ERbeta estrogen compounds and methods of use

Abstract

Estrogen compounds, combinations, pharmaceutical compositions and methods are presented. The estrogen compounds include ER beta selective estrogen compounds and combinations thereof. The novel compositions comprise one or more ER [beta] selective estrogen compounds, and may optionally comprise 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 is not present in nature with the estrogen compound. Methods comprise administering to a patient in need of treatment with an estrogen compound or combination, or a pharmaceutical composition comprising an estrogen compound or combination, a pharmaceutical composition comprising an estrogen compound or combination.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 393,640, filed on July 29, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] field

[0004] 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.

[0005] background

[0006] Estrogen is a steroid hormone involved in controlling a variety of developmental and physiological functions in both males and females. Estrogen is more than just a sex hormone that induces estrus. Estrogens are essential for survival and health in both sexes, although their amounts, adaptive responses, tissue-specific distribution, and receptor affinity vary with different stages of life. Estrogens are essential for glucose homeostasis, immune robustness, bone health, cardiovascular health, fertility, and neurological function.

[0007] Estrogens (estradiol (E2) and related steroid hormones) are biosynthesized primarily in the gonads, but are also biosynthesized in the adrenal cortex and adipose tissue. Estrogen regulation is mediated by nuclear estrogen receptors (ER) and membrane estrogen receptors (GPER1), which are encoded by different genes on different chromosomes, suggesting different functions. However, estrogen is also central to nearly all human pathologies, e.g., infectious conditions, autoimmune conditions, metabolic conditions, and degenerative conditions. Both low and high estrogen levels are associated with chronic and acute diseases. While normal aging leads to a significant decrease in estrogen levels, resulting in tissue degeneration (bone, muscle, nerves, etc.) and metabolite imbalances (glucose, lipids, etc.), the increase in inflammatory agents in daily life elevates the levels of estrogen (or estrogen mimetics), which is of critical pathophysiological importance. The resulting excess estrogen induces fluctuations in estrogen receptors alpha and beta (ERα and ERβ), damaging tissues, leading to autoimmune diseases and neoplasms.

[0008] Estrogen is involved in growth, development and tissue differentiation from the embryonic stage to death. Puberty causes rapid physiological changes, such as growth spurts of bone and muscle mass, and maturation of the gonads and brain. Estrogen is a key signaling contributor to all of these processes and especially to sexual differentiation. Hormone fluctuations and disruptions lead to a variety of pathologies at this age, not all of which are directly related to sexual development and function. The increase in precocious puberty worldwide leads to an increased risk of cancer in women and a reduced quality of life specific to girls.

[0009] During menarche, estrogen levels fluctuate significantly throughout the menstrual cycle. Even at normal levels of cycle fluctuations, early menarche and prolonged menarche can lead to a significantly increased risk of aging diseases such as breast cancer and type 2 diabetes. Most women (up to 91%) experience menstrual pain, and 14% experience heavy bleeding. Female infertility is increasing worldwide, and this is not just due to the increase in the age of first pregnancy. Up to 21% of couples suffer from infertility, of which up to 80% are due to female disorders. Despite improved fertility technologies, such as in vitro fertilization, the success rate of these technologies is very low (IVF is about 9%). Up to 46% of women experience spontaneous abortion (miscarriage). There is an increasing trend in gestational diabetes, premature birth, preeclampsia and anemia during pregnancy, all of which are related to estrogen function.

[0010] In women of childbearing age, estrogen is synthesized primarily in the granulosa cells of the follicles. As women enter menopause, the follicles in the ovaries become depleted during each menstrual cycle due to atresia, and the amount of estrogen produced by the ovaries declines. When estrogen levels begin to decline and fluctuate, short-term symptoms such as hot flashes, night sweats, and mood changes will frequently occur. The ovaries will eventually stop producing estrogen, and the duration of estrogen deficiency will increase, accelerating the risk of chronic diseases such as osteoporosis, cardiovascular disease, obesity, type 2 diabetes, and urogenital atrophy.

[0011] About 70% of all autoimmune diseases are diagnosed in women, including rheumatoid arthritis, lupus, Sjögren's syndrome, scleroderma, Hashimoto's thyroiditis, multiple sclerosis, etc. As women age, the incidence of breast cancer and ovarian cancer increases. Moreover, about 70% of cases of Alzheimer's disease and early dementia are diagnosed in women.

[0012] Estrogen drug intervention, such as menopausal hormone therapy (MHT), is the most common intervention in all women's related disorders. Natural estrogens and synthetic derivatives are used in these prescriptions. The mechanism of action of these estrogens is as a universal agonist, causing activation and inhibition of genes. There are several synthetic steroidal and nonsteroidal estrogen receptor antagonists that are mainly used to treat breast cancer. Although pharmacological intent is to achieve estrogen receptor antagonism, resulting in gene regulation and pathophysiological results different from natural hormones, most steroidal and nonsteroidal drugs result in mixed agonist / antagonist results, so that they cause the opposite results to estrogen in some tissues, and cause similar results in other tissues. These are selective estrogen receptor modulators (SERMs), which are now used to treat breast cancer, osteoporosis, and when combined with estrogen, are used to treat menopausal symptoms.

[0013] Historically, progestins have been prescribed together with estrogens to combat the increased risk of uterine cancer associated with menopausal hormone therapy.Progestins exert their pharmacological actions via the progesterone receptor.

[0014] In recent decades, attempts have been made to develop agonist and antagonist drugs for the receptor subtype since the discovery of ERβ in 1995. None of these drugs have been approved by regulatory agencies.

[0015] Menopausal hormone therapy (MHT) is recommended for only five years for the treatment of menopausal and perimenopausal vasomotor symptoms and vulvovaginal atrophy, because long-term use of MHT has been shown to increase the risk of estrogen-related adverse outcomes, such as breast cancer, stroke, venous thrombosis (VTE) and Alzheimer's disease. Nevertheless, in addition to treating menopausal vasomotor symptoms, treatment with MHT also reduces the risk of osteoporosis and type 2 diabetes (T2DM), cardiovascular disease, obesity and urogenital atrophy. Therefore, there is a need for treatment options that have one or more advantages of MHT and a reduced risk of adverse outcomes, such as the increased risk of cancer associated with traditional MHT (E2, alone or in combination with progestins, or selective estrogen receptor modulators (SERMs)).

[0016] There is a need for additional and novel therapeutic compounds, compositions and methods for treating menopausal and perimenopausal symptoms. Various embodiments disclosed herein address these needs and also provide related advantages.

[0017] Brief description of the invention

[0018] The compounds described herein, alone or in combination with each other or with other estrogens, can be used for a variety of indications because they cause changes in tissues expressing ERβ. Activation of ERα by estradiol results in a dose-dependent increase in proliferation of breast cancer cells and uterine cancer cells and a dose-dependent increase in uterine hyperplasia and uterine weight. Unlike the activity of estradiol on ERα, estradiol inhibits the growth of breast cancer cells and uterine cancer cells expressing ERβ in a dose-dependent manner, and there is no increase in uterine hyperplasia or uterine weight, suggesting that the use of ERβ-specific ligands will enhance estrogen activity and reduce the risk of breast cancer and uterine cancer.

[0019] 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 of any of the foregoing. In some embodiments, the ERβ selective compound is not an agonist, antagonist or mixed agonist / antagonist of estrogen receptor α. A combination of two or more ERβ selective compounds or pharmaceutically acceptable salts thereof of the same group is also provided. A pharmaceutical composition is also provided, which comprises (1) one or more pharmaceutically acceptable ingredients other than ERβ selective compounds and (2) one or more ERβ selective compounds. In some embodiments, at least one pharmaceutically acceptable ingredient other than ERβ selective compounds is estradiol or other compounds that regulate ERα. In some embodiments, at least one pharmaceutically acceptable ingredient other than ERβ selective compounds does not naturally exist in nature with ERβ selective estrogen compounds. 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 progestins, 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 condition or disease state in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder or a cancer.In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0020] Some embodiments described herein include the use of the compounds, combinations or pharmaceutical compositions described in the immediately preceding paragraphs for the manufacture of a medicament for use in treating a condition or disease in a patient requiring treatment with a nuclear receptor reprogramming compound. In some embodiments, the condition or disease state is a developmental condition, a menstrual condition, a fertility condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder or a cancer. In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0021] Some embodiments described herein provide a method of treating a patient in need of such treatment, the method comprising administering to the patient an effective amount of a compound described in any of the two immediately preceding paragraphs, or a pharmaceutically acceptable salt, combination, or pharmaceutical composition thereof. In some embodiments, the condition or disease state is a developmental condition, a menstrual condition, a fertility condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer. In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0022] Other uses and advantages of the various embodiments described herein will be apparent to those skilled in the art upon review of the following disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Features and advantages of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings, in which:

[0025] Figure 1 Luciferase activity of several compounds of the invention in U2OS cells transfected with NKG2E TK-Luc and ERβ is shown.

[0026] Figure 2 Shown is the dose response of several compounds of the invention in U2OS cells transfected with NKG2E TK-Luc and ERβ.

[0027] Figure 3 and Figure 4 The structures of several compounds of the present invention are shown. The basic flavonoid structure with ring position numbering is in Figure 3 and Figure 4 .

[0028] Figure 5 Shown is the dose response of ligands to activate luciferase activity of NKG2E-TK-Luc in U2OS-ERβ cells.

[0029] Figure 6 and Figure 7 Compounds of the invention are shown. The ligands enclosed in the box (liquiritigenin, apigenin, nigericin, naringenin, 7,4'-dihydroxyflavone, 6,4'-dihydroxyflavone, 5,4'-dihydroxyflavone, analog 13, and genistein) activated ERβ at 200 nM. The basic flavonoid structure with ring position numbering is in Figure 6 and Figure 7 .

[0030] Figure 8 Shown is the dose response of ligands for activation of NKG2E ERE in U2OS-ERα cells.

[0031] Fig. 9 and Fig.10 Compounds of the invention are shown. Ligands that activate ERα at 1 μM (apigenin, nigericin, styrax A, 6,4'-dihydroxyflavone, genistein, and equol) are enclosed in double boxes, and ligands that activate ERα at 5 μM (liquiritigenin, 7,4'-dihydroxyflavone, and analog 13) are enclosed in single boxes. The basic flavonoid structure with ring position numbering is in Fig. 9 and Fig.10 .

[0032] Fig.11 : shows keratin 19 mRNA expression in U2OS-ERβ cells by qRT-PCR.

[0033] Fig. 12A Shown are luciferase activities of U2OS-ERβ cells transfected with GRE-TK-Luc and GR in the presence of doxycycline.

[0034] Fig. 12B Shown are the luciferase activities of U2OS WT cells transfected with GRE-TK-Luc and ERβ in the presence of GR.

[0035] Fig.13A Shown is the expression of GILZ in GR-expressing U2OS-ERβ cells in the presence of liquiritigenin and / or doxycycline.

[0036] Fig. 13B Ladinin expression in GR-expressing U2OS-ERβ cells in the presence of liquiritigenin and / or doxycycline is shown.

[0037] Fig.14 Shown are luciferase activities in U2OS-ERβ cells expressing TAT3-TK-Luc and hPRβ in the presence of ERβ ligands and combinations.

[0038] Fig.15The potency of ERβ ligand in MF101 is shown.

[0039] Fig.16A and Fig. 16B Compared with E2, ERβ ligands IATERB5 and IATERB6 ( Fig.16A ) and IATEB7( Fig. 16B Unlike estradiol, three of the ERβ compounds did not increase uterine breast cancer tumorigenesis.

[0040] Fig.17 Shown are dose-response luciferase activities of ERβ ligands alone and in combination with hydrocortisone (HC) in U2OS-ERβ cells transfected with GRE-TK-Luc, GR, and doxycycline. DETAILED DESCRIPTION OF THE INVENTION

[0042] Liquorice root; apigenin; luteolin; galangin; naringenin; calycosin; 6-methoxy luteolin; nifoin; wogonin; burdock 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.

[0043] The compounds of the present invention are summarized in Table 1:

[0044] Table 1: ERβ selective compounds

[0045] Iaterion naming Common Name IATERB1 galangin IATERB2 6-Methoxyluteolin IATERB3 Wogonin IATERB4 Broussonetine A IATERB5 Niacin IATERB6 Isoflavone IATERB7 Licorice IATERB8 Naringenin IATERB9 Apigenin IATERB10 Luteolin IATERB11 5,4′-dihydroxyflavone IATERB12 6,4′-dihydroxyflavone IATERB13 3,4′-dihydroxyflavone IATERB14 7,4′-dihydroxyflavone IATERB15 7,3′-dihydroxyflavone IATERB16 7,2′-dihydroxyflavone IATERB17 Analog 13 (A13)

[0046] Pharmaceutical composition

[0047] The pharmaceutical compositions described herein include one or more compounds selected from the group consisting of: liquiritigenin; apigenin; luteolin; galangin; naringenin; calycosin; 6-methoxyluteolin; nifoin; wogonin; baicalin 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 ingredients may include one or more ingredients that do not naturally occur with the ERβ selective compounds, salts or solvates thereof disclosed herein. Pharmaceutically acceptable ingredients that do not occur naturally with the novel compounds disclosed herein, salts or solvates thereof, may include sterile, isotonic or pyrogen-free excipients.

[0048] A "pharmaceutically acceptable" ingredient is an ingredient that is compatible with the estrogen compound as described herein and with the other ingredients of the composition 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 compound or combination of ERβ compounds as 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.

[0049] In some embodiments, the pharmaceutical composition may be an estrogen composition. The estrogen composition comprises an estrogenically effective amount of an ERβ selective compound or a pharmaceutically acceptable salt thereof, or a combination of an ERβ selective compound as 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 estrogen compound in nature. In particular, the excipient may include at least one compound that does not naturally occur with the ERβ compound or the combination of ERβ compounds as described herein in the human body. 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 in a concentration sufficient to make the composition isotonic. In some embodiments, the additional ingredient may be a flavoring or sweetener that does not exist with the ERβ compound or the combination of ERβ compounds as described herein in nature. In some embodiments, the estrogen composition may be sterile, pyrogen-free and / or isotonic.

[0050] A pharmaceutically acceptable salt can be any salt of an estrogen compound disclosed herein having a suitable solubility in an aqueous solvent of an appropriate pH. Remington, 20th edition, published in 2000, pp. 704-719 provides a method for determining a suitable pharmaceutically acceptable salt. For example, a suitable salt can be selected from Remington's Table 38-2, p. 704. A pharmaceutically acceptable salt can be prepared by dissolving the estrogen compound in a suitable solvent and adding a suitable acid or base or a suitable anti-acid or anti-base (as the case may be) to the solution, and isolating the salt form of the estrogen compound from the solution.

[0051] Pharmaceutical compositions, particularly estrogen compositions, can be formulated for a variety of 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 to aid in the transport of an ERβ compound or combination of ERβ compounds as described herein against one or more external or internal physiological barriers, such as the lung epithelial barrier or the blood-brain barrier.

[0052] 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, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants and buffers.

[0053] Effective dose of estrogen

[0054] 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, may vary depending on a variety of factors, including the route of administration, the age and condition of the patient in need of estrogen therapy, and the like. Typically, an ERβ selective compound or a pharmaceutically acceptable salt thereof, or a combination of ERβ selective compounds or pharmaceutically acceptable salts as described herein, is 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 as described herein may be in the range of 0.01 mg / day to 1000 mg / day. The effective daily dose may be divided into two or more sub-doses, such as 1, 2, 3, 4, 5, 6 or more sub-doses. In the case where the pharmaceutical composition is administered as an infusion, the effective daily dose may be administered as a continuous infusion over the course of several hours (e.g., 1 hour-24 hours). The effective dose may be similar to 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 as described herein, but may be scaled to take into account the greater molecular weight of the compound as compared to the ERβ selective compound or combination of ERβ selective compounds as described herein in terms of the relative biological activity, pharmacokinetics, and pharmacodynamics of the compound, which one of skill in the art will know how to determine by art-recognized methods.

[0055] Those skilled in the art of pharmaceutical formulation and compounding have the knowledge and skill to select appropriate amounts of suitable pharmaceutically acceptable carriers and excipients for use with an ERβ compound or combination of ERβ compounds as described herein. In addition, there are many resources available to those skilled in the art that describe pharmaceutically acceptable carriers and excipients and can be used to select suitable 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).

[0056] Compositions of estrogenic compounds as 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 edition (Mack Publishing Company (2000)).

[0057] In some embodiments, the estrogen composition may include an ERβ compound or a combination of ERβ compounds as 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 compound of the disclosure may be extracted and then administered to a subject, such as as a powder or syrup. Alternatively, the composition may be prepared and packaged in unit dosage form, wherein each physically discrete unit contains an effective amount of an ERβ compound or a combination of ERβ compounds as described herein.

[0058] The ERβ compound or combination of ERβ compounds and a pharmaceutically acceptable carrier or excipient as described herein can be formulated into a dosage form suitable for administration to a subject via a desired route of administration. For example, dosage forms include dosage forms suitable for: (1) oral administration, such as tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; and (2) parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution. Suitable pharmaceutically acceptable carriers or excipients may vary depending on the particular dosage form selected. In addition, suitable pharmaceutically acceptable carriers or excipients may be selected for the specific function they may serve in the composition. For example, certain pharmaceutically acceptable carriers or excipients may be selected for their ability to facilitate the production of a uniform dosage form. Certain pharmaceutically acceptable carriers or excipients may be selected for their ability to facilitate the production of a stable dosage form. Certain pharmaceutically acceptable carriers or excipients may be selected for their ability to facilitate the compounds disclosed herein to be carried or transported from one organ or part of the body to another organ or part of the body after administration to a subject. Certain pharmaceutically acceptable carriers or excipients may be selected for their ability to enhance patient compliance.

[0059] In some embodiments, the estrogen ERβ compound or the combination of ERβ compounds as described herein can be formulated for parenteral administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that can contain suspending agents and thickening agents. The composition can be presented in unit dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier such as water for injection just before use. Instant injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Parenteral preparations can be sterile, pyrogen-free, or sterile and pyrogen-free. Parenteral preparations can be isotonic.

[0060] oral

[0061] The estrogen composition can be an oral estrogen composition comprising an ERβ selective compound as described herein or a pharmaceutically acceptable salt thereof, or a combination of ERβ selective compounds or pharmaceutically acceptable salts thereof and at least one excipient suitable for oral administration. The at least one excipient suitable for oral administration can include a compound that does not naturally occur in nature with the ERβ compound or combination of ERβ compounds as described herein. The at least one excipient suitable for oral administration can include at least one compound other than water. Various dosage forms can 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.

[0062] In some embodiments, the ERβ compound or the combination of ERβ compounds as described herein can be formulated as 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 may also include a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, gum arabic, sodium alginate, alginic acid, tragacanth gum, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The oral solid dosage form may also include a disintegrant. Suitable disintegrants include cross-linked polyvinylpyrrolidone, sodium starch glycolate, cross-linked carboxymethyl cellulose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may also include a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate and talc.

[0063] Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The composition can also be prepared to prolong or sustain the release, for example, by coating or embedding particulate material in polymers, wax, etc.

[0064] The ERβ compounds or combinations of ERβ compounds as described herein can also be combined with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethylaspartame-indophenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, the ERβ compounds or combinations of ERβ compounds as described herein can be combined with a class of biodegradable polymers that can be used to achieve controlled release of drugs, such as cross-linked or amphiphilic block copolymers of polylactic acid, poly-ε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and hydrogels.

[0065] In some embodiments, the ERβ compound or combination of ERβ compounds as described herein can be formulated into a liquid oral dosage form. Oral liquids such as solutions, syrups, and elixirs can be prepared in dosage unit form so that a given amount contains a predetermined amount of the compound disclosed herein. Syrups can be prepared by dissolving the compound of the present disclosure in an appropriately flavored aqueous solution, while elixirs are prepared by using a non-toxic alcohol 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 ethers, preservatives, flavor additives such as peppermint oil or other natural sweeteners or saccharin or other artificial sweeteners, etc. can also be added.

[0066] Intranasal

[0067] The estrogen composition can be an intranasal estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for intranasal administration. At least one excipient suitable for intranasal administration can include at least one compound other than water. For example, the intranasal estrogen composition can include one or more penetration enhancers that increase the absorption and / or bioavailability of the combination of ERβ compounds or ERβ compounds as described herein across the mucosa. In some embodiments, the penetration enhancer can include a mucolytic agent, a degradation enzyme inhibitor, and a compound that increases the permeability of the mucosal cell membrane. Whether a given compound is an "enhancer" can be determined by comparing two formulations (with or without an enhancer) containing a non-associated small polar molecule as a drug in an in vivo or good model test and determining whether the uptake of the drug is enhanced to a clinically significant degree. The enhancer should not cause any problems in terms of chronic toxicity because the enhancer should be non-irritating and / or rapidly metabolized to normal cell components that do not have any significant irritating effects in vivo. In some embodiments, the penetration enhancer can be an alkyl glycoside, such as those disclosed in U.S. Pat. No. 5,661,130, which is incorporated herein by reference in its entirety. Those skilled in the art recognize the need to achieve a suitable hydrophile-lipophile balance (HLB) number, which can be determined as disclosed in U.S. Pre-Grant Publication No. US2009 / 0047347, which is incorporated herein by reference in its entirety.

[0068] The intranasal estrogen composition of the ERβ compound or combination of ERβ compounds as described herein may also include a flavoring agent or fragrance to cover the taste of the ERβ compound or combination of ERβ compounds as described herein. The intranasal composition may also include an isotonicity agent to make the composition isotonic. The intranasal estrogen composition of the ERβ compound or combination of ERβ compounds as described herein may also include a stabilizer.

[0069] In the lungs

[0070] The estrogen composition can be an intrapulmonary estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for intrapulmonary administration. The at least one excipient suitable for intrapulmonary administration can include at least one compound other than water. For example, the intrapulmonary composition can include one or more penetration enhancers that increase the ability of the ERβ compound or a combination of ERβ compounds as described herein to cross the lung epithelium into the bloodstream.

[0071] Intrapulmonary estrogen compositions can be administered to the lungs by inhalation, for example, using an insufflator, an aerosol inhaler, or a conventional or high efficiency nebulizer.

[0072] High-efficiency nebulizer is an inhalation device including a microporous membrane, through which a liquid solution is converted into an aerosol droplet suitable for inhalation by electrical means or mechanical means. High-efficiency nebulizer can deliver most of the loaded dose to the patient. In some embodiments, high-efficiency nebulizer can also utilize one or more active or passively vibrating microporous membranes. In some embodiments, high-efficiency nebulizer can include one or more oscillating membranes. In some embodiments, high-efficiency nebulizer can include a vibrating net or a vibrating plate with multiple holes, and optionally a vibration generator with an aerosol mixing chamber. In some such embodiments, a mixing chamber can be used to collect (or grade) aerosols from an aerosol generator.

[0073] In some embodiments, a high efficiency nebulizer can achieve a lung deposition (deposited lung dose) of at least about 10% based on a nominal dose of an ERβ compound or combination of ERβ compounds as described herein.

[0074] In some embodiments, a high efficiency nebulizer provides lung deposition (deposited lung dose) of an ERβ compound or combination of ERβ compounds as described herein of at least about 5% based on the nominal dose of the ERβ compound or combination of ERβ compounds as described herein.

[0075] According to the present invention, in some embodiments, a nebulizer (such as a high-efficiency nebulizer) may be suitable or may be adapted to operate in conjunction with a unit dosage form (such as an ampoule or vial) containing a single dose of an ERβ compound or a combination of ERβ compounds as described herein for estrogen therapy. The unit dosage form includes a container containing an inhalation solution containing an ERβ compound or a combination of ERβ compounds as described herein. The container is suitable for cooperating with a high-efficiency nebulizer device in such a manner that a nominal dose of inhalation solution is 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 not with other devices or dosage forms. In some specific embodiments, the unit dosage form is configured so that it cooperates with a keyhole-like structure in a high-efficiency nebulizer, but will not operate with other nebulizer devices. In such an embodiment, the high-efficiency nebulizer is configured so that it will accept a unit dosage form containing an ERβ compound or a combination of ERβ compounds as described herein and operate appropriately with the unit dosage form, but will not accept other dosage forms and will not operate with other dosage forms.

[0076] Suitable high-efficiency atomizers with perforated membranes are disclosed in U.S. Pat. Nos. 6,962,151, 5,152,456, 5,261,601, and 5,518,179, each of which is hereby incorporated by reference in its entirety. Suitable high-efficiency atomizers include an oscillating membrane. Features of these high-efficiency atomizers are disclosed in U.S. Pat. Nos. 7,252,085; 7,059,320; 6,983,747, each of which is hereby incorporated by reference in its entirety.

[0077] Commercial high-efficiency nebulizers are available from PARI (Germany) under the trade name Aerogen, Ltd. (Ireland), trade name Go and Pro, Solo and other exploits Nebulizer Technology Nebulizer; Respironics (Murrysville, Calif.), trade name Omron (Bannockburn, Ill.), trade name Activaero (Germany), trade name and AerovecTx (Atlanta, Ga.), trade name

[0078] Conventional nebulizers include, for example, jet nebulizers or ultrasonic nebulizers. Jet nebulizers typically utilize a compressor to generate compressed air that breaks up liquid medication into respirable droplets that form aerosolized (nebulized) mist. In some of these embodiments, when the patient inhales, the valve at the top opens, and the valve then allows air to enter the device, thereby accelerating the generation of mist; when the patient exhales, the top valve closes, thereby slowing down the generation of mist while allowing the patient to exhale through the opening of the mouthpiece fins.

[0079] Some conventional atomizers are disclosed in U.S. Pat. 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 hereby incorporated by reference in its entirety.

[0080] A commercial conventional nebulizer is available from PARI (Germany) under the trade name PARI LC LC and A&H Products, Inc. (Tulsa, Okla.), trade name Hudson RCI (Temecula, Calif.), trade name Intersurgical, Inc. (Liverpool, NY), trade name Salter Labs (Arvin, Calif.), trade name Salter Respironics (Murrysville, Pa.), trade name Bunnell (Salt Lake City, Utah), trade name Whisper Smiths-Medical (Hyth Kent, UK), trade name and DeVilbiss (Somerset, Pa.), trade name

[0081] Intravenous

[0082] The estrogen composition can be an intravenous estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for intravenous administration. The at least one excipient suitable for intravenous administration can include at least one compound other than water. Intravenous compositions of ERβ compounds or a combination of ERβ compounds as described herein are parenteral compositions intended for intravenous administration by injection or infusion. They can contain one or more isotonic agents to make the composition isotonic. They can be and usually are sterile, pyrogen-free, or both.

[0083] Subcutaneous

[0084] The estrogen composition can be a subcutaneous estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for subcutaneous administration. The at least one excipient suitable for subcutaneous administration can include at least one compound other than water. Subcutaneous compositions of ERβ compounds or a combination of ERβ compounds as described herein are parenteral compositions intended for subcutaneous injection. They can contain one or more isotonic agents to make the composition isotonic. They can be and usually are sterile, pyrogen-free, or both.

[0085] Percutaneous

[0086] The estrogen composition can be a transdermal estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for transdermal administration. At least one excipient suitable for transdermal administration may include at least one compound other than water. For example, the transdermal estrogen composition may include one or more penetration enhancers that increase the ability of the ERβ compound or a combination of ERβ compounds as described herein to pass through the dermis into the bloodstream. In addition, the transdermal composition can be delivered by a biasing mechanism (such as an iontophoresis device).

[0087] Sublingual or buccal

[0088] The estrogen composition can be a sublingual or buccal estrogen composition comprising an ERβ compound or a combination of ERβ compounds as 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.

[0089] intraperitoneally

[0090] The estrogen composition can be an intraperitoneal estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for intraperitoneal administration. The at least one excipient suitable for intraperitoneal administration can include at least one compound other than water. The intraperitoneal estrogen composition of the ERβ compound or a combination of ERβ compounds as described herein is a parenteral composition intended for administration to the peritoneum by injection or infusion. They can contain one or more isotonic agents to make the composition isotonic. They can be and usually are sterile, pyrogen-free, or both.

[0091] Intrathecal or intraventricular

[0092] The estrogen composition can be an intrathecal or intraventricular estrogen composition comprising an ERβ compound or a combination of ERβ compounds as described herein and at least one excipient suitable for intrathecal or intraventricular administration. At least one excipient suitable for intrathecal or intraventricular administration may include at least one compound other than a compound naturally occurring in nature with the ERβ compound or a combination of ERβ compounds as described herein (e.g., water). The intrathecal or intraventricular estrogen composition of the ERβ compound or a combination of ERβ compounds as described herein is a parenteral composition intended to be administered into the cerebrospinal fluid by injection or infusion. They may contain one or more isotonic agents to make the composition isotonic. They may be and are typically sterile, pyrogen-free, or both.

[0093] Other routes of administration

[0094] Although 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 thereto without departing from the scope of the appended claims. Therefore, the scope of the compositions and methods described is limited only by the appended claims.

[0095] Estrogen method

[0096] Some embodiments described herein provide a compound of Table 1 described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in treating a condition or disease state in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer. In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0097] Some embodiments described herein provide the use of a compound of Table 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in treating a condition or disease in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer. In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0098] Some embodiments described herein provide a method of treating a patient in need of such treatment, 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 condition or disease state is a developmental condition, a menstrual condition, a fertility condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer. In some embodiments, the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

[0099] Based on the disclosure herein, one skilled in the art understands how to determine an estrogenically effective amount of an ERβ compound or a combination of ERβ compounds as described herein. Typically, an estrogenically effective amount, a therapeutically effective amount, or a prophylactically effective amount of an ERβ compound or a combination of ERβ compounds as described herein can be determined, for example, by extrapolation from in vitro tests. One skilled in the art understands that an effective dose can be extrapolated from an in vitro half-maximal modulatory (inhibitory or activating) concentration of an ERβ compound or a combination of ERβ compounds as described herein. One skilled in the art understands that an effective dose in a human patient will depend on the route of administration, pharmacokinetics, and the like. Taking these factors into account, an estrogenically effective dose of an ERβ compound or a combination of ERβ compounds as described herein can be in the range of 0.1 mg / kg to 150 mg / kg, for example, 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; an effective daily dose of an ERβ compound or a combination of ERβ compounds as described herein can be some multiple of any value within these ranges, for example, one to six (1 to 6) times the values ​​within these ranges.

[0100] Transition Phrases

[0101] In some embodiments, the description of the compositions and methods described herein using the transition word "comprising" indicates that the composition or method is "open" to additional ingredients, components or steps. "Comprising" is intended to include the more restrictive transition phrases "consisting essentially of" and "consisting of". Therefore, the disclosure of the content following the transition phrase "comprising" herein also fully discloses the same content following the transition phrase "consisting essentially of" or "consisting of". The transition phrase "consisting essentially of" has an intermediate effect, indicating that the following subject matter consists only of the listed elements and additions that do not materially affect the novel and basic properties of the claim or claim element. The transition phrase "consisting of" indicates that the following subject matter is limited to the listed steps or ingredients and does not include other steps or ingredients that are not listed. When a transition phrase appears in a clause or subclause after another transition phrase, the embedded transition phrase is intended to affect only the phrase in which it appears. Where "a" or "an" is recited in the specification or claims, the plural is also intended unless the singular is explicitly recited (e.g., "single", "only one", etc.). Example

[0102] The pharmaceutical compositions and estrogenic methods disclosed herein can be further understood with reference to the following examples.

[0103] We present new drugs that selectively modulate ERβ activity pharmacologically. These drugs are selective ERβ subtype agonists. They do not activate reporter genes as estradiol does for ERα. Instead, this class of compounds produces selective ERβ activation of reporter genes and modulates genes on a variety of tissue types differently than estradiol.

[0104] Example 1: ERβ Selective Compounds

[0105] Liquorice root; apigenin; luteolin; galangin; naringenin; calycosin; 6-methoxyluteolin; niacin; wogonin; burdock A; 7,4'-dihydroxyflavone; 6,4'-dihydroxyflavone; 5,4'-dihydroxyflavone; 3,4'-dihydroxyflavone; 7,2'-dihydroxyflavone; 7,3'-dihydroxyflavone; genistein; and equol are obtained from commercial sources such as Sigma-Aldrich.

[0106] Analog 13 (A13) was obtained by the following synthesis method:

[0107] Synthesis of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2. 4-Methoxybenzaldehyde 1 (300 mg, 2.2 mmol) was dissolved in EtOH:H2O (4 mL, V EtOH :V H2O =3:1) was added 1-(4-methoxyphenyl)ethan-1-one a (330 mg, 2.2 mmol) and KOH (246 mg, 4.4 mmol). The resulting solution was stirred at 0°C~rt 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.

[0108]

[0109] Synthesis of (E)-1,3-bis(4-hydroxyphenyl)prop-2-en-1-one analog 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 BBr (0.93 g, 3.73 mmol) dropwise at 0 °C under N2. The resulting mixture was stirred at room temperature for 12 h and then quenched with water at room temperature. Finally, the crude product was purified by a fast reverse phase column (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. 1H NMR (400MHz, DMSO-d6) δ10.30 (s, 1H), 8.03 (d, J = 8.7Hz, 2H), 7.76-7.53 (m, 4H), 6.85 (dd, J = 19.7, 8.6Hz, 4H).

[0110]

[0111] Synthesis of (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) was dissolved in EtOH:H2O (8 mL, V EtOH :V H2O =3:1) was added 3-methoxybenzaldehyde a (387 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol). The resulting solution was stirred at 0°C ~ rt 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.

[0112]

[0113] Synthesis of (E)-3-(3-hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A10. At 0 ° C under N2, BBr3 (762 mg, 3 mmol) was added dropwise to a solution of (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (200 mg, 0.61 mmol) in dry DCM (5 mL). The resulting mixture was stirred at room temperature for 12 h and then quenched with water at room temperature. Finally, the crude product was purified by a fast reverse phase column (MeOH in water = 5% to 100%, 60 min) to give (E)-3-(3-hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-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).

[0114]

[0115] Synthesis of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2. 4-Methoxybenzaldehyde 1 (300 mg, 2.2 mmol) was dissolved in EtOH:H2O (4 mL, V EtOH :V H2O =3:1) was added 1-(4-methoxyphenyl)ethan-1-one a (330 mg, 2.2 mmol) and KOH (246 mg, 4.4 mmol). The resulting solution was stirred at 0°C~rt 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.

[0116]

[0117] Synthesis of (E)-1,3-bis(4-hydroxyphenyl)prop-2-ene-1-one. To a solution of (E)-1,3-bis(4-methoxyphenyl)prop-2-ene-1-one 2 (200 mg, 0.74 mmol) in dry DCM (5 mL) was added dropwise BBr3 (0.93 g, 3.73 mmol) at 0 °C under N2. The resulting mixture was stirred at room temperature for 12 h and then quenched with water at room temperature. Finally, the crude product was purified by a fast reverse phase column (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) δ10.30 (s, 1H), 8.03 (d, J = 8.7Hz, 2H), 7.76-7.53 (m, 4H), 6.85 (dd, J = 19.7, 8.6Hz, 4H).

[0118]

[0119] Synthesis of (E)-3-(4-hydroxyphenyl)-1-phenylprop-2-ene-1-one A15. To a solution of 4-hydroxybenzaldehyde 1 (100 mg, 0.82 mmol) in EtOH (5 mL), acetophenone a (98 mg, 0.82 mmol) and KOH (92 mg, 1.64 mmol) were added. The resulting solution was stirred at room temperature for 12 h. 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-ene-1-one A15 (40 mg, 23%) as a yellow solid. 1H NMR (400 MHz, CHLOROFORM-d) δ 8.06-7.97 (m, 2H), 7.78 (d, J = 15.7 Hz, 1H), 7.62-7.54 (m, 3H), 7.51 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 15.6 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H).

[0120]

[0121] Example 2: Luciferase activity in NKG2E-TK-Luc transfected U2OS-ERβ cells in the presence of doxycycline.

[0122] U2OS cells (wild type) were maintained in 5% charcoal-dextran treated FBS. Cells were transfected by electroporation with 3 μg of a plasmid containing ERE (NKG2E-TK-Luc) upstream of a minimal thymidine kinase luciferase promoter and 1 μg of an ERβ expression vector as described previously (An et al., 2001). The resulting ERE-TK-Luc / ERβU2OS cells were incubated with medium (negative control), E2, and the indicated micromolar concentrations of test compounds (see Figure 3-Figure 4 ) or E2 (10 nM) were incubated for 24 hours. As expected, E2 alone activated ERE-TK-Luc (see U.S. Pat. No. 7,482,029, which is incorporated by reference in its entirety).

[0123] Obtain or prepare Figure 3 and Figure 4 The compounds depicted in Figure 2 and in the presence of E2 alone (10 nM) at different micromolar concentrations Figure 3 and Figure 4 The dose response was tested in doxycycline-dependent U2OS cells transfected with NKG2E-TK-Luc and ERβ in the presence of the compounds. The luciferase activity of each test compound was recorded. Figure 1 and Figure 2 .like Figure 1 and Figure 2 As can be seen in Figure 3, several compounds showed excellent ERβ activity.

[0124] Figure 5 Dose-response activities of ERβ ligands at the NKG2E ERE in U2OS-ERβ cells are summarized. Figure 6 and Figure 7 Those test compounds that activated ERβ at 200 nM concentration are indicated (green box).Such compounds or combinations thereof, including pharmaceutical compositions, are of great interest for use in medicines for treating the symptoms, diseases and disorders described herein.

[0125] Example 3: Dose-response luciferase activity of ligands for activation of NKG2E ERE in U2OS-ERα cells

[0126] U2OS cells (wild type) were maintained in 5% charcoal-dextran treated FBS. Cells were transfected by electroporation with 3 μg of a plasmid containing an ERE (NKG2E-TK-Luc) upstream of a minimal thymidine kinase luciferase promoter and 1 μg of an ERα expression vector as described previously (An et al., 2001). The resulting ERE-TK-Luc / ERβU2OS cells were incubated with medium (negative control), E2, and the indicated micromolar concentrations of the test compounds (see Figure 3-Figure 4 ) or E2 (10 nM) were incubated for 24 hours. As expected, E2 alone activated ERE-TK-Luc (see U.S. Pat. No. 7,482,029, which is incorporated by reference in its entirety).

[0127] Several test compounds showed little or no activity in ERαU2OS cells. Liquorice root; apigenin; luteolin; galangin; naringenin; calycosin; 6-methoxyluteolin; nifolate; wogonin; baicalin A; 7,4'-dihydroxyflavone; 6,4'-dihydroxyflavone; 5,4'-dihydroxyflavone; 3,4'-dihydroxyflavone; 7,2'-dihydroxyflavone; 7,3'-dihydroxyflavone; genistein; and equol. See Figure 8-Figure 10 .

[0128] Example 4: Keratin 19 RNA Isolation and Quantitative Real-Time PCR

[0129] 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, Hercules, CA, USA) and 1 μm total RNA according to the manufacturer's protocol. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the Bio-Rad CFX96 thermal cycler system using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). The results were analyzed by the competitive Ct method (Schmittgen and Livak 2008). The Ct values ​​of the keratin 19 gene were normalized relative to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) run simultaneously to obtain adjusted Ct values ​​(ΔCt). The fold change was calculated by comparing the ΔCt values ​​of the different treatments with the control samples.

[0130] Example 5: Luciferase activity in doxycycline-dependent ERβ-U2OS cells transfected with GRE-TK-Luc and GR in the presence of doxycycline. In order to understand the activity of the ERβ active compounds of the present invention on GRE, doxycycline-dependent ERβ-U2OS cells transfected with GRE-TK-Luc, GR and ERβ in the presence of doxycycline were incubated with the test compounds (see x-axis label, Fig. 12A ) were incubated together. Fig. 12A Listed in.

[0131] 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 on GRE, WT ERβ-U2OS cells transfected with GRE-TK-Luc, GR and ERβ in the presence of doxycycline were incubated with the test compounds (see x-axis label, Fig. 12B ) were incubated together. Fig. 12B Listed in.

[0132] Example 8: Expression of GILZ and Ladinin in U2OS cells. Following a similar 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, licorice and / or dexamethasone (Dex) were added at the indicated concentrations. See x-axis, Fig.13A , Fig. 13B The results are Fig.13A (GILZ) and Fig. 13B (Ladinin)

[0133] Example 9: Luciferase activity in TAT3-TK-Luc ERβ U2OS cells. U2OS cells were transfected with tyrosine aminotransferase 3 (TAT3)-TK-Luc and ERβ following a procedure similar to that described in Example 2. Transfected cells were incubated in the presence of glycyrrhizin or ERβ-41 (positive control) in the absence of progesterone (Prog) and MPA. Fig.14 Shown in.

[0134] Example 10: Isolation of several compounds isolated from MF101 and testing of ERβ activity. Fig.15 The table in rank these compounds by ERβ potency and selectivity. The names of the compounds are listed in Table 1 above.

[0135] Example 11: Effects 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. The names of IATERB5-IATERB7 are shown in Table 1 above.

[0136] MCF-7 cells were plated at a density of 50,000 cells / well in 6-well tissue culture plates in DMEM / F12 supplemented with 5% treated FBS. The next day, cells were cultured in the absence and presence of 2′,3′,4′-THC or Figure 4 Cells were treated with vehicle or E2 for 7 days in the presence of one of the six analogs in . The cells were then detached with trypsin, neutralized with medium containing 5% FBS, and resuspended. An appropriate amount of cell suspension was placed in ISOTON II diluent (Thermo Fisher Scientific, Waltham, MA, USA), and the cell number was then measured using a Coulter counter (Beckman, Brea, CA, USA).

[0137] Flow cytometry was performed based on previously described methods (Pan et al., 2016). Briefly, cells were plated at a density of 500,000 cells / well in DMEM / F-12 supplemented with 5% treated FBS in 6-well tissue culture dishes for 48 h. The medium was then replaced with serum-free DMEM / F12 for 24 h. The cells were then treated with vehicle, the absence or presence of 2′,3′,4′-THC or Figure 4The E2 treatment under the condition of one of the six analogs in the above method lasts for 24h.Then the culture medium is sucked out, and the cells are washed with PBS, separated with trypsin and collected by centrifugation at 1700rpm for 5min.The cell pellet is washed with ice-cold PBS, then centrifuged at room temperature for 10min at 1700rpm.The cell pellet is resuspended in 500μL PBS containing 50μg / mL propidium iodide, 0.1% triton X-100, 0.1% sodium citrate and 10μg / mL RNAse.Then the cell suspension is analyzed by BD LSR II flow cytometer (BD Biosciences, SanJose, CA, USA) in the flow cytometry facility of University of California, Berkeley, and the cell percentage of cell cycle stage is determined by using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0138] Unlike estradiol, the three ERβ compounds (IATERB5, IATERB6, and IATERB7) did not increase uterine weight or induce breast cancer tumor formation. Figure 16A-16B .

[0139] Example 12: Luciferase activity in GRE-TK-Luc transfected U2OS-ERβ cells in the presence of 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 licorice, A10, A13 and A15 (HC, 100 nM). Luciferase activity of each compound at concentrations of 0.5 μM, 1 μM, 2.5 μM and 5 μM was recorded. Each compound tested attenuated the effect of HC on ERβ activity.

[0140] Statistical analysis: All data are expressed as mean ± SE or SD from at least biological triplicates. The statistical significance of the difference between the two groups was assessed by Student's t test. For data sets consisting of more than two groups, the statistical significance of the differences between the various groups (treatments) was analyzed by one-way analysis of variance (single-factor ANOVA) test or two-factor ANOVA as specified in the legend. All ANOVA tests were followed by Tukey or Sidak post hoc multiple comparison tests to analyze the significance of the differences between any two different treatment groups or controls, as shown in the legend. GraphPad Prism version 6 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis and drawing. The statistical significance of the asterisk numbers in the figure is *p<0.05; **p<0.01, ***p<0.001 and ****p<0.0001.

[0141] Although many embodiments of pharmaceutical compositions and estrogenic methods are described herein, it will be appreciated by those skilled in the art that the examples may be altered to provide other embodiments utilizing ERβ compounds or combinations of ERβ compounds as described herein, the compositions and methods described herein. It will be understood, therefore, that the scope of the invention is defined by the appended claims rather than by the specific embodiments presented by way of example.

Claims

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

2. The compound of claim 1, wherein the ERβ selective estrogen compound is a member of the group consisting of: liquiritigenin; apigenin; luteolin; galangin; naringenin; calycosin; 6-methoxyluteolin; nifolate; wogonin; burdock 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 or a pharmaceutically acceptable salt thereof.

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

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

5. A pharmaceutical composition comprising (1) one or more pharmaceutically acceptable ingredients other than an ER[beta] selective compound and (2) a compound according to any one of claims 1 to 3 or a combination according to claim 4.

6. The pharmaceutical composition of claim 5, wherein at least one pharmaceutically acceptable ingredient other than the ER[beta] selective compound is estradiol or other ER[alpha] modulating compound.

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

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 progestins, one or more glucocorticoids, and one or more androgens.

9. The pharmaceutical composition according to any one of claims 5-8, for use in treating a condition or disease in a patient requiring treatment with a nuclear receptor reprogramming compound.

10. The pharmaceutical composition of claim 9, wherein the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer.

11. A pharmaceutical composition according to claim 9 or claim 10, wherein the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, night awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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. A compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, a combination according to claim 4, or a pharmaceutical composition according to any one of claims 5-8, for use in treating a condition or disease state in a patient requiring treatment with a nuclear receptor reprogramming compound.

13. The compound, combination or composition of claim 12, wherein the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder or cancer.

14. A compound, combination or composition according to claim 11 or claim 12, wherein the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, night awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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.

15. Use of a compound, combination or pharmaceutical composition according to any one of claims 1 to 14 for the manufacture of a medicament for use in treating a condition or disease in a patient requiring treatment with a nuclear receptor reprogramming compound.

16. The use of claim 15, wherein the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer.

17. The use of claim 15 or claim 16, wherein the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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. A method of treating a patient in need of such treatment, wherein the patient in need of treatment has a condition or disease state, the method comprising administering to the patient an effective amount of a compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, a combination according to claim 4, or a pharmaceutical composition according to any one of claims 5-8.

19. The method of claim 18, wherein the condition or disease state is a developmental condition, a menstrual condition, a reproductive condition, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer.

20. The method of claim 17 or claim 18, wherein the condition 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, Sjögren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, vasomotor symptoms of menopause and perimenopause, insomnia, nighttime awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, dyspareunia, 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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