Estrogen Compounds and Methods of Use

Abstract

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

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Application No. 63 / 393,635, filed Jul. 29, 2022, the contents of which are hereby incorporated by reference in their entirety.

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

Background Art

[0003] Estrogen is a steroid hormone involved in the regulation of multiple developmental and physiological functions in both males and females. Estrogen is not merely an estrus-inducing sex hormone. Estrogen is important for survival and health in both sexes, but their amounts, adaptive responses, tissue-specific distributions, and receptor affinities vary by various stages of life. Estrogen is essential for glucose homeostasis, immune robustness, bone health, cardiovascular health, fertility, and neurological function.

[0004] Estrogen (estradiol (E2) and related steroid hormones) is mainly biosynthesized in the gonads, but also in the adrenal cortex and adipose tissue. Estrogen regulation is mediated by nuclear estrogen receptors (ERs) and membrane estrogen receptor (GPER1) encoded by different genes on different chromosomes, suggesting differential functions. However, estrogen is also at the center of almost all human pathologies, such as infectious, autoimmune, metabolic, and degenerative diseases. Both low estrogen levels and high estrogen levels have been associated with chronic and acute diseases. Normal aging results in significantly low levels of estrogen, leading to tissue degeneration (bones, muscles, nerves, etc.) and metabolite imbalance (glucose, lipids, etc.), but the increase in inflammatory agents in daily life enhances the level of estrogen (or estrogen mimics), which is pathophysiologically very important. The resulting excess estrogen induces fluctuations in estrogen receptors α and β (ERα and ERβ), damages tissues, and leads to autoimmune diseases and neoplasms.

[0005] Estrogen is involved in growth, development, and tissue differentiation from embryo to death. During puberty, rapid physiological changes occur, such as rapid growth of bone and muscle mass, and maturation of the gonads and brain. Estrogen is an important signaling contributing to all these processes, especially sexual differentiation. Hormonal fluctuations and disruptions lead to multiple pathologies at this age, but not all of them are directly related to sexual development and function. Premature puberty, which increases the risk of cancer in women and reduces the quality of life specific to adolescent girls, is increasing worldwide.

[0006] During menarche, estrogen levels vary significantly throughout the menstrual cycle. Early and extended menarche, even with normal levels of cyclic variation, result in a significant increase in the risk of aging diseases such as breast cancer and type 2 diabetes. Most women (up to 91%) experience painful menstruation, and 14% experience heavy bleeding. Female infertility is increasing globally, but this is not simply due to an increase in the age of first pregnancy. Up to 21% of couples struggle with infertility, and 80% of this is due to female diseases. Despite improved fertility technologies such as in vitro fertilization (IVF), the success rate of these technologies is very low (about 9% for IVF). Up to 46% of women experience spontaneous abortion (miscarriage). The tendency for gestational diabetes, preterm labor, preeclampsia, and gestational anemia is increasing, all of which are related to estrogen function.

[0007] In women of reproductive age, estrogen is mainly synthesized in the granulosa cells of ovarian follicles. When a woman enters menopause, the follicles in the ovaries deplete due to atresia during each menstrual cycle, and the amount of estrogen produced by the ovaries decreases. When estrogen levels decline and begin to fluctuate, short-term symptoms such as hot flashes, night sweats, and mood changes occur frequently. The ovaries eventually stop producing estrogen, the period of estrogen deficiency increases, and the risk of chronic diseases such as osteoporosis, cardiovascular disease, obesity, type 2 diabetes, and urogenital atrophy is accelerated.

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

[0009] Pharmaceutical intervention with estrogen, such as menopausal hormone therapy (MHT), is the most common intervention in all female-related disorders. Natural estrogens and synthetic derivatives are used in these prescriptions. The mechanism of action of these estrogens is as universal agonists, resulting in gene activation and suppression. There are some synthetic steroid estrogen receptor antagonists and synthetic non-steroid estrogen receptor antagonists that are mainly used for the treatment of breast cancer. Unlike natural hormones, most steroid and non-steroid drugs, despite the pharmacological intent to achieve estrogen receptor antagonism, which results in gene regulation and pathophysiological outcome, result in a mixed agonist / antagonist outcome, where they result in an outcome opposite to estrogen in some tissues but a similar outcome in other tissues. These are selective estrogen receptor modulators (SERMs), which are currently used for the treatment of breast cancer and osteoporosis and, when combined with estrogen, for the symptoms of menopause.

[0010] Historically, to counter the increased risk of uterine cancer associated with menopausal hormone therapy, progestogens are co-prescribed with estrogen. Progestogens exert their pharmacological effects via progesterone receptors.

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

[0012] Long-term administration of menopausal hormone therapy has been shown to increase the risk of estrogen-related adverse outcomes such as breast cancer, stroke, venous thrombosis (VTE), and Alzheimer's disease. Therefore, MHT is recommended only for 5 years to treat vasomotor symptoms and vulvovaginal atrophy in menopause and perimenopause. Nevertheless, treatment with MHT, in addition to treating vasomotor symptoms of menopause, also reduces the risks of osteoporosis, type 2 diabetes (T2DM), cardiovascular disease, obesity, and genitourinary atrophy. Thus, there is a need for treatment options that have one or more advantages of MHT, such as a reduced risk of adverse outcomes, including an increased cancer risk associated with traditional MHT (E2, alone or in combination with progestogens or SERMs).

[0013] There is a need for further novel therapeutic compounds, compositions, and methods for treating symptoms of menopause and perimenopause. The various embodiments disclosed herein address these needs and also provide related advantages. SUMMARY OF THE INVENTION

[0014] In the present invention, the inventors propose a new class of drugs for pharmacologically modulating estrogen receptor (ER) activity. These are nuclear receptor reprogramming (NRRP) drugs, which will be described hereinafter in this specification, the appended claims, and the figures. These drugs are neither agonists nor antagonists and do not produce mixed agonist / antagonist activity. They do not activate reporter genes such as estradiol or block the effects of estradiol such as tamoxifen. Instead, this class of compounds produces synergistic activation of reporter genes in the presence of estradiol (E2). NRRP compounds alone have little or no agonist or antagonist effect on estradiol. However, the combination of E2 and NRRP regulates two major classes of genes. One class of genes was synergistically activated by the combination of NRRP / E2. The other class of genes was called reprogrammed genes because they were not regulated by ERα unless NRRP was added in the presence of physiological concentrations of E2. The effect of NRRP is mediated through ER, because the combination with estradiol did not produce a synergistic effect in the absence of ER, or was inhibited by an ER antagonist, or was blocked by a GPER1 antagonist.

[0015] These compounds can be used in a plurality of indications to effect modification of estrogen activity, either alone or in combination with estrogen. Unlike estradiol alone, the combination of NRRP and estradiol does not result in cell proliferation in breast or uterine cancer, suggesting that these uses enhance beneficial estrogen activity and reduce the risk of MHT-related breast and uterine cancers.

[0016] Other uses and advantages of the various embodiments described herein will be apparent to those skilled in the art upon consideration of the following disclosure.

Brief Description of the Drawings

[0017] The features and advantages of the present invention will become apparent from consideration of the following detailed description presented in connection with the accompanying drawings.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

[0018] This specification describes nuclear receptor reprogramming compounds. 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 E2 is present at physiological levels. In some embodiments, a structure of Formula I, or a pharmaceutically acceptable compound thereof, is provided, wherein the compound of Formula I is

[0019] [Chemical Formula] and wherein each

[0020] [Chemical Formula] is a single bond or a double bond, A 1 , A 2 , and A 3 are independently CR 1 or N, A 4 is CR 6 or N, A 5 , A 6 , and A 7 are independently CR2 or N, A 8 is CR 7 or N, A 9 is CR 3 , CHR 3 , N, or NR 3 and each R 1 is independently H, halo, C1-C4 alkyl, OH, NH2, or O-C1-C4 alkyl, each R 2 is independently H or OH, R 3 is H or C1-C4 alkyl, R 4 is O or OH, or, R 7 together with forms a pyrido or pyrimido ring to form a quinoline, isoquinoline or quinazoline bicyclic ring system, R 5 is H, or, R 6 together with forms an -O- bridge to form a benzopyran bicyclic ring system, R 6 is H or HO, or R 5 together with forms an -O- bridge to form a benzopyran bicyclic ring system, R 7 is H, OH, or R 4 together with forms a pyrido or pyrimidine ring to form a quinoline, isoquinoline, or quinazoline bicyclic ring system.

[0021] In some embodiments of the compound of formula I or a salt thereof, each R 1 is independently H, F, CH3, OH, NH2, or O-CH3.

[0022] In some embodiments of the compound of formula I or a salt thereof, A 4 is CH, A 5 is CH, A 6 is CH or COH, A 7is CH or COH, and A 8 is CH or COH.

[0023] In some embodiments of the compound of formula I or a salt thereof, R 4 bonded to

[0024]

Chemical formula

[0025] In some embodiments of the compound of formula I or a salt thereof, A 9 bonded to

[0026]

Chemical formula

[0027] In some embodiments of the compound of formula I or a salt thereof, only one of A 1 , A 2 , and A 3 is N, and the other two are CR 1 .

[0028] In some embodiments of the compound of formula I or a salt thereof, each R 1 is H.

[0029] In some embodiments of the compound of formula I or a salt thereof, at least one R 1 is OH, and the other R 1 is H or OH.

[0030] In some embodiments of the compound of formula I or a salt thereof, one R 1 is OH, and the other R 1 is H.

[0031] In some embodiments of the compound of formula I or a salt thereof, A 3 is N, and A2 is CH, and A 3 is C-OH.

[0032] In some embodiments of the compound of formula I or a salt thereof, A 6 , A 7 , or A 8 at least one of is N.

[0033] In some embodiments of the compound of formula I or a salt thereof, A 6 , A 7 , and A 8 only one of is N.

[0034] In some embodiments of the compound of formula I or a salt thereof, A 1 is C-OH.

[0035] In some embodiments of the compound of formula I or a salt thereof, A 2 and A 3 are CH.

[0036] In some embodiments of the compound of formula I or a salt thereof, A9 is CR 3 , and R 3 is H or C1-C4 alkyl.

[0037] In some embodiments of the compound of formula I or a salt thereof, R 3 is CH3.

[0038] In some embodiments of the compound of formula I or a salt thereof, A 9 bonded to

[0039]

Chemical formula

[0040] In some embodiments of the compound of formula I or a salt thereof, A9 is CHR 3 .

[0041] In some embodiments of the compound of formula I or a salt thereof, R 3 is H or CH3.

[0042] In some embodiments of the compound of formula I or a salt thereof, A 9 is NH.

[0043] In some embodiments of the compound of formula I or a salt thereof, the 4 bonded to R

[0044]

Chemical formula

[0045] In some embodiments of the compound of formula I or a salt thereof, R 4 is OH.

[0046] In some embodiments of the compound of formula I or a salt thereof, R 4 and R 7 together form a pyrido or pyrimidinocyclo fused to the adjacent benzocycle.

[0047] In some embodiments of the compound of formula I or a salt thereof, R 4 and R 7 together form a pyrido ring fused to the adjacent benzocycle to form quinoline or isoquinoline.

[0048] In some embodiments of the compound of formula I or a salt thereof, R 4 and R 7 together form a pyrimidinocyclo fused to the adjacent benzocycle to form quinazoline.

[0049] In some embodiments of the compound of formula I or a salt thereof, A 6 and A 7 are C-OH.

[0050] In some embodiments of the compound of formula I or a salt thereof, A 4 and A 5 are CH.

[0051] In some embodiments of the compound of formula I or a salt thereof, R 5 and R 6 together form an -O- bridge.

[0052] In some embodiments of the compound of formula I or a salt thereof, the compound is one of the following

[0053] [Chemical formula] wherein "E" indicates that the vinyl bond is entgegen.

[0054] In some embodiments of the compound of formula I or a salt thereof, the compound is

[0055] [Chemical formula] is as follows.

[0056] In some embodiments of the compound of formula I, the compound is

[0057] [Chemical formula] wherein "E" indicates that the vinyl bond is entgegen.

[0058] In some embodiments of the compound of formula I, the compound is

[0059] [Chemical formula] is as follows.

[0060] In some embodiments of the compound of formula I, the compound is

[0061] [Chemical formula] wherein, "E" indicates that the vinyl bond is entgegen.

[0062] In some embodiments of the compound of Formula I, the compound is

[0063] [Chemical formula] as follows.

[0064] In some embodiments of the compound of Formula I, the compound is

[0065] [Chemical formula] as follows.

[0066] In some embodiments of the compound of Formula I, the compound is

[0067] [Chemical formula] as follows.

[0068] In some embodiments of the compound of Formula I, the compound is

[0069] [Chemical formula] wherein, "E" indicates that the vinyl bond is entgegen.

[0070] In some embodiments of the compound of Formula I, the compound is

[0071] [Chemical formula] as follows.

[0072] In some embodiments of the compound of Formula I, the compound is

[0073]

Chem.

[0074] In some embodiments of the compound of formula I, the compound is

[0075]

Chem.

[0076] In some embodiments of the compound of formula I, the compound is

[0077]

Chem.

[0078] In some embodiments of the compound of formula I, the compound is

[0079]

Chem.

[0080] In some embodiments of the compound of formula I, the compound is

[0081]

Chem.

[0082] In some embodiments of the compound of formula I, the compound is

[0083]

Chem.

[0084] In some embodiments of the compound of formula I, the compound is

[0085]

Chemical formula

[0086] In some embodiments of the compound of formula I, the compound is

[0087]

Chemical formula

[0088] In some embodiments of the compound of formula I, the compound is

[0089]

Chemical formula

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

[0091]

Chemical formula

[0092] The compound of formula I and its pharmaceutically acceptable salts are NRRP compounds, The NRRP compound enhances the in vitro activity of estrogen receptor α (ERα) in the presence of estradiol (E2), but neither agonizes nor antagonizes ERα in the absence of E2, and reprograms one or more genes under the control of estrogen. The compounds of Formula I are useful in the treatment of one or more disorders, as discussed in more detail hereinbelow.

[0093] 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 agonist / antagonists, one or more selective estrogen receptor modulators (SERMs), one or more progestogens, one or more glucocorticoids, and one or more androgens.

[0094] Synthesis

[0095] The compounds of the present invention can be obtained as follows.

[0096] Variations of the "A" ring: Compounds Formula

[0097]

Chemical formula

[0098]

Chemical formula

[0099]

Chemical formula

[0100] The formula

[0101]

Chemical formula

[0102]

Chemical formula

[0103]

Chemical formula

[0104] A compound having a nitrogen-containing linker group between ring A and ring B can be synthesized by the following synthetic reaction scheme

[0105]

Chem.

[0106] A 4 ~A 8 In some embodiments, where one of them is OH, one or more of the hydroxyl groups can be methylated, in which case a second methyl-group removal step can be used to generate the OH group. For example, the following reaction scheme

[0107]

Chem.

[0108] Compounds restricted in conformationally restricted compounds such as compound A24 can be produced by the following reaction

[0109]

Chem.

[0110] Pharmaceutical composition

[0111] The pharmaceutical compositions described herein are included. At least one of the pharmaceutically acceptable ingredients may include one or more ingredients that do not occur naturally together with the novel compounds, salts or solvates thereof disclosed herein. Pharmaceutically acceptable ingredients that do not occur naturally together with the novel compounds, salts or solvates thereof disclosed herein may include excipients that are sterile, isotonic, or pyrogen-free in nature.

[0112] A "pharmaceutically acceptable" ingredient is one that is compatible with the estrogenic compounds and other ingredients of the compositions described herein and suitable for administration to a patient. Additional ingredients may include carriers, diluents, absorption promoters, stabilizers, preservatives, or other active or inactive ingredients. At least one of the additional ingredients may be an ingredient that does not occur naturally together with the NRRP compounds described herein in nature. 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.

[0113] In some embodiments, the pharmaceutical composition may be an estrogenic composition. An estrogenic composition contains the NRRP compounds and additional ingredients described herein in an estrogenically effective amount. The additional ingredient may be an excipient. The excipient may include at least one compound that does not occur naturally with the NRRP compound in nature. In particular, the excipient may include at least one compound that does not occur naturally with the NRRP compounds described herein in humans. 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 component at a concentration sufficient for the composition to be isotonic. In some embodiments, the additional component may be a flavoring or sweetening agent that is not found in the NRRP compounds described herein in nature. In some embodiments, the estrogen composition may be sterile, pyrogen-free, and / or isotonic.

[0114] A pharmaceutically acceptable salt can be any salt of the estrogen compounds disclosed herein that has appropriate solubility in an aqueous solvent at an appropriate pH. Remington’s, 20th Ed., published 2000, pp. 704 - 719 provides methods for determining appropriate pharmaceutically acceptable salts. For example, appropriate salts can be selected from Table 38 - 2, page 704 of Remington’s. Pharmaceutically acceptable salts can be prepared by dissolving the estrogen compound in an appropriate solvent and adding an appropriate acid or base, or in some cases, an appropriate counter - acid or counter - base to the solution and separating the salt form of the estrogen compound from the solution.

[0115] Pharmaceutical compositions, particularly estrogen compositions, can be formulated for various routes of administration such as oral, intranasal, intrapulmonary (e.g., for inhalation), intravenous, subcutaneous, transdermal, sublingual, buccal, intraperitoneal, or intrathecal administration. The pharmaceutical composition may include one or more enhancers that assist in the transport of the NRRP compounds described herein across one or more external or internal physiological barriers, such as the pulmonary epithelial barrier or the blood brain barrier.

[0116] Suitable pharmaceutically acceptable excipients can include the following types of excipients, diluents, lubricants, binders, disintegrants, fillers, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, flavor masking agents, coloring agents, anti-caking agents, wetting agents, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.

[0117] Estrogenically Effective Doses

[0118] The effective dose of the NRRP compound can vary depending on various factors including the route of administration, the age and condition of the patient in need of estrogen treatment, co-administration with other drugs or compounds, etc. Generally, the NRRP compounds described herein are effective in vitro at nanomolar or micromolar concentrations. The effective daily doses of the NRRP compounds described herein can range from 0.01 mg to 1000 mg per day. The effective daily dose can be divided into two or more divided doses, e.g., 1, 2, 3, 4, 5, 6, or more divided doses. When the estrogen composition is administered as an injection, the effective daily dose can be administered as a continuous infusion over several hours, e.g., 1 to 24 hours. The effective dose can be similar to the effective dose of the NRRP compounds described herein, but can be scaled to account for a larger molecular weight compared to the NRRP compounds described herein in the relative bioactivity, pharmacokinetics, and pharmacodynamics of the compound, knowing how to determine this by methods recognized in the art by those skilled in the art.

[0119] Those of ordinary skill in the art of pharmaceutical formulations and compounding have the knowledge and skill to select appropriate amounts of appropriate pharmaceutically acceptable carriers and excipients for use with the NRRP compounds described herein. In addition, there are several resources available to those of ordinary skill in the art that describe pharmaceutically acceptable carriers and excipients and may be useful in selecting appropriate pharmaceutically acceptable carriers and excipients. Examples include Remington’s Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (The American Pharmaceutical Association and The Pharmaceutical Press).

[0120] The compositions of the estrogen compounds described herein can be prepared using techniques and methods known to those of ordinary skill in the art. Some of the methods commonly used in the art are described in Remington’s Pharmaceutical Sciences, 20th Ed., (Mack Publishing Company (2000)).

[0121] In some embodiments, the estrogenic composition can comprise an NRRP compound described herein and one or more pharmaceutically acceptable carriers or excipients. The composition can be prepared and packaged in bulk form, in which an effective amount of the compound of the present disclosure is extracted and then can be administered to a subject, for example, as a powder or syrup. Alternatively, the composition can be prepared and packaged in unit dosage form, in which each physically distinct unit contains an effective amount of the NRRP compound described herein.

[0122] The NRRP compounds described herein, and pharmaceutically acceptable carriers or excipients, can be formulated into dosage forms suitable for administration to a subject by a desired route of administration. For example, dosage forms include (1) oral administrations such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets, and (2) parenteral administrations such as sterile solutions, suspensions, and powders for reconstitution suitable therefor. Suitable pharmaceutically acceptable carriers or excipients can vary depending on the particular dosage form selected. Further, suitable pharmaceutically acceptable carriers or excipients can be selected for the particular functions they can serve in the composition. For example, a particular pharmaceutically acceptable carrier or excipient can be selected for their ability to facilitate the manufacture of a uniform dosage form. A particular pharmaceutically acceptable carrier or excipient can be selected for their ability to promote the manufacture of a stable dosage form. A particular pharmaceutically acceptable carrier or excipient can be selected for their ability to facilitate the transport or delivery of the compounds disclosed herein from one organ or body part to another organ or another part of the body when administered to a subject. A particular pharmaceutically acceptable carrier or excipient can be selected for their ability to enhance patient compliance.

[0123] In some embodiments, the compositions of the estrogen NRRP compounds described herein can be formulated for parenteral administration. Compositions adapted for parenteral administration can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile injection solutions, and aqueous and non-aqueous sterile suspensions that can contain suspending and thickening agents. The compositions are provided in unit dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze-dried (lyophilized) state that requires only the addition of a sterile liquid carrier, for example water for injection, immediately prior to use. Immediate injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Parenteral formulations can be sterile, pyrogen-free, or both. Parenteral formulations can be isotonic.

[0124] Oral

[0125] The estrogen composition can be an oral estrogenic composition comprising an NRRP compound described herein and at least one excipient suitable for oral administration. At least one excipient suitable for oral administration can include compounds that do not occur naturally with the NRRP compounds described herein in nature. At least one excipient suitable for oral administration can include at least one compound other than water. Various dosage forms such as tablets, capsules, caplets, troches, powders, emulsions, sachets, cachets, gel capsules, elixirs, pills, oral sprays, chewable tablets, sublingual tablets, films or sprays, or buccal films or sprays can be prepared.

[0126] In some embodiments, the NRRP compounds described herein can be formulated as solid oral dosage forms, such as tablets or capsules, containing an effective amount of the compound of the disclosure and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g., corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g., microcrystalline cellulose), calcium sulfate, and calcium hydrogen phosphate. The solid oral dosage form may further contain a binder. Suitable binders include starch (e.g., corn starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g., microcrystalline cellulose). The solid oral dosage form may further contain a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmellose, alginic acid, and sodium carboxymethyl cellulose. The solid oral dosage form may further contain a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0127] When 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 materials in polymers, waxes, and the like.

[0128] The NRRP compounds described herein can be combined with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropylmethacrylamide phenol, polyhydroxyethyl aspartamide-phenol, or polyethylene-oxide polylysine substituted with palmitoyl residues. Further, the NRRP compounds described herein can be combined with classes of biodegradable polymers useful for achieving controlled release of drugs, such as polylactic acid, polycaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyran, polycyanoacrylates, and crosslinked hydrogels or amphipathic block copolymers.

[0129] In some embodiments, the NRRP compounds described herein can be formulated in liquid oral dosage forms. Oral liquids such as solutions, syrups, and elixirs can be prepared in unit dosage forms such that a given quantity 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, and elixirs are prepared by the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compounds disclosed herein in a non-toxic vehicle. Solubilizing and emulsifying agents, preservatives, flavoring agents such as peppermint oil or other natural sweeteners or saccharin or other artificial sweeteners, etc., such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether, can be added.

[0130] Intranasal

[0131] The estrogen composition can be an intranasal estrogen composition comprising the NRRP compound described herein and at least one excipient suitable for intranasal administration. The at least one excipient suitable for intranasal administration can comprise at least one compound other than water. For example, the intranasal estrogen composition can comprise one or more penetration enhancers that increase the absorption of the NRRP compound described herein across the mucosa and / or increase bioavailability. In some embodiments, the penetration enhancer can comprise a mucolytic, a protease 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, in an in vivo or a good model test, two formulations as drugs, with and without the enhancer, containing a non-associated, small polar molecule, and determining whether the uptake of the drug is enhanced to a clinically significant extent. An enhancer in vivo should be non-irritating and / or should be rapidly metabolized by normal cell components without significant stimulatory effects so that the enhancer will not pose a problem with respect to chronic toxicity. In some embodiments, the penetration enhancer can be an alkyl glycoside, such as the alkyl glycoside disclosed in U.S. Patent No. 5,661,130, which is hereby incorporated by reference in its entirety. Those skilled in the art recognize the need to achieve an appropriate hydrophilic-lipophile balance (HLB) number, which can be determined as disclosed in U.S. Patent Publication No. US2009 / 0047347, which is hereby incorporated by reference in its entirety.

[0132] The intranasal estrogen composition of the NRRP compound described herein can also comprise a flavor or fragrance to cover the taste of the NRRP compound described herein. The intranasal composition can also comprise an isotonic agent to make the composition isotonic. The intranasal estrogen composition of the NRRP compound described herein can also comprise a stabilizer.

[0133] In the lungs The estrogen composition can be an intrapulmonary estrogen composition comprising an NRRP compound as described herein and at least one excipient suitable for nasal administration. The at least one excipient suitable for intrapulmonary administration can include at least one compound other than water. For example, the intrapulmonary composition can include one or more penetration enhancers that increase the ability of the NRRP compound as described herein to pass through the lung epithelium and enter the bloodstream.

[0134] The intrapulmonary estrogen composition can be administered to the lungs by inhalation, for example, using an inhaler, an aerosol inhaler, or a conventional or high-efficiency nebulizer.

[0135] High-efficiency nebulizers are inhalation devices that include microperforated membranes through which a liquid solution is converted into aerosol droplets suitable for inhalation by electrical or mechanical means. High-efficiency nebulizers can deliver a majority of the loaded dose to the patient. In some embodiments, high-efficiency nebulizers can also utilize one or more actively or passively vibrating microperforated membranes. In some embodiments, high-efficiency nebulizers can include one or more vibrating membranes. In some embodiments, high-efficiency nebulizers can include a vibrating mesh or plate having a plurality of apertures and, optionally, a vibration generator having an aerosol mixing chamber. In some such embodiments, the mixing chamber can function to collect (or stage) the aerosol from the aerosol generator.

[0136] In some embodiments, high-efficiency nebulizers can achieve at least about 10% lung deposition (deposited lung dose) based on the nominal dose of the NRRP compound as described herein.

[0137] In some embodiments, high-efficiency nebulizers provide at least about 5% of the NRRP compound as described herein (lung deposition (deposited lung dose)) based on the nominal dose of the NRRP compound as described herein.

[0138] According to the present invention, in some embodiments, a nebulizer, such as a high-efficiency nebulizer, can be adapted or can be adaptable to operate in conjunction with a unit dosage form, such as an ampoule or vial, containing a single dosage of an NRRP compound described herein for estrogen therapy. The unit dosage form includes a container containing an inhalation solution comprising the NRRP compound described herein. The container is adapted to cooperate with a high-efficiency nebulizer device so as to be able to administer a nominal dosage of the inhalation solution to a patient in need thereof. In some embodiments, the high-efficiency nebulizer and the unit dosage form are configured such that they can be used together, but not with other devices or dosage forms. In some specific embodiments, the unit dosage form is configured such that it fits into a keyhole-like structure within the high-efficiency nebulizer, but does not operate with other nebulizer devices. In such embodiments, the high-efficiency nebulizer is configured to receive and operate properly with the unit dosage form containing the NRRP compound described herein, but not with other dosage forms.

[0139] Suitable high-efficiency nebulizers having a perforated membrane are disclosed in U.S. Patent 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 nebulizers include a vibratable membrane. The features of these high-efficiency nebulizers are disclosed in U.S. Patent Nos. 7,252,085, 7,059,320, 6,983,747, each of which is hereby incorporated by reference in its entirety.

[0140] Commercially available high-efficiency nebulizers are available under the product name eFlow® from PARI (Germany), AeroNeb® Go and AeroNeb® Pro, AeroNeb® Solo, and other nebulizers using AeroNeb® technology from Aerogen, Ltd. (Ireland), under the product name I-Neb® from Respironics (Marysville, California), under the trademark Micro-Air® from Omron (Bannockburn, Illinois), under the product name Akita® from Activaero (Germany), and under the product name AerovectRx® from AerovectRx (Atlanta, Georgia).

[0141] Conventional nebulizers include, for example, jet nebulizers or ultrasonic nebulizers. Jet nebulizers generally utilize a compressor to generate compressed air, which breaks down a liquid medication into small breathable droplets to form an aerosolized (nebulized) mist. In some of these embodiments, when the patient inhales, an upper valve opens, allowing air to enter the device, thereby accelerating mist generation, and when the patient exhales, the upper valve closes, thereby decelerating mist generation and simultaneously allowing the patient to exhale through the opening of the mouthpiece flap.

[0142] Some conventional nebulizers are disclosed in U.S. Patent Nos. 6,513,727, 6,513,519, 6,176,237, 6,085,741, 6,000,394, 5,957,389, 5,740,966, 5,549,102, 5,461,695, 5,458,136, 5,312,046, 5,309,900, 5,280,784, and 4,496,086, each of which is incorporated herein by reference in its entirety.

[0143] Commercially available conventional nebulizers are available from PARI (Germany) under the product names PARI LC Plus®, LC Star®, and PARI-Jet®, from A&H Products, Inc. (Tulsa, Oklahoma) under the product name AquaTower®, from Hudson RCI (Temecula, California) under the product name AVA-NEB®, from Intersurgical, Inc. (Liverpool, New York) under the trademark Cirrus®, from Salter Labs (Arvin, California) under the product name Salter 8900®, from Respironics (Mariesville, Pennsylvania) under the product name Sidestream®, from Bunnell (Salt Lake City, Utah) under the product name Whisper Jet®, from Smiths-Medical (High Kent, UK) under the product name Downdraft®, and from DeVilbiss (Somerset, Pennsylvania) under the product name DeVilbiss®.

[0144] Intravenous

[0145] The estrogen composition can be an intravenous estrogen composition comprising the NRRP compound 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 described herein is a parenteral composition intended for intravenous administration by injection or infusion. They can contain one or more tonicity agents to make the composition isotonic. They can be sterile, pyrogen-free, or both, and are generally sterile, pyrogen-free, or both.

[0146] Subcutaneous

[0147] The estrogen composition can be a subcutaneous estrogen composition comprising the NRRP compound 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. The subcutaneous composition of the NRRP compound described herein is a parenteral composition intended for injection under the skin. They can contain one or more tonicity agents to make the composition isotonic. They can be sterile, pyrogen-free, or both, and generally are sterile, pyrogen-free, or both.

[0148] Transdermal

[0149] The estrogen composition can be a transdermal estrogen composition comprising the NRRP compound described herein and at least one excipient suitable for transdermal administration. The 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 described herein to pass through the dermis and enter the bloodstream. In addition, the transdermal composition can be delivered by a biasing mechanism such as an iontophoresis device.

[0150] Sublingual or buccal

[0151] The estrogen composition can be a sublingual or buccal estrogen composition comprising the NRRP compound 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.

[0152] Intraperitoneal

[0153] The estrogen composition can be an intraperitoneal estrogen composition comprising the NRRP compound described herein and at least one excipient suitable for intraperitoneal administration. The at least one excipient suitable for intraperitoneal administration can include at least one compound other than water. The intraperitoneal estrogen composition of the NRRP compound described herein is a parenteral composition intended for administration to the peritoneum by injection or infusion. They can contain one or more tonicity agents to make the composition isotonic. They can be sterile, pyrogen-free, or both, and generally are sterile, pyrogen-free, or both.

[0154] Intrathecal or Intracranioventricular

[0155] The estrogen composition can be an intrathecal or intracranio-ventricular estrogen composition comprising the NRRP compound described herein and at least one excipient suitable for intrathecal or intracranio-ventricular administration. The at least one excipient suitable for intrathecal or intracranio-ventricular administration can include at least one compound other than the compounds that naturally occur with the NRRP compound described herein in nature, such as water. The intrathecal or intracranio-ventricular estrogen composition of the NRRP compound described herein is a parenteral composition intended for cerebrospinal fluid administration by injection or infusion. They can contain one or more tonicity agents to make the composition isotonic. They can be sterile, pyrogen-free, or both, and generally are sterile, pyrogen-free, or both.

[0156] Other routes of administration

[0157] Preferred embodiments of the compositions and methods described herein have been shown and described, but it will be readily apparent to those skilled in the art that modifications can be made without departing from the scope of the appended claims. Accordingly, the scope of the described compositions and methods is limited only by the following claims.

[0158] Estrogen method

[0159] Some embodiments described herein provide a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein for use in the treatment of a disease or disorder condition in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the disease or disorder condition is a development condition, a menstruation condition, a fertility condition, a gynecological disease, an autoimmune disorder, a menopause disorder, an aging disorder, or cancer. In some embodiments, the disease or disorder condition is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorder, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, fertility, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, menopause and perimenopausal vasomotor symptoms, insomnia, nocturnal awakening, mood swings, vulvovaginal atrophy, vaginal dryness, sexual intercourse failure, weight gain and obesity during menopause, 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.

[0160] Some embodiments described herein provide for the use of a compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, for the manufacture of a medicament for use in the treatment of a disease or disorder in a patient in need of treatment with a nuclear receptor reprogramming compound. In some embodiments, the disease or disorder condition is a developmental disease, menstrual disease, conception disease, gynecological disease, autoimmune disorder, menopausal disorder, aging disorder, or cancer. In some embodiments, the disease or disorder condition is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorder, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, conception, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, menopause and perimenopausal vasomotor symptoms, insomnia, nocturnal awakening, mood swings, vulvovaginal atrophy, vaginal dryness, sexual intercourse insufficiency, 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.

[0161] Some embodiments described herein provide a method of treating a patient in need of such treatment, the method comprising administering to the patient an effective amount of a compound of formula I described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition. In some embodiments, the disease or disorder state is a developmental disease, a menstrual disease, a fertility disease, a gynecological disease, an autoimmune disorder, a menopausal disorder, an aging disorder, or cancer. In some embodiments, the disease or disorder state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorder, dysmenorrhea, amenorrhea, menorrhagia, estrogen-induced deep vein thrombosis, pulmonary embolism, fertility, fetal implantation, spontaneous abortion, premature birth, endometriosis, polycystic ovary syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, Hashimoto's thyroiditis, multiple sclerosis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, menopause and perimenopausal vasomotor symptoms, insomnia, nocturnal awakening, mood swings, vulvovaginal atrophy, vaginal dryness, sexual dysfunction, 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.

[0162] In light of the disclosure of this specification, those skilled in the art will understand the method for determining the estrogen effective amount of the NRRP compounds described herein. Generally, the estrogen effective amount, therapeutic effective amount, or prophylactic effective amount of the NRRP compounds described herein can be determined, for example, by inference from in vitro tests. Those skilled in the art will understand that the effective dosage can be inferred from the in vitro half maximal modulating (inhibiting or activating) concentration of the NRRP compounds described herein. Those skilled in the art will understand that the effective dosage in human patients depends on factors such as the route of administration and pharmacokinetics. Considering these factors, the estrogen effective dosage of the NRRP compounds described herein can range from 0.1 mg / kg to 150 mg / kg, for example, 0.1 mg / kg to 1 mg / kg, 0.5 mg / kg to 5 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 50 mg / kg, 10 mg / kg to 100 mg / kg, or 50 mg / kg to 150 mg / kg, and the effective daily dosage of the NRRP compounds described herein can be a multiple of any value within these ranges, for example, 1 to 6 (1 to 6) times the value within these ranges.

[0163] Transitional Phrases

[0164] In some embodiments, the description of the compositions and methods described herein using the transitional word "comprising" indicates that the composition or method is "open" to additional ingredients, components or steps. "Comprising" is intended to encompass the more limiting transitional phrases "consisting essentially of" and "consisting of". Thus, the disclosure herein of substances following the transitional phrase "comprising" also fully discloses what follows the transitional phrase "consisting essentially of" or "consisting of". The transitional phrase "consisting essentially of" is an intermediate effect, which indicates that the following subject consists only of the recited elements and additional matters that do not substantially affect the novel and basic characteristics of the claim or the elements of the claim. The transitional phrase "consisting of" indicates that the following subject is limited to the recited steps or components and is closed to other steps or components not recited. When a transitional phrase appears within a clause or subclause that follows another transitional phrase, the embedded transitional phrase is intended to affect only the clause in which it appears. "A" or "an" is also intended to be plural unless explicitly stated in the specification or claims in the singular (e.g., "a single", "only one", etc.).

Examples

[0165] The pharmaceutical compositions and estrogen methods disclosed herein can be further understood with reference to the following examples.

[0166] Example 1: Analogue of 2’,3’,4’-trihydroxy chalcone (CC7).

[0167] Analogues of parental CC7 were prepared as shown in Tables 1A - 1C below.

[0168]

Chem.

[0169]

Table 1 - 1

[0170]

Table 1 - 2

[0171]

Table 1 - 3

[0172] Example 2: Estrogen Receptor (ERα) Stimulation in U2OS Cells

[0173] This specification describes nuclear receptor reprogramming (NRRP) compounds (“Test Compounds”), including those described in Tables 1A, 1B, and 1C, that are neither E2 agonists nor antagonists and do not confer mixed E2 agonist / antagonist activity. They do not activate reporter genes such as E2. They also do not block the effects of E2 on ERα, such as tamoxifen. Instead, these compounds produce synergistic activation of reporter genes in the presence of E2. NRRP compounds alone have little or no estrogen-like agonist or antagonist effect on the ERE. However, despite the lack of this activity in the absence of E2, in the presence of E2, NRRP acts synergistically to regulate two major classes of genes. One class of genes is synergistically activated by the combination of NRRP / E2. The other class of genes is called reprogrammed genes because they are not regulated by ERα unless NRRP is added to physiological concentrations of E2. Since they did not produce a synergistic effect with E2 in the absence of ER, the effects of NRRP are mediated through ER.

[0174] U2OS cells (wild type) were maintained in 5% charcoal-dextran stripped FBS. The cells were transfected by electroporation as previously described (An et al. 2001) with 3 μg of plasmid containing an ERE upstream of the minimal thymidine kinase luciferase promoter (ERE-TK-Luc) and 1 μg of an ERα expression vector. The resulting ERE-TK-Luc / ERα U2OS cells were incubated for 24 hours in the presence of medium (negative control), E2, and the test compound, with or without E2. As expected, E2 alone activated ERE-TK-Luc. (See U.S. Patent No. 7,482,029, which is hereby incorporated by reference in its entirety.) See the red horizontal lines in FIGS. 1-3. As seen in FIGS. 1-3, significant activation of ERE-TK-Luc did not occur with the test compound alone. In contrast, the test compound, when combined with E2, unexpectedly resulted in various levels of synergistic activation of the ERE. These activities are considered to be synergistic because the activities of each of the test compound and E2 exceeded the sum of the activity of E2 and the activity of each test compound.

[0175] Example 3: MCF-7 Cell Proliferation

[0176] Long-term hormone replacement therapy must not promote breast cancer in order to be a viable alternative to currently available treatment options. The growth-promoting properties of some of the compounds of the present invention were studied in MCF-7 breast cancer cells, alone and in combination with E2. MCF-7 cells were incubated for 24 hours in the presence of medium (control), 1 nM of E2, or 5 μM of each of the compounds shown in FIG. 4 (CC7 and six analogs, A9, A10, A11, A13, A15, and A20). The number of viable cells was determined by flow cytometry.

[0177] MCF-7 cells were seeded at a density of 50,000 cells per well in 6-well tissue culture plates in DMEM / F12 supplemented with 5% stripped FBS. The next day, the cells were treated with vehicle, E2, in the absence and presence of 2′,3′,4′-THC or one of the six analogs in Figure 4 for 7 days. The cells were then detached with trypsin, neutralized with medium containing 5% FBS, and resuspended. An appropriate amount of cell suspension was placed into ISOTON II diluent (Thermo Fisher Scientific, Waltham, MA, USA), and then the cell number was measured using a Coulter Counter (Beckman, Brea, CA, USA).

[0178] Flow cytometry was performed based on the previously described method (Pan et al. 2016). Briefly, cells were seeded at a density of 500,000 cells per well in 6-well tissue culture dishes in DMEM / F-12 supplemented with 5% stripped FBS for 48 hours. The culture medium was then replaced with serum-free DMEM / F12 for 24 hours. The cells were then treated with vehicle, E2, in the presence or absence of 2′,3′,4′-THC or one of the six analogs in Figure 4 for 24 hours. The culture medium was then aspirated, the cells were washed with PBS, detached with trypsin, and recovered by centrifugation at 1700 rpm for 5 minutes. The cell pellet was washed with ice-cold PBS and then centrifuged at room temperature at 1700 rpm for 10 minutes. The cell pellet was resuspended in 500 μL of PBS containing 50 μg / mL propidium iodide, 0.1% Triton X-100, 0.1% sodium citrate, and 10 μg / mL RNase. The cell suspension was then analyzed at the Flow Cytometry Facility at the University of California, Berkeley, using a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the percentage of cells in the cell cycle phase was determined using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0179] As shown in Figure 4, E2 alone stimulated the proliferation of breast cancer cells. In contrast, the number of viable cells for each of CC7 and the six analogs was significantly lower than that of E2 alone. The number of cells for A9+E2 was significantly lower than the control and lower than that of E2, while the number of cells for CC7+E2 and A10+E2 was significantly lower than that of E2 alone. These results indicate that CC7 and the six analogs do not stimulate MCF-7 breast cancer cell proliferation like E2. Furthermore, adding each of the six analogs to E2 does not enhance the stimulation of MCF-7 cell proliferation by E2 and, in some cases, even inhibits the pro-stimulatory activity of E2.

[0180] Example 4: Gene Expression Assays

[0181] 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 stripped fetal bovine serum (FBS, Gemini Bio Products, West Sacramento, CA, USA), 100 units / mL penicillin and streptomycin, 50 μg / mL 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.

[0182] U2OS-ERα cells were treated with E2 in the absence or presence 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 “reprogrammed” gene FGR was activated only by the combination of E2 and CC7 and not by either compound alone.

[0183] Similar experiments were performed using analogs of CC7, namely A9, A10, A13, A15, and A20. See Figure 5 for the results.

[0184] Example 5: Luciferase activity of ERE-TK-Luc in Ishikawa cells

[0185] 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. See Figure 6. CC7 and A9 did not affect the luciferase activity of the transfected cells, demonstrating that these compounds do not have estrogenic effects by themselves. However, when combined with E2, CC7 and A9 each showed a synergistic effect with E2, as seen in Figure 6.

[0186] Example 6: Luciferase activity by doxycycline in NKG2E-TK-Luc-transfected U2OS-ERβ cells. Doxycycline-dependent U2OS cells were transfected with NKG2E-TK-Luk and ERβ in the presence of E2 alone or in combination with each of the compounds in Tables 1A, 1B, and 1C. The luciferase activity of each compound was recorded. See Figure 7. As can be seen from Figure 7, A10, A13, A14, and A15 showed exceptional ERβ activity.

[0187] Example 7: Synthesis of the compounds in Tables 1A, 1B, and 1C. The compounds shown in Tables 1A, 1B, and 1C were prepared according to the synthetic procedures shown below.

[0188] A1

[0189] (E)-3-(4-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 was synthesized. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), 4-fluorobenzaldehyde a (354 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-3-(4-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0190]

Chemical formula

[0191] (E)-3-(4-Fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A1 was synthesized. To a solution of (E)-3-(4-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.63 mmol) in dry DCM (5 mL), BB r3 (0.95 g, 3.8 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was obtained and purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-3-(4-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A1 (63 mg, 36%) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 13.35 (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 = 8.8 Hz, 2H), 6.45 (d, J = 8.9 Hz, 1H).

[0192]

Chem.

[0193] A2

[0194] (E)-3-(3-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), 3-fluorobenzaldehyde a (354 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-3-(3-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0195]

Chem.

[0196] (E)-3-(3-Fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A2 synthesis. To a solution of (E)-3-(3-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (500 mg, 1.58 mmol) in dry DCM (5 mL), BB r3(2g, 7.9 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at [temperature] for 12 hours and then quenched with water at [temperature]. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 minutes) to obtain (E)-3-(3-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A2 (65 mg, 15%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.31 (s, 1H), 10.25 (s, 1H), 8.70 (s, 1H), 8.03 (d, J = 15.5 Hz, 1H), 7.88 (dt, J = 10.1, 2.1 Hz, 1H), 7.83 - 7.74 (m, 2H), 7.69 (s, 1H) 7.51 (td, J = 8.0, 6.1 Hz, 1H), 7.30 (td, J = 8.7, 2.6 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H).

[0197]

Chemical Structure

[0198] A3

[0199] (E)-3-(2-Fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), 2-fluorobenzaldehyde a (354 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 hours. The mixture was filtered to obtain (E)-3-(2-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0200]

Chemical Structure

[0201] (E)-3-(2-Fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one synthesis. To a solution of (E)-3-(2-fluorophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (300 mg, 0.95 mmol) in dry DCM (5 mL), BB r3 (1.2 g, 4.74 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was obtained and purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-3-(2-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A3 (235 mg, 90%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 13.27 (d, J = 32.2 Hz, 1H), 10.27 (d, J = 9.0 Hz, 1H), 8.72 (d, J = 9.9 Hz, 1H), 8.15 (t, J = 7.2 Hz, 1H), 8.03 (dd, J = 15.5, 5.1 Hz, 1H), 7.90 (d, J = 15.7 Hz, 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, 3.7 Hz, 1H).

[0202]

Chemical Structure

[0203] (E)-3-(m-Tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. EtOH:H2O (8 mL, V EtOH :V H2OTo a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in 3:1) was added 3-methylbenzaldehyde a (354 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol). The resulting solution was stirred at 0 °C to room temperature 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.

[0204]

Chemical formula

[0205] (E)-3-(m-Tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A4 synthesis. To a solution of (E)-3-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (300 mg, 0.95 mmol) in dry DCM (5 mL) was added BB r3 (1.2 g, 4.74 mmol) dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-3-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A4 (140 mg, 54%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.44 (s, 1H), 10.18 (s, 1H), 8.66 (s, 1H), 7.92 (d, J = 15.5 Hz, 1H), 7.83 - 7.70 (m, 4H), 7.28 (d, J = 8.0 Hz, 2H), 6.45 (d, J = 8.9 Hz, 1H) 2.36 (s, 3H).

[0206]

Chemical formula

[0207] A5

[0208] (E)-3-(m-Tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. In EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) was added with 3-methylbenzaldehyde a (342 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol). The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to obtain (E)-3-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (150 mg, 6.7%) as a white solid.

[0209]

Chemical formula

[0210] (E)-3-(m-Tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A5 synthesis. To a solution of (E)-3-(m-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (150 mg, 0.54 mmol) in dry DCM (5 mL) was added BB r3 (684 mg, 2.73 mmol) dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to obtain (E)-3-(m-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A5 (80 mg, 55%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.41 (s, 1H), 10.22 (s, 1H), 8.69 (s, 1H), 7.93 (s, 1H), 7.83 - 7.71 (m, 3H), 7.67 (d, J = 7.7 Hz, 1H), 7.36 (s, 1H), 7.29 (s, 1H) 6.46 (d, J = 8.9 Hz, 1H), 2.37 (s, 3H).

[0211] [Chemistry]

[0212] A6

[0213] Synthesis of (E)-3-(o-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), 2-methylbenzaldehyde a (354 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-3-(o-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (630 mg, 80%) as a white solid.

[0214] [Chemistry]

[0215] (E)-3-(o-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A6 synthesis. To a solution of (E)-3-(o-tolyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (300 mg, 0.95 mmol) in dry DCM (5 mL), BB r3 (1.2 g, 4.74 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was purified by flash reverse phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-3-(o-tolyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A6 (150 mg, 95%) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 13.37 (s, 1H), 10.23 (s, 1H), 8.70 (s, 1H), 8.12 - 7.95 (m, 2H), 7.86 (d, J = 15.4 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.40 - 7.21 (m, 3H), 6.45 (d, J = 8.9 Hz, 1H), 2.46 (s, 3H).

[0216]

Chem.

[0217] A7

[0218] Synthesis of (E)-3-(pyridin-4-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. EtOH:H2O (8 mL, V EtOH :V H2O = 3:1) of a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) was added isonicotinaldehyde (305 mg, 2.8 mmol) and KOH (156 mg, 2.8 mmol). The resulting solution was stirred at room temperature for 5 minutes. 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.

[0219]

Chem.

[0220] Synthesis of (E)-3-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A7. To a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL) was added BB r3(0.95 g, 3.8 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at room temperature for 6 hours and then quenched with water. The organic layer was concentrated and purified by RP column to obtain the crude product. The crude product was further purified by preparative HPLC to obtain (E)-3-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A7 (35 mg, 20%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.14 (s, 1H), 8.72 - 8.58 (m, 2H), 8.19 (d, J = 15.6 Hz, 1H), 7.89 - 7.81 (m, 2H), 7.82 - 7.68 (m, 2H), 6.47 (d, J = 8.9 Hz, 1H).

[0221]

Chemical Structure

[0222] A8

[0223] Synthesis of (E)-3-(pyridin-3-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1) were added nicotinaldehyde (305 mg, 2.8 mmol) and KOH (156 mg, 2.8 mmol). The resulting solution was stirred at room temperature for 5 minutes. The mixture was concentrated and purified by RP-column to obtain (E)-3-(pyridin-3-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (520 mg, 60%) as a yellow solid.

[0224]

Chemical Structure

[0225] Synthesis of (E)-3-(pyridin-3-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A8. To a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL) was added BB r3 (0.95 g, 3.8 mmol) dropwise at 0 °C under N2. 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 the 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-en-1-one A8 (15 mg, 8.7%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.28 (s, 1H), 9.03 (d, J = 2.1 Hz, 1H), 8.62 (dd, J = 4.7, 1.5 Hz, 1H), 8.37 (dt, J = 8.1, 2.2 Hz, 1H), 8.11 (d, J = 15.6 Hz, 1H), 7.92 - 7.66 (m, 2H) 7.50 (dd, J = 8.0, 4.7 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H).

[0226]

Chemical Structure

[0227] A9

[0228] (E)-3-(pyridin-2-yl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 was synthesized. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1) were added picolinaldehyde (305 mg, 2.8 mmol) and KOH (156 mg, 28 mmol). The resulting solution was stirred at room temperature 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-en-1-one 2 (500 mg, 58%) as a yellow solid.

[0229]

Chem.

[0230] (E)-3-(Pyridin-2-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A9 synthesis. To a solution of 2 (200 mg, 0.67 mmol) in dry DCM (5 mL), BB r3 (0.95 g, 3.8 mmol) was added dropwise at 0 °C under N2. 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 the crude product. The crude was further purified by preparative HPLC to give (E)-3-(pyridin-2-yl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A9 (30 mg, 17%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H), 10.31 (s, 1H), 8.80 - 8.64 (m, 2H), 8.19 (d, J = 15.3 Hz, 1H), 7.98 - 7.86 (m, 2H), 7.78 (d, J = 15.3 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.45 (ddd, J = 6.8, 4.7, 1.9 Hz, 1H), 6.48 (d, J = 8.9 Hz, 1H).

[0231]

Chem.

[0232] A10

[0233] (E)-3-(3-Methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. EtOH:H2O (8 mL, V EtOH :V H2OTo a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in DMF (3 mL), 3-methoxybenzaldehyde a (387 mg, 2.8 mmol) and KOH (1600 mg, 28 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to give (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (600 mg, 64%) as a white solid.

[0234] [Chemical formula]

[0235] (E)-3-(3-Hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A10 synthesis. To a solution of (E)-3-(3-methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.61 mmol) in dry DCM (5 mL), BB r3 (762 mg, 3 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at that temperature for 12 h and then quenched with water at that temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-3-(3-hydroxyphenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A10 (63 mg, 38%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.60 (s, 1H), 10.12 (s, 2H), 8.63 (s, 1H), 7.73 (q, J = 9.0, 8.5 Hz, 5H), 6.84 (d, J = 8.6 Hz, 2H), 6.43 (d, J = 8.9 Hz, 1H).

[0236] [Chemical formula]

[0237] A11

[0238] (E)-3-(4-Aminophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 synthesis. To a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (600 mg, 2.8 mmol) in EtOH:H2O (8 mL, V EtOH :V H2O = 3:1), 4-aminobenzaldehyde (338 mg, 2.8 mmol) and KOH (156 mg, 28 mmol) were added. The resulting solution was stirred at room temperature for 24 h. The mixture was concentrated and purified by FCC to give (E)-3-(4-aminophenyl)-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2 (300 mg, 34%) as a yellow solid.

[0239]

Chem.

[0240] (E)-3-(4-Aminophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A11 synthesis. To a solution of 2 (200 mg, 0.64 mmol) in dry DCM (5 mL), BB r3 (0.95 g, 3.8 mmol) was added dropwise at 0 °C under N2. 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 the crude product. The crude was further purified by preparative HPLC to give (E)-3-(4-aminophenyl)-1-(2,3,4-trihydroxyphenyl)prop-2-en-1-one A11 (30 mg, 17%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 13.86 (s, 1H), 9.99 (s, 1H), 8.55 (s, 1H), 7.76 - 7.62 (m, 2H), 7.62 - 7.52 (m, 3H), 6.59 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 8.8 Hz, 1H), 5.95 (s, 2H).

[0241]

Chem.

[0242] A18

[0243] Synthesis of 4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde 2. A solution of 3,4-dihydro-2H-pyran (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a well-stirred suspension of 4-hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p-toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred overnight at room temperature, then extracted with brine and dried over MgSO4. The solvent was removed under reduced pressure, and the crude compound was purified by column chromatography on silica gel using 20% EtOAc / hexane elution to give 2 as a yellow oil (2 g, 60%).

[0244]

Chem.

[0245] 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) were 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 room temperature for 5 minutes. The mixture was concentrated and purified by RP column to give a crude product. The crude 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.

[0246]

Chem.

[0247] 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 (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.79 (dt, J = 4.7, 1.3 Hz, 1H), 8.14 - 8.00 (m, 3H), 7.79 (d, J = 16.0 Hz, 1H), 7.74 - 7.62 (m, 3H), 7.05 - 6.73 (m, 2H).

[0248]

Chemical Structure

[0249] A13

[0250] Synthesis of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2. To a solution of 4-methoxybenzaldehyde 1 (300 mg, 2.2 mmol) 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 to room temperature for 12 h. The mixture was filtered to give (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (0.57 g, 96%) as a white solid.

[0251]

Chemical Structure

[0252] (E)-1,3-Bis(4-hydroxyphenyl)prop-2-en-1-one synthesis. To a solution of (E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one 2 (200 mg, 0.74 mmol) in dry DCM (5 mL), BB r3 (0.93 g, 3.73 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at the temperature for 12 h and then quenched with water at the temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to obtain N-benzyl-2,3,4-trihydroxybenzamide A13 (150 mg, 83%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.03 (d, J = 8.7 Hz, 2H), 7.76 - 7.53 (m, 4H), 6.85 (dd, J = 19.7, 8.6 Hz, 4H).

[0253]

Chemical formula

[0254] A14

[0255] (E)-1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one 2 synthesis. To a solution of 4-methoxybenzaldehyde 1 (300 mg, 2.2 mmol) in EtOH:H2O (4 mL, V EtOH :V H2O = 3:1), 1-(3,4-dimethoxyphenyl)ethan-1-one a (397 mg, 2.2 mmol) and KOH (246 mg, 4.4 mmol) were added. The resulting solution was stirred at 0 °C to room temperature for 12 h. The mixture was filtered to obtain (E)-1-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one 2 (0.64 g, 97%) as a white solid.

[0256]

Chemical formula

[0257] (E)-1-(3,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one Synthesis of A14. To a solution of (E)-1-(3,4-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one 2 (300 mg, 1 mmol) in dry DCM (5 mL), BB r3 (1.25 g, 5 mmol) was added dropwise at 0 °C under N2. The resulting mixture was stirred at temperature for 12 h and then quenched with water at temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give (E)-1-(3,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2 A14 (115 mg, 45%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 3H), 7.69 (d, J = 8.6 Hz, 2H), 7.63 - 7.54 (m, 3H), 7.49 (d, J = 2.0 Hz, 1H), 6.83 (t, J = 8.8 Hz, 3H).

[0258]

Chemical Structure

[0259] A15

[0260] (E)-3-(4-Hydroxyphenyl)-1-phenylprop-2-en-1-one Synthesis of 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-en-1-one A15 (40 mg, 23%) as a yellow solid. 11H 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).

[0261] [Chemical formula]

[0262] A16

[0263] Synthesis of 4 - ((tetrahydro - 2H - pyran - 2 - yl)oxy)benzaldehyde 2. A solution of 3,4 - dihydro - 2H - pyran (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a well - stirred suspension of 4 - hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p - toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred overnight at room temperature, then extracted with brine and dried over MgSO4. The solvent was removed under reduced pressure, and the crude compound was purified by column chromatography on silica gel using 20% EtOAc / hexane elution to give 2 as a yellow oil (2 g, 60%).

[0264] [Chemical formula]

[0265] (E)-1-(Pyridin-4-yl)-3-(4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 2 synthesis. 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 room temperature for 5 minutes. The mixture was concentrated and purified by RP column to obtain the crude product. The crude product was further purified by preparative HPLC to obtain (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.

[0266] [Chemical formula]

[0267] (E)-3-(4-Hydroxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one synthesis. 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 hours. The mixture was concentrated and purified by RP-column to obtain (E)-3-(4-hydroxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one A16 (60 mg, 83%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 10.20 (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).

[0268] [Chemical formula]

[0269] A17

[0270] Synthesis of 4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde 2. A solution of 3,4-dihydro-2H-pyran (4.1 g, 49 mmol) in dichloromethane (30 mL) was added dropwise to a well-stirred suspension of 4-hydroxybenzaldehyde (2 g, 16 mmol) and pyridinium p-toluenesulfonate (160 mg, 0.6 mmol) in dichloromethane (5 mL). The mixture was stirred overnight at room temperature, then extracted with brine and dried over MgSO4. The solvent was removed under reduced pressure and the crude compound was purified by column chromatography on silica gel using 20% EtOAc / hexane elution to give 2 as a yellow oil (2 g, 60%).

[0271] [Chemical formula]

[0272] (E)-1-(Pyridin-3-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) were 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 room temperature for 5 minutes. The mixture was concentrated and purified by RP column to give a crude product. The crude was further purified by preparative HPLC to give (E)-1-(pyridin-3-yl)-3-(4-((tetrahydro-??H-pyran-2-yl)oxy)phenyl)prop-2-en-1-one 3 (150 mg, 20%) as a yellow solid.

[0273] [Chemical formula]

[0274] Synthesis of (E)-3-(4-hydroxyphenyl)-1-(pyridin-3-yl)prop-2-en-1-one. PTSA (5.5 mg, 0.03 mmol) was added to a solution of 3 (100 mg, 0.32 mmol) in MeOH (1 mL). 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 A 1 7 (30 mg, 41%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.31 - 9.99 (m, 1H), 9.30 (d, J = 2.2 Hz, 1H), 8.81 (dd, J = 4.8, 1.7 Hz, 1H), 8.44 (dt, J = 8.0, 2.0 Hz, 1H), 7.82 - 7.69 (m, 4H), 7.60 (dd, J = 8.0, 4.8 Hz, 1H), 6.93 - 6.79 (m, 2H).

[0275]

Chemical formula

[0276] A20

[0277] Synthesis of (E)-3-phenyl-1-(2,3,4-trimethoxyphenyl)prop-2-en-1-one 2. Benzaldehyde (508 mg, 4.8 mmol) and KOH (537 mg, 9.6 mmol) were added to a solution of 1-(2,3,4-trimethoxyphenyl)ethan-1-one 1 (1.0 g, 4.8 mmol) in EtOH:H2O (10 mL, V EtOH :V H2O = 3:1). The resulting solution was stirred at room temperature 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.

[0278]

Chemical formula

[0279] Synthesis of 3-phenyl-1-(2,3,4-trimethoxyphenyl)propan-1-one 3. To a solution of 2 (500 mg, 1 equiv) in DCM (5 mL) was added 10% Pd / C (50 g). The mixture was stirred at room temperature for 6 h under a H2 atmosphere. Then, it was filtered through celite and the filtrate was concentrated under reduced pressure to obtain 3 as a crude product (450 mg). The crude product was used in the next step without purification.

[0280]

Chemical Structure

[0281] Synthesis of 3-phenyl-1-(2,3,4-trihydroxyphenyl)propan-1-one. To a solution of 3 (300 mg, 1.0 mmol) in dry DCM (5 mL) was added BB r3 (0.95 g, 3.8 mmol) dropwise at 0 °C under N2. 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 obtain 3-phenyl-1-(2,3,4-trihydroxyphenyl)propan-1-one A20 (100 mg, 39%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 10.07 (s, 1H), 8.60 (s, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.27 (d, J = 4.4 Hz, 4H), 7.18 (q, J = 4.4 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H) 3.29 (t, J = 7.6 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H).

[0282]

Chemical Structure

[0283] A21

[0284] 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) were added phenylmethanamine 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.

[0285]

Chemical formula

[0286] Synthesis of N-benzyl-2,3,4-trihydroxybenzamide. To a solution of N-benzyl-2,3,4-trimethoxybenzamide 2 (300 mg, 1 mmol) in dry DCM (5 mL) was added BB r3 (1.25 g, 5 mmol) dropwise at 0 °C under N2. The resulting mixture was stirred at a certain temperature for 12 h and then quenched with water at a certain temperature. Finally, the crude product was purified by flash reverse-phase column (MeOH in water = 5% - 100%, 60 min) to give N-benzyl-2,3,4-trihydroxybenzamide A21 (0.3 g, 70%) as a white solid. 1 1H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 9.50 (s, 1H), 9.10 (t, J = 6.0 Hz, 1H), 8.43 (s, 1H), 7.45 - 7.15 (m, 6H), 6.31 (d, J = 8.8 Hz, 1H), 4.47 (d, J = 5.9 Hz, 2H).

[0287]

Chemical formula

[0288] A24

[0289] 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.

[0290] Synthesis of 7,8-dimethoxyquinolin-4-ol 3. A solution of 2 (900 mg, 1 equiv) 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-dimethoxyquinolin-4-ol 3 (540 mg, 90% yield).

[0291]

Chemical Structure

[0292] 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 h. 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 give 4-chloro-7,8-dimethoxyquinoline 4 (300 mg, 51% yield) as a yellow solid.

[0293] (E)-7,8-Dimethoxy-4-styrylquinoline 5 was synthesized. A mixture of 4 (200 mg, 0.9 mmol), (E)-styrylboronic acid (132 mg, 0.9 mmol), K2CO3 (278 mg, 2.0 mmol) and Pd(dppf)Cl2 (37 g, 0.05 mmol) in 1,4-dioxane (1 mL) / H2O (0.3 mL) was stirred at 95 °C for 10 h. The mixture was cooled to room temperature, filtered through a pad of silica gel (EtOAc), and concentrated. The crude product was separated by chromatography on silica gel to give (E)-7,8-dimethoxy-4-styrylquinoline 5 (130 g, 50%) as a yellow solid.

[0294]

Chem.

[0295] (E)-4-Styrylquinoline-7,8-diol was synthesized. To a solution of 5 (130 mg, 1.0 mmol) in dry DCM (5 mL) was added dropwise BB r3 (0.95 g, 3.8 mmol) at 0 °C under N2. 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 (E)-4-styrylquinoline-7,8-diol A24 (40 mg, 34%) as a yellow solid. 1 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 8.74 (d, J = 4.6 Hz, 1H), 8.14 (s, 1H), 7.99 (d, J = 16.2 Hz, 1H), 7.82 (dd, J = 8.4, 5.1 Hz, 3H), 7.67 (d, J = 4.6 Hz, 1H), 7.56 (d, J = 16.2 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.37 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 9.1 Hz, 1H).

[0296]

Chem.

[0297] Example 8: Synthesis of analogs A9R1, A9R2, A9R3, and A9R4 (Figure 8) was carried out as follows. 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 room temperature for 2 h. The mixture was diluted with water and neutralized with 1 M aqueous HCl solution (pH adjusted to 7). The precipitate was filtered and recovered as the 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 (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.68 (dt, J = 4.7, 1.4 Hz, 1H), 8.14 (d, J = 15.4 Hz, 1H), 8.05 - 8.00 (m, 2H), 7.92 - 7.87 (m, 2H), 7.67 (d, J = 15.4 Hz, 1H), 7.47 - 7.39 (m, 1H), 6.96 - 6.88 (m, 2H).

[0298]

Chemical formula

[0299] Synthesis of (E)-1-(4-hydroxy-2-methylphenyl)-3-(pyridin-2-yl)prop-2-en-1-one. To a stirred solution of 1-(4-hydroxy-2-methylphenyl)ethan-1-one 1 (100 mg, 0.67 mmol, 1 equiv) in EtOH (2 ml), picolinaldehyde 2 (356.63 mg, 3.33 mmol, 5 equiv) and 6 M NaOH (480 mg, 13.32 mmol, 20 equiv) were added. The reaction mixture was stirred at room temperature for 5 h. The mixture was extracted with EA, washed with H2O and brine. The product was rotary evaporated under reduced pressure using a vacuum pump, recrystallized from methanol, and filtered. After suction drying with a vacuum pump, a yellow solid was obtained (26.4 mg, 16.3%). 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 7.91 - 7.70 (m, 4H), 7.53 - 7.38 (m, 2H), 6.73 (d, J = 8.0 Hz, 2H), 2.43 (s, 3H).

[0300]

Chemical Structure

[0301] 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 mixture was stirred at room temperature for 2 h. The mixture was diluted with water and neutralized with 1 M aqueous HCl (pH adjusted to 7). The precipitate was filtered off and recovered as the 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 (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 8.68 (dd, J = 5.0, 1.8 Hz, 1H), 7.92 - 7.88 (m, 1H), 7.86 (t, J = 2.7 Hz, 1H), 7.81 - 7.75 (m, 2H), 7.64 (dd, J = 15.3, 1.6 Hz, 1H), 7.43 (ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 6.77 (dd, J = 8.6, 2.3 Hz, 1H), 6.68 (dd, J = 13.5, 2.3 Hz, 1H).

[0302]

Chemical Structure

[0303] Synthesis of A9R4: 1-(2-Hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one. 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 with 3,4-dihydro-2H-pyran (1.6 g, 20.0 mmol). The reaction mixture was stirred at room temperature for 1 hour. TLC indicated the completion of the reaction. The solution was extracted three times with DCM, and the organic layer was washed three times with H2O and brine, dried over Na2SO4, and concentrated in vacuo. The crude product was purified by silica gel chromatography (PE:EA = 10:1) to obtain 1-(2-Hydroxy-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one 2 (1.3 g, 75%) as an off-white solid.

[0304]

Chem.

[0305] (E)-1-(2,4-Dihydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-one synthesis. 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. NaOH (0.34 g, 8 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. TLC indicated that 50% of the SM remained. 4N HCl was added to the solution to adjust the pH to about 4, then the mixture was stirred at room temperature for 1 h, and the orange solid was collected by filtration, washed three times with EtOH, and dried to give (E)-1-(2,4-dihydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-one C9529-4 (60 mg, 11%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.05 (s, 1H), 10.97 (s, 1H), 8.80 (d, J = 5.0 Hz, 1H), 8.40 (d, J = 15.4 Hz, 1H), 8.22 (d, J = 6.2 Hz, 2H), 8.13 (d, J = 9.0 Hz, 1H) 7.82 (d, J = 15.4 Hz, 1H), 7.73 - 7.64 (m, 1H), 6.47 (dd, J = 8.9, 2.3 Hz, 1H), 6.35 (d, J = 2.3 Hz, 1H).

[0306] [Chemical Structure]

[0307] 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. Refer to the structure in Figure 8. Doxycycline-dependent U2OS cells transfected with ERα and transfected with either ER-TK-Luc or NKG2E-TK-Luc were incubated with E2, A9, A9R1, A9R2, A9R3, or A9R4, or a combination of E2 and A9, A9R1, A9R2, A9R3, or A9R4. The results of luciferase activity are shown in Figure 9 (ER-TK-Luc) and Figure 10 (NKG2E-TK-Luc). Surprisingly, A9 showed the greatest synergistic effect with E2.

[0308] Example 8: MCF-7 cells on the 6th day after treatment

[0309] The growth-promoting properties of A9 and its analogs shown in Figure 8 were studied in MCF-7 breast cancer cells alone and in combination with E2. MCF-7 cells were incubated for 24 hours in medium (control), 1 nM E2, or the presence of 5 μM of each compound shown in Figure 8 (A9 and four analogs, A9R1, A9R2, A9R3, A9R4). The number of viable cells was determined by flow cytometry.

[0310] MCF-7 cells were seeded at a density of 50,000 cells per well in 6-well tissue culture plates in DMEM / F12 supplemented with 5% stripped FBS. The next day, the cells were treated with vehicle or E2 in the absence and presence of A9 or one of the four analogs shown in Figure 8 for 7 days. 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, Massachusetts, USA), and then the cell number was measured using a Coulter Counter (Beckman, Brea, California, USA).

[0311] Flow cytometry was performed based on the previously described method (Pan et al. 2016). Briefly, cells were seeded in 6-well tissue culture plates in DMEM / F-12 supplemented with 5% detached FBS at a density of 500,000 cells per well for 48 hours. Then, the culture medium was replaced with serum-free DMEM / F12 for 24 hours. Then, the cells were treated with vehicle, E2 for 24 hours in the presence or absence of 2′,3′,4′-THC or one of the six analogs in Figure 4. Then, the culture medium was aspirated, the cells were washed with PBS, detached with trypsin, and collected by centrifugation at 1700 rpm for 5 minutes. The cell pellet was washed with ice-cold PBS and then centrifuged at room temperature at 1700 rpm for 10 minutes. The cell pellet was resuspended in 500 μL of PBS containing 50 μg / mL propidium iodide, 0.1% Triton X-100, 0.1% sodium citrate, and 10 μg / mL RNase. Then, the cell suspension was analyzed at the Flow Cytometry Facility of the University of California, Berkeley using a BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the percentage of cells in the different phases of the cell cycle was determined using FlowJo 7.6.5 (FlowJo, LLC, Ashland, OR, USA).

[0312] As can be seen from Figure 11, E2 alone stimulated breast cancer cell proliferation. In contrast, the number of viable cells for each of the four analogs of CC7, A9 and A9 was significantly lower than the number of cells for E2 alone. The number of cells for CC7+E2, A9+E2, A9R1+E2, A9R2+E2 and A9R4+E2 was far below the control, which was lower than the number of cells for E2. These results indicate that the four analogs of CC7, A9, and A9 do not stimulate MCF-7 breast cancer cell proliferation like E2.

[0313] Statistical analysis: All data are presented as mean ± SE or SD from at least biological triplicates. The statistical significance of differences between two groups was evaluated by Student's t-test. For datasets consisting of groups larger than two, the statistical significance of differences between various groups (treatments) was analyzed by one-way analysis of variance (one-way ANOVA) test, or two-way ANOVA as specified in the figure legend. Following all ANOVA tests, Tukey or Sidak multiple comparisons post hoc tests were performed to analyze the significance of differences between any two different treatment groups or controls, as shown in the figure legend. Statistical analysis and graph plotting were performed using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). The statistical significance of the number of asterisks in the figures is *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0314] Although some embodiments of pharmaceutical compositions and estrogen methods are described herein, those skilled in the art will understand that the examples can be modified to provide other embodiments that utilize the NRRP compounds described herein and the methods described herein. Accordingly, it will be understood that the scope of the present invention is defined not by the specific embodiments shown as examples, but by the appended claims.

Claims

1. A nuclear receptor reprogramming compound having the structure of formula I, or a pharmaceutically acceptable salt thereof, wherein the compound of formula I is 【Chemistry 1】 And, During the ceremony, each 【Chemistry 2】 However, it is either a single bond or a double bond. A1, A2, and A3 are independently CR1 or N, A4 is CR6 or N, A5, A6, and A7 are independently CR2 or N, A8 is CR7 or N, A9 is CR3, CHR3, N, or NR3, Each R1 is independently H, halo, C1-C4 alkyl, OH, NH2, or O-C1-C4 alkyl. Each R² is independently either H or OH. R3 is H or C1-C4 alkyl, R4 is either O or OH, or together with R7 to form a pyrido or pyrimidino ring, forming a quinoline, isoquinoline, or quinazoline bicyclic ring system. R5 is either H, or together with R6, forms an -O-bridge to form a benzopyran bicyclic ring system. R 6 is either H or HO, or together with R 5 forms an -O-bridge to form a benzopyran bicyclic ring, and A nuclear receptor reprogramming compound, or a pharmaceutically acceptable salt thereof, wherein R7 is H, OH, or together with R4 to form a pyrido ring or pyrimidine ring, thereby forming a quinoline, isoquinoline, or quinazoline bicyclic ring system.

2. The nuclear receptor reprogramming compound according to Claim 1, or a pharmaceutically acceptable salt thereof, wherein each R1 is independently H, F, CH3, OH, NH2, or O-CH3.

3. A4 is CH, A 5 is CH, A6 is CH or COH, A7 is CH or COH, A nuclear receptor reprogramming compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein A8 is CH or COH.

4. The nuclear receptor reprogramming compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein R4 is OH.

5. The nuclear receptor reprogramming compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein R4 and R7 together form a pyrido ring or pyrimidino ring fused with an adjacent benzo ring.

6. The nuclear receptor reprogramming compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein R4 and R7 combine to form a pyrido ring condensed to an adjacent benzo ring to form a quinoline or isoquinoline.

7. The nuclear receptor reprogramming compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein R4 and R7 combine to form a pyrimidino ring fused with an adjacent benzo ring to form a quinazoline. 【Request Item 8】 【Chemistry 3】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 9】 【Chemistry 4】 And, The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein "E" indicates that the vinyl bond is an entgegen. 【Request Item 10】 【Chemistry 5】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 11】 【Chemistry 6】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 12】 【Chemistry 7】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 13】 【Chemistry 8】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 14】 【Chemistry 9】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 15】 【Chemistry 10】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 16】 【Chemistry 11】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 17】 【Chemistry 12】 The nuclear receptor reprogramming compound according to claim 1, wherein "E" in the formula indicates that the vinyl bond is an entgegen, or a pharmaceutically acceptable salt thereof. 【Request Item 18】 【Chemistry 13】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 19】 【Chemistry 14】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof. 【Request Item 20】 【Chemistry 15】 The nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof.

21. The following structure 【Chemistry 16】 A nuclear receptor reprogramming compound according to claim 1, having one of the structures of the above, or a pharmaceutically acceptable salt thereof.

22. A pharmaceutical composition comprising a nuclear receptor reprogramming compound according to Claim 1, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

23. The pharmaceutical composition according to claim 22, further comprising estradiol (E2), one or more estrogen receptor agonists, one or more estrogen receptor antagonists, one or more mixed estrogen receptor agonist / antagonists, one or more selective estrogen receptor modulators (SERMs), one or more progestogens, one or more glucocorticoids, and one or more androgens.

24. Use of the nuclear receptor reprogramming compound according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a pharmaceutical product for use in the treatment of a disease or disorder in a patient requiring treatment with a nuclear receptor reprogramming compound.

25. The use according to claim 24, wherein the disease or condition is a developmental disorder, menstrual disorder, pregnancy disorder, gynecological disorder, autoimmune disorder, menopausal disorder, aging disorder, or cancer.

26. The use according to claim 24 or 25, wherein the disease or disease state is Turner syndrome, Kallmann syndrome, congenital primary amenorrhea, childhood neuropsychiatric disorder, 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, Krone's disease, menopausal and pre- and post-menopausal vasomotor symptoms, insomnia, nocturnal awakenings, mood swings, vulvovaginal atrophy, vaginal dryness, sexual dysfunction, 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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