Estrogen compounds and methods of use

Abstract

Estrogen compounds, compositions, and methods are provided. The novel estrogen compounds include novel chalcone derivatives and analogs that have little or no measurable estrogen (estradiol, E2) activity but enhance E2 activity without binding to the estrogen receptor (ER). The novel compositions comprise one or more novel 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 pharmaceutical composition a pharmaceutical composition comprising an estrogen compound described herein.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 393,635, 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 techniques, such as in vitro fertilization (IVF), the success rate of these techniques 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 a treatment option that has one or more of the 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 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] In the present invention, we propose a new class of drugs to pharmacologically regulate estrogen receptor (ER) activity. These are nuclear receptor reprogramming (NRRP) drugs, which are described in the following specification and the attached claims and drawings. These drugs are neither agonists nor antagonists, nor do they produce mixed agonist / antagonist activity. They do not activate reporter genes like estradiol, nor do they block the effects of estradiol like tamoxifen. On the contrary, this class of compounds produces synergistic activation of reporter genes in the presence of estradiol (E2). Individually, NRRP compounds have almost no agonistic or antagonistic effects of estradiol. However, the combination of E2 and NRRP regulates two major types of genes. One class of genes is synergistically activated by the NRRP / E2 combination. Another class of genes is called reprogramming genes because they are not regulated by ERα unless NRRP is added in the presence of physiological concentrations of E2. The effect of NRRP is mediated by ER because when ER is not present, they do not cause synergy with estradiol, or they are inhibited by ER antagonists or blocked by GPER1 antagonists.

[0019] These compounds alone or in combination with estrogens may be useful for a variety of indications because they result in alterations in estrogen activity. Unlike estradiol alone, the combination of NRRPs with estradiol did not result in proliferation of breast or uterine cancer cells, suggesting that their use would enhance beneficial estrogen activity and reduce the risk of MHT-associated breast and uterine cancer.

[0020] 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

[0022] Features and advantages of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings, in which:

[0023] Figure 1-Figure 3 Shown are the structure-activity relationships of several chalcone derivatives alone and in combination with E2 in ERE-TK-Luc-transfected U2OS cells.

[0024] Figure 4 MCF-7 cell proliferation of several chalcone derivatives alone and in combination with E2 is shown.

[0025] Figure 5 Shown are the KRT19 and FGR expression levels of several chalcone derivatives in U2OS-ERα cells.

[0026] Figure 6 Shown are the luciferase activities of 2',3',4'-trihydroxychalcone and Compound A9 in the presence and absence of E2 in ERE-TK-Luc-transfected Ishikawa cells.

[0027] Figure 7 Shown are luciferase activities in NKG2E-TK-Luc-transfected U2OS-ERβ cells in the presence of doxycycline and several chalcone derivatives.

[0028] Figure 8 The structures of several chalcone derivatives are provided.

[0029] Fig. 9 and Fig.10 Shown are luciferase activities in NKG2E-TK-Luc-transfected U2OS-ERβ cells in the presence of doxycycline and several chalcone derivatives.

[0030] Fig.11 Shown are the results of a cell proliferation assay in MC7 cells incubated with several chalcone derivatives in the presence and absence of E2. DETAILED DESCRIPTION OF THE INVENTION

[0032] Nuclear receptor reprogramming compounds are described herein. In some embodiments, the nuclear receptor reprogramming compound is not an agonist, antagonist, or mixed agonist / antagonist of the estrogen receptor (ER), and / or the compound modulates estradiol (E2) activity only when estradiol (E2) is present at physiological levels. In some embodiments, the compound has a structure of Formula I or a pharmaceutically acceptable salt thereof, wherein the compound of Formula I is:

[0033]

[0034] in,

[0035] Each is a single bond or a double bond;

[0036] A 1 , A 2 and A 3 Independently CR 1 or N,

[0037] A 4 Yes CR 6 or N;

[0038] A 5 , A 6 and A 7 Independently CR 2 or N,

[0039] A 8 Yes CR 7 or N,

[0040] A 9 Yes CR 3 , CHR 3 , N or NR 3 ,

[0041] Each R 1 are independently H, halogen, C1-C4 alkyl, OH, NH2 or O-C1-C4 alkyl;

[0042] Each R 2 are independently H or OH,

[0043] R 3 is H or C1-C4 alkyl,

[0044] R 4 is O or OH, or with R 7 Together they form a pyrido or pyrimido ring to form a quinoline, isoquinoline or quinazoline bicyclic ring system;

[0045] R 5 is H, or with R 6Together they form an -O-bridge to form a benzopyran bicyclic ring system;

[0046] R 6 is H or HO, or with R 5 together to form an -O- bridge to form a benzopyran bicyclic ring; and

[0047] R 7 is H, OH, or with R 4 Together they form a pyrido or pyrimidine ring to form a quinoline, isoquinoline or quinazoline bicyclic ring system.

[0048] In some embodiments of the compound or salt of Formula I, each R 1 are independently H, F, CH3, OH, NH2 or O-CH3.

[0049] In some embodiments of the compound or salt of Formula I, A 4 It is CH, A 5 It is CH, A 6 is CH or COH, A 7 is CH or COH, A 8 is CH or COH.

[0050] In some embodiments of the compound or salt of Formula I, the 4 of is a double bond, and R 4 It's O.

[0051] In some embodiments of the compound or salt of Formula I, linked to A 9 of It's a double bond.

[0052] In some embodiments of the compound or salt of Formula I, A 1 , A 2 and A 3 One and only one of them is N, and the other two are CR 1 .

[0053] In some embodiments of the compound or salt of Formula I, each R 1 It's H.

[0054] In some embodiments of the compound or salt of Formula I, at least one R 1 is OH, and the other R 1 It is H or OH.

[0055] In some embodiments of the compound or salt of Formula I, one R 1 is OH, and the other R 1 It's H.

[0056] In some embodiments of the compound or salt of Formula I, A 3 Yes N, A 2 is CH, and A 3 It's C-OH.

[0057] In some embodiments of the compound or salt of Formula I, A 6 , A 7 or A 8 At least one of them is N.

[0058] In some embodiments of the compound or salt of Formula I, A 6 , A 7 and A 8 Only one of them is N.

[0059] In some embodiments of the compound or salt of Formula I, A 1 It's C-OH.

[0060] In some embodiments of the compound or salt of Formula I, A 2 and A 3 It is CH.

[0061] In some embodiments of the compound or salt of Formula I, A 9 Yes CR 3 , and R 3 It is H or C1-C4 alkyl.

[0062] In some embodiments of the compound or salt of Formula I, R 3 It is CH3.

[0063] In some embodiments of the compound or salt of Formula I, linked to A 9 of It is a single bond.

[0064] In some embodiments of the compound or salt of Formula I, A 9 It is CHR 3 .

[0065] In some embodiments of the compound or salt of Formula I, R 3 It is H or CH3.

[0066] In some embodiments of the compound or salt of Formula I, A 9 It's NH.

[0067] In some embodiments of the compound or salt of Formula I, the 4 of It is a single bond.

[0068] In some embodiments of the compound or salt of Formula I, R 4 It's OH.

[0069] In some embodiments of the compound or salt of Formula I, R 4 and R 7 Together they form a pyrido ring or a pyrimido ring fused to an adjacent benzo ring.

[0070] In some embodiments of the compound or salt of Formula I, R 4 and R 7 Together they form a pyrido ring which is fused to an adjacent benzo ring to form quinoline or isoquinoline.

[0071] In some embodiments of the compound or salt of Formula I, R 4 and R 7 Together they form a pyrimido ring fused to an adjacent benzo ring to form a quinazoline.

[0072] In some embodiments of the compound or salt of Formula I, A 6 and A 7 It's C-OH.

[0073] In some embodiments of the compound or salt of Formula I, A 4 and A 5 It is CH.

[0074] In some embodiments of the compound or salt of Formula I, R 5 and R 6 Together they form an -O- bridge.

[0075] In some embodiments of the compound or salt of Formula I, the compound is one of:

[0076] Here, "E" indicates that the vinyl bond is trans (entgegen).

[0077] In some embodiments of the compound or salt of Formula I, the compound is:

[0078]

[0079] In some embodiments of the compound of Formula I, the compound is:

[0080]

[0081] wherein "E" indicates that the vinyl bond is trans. In some embodiments of the compound of Formula I, the compound is:

[0082]

[0083] In some embodiments of the compound of Formula I, the compound is:

[0084]

[0085] wherein "E" indicates that the vinyl bond is trans. In some embodiments of the compound of Formula I, the compound is:

[0086]

[0087] In some embodiments of the compound of Formula I, the compound is:

[0088]

[0089] In some embodiments of the compound of Formula I, the compound is:

[0090] In some embodiments of the compound of Formula I, the compound is:

[0091] wherein "E" indicates that the vinyl bond is trans. In some embodiments of the compound of Formula I, the compound is:

[0092]

[0093] In some embodiments of the compound of Formula I, the compound is:

[0094]

[0095]

[0096] wherein "E" indicates that the vinyl bond is trans. In some embodiments of the compound of Formula I, the compound is:

[0097]

[0098] In some embodiments of the compound of Formula I, the compound is:

[0099]

[0100] wherein E represents that the vinyl bond is trans. In some embodiments of the compound of Formula I, the compound is:

[0101]

[0102] In some embodiments of the compound of Formula I, the compound is:

[0103]

[0104] wherein "E" indicates that the vinyl bond is trans, and "Z" indicates that the vinyl bond is cis (zusammen).

[0105] In some embodiments of the compound of Formula I, the compound is:

[0106] The "E" indicates that the vinyl bond is trans.

[0107] In some embodiments of the compound of Formula I, the compound is:

[0108]

[0109] In some embodiments of the compound of Formula I, the compound is:

[0110] Where E indicates that the vinyl bond is trans.

[0111] In some embodiments of the compound of Formula I, the compound is:

[0112]

[0113] In some embodiments of the compound of Formula I, the compound is:

[0114]

[0115] The compounds of formula I and pharmaceutically acceptable salts thereof are NRRP compounds that enhance the in vitro activity of estrogen receptor alpha (ERα) in the presence of estradiol (E2), but neither agonize nor antagonize ERα in the absence of E2, and reprogram one or more genes under the control of estrogen. The compounds of formula I have utility in treating one or more disorders, as discussed in more detail below.

[0116] Some embodiments described herein include pharmaceutical compositions comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may further comprise 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.

[0117] synthesis

[0118] The compound of the present invention can be obtained as follows.

[0119] Variations of the "A" ring: Compounds of the following formula can be synthesized according to the following general method:

[0120] in:

[0121] “A” represents the A ring of chalcone;

[0122] A 1 , A 2 and A 3 Independently CR 1 or N; and

[0123] Each R 1 are independently H, halogen, C1-C4 alkyl, OH, NH2 or O-C1-C4 alkyl,

[0124]

[0125] , where A 1 , A 2 and A 3 has the meaning defined for the compound of formula I. Compounds of formula 1 and compounds of formula 2 are commercially available. For example, following this procedure, the "A" ring can be explored as follows:

[0126]

[0127] The compound of the formula:

[0128]

[0129] Among them A 4 -A 8 Having the definition in formula I, and "B" represents the B ring of chalcone, it can be synthesized by the following reaction:

[0130]

[0131] , where A 4 -A 8 has the meaning defined for compounds of formula I. Compounds of formula 3 and compounds of formula 4 are commercially available. Following this procedure, the "B" ring can be explored as follows:

[0132]

[0133] The compound having a nitrogen-containing linking group between the A ring and the B ring can be synthesized by the following synthetic reaction scheme:

[0134]

[0135] , where A 1 -A8 Defined in Formula I. Compound 5 and Compound 6 are commercially available.

[0136] In some embodiments, wherein A 4 -A 8 One of the hydroxyl groups is OH, and one or more of the hydroxyl groups may be methylated, in which case a second methyl group removal step may be employed to generate an OH group. For example, the following reaction scheme generates compound A21:

[0137]

[0138] Conformationally constrained compounds such as compound A24 can be prepared by the following reaction:

[0139]

[0140] Pharmaceutical composition

[0141] The pharmaceutical compositions described herein include pharmaceutically acceptable ingredients. At least one of the pharmaceutically acceptable ingredients may include one or more ingredients that do not naturally occur with the novel compounds, salts or solvates disclosed herein. Pharmaceutically acceptable ingredients that do not naturally occur with the novel compounds, salts or solvates disclosed herein in nature may include sterile, isotonic or non-pyrogenic excipients.

[0142] 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 NRRP compound 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.

[0143] In some embodiments, the pharmaceutical composition may be an estrogen composition. The estrogen composition comprises an estrogen effective amount of a NRRP 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 NRRP compound in nature. In particular, the excipient may include at least one compound that does not naturally occur in the human body with the NRRP compound as 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 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 NRRP compound as described herein in nature. In some embodiments, the estrogen composition may be sterile, pyrogen-free and / or isotonic.

[0144] 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.

[0145] 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 help NRRP compounds as described herein resist the transport of one or more external or internal physiological barriers such as the lung epithelial barrier or the blood-brain barrier.

[0146] 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.

[0147] Effective dose of estrogen

[0148] The effective dose of the NRRP compound 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, co-administration with other drugs or compounds, etc. Typically, the NRRP compounds described herein are effective in vitro at nanomolar or micromolar concentrations. The effective daily dose of the NRRP compound 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 estrogen 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 of the estrogen composition may be similar to that of the NRRP compound as described herein, but may be scaled to take into account the greater molecular weight compared to the NRRP compound as described herein in terms of the relative biological activity, pharmacokinetics and pharmacodynamics of the compound, and those skilled in the art know how to determine by methods recognized in the art.

[0149] 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 NRRP 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).

[0150] 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)).

[0151] In some embodiments, the estrogen composition may include a NRRP compound 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 in 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 a NRRP compound as described herein.

[0152] NRRP compounds and pharmaceutically acceptable carriers or excipients as described herein can be formulated into dosage forms suitable for administration to a subject by 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 specific dosage form selected. In addition, suitable pharmaceutically acceptable carriers or excipients may be selected for the specific functions they may perform in the composition. For example, certain pharmaceutically acceptable carriers or excipients may be selected for their ability to promote the production of uniform dosage forms. Certain pharmaceutically acceptable carriers or excipients may be selected for their ability to promote the production of stable dosage forms. Certain pharmaceutically acceptable carriers or excipients may be selected for their ability to promote 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 chosen for their ability to enhance patient compliance.

[0153] In some embodiments, estrogen NRRP 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 make 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. Compositions can be presented in unit doses or multi-dose containers, such as sealed ampoules and vials, and can be stored in freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid carriers such as water for injection 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.

[0154] oral

[0155] The estrogen composition can be an oral estrogen composition comprising a NRRP compound as described herein and at least one excipient suitable for oral administration. At least one excipient suitable for oral administration can include a compound that does not naturally occur with the NRRP compound as described herein in nature. 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, pouches, cachets, gel capsules, elixirs, pills, oral sprays, chewable tablets, sublingual tablets, films or sprays, or buccal films or sprays.

[0156] In some embodiments, NRRP compounds as described herein can be formulated as solid oral dosage forms, such as tablets or capsules 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. Oral solid dosage forms can also include adhesives. Suitable adhesives include starch (e.g., corn starch, potato starch, and pregelatinized starch), gelatin, gum arabic, sodium alginate, alginic acid, tragacanth, guar gum, polyvidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). Oral solid dosage forms can also include disintegrants. Suitable disintegrants include cross-linked polyvidone, sodium starch glycolate, cross-linked carboxymethyl cellulose, alginic acid, and sodium carboxymethyl cellulose. Oral solid dosage forms can also include lubricants. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0157] 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.

[0158] NRRP compounds as described herein can also be combined with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamidephenol, polyhydroxyethyl aspartame-indophenol or polyethylene oxide-polylysine substituted by palmitoyl residues. In addition, NRRP 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 polylactic acid, poly-ε-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, crosslinked or amphiphilic block copolymers of polycyanoacrylates and hydrogels.

[0159] In some embodiments, NRRP compounds as described herein can be formulated into liquid oral dosage forms. 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 compounds disclosed herein. Syrups can be prepared by dissolving the compounds 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.

[0160] Intranasal

[0161] The estrogen composition can be an intranasal estrogen composition comprising a NRRP compound 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 of the NRRP compound across the mucosa as described herein and / or increase bioavailability. 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 preparations (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 an alkyl glycoside 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 an appropriate hydrophile-lipophile balance (HLB) number, which can be determined as disclosed in U.S. Pregrant Publication No. US2009 / 0047347, which is incorporated herein by reference in its entirety.

[0162] The intranasal estrogen compositions of NRRP compounds as described herein may also include flavoring agents or fragrances to cover the taste of the NRRP compounds as described herein. The intranasal compositions may also include an isotonicity agent to make the composition isotonic. The intranasal estrogen compositions of NRRP compounds as described herein may also include a stabilizer.

[0163] In the lungs

[0164] The estrogen composition can be an intrapulmonary estrogen composition comprising a NRRP compound as described herein and at least one excipient suitable for intrapulmonary administration. 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 NRRP compound as described herein to pass through the lung epithelium into the bloodstream.

[0165] 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.

[0166] 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.

[0167] 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 a NRRP compound as described herein.

[0168] In some embodiments, a high efficiency nebulizer provides lung deposition of a NRRP compound as described herein (deposited lung dose) of at least about 5% based on the nominal dose of the NRRP compound as described herein.

[0169] 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 a NRRP compound as described herein for estrogen therapy. The unit dosage form includes a container containing an inhalation solution containing a NRRP compound 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 allowed to 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 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 a NRRP compound 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.

[0170] 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.

[0171] 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

[0172] 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.

[0173] 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.

[0174] 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

[0175] Intravenous

[0176] The estrogen composition can be an intravenous estrogen composition comprising a NRRP compound 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. The intravenous composition of the NRRP compound as described herein is a parenteral composition intended to be administered intravenously by injection or infusion. They can include one or more isotonic agents to make the composition isotonic. They can be and usually are sterile, pyrogen-free or both.

[0177] Subcutaneous

[0178] The estrogen composition can be a subcutaneous estrogen composition comprising a NRRP compound 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 NRRP compounds as described herein are parenteral compositions intended for subcutaneous injection. They can include one or more isotonic agents to make the composition isotonic. They can be and are typically sterile, pyrogen-free, or both.

[0179] Percutaneous

[0180] The estrogen composition can be a transdermal estrogen composition comprising a NRRP compound as described herein and at least one excipient suitable for transdermal administration. At least one excipient suitable for transdermal administration can include at least one compound other than water. For example, the transdermal estrogen composition can include one or more penetration enhancers that increase the ability of the NRRP compound 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).

[0181] Sublingual or buccal

[0182] The estrogen composition can be a sublingual or buccal estrogen composition comprising a NRRP compound 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.

[0183] intraperitoneally

[0184] The estrogen composition can be an intraperitoneal estrogen composition comprising a NRRP compound as described herein and at least one excipient suitable for intraperitoneal administration. At least one excipient suitable for intraperitoneal administration can include at least one compound other than water. The intraperitoneal estrogen composition of the NRRP compound as described herein is intended to be administered to the peritoneum by injection or infusion. They can include one or more isotonic agents to make the composition isotonic. They can be and are typically sterile, pyrogen-free or both.

[0185] Intrathecal or intraventricular

[0186] The estrogen composition can be an intrathecal or intraventricular estrogen composition comprising an NRRP compound as described herein and at least one excipient suitable for intrathecal or intraventricular administration. At least one excipient suitable for intrathecal or intraventricular administration can include at least one compound other than a compound (e.g., water) naturally occurring with the NRRP compound as described herein in nature. The intrathecal or intraventricular estrogen composition of the NRRP compound as described herein is a parenteral composition intended to be administered to the cerebrospinal fluid by injection or infusion. They can include one or more isotonic agents to make the composition isotonic. They can be and are typically sterile, pyrogen-free or both.

[0187] Other routes of administration

[0188] 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.

[0189] Estrogen method

[0190] Some embodiments described herein provide a compound of Formula I described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition for use in treating a condition or disease state of 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 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.

[0191] Some embodiments described herein provide the use of a compound of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in treating a condition or disease of 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 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.

[0192] 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 Formula I 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.

[0193] According to the disclosure herein, those skilled in the art understand how to determine the estrogen effective amount of the NRRP compound as described herein. Generally, the estrogen effective amount, therapeutic effective amount or preventive effective amount of the NRRP compound as described herein can be determined, for example, by inferring from an in vitro test. It is understood by those skilled in the art that the effective dose can be inferred from the in vitro half-maximum regulation (inhibition or activation) concentration of the NRRP compound as described herein. It is understood by those skilled in the art that the effective dose in human patients will depend on the route of administration, pharmacokinetics, etc. Taking these factors into account, the estrogen effective dose of the NRRP compound as described herein can be in the range of 0.1 mg / kg to 150 mg / kg, such as 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; the effective daily dose of the NRRP compound as described herein can be some multiples of any value within these ranges, such as one to six (1 to 6) times of the value within these ranges.

[0194] Transition Phrases

[0195] 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

[0196] The pharmaceutical compositions and estrogenic methods disclosed herein can be further understood with reference to the following examples.

[0197] Example 1: Analogs of 2',3',4'-trihydroxychalcone (CC7).

[0198] Analogs of the parent CC7 were prepared as listed in Tables 1A-1C below.

[0199]

[0200]

[0201]

[0202]

[0203] Example 2: Estrogen receptor (ERα) stimulation in U2OS cells

[0204] Nuclear receptor reprogramming (NRRP) compounds are described herein, including those listed in Table 1A, Table 1B, and Table 1C ("test compounds"), which are neither E2 agonists nor E2 antagonists, nor do they produce mixed E2 agonist / antagonist activity. They do not activate reporter genes like E2. They do not block the effect of E2 on ERα like tamoxifen. Instead, these compounds produce synergistic activation of reporter genes in the presence of E2. Individually, NRRP compounds have little or no estrogen-like agonist or antagonist effect on ERE. However, despite the lack of activity in the absence of E2, in the presence of E2, NRRP works synergistically to regulate two major types of genes. One type of gene is synergistically activated by the NRRP / E2 combination. Another type of gene is called reprogramming genes because they are not regulated by ERα unless NRRP is added to the physiological concentration of E2. The effect of NRRP is mediated by ER because they do not produce synergy with E2 when ER is absent.

[0205] 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 (ERE-TK-Luc) upstream of a minimal thymidine kinase luciferase promoter and 1 μg of an ERα expression vector as previously described (An et al., 2001). The resulting ERE-TK-Luc / ERα U2OS cells were incubated in the presence of culture medium (negative control), E2, and test compounds (with and without E2) 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). See Figure 1-Figure 3 The red horizontal line in Figure 1-Figure 3As can be seen in the Figure 1, no significant activation of ERE-TK-Luc occurred using the test compounds alone. In contrast, and unexpectedly, when combined with E2, the test compounds resulted in varying levels of synergistic activation of ERE. These activities are considered synergistic because the activity of each test compound plus E2 is greater than the sum of the activity of E2 plus the activity of the corresponding test compound.

[0206] Example 3: MCF-7 cell proliferation

[0207] In order for long-term menopausal hormone therapy to become a viable alternative to currently available treatment options, it must not promote breast cancer. The growth promoting properties of several compounds of the invention alone and in combination with E2 were studied in MCF-7 breast cancer cells. MCF-7 cells were incubated in medium (control), 1 nM E2 or 5 μM Figure 4 Incubations were continued for 24 hours in the presence of each compound depicted in (CC7 and six analogs, A9, A10, A11, A13, A15 and A20). Viable cell counts were determined by flow cytometry.

[0208] 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 the 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).

[0209] 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 500μLPBS containing 50μg / mL propidium iodide, 0.1% triton X-100, 0.1% sodium citrate and 10μg / mL RNAse.Then the cell suspension is analyzed with the BD LSR II flow cytometer (BD Biosciences, SanJose, CA, USA) in the flow cytometry facility of the University of California, Berkeley, and the cell percentage of the cell cycle stage is determined by using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0210] like Figure 4 As can be seen in the , single E2 stimulates breast cancer cell proliferation. In contrast, the viable cell counts of each of CC7 and six analogs are much lower than the cell count of single E2. The cell count of A9+E2 is much lower than the control, the control is lower than the cell count of E2, and the cell counts of CC7+E2 and A10+E2 are much lower than the cell count of single E2. These results show that CC7 and six analogs do not stimulate MCF-7 breast cancer cell proliferation like E2. In addition, each of the six analogs is added to E2 will not enhance the stimulation of E2 to MCF-7 cell proliferation, and even inhibit the pro-stimulatory activity of E2 in some cases.

[0211] Example 4: Gene expression assay

[0212] Human U2OS (osteosarcoma) cells expressing tetracycline-regulated ERα (U2OS-ERα) were prepared, characterized, and maintained as previously described (Tee et al., 2004). Cells were maintained in phenol red-free Gibco DMEM / F-12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% charcoal-dextran treated fetal bovine serum (FBS, Gemini Bio Products, West Sacramento, CA, USA), 100 units / mL penicillin and streptomycin, 50 μg / mL amphotericin B (Fungizone), and 2 mM glutamine. To maintain stable transfected cells, 50 μg / mL hygromycin B and 500 μg / mL zeocin (Invitrogen, Waltham, MA, USA) were included in the culture medium.

[0213] U2OS-ERα cells were treated with E2 in the absence or combination of 2',3',4'-THC(CC7) and subjected to real-time PCR analysis. E2 alone activated KRT19 ( Figure 5 ), and a large synergistic effect with CC7 was observed. In contrast, the "reprogramming" gene FGR was only activated by the combination of E2 and CC7, but not by either compound by itself.

[0214] Similar experiments were performed with CC7 analogs, namely A9, A10, A13, A15 and A20. The results are shown in Figure 5 .

[0215] Example 5: Luciferase activity of ERE-TK-Luc in Ishikawa cells

[0216] Ishikawa cells transfected with the ERE-TK-Luc reporter gene and ERα were incubated with E2 alone, CC7 alone, A9 alone, CC7+E2, or A9+E2. Figure 6 CC7 and A9 had no effect on the luciferase activity of transfected cells, demonstrating that these compounds themselves have no estrogenic effects. However, when combined with E2, CC7 and A9 each exhibited synergistic effects with E2, as shown in Figure 6 It can be seen in.

[0217] Example 6: Luciferase activity in NKG2E-TK-Luc transfected U2OS-ERβ cells in the presence of doxycycline. Doxycycline-dependent U2OS cells were transfected with NKG2E-TK-Luk and ERβ in the presence of E2 alone or in combination with each compound of Table 1A, Table 1B, and Table 1C. The luciferase activity of each compound was recorded. See Figure 7 .like Figure 7 As can be seen in Figure 3, A10, A13, A14 and A15 showed abnormal ERβ activity.

[0218] Example 7: Synthesis of compounds in Table 1A, Table 1B and Table 1C. The compounds listed in Table 1A, Table 1B and Table 1C were prepared according to the synthetic procedures listed below.

[0219] A1

[0220] Synthesis of (E)-3-(4-fluorophenyl)-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 4-fluorobenzaldehyde a (354 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-(4-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0221]

[0222] Synthesis of (E)-3-(4-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A1. At 0 ° C under N2, a solution of (E)-3-(4-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (200 mg, 0.63 mmol) in dry DCM (5 mL) was added dropwise with BBr3 (0.95 g, 3.8 mmol). 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-(4-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A1 (63 mg, 36%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.35 (s, 1H), 10.16 (s, 1H), 8.64 (s, 1H), 8.05-7.88 (m, 3H), 7.85-7.63 (m, 2H), 7.31 (t, J = 8.8Hz, 2H), 6.45 (d, J = 8.9Hz, 1H).

[0223]

[0224] A2

[0225] Synthesis of (E)-3-(3-fluorophenyl)-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-fluorobenzaldehyde a (354 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-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0226]

[0227] Synthesis of (E)-3-(3-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A2. At 0 ° C under N2, a solution of (E)-3-(3-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (500mg, 1.58mmol) in dry DCM (5mL) was added dropwise with BBr3 (2g, 7.9mmol). The resulting mixture was stirred at room temperature for 12h 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%, 60min) to give (E)-3-(3-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A2 (65mg, 15%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.31 (s, 1H), 10.25 (s, 1H), 8.70 (s, 1H), 8.03 (d, J = 15.5Hz, 1H), 7.88 (dt, J = 10.1, 2.1Hz, 1 H),7.83-7.74(m,2H),7.69(s,1H),7.51(td,J=8.0,6.1Hz,1H),7.30(td,J=8.7,2.6Hz,1H),6.46(d,J=8.9Hz,1H).

[0228]

[0229] A3

[0230] Synthesis of (E)-3-(2-fluorophenyl)-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 2-fluorobenzaldehyde a (354 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-(2-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0231]

[0232] Synthesis of (E)-3-(2-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one. At 0 ° C under N2, BBr3 (1.2 g, 4.74 mmol) was added dropwise to a solution of (E)-3-(2-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (300 mg, 0.95 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-(2-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A3 (235 mg, 90%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.27 (d, J = 32.2Hz, 1H), 10.27 (d, J = 9.0Hz, 1H), 8.72 (d, J = 9.9Hz, 1H), 8.15 (t, J = 7.2Hz, 1H), 8.03 (dd, J = 15.5, 5. 1Hz,1H),7.90(d,J=15.7Hz,1H),7.82-7.75(m,1H),7.74-7.68(m,1H),7.58-7.44(m,1H),7.41-7.24(m,2H),6.46(dd,J=8.9,3.7Hz,1H).

[0233]

[0234] Synthesis of (E)-3-(m-tolyl)-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) in a solution of 3-methylbenzaldehyde a (354 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-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0235]

[0236] Synthesis of (E)-3-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A4. At 0 ° C under N2, a solution of (E)-3-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (300 mg, 0.95 mmol) in dry DCM (5 mL) was added dropwise with BBr3 (1.2 g, 4.74 mmol). 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-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A4 (140 mg, 54%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.44 (s, 1H), 10.18 (s, 1H), 8.66 (s, 1H), 7.92 (d, J = 15.5Hz ,1H),7.83-7.70(m,4H),7.28(d,J=8.0Hz,2H),6.45(d,J=8.9Hz,1H),2.36(s,3H).

[0237]

[0238] A5

[0239] Synthesis of (E)-3-(m-tolyl)-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) in a solution of 3-methylbenzaldehyde a (342 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-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (150 mg, 6.7%) as a white solid.

[0240]

[0241] Synthesis of (E)-3-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A5. At 0 ° C under N2, a solution of (E)-3-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (150 mg, 0.54 mmol) in dry DCM (5 mL) was added dropwise with BBr3 (684 mg, 2.73 mmol). 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-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A5 (80 mg, 55%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.41 (s, 1H), 10.22 (s, 1H), 8.69 (s, 1H), 7.93 (s, 1H), 7.83-7.71 ( m,3H),7.67(d,J=7.7Hz,1H),7.36(s,1H),7.29(s,1H),6.46(d,J=8.9Hz,1H),2.37(s,3H).

[0242]

[0243] A6

[0244] Synthesis of (E)-3-(o-tolyl)-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) in a solution of 2-methylbenzaldehyde a (354 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-(o-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0245]

[0246] Synthesis of (E)-3-(o-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A6. At 0 ° C under N2, a solution of (E)-3-(o-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (300 mg, 0.95 mmol) in dry DCM (5 mL) was added dropwise with BBr3 (1.2 g, 4.74 mmol). 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-(o-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A6 (150 mg, 95%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.37 (s, 1H), 10.23 (s, 1H), 8.70 (s, 1H), 8.12-7.95 (m, 2H), 7.86 (d, J=15.4Hz,1H),7.75(d,J=9.0Hz,1H),7.40-7.21(m,3H),6.45(d,J=8.9Hz,1H),2.46(s,3H).

[0247]

[0248] A7

[0249] Synthesis of (E)-3-(pyridin-4-yl)-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) in a solution of isonicotinaldehyde (305 mg, 2.8 mmol) and KOH (156 mg, 2.8 mmol). The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give (E)-3-(pyridin-4-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (510 mg, 59%) as a yellow solid.

[0250]

[0251] Synthesis of (E)-3-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A7. At 0 ° C under N2, BBr3 (0.95 g, 3.8 mmol) was added dropwise to a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL). The resulting mixture was stirred at room temperature for 6 h and then quenched with water. The organic layer was concentrated and purified by RP column to give a crude product. The crude product was further purified by preparative HPLC to give (E)-3-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A7 (35 mg, 20%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.14(s,1H),8.72-8.58(m,2H),8.19(d,J=15.6Hz,1H),7.89-7.81(m,2H),7.82-7.68(m,2H),6.47(d,J=8.9Hz,1H).

[0252]

[0253] A8

[0254] Synthesis of (E)-3-(pyridin-3-yl)-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) in a solution of (E)-3-(pyridin-3-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (520 mg, 60%) was added. The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give (E)-3-(pyridin-3-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 as a yellow solid (520 mg, 60%).

[0255]

[0256] Synthesis of (E)-3-(pyridin-3-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A8. At 0 ° C under N2, BBr3 (0.95 g, 3.8 mmol) was added dropwise to a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL). The resulting mixture was stirred at room temperature for 6 h and then quenched with water. The organic layer was concentrated and purified by RP column to give a crude product. The crude product was further purified by preparative HPLC to give (E)-3-(pyridin-3-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A8 (15 mg, 8.7%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.28(s,1H),9.03(d,J=2.1Hz,1H),8.62(dd,J=4.7,1.5Hz,1H),8.37(dt,J=8.1, 2.2Hz, 1H), 8.11 (d, J = 15.6Hz, 1H), 7.92-7.66 (m, 2H), 7.50 (dd, J = 8.0, 4.7Hz, 1H), 6.46 (d, J = 8.9Hz, 1H).

[0257]

[0258] A9

[0259] Synthesis of (E)-3-(pyridin-2-yl)-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) in a solution of (E)-3-(pyridin-2-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (500 mg, 58%) was added. The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give (E)-3-(pyridin-2-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (500 mg, 58%) as a yellow solid.

[0260]

[0261] Synthesis of (E)-3-(pyridin-2-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A9. At 0 ° C under N2, BBr3 (0.95 g, 3.8 mmol) was added dropwise to a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL). The resulting mixture was stirred at room temperature for 6 h and then quenched with water. The organic layer was concentrated and purified by RP column to give a crude product. The crude product was further purified by preparative HPLC to give (E)-3-(pyridin-2-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A9 (30 mg, 17%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ13.15(s,1H),10.31(s,1H),8.80-8.64(m,2H),8.19(d,J=15.3Hz,1H),7.98-7.86(m ,2H),7.78(d,J=15.3Hz,1H),7.61(d,J=9.0Hz,1H),7.45(ddd,J=6.8,4.7,1.9Hz,1H),6.48(d,J=8.9Hz,1H).

[0262]

[0263] A10

[0264] 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.

[0265]

[0266] 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) δ13.60 (s, 1H). 10.12 (a, 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).

[0267]

[0268] A11

[0269] Synthesis of (E)-3-(4-aminophenyl)-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) in a solution of 4-aminobenzaldehyde (338 mg, 2.8 mmol) and KOH (156 mg, 28 mmol). The resulting solution was stirred at rt for 24 h. The mixture was concentrated and purified by FCC to give (E)-3-(4-aminophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-ene-1-one 2 (300 mg, 34%) as a yellow solid.

[0270]

[0271] Synthesis of (E)-3-(4-aminophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A11. At 0°C under N2, BBr3 (0.95g, 3.8mmol) was added dropwise to a solution of 2 (200mg, 0.64mmol) in dry DCM (5mL). The resulting mixture was stirred at room temperature for 6h and then quenched with water. The organic layer was concentrated and purified by RP column to give a crude product. The crude product was further purified by preparative HPLC to give (E)-3-(4-aminophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-ene-1-one A11 (30mg, 17%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ13.86(s,1H),9.99(s,1H),8.55(s,1H),7.76-7.62(m,2 H),7.62-7.52(m,3H),6.59(d,J=8.2Hz,2H),6.40(d,J=8.8Hz,1H),5.95(s,2H).

[0272]

[0273] A18

[0274] Synthesis of 4-((tetrahydro-2H-pyrans-2-yl)oxy)benzaldehyde 2. A solution of 3,4-dihydro-2H-pyrans (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a suspension of well-stirred 4-hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p-toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature overnight, then extracted with saline and dried over MgSO. The solvent was removed under reduced pressure, and the crude compound was purified on silica gel using 20% ​​EtOAc / hexane wash-out by column chromatography to give 2 (2 g, 60%) as a yellow oil.

[0275]

[0276] Synthesis of (E)-1-(pyridin-2-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 3. To a solution of 2 (500 mg, 2.4 mmol) in EtOH (8 mL) was added 1-(pyridin-4-yl)ethan-1-one (293 mg, 2.4 mmol) and KOH (134 mg, 2.4 mmol). The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give the crude product. The crude product was further purified by preparative HPLC to give (E)-1-(pyridin-2-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 3 (150 mg, 20%) as a yellow solid.

[0277]

[0278] Synthesis of (E)-3-(4-hydroxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one. To a solution of 3 (200 mg, 0.64 mmol) in MeOH (1 mL) was added PTSA (11.0 mg, 0.06 mmol). The resulting mixture was stirred at room temperature for 5 h. The mixture was concentrated and purified by RP column to give (E)-3-(4-hydroxyphenyl)-1-(pyridin-2-yl)prop-2-en-1-one A18 (6 mg, 8.3%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ10.25 (s, 1H), 8.79 (dt, J = 4.7, 1.3Hz, 1H), 8.14-8.00 (m, 3H), 7.79 (d, J = 16.0Hz, 1H), 7.74-7.62 (m, 3H), 7.05-6.73 (m, 2H).

[0279]

[0280] A13

[0281] 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.

[0282]

[0283] 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).

[0284]

[0285] A14

[0286] Synthesis of (E)-1-(3,4-dimethoxyphenyl)-3-(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-(3,4-dimethoxyphenyl)ethan-1-one a (397 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-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one 2 (0.64 g, 97%) as a white solid.

[0287]

[0288] Synthesis of (E)-1-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-ene-1-one A14. To a solution of (E)-1-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-ene-1-one 2 (300 mg, 1 mmol) in dry DCM (5 mL) was added dropwise BBr (1.25 g, 5 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 (E)-1-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-ene-1-one A14 (115 mg, 45%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.94 (s, 3H), 7.69 (d, J = 8.6Hz, 2H), 7.63-7.54 (m, 3H), 7.49 (d, J = 2.0Hz, 1H), 6.83 (t, J = 8.8Hz, 3H).

[0289]

[0290] A15

[0291] 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. 1 H 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).

[0292]

[0293] A16

[0294] Synthesis of 4-((tetrahydro-2H-pyrans-2-yl)oxy)benzaldehyde 2. A solution of 3,4-dihydro-2H-pyrans (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a suspension of well-stirred 4-hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p-toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature overnight, then extracted with saline and dried over MgSO. The solvent was removed under reduced pressure, and the crude compound was purified on silica gel using 20% ​​EtOAc / hexane wash-out by column chromatography to give 2 (2 g, 60%) as a yellow oil.

[0295]

[0296] Synthesis of (E)-1-(pyridin-4-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 2. To a solution of 2 (500 mg, 2.4 mmol) in EtOH (8 mL) was added 1-(pyridin-4-yl)ethan-1-one (293 mg, 2.4 mmol) and KOH (134 mg, 2.4 mmol). The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give the crude product. The crude product was further purified by preparative HPLC to give (E)-1-(pyridin-4-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 3 (100 mg, 13%) as a yellow solid.

[0297]

[0298] Synthesis of (E)-3-(4-hydroxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one. To a solution of 3 (100 mg, 0.32 mmol) in MeOH (1 mL) was added PTSA (5.5 mg, 0.03 mmol). The resulting mixture was stirred at room temperature for 5 h. The mixture was concentrated and purified by RP column to give (E)-3-(4-hydroxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one A16 (60 mg, 83%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ10.20 (s, 1H), 8.91-8.66 (m, 2H), 8.02-7.91 (m, 2H), 7.82-7.61 (m, 4H), 6.93-6.76 (m, 2H).

[0299]

[0300] A17

[0301] Synthesis of 4-((tetrahydro-2H-pyrans-2-yl)oxy)benzaldehyde 2. A solution of 3,4-dihydro-2H-pyrans (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a suspension of well-stirred 4-hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p-toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature overnight, then extracted with saline and dried over MgSO. The solvent was removed under reduced pressure, and the crude compound was purified on silica gel using 20% ​​EtOAc / hexane wash-out by column chromatography to give 2 (2 g, 60%) as a yellow oil.

[0302]

[0303] Synthesis of (E)-1-(pyridin-3-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 2. To a solution of 2 (500 mg, 2.4 mmol) in EtOH (8 mL) was added 1-(pyridin-4-yl)ethan-1-one (293 mg, 2.4 mmol) and KOH (134 mg, 2.4 mmol). The resulting solution was stirred at rt for 5 min. The mixture was concentrated and purified by RP column to give the crude product. The crude product was further purified by preparative HPLC to give (E)-1-(pyridin-3-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 3 (150 mg, 20%) as a yellow solid.

[0304]

[0305] Synthesis of (E)-3-(4-hydroxyphenyl)-1-(pyridin-3-yl)prop-2-en-1-one. To a solution of 3 (100 mg, 0.32 mmol) in MeOH (1 mL) was added PTSA (5.5 mg, 0.03 mmol). The resulting mixture was stirred at room temperature for 5 h. The mixture was concentrated and purified by RP column to give (E)-3-(4-hydroxyphenyl)-1-(pyridin-3-yl)prop-2-en-1-one A17 (30 mg, 41%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ10.31-9.99(m,1H),9.30(d,J=2.2Hz,1H),8.81(dd,J=4.8,1.7Hz,1H), 8.44(dt,J=8.0,2.0Hz,1H),7.82-7.69(m,4H),7.60(dd,J=8.0,4.8Hz,1H),6.93-6.79(m,2H).

[0306]

[0307] A20

[0308] Synthesis of (E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (1.0 g, 4.8 mmol) was dissolved in EtOH:H2O (10 mL, V EtOH :V H2O =3:1) in a solution of (E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (1.2 g, 85%) was added. The resulting solution was stirred at rt for 36 h. The mixture was concentrated and purified by FCC to give (E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (1.2 g, 85%) as a yellow solid.

[0309]

[0310] Synthesis of 3-phenyl-1-(2,3,4-trimethoxyphenyl)propan-1-one 3. To a solution of 2 (500 mg, 1 equivalent) in DCM (5 mL) was added 10% Pd / C (50 g). The mixture was stirred at room temperature under H2 atmosphere for 6 h. And then filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure to give 3 (450 mg) as a crude product. The crude product was used in the next step without purification.

[0311]

[0312] The synthesis of 3-phenyl-1-(2,3,4-trihydroxyphenyl)propan-1-one. At 0 ° C under N2, BBr3 (0.95 g, 3.8 mmol) was added dropwise to a solution of 3 (300 mg, 1.0 mmol) in dry DCM (5 mL). The resulting mixture was stirred at room temperature for 6 h, and then quenched with water. The organic layer was concentrated and purified by RP column to give 3-phenyl-1-(2,3,4-trihydroxyphenyl)propan-1-one A20 (100 mg, 39%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ12.57 (s, 1H), 10.07 (s, 1H), 7.36 (d, J = 8.9Hz, 1H), 7.27 (d, J = 4.4Hz, 4 H), 7.18 (q, J = 4.4Hz, 1H), 6.38 (d, J = 8.9Hz, 1H), 3.29 (t, J = 7.6Hz, 2H), 2.93 (t, J = 7.6Hz, 2H).

[0313]

[0314] A21

[0315] Synthesis of N-benzyl-2,3,4-trimethoxybenzamide 2. To a solution of 2,3,4-trimethoxybenzoic acid 1 (1.0 g, 4.7 mmol) in dry DMF (10 mL) was added benzylamine a (0.6 g, 5.6 mmol), HOBt (0.75 g, 5.6 mmol) and EDCI (1.27 g, 6.6 mmol). The resulting solution was stirred at 0 ° C under N2 for 2 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 N-benzyl-2,3,4-trimethoxybenzamide 2 (2 g, 95%) as an oil.

[0316]

[0317] Synthesis of N-benzyl-2,3,4-trihydroxybenzamide. At 0 ° C under N2, BBr3 (1.25 g, 5 mmol) was added dropwise to a solution of N-benzyl-2,3,4-trimethoxybenzamide 2 (300 mg, 1 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 N-benzyl-2,3,4-trihydroxybenzamide A21 (0.3 g, 70%) as a white solid. 1 H NMR (400MHz, DMSO-d6) δ12.96 (s, 1H), 9.50 (s, 1H), 9.10 (t, J = 6.0Hz, 1H), 8 .43(s,1H),7.45-7.15(m,6H),6.31(d,J=8.8Hz,1H),4.47(d,J=5.9Hz,2H).

[0318]

[0319] A24

[0320] Synthesis of 5-(((2,3-dimethoxyphenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione 2. To a solution of dimethoxyaniline 1 (1.0 g, 6.5 mmol) in EtOH (10 mL) was added 2,2-dimethyl-1,3-dioxane-4,6-dione (0.94 g, 6.5 mmol). The resulting solution was stirred at 85 ° C for 2.5 h. The mixture was filtered and washed with EtOH to give 5-(((2,3-dimethoxyphenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione 2 (1.5 g, 75%) as a yellow solid. The crude product was used in the next step without purification.

[0321] Synthesis of 7,8-dimethoxyquinoline-4-ol 3. A solution of 2 (900 mg, 1 eq.) in diphenyl ether (15 mL) was stirred at 260 °C for 1 h. The precipitate was filtered and washed with hexane. The brown solid was dried under vacuum to give 7,8-dimethoxyquinoline-4-ol 3 (540 mg, 90% yield).

[0322]

[0323] Synthesis of 4-chloro-7,8-dimethoxyquinoline 4. 3 (540 mg, 2.6 mmol) and the reaction mixture in phosphorus oxychloride (10 mL) were heated to 110 ° C for 1 hour. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude product was diluted with water (20 mL), and the product was extracted with ethyl acetate (2×100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to produce 4-chloro-7,8-dimethoxyquinoline 4 (300 mg, 51% yield) as a yellow solid.

[0324] Synthesis of (E)-7,8-dimethoxy-4-phenylvinylquinoline 5. A mixture of 4 (200 mg, 0.9 mmol), (E)-phenylvinylboronic acid (132 mg, 0.9 mmol), KCO (278 mg, 2.0 mmol) and Pd(dppf)Cl (37 g, 0.05 mmol) in 1,4-dioxane (1 mL) / H0 (0.3 mL) was stirred at 95 °C for 10 hours. The mixture was cooled to room temperature, filtered through a silica gel pad (EtOAc) and concentrated. The crude product was chromatographed on silica gel to provide (E)-7,8-dimethoxy-4-phenylvinylquinoline 5 (130 g, 50%) as a yellow solid.

[0325]

[0326] (E)-4-phenylvinylquinoline-7,8-diol synthesis. At 0 ° C under N2, to 5 (130mg, 1.0mmol) in dry DCM (5mL) solution dropwise added BBr3 (0.95g, 3.8mmol). The resulting mixture was stirred at room temperature for 6h, and then quenched with water. The organic layer was concentrated and purified by RP column to give (E)-4-phenylvinylquinoline-7,8-diol A24 (40mg, 34%) as a yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.29 (s, 1H), 8.74 (d, J = 4.6Hz, 1H), 8.14 (s, 1H), 7.99 (d, J = 16.2Hz, 1H), 7.82 (dd, J = 8.4, 5.1Hz ,3H),7.67(d,J=4.6Hz,1H),7.56(d,J=16.2Hz,1H),7.44(t,J=7.5Hz,2H),7.37(d,J=7.3Hz,1H),7.24(d,J=9.1Hz,1H).

[0327]

[0328] Example 8: Analogs A9R1, A9R2, A9R3 and A9R4 ( Figure 8 ) is synthesized as follows:

[0329] A9R1: Synthesis of (E)-1-(4-hydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-one. To a solution of 1-(4-hydroxyphenyl)ethan-1-one 1 (200 mg, 1.47 mmol) in EtOH (4 mL) was added picolinaldehyde 2 (157 mg, 1.47 mmol) and NaOH (588 mg, 14.7 mmol in 1 mL H2O). The reaction was stirred at RT for 2 h. The mixture was diluted with water and neutralized with 1M HCl aqueous solution (pH adjusted to 7). The precipitate was filtered and collected as a crude product. The crude product was purified by recrystallization from methanol to give the title compound C9529-1 (29 mg, 9%) as a yellow solid. LCMS (ESI): m / z, 226 [M+H]+, RT=2.55 min. HPLC: 96%, RT=3.42 min. 1H NMR (400MHz, DMSO-d6) δ10.49(s,1H),8.68(dt,J=4.7,1.4Hz,1H),8.14(d,J=15.4Hz,1H),8.05 -8.00(m,2H),7.92-7.87(m,2H),7.67(d,J=15.4Hz,1H),7.47-7.39(m,1H),6.96-6.88(m,2H).

[0330]

[0331] A9R2: Synthesis of (E)-1-(4-hydroxy-2-methylphenyl)-3-(pyridin-2-yl)prop-2-ene-1-one. To a stirred solution of 1-(4-hydroxy-2-methylphenyl)ethan-1-one 1 (100 mg, 0.67 mmol, 1 eq.) in EtOH (2 mL) was added picolinaldehyde 2 (356.63 mg, 3.33 mmol, 5 eq.), 6M NaOH (480 mg, 13.32 mmol, 20 eq.). The reaction mixture was stirred at room temperature for 5 h. The mixture was extracted with EA and washed with H2O and brine. The product was rotary evaporated with a vacuum pump under reduced pressure, recrystallized with methanol and filtered. After being evacuated with a vacuum pump, a yellow solid (26.4 mg, 16.3%) was obtained. 1H NMR (400MHz, DMSO-d6) δ 10.18 (s, 1H), 8.66 (d, J = 4.8Hz, 1H), 7.91-7.70 (m, 4H), 7.53-7.38 (m, 2H), 6.73 (d, J = 8.0Hz, 2H), 2.43 (s, 3H).

[0332]

[0333] A9R3: Synthesis of (E)-1-(2-fluoro-4-hydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-one. To a solution of 1-(2-fluoro-4-hydroxyphenyl)ethan-1-one 1 (100 mg, 0.65 mmol) in EtOH (2 mL) was added picolinaldehyde 2 (70 mg, 0.65 mmol) and NaOH (260 mg, 6.5 mmol in 0.5 mL H2O). The reaction was stirred at RT for 2 h. The mixture was diluted with water and neutralized with 1 M HCl aqueous solution (pH adjusted to 7). The precipitate was filtered and collected as a crude product. The crude product was purified by recrystallization from methanol to give the title compound (21 mg, Y=13%) as a yellow solid. LCMS (+ESI): m / z, 244 [M+H]+, RT=2.56 min. HPLC: 95%, RT=3.29 min. 1H NMR (400MHz, DMSO-d6) δ10.91 (s, 1H), 8.68 (dd, J=5.0, 1.8Hz, 1H), 7.92-7.88 (m, 1H), 7.86 (t, J=2.7Hz, 1H), 7.81-7.75 (m, 2H ), 7.64(dd,J=15.3,1.6Hz,1H), 7.43(ddd,J=7.6,4.7,1.2Hz,1H), 6.77(dd,J=8.6,2.3Hz,1H), 6.68(dd,J=13.5,2.3Hz,1H).

[0334]

[0335] A9R4: Synthesis of 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one. To a mixture of 1-(2,4-dihydroxyphenyl)ethan-1-one 1 (1 g, 6 mmol) and PPTS (33 mg, 0.13 mmol) in DCM was added 3,4-dihydro-2H-pyran (1.6 g, 20.0 mmol). The reaction mixture was stirred at RT for 1 h. TLC showed that the reaction was complete. The solution was extracted 3 times with DCM, and the organic layer was washed 3 times with H2O, brine, dried over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel chromatography (PE:EA=10:1) to give 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one 2 (1.3 g, 75%) as an off-white solid.

[0336]

[0337] Synthesis of (E)-1-(2,4-dihydroxyphenyl)-3-(pyridin-2-yl)prop-2-ene-1-one. To a mixture of 1-(2-hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one 2 (0.5 g, 2 mmol) and picolinaldehyde 3 (0.23 g, 2 mmol) in EtOH was added NaOH (0.34 g, 8 mmol). The reaction mixture was stirred at rt for 24 h. TLC showed 50% SM remaining. 4N HCl was added to the solution to adjust pH to 4, and the mixture was then stirred at RT for 1 h. The orange solid was collected by filtration, washed 3 times with EtOH and dried to provide (E)-1-(2,4-dihydroxyphenyl)-3-(pyridin-2-yl)prop-2-ene-1-one C9529-4 (60 mg, 11%) as a yellow solid. 1H NMR (400MHz, DMSO-d6) δ13.05 (s, 1H), 10.97 (s, 1H), 8.80 (d, J = 5.0Hz, 1H), 8.40 (d, J = 15.4Hz, 1H), 8.22 (d, J = 6.2Hz, 2H ), 8.13 (d, J = 9.0Hz, 1H), 7.82 (d, J = 15.4Hz, 1H), 7.73-7.64 (m, 1H), 6.47 (dd, J = 8.9, 2.3Hz, 1H), 6.35 (d, J = 2.3Hz, 1H).

[0338]

[0339] Example 8: Luciferase assay of A9 analogs. To better understand the structure-activity relationship of A9 analogs, analogs A9R1, A9R2, A9R3 and A9R4 were prepared. Figure 8 Doxycycline-dependent U2OS cells transfected with ERα transfected with ER-TK-Luc or NKG2E-TK-Luc were incubated with E2, A9, A9R1, A9R2, A9R3, or A9R4, or with E2 in combination with A9, A9R1, A9R2, A9R3, or A9R4. Luciferase activity results were Fig. 9 (ER-TK-Luc) and Fig.10 (NKG2E-TK-Luc). Surprisingly, A9 showed the greatest synergy with E2.

[0340] Example 8: MCF-7 cells, 6 days after treatment

[0341] The effects of E2 alone and in combination with E2 were studied in MCF-7 breast cancer cells. Figure 8 The growth promoting properties of A9 and analogs are described in. MCF-7 cells were incubated with medium (control), 1 nM E2 or 5 μM Figure 8 Incubations were continued for 24 hours in the presence of each compound depicted in (A9 and the four analogs A9R1, A9R2, A9R3 and A9R4). Viable cell counts were determined by flow cytometry.

[0342] 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 A9 or Figure 8 Cells were treated with vehicle or E2 for 7 days in the presence of one of the four 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).

[0343] 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 4 The 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 500μLPBS containing 50μg / mL propidium iodide, 0.1% triton X-100, 0.1% sodium citrate and 10μg / mL RNAse.Then the cell suspension is analyzed with the BD LSR II flow cytometer (BD Biosciences, SanJose, CA, USA) in the flow cytometry facility of the University of California, Berkeley, and the cell percentage of the cell cycle stage is determined by using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0344] like Fig.11As can be seen in the , E2 alone stimulates breast cancer cell proliferation. In contrast, the viable cell counts of each of the four analogs of CC7, A9, and A9 were much lower than the cell counts of E2 alone. The cell counts of CC7+E2, A9+E2, A9R1+E2, A9R2+E2, and A9R4+E2 were much lower than the control, and the control was lower than the cell count of E2. These results indicate that CC7, A9, and the four analogs of A9 do not stimulate MCF-7 breast cancer cell proliferation like E2.

[0345] 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.

[0346] Although many embodiments of pharmaceutical compositions and estrogenic methods are described herein, it will be appreciated by those skilled in the art that the embodiments may be altered to provide other embodiments utilizing NRRP compounds as described herein, compositions and methods described herein. It will therefore be understood that the scope of the invention is defined by the appended claims rather than by the specific embodiments represented by way of example.

Claims

1. A compound, wherein the compound is a nuclear receptor reprogramming compound.

2. The compound of claim 1, wherein the nuclear receptor reprogramming compound is not an agonist, antagonist, or mixed agonist / antagonist of the estrogen receptor, and wherein the compound modulates estradiol (E2) activity only when E2 is present at physiological levels.

3. The compound according to claim 1 or claim 2, wherein the nuclear receptor reprogramming compound has a structure of Formula I or a pharmaceutically acceptable salt thereof, wherein the compound of Formula I is: in, Each is a single bond or a double bond; A 1 , A 2 and A 3 Independently CR 1 or N, A 4 Yes CR 6 or N; A 5 , A 6 and A 7 Independently CR 2 or N, A 8 Yes CR 7 or N, A 9 Yes CR 3 , CHR 3 , N or NR 3 , Each R 1 are independently H, halogen, C1-C4 alkyl, OH, NH2 or O-C1-C4 alkyl; Each R 2 are independently H or OH, R 3 is H or C1-C4 alkyl, R 4 is O or OH, or with R 7 Together they form a pyrido or pyrimido ring to form a quinoline, isoquinoline or quinazoline bicyclic ring system; R 5 is H, or with R 6 Together they form an -O-bridge to form a benzopyran bicyclic ring system; R 6 is H or HO, or with R 5 together to form an -O- bridge to form a benzopyran bicyclic ring; and R 7 is H, OH, or with R 4 Together they form a pyrido or pyrimidine ring to form a quinoline, isoquinoline or quinazoline bicyclic ring system.

4. The compound according to claim 3, wherein each R 1 are independently H, F, CH3, OH, NH2 or O-CH3.

5. A compound according to claim 3 or claim 4, wherein: A 4 It is CH, A 5 It is CH, A 6 is CH or COH, A 7 is CH or COH, A 8 is CH or COH.

6. A compound according to any one of claims 3 to 5, wherein: Connect to R 4 of is a double bond, and R 4 It's O.

7. A compound according to any one of claims 3 to 6, wherein: Connect to A 9 of It's a double bond.

8. A compound according to any one of claims 3 to 7, wherein A 1 , A 2 and A 3 One and only one of them is N, and the other two are CR 1 .

9. The compound according to claim 8, wherein each R 1 It's H.

10. The compound according to claim 9, wherein at least one R 1 is OH, and the other R 1 It is H or OH.

11. The compound according to claim 10, wherein one R 1 is OH, and the other R 1 It's H.

12. The compound according to claim 11, wherein A 3 Yes N, A 2 is CH, and A 3 It's C-OH.

13. A compound according to any one of claims 3 to 8, wherein A 6 , A 7 or A 8 At least one of them is N.

14. The compound according to claim 13, wherein A 6 , A 7 and A 8 Only one of them is N.

15. A compound according to claim 13 or claim 14, wherein A 1 It's C-OH.

16. The compound according to claim 15, wherein A 2 and A 3 It is CH.

17. A compound according to any one of claims 3 to 8, wherein A 9 Yes CR 3 , and R 3 It is H or C1-C4 alkyl.

18. The compound according to claim 17, wherein R 3 It is CH3.

19. A compound according to any one of claims 3 to 6, wherein the 9 of It is a single bond.

20. The compound according to claim 19, wherein A 9 It is CHR 3 .

21. The compound according to claim 20, wherein R 3 It is H or CH3.

22. The compound according to claim 19, wherein A 9 It's NH.

23. according to the compound described in any one of claims 3-7, wherein connected to R 4 of It is a single bond.

24. The compound according to claim 23, wherein R 4 It's OH.

25. The compound according to claim 23, wherein R 4 and R 7 Together they form a pyrido ring or a pyrimido ring fused to an adjacent benzo ring.

26. The compound according to claim 25, wherein R 4 and R 7 Together they form a pyrido ring which is fused to an adjacent benzo ring to form quinoline or isoquinoline.

27. The compound according to claim 25, wherein R 4 and R 7 Together they form a pyrimido ring fused to an adjacent benzo ring to form a quinazoline.

28. A compound according to any one of claims 25-27, wherein A 6 and A 7 It's C-OH.

29. The compound according to claim 28, wherein A 4 and A 5 It is CH.

30. A compound according to any one of claims 3 to 8, wherein R 5 and R 6 Together they form an -O- bridge.

31. A compound according to any one of claims 1 to 3, which is:

32. A compound according to any one of claims 1 to 3, which is The "E" indicates that the vinyl bond is trans.

33. A compound according to any one of claims 1 to 3, which is:

34. The compound according to claim 33, which is: The "E" indicates that the vinyl bond is trans.

35. A compound according to any one of claims 1 to 3, which is:

36. The compound according to claim 35, which is: The "E" indicates that the vinyl bond is trans.

37. A compound according to any one of claims 1 to 3, which is:

38. The compound according to claim 37, which is:

39. A compound according to any one of claims 1 to 3, which is:

40. The compound according to claim 39, which is: The "E" indicates that the vinyl bond is trans.

41. A compound according to any one of claims 1 to 3, which is:

42. The compound according to claim 41, which is: The "E" indicates that the vinyl bond is trans.

43. A compound according to any one of claims 1 to 3, which is:

44. The compound according to claim 43, which is: Where E indicates that the vinyl bond is trans.

45. A compound according to any one of claims 1 to 3, which is:

46. ​​The compound according to claim 45, which is: wherein "E" indicates that the vinyl bond is trans, and "Z" indicates that the vinyl bond is trans.

47. A compound according to any one of claims 1 to 3, which is: The "E" indicates that the vinyl bond is trans.

48. A compound according to any one of claims 1 to 3, which is:

49. A compound according to any one of claims 1 to 3, which is: Where E indicates that the vinyl bond is trans.

50. A compound according to any one of claims 1 to 3, which is:

51. A compound according to any one of claims 1 to 3, which is:

52. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the compound has one of the following structures:

53. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1-52, wherein the compound or pharmaceutically acceptable salt thereof is estrogenic.

54. A pharmaceutical composition comprising a compound according to any one of claims 1 to 53 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

55. The pharmaceutical composition of claim 54, 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.

56. A compound according to any one of claims 1-53, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 54 or claim 55, for use in treating a condition or disease state in a patient in need of treatment with a nuclear receptor reprogramming compound.

57. The compound of claim 56, 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.

58. A compound according to claim 56 or claim 57, 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.

59. Use of a compound according to any one of claims 1-53, or a pharmaceutically acceptable salt thereof, 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.

60. The use of claim 59, 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.

61. The use of claim 59 or claim 60, 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.

62. A method of treating a patient in need of such treatment, comprising administering to the patient an effective amount of a compound according to any one of claims 1-53 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 54.

63. The method of claim 62, 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.

64. The method of claim 62 or claim 63, 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.

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