Replacement (4'-hydroxyphenyl) cycloalkane and (4'-hydroxyphenyl) cycloalkene compounds and methods of their use as selective agonists of estrogen receptor β isoform for improving memory fixation

Abstract

Disclosed are substituted (4'-hydroxyphenyl)cycloalkane and substituted (4'-hydroxyphenyl)cycloalkene compounds and methods for their use as selective agonists of the estrogen receptor β isoform (ERβ). The disclosed compounds may be formulated as pharmaceutical compositions and administered to subjects in need thereof to treat diseases associated with ER activity, such as neurological diseases and disorders, psychiatric diseases and disorders, and / or cell proliferative diseases and disorders, and to improve memory consolidation.

Description

Technical Field

[0001] Description of Research and Development Funded by the Federal Government This invention was made with government support under R15GM118304 awarded by the National Institute of General Medical Sciences and R01DA038042 awarded by the National Institute on Drug Abuse. The United States government has certain rights in this invention.

[0002] Cross - Reference to Related Patent Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 572,932, filed on October 16, 2017, and U.S. Provisional Application No. 62 / 478,758, filed on March 30, 2017. The contents of these applications are hereby incorporated by reference in their entirety.

[0003] Field of the Invention The field of the invention relates to compounds that function as ligands for estrogen receptors (ER). In particular, the field of the invention relates to substituted (4'-hydroxyphenyl) cycloalkane compounds and (4'-hydroxyphenyl) cycloalkene compounds that are specific agonists for estrogen receptor β (ERβ), and to the use of such compounds in pharmaceutical compositions for treating diseases and disorders related to ER activity in improving memory fixation.

Background Art

[0004] Background Estrogen is an important regulator of many physiological processes including reproduction, cognition, cardiovascular death, and bone metabolism 66Based on its extensive roles in numerous physiological processes, estrogen has been linked to a number of diseases and disorders, including, for example, cell proliferative diseases and disorders (such as breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, and prostate cancer), neurodegenerative diseases and disorders, cardiovascular diseases, and osteoporosis. 66 In many of these diseases and disorders, estrogen mediates its effects through estrogen receptors (ERs).

[0005] ERs exist in two main forms, ERα and ERβ, with different tissue expression patterns. 67 ERα and ERβ are encoded by separate genes, ESR1 and ESR2, found at different chromosomal locations, and there are a very large number of mRNA splice variants for both ERα and ERβ. 68 Because ERα and ERβ have roles in estrogen-related diseases, they have been targeted for the development of specific ligands that regulate the activity of ERα and ERβ. The ligand specificities of ERα and ERβ are different, and a ligand that binds to ERα and functions as an agonist or antagonist of ERα may bind to ERβ and function as an agonist or antagonist of ERβ, or may not bind to ERβ and not function as an agonist or antagonist of ERβ.

[0006] Agonists of ERα and ERβ have a wide range of biological effects involved in diseases such as cancer and central nervous system (CNS) disorders. 17β-estradiol (E2) is an important modulator of hippocampal synaptic plasticity and hippocampus-dependent memory formation in male and female rodents. 6Levels of both E2 decline with age, but decline more rapidly in postmenopausal women. ERβ is the most predominant ER isoform in the hippocampus and plays an important role in mediating the effects of estradiol on neuroplasticity and neuroprotection, which may be important during aging and in Alzheimer's disease (AD). For example, overexpression of ERβ in a rat AD model significantly reduced hippocampal AD lesions and improved learning and memory. 7 Furthermore, a specific allele of the ERβ gene (Esr2), rather than the ERα gene, has been associated with a reduced risk of AD in both men and women. 8 This supports the idea that ERβ is a putative drug target for AD.

[0007] In particular, there are numerous promising clinical applications for ERβ agonists. 1 Current ERβ agonist drug lead molecules have a phenolic ring, and the other half of the molecule typically contains various substituted aromatic ring structures, typically another phenolic ring structure or an indole-like ring structure (Figure 1a). One of these, WAY-200070 (benzoxazole), has shown efficacy as an anxiolytic / antidepressant and has 68-fold selectivity for ERβ over ERα. 1-3 Some ERβ agonists have advanced to human clinical trials for various disease indications ranging from schizophrenia (Eli Lilly; NCT01874756) to fragile X syndrome (Parc de Salut Mar; NCT01855971), amnesia, and hot flashes (National Institutes on Aging; NCT01723917). 4 The studies presented herein focused on one of the more promising new clinical applications of ERβ agonists for treating neuronal symptoms caused by estrogen deficiency during menopause, as exemplified in animal model studies using diarylpropionitrile (DPN). 5 。

[0008] APOE4 is the most established genetic risk factor for Alzheimer's disease (AD). Women with the APOE4 genotype are 2-4 times more likely to develop AD than women without the APOE4 genotype or men with any other APOE genotype. 9-11 APOE4 carriers are also significantly more likely to exhibit symptoms of anxiety and depression. 12 A major factor contributing to these risks in women is estrogen decline during menopause. This is because estrogen mediates cognitive function and neuroprotects brain regions such as the hippocampus and cortex that deteriorate in AD. 13 Therefore, drugs that promote estrogen-mediated effects on cognition, such as the selective ERβ agonists (SERBA) developed herein, may reverse memory loss and reduce anxiety and depression in aging women. However, estrogen-based hormone replacement therapy is associated with a high risk of various diseases, including breast cancer (especially lobular cancer), as well as stroke, gallbladder disease, and venous thromboembolism, which are thought to be related to ERα agonist activity. 14-18 Therefore, any ERβ agonist therapeutic must be selective for ERβ over ERα agonist activity.

[0009] Therefore, new estrogen receptor ligands are desired. In particular, new ligands that exhibit agonist or antagonist activity selective for ERβ compared to ERα are desired. These new ligands should be suitable for treating diseases and disorders related to ER activity, such as cell proliferative diseases and disorders or psychiatric diseases and disorders. Recently, the inventors reported a novel ERβ agonist that is selective for ERβ over ERα activation compared to previously reported clinical candidates. 19 This ERβ agonist is in a unique structural class composed of a phenolic ring tethered to a 4-hydroxymethyl-cycloheptane ring structure. However, the presence of the 4-substituted cycloheptane ring presents synthetic and stereochemical challenges and is therefore not desirable as a drug lead.

[0010] Composed of a 4-hydroxymethyl-cyclohexane ring linked to a phenol ring, and thus belonging to a related molecular class of A-C estrogens that closely resemble natural estrogen molecules but lack the B and D rings. The optimization and characterization of this related molecular class are reported herein (Figures 1b - d). A-CD estrogens have been widely studied and are reported to have up to 15-fold selectivity for ERβ. 16-23 . In contrast, the simpler A-C estrogens reported herein exhibit considerably higher selectivity for ERβ than for ERα. These A-C estrogens are surprisingly simple yet novel isoform-selective ERβ agonists that have the potential to treat age-related memory decline in postmenopausal women.

Summary of the Invention

[0011] Summary Disclosed are substituted (4'-hydroxyphenyl)cycloalkane compounds and (4'-hydroxyphenyl)cycloalkene compounds and their use as selective agonists of estrogen receptor β (ERβ). The disclosed compounds are formulated as pharmaceutical compositions and may be administered to treat diseases related to ER activity.

[0012] In some embodiments, the disclosed compounds have the formula I: TIFF0007708379000001.tif30128 or a hydroxy-protected form thereof; Wherein, (a) Z is a carbon atom; (b) X is selected from the group consisting of hydrogen, hydroxyl, alkyl, hydroxyalkyl, amino, and aminoalkyl; and (c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -CH2CH2- or -OCH2-, and Y and Z form a bridge; or X and Y together form alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime.

[0013] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl)cyclohexane compounds. For example, the disclosed compounds may have the formula Ia: TIFF0007708379000002.tif30128, wherein X, Y, and Z are as defined for formula I.

[0014] The disclosed compounds include the compound 4-hydroxymethyl-(4'-hydroxyphenyl)-cyclohexane, in particular, the enantiomer also known herein by the alias "ISP358-2": TIFF0007708379000003.tif29128.

[0015] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl)cyclohexene compounds. For example, the disclosed compounds may have the formula Ia(i): TIFF0007708379000004.tif31128, wherein X, Y, and Z are as defined for formula I.

[0016] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions. Accordingly, pharmaceutical compositions comprising an effective amount of any of the compounds disclosed herein, or a pharmaceutically acceptable salt of any of the compounds disclosed herein, together with a pharmaceutically acceptable excipient, carrier, or diluent are also disclosed herein.

[0017] The disclosed compounds may be used to prepare pharmaceutical agents for treating diseases or disorders related to estrogen receptor β (ERβ) activity, particularly diseases or disorders that can be treated with ERβ agonists. Thus, the disclosed compounds may exhibit ERβ agonist activity, and preferably, this compound exhibits specificity as an ERβ agonist compared to its activity as an ERβ antagonist and / or compared to its activity as an estrogen receptor α (ERα) agonist or ERα antagonist. The disclosed compounds may be prescribed for use in the treatment of psychiatric diseases or disorders or neurological diseases or disorders. In particular, the disclosed compounds may be prescribed for use in the treatment of subjects in need of improvement in memory consolidation, for example, improvement in memory consolidation under low estrogen conditions observed in postmenopausal women. [Invention 1001] TIFF0007708379000005.tif25128 A compound having the formula and stereochemistry of [Invention 1002] A pharmaceutical composition comprising an effective amount of the compound of Invention 1001 or a pharmaceutically acceptable salt thereof together with a pharmaceutical excipient, carrier, or diluent. [Invention 1003] A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of Invention 1002. [Invention 1004] The method of Invention 1003, wherein the disease or disorder is a neurological disease or disorder. [Invention 1005] The method of Invention 1003, wherein the disease or disorder is a psychiatric disease or disorder. [Invention 1006] The method of Invention 1003, wherein the disease or disorder is cancer. [Invention 1007] The method of Invention 1003, wherein the disease or disorder is associated with amnesia or memory dysfunction. [Invention 1008] A method for improving memory consolidation in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of Invention 1002. [Invention 1009] The method of Invention 1008, wherein the subject is a postmenopausal woman. [Invention 1010] A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject the pharmaceutical composition of Invention 1002. [Invention 1011] The method of Invention 1010, wherein the subject is a postmenopausal woman. [Invention 1012] TIFF0007708379000006.tif34144 A compound having a more selected formula. [Invention 1013] A pharmaceutical composition comprising an effective amount of the compound of Invention 1012 or a pharmaceutically acceptable salt thereof together with a pharmaceutical excipient, carrier, or diluent. [Invention 1014] A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of Invention 1013. [Invention 1015] The method of Invention 1014, wherein the disease or disorder is selected from neurological diseases and disorders, psychiatric diseases and disorders, and cell proliferative diseases and disorders. [Invention 1016] The method of Invention 1014, wherein the disease or disorder is associated with amnesia or memory dysfunction. [Invention 1017] A method for improving memory fixation in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of the present invention 1013. [The present invention 1018] The method of the present invention 1017, wherein the subject is a postmenopausal woman. [The present invention 1019] A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject a pharmaceutical composition of the present invention 1013. [The present invention 1020] The method of the present invention 1019, wherein the subject is a postmenopausal woman. [The present invention 1021] A method for improving memory fixation in a subject in need thereof, the method comprising administering to the subject a compound having the formula: TIFF0007708379000007.tif30128 or a pharmaceutical composition comprising the compound; wherein (a) Z is a carbon atom; (b) X is selected from the group consisting of hydrogen, hydroxyl, alkyl, hydroxyalkyl, amino, and aminoalkyl; and (c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -CH 2 CH 2 - or -OCH 2 -, and Y and Z form a bridge; or X and Y together form alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime, said method. [The present invention 1022] The method of the present invention 1021, wherein the compound has the formula Ia: TIFF0007708379000008.tif30128 [The present invention 1023] In the compound, X is selected from hydrogen, hydroxyl, alkyl-yl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -OCH 2 (c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -OCH -, and Y and Z form a bridge, the method of the present invention 1021. [The present invention 1024] TIFF0007708379000009.tif31128 The method of the present invention 1021, wherein the compound has the formula Ia(i): [The present invention 1025] The method of the present invention 1024, wherein in the compound, X is selected from hydrogen, hydroxyl, alkyl, and hydroxylalkyl, and Y is hydrogen. [The present invention 1026] 2 The method of the present invention 1021, wherein in the compound, X is hydrogen or methyl, and Y is hydroxymethyl (-CH 2 OH) or hydroxyethyl (-CH 2 CH OH). [The present invention 1027] 2 The method of the present invention 1021, wherein in the compound, X is methyl, and Y is hydroxymethyl (-CH OH).

Brief Description of the Drawings

[0018]

Figure 1

[35] . (b) Structure of ISP358-2, (c) estradiol (E2), and (d) ISP538-2 was overlaid on E2 to show its similarity to natural estrogen.

Figure 2

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[38] .

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Figure 13b

Figure 14

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Figure 16b

Mode for Carrying Out the Invention

[0019] Detailed Description As shown below and throughout this application, several definitions are used in this specification to explain the present invention.

[0020] Unless otherwise specified or indicated by the context, the terms "a", "an", and "the" mean "one or more". For example, "a substitution" should be interpreted to mean "one or more substitutions". Similarly, "a substituent group" should be interpreted to mean "one or more substituent groups".

[0021] As used herein, the terms "about", "approximately", "substantially", and "significantly" are understood by those skilled in the art and vary to some extent depending on the context in which they are used. When the use of these terms is not clear to those skilled in the art, considering the context in which these terms are used, "about" and "approximately" mean plus or minus ≤ 10% of a particular term, and "substantially" and "significantly" mean plus or minus > 10% of a particular term.

[0022] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising". The terms "comprise" and "comprising" must be construed as "open" transitional terms that can further include additional elements in addition to the elements recited in the claims. The terms "consist" and "consisting of" must be construed as "closed" transitional terms that cannot include additional elements other than those recited in the claims. The term "consisting essentially of" must be construed as being partially closed and can only include additional elements that do not fundamentally change the subject matter of the present invention.

[0023] As used herein, the "subject in need" may include a human or non-human animal. The term "subject" may be used synonymously with the terms "individual" or "patient".

[0024] As used herein, the term "subject in need" may include subjects in need of treatment with an agonist of estrogen receptor β isoform (ERβ). Subjects in need of treatment with an ERβ agonist may include subjects having a disease or disorder associated with ERβ activity. Diseases or disorders associated with ERβ activity may include, but are not limited to, proliferative diseases and proliferative disorders (e.g., cancer, such as breast cancer, ovarian cancer, and endometrial cancer), psychiatric diseases and psychiatric disorders (e.g., depression, anxiety, and schizophrenia), neurodegenerative diseases or neurodegenerative disorders (e.g., Alzheimer's disease including APOE4-related Alzheimer's disease), memory decline (e.g., memory decline observed under low estrogen conditions, such as those observed in postmenopausal women), bone metabolism diseases or bone metabolism disorders (e.g., osteoporosis), metabolic diseases or metabolic disorders (e.g., obesity or insulin resistance), and cardiovascular diseases or cardiovascular disorders.

[0025] Subjects in need may include subjects exhibiting low estrogen (i.e., estradiol) serum levels. Subjects exhibiting low estrogen serum levels may exhibit low estrogen serum levels associated with menopause (e.g., as observed in postmenopausal women). Subjects exhibiting low estrogen serum levels may include subjects having an estrogen serum level of less than about 60 pg / ml, 55 pg / ml, 50 pg / ml, 45 pg / ml, 40 pg / ml, 35 pg / ml, 30 pg / ml, 25 pg / ml, 20 pg / ml, 15 pg / ml, 10 pg / ml, 5 pg / ml, or less, or within a range bounded by any of these values (e.g., within the range of 15 - 60 pg / ml).

[0026] The subject in need may include a subject showing a low estrogen serum level related to a subject who has been administered a therapy and / or treatment that reduces estrogen serum level and / or estrogen activity in the subject. The subject in need may include a subject who is receiving a therapy for cancer treatment or a therapy after cancer treatment (e.g., a therapy for breast cancer treatment and / or a therapy after breast cancer treatment). The subject in need may include a subject who is receiving hormone therapy (e.g., hormone therapy for cancer, such as breast cancer), and / or a subject who is receiving hormone replacement therapy (e.g., hormone replacement therapy after treatment for cancer, such as breast cancer). The subject in need may include a subject who has received treatment with a drug that may include, but is not limited to, tamoxifen, toremifene (Fareston), fulvestrant (Faslodex), aromatase inhibitors (e.g., letrozole (Femara), anastrozole (Arimidex), and exemestane (Aromasin)), luteinizing hormone-releasing hormone (LHRH) analogs (e.g., goserelin (Zoladex) and leuprolide (Lupron)). The subject in need may include a subject who has undergone oophorectomy and / or hysterectomy.

[0027] The use of substituted (4'-hydroxyphenyl) cycloalkane compounds and (4'-hydroxyphenyl) cycloalkene compounds as selective agonists of estrogen receptor β isoform (ERβ) is disclosed. Some of the compounds of this compound class have been previously described in U.S. Patent Application Publication No. 2016 / 0340279 to Donaldson et al. The content of U.S. Patent Application Publication No. 2016 / 0340279 is hereby incorporated by reference in its entirety. Alternatively, the disclosed compounds may be referred to as substituted 4-cycloalkylphenol compounds or p-cycloalkyl-substituted phenol compounds, which include one or more substitutions on the cycloalkyl substituent, and the cycloalkyl substitution is preferably a cyclohexyl substituent.

[0028] In one aspect, the disclosed compounds include one or more substitutions on the 4-carbon of the cycloalkyl substituent and have the formula I: TIFF0007708379000010.tif30128, and wherein (a) Z is a carbon atom; (b) X is selected from the group consisting of hydrogen, hydroxyl, alkyl, hydroxyalkyl, amino, and aminoalkyl; and (c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -CH2CH2- or -OCH2-, and Y and Z form a bridge; or X and Y together form alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime.

[0029] The alkyl portion of the X substituent or the Y substituent may be C(1-6) alkyl. In certain embodiments, the alkyl portion may be C(1-3) alkyl. The hydroxyalkyl portion of the X substituent or the Y substituent may be hydroxyl-C(1-6) alkyl. In certain embodiments, the hydroxyalkyl portion may be hydroxyl-C(1-3) alkyl. The aminoalkyl portion of the X substituent or the Y substituent may be amino-C(1-6) alkyl. In certain embodiments, the aminoalkyl portion may be amino-C(1-3) alkyl.

[0030] The alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl moiety may be C(1-6) alkyl. In certain embodiments, the alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl may be C(1-3) alkyl. For example, the carboxyalkylidenyl may be carboxy-C(1-6)alkylidenyl or carboxy-C(1-3)alkylidenyl, the esteralkylidenyl may be C(1-6)alkyl-ester-C(1-6)alkylidenyl or C(1-3)alkyl-ester-C(1-3)alkylidenyl, the hydroxyalkylidenyl may be hydroxy-C(1-6)alkylidenyl or hydroxy-C(1-3)alkylidenyl, or the aminoalkylidenyl may be amino-C(1-6)alkylidenyl or amino-C(1-3)alkylidenyl.

[0031] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl)cyclohexane compounds. For example, in the disclosed compounds having formula I, A-B may be -CH2CH2-, A'-B' may be -CH2CH2-, and the compound may have formula Ia TIFF0007708379000011.tif30128.

[0032] wherein X and Y are as defined for formula I. In some embodiments of the compounds having formula Ia, the substituent X is selected from hydrogen, hydroxyl, and hydroxyalkyl, Y is selected from hydrogen, hydroxyl, alkyl, and -OCH2, and Y and Z form a bridge.

[0033] The disclosed compounds having formula Ia may exhibit specific stereochemistry. For example, when X and Y are as defined for formula I, the compounds may include cis and trans isomers of each other. For example, the compounds may have the formula: It may contain cis and trans isomers having TIFF0007708379000012.tif17128.

[0034] In certain embodiments, the compound has the formula It may also be an isomer having TIFF0007708379000013.tif13128.

[0035] In some embodiments of the compound having formula Ia, X may be hydroxyalkyl and Y may be hydrogen. Exemplary compounds have the formula: It may have TIFF0007708379000014.tif13128.

[0036] In some embodiments of the compound having formula Ia, X may be hydroxy and Y may be hydrogen. Exemplary compounds have the formula: It may have TIFF0007708379000015.tif12128.

[0037] In some embodiments of the compound having formula Ia, X may be hydroxyalkyl and Y may be hydroxyl. Exemplary compounds have the formula: It may have TIFF0007708379000016.tif16128.

[0038] In some embodiments of the compound having formula Ia, X may be hydrogen and Y may be -OCH3- and form a bridge with Z. Exemplary compounds have the formula: It may have TIFF0007708379000017.tif10128.

[0039] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl) cyclohexene compounds. For example, in the disclosed compounds having formula I, A-B may be -CH2CH2- and A'-B' may be =CHCH2-, and the compound has the formula Ia(i) It may have TIFF0007708379000018.tif16128.

[0040] Wherein X and Y are as defined for formula I. In some embodiments of the compounds having formula Ia(i), the substituent X is selected from hydrogen, hydroxyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, and alkyl.

[0041] In some embodiments of the compounds having formula Ia(i), X may be hydroxyalkyl and Y may be hydrogen. Exemplary compounds may have the formula: TIFF0007708379000019.tif13128.

[0042] The compounds disclosed herein (e.g., compounds having any of formulae I, Ia, and Ia(i)) may have several chiral centers, and stereoisomers, epimers, and enantiomers of the disclosed compounds are contemplated. The compounds may be optically pure with respect to one or more chiral centers (e.g., some or all of the chiral centers may be completely in the S configuration; and / or some or all of the chiral centers may be completely in the R configuration, etc.). Further or alternatively, one or more of the chiral centers may exist as a mixture of configurations (e.g., a racemic mixture or another mixture of R and S configurations). Compositions comprising substantially purified stereoisomers, epimers, or enantiomers of compounds having any of formulae I, Ia, and Ia(i) (e.g., compositions comprising at least about 90%, 95%, or 99% pure stereoisomers, epimers, or enantiomers) are contemplated herein.

[0043] The compositions contemplated herein are compositions comprising the compound: TIFF0007708379000020.tif25128, and isomers of the compound TIFF0007708379000021.tif25128, which may include compositions in which the isomers of the compound in the composition account for the majority of the isomers of the compound in the composition (e.g., at least about 90%, 95%, or 99% of the isomers of the compound in the composition).

[0044] Compounds that are substantially pure stereoisomers, epimers, or enantiomers, for example, compounds that are at least about 90%, 95%, or 99% pure stereoisomers, epimers, or enantiomers are contemplated herein. Compounds that are substantially pure (e.g., at least about 90%, 95%, or 99%) stereoisomers, epimers, or enantiomers of said compounds: TIFF0007708379000022.tif27128 is contemplated herein.

[0045] Hydroxy-protected derivatives of the compounds disclosed herein are also disclosed herein. For example, the compounds disclosed herein (e.g., compounds having any of Formula I, Ia, and Ia(i)) may contain a hydroxy-protecting group at any hydroxy group. As used herein, the term “protected hydroxy” group refers to a hydroxy group derivatized or protected by any group commonly used for the temporary or permanent protection of hydroxy functionality (e.g., an alkoxycarbonyl, acyl, silyl, or alkoxyalkyl group). A “hydroxy-protecting group” represents any group commonly used for the temporary protection of hydroxy functionality, such as, for example, an alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl group (hereinafter simply referred to as a “silyl” group), and an alkoxyalkyl group. An alkoxycarbonyl protecting group is an alkyl-O-CO-group, for example, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or allyloxycarbonyl. As used herein, the term “alkyl” as used in the description or claims refers to straight-chain or branched alkyl radicals of 1 to 6 carbons of all isomeric forms. “Alkoxy” refers to any alkyl radical attached by oxygen (i.e., a group represented by “alkyl-O-”). Alkoxyalkyl protecting groups are groups such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or groups such as tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl, and similar alkylated silyl radicals. The term “aryl” represents a phenyl group, or an alkyl-substituted phenyl group, a nitro-substituted phenyl group or a halo-substituted phenyl group.The terms "hydroxyalkyl", "deuteroalkyl", and "fluoroalkyl" each refer to an alkyl radical substituted by one or more hydroxy groups, deuterium groups, or fluoro groups. "Alkylidene" refers to a radical having the general formula C. k H 2k - where K is an integer (e.g., 1 to 6). The term "acyl" refers to an alkanoyl group of 1 to 6 carbons, or a carboxyalkanoyl group of 1 to 6 carbons, present in all of its isomeric forms, such as an oxalyl group, a malonyl group, a succinyl group, a glutaryl group, or an aromatic acyl group, such as benzoyl, or a halo-substituted benzoyl group, a nitro-substituted benzoyl group, or an alkyl-substituted benzoyl group.

[0046] The compounds disclosed herein may exhibit binding to estrogen receptors as well as agonist and / or antagonist activity against estrogen receptors. As used herein, "ERα" refers to estrogen receptor-α, particularly human estrogen receptor-α. As used herein, "ERβ" refers to estrogen receptor-β, particularly human estrogen receptor-β. Agonists and antagonists of ERα and ERβ are known in the art, and assays for determining the binding affinity of a compound for ERα and ERβ, as well as assays for determining whether a bound compound is an agonist or antagonist of ERα and ERβ, are also known in the art (see, for example, McCullough et al., "Probing the human estrogen receptor-α binding requirements for phenolic mono- and di-hydroxyl compounds: a combined synthesis, binding and docking study", Biorg. & Med. Chem. (2014) Jan 1;22(1):303-10. doi: 10.1016 / j.bmc.2013.11.024. Epub (2013) Nov 21 and the corresponding Supplementary Information, which are hereby incorporated by reference in their entirety). Assays suitable for determining the binding affinity of a compound for ERα and ERβ and assays suitable for determining whether a bound compound is an agonist or antagonist of ERα and ERβ may include polarization displacement assays and cell-based ERα luminescence activity assays and ERβ luminescence activity assays.

[0047] As used herein, the term "selective agonist" may be used to refer to a compound that selectively binds to and stimulates (agonizes) an estrogen receptor, particularly ERβ, as compared to another estrogen receptor, particularly ERα. For example, a compound that is a selective agonist of ERβ may have an IC 50 (nM) of less than 100 nM, preferably less than 10 nM, and even more preferably less than 1 nM in an assay for examining ERβ receptor agonist activity, and a compound that is a selective agonist of ERβ may have an IC 50 (nM) of greater than 100 nM, preferably greater than 500 nM, and even more preferably greater than 1000 nM in an assay for examining ERα receptor agonist activity.

[0048] As used herein, the term "selective agonist" may be used to refer to a compound that selectively binds to an estrogen receptor, particularly ERβ, as compared to another estrogen receptor, particularly ERα. For example, a compound that is a selective agonist of ERβ may have a binding affinity for the ERβ receptor that is at least 3-fold (or at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold) less than its binding affinity for ERα (when measured, for example, at K d (nM)). Preferably, a selective agonist of ERβ has a K d (nM) for ERβ of less than 100 nM, more preferably less than 10 nM, or even more preferably less than 1 nM. Preferably, a selective agonist of ERβ has a K d (nM) for ERα of greater than 500 nM, more preferably greater than 1000 nM, or even more preferably greater than 2000 nM.

[0049] As used herein, the term "selective agonist" may be used to refer to a compound that does not antagonize estrogen receptors, particularly ERβ, but rather selectively binds to and stimulates estrogen receptors, particularly ERβ. For example, a compound that is a selective agonist of ERβ may have an IC 50 (nM) of less than 100 nM, preferably less than 10 nM, and even more preferably less than 1 nM in an ERβ receptor agonist activity assay. A compound that is a selective agonist of ERβ may have an IC 50 (nM) of greater than 100 nM, preferably greater than 500 nM, and even more preferably greater than 1000 nM in an ERβ receptor agonist activity assay.

[0050] Pharmaceutically acceptable salts of the disclosed compounds are also contemplated herein and may be utilized in the disclosed methods of treatment. For example, substituents of the disclosed compounds may be protonated or deprotonated and may be present together with an anion or cation, respectively, as a pharmaceutically acceptable salt of the compound. As used herein, the term "pharmaceutically acceptable salt" refers to salts of compounds that are substantially non-toxic to an organism. Representative pharmaceutically acceptable salts include salts prepared by reacting the compounds disclosed herein with a pharmaceutically acceptable mineral acid or organic acid or organic base or inorganic base. Such salts are known as acid addition salts and base addition salts. Most, if not all, of the compounds disclosed herein are capable of forming salts, and it will be understood by those skilled in the art that the salt forms of this drug are commonly used. This is because, in many cases, this salt form is more readily crystallized and purified than the free acid or free base.

[0051] Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of suitable pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylate, acrylate, formic acid, hydrochlorides, dihydrochlorides, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, α-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, etc.

[0052] Base addition salts include base addition salts derived from inorganic bases such as hydroxides, carbonates, bicarbonates of ammonium or alkali or alkaline earth metals, etc. Bases useful in the preparation of such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, etc.

[0053] Typically, certain counterions that form part of any salt of a compound disclosed herein are not recognized as being absolutely essential, provided that the entire salt is pharmaceutically acceptable and the counterion does not impart undesirable qualities to the entire salt. Undesirable qualities can include undesirable solubility or toxicity.

[0054] It is further understood that the disclosed compounds can exist in equilibrium with various zwitterions. For example, zwitterions include salts in which the compound contains a deprotonated substituent and a protonated substituent.

[0055] The disclosed compounds may be used in preparing and formulating pharmaceutical compositions. Accordingly, disclosed herein are also pharmaceutical compositions that contain, together with a pharmaceutically acceptable excipient, an effective amount of any compound disclosed herein or a pharmaceutically acceptable salt of any compound disclosed herein. In some embodiments, the disclosed compounds may be used in preparing a medicament for treating a disease or disorder associated with estrogen receptor β (ERβ) activity, particularly a disease or disorder that can be treated using a specific ERβ agonist. Accordingly, the disclosed compounds may exhibit ERβ agonist activity, and preferably, the compound exhibits specificity as an ERβ agonist relative to an ERβ antagonist, an ERα agonist, and / or an ERα antagonist.

[0056] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions for treating diseases related to estrogen ERβ activity. Diseases or disorders related to ERβ activity may include, but are not limited to, cell proliferative diseases and disorders (e.g., cancer, e.g., breast cancer, ovarian cancer, and endometrial cancer), psychiatric diseases and disorders (e.g., depression, anxiety, and / or schizophrenia), neurodegenerative diseases or disorders (e.g., Alzheimer's disease including APOE4-related Alzheimer's disease), memory decline (e.g., memory decline observed under low estrogen conditions as observed in postmenopausal women), bone metabolic diseases or disorders (e.g., osteoporosis), metabolic diseases or disorders (e.g., obesity or insulin resistance), and cardiovascular diseases or disorders. The disclosed pharmaceutical compositions may be administered to a subject in need thereof in a method for treating diseases and disorders related to ERβ activity.

[0057] The compounds and pharmaceutical compositions disclosed herein may be administered to a subject in need thereof for treating a disease or disorder. In some embodiments, for treating a disease or disorder related to ERβ activity, the compounds disclosed herein may be administered at an effective concentration such that the compound functions as an ERβ agonist. In some embodiments, the amount of the disclosed compound effective for the compound to function as an ERβ agonist is about 0.05 - 50 μM (or about 0.05 - 10 μM or about 0.05 - 1 μM).

[0058] As used herein, "subject" may be interchangeable with "patient" or "individual" and means an animal, which may be a human or non-human animal, in need of treatment. Subjects suitable for the disclosed methods may include, for example, mammals such as humans, monkeys, dogs, cats, horses, rats, and mice. Suitable human subjects may include, for example, human subjects having a disease or disorder associated with ERβ activity or human subjects confirmed to be at risk of developing a disease or disorder associated with ERβ activity. Subjects in need of treatment may include postmenopausal women (e.g., postmenopausal women exhibiting low estrogen).

[0059] As used herein, "subject in need of treatment" may include a subject having a disease, disorder, or condition responsive to therapy with an ERβ agonist. For example, "subject in need of treatment" may include a subject having a proliferative disease, disorder, or condition such as cancer (e.g., carcinoma, e.g., breast cancer). Further, "subject in need of treatment" may include a subject having a neurological disease or disorder including psychiatric diseases and psychiatric disorders (e.g., depression, anxiety, and / or schizophrenia). "Subject in need" may include a subject having a neurodegenerative disease or disorder (e.g., Alzheimer's disease including APOE4-related Alzheimer's disease). In particular, subjects in need may include subjects showing memory loss or in need of improvement of memory consolidation (e.g., subjects having a disease or disorder characterized by the need for improvement of memory consolidation under low estrogen conditions). Subjects in need may include postmenopausal women in need of improvement of memory consolidation (e.g., postmenopausal women in need of improvement of memory consolidation under low estrogen conditions).

[0060] As used herein, the terms "treating" or "for treating" each mean reducing a symptom, eliminating the cause of the resulting symptom temporarily or permanently, and / or preventing or delaying the appearance of a symptom resulting from the disorder referred to, or reversing its progression or severity. Accordingly, the methods disclosed herein include therapeutic administration and prophylactic administration.

[0061] As used herein, the term "effective amount" refers to the amount or dosage of the compound when administered as a single dose or multiple doses to a subject, which brings about a desired effect in the subject during diagnosis or treatment. The disclosed method may include administering an effective amount of the disclosed compound (e.g., as present in a pharmaceutical composition) to treat a disease or disorder associated with ERβ activity in a subject, whereby the effective amount induces, promotes, or causes ERβ agonist activity in the subject.

[0062] The effective amount can be readily determined by a responsible diagnostician, who is skilled in the art, by using known techniques and by observing results obtained in similar circumstances. In determining the effective amount or dosage of the compound to be administered, many factors are considered by the responsible diagnostician, such as the species of the subject; its size, age, and general health; the degree of involvement or severity of the disease or disorder involved; the response of the individual subject; the particular compound being administered; the method of administration; the bioavailability characteristics of the preparation being administered; the dosing schedule selected; the use of concomitant medications; and other related circumstances.

[0063] In some embodiments, the daily amount of the disclosed compound may contain each compound used in the treatment method of the present invention from about 0.01 mg / kg to about 100 mg / kg (e.g., from about 0.05 mg / kg to about 50 mg / kg and / or from about 0.1 mg / kg to about 25 mg / kg). This dosage can be administered under any suitable regimen (e.g., weekly, daily, twice a day).

[0064] The pharmaceutical composition for use according to the method disclosed herein may contain one compound as the active ingredient or may contain a combination of compounds as the active ingredient. For example, the method disclosed herein may be carried out using a composition containing one compound that is an ERβ agonist. Alternatively, the disclosed method may be carried out using a composition containing two or more compounds that are ERβ agonists, or a composition containing one compound that is an ERβ agonist together with a compound that is an ERα antagonist.

[0065] Instead of administering a pharmaceutical composition containing a compound that is an ERβ agonist together with a compound that is an ERα antagonist, the disclosed method may be carried out by administering a first pharmaceutical composition (e.g., a pharmaceutical composition containing an ERβ agonist) and administering a second pharmaceutical composition (e.g., a pharmaceutical composition containing an ERα antagonist), where the first pharmaceutical composition may be administered before, simultaneously with, or after the second composition. Thus, the first pharmaceutical composition and the second pharmaceutical composition may be administered simultaneously, regardless of their names, or in any order.

[0066] As will be understood by those skilled in the art, the disclosed pharmaceutical compositions can be prepared using materials (e.g., active excipients, carriers, and diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared using materials having other properties that render the formulation suitable for administration to non-human subjects but not suitable for administration to humans, in terms of purity and / or otherwise.

[0067] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in a solid dosage form, but any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form may be, for example, an immediate-release dosage form, a controlled-release dosage form, a lyophilized dosage form, a delayed-release dosage form, a long-term release dosage form, a pulse-release dosage form, a mixed immediate-release and controlled-release dosage form, or a combination thereof. Alternatively, the compounds utilized in the methods disclosed herein may be formulated as a liquid-based pharmaceutical composition (e.g., an injection solution or a gel).

[0068] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition comprising an excipient, a carrier, or a diluent. For example, the excipient, carrier, or diluent may be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0069] The compounds utilized in the methods disclosed herein may also be formulated as pharmaceutical compositions comprising one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and foaming agents. Fillers may include lactose monohydrate, anhydrous lactose, and various starches. Examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, crystalline cellulose such as Avicel® PH101 and Avicel® PH102, crystalline cellulose, and silicified crystalline cellulose (ProSolv SMCC™). Suitable lubricants that act on the flowability of the powder to be compressed may include colloidal silicon dioxide such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame. Examples of flavoring agents are Magnasweet® (a registered trademark of MAFCO), bubble gum flavoring agents, and fruit flavoring agents. Examples of preservatives may include potassium sorbate, methyl paraben, propyl paraben, benzoic acid and its salts, other para-hydroxybenzoic acid esters such as butyl paraben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0070] Diluents suitable for pharmaceutical compositions can include pharmaceutically acceptable inert bulking agents such as crystalline cellulose, lactose, calcium hydrogen phosphate, sugars, and mixtures of any of the foregoing. Examples of diluents include crystalline cellulose such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, anhydrous lactose, and Pharmatose® DCL21; calcium hydrogen phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0071] The disclosed pharmaceutical compositions may also contain disintegrants. Suitable disintegrants include slightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, corn starch, and modified starch, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

[0072] The disclosed pharmaceutical compositions may also contain effervescent agents. Examples of effervescent agents are effervescent couples such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, as well as anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Or only the sodium bicarbonate component of the effervescent couple may be present.

[0073] The pharmaceutical composition containing the above compound may be adapted for administration by any suitable route, for example, via oral (including buccal or sublingual) route, rectal route, nasal route, topical route (including buccal, sublingual, or transdermal), vaginal route or parenteral route (including subcutaneous, intramuscular, intravenous, or intradermal). Such formulations can be prepared by any method known in the art of pharmacy, for example, by associating the active ingredient with a carrier or excipient.

[0074] Pharmaceutical compositions adapted for oral administration may be presented as discrete units, for example, capsules or tablets; powders or granules; solutions or suspensions dissolved in aqueous liquids or non-aqueous liquids; edible foams or whips; or water-in-oil liquid emulsions or oil-in-water liquid emulsions.

[0075] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches designed to remain in intimate contact with the epidermis of the recipient for a prolonged period. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0076] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols, or oils and may contain suitable conventional additives such as preservatives, solvents to assist drug penetration, and emollients in ointments and creams.

[0077] When applied to the eye or other external tissues, for example, the mouth and skin, the pharmaceutical composition is preferably applied as a topical ointment or cream. When formulated as an ointment, the compound may be used with a paraffinic or water-miscible ointment base. Alternatively, the compound may be formulated in a cream containing a water-in-oil cream base or an oil-in-water base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent.

[0078] Pharmaceutical compositions adapted for local administration in the mouth include lozenges, troches, and mouthwashes.

[0079] A pharmaceutical composition adapted for rectal administration may be presented as a suppository or an enema.

[0080] A pharmaceutical composition adapted for nasal administration, wherein the carrier is solid and comprises a coarse powder having a particle size (e.g., in the range of 20 to 500 microns) adapted to be administered by sniffing tobacco (i.e., by rapid inhalation through the nasal cavity from a powder container held near the nose). When administered as a nasal spray or nasal drops, a suitable formulation wherein the carrier is liquid comprises an aqueous or oily solution of the active ingredient.

[0081] Pharmaceutical compositions adapted for administration by inhalation include fine particle powders or sprays which may be generated by various types of metered dose pressurized aerosols, nebulizers, or inhalers.

[0082] A pharmaceutical composition adapted for vaginal administration may be presented as a pessary, tampon, cream, gel, paste, foam, or spray formulation.

[0083] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending and thickening agents. The formulations may be presented in unit dose containers or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state which requires only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Immediate injection solutions and immediate injection suspensions may be prepared from sterile powders, granules, and tablets.

[0084] Exemplary embodiments The following embodiments are illustrative and should not be construed as limiting the scope of the invention.

[0085] Aspect 1. A compound having the formula and stereochemistry of TIFF0007708379000023.tif25128.

[0086] Aspect 2. The compound of Aspect 1 in substantially pure form.

[0087] Aspect 3. A pharmaceutical composition comprising an effective amount of the compound of Aspect 1, preferably the compound of Aspect 1 in substantially pure form or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent (e.g., stereoisomers corresponding to at least about 90%, 95%, or 99% of the compound in the composition).

[0088] Aspect 4. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject a compound of Aspect 1 or 2 or a pharmaceutical composition of Aspect 3.

[0089] Aspect 5. The method of Aspect 4, wherein the disease or disorder is selected from neurological diseases and disorders, psychiatric diseases and disorders, and cell proliferative diseases and disorders.

[0090] Aspect 6. The method of Aspect 4, wherein the disease or disorder is associated with amnesia or memory impairment.

[0091] Aspect 7. A method for improving memory consolidation in a subject in need thereof, the method comprising administering to the subject a compound of Aspect 1 or 2 or a pharmaceutical composition of Aspect 3.

[0092] Aspect 8. A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject a compound of Aspect 1 or 2 or a pharmaceutical composition of Aspect 3.

[0093] Aspect 9. The method of Aspect 7 or Aspect 8, wherein the subject is a postmenopausal woman.

[0094] Aspect 10. A compound having a formula selected from TIFF0007708379000024.tif34144.

[0095] Aspect 11. A pharmaceutical composition comprising an effective amount of the compound of Aspect 10 or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.

[0096] Aspect 12. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need thereof, the method comprising administering to the subject a compound of Aspect 10 or the pharmaceutical composition of Aspect 11.

[0097] Aspect 13. The method of Aspect 12, wherein the disease or disorder is selected from neurological diseases and disorders, psychiatric diseases and disorders, and cell proliferative diseases and disorders (e.g., cancer, e.g., breast cancer, ovarian cancer, endometrial cancer, etc.).

[0098] Aspect 14. The method of Aspect 12, wherein the disease or disorder is associated with memory loss or memory impairment.

[0099] Aspect 15. A method for improving memory consolidation in a subject in need thereof, the method comprising administering to the subject a compound of Aspect 10 or the pharmaceutical composition of Aspect 11.

[0100] Aspect 16. A method for treating a subject exhibiting low estrogen levels, the method comprising administering to the subject a compound of Aspect 10 or the pharmaceutical composition of Aspect 11.

[0101] Aspect 17. The method of Aspect 15 or Aspect 16, wherein the subject is a postmenopausal woman.

[0102] Aspect 18. A method for improving memory fixation in a subject in need of improved memory fixation, the method comprising administering to the subject a compound having the formula: TIFF0007708379000025.tif30128 or a pharmaceutical composition comprising the compound; wherein (a) Z is a carbon atom; (b) X is selected from the group consisting of hydrogen, hydroxyl, alkyl, hydroxyalkyl, amino, and aminoalkyl; and (c) Y is selected from the group consisting of hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -CH2CH2- or -OCH2-, and Y and Z form a bridge; or X and Y together form alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime, the method.

[0103] Aspect 19. The method of Aspect 18, wherein the compound has the formula Ia: TIFF0007708379000026.tif30128.

[0104] Aspect 20. The method of Aspect 18 or 19, wherein in the compound, X is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl; or Y is -OCH2-, and Y and Z form a bridge.

[0105] Aspect 21. The method of Aspect 18, wherein the compound has the formula Ia(i): TIFF0007708379000027.tif31128.

[0106] Aspect 22. The method of Aspect 21, wherein in the compound, X is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl, and Y is hydrogen.

[0107] Aspect 23. The method of Aspect 18, wherein in the compound, X is hydrogen or methyl, and Y is hydroxymethyl (-CH2OH) or hydroxyethyl (-CH2CH2OH).

[0108] Aspect 24. The method of Aspect 18, wherein in the compound, X is methyl, and Y is hydroxymethyl (-CH2OH).

Examples

[0109] The following examples are illustrative and should not be construed as limiting the scope of the present invention.

[0110] Example 1. A-C estrogen as a potent and selective estrogen receptor-β agonist (SERBA) for improving memory consolidation under low estrogen conditions Overview Estrogen receptor-β (ERβ) is a drug target for memory consolidation in postmenopausal women. A series of potent and selective ERβ agonists (SERBA) that have in vivo efficacy and are A-C estrogens and lack the B and D estrogen rings are reported herein. The most potent and selective A-C estrogens are selective for ER activation compared to seven other nuclear hormone receptors. Surprisingly, the selectivity for the β isoform is 750 times that of the α isoform, and the IC 50 is 20 - 30 nM in cell assays and direct binding assays. Comparison of the potencies in various assays suggests that ER isoform selectivity is related to the ability of the compounds to drive productive conformational changes required for transcriptional activation. The compounds disclosed herein also exhibit in vivo efficacy after microinfusion into the dorsal hippocampus, after intraperitoneal injection (0.5 mg / kg), or after forced oral delivery (5 mg / kg). This simple but novel A-C estrogen is selective, a brain penetrant, and promotes memory consolidation.

[0111] Results Compound synthesis The commercially available 4-(4-hydroxyphenyl)cyclohexanone 1 is converted to alcohol 2 by reacting it with NaBH4, or to alcohol 3 by reacting it with excess methyllithium, or to oxime 4 by condensation with hydroxylamine (Scheme 1). o or converted to alcohol 3 by reacting it with excess methyllithium, o or converted to oxime 4 by condensation with hydroxylamine (Scheme 1).

[0112] TIFF0007708379000028.tif78154

[0113] Scheme 1. Reagents: (a) NaBH4 / MeOH (90%); (b) MeLi / Et2O (37%); (c) H2NOH-HCl, Amberlyst, ethanol (70%); (d) TBSCl, imidazole (83%); (e) Ph3PCH3 + Br - , n-BuLi (84%); (f) TBAF / THF (73 - 78%); (g) H2, Pd / C, (h) paraformaldehyde, MgCl2, NEt3 (40%); (i) H2NOH-HCl, NaHCO3, ethanol (69%); (j) cat. OsO4, NMO (1.4 equiv) (86%); (k) BH3-THF, then 30% H2O2 / 1 N NaOH; (l) 9-BBN, then 30% H2O2 / 1 N NaOH; (m) DDQ (0.5 equiv) / CH2Cl2 (16, 47%; 17, 37%); (n) MgCl2, NEt3 (78%).

[0114] The stereochemistry of 2 and 3 was assigned based on NMR spectral data. In particular, for o alcohol 2, the alcohol methane proton appears as a triplet of triplets at δ 2.38 (J = 11.8, 3.4 Hz). The large coupling is consistent with an axial-axial disposition of this proton, and thus the hydroxyl group is equatorial. For 3, the o alcohol and methyl carbons assigned, 13The signals at δ 69.5 and 31.1 ppm in the 13C NMR spectrum are in good agreement with this type of cis-1,4-alcohol 24 。The t-butyldimethylsilyl ether 5 was olefinated using the ylide generated from methyltriphenylphosphonium bromide to give 6. Treatment of 6 with TBAF to cleave the silyl ether gave 7. Catalytic hydrogenation of 7 gave 8 as a mixture of stereoisomers. Substituted salicaldehyde 9 was obtained by reacting 7 with excess paraformaldehyde, MgCl2, and NEt3, and reaction of this with hydroxylamine gave oxime 10. After dihydroxylation of 6 and cleavage of the silyl ether, 12 was obtained as a single stereoisomer after chromatographic purification. Based on the known stereochemistry of the osmium-catalyzed dihydroxylation of 4-t-butylmethylene cyclohexane, the stereochemistry of 12 was assigned as shown 25 。Hydroboration-oxidation of 6 using BH3-THF gave an inseparable mixture of the stereoisomeric primary alcohols cis-13 and trans-14, which occurred in a 2:1 ratio as determined by integration of the 1H NMR signals of their hydroxymethylene protons (δ 3.60 and 3.39 ppm, respectively). The stereochemistry of the isomers was tentatively assigned based on the relative chemical shifts of these two signals. The signal for the axial hydroxymethylene (i.e., the cis isomer) appears at lower field compared to the signal for the equatorial hydroxymethylene 1 。Alternatively, hydroboration-oxidation using 9-BBN gave a mixture in which trans-14 was present in a larger ratio compared to cis-13 (2:3, cis:trans). The use of these two borane reagents has been previously reported to adjust the cis:trans outcome of 4-substituted methylene cyclohexanes 24 。 26、27Cleavage of the silyl ether using TBAF gave a mixture of the stereoisomers 4-(4-hydroxymethylcyclohexyl)phenol cis-15 / trans-16. Treatment of the mixture of stereoisomers 15 / 16 (2:3, cis:trans) with DDQ (0.5 equiv) gave a separable mixture of the bicyclic ether 17 and trans-16. The tentative structure assignment of trans-16 was confirmed by single crystal X-ray diffraction analysis (Figure 7a). 28 Isolation of the unreacted trans-16 was rationalized based on the faster oxidation rate of cis-15. Oxidation of cis- or trans-4-(4-hydroxymethylcyclohexyl)phenol proceeds via the same benzylic carbocation intermediate (i.e., 18, Scheme 2), and the activation energy for the formation of this intermediate is lower for the less stable cis-15 compared to trans-16, and thus the oxidative cyclization of the cis-isomer is faster. Reaction of 17 with MgCl2 and trimethylamine resulted in an intramolecular elimination reaction, giving cyclohexene (±)-19 (Scheme 1).

[0115] TIFF0007708379000029.tif28154

[0116] Scheme 2. Oxidation of cis- or trans-4-(4-hydroxymethylcyclohexyl)phenol via the same benzylic carbocation intermediate

[0117] The structure of 19 was assigned based on its NMR spectral data. In particular, the signal for the olefinic proton appears as a narrow multiplet at approximately δ 5.95 ppm. This signal is characteristic of other 1-(4-hydroxyphenyl)cyclohexenes 29 。

[0118] Horner-Emmons olefination of the silyl ether 20 (prepared from 1) using triethyl phosphonoacetate gave the unsaturated ester (±)-21. Desilylation using TBAF gave the phenol (±)-22 (Scheme 3).

[0119] TIFF0007708379000030.tif42154

[0120] Scheme 3. Reagents: (a) TBDPSCl, imidazole (93%); (b) EtO2CCH2P(O)(OEt)2, NaH (95%); (c) DIBAL, CH2Cl2, -40 °C (quant.); (d) TBAF (80%); (e) H2 (30 psi), 20% Pd / C (25, 14%; 26, 60%).

[0121] After reducing 21 with DIBAL, the silyl ether was deprotected to give allyl alcohol (±)-24. Catalytic hydrogenation of 24 gave a separable mixture of alcohol 25 and the less abundant ethylcyclohexane derivative 26.

[0122] TR-FRET and cell transcription assays The initial screening of the compounds was performed in a TR-FRET displacement assay. The TR-FRET displacement assay detects binding to ERβLBD (see Figure 2 for the dose-response curves of the selected compounds and Figure 9 for the dose-response curves of all compounds). In this assay, IC 50 was measured for all the synthesized compounds. The IC 50 values were summarized in Table 1.

[0123] (Table 1) Estrogen receptor assay data. Reported values are IC50 and values are in nM TIFF0007708379000031.tif47149TIFF0007708379000032.tif206149TIFF0007708379000033.tif25149* IC 50 is the average of two data sets that are 31 ± 7 nM (Figure 5a) and 23 ± 8 nM (Figure 10). Therefore, the selectivity is 658 - 886 and the average is 750.

[0124] The most potent compounds were 16 (hereinafter referred to as ISP358-2) (hydroxymethyl substitution), 25 (hydroxyethyl substitution), and 8 (methyl substitution). The IC 50 for all of these compounds against ERβ was <30 nM. ISP358-2 is a pure trans isomer and was found to bind with higher affinity to ERβ than a mixture of cis and trans stereoisomers (15 / 16). The presence of a methylene (ISP358-2) or ethylene (25) linker to the hydroxyl group results in efficacy, but direct substitution of the hydroxyl on the cyclohexane ring significantly decreases the affinity (IC 50 of 2 was 7,250 nM). Introduction of unsaturation (24) into the alkyl linker also decreased the affinity (676 nM), but introduction of unsaturation (18) into the cyclohexane ring only moderately decreased the affinity (49 nM). The binding of ISP358-2 to ERα was also tested, and in this TR-FRET assay measuring direct binding to the isolated LBD, ISP358-2 bound with 12-fold higher affinity to ERβ (IC 50 24 nM; Figure 2b).

[0125] Furthermore, ISP358-2 was screened in a nuclear hormone receptor function assay (Figure 3). In this assay, transcriptional activation due to binding and activation of a chimeric receptor composed of the ligand binding domain (LBD) of a hormone receptor of interest (e.g., ERβ) tethered to the DNA binding domain (DBD) of GAL4 was measured. This assay was conducted to evaluate the selectivity of estrogen receptor activation compared to other nuclear hormone receptors. No significant agonist activity of compound ISP358-2 was observed against any nuclear hormone receptor (excluding the estrogen receptor) tested at concentrations of 0.25 - 25 μM (Figure 3a). Thus, ISP358-2 is not an agonist of the following receptors: androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), peroxisome proliferator-activated receptor (PPARδ), progesterone receptor (PR), thyroid hormone receptor (TRβ), and vitamin D receptor (VDR) (see Table 2 for IC50 of control compounds for each nuclear hormone tested). In a follow-up 10-point titration in this same assay, binding and activation of ISP358-2 to full-length chimeric ERβ (357 ± 26 nM) was found to be 2.6-fold more selective compared to full-length chimeric ERα (930 ± 69 nM). This assay (Figure 3) measures transcriptional activation rather than simply binding of an agonist to the ER LBD, as was the case in (Figure 2). However, this assay uses an unnatural chimeric protein (ER LBD fused to GAL4 DBD) that may not accurately reflect activation induced by actual agonists occurring under natural conditions.

[0126] When performing a core-activator type TR-FRET LBD binding assay (Figure 4a), the ISP358-2 compound (Figure 4b) was found to be 15-fold selective compared to ERα (2,940 ± 390 nM) with respect to binding to ER and recruitment of the PPARγ co-activator peptide to ERβ (161 ± 15 nM) (Figure 4c). This assay measures the activation of the ER LBD in that it measures not only the binding of the agonist to the receptor, but also the binding and agonist-induced co-activator peptide recruitment.

[0127] Finally, a cell transcriptional activation assay was performed using native full-length ER (composed of ER LBD and ER DBD). Unlike the previous assays, this assay is cell-based and thus most closely mimics the in vivo situation. The most potent and selective compound tested in this assay was ISP358-2, which had an ERβ agonist efficacy of 31 ± 7 nM (Figure 5a and Figure 10a; this assay was repeated twice and the values obtained were 31 nM and 23 nM, with an average of 27 nM) and an ERα agonist efficacy of 20,419 ± 859 nM (Figure 5b). This gives an ERβ / ERα selectivity ratio of approximately 750 in this more physiologically relevant assay. ISP358-2 showed no antagonist activity for ERβ (Figure 5c) or ERα (Figure 5d) at concentrations up to 10 μM.

[0128] In vitro drugability - CYP450 binding, hERG, and turbidimetric analysis ISP358-2 did not show inhibition of CYP1A2 and CYP2D6 and showed only weak inhibition of CYP2C9 (IC 50 = 34 ± 4.7 μM) and CYP3A4 (IC 50 = 89 ± 18 μM) (Figure 6). ISP358-2 did not bind to hERG and showed only 14% activity at 100 μM. Turbidity analysis (performed on a mixture of ISP171, 15 / 16, isomers) showed no significant aggregation. This indicates good solubility at concentrations up to 300 μM (Figure 11).

[0129] Docking studies ISP358-2 (Figure 7a) was docked to the binding site of agonist-conformation ERα in two conformations with similar docking energies. In one binding mode (Figures 6d and 6e), the phenolic hydroxyl interacts with Arg394 / Glu353 (energy = -7.6 kcal / mol), and in another mode, the ISP358-2 molecule is inverted by 180 degrees (energy = -7.8 kcal / mol), and the aliphatic hydroxyl interacts with Arg394 / Glu353. ISP358-2 binds within the agonist-conformation ERβ pocket (Figures 7c and data not shown), and the phenolic hydroxyl interacts with Arg394 / Glu353 in the minimum energy docking pose (energy = -8.0 kcal / mol). In both cases, there are significant hydrophobic interactions between ISP358-2 and the binding site residues, but the ERβ pocket is smaller, creating a more intimate fit. In both cases, the hydroxymethyl group is close enough (3.0 Å) to His524 to participate in the hydrogen bonding interactions typically observed for ER agonists, but in ERβ, the hydrogen bound to the aliphatic alcohol may be with the backbone carbonyl of Gly472. Similar to the natural estrogen molecule, in ERβ, there are large hydrophobic interactions that force the hydroxymethyl-cyclohexyl ring (ring C) to be nearly planar with the phenolic ring (ring A) (data not shown). This is in contrast to the binding pose in ERα where the two rings are nearly orthogonal (data not shown). The ERβ hydrophobic interactions are with Phe356, Met340, Phe355, Leu298 near the phenolic ring, Leu476, Ile373 near the hydroxymethyl group, and Ala302, Leu298 near the cyclohexane ring (Figures 7c and data not shown). Control docking studies of E2 reproduced the expected binding orientation based on the crystal structure (data not shown).

[0130] Evaluation of memory consolidation Dorsal hippocampal injection First, the inventors examined the effect of direct intra - hippocampal injection of ISP358 - 2 on object recognition and spatial memory consolidation in ovariectomized mice (Figure 8a). Five groups of mice: vehicle (negative control), DPN (positive control), and three doses of ISP358 - 2 (10 pg / hemisphere, 100 pg / hemisphere, and 1 ng / hemisphere) were tested (Figure 8b,c). For object placement (Figure 8b), from a one - sample t - test, it was found that mice given vehicle or 10 pg ISP358 - 2 did not spend significantly more time with the moved object than by chance (ts (7) = 0.44 and 1.19, p>0.05; n = 8), respectively. From this, it can be seen that these groups did not show memory of the training object location. In contrast, mice given DPN, 100 pg ISP358 - 2, or 1 ng ISP358 - 2 spent significantly more time with the moved object than by chance (ts (6) = 4.5, 10.3, and 3.4, p<0.05; n = 7), respectively. This indicates a strong memory of the training object location. Furthermore, a one - way ANOVA performed on the time spent with the moved object showed a significant main effect of treatment (F (4,32) = 2.97, p = 0.034). From Fisher's LSD post - hoc test, it was found that the DPN, 100 pg, and 1 ng groups spent significantly more time with the moved object than the vehicle group, while the vehicle and 10 pg groups did not differ from each other. Overall, these data suggest that dorsal hippocampal injection of 100 pg or 1 ng ISP358 - 2 improved memory consolidation of object placement.

[0131] The results of object recognition (Figure 8c) were almost the same. Neither the vehicle group nor the 10 pg ISP358 - 2 group preferred the novel object (ts (9-10) = 1.08 and 0.88, p>0.05; n = 10 - 11), respectively. However, all of the DPN, 100 pg ISP358 - 2, and 1 ng ISP358 - 2 groups spent significantly more time with the novel object than by chance (ts (9-10)= 2.35, 3.16, and 1.08, p < 0.05; n = 10 - 11). Furthermore, the main effect of the treatment was significant (F (4,48) = 3.69, p = 0.011). Post hoc tests confirmed that the DPN, 100 pg ISP358 - 2, and 1 ng ISP358 - 2 groups were significantly different from the vehicle, while the 10 pg ISP358 - 2 group was not. Similar to the object placement, these data indicate that dorsal hippocampal injection of 100 pg or 1 ng ISP358 - 2 improved memory consolidation of object recognition, while 10 pg ISP358 - 2 did not.

[0132] Intraperitoneal injection Next, the inventors used a new set of mice to examine whether systemic administration of ISP358 - 2 would also produce a memory - enhancing effect similar to that of intra - hippocampal injection (Fig. 8d, e). Intraperitoneal (IP) injection is a common, reliable, and convenient systemic treatment where the injected drug is absorbed into the blood vessels through the peritoneum. 30 Since the drug dose for intra - hippocampal injection is much less than that required to cross the blood - brain barrier, the inventors examined a range of IP doses based on the above cell assay and DH injection results and previous studies showing that IP injection of 0.05 mg / kg DPN improved object recognition memory. 31 In the inventors' cell assay, the IC50 of ISP358 - 2 was approximately 10 times that of DPN. Furthermore, from the inventors' behavioral tests, it was found that ISP358 - 2 improved hippocampal memory at a concentration 10 times that of DPN. Therefore, the inventors' IP doses of ISP358 - 2 were at least 10 times that of DPN (0.5 mg / kg and 5 mg / kg). Thus, the inventors tested four mouse groups as follows: vehicle (negative control), DPN (positive control), and two doses of ISP358 - 2 (0.5 mg / kg and 5 mg / kg).

[0133] For object placement (Fig. 8d), a one - sample t - test showed that mice given the vehicle preferred the non - moved object (t (8)= 0.68, p > 0.05; n = 9). However, all of the DPN group, the 0.5 mg / kg ISP358-2 group, and the 5 mg / kg ISP358-2 group spent significantly more time on the moved object than by chance (ts (9-11) = 3.20, 3.93, and 2.78, p < 0.05; n = 10 - 12), respectively. These results suggest that systemic administration of ISP358-2 improved the memory consolidation of object placement. Furthermore, the main effect of the treatment was significant (F (3,38) = 3.63, p = 0.021), and post hoc tests revealed that the 0.5 mg / kg ISP358-2 group was significantly different from the vehicle. These data demonstrate that IP injection of ISP358-2 improves spatial memory consolidation, similar to dorsal hippocampal infusion.

[0134] Similar results were observed for object recognition (Fig. 8e). One-sample t-test results showed that mice given the vehicle did not spend significantly more time on the novel object than by chance (t (9) = 1.40, p > 0.05; n = 10). In contrast, the DPN group and the 0.5 mg / kg ISP358-2 group preferred the novel object significantly more than by chance (ts (8および11) = 3.52 and 4.17, p < 0.01; n = 12 and 9), respectively. To some extent, the 5 mg / kg ISP358-2 also tended to prefer the novel object (t (12) = 1.65, p = 0.125; n = 13). Furthermore, the main effect of the treatment was significant (F (3,40) = 5.05, p = 0.005), and post hoc tests verified that the DPN group, the 0.5 mg / kg ISP358-2 group, and the 5 mg / kg ISP358-2 group were significantly different from the vehicle group. In summary, data from object placement and object recognition suggest that ISP358-2, especially IP administration at a dose of 0.5 mg / kg, improves object recognition and spatial memory consolidation, similar to dorsal hippocampal infusion. Importantly, these data also demonstrate the brain penetration and behavioral efficacy in ovariectomized mice.

[0135] Forced oral administration Considering the effectiveness of IP injection in enhancing memory, the inventors then evaluated whether oral administration of ISP358-2 could improve memory consolidation (Figure 8f,g). Forced oral administration is a common procedure in scientific experiments to directly deliver drugs to the stomach by syringe. 32 Forced oral administration is much more effective and accurate than other oral administration methods such as administration by delivery in food and / or water, but it is invasive and stressful. 33 Since the inventors observed that IP injection of ISP358-2 improved hippocampal memory consolidation, the same dose as the IP injection was used for forced oral administration (vehicle, 0.5 mg / kg or 5 mg / kg ISP358-2, and 0.05 mg / kg DPN).

[0136] Similar results were observed for oral administration as for IP injection of ISP358-2. For object placement (Figure 8f), from the one-sample t-test results, it was found that mice given the vehicle did not spend significantly more time on the moved object by chance (t (8) = 0.54, p > 0.05; n = 9). However, all of the DPN group, 0.5 mg / kg ISP358-2 group, and 5 mg / kg ISP358-2 group significantly preferred the moved object compared to chance (ts (8-9) = 2.76, 3.65, 5.06, p < 0.05; n = 9 - 10), respectively. This suggests that oral administration of ISP358-2 improved spatial memory consolidation. It was also found that the main effect of the treatment was significant by one-way ANOVA (F (3,33) = 5.04, p = 0.006), and post hoc tests verified that the DPN group, 0.5 mg / kg ISP358-2 group, and 5 mg / kg ISP358-2 group were significantly different from the vehicle group. Similarly, for object recognition (Figure 8g), from the one-sample t-test, it was found that mice given the vehicle did not prefer the novel object (t (8)= 0.25, p > 0.05; n = 9). In contrast, the DPN group, 0.5 mg / kg ISP358-2 group, and 5 mg / kg ISP358-2 group all spent significantly more time on the novel object compared to chance (ts (7-9) = 3.89, 5.37, 2.36, p < 0.05; n = 8 - 10), respectively. Furthermore, the main effect of treatment was significant (F (3,32) = 3.02, p = 0.044), and post hoc tests revealed that the DPN group, 0.5 mg / kg ISP358-2 group, and 5 mg / kg ISP358-2 group were significantly different from the vehicle. In summary, from the results of the object placement and object recognition behaviors, it was demonstrated that oral administration of ISP358-2 improved object recognition and spatial memory consolidation, similar to dorsal hippocampal infusion or IP injection. These data also suggest the oral bioavailability of ISP358-2 in ovariectomized mice.

[0137] Finally, the inventors collected preliminary data to evaluate the effectiveness of forcibly orally administered ISP358-2 on spatial memory consolidation in mice that had experienced long-term estrogen deficiency. Mice injected i.p. with the above vehicle, DPN, or ISP358-2 remained in the inventors' colony for 4 months after ovariectomy. The mice were then trained in an object placement task (using novel objects) and then immediately given the same doses of vehicle, DPN, or ISP358-2 as above via forced oral administration (n = 9 - 12 / group). Different from the mice that were forcibly administered within 1 month after ovariectomy (Figure 8f), DPN or ISP358-2 did not improve spatial memory consolidation in mice treated within 4 months after ovariectomy (Figure 12a). Then, the inventors conducted their previous research 34(ERα, 1:200, Santa Cruz Biotechnology; ERβ, 1:200, Santa Cruz Biotechnology), Western blotting was used to measure ERα and ERβ levels in DH. Tissues were collected approximately 2 and 5 months after ovariectomy. ERβ levels 5 months after ovariectomy were significantly decreased compared to those 2 months after ovariectomy (Figure 12b; t9 = 2.46, p < 0.05). In contrast, ERα levels did not change (Figure 12c). These data suggest that neither DPN nor ISP358-2 improved memory due to decreased ERβ levels after long-term ovariectomy. Extrapolating, these data also support the in vivo selectivity of ISP358-2 for ERβ. That is, if ISP358-2 improved memory by binding to ERα, ERα levels would not have decreased and memory consolidation should have been improved after long-term ovariectomy. However, the fact that ERβ levels were low when ISP358-2 did not improve memory supports the inventors' hypothesis that ISP358-2 regulates memory via ERβ rather than ERα.

[0138] Evaluation of peripheral pathology or cell proliferation by ISP358-2 treatment Generally, tissues from 20 different specimens all appeared similar. Heart : All heart tissues did not draw people's attention. The ventricular wall was intact and had a normal thickness. The atrial wall was intact and had a normal thickness. There was no evidence of congenital defects such as intricate arrangements of muscle fibers or ischemic heart disease or ischemic injury. There was no evidence of inflammation or myocarditis. Kidney : All kidneys did not draw people's attention. The glomeruli were intact. The renal tubules appeared normal. There was no evidence of inflammation involving any structure of the kidney. Liver: The overall structure of the liver was intact and appeared normal, with a large portal-type vein running together with the bile duct and hepatic artery. Central veins were present and appeared normal. Systemic manifestations of low-grade / mild ischemic injury were present in all samples. This appeared to be non-specific, observed in all specimens, and may have been secondary to initial ischemic injury or autolysis occurring postmortem. Small foci of cell necrosis were observed in several animals, most likely secondary to ischemia. Two animals showed areas with small, localized, mild low-grade inflammation. One animal (R15-IP-24V) had multiple foci of organized inflammatory infiltrates composed mainly of mononuclear lymphocytes. Overall, there was no evidence of acute inflammation composed of neutrophils or damage to structures within the liver such as bile ducts. From the chemistry / hematology data of the Bloodwork of the treated animals (Figure 14), there was no significant deviation from the expected reference range compared to the vehicle, except for a moderate effect likely due to hemolysis caused by sample collection via fundic puncture. Finally, doses of 10 μM, 100 μM, and 1,000 μM of E2 caused statistically significant MCF-7 breast cancer cell proliferation, but neither ISP358-2 nor DPN showed any significant proliferation compared to untreated control cells.

[0139] Discussion ERβ has previously been explored as a drug target for a wide range of conditions including anxiety, depression, schizophrenia, and Alzheimer's disease, and representative ERβ drug lead agonist compounds are shown in Figure 1a 35 . The compounds shown herein (Table 1) differ from these previously reported compounds in that they are selective for ERβ over ERα (by about 750-fold) and are A-C estrogens that lack only the B-ring and D-ring and resemble the natural 17β-estradiol molecule in this regard.

[0140] Structure-activity relationship of A-C estrogen The binding affinity of 4-(4-substituted cyclohexyl)phenol was evaluated by a TR-FRET ERβ binding assay (Table 1 and Figure 2). In particular, compounds having a hydroxymethyl functional group attached to the cyclohexyl core showed high affinity in the range of 20 - 200 nM. Of the two components 15 / 16 in a 2:1 mixture of the cis and trans stereoisomers (IC 50 = 184 nM), the trans isomer was found to be more potent than the mixture (ISP358-2, IC 50 = 24 nM). Introduction of unsaturation (18, IC 50 = 49 nM) into the six-membered ring did not significantly reduce the binding affinity compared to ISP358-2. However, the affinity was decreased by conformational constraints as in the case of the exocyclic allyl alcohol (24, IC 50 = 676 nM). The presence of a third hydroxyl group significantly decreased the binding affinity (12, IC 50 = 2,700 nM). Finally, varying the distance between the phenolic OH and the aliphatic alcohol group resulted in a wider range of binding affinities (2, IC 50 = 7250 nM, 25, IC 50 = 11 nM). Furthermore, to evaluate ERβ selectivity in a biologically relevant system in terms of binding affinity and transcriptional activation potency, analogs with IC50 values < 200 nM were tested in a cell transcriptional activation assay. The trans stereoisomers ISP358-2, 8, 18, and 25 showed similar ERβ agonistic efficacy (EC 50 ~ 30 - 75 nM) in the TR-FRET assay, but ISP358-2 was superior as being the most potent and selective in this more biologically relevant cell function assay. Interestingly, the hydroxyethyl analog was not potent in the cell function assay (25, EC 50 = 75 nM vs 11 nM).

[0141] The differences in the effects observed in this assay may be due to what this assay measures. The TR-FRET assay in Figure 2 measures only the displacement of fluorescently labeled estradiol ligand from the ligand-binding domain (LBD) that reflects the binding affinity for ligands that competitively displace the fluorescent probe. In contrast, the cell assay is more complex and measures the entire series of molecular events leading to transcriptional activation. This series of events involves a series of conformational changes (e.g., rotation of helix-12 of the ligand-binding domain) induced by initial hormone binding. 36 . The protein conformational change of the estrogen receptor induced by agonist binding recruits coactivator proteins, also induces protein dimerization, and ultimately DNA binding and transcriptional activation occur. A further interaction between the aliphatic hydroxyl group and the His475 residue of ERβ plays a role in the conformational change involving the nearby helix-12. This conformational change is reflected in the IC 50 value in the cell function assay (Figure 5), but will not be reflected in the TR-FRET binding assay (Figure 2).

[0142] None of the compounds tested showed significant ERβ antagonist activity or ERα antagonist activity (EC 50 > 10,000 nM), and thus also did not show selectivity as agonist activity versus antagonist activity (Table 1 and Figure 5c, d). Among the ERβ agonists, ISP358-2 was the most selective, and the agonist selectivity for ERβ in the cell function assay was approximately 750-fold that of ERα. However, in the TR-FRET binding assay, there was only moderate selectivity (Figure 2b).

[0143] Differences between assays suggest the mechanism of isoform selectivity As described above, from the cell assays targeting ISP358-2, it can be seen that the selectivity of the ERβ agonist activity of ISP358-2 is about 750-fold that of the ERα agonist activity (Figures 5a, b). In contrast, the TR-FRET binding assay shows a moderate 12-fold selectivity for ERβ (Figure 2b). This is because the TR-FRET binding assay simply measures the binding affinity (for the isolated LBD), while the cell assay measures the transcription induced by the binding of the agonist to full-length native ERβ, which causes productive conformational changes in ERβ, including the rotation of helix-12 that leads to coactivator recruitment, dimerization, DNA binding, and then activation of transcription. To test the hypothesis that the differences between the assays are due to these downstream activation events, two other assays were performed. In the first assay, transcriptional activation was measured in different cell assays using, in this case, a non-native chimeric receptor (ER LBD fused to the GLA4 DBD). In this assay (Figure 3b), there was a moderate 2.6-fold selectivity for ERβ. In the second assay, the ability of agonist binding to mobilize the binding of the coactivator peptide to the ER LBD was measured (Figure 4a). In this assay, a 15-fold selectivity for ERβ was observed (Figure 4c). Therefore, the ERβ to ERα selectivity of ISP358-2 varies greatly based on how well the assay incorporates the native downstream activation events that occur after binding to the ERβ binding pocket as a result of hormone-induced conformational changes. Thus, the large ERβ to ERα selectivity shown by ISP358-2 appears to be a function not simply of the binding affinity of the ERβ receptor (as measured by the TR-FRET assay in Figure 2b), but rather of the ability to induce productive conformational changes that result in downstream activation of transcription (Figure 5a).

[0144] Consistent with the above hypothesis that the efficacy and selectivity of ISP358-2 are related to its ability to drive productive conformational changes, docking studies reveal that ISP358-2 docks to the ERβ active site in a conformation that is quite different from the conformation of the ERα binding site. An important difference occurs where the estrogen C-ring is normally positioned (Figure 7c and data not shown), which affects the positioning of aliphatic hydroxyl groups that interact with His524 and / or Gly472 (backbone carbonyl) residues in a region known to be important for driving helix-12 conformational changes that enable coactivator binding (Figure 7c and data not shown). ERβ hydrophobic interactions include π-π stacking between the phenyl ring of Phe356 and the ISP358-2 phenolic ring, along with the cyclohexyl ring in the "C-ring" region and Ala302, Leu298, Leu476, and Ile373 that constrain the attached hydroxymethylmethyl group (Figure 1d). These unique hydrophobic interactions in the ERβ active site may rotate the C (cyclohexane) ring of ISP358-2 by 90° relative to the phenolic ring in the ERβ pocket compared to the ERα pocket (Figure 7c and data not shown), and in such a way, may affect the adjacent coactivator pocket. This 17β-estradiol binding pocket region is known to influence the accessibility and structure of the coactivator binding pocket and may therefore account for the large differences in agonist activity observed for ISP358-2. Future structural characterization studies are planned to address this issue.

[0145] Drugability and preliminary safety toxicity ISP358-2 binds to the ER and activates transcription, but does not show significant off-target activity with seven other nuclear hormone receptors (Figure 3a). ISP358-2 also does not show significant activity against the cardiac potassium ion channel hERG (Figure 11b) and does not show significant inhibition of the major drug-metabolizing cytochrome P450 enzymes, CYP2D6, CYP3A4, CYP2C9, and CYP1A2 (Figure 6). ISP358-2 also has considerable solubility and did not show aggregation in a turbidity assay (Figure 11a).

[0146] To evaluate the potential of ISP358-2 to stimulate breast cancer cell proliferation, an MTT assay was performed using MCF-7 human breast cancer cells (Figure 15). No significant change in MCF-7 cell proliferation was observed after treatment with any concentration of the ERβ agonist ISP358-2 or DPN compared to the untreated control (Figure 15b, c). However, the proliferation of MCF-7 cells treated with 1 μM, 0.1 μM, or 0.01 μM E2 was significantly increased compared to the untreated control (n = 3; p < 0.02, 0.05, 0.00, respectively) (Figure 15a). Furthermore, cell proliferation was significantly less compared to positive control MCF-7 cells treated with 0.01 μM E2 (n = 3; p ≤ 0.04 for both compounds).

[0147] To evaluate the potential peripheral pathology due to ISP358-2 treatment, histological analysis of tissue slices from treated animals was performed (Figure 13). Overall, treatment-induced tissue changes were not remarkable. Mild widespread ischemic changes were noted in the livers of all animals. It is difficult to assign a specific pattern or significance to this finding. These changes are likely to represent low perfusion during the postmortem period and subsequent mild ischemic changes. One animal showed organized lymph hyperplasia in the liver. None of the animals showed significant pathological changes in the heart or kidneys.

[0148] In vivo efficacy The in vivo behavioral assay measures object placement or object recognition (Figure 8a) and showed efficacy for all three routes of administration: microinfusion into the dorsal hippocampus, intraperitoneal injection, or forced oral gavage (Figure 8). Thus, ISP358-2 can improve object recognition and spatial memory consolidation in ovariectomized female mice. Intracerebroventricular infusion of 100 pg and 1 ng of ISP358-2 improved memory consolidation in the object recognition task and object placement task as effectively as the ERβ agonist DPN (Figure 8b, c). In the systemic administration experiments, 0.5 mg / kg ISP358-2 most effectively improved consolidation in both tasks when delivered intraperitoneally (Figure 8d, e), whereas 5 mg / kg ISP358-2 was most effective for forced oral gavage (Figure 8f, g). These data are consistent with previous findings showing that intracerebroventricular or systemic administration of the ERβ agonists DPN or WAY200070 improves hippocampus-dependent memory in tasks including object recognition, object placement, and radial maze in ovariectomized rats and mice. 3,34,39-42 Thus, ISP358-2 may potentially be used to reduce memory impairment in a very large number of neuropsychiatric states with high risk in women, including AD, depression, and schizophrenia, by mimicking the memory enhancing effects of other ERβ agonists with different chemical structures. 43 Furthermore, women have a higher risk of anxiety disorders than men 43 and DPN reduces anxiety-related behavior among rodents tested in the open field and elevated plus maze tasks. 44,45 Thus, ISP358-2 has the potential not only to facilitate memory consolidation but also to reduce anxiety. Although there is promise, including the extent to which the beneficial effects of ISP358-2 will generalize to men, older subjects, rodent models of AD and other disorders, and other forms of memory, a very large number of issues will have to be addressed in future studies.

[0149] Finally, although ISP358-2 was observed to be significantly more selective for ERβ than ERα in biologically relevant cell assays, it is not known whether it has this same selectivity for ERβ in vivo. However, from preliminary studies by the inventors, it has been found that there is a correlation between behavioral outcomes and brain ERβ levels, consistent with the effects being related to ERβ agonist activity (Figure 12). Future studies will be directed at confirming the pharmacological mechanism of ISP358-2 in vivo, including studies of isoform selectivity, effects on signaling cascades, and neuronal morphological changes in the brain, as well as pharmacokinetics and pharmacodynamics.

[0150] Conclusion The results of this study demonstrate that the inventors' lead compound, ISP358-2, is selective for ERβ and does not show obvious signs of peripheral toxicity. Importantly, ISP358-2 also improves multiple types of memory that rely on the hippocampus, a brain region involved in a very large number of disorders, including AD, depression, and schizophrenia. 43,46 ISP358-2 differs from previously reported ERβ agonists in that it has a higher selectivity for ERβ than ERα and in that it closely resembles the natural 17β-estradiol molecule (Figure 1d) as an A-C estrogen. Biological efficacy was also demonstrated in behavioral assays conducted via three routes of administration: direct dorsal hippocampal injection, intraperitoneal injection, and forced oral administration. The last two of direct dorsal hippocampal injection, intraperitoneal injection, and forced oral administration exemplify the brain penetration of effective doses (Figure 8). Overall, these findings suggest that the novel ERβ agonist, ISP358-2, may be a promising drug candidate for improving memory in various disorders characterized by memory dysfunction that occurs under low estrogen conditions such as menopause.

[0151] Experimental section Compound synthesis All chemical substances were purchased from Sigma - Aldrich, Matrix Scientific or Alfa Aesar and used as received. Reactions with reagents sensitive to moisture or air were carried out in an inert nitrogen atmosphere, in oven - dried glassware containing anhydrous solvents. After the reaction, TLC was performed on pre - coated silica plates (60Å, F 254 , EMD Chemicals Inc), and visualized with a UV lamp (UVGL - 25, 254 / 365 nm). Flash column chromatography was performed using flash silica gel (32 - 63μm). NMR spectra were recorded on a Varian UnityInova 400 MHz instrument. CDCl3, d6 - acetone, and CD3OD were purchased from Cambridge Isotope Laboratories. 1 1H NMR spectra were calibrated with respect to residual CHCl3 at δ = 7.26 ppm, d5 - acetone at δ = 2.05 ppm, and residual d3 - CD3OD at δ = 3.30 ppm. 13 13C NMR spectra were calibrated from the central peak at δ = 77.23 ppm for CDCl3, δ = 29.92 ppm for d6 - acetone, and δ = 49.00 ppm for CD3OD. The purity of all compounds was >95% and was determined by chromatography and NMR.

[0152] TIFF0007708379000034.tif10128trans - 4 - (4 - Hydroxycyclohexyl)phenol (2) To a solution of 1 (0.200 g, 5.30 mmol) dissolved in anhydrous methanol (15 mL) at room temperature was added solid NaBH4 (0.400 g, 10.6 mmol). The mixture was stirred for 3 h and then extracted several times with ethyl acetate. The combined extracts were concentrated to give 2 (0.181 g, 90%) as a colorless solid. mp 196 - 208 °C. TIFF0007708379000035.tif32145

[0153] TIFF0007708379000036.tif111284-(4-Hydroxy-4-methylcyclohexyl)phenol (3) At -78 °C under N2, a solution of 1 (0.100 g, 0.526 mmol) dissolved in dry ether (20 mL) was slowly added to a solution of methyl lithium - lithium bromide complex (dissolved in ether to 1.5 M, 0.78 mL, 1.2 mmol). The mixture was stirred at -78 °C for 30 minutes, warmed to room temperature, and stirred for an additional 1 hour. The mixture was cooled to 0 °C and quenched with water. The mixture was extracted several times with ether, the combined extracts were dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexane - ethyl acetate = 4:1) to obtain 3 (0.040 g, 37%) as a colorless solid. mp 126 - 131 °C; TIFF0007708379000037.tif32159

[0154] TIFF0007708379000038.tif111284-(4-Hydroxyphenyl)cyclohexanone oxime (4) To a solution of 1 (0.050 g, 0.26 mmol) dissolved in ethanol (10 mL) were added Amberlyst (0.060 g) and hydroxylamine hydrochloride (0.039 g, 0.560 mmol). The mixture was stirred at room temperature for 2 hours and then filtered. The filtrate was concentrated and extracted several times with ethyl acetate. The combined organic extracts were washed with water, dried (MgSO4), and concentrated to obtain 4 (0.037 g, 70%) as a colorless solid. mp 171 - 174 °C; TIFF0007708379000039.tif46159

[0155] TIFF0007708379000040.tif101284-(4-t-Butyldimethylsilyloxyphenyl)cyclohexan-1-one (5) A solution of 1 (0.500 g, 2.62 mmol) dissolved in anhydrous CH2Cl2 (30 mL) was added to imidazole (0.357 g, 5.24 mmol) at 0 °C under N2. After 30 minutes, tert-butyldimethylsilyl chloride (0.594 g, 3.94 mmol) was added and the mixture was gradually warmed to room temperature overnight. The resulting mixture was diluted with brine (25 mL) and extracted several times with CH2Cl2. The combined organic extracts were dried (Na2SO4) and concentrated. The residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 9:1) to give 5 (0.664, 83%) as a colorless solid. mp 39-42 °C. TIFF0007708379000041.tif26159

[0156] TIFF0007708379000042.tif10128tert-Butyldimethyl(4-(4-methylenecyclohexyl)phenoxy)silane (6) A solution of methyltriphenylphosphonium bromide (0.836 g, 2.34 mmol) dissolved in dry THF (20 mL) was slowly added to n-butyllithium solution (dissolved in hexane at 1.6 M , 1.50 mL, 2.4 mmol) at -10 °C under N2. After 30 minutes, a solution of 5 (0.502 g, 1.17 mmol) dissolved in dry THF (8 mL) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred overnight. After this time, the mixture was diluted with water (20 mL) and extracted several times with ethyl acetate. The combined extracts were dried (Na2SO4) and concentrated. Purification of the crude residue by column chromatography (SiO2, hexane-ethyl acetate = 9:1) gave 6 (1.678 g, 84%) as a colorless oil. TIFF0007708379000043.tif39153

[0157] TIFF0007708379000044.tif111284-(4-Hydroxyphenyl)methylenecyclohexane (7) A solution of 6 (0.739 g, 0.244 mmol) dissolved in anhydrous THF (20 mL) was added to a TBAF solution (dissolved in THF to 1 M , 9.8 mL, 9.8 mmol). The mixture was refluxed and heated for 5 h. After cooling, the solution was partitioned between ethyl acetate and water, and the aqueous layer was extracted several times with ethyl acetate. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (SiO2, hexane-ethyl acetate = 4:1) gave 7 (0.379 g, 83%) as a colorless solid. mp 82~84 °C; TIFF0007708379000045.tif39145

[0158] TIFF0007708379000046.tif111284-(4-Methylcyclohexyl)phenol (8) To a solution of 7 (0.150 g, 0.797 mmol) dissolved in methanol (10 mL) was added 10% Pd / C (85 mg, 10 mol%). The mixture was stirred at room temperature for 12 h under a balloon filled with H2. The reaction mixture was filtered through a single layer of celite, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 4:1) to give 8 (0.121 g, 80%) as a colorless solid. This was 1 confirmed by 1H NMR spectroscopy to be a mixture of cis and trans stereoisomers. mp 93~99 °C; TIFF0007708379000047.tif26145

[0159] TIFF0007708379000048.tif161282-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde (9) To a solution of 7 (0.100 g, 0.531 mmol) dissolved in dry CH3CN (20 mL) were successively added MgCl2 (0.076 g, 0.797), triethylamine (0.28 mL, 2.0 mmol), and then paraformaldehyde (0.108 g, 3.59 mmol). The mixture was heated under reflux for 6 h. The mixture was cooled to room temperature, quenched with 10% HCl (10 mL), and extracted several times with ethyl acetate. The combined extracts were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (SiO2, hexane - diethyl ether = 4:1) gave 9 (0.046 g, 40%) as a colorless oil. TIFF0007708379000049.tif39145

[0160] TIFF0007708379000050.tif211282 - Hydroxy - 5 - (4 - methylenecyclohexyl)benzaldehyde oxime (10) To a solution of 9 (0.050 g, 0.232 mmol) dissolved in absolute ethanol (10 mL) were added sodium bicarbonate (0.024 g, 0.278 mmol) and hydroxylamine hydrochloride (0.025 g, 0.348 mmol). The reaction mixture was heated at 80 °C for 5 h, and the mixture was extracted several times with ethyl acetate. The combined organic extracts were dried (MgSO4) and concentrated. Purification of the residue by column chromatography (SiO2, hexane - ethyl acetate = 13:7) gave 10 (0.037 g, 69%) as a colorless solid. mp 120 - 125 °C; TIFF0007708379000051.tif46145

[0161] TIFF0007708379000052.tif141284 - (4 - ((t - Butyldimethylsilyl)oxy)phenyl)-1 - (hydroxymethyl)cyclohexan - 1 - ol (11) A solution of 6 (0.280 g, 0.926 mmol) and N-methylmorpholine-N-oxide (0.13 mL, 1.3 mmol) dissolved in acetone (6 mL) and distilled water (0.3 mL) was added dropwise a solution of OsO4 dissolved in tert-butanol (2.5%, 90 μL). The mixture was stirred overnight and a saturated aqueous solution of NaHSO3 (10 mL) was added to quench the reaction. The mixture was diluted with ether and washed several times with water. The organic layer was dried (MgSO4), concentrated, and the residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 1:4) to give 11 (0.267 g, 86%) as a colorless solid. mp 80~86 °C; TIFF0007708379000053.tif32153

[0162] TIFF0007708379000054.tif151284-(4-Hydroxy-4-(hydroxymethyl)cyclohexyl)phenol (12) To a solution of 11 (0.230 g, 0.683 mmol) dissolved in anhydrous THF (10 mL) was added a TBAF solution (dissolved in THF at 1 M, 2.8 mL, 2.8 mmol). The mixture was heated to reflux for 6 h and cooled to room temperature. The solution was partitioned between ethyl acetate and water. The combined organic layers were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (SiO2, ethyl acetate-methanol = 9:1) gave 12 (0.118 g, 78%) as a colorless solid. mp 182~188 °C; TIFF0007708379000055.tif32153

[0163] TIFF0007708379000056.tif141284-(4-t-butyldimethylsilyloxyphenyl)cyclohexyl)methanol (13 / 14) A solution of 6 (0.821 g, 2.71 mmol) dissolved in THF (24 mL) was added to a borane-THF complex solution (dissolved in THF to 1 M, 5.4 mL, 5.4 mmol) at 0 °C under N2. The reaction mixture was slowly warmed to room temperature and stirred for 20 h. The mixture was then cooled to 0 °C, and ethanol (50 mL), hydrogen peroxide solution (dissolved in water to 30%, 4.0 mL), and 1 N NaOH solution (20 mL) were successively added. The mixture was warmed to room temperature and stirred for 90 min. The reaction mixture was quenched with saturated sodium bicarbonate solution (10 mL), diluted with water (20 mL), and extracted several times with ethyl acetate. The combined organic extracts were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (SiO2, hexane-ethyl acetate = 7:3) gave a colorless oil (0.572 g, 66%). This was confirmed by 1 1H NMR integration to be a 2:1 mixture of cis-13 and trans-14. TIFF0007708379000057.tif39153Using 9-BBN instead of BH3-THF gave a 2:3 mixture of cis-13:trans-14 (74%).

[0164] TIFF0007708379000058.tif131284-(4-(Hydroxymethyl)cyclohexyl)phenol (15 / 16) To a solution of 13 / 14 (0.594 g, 1.85 mmol, 2:1 mixture c:t) dissolved in dry THF (10 mL) was added a TBAF solution (dissolved in THF to 1 M , 7.5 mL, 7.5 mmol). The reaction mixture was refluxed and heated at 70 °C overnight, then cooled to room temperature. The solution was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 3:2) to give a colorless solid (0.280 g, 73%). This was the signal of the CH2OH groups at δ 3.60 and 3.39 ppm 1It was confirmed by ¹H NMR integration to be a 2:1 mixture of cis-13 and trans-14 stereoisomers. mp 118~122 °C. TIFF0007708379000059.tif39145

[0165] TIFF0007708379000060.tif131281-(4-Hydroxyphenyl)-2-oxabicyclo[2.2.2]octane (17) and trans-(hydroxymethyl)cyclohexyl)phenol (16) At -10 °C, a suspension of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.044 g, 0.194 mmol) dissolved in CH₂Cl₂ (4 mL) was slowly added over 30 minutes to a solution of 15 / 16 (0.080 g, 0.388 mmol, a 2:3 mixture of cis-15:trans-16) dissolved in anhydrous CH₂Cl₂ (20 mL). The green solution was stirred at 0 °C for 2 hours, warmed gradually to room temperature, and stirred for an additional 3 hours. The mixture was quenched by slowly adding saturated sodium bicarbonate solution at 0 °C. After 10 minutes, the layers were separated and the aqueous layer was extracted several times with CH₂Cl₂. The combined organic extracts were washed with brine, dried (Na₂SO₄), and concentrated. The residue was purified by column chromatography (SiO₂, hexane-ethyl acetate = 3:2) to give both 17 (0.029 g, 37%) and 16 (0.038 g, 47%) as colorless solids. The purity of 16 was 1 confirmed by ¹H NMR (Figure 16). TIFF0007708379000061.tif64145

[0166] TIFF0007708379000062.tif111284'-(Hydroxymethyl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-4-ol (±)-19 A solution of 17 (0.103 g, 0.504 mmol) dissolved in dry CH3CN (25 mL) was added with MgCl2 (0.072 g, 0.756 mmol), and then triethylamine (0.26 mL, 1.89 mmol) was added. The mixture was refluxed and heated for 8 h, then cooled and quenched with 10% HCl (15 mL). The mixture was extracted several times with ethyl acetate, and the combined extracts were washed with brine, dried (Na2SO4), and concentrated. Purification of the residue by column chromatography (SiO2, hexane - ethyl acetate = 13:7) gave 19 (0.080 g, 78%) as a colorless solid. mp 177 - 184 °C; TIFF0007708379000063.tif32145

[0167] TIFF0007708379000064.tif111284-(4-((tert-Butyldiphenylsilyl)oxy)phenyl)cyclohexan-1-one (21) At 0 °C, imidazole (0.583 g, 8.57 mmol) was added to a solution of 1 (0.815 g, 4.28 mmol) dissolved in dry CH2Cl2 (30 mL), and then a solution of tert-butyldiphenylsilyl chloride (1.60 mL, 5.57 mmol) dissolved in CH2Cl2 (9 mL) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred for 12 h. The mixture was diluted with water and extracted several times with CH2Cl2. The combined extracts were washed with brine, dried (Na2SO4), and concentrated. The residue was purified by column chromatography (SiO2, hexane - ethyl acetate = 4:1) to give 21 (1.70 g, 93%) as a colorless solid. mp 83 - 84 °C; TIFF0007708379000065.tif26153

[0168] TIFF0007708379000066.tif11128Methyl acetate 2-(4-(4-tert-butyldiphenylsilyloxyphenyl)cyclohexylidene)(±)-22 A solution of trimethyl phosphonoacetate (0.160 mL, 0.980 mmol) dissolved in dry THF (5 mL) at 0 °C was added to NaH (40 mg, dissolved in mineral oil at 55%, 0.980 mmol). After stirring for 45 minutes, a solution of 21 (0.350 g, 0.816 mmol) dissolved in dry THF (5 mL) was added. The mixture was warmed to room temperature and stirred for 8 hours. The mixture was diluted with water and extracted several times with ether. The combined extracts were dried (MgSO4) and concentrated. The residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 9:1) to give 22 (0.376 g, 95%) as a colorless gum. TIFF0007708379000067.tif39153

[0169] TIFF0007708379000068.tif111284-Ethyl [(4-hydroxyphenyl)cyclohexylidene]acetate (±)-23 To a stirred solution of 22 (60 mg, 0.12 mmol) dissolved in dry THF (1 mL) was added tetrabutylammonium fluoride solution (0.247 mL, dissolved in THF at 1.0 M, 0.247 mmol). After 1 hour, the solution was stirred at room temperature. Then the mixture was diluted with water and extracted with ethyl acetate. The combined extracts were washed with brine, dried, and concentrated. The residue was purified by preparative TLC (SiO2, hexane-ethyl acetate = 9:1) to give 23 (20 mg, 64%) as a colorless solid. mp 92-94 °C; TIFF0007708379000069.tif40153

[0170] TIFF0007708379000070.tif111284-(4'-Hydroxyphenyl)(2-hydroxyethylidene)cyclohexane (±)-25 A solution of 22 (275 mg, 0.551 mmol) dissolved in dry CH2Cl2 (2 mL) was added dropwise with a solution of diisobutylaluminum hydride (dissolved in CH2Cl2 to 1.0 M, 1.41 mL, 1.41 mmol) at -40 °C under N2. After 90 minutes, a saturated aqueous solution of potassium sodium tartrate was added, and the reaction mixture was warmed to room temperature. After 2 hours, the layers were separated, and the aqueous layer was extracted several times with CH2Cl2. The combined organic layers were dried, filtered through a single layer of celite, and concentrated to give 4-(4'-t-butyldiphenylsilyloxyphenyl)(2-hydroxyethylidene)cyclohexane (254 mg, quantitative) as a colorless gum. This compound was used without further purification. A solution of crude allyl alcohol (235 mg, 0.514 mmol) dissolved in dry THF (1 mL) was added with tetrabutylammonium fluoride solution (dissolved in THF to 1.0 M , 1.03 mL, 1.03 mmol) at under N2. The solution was stirred for 3 hours, then diluted with water, and the resulting mixture was extracted several times with ethyl acetate. The combined extracts were washed with brine, dried, and concentrated. The residue was purified by column chromatography (SiO2, hexane-ethyl acetate = 4:1) to give 25 (90 mg, 80%) as a colorless solid. mp 165~166 °C; TIFF0007708379000071.tif33159

[0171] TIFF0007708379000072.tif111284-[4-(2-Hydroxyethyl)cyclohexyl]phenol (26) and 4-(4-ethylcyclohexyl)phenol (27) A solution of 25 (50 mg, 0.23 mmol) dissolved in methanol (15 mL) containing a small amount of 20% Pd / C was stirred under H2 (30 psi) for 12 hours. The reaction mixture was filtered through a single layer of celite, concentrated, and the residue was purified by preparative TLC (SiO2, hexane-ethyl acetate = 13:7) to give 27 (28 mg, 60%), and then 26 (7 mg, 14%), both as colorless solids. TIFF0007708379000073.tif58153

[0172] TR-FRET assay The TR-FRET assay was performed using the Thermo Fisher Scientific LanthaScreen® TR-FRET ER Alpha and Beta Competitive Binding Assay kit. These are in a homogeneous mix-and-read assay format and comprise a terbium-labeled anti-GST antibody, a fluorescent small molecule ERα ligand or ERβ ligand as the "tracer", and a human ERα or ERβ ligand binding domain (LBD) tagged with glutathione-S-transferase (GST).

[0173] In the TR-FRET assay, a Tb-anti-GST antibody that binds to the GST tag is used. The fluorescently labeled estrogen (the tracer) binds within the active site pocket. The resulting TR-FRET signal decreases when a competing compound displaces the fluorescently labeled tracer. The assay was performed according to the kit instructions. Briefly, a 1:5 dilution series of the compound was made using DMSO and then diluted with assay buffer such that the highest concentration tested in the assay was 50 μM for ERβ and 50 μM for ERα and DMSO was 1%. The assay was assembled in a 384-well white, small volume plate (Corning® 4512). The assay was incubated for 1 hour at room temperature in the dark and then the plate was placed in a benchtop centrifuge equipped with a swing-out rotor (Eppendorf 5810, rotor A-4-64) and spun at 1000 rpm. The TR-FRET signal was read on a SpectraMax M5 (Molecular Devices) set according to the Thermo Fisher Scientific instrument settings (excitation 332 nm, emission 518 nm and 488 nm and 420 nm cutoff, 50 μs integration delay, 400 μs integration time, and 100 flashes / read). The TR-FRET ratio was calculated using SoftmaxPro software by dividing the emission at 518 nm (fluorescein) by the emission at 488 nm (terbium). The data were normalized to E2. IC 50 was 0.25 ± 0.06 nM in this assay. Data analysis was performed using Prism (GraphPad Software, Inc., La Jolla, CA) and a fit that was typically constrained to be 0 at high concentrations of competing ligand. The standard deviation is the standard deviation of the non-linear least squares fit of the data. Curves were shown when performing iterative assays and fits (Figures 2 and 9). IC 50For the curve where the median value was obtained, the fitted IC 50 was summarized in Table 1).

[0174] Nuclear hormone receptor specificity assay Selectivity was measured using the ThermoFisher SelectScreen™ cell-based nuclear receptor profiling service (Figure 3). This is a FRET-based assay using GeneBLAzer™ technology. It detects the binding of a ligand to the nuclear hormone receptor of interest (ligand binding domain; LBD) fused to the GAL4 DNA binding domain (DBD) that induces β-lactamase expression upon activation, and its activation. This assay has Z' ≥ 0.5 in agonist mode. The compound stock was dissolved in DMSO and diluted to obtain assay concentrations of 0.25 μM, 2.5 μM, and 25 μM. The estrogen receptor data was normalized to E2. The IC 50 was 0.107 nM for ERα and 0.579 nM for ERβ. The data for other receptors was normalized to the appropriate control and listed in Table 2 along with the IC 50 values.

[0175] (Table 2) Control compounds and IC 50 values of the nuclear hormone specificity assay TIFF0007708379000074.tif57149

[0176] Replicate assays of the ERα agonist assay and the ERβ agonist assay in a 10-point curve were also completed (Figure 3b). Again, the data was normalized to E2. The IC 50 was 0.151 nM for ERα and 0.568 nM for ERβ.

[0177] Coactivator assay The ThermoFisher LanthaScreen® TR-FRET assay was used (Figure 4a). This assay is similar to the aforementioned assay (Figure 3) except that the LanthaScreen® assay has a fluorescently labeled coactivator peptide. In this assay, the recruitment of the labeled coactivator peptide to the ERα LBD or ERβ LBD, which is induced by the binding of the ER agonist being assayed, is measured. The coactivator peptide is derived from the PPARγ coactivator protein 1a and is PGC1a containing the LXXLL motif (sequence: TIFF0007708379000075.tif4128). The data were normalized to E2. The IC 50 values for ERα and ERβ were 2.58 nM and 2.79 nM, respectively.

[0178] Cell assay The ERα cell assay and ERβ cell assay for measuring agonist activity and antagonist activity were performed using kits provided by Indigo Biosciences (Figure 5). The assay relied on a luciferase reporter gene that was downstream of an ERα-responsive promoter or an ERβ-responsive promoter and was activated by the added agonist, or agonist activity was blocked by the added antagonist. ER-induced luciferase expression was quantified using chemiluminescence measured with a SpectraMax M5 plate reader. The stock solution of the ligand was prepared by dissolving it in DMSO and diluted to the final concentration (typically low nM to μM) using the Compound Screening Medium provided in the kit so that the DMSO concentration in the assay was kept below the assay limit of 0.4%. A vehicle control was included in both the agonist assay and the antagonist assay. The assay was performed according to the kit instructions. Briefly, cells taken directly from the freezer were diluted with Cell Recovery Media (provided) and warmed at 37°C for 5 minutes. The cell suspension was divided in half. Estradiol, E2, was added to half of the cells for the antagonist assay, while the remaining cells without E2 were used for the agonist assay. The cells were plated and the compounds to be screened were added. The plates were placed in an incubator and incubated at 37°C and 5% CO2 for 22 hours. The assay was typically repeated twice. After removing the medium and adding the detection substrate, luminescence was measured using a SpectraMax M5 plate reader. The data were normalized to E2. The agonist activity IC 50 was 0.31 ± 0.03 nM for ERα and 0.022 ± 0.005 nM for ERβ. The data were fitted to the following equation using GraphPad Prism. TIFF0007708379000076.tif19128

[0179] As described for the TR-FRET assay fitting, IC 50The values and standard deviations are from a non-linear least squares fit of the data. When iterative assays and fits were performed, the median values were reported in Table 1.

[0180] In vitro drugability assay - CYP450 binding, hERG, and turbidimetric analysis CYP450 (cytochrome P450) inhibition was measured using the P450-Glo™ Screening System from Promega Corporation (Madison, WI) as described in the kit instructions. The assay was measured in 96-well white plates (Corning® 3912), and luminescence was measured on a SpectraMax M5 instrument (Figure 6). The luminescence signal is proportional to the amount of luciferin product formed by the CYP reaction. Compounds were prepared by dissolving in DMSO and then making an 8-step 1:2 dilution series in DMSO. This was diluted with water so that the DMSO in the assay did not exceed 0.25% and the highest final concentration of the compound was 62.6 μM. After adding the corresponding cytochrome P450 enzyme, the plates were incubated at 37 °C for 10 minutes to equilibrate the components to the same temperature. Next, an NADPH regeneration system was added to activate the reaction with the luminogenic P450-Glo™ substrate, and each CYP enzyme was incubated at 37 °C for 10 - 30 minutes according to the kit instructions. The enzyme reaction was stopped by adding the luciferin detection reagent and incubated at room temperature for 20 minutes, after which the plate luminescence was read on a Spectramax M5 (Molecular Devices). The data were normalized to positive controls (α-naphthoflavone for CYP1A2, sulfaphenazole for CYP2C9, quinidine for CYP2D6, ketoconazole for CYP3A4). Data analysis was performed using Prism software as described above.

[0181] To confirm the relative tendency of the compound to aggregate in solution, turbidimetric analysis was performed based on the light scattering properties of the molecular aggregates (Figure 11a). Aggregation is a source of common human activities, so compound aggregation in solution is important for measurements in screening campaigns. And aggregation serves as a measure of compound solubility. The compound was tested for aggregation in a clear 96-well plate (Greiner BioOne). Progesterone was used as a positive control for compound aggregation. Data were collected using a BMG NEPHELOStar Plus equipped with a 635 nm laser.

[0182] The hERG assay was performed using the SelectScreen service from ThermoFisher (Figure 11b). This assay is a fluorescence polarization assay that measures the displacement of the fluorescently tagged Predictor™ 47 , as described.

[0183] MTT assay Human breast cancer cells (MCF-7) were provided by Dr. Manish Patankar (Department of Obstetrics and Gynecology, University of Wisconsin-Madison). The cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum and 0.01 mg / mL human recombinant insulin at 37 °C with 5% CO2. A seeding density of 7,000 cells per well was selected and applied to 96-well plates. After 24 hours, treatments with ISP358-2, DPN, or estradiol dissolved in medium containing 0.1% dimethyl sulfoxide (DMSO) were applied to the cells at various concentrations (10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM). Negative control cells, positive control cells, and untreated control cells were given 100% DMSO, 0.01 μM estradiol dissolved in EMEM, or EMEM with 0.1% DMSO content, respectively. The treated cells were incubated for 24 hours, after which the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed by adding 20% MTT dissolved in EMEM solution to each well and incubating for 4 hours. The formazan crystal metabolic product was dissolved using 100% DMSO, and the absorbance was read at OD570 nm as well as the reference 650 nm using a VMax kinetic microplate reader (Molecular Devices, CA) running Softmax Pro version 6.1. The absorbance was converted to cell number using a standard growth curve. A two sample equal variance t-test was performed using Microsoft Excel to determine whether there was a significant difference in cell growth compared to untreated controls or cells treated with 0.1 μM E2.

[0184] Docking Before docking, three-dimensional (3D) conformations were prepared for all ligands (Figure 7). Using AutoDock Tools (ADT) version 1.5.6, ligand files for subsequent AutoDock calculations were prepared and Gasteiger charges were assigned. For the docking calculations, the agonist (pdb code 1ere) 48 and antagonist (pdb code 1err) 49 conformations of the ERα receptor were prepared. For the docking calculations, the agonist (pdb code 2jj3) 50 and antagonist (pdb code 1l2j) 51 conformations of the ERβ receptor were also prepared. Using ADT, hydrogen atoms and partial charges were added to each atom of the protein. The grid box was placed at the center of the co-crystallized ligand and pulled to incorporate the active site amino acids (Arg394, Glu353, and His524 for ERα; Arg346, Glu305, and His475 for ERβ), and the estradiol ligand was removed 52 . AutoDock Vina 53 was used with default parameters except that an energy range of 4 and an exhaustiveness of 8 were used 47, 54-57 . As a control experiment, 17β-estradiol was docked to the ERα structure (pdb code 1ERE) after removing 17β-estradiol. This 17β-estradiol was found to adopt the same binding mode as the originally bound 17β-estradiol (data not shown).

[0185] Evaluation of memory consolidation Subjects. C57BL / 6 female mice (8 - 10 weeks old) were purchased from Taconic Biosciences. Mice were housed individually in a room with a 12-hour light / dark cycle and given free access to food and water. All procedures using live mice were conducted indoors between 9:00 am and 6:00 pm with the dimmer light intensity set above 100 lux. All procedures were approved by the University of Wisconsin - Milwaukee Institutional Animal Care and Use Committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0186] General experimental design A series of three experiments were conducted in mice that had both ovaries removed to eliminate the main source of circulating estrogen. In each experiment, a negative control (dimethyl sulfoxide, DMSO), a positive control (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN), and multiple doses of ISP358-2 were administered to separate groups of mice via one of three routes of administration: direct bilateral dorsal hippocampal injection, intraperitoneal injection, or forced oral gavage. All drugs were administered shortly after training in an object recognition task designed to test hippocampal-dependent object recognition and an object location task designed to test spatial memory consolidation, as described below (Figure 8).

[0187] Surgery Four days after arrival at the facility, as previously described 34,58,59 , both ovaries of the mice were removed. Mice scheduled to receive dorsal hippocampal injection of ISP358-2 were also, as previously described 30-32, a guide cannula was implanted into the dorsal hippocampus (DH). Mice were anesthetized with isoflurane (2% isoflurane in 100% oxygen) and placed in a stereotaxic apparatus (Kopf Instruments). Immediately after ovariectomy, two guide cannulas (22 gauge; C232G, Plastics One) were implanted targeting the dorsal hippocampus of the mice (-1.7 mm AP, ±1.5 mm ML, -2.3 mm DV). To maintain the patency of the guide cannulas, dummy cannulas (C232DC, Plastics One) were placed inside the guide cannulas. Dental cement (Darby Dental) was applied to fix the guide cannulas to the skull, which also helped to close the wound. Mice were allowed to recover for 6 days before behavioral testing.

[0188] Drugs and injections Dorsal hippocampal (DH) infusion or intraperitoneal (IP) injection was performed as previously described 34,58,59 , immediately after training. Mice were gently restrained during the infusion, and the drug was delivered using an infusion cannula (C3131, 28 gauge, extending 0.8 mm beyond a 1.5 mm guide). The infusion cannula was connected to a 10 μl Hamilton syringe using a PE20 polyethylene tube. The infusion was controlled by a microinfusion pump (KDS Legato 180, KD Scientific) at a rate of 0.5 μl / min. After each infusion, the cannula track was flushed back for 1 minute to prevent drug diffusion through the tissue. The negative control (“vehicle”) was 1% DMSO dissolved in 0.9% saline. As a positive control, the ERβ agonist DPN (2,3-bis(4-hydroxyphenyl)-propionitrile, Tocris Bioscience) was dissolved in 1% DMSO in saline and injected at a dose of 10 pg / hemisphere 30 . The affinity of DPN for ERβ is 70-fold higher than that for ERα 60 , and previously, bilateral infusion of 10 pg / hemisphere into the dorsal hippocampus in ovariectomized young adult mice improved memory consolidation in object recognition and object placement tasks34 ISP358-2 was dissolved in 1% DMSO to a concentration of 2 ng / μl and then diluted to administer doses of 1 ng / hemisphere, 100 pg / hemisphere, and 10 pg / hemisphere.

[0189] For intraperitoneal injection, ISP358-2 was dissolved in 10% DMSO dissolved in saline and injected at doses of 0.5 mg / kg or 5 mg / kg in a volume of 10 ml / kg. DPN was dissolved in 10% DMSO dissolved in saline and injected at a dose of 0.05 mg / kg in a volume of 10 ml / kg. Previously, this dose improved memory consolidation of object recognition in ovariectomized young adult mice. 31 For the vehicle control, 10% DMSO in 10 ml / kg of saline was given. For forced oral administration, all drugs were administered at the same doses as for intraperitoneal injection; 0.5 mg / kg or 5 mg / kg ISP358-2 and 0.05 mg / kg DPN in a volume of 10 ml / kg. For the vehicle control, 10% DMSO dissolved in saline was given. In this procedure, the drugs were delivered directly to the stomach using a bulb tipped gastric gavage needle (24GA, 25 mm).

[0190] Memory test As previously described 34,58,59 object recognition and object placement were performed. Object recognition and object placement evaluate object recognition memory and spatial memory, respectively, and require intact dorsal hippocampal function. 39,61-63。First, the mice were handled for 3 days (30 sec / d) to acclimate them to the experiment. On the second day of handling, small Legos were placed in the home cage to acclimate the mice to the objects. These Legos were removed from the cage immediately before training. Three days after handling, the mice were acclimated by allowing them to freely explore an empty white activity area (width 60 cm; length, 60 cm; height 47 cm) for 5 minutes each day for 2 days. On the training day, the mice were acclimated in the activity area for 2 minutes and then removed and placed back in the home cage. Next, two identical objects were placed near the northwest and northeast corners of the activity area. The mice were returned to the activity area and allowed to explore until they had accumulated a total of 30 seconds of object exploration (or until a total of 20 minutes had elapsed). Immediately after this training, the mice were removed from the activity area, injected, and then returned to the home cage. Twenty-four hours after training, object location memory was tested by moving one of the training objects to the southeast or southwest corner of the box. Since mice are innately attracted to novelty, mice that remember the location of the training object will spend more time with the moved object than with the non-moved object. Mice that are acting randomly will spend the same amount of time (15 s) with each object and do not show memory consolidation. Therefore, if the mice spend significantly more time with the moved object than by chance, it is proven that the memory of the training object is consolidated. Object recognition training was performed two weeks after object location. The object recognition task used the same apparatus and general procedure as object location, but instead of changing the location of the objects, during the test, the familiar objects were exchanged for new objects. The object recognition test was performed 48 hours after training. Similar to object location, the mice accumulated 30 seconds of exploration of the novel and familiar objects. Since mice are innately attracted to novelty, if the time spent exploring the novel object is more than by chance, it indicates that there is memory of the familiar training object. To maintain novelty, different objects were used in the object location task and the object recognition task. Female mice injected with vehicle did not remember the location of the training object 24 hours after training but 34 , a 24-hour delay was used to test the memory-enhancing effect of the drug in object location. Similarly, female mice injected with vehicle did not remember the familiar objects 48 hours after training, so 34、To test the memory-enhancing effect of drugs in object recognition, a 48-hour delay was used. For both tasks, the time spent exploring each object and the elapsed time to accumulate 30 s of exploration were recorded using ANYmaze tracking software (Stoelting).

[0191] Analysis of behavioral data One-sample t-tests and one-way analysis of variance (ANOVA) were performed using GraphPad Prism 6 (La Jolla, CA). One-sample t-tests were used to determine whether the mice spent significantly more time investigating a novel or moved object than by chance (15 s). This indicates that each mouse group successfully formed a memory of the identity and location of the training objects. To determine the extent to which DPN treatment or ISP358-2 treatment affected memory consolidation compared to the vehicle, post hoc Fisher's LSD tests were used to compare between groups for each behavioral task after one-way ANOVA. Significance was determined at p > 0.05.

[0192] Evaluation of potential peripheral pathology To evaluate the potential toxicity of ISP358-2 treatment on peripheral organs, ovariectomized mice were injected intraperitoneally once with vehicle or ISP358-2, and liver, kidney, and heart tissues were collected 24 hours later. Similar to the behavioral test, ISP358-2 was injected at a dose of 0.5 mg / kg or 5 mg / kg at a volume of 10 ml / kg, and DPN was injected at a dose of 0.05 mg / kg at a volume of 10 ml / kg. The vehicle control was given 10% DMSO in saline at a volume of 10 ml / kg (Figure 13a). Tissues were fixed in 10% buffered formalin solution for 24 hours. Twenty specimens were processed and analyzed. Each specimen contained three tissue fragments. Tissues from each animal were transferred to labeled cassettes and processed in an automated tissue processor following standard procedures. The tissues were then embedded in paraffin wax. No specific orientation of the tissues was done. Four-micron sections were cut from each paraffin block and placed on slides. The slides were then stained using hematoxylin and eosin (H&E) by an automated stainer (Figure 13b). The slides were then examined by a pathologist (ACM) board-certified in anatomical pathology by the American Board of Pathology. All specimens contained three tissue samples corresponding to the liver, kidney, and heart. In some cases, a portion of adjacent tissue was also present. For example, some specimens had the gallbladder. One specimen had a portion of the spleen. Each organ was examined for specific pathological changes. Three main categories of changes were examined. (1) Structural changes in the organs. For the liver, the central vein, portal triad, and hepatocytes were examined. For the kidney, the glomerulus and renal tubules were examined. For the heart, the myocytes and coronary vessels were examined. (2) Evidence of inflammation, including hepatitis, glomerulonephritis, interstitial nephritis, and myocarditis, was evaluated. (3) Evidence of ischemic changes was examined. See the attached table for an overview of these findings.

[0193] References TIFF0007708379000077.tif105146TIFF0007708379000078.tif210146TIFF0007708379000079.tif204146TIFF0007708379000080.tif199146TIFF0007708379000081.tif203146TIFF0007708379000082.tif208146TIFF0007708379000083.tif208146TIFF0007708379000084.tif201146TIFF0007708379000085.tif225146

[0194] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention exemplified herein can be practiced, as appropriate, without the elements specifically disclosed herein and without limitation. The terms and expressions used have been used as terms of description and not of limitation, and it is not intended to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Accordingly, although the invention has been illustrated by specific embodiments and any features, it is to be understood that modifications and / or variations of the concepts disclosed herein may be used by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0195] Numerous patents and non-patent references have been cited herein. The cited references are hereby incorporated by reference in their entirety. In the event of any conflict between the definitions of terms in the cited references and the definitions of terms in this specification, the terms shall be construed based on the definitions in this specification.

Claims

A compound having the formula and stereochemistry of claim 1, which is a selective agonist of estrogen receptor β (ERβ) and has a binding affinity for ERβ that is at least 500-fold greater than its binding affinity for estrogen receptor α (ERα). **Claim 2** A pharmaceutical composition comprising an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent. **Claim 3** The pharmaceutical composition of claim 2 for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject. **Claim 4** The pharmaceutical composition of claim 3, wherein the disease or disorder is a neurological disease or disorder. **Claim 5** The pharmaceutical composition of claim 3, wherein the disease or disorder is a psychiatric disease or disorder. **Claim 6** The pharmaceutical composition of claim 3, wherein the disease or disorder is cancer. **Claim 7** The pharmaceutical composition of claim 3, wherein the disease or disorder is associated with amnesia or memory dysfunction. **Claim 8** The pharmaceutical composition of claim 2 for improving memory fixation in a subject. **Claim 9** The pharmaceutical composition of claim 8, wherein the subject is a postmenopausal woman. **Claim 10** The pharmaceutical composition of claim 2 for treating a subject exhibiting low estrogen levels. **Claim 11** The pharmaceutical composition of claim 10, wherein the subject is a postmenopausal woman. A compound having a formula selected from claim 12. **Claim 13** A pharmaceutical composition comprising an effective amount of the compound of claim 12 or a pharmaceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent. **Claim 14** The pharmaceutical composition of claim 13 for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject. **Claim 15** The pharmaceutical composition of claim 14, wherein the disease or disorder is selected from neurological diseases and disorders, psychiatric diseases and disorders, and cell proliferative diseases and disorders. **Claim 16** The pharmaceutical composition of claim 14, wherein the disease or disorder is associated with amnesia or memory dysfunction. **Claim 17** The pharmaceutical composition of claim 13 for improving memory fixation in a subject. **Claim 18** The pharmaceutical composition of claim 17, wherein the subject is a postmenopausal woman. **Claim 19** The pharmaceutical composition of claim 13 for treating a subject exhibiting low estrogen levels.

20. The pharmaceutical composition according to claim 19, wherein the subject is a postmenopausal woman.

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