Estrogen Receptor Antagonist Regimens

Abstract

Provided herein is the administration method of estrogen receptor antagonist for use in the treatment of cancer.In some embodiments, provided herein is estrogen receptor antagonist tetrahydropyrido[3,4-b]indole compound.In some embodiments, provided antagonist is complete estrogen receptor antagonist.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 871,191, filed July 7, 2019, and U.S. Provisional Application No. 62 / 871,592, filed July 8, 2019, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002] The estrogen receptor (ER) plays an important role in various cancers, including breast cancer. A variety of therapeutics have been developed to target the estrogen receptor and / or its activity. Summary of the Invention [Means for solving the problem]

[0003] There remains a need for antiestrogens that can fully inhibit estrogen receptors, including those encoded by both wild-type and mutant forms (e.g., those containing activating mutations) of the genes encoding estrogen receptor alpha (ERα), estrogen receptor 1 (ESR1). Selective estrogen receptor modulators (SERMs) or degraders (SERDs) are particularly useful or promising tools for such therapy. Estrogen receptors are tripartite proteins that contain two distinct transcriptional activation functions (AF1 and AF2). Full antiestrogenic activity requires inactivation of both AF1 and AF2. Activating mutations in the gene encoding estrogen receptor 1 allow activation of both AF1 and AF2 even in the absence of estrogen.

[0004] Previous therapies such as tamoxifen, AZD9496, and ARN-810 cannot neutralize both activating functions (i.e., they do not neutralize both AF1 and AF2). Thus, there is still a need for therapies that neutralize both AF1 and AF2 to completely inhibit the estrogen receptor, and there is still a need for therapies that inhibit the estrogen receptor despite activating mutations. The present disclosure establishes, among other things, that certain compounds can be used alone or in combination with other agents as treatments for patients or subjects suffering from cancer, and the patients or subjects carry mutations in estrogen receptor 1 (ESR1). For example, in some embodiments, the compound that is useful for treating patients or subjects suffering from cancer, and the patients or subjects carry mutations in estrogen receptor 1 (ESR1), is compound 1. [ka]

[0005] In some embodiments, the present disclosure provides a method of treating a patient or subject suffering from an estrogen receptor (ER)-associated cancer comprising administration of an estrogen receptor antagonist and a CDK4 / 6 inhibitor.

[0006] In some embodiments, the disclosure provides a method for treating a patient or subject suffering from an estrogen receptor (ER)-associated cancer comprising administration of an estrogen receptor antagonist and a PIK3CA inhibitor.

[0007] In some embodiments, the present disclosure provides a method of treating a patient or subject suffering from an estrogen receptor (ER)-associated cancer comprising administration of an estrogen receptor antagonist and an mTOR inhibitor.

[0008] In some embodiments, the disclosure provides a method of treating a patient or subject suffering from cancer, wherein the cancer has metastasized to the brain, bone, lung, or liver, the method comprising administering Compound 1 to the patient or subject. [ka]

[0009] In some embodiments, the present disclosure provides an assay system for evaluating compounds or compositions for inhibition of estrogen receptor activation function 1 (AF1) and / or activation function 2 (AF2).

[0010] In some embodiments, the disclosure provides a method for treating a patient suffering from cancer, the method comprising oral administration of Compound 1. [ka] [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 is a scatter plot measuring percent estrogenic response as a function of Log[M] for certain estrogen receptor antagonist compounds with no added estrogen. [Figure 1B] FIG. 1 is a scatter plot measuring percent estrogenic response as a function of Log[M] for certain estrogen receptor antagonist compounds with added estrogen. [Diagram 2] 1 is a chart measuring estrogen receptor protein degradation across multiple cell lines for multiple estrogen receptor antagonists. [Figure 3A] FIG. 1 is a scatter plot showing the percent reduction in estrogen response for Compound 1 in combination with various CDK4 / 6 inhibitors. [Figure 3B] FIG. 1 is a scatter plot showing the percent reduction in estrogen response for Compound 1 in combination with various CDK4 / 6 inhibitors. [Figure 3C] FIG. 1 is a scatter plot showing the percent reduction in estrogen response for Compound 1 in combination with various CDK4 / 6 inhibitors. [Figure 4A] FIG. 1 is a scatter plot showing the percent reduction in estrogenic proliferation on MCF-7 cells upon treatment with Compound 1 in combination with a PIK3CA inhibitor. [Figure 4B] FIG. 1 is a scatter plot showing the percent reduction in estrogenic proliferation on MCF-7 cells upon treatment with Compound 1 in combination with a PIK3CA inhibitor. [Figure 5A] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 5B] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 5C] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 5D] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 5E] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 5F] FIG. 13 is a scatter plot of cell lines showing dose response of compound 1 on AF1 inhibition with the most common ESR1 mutations. [Figure 6] FIG. 1 is a scatter plot showing the change in tumor volume for various doses of Compound 1. [Figure 7A] FIG. 1 is a scatter plot measuring drug exposure (ng / ml) over time for mice. [Figure 7B] FIG. 1 is a scatter plot measuring drug exposure (ng / ml) over time for rats. [Figure 7C] FIG. 1 is a scatter plot measuring drug exposure (ng / ml) over time for dogs. [Figure 7D] FIG. 1 is a scatter plot measuring drug exposure (ng / ml) over time for monkeys. [Figure 8A] FIG. 1 is a scatter plot showing the reduction in estrogen concentrations across different cell lines. [Figure 8B] FIG. 1 is a scatter plot showing the reduction in estrogen concentrations across different cell lines. [Figure 8C] FIG. 1 is a scatter plot showing the reduction in estrogen concentrations across different cell lines. [Figure 8D] FIG. 1 is a scatter plot showing the reduction in estrogen concentrations across different cell lines. [Figure 9] 1 is a bar graph showing mutant ER that increases ligand-independent alkaline phosphatase activity (AP) in Ishikawa endometrial cancer cells. [Figure 10] 1 is a bar graph showing that activation domain 1 (AF1) of ER is required for AP activity. [Figure 11] Mammals express two major isoforms of ER, known as ERα and ERβ, each of which is a member of the nuclear hormone receptor family. Figure 1A is a reproduction of Patel & Bihani Pharm & Therap 186:1, 2018. A) The AF domains that make up the estrogen receptor, including the activation function 1 (AF1) domain, DNA binding domain (DBD), hinge region, and ligand binding domain (LBD) / activation function 2 (AF2 domain). B) Effects of endocrine therapy (aromatase inhibitors, SERMs, and SERDs) on the estrogen receptor pathway. Aromatase inhibitors prevent ER signaling by inhibiting the synthesis of estradiol, SERMs prevent ER signaling by binding to the ER and causing an inactive complex, and SERDs prevent ER signaling by causing degradation of the ER. [Figure 12] This is a reproduction of Figure 3 from Hewitt & Korach Endocrine Rev. 39:664-674 (June 12, 2018), showing variations in the basic mechanisms of the E2 response. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] There is a need for estrogen receptor (ER) positive cancer therapy that overcomes the problems associated with previous methods. The present disclosure provides, inter alia, a method for treating a patient or subject suffering from a cancer associated with estrogen receptor, a mutation in the estrogen receptor, comprising administering an estrogen receptor antagonist. In some embodiments, the estrogen receptor antagonist is Compound 1: [ka] or a pharma- ceutically acceptable salt thereof. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method of treating a subject suffering from a cancer characterized by a mutation in estrogen receptor 1 (ESR1), comprising administering to the subject compound 1: [ka] or a pharma- ceutically acceptable salt thereof. (Item 2) 3. The method of claim 2, wherein the mutation is an activating mutation. (Item 3) 1. A method of treating a subject suffering from cancer, comprising administering a compound that is an inhibitor of both activation function 1 and activation function 2 of the estrogen receptor. (Item 4) The compound is Compound 1: [ka] or a pharma- ceutically acceptable version thereof. (Item 5) A method of treating a subject suffering from cancer, comprising administering a compound that is an inhibitor of activation function 2 and a second agent that is an inhibitor of activation function 1. (Item 6) The compound is selected from the group consisting of AZD9496, RAD-1901, ARN-810, endoxifen, fulvestrant, and compound 1; [ka] or a pharma- ceutically acceptable salt thereof. (Item 7) 7. The method of claim 6, wherein the compound is selected from fulvestrant and compound 1. (Item 8) 8. The method of claim 7, wherein the compound is Compound 1. (Item 9) 9. The method of claim 8, wherein the secondary agent is a CDK4 / 6 inhibitor. (Item 10) 10. The method of claim 9, wherein the CDK4 / 6 inhibitor is selected from palvocociclib, ribociclib, abemaciclib, relociclib, and trilaciclib. (Item 11) The method of claim 10, wherein the CDK4 / 6 inhibitor is selected from palvocociclib and abemaciclib. (Item 12) 10. The method of claim 9, wherein the secondary agent is a PIK3CA inhibitor. (Item 13) 13. The method of claim 12, wherein the PIK3CA inhibitor is selected from alpelisib and taselisib. (Item 14) 10. The method of claim 9, wherein the second agent is an mTOR inhibitor. (Item 15) 15. The method of claim 14, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, and everolimus. (Item 16) 1. A method for treating a subject suffering from cancer that has metastasized to the brain, bone, lung, or liver, comprising administering to a subject a compound 1:

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[0013] definition Administration: As used herein, the term "administration" refers to administering a composition to a subject or system, typically to effect delivery of, e.g., an agent that is, is contained in, or is otherwise delivered by, the composition.

[0014] Agent: As used herein, the term "agent" refers to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof (e.g., cell, tissue, organism) or a phenomenon (e.g., heat, electric current or electric field, magnetic force or electric field, etc.).

[0015] Antagonist: As used herein, the term "antagonist" may refer to an agent or condition whose presence, level, degree, type, or form is associated with a decrease in the level or activity of a target. Antagonists may include agents of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity that exhibits relevant inhibitory activity. In some embodiments, an antagonist may be a "direct antagonist" in that it directly binds to its target, and in some embodiments, an antagonist may be an "indirect antagonist" in that it affects the level or activity of a target by means other than directly binding to its target, e.g., by interacting with a regulator of the target, thereby changing the level or activity of the target. In some embodiments, an "antagonist" may be referred to as an "inhibitor."

[0016] Associated: Two events or entities are "associated" with each other, as that term is used herein, if the presence, level, extent, type, and / or form of one correlates with the presence, level, extent, type, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they directly or indirectly interact with each other, such that they are in and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bound to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently bound to each other, but are non-covalently associated by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0017] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or biological fluid. In some embodiments, the biological sample may be or include bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymphatic fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, washings or lavages, such as ductal or bronchoalveolar lavage, aspirates, scrapings, bone marrow specimens, tissue biopsy specimens, surgical specimens, feces, other body fluids, secretions, and / or excretions, and / or cells therefrom, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells from the individual from whom the sample is obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more agents). For example, filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components.

[0018] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously, in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any dose of a second regimen), and in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administering one or more agent(s) or modality(s) to a subject receiving the other agent(s) or modality(s) in combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or necessarily simultaneously), although in some embodiments, two or more agents, or active portions thereof, may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent conjugate).

[0019] Dosage form or unit dosage form: Those skilled in the art will understand that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such amount is a unit dosage (or a whole fraction thereof) appropriate for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., correlates with a therapeutic dosing regimen). Those skilled in the art will understand that the total amount of a therapeutic composition or agent to be administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0020] Dosing regimen or treatment regimen: Those skilled in the art will understand that the terms "dosing regimen" and "treatment regimen" can be used to refer to a series of unit doses (typically more than one) administered individually to a subject, typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of the same length, and in some embodiments, the dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within the dosing regimen are the same unit dose. In some embodiments, the different doses within the dosing regimen are different amounts. In some embodiments, the dosing regimen includes a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen includes a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (ie, is a therapeutic dosing regimen).

[0021] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0022] Oral: As used herein, the phrases "oral administration" and "administered orally" have their art-understood meaning referring to oral administration of a compound or composition.

[0023] Parenteral: As used herein, the phrases "parenteral administration" and "administered parenterally" have their art-understood meaning referring to modes of administration other than oral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion.

[0024] Patient or subject: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition is or can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Exemplary patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient or subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient or subject exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, the patient or subject is undergoing or has undergone a certain therapy to diagnose and / or treat a disease, disorder, or condition.

[0025] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen to a relevant subject (e.g., in an amount that has been demonstrated to exhibit a statistically significant likelihood of achieving a predetermined therapeutic effect when administered), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or in a previous exposure to a condition or agent, etc.). In some embodiments, the comparative term refers to a statistically relevant difference (e.g., a difference of sufficient generality and / or magnitude to achieve statistical relevance). A person skilled in the art will recognize or be able to readily determine the degree and / or generality of difference that is necessary or sufficient to achieve such statistical significance in a given context.

[0026] Pharmaceutically acceptable carrier: As used herein, the term "pharmacologically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates and / or polyanhydrides, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0027] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutical acceptable salts" refers to salts of such compounds that are suitable for use in pharmaceutical contexts, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit-to-risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutical acceptable salts include, but are not limited to, non-toxic acid addition salts. Non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharma- ceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydrogen phosphate, 2-hydroxypropyl ... Examples of the salts include oxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.In some embodiments, pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, as appropriate.

[0028] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria, such as a certain age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is an agent that has been approved or needs to be approved by a government agency before it can be commercially marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a pharmaceutical prescription for administration to humans.

[0029] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0030] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that induces a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As one of ordinary skill in the art would appreciate, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, inhibits, prevents, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose, and in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0031] Estrogen Receptor Estrogen receptors ("ER") are involved in a variety of biological processes related to, for example, the development of the female reproductive system, the maintenance of bone mass, and the protection of cardiovascular and / or central nervous system components (see, e.g., Pearce & Jordan Crit. Rev. Onc / Hem 50:3, 2004; Heldring Phys Rev. 87:905, 2007).

[0032] Mammals express two major isoforms of ER, known as ERα and ERβ, each of which is a member of the nuclear hormone receptor family and has a structural organization as shown in Figure 1A of Patel & Bihani Pharm & Therap 186:1, 2018. This is reproduced in Figure 11. As shown in Figure 11, A) the AF domains that make up the estrogen receptor, including the activation function 1 (AF1) domain, DNA binding domain (DBD), hinge region, and ligand binding domain (LBD) / activation function 2 (AF2 domain). B) The effect of endocrine therapy (aromatase inhibitors, SERMs, and SERDs) on the estrogen receptor pathway. Aromatase inhibitors prevent ER signaling by inhibiting the synthesis of estradiol, SERMs prevent ER signaling by binding to the ER and causing an inactive complex, and SERDs prevent ER signaling by causing degradation of the ER.

[0033] As shown in the figure, six ER "domains" labeled AF are defined in the ER structure. Domain A / B, found at the amino terminus of the ER protein, is the largest domain and contains AF1, one of two so-called "transcriptional activation function" elements, and AF2 is found in the E domain, which also contains the ligand-binding domain and element(s) thought to be involved in ER dimerization and nuclear localization (see, e.g., Hewitt & Korach Endocrine Rev 39:664, 2018). Binding of a ligand to the ligand-binding domain is thought to cause a structural rearrangement of the alpha-helices in the E domain, which may contribute to the activity of AF2 (e.g., in interacting with certain mediator components).

[0034] The C-domain of ER contains its DNA-binding domain, which mediates interaction with so-called "estrogen-responsive element(s)" (ERE(s)) that are operably associated with genes whose transcription is regulated by ER. ERα and ERβ regulate the expression of different ERE-associated genes and show different cellular and tissue distribution patterns. DNA binding by ER appears to be mediated by two zinc finger structures in the C-domain, although additional element(s) may contribute (see, e.g., Hewitt & Korach Endocrine Rev 39:664, 2018). The C-domain of ER may also be involved in or otherwise contribute to ER dimerization.

[0035] The D domain of ER, also called the "hinge region," contains an amino acid element(s) that may be involved in dimerization and / or nuclear localization of ER.

[0036] The F domain of ER may play a role in ER protein stability. This domain appears to be a hallmark of the estrogen receptor compared to other nuclear receptor family members and may contribute to its responsiveness to certain therapies, such as tamoxifen (see, e.g., Arao et al, J. Bio. Chem 293:22, 8495).

[0037] In the presence of a natural ligand (e.g., 17β-estradiol), the ER undergoes a conformational change, homodimerizes, and localizes to the nucleus where it binds to EREs and regulates the transcription of its target genes (see, e.g., Pawlak et al., Kumar & Chambor, Hall & McDonnell). This sequence of events has been described as the "genomic" mechanism of ER gene regulation. Other mechanisms, such as "tethered," "non-genomic," and "ligand-independent," have also been described, as shown in Figure 3 of Hewitt & Korach. This is reproduced in Figure 12.

[0038] As shown in Figure 12, the basic features of the E2 response are changed as reported by Hewitt & Korach. Four different E2 response mechanisms have been described. (1) The genomic mechanism involves the interaction of the ER with the ERE DNA motif. (2) The tether mechanism involves an indirect interaction between the ER and other transcriptional regulators, such as the AP1 DNA motif that binds FOS / JUN dimers. Thus, the ER is "tethered" to the DNA in this example through FOS / JUN binding to its AP1 DNA motif. (3) Non-genomic signaling is so called because it initiates a signal from the extracellular E2, which leads to a rapid signal cascade in the cytoplasm, and therefore this response does not involve interaction with genomic features. This response is mediated by the membrane-associated ER, or GPER, a G protein-coupled receptor. (4) Ligand-independent signaling involves the transduction of extracellular growth factor (GF) activation of cell membrane GF receptors (GFRs) that initiate signaling cascades such as MAPK. This signal is received by the ER and activates its transcriptional regulation of target genes despite the lack of an E2 ligand.

[0039] ER is involved in a variety of cancers. Many tumors express estrogen receptors (i.e., ER + In tumors, active ERα signaling has been demonstrated to lead to cell proliferation (although it has been reported that ERβ signaling can achieve tumor suppressive effects; see, e.g., Nilsson & Gustafson Clin. Pharmacol. Ther. 89:44, 2011). Typically, tumors (e.g., breast tumors) with only 1% of cells staining positive for ER are characterized as "ER + " is classified as such.

[0040] ER-targeted therapy +It is the standard treatment for many patients with tumors (see, e.g., Cardoso et al Annals Onc. https: / / doi.org / 10.1093 / announc / mdmx036,2017; Rugo et al. J. Clin. Oncol. 34:3069,2016; Senkus et al Annal Onc. 26:v8,2015; Sareddy&Vadlamudi Clin. J Nat. Med, 13:801,2015). For example, for early stage breast cancer patients, the recommended therapy typically involves tumor resection followed by ER-targeted therapy (e.g., as discussed below). For advanced breast cancer, including metastatic breast cancer, ER-targeted therapy is the norm.

[0041] Estrogen Receptor Targeted Therapy Given the importance of ER signaling in many cancers, and certain cardiovascular, inflammatory, and neurodegenerative diseases, significant efforts have been invested in the development of therapeutic agents and modalities that target the ER. Although there is some fluidity / flexibility in the terminology used to describe ER-targeting agents, a variety of agents with different mechanisms are being developed and / or investigated.

[0042] Several ER-targeting drugs have been designed and / or demonstrated to reduce the levels of estrogen (ie, 17β-estradiol) production.

[0043] Several ER-targeting drugs have been designed and / or demonstrated to bind directly to the ER, and in some cases such drugs compete with estrogen for binding to the ER and / or prevent the allosteric changes that naturally occur with estrogen binding. Often the term "anti-estrogen" is used to refer to drugs that bind to the ER, and in some cases it is used specifically to denote drugs that compete with estrogen for ER binding.

[0044] The term "selective estrogen receptor modulators" ("SERMs") are used to refer to compounds that are designed and / or demonstrated to alter some aspect of ER activity. Some descriptions refer to "SERMs" as representing a specific class of antiestrogens, however, other descriptions use the term "SERMs" more generally to refer to compounds that specifically affect some characteristic of ER (particularly ERα) expression and / or activity.

[0045] The term "selective estrogen receptor degraders" ("SERDs") is used to refer to compounds that are designed and / or demonstrated to induce or enhance the degradation of ER. Often, a compound may be referred to as a SERD if its presence correlates with a reduction in the level of ER. Some descriptions classify compounds as either SERMs or SERDs, while others refer to SERDs as a particular class or species of compounds that are SERMs.

[0046] Regardless of the mechanism of action of a particular agent, clinical experience to date has demonstrated that incomplete efficacy (e.g., within individual patients and / or across patient populations) and / or the development of resistance remain problems.

[0047] In particular, the presence or expression of certain ER mutations has been reported to affect the efficacy of various ER-targeted therapies (see, e.g., Jeselsohn et al Nature Rev. Clin. Onc. 12,573,2015; Gelsomino et al. Breast Cancer Res. Treat 157:253,2016; Toy et al. 2013). Some particularly problematic mutations are those that "activate" one or more aspects of ER expression and / or function, and some activating mutations have been reported to render ER ligand-independent (i.e., constitutively active). For example, certain mutations in the ER ligand-binding domain, including D538G and Y537S, have been demonstrated to constitutively activate ER, and other mutations, including deletions and / or fusions that remove the ligand-binding domain, may have similar effects (see, e.g., Li et al. Cell Repts 4:1116, 2013; Veeraraghavan et al Breast Cancer Research and Treatment 158, 219-232, 2016; Veeraraghavan, et al. Nature Comms 5:4577, 2014). Some reports indicate that up to 50% of women with metastatic breast cancer may have activating ER mutations detectable in circulating tumor DNA.

[0048] Estrogen receptor antagonists As mentioned above, enormous investments have been and continue to be made in the pursuit of effective therapies targeting the ER (reviewed, for example, in Patel & Bihani Pharmacol. & Therap. 186:1, 2018).

[0049] The most advanced compounds in clinical development include: a. Tamoxifen, an important breast cancer treatment, is credited with "saving the lives of 500,000 women worldwide" (see "Bringing the Investigational Breast Cancer Drug Endoxifen From Bench to Bedside with NCI Support," available at https: / / www.cancer.gov / news-events / cancer-currents-blog / 2017 / endoxifen-breast-cancer-NCI-support (last accessed July 7, 2019) but is known to be less effective in women with low CYP2D6 activity and prone to the development of resistance). b. Endoxifen, the active metabolite of tamoxifen, was originally developed to address tamoxifen failure in women with low CYP2D6 activity, which reduces the ability to convert tamoxifen to endoxifen (see Cancer Currents Blog, National Cancer Institute, August 31, 2017). c.ARN-810 (Brillanestran, GDC-810), which has been described as "a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist / ER degrader that induces tumor regression in tamoxifen-sensitive and resistant ER+BC xenograft models" (see Dickler et al. Cancer Res. 75(15 Suppl):Abstract nr CT231, 2015), has been brought into Phase II clinical trials for the treatment of ER+ breast cancer patients who have failed other hormonal agents but may have subsequently experienced reduced further growth (see, e.g., Biospace April 27, 2017). d. AZD9496 has been described as "an oral nonsteroidal small molecule inhibitor of estrogen receptor alpha (ERα) and a potent and selective antagonist and degrader of ERα" (see Hamilton et al Clin Cancer Res 1:3519, 2018). AZD9496 has been reported as "antagonizing[ze] and degrading[e]ER with antitumor activity in both endocrine-sensitive and endocrine-resistant models" and has been described as "comparable to fulvestrant in antagonizing ER and circumventing endocrine resistance" (see Nardone et al. Br. J. Cancer 120:331, 2019). e.RAD-1901 (elacestrant) has been described as a "novel nonsteroidal oral SERD that demonstrated single-agent activity in heavily pretreated patients with ER+ advanced breast cancer" (see de Vries et al, Cancer Res. Abstract P1-10-04, 2018; see also Bardia et al. J. Clin. Onc. 35:15_suppl, 1014, 2017). Also, preclinical studies have reported that "elacestrant significantly inhibited the growth of xenograft models harboring ESR1 mutations, including those with Y537S or D538G mutations, as well as models that were insensitive to fulvestrant and tamoxifen" (see Patel et al. Cancer Res 79: Abstract nr P6-20-08, 2019). f. Fulvestrant (Faslodex™) was the first FDA-approved SERD and is approved for the treatment of certain ER+ cancers, including in combination with palbociclib or abemaciclib. Fulvestrant is a "selective estrogen receptor degrader that binds to, blocks, and degrades the estrogen receptor (ER), resulting in complete inhibition of estrogen signaling through the ER" (see Nathan & Schmid Oncol Ther 5:17, 2017). Fulvestrant has had significant clinical success and is often considered the "gold standard" against which ER-targeted therapies are compared. However, fulvestrant is administered by injection rather than orally, and in practice (after the first dose) requires monthly administration of 500 mg by intramuscular injection. Also, certain retrospective analyses have offered hope that fulvestrant may have some utility in treating patients with ER mutants, but have not achieved confirmed activity (see, e.g., Fribbens et al. J Clin Oncol. 34:2961, 2916; Spoerke et al. Nat Commun 7:11579, 2016). [ka]

[0050] The present disclosure demonstrates that the success of fulvestrant is due to its ability to (1) inhibit both AF1 and AF2, thereby inhibiting AF1 activity that persists in constitutively active ER mutants, (2) promote ER degradation, and (3) lack the partial ER agonist activity observed with certain other agents (see, e.g., FIG. 1A, among others, which establishes that ARN-810, AZD-9496, and endoxifen each increase ER activity in the absence of added estrogen, while reducing ER activity when estrogen is present therein). For example, compared to therapies that limit estrogen production (e.g., anastrozole) or partial antagonists (e.g., tamoxifen), fulvestrant exhibits superior activity and is a preferred treatment option for patients with hormone receptor-positive locally advanced or metastatic breast cancer. See Robertson, et al. The Lancet, 388(10063):2997-3005 (Dec.17, 2016). Without wishing to be bound by any particular theory, it is proposed that the ability of fulvestrant to inhibit both AF1 and AF2 may be due to its recruitment of corepressors to the ER complex.

[0051] Nonetheless, the present disclosure further recognizes that many other compounds, including, for example, ARN-810, AZD9496, tamoxifen, and others, are less effective than fulvestrant, at least in part, because they only partially antagonize the ER, specifically inhibiting activation of AF2 but not AF1.

[0052] The present disclosure relates to the aforementioned compound, (1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1-(4-((1-propylazetidin-3-yl)oxy)phenyl)-2,3,4,4a,9,9a- Tetra Hydro-1H-pyrido[3,4-b]indole ("Compound 1", see PCT Publication No. 2017 / 059139, which is incorporated by reference in its entirety) [ka] This provides important and unexpected insights that are consistent with the CERAN attributes of fulvestrant, and further provide additional valuable properties, including (i) being orally bioavailable, having a long half-life, and (ii) exhibiting good blood-brain barrier penetration. In particular, the present disclosure demonstrates that compound 1 is uniquely useful in certain contexts, including (a) treating cancers associated with ER mutations, including ligand-independent / constitutive mutations, (b) treating cancers with CNS (e.g., brain) metastases or tumors, and (c) in combination with certain other agents, including certain agents that have been shown or proposed to be useful with fulvestrant.

[0053] For example, among other things, the present disclosure reports the discovery that Compound 1 exhibits full estrogen receptor antagonism, and further, that such antagonism is comparable to fulvestrant in various assays. As reported in Example 4, among other things, the present disclosure demonstrates that Compound 1 is characterized by its ability to inhibit AF1 and AF2, and thus is appropriately described as a "full estrogen receptor antagonist."

[0054] The present disclosure teaches that Compound 1 may be particularly useful or effective in treating diseases, disorders, or conditions (e.g., cancer) associated with the presence of one or more ER mutants, including specifically ligand-independent ER mutants. Thus, in some embodiments, the present disclosure provides methods of treatment in which Compound 1 is administered to a subject expressing (e.g., in relevant cells or tissues) one or more ERs, and a particular ligand-independent ER or a plurality of ERs, and in some embodiments, such subject(s) have been demonstrated to express such mutant ER(s) prior to administration.

[0055] Those skilled in the art will recognize various techniques that allow for the detection of mutant ER in a sample from a subject. In some embodiments, mutant ER protein is detected, and in some embodiments, the nucleic acid encoding the mutant protein (e.g., mutant ESR1 gene) is detected.

[0056] The ability of Compound 1 to inhibit both AF1 and AF2 allows Compound 1 to function as a CERAN despite activating mutations in estrogen receptors (eg, ESR1).

[0057] Thus, in some embodiments, the disclosure provides a method of treating a subject (or a population of subjects) suffering from cancer, wherein the subject carries an ESR1 mutation (and / or expresses a mutant ER protein), and the method comprises administering to the subject Compound 1, [ka] or a pharma- ceutically acceptable salt thereof.

[0058] Evaluation of ER antagonists Among other things, the present disclosure teaches that useful ER antagonist agents are those that have CERAN activity as described herein.

[0059] One aspect of the present disclosure is the insight that conventional strategies for evaluating or characterizing ER antagonist (and / or potential antagonist) agents have been deficient, at least in that they typically do not distinguish between SERD and CERAN. In particular, most such conventional strategies do not evaluate the ability of agents to specifically affect AF1.

[0060] In particular, the present disclosure teaches that particularly useful ER antagonist agents are those that can inhibit ligand-independent ER activity, which in some embodiments includes activity observed with constitutive ER variant(s), such as, for example, AF2 deletions or truncations, and / or LBD mutants (e.g., D538G and Y537S).

[0061] Further, the present disclosure provides that particularly useful ER antagonist agents include a. Inhibition of AF1 (e.g., inhibition of at least one, and preferably all, known constitutive ER variants) b. Inhibition of AF2 (e.g., inhibition of ligand-dependent ER activity) c. Promotion of ER degradation.

[0062] Additionally, in some embodiments, particularly useful ER antagonist agents are: a. Oral bioavailability and long half-life. b. Penetration of the blood-brain barrier.

[0063] In certain embodiments, the activity of the ER antagonist agent(s) may be evaluated in comparison to the activity of one or more of ARN-810, AZD9496, endoxifen, fulvestrant, RAD1901, tamoxifen, and / or compound 1, and in some such embodiments, the comparison may be made contemporaneously or, in some embodiments, to historical records or future results.

[0064] Combination therapy The present disclosure further provides insight that the unique ability of compound 1 to function as an inhibitor of both AF1 and AF2 makes it particularly attractive for use in certain combination therapies. As shown in Figures 3A-3B (for CDK4 / 6 inhibitors) and Figures 4A-4B (for PIK3CA inhibitors), compound 1, an estrogen receptor antagonist, and in fact a complete estrogen receptor antagonist, can be combined with a second agent to substantially eliminate cell proliferation. The present disclosure encompasses the recognition that certain drug combinations can be beneficially used to completely antagonize the estrogen receptor by inactivating both AF1 and AF2. Thus, in some embodiments, the present disclosure provides a method of treating a subject suffering from cancer, comprising administering a compound that is an inhibitor of activation function 2 and a second agent that is an inhibitor of activation function 1. In some embodiments, the compound is an estrogen receptor antagonist selected from AZD9496, RAD-1901, ARN-810, endoxifen, fulvestrant, and compound 1. In some embodiments, the compound is selected from fulvestrant and compound 1.

[0065] In some embodiments, the disclosure provides a method of treating a patient or subject suffering from cancer, the method comprising administering Compound 1 and a second agent selected from a CDK2, CDK4, CDK6, or CDK7 inhibitor. In some embodiments, the second agent is a CDK2 inhibitor. In some embodiments, the second agent is a CDK4 inhibitor. In some embodiments, the second agent is a CDK6 inhibitor. In some embodiments, the second agent is a CDK7 inhibitor. In some embodiments, the second agent is a CDK4 / 6 inhibitor (i.e., inhibits one or both of CDK4 and CDK6). In some embodiments, the second agent is a CDK2 / 4 / 6 inhibitor (i.e., inhibits one or more of CDK2, CDK4, and CDK6).

[0066] In some embodiments, the second agent is a CDK4 / 6 inhibitor selected from palvocociclib, ribociclib, abemaciclib, relociclib, trilaciclib, and SHR6390. In some embodiments, the CDK4 / 6 inhibitor is palvocociclib. In some embodiments, the CDK4 / 6 inhibitor is ribociclib. In some embodiments, the CDK4 / 6 inhibitor is abemaciclib. In some embodiments, the CDK4 / 6 inhibitor is relociclib. In some embodiments, the CDK4 / 6 inhibitor is trilaciclib. In some embodiments, the CDK4 / 6 inhibitor is SHR6390.

[0067] In some embodiments, the disclosure provides a method of treating a patient or subject suffering from cancer, the method comprising administering Compound 1 and a second agent, wherein the second agent is a PIK3CA inhibitor. In some embodiments, the PIK3CA inhibitor is selected from alpelisib, taselisib, and LY3023414. In some embodiments, the PIK3CA inhibitor is alpelisib. In some embodiments, the PIK3CA inhibitor is taselisib. In some embodiments, the PIK3CA inhibitor is LY3023414.

[0068] In some embodiments, the present disclosure provides a method of treating a patient or subject suffering from cancer, the method comprising administering compound 1 and a second agent, the second agent being an mTOR inhibitor. In some embodiments, the mTOR inhibitor is selected from sirolimus, temsirolimus, everolimus, and LY3023414. In some embodiments, the mTOR inhibitor is sirolimus. In some embodiments, the mTOR inhibitor is temsirolimus. In some embodiments, the mTOR inhibitor is everolimus. In some embodiments, the mTOR inhibitor is LY3023414.

[0069] Administration The present disclosure encompasses the recognition that certain disorders or conditions, such as cancer, can be effectively treated using less active compound than other compounds of similar activity.For example, as shown in Figure 6, compound 1 has been found to reduce tumor volume more efficiently than other CERANs, such as fulvestrant.In addition, compound 1 is suitable for oral administration, which is advantageous over other CERANs, such as fulvestrant, that need to be administered parenterally.

[0070] Thus, the present disclosure provides a method of treating a patient or subject suffering from cancer, the method comprising administering a composition comprising Compound 1. In some embodiments, the composition comprises Compound 1 and a pharma- ceutically acceptable excipient, carrier, or diluent. The aforementioned compositions may be administered orally, parenterally, by inhalation or nasal spray, topically (e.g., by powder, ointment, or drops), rectally, bucally, vaginally, intraperitoneally, intravesically, or via an implanted reservoir, depending on the severity of the condition being treated. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. In certain embodiments, the provided compounds are administered orally or parenterally at a dosage level of about 0.01 mg / kg to about 50 mg / kg of the subject's body weight per day, one or more times per day, to obtain the desired therapeutic effect.

[0071] The pharmaceutically acceptable compositions described herein can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.In such solid dosage forms, active compound may be mixed with at least one inert diluent, such as sucrose, lactose or starch.Such dosage forms may also contain additional substances other than inert diluents, such as lubricants and other tableting aids, as is customary, such as magnesium stearate and microcrystalline cellulose.When aqueous suspensions are required for oral use, active ingredient is combined with emulsifiers and suspending agents.If desired, certain sweeteners, flavors or colorants can also be added.

[0072] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharma- ceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) humectants such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and / or i) talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. The active compounds can also be in microencapsulated form with one or more excipients as noted above.

[0073] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings (i.e., buffers) and other coatings well known in the pharmaceutical formulating art. These may optionally contain opacifying agents and may also be of a composition that optionally releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0074] Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed flour, peanut, corn, germ, olive, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and aromatic agents.

[0075] Alternatively, the pharma- ceutically acceptable compositions disclosed herein can be administered in the form of suppositories for rectal or vaginal administration. These can be prepared by mixing the compounds of the present application with suitable non-irritating excipients or carriers, which are solid at room temperature but liquid at body temperature (e.g., rectum or vagina), and therefore melt in the rectum or vaginal cavity to release the active compound. Such materials include cocoa butter, suppository waxes (e.g., beeswax), and polyethylene glycols.

[0076] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered in an amount of 30 mg / kg or less of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount of 10 mg / kg or less of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / kg of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount of 3 mg / kg or less of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount of 1 mg / kg or less of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / kg of the patient's or subject's body weight. In some embodiments, the composition is administered in an amount that is 0.1 mg / kg of the patient's body weight.

[0077] In some embodiments, Compound 1 is administered as a unit dosage form. In some embodiments, Compound 1 is administered in the form of a capsule. In some embodiments, Compound 1 is administered in the form of a tablet. In some embodiments, Compound 1 is administered as a suspension. In some embodiments, Compound 1 is administered as a solution.

[0078] In some embodiments, Compound 1 is administered as a daily dose (QD). In some embodiments, Compound 1 is administered as a twice-daily dose (BID). In some embodiments, Compound 1 is administered every other day (QOD). In some embodiments, Compound 1 is administered as a weekly dose (QW). In some embodiments, Compound 1 is administered as a monthly dose (Q4W).

[0079] As shown in Figures 7A-7D, drug exposure (ng / ml) over time is also high in mouse (Figure 7A), rat (Figure 7B), dog (Figure 7C), and monkey (Figure 7D) subjects.

[0080] Fault or condition The present disclosure also includes the recognition that compound 1 can be advantageously used to treat metastatic cancer, for example, cancer that spreads to the brain, bone, lung, liver, or central nervous system.As illustrated in the table below, compound 1 can penetrate the blood-brain barrier when administered at a single oral dose of 300mg / kg.Other estrogen receptor antagonists, such as fulvestrant, cannot penetrate the blood-brain barrier at similar doses. [Table 1]

[0081] Thus, the present disclosure provides a method for treating a patient or subject suffering from cancer that has metastasized to the brain, bone, lung, liver, or central nervous system, comprising administering Compound 1. [ka]

[0082] In some embodiments, the cancer comprises one or more CNS tumors (e.g., metastases), and in some embodiments, the cancer has metastasized to the brain, bone, lung, or liver, hi some embodiments, the cancer has metastasized to the central nervous system.

[0083] Example The examples provided herein demonstrate and support certain aspects of the present disclosure, but are not intended to limit the scope of any claims. Unless specifically presented in the past tense, the inclusion of an example is not intended to imply that the described work has been completed or even performed. The following non-limiting examples are provided to further illustrate certain teachings provided by the present disclosure. Those skilled in the art will understand in light of this application that various changes can be made in the specific embodiments illustrated in the examples without departing from the spirit and scope of the teachings of the present invention.

[0084] The following abbreviations may be used in the following examples: aq. (aqueous), ACN (acetonitrile), CSA (camphorsulfonic acid), d (day or days), DCM (dichloromethane), DEA (diethylamine), DHP (dihydropyran), DMF (N,N-dimethylformamide), DIPEA (N,N-diisopropylethylamine), DMAP (4-dimethylaminopyridine), DMSO (dimethylsulfoxide), EA (ethyl acetate), ee (enantiomeric excess), equiv. (equivalents), ethanol (EtOH), h (hour or hours), Hex (hexane), HPLC (high performance liquid chromatography), IPA (isopropyl alcohol), KHMDS (potassium bis(trimethylsilyl)sulfate ... (trimethylsilyl)amide, LAH (lithium aluminum hydride), LCMS (liquid chromatography mass spectrometry), LDA (lithium diisopropylamide), LiHMDS (lithium bis(trimethylsilyl)amide), MeOH (methanol), min (minute or minutes), NMR (nuclear magnetic resonance), Pd / C (palladium on carbon), PPhO (triphenylphosphine oxide), Pt / C (platinum on carbon), rb (round bottom), Rf (retention factor), rt or RT (room temperature), SM (starting material), TEA (triethylamine), THF (tetrahydrofuran), THP (tetrahydropyran), TLC (thin layer chromatography), TsOH (p-toluenesulfonic acid or tosylic acid), and UV (ultraviolet).

[0085] Example 1: Synthesis of compound 1 The complete synthesis of compound 1 is presented in PCT Publication No. 2017 / 059139, which is incorporated herein by reference and repeated below. Preparation of 4-((1-propylazetidin-3-yl)oxy)benzaldehyde [ka] Step 1: Preparation of 1-propionylazetidin-3-one [ka]

[0086] Compound 3-azetidinone hydrochloride (10.000 g, 93.0 mmol, 1.0 equiv), anhydrous 1,2-dichloroethane (200 mL), and diisopropylethylamine (38.9 mL, 223 mmol, 2.4 equiv) were added to a round bottom flask (500 mL) to give a pale yellow suspension. The suspension was sonicated for 1 h and then cooled to -10 °C (dry ice / MeOH) for 10 min. Propionyl chloride (9.8 mL, 112 mmol, 1.2 equiv) was added dropwise to this cooled suspension to give an orange solution. The reaction was removed from the bath and stirred at room temperature for 16 h. The solvent was removed to give a semi-solid. The semi-solid was suspended in EA (300 mL) and the suspension was filtered. The semi-solid was rinsed with EA (2x100 mL). TLC analysis (10% MeOH / DCM, KMnO7 stain / heat) showed there were three spots: Rf: 0.2, 0.5, 0.7. TLC (50% EA / Hex, KMnO7 stain / heat) showed there were two spots: Rf: 1, 0.3. The filtrate was concentrated, adsorbed onto silica gel (25 g) and chromatographed through silica gel (100 g cartridge) with DCM (5 min), then 0-10% MeOH over 15 min. The product separated early from the column in DCM and continued to elute from the column with up to 10% MeOH. TLC was performed in both solvent systems to determine if any propionyl chloride was present in the initial fractions. Fractions containing the product were pooled and concentrated to give the title compound as a yellow liquid (11.610 g, 98.2%).

[0087] 1 H NMR (300 MHz, CDCl3) δ: 4.80 (d, J = 5.6 Hz, 4H), 2.29 (q, J = 7.5 Hz, 2H) ,2.01 (s, 3H), 1.18 (t, J = 7.5 Hz, 3H). Step 2. Preparation of 1-propylazetidin-3-ol [ka]

[0088] Lithium aluminum hydride (10.397 g, 273.9 mmol, 3.0 equiv) was suspended in THF (200 mL) and cooled in an ice bath. To this reaction mixture was added a solution of 1-propionylazetidin-3-one (11.610 g, 91.3 mmol, 1.0 equiv) in THF (100 mL) dropwise over 30 min via a pressure equalizing addition funnel. The addition funnel was removed. The flask was then fitted with a condenser and the reaction was heated to reflux in a 75° C. oil bath for 16 h. The reaction was cooled in an ice bath for 20 min and sodium sulfate decahydrate (Glauber's salt, 25 g) was added in portions over 20 min. After complete addition, the mixture was stirred at room temperature for 2 h. The mixture was filtered through a bed of Celite® (2 cm) and the solids were rinsed with EA (2×250 mL). The clear solution was concentrated to a pale yellow liquid (9.580 g, 91.1%). NMR indicated the presence of THF and EA. This material was used in the preparation of the following example compounds without further purification.

[0089] 1 H NMR (300 MHz, CDCl3) δ: 4.39 (quintet, J = 6Hz, 1H), 3.62 - 3.56 (m, 2H), 2.90 - 2.85 (m, 2H), 2.41 (t, J = 7.5 Hz,2H), 1.34(sextet, J = 7.2 Hz, 2H), 0.87 (t, J = 7.8 Hz, 3H). Preparation of (R)-1-(1H-indol-3-yl)-N-((R)-1-phenylethyl)propan-2-amine: [ka]

[0090] Indole-3-acetone (25.0 g, 144 mmol, 1.0 equiv) was added to a solution of (R)-(+)-1-phenylethylamine (23.0 mL, 181 mmol, 1.3 equiv) in dichloromethane (600 mL) at 25 °C under N2 and the mixture was stirred for 1 h. The reaction was cooled to 0-5 °C and to this ice-cooled solution was added sodium triacetoxyborohydride (100 g, 472 mmol, 3.3 equiv) via a powder addition funnel over 30 min. The orange solution was stirred at 0 °C for 1 h and then allowed to warm to room temperature. The reaction was stirred at room temperature for 19 h. At this point, ESI+ indicated the absence of indole starting material. Saturated NaHCO3 solution (100 mL) was added in 5 mL portions over 15 min at 10 °C with vigorous stirring. The solution was stirred for 15 min and saturated Na2CO3 solution (200 mL) was added over 15 min. Solid K2CO3 (9 g) was added in 3 g portions at which point the aqueous layer was pH 12 and bubbles had stopped forming. The aqueous layer was filtered and separated. The red organic layer was washed with saturated aqueous NaHCO3 (2x100 mL). The aqueous layers were combined and extracted with DCM (2x100 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated to give the crude product (49 g). TLC (90:10 DCM:MeOH) showed four spots (Rf = 0.63, 0.50, 0.16, 0.26), two of which were separated diastereomeric major products (Rf = 0.16 and 0.26). The crude was adsorbed onto silica gel and purified via flash chromatography (330 g cartridge, 0-100% EA:Hex). Fractions containing the R,R diastereomer were pooled and purified a second time using the same flash chromatography conditions to give 24 g of product (approximately 82% ee). Since the previous successful separation was achieved with a silica gel:crude ratio of 40:1, the mixture was split into three portions and separated on 3 × 330 g silica gel cartridges (0-40% EA / Hex, 20 min, isocratic 40% EA / Hex, 40 min). All fractions containing the desired product were >99% diastereomeric purity.Pure fractions were concentrated and pooled to give (R)-1-(1H-indol-3-yl)-N-((R)-1-phenylethyl)-propan-2-amine as an orange semi-solid (11.91 g, 29.6%).

[0091] 1 H NMR (CDCl3, 300 MHz) R,R diastereomer: δ 0.96 (d, J = 6.6 Hz, 3H), 1.30 (d, J = 6.6 Hz, 3H), 2.68 (q, J = 7.2 Hz, 1H), 2.97 (m, 2H) 4.00 (q, J = 6.3 Hz, 1H), 7.43-6.97 (m, 10H), 7.96 (br s, 1H). R,S diastereomer: δ 1.11 (d, J = 5.7 Hz, 3H), 1.30 (d, J = 5.4 Hz, 3H) 2.80 (m, 3H), 3.92 (q, J = 6.9 Hz, 1H), 6.93-7.40 (m, 10H), 8.13 (br s, 1H); the aromatic region was difficult to distinguish from the R,R diastereomer due to lack of purity.

[0092] LCMS: ES+[M+H]+279.0. Preparation of (2R)-1-(1H-indol-3-yl)propan-2-amine [ka]

[0093] The compound (R)-1-(1H-indol-3-yl)-N-((R)-1-phenylethyl)propan-2-amine (11.91 g, 42.8 mmol, 1.0 equiv.) was dissolved in methanol (250 mL) and added to a 2 L Parr bottle and the solution was sparged with N2 for 10 min. Water-wet 20% Pd(OH)2 on carbon (10.71 g, 76.3 mmol, 1.8 equiv.) was added and the bottle was pressurized with 50 psi of hydrogen and shaken in a Parr apparatus for 22 h, LCMS analysis showed the reaction was complete. The suspension was filtered through Celite® and concentrated to remove MeOH. The crude was dissolved in DCM and washed with saturated Na2CO3 solution (50 mL) and the aqueous layer was extracted with DCM (2 x 50 mL). The organic layers were combined, dried and concentrated to give (2R)-1-(1H-indol-3-yl)propan-2-amine as a light brown solid (6.68 g, 89.6%) which required no further purification.

[0094] 1 H NMR (CDCl3, 300 MHz) δ 1.17 (d, J = 6.6 Hz, 3H), 2.66 (dd, J = 8.4, 14.7 Hz, 1H), 2.88(dd, J = 5.4, 14.1 Hz, 1H), 3.27 (sextet, J = 1.5 Hz, 1H), 7.05-7.22 (m, 3H), 7.37 (d, J = 7.5 Hz, 1H), 7.62 (d, J = 8.7 Hz, 1H), 8.00 (br s, 1H).

[0095] LCMS: ES+[M+H]+174.9. Preparation of 2-fluoro-2-methylpropanol [ka]

[0096] To a stirred suspension of lithium aluminum hydride (2.50 g, 65.9 mmol, 1.6 equiv) in anhydrous diethyl ether (100 mL) cooled in an ice bath, methyl 2-fluoro-2-methylpropionate (5.01 g, 40.5 mmol, 1.0 equiv) was added dropwise over 15 min. After 2 h, 2.0 mL of water, 2.0 mL of 15% w / v NaOH, and 5.0 mL of water were added dropwise in sequence. After 15 min, the white suspension was diluted with DCM and gravity filtered through Celite®, washing the solid with DCM. The filtrate was concentrated (200 mbar, 25° C.) to give 2-fluoro-2-methylpropanol as a colorless oil (2.09 g, 56.1%).

[0097] 1 H NMR (300 MHz, CDCl3) δ 1.34 (d, J = 21.3 Hz, 6H), 1.95 (br t, 1H), 3.56 (dd, J = 6.6, 20.7 Hz, 2H). Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate [ka]

[0098] Trifluoromethanesulfonic anhydride (5.0 mL, 29.7 mmol, 1.3 equiv) was added dropwise over 30 min to a 0° C. solution of 2-fluoro-2-methylpropanol (2.090 g, 22.7 mmol, 1.0 equiv) and 2,6 lutidine (3.40 mL, 29.4 mmol, 1.3 equiv) in DCM (25 mL). After 2 h, the red solution turned light brown. TLC (20:80 EA:Hex, KMnO4 stain) showed no starting material. The reaction mixture was washed with 1 M HCl solution (2×20 mL) and saturated NaHCO3 solution (2×20 mL). The aqueous layers were back-extracted with DCM (20 mL) respectively. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure (150 mbar, 25 C) to give 2-fluoro-2-methylpropyl trifluoromethanesulfonate as a red oil (4.39 g, 86.3%).

[0099] 1 H NMR (300 MHz, CDCl3) δ 1.46 (d, J = 20.4 Hz, 6H), 4.41 (d, J = 18.6 Hz, 2H). 19 F NMR (282 MHz, CDCl3) δ -147.1, -74.5. Preparation of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine: [ka]

[0100] Compound 2-fluoro-2-methylpropyl trifluoromethanesulfonate (9.587 g, 42.8 mmol, 1.1 eq) (solution in DCM, 16% by weight DCM, 11.4384 g) was added to a solution of (2R)-1-(1H-indol-3-yl)propan-2-amine (6.680 g, 38.3 mmol, 1.0 eq), anhydrous 1,4-dioxane (60.000 ml, 701.4 mmol, 18.3 eq), and freshly distilled diisopropylethylamine (8.500 ml, 48.8 mmol, 1.3 eq). The dark brown solution was heated at 90° C. for 3 hours. After 3 hours, LCMS showed that a small amount of indoleamine starting material was still present. TLC (10% MeOH / DCM) showed that the triflate (Rf=0.54) was consumed. NMR of unused triflate SM (286-30) showed that the triflate had not decomposed overnight, so another 0.1 equivalent (0.9883 g, 13 wt % DCM, 0.8563 g triflate SM) was added and the reaction was heated at 90 °C for 2 h. LCMS showed the reaction was complete and TLC (10% MeOH / DCM) showed one spot (Rf = 0.24) (TLC with 50% EA / Hex, one streaky spot Rf ≤ 0.12, another spot at Rf = 0). EtOAc (50 mL) was added, the solution was washed with NaHCO3 (2 x 50 mL) and the combined aqueous layers were washed with EtOAc (50 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude material (brown oil, 14.8 g) was purified by flash silica chromatography (240 g cartridge, 0-100% EA / Hex). The desired product eluted as a long tailing peak. Pure fractions were concentrated to give (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (4.211 g, 17.0 mmol) as a dark yellow oil.

[0101] 1H NMR (300 MHz, CDCl3) δ 1.10 (d, J = 6.3 Hz, 3H), 1.34 (dd, J = 3.0, 21.9 Hz, 6H), 2.68-2.95 (m, 4H), 3.02 (sextet, J = 6.6 Hz, 1H), 7.05 (d, J = 2.4 Hz, 1H), 7.26-7.11 (m, 2H), 7.36 (d, J = 6.9 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 8.18 (br s, 1H). 19 F NMR (282MHz, CDCl3) δ -144.2. m / z:ES+[M+H]+ 249.0.

[0102] Preparation of Compound 1 To a solution of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (0.070 g, 0.3 mmol, 1.0 equiv.) in anhydrous toluene (1.50 mL) and glacial acetic acid (0.100 mL, 1.7 mmol, 6.2 equiv.) was added 4-((1-propylazetidin-3-yl)oxy)benzaldehyde (0.096 g, 0.4 mmol, 1.3 equiv.). Molecular sieves were added and the solution was stirred at 80° C. in the dark under N2 for 8 h. The reaction solution was diluted with DCM, filtered and washed with saturated Na2CO3 solution. The aqueous layer was extracted with DCM and the combined organic layers were dried over Na2SO4. The solution was filtered and concentrated. The residue was dissolved in acetonitrile (2 mL) and filtered through a syringe filter before being purified via preparative LC (40-90% ACN:HO for 18 min followed by isocratic 90% ACN for 7 min). Pure fractions were concentrated and dried to give (1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1-(4-((1-propylazetidin-3-yl)oxy)phenyl)-2,3,4,4a,9,9a- as a white powder. Tetra Hydro-1H-pyrido[3,4-b]indole was obtained.

[0103] Example 2 Assay of estrogen receptor protein levels This example describes the evaluation of various compounds (ARN-810, AZD9496, Compound 1, endoxifen, and fulvestrant) on ERα protein levels in various cell lines. Depending on the cell type, 90,000 to 500,000 cells per well were plated in each well of a 12-well dish and incubated for at least 24 hours in phenol red-free medium containing 5% charcoal dextran stripped fetal bovine serum (treated FBS) (HyClone). Cells were treated with 300 nM antiestrogens for 4 hours in serum-free medium, followed by lysis of lysates in RIPA buffer supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific). Total protein extracts were separated on 10% SDS-PAGE TGX gels and transferred to nitrocellulose membranes (BioRad). Blots were incubated with mouse monoclonal anti-ERα, either D12 (#sc-8005, SantaCruz Biotechnology) or SP1 (#MA5-14501, ThermoFisher Scientific). β-actin monoclonal antibody (#MA5-15739 or #MA5-16410 (ThermoFisher Scientific) or #sc-47778 (SantaCruz Biotechnology)) was used as a loading control. Blots were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (ThermoFisher Scientific). Signals were detected with Super Signal Femto chemiluminescence reagent (ThermoFisher Scientific). The results are shown in Figure 2. As can be seen, all compounds except endoxifen showed a remarkable ability to reduce ER protein levels in most cell lines, with compound 1 and fulvestrant being the most effective in reducing ER protein levels and showing comparable activity in this regard.

[0104] Example 3 Cell proliferation assay In this example, human ER + An assay is described to evaluate the effect of test compounds on human MCF-7 cells, a breast cancer cell line. Specifically, 1000 MCF-7 cells per well (Cheryl Walker, Baylor College of Medicine) were plated in 96-well plates in phenol red-free medium (ThermoFisher Scientific) containing 5% treated FBS. After at least 4 hours, cells were treated with antiestrogen and medium was diluted to 2.5% treated FBS in the presence of 100 pM E2 for 6-8 days. Proliferation was measured using a CyQuant fluorescent DNA binding dye kit (ThermoFisher Scientific) using 1:200 GR dye and an excitation of 485 nm and a fluorescence read at 538 nm.

[0105] Example 4 Transient transfection of estrogen receptors and variants In this example, we describe a study in which Ishikawa cells, a human endometrial cancer cell line, were transfected with certain estrogen receptor constructs and endogenous alkaline phosphatase was assayed. 15,000 Ishikawa cells per well were plated in 96-well plates in phenol red medium containing 5% treated FBS. At the time of plating, cells in each well were transiently transfected with 75-100 ng of estrogen receptor construct (or empty vector, pSG5) using Lipofectamine LTX (ThermoFisher Scientific). Approximately 4 hours later, cells were treated with the indicated amount of anti-estrogen (in the absence of E2) or 500 pM E2 (Figure 9), and the medium was diluted in 2.5% treated FBS. Cells were incubated for 3 days, medium was removed, and plates were frozen at -80°C. Thawed plates were incubated with p-nitrophenyl phosphate (ThermoFisher Scientific), a chromogenic substrate for AP and thus revealing AP activity levels. After 40–80 min at 40°C, absorbance was read at 405 nm.

[0106] Compound 1 has ER antagonist, no agonist, and activity The AP activity of endogenous wild-type ER in untransfected Ishikawa cells was assayed as described above. Cells were treated with the indicated compounds (ARN-810, AZD-9496, compound 1, endoxifen, or fulvestrant) alone (agonist mode) or in the presence of 500 pM 17β-estradiol (E2) (antagonist mode). The results are shown in Figure 1A (agonist mode) and Figure 1B (antagonist mode). As shown in the figures, all compounds showed significant antagonist activity, with compound 1 and fulvestrant being the most potent. All compounds other than compound 1 and fulvestrant also showed significant agonist activity.

[0107] Certain ER mutants increase ligand-independent ER activity Wild-type ER (HEGO), empty vector (pSG5) or the indicated LBD mutant ER were transiently transfected into Ishikawa cells as described above. Empty vector alone was treated with 500 pM 17β-estradiol (E2). After 72 hours, cells were assayed for AP activity. The results are shown in Figure 9. Bars represent the mean absorbance at 405 nm from triplicate wells + standard error. As shown in the figure, the various tested ER mutants were observed to be "activating mutants" in that they exhibited more activity than wild-type ER in the absence of ligand.

[0108] Activation domain 1 (AF1) is required for ligand-independent activity observed in certain ER mutants AF1 wild-type ER (HEGO, AA1-595), empty vector (pSG5), or the indicated ER lacking activation domain 2 ("AF2") (AA1-282) or lacking activation domain 1 ("AF1") (AA178-595, with or without the Y537S mutation) were transiently transfected into Ishikawa cells as described above. After 72 hours, cells were assayed for AP activity. Bars represent the mean absorbance at 405 nm from quadruplicate wells + standard error. As shown, even in the presence of the activating Y537S mutation (ΔAF1 / Y5372), F1AF1 is required for the ligand-independent ER activity observed when the ER is truncated (ΔAF2).

[0109] Compound 1 inhibits the activity of ligand-independent ER mutants Wild-type ER or the indicated ER variants were transiently transfected into Ishikawa cells as described above, and activity was assayed in the presence of compound 1 or fulvestrant. Results are shown in Figures 5A-5F (each of Figures 5A-5F reports the specific cell line as indicated in each figure). Points represent the mean AP activity normalized to vehicle from duplicate wells + / - standard error. Dose-response curves for compound 1 and fulvestrant were fitted using least-squares fitting methods, and pIC 50 (-Log I C 50 ) was calculated using a variable slope sigmoidal dose-response model. The lines represent the normalized AP activity of endogenous receptors (transfected with empty vector (pSG5)). As shown in the figure, compound 1 has an IC comparable to that of fulvestrant. 50 The results showed that the ER inhibitor α-amyloid β-terminal kinase inhibited the activity of each of the ligand-independent ER mutants with α-amyloid β-terminal kinase.

[0110] Certain clinical candidates fail to inhibit the activity of ligand-independent ER mutants Wild-type ER or the indicated ER variants were transiently transfected into Ishikawa cells as described above and activity was assayed in the presence of compound 1 or fulvestrant compared to endoxifen, RAD-1901, ARN-810 (GDC-0810) or AZD-9496 (results are shown in Figures 8A-8B, where compound 1 and fulvestrant are compared to endoxifen and RAD-1901 in Figures 8A and 8B, or ARN in Figures 8C-8D). -810 (GDC-0810) and AZD-9496. Points represent the mean absorbance at 405 nm from triplicate wells + standard error. Lines represent AP activity of endogenous receptors (transfected with empty vector (pSG5)). As can be seen, neither endoxifen, RAD-1901, ARN-810 (GDC-0810), nor AZD-9496 are able to inhibit the activity of the ligand-independent ER variant as Compound 1 and Fulvestrant do.

Claims

1. 1. A composition for treating cancer in a subject characterized by a mutation in estrogen receptor 1 (ESR1), the composition comprising compound 1: 【Chemistry 1】 Compound 1 or a pharma- ceutically acceptable salt thereof, wherein said mutation is an activating mutation.

2. The composition described in claim 1, wherein the activating mutation is a mutation of activating function 2.

3. The composition described in claim 1 or 2, wherein the activating mutation is D538G or Y537S.

4. The composition described in claim 3, wherein the activating mutation is D538G.

5. The composition described in claim 3, wherein the activating mutation is Y537S.

6. The composition described in any one of claims 1 to 5, wherein the cancer is breast cancer.

7. The method of claim 1, wherein the treatment comprises administering to a patient a compound of formula I: Compound 1 or a pharma- ceutically acceptable salt thereof.

8. The composition of claim 7, wherein the composition is a tablet.

9. The composition of claim 7, wherein the composition is a capsule.

10. A composition described in any one of claims 1 to 9, wherein the subject is being administered or has been administered a secondary agent.

11. The composition of claim 10 , wherein the second agent is a CDK4 / 6 inhibitor.

12. 12. The composition of claim 11, wherein the CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, abemaciclib, relociclib, and trilaciclib.

13. 13. The composition of claim 12, wherein the CDK4 / 6 inhibitor is palbociclib.

14. 13. The composition of claim 12, wherein the CDK4 / 6 inhibitor is ribociclib.

15. The composition of claim 12, wherein the CDK4 / 6 inhibitor is abemaciclib.

16. The composition of claim 10 , wherein the secondary agent is a PIK3CA inhibitor.

17. 17. The composition of claim 16, wherein the PIK3CA inhibitor is selected from alpelisib and taselisib.

18. The composition of claim 10 , wherein the second agent is an mTOR inhibitor.

19. 19. The composition of claim 18, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, and everolimus.

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