U.S. patent application number 10/206430 was filed with the patent office on 2003-05-01 for 1,3-dihydroxy-20,20-dialkyl-vitamin d3 analogs.
This patent application is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Manchand, Percy S., Uskokovic, Milan R..
Application Number | 20030083319 10/206430 |
Document ID | / |
Family ID | 22014900 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030083319 |
Kind Code |
A1 |
Manchand, Percy S. ; et
al. |
May 1, 2003 |
1,3-Dihydroxy-20,20-dialkyl-vitamin D3 analogs
Abstract
This invention relates to 1,3-dihydroxy-20,20-dialkyl-vitamin D3
analogs of Formula (I): 1 compositions comprising the analogs,
methods of preparing the analogs and methods of treatment of
osteoporosis, secondary hyperparathyroidism, cancer and autoimmune
diseases using such analogs.
Inventors: |
Manchand, Percy S.;
(Montclair, NJ) ; Uskokovic, Milan R.; (Upper
Montclair, NJ) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Hoffmann-La Roche Inc.
Nutley
NJ
|
Family ID: |
22014900 |
Appl. No.: |
10/206430 |
Filed: |
July 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10206430 |
Jul 25, 2002 |
|
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09148018 |
Sep 3, 1998 |
|
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60058132 |
Sep 8, 1997 |
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Current U.S.
Class: |
514/167 ;
552/653 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61K 31/593 20130101; C07C 401/00 20130101;
A61P 19/10 20180101 |
Class at
Publication: |
514/167 ;
552/653 |
International
Class: |
C07C 401/00; A61K
031/59 |
Claims
We claim:
1. A compound selected from the group of compounds represented by
Formula (I) 17wherein X is hydrogen or .dbd.CH.sub.2; R.sup.1 and
R.sup.2 are, independently of each other, a (C.sub.1-C.sub.4)alkyl
or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2; R.sup.3 and R.sup.4 are, independently of each
other, a (C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or
R.sup.3 and R.sup.4 together with C25 form a
(C.sub.3-C.sub.9)cycloalkyl or (C.sub.3-C.sub.9)cyclofluoroalkyl; A
is a single or a double bond; and B is a single, double or triple
bond; and prodrugs thereof, provided that: (i) when R.sup.1 and
R.sup.2 are (C.sub.1-C.sub.4)alkyl or R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group or .dbd.CH.sub.2, R.sup.3 and
R.sup.4 are (C.sub.1-C.sub.4)alkyl, trifluoromethyl or R.sup.3 and
R.sup.4 together with C25 form (C.sub.3-C.sub.6)cycloalkyl and A is
a single bond, then B is not a trans double bond; (ii) when B is a
single bond, then R.sup.1 and R.sup.2 together with C20 form a
(C.sub.3-C.sub.6)cycloalkyl or (C.sub.3-C.sub.6)cyclofluoroalkyl
group; and (iii) when R.sup.1 and R.sup.2 are
(C.sub.1-C.sub.4)alkyl, R.sup.3 and R.sup.4 are
(C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and A is a single bond, then
B is not a double bond.
2. The compound of claim 1, wherein: B is a triple bond.
3. The compound of claim 2, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sub.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; X is .dbd.CH.sub.2; and A is a single
bond.
4. The compound of claim 3, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
5. The compound of claim 4, wherein R.sup.3 and R.sup.4 are methyl
namely,
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-cholecalciferol.
6. The compound of claim 4, wherein R.sup.3 and R.sup.4 are
trifluoromethyl, namely
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-c-
yclopropyl-cholecalciferol.
7. The compound of claim 2, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; X is hydrogen; and A is a single
bond.
8. The compound of claim 7, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
9. The compound of claim 8, wherein R.sup.3 and R.sup.4 are methyl,
namely
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-19-nor-cholecalciferol.
10. The compound of claim 8, wherein R.sup.3 and R.sup.4 are
trifluoromethyl, namely
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-c-
yclopropyl-19-nor-cholecalciferol.
11. The compound of claim 2, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; X is .dbd.CH.sub.2; and A is a double
bond.
12. The compound of claim 11, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.44 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
13. The compound of claim 2, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; X is H.sub.2; and A is a double
bond.
14. The compound of claim 13, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
15. The compound of claim 1, wherein: A is a double bond; and B is
a double bond.
16. The compound of claim 15, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; and X is .dbd.CH.sub.2.
17. The compound of claim 16, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
18. The compound of claim 15, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; and X is H.sub.2.
19. The compound of claim 18, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
20. The compound of claim 1, wherein: A is a single bond; and B is
a cis double bond.
21. The compound of claim 20, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; and X is .dbd.CH.sub.2.
22. The compound of claim 21, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
23. The compound of claim 22, wherein R.sup.3 and R.sup.4 are
trifluoromethyl namely,
1,25-dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,-
28-cyclopropyl-cholecalciferol.
24. The compound of claim 20, wherein: R.sup.1 and R.sup.2 together
with C20 form a (C.sub.3-C.sub.6)cycloalkyl; R.sup.3 and R.sup.4
are, independently of each other, a (C.sub.1-C.sub.4)alkyl or a
(C.sub.1-C.sub.4)fluoroalkyl; and X is H.sub.2.
25. The compound of claim 24, wherein: R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group; and R.sup.3 and R.sup.4 are,
independently of each other, methyl, ethyl, trifluoromethyl,
1,1-difluoroethyl or 2,2,2-trifluoroethyl.
26. The compound of claim 25, wherein R.sup.3 and R.sup.4 are
trifluoromethyl namely,
1,25-dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,-
28-cyclopropyl-19-nor-cholecalciferol.
27. A method of treating osteoporosis via administration of a
therapeutically effective amount of a compound of Formula (I)
18wherein: X is hydrogen or .dbd.CH.sub.2; R.sup.1 and R.sup.2 are,
independently of each other, a (C.sub.1-C.sub.4)alkyl or
(C.sub.1-C.sub.4)fluoroalkyl, or R and R.sup.2 together with C20
form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2; R.sup.3 and R.sup.4 are, independently of each
other, a (C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or
R.sup.3 and R.sup.4 together with C25 form a
(C.sub.3-C.sub.9)cycloalkyl or (C.sub.3-C.sub.9)cyclofluoroalkyl; A
is a single or a double bond; and B is a single, double or triple
bond; and prodrugs thereof.
28. A method of treating cancer via administration of a
therapeutically effective amount of a compound of Formula (I)
19wherein: X is hydrogen or .dbd.CH.sub.2; R.sup.1 and R.sup.2 are,
independently of each other, a (C.sub.1-C.sub.4)alkyl or
(C.sub.1-C.sub.4)fluoroalkyl, or R and R.sup.2 together with C20
form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2; R.sup.3 and R.sup.4 are, independently of each
other, a (C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or
R.sup.3 and R.sup.4 together with C25 form a
(C.sub.3-C.sub.9)cycloalkyl or (C.sub.3-C.sub.9)cyclofluoroalkyl; A
is a single or a double bond; and B is a single, double or triple
bond; and prodrugs thereof, provided that: (i) when R.sup.1 and
R.sup.2 are (C.sub.1-C.sub.4)alkyl or R.sup.1 and R.sup.2 together
with C20 form a cyclopropyl group or .dbd.CH.sub.2, R.sup.3 and
R.sup.4 are (C.sub.1-C.sub.4)alkyl, trifluoromethyl or R.sup.3 and
R.sup.4 together with C25 form (C.sub.3-C.sub.6)cycloalkyl and A is
a single bond, then B is not a trans double bond; (ii) when B is a
single bond, then R.sup.1 and R.sup.2 together with C20 form a
(C.sub.3-C.sub.6)cycloalkyl or (C.sub.3-C.sub.6)cyclofluoroalkyl
group; and (iii) when R.sup.1 and R.sup.2 are
(C.sub.1-C.sub.4)alkyl, R.sup.3 and R.sup.4 are
(C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and A is a single bond, then
B is not a double bond.
29. A method of treating secondary hyperparathyroidism via
administration of a therapeutically effective amount of a compound
of Formula (I) 20wherein: X is hydrogen or .dbd.CH.sub.2; R.sup.1
and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2; R.sup.3 and R.sup.4 are, independently of each
other, a (C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or
R.sup.3 and R.sup.4 together with C25, form a
(C.sub.3-C.sub.9)cycloalkyl or (C.sub.3-C.sub.9)cyclofluoroalkyl; A
is a single or a double bond; and B is a single, double or triple
bond; and prodrugs thereof.
30. A pharmaceutical composition comprising an effective amount of
a compound of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 119 (e)
of U.S. provisional patent application No. 60/058,132, filed Sep.
8, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to
1,3-dihydroxy-20,20-dialkyl-vitamin D.sub.3 analogs, compositions
comprising the analogs and methods of treatment of osteoporosis,
secondary hyperparathyroidism, cancer and autoimmune diseases using
such analogs.
[0004] 2. Description of Related Art
[0005] a. Osteoporosis
[0006] Osteoporosis is the most common form of metabolic bone
disease and may be considered the symptomatic, fracture stage of
bone loss (osteopenia). Although osteoporosis may occur secondary
to a number of underlying diseases, 90% of all cases appear to be
idiopathic. Postmenopausal women are at risk for idiopathic
osteoporosis (postmenopausal or Type I osteoporosis); another
particularly high risk group for idiopathic osteoporosis is the
elderly of either sex (senile or Type II osteoporosis).
Osteoporosis has also been related to corticosteroid use,
immobilization or extended bed rest, alcoholism, diabetes,
gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa,
primary and secondary amenorrhea, transplant immunosuppression, and
oophorectomy. Postmenopausal osteoporosis is characterized by
fractures of the spine, while femoral neck fractures are the
dominant features of senile osteoporosis.
[0007] The mechanism by which bone is lost in osteoporotics is
believed to involve an imbalance in the process by which the
skeleton renews itself. This process has been termed bone
remodeling. It occurs in a series of discrete pockets of activity.
These pockets appear spontaneously within the bone matrix on a
given bone surface as a site of bone resorption. Osteoclasts (bone
dissolving or resorbing cells) are responsible for the resorption
of a portion of bone of generally constant dimension. This
resorption process is followed by the appearance of osteoblasts
(bone forming cells) which then refill with new bone the cavity
left by the osteoclasts.
[0008] In a healthy adult subject, osteoblasts and osteoclasts
function so that bone formation and bone resorption are in balance.
However, in osteoporotics an imbalance in the bone remodeling
process develops which results in bone being replaced at a slower
rate than it is being lost. Although this imbalance occurs to some
extent in most individuals as they age, it is much more severe and
occurs at a younger age in postmenopausal osteoporotics, following
oophorectomy, or in iatrogenic situations such as those resulting
from corticosteroid therapy or the immunosuppression practiced in
organ transplantation.
[0009] Various approaches have been suggested for increasing bone
mass in humans afflicted with osteoporosis, including
administration of androgens, fluoride salts, and parathyroid
hormone and modified versions of parathyroid hormone. It has also
been suggested that bisphosphonates, calcitonin, calcium,
1,25-dihydroxy vitamin D.sub.3 and some of its analogs, and/or
estrogens, alone or in combination, may be useful for preserving
existing bone mass.
[0010] Vitamin D.sub.3 is a critical element in the metabolism of
calcium, promoting intestinal absorption of calcium and phosphorus,
maintaining adequate serum levels of calcium and phosphorus, and
stimulating flux of calcium into and out of bone. The D vitamins
are hydroxylated in vivo, with the resulting 1:,25-dihydroxy
metabolite being the active material. Animal studies with
1,25-(OH).sub.2 vitamin D.sub.3 have suggested bone anabolic
activity. Aerssens et al., in Calcif Tissue Int, 55:443-450 (1994),
reported upon the effect of 1.alpha.-hydroxy vitamin D.sub.3 on
bone strength and composition in growing rats with and without
corticosteroid treatment. However, human usage is restricted to
antiresorption due to the poor therapeutic ratio (hypercalciuria
and hypercalcemia as well as nephrotoxicity).
[0011] Dechant and Goa, in "Calcitriol. A review of its use in the
treatment of postmenopausal osteoporosis and its potential in
corticosteroid-induced osteoporosis", Drugs Aging [NEW ZEALAND 5
(4): 300-17 (1994)], reported that 1,25-dihydroxy vitamin D.sub.3
(calcitriol) has shown efficacy in the treatment of postmenopausal
osteoporosis (and promise in corticosteroid-induced osteoporosis)
based upon a clinical trial in 622 women with postmenopausal
osteoporosis. Patients with mild to moderate disease (but not those
with more severe disease) who received calcitriol (0.25 microgram
twice daily) had a significant 3-fold lower rate of new vertebral
fractures after 3 years of treatment compared with patients
receiving elemental calcium 1000 mg/day. In patients commencing
long term treatment with prednisone or prednisolone, calcitriol 0.5
to 1.0 micrograms/day plus calcium 1000 mg/day, administered with
or without intranasal calcitonin 400 IU/day, prevented
steroid-induced bone loss. Overall, calcitriol was well tolerated.
At recommended dosages hypercalcaemia was infrequent and mild,
generally responding to reductions in calcium intake and/or
calcitriol dosage. The narrow therapeutic window of calcitriol
required that its use be adequately supervised, with periodic
monitoring of serum calcium and creatinine levels. This study
clearly identifies the key limitation of calcitriol therapy as the
close proximity of therapeutic and toxic doses.
[0012] This invention provides novel vitamin D.sub.3 derivatives
which have more favorable therapeutic doses.
[0013] b. Cancer
[0014] Epidemiologic studies have correlated sun or UV light
exposure with a lower incidence of a variety of malignancies,
including breast, colon and prostate cancer. Evidence from receptor
studies demonstrates that besides the classic target organs, such
as intestine, kidney and bone, vitamin D receptors (VDR) are
present on a wide variety of human normal and cancer cell lines and
fresh tissue. Growth inhibition with vitamin D or
1,25-dihydroxycholecalciferol does not always translate into
potential therapeutic efficacy in vivo. Early in vivo studies have
focused on the anti-proliferative effects of
1,25-dihydroxycholecalciferol and its analogues in murine leukemia
model systems where 1,25-dihydroxycholecalci- ferol has been shown
to induce not only an anti-proliferative effect, but also a
differentiating effect. Therapeutic efficacy in vivo has its
limitations due to the hypercalcemia observed with high dose
treatment of the parent 1,25-dihydroxycholecalciferol. As a result,
a number of analogues have been developed that produce significant
anti-tumor effects without hypercalcemia.
[0015] Steinmeyer et al in U.S. Pat. No. 5,585,368 discloses
Ice-25-dihydroxy-20-disubstituted vitamin D.sub.3 analogs for the
treatment of hyperproliferative disorders of the-skin, malignant
tumors such as leukemia, colon and breast cancers, autoimmune
diseases such as diabetes and for the treatment of sebaceous gland
diseases. Danielsson, C. et al in J. Cell Biochem., 63, No. 2,
199-206 (1996) disclose 20-methyl analogues of 1,25-dihydroxy
vitamin D.sub.3, including 1.alpha.-25-dihydroxy-20,methyl-23
(E)-ene-cholecalciferol for the treatment of hyperproliferative
disorders. This invention provides novel vitamin D.sub.3
derivatives for the treatment of hyperproliferative disorders of
the skin, malignant tumors such as leukemia, colon and breast
cancers, autoimmune diseases such as diabetes and for the treatment
of sebaceous gland diseases which have more favorable therapeutic
ratios or margins.
[0016] c. Hyperparathyroidism
[0017] Secondary hyperparathyroidism is routine in patients with
chronic renal failure. It is established that the reduction of
renal 1,25 (OH).sub.2 vitamin D.sub.3 (calcitriol) synthesis is one
of the principal mechanisms leading to the secondary
hyperparathyroidism in these patients and it has been shown that
calcitriol possesses direct suppressive action on PTH synthesis.
Therefore, administration of calcitriol has been recommended for
the treatment of secondary hyperparathyroidism in these patients.
However, as described above, calcitriol has potent hypercalcemic
effects giving it a narrow therapeutic window which limits its
usage, especially at high doses. It would therefore be desirable to
have an alternative means of treating hyperparathyroidism and
repleting circulating vitamin 13 activity without incurring these
undesirable hypercalcemic effects.
[0018] This invention provides novel vitamin D.sub.3 derivatives
which have more favorable therapeutic windows.
SUMMARY OF THE INVENTION
[0019] One aspect of the invention concerns Vitamin D.sub.3 analogs
of the Formula (I): 2
[0020] wherein:
[0021] X is hydrogen or .dbd.CH.sub.2;
[0022] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.12 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl
or (C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2
together form .dbd.CH.sub.2;
[0023] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0024] A is a single or a double bond; and
[0025] B is a single, double or triple bond;
[0026] and prodrugs thereof, provided that:
[0027] (i) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl or
R.sup.1 and R.sup.2 together with C20 form a cyclopropyl group or
.dbd.CH.sub.2, R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl,
trifluoromethyl or R.sup.3 and R.sup.4 together with C25 form
(C.sub.3-C.sub.6)cycloalkyl and A is a single bond, then B is not a
trans double bond;
[0028] (ii) when B is a single bond, then R.sup.1 and R.sup.2
together with C20 form a (C.sub.3-C.sub.6)-- cycloalkyl or
(C.sub.3-C.sub.6)cyclof- luoroalkyl group; and
[0029] (iii) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl,
R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and
A is a single bond, then B is not a double bond.
[0030] A second aspect of this invention relates to a method for
treating osteoporosis or secondary hyperparathyroidism via
administration of a compound of Formula (I), wherein:
[0031] X is hydrogen or .dbd.CH.sub.2;
[0032] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2;
[0033] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0034] A is a single or a double bond; and
[0035] B is a single, double or triple bond;
[0036] and prodrugs thereof, in an amount therapeutically effective
to restore bone density to an asymptomatic level, without inducing
hypercalciuria, hypercalcemia, or nephrotoxicity.
[0037] A third aspect of this invention relates to a method for
treating cancer via administration of a compound of Formula (I),
wherein:
[0038] X is hydrogen or .dbd.CH.sub.2;
[0039] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2;
[0040] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0041] A is a single or a double bond; and
[0042] B is a single, double or triple bond;
[0043] and prodrugs thereof, in an amount therapeutically
effective, without inducing hypercalciuria, hypercalcemia, or
nephrotoxicity provided that:
[0044] (i) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl or
R.sup.1 and R.sup.2 together with C20 form a cyclopropyl group or
.dbd.CH.sub.2, R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl,
trifluoromethyl or R.sup.3 and R.sup.4 together with C25 form
(C.sub.3-C.sub.6)cycloalkyl and A is a single bond, then B is not a
trans double bond;
[0045] (ii) when B is a single bond, then R.sup.1 and R.sup.2
together with C20 form a (C.sub.3-C.sub.6)-- cycloalkyl or
(C.sub.3-C.sub.6)cyclof- luoroalkyl group; and
[0046] (iii) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl,
R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and
A is a single bond, then B is not a double bond.
[0047] A fourth aspect of this invention relates to pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and a
vitamin D.sub.3 analog of Formula (I).
DETAILED DESCRIPTION OF THE INVENTION
[0048] The vitamin D.sub.3 analogs of the present invention have
the following general structure: 3
[0049] wherein:
[0050] X is hydrogen or .dbd.CH.sub.2;
[0051] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R and
R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2;
[0052] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0053] A is a single or a double bond; and
[0054] B is a single, double or triple bond;
[0055] and prodrugs thereof, provided that:
[0056] (i) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl or
R.sup.1 and R.sup.2 together with C20 form a cyclopropyl group or
.dbd.CH.sub.2, R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl,
trifluoromethyl or R.sup.3 and R.sup.4 together with C25 form
(C.sub.3-C.sub.6)cycloalkyl and A is a single bond, then B is not a
trans double bond;
[0057] (ii) when B is a single bond, then R.sup.1 and R.sup.2
together with C20 form a (C.sub.3-C.sub.6)-- cycloalkyl or
(C.sub.3-C.sub.6)cyclof- luoroalkyl group; and
[0058] (iii) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl,
R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and
A is a single bond, then B is not a double bond.
[0059] A method for treating osteoporosis or secondary
hyperparathyroidism via administration of a compound of Formula
(I), wherein:
[0060] X is hydrogen or .dbd.CH.sub.2;
[0061] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2;
[0062] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0063] A is a single or a double bond; and
[0064] B is a single, double or triple bond;
[0065] and prodrugs thereof, in an amount therapeutically effective
to restore bone density to an asymptomatic level, without inducing
hypercalciuria, hypercalcemia, or nephrotoxicity.
[0066] A method for treating cancer via administration of a
compound of Formula (I), wherein:
[0067] X is hydrogen or .dbd.CH.sub.2;
[0068] R.sup.1 and R.sup.2 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.1
and R.sup.2 together with C20 form a (C.sub.3-C.sub.6)cycloalkyl or
(C.sub.3-C.sub.6)cyclofluoroalkyl, or R.sup.1 and R.sup.2 together
form .dbd.CH.sub.2;
[0069] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)fluoroalkyl, or R.sup.3
and R.sup.4 together with C25 form a (C.sub.3-C.sub.9)cycloalkyl or
(C.sub.3-C.sub.9)cyclofluoroalkyl;
[0070] A is a single or a double bond; and
[0071] B is a single, double or triple bond;
[0072] and prodrugs thereof, in an amount therapeutically
effective, without inducing hypercalciuria, hypercalcemia, or
nephrotoxicity, provided that:
[0073] (i) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl or
R.sup.1 and R.sup.2 together with C20 form a cyclopropyl group or
.dbd.CH.sub.2, R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl,
trifluoromethyl or R.sup.3 and R.sup.4 together with C25 form
(C.sub.3-C.sub.6)cycloalkyl and A is a single bond, then B is not a
trans double bond;
[0074] (ii) when B is a single bond, then R.sup.1 and R.sup.2
together with C20 form a (C.sub.3-C.sub.6)-- cycloalkyl or
(C.sub.3-C.sub.6)cyclof- luoroalkyl group; and
[0075] (iii) when R.sup.1 and R.sup.2 are (C.sub.1-C.sub.4)alkyl,
R.sup.3 and R.sup.4 are (C.sub.1-C.sub.4)alkyl, X.dbd.CH.sub.2 and
A is a single bond, then B is not a double bond.
Definitions
[0076] As used herein, the term (C.sub.1-C.sub.4) alkyl means a
fully-saturated hydrocarbon radical having one to four carbon
atoms; a (C.sub.1-C.sub.4) fluoroalkyl is an alkyl radical, as
defined above, in which one or more hydrogen atoms attached to the
carbon backbone have been substituted with one or more fluorine
atoms. A (C.sub.3-C.sub.6) cycloalkyl is a cyclic saturated
hydrocarbon radical having three to six ring carbon atoms; a
(C.sub.3-C.sub.6) cyclofluoroalkyl is a cycloalkyl radical, as
defined above, in which one or more hydrogen atoms attached to the
carbon backbone have been substituted with one or more fluorine
atoms. A (C.sub.3-C.sub.9) cycloalkyl is a cyclic saturated
hydrocarbon radical having three to nine ring carbon atoms; a
(C.sub.3-C.sub.9) cyclofluoroalkyl is a cyclic saturated
hydrocarbon radical having three to nine carbon atoms in which one
or more hydrogen atoms attached to the carbon backbone have been
substituted with one or more fluorine atoms.
[0077] Further as used herein, by double bond it is meant an
unsaturated linkage between two adjacent carbon atoms in which two
pairs of electrons are shared equally, and wherein each carbon atom
bears two single-bonded substituents in either a cis (Z) or a trans
(E) configuration about the double bond.
[0078] "Pro-drugs" means any compound which releases an active
parent drug according to Formula (I) in vivo when such prodrug is
administered to a mammalian subject. Prodrugs of a compound of
Formula (I) are prepared by modifying functional groups present in
the compound of Formula (I) in such a way that the modifications
may be cleaved in vivo to release the parent compound. Prodrugs
include compounds of Formula (I) wherein a hydroxy group in
compound (I) is bonded to any group that may be cleaved in vivo to
regenerate the free hydroxyl group. Examples of prodrugs include,
but are not limited to esters (e.g., acetate, formate, and benzoate
derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) and
ethers of hydroxy functional groups in compounds of Formula (I),
and the like. Such compounds are routinely made by one of skill in
the art by acylating or etherifying the hydroxy group in the parent
molecule.
[0079] A "therapeutically effective amount" means the amount of a
compound that, when administered to a mammal for treating or
preventing a disease, is sufficient to effect such treatment or
prevention for the disease. The "therapeutically effective amount"
will vary depending on the compound, the disease and its severity
and the age, weight, etc., of the mammal to be treated.
[0080] The compounds of the present invention may be generically
described as 1.alpha.,25-dihydroxy-20,20-dialkyl and
1.alpha.,25-dihydroxy-20,20-di- alkyl-19-nor analogs of vitamin
D.sub.3.
Nomenclature
[0081] The compounds of the invention are named using the numbering
system shown in Figure (1) below. 4
[0082] For example, a compound of the invention where X is
.dbd.CH.sub.2, R.sup.1 and R.sup.2 together form a cyclopropyl
group, A is a single bond and B is a triple bond is named as
1.alpha.-25-dihydroxy-23-yne-20,21,28--
cyclopropyl-cholecalciferol.
[0083] The following Table I provides some representative examples
of compounds of the present invention:
1TABLE I CPD # A B R.sup.1 R.sup.2 R.sup.3 R.sup.4 X 1 -- .ident.
--CH.sub.2CH.sub.2-- CH.sub.3 CH.sub.3 .dbd.CH.sub.2 2 -- .ident.
--CH.sub.2CH.sub.2-- CH.sub.3 CH.sub.3 H.sub.2 3 -- .ident.
--CH.sub.2CH.sub.2-- CF.sub.3 CF.sub.3 .dbd.CH.sub.2 4 -- .ident.
--CH.sub.2CH.sub.2-- CF.sub.3 CF.sub.3 H.sub.2 5 -- cis = bond
--CH.sub.2CH.sub.2-- CF.sub.3 CF.sub.3 .dbd.CH.sub.2 6 -- cis =
bond --CH.sub.2CH.sub.2-- CF.sub.3 CF.sub.3 H.sub.2
[0084] and are named as:
[0085] 1.
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-cholecalciferol.
[0086] 2.
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-19-nor-cholecalcifero-
l.
[0087] 3.
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-chol-
ecalciferol.
[0088] 4.
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-19-n-
or-cholecalciferol.
[0089] 5.
1,25-dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,28-cyclopropyl--
cholecalciferol.
[0090] 6.
1,25-dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,28-cyclopropyl--
19-nor-cholecalciferol.
Preferred Embodiments
[0091] While the broadest definition of this invention is set forth
in the Summary of the Invention, certain compounds of Formula (I)
are preferred.
[0092] A preferred group of compounds are those wherein:
[0093] A is a single or a double bond, preferably a single bond;
and
[0094] B is a triple bond.
[0095] Another preferred group of compounds are those wherein:
[0096] A is a double bond; and
[0097] B is a double bond.
[0098] Yet another preferred group of compounds are those
wherein:
[0099] A is a single or a double bond, preferably a single bond;
and
[0100] B is a cis double bond.
[0101] Within these preferred groups of compounds, more preferred
groups are those wherein:
[0102] R.sup.1 and R.sup.2 together with C20 form a
(C.sub.3-C.sub.6)cycloalkyl, preferably a cyclopropyl; and
[0103] R.sup.3 and R.sup.4 are, independently of each other, a
(C.sub.1-C.sub.4)alkyl or a (C.sub.1-C.sub.4)fluoroalkyl,
preferably methyl, ethyl, trifluoromethyl, 1,1-difluoroethyl or
2,2,2-trifluoroethyl, more preferably methyl or
trifluoromethyl.
General Synthesis
[0104] Analogs of this invention may generally be prepared by
reaction and combination of fragments of Vitamin D.sub.3 molecules
(see e.g., Shiuey et al., J. Org. Chem, 55:243 (1990); Wovkulich,
P. M. et al., Tetrahedron, 40, 2283 (1984); Baggiolini E. B. et al
J. Org. Chem., 51, 3098-3108, (1986) and Steinmeyer et al., U.S.
Pat. No. 5,585,368.
[0105] The starting materials and reagents used in preparing these
compounds are either available from commercial suppliers such as
Aldrich Chemical Co., (Milwaukee, Wis.), or Sigma (St. Louis, Mo.)
or they can be prepared by methods known to those skilled in the
art following procedures set forth in references such as Fieser and
Fieser's Reagents for Organic Synthesis, Vol. 1-15 (John Wiley and
Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and
Sons 4th Edition) and Larock's Comprehensive Organic
Transformations (VCH Publishers Inc., 1989).
[0106] The starting materials and the intermediates of the reaction
may be isolated and purified if desired using conventional
techniques, including but not limited to filtration, distillation,
crystallization, chromatography and the like. Such materials may be
characterized using conventional means, including physical
constants and spectral data.
[0107] The preparation of compounds of Formula (I) and the
intermediates used in their preparation is illustrated by the
reaction schemes below.
Preparation of Compounds of Formula (I)
[0108] In general, a compound of Formula (I) is prepared by
coupling a 4H-inden-4-one derivative of Formula (II) where R.sup.1,
R.sup.2, R.sup.3, R.sup.4, A and B are as described in the Summary
of the Invention and R.sup.5 is hydrogen or a hydroxy protecting
group (e.g., trialkylsilyl, preferably trimethylsilyl) with a
diphenylphosphine oxide derivative of a compound of Formula (III)
where X is hydrogen or .dbd.CH.sub.2, as shown in Scheme I below.
5
[0109] The coupling reaction is carried out in the presence of a
strong base such as an alkyllithium like n-butyllithium in a
mixture of hexane and tetrahydrofuran at -78.degree. C. to give a
trisilyl derivative of compound of Formula (I). Removal of the
silyl protecting groups with tetrabutylammonium fluoride in a
suitable polar organic solvent such as tetrahydrofuran provides a
compound of Formula (I).
[0110] It should be noted that although the shown intermediates
have hydroxy groups typically protected as silylethers, the scope
of the invention includes the use of alternative hydroxyl
protecting groups known in the art as described in T. W. Greene,
"Protective Groups in Organic Synthesis," Wiley, New York (1991)
and J. F. McOmie, "Protective Groups in Organic Chemistry," Plenum
Press, London (1973), together with alternative methods for
deprotection.
[0111] Synthesis of compounds of Formula (III) are known and
conventional in this art. See, for example, U.S. Pat. Nos.
5,585,368 to Steinmeyer et al., 5,384,314 to Doran et al.,
5,428,029 to Doran et al., 5,451,574 to Baggiolini et al.; pending
U.S. patent application Serial No. 60/018,219; Shiuey et al., J.
Org. Chem., 55:243-247 (1990), Kiegel, J. et al. and Tetr. Lett.,
32:6057-6060 (1991), Perlman, K. L., et al., Tetr. Lett.,
32:7663-7666 (1991).
[0112] Synthesis of compounds of Formula (II) is described in
Scheme II below.
[0113] Detailed descriptions of the synthesis of compounds of
Formula (I) where R.sup.1 and R.sup.2 together form a cyclopropyl
ring, X is .dbd.CH.sub.2 or H.sub.2, A is a single bond, B is a
triple bond and R.sup.3 and R.sup.4 are either methyl or
trifluoromethyl are described in Examples 2, 3, 5 and 6.
[0114] Detailed descriptions of the synthesis of compounds of
Formula (I) where R.sup.1 and R.sup.2 together form a cyclopropyl
ring, X is .dbd.CH.sub.2 or H.sub.2, A is a single bond, B is a cis
double bond and R.sup.3 and R.sup.4 are trifluoromethyl are
described in Examples 8 and 9.
Preparation of Compounds of Formula (II)
[0115] The 4H-inden-4-one derivatives of Formula (II) are prepared
as shown in Scheme II below. 6
[0116] As shown above, preparation of a compound of Formula (II)
involves preparation of a common intermediate,
1-[(5-hydroxy)-3-alkynyl]-inden-4-o- l derivative (VII), which is
then converted to a compound of Formula (II) where B is either a
double or a triple bond by following method (a) or method (b)
respectively.
[0117] Compound (VII) is prepared by condensation of lithium
acetylide derived from a
1-(3-alkynyl)-4-tert-butyldimethylsilyloxy-7a-methyl-inden- e
derivative (IV) with a ketone of Formula (V) where R.sup.3 and
R.sup.4 are as defined in the Summary of the Invention to give a
1-[(5-hydroxy)-3-alkynyl]-4-tert-butyldimethylsilyloxy-7a-methyl-indene
derivative (VI). The condensation reaction is carried out in the
presence of a strong base such as n-butyllithium in an aprotic
organic solvent such as tetrahydrofuran and at low temperatures
ranging between -50 to -100.degree. C. Removal of the silyl group
with tetrabutylammonium fluoride in an suitable organic solvent
such as tetrahydrofuran gives the
1-[(5-hydroxy)-3-alkynyl]-inden-4-ol derivative (VII).
[0118] A detailed description of the synthesis of compounds of
Formula (IV) where R.sup.1 and R.sup.2 together form a cyclopropyl
ring and A is a single bond is given in Example 1. Synthesis of
other compounds of Formula (IV) and alternative methods for
preparing compounds of Formula (VII) have been described in
copending U.S. application Ser. No. 08/857,569, published as EP 0
808,832 A2, whose disclosure is hereby incorporated by
reference.
[0119] A compound of Formula (II) where B is a triple bond and
R.sup.5 is hydrogen is prepared, as shown in method (a), by
oxidation of the hydroxy group at the 4-position in compound (VII)
to the keto group with a suitable oxidizing agent such as
pyridinium dichromate at room temperature. The oxidation reaction
is carried out in a chlorinated hydrocarbon solvent such as
methylene chloride, chloroform and the like. A compound of Formula
(II) where W is hydrogen is converted to the corresponding compound
of Formula (II) where R is trialkylsilyl, preferably
trimethylsilyl, by reacting it with a suitable silylating agent
such as 1-trimethylsilylimidazole in a non-alcoholic organic
solvent such as tetrahydrofuran, methylene chloride, preferably
methylene chloride, and the like.
[0120] Synthesis of compounds of Formula (II) where A is a single
bond, B is a triple bond, R.sup.1 and R.sup.2 together form a
cyclopropyl ring, R.sup.5 is trimethylsilyl or hydrogen and R.sup.3
and R.sup.4 are methyl or trifluoromethyl are described in Examples
1 and 4.
[0121] Alternatively, a compound of Formula (II) where B is a
double bond is prepared, as shown in method (b), by partial
reduction of the triple bond in compound (VII) with a suitable
reducing agent to give a 3-alkene-4H-inden-4-ol of Formula (VIII).
The choice of the reducing agent depends on the configuration about
the double bond. If the E configuration is desired, then the
reduction is carried out with lithium aluminum hydride in the
presence of an alkali metal alkoxide, such as sodium methoxide, and
in an aprotic organic solvent like ether or more preferably
tetrahydrofuran. If the Z configuration is desired, then the
reduction is carried out with Lindlar's catalyst. Compound (VIII)
is then converted to a compound of Formula (II) where B is a double
bond and R.sup.5 is hydrogen or a silyl group by carrying out the
oxidation and silylation steps as described above. Synthesis of a
compound of Formula (II) where A is a single bond, B is a cis
double bond, R.sup.1 and R.sup.2 together form a cyclopropyl ring,
R.sup.5 is trimethylsilyl and R.sup.3 and R.sup.4 are
trifluoromethyl is described in Example 7.
[0122] A reaction scheme showing the preparation of a compound of
Formula (I) where A is a single bond, B is a single bond, R.sup.1
and R.sup.2 form a cyclopropyl ring and X is .dbd.CH.sub.2 is shown
below in Scheme III and is described further in Example 10. 7
[0123] The starting material is reduced, preferably by catalytic
hydrogenation, to give the completely saturated side chain
derivative which is then oxidized to give the corresponding ketone.
Condensation with a diphenylphosphine oxide as previously described
in Scheme I followed by removal of the silyl protecting groups
gives the desired compound. One of skill in the art will recognize
that similar procedures may be used to form other compounds of the
invention where R.sup.1-R.sup.4 and X may vary as described in the
Summary of the Invention.
Utility
[0124] The compounds of this invention are useful for the
prevention and treatment of a variety of mammalian conditions
manifested by loss of bone mass. In particular, the compounds of
this invention are anabolic agents and are indicated for the
prophylaxis and therapeutic treatment of osteoporosis and
osteopenia in mammals without inducing hypercalciuria,
hypercalcemia, or nephrotoxicity. As used herein, "hypercalciuria"
is excessive calcium in the urine, in humans corresponding to an
excretion of greater than about 4 mg/kg/day. This often results in
nephrolithiasis (renal calculi). "Hypercalcemia" is an excessive
concentration of calcium in the serum; in humans (and rats) this
corresponds to greater than about 10.5 mg/dl. "Intolerable
hypercalcemia", usually occurring at serum calcium concentrations
greater than about 12 mg/dl, is associated with emotional lability,
confusion, delirium, psychosis, stupor, and coma.
[0125] The compounds of this invention are expected to be useful in
the treatment of Type I (postmenopausal), Type II (senile), and
Type III (iatrogenic) osteoporosis, including that associated with
immunosuppressive drugs used in organ transplantation, as well in
the treatment of osteodystrophy due to renal dialysis and secondary
hyperparathyroidism.
[0126] Compounds of this invention are also useful in treating
diseases caused by elevated levels of parathyroid hormone. In one
aspect, compounds of the invention are used in treating secondary
hyperparathyroidism associated with renal failure and in particular
with reversing or reducing the bone loss associated with renal
insufficiency. Other aspects include the treatment of renal
osteodystrophy associated with late stage secondary
hyperparathyroidism. Other aspects include the treatment of primary
hyperparathyroidism.
[0127] Compounds of Formula (I) are also useful in treating
neoplastic diseases such as leukemia, colon cancer, breast cancer
and prostate cancer.
[0128] Compounds of Formula (I) are also useful in treating
immunosuppressive and autoimmune diseases. Such diseases include,
but are not limited to, multiple sclerosis, systemic lupus
erythematosus, diabetes, thyroiditis and allograft rejection. In
particular, compounds of Formula (I) are useful to treat diseases
via modulation of the activity of the vitamin D.sub.3 receptor
(VDR). The utility of these compounds is demonstrated in vivo using
murine models for these diseases as is well known in the art. See,
e.g., Lemire et al., Autoimmunity, 12:143-148 (1992); Lemire et.
al., J. Clin. Invest., 87:1103-1107 (1991), Lemire et al.,
Endocrinology, 135:2818 (1994), and Lemire et al., J. Cellular
Biochem., 49:26-31 (1992).
Testing
[0129] The bone anabolic activity of the compounds of the invention
was demonstrated in vivo in the ovariectomized rat model as
described in detail in Example 10. The anti-cell proliferation
activity of the compounds of the invention was demonstrated in
vitro as described in detail in Examples 11 and 12. The parathyroid
hormone suppressive activity of the compounds of the invention was
demonstrated in vivo as described in detail in Example 13.
Administration & Pharmaceutical Compositions
[0130] In general, the compound of this invention may be
administered in amounts between about 0.0002 and 5 .mu.g per day,
preferably from about 0.001 to about 2 .mu.g per day, most
preferably from about 0.002 to about 1 .mu.g per day. For a 50 kg
human subject, the daily dose of active ingredient may be from
about 0.01 to about 250 .mu.g, preferably from about 0.05 to about
100 .mu.g, most preferably from about 0.1 to about 50 .mu.g per
day. In other mammals, such as horses, dogs, and cattle, other
doses may be required. This dosage may be delivered in a
conventional pharmaceutical composition by a single administration,
by multiple applications, or via controlled release, as needed to
achieve the most effective results, preferably once or twice daily
by mouth. In certain situations, alternate day dosing may prove
adequate to achieve the desired therapeutic response.
[0131] The selection of the exact dose and composition and the most
appropriate delivery regimen will be influenced by, inter alia, the
pharmacological properties of the formulation, the nature and
severity of the condition being treated, and the physical condition
and mental acuity of the recipient. In the treatment of
corticosteroid induced osteopenia, it is expected that the
requisite dose will be greater for higher doses of
corticosteroids.
[0132] Representative delivery regimens include oral, parenteral
(including subcutaneous, intramuscular and intravenous), rectal,
buccal (including sublingual), pulmonary, transdermal, and
intranasal, most preferably oral.
[0133] A further aspect of the present invention relates to
pharmaceutical compositions comprising as an active ingredient a
compound of the present invention, in admixture with a
pharmaceutically acceptable, non-toxic carrier. As mentioned above,
such compositions may be prepared for parenteral (subcutaneous,
intramuscular or intravenous) administration, particularly in the
form of liquid solutions or suspensions; for oral or buccal
administration, particularly in the form of tablets or capsules;
for pulmonary or intranasal administration, particularly in the
form of powders, nasal drops or aerosols; and for rectal or
transdermal administration.
[0134] The compositions may conveniently be administered in unit
dosage form and may be prepared by any of the methods well-known in
the pharmaceutical art, for example as described in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., (1985). Formulations for parenteral administration may contain
as excipients sterile water or saline, alkylene glycols such as
propylene glycol, polyalkylene glycols such as polyethylene glycol,
oils of vegetable origin, hydrogenated naphthalenes and the like.
Formulations for nasal administration may be solid and may contain
excipients, for example, lactose or dextran, or may be aqueous or
oily solutions for use in the form of nasal drops or metered spray.
For buccal administration typical excipients include sugars,
calcium stearate, magnesium stearate, pregelatinated starch, and
the like.
[0135] Orally administrable compositions may comprise one or more
physiologically compatible carriers and/or excipients and may be in
solid or liquid form, including, for example, tablets, coated
tablets, capsules, lozenges, aqueous or oily suspensions,
solutions, emulsions, elixirs, and powders suitable for
reconstitution with water or another suitable liquid vehicle before
use. Tablets and capsules may be prepared with binding agents, for
example, syrup, acacia, gelatin, sorbitol, tragacanth, or
poly-vinylpyrollidone; fillers, such as lactose, sucrose, corn
starch, calcium phosphate, sorbitol, or glycine; lubricants, such
as magnesium stearate, talc, polyethylene glycol, or silica; and
surfactants, such as sodium lauryl sulfate. Liquid compositions may
contain conventional additives such as suspending agents, for
example sorbitol syrup, methyl cellulose, sugar syrup, gelatin,
carboxymethylcellulose, or edible fats; emulsifying agents such as
lecithin, or acacia; vegetable oils such as almond oil, coconut
oil, cod liver oil, or peanut oil; preservatives such as butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Liquid
compositions may be encapsulated in, for example, gelatin to
provide a unit dosage form.
[0136] Preferred solid oral dosage forms include tablets, two-piece
hard shell capsules and soft elastic gelatin (SEG) capsules. SEG
capsules are of particular interest because they provide distinct
advantages over the other two forms (see Seager, H., "Soft gelatin
capsules: a solution to many tableting problems"; Pharmaceutical
Technology, 9, (1985). Some of the advantages of using SEG capsules
are: a) dose-content uniformity is optimized in SEG capsules
because the drug is dissolved or dispersed in a liquid that can be
dosed into the capsules accurately, b) drugs formulated as SEG
capsules show good bioavailability because the drug is dissolved,
solubilized or dispersed in an aqueous-miscible or oily liquid and
therefore when released in the body produce drug dispersions of
high surface area and c) degradation of drugs that are sensitive to
oxidation during long-term storage is prevented because the dry
shell of soft gelatin provides a barrier against the diffusion of
oxygen.
[0137] The dry shell formulation typically comprises of about 40%
to 60% concentration of gelatin, about a 20% to 30% concentration
of plasticizer (such as glycerin, sorbitol or propylene glycol) and
about a 30 to 40% concentration of water. Other materials such as
preservatives, dyes, opacifiers and flavours also may be present.
The liquid fill material comprises a solid drug that has been
dissolved, solubilized or dispersed (with suspending agents such as
beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a
liquid drug in vehicles or combinations of vehicles such as mineral
oil, vegetable oils, triglycerides, glycols, polyols and
surface-active agents.
EXAMPLES
[0138] The following examples are given to enable those skilled in
the art to more clearly understand and to practice the present
invention. They should not be considered as limiting the scope of
the invention, but merely as being illustrative and representative
thereof.
Example 1
[1R-(1.alpha.,3a.beta.,7a.alpha.)]-Octahydro-7a-methyl-1-[1-[4-methyl-4-[t-
rimethylsilyloxy]-2-pentynyl]cyclopropyl]-4H-inden-4-one
[0139] 8
[0140] Step 1
[0141] Cold dimethylaluminum chloride (34.5 ml, 34.5 mmol, 1M
solution in hexanes) was added dropwise to a suspension of
[1R-(1.alpha.,3a.beta.,4.a-
lpha.,7a.alpha.)](1,1-dimethylethyl)dimethyl-[[(octahydro-7a-methyl-1-(1-m-
ethylethenyl)-1H-inden-4-yl]oky]silane (9.25 g, 30 mmol) and
paraformaldehyde (1.03 g, 34.5 mmol) in dichloromethane (90 ml) at
-20.degree. C. The reaction mixture was stirred at 10.degree. C.
for 1 h and then poured on ice and acidified with 0.1N hydrochloric
acid (100 ml). After 15 min., the reaction mixture was extracted
into hexanes and the extracts were washed with brine, dried over
magnesium sulfate and concentrated in vacuo to give 9.67 g of a
colorless gum. Flash chromatography on silica gel with 25% ethyl
acetate/hexanes as the eluant gave 8.93 g of a colorless gum. 1.0 g
of this material was purified by HPLC to give
[1R-(1.alpha.,3a.alpha.,4.alpha.,7a.alpha.)]-4-[[(1,1-dimeth-
ylethyl)dimethylsilyl]oxy]octahydro-7a-methyl-.gamma.-methylene-1H-indene--
1-propanol (0.93 g): mp 38-40.degree. C.;
[.alpha.].sup.25D=+26.7.degree.; IR (CHCl.sub.3) 3630 and 1635
cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.01 (3H, s), 0.011
(3H, s), 0.80 (3H, s), 0.88 (9H, s), 1.15 (1H, m), 1.3-1.63 (5H,
m), 1.65-1.76 (6H, m), 2.0 (1H, m), 2.27 (2H, m), 3.68 (2H, t,
J=6.0 Hz), 4.01 (1H, s), 4.91 (1H, s), 4.95 (1H, s); MS m/z 339
(M.sup.++H, 40). Anal. Calcd. for C.sub.20H.sub.38O.sub.2Si: C,
70.94; H, 11.31; Si, 8.29. Found: C, 70.95; H, 11.62; Si, 8.30.
[0142] Step 2
[0143] To a mixture of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-4-[[(1,-
1-dimethylethyl)dimethylsilyl]oxy]-octahydro-7a-methyl-.gamma.-methylene-1-
H-indene-1-propanol (5.07 g, 15 mmol), [prepared as described in
Step 1 above] and diiodomethane (13.39 g, 50 mmol) in
dichloromethane (45 ml) at -10.degree. C. was added a cold
(-1.degree. C.) solution of diethylzinc (45 ml, 45 mmol, 1 M
solution in toluene). The reaction mixture was stirred at
5-7.degree. C. for 4.25 h and then poured into a mixture of hexanes
(25 ml) and 0.1 N sulfuric acid (150 ml). The product was extracted
into hexanes and the extracts were washed with brine, dried over
magnesium sulfate and evaporated in vacuo to give 5.34 g of a pale
yellow gum. Flash chromatography on silica gel with 30% ethyl
acetate/hexanes as the eluant gave 4.91 g of crude product which
was further purified by HPLC (15-30 .mu.m mesh silica, 50.times.50
mm column, 70 ml/min, flow rate) with 20% ethyl acetate in hexanes
as eluant to give pure
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-1-[4-[[1,1-dimethylethyl-
)dimethylsilyl]oxy]octahydro-7a-methyl-1H-inden-1-yl]cyclopropaneethanol
(4.13 g): mp 65-66.degree. C.; [.alpha.].sup.25D=+69.17.degree.
(CHCl.sub.3, c=1.33); IR (CHCl.sub.3) 3620 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.04 (3H, s), 0.06 (3H, s), 0.80 (1H, m), 0.22
(2H, m), 0.69 (1H, m), 0.88 (9H, s), 0.92 (2H, m), 0.94 (3H, s).
1.20-1.55 (8H, m), 1.64 (1H, br d), 1.78-1.92 (2H, m), 2.02 (1H, d,
J=12 Hz), 2.30 (1H, m), 3.76 (2H, br, t of t), 3.97 (1H, s); MS m/z
353 (M.sup.++H, 70). Anal. Calcd. for C.sub.21H.sub.40O.sub.2Si: C,
71.53; H, 11.43; Si, 7.96. Found: C, 71.48; H, 11.67; Si, 7.93.
[0144] Step 3
[0145]
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-1-[4-[[1,1-dimethylethy-
l)dimethylsilyl]oxy]octahydro-7a-methyl-1H-inden-1-yl]cyclopropaneethanol
(1.056 g, 3.0 mmol), [prepared as described in Step 2 above] was
added to a suspension of pyridinium chlorochromate (1.132 g, 5.246
mmol) and anhydrous sodium acetate (0.430 g, 5.244 mmol) in
dichloromethane (15 ml) and the reaction mixture was stirred at
room temperature. After 2 h, the reaction mixture was diluted with
ether (35 ml), stirred for an additional 15 min., and then filtered
through a pad of Florisil. The Florisil pad was washed with ether
and the combined filtrate was concentrated in vacuo to give 0.93 g
of a solid. Flash chromatography of the solid on a silica gel
column with 10% ethyl acetate in hexanes as the eluant gave
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-1-[4-[[1,1-dimeth-
ylethyl)dimethylsilyl]oxy]octahydro-7a-methyl-1H-inden-1-yl]cyclopropaneac-
etaldehyde (0.86 g): mp 74-75.degree. C.;
[.alpha.].sup.25D=93.73.degree. (CHCl.sub.3, c=1.18); IR
(CHCl.sub.3) 1720 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.00
(3H, s), 0.01 (3H, s), 0.27 (1H, m), 0.32 (1H, m), 0.50 (1H, m),
0.80 (1H, m), 0.88 (9H, s), 0.97 (3H, s), 1.03 (1H, m), 1.15 (1H,
m), 1.20-1.55 (6H, m), 1.60-1.75 (2H, m), 1.80 (H, d, J=16 Hz),
1.95 (1H, d, J=12 Hz), 2.89 (1H, d, J=16 Hz), 3.97 (1H, s), 9.81
(1H, d, J=3 Hz); MS n/z 351 (M.sup.++H, 20). Anal. Calcd. for C2
1H.sub.38O.sub.2Si: C, 71.94; H, 10.92; Si, 8.01. Found: C, 71.71;
H, 11.15; Si, 8.23.
[0146] Step 4
[0147] A solution of diethyldiazomethyl phosphonate (1.78 g, 10
mmol) in anhydrous tetrahydrofuran (6 ml) was added to a solution
of potassium t-butoxide (1.234 g, 10.9 mmol) in anhydrous
tetrahydrofuran (20 ml) at -70.degree. C. After 25 min., a solution
of [1R-(1.alpha.,3a.beta.,4.alph-
a.,7a.alpha.)]-1-[4-[[1,1-dimethylethyl)dimethylsilyl]oxy]octahydro-7a-met-
hyl-1H-inden-1-yl]cyclopropaneacetaldehyde (2.103 g, 6.0 mmol),
[prepared as described in Step 3 above] in tetrahydrofuran (6.0 ml)
was added. After 1 h, the cooling bath was removed and the stirring
was continued for an additional 1.5 h. A solution of saturated
ammonium chloride (10 ml) was added. After 15 min., the reaction
mixture was poured into a mixture of ether (100 ml) and saturated
ammonium chloride (60 ml). The organic phase was separated and
washed with brine, dried over magnesium sulfate and evaporated in
vacuo to give 2.18 g of a gum. Purification by flash chromatography
on silica gel column with 2.5% ethyl acetate in hexanes as the
eluant gave as a crystalline solid which was slurried with methanol
and filtered to give [1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-
[(1,1-dimethylethyl)dimethyl[[octahydro-7a-methyl-1-[1-(2-propynyl)cyclopr-
opyl]-1H-inden-4-yl]oxy]silane (1.77 g): mp 49-50.degree. C.;
[.alpha.].sup.25D=58.64.degree. (CHCl.sub.3, c=1.03); IR
(CHCl.sub.3) 3307 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.-0.01
(3H, s), 0.00 (3H, s), 0.22 (2H, m), 0.38 (1H, m), 0.69 (1H, m),
0.87 (9H, s), 0.94 (3H, s), 0.96 (1H, m), 1.25-1.55 (7H, m), 1.64
(1H, br d, J=12 Hz), 1.75-1.90 (3H, m), 1.95 (1H, d, J=16 Hz), 1.96
(1H, s), 2.69 (1H, d, J=16 Hz), 3.98 (1H, s); MS m/z 346 (M.sup.+,
20). Anal. Calcd. for C.sub.21H.sub.38OSi: C, 76.23; H, 11.05; Si,
8.10. Found: C, 76.03; H, 10.84; Si, 8.12.
[0148] Step 5
[0149] n-Butyllithium (5.5 ml, 8.8 mmol, 1.6 M solution in hexanes)
was added to a solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)][(1,1-d-
imethylethyl)dimethyl[[octahydro-7a-methyl-1-[1-(2-propynyl)cyclopropyl]-1-
H-inden-4-yl]oxy]silane (1.73 g, 5.0 mmol), [prepared as described
in Step 4 above] in anhydrous tetrahydrofuran (18 ml) at
-78.degree. C. After 30 min., acetone (5.8 g, 100 mmol) was added
and the stirring was continued for an additional 30 min. The
cooling bath was removed and after 3 h additional amounts of
acetone (2.9 g, 50 mmol) was added. After 1.5 h, the reaction
mixture was quenched with saturated ammonium chloride (15 ml) and
then poured into a mixture of ether (100 ml) and saturated ammonium
chloride (60 ml). The organic layer was separated and washed with
brine, dried over sodium sulfate and evaporated in vacuo to give
2.12 g of a colorless gum. Purification by flash chromatography on
a silica gel column using 15% ethyl acetate in hexanes as eluant
gave
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-5-[1-[4-[[1,1-dimethylethyl)d-
imethylsilyl]oxy]octahydro-7a-methyl-1H-inden-1-yl]cyclopropyl]-2-methyl-3-
-pentyn-2-ol as a colorless gum (1.67 g):
[.alpha.].sup.25D=+39.09.degree. (EtOH, c=1.036); IR (CHCl.sub.3)
3602 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.-0.01 (3H, s), 0.00
(3H, s), 0.20 (2H, m), 0.40 (1H, m), 0.62 (1H, m), 0.87 (9H, s),
0.93 (3H, s), 1.00 (1H, m), 1.2-1.4 (13H, m), 1.49 (6H, s),
1.62-1.95 (5H, m), 2.03 (1H, d, J=17 Hz), 2.62 (1H, d, J=17 Hz),
3.97 (1H, s); MS m/z 404 (M.sup.+, 18). Anal. Calcd. for
C.sub.25H.sub.44O.sub.2Si: C, 74.28; H, 10.96; Si, 6.94. Found: C,
73.92; H, 11.22; Si, 6.87.
[0150] Step 6
[0151] Fluorosilicic acid (3.75 ml, 30% aqueous solution prepared
as described in Pilcher, A. S. and DeShong, P. J. Org. Chem., 58,
5130 (1993)) was added to a solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alp-
ha.)]-5-[1-[4-[[1,1-dimethylethyl)dimethylsilyl]oxy]octahydro-7a-methyl-1H-
-inden-1-yl]cyclopropyl]-2-methyl-3-pentyn-2-ol (0.8 g, 2.0
mmol),), [prepared as described in Step 5 above] in acetonitrile
(12 ml) at 0.degree. C. and the reaction mixture was allowed to
warm to 15.degree. C. After 3.5 h, the reaction mixture was diluted
with water (10 ml) and ethyl acetate (10 ml) and then poured into a
mixture of ethyl acetate (100 ml) and water (50 ml). The organic
layer was separated and washed with brine, saturated sodium
bicarbonate, dried over sodium sulfate and evaporated in vacuo to
give a gum. Purification by flash chromatography on a silica gel
column using 25% ethyl acetate in hexanes as eluant gave
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]octahydro-1-[1-(4-hydroxy-4-me-
thyl-2-pentynyl)cyclopropyl]-7a-methyl-4H-inden-4-ol as a
crystalline solid (0.5 g): mp 97-98.degree. C.;
[.alpha.].sup.25D=36.degree. (MeOH, c=1.03); IR (CHCl.sub.3)
3604,2230 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.22 (2H, m),
0.39 (1H, m), 0.63 (1H, m), 0.97 (3H, s), 1.07 (1H, m), 1.2-1.45
(8H, m), 1.50 (6H, s), 1.79-1.90 (3H, m), 2.01 (1H, m), 2.03 (1H,
d, J=17 Hz), 2.62 (1H, d, J=17 Hz), 4.06 (1H, s); MS n/z 581
(2.times. M.sup.++H). Anal. Calcd. for C.sub.19H.sub.30O.sub.2: C,
78.57; H, 10.41. Found: C, 78.54; H, 10.54.
[0152] Step 7
[0153] Pyridinium dichromate (3.30 g, 8.77 mmol) was added to a
solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]octahydro-1-[1-(4-hydroxy-4-
-methyl-2-pentynyl)cyclopropyl]-7a-methyl-4H-inden-4-ol (0.8 g,
2.75 mmol),), [prepared as described in Step 6 above] in
dichloromethane (16 ml) and the reaction mixture was stirred at
room temperature. After 3.5 h, additional amounts of
dichloromethane (2.5 ml) and pyridinium dichromate (2.0 g, 5.3
mmol) were added and the stirring was continued for an additional
2.5 h. The reaction mixture was diluted, with ether (25 ml),
stirred for 30 min., and then filtered through a pad of Celite. The
Celite pad was washed with ether and the filtrate was concentrated
in vacuo to give 0.75 g of a pale yellow gum. Purification by flash
chromatography on a silica gel column using 50% ethyl acetate in
hexanes as eluant gave
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]octahydro-1-[1--
[4-hydroxy-4-methyl-2-pentynyl)cyclopropyl]-7a-methyl-4H-inden-4-one
(0.70 g) as a colorless gum: [.alpha.].sup.25D=5.5 (MeOH, c=1.2);
IR (CHCl.sub.3) 3602, 2232, 1706 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.31 (2H, m), 0.44 (1H, m), 0.62 (1H, m), 0.68
(3H, s), 1.14 (1H, m), 1.53 (6H, s), 1.73 (2H, m), 1.83 (1H, s,
OH), 1.96 (1H, m), 2.04 (1H, m), 2.05 (1H, d, J=17 Hz), 2.16-2.29
(4H, m), 2.50 (1H, dd, J=7.6 Hz), 2.62 (1H, d, J=17 Hz); MS (E/I)
m/z 288.2092. Anal. Calcd. for C.sub.19H.sub.28O.sub.2: C, 79.12;
H, 9.78. Found: C, 78.93; H, 9.80.
[0154] Step 8
[0155] A solution of
[1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-1-[1-[4-h-
ydroxy-4-methyl-2-pentynyl) cyclopropyl]-7a-methyl-4H-inden-4-one
(0.7 g, 2.426 mmol), [prepared as described in Step 7 above] and
1-(trimethylsilyl)imidazole (2.6 ml, 17.7 mmol) in methylene
chloride (15 ml) was stirred under an argon atmosphere at room
temperature for 18 h and then quenched with water (10 ml). After 25
min., the reaction mixture was poured into a mixture of ether (100
ml) and water (50 ml). The organic phase was collected and the
aqueous phase was re-extracted with ether. The combined organic
extracts were washed with brine, dried over sodium sulfate and
evaporated to give 0.82 g of a colorless oil. Purification by flash
chromatography on silica gel with 20% ethyl acetate in hexane as
eluant gave [1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-7a-m-
ethyl-1-[1-[4-methyl-4-[trimethylsilyloxy]-2-pentynyl]cyclopropyl]-4H-inde-
n-4-one (0.79 g, 90%) as an oil: [.alpha.].sup.25D=-10.69.degree.
(EtOH, c=0.8151); IR (CHCl.sub.3) 2250 and 1706 cm .sup.1; .sup.1H
NMR (CDCl.sub.3) .delta. 0.18 (9H, s), 0.28 (2H, m), 0.32 (1H, m),
0.62 (1H, m), 0.69 (3H, s), 1.13 (1H, m), 1.47 (3H, s), 1.48 (3H,
s) 1.50-1.58 (2H, m), 1.70-1.76 (2H, m), 1.92-1.99 (1H, m), 2.00
(1H, d, J=17 Hz), 2.05 (1H, m), 2.51 (1H, m), 2.64 (1H, d, J=17
Hz); MS n/z 361 (11). Anal. Calcd. for C.sub.22H.sub.36O.sub.2Si:
C, 73.28; H, 10.06; Si, 7.79. Found: C, 73.28; H, 10.10; Si,
7.79.
Example 2
1,25-Dihydroxy-23-yne-20,21,28-cyclopropyl-cholecalciferol
[0156] 9
[0157] Step 1
[0158] n-Butyllithium (0.5 ml, 0.8 mmol, 1.6 M solution in hexane)
was added to a solution of
[3S-(1Z,3.alpha.,5.beta.)]-[2-[3,5-bis[[1,1-dimeth-
ylethyl)dimethylsilyl]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosph-
ine oxide (0.465 g, 0.79 mmol) (see Kiegel, J. et al. Tetr. Lett.,
32:6057-6060 (1991)), in anhydrous tetrahydrofuran (5.0 ml) at
-78.degree. C. The resultant deep red solution was stirred at
-72.degree. C. for 7 min., and then treated with a solution of
[1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-7a-methyl-1-[1-[4-methyl-4-[(-
trimethylsilyl)oxy]-2-pentynyl]-cyclopropyl]-4H-inden-4-one (0.18
g, 0.5 mmol) [prepared as described in Example 1] in anhydrous
tetrahydrofuran (4.0 ml). After 3 h, the reaction mixture was
quenched with a 1:1 mixture of 2 N Rochelle salt solution and 2 N
potassium bicarbonate solution (10 ml). The reaction mixture was
allowed to warm to room temperature and then poured into ethyl
acetate (100 ml) and a 1:1 mixture of Rochelle salt solution and 2
N potassium bicarbonate solution (50 ml). The organic phase was
separated and the aqueous phase was extracted with ethyl acetate.
The combined organic extracts were washed with brine, dried over
sodium sulfate and evaporated to give 0.89 g of residue.
Purification by flash chromatography on silica gel with 5% ethyl
acetate-hexanes as eluant gave a trisilyl intermediate (0.34 g),
which was used directly in the next step.
[0159] Step 2
[0160] A solution of the trisilyl intermediate (0.33 g, 0.455
mmol), [prepared as described in Step 1 above] and
tetrabutylammonium fluoride (3.3 ml, 3.3 mmole, 1.0 M solution in
tetrahydrofuran) in anhydrous tetrahydrofuran (3.3 ml) was stirred
at room temperature under argon atmosphere. After 17 h, the
reaction mixture was diluted with water (10 ml). After 10 min., the
reaction mixture was poured into a 1:1 mixture of brine and water
and the organic phase was collected. The aqueous phase was
re-extracted with ethyl acetate and the combined organic extracts
were washed with brine, dried over sodium sulfate, and evaporated
to give 0.19 g of a gum. Purification by flash chromatography on
silica gel column with ethyl acetate as eluant gave 0.144 g of a
colorless residue which was dissolved in anhydrous methyl formate
(5 ml) and filtered through a 0.45 .mu.m filter. The filtrate was
evaporated at 40.degree. C. and the residue was kept under high
vacuum (0.2 mm of Hg) for 4 h to give
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-cholecalciferol (0.13 g)
as a colorless foam: [.alpha.].sub.D.sup.23=-10.23.degree. (EtOH,
c=0.38); .lambda..sub.max (MeOH) 264 (.epsilon.=16859), 248 (sh,
15198), 212 (.epsilon.=15127); .sup.1H NMR (CDCl.sub.3) .delta.
0.26 (2H, m), 0.41 (1H, m), 0.56 (1H, m), 0.59 (3H, s), 1.1 (1H,
m), 1.40-1.49 (8H, m), 1.50 (6H, s), 1.70 (2H, m), 1.84 (1H, s,
OH), 1.96 (1H, m), 2.0 (4H, m), 2.05 (1H, d, J=17 Hz), 2.31 (H, m),
2.60 (1H, d, J=17 Hz), 2.61 (H, m), 2.80 (1H, m), 4.23 (1H, br s),
4.42 (1H, br s), 4.99 (1H, s), 5.33 (1H, s), 5.98 (1H, d, J=11 Hz),
6.37 (1H, d, J=11 Hz); MS (FAB) m/z 424 (M.sup.+52).
Example 3
1,25-Dihydroxy-23-yne-20,21,28-cyclopropyl-19-nor-cholecalciferol
[0161] 10
[0162] Step 1
[0163] n-Butyllithium (0.55 ml, 0.81 mmol, 1.6 M solution in
hexanes) was added to a solution of
[3R-(3.alpha.,5.beta.,Z)-3,5-bis[[1,1-dimethylethy-
l)dimethylsilyl]oxy]cyclohexylidene]-ethyl]diphenylphosphine oxide
(0.51 g, 0.79 mmol) (see Perlman, K. L., et al., Tetr. Lett.,
32:7663-7666 (1991)), in anhydrous tetrahydrofuran (5 ml) at
-78.degree. C. under an argon atmosphere. The resultant deep red
solution was stirred at -68.degree. C. for 10 min., and then
treated with a solution of
[1R-(1.alpha.,3a.beta.,7a.alpha.)octahydro-7a-methyl-1-[1-[4-methyl-4-[tr-
imethylsily; oxy]-2-pentynyl]cyclopropyl]-4H-inden-4-one (0.18 g,
0.5 mmol), [prepared as described in Example 1] in anhydrous
tetrahydrofuran (4.0 ml). The reaction mixture was stirred at
-78.degree. C. for 4 h, then allowed to warm to 20.degree. C. and
quenched with a 1:1 mixture of 1 N Rochelle salt solution and 1 N
potassium bicarbonate solution (10 ml). After 10 min., the reaction
mixture was poured into ethyl acetate (100 ml) and 1:1 mixture of
1N Rochelle salt solution and 1 N potassium bicarbonate solution
(50 ml). The organic phase was collected and the aqueous phase was
re-extracted with ethyl acetate. The combined organic extracts were
dried over sodium sulfate and evaporated to give 0.59 g of a gum.
Purification by flash chromatography on silica gel column with 5%
ethyl acetate in hexane as eluant gave the trisilyl intermediate
(0.31 g) which was used in the next step without further
purification.
[0164] Step 2
[0165] The trisilyl intermediate (0.30 g, 0.42 mmol), [prepared as
described in Step 1 above] was dissolved in anhydrous
tetrahydrofuran (3.0 ml) and treated with tetrabutylammonium
fluoride (3.5 ml, 3.5 mmol, 1M solution in tetrahydrofuran). The
reaction mixture was stirred at room temperature under an argon
atmosphere for 48 h, then diluted with water (10 ml) and stirred
for an additional 10 min. The reaction mixture was then poured into
ethyl acetate (75 ml) and a 1:1 mixture of brine/water (50 ml). The
organic phase was collected and the aqueous phase was re-extracted
with ethyl acetate. The combined organic extracts were dried over
sodium sulfate and evaporated to give 0.24 g of a residue.
Purification of the residue by flash chromatography on a silica gel
column using ethyl acetate as eluant gave crude product which was
dissolved in methyl formate (5.0 ml) and filtered through a 0.45
.mu.m filter. The organics were evaporated and the residue was
dried under high vacuum (0.2 Torr) at room temperature for 5 h to
give
1,25-dihydroxy-23-yne-20,21,28-cyclopropyl-19-nor-cholecalciferol
(0.15 g) as a colorless foam: [.alpha.].sub.D.sup.23=+52.3.degree.
(EtOH, c=0.45); .lambda..sub.max (MeOH) 261 (.epsilon.=25929), 251
(E=38263), 243 (.epsilon.=22011), 227 (sh, .epsilon.=13396);
.sup.1H NMR (CDCl.sub.3) .delta. 0.28 (2H, m), 0.40 (1H, m), 0.55
(1H, m), 0.58 (3H, s), 1.11 (1H, m), 1.40-1.48 (2H, m), 1.50 (6H,
s), 1.55-1.60 (5H, m), 1.68 (2H, m), 1.80 (2H, m), 1.90-2.05 (4H,
m), 2.10 (1H, d, J=17 Hz), 2.20 (2H, m), 2.50 (1H, d, J=16 Hz),
2.60 (1H, d, J=17 Hz), 4.06 (1H, br s), 4.11 (1H, br s), 5.82 (1H,
d, J=11 Hz), 6.30 (1H, d, J=11 Hz); MS (FAB) m/z 412 (M.sup.+).
Example 4
[(1R-(1.alpha.,3a.beta.,7a.alpha.)]Octahydro-7a-methyl-1-[1-[5,5,5-trifluo-
ro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl]cyclopropyl]-4H-inden-4-one
[0166] 11
[0167] Step 1
[0168] n-Butyllithium (7.5 ml, 12 mmol, 1.6 M solution in hexanes)
was added to a solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]
[(1,1-dimethylethyl)dimethyl[[octahydro-7a-methyl-1-[1-(2-propynyl)cyclop-
ropyl]-1H-inden-4-yl]oxy]silane (2.36 g, 6.25 mmol), [prepared as
described in Example 1, Step 4] in anhydrous tetrahydrofuran (25
ml) at -70.degree. C. After 45 min., hexafluoroacetone (5.8 ml, 100
mmol) that had been condensed into an addition funnel capped with a
dry-ice condenser was added and stirring was continued. After 1.5
h, the reaction mixture was quenched with 2 N Rochelle salt
solution (20 ml), the reaction mixture was allowed to warm to room
temperature and then poured into a mixture of ethyl acetate (125
ml) and 50% brine (75 ml). The organic layer was separated and
washed with brine, dried over sodium sulfate and evaporated in
vacuo to give 5.8 g of a colorless gum. Purification by flash
chromatography on a silica gel column using 15% ethyl acetate in
hexanes as eluant gave [1R-(1.alpha.,3a.beta.,4.alpha.,7-
a.alpha.)]-5-[1-[4-[[1,1-dimethylethyl)dimethylsilyl]-oxy]octahydro-7a-met-
hyl-1H-inden-1-yl]cyclopropyl]-1,1,1-trifluoro-2-(trifluoromethyl)-3-penty-
n-2-ol (3.6 g) as a colorless oil: [.alpha.].sup.25D=+7.69.degree.
(EtOH, c=4.0); IR (CHCl.sub.3) 3588, 2241 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta.-0.04 (6H, s), 0.19 (1H, m), 0.28 (1H, m), 0.36
(1H, m), 0.70 (1H, m), 0.86 (9H, s), 0.93 (3H, s), 1.00 (1H, q,
J=11 Hz), 1.2-1.59 (7H, m), 1.64-1.92 (4H, m), 2.06 (1H, d, J=17
Hz), 2.75 (1H, d, J=17 Hz), 3.13 (1H, s, OH), 3.96 (1H, s); MS m/z
512 (M.sup.+, 18). Anal. Calcd. for
C.sub.25H.sub.38F.sub.6O.sub.2Si: C, 58.57; H, 7.47; F, 22.24; Si,
5.48. Found: C, 58.39; H, 7.57; F, 22.34; Si, 5.41.
[0169] Step 2
[0170] Fluorosilicic acid (6.0 ml, 30% aqueous solution prepared as
described in Pilcher, A. S. and DeShong, P. J. Org. Chem., 58, 5130
(1993)) was added to a solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alp-
ha.)]-5-[1-[4-[[11,1-dimethylethyl)dimethylsilyl]oxy]octahydro-7a-methyl-1-
H-inden-1-yl]cyclopropyl]-1,1,1-trifluoro-2-(trifluoromethyl)-3-pentyn-2-o-
l (1.24 g, 2.40 mmol), [prepared as described in Step 1 above] in
acetonitrile (18 ml) and the reaction mixture was stirred at room
temperature under an argon atmosphere. After 2.5 h, the reaction
mixture was poured into a mixture of ethyl acetate (100 ml) and
saturated sodium bicarbonate (50 ml). The organic layer was
separated and washed with brine, dried over sodium sulfate and
evaporated in vacuo to give 0.9 g of a partially crystalline solid.
Purification by flash chromatography on a silica gel column using
25% ethyl acetate in hexanes as eluant gave
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-octahydro-7a-methyl-1-[1-[5,5-
,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl]-cyclopropyl]-4H-ind-
en-4-ol as crystalline solid (0.65 g): mp 96-970C;
[.alpha.].sup.25D=25.39- .degree. (EtOH, c=0.957); IR (CHCl.sub.3)
3590, 2266, 2241 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.22
(1H, m), 0.32 (1H, m), 0.42 (1H, m), 0.71 (1H, m), 0.98 (3H, s),
1.04 (1H, m), 1.26-1.33 (2H, m), 1.40-1.60 (6H, m), 1.84-1.98 (4H,
m), 2.01 (H, d, J=17 Hz), 2.76 (1H, d, J=17 Hz), 3.86 (1H, s, OH),
4.08 (1H, s); MS m/z 397 (M.sup.+-H). Anal. Calcd. for
C.sub.19H.sub.24F.sub.6O.sub.2: C, 57.28; H, 6.07; F, 28.61. Found:
C, 57.34; H, 5.97; F, 28.66.
[0171] Step 3
[0172] To a stirred solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)-
]octahydro-7a-methyl-1-[5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pe-
ntynyl]cyclopropyl]-4H-inden-4-ol (0.66 g, 1.65 mmol), [prepared as
described in Step 2 above] in methylene chloride (14 ml) was added
pyridinium dichromate (4.0 g, 10.63 mmol) and the reaction mixture
was stirred at room temperature for 4 h. Additional amounts of
pyridinium dichromate (0.5 g, 1.32 mmol) was added and the stirring
was continued for 30 min. Diethyl ether (25 ml) was added and the
reaction mixture was filtered over Celite pad, and the Celite pad
was then washed with diethyl ether. The combined filtrate and
washings were washed with 1N potassium bicarbonate (100 ml),
followed by a 1:1 mixture of brine/water. The aqueous washings were
back-extracted with ethyl acetate and the combined organic extracts
were dried over sodium sulfate and evaporated to give 0.64 g of
partially crystalline material. Flash chromatography on silica gel
column with 25% ethyl acetate in hexanes as the eluant gave
[(1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-7a-methyl-1-[1-[5,5,5-triflu-
oro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl]cyclopropyl]-4H-inden-4-one
(0.56 g, 86%) as colorless crystals. Crystallization of 65 mgs of
the product from 50% ether in hexane gave
[(1.alpha.,3a.beta.,7a.alpha.)]octa-
hydro-7a-methyl-1-[1-[5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pent-
ynyl]cyclopropyl]-4H-inden-4-one (51 mg) as colorless needles: mp
145-146.degree. C.; [.alpha.].sup.26.sub.D=-8.52.degree. (EtOH,
c=0.704); IR (CHCl.sub.3) 3588, 2268, 2242, and 1707 cm.sup.-1;
.sup.1H NMR(CDCl.sub.3) .delta. 0.30 (1H, m), 0.38 (1H, m), 0.45
(1H, m), 0.68 (3H, s), 1.13 (1H, q, J=14 Hz), 1.55 (2H, m), 1.73
(2H, m), 1.95 (1H, m), 2.11-2.31 (4H, m), 2.13 (1H, d, J=17 Hz),
2.50 (1H, m), 2.75 (1H, d, J=17 Hz), 3.88 (1H, s, OH); MS m/z 396.
Anal. Calcd for C.sub.19H.sub.22F.sub.6O.sub.2: C, 57.47; H, 5.59;
F, 28.76. Found: C, 57.60, H, 5.65; F, 28.66.
Example 5
1,25-Dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-cholecalcifero-
l
[0173] 12
[0174] Step 1
[0175] n-Butyllithium (0.52 ml, 0.81 mmol, 1.6 M solution in
hexane) was added to a stirred solution of
[3S-(1Z,3.alpha.,5.beta.)]-[2-[3,5-bis[[1,-
1-dimethylethyl)dimethylsilyl]oxy]-2-methylene
cyclohexylidene]ethyl]diphe- nylphosphine oxide (0.475 g, 0.81
mmol) in anhydrous tetrahydrofuran (5.0 ml) at -78.degree. C. The
resultant deep red solution was stirred at -78.degree. C. under
argon for 8 min and then treated with a solution of
[1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-7a-methyl-1-[1-[5,5,5-trifluo-
ro-4-hydroxy-4-(trifluoromethyl)-2-pentynyl]cyclopropyl]-4H-inden-4-one
(0.16 g, 0.4 mmol), [prepared as described in Example 4] in
anhydrous tetrahydrofuran (2.0 ml). After 3 h, the reaction mixture
was allowed to warm to 10.degree. C. and then quenched with a 1:1
mixture of 1 N Rochelle salt solution and 1 N potassium bicarbonate
solution (10 ml). After 10 min., the reaction mixture was poured
into ethyl acetate (100 ml) and a 1:1 mixture of 1 N Rochelle salt
solution and 1 N potassium bicarbonate solution (50 ml). The
organic phase was collected and the aqueous phase was re-extracted
with ethyl acetate. The combined organic extracts were dried over
sodium sulfate and evaporated to give 0.55 g of a gum. Purification
by flash chromatography on silica gel column with 20% ethyl acetate
in hexane as eluant gave 0.15 g of the trisilyl intermediate as
colorless gum, which was used without purification in the next
step.
[0176] Step 2
[0177] Tetrabutylammonium fluoride (2.5 ml, 2.5 mmol, 1M solution
in tetrahydrofuran) was added to a solution of the trisilyl
intermediate (0.145 g, 0.19 mmol) in anhydrous tetrahydrofuran (3.0
ml) and the reaction mixture was stirred at room temperature under
an argon atmosphere. After 19 h, the reaction mixture was diluted
with water (10 ml), stirred for an additional 10 min. and then
poured into a mixture of ethyl acetate (75 ml) and a 1:1 mixture of
brine/water (50 ml). The organic phase was collected and the
aqueous phase was re-extracted with ethyl acetate. The combined
organic extracts were dried over sodium sulfate and evaporated to
give 0.16 g of residue. Purification by flash chromatography on
silica gel column with ethyl acetate as eluant gave a solid which
was dissolved in methyl formate (2.0 ml) and filtered through a
0.45 .mu.m filter. The filtrate was evaporated at 40.degree. C. and
kept under high vacuum at room temperature for 6 h to give
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-cholecalcifer-
ol (93 mgs) as a colorless foam: [.alpha.].sub.D.sup.25=-1.12
(EtOH, c=0.50); .lambda..sub.max (MeOH) 264 (.epsilon.=16762), 247
(sh, .epsilon.=14746), 213 (.epsilon.=13727); .sup.1H NMR
(CDCl.sub.3) .delta. 0.28 (1H, m), 0.35 (1H, m), 0.41 (1H, m), 0.59
(3H, s), 0.64 (1H, m), 1.09 (1H, m), 1.40-1.60 (7H, m), 1.65-1.78
(2H, m), 1.90-2.05 (5H, m), 2.18 (2H, d, J=17 Hz), 2.31 (1H, dd,
J=14, 7 Hz), 2.63 (1H, d, J=14 Hz), 2.73 (1H, d, J=17 Hz), 2.85
(1H, m), 3.45 (1H, s, OH), 4.23 (1H, br s), 4.43 (1H, br s), 5.00
(1H, s), 5.32 (1H, s), 6.00 (1H, d, J=11 Hz), 6.37 (1H, d, J=11
Hz); MS (FAB) m/z 532 (M.sup.+50).
Example 6
1,25-Dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-19-nor-choleca-
lciferol
[0178] 13
[0179] Step 1
[0180] n-Butyllithium (0.5 ml, 0.80 mmol, 1.6 M solution in hexane)
was added to a stirred solution of
[3R-(3.alpha.,5.beta.,Z)-3,5-bis[[1,1-(dim-
ethylethyl)dimethylsilyl]oxy]cyclohexylidene]-ethyl]diphenylphosphine
oxide (0.45 g, 0.79 mmol) in anhydrous tetrahydrofuran (5.0 ml) at
-78.degree. C. The resultant deep red solution was stirred at
-78.degree. C. under argon for 8 min., and then treated with a
solution of
[1R-(1.alpha.,3a.beta.,7a.alpha.)]octahydro-7a-methyl-1-[5,5,5-trifluoro--
4-hydroxy-4-(trifluoromethyl)-2-pentynyl]cyclopropyl]-4H-inden-4-one
(0.16 g, 0.40 mmol), [prepared as described in Example 4 above] in
anhydrous tetrahydrofuran (3.0 ml). The reaction mixture was
stirred at -78.degree. C. for 3 h, then allowed to warn to
10.degree. C., and quenched with a 1:1 mixture of 1 N Rochelle salt
solution and 1 N potassium bicarbonate solution (10 ml). After 10
min., the reaction mixture was poured into ethyl acetate (100 ml)
and a 1:1 mixture of 1 N Rochelle salt solution and 1 N potassium
bicarbonate solution (50 ml). The organic phase was collected and
the aqueous phase was re-extracted with ethyl acetate. The combined
organic extracts were dried over sodium sulfate and evaporated to
give 0.54 g of residue. Purification by flash chromatography on a
silica gel column with 20% ethyl acetate in hexane as eluant gave
0.15 g of trisilyl intermediate as a colorless gum, which was used
without further purification in the next step.
[0181] Step 2
[0182] Tetrabutylammonium fluoride (2.5 ml, 2.5 mmol, 1M solution
in tetrahydrofuran) was added to a solution of above trisilyl
intermediate (0.15 g, 0.20 mmol) in anhydrous tetrahydrofuran (3.0
ml). The reaction mixture was stirred under argon at room
temperature for 40 h, then diluted with water (10 ml) and poured
into ethyl acetate (75 ml) and a 1:1 mixture of brine/water (50
ml). The organic phase was collected and the aqueous phase was
re-extracted with ethyl acetate. The combined organic extracts were
dried over sodium sulfate and evaporated to give 0.10 g of crude
product. Purification by flash chromatography on a silica gel
column with ethyl acetate as eluant gave a residue, which was
dissolved in methyl formate (5.0 ml), filtered through a 0.45 .mu.m
filter. The filtrate was evaporated at 40.degree. C. and dried
under high vacuum (0.2 Torr) at room temperature for 6 h to give
1,25-dihydroxy-23-yne-26,27-hexafluoro-20,21,28-cyclopropyl-19-nor-cholec-
alciferol (95 mg) as a colorless foam:
[.alpha.].sub.D.sup.23=+36.20.degre- e. (EtOH, c=0.32);
.lambda..sub.max (MeOH) 243 (.epsilon.=31322), 251
(.epsilon.=37316), 260 (.epsilon.=25430) nm; IR(CHCl.sub.3) 3603,
2242 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.28 (1H, m), 0.35
(I H, m), 0.42 (1H, m), 0.60 (3H, s), 0.65 (1H, m), 1.10 (1H, m),
1.40-1.72 (9H, m), 1.80 (1H, m), 1.97 (4H, m), 2.19 (1H, d, J=17
Hz), 2.49 (1H, m), 2.72 (1H, d, J=17 Hz), 2.75 (2H, m), 3.40 (1H,
br s, OH), 4.05 (1H, br, s), 4.12 (1H, br s), 5.82 (1H, d, J=1 Hz),
6.30 (1H, d, J=11 Hz); MS (FAB) n/z 520 (M.sup.+80).
Example 7
[1R-[1.alpha.,3a.beta.,7a.alpha.]]-Octahydro-7a-methyl-1-[1-[5,5,5-trifluo-
ro-4-(trifluormethyl)-4-[(trimethylsilyl)oxy]-2-pentenyl]-cyclopropyl]-4H--
inden-4-one
[0183] 14
[0184] Step 1
[0185] A solution of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]octahydro--
7a-methyl-1-[1-[5,5,5-trifluoro-4-hydroxy-4-(trifluormethyl)-2-pentynyl]cy-
clopropyl]-4H-inden-4-ol (1.195 g) in ethyl acetate (12 ml), hexane
(30.0 ml), absolute ethanol (1.2 ml), and quinoline (60 ml) was
hydrogenated over Lindlar's catalyst (240 mg) at atmospheric
pressure and room temperature. After 2.0 h, the reaction mixture
was filtered through a pad of Celite. The Celite pad was washed
with ethyl acetate and the combined filtrates were washed with 1.0
N hydrochloric acid (50 ml), brine, dried over magnesium sulfate
and evaporated to give 1.16 g of a colorless gummy residue. The
residue was purified by flash chromatography on a silica gel column
with 40% ethyl acetate in hexanes as eluant to give 1.09 g of a
colorless gum which was triturated with hexane to give
[1R-[1.alpha.(Z),3a.beta.,4.alpha.,7a.alpha.]]octahydro-7a-methyl-1-[1-[5-
,5,5-trifluoro-4-hydroxy-4-(trifluormethyl)-2-pentenyl]cyclpropyl]-4H-inde-
n-4-ol (84 mg): mp 99-100.degree. C.;
[.alpha.].sub.D.sup.25+24.49.degree. (MeOH, c=1.03); IR
(CHCl.sub.3) 3619, 3569, 1659 cm.sup.-1; .sup.1H NMR (CDCl.sub.3)
.delta. 0.09 (1H, m), 0.23 (1H, m), 0.34 (1H, m), 0.67 (1H, m),
1.00 (3H, s), 1.11 (1H, m), 1.19-1.30 (2H, m), 1.37-1.56 (6H, m),
1.76-1.88 (3H, m), 2.03 (1H, d, J=16 Hz), 2.17 (1H, ddd, J=16, 7, 6
Hz), 2.95 (1H, ddd, J=16, 7, 6), 3.13 (1H, s, OH), 4.06 (1H, s),
5.40 (1H, d, J=12 Hz), 6.10 (1H, ddd, J=12, 7, 6 Hz); MS m/z 400
(M.sup.+, 10). Anal. Calcd for C.sub.19H.sub.26F.sub.6O.sub.2: C,
56.99; H, 6.55; F, 28.47. Found: C, 57.10; H, 6.57; F, 28.31.
[0186] Step 2
[0187] Pyridinium dichromate (3.3 g, 8.7 mmol) was added to a
stirred solution of
[1R-[1.alpha.(Z),3a.beta.,4.alpha.,7a.alpha.]]octahydro-7a-me-
thyl-1-[1-[5,5,5-trifluoro-4-hydroxy-4-(trifluormethyl)-2-pentenyl]cyclpro-
pyl]-4H-inden-4-ol (1.00 g, 2.5 mmol) in dichloromethane (25 ml)
and the resultant heterogeneous mixture was stirred at room
temperature. After 5 h, the reaction mixture was diluted with
diisopropyl ether (30 ml), stirred for an additional 15 min., and
then filtered through a pad of Celite. The filtrate was evaporated
to give 0.984 g of a pale yellow solid. Purification by flash
chromatography on a silica gel column with 30% ethyl acetate in
hexanes as eluant, gave 0.84 g of a colorless solid. The solid was
dissolved in dichloromethane (4 ml) and filtered through a 0.45 mm
filter (Millex-HV). The filtrate was diluted with hexane (7.0 ml)
and then concentrated to about 6 ml and left at -2.degree. C.
overnight. The solid was filtered off to give
[1R-[1.alpha.(Z),3a.beta.,7a.alpha.]]o-
ctahydro-7a-methyl-1-[1-[5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-p-
entenyl]cyclopropyl]-4H-inden-4-one (0.8 g) as colorless crystals:
mp 124-125.degree. C.; [.alpha.].sub.D.sup.25-2.6.degree. (EtOH,
c=1.00); IR (CHCl.sub.3) 3568, 1706 cm.sup.-1; .sup.1H NMR
(CDCl.sub.3) .delta. 0.15 (1H, m), 0.36 (2H, m), 0.60 (1H, m), 0.65
(3H, s), 1.15 (1H, m), 1.50-1.80 (4H, m), 1.85-2.3 (4H, m), 2.45
(1H, dd, J=7.6, 6.8 Hz), 2.91 (1H, ddd, J=16, 7.6, 5.9 Hz), 2.98
(1H, s, OH), 5.42 (1H, d, J=12 Hz), 6.10 (1H, ddd, J=12, 7.6, 6.8
Hz). MS m/z 398 (M.sup.+, 22). Anal. Calcd for
C.sub.19H.sub.24F.sub.6O.sub.2: C, 57.28; H, 6.07; F, 28.61. Found:
C, 57.39; H, 6.01; F, 28.75.
[0188] Step 3
[0189] A solution of
[1R-[1.alpha.(Z),3a.beta.,7a.alpha.]]octahydro-7a-met-
hyl-1-[1-[5,5,5-trifluoro-4-hydroxy-4-(trifluoromethyl)-2-pentenyl]cyclopr-
opyl]-4H-inden-4-one (0.75 g, 1.88 mmol) and
1-(trimethylsilyl)imidazole (2.6 ml, 17.75 mmol) in dichloromethane
(20 ml) was stirred under argon for 7 hr and then diluted with
water (10 ml). After stirring for 15 min., the reaction mixture was
poured into dichloromethane (60 ml) and water (50 ml). The organic
phase was collected and the aqueous phase was re-extracted with
dichloromethane. The combined organic extracts were washed with
water, dried over magnesium sulfate and evaporated to give 0.87 g
of a partially crystalline solid. Purification by flash
chromatography on a silica gel with 20% ethyl acetate in hexanes as
eluant gave 0.83 g of colorless crystals. The crystals were
dissolved in ether (5 ml), filtered through a 0.45 mm filter
(Millex-HV) and the filtrate was diluted with hexane (5 ml). The
ether was evaporated and the solution was left at -1.degree. C.
overnight. Filtration of the solid gave
[1R-[1.alpha.(Z),3a.beta.,7a.alpha.]]octahydro-7a-methyl-1-[1-[5,5,5-
-trifluoro-4-(trifluormethyl)-4-[(trimethylsilyl)oxy]-2-pentenyl]cycloprop-
yl]-4H-inden-4-one (0.80 g) as colorless crystals: mp 70-71.degree.
C.; [.alpha.].sub.D.sup.25+0.9.degree. (MeOH, c=1.00); IR
(CHCl.sub.3) 1706 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.11
(1H, m); 0.22 (9H, s), 0.32 (2H, m), 0.65 (1H, m), 0.69 (3H, s),
1.12 (1H, in), 1.50-1.73 (4H, m), (1.90-2.30 (7H in), 2.47 (1H, dd,
J=17, 7 Hz), 2.94 (1H, ddd, J=12, 7, 6 Hz), 5.41 (1H, d, J=12 Hz),
6.05 (1H, ddd, J=12, 7, 6 Hz). MS m/z 471 (M.sup.++H, 100). Anal.
Calcd for C.sub.22H.sub.32F.sub.6O.sub.2Si: C, 56.15; H, 6.85; F,
24.22; Si, 5.97. Found: C, 56.26; H, 6.72; F, 24.29; Si, 5.80.
Example 8
1,25-Dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,28-cyclopropyl-cholecalci-
ferol
[0190] 15
[0191] Step 1
[0192] n-Butyllithium (0.5 ml, 8 mmol, 1.6 M solution in hexanes)
was added to a solution of
[3S-(1Z,3.alpha.,5.beta.)]-2-[3,5-bis[[1,1-dimethy-
lethyl)dimethylsilyl]oxy-2-methylenecyclohexylidene]ethyl]diphenylphosphin-
e oxide (0.47 g, 0.8 mmol) in anhydrous tetrahydrofuran (4 ml) at
-78.degree. C. The resulting deep red solution was stirred at
-78.degree. C. for 7 minutes and then treated with a solution of
[1R-1.alpha.(Z),3a.beta.,7a.alpha.]octahydro-7a-methyl-1-[1-[5,5,5-triflu-
oro-4-(trifluoromethyl)-4-[trimethylsilyl)oxy]-2-pentenyl]cyclopropyl]-4H--
inden-4-one (0.19 g, 0.4 mmol) in anhydrous tetrahydrofuran (3 ml).
After 2 h, the reaction mixture was allowed to warm to -10.degree.
C. and then quenched with a 1:1 mixture of 2 N Rochelle salt
solution and 2 N potassium bicarbonate solution (5.0 ml). After 20
min., the reaction mixture was poured into a mixture of ethyl
acetate (60 ml) and a 1:1 mixture of 2 N Rochelle salt solution and
2 N potassium bicarbonate solution (50 ml). The organic phase was
collected and the aqueous phase was re-extracted with ethyl
acetate. The combined organic extracts were washed with 50% brine
(100 ml), dried over sodium sulfate and evaporated to give a gum.
Purification by flash chromatography on a silica gel column with
20% ethyl acetate in hexanes as eluant gave 0.11 g of the trisilyl
intermediate as colorless gum, which was without further
purification in the next step.
[0193] Step 2
[0194] Tetrabutylammonium fluoride (3.0 ml, 3.0 mmol, 1.0 M
solution in tetrahydrofuran) was added to a solution of the
trisilyl intermediate (0.11 g) in tetrahydrofuran {3 ml) and the
reaction mixture was stirred at room temperature. After 17 h, the
reaction mixture was diluted with water (5 ml), stirred for
additional 15 min., and then poured into a mixture of ethyl acetate
(50 ml) and 50% brine (40 ml). The organic phase was collected and
the aqueous phase was re-extracted with ethyl acetate. The combined
organic extracts were washed with water, dried over sodium sulfate
and evaporated to give 86 mg of a gum. Purification by flash
chromatography on a silica gel column with ethyl acetate as eluant
gave a gum, which was dissolved in anhydrous methyl formate (7 ml),
filtered through a 0.4 gm filter, and evaporated to give
1,25-dihydroxy-23-(Z)-ene-
-26,27-hexafluoro-20,21,28-cyclopropyl-cholecalciferol (69 mg) as a
colorless foam: [.alpha.].sub.D.sup.25=-4.0.degree. [MeOH, c=0.35);
.lambda..sub.max (MeOH) 265 (.epsilon. 15837), 211
(.epsilon.=14458) nm; IR (CHCl.sub.3) 3598, 1651 cm.sup.-1; .sup.1H
NMR (CDCl.sub.3) .delta. 0.11 (1H, m), 0.29 (2H, m), 0.60 (3H, s),
0.61 (1H, m), 1.10 (1H, m), 1.21-1.35 (1H, m), 1.50 (6H m), 1.70
(2H, m), 1.90 (2H, m), 2.00 (3H, m), 2.30 (2H, m), 2.60 (1H, d,
J=12 Hz), 2.85 (2H, m), 2.90 (1H, s, OH), 4.22 (1H, s), 4.42 (1H,
s), 4.99 (1H, s), 5.32 (1H, s), 5.42 (1H, d, J=12 Hz), 5.99 (1H,
d=11 Hz), 6.10 (1H, ddd, J=12, 7, 6), 6.36 (1H, d, J=11 Hz); MS
(FAB) m/z 535 (M.sup.++H).
Example 9
1,25-Dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,28-cyclopropyl-19-nor-cho-
lecalciferol
[0195] 16
[0196] Step 1
[0197] n-Butyllithium (0.32 m, 0.5 mmol, 1.6 M solution in hexanes)
was added to a solution of
[3R-(3.alpha.,5.beta.,Z)]-3,5-bis[[1,1-dimethyleth- yl)
dimethyl-silyl]oxy]cyclohexylidene]ethyl} diphenylphosphine oxide
(0.285 g, 0.5 mmol) in anhydrous tetrahydrofuran (3 ml) at
-78.degree. C. The resulting deep red solution was stirred at
-78.degree. C. for 6 minutes and then treated with
[1R-1.alpha.((Z),3a.beta.,7a.alpha.]]octahy-
dro-7a-methyl-1-[1-[5,5,5-trifluoro-4-(trifluoromethyl)-4-[trimethylsilyl)-
oxy]-2-pentenyl]cyclopropyl]-4H-inden-4-one (0.12 g, 0.25 mmol) in
anhydrous tetrahydrofuran (2 ml). After 3.0 h, the reaction mixture
was allowed to warm to 15.degree. C. and quenched with 1:1 mixture
of 2 N Rochelle salt solution and 2 N potassium bicarbonate
solution (5 ml). After 20 min., the reaction mixture was diluted
with ethyl acetate (15 ml), poured into a mixture of ethyl acetate
(50 ml) and a 1:1 mixture of 2 N Rochelle salt solution and 2 N
potassium bicarbonate solution (50 ml). The organic phase was
collected and the aqueous phase was re-extracted with ethyl
acetate. The combined organic extracts were washed with 50% brine,
dried over sodium sulfate and evaporated to give 0.58 g of a gum.
Purification by flash chromatography on a silica gel column with
20% ethyl acetate in hexanes as eluant, gave 0.18 g of the trisilyl
intermediate as a colorless gum, which was without further
purification in the next step.
[0198] Step 2
[0199] Tetrabutylammonium fluoride (3.0 ml, 3.0 mmol, 1.0 M
solution in tetrahydrofuran) was added to a solution of the
trisilyl intermediate (0.18 g) in tetrahydrofuran (3 ml) and the
reaction mixture was stirred at room temperature. After 42 h, the
reaction mixture was diluted with water (5 ml), stirred for an
additional 15 min., and then poured into a mixture of ethyl acetate
(50 ml) and 50% brine (40 ml). The organic phase was collected and
the aqueous phase was re-extracted with ethyl acetate. The combined
organic extracts were washed with water, dried over sodium sulfate
and evaporated to give 0.12 g of a gum. Purification by flash
chromatography on a silica gel column with ethyl acetate as eluant
gave a gum, which was dissolved in anhydrous methyl formate (8 ml),
filtered through a 0.4 .mu.m filter, and evaporated to give
1,25-dihydroxy-23-(Z)-ene-26,27-hexafluoro-20,21,28-cyclopropyl-19-nor-ch-
olecalciferol (98 mg) as a colorless foam:
[.alpha.].sub.D.sup.25+47.4.deg- ree. [MeOH, c=0.35);
.lambda..sub.max (MeOH) 260 (E 28200), 251 (.epsilon.=41760), 243
(.epsilon.=34747), 235 (sh, .epsilon.=23594) nm; IR (CHCl.sub.3)
3603 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.12 (1H, m), 0.32
(2H, m), 0.60 (3H, s), 0.62 (1H, m), 1.14 (1H, m), 1.35 (1H, m),
1.41 (2H m), 1.52 (4H, m), 1.70 (2H, m), 1.82 (1H, m), 1.88-2.00
(2H, m), 2.04 (2H, m), 2.23 (3H, m), 2.47 (1H, d, J=12 Hz), 2.82
(3H, m), 2.96 (1H, s, OH), 4.04 (1H, s), 4.12 (1H, s), 5.42 (1H, d,
J=12 Hz), 5.82 (1H, d=11 Hz), 6.12 (1H, ddd, J=12, 7, 6), 6.30 (1H,
d, J=11 Hz); MS (EI) m/z 522 (M, 60).
Example 10
1,25-Dihydroxy-20,21,28-cyclopropyl-cholecalciferol
[0200] Step 1
[0201]
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-Octahydro-1-[1-(4-hydro-
xy-4-methylpentyl)cyclopropyl]-7a-methyl-4H-inden-4-ol
[0202] A solution of 250 mg (0.86 mmol) of
[1R-(1.alpha.,3a.beta.,4.alpha.-
,7a.alpha.)]octahydro-1-[1-(4-hydroxy-4-methyl-2-pentynyl)-cyclopropyl]-7a-
-methyl-4H-inden-4-ol (Ro 27-3152) in 4.0 mL of ethyl acetate, 10
mL of hexane, 0.5 mL of ethanol, and 20 .mu.L of quinoline was
hydrogenated over 75 mg of Lindlar's catalyst (5% Pd+3.5% Pb on
CaCO3) at room temperature and atmospheric pressure for 2.5 hrs.
The mixture was diluted with 50 mL of ethyl acetate and filtered
over a pad of Celite, which was washed with 3.times.20 mL of ethyl
acetate. The combined filtrate and washings were washed with 50 mL
of 0.1N HCl then 50 mL of water, dried (Na.sub.2SO.sub.4) and
evaporated to give 244 mg of a colorless gum. Flash chromatography
on 50 g of silica gel (40-65 .mu.m; 3.5 cm diameter column) with
50% ethyl acetate, taking 12-mL fractions gave, after evaporation
of fractions 10-18, 230 mg of a colorless gum. .sup.1H NMR
(CDCl.sub.3) indicated it to be a mixture of the title compound and
the 23,24 (Z)-ene product. The mixture was dissolved in 25 mL of
CH.sub.2Cl.sub.2 and hydrogenated with 40 mg of
[1,4-bis(diphenylphosphin- o) butane] (1,5-cyclooctadiene)-rhodium
(1) tetrafluoroborate as catalyst in the presence of 1 drop of
mercury in a Parr hydrogenator at room temperature and 50 psi for 3
hrs. After dilution with 30 mL of CH.sub.2Cl.sub.2, the mixture was
filtered over a pad of Celite, which was washed with 3.times.40 mL
of ethyl acetate. The filtrate and washings were evaporated to give
an orange-colored gum, which was purified by flash chromatography
on 45 g of silica gel (40-65 .mu.m mesh; 3.5 cm diameter column)
with 50% ethyl acetate in hexanes as eluent, taking 12-mL
fractions. Fractions 13-17 were combined and evaporated to give a
partially crystalline solid, which was triturated with hexane to
give 204 mg of the title compound as colorless crystals, mp
126-128.degree. C.; [.alpha.].sub.D.sup.25+42.6.degree. (MEOH,
c=0.3); IR 3611 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta.-0.06
(1H, m) 0.18 (2H, m), 0.54 (1H, m), 0.59 (1H, m), 0.98 (3H, m),
1.21 (6H, s), 1.2-1.6 (15H, m), 1.75-2.10 (5H, m), 4.06 (1H, s); MS
(+FAB) m/z (295, M++1, 10). Anal. Calcd for
C.sub.19H.sub.34O.sub.2: C, 77.50; H, 11.64. Found C: 77.40; H,
11.89.
[0203] Step 2
[0204]
[1R-(1.alpha.,3a.beta.,7a.alpha.)]-Octahydro-7a-methyl-1-[1-[5-meth-
yl-5-[(trimethylsilyl)oxy]pentyl]cyclopropyl]-4H-inden-4-one
[0205] To a stirred solution of 190 mg (0.64 mmol) of
[1R-(1.alpha.,3a.beta.,4.alpha.,7a.alpha.)]-octahydro-1-[1-(4-hydroxy-4-m-
ethylpentyl)cyclopropyl]-7a-methyl-4H-inden-4-ol (Ro 27-5155) in
8.0 mL of CH.sub.2Cl.sub.2 was added 2.0 g (5.3 mmol) of pyridinium
dichromate and the mixture was stirred at room temperature for 5.0
hrs. It was diluted with 20 mL of diisopropyl ether, stirred for 15
minutes and filtered over a pad of Celite, which was washed with
4.times.25 mL of diisopropyl ether. Evaporation of the filtrate and
washings gave 184 mg of a pale yellow gum, which was purified by
flash chromatography on 45 g of silica gel (40-65 .mu.m mesh; 3.5
cm diameter column) with 45% of ethyl acetate on hexanes as eluent
taking 12-mL fractions. Fractions 19-25 were combined and
evaporated to give 158 mg of a colorless gum. The latter was
dissolved in 5.0 mL of CH.sub.2Cl.sub.2 and treated with 1.0 .mu.L
(6.8 mmol) of 1-trimethylsilylimidazole and the mixture was stirred
at room temperature for 2.0 hrs. It was diluted with 15 mL of water
and 15 mL of CH.sub.2Cl.sub.2, stirred for a further 15 mins, and
poured into a mixture of 40 mL of CH.sub.2Cl.sub.2 and 20 mL of 10%
brine. The organic phase was separated and the aqueous phase was
re-extracted with 3.times.50 mL of CH.sub.2Cl.sub.2. The combined
organic extracts were washed with 3.times.60 mL of 10% brine, dried
(Na.sub.2SO.sub.4) and evaporated to give 180 mg of a colorless
gum, which was purified by flash chromatography on 45 g of silica
gel (40-65 Mm mesh; 3.5 cm diameter column) with 15% ethyl acetate
in hexanes as eluent, taking 12-mL fractions. Fractions 10-14 were
combined and evaporated to give 137 mg of a colorless gum, which
was further purified by HPLC on silica gel (15-30 .mu.m mesh; 50
mm.times.50 cm column; 70 mL/min) with 7.5% ethyl acetate in
hexanes as eluent. The material eluting at 18.5 minutes was
collected and evaporated to give 114 mg of the title compound as a
gum, which solidified on keeping at 0.degree. C. overnight;
[.alpha.].sub.D.sup.25+7- .8.degree. (CHCl.sub.3, c=0.41); IR 1707
cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.00 (1H, m), 0.11 (9H,
s), 0.20 (2H, m), 0.63 (2H, m), 0.68 (3H, s), 1.01 (1H m), 1.21
(6H, s), 1.30-1.72 (7H, m), 1.90-2.10 (3H, m), 2.15-2.3 (5H, m),
2.5 (1H, m); MS (+FAB) m/z 349.252 (M.sup.+-15, 48).
[0206] Step 3
[0207] 1,25-Dihydroxy-20,21,28-cyclopropyl-cholecalciferol
[0208] To a cooled (-78.degree. C.), stirred solution of 335 mg
(0.57 mmol) of
[3S-(1Z,3.alpha.,5.beta.)]-[2-[3,5-bis[(1,1-dimethylethyl)dimeth-
ylsilyl)oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine
oxide in 4.0 mL of anhydrous THF was added 0.35 mL (0.56 mmol) of a
1.6 M solution of n-butyllithium in hexanes and the resultant deep
red solution was stirred at -78.degree. C. for 7 minutes. A
solution of 105 mg (0.28 mmol) of
[1R-(1.alpha.,3a.beta.,7a.alpha.)]-octahydro-7a-methyl-1-[1-[5-methyl--
5-[(trimethylsilyl)oxy]pentyl]cyclopropyl]-4H-inden-4-one (Ro
27-5156) in 1.5 mL of anhydrous THF was added and the mixture was
stirred at -78.degree. C. for 3 hours and then at room temperature
for 15 minutes. To the mixture was added 5 mL of a 1:1 mixture of
1.0 M Rochelle salt solution and 1.0 N KHCO.sub.3 solution. After
15 minutes the mixture was poured into 50 mL of ethyl acetate and
40 mL of a 1:1 mixture of 1.0 M Rochelle salt solution and 1.0 N
KHCO.sub.3 solution. The organic phase was separated and the
aqueous phase was re-extracted with 3.times.50 mL of ethyl acetate.
The combined organic extracts were dried (Na.sub.2SO.sub.4) and
evaporated to give 440 mg of a gum, which was chromatographed on 40
g of silica gel (40-65 .mu.m mesh; 3.5 cm diameter column) with 5%
ethyl acetate in hexanes as eluent, taking 12-mL fractions.
Fractions 5-8 were combined and evaporated to give 131 mg of a
colorless gum. The latter was dissolved in 3.0 mL of THF, treated
with 1.5 mL (1.5 mmol) of a 1.0 M solution of tetra-n-butylammonium
fluoride in THF, and stirred at room temperature for 17 hours. The
mixture was diluted with 10 mL of water, stirred for 15 minutes,
and poured into a mixture of 60 mL of ethyl acetate and 40 mL of
10% brine. The organic phase was separated and the aqueous phase
was re-extracted with 3.times.60 mL of ethyl acetate. The combined
organic extracts were washed with 4.times.100 mL of water, dried
(Na.sub.2SO.sub.4), and evaporated to give 78 mg of a colorless
gum, which was purified by flash chromatography on 40 g of silica
gel (40-65 .mu.m mesh; 3.2 cm diameter column) with ethyl acetate
as eluent, taking 10-mL fractions. Fractions 10-12 were combined
and evaporated to give a gum, which was dissolved in 10 mL of
anhydrous methyl formate. The solution was filtered through a 0.4
.mu.m filter and the filtrate was evaporated to give 64 mg of the
title compound as a colorless foam:
[.alpha.].sub.D.sup.25+18.3.degree. (MeOH, c=0.18); IR (CHCl.sub.3)
3608 cm.sup.-1; .sup.1H NMR (CDCl.sub.3) .delta. 0.00 (2H, m), 0.20
(2H, m), 0.59 (3H, s), 0.63 (2H, m), 0.90 (2H, m), 1.22 (6H, s),
1.30-1.70 (20H, m), 1.90-2.12 (5H, n), 2.60 (1H, d), 2.81 (1H, d),
4.22 (1H, br s), 4.43 (1H, br s), 4.99 (1H, s), 5.32 (1H, s), 5.99
(1H, d, J=11 Hz) 6.37 (1H, d, J=11 Hz); MS (EI) Calcd. for
C.sub.28H.sub.44O.sub.3: m/Z 428.3290. Found n/z 428.3297.
Example 11
Bone Anabolism in the Rat
[0209] The compounds of the present invention are more effective
than 1,25-dihydroxy vitamin D.sub.3 at bone accretion and do not
induce hypercalciuria, nephrotoxicity, or hypercalcemia at
therapeutically effective doses. This has been demonstrated as
follows:
[0210] Three month old rats are ovariectomized (Ovx) and
administered either 1,25-dihydroxy vitamin D.sub.3 (vit. D in
Table) or one of the compounds of the present invention once a day
by mouth starting at 3 weeks post-ovariectomy and continuing until
final sacrifice at 6 weeks post-ovariectomy. Control groups, both
sham (rats that were not ovariectomized) and Ovx, received vehicle
only. Blood and urine samples were collected twice, at 4 weeks
post-ovariectomy and again at the 6 week mark and the amount of
serum and urine calcium was determined. The final femoral calcium
was determined upon sacrifice 6 weeks post-ovariectomy.
[0211] The bone mineral density of the right femur was determined
by using a High Resolution Software Package on a QDR-1000W Bone
Densitometer.TM. (Hologic, Walthan, Mass.). The animals were
scanned by placing them on a scanning block in a supine position
such that the right leg was perpendicular to the main body and the
tibia was perpendicular to the femur. The increase in the bone
mineral density and the amount of calcium in the urine and the
serum for some of the compounds of this invention in this assay are
given in the table below:
2 Urine Whole Serum Calcium/ CPD # Femur Calcium Creatinine see
Dose BMD mg/dl mg/dl Table I Surgery Treatment .mu.g/kg/day
mg/cm.sup.2 (6th Week) (6th Week) 3 Sham Vehicle 0.000 0.2457 9.17
0.29 Ovx Vehicle 0.000 0.2330 9.45 0.23 Ovx Vit D 0.200 0.2368
10.65 1.71 Ovx Cpd #3 0.010 0.2396 10.25 0.83 4 Sham Vehicle 0.000
0.2435 8.26 0.26 Ovx Vehicle 0.000 0.2228 8.57 0.31 Ovx Vit D 0.200
0.2349 9.49 1.37 Ovx Cpd #4 0.005 0.2413 8.94 0.60
Example 12
Cell Proliferation Assay in MCF-7 Breast Cancer Cells
[0212] MCF-7 cells are human mammary carcinoma cells that are
positive for estrogen receptors. The potential activity of vitamin
D.sub.3 analogs against breast cancer was assessed from inhibition
of proliferation of MCF-7 cells in culture.
[0213] MCF-7 cells were plated at 9000 cells/well in 24-well plates
and incubated at 37.degree. C. in 5% CO.sub.2/95% air in Dulbecco's
Modified Eagle Medium containing 10% fetal bovine serum, 700 nM
insulin, 2 mM glutamine, 0.1 mM MEM non-essential amino acids and 1
mM sodium pyruvate. Stock solutions of vitamin D.sub.3 analogs were
prepared at a concentration of 10 mM in absolute ethanol and stored
at -20.degree. C. under argon. Four days after plating, the number
of MCF-7 were counted by removing the medium in 8 wells, rinsing
the cells with 0.5 ml PBS without Ca/Mg and then incubating the
cells with 0.3 ml of trypsin-EDTA. After 15 min., the
trypsinization was stopped by adding 0.3 ml of medium. 0.2 ml
aliquot was transferred from each well into dilu-vials containing
10 ml isoton and the number of cells were counted on Coulter
Counter.TM. (Coulter, Miami, Fla.).
[0214] MCF-7 cells in the remaining wells were refed with either
control medium or medium containing varying concentrations of the
vitamin D.sub.3 analog. After a further 7 days of culture, the
number of MCF-7 cells in each well was assessed by removing the
medium, rinsing the cells with 0.5 ml PBS without Ca/Mg and then
incubating the cells with 0.5 ml of trypsin-EDTA for 15 min. The
trypsinization was stopped by adding 0.5 ml of medium and 0.1 ml
aliquot from each well was transferred into dilu-vials containing
10 ml isoton and the number of cells were counted on Coulter
Counter.TM..
[0215] The anti-cell proliferation activities (expressed as
IC.sub.50, the concentration causing 50% reduction in MCF-7 cell
growth in culture) of some compounds of the invention and
1,25-dihydroxy-cholecalciferol as a comparator, were:
3 CPD # see Table 1 IC.sub.50 (nm) 1,25-di(OH)-cholecalciferol 149
1 0.50 2 0.30 3 0.03 4 0.05 5 0.03 6 0.03
[0216] The results of the above test show that compounds of this
invention are more potent than 1,25-dihydroxy-cholecalciferol in
inhibition of MCF-7 breast cells growth in culture.
Example 13
Cell Proliferation Assay in ZR-75 Breast Cancer Cells
[0217] ZR-75 cells are human mammary carcinoma cells that are
positive for estrogen receptors. The potential activity of vitamin
D.sub.3 analogs against breast cancer was assessed from inhibition
of proliferation of ZR-75 cells in culture.
[0218] ZR-75 cells were plated at 12,500 cells/well in 24-well
plates and incubated at 37.degree. C. in 5% CO.sub.2/95% air in
RPMI medium containing 10% fetal bovine serum and 2 mM L-glutamine.
Stock solutions of vitamin D.sub.3 analogs were prepared at a
concentration of 10 mM in absolute ethanol and stored at
-20.degree. C. under argon. One day after plating, ZR-75 cells were
refed with either control medium or medium containing varying
concentrations of the vitamin D.sub.3 analog. After a further 10
days of culture, the number of ZR-75 cells in each well was
assessed from the reduction of the dye MTT
(3-(4,5-dimethylthiazol-2-yl)-- 2,5 diphenyltetrazolium bromide),
as described by F. Denizot and R. Lang, J. Immunological Methods,
Vol. 89:271-277 (1986). MTT was added to each well to a final
concentration of 1 mg/ml and the cells were incubated for a period
of three hours, after which reduced MTT was extracted using 95%
ethanol and the optical density was measured at a wavelength of 570
nm.
[0219] For each vitamin D.sub.3 analog, the IC.sub.50 value was
determined from a graph relating the optical density of 570 nm to
the concentration used.
[0220] The anti-cell proliferation activities (expressed as
IC.sub.50, the concentration of the vitamin D.sub.3 analog
corresponding to half-maximal reduction in 570 nm absorbance) of
some compounds of the invention and 1,25-dihydroxy-cholecalciferol
as a comparator, were:
4 CPD # see Table 1 IC.sub.50 (nm) 1,25-di(OH)-cholecalciferol 13 2
0.90 3 0.01 4 0.15 5 0.10 6 0.10
[0221] The results of the above test show that compounds of this
invention are more potent than 1,25-dihydroxy-cholecalciferol in
inhibition of ZR-75 breast cells growth in culture.
Example 14
Effect of Vitamin D.sub.3 Analogs on Secondary Hyperparathyroidism
in the Rat Renal Insufficiency Model
[0222] The parathyroid hormone suppressive activity of the vitamin
D.sub.3 analogs of this invention was demonstrated in rats with
secondary hyperparathyroidism due to renal failure using the 7/8
nephrectomy induced rat model of renal failure (Kidney
International, M. Fukugawa et al., 39:874-881 (1991).
[0223] Test Materials:
[0224] Compound of Formula (I)
[0225] 1,25 (OH).sub.2 vitamin D.sub.3 (control)
[0226] Vehicle--Miglyol 812
[0227] Female Sprague Dawley rats were anesthetized, their right
kidney removed and 2-3 branches of the left renal artery were
ligated to achieve 7/8 nephrectomy. They were placed on a high
phosphorous diet (0.6% Ca and 0.8 phosphorous). Approximately 3-6
weeks after surgery, rats were bled to screen serum PTH levels and
rats with PTH levels between 100-500 pg/ml were selected for the
study.
[0228] There was a pre-bleed (T=0) and each group was dosed daily
for seven days by oral lavage with either the compound of Formula
(I) (0.1 .mu.g/kg/day), vehicle control or 1, 25-(OH).sub.2 vitamin
D.sub.3 positive control. Compounds were predissolved in ethanol
and diluted with vehicle (Miglyol 812) followed by evaporation of
the ethanol.
[0229] After the last day of dosing, the animals were bled again
(T=1) and sacrificed. Serum PTH assays were done with Nichols
Institute Diagnostic Kit #40-2240. Serum calcium assays were done
with Sigma Diagnostic Kit #587 with o-cresophthalein. Serum
creatinine assays were done with Sigma Diagnostic Kit #1600-320
with ammonium molybdate.
5 CPD # PTH pg/ml Final Ca Levels see Table 1 T = 1 - T = 0 (mg/ml)
Vehicle 145 9.04 1,25(OH).sub.2 vit. D.sub.3 (0.1 .mu.g/kg) -18
9.75 3 (0.1 .mu.g/kg) -70 10.65 4 (0.1 .mu.g/kg) -137 10.08
[0230] The results show that the compounds of Formula (I) are more
effective than 1,25 (OH).sub.2 vit. D.sub.3 in suppressing the
elevated levels of parathyroid hormone.
Example 15
Oral Dosage Form Soft Gelatin Capsule
[0231] A capsule for oral administration is formulated under
nitrogen in amber light from 0.01 to 25.0 mg of one of the
compounds of the present invention in 150 mg of fractionated
coconut oil, with 0.015 mg butylated hydroxytoluene (BHT) and 0.015
mg butylated hydroxyanisole (BHA), filled in a soft gelatin
capsule.
[0232] The foregoing invention has been described in some detail by
way of illustration and example, for the purposes of clarity and
understanding. It will be obvious to one of ordinary skill in the
art that changes and modifications may be practiced within the
scope of the appended claims. Therefore, it is to be understood
that the above description is intended to be illustrative and not
restrictive. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the following appended
claims, along with the full scope of equivalents to which such
claims are entitled.
[0233] The patents, patent applications and publications cited in
this application are hereby incorporated by reference in their
entirety for all purposes to the same extent as if each individual
patent, patent application or publication were so individually
denoted.
* * * * *