Estrogen receptor antagonist therapy

Abstract

The present specification provides a method for administering an estrogen receptor antagonist for use in the treatment of cancer. In some embodiments, the estrogen receptor antagonist provided in the present specification is a tetrahydropyrido[3,4-b]indole compound. In some embodiments, the provided antagonist is a complete estrogen receptor antagonist.

Description

Technology Field

[0001] Cross-references regarding related applications

[0002] 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 herein by reference in its entirety. Background Technology

[0003] Estrogen receptors (ERs) play an important role in various cancers, including breast cancer. Various therapies targeting estrogen receptors and / or their activity are being developed.

[0004] There is still a need for anti-estrogen agonists capable of completely inhibiting estrogen receptors, including estrogen receptor 1 (ESR1), encoded by both wild-type and mutant forms (e.g., those containing activating mutations) of the gene encoding estrogen receptor-alpha (ERα). Selective estrogen receptor modulators (SERMs) or degraders (SERDs) are particularly useful or promising tools for these therapies. The estrogen receptor is a tripartite protein containing two distinct transcriptional activating functions (AF1 and AF2). Complete anti-estrogen activity requires the inactivation of both AF1 and AF2. Activating mutations in the gene encoding estrogen receptor 1 can activate both AF1 and AF2 in the absence of estrogen.

[0005] Previous therapies, such as tamoxifen, AZD9496, and ARN-810, fail to neutralize both activating functions (i.e., fail to neutralize both AF1 and AF2). As such, there is still a need for therapies that neutralize both AF1 and AF2 to completely inhibit estrogen receptors, and there is also a need for therapies that inhibit estrogen receptors despite activating mutations. In particular, this disclosure describes that certain compounds may be used as a treatment for patients or subjects suffering from cancer, either alone or in combination with other agents, wherein the patient or subject has a mutation in Estrogen Receptor 1 (ESR1). For example, a compound useful for the treatment of a patient or subject suffering from cancer is the following compound 1, wherein the patient or subject has a mutation in Estrogen Receptor 1 (ESR1):

[0006]

[0007] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer associated with estrogen receptors (ER), comprising the step of administering an estrogen receptor antagonist and a CDK4 / 6 inhibitor.

[0008] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer associated with estrogen receptors (ER), comprising the step of administering an estrogen receptor antagonist and a PIK3CA inhibitor.

[0009] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer associated with estrogen receptors (ER), comprising the step of administering an estrogen receptor antagonist and an mTOR inhibitor.

[0010] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer that has metastasized to the brain, bone, lung, or liver, comprising the step of administering the following compound 1 to the patient or subject:

[0011]

[0012] In some embodiments, the present disclosure provides a testing system for evaluating a compound or composition for the inhibition of estrogen receptor activating function 1 (AF1) and / or activating function 2 (AF2).

[0013] In some embodiments, the present disclosure provides a method for treating a patient suffering from cancer, comprising the step of orally administering the following compound 1:

[0014] Brief explanation of the drawing

[0015] Figure 1a is a scatter plot measuring the percentage of estrogenic response as a function of Log[M] for a specific estrogen receptor antagonist compound without added estrogen. Figure 1b is a scatter plot measuring the percentage of estrogenic response as a function of Log[M] for a specific estrogen receptor antagonist compound to which estrogen has been added. Figure 2 is a chart measuring the degradation of estrogen receptor proteins across multiple cell lines for multiple estrogen receptor antagonists. Figures 3a to 3c are scatter plots illustrating the percentage reduction in the estrogen response to compound 1 with various CDK4 / 6 inhibitors. Figures 4a and 4b are scatter plots illustrating the percentage reduction in estrogen proliferation in MCF-7 cells when treated with compound 1 along with a PIK3CA inhibitor. Figures 5a to 5f are scatter plots for cell lines illustrating the dose-response of compound 1 to AF1 inhibition with the most common ESR1 mutations. Figure 6 is a scatter plot illustrating changes in tumor volume for various doses of compound 1. Figures 7a to 7d are scatter plots measuring drug exposure (ng / mL) over time for mice (Fig. 7a), rats (Fig. 7b), dogs (Fig. 7c), and monkeys (Fig. 7d). Figures 8a to 8d are scatter plots illustrating the decrease in estrogen concentration across different cell lines. Figure 9 is a bar graph illustrating that mutant ER increases ligand-independent alkaline phosphatase activity (AP) in Ishikawa endometrial cancer cells. Figure 10 is a bar graph illustrating that the activation domain 1 (AF1) of the ER is required for AP activation. Figure 11 illustrates that mammals express two major isoforms of the ER, known as ERα and ERβ, each of which are members of the nuclear hormone receptor family, according to the literature [Patel & Bihani]. Pharm & Therap This is a reproduction of Fig. 1a of [186:1, 2018]. A) An AF domain constituting an estrogen receptor, comprising an activating function 1 (AF1) domain, a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD) / activating 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 inducing an inactive complex, and SERDs prevent ER signaling by inducing the degradation of the ER. Figure 12 illustrates a variation of the basic mechanism of the E2 reaction, from the literature [Hewitt & Korach Endocrine Rev. This is a reproduction of Fig. 3 of 39:664-674 (June 12, 2018). Specific details for implementing the invention

[0016] There is still a need for a treatment for estrogen receptor (ER)-positive cancer types that overcomes the problems associated with conventional methods. The present disclosure provides, in particular, a method for treating a patient or subject suffering from cancer associated with estrogen receptors and mutations in estrogen receptors, comprising the step of administering an estrogen receptor antagonist. In some embodiments, the estrogen receptor antagonist is the following compound 1 or a pharmaceutically acceptable salt thereof:

[0017]

[0018] definition

[0019] administration As used herein, the term "administration" typically refers to the administration of a composition to a target or system to achieve the delivery of an agent that is, for example, a composition, is included in a composition, or otherwise delivered by a composition.

[0020] agent As used herein, the term "agent" refers to an entity (e.g., lipids, metals, nucleic acids, polypeptides, polysaccharides, small molecules, etc., or complexes, combinations, mixtures, or systems thereof [e.g., cells, tissues, organisms]), or a phenomenon (e.g., heat, electric current or electric field, magnetic force or magnetic field, etc.).

[0021] antagonist As used herein, the term “antagonist” may refer to an agent or state in which the presence, level, degree, type, or form is associated with a reduction in the level or activity of a target. Antagonists may include agents of any chemical class, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity exhibiting related inhibitory activity. In some embodiments, an antagonist may be a “direct antagonist” in that it binds directly to its target; in some embodiments, an antagonist may be an “indirect antagonist” in that it exerts its effect by means other than directly binding to its target, for example, by interacting with a modifier of the target to alter the level or activity of the target. In some embodiments, an “antagonist” may be referred to as an “inhibitor.”

[0022] associated : Where one existence, level, degree, type and / or form is correlated with the existence, level, degree, type and / or form of another, two events or entities are “associated” with each other as such terms are used herein. For example, a specific entity (e.g., polypeptide, genetic trait, metabolite, microorganism, etc.) is considered associated with a specific disease, disorder, or condition if its existence, level and / or form is correlated with the incidence and / or susceptibility to the disease, disorder, or condition (e.g., across relevant populations). In some embodiments, two or more entities are physically “associated” with each other if they interact directly or indirectly such that they are physically close to each other and / or remain close to each other. In some embodiments, two or more entities physically associated with each other are covalently bonded to each other; In some embodiments, two or more entities physically associated with each other are not covalently bonded but are non-covalently associated by, for example, hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0023] biological samples As used herein, the term “biological sample” refers to a sample obtained from or derived from a biological source considered (e.g., tissue or organism or cell culture) as typically described herein. In some embodiments, the source considered includes an organism, e.g., an animal or a human. In some embodiments, a biological sample is or includes biological tissue or fluid. In some embodiments, a biological sample includes bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free-suspended nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washing or lavage, e.g., tubal lavage or bronchoalveolar lavage; aspirates; scraping; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells derived therefrom, etc., may be or include therefrom. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells from an individual from which the sample is obtained. In some embodiments, the sample is a “primary sample” obtained directly from a source considered 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 body fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as is evident from the context, the term “sample” refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or adding one or more agents to the primary sample), for example, by filtering using a semipermeable membrane.These “processed samples” may include nucleic acids or proteins obtained, for example, by extracting from a sample or by processing a primary sample with techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components.

[0024] Combination therapy As used herein, the term “combination therapy” refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, these regimens may be administered sequentially (e.g., a first regimen of any “dose” is administered before the administration of a second regimen of any dose); in some embodiments, these agents are administered as an overlapping administration regimen. In some embodiments, “administration” of combination therapy may include the administration of one or more agent(s) or modality(s) to a subject receiving other agent(s) or modality(s) in combination. For clarity, combination therapy does not require that individual agents be administered together (or even necessarily simultaneously) in a single composition, and in some embodiments, two or more agents, or their active metabolites, may be administered together in a combination composition or even a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0025] Dosage form or unit dosage form : Those skilled in the art will recognize that the term may be used to refer to physically individual units of an active agent (e.g., therapeutic or diagnostic agent) to be administered to a subject. Typically, each of these units contains a predetermined amount of active agent. In some embodiments, this amount is a unit dose (or whole fraction thereof) appropriate for administration according to a dosing regimen (i.e., therapeutic dosing regimen) determined to correlate with the desired or beneficial result when administered to the relevant population. Those skilled in the art recognize that the total amount of a therapeutic composition or agent administered to a specific subject is determined by one or more attending physicians and may involve administration in multiple forms of dosage.

[0026] Administration therapy or treatment therapy Those skilled in the art will recognize that the terms “administration regimen” and “therapeutic regimen” may be used to refer to a set of unit doses (typically more than once) administered individually to a subject, which are typically separated by periods of time. In some embodiments, the provided therapeutic agent has the proposed administration regimen, which may include one or more doses. In some embodiments, the administration regimen includes multiple doses, each of which is temporally separated from the other doses. In some embodiments, individual doses are separated from each other by periods of the same length, and in some embodiments, the administration regimen includes at least two different periods of time separating the multiple doses and individual doses. In some embodiments, all doses within the administration regimen are the same unit dose. In some embodiments, different doses within the administration regimen are different amounts. In some embodiments, the administration regimen includes a first dose of a first dose, followed by one or more additional doses of a second dose different from the first dose. In some embodiments, the administration regimen comprises a first administration of a first dose, followed by one or more additional administrations of a second dose equal to the first dose. In some embodiments, the administration regimen correlates with a desired or beneficial outcome when administered across relevant populations (i.e., it is a therapeutic administration regimen).

[0027] Brothers : As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition to provide or contribute to a desired consistency or stabilizing effect, for example. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc.

[0028] oral- As used herein, the terms "oral administration" and "orally administered" have the meanings understood in the art to refer to the administration of a compound or composition by mouth.

[0029] Non-parenteral As used herein, the terms “parenteral administration” and “parenterally administered” have the meaning understood in the art to refer to modes of administration other than intestinal and local administration, generally by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, and intrasternal injections and infusions.

[0030] Patient or subject As used herein, the terms “patient” or “subject” refer to any organism to which the provided composition may be administered, for example, for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. A typical patient or subject includes animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient or subject may be 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 or has received a specific therapy to diagnose and / or treat a disease, disorder, or condition.

[0031] Pharmaceutical composition As used herein, the term “pharmaceutical composition” refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen for a relevant subject (e.g., in an amount proven to represent a statistically significant probability of achieving a predetermined therapeutic effect upon administration), or in a different similar subject (e.g., in a similar subject or system different from the subject or system in the presence of one or more indicators of the specific disease, disorder, or condition under consideration, or in the presence of prior exposure to a condition or agent). In some embodiments, the term “comparison” refers to a statistically significant difference (e.g., a predominance and / or magnitude sufficient to achieve statistical significance). A person skilled in the art will recognize or readily determine, in the provided context, the degree and / or prevalence of the difference required or necessary to achieve such statistical significance.

[0032] Pharmaceutically acceptable carrier As used herein, the term “pharmaceuticalally acceptable carrier” means a pharmaceutically acceptable substance, composition, or vehicle involved in carrying or transporting a target compound from one organ or part of the body to another organ or part of the body, e.g., liquid or solid fillers, diluents, excipients, or solvent-encapsulated materials. Each carrier must be “acceptable” in the sense that it is miscible with other components of the formulation and is not harmful to the patient. Some examples of substances that may serve as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose, and derivatives thereof, e.g., sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository wax; Oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solution; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic miscible materials used in pharmaceutical formulations.

[0033] Pharmaceutically acceptable salt As used herein, the term “pharmaceuticalally acceptable salt” refers to a salt of such compound suitable for use in a pharmaceutical setting, that is, a salt of such compound suitable for use in contact with human or lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and corresponding to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are widely known in the art. For example, SM Berge et al. [ J. Pharmaceutical SciencesPharmaceutically acceptable salts are described in detail in [66: 1-19 (1977)]. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts of amino groups formed with inorganic acids, e.g., hydrochloric acid, hydrobromide, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, e.g., 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, pharmaceutically acceptable salts are adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, campersulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, Includes, but is not limited to, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.In some embodiments, pharmaceutically acceptable salts comprise, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions, e.g., halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and aryl sulfonates.

[0034] remedy As used herein, the term “therapeutic agent” generally refers to any agent that elicits 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 over 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, e.g., a specific age group, sex, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that may be used to alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce severity, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or pathological condition. In some embodiments, a “therapeutic agent” is an agent that has been or must be approved by a government agency before being sold for administration to humans. In some embodiments, a “therapeutic agent” is an agent that requires a medical prescription for administration to humans.

[0035] treat As used herein, the terms “treat,” “treat,” or “treating” refer to any method used to partially or completely alleviate, improve, reduce, inhibit, prevent, delay onset, reduce severity, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. The treatment may be administered to subjects who do not exhibit signs of the disease, disorder, and / or condition. In some embodiments, the treatment may be administered to subjects who exhibit only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0036] Therapeutic effective amount As used herein, the term “therapeutic effective dose” refers to an amount of a substance (e.g., therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, the therapeutic effective dose 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 who is suffering from or may be susceptible to such disease, disorder, and / or condition. As recognized by those skilled in the art, the effective dose of a substance may vary depending on factors such as the desired biological endpoint, the substance being delivered, the target cell or tissue, etc. For example, the effective dose of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, reduces, inhibits, prevents, delays the onset, reduces severity, and / or reduces the incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, the therapeutic effective dose is administered as a single dose, and in some embodiments, multiple unit doses are required to deliver the therapeutic effective dose.

[0037] Estrogen receptors

[0038] Estrogen receptors ("ER") are involved in various biological processes related, for example, the development of the female reproductive system, the maintenance of bone mass, and the protection of cardiovascular and / or central nervous system elements (see, e.g., literature [Pearce & Jordan]). Crit. Rev. Onc / Hem 50:3, 2004; Heldring Phys. Rev. [Refer to 87:905, 2007]).

[0039] Mammals express two major isoforms of the ER, known as ERα and ERβ, each of which is a member of the nuclear hormone receptor family, as reproduced in Fig. 11, [Patel & Bihani Pharm & Therap It has a structural organization as illustrated in Fig. 1A of [186:1, 2018]. As illustrated in Fig. 11, A) an AF domain constituting an estrogen receptor comprising an activating function 1 (AF1) domain, a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD) / activating 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 inducing an inactive complex, and SERDs prevent ER signaling by inducing the degradation of the ER.

[0040] As can be seen, the six ER "domains" labeled as AF were defined in the ER structure. Domains A / B, found at the amino-terminal ends of the ER protein, are the largest domains and contain AF1, one of the two so-called "transcriptional activating functions," while AF2 is found in the E domain and also contains ligand-binding domains and element(s) believed to participate in ER dimerization and nuclear localization (e.g., the literature [Hewitt & Korach Endorine Rev[Refer to 39:664, 2018]). It is believed that the binding of a ligand to the ligand-binding domain triggers structural reorganization of the alpha-helical within the E domain, and that this reorganization may contribute to the activity of AF2 (e.g., interacting with specific mediating components).

[0041] The C domain of the ER contains its DNA binding domain, which mediates interactions with so-called "estrogen-responsive element(s)" (ERE(s)) that are operably associated with genes whose transcription is regulated by the ER. ERα and ERβ regulate the expression of different ERE-associated genes and exhibit different cellular and tissue distribution patterns. DNA binding by the ER appears to be mediated by two zinc finger structures within the C domain, and additional element(s) may contribute (e.g., the literature [Hewitt & Korach Endocrine Rev [Refer to 39:664, 2018]). The C domain of ER can also participate in or otherwise contribute to ER dimerization.

[0042] The D domain of the ER is also called the "hinge region" and contains amino acid element(s) that can participate in ER dimerization and / or nuclear localization.

[0043] The F domain of the ER may play an important role in ER protein stability. Compared to other members of the nuclear receptor family, this domain appears to be a characteristic of estrogen receptors and may contribute to responsiveness to specific therapies (e.g., tamoxifen) (e.g., literature [Arao et al , J. Bio. Chem [See 293:22, 8495]).

[0044] In the presence of natural ligands (e.g., 17β-estradiol), the ER undergoes structural changes, homodimerizes, and localizes in the nucleus, where it binds to the ERE and regulates the transcription of its target genes (e.g., literature [Pawlak et al .; Kumar & Chambor; Hall & McDonnell); The events of this series have been described as "genomic" mechanisms of ER gene regulation. Other mechanisms, e.g., "tethered," "non-genomic," and "ligand-independent" have also been described, as illustrated in Fig. 3 of the literature (Hewitt & Korach), reproduced in Fig. 12.

[0045] Variations in the underlying mechanisms of the E2 response as exemplified in Fig. 12 and reported in the literature (Hewitt & Korach). Four different E2 response mechanisms have been described. (1) The genomic mechanism involves interactions between the ER and the CRE DNA motif. (2) The tethering mechanism involves indirect interactions between the ER and the AP1 DNA motif binding to other transcription regulators, e.g., the FOS / JUN dimer. Accordingly, in this example, the ER is "tethered" to DNA through the binding of FOS / JUN to its AP1 DNA motif. (3) This is called non-genomic signaling because it initiates signals from extracellular E2 leading to a rapid signal cascade in the cytoplasm; accordingly, the response does not involve interactions with genomic features. The response is mediated by membrane-associated ERs or by GPERs, G protein-coupled receptors. (4) Ligand-independent signaling involves the transduction of extracellular growth factor (GF) activation of the cell membrane CF receptor (CFR), which initiates a signaling cascade such as MAPK. The signal is received by the ER and activates the transcriptional regulation of target genes despite the lack of E2 ligand.

[0046] The ER is implicated in various cancers. Several tumors expressing estrogen receptors (i.e., ER + In tumors, active ERα signaling has been demonstrated to induce cell proliferation (ERβ signaling has been reported to achieve tumor suppressive effects (e.g., in the literature [Nilsson & Gustafson]). Clin. Pharmacol. Ther. [Refer to 89:44, 2011]). Typically, tumors with less than 1% cell staining positive for the ER (e.g., breast tumors) are "ER + It is classified as ".

[0047] Therapeutics that target the ER are ER + It is the standard treatment for many patients with tumors (e.g., literature [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 [See , 13:801, 2015]). For patients with early-stage breast cancer, for example, recommended treatment typically includes ER-targeted therapy after tumor resection (for example, discussed below). For advanced breast cancer, including metastatic breast cancer, ER-targeted therapy is the mainstream.

[0048] Estrogen-receptor-targeted therapy

[0049] Given the importance of ER signaling in various cancers as well as specific cardiovascular, inflammatory, and neurodegenerative diseases, significant effort has been invested in developing therapeutic agents and modalities that target the ER. While there is some fluidity in the terminology used to describe ER-targeting agents, various agents with different mechanisms have been developed and / or studied.

[0050] Some ER-targeting agonists are designed and / or documented to reduce the production of estrogen (i.e., 17β-estradiol).

[0051] Some ER-targeting agonists are designed and / or documented to bind directly to the ER, and in some cases, these agonists compete with estrogen for binding to the ER and / or interfere with the allosteric changes that naturally occur in estrogen binding. Often, the term "anti-estrogen" is used to refer to agonists that bind to the ER, and more specifically, to refer to such agonists that compete with estrogen for binding to the ER.

[0052] The term "selective estrogen receptor modulator" (“SERM”) has been used to refer to compounds designed or documented to modify some aspect of ER activity. While some texts refer to “SERM” as indicating a specific type of anti-estrogen, others use the term “SERM” more generally to refer to compounds that specifically affect some characteristics of ER (particularly ERα) expression and / or activity.

[0053] The term "selective estrogen receptor degrader" ("SERD") has been used to refer to compounds designed and / or documented to trigger or enhance the degradation of the ER. In many cases, if the presence of a compound is correlated with a reduction in ER levels, the compound may be referred to as a SERD. In some texts, compounds are classified as SERMs or SERDs, while in others, SERDs are referred to as a specific type or species of compound that is a SERM.

[0054] Regardless of the mechanism of action of specific agents, clinical experience to date indicates that incomplete efficacy (e.g., within individual patients and / or across patient populations) and / or the development of resistance remain issues.

[0055] In particular, the presence or development of specific ER mutations has been reported to affect the efficacy of various ER-targeted therapies (e.g., literature [Jeselsohn et al Nature Rev. Clin. Onc. 12, 573, 2015; Gelsomino et al. Breast Cancer Res. Treat 157:253, 2016; Toy et al. [Refer to 2013]). It has been reported that some particularly problematic mutations "activate" one or more modes of ER expression and / or function, and that some activating mutations can provide ER ligand-independent (i.e., constitutively active). For example, specific mutations of the ER ligand binding domain, including D538G and Y537S, have been shown to constitutively activate the ER, and other mutations involving deletions and / or fusions that remove the ligand binding domain may have similar effects (e.g., Literature [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 [See 5:4577, 2014]). Some reports indicate that 50% of women with metastatic breast cancer may have detectable activating ER mutations in circulating tumor DNA.

[0056] Estrogen receptor antagonists

[0057] As discussed above, there has been and continues to be significant investment in pursuing effective therapies targeting the ER (e.g., literature [Patel & Bihani]). Pharmacol. & Therap. Reviewed in 186:1, 2018).

[0058] Among the most advanced compounds currently in clinical development are the following:

[0059] a. Although it is an important breast cancer treatment recognized for having "saved 500,000 women worldwide" ( https: / / www.cancer.gov / news-events / cancer-currents-blog / 2017 / endoxifen-breast-cancer-NCI-support Tamoxifen, known to be less effective and also susceptible to resistance development in women with low CYP2D6 activity, as available in the literature [Refer to Bringing the Investigational Breast Cancer Drug Endoxifen From Bench to Bedside with NCI Support], last accessed on July 7, 2019.

[0060] b. Endoxifene, an active metabolite of tamoxifen, originally developed to resolve tamoxifen failure in women with low CYP2D6 activity, which reduces their ability to convert tamoxifen to endoxifen (literature[ Cancer Currents Blog [Refer to , National Cancer Institute, Aug 31, 2017]).

[0061] c. Described as "a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist / ER degrader inducing tumor regression in tamoxifen-sensitive and resistant ER+ BC xenograft models" (reference [Dickler et al. Cancer Res. [Refer to 75(15 Suppl): Abstract nr CT231, 2015]), ARN-810 (Brillastrant; GDC-810) which launched a Phase II clinical trial for the treatment of patients with ER+ breast cancer for whom other hormone agonists have failed but further development may be subsequently discontinued (e.g., literature [ Biospace [See April 27, 2017]).

[0062] d. AZD9496 described as "oral nonsteroidal small molecule inhibitors of estrogen receptor alpha (ERα) and potent and selective antagonists and degraders of ERα" (Reference [Hamilton et al Clin Cancer Res[Refer to 1:3519, 2018]); AZD9496 has been reported to "antagonize and degrade ER with antitumor activity in both endocrine-sensitive and endocrine-resistant models" and has been described as "similar to fulvestrant in antagonizing ER and bypassing endocrine resistance" (Reference [Nardone et al. Br. J. Cancer [See 120:331, 2019]).

[0063] e. RAD-1901 (Elacestrant) was described as a "novel, non-steroidal oral SERD" exhibiting monoagonist activity in severe, pre-treatment patients with ER+ advanced breast cancer (literature [de Vries et al, Cancer Res. Abstract P1-10-04, 2018; also, Bardia et al. J. Clin. Onc. (See [35:15_suppl, 1014, 2017]). Preclinical studies have also reported that "elacestrant significantly inhibited the growth of xenograft models containing ESR1 mutations, including models containing Y537S or D538G mutations and models insensitive to fulvestrant and tamoxifen" (Patel et al. Cancer Res [Refer to 79:Abstract nr P6-20-08, 2019]).

[0064] f. Fullvestrant (Faslodex TM ) was the first FDA-approved SERD, and certain ERs including combination with palbociclib or abemaciclib +It is approved for the treatment of cancer. Fulvestrant is a "selective estrogen receptor degrader" that binds to, blocks, and degrades the estrogen receptor (ER), leading to the complete inhibition of estrogen signaling through the ER (see literature [Nathan & Schmid Oncol Ther 5:17, 2017]). Fulvestrant has achieved significant clinical success and is often considered the "gold standard" compared to ER-targeted therapies. However, fulvestrant is administered by injection rather than orally, and in practice, requires a monthly administration of 500 mg via intramuscular injection (after initial administration). Additionally, while certain retrospective analyses have offered hope that fulvestrant may have some utility in the treatment of patients with ER mutations, definitive evidence of activity has not been achieved (e.g., literature [Fribbens et al. J Clin Oncol. 34:2961, 2916; Spoerke et al. Nat Commun [Refer to 7:11579, 2016]):

[0065]

[0066]

[0067] The present disclosure recognizes that the success of fulvestrant is due to its ability to (1) inhibit both AF1 and AF2 so as to inhibit AF1 activity present in constitutively active ER mutants; (2) promote ER degradation; and (3) function as a complete estrogen receptor antagonist ("CERAN") lacking the partial ER agonist activity observed with certain other agonists (e.g., see FIG. 1a, which shows that ARN-810, AZD-9496, and endoxifen each increase ER activity in the absence of added estrogen, even though they decrease ER activity in the presence of estrogen). 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 (Reference [Robertson, et al., The Lancet [See , 388(10063):2997-3005 (Dec. 17, 2016]). Without being bound to any specific theory, it is suggested that the ability of fulvestrant to inhibit both AF1 and AF2 may be due to its recruitment of a co-repressor of gene expression to the ER complex.

[0068] Nevertheless, the present disclosure also recognizes that various other compounds, including, for example, ARN-810, AZD9496, tamoxifen, etc., are less effective than fulvestrant because they at least partially antagonize the ER, and specifically, inhibit the activation of AF2 but not the activation of AF1.

[0069] The present disclosure provides an important and unexpected insight that a previously described compound, (1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1-(4-((1-propylazetidin-3-yl)oxy)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (“Compound 1”; see PCT Application Publication WO 2017 / 059139, the entirety of which is by reference) is consistent with the CERAN properties of polvestrant and also provides additional valuable properties, including, for example, (i) being bioavailable orally and having a long half-life, and ii) exhibiting good blood-brain barrier penetration:

[0070]

[0071] In particular, the present disclosure indicates that compound 1 is uniquely useful in certain environments, including (a) the treatment of cancer associated with ER mutations including ligand-independent / constitutive mutations; (b) the treatment of cancer having CNS (e.g., brain) metastases or tumors; and (c) use in combination with certain other agents, including certain agents that have been proven or suggested to be useful with fulvestrant.

[0072] For example, in particular, the present disclosure reports the finding that Compound 1 exhibits complete estrogen receptor antagonism and that this antagonism is similar to that of fulvestrant in various assays. In particular, as reported in Example 4, the present disclosure indicates that Compound 1 is characterized by its ability to inhibit AF1 and AF2, and is accordingly appropriately described as a "complete estrogen receptor antagonist."

[0073] The present disclosure teaches that Compound 1 may be particularly useful or effective for the treatment of a disease, disorder, or pathological condition (e.g., cancer) associated with the presence of one or more ER mutants, specifically including ligand-independent ER mutants. Accordingly, in some embodiments, the present disclosure provides a therapeutic method in which Compound 1 is administered to a subject expressing one or more ERs (e.g., in a relevant cell or tissue), and in particular one or more ligand-independent ERs; and in some embodiments, such subject(s) express these mutant ER(s) prior to administration.

[0074] Those skilled in the art will be aware of various techniques for detecting mutant ER in a sample from a subject. In some embodiments, mutant ER protein is detected; in some embodiments, nucleic acid encoding mutant protein (e.g., mutant ESR1 gene) is detected.

[0075] The ability of compound 1 to inhibit both AF1 and AF2 allows compound 1 to function as CERAN despite activating mutations of the estrogen receptor (e.g., ESR1).

[0076] Accordingly, in some embodiments, the present disclosure provides a method for treating a subject (or group of subjects) suffering from cancer, wherein the subject has an ESR1 mutation (and / or expresses a mutant ER protein), and the method comprises the step of administering the following compound 1 or a pharmaceutically acceptable salt thereof to the subject:

[0077]

[0078] Evaluation of ER antagonists

[0079] In particular, the present disclosure teaches that a useful ER antagonist agent has CERAN activity as described in this specification.

[0080] One aspect of the present disclosure is the insight that existing strategies for evaluating or characterizing ER antagonist (and / or potential antagonist) agents are insufficient in that they do not, at least typically, distinguish between SERD and CERAN. In particular, most of these existing strategies fail to evaluate the ability of agents to specifically affect AF1.

[0081] In particular, the present disclosure teaches that particularly useful ER antagonist agents can inhibit ligand-independent ER activity, including activity observed in some embodiments as constitutive ER variant(s), such as AF2 deletion or cleavage and / or LBD mutants (e.g., D538G and Y537S).

[0082] Furthermore, the present disclosure teaches that a particularly useful ER antagonist agent is characterized by each of the following:

[0083] a. Inhibition of AF1 (e.g., inhibition of at least one, and preferably all, known constitutive ER variants)

[0084] b. Inhibition of AF2 (e.g., inhibition of ligand-dependent ER activity)

[0085] c. Enhancement of ER decomposition.

[0086] Additionally, in some embodiments, particularly useful ER antagonist agents are further characterized by one or more of the following:

[0087] a. Oral bioavailability and long half-life.

[0088] b. Penetration of the blood-brain barrier.

[0089] In certain embodiments, the activity of the ER antagonist(s) may be evaluated in comparison with the activity of one or more of ARN-810, AZD9496, endoxifen, fulvestrant, RAD1901, tamoxifen, and / or compound 1, and in some of these embodiments, the comparison may be simultaneous or alternatively, in some embodiments, this may be with historical records or future results.

[0090] Combination therapy

[0091] 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 illustrated in FIGS. 3a and 3b (for CDK4 / 6 inhibitors) and FIGS. 4a and 4b (for PIK3CA inhibitors), Compound 1, an estrogen receptor antagonist, and indeed, a complete estrogen receptor antagonist, in combination with a second agonist, can substantially eliminate cell proliferation. The present disclosure includes the recognition that a combination of certain agonists can be advantageously used to completely antagonize estrogen receptors by inactivating both AF1 and AF2. Accordingly, in some embodiments, the present disclosure provides a method for treating a subject suffering from cancer, comprising the step of administering a compound that is an inhibitor of activating function 2 and a second agonist that is an inhibitor of activating 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.

[0092] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer, comprising the step of administering compound 1 and a second agent selected from CDK2, CDK4, CDK6, or CDK7 inhibitors. 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., inhibiting one or both of CDK4 and CDK6). In some embodiments, the second agent is a CDK2 / 4 / 6 inhibitor (i.e., inhibiting one or more of CDK2, CDK4, and CDK6).

[0093] In some embodiments, the second agent is a CDK4 / 6 inhibitor selected from palbocociclib, ribociclib, abemaciclib, lerosiclib, trilaciclib, and SHR6390. In some embodiments, the CDK4 / 6 inhibitor is palbocociclib. 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 lerosiclib. In some embodiments, the CDK4 / 6 inhibitor is trilaciclib. In some embodiments, the CDK4 / 6 inhibitor is SHR6390.

[0094] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer, 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, tasselisib, and LY3023414. In some embodiments, the PIK3CA inhibitor is alpelisib. In some embodiments, the PIK3CA inhibitor is tasselisib. In some embodiments, the PIK3CA inhibitor is LY3023414.

[0095] In some embodiments, the present disclosure provides a method for treating a patient or subject suffering from cancer, comprising administering compound 1 and a second agent, wherein the second agent is 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.

[0096] dosage

[0097] The present disclosure includes the recognition that certain disorders or conditions, e.g., cancer, can be effectively treated using a smaller amount of an active compound than other compounds of similar activity. For example, as illustrated in FIG. 6, Compound 1 was found to reduce tumor volume more effectively than other CERANs, e.g., fulvestrant. Additionally, Compound 1 is suitable for oral administration, which is advantageous compared to other CERANs, e.g., fulvestrant, which must be administered parenterally.

[0098] Accordingly, the present disclosure provides a method for treating a patient or subject suffering from cancer, comprising the step of administering a composition comprising compound 1. In some embodiments, the composition comprises compound 1 and pharmaceutically acceptable excipients, carriers, or diluents. Depending on the severity of the condition being treated, the composition may be administered orally, parenterally, by inhalation or nasal spray, topically (e.g., by powder, ointment, or drop), rectally, buccally, vaginally, intraperitoneally, into a water bath, or through an implanted reservoir. Preferably, the composition is administered orally, intraperitoneally, or intravenously. In certain embodiments, the provided compound is administered orally or parenterally at a dosage level of about 0.01 mg to about 50 mg per kg of body weight per day, at least once a day, to achieve the desired therapeutic effect.

[0099] The pharmaceutically acceptable compositions described herein may be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In these solid dosage forms, the active compound may be mixed with at least one inert diluent, e.g., sucrose, lactose, or starch. These dosage forms may also, as in common practice, include additional substances other than the inert diluent, e.g., lubricants and other tableting aids, e.g., magnesium stearate and microcrystalline cellulose. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifying and suspending agent. If desired, certain sweeteners, flavorings, or colorings may also be added.

[0100] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound comprises at least one inert pharmaceutically acceptable excipient or carrier, e.g., sodium citrate or dicalcium phosphate and / or a) fillers or extenders, e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, e.g., glycerol; d) disintegrants, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents, e.g., paraffin; f) absorption promoters, e.g., quaternary ammonium compounds; g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; h) absorbents, e.g., Kaolin and bentonite clay, and / or i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof are mixed. In the case of capsules, tablets, and pills, the dosage form may also include a buffer. The active compound may also be present in a micro-encapsulated form with one or more excipients as noted above.

[0101] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin using excipients, e.g., lactose or lactose, as well as high molecular weight polyethylene glycol. Solid dosage forms of tablets, coated tablets, capsules, pills, and granules may be prepared with coatings and shells, e.g., intestinal coatings (i.e., buffers) and other coatings widely known in the field of pharmaceutical formulations. These may optionally contain opacifying agents and may also be compositions that release only the active ingredient(s), or preferentially, optionally, release them to specific parts of the intestinal tract in a delayed manner. Examples of embedding compositions that may be used include polymer materials and waxes.

[0102] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, e.g., water or other solvents, solubilizers and emulsifiers, e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof. In addition to inert diluents, the oral composition may also include adjuvants, e.g., humectants, emulsifying and suspending agents, sweeteners, flavoring agents, and perfumes.

[0103] Alternatively, the pharmaceutically acceptable compositions described herein may be administered in the form of suppositories for rectal or vaginal administration. These may be prepared by mixing the compounds of the present application with suitable non-irritating excipients or carriers that are solid at room temperature but liquid at body (e.g., rectal or vaginal) temperature and thus melt in the rectal or vaginal cavity to release the active compound. Such materials include cocoa butter, suppository wax (e.g., beeswax), and polyethylene glycol.

[0104] In some embodiments, the composition is administered orally. In some embodiments, the composition is administered in an amount of 30 mg or less per 1 kg of body weight of a patient or subject. In some embodiments, the composition is administered in an amount of 10 mg or less per 1 kg of body weight of a patient or subject. In some embodiments, the composition is administered in an amount of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg per 1 kg of body weight of a patient or subject. In some embodiments, the composition is administered in an amount of 3 mg per 1 kg of body weight of a patient or subject. In some embodiments, the composition is administered in an amount of 1 mg per 1 kg of body weight of a patient or subject. 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 per 1 kg of body weight of a patient or subject. In some embodiments, the composition is administered in an amount of 0.1 mg per 1 kg of body weight of a patient or subject.

[0105] In some embodiments, compound 1 is administered as a unit dose. In some embodiments, compound 1 is administered in capsule form. In some embodiments, the compound is administered in tablet form. In some embodiments, compound 1 is administered as a suspension. In some embodiments, compound 1 is administered as a solution.

[0106] In some embodiments, compound 1 is administered as a daily dose (QD). In some embodiments, compound 1 is administered twice a day (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).

[0107] As illustrated in FIGS. 7a through 7d, drug exposure over time (ng / mL) is high and much higher for mouse (Fig. 7a), rat (Fig. 7b), dog (Fig. 7c), and monkey (Fig. 7d) subjects.

[0108] Disability or pathological condition

[0109] The present disclosure also includes the recognition that Compound 1 may be used to treat cancer that has advantageously metastasized, e.g., cancer that has spread to the brain, bone, lungs, 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 300 mg / kg. Other estrogen receptor antagonists, e.g., fulvestrant, cannot penetrate the blood-brain barrier at similar amounts.

[0110]

[0111] Accordingly, the present disclosure provides a method for treating a patient or subject suffering from cancer that has metastasized to the brain, bone, lungs, liver, or central nervous system, comprising the step of administering the following compound 1:

[0112]

[0113] In some embodiments, the cancer comprises one or more CNS tumors (e.g., metastases); in some embodiments, the cancer has metastasized to the brain, bone, lung, or liver. In some embodiments, the cancer has metastasized to the central nervous system.

[0114] Examples

[0115] The embodiments provided herein document and support specific aspects of the disclosure, but are not intended to limit any claims. Unless specifically presented in the past tense, what is included in the embodiments is not intended to imply that the described work has been completed or even performed. The following non-limiting embodiments are provided to further illustrate specific teachings provided by the disclosure. Those skilled in the art will recognize that, in light of this application, various modifications may be made to specific embodiments illustrated herein without departing from the spirit and scope of the teachings.

[0116] In the following examples, the following abbreviations may be used: aq. (aqueous); ACN (acetonitrile); CSA (campersulfonic acid); d (one or ones); DCM (dichloromethane); DEA (diethylamine); DHP (dihydropyran); DMF (N,N-dimethylformamide); DIPEA (N,N-diisopropylethylamine); DMAP (4-dimethylaminopyridine); DMSO (dimethyl sulfoxide); EA (ethyl acetate); ee (excess of enantiomer); equiv. (equivalent); ethanol (EtOH); h (hour or hours); Hex (hexane); HPLC (high-performance liquid chromatography); IPA (isopropyl alcohol); KHMDS (potassium bis(trimethylsilyl)amide); LAH (lithium aluminum hydride); LCMS (liquid chromatography-mass spectrometry); LDA (lithium diisopropylamide); LiHMDS (lithium bis(trimethylsilyl)amide); MeOH (methanol); min (min or min); NMR (nuclear magnetic resonance); Pd / C (palladium carbon); PPh3O (triphenylphosphine oxide); Pt / C (platinum carbon); rb (round bottom); Rf (retention factor); rt or RT (room temperature); SM (starting material); TEA (triethylamine); THF (tetrahydrofuran); THP (tetrahydropyran); TLC (thin film chromatography); TsOH (p-toluenesulfonic acid or tosylic acid); and UV (ultraviolet).

[0117] Example 1:

[0118] Synthesis of Compound 1

[0119] The complete synthesis of Compound 1 is provided in PCT Application Publication WO 2017 / 059139, which is incorporated by reference herein and repeated below.

[0120] Preparation of 4-((1-propylazetidin-3-yl)oxy)benzaldehyde

[0121]

[0122] Step 1: Preparation of 1-propionylazetidine-3-one

[0123]

[0124] 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 provide a bright yellow suspension. The suspension was sonicated for 1 hour and then cooled to -10°C (dry ice / MeOH) for 10 minutes. Propionyl chloride (9.8 mL, 112 mmol, 1.2 equiv.) was added dropwise to the cooled suspension to provide an orange solution. The reaction was removed from the bath and stirred at room temperature for 16 hours. The solvent was removed to provide a semi-solid. The semi-solid was suspended in EA (300 mL) and the suspension was filtered. The solid was washed with EA (2 × 100 mL). TLC analysis (10% MeOH / DCM, KMnO7 staining column) indicated the presence of 3 spots: Rf: 0.2, 0.5, 0.7. TLC (50% EA / Hex, KMnO7 staining / column) indicated the presence of 2 spots: Rf: 1, 0.3. The filtrate was concentrated, adsorbed onto silica gel (25 g), and chromatographed through silica gel (100 g cartridge) with 0 to 10% MeOH over 5 mins, followed by 15 mins in DCM. The product was initially removed from the column in DCM and then eluted from the column with up to 10% MeOH. TLC was performed on both solvent systems to determine if any propionyl chloride was present in the initial fraction. The fraction containing the product was pooled and concentrated to obtain the title compound as a yellow liquid (11.610 g, 98.2%).

[0125] 1H 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).

[0126] Step 2. Preparation of 1-propylazetidin-3-ol

[0127]

[0128] Lithium aluminum hydride (10.397 g, 273.9 mmol, 3.0 equiv.) was suspended in THF (200 mL) and cooled in an ice bath. A solution of 1-propionylazetidin-3-one (11.610 g, 91.3 mmol, 1.0 equiv.) in THF (100 mL) was added dropwise to the reaction mixture over 30 minutes using a pressure equalization addition funnel. The addition funnel was removed. Subsequently, a condenser was fitted to the flask, and the reaction mixture was heated in an oil bath under reflux at 75°C for 16 hours. The reaction was cooled in an ice bath for 20 minutes, and sodium sulfate decahydrate (Glauber salt, 25 g) was added dropwise over 20 minutes. After complete addition, the mixture was stirred at room temperature for 2 hours. The mixture was then [prepared] in Celite ® The solid was filtered through a layer (2 cm) and washed with EA (2 × 250 ml). The clear solution was concentrated into a pale yellow liquid (9.580 g, 91.1%). NMR indicated the presence of THF and EA. These substances were used without further purification in the preparation of the compounds of the following examples.

[0129] 1 H NMR (300 MHz, CDCl3) δ: 4.39 (pent, J = 6 Hz, 1H), 3.62 - 3.56 (m, 2H), 2.90 - 2.85 (m, 2H), 2.41 (t, J = 7.5 Hz, 2H), 1.34 (hextet, J = 7.2 Hz, 2H), 0.87 (t, J = 7.8 Hz, 3H).

[0130] Preparation of (R)-1-(1H-indole-3-yl)-N-((R)-1-phenylethyl)propane-2-amine:

[0131]

[0132] 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) under N2 at 25°C, and the mixture was stirred for 1 hour. The reaction was cooled to 0 to 5°C, and sodium triacetoxyborohydride (100 g, 472 mmol, 3.3 equiv.) was added to the ice-cooled solution over 30 minutes using a powder addition funnel. The orange solution was stirred at 0°C for 1 hour, and then heated to room temperature. The reaction was stirred at room temperature for 19 hours. At this time, it was indicated that the indole starting material was not present in ESI+. A saturated solution of NaHCO3 (100 ml) was added in 5 ml portions over 15 minutes at 10°C with vigorous stirring. The solution was stirred for 15 minutes, and a saturated solution of Na2CO3 (200 ml) was added over 15 minutes. A 3 g portion of solid K2CO3 (9 g) was added, at which point the aqueous layer had a pH of 12 and bubble formation stopped. The layers were filtered and separated. The reddish-orange layer was washed with sat. aq. NaHCO3 (2 × 100 ml). The aqueous layers were combined and extracted with DCM (2 × 100 ml). The combined organic layer was dried over Na2SO4, filtered, and concentrated to obtain an unpurified product (49 g). Four spots appeared on TLC (90:10 DCM:MeOH) (Rf = 0.63, 0.50, 0.16, 0.26), two of which were the major products of the separated diastereomers (Rf = 0.16 and 0.26). The crude material was adsorbed onto silica gel and purified by flash chromatography (330 g cartridge, 0 to 100% EA:Hex). The fraction containing the R and R diastereomers was pooled and purified a second time under the same flash chromatography conditions to obtain 24 g of product (approx. 82% ee).Previous successful separation was achieved by a 40:1 silica gel:unpurified non-liquidation ratio, and accordingly, the mixture was divided into three parts and separated on 3 × 330 g silica gel cartridges (0 to 40% EA / Hex for 20 minutes, isosolvent 40% EA / Hex for 40 minutes). All fractions containing the desired product are > 99% diastereomericly pure. The pure fractions were concentrated and pooled to obtain (R)-1-(1H-indole-3-yl)-N-((R)-1-phenylethyl)-propane-2-amine as an orange semi-solid (11.91 g, 29.6%).

[0133] 1 ¹H NMR (CDCl3, 300 MHz) R,R Diastereoisomers: 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 diastereomers: 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 is due to a lack of purity R,R It was difficult to distinguish from the diastereomer.

[0134] LCMS: ES+ [M+H]+ 279.0.

[0135] Preparation of (2R)-1-(1H-indole-3-yl)propane-2-amine

[0136]

[0137] Compound ( R )-1-(1H-Indol-3-day)- N -(( R)-1-phenylethyl)propane-2-amine (11.91 g, 42.8 mmol, 1.0 equiv.) was dissolved in methanol (250 ml), added to a 2 L Parr bottle, and N2 was sprayed into the solution for 10 minutes. 20% carbonaceous Pd(OH)2 (10.71 g, 76.3 mmol, 1.8 equiv.) moistened with water was added, the bottle was compressed with hydrogen at 50 psi, and shaken in a Parr apparatus for 22 hours; LCMS analysis indicated that the reaction was complete. The suspension was filtered through Celite® and concentrated to remove MeOH. The crude material was dissolved in DCM, washed with a saturated Na2CO3 solution (50 ml), and the aqueous layer was extracted with DCM (2 × 50 ml). The organic layers were combined, dried, and concentrated (2 R )-1-(1H-indole-3-yl)propane-2-amine was obtained as a light brown solid, which did not require further purification (6.68 g, 89.6 %).

[0138] 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).

[0139] LCMS: ES+ [M+H]+ 174.9.

[0140] Preparation of 2-fluoro-2-methylpropanol

[0141]

[0142] Methyl 2-fluoro-2-methylpropionate (5.01 g, 40.5 ml, 1.0 equiv.) was added dropwise to a stirred suspension of lithium aluminum hydride (2.50 g, 65.9 ml, 1.6 equiv.) in anhydrous diethyl ether (100 ml) cooled in an ice bath over 15 minutes. After 2 hours, 2.0 ml of water, 2.0 ml of 15% w / v NaOH, and 5.0 ml of water were added sequentially. After 15 minutes, the white suspension was diluted with DCM, gravity filtered through Celite®, and the solid was washed with DCM. The filtrate was concentrated (200 mbar, 25°C) to obtain 2-fluoro-2-methylpropanol as a colorless oil (2.09 g, 56.1%).

[0143] 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).

[0144] Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate

[0145]

[0146] Trifluoromethanesulfonic acid anhydride (5.0 mL, 29.7 mmol, 1.3 equiv.) was added dropwise over 30 minutes 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 hours, the red solution turned light brown. TLC (20:80 EA:Hex, KMnO4 staining) indicated the absence of the starting material. The reaction mixture was washed with 1 M HCl solution (2 × 20 mL) and sat. NaHCO3 solution (2 × 20 mL). The aqueous layers were each back-extracted with DCM (20 mL). The combined organic layer was dried with Na2SO4, filtered, and concentrated under reduced pressure (150 mbar, 25℃) to obtain 2-fluoro-2-methylpropyl trifluoromethanesulfonate as a red oil (4.39 g, 86.3%).

[0147] 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.

[0148] Preparation of (R)-N-(1-(1H-indole-3-yl)propane-2-yl)-2-fluoro-2-methylpropane-1-amine:

[0149]

[0150] Compound 2-fluoro-2-methylpropyl trifluoromethanesulfonate (9.587 g, 42.8 mmol, 1.1 equiv.) (solution in DCM, 16 wt% DCM, 11.4384 g) was added to a solution of (2R)-1-(1H-indole-3-yl)propane-2-amine (6.680 g, 38.3 mmol, 1.0 equiv.), anhydrous 1,4-dioxane (60.000 ml, 701.4 mmol, 18.3 equiv.), and freshly distilled diisopropylethylamine (8.500 ml, 48.8 mmol, 1.3 equiv.). The dark brown solution was heated at 90°C for 3 hours. After 3 hours, the LCMS indicated that a small amount of indoleamine starting material was still present. TLC (10% MeOH / DCM) indicated that a triplate (Rf = 0.54) was used. NMR of the unused triplate SM (286-30) indicated that the triplate had not decomposed overnight, so, another 0.1 equivalent (0.9883 g, 13% DCM wt%, 0.8563 g triplate SM) was added, and the reaction was heated at 90°C for 2 hours. LCMS indicated that the reaction was complete, and TLC (10% MeOH / DCM) showed one spot (Rf = 0.24) (TLC with 50% EA / Hex, one striped spot Rf <= 0.12, another spot at Rf = 0). EtOAc (50 ml) was added, the solution was washed with NaHCO3 (2 × 50 ml), and the combined aqueous layer was washed with EtOAc (50 ml). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product (brown oil, 14.8 g) was purified by flash silica chromatography (240 g cartridge, 0 to 100% EA / Hex). The desired product was eluted as a long tailing peak. The pure fraction was concentrated to obtain (R)-N-(1-(1H-indole-3-yl)propane-2-yl)-2-fluoro-2-methylpropane-1-amine (4.211 g, 17.0 mmol) as a dark yellow oil.

[0151] 1 H 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 (282 MHz, CDCl3) δ -144.2. m / z: ES+ [M+H]+ 249.0.

[0152] Preparation of Compound 1

[0153] 4-((1-propylazetidine-3-yl)oxy)benzaldehyde (0.096 g, 0.4 ml, 1.3 equiv.) in anhydrous toluene (1.50 ml) RIt was added to a solution of )-N-(1-(1H-indole-3-yl)propane-2-yl)-2-fluoro-2-methylpropane-1-amine (0.070 g, 0.3 ml, 1.0 equiv.) and glacial acetic acid (0.100 ml, 1.7 ml, 6.2 equiv.). A molecular sieve was added, and the solution was stirred in the dark under N2 at 80°C for 8 hours. The reaction solution was diluted in DCM, filtered, and washed with a saturated Na2CO3 solution. The aqueous layer was extracted with DCM, and the combined organic layer was dried over Na2SO4. The solution was filtered and concentrated. The remainder was dissolved in acetonitrile (2 ml) and filtered through a syringe filter before purification via prep LC (40 to 90% ACN:H2O over 18 minutes, followed by 90% ACN isosolvent over 7 minutes). The pure fraction was concentrated and dried to obtain (1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-1-(4-((1-propylazetidin-3-yl)oxy)phenyl)-2,3,4,9-tetrahydro-1H-pyridodo[3,4-b]indole as a white powder.

[0154] Example 2

[0155] Estrogen receptor protein level test

[0156] This example describes the evaluation of various compounds (ARN-810, AZD9496, Compound 1, Endoxifene, and Fulvestrant) on ERα protein levels in various cell lines. Depending on the cell type, 90,000 to 500,000 cells per well were pre-plated into each well of a 12-well dish and incubated in phenol red-free medium containing 5% charcoal dextran-stripped fetal bovine serum (stripped FBS) (HyClone) for at least 24 hours. Cells were treated with 300 nM anti-estrogen for 4 hours in serum-free medium, and the eluent was subsequently eluted with RIPA buffer supplemented with a protease and phosphatase inhibitor (ThermoFisher Scientific). The total protein extract was separated on a 10% SDS-PAGE TGX gel and transferred to a nitrocellulose membrane (BioRad). The blots were incubated with mouse monoclonal anti-ERα, D12 (#sc-8005, SantaCruz Biotechnology) or SP1 (#MA5-14501, ThermoFisher Scientific). β-actin monoclonal antibodies (#MA5-15739 or #MA5-16410 (ThermoFisher Scientific) or #sc-47778 (SantaCruz Biotechnology)) were used as loading controls. The blots were incubated with an appropriate secondary antibody (ThermoFisher Scientific) conjugated to male radish peroxidase. The signal was detected using Super Signal Femto chemiluminescence reagent (ThermoFisher Scientific). The results are shown in Figure 2. As can be seen, all compounds except endoxifen showed the ability to significantly reduce ER protein levels in most cell lines, and compounds 1 and fulvestrant were the most effective in reducing ER protein levels and showed similar activity in this respect.

[0157] Example 3

[0158] Cell proliferation test

[0159] This embodiment is a human ER + This describes an assay to evaluate the effect of a tested compound on human MCF-7 cells, a breast cancer cell line. Specifically, 1,000 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% stripped FBS. After at least 4 hours, the cells were treated with anti-estrogen, and the medium was diluted with 2.5% stripped FBS in the presence of 100 pM E2 for 6 to 8 days. Growth was measured using the CyQuant fluorescent DNA-binding dye kit (ThermoFisher Scientific) with a 1:200 GR dye and fluorescence excitation at 485 nm and readout at 538 nm.

[0160] Example 4

[0161] Transient transfection of estrogen receptors and variants

[0162] This example describes a study in which specific estrogen receptor constructs were transfected into human endometrial cancer cell line Ishikawa cells, and endogenous alkaline phosphatase was evaluated. 15,000 Ishikawa cells per well were plated in 96-well plates in phenol-red medium containing 5% stripped FBS. At the time of plating, cells in each well were transiently transfected with 75 to 100 ng of estrogen receptor construct (or empty vector, pSG5) using Lipofectamine LTX (ThermoFisher Scientific). Approximately 4 hours later, the cells were treated with a specified amount of anti-estrogen (in the absence of E2) or 500 pM E2 (Fig. 9), and the medium was diluted with 2.5% stripped FBS. The cells were incubated for 3 days, the medium was removed, and the plates were frozen at -80°C. Thawed plates were incubated with p-nitrophenyl phosphate (ThermoFisher Scientific), which is a chromogenic substrate for AP and thus indicates the level of AP activity. After 40 to 80 minutes at 40°C, absorbance was read at 405 nm.

[0163] Compound 1 has ER antagonist activity rather than agonist activity.

[0164] AP activity of endogenous wild-type ER in transfected Ishikawa cells was tested as described above. Cells were treated with the specified compounds (ARN-810, AZD-9496, Compound 1, Endoxifene, or Fulvestrant) alone (agonist mode) or in the presence of 500 pM 17β-estradiol (E2) (antagonist mode). The results are presented in Figures 1a (agonist mode) and 1b (antagonist mode). As can be seen, all compounds exhibited significant antagonist activity, with Compound 1 and Fulvestrant being the most potent. All compounds except Compound 1 and Fulvestrant also exhibited significant agonist activity.

[0165] Certain mutant ERs increase ligand-independent ER activity

[0166] Wild-type ER (HEGO), empty vector (pSG5), or the specified LBD mutant ER were transiently transfected into Ishikawa cells as described above. Only the empty vector was treated with 500 pM 17β-estradiol (E2). After 72 hours, cells were tested for AP activity. The results are presented in Figure 9. Bars represent the average absorbance+sem at 405 nm from three wells. As can be seen, the various tested ER mutants were observed to be "activating mutants" in that they exhibited greater activity than the wild-type ER in the absence of a ligand.

[0167] Activation domain 1 (AF1) is required for the ligand-independent activity observed in certain ER mutants.

[0168] As previously described, Ishikawa cells were transiently transfected with specified ERs lacking AF1 wild-type ER (HEGO, AA 1-595), an empty vector (pSG5), or activation domain 2 ("AF2") (AA 1-282) or activation domain 1 ("AF1") (AA 178-595, with or without the Y537S mutation). After 72 hours, cells were tested for AP activity. Bars represent the average absorbance+ SEM at 405 nm from four wells. As can be seen, F1AF1 is required for the ligand-independent ER activity observed when the ER is cleaved (ΔAF2), even in the presence of the activating Y537S mutation (ΔAF1 / Y5372).

[0169] Compound 1 inhibits the activity of ligand-independent ER mutants.

[0170] Wild-type ER or the specified ER variants were transiently transfected into Ishikawa cells as described above, and activity was tested in the presence of Compound 1 or fulvestrant. The results are presented in Figures 5a through 5f (Figures 5a through 5f each report a specific cell line as specified in the figure). Points represent the mean AP activity + / - SEM normalized to the vehicle from two wells. Dose-response curves for Compound 1 and fulvestrant were fitted using the least squares fit method, and pIC 50 (-Log IC 50 ) was calculated using a variable-gradient S-shaped dose-response model. The line represents the normalized AP activity of the endogenous receptor (transformed with the empty vector (pSG5)). As can be seen, Compound 1 has an IC50 similar to that of fulvestrant. 50 Each ligand-independent ER mutant having inhibited the activity of.

[0171] Certain clinical candidates fail to inhibit the activity of ligand-independent ER mutants

[0172] Wild-type ER or specified ER variants were transiently transfected into Ishikawa cells as described above, and activity was evaluated in the presence of Compound 1 or fulvestrant in comparison to endoxifen, RAD-1901, ARN-810 (GDC-0810), or AZD-9496 (results are shown in Figs. 8a and 8b, where Compound 1 and fulvestrant are compared with endoxifen and RAD-1901 in Figs. 8a and 8b, or with ARN-810 (GDC-0810) and AZD-9496 in Figs. 8c and 8d). Points represent the average absorbance + SEM at 405 nm from three wells. Lines represent the AP activity of the endogenous receptor (transfected with the empty vector (pSG5)). As can be seen, none of endoxifen, RAD-1901, ARN-810 (GDC-0810) or AZD-9496 may inhibit the activity of ligand-independent ER variants as performed by compound 1 and fulvestrant.

Claims

Claim 1 A composition for use in a method of treating a subject suffering from breast cancer characterized by a mutation in estrogen receptor 1 (ESR1), comprising the following compound 1 or a pharmaceutically acceptable salt thereof, wherein the mutation is an activating mutation and the activating mutation is one or more selected from the group consisting of D538G and Y537S. [Compound 1] Claim 2 A composition according to claim 1, wherein the activating mutation is D538G. Claim 3 A composition according to claim 1, wherein the activating mutation is Y537S. Claim 4 A composition according to claim 1, wherein the composition is formulated for oral administration. Claim 5 In paragraph 4, the composition is in the form of a tablet. Claim 6 In paragraph 4, the composition is in the form of a capsule. Claim 7 A composition according to claim 1, wherein the subject is or has been administered a second agent. Claim 8 A composition according to claim 7, wherein the second agent is a CDK4 / 6 inhibitor selected from palbociclib, ribociclib, abemaciclib, lerociclib, and trilaciclib. Claim 9 A composition according to claim 8, wherein the CDK4 / 6 inhibitor is palbociclib. Claim 10 A composition according to claim 8, wherein the CDK4 / 6 inhibitor is ribociclib. Claim 11 A composition according to claim 8, wherein the CDK4 / 6 inhibitor is abemaciclib. Claim 12 A composition according to claim 7, wherein the second agent is a PIK3CA inhibitor selected from alpelisib and taselisib. Claim 13 A composition according to claim 7, wherein the second agent is an mTOR inhibitor selected from sirolimus, temsirolimus, and everolimus. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete

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