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Definitions

• This invention relates to 6H-[1]benzopyrano[4,3-b]quinoline compounds, their use as estrogenic agents, and methods of their preparation.
• Estrogens can exert effects on tissues in several ways, and the most well characterized mechanism of action is their interaction with estrogen receptors leading to alterations in gene transcription.
• Estrogen receptors are ligand-activated transcription factors and belong to the nuclear hormone receptor superfamily. Other members of this family include the progesterone, androgen, glucocorticoid and mineralocorticoid receptors.
• these receptors Upon binding ligand, these receptors dimerize and can activate gene transcription either by directly binding to specific sequences on DNA (known as response elements) or by interacting with other transcription factors (such as AP1), which in turn bind directly to specific DNA sequences (see Moggs and Orphanides, EMBO Reports 2: 775-781 (2001), Hall, et al., Journal of Biological Chemistry 276: 36869-36872 (2001), McDonnell, Principles of Molecular Regulation 351-361 (2000)).
• a class of "coregulatory" proteins can also interact with the ligand-bound receptor and further modulate its transcriptional activity (see McKenna, et al., Endocrine Reviews 20: 321-344 (1999)).
• estrogen receptors can suppress NF ⁇ B-mediated transcription in both a ligand-dependent and independent manner (see Quaedackers, et al., Endocrinology 142: 1156-1166 (2001), Bhat, et al., Journal of Steroid Biochemistry & Molecular Biology 67: 233-240 (1998), Pelzer, et al., Biochemical & Biophysical Research Communications 286: 1153-7 (2001)).
• Estrogen receptors can also be activated by phosphorylation. This phosphorylation is mediated by growth factors such as EGF and causes changes in gene transcription in the absence of ligand (see Moggs and Orphanides, EMBO
• estrogens can affect cells through a so-called membrane receptor.
• membrane receptor A less well-characterized means by which estrogens can affect cells is through a so-called membrane receptor.
• the existence of such a receptor is controversial, but it has been well documented that estrogens can elicit very rapid non-genomic responses from cells.
• the molecular entity responsible for transducing these effects has not been definitively isolated, but there is evidence to suggest it is at least related to the nuclear forms of the estrogen receptors (see Levin, Journal of Applied Physiology 91 : 1860-1867 (2001), Levin, Trends in Endocrinology & Metabolism 10: 374-377 (1999)).
• ERa The first estrogen receptor was cloned about 15 years ago and is now referred to as ERa (see Green, et al., Nature 320: 134-9 (1986)).
• the second form of the estrogen receptor was found comparatively recently and is called ER ⁇ (see Kuiper, et al., Proceedings of the National Academy of Sciences of the United States of America 93: 5925-5930 (1996)).
• ER ⁇ Early work on ER ⁇ focused on defining its affinity for a variety of ligands and indeed, some differences with ERa were seen. The tissue distribution of ER ⁇ has been well mapped in the rodent and it is not coincident with ERa.
• Tissues such as the mouse and rat uterus express predominantly ERa, whereas other tissues such as the mouse and rat lung express predominantly ER ⁇ (see Couse, et al., Endocrinology 138: 4613-4621 (1997), Kuiper, et al., Endocrinology 138: 863-870 (1997)). Even within the same organ, the distribution of ERa and ER ⁇ can be compartmentalized.
• ER ⁇ is highly expressed in the granulosa cells and ERa is restricted to the thecal and stromal cells (see Sar and Welsch, Endocrinology 140: 963-971 (1999), Fitzpatrick, et al., Endocrinology 140: 2581-2591 (1999)).
• the receptors are 5 031696
• estradiol A large number of compounds have been described that either mimic or block the activity of 17 ⁇ -estradiol.
• Estrogen receptor antagonists Those which, when given in combination with 17 ⁇ -estradiol, block its effects are called “estrogen receptor antagonists”.
• Estrogen receptor antagonists In reality there is a continuum between estrogen receptor agonist and estrogen receptor antagonist activity, and indeed, some compounds behave as estrogen receptor agonists in some tissues and estrogen receptor antagonists in others.
• SERMS selective estrogen receptor modulators
• EVISTA ®
• McDonnell Journal of the Society for Gynecologic Investigation 7: S10-S15 (2000), Goldstein, et al., Human Reproduction Update 6: 212-224 (2000).
• the precise reason why the same compound can have cell-specific effects has not been elucidated, but the differences in receptor conformation and/or in the milieu of coregulatory proteins have been suggested.
• phage display has been used to identify peptides that interact with estrogen receptors in the presence of different ligands (see Paige, et al., Proceedings of the National Academy of Sciences of the United States of America 96: 3999-4004 (1999)). For example, a peptide was identified that distinguished between ERa bound to the full estrogen receptor agonists 17 ⁇ -estradiol and diethylstilbesterol. A different peptide was shown to distinguish between clomiphene bound to ERa and ER ⁇ . These data indicate that each ligand potentially places the receptor in a unique and unpredictable conformation that is likely to have distinct biological activities.
• estrogens affect a panoply of biological processes.
• gender differences e.g. disease frequencies, responses to challenge, etc.
• the explanation involves the difference in estrogen levels between males and females.
• This invention is directed to these, as well as other, important ends.
• the present invention provides 6H-[1]benzopyrano[4,3-b]quinoline compounds that find use as estrogenic agents.
• the compounds have the formula I:
• R 1 and R 2 are independently selected from the group consisting of H, halogen, C 1 -C 6 alkyl, C 1 -C 6 perhaloalkyl, CF 3 , C 2 -C 7 alkenyl and C 1 -C 6 alkoxy;
• R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of H, halogen, CF 3 , C 1 -C 6 perhaloalkyl, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, C 3 - C 7 cycloalkyl, C 1 -C 6 alkoxy, CN, -CHO, acyl, phenyl, aryl and heteroaryl; wherein the alkyl or alkenyl moieties of R 3 , R 4 , R 5 and R 6 can each be optionally substituted with up to three substituents independently selected from halogen, OH, CN, trifluoroalkyl, trifluoroalkoxy, NO 2 or phenyl, wherein said phenyl is optionally substituted with up to three independently selected R 10 groups; wherein the alkynyl moiety of R 3 , R 4 , R 5 and R 6 can each
• the compounds of the invention are estrogen receptor modulators useful in the treatment or inhibition of conditions, disorders, or disease states that are at least partially mediated by an estrogen deficiency or excess, or which may be treated or inhibited through the use of an estrogenic agent.
• the invention is directed to the use of the compounds of the invention in the treatment or prevention of diseases such as osteoporosis, inflammatory bowel diseases, Crohn's disease, ulcerative proctitis, colitis, estrogen dependent cancers, hypercholesteremia, hyperlipidemia, cardiovascular disease, atherosclerosis, senile dementias, Alzheimer's disease, anxiety disorders, neurodegenerative disorders, infertility, or arthritis.
• diseases such as osteoporosis, inflammatory bowel diseases, Crohn's disease, ulcerative proctitis, colitis, estrogen dependent cancers, hypercholesteremia, hyperlipidemia, cardiovascular disease, atherosclerosis, senile dementias, Alzheimer's disease, anxiety disorders, neurodegenerative disorders, infertility, or arthritis.
• the present invention provides 6H-[1]benzopyrano[4,3-b]quinoline compounds, compositions containing the compounds, and methods for use of the compounds as estrogenic agents.
• the compounds of the invention are useful in the treatment and prevention of diseases associated with the estrogen receptor, particularly ER ⁇ .
• the estrogenic compounds of the invention have the formula I:
• R 1 and R 2 are independently selected from the group consisting of H, halogen, C 1 -C 6 alkyl, C 1 -C 6 perhaloalkyl, CF 3 , C 2 -C 7 alkenyl and C 1 -C 6 alkoxy;
• R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of H, halogen, CF 3 , C 1 -C 6 perhaloalkyl, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, C 3 - C 7 cycloalkyl, C 1 -C 6 alkoxy, CN, -CHO, acyl, phenyl, aryl and heteroaryl; wherein the alkyl or alkenyl moieties of R 3 , R 4 , R 5 and R 6 can each be optionally substituted with up to three substituents independently selected from halogen, OH, CN, trifluoroalkyl, trifluoroalkoxy, NO 2 or phenyl, wherein said phenyl is optionally substituted with up to three independently selected R 10 groups; wherein the alkynyl moiety of R 3 , R 4 , R 5 and R 6 can each
• a and A' are each OH. In some further embodiments, one of A and A' is OH, and the other of A and A' is OR. In some further embodiments, one of A and A' is OH, and the other of A and A' is 0-C 1 -C 6 alkyl. In some further embodiments, A and A' are each OR. In still further embodiments, A and A' are each -0-C 1 -C 6 alkyl. In still further embodiments, one of A and A' is H, and the other of A and A' is OH or OR. In further embodiments, one of A and A' is H, and the other of A and A' is OH or 0-C 1 -C 6 alkyl.
• R 3 and R 5 are each, independently, H, halogen, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, -CN, -CHO, acyl or optionally substituted phenyl, as previously described. In some such embodiments, R 3 is other than H.
• R 3 is halogen, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, -CN, -CHO, or phenyl optionally substituted with up to three groups selected from halogen, -0-C 1 -C 6 alkyl (i.e., C 1 -C 6 alkoxy), perfluoroalkyl and CN; and R 5 is H, halogen, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, -CN, -CHO, or phenyl optionally substituted with up to three groups selected from halogen, C 1 -C 6 alkoxy, perfluoroalkyl and CN.
• the phenyl of R 3 is optionally substituted with up to three substituents selected from F, Cl, Br, CN, OCH 3 and CF 3 .
• R 3 is halogen, C 2 -C 7 alkynyl or -CN.
• R 3 and R 5 are each independently halogen, C 2 -C 7 alkynyl or -CN.
• one of R 1 and R 2 is halogen. In some preferred embodiments, one of R 1 and R 2 is fluorine. In some further embodiments, one of R 1 and R 2 is halogen, and the other of R 1 and R 2 is H. In some further embodiments, one of R 1 and R 2 is fluorine, and the other of R 1 and R 2 is H, In some further embodiments, R 1 and R 2 are each independently halogen. In some further embodiments, R 1 and R 2 are each fluorine. In some further embodiments, R 1 and R 2 are each H. In some embodiments, R 4 is H, halogen or -CN, preferably H.
• R 3 is halogen, C 1 -C 6 alkyl, C 2 -C 7 alkenyl, C 2 -C 7 alkynyl, -CN, -CHO, or phenyl optionally substituted with up to three groups selected from halogen, C 1 -C 6 alkoxy, perfluoroalkyl and CN;
• R 5 is H, halogen, C 1 -C 6 alkyl, C 2 - C 7 alkenyl, C 2 -C 7 alkynyl, -CN, -CHO, or phenyl optionally substituted with up to three groups selected from halogen, C 1 -C 6 alkoxy, perfluoroalkyl and CN; and one of R 1 and R 2 is halogen; and R 4 is H, halogen or -CN.
• R 2 , R 4 , R 5 and R 6 are hydrogen, and R 3 is halogen, or R 3 is OH, or R 3 is C 2 -C 7 alkenyl, or R 3 is CN, or R 3 is C 2 -C 7 alkynyl, or R 3 is C 1 -C 6 alkyl, or R 3 is optionally substituted phenyl, preferably wherein the substituents of the phenyl are halogen, C r
• n 1
• the invention provides compositions containing one or more compounds of the invention, or pharmaceutically acceptable salts, chelates, complexes or prodrugs thereof.
• Pharmaceutically acceptable salts can be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable acids when a compound of this invention contains a basic moiety.
• organic and inorganic acids for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluen
• Salts may also be formed from organic and inorganic bases, such as alkali metal salts (for example, sodium, lithium, or potassium), alkaline earth metal salts, ammonium salts, alkylammonium salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in-each alkyl group, when a compound of this invention contains an acidic moiety.
• alkali metal salts for example, sodium, lithium, or potassium
• alkaline earth metal salts such as sodium, lithium, or potassium
• ammonium salts for example, sodium, lithium, or potassium
• alkylammonium salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon atoms in each alkyl group such as sodium, lithium, or potassium
• alkaline earth metal salts such as sodium, lithium, or potassium
• ammonium salts for example, sodium
• Exemplary salts further include acid-addition salts, e.g., HCI, H 2 SO 4 , HBr, HI, HNO 3 , H 3 PO 4 , NaH 2 PO 4 , Na 2 HPO 4 , H 3 PO 3 , NaH 2 PO 3 , Na 2 HPO 4 , H 2 SO 4 , NaHSO 4 , carboxylic acids, such as acetic acid, malonic acid, capric acid, lauric acid, dichloroacetic acid, trichloroacetic acid, etc., and other pharmacologically tolerated salts. Hydrates include hemihydrates, monohydrates, dihydrates, etc. Unless otherwise modified herein, the use of a free base formula is intended to include the salt and/or hydrate thereof.
• the instant invention also encompasses N-oxide derivatives of the compounds disclosed herein.
• These N-oxides can be prepared by methods known for preparing analogous compounds.
• the compounds maybe oxidized with a peracid, hydrogen peroxide, an alkali metal peroxide or an alkyl peroxide.
• One useful N-oxide derivative is a composition where the nitrogen atom of the quinoline ring forms the N-oxide group.
• the instant invention also encompasses prodrug derivatives.
• Prodrug derivative or “prodrug” means derivatives of the instant compounds that are converted in vivo to the corresponding non-derivatized form of the instant compounds.
• alkyl refers to an aliphatic hydrocarbon chain and includes, but is not limited to, straight and branched chains containing from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms, unless explicitly specified otherwise.
• methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc. are encompassed by the term "alkyl.”
• the number of carbon atoms as used in the definitions herein refers to the carbon backbone and carbon branching of the moiety, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like, of the moiety.
• alkenyl refers to an aliphatic hydrocarbon chain and includes, but is not limited to, straight and branched chains having 2 to 8 carbon atoms, e.g., 2-7 carbon atoms and containing at least one double bond.
• the alkenyl moiety has 1 or 2 double bonds.
• vinyl, allyl, 1-methyl vinyl, etc. are encompassed by the term "alkenyl”.
• alkenyl moieties may exist in the E or Z conformations and the compounds of this invention include both conformations.
• alkynyl refers to an aliphatic hydrocarbon chain and includes, but is not limited to, straight and branched chains having 2 to 8 carbon atoms, e.g., 2-7 carbon atoms and containing at least one triple bond. Preferably, the alkynyl moiety has 1 or 2 triple bonds. For example, ethynyl, propynyl, etc. are encompassed by the term “alkynyl”.
• acyl refers to alkylcarbonyl groups, e.g., where alkyl is as defined herein.
• benzyl has its accustomed meaning as a phenylmethyl group.
• aroyl refers to an aryl moiety connected through a carbonyl group, such as a benzoyl group.
• the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, aroyl, acyl and phenyl groups that are described herein for variables R 1 , R 2 R 3 , R 4 , R 5 , R 6 , A and A' can be optionally substituted with one or more substituents, preferably, with up to three substituents.
• preferred substituents include halogen, OH, CN, trifluoroalkyl, trifluoroalkoxy, perfluoroalkyl, perfluoroalkoxy, arylalkyl, alkylaryl, NO 2 and phenyl, wherein said phenyl is optionally substituted with up to three independently selected R 10 groups as described herein.
• alkyl or alkenyl moieties when substituted, they can typically be mono-, di-, tri- or persubstituted.
• a halogen substituent include 1- bromo vinyl, 1-fluoro vinyl, 1 ,2-difluoro vinyl, 2,2-difluorovinyl, 1 ,2,2-trifluorovinyl, 1 ,2- dibromo ethane, 1 ,2-fluoro ethane, 1-fluoro-2-bromo ethane, CF 2 CF 3 , CF 2 CF 2 CF 3 , and the like.
• halogen includes fluorine, chlorine, bromine, and iodine.
• exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, etc.
• the cycloalkyl groups have 3-10 carbon atoms.
• cycloalkyl groups have 3-7 carbon atoms.
• cycloalkyl further includes unsaturated cycloalkyl groups, i.e., cycloalkenyl groups.
• Exemplary unsaturated cycloalkyl groups include cyclopentenyl, cyclohexenyl, cycloheptenyl, etc.
• Aryl groups are moieties that possess at least one aromatic ring containing no hetero (i.e., non-carbon) ring atoms.
• aryl includes mono- and polycyclic aromatic ring systems, e.g., of 6-15 carbon atoms, for example, phenyl, naphthyl, etc.
• Aryl groups can have fully or partially saturated rings fused to the aromatic ring.
• exemplary aryl groups include phenyl, naphthyl, pyrenyl, 5,6,7,8- tetrahydronaphth-1-yl, and the like.
• heteroaryl is intended to mean an aromatic ring system that contains at least one non-carbon ring atom (e.g., one to three heteroatoms) selected from O, N and S and having for example five to 14 ring atoms.
• exemplary heteroaryl groups include pyrrolyl, imidazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, quinolyl, quoxalinyl, quinazolinyl, thiophenyl, furanyl, oxazolyl, thiazolyl, thienyl, pyranyl, thiopyranyl, benzofuranyl, indolyl, indazolyl, benzimidazolyl, benzothiazolyl, benzopyranyl, benzothiopyranyl, indazolyl, pyridopyrrolyl, and the like.
• the term "providing,” with respect to providing a compound or substance covered by this invention means either directly administering such a compound or substance, or administering a prodrug, derivative, or analog that will form the effective amount of the compound or substance within the body.
• the compounds of the invention are estrogen receptor modulators useful in the treatment or inhibition of conditions, disorders, or disease states that are at least partially mediated by an estrogen deficiency or excess, or which may be treated or inhibited through the use of an estrogenic agent.
• the compounds of this invention are particularly useful in treating a peri-menopausal, menopausal, or postmenopausal patient in which the levels of endogenous estrogens produced are greatly diminished.
• Menopause is generally defined as the last natural menstrual period and is characterized by the cessation of ovarian function, leading to the substantial diminution of circulating estrogen in the bloodstream.
• menopause also includes conditions of decreased estrogen production that may be caused surgically, chemically, or by a disease state that leads to premature diminution or cessation of ovarian function.
• the compounds of this invention are useful in treating or inhibiting osteoporosis and in the inhibition of bone demineralization, which may result from an imbalance in formation of new bone tissues and the resorption of older tissues in an individual, leading to a net loss of bone.
• bone depletion results in a range of individuals, particularly in post-menopausal women, women who have undergone bilateral oophorectomy, those receiving or who have received extended corticosteroid therapies, those experiencing gonadal dysgenesis, and those suffering from Cushing's syndrome.
• Special needs for bone replacement, including teeth and oral bone can also be addressed using these compounds in individuals with bone fractures, defective bone structures, and those receiving bone-related surgeries and/or the implantation of prosthesis.
• these compounds can be used in treatment or inhibition for osteoarthritis, spondyloarthropathies, hypocalcemia, hypercalcemia, Paget's disease, osteomalacia, osteohalisteresis, multiple myeloma and other forms of cancer having deleterious effects on bone tissues.
• the compounds of this invention are further useful in treating or inhibiting joint damage secondary to arthroscopic or surgical procedures.
• the compounds of this invention are also useful in treating or inhibiting benign or malignant abnormal tissue growth, including prostatic hypertrophy, uterine leiomyomas, breast cancer, endometriosis, endometrial cancer, polycystic ovary syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian cancer, melanoma, prostate cancer, cancers of the colon, CNS cancers, such as glioma or astioblastomia.
• benign or malignant abnormal tissue growth including prostatic hypertrophy, uterine leiomyomas, breast cancer, endometriosis, endometrial cancer, polycystic ovary syndrome, endometrial polyps, benign breast disease, adenomyosis, ovarian cancer, melanoma, prostate cancer, cancers of the colon, CNS cancers, such as glioma or astioblastomia.
• the compounds of this invention are cardioprotective and they are useful in lowering cholesterol, triglycerides, Lp(a) lipoprotein, and low density lipoprotein (LDL) levels; inhibiting or treating hypercholesteremia; hyperlipidemia; cardiovascular disease; atherosclerosis; peripheral vascular disease; restenosis, and vasospasm; and inhibiting vascular wall damage from cellular events leading toward immune mediated vascular damage.
• LDL low density lipoprotein
• the compounds of this invention are also antioxidants, and therefore, are useful in treating or inhibiting free radical induced disease states.
• Specific situations in which antioxidant therapy is indicated to be warranted are with cancers, central nervous system disorders, Alzheimer's disease, bone disease, aging, inflammatory disorders, peripheral vascular disease, rheumatoid arthritis, autoimmune diseases, respiratory distress, emphysema, asthma, pleurisy, uveitis, sepsis, hemorrhagic shock, prevention of reperfusion injury, viral hepatitis, chronic active hepatitis, tuberculosis, psoriasis, systemic lupus erythematosus, adult respiratory distress syndrome, central nervous system trauma and stroke.
• the compounds of this invention are also useful in providing cognition enhancement, and in treating or inhibiting senile dementias, Alzheimer's disease, cognitive decline, neurodegenerative disorders, providing neuroprotection or cognition enhancement.
• the compounds of this invention are also useful in treating or inhibiting inflammatory bowel disease, ulcerative proctitis, Crohn's disease, and colitis; menopausal related conditions, such as vasomotor symptoms, including hot flushes, vaginal or vulvar atrophy, atrophic vaginitis, vaginal dryness, pruritus, dyspareunia, dysuria, frequent urination, urinary incontinence, urinary tract infections, myalgia, arthralgia, insomnia, irritability, and the like; male pattern baldness; skin atrophy; acne; type Il diabetes; dysfunctional uterine bleeding; and infertility.
• menopausal related conditions such as vasomotor symptoms, including hot flushes, vaginal or vulvar atrophy, atrophic vaginitis, vaginal dryness, pruritus, dyspareunia, dysuria, frequent urination, urinary incontinence, urinary tract infections,
• the compounds of this invention are useful in disease states where amenorrhea is advantageous, such as leukemia, endometrial ablations, chronic renal or hepatic disease or coagulation diseases or disorders.
• the compounds of this invention can be used as a contraceptive agent, particularly, when combined with a progestin.
• the effective dosage may vary depending upon the particular compound utilized, the mode of administration, the condition being treated, and severity thereof, as well as, the various physical factors related to the individual being treated.
• Effective administration of the compounds of this invention may be given at an oral dose of from about 0.1 mg/day to about 1,000 mg/day.
• administration will be from about 10 mg/day to about 600 mg/day, more preferably from about 50 mg/day to about 600 mg/day, in a single dose or in two or more divided doses.
• the projected daily dosages are expected to vary with route of administration.
• Such doses may be administered in any manner useful in directing the active compounds herein to the recipient's bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, intranasally, vaginally, and transdermally.
• Oral formulations containing the active compounds of this invention may comprise any conventionally used oral forms, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions.
• Capsules may contain mixtures of the active compound(s) with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g., com, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc.
• Useful tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar.
• pharmaceutically acceptable diluents including, but not limited to, magnesium stearate, stearic acid, talc, sodium lau
• Preferred surface modifying agents include nonionic and anionic surface modifying agents.
• Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.
• Oral formulations herein may utilize standard delay or time release formulations to alter the absorption of the active compound(s).
• the oral formulation may also consist of administering the active ingredient in water, or a fruit juice, containing appropriate solubilizers or emulsifiers, as needed.
• the compounds of this invention may also be administered parenterally or intraperitoneally.
• Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• transdermal administrations are understood to include all administrations across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administrations may be carried out using the present compounds, N-oxides thereof, prodrugs thereof, or pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).
• Transdermal administration may be accomplished through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin.
• the carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices.
• the creams and ointments may be viscous liquids or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable.
• occlusive devices may be used to release the active ingredient into the blood stream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.
• Other occlusive devices are known in the literature.
• Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin.
• Water soluble suppository bases such as polyethylene glycols of various molecular weights, may also be used.
• the compounds of this invention can be prepared by methods known in the art of organic chemistry.
• the reagents used in the preparation of the compounds of this invention can be either commercially obtained or can be prepared by standard procedures described in the literature.
• R 3 4-CF 3 -phenyl
• ⁇ Chemical shifts ( ⁇ ) are given in parts per million (ppm) down field from tetramethylsilane (TMS). Melting points were determined on a Thomas-Hoover apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer diffraction grating or Perkin- Elmer 784 spectrophotometers (Perkin-Elmer, Shelton, CT). Mass spectra were recorded on a Kratos MS 50 or Finnigan 8230 mass spectrometers. Compound nomenclature was generally arrived at by use of the Beilstein AutonomTM program.
• Method B To a reaction vessel containing 3-(3-methoxy-phenoxy)-propionic acid (1) (7.00 g, 35.6 mmol) at 0 0 C was added slowly trifluoromethanesulfonic acid (15 ml_). The reaction mixture was stirred for 3 hours while allowing to warm up to room temperature. After cooling to 0 0 C, the reaction mixture was quenched with crushed ice, then extracted with Et 2 O (2 x 300 ml_). The organic layer was washed with water (2x), aqueous NaHCO 3 , water, brine, then dried (Na 2 SO 4 ), filtered and concentrated to give a crude oil, which was purified by silica gel chromatography to give a pure product as a yellow solid.
• Method C A mixture of 2-amino-5-methoxybenzoic acid (1.839 g, 11.00 mmol) and 7-methoxy-chroman-4-one (2) (1.960 g, 11.00 mmol) in Ph 2 O (10 mL) was heated at 170 0 C for 1 hour and at 200 "C for 7 hours. After cooling to room temperature, hexane was added. The yellow precipitate formed was collected by filtration and washed successfully with hexane and Et 2 O and dried in vacuo. Yield: 2.171 g (64%).
• Method C A mixture of 3,9-dimethoxy-6H-chromeno[4,3-b]quinolin-7-ol (3) (124 mg, 0.400 mmol) and POCI 3 (1 ml_) was heated at reflux for 1 hour. After cooling, excess POCI 3 was removed under reduced pressure. Water and then aqueous K 2 CO 3 were slowly added to the solid residue and the reaction mixture was extracted with EtOAc. The organic layer was washed with brine, then dried (Na 2 SO 4 ), filtered and concentrated to give a crude solid, which was passed through a short pad of silica gel and recrystallized (hot heptane/-20 0 C) to give a pure product as a yellow powder.
• Method E A mixture of 3,9-dimethoxy-6H-chromeno[4,3-b]quinolin-7-ol (3) (1.025 g, 3.31 mmol) and POBr 3 (1.430 g, 5.00 mmol, 1.5 equiv.) in DMF (15 mL) was heated at 70 0 C for 30 mins. After cooling to room temperature, water and then aqueous K 2 CO 3 were slowly added, and the reaction mixture was extracted with warm CHCI 3 (2x).
• Method H A mixture of 7-bromo-3,9-dihydroxy-6H-chromeno[4,3-b]quinoline (7) (34.5 mg, 0.100 mmol), tributyl(vinyl)tin (38 mg, 0.120 mmol, 1.2 equiv.), and Pd(PPh 3 ) 4 (11.6 mg, 0.0100 mmol, 10 mol%) in toluene (1.5 mL) was refluxed under nitrogen until all starting material was consumed (1-2 hour(s)). Filtration through Celite ® and purification by passing through a short pad of silica gel gave a pure product as an orange powder.
• Method I A mixture of 7-bromo-3,9-dihydroxy-6H-chromeno[4,3-b]quinoline (7) (51.6 mg, 0.150 mmol), (trimethylsilylethynyl)tributyltin (70 mg, 0.180 mmol, 1.2 equiv.), and Pd(PPh 3 ) 4 (17 mg, 0.015 mmol, 10 mol%) in toluene (2 mL) was refluxed under nitrogen until all starting material was consumed (1-2 hour(s)). Filtration through Celite ® and purification by passing through a short pad of silica gel gave a pure product as a red solid.
• Method L A mixture of 7-bromo-3,9-dihydroxy-6H-chromeno[4,3-b]quinoline (7) (47 mg, 0.14 mmol), CuCN (370 mg, 4.13 mmol) in anhydrous DMF (2 mL) was heated at 200 0 C in a sealed tube until all starting material was consumed (5 hours). After cooling to room temperature, the reaction mixture was filtered through Celite ® and rinsed with EtOAc. Water was added to the filtrate and the reaction mixture was extracted with EtOAc. The organic layer was washed with water (2x) and brine, then dried (Na 2 SO 4 ), filtered and concentrated to give a crude solid, which was purified by silica gel chromatography to give a pure product as a brown powder.
• Representative examples of the invention were evaluated for their ability to compete with 17 ⁇ -estradiol for both ERa and ER ⁇ .
• the test procedure used allows one to determine whether a particular compound binds to the estrogen receptor (and is therefore "estrogenic") and whether there is selectivity for ERa or ER ⁇ .
• Results of representative compound examples are shown in Table 1 below, with the values obtained reported as IC 50 S.
• the IC 50 is defined as the concentration of compound that decreases total 17 ⁇ -estradiol binding by 50%.
• the procedure used is briefly described. A crude lysate of E. coli expressing the estrogen receptor ligand binding domains (D, E, and F) of human ERa or ER ⁇ was prepared.
• Both receptors and compounds were diluted in 1X Dulbecco's phosphate buffered saline (DPBS) supplemented with 1 mM ethylenediamine tetraacetic acid (EDTA).
• DPBS Dulbecco's phosphate buffered saline
• EDTA ethylenediamine tetraacetic acid
• 100 uL of receptor (1 uG/well) was combined with 2 nM [ 3 H]-17 ⁇ -estradiol and various concentrations of compound. After between 5 and 15 hours at room temperature, the plates were washed with DPBS/1 mM EDTA and bound radioactivity determined by liquid scintillation counting.
• the compounds of this invention are estrogenic compounds, many with strong preferential affinity for the ER ⁇ receptor.
• the compounds of this invention range from having high preferential affinity for ER ⁇ over ERa to almost equal affinity for both receptors.
• compounds of this invention will span a range of activity based, at least partially, on their receptor affinity selectivity profiles.
• compounds of this invention will display different modulatory behavior depending on the cellular context they are in. For example, in some cell-types, it is possible for a compound to behave as an estrogen agonist while in other tissues, as an antagonist.
• SERMs Selective Estrogen Receptor Modulators
• a mammal e.g., a human such as a woman
• disease states or syndromes which are caused or associated with an estrogen deficiency (e.g., in certain tissues such as bone or cardiovascular) or an excess of estrogen (e.g., in the uterus or mammary glands).
• compounds of this invention also have the potential to behave as agonists on one receptor type while behaving as antagonists on the other.
• compounds can be an antagonist on ER ⁇ while being an agonist on ERa.
• ERSAA Estrogen Receptor Selective Agonist Antagonist
• the estrogenic and antiestrogenic properties of the compounds can be determined in an immature rat uterotrophic assay (4 days) (see L.J. Black and R. L. Goode, Life Sciences 26: 1453 (1980)). Immature Sprague-Dawley rats (female, 18 days old) were tested in groups of six. The animals were treated by daily intraperitoneal (ip) injection with 10 ⁇ g compound, 100 ⁇ g compound, 100 ⁇ g compound + 1 ⁇ g 17 ⁇ -estradiol (to check antiestrogenicity), and 1 ⁇ g 17 ⁇ -estradiol, with 50% DMSO/50% saline as the injection vehicle.
• ip intraperitoneal
• mice Female Sprague Dawley CD rats, ovx or sham ovx, are obtained 1 day after surgery from Taconic (Greenwood, NY) (weight range 240-275 g). They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark) schedule and provided with food (Purina Mills ® 5K96C rat chow) and water ad libitum. Treatment for all studies begin 1 day after the animals' arrival and dosed 7 days per week as indicated for 6 weeks. A group of age matched sham operated rats not receiving any treatment serve as an intact, estrogen replete control group for each study.
• All treatments are prepared in 1% Tween 80 in normal saline at defined concentrations so that the treatment volume is 0.1 mL/100 g body weight. 17 ⁇ estradiol is dissolved in corn oil (20 ⁇ g/mL) and delivered subcutaneously, 0.1 mL/rat. All dosages are adjusted at three week intervals according to group mean body weight measurements. Five weeks after the initiation of treatment and one week prior to the termination of the study, each rat is evaluated for bone mineral density (BMD). The total and trabecular density of the proximal tibia are evaluated in anesthetized rats using an XCT-960M (pQCT ( ); Stratec Medizintechnik, Pforzheim, Germany). The measurements are performed as follows: Fifteen minutes prior to scanning, each rat is anesthetized with an ip injection of 45 mg/kg ketamine, 8.5 mg/kg xylazine, and 1.5 mg/kg acepromazine.
• BMD bone mineral density
• the right hind limb is passed through a polycarbonate tube with a diameter of 25 mm and taped to an acrylic frame with the ankle joint at a 90° angle and the knee joint at 180°.
• the polycarbonate tube is affixed to a sliding platform that maintains it perpendicular to the aperture of the pQCT.
• the platform is adjusted so that the distal end of the femur and the proximal end of the tibia would be in the scanning field.
• a two dimensional scout view is run for a length of 10 mm and a line resolution of 0.2 mm. After the scout view is displayed on the monitor, the proximal end of the tibia is located.
• the pQCT scan is initiated 3.4 mm distal from this point.
• the pQCT scan is 1 mm thick, has a voxel (three dimensional pixel) size of 0.140 mm, and consists of 145 projections through the slice.
• the image is displayed on the monitor.
• a region of interest including the tibia but excluding the fibula is outlined.
• the soft tissue is automatically removed using an iterative algorithm.
• the density of the remaining bone is reported in mg/cm 3 .
• the outer 55% of the bone is peeled away in a concentric spiral.
• the density of the remaining bone is reported in mg/cm 3 .
• One week after BMD evaluation the rats are euthanized by carbon dioxide suffocation and blood collected for cholesterol determination.
• the uteri are removed and the weights taken.
• Total cholesterol is determined using a Boehringer-Mannheim Hitachi 911 clinical analyzer using the Cholesterol/HP kit. Statistics were compared using one-way analysis of variance (ANOVA) with Dunnet's test.
• test compounds (usually 0.1 M) are prepared in DMSO and then diluted 10 to 100-fold with DMSO to make working solutions of 1 or 10 mM.
• the DMSO stocks are stored at either 4 0 C (0.1 M) or -20 0 C ( ⁇ 0.1M).
• MCF-7 cells are passaged twice a week with growth medium [D-MEM/F-12 medium containing 10% (v/v) heat-inactivated fetal bovine serum, 1% (v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1].
• the cells are maintained in vented flasks at 37 0 C inside a 5% C ⁇ 2/95% humidified air incubator.
• the cells are plated with growth medium at 25,000/well into 96 well plates and incubated at 37 0 C overnight.
• the cells are infected for 2 hours at 37 0 C with 50 ⁇ l/well of a 1:10 dilution of adenovirus 5-ERE-tk-luciferase in experimental medium [phenol red-free D-MEM/F- 12 medium containing 10% (v/v) heat-inactivated charcoal-stripped fetal bovine serum, 1% (v/v) Penicillin-Streptomycin, 2 mM glutaMax-1 , 1 mM sodium pyruvate]. The wells are then washed once with 150 ⁇ l of experimental medium. Finally, the cells are treated for 24 hours at 37 0 C in replicates of 8 wells/treatment with 150 ⁇ l/well of vehicle ( ⁇ 0.1% v/v DMSO) or compound that is diluted ⁇ 1000-fold into experimental medium.
• vehicle ⁇ 0.1% v/v DMSO
• Initial screening of test compounds is done at a single dose of 1 ⁇ M that is tested alone (agonist mode) or in combination with 0.1 nM 17 ⁇ -estradiol (EC 80 ; antagonist mode).
• Each 96-well plate also includes a vehicle control group (0.1% v/v DMSO) and an agonist control group (either 0.1 or 1 nM 17 ⁇ -estradiol).
• Dose- response experiments are performed in either the agonist and/or antagonist modes on active compounds in log increases from 10 "14 to 10 '5 M. From these dose- response curves, EC 5 O and IC 50 values, respectively, are generated.
• the final well in each treatment group contains 5 ⁇ l of 3 x 10 "5 M ICI-182,780 (10 "6 M final concentration) as an ER antagonist control. After treatment, the cells are lysed on a shaker for 15 mins. with 25 ⁇ l/well of
• 1X cell culture lysis reagent Promega Corporation.
• the cell lysates (20 ⁇ l) are transferred to a 96-well luminometer plate, and luciferase activity is measured in a MicroLumat LB 96 P luminometer (EG & G Berthold) using 100 ⁇ l/well of luciferase substrate (Promega Corporation).
• a 1 second background measurement is made for each well.
• luciferase activity is measured for 10 seconds after a 1 second delay.
• the data are transferred from the luminometer to a Macintosh personal computer and analyzed using the JMP software (SAS Institute); this program subtracts the background reading from the luciferase measurement for each well and then determines the mean and standard deviation of each treatment.
• the luciferase data are transformed by logarithms, and the Huber M- estimator is used to down-weight the outlying transformed observations.
• the JMP software is used to analyze the transformed and weighted data for one-way ANOVA (Dunnett's test). The compound treatments are compared to the vehicle control results in the agonist mode, or the positive agonist control results (0.1 nM 17 ⁇ - estradiol) in the antagonist mode. For the initial single dose experiment, if the compound treatment results are significantly different from the appropriate control (p ⁇ 0.05), then the results are reported as the percent relative to the 17 ⁇ -estradiol control [i.e., ((compound - vehicle control)/(17 ⁇ -estradiol control - vehicle control)) x 100]. The JMP software is also used to determine the EC 50 and/or IC C 50 values from the non-linear dose-response curves.
• Porcine aortas are obtained from an abattoir, washed, transported in chilled phosphate buffered saline (PBS), and aortic endothelial cells are harvested. To harvest the cells, the intercostal vessels of the aorta are tied off and one end of the aorta clamped. Fresh, sterile filtered, 0.2% collagenase (Sigma Type I) is placed in the vessel and the other end of the vessel then clamped to form a closed system. The aorta is incubated at 37 0 C for 15-20 mins., after which the collagenase solution is collected and centrifuged for 5 mins. at 2000 x g. Each pellet is suspended in 7 ml.
• PBS chilled phosphate buffered saline
• endothelial cell culture medium consisting of phenol red free DMEM/Ham's F12 media supplemented with charcoal stripped fetal bovine serum (FBS; 5%), NuSerum ® (5%), L-glutamine (4 mM), penicillin-streptomycin (1000 U/ml, 100 ⁇ g/ml) and gentimicin (75 ⁇ g/ml), seeded in a 100 mm petri dish and incubated at 37 0 C in 5% CO 2 . After 20 minutes, the cells are rinsed with PBS and fresh medium added, and again, at 24 hours. The cells are confluent after approximately 1 week.
• the endothelial cells are routinely fed twice a week and, when confluent, trypsinized and seeded at a 1:7 ratio.
• Cell mediated oxidation of 12.5 ⁇ g/mL LDL is allowed to proceed in the presence of the compound to be evaluated (5 ⁇ M) for 4 hours at 37 0 C.
• Results are expressed as the percent inhibition of the oxidative process as measured by the TBARS (thiobarbituric acid reactive substances) method for analysis of free aldehydes (Yagi K., Biochem Med 15:212-216 (1976)).
• D12 rat hypothalamic cells are subcloned from the RCF17 parental cell line and stored frozen. They are routinely grown in DMEM:F12 (1 :1), glutaMAX-1 (2 mM), penicillin (100 U/ml)-streptomycin (100 mg/ml), plus 10% FBS. The cells are plated in phenol red-free medium (DMEM:F12, glutaMAX, penicillin-streptomycin) containing 2-10% charcoal stripped FBS at a subconfluent density (1-4 x 10 6 cells/ 150 mm dish). The cells are refed 24 hours later with medium containing 2% stripped serum.
• cells are treated with 10 nM 17 ⁇ -estradiol or various doses of test compound (1 mM or a range from 1 pM to 1 mM).
• test compound 1 mM or a range from 1 pM to 1 mM.
• antagonist activity the cells are treated with 0.1 nM 17 ⁇ -estradiol in the absence or presence of varying doses (100 pM to 1 mM) of test compound.
• Control dishes are also treated with DMSO as a negative control. Forty-eight hours after hormone addition, the cells are lysed and a binding test procedure performed.
• Ovariectomized animals are randomly divided into groups that are injected with vehicle (50% DMSO, 40% PBS, 10% ethanol vehicle), 17 ⁇ -estradiol (200 ng/kg) or the compound to be tested. Additional animals are injected with the test compound 1 hour prior to injection of 17 ⁇ -estradiol to evaluate the antagonistic properties of this compound. Six hours after subcutaneous (sc) injection, the animals are euthanized with a lethal dose of CO 2 and their brains collected and frozen.
• vehicle 50% DMSO, 40% PBS, 10% ethanol vehicle
• 17 ⁇ -estradiol 200 ng/kg
• Additional animals are injected with the test compound 1 hour prior to injection of 17 ⁇ -estradiol to evaluate the antagonistic properties of this compound.
• sc subcutaneous
• Tissue collected from the animals is cut on a cryostat at -16 0 C and collected on Silane-coated microscope slides.
• the section-mounted slides are then dried on a slide warmer maintained at 42 0 C and stored in desiccated slide boxes at -80 0 C.
• the desiccated slide boxes Prior to processing, the desiccated slide boxes are slowly warmed to room temperature (-20 °C for 12-16 hours; 4 0 C for 2 hours; room temperature for 1 hour) to eliminate condensation formation on slides and thus, minimize tissue and RNA degradation.
• the dry slides are loaded into metal racks, post-fixed in 4% paraformaldehyde (pH 9.0) for 5 mins. and processed, as previously described.
• a plasmid containing a 815bp fragment of the rat PR cDNA 9 (ligand binding domain) is linearized and used to generate a S 35-UTP labeled probe that is complimentary to a portion of the rat PR mRNA.
• Processed section-mounted slides are hybridized with 200 ml_ of hybridization mix containing the riboprobe (4-6x10 6 DPM/slide) and 50% formamide, and incubated overnight in a 55 0 C humidified chamber. In the morning, the slides are placed in metal racks that are immersed in 2xSSC (0.15M NaCI, 0.015M sodium citrate; pH 7.0)/10 mM DTT.
• 2xSSC 0.15M NaCI, 0.015M sodium citrate; pH 7.0
• the racks are all transferred to a large container and washed in 2xSSC/10 mM DTT for 15 mins. at room temperature with gentle agitation. Slides are then washed in RNase buffer at 37 0 C for 30 mins., treated with RNase A (2 mg/ml) for 30 mins. at 37 0 C, and washed for 15 mins. in room temperature IxSSC. Subsequently, the slides are washed (2x 30 mins.) in 65 0 C in 0. IxSSC to remove nonspecific label, rinsed in room temperature 0. IxSSC for 15 mins. and dehydrated with a graded series of alcohol: ammonium acetate (70%, 95%, and 100%). Air dried slides are opposed to x-ray film for 3 days and then photographically processed. The slides from all animals are hybridized, washed, exposed and photographically processed together to eliminate differences due to interassay variation in conditions.
• Ovariectomized-female, 60 days-old, Sprague-Dawley rats are obtained following surgery. The surgeries are done a minimum of 8 days prior to the first treatment. The animals are housed individually under a 12/12 hours light/dark cycle and given standard rat chow and water ad libitum.
• mice Two control groups are included in every study. Doses are prepared based on mg/kg mean group body weight in either 10% DMSO in sesame oil (sc studies) or in 1.0% Tween 80 in saline ((po) studies). Animals are administered test compounds at doses ranging from 0.01 to 10 mg/kg mean group body weight. Vehicle and ethinyl estradiol (EE) controls (0.1 mg/kg, sc or 0.3 mg/kg, po) control groups are included in each test. When the compounds are tested for their antagonist activity, EE is coadministered at 0.1 or 0.3 mg/kg for sc or po studies, respectively. The test compounds are administered up to the day that tail skin temperature is measured.
• sc studies 10% DMSO in sesame oil
• Tween 80 in saline
• Animals are administered test compounds at doses ranging from 0.01 to 10 mg/kg mean group body weight.
• Vehicle and ethinyl estradiol (EE) controls 0.1 mg/
• the animals are treated once daily with the compound(s) of interest. There are 10 animals/treatment group. Administration of the compound is either by sc injection of 0.1 ml in the nape of the neck or po in a volume of 0.5 ml. On the 3rd day of treatment, a morphine pellet (75 mg morphine sulfate) is implanted subcutaneously. On the 5th day of treatment, one or two additional morphine pellets are implanted.
• Sprague-Dawley rats (240-260 grams) are divided into 4 groups:
• the animals are ovariectomized approximately 3 weeks prior to treatment. Each animal receives 1 mg/kg/day of either 17- ⁇ estradiol sulfate or test compound suspended in distilled, deionized water with 1 % Tween-80 by gastric gavage. Vehicle treated animals received an appropriate volume of the vehicle used in the drug treated groups.
• a resting tension of 1 gram is placed on the rings. Rings are equilibrated for 1 hour, while signals are acquired and analyzed. After equilibration, the rings are exposed to increasing concentrations of phenylephrine (10" ⁇ to 10" 4 M) and the tension recorded. Baths are then rinsed 3 times with fresh buffer. After washout, 200 mM L-NAME is added to the tissue bath and equilibrated for 30 minutes. The phenylephrine concentration response curve is then repeated.
• mice Male, Sprague-Dawley, CD rats (Charles River, Springfield, NY) weighing 200- 250 g on arrival are used. For one week, the rats are housed, six per cage, with standard laboratory chow and water available ad libitum. Housing is in a colony room maintained at 22 0 C with a 12/12 hours light/dark cycle with lights on at 6:00 AM. Following habituation to the facility, the animals are individually housed and maintained at 85% of free-feeding weight. Once stable weights are attained, the rats are acclimated to the 8-arm radial maze.
• the structure of the maze is an adaptation from that of Peele and Baron ⁇ Pharmacology, Biochemistry, and Behavior 29:143-150 (1988)).
• the maze is elevated to a height of 75.5 cm and composed of a circular area surrounded by 8 arms radiating away from the center, equidistant from one another. Each arm is 58 cm long x 13 cm high.
• a clear plexiglass cylinder is lowered to enclose the animal in the center portion of the maze prior to the start of each session.
• Each arm of the maze is equipped with 3 sets of photocells interfaced to a data acquisition unit, which in turn is interfaced to a computer. The photocells are used to track the movement of the rat in the maze.
• Pellet feeders located above food cups at the end of each arm, dispensed two 45 mg chocolate pellets when the outer photocell of the arm is activated for the first time in a given session.
• the maze is located in a testing room with black and white geometric posters on each wall to serve as visual cues. During all training and testing procedures, white noise is audible ( ⁇ 70 db).
• the training procedure consists of five phases, each with daily sessions lasting 5 or 10 minutes. A 10 second delay is imposed between the time the rat is placed in the center portion of the maze and when the cylinder is raised to begin the session.
• food-restricted pairs of rats are placed on the maze for 10 minutes with 45 mg chocolate food pellets scattered throughout the 8 arms of the maze.
• each rat is placed individually on the maze for a 10 minute period, with pellets scattered from the middle photocell to the food cup of each arm.
• each rat is placed on the maze for a 10 minute period, with food pellets located only in and around the food cups in each arm.
• each rat is allowed 10 minutes to collect two pellets from each arm. Re-entry into an arm is considered an error. Rats are trained daily in this manner until they achieved criterion performance with less than or equal to 2 total errors on three consecutive days of training. Total habituation and training time is approximately 3 weeks.
• Test compound is prepared in phosphate buffered saline and administered in a volume of 1 ml/kg.
• Scopolamine HBr (0.3 mg/kg sc) served as the impairing agent, producing an increase in error rate (loss of memory).
• Test compound is given intraperitoneally, simultaneously with scopolamine, 30 minutes prior to the first maze exposure on any given test day.
• DMEM/10% PDHS pregnant donor horse serum
• ARC cytosine arabinoside
• Control primary neuronal cultures show progressive cell death between days 12 and 18 in culture. Twelve cultures were evaluated on days 12 and 16 for levels of the enzyme lactate dehydrogenase (LD) after adding test compound to 6 cultures maintained in DMEM and 10% PDHS on day 9 and maintaining the remaining cultures as controls. LD was assayed using a variation of the method by Wroblewski et al., Proc. Soc. Exp. Biol. Med. 90:210-213 (1955). LD is a cytosolic enzyme that is commonly used in both clinical and basic research to determine tissue viability. An increase in media LD is directly related to cell death.
• LD lactate dehydrogenase
• C6 glioma cells obtained from American Tissue Culture Center were plated in RPMI media with FBS at a concentration of 1 x 10 6 cells/mL in FALCON ® 25 cm 2 tissue culture flasks.
• FALCON ® 25 cm 2 tissue culture flasks Four hours prior to the onset of hypoglycemia, the maintenance media was discarded, monolayers were washed twice in the appropriate media and then incubated for four hours at 37 °C in either serum free or serum free plus test compound.
• Kreb's Ringer Phosphate buffer was used to wash the monolayers twice before the addition of appropriate glucose treatment.
• RPMI medium contains 2 mg glucose/mL; flasks were divided into groups of 6 with each receiving 100% glucose (2 mg/ml), 80% glucose (1.6 mg/ml), 60% glucose (1.2 mg/ml) or 0% glucose (buffer) or supplemented with test compound. All flasks were incubated for 20 hours and then evaluated for total, live, and dead cell number utilizing trypan blue.
• Cortical neurons are prepared from E18 rat fetus and plated in 8-well chamber slides pre-coated with poly-D-lysine (10 ng/ml) and serum at a density of 100,000 cells/well.
• Cells are plated in high glucose DMEM containing 10% FCS and kept in the incubator at 37 0 C with 10% CO 2 /90% air.
• serum is removed by replacing culture media with high glucose DMEM containing B27 supplement, and cells are kept in the incubator without further media change until the day of experiment.
• slides are divided into two groups: a control group and a OGD group.
• Cells in the control group receive DMEM with glucose and custom B27 (without antioxidants).
• Cells in the OGD group receive no-glucose DMEM with custom B27, which has been degassed under vacuum for 15 mins.
• Cells are flushed with 90% N 2 /10% CO 2 for 10 mins. in an airtight chamber and incubated at 37 0 C for 6 hours.
• both control and OGD cells are subject to replacement of media containing either vehicle (DMSO) or test compound in glucose-containing DMEM with custom B27.
• Cells are returned to a normoxic incubator at 37 0 C.
• cells are fixed in 4% PFA for 10 mins. at 4 0 C and stained with Topro (fluorescent nuclear binding dye). Apoptosis is assessed using a laser scanning cytometer by measuring pyknotic nuclei.
• Cortical neurons are prepared from E18 rat fetus and plated in 48-well culture plates pre-coated with poly-D-lysine (10 ng/ml) and serum at a density of 150,000 cells/well.
• Cells are plated in high glucose DMEM containing 10% FCS and kept in the incubator at 37 0 C with 10% CO 2 /90% air.
• serum is removed by replacing culture media with high glucose DMEM containing B27 supplement.
• cells are divided into two groups: a control group and a OGD group. Cells in the control group receive DMEM with glucose and custom B27 (without antioxidants).
• Cells in the OGD group receive no-glucose DMEM with custom B27, which has been degassed under vacuum for 15 mins. Cells are flushed with 90% N 2 /10% CO 2 for 10 mins. in an airtight chamber and incubated at 37 0 C for 6 hours. After 6 hours, both control and OGD cells are subject to replacement of media containing either vehicle (DMSO) or test compound in glucose-containing DMEM with custom B27. Cells are returned to a normoxic incubator at 37 0 C. After 24 hours, cell death is assessed by measuring cellular release of LDH (lactate dehydrogenase) into the culture medium. For the LDH assay, an aliquot of 50 ⁇ l culture medium is transferred into the 96-well plate.
• LDH lactate dehydrogenase
• the plate After the addition of 140 ⁇ l 0.1 M potassium phosphate buffer (pH 7.5) and 100 ⁇ l 0.2 mg/ml NADH, the plate is allowed to sit in the dark at room temperature for 20 mins. The reaction is initiated by the addition of 10 ⁇ l of sodium pyruvate. The plate is read immediately at 340 nM in a Thermomax plate reader (Molecular Devices). The absorbance, an index of NADH concentration, is recorded every 6 seconds for 5 minutes and the slope indicating the rate of NADH disappearance is used to calculate LDH activity:
• Male HLA-B27 rats are obtained from Taconic and provided unrestricted access to a food (Purina Mills ® LabDiet ® 5001) and water. At the start of the study, rats are 22-26 weeks old.
• Rats are dosed subcutaneously once per day for seven days with one of the formulations listed below. There are five rats in each group and the last dose is administered two hours before euthanasia.
• serum is collected and stored at -70 °C.
• a section of colon is prepared for histological analysis and an additional segment is analyzed for myeloperoxidase activity.
• Colon tissue is harvested and flash frozen in liquid nitrogen. A representative sample of the entire colon is used to ensure consistency between samples. The tissue is stored at -80 0 C until use. Next, the tissue is weighed (approximately 500mg) and homogenized in 1 :15 w/v of 5 mM H 2 KPO 4 (pH 6) washing buffer. The tissue is spun down at 20,000 x g in a Sorvall RC 5B centrifuge for 45 minutes at 2-8 0 C. Supernatant is then discarded.
• Tissue is resuspended and homogenized in 2.5 ml_ (1 :5 w/v) of 50 mM H 2 KPO 4 with 10 mM EDTA and 0.5% Hex Ammonium Bromide to help solubilize the intracellular MPO.
• Tissue is frozen in liquid nitrogen, thawed in a 37 °C-water bath and sonicated for 15 seconds to ensure membrane lysis. This procedure is repeated 3 times. Samples are then kept on ice for 20 minutes and centrifuged at 12,000 x g for 15 minutes at 2-8 0 C. The supernatant is analyzed following these steps.
• the test mixture is prepared by adding 2.9 ml_ of 50 mM H 2 KPO 4 with 0.167 O-Dianisidine/ml with 0.0005% H 2 O 2 into a reaction tube.
• O-Dianisidine When hydrogen peroxide is degraded, O-Dianisidine is oxidized and absorbs at 460 nm in a concentration dependent manner.
• the mixture is heated to 25 0 C.
• One hundred (100) ⁇ l_ of the tissue supernatant is added to the reaction tube and incubated for one minute at 25 0 C, then 1 ml is transferred to a disposable plastic cuvette.
• Optical density (OD) is measured every 2 minutes of reaction time at 460 nm against a blank containing 2.9 ml_ of reaction mixture and 100 ⁇ L of the 0.5% ammonium bromide solution.
• Enzyme activity units are quantified by comparison of absorbance at 460 nm to a standard curve prepared with purified human MPO 31.1 units/vial.
• the MPO is reconstituted and serially diluted using 5OmM H 2 KPO 4 with 10 mM EDTA and 0.5% Hex Ammonium Bromide to four known concentrations. Sample absorbancies are compared against this curve to determine activity.
• Histological analysis is performed as follows. Colonic tissue is immersed in 10% neutral buffered formalin. Each specimen of colon is separated into four samples for evaluation. The formalin-fixed tissues are processed in a vacuum infiltration processor for paraffin embedding. The samples are sectioned at 5 ⁇ m and then stained with hematoxylin and eosin (H&E) for blinded histologic evaluations using a scale modified after Boughton-Smith. After the scores are completed the samples are unblinded, and data are tabulated and analyzed by ANOVA linear modeling with multiple mean comparisons.
• H&E hematoxylin and eosin
• the compounds of this invention are estrogen receptor modulators useful in the treatment or inhibition of conditions, disorders, or disease states that are at least partially mediated by an estrogen deficiency or excess, or which may be treated or inhibited through the use of an estrogenic agent.

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