ER-beta estrogen compounds and methods of use

Abstract

Estrogen compounds, combinations, pharmaceutical compositions and methods are presented. Estrogen compounds include ERβ selective estrogen compounds and their combinations. The novel compositions include one or more ERβ selective estrogen compounds and may optionally include one or more excipients. The one or more excipients may be pharmaceutically acceptable. The one or more excipients may include at least one compound that does not naturally occur with the estrogen compound. The method includes administering a pharmaceutical composition comprising the estrogen compound described herein to a patient in need of treatment with an estrogen compound or combination, or a pharmaceutical composition comprising an estrogen compound or combination.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 393,640, filed July 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] TECHNICAL FIELD The present invention relates to estrogenic compounds, compositions, and methods, including compositions, for use in preventing, treating, or reducing the severity and / or frequency of menopausal symptoms. TECHNICAL BACKGROUND

[0003] Estrogens are steroid hormones involved in the regulation of multiple developmental and physiological functions in both males and females. Estrogens are not simply estrus-inducing sex hormones. Estrogens are important for survival and health in both sexes, but their amounts, adaptive responses, tissue-specific distribution, and receptor affinities vary at different stages of life. Estrogens are essential for glucose homeostasis, immune robustness, bone health, cardiovascular health, fertility, and neurological function.

[0004] Estrogens (estradiol (E2) and related steroid hormones) are primarily biosynthesized in the gonads, but also in the adrenal cortex and adipose tissue. Estrogen regulation is mediated by nuclear estrogen receptors (ERs) and membrane estrogen receptors (GPER1), which are encoded by different genes on different chromosomes, suggesting differential functions. However, estrogens are also central to almost all human pathologies, including infectious, autoimmune, metabolic, and degenerative diseases. Both low and high estrogen levels are associated with chronic and acute diseases. While normal aging results in significantly lower levels of estrogen, leading to tissue degeneration (bone, muscle, nerve, etc.) and metabolic imbalance (glucose, lipids, etc.), increased inflammatory agents in daily life enhance levels of estrogen (or estrogen mimics), which is of great pathophysiological importance. The resulting excess estrogen induces modulation of estrogen receptor α and β (ERα and ERβ), damaging tissues and leading to autoimmune diseases and neoplasia.

[0005] Estrogens are involved in growth, development, and tissue differentiation from embryonic life through death. Rapid physiological changes occur during puberty, including rapid growth in bone and muscle mass and maturation of the gonads and brain. Estrogen is a key signaling pathway contributing to all of these processes, particularly sexual differentiation. Hormonal fluctuations and disruptions lead to multiple pathologies at this age, not all of which are directly related to sexual development and function. Precocious puberty, which increases women's cancer risk and reduces girls' quality of life, is on the rise worldwide.

[0006] During menarche, estrogen levels fluctuate significantly throughout the menstrual cycle. Early and delayed menstruation, even with normal cycle variations, significantly increase the risk of aging diseases such as breast cancer and type 2 diabetes. The majority of women (up to 91%) experience painful menstruation, and 14% experience heavy bleeding. Female infertility is increasing worldwide, and this is not simply due to an increasing age at first pregnancy. Up to 21% of couples experience infertility, 80% of which are due to female disorders. Despite improved fertility technologies such as in vitro fertilization, the success rate of these techniques is very low (approximately 9% for IVF). Up to 46% of women experience spontaneous abortion (miscarriage). There is an increasing trend towards gestational diabetes, preterm labor, preeclampsia and gestational anemia, all of which are related to estrogen function.

[0007] In women of reproductive age, estrogen is primarily synthesized in the granulosa cells of ovarian follicles. As women enter menopause, the follicles in their ovaries are depleted by atresia during each menstrual cycle, and the amount of estrogen produced by the ovaries declines. As estrogen levels decline and begin to fluctuate, short-term symptoms such as hot flashes, night sweats, and mood changes frequently occur. The ovaries eventually stop producing estrogen, and periods of estrogen deficiency increase, accelerating the risk of chronic diseases such as osteoporosis, cardiovascular disease, obesity, type 2 diabetes, and urogenital atrophy.

[0008] Approximately 70% of all autoimmune diseases are diagnosed in women, including rheumatoid arthritis, lupus, Sjogren's syndrome, scleroderma, Hashimoto's thyroiditis, and multiple sclerosis, among others. As women age, the incidence of breast and ovarian cancer increases. Additionally, approximately 70% of cases of Alzheimer's disease and early onset dementia are diagnosed in women.

[0009] Estrogen pharmaceutical interventions, such as menopausal hormone therapy (MHT), are the most common interventions for all female-related disorders. Natural estrogens and synthetic derivatives are used in these prescriptions. The mechanism of action of these estrogens is universal agonism, resulting in gene activation and repression. Several synthetic steroidal and nonsteroidal estrogen receptor antagonists are primarily used in the treatment of breast cancer. Unlike natural hormones, despite the pharmacological intent of achieving estrogen receptor antagonism, which results in gene regulation and pathophysiological outcomes, most steroidal and nonsteroidal drugs result in mixed agonist / antagonist outcomes, which produce outcomes opposite to those of estrogen in some tissues but similar to those in others. These are selective estrogen receptor modulators (SERMs) that are currently used to treat breast cancer, osteoporosis, and, in combination with estrogen, for menopausal symptoms.

[0010] Historically, progestogens have been co-prescribed with estrogens to counter the increased risk of uterine cancer associated with menopausal hormone therapy. Progestogens exert their pharmacological actions via the progesterone receptor.

[0011] In the past few decades, attempts have been made to develop receptor subtype agonist and antagonist drugs since the discovery of ERβ in 1995. None of these drugs have been approved by regulatory authorities.

[0012] Because long-term administration of menopausal hormone therapy has been shown to increase the risk of estrogen-related adverse outcomes, such as breast cancer, stroke, venous thromboembolism (VTE), and Alzheimer's disease, MHT is only recommended for 5 years to treat menopausal and perimenopausal vasomotor symptoms and vulvovaginal atrophy. Nevertheless, in addition to treating menopausal vasomotor symptoms, MHT treatment also reduces the risk of osteoporosis and type 2 diabetes mellitus (T2DM), cardiovascular disease, obesity, and urogenital atrophy. Therefore, there is a need for treatment options that offer one or more of the benefits of traditional MHT (E2, alone or in combination with progestogens or selective estrogen receptor modulators (SERMs)) with a reduced risk of adverse outcomes, such as increased cancer risk, associated with traditional MHT.

[0013] There is a need for additional novel therapeutic compounds, compositions, and methods for treating menopausal and perimenopause symptoms. The various embodiments disclosed herein address these needs and also provide related advantages. Summary of the Invention

[0014] The compounds described herein can be used alone or in combination with each other or other estrogens to modify the tissues that express ERβ, and thus can be used for multiple indications.The activation of ERα by estradiol leads to a dose-dependent increase in the proliferation of breast cancer cells and uterine cancer cells, as well as uterine hyperplasia and a dose-dependent increase in body weight.Unlike the activity of estradiol on ERα, estradiol inhibits the growth of breast cancer cells and uterine cancer cells that express ERβ in a dose-dependent manner and without uterine hyperplasia or an increase in uterine weight, suggesting that the use of ERβ-specific ligands can enhance estrogen activity and reduce the risk of breast cancer and uterine cancer.

[0015] Thus, provided herein are compounds and pharmaceutically acceptable salts thereof, wherein the compounds are ERβ-selective estrogen compounds. In some embodiments, each ERβ-selective estrogen compound is a member of the group consisting of liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4'-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, analog 13, genistein, or equol, or a pharmaceutically acceptable salt thereof. In some embodiments, the ERβ-selective compound is not an agonist, antagonist, or mixed agonist / antagonist of estrogen receptor α. Also provided are combinations comprising two or more ERβ-selective compounds of the same group, or pharmaceutically acceptable salts thereof. Also provided are pharmaceutical compositions comprising (1) one or more pharmaceutically acceptable ingredients other than an ERβ-selective compound, and (2) one or more ERβ-selective compounds. In some embodiments, the at least one pharmaceutically acceptable ingredient other than an ERβ-selective compound is estradiol or another ERα-modulating compound. In some embodiments, the at least one pharmaceutically acceptable ingredient other than an ERβ-selective compound does not naturally occur with the ERβ-selective estrogen compound in nature. In some embodiments, the pharmaceutical composition comprises a member of the group consisting of estradiol (E2), one or more estrogen receptor agonists, one or more estrogen receptor antagonists, one or more mixed estrogen receptor agonists / antagonists, one or more selective estrogen receptor modulators (SERMs), one or more progestogens, one or more glucocorticoids, and one or more androgens.In some embodiments, the compound, combination, or pharmaceutical composition is intended for use in treating a disease or disease state in a patient in need of treatment with a nuclear receptor reprogramming compound, hi some embodiments, the disease or disease state is a developmental disease, a menstrual disorder, a fertility disorder, a gynecological disorder, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0016] Some embodiments described herein include the use of a compound, combination, or pharmaceutical composition described in the immediately preceding paragraph for the manufacture of a medicament for use in treating a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the disease or disorder is a developmental disorder, a menstrual disorder, a fertility disorder, a gynecological disorder, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0017] Some embodiments described herein provide methods of treating a patient in need of such treatment, comprising administering to the patient an effective amount of a compound described in either of the two immediately preceding paragraphs, or a pharmaceutically acceptable salt, combination, or pharmaceutical composition thereof. In some embodiments, the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0018] Other uses and advantages of the various embodiments described herein will be apparent to those of ordinary skill in the art upon review of the following disclosure. [Brief explanation of the drawings]

[0019] The features and advantages of the present invention will become apparent from consideration of the following detailed description, presented in conjunction with the accompanying drawings. [Figure 1] Figure 1 shows the luciferase activity of several compounds of the present invention in U2OS cells transfected with NKG2E TK-Luc and ERβ. [Figure 2] 1 shows the dose response of several compounds of the invention in U2OS cells transfected with NKG2E TK-Luc and ERβ. [Figure 3]The structures of some compounds of the invention are shown in Figure 3. The basic flavone structure, with the ring positions numbered, is in the upper left corner of Figure 3. [Figure 4] The structures of some compounds of the invention are shown in Figure 4. The basic flavone structure, with ring positions numbered, is in the upper left corner of Figure 4. [Figure 5] 1 shows the luciferase activity dose response of ligands to activation of NKG2E-TK-Luc in U2OS-ERβ cells. [Figure 6] Compounds of the present invention are shown. Ligands enclosed in boxes (liquiritigenin, apigenin, Nyasol, naringenin, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, analog 13, and genistein) activate ERβ at 200 nM. The basic flavone structure with ring positions numbered is in the upper left corner of Figure 6. [Figure 7] Compounds of the present invention are shown. Ligands enclosed in boxes (liquiritigenin, apigenin, Nyasol, naringenin, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, analog 13, and genistein) activate ERβ at 200 nM. The basic flavone structure with ring positions numbered is in the upper left corner of Figure 7. [Figure 8] 1 shows the ligand dose response for NKG2E ERE activation in U2OS-ERα cells. [Figure 9]Compounds of the present invention are shown. Ligands that activate ERα at 1 μM (apigenin, Nyasol, Broussonin A, 6,4'-dihydroxyflavone, genistein, and equol) are double-boxed, and ligands that activate ERα at 5 μM (liquiritigenin, 7,4'-dihydroxyflavone, and analog 13) are single-boxed. The basic flavone structure with ring positions numbered is in the upper left corner of Figure 9. [Figure 10] Compounds of the present invention are shown. Ligands that activate ERα at 1 μM (apigenin, Nyasol, Broussonin A, 6,4'-dihydroxyflavone, genistein, and equol) are double-boxed, and ligands that activate ERα at 5 μM (liquiritigenin, 7,4'-dihydroxyflavone, and analog 13) are single-boxed. The basic flavone structure with ring positions numbered is in the upper left corner of Figure 10. [Figure 11] 1 shows keratin 19 mRNA expression in U2OS-ERβ cells by qRT-PCR. [Figure 12A] Luciferase activity of U2OS-ERβ cells transfected with GRE-TK-Luc and GR in the presence of doxycycline is shown. [Figure 12B] Luciferase activity of U2OS WT cells transfected with GRE-TK-Luc and ERβ in the presence of GR is shown. [Figure 13A] 1 shows GILZ expression in the presence of liquiritigenin and / or doxycycline in GR-expressing U2OS-ERβ cells. [Figure 13B] Figure 1 shows rhazinin expression in the presence of liquiritigenin and / or doxycycline in GR-expressing U2OS-ERβ cells. [Figure 14] Luciferase activity in U2OS-ERβ cells expressing TAT3-TK-Luc and hPRβ in the presence of ERβ ligands and combinations is shown. [Figure 15] 1 shows the efficacy of ERβ ligands in MF101. [Figure 16A] Histological results for the ERβ ligands IATERB5, IATERB6 (FIG. 16A), and IATERB7 (FIG. 16B) compared to E2 are shown. Unlike estradiol, three of the ERβ compounds do not increase uterine breast cancer tumorigenesis. [Figure 16B] Histological results for the ERβ ligands IATERB5, IATERB6 (FIG. 16A), and IATERB7 (FIG. 16B) compared to E2 are shown. Unlike estradiol, three of the ERβ compounds do not increase uterine breast cancer tumorigenesis. [Figure 17] Dose-response luciferase activity of ERβ ligands alone and in combination with hydrocortisone (HC) is shown in U2OS-ERβ cells transfected with GRE-TK-Luc, GR, and doxycycline. DETAILED DESCRIPTION OF THE INVENTION

[0020] Liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, genistein, and equol can be obtained from commercial sources, such as Sigma-Aldrich. Analog 13 can be obtained by the synthetic method described in the Examples below.

[0021] The compounds of the present invention are summarized in Table 1.

[0022] [Table 1]

[0023] Pharmaceutical Composition

[0024] The pharmaceutical compositions described herein include one or more compounds, e.g., two or more compounds, three or more compounds, four or more compounds, five or more compounds, six or more compounds, or seven or more compounds, selected from the group consisting of liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4'-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, Analog 13, genistein, or equol, or a pharmaceutically acceptable salt of any of the foregoing. At least one of the pharmaceutically acceptable components may include one or more ingredients that do not naturally occur with the ERβ-selective estrogen compounds, salts, or solvates thereof disclosed herein. Pharmaceutically acceptable ingredients that do not naturally occur with the novel compounds disclosed herein, their salts or solvates, may include excipients that are sterile, isotonic, or pyrogen-free in nature.

[0025] A "pharmaceutically acceptable" ingredient is one that is compatible with the estrogen compounds and other ingredients of the compositions described herein and is suitable for administration to a patient. Additional ingredients may include carriers, diluents, absorption enhancers, stabilizers, preservatives, or other active or inactive ingredients. At least one of the additional ingredients may be an ingredient that does not naturally occur in nature with the ERβ compounds or combinations of ERβ compounds described herein. At least one of the additional ingredients may be an ingredient other than water. In some embodiments, the pharmaceutical composition may be sterile, pyrogen-free, and / or isotonic. In some embodiments, the pharmaceutical composition is sterile or pyrogen-free. In some embodiments, the pharmaceutical composition is sterile and pyrogen-free. In some preferred embodiments, the pharmaceutical composition is sterile, pyrogen-free, and isotonic.

[0026] In some embodiments, the pharmaceutical composition may be an estrogenic composition. The estrogenic composition comprises an estrogenically effective amount of an ERβ-selective compound or a pharmaceutically acceptable salt thereof, or a combination of ERβ-selective compounds described herein, and an additional ingredient. The additional ingredient may be an excipient. The excipient may include at least one compound that does not naturally occur with the estrogenic compound in nature. In particular, the excipient may include at least one compound that does not naturally occur in humans with the ERβ compound or combination of ERβ compounds described herein. In some embodiments, the excipient may include at least one compound other than water. In some embodiments, the additional compound may be a salt or other ingredient at a concentration sufficient to make the composition isotonic. In some embodiments, the additional ingredient may be a flavoring or sweetener not found in nature with the ERβ compound or combination of ERβ compounds described herein. In some embodiments, the estrogenic composition may be sterile, pyrogen-free, and / or isotonic.

[0027] Pharmaceutically acceptable salts can be any salt of the estrogen compound disclosed herein that has suitable solubility in aqueous solvents at suitable pH. Remington's, 20th Ed., published 2000, pp. 704-719, provides a method for determining suitable pharmaceutically acceptable salts. For example, suitable salts can be selected from Table 38-2, p. 704, of Remington's. Pharmaceutically acceptable salts can be prepared by dissolving the estrogen compound in a suitable solvent, adding a suitable acid or base, or optionally a suitable counter-acid or counter-base, to the solution, and separating the salt form of the estrogen compound from the solution.

[0028] Pharmaceutical compositions, particularly estrogen compositions, can be formulated for various routes of administration, such as oral, intranasal, intrapulmonary (e.g., for inhalation), intravenous, subcutaneous, transdermal, sublingual, buccal, intraperitoneal, or intrathecal administration. Pharmaceutical compositions can include one or more enhancers that aid in the transport of an ERβ compound or combination of ERβ compounds described herein across one or more external or internal physiological barriers, such as the pulmonary epithelial barrier or the blood-brain barrier.

[0029] Suitable pharmaceutically acceptable excipients may include the following types of excipients: diluents, lubricants, binders, disintegrants, fillers, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, flavor masking agents, coloring agents, anti-caking agents, wetting agents, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers.

[0030] Estrogenically Effective Dose

[0031] The effective dose of an ERβ-selective compound, or a pharmaceutically acceptable salt thereof, or a combination of ERβ-selective compounds or pharmaceutically acceptable salts thereof, can vary depending on various factors, including the route of administration, the age and disease of the patient requiring estrogen treatment, etc. Generally, the ERβ-selective compounds, or a pharmaceutically acceptable salt thereof, or a combination of ERβ-selective compounds or pharmaceutically acceptable salts described herein are effective in vitro at nanomolar or micromolar concentrations. An effective daily dose of an ERβ-selective compound, or a pharmaceutically acceptable salt thereof, or a combination of ERβ-selective compounds or pharmaceutically acceptable salts described herein can range from 0.01 mg to 1000 mg per day. An effective daily dose can be divided into two or more divided doses, e.g., 1, 2, 3, 4, 5, 6, or more divided doses. When the pharmaceutical composition is administered as an infusion, the effective daily dose can be administered as a continuous infusion over several hours, e.g., 1 to 24 hours. An effective dose may be similar to the dose of an ERβ-selective compound, or a pharmaceutically acceptable salt thereof, or a combination of ERβ-selective compounds, or pharmaceutically acceptable salts thereof, described herein, but may be scaled to account for the relative biological activity, pharmacokinetics, and pharmacodynamics of the compound, as compared to the ERβ-selective compound or combination of ERβ-selective compounds described herein, and one of ordinary skill in the art would know how to determine this by art-recognized methods.

[0032] Those skilled in the art of pharmaceutical formulation and compounding have the knowledge and skill to select appropriate pharmaceutically acceptable carriers and excipients in appropriate amounts for use with the ERβ-compounds or ERβ-compound combinations described herein. In addition, there are several resources available to those skilled in the art that describe pharmaceutically acceptable carriers and excipients and may be useful in selecting appropriate pharmaceutically acceptable carriers and excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (The American Pharmaceutical Association and The Pharmaceutical Press).

[0033] The compositions of the estrogen compounds described herein can be prepared using techniques and methods known to those skilled in the art. Some methods commonly used in the art are described in Remington's Pharmaceutical Sciences, 20th Ed., (Mack Publishing Company (2000)).

[0034] In some embodiments, the estrogen composition may comprise an ERβ compound or a combination of ERβ compounds described herein and one or more pharmaceutically acceptable carriers or excipients. The composition may be prepared and packaged in bulk form, from which an effective amount of the disclosed compound may be extracted and then administered to a subject, such as in a powder or syrup. Alternatively, the composition may be prepared and packaged in a unit dosage form, with each physically separate unit containing an effective amount of an ERβ compound or a combination of ERβ compounds described herein.

[0035] The ERβ compound or combination of ERβ compounds described herein and a pharmaceutically acceptable carrier or excipient can be formulated into a dosage form adapted for administration to a subject via a desired route of administration. For example, dosage forms include (1) oral administration forms such as tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets, and (2) parenteral administration forms such as sterile solutions, suspensions, and powders for reconstitution. The appropriate pharmaceutically acceptable carrier or excipient can vary depending on the specific dosage form selected. Furthermore, the appropriate pharmaceutically acceptable carrier or excipient can be selected for the specific function they may serve in the composition. For example, certain pharmaceutically acceptable carriers or excipients can be selected for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable carriers or excipients can be selected for their ability to promote the production of stable dosage forms. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to facilitate the transport or transport of the compounds disclosed herein from one organ or part of the body to another organ or part of the body when administered to a subject. Certain pharmaceutically acceptable carriers or excipients may be chosen for their ability to enhance patient compliance.

[0036] In some embodiments, the estrogen ERβ compound or combination of ERβ compounds described herein can be formulated for parenteral administration. Compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The compositions 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 prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Parenteral formulations may be sterile, pyrogen-free, or both. Parenteral formulations may be isotonic.

[0037] Oral

[0038] The estrogen composition may be an oral estrogen composition comprising an ERβ selective compound or a pharmaceutically acceptable salt thereof, or a combination of an ERβ selective compound or a pharmaceutically acceptable salt thereof described herein, and at least one excipient suitable for oral administration.The at least one excipient suitable for oral administration may comprise a compound that does not naturally occur in nature with the ERβ compound or combination of ERβ compounds described herein.The at least one excipient suitable for oral administration may comprise at least one compound other than water.Various dosage forms may be prepared, such as tablets, capsules, caplets, lozenges, powders, emulsions, sachets, cachets, gel capsules, elixirs, pills, oral sprays, chewable tablets, sublingual tablets, films or sprays, or buccal films or sprays.

[0039] In some embodiments, the ERβ compound or combination of ERβ compounds described herein can be formulated into a solid oral dosage form, such as a tablet or capsule, containing an effective amount of a compound of the present disclosure and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pregelatinized starch), cellulose and its derivatives (e.g., microcrystalline cellulose), calcium sulfate, and calcium hydrogen phosphate. The oral solid dosage form can further include a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The oral solid dosage form can further include a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmellose, alginic acid, and sodium carboxymethylcellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0040] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The compositions can also be prepared so as to prolong or sustain release, for example, by coating or embedding particulate material in polymers, wax, or the like.

[0041] The ERβ compounds or combinations of ERβ compounds described herein can also be combined with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartame-idephenol, or polyethylene-oxide polylysine substituted with palmitoyl residues. Furthermore, the ERβ compounds or combinations of ERβ compounds described herein can be combined with biodegradable polymer classes useful for achieving controlled release of drugs, such as polylactic acid, porphyrin caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0042] In some embodiments, the ERβ compound or combination of ERβ compounds described herein can be formulated into a liquid oral dosage form. Oral liquids such as solutions, syrups, and elixirs can be prepared in unit dosage forms, with each containing a predetermined amount of the compounds disclosed herein. Syrups can be prepared by dissolving the compounds disclosed herein in a suitable flavored aqueous solution, and elixirs can be prepared by using a non-toxic alcoholic vehicle. Suspensions can be prepared by dispersing the compounds disclosed herein in a non-toxic vehicle. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, preservatives, flavor additives, such as peppermint oil or other natural sweeteners or saccharin or other artificial sweeteners, can be added.

[0043] intranasal

[0044] The estrogen composition may be an intranasal estrogen composition comprising an ERβ compound or combination of ERβ compounds described herein and at least one excipient suitable for intranasal administration. The at least one excipient suitable for intranasal administration may comprise at least one compound other than water. For example, the intranasal estrogen composition may comprise one or more penetration enhancers that increase absorption and / or bioavailability of the ERβ compound or combination of ERβ compounds described herein across mucous membranes. In some embodiments, penetration enhancers may include mucolytic agents, degradative enzyme inhibitors, and compounds that increase the permeability of mucosal cell membranes. Whether a given compound is an "enhancer" can be determined in vivo or in a good model test by comparing two formulations containing a non-associated, small polar molecule as the drug with and without the enhancer and determining whether drug uptake is enhanced to a clinically significant extent. In vivo enhancers should be non-irritating and / or be rapidly metabolized into normal cellular components that do not have significant irritating effects, so enhancers will not cause problems with chronic toxicity. In some embodiments, the penetration enhancer can be an alkylglycoside, such as the alkylglycoside disclosed in U.S. Patent No. 5,661,130, which is incorporated herein by reference in its entirety. Those skilled in the art will recognize the need to achieve an appropriate hydrophilic-lipophilic balance (HLB) number, which can be determined as disclosed in U.S. Patent Application Publication No. US2009 / 0047347, which is incorporated herein by reference in its entirety.

[0045] The intranasal estrogen compositions of the ERβ compounds or combinations of ERβ compounds described herein may also include a flavoring or fragrance to mask the taste of the ERβ compounds or combinations of ERβ compounds described herein.The intranasal compositions may also include a tonicity agent to make the composition isotonic.The intranasal estrogen compositions of the ERβ compounds or combinations of ERβ compounds described herein may also include a stabilizer.

[0046] Intrapulmonary

[0047] The estrogen composition can be an intrapulmonary estrogen composition, comprising an ERβ compound or a combination of ERβ compounds described herein and at least one excipient suitable for intranasal administration.The at least one excipient suitable for intrapulmonary administration can comprise at least one compound other than water.For example, the intrapulmonary composition can comprise one or more penetration enhancers that increase the ability of the ERβ compound or ERβ compound described herein to pass through the pulmonary epithelium and enter the bloodstream.

[0048] Pulmonary estrogen compositions can be administered to the lungs by inhalation, for example, using an inhaler, an aerosol inhaler, or a conventional or high efficiency nebulizer.

[0049] High-efficiency nebulizers are inhalation devices that contain a microperforated membrane through which a liquid solution is converted by electrical or mechanical means into aerosol droplets suitable for inhalation. High-efficiency nebulizers can deliver a large percentage of the loaded dose to the patient. In some embodiments, high-efficiency nebulizers can also utilize one or more actively or passively vibrating microperforated membranes. In some embodiments, high-efficiency nebulizers can include one or more vibrating membranes. In some embodiments, high-efficiency nebulizers can include a vibrating mesh or plate with multiple openings and, optionally, a vibration generator with an aerosol mixing chamber. In some such embodiments, the mixing chamber can function to collect (or stage) the aerosol from the aerosol generator.

[0050] In some embodiments, a high-efficiency nebulizer can achieve at least about 10% lung deposition (deposited lung dose) based on a nominal dose of an ERβ compound or combination of ERβ compounds described herein.

[0051] In some embodiments, the high-efficiency nebulizer provides a pulmonary deposition (pulmonary deposited dose) of at least about 5% of the ERβ compound or combination of ERβ compounds described herein, based on the nominal dose of the ERβ compound or combination of ERβ compounds described herein.

[0052] According to the present invention, in some embodiments, a nebulizer, such as a high-efficiency nebulizer, can be adapted or compatible to operate in conjunction with a unit dosage form, such as an ampoule or vial containing a single dose of a compound described herein for an ERβ compound or combination of ERβ compounds for estrogen therapy. The unit dosage form includes a container containing an inhalation solution containing an ERβ compound or combination of ERβ compounds described herein. The container is adapted to work with a high-efficiency nebulizer device so that a nominal dose of the inhalation solution can be administered to a patient in need thereof. In some embodiments, the high-efficiency nebulizer and the unit dosage form are configured so that they can be used together but cannot be used with other devices or dosage forms. In some specific embodiments, the unit dosage form is configured so that it fits into a keyhole-like structure in the high-efficiency nebulizer but does not operate with other nebulizer devices. In such embodiments, the high-efficiency nebulizer is configured to receive and operate appropriately with a unit dosage form containing an ERβ compound or combination of ERβ compounds described herein, but not with other dosage forms.

[0053] Suitable high-efficiency nebulizers with perforated membranes are disclosed in U.S. Patent Nos. 6,962,151, 5,152,456, 5,261,601, and 5,518,179, each of which is incorporated herein by reference in its entirety. Suitable high-efficiency nebulizers include vibratable membranes. Features of these high-efficiency nebulizers are disclosed in U.S. Patent Nos. 7,252,085, 7,059,320, and 6,983,747, each of which is incorporated herein by reference in its entirety.

[0054] Commercially available, highly efficient nebulizers are available from PARI GmbH (Germany) under the trade name eFlow®, Aerogen, Ltd. (Ireland) under the trade names AeroNeb® Go and AeroNeb® Pro, AeroNeb® Solo, and other nebulizers utilizing OnQ® nebulizer technology, Respironics (Marysville, California) under the trade name I-Neb®, Omron (Bannockburn, Illinois) under the trade name Micro-Air®, Activaero (Germany) under the trade name Akita®, and AerovectRx (Atlanta, Georgia) under the trade name AerovectRx®.

[0055] Conventional nebulizers include, for example, jet nebulizers or ultrasonic nebulizers. Jet nebulizers generally utilize a compressor to generate compressed air, which breaks down a liquid medication into small, respirable droplets and forms an aerosolized (atomized) mist. In some of these embodiments, when the patient inhales, a valve at the top opens, allowing air to enter the device, thereby accelerating mist generation; when the patient exhales, the valve at the top closes, thereby slowing mist generation and simultaneously allowing the patient to exhale through an opening in a mouthpiece flap.

[0056] Some conventional nebulizers are disclosed in U.S. Patent Nos. 6,513,727, 6,513,519, 6,176,237, 6,085,741, 6,000,394, 5,957,389, 5,740,966, 5,549,102, 5,461,695, 5,458,136, 5,312,046, 5,309,900, 5,280,784, and 4,496,086, each of which is incorporated herein by reference in its entirety.

[0057] Commercially available conventional nebulizers include those manufactured by PARI (Germany) under the trade names PARI LC Plus®, LC Star®, and PARI-Jet®, A&H Products, Inc. (Tulsa, Oklahoma) under the trade name AquaTower®, Hudson RCI (Temecula, California) under the trade name AVA-NEB®, Intersurgical, Inc. (Liverpool, New York) under the trade name Cirrus®, Salter Labs (Arvin, California) under the trade name Salter 8900®, Respironics (Marysville, Pennsylvania) under the trade name Sidestream®, Bunnell (Salt Lake City, Utah) under the trade name Whisper Jet®, and Smiths-Medical (Hythe, United Kingdom) under the trade name Downdraft®. and DeVilbiss (Somerset, PA) under the trade name DeVilbiss®.

[0058] Intravenous

[0059] The estrogen composition may be an intravenous estrogen composition comprising an ERβ compound or a combination of ERβ compounds described herein and at least one excipient suitable for intravenous administration. The at least one excipient suitable for intravenous administration may include at least one compound other than water. The intravenous composition of an ERβ compound or a combination of ERβ compounds described herein is a parenteral composition intended for intravenous administration by injection or infusion. They may contain one or more tonicity adjusting agents to make the composition isotonic. They may be sterile, pyrogen-free, or both, and are generally sterile, pyrogen-free, or both.

[0060] subcutaneous

[0061] The estrogen composition may be a subcutaneous estrogen composition comprising an ERβ compound or a combination of ERβ compounds described herein and at least one excipient suitable for subcutaneous administration. The at least one excipient suitable for subcutaneous administration may include at least one compound other than water. The subcutaneous compositions of the ERβ compound or a combination of ERβ compounds described herein are parenteral compositions intended for subcutaneous injection. They may contain one or more tonicity adjusting agents to make the composition isotonic. They may be sterile, pyrogen-free, or both, and are generally sterile, pyrogen-free, or both.

[0062] percutaneous

[0063] The estrogen composition may be a transdermal estrogen composition comprising an ERβ compound or a combination of ERβ compounds described herein and at least one excipient suitable for transdermal administration. The at least one excipient suitable for transdermal administration may comprise at least one compound other than water. For example, the transdermal estrogen composition may comprise one or more penetration enhancers that increase the ability of the ERβ compound or a combination of ERβ compounds described herein to pass through the dermis and enter the bloodstream. In addition, the transdermal composition may be delivered by a biasing mechanism, such as an iontophoresis device.

[0064] Sublingual or buccal

[0065] The estrogen composition can be a sublingual or buccal estrogen composition comprising an ERβ compound or combination of ERβ compounds described herein and at least one excipient suitable for sublingual or buccal administration. The at least one excipient suitable for sublingual or buccal administration can include at least one compound other than water.

[0066] intraperitoneal

[0067] The estrogen composition may be an intraperitoneal estrogen composition comprising an ERβ compound or a combination of ERβ compounds described herein and at least one excipient suitable for intraperitoneal administration.The at least one excipient suitable for intraperitoneal administration may comprise at least one compound other than water.The intraperitoneal estrogen composition of an ERβ compound or a combination of ERβ compounds described herein is a parenteral composition intended for peritoneal administration by injection or infusion.They may contain one or more tonicity adjusting agents to make the composition isotonic.They may be sterile, pyrogen-free, or both, and are generally sterile, pyrogen-free, or both.

[0068] Intrathecal or intracranial

[0069] The estrogen composition may be an intrathecal or intracranial estrogen composition comprising an ERβ compound or an ERβ compound described herein and at least one excipient suitable for intrathecal or intracranial administration.The at least one excipient suitable for intrathecal or intracranial administration may comprise at least one compound other than the compound naturally present in nature with the ERβ compound or combination of ERβ compounds described herein, such as water.The intrathecal or intracranial estrogen composition of the ERβ compound or combination of ERβ compounds described herein is a parenteral composition intended for administration into cerebrospinal fluid administration by injection or infusion.They may contain one or more tonicity agents to make the composition isotonic.They may be sterile, pyrogen-free, or both, and are generally sterile, pyrogen-free, or both.

[0070] Other routes of administration

[0071] While preferred embodiments of the compositions and methods described herein have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made therein that do not exceed the scope of the appended claims. Accordingly, the scope of the compositions and methods described is limited only by the following claims.

[0072] Estrogen Method

[0073] Some embodiments described herein provide a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, for use in treating a disease or disease state in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the disease or disease state is a developmental disease, a menstrual disorder, a fertility disorder, a gynecological disorder, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0074] Some embodiments described herein provide for the use of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, for the manufacture of a medicament for use in treating a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the disease or disorder is a developmental disorder, a menstrual disorder, a fertility disorder, a gynecological disorder, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0075] Some embodiments described herein provide methods of treating a patient in need of such treatment, the method comprising administering to the patient an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein. In some embodiments, the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer. In some embodiments, the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

[0076] In light of the disclosure herein, those skilled in the art will understand how to determine the estrogenically effective amount of the ERβ compound or combination of ERβ compounds described herein. Generally, the estrogenically effective amount, therapeutically effective amount, or prophylactically effective amount of the ERβ compound or combination of ERβ compounds described herein can be determined, for example, by inference from in vitro tests. Those skilled in the art will understand that the effective dose can be estimated from the half-maximal modulating (inhibitory or activating) concentration of the ERβ compound or combination of ERβ compounds described herein in vitro. Those skilled in the art will understand that the effective dose in human patients depends on the route of administration, pharmacokinetics, etc. Taking these factors into consideration, an effective estrogenic dose of an ERβ compound or combination of ERβ compounds described herein can be in the range of 0.1 mg / kg to 150 mg / kg, e.g., 0.1 mg / kg to 1 mg / kg, 0.5 mg / kg to 5 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 50 mg / kg, 10 mg / kg to 100 mg / kg, or 50 mg / kg to 150 mg / kg, and an effective daily dose of an ERβ compound or combination of ERβ compounds described herein can be a multiple of any of the values within these ranges, e.g., 1 to 6 (one to six) times the values within these ranges.

[0077] Transitional Phrases

[0078] In some embodiments, descriptions of compositions and methods described herein using the transitional word "comprising" indicate that the composition or method is "open" to additional ingredients, components, or steps. "Comprising" is intended to encompass the more restrictive transitional phrases "consisting essentially of" and "consisting of." Thus, disclosure herein of a material following the transitional phrase "comprising" also fully discloses that which follows the transitional phrase "consisting essentially of" or "consisting of." The transitional phrase "consisting essentially of" has intermediate effect, indicating that the following subject matter consists only of the recited elements and additional matters that do not materially affect the novel and basic characteristics of the claim or claim element. The transitional phrase "consisting of" indicates that the following subject matter is limited to the recited steps or ingredients and closed to other unrecited steps or ingredients. When a transitional phrase appears within a clause or subclause following another transitional phrase, the embedded transitional phrase is intended to affect only the clause in which it appears. Where "a" or "an" appears in the specification or claims, the plural is also intended unless the singular is expressly stated (e.g., "a single," "only one," etc.). [Example]

[0079] The pharmaceutical compositions and estrogen methods disclosed herein can be further understood with reference to the following examples.

[0080] We propose a new class of drugs for selectively modulating ERβ activity pharmacologically. These drugs are selective ERβ subtype agonists. They do not activate reporter genes like estradiol does on ERα. Instead, this class of compounds selectively activates reporter genes on ERβ and regulates genes in various tissue types differently from estradiol.

[0081] Example 1: ERβ-selective compounds.

[0082] Liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, genistein, and equol were obtained from commercial sources, e.g., Sigma-Aldrich.

[0083] Analog 13 (A13) was obtained by the following synthetic method.

[0084] Synthesis of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2. EtOH:HO (4 mL, V EtOH :V H2O To a solution of 4-methoxybenzaldehyde 1 (300 mg, 2.2 mmol) in HCl (3:1), 1-(4-methoxyphenyl)ethan-1-one a (330 mg, 2.2 mmol) and KOH (246 mg, 4.4 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (0.57 g, 96%) as a white solid.

[0085] [ka]

[0086] Synthesis of (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one analogue 13. To a solution of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.74 mmol) in dry DCM (5 mL) was added BB r3 (0.93 g, 3.73 mmol) was added dropwise at 0 °C under N. The resulting mixture was stirred at RT for 12 h and then quenched with water at RT. Finally, the crude product was purified by flash reverse-phase column chromatography (MeOH in water = 5% to 100%, 60 min) to give N-benzyl-2,3,4-trihydroxybenzamide CAT8511-13 (150 mg, 83%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ 10.30 (s, 1H), 8.03 (d, J = 8.7Hz, 2H), 7.76-7.53 (m, 4H), 6.85 (dd, J = 19.7, 8.6Hz, 4H).

[0087] [ka]

[0088] A10

[0089] Synthesis of (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. EtOH:HO (8 mL, V EtOH :V H2O To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in HCl (3:1), 3-methoxybenzaldehyde a (387 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (600 mg, 64%) as a white solid.

[0090] [ka]

[0091] Synthesis of (E)-3-(3-hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A10. To a solution of (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.61 mmol) in dry DCM (5 mL) was added BB r3 (762 mg, 3 mmol) was added dropwise at 0 °C under N. The resulting mixture was stirred at RT for 12 h and then quenched with water at RT. Finally, the crude product was purified by flash reverse-phase column chromatography (MeOH in water = 5% to 100%, 60 min) to give (E)-3-(3-hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A10 (63 mg, 38%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 13.60(s,1H),10.12(s,2H),8.63(s,1H),7.73(q,J=9.0,8.5Hz,5H),6.84(d,J=8.6Hz,2H),6.43(d,J=8.9Hz,1H).

[0092] [ka]

[0093] A13

[0094] Synthesis of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2. EtOH:HO (4 mL, V EtOH :V H2OTo a solution of 4-methoxybenzaldehyde 1 (300 mg, 2.2 mmol) in HCl (3:1), 1-(4-methoxyphenyl)ethan-1-one a (330 mg, 2.2 mmol) and KOH (246 mg, 4.4 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (0.57 g, 96%) as a white solid.

[0095] [ka]

[0096] Synthesis of (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one. To a solution of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.74 mmol) in dry DCM (5 mL) was added BB r3 (0.93 g, 3.73 mmol) was added dropwise at 0 °C under N. The resulting mixture was stirred at RT for 12 h and then quenched with water at RT. Finally, the crude product was purified by flash reverse-phase column chromatography (MeOH in water = 5% to 100%, 60 min) to give N-benzyl-2,3,4-trihydroxybenzamide A13 (150 mg, 83%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 10.30(s,1H),8.03(d,J=8.7Hz,2H),7.76-7.53(m,4H),6.85(dd,J=19.7,8.6Hz,4H).

[0097] [ka]

[0098] A15

[0099] Synthesis of (E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one A15. To a solution of 4-hydroxybenzaldehyde 1 (100 mg, 0.82 mmol) in EtOH (5 mL) was added acetophenone a (98 mg, 0.82 mmol) and KOH (92 mg, 1.64 mmol). The resulting solution was stirred at room temperature for 12 hours. The mixture was diluted with EA (50 mL), washed with brine, concentrated under reduced pressure, and purified by column chromatography on silica gel with PE / EA = 5 / 1 to give (E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one A15 (40 mg, 23%) as a yellow solid. 1 H NMR(400MHz,chloroform-d)δ8.06-7.97(m,2H),7.78(d,J=15.7Hz,1H),7.62-7.5 4(m,3H),7.51(t,J=7.5Hz,2H),7.41(d,J=15.6Hz,1H),6.89(d,J=8.6Hz,2H).

[0100] [ka]

[0101] Example 2: Doxycycline-induced luciferase activity in NKG2E-TK-Luc-transfected U2OS-ERβ cells.

[0102] U2OS cells (wild-type) were maintained in 5% charcoal-dextran stripped FBS. Cells were transfected with 3 μg of a plasmid containing an ERE upstream of a minimal thymidine kinase luciferase promoter (NKG2E-TK-Luk) and 1 μg of an ERα expression vector by electroporation as previously described (An et al. 2001). The resulting ERE-TK-Luc / ERβ U2OS cells were incubated for 24 hours in the presence of medium (negative control), E2, and the indicated micromolar concentrations of test compounds (see Figures 3-4) or E2 (10 nM). As expected, E2 alone activated ERE-TK-Luc. (See U.S. Patent No. 7,482,029, incorporated by reference in its entirety.)

[0103] The compounds shown in Figures 3 and 4 were obtained or prepared, and dose responses were tested in doxycycline-dependent U2OS cells transfected with NKG2E-TK-Luk and ERβ in the presence of various micromolar concentrations of the compounds described in Figures 3 and 4, and in the presence of E2 (10 nM) alone. The luciferase activity of each test compound was recorded. See Figures 1 and 2. As can be seen in Figures 1 and 2, several compounds exhibited exceptional ERβ activity.

[0104] Figure 5 summarizes the dose response activities of ERβ ligands at the NKG2E ERE in U2OS-ERβ cells. Figures 6 and 7 show test compounds that activate ERβ at a concentration of 200 nM (green box). Such compounds or combinations thereof, including pharmaceutical compositions, are of great interest for use in drugs for the treatment of the conditions, diseases, and disorders described herein.

[0105] Example 3: Dose-response luciferase activity of ligands on activation of NKG2E ERE in U2OS-ERα cells

[0106] U2OS cells (wild-type) were maintained in 5% charcoal-dextran stripped FBS. Cells were transfected with 3 μg of a plasmid containing an ERE upstream of a minimal thymidine kinase luciferase promoter (NKG2E-TK-Luk) and 1 μg of an ERα expression vector by electroporation as previously described (An et al. 2001). The resulting ERE-TK-Luc / ERβ U2OS cells were incubated for 24 hours in the presence of medium (negative control), E2, and the indicated micromolar concentrations of test compounds (see Figures 3-4) or E2 (10 nM). As expected, E2 alone activated ERE-TK-Luc. (See U.S. Patent No. 7,482,029, incorporated by reference in its entirety.)

[0107] Several test compounds showed little or no activity in ERα U2OS cells: liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4'-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, genistein, and equol. See Figures 8-10.

[0108] Example 4: Keratin 19 RNA isolation and quantitative real-time PCR

[0109] Total cellular RNA was extracted using the Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's protocol. Reverse transcription reactions were performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) with 1 μM total RNA according to the manufacturer's protocol. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed on a Bio-Rad CFX96 Thermal Cycler System using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). Results were analyzed by the competitive Ct method (Schmittgen and Livak 2008). Ct values of the keratin 19 gene were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), run simultaneously, to obtain adjusted Ct values (ΔCt). Fold changes were calculated by comparing the ΔCt values from the various treatments with the control sample.

[0110] Example 5: Luciferase activity in doxycycline-dependent ERβ-U2OS cells transfected with GRE-TK-Luc and GR in the presence of doxycycline. To understand the activity of the ERβ-active compounds of the present invention against GRE, doxycycline-dependent ERβ-U2OS cells transfected with GRE-TK-Luc, GR, and ERβ in the presence of doxycycline were incubated with test compounds (see x-axis label in Figure 12A) in the absence and presence of 100 nM hydrocortisone (HC). The results are shown in Figure 12A.

[0111] Example 6: Luciferase activity in wild-type (WT) U2OS cells transfected with GRE-TK-Luc, GR, and ERβ. To understand the activity of the ERβ-active compounds of the present invention against GRE, WT ERβ-U2OS cells transfected with GRE-TK-Luc, GR, and ERβ in the presence of doxycycline were incubated with test compounds in the absence and presence of 100 nM hydrocortisone (HC) (see x-axis label in Figure 12B). The results are shown in Figure 12B.

[0112] Example 8: Expression of GILZ and Ladinin in U2OS cells. Following the same protocol as in Example 4, the expression of GILZ and Ladinin was determined in U2OS cells transfected with GRE-TK-Luc, GR, and ERβ in the presence of doxycycline. Vehicle, liquiritigenin, and / or dexamethasone (Dex) were added at the indicated concentrations. See the x-axis of Figures 13A and 13B. The results are shown in Figure 13A (GILZ) and Figure 13B (Ladinin).

[0113] Example 9: Luciferase activity in TAT3-TK-Luc ERβ U2OS cells. U2OS cells were transfected with tyrosine aminotransferase 3 (TAT3)-TK-Luc and ERβ according to the same procedure as described in Example 2. The transfected cells were incubated in the presence of liquiritigenin or ERβ-41 (positive control) in the absence of progesterone (Prog) and MPA. The results are shown in Figure 14.

[0114] Example 10: Several compounds isolated from MF101 were isolated and tested for ERβ activity. The table in Figure 15 ranks these compounds by ERβ potency and selectivity. The compound names are listed in Table 1 above.

[0115] Example 11: Effect of ERβ compounds on uterine cancer cells and breast cancer cells. Uterine cancer cells and MCF-7 breast cancer cells were incubated in the presence of vehicle (control), estradiol (E2), IATERB5, IATERB6, and IATERB7. For the names of IATERB5-7, see Table 1 above.

[0116] MCF-7 cells were seeded at a density of 50,000 cells per well in 6-well tissue culture plates in DMEM / F12 supplemented with 5% detached FBS. The following day, cells were treated with vehicle, E2, in the absence and presence of 2',3',4'-THC or one of the six analogs in Figure 4 for 7 days. Cells were then detached with trypsin, neutralized, and resuspended in medium containing 5% FBS. An appropriate amount of the cell suspension was placed in ISOTON II diluent (Thermo Fisher Scientific, Waltham, MA, USA), and cell number was then determined using a Coulter Counter (Beckman, Brea, CA, USA).

[0117] Flow cytometry was performed as previously described (Pan et al. 2016). Briefly, cells were seeded at a density of 500,000 cells per well in six-well tissue culture dishes in DMEM / F-12 supplemented with 5% detached FBS for 48 hours. The culture medium was then replaced with serum-free DMEM / F12 for 24 hours. Cells were then treated with vehicle, E2, with or without 2',3',4'-THC, or one of the six analogs listed in Figure 4 for 24 hours. The culture medium was then aspirated, and cells were washed with PBS, detached with trypsin, and collected by centrifugation at 1700 rpm for 5 minutes. The cell pellet was washed with ice-cold PBS and subsequently centrifuged at 1700 rpm for 10 minutes at room temperature. The cell pellet was resuspended in 500 μL of PBS containing 50 μg / mL propidium iodide, 0.1% Triton X-100, 0.1% sodium citrate, and 10 μg / mL RNase A. The cell suspension was then analyzed at the University of California, Berkeley, Flow Cytometry Facility using a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the percentage of cells in cell cycle phase was determined using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0118] Unlike estradiol, three of the ERβ compounds (IATERB5, IATERB6, and IATERB7) do not increase uterine weight or induce breast cancer tumor formation. See Figures 16A-B for histology.

[0119] Example 12: Luciferase activity in GRE-TK-Luc-transfected U2OS-ERβ cells by doxycycline. Doxycycline-dependent U2OS cells were transfected with GRE-TK-Luk, GR, and ERβ in the presence of hydrocortisone (HC, 100 nM) alone or in combination with liquiritigenin, A10, A13, and A15. The luciferase activity of each compound was recorded at concentrations of 0.5, 1, 2.5, and 5 μM. Each of the test compounds attenuated the effect of HC on ERβ activity.

[0120] Statistical analysis: All data are presented as the mean ± SE or SD from at least biological triplicates. The statistical significance of differences between two groups was assessed by Student's t-test. For data sets consisting of more than two groups, the statistical significance of differences between various groups (treatments) was analyzed by one-way analysis of variance (one-way ANOVA) or two-way ANOVA as specified in the figure legends. All ANOVA tests were followed by Tukey's or Sidak's multiple comparisons post hoc tests to analyze the significance of differences between any two different treatment groups or controls, as indicated in the figure legends. Statistical analysis and graph plots were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). The number of asterisks in the figures indicates statistical significance: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0121] Although several embodiments of pharmaceutical compositions and estrogen methods are described herein, those skilled in the art will understand that the examples may be modified to provide other embodiments that utilize the compounds and methods described herein. Accordingly, it will be understood that the scope of the present invention is defined by the appended claims, rather than by the specific embodiments set forth by way of example.

Claims

1. A compound, or a pharmaceutically acceptable salt thereof, which is an ERβ selective estrogen compound.

2. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the ERβ selective estrogen compound is a member of the group consisting of liquiritigenin, apigenin, luteolin, galangin, naringenin, calycosin, 6-methoxyluteolin, Nyasol, wogonin, Broussonin A, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, 3,4'-dihydroxyflavone, 7,2'-dihydroxyflavone, 7,3'-dihydroxyflavone, Analog 13, genistein, or equol, A10, A13, A15.

3. 3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein the ERβ selective estrogen compound is not an agonist, antagonist or mixed agonist / antagonist of estrogen receptor alpha.

4. An estrogen combination comprising two or more ERβ selective compounds according to any one of claims 1 to 3, or pharmaceutically acceptable salts thereof.

5. A pharmaceutical composition comprising: (1) one or more pharmaceutically acceptable ingredients other than an ERβ selective compound; and (2) a compound according to any one of claims 1 to 3, or an estrogen combination according to claim 4.

6. 6. The pharmaceutical composition of claim 5, wherein at least one pharmaceutically acceptable ingredient other than the ERβ selective compound is estradiol or another ERα modulating compound.

7. 6. The pharmaceutical composition of claim 5, wherein the at least one pharmaceutically acceptable ingredient other than the ERβ selective compound does not naturally occur in nature with the ERβ selective estrogen compound.

8. 8. The pharmaceutical composition of claim 7, further comprising a member of the group consisting of estradiol (E2), one or more estrogen receptor agonists, one or more estrogen receptor antagonists, one or more mixed estrogen receptor agonists / antagonists, one or more selective estrogen receptor modulators (SERMs), one or more progestogens, one or more glucocorticoids, and one or more androgens.

9. A pharmaceutical composition according to any one of claims 5 to 8 for the treatment of a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound.

10. 10. The pharmaceutical composition of claim 9, wherein the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer.

11. 11. The pharmaceutical composition of claim 9 or claim 10, wherein the disease or disorder condition is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nocturnal awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

12. 10. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, an estrogen combination according to claim 4, or a pharmaceutical composition according to any one of claims 5 to 8, for use in treating a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound.

13. 13. The compound of claim 12, or a pharmaceutically acceptable salt thereof, estrogen combination, or pharmaceutical composition thereof, wherein the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer.

14. The disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovarian syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor disorders. symptoms, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer, or a pharmaceutically acceptable salt thereof.

15. 15. Use of a compound according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, estrogen combination, or pharmaceutical composition thereof, for the manufacture of a medicament for use in treating a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound.

16. 16. The use of claim 15, wherein the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer.

17. 17. The use of claim 15 or claim 16, wherein the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nocturnal awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and non-small cell lung cancer.

18. 10. A method of treating a patient in need thereof, wherein the patient in need thereof has a disease or condition, the method comprising administering to the patient an effective amount of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, an estrogen combination according to claim 4, or a pharmaceutical composition according to any one of claims 5 to 8.

19. 20. The method of claim 18, wherein the disease or disease state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an age-related disorder, or cancer.

20. 19. The method of claim 17 or claim 18, wherein the disease or disorder condition is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorders, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Krone's disease, menopausal and perimenopausal vasomotor symptoms, insomnia, nocturnal awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dysfunction of sexual intercourse, menopausal weight gain and obesity, osteoporosis, type 2 diabetes, estrogen-induced deep vein thrombosis and pulmonary embolism, Alzheimer's disease, early dementia, breast cancer, uterine cancer, ovarian cancer, prostate cancer, non-small cell lung cancer, and colon cancer.

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