), 2.33 (s, 6H, (CH.sub.3).sub.2N), 2.71 (t, J=5.8 Hz, 2H,
NCH.sub.2CH.sub.2e), 2.83 (m, 2H, 6-CH.sub.2), 4.04 (t, J=5.8 Hz,
2H, NCH.sub.2CH.sub.2O), 6.65 (d, J=2.4 Hz, 1H, 4-CH), 6.73 (dd,
J.sub.1=2.7 Hz and J.sub.2=8.6 Hz, 1H, 2-CH), 7.19 (d, J=8.6 Hz,
1H, 1-CH); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.45, 23.30,
25.62, 26.11, 27.46, 27.67, 27.77, 29.73, 31.64, 32.01, 34.86,
37.79, 39.16, 43.70, 45.87, 47.30, 48.72, 58.35, 65.92, 93.71,
112.13, 114.59, 126.21, 132.34, 137.79, 156.79, 177.85; HRMS
calculated for C.sub.28H.sub.42O.sub.3N (M.sup.++H): 440.31647,
found: 440.31520.
[0738]
3-(N-piperidyl-ethyl)-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-di-
methyl-6'-oxo)-tetrahydropyran (EM-1390-CS)
[0739] White solid; 69% yield; IR (film NaCl) .nu. 2933, 2871,
2783, 1724, 1609, 1574, 1499, 1455, 1385, 1308, 1290, 1256, 1236,
1202, 1148, 1136, 1033; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
1.01 (s, 3H, 18-CH3), 1.28 (2s, 6H, 2.times.CH.sub.3), 1.30 to 2.40
(m, 17H), 2.50 (m, 4H, (CH.sub.2).sub.2N), 2.76 (t, J=6.2 Hz, 2H,
NCH.sub.2CH.sub.2O), 2.84 (m, 2H, 6CH2), 4.08 (t, J=6.2 Hz, 2H,
NCH.sub.2CH.sub.2O), 6.63 (d, J=2.6 Hz, 1H, 4-CH), 6.71 (dd,
J.sub.1=2.6 Hz and J.sub.2=8.6 Hz, 1H, 2-CH), 7.18 (d, J=8.6 Hz,
1H, 1-CH); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.46, 23.31,
24.18, 25.63, 25.92, 26.13, 27.47, 27.68, 27.79, 29.74, 31.65,
32.02, 34.87, 37.81, 39.17, 43.71, 47.32, 48.73, 55.00, 57.98,
65.83, 93.70, 112.14, 114.60, 126.23, 132.32, 137.81, 156.72,
177.87; HRMS calculated for C.sub.31H.sub.46O.sub.3N (M.sup.++H):
480.34778, found: 480.34550.
Example 22
Synthesis of 2-chloro-1,3,5(10)-estratrien-17-spiro-.delta.-lactone
Derivatives
[0740] These syntheses are described in Scheme 19. 94
[0741] a PhSeCl, CHCl.sub.3 b. NCS, CHCl.sub.3 c. Cs.sub.2CO.sub.3,
CH.sub.3OCH.sub.2CH.sub.2Cl, CH.sub.3CN, NaI
Example 22A
[0742]
3-Hydroxy-2-phenylselenenyl-1,3,5(10)-estratrien-17(R)-spiro-2'-(5'-
,5'-dimethyl-6'-oxo) tetrahydropyran (90). A solution of
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetr-
ahydropyran (89) (406 mg, 1.10 mmol) and phenylselenenyl chloride
(253 mg, 1.32 mmol) in dry CHCl.sub.3 (24 mL) under Ar (g) was
stirred at 0.degree. C. for 1 h then at room temperature overnight.
The resulting yellow solution was poured onto ice/H.sub.2O then
extracted with CH.sub.2Cl.sub.2 (3.times.). The combined organic
phase was dried (cotton plug) then rotary evaporated to give a
crude foamy solid. Purification by flash chromatography (SiO.sub.2)
using 1:9 EtOAc/Hexanes as eluent gave compound 90 (353 mg, 61%)
with the 4-isomer (86 mg, 15%). Compound 90:
[.alpha.].sup.25.sub.D+77.7.degree. (c 1.14, CHCl.sub.3); IR .nu.
3366, 3050, 2965, 2928, 2869, 1709, 1603, 1576, 1550, 1458, 1438,
1384, 1349, 1310, 1294, 1262, 1202, 1157, 1141, 1114, 1065, 1017,
984, 892, 845, 736, 689, 665, 593, 555, 498, 460 cm.sup.-1; .sup.1H
NMR(CDCl.sub.3) .delta. 1.02 (3H, s), 1.27 (9) (3H, s), 1.28 (4)
(3H, s), 1.27-1.80 (11H, m), 1.88-2.28 (6H, m), 2.87 (2H, t, J=4.8
Hz), 6.24 (1H, s, OH), 6.80 (1H, s), 7.21 (5H, br s), 7.52 (1H, s);
.sup.13C NMR(CDCl.sub.3) .delta. 14.4, 23.3, 25.5, 26.1, 27.2,
27.6, 27.7, 29.0, 31.5, 31.8, 34.7, 37.7, 38.9, 43.4, 47.2, 48.6,
93.6, 111.6, 114.7, 126.5, 129.2 (6), 129.3 (4), 131.2, 133.3,
134.7, 141.4, 154.4, 177.8.
Example 22B
[0743]
2-Chloro-3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimet-
hyl-6'-oxo) tetrahydropyran (91). A solution of compound 90 (347
mg, 0.66 mmol) and N-chlorosuccinimide (177 mg, 1.33 mmol) in dry
CHCB (30 mL)under Ar(g) at 0.degree. C. were stirred for 30 min.
The solution was poured onto ice/H.sub.2O then was extracted with
CH.sub.2Cl.sub.2 (3.times.). The combined organic phase was dried
(cotton plug) then rotary evaporated to give a crude solid.
Purification by flash chromatography (SiO.sub.2) using 1:9
EtOAc/Hexanes as eluent gave compound 91 (104 mg, 39%) as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 1.01 (3H, s), 1.28 (6H, s),
1.25-1.75 (11H, m), 1.85-2.28 (6H, m), 2.80 (2H, dd, J'=8.7 Hz,
J'=3.9 Hz), 5.39 (1H, br s, OH), 6.73 (1H, s), 7.19 (1H, s);
.sup.13C NMR(CDCl.sub.3) 614.4, 23.3, 25.6, 26.1, 27.2, 27.7, 27.8,
29.1, 31.6, 31.9, 34.8, 37.8, 38.8, 43.5, 47.3, 48.6, 93.6, 116.0,
117.1, 125.6, 133.5, 137.2, 149.0, 177.8.
Example 22C
[0744]
2-Chloro-3-methyloxyethyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5-
',5'-dimethyl-6'-oxo) tetrahydropyran (EM-1371). A mixture of
compound 91 (95 mg, 0.24 mmol), Cs.sub.2CO.sub.3 (230 mg, 0.70
mmol) 2-chloroethylmethyl ether (1.72 mL, 1.78 g, 18.86 mmol) and
NaI (4 mg, 0.02 mmol) in acetonitrile (45 mL) were reflexed for 4
h. The solvent was rotary evaporated and the residue was taken into
H.sub.2O/CH.sub.2Cl.sub.- 2. The aqueous phase was extracted with
CH.sub.2C.sub.2 (3.times.). The combined organic phase was dried
(MgSO.sub.4), filtered then rotary evaporated. The crude solid was
purified by flash chromatography (SiO.sub.2) Using 1:9 to 2:8
EtOAc/Hexanes as eluent to give EM-1371 (73 mg, 67%) as a white
solid. IR v 2982, 2963, 2927, 2880, 1718, 1654, 1598, 1499, 1458,
1397, 1387, 1364, 1323, 1307, 1286, 1259, 1247, 1210, 1151, 1125,
1059, 1032, 1018, 987, 928, 885, 866, 738, 669 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) .delta. 1.02 (3H, s), 1.28 (6H, s), 1.29-1.75
(11H, m), 1.85-2.35 (6H, m), 2.80 (2H, br t, J=5.0 Hz), 3.48 (3H,
s), 3.78 (2H, t, J=5.2 Hz), 4.14 (2H, t, J=5.2 Hz), 6.66 (1H; s),
7.25 (1H, s); .sup.13C NMR(CDCl.sub.3) .delta. 14.4, 23.2, 25.5,
26.0, 27.2, 27.6, 27.7, 29.3, 31.5, 31.9, 34.7, 37.7, 38.7, 43.4,
47.2, 48.5, 59.3, 68.9, 70.8, 93.5, 114.5, 120.3, 127.0, 133.7,
136.1, 152.1, 177.7.
Example 23
3-hydroxy-2,4dihalo-1,3,5(10)-estratrien-17-spiro-(dimethyl-.delta.-lacton-
e) Derivatives
[0745] These syntheses are described in Scheme 20. 95
Example 23A
[0746]
2,4dihalo-3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dime-
thyl-6'-oxo)tetrahydropyran (92). Under argon atmosphere, a
solution of
3-hydroxy-1,3,5(10)-estratriene-17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tet-
rahydropyran (89) and N-halosuccinimide (2 equiv) in anhydrous
chloroform (1.3% W/V) was stirred at room temperature for 1.5 h.
The reaction mixture was diluted with dichloromethane, washed with
brine, dried over magnesium sulfate, filtered, and evaporated. The
crude mixture was purified by flash chromatography (hexanes-ethyl
acetate 32-1 to hexanes-ethyl acetate 9-1) to provide compound 92
(e.g., EM-1382-CS, X=Br, 62%): IR (NaCl) 3197, 2936, 2872, 1694,
1466, 1387, 1297, 1154 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.01 (s, 3H), 1.29 (s, 6H), 1.35-2.35 (m, 17H), 2.65 (m,
1H), 2.88 (dd, J=6.1 and 18.0 Hz, 1H), 5.83 (s, 1H), 7.40 (s, 1H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.35, 23.25, 25.55,
26.29, 27.39, 27.69, 27.76, 30.98, 31.58, 31.77, 34.76, 37.80,
38.12, 43.59, 47.11, 48.50, 93.44, 106.36, 113.21, 128.45, 135.23,
136.51, 147.17, 177.75.
Example 23B
[0747]
3-substituted-2,4dihalo-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'--
dimethyl-6'-oxo)tetrahydropyran (93)
[0748] Method A: Under argon atmosphere, a solution of compound 92,
anhydrous potassium carbonate (1.2 equiv) and alkyliodide (2.0
equiv) in dimethylformamide (3.3% W/V) was stirred at 30.degree. C.
for 1 h. The reaction mixture was cooled at room temperature,
quenched with saturated ammonium chloride, and extracted with ethyl
acetate. The organic phase was quenched with brine, dried over
magnesium sulfate, filtered, and evaporated. The crude mixture was
purified by flash chromatography (hexanes-ethyl acetate 49-1 to
hexanes-ethyl acetate 19-1) to provide compound 93 (e.g.,
EM-1385-CS, X=Br, R=Me, 78%): IR (NaCl) 2935, 2870, 1720, 1462,
1384, 1298, 1150 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.01 (s, 3H), 1.29 (s, 6H), 1.30-2.35 (m, 17H), 2.68 (m,
1H), 2.88 (dd, J=5.6 and 18.2 Hz, 1H), 3.86 (s, 3H), 7.45 (s, 1H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.36, 23.28, 25.59,
26.24, 27.45, 27.72, 27.80, 31.07, 31.62, 31.84, 34.79, 37.84,
37.99, 43.87, 47.11, 48.63, 60.38, 93.45, 114.48, 121.45, 129.07,
137.31, 139.26, 151.96, 177.75.
[0749] Method B: Under argon atmosphere, a solution of compound 92,
triphenylphosphine (6 equiv) and alcohol (6 equiv) in THF (1.5%
W/V) was cooled at 0.degree. C. and treated with diethyl
azodicarboxylate (6 equiv). The solution was warmed at room
temperature, stirred for 2 h, and evaporated. The crude mixture was
purified by flash chromatography (hexanes-ethyl acetate 19-1) to
provide compound 93 (e.g., EM-1387, X=Br, R-allyl, 72%): IR (neat)
2937, 2871, 1722, 1453, 1386 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) a 1.01 (s, 3H), 1.28 (s, 6H), 1.30-2.35 (m, 17H), 2.68
(m, 1H), 2.88 (dd, J=5.8 and 18.0 Hz, 1H), 4.50 (d, J=5.6 Hz, 2H),
5.30 (dd, J=1.3 and 10.3 Hz, 1H), 5.46 (dd, J--1.3 and 17.0 Hz,
1H), 6.18 (m, 1H), 7.45 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 14.35, 23.26, 25.56, 26.20, 27.42, 27.69, 27.77, 31.09,
31.59, 31.81, 34.76, 37.81, 37.93, 43.84, 47.08, 48.60, 73.79,
93.42, 114.76, 118.41, 121.73, 129.04, 133.20, 137.24, 139.21,
150.86, 177.72.
Example 24
Synthesis of
2-fluoro-1,3,5(10)estratrien-17-dimethyl-.delta.-lactone
Derivatives
[0750] These syntheses are described in Scheme 21. 9697
Example 24A
[0751] 2-Nitro-3-methyloxyethyloxy-1,3,5(10)-estratrien-17-one
(94). A mixture of compound 79a (905 mg, 2.87 mmol),
K.sub.2CO.sub.3 (793 mg, 5.74 mmol), 2-chloroethylmethyl ether (17
mL, 18 g, 189 mmol) and NaI (43 mg, 0.29 mmol) in acetonitrile (60
mL) were refluxed for 24 h. The solvent was rotary evaporated and
the residue was taken into H.sub.2O/EtOAc. The aqueous phase was
extracted with EtOAc (3.times.). The combined organic phase was
washed with brine, dried (MgSO.sub.4), filtered then rotary
evaporated. The crude solid was purified by flash chromatography
(SiO.sub.2) using 1:9 to 2:8 EtOAc/Hexanes as eluent to give
recovery of the starting material (416 mg, 46%) and the title
compound (520 mg, 49%) as a yellow solid.
[.alpha.].sup.25.sub.D+135.8.de- gree. (c 1.28, CHCl.sub.3); IR
.nu. 3448 (w), 2930, 2890, 1737 (CO), 1617, 1568, 1518, 1498, 1454,
1409, 1373, 1351, 1339, 1287, 1194, 1129, 1070, 1031, 1006, 960,
916, 895, 874, 833, 795, 758, 733, 669, 594, 588 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) .delta. 0.91 (3H, s), 1.42-1.66 (6H, m), 1.95-2.42
(6H, m), 2.51 (1H, dd, J=18.9 Hz, J'=8.9 Hz), 2.92 (2H, br t, J=9.5
Hz), 3.45 (3H, s), 3.78 (2H, t, J=5.1 Hz), 4.20 (2H, t, J=4.8 Hz),
6.80 (1H, s), 7.79 (1H, s); .sup.13C NMR(CDCl.sub.3) .delta. 13.8,
21.5, 25.7, 26.0, 29.7, 31.3, 35.7, 37.8, 43.5, 47.8, 50.2, 59.4,
69.7, 70.7, 115.4, 122.9, 132.9, 137.9, 144.1, 150.4, 220.1.
Example 24B
[0752] 2-Amino-3-methyloxyethyloxy-1,3,5(10)-estratrien-17-one
(95). To a refluxing solution of compound 94 (506 mg, 1.35 mmol) in
acetone (120 mL), H.sub.2O (24 mL) and aqueous 1 M NaOH (24 mL,
24.0 mmol) was portionwise added with care solid
Na.sub.2S.sub.2O.sub.4 (3.89 g, 18.97 mmol). The reflux was
continued for 2 h then the acetone was removed under vacuum. More
H.sub.2O (50 mL) was added and the aqueous phase was extracted with
CH.sub.2Cl.sub.2 (4.times.). The organic phase was dried
(MgSO.sub.4), filtered then rotary evaporated. The crude solid was
purified by flash chromatography (SiO.sub.2) using 3:7 to 1:1
EtOAc/Hexanes as eluent to give compound 95 (228 mg, 49%) as a
foamy solid. .sup.1H NMR (CDCl.sub.3) .delta. 0.88 (3H, s),
1.37-2.78 (14H, m), 3.41 (3H, s), 3.71 (2H, t, J=5.0 Hz), 4.09 (2H,
2.9 Hz), 6.52 (1H, s), 6.65 (1H, s).
Example 24C
[0753] 2-Fluoro-3-methyloxyethyloxy-1,3,5(10)-estratrien-17-one
(96). To neat BF.sub.3-Et.sub.2O (126 .mu.L, 141 mg, 1.00 mmol) at
-15.degree. C. under Ar (g) was added a solution of compound 95
(228 mg, 0.66 mmol) in dry CH.sub.2Cl.sub.2 (4 mL) followed by the
dropwise addition t-butyl nitrite (95 .mu.L, 82 mg, 0.80 mmol).
This solution was stirred at -15.degree. C. for 10 min then at
0.degree. C. for 1.5 h. Pentane (16 mL) was added to the cold
solution and a red gummy solid precipitated. The solvent was
decanted and the residue was rinced with cold Et.sub.2O (5 mL). The
residue was dried under vacuum to give a foamy orange-red solid.
This solid was heated under vacuum with a heatgum for 5 min. The
residue was purified by flash chromatography (SiO.sub.2) using 3:7
to 1:1 EtOAc/Hexanes as eluent to give compound 96 (32 mg, 14%) as
a solid. .sup.1H NMR (CDCl.sub.3) .delta. 0.90 (3H, s), 1.25-1.64
(m, 6H), 1.92-2.29 (6H, m), 2.50 (1H, dd, J=18.9 Hz, 8.9 Hz), 2.82
(2H, dd, J=8.4 Hz, J=3.5 Hz), 3.45 (3H, s), 3.75 (2H, t, J=4.7 Hz),
4.15 (2H, t, J=4.5 Hz), 6.71 (1H, d, J=8.7 Hz), 6.98 (1H, d, J=13.2
Hz); .sup.13C NMR(CDCl.sub.3) .delta. 13.8, 21.5, 25.9, 26.5, 29.0,
29.7, 31.5, 35.8, 38.0, 43.9, 47.9, 50.3, 59.2, 69.2, 71.0, 113.1
(d, J=18.7 Hz), 116.1, 132.6 (d, J=87.0 Hz), 144.5, 149.5, 152.7,
220.6.
Example 24D
[0754]
2-Fluoro-17.beta.-hydroxy-3-methyloxyethyloxy-17.alpha.-(4'-(2"-tet-
rahydro-2"H-pyranyloxy)-butynyl)-1,3,5(10)-estratriene (97). To a
stirred solution of 2-(3-butynyloxy)tetrahydro-2H-pyran (58 IL, 57
mg, 0.37 mmol) in dry THF (4 mL) at -30.degree. C. under Ar(g) was
dropwise added MeLi (1.4 M in Et.sub.2O, 5.39 mL, 7.55 mmol). The
solution was stirred at room temperature for 25 min. then cooled
down to -30.degree. C. A solution of compound 96 (32 mg, 0.09 mmol)
in dry THF (4 mL) was added and the solution was stirred for a
further 1 h. The solution was quenched with ice and aqueous sat.
NaHCO.sub.3 and extracted with EtOAc. The organic layer was washed
with brine, dried (MgSO.sub.4), filtered then rotary evaporated to
give compound 97 as a crude oil (73 mg). This compound was used
without further purification.
Example 24E
[0755]
2-Fluoro-17.beta.-hydroxy-3-methyloxyethyloxy-17.alpha.-(4'-(2"-tet-
rahydro-2"H-pyranyloxy)-butyl)-1,3,5(10)-estratriene (98). A
mixture of crude compound 97 (73 mg, 0.09 mmol) and 5%
Pd/CaCO.sub.3 (35 mg) in MeOH (10 mL) was stirred under H.sub.2 (g)
(balloon) at room temperature for 2 h. The mixture was filtered
through celite and the solvent evaporated under vacuum to give
compound 98 (52 mg) as a crude oil. This compound was used without
further purification.
Example 24F
[0756]
2-Fluoro-1713-hydroxy-3-methyloxyethyloxy-17.alpha.-(4'-hydroxybuty-
l)-1,3,5(10)-estratriene (99). Crude compound 98 (52 mg, 0.09 mmol)
and PTSA monohydrate (7 mg, 0.04 mmol) were stirred in MeOH (5 mL)
at room temperature for 3 h. The solvent was reduced to about 1-2
mL, EtOAc was added, and the organic phase was washed with cold
aqueous saturated NaHCO.sub.3, H.sub.2O, brine and was dried
(MgSO.sub.4), filtered and then evaporated to give compound 99 (42
mg) as a crude solid. This compound was used without further
purification.
Example 24G
[0757]
2-Fluoro-3-methyloxyethyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(6-
'-oxo) tetrahydropyran (100). To a stirred solution of crude
compound 99 (42 mg, .about.0.09 mmol) in acetone (6 mL) at
0.degree. C. was added 1.25 M H.sub.2CrO.sub.4 Gones' reagent, 326
.mu.L, 0.4 mmol). The solution was stirred for 20 min. then more
1.25 M H.sub.2CrO.sub.4 Gones' reagent, 326 .mu.L, 0.4 mmol) was
added. After 20 min, iso-propanol (1 mL) was added and the solution
was stirred for a further 10 min. The precipitated formed was
decanted and washed with acetone (4.times.). The acetone volume was
reduced to about 2 mL and was then poured onto cold aqueous
saturated NaHCO.sub.3 with the help of EtOAc. The organic phase was
washed with H.sub.2O, brine, dried (MgSO.sub.4), filtered and then
evaporated to give compound 100 (33 mg) as a crude solid. This
compound was used without further purification. .sup.1H NMR
(CDCl.sub.3) .delta. 1.01 (3H, s), 1.24-2.60 (19H, m), 2.78 (2H, br
s), 3.45 (3H, s), 3.74 (2H, dd, J=5.0 Hz, J=4.5 Hz), 4.14 (2H, dd,
J=5.0 Hz, J=4.5 Hz), 6.69 (1H, d, J=8.7 Hz), 6.97 (1H, d, J=13.2
Hz); .sup.13C NMR (CDCl.sub.3) .delta. 14.3, 15.9, 23.4, 26.0,
27.4, 27.9, 29.0, 29.5, 31.9, 34.0, 38.7, 43.6, 47.2, 48.8, 59.2,
69.2, 71.0, 93.2, 113.1 (d, J=18.6 Hz), 116.1, 132.0, 133.3, 144.4,
151.1 (d, J.sub.C-F=243.4 Hz), 172.0.
Example 24H
[0758]
2-Fluoro-3-methyloxyethyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5-
',5'-dimethyl-6'-oxo) tetrahydropyran (EM-1393). To a stirred
solution of compound 100 (33 mg, .about.0.08 mmol) in dry THF (3
mL) at 0.degree. C. under Ar(g) was dropwise added HMDSLi (1.0 M in
THF, 198 .mu.L, 0.20 mmol). After 25 min, the solution was cooled
down to -60.degree. C. and MeI (99 .mu.L, 225 mg, 1.58 mmol) was
added. The solution temperature was allowed to slowly rise from -60
to 0.degree. C. over 40 min. The reaction was quenched with aqueous
NH.sub.4Cl, extracted with EtOAc (4.times.), dried (MgSO.sub.4),
filtered and then evaporated. The crude compound was purified on
silica gel using 1:9 EtOAc/Hexanes as eluent to give EM-1393 (8 mg,
19% yield from compound 96, 5 steps) as a solid. IR (.nu.) 2924,
2871, 2832, 1722 (CO), 1620, 1586, 1513, 1454, 1384, 1357, 1306,
1290, 1268, 1201, 1149, 1133, 1116, 1032, 1018, 931, 874, 810, 789,
722 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 1.02 (3H, s), 1.28
(0) (3H, s), 1.28 (3) (3H, s), 1.29-2.30 (17H, m), 2.80 (2H, br s),
3.45 (3H, s), 3.75 (2H, t, J=4.8 Hz), 4.15 (2H, dd, J=5.0 Hz,
J'=4.6 Hz), 6.69 (1H, d, J=8.6 Hz), 6.98 (1H, d, J=13.2 Hz);
.sup.13C NMR (CDCl.sub.3) .delta. 14.4, 23.3, 25.6, 26.1, 27.4,
27.6, 27.8, 29.1, 29.7, 31.6, 34.9, 37.8, 38.8, 43.6, 47.3, 48.6,
59.9, 69.2, 71.0, 93.6, 113.1 (d, J=17.7 Hz), 116.1, 132.1, 133.4,
144.4, 151.1 (d, J=248.6 Hz), 177.8.
Example 25
Synthesis of
3-methylthioethyloxy-1,3,5(10)-estratrien-.delta.-lactone
[0759] These syntheses are described in Scheme 22. 98
Example 25A
[0760]
3Methylthioethyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(6'-oxo)
tetrahydropyran (CS-242). To a solution of
1,3,5(10)-estratrien-3-ol-17(R- )-spiro-2'(6'-oxo)tetrahydropyran
(EM-919, 40 mg, 0.117 mmol) in anhydrous acetonitrile (20 mL) at
room temperature under Ar(g) was added K.sub.2CO.sub.3 (16 mg,
0.117 mmol) and chloroethylmethyl sulfide (39 mg, 35 mL, 0.350
mmol). The solution was refluxing for 44 h. The acetonitrile
solution was evaporated to dryness and ethyl acetate (40 mL) was
then added. The organic phase was washed with water, brine and
dried with MgSO.sub.4 to gave crude product (49 mg). Purification
by flash chromatography on silica gel (SiO.sub.2, 3 g) using ethyl
acetate/hexanes (2:8) as eluent gave desired product (35 mg, 70%).
IR (.nu.) 2921, 1727, 1608, 1576, 1499, 1466, 1438, 1382, 1348,
1330, 1310, 1280, 1236, 1187, 1159, 1100, 1067, 1036, 992, 931,
914, 860, 818, 731 cm.sup.-1; .sup.1H NMR(CDCl.sub.3) .delta. 1.01
(3H, s), 1.25-2.57 (19H, m), 2.20 (3H, s) 2.85 (4H, t, J=6.88 Hz),
4.12 (2H, t, J=6.74 Hz), 6.62 (1H, d, J-2.49 Hz), 6.71 (1H, dd,
J1=2.58 Hz J2=5.89 Hz), 7.19 (1H, d, J=8.54 Hz); .sup.13C
NMR(CDCl.sub.3) .delta. 14.8, 15.9, 16.2, 23.5, 26.1, 27.5, 28.0,
29.5, 29.7, 32.0, 33.1, 34.0, 39.1, 43.7, 47.3, 48.9, 67.4, 93.3,
112.1, 114.6, 126.3, 132.6, 137.9, 156.5, 172.1 ppm. HRMS: FAB M/S
[M].sup.+ calculated for C25H.sub.35O.sub.3S: 415.23068, found
415.23250.
Example 26
Synthesis of 3-fluoro-1,3,5(10)
estratrien-17-spiro(dimethyl-.delta.-lacto- ne) Derivatives
[0761] These syntheses are described in Scheme 22. 99100
Example 26A
[0762] 2-Amino-1,3,5(10)-estratriene (101). The title compound was
synthesized from estrone according to the procedure reported by
Marrow and Hofer (J. Med. Chem. 9, 249-51, 1966).
Example 26B
[0763] 3-Fluoro-1,3,5(10)-estratriene (102). 3-Fluoroestrone was
synthesized from 2-aminoestrone by adapting the procedure reported
by Doyle and Bryker (U. Org. Chem. 44,1572-1574, 1979) for the
synthesis of arenediaxonium tetrafluoroborate salts from aromatic
amines. The procedure is as followed: To neat stirred boron
trifluoride etherate (642 .mu.L, 719 mg, 5.07 mmol) at -15.degree.
C. under Ar (g) was added a solution of 3-aminoestrone (101) (910
mg, 3.38 mmol) in dry CH.sub.2Cl.sub.2 (10 mL). After 15 min, a
solution of t-butylnitrite (482 .mu.L, 418 mg, 4.05 mmol) in dry
CH.sub.2Cl.sub.2 (5 mL) was dropwise added. The now dark brown
solution was stirred at -15.degree. C. for 15 min, then at
0.degree. C. for 30 min. Pentane was added to the solution and a
gummy solid precipitated. The solvent was decanted and the residue
was dried under vacuum to give a crude light brown solid. The neat
solid was heated under vacuum at 70-80.degree. C. in an oil bath
for 15 min to give a crude orange solid. Purification by flash
chromatography on SiO.sub.2 using 1:9 EtOAc/Hexanes gave
3-fluoroestrone (102) as a white solid (437 mg, 47%). M.p.
178.degree. C.; [.alpha.].sup.25.sub.D+152.0.de- gree. (c 1.03,
CDCl.sub.3); IR (.nu.) 3044, 3039, 2928, 1740 (CO), 1611, 1585,
1495, 1474, 1458, 1428, 1405, 1377, 1340, 1272, 1245, 1230, 1212,
1148, 1094, 1084, 1053, 1008, 908, 889, 817, 784, 718, 704, 642,
620, 580, 564, 467 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.92
(3H, s), 1.37-1.70 (6H, m), 1.93-2.44 (6H, m), 2.52 (1H, dd, J=18.8
Hz, J'=9.0 Hz), 2.91 (2H, dd, J=8.4 Hz, J'=3.8 Hz), 6.82 (2H, dq,
J=8.3 Hz, J'=2.8 Hz), 7.23 (1H, dd, J=8.5 Hz, J'=5.8 Hz); .sup.13C
NMR (CDCl.sub.3) .delta. 13.8, 21.6, 25.9, 26.3, 29.5, 31.5, 35.9,
38.1, 44.0, 47.9, 50.4, 112.5 (d, J=21.6 Hz), 115.1 (d, J=19.7 Hz),
126.8 (d, J=7.5 Hz), 135.3, 138.7, 161.0 (d, J=244.3 Hz), 20.7.
Example 26C
[0764]
3-Fluoro-17.beta.-hydroxy-17.alpha.-(4'-(2"-tetrahydro-2"H-pyranylo-
xy-1'-butynyl)-1,3,5(10)-estratriene (103). To a stirred solution
of 2-(3-butynyloxy)tetrahydro-2H-pyran (1.21 mL, 1.199 g, 7.77
mmol) in dry THF (45 mL) at -30.degree. C. under Ar(g) was dropwise
added MeLi (1.4 M in Et.sub.2O, 5.39 mL, 7.55 mmol). After 30 min.,
a solution of 3-fluoroestrone (102) in dry THE was added and the
solution was stirred for a further 2 h. The solution was quenched
with ice and aqueous sat NaHCO.sub.3 and extracted with EtOAc. The
organic layer was washed with H.sub.2O, brine, dried (MgSO.sub.4),
filtered then rotary evaporated to give a crude orange oil.
Purification by flash chromatography on silica gel using
EtOAc/Hexanes (1:9->2:8-->3:7) gave pure compound 103 (789
mg, 83%) as a solid. [.alpha.].sup.25.sub.D-4.7.degree. (c 1.14,
CDCl.sub.3); IR (.nu.) 3432 (br, OH), 2937, 2870, 1611, 1589, 1495,
1458, 1438, 1420, 1380, 1354, 1285, 1233, 1201, 1183, 1122, 1073,
1032, 970, 911, 870, 846, 815, 783, 728, 692, 563, 466 cm.sup.-1;
.sup.1H NMR (CDCl.sub.3) .delta. 0.87 (3H, s), 1.25-2.05 (17H, m),
2.22-2.36 (3H, m), 2.56 (2H, t, J=7.0 Hz), 2.84 (2H, br t, J=4.8
Hz), 3.49-3.59 (2H, m), 3.79-3.88 (2H, m), 4.66 (1H, br s), 6.80
(2H, dq, J=9.8 Hz, J'=2.8 Hz), 7.24 (1H, dd, J=8.4 Hz, P=5.8 Hz);
.sup.13C NMR (CDCl.sub.3) .delta. 12.7, 19.1, 20.2, 22.7, 25.3,
26.3, 26.9, 29.5, 30.4, 32.7, 38.9, 39.1, 43.5, 46.9, 49.3, 61.8,
65.7, 79.6, 82.9, 84.6, 98.4, 112.1 (d, J=20.9 Hz), 114.9 (d,
J=20.6 Hz), 126.6 (d, J=8.5 Hz), 135.7, 138.7 (d, J=6.7 Hz), 160.7
(d, J=244.8 Hz).
Example 26D
[0765]
3Fluoro-17.beta.-hydroxy-17.alpha.-(4'-(2"-tetrahydro-2"H-pyranylox-
y-1'-butyl)-1,3,5(10)-estratriene (104). A suspension of compound
103 (720 mg, 1.69 mmol) and 5% Pd/CaCO.sub.3 (123 mg) in MeOH (40
mL) was stirred under H.sub.2 (g) atmosphere (balloon) at room
temperature for 2 h. The mixture was filtered through celite and
the solvent rotary evaporated to give compound 104 (692 mg, 95%) as
a solid. This compound was used without further purification for
the next step. [.alpha.].sup.25.sub.D+28- .8.degree. (c 0.59,
CDCl.sub.3); IR (.nu.) 3464 (br w, OH), 2938, 2870, 1611, 1588,
1494, 1453, 1440, 1380, 1271, 1252, 1234, 1200, 1142, 1119, 1076,
1024, 989, 971, 930, 910, 868, 815, 782, 731 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.91 (3H, s), 1.31-2.35 (20H, m), 2.84 (2H, br
t, J=4.8 Hz), 3.43-3.52 (2H, m), 3.78-3.88 (2H, m), 6.80 (2H, dq,
J=8.3 Hz, J'=2.7 Hz), 7.23 (1H, dd, J=8.4 Hz, J'=6.1 Hz) ppm;
.sup.13C NMR (CDCl.sub.3) .delta. 14.4, 19.7, 20.5, 23.4, 25.5,
26.3, 27.3, 29.7, 30.4, 30.8, 31.6, 34.3, 36.4, 39.2, 39.4, 43.8,
46.7, 62.4, 67.7, 83.4, 98.4 (d, J=4.5 Hz), 112.3 (d, J=21.0 Hz),
115.0 (d, J=20.3 Hz), 126.7 (d, J=8.1 Hz), 136.0, 138.9 (d, J=6.6
Hz), 160.9 (d, J=244.1 Hz).
Example 26E
[0766]
3-Fluoro-17.beta.-hydroxyl-17.alpha.-(4'-hydroxybutyl)-1,3,5(10)-es-
tratriene (105). A solution of compound 104 (692 mg, 1.61 mmol),
PTSA monohydrate (31 mg, 0.16 mmol) in MeOH was stirred overnight
at room temperature. The volume of solvent was reduced to about 10
mL and EtOAc (125 mL) was added. This solution was washed with
aqueous saturated NaHCO.sub.3, H.sub.2O, brine, dried (MgSO.sub.4),
filtered then rotary evaporated to give a crude solid. Purification
on silica gel (3:7 to 1:1 EtOAc/Hexanes) gave compound 105 (469 mg,
84%) as a solid. [.alpha.].sup.25.sub.D+42.6.degree. (c 1.07,
CDCl.sub.3); IR (.nu.) 3362 (br s, OH), 2933, 2866, 1740 (w), 1611,
1589, 1495, 1458, 1420, 1376, 1303, 1271, 1236, 1144, 1036, 1008,
931, 912, 870, 816, 783, 728, 562, 468 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.91 (3H, s), 1.25-1.65 (17H, m), 1.85-2.35
(4H, m), 2.85 (2H, br t, J=4.6 Hz), 3.70 (2H, br t, J=5.9 Hz), 6.81
(2H, dq, J=8.4 Hz, J'=1.1 Hz), 7.22 (1H, dd, J=8.4 Hz, J'=6.0 Hz);
.sup.13C NMR (CDCl.sub.3) .delta. 14.3, 19.8, 23.4, 26.3, 27.3,
29.6, 31.6, 33.3, 34.4, 36.1, 39.4, 43.8, 46.7, 49.5, 62.7, 83.5,
112.3 (d, J=19.9 Hz) 115.1 (d. J20.5 Hz), 126.7 (d J=8.1 Hz), 135.9
138.9 (d, J=6.9 Hz), 160.9 (d, J=243.8 Hz).
Example 26F
[0767]
3-Fluoro-1,3,5(10)-estratrien-17(R)-spiro-2'-(6'-oxo)tetrahydropyra-
n (EM-1170). To a stirred solution of compound 105 (410 mg, 1.18
mmol) in acetone (40 mL) at 0.degree. C. was added Jones' reagent
(1.25 M, 1.04 mL, 1.30 mmol). The solution was stirred for 15 min.
then more Jones' reagent (1.25 M, 1.04 mL, 1.30 mmil) was added.
After 50 min., the excess reagent was quenched with isopropanol and
the solution was stirred for a further 10 min. The precipitate was
filtered through celite and the acetone rotary evaporated. The
residue was taken in EtOAc and the solution was washed with aqueous
saturated NaHCO.sub.3, H.sub.2O, brine, dried (MgSO.sub.4),
filtered, then rotary evaporated to give a crude solid.
Purification on silica gel (2:8-->3:7 EtOAC/Hexanes) gave
EM-1170 (298 mg, 72%) as a solid.
[.alpha.].sup.25.sub.D+47.0.degree. (c 1.09, CDCl.sub.3); IR (.nu.)
3435, 3040, 2955, 2933, 2906, 2879, 2836, 2815, 1885, 1736 (CO),
1611, 1586, 1494, 1467, 1435, 1419, 1383, 1366, 1356, 1328, 1312,
1290, 1263, 1232, 1182, 1140, 1103, 1067, 1026, 990, 964, 933, 912,
861, 822, 782, 718, 670, 636, 584, 563, 536, 508 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) .delta. 1.02 (3H, s), 1.23-2.58 (19H, m), 2.85
(2H, br t, J=5.2 Hz), 6.80 (2H, dq, J=8.4 Hz, J'=2.7), 7.23 (1H,
dd, J=8.4 Hz, J'=6.0 Hz); .sup.13C NMR (CDCl.sub.3) .delta. 14.2,
15.7, 23.4, 25.9, 27.1, 27.8, 29.4, 31.8, 33.8, 38.7, 43.5, 47.1,
48.7, 93.1, 112.2 (d, J=20.5 Hz), 114.9 (d, J=20.1 Hz), 126.6 (d,
J=8.5 Hz), 135.4, 138.6 (d, J=6.7 Hz), 160.8 (d, J=243.8 Hz),
171.9.
Example 26G
[0768]
3-Fluoro-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo-
)tetrahydropyran (EM-1157). LDA was prepared as follows. To a
stirred solution of diisopropylamine (501 .mu.L, 386 mg, 3.82 mmol)
in dry THF (20 mL) at -78.degree. C. under Ar(g) was dropwide added
n-BuLi (1.6 M in Hexane, 2.38 mL, 3.82 mmol). The solution was
stirred at room temperature for 30 min then was cooled down to
-78.degree. C. for the addition of a solution of EM-1170 (218 mg,
0.64 mmol) in dry THF (15 mL). The solution was then stirred at
room temperature for 1 h. then cooled down to -78.degree. C. MeI
(476 .mu.L, 1.084 g, 0.64 mmol)was added and the solution was
stirred at -78.degree. C. for 5 h and was allowed to rise to room
temperature overnight. The reaction was quenched with ice/H.sub.2O,
extracted with EtOAc, washed with aqeous saturated NH.sub.4Cl, 1M
aqueous Na.sub.2SO.sub.3, H.sub.2O, brine, dried (MgSO.sub.4),
filtered then rotary evaporated to give a crude solid. Purification
on silica gel (1:9 EtOAc/Hexanes) gave EM-1157 (40 mg, 17%) as a
solid. [.alpha.].sup.25.sub.D+43.3.degree. (c 1.09, CHCl.sub.3); IR
(.nu.) 3430, 2969, 2941, 2867, 1734 (CO), 1492, 1458, 1387, 1306,
1235, 1201, 1148, 1132, 1062, 1032, 1017, 979, 915, 874, 818, 785,
727, 597, 580, 567, 530, 508 cm.sup.-1; .sup.1H NMR (CDCl.sub.3)
.delta. 1.02 (3H, s), 1.28 (3H, s), 1.28 (3H, s), 1.29-1.74 (10H,
m), 1.83-2.36 (7H, m), 2.85 (2H, t, J=5.1 Hz), 6.80 (2H, dq, J=8.6
Hz, J'=2.7 Hz), 7.22 (1H, dd, J=8.6 Hz, J=6.2 Hz); .sup.13C NMR
(CDCl.sub.3) .delta. 14.4, 23.3, 25.6, 26.1, 27.2, 27.7, 27.8,
29.5, 31.6, 31.9, 34.8, 37.8, 38.9, 43.7, 47.2, 48.7, 93.6, 112.3
(d, J=19, 9 Hz), 115.1 (d, J--19.5 Hz), 126.7 (d, J--8.4 Hz),
135.5, 138.7 (d, J=6.8 Hz), 160.9 (d, J=244.2 Hz), 177.8.
Example 27
3-sulfonyl derivatives of 1,3,5(10)-estatrien-17-spiro-(dimethyl
.delta.-lactone)
[0769] These syntheses are described in Scheme 23. 101
Example 27A
[0770]
3-alkylsulfonyloxy-1,3,5(10)estratrien-17(R)-spiro-2'-(5',5'-dimeth-
yl-6'-oxo)tetrahydropyran (106). A solution of compound 89 in
dichloromethane (3.0% W/V) was treated with alkylsulfonyl chloride
(1.1 equiv) and triethylamine (1.5 equiv), and stirred for 2 h. The
reaction mixture was quenched with distilled water and diluted with
dichloromethane. The organic phase was washed with brine and 5%
sodium bicarbonate, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 49-1 to hexanes-ethyl acetate 9-1) and
trituration (hexanes-acetone 19-1) to provide compound 106 (e.g.,
EM-1364-CS, R=Et, 90%): IR (CHCB) .nu. 3025, 2944, 2874, 1708,
1605, 1492, 1366, 1213, 1136 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 1.02 (s, 3H), 1.28 (s, 3H), 1.29 (s, 3H),
1.30-1.80 (m, 13H), 1.80-2.05 (m, 5H), 2.18 (m, 1H), 2.34 (m, 1H),
2.88 (m, 2H), 3.26 (q, J=14.8 Hz, 2H), 6.99-7.05 (m, 2H), 7.30 (d,
J=8.5 Hz, 1H); .sup.13C NMR (75 Hz, CDCl.sub.3) .delta. 8.23,
14.41, 23.29, 25.58, 25.92, 27.67, 27.76, 29.46, 31.61, 31.93,
34.79, 37.80, 38.68, 43.81, 44.89, 47.20, 48.72, 93.53, 118.89,
121.90, 126.77, 138.84, 139.13, 146.99, 177.76.
Example 28
3-carbonyl Substituted
1,3,5(10)-estatrien-17-spiro-(dimethyl-.delta.-lact- one)
[0771] These syntheses are described in Scheme 24. 102
Example 28A
[0772]
3-trifluoromethanesulfonyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(-
5',5'-dimethyl-6'-oxo)tetrahydropyran (107). Under argon
atmosphere, a solution of compound 89 (500 mg, 1.35 mmol),
2,6-lutidine (0.355 mL, 3.05 mmol) and 4-dimethylaminopyridine (33
mg, 0.27 mmol) in dry dichloromethane (25 mL) was cooled at
0.degree. C., treated with trifluoromethanesulfonic anhydride
(0.308 mL, 1.83 mmol) and stirred for 45 min. The reaction mixture
was quenched with water and extracted with dichloromethane. The
organic phase was washed with 2% hydrochloric acid, saturated
sodium bicarbonate and water, dried over magnesium sulfate,
filtered, and evaporated. The crude oil was purified by flash
chromatography (hexanes-ethyl acetate 49-1 to hexanes-ethyl acetate
4-1) to provide trifluoromethanesulfonate 107 (EM-1399) (540 mg,
80%): IR (CHCl.sub.3) 2957, 2872, 1711, 1490, 1426, 1248, 1214,
1141, 926, 846, 621 cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 1.03 (s, 3H), 1.28 (s, 3H), 1.29 (s, 3H), 1.35-2.40 (m,
17H), 2.88 (m, 2H), 6.98 (s 1H), 7.02 (d J=8 Hz, 1H), 7.33 (d,
J=8.7 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.32,
23.22, 25.48, 25.80, 26.89, 27.57, 27.68, 29.37, 31.49, 31.80,
34.69, 37.72, 38.46, 43.66, 47.10, 48.59, 93.43, 116.54, 118.08,
120.80, 121.07, 127.05, 139.31, 140.43, 147.46, 177.70.
Example 28B
[0773]
3-carboxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'-ox-
o)tetrahydropyran (EM-1401). Method A: A mixture of compound 107
(560 mg, 1.12 mmol), potassium acetate (440 mg, 4.48 mmol),
palladium acetate (12.6 mg, 0.056 mmol), and
1,1'-bis(diphenylphosphino)ferrocene (125 mg, 0.255 mmol) in
dimethyl sulfoxide (20 mL) was purged with carbon monoxide for 20
min and stirred over under a carbon monoxide balloon at 80.degree.
C. over a 3 h period. The reaction mixture was diluted with 0.5 N
hydrochloric acid and extracted with dichloromethane. The organic
phase was washed with water, dried over magnesium sulfate,
filtered, and evaporated. The reaction mixture was purified by
flash chromatography (dichloromethane-methanol 19-1 to
dichloromethane-methanol 4-1) to provide the carboxylic acid
EM-1401 (300 mg, 68%): IR (KBr) 2937, 2872, 1718, 1676, 1388, 1314,
1230, 1180, 1160 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 0.75 (s, 3H), 1.01 (s, 6H),
1.10-2.17 (m, 17H), 2.65 (m, 2H), 7.09 (d, J=8.1 Hz, 1H), 7.48 (s,
1H), 7.51 (d, J=8.5 Hz, 1H); .sup.13C NMR (75 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 13.71, 22.75, 24.98, 25.27, 26.65,
26.87, 28.76, 30.84, 31.46, 34.21, 37.33, 38.22, 43.84, 46.74,
93.92, 124.84, 126.52, 127.32, 129.91, 136.31, 144.94, 168.70,
178.97.
Example 28C
[0774]
3-alkoxycarbonyl-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethy-
l-6'-oxo)tetrahydropyran (108). A mixture of compound 107,
triethylamine (3.25 equiv), palladium acetate (0.07 equiv),
1,3-bis(diphenylphosphino)p- ropane (0.06 equiv), and alcohol (1.5
equiv to large excess) in DMF (10% W/V) was purged with carbon
monoxide for 20 min and stirred under a carbon monoxide balloon at
90.degree. C. over a 16 h period. The reaction mixture was cooled
at room temperature, diluted with water and extracted with
dichloromethane. The organic phase was washed with brine, dried
over magnesium sulfate, filtered, and evaporated. The reaction
mixture was purified by 3 flash chromatographies (2 times with
benzene-acetone 4-1 and hexanes-ethyl acetate 7-3) to provide
compound 108 (e.g., EM-1398, R=benzyl, 70%): IR (CHCl.sub.3) 2938,
1716, 1293, 1262, 1177, 1152, 1130, 1109, 732 cm.sup.-1; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 1.02 (s, 3H), 1.28 (s, 3H), 1.29
(s, 3H), 1.34-1.41 (m, 17H), 2.91 (m, 2H), 5.35 (s, 2H), 7.33-7.45
(m, 6H), 7.79 (s, 1H), 7.83 (d, J=8.1 Hz, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 14.39, 23.28, 25.55, 25.74, 27.14, 27.64,
27.75, 29.25, 31.56, 31.93, 34.75, 37.77, 38.56, 44.34, 47.16,
48.82, 66.42, 93.50, 125.34, 126.90, 127.45, 128.05, 128.10,
128.52, 130.23, 136.22, 136.81, 145.49, 166.55, 177.75.
Example 28D
[0775]
3-carboxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6'-ox-
o)tetrahydropyran (EM-1401). Method B: A mixture of compound 108
(350 mg, 0.72 mmol) and 10% palladium on activated carbon (50 mg)
in ethyl acetate (40 mL) was stirred under an hydrogen balloon over
a 3 h period. The reaction mixture was filtered on celite and
evaporated. The crude mixture was purified by flash chromatography
(dichloromethane-THF 19-1 to dichloromethane-THF 3-1) to provide
the carboxylic acid EM-1401 (240 mg, 84%). A sample was
recrystallized in methanol-THF (the characterization was described
previously).
Example 28E
[0776]
3-carboxamido-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethyl-6-
'-oxo)tetrahydropyran (109). Under argon atmosphere, a solution of
EM-1401 and pyridine (15 equiv) in dry dichloromethane (1.6% W/V)
was cooled at 0.degree. C., treated with oxalyl chloride (6 equiv)
and stirred for 0.5 h. The reaction mixture was allowed to reach
room temperature and stirred over a 4 h period. The reaction
mixture was evaporated, dissolved in dry THF (1.6% W/V), cooled at
0.degree. C., treated with 10 equiv of amine and stirred for 15
min. The reaction mixture was quenched with water, extracted with
dichloromethane, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes-acetone 19-1 to hexanes-acetone 3-2) to provide compound
109 (e.g., EM-1404, R.sup.1=R.sup.2=H, 65%): IR (CHCl.sub.3) 3433,
3350, 2941, 2873, 1702, 1664, 1611, 1388, 1310, 1159 cm.sup.-1;
.sup.1H NMR (300 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 0.73 (s, 3H),
0.99 (s, 6H), 1.10-2.16 (m, 17H), 2.64 (m, 2H), 7.08 (d, J--8.0 Hz,
1H), 7.30 (s, 1H), 7.32 (d, J.apprxeq.9 Hz, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3+CD.sub.3OD) .delta. 13.69, 22.72, 24.96, 25.27,
26.64, 26.84, 28.78, 29.09, 30.81, 31.44, 34.19, 37.31, 38.27,
43.72, 46.74, 93.92, 124.20, 124.93, 127.70, 130.00, 136.45,
143.88, 170.63, 178.96.
Example 29
Synthesis of
2-carboxy/alkoxycarbonyl/carboxamide-3-alkoxy-1,3,5(10)-estra-
trien-17spiro-(dimethyl-.delta.-lactone) Derivatives
[0777] These syntheses are described in Scheme 25. 103
[0778]
2-formyl-1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-dimethyl-6-
'-oxo)tetrahydropyran (110). Lactone 89 (1.0 g, 2.72 mmol) was
dissolved in dry 1,2-dichloroethane (9 mL) under argon atmosphere.
SnCl.sub.4 (0.16 mL, 1.37 mmol) and Bu.sub.3N (0.52 mL, 2.18 mmol)
were added successively. The mixture was stirred at room
temperature for 20 min. Formaldehyde (0.23 g, 7.84 mmol) was added
and the mixture was stirred at reflux for 6 h. The reaction mixture
was poured into aq acid (pH=2) and, was extracted with
CH.sub.2Cl.sub.2. The organic layers were washed with brine
solution, dried (Na.sub.2SO.sub.4) filtered and concentrated in
vacuo. The crude product was purified by flash chromatography on
silica gel, eluting with (95:5 to 80:20) hexanes-acetone to yield
0.74 g (69%) of the product; IR (NaCl cm.sup.-1): 3164, 2937, 2872,
1716, 1652, 1571, 1487, 1466, 1386, 1298, 1152, 1017, 914, 731;
.sup.1H NMR (CDCl.sub.3) 1.00 (s, 3H), 1.26 (s, 6H) 1.23-2.40 (m,
17H), 2.80-2.90 (m, 2H), 6.66 (s, 1H), 7.39 (s, 1H), 9.79 (s, 1H),
10.77 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta. 14.3, 23.2, 25.4,
25.9, 26.8, 27.6, 27.7, 30.0, 31.4, 31.6, 34.6, 37.7, 38.6, 42.9,
47.0, 48.5, 93.4, 116.9, 118.9, 130.3, 132.2, 147.8, 159.2, 177.7,
196.0.
[0779]
2-formyl-3-(2"-methoxyethyloxy)-1,3,5(10)-estratrien-17(R)-spiro-2'-
-(5',5'-dimethyl-6'-oxo)tetrahydropyran (111). To a solution of
aldehyde 110 (0.74 g, 1.87 mmol) in CH.sub.3CN (19 mL),
Cs.sub.2CO.sub.3 (0.96 g, 2.95 mmol), NaI (55 mg, 0.37 mmol) and
2-chloroethyl methyl ether (0.86 mL, 9.42 mmol) were added, and the
mixture was stirred at reflux for 20 h. The reaction mixture was
quenched with brine solution, and was extracted with
CH.sub.2Cl.sub.2. The organic layers were dried (Na.sub.2SO.sub.4)
filtered and evaporated to crude product, which was purified by
flash column chromatography (CH.sub.2Cl.sub.2: Acetone, 95:5) to
give 0.62 g (73%) of the desired compound; IR (NaCl, cm.sup.-1):
2942, 2879, 1714, 1675, 1609, 1459, 1394, 1308, 1269, 1152, 874;
.sup.1H NMR (CDCl.sub.3) .delta. 1.01 (s, 3H), 1.28 (s, 6H)
1.25-2.20 (m, 16H), 2.35-2.45 (m, 1H), 2.85-2.95 (m, 2H), 3.45 (s,
3H), 3.79 (t, 2H, J=4.7 Hz), 4.20 (t, 2H, J=4.6 Hz), 6.68 (s, 1H),
7.76 (s, 1H), 10.45 (s, 1H); .sup.13C NMR (CDCl.sub.3) .delta.
14.4, 23.3, 25.6, 26.0, 27.1, 27.7, 27.8, 30.4, 31.6, 31.9, 34.9,
37.8, 38.9, 43.4, 47.3, 48.7, 59.4, 68.3, 70.9, 93.6, 113.0, 123.0,
125.2, 133.1, 146.0, 159.2, 177.8, 189.6.
[0780]
2-carboxy-3-(2"-methoxyethyloxy)-1,3,5(10)-estratrien-17(R)-spiro-2-
'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1405). The aldehyde
(130 mg, 0.29 mmol) was solubilized in pyridine (4 mL) and
n-Bu.sub.4NMnO.sub.4 (140 mg, 2 eq., 0.38 mmol) (prepared by mixing
the aquous solution of KMnO.sub.4 and n-Bu.sub.4NBr in water and
filtering the precipitate) was added to the above solution. After
16 h the mixture was poured in a solution of 300 mg of NaHSO.sub.3
in 30 ml of 1 N HCl. The final product was then extracted with
ethyl acetate and dried (Na.sub.2SO.sub.4). Evaporation of solvents
under vacuum gave 140 mg of a semi-solid mixture, which was
purified on C.sub.18 reverse phase gel using MeCN:MeOH:H.sub.2O in
a proportion of 35:35:30 and then 40:35:25 to give the acid (44 mg,
32%); IR (KBr, cm.sup.-1) 3274, 2944, 2890, 1723, 1613, 1422;
.sup.1H NMR(CDCl.sub.3) .delta. 10.96 (bs, 1H), 8.08 (s, 1H), 6.73
(s, 1H), 4.32 (t, 2H, J=5.2 Hz), 3.78 (t, 2H, J=4.4 Hz), 3.45 (s,
3H), 2.87-2.88 (m, 2H), 1.28 (s, 6H), 1.02 (s, 3H); .sup.13C NMR
(CDCl.sub.3) .delta. 177.7, 165.7, 155.2, 144.6, 134.6, 130, 115.7,
113.6, 93.5, 69.9, 69.2, 59.1, 48.6, 47.2, 43.4, 38.7, 37.7, 34.8,
31.7, 31.5, 29.9, 27.7, 27.6, 26.9, 25.9, 25.5, 23.2, 14.4.
[0781] 2-carbomethoxy-3-(2"-methoxyethyloxy)-1,3,5(10)-estratrien
17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1402).
The acid EM-1405 (44 mg, 0.094 mmol) was solubilized in a mixture
of ahydrous methanol (2 mL) and anhydrous benzene (4 mL). Then, a
2M solution of TMSCHN.sub.2 (250 mL, 0.5 mmol, 5 eq.) in hexane was
added and the mixture was stirred for 4 h at room temperature. The
solvants were removed under vacuum and the oil was purified on
silica gel column using acetone:hexanes as an eluent to give the
methyl ester (32 mg, 70%); IR (KBr, cm.sup.-1) 2941, 2813, 1716,
1611, 1271; .sup.1H NMR (CDCl.sub.3) .delta. 7.73 (s, 1H), 6.69 (s,
1H), 4.15 (t, 2H, J=4.8 Hz), 3.86 (s, 3H, J--4.5 Hz), 3.79 (t, 2H),
3.47 (s, 3H), 2.82-2.90 (m, 2H), 1.28 (s, 6H), 1.02 (s, 3H);
.sup.13C NMR (CDCl.sub.3) .delta. 177.7, 166.8, 156.5, 142.8,
132.5, 128.8, 118.0, 114.6, 93.5, 71.0, 69.1, 59.3, 51.7, 48.6,
47.2, 43.4, 38.9, 37.7, 34.7, 318, 31.5, 29.8, 27.7, 27.6, 27.1,
25.9, 25.5, 23.2, 14.4.
[0782] 2-dimethylcarbamoyl-3-(2"-methoxyethyloxy)
1,3,5(10)-estratrien
17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1413): To
a solution of the acid EM-1405 (50 mg, 0.11 mmol) in anhydrous
CH.sub.2Cl.sub.2 (5 mL) at 0.degree. C., were added pyridine (40
.mu.L, 0.49 mmol) oxalyl chloride (30 .mu.L, 0.34 mmol) and DMF (10
.mu.L, 0.13 mmol). The mixture was allowed to warm to room
temperature and stirred over a period of 3 h. The volatiles were
removed under vacuum. The dry residue was dissolved in anhydrous
CH.sub.2C.sub.2 (10 mL) under argon and cooled to 0.degree. C. A 2M
solution of dimethylamine (1 mL, 2.1 mmol) in THF was added and the
temperature was raised to 25.degree. C., and the mixture was
stirred for 1 h. The reaction mixture was diluted with
CH.sub.2Cl.sub.2, washed with brine, dried (MgSO.sub.4) and
concentrated to dryness. The residue was purified by silica gel
column chromatography with hexanes/acetone (7/3) to afford 35 mg
(70%) of the dimethylamide. IR (NaCl, cm.sup.-1): 2927, 2871, 1719,
1630, 1148, 1134; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.18
(s, 1H), 6.59 (s, 1H), 4.09 (m, 2H), 3.69 (m, 2H), 3.41 (s, 3H),
3.09 (s, 3H), 2.88 (s, 3H), 2.87-2.80 (m, 2H), 2.35-2.22 (m, 1H),
1.98-1.80 (m, 3H), 1.77-1.31 (m, 13H), 1.28 (s, 6H, 2.times.CH),
0.88 (s, 3H, 18-Me); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
177.8, 169.7, 152.4, 138.9, 133.1, 125.3, 124.4, 112.7, 93.6, 71.0,
68.2, 59.2, 48.7, 47.3, 43.6, 39.0, 38.3, 37.8, 34.9, 34.7, 31.9,
31.6, 29.8, 27.8, 27.6, 27.4, 26.0, 25.6, 23.3, 14.4.
[0783] 2-carbamoyl-3-(2"-methoxyethyloxy)-1,3,5(10)-estratrien
17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1424).
The acid of EM-1405 (0.11 mmol) was dissolved in a mixture of
CH.sub.2Cl.sub.2 (5 mL) and 1 (5 mL) and cooled to 0.degree. C. 28%
of Aq. ammonium hydroxide (260 .mu.L, 23 mmol) was added, and the
mixture was allowed to warm to room temperature and stirred over a
period of 3 h. The reaction mixture was then diluted with
CH.sub.2Cl.sub.2, washed with brine, dried (MgSO.sub.4) and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography with hexanes/acetone (6/4) to
afford 27 mg (45%) of the amide; IR (NaCl, cm.sup.-1): 3446, 3335,
3178, 2926, 2872, 1717, 1664, 1589, 1427, 1151, 1134; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 8.11 (s, 1H), 7.98 (br.s, 1H), 6.67
(s, 1H), 5.64 (br.s, 1H), 4.23-4.20 (m, 2H), 3.79-3.76 (m, 2H),
3.42 (s, 3H), 2.89-2.86 (m, 2H), 2.52-2.45 (m, 1H), 2.16-1.30 (m,
16H), 1.28 (s, 6H, 2.times.CH.sub.3), 1.01 (s, 3H, 18-Me); .sup.13C
NMR (75 NM, CDCl.sub.3) a 177.8, 167.2, 154.9, 142.5, 133.5, 129.6,
118.9, 113.3, 93.6, 70.5, 68.1, 58.9, 48.7, 47.3, 43.6, 39.0, 37.8,
34.9, 31.9, 31.6, 29.8, 27.8, 27.7, 27.1, 26.1, 25.6, 23.3,
14.4.
Example 30
3-hydroxy derivatives of
2-cyano-1,3,5(10)-estratrien-17-spiro-(dimethyl-.-
delta.-lactone)
[0784] These syntheses are described in Scheme 26. 104
Example 30A
[0785]
2-oximino-1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-dimethyl--
6'-oxo)tetrahydropyran (113). Under argon atmosphere, a solution of
compound 112 (215 mg, 0.54 mmol) in anhydrous ethanol-pyridine 1-1
(4 mL) was treated with hydroxylamine hydrochloride (56.6 mg, 0.814
mmol) and stirred at room temperature for 25 min. The reaction
mixture was evaporated, diluted with water, and extracted 3 times
with dichloromethane. The combined organic phase was washed with
brine, dried over sodium sulfate, filtered, and evaporated to
provide the oxime 113 (217 mg, 98%): .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 1.02 (s, 3H), 1.29 (s, 6H), 1.43-1.70 (m, 10H),
1.89-2.12 (m, 6F), 2.22-2.37 (m, 1H), 2.80-2.87 (m, 2H), 6.69 (s,
1H), 7.05 (s, 1H), 8.15 (broad s, 1H), 8.20 (s, 1H), 9.61 (s,
1H).
Example 30B
[0786]
3-acetoxy-2-cyano-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimeth-
yl-6'-oxo)tetrahydropyran (114a). A solution of compound 113 (180
mg, 0.44 mmol) and acetic anhydride (125 .mu.L, 1.32 mmol) in
pyridine (3.5 mL) was refluxed for 1 h. The reaction mixture was
evaporated, diluted with dichloromethane, and washed 3 times with
water, 1 time with saturated sodium bicarbonate and 1 time with
brine. The organic phase was dried over magnesium sulfate,
filtered, and evaporated. The crude mixture was purified by flash
chromatography (dichloromethane to dichloromethane-ethyl acetate
19-1) to provide acetate 114a (145 mg, 76%): IR (CHCl.sub.3) 2933,
2872, 2229, 1773, 1718, 1613, 1494, 1183 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 1.02 (s, 3H), 1.28 (s, 6H), 1.34-1.89
(m, 11H), 1.94-2.33 (m, 6H), 2.37 (s, 3H), 2.89-2.94 (m, 2H), 6.95
(s, 1H), 7.55 (s, 1H).
Example 30C
[0787]
2-cyano-1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-dimethyl-6'-
-oxo)tetrahydropyran (114b). A solution of compound 114a (135 mg,
0.34 mmol) in methanol (10 mL) was treated with 10% potassium
carbonate (1 mL) and stirred 30 min. The reaction mixture was
acidified to pH 2 with 1 N hydrochloric acid and extracted 3 times
with dichloromethane. The combined organic phase was washed with
water, saturated sodium bicarbonate, and brine, dried over
magnesium sulfate, filtered, and evaporated. The crude phenol 114b
(115 mg, 85%) was directly used for the next step: .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 0.97 (s, 3H), 1.29 (s, 6H), 1.26-2.14 (m,
16H), 2.21-2.28 (m, 1H), 2.82-2.86 (m, 2H), 6.69 (s, 1H), 6.91 (s,
1H), 7.35 (s, 1H).
Example 30D
[0788]
3-alkyloxy-2cyano-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimeth-
yl-6'-oxo)tetrahydropyran (114c). Under argon atmosphere, a
suspension of compound 114b, alkyl iodide (5 equiv) and cesium
carbonate (1.5 equiv) in anhydrous acetonitrile (1% W/V) was
stirred for 16 h with refluxing condition if necessary. The
reaction mixture was quenched with brine and extracted 3 times with
dichloromethane. The combined organic phase was washed with brine,
dried over magnesium sulfate, filtered, and evaporated. The crude
mixture was purified by flash chromatography (dichloromethane to
dichloromethane-ethyl acetate 10-1) and recrystallization
(methanol) to provide compound 114c (e.g., EM-1396,
R=(CH.sub.2).sub.2OCH.sub.3, 75%): IR (CHCl.sub.3) 3013, 2941,
2881, 2229, 1710, 1610, 1500, 1304, 1136 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 1.01 (s, 3H), 1.28 (s, 6H), 1.20-1.75
(m, 10H), 1.80-2.20 (m, 6H), 2.20-2.35 (m, 1H), 2.80-2.95 (m, 2H),
3.47 (s, 3), 3.79 (t, J=4.8 Hz, 2H), 4.17 (t, J=4.8 Hz, 2H), 6.67
(s, 1H), 7.44 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
14.41, 23.25, 25.58, 25.90, 26.96, 27.66, 27.74, 30.24, 31.59,
31.78, 34.79, 37.80, 38.68, 43.16, 47.20, 48.60, 59.55, 68.78,
70.71, 93.43, 99.55, 112.80, 116.98, 130.72, 133.31, 144.09,
158.29, 177.70.
Example 31
Synthesis of
1,3,5(10)-estratrien-6-one-17-spiro-(dimethyl-.delta.-lactone-
s)
[0789] These syntheses are described in Schemes 27 and 28. 105106
107
Example 31A
[0790]
3-alkoxy-1,3,5(10)-estratrien-6-one-17(R)-spiro-2'-(5',5'-dimethyl--
6'-oxo)tetrahydropyran (120). Under argon atmosphere, a solution of
chromium (VI) oxide (10 equiv) in anhydrous dichloromethane (22%
W/V) was treated with 3,5-dimethylpyrazole (10 equiv), cooled at
-20.degree. C., and stirred 20 min. The reaction mixture was
treated with a cool solution (-20.degree. C.) of
3-alkoxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-d-
imethyl-6'-oxo)tetrahydropyran (89a) in anhydrous dichloromethane
(29% W/V) and stirred 1.5 h. The reaction mixture was then poured
on silica gel and eluted with dichloromethane to
dichloromethane-ethyl acetate 3-1. The crude mixture was purified
by flash chromatography (hexanes to hexanes-acetone 5-1) to provide
compound 120 (e.g., EM-1394, R=Me, 11%): IR (CHCl.sub.3) 2990,
2963, 1709, 1678, 1608, 1493, 1217, 1152 cm.sup.-1; .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 1.03 (s, 3H), 1.29 (s, 6H), 1.47-2.25
(m, 14H), 2.43-2.47 (m, 2H), 2.76 (dd, J=2.9 and 16.4 Hz, 1H), 3.85
(s, 3H), 7.11 (dd, J=2.8 and 8.6 Hz, 1H), 7.34 (d, J=8.6 Hz, 1H),
7.56 (d, J=2.8 Hz, 1H); .sup.13C NMR (75 Hz, CDCl.sub.3) .delta.
14.32, 23.03, 25.37, 25.55, 27.67, 27.76, 31.53, 31.62, 34.61,
37.82, 40.39, 42.65, 44.15, 47.07, 48.61, 55.51, 93.20, 109.69,
121.58, 126.52, 133.29, 139.18, 158.27, 177.60, 197.53.
Example 31B
[0791]
3-alkoxy-1,3,5(10)-estratrien-9.alpha.-ol-6-one-17(R)-spiro-2'-(5',-
5'-dimethyl-6'-oxo)tetrahydropyran (121). Under argon atmosphere,
compound 89a in acetonitrile (5.5% W/V) was treated with copper
iodide (0.01 equiv) and t-butyl hydroperoxide (6.7 equiv), and
stirred and heated at 50.degree. C. for 20 h. The reaction mixture
was poured into 10% sodium sulfite and extracted with ethyl
acetate. The organic phase was washed with saturated sodium
bicarbonate, brine and water, dried over magnesium sulfate,
filtered, and evaporated. The crude mixture was purified by flash
chromatography (hexanes-ethyl acetate 19-1 to hexanes-ethyl acetate
7-3) to provide compound 121 (e.g., EM-1386, R.sup.1=CH.sub.3,
44%); IR (CHCl.sub.3) 3603, 3486, 3012, 2985, 2872, 1710, 1682,
1604, 1494, 1288, 1248, 1144, 1030 cm.sup.-1; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 1.04 (s, 3H), 1.29 (s, 6H), 1.40-1.80 (m, 5H),
1.80-2.23 (m, 8H), 2.35 (m, 1H), 2.43 (dd, J=2.5 and J=9.5 Hz, 1H),
2.52 (dd, J=3.7 and 17.6 Hz, 1H), 2.78 (dd, J=8.7 and 12.4 Hz, 1H),
3.86 (s, 3H), 7.13 (dd, J=2.9 and 8.7 Hz, 1H), 7.51 (d, J=8.7 Hz,
1H), 7.56 (d, J=2.9 Hz, 1H); .sup.13C NMR (75 Hz, CDCl.sub.3)
.delta. 13.51, 22.75, 25.75, 27.54, 27.60, 27.70, 31.47, 32.18,
34.75, 37.77, 41.73, 41.96, 46.98, 55.49, 68.96, 93.30, 110.37,
121.24, 125.53, 132.75, 139.96, 159.46, 177.79, 197.85.
Example 31C
[0792]
3,9.alpha.-dialkoxy-1,3,5(10)-estratrien-6-one-17(R)-spiro-2'-(5',5-
'-dimethyl-6'-oxo)tetrahydropyran (122). A solution of compound 121
in methanol (0.8% W/V) was treated with oxalic acid (4 equiv) and a
drop of water and refluxed for 0.5 h. The reaction mixture was
cooled at room temperature, evaporated and diluted with ethyl
acetate. The obtained solution was washed with 5% sodium
bicarbonate and brine, dried over magnesium sulfate, filtered, and
evaporated. The crude mixture was purified by flash chromatography
(hexanes-ethyl acetate 9-1 to hexanes-ethyl acetate 7-3) to provide
compound 122 (e.g., R.sup.1,R.sup.2=CH.sub.3, 35%): IR (CHCl.sub.3)
2955, 2874, 2824, 1720, 1683, 1604, 1493, 1286, 1268, 1248, 1141
cm.sup.-1; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.04 (s, 3H),
1.28 (s, 6H), 1.35-2.20 (m, 12H), 2.35 (m, 1H), 2.43 (dd, J=4.6 and
17.9 Hz, 1H), 2.64 (m, 1H), 2.80 (m, 1H), 2.83 (s, 3H), 3.86 (s,
3H), 7.08 (dd, J=2.8 and 8.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.58
(d, J=2.8 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 13.79,
22.92, 24.48, 25.81, 27.37, 27.71, 27.77, 31.50, 34.74, 37.29, 37
82, 42 02, 42.74, 46.84, 49.27, 55.52, 72.96, 93.34, 110.89,
119.61, 127.56, 133.75, 136.72, 159.54, 177.79, 198.26.
Example 32
2,3-oximino-1,3,5(10)-estratrien-17-spiro-(dimethyl-.delta.-lactone)
[0793] This synthesis is described in Scheme 29. 108
Example 32A
[0794] Synthesis of
1,3,5(10)-estratrien-(2,3-oximino)-17(R)-spiro-2'-(5',-
5'-dimethyl-6'-oxo)tetrahydropyran (EM-1407-CS). A solution of
hydroxylamine-O-sulfonic acid (14 mg, 0.12 mmol) and sodium sulfate
(2 mg, 0.014 mmol) in methanol-water 2-1 (1.5 mL) was treated with
compound 110 (40 mg, 0.10 mmol) and stirred for 30 min. The
reaction mixture was diluted with water (5 mL) and dichloromethane
(5 mL), cooled at -10.degree. C., vigorously stirred for 15 min,
treated with sodium bicarbonate (17 mg, 0.20 mmol), and stirred for
30 min. The reaction mixture was allowed to reach room temperature
and stirred for 1 h. The two phases were separated, then the
aqueous phase was extracted 4 times with dichloromethane. The
combined organic phase was dried over magnesium sulfate, filtered,
and evaporated. The crude mixture was purified by flash
chromatography (hexanes to hexanes-acetone 19-1) to provide the
1,2-benzisoxazole EM-1407-CS (30 mg, 77%): IR (CHCl.sub.3) 3013,
2937, 2872, 1708, 1625, 1507, 1299, 1153, 1017 cm.sup.-1; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 1.03 (s, 3H), 1.29 (s, 6H),
1.30-180 (m, 10H), 1.80-2.20 (m, 5H), 2.20-245 (m, 2H), 3.01-3.06
(m, 2H), 7.32 (s, 1H), 7.61 (s, 1H), 8.61 (s, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 14.41, 23.38, 25.59, 26.11, 27.10, 27.67,
27.78, 330.15, 31.59, 31.84, 34.78, 37.81, 38.76, 43.71, 47.20,
49.00, 93.52, 108.70, 117.57, 119.62, 137.00, 140.62, 146.02,
160.96, 177.78.
Example 33
Synthesis of
2-methyl/2-trifluoromethyl-3-alkoxy-1,3,5(10)-estratrien-17-s-
piro-(dimethyl-6-lactone) Derivatives
[0795] These syntheses are described in Schemes 30 and 31. 109
110111
Example 33A
[0796]
2-iodo-3-methoxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimethy-
l-6'-oxo)tetrahydropyran (123). Compound 89a (400 mg, 1.05 mmol),
silver trifluoroacetate (254 mg, 1.15 mmol) and sodium bicarbonate
(439 mg, 5.23 mmol) were mixed in dichloromethane (4 mL) at
-30.degree. C. Crushed iodine (278 mg 1.10 mmol) was added and the
resulting mixture was stirred vigorously for 1 h during which the
red color completely disappeared. The reaction mixture was then
filtered and washed with dichloromethane (50 mL), and concentrated.
The residue was purified by chromatography on silica gel using
hexanes/ethyl acetate (80/20) to afford 412 mg (77%) of the 2-iodo
compound as a white solid along with 10% of the 4-iodo compound; IR
(KBr, cm.sup.-1): 2955, 1712, 1461, 1288, 1140; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 1.0 (s, 3H), 1.25-1.78 (m, 10H), 1.28 (s,
6H), 1.82-2.40 (m, 7H), 2.80-2.84 (m, 2H), 3.83 (s, 3H), 6.54 (s,
1H), 7.63 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.4,
23.3, 25.6, 26.11, 27.3, 27.7, 27.8, 29.6, 31.6, 31.8, 34.8, 37.8,
38.9, 43.3, 47.2, 48.6, 56.3, 82.6, 93.5, 111.4, 134.6, 136.3,
138.2, 156.0, 177.8.
Example 33B
[0797]
3-methoxy-2-methyl-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-dimet-
hyl-6'-oxo)tetrahydropyran (EM-1412). The 2-iodoestrone derivative
123 (300 mg, 0.590 mmol) was dissolved in anhydrous THF (4 mL) and
then cooled to -78.degree. C. A 1.6 M solution of BuLi in hexane
(390 .mu.L, 0.620 mmol) was added following after 10 min.,
iodomethane (180 .mu.L, 2.95 mmol) and after 5 min., carbon
disulfide (39 .mu.L, 0.65 mmol). The resulting mixture was stirred
at -78.degree. C. for 1 h then warmed to room temperature. After 1
h, the solvent was removed and the residue was purified by column
chromatography using hexanes/ethyl acetate (85/15) to give a white
solid (104 mg, 44%). IR (KBr, cm.sup.-1): 2932, 1718, 1507, 1202,
1154; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.02 (s, 3H),
1.20-1.80 (m, 10H), 1.29 (s, 6H), 1.80-2.20 (m, 6H), 2.18 (s, 3H),
2.30-2.40 (m, 1), 2.75-2.90 (m, 2H), 3.79 (s, 3H), 6.55 (s, 1H),
7.05 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.4, 15.9,
23.3, 25.6, 26.2, 27.6, 27.6, 27.8, 29.6, 31.6, 32.1, 34.8, 37.8,
39.3, 43.6, 47.3, 48.7, 55.3, 93.7, 110.4, 123.8, 127.6, 131.4,
134.8, 155.7, 177.9.
Example 33C
[0798]
3-methoxy-2-trifluoromethyl-1,3,5(10)-estratrien-17(R)-spiro-2'-(5'-
,5'-dimethyl-6'-oxo)tetrahydropyran (EM-1409). A pressure reaction
bottle was charged with activated copper powder (90 mg, 1.4 mmol),
as a suspension in DMF (1 mL) and PA (0.8 mL). 2-Iodoestrone
derivative 123 (180 mg, 0.354 mmol) was added and the resulting
mixture was cooled to -78.degree. C., and an excess of
trifluoromethyl iodide was condensed. The pressure bottle was
tightly closed and warmed to 130.degree. C. Stirring was maintained
for 20 h. The mixture was cooled to room temperature and filtered
on silica gel, and washed with a mixture of 25 mL of
dichloromethane and 75 mL of ethyl acetate. Evaporation of solvents
and column chromatography using hexanes/ethyl acetate (80/20) gave
a white solid (104 mg, 65%); IR (KBr, cm.sup.4l): 2938, 1713, 1623,
1509, 1299, 1120; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 1.02
(s, 3H),1.25-1.80 (m, 10H), 1.29 (s, 3H), 1.80-2.25 (m, 6H),
2.34-2.42 (m, 1H), 2.80-2.96 (m, 2H), 3.86 (s, 3H), 6.70 (s, 1H),
7.45 (s, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 14.4, 23.2,
25.5, 26.0, 27.1, 27.6, 27.7, 29.8, 31.5, 31.9, 34.7, 37.7, 38.9,
43.3, 47.2, 48.5, 93.5, 112.3, 116.0 (q, J.sub.C-F=31 Hz), 123.9
(q, J.sub.C-F=272 Hz), 123.9 (d, J.sub.C-F=6 Hz), 131.7, 142.3,
155.2, 177.8, 196.7.
Example 33D
[0799] 3-(2'-methoxyethoxy)-1,3,5(10)-estratrien-17-one (124). A
mixture of estrone (10 g; 37 mmol), NaI (1.1 g, 7.4 mmol),
Cs.sub.2CO.sub.3 (18.08 g; 5.55 mmol) and
Br(CH.sub.2).sub.2OCH.sub.3 (15 mL; 156 mmol) in CH.sub.3CN (200
mL) was refluxed for 1 h. The mixture was filtered over silica gel
and washed successively with CH.sub.2C.sub.2 and EtOAc to give the
ether (121 g; 99.4%); .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.23 (d, 1H, J=8.6 Hz), 6.75 (dd, 1H, J=2.6, 8.6 Hz), 6.72 (s, 1H),
4.1 (t, 2H, J=4.4 Hz), 3.74 (t, 2H, J=3.7 Hz), 3.44 (s, 3H), 2.87
(dd, 2H, J=4.25, 8.5 Hz), 2.48 (dd, 1H, J=8.4, 18.7 Hz), 0.91 (s,
3H).
Example 33E
[0800] 2-iodo-3-(2'-methoxyethyl)-1,3,5(10)-estratrien-17-one
(125). The iodo compound was prepared in 73.5% yield (3.04 g) by
following the method, described above; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.64 (s, 1H), 6.57 (s, 1H), 4.12 (t, 2H, J=5.1
Hz), 3.8 (t, 2H, J=4.9 Hz), 3.5 (s, 3H), 2.85 (dd, 2H, J=3.9, 8.5
Hz), 2.5 (dd, 1H, 8.8, 18.8 Hz), 0.90 (s, 3H).
Example 33F
[0801]
3-(2'-methoxyethoxy)-2-trifluoromethyl-1,3,5(10)-estratrien-17-one
(126) was prepared in 66.6% yield (581 mg) by the method, described
above; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.45 (s, 1H), 6.72
(s, 1H), 4.15 (t, 2H, J=5.1 Hz), 3.77 (t, 2H, J=5.1 Hz), 3.45 (s,
3H), 2.91 (m, 2H), 2.5 (dd, 1H, 8.9, 18.8 Hz), 0.91 (s, 3H).
Example 33G
[0802]
17.beta.-Hydroxy-3-(2'-methoxyethoxy)-17.alpha.-{4'-(2"-tetrahydro--
2"H-pyranyl)butyn-1'-yl}-2-trifluoromethyl-1,3,5(10)-estratriene
(127) was prepared in 63% yield (523 mg) by the method, described
above; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.42 (s, 1H), 6.67
(s, 1H), 4.64 (bs, 1H), 4.11 (t, 2H, J=4.5 Hz), 3.84-3.75 (m, 2H),
3.73 (t, 2H, J=4.7 Hz), 3.54-3.46 (m, 3H), 3.41 (s, 3H), 2.82 (bs,
2H), 2.71 (bs, 1H), 2.52 (t, 2H, 6.8 Hz), 0.81 (s, 3H).
Example 33H
[0803]
17.beta.-Hydroxy-3-(2'-methoxyethoxy)-17.alpha.-{4'-(2"-tetrahydro--
2"H-pyranyl)butan-1'-yl}-2-trifluoromethyl-1,3,5(10)-estratriene
(128) was prepared (the crude; 455 mg) by the method. described
above; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.44 (s, 1H), 6.7
(s, 1H), 4.59 (bs, 1H), 4.14 (t, 2H, J=5.1 Hz), 3.90-3.80 (m, 2H),
3.76 (t, 2H, J=5 Hz), 3.52-3.43 (m, 2H), 3.45 (s, 3H), 2.87-2.85
(m, 2H), 0.89 (s, 3H).
Example 331
[0804]
17.beta.-Hydroxy-3-(2'-methoxyethoxy)-17.alpha.-(4'-hydroxybutan-1'-
-yl)-2-trifluoromethyl-1,3,5(10)-estratriene 17 (129) was prepared
(the crude; 386 mg) by the method described above; .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 7.44 (s, 1H), 6.7 (s, 1H), 4.14 (t, 2H,
J=4.9 Hz), 3.77 (t, 2H, J=5.1 Hz), 3.70 (t, 2H, J=5.8 Hz), 3.46 (s,
3H), 2.86 (m, 2H), 0.90 (s, 3H).
Example 33J
[0805] 3-(2'-methoxyethoxy)-2-trifluoromethyl-1,3,5(10)-estratrien
17(R)-spiro-2'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (130) was
perpared in 59% yield (for 3 steps) by the method, described above;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.44 (s, 1H), 6.7 (s,
1H), 4.14 (t, 2H, J=4.2 Hz), 3.77 (t, 2H, J=5.1 Hz), 3.45 (s, 3H),
2.91-2.90 (m, H), 1.02 (s, 3H).
Example 33H
[0806]
3-(2'-methoxyethoxy)-2-trifluoromethyl-1,3,5(10)-estratrien-17(R)-s-
piro-2'-(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1438) was
prepared in 88.7% yield by the method described above; IR (KBr,
cm.sup.-1), 3419, 2988, 2939, 2879, 2818, 1718, 1622, 1508, 1298,
1154, 1052; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.43 (s, 1H),
6.7 (s, 1H), 4.13 (t, 2H, J=5.2 Hz), 3.76 (t, 2H, J=4.77 Hz), 3.44
(s, 3H), 2.87 (m, 2H), 2.36-3.32 (m, 1H), 1.27 (s, 6H), 1.01 (s,
3H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 177.7, 154.5, 142.2,
132.1, 123.9 (d, J.sub.C-F=5.3 Hz), 123.8 (q, J.sub.C-F=272 Hz),
116.6 (q, J.sub.C-F=31 Hz), 113.7, 93.5, 70.8, 68.8, 59.3, 48.6,
47.2, 43.3, 38.9, 37.8, 34.8, 31.8, 31.6, 29.8, 27.7, 27.6, 27.1,
26.0, 25.5, 23.2, 14.4.
Example 34
Synthesis of
1,3,5(10)-estratrien-17-spiro-(diethyl-.delta.-lactone)
Derivatives
[0807] These syntheses are described in Scheme 32. 112
Example 34A
[0808]
3-t-butyldimethylsilyloxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5-
'-diethyl-6t-oxo)tetrahydropyran (131). In a dry 25 mL flask under
argon the lactone 87 (200 mg, 0.44 mmol) in THF (5 mL) was charged
and cooled to 0.degree. C. LiHMDS (1.1 mL, 1.1 mmol) was added
dropwise. The mixture was stirred 15 minutes at 0.degree. C. and
the cooled to -78.degree. C. Ethyl iodide (176 .mu.L, 2.2 mmol) was
added, and stirred 1 h at this temperature, and then allowed to
warm to room temperature over a period of 2 h. A saturated solution
of NH.sub.4Cl (5 mL) was added and the mixture was extracted with
CH.sub.2Cl.sub.2. The organic layer was washed with a saturated
solution of Na.sub.2S.sub.2O.sub.3 and brine, dried (MgSO.sub.4),
and concentrated under reduced pressure. The residue was purified
by silica gel column chromatography with hexanes/acetone (9/1) as
an eluent to afford 193 mg (86%) of diethyl compound; IR (NaCl,
cm.sup.-1): 2933, 2857, 1719, 1496, 1259, 1143, 957, 839; .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.11 (d, 1H, J=8.4 Hz), 6.62 (dd,
1H, J=2.1, 8.4 Hz), 6.56 (s, 1H), 2.81-2.78 (m, 2H), 2.36-2.25 (m,
1H), 2.19-1.22 (m, 20H), 1.02 (s, 3H, 18-Me), 0.98 (s, 9H), 0.90
(app.q, 6H, J=7.5 Hz), 0.19 (s, 6H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 176.6, 153.3, 137.6, 132.6, 125.9, 119.8,
117.1, 93.0, 48.6, 47.2, 45.3, 43.6, 39.0, 34.5, 31.9, 30.5, 29.5,
27.4, 25.9, 25.6, 24.3, 23.4, 18.1, 14.3, 8.2, -4.5.
Example 34B
[0809]
1,3,5(10)-estratrien-3-ol-17(R)-spiro-2'-(5',5'-diethyl-6'-oxo)tetr-
ahydropyran (EM-1416). The silyl ether (193 mg, 0.38 mmol) was
treated with a 1M solution of TBAF (455 .mu.L) to give the hydroxy
compound (143 mg, 95%); IR (NaCl, cm.sup.-1): 3349, 2967, 2930,
2878, 1696, 1682, 1503, 1454, 1146, 910, 732; .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 7.10 (d, 1H, J=8.4 Hz), 6.76 (s, 1H, OH), 6.66
(d, 1H, J=8.4 Hz), 6.60 (s, 1H), 2.82-2.77 (m, 2H), 2.36-2.28 (m,
1H), 2.17-1.22 (m, 20H), 0.98 (s, 3H, 18-Me), 0.91 (app.q, 6H,
J=7.6 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 177.8, 154.0,
137.6, 137.6, 131.4, 126.1, 115.3, 112.9, 93.5, 48.5, 47.2, 45.4,
43.5, 39.1, 34.5, 31.9, 31.7, 30.6, 29.4, 27.3, 26.0, 25.6, 24.1,
23.3, 14.2, 8.7.
Example 34C
[0810]
3-methoxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(5',5'-diethyl-6'-oxo-
)tetrahydropyran (EM-1419). To the alcohol (19 mg, 0.063 mmol) and
Cs.sub.2CO.sub.3 (31 mg, 0.094 mmol) in acetonitrile (2 mL), was
added MeI (40 .mu.L, 0.63 mmol) at room temperature. after 1 h,
another MeI (40 .mu.L) was added. The solution was diluted with
CH.sub.2Cl.sub.2, washed with a 10% aq HCl and brine, dried
(MgSO.sub.4), and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography with hexanes/EtOAc
(9/1) to afford 19 mg (97%) of the 3-methoxy compound; IR (NaCl,
cm.sup.-1): 2935, 2877, 1718, 1500, 1462, 1256, 1142, 1037, 731;
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.20 (d, 1H, J=8.6 Hz),
6.71 (dd, 1H, J=2.8, 8.6 Hz), 6.63 (d, 1H, J=2.4 Hz), 3.78 (s, 3H),
2.87-2.84 (m, 2H), 2.35-2.31 (m, 1H), 2.17-1.26 (m, 20H), 1.02 (s,
3H, 18-Me), 0.90 (app.q, 6H, J=7.5 Hz); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 176.6, 157.5, 137.8, 132.2, 126.2, 113.8,
111.5, 93.0, 55.2, 48.7, 47.2, 45.4, 43.7, 39.1, 34.6, 31.9, 30.6,
29.7, 27.5, 26.1, 25.7, 24.4, 23.4, 14.4, 8.7.
Example 34D
[0811]
3-[2'-(N-piperidinyl)ethyl]oxy-1,3,5(10)-estratrien-17(R)-spiro-2'--
(5',5'-dimethyl-6'-oxo)tetrahydropyran (EM-1434). The mixture of
alcohol EM-1416 (60 mg, 0.15 mmol), K.sub.2CO.sub.3 (668 mg, 4.8
mmol) and 1-(2-chloroethyl)-piperidine monohydrochloride (834 mg,
4.5 mmol) in acetonitrile (25 mL) was refluxed for a period of 15
h. The pH was adjusted to a neutral with a 10% aq HCl and then, the
mixture was extracted with EtOAc, washed with brine, dried
(MgSO.sub.4) and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography with
hexanes/acetone (5/1) to afford 35 mg (46%) of compound; IR (NaCl,
cm.sup.-1): 2934, 1719, 1499, 1256, 1142, 1037; .sup.1H NMR (300
MHz, CDCl.sub.3): 7.18 (d, 1H, J=8.6 Hz), 6.70 (dd, 1H, J=2.6, 8.6
Hz), 6.63 (d, 1H, J=2.3 Hz), 4.07 (t, 2H, J=6.0 Hz), 2.87-2.81 (m,
2H), 2.75 (t, 2H, J=6.0 Hz), 2.51-2.48 (m, 4H), 2.34-2.30 (m, 1H),
2.17-1.25 (m, 26H), 1.01 (s, 3H, 18-Me), 0.90 (app.q, 6H, J=7.5
Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 176.7, 156.7, 137.8,
132.3, 126.2, 114.6, 112.1, 93.0, 65.8, 57.9, 54.9, 48.6, 47.2,
45.4, 43.6, 39.1, 34.6, 31.9, 30.6, 29.7, 27.5, 26.1, 25.9, 25.7,
24.4, 24.2, 23.2, 14.4, 8.7.
Example 35
Synthesis of
1,3,5(10)-estratrien-17-spiro-(5"-allyl/5"-methoxycarbonyl/4"-
-methyl)-.delta.-lactone and Lactenone
[0812] These syntheses are described in Scheme 33 113
[0813] a: NH(isopropyl).sub.2, nBuLi, HMPA, THF,
CH.sub.2.dbd.CH--CH.sub.2- Br, -78.degree. C..fwdarw.room
temperature
[0814] b: 1M Bu.sub.4NF, 2 h, room temperature.
[0815] c: NH(isopropyl).sub.2, nBuLi, HMPA, THF, NC COOCH.sub.3,
-78.degree. C.
[0816] d: NH(isopropyl).sub.2, nBuLi, THF, 0.degree. C.
[0817] e: PhSeCl, -78.degree. C. room temperature
[0818] f: H.sub.2O CH.sub.2Cl.sub.2, room temperature.
[0819] g: 1M Bu.sub.4NF, 0.degree. C.
[0820] h: CuCN, Et.sub.2O, room temperature, CH.sub.3I.
Example 35A
[0821]
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(4',5'-dihydro-6'-oxo-
)-pyran. To a solution of 1.6 mmol of LDA in 20 mL of dry THF was
added 380 mg (0.837 mmol) of lactone 87 in 20 mL of dry THF at
0.degree. C. and the mixture was stirred for 30 min. Then, the
temperature was dropped to -78.degree. C. and 248 mg (1.29 mmol) of
PhSeCl was added. The solution was stirred overnight and let warm
to room temperature. The reaction mixture was quenched with water
and extracted with EtOAc. The organic phase was washed with water
and dried over MgSO.sub.4. After evaporation of solvent, the crude
compound was dissolved in 10 mL of CH.sub.2Cl.sub.2 and 0.5 mL of
H.sub.2O.sub.2 (30% w/v). After 30 min at room temperature, water
was added, and aqueous phase extracted with CH.sub.2Cl.sub.2. The
organic phase was washed with water and dried over MgSO.sub.4. The
crude compound was purified by flash chromatography with
hexanes/EtOAc (95:5) as eluent to give 133 mg (35%) of
3-(tert-butyldimethylsilyloxy)-1,3,5(10-
)-estratrien-17(R)-spiro-2'-(4',5'-dihydro-6'-oxo)-tetrahydropyran.
White solid; IR .nu. (KBr): 1722 (C.dbd.O, lactone); .sup.1H NMR
(CDCl.sub.3) .delta. 0.18 (s, 6H, Si(CH.sub.3).sub.2), 0.97 (s, 9H,
SiC(CH.sub.3).sub.3), 1.01 (s, 3H, 18-CH3), 2.81 (m, 2H,
6-CH.sub.2), 6.03 (d, J.sub.1=9.8 Hz, 1H, 3'-CH), 6.55 (d, 1H,
J=2.1 Hz, 4-CH), 6.60 (dd, J.sub.1=2.5 Hz and J.sub.2=8.5 Hz, 2H,
2-CH), 6.80 (m, 1H, 2'-CH), 7.09 (d, J=8.4 Hz, 1H, 1-CH); .sup.13C
NMR (CDCl.sub.3) .delta. -4.42 (Si(CH.sub.2).sub.2), 14.06 (C-18),
22.94 (C-15), 25.68 (SiC(CH.sub.3).sub.3), 26.23 (C-11), 27.42
(C-7), 29.49 (C-6), 31.35 (C-12), 34.26 (C-16), 36.13 (C-2'), 38.98
(C-8), 43.63 (C-9), 47.22 (C-13), 49.17 (C-14), 91.91 (C-17),
117.22 (C-2), 119.95 (C-4), 121.54 (C-1'), 126.01 (C-1), 132.44
(C-10), 137.59 (C-5), 144.21 (C-3'), 154.41 (C-3), 164.56 (C.dbd.O,
lactone).
[0822] The TBDMS group of the above compound (133 mg, 0.294 mmol)
was cleaved at 0.degree. C. using 0.16 mL of 1 M Bu.sub.4NF
solution in THF (0.16 mmol). After evaporation of solvent, the
crude compound was purified by flash chromatography with
hexanes/EtOAc (7:3) as eluent to give 97 mg (97%) of
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(4',5'--
dihydro-6'-oxo)pyran (PB-132-140). White solid; IR .nu. (KBr): 3235
(OH, phenol), 1691 (C.dbd.O, lactone); .sup.1H NMR (CDCl.sub.3 90%
and CD.sub.3OD 10%) .delta. 0.84 (s, 3H, 18-CH.sub.3), 2.62 (m, 2H,
6-CH2), 5.86 (d, J=9.9 Hz, 1H, 3'-CH), 6.40 (d, 1H, J=2.2 Hz,
4-CH), 6.45 (dd, J.sub.1=.times.Hz and J.sub.2=.times.Hz, 2H,
2-CH), 6.75 (m, 1H, 21-CH), 6.93 (d, J=8.4 Hz, 1H, 1-CH); .sup.13C
NMR (CDCl.sub.3 90% and CD.sub.3OD 10%) .delta. 13.73 (C-18), 22.66
(C-15), 26.09 (C-11), 27.17 (C-7), 29.29 (C-6), 31.12 (C-12), 34.05
(C-16), 35.84 (C-1), 38.91 (C-8), 43.34 (C-9), 47.82 (C-13), under
solvent pics (C-14), 92.15 (C-17), 112.58 (C-2), 115.02 (C4),
120.82 (C-2'), 126.00 (C-1), 130.80 (C-10), 137.56 (C-5), 145.09
(C-3'), 154.28 (C-3), 165.36 (C.dbd.O, lactone). EI-HRMS: calcd for
C.sub.21H.sub.26O.sub.3 (M.sup.+) 338.18820, found 338.18638
Example 35B
[0823]
3-hydroxy-1,3,5(10)-estratrien-17(R)-spiro-2'-(4'-methyl-6'-oxo)tet-
rahydropyran (PB-132-142). To a solution of 24 mg (0.271 mmol) of
CuCN in 5 mL of dry Et.sub.2O at room temperature was added 0.377
mL (0.528 mmol) of CH.sub.3I. After 30 min, a solution of 30 mg
(0.066 mmol) of TBDMS-derivative of compound PB-132-140 in dry
Et.sub.2O was added at -78.degree. C. The solution was stirred
overnight and let reheat to room temperature. The solution was then
quenched at -20.degree. C. with aqueous NH.sub.4Cl and extracted
with EtOAc. The organic phase was washed with water and dried over
MgSO.sub.4. The crude compound was purified by flash chromatography
with hexanes/EtOAc (9:1) as eluent to give 24 mg (78%) of
intermediate compound. As described above the TBDMS group of this
later compound was cleaved using 0.07 mL of 1 M Bu.sub.4NF solution
in THF (0.07 mmol) at 0.degree. C. After evaporation of solvent,
the crude compound was purified by flash chromatography with
hexanes/EtOAc (7:3) as eluent to give 17 mg (73% two steps) of
P-methylated lactone PB-132-142. White solid; IR .nu. (film): 3330
(OH, phenol), 1694 (C.dbd.O, lactone); .sup.1H NMR (CDCl.sub.3)
.delta. 0.99 (s, 3H, 18-CH.sub.3), 1.04 (d, J=6.2 Hz, 3H, CH.sub.3
of lactone ring), 2.56 (dm, J=13.9 Hz, 1H), 2.80 (m, 2H,
6-CH.sub.2), 6.58 (s.sub.app, 1H, 4-CH), 6.63 (dd, J.sub.1=8.4 Hz
and J.sub.2=2.5 Hz, 1H, 2-CH), 7.12 (d, J=8.4 Hz, 1H, 1-CH);
.sup.13C NMR (CDCl.sub.3) .delta. 14.78 (C-18), 21.76 (C-1'), 22.90
(C-15), 25.05 (C-3'), 26.22 (C-11), 27.36 (C-7), 29.55 (C-6), 29.67
(CH.sub.3 of lactone), 33.21 (C-12), 38.28 (C-16), 39.25 (C-8),
40.19 (C-2'), 43.45 (C-9), 48.14 (C-13), 49.37 (C-14), 93.63
(C-17), 112.82 (C-2), 115.31 (C-4), 126.37 (C-1), 131.87 (C-10),
138.01 (C-5), 153.73 (C-3),172.23 (C.dbd.O, lactone). El-HRMS:
calcd for C.sub.23H.sub.30O.sub.3 (M.sup.+) 354.21948, found
354.21791.
Example 35C
[0824]
3-hydroxy-1,3,5(10)-estratrien-17(R)-2'-(5'-allyl-6'-oxo)tetrahydro-
pyran (PB-132-152). The allylation of lactone 87 (50 mg, 0.11 mmol)
was conducted at -78.degree. C. with LDA (0.45 mmol), HMPA (0.12
mmol) and allylbromide (0.72 mmol). The mixture was stirred
overnight and let warm to room temperature. Wihtout purification
the crude lactone was treated for 2 h with a solution in THF of
0.12 ml of 1 M Bu.sub.4NF (0.12 mmol). Then, the crude allyl
derivative was purified by flash chromatography with hexanes/EtOAc
(9:1) as eluent to give 33 mg (79% two step) of allyl-lactone
PB-132-152. White solid; IR .nu. (film): 3348 (OH, phenol), 1699
(C.dbd.O, lactone); .sup.1H NMR (CDCl.sub.3) .delta. 1.00 (s, 3H,
18-CH.sub.3), 2.82 (m, 2H, 6-CH2), 5.09 (d, J=8.8 Hz, 1H,
CH.sub.2CH.dbd.CH.sub.2), 5.11 (d, J=19.6 Hz, 1H,
CH.sub.2CH.dbd.CH.sub.2- ), 5.79 (m, 1H, CH.sub.2CH.dbd.CH.sub.2),
6.57 (d, J=2.5 Hz, 1H, 4-CH), 6.64 (dd, J.sub.1=8.4 Hz and
J.sub.2=2.5 Hz, 1H, 2-CH), 7.13 (d, J=8.4 Hz, 1H, 1-CH); .sup.13C
NMR (CDCl.sub.3) .delta. 14.34 (C-18), 20.25, 21.30 (C-2'), 21.63,
23.40 and 23.58 (C-15), 25.61, 26.09 (C-11), 27.28, 27.40, 28.27
(C-1'), 29.53 (C-6), 29.67 CC-1"), 31.95 (C-12), 34.00 (C-3'),
34.50 (C-16), 35.96, 38.13, 39.12 (C-8), 40.50, 43.63 (C-9), 47.22
(C-13), 48.62 and 48.76 (C-14), 92.85 and 93.57 (C-17), 112.81
(C-2), 115.30 (C4), 117.35 and 117.63 (C-2"), 126.35 (C-1), 131.91
(C-10), 135.04 and 135.29 (C-3"), 137.96 (C-3), 153.78 (C-3),
173.87 and 174.79 (C.dbd.O, lactone).
Example 35D
[0825]
3-hydroxy-1,3,5(10)-estratrien-17(R)-2'-(5'-methoxycarbonyl-6'-oxo)-
tetrahydropyran (PB-132-146). At 0.degree. C., a solution of
lactone 87 (50 mg, 0.11 mmol) in dry THF was added to 0.41 mmol of
LDA and the mixture was cooled to -78.degree. C. Then, HMPA (20
.mu.L, 0.12 mmol) and NCCOOCH.sub.3 (31 .mu.L, 0.45 mmol) were
added. Without purification the crude lactone was treated for 2 h
with a solution in THF of 0.13 ml of 1 M Bu.sub.4NF (0.13 mmol).
The crude phenol was purified by flash chromatography with
hexanes/EtOAc (6:4) as eluent to give 32 mg (73% two steps) of
methoxycarbonyl-lactone PB-132-146. White solid; IR .nu. (film):
3372 (OH, phenol), 1744, 1720, 1710 (C.dbd.O, lactone and
COOCH.sub.3); .sup.1H NMR (CDCl.sub.3) .delta. 1.02 (s, 3H,
18-CH.sub.3), 2.82 (m, 2H, 6-CH.sub.2), 3.46 (dd, J.sub.1=7.5 Hz
and J.sub.2=10.7 Hz, 0.5H, CH of lactone), 3.54 (dd, J=5.9 Hz and
J.sub.2=7.8 Hz, 0.5H, CH of lactone), 3.79 and 3.80 (2s, 3H,
COOCH.sub.3), 6.57 (s.sub.app, 1H, 4-CH), 6.63 (dd, J.sub.1=8.1 Hz
and J.sub.2=2.2 Hz, 1H, 2-CH), 7.13 (d, J=8.4 Hz, 1H, 1-CH);
.sup.13C NMR (CDCl.sub.3) .delta. 14.33 (C-18), 20.00 and 20.68
(C-2'), 23.46 (C-15), 26.02 (C-11), 26.26 (C-1'*), 27.39 (C-7),
29.52 (C-6), 29.68, 31.85 and 31.97 (C-12), 34.03 and 34.34 (C-16),
39.10 (C-8), 43.61 (C-9), 46.30, 47.43 and 47.93 (C-13), 48.72 and
48.83 (C-14), 52.79 (COOCH.sub.3), 94.36 and 94.55 (C-17), 112.79
(C-2), 115.28 (C4), 126.40 (C-1), 131.99 (C-10), 138.01 (C-5),
153.62 (C-3), 167.28 and 167.94 (COOCH.sub.3), 169.93 (C.dbd.O,
lactone); EI-HRMS: calcd for C.sub.24H.sub.30O.sub.5 (M.sup.+)
398.20932, found 398.21077.
Synthesis of Preferred Type 3 17.beta.-HSD Inhibitors
Example 36
Example 36A
[0826] Syntheses of 3-thio-3-deoxy Estrone Derivatives
[0827] General procedure for 3-alkylation of
3-thio-3-deoxy-estrone. To a solution of 3-thio-3-deoxyestrone in
DMF under Ar(g) was added 1.05 molar equivalent of NaH (60% in oil)
at 0.degree. C. After 1 h, 1.2 molar equivalent of the appropriate
alkyl halide chain was added and the solution was stirred at room
temperature overnight. The solution was poured onto cold water then
was extracted with EtOAc. The organic phase was washed with brine,
dried with MgSO.sub.4, filtered then evaporated under reduced
pressure to give the crude product which was purified by flash
chromatography on SiO.sub.2 using a mixture of EtOAc/Hexanes.
[0828] 3-Methoxymethylthio-Estra-1,3,5(10)-trien-17-one (EM-1064).
Using 3-thioestrone (600 mg, 2.1 mmol) and chloromethyl methyl
ether (202 mg, 2.51 mmol) to give EM-1064 as white solid (488 mg,
72%), Mp: 55.degree. C., [.alpha.].sup.25.sub.D+88.9.degree. (c
1.6, CH.sub.2Cl.sub.2); IR: 2929, 2820, 2249, 1970, 1738, 1594,
1557, 1485, 1453, 1404, 1373, 1305, 1259, 1182, 1083, 1052, 1008,
951, 898, 860, 820, 777, 732, 693, 679, 582, 559, 483 cm.sup.-1;
.sup.1H NMR(CDCl.sub.3) .delta. 0.91 (3H, s), 1.43-1.63 (6H, m),
1.95-2.49 (7H, m), 2.90 (2H, dd, J1=4.6 Hz, J2=4.0 Hz), 3.43 (3H,
s), 4.93 (2H, s), 7.21-7.26 (3H, m). .sup.13C NMR(CDCl.sub.3)
.delta. 13.8, 21.6, 25.7, 26.4, 29.3, 31.6, 35.9, 38.1, 44.3, 48.0,
50.5, 56.0, 76.6, 126.0, 128.0, 131.0, 132.5, 137.4, 138.7, 220.8.
HRMS: 330.16534 C.sub.20H.sub.260.sub.2S.
[0829] 3-Methylthioethylthio-Estra-1,3,5(10)-trien-17-one
(EM-1065). Using 3-thioestrone (406 mg, 1.4 mmol) and 2chloroethyl
methyl sulfide (188 mg, 1.7 mmol) to give EM-1065 as a white solid
(187 mg, 37%), Mp: 74.degree. C., [.alpha.].sup.25 D-58.2.degree.
(c 2.0, CH.sub.2Cl.sub.2); IR: 2927, 2859, 2247, 1737, 1593, 1556,
1484, 1453, 1436, 1404, 1373, 1339, 1260, 1202, 1120, 1082, 1051,
1007, 964, 912, 859, 821, 777, 733, 646, 581, 559 cm.sup.-1;
.sup.1H NMR(CDCl.sub.3) .delta. 0.91 (3H, s), 1.44-1.63 (6H, m),
1.95-2.55 (7H, m), 2.13 (3H, s), 2.70 (2H, dd, J1=1.9 Hz, J2=4.4
Hz), 2.89 (2H, dd, J1=4.7 Hz, J2=4.0 Hz), 3.08 (2H, dd, J1=4.9 Hz,
J2=5.5 Hz), 7.12 (1H, d, J=6.7 Hz), 7.16 (1H, s), 7.22 (1H, d,
J=8.2 Hz). .sup.13C NMR(CDCl.sub.3) .delta. 13.8, 15.5, 21.6, 25.7,
26.4, 29.3, 31.6, 33.7, 35.8, 38.1, 44.2, 48.0, 50.5, 126.1, 127.6,
130.7, 132.2, 137.5, 138.5, 220.7 ppm. HRMS: 360.15817
(C.sub.21H.sub.28OS.sub.2).
Example 36B
[0830] Synthesis of Estrone Derivatives.
[0831] 3-Methylthiomethyloxy-1,3,5(10)-estratrien-17-one (EM-1066):
To a solution of estrone (20.0 g, 7.39 mmol) in anhydrous DMF under
Ar (g), was added NaH (60% in oil, 0.26 g, 11.0 mmol). After the
evolution of hydrogen had ceased, the solution was cooled in an ice
bath for the addition of CH.sub.3SCH.sub.2Cl (3.56 g, 36.9 mmol,
3.08 mL) and DMAP (0.09 g, 7.0 mmol). The solution was stirred in a
cold room (4.degree. C.) for 16 hours. The reaction was stopped
with cold water, extracted with ethyl acetate, wash with water and
brine, dry over MgSO.sub.4, filtred and concentrated under reduce
pressure. The crude extract was purified by flash chromatography on
silica gel column and eluted with EtOAc:hexanes (1:9), to give
EM-1066 (0.7 g, 30%) as a white solide; Rf 0.5 (3:7 EtOAc/Hexanes);
M.p.65.degree. C.; [.alpha.].sub.D.sup.26+116.4.- degree. (c 1.06,
CHCl.sub.3); IR (.nu.) 2919, 2864, 1735, 1604, 1577, 1497, 1448,
1375, 1282, 1255, 1211, 1151, 1051, 989, 885, 823, 787, 739,
640-690, 579 cm.sup.-1; .sup.1H NMR .delta. 2.24 (3H, s,
CH.sub.3--S), 5.12 (2H, s, S--CH.sub.2--O), 7.21 (1H, d, J=2.8 Hz,
1-CH), 6.76 (2H, dd, J.sub.1=2.8 Hz and J.sub.2=8.6 Hz, 2'-CH),
6.70 (1H, d, J=8.0 Hz, 4'-CH).
Example 36C
[0832] 3-Methyloxymethyloxy-1,3,5(10)-estratrien-17-one (EM-1070).
To a solution of estrone (55 mg, 0.203 mmole) in anhydrous
dichloromethane (20 mL) under Ar (g) was added
diisopropylethylamine (580.5 .mu.L, 3.33 mmole). The solution was
cooled down to 0.degree. C. and chloromethylmethylether (110 .mu.L,
1.45 mmole) was added. The solution was refluxed lightly overnight
after which was added aqueous 1M HCl (40 mL). The solution was
extracted 2 times with dichloromethane, washed with brine, dried
with magnesium sulfate, filtered and concentrated in vacuo. The
yellow solid was purified on silica gel (20 g, 1:9 ethyl
acetate/hexanes) to give EM-1070 as yellowish crystals (62 mg,
96%). m.p.; [.alpha.].sub.D.sup.25+103.9.degree. (c 1.12,
CDCl.sub.3); IR (NaCl) 2926, 1737 (s, C.dbd.O), 1669, 1498, 1243,
1152, 1077, 1006 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.91
(3H, s, 18-CH.sub.3), 1.25-1.78 (6H, m), 1.90-2.38 (5H, m).
2.40-2.55 (2H, m), 2.89 (2H, in, 6-CH2), 3.48 (3H, s, CH30), 5.15
(2H, s, CH30CH.sub.2), 6.79 (1H, d, J=2.4 Hz, 4CH), 6.84 (1H, dd,
J1=8.4 Hz and J2=2.6 Hz, 2-CH), 7.21 (1H, d, J=8.6 Hz, 1-CH).
Example 36D
[0833] 3-Ethyloxyethyloxy-1,3,5(10)-estratrien-17-one (EM-1071). To
a solution of estrone (495 mg, 1.83 mmole) in anhydrous
acetonitrile (150 mL) under Ar (g) was added dry potassium
carbonate (291 mg, 2.11 mmole) and 2-chloroethylethylether (2.6 mL,
23.77 mmole). The solution was refluxed for 36 hours then
concentrated and ethyl acetate (175 mL) was added. The solution was
washed with water (2 times), with brine (2 times), dried with
magnesium sulfate, filtered and then concentrated in vacuo. The
white solid was purified on RP-18 (40-63 .mu.m) gel (40 g, 100%
methanol) to give EM-1071 as white crystals (588 mg, 94%). m.p.;
[.alpha.].sub.D.sup.25+129.6.degree. (c 3.06, CDCl.sub.3); IR
(NaCl) 2928, 2867, 1739 (s, C.dbd.O), 1609, 1574, 1499, 1454, 1373,
1310, 1281, 1255, 1158, 1122, 1064, 1007, 957, 924, 875, 818, 781,
650, 581 cm.sup.-; .sup.1H NMR (CDCl.sub.3) .delta. 0.91 (3H, s,
18-CH3), 1.24 (3H, t, J=6.7 Hz, CH.sub.3CH.sub.2), 1.24-1.78 (6H,
m), 1.90-2.38 (5H, m). 2.40-2.55 (2H, m), 2.88 (2H, m, 6-CH2), 3.60
(2H, q, J=7.2 Hz, CH.sub.3CH.sub.2), 3.78 (2H, t, J=5.2 Hz,
CH.sub.2CH.sub.2O), 4.10 (2H, t, J=4.8 Hz, CH.sub.2OPh), 6.68 (1H,
d, J=2.5 Hz, 4-CH), 6.74 (1H, dd, J.sub.1=8.5 Hz and J.sub.2=2.7
Hz, 2-CH), 7.19 (1H, d, J=8.6 Hz, 1-CH).
Example 36E
[0834] 3-Methyloxyethyloxy-1,3,5(10)-estratrien-17-one (EM-1073).
To a solution of estrone (502 mg, 1.86 mmole) in anhydrous
acetonitrile (250 mL) under Ar (g) was added potassium carbonate
(547 mg, 3.96 mmole) and 2-chloroethylmethylether (16.6 mL, 182
mmole). The solution was refluxed for 72 hours, concentrated and
ethyl acetate (175 mL) was added. The solution was washed with
water (2 times) with brine (2 times), dried with magnesium sulfate,
filtered and then concentrated in vacuo. The brownish powder was
purified on silica gel (40 g, 1:15 ethyl acetate/hexanes) to give
EM-1073 as white crystals (232 mg, 38%). m.p.
[.alpha.].sub.D.sup.25+134.0.degree. (c 1.06, CHCl.sub.3); IR
(NaCl) 2927, 2872, 1738 (s, C.dbd.O), 1609, 1574, 1499, 1454, 1406,
1372, 1338, 1311, 1281, 1256, 1198, 1158, 1128, 1065, 1036, 1007,
954, 868, 818, 781 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.90
(3H, s, 18-CH.sub.3), 1.25-1.78 (6H, m), 1.90-2.38 (5H, m).
2.40-2.55 (2H, m), 2.87 (2H, m, 6-CH2), 3.44 (3H, s, CH.sub.3O),
3.73 (2H, t, J=5.1 Hz, CH.sub.3OCH.sub.2), 4.09 (2H, t, J=4.4 Hz,
CH.sub.2OPh), 6.68 (1H, d, J=2.8 Hz, 4-CH), 6.74 (1H, dd,
J.sub.1=8.5 Hz and J.sub.2=2.8 Hz, 2-CH), 7.19 (1H, d, J=8.6 Hz,
1-CH).
Example 36F
[0835] 3Butyloxy-1,3,5(10)-estratrien-17-one (EM-1074). To a
solution of estrone (508 mg, 1,88 mmole) in anhydrous
dimethylformamide (75 mL) under Ar (g) was added 60% sodium hydride
in oil (89 mg, 2.23 mmole). After the evolution of hydrogen had
ceased bromobutane (596 .mu.L, 5.55 mmole) was added. The reaction
mixture was keep at 80.degree. C. overnight after which was added
100 g of ice. The solution was extracted 3 times with ethyl
acetate, washed 4 times with brine, dried with magnesium sulfate,
filtered and then concentrated in vacuo. The yellowish solid was
purified on silica gel (40 g, 1:9 ethyl acetate/hexanes) to give
EM-1074 as white crystals (559 mg, 91%). m.p.;
[.alpha.].sub.D.sup.25+138.5.degree. (c 0.53, CHCl.sub.3); IR
(NaCl) 3448, 2960, 2936, 2867, 1735 (s, C.dbd.O), 1612, 1571, 1493,
1474, 1439, 1390, 1349, 1280, 1257, 1222, 1185, 1156, 1115, 1055,
1007, 969, 918, 872, 821, 782, 738, 657, 581 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.90 (3H, s, 18-CH3), 0.96 (3H, t, J=7.3 Hz,
CH.sub.3CH2), 1.18-1.78 (10H, m), 1.90-2.30 (5H, m). 2.36-2.55 (2H,
m), 2.88 (2H, m, 6-CH.sub.2), 3.93 (2H, t, J=6.6 Hz, CH.sub.2O),
6.64 (1H, s, 4-CH), 6.70 (1H, d, J=8.5 Hz, 2-CH), 7.18 (1H, d,
J=8.6 Hz, 1-CH).
Example 37
Synthesis of Androstane Derivatives
[0836] These syntheses are described in Scheme 34. 114
Example 37A
[0837] Protection of the 17.beta.-alcohol with TBDMS. To a solution
of dihydrotestosterone (DHT, 132) (5 g, 17.2 mmol) in DMF was added
imidazole (6 eq.) and TBDMSCI (5 eq.). The reaction was stirred
overnight at room temperature. The mixture was poured onto ice and
filtered. The resulting white precipitate was washed with water,
dried over phosphorous pentoxide under reduced pressure for 24 h. A
85 to 90% yield was obtained.
[0838]
17.beta.-[(tert-butyldimethylsilyl)oxy]-5.alpha.-androstane-3-one
(133). White solid; IR (KBr) .nu. 1719 (C.dbd.O, ketone); .sup.1H
NMR (CDCl.sub.3) 5-0.001 and 0.005 (s, 6H, Si(CH.sub.3).sub.2),
0.71 (s, 3H, CH.sub.3-18), 0.87 (s, 9H, SiC(CH.sub.3).sub.3), 1.01
(s, 3H, CH.sub.3-19), 3.54 (t, J=8.2 Hz, 1H, CH-17); .sup.13C NMR
(CDCl.sub.3) .delta. 4.80 and -4.47, 11.41, 11.52, 18.11, 21.13,
23.56, 25.87, 28.98, 30.94, 31.36, 35.54, 35.78, 37.13, 38.21,
38.65, 43.36, 44.74, 46.84, 50.55, 54.15, 81.79, 212.03.
Example 37B
[0839] Alkylation of the carbonyl at position 3. To a solution of
compound 133 (500 mg, 1.23 mmol) in dry THF (100 mL) at 0.degree.
C. was added dropwise 3 eq. of commercially available Grignard's
reagent, in dry THF. The mixture was allowed to react for 3 h at
0.degree. C., then left over night at room temperature. A solution
of saturated NH.sub.4Cl was added and the crude product was
extracted with EtOAc. The organic phase was washed with a saturated
NaCl solution, dried over MgSO.sub.4 and evaporated under reduced
pressure. The 3.beta.-alkylated stereoisomer was easily separated
from the 3.alpha.-alkylated stereoisomer by flash chromatography on
silica gel, using a mixture of hexanes and ethyl acetate as eluent.
When the Grignard's reagent was generated in situ as in the case of
ethylphenyl magnesium bromide, 5 eq. was prepared, by a well-known
procedure, using the corresponding bromide, activated magnesium and
iodide. The steroid was then dissolved in dry diethyl ether and
added dropwise to the solution of reagent. The yields obtained were
around 60% for the two stereoisomers.
[0840]
3.beta.-benzyl-17.beta.[(tert-butyldimethylsilyl)oxy]-3.alpha.-hydr-
oxy-5.alpha.-androstane (134a). White solid (24%); IR (KBr) .nu.
3585 and 3460 (OH, alcohol); .sup.1H NMR (CDCl.sub.3) .delta. 0.002
and 0.009 (s, 6H, Si(CH.sub.3).sub.2), 0.69 (s, 3H, CH.sub.3-18),
0.75 (s, 3H, CH.sub.3-19), 0.88 (s, 9H, SiC(CH.sub.3).sub.3), 2.71
(s, 2H, CH.sub.2Ph), 3.54 (t, J=8.2 Hz, 1H, CH-17), 7.20 to 7.34
(5H, Ph); .sup.13C NMR (CDCl.sub.3) .delta. -4.82 and -4.50
(SiC(CH.sub.3).sub.3), 11.25, 11.40, 18.08, 20.62, 23.30, 25.85,
28.41, 30.91, 31.62, 33.27, 33.81, 35.60, 35.84, 37.19, 40.10,
40.84, 43.30, 50.43, 50.69, 54.43, 71.22, 81.82 (C-17), 126.37,
128.09 (2.times.), 130.56 (2.times.), 137.06.
[0841]
3.alpha.-hydroxy-3.beta.-(phenylethyl)-17.beta.[(tert-butyldimethyl-
silyl)oxy]-5.alpha.-androstane (134b). White solid (38%); IR (film)
.nu. 3447 (OH, alcohol); .sup.1H NMR (CDCl.sub.3) .delta. 0.018 and
0.025 (s, 6H, Si(CH.sub.3).sub.2), 0.71 (s, 3H, CH.sub.3-18), 0.78
(s, 3H, CH.sub.3-19), 0.89 (s, 9H, SiC(CH.sub.3).sub.3), 2.73 (m,
2H, Ph-CH.sub.2), 3.56 (t, J=8.1 Hz, 1H, CH-17), 7.18 to 7.31 (5H,
Ph); .sup.13C NMR (CDCl.sub.3) .delta. -4.77 and -4.46
(Si(CH.sub.3).sub.3), 11.28, 11.44, 18.12 (SiC(CH.sub.3).sub.3),
20.67, 23.54, 25.89 (SiC(CH.sub.3).sub.3), 28.52, 29.60, 30.97,
31.66, 33.31, 33.92, 35.66, 36.04, 37.25, 40.03, 41.05, 43.35,
46.47, 50.76, 34.55, 71.50 (C-3), 81.86 (C-17), 125.68, 128.38
(4.times.), 142.82.
Example 37C
[0842] Procedure for hydrolysis of TBDMS group and oxidation of the
resulting alcohol. The silylated ether was dissolved in a
methanolic solution of HO (2%, v/v) and the resulting mixture was
stirred at room temperature for 3 h. Water was then added and MeOH
evaporated under vacuum. The resulting white precipitate was
submitted to Jones' oxidation without purification. To a stirred
solution of crude alcohol in acetone at 0.degree. C., Jones'
reagent (2.7M chromic acid solution) was added dropwise. After 30
to 45 minutes, the reaction was completed. Isopropanol and water
were added and acetone was removed in vacuo. The remaining aqueous
layer was extracted with EtOAc. The combined organic phases were
washed with brine, dried over MgSO.sub.4, filtered and evaporated
under reduced pressure. The purification was done on silica gel,
using HPLC grade solvents, EtOAc and hexanes as eluents.
[0843] 3.beta.-benzyl-3.alpha.-Hydroxy-5.alpha.-androstane-17-one
(CS-213). White solid (88% for the two steps); IR (KBr) .nu. 3408
(OH, alcohol), 1732 (C.dbd.O, ketone); .sup.1H NMR (CDCl.sub.3)
.delta. 0.75 (s, 3H, CH.sub.3-19), 0.84 (s, 3H, CH.sub.3-18), 2.69
(s, 2H, CH.sub.2Ph), 7.18 to 7.32 (5H, Ph); .sup.13C NMR
(CDCl.sub.3) .delta. 11.18, 13.78, 20.20, 21.71, 28.16, 30.79,
31.52, 33.18, 33.70, 35.64, 35.79, 35.88, 39.97, 40.69, 47.75,
50.39, 51.41, 54.22, 71.12, 126.40, 128.09 (2.times.), 130.51
(2.times.), 136.93, 221.27
[0844]
3.alpha.-hydroxy-3.beta.-phenylethyl-5.beta.-androstane-17-one
(EM-1324CS). White solid (82% for the two steps); IR (film) .nu.
3486 (OH, alcohol), 1737 (C.dbd.O, ketone); .sup.1H NMR
(CDCl.sub.3) .delta. 0.79 (s, 3H, CH.sub.3-19), 0.86 (s, 3H,
CH.sub.3-18), 2.71 (m, 2H, Ph-CH.sub.2), 7.18 to 7.30 (5H, Ph);
.sup.13C NMR (CDCl.sub.3) .delta. 11.21, 13.82, 20.26, 21.76,
28.26, 29.54, 30.87, 31.58, 33.27, 33.80, 35.10, 35.84, 36.07,
39.89, 40.90, 46.43, 47.80, 51.49, 54.35, 71.42, 125.69, 128.31
(2.times.), 128.39 (2.times.), 142.70, 221.31.
Pharmaceutical Composition Examples
[0845] Set forth below, by way of example and not of limitation,
are several pharmaceutical compositions utilizing a preferred
active compound EM-1404. Other compounds of the invention or
combination thereof, may be used in place of (or in addition to)
EM-1404. The concentration of active ingredient may be varied over
a wide range as discussed herein. The amounts and types of other
ingredients that may be included are well known in the art.
Example A
Composition Suitable for Injection
[0846]
16 Weight % Ingredient (by weight of total composition) EM-1404 0.4
Ethanol 6.4 NaCl 0.8 Water 91.5 Benzyl alcohol 0.9
Example B
Composition Suitable for use as Topical Lotion
[0847]
17 Weight % Ingredient (by weight of total composition) EM-1404 1.0
Ethanol 70.0 Propylene glycol 29.0
Example C
Composition Suitable for use as Topical Gel
[0848]
18 Weight % Ingredient (by weight of total composition) EM-1404 1.0
Krucel 1.5 Ethanol 70.0 Propylene glycol 27.5
Example D
Tablet
[0849]
19 Weight % Ingredient (by weight of total composition) EM-1404 1.0
Gelatin 5.0 Lactose 67.5 Starch 26.5
Example E
Gelatin Capsule
[0850]
20 Weight % Ingredient (by weight of total composition) EM-1404 2.0
Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 12.8
Magnesium stearate 0.4
Example F
Composition Suitable for use as Topical Gel
[0851]
21 Weight % Ingredient (by weight of total composition) EM-1404 1.0
Ethanol 4.0 Polyethylene glycol 4.0 Gelatin 1.0 NaCl 0.9 Benzyl
alcohol 1.0 Water USP 88.1
[0852] Other inhibitors of type 5 17.beta.-hydroxysteroid
dehydrogenase may be substituted for EM-1404 in the above
formulations, as may an inhibitor of type 3 17.beta.-hydroxysteroid
dehydrogenase. Both type 3 and type 5 may be included together, in
which case the combined weight percent of the two is preferably
double that of EM-1404 alone, with a corresponding reduction in the
weight of the most prevalent excipient (e.g., water lactose,
ethanol or the like).
[0853] The invention has been described in terms of preferred
embodiments and examples,-but is not limited thereby. Those of
skill in the art will readily recognize the broader applicability
and scope of the invention which is limited only by the patent
claims herein.
* * * * *