U.S. patent application number 10/412997 was filed with the patent office on 2004-05-13 for benzopyranone compounds, compositions thereof, and methods of treatment therewith.
Invention is credited to Bhagwat, Shripad S., McKie, Jeffrey A., Missbach, Martin, Renaud, Johanne.
Application Number | 20040092572 10/412997 |
Document ID | / |
Family ID | 27804483 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040092572 |
Kind Code |
A1 |
Renaud, Johanne ; et
al. |
May 13, 2004 |
Benzopyranone compounds, compositions thereof, and methods of
treatment therewith
Abstract
Benzopyranone compounds having the following structure: 1
wherein R.sub.1, X, Y and n are as defined here, are disclosed. The
compounds of formula (I), wherein R.sub.1 is H, can be prepared by
demethylation of the corresponding phenolic methyl ether. The
compounds are useful for treating a bone-resorbing disease, cancer,
arthritis or an estrogen-related condition such as breast cancer,
osteoporosis, endometriosis, cardiovascular disease,
hypercholesterolemia, prostatic hypertrophy, prostatic carcinomas,
obesity, hot flashes, skin effects, mood swings, memory loss, and
adverse reproductive effects associated with exposure to
environmental chemicals or natural hormonal imbalances.
Inventors: |
Renaud, Johanne; (Basel,
CH) ; Missbach, Martin; (Basel, CH) ; McKie,
Jeffrey A.; (San Diego, CA) ; Bhagwat, Shripad
S.; (San Diego, CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST STREET
NEW YORK
NY
10017
US
|
Family ID: |
27804483 |
Appl. No.: |
10/412997 |
Filed: |
April 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10412997 |
Apr 14, 2003 |
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10125965 |
Apr 19, 2002 |
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6620838 |
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Current U.S.
Class: |
514/422 ;
514/320 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
19/02 20180101; A61P 35/04 20180101; A61P 25/00 20180101; C07D
311/16 20130101; A61P 5/18 20180101; A61P 27/12 20180101; A61P
35/00 20180101; A61P 13/00 20180101; A61P 19/10 20180101; A61P
17/10 20180101; A61P 25/28 20180101; A61P 19/08 20180101; A61P 9/00
20180101 |
Class at
Publication: |
514/422 ;
514/320 |
International
Class: |
A61K 031/4025; A61K
031/454 |
Claims
What is claimed is:
1. A method for modulating gene expression in a cell expressing ER,
comprising contacting the cell with an effective amount of a
compound having the structure: 41or a pharmaceutically acceptable
salt thereof, wherein: n is 2, 3 or 4; R.sub.1 is hydrogen,
C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2,
C(.dbd.O)NR.sub.2R.sub.3, or S(.dbd.O.sub.2)NR.sub.2R.sub.3;
R.sub.2 and R.sub.3 are independently C.sub.1-8alkyl,
C.sub.6-12aryl, C.sub.7-12arylalkyl, or a five- or six-membered
heterocycle containing up to two heteroatoms selected from O,
NR.sub.4 and S(O).sub.q, wherein each of the above groups are
optionally substituted with one to three substituents independently
selected from R.sub.5 and q is 0, 1 or 2; R.sub.4 is hydrogen or
C.sub.1-4 alkyl; R.sub.5 is hydrogen, halogen, hydroxy,
C.sub.1-6alkyl, C.sub.1-4alkoxy, C.sub.1-4acyloxy, C.sub.1-4thio,
C.sub.1-4alkylsulfinyl, C.sub.1-4alkylsulfonyl,
(hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl, C.sub.7-12aralkyl, COOH,
CN, CONHOR.sub.6, SO.sub.2NHR.sub.6, NH.sub.2, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, NHSO.sub.2R.sub.6, NO.sub.2, or a five- or
six-membered heterocycle, where each occurrence of R.sub.6 is
independently C.sub.1-6alkyl; X is hydrogen, halogen or
trifluoromethyl; and Y is halogen or trifluoromethyl.
2. The method of claim 1 wherein ER is ER-.alpha. or ER-.beta..
3. The method of claim 1 wherein the cell preferentially expresses
ER-.beta. over ER-.alpha..
4. The method of claim 1 wherein the cell is of bone, bladder,
uterus, ovary, prostate, testis, epididymis, gastrointestinal
tract, kidney, breast, eye, heart, vessel wall, immune system,
lung, pituitary, hippocampus or hypothalamus.
5. A method of modulating ER in tissue expressing ER, comprising
contacting the tissue with an effective amount of a compound having
the structure: 42or a pharmaceutically acceptable salt thereof,
wherein: n is 2, 3 or 4; R.sub.1 is hydrogen, C(.dbd.O)R.sub.2,
C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R- .sub.3; R.sub.2 and R.sub.3 are
independently C.sub.1-8alkyl, C.sub.6-12aryl, C.sub.7-12arylalkyl,
or a five- or six-membered heterocycle containing up to two
heteroatoms selected from O, NR.sub.4 and S(O).sub.q, wherein each
of the above groups are optionally substituted with one to three
substituents independently selected from R.sub.5 and q is 0, 1 or
2; R.sub.4 is hydrogen or C.sub.1-4 alkyl; R.sub.5 is hydrogen,
halogen, hydroxy, C.sub.1-6alkyl, C.sub.1-4alkoxy,
C.sub.1-4acyloxy, C.sub.1-4thio, C.sub.1-4alkylsulfinyl,
C.sub.1-4alkylsulfonyl, (hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl,
C.sub.7-12aralkyl, COOH, CN, CONHOR.sub.6, SO.sub.2NHR.sub.6,
NH.sub.2, C.sub.1-4alkylamino, C.sub.1-4dialkylamino,
NHSO.sub.2R.sub.6, NO.sub.2, or a five- or six-membered
heterocycle, where each occurrence of R.sub.6 is independently
C.sub.1-6alkyl; X is hydrogen, halogen or trifluoromethyl; and Y is
halogen or trifluoromethyl.
6. The method of claim 5 wherein ER is ER-.alpha. or ER-.beta..
7. The method of claim 5 wherein the tissue preferentially
expresses ER-.beta. over ER-.alpha..
8. The method of claim 5 wherein the tissue is of bone, bladder,
uterus, ovary, prostate, testis, epididymis, gastrointestinal
tract, kidney, breast, eye, heart, vessel wall, immune system,
lung, pituitary, hippocampus or hypothalamus.
9. A method for obtaining a compound having the structure: 43or a
pharmaceutically acceptable salt thereof, wherein: n is 2, 3 or 4;
R.sub.1 is hydrogen; X is hydrogen, halogen or trifluoromethyl; and
Y is halogen or trifluoromethyl; comprising the step of
demethylating of a compound having the structure: 44or a
pharmaceutically acceptable salt thereof, wherein: n is 2, 3 or 4;
X is hydrogen, halogen or trifluoromethyl; and Y is halogen or
trifluoromethyl.
10. A method for activating the function of ER in a bone cell,
comprising contacting a bone cell with an effective amount of a
compound having the structure: 45or a pharmaceutically acceptable
salt thereof, wherein: n is 2, 3 or 4; R.sub.1 is hydrogen,
C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2,
C(.dbd.O)NR.sub.2R.sub.3, or S(.dbd.O.sub.2)NR.sub.2R- .sub.3;
R.sub.2 and R.sub.3 are independently C.sub.1-8alkyl,
C.sub.6-12aryl, C.sub.7-12arylalkyl, or a five- or six-membered
heterocycle containing up to two heteroatoms selected from O,
NR.sub.4 and S(O).sub.q, wherein each of the above groups are
optionally substituted with one to three substituents independently
selected from R.sub.5 and q is 0, 1 or 2; R.sub.4 is hydrogen or
C.sub.1-4 alkyl; R.sub.5 is hydrogen, halogen, hydroxy,
C.sub.1-6alkyl, C.sub.1-4alkoxy, C.sub.1-4acyloxy, C.sub.1-4thio,
C.sub.1-4alkylsulfinyl, C.sub.1-4alkylsulfonyl,
(hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl, C.sub.7-12aralkyl, COOH,
CN, CONHOR.sub.6, SO.sub.2NHR.sub.6, NH.sub.2, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, NHSO.sub.2R.sub.6, NO.sub.2, or a five- or
six-membered heterocycle, where each occurrence of R.sub.6 is
independently C.sub.1-6alkyl; X is hydrogen, halogen or
trifluoromethyl; and Y is halogen or trifluoromethyl.
11. The method of claim 10 wherein the cell is an osteosarcoma
cell.
12. A method for inhibiting the function of ER in a breast cancer
cell, ovary cancer cell, endometrial cancer cell, uterine cancer
cell, prostate cancer cell or hypothalamus cancer cell comprising
contacting said cell with an effective amount of a compound having
the structure: 46or a pharmaceutically acceptable salt thereof,
wherein: n is 2, 3 or 4; R.sub.1 is hydrogen, C(.dbd.O)R.sub.2,
C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R- .sub.3; R.sub.2 and R.sub.3 are
independently C.sub.1-8alkyl, C.sub.6-12aryl, C.sub.7-12arylalkyl,
or a five- or six-membered heterocycle containing up to two
heteroatoms selected from O, NR.sub.4 and S(O).sub.q, wherein each
of the above groups are optionally substituted with one to three
substituents independently selected from R.sub.5 and q is 0, 1 or
2; R.sub.4 is hydrogen or C.sub.1-4 alkyl; R.sub.5 is hydrogen,
halogen, hydroxy, C.sub.1-6alkyl, C.sub.1-4alkoxy,
C.sub.1-4acyloxy, C.sub.1-4thio, C.sub.1-4alkylsulfinyl,
C.sub.1-4alkylsulfonyl, (hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl,
C.sub.7-12aralkyl, COOH, CN, CONHOR.sub.6, SO.sub.2NHR.sub.6,
NH.sub.2, C.sub.1-4alkylamino, C.sub.1-4dialkylamino,
NHSO.sub.2R.sub.6, NO.sub.2, or a five- or six-membered
heterocycle, where each occurrence of R.sub.6 is independently
C.sub.1-6alkyl; X is hydrogen, halogen or trifluoromethyl; and Y is
halogen or trifluoromethyl.
13. A method for inhibiting the expression of IL-6, comprising
contacting a cell capable of expressing ER and IL-6 with an
effective amount of a compound having the structure: 47or a
pharmaceutically acceptable salt thereof, wherein: n is 2, 3 or 4;
R.sub.1 is hydrogen, C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2,
C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R.sub.3; R.sub.2 and R.sub.3 are
independently C.sub.1-8alkyl, C.sub.6-12aryl, C.sub.7-12arylalkyl,
or a five- or six-membered heterocycle containing up to two
heteroatoms selected from O, NR.sub.4 and S(O).sub.q, wherein each
of the above groups are optionally substituted with one to three
substituents independently selected from R.sub.5 and q is 0, 1 or
2; R.sub.4 is hydrogen or C.sub.4 alkyl; R.sub.5 is hydrogen,
halogen, hydroxy, C.sub.1-6alkyl, C.sub.1-4alkoxy,
C.sub.1-4acyloxy, C.sub.1-4thio, C.sub.1-4alkylsulfinyl,
C.sub.1-4alkylsulfonyl, (hydroxy)C.sub.1-4alkyl, C6-12aryl,
C.sub.7-12aralkyl, COOH, CN, CONHOR.sub.6, SO.sub.2NHR.sub.6,
NH.sub.2, C.sub.1-4alkylamino, C.sub.1-4dialkylamino,
NHSO.sub.2R.sub.6, NO.sub.2, or a five- or six-membered
heterocycle, where each occurrence of R.sub.6 is independently
C.sub.1-6alkyl; X is hydrogen, halogen or trifluoromethyl; and Y is
halogen or trifluoromethyl.
14. The method of claim 13 wherein the cell is a bone cell.
15. A method for inhibiting the growth of a cancer or neoplastic
cell comprising contacting a cancer or neoplastic cell capable of
expressing ER with an effective amount of a compound having the
structure: 48or a pharmaceutically acceptable salt thereof,
wherein: n is 2, 3 or 4; R.sub.1 is hydrogen, C(.dbd.O)R.sub.2,
C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R- .sub.3; R.sub.2 and R.sub.3 are
independently C.sub.1-8alkyl, C.sub.6-12aryl, C.sub.7-12arylalkyl,
or a five- or six-membered heterocycle containing up to two
heteroatoms selected from O, NR.sub.4 and S(O).sub.q, wherein each
of the above groups are optionally substituted with one to three
substituents independently selected from R.sub.5 and q is 0, 1 or
2; R.sub.4 is hydrogen or C.sub.1-4 alkyl; R.sub.5 is hydrogen,
halogen, hydroxy, C.sub.1-6alkyl, C.sub.1-4alkoxy,
C.sub.1-4acyloxy, C.sub.1-4thio, C.sub.1-4alkylsulfinyl,
C.sub.1-4alkylsulfonyl, (hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl,
C.sub.7-12aralkyl, COOH, CN, CONHOR.sub.6, SO.sub.2NHR.sub.6,
NH.sub.2, C.sub.1-4alkylamino, C.sub.1-4dialkylamino,
NHSO.sub.2R.sub.6, NO.sub.2, or a five- or six-membered
heterocycle, where each occurrence of R.sub.6 is independently
C.sub.1-6alkyl; X is hydrogen, halogen or trifluoromethyl; and Y is
halogen or trifluoromethyl.
16. A method for reducing a patient's serum level comprising
administering to a patient in need thereof an effective amount of a
compound having the structure: 49or a pharmaceutically acceptable
salt thereof, wherein: n is 2, 3 or 4; R.sub.1 is hydrogen,
C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2,
C(.dbd.O)NR.sub.2R.sub.3, or S(.dbd.O.sub.2)NR.sub.2R- .sub.3;
R.sub.2 and R.sub.3 are independently C.sub.1-8alkyl,
C.sub.6-12aryl, C.sub.7-12arylalkyl, or a five- or six-membered
heterocycle containing up to two heteroatoms selected from O,
NR.sub.4 and S(O).sub.q, wherein each of the above groups are
optionally substituted with one to three substituents independently
selected from R.sub.5 and q is 0, 1 or 2; R.sub.4 is hydrogen or
C.sub.1-4alkyl; R.sub.5 is hydrogen, halogen, hydroxy,
C.sub.1-6alkyl, C.sub.1-4alkoxy, C.sub.1-4acyloxy, C.sub.1-4thio,
C.sub.1-4alkylsulfinyl, C.sub.1-4alkylsulfonyl,
(hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl, C.sub.7-12aralkyl, COOH,
CN, CONHOR.sub.6, SO.sub.2NHR.sub.6, NH.sub.2, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, NHSO.sub.2R.sub.6, NO.sub.2, or a five- or
six-membered heterocycle, where each occurrence of R.sub.6 is
independently C.sub.1-6alkyl; X is hydrogen, halogen or
trifluoromethyl; and Y is halogen or trifluoromethyl.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/125,965 filed Apr. 19, 2002 which is
incorporated by reference herein in its entirety.
1. FIELD OF THE INVENTION
[0002] This invention is generally directed to benzopyranone
compounds, compositions comprising the benzopyranone compounds and
methods for treating a bone-resorbing disease, cancer, arthritis or
an estrogen-related condition, comprising administering an
effective amount of a benzopyranone compound to a patient in need
thereof.
2. BACKGROUND OF THE INVENTION
[0003] The estrogen hormone has a broad spectrum of effects on
tissues in both females and males. Many of these biological effects
are positive, including maintenance of bone density, cardiovascular
protection, central nervous system (CNS) function, and the
protection of organ systems from the effects of aging. However, in
addition to its positive effects, estrogen also is a potent growth
factor in the breast and endometrium that increases the risk of
cancer.
[0004] Until recently, it was assumed that estrogen binds to a
single estrogen receptor (ER) in cells. As discussed below, this
simple view changed significantly when a second ER (ER-.beta.) was
cloned (with the original ER being renamed ER-.alpha.), and when
co-factors that modulate the ER response were discovered. Ligands
can bind to two different ERs which, in the presence of
tissue-specific co-activators and/or co-repressors, bind to an
estrogen response element in the regulatory region of genes or to
other transcription factors. Given the complexity of ER signaling,
along with the tissue-specific expression of ER-.alpha. and
ER-.beta. and its co-factors, it is now recognized that ER ligands
can act as estrogen agonists and antagonists that mimic the
positive effects, or block the negative effects, of estrogen in a
tissue-specific manner. This has given rise to the discovery of an
entirely new class of drugs, referred to as Selective Estrogen
Receptor Modulators or SERMs. These drugs have significant
potential for the prevention and/or treatment of cancer and
osteoporosis, as well as cardiovascular diseases and
neurodegenerative diseases such as Alzheimer's disease.
[0005] Bone-resorbing diseases, such as osteoporosis, are
debilitating conditions which affect a wide population, and to
which there is only limited treatment. For example, osteoporosis
affects about 50% of women, and about 10% of men, over the age of
50 in the United States. In individuals with osteoporosis,
increased loss of bone mass results in fragile bones and, as a
result, increased risk of bone fractures. Other bone-resorption
diseases, such as Paget's disease and metastatic bone cancer,
present similar symptoms.
[0006] Bone is a living tissue which contains several different
types of cells. In healthy individuals, the amount of bone made by
the osteoblastic cells is balanced by the amount of bone removed or
resorbed by the osteoclastic cells. In individuals suffering from a
bone-resorbing disease, there is an imbalance in the function of
these two types of cells. Perhaps the most well known example of
such an imbalance is the rapid increase in bone resorption
experienced by postmenopausal women. Such accelerated bone lose is
attributed to estrogen deficiency associated with menopause.
However, the mechanism of how the loss of estrogen results in
increased bone resorption has long been debated.
[0007] Recently, investigators have suggested that an increase in
bone-resorbing cytokines, such as interleukin-1 (IL-1) and tumor
necrosis factor (TNF), may be responsible for postmenopausal bone
loss (Kimble et al., J. Biol. Chem. 271:28890-28897, 1996), and
that inhibitors of these cytokines can partially diminish bone loss
following ovariectomy in rodents (Pacifici, J. Bone Miner Res. 11:
1043-1051, 1996). Further, discontinuation of estrogen has been
reported to lead to an increase in IL-6 secretion by murine bone
marrow and bone cells (Girasole et al., J. Clin. Invest.
89:883-891, 1992; Jilka et al., Science 257:88-91, 1992; Kimble et
al., Endocrinology 136:3054-3061, 1995; Passseri et al.,
Endocrinology 133:822-828, 1993), antibodies against IL-6 can
inhibit the increase in osteoclast precursors occurring in
estrogen-depleted mice (Girasole et al, supra), and bone loss
following ovariectomy does not occur in transgenic mice lacking
IL-6 (Poli et al., EMBO J. 13:1189-1196, 1994).
[0008] Existing treatments for slowing bone loss generally involves
administration of compounds such as estrogen, bisphosphonates,
calcitonin, and raloxifene. These compounds, however, are generally
used for long-term treatments, and have undesirable side effects.
Further, such treatments are typically directed to the activity of
mature osteoclasts, rather than reducing their formation. For
example, estrogen induces the apoptosis of osteoclasts, while
calcitonin causes the osteoclasts to shrink and detach from the
surface of the bone (Hughes et al., Nat. Med. 2:1132-1136, 1996;
Jilka et al., Exp. Hematol. 23:500-506, 1995). Similarly,
bisphosphonates decrease osteoclast activity, change their
morphology, and increase the apoptosis of osteoclasts (Parfitt et
al., J. Bone Miner Res. 11:150-159, 1996; Suzuki et al.,
Endocrinology 137:4685-4690, 1996).
[0009] Cytokines are also believed to play an important role in a
variety of cancers. For example, in the context of prostate cancer,
researchers have shown iL-6 to be an autocrine/paracrine growth
factor (Seigall et al., Cancer Res. 50:7786, 1999), to enhance
survival of tumors (Okamoto et al., Cancer Res. 57:141-146, 1997),
and that neutralizing IL-6 antibodies reduce cell proliferation
(Okamoto et al., Endocrinology 138:5071-5073, 1997; Borsellino et
al., Proc. Annu. Meet. Am. Assoc. Cancer Res. 37:A2801, 1996).
Similar results have been reported for IL-6 with regard to multiple
myeloma (Martinez-Maza et al., Res. Immunol. 143:764-769, 1992;
Kawano et al., Blood 73:517-526, 1989; Zhang et al., Blood
74:11-13, 1989; Garrett et al., Bone 20:515-520, 1997; and Klein et
al., Blood 78:1198-12-4, 1991), renal cell carcinoma (Koo et al.,
Cancer Immunol. 35:97-105, 1992; Tsukamoto et al., J. Urol.
148:1778-1782, 1992; and Weissglas et al., Endocrinology
138:1879-1885, 1997), and cervical carcinoma (Estuce et al.,
Gynecol. Oncol. 50:15-19, 1993; Tartour et al., Cancer Res.
54:6243-6248, 1994; and Iglesias et al., Am. J. Pathology
146:944-952, 1995).
[0010] Furthermore, IL-6 is also believed to be involved in
arthritis, particularly in adjuvant-, collagen- and antigen-induced
arthritis (Alonzi et al., J. Exp. Med. 187:146-148, 1998; Ohshima
et al., Proc. Natl. Acad. Sci. USA 95:8222-8226, 1998; and Leisten
et al., Clin. Immunol. Immunopathol 56:108-115, 1990), and
anti-IL-6 antibodies have been reported for treatment of arthritis
(Wendling et al., J. Rheumatol. 20:259-262, 1993). In addition,
estrogen has been shown to induce suppression of experimental
autoimmune encephalomyelitis and collagen-induced arthritis in mice
(Jansson et al., Neuroimmunol. 53:203-207, 1994).
[0011] The cytokine IL-6 has also been shown to be an important
factor in inducing the formation of osteoclasts (Girasole et al.,
supra; Jilka et al. (1992), supra; Jilka et al. (1995), supra;
Kimble et al. (1995), supra; Pacifici et al., supra; and Passeri et
al., supra). Other investigators have shown that administration of
the neutralizing antibody, antisense oligos, or the Sant 5
antagonist against IL-6, reduces the number of osteoclasts in
trabecular bone of ovariectomized mice (Devlin et al., J. Bone
Miner 13:393-399, 1998; Girasole et al., supra; Jilka et al.
(1992), supra; and Schiller et al., Endocrinology 138:4567-4571,
1997), the ability of human giant cells to resorb dentine (Ohsaki
et al., Endocrinology 131:2229-2234, 1993; and Reddy et al., J.
Bone Min. Res. 9:753-757, 1994), and the formation of osteoclasts
in normal human bone marrow culture. It has also been found that
estrogen downregulates the IL-6 promoter activity by interactions
between the estrogen receptor and the transcription factors
NF-.kappa.B and C/EBP.beta. (Stein et al., Mol. Cell Biol.
15:4971-4979, 1995).
[0012] Granulocyte-macrophage colony-stimulating factor (GM-CSF)
has been suggested to play a role in the proliferation of
osteoclastic precursor cells. In long term cultures of human or
mouse bone marrow cells or peripheral blood cells, GM-CSF promotes
the formation of osteoclastic cells (Kurihara et al., Blood
74:1295-1302, 1989; Lorenzo et al., J. Clin. Invest. 80:160-164,
1987; MacDonald et al., J. Bone Miner 1:227-233, 1986; and Shinar
et al, Endocrinology 126:1728-1735, 1990). Bone marrow cells
isolated from postmenopausal women, or women who discontinued
estrogen therapy, expressed higher levels of GM-CSF than cells from
premenopausal women (Bismar et al., J. Clin. Endocrinol. Metab.
80:3351-3355, 1995). Expression of GM-CSF has also been shown to be
associated with the tissue distribution of bone-resorbing
osteoclasts in patients with erosion of orthopedic implants
(Al-Saffar et al., Anatomic Pathology 105:628-693, 1996).
[0013] As noted above, it had previously been assumed that estrogen
binds to a single estrogen receptor (ER) in cells, causing
conformational changes that result in release from heat shock
proteins and binding of the receptor as a dimer to the so-called
estrogen response element in the promoter region of a variety of
genes. Further, pharmacologists have generally believed that
non-steroidal small molecule ligands compete for binding of
estrogen to ER, acting as either antagonists or agonists in each
tissue where the estrogen receptor is expressed. Thus, such ligands
have traditionally been classified as either pure antagonists or
agonists. This is no longer believed to be correct.
[0014] Rather, it is now known that estrogen modulates cellular
pharmacology through gene expression, and that the estrogen effect
is mediated by estrogen receptors. As noted above, there are
currently two estrogen receptors, ER-.alpha. and ER-.beta.. The
effect of estrogen receptor on gene regulation can be mediated by a
direct binding of ER to the estrogen response element
(ERE)--"classical pathway" (Jeltsch et al., Nucleic Acids Res.
15:1401-1414, 1987; Bodine et al., Endocrinology 139:2048-2057,
1998), binding of ER to other transcription factors such as
NF-.kappa.B, C/EBP-.beta. or AP-1- "non-classical pathway" (Stein
et al., Mol. Cell Biol. 15:4971-4979, 1995; Paech et al., Science
277:1508-1510, 1997; Duan et al., Endocrinology 139:1981-1990,
1998), and through non-genomic effects via extra-nuclear estrogen
receptor signaling that potentially include plasma membrane ER
(Nadal, A. et al., Trends in Pharmacological Sciences 22:597-599,
2001; Wyckoff, M. H. et al., J. Biol. Chem. 276: 27071-27076, 2001;
Chung, Y-L. et al., Int. J. of Cancer 97:306-312, 2002; Kelly, M.
J. et al., Trends Endocrinol. Metab. 10:369-374, 1999; Levin, E. R.
et al., Trends Endocrinol. Metab. 10:374-377, 1999).
[0015] Progress over the last few years has shown that ER
associates with co-activators (e.g., SRC-I, CBP and SRA) and
co-repressors (e.g., SMRT and N-CoR), which also modulate the
transcriptional activity of ER in a tissue-specific and
ligand-specific manner. In such cases, ER interacts with the
transcription factors critical for regulation of these genes.
Transcription factors known to be modulated in their activity by ER
include, for example, AP-1, NF-.kappa.B, C/EBP and Sp-1. In
addition, orphan nuclear receptors, such as estrogen
receptor-related receptors .alpha., .beta., .gamma. (ERR-.alpha.,
ERR-.beta., ERR-.gamma.), have been identified. Although estradiol
does not appear to be a ligand for the ERRs, some SERMs and other
traditional ER-ligands have been shown to bind to the receptors
with high affinity (Coward, P. et al., Proc. Natl. Acad. Sci.
98:8880-8884, 2001; Lu, D. et al., Cancer Res. 61:6755-6761, 2001;
Tremablay, G. B. et al., Endocrinology 142:4572-4575, 2001; Chen,
S. et al., J. Biol. Chem. 276:28465-28470, 2001).
[0016] Furthermore, ER-.alpha. and ER-.beta. have both overlapping
and different tissue distributions, as analyzed predominantly by
RT-PCR or in-situ hybridization due to a lack of good ER-.beta.
antibodies. Some of these results, however, are controversial,
which may be attributable to the method used for measuring ER, the
species analyzed (rat, mouse, human) and/or the differentiation
state of isolated primary cells. Very often tissues express both
ER-.alpha. and ER-.beta., but the receptors are localized in
different cell types. In addition, some tissues (such as kidney)
contain exclusively ER-.alpha., while other tissues (such as
uterus, pituitary and epidymis) show a great predominance of ER-1
(Couse et al., Endocrinology 138, 4613-4621, 1997; Kuiper et al.,
Endocrinology 138, 863-870, 1997). In contrast, tissues expressing
high levels of ER-.beta. include prostate, testis, ovaries and
certain areas of the brain (Brandenberger et al., J. Clin.
Endocrinol. Metab. 83, 1025-8, 1998; Enmark et al., J. Clinic.
Endocrinol. Metabol. 82, 4258-4265, 1997; Laflamme et al., J.
Neurobiol. 36, 357-78, 1998; Sar and Welsch, Endocrinology 140,
963-71, 1999; Shughrue et al., Endocrinology 138, 5649-52, 1997a;
Shughrue et al., J. Comp. Neurol. 388, 507-25, 1997b).
[0017] The development of ER-.alpha. (Korach, Science 266,
1524-1527, 1994) and ER-.beta. (Krege et al., Proc. Natl. Acad.
Sci. USA 95, 15677-82, 1998) knockout mice further demonstrate that
ER-.beta. has different functions in different tissues. For
example, ER-.alpha. knockout mice (male and female) are infertile,
females do not display sexual receptivity and males do not have
typical male-aggressive behavior (Cooke et al., Biol. Reprod. 59,
470-5, 1998; Das et al., Proc. Natl. Acad. Sci. USA 94,
12786-12791, 1997; Korach, 1994; Ogawa et al., Proc. Natl. Acad.
Sci. USA 94, 1476-81, 1997; Rissman et al., Endocrinology 138,
507-10, 1997a; Rissman et al., Horm. Behav. 31, 232-243, 1997b).
Further, the brains of these animals still respond to estrogen in a
pattern that is similar to that of wild-type animals (Shughrue et
al., Proc. Natl. Acad. Sci. USA 94, 11008-12, 1997c), and estrogen
still inhibits vascular injury caused by mechanical damage (Iafrati
et al., Nature Med. 3, 545-8, 1997). In contrast, mice lacking the
ER-.beta. develop normally, are fertile and exhibit normal sexual
behavior, but have fewer and smaller litters than wild-type mice
(Krege et al., 1998), have normal breast development and lactate
normally. The reduction in fertility is believed to be the result
of reduced ovarian efficiency, and ER-.beta. is the predominant
form of ER in the ovary, being localized in the granulosa cells of
maturing follicles.
[0018] In summary, compounds which serve as estrogen antagonists or
agonists have long been recognized for their significant
pharmaceutical utility in the treatment of a wide variety of
estrogen-related conditions, including conditions related to the
brain, bone, cardiovascular system, skin, hair follicles, immune
system, bladder and prostate (Barkhem et al., Mol. Pharmacol. 54,
105-12, 1998; Farhat et al., FASEB J. 10, 615-624, 1996;
Gustafsson, Chem. Biol. 2, 508-11, 1998; Sun et al., 1999; Tremblay
et al., Endocrinology 139, 111-118, 1998; Turner et al.,
Endocrinology 139, 3712-20, 1998). In addition, a variety of breast
and non-breast cancer cells have been described to express ER, and
serve as the target tissue for specific estrogen antagonists
(Brandenberger et al., 1998; Clinton and Hua, Crit. Rev. Oncol.
Hematol. 25, 1-9, 1997; Hata et al., Oncology 55 Suppl 1, 35-44,
1998; Rohlffet al., Prostate 37, 51-9, 1998; Simpson et al., J
Steroid Biochem Mol Biol 64, 137-45,1998; Yamashita et al.,
Oncology 55 Suppl 1,17-22, 1998).
[0019] In recent years a number of both steroidal and nonsteroidal
compounds which interact with ER have been developed. For example,
Tamoxifen was originally developed as an anti-estrogen and used for
the treatment of breast cancer, but more recently has been found to
act as a partial estrogen agonist in the uterus, bone and
cardiovascular system. Raloxifene is another compound that has been
proposed as a SERM, and has been approved for treatment of
osteoporosis. 2
[0020] Analogs of Raloxifene have also been reported (Grese et al.,
J. Med. Chem. 40:146-167, 1997).
[0021] As for coumarin-based compounds, a number of structures have
been proposed, including the following: Roa et al., Synthesis
887-888, 1981; Buu-Hoi et al., J. Org. Chem. 19:1548-1552, 1954;
Gupta et al., Indian J. Exp. Biol. 23:638-640, 1985; Published PCT
Application No. WO 96/31206; Verma et al., Indian J. Chem.
32B:239-243, 1993; Lednicer et al., J. Med. Chem. 8:725-726, 1965;
Micheli et al., Steroids 5:321-335, 1962; Brandt et al., Int. J.
Quantum Chemistry: Quantum Biol. Symposia 13:155-165, 1986; Wani et
al., J. Med. Chem. 18:982-985, 1975; Pollard et al., Steroids
11:897-907, 1968.
[0022] Accordingly, there is a need in the art for compounds useful
for treating a bone-resorbing disease, cancer, arthritis or an
estrogen-related condition.
[0023] Citation or identification of any reference in Section 2 of
this application is not to be construed as an admission that the
reference is prior art to the present application.
3. SUMMARY OF THE INVENTION
[0024] The invention relates to compounds having the following
general structure (I): 3
[0025] and pharmaceutically acceptable salts thereof, wherein:
[0026] n is 2, 3 or 4;
[0027] R.sub.1 is hydrogen, C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2,
C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R- .sub.3;
[0028] R.sub.2 and R.sub.3 are independently C.sub.1-8alkyl,
C.sub.6-12aryl, C.sub.7-12arylalkyl, or a five- or six-membered
heterocycle containing up to two heteroatoms selected from O,
NR.sub.4 and S(O).sub.q, wherein each of the above groups are
optionally substituted with one to three substituents independently
selected from R.sub.5 and q is 0, 1 or 2;
[0029] R.sub.4 is hydrogen or C.sub.1-4 alkyl;
[0030] R.sub.5 is hydrogen, halogen, hydroxy, C.sub.1-6alkyl,
C.sub.1-4alkoxy, C.sub.1-4acyloxy, C.sub.1-4thio,
C.sub.1-4alkylsulfinyl, C.sub.1-4alkylsulfonyl,
(hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl, C.sub.7-12aralkyl, COOH,
CN, CONHOR.sub.6, SO.sub.2NHR.sub.6, NH.sub.2, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, NHSO.sub.2R.sub.6, NO.sub.2, or a five- or
six-membered heterocycle, where each occurrence of R.sub.6 is
independently C.sub.1-6alkyl;
[0031] X is hydrogen, halogen or trifluoromethyl; and
[0032] Y is halogen or trifluoromethyl.
[0033] The invention also relates to a method of obtaining a
compound of formula (I), wherein R.sub.1 is H, by demethylation of
a compound of formula (II).
[0034] The invention further relates to a method for inhibiting a
cytokine in a patient, comprising administering to a patient in
need thereof an effective amount of a compound of formula (I), (II)
or a pharmaceutically acceptable salt of the compound.
[0035] The invention further relates to a method for treating or
preventing a bone-resorbing disease in a patient, comprising
administering to a patient in need thereof an effective amount of a
compound of formula (I), (II) or a pharmaceutically acceptable salt
of the compound.
[0036] The invention further relates to a method for treating or
preventing cancer in a patient, comprising administering to a
patient in need thereof an effective amount of a compound of
formula (I), (II) or a pharmaceutically acceptable salt of the
compound.
[0037] The invention further relates to a method for treating or
preventing arthritis in a patient, comprising administering to a
patient in need thereof an effective amount of a compound of
formula (I), (II) or a pharmaceutically acceptable salt of the
compound.
[0038] The invention further relates to a method for modulating
gene expression in a cell expressing ER, comprising contacting the
cell with an effective amount of a compound of formula (I), (II) or
a pharmaceutically acceptable salt of the compound.
[0039] The invention further relates to a method for modulating
gene expression in a tissue expressing ER, comprising contacting
the cell with an effective amount of a compound of formula (I),
(II) or a pharmaceutically acceptable salt of the compound.
[0040] The invention further relates to a method for activating the
function of ER in a bone cell, comprising contacting bone a cell
with an effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound.
[0041] The invention further relates to a method for inhibiting the
function of ER in a breast cancer cell, an ovarian cancer cell, an
endometrial cancer cell, a uterine cancer cell, a prostate cancer
cell or a hypothalamus cancer cell, comprising contacting the cell
with an effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound.
[0042] The invention further relates to a method for inhibiting the
expression of IL-6 in a cell, comprising contacting a cell capable
of expressing ER and IL-6 with an effective amount of a compound of
formula (I), (II) or a pharmaceutically acceptable salt of the
compound.
[0043] The invention further relates to methods for inhibiting
proliferation of a cancer or neoplastic cell, comprising contacting
a cancer or neoplastic cell capable of expressing ER with an
effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound.
[0044] The methods of the invention further comprise the
administration of an effective amount of another therapeutic agent.
Examples of other therapeutic agents include, but are not limited
to, an agent useful for the treatment or prevention of an
estrogen-related condition, an agent useful for the treatment or
prevention of a bone-loss disease, an agent useful for the
reduction of a patient's serum cholesterol level and an agent
useful for the treatment or prevention of cancer or a neoplastic
disease.
[0045] The present invention may be understood more fully by
reference to the detailed description and examples, which are
intended to exemplify non-limiting embodiments of the
invention.
4. DETAILED DESCRIPTION OF THE INVENTION
[0046] The invention relates to compounds of formula (I): 4
[0047] and pharmaceutically acceptable salts thereof,
[0048] wherein:
[0049] n is 2, 3 or 4;
[0050] R.sub.1 is hydrogen, C(.dbd.O)R.sub.2, C(.dbd.O)OR.sub.2,
C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R- .sub.3;
[0051] R.sub.2 and R.sub.3 are independently C.sub.1-8alkyl,
C.sub.6-12aryl, C.sub.7-12arylalkyl, or a five- or six-membered
heterocycle containing up to two heteroatoms selected from O,
NR.sub.4 and S(O).sub.q, wherein each of the above groups are
optionally substituted with one to three substituents independently
selected from R.sub.5 and q is 0, 1 or 2;
[0052] R.sub.4 is hydrogen or C.sub.1-4 alkyl;
[0053] R.sub.5 is hydrogen, halogen, hydroxy, C.sub.1-6alkyl,
C.sub.1-4alkoxy, C.sub.1-4acyloxy, C.sub.1-4thio,
C.sub.1-4alkylsulfinyl, C.sub.1-4alkylsulfonyl,
(hydroxy)C.sub.1-4alkyl, C.sub.6-12aryl, C.sub.7-12aralkyl, COOH,
CN, C(.dbd.O)NHOR.sub.6, S(.dbd.O.sub.2)NHR.sub.- 6, NH.sub.2,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, NHSO.sub.2R.sub.6,
NO.sub.2, or a five- or six-membered heterocycle, where each
occurrence of R.sub.6 is independently C.sub.1-6alkyl;
[0054] X is hydrogen, halogen or trifluoromethyl; and
[0055] Y is halogen or trifluoromethyl.
[0056] In a preferred embodiment, the compounds of formula (I) are
those wherein n=2 and R.sub.1 is hydrogen.
[0057] The invention further relates to a method for obtaining
compounds of formula (I), wherein R.sub.1 is H, comprising the step
of demethylating a compound of formula (II) shown below: 5
[0058] or a pharmaceutically acceptable salt thereof, wherein n is
2, 3 or 4 and X and Y are as defined above.
[0059] The demethylation of compounds of formula (II) can be
achieved using any method known in the art useful in the
deprotection of phenolic methyl ethers. Examples of such methods
can be found in Greene, T. W., Protective Groups in Organic
Synthesis, Chapter 3, John Wiley and Sons, New York, 1981, pp.
88-92, which is incorporated herein by reference in its entirety.
Preferably, demethylation proceeds by a method comprising
contacting a compound of formula (II) with about 1.0 to about 50.0
molar equivalents of a demethylating agent such as
iodotrimethylsilane, pyridine hydrochloride, hydrobromic acid,
hydrochloric acid, hydroiodic acid, a Grignard reagent, a Lewis
acid or a strong nucleophile. More preferably, the demethylating
agent is aqueous HBR, more preferably as a mixture in acetic acid.
In a more preferred embodiment, demethylation is achieved by
heating the compound of formula (II), or a pharmaceutically
acceptable salt thereof, in the presence of the demethylating
agent, optionally in the presence of a solvent, preferably a
carboxylic acid, at a temperature of about room temperature to
about 200.degree. C., preferably at a temperature of about
100.degree. C. to about 160.degree. C. for 15 minutes to about 24
hours. In one embodiment, the demethylation reaction vessel is
sealed, for example a sealed tube, to prevent solvent evaporation,
particularly where the boiling point of the solvent is lower than
the temperature of the demethylation reaction. The acid salt of
compounds of formula (I), wherein R.sub.1 is H, can be obtained by
isolating the compound directly from the demethylation reaction
which can then be used to prepare the corresponding
pharmaceutically acceptable salt. The free base form is available
upon washing the acid salt with an appropriate base such as sodium
hydroxide and isolating the compound.
[0060] The resulting compounds of formula (I), wherein R.sub.1 is
H, that are produced by demethylation of compounds of formula (II),
are useful as cytokine inhibitors as well as for the treatment or
prevention of a bone-resorbing disease, cancer, arthritis or an
estrogen-related condition. The compounds of formula (I), wherein
R.sub.1 is H, that are produced by demethylation of compounds of
formula (II) are also useful as intermediates in the synthesis of
compounds of formula (I) wherein R.sub.1 is C(.dbd.O)R.sub.2,
C(.dbd.O)OR.sub.2, C(.dbd.O)NHR.sub.2, C(.dbd.O)NR.sub.2R.sub.3, or
S(.dbd.O.sub.2)NR.sub.2R.sub.3.
[0061] The compounds of formula (I) and pharmaceutically acceptable
salts thereof (collectively, the "benzopyranone compounds"), are
useful for treating or preventing a bone-resorbing disease, cancer,
arthritis or an estrogen-related condition. The benzopyranone
compounds are also useful for inhibiting a cytokine in a patient
and modulating gene expression in a cell and/or tissue expressing
ER. Thus, the compounds of this invention may be administered as a
therapeutic and/or prophylactic agent.
[0062] As used herein, a "C.sub.6-12aryl" is an aromatic moiety
containing from 6 to 12 carbon atoms. In one embodiment, the
C.sub.6-12aryl is selected from (but not limited to) phenyl,
tetralinyl, and napthalenyl.
[0063] A "C.sub.7-12aralkyl" is an arene containing from 7 to 12
carbon atoms, and has both aliphatic and aromatic units. In one
embodiment, the C.sub.7-12aralkyl is an aryl group bonded directly
through an alkyl group, such as (but not limited to) benzyl,
ethylbenzyl (i.e.,--(CH.sub.2).sub.2phenyl), propylbenzyl and
isobutylbenzyl.
[0064] A "C.sub.3-12heterocycle" is a compound that contains a ring
made up of more than one kind of atom, and which contains 3 to 12
carbon atoms, including (but not limited to) pyrrolidinyl,
pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl,
imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl,
isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl,
isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl,
piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,
2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl,
N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl
sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide,
thiomorpholinyl sulfone, 1,3-dioxolane and
tetrahydro-1,1-dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl,
thiiranyl, triazinyl, and triazolyl.
[0065] A "C.sub.4-16heterocyclealkyl" is a compound that contains a
C.sub.3-12heterocycle as listed above linked to a
C.sub.1-8alkyl.
[0066] A "C.sub.1-8alkyl" is a straight chain or branched carbon
chain containing from 1 to 8 carbon atoms, including (but not
limited to) methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,
and the like. Similarly, a "C.sub.1-x alkyl has the same meaning,
but wherein "x" represents the number of carbon atoms less than
eight, such as C.sub.1-6alkyl.
[0067] A "substituted" C.sub.1-x alkyl, C.sub.6-12aryl,
C.sub.7-12aralkyl, C.sub.3-12heterocycle, or
C.sub.4-16heterocyclealkyl moiety is a C.sub.1-x alkyl,
C.sub.6-12aryl, C.sub.7-12aralkyl, C.sub.3-12heterocycle, or
C.sub.4-16heterocyclealkyl moiety having at least one hydrogen atom
replaced with a substituent.
[0068] A "substituent" is a moiety selected from halogen, --OH,
--R', --OR', --COOH, --COOR', --COR', --CONH.sub.2, --NH.sub.2,
--NHR', --NR'R', --SH, --SR', --SOOR', --SOOH and --SOR', where
each occurrence of R' is independently selected from an
unsubstituted or substituted C.sub.1-8alkyl, C.sub.6-12aryl,
C.sub.7-12aralkyl, C.sub.3-12heterocycle or
C.sub.4-16heterocyclealkyl.
[0069] A "halogen" is fluorine, chlorine, bromine or iodine.
[0070] The benzopyranone compounds can have chiral centers and can
occur as racemates, racemic mixtures and as individual enantiomers
or diastereomers. All such isomeric forms are included within the
present invention, including mixtures thereof. Furthermore, some of
the crystalline forms of the benzopyranone compounds can exist as
polymorphs, which are included in the present invention. In
addition, some of the benzopyranone compounds can also form
solvates with water or other organic solvents. Such solvates are
similarly included within the scope of this invention.
[0071] An estrogen "agonist" is a compound that binds to ER and
mimics the action of estrogen in one or more tissues, while an
"antagonist" binds to ER and blocks the action of estrogen in one
or more tissues. Further, the term "estrogen-related condition"
encompasses any condition associated with elevated or depressed
levels of estrogen, a selective estrogen receptor modulator (SERM)
or ER. In this context, ER includes both ER-.alpha. and/or
ER-.beta., as well as any isoforms, mutations and proteins with
significant homology to ER.
[0072] A "patient" is an animal, including, but not limited to, an
animal such a cow, monkey, horse, sheep, pig, chicken, turkey,
quail, cat, dog, mouse, rat, rabbit, and guinea pig, and is more
preferably a mammal, and most, preferably a human.
[0073] Although not intending to be limited by the following
theory, particularly in the context of bone-resorbing diseases, it
is believed that the benzopyranone compounds function by blocking
cytokine production and/or by inhibiting formation of
osteoclasts.
[0074] The present invention also relates to pharmaceutical
compositions comprising an effective amount of a benzopyranone
compound and optionally a pharmaceutically acceptable carrier or
vehicle, wherein a pharmaceutically acceptable carrier or vehicle
can comprise an excipient, diluent, or a mixture thereof. Other
embodiments of the present invention include methods for treating
or preventing bone-resorbing diseases, including, but not limited
to, osteoporosis, metastatic bone cancer and hypercalcemia,
osteolytic lesions with orthopedic implants, Paget's disease, and
bone loss associated with hyperparathyroidism; conditions
associated with IL-6, including various cancers and arthritis;
cancer, including breast cancer, prostrate cancer, colon cancer,
endometrial cancer, multiple myeloma, renal cell carcinoma and
cervical carcinoma; and arthritis, including adjuvant-, collagen-,
bacterial- and antigen-induced arthritis, particularly rheumatoid
arthritis. These methods comprise administering an effective amount
of a benzopyranone compound to a patient in need thereof.
[0075] In addition, the benzopyranone compounds are useful for
treating or preventing a wide range of estrogen-related conditions,
including, but not limited to, breast cancer, osteoporosis,
endometriosis, cardiovascular disease, hypercholesterolemia,
prostatic hypertrophy, prostatic carcinomas, obesity, hot flashes,
skin effects, mood swings, memory loss, prostate cancer, menopausal
syndromes, hair loss (alopecia), type-II diabetes, Alzheimer's
disease, urinary incontinence, GI tract conditions,
spermatogenesis, vascular protection after injury, endometriosis,
learning and memory, CNS effects, plasma lipid levels, acne,
cataracts, hirsutism, other solid cancers (such as colon, lung,
ovarian, melanoma, CNS, and renal), multiple myeloma, lymphoma, and
adverse reproductive effects associated with exposure to
environmental chemicals or natural hormonal imbalances.
[0076] The benzopyranone compounds are also useful for oral
contraception; relief for the symptoms of menopause; prevention of
threatened or habitual abortion; relief of dysmenorrhea; relief of
dysfunctional uterine bleeding; relief of endometriosis; an aid in
ovarian development; treatment of acne; diminution of excessive
growth of body hair in women (hirsutism); the prevention or
treatment of cardiovascular disease; prevention and treatment of
atherosclerosis; prevention and treatment of osteoporosis;
treatment of benign prostatic hyperplasia and prostatic carcinoma
obesity; and suppression of postpartum lactation. The benzopyranone
compounds also have a beneficial effect on plasma lipid levels and
as such are useful in treating and preventing hypercholesterolemia.
The benzopyranone compounds are further useful in the treatment and
prevention of breast and ovarian cancer.
[0077] In another embodiment, the invention relates to a method for
inhibiting a cytokine in a patient, comprising administering to a
patient in need thereof an effective amount of a compound of
formula (I), (II) or a pharmaceutically acceptable salt of the
compound.
[0078] In a further embodiment, the invention relates to a method
for modulating gene expression in a cell expressing ER, either
ER-.alpha. or ER-.beta., comprising contacting the cell with an
effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound.
[0079] In a further embodiment, the invention relates to a method
for modulating gene expression in a tissue expressing ER, either
ER-.alpha. or ER-.beta., comprising contacting the cell with an
effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound.
[0080] In a further embodiment, the invention relates to methods
for activating the function of ER in a bone cell, comprising
contacting a bone cell with an effective amount of a compound of
formula (I), (II) or a pharmaceutically acceptable salt of the
compound. Activating the function of ER in a bone cell is useful
for treating or preventing osteoporosis.
[0081] In a further embodiment, the invention relates to methods
for inhibiting the function of ER in a breast cancer cell, an
ovarian cancer cell, an endometrial cancer cell, a uterine cancer
cell, a prostate cancer cell or a hypothalamus cancer cell,
comprising contacting the cell with an effective amount of a
compound of formula (I), (II) or a pharmaceutically acceptable salt
of the compound. Inhibiting the function of ER in a breast cancer
cell, ovarian cancer cell, endometrial cancer cell, uterine cancer
cell, prostate cancer cell or hypothalamus cancer cell is useful
for inhibiting the growth of said cell and accordingly for treating
or preventing cancer. In one embodiment, the breast cancer cell is
MCF-7. In one embodiment, the ovarian cancer cell is BG-1.
[0082] In a further embodiment, the invention relates to methods
for inhibiting the expression of IL-6 in a cell, comprising
contacting a cell capable of expressing ER and IL-6 with an
effective amount of a compound of formula (I), (II) or a
pharmaceutically acceptable salt of the compound. In one
embodiment, the cell that expresses ER and IL-6 is a bone cell. In
another embodiment, the cell the expresses ER and IL-6 is a human
U-2 OS osteosarcoma cell stably transfected with human ER-.alpha..
Inhibiting the expression of IL-6 in a cell in vivo is useful for
the treatment of a bone-loss disease or bone cancer. In one
embodiment, the bone-loss disease is osteoporosis. Inhibiting the
expression of iL-6 in a cell in vitro is useful in a biological
activity screening assay (e.g., as a standard) for the screening of
a compound that inhibits the expression of IL-6.
[0083] In a further embodiment, the invention relates to methods
for inhibiting cell proliferation of a cancer or neoplastic cell,
comprising contacting a cancer or neoplastic cell capable of
expressing ER with an effective amount of a compound of formula
(I), (II) or a pharmaceutically acceptable salt of the compound.
Examples of cancer or neoplastic cells capable of expressing ER
include, but are not limited to, breast cells, ovarian cells,
endometrial cells, uterine cells, prostate cells and hypothalamus
cells. Inhibiting the proliferation of such cancer or neoplastic
cells in vivo is useful for the treatment or prevention of cancer.
Inhibiting the proliferation of such cancer or neoplastic cells in
vitro is useful in a biological activity screening assay (e.g., as
a standard) for anti-cancer or ant-ineoplastic agents or in a
diagnostic assay.
[0084] In a further embodiment, the invention involves methods for
reducing a patient's serum cholesterol level, comprising
administering to a patient in need thereof an effective amount of a
compound of formula (I), (II) or a pharmaceutically acceptable salt
of the compound. The reduction of a patient's serum cholesterol
level is useful for treating or preventing a cardiovascular disease
or reducing the risk of cardiovascular disease.
[0085] In a further embodiment, the methods of the invention
further comprise the administration of an effective amount of
another therapeutic agent. In one embodiment, the other therapeutic
agent is administered before, after or concurrently with the
compound of formula (I), (II) or a pharmaceutically acceptable salt
of the compound. In one embodiment, the time at which the compound
of formula (I), (II) or a pharmaceutically acceptable salt of the
compound exerts its therapeutic effect on the patient overlaps with
the time at which the other therapeutic agent exerts its
therapeutic effect on the patient.
[0086] In a further embodiment, the other therapeutic agent is
useful for the treatment or prevention of an estrogen-related
condition. Other therapeutic agents that are useful for the
treatment or prevention of an estrogen-related condition include,
but are not limited to, tamoxifen, raloxifene, medroxyprogesterone,
danizol and gestrinone.
[0087] In a further embodiment, the other therapeutic agent is
useful for the treatment or prevention of a bone-loss disease
(e.g., osteoporosis). Other therapeutic agents useful for the
treatment or prevention of a bone-loss disease include, but are not
limited to, cathepsin K inhibitors (e.g., a pro-peptide of
cathepsin K), bisphosphonates (e.g., eitodronate, pamidronate,
alendronate, risedronate, zolendronate, ibandronate, clodronate or
tiludronate), parathryoid hormone ("PTH") or fragments thereof,
compounds that release endogenous PTH (e.g., a PTH releasing
hormone) and calcitonin or fragments thereof.
[0088] In a further embodiment, the other therapeutic agent is
useful for the reduction of a patient's serum cholesterol level.
Other therapeutic agents useful for the reduction of a patient's
serum cholesterol level include, but are not limited to, statins
(e.g., lovastatin, atorvastatin, pravastatin) or a acyl-Coenzyme-A
mimic.
[0089] In a further embodiment, the other therapeutic agent is
useful for the treatment or prevention of cancer or a neoplastic
disease (e.g., cancer of the breast, ovary, uterine, prostate or
hypothalamus). Other therapeutic agents useful for the treatment or
prevention of cancer or a neoplastic disease include, but are not
limited to, alkylating agents (e.g., nitrosoureas), an
anti-metabolite (e.g., methotrexate or hydroxyurea), etoposides,
campathecins, bleomycin, doxorubicin, daunorubicin, colchicine,
irinotecan, camptothecin, cyclophosphamide, 5-fluorouracil,
cisplatinum, carboplatin, methotrexate, trimetrexate, erbitux,
thalidomide, taxol, a vinca alkaloid (e.g., vinblastine or
vincristine) or a microtubule stabilizer (e.g., an epothilone).
[0090] Further illustrative examples of therapeutic agents useful
for the treatment or prevention of cancer include, but are not
limited to: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; aminoglutethimide; amsacrine; anastrozole;
anthramycin; asparaginase; asperlin; azacitidine; azetepa;
azotomycin; batimastat; benzodepa; bicalutamide; bisantrene
hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;
brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; ImiDs; interleukin II (including recombinant
interleukin II, or rIL2), interferon-2a; interferon alpha-2b;
interferon alpha-n1 interferon alpha-n3; interferon beta-I a;
interferon gamma-I b; iproplatin; irinotecan hydrochloride;
lanreotide acetate; letrozole; leuprolide acetate; liarozole
hydrochloride; lometrexol sodium; lomustine; losoxantrone
hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; rogletimide; safingol; safingol hydrochloride; SelCid;
semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin;
streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan
sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
temozolomide; temodar; thiotepa; tiazofurin; tirapazamine;
toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin;
vinblastine sulfate; vincristine sulfate; vindesine; vindesine
sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine
sulfate; vorozole; zeniplatin; zinostatin; zorubicin
hydrochloride.
[0091] Other therapeutic agents useful for the treatment or
prevention of cancer include, but are not limited to: 20-epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;
acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
aphidicolin glycinate; apoptosis gene modulators; apoptosis
regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2;
axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III
derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine; calcipotriol; calphostin C; camptothecin
derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); cell-cycle inhibitors (e.g., flavopiridol A,
tryprostatin B, p19ink4D); cyclin-dependent kinase inhibitors
(e.g., roscovitine, olomucine and purine analogs); MAP kinase
inhibitors (CNI-1493); castanospermine; cecropin B; cetrorelix;
chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflomithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; retinoic acid (e.g., 9-cis
RA); histone deacetylase inhibitors (e.g., sodium butyrate,
suberoylanilide hydroxamic acid); TRAIL; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonennin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
Preferred additional anti-cancer drugs are 5-fluorouracil and
leucovorin.
[0092] The benzopyranone compounds can be prepared according to the
general reaction schemes (Route 1 and Route 2) shown below. 67
[0093] Step 1: Fries Reaction
[0094] Reaction yields are 40% to 55% and the reaction has been run
on gram to multiple kilogram scale. On smaller scale reactions
POCl.sub.3 (solvent) and ZnCl.sub.2 have been used in place of the
BF.sub.3 diethyl etherate.
[0095] Step 2: Coumarin Formation Reaction Summary
[0096] Reaction yields are typically 10% to 90% and the reactions
have been run on a multiple gram scale. Powdered K.sub.2CO.sub.3 is
essential for efficient reaction. Reactions have also been run by
adding all reagents simultaneously instead of preactivating the
acid as described above. Under these conditions slightly lower
yields are obtained.
[0097] Step 3: Side-Chain Introduction Reaction Summary
[0098] Reaction yields are typically 30% to 70% and the reactions
have been run on multiple gram scale. Powdered K.sub.2CO.sub.3 is
essential and granular material results in incomplete or prolonged
reaction times. The reaction yield in the examples provided are our
most recent efforts and the yields were lower than expected. In the
case of the dichloro analog, product precipitated on the column
during flash chromatography In general this is the highest yielding
step of the reaction sequence. The side-chain has also been
introduced as described in the alternative synthesis scheme
[0099] Step 4: Demethylation Reaction Summary
[0100] Reaction yields are typically 60% to 75%. Sealed tube
reaction minimizes HBr escape and greatly facilitates the reaction
rate. Reactions run at atmospheric pressure require one day or more
for completion. 89
[0101] Methods of this invention involve administering an effective
amount of a benzopyranone compound, or a pharmaceutical composition
containing one or more of the same, to a patient in need thereof in
an amount sufficient to treat the disease or condition of interest.
To that end, the term "treat" (or the related terms "treating" and
"treatment") means administration of a compound, typically in
combination with an appropriate delivery vehicle or agent, to a
patient that does not show signs of a disease or condition (e.g.,
prophylactic or preventative administration) or that does show
signs of a disease or condition (e.g., curative or treatment
administration). Further, the phrase "effective amount" means a
benzopyranone compound dose, or other active agent dose, that,
after a given time, results in the desired effect. For example, in
the context of bone-resorbing disease, an effective amount results
in bones mass that is statistically different from that of animals
treated with placebo. Similarly, for cancer and arthritis, an
effective amount is an amount sufficient to produce the desired
effect on the cancerous or arthritic tissue. In one embodiment, the
"effective amount" is a dose capable of: treating or preventing a
bone-resorbing disease; treating or preventing cancer; treating or
preventing arthritis; modulating gene expression in a cell or
tissue expressing ER; activating the function of ER in a bone cell;
inhibiting the function of ER in a breast cancer cell, an ovarian
cancer cell, an endometrial cell, a uterine cell, a prostate cell
or a hypothalamus cell; inhibiting the function of ER in a cell
that expresses ER and IL-6; inhibiting cell proliferation in a
cancer or neoplastic cell; or reducing a patient's serum
cholesterol level.
[0102] The benzopyranone compounds can exist as a pharmaceutically
acceptable salt of a compound of structure (I) or (II). The
pharmaceutically acceptable acid addition salts of the
benzopyranone compounds can be formed of the compound itself, or of
any of its esters, and include the pharmaceutically acceptable
salts which are often used in pharmaceutical chemistry. For
example, salts may be formed with organic or inorganic acids.
Suitable organic acids include maleic, fumaric, benzoic, ascorbic,
succinic, methanesulfonic, benzenesulfonic, toluenesulfonic,
acetic, oxalic, trifluoroacetic, propionic, tartaric, salicylic,
citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic,
palmitic, formic, glycolic, glutamic, and benzenesulfonic acids.
Suitable inorganic acids include hydrochloric, hydrobromic,
sulfuric, phosphoric, and nitric acids. Additional salts include
chloride, bromide, iodide, bisulfate, acid phosphate,
isonicotinate, lactate, acid citrate, oleate, tannate,
pantothenate, bitartrate, gentisinate, gluconate, glucaronate,
saccharate, ethanesulfonate, p-toluenesulfonate, and pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The term
"pharmaceutically acceptable salt" is intended to encompass any and
all acceptable salt forms.
[0103] Pharmaceutically acceptable salts can be formed by
conventional and known techniques, such as by reacting a compound
of this invention with a suitable acid as disclosed above. Such
salts are typically formed in high yields at moderate temperatures,
and often are prepared by merely isolating the compound from a
suitable acidic wash in the final step of the synthesis. The
salt-forming acid may dissolved in an appropriate organic solvent,
or aqueous organic solvent, such as an alkanol, ketone or ester. On
the other hand, if the benzopyranone compound is desired in the
free base form, it may be isolated from a basic final wash step,
according to known techniques. For example, a typical technique for
preparing hydrochloride salt is to dissolve the free base in a
suitable solvent, and dry the solution thoroughly, as over
molecular sieves, before bubbling hydrogen chloride gas through
it.
[0104] The benzopyranone compounds can be administered to a patient
orally or parenterally in the conventional form of preparations,
such as capsules, microcapsules, tablets, granules, powder,
troches, pills, suppositories, injections, suspensions and syrups.
Suitable formulations can be prepared by methods commonly employed
using conventional, organic or inorganic additives, such as an
excipient (e.g., sucrose, starch, mannitol, sorbitol, lactose,
glucose, cellulose, talc, calcium phosphate or calcium carbonate),
a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose,
polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic,
polyethyleneglycol, sucrose or starch), a disintegrator (e.g.,
starch, carboxymethylcellulose, hydroxypropylstarch, low
substituted hydroxypropylcellulose, sodium bicarbonate, calcium
phosphate or calcium citrate), a lubricant (e.g., magnesium
stearate, light anhydrous silicic acid, talc or sodium lauryl
sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or
orange powder), a preservative (e.g, sodium benzoate, sodium
bisulfite, methylparaben or propylparaben), a stabilizer (e.g.,
citric acid, sodium citrate or acetic acid), a suspending agent
(e.g., methylcellulose, polyvinyl pyrroliclone or aluminum
stearate), a dispersing agent (e.g., hydroxypropylmethylcellu-
lose), a diluent (e.g., water), and base wax (e.g., cocoa butter,
white petrolatum or polyethylene glycol). The effective amount of
the benzopyranone compound in the pharmaceutical composition may be
at a level that will exercise the desired effect; for example,
about 0.1 mg to 100 mg in unit dosage for both oral and parenteral
administration.
[0105] The benzopyranone compound can be usually administered one
to four times a day with a unit dosage of 0.1 mg to 100 mg in human
patients, but the above dosage may be properly varied depending on
the age, body weight and medical condition of the patient and the
type of administration. A preferred dose is 0.25 mg to 25 mg in
human patients. One dose per day is preferred.
[0106] The dose of a benzopyranone compound to be administered to a
human is rather widely variable and subject to the judgment of the
attending physician. It should be noted that it may be necessary to
adjust the dose of a benzopyranone compound when it is administered
in the form of a salt, such as a laureate, the salt forming moiety
of which has an appreciable molecular weight. The general range of
effective administration rates of the benzopyranone compounds is
from about 0.05 mg/day to about 100 mg/day. A preferred rate range
is from about 0.25 mg/day to 25 mg/day. Of course, it is often
practical to administer the daily dose of a benzopyranone compound
in portions, at various hours of the day. However, in any given
case, the amount of benzopyranone compound administered will depend
on such factors as the solubility of the active component, the
formulation used and the route of administration.
[0107] It is usually preferred to administer a benzopyranone
compound orally for reasons of convenience. However, the
benzopyranone compounds may equally effectively be administered
percutaneously, or as suppositories for absorption by the rectum,
if desired in a given instance.
[0108] The benzopyranone compounds can be administered as
pharmaceutical compositions. The compositions can be in the form of
tablets, chewable tablets, capsules, solutions, parenteral
solutions, troches, suppositories and suspensions. Compositions can
be formulated to contain a daily dose, or a convenient fraction of
a daily dose, in a dosage unit, which may be a single tablet or
capsule or convenient volume of a liquid.
[0109] The compositions can be readily formulated as tablets,
capsules and the like; it is preferable to prepare solutions from
water-soluble salts, such as the hydrochloride salt. In general,
all of the compositions are prepared according to known methods in
pharmaceutical chemistry. Capsules are prepared by mixing the
benzopyranone compound with a suitable diluent and filling the
proper amount of the mixture in capsules. The usual diluents
include inert powdered substances such as starch of many different
kinds, powdered cellulose, especially crystalline and
microcrystalline cellulose, sugars such as fructose, mannitol and
sucrose, grain flours and similar edible powders.
[0110] Tablets are prepared by direct compression, by wet
granulation, or by dry granulation. Their formulations usually
incorporate diluents, binders, lubricants and disintegrators as
well as the compound. Typical diluents include, for example,
various types of starch, lactose, mannitol, kaolin, calcium
phosphate or sulfate, inorganic salts such as sodium chloride and
powdered sugar. Powdered cellulose derivatives are also useful.
Typical tablet binders are substances such as starch, gelatin and
sugars such as lactose, fructose, glucose and the like. Natural and
synthetic gums are also convenient, including acacia, alginates,
methylcellulose, polyvinylpyrrolidine and the like. Polyethylene
glycol, ethylcellulose and waxes can also serve as binders.
[0111] A lubricant might be necessary in a tablet formulation to
prevent the tablet and punches from sticking in the die. The
lubricant can be chosen from such slippery solids as talc,
magnesium and calcium stearate, stearic acid and hydrogenated
vegetable oils. Tablet disintegrators are substances that swell
when wetted to break up the tablet and release the compound. They
include starches, clays, celluloses, algins and gums. More
particularly, corn and potato starches, methylcellulose, agar,
bentonite, wood cellulose, powdered natural sponge, cation-exchange
resins, alginic acid, guar gum, citrus pulp and carboxymethyl
cellulose, for example, can be used as well as sodium lauryl
sulfate. Tablets can be coated with sugar as a flavor and sealant,
or with film-forming protecting agents to modify the dissolution
properties of the tablet. The compositions can also be formulated
as chewable tablets, for example, by using substances such as
mannitol in the formulation.
[0112] When it is desired to administer a benzopyranone compound as
a suppository, typical bases can be used. Cocoa butter is a
traditional suppository base, which can be modified by addition of
waxes to raise its melting point slightly. Water-miscible
suppository bases comprising, particularly, polyethylene glycols of
various molecular weights are in wide use.
[0113] The effect of the benzopyranone compounds can be delayed or
prolonged by proper formulation. For example, a slowly soluble
pellet of the benzopyranone compound can be prepared and
incorporated in a tablet or capsule, or as a slow-release
implantable device. The technique also includes making pellets of
several different dissolution rates and filling capsules with a
mixture of the pellets. Tablets or capsules can be coated with a
film that resists dissolution for a predictable period of time.
Even the parenteral preparations can be made long-acting, by
dissolving or suspending the benzopyranone compound in oily or
emulsified vehicles that allow it to disperse slowly in the
serum.
5. EXAMPLES
[0114] The following Examples are presented by way of illustration,
not limitation.
Example 1
3-(2-CHLORO-4-TRIFLUOROMETHYLPHENYL)-7-HYDROXY-4-(4-(2-PYRROLIDIN-1-YL-ETH-
OXY)-BENZYL)-CHROMEN-2-ONE
[0115] A. (2-Chloro-4-trifluoromethylphenyl)-acetic Acid 10
[0116] A solution of LiHMDS in toluene was prepared by the addition
of n-BuLi (357 mL of a 1.6 M solution in hexanes, 571 mmol) to a
cold (-78.degree. C.) solution of HMDS (120.5 mL, 571 mmol) in
toluene (700 mL). After 30 min, the reaction mixture was allowed to
warm up to 10.degree. C. over 1 h. The solution was then
transferred via a cannula to a flame-dried, three-neck flask under
N.sub.2 containing Pd.sub.2 dba.sub.3 (4.18 g, 4.57 mmol) and
(2'-dicyclohexylphosphanylbiphenyl-2-yl- )-dimethylamine (3.77 g,
9.59 mmol). The mixture was stirred for 15 min at 15.degree. C.,
cooled to -10.degree. C. and t-butylacetate (70.5 mL, 525.4 mmol)
was added dropwise. After 10 min, 3-chloro-4-iodobenzotrifluo- ride
(70 g, 228.4 mmol) was added and the reaction mixture was warmed up
to 28.degree. C. After stirring at this temperature for 1.5 h, the
mixture was filtered through silica gel, using toluene as eluent,
and the solvent was removed in vacuo. The residue was purified
using flash chromatography (silica gel, 98:2 hexanes:EtOAc) to
yield (2-chloro-4-trifluoromethylphenyl)-acetic acid tert-butyl
ester as a solid.
[0117] A solution of (2-chloro-4-trifluoromethylphenyl)-acetic acid
tert-butyl ester (40 g, 135.7 mmol) in dioxane (100 mL) containing
conc. HCl (31.3 mL) was stirred at 50.degree. C. for 5 h. After
cooling the mixture to r.t., it was diluted with Et.sub.2O and the
organic layer was washed with H.sub.2O (3.times.). The organic
phase was dried (MgSO.sub.4) and the solvent was removed in vacuo.
Recrystallization of the residue with AcOEt-hexane yielded the
title compound as a solid showing: .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.71 (s, 1H), 7.55 (dd, 1H, J=1.0, 8.0 Hz),
7.47 (d, 1H, J=8.0 Hz), 3.92 (s, 2H). MS (ESI) m/z 237
(M-H).sup.-.
[0118] B.
3-(2-Chloro-4-trifluoromethylphenyl)-4-(4-hydroxybenzyl)-7-metho-
xy-chromen-2-one 11
[0119] A mixture of 2-(chloro-4-trifluoromethylphenyl)-acetic acid
(3.2 g, 13.41 mmol) and 1,1'-carbonyldiimidazole (2.72 g, 16.77
mmol) in DMF (15 mL) was heated to 70.degree. C. for 25 min. The
reaction mixture was cooled to 10.degree. C. and K.sub.2CO.sub.3
(2.78 g, 20.1 mmol),
1-(2-hydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl)-ethan-1-one (1.73
g, 6.7 mmol, prepared as described in Example 4A), DMAP (164 mg,
1.34 mmol) and DMF (10 mL) were added. After stirring the reaction
mixture at 115.degree. C. for 1.5 h, the resulting suspension was
cooled to r.t., poured onto AcOEt/H.sub.2O and the layers were
separated. The organic layer was washed with H.sub.2O, aq 1N HCl
and brine, was dried (MgSO.sub.4) and the solvent was removed in
vacuo. The resultant red residue was purified using flash
chromatography (silica gel, 2:1 to 1:2 hexanes:Et.sub.2O) to
provide the title compound as a white solid showing: .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.75 (s, 1H), 7.53 (br d, 1H, J=8.0
Hz), 7.47 (d, 1H, J=9.0 Hz), 7.33 (d, 1H, J=8.0 Hz), 6.92-6.85 (m,
3H), 6.80 (d, 1H, J=2.5, 9.0 Hz), 6.70 (d, 2H, J=8.5 Hz), 4.95-4.64
(verybr s, 1H), 4.02 (d, 1H, J=15.5 Hz), 3.88 (s, 3H), 3.76 (d, 1H,
J=15.5 Hz). MS (ESI) m/z 461 (M+H).sup.+.
[0120] C.
3-(2-Chloro-4-trifluoromethylphenyl)-7-methoxy-4-(4-(2-pyrrolidi-
n-1-yl-ethoxy)-benzyl)-chromen-2-one 12
[0121] A mixture of
3-(2-chloro-4-trifluoromethylphenyl)-4-(4-hydroxybenzy-
l)-7-methoxychromen-2-one (460 mg, 1 mmol),
1-(2-chloroethyl)pyrrolidine hydrochloride (254.7 mg, 1.5 mmol) and
K.sub.2CO.sub.3 (413.9 mg, 2.99 mmol) in EtOH (5 mL) was stirred
for 2 min prior to the addition of H.sub.2O (0.5 mL). The mixture
was stirred at 55.degree. C. for 2.5 h, after which time it was
cooled to r.t. and poured into CHCl.sub.3--H.sub.2O. The layers
were separated and the aqueous phase was extracted with CHCl.sub.3
(3.times.). The combined organic layers were washed with brine,
dried (MgSO.sub.4) and the solvent was removed in vacuo. The
resultant brown foam was purified using flash chromatography
(silica gel, 19:1 CH.sub.2Cl.sub.2:MeOH) to provide the title
compound as a pale brown foam which displayed: .sup.1H NMR (300
MHz, CD.sub.3OD) .delta. 7.83 (s, 1H), 7.65 (d, 1H, J=9.0 Hz), 7.63
(d, 1H, J=8.0 Hz), 7.51 (d, 1H, J=8.0 Hz), 7.00 (d, 1H, J=2.5 Hz),
6.95 (d, 2H, J=8.5 Hz), 6.89 (dd, 1H, J=2.5, 9.0 Hz), 6.79 (d, 2H,
J=8.5 Hz), ), 4.07 (d, 1H, J=15.5 Hz), 4.05 (t, 2H, J=5.5 Hz), 3.90
(s, 3H), 3.83 (d, 1H, J=15.5 Hz), 2.89 (t, 2H, J=5.5 Hz), 2.72-2.60
(m, 4H), 1.90-1.75 (m, 4H). MS (ESI) m/z 558 (M+H).sup.+.
[0122] D.
3-(2-Chloro-4-trifluoromethylphenyl)-7-hydroxy-4-(4-(2-pyrrolidi-
n-1-yl-ethoxy)-benzyl)-chromen-2-one 13
[0123]
3-(2-Chloro-4-trifluoromethylphenyl)-7-methoxy-4-(4-(2-pyrrolidin-1-
-yl-ethoxy)-benzyl)-chromen-2-one (330 mg, 0.59 mmol) was dissolved
in AcOH (2.4 mL)-48% aq HBr (2.4 mL). The mixture was stirred at
130.degree. C. for 15 h. After cooling the mixture to r.t., it was
poured onto EtOAc/aq NaHCO.sub.3. 1M aq NaOH was then added to
bring the pH to 8. The layers were separated and the aqueous layer
was back-extracted with EtOAc (3.times.). The combined organic
layers were washed with brine, dried (MgSO.sub.4) and the solvent
was removed in vacuo. The residue was purified using flash
chromatography (silica gel, 5:1 CH.sub.2Cl.sub.2--MeOH) to provide
the title compound as a yellow foam showing: IR (KBr)
.nu.=3670-2140, 1709, 1611, 1569, 1511, 1367, 1323, 1247, 1172,
1133, 1081, 1067, 1044, 1012 cm.sup.-1. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.81 (d, 1H, J=1.5 Hz), 7.61 (dd, 1H, J=1.5,
8.0 Hz), 7.56 (d, 1H, J=8.8 Hz), 7.48 (d, 1H, J=8.0 Hz), 6.93 (d,
2H, J=8.5 Hz), 6.79 (d, 2H, J=8.5 Hz), 6.76 (d, 1H, J=2.5 Hz), 6.73
(dd, 1H, J=2.5, 8.8 Hz), 4.09 (t, 2H, J=5.5 Hz), 4.05 (d, 1H,
J=15.5 Hz), 3.80 (d, 1H, J=15.5 Hz), 3.04 (t, 2H, J=5.5 Hz),
2.86-2.78 (m, 4H), 1.92-1.82 (m, 4H). HRMS (ESI) calcd for
C.sub.29H.sub.25ClF.sub.3NO.sub.4 (M+H).sup.+: 544.1502; found:
544.1504.
Example 2
3-(4-CHLORO-2-TRIFLUOROMETHYLPHENYL)-7-HYDROXY-4-(4-(2-PYRROLIDIN-1-YL-ETH-
OXY)-BENZYL)-CHROMEN-2-ONE
[0124] A. (4-Chloro-2-trifluoromethylphenyl)-acetic Acid 14
[0125] A solution containing
4-chloro-1-iodo-2-trifluoromethylbenzene (14.98 g, 48.9 mmol),
Bu.sub.3SnCH.dbd.CH.sub.2 (15.7 mL, 53.7 mmol) and
(Ph.sub.3P).sub.4Pd (2.26 g, 1.955 mmol) in anhyd toluene (200 mL)
was deoxygenated using vacuum-N.sub.2 flush (3.times.). After
refluxing the reaction mixture for 17 h, it was cooled to 0.degree.
C. and a solution of disiamylborane-methyl sulfide complex in
toluene (.about.1.95 M, 47 mL) was added dropwise over a period of
5 min. The disiamylborane-methyl sulfide complex solution was
prepared by adding 2-methyl-2-butene (26 mL, 245 mmol) to a cold
(0.degree. C.) solution of borane-methyl sulfide complex (11.6 mL,
122.3 mmol) in anhyd toluene (25 mL) and stirring the resultant
mixture at r.t. for 2 h. The bath was removed and the reaction
mixture was stirred at r.t. for 3 h. After that period of time, the
mixture was cooled to 0.degree. C., EtOH (75 mL) was added slowly,
followed by 2 N aq NaOH (37.5 mL) and 30% aq H.sub.2O.sub.2 (30
mL). The solution was stirred at r.t. for 1.5 h and was then poured
onto Et.sub.2O--H.sub.2O. The layers were separated and the organic
layer was washed with H.sub.2O and brine, was dried (MgSO.sub.4)
and the solvent was removed in vacuo, while the bath temperature
was maintained below 30.degree. C. The black residue was purified
using flash chromatography (silica gel; 4:1 to 3:1 hexanes:AcOEt)
to afford 2-(4-chloro-2-trifluorom- ethyl-phenyl)-ethanol as a
brown oil which was used directly in the next step.
[0126] To a solution of
2-(4-chloro-2-trifluoromethylphenyl)-ethanol in acetone (50 mL) at
0.degree. C. was added dropwise a solution of Jones reagent (40.3
mL of a 2.67 M solution in H.sub.2SO.sub.4). After 25 min, the
mixture was poured onto Et.sub.2O/H.sub.2O and the layers were
separated. The organic layer was washed with H.sub.2O and brine,
dried (MgSO.sub.4) and the solvent was removed in vacuo. The
resultant orange solid was crystallized from hexane and heptane to
furnish the title compound as a solid which showed: .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.67 (d, 1H, J=2.0 Hz), 7.51 (dd, 1H,
J=2.0, 8.0 Hz), 7.34 (d, 1H, J=8.0 Hz), 3.84 (s, 2H).
[0127] B.
3-(4-Chloro-2-trifluoromethylphenyl)-4-(4-hydroxybenzyl)-7-metho-
xy-chromen-2-one 15
[0128] This compound was prepared using the methodology described
above in Example 1B. The resultant residue was purified using flash
chromatography (silica gel, 1:1 to 55:45 to 3:2 Et.sub.2O:hexanes)
to provide the title compound as a beige solid showing: .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.76 (d, 1H, J=1.5 Hz), 7.50 (dd, 1H,
J=1.5, 8.0 Hz), 7.41 (d, 1H, J=9.0 Hz), 7.15 (d, 1H, J=8.0 Hz),
6.89 (d, 1H, J=2.5 Hz), 6.85 (d, 2H, J=8.5 Hz), 6.77 (dd, 1H,
J=2.5, 9.0 Hz), 6.70 (d, 2H, J=8.5 Hz), 4.00 (d, 1H, J=15.5 Hz),
3.87 (s, 3H), 3.61 (d, 1H, J=15.5 Hz). MS (ESI) m/z 461
(M+H).sup.+.
[0129] C.
3-(4-Chloro-2-trifluoromethylphenyl)-7-methoxy-4-(4-(2-pyrrolidi-
n-1-yl-ethoxy)-benzyl)-chromen-2-one 16
[0130] This compound was prepared using the methodology described
above in Example 1C. The resultant brown foam was purified using
flash chromatography (silica gel, 94:6 CH.sub.2Cl.sub.2:MeOH) to
provide the title compound as a light yellow foam which displayed:
.sup.1H NMR (300 MHz, CD.sub.3OD) .delta. 7.83 (d, 1H, J=1.7 Hz),
7.61 (dd, 1H, J=1.7, 8.5 Hz), 7.55 (d, 1H, J=9.0 Hz), 7.34 (d, 1H,
J=8.5 Hz), 7.00-6.93 (m, 3H), 6.85 (dd, 1H, J=2.5, 9.0 Hz), 6.81
(d, 2H, J=9.0 Hz), 4.11 (d, 1H, J=15.5 Hz), 4.05 (t, 2H, J=5.5 Hz),
3.88 (s, 3H), 3.61 (d, 1H, J=15.5 Hz), 2.90 (t, 2H, J=5.5 Hz),
2.71-2.61 (m, 4H), 1.86-1.77 (m, 4H). MS (ESI) m/z 558
(M+H).sup.+.
[0131] D.
3-(4-Chloro-2-trifluoromethylphenyl)-7-hydroxy-4-(4-(2-pyrrolidi-
n-1-yl-ethoxy)-benzyl)-chromen-2-one 17
[0132] This compound was prepared using the methodology described
above in Example 1D. The residue was purified using flash
chromatography (silica gel, 5:1 CH.sub.2Cl.sub.2--MeOH) to provide
the title compound as a yellow solid showing: IR (KBr)
.nu.=3700-2100, 1721, 1597, 1512, 1467, 1377, 1305, 1265, 1247,
1182, 1136, 1108, 1061, 1046, 1016, 843 cm-1. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.82 (d, 1H, J=2.0 Hz), 7.59 (dd, 1H, J=2.0,
8.0 Hz), 7.47 (d, 1H, J=9.0 Hz), 7.31 (d, 1H, J=8.0 Hz), 6.96 (d,
2H, J=8.5 Hz), 6.82 (d, 2H, J=8.5 Hz), 6.75 (d, 1H, J=2.5 Hz), 6.69
(dd, 1H, J=2.5, 9.0 Hz), 4.12-4.06 (m, 3H), 3.59 (d, 1H, J=15.5
Hz), 3.05 (t, 2H, J=5.5 Hz), 2.86-2.81 (m, 4H), 1.92-1.84 (m, 4H).
HRMS (ESI) calcd for C.sub.29H.sub.25ClF.sub.3NO.sub.4 (M+H).sup.+:
544.1502; found: 544.1505.
Example 3
3-(2,4-BIS-TRIFLUOROMETHYLPHENYL)-7-HYDROXY-4-(4-(2-PYRROLIDIN-1-YL-ETHOXY-
)-BENZYL)-CHROMEN-2-ONE
[0133] A.
3-(2,4-bistrifluoromethylphenyl)-4-(4-hydroxybenzyl)-7-methoxych-
romen-2-one 18
[0134] This compound was prepared using the methodology described
above in Example 1B. The resultant residue was purified using flash
chromatography (silica gel, 1:1 to 3:2 Et.sub.2O:hexanes) to
provide the title compound as a yellow foam showing: .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 8.03 (s, 1H), 7.78 (d, 1H, J=8.0 Hz),
7.42 (d, 1H, J=8.8 Hz), 7.36 (d, 1H, J=8.0 Hz), 6.90 (d, 1H, J=2.5
Hz), 6.84 (d, 2H, J=8.5 Hz), 6.78 (dd, 1H, J=2.5, 8.8 Hz), 6.70 (d,
2H, J=8.5 Hz), 4.76 (s, 1H), 4.01 (d, 1H, J=16.0 Hz), 3.88 (s, 3H),
3.57 (d, 1H, J=16.0 Hz).
[0135] B.
3-(2,4-bistrifluoromethylphenyl)-7-methoxy-4-(4-(2-pyrrolidin-1--
yl-ethoxy)benzyl)-chromen-2-one 19
[0136] This compound was prepared using the methodology described
above in Example 1C. The resultant brown foam was purified using
flash chromatography (silica gel, 96:4 CH.sub.2Cl.sub.2:MeOH) to
provide the title compound as a beige foam which displayed: .sup.1H
NMR (300 MHz, CD.sub.3OD) .delta. 8.09 (s, 1H), 7.93 (d, 1H, J=8.5
Hz), 7.59 (d, 1H, J=8.5 Hz), 7.57 (d, 1H, J=9.0 Hz), 6.99 (d, 1H,
J=2.5 Hz), 6.97 (d, 2H, J=8.5 Hz), 6.87 (dd, 1H, J=2.5, 9.0 Hz),
6.81 (d, 2H, J=8.5 Hz), 4.13 (d, 1H, J=16.0 Hz), 4.05 (t, 2H, J=5.5
Hz), 3.89 (s, 3H), 3.59 (d, 1H, J=16.0 Hz), 2.89 (t, 2H, J=5.5 Hz),
2.73-2.58 (m, 4H), 1.87-1.77 (m, 4H). MS (ESI) m/z 592
(M+H).sup.+.
[0137] C.
3-(2,4-bistrifluoromethylphenyl)-7-hydroxy-4-(4-(2-pyrrolidin-1--
ylethoxy)-benzyl)-chromen-2-one 20
[0138] This compound was prepared using the methodology described
above in Example 1D. The residue was purified using flash
chromatography (silica gel, 3:1 CH.sub.2Cl.sub.2--MeOH) to provide
the title compound as a yellow foam showing: IR (KBr)
.nu.=3700-2300, 1714, 1615, 1512, 1462, 1368, 1346, 1300, 1272,
1179, 1133, 1082, 1062, 1045, 1014, 846 cm-1. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.07 (s, 1H), 7.91 (br d, 1H, J=8.5 Hz), 7.56
(d, 1H, J=8.5 Hz), 7.49 (d, 1H, J=8.8 Hz), 6.96 (d, 2H, J=9.0 Hz),
6.82 (d, 2H, J=9.0 Hz), 6.77 (d, 1H, J=2.5 Hz), 6.71 (dd, 1H,
J=2.5, 8.8 Hz), 4.11 (d, 1H, J=16.0 Hz), 4.11 (t, 2H, J=5.5 Hz),
3.57 (d, 1H, J=16.0 Hz), 3.08 (t, 2H, J=5.5 Hz), 2.90-2.83 (m, 4H),
1.92-1.86 (m, 4H). HRMS (ESI) calcd for
C.sub.30H.sub.25F.sub.6NO.sub.4 (M+H).sup.+: 578.1766; found:
578.1762.
Example 4
3-(4-TRIFLUOROMETHYLPHENYL)-4-(4-(2-PYRROLIDIN-1-YL-ETHOXY)BENZYL)-7-METHO-
XYCHROMEN-2-ONE
[0139] A.
1-(2-hydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl)ethan-1-one 21
[0140] A suspension of 3-methoxyphenol (44.69 kg, 360 mol) and
4-hydroxyphenylacetic acid (68.5 kg, 450 mol) in 144 L of
chlorobenzene was purged with nitrogen gas. Boron trifluoride
diethyl etherate (177 L, 1440 mol) was added at 20 to 25.degree. C.
The suspension was heated to 80.degree. C. and stirred for 4 to 5 h
then cooled to 5 to 10.degree. C. and stirred overnight.
[0141] The precipitated red/orange solid (undesired isomer) was
filtered with N.sub.2 pressure and the filtrate was quenched by
pouring onto ice/H.sub.2O. The filter cake was washed with
CH.sub.2Cl.sub.2. The boron trifluoride etherate was quenched by
the slow addition of 80% Na.sub.2CO.sub.3 (aq) until the pH of the
aqueous solution reached 6 to 7. Gas evolution was observed and the
product precipitated from solution.
[0142] The orange suspension was stirred at 20.degree. C. overnight
and subsequently filtered. The filter cake was washed with H.sub.2O
and MTBE and dried overnight to provide the desired product (38 kg,
42% yield, HPLC purity 95.1% a/a). .sup.1H NMR (300 MHz, DMSO-d6)
.delta. 12.30 (s, 1H), 9.31 (s, 1H), 7.99 (d, 1H, J=9.1 Hz), 7.08
(d, 2H, J=8.4 Hz), 6.70, (d, 2H, J=8.4 Hz), 6.53 (dd, 1H, J=2.5,
9.1 Hz), 6.47 (d, 1H, J=2.5 Hz), 4.18 (s, 2H), 3.81 (s, 3H). MS
(ESI) m/z 259 (M+H).sup.+.
[0143] B.
3-(4-trifluoromethylphenyl)-4-(4-hydroxybenzyl)-7-methoxychromen-
-2-one 22
[0144] A solution of 4-trifluoromethylphenylacetic acid (15.2 g,
74.45 mmol) in 120 mL of DMF at 25.degree. C. was treated with CDI
(13.2 g, 82 mmol) in several portions over 5 minutes. The reaction
mixture was warmed to 40.degree. C. for 10 minutes then cooled to
room temperature.
1-(2-hydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl)ethan-1-one (9.81
g, 38 mmol), K.sub.2CO.sub.3 (15.7 g, 114 mmol), and DMAP (0.93 g,
7.6 mmol) were added and the reaction mixture was warmed to
80.degree. C. for 2 hours.
[0145] The suspension was cooled to room temperature and 200 mL of
water was added. The aqueous layer was extracted with
CH.sub.2Cl.sub.2 and the combined organic layer was dried
(MgSO.sub.4) then concentrated under vacuum. The resulting solid
was purified using flash chromatography (CH.sub.2Cl.sub.2:EtOAc) to
provide the desired product (10.2 g, 63%). .sup.1H NMR (300 MHz,
DMSO-d6) .delta. 9.29 (s, 1H), 7.79 (d, 2H, J=8.7 Hz), 7.57 (d, 2H,
J=8.7 Hz), 7.53 (d, 1H, J=8.5 Hz), 7.04 (d, 1H, J=2.3 Hz), 6.93 (d,
2H, J=8.9 Hz), 6.87 (dd, 1H, J=8.5, 2.3 Hz), 6.61 (d, 2H, J=8.9
Hz), 3.90 (s, 2H), 3.84 (s, 3H). MS (ESI) m/z 427 (M+H).sup.+.
[0146] C.
3-(4-trifluoromethylphenyl)-4-(4-(2-pyrrolidin-1-ylethoxy)benzyl-
)-7-methoxychromen-2-one 23
[0147] A solution of
3-(4-trifluoromethylphenyl)-4-(4-hydroxybenzyl)-7-met-
hoxychromen-2-one (6.0 g, 14 mmol), 1-(2-chloroethyl)pyrrolidine
hydrochloride (3.3 g, 22.5 mmol), and K.sub.2CO.sub.3 (6.6 g, 47.8
mmol) in 30 mL of DMF was warmed at 120.degree. C. for 2 hours.
Solvent was removed under reduced pressure. Water was added and the
aqueous layer was extracted with ethyl acetate. The combined
organic layer was dried and concentrated to provide a dark brown
oil. Flash chromatography (CH.sub.2Cl.sub.2:EtOAc:MeOH:TEA)
provided the desired product (4.7 grams, 64%). .sup.1H NMR (300
MHz, DMSO-d6) .delta. 7.79 (d, 2H, J=8.1 Hz), 7.58 (d, 2H, J=8.1
Hz), 7.51 (d, 1H, J=9.0 Hz), 7.08 (d, 2H, J=8.9 Hz), 7.06 (d, 1H,
J=2.5 Hz), 6.87 (dd, 1H, J=2.5, 9.0 Hz), 6.82 (d, 2H, J=8.9 Hz),
4.08 (t, 2H, J=5.0 Hz), 3.96 (s, 2H), 3.84 (s, 3H), 3.17-3.12 (m,
2H), 2.94-2.88 (m, 4H), 1.83-1.78 (m, 4H). MS (ESI) m/z 524
(M+H).sup.+.
[0148] D.
3-(4-trifluoromethylphenyl)-4-(4-(2-pyrrolidin-1-yl-ethoxy)benzy-
l)-7-methoxychromen-2-one 24
[0149] A solution of
3-(4-trifluoromethylphenyl)-4-(4-(2-pyrrolidin-1-yl-e-
thoxy)benzyl)-7-methoxychromen-2-one (4.2 grams, 8.02 mmol) and 25
mL of 30% HBr/HOAc in a sealed tube was warmed at 120.degree. C.
for 3 h. The solvent was removed under reduced pressure and the
residue was quenched with NaHCO.sub.3 (aq). The aqueous layer was
extracted with CH.sub.2Cl.sub.2 and the combined organic layer was
concentrated. The crude product was purified by passage through a
short column of silica gel followed by reverse phase preparative
HPLC to provide the title compound (2.9 g, 71%). .sup.1H NMR (300
MHz, DMSO) .delta. 7.77 (d, 2H, J=8.0 Hz), 7.55 (d, 2H, J=8.0 Hz),
7.44 (d, 1H, J=8.8 Hz), 7.03 (d, 2H, J=8.0 Hz), 6.79 (d, 2H, 8.0
Hz), 6.76 (s, 1H), 6.70 (d, 1H, J=8.5 Hz), 3.97 (t, 2H, J=5.8 Hz),
3.92 (s, 2H), 2.72 (d, 2H, J=5.8 Hz), 2.50-2.47 (m, 4H), 1.66-1.64
(m, 4H). MS (ESI) m/z 510 (M+H).sup.+.
Example 5
3-(4-CHLOROPHENYL)-4-(4-(2-PYRROLIDIN-1-YL-ETHOXY)BENZYL)-7-HYDROXYCHROMEN-
-2-ONE HYDROCHLORIDE
[0150] A.
1-(2-hydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl)ethan-1-one 25
[0151] This compound was prepared using the methodology described
above in Example 4A.
[0152] B.
3-(4-chlorophenyl)-4-(4-hydroxybenzyl)-7-methoxychromen-2-one
26
[0153] This compound was prepared using the methodology described
above in Example 4B. .sup.1H NMR (300 MHz, DMSO-d6) .delta. 9.30
(s, 1H), 7.51 (d, 1H, J=9.1 Hz), 7.47 (d, 2H, J=8.2 Hz), 7.34 (d,
2H, J=8.2 Hz), 7.02 (d, 1H, J=2.2 Hz), 6.91 (d, 2H, J=8.5 Hz), 6.85
(dd, 1H, J=9.1, 2.2 Hz), 6.61 (d, 2H, J=8.5 Hz), 3.91 (s, 2H), 3.83
(s, 3H). MS (ESI) m/z 393 (M+H).sup.+.
[0154] C.
3-(4-chlorophenyl)-4-(4-(2-bromoethoxy)benzyl)-7-methoxychromen--
2-one 27
[0155] A solution of
3-(4-chlorophenyl)-4-(4-hydroxybenzyl)-7-methoxychrom- en-2-one
(21.2 g, 54 mmol), dibromoethane (50.7 g, 270 mmol), and
K.sub.2CO.sub.3 (8.3 g, 60 mmol) in 200 mL of acetone was heated at
reflux for 12 h. The reaction mixture was cooled to room
temperature and volatiles were removed under reduced pressure.
Hexanes (500 mL) was added with stirring and the resulting solid
that formed was collected by filtration. The material was rinsed
with hexanes (2.times.100 mL), collected and dried under vacuum to
provide the desired product (22.5 g, 83%). .sup.1H NMR (300 MHz,
DMSO) .delta. 7.50 (d, 1H, J=9.1 Hz), 7.48 (d, 2H, J=8.2 Hz), 7.35
(d, 2H, J=8.2 Hz), 7.07 (d, 2H, J=8.5 Hz), 7.04 (d, 1H, J=2.6 Hz),
6.86 (dd, 1H, J=9.1, 2.6 Hz), 6.82 (d, 2H, J=8.5 Hz), 4.24 (t, 2H,
J=5.8 Hz), 3.98 (s, 2H), 3.84 (s, 3H), 3.76 (t, 2H, J=5.8 Hz). MS
(ESI) m/z 500 (M+H).sup.+.
[0156] D.
3-(4-chlorophenyl)-4-(4-(2-bromoethoxy)benzyl)-7-hydroxychromen--
2-one 28
[0157] A solution of
3-(4-chlorophenyl)-4-(4-(2-bromoethoxy)benzyl)-7-meth-
oxychromen-2-one (16.5 grams, 33 mmol) and 150 mL of 30% HBr/HOAc
in a sealed tube was warmed at 100.degree. C. for 8 h. The reaction
mixture was cooled to room temperature and poured into 300 mL of
water. The resulting solid was collected by filtration and purified
using flash chromatography to provide the desired product (12.5 g,
78%). .sup.1H NMR (300 MHz, DMSO) .delta. 10.55 (s, 1H), 7.47 (d,
2H, J=8.5 Hz), 7.43 (d, 1H,1=8.8 Hz), 7.33 (d, 2H, J=8.5 Hz), 7.05
(d, 2H, J=8.5 Hz), 6.83 (d, 2H, J=8.5 Hz), 6.75 (d, 1H, J=2.2 Hz),
6.70 (dd, 1H, J=8.8, 2.2 Hz), 4.24 (t, 2H, J=5.7 Hz), 3.94 (s, 2H),
3.76 (t, 2H, J=5.7 Hz). MS (ESI) m/z 486 (M+H).sup.+.
[0158] E.
3-(4-chlorophenyl)-4-(4-(2-pyrrolidin-1-ylethoxy)benzyl)-7-hydro-
xychromen-2-one Hydrochloride 29
[0159] A solution of
3-(4-chlorophenyl)-4-(4-(2-bromoethoxy)benzyl)-7-hydr-
oxychromen-2-one (8.3 g, 17.2 mmol) in 200 mL of THF was treated
with 8 mL of pyrrolidine and the reaction mixture was heated at
reflux for 5 h. The reaction mixture was concentrated and the crude
product was purified using flash chromatography. The product was
suspended in 250 mL of acetone and 4 mL of 5 M HCl(aq) was added.
The mixture was stirred at room temperature overnight and the
resulting solid was collected by filtration. The solid was
suspended in 200 mL of ethyl acetate and the suspension was heated
at reflux for 2 h. The solution was cooled to room temperature and
the final product was collected by filtration and dried under
vacuum. The final yield was 4.96 grams (56%). .sup.1H NMR (300 MHz,
DMSO) .delta. 10.62 (s, 1H), 10.42 (s, 1H), 7.47 (d, 2H, J=8.5 Hz),
7.43 (d, 1H, J=8.8 Hz), 7.34 (d, 2H, J=8.5 Hz), 7.09 (d, 2H, J=8.5
Hz), 6.87 (d, 2H, J=8.5 Hz), 6.77 (d, 1H, J=2.5 Hz), 6.71 (dd, 1H,
J=2.5, 8.8 Hz), 4.26 (t, 2H, 4.9 Hz), 3.96 (s, 2H), 3.59-3.51 (m,
4H), 3.15-3.02 (m, 2H), 2.03-1.88 (m, 4H). MS (ESI) m/z 476
(M+H).sup.+.
Example 6
3-(2,4-DICHLOROPHENYL)-4-(4-(2-PYRROLIDIN-1-YL-ETHOXY)BENZYL)-7-HYDROXYCHR-
OMEN-2-ONE
[0160] A.
1-(2-hydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl)ethan-1-one 30
[0161] This compound was prepared using the methodology described
above in Example 4A.
[0162] B.
3-(2,4-dichlorophenyl)-4-(4-hydroxybenzyl)-7-methoxychromen-2-on- e
31
[0163] This compound was prepared using the methodology described
above in Example 4B. 20 grams ketone (77.5 mmol) and 31.6 grams
acid (155 mmol) provided 27.52 grams product (83%). .sup.1H NMR
(300 MHz, DMSO-d6) .delta. 9.26 (s, 1H), 7.58 (d, 1H, J=8.8 Hz),
7.50 (dd, 1H, J=1.9, 8.2 Hz), 7.45 (d, 1H, J=8.2 Hz), 7.06 (d, 1H,
J=2.2 Hz), 6.90 (d, 3H, J=8.2 Hz), 6.59 (d, 2H, J=8.2 Hz), 3.98 (d,
1H, J=15.4 Hz), 3.85 (s, 3H), 3.69 (d, 1H, J=15.4 Hz). MS (ESI) m/z
428 (M+H).sup.+.
[0164] C.
3-(2,4-dichlorophenyl)-4-(4-(2-pyrrolidin-1-ylethoxy)benzyl)-7-m-
ethoxychromen-2-one 32
[0165] This compound was prepared using the methodology described
above in Example 4C. (27.5 grams (64 mmol) of
3-(2,4-dichlorophenyl)-4-(4-hydroxyb- enzyl)-7-methoxychromen-2-one
provided 13.5 grams product, 40% yield). .sup.1H NMR (300 MHz,
DMSO-d6) .delta. 7.75 (d, 1H, J=1.8 Hz), 7.57 (d, 1H, J=8.9 Hz),
7.51 (dd, 1H, J=1.8, 8.2 Hz), 7.47 (d, 1H, J=8.2 Hz), 7.07 (d, 1H,
2.5 Hz), 7.02 (d, 2H, J=8.7 Hz), 6.89 (dd, 1H, J=2.5, 8.9 Hz), 6.78
(d, 2H, J=8.7 Hz), 4.04 (d, 1H, J=15.4 Hz), 3.98 (t, 2H, J=5.8 Hz),
3.85 (s, 3H), 3.74 (d, 1H, J=15.4 Hz), 2.79 (t, 2H, J=5.8 Hz),
2.57-2.52 (m, 4H), 1.69-1.65 (m, 4H). MS (ESI) m/z 525
(M+H).sup.+.
[0166] D.
3-(2,4-dichlorophenyl)-4-(4-(2-pyrrolidin-1-yl-ethoxy)benzyl)-7--
hydroxychromen-2-one 33
[0167] This compound was prepared using the methodology described
above in Example 4D. (4.5 grams of
3-(2,4-dichlorophenyl)-4-(4-(2-pyrrolidin-1-yle-
thoxy)benzyl)-7-methoxychromen-2-one (8.5 mmol) provided 3.2 grams
product, 73% yield). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.49
(d, 1H, J=1.9 Hz), 7.35 (d, 1H, J=8.5 Hz), 7.27 (s, 1H) 7.23 (dd,
1H, J=2.2, 8.2 Hz), 7.04 (d, 1H, J=8.2 Hz), 6.81 (d, 2H, J=8.5 Hz),
6.67 (s, 1H), 6.65 (dd, 1H, J=2.2, 8.5 Hz), 6.56 (d, 2H, J=8.5 Hz),
3.99 (d, 1H, J=15.6 Hz), 3.97 (t, 2H, J=5.8 Hz), 3.71 (d, 1H,
J=15.6 Hz), 2.73 (t, 2H, J=6.0 Hz), 2.51-2.46 (m, 4H), 1.68-1.63
(m, 4H). MS (ESI) m/z 511 (M+H).sup.+.
Example 7
Additional Representative Compounds
[0168] Table 1, below, discloses representative benzopyranone
compounds. These benzopyranone compounds can be obtained using the
methods disclosed herein.
1TABLE 1 Representative Benzopyranone Compounds (I) 34 No. R.sub.1
X Y n 1 H F CF.sub.3 2 2 H Br CF.sub.3 2 3 H I CF.sub.3 2 4
C(.dbd.O)CH.sub.3 H CF.sub.3 2 5 C(.dbd.O)CH.sub.3 Cl CF.sub.3 2 6
C(.dbd.O)CH.sub.3 F CF.sub.3 2 7 C(.dbd.O)CH.sub.3 Br CF.sub.3 2 8
C(.dbd.O)CH.sub.3 I CF.sub.3 2 9 C(.dbd.O)CH.sub.3 CF.sub.3
CF.sub.3 2 10 H F Cl 2 11 H Br Cl 2 12 H I Cl 2 13
C(.dbd.O)CH.sub.3 H Cl 2 14 C(.dbd.O)CH.sub.3 Cl Cl 2 15
C(.dbd.O)CH.sub.3 F Cl 2 16 C(.dbd.O)CH.sub.3 Br Cl 2 17
C(.dbd.O)CH.sub.3 I Cl 2 18 C(.dbd.O)CH.sub.3 CF.sub.3 Cl 2
Example 8
Inhibition OF IL-6 Release
[0169] Illustrative benzopyranone compounds were tested for their
ability to inhibit IL-6 release from human U-2 OS osteosarcoma
cells stably transfected with human ER-.alpha.. (Stein, B.; Yang,
M. X. Mol. Cell. Biol 15: 4971-4979, 1995; Poli, V. et. al., EMBO
J. 13:1189-1196, 1994). As a control, IL-6 release was determined
from the parental non-transfected U-2 OS cell line, which does not
express detectable levels of ER-.alpha.. Benzopyranone compounds
having an IC.sub.50<100 nM are particularly useful as bone
resorption inhibitors in vivo. Accordingly, the compounds of this
assay, illustrative benzopyranone compounds, are particularly
useful for the treatment of osteoporosis, Paget's disease and
metastatic bone cancer. These compounds are also useful as
anti-cancer agents as elevated IL-6 levels are responsible for
certain cancers such as multiple myeloma, prostate cancer, ovarian
cancer, renal carcinoma and cervical carcinoma.
[0170] Human U-2 OS osteosarcoma cells (ATCC) were stably
transfected with expression vectors for human full-length
ER-.alpha. using standard molecular biology techniques. Stable
subclones were generated that expressed high levels of ER-.alpha.
mRNA. The expression of ER-.alpha. was confirmed using RNase
protection analysis. The parental U-2 OS cells did not express any
measurable amounts of ER-.alpha..
[0171] Cells were plated into 96-well plates at a density of 80,000
cells per well in phenol red-free media with charcoal-stripped
fetal calf serum. Twenty four hours later, cells were either
treated with vehicle (0.2% DMSO) or test compound (0.01-1000 nm in
0.2% DMSO). Thirty minutes later cells were stimulated with 2.5
ng/ml TNF.alpha. and 1 ng/ml IL-1.beta.. Twenty-four hours later
the media supernatant was analyzed for cytokine production (IL-6)
using commercially available ELISA kits following the
manufacturer's instructions. Cytokine production in the presence of
vehicle (0.2% DMSO) was set to 100%. The results are expressed as
IC.sub.50 (nM) values (Table 2) which is the concentration of the
benzopyranone compound necessary to inhibit the production of IL-6
50% relative to the amount of IL-6 produced in the presence of
vehicle. The results show that all of the illustrative
benzopyranone compounds assayed show activity and, accordingly, are
useful for treating or preventing bone-resorbing diseases such as
osteoporosis, Paget's disease and metastatic bone cancer, and
cancers such as multiple myeloma, prostate and ovarian cancer.
Example 9
Inhibition of MCF-7 Breast Cancer Cell Proliferation
[0172] This example shows the ability of illustrative benzopyranone
compounds to inhibit 17.beta.-estradiol-dependent growth of MCF-7
breast cancer cells in vitro and compares their activity to that of
reference SERMs. MCF-7 cells represent an excellent in vitro system
to study the effects of compounds on estrogen-dependent breast
cancer growth. (May, F. E. B.; Westley, B. R. J. Biol. Chem.
262:15894-15899, 1987). Benzopyranone compounds having an
IC.sub.50<100 nM are particularly useful as anti-breast cancer
agents in vivo.
[0173] MCF-7 breast carcinoma cells were plated in 24-well dishes
at a density of 5.times.10.sup.3 cells/well in phenol-red free
DMEM:F-12 (1:1) medium containing 1% antibiotics, 0.05%
mercaptoethanol, 0.01% ethanolamine, 0.42 ng/mL sodium selenite and
5% charcoal-stripped FCS.
[0174] Illustrative benzopyranone compounds (0.1-1000 nM in 0.2%
DMSO) and 0.1 nM 17.beta.-estradiol were added to the cultured
MCF-7 breast cancer cells for 72 h. Subsequently, .sup.3H-labeled
thymidine was added and its incorporation into cells was measured
following 4 h incubation. The results are expressed as IC.sub.50
(nM) values (Table 2) which is the concentration of the
benzopyranone compound necessary to inhibit the growth of MCF-7
breast cancer cells by 50% relative to controls. The results show
that all the illustrative benzopyranone compounds assayed show
activity and, accordingly, are useful for treating or preventing
breast cancer in a patient.
Example 10
Inhibition of BG-1 Ovarlan Carcinoma Cell Proliferation
[0175] This assay shows the ability of illustrative benzopyranone
compounds to inhibit 17.beta.-estradiol-dependent growth of BG-1
ovarian carcinoma cells in vitro and compares their ability to that
of reference SERMs. BG-1 cells serve as a useful in vitro model for
the evaluation of the effects of antiestrogenic compounds on
ovarian tumor growth (Greenberger, L. M. et. al., Clin. Cancer Res.
7:3166-3177, 2001). Benzopyranone compounds having an
IC.sub.50<100 nM are particularly useful as anti-ovarian cancer
agents in vivo.
[0176] BG-1 ovarian carcinoma cells were plated in 24-well dishes
at a density of 5.times.10.sup.3 cells/well in phenol-red free
DMEM:F-12 (1:1) medium containing 1% antibiotics, 0.05%
mercaptoethanol, 0.01% ethanolamine, 0.42 ng/mL sodium selenite and
5% charcoal-stripped FCS. Illustrative benzopyranone compounds
(0.1-1000 nM in 0.2% DMSO) and 0.1 nM 17.beta.-estradiol were added
to the cultured BG-1 ovarian carcinoma cells and incubated for 72
h. Subsequently, .sup.3H-labeled thymidine was added and its
incorporation into cells was measured following 4 h incubation. The
results are expressed as IC.sub.50 (nM) values (Table 2) which is
the concentration of the benzopyranone compound necessary to
inhibit the growth of BG-1 ovarian carcinoma cells by 50% relative
to controls. The results show that all the illustrative
benzopyranone compounds assayed show activity and, accordingly, are
useful for treating or preventing ovarian cancer in a patient.
2TABLE 2 In vitro data IC.sub.50(nM) Structure IL-6 (ER-.alpha.)
MCF-7 BG-1 35 1.5 13.6 13.6 36 0.5 4.5 4.65 37 1.0 26 5.5 38 0.40
3.0 2.3 39 0.40 26.0 5.8 40 0.29 6.5 1.4
[0177] Accordingly, the in vitro results of Examples 8-10 as
illustrated in Table 2 above, show that the benzopyranone compounds
of the present invention are useful for the treatment or prevention
of bone-resorbing diseases and various cancers.
Example 11
Rat Pharmacokinetic (PK) Analysis
[0178] Rat PK Cassette Standard Assay
[0179] An illustrative compound of formula (I), (II) or a
pharmaceutically acceptable salt thereof, and an internal standard
raloxifene is administered by oral gavage at a dose level of 5
mg/kg body weight. Blood is sampled over the time period from 15
min to 24 h postdose. Blood samples are prepared by acetonitrile
precipitation, centrifuged, and supernatants are evaporated in a
vacuum centrifuge. Dried residuals are dissolved in methanol/water
(60:40 v/v) containing 1% formic acid and analyzed by HPLC on an
UPTISPHERE.TM. C18 reversed-phase HPLC column (particle size: 3
.mu.m; column dimensions: 2.times.50 mm). Eluent A is 10%
acetonitrile in water with 0.1% formic acid (pH 2.1), eluent B is
90% acetonitrile with 10% water and 0.1% formic acid (pH 2.1). A
linear gradient is run from 5 to 100% B over 7 min followed by a 3
min hold at 100% B at a constant temperature of 50.degree. C. in
the column compartment. The flow rate is held constant at 0.4
mL/min. Sample injection volume is 10 .mu.L. The flow from the HPLC
system is directly introduced into the ion source of an Agilent
1100 series MS-detector (single quadrupole mass analyzer) and
subjected to atmospheric pressure electrospray ionization (positive
mode). All compounds are detected as protonated quasi-molecular
ions [M+H].sup.+. A structurally closely related SERM is used as an
analytical internal standard. Quantification of blood levels of the
compounds is based on a 7-level calibration curve (in triplicate)
using blank rat blood samples to which have been added stock
solutions of external and internal standards.
[0180] Rat PK Cassette Validation
[0181] Raloxifene alone is administered p.o. (3 mg/kg) to four
female rats each. Blood samples are taken and analyzed as described
above. The pharmacokinetic data generated from this validation
study is compared with the data for raloxifene obtained in cassette
dosing experiments to check for potential pharmacokinetic
interactions. Deviations exceeding the typical range of biological
variability (approx. .+-.50% max. for individual parameters) are
considered strongly indicative for pharmacokinetic interactions
between compounds in the cassette, and the respective data are
discarded.
[0182] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a few aspects of the invention and any
embodiments that are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within the scope of the appended claims.
[0183] A number of references have been cited, the entire
disclosures of which are incorporated herein by reference in their
entirety.
* * * * *