U.S. patent application number 11/285779 was filed with the patent office on 2006-09-21 for 6,7-oxygenated steroids and uses related thereto.
This patent application is currently assigned to Inflazyme Pharmaceuticals Ltd.. Invention is credited to David L. Burgoyne, Joseph H. L. Chau, John M. Langlands, Edward Piers, Christine Rogers, Hassan Salari, Yaping Shen.
Application Number | 20060211857 11/285779 |
Document ID | / |
Family ID | 26697171 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060211857 |
Kind Code |
A1 |
Burgoyne; David L. ; et
al. |
September 21, 2006 |
6,7-Oxygenated steroids and uses related thereto
Abstract
Steroid compounds having various oxygen substitution on the
steroid nucleus are disclosed. A specific functionality present on
many of the steroid compounds is oxygen substitution at both of
positions 6 and 7. Thus, certain steroids have oxygen substitution
at C6 and C7, and some have specific stereochemistries such as
6.alpha. and 7.beta. oxygen substitution, and an alpha hydrogen at
the 5 position in addition to having 6.alpha. and 7.beta. oxygen
substitution. Steroids having 3,4-epoxy functionality are also
disclosed. In addition, steroids having C17 pyran and
.delta.-lactone functionality, with oxygen substitution at C6 and
C7, or at C15, of the steroid nucleus, are disclosed.
Inventors: |
Burgoyne; David L.; (Delta,
CA) ; Shen; Yaping; (Port Coquitlam, CA) ;
Langlands; John M.; (Richmond, CA) ; Rogers;
Christine; (Vancouver, CA) ; Chau; Joseph H. L.;
(Vancouver, CA) ; Piers; Edward; (Richmond,
CA) ; Salari; Hassan; (Tswassen, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Inflazyme Pharmaceuticals
Ltd.
Richmond
CA
The University of British Columbia
Vancouver
CA
The University of Alberta
Edmonton
CA
|
Family ID: |
26697171 |
Appl. No.: |
11/285779 |
Filed: |
November 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10703155 |
Nov 6, 2003 |
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11285779 |
Nov 22, 2005 |
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09471827 |
Dec 23, 1999 |
6706701 |
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10703155 |
Nov 6, 2003 |
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08893575 |
Jul 10, 1997 |
6046185 |
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09471827 |
Dec 23, 1999 |
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08679642 |
Jul 12, 1996 |
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08893575 |
Jul 10, 1997 |
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60023450 |
Jul 11, 1996 |
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Current U.S.
Class: |
540/114 |
Current CPC
Class: |
C07J 51/00 20130101;
C07J 41/0016 20130101; C07J 7/002 20130101; C07J 1/0007 20130101;
C07J 1/0022 20130101; C07J 3/00 20130101; C07J 1/0011 20130101;
C07J 71/0026 20130101; Y10S 514/825 20130101; C07J 17/00 20130101;
C07J 21/008 20130101; C07J 13/005 20130101; C07J 71/001 20130101;
Y02P 20/55 20151101; C07J 31/006 20130101; C07J 13/007 20130101;
C07J 9/00 20130101; Y10S 514/826 20130101 |
Class at
Publication: |
540/114 |
International
Class: |
C07J 17/00 20060101
C07J017/00 |
Claims
1. A compound of the formula: ##STR157## or a pharmaceutically
acceptable salt or solvate thereof, wherein: numerals 1 through 17
each represent a carbon; .sub.each of C1, C2, C3, C4, C11, C12, C15
and C16 is independently substituted according to any of (a) and
(b): (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected; each of C5, C6, C7, C8, C9, C10, C13 and
C14 is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; C17 is substituted according to any of (c), (d), (e),
(f), (g), (h) and (i): (c) one of: .dbd.O,
.dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6, as long as one of the following conditions i), ii), iii)
or iv) apply: i) C5 is substituted with a hydrogen in the alpha
configuation, and C3 is not bonded to oxygen, and when C3 is
substituted with two hydrogen atoms then C17 is not substituted
with either --CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2 or
--CH(CH.sub.3)(CH.sub.2).sub.2C(.dbd.O)OCH.sub.3; ii) C10 and C13
are not simultaneously substituted with methyl, and when C10 is
substituted with methyl, then C14 is not substituted with a methyl,
and the A ring is never aromatic; iii) if C3 and C4 are bonded to
oxygen atoms, and the C6 --OR.sup.1 substituent has the alpha
configuration, and the C7 --OR.sup.1 substituent has the beta
configuration, then C17 is not substituted with any of the
following: ##STR158## iv) C3 and C4 are each bonded to the same
oxygen atom so as to form an oxirane ring, with the proviso that C7
does not have carbonyl substitution when C5 has hydroxyl or
--OR.sup.1 substitution; (d) a cyclic structure of the formula
##STR159## wherein G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--, as long as
C3 and C4 are not simultaneously substituted with hydroxyl or
protected hydroxyl; (e) two hydrogen atoms, as long as C3 is not
substituted with a carbonyl group; the A, B, C and D rings may
independently be fully saturated, partially saturated or fully
unsaturated; R.sup.1 is H or a protecting group such that
--OR.sup.1 is a protected hydroxyl group, where vicinal --OR.sup.1
groups may together form a cyclic structure which protects vicinal
hydroxyl groups, and where geminal --OR.sup.1 groups may together
form a cyclic structure which protects a carbonyl group, with the
proviso that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group; R.sup.2, R.sup.3 and R.sup.4
at each occurrence is independently selected from H and C.sub.1-30
organic moiety that may optionally contain at least one heteroatom
selected from the group consisting of boron, halogen, nitrogen,
oxygen, silicon and sulfur, where two geminal R.sup.4 groups may
together form a ring with the carbon atom to which they are both
bonded; and X represents fluoride, chloride, bromide and
iodide.
2. A compound of claim 1 having the formula ##STR160## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C4, C11, C12, C15 and C16 is independently substituted
with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; each of C5, C8, C9, C10
and C13 is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; C3 is substituted with .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; each of C6 and C7 is independently substituted with
hydrogen or --OR.sup.1; C14 is substituted with --X, --OR.sup.1 or
--R.sup.4 excluding methyl; the A, B, C and D rings may
independently be fully saturated, partially saturated or fully
unsaturated; R.sup.1 is H or a protecting group such that
--OR.sup.1 is a protected hydroxyl group, where vicinal --OR.sup.1
groups may together form a cyclic structure which protects vicinal
hydroxyl groups, and where geminal --OR.sup.1 groups may together
form a cyclic structure which protects a carbonyl group, with the
proviso that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group; R.sup.2, R.sup.3 and R.sup.4
at each occurrence is independently selected from H and C.sub.1-30
organic moiety that may optionally contain at least one heteroatom
selected from the group consisting of boron, halogen, nitrogen,
oxygen, silicon and sulfur, where two geminal R.sup.4 groups may
together form a ring with the carbon atom to which they are both
bonded; and X represents fluoride, chloride, bromide and
iodide.
3. (canceled)
4. A compound of claim 1 having the formula ##STR161## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C4, C11, C12, C15, C16 and C17 is independently
substituted with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; each of C8, C9, C10, C13
and C14 is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; C3 is substituted with one of .dbd.C(R.sup.4)(R.sup.4)
and --C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- wherein n
ranges from 1 to about 6; the A, B, C and D rings may independently
be fully saturated, partially saturated or fully unsaturated;
R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represent a carbonyl
or protected carbonyl group; R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur; where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and X
represents fluoride, chloride, bromide and iodide.
5. A compound of claim 1 having the formula ##STR162## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is independently
substituted with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; each of C5, C8, C9, C10,
C13 and C14 is independently substituted with one of --X, --R.sup.4
or --OR.sup.1; with the provisos that (a) C10 and C13 are not
simultaneously substituted with methyl, and (b) when C10 is
substituted with methyl, then C14 is not substituted with a methyl;
the A, B, C and D rings may independently be fully saturated,
partially saturated or fully unsaturated with the proviso that the
A ring is not aromatic; R.sup.1 is H or a protecting group such
that --OR.sup.1 is a protected hydroxyl group, where vicinal
--OR.sup.1 groups may together form a cyclic structure which
protects vicinal hydroxyl groups, and where geminal --OR.sup.1
groups may together form a cyclic structure which protects a
carbonyl group, with the proviso that either or both of --OR.sup.1
at C6 and C7 represent a carbonyl or protected carbonyl group;
R.sup.4 at each occurrence is independently selected from H and
C.sub.1-30 organic moiety that may optionally contain at least one
heteroatom selected from the group consisting of boron, halogen,
nitrogen, oxygen, silicon and sulfur; where two geminal R.sup.4
groups may together form a ring with the carbon atom to which they
are both bonded; and X represents fluoride, chloride, bromide and
iodide.
6. A compound of claim 1 having the formula ##STR163## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C11, C12, C15, and C16 and G17 is independently
substituted with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; C17 is independently
substituted with .dbd.O,
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; each of C5, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1; C8 is
substituted with --X or --R.sup.4 and is preferably not bonded
directly to oxygen; the A, B, C and D rings may independently be
fully saturated, partially saturated or fully unsaturated; R.sup.1
is H or a protecting group such that --OR.sup.1 is a protected
hydroxyl group, where vicinal --OR.sup.1 groups may together form a
cyclic structure which protects vicinal hydroxyl groups, and where
geminal --OR.sup.1 groups may together form a cyclic structure
which protects a carbonyl group; R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur; where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and X
represents fluoride, chloride, bromide and iodide.
7. A compound of claim 1 having the formula ##STR164## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C3, C4, C11, C12, C15 and C16 is independently
substituted with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; with the proviso that C3
and C4 are not simultaneously substituted with hydroxyl or
protected hydroxyl, and are preferably not simultaneously
substituted with oxygen atoms; each of C5, C8, C9, C10, C13 and C14
is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--; the A, B, C
and D rings may independently be fully saturated, partially
saturated or fully unsaturated; R.sup.1 is H or a protecting group
such that --OR.sup.1 is a protected hydroxyl group, where vicinal
--OR.sup.1 groups may together form a cyclic structure which
protects vicinal hydroxyl groups, and where geminal --OR.sup.1
groups may together form a cyclic structure which protects a
carbonyl group, with the proviso that either or both of --OR.sup.1
at C6 and C7 represents a carbonyl or protected carbonyl group;
R.sup.4 at each occurrence is independently selected from H and
C.sub.1-30 organic moiety that may optionally contain at least one
heteroatom selected from the group consisting of boron, halogen,
nitrogen, oxygen, silicon and sulfur, where two geminal R.sup.4
groups may together form a ring with the carbon atom to which they
are both bonded; and X represents fluoride, chloride, bromide and
iodide.
8. A compound of claim 1 having the formula ##STR165## or a
pharmaceutically acceptable salt or solvate thereof, wherein: each
of C1, C2, C11, C12, C15, C16 and C17 is independently substituted
with (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or (b) two of the following, which are independently
selected: --X, --R.sup.4 and --OR.sup.1; each of C5, C8, C9, C10,
C13 and C14 is independently substituted with one of --X, --R.sup.4
or --OR.sup.1; the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated; R.sup.1 is H
or a protecting group such that --OR.sup.1 is a protected hydroxyl
group, where vicinal --OR.sup.1 groups may together form a cyclic
structure which protects vicinal hydroxyl groups, and where geminal
--OR.sup.1 groups may together form a cyclic structure which
protects a carbonyl group, with the proviso that either or both of
--OR.sup.1 at C6 and C7 represents a carbonyl or protected carbonyl
group; R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and X represents fluoride, chloride,
bromide and iodide; with the proviso that C7 does not have carbonyl
substitution when C5 has hydroxy or --OR.sup.1 substitution.
9. A compound of claim 1 having a formula selected from: ##STR166##
or a pharmaceutically acceptable salt or solvate thereof, wherein:
each of C1, C2, C3, C4, C11, C12 and C16 is independently
substituted according to (a) or (b): (a) one of: .dbd.O,
.dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6, (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected; C5 is substituted with a hydrogen atom;
each of C6, C7, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1; and C17 is
substituted according to (c), (d), (e) or (f): (c) two substituents
selected from hydrogen, halogen, C.sub.1-C.sub.30 saturated
hydrocarbyl excluding
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2, halogen
substituted C.sub.1-C.sub.30 saturated hydrocarbyl,
C.sub.1-C.sub.30 unsaturated hydrocarbyl, and halogen substituted
C.sub.1-C.sub.30 unsaturated hydrocarbyl; (d) one substituent
selected from .dbd.C(R.sup.4)(R.sup.4) with the proviso that C14 is
not substituted with methyl; (e) at least one oxygen
atom-containing substituent selected from .dbd.O,
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6, --OH, and --OR.sup.1; (f) at least one nitrogen
atom-containing substituent selected from --N(R.sup.4)(R.sup.4)
wherein the two R.sup.4 groups may together with the nitrogen atom
form one or more rings, so that the nitrogen atom-containing
substituent includes nitrogen atom-containing heterocyclic groups;
wherein the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated; R.sup.1 is H
or a protecting group such that --OR.sup.1 is a protected hydroxyl
group, where --OR.sup.1 groups bonded to adjacent carbon atoms may
together form a cyclic structure which protects both hydroxyl
groups; R.sup.4 at each occurrence is independently selected from H
and R.sup.5; R.sup.5 is a C.sub.1-30 organic moiety that may
optionally contain at least one heteroatom selected from the group
consisting of boron, halogen, nitrogen, oxygen, silicon and sulfur;
where two geminal R.sup.1 groups may together form a ring with the
carbon atom to which they are both bonded; and X represents
fluoride, chloride, bromide or iodide.
10. A compound of claim 1 having the formula: ##STR167##
11. A compound of claim 1 having the formula ##STR168##
12. A compound of claim 1 wherein C10 and C13 are each substituted
with methyl.
13. A compound of claim 1 wherein C11 and C12 are each substituted
only with hydrogen.
14. (canceled)
15. (canceled)
16. A compound of claim 1 wherein C17 is substituted with a
substituent selected from carbonyl or protected carbonyl.
17. A compound of claim 1 wherein C3 is substituted with a
substituent selected from halogen, hydroxyl and protected
hydroxyl.
18. (canceled)
19. A compound of claim 1 wherein C3 and C4 are both substituted
with oxygen and together form an epoxide, acetal or ketal.
20. A compound of claim 1 wherein the A, B, C and D rings are
saturated.
21. A compound of claim 1 wherein the A ring is unsaturated.
22. A compound of claim 1 wherein a double bond is present between
C4 and C5.
23. A compound of claim 1 wherein C6 and C7 are each substituted
with hydrogen.
24. A compound of claim 1 wherein C6 and C7 are both substituted
with hydroxyl groups.
25. (canceled)
26. A pharmaceutical composition comprising a compound of claim 1
in combination with a pharmaceutically acceptable carrier or
diluent.
27-60. (canceled)
61. A process for treating at least one of asthma, allergy,
arthritis and thrombosis comprising administering to a subject in
need thereof an effective amount of the compound or salt thereof of
according to claim 1.
62. A process for treating at least one of asthma, allergy,
arthritis and thrombosis comprising administering to a subject in
need thereof an effective amount of the pharmaceutical composition
of claim 26.
63. (canceled)
64. A process for treating a condition associated with an elevated
level of NF.kappa.B activity in a subject, comprising administering
to a subject in need thereof an amount of a compound effective to
lower the NF.kappa.B activity, wherein the compound has the formula
of the compounds of claim 1.
65. A process for treating a condition associated with an elevated
level of NF.kappa.B activity in a subject, comprising administering
to a subject in need thereof an amount of a composition effective
to lower the NF.kappa.B activity, wherein the composition is
described in claim 26.
66. (canceled)
67. A process for introducing an exocyclic olefin group to the C17
position of a 6,7-dioxygenated steroid comprising providing a
compound of Formula (10), reacting the compound of Formula (10)
with a Wittig reagent of Formula (11) in the presence of a base, to
provide an olefin compound of Formula (12) ##STR169## wherein each
of the compounds of Formulas (10) and (12) include pharmaceutically
acceptable salts and solvates thereof, and wherein: each of C1, C2,
C3, C4, C11, C12, C15 and C16 is independently substituted
according to any of (a) and (b): (a) one of: .dbd.O,
.dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected; each of C5, C6, C7, C8, C9, C10, C13 and
C14 is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; R.sup.1 is H or a protecting group such that --OR.sup.1
is a protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group; Ra, Rb and R.sup.4 at each
occurrence is independently selected from H and C.sub.1-30 organic
moiety that may optionally contain at least one heteroatom selected
from the group consisting of boron, halogen, nitrogen, oxygen,
silicon and sulfur, where two geminal R.sup.4 groups may together
form a ring with the carbon atom to which they are both bonded; and
X represents fluoride, chloride, bromide and iodide, which is
independently selected at each occurrence.
68. The process of claim 67 wherein the base is selected from
sodium t-butoxide, potassium t-butoxide and sodium hydride, and the
base is in admixture with an aprotic solvent including toluene,
tetrahydrofuran, methylene chloride, dimethylformamide,
dimethylsulfoxide, benzene and diethyl ether.
69. The process of claim 67 wherein Ra and Rb are independently
selected from hydrogen and C.sub.1-C.sub.7alkyl, and X is selected
from chloride, bromide and iodide.
70-72. (canceled)
73. A process for a stereocontrolled introduction of a hydroxyl
group at C3 of a steroid nucleus, comprising providing a steroid
compound of Formula (15) having a carbonyl group at C3, and
reducing the carbonyl group to a hydroxyl group with a reducing
agent so as to provide at least one compound of Formulas (16) and
(17) ##STR170## wherein each of the compounds of Formulas (15),
(16) and (17) include pharmaceutically acceptable salts and
solvates thereof, and wherein: each of C1, C2, C4, C11, C12, C15,
C16 and C17 is independently substituted according to any of (a)
and (b): (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected; each of C5, C6, C7, C8, C9, C10, C13 and
C14 is independently substituted with one of --X, --R.sup.4 or
--OR.sup.1; R.sup.1 is H or a protecting group such that --OR.sup.1
is a protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group; R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur, where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and X
represents fluoride, chloride, bromide and iodide.
74. The process of claim 73 wherein the reducing agent is selected
from lithium trisiamylborohydride, lithium tri-sec-butylborohydride
and potassium tri-sec-butylborohydride, to provide predominantly
the hydroxyl compound of Formula (16).
75. The process of claim 73 wherein the reducing agent is selected
from sodium borohydride and lithium aluminum hydride, to provide
predominantly the hydroxyl compound of Formula (17).
76. The process of claim 73 wherein the ratio of Formula (16) to
Formula (17) compounds following the reduction is other than 1:1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/703,155 filed Nov. 6, 2003 (now allowed);
which is a continuation of U.S. patent application Ser. No.
09/471,827 filed Dec. 23, 1999 (U.S. Pat. No. 6,706,701); which is
a division of U.S. patent application Ser. No. 08/893,575 filed
Jul. 10, 1997 (now U.S. Pat. No. 6,046,185); which is a
continuation of U.S. patent application Ser. No. 08/679,642 filed
Jul. 12, 1996 (now abandoned); which claims the benefit of U.S.
Provisional Application No. 60/023,450, filed Jul. 11, 1996. All of
these applications are incorporated herein by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention is directed to steroid compounds, and in
particular to 6,7-oxygenated steroid compounds and therapeutic uses
related thereto.
[0004] 2. Description of the Related Art
[0005] Asthma and allergy are closely related with good evidence
from clinical studies demonstrating a strong correlation between
the severity of asthma and the degree of atopy (allergy).
Sensitization to allergens is believed to be the most important
risk factor for asthma in both children and adults, with
approximately 90% of asthma cases exhibiting atopy.
[0006] Allergy is characterized by an increased blood serum IgE
(antibody) level. Repeated exposure to allergens, in a process
called sensitization, is normally required to elicit sufficient B
cell production of IgE specific to a given allergen or series of
allergens to trigger atopy and the subsequent asthmatic or allergic
response. Once B cells are exposed to allergens, they produce
antibodies which bind to the surface of mast cells. The
crosslinking of 2 antibodies by the antigen causes a series of
reactions causing degranulation and the release of a number of
mediators which modulate the inflammatory response. Mediators that
are released or generated during the asthmatic and allergic
response include histamine, leukotrienes, prostaglandins, cytokines
and tryptase.
[0007] Asthma is characterized by hyperresponsiveness of the
airways, episodic periods of bronchospasm and chronic inflammation
of the lungs. Obstruction of the airways is reversible with time or
in response to drug therapies. Patients exhibiting normal airflow
may be hyperreactive to a variety of naturally occurring stimuli,
e.g., cold air, exercise, chemicals and allergen. The most common
event initiating an asthmatic response is an immediate
hypersensitivity to common allergens including ragweed pollen,
grass pollen, various fungi, dust mites, cockroaches and domestic
animals. The symptoms of the disease include chest tightness,
wheezing, shortness of breath and coughing. Mild forms of the
disease occur in up to 10% of the U.S. population, while the U.K.,
Australia and New Zealand report higher prevalences. Asthma
incidence and mortality has been increasing worldwide, doubling
over the past 20 years despite modern therapies.
[0008] The response of the airways to allergen is complex and
consists of an early asthmatic response (EAR) which peaks 20-30 min
after exposure to the stimuli, is characterized by
bronchoconstriction and normally resolves after 11/2 to 2 hours.
The late asthmatic response (LAR) generally occurs 3-8 hours after
initial exposure, and involves both bronchoconstriction and the
development of inflammation and edema in the lung tissue. This
inflammation often becomes chronic, with epithelial damage
occurring and infiltration of the lungs with inflammatory cells
such as eosinophils and neutrophils.
[0009] Current Treatments for Asthma
[0010] Glucocorticosteroids (steroids) are the most effective
long-term therapy for the treatment of asthma. Oral steroids are
not very useful for the control of acute asthma attacks and their
chronic use in the control of asthma is minimal due to the
introduction of inhaled steroids. Due to the presence of airway
inflammation even in mild asthma, inhaled steroids are used even in
early stage drug therapy. As effective as inhaled steroids are,
side effects limit their use and combination therapy is often
employed. Combination therapy is divided into the following areas:
anti-inflammatory drugs (e.g., inhaled and oral steroids),
bronchodilators, (e.g., .beta..sub.2-agonists, xanthines,
anticholinergics), and mediator inhibitors (e.g., cromolyns and
leukotriene antagonists).
[0011] Cromolyns (e.g., disodium cromoglycate and nedocromil)
inhibit the release of histamine in vitro and prevent bronchial
hyperreactivity, while displaying few side effects. They are not
effective orally and have no bronchodilator effect. Usually chronic
treatment (several days) is required to achieve optimal
anti-inflammatory effect, though cromolyns exhibit beneficial
effects against exercise-induced asthma when administered only 10
minutes prior to exercise. Cromolyns are, at best, only marginally
effective against moderate to severe asthma.
[0012] Glucocorticosteroids (steroids) have profound effects
against lung inflammation, and are by far the most effective drugs
for the treatment of asthma and allergies. In mast cells they
inhibit the production of arachidonic acid metabolites
(leukotrienes and prostaglandins) and cytokines. Responses to
inhaled steroids or systemic steroids can occur within 4 hours but
may take several days depending on the severity of the disease
state. Symptoms often return without regular chronic treatment.
Side effects of inhaled steroids used on a continual basis include
dysphonia, local irritation and oral candidiasis (a fungal
infection). Higher doses of inhaled steroids cause suppression of
the HPA-axis which is responsible for the regulation of serum
cortisol levels, metabolism, stress, CNS function and immunity.
Continuous use of high dose inhaled steroids or oral steroids
induce more severe side effects: severe suppression of the HPA
axis, causing effects on the immune system, hypertension,
osteoporosis, peptic ulcers, growth retardation in children,
behavioral problems, reproductive problems, cataracts and
hematological disorders.
[0013] Beta-agonists reverse the bronchospasm produced during an
asthmatic attack and have a modest activity against the onset of
the response. Their routes of administration and duration of action
are variable. Prolonged use of these agents can cause decreased
response to the therapy itself with the development of tolerance.
These compounds have no effect on the inflammatory response
itself.
[0014] Xanthines, which are cyclic AMP phosphodiesterase
inhibitors, are also used in bronchodilator therapy. Though
effective, xanthine activity is influenced by a number of factors
including food, age, smoking, etc. The therapeutic window is
relatively narrow and side effects include gastrointestinal
disorders, CNS disturbances, headache, anxiety and cardiac
arrhythmias. The importance of treatment of inflammation in asthma
and allergy has led to a decline in the use of xanthines for
therapy.
[0015] Anticholinergic agents such as ipratropium bromide are used
to block the contraction of bronchial smooth muscle induced by
acetylcholine released as a neurotransmitter. Some positive effects
are reported in asthma, with these drugs being most effective
against chronic obstructive pulmonary disease. A large number of
side effects are seen with these drugs including urinary retention,
dry mouth, tachycardia, nausea, vomiting, flushing and
hypertension.
[0016] Inhibitors of 5-lipoxygenase inhibit the generation of
leukotrienes, while leukotriene antagonists prevent the action of
leukotrienes, which are potent bronchospastic mediators released
during an asthmatic reaction. Use of leukotriene synthesis
inhibitors has been associated with increased liver enzymes,
indicating the need to monitor liver function closely in certain
patient populations. Leukotriene inhibitors have shown comparative
activity to the cromolyns, and activity equivalent to low dose
corticosteroids.
[0017] In general, moderate to severe asthma patients are poorly
served by the present armamentarium of drugs. Drugs that are safe
are only marginally effective, while effective drugs have
unacceptable side effects with extensive monitoring of patients
required. There is a significant need for therapeutic agents that
achieve safe and effective relief of asthma and allergy symptoms.
The present invention provides these and related benefits as
described herein.
SUMMARY OF THE INVENTION
[0018] One aspect of the invention provides compounds of the
formula: ##STR1## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0019] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted according to any of (a) and (b):
[0020] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0021] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0022] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0023] C17 is substituted according to any of (c), (d), (e), (f),
(g), (h) and (i):
[0024] (c) .dbd.C(R.sup.2)(R.sup.3) except when C14 is substituted
with methyl;
[0025] (d) --R.sup.5 and --OR.sup.6 so long as the A and B rings
are not aromatic, and when C10 is substituted with methyl then C5
is not bonded directly to oxygen, where R.sup.5 and R.sup.6 may
together form a direct bond so C17 is a carbonyl group, or may
together with C17 form a cyclic 3-6 membered ether or 4-6 membered
lactone; otherwise R.sup.5 is R.sup.4 or --OR.sup.6 and R.sup.6 is
R.sup.1 or R.sup.4.
[0026] (e) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6, as long as one of the following conditions i), ii), iii)
or iv) apply: [0027] i) C5 is substituted with a hydrogen in the
alpha configuation, and C3 is not bonded to oxygen, and when C3 is
substituted with two hydrogen atoms then C17 is not substituted
with either --CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2 or
--CH(CH.sub.3)(CH.sub.2)2C(.dbd.O)OCH.sub.3; [0028] ii) C10 and C13
are not simultaneously substituted with methyl, and when C10 is
substituted with methyl, then C14 is not substituted with a methyl,
and the A ring is never aromatic; [0029] iii) if C3 and C4 are
bonded to oxygen atoms, and the C6--OR.sup.1 substituent has the
alpha configuration, and the C7 --OR.sup.1 substituent has the beta
configuration, then C17 is not substituted with any of the
following: ##STR2## [0030] iv) C3 and C4 are each bonded to the
same oxygen atom so as to form an oxirane ring, with the proviso
that C7 does not have carbonyl substitution when C5 has hydroxyl or
--OR.sup.1 substitution;
[0031] (f) two of the following substituents, which are
independently selected: --X, --R.sup.4 and --OR.sup.1, as long as
one of the above conditions i), ii), iii) or iv) apply;
[0032] (g) a cyclic structure of the formula ##STR3##
[0033] wherein G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--, as long as
C3 and C4 are not simultaneously substituted with hydroxyl or
protected hydroxyl;
[0034] (h) two hydrogen atoms, as long as C3 is not substituted
with a carbonyl group;
[0035] (i) one hydrogen atom and one group selected from
C.sub.1-C.sub.30 hydrocarbyl groups and C.sub.1-C.sub.30 halogen
substituted hydrocarbyl groups, excluding
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2;
[0036] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0037] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0038] R.sup.2, R.sup.3 and R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur, where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and
[0039] X represents fluoride, chloride, bromide and iodide.
[0040] In a preferred embodiment, the compounds have the formula
##STR4## including pharmaceutically acceptable salts and solvates
thereof, wherein:
[0041] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0042] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0043] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0044] each of C5, C8, C9, C10 and C13 is independently substituted
with one of --X, --R.sup.4 or --OR.sup.1;
[0045] C14 is substituted with --X, --OR.sup.1, or --R.sup.4
excluding methyl;
[0046] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0047] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0048] R.sup.2, R.sup.3 and R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur, where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and
[0049] X represents fluoride, chloride, bromide and iodide.
[0050] In another preferred embodiment, the compounds have the
formula ##STR5## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0051] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0052] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0053] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0054] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0055] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated, with the
proviso that neither the A nor B ring is aromatic;
[0056] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0057] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0058] R.sup.5 and R.sup.6 may together form a direct bond so C17
is a carbonyl group, or may together with C17 form a cyclic 3-6
membered ether or 4-6 membered lactone; otherwise R.sup.5 is
R.sup.4 or --OR.sup.6 and R.sup.6 is R.sup.1 or R.sup.4; and
[0059] X represents fluoride, chloride, bromide and iodide.
[0060] with the proviso that when C10 is substituted with methyl,
then C5 is not directly bonded to an oxygen atom.
[0061] In another preferred embodiment, the compounds have the
formula ##STR6## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0062] each of C1, C2, C4, C11, C12, C15, C16 and C17 is
independently substituted with
[0063] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0064] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1 each of C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0065] C3 is substituted with one of .dbd.C(R.sup.4)(R.sup.4) and
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- wherein n ranges
from 1 to about 6, or two of --X, and --R.sup.4 with the proviso
that C3 is not bonded to an oxygen atom, and when C3 is substituted
with two hydrogen atoms then C17 is not substituted with either
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2 or
--CH(CH.sub.3)(CH.sub.2)2C(.dbd.O)OCH.sub.3;
[0066] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0067] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represent a carbonyl
or protected carbonyl group;
[0068] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0069] X represents fluoride, chloride, bromide and iodide.
[0070] In another preferred embodiment, the compounds have the
formula ##STR7## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0071] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted with
[0072] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0073] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0074] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0075] with the provisos that (a) C10 and C13 are not
simultaneously substituted with methyl, and (b) when C10 is
substituted with methyl, then C14 is not substituted with a
methyl;
[0076] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated with the
proviso that the A ring is not aromatic;
[0077] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represent a carbonyl or
protected carbonyl group;
[0078] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0079] X represents fluoride, chloride, bromide and iodide.
[0080] In another preferred embodiment, the compounds have the
formula ##STR8## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0081] each of C1, C2, C11, C12, C15, C16 and C17 is independently
substituted with
[0082] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0083] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0084] with the proviso that C17 is not substituted with any of the
following: ##STR9##
[0085] each of C5, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0086] C8 is substituted with --X or --R.sup.4 and is preferably
not bonded directly to oxygen;
[0087] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0088] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group;
[0089] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0090] X represents fluoride, chloride, bromide and iodide.
[0091] In another preferred embodiment, the compounds have the
formula ##STR10## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0092] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0093] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0094] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0095] with the proviso that C3 and C4 are not simultaneously
substituted with hydroxyl or protected hydroxyl, and are preferably
not simultaneously substituted with oxygen atoms;
[0096] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0097] G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--;
[0098] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0099] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0100] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0101] X represents fluoride, chloride, bromide and iodide.
[0102] In another preferred embodiment, the compounds have the
formula ##STR11## including pharmaceutically acceptable salts and
solvates thereof, wherein:
[0103] each of C1, C2, C11, C12, C15, C16 and C17 is independently
substituted with
[0104] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0105] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0106] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0107] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0108] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR groups may together
form a cyclic structure which protects vicinal hydroxyl groups, and
where geminal --OR.sup.1 groups may together form a cyclic
structure which protects a carbonyl group, with the proviso that
either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0109] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0110] X represents fluoride, chloride, bromide and iodide;
[0111] with the proviso that C7 does not have carbonyl substitution
when C5 has hydroxyl or --OR.sup.1 substitution.
[0112] In another preferred embodiment, the compounds have one of
the formulas ##STR12## including pharmaceutically acceptable salts
and solvates thereof, wherein:
[0113] each of C1, C2, C3, C4, C11, C12 and C16 is independently
substituted according to (a) or (b):
[0114] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)_wherein n ranges from 1 to about
6,
[0115] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0116] C5 is substituted with a hydrogen atom;
[0117] each of C6, C7, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1; and
[0118] C17 is substituted according to (c), (d), (e) or (f):
[0119] (c) two substituents selected from hydrogen, halogen,
C.sub.1-C.sub.30 saturated hydrocarbyl excluding
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2, halogen
substituted C.sub.1-C.sub.30 saturated hydrocarbyl,
C.sub.1-C.sub.30 unsaturated hydrocarbyl, and halogen substituted
C.sub.1-C.sub.30 unsaturated hydrocarbyl;
[0120] (d) one substituent selected from .dbd.C(R.sup.4)(R.sup.4)
with the proviso that C14 is not substituted with methyl;
[0121] (e) at least one oxygen atom-containing substituent selected
from .dbd.O, --(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges
from 1 to about 6, --OH, and --OR.sup.1;
[0122] (f) at least one nitrogen atom-containing substituent
selected from --N(R.sup.4)(R.sup.4) wherein the two R.sup.4 groups
may together with the nitrogen atom form one or more rings, so that
the nitrogen atom-containing substituent includes nitrogen
atom-containing heterocyclic groups; wherein
[0123] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0124] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where --OR.sup.1 groups bonded to
adjacent carbon atoms may together form a cyclic structure which
protects both hydroxyl groups;
[0125] R.sup.4 at each occurrence is independently selected from H
and R.sup.5;
[0126] R.sup.5 is a C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur; where two
geminal R.sup.5 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0127] X represents fluoride, chloride, bromide or iodide.
[0128] In another aspect, the invention provides a pharmaceutical
composition comprising a compound according any of the descriptions
provided above, in combination with a pharmaceutically acceptable
carrier or diluent.
[0129] In another aspect, the invention provides a pharmaceutical
composition comprising a compound in combination with a
pharmaceutically acceptable carrier or diluent, the compound having
the formula ##STR13## including pharmaceutically acceptable salts
and solvates thereof, wherein:
[0130] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with --X, --R.sup.4 and --OR.sup.1;
[0131] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted with a substituent selected from (a) or
(b), wherein
[0132] (a) represents one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)--, wherein n ranges from 1 to
about 6; and
[0133] (b) represents two of: --X, --R.sup.4 and --OR.sup.1, which
are independently selected at each occurrence;
[0134] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0135] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where the C6 and C7-OR.sup.1 groups may
together form a cyclic structure which protects both hydroxyl
groups;
[0136] R.sup.4 at each occurrence is independently selected from H
and R.sup.5;
[0137] R.sup.5 is a C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur; where two
geminal R.sup.4 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0138] X represents fluoride, chloride, bromide or iodide;
[0139] with the proviso that C15 is not bonded to an oxygen
atom.
[0140] In another aspect, the invention provides for the use of the
above compounds (any one or mixture thereof) for manufacture of a
medicament for the treatment of asthma, allergy, inflammation
including arthritis, and/or thrombosis, or for treating a condition
associated with an elevated level of NFKB.
[0141] In another aspect, the invention provides a process for
treating asthma comprising administering to a subject in need
thereof an effective amount of the compound or salt thereof, or a
pharmaceutical composition, each as described above.
[0142] In another aspect, the invention provides a process for
treating allergy comprising administering to a subject in need
thereof an effective amount of the compound or salt thereof, or a
pharmaceutical composition, each as described above.
[0143] In another aspect, the invention provides a process for
treating inflammation due to arthritis comprising administering to
a subject in need thereof an effective amount of the compound or
salt thereof, or a pharmaceutical composition, each as described
above.
[0144] In another aspect, the invention provides a process for
treating thrombosis comprising administering to a subject in need
thereof an effective amount of the compound or salt thereof, or a
pharmaceutical composition, each as described above.
[0145] In another aspect, the invention provides a process for
treating a condition associated with an elevated level of
NF.kappa.B activity in a subject, comprising administering to a
subject in need thereof an effective amount of the compound or salt
thereof, or a pharmaceutical composition, each as described
above.
[0146] In another aspect, the invention provides a process for
introducing an exocyclic olefin group to the C17 position of a
6,7-dioxygenated steroid comprising providing a compound of Formula
(10), reacting the compound of Formula (10) with a Wittig reagent
of Formula (11) in the presence of a base, to provide an olefin
compound of Formula (12) ##STR14##
[0147] wherein each of the compounds of Formulas (10) and (12)
include pharmaceutically acceptable salts and solvates thereof, and
wherein:
[0148] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted according to any of (a) and (b):
[0149] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0150] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0151] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0152] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0153] Ra, Rb and R.sup.4 at each occurrence is independently
selected from H and C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur, where two
geminal R.sup.4 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0154] X represents fluoride, chloride, bromide and iodide, which
is independently selected at each occurrence.
[0155] In another aspect, the invention provides a process for
introducing 6.alpha.,7.beta.-dioxygenation into a steroid,
comprising providing a steroid of Formula (13) having a carbonyl
group at C7 and a double bond between C5 and C6, comprising a
reduction the carbonyl group to a hydroxyl group, followed by a
hydroboration of the double bond to provide a hydroxyl group at C6,
wherein the C6 hydroxyl group has the .alpha.-configuration and the
C7 hydroxyl group has the .beta.-configuration, ##STR15## wherein
each of the compounds of Formulas (13) and (14) include
pharmaceutically acceptable salts and solvates thereof, and
wherein:
[0156] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted according to any of (a) and (b):
[0157] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0158] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0159] each of C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0160] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0161] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0162] X represents fluoride, chloride, bromide and iodide.
[0163] In another aspect, the invention provides a process for a
stereocontrolled introduction of a hydroxyl group at C3 of a
steroid nucleus, comprising providing a steroid compound of Formula
(15) having a carbonyl group at C3, and reducing the carbonyl group
to a hydroxyl group with a reducing agent so as to provide at least
one compound of Formulas (16) and (17) ##STR16## wherein each of
the compounds of Formulas (15), (16) and (17) include
pharmaceutically acceptable salts and solvates thereof, and
wherein:
[0164] each of C1, C2, C4, C11, C12, C15, C16 and C17 is
independently substituted according to any of (a) and (b):
[0165] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0166] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0167] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0168] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which, protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0169] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0170] X represents fluoride, chloride, bromide and iodide.
DETAILED DESCRIPTION OF THE INVENTION
[0171] The invention is directed to various steroid derivatives
having specific functionality as described in detail herein. The
compounds described herein demonstrate effectiveness as good
controllers of the asthmatic and allergic responses in that they
show efficacy against mast cell degranulation, inhibition of
allergen-induced bronchospasm (acute phase) and inhibition of
allergen-induced lung inflammation (late phase). This group of
compounds represents a new series of agents which have potential
therapeutic benefit in the treatment of asthma and allergies, with
high potency, a broad spectrum of activity and the reduced
probability of side effects.
[0172] For convenience in identifying the novel features of the
invented compounds, an unsubstituted steroid nucleus having each
ring carbon thereof identified with a unique number is shown below
as Structure 1. This numbering system will be used consistently
herein. ##STR17##
[0173] The compounds of the present invention contain at least two
asymmetric carbon atoms and thus exist as enantiomers and
diastereomers. Unless otherwise noted, the present invention
includes all enantiomeric and diastereomeric forms of the compounds
of the above formula. Pure stereoisomers, mixtures of enantiomers
and/or diastereomers, and mixtures of different compounds of the
above formulae are included within the present invention.
[0174] The synthesis procedures described herein, especially when
taken with the general knowledge in the art, provide sufficient
guidance to those of ordinary skill in the art to perform the
synthesis, isolation, and purification of the preferred compounds
described herein and other analogous compounds. Individual
enantiomers may be obtained, if desired, from mixtures of the
different forms by known methods of resolution, such as the
formation of diastereomers, followed by recrystallization.
[0175] The compounds of the above formula may be in the form of a
solvate or a pharmaceutically acceptable salt, e.g., an acid
addition salt. Such salts include hydrochloride, sulfate,
phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate,
maleate, lactate, mandelate, salicylate, succinate and other salts
known in the art.
[0176] A compound of the present invention may be prepared as a
composition by combining it with a pharmaceutically acceptable
carrier or diluent. Suitable carriers or diluents include
physiological saline. It will be evident to those of ordinary skill
in the art that a composition of the present invention may contain
more than one steroid compound or one or more steroid compounds in
combination with one or more non-steroid compounds.
[0177] A specific functionality present on many of the steroid
compounds of the invention is oxygen substitution at both of
positions 6 and 7. Thus, certain steroids of the invention have the
oxygen substitution pattern shown in Structure 2 below. Some of
these steroids are additionally characterized by having specific
stereochemistries. For example, steroids having 6.alpha. and
7.beta. oxygen substitution, as shown in Structure 3, and steroids
having an alpha hydrogen at the 5 position in addition to having
6.alpha. and 7.beta. oxygen substitution, as shown in Structure 4
below, fall within the scope of the invention. ##STR18##
[0178] In Structures 2, 3 and 4, each of the oxygen atoms that are
bonded to carbons 6 and 7 are simultaneously bonded to an "R.sup.1"
group. The R.sup.1 group is hydrogen or a protecting group for an
hydroxyl group. Suitable protecting groups are set forth in Greene,
"Protective Groups in Organic Chemistry", John Wiley & Sons,
New York N.Y. (1981). When a compound of Structures 2-4 contains
vicinal --OR.sup.1 groups (i.e., --OR.sup.1 groups on neighboring
carbon atoms), those vicinal --OR.sup.1 groups may together form a
cyclic structure which protects vicinal hydroxyl groups. A ketal is
an example of protected vicinal --OR.sup.1 group. Geminal
--OR.sup.1 groups (i.e., two --OR.sup.1 groups on the same carbon
atom) may together form a cyclic structure which protects a
carbonyl group. A ketal is an example of such a cyclic structure.
It should be understood that either or both of --OR.sup.1 at C6 and
C7 represents a carbonyl or protected carbonyl group, and thus at
C6 and C7, R.sup.1 may be a direct bond between the oxygen atom and
the carbon (C6 or C7) to which the oxygen atom is bonded.
[0179] Steroids of the invention may have substituents with either
the .alpha. or .beta. stereochemistry at the C8 and/or C9
positions. A hydrogen atom at C8 of the steroids of the invention
is typically in the .beta. configuration. In addition, preferred
steroids of the invention may have methyl substituents with .beta.
stereochemistry at the C10 and/or C13 positions. Compounds of the
invention preferably have a C14 hydrogen with the a stereochemistry
when C15 is not a ketone. In preferred steroids of the invention
that have a substituent at C17, the C17 substituent has .beta.
stereochemistry.
[0180] Steroids having 6,7-dioxygenation in the B-ring according to
Structure 2 can be synthesized from a number of commercially
available steroidal precursors having an .alpha.,.beta.-unsaturated
carbonyl group in the A-ring, including 4-androsten-3,17-dione
(compound 1 below) and dehydroisoandrosterone (compound 247 below).
These specific steroid precursors are available from Steraloids
Inc., Wilton, N.H. Other suitable steroid precursors having C3
oxygen functionalities and .DELTA..sup.5 carbon-carbon double bonds
may be obtained from, e.g., Aldrich Chemical Co., Milwaukee,
Wis.
[0181] An exemplary synthetic sequence to prepare a compound of
Structure 2 from 4-androsten-3,17-dione is summarized in Scheme 1
below. ##STR19## ##STR20##
[0182] Initially, the carbonyl functionalities of
4-androsten-3,17-dione are protected by carbonyl protecting groups.
As shown in Scheme 1, this may be accomplished by reacting compound
1 with a benzene solution of (CH.sub.2OH).sub.2 and p-TsOH, thereby
converting the carbonyl groups to ketal groups. Other suitable
carbonyl protecting groups are listed in Greene, "Protective Groups
in Organic Chemistry", John Wiley & Sons, New York, N.Y.
(1981). Under the acidic conditions which form the protected ketone
groups, there occurs concomitant migration of the C4-C5
carbon-carbon double bond to the C5-C6 position, to ultimately form
compound 2.
[0183] Allylic oxidation of the C5-C6 carbon-carbon double bond of
compound 2 introduces a carbonyl oxygen at C7, to thereby form
compound 3. A number of oxidizing agents and experimental
conditions can be used for this allylic oxidation, including
chromium trioxide/3,5-dimethylpyrazole complex, pyridinium
chlorochromate (PCC), pyridinium dichromate (PDC), or RuCl.sub.3
and t-butylhydroperoxide.
[0184] Reduction of the resultant C7 ketone with an appropriate
reducing agent gives the hydroxyl functionality at C7, as shown in
compound 4. Any of several metal hydride reducing agents can be
used for this task including sodium borohydride or lithium aluminum
hydride. Generally, reduction of the C7 ketone produces the
.beta.--OH configuration by hydride attack from the least hindered
face of the steroid. The C7 hydroxyl group is then preferably
protected with an hydroxylprotecting group, e.g.,
t-butyldimethylsilane (TBDMS), to provide a protected allylic
alcohol as in compound 5. Other suitable hydroxylprotecting groups
are listed in Greene, supra.
[0185] Introduction of the C6 oxygen can be achieved, before or
after protection of the C7 hydroxyl group, by methods such as
hydroboration/oxidation or epoxidation followed by ring opening.
For example, the .DELTA..sup.5 carbon-carbon double bond of
compound 5 can be epoxidized with any of a number of peracids
including m-chloroperbenzoic acid, trifluoroperacetic acid or
3,5-dinitroperoxybenzoic acid, to provide an epoxide such as in
compound 6. Generally, the epoxide introduced has the
.alpha.-configuration arising from attack on the least hindered
face of the steroid ring structure. Subsequent ring opening of the
epoxide can be accomplished under acidic conditions, such as 80%
aqueous acetic acid at 60.degree. C. The crude mixture contains
both compound 7 (having an allylic alcohol at the C6 position with
the o-configuration) and the C7 silyl derivative thereof. This
crude mixture can be treated with tetrabutylammonium fluoride
(TBAF) in tetrahydrofuran (THF) to give a single compound (7).
Alternatively, hydroboration of the .DELTA..sup.5 double bond with
an appropriate borane complex followed by oxidation using reagents
such as basic hydrogen peroxide will also introduce an hydroxyl
group in the .alpha.-configuration at C6.
[0186] Compound 7 is exemplary of compounds having the oxygenation
pattern of Structures 2 and 3. The methodology by which compound 1
may be converted to a compound of Structures 2 and/or 3 is
generally applicable to a wide variety of compounds having an
.alpha.,.beta.-unsaturated carbonyl group in the A-ring of a
steroid. Additional compounds of Structures 2 and/or 3 may be
prepared by modification of a dihydroxy compound such as compound
7. In such case, it may be necessary to protect each of the C6 and
C7 hydroxyl groups, and methodology to achieve such protection is
described later herein.
[0187] Compound 7 or an analog thereof may be converted to a
compound of Structure 4. Essentially, this may be accomplished by
protecting the C6 and C7 hydroxyl groups and the C17 carbonyl
group, and then reducing the .DELTA..sup.4 carbon-carbon double
bond. Lithium in ammonia/THF is an example of a suitable reducing
agent. Such a reduction provides an enolate, which may be trapped
with a suitable electrophile, e.g., trimethylsilyl chloride or
diethylchlorophosphate.
[0188] An example of such a conversion is shown in Scheme 2. Thus,
protection of the C6 and C7 hydroxyl groups of compound 7 may be
accomplished by treatment with 2,2-dimethoxypropane and a catalytic
amount of (1S)-(+)-10-camphorsulfonic acid (CSA) to produce
acetonide 8. The C17 carbonyl group of compound 8 may be protected
by converting it to an hydroxyl group, and then protecting the
hydroxyl group. Chemoselective reduction of the C17 carbonyl group
may be accomplished by use of NaBH.sub.4 in methanol to provide
compound 9, which in turn is reacted with a suitable
hydroxylprotecting group, e.g., t-butyldimethylsilyl chloride, to
provide silyl ether compound 10. Compound 10 may be reacted with
lithium in liquid ammonia/THF, followed by quenching with
diethylchlorophosphate, to provide compound 11. Compound 11 has a
5.alpha. hydrogen, as well as C6 and C7 dihydroxylation, and thus
is a representative compound of Structure 4. ##STR21##
[0189] In an aspect of the present invention, olefinic steroids
having an exocyclic olefin at C17 and oxygen atoms at both C6 and
C7 are provided. In one embodiment, the olefinic steroid has the
Structure 5, including individual enantiomeric or geometric isomers
thereof, and further including a solvate or pharmaceutically
acceptable salt thereof. Structure 5 is defined as follows:
[0190] A compound of the formula ##STR22## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0191] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0192] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0193] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0194] each of C5, C8, C9, C10 and C13 is independently substituted
with one of --X, --R.sup.4 or --OR.sup.1;
[0195] C14 is substituted with --X, --OR.sup.1, or --R.sup.4
excluding methyl;
[0196] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0197] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0198] R.sup.2, R.sup.3 and R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur, where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and
[0199] X represents fluoride, chloride, bromide and iodide.
[0200] Providing an exocyclic double bond at C17 is readily
accomplished by the Wittig reaction, starting with a C17 carbonyl
compound. Steroids of the invention having C17 carbonyl
functionality are readily available, e.g., in compound 7 as
prepared according to Scheme 1, or by the synthetic sequence
summarized in Scheme 3 below, which starts from compound 10 (as
prepared in Scheme 2). ##STR23## ##STR24##
[0201] Thus, the A-ring of compound 10 can be reduced to afford a
C3 carbonyl group as the only functionality in the A-ring. Scheme 3
illustrates a two-step sequence to achieve this reduction, wherein
compound 10 is reduced with lithium in liquid ammonia and an ether
solvent, e.g., diethyl ether or THF, to provide a mixture of
compounds 12 and 13. This mixture may then be oxidized with a
suitable oxidizing agent, for example PDC, to give exclusively
compound 13. Compound 13 may then be reduced with
LS-Selectride.RTM. (Aldrich Chemical Co., Milwaukee, Wis.) or other
selective reducing agent, to provide compound 14 having the
indicated stereochemistry.
[0202] The 3.alpha.-hydroxyl group of compound 14 may then be
protected as the acetate using acetic anhydride and pyridine to
give compound 15. Other suitable hydroxyl protecting groups could
be used instead of the acetate group. Removal of the silyl
protecting group at C17 can be achieved under standard conditions
known in the art for removing this silyl protecting group, e.g.,
using tetrabutylammonium fluoride (TBAF), to afford a C17 hydroxyl
compound such as compound 16. The C17 hydroxyl group can be
oxidized to a carbonyl group under typical oxidation conditions,
e.g., using oxalyl chloride in DMSO and Et.sub.3N, to provide
ketone compound 17.
[0203] Compound 17 can be used in a multitude of olefination
reactions, including Wittig-type reactions, to provide compounds of
Structure 5 having an olefin at C17. For example, compound 17 may
be reacted with ethyltriphenylphosphonium bromide to provide the
ethylidene compound 18. Other starting ketones may be used to
provide other steroids having an exocyclic double bond at C17.
[0204] As described previously, compounds containing a carbonyl at
C17 (or those that contain functionality that is readily converted
to a carbonyl group) can be transformed into compounds containing a
carbon-carbon double bond at C17 using Wittig chemistry. For
example, as outlined in Scheme 4 below, compound 19 may be
transformed into the corresponding C17 ethylidene compound 23 in a
five step process. Thus, the 2.alpha.,3.beta.-dihydroxy
functionality of compound 19 may be protected with
hydroxylprotecting groups, (e.g., using 2,2-dimethoxy propane and
camphor sulfonic acid (CSA) in N,N-dimethylformamide (DMF) to give
a compound such as compound 20. Deprotection of the C17 hydroxyl
may be achieved using reaction conditions suitable for the
particular hydroxyl protecting group (in this instance, TBAF in THF
may be used) followed by oxidation of the resulting hydroxyl group
(e.g., using PDC in CH.sub.2Cl.sub.2) yields the compound
containing the C17 ketone (21). Reaction of compound 21 with a
Wittig reagent, e.g., ethyl triphenyl phosphonium bromide and
potassium t-butoxide in toluene, gives compound 22. Deprotection of
the hydroxyl groups in olefin 22 affords the tetrahydroxy compound
23. ##STR25##
[0205] Protection steps may be required prior to derivatization at
C17 in some cases. For example, in compound 24 (prepared according
to Scheme 14) the C3 ketone should first be protected before
proceeding with the transformations at C17 (see Scheme below).
Thus, compound 24 may first be reduced (e.g., by reaction with
NaBH.sub.4 in ethanol) then acylated (e.g., using acetic anhydride
in pyridine) to yield the C3,C5-acetoxy derivative 25.
Deprotection, oxidation and Wittig chemistry at C17, analogous to
that described in Scheme 4 may be used to provide compound 27.
Subsequent deprotection of the C6 and C7 hydroxyl groups (80%
acetic acid is conveniently used to remove the ketal group of
compound 27) gives compound 28 which contains the exocyclic
.DELTA..sup.17 olefin. ##STR26##
[0206] In an aspect of the present invention, steroids having C17
oxygenation as well as oxygenation at C6 and C7 are provided. In
one embodiment, the steroid has the Structure 6, including
individual enantiomeric or geometric isomers thereof, and further
including a solvate or pharmaceutically acceptable salt thereof.
Structure 6 is defined as follows:
[0207] A compound of the formula ##STR27## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0208] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0209] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0210] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0211] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0212] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0213] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0214] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0215] R.sup.5 and R.sup.6 may together form a direct bond so C17
is a carbonyl group, or may together with C17 form a cyclic 3-6
membered ether or 4-6 membered lactone; otherwise R.sup.5 is
R.sup.4 or --OR.sup.6 and R.sup.6 is R.sup.1 or R.sup.4; and
[0216] X represents fluoride, chloride, bromide and iodide.
[0217] Preferably, neither the A nor B ring in compounds of
Structure 6 is aromatic. In another preferred embodiment, when C10
is substituted with methyl, then C5 is not directly bonded to an
oxygen atom.
[0218] Many examples of compounds of Structure 6 and their
synthesis have already been provided above. For instance, compounds
7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 19, 20, 21, 24, 25 and 26 are
representative compounds of Structure 6. Many additional compounds
of Structure 6, including the synthesis thereof, are provided
herein in connection with other compounds of the invention.
Therefore, one of ordinary skill in the art is able to prepare many
compounds of Structure 6 in view of the disclosure herein.
[0219] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C1. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C1 for a compound of
Structure 6 is provided below and outlined in Schemes 6, 7, and 8.
It should be recognized that the same or analogous synthetic
methodology can be applied to provide oxygen and/or hydrocarbon
substitution at C1 for any compound of Structures 5-12 where C1
oxygen and/or hydrocarbon substitution is desired.
[0220] Introduction of an oxygen functionality at C1 of the steroid
carbon skeleton can be accomplished by first generating the
1-ene-3-one functionalization pattern in the A-ring of a steroid,
followed by Michael addition chemistry using any of a number of
alkoxide anions, as outlined in Scheme 6. For example, the enone 29
may be produced from compound 13 using standard methodology. The
benzyloxy compound 30 may then be produced by reacting the enone
(29) with benzyl alcohol and KOH. Reduction of the C3 ketone of
compound 30, and protection of the resultant secondary alcohol as
the silyloxy derivative (to provide compound 31) may be followed by
catalytic hydrogenation to yield the C1 hydroxyl functionality in
compound 32. Oxidation of this secondary alcohol using, e.g., PDC
in CH.sub.2Cl.sub.2 may produce compound 33 having a C1 ketone.
##STR28##
[0221] Compounds containing both an alkyl group and an hydroxyl
group at C1 may be produced by reaction of compound 33 with an
alkyl lithium reagent. For example, reaction of compound 33 with
CH.sub.3Li in ether will provide the tertiary alcohol in compound
34 (Scheme 7). ##STR29##
[0222] Michael addition chemistry similar to that described in
Scheme 6 can be used to add an alkyl group to the C1 position. This
can be accomplished using a number of reagents including
R.sub.2CuLi where R may be alkyl, vinyl or aryl. For example,
compound 29 may be reacted with Me.sub.2CuLi in ether to yield the
C1 methyl substituted derivative 35 (Scheme 8). ##STR30##
[0223] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C2. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C2 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C2 for any compound of
Structures 5-12 where C2 oxygen and/or hydrocarbon substitution is
desired.
[0224] Compounds containing oxygen at C2 may be prepared in a
number of ways including hydroboration of a silyl enol ether as
shown in Scheme 9. The silyl enol ether may be prepared from the
enone 29 via Li/NH.sub.3 reduction followed by trapping of the
resultant enolate using TMSCI to yield compound 36 (or other
R.sub.3SiCl reagents to produce an analogous silyl enol ether).
Hydroboration of the carbon-carbon double bond in 36 may give the
2.alpha.,3.beta.-dihydroxy functionalization pattern (compound 19).
Oxidation of this dihydroxy compound using PDC in CH.sub.2Cl.sub.2
may provide the diketone 38. ##STR31##
[0225] Preparation of C2 hydrocarbon substituted compounds can be
produced, e.g., by .alpha.-alkylation of a compound containing a C3
ketone functionality. For example, Li/NH.sub.3 reduction of the
enone 29 followed by trapping the resultant anion with an
alkylating agent provides C2 alkylation. Treatment of the resultant
enol ate with methyl iodide may yield the C2 methylated compound 39
(Scheme 10 below). This methodology can be applied to a variety of
different compounds using a number of different alkyl halides.
##STR32##
[0226] Compounds of Structure 6 may have hydrocarbon substitution
at C3. Exemplary synthetic methodology to provide hydrocarbon
substitution at C3 for a compound of Structure 6 is provided below.
It should be recognized that the same or analogous synthetic
methodology can be applied to provide hydrocarbon substitution at
C3 for any compound of Structures 5-12 where C3 hydrocarbon
substitution is desired.
[0227] Wittig chemistry on compound 13 followed by reduction of the
double bond or alternative modifications will provide the alkyl or
dialkyl derivative at C3. For example, reaction of compound 13 with
methyl triphenylphosphonium bromide and tBuOK in toluene may be
used to give compound 40 (Scheme 11). A Simmons-Smith reaction on
compound 40 with CH.sub.2I.sub.2 and Zn--Cu followed by catalytic
hydrogenolysis of the cyclopropane derivative 41 using H.sub.2,
Pd/C in ethanol can be used to give the dialkyl derivative 42
(Scheme 11). ##STR33##
[0228] Compounds of Structure 6 may have hydrocarbon substitution
at C4. Exemplary synthetic methodology to provide hydrocarbon
substitution at C4 for a compound of Structure 6 is provided below.
It should be recognized that the same or analogous synthetic
methodology can be applied to provide hydrocarbon substitution at
C4 for any compound of Structures 5-12 where C4 hydrocarbon
substitution is desired.
[0229] Alkylation at C4 may be achieved by first producing the
enolate anion from the enone in compound 10 (using, for example,
reduction with lithium in liquid ammonia) followed by treatment
with an appropriate alkyl halide as shown in Scheme 12.
##STR34##
[0230] Alternatively, Compounds of Structure 6 may have carbonyl
functionality at C4. Exemplary synthetic methodology to provide
carbonyl functionality at C4 for a compound of Structure 6 is
provided below. It should be recognized that the same or analogous
synthetic methodology can be applied to provide carbonyl
functionality at C4 for any compound of Structures 5-12 where a C4
carbonyl group is desired. As described below, the carbonyl
functionality at C4 provides a convenient entry into compounds
having a tertiary alcohol and a hydrocarbyl group at C4.
[0231] Compounds with a ketone (carbonyl) functionality at C4 may
be prepared from compound 44 (which in turn is prepared from a
deacetylation of acetate 147 from Scheme 44) by selectively
tosylating, epoxidation and then epoxide ring opening followed by
oxidation of the resultant 4.beta.-hydroxyl functionality. For
example, as illustrated in Scheme 13, treatment of the diol 44 with
p-toluenesulfonyl chloride in pyridine and DMF followed by reaction
of the resultant tosylate 45 with tBuOK can introduce the
3.beta.,4.beta.-epoxide (compound 46). Treatment of the epoxide
with Me.sub.2CuLi gives the 3.alpha.-methyl derivative 47 and
subsequent oxidation using, for example, PDC in CH.sub.2Cl.sub.2
gives the desired ketone (carbonyl) at C4 (compound 48).
Epimerization to the 3.beta.-methyl derivative can be achieved
using tBuOK in tBuOH and subsequent treatment of the ketone with a
methyl lithium in THF can provide the tertiary alcohol at C4
(compound 49). ##STR35##
[0232] Alternatively, compounds of Structure 6 may have oxygen or
hydrocarbon substitution at C5. Exemplary synthetic methodology to
provide oxygen or hydrocarbon substitution at C5 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen or hydrocarbon substitution at C5 for any compound of
Structures 5-12 where C5 oxygen or hydrocarbon substitution is
desired.
[0233] Epoxidation of compound 10 followed by ring opening can be
used to generate a hydroxy and subsequently an alkoxy substitution
at C5 of the carbon skeleton. For example, epoxidation of the
double bond in compound 10 can yield the corresponding epoxide
derivative 50 which may be readily converted to the tertiary
hydroxyl compound 24 (Scheme 14 below). Subsequent reduction of
compound 24 using NaBH.sub.4 in THF and methylation using MeI in
the presence of tBuOK in THF can give the diacetoxy compound 51
(Scheme 15 below). Alkyl substitution at C5 may be achieved using
an appropriate alkyl copper lithium reagent. For example, treatment
of compound 10 with (CH.sub.3).sub.2CuLi in ether may produce the
C5 methyl derivative 52 (Scheme 16). ##STR36## ##STR37##
##STR38##
[0234] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C9. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C9 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C9 for any compound of
Structures 5-12 where C9 oxygen and/or hydrocarbon substitution is
desired.
[0235] Hydroxylation at the C9 position may be achieved by reaction
of a .DELTA..sup.9,11 olefinic compound with m-chloroperbenzoic
acid followed by reduction with LiAlH.sub.4, as outlined in Scheme
17. For example, using this procedure, compound 53 (prepared from
the dehydration of compound 60; e.g., NaH, CS.sub.2, MeI, heat) may
be used as the starting material to produce compound 54 which, upon
reduction of the epoxide can produce the C9 hydroxyl-containing
derivative 55. Subsequent reaction of the tertiary alcohol in
compound 55 with dimethyl sulfate in aqueous sodium hydroxide may
be used to give the corresponding alkoxy derivative, compound 56.
##STR39##
[0236] Alternatively, compounds of Structure 6 may have hydrocarbon
substitution at C9. Exemplary synthetic methodology to provide
hydrocarbon substitution at C9 for a compound of Structure 6 is
provided below. It should be recognized that the same or analogous
synthetic methodology can be applied to provide hydrocarbon
substitution at C9 for any compound of Structures 5-12 where C9
hydrocarbon substitution is desired.
[0237] Cyclopropanation of compound 53 using CH.sub.2I.sub.2 and
Zn--Cu followed by catalytic hydrogenation may provide the
corresponding C9-alkyl substituted compound 57 (Scheme 18).
##STR40##
[0238] Alternatively, compounds of Structure 6 may have halide
substitution at C9. Exemplary synthetic methodology to provide
halide substitution at C9 for a compound of Structure 6 is provided
below. It should be recognized that the same or analogous synthetic
methodology can be applied to provide halide substitution at C9 for
any compound of Structures 5-12 where C9 halide substitution is
desired.
[0239] Introduction of a halogen atom at C9 can be achieved in a
number of ways including reaction of a C9 tertiary alcohol (see,
e.g., compound 55 in Scheme 17) with thionyl chloride. Thus,
reaction of compound 55 with SOCl.sub.2 in CH.sub.2Cl.sub.2 may be
used to provide the chloro derivative 59 as shown in Scheme 19.
##STR41##
[0240] Compounds of Structure 6 preferably have a methyl
substituent at C10. However, the C10 position may be derivatized so
as to have many functional groups other than methyl. Exemplary
synthetic methodology to provide oxygen and/or hydrocarbon
substitution at C10 for a compound of Structure 6 is provided
below. It should be recognized that the same or analogous synthetic
methodology can be applied to provide oxygen and/or hydrocarbon
substitution at C10 for any compound of Structures 5-12 where C10
oxygen and/or hydrocarbon substitution is desired.
[0241] The derivatization of the C10 position may be achieved via
the route shown in Scheme 20. A 10.beta.-hydroxy steroid 60
(prepared, for example, as outlined in Scheme 22 below) may be
derivatized using nitrosyl chloride (NOCl) in pyridine to yield a
nitrite derivative such as 61. Irradiation of the nitrite 61 then
can lead to a mixture of the oximes 62 and 63. Compound 63 is
reduced to the corresponding imine 64 by treatment with aqueous
TiCl.sub.3 in dioxane and acetic acid. The hemiacetal acetate 65
may be produced upon treatment of 64 with NaNO.sub.2 in aqueous
acetic acid. This can also lead to deprotection of the 6,7-hydroxyl
groups. The acetonide can be reintroduced by reaction of the crude
product with 2,2-dimethoxypropane and camphor sulfonic acid.
Alkaline hydrolysis (NaOH, MeOH) to give the hydroxy aldehyde 66 is
followed by protection of the secondary alcohol at C11 as the
benzyl ether using BnBr, NaH in DMF to afford compound 67.
##STR42##
[0242] A Grignard reaction of compound 67 with CH.sub.3MgBr
followed by PDC oxidation in CH.sub.2Cl.sub.2 then by a
Bayer-Williger oxidation with m-chloroperbenzoic acid in methylene
chloride can give the C10 acetoxy derivative 68. Removal of the
acetate group may be accomplished with base, for example, sodium
methoxide in methanol, to give the C10-.beta. alcohol 69. This C10
hydroxyl group may then be further derivatized to the alkoxide
analogue 70, using, for example, sodium hydride in THF followed by
treatment with an alkylating agent such as methyl iodide.
Alternatively, conversion of the C10 hydroxyl group in compound 69
to the corresponding chloride derivative 71 is achieved using a
chlorinating agent, e.g., thionyl chloride, as shown in Scheme 21.
##STR43##
[0243] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C11. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C11 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C11 for any compound of
Structures 5-12 where C11 oxygen and/or hydrocarbon substitution is
desired.
[0244] The preparation of compounds of Structure 6 containing an
oxygen function at the C11 position may be achieved according to
the pathway shown in Scheme 22 from the commercially available
starting material 72 and related compounds. ##STR44##
[0245] If starting with a steroid having a hydroxyl group in the
A-ring, as in compound 75 (prepared from the commercially available
compound 72 (Scheme 22)), removal of the C3 hydroxyl may be
achieved using a two step procedure involving preparation of the
methyl xanthate using NaH, CS.sub.2 and CH.sub.3I in THF followed
by nBu.sub.3SnH reduction and deprotection (80% AcOH) to yield
compound 77. After reduction and protection of the C17 ketone using
NaBH.sub.4 in methanol followed by TBDMSCI and imidazole in DMF,
oxidation of the C7 position can be achieved using a number of
oxidizing conditions such as CrO.sub.3 and 3,5-dimethylpyrazole in
CH.sub.2Cl.sub.2 or RuCl.sub.3 and tBuOOH in H.sub.2O and
cyclohexane. Subsequent reduction (NaBH.sub.4, CeCl.sub.3,
THF-MeOH) of the C7 ketone and acetylation can provide the C7
acetoxy derivative 80. Hydroboration of compound 80 provides a
product with the 6.alpha.,7.beta.,11.beta.-hydroxylation pattern as
in triol 81. Protection of the 6.alpha.,7.beta. hydroxyls in
compound 81 using 2,2-dimethoxypropane in the presence of camphor
sulfonic acid (CSA) followed by oxidation using PDC in
CH.sub.2Cl.sub.2 gives compound 82 which contains the C11
ketone.
[0246] Compounds of Structure 6 may alternatively or additionally
have hydrocarbon substitution at C11. Exemplary synthetic
methodology to provide hydrocarbon substitution at C11 for a
compound of Structure 6 is provided below. It should be recognized
that the same or analogous synthetic methodology can be applied to
provide hydrocarbon substitution at C11 for any compound of
Structures 5-12 where C11 hydrocarbon substitution is desired.
[0247] Conversion of the compound 82 C11-ketosteroid to a
quaternary alkyl center may be accomplished as shown in Scheme 23
below. ##STR45##
[0248] Thus, the C11-ketosteroid 82 in toluene may be added to a
solution of methyl triphenylphosphonium bromide and tBuOK to afford
compounds with a .DELTA..sup.11 carbon-carbon double bond such as
83. Subsequent treatment of the compound 83 with CH.sub.2I.sub.2,
Zn--Cu may give the cyclopropyl derivative 84. Hydrogenation of the
cyclopropane ring (H.sub.2, Pd/C in ethanol) may give the dialkyl
derivative 85. Other Wittig reagents may be employed to make
analogous alkyl-substituted steroids.
[0249] Monoalkylation of the C11 position may be achieved by
application of Wittig chemistry on compounds with a C11 ketone, as
described above, followed directly by catalytic hydrogenation (as
illustrated in Scheme 24). For example, catalytic hydrogenation
(H.sub.2, Pd/C in ethanol) on compound 83 affords the C11
methylated steroid 86. ##STR46##
[0250] Compounds of Structure 6 may have halide substitution at
C11. Exemplary synthetic methodology to provide halide substitution
at C11 for a compound of Structure 6 is provided below. It should
be recognized that the same or analogous synthetic methodology can
be applied to provide halide substitution at C11 for any compound
of Structures 5-12 where C11 halide substitution is desired.
[0251] Thus, halogenation of the C11 position may be achieved
according to the route shown in Scheme 25. For example, treatment
of compound 60 with a halogenating agent, e.g., thionyl chloride in
CH.sub.2Cl.sub.2, gives the corresponding 11.beta.-chloro
derivative 87. In general, hydroxyl functionality may serve as a
precursor to halide functionality. ##STR47##
[0252] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C12. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C12 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C12 for any compound of
Structures 5-12 where C12 oxygen and/or hydrocarbon substitution is
desired.
[0253] Placement of an oxygen function at the C12 position may be
achieved as illustrated in Scheme 26. ##STR48##
[0254] Thus, a C11 ketosteroid, such as compound 82, may be reacted
with LDA in THF followed by trapping of the enolate anion with
(Me.sub.2N).sub.2P(O)Cl followed by reduction of the enolphosphate
using Li and EtNH.sub.2 to provide a compound, such as 88, with a
.DELTA..sup.11,12 carbon-carbon double bond. Epoxidation may be
achieved using an epoxidizing agent, e.g., mCPBA in
CH.sub.2Cl.sub.2, to give the corresponding
11.alpha.,12.alpha.-epoxide derivative 89. Subsequent LiAlH.sub.4
reduction of the epoxide can form the 12.alpha.-hydroxy derivative
(90) which can be oxidized using the appropriate oxidizing agent,
for example, pyridinium dichromate (PDC) in methylene chloride, to
give the desired C12 ketosteroid 91.
[0255] Compounds of Structure 6 may have hydrocarbon substitution
at C12. Exemplary synthetic methodology to provide hydrocarbon
substitution at C12 for a compound of Structure 6 is provided
below. It should be recognized that the same or analogous synthetic
methodology can be applied to provide hydrocarbon substitution at
C12 for any compound of Structures 5-12 where C12 hydrocarbon
substitution is desired.
[0256] Alkyl groups, such as methyl, may be introduced into the C12
position as shown in Scheme 27 below. The C11 ketosteroid 82,
(prepared, for example, according to Scheme 22), and a strong base,
e.g., lithium diisopropylamide in THF, are combined and treated
with an alkylating agent, e.g., methyl iodide, to afford the C12
methylated product 92. At this stage, the C11 ketone can be removed
using a number of methods including those described in connection
with Scheme 26 to give the monomethylated product 93. Further
treatment with strong base and an alkylating agent, e.g., lithium
diisopropylamide and methyl iodide, gives the C12 dimethylated
product 94. Again, this compound may be subjected to reducing
conditions to remove the C11 ketone group thus giving the C12
dimethyl derivative 95. ##STR49##
[0257] Compounds of Structure 6 may have oxygen and hydrocarbon
substitution at C12. Exemplary synthetic methodology to provide
oxygen and hydrocarbon substitution at C12 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and hydrocarbon substitution at C12 for any compound of
Structures 5-12 where C12 oxygen plus hydrocarbon substitution is
desired.
[0258] Scheme 28 shows the preparation of a tertiary alcohol at the
C12 position from the corresponding C12 ketone. In Scheme 28, the
C12 ketone 91 is treated with a alkyl lithium reagent, e.g., methyl
lithium in diethyl ether, to give the desired tertiary alcohol 96.
##STR50##
[0259] Compounds of Structure 6 may have carbon, oxygen or halogen,
to name a few atoms, bonded to C13. Exemplary synthetic methodology
to provide such substitution at C13 for a compound of Structure 6
is provided below. It should be recognized that the same or
analogous synthetic methodology can be applied to provide the same
or analogous substitution at C13 for any compound of Structures
5-12 where such C13 substitution is desired.
[0260] Substituents at the C13 position may be introduced according
to the pathway shown in Scheme 29 below. In a fashion similar to
that previously described in Scheme 20, the C13 position can be
substituted with a hydrocarbyloxy moiety, e.g., a methoxy moiety.
Thus, the oxime derivative 62, (prepared, for example, as described
in Scheme 20), is reduced to the corresponding imine 97 by
treatment with aqueous TiCl.sub.3 in dioxane and acetic acid. The
hemiacetal acetate 98 may be produced upon treatment of compound 97
with NaNO.sub.2 in aqueous acetic acid. Alkaline hydrolysis (NaOH,
MeOH) to give the hydroxy aldehyde 99 is followed by protection of
the secondary alcohol at C11 as the benzyl ether using BnBr, NaH in
DMF to afford compound 100. ##STR51##
[0261] A Grignard reaction on compound 100 may be used to introduce
additional functionality at C13. For example, treatment of compound
100 with methyl magnesium bromide, followed by oxidation of the
resulting C13 secondary alcohol gives the methyl ketone substituent
at C13. This may be oxidized, e.g., using Bayer-Williger oxidation
with m-chloroperbenzoic acid in methylene chloride, to give the C13
acetoxy derivative 101. This ester may be hydrolyzed by treatment
with sodium methoxide in methanol to produce the tertiary alcohol
102. Subsequent reaction of the alcohol with sodium hydride in THF
followed by its quenching with methyl iodide may be used to produce
the C13 methoxysteroid 103. Other alkylating agents could be used
to prepare other hydrocarbyloxy derivatives. The C13 hydroxyl
moiety may then be converted to a halide, for example a chloride,
by the reaction of alcohol 102 with thionyl chloride, thus
affording the C13 chlorosteroid 104, as shown in Scheme 30 below.
##STR52##
[0262] Compounds of Structure 6 may have hydrocarbon substitution
at C14. Exemplary synthetic methodology to provide hydrocarbon
substitution at C14 for a compound of Structure 6 is provided
below. It should be recognized that the same or analogous synthetic
methodology can be applied to provide hydrocarbon substitution at
C14 for any compound of Structures 5-12 where C14 hydrocarbon
substitution is desired.
[0263] For example, introduction of an alkyl group at C14 of the
steroid carbon skeleton could be accomplished by alkylation at the
C14 position. One approach to achieve such an alkylation is shown
in Scheme 31 below. Initially, preparation of the enone 107 can be
accomplished by deprotection (TBAF, THF) of compound 105 followed
by oxidation of the secondary alcohol using PDC in CH.sub.2Cl.sub.2
to give the C17 ketone derivative 106. Conversion of the ketone 106
to the enone 107 may be achieved using isopropenyl acetate and
pTsOH to produce the intermediate enol acetate followed by
production of the enone using reagents set forth in Scheme 31. This
is followed by conversion of the enone 107 to the silyl enol ether
108 by reacting enone 107 with lithium diethylamide in THF followed
by reaction of the resultant anion with triisopropylsilyl triflate
(TIPSOTf). The cyclopropane derivative 109 is then prepared from
silyl ether 108 using CH.sub.2I.sub.2 and Zn--Cu. Deprotection of
the silyl enol ether and cleavage of the cyclopropane ring is
achieved using TBAF in THF followed by tBuOK in DMSO and aqueous
work-up procedures. ##STR53##
[0264] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C15. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C15 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C15 for any compound of
Structures 5-12 where C15 oxygen and/or hydrocarbon substitution is
desired.
[0265] For example, introduction of an oxygen functionality at C15
of the steroid carbon skeleton could be accomplished by Michael
addition type chemistry using any of a number of alkoxide anions.
As outlined in Scheme 32 below, the 4-methoxybenzyloxy compound 111
(a representative C15-hydrocarbyloxy steroid derivative of the
invention, where the 4-methoxybenzyloxy group (MPMO) serves as an
hydroxylprotecting group) could be produced by reacting the enone
107 with 4-methoxybenzyl alcohol and base (e.g., powdered KOH). The
4-methoxybenzyl protecting group may be removed under oxidizing
conditions, e.g., by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) oxidation, to yield the C15 hydroxyl group (compound 112).
When this is followed by oxidation of the secondary alcohol (using,
for example, PDC in CH.sub.2Cl.sub.2), the corresponding C15 ketone
(compound 113) may be produced. ##STR54##
[0266] Compounds containing an alkyl group at C15 may also be
produced by a Michael type conjugate addition. For example,
reaction of compound 107 with an organolithium cuprate (e.g.,
Me.sub.2CuLi) in Et.sub.2O may be used to produce the methyl
derivative 114 as shown in Scheme 33. ##STR55##
[0267] Compounds containing both a hydrocarbyl (e.g., an alkyl)
group and a hydrocarbyloxy (e.g., an alkoxy) group at C15 can be
produced by using Grignard chemistry on compound 117, as outlined
in Scheme 34 below. Compound 117 may be prepared in a three step
process involving the reduction (e.g., nBu.sub.3SnH reduction of a
methyl xanthate prepared from the C.sub.1-7 hydroxy analog of
compound 111) to afford steroid 115, followed by oxidative removal
(e.g., using DDQ) of the MPM protecting group yielding the
secondary alcohol derivative 116. Subsequent oxidation of compound
116 to the corresponding ketone yields compound 117. A Grignard
reaction on compound 117 using an alkylmagnesium bromide reagent
(e.g., CH.sub.3MgBr) in ether produces the tertiary alcohol 118.
Methylation of the tertiary alcohol in 118 with an alkylating agent
(e.g., CH.sub.3I (note that an acylating agent can be used in place
of an alkylating agent, in Scheme 34 and in every Scheme herein
having an alkylating agent)) in the presence of base (e.g.,
K.sub.2CO.sub.3) yields the tertiary methoxy compound 119.
##STR56##
[0268] Compounds of Structure 6 may have oxygen and/or hydrocarbon
substitution at C16. Exemplary synthetic methodology to provide
oxygen and/or hydrocarbon substitution at C16 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
oxygen and/or hydrocarbon substitution at C16 for any compound of
Structures 5-12 where C16 oxygen and/or hydrocarbon substitution is
desired.
[0269] Introduction of a tertiary hydroxyl group at C16 of the
steroid carbon skeleton may be accomplished using Grignard
chemistry on compound 121 as shown in Scheme 35 below. The ketone
121 may be produced via hydroboration of the olefin (using, for
example, Sia.sub.2BH in THF then aqueous NaOH, H.sub.2O.sub.2) of
compound 308 (prepared from compound 106 as outlined in Scheme 35)
to afford alcohol 120. The desired C16 ketone functionality may
then generated by oxidizing the secondary alcohol at C16, using,
for example, PDC in CH.sub.2Cl.sub.2, to yield compound 121.
Reaction of the ketone 121 with a Grignard reagent, e.g.,
CH.sub.3MgBr in ether, may be used to produce the corresponding
tertiary alcohol derivative, in this example, compound 122. The
corresponding alkoxy derivative 123 could then be produced directly
from compound 122 using the appropriate base and alkyl halide.
##STR57##
[0270] Alkoxy groups at C16 may be produced directly from the
corresponding C16 hydroxyl compound. For example, compound 124 may
be produced by reacting compound 120 with an reagent, e.g.,
CH.sub.3I, and a base, e.g., K.sub.2CO.sub.3 (Scheme 36).
##STR58##
[0271] C16 alkyl groups may be introduced by the direct alkylation
of compounds that contain a C17 carbonyl. For example, reaction of
compound 106 with CH.sub.3I and LDA (other strong bases and
alkylating agents could be used) in THF yields the C16 methyl
compound 125 (Scheme 37). ##STR59##
[0272] Compounds of Structure 6 have oxygen and/or hydrocarbon
substitution at C17, including tertiary alcohol and hydroxyl
functionality. Exemplary synthetic methodology to provide tertiary
alcohol and hydroxyl substitution at C17 for a compound of
Structure 6 is provided below. It should be recognized that the
same or analogous synthetic methodology can be applied to provide
tertiary alcohol and hydroxyl substitution at C17 for any compound
of Structures 5-12 where C17 tertiary alcohol or hydroxyl
substitution is desired.
[0273] Thus, Grignard chemistry similar to that described in Scheme
34 may be used to add a tertiary alcohol functionality to the C17
position. For example, as outlined in Scheme 38 below, compound 106
may be reacted with CH.sub.3MgBr in ether to yield the tertiary
alcohol derivative 126. Methylation of the resultant tertiary
alcohol gives the corresponding C17 methoxy compound 127. Of
course, other alkylating agents could be used to provide a wide
range of hydrocarbyloxy compounds. ##STR60##
[0274] In an aspect of the present invention, C5 stereodefined
steroids having hydroxylation at C6 and C7, a 5.alpha. hydrogen and
no oxygen atom bonded to C3 is provided. In one embodiment, the
stereodefined steroid has the Structure 7, including individual
enantiomeric or geometric isomers thereof, and further including a
solvate or pharmaceutically acceptable salt thereof. Structure 7 is
defined as follows:
[0275] A compound of the formula ##STR61## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0276] each of C1, C2, C4, C11, C12, C15, C16 and C17 is
independently substituted with
[0277] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0278] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0279] each of C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0280] C3 is substituted with one of .dbd.C(R.sup.4)(R.sup.4) and
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- wherein n ranges
from 1 to about 6, or two of --X, and --R.sup.4;
[0281] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0282] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represent a carbonyl
or protected carbonyl group;
[0283] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0284] X represents fluoride, chloride, bromide and iodide.
[0285] In a preferred embodiment of compounds of Structure 7, C3 is
not bonded to an oxygen atom. In another preferred embodiment, when
C3 is substituted with two hydrogen atoms then C17 is not
substituted with either
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2 or
--CH(CH.sub.3)(CH.sub.2)2C(.dbd.O)OCH.sub.3.
[0286] Compounds of Structure 7 have hydroxylation at C6 and C7 and
a 5.alpha. hydrogen. A synthetic sequence for the preparation of
compounds having these structural features has been set forth
above, in Scheme 2, which shows the preparation of compound 11.
While compound 11 has an oxygen atom bonded to C3, and is thus not
a representative compound of Structure 7, compound 11 can be
converted to a compound of Structure 7. Thus, as shown in Scheme 39
below, compound 11 may be reduced to compound 128, where lithium in
liquid ammonia/t-butanol may be used to afford the desired
reduction. Hydrogenation of compound 128 can provide compound 105,
having a --CH.sub.2-- group at C3, as also shown in Scheme 39.
##STR62##
[0287] As illustrated in Scheme 40, compound 128 may alternatively
be converted to additional compounds of Structure 7. Thus, the C17
protected hydroxyl group of compound 128 may be deprotected to
yield compound 129, and then the C17 hydroxyl group of compound 129
may be oxidized to a C17 carbonyl group as in compound 106.
##STR63##
[0288] Compounds of Structure 7 containing a methylene at C3 may be
obtained from compounds with an hydroxyl, protected hydroxyl or
ketone functionality at C3. The same or analogous synthetic
methodology may be used to prepare compounds of any of Structures
5-12 wherein a methylene group at C3 is desired.
[0289] For example, Scheme 39 above describes the conversion of
compound 11 to compound 105 using chemistry described earlier.
Thus, the chemistry described in connection with the Schemes herein
can be extended to include compounds containing a methylene rather
than an hydroxyl or carbonyl group at C3. In some cases, however, a
series of protection and/or deprotection steps is first
required.
[0290] Scheme 41 shows an example where the C3 silyloxy
functionality must first be deprotected prior to the deoxygenation
reaction. The TBDMS group in compound 31 may be removed using TBAF
in THF. Preparation of the methyl xanthate derivative of compound
130 using KH, CS.sub.2 and MeI is followed by nBu.sub.3SnH
reduction gives the compound (131) containing a methylene group at
C3 and a protected hydroxyl group at C1. Oxidation to the C1 ketone
is then achieved by removal of the C1 protecting group followed by
using a suitable oxidizing agent, e.g., PDC in CH.sub.2Cl.sub.2, to
give compound 132. ##STR64##
[0291] Compounds of Structures 5-12, including Structure 7, having
C3 alkyl functionality may be obtained by Wittig chemistry
(prepared from the C3 ketone as described in connection with Scheme
11). A number of Wittig reagents can be used for this purpose
giving rise to substituents with various chain lengths and
branching.
[0292] In an aspect of the present invention, demethylated steroids
are provided which have oxygen and/or hydrocarbon substitution at
C6 and C7, however do not have methyl groups at both of C10 and
C13. In one embodiment, the demethylated steroid has the Structure
8, including individual enantiomeric or geometric isomers thereof,
and further including a solvate or pharmaceutically acceptable salt
thereof. Structure 8 is defined as follows:
[0293] A compound of the formula ##STR65## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0294] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted with
[0295] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0296] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0297] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0298] with the provisos that (a) C10 and C13 are not
simultaneously substituted with methyl, and (b) when C10 is
substituted with methyl, then C14 is not substituted with a
methyl;
[0299] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0300] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represent a carbonyl
or protected carbonyl group;
[0301] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0302] X represents fluoride, chloride, bromide and iodide.
[0303] In a preferred embodiment, compounds of Structure 8 do not
have aromatic A rings.
[0304] A number of examples of compounds containing substituents
other than methyl at C10 or C13 are described herein in connection
with Schemes 20, 29 and 30. The substituents include carbonyl,
hydroxymethylene, methoxy, ketal, lactone carbonyl, aldehyde,
hydroxy, etc. Not described in connection with Schemes 20, 29 and
30 are compounds containing no substitution (i.e., merely hydrogen
substitution) at C10 and/or C13. Below are examples discussing
synthetic approaches to producing
19-nor-6.alpha.,7.beta.-dioxygenated steroids.
[0305] The synthesis of many of the various compounds of the
present invention has been detailed in connection with compounds 1
and 247, both commercially available starting materials. However,
the preparation of analogous compounds, e.g., compound 141, that
differs only in the lack of a C10 methyl substituent, can be
achieved according to Scheme 42 shown below.
[0306] In Scheme 42, the starting material is the commercially
available 19-nor-testosterone (133) (Steraloids Inc., Wilton, N.H.,
or Aldrich Chemical Company, Milwaukee, Wis.). Reduction of
compound 133 using NaBH.sub.4 in ethanol may give compound 134
which contains the 3.beta.-hydroxyl group. After protection of the
3.beta.-hydroxyl group using TBDMSCI and imidazole in DMF, allylic
oxidation on the resultant diprotected compound (135) may be used
to afford the enone derivative 136. Reduction and acetylation, as
described in previous Sections (Scheme 1), followed by
hydroboration using BH.sub.3-THF and oxidative work up
(H.sub.2O.sub.2, 30% NaOH), gives compound 138 which contains the
6.alpha.,7.beta.,17.beta.-hydroxylation pattern. Protection of the
6.alpha.,7.beta.-hydroxyls using 2,2-dimethoxypropane and camphor
sulfonic acid can be followed by oxidation of the C17 hydroxyl
group using PDC in CH.sub.2Cl.sub.2 to afford compound 140
containing the C17 ketone functionality. Reaction of compound 140
with the Wittig reagent prepared from ethyltriphenylphosphonium
bromide and tBuOK in toluene gives the ethylidene derivative which
can be deprotected in 80% acetic acid to yield the trihydroxy
compound 141 which is identical to compound 333 except for the lack
of a C10 methyl substituent. ##STR66##
[0307] In an aspect of the present invention, polyoxygenated
steroids having oxygen and/or hydrocarbon substitution at each of
C3, C4, C6 and C7, where the oxygen and/or hydrocarbon substitution
at C6 has the alpha stereochemistry and the oxygen and/or
hydrocarbon substitution at C7 has the beta stereochemistry, are
provided. In one embodiment, the polyoxygenated steroid has the
Structure 9, including individual enantiomeric or geometric isomers
thereof, and further including a solvate or pharmaceutically
acceptable salt thereof. Structure 9 is defined as follows:
[0308] A compound of the formula ##STR67## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0309] each of C1, C2, C11, C12, C15, C16 and C17 is independently
substituted with
[0310] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0311] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0312] with the proviso that C17 is not substituted with any of the
following: ##STR68## each of C5, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0313] C8 is substituted with --X or --R.sup.4 and is preferably
not bonded directly to oxygen;
[0314] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0315] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group;
[0316] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur; where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0317] X represents fluoride, chloride, bromide and iodide.
[0318] Compounds having the oxygen and/or hydrocarbon substitution
shown in Structure 9 may be prepared from compound 142, which was
prepared as described below in Scheme 52. Thus, as shown in Scheme
43, compound 142 may be epoxidized with any number of epoxidization
conditions, e.g., using m-chloroperbenzoic acid (m-CPBA) in
dichloromethane, to provide the epoxide compound 143. Ring opening
of the epoxide group using a mild organic acid (e.g., anhydrous
acetic acid, which is preferred) provides compound 144, which is a
representative compound of Structure 9. ##STR69##
[0319] From compound 144, many other compounds of Structure 9 may
be prepared. For example, as illustrated in Scheme 43, compound 144
may be deacetylated to provide the tetrahydroxy ketone compound
145. The ketone group at C17 may be subject to Wittig chemistry as
discussed above, to provide entry into a large class of
tetrahydroxy olefin compounds of Structure 9.
[0320] Structure 9, which has a 3,4,6,7-tetraoxygenation pattern,
may additionally contain further oxygen-containing substituents.
For example, compounds of Structure 9 may have an oxygen atom at
C11. Synthetic methodology to introduce a C11 oxygen atom, which
may be employed to prepare compounds of Structures 5-12 including
Structure 9, may be achieved by chemistry shown in Scheme 44, or by
chemistry analogous to that shown in Scheme 44.
[0321] For example, rather than using a commercially available
starting material with a C11 hydroxyl functionality or
.DELTA..sup.9,11 carbon-carbon double bond, the formation of the
m-bischloroiodosobenzylformyl ester followed by its photolysis
generates the desired unsaturation at the .DELTA..sup.9,11 position
(compound 149). Thus, the C6 and C7 hydroxyls in compound 146
(prepared according to Scheme 61) may be protected using
2,2-dimethoxypropane and camphor sulfonic acid to give compound
147. Subsequent reaction of 147 with m-bischloroiodosobenzylformyl
chloride in pyridine followed by photolysis in CCl.sub.4 gives
compound 149. Protection of the A-ring hydroxyl groups followed by
hydroboration/oxidation yields the C11 hydroxy derivative 151.
Complete deprotection using 80% acetic acid gives the hexol 152.
##STR70##
[0322] In an aspect of the present invention, steroid ketones
having a pyran or 8-lactone ring in the C17 sidechain are provided.
In one embodiment, the steroid ketone has the Structure 10,
including individual enantiomeric or geometric isomers thereof, and
further including a solvate or pharmaceutically acceptable salt
thereof. Structure 10 is defined as follows:
[0323] A compound of the formula ##STR71## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0324] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0325] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0326] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0327] with the proviso that C3 and C4 are not simultaneously
substituted with hydroxyl or protected hydroxyl, and are preferably
not simultaneously substituted with oxygen atoms;
[0328] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0329] G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--;
[0330] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0331] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0332] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0333] X represents fluoride, chloride, bromide and iodide.
[0334] Convenient access to the C17 sidechain in compounds of
Structure 10 begins with L-carvone, as shown in Scheme 45.
##STR72##
[0335] L-Carvone (153) may be converted to compound 154 according
to literature procedures. See, e.g., Tetrahedron Letters
25(41):4685-4688 (1984). The primary alcohol in compound 154 is
then protected by, e.g., conversion to an acetate ester. Removal of
the ketal protecting group in compound 155 using acidic conditions
provides aldehyde 156.
[0336] The compound 156 can provide access to the C17 sidechain in
compounds of Structure 10 as shown in Scheme 46 below. Thus,
compound 145 as prepared in Scheme 43, may be treated with the ylid
prepared from ethyltriphenylphosphonium bromide and base to afford
compound 157 (used as starting material in Scheme 46). Thereafter,
the four hydroxyl groups may be converted to protected hydroxyl
groups, for example benzyloxy groups, as shown in compound 158.
Compound 158 is then coupled with the aldehyde 156 (Scheme 45) in
the presence of a Lewis acid, to provide compound 159. Deprotection
of the C29 acetoxy group may then be accomplished with base, to
provide diol compound 160, which may then be oxidized to the
.delta.-lactone compound 161. Allylic oxidation of compound 161 may
introduce a carbonyl moiety at C15 with concurrent oxidation of the
benzyl groups (Bn) to benzoate (Bz) groups, to form compound
162.
[0337] Reduction of the conjugated .DELTA..sup.16 carbon-carbon
double bond in the D-ring of compound 162 gives compound 163.
Removal of the benzoate groups in 163 may be achieved using basic
conditions (for example NaOMe in MeOH) with concurrent
epimerization at C14 to yield product 164 which contains an
epimeric mixture of the compounds containing the cis C/D ring
junction and the trans C/D ring junction. Finally, protection of
the C15 ketone followed by reduction of the .delta.-lactone to the
lactol and deprotection (80% acetic acid) may be accomplished to
give 22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(compound 165) and it's C14 epimer
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.alpha.-stigmastan-15-one.
##STR73## ##STR74##
[0338] Compounds of Structure 10 may have a C15 ketone and C22,29
epoxy functionality. In fact, compounds containing a variety of
functionality in the A-D rings in addition to a C15 ketone and a
sidechain hemiacetal may be produced using a combination of
methodology described herein.
[0339] For example, as illustrated in Scheme 47, compound 176,
which contains a methylene at C3, a carbonyl at C15 and a sidechain
hemiacetal, may be produced by using methodology described herein.
The C15 ketone and the sidechain hemiacetal may then be
incorporated using methodology described in detail above (in
connection with Schemes 45 and 46). ##STR75##
[0340] As shown in Scheme 47, compound 76 can be deprotected using
H.sub.2, Pd/C in ethanol to give a compound containing the C11
hydroxyl functionality which, upon heating in POCl.sub.3 and
pyridine, may produce compound 167 containing the .DELTA..sup.9,11
double bond and its .DELTA..sup.11,12 isomer. Epoxidation (of 167)
using mCPBA followed by LiAlH.sub.4 reduction may be used to afford
compound 169 which contains the C9 hydroxyl functional group.
Protection of this hydroxyl group followed by removal of the ketal
protecting group and Wittig chemistry may be done to yield the
olefinic product 171. Conversion of compound 171 to lactol 176 may
be accomplished using standard methods described herein.
[0341] A second example involves the preparation of derivative 186,
a compound that contains the C15 ketone and the sidechain
hemiacetal as well as a C1 hydroxyl functionality. Compound 186 may
be produced in a multi-step procedure from the commercially
available starting material 177 as shown in Scheme 48. The first
step involves protection of compound 178 using e.g., ethylene
glycol, pTsOH in benzene. Subsequent Michael addition using, e.g.,
benzyl alcohol and potassium hydroxide gives the C1 benzyloxy
derivative 179. LS-Selectride.RTM. reduction of the ketone 179
followed by protection of the resultant alcohol as the benzyloxy
derivative may be used to give compound 180. The conversion of
compound 180 to lactol 186 may then be achieved using methods
described in Scheme 47 and described in detail in other previous
examples. ##STR76##
[0342] Thus, the methodology described herein may be used to
produce compounds with functionality at carbons in the steroidal
ring structure as well as both the C15 ketone functionality and
sidechain hemiacetal.
[0343] In a related aspect of the present invention, steroids
having oxygenation at C6 and C7, with a pyran-or
.delta.-lactone-containing sidechain at C17 are provided. In one
embodiment, the steroid has the Structure 11, including individual
enantiomeric or geometric isomers thereof, and further including a
solvate or pharmaceutically acceptable salt thereof. Structure 11
is defined as follows:
[0344] A compound of the formula ##STR77## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0345] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted with
[0346] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0347] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0348] with the proviso that C3 and C4 are not simultaneously
substituted with hydroxyl or protected hydroxyl, and are preferably
not simultaneously substituted with oxygen atoms;
[0349] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0350] G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--;
[0351] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0352] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0353] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0354] X represents fluoride, chloride, bromide and iodide.
[0355] The preparation of compounds of Structure 11 may be achieved
using methodology set forth many places herein. For example,
compounds 196 (Scheme 49) and 207 (Scheme 50) may be synthesized
from compounds 30 and 55 in multi-step processes. Methods used to
convert the C17 silyloxy group in compound 30 to the olefin 190 are
analogous to those described in detail in previous examples, as are
the methods used to convert compound 190 to compound 196. The same
holds true for the conversions of compounds 55 to 200 and 200 to
207, respectively. ##STR78## ##STR79##
[0356] The chemistry described in Schemes 49 and 50 above are just
two examples of how the methods discussed herein may be applied to
produce compounds containing a 6,7-dioxygenation pattern and the
hemiacetal or 6-lactone sidechain. Thus, the methodology described
previously may be used to produce compounds with functionality at
C2, C4, C8, etc.
[0357] In an aspect of the present invention, steroid epoxides are
provided. In one embodiment, the steroid epoxide has the Structure
12, including individual enantiomeric or geometric isomers thereof,
and further including a solvate or pharmaceutically acceptable salt
thereof. Structure 12 is defined as follows:
[0358] A compound of the formula ##STR80## including
pharmaceutically acceptable salts and solvates thereof,
wherein:
[0359] each of C1, C2, C1, C12, C15, C16 and C17 is independently
substituted with
[0360] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6; or
[0361] (b) two of the following, which are independently selected:
--X, --R.sup.4 and --OR.sup.1;
[0362] each of C5, C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R or --OR.sup.1;
[0363] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0364] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0365] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0366] X represents fluoride, chloride, bromide and iodide.
[0367] Preferably, in compounds of Structure 12, C7 does not have
carbonyl substitution when C5 has hydroxy or --OR.sup.1
substitution.
[0368] As with the previous examples, introduction of functional
groups at various positions within the steroid ring structure of
compounds containing a 3,4-epoxide group of Structure 12 may be
achieved using methods described herein. For example, as shown in
Scheme 51, an oxygen atom may be placed at C9 and/or C11, via
epoxidation of .DELTA..sup.9,11 double bond.
[0369] Thus, LS-selectride reduction followed by remote oxidation
using reagents described earlier on a compound such as compound 10
may provide an olefinic compound 208 (Scheme 51). Transformations
to the .DELTA..sup.9,11 olefin can be achieved using standard
methodology and concurrent reaction of both the .DELTA..sup.3,4 and
.DELTA..sup.9,11 double bonds provide the desired epoxides at C3-C4
and C9-C11. Oxidation of the C3 hydroxyl moiety with PDC in
CH.sub.2Cl.sub.2 then may be used to give the desired unsaturated
A-ring (and optionally ring-opening the epoxide rings will provide
a 3,6,7,9-polyhydroxylated steroid 215). ##STR81##
[0370] The introduction of an alkyl group in the C16 position may
also be achieved using similar chemistry described above. In the
following example (Scheme 52), a methyl group is incorporated into
this position from the condensation of the D-ring enolate with
methyl iodide. This methodology is analogous to that described in
connection with Scheme 37. As shown in Scheme 52, the alkylated
epoxide 218 may be subjected to epoxide-ring-opening conditions to
afford a 3,4,6,7-tetrahydroxy steroid 220. ##STR82##
[0371] Compounds having 6.alpha.,7.beta.-hydroxylation pattern have
been discussed in detail in previous sections. Alternatively,
compounds containing other stereochemistries at C6 and C7 may also
be produced as discussed in the following section. For example,
selective tosylation of compound 221 (prepared according to Scheme
73) using pTsCl in pyridine followed by treatment with potassium
carbonate may yield the epoxide containing compound 223. Subsequent
ring opening using aqueous acid may yield compounds with the
6.beta.,7.alpha. stereochemistry as shown in Scheme 53.
##STR83##
[0372] Compounds with the 6.alpha.,7.alpha. stereochemistry can be
prepared from commercially available starting materials as shown in
Scheme 54. Thus, cholesteryl acetate may be oxidized using
RuCl.sub.3 and tBuOOH in CH.sub.2Cl.sub.2 to afford the enone
containing compound 229. Exchange of the protecting group at C3 to
the tBDMS derivative is followed by lithium ammonia reduction and
trapping of the enolate anion with (MeO).sub.2PCl giving the enol
phosphate 231. A second lithium-ammonia reduction gives the
.DELTA..sup.6,7 double bond which may be oxidized with OsO.sub.4 to
afford compound 233 containing the
3.beta.,6.alpha.,7.alpha.-trihydroxylation pattern. ##STR84##
[0373] More generally, compounds of the present invention may be
characterized by the following formula: ##STR85##
[0374] including pharmaceutically acceptable salts and solvates
thereof, wherein:
[0375] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted according to any of (a) and (b):
[0376] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0377] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0378] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0379] C17 is substituted according to any of (c), (d), (e), (f),
(g), (h) and (i):
[0380] (c) .dbd.C(R.sup.2)(R.sup.3) except when C14 is substituted
with methyl;
[0381] (d) --R.sup.5 and --OR.sup.6 so long as the A and B rings
are not aromatic, and when C10 is substituted with methyl then C5
is not bonded directly to oxygen, where R.sup.5 and R may together
form a direct bond so C17 is a carbonyl group, or may together with
C17 form a cyclic 3-6 membered ether or 4-6 membered lactone;
otherwise R.sup.5 is R.sup.4 or --OR.sup.6 and R.sup.6 is R.sup.1
or R.sup.4.
[0382] (e) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6, as long as one of the following conditions i), ii), iii)
or iv) apply: [0383] i) C5 is substituted with a hydrogen in the
alpha configuation, and C3 is not bonded to oxygen, and when C3 is
substituted with two hydrogen atoms then C17 is not substituted
with either --CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2 or
--CH(CH.sub.3)(CH.sub.2)2C(.dbd.O)OCH.sub.3; [0384] ii) C10 and C13
are not simultaneously substituted with methyl, and when C10 is
substituted with methyl, then C14 is not substituted with a methyl,
and the A ring is never aromatic; [0385] iii) if C3 and C4 are
bonded to oxygen atoms, and the C6--OR.sup.1 substituent has the
alpha configuration, and the C7 --OR.sup.1 substituent has the beta
configuration, then C17 is not substituted with any of the
following: ##STR86## [0386] iv) C3 and C4 are each bonded to the
same oxygen atom so as to form an oxirane ring, with the proviso
that C7 does not have carbonyl substitution when C5 has hydroxyl or
--OR.sup.1 substitution;
[0387] (f) two of the following substituents, which are
independently selected: --X, --R.sup.4 and --OR.sup.1, as long as
one of the above conditions i), ii), iii) or iv) apply;
[0388] (g) a cyclic structure of the formula ##STR87##
[0389] wherein G is --C(.dbd.O)--, --CH(OR.sup.1)--,
--C(R.sup.4)(OR.sup.1)-- or --C(OR.sup.1)(OR.sup.1)--, as long as
C3 and C4 are not simultaneously substituted with hydroxyl or
protected hydroxyl;
[0390] (h) two hydrogen atoms, as long as C3 is not substituted
with a carbonyl group;
[0391] (i) one hydrogen atom and one group selected from
C.sub.1-C.sub.30 hydrocarbyl groups and C.sub.1-C.sub.30 halogen
substituted hydrocarbyl groups, excluding
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2;
[0392] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0393] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0394] R.sup.2, R.sup.3 and R.sup.4 at each occurrence is
independently selected from H and C.sub.1-30 organic moiety that
may optionally contain at least one heteroatom selected from the
group consisting of boron, halogen, nitrogen, oxygen, silicon and
sulfur, where two geminal R.sup.4 groups may together form a ring
with the carbon atom to which they are both bonded; and
[0395] X represents fluoride, chloride, bromide and iodide.
[0396] In a preferred embodiment, the compounds of the invention
have one of the structure set forth below, and mixtures thereof:
##STR88##
[0397] including pharmaceutically acceptable salts and solvates
thereof, wherein:
[0398] each of C1, C2, C3, C4, C11, C12 and C16 is independently
substituted according to (a) or (b):
[0399] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6,
[0400] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0401] C5 is substituted with a hydrogen atom;
[0402] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or --OR.sup.1,
although C5 is preferably substituted with hydrogen; and
[0403] C17 is substituted according to (c), (d), (e) or (f):
[0404] (c) two substituents selected from hydrogen, halogen,
C.sub.1-C.sub.30 saturated hydrocarbyl excluding
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2, halogen
substituted C.sub.1-C.sub.30 saturated hydrocarbyl,
C.sub.1-C.sub.30 unsaturated hydrocarbyl, and halogen substituted
C.sub.1-C.sub.30 unsaturated hydrocarbyl;
[0405] (d) one substituent selected from .dbd.C(R.sup.4)(R.sup.4)
with the proviso that C14 is not substituted with methyl;
[0406] (e) at least one oxygen atom-containing substituent selected
from .dbd.O, --(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges
from 1 to about 6, --OH, and --OR.sup.1;
[0407] (f) at least one nitrogen atom-containing substituent
selected from --N(R.sup.4)(R.sup.4) wherein the two R.sup.4 groups
may together with the nitrogen atom form one or more rings, so that
the nitrogen atom-containing substituent includes nitrogen
atom-containing heterocyclic groups; wherein
[0408] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated, however fully
saturated rings are preferred;
[0409] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where --OR.sup.1 groups bonded to
adjacent carbon atoms may together form a cyclic structure which
protects both hydroxyl groups;
[0410] R.sup.4 at each occurrence is independently selected from H
and R.sup.5;
[0411] R.sup.5 is a C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur; where two
geminal R.sup.5 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0412] X represents fluoride, chloride, bromide or iodide.
[0413] The compounds of general and preferred structures as
disclosed herein may be prepared by synthetic methodology as set
forth in the Schemes 1-54, the references cited herein and the
Examples provided herein, as well as knowledge of the skilled
artisan. The following are preferred synthetic procedures useful in
preparing compounds of the present invention.
[0414] In one aspect, the invention provides a process for
introducing an exocyclic olefin group to the C17 position of a
6,7-dioxygenated steroid. The process includes the step of
providing a compound of Formula (10) (such a compounds may be
commercially available or may be prepared by techniques disclosed
herein), and then reacting the compound of Formula (10) with a
Wittig reagent of Formula (11) in the presence of a base, to
provide an olefin compound of Formula (12) ##STR89##
[0415] Each of the compounds of Formulas (10) and (12) include
pharmaceutically acceptable salts and solvates thereof. In Formula
(10), (11) and (12):
[0416] each of C1, C2, C3, C4, C11, C12, C15 and C16 is
independently substituted according to any of (a) and (b):
[0417] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0418] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0419] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R or --OR.sup.1;
[0420] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR.sup.1 at C6 and C7 represents a
carbonyl or protected carbonyl group;
[0421] Ra, Rb and R.sup.4 at each occurrence is independently
selected from H and C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur, where two
geminal R.sup.4 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0422] X represents fluoride, chloride, bromide and iodide, which
is independently selected at each occurrence.
[0423] In preferred embodiments of the process, the base is
selected from sodium t-butoxide, potassium t-butoxide and sodium
hydride and the like. The base is preferably in admixture with an
aprotic solvent. Suitable aprotic solvents include toluene,
tetrahydrofuran, methylene chloride, dimethylformamide,
dimethylsulfoxide, benzene and diethyl ether. In another preferred
embodiment, Ra and Rb are independently selected from hydrogen and
C.sub.1-C.sub.7alkyl, and X is selected from chloride, bromide and
iodide.
[0424] In another aspect, the invention provides a process for
introducing 6.alpha.,7.beta.-dioxygenation into a steroid. The
process includes the steps of providing a steroid of Formula (13)
having a carbonyl group at C7 and a double bond between C5 and C6.
Steroids of Formula (13) may be prepared by, for example, synthetic
methodology disclosed herein. In a subsequent step, the carbonyl
group is reduced to a hydroxyl group, followed by a hydroboration
of the double bond to provide a hydroxyl group at C6, wherein the
C6 hydroxyl group has the .alpha.-configuration and the C7 hydroxyl
group has the .beta.-configuration, ##STR90##
[0425] The compounds of Formulas (13) and (14) include
pharmaceutically acceptable salts and solvates thereof. In Formula
(13) and (14):
[0426] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted according to any of (a) and (b):
[0427] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0428] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0429] each of C8, C9, C10, C13 and C14 is independently
substituted with one of --X, --R.sup.4 or --OR.sup.1;
[0430] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0431] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0432] X represents fluoride, chloride, bromide and iodide.
[0433] In preferred embodiments of the process, the reduction is
accomplished with sodium borohydride in combination with
cerium(III) chloride heptahydrate. In another preferred embodiment
of the process, the hydroboration is conducted with a hydroboration
reagent selected from BH.sub.3 and 9-BBN, and preferably in the
presence of an aprotic solvent. Suitable aprotic solvents include
tetrahydrofuran, methylene chloride, diethyl ether, dimethyl
sulfide and carbon disulfide. The hydroboration is preferably
immediately followed by treatment with a peroxide, such as hydrogen
peroxide or t-butylperoxide, and a base, such as sodium hydroxide
and potassium hydroxide.
[0434] Another aspect of the invention provides a process for a
stereocontrolled introduction of a hydroxyl group at C3 of a
steroid nucleus. The process includes the step of providing a
steroid compound of Formula (15) having a carbonyl group at C3.
Steroid compounds of Formula (15) may be prepared by, for example,
synthetic methods disclosed herein. This is followed by reducing
the carbonyl group to a hydroxyl group with a reducing agent so as
to provide at least one compound of Formulas (16) and (17)
##STR91##
[0435] Each of the compounds of Formulas (15), (16) and (17)
include pharmaceutically acceptable salts and solvates thereof. In
the compounds of Formulas (15), (16) and (17):
[0436] each of C1, C2, C4, C11, C12, C15, C16 and C17 is
independently substituted according to any of (a) and (b):
[0437] (a) one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)-- wherein n ranges from 1 to
about 6;
[0438] (b) two of: --X, --R.sup.4 and --OR.sup.1, each
independently selected;
[0439] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with one of --X, --R.sup.4 or
--OR.sup.1;
[0440] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where vicinal --OR.sup.1 groups may
together form a cyclic structure which protects vicinal hydroxyl
groups, and where geminal --OR.sup.1 groups may together form a
cyclic structure which protects a carbonyl group, with the proviso
that either or both of --OR, at C6 and C7 represents a carbonyl or
protected carbonyl group;
[0441] R.sup.4 at each occurrence is independently selected from H
and C.sub.1-30 organic moiety that may optionally contain at least
one heteroatom selected from the group consisting of boron,
halogen, nitrogen, oxygen, silicon and sulfur, where two geminal
R.sup.4 groups may together form a ring with the carbon atom to
which they are both bonded; and
[0442] X represents fluoride, chloride, bromide and iodide.
[0443] In a preferred embodiment of this process, the reducing
agent is selected from lithium trisiamylborohydride, lithium
tri-sec-butylborohydride and potassium tri-sec-butylborohydride,
and will predominantly provide the hydroxyl compound of Formula
(16) (relative to the hydroxyl compound of Formula (17)). In
another preferred embodiment, the reducing agent is selected from
sodium borohydride and lithium aluminum hydride, and will
predominantly provide the hydroxyl compound of Formula (17). In
general, the inventive process will achieve a reduction of
compounds of Formula (15) such that the product mixture contains a
ratio of Formula (16) to Formula (17) compounds of other than
1:1.
[0444] As used herein, the term organic moiety of an indicated
carbon number range refers to a stable arrangement of atoms
composed of at least one and not more than about the maximum carbon
number set forth in the range, typically not more than about 30
carbon atoms, and any number of non-carbon atoms.
[0445] The C.sub.1-30 organic moiety may be a saturated or
unsaturated hydrocarbyl radical. A saturated hydrocarbyl radical is
defined according to the present invention as any radical composed
exclusively of carbon and hydrogen, where single bonds are
exclusively used to join carbon atoms together. Thus, any stable
arrangement of carbon and hydrogen atoms, having at least one
carbon atom, is included within the scope of a saturated
hydrocarbon radical according to the invention. Some specific
terminology that may be used to refer to specific carbon atom
arrangements will be discussed below.
[0446] The carbon atoms may form an alkyl group, i.e., an acyclic
chain of carbon atoms which may be branched or unbranched (linear).
Methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl
and t-butyl are alkyl groups having 1 to 4 carbon atoms (commonly
referred to as lower alkyl groups), and are exemplary of alkyl
groups of the invention. The carbon atoms may form a cycloalkyl
group, i.e., a cyclic arrangement of carbon atoms, where
cyclopropyl, cyclobutyl, cyclopentyl are cycloalkyl groups of the
invention having 3-5 carbon atoms. Additional groups within the
scope of "ccycloalkyl" as defined herein are polycycloalkyl groups,
defined below.
[0447] A polycycloalkyl group is an arrangement of carbon atoms
wherein at least one carbon atom is a part of at least two
separately identifiable rings. The polycycloalkyl group may contain
bridging between two carbon atoms, where bicyclo[1.1.0]butyl,
bicyclo[3.2.1]octyl, bicyclo[5,2.0]nonyl,
tricycl[2.2.1.0.sup.1]heptyl, norbornyl and pinanyl are
representative examples. The polycycloalkyl group may contain one
or more fused ring systems, where decalinyl (radical from decalin)
and perhydroanthracenyl are representative examples. The
polycycloalkyl group may contain a spiro union, in which a single
atom is the only common member of two rings. Spiro[3.4]octyl,
spiro[3.3]heptyl and spiro[4.5]decyl are representative
examples.
[0448] In addition, the saturated hydrocarbyl radical can be
composed of any combination of two or more of the above, i.e., any
combination of alkyl and cycloalkyl groups. Thus, the R.sup.4 or
R.sup.5 groups may be an alkyl group (e.g., methyl) with a
cycloalkyl (e.g., cyclohexyl) substituent, so that R.sup.4 or
R.sup.5 is a cyclohexylmethyl group. As another example, R.sup.4 or
R.sup.5 may be a cycloalkyl group (e.g., cyclooctyl) having two
alkyl substituents (e.g., a methyl and ethyl substituent), so that
R.sup.4 or R.sup.5 is a methylethylcyclooctyl group. As a final
example, R.sup.4 or R.sup.5 may be a cycloalkyl group with an alkyl
substituent, where the alkyl substituent is substituted with a
polycycloalkly substituent.
[0449] As indicated above, R.sup.4 or R.sup.5 may be an unsaturated
hydrocarbyl radical. Such an R.sup.4 or R.sup.5 group is defined as
having a carbon arrangement as set forth above for saturated
hydrocarbyl radicals, with the additional feature that at least one
bond between any two carbon atoms is other than a single bond. An
alkyl group with a single double bond is referred to as an alkenyl
group, while an alkyl group having more than one double bond is
referred to as an alkapolyenyl group, where alkadienyl (2 double
bonds) and alkatrienyl (3 double bonds) are exemplary. An alkyl
group with a single triple bond is referred to as an alkynyl group,
while an alkyl group having more than one triple bond is referred
to as a alkapolyynyl group, where alkydiynyl (2 triple bonds) and
alkatriynyl (3 triple bonds) are exemplary.
[0450] Likewise, the cycloalkyl group may have one or more double
or triple bonds, and be included within the scope of an unsaturated
hydrocarbyl radical according to the invention. Cycloalkenyl and
cycloalkynyl are general names given to groups having a single
carbon-based ring with a single double and triple bond in the ring,
respectively. Cycloalkadienyl groups are cycloalkyl groups with two
double bonds contained in the ring structure. The double bond may
be exocyclic to the ring, e.g., a carbon atom of the ring may have
a .dbd.CH.sub.2 group (i.e., a methylidene group) or higher
homologue bonded to it.
[0451] A ring may be unsaturated to the extent of being aromatic,
and still be included within the scope of an unsaturated
hydrocarbyl radical. Thus, an aryl group, for example, phenyl and
naphthyl, are included within the scope of such hydrocarbyl groups.
As any combination of the above is also included within the scope
of an unsaturated hydrocarbyl radical, aralkyl (R.sup.4 or R.sup.5
is an alkyl group with at least one aryl substituent, e.g., benzyl)
and alkylaryl (R.sup.4 or R.sup.5 is an aryl ring with at least one
alkyl substituent, e.g., tolyl) groups are included within the
scope of R.sup.4 or R.sup.5. C.sub.6 aryls are a preferred
component of organic moieties of the invention.
[0452] R.sup.4 or R.sup.5 includes organic moieties that contain a
heteroatom. Heteroatoms according to the invention are any atom
other than carbon and hydrogen. A preferred class of heteroatoms
are naturally occurring atoms (other than carbon and hydrogen).
Another preferred class are non-metallic (other than carbon and
hydrogen). Another preferred class consists of boron, nitrogen,
oxygen, silicon, phosphorous, sulfur, selenium and halogen (i.e.,
fluorine, chlorine, bromine and iodine, with fluorine and chlorine
being preferred). Another preferred class consists of nitrogen,
oxygen, sulfur and halogen. Another preferred class consists of
nitrogen, oxygen and sulfur. Oxygen is a preferred heteroatom.
Nitrogen is a preferred heteroatom.
[0453] For example, R.sup.4 or R.sup.5 may be a hydrocarbyl radical
as defined above, with at least one substituent containing at least
one heteroatom. In this paragraph, R.sup.4 will be used to refer to
both R.sup.4 and R.sup.5. In other words, R.sup.4 may be a
hydrocarbyl radical as defined above, wherein at least one hydrogen
atom is replaced with a heteroatom. For example, if the heteroatom
is oxygen, the substituent may be a carbonyl group, i.e., two
hydrogens on a single carbon atom are replaced by an oxygen, to
form either a ketone or aldehyde group. Alternatively, one hydrogen
may be replaced by an oxygen atom, in the form of an hydroxy,
alkoxy, aryloxy, aralkyloxy, alkylaryloxy (where alkoxy, aryloxy,
aralkyloxy, alkylaryloxy may be collectively referred to as
hydrocarbyloxy), heteroaryloxy, --OC(O)R.sup.4, ketal, acetal,
hemiketal, hemiacetal, epoxy and --OSO.sub.3M. The heteroatom may
be a halogen. The heteroatom may be a nitrogen, where the nitrogen
forms part of an amino (--NH.sub.2, --NHR.sup.4,
--N(R.sup.4).sub.2), alkylamido, arylamido, arylalkylamido,
alkylarylamido, nitro, --N(R.sup.4)SO.sub.3M or aminocarbonylamide
group. The heteroatom may be a sulfur, where the sulfur forms part
of a thiol, thiocarbonyl, --SO.sub.3M, sulfonyl, sulfonamide or
sulfonhydrazide group. The heteroatom may be part of a
carbon-containing substituent such as formyl, cyano,
--C(O)OR.sup.4, --C(O)OM, --C(O)R.sup.4, --C(O)N(R.sup.4).sub.2,
carbamate, carbhydrazide and carbohydroxamic acid.
[0454] In the above exemplary heteroatom-containing substituents, M
represents proton or a metal ion. Preferred metal ions, in
combination with a counterion, form physiologically tolerated
salts. A preferred metal from which a metal ion may be formed
include an alkali metal [for example, lithium (Li), sodium (Na),
potassium (K), rubidium (Rb) and cesium (Cs)] an alkaline earth
metal (for example, magnesium (Mg), calcium (Ca) and strontium
(Sr)], or manganese (Mn), iron (Fe), zinc (Zn) or silver (Ag). An
alkali metal or an alkaline earth metal are preferred M groups.
Sodium, potassium, magnesium and calcium are preferred M groups.
Sodium and potassium are preferred M groups.
[0455] Another class of organic moieties according to the invention
are hydrocarbyl radicals as defined above, wherein at least one
carbon is substituted for at least one heteroatom. Examples of such
organic moieties are heterocycloalkyl (a cycloalkyl group having at
least one carbon replaced with at least one heteroatom),
heterocycloalkenyl, heteroaryl, heteroaryloxy, heteroaralkyl,
heteroaralkenyl, etc. Collectively, this class of organic moieties
may be referred to as heterohydrocarbyls. Another example of such
organic moieties have a heteroatom bridging (a) the radical to
which the organic moiety is bonded and (b) the remainder of the
organic moiety. Examples include alkoxy, aryloxy, arylalkyloxy and
alkylaryloxy radicals, which may collectively be referred to herein
as hydrocarbyloxy radicals or moieties. Thus, --OR.sup.4 is an
exemplary R.sup.4 group of the invention. Another example is
--NHR.sup.4.
[0456] Examples of heterocycloalkylene are pyrrolidinylene,
piperidinylene, tetrahydrofuranylene, di and tetrahydropyranylene.
Examples of heterocycloalkyl are radicals derived from pyrrolidine,
imidazolidine, oxazolidine, pyrazolidine, piperidine, piperazine
and morpholine. Examples of heterocycloalkenyl substituents are
radicals derived by removal of a hydrogen from 2- and 3-pyrroline,
oxazoline, 2- and 4-imidazoline and 2- and 3-pyrazoline.
[0457] While the organic moiety may have up to about 30 carbon
atoms, preferred organic moieties of the invention have fewer than
30 carbon atoms, for example, up to about 25 carbon atoms, more
preferably up to about 20 carbon atoms. The organic moiety may have
up to about 15 carbon atoms, or up to about 12 or 10 carbon atoms.
A preferred category of organic moieties has up to about 8 or 6
carbon atoms.
[0458] The following are exemplary R.sup.4 and R.sup.5 organic
moieties where R.sup.4 or R.sup.5 is joined to the steroid nucleus
through a carbon atom: alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl,
arylcarbonyl, heterocyclylcarbonyl, alkyloxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, cycloalkyloxycarbonyl,
cycloalkenyloxycarbonyl, aryloxycarbonyl, heterocyclyloxycarbonyl,
carboxylic acid, cyano and formyl.
[0459] The following are exemplary R.sup.4 and R.sup.5 organic
moieties where R.sup.4 or R.sup.5 is joined to the steroid nucleus
through an oxygen atom: hydroxy, oxo, alkoxy, alkenyloxy,
alkynyloxy, cycloalkoxy, cycloalkenyloxy, aryloxy,
alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,
cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy, arylcarbonyloxy and
heterocyclyloxy.
[0460] R.sup.4 and R.sup.5 organic moieties may contain a nitrogen
atom through which the R.sup.4 or R.sup.5 organic moiety is joined
to the steroid nucleus. Examples are nitro and organic moieties of
the formula --NL.sup.2L.sup.3 wherein L.sup.2 and L.sup.3 are
independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, formyl, heterocyclyl, alkylcarbonyl,
alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,
cycloalkenylcarbonyl, arylcarbonyl and heterocyclylcarbonyl, such
that L.sup.2 and L.sup.3 together may be alkylene or alkenylene to
thereby form a 3- to 8-membered saturated or unsaturated ring in
combination with the nitrogen atom to which they are attached.
[0461] The following are exemplary R.sup.4 and R.sup.5 organic
moieties where the R.sup.4 or R.sup.5 moiety is joined to the
steroid nucleus through a sulfur atom: alkylsulfide,
alkenylsulfide, alkynylsulfide, cycloalkylsulfide,
cycloalkenylsulfide, arylsulfide, heterocyclylsulfide,
alkylcarbonylsulfide, alkenylcarbonylsulfide,
alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,
cycloalkenylcarbonylsulfide, arylcarbonylsulfide,
heterocyclylcarbonylsulfide, and groups of the formulas:
--S(O).sub.nH, --S(O).sub.nL.sup.4, S(O).sub.mOH,
S(O).sub.mOL.sup.4, OS(O).sub.mOL.sup.4, and --O(S).sub.nOH,
wherein L.sup.4 is selected from alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, aryl and heterocyclyl.
[0462] In the above R.sup.4 and R.sup.5 organic moieties, the
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and aryl groups
(collectively referred to as the R.sup.4 or R.sup.5 hydrocarbyl
groups) may be fully or partially halogenated, and/or substituted
with up to five L.sup.5 groups. The heterocyclyl, heterocyclyloxy,
heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylcarbonyloxy groups (collectively referred to as the
R.sup.4 heterocyclyl groups) may likewise be fully or partially
halogenated and/or substituted with up to five L.sup.5 groups.
[0463] L.sup.5 groups contain a carbon, oxygen, nitrogen or sulfur
atom through which they are joined to a carbon atom of the R.sup.4
or R.sup.5 hydrocarbyl groups or a carbon or nitrogen atom of the
R.sup.4 or R.sup.5 heterocyclyl groups.
[0464] The following are exemplary L.sup.5 groups wherein a carbon
atom of L.sup.5 is joined to the R.sup.4 hydrocarbyl or
heterocyclyl group: alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, alkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl,
arylcarbonyl, alkyloxycarbonyl, alkenyloxycarbonyl,
alkynyloxycarbonyl, cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl
and aryloxycarbonyl.
[0465] The following are exemplary L.sup.5 groups wherein an oxygen
atom of L.sup.5 is joined to the R.sup.4 hydrocarbyl or
heterocyclyl group: hydroxy, oxo, alkoxy, alkenyloxy, alkynyloxy,
cycloalkoxy, cycloalkenyloxy, aryloxy, alkylcarbonyloxy,
alkenylcarbonyloxy, alkynylcarbonyloxy, cycloalkylcarbonyloxy,
cycloalkenylcarbonyloxy and arylcarbonyloxy.
[0466] The L.sup.5 group may contain a nitrogen atom through which
the L group is joined to the R.sup.4 or R.sup.5 hydrocarbyl or
heterocyclyl group. Examples include nitro and nitrogen-containing
groups of the formula --NL.sup.6L.sup.7 wherein L.sup.6 and L.sup.7
are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, formyl, alkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl and
arylcarbonyl such that L.sup.6 and L.sup.7 together may be alkylene
or alkenylene to thereby form a 3- to 8-membered saturated or
unsaturated ring in combination with the nitrogen atom to which
they are attached.
[0467] The following are exemplary L.sup.5 groups wherein a sulfur
atom of L.sup.5 is joined to the R.sup.4 or R.sup.5 hydrocarbyl or
heterocyclyl group: alkylsulfide, alkenylsulfide, alkynylsulfide,
cycloalkylsulfide, cycloalkenylsulfide, arylsulfide,
alkylcarbonylsulfide, alkenylcarbonylsulfide,
alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,
cycloalkenylcarbonylsulfide, arylcarbonylsulfide, and groups of the
formulas: --S(O).sub.nL.sup.8, --S(O).sub.mOR,
--S(O).sub.mOL.sup.8, --OS(O).sub.mOL.sup.8, and --O(S).sub.mOH,
wherein L.sup.8 is selected from alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, aryl and heterocyclyl.
[0468] In the exemplary R.sup.4 and R.sup.5 organic moieties, the
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and aryl groups
which form part of L.sup.5 (collectively referred to as the L.sup.5
hydrocarbyl groups) may be fully or partially halogenated, and/or
substituted with up to three L.sup.9 groups. The heterocyclyl,
heterocyclyloxy, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylcarbonyloxy groups (collectively referred to as the
L.sup.5 heterocyclyl groups) may likewise be fully or partially
halogenated, and/or substituted with up to three L.sup.5
groups.
[0469] L.sup.9 groups contain a carbon, oxygen, nitrogen or sulfur
atom through which they are joined to the L.sup.5 hydrocarbyl group
or the L.sup.5 heterocyclyl group.
[0470] The following are exemplary L.sup.9 groups wherein a carbon
atom of L.sup.9 is joined to the L.sup.5 hydrocarbyl or
heterocyclyl group: alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl,
arylcarbonyl, heterocyclylcarbonyl, alkyloxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, cycloalkyloxycarbonyl,
cycloalkenyloxycarbonyl, aryloxycarbonyl and
heterocyclyloxycarbonyl.
[0471] The following are exemplary L.sup.9 groups wherein an oxygen
atom of L.sup.9 is joined to the L.sup.5 hydrocarbyl or
heterocyclyl group: hydroxy, oxo, alkoxy, alkenyloxy, alkynyloxy,
cycloalkoxy, cycloalkenyloxy, aryloxy, heterocyclyloxy,
alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,
cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy, arylcarbonyloxy and
heterocyclylcarbonyloxy.
[0472] The L.sup.9 group may contain a nitrogen atom through which
the L.sup.9 group is joined to the L.sup.5 hydrocarbyl or
heterocyclyl group. Such nitrogen-containing L.sup.9 groups include
nitro and groups having the formula --N L.sup.10L.sup.11 wherein
L.sup.10 and L.sup.11 are independently hydrogen, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, formyl,
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, arylcarbonyl and
heterocyclylcarbonyl such that L.sup.10 and L.sup.11 together may
be alkylene or alkenylene to thereby form a 3- to 8-membered
saturated or unsaturated ring in combination with the nitrogen atom
to which they are attached.
[0473] The following are exemplary L.sup.9 groups wherein an sulfur
atom of L.sup.9 is joined to the L.sup.5 hydrocarbyl or
heterocyclyl group: alkylsulfide, alkenylsulfide, alkynylsulfide,
cycloalkylsulfide, cycloalkenylsulfide, arylsulfide,
heterocyclylsulfide alkylcarbonylsulfide, alkenylcarbonylsulfide,
alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,
cycloalkenylcarbonylsulfide, arylcarbonylsulfide,
heterocyclylcarbonylsulfide and groups of the formulas:
--S(O).sub.nL.sup.12, --S(O).sub.mOH, S(O).sub.mOL.sup.12,
OS(O).sub.nOL.sup.12, and --O(S).sub.mOH, wherein L.sup.12 is
selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
aryl and heterocyclyl.
[0474] In the exemplary R.sup.4 and R.sup.5 organic moieties, the
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and aryl groups
which form part of L.sup.9 (collectively referred to as the L.sup.9
hydrocarbyl groups) may be fully or partially halogenated, and/or
substituted with up to three L.sup.13 groups. The heterocyclyl,
heterocyclyloxy, heterocyclylcarbonyl, heterocyclyloxycarbonyl,
heterocyclylcarbonyloxy groups (collectively referred to as the
L.sup.9 heterocyclyl groups) may likewise be fully or partially
halogenated, and/or substituted with up to three L.sup.13
groups.
[0475] An L.sup.13 group contains a carbon, oxygen, nitrogen or
sulfur atom through which the L.sup.13 group is joined to the
L.sup.9 hydrocarbyl group or L.sup.9 heterocyclyl group.
[0476] The following are exemplary L.sup.13 groups wherein a carbon
atom of L.sup.13 is joined to the L.sup.9 hydrocarbyl or
heterocyclyl group: alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, aryl, heterocyclyl, alkylcarbonyl, alkenylcarbonyl,
alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl,
arylcarbonyl, heterocyclylcarbonyl, alkyloxycarbonyl,
alkenyloxycarbonyl, alkynyloxycarbonyl, cycloalkyloxycarbonyl,
cycloalkenyloxycarbonyl, aryloxycarbonyl and
heterocyclyloxycarbonyl.
[0477] The following are exemplary L.sup.13 groups wherein an
oxygen atom of L.sup.13 is joined to the L.sup.9 hydrocarbyl or
heterocyclyl group: hydroxy, oxo, alkoxy, alkenyloxy, alkynyloxy,
cycloalkoxy, cycloalkenyloxy, aryloxy, heterocyclyloxy,
alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,
cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy, arylcarbonyloxy and
heterocyclylcarbonyloxy.
[0478] The L.sup.13 group may contain a nitrogen atom through which
the L.sup.13 group is joined to the L.sup.9 hydrocarbyl or
heterocyclyl group. Such nitrogen-containing L.sup.13 groups
include nitro and groups of the formula --NL.sup.14L.sup.15 wherein
L.sup.14 and L.sup.15 are independently hydrogen, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, formyl,
alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl, arylcarbonyl and
heterocyclylcarbonyl such that L.sup.15 and L.sup.15 together may
be alkylene or alkenylene to thereby form a 3- to 8-membered
saturated or unsaturated ring in combination with the nitrogen atom
to which they are attached.
[0479] The following are exemplary L.sup.13 groups wherein an
sulfur atom of L.sup.13 is joined to the L.sup.9 hydrocarbyl or
heterocyclyl group: alkylsulfide, alkenylsulfide, alkynylsulfide,
cycloalkylsulfide, cycloalkenylsulfide, arylsulfide,
heterocyclylsulfide, alkylcarbonylsulfide, alkenylcarbonylsulfide,
alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,
cycloalkenylcarbonylsulfide, arylcarbonylsulfide,
heterocyclylcarbonylsulfide and groups of the formulas:
--S(O).sub.nL.sup.14, --S(O).sub.mOH, --S(O).sub.mOL.sup.14,
--OS(O).sub.mOL.sup.14, and --O(S).sub.mOH, wherein L.sup.14 is
selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
aryl and heterocyclyl.
[0480] In the groups set forth above, m is independently 1 or 2,
and n is independently 0, 1 or 2.
[0481] Certain of the R.sup.4 and R.sup.5 substituents may contain
asymmetric carbon atoms. Compounds containing such substituents may
therefore exist in enantiomeric and diastereomeric forms and in
racemic mixtures thereof. All are within the scope of the present
invention. A racemate or racemic mixture does not imply a 50:50
mixture of stereoisomers.
[0482] In accordance with the description of exemplary R.sup.4 or
R.sup.5 organic moieties, the following terms have the designated
meanings, unless explicitly stated otherwise:
[0483] Alkyl, alkenyl and alkynyl refer to straight or branched
chain hydrocarbons having 1 to 30 carbon atoms (at least two carbon
atoms for an alkynyl group) and no unsaturation, at least one
double bond or at least one triple bond, respectively. Preferred
carbon number ranges are 1 to 20 and 1 to 10.
[0484] Cycloalkyl and cycloalkenyl refer to cyclic hydrocarbon
groups of 3 to 8 carbon atoms, where a cycloalkyl group is
saturated, and a cycloalkenyl group has at least one double bond
within the cyclic structure. Suitable cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl.
[0485] Aryl refers to refers to aromatic groups which have at least
one ring having a conjugated pi electron system and includes
carbocyclic aryl, heterocyclic aryl and biaryl groups.
[0486] Carbocyclic aryl refers to aromatic groups wherein the ring
atoms of the aromatic ring are carbon atoms. Carbocyclic aryl
groups include phenyl, naphthyl and indenyl groups.
[0487] Heterocyclic aryl refers to a mono- or bicyclic ring system
of about 5 to about 12 carbon atoms, where each monocyclic ring may
possess from 0 to about 4 heteroatoms, and each bicyclic ring may
possess about 0 to about 5 heteroatoms selected from N, O, and S
provided said heteroatoms are not vicinal oxygen and/or sulfur
atoms. Examples of such mono- and bicyclic ring systems include,
without limitation, benzofuran, benzothiophene, indole,
benzopyrazole, coumarin, isoquinoline, pyrrole, thiophene, furan,
thiazole, imidazole, pyrazole, triazole, quinoline, pyrimidine,
pyridine, pyridone, pyrazine, pyridazine, isothiazole, isoxazole
and tetrazole.
[0488] Biaryl refers to phenyl substituted by carbocyclic aryl or
heterocyclic aryl as defined herein, ortho, meta or para to the
point of attachment of the phenyl ring.
[0489] Heterocyclyl refers to a stable 5- to 7-membered mono- or
bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring
system any ring of which may be saturated or unsaturated, and which
consists of carbon atoms and from one to three heteroatoms selected
from the group consisting of N, O and S, and wherein the nitrogen
and sulfur heteroatoms may optionally be oxidized, and the nitrogen
heteroatom may optionally be quaternized, and including any
bicyclic group in which any of the above-defined heterocyclic rings
is fused to a benzene ring. The heterocyclic ring may be attached
to the steroid nucleus through any heteroatom or carbon atom of the
heterocyclic ring which results in the creation of a stable
structure. Examples of such heterocyclic groups include
piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxopyrrolodinyl, 2-oxcazepinyl, azepinyl, pyrrolyl,
4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl,
imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl,
pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl,
morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl,
isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl,
benzimidazolyl, thiadiazoyl, benzopyranyl. benzothiazolyl,
benzoxazolyl, furyl, tetrahydrofuryl. tetrahydropyranyl, thienyl,
benzothienyl, thiarnorpholinyl, thiamorpholinyl sulfoxide.
thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as
morpholinyl.
[0490] Heterocyclyloxy and heterocyclylcarbonyl refer to
heterocyclyl groups bonded through an oxygen atom or a carbonyl
group, respectively, to one of the to one or more of the steroid
nucleus, R.sup.4 hydrocarbyl group, L.sup.5 hydrocarbyl group or
L.sup.9 hydrocarbyl group.
[0491] Heterocyclyloxycarbonyl refers to a heterocyclyloxy group
bonded through a carbonyl group to one or more of the steroid
nucleus, R4 hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9
hydrocarbyl group.
[0492] Heterocyclylcarbonyloxy refers to a heterocyclylcarbonyl
group bonded through an oxygen atom to one or more of the steroid
nucleus, R4 hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9
hydrocarbyl group.
[0493] Alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl,
cycloalkylcarbonyl, cycloalkenylcarbonyl and arylcarbonyl refer to
moieties wherein a carbonyl group (C.dbd.O) provides the carbon
atom through which the moiety is joined to one of the steroid
nucleus, R4 hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9
hydrocarbyl group, and an alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl or aryl group, respectively, is also joined to the
carbonyl group.
[0494] Alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl,
cycloalkyloxycarbonyl, cycloalkenyloxycarbonyl and aryloxycarbonyl
refer to moieties wherein a carbonyl group (C.dbd.O) provides the
carbon atom through which the moiety is joined to one of the
steroid nucleus, R4 hydrocarbyl group, Ls hydrocarbyl group or
L.sup.9 hydrocarbyl group, and an alkoxy, alkenyloxy, alkynyloxy,
cycloalkoxy, cycloalkenyloxy or aryloxy group, respectively, is
also joined to the carbonyl group.
[0495] Alkoxy, alkenyloxy, alkynyloxy, cycloalkoxy, cycloalkenyloxy
and aryloxy refer to groups wherein oxygen is bonded to an alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl or aryl group,
respectively, and that oxygen is also bonded to one of the steroid
nucleus, R4 hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9
hydrocarbyl group.
[0496] Alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy,
cycloalkylcarbonyloxy, cycloalkenylcarbonyloxy and arylcarbonyloxy
refer to groups wherein oxygen is bonded to an alkylcarbonyl,
alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,
cycloalkenylcarbonyl and arylcarbonyl group, respectively, and that
oxygen is also bonded to one of the steroid nucleus, R4 hydrocarbyl
group, L.sup.5 hydrocarbyl group or L.sup.9 hydrocarbyl group.
[0497] Alkylsulfide, alkenylsulfide, alkynylsulfide,
cycloalkylsulfide, cycloalkenylsulfide and arylsulfide refer to
groups wherein sulfur is bonded to an alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl or aryl group, respectively, and that
sulfur atom is also bonded to one of the steroid nucleus, R4
hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9 hydrocarbyl
group.
[0498] Alkylcarbonylsulfide, alkenylcarbonylsulfide,
alkynylcarbonylsulfide, cycloalkylcarbonylsulfide,
cycloalkenylcarbonylsulfide and arylcarbonylsulfide refer to groups
wherein a sulfur atom is bonded to a alkylcarbonyl,
alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl,
cycloalkenylcarbonyl or arylcarbonyl group, respectively, and that
sulfur atom is also bonded to one of the steroid nucleus, R4
hydrocarbyl group, L.sup.5 hydrocarbyl group or L.sup.9 hydrocarbyl
group.
[0499] Alkylene refers to a straight chain bridge of 1 to 5 carbon
atoms, which may be substituted with 1 to 3 lower alkyl groups or
fully or partially halogenated lower alkyl groups.
[0500] Alkenylene refers to a straight chain bridge of 2 to 5
carbon atoms having one or two double bonds, which may be
substituted with 1 to 3 lower alkyl groups or fully or partially
halogenated lower alkyl groups.
[0501] Alkynylene refers to a straight chain bridge of 2 to 5
carbon atoms having one or two triple bonds, which may be
substituted with 1 to 3 lower alkyl group or fully or partially
halogenated lower alkyl groups.
[0502] A lower alkyl group refers to C1-C5 alkyl groups, e.g.,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, sec-butyl,
iso-butyl, n-pentyl, iso-pentyl, etc.
[0503] Halogen refers to fluorine, chlorine, bromine and iodine,
and a halogenated group refers to a carbon atom having at least one
halogen bonded thereto.
[0504] Formyl refers to --C(.dbd.O)H; hydroxyl refers to --OH; and
oxo refers to an oxygen atom which forms part of a carbonyl
group.
[0505] A pharmaceutically acceptable salt includes acid addition
salts and base addition salts.
[0506] Acid addition salts refer to those salts formed from steroid
compounds of the invention and inorganic acids such as hydrochloric
acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid
and the like, and/or organic acids such as acetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,
malonic acid, succinic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid
and the like.
[0507] Base addition salts include those salts derived from
steroids of the invention and inorganic bases such as sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
copper, manganese, aluminum salts and the like. Suitable salts
include the ammonium, potassium, sodium, calcium and magnesium
salts derived from pharmaceutically acceptable organic non-toxic
bases include salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, 2-dimethylaminoethanol,
2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,
arginine, histidine, caffeine, procaines, hydrabamine, choline,
betaine, ethylenediamine, glucosamine, methylglucamine,
theobromine, purines, piperazine, piperidine, N-ethylpiperidine,
and the like.
[0508] In another embodiment, the present invention provides
compositions which include a 6,7-dioxygenated steroid compound as
described above in admixture or otherwise in association with one
or more inert carriers, as well as optional ingredients if desired.
A pharmaceutical composition comprising a compound in combination
with a pharmaceutically acceptable carrier or diluent, the compound
having the formula ##STR92## including pharmaceutically acceptable
salts and solvates thereof, wherein:
[0509] each of C5, C6, C7, C8, C9, C10, C13 and C14 is
independently substituted with --X, --R.sup.4 and --OR.sup.1;
[0510] each of C1, C2, C3, C4, C11, C12, C15, C16 and C17 is
independently substituted with a substituent selected from (a) or
(b), wherein
[0511] (a) represents one of: .dbd.O, .dbd.C(R.sup.4)(R.sup.4),
--C(R.sup.4)(R.sup.4)(C(R.sup.4)(R.sup.4)).sub.n-- and
--(O(C(R.sup.4)(R.sup.4)).sub.nO)--, wherein n ranges from 1 to
about 6; and
[0512] (b) represents two of: --X, --R.sup.4 and --OR.sup.1, which
are independently selected at each occurrence;
[0513] the A, B, C and D rings may independently be fully
saturated, partially saturated or fully unsaturated;
[0514] R.sup.1 is H or a protecting group such that --OR.sup.1 is a
protected hydroxyl group, where the C6 and C7 --OR.sup.1 groups may
together form a cyclic structure which protects both hydroxyl
groups;
[0515] R.sup.4 at each occurrence is independently selected from H
and R.sup.5;
[0516] R.sup.5 is a C.sub.1-30 organic moiety that may optionally
contain at least one heteroatom selected from the group consisting
of boron, halogen, nitrogen, oxygen, silicon and sulfur; where two
geminal R.sup.4 groups may together form a ring with the carbon
atom to which they are both bonded; and
[0517] X represents fluoride, chloride, bromide or iodide;
[0518] with the proviso that C15 is not bonded to an oxygen
atom.
[0519] In preferred compositions: C17 is substituted with a
hydrocarbyl group; such as a C.sub.1-C.sub.7 alkyl group; or such
as an olefinic group of the formula .dbd.C(R.sup.4)(R.sup.4), where
preferably R.sup.4 is hydrogen or C.sub.1-C.sub.10 alkyl; in a
preferred embodiment the C17 hydrocarbyl group excludes
--CH(CH.sub.3)(CH.sub.2).sub.3CH(CH.sub.3).sub.2. In other
preferred compositions, C17 is substituted with two atoms
independently selected from hydrogen and halogen atoms; or C17 is
substituted with at least one oxygen atom; or C17 is substituted
with a hydroxyl or protected hydroxyl group; or C17 is substituted
with a carbonyl or protected carbonyl group; or C17 is substituted
with an alkoxy group. In preferred compositions, the substituent at
C17 excludes ##STR93##
[0520] In other preferred composition, C15 is substituted with two
hydrogen atoms; and/or C4 is substituted with hydrogen and one of
--X, --R.sup.5 or --OR.sup.1; and/or C5 is substituted with
hydrogen; and/or C4 is bonded to at least one hydrogen such that
when C4 is bonded to two hydrogen atoms then C3 is not bonded to
either oxygen or to two hydrogen atoms. In other preferred
composition, C4 is bonded to two hydrogen atoms only when C3 is not
bonded to either oxygen or to two hydrogen atoms. In another
preferred composition, C4 is bonded to methyl only when C4 is not
bonded to two methyls or formyl. In other preferred compositions,
the compounds have a hydrogen at C5 in the alpha configuration. In
another preferred composition, the compounds have an --OR.sup.1
group at C6 with the alpha configuration. In another preferred
composition, the compounds have an --OR.sup.1 group at C7 with the
beta configuration. In another preferred composition, the compounds
have an --OR.sup.1 substituent at C6 with the alpha configuration
and an --OR.sup.1 substituent at C7 with the beta configuration. In
other preferred compositions, the compounds have at least one of C3
and C4 bonded to an oxygen atom, and in a preferred embodiment,
both C3 and C4 are bonded to an oxygen atom. In another preferred
composition, C10 of the compound is substituted with a methyl
group; and/or C13 of the compound is substituted with a methyl
group; or both C10 and C13 of the compounds are substituted with
methyl groups. In a preferred composition, both C6 and C7 are
bonded to hydrogen atoms. In another preferred composition, at
least one of C1, C2, C3, C4, C5, C8, C9, C10, C11, C12, C13, C14,
C15, C16 and C17 is substituted exclusively with hydrogen atoms,
and more preferably C1 and C2 are substituted exclusively with
hydrogen atoms; and/or C11 and C12 are substituted exclusively with
hydrogen atoms; and/or C15 and C16 are substituted exclusively with
hydrogen atoms. In a preferred composition, the compounds have a
saturated A ring; and/or a saturated B ring; and/or a saturated C
ring; and/or a saturated D ring. Compositions with compounds having
a saturated A ring are preferred, and compositions with compounds
having fully saturated A,B,C and D rings are more preferred. In
another preferred composition, the A ring of the compound does not
contain a bicyclic structure. In yet another preferred composition,
C3 and C4 of the compound are not both substituted solely with
hydrogen atoms. These compositions may be used for the treatment of
asthma, allergy, inflammation including arthritis, and thrombosis.
These compositions may also be formed into a medicament, which may
used in the treatment of, for example, asthma, allergy,
inflammation including arthritis, and thrombosis.
[0521] These compositions are useful as, for example, assay
standards, convenient means of making bulk shipments, or
pharmaceutical compositions. An assayable amount of a compound of
the invention is an amount which is readily measurable by standard
assay procedures and techniques as are well known and appreciated
by those skilled in the art. Assayable amounts of a compound of the
invention will generally vary from about 0.001 wt % to about 80 wt
% of the entire weight of the composition. Inert carriers include
any material which does not degrade or otherwise covalently react
with a compound of the invention. Examples of suitable inert
carriers are water; aqueous buffers, such as those which are
generally useful in High Performance Liquid Chromatography (HPLC)
analysis; organic solvents, such as acetonitrile, ethyl acetate,
hexane and the like; and pharmaceutically acceptable carriers.
[0522] Thus, the present invention provides a pharmaceutical or
veterinary composition (hereinafter, simply referred to as a
pharmaceutical composition) containing a 6,7-dioxygenated steroid
compound as described above, in admixture with a pharmaceutically
acceptable carrier. The invention further provides a pharmaceutical
composition containing an effective amount of a 6,7-dioxygenated
steroid compound as described above, in association with a
pharmaceutically acceptable carrier.
[0523] The pharmaceutical compositions of the present invention may
be in any form which allows for the composition to be administered
to a patient. For example, the composition may be in the form of a
solid, liquid or gas (aerosol). Typical routes of administration
include, without limitation, oral, topical, parenteral, sublingual,
rectal, vaginal, and intranasal. The term parenteral as used herein
includes subcutaneous injections, intravenous, intramuscular,
intrasternal injection or infusion techniques. Pharmaceutical
composition of the invention are formulated so as to allow the
active ingredients contained therein to be bioavailable upon
administration of the composition to a patient. Compositions that
will be administered to a patient take the form of one or more
dosage units, where for example, a tablet may be a single dosage
unit, and a container of steroid in aerosol form may hold a
plurality of dosage units.
[0524] Materials used in preparing the pharmaceutical compositions
should be pharmaceutically pure and non-toxic in the amounts used.
It will be evident to those of ordinary skill in the art that the
optimal dosage of the active ingredient(s) in the pharmaceutical
composition will depend on a variety of factors. Relevant factors
include, without limitation, the type of subject (e.g., human), the
particular form of the active ingredient, the manner of
administration and the composition employed.
[0525] In general, the pharmaceutical composition includes an
active 6,7-dioxygenated steroid compounds as described herein, in
admixture with one or more carriers. The carrier(s) may be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) may be liquid, with the compositions
being, for example, an oral syrup or injectable liquid. In
addition, the carrier(s) may be gaseous, so as to provide an
aerosol composition useful in, e.g., inhalatory administration.
[0526] When intended for oral administration, the composition is
preferably in either solid or liquid form, where semi-solid,
semi-liquid, suspension and gel forms are included within the forms
considered herein as either solid or liquid.
[0527] As a solid composition for oral administration, the
composition may be formulated into a powder, granule, compressed
tablet, pill, capsule, chewing gum, wafer or the like form. Such a
solid composition will typically contain one or more inert diluents
or edible carriers. In addition, one or more of the following
adjuvants may be present: binders such as carboxymethylcellulose,
ethyl cellulose, microcrystalline cellulose, or gelatin; excipients
such as starch, lactose or dextrins, disintegrating agents such as
alginic acid, sodium alginate, Primogel, corn starch and the like;
lubricants such as magnesium stearate or Sterotex; glidants such as
colloidal silicon dioxide; sweetening agents such as sucrose or
saccharin, a flavoring agent such as peppermint, methyl salicylate
or orange flavoring, and a coloring agent.
[0528] When the composition is in the form of a capsule, e.g., a
gelatin capsule, it may contain, in addition to materials of the
above type, a liquid carrier such as polyethylene glycol or a fatty
oil.
[0529] The composition may be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid may be
for oral administration or for delivery by injection, as two
examples. When intended for oral administration, preferred
composition contain, in addition to the present compounds, one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition intended to be administered by
injection, one or more of a surfactant, preservative, wetting
agent, dispersing agent, suspending agent, buffer, stabilizer and
isotonic agent may be included.
[0530] The liquid pharmaceutical compositions of the invention,
whether they be solutions, suspensions or other like form, may
include one or more of the following adjuvants: sterile diluents
such as water for injection, saline solution, preferably
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or digylcerides which may serve
as the solvent or suspending medium, polyethylene glycols,
glycerin, propylene glycol or other solvents; antibacterial agents
such as benzyl alcohol or methyl paraben; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic. Physiological saline is a preferred
adjuvant. An injectable pharmaceutical composition is preferably
sterile.
[0531] A liquid composition intended for either parenteral or oral
administration should contain an amount of the inventive compound
such that a suitable dosage will be obtained. Typically, this
amount is at least 0.01% of a compound of the invention in the
composition. When intended for oral administration, this amount may
be varied to be between 0.1 and about 70% of the weight of the
composition. Preferred oral compositions contain between about 4%
and about 50% of the active steroid compound. Preferred
compositions and preparations according to the present invention
are prepared so that a parenteral dosage unit contains between 0.01
to 1% by weight of active compound.
[0532] The pharmaceutical composition may be intended for topical
administration, in which case the carrier may suitably comprise a
solution, emulsion, ointment or gel base. The base, for example,
may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers. Thickening agents may
be present in a pharmaceutical composition for topical
administration. If intended for transdermal administration, the
composition may include a transdermal patch or iontophoresis
device. Topical formulations may contain a concentration of the
inventive compound of from about 0.1 to about 10% w/v (weight per
unit volume).
[0533] The composition may be intended for rectal administration,
in the form, e.g., of a suppository which will melt in the rectum
and release the drug. The composition for rectal administration may
contain an oleaginous base as a suitable nonirritating excipient.
Such bases include, without limitation, lanolin, cocoa butter and
polyethylene glycol.
[0534] The composition may include various materials which modify
the physical form of a solid or liquid dosage unit. For example,
the composition may include materials that form a coating shell
around the active ingredients. The materials which form the coating
shell are typically inert, and may be selected from, for example,
sugar, shellac, and other enteric coating agents. Alternatively,
the active ingredients may be encased in a gelatin capsule.
[0535] The composition in solid or liquid form may include an agent
which binds to the active steroid component(s) and thereby assists
in the delivery of the active components. Suitable agents which may
act in this capacity include a monoclonal or polyclonal antibody, a
protein or a liposome.
[0536] The pharmaceutical composition of the present invention may
consist of gaseous dosage units, e.g., it may be in the form of an
aerosol. The term aerosol is used to denote a variety of systems
ranging from those of colloidal nature to systems consisting of
pressurized packages. Delivery may be by a liquefied or compressed
gas or by a suitable pump system which dispenses the active
ingredients. Aerosols of compounds of the invention may be
delivered in single phase, bi-phasic, or tri-phasic systems in
order to deliver the active ingredient(s). Delivery of the aerosol
includes the necessary container, activators, valves,
subcontainers, spacers and the like, which together may form a kit.
Preferred aerosols may be determined by one skilled in the art,
without undue experimentation.
[0537] Whether in solid, liquid or gaseous form, the pharmaceutical
composition of the present invention may contain one or more known
pharmacological agents used in the treatment of asthma, allergy,
inflammation (including arthritis) or thrombosis.
[0538] The pharmaceutical compositions may be prepared by
methodology well known in the pharmaceutical art. Various steroid
compounds are, and have been widely used as active ingredients in
pharmaceutical composition intended for therapeutic use, and
accordingly one of ordinary skill in the art is familiar with
preparing such compositions. The steroid compounds of the present
invention may be formulated into pharmaceutical compositions in a
like manner.
[0539] A composition intended to be administered by injection can
be prepared by combining the 6,7-dioxygenated steroid with water so
as to form a solution. A surfactant may be added to facilitate the
formation of a homogeneous solution or suspension. Surfactants are
compounds that non-covalently interact with the steroid so as to
facilitate dissolution or homogeneous suspension of the steroid in
the aqueous delivery system.
[0540] The compounds and compositions described above have utility
in treating allergy and asthma, arthritis and/or thrombosis. The
compounds and composition described above may also be used to treat
a condition associated with an elevated level of NF.kappa.B,
wherein a subject in need thereof is administered an amount of the
compound (or composition containing the compound) effective to
lower the NF.kappa.B activity. As used herein, "treating allergy
and asthma, arthritis and/or thrombosis" refers to both therapy for
allergy and asthma, arthritis and thrombosis, and for the
prevention of the development of the allergic response,
bronchoconstriction, inflammation and the formation of blood clots
that cause thrombosis and associated diseases. As also used herein,
NF-kB activity refers to any increase or decrease in the
transcriptional activity of genes that is attributable to, directly
or indirectly, the binding of any members of the NF-kB family of
proteins to all DNA sequences recognized by this family of
proteins.
[0541] An effective amount of a compound or composition of the
present invention is used to treat allergy, asthma, arthritis or
thrombosis in a warm-blooded animal, such as a human. Methods of
administering effective amounts of anti-allergy, anti-asthma,
anti-arthritis and anti-thrombotic agents are well known in the art
and include the administration of inhalation, oral or parenteral
forms. Such dosage forms include, but are not limited to,
parenteral solutions, tablets, capsules, sustained release implants
and transdermal delivery systems; or inhalation dosage systems
employing dry powder inhalers or pressurized multi-dose inhalation
devices. Generally, oral or intravenous administration is preferred
for the treatment of arthritis and thrombosis, while oral or
inhalation/intranasal are preferred for asthma and allergy. The
dosage amount and frequency are selected to create an effective
level of the agent without harmful effects. It will generally range
from a dosage of about 0.1 to 100 mg/kg/day, and typically from
about 0.1 to 10 mg/Kg/day where administered orally or
intravenously, for anti-allergy, anti-asthma, anti-arthritis or
anti-thrombotic effects. Also, the dosage range will be typically
from about 0.01 to 1 mg/Kg/day where administered intranasally or
by inhalation for anti-asthma and anti-allergy effects.
[0542] Administration of compounds or compositions of the present
invention may be carried out in combination with the administration
of other agents. For example, it may be desired to administer a
bronchodilator or a glucocorticoid agent for effects on asthma, a
glucocorticoid for effects on arthritis, or an anti-histamine for
effects on allergy. Non-steroid compounds may be co-administered
with the steroids of the present invention, and/or non-steroid
compounds may used in combination with the steroid compounds of the
invention to provide a therapy for one or more of asthma,
allergies, arthritis and thrombosis.
[0543] The following examples are offered by way of illustration
and not by way of limitation.
[0544] Unless otherwise stated, flash chromatography and column
chromatography may be accomplished using Merck silica gel 60
(230-400 mesh). Flash chromatography may be carried out according
to the procedure set forth in: "Purification of Laboratory
Chemicals", 3rd. edition, Butterworth-Heinemann Ltd., Oxford
(1988), Eds. D. D. Perrin and W. L. F. Armarego, page 23. Column
chromatography refers to the process whereby the flow rate of
eluent through a packing material is determined by gravity. In all
cases flash chromatography and radial chromatography may be used
interchangeably. Radial chromatography is performed using silica
gel on a Chromatotron Model # 7924T (Harrison Research, Palo Alto,
Calif.).
[0545] A typical work-up procedure for a reaction mixture involves
dilution of the reaction mixture with an organic solvent (ethyl
acetate or diethyl ether) and washing of the organic mixture with
saturated sodium bicarbonate followed by saturated sodium chloride.
The organic layer is then dried over MgSO.sub.4, the mixture is
filtered and the filtrate evaporated to dryness in vacuo to yield
the crude product which may or may not require further
purification.
[0546] A typical work-up procedure for a Wittig reaction involves
first quenching by the dropwise addition of water. The mixture is
then diluted with ethyl acetate and washed with saturated sodium
bicarbonate and then sodium chloride. The organic layer is dried
over magnesium sulphate, filtered and evaporated to dryness.
[0547] A typical work-up procedure for a hydroboration reaction
involves pouring the reaction mixture into saturated sodium
chloride solution (200 ml) followed by extraction of the aqueous
slurry with methylene chloride and then washing the combined
organic layers with aqueous 25% sodium thiosulphate solution. The
organic layer is then dried over magnesium sulphate, filtered and
evaporated to dryness.
[0548] Reactions may typically be monitored with thin layer
chromatography (TLC) using Silica gel 60 F.sub.254 plates (EM
Science, Gibbstown, N.J.) and an appropriate solvent system. Thin
layer chromatography may be carried out according to the procedure
set forth in: "Purification of Laboratory Chemicals", 3rd. edition,
Butterworth-Heinemann Ltd., Oxford (1988), Eds. D. D. Perrin and W.
L. F. Arnarego, page 30. After elution is complete, the TLC plate
is dried, lightly sprayed with a 10% solution of H.sub.2SO.sub.4 in
ethanol and then heated until the spots corresponding to the
compounds appear. Unless otherwise stated, filtrations are carried
out using a Whatman (type 1) filter paper.
EXAMPLES
Section 1
Synthesis of 3,4,6,7-polyhydroxylated Steroids
[0549] Steroids with the same or closely related ring-structure
hydroxylation pattern as compound 237 (shown by the structure
below) can be synthesized starting from a number of steroid
precursors including 4-androsten-3,17-dione (1) and others with C3
oxygen functionalities and .DELTA..sup.5 carbon-carbon double bonds
such as dehydroisoandrosterone (247). ##STR94## 237
(3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.) where
R.sup.1.dbd.R.sup.3.dbd.R.sup.4.dbd.H, and
R.sup.2.dbd.R.sup.5.dbd.OH
[0550] For example, after protection of the ketone functionalities
of androsten-3,17-dione (1) (Example 1, Scheme 55) with any one of
a number of appropriate carbonyl protecting groups and concomitant
migration of the carbon-carbon double bond, allylic oxidation
introduces a C7 oxygen. A number of oxidizing agents and
experimental conditions can be used for this oxidation step
including but not limited to chromium trioxide/3,5-dimethylpyrazole
complex, pyridinium chlorochromate (PCC), pyridinium dichromate
(PDC), and tBuOOH with RuCl.sub.3. Reduction of the resultant
ketone with an appropriate reducing agent gives the hydroxyl
functionality at C7. Several metal hydride reducing agents can be
used for this task including sodium borohydride and lithium
aluminum hydride. Generally, reduction of the C7 carbonyl produces
the .beta.OH configuration by hydride attack from the least
hindered face of the steroid.
[0551] Introduction of the C6 oxygen can be achieved, after
protection of the C7 hydroxyl with an appropriate protecting group,
by methods such as hydroboration/oxidation or epoxidation followed
by ring opening. The .DELTA..sup.5 carbon-carbon double bond can be
epoxidized with any of a number of peracids including
m-chloroperbenzoic acid, trifluoroperacetic acid or
3,5-dinitroperoxybenzoic acid. Generally, the epoxide introduced
has the .alpha.-configuration arising from attack on the least
hindered face of the steroid ring structure. Subsequent ring
opening of the epoxide can be accomplished under acidic conditions
such as 80% aqueous acetic acid at 60.degree. C. This produces an
allylic alcohol at the C6 position with the .alpha.-configuration.
Alternatively, hydroboration of the .DELTA..sup.5 double bond with
an appropriate borane complex followed by oxidation using reagents
such as basic hydrogen peroxide will also introduce an hydroxyl
group in the .alpha.-configuration at C6.
[0552] Hydroxyl groups can be introduced at the C3 and C4 positions
starting from the A-ring 4-ene-3-one functionalization pattern.
Reduction of the .alpha.,.beta.-unsaturated ketone can be
accomplished using lithium dissolved in liquid ammonia. The
resultant enolate can be trapped with an electrophile such as
trimethylsilylchloride or diethylchlorophosphate.
Hydroboration-oxidation of the silyl enol ether results in the
introduction of an oxygen at C4. This method generates the 3.beta.,
4.alpha. hydroxylation pattern. Alternatively, a second reduction
using lithium in liquid ammonia on the enol phosphate produces the
.DELTA..sup.3 carbon-carbon double bond. Epoxidation of the
.DELTA..sup.3 double bond with a peracid such as m-chloroperbenzoic
acid produces the .alpha.-epoxide. Ring opening of this epoxide
could be achieved using a number of acidic or basic conditions. For
example, treatment of the 3.alpha., 4.alpha.-epoxy functionality
with glacial acetic acid in compound 238 (Example 3, Scheme 61)
produces the 3.alpha.-hydroxy, 4.beta.-acetoxy pattern. Removal of
the acetate group using any of a number of reagents including
potassium carbonate (or sodium methoxide) in methanol gives the
3.alpha.,4.beta.-hydroxylation pattern.
Example 1
The Steroid
3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androsta-
ne (237) can be Synthesized According to the Reaction Sequence
Illustrated by Scheme 55
[0553] ##STR95## ##STR96## ##STR97## Key: (i) p-TsOH,
(CH.sub.2OH).sub.2, benzene (ii) CrO.sub.3, 3,5-dimethylpyrazole,
CH.sub.2Cl.sub.2 (iii) NaBH.sub.4, CeCl.sub.3, THF-MeOH (iv)
TBDMSCl, imidazole, DMF (v) m-CPBA, CH.sub.2Cl.sub.2 (vi) 80% AcOH
(vii) TBAF, THF (viii) 2,2-dimethoxypropane, CSA, DMF (ix)
NaBH.sub.4, MeOH (x) TBDMSC1, imidazole, DMF (xi) 1.
LiBNH.sub.3-THF, 2. TMSCl, Et.sub.3N (xii) 1. BH.sub.3-THF complex,
2.30% NaOH, 30% H.sub.2O.sub.2, THF (xiii) TBAF, THF (xiv) p-TsOH,
H2O, THF.
[0554] Commercially available 4-androsten-3,17-dione (1) (20.0 g,
62.8 mmol) is stirred with ethylene glycol (10 mL) and a catalytic
amount of p-toluenesulfonic acid (1.0 g, 5.2 mmol) in benzene (500
mL) at reflux under nitrogen for 26 hours (Scheme 56). The water
generated by the reaction is removed during this time using a
Dean-Stark apparatus. The mixture is then cooled to room
temperature and Et.sub.2O is added. The mixture is washed with
sodium bicarbonate, then water and dried over magnesium sulfate.
Filtration and concentration gives a pale yellow solid that is
washed with methanol to give the diketal 2 as a white powder (14.6
g, 39.0 mmol, 62%). A monoketal byproduct (5.13 g, 15.0 mmol) is
recovered from the filtrate and recycled. The overall yield,
accounting for the byproduct, is 86% of diketal 2.
[0555] Allylic oxidation at the C7 position of the diketal 2, using
a chromium trioxide-3,5-dimethylpyrazole complex in
dichloromethane, affords compound 3 (Scheme 56). Chromium trioxide
(46.7 g; 467 mmol) and dry dichloromethane (450 mL) are added to a
flask under nitrogen, and then cooled to -20.degree. C. using a
dry-ice/CaCl.sub.2 solution. 3,5-Dimethylpyrazole (44.9 g, 467
mmol) is added and the mixture is stirred at -20 to -30.degree. C.
for 1.5 hours. This is followed by the addition of diketal 2 (7.00
g, 18.7 mmol) and continued stirring for 7 hours at -20.degree. C.
The reaction is quenched with water and filtered. The filtrate is
washed with water, the volume reduced to 200 mL and then dried over
MgSO.sub.4. Filtration and concentration gives a dark brown oil
that is purified using silica gel column chromatography
(CH.sub.2Cl.sub.2/EtOAc) to give the enone 3 in 68% yield (4.95 g,
12.8 mmol).
[0556] Reduction of enone 3 to the allylic alcohol 4 (Scheme 56) is
carried out using sodium borohydride and cerium (III) chloride
heptahydrate in THF and methanol. Freshly distilled THF (200 mL)
and methanol (50 mL) are added to a flask containing the enone 3
(15.3 g, 39.4 mmol) and CeCl3-7H.sub.2O (16.0 g, 42.9 mmol) under
nitrogen. NaBH.sub.4 (3.20 g, 84.6 mmol) is added in portions, and
stirring is continued for 1 hour at room temperature.
Dichloromethane is added, the mixture is washed with NaOH (0.6 N)
then water and the organic layer is dried over MgSO.sub.4.
Filtration and concentration yields compound 4 as a white solid
(14.1 g, 36.1 mmol, 92%). ##STR98##
[0557] The C-7 hydroxyl moiety of alcohol 4 is then protected as a
silyl ether (Scheme 57). Compound 4 (14.1 g, 36.1 mmol) is
dissolved in dry DMF (50 mL) then imidazole (5.9 g, 86.7 mmol) and
TBDMSCl (6.7 g, 44.5 mmol) are added, and the mixture is stirred
under nitrogen for 5 hours. Dichloromethane is added, the mixture
is washed with water, and the organic layer is dried over
MgSO.sub.4. Filtration and concentration gives a light yellow solid
that is recrystallized from methanol to give compound 5 as a white
solid in 61% yield (11.2 g, 22.2 mmol).
[0558] The subsequent epoxidation of compound 5 (Scheme 57) is
carried out using meta-chloroperbenzoic acid (m-CPBA). Compound 5
(0.72 g, 1.4 mmol) is dissolved in dry dichloromethane (5 mL),
m-CPBA (0.50 g, 2.9 mmol) is added and the mixture is stirred
vigorously for 20 minutes. Saturated sodium bicarbonate is added
and the aqueous slurry is extracted with dichloromethane. The
combined organic extracts are washed sequentially with sodium
carbonate solution, water, 10% sodium thiosulfate, then again with
water. Drying (MgSO.sub.4), filtration and concentration gives a
white solid that is purified using flash chromatography to yield
compound 6 in 77% yield (0.57 g, 1.1 mmol).
[0559] Compound 6 is treated with acid to deprotect the C3 and C17
ketones and to open the epoxide moiety to yield the
6-hydroxy-7-silyloxy compound 310 (Scheme 57). Aqueous acetic acid
(80%, 1 mL) is added to a flask containing compound 6. The mixture
is heated at 65.degree. C. for 5 hours, cooled and poured onto
dichloromethane. The mixture is washed with sodium bicarbonate and
dried over MgSO.sub.4. After filtration and concentration, the
resultant crude product 310 is used in the next step without
further purification.
[0560] Compound 310 (2.30 mg, 5.32 mmol) in THF (10 mL) is treated
with tetrabutylammonium fluoride (TBAF) (8 mL, 1 M solution in THF)
at room temperature under nitrogen for ten minutes in order to
remove the silyl protecting group (Scheme 57). The reaction mixture
is concentrated and then purified by flash chromatography (3:1
CH.sub.2Cl.sub.2/EtOAc) to give compound 7 (1.37 mg, 4.31 mmol) in
81% yield. ##STR99##
[0561] Protection of the 6,7-diol of compound 7 is accomplished by
treatment with 2,2-dimethoxypropane and a catalytic amount of
(1S)-(+)-10-camphorsulfonic acid (CSA) to produce acetonide 8
(Scheme 58). Compound 7 (1 g, 3.14 mmol) and a catalytic amount of
CSA are dissolved in dry DMF (2 mL) and 2,2-dimethoxypropane (10
mL). The mixture is heated at 100.degree. C. for 0.5 hours.
Dichloromethane is added and the mixture is washed with water. The
organic layer is dried over MgSO.sub.4, filtered and concentrated
to yield compound 8 (1.10 g, 3.07 mmol, 98%) which is used directly
in the next reaction without further purification.
[0562] Chemoselective reduction of the C-17 carbonyl moiety (Scheme
58), followed by protection of the resultant alcohol as a silyl
ether is necessary. Compound 8 (83 mg, 0.23 mmol) is dissolved in
methanol (250 mL) under nitrogen and NaBH.sub.4 (15 mg) is added in
portions over a period of 1.5 hours. After an additional 30
minutes, the reaction is quenched with acetic acid, then
neutralized with NaHCO.sub.3. The methanol is evaporated, and the
residue taken up in dichloromethane. The mixture is washed with
water and dried over MgSO.sub.4. Flash chromatography (2:1
CH.sub.2Cl.sub.2/EtOAc) gives compound 9 (72 mg, 0.20 mmol, 87%) as
the major product.
[0563] Protection of the C17 hydroxyl group as a silyl ether to
produce compound 10 is followed by reduction of the
.alpha.,.beta.-unsaturated ketone in the A-ring using lithium in
liquid ammonia-THF, with trapping of the enolate by trimethylsilyl
chloride (Scheme 58). A solution of compound 10 (75.2 mg, 0.158
mmol) in t-BuOH (0.020 mL) and THF (1.5 mL) is transferred to a
flask containing lithium metal in dry, distilled ammonia (11.4 mg
in 10 mL) at -78.degree. C. After 20 minutes at -78.degree. C.,
isoprene (0.5 mL) is added to destroy the excess lithium. The
mixture is then warmed to room temperature and the solvent is
evaporated in vacuo. The residue is dissolved in THF (5 mL), cooled
to -78.degree. C. and Et.sub.3N (1.1 mL, 0.30 mmol) and TMSCl (0.80
mL, 0.30 mmol) are added. The cooling bath is removed and the
mixture is stirred for 15 minutes. Saturated NaHCO.sub.3 is added
and this aqueous layer is extracted with Et.sub.2O and
dichloromethane. The combined organic layers are washed twice with
brine, dried over MgSO.sub.4 and concentrated. The crude product is
purified by radial chromatography to give compound 235 in 67% yield
(58.5 mg, 0.10 mmol).
[0564] The C4 hydroxyl is introduced by hydroboration-oxidation of
the silyl enol ether 235 (Scheme 58). Compound 235 (58.5 mg, 0.106
mmol) is dissolved in dry THF (15 mL) and cooled in an ice-bath.
Borane (1.0M THF complex: 0.32 mL, 0.32 mmol) is added and the
mixture is warmed to room temperature and stirred for 45 minutes.
More BH.sub.3-THF complex (0.16 mL) is added and stirring is
continued for 2 hours. The mixture is then cooled in an ice-bath
and 15% NaOH (0.5 mL) and 30% H.sub.2O.sub.2 (0.5 mL) are added.
Vigorous stirring is continued for 2 hours. The aqueous layer is
then extracted with dichloromethane, then Et.sub.2O, and the
combined organic extracts are washed with 10% aqueous
Na.sub.2S.sub.2O.sub.3, then brine and dried over MgSO.sub.4. The
crude product is purified using radial chromatography to yield
compound 236 (34.0 mg, 0.0688 mmol, 65%) and the corresponding
3.beta.-silyl ether (11.8 mg, 0.0208 mmol, 20%).
[0565] Two step deprotection of compound 236 using TBAF in THF then
aqueous acidic-THF, gives
3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androsta-
ne (237). ##STR100## ##STR101##
Example 2
The Steroid
3.alpha.,4.alpha.-epoxy-6.alpha.,7.beta.,17.beta.-trihydroxy-5.alpha.-and-
rostane (239) can be Synthesized According to the Reaction Sequence
Shown in Scheme 59
[0566] ##STR102## ##STR103## ##STR104##
[0567]
3.alpha.,4.alpha.-Epoxy-6.alpha.,7.beta.,17.beta.-trihydroxy-5.alp-
ha.-androstane (239) can be produced from intermediate 10 in the
synthesis of
3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-andro-
stane (258) (Scheme 60). A solution of compound 10 (111 mg, 0.234
mmol) in THF (4 mL) is transferred to a flask containing lithium
metal in liquid ammonia (6.4 mg in 10 mL) at -78.degree. C. under
argon. After 30 minutes at -78.degree. C., isoprene (0.5 mL) is
added to destroy the excess lithium. The mixture is warmed to room
temperature and the solvent is evaporated in vacuo. The residue is
dissolved in THF (5 mL) and cooled to -78.degree. C., then
ClP(O)(OEt).sub.2 (0.044 mL, 0.30 mmol) is added and the mixture is
stirred for 1 hour. Water and dichloromethane are added, the
mixture acidified, and the aqueous layer extracted with
dichloromethane and Et.sub.2O. The combined organic layers are
washed with water and dried over MgSO.sub.4. The crude product is
purified by radial chromatography to give compound 11(85.1 mg,
0.139 mmol) in 59% yield.
[0568] Reduction of compound 11 with lithium in liquid ammonia in
the presence of t-butyl alcohol produces the olefin 128. A solution
of compound 11 (85.1 mg, 0.139 mmol) and t-BuOH (0.05 mL) in THF (6
mL) is transferred to a flask containing lithium metal (16 mg) in
liquid ammonia at -30.degree. C. under argon. After 30 minutes, the
ammonia is allowed to evaporate, then water is added. After
acidification with aqueous HCl, the mixture is extracted with
Et.sub.2O, then EtOAc and the combined organic layers are washed
with water and dried over MgSO.sub.4. The crude product is purified
by radial chromatography to give compound 128 (50.3 mg, 0.109 mmol)
in 78% yield.
[0569] Epoxidation of 128 with meta-chloroperbenzoic acid (m-CPBA)
in dichloromethane gives the epoxide 238. Compound 128 (50.3 mg,
0.109 mmol) is dissolved in dry dichloromethane (1.5 mL) and m-CPBA
(43.0 mg) is added. The mixture is stirred at room temperature for
1.5 hours and then transferred to a separatory funnel and washed
with 10% Na.sub.2S.sub.2O.sub.3, saturated NaHCO.sub.3 and water
and dried over MgSO.sub.4. Filtration and evaporation of the
filtrate gives compound 238 in 78% yield (52.0 mg, 0.109 mmol)
which is used in the next step without further purification.
[0570] Two step deprotection of compound 238 with TBAF in refluxing
THF and then acidic aqueous THF gives compound 239. ##STR105##
Example 3
The Steroid
3.alpha.,4.beta.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androsta-
ne (241) can be Synthesized According to the Following Reaction
Sequence (Scheme 61)
[0571] ##STR106## ##STR107## ##STR108##
[0572] The synthesis of
3.alpha.,4.beta.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androsta-
ne (241) is carried out to the intermediate 238 using the same
conditions as in the synthesis of
3.alpha.,4.alpha.-epoxy-6.alpha.,7.beta.,17.beta.-trihydroxy-5.alpha.-and-
rostane (239). The subsequent epoxide ring opening with concurrent
removal of the acetonide functionality (Scheme 62) is achieved by
heating with glacial acetic acid to yield compound 146, which
contains an acetoxy functionality at C4. To a flask containing
compound 238 (18.5 mg, 0.038 mmol) is added acetic acid (0.30 mL).
The mixture is stirred with heating at 60.degree. C. for 24 hours,
then at room temperature for 2 days. The acetic acid is removed in
vacuo to give compound 146 in 93% yield (18 mg, 0.036 mmol).
[0573] Deprotection of the C4 hydroxyl using K.sub.2CO.sub.3 in
refluxing methanol, and deprotection of the C17 hydroxyl using TBAF
yields the pentahydroxy compound 241. ##STR109##
Example 4
3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androstan-
e 6,7-acetonide (246)
[0574] The steroid
3.beta.,4.alpha.,6.alpha.,7.beta.,17.beta.-pentahydroxy-5.alpha.-androsta-
ne 6,7-acetonide (246) can be synthesized according to the reaction
sequence illustrated by Scheme 63. ##STR110## ##STR111##
[0575] Compound 10 is prepared as described in Section 1, Example
1. Deprotection of the 6,7 and 17 hydroxyl moieties is achieved
with 80% acetic acid solution and, after appropriate work-up,
reprotection of these groups as the silyl ether derivative is
accomplished to afford 242. Compound 10 is dissolved in 80% acetic
acid and the mixture is concentrated in vacuo after stirring for 8
hours at room temperature. The residue is placed in dry DMF
containing imidazole and TBDMSCl and stirred for 20 hours at room
temperature under a nitrogen atmosphere. Ether is added and the
mixture washed with 5% HCl aqueous solution, saturated NaHCO.sub.3
solution and saturated NaCl solution. The organic mixture is dried
over MgSO.sub.4, filtered and evaporated. Purification of this
residue by chromatography over silica gel gives compound 242.
[0576] Reduction of the .alpha.,.beta.-unsaturated ketone in the
A-ring is achieved using lithium in liquid ammonia-THF, with
trapping of the enolate by trimethylsilyl chloride. A solution of
compound 242 in a 1:4 mixture of t-BuOH and THF is transferred to a
flask containing lithium metal in dry, distilled ammonia at
-78.degree. C. After 20 minutes at -78.degree. C., isoprene is
added to destroy the excess lithium. The mixture is then warmed to
room temperature and the solvent is evaporated in vacuo. The
residue is dissolved in THF, cooled to -78.degree. C. and Et.sub.3N
and TMSCl are added. The cooling bath is removed and the mixture is
stirred for 15 minutes. Saturated NaHCO.sub.3 is added and this
aqueous layer is extracted with Et.sub.2O and dichloromethane. The
combined organic layers are washed twice with brine, dried over
MgSO.sub.4 and concentrated. The crude product is purified by
radial chromatography to give compound 243.
[0577] The C4 hydroxyl is introduced by hydroboration-oxidation of
the silyl enol ether 243. Compound 243 is dissolved in dry THF and
cooled in an ice-bath. Borane (1.0 M THF complex) is added and the
mixture is warmed to room temperature and stirred for 45 minutes.
More BH.sub.3-THF complex is added and stirring is continued for 2
hours. The mixture is then cooled in an ice-bath and 30% NaOH and
30% H.sub.2O.sub.2 are added. Vigorous stirring is continued for 12
hours. The aqueous layer is then extracted with dichloromethane,
then Et.sub.2O, and the combined organic extracts are washed with
10% aqueous Na.sub.2S.sub.2O.sub.3, then brine and dried over
MgSO.sub.4. The crude product is purified using radial
chromatography to yield compound 244.
[0578] Protection of the 3,4-diol of compound 244 is accomplished
by treatment with 2,2-dimethoxypropane and a catalytic amount of
(1S)-(+)-10-camphorsulfonic acid (CSA) to produce acetonide 245.
Compound 244 and a catalytic amount of CSA are dissolved in dry DMF
and 2,2-dimethoxypropane. The mixture is heated at 100.degree. C.
for 0.5 hours. Dichloromethane is added and the mixture is washed
with saturated NaHCO.sub.3 solution. The organic layer is dried
over MgSO.sub.4, filtered and concentrated to yield compound 245
which is used directly in the next reaction without further
purification.
[0579] Compound 245 is then converted to the triol 246 with TBAF.
Thus, the trisilyl ether 245 is dissolved in THF and treated with
tetrabutylammonium fluoride (TBAF) (1 M solution in THF) at room
temperature under nitrogen for 5 hours. The reaction mixture is
poured into CH.sub.2Cl.sub.2 and washed with brine, dried
(MgSO.sub.4) and concentrated in vacuo. The residue is then
purified by flash chromatography (3:1 CH.sub.2Cl.sub.2/EtOAc) to
give compound 246.
Section 2
The Synthesis of 22,29-epoxy-15-one Steroids
[0580] Steroids that are related to
22,29-epoxy-3,4,6,7,29-pentahydroxy-140-stigmastan-15-one (165) by
the presence of a C15 ketone and a cyclic hemiacetal functionality
in the steroid side chain (i.e., a C22 hydroxyl functionality
condensing with a C29 aldehyde functionality to form a
tetrahydropyran ring and a C29 hydroxyl group) can be synthesized
by a number of methods. The key steps include the introduction of
the C15 oxygen, the synthesis of the appropriate side chain
(described in Section 3), and the coupling of the side chain. A
number of commercially available steroids containing either a
ketone or an acetyl moiety at C17 (e.g., pregnenolone (C17 acetyl),
4-androsten-3,17-dione (C17 ketone), dehydroisoandrosterone (C17
ketone) can be used as starting materials. ##STR112##
[0581] In one method (Example 5, Scheme 64), the C17 ketone is
treated with the Wittig reagent prepared by the reaction of
ethyltriphenylphosphonium bromide and potassium t-butoxide in THF
in order to introduce a (Z)-17(20) ethylidene moiety. Reaction of
the Wittig product with an aldehyde-containing compound such as
5-acetoxy-3-(1'-methylethyl)-pentanal (156, Section 3, Example 8,
Scheme 71) in the presence of a Lewis acid gives products that
contain the required C22 oxygen, present as an hydroxyl
functionality, and the C29 oxygen protected as the acetate. The
latter ene reaction gives both stereoisomers at C22, the ratios of
which are dependent on reaction conditions and choice of the
aldehyde starting material. Therefore, the four possible
diastereomers arising from the configurations at C22 and C24 can be
synthesized by utilizing either the 3R or 3S isomer of compound
156.
[0582] The ene reaction described above results in a .DELTA..sup.16
carbon-carbon double bond which can be utilized to introduce a C15
oxygen via allylic oxidation. For example, after protection of the
C22 hydroxyl group of the ene product with an appropriate
protecting group such as t-butyldimethylsilyl, allylic oxidation,
using a reagent such as chromium trioxide/3,5-dimethylpyrazole
complex, introduces a ketone functionality at the C15 position.
Reduction of the .DELTA..sup.16 double-bond produces a steroidal
compound that has the identical D-ring functionality as
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(165).
[0583] Removal of the C29 protecting group, typically an acetate
moiety, followed by oxidation of the resultant primary alcohol
produces the required aldehyde at C29. Removal of the C22
protecting group, generally a t-butyldimethylsilyl group, results
in the formation of the side chain hemiacetal moiety found in
compound 165.
[0584] The second strategy for the attachment of the required
hemiacetal side chain and C15 ketone functionality involves
introduction of the C15 oxygen before side chain coupling (Example
6, Scheme 67). In this method the first step involves conversion of
the C17 ketone functionality of a steroidal intermediate to the
enol acetate by treatment with isopropenyl acetate and
p-toluenesulfonic acid. Conversion of the enol acetate to the
.alpha.,.beta.-unsaturated ketone is accomplished by treatment with
palladium acetate and tributyltin methoxide. ##STR113##
[0585] Introduction of the C15 oxygen is then accomplished using a
Michael-type addition to the enone by an alkoxide derived from
p-methoxybenzyl alcohol in the presence of potassium hydroxide.
This functionality can later be selectively deprotected and the
resultant alcohol oxidized to the C15 ketone.
[0586] Elaboration of the C17 ketone functionality to an acetyl
group is a process that begins with the attack of an acetylide
anion. A reagent such as commercially available lithium
acetylide-ethylene diamine complex can be used for this task.
Dehydration of the product yields the compound with a conjugated
.DELTA..sup.16 carbon-carbon double bond. Hydration of the
acetylene moiety using reagents such as mercury-impregnated Dowex
resin in methanol, THF and water produces an acetyl group at C17
(.DELTA..sup.16, C20 ketone). The .DELTA..sup.16 carbon-carbon
double-bond is reduced using sodium dithionite and bicarbonate
under phase transfer conditions.
[0587] The methyl ketone is then converted to an epoxide by
treatment with a sulphur ylid prepared from trimethylsulphonium
iodide and n-butyllithium in THF. The epoxide is then opened
stereoselectively using a Lewis acid such as magnesium bromide
etherate to give the C22 aldehyde. An alkyl anion generated via a
lithium-halide exchange reaction from an appropriate halogen, such
as the iodide 284 (Example 9, Scheme 72), is then used to attack
the steroidal aldehyde to generate the same side chain, in a
protected form, as found in compound 165. Deprotection of the C29
aldehyde produces the desired side chain.
[0588] The details of these two strategies are presented in
Examples 5 and 6, which follow.
Example 5
22,29-epoxy-3,29-dihydroxy-14.beta.-stigmastan-15-one (260)
[0589] As an example of the first method described in the
introduction to section 2, the steroid
22,29-epoxy-3,29-dihydroxy-14.beta.-stigmastan-15-one (260) can be
synthesized according to the following reaction sequence outlined
in Scheme 64. ##STR114## ##STR115##
[0590] The synthesis of
22,29-epoxy-3,29-dihydroxy-14.beta.-stigmastan-15-one (260) can be
accomplished in ten steps from dehydroisoandrosterone (247).
Catalytic hydrogenation of the .DELTA..sup.5 carbon-carbon double
bond in compound 247 yields compound 250, which contains a
trans-fused A/B ring-system. Compound 247 is dissolved in EtOAc and
10% Pd/C is added. The mixture is stirred under H.sub.2 at room
temperature overnight. Filtration through celite and concentration
yields compound 250, which can be used directly in the next
reaction.
[0591] A Wittig reaction on compound 250 using the phosphorous ylid
prepared from ethyltriphenylphosphonium bromide and potassium
t-butoxide in THF gives compound 251. Ethyltriphenylphosphonium
bromide is stirred as a suspension in THF. Potassium t-butoxide is
added under a stream of nitrogen and the mixture stirred at room
temperature for 1 hour. Compound 250 is added to the anion thus
formed as a solution in THF. The mixture is refluxed for 2 hours
under nitrogen then cooled to room temperature and quenched by the
dropwise addition of water. Saturated ammonium chloride is added
and this aqueous layer is extracted with EtOAc and the combined
organic phases washed with water and brine and dried over
MgSO.sub.4. Filtration and concentration gives the crude product
251 which is purified using silica flash chromatography with a
step-gradient of hexanes and EtOAc.
[0592] Protection of the 3.beta.-hydroxyl is accomplished using
t-butyldimethylsilyl chloride and imidazole in DMF to yield 252.
Compound 251 is dissolved in DMF and imidazole is added. After
addition of TBDMSCl, the mixture is stirred overnight at room
temperature. Dichloromethane is then added and the mixture is
washed with water and the organic phase dried over MgSO.sub.4.
Filtration and concentration yields the crude product 252 which can
be used without further purification.
[0593] Compound 252 is then coupled with the aldehyde 156 in the
presence of an appropriate Lewis acid to yield compound 253. The
aldehyde 156 is dissolved in dichloromethane and Me.sub.2AlCl
(11.0M in hexane) is added at -78.degree. C. After 5 minutes a
solution of compound 252 in dichloromethane is added. The mixture
is then warmed to room temperature over 16 hours. It is then cooled
to -78.degree. C. and quenched with a methanol/water mixture. The
layers are separated and the aqueous phase is extracted with
Et.sub.2O. The combined organic layers are then washed sequentially
with 1N HCl, saturated aqueous NaHCO.sub.3 and brine and then dried
over MgSO.sub.4. After filtration and evaporation to dryness, the
C22 isomers are separated using silica flash chromatography to
yield compounds 253a and 253b.
[0594] Compound 253a (or 253b) is then dissolved in dry DMF and
imidazole is added. TBDMSCl is then added and the mixture is
stirred at room temperature for 14 hours and then at 60.degree. C.
for 3 hours. The mixture is diluted with Et.sub.2O, washed with
water and then dried over MgSO.sub.4. After filtration and
evaporation, the crude product is purified using silica flash
chromatography with EtOAc and hexane mixtures as the eluent to give
compound 254.
[0595] Allylic oxidation at C15 of compound 254 with CrO.sub.3 and
3,5-dimethylpyrazole in dichloromethane gives compound 255.
CrO.sub.3 and dichloromethane are added to a flask and cooled to
-20.degree. C., and are allowed to stir at this temperature for 15
minutes. 3,5-Dimethylpyrazole is added and then the reaction
stirred for an additional 1.5 hours. Compound 254 in
dichloromethane is added and the mixture is kept at -20.degree. C.
for 5 days. The mixture is then warmed to room temperature and
filtered through silica gel, washing with EtOAc. Evaporation of the
solvent gives the crude product which is purified using flash
chromatography (EtOAc/hexane) to yield pure compound 255.
[0596] Reduction of the .DELTA..sup.16 carbon-carbon double bond in
compound 255 can then be achieved using hydrogen and palladium on
carbon in EtOAc. Compound 255 is dissolved in EtOAc and a catalytic
amount of 10% Pd/C is added. The mixture is stirred under a
hydrogen atmosphere overnight, then filtered through Celite and
concentrated to yield, after purification, compound 256.
[0597] Removal of the acetate protecting group in the side chain of
compound 256 can then be carried out using potassium carbonate in
methanol (or NaOMe in MeOH) to give compound 257. Compound 256 is
dissolved in methanol and K.sub.2CO.sub.3 is added. The mixture is
refluxed for 3 hours, cooled to room temperature and poured onto
dichloromethane. Aqueous (10%) NaHCO.sub.3 is added and the layers
are separated. The aqueous layer is extracted with dichloromethane
and the combined organic extracts are washed with water and dried
over MgSO.sub.4. Filtration, evaporation and purification yields
compound 257.
[0598] Oxidation of the resultant primary alcohol 257 to the
aldehyde 258 can be achieved using PCC. Compound 257 and NaOAc are
stirred in dichloromethane and PCC is added. The mixture is stirred
at room temperature for 3 hours and then filtered through Celite.
The filtrate is concentrated and the residue purified using flash
chromatography to give compound 258.
[0599] Deprotection of both hydroxyl moieties in compound 258 can
be achieved in one step using tetrabutylammonium fluoride. Compound
258 is dissolved in THF and tetrabutylammonium fluoride in THF is
added. The mixture is stirred overnight at room temperature and
then concentrated in vacuo and purified to give
22,29-epoxy-3,29-dihydroxy-14.alpha.-stigmastan-15-one (248).
[0600] Epimerization of compound 248 at the C14 position using KOH
in MeOH yields
22,29-epoxy-3,29-dihydroxy-14.beta.-stigmastan-15-one (260).
Compound 248 is dissolved in MeOH and a solution of KOH in MeOH (25
mg/ml) is added. The mixture is refluxed for 15 minutes then cooled
to room temperature. Water is added and the aqueous slurry is
extracted with chloroform and then dried over MgSO.sub.4.
Filtration and concentration gives the crude product that contains
an epimeric mixture of compounds 248 and 260. Separation of 248 and
260 is achieved using column chromatography.
[0601] An alternative route to compound 260 (Scheme 65) involves
preparation of the .delta.-lactone in the sidechain and subsequent
Dibal-H reduction to yield the compound containing the C29
hemiacetal functionality. For example, the 3.beta.-hydroxyl in
compound 251 is protected as the benzyloxy functionality followed
by the standard ene reaction described in example 5. Deprotection
of the C29 acetoxy group is then accomplished using sodium
methoxide in methanol. The resultant diol 263 is then oxidized to
the .delta.-lactone using silver carbonate on celite in refluxing
benzene. Compound 263 is dissolved in benzene and silver carbonate
embedded on celite is added and the mixture refluxed for 12 hours.
The reaction mixture is then filtered, evaporated and the residue
purified by flash chromatography to yield lactone 264. Allylic
oxidation of compound 264 using chromium trioxide and
3,5-dimethylpyrazole in dichloromethane introduces a carbonyl
moiety at C15 (compound 265). Reduction of the conjugated
.DELTA..sup.16 carbon-carbon double bond using hydrogen and
palladium on carbon in EtOAc followed by removal of the benzoate
groups using NaOMe in 1:1 CHCl.sub.3/MeOH, yields product 266.
Finally, protection of the C15 ketone as the ethylene ketal
followed by selective reduction of the .delta.-lactone to the
lactol is then accomplished using DIBAL at -78.degree. C. and
deprotection using 80% aqueous acetic acid to give compound 260.
##STR116##
Example 6
[0602] As stated at the beginning of this section, a second method
for the introduction of the C15 oxygen involves using a Michael
addition as described by Cantral et al., J. Org. Chem., 29:64,
1963. Selective removal of the protecting group followed by
oxidation of the resultant secondary alcohol to a ketone at C15 is
required. It has been shown by Horita et al., Tetrahedron,
42(11):3021-3028, 1986 that p-methoxybenzyl protecting groups can
be removed in the presence of many other protecting groups,
including benzyl functionalities, using
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). Therefore, the alkoxide
produced from p-methoxybenzyl alcohol in KOH, is used instead of
the analogous benzyloxy alkoxide used by Cantral et al. Scheme 66
below shows an example of this chemistry on the enone in compound
270, which is produced in three steps from transdehydroandrosterone
(247) using procedures described by Takahashi et al., Tetrahedron,
41(24):5747-5754, 1985. ##STR117##
[0603] The reaction procedures for the Michael Addition illustrated
in Scheme 67 are as follows. Compound 270 is dissolved in
p-methoxybenzyl alcohol and powdered KOH is added. The mixture is
stirred under nitrogen at room temperature for 4 hours. The mixture
is diluted with Et.sub.2O and washed with water. The organic layer
is dried over MgSO.sub.4, filtered and concentrated. The crude
residue is purified using flash chromatography with a step gradient
(EtOAc/hexane) elution to yield compound 271. ##STR118##
[0604] Attachment of the sidechain can be accomplished on compounds
containing a C17 ketone functionality using a Wittig/ene procedure
as illustrated earlier (Example 5), or by using a procedure
involving a compound containing a methyl ketone at C17, such as
ketone 275. The conversion of compounds with C17 ketone
functionalities to methyl ketone derivatives is accomplished in a
four step process using procedures described in the literature. An
example of this methodology is illustrated in Scheme 68.
##STR119##
[0605] The first step in the above process involves conversion of
compound 271 to the acetylenic alcohol compound 272 using lithium
acetylide-ethylenediamine complex. The acetylide complex is
suspended in THF, and after cooling to -20.degree. C., a solution
of compound 271 in THF is added. The mixture is stirred at room
temperature for 6 hours, cooled to 0.degree. C. and then quenched
with water. The mixture is extracted with dichloromethane then the
combined organic extracts are washed with brine and water and then
dried over MgSO.sub.4. After filtration and concentration the
residue is purified using silica flash chromatography (1:6
EtOAc/hexane) to yield compound 272.
[0606] Dehydration of compound 272 yields the conjugated
carbon-carbon double bond in the D-ring of compound 273. Compound
272 is dissolved in dry pyridine and phosphorus oxychloride is
added. The mixture is stirred for 30 minutes at room temperature
and then poured onto ice-water. The aqueous slurry is extracted
with dichloromethane and the combined organic extracts are washed
with aqueous NaHCO.sub.3 and water and dried over MgSO.sub.4. After
filtration and concentration the crude product is purified using
silica flash chromatography (1:6 EtOAc/hexane) to yield compound
273.
[0607] Mercury (2+) impregnated Dowex-50W resin, in a solvent
mixture of methanol, THF and water is then used to produce the
conjugated methyl ketone 274 from compound 273. Compound 273 is
dissolved in methanol/THF (2:1) and two drops of water are added.
Hg.sup.2+/Dowex resin is added and the mixture is stirred at
60.degree. C. overnight. Filtration and concentration yields a
crude residue which is purified by silica flash chromatography (1:2
EtOAc/hexane) to give methyl ketone 274.
[0608] The .DELTA..sup.16 carbon-carbon double bond in compound 274
is reduced using sodium dithionite. Compound 274 in toluene is
added to a mixture of sodium dithionite and sodium bicarbonate in
water. The phase transfer catalyst Aliquat.RTM. 336 is added and
the mixture is refluxed for 3 hours. The mixture is extracted with
dichloromethane and the organic phase is dried over MgSO.sub.4.
Filtration and concentration of the filtrate in vacuo gives a crude
residue, containing compound 275.
[0609] Selective deprotection of the C15 hydroxyl in 275 can be
accomplished, at this stage or after coupling of the appropriate
sidechain, according to the procedures described by Horita et al.
(Tetrahedron, 1986, 42(1]), 3021-3028) which involve oxidation of
the p-methoxybenzyl ether with 2,3-dichloro-5,6-dicyanobenzoquinone
(DDQ) in dichloromethane and water. Oxidation of the resultant C15
hydroxyl group to the ketone functionality can be accomplished
using a number of methods including PCC in dichloromethane.
Example 7
[0610] As described in the introduction to this section, the
hemiacetal sidechain functionality in compounds such as 165 can be
introduced using Grignard methodology. This first involves a two
step conversion from the methyl ketone functionality at C17 to a
C22 aldehyde containing compound. The methodology for such a
conversion, illustrated in Scheme 69, has previously been described
by a number of groups including Koreeda et al., Tetrahedron
Letters, No. 19, 1641-1644, 1978. ##STR120##
[0611] The coupling (Scheme 70) of the desired sidechain to the
steroidal aldehyde 278 is achieved by nucleophilic attack of a
carbanion. Compound 28 is prepared in situ by treatment of the
iodide 284 with t-BuLi in Et.sub.2O at -78.degree. C. (see Section
3, Example 9, Scheme 72) and the solution of the aldehyde 278 in
Et.sub.2O is then added. The mixture is stirred for 1.5 hours at
-78.degree. C. then warmed to room temperature and EtOAc is added.
The organic layer is washed sequentially with water, 1N HCl, and
water, then dried over MgSO.sub.4. After filtration and
concentration, the crude product is purified using silica flash
chromatography to yield compound 281. Deprotection of the
sidechain, using standard procedures such as 80% aqueous acetic
acid at 60.degree. C., gives the desired sidechain hemiacetal.
##STR121##
Section 3
The Synthesis of Various Side Chain Carbon Skeletons for Coupling
to Steroid Nuclei
[0612] The synthesis of compound 165, and analogues outlined are
convergent (i.e., a highly functionalized side chain is coupled
with a functionalized steroid nucleus). The two methods employed to
couple the side chain carbon skeleton to the steroid ring structure
were described in Section 2. Below is a description of the
production of the side chain carbon skeletons to be used in the
coupling reactions.
[0613] The first method involves the synthesis of
5-acetoxy-3-(1'-methylethyl)-pentanal (156) and related aldehydes
from the primary alcohol 154 (produced from L-carvone, which is
available from, e.g., Aldrich Chemical Co., Milwaukee, Wis.). This
sequence gives rise to compound 156 with the S-configuration. The
compound with the R-configuration is synthesized from D-carvone. It
is also noteworthy that any appropriate alcohol protecting group
can be used in place of the acetate ester to generate an aldehyde
compound suitable for coupling to the steroid via an ene-type
reaction on compound 252 and related steroids.
[0614] The second method (Scheme 72) involves the synthesis of
compound 284 and related alkyl halides from the carboxylic acid
compound 282. This sequence also generates a product (280) with the
S-configuration. Again, the enantiomer of compound 280 is
synthesized from D-carvone. As above, any appropriate aldehyde
protecting group can be used in place of the ketal functionality in
280, for the steps in the production of the carbanion used in the
coupling reaction (generated from a lithium/halide exchange
reaction on the corresponding alkyl halide).
Example 8
5-acetoxy-3-(1-methylethyl)-pentanal (156)
[0615] The intermediate 5-acetoxy-3-(1'-methylethyl)-pentanal
(156), used in the Wittig reaction described in previous sections,
can be synthesized according to the reaction sequence shown in
Scheme 71. ##STR122##
[0616] Conversion of L-carvone (153) to compound 154 is
accomplished following literature procedures (Tetrahedron Letters,
1984, 25(41), 4685-4688). Protection of the primary alcohol in
compound 154 is accomplished by conversion to an acetate ester.
Compound 154 (207 mg, 1.10 mmol) is dissolved in pyridine (2 mL)
and DMAP (10 mg) and acetic anhydride (2 mL) are added. The mixture
is stirred at room temperature overnight and then diluted with
Et.sub.2O. The mixture is washed with 10% aqueous NaHCO.sub.3 then
water and dried over MgSO.sub.4. Purification of the crude product
is achieved using silica flash chromatography (1:4 EtOAc/hexane) to
give compound 155 (249 mg, 1.08 mmol) in 98% yield.
[0617] Removal of the ketal protecting group in ketal 155 using 80%
acetic acid gives the desired aldehyde 156. Compound 155 (150 mg,
0.652 mmol) is suspended in 80% AcOH (5 mL) and the mixture is
heated at 70.degree. C. for 2 hours. The solvent is removed in
vacuo and the residue is taken up in Et.sub.2O (50 mL). The mixture
is then washed with aqueous NaHCO.sub.3 and brine and then dried
over MgSO.sub.4. Filtration and evaporation of the filtrate gives
pure compound 156 (110 mg, 0.591 mmol) in 91% yield.
Example 9
[0618] The intermediate 280, used in the coupling reaction
described in previous sections, can be synthesized according to the
reaction sequence summarized in Scheme 72. ##STR123##
[0619] Compound 282 is first synthesized using standard literature
procedures (Tetrahedron Letters, 25(41), 4685-4688, 1984) and then
converted to 280 in a three step procedure. Compound 282 (1.04 g,
5.12 mmol) is dissolved in dry CCl.sub.4 (120 mL), and 12 (2.57 g)
and iodobenzenediacetate (IDBA) (3.28 g) are added. The mixture is
refluxed while irradiating with a 100 Watt bulb for 10 minutes.
After cooling to room temperature, 5% aqueous
Na.sub.2S.sub.2O.sub.3 solution (300 mL) is added until the
solution is colorless then the layers are separated. The organic
phase is washed with water and dried over MgSO.sub.4. Purification
gives pure compound 283 (657 mg, 2.30 mmol, 45% yield).
[0620] Exchange of the aldehyde protecting groups is achieved to
produce compound 284 from compound 283 using
2,2-dimethyl-1,3-propanediol and p-toluenesulphonic acid as a
catalyst. Compound 283 (42 mg, 0.18 mmol) is dissolved in benzene
(5 mL) and 2,2-dimethyl-1,3-propanediol (300 mg) and
p-toluenesulphonic acid (3 mg) are added. The mixture is heated at
reflux overnight then cooled and evaporated to dryness in vacuo.
The residue is purified using silica flash chromatography (19:1
CH.sub.2Cl.sub.2/Hexanes) to yield compound 284 (40 mg, 0.14 mmol,
80%).
[0621] Finally, a lithium iodine exchange reaction on 284 using
t-butyllithium in THF gives the desired product 280. t-BuLi (1.7 M
solution in pentane, 0.25 mL) is added to a solution of compound
284 (52.1 mg, 0.160 mmol) in dry Et.sub.2O (2.0 mL) at -78.degree.
C. The solution is stirred at -78.degree. C. until no starting
material remains by GC analysis. This solution is used directly in
the coupling reaction with the aldehyde 278 (Example 7, Scheme
70).
Section 4
The Synthesis of 3,4,6,7-polyhydroxy-22,29-epoxy-15-one
Steroids
[0622] The synthesis of polyhydroxylated steroids containing one or
both of the C15 ketone and the side chain hemiacetal found in
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(165) can be accomplished using a combination of the methods
described in the first two sections. One can either begin with the
functionalization of the A and B rings, then the functionalization
of the D-ring and coupling of the side chain or the converse. In
the compounds containing a C15 ketone and/or a C29 hemiacetal, the
A/B ring can contain 2, 3 or 4 hydroxyl groups at carbons 3, 4, 6
and/or 7 in any combination and in any combination of
configurations.
[0623] The following describes synthetic routes to the compounds
22,29-epoxy-3,6,7,29-tetrahydroxy-14.beta.-stigmastan-15-one (304)
and 22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(165) and the corresponding H14.alpha. epimers. These are examples
of compounds containing the C15 ketone and the side chain C29
hemiacetal that are derived from synthetic transformations
described in the first two sections and the same methodology can be
applied in the production of compounds containing other
hydroxylation patterns at carbons 3, 4, 6 and 7 in the A/B ring
system.
[0624] Also included below is the synthetic route to
22,29-epoxy-3,6,7,29-tetrahydroxystigmastanol (306), a compound
that contains a polyhydroxylated A/B ring system and a hemiacetal
side chain but lacks the C15 ketone functionality found in compound
165.
Example 10
22,29-epoxy-3,6,7,29-tetrahydroxy-14.beta.-stigmastan-15-one
(304)
[0625] The steroid
22,29-epoxy-3,6,7,29-tetrahydroxy-14.beta.-stigmastan-15-one (304)
can be synthesized according to Scheme 73. ##STR124## ##STR125##
##STR126## ##STR127## ##STR128##
[0626] The steroid
22,29-epoxy-3,6,7,29-tetrahydroxy-14.beta.-stigmastan-15-one (304)
can be synthesized starting from the commercially available steroid
dehydroisoandrosterone (247). The C17 ketone of compound 247 is
first protected as the ketal by treatment with ethylene glycol and
a catalytic amount of p-toluenesulfonic acid in refluxing benzene.
Protection of the 3, hydroxyl as a silyl ether is then achieved
using t-butyldimethylsilyl chloride and imidazole in DMF to give
compound 286. Allylic oxidation of compound 286 using chromium
trioxide and 3,5-dimethylpyrazole introduces a carbonyl moiety at
the C7 position (compound 287). Chemoselective 1,2-reduction of
this ketone using sodium borohydride and cerium chloride yields the
allylic alcohol 288 in a THF-methanol solvent system.
[0627] Introduction of the C6 alcohol can be accomplished using the
sequence described in Example 1, or by hydroboration using a
borane-THF complex followed by oxidative hydrolysis with basic
hydrogen peroxide. In the second method, the allylic alcohol in
compound 288 is first protected as the acetate using acetic
anhydride and pyridine and the product 289 is dissolved in dry THF
and at 0.degree. C. a solution of 1.0 M borane in THF is added. The
mixture is stirred at 0.degree. C. for 30 minutes and then at room
temperature for 2.5 hours. A 3N NaOH solution is then added
dropwise followed by 30% aqueous H.sub.2O.sub.2. The mixture is
stirred at room temperature for 16 hours and then poured into
saturated sodium chloride solution. The aqueous slurry is extracted
with chloroform and the combined organic layers are dried over
MgSO.sub.4. Filtration and evaporation of the filtrate gives a
crude product which is purified by flash chromatography to yield
compound 221.
[0628] Protection of the vicinal 6,7-diol 221 is then achieved as
the dibenzyloxy derivative using benzyl bromide and sodium hydride
in dimethylformamide. Compound 221 is dissolved in
dimethylformamide and sodium hydride is added. The mixture is
stirred for 1 hour at room temperature then benzyl bromide is
added. Stirring is continued for 2.5 hours then the reaction is
quenched by the addition of water and stirring is continued for 30
minutes. The mixture is extracted with diethyl ether and then
washed successively with 5% HCl, saturated sodium bicarbonate and
saturated sodium chloride. The organic layer is dried over
magnesium sulphate, filtered and evaporated to dryness. The
resultant crude residue is purified using flash chromatography to
yield compound 291.
[0629] Deprotection of the C3 alcohol in compound 291 is achieved
using TBAF in THF at reflux for 2 hours to yield compound 292.
Subsequent oxidation using PDC gives the ketone 293. To affect this
transformation, the crude product 292 is dissolved in
CH.sub.2Cl.sub.2 and PDC is added. The mixture is stirred at room
temperature for 22 hours. Filtration through a bed of celite
followed by purification of the evaporated filtrate using flash
chromatography gives the product ketone 293 as a white solid.
[0630] Selective reduction of 293 gives the .alpha.-hydroxyl group
at C3 in 83% yield. To achieve this, a solution of compound 293 in
THF is cooled to -78.degree. C. and LS-Selectride.RTM. is added
slowly and stirring is continued at -78.degree. C. for 1 hour under
nitrogen. Methanol is added and the reaction mixture is warmed to
room temperature. Standard work-up followed by purification by
flash chromatography gave compound 294 in approximately 80% yield.
The 3.beta.-epimer is obtained in approximately 10% yield.
Protection of the .alpha.-hydroxyl group in compound 294 is
achieved as the benzyloxy derivative. Thus, compound 294 is
dissolved in dimethylformamide and sodium hydride is added. The
mixture is stirred for 1 hour at room temperature then benzyl
bromide is added. Stirring is continued for 16 hours then the
reaction is quenched by the slow addition of water and stirring is
continued for 30 minutes. The mixture is extracted with diethyl
ether and then washed successively with 5% HCl, saturated sodium
bicarbonate and saturated sodium chloride. The organic layer is
dried over magnesium sulphate, filtered and evaporated to dryness.
The resultant crude residue is purified using flash chromatography
to yield compound 295.
[0631] Deprotection of the C17 ketal in compound 295 using a
mixture of acetic acid, water and acetone (2:1:2) for 14 hours at
reflux gives compound 296 in 99% yield after purification. Compound
296 is converted to olefin 297 using the ylid prepared by the
treatment of ethyltriphenylphosphonium bromide with potassium
t-butoxide in THF. The coupling of the carbon structure of the side
chain is achieved using the aldehyde 156 (Scheme 45) and the Lewis
acid, dimethylaluminum chloride, in dichloromethane to yield the
C22 epimers 298a and 298b after purification. Deprotection of the
C29 acetoxy group is then accomplished using sodium methoxide in
methanol. The resultant diol 299 is then oxidized to the
.delta.-lactone using silver carbonate on celite in refluxing
benzene. Compound 299 is dissolved in benzene and silver carbonate
embedded on celite is added and the mixture refluxed for 12 hours.
The reaction mixture is then filtered, evaporated and the residue
purified by flash chromatography to yield lactone 300. Allylic
oxidation of compound 300 using chromium trioxide and
3,5-dimethylpyrazole in dichloromethane introduces a carbonyl
moiety at C15 (compound 301). Reduction of the conjugated
.DELTA..sup.16 carbon-carbon double bond using hydrogen and
palladium on carbon in EtOAc gives compound 302. Removal of the
benzoate groups in ester 302 is achieved with concurrent
epimerization at C14 to yield the trihydroxy product 303 which
contains the cis C/D ring junction in addition to the trihydroxy
product containing the trans C/D ring junction which are separable
by chromatography. Finally, protection of the C15 ketone as the
ethylene ketal ((CH.sub.2OH).sub.2, pTsOH, benzene) followed by
selective reduction of the 8-lactone to the lactol using DIBAL at
-78.degree. C. and deprotection (80% AcOH) can give
22,29-epoxy-3,6,7,29-tetrahydroxy-14.beta.-stigmastan-15-one
(304).
[0632] As an alternative, compound 221 may be treated to remove all
of the protecting group, for example using 80% acetic acid.
Thereafter, the hydroxyl groups in the B-ring may be selectively
protected. This may be done with 2,2-dimethoxypropane and
camphorsulfonic acid. The ketone group at C17 may then be
elaborated to an exocyclic double bond using Wittig chemistry, for
example, using ethyltriphenylphosphoniium bromide, t-BuOK and THF
affords ethylidene substitution at C17. Thereafter, the C3 hydroxyl
group may be oxidized to a carbonyl group with, e.g., oxalyl
chloride, DMSO, Et.sub.3N in methylene chloride, followed by
reduction of the resulting C3 carbonyl with LS-Selectride.RTM.t
(Aldrich Chemical Co., Milwaukee, Wis.) to afford the 3-.alpha.
hydroxy group. Deprotection of the hydroxyl groups in the B-ring
then affords compound 330. This is an alternative route to compound
330 from what is shown in Scheme 79.
Example 11
[0633] Compound 306 can be synthesized from compound 300 according
to the reaction sequence in Scheme 74. ##STR129##
[0634] Compound 306 can be synthesized from compound 300 in a two
step process with the first step being the deprotection of the
hydroxyl groups using hydrogen and palladium on carbon in EtOAc and
EtOH with concurrent reduction of the .DELTA..sup.16 carbon-carbon
double bond. The above mixture is stirred at room temperature for
12 days, filtered and purified by flash chromatography to yield
305. Selective reduction of the .delta.-lactone to the lactol is
then accomplished as follows. Compound 305 is dissolved in THF and
cooled to -78.degree. C. DIBAL is added and the mixture is stirred
at -78.degree. C. for 3 hours. Standard workup and purification
using silica flash chromatography gives give
22,29-epoxy-3,6,7,29-tetrahydroxystigmastanol (306).
Example 12
[0635] Reaction conditions described in the previous sections can
be applied to the synthesis of compound 165 as illustrated in
Scheme 75. ##STR130## ##STR131## ##STR132## ##STR133## ##STR134##
##STR135##
[0636] The synthesis of
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(165) is carried out starting with the commercially available
steroid 4-androstene-3,17-dione (1). The synthesis from compound 1
to the intermediate 128 has already been described (Example 3,
Scheme 61) for the synthesis of
3.alpha.,4.beta.,6.alpha.,7.beta.-pentahydroxy-5.alpha.-androstanol
(241).
[0637] Preparing the D-ring of compound 128 for the side chain
coupling procedures described in subsequent examples (Example 16,
Scheme 79) begins with the removal of the silyl group at C17 of
steroid 128. Compound 128 is dissolved in THF and TBAF (1.0 M in
THF) is added. The mixture is heated at reflux for 2 hours and then
concentrated in vacuo. The residue is purified by flash
chromatography to give alcohol 129.
[0638] The hydroxyl moiety in alcohol 129 is convert to the ketone
using oxalyl chloride in CH.sub.2Cl.sub.2. Compound 129 is
dissolved in CH.sub.2Cl.sub.2 and added to a solution of oxalyl
chloride in CH.sub.2Cl.sub.2 at -78.degree. C. After stirring at
-78.degree. C. for 15 minutes, triethylamine is added and stirring
is continued for 5 minutes. Standard work-up and purification gave
compound 142 as a white solid.
[0639] The conversion of compound 142 to compound 145 is
accomplished using the same procedures described in Example 3
(Scheme 61) for the conversion of compound 128 to compound 241.
Epoxidation of compound 142 using m-CPBA in dichloromethane to
yield epoxide 143 is followed by epoxide opening using acetic acid
to give the 3,6,7-trihydroxy-4-acetoxy compound 144. and removal of
the acetate group attached to the oxygen on C4 using
K.sub.2CO.sub.3 in methanol to yield compound 145.
[0640] Synthesis of compound 165 from compound 145 is accomplished
using the same types of reactions described in previous sections.
Compound 145 is converted to ethylidene 157 using the ylid prepared
from ethyltriphenylphosphonium bromide and potassium t-butoxide in
THF. The four hydroxyl groups are then protected as benzyl moieties
to yield the tetrabenzyloxy compound 158. Coupling of the side
chain is achieved using the aldehyde 156 (Scheme 71) and a Lewis
acid such as dimethylaluminum chloride in dichloromethane to yield
compound 159. Deprotection of the C29 acetoxy group is then
accomplished using sodium methoxide in methanol. The resultant diol
is then oxidized to the .delta.-lactone 161 using silver carbonate
on celite in benzene. Allylic oxidation of compound 161 using
chromium trioxide and 3,5-dimethylpyrazole in dichloromethane
introduces a carbonyl moiety at C15 with concurrent oxidation of
the benzyl groups to benzoate groups (compound 162). Reduction of
the conjugated .DELTA..sup.16 carbon-carbon double bond using
hydrogen and palladium on carbon in EtOAc and EtOH gives compound
163. Removal of the benzoate groups in ester 163 is achieved using
basic conditions (for example KOH in MeOH) with concurrent
epimerization at C14 to yield product 164 which contains an
epimeric mixture of the compounds containing the cis C/D ring
junction and the trans C/D ring junction. Finally, protection of
the C15 ketone as the ethylene ketal ((CH.sub.2OH).sub.2, pTsOH,
benzene) followed by selective reduction of the 6-lactone to the
lactol using DIBAL at -78.degree. C. and deprotection (80% AcOH)
can give
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.beta.-stigmastan-15-one
(165) and it's C14 epimer give
22,29-epoxy-3,4,6,7,29-pentahydroxy-14.alpha.-stigmastan-15-one.
Section 5
Additional Examples of the Synthesis of Novel Polyhydroxylated
Steroids with Biological Activities
[0641] In addition to the compounds described in previous sections,
a number of related compounds with biological activities have been
produced. These include, among others, compounds containing the
3.alpha.,6.alpha.,7.beta.-hydroxylation pattern with varying
functionalities at the C17 position, as well as compounds
containing the 3.beta.,6.alpha.,7.beta.-hydroxylation pattern with
varying functionalities at the C17 position. Some of the procedures
used for the production of these compounds are described in the
following examples.
Example 13
[0642] A number of 3,6,7-hydroxylated compounds can be prepared
from the intermediate compound 221. As described in Scheme 73,
compound 221 may be prepared from the commercially available
starting material dehydroisoandrosterone (247). Specifically,
compound 247 (20.0 g, 69.3 mmol) is dissolved in benzene (200 ml)
in a 500 ml round bottom flask which is then connected to a
Dean-Stark apparatus. p-Toluenesulfonic acid monohydrate (0.501 g,
2.64 mmol) is added followed by ethylene glycol (20 ml) and the
mixture is refluxed for 4.5 hours. The mixture is cooled to room
temperature and diluted with diethyl ether (200 ml). The organic
layer is washed with saturated sodium bicarbonate (2.times.100 ml)
then by saturated sodium chloride (2.times.100 ml). The organic
layer is dried with MgSO.sub.4, filtered and evaporated to dryness
to yield the product 285 (22.8 g, 68.6 mmol, 99%) which is used in
the next reaction without further purification. Protection of the
30-hydroxyl in compound 285 as a silyl ether may then be achieved
as follows. Compound 285 (22.5 g, 67.7 mmol) is dissolved in a
mixture of dimethylformamide (112.5 ml) and dichloromethane (112.5
ml). Imidazole (11.3 g, 166.0 mmol) is added followed by
t-butyldimethylsilyl chloride (15.8 g, 104.8 mmol). The mixture is
stirred at room temperature for 6 hours under Argon then diluted
with diethyl ether (675 ml). The organic mixture is washed with
aqueous 5% HCl (2.times.135 ml) followed by saturated sodium
bicarbonate (2.times.135 ml) then by saturated sodium chloride
(2.times.135 ml). The organic layer is dried with MgSO.sub.4,
filtered and evaporated to dryness to yield the crude product 286.
The crude product is then recrystallized from ethyl
acetate/methanol (3:2) to give compound 286 (25.9 g, 58.0 mmol, 83%
over two steps) as white crystals. Allylic oxidation of compound
286 may then introduce a carbonyl moiety at the C7 position
(compound 287). Compound 286 (15.0 g, 33.6 mmol) is dissolved in
cyclohexane (60 ml) and H.sub.2O (7.3 ml) is added. Ruthenium (III)
chloride hydrate (0.0561 g, 0.27 mmol) is added followed by the
dropwise addition of t-butylhydroperoxide (37.6 ml). The mixture is
stirred at room temperature for 24 hours and then diluted with
ethyl acetate (376 ml). The organic mixture is washed with
saturated sodium chloride (2.times.188 ml) then with 25% sodium
thiosulphate (2.times.188 ml). The organic layer is dried with
MgSO.sub.4, filtered and evaporated to dryness to yield the crude
product. The crude product is recrystallized from ethyl acetate to
yield compound 287 (8.6 g, 18.7 mmol, 56%). Chemoselective
1,2-reduction of the ketone in compound 287 may yield 288. Thus,
compound 287 (14.7 g, 31.9 mmol) is dissolved in tetrahydrofuran
(118 ml) and cerium (111) chloride heptahydrate (17.6 g, 47.2 mmol)
in methanol (35 ml) is added. The mixture is cooled using an
ice-bath and sodium borohydride (2.5 g, 66.1 mmol) is added slowly.
The mixture is warmed to room temperature and then stirred for 2.5
hours. Aqueous 5% HCl (44 ml) is then slowly added to the mixture
followed by ethyl acetate (588 ml). The reaction is washed with
aqueous 5% HCl (120 ml) followed by saturated sodium bicarbonate
(120 ml) then by saturated sodium chloride (120 ml). The organic
layer is dried with MgSO.sub.4, filtered and evaporated to dryness
to yield the product 288 (14.8 g) which is used in the next
reaction without further purification. Compound 288 (14.8 g) is
then dissolved in pyridine (30 ml) and acetic anhydride (15 ml) and
a catalytic amount of 4-dimethylaminopyridine (30 mg) is added. The
mixture is stirred at room temperature for 16 hours and then
diluted with ethyl acetate (300 ml). The organic mixture is washed
with saturated sodium bicarbonate (2.times.60 ml) then by saturated
sodium chloride (2.times.60 ml). The organic layer is dried with
MgSO.sub.4, filtered and evaporated to dryness to yield the crude
product. Recrystallization from methanol gives compound 289 (12.6
g, 25.0 mmol, 78% yield over two steps). Hydroboration of compound
289 affords the 6.alpha.,7.beta.-hydroxylation pattern. Thus,
compound 289 (8.4 g, 16.6 mmol) is dissolved in dry THF (50 ml) and
the mixture is cooled to 0.degree. C. A solution of 1.0 M borane in
THF (20 ml) is added and the mixture is stirred at 0.degree. C. for
30 minutes and then at room temperature for 2.5 hours. An aqueous
10N NaOH solution (10 ml) is then added dropwise followed by 30%
aqueous H.sub.2O.sub.2 (10 ml). The mixture is stirred at room
temperature for 18 hours and then poured into saturated sodium
chloride solution (200 ml). The aqueous slurry is extracted with
methylene chloride (2.times.250 ml) and the combined organic layers
are washed with aqueous 25% sodium thiosulphate solution
(2.times.250 ml) and the organic layer is dried over MgSO.sub.4.
Filtration and evaporation of the filtrate gives a crude product
which is purified by silica gel flash chromatography (3:1
hexane/ethyl acetate) to yield compound 221 (5.9 g, 12.3 mmol,
74%).
[0643] Scheme 76 illustrates the synthesis of compounds 326 and 327
from compound 221. Compound 221 (1.2 g, 2.4 mmol) is dissolved in
acetic anhydride (3 ml) and pyridine (3 ml) and a catalytic amount
of 4-dimethylaminopyridine (40 mg) is added. The mixture is stirred
at room temperature for 3 hours then diluted with ethyl acetate
(100 ml). The organic mixture is washed with aqueous 5% HCl then
saturated sodium bicarbonate (100 ml) and saturated sodium chloride
(100 ml). The organic layer is dried with MgSO.sub.4, filtered and
evaporated to dryness to yield the product 321 (1.3 g) which is
used in the next reaction without further purification. Removal of
the silyl protecting group using TBAF in THF gives compound 322
which contains the 3.beta.-hydroxyl group. The crude product 321 is
dissolved in THF (10 ml) and 1.0 M tetrabutylammonium fluoride (4
ml) is added. The mixture is refluxed for 1 hour, cooled to room
temperature then poured into saturated sodium chloride solution (50
ml). The aqueous slurry is extracted with methylene chloride
(5.times.40 ml) and the organic layer is dried over MgSO.sub.4.
Filtration and evaporation of the filtrate gives a crude product
which is purified by silica gel flash chromatography (1:1
hexane/ethyl acetate) to yield compound 322 (0.85 g, 1.9 mmol, 76%
yield over two steps). Inversion of the stereochemistry at C3 is
then accomplished by oxidation using PDC in CH.sub.2Cl.sub.2 to
give the ketone 323 followed by L-Selectride.RTM. reduction to
yield predominantly the 3.alpha.-hydroxyl compound 324. Thus,
compound 322 (0.84 g, 1.9 mmol) is dissolved in CH.sub.2Cl.sub.2
(15 ml) and PDC (1.2 g, 3.2 mmol) is added. The mixture is stirred
for 40 hours at room temperature and then diluted with diethyl
ether (50 ml). Filtration and evaporation to dryness gives the
crude product which is purified by silica gel flash chromatography
(9:1 hexanes/ethyl acetate) to yield compound 323 (0.81 g, 1.8
mmol, 95%). Compound 323 (0.34 g, 0.75 mmol) is then dissolved in
THF (10 ml) and then cooled to -78.degree. C. L-Selectride (1.0 M
in THF, 1.6 ml) is added and the mixture is stirred at -78.degree.
C. for 1 hour. The mixture is warmed to room temperature and an
aqueous 10N NaOH solution (1 ml) is then added dropwise followed by
30% aqueous H.sub.2O.sub.2 (1 ml). The mixture is stirred at room
temperature for 1 hour and then poured into ethyl acetate (50 ml).
The organic mixture is washed with aqueous 5% HCl (2.times.25 ml)
then saturated sodium bicarbonate (2.times.25 ml) and saturated
sodium chloride (2.times.25 ml). The organic layer is dried with
MgSO.sub.4, filtered and evaporated to dryness and purified using
silica gel flash chromatography (3:1 hexane/ethyl acetate) to yield
compound 324 (0.214 g, 0.48 mmol, 64%).
[0644] Removal of the acetate protecting groups may then be
accomplished. Compound 324 (0.25 g, 0.56 mmol) is dissolved in
methanol (10 ml) and sodium methoxide (250 mg) is added. The
mixture is stirred at room temperature for 3 hours and then diluted
with ethyl acetate (50 ml). The organic mixture is washed with
aqueous 5% HCl (2.times.25 ml) then saturated sodium bicarbonate
(2.times.25 ml) and saturated sodium chloride (2.times.25 ml). The
organic layer is dried with MgSO.sub.4, filtered and evaporated to
dryness and purified using silica gel flash chromatography (ethyl
acetate) to yield compound 325 (0.185 g, 0.51 mmol, 91%). Compound
325 (141 mg, 0.385 mmol) is then dissolved in 80% acetic acid (10
ml) and stirred at 70.degree. C. for 14 hours. The mixture is
diluted with ethyl acetate (50 ml) and washed with saturated sodium
bicarbonate (2.times.25 ml) and saturated sodium chloride
(2.times.25 ml). The organic layer is dried with MgSO.sub.4,
filtered and evaporated to dryness and purified using silica gel
flash chromatography (ethyl acetate) to yield compound 326 (0.054
g, 0.17 mmol, 44%). Finally, reduction of the ketone 326 (0.023 g,
0.072 mmol) by NaBH.sub.4 (0.034 g) in 95% ethanol (1 ml) at room
temperature for 2 hours produced the tetrahydroxy compound 327
(0.018 g, 0.056 mmol, 78%). ##STR136## ##STR137##
Example 14
[0645] As illustrated in Scheme 77, compound 329 can be produced
from compound 322 in a two step process. Removal of the acetate
protecting groups is done by stirring ester 322 in sodium methoxide
and methanol for 15 hours at room temperature. Deketalization is
then accomplished on compound 328 using 80% acetic acid to give the
trihydroxy compound 329. ##STR138##
Example 15
[0646] Compound 329 can also be produced directly from compound 221
in a single step using 80% AcOH as shown in Scheme 78. Thus, ketal
221 (1.3 g, 2.7 mmol) is dissolved in 80% aqueous acetic acid (20
ml) and the mixture is stirred for 3 hours at room temperature.
Evaporation to dryness provides compound 329 (0.79 g, 2.5 mmol,
93%) which is used in subsequent reactions without further
purification. ##STR139##
Example 16
[0647] The steroid
3.alpha.,6.alpha.,7.beta.-trihydroxy-17(20)-pregnene (330) can be
synthesized according to the reaction sequence shown in Scheme 79.
##STR140## ##STR141## ##STR142## ##STR143##
[0648] Compound 330 can be produced from compound 10 (as shown in
Scheme 79). A solution of compound 10 (0.82 g, 1.73 mmol) in
diethyl ether (15 ml) is transferred to a flask containing lithium
metal (55 mg) in liquid ammonia (30 ml) at -78.degree. C. under
argon. After 30 minutes at -78.degree. C., NH.sub.4Cl (2.0 g) is
added and the ammonia is evaporated. Water (10 ml) is added and the
layers are separated. The aqueous layer is extracted with
CH.sub.2Cl.sub.2 (2.times.25 ml) and the combined organic layers
are washed with water (25 ml) and dried over magnesium sulphate.
After filtration and evaporation to dryness, the residue is
dissolved in CH.sub.2Cl.sub.2 (15 ml) and PDC (600 mg, 1.59 mmol)
is added. The mixture is stirred at room temperature for 18 hours
then filtered through a bed of celite. The filtrate is then
purified using silica gel flash chromatography (5:1 hexane/ethyl
acetate) to give compound 13 (653 mg, 1.40 mmol, 81%).
[0649] The ketone 13 is then reduced by the following procedure.
Compound 13 (1.2 g, 2.53 mmol) is dissolved in THF (30 ml) then the
mixture is cooled to -78.degree. C. and L-Selectrideg (11.0M in
THF, 3.8 ml) is added. The mixture is stirred at -78.degree. C. for
2.5 hours and then warmed to 0.degree. C. Aqueous 10% NaOH (10 ml)
is added followed by 30% H.sub.2O.sub.2 (10 ml). After stirring for
2 hours, water (20 ml) is added and the aqueous slurry is extracted
with CH.sub.2Cl.sub.2 (4.times.100 ml). The combined organic
extracts are then washed with 10% Na.sub.2S.sub.2O.sub.3
(2.times.100 ml) and saturated soldium chloride (2.times.100 ml).
The organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness to yield the product 14 which is used in the
next step without further purification. The 3.alpha.-hydroxyl group
is then protected as the acetate using acetic anhydride and
pryidine to give acetate 15. Thus, compound 14 is dissolved in
pyridine (15 ml) and acetic anhydride (10 ml) and the mixture is
stirred for 12 hours. Ethyl acetate and diethyl ether (1:1, 150 ml)
are added and the mixture is washed with 5% HCl (2.times.50 ml)
then by saturated sodium bicarbonate (2.times.50 ml). The organic
layer is dried over magnesium sulphate, filtered and evaporated to
dryness and the residue is purified by silica gel flash
chromotography (10:1 hexane/ethyl acetate) to give compound 15
(0.89 g, 1.73 mmol, 68% over two steps). Removal of the silyl
protecting group at C17 of compound 15 (0.85 g, 1.63 mmol) is
accomplished by refluxing in THF (30 ml) and TBAF (11.0M in THF,
3.6 ml) for three hours followed by evaporation to dryness. The
residue is then dissolved in CH.sub.2Cl.sub.2 (100 ml) and washed
with H.sub.2O (3.times.30 ml). The organic layer is dried over
magnesium sulphate, filtered and evaporated to dryness and the
residue is purified by silica gel flash chromatography (1:1
hexane/ethyl acetate) to give compound 16 (0.59 g, 1.45 mmol, 89%).
Oxidation of the C17 hydroxyl in compound 16 is then accomplished
using oxalyl chloride in DMSO. Thus, compound 16 (0.57 g, 1.40
mmol) is dissolved in CH.sub.2Cl.sub.2 (5 ml) and is added to a
solution of oxalyl chloride (0.15 ml, 1.68 mmol) and DMSO (0.24 ml,
3.36 mmol) in CH.sub.2Cl.sub.2 (10 ml) at -78.degree. C. After
stirring at -78.degree. C. for 15 minutes, triethylamine (0.98 ml)
is added and stirring is continued for 5 minutes. The mixture is
warmed to room temperature and H.sub.2O (10 ml) is added. The
layers are separated and the organic layer is washed with 5% HCl
(2.times.5 ml) then by saturated sodium bicarbonate (2.times.5 ml).
The organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness and the residue is purified by silica gel
flash chromatography (2:1 hexane/ethyl acetate) to give compound 17
(0.54 g, 1.33 mmol, 95%).
[0650] A Wittig reaction on compound 17 using the phosphorous ylid
prepared from ethyltriphenylphosphonium bromide and potassium
t-butoxide in THF gives compound 18. Potassium t-butoxide (0.59 g,
5.23 mmol) is added under a stream of nitrogen to a suspension of
ethyltriphenylphosphonium bromide (1.94 g, 5.23 mmol) in THF (15
ml) and the mixture is stirred at room temperature for 1 hour.
Compound 17 (0.53 g, 1.31 mmol) is dissolved in THF (10 ml) and the
solution is added to the ylid in THF. The resultant mixture is
refluxed for 12 hours under nitrogen then cooled to room
temperature. The mixture is filtered through celite and the
filtrate is evaporated to dryness. The residue is dissolved in
CH.sub.2Cl.sub.2 (100 ml) and washed with saturated NH.sub.4Cl
solution (2.times.30 ml) and H.sub.2O (2.times.30 ml). The organic
layer is dried over magnesium sulphate, filtered and evaporated to
dryness and the residue is purified by silica gel flash
chromatography (3:1 hexane/ethyl acetate) to give compound 18 (0.33
g, 0.88 mmol, 67%).
[0651] Finally, removal of the acetonide group is accomplished.
Thus, compound 18 (20 mg, 0.053 mmol) is dissolved in 80% aqueous
acetic acid (1.5 ml) and stirred at 60.degree. C. for 1 hour. The
mixture is evaporated to dryness to yield compound 330 (17.8 mg,
0.053 mmol, 99%).
Example 17
[0652] A number of compounds with important biological activities
can be synthesized from compound 329. For example, compound 333,
which contains the 3.beta.,6.alpha.,7.beta.-hydroxylation pattern
and an ethylidene residue at C17, is prepared in three steps from
compound 329 (Scheme 80). Thus, compound 329 (1.81 g, 5.6 mmol) is
dissolved in 2,2-dimethoxypropane (25 ml) and a catalytic amount of
camphor sulfonic acid (CSA) (0.03 g) is added and the mixture is
stirred at room temperature for 3 hours. Ethyl acetate (200 ml) is
added and the mixture is washed with 5% aqueous HCl (50 ml) then by
saturated sodium bicarbonate (2.times.100 ml) and by saturated
sodium chloride (2.times.100 ml). The organic layer is dried over
magnesium sulphate, filtered and evaporated to dryness and the
residue is purified by silica gel flash chromatography (1:1
hexane/ethyl acetate) to give compound 331 (1.54 g, 4.3 mmol, 76%).
A Wittig reaction on compound 331 using the phosphorous ylid
described in previous sections produces compound 332. Thus,
potassium t-butoxide (7.15 g, 63.7 mmol) is added under a stream of
nitrogen to a stirring solution of ethyltriphenylphosphonium
bromide (23.7 g, 63.7 mmol) in toluene (360 ml). The mixture is
then stirred for 1 hour at room temperature then compound 331 (7.7
g, 21.2 mmol) in toluene (210 ml) is added. The mixture is stirred
at room temperature for 24 hours under nitrogen then quenched by
the dropwise addition of water (120 ml). The mixture is diluted
with ethyl acetate (900 ml) and washed with saturated sodium
bicarbonate (2.times.200 ml) sodium chloride (2.times.200 ml). The
organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness and the residue is purified by silica gel
flash chromatography (2:1 hexane/ethyl acetate) to give compound
332 (7.2 g, 19.2 mmol, 90%). Deprotection of the hydroxyl groups in
compound 332 is then achieved by stirring 332 in 80% acetic acid.
Thus, compunds 332 (7.2 g, 19.2 mmol) is dissolved in 80% acetic
acid (115 ml) and the mixture is stirred at room temperature for 3
hours. Evaporation to dryness followed by purification by silica
gel flash chromatography (9:1 CH.sub.2Cl.sub.2/MeOH) gives compound
333 (5.81 g, 17.4 mmol, 90%). ##STR144##
Example 18
[0653] Compounds containing a ketone at C3 and a 6,7-hydroxylation
pattern can be obtained from compound 332. For example, oxidation
of compound 332 using Swern conditions produces compound 334 which
can then be deprotected to compound 335 (Scheme 81). Compound 332
(1.01 g, 2.70 mmol) is dissolved in CH.sub.2Cl.sub.2 (10 ml) and
then added to a solution of DMSO (2.5 ml) and 2.0M oxalyl chloride
in CH.sub.2Cl.sub.2 (8.1 ml) at -78.degree. C. After stirring at
-78.degree. C. for 15 minutes, triethylamine (4.6 ml) is added and
stirring is continued for 15 minutes followed by stirring at room
temperature for 30 minutes. The mixture is diluted with ethyl
acetate (100 ml) and washed with saturated sodium bicarbonate
(2.times.50 ml) then saturated sodium chloride (2.times.50 ml). The
organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness and the residue is purified by silica gel
(pretreated with 1% triethylamine in hexanes) flash chromatography
(19:1 hexane/ethyl acetate) to give compound 334 (0.77 g, 2.07
mmol, 77%). Deprotection of the hydroxyl groups in compound 334 is
achieved, as in previous examples, by stirring the compound 334 (11
mg, 0.030 mmol) in 80% aqueous acetic acid (1.25 ml) at room
temperature for 1 hour to give, after evaporation to dryness and
purification by silica gel flash chromatography (ethyl acetate),
compound 335 (9.8 mg, 0.029 mmol, 97%). ##STR145##
Example 19
[0654] A by-product of the Swern oxidation in Scheme 81 is the
chloro derivative 336. Compound 336 can be deprotected by treatment
with 80% aqueous acetic acid as shown in Scheme 82. Thus, compound
336 (0.028 g, 0.072 mmol) is dissolved in 80% aqueous acetic acid
(2 ml) and stirred at room temperature for 1 hour. The mixture is
evaporated to dryness and the residue is purified by silica gel
flash chromatography (3:2 hexane/ethyl acetate) to give compound
337 (0.024 g, 0.067 mmol, 94%). ##STR146##
Example 20
[0655] As described in an earlier section, compound 330 can be
prepared by reduction of the C3 carbonyl in compound 334 (e.g.,
with LS-Selectride.RTM., Aldrich Chemical Co., Milwaukee, Wis.) to
afford the 3.alpha. hydroxy group followed by deprotection. Thus,
compound 334 (0.85 g, 2.3 mmol) is dissolved in THF (25 ml) and
cooled to -78.degree. C. LS-Selectride (11.0M in THF, 3.0 ml) is
added and the mixture is stirred at -78.degree. C. for 3 hours.
Aqueous 10N NaOH (2 ml) and 30% H.sub.2O.sub.2 (2 ml) are added and
the mixture is warmed to 0.degree. C. The mixture is diluted with
ethyl acetate (150 ml) and washed with saturated sodium bicarbonate
(2.times.50 ml) then saturated sodium chloride (2.times.50 ml). The
organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness and the residue is purified by silica flash
chromatography (3:1 hexane/ethyl acetate) to give compound 338
(0.703 g, 1.88 mmol, 80%). Deprotection of the hydroxyl groups in
the B-ring then affords compound 330 (Scheme 83). Thus, compound
338 (1.44 g, 3.85 mmol) is dissolved in 80% aqueous acetic acid (25
ml) and stirred at room temperature for 3 hours. The mixture is
evaporated to dryness to give compound 330 (1.25 g, 3.74 mmol,
97%). This is an alternative route to compound 330 from what is
shown in Scheme 79. ##STR147##
Example 21
[0656] Compounds containing an ethyl residue or another alkyl chain
at C17 can be prepared from the corresponding compound containing
an exocyclic double at C17. For example, compound 339 is obtained
by catalytic hydrogenation of the C17-C20 double bond of compound
338, followed by the deprotection as shown below. Thus, compound
338 (0.15 g, 0.40 mmol) is dissolved 1:1 in acetic acid and ethanol
(4 ml) and 10% Pd--C (15 mg) is added. The mixture is stirred under
H.sub.2 for 16 hours followed by filtration and evaporation to
yield the desired product 339 (0.115 g, 0.340 mmol, 85%) (Scheme
84a). The intermediate 17-ethyl-6,7-acetonide 361 is produced if
the hydrogenation reaction is carried out in ethyl acetate (Scheme
84b). Thus, compound 338 (0.019 g, 0.050 mmol) is dissolved in
ethyl acetate (5 ml) and 10% Pd--C (9 mg) is added. The mixture is
stirred under H.sub.2 for 14 hours followed by filtration and
evaporation to yield the desired product 361 (0.018 g, 0.048 mmol,
96%).
[0657] Likewise, compound 332 in acetic acid is hydrogenated using
H.sub.2 and 10% Pd--C to yield the product 340 (Scheme 85).
##STR148## ##STR149## ##STR150##
Example 22
[0658] Compounds with alternative alkyl groups at C17 can be
prepared using different Wittig reagents. For example, compound 342
is easily obtained from a Wittig reaction, similar to the one
described previously but using MePh.sub.3PBr as the Wittig reagent
rather than EtPh.sub.3PBr followed by the deprotection of triol 341
as shown below (Scheme 86). Thus, methyltriphenylphosphonium
bromide (1.97 g, 5.51 mmol) and tBuOK (0.61 g, 5.4 mmol) are
stirred in THF (10 ml) for 1 hour. Ketone 331 (0.411 g, 1.14 mmol)
in THF (5 ml) is added to the ylid and the mixture is refluxed for
3 hours. After standard work-up, as described earlier, the crude
product is purified using silica gel flash chromatography (3:1
hexane/ethyl acetate) to give compound 341 (0.332 g, 0.920 mmol,
81%). Product 341 (0.204 g, 0.567 mmol) is then treated with 80%
acetic acid solution (4 ml) for 1.5 hours at room temperature. The
mixture is evaporated in vacuo to give the desired triol 342 (0.173
g, 0.540 mmol, 95%). The corresponding C17 methyl derivative is
then prepared by hydrogenation of compound 341 (0.023 g, 0.063
mmol) and 10% Pd--C (15 mg) in ethyl acetate (5 ml) under H.sub.2
atmosphere followed by, after filtration and evaporation to
dryness, deprotection with 80% acetic acid solution (2.5 ml) to
afford compound 343 (0.018 g, 0.056 mmol, 88%) (Scheme 87).
##STR151## ##STR152##
Example 23
[0659] Compounds containing a methylene carbon at C3 can be
synthesized in a number of different ways. One example involves a
modified Barton procedure (Robins et al., J. Am. Chem. Soc.,
105:4059-4065, 1983) as shown in Scheme 88. The alcohol 332 (0.10
g, 0.27 mmol) is treated with phenyl chlorothionoformate (0.45 ml,
3.3 mmol) in pyridine (2 ml) and methylene chloride (3 ml) at room
temperature for 2 hours. The mixture is evaporated to dryness and
the residue is purified by silica gel flash chromatography (30:1
hexane/ethyl acetate) to give the thionester 344 (0.12 g, 0.22
mmol, 84%). The ester 334 (0.091 g, 0.17 mmol) is then treated with
nBu.sub.3SnH (60 .mu.l, 0.22 mmol) and a catalytic amount of AIBN
(4 mg) in toluene (3 ml) at 75.degree. C. for 3 hours under an
inert atmosphere. The mixture is evaporated to dryness and compound
345 (0.035 g, 0.1 mmol, 57%) is obtained upon purification using
silica gel flash chromatography (30:1 hexane/ethyl acetate).
Treatment of compound 345 (0.025 g, 0.069 mmol) with 80% aqueous
acetic acid (2 ml) for 1 hour at room temperature followed by
evaporation to dryness and purification by silica gel flash
chromatography (3:1 hexane/ethyl acetate) gives the diol 346 (0.021
g, 0.066 mmol, 96%). ##STR153##
Example 24
[0660] Compounds containing a methylene carbon at C17 can be
produced using similar chemistry to that described in Example 23.
For example, the alcohol group of compound 331 (0.15 g, 0.42 mmol)
is initially protected as a silyl ether by treatment with
t-butyldimethylsilyl chloride (0.095 g, 0.63 mmol) and imidazole
(0.057 g, 0.84 mmol) in dimethylformamide (4 ml) at room
temperature for 4 hours. The mixture is diluted with diethyl ether
(75 ml) and washed with saturated sodium bicarbonate (2.times.25
ml) then H.sub.2O (2.times.25 ml). The organic layer is dried over
magnesium sulphate, filtered and evaporated to dryness and the
residue is purified by silica flash chromatography (9:1
hexane/ethyl acetate) to give compound 347 (0.18 g, 0.38 mmol, 90%)
(Scheme 89). The ketone function of compound 347 is then reduced
with lithium aluminum hydride in diethyl ether to afford the
17.beta.-alcohol 348. Thus, compound 347 (0.18 g, 0.37 mmol) is
dissolved in diethyl ether (5 ml), cooled to 0.degree. C. and
lithium aluminum hydride (0.018 g, 0.48 mmol) is added. The mixture
is stirred at 0.degree. C. for 30 minutes then saturated sodium
bicarbonate (1 ml) is added dropwise. The mixture is diluted with
diethyl ether (50 ml) and washed with saturated sodium bicarbonate
(2.times.15 ml) then by H.sub.2O (2.times.15 ml). The organic layer
is dried over magnesium sulphate, filtered and evaporated to
dryness and the residue is purified by silica flash chromatography
(4:1 hexane/ethyl acetate) to give compound 348 (0.15 g, 0.31 mmol,
85%). Using a Barton type reaction, compound 348 is treated with
NaH, CS.sub.2 and MeI in THF to produce the methyl xanthate 349
after work-up and purification. Thus, compound 348 (0.052 g, 0.11
mmol) is dissolved in THF (5 ml) and NaH (17.4 mg (60% in oil),
0.43 mmol) and imidazole (5 mg, 0.074 mmol) are added. The mixture
is stirred at room temperature for 30 minutes then carbon disulfide
(0.2 ml) is added and stirring is continued for 2 hours followed by
refluxing for 30 minutes. MeI (0.2 ml) is added and refluxing is
continued for an addition 30 minutes. H.sub.2O (1 ml) is added
dropwise and the mixture is diluted with diethyl ether (100 ml) and
washed with 5% HCl (2.times.30 ml) then saturated sodium
bicarbonate (2.times.30 ml) and then H.sub.2O (2.times.30 ml). The
organic layer is dried over magnesium sulphate, filtered and
evaporated to dryness and the residue is purified by silica gel
flash chromatography (15:1 hexane/ethyl acetate) to give compound
349 (0.054 g, 0.09 mmol, 85%). In the next reaction AIBN is
typically used as the radical initiator. Thus, compound 349 (0.05
g, 0.087 mmol) is dissolved in toluene (15 ml) and nBu.sub.3SnH
(0.051 g, 0.17 mmol) and a catalytic amount of AIBN (10 mg) are
added and the mixture is refluxed for 22 hours under an inert
atmosphere. The mixture is cooled to room temperature, evaporated
to dryness and purified using silica flash chromatography (40:1
hexane/ethyl acetate) to give compound 350 (0.010 g, 0.022 mmol,
25%). Treatment of compound 350 (0.010 g, 0.022 mmol) with 80%
acetic acid (2 ml) for 18 hours at room temperature followed by
evaporation to dryness and purification using silica gel flash
chromatography (20:1 CHCl.sub.3/MeOH) gives compound 351 (0.0065 g,
0.021 mmol, 96%). ##STR154##
Example 25
[0661] Compounds with higher alkyl chains attached to C17 can be
produced using similar chemistry to that described in previous
examples. For example, compound 354 can be produced in 4 steps from
commercially available cholesteryl acetate (228), as shown in
Scheme 90. Methodology previously described involving C7 oxidation
using RuCl.sub.3 and tBuOOH followed by reduction of the C7 ketone
using NaBH.sub.4/CeCl.sub.3 affords alcohol 352. Acetylation of
compound 352 followed by hydroboration (and subsequent
alkaline-peroxide workup) produces the desired compound 354.
[0662] Specifically, compound 228 (0.431 g, 1.01 mmol), RuCl.sub.3
(0.020 g, 0.098 mmol), cyclohexane (5 mL), water (0.25 mL) and 70%
tBuOOH in water (1.5 mL, 11.0 mmol) are stirred for 24 hours at
room temperature. The mixture is diluted with ethyl acetate (125
ml) and washed with an aqueous solution of 10% Na.sub.2SO.sub.3
(2.times.50 ml) and saturated NaCl (2.times.50 ml). The organic
layer is dried with MgSO.sub.4 and evaporated to dryness.
Purification by silica gel flash chromatography with 9:1
hexanes-ethyl acetate affords compound 229 (0.263 g, 0.591 mmol,
59%). The reduction of the C7 ketone proceeds as follows. A mixture
of CeCl.sub.37H.sub.2O (2.00 g, 5.368 mmol) in methanol (5 ml) is
added to a solution of ketone 229 (1.11 g, 2.52 mmol) in THF (5 ml)
and the mixture is cooled to 0.degree. C. NaBH.sub.4 (0.119 g, 5.14
mmol) is added and the mixture stirred at 0.degree. C. for 1 hour
followed by warming to room temperature and continued stirring for
2 hours. The mixture is cautiously quenched with aqueous 5% HCl (10
ml) and diluted with ethyl acetate (250 ml). The emulsion is then
washed with aqueous 5% HCl (2.times.100 ml), saturated aqueous
NaHCO.sub.3 (2.times.100 ml), and saturated aqueous NaCl
(2.times.100 ml). The organic phase is dried with MgSO.sub.4 and
evaporated to dryness. The residue is purified by silica gel flash
chromatography with 9:1 hexanes-ethyl acetate giving alcohol 352
(0.850 g, 1.91 mmol, 76%). Protection and hydroboration are then
accomplished. Thus, compound 352 (0.850 g, 1.91 mmol), pyridine (5
ml) and acetic anhydride (5 mL) are stirred at room temperature for
16 hours. The mixture is diluted with ethyl acetate (150 ml) and
washed with aqueous 5% HCl (3.times.50 ml), saturated aqueous
NaHCO.sub.3 (2.times.50 ml), and saturated aqueous NaCl (2.times.50
ml). The organic phase is dried with MgSO.sub.4 and evaporated to
dryness. The residue is purified by silica gel flash chromatography
with 19:1 hexanes-ethyl acetate giving product 353 (0.823 g, 1.70
mmol, 99%). The diacetate 353 (0.275 g, 0.5648 mmol) in THF (5 ml)
is cooled to 0.degree. C. and BH.sub.3 in THF (1.0 M, 2.5 mL, 2.5
mmol) is added. The mixture is stirred for 3 hours at 0.degree. C.,
then cautiously quenched with an aqueous 10 N NaOH solution (1 mL)
and an aqueous 30% H.sub.2O.sub.2 (1 ml) solution. The resultant
mixture is stirred for 16 hours, diluted with ethyl acetate (100
ml) and washed with an aqueous 10% Na.sub.2SO.sub.3 solution
(2.times.50 ml), a saturated aqueous NaHCO.sub.3 solution
(2.times.50 ml) and saturated NaCl solution (2.times.50 ml). The
organic phase is dried over MgSO.sub.4 and evaporated to dryness.
Purification by silica gel flash chromatography (3:1 hexanes-ethyl
acetate) affords the product
313-acetoxy-6.alpha.,7.beta.-dihydroxy-5.alpha.-cholestane (0.032
g, 0.069 mmol, 13%) which is deprotected by treatment with sodium
methoxide (prepared from sodium metal (0.262 g, 11.4 mmol) and
methanol (10 ml)) at room temperature for 1.5 hours. The mixture is
diluted with ethyl acetate (30 ml) and then washed with a saturated
aqueous NaHCO.sub.3 solution (2.times.15 ml) and saturated aqueous
NaCl solution (2.times.15 ml). The organic layer is dried with
MgSO.sub.4 and evaporated to dryness. The residue is purified by
silica gel flash chromatography with 1:1 hexanes-ethyl acetate
giving triol 354 (0.029 g, 0.069 mmol, 99%). ##STR155##
Example 26
[0663] Compounds containing additional functional groups in the
A-ring have been prepared and tested for biological activity. For
example, compound 360 can be prepared in a multi-step synthesis
from compound 335, as illustrated in Scheme 91. Acetylation of
compound 335 using acetic anhydride and pyridine and DMAP to give
the diacetoxy compound 355. Thus, compound 335 (1.5 g, 4.5 mmol)
pyridine (10 ml), acetic anhydride (5 mL) and
4-dimethylaminopyridine (0.028 g, 0.23 mmol) are stirred at room
temperature for 12 hours. The mixture is diluted with ethyl acetate
(300 ml) and washed with aqueous 5% HCl (3.times.50 ml), saturated
aqueous NaHCO.sub.3 (3.times.50 ml), and H.sub.2O (3.times.50 ml).
The organic phase is dried with MgSO.sub.4 and evaporated to
dryness to yield a crude residue 355 (1.9 g) which is used in the
next reaction without further purification. Thus, crude product
355(0.800 g, 1.9 mmol) is dissolved in AcOH and 10% Pd--C (80 mg)
is added. The mixture is then stirred under H.sub.2 atmosphere for
16 hours at room temperature. The mixture is filtered and
evaporated to dryness to yield a crude residue which is purified by
silica gel flash chromatography (5:1 hexane/ethyl acetate) to give
compound 356 (0.702 g, 1.67 mmol, 88%). The oxime 357 is then
obtained by refluxing compound 356 with HONH.sub.2--HCl in a
MeOH-pyridine solution. Thus, compound 356 (0.05 g, 0.12 mmol) is
dissolved in a mixture of pyridine (2 ml) and methanol (2 ml) and
HONH.sub.2--HCl (0.017 g, 0.24 mmol) is added. The mixture is
refluxed for 1.5 hours and the mixture is diluted with ethyl
acetate (50 ml) and washed with 5% HCl (3.times.15 ml) then by
saturated sodium bicarbonate (3.times.15 ml) and then H.sub.2O
(3.times.15 ml). The organic layer is dried over magnesium
sulphate, filtered and evaporated to dryness to yield compound 357
(0.052 g, 0.12 mmol, 99%) which is used in the next reaction
without further purification. Product 357 (0.071 g, 0.16 mmol) is
then dissolved in pyridine (13 mg) in acetic anhydride (3 ml),
cooled to 0.degree. C. and acetyl chloride (15.5 mg, 0.20 mmol) is
added. The mixture is then heated for 8 hours at 100.degree. C.
H.sub.2O (0.5 ml) is added and heating is continued for 30 minutes.
The mixture is then cooled to room temperature, diluted with
H.sub.2O (10 ml), and extracted with CH.sub.2Cl.sub.2 (3.times.15
ml). The combined organic extracts are then washed with H.sub.2O
(2.times.10 ml). The organic layer is dried over magnesium
sulphate, filtered and evaporated to dryness and the residue is
purified by silica gel flash chromatography (2:1 hexane/ethyl
acetate) to give compound 358 (0.41 g, 0.086 mmol, 52%). The C3
ketone in 358 (0.020 g, 0.042 mmol) is then reduced with NaBH.sub.4
(2.4 mg) in THF (2 ml) at room temperature for 1 hour. AcOH (2
drops) is added and the mixture is diluted with ethyl acetate (50
ml) and washed with saturated sodium bicarbonate (2.times.15 ml)
then H.sub.2O (2.times.50 ml). The organic layer is dried over
magnesium sulphate, filtered and evaporated to dryness and the
crude product 359 is dissolved in methanol (1.5 ml). NaOMe (10 mg)
is added and the mixture is stirred at room temperature for 48
hours. Amberlite IR-120 ion exchange resin is added until pH 6. The
mixture is filtered and evaporated to dryness and purified by
silica gel flash chromatography (10:1 CHCl.sub.3/MeOH) to give
compound 360 (0.010 g, 0.028 mmol, 67% over two steps).
##STR156##
[0664] The following examples are offered by way of illustration
and not by way of limitation.
Utility Examples
[0665] The compounds described above have utility in treating
allergy and asthma, arthritis and/or thrombosis. As used herein,
"treating allergy and asthma, arthritis and/or thrombosis" refers
to both therapy for allergy and asthma, arthritis and thrombosis,
and for the prevention of the development of the allergic response,
bronchoconstriction, inflammation and the formation of blood clots
that cause thrombosis and associated diseases. An effective amount
of a compound or composition of the present invention is used to
treat allergy, asthma, arthritis or thrombosis in a warm-blooded
animal, such as a human. Methods of administering effective amounts
of anti-allergy, anti-asthma, anti-arthritis and anti-thrombotic
agents are well known in the art and include the administration of
inhalation, oral or parenteral forms. Such dosage forms include,
but are not limited to, parenteral solutions, tablets, capsules,
sustained release implants and transdermal delivery systems; or
inhalation dosage systems employing dry powder inhalers or
pressurized multi-dose inhalation devices. Generally, oral or
intravenous administration is preferred for the treatment of
arthritis and thrombosis, while oral or inhalation/intranasal are
preferred for asthma and allergy. The dosage amount and frequency
are selected to create an effective level of the agent without
harmful effects. It will generally range from a dosage of about
0.01 to 100 mg/kg/day, and typically from about 0.1 to 10 mg/Kg/day
where administered orally or intravenously, for anti-allergy,
anti-asthma, anti-arthritis or anti-thrombotic effects. Also, the
dosage range will be typically from about 0.01 to 1 mg/Kg/day where
administered intranasally or by inhalation for anti-asthma and
anti-allergy effects.
[0666] Administration of compounds or compositions of the present
invention may be carried out in combination with the administration
of other agents. For example, it may be desired to administer a
bronchodilator or a glucocorticoid agent for effects on asthma, a
glucocorticoid for effects on arthritis, or an anti-histamine for
effects on allergy. Non-steroid compounds may be co-administered
with the steroids of the present invention, and/or non-steroid
compounds may used in combination with the steroid compounds of the
invention to provide a therapy for one or more of asthma,
allergies, arthritis and thrombosis.
[0667] For example, provided below are several examples of the
biological activity of various compounds described in Synthesis
Examples, Sections 1-5.
[0668] Anti-thrombolytic Activity of Polyhydroxylated Steroids
[0669] Within the present invention, it was discovered that the
polyhydroxylated steroids as well as intermediates described in
previous sections inhibited the aggregation of platelets caused by
platelet activating factor (PAF). PAF is a local mediator of
thrombosis and prevention of the formation of blood clots has
direct implication in the treatment of thrombosis and associated
cardiovascular diseases. The assay system used to evaluate the
ability of compounds to inhibit the aggregation of platelets in
response to exogenous stimuli is indicative of anti-thrombotic or
thrombolytic activity.
[0670] Platelets were isolated from rabbit blood and prepared at a
density of 2.4.times.10.sup.8 cells/ml in Tyrodes buffer (pH 7.2)
containing Ca.sup.2+. Platelets were incubated with each compound
fro 5 min at 37.degree. C. prior to stimulation. Platelets were
stimulated with 1 nM platelet activating factor (PAF; EC.sub.75) in
the presence of each of the compounds and aggregation was monitored
for 5 min. Compounds were solubilized in dimethylsulfoxide (DMSO)
and aggregation was measured as a percentage of the response to 1
nM PAF obtained in the presence of the appropriate concentration of
DMSO. The degree of inhibition caused by each sample was calculated
using the control response in the presence of DMSO equal to
100%.
[0671] Table 1 shows examples of some of the compounds that inhibit
platelet aggregation in response to PAF. TABLE-US-00001 TABLE 1 THE
EFFECT OF VARIOUS COMPOUNDS ON THE AGGREGATION OF RABBIT PLATELETS
STIMULATED WITH 0.1 NM PAF* Sample % Inhibition Number at 80 .mu.M
7 12.3 8 11.6 165 51.2 236 100 241 93.1 246 21 330 20.9 *Platelets
were incubated with 80 .mu.M of each compound for 5 minutes prior
to stimulation. Responses were measured as a percentage of the
inhibition of the PAF-induced response obtained in the presence of
the appropriate concentration of DMSO alone.
[0672] Effects of Compounds on the Release of Hexosaminidase from a
Rat Mast Cell Line (RBL-2H3)
[0673] The anti-allergic effects of various polyhydroxylated
steroids of the present invention were evaluated by measuring their
effect on antigen-induced secretion of hexosaminidase from a
passively sensitized rat mast cell line (RBL-2H3) and a murine mast
cell line (MC/9). The ability of agents to inhibit the release of
mast cell granule contents, e.g., histamine and hexosaminidase, is
indicative of anti-allergy and/or anti-asthma activity.
[0674] Hexosaminidase is released from the mast cell granule along
with histamine and other mediators during antigen challenge.
RBL-2H3 and MC/9 cells were grown in culture and passively
sensitized to dinitrophenol (DNP) using anti-human-DNP (IgE). Cells
were incubated with each compound (25 .mu.M) for 1 hour at
37.degree. C. and then stimulated with 0.1 mg/ml DNP-HSA (antigen)
for 15 min. Aliquots of the supernatant were removed and used to
measure the amount of hexosaminidase released during challenge with
the antigen. The amount of hexosaminidase present in the
supernatant was determined calorimetrically by monitoring the
enzymatic metabolism of
p-nitrophenyl-N-acetyl-.beta.-D-glucosaminide (p-NAG) over a period
of 1 hour at 410 nm. The effect of each compound was determined as
a percentage of the antigen-induced response (minus background
release) obtained in the presence of DMSO alone, as set forth in
Tables 2 and 3. These values were used to determine the degrees of
inhibition of antigen-induced hexosaminidase release from the
cells. TABLE-US-00002 TABLE 2 THE EFFECT OF VARIOUS COMPOUNDS (EACH
25 .mu.M) ON ANTIGEN-INDUCED RELEASE OF HEXOSAMINIDASE FROM
PASSIVELY SENSITIZED RAT MAST CELLS (RBL-2H3) AND MOUSE MAST CELLS
(MC/9)* Percentage Inhibition (mean) Compound Number RBL-2H3 Cells
MC/9 cells 333 69 71 335 56 69 339 79 67 337 44 56 339 76 55 330 59
49 343 52 40 342 57 39 361 ND 13 331 ND 5 354 30 76 346 16 61 ND =
not determined.
[0675] TABLE-US-00003 TABLE 3 THE EFFECT OF VARIOUS COMPOUNDS (EACH
25 .mu.M) ON ANTIGEN-INDUCED RELEASE OF HEXOSAMINIDASE FROM
PASSIVELY SENSITIZED RAT MAST CELLS (RBL-2H3)* Percentage
Inhibition (mean) RBL-2H3 Cells 7 21.7 165 51.8 236 31 241 22 246
29.4 306 40.5 322 25.6 327 35.2 328 34.3 266 -12.5 239 9.6 351 33.6
360 76.0 *Values represent the percentage inhibition produced by
each compound compared to the response obtained in the presence of
DMSO alone.
Effects of Selected Compounds on Allergen-Induced Contraction of
Ileum Smooth Muscle
[0676] The ability of compounds to inhibit allergen-induced
contraction of ileum from sensitized animals is indicative of
antiallergic activity. Sensitized guinea pig ileum is particularly
useful in measuring the immediate allergic response.
[0677] The guinea pig ileum has been used to evaluate the ability
of compounds to inhibit allergen-induced histamine and mediator
release causing smooth muscle contraction. Guinea pigs were
sensitized by an intraperitoneal injection of 100 mg ovalbumin and
an intramuscular injection of 50 mg ovalbumin on day 0, followed by
a second intramuscular injection of 50 mg ovalbumin on day 1.
Twenty one days after the initial immunization, the animals were
found to be sensitized, in that an anaphylactic response was
obtained upon challenge with allergen. Segments of ileum were
prepared and suspended, with muscle contractions being measured in
the longitudinal plane, in Tyrode's buffer at 37.degree. C. and
aerated with 5% CO.sub.2 in O.sub.2. Tissues were suspended under a
resting tension of 2 grams and isometric contractions were measured
using force-displacement transducers coupled to a polygraph.
Tissues were stimulated with 3 .mu.M histamine 3 times to ensure
reproducible contractions were obtained. Tissues were then
incubated with each compound (30 .mu.M) or 0.15% dimethylsulphoxide
(DMSO) as a control, for 20 min, after which time the tissues were
challenged with 100 .mu.g/ml ovalbumin. The magnitude of the
contraction induced by OA in the presence of each compound was
expressed as a percentage of the contraction obtained to 3 .mu.M
histamine. The protective effects of the various compounds on
OA-induced contraction of guinea pig ileum from sensitized animals
are summarized in Table 4. TABLE-US-00004 TABLE 4 THE EFFECT OF
VARIOUS COMPOUNDS (EACH 30 .mu.M) ON ANTIGEN-INDUCED CONTRACTION OF
ILEUM FROM SENSITIZED GUINEA PIGS. ANTIGEN-INDUCED CONTRACTIONS
WERE EXPRESSED AS A PERCENTAGE OF THE CONTRACTION INDUCED BY 3
.mu.M HISTAMINE*. Compound Number Percentage Inhibition 330 70.0
221 60.7 338 64.0 7 54.0 333 63.0 343 48.9 336 79.3 342 28.3 339
40.6 335 30.7 337 50.7 165 27.0 251 -10.5(stim) 361 55.0 331 14.5
339 36.3 346 62.5 334 74.0 266 17.4 351 43.6 360 50.5 *Values
represent the mean percentage inhibition produced by each compound
compared to the response obtained in the presence of DMSO alone, n
= 3-4.
[0678] Effects of Selected Compounds on Allergen-Induced
Bronchoconstriction In Vitro and In Vivo
[0679] The effects of a number of the compounds described herein on
allergen-induced bronchoconstriction were evaluated for anti-asthma
activity. The ability of a compound to inhibit allergen-induced
decreases in lung function in sensitized guinea pigs in response to
antigen-challenge is indicative of anti-asthma activity. In
particular, the model system is useful in the evaluation of the
potential effects of a compound in the treatment of the early
asthmatic reaction (EAR) when severe bronchoconstriction
occurs.
[0680] Guinea pigs were exposed to a nebulized solution of 1%
ovalbumin (OA) in saline for 15 min. After 10 days the animals were
found to be sensitized, i.e., the tracheal tissue responded with
anaphylactic bronchospasm to further antigen (OA) challenge.
Trachea from these animals were found to respond in a similar
manner to the in vivo situation. Tracheal rings were prepared and
bathed in Krebs-Henseleit solution at 37.degree. C. and aerated
with 5% CO.sub.2 in O.sub.2. Tissues were suspended under a resting
tension of 2 g and isometric contractions were measured using
force-displacement transducers coupled to a polygraph. Tissues were
incubated with each compound or 0.1% dimethylsulfoxide (as a
control) for 20 min, after which increasing concentrations of OA
(0.001-100 .mu.g/ml) were added to the tissue. After the final
concentration of OA was added and the response was recorded, the
tissues were stimulated with 100 .mu.M methacholine which caused
maximum contraction of the trachea. The magnitude of the
contraction induced by OA in the presence of each compound was
expressed as a percentage of the maximum contraction obtained using
methacholine (100 .mu.M). The protective effects of various
compounds on OA-induced contraction of tracheal tissue are
summarized in Tables 5-7. TABLE-US-00005 TABLE 5 EFFECTS OF
SELECTED COMPOUNDS (EACH 20 .mu.M) ON ALLERGEN-INDUCED CONTRACTIONS
OF ISOLATED TRACHEA* (STUDY 1) .mu.g/ml OA 0.001 0.003 0.01 0.03
0.1 0.3 1.0 3.0 10.0 30.0 Ctrl 2.9 7.7 11.4 17.4 19.7 26.4 30.9
37.2 43.6 46.8 241 8.5 12.6 17.1 25.8 31.0 33.4 36.3 34.4 35.0 37.0
236 1.9 5.6 5.6 5.6 11.1 16.7 39 28 39 41 145 0.8 6.2 5.9 7.6 8.3
13.6 14.8 20.0 28.0 31.0 246 2.6 4.0 6.5 9.7 12.2 15.3 20.0 21.0
23.0 22.0 *Values represent percentage contraction compared to that
obtained with 100 .mu.M methacholine (100%), Ctrl = control (0.1%
DMSO)
[0681] TABLE-US-00006 TABLE 6 EFFECTS OF VARIOUS COMPOUNDS (EACH 20
.mu.M) ON ALLERGEN-INDUCED CONTRACTIONS OF ISOLATED TRACHEA* (STUDY
2) OA .mu.g/ml Sample 0.01 0.1 1 10 100 Ctrl 0.95 9.0 25.7 44.7
54.6 326 0 10.25 23.4 43.9 49.1 327 0 0 9.1 27.3 40.9 *Values
represent percentage contraction compared to that obtained with 100
.mu.M methacholine (100%), Ctrl = control (0.1% DMSO).
[0682] TABLE-US-00007 TABLE 7 EFFECTS OF COMPOUND 330 (EACH 30
.mu.M) ON ALLERGEN-INDUCED CONTRACTIONS OF ISOLATED TRACHEA* (STUDY
3) OA .mu.g/ml Sample 0.001 0.01 0.1 1 10 100 Ctrl 6.0 12.0 26.0
41.0 54.0 59.0 330 0 1.90 9.00 17.5 26.0 30.0 *Values represent
percentage contraction compared to that obtained with 100 .mu.M
methacholine (100%), Ctrl = control (0.1% DMSO).
[0683] In addition, the effect of compounds of the invention on
lung function in vivo was determined in sensitized animals as
follows:
[0684] Female Cam Hartley guinea pigs (350-400 g) are sensitized to
ovalbumin by exposure of the guinea pigs to a nebulized solution of
1% ovalbumin in saline for 15 minutes. After 10-12 days, the
animals are found to be acutely sensitized to the allergen
(ovalbumin). Animals are treated by oral gavage, under light
halothane anesthesia, with 300 .mu.l polyethyleneglycol-200 (PEG)
or 5 mg/Kg of test compound in 300 .mu.l of PEG. Animals are
treated once daily for 4 days with the final dose administered 2
hours prior to allergen challenge. Alternatively, compounds were
delivered by inhalation, using a Hudson nebulizer driven by 6 psi
oxygen, providing a single dose of 50 .mu.g/Kg 20 min prior to
challenge.
[0685] An animal is anaesthetized using ketamine (50 mg/ml; i.p.)
and xylazine (10 mg/Kg; i.p.) and 1% halothane during the surgical
procedure. A tracheostomy is performed and a water-filled
esophageal cannula is inserted prior to positioning the animal in a
body plethysmograph. The tracheal cannula is attached to a fixed
tracheal cannula in the plethysmograph. Cardiac function is
monitored using electrocardiography. The animal is paralyzed using
pancuronium bromide (0.8 mg/Kg; i.m.) and ventilated with 3 ml
tidal breaths using a Harvard small animal ventilator, at a
frequency of 60 breaths per minute. Pulmonary resistance and
dynamic lung compliance data are obtained from volume, flow and
transpulmonary pressure signals using multipoint analysis.
[0686] Pulmonary function is continually monitored throughout the
experiment and measurements of lung resistance and lung compliance
are made at various time-points (e.g., 0, 1, 2, 3, 4, 5, 10, 20 and
30 min) following antigen challenge. Data is collected on a
computer-linked physiological measurement system using DIREC
Physiological recording software and analyzed using ANADAT software
designed for lung mechanics measurements. This software was
obtained from RHT-InfoDat Inc., Montreal, Quebec, Canada.
[0687] Once baseline resistance and compliance measurements are
obtained, the animal is challenged with 6 breaths of saline. After
10 minutes, during which time no alterations in lung function
should occur, the animal is challenged with 6 breaths of 2 or 3%
ovalbumin in saline (as the antigen stimulus). Saline and antigen
are delivered in each breath using a Hudson nebulizer. The
protective effects of compound 330 administered orally on
OA-induced contraction of tracheal tissue are summarized in Tables
8 and 9 below. TABLE-US-00008 TABLE 8 THE EFFECT OF COMPOUND 330 (5
MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED INCREASE IN LUNG
RESISTANCE IN SENSITIZED GUINEA PIGS Lung Resistance Time Interval
(cm H.sub.2O/ml/sec) after challenge Control 330 Baseline 0.287
.+-. 0.020 0.275 .+-. 0.036 OA/10 s 0.295 .+-. 0.024 0.260 .+-.
0.036 1 min 0.982 .+-. 0.209 0.560 .+-. 0.101 2 min 2.390 .+-.
0.728 0.845 .+-. 0.201 3 min 2.627 .+-. 0.714 0.887 .+-. 0.160 (P
< 0.06) 4 min 2.801 .+-. 1.042 0.778 .+-. 0.119* 5 min 2.514
.+-. 0.952 0.791 .+-. 0.139* 10 min 1.329 .+-. .209 0.661 .+-.
0.141* 20 min 1.352 .+-. 0.494 0.366 .+-. 0.046* 30 min 1.00 .+-.
0.434 0.340 .+-. 0.037* *Significant difference from control, P
< 0.05.
[0688] TABLE-US-00009 TABLE 9 THE EFFECT OF COMPOUND 330 (5
MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED DECREASES IN LUNG
COMPLIANCE IN SENSITIZED GUINEA PIGS Lung Compliance Time Interval
(ml/cm H.sub.2O) after challenge Control 330 Baseline 0.412 .+-.
0.053 0.318 .+-. 0.042 OA/10 s 0.573 .+-. 0.083 0.435 .+-. 0.041 1
min 0.077 .+-. 0.016 0.182 .+-. 0.093 2 min 0.029 .+-. 0.006 0.145
.+-. 0.092 3 min 0.024 .+-. 0.003 0.133 .+-. 0.095 4 min 0.023 .+-.
0.001 0.124 .+-. 0.088* 5 min 0.026 .+-. 0.002 0.125 .+-. 0.087* 10
min 0.042 .+-. 0.002 0.150 .+-. 0.082* 20 min 0.059 .+-. 0.007
0.184 .+-. 0.061* 30 min 0.077 .+-. 0.010 0.196 .+-. 0.061*
*Significant difference from control, P < 0.05.
[0689] The protective effects of compound 330 administered by
inhalation on OA-induced contraction of tracheal tissue are
summarized in Tables 10-11 below. TABLE-US-00010 TABLE 10 THE
EFFECT OF COMPOUND 330 (50 .mu.G/KG; INHALATION) ON
ALLERGEN-INDUCED INCREASE IN LUNG RESISTANCE IN SENSITIZED GUINEA
PIGS Lung Resistance Time Interval (cm H.sub.2O/ml/sec) after
challenge Control 330 Baseline 0.257 .+-. 0.019 0.300 .+-. 0.025
OA/10 s 0.257 .+-. 0.046 0.288 .+-. 0.036 1 min 0.557 .+-. 0.118
0.382 .+-. 0.033 2 min 1.323 .+-. 0.344 0.420 .+-. 0.044* 3 min
1.987 .+-. 0.572 0.420 .+-. 0.051* 4 min 1.625 .+-. 0.248 0.455
.+-. 0.047* 5 min 1.395 .+-. 0.193 0.446 .+-. 0.124* 10 min 0.949
.+-. 0.165 0.436 .+-. 0.036* 20 min 0.589 .+-. 0.091 0.413 .+-.
0.076 30 min 0.493 .+-. 0.067 0.412 .+-. 0.072 *Significant
difference from control, P < 0.05.
[0690] TABLE-US-00011 TABLE 11 THE EFFECT OF COMPOUND 330 (50
.mu.G/KG; INHALATION) ON ALLERGEN-INDUCED DECREASE IN LUNG
COMPLIANCE IN SENSITIZED GUINEA PIGS Lung Compliance Time Interval
(ml/cm H.sub.2O) after challenge Control 330 Baseline 0.515 .+-.
0.169 0.463 .+-. 0.129 OA/10 s 0.526 .+-. 0.042 0.565 .+-. 0.062 1
min 0.095 .+-. 0.015 0.349 .+-. 0.059* 2 min 0.044 .+-. 0.010 0.213
.+-. 0.046* 3 min 0.031 .+-. 0.007 0.176 .+-. 0.045* 4 min 0.037
.+-. 0.009 0.145 .+-. 0.046* 5 min 0.047 .+-. 0.007 0.146 .+-.
0.031* 10 min 0.127 .+-. 0.053 0.138 .+-. 0.022 20 min 0.096 .+-.
0.009 0.193 .+-. 0.054 30 min 0.110 .+-. 0.010 0.181 .+-. 0.046
*Significant difference from control, P < 0.05.
[0691] The protective effects of compound 339 administered orally
on OA-induced contraction of tracheal tissue are summarized in
Tables 12-13 below. TABLE-US-00012 TABLE 12 THE EFFECT OF COMPOUND
339 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED INCREASE IN
LUNG RESISTANCE IN SENSITIZED GUINEA PIGS Lung Resistance Time
Interval (cm H.sub.2O/ml/sec) after challenge Control 339 Baseline
0.25 .+-. 0.008 0.249 .+-. 0.017 OA/10 s 0.261 .+-. 0.011 0.239
.+-. 0.013 1 min 1.781 .+-. 0.737 0.326 .+-. 0.041* 2 min 3.079
.+-. 1.066 0.522 .+-. 0.187* 3 min 3.623 .+-. 0.806 1.102 .+-.
0.047* 4 min 1.699 .+-. 0.342 0.996 .+-. 0.380 5 min 2.783 .+-.
1.010 1.014 .+-. 0.413 10 min 1.115 .+-. 0.348 0.440 .+-. 0.099 20
min 0.624 .+-. 0.178 0.296 .+-. 0.031 30 min 0.465 .+-. 0.126 0.291
.+-. 0.037 *Significant difference from control, P < 0.05.
[0692] TABLE-US-00013 TABLE 13 THE EFFECT OF COMPOUND 339 (5
MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED DECREASE IN LUNG
COMPLIANCE IN SENSITIZED GUINEA PIGS Lung Compliance Time Interval
(ml/cm H.sub.2O) after challenge Control 339 Baseline 0.548 .+-.
0.116 0.463 .+-. 0.026 OA/10 s 0.598 .+-. 0.129 0.442 .+-. 0.025 1
min 0.026 .+-. 0.005 0.172 .+-. 0.027* 2 min 0.018 .+-. 0.002 0.088
.+-. 0.018* 3 min 0.016 .+-. 0.002 0.060 .+-. 0.017* 4 min 0.019
.+-. 0.002 0.050 .+-. 0.013 5 min 0.021 .+-. 0.003 0.051 .+-. 0.011
10 min 0.043 .+-. 0.005 0.084 .+-. 0.012* 20 min .074 .+-. 0.007
0.123 .+-. 0.015* 30 min 0.093 .+-. 0.010 0.150 .+-. 0.012*
*Significant difference from control, P < 0.05.
[0693] The protective effects of compound 342 administered orally
on OA-induced contraction of tracheal tissue are summarized in
Tables 14-15 below. TABLE-US-00014 TABLE 14 THE EFFECT OF COMPOUND
342 (5 MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED INCREASE IN
LUNG RESISTANCE IN SENSITIZED GUINEA PIGS Lung Resistance Time
Interval (cm H.sub.2O/ml/sec) after challenge Control 342 Baseline
0.214 .+-. 0.010 0.212 .+-. 0.020 OA/10 s 0.204 .+-. 0.010 0.223
.+-. 0.020 1 min 2.380 .+-. 0.83 0.453 .+-. 0.120* 2 min 4.241 .+-.
1.04 1.786 .+-. 0.82* 3 min 4.657 .+-. 1.21 1.930 .+-. 0.55* 4 min
4.088 .+-. 1.42 1.621 .+-. 0.36* 5 min 4.519 .+-. 1.65 1.4816 .+-.
0.32* 10 min 1.821 .+-. 0.38 1.002 .+-. 0.14* 20 min 0.979 .+-.
0.23 0.524 .+-. 0.08* 30 min 0.703 .+-. 0.24 0.354 .+-. 0.04
*Significant difference from control, P < 0.05.
[0694] TABLE-US-00015 TABLE 15 THE EFFECT OF COMPOUND 342 (5
MG/KG/DAY FOR 4 DAYS; P.O.) ON ALLERGEN-INDUCED DECREASE IN LUNG
COMPLIANCE IN SENSITIZED GUINEA PIGS Lung Compliance Time Interval
(ml/cm H.sub.2O) after challenge Control 342 Baseline 0.441 .+-.
0.034 0.444 .+-. 0.037 OA/10 s 0.509 .+-. 0.057 0.464 .+-. 0.031 1
min 0.028 .+-. 0.007 0.154 .+-. 0.055* 2 min 0.027 .+-. 0.012 0.073
.+-. 0.038* 3 min 0.016 .+-. 0.004 0.044 .+-. 0.022* 4 min 0.017
.+-. 0.004 0.044 .+-. 0.022 5 min 0.018 .+-. 0.004 0.038 .+-. 0.015
10 min 0.034 .+-. 0.004 0.048 .+-. 0.005 20 min 0.054 .+-. 0.005
0.084 .+-. 0.005 30 min 0.074 .+-. 0.008 0.109 .+-. 0.006
*Significant difference from control, P < 0.05.
[0695] Effect of Selected Compounds on Allergen Induced Lung
Inflammation
[0696] The ability of a compound to inhibit the allergen-induced
accumulation of inflammatory cells such as eosinophils and
neutrophils in the lavage fluid obtained from sensitized animals is
indicative of anti-asthma activity. In particular, the model system
is useful in the evaluation of the effects of compounds in the
treatment of the late-phase response of asthma, when lung
inflammation and the second phase of bronchoconstriction is
apparent.
[0697] Male Brown Norway rats (200-250 g) are sensitized to
ovalbumin by an intraperitoneal injection of 1 mg ovalbumin and 100
mg aluminum hydroxide in 1 ml of sterile saline. After 21 days the
animals are found to be sensitized to ovalbumin. Animals are
treated with drug or vehicle (0.3 ml PEG-200) once daily for 4 days
by oral gavage. The animals are challenged by exposure, for a
period of 60 minutes, to nebulized solution of 0.5% ovalbumin in
saline generated using a Devillbis nebulizer. The final dose of
drug is given 24 hours after challenge. Forty eight hours after
challenge the animals are euthanized by an overdose of halothane
and the lungs are lavaged with 7.times.2 mLs of sterile saline
(room temperature). The recovered lavage fluid is placed on ice and
centrifuged at 1200 rpm to separate the cells from the supernatant.
The cells are exposed briefly to Tris/ammonium chloride, pH 7.3 to
remove any red cells, and washed in phosphate buffered saline.
Cytospins of each cell sample are prepared and stained for the
presence of cells containing peroxidase and for the determination
of the numbers of eosinophils and neutrophils. The numbers of
inflammatory cells are expressed as a percentage of the total
number of cells recovered in the lavage fluid. The protective
effect of compound 330 on allergen-induced lung inflammation is
summarized in Table 16. TABLE-US-00016 TABLE 16 EFFECT OF COMPOUND
330 (5 MG/KG/DAY FOR 4 DAYS, P.O.) ON OVALBUMIN-INDUCED
ACCUMULATION OF INFLAMMATORY CELLS IN THE LUNG LAVAGE FLUID
OBTAINED FROM SENSITIZED BROWN NORWAY RATS# Percentage Total Cells
Recovered in Lavage Fluid Cells Stained Positive Treatment for
Peroxidase Eosinophils Neutrophils Control 0.55 .+-. 0.27 0.69 .+-.
0.30 0.665 .+-. 0.31 OA Alone 36.03 .+-. 5.55 20.0 .+-. 2.65 11.58
.+-. 1.53 Cpd 330 + OA 5.65 .+-. 2.44* 1.98 .+-. 0.78* 6.16 .+-.
4.54 #Drug was administered in 300 .mu.L polyethyleneglycol-200
which was used as a vehicle. No-drug-treated animals received 300
.mu.L polyethyleneglycol-200 alone. *Significant difference from OA
alone, P < 0.05.
[0698] Effect of Selected Compounds in the Allergic Sheep Model of
Asthma
[0699] Effect of selected compounds in the allergic sheep model of
asthma were studied.
[0700] The allergic sheep model was used as it exhibits the
cardinal features associated with asthma. Such a model exhibits
natural allergy, early (acute) bronchoconstriction, late phase
bronchoconstriction, lung inflammation and bronchial
hyperresponsiveness. The model is conscious, where the animals are
breathing spontaneously, allowing measurement of airways
bronchoconstriction and acute airway hyperresponsiveness.
[0701] Sheep which were naturally sensitized to Ascaris suum (30-40
Kg) were intubated with an endotracheal tube and a balloon
catheter, positioned in the lower esophagus. Pleural pressure was
estimated with the esophageal catheter, while later pressure was
measured with a side-hole catheter advanced through and positioned
distal to the tip of the endotracheal tube. Transpulmonary pressure
differences between the trachea and pleural pressures were measured
with a differential pressure transducer catheter system.
[0702] The proximal end of the endotracheal tube was connected to a
Fleisch pneumotachograph in order to measure flow changes.
Pulmonary resistance (R.sub.L) was calculated from pressure
measurements of transpulmonary pressure, respiratory volume (from
digital integration of the flow signal) and flow by the mid-flow
technique. SR.sub.L was calculated as R.sub.LV.sub.tg
(V.sub.tg=thoracic gas volume).
[0703] Aerosols generated using a disposable nebulizer, were
directed into a T-piece connected to a Harvard respirator and the
tracheal tube in series. The aerosol delivery was controlled using
a dosimeter system, consisting of a solenoid valve and compressed
air (20 psi) activated at the start of each inspiratory cycle.
Aerosols were delivered in tidal volumes of 500 ml at 20 Hz.
[0704] Selected compounds from the invention were dissolved as a
stock solution in DMSO and diluted in saline. Animals received
either, 400 .mu.g/Kg of compound 30 min prior to challenge and 4
hours after challenge, or 400 .mu.g/Kg of compound for 4 days with
the last dose 2 hours before challenge. Ascaris suum extract was
diluted in phosphate buffered saline to a concentration of 82000
protein nitrogen units/ml and delivered by aerosol over 20 min.
Carbachol was dissolved in PBS to concentrations of 0.25, 0.5 1.0,
2.0, 4.0% wt/vol. Each animal served as its own control throughout
the study.
[0705] Specific lung resistance (SRL) was measured every 60 min for
8 hours after antigen challenge. Airways hyperresponsiveness to
carbachol was measured 24 hours after the initial challenge.
[0706] The protective effects of compound 330 administered acutely
(30 min prior to challenge and 4 hours after challenge; 400
.mu.g/Kg) on specific lung resistance and hyperresponsiveness are
summarized in Tables 17-18. TABLE-US-00017 TABLE 17 EFFECT OF
COMPOUND 330 (400 .mu.G/KG 30 MIN PRIOR TO CHALLENGE AND 4 HRS
AFTER CHALLENGE, BY INHALATION) ON ALLERGEN-INDUCED CHANGES IN
SPECIFIC LUNG RESISTANCE IN ASCARIS SUUM SENSITIZED SHEEP# Specific
Lung Time Interval Resistance after (% Baseline) Challenge (hr)
Control 330 Baseline 7 .+-. 10 2 .+-. 5 -0.5 7 .+-. 10 0 .+-. 4
Challenge (0) 266 .+-. 33 268 .+-. 35 1 197 .+-. 51 117 .+-. 11 2
77 .+-. 21 49 .+-. 11 3 68 .+-. 36 32 .+-. 6 4 23 .+-. 7 20 .+-. 6
5 73 .+-. 12 14 .+-. 4* 6 132 .+-. 29 14 .+-. 5* 6.5 126 .+-. 15 18
.+-. 6* 7 129 .+-. 16 10 .+-. 2* 7.5 156 .+-. 13 17 .+-. 8* 8 123
.+-. 27 5 .+-. 3* #Drug was administered in 3 mLs (66% DMSO in
saline). Vehicle alone had no effect. Animals were treated once 30
min prior to challenge and 4 hours post challenge. *Significantly
different from control, P < 0.05.
[0707] TABLE-US-00018 TABLE 18 EFFECT OF COMPOUND 330 (400 .mu.G/KG
30 MIN PRIOR TO CHALLENGE AND 4 HOURS AFTER CHALLENGE, BY
INHALATION) ON BRONCHIAL HYPERRESPONSIVENESS TO CARBACHOL IN
ASCARIS SUUM SENSITIZED SHEEP# PC.sub.400 (Breath Units)
Hyperresponsiveness Control 330 Baseline 26.65 .+-. 3.08 26.01 .+-.
3.06 Post-Challenge 12.28 .+-. 1.49 22.79 .+-. 6.11* #Drug was
administered in 3 mLs (66% DMSO in saline). Vehicle alone had no
effect. Animals were treated once 30 min prior to challenge and 4
hours post challenge. Hyperresponsiveness to carbachol was measured
24 hours after the initial challenge. *Significantly different from
control, P < 0.05.
[0708] Further studies demonstrated the protective effects of
compound 330 administered for 4 days (400 .mu.g/Kg) on specific
lung resistance and hyperresponsiveness are summarized in Tables
19-20. TABLE-US-00019 TABLE 19 EFFECT OF COMPOUND 330 (400
.mu.G/KG/DAY FOR 4 DAYS, BY INHALATION) ON ALLERGEN-INDUCED CHANGES
IN SPECIFIC LUNG RESISTANCE IN ASCARIS SUUM SENSITIZED SHEEP# Time
Interval Specific Lung after Resistance (% Baseline) Challenge (hr)
Control 330 Baseline 2.00 .+-. 2.00 5.25 .+-. 4.50 -0.5 2.00 .+-.
2.00 -3.75 .+-. 4.29 Challenge (0) 248.25 .+-. 85.71 126.00 .+-.
19.11 1 170.75 .+-. 58.62 34.00 .+-. 10.75 2 74.25 .+-. 17.10 11.50
.+-. 6.18* 3 82.00 .+-. 6.82 -15.00 .+-. 37.67* 4 21.25 .+-. 5.41
4.00 .+-. 1.78* 5 57.50 .+-. 7.51 -4.50 .+-. 4.17* 6 132.00 .+-.
9.68 7.75 .+-. 9.75* 6.5 153.75 .+-. 21.93 5.50 .+-. 4.87* 7 173.75
.+-. 21.74 10.75 .+-. 4.91* 7.5 148.00 .+-. 20.96 4.00 .+-. 2.35* 8
124.75 .+-. 28.53 3.25 .+-. 4.73* #Drug was administered in 3 mLs
(66% DMSO in saline). Vehicle alone had no effect. Animals were
treated for 4 days with the final dose 30 min prior to challenge.
*Significantly different from control, P < 0.05.
[0709] TABLE-US-00020 TABLE 20 EFFECT OF COMPOUND 330 (400
.mu.G/KG/DAY FOR 4 DAYS, BY INHALATION) ON BRONCHIAL
HYPERRESPONSIVENESS TO CARBACHOL IN ASCARIS SUUM SENSITIZED SHEEP#
PC.sub.400 (Breath Units) Hyperresponsiveness Control 330 Baseline
25.1 .+-. 1.54 21.26 .+-. 2.75 Post-Challenge 11.9 .+-. 1.09 21.21
.+-. 3.10* #Drug was administered in 3 mLs (66% DMSO in saline).
Vehicle alone had no effect. Animals were treated for 4 days with
the final dose 30 min prior to challenge. Hyperresponsiveness to
carbachol was measured 24 hours after the initial challenge.
*Significantly different from control, P < 0.05.
[0710] Effects of Selected Compounds on Transcription Factors
Involved in the Inflammatory Process
[0711] The hallmark of a number of chronic inflammatory diseases is
the activation of a number of genes known to be integral in
maintaining the inflammation state. Among these are cytokines,
chemokines, adhesion molecules, transcription factors and
proteases. Pivotal to the induced expression of many of these
pro-inflammatory molecules are a class of proteins called
transcription factors. One family of transcription factors known to
be key to a pro-inflammatory state is NF-.kappa.B. A number of
clinical disease states are associated with elevated levels of
activated NF-.kappa.B. These include atherosclerosis, cancers,
infectious diseases, and various inflammatory based diseases
including asthma, inflammatory bowel disease, arthritis,
ischemia/perfusion and inflammatory skin conditions. It was
discovered that compounds described in the invention caused
inhibition of NF-.kappa.B activation caused by phorbol esters
(activators of NF-.kappa.B).
[0712] Gel shift assays were used to examine the effect of selected
compounds in the invention on the activation of NF-.kappa.B, by
determining the level of binding of NF-.kappa.B to specific sites
on DNA. Oligonucleotides used to measure binding of NF-.kappa.B
were labeled by the following procedure. 5 .mu.l NF-.kappa.B
oligonucleotide (8.9 pmol), 2 .mu.l 10.times. T4-polynucleotide
kinase buffer, 10 units T4 polynucleotide kinase, and 1 ul
.gamma.-P-32-dATP (10 .mu.Ci) were made up to a final volume of 20
.mu.l with H.sub.2O. The reaction was incubated at 37.degree. C.
for 30 minutes. At this time the reaction was quenched with 2 .mu.l
0.5 M EDTA and 2 .mu.l 3M NaOAc (pH 5.2). 2.5.times. vol of 100%
EtOH was added and the resultant mixture centrifuged at 15,000 g
(eppendorf microfuge) for 10 minutes. The pellet was then washed
several times in 70% ethanol, air dried at room temperature for 10
minutes, and resuspended in double distilled H.sub.2O (final conc.
of 0.75 pmol/2 .mu.l). Cells (RBL-2H3 and A-549) were washed twice
in phosphate buffered saline (PBS) at room temperature. They were
scraped off the tissue culture dishes into 5 ml PBS using a cell
scraper and centrifuged (1500 rpm at room temp; Beckman GPR
centrifuge). Following the removal of the supernatant the cells
were resuspended in 2.times. pellet volume of Buffer A (0.25M
Sucrose, 20 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl.sub.2, 0.5 mM
DTT, 0.5 mM Spermidine, 0.15 mM Spermine). This was re-centrifuged
and the cells resuspended in the same buffer at a concentration of
10.sup.8 cells/ml. The cells were allowed to incubate at room
temperature for 5 minutes. Lysolecithin (10 mg/ml in Buffer A) was
added to a final concentration of 400 .mu.g/ml (4 .mu.l/100 .mu.l
Buffer A) and the suspension was incubated with gentle inversion
for no longer than 90 seconds. Cell lysis was rapidly stopped by
the addition of twice the vol. of ice-cold Buffer A containing 3%
BSA. Nuclei were collected by centrifugation at 4000 rpm for 1
minute at 4.degree. C. in a microfuge. The supernatant was removed
and the pellet resuspended in Buffer A containing 3% BSA before
centrifugation at 30,000 g for 60 seconds at 4.degree. C. (Beckman,
TL-100). The nuclei were resuspended in ice-cold Buffer B (20 mM
Hepes (pH 7.9), 25% v/v glycerol, 0.6 M KCl.sub.2, 1.5 mM
MgCl.sub.2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) at approximately
10.sup.7 nuclei/ml. The nuclei were disrupted by sonication on ice
with 2.times. five second pulses (40% intensity setting, MICROSON:
Ultrasonic cell disrupter). The homogenate was gently stirred on
ice for 30 minutes before centrifugation at 25,000 g at 4.degree.
C. in a Beckman TL-100. The supernatant was then removed and frozen
at -70.degree. C. if not used immediately. Determinations of NF-KB
DNA binding activity were conducted as follows; 2 .mu.l of
10.times. binding buffer {20 mM HEPES (pH 7.5), 50 mM KCl, 5 mM
MgCl.sub.2, 200 .mu.g/mg BSA (Sigma # B-2185), 8% glycerol}, 0.4
.mu.l Poly dI-dC (0.5 mg/ml stock), 2.0 .mu.l .sup.32P-labelled
oligo were mixed with 5 .mu.g of protein isolated from cell nuclei.
The resulting mix was made up to a final volume of 20 .mu.l with
distilled H.sub.2O. This was then incubated on ice for 5 minutes to
allow binding to occur. A further incubation (20 to 30 minutes) at
room temperature followed. Samples are then loaded onto a 4.5%
acrylamide gel {6 ml (29:1) acrylamide:bis, 2 ml 5.times. TBE
buffer, 800 .mu.l 150% glycerol, 31 ml distilled H.sub.2O, 150
.mu.l 10% APS (ammonium persulphate), 40 .mu.l TEMED}. Acrylamide
gels were pre-run in 0.25.times. TBE buffer for 1.5 hrs (10
volts/cm), followed by a buffer change prior to loading and running
of the actual samples.
[0713] The effect of selected compounds from the invention on
NF-.kappa.B activity, as determined using the gel-shift binding
assay, are shown in Table 21. TABLE-US-00021 TABLE 21 EFFECT OF
SELECTED COMPOUNDS ON NF-.kappa.B BINDING IN RBL-2H3 CELLS
STIMULATED WITH TPA (0.1 .mu.M)# Percent Inhibition of Treatment
Response to TPA (0.1 .mu.M) 165 (10 .mu.M) 54 330 (1 .mu.M) 66 333
(1 .mu.M) 34 339 (1 .mu.M) 65 #Compounds or vehicle (0.1% DMSO)
were preincubated with cells (RBL-2H3) for 2 hours prior to
stimulation with TPA. Cells were stimulated with 0.1 .mu.M TPA for
2.5 hours to activate NF-.kappa.B. All values shown are as a
percent inhibition of control (0.1 .mu.M TPA in the presence of
vehicle).
[0714] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually incorporated by reference.
[0715] From the foregoing, it will be evident that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
* * * * *