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  • C07D411/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D411/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D411/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms
  • C07D411/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D411/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • A—HUMAN NECESSITIES
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  • A61P19/00—Drugs for skeletal disorders
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P3/00—Drugs for disorders of the metabolism
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
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  • A61P5/00—Drugs for disorders of the endocrine system
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  • A61P5/30—Oestrogens
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  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/32—Antioestrogens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D327/00—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms
  • C07D327/02—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms one oxygen atom and one sulfur atom
  • C07D327/06—Six-membered rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D339/00—Heterocyclic compounds containing rings having two sulfur atoms as the only ring hetero atoms
  • C07D339/08—Six-membered rings

Definitions

• Naturally occurring and synthetic estrogens have broad therapeutic utility, including: relief of menopausal symptoms, treatment of acne, treatment of dysmenorrhea and dysfunctional uterine bleeding, treatment of osteoporosis, treatment of hirsutism, treatment of prostatic cancer, treatment of hot flashes and prevention of cardiovascular disease. Because estrogen is very therapeutically valuable, there has been great interest in discovering compounds that mimic estrogen-like behavior in estrogen responsive tissues.
• Bone loss occurs in a wide range of subjects, including women that are post-menopausal or have had a hysterectomy, patients who were or are currently being treated with corticosteroids, and patient's having gonadal dysgenesis.
• the current major bone diseases of public concern are osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget's disease, immobilization-induced osteopenia, and glucocorticoid-induced osteoporosis.
• All of these conditions are characterized by bone loss, resulting from an imbalance between bone resorption, i.e. breakdown, and bone formation, which continues throughout life at the rate of about 14% per year on the average.
• the rate of bone turnover differs from site to site, for example, it is higher in the trabecular bone of the vertebrae and the alveolar bone in the jaws than in the cortices of the long bones.
• the potential for bone loss is directly related to turnover and can amount to over 5% per year in vertebrae immediately following menopause, a condition which leads to increased fracture risk.
• Osteoporosis affects approximately 20 to 25 million post-menopausal women in the U.S. alone. It has been theorized that the rapid loss of bone mass in these women is due to the cessation of estrogen production of the ovaries. Since studies have shown that estrogen slows the reduction of bone mass due to osteoporosis, estrogen replacement therapy is a recognized treatment for post- menopausal osteoporosis.
• estrogen replacement therapy could be an effective treatment for such disease.
• side effects associated with long term estrogen use limit the use of this alternative.
• tamoxifen a dual antagonist and agonist of estrogen receptors
• tamoxifen a dual antagonist and agonist of estrogen receptors
• treatment with tamoxifen is less than ideal because tamoxifen's agonist behavior enhances its unwanted estrogenic side effects.
• tamoxifen and other compounds that agonize estrogen receptors tend to increase cancer cell production in the uterus.
• a better therapy for such cancers would be an anti-estrogen compound that has negligible or nonexistent agonist properties.
• estrogen can be beneficial for treating pathologies such as bone loss, increased lipid levels, and cancer
• long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine and endometrial cancers.
• Prostatic cancer In addition to post-menopausal women, men suffering from prostatic cancer can also benefit from anti-estrogen compounds.
• Prostatic cancer is often endocrine-sensitive; androgen stimulation fosters tumor growth, while androgen suppression retards tumor growth.
• the administration of estrogen is helpful in the treatment and control of prostatic cancer because estrogen administration lowers the level of gonadotropin and, consequently, androgen levels.
• the estrogen receptor has been found to have two forms: ER ⁇ and
• ER ⁇ Ligands bind differently to these two forms, and each form has a different tissue specificity to binding ligands.
• compounds that are selective for ER ⁇ or ER ⁇ and therefore confer a degree of tissue specificity to a particular ligand.
• What is needed in the art are compounds that can produce the same positive responses as estrogen replacement therapy without the negative side effects. Also need are estrogen-like compounds that exert selective effects on different tissues of the body.
• the compounds of the instant invention are ligands for estrogen receptors and as such may be useful for treatment or prevention of a variety of conditions related to estrogen functioning including: bone loss, bone fractures, osteoporosis, cartilage degeneration, endometriosis, uterine fibroid disease, hot flashes, increased levels of LDL cholesterol, cardiovascular disease, impairment of cognitive functioning, cerebral degenerative disorders, restinosis, gynacomastia, vascular smooth muscle cell proliferation, obesity, incontinence, and cancer, in particular of the breast, uterus and prostate.
• the present invention relates to compounds of the following chemical formula:
• Ri, R2 ; R3 ; and R4 are each independently selected from the group consisting of hydrogen, C ⁇ _5 alkyl, C3-.8 cycloalkyl, CJ-5 alkenyl, C2- 5 alkynyl, C3_8 cycloalkenyl, phenyl, heteroaryl, heterocyclical, CF3, - OR6, halogen, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H, -COOC ⁇ -5 alkyl, -COC1.5 alkyl, -CONZ2, -SO2NZ2, and -S ⁇ 2C ⁇ _5 alkyl, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, phenyl, heteroaryl, heterocyclical groups can be optionally substituted with C ⁇ _ 5 alkyl, C3..8 cycloalkyl, CF3, phenyl, heteroaryl,
• R5 is selected from the group consisting of C ⁇ _5 alkyl, C3-8 cycloalkyl, C2-5 alkenyl, C2-5 alkynyl, C3-8 cycloalkenyl, phenyl, heteroaryl, heterocyclical groups wherein said groups can be optionally substituted with C ⁇ .5 alkyl, C3-8 cycloalkyl, CF3, phenyl, heteroaryl, heterocyclical, -OR6, halogen, amino, C ⁇ .5 alkylthio, thiocyanato, cyano, -CO2H, -COOC ⁇ - 5 alkyl, -COC ⁇ -5 alkyl, -CONZ2, -SO2NZ2, and -SO2C 1-5 alkyl: X and Y are each independently selected from the group consisting of oxygen, sulfur,
• both Zs and the nitrogen to which they are attached may be taken together to form a 3-8 membered ring
• said ring may optionally contain atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen, wherein said ring may either be saturated or unsaturated, and the carbon atoms of said ring maybe optionally substituted with one to three substituents selected from the group consisting of Cf-5 alkyl, CF3, -OR6, halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano,
• Each V is independently selected from the group consisting of C ⁇ -5 alkyl, CF3, -OR6, halogen, amino, C1 -.5 alkylthio, thiocyanato, cyano, -CO2H -COOC ⁇ _ 5 alkyl, -COC]_5 alkyl, and -SO2CX.5 alkyl;
• n is independently an integer from one to five; and the pharmaceutically acceptable salts thereof.
• the present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and a pharmaceutically acceptable carrier.
• the present invention also relates to methods for making the pharmaceutical compositions of the present invention.
• the present invention also related to processes and intermediates useful for making the compounds and pharmaceutical compositions of the present invention.
• the present invention also relates to methods for eliciting an estrogen receptor modulating effect in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.
• the present invention also relates to methods for eliciting an estrogen receptor antagonizing effect in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.
• the estrogen receptor antagonizing effect can be either an ER ⁇ antagonizing effect, and ER ⁇ antagonizing effect or a mixed ER ⁇ and ER ⁇ antagonizing effect.
• the present invention also relates to methods for eliciting an estrogen receptor agonizing effect in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.
• the estrogen receptor agonizing effect can be either an ER ⁇ agonizing effect, and ER ⁇ agonizing effect or a mixed ER ⁇ and ER ⁇ agonizing effect.
• the present invention also relates to methods for treating or preventing disorders related to estrogen functioning, bone loss, bone fractures, osteoporosis, cartilage degeneration, endometriosis, uterine fibroid disease, cancer of the breast, uterus or prostate, hot flashes, cardiovascular disease, impairment of cognitive function, cerebral degenerative disorders, restenosis, gynacomastia, vascular smooth muscle cell proliferation, obesity and incontinence in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.
• the present invention also relates to methods for reducing bone loss, lowering LDL cholesterol levels and eliciting a vasodilatory effect, in a mammal in need thereof by administering the compounds and pharmaceutical compositions of the present invention.
• the present invention relates to compounds useful as estrogen receptor modulators.
• Compounds of the present invention are described by the following chemical formula:
• Rl, R2, R3 ? and R4 are each independently selected from the group consisting of hydrogen, C _5 alkyl, C3.8 cycloalkyl, C2-5 alkenyl, C2-
• X and Y are each independently selected from the group consisting of oxygen, sulfur, sulfoxide and sulfone;
• R6 is selected from the group consisting of hydrogen, C ⁇ _5 alkyl, benzyl, methoxymethyl, triorganosilyl, C ⁇ _5 alkylcarbonyl, alkoxycarbonyl and CONZ2;
• Each Z is independently selected from the group consisting of hydrogen, C ⁇ _5 alkyl, trifluoromethyl, wherein said alkyl group can be optionally substituted with C ⁇ _5 alkyl, CF3, -OR6, halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H -COOC ⁇ -5 alkyl, -COC ⁇ -5 alkyl, - CONV2, -SO2NV2, and -S ⁇ 2C ⁇ _5 alkyl;
• both Zs and the nitrogen to which they are attached may be taken together to form a 3-8 membered ring
• said ring may optionally contain atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen, wherein said ring may either be saturated or unsaturated, and the carbon atoms of said ring maybe optionally substituted with one to three substituents selected from the group consisting of C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ -.5 alkylthio, thiocyanato, cyano, -CO2H, -COOC1-5 alkyl, -COC ⁇ .5 alkyl, -CONV2, -SO2NV2, and - SO2Cj_5 alkyl; Each V is independently selected from the group consisting of C 1-5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H -COOC
• Rl ,R2 ,R3 and R are selected from the group consisting of hydrogen, C ⁇ _5 alkyl, C3-8 cycloalkyl, C ⁇ _5 alkenyl, C l-5 alkynyl, -OR6 and halogen.
• R5 is selected from the group consisting of C3-8 cycloalkyl, phenyl, heteroaryl and heterocyclical groups wherein said groups can be optionally substituted with -OR6 and halogen.
• R° is preferably selected from the group consisting of hydrogen, C ⁇ _5 alkyl, benzyl, methoxymethyl and triisopropylsilyl.
• the present invention also relates to a process for preparing a compound of formula I
• Ri is H, F, or Cl;
• R2 is H or OR6 ;
• R3 is H or OR6;
• R4 is H or CH3;
• R5 is C ⁇ _5 alkyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, phenyl, heteroaryl, or heterocyclical groups wherein said groups can be optionally substituted with C l-5 alkyl, C3-8 cycloalkyl, CF3, phenyl, heteroaryl, heterocyclical, -OR6, halogen, amino, C1.5 alkylthio, thiocyanato, cyano, carboxyl (-CO2H), carboalkoxyl (- COOC 1-5 alkyl), carbonyl (-COCl-5 alkyl, carboxamido (-CONZ2), sulfonamido (- SO2NZ2), and sulfonyl (-SO2C 1-5 alkyl);
• X and Y are each independently selected from the group consisting of oxygen, sulfur, sulfoxide and sulfone;
• Each Z is independently selected from the group consisting of hydrogen, C 1-5 alkyl, trifluoromethyl, wherein said alkyl group can be optionally substituted with C 1.5 alkyl, CF3, -OR° ⁇ halogen, amino, C 1-.5 alkylthio, thiocyanato, cyano, -CO2H - COOC ⁇ -5 alkyl, -COC ⁇ -5 alkyl, -CONV2, -SO2NV2, and -SO2C1-5 alkyl; Or both Zs and the nitrogen to which they are attached may be taken together to form a 3-8 membered ring, said ring may optionally contain atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen, wherein said ring may either be saturated or unsaturated, and the carbon atoms of said ring maybe optionally substituted with C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyan
• Each V is independently selected from the group consisting of C ⁇ _5 alkyl, CF3, -OR6, halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H, -COOC ⁇ _5 alkyl, -
• n is an integer from one to five; and the stereoisomer is cis; or a pharmaceutically acceptable salt thereof, comprising the steps of a) reacting a compound of formula II with a compound of formula III under basic conditions
• step b cyclizing IV, of step a, under acidic conditions in the presence of a reducing agent, to provide the cis compound of formula V
• the present invention also relates to a process for preparing a compound of formula ID
• Rl is H, F, or Cl
• R3 is H
• R+ is H or CH3
• the stereoisomer is cis
• the optical isomer is dextrorotatory (+), having the absolute configuration: (2S,
• step b cyclizing IVD, of step a, under acidic conditions in the presence of a reducing agent to provide the racemic, cis compound of formula VD
• step c) performing a chiral chromatography with VD, from step b, to resolve the enantiomeric forms to provide the dextrorotatory (+) isomer VID;
• step d removing either protecting group from VIID, from step d, to afford either a compound of formula VIIID or a compound of formula IXD
• the present invention also comprises a process according for preparing a compound of formula IE
• Ri is selected from the group consisting of H, F, or Cl;
• R3 and R are each H
• R7 is selected from the group consisting of H or OH; the stereoisomer is cis, and the optical isomer is dextrorotatory (+), having the absolute configuration (2S, 3R); or a pharmaceutically acceptable salt thereof comprising the steps of
• step a cyclizing IVE, of step a, under acidic conditions in the presence of a reducing agent to provide the racemic, cis compound of formula VE
• the present invention also relates to novel intermediates useful for preparing compounds and compositions described herein, i.e compounds of formula I, IA, IB, IC, ID and IE.
• An emobidment of the invention is an intermediate of the formula:
• Rl is H, F, or Cl
• R2 is H or OR 0
• R3 is H or OR6
• R is H or CH3;
• R5 is Ci-5 alkyl, C3-8 cycloalkyl, C3-8 Gycloalkenyl, phenyl, heteroaryl, or heterocyclical groups wherein said groups can be optionally substituted with C ⁇ -5 alkyl, C3-8 cycloalkyl, CF3, phenyl, heteroaryl, heterocyclical, -OR6, halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, carboxyl (-CO2H), carboalkoxyl (-COOCf-5 alkyl), carbonyl (-
• COC ⁇ -5 alkyl carboxamido (-CONZ2), sulfonamido (-SO2NZ2), and sulfonyl (-SO2CX-5 alkyl);
• 6 is H, benzyl, methyl, methoxymethyl, or trisopropylsilyl, with the proviso that when OR6 exists elsewhere, it is chemically differentiable;
• Each Z is independently selected from the group consisting of hydrogen, C ⁇ -5 alkyl, trifluoromethyl, wherein said alkyl group can be optionally substituted with C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H -COOC ⁇ -5 alkyl, -COC i_5 alkyl, -CONV2, -SO2NV2, and -S ⁇ 2C ⁇ protagonist5 alkyl; Or both Zs and the nitrogen to which they are attached may be
• Ri is H, F, or Cl
• R2 is H or OR ⁇
• R3 is H or OR6;
• R4 is H or CH3;
• R5 is C ⁇ _5 alkyl, C3_8 cycloalkyl, C3-8 cycloalkenyl, phenyl, heteroaryl, or heterocyclical groups wherein said groups can be optionally substituted with C ⁇ _5 alkyl, C3_8 cycloalkyl, CF3, phenyl, heteroaryl, heterocyclical, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, carboxyl (-CO2H), carboalkoxyl (-COOC ⁇ -5 alkyl), carbonyl (-
• COC ⁇ -5 alkyl carboxamido (-CONZ2), sulfonamido (-SO2NZ2), and sulfonyl (-S ⁇ 2C ⁇ _5 alkyl);
• R6 is H, benzyl, methyl, methoxymethyl, or triisopropylsilyl, with the proviso that when OR6 exists elsewhere, it is chemically differentiable;
• Each Z is independently selected from the group consisting of hydrogen, C ⁇ _5 alkyl, trifluoromethyl, wherein said alkyl group can be optionally substituted with C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, -CO2H -COOC1-5 alkyl, -COCj-5 alkyl, -CONV2, -SO2NV2, and -SO2C1-5 alkyl; Or both Zs may be taken together form a 3-8 membered ring, said ring may optionally contain atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen, wherein said ring may either be saturated or unsaturated, and the carbon atoms of said ring maybe optional
• Each V is independently selected from the group consisting of C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C 1-.5 alkylthio. thiocyanato, cyano, -CO2H -COOC i-
• Ri is H, F, or Cl
• R6 is H, benzyl, methyl, methoxymethyl, or triisopropylsilyl, with the proviso that all existing R6 groups are chemically differentiable.
• Ri is H, F, or Cl
• R6 is H, benzyl, methyl, methoxymethyl, or trisopropylsilyl, with the proviso that all existing R6 groups are chemically differentiable.
• R is H o CH3
• R5 is Ci-5 alkyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, phenyl, heteroaryl, or heterocyclical groups wherein said groups can be optionally substituted with C ⁇ _5 alkyl, C3-8 cycloalkyl, CF3, phenyl, heteroaryl, heterocyclical, -OR° ⁇ halogen, amino, C ⁇ _5 alkylthio, thiocyanato, cyano, carboxyl (-CO2H), carboalkoxyl (-COOC i-5 alkyl), carbonyl (- COC ⁇ -5 alkyl, carboxamido (-CONZ2), sulfonamido (-SO2NZ2), and sulfonyl (-S ⁇ 2C ⁇ _5 alkyl); R6 is H, benzyl, methyl, methoxymethyl, or triisopropylsilyl, with the proviso that when OR6 exists elsewhere, it is chemically different
• Each Z is independently selected from the group consisting of hydrogen, C ⁇ _5 alkyl, trifluoromethyl, wherein said alkyl group can be optionally substituted with C ⁇ _5 alkyl, CF3, -OR° ⁇ halogen, amino, C ⁇ -5 alkylthio, thiocyanato, cyano, -CO2H -COOCj-5 alkyl, -COCj-5 alkyl, -CONV2, -SO2NV2, and -SO2C ⁇ .5 alkyl;
• both Zs and the nitrogen to which they are attached may be taken to together form a 3-8 membered ring, said ring may optionally contain atoms selected from the group consisting of carbon, oxygen, sulfur, and nitrogen, wherein said ring may either be saturated or unsaturated, and the carbon atoms of said ring maybe optionally substituted with
• Each V is independently selected from the group consisting of C I _5 alkyl, CF3, -OR° ⁇ halogen, amino, C 1-.5 alkylthio, thiocyanato, cyano, -CO2H, -COOC )_ 5 alkyl, -COCj-5 alkyl, and -SO2C 1-.5 alkyl.
• Another embodiment of the invention is an intermediate of the formula: wherein Ri is H, F, or Cl;
• R6 is H, benzyl, methyl, methoxymethyl, or triisopropylsilyl, with the proviso that all existing R6 groups are chemically differentiable.
• Ri is H, F, or Cl
• R6 is H, benzyl, methyl, methoxymethyl, or triisopropylsilyl, with the proviso that all existing R6 groups are chemically differentiable.
• Non-limiting examples of the present invention include:
• An embodiment of the invention is a method of eliciting an estrogen receptor modulating effect in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of any of the compounds or any of the above pharmaceutical compositions described above.
• a class of the embodiment is the method wherein the estrogen receptor modulating effect is an antagonizing effect.
• a subclass of the embodiment is the method wherein the estrogen receptor is an ER ⁇ receptor.
• a second subclass of the embodiment is the method wherein the estrogen receptor is an ER ⁇ receptor,
• a third subclass of the embodiment is the method wherein the estrogen receptor modulating effect is a mixed ER ⁇ and ER ⁇ receptor antagonizing effect.
• a second class of the embodiment is the method wherein the estrogen receptor modulating effect is an agonizing effect.
• a subclass of the embodiment is the method wherein the estrogen receptor is an ER ⁇ receptor.
• a second subclass of the embodiment is the method wherein the estrogen receptor is an ER ⁇ receptor.
• a third subclass of the embodiment is the method wherein the estrogen receptor modulating effect is a mixed ER ⁇ and ER ⁇ receptor agonizing effect.
• Another embodiment of the invention is a method of treating or preventing post-menopausal osteoporosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
• Another embodiment of the invention is a method of treating or preventing uterine fibroids in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above,
• Another embodiment of the invention is a method of treating or preventing restenosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
• Another embodiment of the invention is a method of treating or preventing endometriosis in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
• Another embodiment of the invention is a method of treating or preventing hyperlipidemia in a mammal in need thereof by administering to the mammal a therapeutically effective amount of any of the compounds or pharmaceutical compositions described above.
• Exemplifying the invention is a pharmaceutical composition comprising any of the compounds described above and a pharmaceutically acceptable canicr. Also exemplifying the invention is a pharmaceutical composition made by combining any of the compounds described above and a pharmaceutically acceptable carrier. An illustration of the invention is a process for making a pharmaceutical composition comprising combining any of the compounds described above and a pharmaceutically acceptable carrier. Further exemplifying the invention is the use of any of the compounds described above in the preparation of a medicament for the treatment and/or prevention of osteoporosis in a mammal in need thereof.
• Still further exemplifying the invention is the use of any of the compounds described above in the preparation of a medicament for the treatment and or prevention of: bone loss, bone resorption, bone fractures, cartilage degeneration, endometriosis, uterine fibroid disease, breast cancer, uterine cancer, prostate cancer, hot flashes, cardiovascular disease, impairment of cognitive functioning, cerebral degenerative disorder, restenosis, vascular smooth muscle cell proliferation, incontinence, and/or disorders related to estrogen functioning.
• the present invention is also directed to combinations of any of the compounds or any of the pharmaceutical compositions described above with one or more agents useful in the prevention or treatment of osteoporosis.
• the compounds of the instant invention may be effectively administered in combination with effective amounts of other agents such as an organic bisphosphonate or a cathepsin K inhibitor.
• organic bisphosphonates include alendronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, risedronate, piridronate, pamidronate, tiludronate, zoledronate, pharmaceutically acceptable salts or esters thereof, and mixtures thereof.
• Preferred organic bisphosphonates include alendronate and pharmaceutically acceptable salts and mixtures thereof. Most preferred is alendronate monosodium tri hydrate.
• the precise dosage of the bisphosphonate ill vary with the dosing schedule, the oral potency of the particular bisphosphonate chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies. Generally, an appropriate amount of bisphosphonate is chosen to obtain a bone resorption inhibiting effect, i.e. a bone resorption inhibiting amount of the bisphosphonate is administered. For humans, an effective oral dose of bisphosphonate is typically from about 1.5 to about 6000 jttg kg body weight and preferably about 10 to about 2000 ⁇ g/kg of body weight.
• a unit dosage typically comprises from about 8J5 mg to about 140 mg of the alendronate compound, on an alendronic acid active weight basis, i.e. on the basis of the corresponding acid.
• salts of the compounds of this invention refer to non-toxic “pharmaceutically acceptable salts.”
• Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.
• salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts which are generally prepared by reacting the free base with a suitable organic or inorganic acid.
• Representative salts include but are not limited to the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, ox
• suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
• alkali metal salts e.g., sodium or potassium salts
• alkaline earth metal salts e.g., calcium or magnesium salts
• suitable organic ligands e.g., quaternary ammonium salts.
• the compounds of the present invention can have chiral centers and occur as racemates, racemic mixtures, diastereomeric mixtures, and as individual diastereomers, or enantiomers with all isomeric forms being included in the present invention. Therefore, where a compound is chiral, the separate enantiomers, substantially free of the other, are included within the scope of the invention; further included are all mixtures of the two enantiomers. Also included within the scope of the invention are polymorphs, hydrates and solvates of tiie compounds of the instant invention.
• the present invention includes within its scope prodrugs of the compounds of this invention.
• prodrugs will be functional derivatives of the compounds of this invention which are readily convertible in vivo into the required compound.
• the term “administering” shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient.
• Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985, which is incoiporated by reference herein in its entirety.
• Metabolites of these compounds include active species produced upon introduction of compounds of this invention into the biological milieu.
• the term "therapeutically effective amount” shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician.
• a base is a substance that accepts a proton; or according to the Lewis definition, a base is a substance that can furnish an electron pair to form a covalent bond.
• bases used herein are tertiary amine bases such as triethylamine, diisopropylethylamine, or the like.
• an acid is a substance that gives up a proton; or according to the Lewis definition, an acid is a substance that can take up an electron pair to form a covalent bond.
• acids used herein are strong carboxylic acids such as trifluoroacetic acid, or the like, strong sulfonic acids, such as trifluoromethane sulfonic acid, or the like, and Lewis acids, such as boron trifluoride etherate, or stannous chloride, or the like.
• reducing agent refers to a reagent capable of performing a reduction.
• a reduction is the conversion of a functional group or an intermediate from one category to a lower one.
• reducing agents used herein but arc not limited to, are triorganosilanes or stannanes, such as triethylsilanc, triphenylsilane, and tri-n-butyl tin hydride, or the like.
• chemically differentiable refers to two or more non- identical R6 substituents whose unique structures are such that one of ordinary skill in the art could choose reaction conditions which would convert one of the non-identical R6 substituents to H, without affecting the other R6 substituent.
• alkyl shall mean a substituting univalent group derived by conceptual removal of one hydrogen atom from a straight or branched-chain acyclic saturated hydrocarbon (i.e., -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH 2 CH 2 CH 3 , -CH 2 CH(CH 3 ) 2 , -C(CH 3 ) 3, etc.).
• cycloalkyl shall mean a substituting univalent group derived by conceptual removal of one hydrogen atom from a saturated monocyclic hydrocarbon (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl).
• cycloalkenyl shall mean a substituting univalent group derived by conceptual removal of one hydrogen atom from an unsaturated monocyclic hydrocarbon containing a double bond (i.e., cyclopentenyl or cyclohexenyl).
• heterocyclical shall mean a substituting univalent group derived by conceptual removal of one hydrogen atom from a heterocycloalkane wherein said heterocycloalkane is derived from the corresponding saturated monocyclic hydrocarbon by replacing one or two carbon atoms with atoms selected from N, 0 or S.
• heterocyclical groups include, but are not limited to, oxiranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl.
• Heterocyclical substituents can be attached at a carbon atom. If the substituent is a nitrogen containing heterocyclical substituent, it can be attached at the nitrogen atom.
• heteroaryl refers to a substituting univalent group derived by the conceptual removal of one hydrogen atom from a monocyclic or bicyclic aromatic ring system containing 1 , 2, 3, or 4 heteroatoms selected from N, O, or S.
• heteroaryl groups include, but are not limited to, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, benzimidazolyl, indolyl, and purinyl.
• Heteraryl substituents can be attached at a carbon atom or through the heteroatom.
• triorganosilyl means those silyl groups trisubstituted by lower alkyl groups or aryl groups or combinations thereof and wherein one substituent may be a lower alkoxy group.
• examples of triorganosilyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, triisopropylsilyl, triphenylsilyl, dimethylphenylsilyl, t-butyldiphenylsilyl, phenyl-t-butylmethoxysilyl and the like.
• alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclical and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms be alternative non-hydrogen groups.
• hydrogen atoms include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
• alkyl or aryl or either of their prefix roots appear in a name of a substituent (e.g., aryl Q)-8 alkyl) it shall be interpreted as including those limitations given above for "alkyl” and "aryl.”
• Designated numbers of carbon atoms e.g., C ⁇ - ⁇ n shall refer independently to the number of carbon atoms in an alkyl or cyclic alkyl moiety or to the alkyl portion of a larger substituent in which alkyl appears as its prefix root.
• arylalkyl and “alkyl aryl” include an alkyl portion where alkyl is as defined above and to include an aryl portion where aryl is as defined above.
• arylalkyl include, but are not limited to, benzyl, fluorobenzyl, chlorobenzyl, phenylethyl, phenylpropyl, fluorophenylethyl, chlorophenylethyl, thienylmethyl, thienylethyl, and thienylpropyl.
• alkylaryl include, but are not limited to, toluyl, ethylphenyl, and propylphenyl.
• heteroarylalkyl shall refer to a system that includes a heteroaryl portion, where heteroaryl is as defined above, and contains an alkyl portion.
• heteroarylalkyl include, but are limited to, pyridylmethyl, pyridylethyl and imidazoylmethyl.
• halo shall include iodo, bromo, chloro and fluoro.
• oxy means an oxygen (O) atom.
• thio means a sulfur (S) atom.
• substituted shall be deemed to include multiple degrees of substitution by a named substitutent. Where multiple substituent moieties arc disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
• Representative compounds of the present invention typically display submicromolar affinity for alpha and/or beta estrogen receptors. Compounds of this invention are therefore useful in treating mammals suffering from disorders related to estrogen functioning. Pharmacologically effective amounts of the compound, including the pharmaceutically effective salts thereof, are administered to the mammal, to treat disorders related to estrogen functioning, such as bone loss, hot flashes and cardiovascular disease.
• the compounds of the present invention are available in racemic form or as individual enantiomers. For convenience, some structures are graphically represented as a single enantiomer but, unless otherwise indicated, is meant to include both racemic and enantiomeric forms. Where cis and trans sterochemistry is indicated for a compound of the present invention, it should be noted that the stereochemistry can be construed as relative, unless indicated otherwise.
• Racemic mixtures can be separated into their individual enantiomers by any of a number of conventional methods. These include chiral chromatography, derivatization with a chiral auxiliary followed by separation by chromatography or crystallization, and fractional crystallization of d ⁇ astereomei ⁇ c salts.
• the compounds of the present invention can be used in combination with other agents useful for treating estrogen-mediated conditions.
• the individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.
• the instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. It will be understood that the scope of combinations of the compounds of this invention with other agents useful for treating estrogen-mediated conditions includes in principle any combination with any pharmaceutical composition useful for treating disorders related to estrogen functioning.
• composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
• the compounds of the present invention can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions. Likewise, they may also be administered in intravenous (bolus or infusion), intraperitoneal, topical (e.g., ocular eyedrop), subcutaneous, intramuscular or transdcrmal (e.g., patch) form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
• the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
• An ordinarily skilled physician, veterinarian or clinician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
• Oral dosages of the present invention when used for the indicated effects, will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day, and most preferably 0J to 5.0 mg/kg/day.
• compositions are preferably provided in the form of tablets containing 0.01, 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably, from about 1 mg to about 100 mg of active ingredient.
• the most preferred doses will range from about 0J to about 10 g/kg/minute during a constant rate infusion.
• compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
• preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
• the dosage administration will, of course, be continuous rather than intermittant throughout the dosage regimen.
• the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or earners (collectively referred to herein as 'earner' materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
• the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral drug components can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
• suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
• Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
• Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
• Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
• the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
• Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
• Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual earners to which the compound molecules are coupled.
• the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
• Such polymers can include polyvinylpy ⁇ olidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
• the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
• the novel compounds of the present invention can be prepared according to the procedure of the following schemes and examples, using appropriate materials and are further exemplified by the following specific examples.
• Intermediate IV was reductively cyclized in the presence of an organic acid such as trifluoroacetic acid, triflic acid, or the like, or a Lewis acid such as boron trifluoride etherate, stannous chloride, or the like, and a reducing agent such as a trisubstituted silane, such triethylsilane, or the like, in a solvent such as dichloromethane, chloroform, THF, toluene, or the like at a temperature of from - 40OC to 100°C for as long as it takes for the reaction to complete to provide the cyclized product V, in which the stereochemistry of the aryl substituent and R5 in the newly created ring is exclusively cis.
• an organic acid such as trifluoroacetic acid, triflic acid, or the like, or a Lewis acid such as boron trifluoride etherate, stannous chloride, or the like
• a reducing agent such as a trisubstituted
• R6 is a methoxymethyl (MOM) group
• R is -OR° ⁇ wherein R6 is a benzyl (Bn) group
• R5 is a phenyl ring substituted by R7 where R is OR° ⁇ wherein R6 is a triisopropylsilyl (TIPS) group
• all unspecified substitutents are hydrogen.
• the alcohol intermediate VI was then reacted with a reagent HO(CH2)n Z2 in a Mitsunobu reaction protocol, in which they are combined with a trisubstituted phosphine, such as triphenylphosphine and a diazodicarboxylate, such as diisopropylazodicarboxylate, in a suitable solvent such as THF at from 0°C to 80°C for as long as it takes for the reaction to complete to provide the coupled product I.
• a suitable solvent such as THF
• the ketone intermediate IV from Scheme I was reduced with sodium borohydride, super hydride, or the like, in a mixture of methanol and dichloromethane, or THF or the like at from 0°C to ambient temperature for from a few minutes to a few hours to provide the analogous hydroxyl intermediate VII.
• Cyclization of intermediate VII was accomplished in the presence of an acid catalyst such as amberlyst 15, or triflic acid or the like, in a solvent such toluene, or dichloromethane or the like, at a temperature of from ambient to reflux to afford the trans compound VIII as the major isomer.
• the compounds I of the invention are pcroxidized with an oxidant such as ///-chloroperbenzoic acid, or per-tr ⁇ fluoroacctic acid, or the like, in a solvent such dichloromethane or the like, at a temperature of from OOC to reflux to produce the trioxide intermediate X.
• oxidant such as ///-chloroperbenzoic acid, or per-tr ⁇ fluoroacctic acid, or the like
• a solvent such dichloromethane or the like
• X is preferably O
• Y is preferably S.
• Ri ,R2 ,R3 and R4 are preferably selected from the group consisting of hydrogen, C ⁇ _5 alkyl, C3-8 cycloalkyl, C ⁇ -5 alkenyl, C ⁇ -5 alkynyl, -OR6 and halogen.
• R5 is preferably selected from the group consisting of C3_8 cycloalkyl, phenyl, and substituted phenyl.
• R° is preferably selected from the group consisting of hydrogen, C ⁇ -5 alkyl, benzyl, methoxymethyl and trisopropylsilyl.
• Ri and R4 are hydrogen
• R2 and R are independently -OH
• R5 is independently selected from the group consisting of phenyl and substituted phenyl.
• Ri is independently selected fluorine and chlorine
• R is hydrogen
• R2 and R are independently -OH
• R5 is independently selected from the group consisting of phenyl and substituted phenyl.
• Rl and R are hydrogen and, R2 is -OH, and R5 is independently selected from the group consisting of phenyl and /wra-hydroxy-phenyl.
• SCHEME IV GENERAL SYNTHESIS FOR DIHYDROBENZOXATHIIN OXIDES
• X is preferably O
• Y is preferably S.
• Ri ,R2 ,R3 and R are preferably selected from the group consisting of hydrogen, C ⁇ -5 alkyl, C3 relieve8 cycloalkyl. C ⁇ _5 alkenyl, Cj-5 alkynyl, -OR6 and halogen.
• R5 is preferably selected from the group consisting of C3-8 cycloalkyl, phenyl, and substituted phenyl.
• R° is preferably selected from the group consisting of hydrogen, C ⁇ _5 alkyl, benzyl, methoxymethyl and trisopropylsilyl.
• Rl and R4 are hydrogen, R2 and R are independently -OH, and R5 is independently selected from the group consisting of phenyl and substituted phenyl.
• Rl is independently selected fluorine and chlorine
• R is hydrogen
• R2 and R3 are independently -OH
• R5 is independently selected from the group consisting of phenyl and substituted phenyl.
• Ri and R are hydrogen and, R2 is -OH, and R5 is independently selected from the group consisting of phenyl, metfl-hydroxy-phenyl, and para- hydroxy-phenyl.
• 2-cyclohexyl- l-(4-hydroxy-phenyl)-ethanone 70% yield using methylene chloride- ethyl acetate(50:l) as the chromatography eluant.
• 2-cyclopentyl- l -(4-hydroxy-phenyl)-ethanone 74% yield using methylene chloride- ethyl acctate(50: 1 ) as the chromatography eluant.
• 2-cyclopentyl-l-(4-triisopropylsilyloxy-phenyl)-ethanone use methylene chlor ⁇ de- hexanes(l: 1) as the chromatography eluant.
• 2-cyclopentyl-2-bromo-l-(4-triisopropylsiIyloxy-phenyl)-ethanone use methylene chloride-hexanes(lJ) as the chromatography eluant.
• 2-(4-pyridyl)-2-bromo-l-(4-triisopropylsiIyIoxy-phenyl)-ethanone added 2 equivalents of trimethylammoniumphenyl perbromide and stirred at 0 °C for 1 h; isolated as an orange/yellow oil and used in the next reaction without purification.
• Step B To a stirred solution of the product obtained from Step A (423mg, 1.55mmole) and imidazole (21 l mg, 3Jmmole) in 20mL of dry DMF at 0°C was added triisopropylsilyl chloride (3J mmole) and the reaction mixture was allowed to warm to room temperature and stirred further for 2-3 hours. The reaction was quenched by the addition of aqueous NaHCO 3 solution and extracted with EtOAc. The organic layer was washed with brine and dried with MgS0 4 . Chromatography (10%
• Bromoketone with MOM group ⁇ NMR (400 MHz, CDC1 3 ) ⁇ (ppm): 8.0 (d, 2H), 7.4 (d, 2H), 6.88 (d, 2H), 6.86 (d, 2H), 6.36 (s, IH), 1.24 (m, 3H), 1J (d, 18H); Bromoketone without MOM group: ⁇ NMR (400 MHz, CDC1 3 ) ⁇ (ppm): 7.94 (d, 2H), 7.4 (d, 2H), 6.88 (d, 2H), 6.86 (d, 2H), 6.36 (s, IH), 1.24 (m, 3H), 1J (d, 18H).
• Example 16 Following the procedure outlined in Example 16 and using 0.8g (1.57mmole) of the bromoketone from Example 12 (Step C) with the thiophenol derivative prepared from Example 1, the desired product was obtained after silica gel chromatography using EtOAc/hexane (1/5) as eluant.
• Example 16 Following the procedure outlined in Example 16 and using 0.56g (l. lmmole) of the bromoketone from Example 12 (Step C) with 0J9g (0J3mmole) of thiophenol derivative prepared from Example 1 , the desired product was obtained after silica gel chromatography using EtOAc/hexane (1/5) as eluant.
• Example 16 Following the procedure outlined in Example 16 and using 2.6g (5.82mmole) of the bromoketone from Example 13 with the thiophenol derivative prepared from Example 1, the desired product was obtained after silica gel chromatography using
• Example 16 Following the procedure outlined in Example 16 and using the bromoketone from Example 13 with the appropriate thiophenol derivative prepared from Example 1, the desired product was obtained after silica gel chromatography using EtOAc/hexane (1/5) as the eluant.
• 1H NMR 500 MHz, CDC1 3 ) ⁇ (ppm) 8.28 (s, IH), 7.82 (d, 2H), 7.40 (m, 5H), 7.22 (m, 5H), 6.80 (d, 2H), 6.40 (d, IH), 6.21 (dd, IH), 5.80 (s, IH), 5.00 (s, 2H), 1.24 (m, 3H), HO (d, 18H).
• Example 16 Following the procedure outlined in Example 16 and using the bromoketone from Example 12 with the thiophenol derivative prepared from Example 1 , the desired product was obtained after silica gel chromatography using EtOAc/hexane (1/5) as eluant.
• Example 16 Following the procedure outlined in Example 16 and using the bromoketone from Example 12 with the thiophenol derivative prepared from Example 1 , the desired product was obtained after silica gel chromatography using EtOAc/hexane ( 1/5) as eluant.
• Example 16 Following the procedure outlined in Example 16 and the bromoketone from Example 13 with the thiophenol derivative prepared from Example 1 , the desired product was obtained after silica gel chromatography using EtOAc/hexane ( 1/5) as eluant.
• Example 1 the two desired products I and II were obtained after silica gel chromatography using EtOAc/hexane (1/5) as eluant.
• Cyclohexyl derivative use methylene chloride/hexanes(3:l) as the chromatography eluant. ⁇ 500MHz NMR(CDCb) ppm( ⁇ ): l J2 (d, 18H), l. H-2.34 (m, 15H), 4J (d, I H), 5.0 (s, 2H), 6.44 (dd, IH), 6.54 (d, IH), 6.86 (m, 3H), 1.25-1.12 (m, 7H). Cyclopentyl derivative: use methylene chloride/hexanes(2: l ) as the chromatography eluant.
• Example 10 Utilizing the bromides prepared in Example 10 and the appropriate mercaptan prepared in Example 1 and employing the procedure outlined in Example 16 the following compounds were prepared:
• Cyclohexyl derivative use methylene chloride/hexanes(3: 1) as the chromatography eluant. ⁇ 500MHz NMR(CDC1 ) ppm( ⁇ ): 1 J2 (d, 18H), 1 J 1-2.3 (m, 15H), 4.24 (d, IH), 4.89 (m, 2H), 6.8-7.6 (m, I2H).
• Cyclopentyl derivative use methylene chloride/hexanes(2: l) as the chromatography eluant.
• 'H 500MHz NMR(CDC1 3 ) ppm( ⁇ )J J2 (d, 18H), 1.26-2J2 (m, UH), 2.5 (m, IH), 4.24 (d, IH), 4.9 (m, 2H), 6.8-7.69 (m, 12H).
• Example 10 Utilizing the appropriate bromide prepared in Example 10 and the mercaptoquinol [prepared according to the method of Burton, etal, J. Chem. Sac, 1952, 2193] and employing the procedure outlined in Example 16, the desired product was obtained as an orange/red oil after silica gel chromatography with 30% EtOAc/hexane as the eluant.
• Example 17 The ketone generated in Example 17 was converted to the desired product following the procedure described in Example 44 with the exception that 5 equivalents of TEA and 2 equivalents of Et 3 SiH was necessary to drive the reaction to completion.
• the MOM group was removed with mild acid treatment (2N-HCI, 75°C) to give the desired product.
• Example 18 The ketone generated in Example 18 was converted to the dihydrobenzoxathiin utilizing the procedure from Example 44 with the exception that 20 equivalents of TFA and 15 equivalents of EtiSiH were necessary to drive the reaction to completion.
• the desired product was isolated after purification by silica gel chromatography using 10% EtOAc/hexane as eluant.
• Example 19 The ketone generated in Example 19 was converted to the dihydrobenzoxathiin utilizing the procedure from Example 44 with the exception that the reaction was run at -10X ⁇ for 48 hours in the presence of 20 equivalents of TFA and 2 equivalents of Et 3 SiH.
• the desired product [with 20% recovered starting material] was isolated after purification by silica gel chromatography using 10% EtOAc/hexane as eluant.
• Example 71 Following the procedure outlined in Example 44 and using the ketone derivative obtained from Example 27, the desired product was obtained, which was subsequently desilylated using the procedure described in Example 71 (Step C).
• the desired product was obtained as an oil after purification by silica gel chromatography using 15% EtOAc/hexane as eluant.
• Step A To a stirred solution of a mixture of dihydrobenzoxathiin (60mg, 0J mmole), obtained from Example 48 (which was dried by the azeotropic method prior to use), triphenylphosphine (157mg, 0.6mmole), and 1-piperidineethanol (0.08mL, O. ⁇ mmole) in 4mL of anhydrous THF at 0°C was added dropwise 0J18mL (0.6mmole)of diisopropyl azodicarboxylate (DIAD) over 0.2 hours. The resulting pale yellow solution was stin-ed at room temperature for 2-3 hours.
• DIAD diisopropyl azodicarboxylate
• Example 71 Using the procedure described in Example 71 (Step A), the dihydrobenzoxathiin obtained from Example 53 was coupled with l-piperidineethanol. After purification by silica gel chromatography, using 3% MeOH CHTC as eluant, the desired adduct was obtained.
• Step A The adduct generated in Step A was debenzylated using the procedure described in Example 71 (Step B) to give the desired product.
• Step A The dihydrobenzoxathiin obtained from Example 45 was coupled with I- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography using 3% MeOH/CH-Cb as eluant, the desired adduct was obtained.
• Example 46 The dihydrobenzoxathiin obtained from Example 46 was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOHyCH-CL, the desired adduct was obtained as an oil.
• Step A The dihydrobenzoxathiin generated from Example 49 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Step A The dihydrobenzoxathiin obtained from Example 50 was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CHiC , the desired adduct was obtained.
• Step A The adduct generated in Step A was debenzylated using the procedure described in Example 71 (Step B).
• Step A The dihydrobenzoxathiin obtained from Example 51 was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH ⁇ Cb, the desired adduct was obtained.
• Step A The adduct generated in Step A was debenzylated using the procedure described in Example 71 (Step B). After purification by silica gel chromatography using 5% MeOH/QLCb as the eluant, the desired product was obtained as an oil.
• Example 53 The dihydrobenzoxathiin obtained from Example 53 was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH-Cb, the desired adduct was obtained.
• the debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C).
• the desired product was obtained as a white solid after silical gel chromatography with 5% MeOH/CJJXb as eluant.
• Example 54 The dihydrobenzoxathiin obtained from Example 54 was coupled with I- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH-C , the desired adduct was obtained.
• Step B The adduct generated in Step A was debenzylated using the procedure described in Example 71 (Step B). After purification by silica gel chromatography using 5% MeOH/CH-Cb as eluant, the desired product was obtained as an oil.
• Step C The debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Example 55 The dihydrobenzoxathiin generated from Example 55 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Step B The MOM protecting group was removed following the procedure outlined in
• Step B The silyl protecting group was removed following the procedure outlined in Example 71 (Step C).
• the desired product was isolated after purification by silica gel chromatography using 5% MeOH/CH 2 Cb as the eluant.
• EXAMPLE 85 PREPARATION OF
• Example 83 Utilizing the procedure from Example 83 (Step A), the dihydrobenzoxathiin (20mg, 0.028 mmole) obtained from Example 71 (Step A), was oxidized by m-CPBA at room temperature. The crude material was used for next step without further purification.
• Step A The product from Step A was deblocked using the standard procedure described in Example 71 (Step B) to afford the debenzylated product, which was used without further purification.
• the desilylated product (80mg, 0J65mmole) obtained from Step B was coupled with 1-piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH-CL, the desired adduct was obtained.
• Step E The crude product from Step D was deblocked using the standard procedure described in Example 71 (Step B) to afford the final product, after purification by silica gel chromatography using 5% MeOH CH-Cb as the eluant.
• Example 81 The racemic dihydrobenzoxathiin obtained from Example 81 (Step C) was resolved via chiral chromatography on a Chiralpak AD column, using 20% EtOH in hexane as the eluant.
• Example 82 The racemic dihydrobenzoxathiin obtained from Example 82 (Step C) was resolved via chiral chromatography on a Chiralpak AD column, using 20% EtOH in hexane as the eluant.
• Example 58 The dihydrobenzoxathiin generated from Example 58 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• 'H NMR 500 MHz, CDC1 3 ) ⁇ (ppm): 7.5-7.3 (m, 5H), 7.2-7 J (m, 3H), 6.85 (2d, 4H), 6.68 (d, 2H), 6.55 (d, 2H), 5.55 (d, IH), 5.04 (s, 2H), 4.40(d, IH).
• Step B The desilylated product obtained from Step A was coupled with 1-piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH 2 Cl 2 , the desired adduct was obtained.
• Step C A mixture of the adduct (80mg, 0J44mmole), generated in Step B, 20 mg of palladium black and 5 drops of AcOH in 4 mL of ethanol, was stirred under a balloon of hydrogen gas and monitored by TLC. After 18 hours, the reaction mixture was filtered through a pad of Celite to remove the catalyst, and the filtrate was neutralized by the addition of saturated, aqueous NaHC0 solution and extracted by EtOAc. The organic layer was separated, dried over MgSOj and concentrated in vacuo to give the desired product.
• Example 59 The dihydrobenzoxathiin generated from Example 59 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Example 60 The dihydrobenzoxathiin, obtained from Example 60, was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CTbCb the desired adduct was obtained.
• Step C The debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid. 'HNMR
• Example 61 The dihydrobenzoxathiin, obtained from Example 61, was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/QLCb the desired adduct was obtained.
• Step B The adduct, generated in Step A, was debenzylated using the procedure described in
• Example 62 The dihydrobenzoxathiin, obtained from Example 62, was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/HLCb the desired adduct was obtained.
• Example 63 The dihydrobenzoxathiin, obtained from Example 63, was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CH-Cb the desired adduct was obtained.
• the debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C).
• the desired product was obtained as a white solid after purification by silica gel chromatography with 5% MeOH/CH-Cb as eluant.
• Example 64 The dihydrobenzoxathiin, obtained from Example 64, was coupled with 1- piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/C ⁇ Cb the desired adduct was obtained.
• Step B The adduct, generated in Step A, was debenzylated using the procedure described in
• Example 65 The dihydrobenzoxathiin generated from Example 65 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Example 66 The dihydrobenzoxathiin generated from Example 66 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Example 67 The dihydrobenzoxathiin generated from Example 67 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Step B The desilylated product obtained from Step A was coupled with 1-piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CTbCb, the desired adduct was obtained.
• Step C The adduct, generated in Step B, was debenzylated using the procedure described in Example 71 (Step B) to afford the desired product.
• EXAMPLE 100 PREPARATION OF
• Example 68 The dihydrobenzoxathiin generated from Example 68 was desilylated using the procedure described in Example 71 (Step C). The desired product was obtained as a white solid.
• Example 100 The racemic dihydrobenzoxathiin obtained from Example 100 (Step C) was resolved via chiral chromatography on a Chiralpak AD column, using 20% EtOH in hexane as the eluant.
• the debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C).
• the desired product was obtained as a white solid after purification by silica gel chromatography with 5% MeOH/CH-Cb as eluant.
• Example 70 The slow moving (-)-dihydrobenzoxathiin obtained from Example 70 was coupled with 1-piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography with 3% MeOH/CHXb, the desired adduct was obtained. Step B
• the debenzylated product from Step B was desilylated using the procedure described in Example 71 (Step C).
• the desired product was obtained as a white solid after purification by silica gel chromatography with 5% MeOH/CH-Cb as eluant.
• reaction mixture was partitioned between ethyl acetate/saturated NaHC0 3 /ice/ brine, and the organic phase was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated.
• the residue was purified by silica gel chromatography using methylene chloride/hexanes( 1: 1) as eluant to provide the cis- cyclopentyl-dihydrobenzooxathiin derivative.
• the mixture was partitioned between ethyl acetate/2N HCl/ice/ brine, and the organic phase was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated.
• the residue was purified by silica gel chromatography using ethyl acetate-methanoI(9: 1) as eluant to provide the adduct.
• Example 105 Starting with the isopropyl adduct (0.0208 g, 0.049 mmol) prepared in Example 42 and utilizing the procedure outlined in Example 105 (Step A), the crude product was isolated after stimng at -23 °C for 6 h 20 min. Purification by silica gel chromatography with 30% EtOAc/hexane as the eluant afforded the desired product as a yellow oil.
• Step D the con-esponding c/Visopropyl-benzoxathiin adduct was prepared after silica gel chromatography with 10% MeOH/CH 2 Cb as the eluant.
• Step C Following the procedure detailed in Example 105 (Step C) with the exception that the reaction was allowed to warm from 0 °C to ambient temperature over 4 h, the material prepared in the previous step was converted to the desired product after silica gel chromatography (one elution with 30% EtOAc/hexane followed by a second elution with 10% MeOH/CH 2 Cb).
• Example 44 0.0792 g of the 3-pyridyl derivative prepared in Example 41 was converted to its con-esponding benzoxathiin after stirring at ambient temperature for 5 h.
• the desired product was isolated from the reaction mixture after silica gel chromatography using 30% EtOAc/hexane as the eluant.
• Step B the dihydrobenzoxathiin generated in Step A above was desilylated to afford the desired product after silica gel chromatography (one elution with 50% EtOAc/hexane followed by a second elution with 30% EtOAc/hexane).
• Step A Reductive Cyclization Following the procedure outlined in Example 44, 0J87I g of the 4-pyridyl derivative prepared in Example 41 was converted to its corresponding dihydrobenzoxathiin after stimng at ambient temperature for 30 h. The desired product was isolated from the reaction mixture after silica gel chromatography using 30% EtOAc/hexane as the eluant. ⁇ 500MHz NMR(CDCb) ppm( ⁇ ):l.U (d, 18H), 1.24 (m, 3H), 4.32 (d, IH), 5.08 (s, 2H), 5.50 (d, IH), 6.60-8.39 (m, I6H).
• Step B the dihydrobenzoxathiin generated in Step A above was desilylated to afford the desired product after silica gel chromatography (one elution with 50% EtOAc/hexane followed by a second elution with 30% EtOAc/hexane).
• Step C Mitsunobu reaction Following the procedure detailed in Example 105 (Step C) with the exception that the reaction was allowed to warm from 0 °C to ambient temperature over 5 h, the material prepared in the previous step was converted to the desired product after silica gel chromatography (one elution with 10% MeOH/CH . Cb followed by a second elution with 20% EtOAc/CH-Cb).
• Step D Debenzylation Starting with the material prepared in Step C above, and utilizing the procedure outlined in Example 105 (Step D), the desired product was obtained as a 4:1 cis/trans mixture after silica gel chromatography (IX elution with 30% EtOAc/hexane followed by a second elution with 10% MeOH/CH 2 Cb).
• Step B the material (0.0366 g, 0.089 mmol) generated in the previous step was converted to its corresponding trans- dihydrobenzoxathiin after stirring for 5 h 15 min. at ambient temperature. Purification by silica gel chromatography using 30% EtOAc/hexane as the eluant afforded the desired product as a white solid.
• the material (0.0266 g, 0.068 mmol) generated in the previous step was converted to its corresponding trans- isopropyl-dihydrobenzoxathiin adduct after warming from 0 °C to ambient temperature over 4 h 20 min. Purification by silica gel chromatography (one elution with 10% MeOH/CH 2 Cl 2 followed by a second elution with 30% EtOAc/hexane) afforded the desired product as a white solid.
• Step D the material (0.0395 g, 0.068 mmol) generated in the previous step was converted to its corresponding trans- isopropyl-dihydrobenzoxathiin product. Purification was accomplished by silica gel chromatography using 10% MeOH/CH 2 Cl 2 as the eluant.
• Example 40 Following the procedure outlined in Example 1 12 (Step A), the isopropyl-thio-ketone (0.6314 g, 1.5 mmol) generated in Example 40 was silylated. Purification by silica gel chromatography using 30% EtOAc/hexane as the eluant afforded the desired product as a yellow oil. ⁇ 500MHz NMR(CDCb) ppm( ⁇ ): 0.98-1.30 (m, 49H), 2.35 (m, IH), 4.38 (d, IH), 4.99 (q, 2H), 6.33-7J9 (m, 12H).
• Step B Reduction Following the procedure outlined in Example 112 (Step B), the material (0.8009 g, IJ mmol) isolated in Step A above was reduced to its corresponding alcohol and used without further purification in the next step.
• Step C Following the procedure outlined in Example 112 (Step C), the material (0.022 mmol) isolated in Step B above was deprotected to afford the desired product which was used in the next step without purification.
• Step E Mitsunobu reaction Following the procedure detailed in Example 105 (Step C), the material (0.008 g,
• Step D the material (0.0085 g, 0.017 mmol) generated in the previous step was converted to its corresponding trans- isopropyl-dihydrobenzoxathiin product. Purification was accomplished by silica gel chromatography using 10% MeOH/CH 2 Cl 2 as the eluant.
• Example xx The thiin obtained from Example xx was coupled with 1-piperidineethanol using the procedure described in Example 71 (Step A). After purification by silica gel chromatography using 3% MeOH/CH ⁇ Cb as eluant, the desired adducts were obtained as a mixture.
• the estrogen receptor ligand binding assays are designed as scintillation proximity assays employing the use of tritiated estradiol and recombinant expressed estrogen receptors.
• the full length recombinant human ER- ⁇ and ER- ⁇ proteins are produced in a bacculoviral expression system.
• ER- ⁇ or ER- ⁇ extracts are diluted 1:400 in phosphate buffered saline containing 6 mM ⁇ -monothiolglycerol. 200 ⁇ L aliquots of the diluted receptor preparation are added to each well of a 96-well Flashplate. Plates are covered with Saran Wrap and incubated at 4 ° C overnight.
• Test compounds are evaluated over a range of concentrations from 0.01 nM to 1000 nM.
• the test compound stock solutions should be made in 100% DMSO at 100X the final concentration desired for testing in the assay.
• the amount of DMSO in the test wells of the 96 well plate should not exceed 1 %.
• the final addition to the assay plate is a 2 ul aliquot of the test compound which has been made up in 100% DMSO. Seal the plates and allow them to equilibrate at room temperature for 3 hours. Count the plates in a scintillation counter equipped for counting 96 well plates. Ovariectomized Rat Assay
• OVX Rat Assay In the ovariectomized (OVX) Rat Assay, estrogen-deficiency is used to induce cancel lous osteopenia (e.g. low bone mineral density [BMD; mg/cm"]), associated with accelerated bone resoiption and formation. Both the BMD and bone resoi tion/formation outcomes are used to model the changes in bone that occur as women pass through menopause.
• BMD bone mineral density
• the OVX Rat Assay is the principal in vivo assay used by all major academic and industrial laboratories studying the efficacy of new chemical entities in preventing estrogen-deficiency bone loss. Sprague-Dawley female rats aged 6-8 months are OVXd and, within
• Test compounds may be administered orally, subcutaneously, or by infusion through subcutaneously-implanted minipump. Before necropsy, in vivo dual labeling with calcein (8 mg/kg by subcutaneous injection), a bone seeking fluorochrome, is completed. At necropsy, blood, femurs, a vertebral body segment, and the uterus, are obtained.
• the routine endpoints for the OVX Rat Assay include assessments of bone mass, bone resorption, and bone formation. For bone mass, the endpoint is
• BMD of the distal femoral metaphysis a region that contains about 20% cancellous bone.
• the vertebral segment, a region with -25% cancellous bone may also be used for BMD determination.
• the BMD measurement is made by dual energy x-ray absorptiometry (DXA, Hologic 4500A; Waltham, MA).
• DXA dual energy x-ray absorptiometry
• Hologic 4500A Hologic 4500A
• the endpoint is urinary deoxypyridinoline crosslinks, a bone collagen breakdown product (uDPD; expressed as nM DPD/ nM creatinine). This measurement is made with a commercially available kit (Pyrilinks; Metra Biosystems, Mountain View, CA).
• the endpoints are mineralizing surface and mineral apposition rate, histomorphometi ⁇ c measures of osteoblast number and activity. This measurement is done on 5 ⁇ m sections of the non-decalcified proximal tibial metaphysis, using a semi- automated system (Bioquant; R&M Biometrics; Nashville, TN). Similar endpoints and measuring techniques for each endpoint are commonly used in postmenopausal women. Rat Cholesterol Lowering Assay
• Sprague-Dawley rats (5 per group) weighing about 250g were subcutaneously dosed with compounds of the present invention dissolved in propylene glycol for 4 days. A group of 5 rats were dosed with vehicle only. On the fifth day, rats were euthanized with carbon dioxide and their blood samples were obtained. Plasma levels of cholesterol were assayed from these samples with commercially available cholesterol determination kits from Sigma.
• MCF-7 Estrogen Dependent Proliferation Assay MCF-7 cells are human mammary gland adenocarcinoma cells that require estrogen for growth.
• the growth media (GM) for the MCF-7 cells is Minimum Essential Media (without phenol red) supplemented with fetal bovine serum(FBS) to 10%.
• FBS fetal bovine serum
• the FBS serves as the sole source of estrogen and this GM supports the full growth of the cells and is used for the routine growth of the cell cultures.
• CD-FBS Charcoal- Dextran treated fetal bovine serum
• the CD-FBS does not contain detectable levels of estrogen and the media containing this sera is refen'ed to as Estrogen Depleted Media (EDM).
• EDM Estrogen Depleted Media
• the culture supernatant is aspirated and replaced with fresh EDM and test compound dilutions as above.
• the assay is terminated at day 8-10 when the appropriate controls reach 80-90% confiuency.
• the culture supernatants are aspirated, the cells washed 2X with PBS, the wash solution aspirated and the protein content of each well determined.
• Each drug dilution is evaluated on a minimum of 5 wells and the range of dilution of the test compounds in the assay is 0.00 InM to lOOOnM.
• the assay in the above format is employed to determine the estradiol agonist potential of a test compound.
• the MCF-7 cells are maintained in EDM for a minimum of 6 days. Then on day 0 (at the start of the assay), these estrogen depleted cells are plated into 96-well cell culture plates at a density of 1000 cells/well in EDM in a volume of 180ul/well. On day 1 the test compounds in fresh media containing 3 pM estradiol are applied to the cells. On days 4 and 7 of the assay, the culture supernatant is aspirated and replaced with fresh EDM containing 3 pM estradiol and the test compound. The assay is terminated at day 8-10 when the appropriate controls reach 80-90% confiuency and the protein content of each well is determined as above.
• Rattus norvegicus Strain Sprague-Dawley CD
• Rats are single-housed in polycarbonate cages and are provided Teklad Global Diet 2016 (Madison, WI) and bottled reverse osmosis purified H20 ad libitum. They are maintained on ai2/12 light/dark cycle.
• Rats are anesthetized with TelazolTM (20 mg/kg, ip) and oxymoiphone (0.2 mg/kg sc) and positioned dorsoventrally on a sterile drape. Body temperature is maintained using a underlying circulating water blanket. The surgical sites are shaved with clippers and cleaned using three cycles of betadine/ isopropyl alcohol or Duraprep® (3M). The incisional area is covered with a sterile drape.
• a 5 cm midline lower abdominal incision is made through the skin, subcutaneous and muscle layers.
• a bilateral ovariectomy is performed.
• the left uterine blood vessels are ligated and a 7 mm segment of the left uterine horn is excised.
• the uterus is closed with 4-0 gut suture.
• the myometrium is aseptically separated from the endometrium and trimmed to 5X5 mm.
• the trimmed section of the endometrium is transplanted to the ventral peritoneal wall with the epithelial lining of the segment opposed to the peritoneal wall.
• the explanted endometrial tissue is sutured at its four comers to the body wall using sterile 6-0 silk.
• the abdominal muscular layer is closed using sterile 4-0 chromic gut.
• the skin incision is closed using sterile stainless surgical clips.
• a sterile 90-day sustained release estrogen pellet (Innovative Research of America, 0J2 ng/pellet; circulating estrogen equivalent of 200-250 pg/mL) is implanted subcutaneously in the dorsal lateral scapular area.
• a sterile implantable programmable temperature transponder (IPTT) (BMDS, Seaford, DE) is injected subcutaneously in the dorsoscapular region. The rats are observed until fully ambulatory, and allowed to recover from surgery undisturbed for 3 weeks.
• the animals undergo a repeat laparotomy using aseptic surgical site preparation and technique.
• the explant is evaluated for graft acceptance, and the area is measured with calipers and recorded.
• the animals with rejected grafts are removed from the study. Animals are sorted to create similar average explant volume per group.
• Drug or vehicle(control) treatment is initiated one day after the second laparotomy and continued for 14 days. Body temperature is recorded every other day at 10:00 am using the BMDS scanner.
• the animals are euthanized by CO 2 overdose. Blood is collected by cardiocentesis for circulating estrogen levels. The abdomen is opened, the explant is examined, measured, excised, and wet weight is recorded. The right uterine horn is excised, and wet and dry weights are recorded.
• 25 mg of the compound from Example 71 is formulated with sufficient finely divided lactose to provide a total amound of 580 to 590 mg to fill a size 0, hard-gelatin capsule.

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