Substituted (4'-hydroxyphenyl)cycloalkane and (4'-hydroxyphenyl)cycloalkene compounds and uses thereof as selective agonists of the estrogen receptor β isoform for enhanced memory consolidation

Abstract

To provide a compound that functions as a ligand for the estrogen receptor (ER).SOLUTION: Disclosed are substituted (4'-hydroxylphenyl)cycloalkane compounds and substituted (4'-hydroxylphenyl)cycloalkene compounds and use thereof as selective agonists of the estrogen receptor β isoform (ERβ). The disclosed compounds may be formulated as pharmaceutical compositions and administered for treating diseases associated with ER activity, such as neurological diseases and disorders, psychiatric diseases and disorders, and / or cell proliferative diseases and disorders as well as for enhancing memory consolidation in subjects in need thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under awards R15GM118304 from the National Institute of General Medical Sciences and R01DA038042 from the National Institute on Drug Abuse. The U.S. 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 October 16, 2017, and U.S. Provisional Application No. 62 / 478,758, filed March 30, 2017, the contents of which are incorporated herein by reference in their entireties.

[0003] FIELD OF THEINVENTION The field of the invention relates to compounds that function as ligands for the estrogen receptor (ER). In particular, the field of the invention relates to substituted (4'-hydroxyphenyl)cycloalkane and (4'-hydroxyphenyl)cycloalkene compounds that are specific agonists of the estrogen receptor β (ERβ) and the use of such compounds in pharmaceutical compositions for treating diseases and disorders associated with ER activity in improving memory consolidation. [Background technology]

[0004] background Estrogen is a key regulator of many physiological processes, including reproduction, cognition, cardiovascular mortality, and bone metabolism. 66Based on their widespread role in numerous physiological processes, estrogens have been implicated in numerous diseases and disorders, including cell proliferative diseases and disorders (e.g., breast, ovarian, endometrial, colorectal, and prostate cancer), neurodegenerative diseases and disorders, cardiovascular disease, and osteoporosis, to name a few. 66 In many of these diseases and disorders, estrogen mediates its effects through the estrogen receptor (ER).

[0005] ER exists in two major forms, ERα and ERβ, which have different tissue expression patterns. 67 ERα and ERβ are encoded by separate genes, ESR1 and ESR2, respectively, that are found at different chromosomal locations, and numerous mRNA splice variants exist for both ERα and ERβ. 68 Because of their role in estrogen-related diseases, ERα and ERβ have been targeted to develop specific ligands to modulate their activity. The ligand specificities of ERα and ERβ differ, and ligands that bind to ERα and function as agonists or antagonists of ERα may also bind to ERβ and function as agonists or antagonists of ERβ, or may not bind to ERβ and function as agonists or antagonists of ERβ.

[0006] Agonists of ERα and ERβ have a wide range of biological actions that are implicated in diseases such as cancer and central nervous system (CNS) disorders. 2 ) is a key modulator of hippocampal synaptic plasticity and hippocampus-dependent memory formation in male and female rodents 6 As they age, both sexes have 2Levels decrease in the hippocampus, but more rapidly in menopausal women. ERβ is the most predominant ER isoform in the hippocampus and plays an important role in mediating estradiol's effects 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 pathology and improved learning and memory. 7 Furthermore, certain alleles of the ERβ gene (Esr2), but not the ERα gene, are associated with a reduced risk of AD in both men and women. 8 This supports ERβ as a putative drug target for AD.

[0007] In particular, ERβ agonists have many promising clinical applications. 1 Current ERβ agonist drug lead molecules contain a phenol ring, with the other half of the molecule typically containing another phenolic or indole-like ring structure and various substituted aromatic ring structures (Figure 1a). One of these, WAY-200070 (a benzoxazole), has shown potency as an anxiolytic / antidepressant, with 68-fold selectivity for ERβ over ERα. 1-3 Several ERβ agonists have advanced into human clinical trials for a variety of disease indications ranging from schizophrenia (Eli Lilly; NCT01874756) to fragile X syndrome (Parc de Salut Mar; NCT01855971) to memory loss and hot flashes (National Institutes on Aging; NCT01723917). 4 The work presented herein focuses 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 with diarylpropionitrile (DPN). 5 .

[0008] APOE4 is the best-established genetic risk factor for Alzheimer's disease (AD). Women with the APOE4 genotype are two to four 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 experience symptoms of anxiety and depression. 12 In women, a major contributing factor to these risks is the decline in estrogen during menopause, as estrogen mediates cognitive function and is neuroprotective in brain regions such as the hippocampus and cortex that are impaired in AD. 13 Thus, drugs that enhance estrogen-mediated effects on cognition, such as the selective ERβ agonists (SERBAs) developed herein, may reverse memory loss and reduce anxiety and depression in aging women. However, estrogen-based hormone replacement therapy is associated with an increased risk of a variety of diseases, including breast cancer (especially lobular carcinoma), stroke, gallbladder disease, and venous thromboembolism, which are believed 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 desirable. In particular, new ligands that exhibit selective agonistic or antagonistic activity for ERβ compared to ERα are desirable. These new ligands should be suitable for treating diseases and disorders associated with ER activity, such as cell proliferation and disorders or psychiatric diseases and disorders. Recently, the present inventors have reported a novel ERβ agonist that is more selective for ERβ versus ERα activation than previously reported clinical candidates. 19 This ERβ agonist is in a unique structural class consisting of a phenol ring tethered to a 4-hydroxymethyl-cycloheptane ring structure. However, the presence of the 4-substituted cycloheptane ring poses synthetic and stereochemical challenges and is therefore undesirable as a drug lead.

[0010] Reported herein is the optimization and characterization of a related class of molecules that consist of a 4-hydroxymethyl-cyclohexane ring tethered to a phenol ring, thus resulting in AC estrogens that closely resemble natural estrogen molecules but lack the B and D rings (Figure 1b-d). A-CD estrogens have been extensively studied and are reported to have up to 15-fold selectivity for ERβ. 16-23 In contrast, the simpler AC estrogens reported here show considerably higher selectivity for ERβ over ERα. These AC estrogens represent a surprisingly simple yet novel class of isoform-selective ERβ agonists with the potential to treat age-related memory decline in postmenopausal women. Summary of the Invention

[0011] overview Substituted (4'-hydroxyphenyl)cycloalkane and (4'-hydroxyphenyl)cycloalkene compounds and their use as selective agonists of estrogen receptor beta (ERβ) are disclosed. The disclosed compounds may be formulated as pharmaceutical compositions and administered to treat diseases associated with ER activity.

[0012] In some embodiments, the disclosed compounds have formula I: TIFF2025081398000001.tif30128 or its hydroxy-protected form; During the ceremony, (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 an 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 have the formula Ia: TIFF2025081398000002.tif30128, where 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 referred to herein as “ISP358-2”: Includes TIFF2025081398000003.tif29128.

[0015] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl)cyclohexene compounds. For example, the disclosed compounds may have the formula Ia(i): TIFF2025081398000004.tif31128, where X, Y, and Z are as defined for formula I.

[0016] The disclosed compounds can be used to prepare and formulate pharmaceutical compositions.Therefore, also disclosed herein is a pharmaceutical composition that comprises an effective amount of any compound disclosed herein or any pharmaceutically acceptable salt of any compound disclosed herein, together with pharmaceutically acceptable excipient, carrier or diluent.

[0017] The disclosed compounds may be used to prepare pharmaceutical agents for treating diseases or disorders related to estrogen receptor beta (ERβ) activity, particularly diseases or disorders that can be treated with ERβ agonists.Thus, the disclosed compounds may exhibit ERβ agonist activity, and preferably, the compounds exhibit specificity as ERβ agonists compared to activity as ERβ antagonists and / or activity as estrogen receptor alpha (ERα) agonists or activity as ERα antagonists.The disclosed compounds may be prescribed for use in treating psychiatric or neurological diseases or disorders.In particular, the disclosed compounds may be prescribed for use in treating subjects who require improved memory consolidation, for example, improved memory consolidation under low estrogen conditions observed in postmenopausal women. [The present invention 1001] The compound has the formula and stereochemistry of TIFF2025081398000005.tif25128. [The present invention 1002] A pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharma- ceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent. [The present invention 1003] A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition of the present invention 1002. [The present invention 1004] The method of the present invention 1003, wherein the disease or disorder is a neurological disease or disorder. [The present invention 1005] The method of the present invention 1003, wherein the disease or disorder is a psychiatric disease or disorder. [The present invention 1006] The method of claim 1003, wherein the disease or disorder is cancer. [The present invention 1007] The method of claim 1003, wherein the disease or disorder is associated with memory loss or memory dysfunction. [The present invention 1008] A method for improving memory consolidation in a subject in need thereof, comprising administering to said subject a pharmaceutical composition of the present invention. [The present invention 1009] The method of claim 1008, wherein the subject is a postmenopausal woman. [The present invention 1010] A method for treating a subject exhibiting low estrogen levels, comprising administering to said subject a pharmaceutical composition of the present invention 1002. [The present invention 1011] The method of claim 1010, wherein the subject is a postmenopausal woman. [The present invention 1012] A compound having a formula selected from TIFF2025081398000006.tif34144. [The present invention 1013] A pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharma- ceutically acceptable salt thereof together with a pharmaceutical excipient, carrier, or diluent. [The present invention 1014] A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition of the present invention. [The present invention 1015] The method of the present invention, wherein the disease or disorder is selected from neurological diseases and disorders, psychiatric diseases and disorders, and cell proliferative diseases and disorders. [The present invention 1016] The method of claim 1014, wherein the disease or disorder is associated with memory loss or memory dysfunction. [The present invention 1017] A method for improving memory consolidation in a subject in need thereof, comprising administering to said subject a pharmaceutical composition of the present invention. [The present invention 1018] The method of claim 1017, wherein the subject is a postmenopausal woman. [The present invention 1019] A method for treating a subject exhibiting low estrogen levels, comprising administering to said subject a pharmaceutical composition of the present invention. [The present invention 1020] The method of the present invention, wherein the subject is a postmenopausal woman. [The present invention 1021] 1. A method for improving memory consolidation in a subject in need thereof, the method comprising administering to a subject a compound of the formula: administering to a subject a compound having the sequence of any one of the preceding claims or a pharmaceutical composition comprising the compound; During the ceremony, (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 an alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime, The method. [The present invention 1022] The compound has formula Ia: The method of the present invention 1021 having TIFF2025081398000008.tif30128. [The present invention 1023] In the compound, X is selected from hydrogen, hydroxyl, alkylyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl, or Y is -OCH 2- and Y and Z form a bridge. [The present invention 1024] 3. The compound of formula Ia(i): The method of the present invention 1021 having TIFF2025081398000009.tif31128. [The present invention 1025] The method of claim 1024, wherein in the compound, X is selected from hydrogen, hydroxyl, alkyl, hydroxylalkyl, and Y is hydrogen. [The present invention 1026] In the compound, X is hydrogen or methyl and Y is hydroxymethyl (-CH 2 OH) or hydroxyethyl (-CH 2 CH 2 1021. The method of claim 1021, wherein [The present invention 1027] In the compound, X is methyl and Y is hydroxymethyl (-CH 2 1021. The method of claim 1021, wherein [Brief description of the drawings]

[0018] [Figure 1] Estrogen receptor agonist structures. (a) Previously reported ERβ agonists. Diagram adapted from

[35] . (b) Structure of ISP358-2, (c) estradiol (E2), and (d) ISP538-2 superimposed on E2 to show similarity to natural estrogens. [Diagram 2] Estrogen receptor binding assays. (a) TR-FRET binding assay examining binding to the ligand binding domain (LBD) of ERβ. (b) Binding of ISP358-2 to the LBD of ERβ and ERα. ISP-358-2 has moderate 12-fold selectivity for ERβ (IC50=24±5nM) compared to ERα (IC50=289±92nM) in this assay. [Diagram 3]Nuclear hormone receptor specificity assay for ISP358-2. (a) Agonist activity was measured at three concentrations of ISP358-2 in a GeneBLAzer™ cellular transcription activation assay using chimeric nuclear hormone receptors (NRs) composed of the relevant NR ligand binding domain (LBD) and the DNA binding domain (DBD) from GAL4. Nine different NRs were assayed: 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). ISP358-2 has high selectivity for binding to ER compared to other nuclear receptors (NRs). (b) Agonist activity dose-response curves in the GeneBLAzer™ assay for ERβ and ERα (open symbols). A moderate 2.6-fold selectivity for ERβ (IC50=357±26 nM) over ERα (IC50=930±69 nM) was demonstrated. Data from panel (a) are included for comparison (filled symbols). [Figure 4] Specificity assay for ISP-358-2 binding in a coactivator assay. (a) The assay measures the recruitment of a labeled coactivator peptide to the ERα or ERβ LBD induced by binding of an ER agonist (in this case, ISP358-2). The coactivator peptide is derived from the PPARγ coactivator protein 1a. Figure adapted from ThermoFisher manual. (b) Chemical structure of ISP358-2. (c) Dose-response curve for ISP358-2 in a coactivator assay. The curves yield an IC50 of 161 ± 15 nM for ERβ and 2,940 ± 390 nM for ERα, providing 15-fold selectivity for ERβ. [Diagram 5]Specificity assays for ISP358-2 in cellular assays. (a) ERβ agonist activity and (b) ERα agonist activity based on transcriptional activation by full-length estrogen receptor. (c) ERβ antagonist activity and (d) ERα antagonist activity based on estradiol-induced transcriptional inhibition by antagonist compounds. The average ERβ agonist potency is 27±4 nM in panel (a) (IC50 of 31±7 for the data here) and ERα agonist potency is 20,400±860 nM. This results in approximately 750-fold selectivity for ERβ. No measurable antagonist activity was observed for ERβ or ERα in panels (c) and (d) at ISP358-2 concentrations up to 10 μM. [Figure 6] Cytochrome P450 Inhibition by ISP358-2. Inhibition of CYP450 activity by ISP358-2 (Promega P450-Glo™ assay) for CYP2D6, CYP3A4 (IC50=89±18 μM), (c) CYP1A2, and (d) CYP2C9 (IC50=34±4.7 μM). [Figure 7a] Structural analysis of ISP358-2. (a) ISP358-2 crystal structure (Ortep rendering) showing the trans stereochemistry of the cyclohexane ring. (b) Docked structure of ISP358-2 in the ERα binding pocket showing interactions with active site residues including hydrogen bonds with Arg394, Glu353, and His524. (c) Same as panel b, but with ISP358-2 bound to ERβ. Hydrophobic interactions in ERβ were observed between the phenol ring of ISP358-2 and Phe356, Phe355, and Met340, and between the cyclohexane ring and Leu476, Ala302, Ile373, and Leu298. Docking energies are −7.6 kcal / mol for ERα and −8.0 kcal / mol for ERβ. [Figure 7b] See legend to Figure 7a. [Figure 7c] See legend to Figure 7a. [Figure 8a]Behavioral assays. (a) Outline of OR and OP test procedures. DPN and ISP358-2 at 100 pg / hemisphere and 1 ng / hemisphere doses injected into the DH significantly increased the time spent on the displaced object (b) or the novel object (c) compared to chance (15 s; *p<0.05; **p<0.01) and vehicle (#p<0.05;##p<0.01), suggesting that ISP358-2 improved memory consolidation to a similar extent as the positive control DPN. When injected IP, DPN and 0.5 mg / kg ISP358-2 improved memory consolidation in the OP (d) and OR (e) tests (###p<0.001). 5 mg / kg ISP358-2 also improved OP memory consolidation. Similarly, oral gavage treatment with 0.5 mg / kg and 5 mg / kg ISP358-2 also improved memory consolidation in the OP(f) and OR(g) tests. Panel (a) is adapted from

[38] . [Figure 8b] See legend to Figure 8a. [Figure 8c] See legend to Figure 8a. [Figure 8d] See legend to Figure 8a. [Figure 8e] See legend to Figure 8a. [Figure 8f] See legend to Figure 8a. [Figure 8g] See legend to Figure 8a. [Figure 9] ERβ binding assay. TR-FRET binding assay examining binding to the ligand binding domain (LBD) of ERβ, including E2 and the ERβ agonist DPN as a control. [Figure 10]Cellular assays comparing ISP358-2 to known compounds. Estrogen receptor agonist activity based on transcription with full-length estrogen receptor. (a) ERα IC50 values ​​are 0.31±0.03nM for E2, 2,300±86nM for DPN, 2,103±414nM for WAY200070, and 18,615±939 for ISP358-2. (b) ERβ IC50 values ​​are 0.022±0.005nM for E2, 1.1±0.12nM for DPN, 1.5±0.57nM for WAY200070, and 23±8nM for ISP358-2. [Figure 11] Evaluation of in vitro druggability parameters. (a) Turbidimetric analysis shows good solubility of 15:16. Note: all compounds with a turbidimetric inflection point greater than 50 μM are considered soluble.1 (b) hERG assay of ISP358-2 showed only 13% inhibition at 100 μM, suggesting no significant hERG activity. [Figure 12]In vivo correlation of behavioral effects and ERβ levels. As shown in Figure 8, administration of DPN or ISP358-2 via oral gavage improved spatial memory consolidation in mice ovariectomized within 1 month of OP testing. However, when treatment was delayed until 4 months after ovariectomy (ovx), neither DPN nor ISP358-2 affected memory (panel a). Western blot analysis of ERα and ERβ levels in dorsal hippocampal tissue from these mice shows less ERβ (but not ERα) levels after 5 months of ovx compared to 2 months of ovx (panels b, c). This suggests that the lack of effect of DPN and ISP358-2 on memory in long-term ovx mice is due to reduced ERβ levels. These data also suggest a specific effect of the compounds on ERβ, since ERα levels were not reduced 4-5 months after ovx, at which time either compound could have improved memory if it acted through ERα. The fact that neither compound affected memory 4 months after ovx is consistent with our hypothesis that DPN-like ISP358-2 is selective for ERβ over ERα in vivo. [Figure 13a] Histopathological analysis of ISP358-2. (a) Tissue samples analyzed were from 4 groups with 5 mice per group. Labels indicated as VDLH: Vehicle (V), DPN (D), 0.5 mg / kg ISP358-2 (L), and 5 mg / kg ISP358-2 (H). [Figure 13b] Histopathological analysis of ISP358-2. (b) Representative images of hematoxylin and eosin (H&E) stained tissues collected from vehicle control animals (A–C), DPN-treated animals (D–F), low-dose ISP358-2 0.5 mg / kg-treated animals (G–I), and high-dose ISP358-2 5.0 mg / kg-treated animals (J–L). Examples of portal vein and hepatic ducts (A, D, G, J), glomeruli and renal tubules (B, E, H, K), and myocytes derived from the ventricular septum (C, F, I, L) are shown. No histological abnormalities were detected in control animals or in any of the treatment groups. [Figure 14] Bloodwork panel for histopathology analysis targeting ISP358-2. Bloodwork hematology analysis was performed by Animal Reference Pathology, LLC (animalreferencepathology.com), Salt Lake City, UT. Reference ranges were from CrL:Wi(Han) female 8-16 week old rats from Charles River Laboratories. Blood samples were collected by cardiac puncture at the same time points and treatment conditions as in Figure 13. Ovariectomized 9 week old (n-8) mice were injected i.p. with vehicle, DPN, 0.5 mg / kg ISP358-2, or 5 mg / kg ISP358-2. In some cases, hemolysis occurred, likely due to the blood collection procedure. Hemolysis excluded analysis of these samples, and thus the sample size for this analysis was 6-8. The mean and standard error of the mean for each group are listed in the table along with the results from the one-way ANOVA statistical analysis. In all cases of significant ANOVA, Fisher's post-hoc test showed that the vehicle group was significantly different from all drug groups (p<0.05). Blood samples were analyzed by a SysmexXT-2000iV using veterinary software and rat species settings. Reagents for individual assays were sourced from Seimens, RandOx, or Sekisui. [Figure 15a] MTT assay for proliferation of MCF-7 cells. Cells were seeded in 96-well plates and incubated for 24 hours before treatments were applied. All wells contained 0.1% DMSO, which did not significantly affect proliferation2. Test compounds were dissolved in medium and applied to cells, and cells were incubated for an additional 24 hours at which point the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. Absorbance values ​​were converted to cell numbers using a standard growth curve. Treatments were (a) E2, (b) ISP358-2, and (c) DPN. *Indicates significantly different cell numbers compared to untreated control (Student's t-test, p-value <0.05). [Figure 15b] See legend to Figure 15a. [Figure 15c] See legend to Figure 15a. [Figure 16a] Purity analysis of ISP358-2 (16). 1H NMR (400 MHz) spectra of (a) the 16 / 15 mixture (approximately 2:1 ratio) and (b) the material sent for combustion analysis (16:15>98:2). The stereochemical configuration of 16 (also known as ISP358-2) was confirmed by X-ray crystallography. The spectrum only shows the range from 4.0 to 3.0 ppm for clarity in comparison with the signal of CH2OH (which also contains the solvent peak at 3.31 ppm used as a reference). [Figure 16b] See legend to Figure 16a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description The present invention is described herein using several definitions, as set forth below and throughout the application.

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

[0021] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary, to an extent, depending on the context in which the terms are used. If there are uses of these terms that are not clear to those of ordinary skill in the art, given the context in which the terms are used, "about" and "approximately" will mean plus or minus ≦10% of the particular term, and "substantially" and "significantly" will mean plus or minus >10% of the particular term.

[0022] The terms "include" and "including" as used herein have the same meaning as the terms "comprise" and "comprising". The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that may further include additional elements to the elements recited in the claims. The terms "consist" and "consisting of" should be interpreted as "closed" transitional terms that may not include additional elements other than the elements recited in the claims. The term "consisting essentially of" should be interpreted as being partially closed and may only include additional elements that do not fundamentally change the content of the invention.

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

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

[0025] Subjects in need may include those 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 those exhibiting estrogen serum levels 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 any of the bounded ranges (e.g., within the range of 15-60 pg / ml).

[0026] The subject in need may include the subject that shows low estrogen serum level related to the subject that has been administered therapy and / or treatment that reduces estrogen serum level and / or estrogen activity in the subject.The subject in need may include the subject that is undergoing therapy for cancer treatment or therapy after cancer treatment (e.g., therapy for breast cancer treatment and / or therapy after breast cancer treatment).The subject in need may include the subject that is undergoing hormone therapy (e.g., hormone therapy for cancer, e.g., breast cancer) and / or hormone replacement therapy (e.g., hormone replacement therapy after cancer, e.g., breast cancer treatment). Subjects in need may include those undergoing treatment with drugs that may include, but are 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)). Subjects in need may include those who have undergone oophorectomy and / or hysterectomy.

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

[0028] In some embodiments, the disclosed compounds include one or more substitutions on the 4-carbon of the cycloalkyl substituent and have formula I: TIFF2025081398000010.tif30128, During the ceremony, (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 an alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime.

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

[0030] The alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl moiety can be C(1-6)alkyl. In certain embodiments, the alkyl portion of the carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, or aminoalkylidenyl can be C(1-3)alkyl. For example, carboxyalkylidenyl can be carboxy-C(1-6)alkylidenyl or carboxy-C(1-3)alkylidenyl, esteralkylidenyl can be C(1-6)alkyl-ester-C(1-6)alkylidenyl or C(1-3)alkyl-ester-C(1-3)alkylidenyl, hydroxyalkylidenyl can be hydroxy-C(1-6)alkylidenyl or hydroxy-C(1-3)alkylidenyl, or aminoalkylidenyl can 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, AB is -CH 2 CH 2 -, and A'-B' is -CH 2 CH 2 -, and said compound may be of formula Ia TIFF2025081398000011.tif30128 may be used.

[0032] wherein X and Y are as defined for formula I. In some embodiments of compounds having formula Ia, the substituent X is selected from hydrogen, hydroxyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and -OCH 2 and Y and Z form a bridge.

[0033] The disclosed compounds having formula Ia may exhibit a particular 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 with TIFF2025081398000012.tif17128.

[0034] In certain embodiments, the compound has the formula It may also be an isomer having TIFF2025081398000013.tif13128.

[0035] In some embodiments of compounds having formula Ia, X can be hydroxyalkyl and Y can be hydrogen. Exemplary compounds have the formula: TIFF2025081398000014.tif13128 may be used.

[0036] In some embodiments of compounds having formula Ia, X can be hydroxy and Y can be hydrogen. Exemplary compounds have the formula: TIFF2025081398000015.tif12128 may be used.

[0037] In some embodiments of compounds having formula Ia, X can be hydroxyalkyl and Y can be hydroxyl. Exemplary compounds have the formula: TIFF2025081398000016.tif16128 may be used.

[0038] In some embodiments of the compounds having formula Ia, X can be hydrogen and Y can be -OCH 3 -, forming a bridge with Z. Exemplary compounds have the formula: TIFF2025081398000017.tif10128 may be used.

[0039] The disclosed compounds may include 4-substituted-(4'-hydroxyphenyl)cyclohexene compounds. For example, in the disclosed compounds having formula I, AB is -CH 2 CH 2 - is also acceptable, and A'-B' is =CHCH 2 -, said compound having formula Ia(i) TIFF2025081398000018.tif16128 may be used.

[0040] wherein X and Y are as defined for formula I. In some embodiments of 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 compounds having formula Ia(i), X can be hydroxyalkyl and Y can be hydrogen. Exemplary compounds have the formula: TIFF2025081398000019.tif13128 may be used.

[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 entirely in the S configuration; and / or some or all of the chiral centers may be entirely in the R configuration, etc.). Additionally or alternatively, one or more of the chiral centers may be present as a mixture of configurations (e.g., a racemic or other mixture of R and S configurations). Compositions comprising substantially purified stereoisomers, epimers, or enantiomers of the 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 include the compound: A composition comprising TIFF2025081398000020.tif25128, TIFF2025081398000021.tif25128 may include a composition that accounts for a 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 are contemplated herein, e.g., compounds that are at least about 90%, 95%, or 99% pure stereoisomers, epimers, or enantiomers. Compounds that are substantially pure (e.g., at least about 90%, 95%, or 99%) stereoisomers, epimers, or enantiomers of the compounds: TIFF2025081398000022.tif27128 is contemplated herein.

[0045] Also disclosed herein are hydroxy-protected derivatives of the compounds disclosed herein. For example, the compounds disclosed herein (e.g., compounds having any of formulas I, Ia, and Ia(i)) may include a hydroxy-protecting group at any hydroxy group. A "protected hydroxy" group as intended herein is a hydroxy group that is derivatized or protected by any group (e.g., alkoxycarbonyl, acyl, silyl, or alkoxyalkyl group) that is commonly used for temporary or permanent protection of hydroxy function. A "hydroxy-protecting group" refers to any group that is commonly used for temporary protection of hydroxy function, such as, for example, alkoxycarbonyl, acyl, alkylsilyl, or alkylarylsilyl group (hereinafter simply referred to as "silyl" group), and alkoxyalkyl group. Alkoxycarbonyl protecting groups are alkyl-O-CO- groups, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or allyloxycarbonyl. As intended herein, the term "alkyl" as used in the description or claims refers to a straight or branched alkyl radical of 1 to 6 carbons in all isomeric forms. "Alkoxy" refers to any alkyl radical attached by an oxygen (i.e., a group represented by "alkyl-O-"). Alkoxyalkyl protecting groups are groups such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or 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" refers to a phenyl group, or an alkyl-, nitro- or halo-substituted phenyl group.The terms "hydroxyalkyl", "deuteroalkyl", and "fluoroalkyl" refer to an alkyl radical substituted with one or more hydroxy, deuterium, or fluoro groups, respectively. An "alkylidene" has 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 in all its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as oxalyl, malonyl, succinyl, glutaryl, or an aromatic acyl group, such as benzoyl, or a halo-, nitro-, or alkyl-substituted benzoyl group.

[0046] The compounds disclosed herein can exhibit binding to estrogen receptor and agonistic and / or antagonistic activity to estrogen receptor.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, as are assays for determining the binding affinity of compounds to ERα and ERβ and whether a bound compound is an agonist or antagonist of ERα and ERβ (see, e.g., 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 corresponding Supplementary Information, the contents of which are incorporated herein by reference in their entireties). Suitable assays for determining the binding affinity of a compound to ERα and ERβ and whether a bound compound is an agonist or antagonist of ERα and ERβ may include a fluorescence polarization displacement assay and a cell-based ERα luminescence activity assay and an ERβ luminescence activity assay.

[0047] As used herein, the term "selective agonist" may be used to refer to a compound that selectively binds to and agonizes an estrogen receptor, particularly ERβ, relative to another estrogen receptor, particularly ERα. For example, a compound that is a selective agonist of ERβ has an IC50 of less than 100 nM, preferably less than 10 nM, and even more preferably less than 1 nM in an assay for ERβ receptor agonist activity. 50 (nM), and compounds that are selective agonists of ERβ have an IC of greater than 100 nM, preferably greater than 500 nM, and even more preferably greater than 1000 nM in an assay for ERα receptor agonist activity. 50 (nM).

[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, relative to another estrogen receptor, particularly ERα. For example, a compound that is a selective agonist of ERβ (e.g., K d It 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) greater than its binding affinity for ERα (measured in nM). Preferably, a selective agonist of ERβ has a K for ERβ of less than 100 nM, more preferably less than 10 nM, or even more preferably less than 1 nM. d Preferably, the selective agonist of ERβ has a K for ERα of greater than 500 nM, more preferably greater than 1000 nM, or even more preferably greater than 2000 nM. d (nM).

[0049] As used herein, the term "selective agonist" may be used to refer to a compound that selectively binds to and stimulates estrogen receptors, particularly ERβ, rather than antagonizing estrogen receptors, particularly ERβ. For example, a compound that is a selective agonist of ERβ has an IC 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. 50 Compounds that are selective agonists of ERβ may have an IC50 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. 50 (nM).

[0050] Pharmaceutically acceptable salts of the disclosed compounds are also contemplated herein and may be utilized in the disclosed treatment methods. For example, the substituents of the disclosed compounds may be protonated or deprotonated and may be present with an anion or cation, respectively, as pharma-ceutically acceptable salts of the compounds. The term "pharma-ceutically acceptable salts" as used herein refers to salts of compounds that are substantially non-toxic to living organisms. Representative pharma-ceutically acceptable salts include salts prepared by reacting the compounds disclosed herein with pharma-ceutically acceptable mineral or organic acids or organic or inorganic bases. Such salts are known as acid addition salts and base addition salts. It will be understood by the skilled reader that most or all of the compounds disclosed herein can form salts, and that the salt form of the drug is commonly used. This is because in many cases, the salt form is easier to crystallize and purify than the free acid or free base.

[0051] Acids commonly used to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, 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, and the like. Examples of suitable pharma- ceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyrate-1,4 diacid salt. yne-.1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

[0052] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, 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, and the like.

[0053] It will generally be recognized that the particular counterion forming a part of any salt of the compounds disclosed herein is not essential, so long as the overall salt is pharmacologically acceptable, and so long as the counterion does not impart undesirable qualities to the overall salt, which may include undesirable solubility or toxicity.

[0054] It is further understood that the disclosed compounds may be in equilibrium with various internal salts. For example, internal salts include salts in which the compounds contain a deprotonated and a protonated substituent.

[0055] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions.Accordingly, also disclosed herein is a pharmaceutical composition comprising an effective amount of any compound disclosed herein or a pharma- ceutically acceptable salt of any compound disclosed herein together with a pharmaceutical excipient.In some embodiments, the disclosed compounds may be used to prepare a medicament for treating diseases or disorders related to estrogen receptor beta (ERβ) activity, particularly diseases or disorders that can be treated with specific ERβ agonists.Accordingly, the disclosed compounds may exhibit ERβ agonist activity, and preferably, the compounds exhibit specificity as ERβ agonists relative to ERβ antagonists, ERα agonists, and / or ERα antagonists.

[0056] The disclosed compounds may be used to prepare and formulate pharmaceutical compositions for treating diseases associated with estrogen ERβ activity.Diseases or disorders associated with ERβ activity may include, but are not limited to, cell proliferation diseases and disorders (e.g., cancer, such as 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, such 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 in a method for treating diseases and disorders associated with ERβ activity.

[0057] The compounds and pharmaceutical compositions disclosed herein may be administered to a subject in need thereof to treat a disease or disorder. In some embodiments, to treat a disease or disorder associated with 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 refers to an animal, which may be a human or a 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 include, for example, human subjects with a disease or disorder associated with ERβ activity or identified as 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, a "subject in need of treatment" may include a subject with a disease, disorder, or condition that responds to therapy with an ERβ agonist. For example, a "subject in need of treatment" may include a subject with a cell proliferative disease, disorder, or condition, such as cancer (e.g., cancer, e.g., breast cancer). In addition, a "subject in need of treatment" may include a subject with a neurological disease or disorder, including psychiatric diseases and disorders (e.g., depression, anxiety, and / or schizophrenia). A "subject in need" may include a subject with a neurodegenerative disease or disorder (e.g., Alzheimer's disease, including APOE4-associated Alzheimer's disease). In particular, a subject in need may include a subject that exhibits memory loss or a need for improved memory consolidation (e.g., a subject with a disease or disorder characterized by a need for improved memory consolidation under low estrogen conditions). A subject in need may include a postmenopausal woman in need of improved memory consolidation (e.g., a postmenopausal woman in need of improved memory consolidation under low estrogen conditions).

[0060] As used herein, the terms "treat" or "to treat" refer to, respectively, alleviating symptoms, eliminating temporarily or permanently the cause of the resulting symptoms, and / or preventing or slowing the appearance of, or reversing the progression or severity of, the resulting symptoms of the referenced disorder. Thus, the methods disclosed herein include therapeutic and prophylactic administration.

[0061] The term "effective amount" as used herein refers to an amount or dose of the compound, either in a single dose or in multiple doses administered to a subject, that produces a desired effect in the subject being diagnosed or treated. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) to treat a disease or disorder associated with ERβ activity in the subject, whereby the effective amount induces, promotes, or causes ERβ agonist activity in the subject.

[0062] Effective amount can be easily determined by the attending diagnostician who is skilled in the art by using known techniques and observing the results obtained under similar circumstances.When determining the effective amount or dosage of the compound to be administered, the attending diagnostician may take into account many factors, such as the species of the subject; its size, age and overall health; the degree of involvement or severity of the disease or disorder involved; the response of the individual subject; the specific compound to be administered; the method of administration; the bioavailability characteristics of the preparation to be administered; the selected dosing schedule; the use of concomitant drugs; and other relevant circumstances.

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

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

[0065] Instead of administering a pharmaceutical composition comprising a compound that is an ERβ agonist together with a compound that is an ERα antagonist, the disclosed methods may be practiced by administering a first pharmaceutical composition (e.g., a pharmaceutical composition comprising an ERβ agonist) and a second pharmaceutical composition (e.g., a pharmaceutical composition comprising 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, regardless of their names, may be administered simultaneously or in any order.

[0066] As one of skill in the art would understand, the disclosed pharmaceutical compositions can be prepared using materials (e.g., active excipients, carriers, diluents, etc.) that have properties (e.g., purity) that make the formulation suitable for administration to humans, or the formulations can be prepared using materials that have purity and / or other properties that make the formulation suitable for administration to non-human subjects, but not for administration to humans.

[0067] The compound utilized in the method disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, but can utilize any pharma- ceutically acceptable dosage form.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, a fast-dissolving dosage form, a controlled release dosage form, a lyophilized dosage form, a delayed release dosage form, a prolonged release dosage form, a pulsed release dosage form, a mixed immediate release and controlled release dosage form, or a combination thereof.Alternatively, the compound utilized in the method disclosed herein may be formulated as a pharmaceutical composition in liquid form (e.g., injection solution or gel).

[0068] The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions containing excipients, carriers, or diluents.For example, the excipients, carriers, or diluents may be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0069] The compound utilized in the method disclosed herein may also be formulated as a pharmaceutical composition that includes one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers may include lactose monohydrate, anhydrous lactose, and various starches. Examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC®). Suitable lubricants, including agents that affect 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 can include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents include Magnasweet® (registered trademark of MAFCO), bubble gum flavoring, and fruit flavoring. Examples of preservatives can include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other parahydroxybenzoic acid esters, such as butylparaben, alcohol, such as ethyl alcohol or benzyl alcohol, phenolic compounds, such as phenol, or quaternary compounds, such as benzalkonium chloride.

[0070] Suitable diluents for pharmaceutical compositions can include pharmaceutically acceptable inactive fillers, such as crystalline cellulose, lactose, calcium hydrogen phosphate, sugars, and any mixture of the above.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 include disintegrants.Suitable disintegrants include slightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

[0072] The disclosed pharmaceutical composition may also include an effervescent agent. An example of an effervescent agent is an effervescent couple, such as an organic acid and a carbonate or bicarbonate. 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. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0073] Pharmaceutical compositions containing the compounds may be adapted for administration by any suitable route, for example, oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes. Such formulations may be prepared by any method known in the art of pharmacy, for example, by bringing into association the active ingredient with the carrier or excipient.

[0074] Pharmaceutical compositions adapted for oral administration may be presented as discrete units, for example, capsules or tablets; powders or granules; a solution or suspension in an aqueous liquid or a non-aqueous liquid; edible foams or whips; or an oil-in-water or water-in-oil liquid emulsion.

[0075] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time, 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 appropriate 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, such as mouth and skin, the pharmaceutical composition is preferably applied as a topical ointment or cream.When formulated in 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 an oil-in-water cream base or a water-in-oil 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 topical administration in the mouth include lozenges, pastilles, and mouthwashes.

[0079] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0080] A pharmaceutical composition adapted for nasal administration wherein the carrier is a solid and comprises a coarse powder having a particle size (for example in the range 20 to 500 microns) which can be administered in the manner of snuff (i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose). Suitable formulations wherein the carrier is a liquid, when administered as a nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient.

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

[0082] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations.

[0083] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents.The preparations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

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

[0085] Aspect 1. The compound has the formula and stereochemistry of TIFF2025081398000023.tif25128.

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

[0087] Embodiment 3. A pharmaceutical composition comprising an effective amount of a compound of embodiment 1, preferably in substantially pure form (e.g., a stereoisomer representing at least about 90%, 95%, or 99% of the compound in the composition), together with a pharmaceutical excipient, carrier, or diluent.

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

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

[0090] Embodiment 6 The method of embodiment 4, wherein the disease or disorder is associated with memory loss or memory dysfunction.

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

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

[0093] Embodiment 9. The method of embodiment 7 or embodiment 8, wherein the subject is a postmenopausal female.

[0094] Aspect 10. A compound having a formula selected from TIFF2025081398000024.tif34144.

[0095] Embodiment 11. A pharmaceutical composition comprising an effective amount of a compound of embodiment 10, or a pharma- ceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.

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

[0097] Embodiment 13. The method of embodiment 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] Embodiment 14 The method of embodiment 12, wherein the disease or disorder is associated with memory loss or memory dysfunction.

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

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

[0101] Embodiment 17 The method of embodiment 15 or embodiment 16, wherein the subject is a postmenopausal female.

[0102] Embodiment 18. A method for improving memory consolidation in a subject in need thereof, the method comprising administering to a subject a compound of the formula: administering to the subject a compound having the sequence TIFF2025081398000025.tif30128 or a pharmaceutical composition comprising the compound; During the ceremony, (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 an alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime, The method.

[0103] Embodiment 19. The compound has formula Ia: The method of embodiment 18, having TIFF2025081398000026.tif30128.

[0104] 20. The compound wherein X is selected from hydrogen, hydroxyl, alkylyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl, or Y is -OCH 2 and Y and Z form a bridge.

[0105] Embodiment 21. The compound has formula Ia(i): The method of embodiment 18, having TIFF2025081398000027.tif31128.

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

[0107] 23. A compound in which X is hydrogen or methyl and Y is hydroxymethyl (-CH 2 OH) or hydroxyethyl (-CH 2 CH 2 19. The method of embodiment 18, wherein

[0108] Aspect 24. A compound in which X is methyl and Y is hydroxymethyl (-CH 2 19. The method of embodiment 18, wherein EXAMPLES

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

[0110] Example 1. AC Estrogen as a Potent and Selective Estrogen Receptor-β Agonist (SERBA) to Improve 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 (SERBAs) that have in vivo efficacy, are AC estrogens, and lack the B and D estrogen rings are reported herein. The most potent and selective AC estrogen is selective for ER activation compared to seven other nuclear hormone receptors, and surprisingly, is 750-fold more selective for the β isoform than the α isoform, with IC 50is 20-30 nM in cellular and direct binding assays. Comparison of potency in various assays suggests that ER isoform selectivity is related to the ability of the compound to drive productive conformational changes required for transcriptional activation. Compounds disclosed herein also demonstrate in vivo potency following microinfusion into the dorsal hippocampus, intraperitoneal injection (0.5 mg / kg), or oral gavage delivery (5 mg / kg). This simple yet novel AC estrogen is selective, brain penetrant, and promotes memory consolidation.

[0111] result compound synthesis Commercially available 4-(4-hydroxyphenyl)cyclohexanone 1 was dissolved in NaBH 4 By reacting with o This can be converted to alcohol 2 or reacted with excess methyllithium to give 3. o This was converted to the alcohol 3 or by condensation with hydroxylamine to the oxime 4 (Scheme 1).

[0112] TIFF2025081398000028.tif78154

[0113] Scheme 1. Reagents: (a) NaBH 4 / MeOH (90%); (b) MeLi / Et 2 O (37%); (c) H 2 NOH-HCl, Amberlyst, ethanol (70%); (d) TBSCl, imidazole (83%); (e) Ph 3 PCH 3 + Br - , n-BuLi (84%); (f) TBAF / THF (73~78%); (g) H 2 , Pd / C, (h) paraformaldehyde, MgCl 2 , NEt 3 (40%);(i)H 2 NOH-HCl, NaHCO3 , ethanol (69%); (j) cat.OsO 4 , NMO (1.4 equivalents) (86%); (k)BH 3 -THF, then 30% H 2 O 2 / 1 N NaOH; (l) 9-BBN, then 30% H 2 O 2 / 1 N NaOH;(m)DDQ(0.5 equivalent) / CH 2 Cl 2 (16, 47%; 17, 37%); (n) MgCl 2 , NEt 3 (78%).

[0114] The stereochemistry of 2 and 3 was assigned based on NMR spectroscopic data. o In the case of 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 therefore the hydroxyl group is equatorial. 3 o 3, assigned to the alcohol and methyl carbons 13 The signals at δ 69.5 and 31.1 ppm in the C NMR spectrum are consistent with this type of cis-1,4-alcohol. 24 Olefination of t-butyldimethylsilyl ether 5 with the ylide generated from methyltriphenylphosphonium bromide gave 6. Cleavage of the silyl ether with TBAF gave 7. Catalytic hydrogenation of 7 gave 8 as a mixture of stereoisomers. 7 was reacted with excess paraformaldehyde, MgCl 2 , and NEt 3Reaction of 6 with 4-(2-methylphenyl)-1,1-dihydro-1,1-tri ...dihydro-1,1-trimethylphenyl)-1,1-dihydro-1 25 .BH 3 Hydroboration-oxidation of 6 with -THF afforded an inseparable mixture of stereoisomeric primary alcohols cis-13 and trans-14, with the respective hydroxymethylene protons being substituted. 1 They occur in a 2:1 ratio as confirmed by integration of the H NMR signals (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 signals of the axial hydroxymethylene (i.e., the cis isomer) appear downfield compared to the signals of the equatorial hydroxymethylene. 24 Alternatively, hydroboration-oxidation with 9-BBN afforded a mixture in which trans-14 was present in a greater ratio compared to cis-13 (2:3, cis:trans). The use of these two borane reagents to tailor the cis:trans outcome of 4-substituted methylenecyclohexanes has been previously reported. 26、27 Cleavage of the silyl ether with TBAF afforded a mixture of stereoisomeric 4-(4-hydroxymethylcyclohexyl)phenols cis-15 / trans-16. Treatment of the mixture of stereoisomers 15 / 16 (2:3, cis:trans) with DDQ (0.5 equiv.) afforded a separable mixture of bicyclic ethers 17 and trans-16. The tentative structural assignment of trans-16 was confirmed by single crystal X-ray diffraction analysis (Figure 7a). 28The isolation of unreacted trans-16 was rationalized based on the faster oxidation rate of cis-15. Because the oxidation of cis- or trans-4-(4-hydroxymethylcyclohexyl)phenol proceeds via the same benzylic carbocation intermediate (i.e., 18, Scheme 2), the activation energy for the formation of this intermediate is lower for the less stable cis-15 compared to trans-16, thus allowing for faster oxidative cyclization of the cis-isomer. 17 was dissolved in MgCl 2 and trimethylamine, an intramolecular elimination reaction occurred, which gave cyclohexene (±)-19 (Scheme 1).

[0115] TIFF2025081398000029.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 on the basis of its NMR spectral data. In particular, the olefinic proton signal appears as a narrow multiplet at about 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) with triethyl phosphonoacetate afforded the unsaturated ester (±)-21. Desilylation with TBAF afforded the phenol (±)-22 (Scheme 3).

[0119] TIFF2025081398000030.tif42154

[0120] Scheme 3. Reagents: (a) TBDPSCl, imidazole (93%); (b) EtO 2 CCH 2 P(O)(OEt) 2, NaH(95%);(c)DIBAL, CH 2 Cl 2 , -40℃(quant.);(d)TBAF(80%);(e)H 2 (30psi), 20%Pd / C(25, 14%;26, 60%).

[0121] Reduction of 21 with DIBAL followed by deprotection of the silyl ether afforded the allylic alcohol (±)-24. Catalytic hydrogenation of 24 afforded a separable mixture of alcohol 25 and a reduced excess of the ethylcyclohexane derivative 26.

[0122] TR-FRET and cell transcription assays Initial screening of compounds was performed in a TR-FRET displacement assay, which detects binding to the ERβ LBD (see Figure 2 for dose-response curves of selected compounds and Figure 9 for dose-response curves of all compounds). All synthesized compounds showed IC 50 The IC 50 The values ​​are summarized in Table 1.

[0123] Table 1: Estrogen receptor assay data. Values ​​reported are IC50 and values ​​are in nM. TIFF2025081398000031.tif47149TIFF2025081398000032.tif206149TIFF2025081398000033.tif25149* IC 50 The average of the two data sets is 31 ± 7 nM (Figure 5a) and 23 ± 8 nM (Figure 10). The selectivity is therefore 658–886, with an average of 750.

[0124] The most potent compounds were 16 (hereafter referred to as ISP358-2) (hydroxymethyl-substituted), 25 (hydroxyethyl-substituted), and 8 (methyl-substituted). The IC values ​​for ERβ of all these compounds were 50The IC value for ISP358-2 was <30 nM. ISP358-2 is a pure trans isomer and was found to bind with higher affinity to ERβ than the mixture of cis and trans stereoisomers (15 / 16). Methylene (ISP358-2) or ethylene (25) linkers to the hydroxyl group also provided efficacy, but direct substitution of the hydroxyl on the cyclohexane ring significantly reduced affinity (IC value for 2). 50 The affinity was also reduced by introducing an unsaturation into the alkyl linker (24) (676 nM), whereas the affinity was only moderately reduced by introducing an unsaturation into the cyclohexane ring (18) (49 nM). Binding of ISP358-2 to ERα was also examined, and ISP358-2 bound with 12-fold higher affinity (IC) than ERβ in this TR-FRET assay, which measures direct binding to the isolated LBD. 50 24 nM; Figure 2b).

[0125] Additionally, ISP358-2 was screened in a nuclear hormone receptor functional assay (Figure 3). This assay measures transcriptional activation by binding and activation of a chimeric receptor composed of the LBD of a hormone receptor of interest (e.g., ERβ) tethered to the DNA binding domain (DBD) of GAL4. This assay was performed to evaluate the selectivity of estrogen receptor activation over other nuclear hormone receptors. No significant agonist activity of compound ISP358-2 was observed against any nuclear hormone receptors (except estrogen receptor) tested at concentrations between 0.25 and 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, ISP358-2 was found to be 2.6-fold more selective in binding and activation to full-length chimeric ERβ (357±26 nM) compared to full-length chimeric ERα (930±69 nM). This assay (FIG. 3) measures transcriptional activation rather than simply binding (as in FIG. 2) to the agonist binding to the ER LBD. However, this assay uses a non-native chimeric protein (ER LBD fused to GAL4 DBD) that may not accurately reflect the actual agonist-induced activation that occurs under natural conditions.

[0126] When performing a coactivator-type TR-FRET LBD binding assay (Fig. 4a), the ISP358-2 compound (Fig. 4b) was found to be 15-fold more selective for binding to the ER and recruiting the PPARγ coactivator peptide to ERβ (161±15 nM) compared to ERα (2,940±390 nM) (Fig. 4c). This assay measures activation of the ER LBD by measuring binding and agonist-induced coactivator peptide recruitment rather than simply measuring agonist-receptor binding.

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

[0128] In vitro druggability - CYP450 binding, hERG, and turbidimetric analysis ISP358-2 showed no inhibition of CYP1A2 and CYP2D6, but showed no 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. Turbidimetric analysis (performed on ISP171, 15 / 16, isomer mixture) showed no significant aggregation, indicating good solubility at concentrations up to 300 μM (Figure 11).

[0129] Docking studies ISP358-2 (Fig. 7a) was docked into the binding site of agonist-conformation ERα in two conformations with similar docking energies. In one binding configuration (Figs. 6d and 6e), the phenolic hydroxyl interacts with Arg394 / Glu353 (energy = -7.6 kcal / mol), while in the other, the ISP358-2 molecule is flipped 180 degrees (energy = -7.8 kcal / mol) and the aliphatic hydroxyl interacts with Arg394 / Glu353. ISP358-2 is bound in the agonist-conformation ERβ pocket (Fig. 7c and data not shown), with the phenolic hydroxyl interacting with Arg394 / Glu353 in the lowest energy docking pose (energy = -8.0 kcal / mol). In both cases, there are significant hydrophobic interactions between the binding site residues and bound ISP358-2, but they are smaller in the ERβ pocket, creating a closer fit. In both cases, the hydroxymethyl group is close enough to His524 (3.0 Å) to participate in hydrogen-bonding interactions typically seen for ER agonists, but in ERβ, the hydrogen attached to the aliphatic alcohol can be with the backbone carbonyl of Gly472. As with natural estrogen molecules, in ERβ, there are large hydrophobic interactions that force the hydroxymethyl-cyclohexyl ring (ring C) to be nearly planar with the phenol ring (ring A) (data not shown). This contrasts with the nearly orthogonal binding pose of the two rings in ERα (data not shown). The ERβ hydrophobic interactions are with Phe356, Met340, Phe355, and Leu298 near the phenol ring, Leu476 and Ile373 near the hydroxymethyl group, and Ala302 and Leu298 near the cyclohexane ring (Figure 7c and data not shown). E 2 Control docking studies of recapitulated the predicted binding orientation based on the crystal structure (data not shown).

[0130] Assessment of memory consolidation Dorsal hippocampal injection First, we investigated the effect of direct intrahippocampal injection of ISP358-2 on object recognition and spatial memory consolidation in ovariectomized mice (Fig. 8a). Five groups of mice were tested: vehicle (negative control), DPN (positive control), and three doses of ISP358-2 (10 pg / hemisphere, 100 pg / hemisphere, and 1 ng / hemisphere) (Fig. 8b,c). For object placement (Fig. 8b), one-sample t-tests showed that mice given vehicle or 10 pg ISP358-2 did not spend significantly more time on the displaced object than chance (ts (7) = 0.44 and 1.19, p > 0.05; n = 8), indicating that these groups did not show memory for the training object location. In contrast, mice given DPN, 100 pg ISP358-2, or 1 ng ISP358-2 spent significantly more time at the displaced object than chance (ts (6) = 4.5, 10.3, and 3.4, p < 0.05; n = 7), indicating robust memory for the training object location. Furthermore, a one-way ANOVA performed on the time spent at the displaced object revealed a significant main effect of treatment (F (4,32) = 2.97, p = 0.034). Fisher's LSD post-hoc tests revealed that the DPN, 100 pg, and 1 ng groups spent significantly more time with the displaced object than the vehicle group, whereas the vehicle and 10 pg groups did not differ from each other. Taken together, these data suggest that dorsal hippocampal injections of 100 pg or 1 ng ISP358-2 improved memory consolidation of object placement.

[0131] The results for object recognition (Figure 8c) were nearly identical. Neither the vehicle 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). However, the DPN, 100 pg ISP358-2, and 1 ng ISP358-2 groups all spent significantly more time on the novel object than chance (ts (9-10)= 2.35, 3.16, and 1.08, p < 0.05; n = 10–11). Furthermore, the main effect of treatment was significant (F (4,48) = 3.69, p = 0.011), and post-hoc tests confirmed that the DPN, 100 pg ISP358-2, and 1 ng ISP358-2 groups were significantly different from vehicle, but the 10 pg ISP358-2 group was not. As with object placement, these data indicate that dorsal hippocampal injections of 100 pg or 1 ng ISP358-2, but not 10 pg ISP358-2, improved memory consolidation of object recognition.

[0132] intraperitoneal injection Next, we used a new set of mice to examine whether systemic administration of ISP358-2 also produced similar memory-enhancing effects as intrahippocampal injection (Fig. 8d, e). Intraperitoneal (IP) injection is a common, reliable, and convenient systemic procedure in which the injected drug is absorbed into the blood vessels through the peritoneum. 30 Because the drug dose for intrahippocampal infusion is considerably less than the dose required to cross the blood-brain barrier, we examined a range of IP doses based on the cell assay and DH infusion results described above and on previous studies showing that IP injection of 0.05 mg / kg DPN improved object recognition memory. 31 In our cell assays, the IC50 of ISP358-2 was approximately 10-fold higher than that of DPN. Furthermore, our behavioral studies showed that ISP358-2 improved hippocampal memory at a concentration 10-fold higher than DPN. Thus, our IP dose of ISP358-2 was at least 10-fold higher than DPN (0.5 mg / kg and 5 mg / kg). Thus, we tested four groups of mice 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), one-sample t-tests showed that mice given vehicle did not prefer the displaced object (t (8)= 0.68, p > 0.05; n = 9). However, the DPN, 0.5 mg / kg ISP358-2, and 5 mg / kg ISP358-2 groups all spent significantly more time on the displaced object than chance (ts (9-11) = 3.20, 3.93, and 2.78, p < 0.05; n = 10–12). This suggests that systemic administration of ISP358-2 improved memory consolidation of the object placement. Furthermore, the main effect of treatment was significant (F (3,38) = 3.63, p = 0.021), and post-hoc tests showed that the 0.5 mg / kg ISP358-2 group was significantly different from vehicle. These data demonstrate that IP injection of ISP358-2 improves spatial memory consolidation similar to dorsal hippocampal injection.

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

[0135] Oral gavage Given the memory-enhancing efficacy of IP injection, we next assessed whether oral administration of ISP358-2 could improve memory consolidation (Fig. 8f, g). Oral gavage is a common procedure in scientific experiments in which drugs are delivered directly to the stomach by syringe. 32 Oral gavage is highly effective and accurate, but invasive and stressful, compared to other oral administration methods such as delivery in food and / or water. 33 Because we observed that IP injection of ISP358-2 improved hippocampal memory consolidation, we used the same doses for oral gavage as for IP injection (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 (FIG. 8f), one-sample t-test results showed that mice given vehicle did not spend significantly more time on the displaced object than chance (t (8) =0.54, p>0.05; n=9). However, the DPN, 0.5 mg / kg ISP358-2, and 5 mg / kg ISP358-2 groups all showed a significant preference for the displaced object compared to chance (ts (8-9) = 2.76, 3.65, and 5.06, p < 0.05; n = 9–10). This suggests that oral administration of ISP358-2 improved spatial memory consolidation. One-way ANOVA also revealed that the main effect of treatment was significant (F (3,33) =5.04, p=0.006), and post-hoc tests verified that the DPN, 0.5mg / kg ISP358-2, and 5mg / kg ISP358-2 groups were significantly different from the vehicle group. Similarly, for object recognition (Figure 8g), one-sample t-tests showed that mice given vehicle had no preference for the novel object (t (8)=0.25, p>0.05; n=9). In contrast, the DPN, 0.5 mg / kg ISP358-2, and 5 mg / kg ISP358-2 groups all spent significantly more time on the novel object compared to chance (ts (7-9) = 3.89, 5.37, and 2.36, p < 0.05; n = 8–10). Furthermore, the main effect of treatment was significant (F (3,32) = 3.02, p = 0.044), and post-hoc tests showed that the DPN, 0.5 mg / kg ISP358-2, and 5 mg / kg ISP358-2 groups were significantly different from vehicle. Collectively, the results of object placement and object recognition behavior demonstrate that oral administration of ISP358-2 improves object recognition and spatial memory consolidation similar to dorsal hippocampal or IP injection. These data also suggest oral bioavailability of ISP358-2 in ovariectomized mice.

[0137] Finally, we collected preliminary data to evaluate the efficacy of gavaged ISP358-2 on spatial memory consolidation in mice that had experienced long-term estrogen deprivation. Mice ip-injected with vehicle, DPN, or ISP358-2 as described above remained in our colony for 4 months after ovariectomy. Mice were then trained in an object placement task (with novel objects) and then immediately given the same doses of vehicle, DPN, or ISP358-2 as described above via gavage (n=9-12 / group). Unlike mice gavaged within 1 month of ovariectomy (Fig. 8f), DPN or ISP358-2 did not improve spatial memory consolidation in mice treated within 4 months of ovariectomy (Fig. 12a). We then compared the efficacy of gavaged ISP358-2 with that of ISP358-2 in mice treated within 4 months of ovariectomy (Fig. 12b). We then compared the efficacy of gavaged ISP358-2 with that of ISP358-2 in mice treated within 4 months of ovariectomy (Fig. 12c). 34ERα and ERβ levels in DH were measured using Western blotting according to the protocol described in (ERα, 1:200, Santa Cruz Biotechnology; ERβ, 1:200, Santa Cruz Biotechnology). Tissues were collected approximately 2 and 5 months after ovariectomy. ERβ levels 5 months after ovariectomy were significantly decreased compared to 2 months after ovariectomy (Figure 12b; t 9 =2.46, p<0.05). In contrast, ERα levels were unchanged (Fig. 12c). These data suggest that neither DPN nor ISP358-2 improved memory after chronic ovariectomy due to reduced ERβ levels. By extension, these data also support the in vivo selectivity of ISP358-2 for ERβ, because if ISP358-2 improved memory by binding to ERα, it should have been able to improve memory consolidation after chronic ovariectomy because ERα levels were not reduced. However, the fact that ERβ levels were low when ISP358-2 did not improve memory supports our hypothesis that ISP358-2 regulates memory through ERβ and not ERα.

[0138] Assessment of peripheral pathology or cell proliferation following ISP358-2 treatment In general, the tissues from the 20 different specimens all appeared similar. heart : All cardiac tissue was unremarkable. The ventricular walls were intact and of normal thickness. The atrial walls were intact and of normal thickness. There was no evidence of muscle fiber disarray or congenital defects such as ischemic heart disease or ischemic injury. There was no evidence of inflammation or myocarditis. kidney The kidneys were entirely unremarkable. The glomeruli were intact. The tubules appeared normal. There was no evidence of inflammation involving any of the renal structures. liverThe gross architecture of the liver was intact and appeared normal, with large portal-type veins co-running with the hepatic ducts and hepatic artery. The central vein was present and appeared normal. Generalized appearance of low-grade / mild ischemic injury was present in all specimens. This appeared to be nonspecific and was present in all specimens and may have been secondary to the initial ischemic injury or autolysis occurring postmortem. Small foci of cellular necrosis were observed in several animals, likely secondary to ischemia. Two animals showed small, focal areas of mild low-grade inflammation. One animal (R15-IP-24V) had multifocal areas of organized inflammatory infiltrates composed primarily of mononuclear lymphocytes. Overall, there was no evidence of acute inflammation composed of neutrophils or damage to intrahepatic structures such as the hepatic ducts. Bloodwork chemistry and hematology data for treated animals (Figure 14) show no significant deviations from the expected reference range compared to vehicle, except for a moderate effect due to hemolysis likely due to sample collection via cardia puncture. Finally, E 2 induced statistically significant proliferation of MCF-7 breast cancer cells, whereas neither ISP358-2 nor DPN showed any significant proliferation compared to untreated control cells.

[0139] Consideration 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 presented herein (Table 1) differ from these previously reported compounds in that they are selective for ERβ over ERα (approximately 750-fold) and are AC estrogens, resembling the native 17β-estradiol molecule and lacking only the B and D rings.

[0140] Structure-activity relationships of AC estrogens The binding affinities of 4-(4-substituted cyclohexyl)phenols were evaluated in a TR-FRET ERβ binding assay (Table 1 and Figure 2). In particular, compounds bearing a hydroxymethyl functional group attached to the cyclohexyl core showed high affinities in the range of 20-200 nM. The IC of the two components 15 / 16 in a 2:1 mixture of cis and trans stereoisomers was 0.015 / 0.015 (IC 50 =184 nM), the trans isomer was found to be more potent than the mixture (ISP358-2, IC 50 =24nM). Unsaturated in the six-membered ring (18, IC 50 = 49 nM) did not significantly reduce the binding affinity compared to ISP358-2. However, conformational constraints such as those present in the exocyclic allylic alcohol reduced the affinity (24, IC 50 = 676 nM). The presence of a third hydroxyl group significantly reduced 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 larger range of binding affinities (2, IC 50 =7250nM, 25, IC 50 =11 nM). Furthermore, to assess 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 cellular transcriptional activation assay. Trans stereoisomers ISP358-2, 8, 18, and 25 showed similar ERβ agonist potency (EC 50 30-75 nM), with ISP358-2 standing out as the most potent and selective in this more biologically relevant cellular functional assay. Interestingly, the hydroxyethyl analogs were not as potent in the cellular functional assay (25, EC 50 =75nM vs. 11nM).

[0141] The difference in efficacy observed in this assay may be due to what the assay measures. The TR-FRET assay in Figure 2 measures only the displacement of a fluorescently labeled estradiol ligand from the ligand-binding domain (LBD), which reflects the binding affinity for the ligand that competitively displaces the fluorescent probe. In contrast, the cellular assay is more complex and measures the entire series of molecular events that lead 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 the initial hormone binding. 36 Agonist binding-induced protein conformational changes in the estrogen receptor recruit coactivator proteins and also induce protein dimerization, ultimately resulting in DNA binding and transcriptional activation. An additional interaction between the aliphatic hydroxyl group and the His475 residue of ERβ plays a role in a conformational change involving nearby helix-12. This conformational change is responsible for the IC2016 signaling in cellular functional assays. 50 values ​​(Figure 5), but would not be reflected in the TR-FRET binding assay (Figure 2).

[0142] All the compounds tested showed no significant ERβ or ERα antagonist activity (EC 50 >10,000 nM) and therefore showed no selectivity for agonist versus antagonist activity (Table 1 and Fig. 5c, d). Among the ERβ agonists, ISP358-2 was the most selective, with an agonist selectivity for ERβ of approximately 750-fold higher than that for ERα in the cellular functional assay, but only moderate selectivity in the TR-FRET binding assay (Fig. 2b).

[0143] Differences between assays suggest mechanisms of isoform selectivity As mentioned above, the cellular assay with ISP358-2 reveals that the selectivity of ISP358-2 ERβ agonist activity is approximately 750-fold over its ERα agonist activity (Fig. 5a,b). In contrast, the TR-FRET binding assay shows a moderate 12-fold selectivity for ERβ (Fig. 2b). This may be because the TR-FRET binding assay simply measures binding affinity (to isolated LBD), whereas the cellular assay measures transcription induced by agonist binding to full-length native ERβ, which triggers a productive conformational change in ERβ involving the rotation of helix-12 (resulting in coactivator recruitment, dimerization, DNA binding, and then activating transcription). To test the hypothesis that the differences between the assays are due to these downstream activation events, two other types of assays were performed. In the first assay, transcription activation was measured in a different cellular assay, in this case with a non-native chimeric receptor (ER LBD fused to GLA4 DBD). In this assay (Fig. 3b), there was a moderate 2.6-fold selectivity for ERβ. In a second assay, we measured the ability of agonist binding to recruit coactivator peptides to the ER LBD (Fig. 4a). In this assay, a 15-fold selectivity for ERβ was observed (Fig. 4c). Thus, the ERβ vs. ERα selectivity of ISP358-2 varies greatly based on how well the assay incorporates the natural downstream activation events that occur after binding to the ERβ binding pocket as a result of hormone-induced conformational changes. Thus, the large ERβ vs. ERα selectivity exhibited by ISP358-2 appears not simply to be a function of binding affinity for the ERβ receptor (as measured by the TR-FRET assay in Fig. 2b), but rather a function of its ability to induce productive conformational changes that result in downstream activation of transcription (Fig. 5a).

[0144] Consistent with the above hypothesis that ISP358-2 potency and selectivity are related to its ability to drive productive conformational changes, docking studies show that ISP358-2 docks into the ERβ active site in a conformation that is significantly different from that of the ERα binding site. Important differences occur in where the estrogen C-ring is normally located (Figure 7c and data not shown), thereby affecting the positioning of the aliphatic hydroxyl groups that interact with His524 and / or Gly472 (backbone carbonyl) residues in regions known to be important for driving the helix-12 conformational changes that enable coactivator binding (Figure 7c and data not shown). ERβ hydrophobic interactions involve π-π stacking between Phe356 and the ISP358-2 phenol ring, with Ala302, Leu298, Leu476, and Ile373 constraining the cyclohexyl ring in the "C-ring" region and the hydroxymethyl methyl groups attached to it (Figure 1d). These unique hydrophobic interactions in the ERβ active site may result in a 90° rotation of the C (cyclohexane) ring of ISP358-2 relative to the phenol ring in the ERβ pocket compared to the ERα pocket (Figure 7c and data not shown), and in this way may affect the adjacent coactivator pocket. This 17β-estradiol binding pocket region is known to affect the accessibility and structure of the coactivator binding pocket and may therefore be the reason for the large difference in agonist activity observed for ISP358-2. Future structural characterization studies are planned to address this issue.

[0145] Druggability and preliminary safety toxicity ISP358-2 binds to the ER and activates transcription but does not exhibit significant off-target activity with seven other nuclear hormone receptors (Figure 3a). ISP358-2 also showed no significant activity against the cardiac potassium ion channel hERG (Figure 11b), nor did it show significant inhibition of the major drug metabolizing cytochrome P450 enzymes, CYP2D6, CYP3A4, CYP2C9, and CYP1A2 (Figure 6). ISP358-2 also had considerable solubility and did not show aggregation in turbidimetric assays (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 changes in MCF-7 cell proliferation were observed after treatment with any concentration of the ERβ agonist ISP358-2 or DPN compared to untreated controls (Figure 15b,c). However, there was a significant increase in proliferation of MCF-7 cells after treatment with 1 μM, 0.1 μM, or 0.01 μM ERβ agonist ISP358-2 compared to untreated controls. 2 The proliferation of MCF-7 cells treated with 0.01 μM E was significantly increased (n=3; p<0.02, 0.05, 0.00, respectively) (FIG. 15a). Furthermore, cell proliferation was significantly increased with 0.01 μM E 2 This was significantly less than that in the positive control MCF-7 cells treated with α-lactam (n=3; p≦0.04 for both compounds).

[0147] To evaluate potential peripheral pathology due to ISP358-2 treatment, histological analysis of tissue slices from treated animals was performed (Figure 13). Overall, tissue changes due to treatment did not attract human attention. Mild widespread ischemic changes were noted in the liver of all animals. It is difficult to assign a specific pattern or significance to this finding. These changes likely represent hypoperfusion during the postmortem period followed by mild ischemic changes. One animal showed organized lymphoid hyperplasia in the liver. No animal showed significant pathological changes in the heart or kidneys.

[0148] In vivo efficacy In vivo behavioral assays measuring object placement or object recognition (Fig. 8a) showed efficacy for all three routes of administration: microinfusion into the dorsal hippocampus, intraperitoneal injection, or oral gavage (Fig. 8). Thus, ISP358-2 can improve object recognition and spatial memory consolidation in ovariectomized female mice. Intrahippocampal injection of 100 pg and 1 ng of ISP358-2 improved memory consolidation in object recognition and object placement tasks as effectively as the ERβ agonist DPN (Fig. 8b,c). In systemic administration experiments, 0.5 mg / kg ISP358-2 most effectively improved consolidation in both tasks when delivered intraperitoneally (Fig. 8d,e), whereas 5 mg / kg ISP358-2 was most effective when administered orally (Fig. 8f,g). These data are consistent with previous findings showing that intrahippocampal or systemic administration of the ERβ agonists DPN or WAY200070 improves hippocampal-dependent memory in tasks including object recognition, object placement, and the radial arm maze in ovariectomized rats and mice. 3,34,39-42 Thus, ISP358-2 mimics the memory-enhancing effects of other ERβ agonists with different chemical structures and may potentially be used to reduce memory dysfunction in a large number of neuropsychiatric conditions for which women are at increased risk, including AD, depression, and schizophrenia. 43 Additionally, women are at higher risk for anxiety disorders than men. 43 DPN reduces anxiety-related behaviors in rodents tested in open field and elevated plus maze tasks 44,45 Thus, ISP358-2 has the potential to not only facilitate memory consolidation but also reduce anxiety. Although promising, many challenges remain to be addressed in future studies, including the extent to which the beneficial effects of ISP358-2 generalize to males, older subjects, rodent models of AD and other disorders, and to other forms of memory.

[0149] Finally, although ISP358-2 was observed to be significantly more selective for ERβ than ERα in biologically relevant cell assays, it is unknown whether it has this same selectivity for ERβ in vivo. However, our preliminary studies have shown 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 ascertaining the pharmacological mechanisms of ISP358-2 in vivo, including isoform selectivity, effects on signaling cascades, and brain neuromorphological changes, as well as pharmacokinetic and pharmacodynamic studies.

[0150] conclusion The results of this study demonstrate that our lead compound, ISP358-2, is selective for ERβ and shows no obvious signs of peripheral toxicity. Importantly, ISP358-2 also improves multiple types of memory that are dependent on the hippocampus, a brain region implicated in numerous disorders, including AD, depression, and schizophrenia. 43,46 ISP358-2 differs from previously reported ERβ agonists in its high selectivity for ERβ over ERα and in its close resemblance to the natural 17β-estradiol molecule (Figure 1d) as an AC estrogen. Our study also demonstrated biological efficacy in behavioral assays conducted via three routes of administration: direct dorsal hippocampal injection, intraperitoneal injection, and oral gavage. The last two of these routes illustrate the brain penetration of an effective dose (Figure 8). Overall, these findings suggest that ISP358-2, a novel ERβ agonist, may be a promising drug candidate for improving memory in various disorders characterized by memory dysfunction occurring under low estrogen conditions such as menopause.

[0151] Experimental Section compound synthesis All chemicals were purchased from Sigma-Aldrich, Matrix Scientific, or Alfa Aesar and used as received. Reactions with moisture- or air-sensitive reagents were carried out in oven-dried glassware containing anhydrous solvents under an inert nitrogen atmosphere. After reactions, the reaction mixture was transferred to pre-coated silica plates (60 Å, F 254 TLC was performed on a column chromatography column (Eds., EMD Chemicals Inc.) and visualized by UV lamp (UVGL-25, 254 / 365 nm). Flash column chromatography was performed using flash silica gel (32-63 μ). NMR spectra were recorded on a Varian UnityInova 400 MHz instrument. CDCl 3 , d 6 -Acetone, and CD 3 OD was purchased from Cambridge Isotope Laboratories. 1 H NMR spectra were analyzed using residual CHCl 3 For δ = 7.26 ppm, d 5 - Acetone is calibrated to δ = 2.05 ppm, and residual d 3 -CD 3 The OD was calibrated to δ=3.30 ppm. 13 C NMR spectra were obtained using CDCl 3 For δ = 77.23 ppm, d 6 - acetone δ = 29.92 ppm, CD 3 OD was calibrated from the central peak at δ=49.00 ppm. All compounds were >95% pure as determined by chromatography and NMR.

[0152] TIFF2025081398000034.tif10128trans-4-(4-hydroxycyclohexyl)phenol (2) A solution of 1 (0.200 g, 5.30 mmol) in anhydrous methanol (15 mL) at room temperature was added to solid NaBH 4(0.400 g, 10.6 mmol) was added. 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. TIFF2025081398000035.tif32145

[0153] TIFF2025081398000036.tif111284-(4-Hydroxy-4-methylcyclohexyl)phenol (3) -78°C, N 2 To a solution of 1 (0.100 g, 0.526 mmol) in dry ether (20 mL) was slowly added a solution of methyllithium-lithium bromide complex (1.5 M in ether, 0.78 mL, 1.2 mmol) under reduced pressure. The mixture was stirred at -78 °C for 30 min, warmed to room temperature, and stirred for an additional 1 h. The mixture was cooled to 0 °C and quenched with water. The mixture was extracted several times with ether and the combined extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 , hexane-ethyl acetate=4:1) to give 3 (0.040g, 37%) as a colorless solid. mp 126~131℃; TIFF2025081398000037.tif32159

[0154] TIFF2025081398000038.tif111284-(4-Hydroxyphenyl)cyclohexanone oxime (4) To a solution of 1 (0.050 g, 0.26 mmol) dissolved in ethanol (10 mL) was added Amberlyst (0.060 g) and hydroxylamine hydrochloride (0.039 g, 0.560 mmol). The mixture was stirred at room temperature for 2 h and then filtered. The filtrate was concentrated and extracted several times with ethyl acetate. The combined organic extracts were washed with water and dried (MgSO 4 ), and concentrated to give 4 (0.037 g, 70%) as a colorless solid. mp 171-174°C; TIFF2025081398000039.tif46159

[0155] TIFF2025081398000040.tif101284-(4-t-Butyldimethylsilyloxyphenyl)cyclohexan-1-one (5) 0°C, N 2 Under the conditions below, anhydrous CH 2 Cl 2 To a solution of 1 (0.500 g, 2.62 mmol) in 1H2O (30 mL) was added imidazole (0.357 g, 5.24 mmol). After 30 min, t-butyldimethylsilyl chloride (0.594 g, 3.94 mmol) was added and the mixture was allowed to gradually warm to room temperature overnight. The resulting mixture was diluted with brine (25 mL) and diluted with CH 2 Cl 2 The combined organic extracts were dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 The residue was purified by elution with hexane-ethyl acetate (9:1) to give 5 (0.664, 83%) as a colorless solid. mp 39-42°C. TIFF2025081398000041.tif26159

[0156] TIFF2025081398000042.tif10128t-Butyldimethyl(4-(4-methylenecyclohexyl)phenoxy)silane(6) -10℃, N 2 A solution of methyltriphenylphosphonium bromide (0.836 g, 2.34 mmol) dissolved in dry THF (20 mL) was added with n-butyllithium solution (1.6% dissolved in hexane) under the conditions below. M , 1.50 mL, 2.4 mmol) was slowly added. After 30 min, a solution of 5 (0.502 g, 1.17 mmol) in dry THF (8 mL) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature and stirred overnight. After this time, the mixture was diluted with water (20 mL), extracted several times with ethyl acetate, and the combined extracts were dried (Na 2 SO 4) and concentrated. 2 Purification of the crude residue with hexane-ethyl acetate=9:1) gave 6 (1.678 g, 84%) as a colorless oil. TIFF2025081398000043.tif39153

[0157] TIFF2025081398000044.tif111284-(4-hydroxyphenyl)methylenecyclohexane(7) A solution of 6 (0.739 g, 0.244 mmol) in anhydrous THF (20 mL) was added to a solution of TBAF (1 dissolved in THF). M , 9.8 mL, 9.8 mmol) was added. The mixture was heated at reflux 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 (Na 2 SO 4 ) and concentrated. 2 Purification of the residue with hexane-ethyl acetate (4:1) gave 7 (0.379 g, 83%) as a colorless solid. mp 82-84°C; TIFF2025081398000045.tif39145

[0158] TIFF2025081398000046.tif111284-(4-Methylcyclohexyl)phenol (8) To a solution of 7 (0.150 g, 0.797 mmol) in methanol (10 mL) was added 10% Pd / C (85 mg, 10 mol%). 2 The mixture was stirred at room temperature for 12 h under a balloon filled with . The reaction mixture was filtered through a sheet of Celite and dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 The product was purified by elution with hexane-ethyl acetate (4:1) to give 8 (0.121 g, 80%) as a colorless solid. 1H NMR spectroscopy confirmed it to be a mixture of cis and trans stereoisomers. mp 93-99 °C; TIFF2025081398000047.tif26145

[0159] TIFF2025081398000048.tif161282-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde (9) Dry CH 3 A solution of 7 (0.100 g, 0.531 mmol) in CN (20 mL) was added to MgCl 2 (0.076 g, 0.797), triethylamine (0.28 mL, 2.0 mmol), followed by paraformaldehyde (0.108 g, 3.59 mmol) were added successively. The mixture was heated at 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 (Na 2 SO 4 ) and concentrated. 2 Purification of the residue with hexane-diethyl ether=4:1) gave 9 (0.046 g, 40%) as a colorless oil. TIFF2025081398000049.tif39145

[0160] TIFF2025081398000050.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) was added sodium bicarbonate (0.024 g, 0.278 mmol) and hydroxylamine hydrochloride (0.025 g, 0.348 mmol). The reaction was heated at 80 °C for 5 h and the mixture was extracted several times with ethyl acetate. The combined organic extracts were dried (MgSO 4 ) and concentrated. 2Purification of the residue with hexane-ethyl acetate (13:7) gave 10 (0.037 g, 69%) as a colorless solid. mp 120-125°C; TIFF2025081398000051.tif46145

[0161] TIFF2025081398000052.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) in acetone (6 mL) and distilled water (0.3 mL) was added to a solution of OsO dissolved in tert-butanol (2.5%, 90 μL). 4 A solution of NaHSO was added to the mixture. The mixture was stirred overnight and the reaction was quenched with 3 Saturated aqueous solution (10 mL) was added. The mixture was diluted with ether and washed several times with water. The organic layer was dried (MgSO 4 ), concentrated, and the residue was purified by column chromatography (SiO 2 , hexane-ethyl acetate=1:4) to give 11 (0.267 g, 86%) as a colorless solid. mp 80~86℃; TIFF2025081398000053.tif32153

[0162] TIFF2025081398000054.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 TBAF solution (1 M in THF, 2.8 mL, 2.8 mmol). The mixture was heated at 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 (Na 2 SO 4 ) and concentrated. 2Purification of the residue with ethyl acetate-methanol (9:1) gave 12 (0.118 g, 78%) as a colorless solid. mp 182-188°C; TIFF2025081398000055.tif32153

[0163] TIFF2025081398000056.tif141284-(4-t-butyldimethylsilyloxyphenyl)cyclohexyl)methanol (13 / 14) 0°C, N 2 Borane-THF complex solution (1M in THF, 5.4 mL, 5.4 mmol) was added to a solution of 6 (0.821 g, 2.71 mmol) in THF (24 mL) under reduced pressure. The reaction mixture was slowly warmed to room temperature and stirred for 20 h. The mixture was then cooled to 0° C. before ethanol (50 mL), hydrogen peroxide solution (30%, in water, 4.0 mL), and 1N NaOH solution (20 mL) were added successively. The mixture was allowed to warm 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 (Na 2 SO 4 ) and concentrated. 2 Purification of the residue with hexane-ethyl acetate (7:3) gave a colorless oil (0.572 g, 66%), which was enriched in CH 2 OH group signal 1 H NMR integration confirmed a 2:1 mixture of cis-13 and trans-14. TIFF2025081398000057.tif39153BH 3 Using 9-BBN instead of -THF gave a 2:3 mixture of cis-13:trans-14 (74%).

[0164] TIFF2025081398000058.tif131284-(4-(hydroxymethyl)cyclohexyl)phenol (15 / 16) A solution of 13 / 14 (0.594 g, 1.85 mmol, 2:1 mixture c:t) in dry THF (10 mL) was added to a solution of TBAF (10 mL). M , 7.5 mL, 7.5 mmol) was added. The reaction mixture was heated at reflux at 70° C. overnight and cooled to room temperature. The solution was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 , hexane-ethyl acetate=3:2) to give a colorless solid (0.280 g, 73%), which was purified by elution with CH 2 OH group signal 1 H NMR integration confirmed a 2:1 mixture of cis-13 and trans-14 stereoisomers. mp 118~122 °C. TIFF2025081398000059.tif39145

[0165] TIFF2025081398000060.tif131281-(4-Hydroxyphenyl)-2-oxabicyclo[2.2.2]octane (17) and trans-(hydroxymethyl)cyclohexyl)phenol (16) At -10 °C, anhydrous CH 2 Cl 2 A solution of 15 / 16 (0.080 g, 0.388 mmol, a 2:3 mixture of cis-15:trans-16) in 20 mL of CH 2 Cl 2 A suspension of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.044 g, 0.194 mmol) in 1,2,3-dichloro-5,6-dicyano-1,4-benzoquinone (4 mL) was added slowly over 30 min. The green solution was stirred at 0 °C for 2 h, gradually warmed to room temperature and stirred for an additional 3 h. The mixture was quenched by the slow addition of saturated sodium bicarbonate solution at 0 °C. After 10 min, the layers were separated and the aqueous layer was washed with CH 2 Cl 2 The combined organic extracts were washed with brine, dried (Na 2 SO 4) and concentrated. The residue was purified by column chromatography (SiO 2 The mixture was purified by hexane-ethyl acetate (3:2) to give 17 (0.029 g, 37%) followed by 16 (0.038 g, 47%), both as colorless solids. 1 This was confirmed by 1 H NMR (Figure 16). TIFF2025081398000061.tif64145

[0166] TIFF2025081398000062.tif111284'-(Hydroxymethyl)-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-4-ol (±)-19 Dry CH 3 A solution of 17 (0.103 g, 0.504 mmol) in CN (25 mL) was added to MgCl 2 (0.072 g, 0.756 mmol) was added followed by triethylamine (0.26 mL, 1.89 mmol). The mixture was heated at reflux 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 (Na 2 SO 4 ) and concentrated. 2 Purification of the residue with hexane-ethyl acetate (13:7) gave 19 (0.080 g, 78%) as a colorless solid. mp 177-184°C; TIFF2025081398000063.tif32145

[0167] TIFF2025081398000064.tif111284-(4-((t-butyldiphenylsilyl)oxy)phenyl)cyclohexan-1-one (21) 0 °C, dry CH 2 Cl 2 To a solution of 1 (0.815 g, 4.28 mmol) in 1H2O (30 mL), imidazole (0.583 g, 8.57 mmol) was added, followed by CH 2 Cl 2A solution of t-butyldiphenylsilyl chloride (1.60 mL, 5.57 mmol) in 1,2-dichloro-2,4-tetramethylphenylsilane (9 mL) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature and stirred for 12 h. The mixture was diluted with water and diluted with CH 2 Cl 2 The combined extracts were washed with brine, dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 , hexane-ethyl acetate=4:1) to give 21 (1.70 g, 93%) as a colorless solid. mp 83-84℃; TIFF2025081398000065.tif26153

[0168] TIFF2025081398000066.tif11128 Methyl acetate 2-(4-(4-t-butyldiphenylsilyloxyphenyl)cyclohexylidene) (±)-22 To a solution of trimethyl phosphonoacetate (0.160 mL, 0.980 mmol) in dry THF (5 mL) was added NaH (40 mg, 55% in mineral oil, 0.980 mmol) at 0 °C. After stirring for 45 min, a solution of 21 (0.350 g, 0.816 mmol) in dry THF (5 mL) was added and the mixture was allowed to warm to room temperature and stirred for 8 h. The mixture was diluted with water and extracted several times with ether. The combined extracts were dried (MgSO 4 ) and concentrated. The residue was purified by column chromatography (SiO 2 , hexane-ethyl acetate=9:1) to give 22 (0.376 g, 95%) as a colorless gum. TIFF2025081398000067.tif39153

[0169] TIFF2025081398000068.tif111284-[(4-hydroxyphenyl)cyclohexylidene]acetic acid ethyl ester (±)-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. 1.0 M in THF. 0.247 mmol). After 1 h 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 analyzed by preparative TLC (SiO 2 , hexane-ethyl acetate=9:1) to give 23 (20 mg, 64%) as a colorless solid. mp 92-94℃; TIFF2025081398000069.tif40153

[0170] TIFF2025081398000070.tif111284-(4'-Hydroxyphenyl)(2-hydroxyethylidene)cyclohexane(±)-25 -40°C, N 2 Under dry CH 2 Cl 2 A solution of 22 (275 mg, 0.551 mmol) in 1000 mL of diisobutylaluminum hydride (CH 2 Cl 2 (1.0 M, 1.41 mL, 1.41 mmol) was added. After 90 min, saturated aqueous potassium sodium tartrate was added and the reaction mixture was allowed to warm to room temperature. After 2 h, the layers were separated and the aqueous layer was washed with CH 2 Cl 2 The mixture was extracted several times with 500 ml of tetrabutylammonium fluoride (1.0 mL). The combined organic layers were dried, filtered through a sheet 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 the crude allyl alcohol (235 mg, 0.514 mmol) in dry THF (1 mL) under nitrogen was diluted with tetrabutylammonium fluoride solution (1.0 mL, dissolved in THF) and diluted with tetrabutylammonium fluoride solution (1.0 mL, dissolved in THF). M, 1.03mL, 1.03mmol) was added. 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 (SiO 2 , hexane-ethyl acetate=4:1) to give 25 (90 mg, 80%) as a colorless solid. mp 165-166℃; TIFF2025081398000071.tif33159

[0171] TIFF2025081398000072.tif111284-[4-(2-hydroxyethyl)cyclohexyl]phenol (26) and 4-(4-ethylcyclohexyl)phenol (27) A solution of 25 (50 mg, 0.23 mmol) in methanol (15 mL) containing a small amount of 20% Pd / C was heated with H 2 (30 psi) for 12 h. The reaction mixture was filtered through a sheet of Celite, concentrated, and the residue was purified by preparative TLC (SiO 2 , hexane-ethyl acetate=13:7) to give 27 (28 mg, 60%) followed by 26 (7 mg, 14%) both as colorless solids. TIFF2025081398000073.tif58153

[0172] TR-FRET assay TR-FRET assays were performed using Thermo Fisher Scientific's LanthaScreen® TR-FRET ER Alpha and Beta Competitive Binding Assay kits, which comprised terbium-labeled anti-GST antibodies, a "tracer" fluorescent small molecule ERα or ERβ ligand, and glutathione-S-transferase (GST)-tagged human ERα or ERβ ligand-binding domains (LBDs) in a homogenous mix-and-read assay format.

[0173] The TR-FRET assay uses a Tb-anti-GST antibody that binds to the GST tag. Fluorescently labeled estrogen (tracer) binds in 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's instructions. Briefly, a 1:5 dilution series of compounds was made with DMSO and then diluted in assay buffer such that the highest concentrations tested in the assay were 50 μM for ERβ and 50 μM for ERα, with 1% DMSO. The assay was assembled in a 384-well white, small volume plate (Corning® 4512). The assay was incubated in the dark at room temperature for 1 hour, after which the plate was spun at 1000 rpm in a tabletop centrifuge equipped with a swing-out rotor (Eppendorf 5810, rotor A-4-64). The TR-FRET signal was read on a SpectraMax M5 (Molecular Devices) set according to Thermo Fisher Scientific machine settings (excitation 332 nm, emission 518 nm and 488 nm with 420 nm cutoff, 50 μs integration delay, 400 μs integration time, and 100 flashes / read). The TR-FRET ratio was calculated by dividing the emission at 518 nm (fluorescein) by the emission at 488 nm (terbium) using SoftmaxPro software. Data were analyzed using Eq. 2 Normalized to IC 50 was 0.25 ± 0.06 nM in this assay. Data analysis was performed using Prism (GraphPad Software, Inc., La Jolla, CA) with the fit typically constrained to be 0 at high concentrations of competing ligand. Standard deviations are those of a nonlinear least-squares fit of the data. Replicate assays and fits are shown in Figures 2 and 9. IC 50The fitted IC for the curve from which the median value was obtained 50 are summarized in Table 1).

[0174] Nuclear hormone receptor specificity assay Selectivity was measured using ThermoFisher's SelectScreen™ cell-based nuclear receptor profiling service (Figure 3). This is a FRET-based assay that uses GeneBLAzer™ technology. It detects the binding and activation of a ligand to a nuclear hormone receptor of interest (ligand binding domain; LBD) fused to a GAL4 DNA binding domain (DBD), which induces β-lactamase expression upon activation. The assay has a Z' ≥ 0.5 in agonist mode. Compound stocks were dissolved in DMSO and diluted to obtain assay concentrations of 0.25 μM, 2.5 μM, and 25 μM. Estrogen receptor data was analyzed using E 2 Normalized to IC 50 The IC values ​​for ERα and ERβ were 0.107 nM and 0.579 nM, respectively. Data for other receptors were normalized to appropriate controls and are shown in Table 2. 50 Enumerated with values.

[0175] Table 2. Control compounds and ICs for nuclear hormone specificity assays 50 value TIFF2025081398000074.tif57149

[0176] Replicate assays of ERα and ERβ agonist assays in 10-point curves were also completed (Figure 3b). Again, the data were analyzed using E 2 Normalized to IC 50 was 0.151 nM for ERα and 0.568 nM for ERβ.

[0177] Coactivator assay The LanthaScreen® TR-FRET assay from ThermoFisher was used (Figure 4a). This assay is similar to the previous assay (Figure 3) except that the LanthaScreen® assay has a fluorescently labeled coactivator peptide. This assay measures the recruitment of a labeled coactivator peptide to the ERα or ERβ LBD induced by binding of the ER agonist being assayed. The coactivator peptide is derived from the PPARγ coactivator protein 1a and contains a LXXLL motif (sequence: TIFF2025081398000075.tif4128) is PGC1a. 2 Normalized to IC for ERα and ERβ 50 were 2.58 nM and 2.79 nM, respectively.

[0178] Cellular assays ERα and ERβ cell assays to measure agonist and antagonist activity were performed using kits provided by Indigo Biosciences (Figure 5). The assays relied on luciferase reporter genes downstream of ERα or ERβ responsive promoters that were activated by added agonists or agonist activity was blocked by added antagonists. ER-induced luciferase expression was quantified using chemiluminescence measured using a SpectraMax M5 plate reader. Ligand stock solutions were prepared in DMSO and diluted to final concentrations (typically low nM to μM) using Compound Screening Medium provided in the kit such that the DMSO concentration in the assay was kept below the assay limit of 0.4%. Vehicle controls were included in both agonist and antagonist assays. Assays were performed according to the kit instructions. Briefly, cells directly from the freezer were diluted in Cell Recovery Media (provided) and warmed to 37°C for 5 min. The cell suspension was divided in half. Estradiol, E2 was added to half of the cells for antagonist assays, whereas E 2 The remaining cells without any IgG were used. The cells were plated and the compounds to be screened were added. The plates were placed in an incubator at 37°C and 5% CO 2 The plates were incubated at 4°C for 22 hours. Assays were typically performed in duplicate. After removing the medium and adding detection substrate, luminescence was measured using a SpectraMax M5 plate reader. Data were analyzed using E 2 Agonist activity was normalized to IC 50 was 0.31±0.03 nM for ERα and 0.022±0.005 nM for ERβ. Data were fitted to the following equation using GraphPad Prism: TIFF2025081398000076.tif19128

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

[0180] In vitro druggability assays-CYP450 binding, hERG, and turbidimetry 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 a 96-well white plate (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 in DMSO and then an eight-step 1:2 dilution series was made in DMSO. This was diluted in water so that the assay did not exceed 0.25% DMSO and the highest final concentration of compound was 62.6 μM. After addition of the appropriate cytochrome P450 enzyme, the plate was incubated at 37°C for 10 minutes to allow the components to equilibrate to temperature. An NADPH regenerating system was then added to activate the reaction for the luminogenic P450-Glo™ substrate and incubated for 10-30 min at 37°C according to the kit instructions for each CYP enzyme. The enzyme reaction was stopped by adding luciferin detection reagent and the plates were read for luminescence on a Spectramax M5 (Molecular Devices) after 20 min incubation at room temperature. Data were normalized to positive controls (α-naphthoflavone for CYP1A2, sulfaphenazole for CYP2C9, quinidine for CYP2D6, and ketoconazole for CYP3A4). Data analysis was performed using Prism software as previously described.

[0181] To ascertain the relative tendency of compounds to aggregate in solution, turbidimetric analysis was performed based on the light scattering properties of molecular aggregates (Figure 11a). Compound aggregation in solution is important to measure in screening campaigns, as aggregation is a common source of artifactual activity, and aggregation is a measure of compound solubility. Compounds were tested for aggregation in clear 96-well plates (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). The assay was performed as described 47 , a fluorescence polarization assay that measures the displacement of fluorescently tagged Predictor™.

[0183] MTT assay Human breast cancer cells (MCF-7) were provided by Dr. Manish Patankar (Department of Obstetrics and Gynecology, University of Wisconsin-Madison). 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 for 24 h at 20 °C under 5% CO. 2The cells were cultured at 37°C for 24 h. A seeding density of 7,000 cells per well was selected and applied to a 96-well plate. After 24 h, treatment with ISP358-2, DPN, or estradiol dissolved in medium containing 0.1% dimethyl sulfoxide (DMSO) was 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 received 100% DMSO, 0.01 μM estradiol dissolved in EMEM, or EMEM with 0.1% DMSO content, respectively. Treated cells were incubated for 24 h, after which 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 h. The formazan crystalline metabolite was dissolved with 100% DMSO and absorbance was read at OD570nm as well as at a reference 650nm using a VMax kinetic microplate reader (Molecular Devices, CA) running Softmax Pro version 6.1. Absorbance was converted to cell number using a standard growth curve. Cell proliferation was compared to untreated controls or 0.1 μM E 2 To determine whether there was a significant difference between cells treated with , a two sample equal variance t-test was performed using Microsoft Excel.

[0184] docking Prior to docking, three-dimensional (3D) conformations were prepared for all ligands (Figure 7). AutoDock Tools (ADT) version 1.5.6 was used to prepare ligand files for subsequent AutoDock calculations and to assign Gasteiger charges. For docking calculations, the agonist (pdb code 1ere) 48 and antagonist (pdb code 1err) 49 The conformation of the ERα receptor was prepared. For docking calculations, the agonist (pdb code 2jj3)50 and antagonist (pdb code 1l2j) 51 The ERβ receptor was also prepared for conformation. Hydrogen atoms and partial charges were added to each atom of the protein using ADT. A grid box was placed in the center of the co-crystallized ligand and the box was pulled to incorporate the active site amino acids (Arg394, Glu353, and His524 for ERα and Arg346, Glu305, and His475 for ERβ) and remove the estradiol ligand. 52 Docking calculations were performed using 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 into the ERα structure (pdb code 1ERE) after removal of 17β-estradiol and was found to adopt the same binding mode as the initially bound 17β-estradiol (data not shown).

[0185] Assessment of memory consolidation Subjects. C57BL / 6 female mice (8-10 weeks old) were purchased from Taconic Biosciences. Mice were housed singly in a room with a 12-h light / dark cycle and were provided with food and water ad libitum. All procedures with live mice were performed in a room with a dimmer intensity of >100 lux between 9:00 am and 6:00 pm. All procedures were approved by the University of Wisconsin-Milwaukee Institutional Animal Care and Use Committee and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

[0186] Rough experimental design A series of three experiments was performed in mice that had been bilaterally ovariectomized to remove the primary 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 oral gavage. All drugs were administered immediately and briefly following training in an object recognition task designed to test hippocampal-dependent object recognition as described below, and an object place task designed to test spatial memory consolidation (Figure 8).

[0187] surgery Four days after arrival at the laboratory, as previously described 34,58,59 Mice were then bilaterally ovariectomized. Mice destined to receive dorsal hippocampal injections of ISP358-2 were also cultured as previously described. 30-32 A guide cannula was implanted in the dorsal hippocampus (DH). Mice were anesthetized with isoflurane gas (2% isoflurane in 100% oxygen) and placed in a stereotaxic apparatus (Kopf Instruments). Immediately after ovariectomy, mice were implanted with two guide cannulas (22 gauge; C232G, Plastics One) aimed (-1.7 mm AP, ±1.5 mm ML, -2.3 mm DV) in the dorsal hippocampus. A dummy cannula (C232DC, Plastics One) was placed inside the guide cannula to keep it patent. Dental cement (Darby Dental) was applied to fix the guide cannula to the skull, which also served to close the wound. Mice were allowed to recover for 6 days before behavioral testing.

[0188] Drugs and Infusions Dorsal hippocampal (DH) or intraperitoneal (IP) injections were performed as previously described. 34,58,59, immediately after training. Mice were lightly restrained during the infusions, and drugs were delivered using an infusion cannula (C3131, 28 gauge, extending 0.8 mm beyond the 1.5 mm guide). The infusion cannula was connected to a 10 μl Hamilton syringe using PE20 polyethylene tubing. Infusions were controlled by a microinfusion pump (KDS Legato 180, KD Scientific) at a rate of 0.5 μl / min. After each infusion, we waited 1 min to allow the drug to diffuse back down the cannula track and not through the tissue. The negative control ("vehicle") was 1% DMSO 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 infused at a dose of 10 pg / hemisphere. 30 The affinity of DPN for ERβ is 70-fold higher than that for ERα. 60 , previously reported that bilateral injections of 10 pg / hemisphere into the dorsal hippocampus in ovariectomized young adult mice improved memory consolidation in object recognition and object placement tasks. 34 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 injections, ISP358-2 was dissolved in 10% DMSO in saline and injected at a dose of 0.5 mg / kg or 5 mg / kg in a volume of 10 ml / kg. DPN was dissolved in 10% DMSO in saline and injected at a dose of 0.05 mg / kg in a volume of 10 ml / kg. This dose has previously been shown to improve object recognition memory consolidation in ovariectomized young adult mice. 31Vehicle controls received 10% DMSO in saline at 10 ml / kg. For oral gavage, all drugs were administered in a volume of 10 ml / kg at the same doses as for intraperitoneal injection; 0.5 mg / kg or 5 mg / kg ISP358-2 and 0.05 mg / kg DPN. Vehicle controls received 10% DMSO in saline. In this procedure, drugs were delivered directly to the stomach using a bulb tipped gastric gavage needle (24 GA, 25 mm).

[0190] Memory Test As previously explained 34,58,59 Object recognition and object placement were performed. Object recognition and object placement assess object recognition memory and spatial memory, respectively, and require intact dorsal hippocampal function. 39,61-63First, mice were cared for for 3 days (30 sec / d) to familiarize them with the experiment. On the second day of care, small Lego pieces were placed in the home cage to familiarize the mice with the objects. The Lego pieces were removed from the cage immediately before training. After 3 days of care, mice were habituated to an empty white arena (width, 60 cm; length, 60 cm; height, 47 cm) by allowing them to freely explore for 5 min each day for 2 days. On the training day, mice were allowed to habituate for 2 min in the arena, then removed and placed in the home cage. Two identical objects were then placed near the northwest and northeast corners of the arena. Mice were returned to the arena and allowed to explore until they had accumulated a total of 30 s exploring the objects (or until a total of 20 min had elapsed). Immediately after this training, mice were removed from the arena, injected, and then returned to the home cage. Object placement memory was tested 24 h after training by moving one of the training objects to the southeast or southwest corner of the box. Since mice have an innate preference for novelty, mice that remember the location of the training object will spend more time on the moved object than on the non-moved object. Mice operating by chance will spend the same amount of time (15 s) on each object and will not show memory consolidation. Thus, if the mice spend significantly more time on the moved object than by chance, memory consolidation of the training object is evidenced. Object recognition training was performed 2 weeks after object placement. The object recognition task used the same apparatus and general procedure as object placement, but instead of changing the location of the object, the familiar object was replaced with a novel object during testing. The object recognition test was performed 48 hours after training. As with object placement, mice were allowed 30 s to accumulate exploration of the novel and familiar objects. Since mice are innately attracted to novelty, if the time spent exploring the novel object was greater than by chance, memory of the familiar training object was indicated. To maintain novelty, a variety of objects were used in the object placement task and the object recognition task. Female mice injected with vehicle did not remember the location of the training object 24 hours after training. 34 A 24-h delay was used to test the memory-enhancing effects of drugs on object placement. Similarly, female mice injected with vehicle did not remember the familiar object 48 h after training. 34A 48-h delay was used to test the memory-enhancing effects of drugs on object recognition. For both tasks, the time spent exploring each object and the elapsed time to accumulate 30 s of exploration were recorded using the ANYmaze tracking software (Stoelting).

[0191] Behavioral Data Analysis 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 mice spent significantly more time than chance (15 s) investigating the novel or displaced objects, indicating that each group of mice successfully formed memory for the identity and location of the training objects. To determine the extent to which DPN or ISP358-2 treatment affected memory consolidation compared to vehicle, between-group comparisons were performed for each behavioral task using one-way ANOVA followed by Fisher's LSD post-hoc test. Significance was determined at p>0.05.

[0192] Assessment of potential peripheral pathology To evaluate the possible 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. As in the behavioral tests, ISP358-2 was injected at a dose of 0.5 mg / kg or 5 mg / kg in a volume of 10 ml / kg, and DPN was injected at a dose of 0.05 mg / kg in a volume of 10 ml / kg. Vehicle controls received 10 ml / kg of 10% DMSO in saline (Figure 13a). Tissues were fixed in 10% formalin buffer solution for 24 hours. Twenty specimens were processed and analyzed. Each specimen contained three tissue sections. Tissues from each animal were transferred to labeled cassettes and processed in an automated tissue processor according to standard procedures. Tissues were then embedded in paraffin wax. No specific orientation of the tissues was performed. Four-micron sections were cut from each paraffin block and placed on slides. The slides were then stained with hematoxylin and eosin (H&E) by an automated stainer (Figure 13b). The slides were then examined by a pathologist board-certified in anatomic pathology by the American Board of Pathology (ACM). All specimens contained three tissue samples corresponding to the liver, kidney, and heart. In some cases, parts of adjacent tissues were also present. For example, some specimens had the gallbladder. Some specimens had parts of the spleen. Each organ was examined for specific pathological changes. Three main categories of changes were examined: (1) Structural changes in the organ. For the liver, the central vein, portal triad, and hepatocytes were examined. For the kidney, the glomeruli and tubules were examined. For the heart, the myocytes and coronary vessels were examined. (2) Evidence of inflammation was evaluated, including hepatitis, glomerulonephritis, interstitial nephritis, and myocarditis. (3) Evidence of ischemic changes was examined. Please see the attached table for a summary of the findings.

[0193] References TIFF2025081398000077.tif105146TIFF2025081398000078.tif210146TIFF202 5081398000079.tif204146TIFF2025081398000080.tif199146TIFF20250813980 00081.tif203146TIFF2025081398000082.tif208146TIFF2025081398000083.t if208146TIFF2025081398000084.tif201146TIFF2025081398000085.tif225146

[0194] In the above description, it is readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may be implemented without elements or limitations not specifically disclosed herein, as appropriate. The terms and expressions used are used as terms of description and not as terms of limitation, and in using such terms and expressions, it is not intended to exclude any equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, although the present invention has been illustrated by specific embodiments and optional features, it should 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 present invention.

[0195] A number of patent and non-patent references have been cited herein. The cited references are incorporated herein by reference in their entirety. In the unlikely event that there is a discrepancy in the definition of a term in this specification compared to the definition of a term in a cited reference, the term shall be interpreted in accordance with the definition in this specification.

[0196] Sequence information SEQUENCE LISTING <110> Marquette University Concordia University, Inc. UWM Research Foundation, Inc. <120> SUBSTITUTED (4'-HYDROXYPHENYL)CYCLOALKANE AND (4'-HYDROXYPHENYL)CYCLOALKENE COMPOUNDS AND USES THEREOF AS SELECTIVE AGONISTS OF THE ESTROGEN RECEPTOR BETA ISOFORM FOR ENHANCED MEMORY CONSOLIDATION <150> US 62 / 572,932 <151> 2017-10-16 <150> US 62 / 478,758 <151> 2017-03-30 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys Leu Leu Leu Ala Pro Ala 1 5 10 15 Asn Thr Gln

Claims

1. A compound having the formula and stereochemistry:

2. 13. A pharmaceutical composition comprising an effective amount of the compound of claim 1 or a pharma- ceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.

3. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described in claim 2.

4. 4. The method of claim 3, wherein the disease or disorder is a neurological disease or disorder.

5. 4. The method of claim 3, wherein the disease or disorder is a psychiatric disease or disorder.

6. 4. The method of claim 3, wherein the disease or disorder is cancer.

7. 4. The method of claim 3, wherein the disease or disorder is associated with memory loss or memory dysfunction.

8. 13. A method for improving memory consolidation in a subject in need thereof, comprising administering to said subject the pharmaceutical composition of claim 2.

9. 9. The method of claim 8, wherein the subject is a postmenopausal woman.

10. 11. A method for treating a subject exhibiting low estrogen levels, comprising administering to the subject the pharmaceutical composition of claim 2.

11. 11. The method of claim 10, wherein the subject is a postmenopausal woman.

12. A compound having a formula selected from:

13. 13. A pharmaceutical composition comprising an effective amount of a compound of claim 12 or a pharma- ceutically acceptable salt thereof, together with a pharmaceutical excipient, carrier, or diluent.

14. A method for treating a disease or disorder associated with estrogen receptor β (ERβ) activity in a subject in need of such treatment, comprising administering to the subject a pharmaceutical composition described in claim 13.

15. 15. The method 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.

16. 15. The method of claim 14, wherein the disease or disorder is associated with memory loss or memory dysfunction.

17. 14. A method for improving memory consolidation in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 13.

18. 18. The method of claim 17, wherein the subject is a postmenopausal woman.

19. 14. A method for treating a subject exhibiting low estrogen levels, comprising administering to said subject the pharmaceutical composition of claim 13.

20. 20. The method of claim 19, wherein the subject is a postmenopausal woman.

21. 1. A method for improving memory consolidation in a subject in need thereof, the method comprising administering to a subject a compound of the formula: or a pharmaceutical composition comprising said compound; During the ceremony, (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 an alkylidenyl, carboxyalkylidenyl, esteralkylidenyl, hydroxyalkylidenyl, hydroxyalkylalkylidenyl, aminoalkylidenyl, oxo, or oxime, The method.

22. The compound has formula Ia:

22. The method of claim 21, comprising:

23. In the compound, X is selected from hydrogen, hydroxyl, alkylyl, and hydroxyalkyl, and Y is selected from hydrogen, hydroxyl, alkyl, and hydroxyalkyl, or Y is -OCH 2 - and Y and Z form a bridge.

24. 3. The compound of formula Ia(i):

22. The method of claim 21, comprising:

25. 25. The method of claim 24, wherein in the compound, X is selected from hydrogen, hydroxyl, alkyl, hydroxylalkyl, and Y is hydrogen.

26. In the compound, X is hydrogen or methyl and Y is hydroxymethyl (-CH 2 OH) or hydroxyethyl (-CH 2 CH 2 22. The method of claim 21, wherein said compound is selected from the group consisting of aryl, ... and

27. In the compound, X is methyl and Y is hydroxymethyl (-CH 2 22. The method of claim 21, wherein said compound is selected from the group consisting of aryl, ... and

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