U.S. patent application number 12/171108 was filed with the patent office on 2010-01-14 for 19-nor-vitamin d analogs with 3,2-dihydrofuran ring.
Invention is credited to Margaret Clagett-Dame, Hector F. DeLuca, Agnieszka Glebocka, Lori A. Plum, Rafal R. Sicinski, Katarzyna Sokolowska.
Application Number | 20100009945 12/171108 |
Document ID | / |
Family ID | 41395876 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009945 |
Kind Code |
A1 |
DeLuca; Hector F. ; et
al. |
January 14, 2010 |
19-NOR-VITAMIN D ANALOGS WITH 3,2-DIHYDROFURAN RING
Abstract
19-nor-vitamin D analogs having an additional dihydrofuran ring
connecting the 3.beta.-oxygen and carbon-2 of the A-ring of the
analog, and pharmaceutical uses therefore, are described. These
compounds exhibit selective in vitro and in vivo activities, making
them therapeutic agents for the treatment or prophylaxis of
autoimmune diseases, some types of cancers, metabolic bone
diseases, osteomalacia, osteopenia, secondary hyperparathyroidism,
psoriasis, or other skin diseases.
Inventors: |
DeLuca; Hector F.;
(Deerfield, WI) ; Glebocka; Agnieszka; (Madison,
WI) ; Sokolowska; Katarzyna; (Lomza, PL) ;
Sicinski; Rafal R.; (Warsaw, PL) ; Plum; Lori A.;
(Arena, WI) ; Clagett-Dame; Margaret; (Deerfield,
WI) |
Correspondence
Address: |
Thomas M. Wozny;ANDRUS, SCEALES, STARKE & SAWALL, LLP
Suite 1100, 100 East Wisconsin Avenue
Milwaukee
WI
53202
US
|
Family ID: |
41395876 |
Appl. No.: |
12/171108 |
Filed: |
July 10, 2008 |
Current U.S.
Class: |
514/167 ;
549/462 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
19/08 20180101; A61P 29/00 20180101; A61P 37/02 20180101; A61P
17/06 20180101; C07D 307/79 20130101; A61P 13/12 20180101; A61P
17/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/167 ;
549/462 |
International
Class: |
A61K 31/59 20060101
A61K031/59; C07D 307/77 20060101 C07D307/77; A61P 17/06 20060101
A61P017/06; A61P 35/00 20060101 A61P035/00; A61P 37/02 20060101
A61P037/02; A61P 29/00 20060101 A61P029/00; A61P 17/00 20060101
A61P017/00; A61P 13/12 20060101 A61P013/12; A61P 3/04 20060101
A61P003/04; A61P 19/08 20060101 A61P019/08 |
Claims
1. A compound having the formula: ##STR00009## where Y is selected
from the group consisting of hydrogen and a hydroxy-protecting
group, and where the group R represents an alkyl, hydrogen,
hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain
of the formula: ##STR00010## where Z in the above side chain
structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and
--CH.dbd.CHY, where the double bond in the side chain may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00011##
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 having a general formula C.sub.kH.sub.2k--
where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
2. The compound of claim 1 wherein Y is hydrogen.
3. A pharmaceutical composition containing an effective amount of
at least one compound as claimed in claim 1 together with a
pharmaceutically acceptable excipient.
4. The pharmaceutical composition of claim 3 wherein said effective
amount comprises from about 0.01 .mu.g to about 1000 .mu.g per gram
of composition.
5. The pharmaceutical composition of claim 3 wherein said effective
amount comprises from about 0.1 .mu.g to about 500 .mu.g per gram
of composition.
6. 3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00012##
7. A pharmaceutical composition containing an effective amount of
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
together with a pharmaceutically acceptable excipient.
8. The pharmaceutical composition of claim 7 wherein said effective
amount comprises from about 0.01 .mu.g to about 1000 .mu.g per gram
of composition.
9. The pharmaceutical composition of claim 7 wherein said effective
amount comprises from about 0.1 .mu.g to about 500 .mu.g per gram
of composition.
10. A method of treating psoriasis comprising administering to a
subject with psoriasis an effective amount of a compound having the
formula: ##STR00013## where Y is selected from the group consisting
of hydrogen and a hydroxy-protecting group, and where the group R
represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group,
or R may represent a side chain of the formula: ##STR00014## where
Z in the above side chain structure is selected from Y, --OY,
--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond
in the side chain 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
11. The method of claim 10 wherein the compound is administered
orally.
12. The method of claim 10 wherein the compound is administered
parenterally.
13. The method of claim 10 wherein the compound is administered
transdermally.
14. The method of claim 10 wherein the compound is administered
topically.
15. The method of claim 10 wherein the compound is administered
rectally.
16. The method of claim 10 wherein the compound is administered
nasally.
17. The method of claim 10 wherein the compound is administered
sublingually.
18. The method of claim 10 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
19. The method of claim 10 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00016##
20. A method of treating a disease selected from the group
consisting of leukemia, colon cancer, breast cancer, skin cancer or
prostate cancer comprising administering to a subject with said
disease an effective amount of a compound having the formula:
##STR00017## where Y is selected from the group consisting of
hydrogen and a hydroxy-protecting group, and where the group R
represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group,
or R may represent a side chain of the formula: ##STR00018## where
Z in the above side chain structure is selected from Y, --OY,
--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond
in the side chain may have the cis or trans geometry, and where Y
is selected from hydrogen, methyl, --COR.sup.5 and a radical of the
structure: ##STR00019## 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
21. The method of claim 20 wherein the compound is administered
orally.
22. The method of claim 20 wherein the compound is administered
parenterally.
23. The method of claim 20 wherein the compound is administered
transdermally.
24. The method of claim 20 wherein the compound is administered
rectally.
25. The method of claim 20 wherein the compound is administered
nasally.
26. The method of claim 20 wherein the compound is administered
sublingually.
27. The method of claim 20 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
28. The method of claim 20 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00020##
29. A method of treating an autoimmune disease selected from the
group consisting of multiple sclerosis, lupus, diabetes mellitus,
host versus graft rejection, and rejection of organ transplants,
comprising administering to a subject with said disease an
effective amount of a compound having the formula: ##STR00021##
where Y is selected from the group consisting of hydrogen and a
hydroxy-protecting group, and where the group R represents an
alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may
represent a side chain of the formula: ##STR00022## where Z in the
above side chain structure is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond in the side
chain may have the cis or trans geometry, and where Y is selected
from hydrogen, methyl, --COR.sup.5 and a radical of the structure:
##STR00023## 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
30. The method of claim 29 wherein the compound is administered
orally.
31. The method of claim 29 wherein the compound is administered
parenterally.
32. The method of claim 29 wherein the compound is administered
transdermally.
33. The method of claim 29 wherein the compound is administered
rectally
34. The method of claim 29 wherein the compound is administered
nasally.
35. The method of claim 29 wherein the compound is administered
sublingually.
36. The method of claim 29 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
37. The method of claim 29 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00024##
38. A method of treating an inflammatory disease selected from the
group consisting of rheumatoid arthritis, asthma, and inflammatory
bowel diseases, comprising administering to a subject with said
disease an effective amount of a compound having the formula:
##STR00025## where Y is selected from the group consisting of
hydrogen and a hydroxy-protecting group, and where the group R
represents an alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group,
or R may represent a side chain of the formula: ##STR00026## where
Z in the above side chain structure is selected from Y, --OY,
--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond
in the side chain may have the cis or trans geometry, and where Y
is selected from hydrogen, methyl, --COR.sup.5 and a radical of the
structure: ##STR00027## 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
39. The method of claim 38 wherein the compound is administered
orally.
40. The method of claim 38 wherein the compound is administered
parenterally.
41. The method of claim 38 wherein the compound is administered
transdermally.
42. The method of claim 38 wherein the compound is administered
rectally.
43. The method of claim 38 wherein the compound is administered
nasally.
44. The method of claim 38 wherein the compound is administered
sublingually.
45. The method of claim 38 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
46. The method of claim 38 wherein the compound
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00028##
47. A method of treating a skin condition selected from the group
consisting of wrinkles, lack of adequate skin firmness, lack of
adequate dermal hydration and insufficient sebum secretion which
comprises administering to a subject with said skin condition an
effective amount of a compound having the formula: ##STR00029##
where Y is selected from the group consisting of hydrogen and a
hydroxy-protecting group, and where the group R represents an
alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may
represent a side chain of the formula: ##STR00030## where Z in the
above side chain structure is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond in the side
chain may have the cis or trans geometry, and where Y is selected
from hydrogen, methyl, --COR.sup.5 and a radical of the structure:
##STR00031## 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
48. The method of claim 47 wherein the compound is administered
orally.
49. The method of claim 47 wherein the compound is administered
parenterally.
50. The method of claim 47 wherein the compound is administered
transdermally.
51. The method of claim 47 wherein the compound is administered
topically.
52. The method of claim 47 wherein the compound is administered
rectally.
53. The method of claim 47 wherein the compound is administered
nasally.
54. The method of claim 47 wherein the compound is administered
sublingually.
55. The method of claim 47 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
56. The method of claim 47 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00032##
57. A method of treating renal osteodystrophy comprising
administering to a subject with renal osteodystrophy an effective
amount of a compound having the formula: ##STR00033## where Y is
selected from the group consisting of hydrogen and a
hydroxy-protecting group, and where the group R represents an
alkyl, hydrogen, hydroxyalkyl, or fluoroalkyl group, or R may
represent a side chain of the formula: ##STR00034## where Z in the
above side chain structure is selected from Y, --OY, --CH.sub.2OY,
--C.ident.CY and --CH.dbd.CHY, where the double bond in the side
chain 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 having a general
formula C.sub.kH.sub.2k-- where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
58. The method of claim 57 wherein the compound is administered
orally.
59. The method of claim 57 wherein the compound is administered
parenterally.
60. The method of claim 57 wherein the compound is administered
transdermally.
61. The method of claim 57 wherein the compound is administered
rectally.
62. The method of claim 57 wherein the compound is administered
nasally.
63. The method of claim 57 wherein the compound is administered
sublingually.
64. The method of claim 57 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
65. The method of claim 57 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00036##
66. A method of treating or preventing obesity of an animal,
inhibiting adipocyte differentiation, inhibiting SCD-1 gene
transcription, and/or reducing body fat in an animal comprising
administering to an animal in need thereof an effective amount of a
compound having the formula: ##STR00037## where Y is selected from
the group consisting of hydrogen and a hydroxy-protecting group,
and where the group R represents an alkyl, hydrogen, hydroxyalkyl,
or fluoroalkyl group, or R may represent a side chain of the
formula: ##STR00038## where Z in the above side chain structure is
selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY,
where the double bond in the side chain may have the cis or trans
geometry, and where Y is selected from hydrogen, methyl,
--COR.sup.5 and a radical of the structure: ##STR00039## 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 having a general formula C.sub.kH.sub.2k-- where k
is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
67. The method of claim 66 wherein the compound is administered
orally.
68. The method of claim 66 wherein the compound is administered
parenterally.
69. The method of claim 66 wherein the compound is administered
transdermally.
70. The method of claim 66 wherein the compound is administered
rectally.
71. The method of claim 66 wherein the compound is administered
nasally.
72. The method of claim 66 wherein the compound is administered
sublingually.
73. The method of claim 66 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 .mu.g/day.
74. The method of claim 66 wherein the compound is
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
having the formula: ##STR00040##
75. The method of claim 66 wherein the animal is a human.
76. The method of claim 66 wherein the animal is a domestic
animal.
77. The method of claim 66 wherein the animal is an agricultural
animal.
78. A method of treating a metabolic bone disease comprising
administering to a subject with said disease an effective amount of
a compound having the formula: ##STR00041## where Y is selected
from the group consisting of hydrogen and a hydroxy-protecting
group, and where the group R represents an alkyl, hydrogen,
hydroxyalkyl, or fluoroalkyl group, or R may represent a side chain
of the formula: ##STR00042## where Z in the above side chain
structure is selected from Y, --OY, --CH.sub.2OY, --C.ident.CY and
--CH.dbd.CHY, where the double bond in the side chain may have the
cis or trans geometry, and where Y is selected from hydrogen,
methyl, --COR.sup.5 and a radical of the structure: ##STR00043##
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 having a general formula C.sub.kH.sub.2k--
where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
79. The method of claim 78 where the disease is senile
osteoporosis.
80. The method of claim 78 where the disease is postmenopausal
osteoporosis.
81. The method of claim 78 where the disease is steroid-induced
osteoporosis.
82. The method of claim 78 where the disease is low bone turnover
osteoporosis.
83. The method of claim 78 where the disease is osteomalacia.
84. The method of claim 78 wherein the compound is administered
orally.
85. The method of claim 78 wherein the compound is administered
parenterally.
86. The method of claim 78 wherein the compound is administered
transdermally.
87. The method of claim 78 wherein the compound is administered
rectally.
88. The method of claim 78 wherein the compound is administered
nasally.
89. The method of claim 78 wherein the compound is administered
sublingually.
90. The method of claim 78 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 mg/day.
91. The method of claim 78 wherein the compound is a
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
analog having the formula: ##STR00044##
92. A method of treating osteopenia comprising administering to a
subject with osteopenia an effective amount of a compound having
the formula: ##STR00045## where Y is selected from the group
consisting of hydrogen and a hydroxy-protecting group, and where
the group R represents an alkyl, hydrogen, hydroxyalkyl, or
fluoroalkyl group, or R may represent a side chain of the formula:
##STR00046## where Z in the above side chain structure is selected
from Y, --OY, --CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where
the double bond in the side chain 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 having a general formula C.sub.kH.sub.2k-- where k
is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
93. The method of claim 92 wherein the compound is administered
orally.
94. The method of claim 92 wherein the compound is administered
parenterally.
95. The method of claim 92 wherein the compound is administered
transdermally.
96. The method of claim 92 wherein the compound is administered
rectally.
97. The method of claim 92 wherein the compound is administered
nasally.
98. The method of claim 92 wherein the compound is administered
sublingually.
99. The method of claim 92 wherein the compound is administered in
a dosage of from about 0.01 .mu.g/day to about 1000 mg/day.
100. The method of claim 92 wherein the compound is a
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 analog
having the formula: ##STR00048##
101. A compound having the formula: ##STR00049## where Y is
selected from the group consisting of hydrogen and a
hydroxy-protecting group.
102. The compound of claim 101 wherein Y is hydrogen.
103. The compound of claim 101 wherein Y is t-butyldimethylsilyl.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to vitamin D compounds, and more
particularly to 3,2-dihydrofuran- 19-nor-vitamin D analogs and
their pharmaceutical uses.
[0002] The natural hormone, 1.alpha.,25-dihydroxyvitamin D.sub.3
and its analog in 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 more
recently their activity in cellular differentiation has 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.
[0003] In 1990, a new class of vitamin D analogs was discovered,
i.e. the so called 19-nor-vitamin D compounds, characterized by the
replacement of the ring A 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, with 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 Letters 31, 1823 (1990); Perlman et al., Tetrahedron
Letters 32, 7663 (1991), and DeLuca et al., U.S. Pat. No.
5,086,191). A few years later, analogs of
1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 substituted at
2-position with hydroxy or alkoxy groups (DeLuca et al., U.S. Pat.
No. 5,536,713) were synthesized. Other 2-substituted analogs of
1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 have also been
synthesized, e.g. compounds substituted at 2-position 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). It has
been established that they 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.
[0004] 17-ene vitamin D compounds as well as vitamin D compounds
having a double bond in the side chain thereof are also known, and
have been proposed for various pharmacological uses. Bone diseases
such as osteoporosis, skin disorders such as psoriasis, cancers
such as leukemia and cosmetic conditions such as wrinkles are just
some of the applications proposed for such compounds. 17-ene
compounds are described in U.S. Pat. Nos. 5,545,633; 5,929,056 and
6,399,797 while 2-alkylidene compounds having a side chain with a
double bond therein are described in, for example, U.S. Pat. No.
5,843,928.
[0005] 19-nor vitamin D compounds substituted at the carbon-2
position of ring A with an alkyl group such as methyl, or an
alkylidene group such as methylene, and having a side chain lacking
one or more of the standard vitamin D.sub.3 substitutents, are also
known, and have been proposed for various pharmacological uses. For
example, numerous 2.alpha.-methyl-19,26,27-trinor analogs are
described in published U.S. Application No. 2007/028704 and in
published U.S. Application No.2007/0270391, and numerous
2-methylene-19,26,27-trinor analogs are described in published U.S.
Application No. 2007/0249567. In addition,
2.alpha.-methyl-19-nor-(20S)-1.alpha.-hydroxy-bishomopregnacalciferol
is described in published U.S. Application No. 2007/0254857, and
numerous 2-methylene-19,26-dinor vitamin D analogs are described in
published U.S. Application No.2007/0191317 and in published U.S.
Application No. 2007/0191316.
[0006] 19-nor-vitamin D analogs which are characterized by the
presence of a methylene substituent at carbon 2 (C-2), a hydroxyl
group at carbon 1 (C-1), and a shortened side chain attached to
carbon 20 (C-20) have also been synthesized and tested.
1.alpha.-hydroxy-2-methylene-19-nor-pregnacalciferol is described
in U.S. Pat. No. 6,566,352 while
1.alpha.-hydroxy-2-methylene-19-nor-homopregnacalciferol is
described in U.S. Pat. No. 6,579,861 and
1.alpha.-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is
described in U.S. Pat. No. 6,627,622. Their biological activities
make these compounds excellent candidates for a variety of
pharmaceutical uses, as set forth in the above patents and
published patent applications.
[0007] In a continuing effort to explore the 19-nor class of
pharmacologically important vitamin D compounds, analogs which are
characterized by the transposition of the ring A exocyclic
methylene group from carbon 10 (C-10) to carbon 2 (C-2), i.e.
2-methylene-19-nor-vitamin D compounds have been recently
synthesized and tested (Sicinski et al., J. Med. Chem., 41, 4662
(1998); Sicinski et al., Steroids 67, 247 (2002); DeLuca et al.,
U.S. Pat. No. 5,843,928, 5,936,133 and 6,382,071). Molecular
mechanics studies, performed on these analogs, showed that a change
of ring-A conformation can be expected resulting in the
"flattening" of the cyclohexanediol ring. From molecular mechanics
calculations and NMR studies their A-ring conformational
equilibrium was established to be ca. 6:4 in favor of the conformer
that has an equatorial 1.alpha.-OH. Introduction of the 2-methylene
group into 19-nor-vitamin D carbon skeleton changes the character
of its (1.alpha.- and 3.beta.-) A-ring hydroxyls; they are both now
in the allylic positions, similar to the 1.alpha.-hydroxyl group
(crucial for biological activity) in the molecule of the natural
hormone, 1.alpha.,25-(OH).sub.2D.sub.3. It was found that
1.alpha.,25-dihydroxy-2-methylene-19-norvitamin D analogs are
characterized by significant biological potency, enhanced
dramatically in compounds with an "unnatural"
(20S)-configuration.
[0008] Very recently, 2-ethylidene analogs of
1.alpha.,25-dihydroxy-19-norvitamin D.sub.3 have been synthesized.
It turned out that such modification of the ring A results in
significant biological potency of compounds, especially enhanced in
the E-geometrical isomers, Sicinski et al., J. Med. Chem., 45, 3366
(2002). Interestingly, it has been established that E-isomers have
A-ring conformational equilibrium considerably shifted to one
particular chair form, that possessing 1.alpha.-hydroxyl in an
equatorial orientation. Also, the analogs which are characterized
by the presence of substituted propylidene moiety at C-2 have also
been synthesized and preliminary biological tests indicated strong
and selective (intestinal) calcemic activity of the E-geometrical
isomers.
[0009] A-ring conformational equilibrium in vitamin D compounds has
attracted considerable research interest for more than 30 years.
Development of NMR spectroscopy and force field calculation methods
made it possible to establish, or even predict, the proportion of
equilibrating .alpha.- and .beta.-chair A-ring forms. Parallel to
these studies another, closely related problem has been discussed
in the literature, namely the correlation of A-ring conformation
with biological activities of vitamin D compounds. As early as in
1974 it was proposed [Okamura et al., Proc. Natl. Acad. Sci. USA,
71, 4194 (1974)] that equatorial orientation of 1.alpha.-hydroxy
group (i.e., the .beta.-chair form) is necessary for the calcium
regulation ability. Recently, Moras reported the crystal structures
of hVDR ligand binding domain (LBD) bound to the natural hormone
[Moras et al, Moll. Cell, 5, 173 (2000)] and the ligands with
unnatural configuration at C-20, [Moras et al, Proc. Natl. Acad.
Sci. USA, 98, 5491 (2001)] and it became clear that vitamin D
receptor binds (at least in the crystalline state) to vitamin D
analogs having their A-rings in .beta.-chair conformation. It
seemed, therefore, interesting to synthesize a vitamin D analog
that could only assume the opposite .alpha.-chair conformation of
its ring A, and as a consequence, possesses 1.alpha.-hydroxy group
in the axial orientation.
[0010] As a continuation of the search for biologically active
2-alkylidene-19-norvitamin D compounds, analogs which are
characterized by the presence of an additional ring and "flattening
bond" system [Corey et al, J. Org. Chem., 45, 757 (1980)] have also
been synthesized and tested. Such 19-norvitamin D compounds seemed
interesting targets because structural constrains of their
molecules would prevent their ring A from flipping over to the
alternative .beta.-chair form, effectively "freezing" the A-ring
.alpha.-chair conformation.
SUMMARY OF THE INVENTION
[0011] The present invention is directed toward
3,2-dihydrofuran-19-nor-vitamin D analogs, their biological
activity, and various pharmaceutical uses for these compounds.
[0012] A class of vitamin D compounds not known heretofore are the
vitamin D isomers having the A-ring exocyclic methylene moiety at
C-10 removed and possessing an additional fused dihydrofuran ring
connecting 3.beta.-oxygen and C-2. Structurally these novel analogs
are characterized by the general formula I shown below:
##STR00001##
where Y is selected from the group consisting of hydrogen and a
hydroxy-protecting group, and where the group R represents any of
the typical side chains known for vitamin D type compounds. Thus, R
may be an alkyl, hydrogen, hydroxyalkyl or fluoroalkyl group, or R
may represent a side chain of the formula:
##STR00002##
where Z in the above side chain structure is selected from Y, --OY,
--CH.sub.2OY, --C.ident.CY and --CH.dbd.CHY, where the double bond
in the side chain 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##
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.5, 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 having a general formula C.sub.kH.sub.2k--
where k is an integer, the 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--,
--CR.sub.1R.sub.2-- or --(CH.sub.2).sub.n-- at positions 20, 22,
and 23, respectively, may be replaced by an oxygen or sulfur
atom.
[0013] The wavy line to the carbon 20 indicates that carbon 20 may
have either the R or S configuration.
[0014] Specific important examples of side chains with natural
20R-configuration are the structures represented by formulas (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##
[0015] The preferred analog of formula I is
3,2-dihydrofuran-1.alpha.,25-dihydroxy -19-nor-vitamin D.sub.3
(hereinafter referred to as "B-REV5") which has the following
formula Ia:
##STR00005##
The above compounds I, and particularly Ia, exhibit a desired, and
highly advantageous, pattern of biological activity. This compound
has markedly reduced in vitro activities. The VDR binding affinity
of B-REV5 is approximately 4 orders of magnitude lower than the
native hormone. The potency of B-REV to promote HL60 cell
differentiation is 3 logs less than calcitriol. A similar reduction
in potency is observed for the in vitro transcription assay
conducted in bone cells. Importantly, in vivo this compound
exhibits biological activities in bone and intestine that are of
only slightly lower potency than 1,25-(OH).sub.2 D.sub.3. This in
vitro and in vivo disparity can be explained by metabolic
conversion to a more active compound only observable in the whole
animal system. It is likely that this compound might serve as a
slow-release drug or be used as a local-acting drug coupled with an
appropriate delivery method. Such compound might be a useful agent
for all therapeutic targets of vitamin D analogs, such as
autoimmune diseases, some types of cancer, secondary
hyperparathyroidism, psoriasis or other skin diseases, or bone
diseases.
[0016] One or more of the compounds may be present in a
pharmaceutical composition to treat or prevent the above-noted
diseases and disorders in an amount from about 0.01 .mu.g/gm to
about 1000 .mu.g/gm of the composition, preferably from about 0.1
.mu.g/gm to about 500 .mu.g/gm of the composition, and may be
administered topically, transdermally, orally, rectally, nasally,
sublingually, or parenterally in dosages of from about 0.01
.mu.g/day to about 1000 .mu.g/day, preferably from about 0.1
.mu.g/day to about 500 .mu.g/day.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1-5 illustrate various biological activities of
3,2-dihydrofuran-1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3
analog 10, referred to as "B-REV5," as compared to the native
hormone 1.alpha.,25-dihydroxyvitamin D.sub.3, hereinafter
"1,25(OH).sub.2D.sub.3."
[0018] FIG. 1 is a graph illustrating the relative activity of
B-REV5 and 1,25-(OH).sub.2D.sub.3 to compete for binding with
[.sup.3H]-1,25-(OH).sub.2-D.sub.3 to the full-length recombinant
rat vitamin D receptor;
[0019] FIG. 2 is a graph illustrating the percent HL-60 cell
differentiation as a function of the concentration of B-REV5 and
1,25-(OH).sub.2D.sub.3;
[0020] FIG. 3 is a graph illustrating the in vitro transcription
activity of 1,25-(OH).sub.2D.sub.3 as compared to B-REV5;
[0021] FIG. 4 is a bar graph illustrating the bone calcium
mobilization activity of 1,25-(OH).sub.2D.sub.3 as compared to
B-REV5, and
[0022] FIG. 5 is a bar graph illustrating the intestinal calcium
transport activity of 1,25-(OH).sub.2D.sub.3 as compared to
B-REV5.
DETAILED DESCRIPTION OF THE INVENTION
[0023] 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.
"Alkoxy" refers to any alkyl radical which is attached by oxygen,
i.e. a group represented by "alkyl-O--." 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.
[0024] 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. An "alkylidene" refers to
a radical having the general formula C.sub.kH.sub.2k-- where k is
an integer.
[0025] The preparation of 19-nor-vitamin D compounds of the basic
structures I and II can be accomplished by a common general method,
i.e. the Julia olefination involving a coupling of an unsaturated
sulfone IV, easily prepared from a bicyclic Windaus-Grundmann type
ketone III, with the bicyclic ketone V:
##STR00006##
[0026] In the structures III, IV and V groups Y and R represent
groups defined above whereas Ar represents phenyl, substituted
phenyl (preferably phenylthiazoline group) and other aromatic
groups that can be suitable for the Julia olefination process, it
being also understood that any functionalities in Ar that might be
sensitive, or that interfere with the condensation reaction, should
be avoided. 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.
Kittaka et al, Synlett, 8, 1175 (2003), and J. Org. Chem., 68, 7407
(2003).
[0027] Hydrindanones of the general structure III are known, or can
be prepared by known methods. Specific important examples of such
known bicyclic ketones are the structures with the side chains (a),
(b), (c) and (d) described above, i.e. 25-hydroxy Grundmann's
ketone (e) [Baggiolini et al., J. Org. Chem, 51, 3098 (1986)];
Grundmann's ketone (f) [Inhoffen et al., Chem. Ber. 90, 664
(1957)]; 25-hydroxy Windaus ketone (g) [Baggiolini et al., J. Org.
Chem., 51, 3098 (1986)] and Windaus ketone (h) [Windaus et al.,
Ann., 524, 297 (1936)]:
##STR00007##
[0028] For the preparation of the required bicyclic ketones of
general structure V, a new synthetic route has been developed
starting from cyclohexanedione derivative 1 that was obtained from
commercial (1R,3R,4S,5R)-(-)-quinic acid as described previously
[Sicinski et al., J. Med. Chem., 45, 3366 (2002)]. Process of
transformation of the starting diketone 1 into the desired A-ring
synthon is shown on the SCHEME I. Due to a considerable difference
in the steric hindrance between the two carbonyl groups, it was
possible to achieve selective protection of one of them providing
mono thioacetal 2 in 53% yield. The following Wittig reaction with
an ylide, generated from methyltriphenylphosphonium bromide and
n-BuLi, proved to be more efficient (84%) and resulted in the
methylenation of the remaining carbonyl carbon. Then, one of the
two secondary hydroxy groups of the obtained product 3 was
selectively deprotected and the alcohol 4, formed in 55% yield, was
then subjected to Williamson reaction. Thus, an anion of 4
generated with sodium hydride reacted with allyl bromide to form an
allylic ether 5 in 69% yield. Ring closing methatesis of compound 5
was performed in a presence of commercially available Grubb's II
generation catalyst 6. The expected tricyclic compound 7 was
obtained in 81% yield. Deprotection of the carbonyl group in 7 was
achieved by its treatment with thallium(III) trifluoroacetate. The
desired A-ring fragment 8, formed in 70% yield, was then subjected
to modified Julia olefination with the anion generated from
thiazoline sulphone 9 [prepared from the corresponding Grundmann
ketone according to the known procedure, Sicinski et al., J. Med.
Chem., 50, 6154 (2007)] accepted for publication) and lithium
bis(trimethylsilyl)amide. Removal of the silyl protecting groups in
the obtained 19-norvitamins gave the expected mixture of two
19-norvitamin D analogs 10 and 11 which were purified and separated
by straight- and reversed-phase HPLC. Analysis of their .sup.1H NMR
spectra confirmed that ring A in these compounds, due to the
presence of an exocyclic double bond being a part of additional
five-membered ring, is prevented from flipping and held in the
single chair conformation.
[0029] Several other 19-nor-vitamin D compounds may be synthesized
by the method disclosed herein using the A-ring synthon 8 and the
appropriate C,D-fragments derived from the Windaus-Grundmann
ketones having the desired side chain structure.
[0030] 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 the SCHEME I.
EXAMPLES
[0031] Chemistry. Melting points (uncorrected) were determined on a
Thomas-Hoover capillary melting-point apparatus. Ultraviolet (UV)
absorption spectra were recorded with a Perkin-Elmer Lambda 3B
UV-VIS spectrophotometer in ethanol. .sup.1H nuclear magnetic
resonance (NMR) spectra were recorded at 200, 400 and 500 MHz with
a Bruker Instruments DMX-400 and DMX-500 Avance console
spectrometers in deteriochloroform. .sup.13C nuclear magnetic
resonance (NMR) spectra were recorded at 125 MHz with a Varian
Unity plus 200 spectrometer, and Bruker Instruments DMX-500 Avance
console spectrometer in deuteriochloroform. Chemical shifts
(.delta.) are reported downfield from internal Me.sub.4Si (.delta.
0.00). Electron impact (EI) mass spectra were obtained with a
Micromass AutoSpec (Beverly, Mass.) instrument. High-performance
liquid chromatography (HPLC) was performed on a Waters Associates
liquid chromatograph equipped with a Model 6000A solvent delivery
system, a Model U6K Universal injector, and a Model 486 tunable
absorbance detector. THF was freshly distilled before use from
sodium benzophenone ketyl under argon.
Example 1
Preparation of 19-norvitamin D.sub.3 analogues 10 and 11
[0032] Referring to SCHEME I the starting cyclohexanedione 1 was
obtained from commercial (-)-quinic acid according to the described
procedure, Sicinski et al., J. Med. Chem. 45, 3366 (2002).
[0033] (a) Selective Protection of Carbonyl Group in Diketone 1
[0034]
(2R,6R)-2,6-Bis[(tert-butyldimethylsilyl)oxy]-4,4-ethylenedithio-cy-
clohexanone (2). To a stirred solution of 1,2-ethanedithiol (0.25
mL, 3.0 mmol) and Zn(OTf).sub.2 (646 mg, 1.78 mmol) in anhydrous
methylene chloride (7.2 mL) was transferred a solution of 1 (964
mg, 2.24 mmol) in anhydrous methylene chloride (9.6 mL) at
0.degree. C. under argon. The mixture was stirred at 0.degree. C.
for 1 h, and at room temperature for 1.5 h and then it was poured
into brine and extracted with ethyl acetate. The extract was washed
with saturated NaHCO.sub.3, water, 5% HCl, again water, dried
(MgSO.sub.4) and evaporated. The residue was purified by column
chromatography on silica. Elution with hexane/ethyl acetate (96:4)
afforded an oily ketone 2 (729 mg, 53%). 2: [.alpha.].sup.20.sub.D
-52.degree. (c 0.1, CHCl.sub.3); .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 0.078 and 0.085 (2.times.6H, 2.times.s,
4.times.SiCH.sub.3), 0.92 (2.times.9H, s, 2.times.Si-t-Bu), 2.35
(2H, ddd, J=14.0, 7.4, 1.5 Hz, 3.alpha.- and 5.beta.-H), 2.57 (2H,
ddd, J=14.0, 4.6, 1.5 Hz, 3.beta.- and 5.alpha.-H), 3.23-3.38 (4H,
m, S--CH.sub.2CH.sub.2--S), 4.63 (2H, dd, J=7.4, 4.6 Hz, 2.beta.-
and 6.alpha.-H).
[0035] (b) Wittig Reaction of Ketone 2
[0036]
(1R,3R)-1,3-Bis[(tert-butyldimethylsilyl)oxy]-5,5-ethylenedithio-2--
methylenecyclohexane (3). To the methyltriphenylphosphonium bromide
(59 mg, 0.167 mmol) in anhydrous THF (0.8 mL) was added at
0.degree. C. n-BuLi (1.6 M in cyclohexane, 105 .mu.L, 0.167 mmol)
under argon with stirring. The orange-red mixture was cooled to
-78.degree. C. and siphoned to a stirred solution of ketone 2 (30
mg, 0.067 mmol) in anhydrous THF (0.5 mL). The mixture was stirred
at -78.degree. C. for 2 h, and the reaction was quenched by the
addition of brine containing 1% HCl. Saturated NaHCO.sub.3 (3 mL),
water (3 mL), diethyl ether(3 mL) and ethyl acetate (6 mL) were
added and the mixture was vigorously stirred at room temperature.
After 16 h the layers were separated, organic phase was washed with
brine, dried (MgSO.sub.4), and evaporated. The residue was purified
by column chromatography on silica. Elution with hexane/ethyl
acetate (99:1) gave an oily compound 3 (25 mg, 84%). 3:
[.alpha.].sup.20.sub.D -24.degree. (c 0.1, CDCl.sub.3); .sup.1H NMR
(200 MHz, CDCl.sub.3) .delta. 0.056 and 0.079 (2.times.6H,
2.times.s, 4.times.SiCH.sub.3), 0.92 (2.times.9H, s,
2.times.Si-t-Bu), 2.07 (2H, br dd, J=13.2, 7.1 Hz, 4.alpha.- and
6.beta.-H), 2.39 (2H, ddd, J=13.2, 4.0, 1.0 Hz, 4.beta.- and
6.alpha.-H), 3.07-3.30 (4H, m, S--CH.sub.2CH.sub.2--S), 4.59 (2H,
dd, J=7.1, 4.0, Hz, 1.alpha.- and 3.beta.-H), 4.94 (2H, s,
.dbd.CH.sub.2).
[0037] (c) Selective Deprotection of Hydroxyl Group in Compound
3
[0038]
(3R)-3-[(tert-Butyldimethylsilyl)oxy]-5,5-ethylenedithio-2-methylen-
ecyclohexanol (4). To a solution of compound 3 (93 mg, 208 .mu.mol)
in anhydrous THF (7.8 mL) was added tetrabutylammonium fluoride (1M
in THF; 208 .mu.L, 208 .mu.mol) and the mixture was stirred for 18
h. It was then poured into brine and extracted with ethyl acetate.
Organic phase was washed with brine, dried (MgSO.sub.4), and
evaporated. The residue was purified by column chromatography on
silica. Elution with hexane/ethyl acetate (96:4) gave an oily
alcohol 4 (38 mg, 55%). 4: [.alpha.].sup.20.sub.D -44.6.degree. (c
0.15, CDCl.sub.3); .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 0.082
and 0.100 (2.times.3H, 2.times.s, 2.times.SiCH.sub.3), 0.92 (9H, s,
Si-t-Bu), 2.14 (2H, m, 4.beta.- and 6.alpha.-H), 2.41 (2H, m,
4.alpha.- and 6.beta.-H), 3.32 (4H, s, S--CH.sub.2CH.sub.2--S),
4.60 (2H, m, 1.beta.-i 3.alpha.-H), 5.03 and 5.06 (2H, 2.times.s,
.dbd.CH.sub.2).
[0039] (d) Williamson Reaction of Compound 4 with Allyl Bromide
[0040]
(1R,3R)-1-Allyloxy-3-[(tert-Butyldimethylsilyl)oxy]-5,5-ethylenedit-
hio-2-methylenecyclohexane (5). Sodium hydride (60% suspension in
oil; 8.5 mg, 0.213 mmol) was washed with anhydrous hexane (1 mL)
under argon, 18-crown-6 (8.5 mg, 32 .mu.mol) was added, and then a
solution of cyclohexanol 4 (22 mg, 66 .mu.mol) in anhydrous DMF
(0.63 mL) was siphoned to the reaction flask. After 5 min of
stirring allyl bromide (87.8 mg, 89 .mu.l, 0.726 mmol) was added.
The mixture was stirred at room temperature for 1.5 h, water was
added and the mixture was extracted with ethyl acetate. Organic
phase was separated, washed with water, dried (MgSO.sub.4), and
evaporated. The residue was purified by column chromatography on
silica. Elution with hexane/ethyl acetate (99:1) gave an oily
compound 5 (17 mg, 69%). 5: [.alpha.].sup.20.sub.D -45.degree. (c
0.6, CDCl.sub.3); .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 0.079
and 0.088 (2.times.3H, 2.times.s, 2.times.SiCH.sub.3), 0.93 (9H, s,
Si-t-Bu), 2.00 (1H, dd, J=12.7, 9.3 Hz, 4.beta.-H), 2.37 (2H, m,
6.alpha.- and 6.beta.-H), 2.41 (1H, dd, J=12.7, 4.6 Hz,
4.alpha.-H), 3.22-3.35 (4H, narr m, S--CH.sub.2CH.sub.2--S), 3.86
(2H, m, OCH.sub.2CH.dbd.CH.sub.2), 4.16 (1H, t, J=4.4 Hz,
1.beta.-H), 4.57 (1H, m, 3.alpha.-H), 4.97 [(1H, br s, one of
C(2).dbd.CH.sub.2], 5.15 [1H, m, C(2')H.dbd.CH.sub.trans], 5.17
[(1H, br s, one of C(2).dbd.CH.sub.2], 5.35 [1H, br ddd, J=17.0,
4.0, 2.0 Hz, C(2')H.dbd.CH.sub.cis], 5.93 (1H, br m,
CH.sub.2--CH.dbd.).
[0041] (e) Ring Closing Methatesis of Compound 5
[0042]
(4R,7aR)-4-[(tert-Butyldimethylsilyl)oxy]-6,6-etylenodithio-2,4,5,6-
,7,7a-hexahydro-benzofuran (7). To a stirred solution of compound 5
(12 mg, 0.032 mmol) in anhydrous toluene (8 mL) at 80.degree. C.
Grubb's II generation catalyst 6 (2.7 mg, 3 1.mu.mol) was added.
The mixture was for 40 min, and then it was cooled to room
temperature. The mixture was applied on Waters silica Sep-Pak to
remove the catalyst and eluted with anhydrous toluene. The residue
was purified by column chromatography on silica. Elution with
hexane/ethyl acetate (99:1) gave an oily compound 7 (9 mg, 81%). 7:
[.alpha.].sup.20.sub.D -29.5.degree. (c 0.4, CDCl.sub.3); .sup.1H
NMR (500 MHz,CDCl.sub.3) .delta. 0.069 and 0.084 (3H and 3H,
2.times.s, 2.times.SiCH.sub.3), 0.91 (9H, s, Si-t-Bu), 1.77 (1H,
dd, J=12.5, 10.5 Hz, 7.alpha.-H), 2.27 (1H, dd, J=14.0, 2.5 Hz,
5.alpha.-H), 2.41 (1H, dt, J=14.0, 2.5 Hz, 5.beta.-H), 2.63 (1H,
ddd, J=12.5, 5.5, 2.5, 7.beta.-H), 3.25 (4H, br m,
S--CH.sub.2CH.sub.2--S), 4.62 (1H, ddd, J=13.0, 4.5, 1.5 Hz,
2.alpha.-H), 4.67 (1H, ddd, J=13.0, 3.0, 1.5 Hz, 2.beta.-H), 4.72
(1H, t, J=2.5 Hz, 4.alpha.-H), 5.09 (2H, br m, w/2=22 Hz,
7a.beta.-H), 5.54 (1H, m, 3-H); MS (ESI) exact mass calculated for
C.sub.16H.sub.28O.sub.2S.sub.2Si.sub.2Na 367.1 (M.sup.++Na), found
367.2.
[0043] (f) Deprotection of Carbonyl Group in Compound 7
[0044]
(4R,7aR)-4-[(tert-Butyldimethylsilyl)oxy]-4,5,7,7a-tetrahydro-2H-be-
nzofuran-6-one (8). Thallium trifluoroacetate (39 mg, 60 mmol) was
added at room temperature to a solution of 7 (20 mg, 58 mmol) in
anhydrous THF (1 mL). After stirring for 40 min saturated
Na.sub.2SO.sub.3 was added, the mixture was poured into water and
extracted with ethyl acetate. Organic phase was washed with water,
dried (MgSO.sub.4), and evaporated to give a colorless residue
which was purified by column chromatography on silica. Elution with
hexane/ethyl acetate (95:5) gave ketone 8 (6 mg, 38%; 70% based on
recovered substrate). The column was then washed with hexane/ethyl
acetate (9:1) to afford the unreacted thioacetal 7 (9 mg). 8:
[.alpha.].sup.24.sub.D -92.degree. (c 0.28, CHCl.sub.3); .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 0.034 and 0.073 (3H and 3H, each
s, 2.times.SiCH.sub.3), 0.843 [9H, s, Si-t-Bu], 2.42 (1H, br d,
J=14.3 Hz, 5.beta.-H), 2.44 (1H, dd, J=13.4, 10.0 Hz, 7.alpha.-H),
2.51 (1H, dd, J=14.3, 3.4 Hz, 5.alpha.-H), 3.00 (1H, ddd, J=13.4,
6.5, 1.6 Hz, 7.beta.-H), 4.77 (2H, d, J=4.5 Hz, 2.alpha.- and
2.beta.-H), 4.92 (1H, narr m, 4.alpha.-H), 5.17 (1H, br m w/2=19
Hz, 7a.beta.-H), 5.76 (1H, br s, 3-H); .sup.13C NMR (125 MHz)
.delta. -5.1 (Si--CH.sub.3), -4.9 (Si--CH.sub.3), 18.0
[C(CH.sub.3).sub.3], 25.7 [C(CH.sub.3).sub.3], 50.5 and 50.8
(C.sub.5 and C.sub.7), 65.7 (C.sub.7a), 75.9 (C.sub.2), 81.7
(C.sub.4), 119.7 (C.sub.3), 141.1 (C.sub.3a), 206.5 (C.sub.6).
[0045] (g) Julia Coupling of Ketone 8 and Sulfone 9
[0046] 1.alpha.,25-Dihydroxy- and 25-hydroxy-19-norvitamin D.sub.3
analogues (10 and 11). To a solution of sulfone 9 (30.0 mg, 48
.mu.mol) in dry THF (200 .mu.L) was added LiHMDS (1 M in THF, 48
.mu.L, 48 .mu.mol) at -78.degree. C. under argon. The solution
turned deep red. The mixture was stirred at -78.degree. C. for 20
min and a solution of the ketone 8 (5.4 mg, 20 .mu.mol) in THF
(100+80 .mu.L) was added. The stirring was continued at -78.degree.
C. for 1.5 h, and the reaction mixture was allowed to warm to
-10.degree. C. during ca. 1.5 h. Then it was poured into saturated
NH.sub.4Cl and extracted with ether. The extract was washed with
brine, dried (Na.sub.2SO.sub.4) and evaporated. The yellow oily
residue was applied on silica Sep-Pak, eluted with hexane/ethyl
acetate (98:2), concentrated under vacuum, dissolved in anhydrous
methanol (1 mL) and treated with (+)-10-camphorosulfonic acid (15
mg, 64.5 .mu.mol). The solution was stirred at room temperature
under argon for 19 h, poured into brine, and extracted with ethyl
acetate. The extract was washed with diluted NaHCO.sub.3 and brine,
dried (Na.sub.2SO.sub.4) and evaporated. The residue was purified
by HPLC (9.4 mm.times.25 cm Zorbax-Sil column, 4 mL/min) using
hexane/2-propanol (87:13) solvent system. Isomeric 19-norvitamins
10 (B-REV5) (1.4 mg, 17%) and 11 (A-REV5) (0.5 mg, 6%) were
collected at R.sub.V 30 mL and R.sub.v 35 mL, respectively. Final
purification and separation of both isomers was achieved by
reversed-phase HPLC (9.4 mm.times.25 cm Zorbax-ODS column, 4
mL/min) using methanol/water (95:5) solvent system:
25-hydroxyvitamin D analogue 11 was collected at R.sub.v 19 mL and
isomeric 1.alpha.,25-dihydroxyvitamin D analogue 10 at R.sub.V 23
mL.
[0047] 10 (B-REV5): UV (in EtOH) .lamda..sub.max 245.5, 253.0,
262.0 nm; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.549 (3H, s,
18-H3), 0.939 (3H, d, J=6.3 Hz, 21-H.sub.3), 1.219 (6H, s, 26- and
27-H.sub.3), 2.81 (2H, m, 4.alpha.-H overlapped with 9.beta.-H),
3.09 (1H, d, J=14.0 Hz, 10.alpha.-H), 4.69 (2H, narr m,
CH.sub.2--O), 4.76 (1H, br s, 1.beta.-H), 4.90 (1H, m, w/2 19 Hz,
3.alpha.-H), 5.71 (1H, narr m, O--CH.sub.2--HC.dbd.C), 5.85 and
6.48 (1H and 1H, each d, J=11.2 Hz, 7- and 6-H.
[0048] 11 (A-REV5): UV (in EtOH) .lamda..sub.max 245.5, 253.0,
262.5 nm; .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 0.551 (3H, s,
18-H.sub.3), 0.940 (3H, d, J=6.3 Hz, 21-H.sub.3), 1.22 (6H, s, 26-
and 27-H.sub.3), 1.85 (1H, t, J.about.11.5 Hz, 10.alpha.-H), 2.47
(2H, narr m, 4.alpha.- and 4.beta.-H), 2.82 (1H, br d, J=13.0 Hz,
9.beta.-H), 3.36 (1H, dd, J=11.9, 5.9 Hz, 10.beta.-H), 4.70 (3H,
narr m, 3.alpha.-H overlapped with CH.sub.2--O), 4.82 (1H, m,
w/2=25 Hz, 1.beta.-H), 5.70 (1H, br s, O--CH.sub.2--HC.dbd.C), 5.90
and 6.36 (1H and 1H, each d, J=11.2 Hz, 7- and 6-H); HRMS (ESI)
exact mass calcd for C.sub.28H.sub.44O.sub.3Na
(M+Na).sup.+451.3188, measured 451.3197.
##STR00008##
Biological Activity of
3,2-Dihydrofuran-1.alpha.,25-Dihydroxy-19-Nor Vitamin D.sub.3
Compound (Analog 10, B-REV5)
[0049] The introduction of a fused dihydrofuran ring connecting
3.beta.-oxygen and carbon-2 (analog 10, B-REV5) diminished binding
to the full length recombinant rat vitamin D receptor, as compared
to 1.alpha.,25-dihydroxyvitamin D.sub.3 by about 1 log or 10-fold
(FIG. 1). Despite a reduced receptor binding activity in vitro,
this compound in vivo had significant intestinal calcium transport
activity with no activity in bone mobilization. Thus, B-REV5 is an
analog with highly unique selective biological activity.
[0050] FIG. 5 shows that B-REV5 has very significant ability to
increase intestinal calcium transport activity in vivo. It clearly
has about the same potency in vivo as compared to that of
1,25-dihydroxyvitamin D.sub.3 (1,25-(OH).sub.2D.sub.3), the natural
hormone, in stimulating intestinal calcium transport.
[0051] FIG. 4 demonstrates that B-REV5 also has very significant
bone calcium mobilization activity, as compared to
1,25-(OH).sub.2D.sub.3. B-REV5 demonstrated only slightly less bone
calcium mobilization activity at the doses as compared to of
1,25-(OH).sub.2D.sub.3. Thus, B-REV5 clearly has significant
potency in mobilizing calcium from bone as compared to
1,25-(OH).sub.2D.sub.3, at the recommended doses.
[0052] FIGS. 4 and 5 thus illustrate that B-REV5 may be
characterized as having substantially high intestinal calcium
transport activity, as well as substantially high bone calcium
mobilization activity, i.e. as having significant calcemic
activity. The preferential activity on bone calcium mobilizing
activity allows the in vivo administration of this compound for the
treatment and prophylaxis of metabolic bone diseases. Because of
its preferential activity on bone, this compound would be a
preferred therapeutic agent for the treatment and prophylaxis of
diseases such as osteoporosis, especially low bone turnover
osteoporosis, steroid induce osteoporosis, senile osteoporosis or
postmenopausal osteoporosis, as well as osteomalacia, osteopenia
and renal osteodystrophy.
[0053] FIG. 2 illustrates that B-REV5 is considerably less active
than 1,25-(OH).sub.2D.sub.3 on HL-60 cell differentiation. The data
show it is about 1,000 times less active than
1,25(OH).sub.2D.sub.3.
[0054] FIG. 3 illustrates that the compound B-REV5 has much less
transcriptional activity (about 1000 times less potent) than
1.alpha.,25-dihydroxyvitamin D.sub.3 in bone cells.
[0055] Although not as potent as 1.alpha.,25-(OH).sub.2D.sub.3,
B-REV5 still has cell differentiation activity, and thus the
activity of B-REV5 on cell differentiation suggests that B-REV5 may
be an excellent candidate for the treatment of psoriasis and
cancer, especially against leukemia, colon cancer, breast cancer,
skin cancer and prostate cancer. In addition, due to its relatively
high cell differentiation activity, this compound provides a
therapeutic agent for the treatment of various skin conditions
including wrinkles, lack of adequate dermal hydration, i.e. dry
skin, lack of adequate skin firmness, i.e. slack skin, and
insufficient sebum secretion. Use of this compound thus not only
results in moisturizing of skin but also improves the barrier
function of skin.
[0056] FIG. 3 illustrates that in bone cells the compound B-REV5
has some ability to increase transcription of the 24-hydroxylase
gene. This result, together with the cell differentiation activity
of FIG. 2, suggests that B-REV5 will be very effective in psoriasis
because it has direct cellular activity in causing cell
differentiation, gene transcription, and in suppressing cell
growth. These data also indicate that B-REV5 may have significant
activity as an anti-cancer agent, especially against leukemia,
colon cancer, breast cancer, skin cancer and prostate cancer.
[0057] The activity of B-REV5 on HL-60 differentiation suggests it
will be active in suppressing growth of parathyroid glands and in
the suppression of the preproparathyroid gene. It is undesirable to
raise serum calcium to supraphysiologic levels when suppressing the
preproparathyroid hormone gene (Darwish & DeLuca, Arch.
Biochem. Biophys. 365, 123-130, 1999) and parathyroid gland
proliferation. These analogs having relatively low bone calcium
mobilization activity while active on cell differentiation are
expected to be useful as a therapy for suppression of secondary
hyperparathyroidism of renal osteodystrophy.
Experimental Methods
[0058] Vitamin D Receptor Binding
[0059] Test Material
[0060] Protein Source
[0061] Full-length recombinant rat receptor was expressed in E.
coli BL21 (DE3) Codon Plus RIL cells and purified to homogeneity
using two different column chromatography systems. The first system
was a nickel affinity resin that utilizes 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 in
binding assays, the protein was diluted in TEDK.sub.50 (50 mM Tris,
1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCl) with 0.1% Chaps
detergent. The receptor protein and ligand concentration were
optimized such that no more than 20% of the added radiolabeled
ligand was bound to the receptor.
[0062] Study Drugs
[0063] Unlabeled ligands were dissolved in ethanol and the
concentrations determined using UV spectrophotometry
(1,25-(OH).sub.2D.sub.3: molar extinction coefficient=18,200 and
.lamda..sub.max=265 nm; Analogs: molar extinction
coefficient=42,000 and .lamda..sub.max=252 nm). Radiolabeled ligand
(.sup.3H-1,25-(OH).sub.2D.sub.3, .about.159 Ci/mmole) was added in
ethanol at a final concentration of 1 nM.
[0064] Assay Conditions
[0065] Radiolabeled and unlabeled ligands were added to 100 mcl of
the diluted protein at a final ethanol concentration of
.ltoreq.10%, 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 hydroxylapaptite was collected by centrifugation
and then washed three times with Tris-EDTA buffer (50 mM Tris, 1.5
mM EDTA, pH 7.4) containing 0.5% Titron 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.
[0066] HL-60 Differentiation
[0067] Test Material
[0068] Study Drugs
[0069] The study drugs were dissolved in ethanol and the
concentrations determined using UV spectrophotometry. Serial
dilutions were prepared so that a range of drug concentrations
could be tested without changing the final concentration of ethanol
(.ltoreq.0.2%) present in the cell cultures.
[0070] Cells
[0071] 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.
[0072] Assay Conditions
[0073] HL60 cells were plated at 1.2.times.10.sup.5 cells/ml.
Eighteen hours after plating, cells in duplicate were treated with
drug. Four days later, the cells were harvested and a nitro blue
tetrazolium reduction assay was performed (Collins et al., 1979; J.
Exp. Med. 149:969-974). 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).
[0074] In Vitro Transcription Assay
[0075] Transcription 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.
[0076] RLU=Relative Luciferase Units.
[0077] Intestinal Calcium Transport and Bone Calcium
Mobilization
[0078] Male, weanling Sprague-Dawley rats were placed on Diet 11
(0.47% Ca) diet+AEK oil for one week followed by Diet 11 (0.02%
Ca)+AEK oil for 3 weeks. The rats were then switched to a diet
containing 0.47% Ca for one week followed by two weeks on a diet
containing 0.02% Ca. Dose administration began during the last week
on 0.02% calcium diet. Four consecutive ip doses were given
approximately 24 hours apart. Twenty-four hours after the last
dose, blood was collected from the severed neck and the
concentration of serum calcium determined as a measure of bone
calcium mobilization. The first 10 cm of the intestine was also
collected for intestinal calcium transport analysis using the
everted gut sac method.
Interpretation of Data
[0079] VDR binding, HL60 cell differentiation, and transcription
activity. B-REV5 (K.sub.i=8.times.10.sup.-7M) has much lower
ability than the natural hormone 1 (1.alpha.,25-dihydroxyvitamin
D.sub.3 (K.sub.i=7.times.10.sup.-11M) in its ability to compete
with [.sup.3H]-1,25(OH).sub.2D.sub.3 for binding to the full-length
recombinant rat vitamin D receptor (FIG. 1). B-REV5 is about 4
logs, or 40 times, less potent than 1,25-(OH).sub.2D.sub.3 in its
affinity for the VDR. B-REV5 (EC.sub.50=1.times.10.sup.-6M) is also
considerably lower in its ability to promote HL60 differentiation
as compared to 1.alpha.,25-dihydroxyvitamin D.sub.3
(EC.sub.50=3.times.10.sup.-9M) (See FIG. 2). The ability of the
vitamin B-REV5 to induce differentiation of human promyelocyte
HL-60 cells into monocytes is about 1,000 times less potent than
the natural hormone (FIG. 2). Also, compound B-REV5
(EC.sub.50=3.times.10.sup.-7M) has much lower transcriptional
activity in bone cells than 1.alpha.,25-dihydroxyvitamin D.sub.3
(EC.sub.50=2.0.times.10.sup.-10M) (see FIG. 3). Thus, B-REV5 has
weak transcriptional activity, indicated in the 24-hydroxylase
(CYP-24) promoter driving luciferase reporter gene system, and is
about 1,000 times less potent than 1,25-(OH).sub.2D.sub.3 in
increasing transcription of the 24-hydroxylase gene (see FIG.
3).
[0080] Calcium mobilization from bone in vitamin D-deficient
animals. Using vitamin D-deficient mice on a low calcium diet
(0.02%), the activities of B-REV5 and 1,25-(OH).sub.2D.sub.3 in
bone were tested. As expected, the native hormone
(1,25-(OH).sub.2D.sub.3) increased serum calcium levels at all the
dosages tested (FIG. 4).
[0081] The study reported in FIG. 4 shows that B-REV5 has
relatively high activity in mobilizing calcium from bone. B-REV5 is
only slightly less potent than 1.alpha.,25-(OH).sub.2D.sub.3 as the
administration of 7,020 pmol/day of B-REV5 for 4 consecutive days
resulted in a serum calcium value of 6.1 mg/dL whereas the native
hormone 1,25-(OH).sub.2D.sub.3 had a value of 7.9 mg/dL at 7,020
pmol/day.
[0082] Intestinal calcium transport was evaluated in the same group
of animals using the everted gut sac method (FIG. 5). The study
reported in FIG. 5 shows B-REV5 has relatively significant
intestinal calcium transport activity which is similar to
1,25-(OH).sub.2D.sub.3. Administration of 780 pmol/day of B-REV5
for 4 consecutive days resulted in almost the same activity as
1,25-(OH).sub.2D.sub.3 at 780 pmol/day.
[0083] These results show that the compound B-REV5 promotes
intestinal calcium transport and bone calcium mobilization. Thus,
it may be concluded that B-REV5 has similar intestinal calcium
transport activity and is only slightly less potent in bone calcium
mobilization activity to that of 1,25-(OH).sub.2D.sub.3 at the
recommended doses.
[0084] These results illustrate that B-REV5 is an excellent
candidate for numerous human therapies as described herein, and
that it may be particularly useful in a number of circumstances
such as suppression of secondary hyperparathyroidism of renal
osteodystrophy, autoimmune diseases, cancer, numerous types of skin
conditions, and psoriasis, metabolic bone diseases, osteomalcia and
osteopenia. B-REV5 is an excellent candidate for treating psoriasis
because: (1) it has VDR binding, transcription activity and
cellular differentiation activity; (2) it has low hypercalcemic
liability, unlike 1,25-(OH).sub.2D.sub.3; and (3) it is easily
synthesized. Since B-REV5 has binding activity to the vitamin D
receptor, but has relatively low ability to raise blood serum
calcium, it may also be particularly useful for the treatment of
secondary hyperparathyroidism of renal osteodystrophy.
[0085] The compounds of the invention of formula I, and
particularly B-REV5 of formula Ia, are characterized as having
significant calcemic activity. Their preferential activity on bone
calcium mobilizing activity allows the in vivo administration of
these compounds for the treatment and prophylaxis of metabolic bone
diseases. Because of their preferential activity on bone, these
compounds would be preferred therapeutic agents for the treatment
and prophylaxis of diseases such as osteoporosis, especially low
bone turnover osteoporosis, steroid induced osteoporosis, senile
osteoporosis or postmenopausal osteoporosis, as well as
osteomalacia, osteopenia and renal osteodystrophy.
[0086] These data also indicate that the compound B-REV5 of the
invention may be 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, lupus, diabetes mellitus, host versus graft rejection,
and rejection of organ transplants; and additionally for the
treatment of inflammatory diseases, such as rheumatoid arthritis,
asthma, and inflammatory bowel diseases such as celiac disease,
ulcerative colitis and Crohn's disease. Acne, alopecia and
hypertension are other conditions which may be treated with the
compound B-REV5 of the invention.
[0087] The compounds of the invention of formula I, and
particularly B-REV5 of formula Ia, are also useful in preventing or
treating obesity, inhibiting adipocyte differentiation, inhibiting
SCD-1 gene transcription, and/or reducing body fat in animal
subjects. Therefore, in some embodiments, a method of preventing or
treating obesity, inhibiting adipocyte differentiation, inhibiting
SCD-1 gene transcription, and/or reducing body fat in an animal
subject includes administering to the animal subject, an effective
amount of one or more of the compounds or a pharmaceutical
composition that includes one or more of the compounds of formula
I. Administration of the compound or the pharmaceutical
compositions to the subject inhibits adipocyte differentiation,
inhibits gene transcription, and/or reduces body fat in the animal
subject. The animal may be a human, a domestic animal such as a dog
or a cat, or an agricultural animal, especially those that provide
meat for human consumption, such as fowl like chickens, turkeys,
pheasant or quail, as well as bovine, ovine, caprine, or porcine
animals.
[0088] For prevention and/or treatment purposes, the compounds of
this invention defined by formula I, particularly B-REV5, 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.
[0089] The compounds of formula I and particularly B-REV5, may be
administered orally, topically, parenterally, rectally, nasally,
sublingually or transdermally. The compound is 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. A dose of
from 0.01 .mu.g to 1000 .mu.g per day of the compounds I,
particularly B-REV5, preferably from about 0.1 .mu.g to about 500
.mu.g per day, is appropriate for prevention and/or treatment
purposes, such dose 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 compound exhibits 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.
[0090] Compositions for use in the above-mentioned treatments
comprise an effective amount of the compounds I, particularly
B-REV5, as defined by the above formula I and Ia as the active
ingredient, and a suitable carrier. An effective amount of such
compound for use in accordance with this invention is from about
0.01 .mu.g to about 1000 .mu.g per gm of composition, preferably
from about 0.1 .mu.g to about 500 .mu.g per gram of composition,
and may be administered topically, transdermally, orally, rectally,
nasally, sublingually, or parenterally in dosages of from about
0.01 .mu.g/day to about 1000 .mu.g/day, and preferably from about
0.1 .mu.g/day to about 500 .mu.g/day.
[0091] The compounds I, particularly B-REV5, may be formulated as
creams, lotions, ointments, topical patches, pills, capsules or
tablets, suppositories, aerosols, 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.
[0092] The compounds I, particularly B-REV5, may be 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] For nasal administration, 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..
[0099] 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.
* * * * *