U.S. patent application number 12/709181 was filed with the patent office on 2010-07-15 for 2-alkylidene-18,19-dinor-vitamin d compounds.
This patent application is currently assigned to WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to Rafal Barycki, Hector F. DeLuca, Pawel Grzywacz, Lori A. Plum, Rafal R. Sicinski.
Application Number | 20100179344 12/709181 |
Document ID | / |
Family ID | 34960524 |
Filed Date | 2010-07-15 |
United States Patent
Application |
20100179344 |
Kind Code |
A1 |
DeLuca; Hector F. ; et
al. |
July 15, 2010 |
2-Alkylidene-18,19-Dinor-Vitamin D Compounds
Abstract
2-alkylidene-18,19-dinor-vitamin D compounds are disclosed as
well as pharmaceutical uses for these compounds and methods of
synthesizing these compounds. These compounds are characterized by
low bone calcium mobilization activity and high intestinal calcium
transport activity. This results in novel therapeutic agents for
the treatment and prophylaxis of diseases where bone formation is
desired, particularly osteoporosis, as well as autoimmune diseases
such as multiple sclerosis, diabetes mellitus and lupus. These
compounds also exhibit pronounced activity in arresting the
proliferation of undifferentiated cells and inducing their
differentiation to the monocyte thus evidencing use as an
anti-cancer agent and for the treatment of skin diseases such as
psoriasis. These compounds also increase both breaking strength and
crushing strength of bones evidencing use in conjunction with bone
replacement surgery such as hip and knee replacements.
Inventors: |
DeLuca; Hector F.;
(Deerfield, WI) ; Barycki; Rafal; (Madison,
WI) ; Grzywacz; Pawel; (Madison, WI) ; Plum;
Lori A.; (Arena, WI) ; Sicinski; Rafal R.;
(Warsaw, PL) |
Correspondence
Address: |
Andrus, Sceales, Starke & Sawall, LLP
100 East Wisconsin Avenue, Suite 1100
Milwaukee
WI
53202-4178
US
|
Assignee: |
WISCONSIN ALUMNI RESEARCH
FOUNDATION
Madison
WI
|
Family ID: |
34960524 |
Appl. No.: |
12/709181 |
Filed: |
February 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10821828 |
Apr 9, 2004 |
7713951 |
|
|
12709181 |
|
|
|
|
Current U.S.
Class: |
558/428 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 35/00 20180101; C07C 401/00 20130101; A61P 3/10 20180101; Y10S
514/825 20130101; Y02P 20/55 20151101; A61P 19/10 20180101; A61P
17/00 20180101; A61P 25/00 20180101; A61P 35/02 20180101; A61P
37/06 20180101; A61P 3/14 20180101; A61P 17/06 20180101; A61P 1/04
20180101 |
Class at
Publication: |
558/428 |
International
Class: |
C07C 255/53 20060101
C07C255/53 |
Claims
1. A compound having the formula: ##STR00013## where Y.sub.1 and
Y.sub.2, which may be the same or different, are each selected from
the group consisting of hydrogen and a hydroxy-protecting group,
R.sub.6 and R.sub.8, which may be the same or different, are each
selected from the group consisting of hydrogen, alkyl, hydroxyalkyl
and fluoroalkyl, or, when taken together represent the group
--(CH.sub.2).sub.x-- where x is an integer from 2 to 5, and where
the group R is represented by the structure: ##STR00014## where the
stereochemical center at carbon 20 may have the R or S
configuration, and where Z is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00015##
where m and n, independently, represent the integers from 0 to 5,
where R.sup.1 is selected from hydrogen, deuterium, hydroxy,
protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl,
which may be straight chain or branched and, optionally, bear a
hydroxy or protected-hydroxy substituent, and where each of
R.sup.2, R.sup.3, and R.sup.4, independently, is selected from
deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and
C.sub.1-5 alkyl, which may be straight-chain or branched, and
optionally, bear a hydroxy or protected-hydroxy substituent, and
where R.sup.1 and R.sup.2, taken together, represent an oxo group,
or an alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2)m-,
--(CH.sub.2)n-, or --(CR.sub.1R.sub.2)-- at positions 20, 22, and
23, respectively, may be replaced by an oxygen or sulfur atom.
2. The compound of claim 1 where R is a side chain of the formula
##STR00016##
3. The compound of claim 1 where R is a side chain of the formula
##STR00017##
4. The compound of claim 1 where R is a side chain of the formula
##STR00018##
5. The compound of claim 1 where R is a side chain of the formula
##STR00019##
6. The compound of claim 1 where R is a side chain of the formula
##STR00020##
7. The compound of claim 1 where R is a side chain of the formula
##STR00021##
8. The compound of claim 1 where R is a side chain of the formula
##STR00022##
9. The compound of claim 1 where R is a side chain of the formula
##STR00023##
10. The compound of claim 1 where R is a side chain of the formula
##STR00024##
11. The compound of claim 1 where R is a side chain of the formula
##STR00025##
12. (20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
13. A pharmaceutical composition containing an effective amount of
at least one compound as claimed in claim 1 together with a
pharmaceutically acceptable excipient.
14. The pharmaceutical composition of claim 13 wherein said
effective amount comprises from about 0.01 .mu.g to about 100 .mu.g
per gram of composition.
15. The pharmaceutical composition of claim 13 wherein said
effective amount comprises from about 0.1 .mu.g to about 50 .mu.g
per gram of composition.
16. The pharmaceutical composition of claim 13 containing
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
in an amount from about 0.01 .mu.g to about 100 .mu.g.
17. The pharmaceutical composition of claim 13 containing
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
in an amount from about 0.1 .mu.g to about 50 .mu.g.
18. A compound having the formula: ##STR00026##
19. A compound having the formula: ##STR00027## where X.sup.2 is
--H or a hydroxy protecting group.
20. A compound having the formula: ##STR00028##
21. A compound having the formula: ##STR00029## where X.sup.3 is
--H or a hydroxy protecting group.
22. A method of treating metabolic bone disease where it is desired
to maintain or increase bone mass comprising administering to a
patient with said disease an effective amount of a compound having
the formula: ##STR00030## where Y.sub.1 and Y.sub.2, which may be
the same or different, are each selected from the group consisting
of hydrogen and a hydroxy-protecting group, R.sub.6 and R.sub.8,
which may be the same or different, are each selected from the
group consisting of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl,
or, when taken together represent the group --(CH.sub.2).sub.x--
where x is an integer from 2 to 5, and where the group R is
represented by the structure: ##STR00031## where the stereochemical
center at carbon 20 may have the R or S configuration, and where Z
is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and
--CH.dbd.CHY, where the double bond may have the cis or trans
geometry, and where Y is selected from hydrogen, methyl,
--COR.sup.5 and a radical of the structure: ##STR00032## where m
and n, independently, represent the integers from 0 to 5, where
R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected
hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl, which may be
straight chain or branched and, optionally, bear a hydroxy or
protected-hydroxy substituent, and where each of R.sup.2, R.sup.3,
and R.sup.4, independently, is selected from deuterium,
deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5
alkyl, which may be straight-chain or branched, and optionally,
bear a hydroxy or protected-hydroxy substituent, and where R.sup.1
and R.sup.2, taken together, represent an oxo group, or an
alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,
--(CH.sub.2).sub.n-- or --(CR.sub.1R.sub.2)-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
23. The method of claim 22 where the disease is senile
osteoporosis.
24. The method of claim 22 where the disease is postmenopausal
osteoporosis.
25. The method of claim 22 where the disease is steroid-induced
osteoporosis.
26. The method of claim 22 where the disease is low bone turnover
osteoporosis.
27. The method of claim 22 where the disease is osteomalacia.
28. The method of claim 22 where the disease is renal
osteodystrophy.
29. The method of claim 22 wherein the compound is administered
orally.
30. The method of claim 22 wherein the compound is administered
parenterally.
31. The method of claim 22 wherein the compound is administered
transdermally.
32. The method of claim 22 wherein the compound is administered in
a dosage of from 0.01 .mu.g to 100 .mu.g per day.
33. The method of claim 22 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
34. A method of treating psoriasis comprising administering to a
patient with psoriasis an effective amount of a compound having the
formula: ##STR00033## where Y.sub.1 and Y.sub.2, which may be the
same or different, are each selected from the group consisting of
hydrogen and a hydroxy-protecting group, R.sub.6 and R.sub.8, which
may be the same or different, are each selected from the group
consisting of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or,
when taken together represent the group --(CH.sub.2).sub.x-- where
x is an integer from 2 to 5, and where the group R is represented
by the structure: ##STR00034## where the stereochemical center at
carbon 20 may have the R or S configuration, and where Z is
selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY,
where the double bond may have the cis or trans geometry, and where
Y is selected from hydrogen, methyl, --COR.sup.5 and a radical of
the structure: ##STR00035## where m and n, independently, represent
the integers from 0 to 5, where R.sup.1 is selected from hydrogen,
deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and
C.sub.1-5-alkyl, which may be straight chain or branched and,
optionally, bear a hydroxy or protected-hydroxy substituent, and
where each of R.sup.2, R.sup.3, and R.sup.4, independently, is
selected from deuterium, deuteroalkyl, hydrogen, fluoro,
trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or
branched, and optionally, bear a hydroxy or protected-hydroxy
substituent, and where R.sup.1 and R.sup.2, taken together,
represent an oxo group, or an alkylidene group,
.dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is
an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken
together, represent an oxo group, or the group
--(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where
R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,
--(CH.sub.2).sub.n-- or (CR.sub.1R.sub.2)-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
35. The method of claim 34 wherein the compound is administered
orally.
36. The method of claim 34 wherein the compound is administered
parenterally.
37. The method of claim 34 wherein the compound is administered
transdermally.
38. The method of claim 34 wherein the compound is administered
topically.
39. The method of claim 34 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
40. The method of claim 34 wherein said effective amount comprises
about 0.01 .mu.g/day to about 100 .mu.g/day of said compound.
41. A method of treating leukemia, colon cancer, breast cancer,
skin cancer or prostate cancer comprising administering to a
patient an effective amount of a compound having the formula:
##STR00036## where Y.sub.1 and Y.sub.2, which may be the same or
different, are each selected from the group consisting of hydrogen
and a hydroxy-protecting group, R.sub.6 and R.sub.8, which may be
the same or different, are each selected from the group consisting
of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or, when taken
together represent the group --(CH.sub.2).sub.x-- where x is an
integer from 2 to 5, and where the group R is represented by the
structure: ##STR00037## where the stereochemical center at carbon
20 may have the R or S configuration, and where Z is selected from
Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the
double bond may have the cis or trans geometry, and where Y is
selected from hydrogen, methyl, --COR.sup.5 and a radical of the
structure: ##STR00038## where m and n, independently, represent the
integers from 0 to 5, where R.sup.1 is selected from hydrogen,
deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and
C.sub.1-5-alkyl, which may be straight chain or branched and,
optionally, bear a hydroxy or protected-hydroxy substituent, and
where each of R.sup.2, R.sup.3, and R.sup.4, independently, is
selected from deuterium, deuteroalkyl, hydrogen, fluoro,
trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or
branched, and optionally, bear a hydroxy or protected-hydroxy
substituent, and where R.sup.1 and R.sup.2, taken together,
represent an oxo group, or an alkylidene group,
.dbd.CR.sup.2R.sup.3, or the group --(CH.sub.2).sub.p--, where p is
an integer from 2 to 5, and where R.sup.3 and R.sup.4, taken
together, represent an oxo group, or the group
--(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and where
R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,
--(CH.sub.2).sub.n-- or (CR.sub.1R.sub.2)-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
42. The method of claim 41 wherein the compound is administered
orally.
43. The method of claim 41 wherein the compound is administered
parenterally.
44. The method of claim 41 wherein the compound is administered
transdermally.
45. The method of claim 41 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 100 .mu.g/day.
46. The method of claim 41 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
47. A method of increasing the strength of a bone comprising
administering to a patient in need of such treatment an effective
amount of a compound having the formula: ##STR00039## where Y.sub.1
and Y.sub.2, which may be the same or different, are each selected
from the group consisting of hydrogen and a hydroxy-protecting
group, R.sub.6 and R.sub.8, which may be the same or different, are
each selected from the group consisting of hydrogen, alkyl,
hydroxyalkyl and fluoroalkyl, or, when taken together represent the
group --(CH.sub.2).sub.x-- where X is an integer from 2 to 5, and
where the group R is represented by the structure: ##STR00040##
where the stereochemical center at carbon 20 may have the R or S
configuration, and where Z is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00041##
where m and n, independently, represent the integers from 0 to 5,
where R.sup.1 is selected from hydrogen, deuterium, hydroxy,
protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl,
which may be straight chain or branched and, optionally, bear a
hydroxy or protected-hydroxy substituent, and where each of
R.sup.2, R.sup.3, and R.sup.4, independently, is selected from
deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and
C.sub.1-5 alkyl, which may be straight-chain or branched, and
optionally, bear a hydroxy or protected-hydroxy substituent, and
where R.sup.1 and R.sup.2, taken together, represent an oxo group,
or an alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,
--(CH.sub.2).sub.n-- or (CR.sub.1R.sub.2)-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
48. The method of claim 47 wherein the bone strength is cortical
strength.
49. The method of claim 47 wherein the bone strength is trabecular
strength.
50. The method of claim 47 wherein the compound is administered
orally.
51. The method of claim 47 wherein the compound is administered
parenterally.
52. The method of claim 47 wherein the compound is administered
transdermally.
53. The method of claim 47 wherein the compound is administered in
a dosage of from 0.01 .mu.g to 100 .mu.g per day.
54. The method of claim 47 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
55. A method of treating an autoimmune disease comprising
administering to a patient with said disease an effective amount of
a compound having the formula ##STR00042## where Y.sub.1 and
Y.sub.2 which may be the same or different, are each selected from
the group consisting of hydrogen and a hydroxy-protecting group,
R.sub.6 and R.sub.8, which may be the same or different, are each
selected from the group consisting of hydrogen, alkyl, hydroxyalkyl
and fluoroalkyl, or, when taken together represent the group
--(CH.sub.2).sub.x-- where x is an integer from 2 to 5, and where
the group R is represented by the structure: ##STR00043## where the
stereochemical center at carbon 20 may have the R or S
configuration, and where Z is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00044##
where m and n, independently, represent the integers from 0 to 5,
where R.sup.1 is selected from hydrogen, deuterium, hydroxy,
protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl,
which may be straight chain or branched and, optionally, bear a
hydroxy or protected-hydroxy substituent, and where each of
R.sup.2, R.sup.3, and R.sup.4, independently, is selected from
deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and
C.sub.1-5 alkyl, which may be straight-chain or branched, and
optionally, bear a hydroxy or protected-hydroxy substituent, and
where R.sup.1 and R.sup.2, taken together, represent an oxo group,
or an alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2)m-,
--(CH.sub.2)n-, or --(CR.sub.1R.sub.2)-- at positions 20, 22, and
23, respectively, may be replaced by an oxygen or sulfur atom.
56. The method of claim 55 where the disease is multiple
sclerosis.
57. The method of claim 55 where the disease is diabetes
mellitus.
58. The method of claim 55 where the disease is lupus.
59. The method of claim 55 wherein the compound is administered
orally.
60. The method of claim 55 wherein the compound is administered
parenterally.
61. Them method of claim 55 wherein the compound is administered
transdermally.
62. The method of claim 55 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 100 .mu.g/day.
63. The method of claim 55 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
64. A method of treating an inflammatory bowel disease comprising
administering to a patient with said disease an effective amount of
a compound having the formula ##STR00045## where Y.sub.1 and
Y.sub.2 which the same or different, are each selected from the
group consisting of hydrogen and a hydroxy-protecting group,
R.sub.6 and R.sub.8, which may be the same or different, are each
selected from the group consisting of hydrogen, alkyl, hydroxyalkyl
and fluoroalkyl, or, when taken together represent the group
--(CH.sub.2).sub.x-- where x is an integer from 2 to 5, and where
the group R is represented by the structure: ##STR00046## where the
stereochemical center at carbon 20 may have the R or S
configuration, and where Z is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00047##
where m and n, independently, represent the integers from 0 to 5,
where R.sup.1 is selected from hydrogen, deuterium, hydroxy,
protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl,
which may be straight chain or branched and, optionally, bear a
hydroxy or protected-hydroxy substituent, and where each of
R.sup.2, R.sup.3, and R.sup.4, independently, is selected from
deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and
C.sub.1-5 alkyl, which may be straight-chain or branched, and
optionally, bear a hydroxy or protected-hydroxy substituent, and
where R.sup.1 and R.sup.2, taken together, represent an oxo group,
or an alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2)m-,
--(CH.sub.2)n-, or --(CR.sub.1R.sub.2)-- at positions 20, 22, and
23, respectively, may be replaced by an oxygen or sulfur atom.
65. The method of claim 64 wherein the disease is Crohn's
disease.
66. The method of claim 64 wherein the disease is ulcerative
colitis.
67. The method of claim 64 wherein the compound is administered
orally.
68. The method of claim 64 wherein the compound is administered
parenterally.
69. The method of claim 64 wherein the compound is administered
transdermally.
70. The method of claim 64 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 100 .mu.g/day.
71. The method of claim 64 wherein the compound is
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3.
Description
[0001] This application is a division of application Ser. No.
10/821,828 filed Apr. 9, 2004, now U.S. Pat. No. ______.
BACKGROUND OF THE INVENTION
[0002] This invention relates to vitamin D compounds, and more
particularly to 2-alkylidene-18,19-dinor-vitamin D compounds,
pharmaceutical uses for these compounds and a general method for
chemically synthesizing these compounds.
[0003] The natural hormone, 1.alpha.,25-dihydroxyvitamin D.sub.3
and its analog in the ergosterol series, i.e.
1.alpha.,25-dihydroxyvitamin D.sub.2 are known to be highly potent
regulators of calcium homeostasis in animals and humans, and their
activity in cellular differentiation has also been established,
Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Many
structural analogs of these metabolites have been prepared and
tested, including 1.alpha.-hydroxyvitamin D.sub.3,
1.alpha.-hydroxyvitamin D.sub.2, various side chain homologated
vitamins and fluorinated analogs. Some of these compounds exhibit
an interesting separation of activities in cell differentiation and
calcium regulation. This difference in activity may be useful in
the treatment of a variety of diseases as renal osteodystrophy,
vitamin D-resistant rickets, osteoporosis, psoriasis, and certain
malignancies.
[0004] A particularly interesting class of vitamin D analogs are
referred to as the 19-nor-vitamin D compounds. The 19-nor-vitamin D
compounds are characterized by the replacement of the A-ring
exocyclic methylene group (carbon 19), typical of the vitamin D
system, by two hydrogen atoms. Biological testing of such
19-nor-analogs (e.g., 1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3)
revealed a selective activity profile with high potency in inducing
cellular differentiation, and very low calcium mobilizing activity.
Thus, these compounds are potentially useful as therapeutic agents
for the treatment of malignancies, or the treatment of various skin
disorders. Two different methods of synthesis of such
19-nor-vitamin D analogs have been described (Perlman et al.,
Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron
Lett. 32, 7663 (1991), and DeLuca et al., U.S. Pat. No.
5,086,191).
[0005] In U.S. Pat. No. 4,666,634, 2.beta.-hydroxy and alkoxy
(e.g., ED-71) analogs of 1.alpha.,25-dihydroxyvitamin D.sub.3 have
been described and examined by Chugai group as potential drugs for
osteoporosis and as antitumor agents. See also Okano et al.,
Biochem. Biophys. Res. Commun. 163, 1444 (1989). Other
2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkyl
groups) A-ring analogs of 1.alpha.,25-dihydroxyvitamin D.sub.3 have
also been prepared and tested (Miyamoto et al., Chem. Pharm. Bull.
41, 1111 (1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190
(1993); Posner et al., J. Org. Chem. 59, 7855 (1994), and J. Org.
Chem. 60, 4617 (1995)).
[0006] Recently, 2-substituted analogs of
1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 have also been
synthesized, i.e. compounds substituted at the 2-position of the
A-ring with hydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No.
5,536,713), with 2-alkyl groups (DeLuca et al U.S. Pat. No.
5,945,410), and with 2-alkylidene groups (DeLuca et al U.S. Pat.
No. 5,843,928), which exhibit interesting and selective activity
profiles. All these studies indicate that binding sites in vitamin
D receptors can accommodate different substituents at C-2 in the
synthesized vitamin D analogs.
[0007] Another class of known vitamin D compounds are the
18,19-dinor analogs. These analogs have both the C-18 angular
methyl substituent (carbon 18) normally attached to carbon 13 of
the CD-ring structure and the C-19 exocyclic methylene group
(carbon 19) normally attached to carbon 10 of the A-ring, which are
typical of all vitamin D compounds, removed and replaced by
hydrogen atoms. Reference should be made to the U.S. Pat. No.
5,843,927 as well as U.S. Pat. Nos. 5,756,489 and 5,721,225 for a
more complete description of these compounds, their pharmaceutical
uses, and their synthesis.
SUMMARY OF THE INVENTION
[0008] In a continuing effort to explore the 19-nor class of
pharmacologically important vitamin D compounds, the present
invention is directed toward 2-alkylidene-18,19-dinor-vitamin D
analogs, various pharmaceutical uses for these compounds, and a
general method for chemically synthesizing these compounds. In
particular, the present invention is directed toward
(20S)-2-methylene-1.alpha.,25-dihydroxy-18,19-dinor-vitamin
D.sub.3, its biological activity, and various pharmaceutical uses
for this compound.
[0009] Structurally these novel 2-alkylidene-18,19-dinor-vitamin D
analogs are characterized by the general formula I shown below:
##STR00001##
[0010] where Y.sub.1 and Y.sub.2, which may be the same or
different, are each selected from the group consisting of hydrogen
and a hydroxy-protecting group, R.sub.6 and R.sub.8, which may be
the same or different, are each selected from the group consisting
of hydrogen, alkyl, hydroxyalkyl and fluoroalkyl, or, when taken
together represent the group --(CH.sub.2).sub.x-- where X is an
integer from 2 to 5, and where the group R represents any of the
typical side chains known for vitamin D type compounds.
[0011] More specifically R can represent a saturated or unsaturated
hydrocarbon radical of 1 to 35 carbons, that may be straight-chain,
branched or cyclic and that may contain one or more additional
substituents, such as hydroxy- or protected-hydroxy groups, fluoro,
carbonyl, ester, epoxy, amino or other heteroatomic groups.
Preferred side chains of this type are represented by the structure
below
##STR00002##
[0012] where the stereochemical center (corresponding to C-20 in
steroid numbering) may have the R or S configuration, (i.e. either
the natural configuration about carbon 20 or the 20-epi
configuration), and where Z is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure:
##STR00003##
[0013] where m and n, independently, represent the integers from 0
to 5, where R.sup.1 is selected from hydrogen, deuterium, hydroxy,
protected hydroxy, fluoro, trifluoromethyl, and C.sub.1-5-alkyl,
which may be straight chain or branched and, optionally, bear a
hydroxy or protected-hydroxy substituent, and where each of
R.sup.2, R.sup.3, and R.sup.4, independently, is selected from
deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyl and
C.sub.1-5 alkyl, which may be straight-chain or branched, and
optionally, bear a hydroxy or protected-hydroxy substituent, and
where R.sup.1 and R.sup.2, taken together, represent an oxo group,
or an alkylidene group, .dbd.CR.sup.2R.sup.3, or the group
--(CH.sub.2).sub.p--, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q--, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --(CH.sub.2).sub.m--,
(CH.sub.2).sub.n or --CR.sup.1R.sup.2-- at positions 20, 22, and
23, respectively, may be replaced by an oxygen or sulfur atom.
[0014] The wavy line to the methyl substituent at C-20 indicates
that carbon 20 may have either the R or S configuration, i.e. the
natural configuration (20R) or the unnatural 20-epi configuration
(20S).
[0015] Specific important examples of side chains with natural
20R-configuration are the structures represented by formulae (a),
b), (c), (d) and (e) below. i.e. the side chain as it occurs in
25-hydroxyvitamin D.sub.3 (a); vitamin D.sub.3 (b);
25-hydroxyvitamin D.sub.2 (c); vitamin D.sub.2 (d); and the C-24
epimer of 25-hydroxyvitamin D.sub.2 (e):
##STR00004##
[0016] The above novel 2-alkylidene-18,19-dinor vitamin D compounds
exhibit a desired, and highly advantageous, pattern of biological
activity. These compounds are characterized by relatively high
intestinal calcium transport activity, i.e. similar to that of
1.alpha.,25-dihydroxyvitamin D.sub.3, while also exhibiting
relatively low activity, as compared to
1.alpha.,25-dihydroxyvitamin D.sub.3, in their ability to mobilize
calcium from bone. Hence, these compounds are highly specific in
their calcemic activity. Their preferential activity on intestinal
calcium transport and reduced calcium mobilizing activity allows
the in vivo administration of these compounds for the treatment and
prophylaxis of metabolic bone diseases where bone loss is a major
concern. Because of their preferential calcemic activity on gut
calcium transport, these compounds would be preferred therapeutic
agents for the treatment and prophylaxis of diseases where bone
formation is desired, such as osteoporosis, especially low bone
turnover osteoporosis, steroid induced osteoporosis, senile
osteoporosis or postmenopausal osteoporosis, as well as
osteomalacia and renal osteodystrophy. The compounds may be
administered transdermally, orally or parenterally. The compounds
may be present in a pharmaceutical composition in an amount from
about 0.01 .mu.g/gm to about 100 .mu.g/gm of the composition,
preferably from about 0.1 .mu.g/gm to about 50 .mu.g/gm of the
composition, and may be administered in dosages of from about 0.01
.mu.g/day to about 100 .mu.g/day, preferably from about 0.1
.mu.g/day to about 50 .mu.g/day.
[0017] The compounds of the invention are also especially suited
for treatment and prophylaxis of human disorders which are
characterized by an imbalance in the immune system, e.g. in
autoimmune diseases, including multiple sclerosis, diabetes
mellitus, lupus, host versus graft reaction, and rejection of
transplants; and additionally for the treatment and prophylaxis of
inflammatory diseases, such as rheumatoid arthritis, asthma, and
inflammatory bowel diseases such as Crohn's disease or ulcerative
colitis, as well as the improvement of bone fracture healing and
improved bone grafts. It has also been discovered that these
compounds increase breaking strength (cortical strength) as well as
crushing strength (trabecular strength) of bones. Thus, these
compounds could also be used in conjunction with bone replacement
procedures such as hip replacements, knee replacements, and the
like. Acne, alopecia, skin conditions such as dry skin (lack of
dermal hydration), undue skin slackness (insufficient skin
firmness), insufficient sebum secretion and wrinkles, and
hypertension are other conditions which may be treated with the
compounds of the invention.
[0018] The above compounds are also characterized by high cell
differentiation activity. Thus, these compounds also provide
therapeutic agents for the treatment of psoriasis, or as an
anti-cancer agent, especially against leukemia, colon cancer,
breast cancer, skin cancer and prostate cancer. The compounds may
be present in a composition to treat psoriasis in an amount from
about 0.01 .mu.g/gm to about 100 .mu.g/gm of the composition,
preferably from about 0.1 .mu.g/gm to about 50 .mu.g/gm of the
composition, and may be administered topically, transdermally,
orally or parenterally in dosages of from about 0.01 .mu.g/day to
about 100 .mu.g/day, preferably from about 01 .mu.g/day to about 50
.mu.g/day.
[0019] In particular,
2-methylene-18,19-dinor-(20S)-1.alpha.,25-dihydroxy-vitamin D.sub.3
has been synthesized and its binding, transcriptional, calcemic
(both intestinal calcium transport and bone calcium mobilization)
and differentiation activities determined. Structurally this
18,19-dinor analog is characterized by the general formula Ia shown
below:
##STR00005##
[0020] The invention also provides a novel synthesis for the
production of the end products of formula I, and specifically of
formula Ia. In addition, this invention provides novel intermediate
compounds formed during the synthesis of the end products.
Structurally, these novel intermediates are characterized by the
general formulae IV, V, VI and VII, below where X.sup.1 may be --H
or --NO, and X.sup.2 and X.sup.3 may be --H or a hydroxy protecting
group.
##STR00006##
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a graph illustrating the relative activity of
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001) as well as the herein
described and claimed
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) in binding to the 1.alpha.,25-dihydroxyvitamin D pig
intestinal nuclear receptor;
[0022] FIG. 2 is a graph illustrating the percent HL-60 cell
differentiation as a function of the concentration of
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001),
(20S)-2-methylene-19-nor-1.alpha.,25-dihydroxyvitamin D.sub.3 (2MD)
and (20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3 (DP035);
[0023] FIG. 3 is a graph illustrating the transcriptional activity
as a function of the concentration of 1.alpha.,25-dihydroxyvitamin
(C001), (20S)-2-methylene-19-nor-1.alpha.,25-dihydroxyvitamin
D.sub.3 (2MD) and
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035);
[0024] FIG. 4 is a bar graph illustrating the intestinal calcium
transport activity of
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) at various dosages as compared to control (vehicle) and
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001);
[0025] FIG. 5 is a bar graph illustrating the bone calcium
mobilization activity of
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) at various dosages as compared to control (vehicle) and
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001); and
[0026] FIG. 6 is a bar graph illustrating the bone calcium
mobilization activity of
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) as compared to 1.alpha.,25-dihydroxyvitamin D.sub.3 (C001)
at various dosages.
DETAILED DESCRIPTION OF THE INVENTION
[0027] As used in the description and in the claims, the term
"hydroxy-protecting group" signifies any group commonly used for
the temporary protection of hydroxy functions, such as for example,
alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups
(hereinafter referred to simply as "silyl" groups), and alkoxyalkyl
groups. Alkoxycarbonyl protecting groups are alkyl-O--CO--
groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,
tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The
term "acyl" signifies an alkanoyl group of 1 to 6 carbons, in all
of its isomeric forms, or a carboxyalkanoyl group of 1 to 6
carbons, such as an oxalyl, malonyl, succinyl, glutaryl group, or
an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl
substituted benzoyl group. The word "alkyl" as used in the
description or the claims, denotes a straight-chain or branched
alkyl radical of 1 to 10 carbons, in all its isomeric forms.
Alkoxyalkyl protecting groups are groupings such as methoxymethyl,
ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and
tetrahydropyranyl. Preferred silyl-protecting groups are
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl,
diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The
term "aryl" specifies a phenyl-, or an alkyl-, nitro- or
halo-substituted phenyl group.
[0028] A "protected hydroxy" group is a hydroxy group derivatised
or protected by any of the above groups commonly used for the
temporary or permanent protection of hydroxy functions, e.g. the
silyl, alkoxyalkyl, acyl or alkoxycarbonyl groups, as previously
defined. The terms "hydroxyalkyl", "deuteroalkyl" and "fluoroalkyl"
refer to an alkyl radical substituted by one or more hydroxy,
deuterium or fluoro groups respectively.
[0029] It should be noted in this description that the term
"24-homo" refers to the addition of one methylene group and the
term "24-dihomo" refers to the addition of two methylene groups at
the carbon 24 position in the side chain. Likewise, the term
"trihomo" refers to the addition of three methylene groups. Also,
the term "26,27-dimethyl" refers to the addition of a methyl group
at the carbon 26 and 27 positions so that for example R.sup.3 and
R.sup.4 are ethyl groups. Likewise, the term "26,27-diethyl" refers
to the addition of an ethyl group at the 26 and 27 positions so
that R.sup.3 and R.sup.4 are propyl groups.
[0030] In the following lists of side chain unsaturated and side
chain saturated compounds, if the methyl group attached at the
carbon 20 position is in its epi or unnatural configuration, the
term "20(S)" or "20-epi" should be included in each of the
following named compounds. Also, if the side chain contains an
oxygen atom substituted at any of positions 20, 22 or 23, the term
"20-oxa," "22-oxa" or "23-oxa," respectively, should be added to
the named compound. The named compounds could also be of the
vitamin D.sub.2 type if desired.
[0031] Specific and preferred examples of the
2-alkylidene-18,19-dinor-vitamin D compounds of structure I when
the side chain is unsaturated are: [0032]
2-methylene-18,19-dinor-1.alpha.-hydroxy-22-dehydrovitamin D.sub.3;
[0033] 2-methylene-18,19-dinor-25-hydroxy-22-dehydrovitamin
D.sub.3; [0034]
2-methylene-18,19-dinor-1.alpha.,25-dihydroxy-22-dehydrovitamin
D.sub.3; [0035]
2-methylene-18,19-dinor-24-homo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3; [0036]
2-methylene-18,19-dinor-24-dihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3; [0037]
2-methylene-18,19-dinor-24-trihomo-1,25-dihydroxy-22-dehydrovitamin
D.sub.3; [0038]
2-methylene-18,19-dinor-26,27-dimethyl-24-homo-1,25-dihydroxy-22-dehydrov-
itamin D.sub.3; [0039]
2-methylene-18,19-dinor-26,27-dimethyl-24-dihomo-1,25-dihydroxy-22-dehydr-
ovitamin D.sub.3; [0040]
2-methylene-18,19-dinor-26,27-dimethyl-24-trihomo-1,25-dihydroxy-22-dehyd-
rovitamin D.sub.3; [0041]
2-methylene-18,19-dinor-26,27-diethyl-24-homo-1,25-dihydroxy-22-dehydrovi-
tamin D.sub.3; [0042]
2-methylene-18,19-dinor-26,27-diethyl-24-dihomo-1,25-dihydroxy-22-dehydro-
vitamin D.sub.3; [0043]
2-methylene-18,19-dinor-26,27-diethyl-24-trihomo-1,25-dihydroxy-22-dehydr-
ovitamin D.sub.3; [0044]
2-methylene-18,19-dinor-26,27-dipropyl-24-homo-1,25-dihydroxy-22-dehydrov-
itamin D.sub.3; [0045]
2-methylene-18,19-dinor-26,27-dipropyl-24-dihomo-1,25-dihydroxy-22-dehydr-
ovitamin D.sub.3; and [0046]
2-methylene-18,19-dinor-26,27-dipropyl-24-trihomo-1,25-dihydroxy-22-dehyd-
rovitamin D.sub.3.
[0047] With respect to the above unsaturated compounds, it should
be noted that the double bond located between the 22 and 23 carbon
atoms in the side chain may be in either the (E) or (Z)
configuration. Accordingly, depending upon the configuration, the
term "22,23(E)" or "22,23(Z)" could be included in each of the
above named compounds. Also, it is common to designate the double
bond located between the 22 and 23 carbon atoms with the
designation ".DELTA..sup.22". Thus, for example, the fourth named
compound above could also be written as
2-methylene-18,19-dinor-24-homo-22,23(E)-.DELTA..sup.22-1,25-(OH).sub.2D.-
sub.3 where the double bond is the (E) configuration. Similarly, if
the methyl group attached at carbon 20 is in the unnatural
configuration, this compound could be written as
2-methylene-18,19-dinor-20(S)-24-homo-22,23(E)-.DELTA..sup.22-1,25-(OH).s-
ub.2D.sub.3.
[0048] Specific and preferred examples of the
2-alkylidene-18,19-dinor-vitamin D compounds of structure I when
the side chain is saturated are: [0049]
2-methylene-18,19-dinor-1.alpha.-hydroxyvitamin D.sub.3; [0050]
2-methylene-18,19-dinor-25-hydroxyvitamin D.sub.3; [0051]
2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3;
[0052] 2-methylene-18,19-dinor-24-homo-1,25-dihydroxyvitamin
D.sub.3; [0053]
2-methylene-18,19-dinor-24-dihomo-1,25-dihydroxyvitamin D.sub.3;
[0054] 2-methylene-18,19-dinor-24-trihomo-1,25-dihydroxyvitamin
D.sub.3; [0055]
2-methylene-18,19-dinor-26,27-dimethyl-24-homo-1,25-dihydroxyvitamin
D.sub.3; [0056]
2-methylene-18,19-dinor-26,27-dimethyl-24-dihomo-1,25-dihydroxyvitamin
D.sub.3; [0057]
2-methylene-18,19-dinor-26,27-dimethyl-24-trihomo-1,25-dihydroxyvitamin
D.sub.3; [0058]
2-methylene-18,19-dinor-26,27-diethyl-24-homo-1,25-dihydroxyvitamin
D.sub.3; [0059]
2-methylene-18,19-dinor-26,27-diethyl-24-dihomo-1,25-dihydroxyvitamin
D.sub.3; [0060]
2-methylene-18,19-dinor-26,27-diethyl-24-trihomo-1,25-dihydroxyvitamin
D.sub.3; [0061]
2-methylene-18,19-dinor-26,27-dipropyl-24-homo-1,25-dihydroxyvitamin
D.sub.3; [0062]
2-methylene-18,19-dinor-26,27-dipropyl-24-dihomo-1,25-dihydroxyvitamin
D.sub.3; and [0063]
2-methylene-18,19-dinor-26,27-dipropyl-24-trihomo-1,25-dihydroxyvitamin
D.sub.3.
[0064] The preparation of 2-alkylidene-18,19-dinor vitamin D
compounds having the structure I is based on the Wittig-Horner
reaction of an 18-nor-CD-ring ketone (see (a) Baggiolini et al, J.
Org. Chem., 1986, 51, 3098-3108; (b) Baggiolini et al, J. Am. Chem.
Soc., 1982, 104, 2945-2948; and (c) Cohen et al, J. Org. Chem.,
1979, 44, 3077-3080) and a phosphine oxide, i.e. the condensation
of a bicyclic 18-nor-CD-ring type ketone II with an allylic
phosphine oxide III to the corresponding 2-alkylidene-18,19-dinor
vitamin D analog I followed by deprotection at C1 and C-3 in the
latter compounds:
##STR00007##
[0065] In the structures I, II and III groups R.sub.6 and R.sub.8,
Y.sub.1 and Y.sub.2, and R represent groups defined above; Y.sub.1
and Y.sub.2 are preferably hydroxy-protecting groups such as
tert-butyldimethylsilyl (TBDMS), it being also understood that any
functionalities in R that might be sensitive, or that interfere
with the condensation reaction, be suitably protected as is
well-known in the art. The process shown above represents an
application of the convergent synthesis concept, which has been
applied effectively for the preparation of vitamin D compounds
[e.g. Lythgoe et al., J. Chem. Soc. Perkin Trans. I, 590 (1978);
Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Toh et al., J. Org. Chem.
48, 1414 (1983); Baggiolini et al., J. Org. Chem. 51, 3098 (1986);
Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org. Chem. 51,
1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca et al.,
U.S. Pat. No. 5,536,713].
[0066] Hydrindanones of the general structure II can be prepared
starting from vitamin D.sub.2 by the method of SCHEME 1 disclosed
hereinafter. Specific important examples of such bicyclic 18-nor-CD
ketones are the structures with the side chains (a), (b), (c), (d)
and (e) described above, i.e. 25-hydroxy ketone (f); ketone (g);
25-hydroxy ketone (h); ketone (i) and 24-epi ketone (j). Other
important 18-nor CD ketones of general structure II are the
structures with the side chains (f) through (j) wherein the
20-methyl group is in its unnatural 20-epi configuration, i.e.
ketones (k) through (o).
##STR00008## ##STR00009##
[0067] For the preparation of the required phosphine oxides of
general structure III, a synthetic route has been developed
starting from a diol, easily obtained from commercial
(1R,3R,4S,5R)-(-)-quinic acid as described by Sicinski et al, J.
Med. Chem., 1998, 41, 4462-4674. The overall process of
transformation of the starting diol into the desired
2-alkylidene-A-ring synthon of general structure III, and more
particularly, the 2-methylene-A-ring synthon 15 shown in SCHEME 1,
is summarized and illustrated in U.S. Pat. No. 6,843,928, the
description of which is specifically incorporated herein by
reference. Thus, the starting diol will be oxidized with ruthenium
tetroxide to the corresponding hydroxyketone. The latter compound
will be treated with an ylide prepared from
methyltriphenylphosphonium bromide and n-butyllithium. The product
of the Wittig reaction will be reduced by lithium aluminum hydride
to a vicinal diol, which will be cleaved by sodium periodate, and
the resulting ketone will be converted to an unsaturated ester by
the Peterson olefination with methyl (trimethylsilyl)acetate. The
ester will then be reduced with DIBALH to an allylic alcohol which
will be in situ tosylated with n-butyllithium and p-toluenesulfonyl
chloride, converted into the corresponding phosphine by a reaction
with diphenylphosphine lithium salt, and oxidized with hydrogen
peroxide to the desired A-ring phosphine oxide 15. The
Wittig-Horner coupling of the two fragments 14 and 15, to give the
protected vitamin compound 16, followed by the deprotection of
hydroxy groups in any known manner such as with tetrabutylammonium
fluoride, will give the final analog 17.
[0068] Numerous 2-alkylidene-18,19-dinor-vitamin D compounds of the
general structure I may be synthesized using the A-ring synthon III
and the appropriate 18-nor-CD-ring ketone II having the desired
side chain structure R. Thus, for example, Wittig-Horner coupling
of the A-ring phosphine oxide 15 with n-butyllithium and any of the
ketones (f), (g), (h), (i), (j), (k), (l), (m), (n) and (o)
previously illustrated herein (or any other ketone with the desired
side chain defined by R) may be performed as illustrated in SCHEME
1 to give the respective protected vitamin compound. This, after
deprotection then affords the desired
2-methylene-18,19-dinor-vitamin D analog having the desired side
chain structure R.
[0069] The C-20 epimerization may be accomplished by the analogous
coupling of the phosphine oxide of structure III with the
appropriate protected (20S)--CD-ring ketone of structure II which
after hydrolysis of the hydroxy-protecting groups will give the
desired (20S)-2-alkylidene-18,19-dinor-vitamin D analog having the
desired side chain structure R.
[0070] As noted above, other 2-alkylidene-18,19-dinor-vitamin D
analogs may be synthesized by the method disclosed herein. For
example, 1.alpha.-hydroxy-2-methylene-18,19-dinor-vitamin D.sub.3
can be obtained by providing the CD-ring ketone (g).
[0071] This invention is described by the following illustrative
examples. In these examples specific products identified by Arabic
numerals (e.g. 1, 2, 3, etc) refer to the specific structures so
identified in the preceding description and in SCHEME 1.
Example 1
Preparation of
(20S)-2-methylene-1.alpha.,25-dihydroxy-18,19-dinor-vitamin D.sub.3
(17) via condensation (SCHEME I)
[0072] Des-A,B-23,24-dinorcholane-8.beta.,22-diol (1). A solution
of vitamin D.sub.2 (5 g, 12.7 mmol) in methanol (400 mL) and
pyridine (5 mL) was cooled to -78.degree. C. while purging with
argon. The argon stream was stopped and a stream of ozone was
passed until a blue color appeared. The solution was purged with
oxygen until blue color disappeared and treated with NaBH.sub.4
(1.2 g, 32 mmol). After 20 min. the second portion of NaBH.sub.4
(1.2 g, 32 mmol) was added and reaction was allowed to warm to room
temperature. The third portion of NaBH.sub.4 (1.2 g, 32 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. The reaction was quenched with 70 mL of water and
concentrated under vacuum. The residue was extracted with methylene
chloride (3.times.100 mL). The organic phase was washed with 1M
aqueous solution of HCl (2.times.100 mL), saturated aqueous
solution of NaHCO.sub.3 (100 mL), dried over anhydrous MgSO.sub.4
and concentrated under vacuum. The residue was purified by flash
chromatography (25% ethyl acetate/hexane) to yield 1.875 g (8.84
mmol, 70% yield) of diol 1 as white crystals. [.alpha.].sub.D+56.0
(c 0.95, CHCl.sub.3); mp 110-111.degree. C.; .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 0.96 (3H, s), 1.03 (3H, d, J=6.6 Hz), 3.38 (1H,
dd, J=10.5 Hz, J=6.8 Hz), 3.64 (1H, dd, J=10.5 Hz, J=3.2 Hz), 4.09
(1H, d, J=2.3 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) 13.6, 16.6,
17.4, 22.6, 26.6, 33.5, 38.2, 40.2, 41.3, 52.3, 52.9, 67.8, 69.2;
MS (EI) m/z 212 (2, M.sup.+), 194 (17), 179 (18), 163 (10), 135
(19), 125 (34), 111 (100); exact mass calculated for
C.sub.13H.sub.22O ([M-H.sub.2O].sup.+) 194.1671, found
194.1665.
[0073] Des-A,B-8.beta.-(benzoyloxy)-23,24-dinorcholane-22-ol (2).
Diol 1 (1.85 g, 8.79 mmol) was dissolved in pyridine (30 mL) and
DMAP (45 mg, 0.3 mmol) was added. The solution was cooled to
0.degree. C. then benzoyl chloride (3 mL, 3.6 g, 25 mmol) was added
dropwise. The reaction mixture was kept at 5.degree. C. for 24 h.
Methylene chloride (100 mL) was added and the resulting mixture was
washed with 5% aqueous solution of HCl (100 mL), saturated aqueous
solution of CuSO.sub.4 (2.times.80 mL), saturated aqueous solution
of NaHCO.sub.3 (80 mL) and water (100 mL). The extract was dried
over anhydrous MgSO.sub.4. Removal of the solvent in vacuo afforded
a crude dibenzoate.
[0074] The crude dibenzoate (5.05 g) was added at room temperature
to a solution of KOH (87%, 1.5 g, 23.3 mmol) in absolute ethanol
(30 mL). The resulting reaction mixture was stirred at room
temperature for 3 h 20 min. Then the reaction mixture was quenched
with ice and neutralized with 5% aqueous solution of HCl. The
reaction mixture was extracted with methylene chloride (3.times.60
mL) The combined organic phases were washed with saturated aqueous
solution of NaHCO.sub.3 (50 mL) and dried over anhydrous
MgSO.sub.4. Drying agent was removed and solvent was evaporated in
vacuo. Pure product was obtained by column chromatography (25%
ethyl acetate/hexane) to give 2.58 g (8.16 mmol, 93% yield from
diol 1) of monobenzoate 2. [.alpha.].sub.D+65.2 (c 1.15,
CHCl.sub.3); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.39 (1H,
dd, J=10.4 Hz, J=6.8 Hz), 3.65 (1H, dd, J=10.5 Hz, J=3.2 Hz), 5.42
(1H, br d, J=22.2 Hz), 7.45 (2H, m), 7.56 (1H, m), 8.05 (2H, m);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.6, 16.6, 18.0, 22.7,
26.6, 30.5, 38.4, 39.8, 41.9, 51.4, 52.7, 67.7, 72.1, 128.3, 129.5,
130.8, 166.5; MS (EI) m/z 211 (4), 194 (52), 179 (11), 135 (41),
108 (23), 105 (100); exact mass (ESI) calculated for
C.sub.20H.sub.28O.sub.3Na ([M+Na].sup.+) 339.1936, found
339.1941.
[0075] Des-A,B-8.beta.-(benzoyloxy)-23,24-dinorcholane-22-al (3).
Sulfur trioxide pyridine complex (7.02 g, 44.1 mmol) was added to a
solution of alcohol 2 (2.32 g, 7.34 mmol) and triethylamine (5.15
mL, 3.71 g, 36.7 mmol) in anhydrous methylene chloride (30 mL) and
DMSO (8 mL) at 0.degree. C. The reaction mixture was stirred under
argon for 20 min. at 0.degree. C. and then concentrated in vacuo.
The residue was purified by column chromatography (5% ethyl
acetate/hexane) to give 2.05 g (6.53 mmol, 90% yield) of aldehyde
3. [.alpha.].sub.D+67.4 (c 0.95, CHCl.sub.3); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 1.10 (3H, s), 1.15 (3H, d, J=6.8 Hz), 5.44 (1H,
br d, J=2.2 Hz), 7.45 (2H, m), 7.56 (1H, m), 8.05 (2H, m), 9.60
(1H, d, J=3.2 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.6,
14.1, 18.1, 23.1, 26.2, 30.7, 39.8, 42.6, 49.2, 51.2, 51.5, 128.6,
129.7, 130.9, 133.0, 205.0; MS (EI) m/z 285 (3), 216 (3), 208 (9),
180 (17), 162 (47), 147 (21), 135 (46), 122 (16), 105 (100), 95
(22), 77 (49); exact mass (ESI) calculated for
C.sub.19H.sub.25O.sub.2 ([M-CHO].sup.+) 285.1855, found
285.1848.
[0076] (20R)-Des-A,B-8.beta.-(benzoyloxy)-23,24-dinorcholane-22-ol
(4). To a solution of aldehyde 3 (2.05 g, 6.53 mmol) in methylene
dichloride (25 mL), 40% aqueous solution of n-Bu.sub.4NOH (8.4 mL,
12.9 mmol) was added. The resulting reaction mixture was vigorously
stirred overnight. Methylene dichloride (30 mL) was then added and
the mixture was washed with water (20 mL), dried over anhydrous
MgSO.sub.4 and concentrated under reduced pressure. The residue was
purified by column chromatography (5% ethyl acetate/hexane) to give
1.50 g (4.78 mmol) of the mixture of diastereoisomeric
aldehydes.
[0077] The mixture of aldehydes was dissolved in ethanol (15 mL)
and NaBH.sub.4 (350 mg, 9.2 mmol) was added. The resulting mixture
was stirred for 30 min. The reaction mixture was quenched with
saturated aqueous solution of NH.sub.4Cl (30 mL). The mixture was
extracted with methylene dichloride (3.times.40 mL) and the
combined organic phases were washed with water (30 mL), dried over
anhydrous MgSO.sub.4 and concentrated under reduced pressure. The
residue was purified by column chromatography (5% ethyl
acetate/hexane) to give 870 mg (2.75 mmol, 42% yield) of 4 and 437
mg (1.38 mmol, 21% yield) of 2. [.alpha.].sub.D+50.0 (c 1.10,
CHCl.sub.3); .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.97 (3H, d,
J=6.7 Hz), 1.07 (3H, s), 3.48 (1H, dd, J=10.5 Hz, J=7.1 Hz), 3.76
(1H, dd, J=10.6 Hz, J=3.5 Hz), 5.42 (1H, s), 7.45 (2H, m), 7.55
(1H, m), 8.05 (2H, m); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.
13.9, 16.5, 18.0, 22.5, 26.4, 30.5, 37.5, 39.3, 41.7, 51.5, 52.7,
66.9, 72.0, 128.3, 129.5, 130.8, 166.5; MS (EI) m/z 316 (16,
M.sup.+), 301 (5), 285 (9), 242 (11), 194 (60), 147 (71), 105
(100); exact mass (ESI) calculated for C.sub.20H.sub.28O.sub.3Na
([M+Na].sup.+) 339.1936, found 339.1948.
[0078]
(20R)-Des-A,B-8.beta.-(benzoyloxy)-23,24-dinor-22-(tosyloxy)cholane
(5). To a mixture of alcohol 4 (870 mg, 2.75 mmol), triethylamine
(1.5 mL, 10.8 mmol) and DMAP (20 mg) in anhydrous methylene
dichloride (20 mL) tosyl chloride (710 mg, 3.73 mmol) was added at
0.degree. C. The reaction mixture was allowed to stand at room
temperature for 16 h. Then methylene dichloride (100 mL) was added
and the mixture was washed with saturated aqueous solution of
NaHCO.sub.3 (2.times.50 mL), dried over anhydrous MgSO.sub.4 and
concentrated under reduced pressure. The residue was purified by
column chromatography (5% ethyl acetate/hexane) to give 1162 mg
(2.47 mmol, 90% yield) of 5. [.alpha.].sub.D+14.2 (c 0.95,
CHCl.sub.3); mp. 100-102.degree. C.; NMR (500 MHz, CDCl.sub.3)
.delta. 0.90 (3H, d, J=6.6 Hz), 0.98 (3H, s), 2.46 (3H, s), 3.83
(1H, dd, J=9.2 Hz, J=7.2 Hz), 4.15 (1H, dd, J=9.3 Hz, J=3.3 Hz),
7.35 (2H, d, J=8.1 Hz), 7.44 (2H, m), 7.55 (1H, m), 7.80 (2H, d,
J=8.1 Hz), 8.02 (2H, m); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.
13.9, 16.6, 17.9, 21.6, 22.3, 26.3, 30.4, 34.8, 39.1, 41.6, 71.8,
74.0, 127.9, 128.4, 129.5, 129.7, 130.7, 132.8, 133.1, 144.6,
166.7; MS (EI) m/z 365 (12), 348 (61), 193 (9), 176 (32), 161 (13),
134 (19), 105 (100), 91 (17), 77 (20); exact mass (ESI) calculated
for C.sub.27H.sub.34O.sub.5SNa ([M+Na].sup.+) 493.2025, found
493.2032.
[0079] (20S)-Des-A,B-cholestan-8.beta.-ol (7). Magnesium turnings
(4.41 g, 184 mmol) were stirred with a magnetic stir bar overnight
under argon. Anhydrous THF (50 mL) and 1-chloro-3-methylbutane (11
mL, 90.8 mmol) were then added. The mixture was refluxed for 6 h.
The resulting solution of Grignard reagent 6 was then added via
cannula to a stirred solution of 5 in anhydrous THF (15 mL) at
-78.degree. C. followed by addition of a solution of dilithium
tetrachlorocuprate (620 mg, 2.73 mmol) in anhydrous THF (27 mL).
The cooling bath was removed and the reaction mixture was stirred
overnight. The reaction mixture was poured into a stirred mixture
of ice (15 mL) and saturated aqueous solution of NH.sub.4Cl (40
mL). The mixture was then extracted with ethyl acetate (3.times.100
mL), washed with water and dried over anhydrous Na.sub.2SO.sub.4.
The residue was purified by column chromatography (5 to 25% ethyl
acetate/hexane) to give 389 mg (1.46 mmol, 58% yield) of 7.
[.alpha.].sub.D+9.6 (c 1.15, CHCl.sub.3); .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 0.82 (3H, d, J=6.6 Hz), 0.87 (6H, d, J=6.6 Hz),
0.93 (3H, s), 4.07 (1H, s); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 13.8, 17.5, 18.5, 22.4, 22.5, 22.6, 22.7, 24.0, 27.1, 28.0,
29.7, 33.6, 34.8, 35.5, 39.4, 40.3, 41.9, 52.7, 56.3, 69.5; MS (EI)
m/z 266 (45, M.sup.+), 251 (19), 233 (8), 177 (9), 163 (11), 152
(20), 135 (30), 125 (37), 111 (100); exact mass calculated for
C.sub.18H.sub.34O 266.26310, found 266.2623.
[0080] (20S)-Des-A,B-cholestan-8.beta.-yl nitrite (8). A solution
of 7 (185 mg, 0.69 mmol) in chloroform (5 mL) was treated with
tert-butyl nitrite (1 mL) for 1 h in darkness. Benzene (10 mL) was
then added and solvents were removed under reduced pressure,
protecting the mixture from light. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 0.76 (3H, s), 0.81 (3H, d, J=6.5 Hz), 0.87 (6H,
d, J=6.6 Hz), 5.78 (1H, s); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 13.1, 17.9, 18.5, 22.2, 22.6, 22.7, 23.9, 27.1, 28.0, 31.5,
34.9, 35.3, 39.3, 39.7, 41.9, 51.9, 56.0.
[0081] (18E)-(20S)-18-(Hydroxyimino)-des-A,B-cholestan-8.beta.-ol
(9). Crude nitrite was dissolved in anhydrous benzene (150 mL) and
irradiated in an apparatus consisting of a Pyrex vessel with a
watercooled immersion well and Hanovia high-pressure mercury arc
lamp equipped with Pyrex filter. A slow stream of argon was passed
through solution and temperature was maintained at about 10.degree.
C. A reaction progress was monitored by TLC. After 30 min. reaction
was completed. Benzene was removed under reduced pressure and the
residue was dissolved in 2-propanol (5 mL) and refluxed for 2 h,
cooled and allowed to stand overnight to accomplish isomerisation
of a nitroso compound to an oxime. The solvent was evaporated and
the residue was purified on Waters silica gel Sep-Pack cartridge
(25% ethyl acetate/hexane) to give 102 mg (0.35 mmol, 51% yield
from 7) of the oxime 9. [.alpha.].sub.D+8.2 (c 0.80, CHCl.sub.3);
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.84 (3H, d, J=6.3 Hz),
0.87 (6H, d, J=6.6 Hz), 2.20 (1H, br d, J=13.1 Hz), 4.04 (1H, br d,
J=2.6 Hz), 7.33 (1H, s), 10.8 (1H, br s); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 17.5, 18.6, 21.8, 22.6, 22.7, 24.1, 27.2, 28.0,
34.3, 35.0, 35.6, 39.3, 49.5, 52.6, 56.7, 67.6, 152.2; MS (EI) m/z
295 (2, M.sup.+), 278 (28), 260 (20), 245 (8), 206 (19), 183 (38),
165 (13), 148 (15), 121 (100); exact mass calculated for
C.sub.18H.sub.33NO.sub.2Na ([M+Na].sup.+) 318.2409, found
318.2412.
[0082] (20S)-8.beta.-(Acetoxy)-des-A,B-cholestan-18-nitrile (10). A
solution of 9 (100 mg, 0.34 mmol) in acetic anhydride (5 mL) was
refluxed for 1.5 h. The reaction mixture was cooled, poured
carefully into ice and extracted with benzene (3.times.40 mL). The
combined organic phases were washed with saturated aqueous solution
of NaHCO.sub.3 (2.times.40 mL), water (30 mL), dried over anhydrous
Na.sub.2SO.sub.4 and evaporated. The residue was purified on a
Waters silica gel Sep-Pack cartridge (5% ethyl acetate/hexane) to
give 91 mg (0.28 mmol, 84% yield) of 9. [.alpha.].sub.D-26.4 (c
0.75, CHCl.sub.3); IR (CHCl.sub.3) 2228, 1741, 1241; .sup.1H NMR
(500 MHz, CDCl.sub.3) .delta. 0.87 (6H, d, J=6.6 Hz), 0.91 (3H, d,
J=6.6 Hz), 2.15 (3H, s), 2.46 (1H, br d, J=3.2 Hz), 5.20 (1H, s);
.sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 17.9, 18.8, 22.6, 22.7,
23.3, 23.8, 27.1, 28.0, 29.9, 35.6, 36.2, 36.3, 39.1, 45.6, 51.9,
54.1, 68.7, 121.2, 171.0; MS (EI) m/z 319 (18, M.sup.+), 304 (10),
290 (3), 277 (84), 259 (100), 244 (54), 234 (27), 216 (40), 202
(33), 188 (60), 174 (47), 147 (39), 134 (34), 121 (95); exact mass
(ESI) calculated for C.sub.20H.sub.33NO.sub.2Na ([M+Na].sup.+)
342.2409, found 342.2413.
[0083] (20S)-Des-A,B-cholestan-18-nitrile-8.beta.-ol (11). 10 (90
mg, 0.28 mmol) was dissolved in methanol (3 mL) and treated with 5%
solution of MeONa in methanol (3 mL) for 2 h. The reaction mixture
was quenched with a saturated aqueous solution of NH.sub.4Cl (5
mL), water (10 mL), extracted with methylene dichloride (5.times.40
mL), dried over anhydrous Na.sub.2SO.sub.4 and evaporated. The
residue was purified on a Waters silica gel Sep-Pack cartridge (20%
ethyl acetate/hexane) to give 73 mg (0.26 mmol, 94% yield) of 10.
[.alpha.].sub.D-6.1 (c 0.75, CHCl.sub.3); IR (CHCl.sub.3) 3486,
2228; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.87 (6H, d, J=6.6
Hz), 0.92 (3H, d, J=6.7 Hz), 2.43 (1H, br d, J=3.1 Hz), 4.10 (1H,
s); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 17.9, 22.6, 22.7,
22.9, 23.9, 27.1, 28.0, 32.8, 35.7, 36.2, 36.3, 44.7, 53.4, 54.2,
122.5; MS (EI) m/z 277 (28, M.sup.+), 262 (34), 259 (18), 248 (16),
244 (24), 220 (30), 216 (18), 206 (100); exact mass calculated for
C.sub.18H.sub.31NO 277.2496, found 277.2395.
[0084] (20S)-Des-A,B-18-norcholestan-8.beta.-ol (12). To a stirred
mixture of potassium (110 mg, 2.82 mmol) in HMPA (280 d, 1.62 mmol)
and diethyl ether (700 .mu.l) a solution of 11 (70 mg, 0.25 mmol)
in tert-butyl alcohol (65 .mu.l) and diethyl ether (250 .mu.l) was
added dropwise at 0.degree. C. under argon. The mixture was allowed
to warm up to room temperature and stirred for 5 h. Remaining
potassium was removed, a few drops of 2-propanol and benzene (20
mL) were added. Organic phase was washed with water (10 mL), dried
over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced
pressure. The residue was purified on Waters silica gel Sep-Pack
cartridge (10% ethyl acetate/hexane) to give 54 mg (0.21 mmol, 85%
yield) of 12. [.alpha.].sub.D+32.6 (c 0.90, CHCl.sub.3); .sup.1H
NMR (500 MHz, CDCl.sub.3) .delta. 0.78 (3H, d, J=6.8 Hz), 0.87 (6H,
d, J=6.6 Hz), 4.06 (1H, s); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 14.7, 20.2, 22.7, 22.9, 24.7, 25.3, 28.0, 30.8, 33.1, 33.5,
36.3, 39.3, 39.7, 48.6, 50.3, 67.9; MS (EI) m/z 252 (6, M.sup.+),
234 (21), 219 (23), 209 (26), 191 (8), 179 (4), 167 (13), 149 (89),
139 (47), 122 (90), 107 (35), 95 (80), 79 (87), 67 (88), 58 (100);
exact mass calculated for C.sub.17H.sub.32O 252.2453, found
252.2448.
[0085] (20S)-Des-A,B-25-hydroxy-18-norcholestane-8-one (13). To a
stirred solution of RuCl.sub.3.times.H.sub.2O (10 mg, 0.05 mmol)
and NaIO.sub.4 (227 mg, 1.06 mmol) in water (1 mL) a solution of 12
(74 mg, 0.29 mmol) in tetrachloromethane (0.75 mL) and acetonitrile
(0.75 mL) was added. The reaction mixture was vigorously stirred
for 3 days. Then a few drops of 2-propanol and water (10 mL) were
added. Reaction products were extracted with methylene dichloride
(3.times.20 mL). Organic phase was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated under reduced pressure. The
residue was purified on Waters silica gel Sep-Pack cartridge (10 to
30% ethyl acetate/hexane) to give 13 mg (0.05 mmol, 17% yield) of
13. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.78 (3H, d, J=6.7
Hz), 1.22 (6H, s), 2.01 (1H, br d, J=12.3 Hz); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 14.3, 21.3, 22.2, 22.6, 27.8, 29.3, 29.7,
33.0, 36.5, 41.6, 44.1, 49.6, 51.0, 58.0, 71.0, 212.0; MS (EI) m/z
264 (3), 248 (57), 233 (19), 215 (4), 208 (15), 163 (29), 137
(100); exact mass (ESI) calculated for C.sub.17H.sub.30O.sub.2Na
([M+Na].sup.+) 289.2144, found 289.2136.
[0086] (20S)-25-[(Triethylsilyl)oxy]-des-A,B-18-norcholestane-8-one
(14). To a stirred solution of 13 (12 mg, 45 .mu.mol) and
2,6-lutidine (13 .mu.l, 100 .mu.mol) in anhydrous methylene
dichloride (250 .mu.l) triethylsilyl trifluoromethanesulfonate was
added dropwise at -50.degree. C. under argon. After 20 min. a few
drops of wet methylene dichloride and water (7 mL) were added.
Reaction mixture was extracted with methylene dichloride (3.times.7
mL). Organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated under reduced pressure. The residue was purified on
Waters silica gel Sep-Pack cartridge (3% ethyl acetate/hexane) and
on HPLC (5% ethyl acetate/hexane, 4 mL/min., Zorbax-silica
10.times.250 mm) to give 13 mg (34 .mu.mol, 76% yield) of 14.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.56 (6H, q, J=7.9 Hz),
0.77 (3H, d, J=6.8 Hz), 0.94 (9H, t, J=7.9 Hz), 1.19 (6H, s);
.sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 6.8, 7.1, 14.3, 21.4,
22.2, 22.7, 27.8, 29.7, 29.8, 29.9, 32.9, 36.4, 41.6, 45.2, 49.6,
51.1, 58.0, 73.4, 212.1; MS (EI) m/z 365 (8), 351 (100), 322 (6),
239 (2), 231 (25), 220 (4), 205 (15), 189 (4), 173 (92); exact mass
(ESI) calculated for C.sub.23H.sub.44O.sub.2SiNa ([M+Na].sup.+)
403.3008, found 403.2995.
[0087] (20S)-2-Methylene-1.alpha.,25-dihydroxy-18,19-dinorvitamin
D.sub.3 (17). To a stirred solution of phosphine oxide 15 (46 mg,
79 .mu.mol) in anhydrous THF (600 .mu.l) a 1.5 M solution of phenyl
lithium in THF (63 .mu.l, 95 .mu.mol) was added at -20.degree. C.
under argon. The mixture was stirred for 20 min. and then cooled to
-78.degree. C. A precooled solution of 14 (13 mg, 34 .mu.mol) in
anhydrous THF (300 was added via cannula and the reaction mixture
was stirred for 3 h at -78.degree. C. After that the reaction
mixture was stirred at 4.degree. C. overnight. Then ethyl acetate
was added and organic phase was washed with brine, dried over
anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure.
The residue was purified on Waters silica gel Sep-Pack cartridge
(hexane to 2% ethyl acetate/hexane) and then on HPLC (0.05%
2-propanol/hexane, 4 mL/min., Zorbax-silica 10.times.250 mm) to
give 13.5 mg (18 .mu.mol, 53% yield) of protected vitamin D.sub.3
16. UV (hexane) .lamda..sub.max=242, 251, 261 nm; .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 0.06 (3H, s), 0.11 (3H, s), 0.17 (3H, s),
0.19 (3H, s), 0.56 (6H, q, J=8.0 Hz), 0.76 (3H, d, J=6.7 Hz), 0.94
(9H, t, J=8.0 Hz), 2.18 (1H, dd, J=12.5 Hz, J=8.1 Hz), 2.86 (1H, br
d, J=13.8 Hz), 4.42 (2H, m), 4.93 (1H, s), 4.96 (1H, s), 5.92 (1H,
d, J=11.1 Hz), 6.19 (1H, d, J=11.1 Hz); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. -5.1, -4.9, -4.9, -4.8, 6.8, 7.1, 18.2, 18.2,
22.3, 23.1, 25.8, 25.8, 27.8, 29.0, 29.7, 29.8, 29.9, 31.3, 33.6,
36.5, 38.7, 45.3, 47.5, 49.0, 50.2, 52.3, 71.9, 72.3, 73.4, 106.3,
113.7, 122.4, 132.9, 143.8, 152.9; MS (EI) m/z 687 (6), 628 (2),
612 (100), 583 (6), 555 (4), 480 (29), 366 (44); exact mass
calculated for C.sub.40H.sub.75O.sub.3Si.sub.3 ([M-t-Bu].sup.+)
687.5024, found 687.5028.
[0088] 16 (13 mg, 17 .mu.mol) was dissolved in anhydrous THF (5
mL). Then a 1 M solution of tetrabutyl ammonium fluoride in THF
(260 .mu.l, 260 .mu.mol) was added dropwise followed by addition of
activated molecular sieves 4A (200 mg). The reaction mixture was
stirred under argon for 2 h. Then solvent was removed under reduced
pressure and the residue was purified on Waters silica gel Sep-Pack
cartridge (40 to 50% ethyl acetate/hexane). Crude 17 was then
purified on HPLC (20% 2-propanol/hexane, 4 mL/min., Zorbax-silica
10.times.250 mm) to give 3.8 mg (9.5 .mu.mol, 56% yield) of 17 at
R.sub.t=5.58 min.; UV (EtOH) .lamda..sub.max=242, 250, 260 nm; NMR
(500 MHz, CDCl.sub.3) .delta. 0.77 (3H, d, J=6.6 Hz), 1.21 (6H, s),
2.58 (1H, dd, J=13.2 Hz, J=3.9 Hz), 2.81 (1H, dd, J=13.3 Hz, J=4.4
Hz), 2.87 (1H, br d, J=13.9 Hz), 4.48 (2H, m), 5.10 (1H, s), 5.11
(1H, s), 5.97 (1H, d, J=11.3 Hz), 6.35 (1H, d, J=11.3 Hz); MS (EI)
m/z 402 (39, M.sup.+), 384 (41), 366 (14), 351 (11), 299 (58), 231
(36), 142 (58), 69 (100); exact mass calculated for
C.sub.26H.sub.42O.sub.3 402.3134, found 402.3121.
[0089] For treatment purposes, the novel compounds of this
invention defined by formula I may be formulated for pharmaceutical
applications as a solution in innocuous solvents, or as an
emulsion, suspension or dispersion in suitable solvents or
carriers, or as pills, tablets or capsules, together with solid
carriers, according to conventional methods known in the art. Any
such formulations may also contain other
pharmaceutically-acceptable and non-toxic excipients such as
stabilizers, anti-oxidants, binders, coloring agents or emulsifying
or taste-modifying agents.
[0090] The compounds may be administered orally, topically,
parenterally or transdermally. The compounds are advantageously
administered by injection or by intravenous infusion or suitable
sterile solutions, or in the form of liquid or solid doses via the
alimentary canal, or in the form of creams, ointments, patches, or
similar vehicles suitable for transdermal applications. Doses of
from 0.01 .mu.g to 100 .mu.g per day of the compounds, preferably
from about 0.1 .mu.g/day to about 50 .mu.g/day, are appropriate for
treatment purposes, such doses being adjusted according to the
disease to be treated, its severity and the response of the subject
as is well understood in the art. Since the new compounds exhibit
specificity of action, each may be suitably administered alone, or
together with graded doses of another active vitamin D
compound--e.g. 1.alpha.-hydroxyvitamin D.sub.2 or D.sub.3, or
1.alpha.,25-dihydroxyvitamin D.sub.3--in situations where different
degrees of bone mineral mobilization and calcium transport
stimulation is found to be advantageous.
[0091] Compositions for use in the above-mentioned treatment of
psoriasis and other malignancies comprise an effective amount of
one or more 2-alkylidene-18,19-dinor-vitamin D compound as defined
by the above formula I as the active ingredient, and a suitable
carrier. An effective amount of such compounds for use in
accordance with this invention is from about 0.01 .mu.g to about
100 .mu.g per gm of composition, preferably from about 0.01
.mu.g/gm to about 50 .mu.g/gm of the composition, and may be
administered topically, transdermally, orally or parenterally in
dosages of from about 0.01 .mu.n/day to about 100 .mu.g/day,
preferably from about 0.1 .mu.g/day to about 50 .mu.g/day.
[0092] The compounds may be formulated as creams, lotions,
ointments, topical patches, pills, capsules or tablets, or in
liquid form as solutions, emulsions, dispersions, or suspensions in
pharmaceutically innocuous and acceptable solvent or oils, and such
preparations may contain in addition other pharmaceutically
innocuous or beneficial components, such as stabilizers,
antioxidants, emulsifiers, coloring agents, binders or
taste-modifying agents.
[0093] The compounds are advantageously administered in amounts
sufficient to effect the differentiation of promyelocytes to normal
macrophages. Dosages as described above are suitable, it being
understood that the amounts given are to be adjusted in accordance
with the severity of the disease, and the condition and response of
the subject as is well understood in the art.
[0094] The formulations of the present invention comprise an active
ingredient in association with a pharmaceutically acceptable
carrier therefore and optionally other therapeutic ingredients. The
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulations and not deleterious to
the recipient thereof.
[0095] Formulations of the present invention suitable for oral
administration may be in the form of discrete units as capsules,
sachets, tablets or lozenges, each containing a predetermined
amount of the active ingredient; in the form of a powder or
granules; in the form of a solution or a suspension in an aqueous
liquid or non-aqueous liquid; or in the form of an oil-in-water
emulsion or a water-in-oil emulsion.
[0096] Formulations for rectal administration may be in the form of
a suppository incorporating the active ingredient and carrier such
as cocoa butter, or in the form of an enema.
[0097] Formulations suitable for parenteral administration
conveniently comprise a sterile oily or aqueous preparation of the
active ingredient which is preferably isotonic with the blood of
the recipient.
[0098] Formulations suitable for topical administration include
liquid or semi-liquid preparations such as liniments, lotions,
applicants, oil-in-water or water-in-oil emulsions such as creams,
ointments or pastes; or solutions or suspensions such as drops; or
as sprays.
[0099] For asthma treatment, inhalation of powder, self-propelling
or spray formulations, dispensed with a spray can, a nebulizer or
an atomizer can be used. The formulations, when dispensed,
preferably have a particle size in the range of 10 to 100.mu..
[0100] The formulations may conveniently be presented in dosage
unit form and may be prepared by any of the methods well known in
the art of pharmacy. By the term "dosage unit" is meant a unitary,
i.e. a single dose which is capable of being administered to a
patient as a physically and chemically stable unit dose comprising
either the active ingredient as such or a mixture of it with solid
or liquid pharmaceutical diluents or carriers.
2-Alkylidene-18,19-Dinor Slow Release Compounds
[0101] Modified vitamin D compounds that exhibit a desirable and
highly advantageous pattern of biological activity in vivo, namely,
the more gradual onset and more prolonged duration of activity, may
also be used herein.
[0102] Structurally, the key feature of the modified vitamin D
compounds having these desirable biological attributes is that they
are derivatives of 2-alkylidene-18,19-dinor-vitamin D analogs, in
which a hydrolyzable group is attached to the hydroxy group at
carbon 25 and, optionally, to any other of the hydroxy groups
present in the molecule. Depending on various structural
factors--e.g. the type, size, structural complexity--of the
attached group, these derivatives hydrolyze to the active
2-alkylidene-18,19-dinor-vitamin D analog, at different rates in
vivo, thus providing for the "slow release" of the biologically
active vitamin D compound in the body.
[0103] The "slow release" in vivo activity profiles of such
compounds can, of course, be further modulated by the use of
mixtures of derivatives or the use of mixtures consisting of one or
more vitamin D derivative together with underivatized vitamin D
compounds.
[0104] It is important to stress that the critical structural
feature of the vitamin derivatives identified above is the presence
of a hydrolyzable group attached to the hydroxy group at carbon 25
of the molecule. The presence of a hydrolyzable group at that
position imparts on the resulting derivatives the desirable
"slow-release" biological activity profile mentioned above. Other
hydroxy functions occurring in the molecule (e.g. hydroxy functions
at carbons 1 or 3) may be present as free hydroxy groups, or one or
more of them may also be derivatised with a hydrolyzable group.
[0105] The "hydrolyzable group" present in the above-mentioned
derivatives is preferably an acyl group, i.e. a group of the type
Q.sup.1CO--, where Q.sup.1 represents hydrogen or a hydrocarbon
radical of from 1 to 18 carbons that may be straight chain, cyclic,
branched, saturated or unsaturated. Thus, for example, the
hydrocarbon radical may be a straight chain or branched alkyl
group, or a straight chain or branched alkenoyl group with one or
more double bonds, or it may be an optionally substituted
cycloalkyl or cycloalkenyl group, or an aromatic group, such as
substituted or unsubstituted phenyl, benzyl or naphthyl. Especially
preferred acyl groups are alkanoyl or alkenoyl groups, of which
some typical examples are formyl, acetyl, propanoyl, hexanoyl,
isobutyryl, 2-butenoyl, palmitoyl or oleoyl. Another suitable type
of hydrolyzable group is the hydrocarbyloxycarbonyl group, i.e. a
group of the type Q.sup.2-O--CO--, where Q.sup.2 is a C.sub.1 to
C.sub.18 hydrocarbon radical as defined above. Exemplary of such
hydrocarbon radicals are methyl, ethyl, propyl, and higher straight
chain or branched alkyl and alkenoyl radicals, as well as aromatic
hydrocarbon radicals such as phenyl or benzoyl.
[0106] These modified vitamin D compounds are hydrolyzable in vivo
to the active analog over a period of time following
administration, and as a consequence regulate the in vivo
availability of the active analog, thereby also modulating their
activity profile in vivo. The term "activity profile" refers to the
biological response over time of vitamin D compounds. Individual
modified compounds, or mixtures of such compounds, can be
administered to "fine tune" a desired time course of response.
[0107] As used herein the term "modified vitamin D compound"
encompasses any vitamin D compound in which one or more of the
hydroxy functions present in such a compound are modified by
derivatization with a hydrolyzable group. A "hydrolyzable group" is
a hydroxy-modifying group that can be hydrolyzed in vivo, so as to
regenerate the free hydroxy functions.
[0108] In the context of this disclosure, the term hydrolyzable
group preferably includes acyl and hydrocarbyloxycarbonyl groups,
i.e. groups of the type Q.sup.1CO-- and Q.sup.2-O--CO,
respectively, where Q.sup.1 and Q.sup.2 have the meaning defining
earlier.
[0109] Structurally, the modified vitamin D compounds encompassed
may be represented by the formula I shown below:
##STR00010##
where Y.sub.1, Y.sub.2, and R are as previously defined herein with
respect to formula I with the exception that R.sup.5 in the side
chain is --OY.sub.3 and Y.sub.3 is an acyl group or a
hydrocarbyloxycarbonyl group, as previously defined herein.
[0110] Some specific examples of such modified vitamin D compounds
include 2-methylene-18,19-dinor derivatives such as: [0111]
2-methylene-18,19-dinor-1.alpha.,25(OH).sub.2-D.sub.3-1,3,25-Triacetate
where Y.sub.1.dbd.Y.sub.2.dbd.Y.sub.3 and is CH.sub.3CO; [0112]
2-methylene-18,19-dinor-1.alpha.,25(OH).sub.2-D.sub.3-1,3,25-Trihexanoate
where Y.sub.1.dbd.Y.sub.2.dbd.Y.sub.3 and is
CH.sub.3(CH.sub.2).sub.4CO; [0113]
2-methylene-18,19-dinor-1.alpha.,25(OH).sub.2-D.sub.3-1,3,25-Trino-
nanoate where Y.sub.1.dbd.Y.sub.2.dbd.Y.sub.3 and is
CH.sub.3(CH.sub.2).sub.7CO;
[0114]
2-methylene-18,19-dinor-1.alpha.,25(OH).sub.2-D.sub.3-25-Acetate
where Y.sub.1.dbd.Y.sub.2 and is H and Y.sub.3 is CH.sub.3CO.
[0115] These compounds can be prepared by known methods. See for
example U.S. Pat. No. 5,843,927.
##STR00011## ##STR00012##
Biological Activity of 2-Alkylidene-18,19-Dinor-Vitamin D
Compounds
[0116] FIG. 1--Competitive VDR Binding
[0117] Competitive binding of the analogs to the porcine intestinal
receptor was carried out by the method described by Dame et al
(Biochemistry 25, 4523-4534, 1986).
[0118] Test Material
[0119] Protein Source
[0120] Full-length recombinant rat receptor was expressed in E.
coli BL21 (DE3) Codon Plus RIL cells and purified using two
different column chromatography systems. The first system was a
nickel affinity resin that utilized the C-terminal histidine tag on
this protein. The protein that was eluted from this resin was
further purified using ion exchange chromatography (S-Sepharose
Fast Flow). Aliquots of the purified protein were quick frozen in
liquid nitrogen and stored at -80.degree. C. until use. For use,
the protein was diluted in TEDK50 (50 mM Tris, 1.5 mM EDTA, pH7.4,
5 mM DTT, 150 mM KCl) with 0.1% Chaps detergent so that no more
than 20% of the added radiolabeled ligand was bound to the
receptor.
[0121] Study Drugs
[0122] Unlabeled ligands were dissolved in ethanol and the
concentrations determined using UV spectrophotometry. Serial
dilutions were prepared so that a range of unlabeled ligands could
be added to the protein without changing the final concentration of
ethanol (<10%) present in the assay mixture. Radiolabeled ligand
(3H-1,25(OH).sub.2D.sub.3) was added in ethanol at a final
concentration of 1 nM.
[0123] Assay Conditions
[0124] Radiolabeled and unlabeled ligands were added to 100 mcl of
the diluted protein, mixed and incubated overnight on ice to reach
binding equilibrium. The following day, 100 mcl of hydroxylapatite
slurry (50%) was added to each tube and mixed at 10-minute
intervals for 30 minutes. The hydroxylapatite was pelleted by
centrifugation and then washed three times with Tris-EDTA buffer
(50 mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Triton X-100.
After the final wash, the pellets were transferred to scintillation
vials containing 4 ml of Biosafe II scintillation cocktail, mixed
and placed in a scintillation counter. Total binding was determined
from the tubes containing only radiolabeled ligand. The percentage
of competition was calculated by subtracting the number of dpm
remaining in the hydroxylapatite pellet from the total number of
dpm bound, dividing by the total number of dpm bound and
multiplying by one hundred. Duplicate tubes were prepared and
analyzed for each test concentration.
[0125] FIG. 2--HL-60 Cell Differentiation
[0126] The differentiation of HL-60 promyelocytic into monocytes
was determined as described by Ostrem et al (J. Biol. Chem. 262,
14164-14171, 1987).
[0127] Test Material
[0128] Study Drugs
[0129] The study drugs were dissolved in ethanol and the
concentrations determined using UV spectrophotometry (2MD:molar
extinction coefficient=42,000 and Imax=252 nm;
1,25(OH).sub.2D.sub.3:molar extinction=18,200 and Imax=265 nm).
Serial dilutions were prepared so that a range of drug
concentrations could be tested without changing the final
concentration of ethanol (<0.2%) present in the cell
cultures.
[0130] Cells
[0131] Human promyelocytic leukemia (HL60) cells were grown in
RPMI-1640 medium containing 10% fetal bovine serum. The cells were
incubated at 37.degree. C. in the presence of 5% CO.sub.2.
[0132] Assay Conditions
[0133] HL60 cells were plated at 1.2.times.105 cells/ml. Eighteen
hours after plating, the cells were administered the drug in
ethanol. Four days post-dose, the cells were harvested and a nitro
blue tetrazolium reduction assay was performed (Collins et al.,
1979; J. Exp. Med. 149:969-974, Appendix A). The percentage of
differentiated cells was determined by counting a total of 200
cells and recording the number that contained intracellular
black-blue formazan deposits. Verification of differentiation to
monocytic cells was determined by measuring phagocytic activity
(data not shown). All drug concentrations were tested in
duplicate.
[0134] FIG. 3--Transcription Activation
[0135] Transcriptional activity was measured in ROS 17/2.8 (bone)
cells that were stably transfected with a 24-hydroxylase (24OHase)
gene promoter upstream of a luciferase reporter gene (Arbour et
al., 1998). Cells were given a range of doses. Sixteen hours after
dosing the cells were harvested and luciferase activities were
measured using a luminometer.
[0136] "RLU" in FIG. 3 refers to relative luciferase units.
[0137] FIG. 4--Intestinal Calcium Transport
[0138] Weanling, male Sprague-Dawley rats were purchased from
Harlan. Upon receipt, the animals were identified by individual
tail marks and fed a calcium containing (0.47%) diet (Suda et al.,
Purified Rodent Diet-Diet 11; Appendix A) for one week before
switching to the same diet devoid of calcium (0.02%). Water and a
purified rodent diet (Diet 11; Appendix A) containing either 0.47%
or 0.02% calcium and 0.3% phosphorus were provided ad libitum.
Animals were fed the purified diet containing 0.47% calcium for the
first week and then the 0.02% calcium containing diet for the next
three weeks of the study. The rats were then fed 0.47% calcium
containing diet for one week before switching back to 0.02% calcium
containing diet for the remainder of the study. During the second
week back on 0.02% calcium containing diet, dose administration
began. All doses were administered intraperitoneally in 100
microliters of propylene glycol. Four consecutive doses were given
approximately 24 hours apart. Twenty-four hours after the last
dose, blood was collected from the tail artery of each experimental
animal. The blood was allowed to coagulate at room temperature and
then centrifuged at 3000.times.g for 15 minutes. The serum was
transferred to a polypropylene tube and stored frozen at
-20.degree. C. The level of calcium was determined by diluting the
serum into 0.1% lanthum chloride and measuring the absorbance on an
atomic absorption spectrophotometer (Perkin Elmer Model 3110,
Shelton, Conn.). Twenty-four hours after the last dose, intestinal
calcium transport was assessed ex vivo using the everted gut sac
technique.
[0139] FIGS. 5 and 6--Bone Calcium Mobilization
[0140] Weanling, male Sprague-Dawley rats were purchased from
Harlan. Upon receipt, the animals were identified by individual
tail marks and fed a calcium containing (0.47%) diet (Suda et al.,
Purified Rodent Diet-Diet 11; Appendix A) for one week before
switching to the same diet devoid of calcium (0.02%). Water and a
purified rodent diet (Diet 11; Appendix A) containing either 0.47%
or 0.02% calcium and 0.3% phosphorus were provided ad libitum.
Animals were fed the purified diet containing 0.47% calcium for the
first week and then the 0.02% calcium containing diet for the next
three weeks of the study. The rats were then fed 0.47% calcium
containing diet for one week before switching back to 0.02% calcium
containing diet for the remainder of the study. During the second
week back on 0.02% calcium containing diet, the animals were
tail-bled (baseline serum calcium) and then dose administration was
initiated. All doses were administered intraperitoneally in 100
microliters of propylene glycol. Four consecutive doses were given
approximately 24 hours apart. Twenty-four hours after the last
dose, blood was collected from the tail artery of each experimental
animal. The blood was allowed to coagulate at room temperature and
then centrifuged at 3000.times.g for 15 minutes. The serum was
transferred to a polypropylene tube and stored frozen at
-20.degree. C. The level of calcium was determined by diluting the
serum into 0.1% lanthum chloride and measuring the absorbance on an
atomic absorption spectrophotometer (Perkin Elmer Model 3110,
Shelton, Conn.).
Interpretation of Biological Data
[0141] FIG. 1 illustrates the relative activity of
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(also herein referred to as "DP035"),
(20S)-2-methylene-19-nor-1.alpha.,25-dihydroxyvitamin D.sub.3,
(also herein referred to as "2MD") and 1.alpha.,25-dihydroxyvitamin
D.sub.3 (also herein referred to as "C001") in binding to the
1.alpha.,25-dihydroxyvitamin D pig intestinal nuclear receptor.
FIG. 1 shows that
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
is very active in binding to the 1.alpha.,25-hydroxyvitamin D.sub.3
receptor from porcine intestinal nuclei.
[0142] The 2-alkylidene-18,19-dinor compounds of this invention
exhibit a pattern of biological activity having high potency in
promoting the differentiation of malignant cells, relatively high
intestinal calcium transport activity and a relatively low ability
to mobilize calcium from bone. This is illustrated by the
biological assay results obtained for
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxy-vitamin D.sub.3
which is summarized in FIGS. 2 through 6. FIG. 2 shows a comparison
of the activity of the known active metabolite
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001) as well as analog 2MD
and the presently claimed
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) in inducing the differentiation of human leukemia cells
(HL-60 cells) in culture to monocytes. Differentiation activity was
assessed by a standard differentiation assay, abbreviated as NBT
reduction (nitroblue tetrazolium reduction). The assay was
conducted according to known procedures, as given, for example, by
DeLuca et al U.S. Pat. No. 4,717,721 and Ostrem et al, J. Biol.
Chem. 262, 14164, 1987. For the assay, the differentiation activity
of the test compounds is expressed in terms of the percent of HL-60
cells having differentiated to normal cells in response to a given
concentration of test compound.
[0143] The results summarized in FIG. 2 clearly show that the
analog, (20S)-2-methylene-1.alpha.,25-dihydroxy-18,19-dinor-vitamin
D.sub.3 (DP035) is more potent than 1.alpha.,25-dihydroxyvitamin
D.sub.3 (C001) in promoting the differentiation of leukemia cells.
Thus, in the NBT assay close to 90% of the cells are induced to
differentiate by 1.alpha.,25-dihydroxyvitamin D.sub.3 (C001) at a
concentration of 1.times.10.sup.-7M, and the same degree of
differentiation is achieved by the (20S)-2-methylene-18,19-dinor
analog (DP035) at 1.times.10.sup.-7M.
[0144] FIG. 3 illustrates that
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) has higher transcriptional activity than
1.alpha.,25-dihydroxyvitamin D.sub.3 in bone cells. This result,
together with the cell differentiation activity of FIG. 2, suggests
that DP035 will be very effective in psoriasis because it has
direct cellular activity in causing cell differentiation and in
suppressing cell growth. These data also indicate that DP035 may
have significant activity as an anti-cancer agent, especially
against leukemia, colon cancer, breast cancer, skin cancer and
prostate cancer.
[0145] FIGS. 4 through 6 show a comparison of the calcemic activity
of the known active metabolite 1.alpha.,25-dihydroxyvitamin D.sub.3
(C001), and the 19-nor analog 2MD and the presently claimed
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035). FIG. 4 shows that
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) is as active as 1.alpha.,25-dihydroxyvitamin D.sub.3 (C001)
in intestinal calcium transport activity. Also, FIGS. 5 and 6 show
that although
(20S)-2-methylene-18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
(DP035) has some ability to mobilize calcium from bone, it is
clearly not as active in this regard as
1.alpha.,25-dihydroxyvitamin D.sub.3 (C001). Thus, in summary, the
(20S)-2-methylene-18,19-dinor analog (DP035) shows a selective
activity profile combining high potency in inducing the
differentiation of malignant cells, relatively high intestinal
calcium transport activity and relatively low bone calcium
mobilization activity.
* * * * *