Method for treating estrogen receptor-related diseases

Abstract

The present disclosure provides methods of treating estrogen receptor-associated diseases, disorders, and conditions.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 985,929, filed on March 6, 2020, which is incorporated herein by reference in its entirety.

Background Art

[0002] Estrogen receptor (ER) plays an important role in various cancers, including breast cancer. Various treatments have been developed to target the estrogen receptor and / or its activity. Cancer cells in the brain are particularly problematic. In the case of hormone - positive diseases, such as ER - positive breast cancer, the incidence of breast cancer brain metastases (BCBM) is 14%, and the median overall survival after the onset of brain metastases is 9 - 10 months. Brosnan, Ann Transl Med., 2016;6(9):163.

Prior Art Documents

Non - Patent Literature

[0003]

Non - Patent Literature 1

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides new findings regarding compounds and / or regimens useful for the treatment of estrogen receptor (ER) - related diseases, disorders, and conditions (e.g., cancer cells) and / or for modulating (e.g., inhibiting) estrogen receptors in the brain. In particular, the present disclosure defines certain structural and / or functional attributes that characterize compounds useful for such treatment and / or modulation.

[0005] In some embodiments, the present disclosure provides certain non-invasive techniques for the treatment of ER-related diseases, disorders, and conditions (e.g., cancer cells) and / or for otherwise modulating (e.g., inhibiting) estrogen receptors in the brain. For example, in some embodiments, the present disclosure defines compounds that inhibit estrogen receptors in the brain when administered systemically (e.g., orally). In particular, the present disclosure defines complete estrogen receptor antagonist compounds that are useful as described herein (e.g., orally bioavailable complete receptor antagonists that cross the blood-brain barrier).

[0006] There remains a need for anti-estrogen agents that can completely inhibit estrogen receptors, including those encoded by both the wild-type and mutant forms of the gene encoding estrogen receptor alpha (ERα), estrogen receptor 1 (ESR1) (e.g., those containing activating mutations). Selective estrogen receptor modulators (SERMs) or degraders (SERDs) are particularly useful or promising tools for such therapies. Estrogen receptors are three-part proteins that contain two distinct transcriptional activation functions (AF1 and AF2). Complete anti-estrogen 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.

[0007] Furthermore, there remains a need for the identification, characterization, and / or treatment of certain brain lesions (e.g., brain tumors such as brain metastases) that do not require invasive techniques such as whole-brain radiation therapy and / or surgery for estrogen receptor (ER)-related diseases, disorders, or conditions such as ER-positive cancer. The present application provides techniques related to such identification, characterization, and / or treatment. In some embodiments, the techniques provided include and / or deliver compositions that administer a complete estrogen receptor antagonist as described herein (e.g., a complete receptor antagonist that is orally bioavailable and / or crosses the blood-brain barrier).

[0008] Those skilled in the art recognize that many existing strategies for treating ER-related diseases, such as cancer, include first-line therapies such as tamoxifen and / or endoxifen, against which patients ultimately develop resistance. Certain second-line therapies, such as fulvestrant, have been developed, but research continues to pursue improved treatment strategies.

[0009] Previous therapies, such as tamoxifen, AZD9496, and ARN-810, are not complete estrogen receptor antagonists because they cannot inactivate both activation functions (i.e., they cannot inactivate both AF1 and AF2). Thus, there is still a need for therapies that inactivate 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 demonstrates, inter alia, that certain compounds can be used, alone or in combination with other agents, as a treatment for patients or subjects suffering from cancer, and that the patient or subject harbors a mutation in estrogen receptor 1 (ESR1).

[0010] Fulvestrant, unlike other approved ER antagonists (such as anti-estrogens), has been hailed as a so-called "complete" estrogen receptor antagonist because (1) it inhibits both activation function 1 (AF1) and activation function 2 (AF2) since complete anti-estrogenic activity requires inactivation of both AF1 and AF2, (2) it promotes ER degradation, and (3) it is characterized by its ability to avoid partial ER agonist activity. Fulvestrant is currently the only such "complete" ER antagonist approved for use by intramuscular injection in certain hormone receptor (HR)-positive breast cancers (specifically, including HR-positive metastatic breast cancer in postmenopausal women in whom the disease has progressed after endocrine therapy). Fulvestrant can be used in combination with cyclin-dependent kinase 4 / 6 inhibitors such as palbociclib, ribociclib, and abemaciclib for either initial treatment or after progression in the endocrine therapy of metastatic or locally advanced breast cancer.

[0011] However, fulvestrant has poor oral bioavailability and needs to be administered parenterally. Furthermore, fulvestrant does not cross the blood-brain barrier and is not useful for treating brain tumors (e.g., metastases).

[0012] The present disclosure provides findings regarding complete estrogen receptor antagonists that cross the blood-brain barrier and / or are orally available, and further describes the usefulness of such compounds in the detection, evaluation, and / or treatment of one or more ER-related diseases, disorders, or conditions (such as metastatic ER-positive breast cancer). The present disclosure specifically exemplifies the crossing of the blood-brain barrier by certain orally bioavailable complete estrogen receptor antagonists, and further provides findings regarding specific structural features and / or combinations of features that may contribute to and / or be involved in blood-brain barrier crossing and / or complete estrogen receptor antagonist activity. Without wishing to be bound by any particular theory, the present disclosure defines structure-function correlations regarding complete estrogen receptor antagonists that cross the blood-brain barrier and / or are orally bioavailable.

[0013] In particular, the present disclosure provides methods of using such complete estrogen receptor antagonists (i.e., those that cross the blood-brain barrier and / or are orally bioavailable) that specifically include detecting, evaluating, and / or treating brain lesions (such as tumors, e.g., metastases). In some embodiments, the present disclosure provides such methods with respect to ER-related diseases, disorders, or conditions (such as ER-related cancer). In some embodiments, the methods provided include administering to a subject afflicted with ER-related cancer a composition comprising and / or delivering to the subject's brain (e.g., upon oral administration) a complete estrogen receptor antagonist, wherein the subject has been determined to have or is suspected of having brain metastases.

[0014] The present disclosure defines the structural features of a complete receptor antagonist that passes through the blood-brain barrier (e.g., upon oral administration). For example, in some embodiments, without wishing to be bound by any particular theory, the present disclosure defines a structure-function correlation(s) between one or more of complete ER antagonism (as defined herein), the ability to pass through the blood-brain barrier, and / or oral bioavailability. In some embodiments, the present disclosure defines a structure-function correlation(s) for compounds that exhibit all of these (e.g., defining the characteristics of the structural properties). For example, without wishing to be bound by any particular theory, the present disclosure provides the finding that certain structural element(s) contribute to this combination of desired activities and, in certain embodiments, defines a potential structure-function correlation therewith.

[0015] In some embodiments, the present disclosure teaches that compounds having a pyrido[3,4-b]indole group and / or an azetidinyl group, and / or a substituted or unsubstituted phenyl moiety are particularly useful as fully estrogen receptor antagonists that are orally bioavailable and can cross the blood-brain barrier. For example, in some embodiments, the present disclosure teaches that compounds having a pyrido[3,4-b]indole group covalently linked to a phenyl linker and an azetidinyl moiety linked to the phenyl linker via a heteroatom or heteroalkyl linker are particularly useful fully estrogen receptor antagonists that are orally bioavailable and can cross the blood-brain barrier. In some embodiments, the phenyl moiety is additionally substituted with one or more halogen atoms (e.g., F, Br, Cl, I). In some embodiments, the phenyl moiety is not additionally substituted (e.g., contains no additional substitution other than the linkage to the pyrido[3,4-b]indole group and the heteroatom or heteroalkyl that links the phenyl group and the azetidinyl moiety). In some embodiments, the azetidinyl moiety is distal to the phenyl group. In some embodiments, the azetidinyl moiety is proximal to the phenyl group. In some embodiments, the azetidinyl moiety is further substituted with alkyl or haloalkyl.

[0016] In some embodiments, the present disclosure teaches that compounds having the structure described in Formula I may be particularly useful as described herein:

Chemical formula

[0017] In certain embodiments, the present disclosure provides the insight that one or more features of the pyrido[3,4-b]indole group and / or the X-Y group of Formula I, and / or combinations thereof, can provide or participate in the described function(s) (e.g., complete estrogen receptor antagonism, ability to cross the blood-brain barrier, oral bioavailability, and / or combinations thereof).

[0018] In some embodiments, the present disclosure teaches that a compound having the structure described in Formula I, wherein the compound contains an azetidinyl moiety, can provide or be involved in the desired function(s) (e.g., complete estrogen receptor antagonism, oral bioavailability, ability to cross the blood-brain barrier, and / or combinations thereof).

[0019] In some embodiments, the present disclosure teaches that a compound having the structure described in Formula I, wherein the compound does not contain a difluorophenyl moiety, can provide or be involved in the desired function(s) (e.g., complete estrogen receptor antagonism, oral bioavailability, ability to cross the blood-brain barrier, and / or combinations thereof).

[0020] In some embodiments, the present disclosure teaches that a compound having the structure described in Formula I, wherein the compound contains a difluorophenyl moiety, can provide or be involved in the desired function(s) (e.g., complete estrogen receptor antagonism, oral bioavailability, ability to cross the blood-brain barrier, and / or combinations thereof).

[0021] In some embodiments, the present disclosure teaches that one or more of the following compounds may be particularly useful, as described herein: GDC-9545, SAR439859, AZD9833,

Chemical Structure

[0022] Furthermore, the present disclosure demonstrates that certain complete estrogen antagonists (e.g., having the structure within Formula I) described herein can preferentially accumulate in tumors (s) over plasma to a significantly greater extent than that observed for fulvestrant when administered according to an appropriate regimen.

[0023] In some embodiments, the present disclosure provides a method of treating an ER-related disease or condition (e.g., a tumor (s) in the brain such as a brain metastasis, or an ER-related cancer including but not limited to tumors (s) that include tumors (s)) by administering a specific complete estrogen receptor antagonist according to a regimen that achieves preferential accumulation in the tumor compared to the patient's plasma (i.e., achieving accumulation in the tumor to a concentration higher than the concentration in the plasma). In some such embodiments, such accumulation is greater than that observed for fulvestrant. Alternatively or additionally, in some embodiments, such accumulation is at least about 30-fold higher than that observed in plasma.

[0024] Still further, the present disclosure provides various effective dosing regimens for certain complete estrogen antagonists (e.g., having the structure within Formula I) described herein. The present disclosure provides a method of treating an ER-related disease or condition (e.g., a tumor (s) in the brain such as a brain metastasis, or an ER-related cancer including but not limited to tumors (s) in the brain, by administering such specific complete estrogen receptor antagonist (s) according to such regimen (s). BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0060] There is a need for the treatment of certain brain metastases that do not require invasive techniques such as whole brain radiation therapy and surgery, particularly for estrogen receptor (ER)-positive cancer types. This application provides a method for treating brain metastases associated with ER-related diseases (including cancers related to estrogen receptors and mutations to estrogen receptors) in a subject by administering a composition comprising a complete estrogen receptor antagonist.

[0061] Definition Administration: As used herein, the term "administration" refers to the administration of a composition, typically, to a subject or system, to achieve delivery of an agent that is a composition, is included in a composition, or otherwise is delivered by a composition.

[0062] 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., a cell, tissue, organism]), or a phenomenon (e.g., heat, an electric current or field, a magnetic force or field, etc.).

[0063] Alkyl: The term "alkyl", used alone or as part of a larger moiety, refers to (unless otherwise specified) a saturated, optionally substituted, straight-chain or branched, or cyclic hydrocarbon group having from 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms (e.g., C1-C 12 、C1-C 10 、C1-C8, C1-C6, C1-C4, C1-C3, or C1-C2). Exemplary alkyl groups include methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, sec-butyl, isobutyl, t-butyl, etc.), pentyl, hexyl, and heptyl. The term "cycloalkyl" refers to a saturated ring system of from about 3 to about 10 ring carbon atoms, optionally substituted. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0064] Alkylene: The terms "alkylene" and "alkylenyl" are used interchangeably and refer to a divalent alkyl group. In some embodiments, "alkylene" is a divalent straight-chain or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2) n- and wherein n is a positive integer, for example 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are optionally replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups and those described herein. It will be understood that two substituents of the alkylene group can together form a ring system. In certain embodiments, two substituents can together form a 3- to 7-membered ring. The substituents can be on the same or different atoms.

[0065] Alkenyl: The term "alkenyl" used alone or as part of a larger moiety refers to (unless otherwise specified) an optionally substituted straight-chain, branched-chain, or cyclic hydrocarbon group having at least one double bond and having 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C2-C 12 , C2-C 10 , C2-C8, C2-C6, C2-C4, or C2-C3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0066] Antagonist: As used herein, the term "antagonist" can 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. An antagonist may include, for example, agents of any chemical class, including small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity exhibiting related inhibitory activity. In some embodiments, an antagonist can be a "direct antagonist" in that it binds directly to its target, and in some embodiments, an antagonist can be an "indirect antagonist" in that it affects the level or activity of the target by means other than direct binding to the target, for example, by interacting with a regulator of the target. In some embodiments, "antagonist" can be referred to as "inhibitor".

[0067] Aryl: The term "aryl" refers to monocyclic and bicyclic ring systems having a total of 5 to 14 ring members (e.g., C 5~14 ) such that at least one ring within the system is aromatic and each ring within the system contains 3 to 7 ring members. The term "aryl" can be used synonymously with the term "aryl ring". In certain embodiments of the present invention, "aryl" refers to aromatic ring systems such as phenyl, biphenyl, naphthyl, anthracyl, etc., which may have one or more substituents, but are not limited thereto. Unless otherwise specified, an "aryl" group is a hydrocarbon.

[0068] Related: As used herein, two events or entities are "related" to each other if the presence, level, degree, type, and / or form of one correlates with the presence, level, degree, type, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence and / or susceptibility to the disease, disorder, or condition (e.g., across the relevant population). In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly, such that they are physically proximate to each other and / or remain physically proximate to each other. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other, and in some embodiments, two or more entities that are physically related to each other are not covalently bonded to each other, but are non-covalently related by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0069] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained from, or derived from, a biological source of interest (e.g., a tissue or an organism or a 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 a 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, cell-free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph fluid, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or wash fluid, e.g., tube wash fluid or bronchoalveolar lavage fluid, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluids, secretions, and / or excretions, and / or cells therefrom, or may include them. 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 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 body fluid (e.g., blood, lymph fluid, feces, etc.). In some embodiments, as apparent 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 by adding one or more agents). For example, filtration using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins obtained by extracting from the sample or subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components.

[0070] Combination Therapy: As used herein, the term "combination therapy" refers to a situation where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously, and in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before the administration of any dose of the second regimen), and in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve administering one or more agent(s) or modality(ies) in combination to a subject who is receiving another agent(s) or modality(ies). For clarity, combination therapy does not necessarily require that the individual agents be administered together (or even simultaneously) in a single composition, but in some embodiments, two or more agents, or their active moieties, may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or conjugate).

[0071] Dosage Form or Unit Dosage Form: One of ordinary skill in the art will understand that the term "dosage form" can be used to refer to a physically distinct 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 an appropriate unit dosage (or an integral part thereof) for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to the relevant population (i.e., correlates with a therapeutic dosing regimen). One of ordinary skill in the art understands that the total amount of a therapeutic composition or agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0072] Dosing regimen or treatment regimen: Those skilled in the art will understand that the terms "dosing regimen" and "treatment regimen" can typically be used to refer to a series of unit doses (typically more than one) that are administered individually to a subject and are separated by a period. 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 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 separating the individual doses and at least two different periods. 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 at a first dosage, followed by one or more additional doses at a second dosage different from the first dosage. In some embodiments, the dosing regimen includes a first dose at a first dosage, followed by one or more additional doses at a second dosage the same as the first dosage. In some embodiments, the dosing regimen is correlated with a desired or beneficial result when administered across the relevant population (i.e., it is a therapeutic dosing regimen).

[0073] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that can 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.

[0074] Halo or halogen: As used herein, the terms "halogen" or "halo" refer to fluorine, chlorine, bromine, and iodine. "Halo" can modify another group to indicate an optional substitution of a hydrogen atom by a halogen atom. For example, as used herein, "haloalkyl" is a branched, straight-chain, or cyclic alkyl group substituted with one or more halogen atoms (e.g., a "C1-C6 haloalkyl" group has 1 to 6 carbon atoms and one or more hydrogen atoms are substituted with halogen atoms). Examples of haloalkyl groups include, but are not limited to, mono-, di-, or tri-fluoromethyl, -CH2-CH2-fluoro, -CH2-CH2-CH2-fluoro, -CH2-CH2-CH2-CH2-fluoro, and -CH2-CF3, -CH2-C(CH3)2-F.

[0075] Heteroaryl: The terms "heteroaryl" and "heteroal-", used alone or as part of a larger moiety, such as "heteroalkyl" or "heteroalkoxy", refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl), having 6, 10, or 14 π electrons shared in a cyclic array, and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. Examples of heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thieno[2,3-d]pyrimidinyl, triazolo[4,5-d]pyridinyl, and benzisoxazolyl. The terms "heteroaryl" and "heteroal-" as used herein also include groups in which the heteroaromatic ring is fused to one or more aryl rings, cycloaliphatic rings, or heterocyclyl rings and the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and benzisoxazolyl. A heteroaryl group can be monocyclic or bicyclic.The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "heteroaromatic", and any of these terms includes an optionally substituted ring. The term "heteroalkyl" refers to an alkyl group substituted by heteroaryl, and the alkyl portion and the heteroaryl portion are each independently optionally substituted.

[0076] Heteroatom: As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen.

[0077] Heterocyclic ring: As used herein, the terms "heterocyclic ring", "heterocyclyl", "heterocyclic radical", and "heterocycle" are used interchangeably and refer to a stable 3- to 8-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, such as 1 to 4, heteroatoms in addition to carbon atoms. When used with respect to the ring atoms of a heterocyclic ring, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen is N (such as in 3,4-dihydro-2H-pyrrolyl), NH (such as in pyrrolidinyl), or NR +(such as in N-substituted pyrrolidinyl). The heterocycle can be attached to the pendant group by any heteroatom or carbon atom that provides a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiomorpholinyl. The heterocyclyl group may be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, more preferably monocyclic or bicyclic. The term "heterocycloalkyl" refers to an alkyl group substituted by a heterocyclyl, and the alkyl portion and the heterocyclyl portion are each independently optionally substituted. The bicyclic heterocycle also includes a group in which the heterocycle is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, tetrahydroquinolinyl, and

Chemical Structure

[0078] Oral: As used herein, the terms "oral administration" and "administered orally" have the meaning understood in the art and refer to the oral administration of a compound or composition.

[0079] Parenteral: As used herein, the terms "parenteral administration" and "administered parenterally" have the meaning understood in the art and refer to a mode 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, subepidermal, intraarticular, subcapsular, subdural, intramedullary, and intrasternal injections and infusions.

[0080] Patient or subject: As used herein, the terms "patient" or "subject" refer to any organism to which a provided composition is, or can be, administered for, e.g., experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical 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 human. In some embodiments, the patient or subject has, or is 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 has received, or has been the subject of, a particular therapy for diagnosing and / or treating a disease, disorder, or condition.

[0081] 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 in a unit dose appropriate for administration in a treatment regimen to a relevant subject (e.g., an amount that has been demonstrated to show a statistically significant likelihood of achieving a predetermined therapeutic effect when administered), or is present in a different, equivalent subject (e.g., in an equivalent subject or system different from the subject or system of interest, in the presence of one or more indicators of one or more of a particular disease, disorder or target condition, or due to prior exposure to a condition or agent). In some embodiments, the comparative terms refer to a statistically relevant difference (e.g., a difference of sufficient prevalence and / or magnitude to achieve statistical relevance). One of ordinary skill in the art will recognize, or be able to readily determine, the degree and / or prevalence of difference necessary or sufficient to achieve such statistical significance in a given context.

[0082] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material involved in carrying or transporting a subject compound from one organ or part of the body to another organ or another 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 that can function as pharmaceutically acceptable carriers include saccharides such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffering solutions; polyesters, polycarbonates, and / or polyanhydrides, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0083] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in the pharmaceutical context, i.e., within the scope of sound medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reaction, etc., and are salts that meet a reasonable benefit-to-risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically 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, pharmaceutically 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-hydroxy-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, pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.

[0084] Substituted or optionally substituted: As described herein, the compounds of the present invention may contain "optionally substituted" moieties. Generally, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. "Substituted" is explicit or implicit from the structure (e.g.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0085] Suitable monovalent substituents on the replaceable carbon atoms of an "optionally substituted" group are, independently, halogen; -(CH2)0-4R°; -(CH2)0-4OR°; -O(CH2)0-4R o, -O-(CH2)0-4C(O)OR°; -(CH2)0-4CH(OR°)2; -(CH2)0-4SR°; -(CH2)0-4Ph (which may be substituted with R°); -(CH2)0-4O(CH2)0-1Ph (which may be substituted with R°); -CH=CHPh (which may be substituted with R°); -(CH2)0-4O(CH2)0-1-pyridyl (which may be substituted with R°); -NO2; -CN; -N3; -(CH2)0-4N(R°)2; -(CH2)0-4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2)0-4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)0-4N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)R°; C(S)R°; -(CH2)0-4C(O)OR°; -(CH2)0-4C(O)SR°; -(CH2)0-4C(O)OSiR°3; -(CH2)0-4OC(O)R°; -OC(O)(CH2)0-4SR°; -(CH2)0-4SC(O)R°; -(CH2)0-4C(O)NR°2; -C(S)NR°2; -C(S)SR°; -SC(S)SR°, -(CH2)0-4OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)0-4SSR°; -(CH2)0-4S(O)2R°; -(CH2)0-4S(O)2OR°; -(CH2)0-4OS(O)2R°; -S(O)2NR°2; -(CH2)0-4S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; -(C1-4 linear or branched alkylene)O-N(R°)2;or -(C1-4 linear or branched alkylene)C(O)O-N(R°)2, where each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -O(CH2)0-1Ph, -CH2-(5-6 membered heteroaryl ring), or a 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independent occurrences of R° together with the intervening atom(s) form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and which may be substituted as defined below.;

[0086] Suitable monovalent substituents on R° (or the ring formed by two independent occurrences of R° together with the intervening atoms) are independently halogen, -(CH2)0-2R●, -(haloR●), -(CH2)0-2OH, -(CH2)0-2OR●, -(CH2)0-2CH(OR●)2, -O(haloR●), -CN, -N3, -(CH2)0-2C(O)R●, -(CH2)0-2C(O)OH, -(CH2)0-2C(O)OR●, -(CH2)0-2SR●, -(CH2)0-2SH, -(CH2)0-2NH2, -(CH2)0-2NHR●, -(CH2)0-2NR●2, -NO2, -SiR●3, -OSiR●3, -C(O)SR●, -(C1-4 linear or branched alkylene)C(O)OR●, or -SSR● (each R● is unsubstituted or, in the preceding case) "halo" is substituted with only one or more halogens and is independently selected from C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 3-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on the saturated carbon atoms of R° include =O and =S.

[0087] Suitable divalent substituents on a saturated carbon atom of a "optionally substituted" radical include the following: =O ("oxo"), =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, =NR*, =NOR*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, where each independent occurrence of R* is hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents bonded to an adjacent replaceable carbon of an "optionally substituted" radical include -O(CR*2)2-3O-, where each independent occurrence of R* is hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0088] Suitable substituents for the aliphatic group of R* include halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, where each R● is unsubstituted or, when preceded by "halo", substituted by only one or more halogens and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0089] Suitable substituents on replaceable nitrogen of the "may be replaced" group include -R†, -NR†2, -C(O)R†, -C(O)OR†, -C(O)C(O)R†, -C(O)CH2C(O)R†, -S(O)2R†, -S(O)2NR†2, -C(S)NR†2, -C(NH)NR†2, or -N(R†)S(O)2R†, where each R† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having 3 to 6 members, saturated, partially unsaturated, or aromatic, or notwithstanding the above definition, two independent occurrences of R†, together with intervening atom(s), form an unsubstituted monocyclic or bicyclic ring having 3 to 12 members, saturated, partially unsaturated, or aromatic and having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0090] Suitable substituents for the aliphatic group of R† include, independently, halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, where each R● is unsubstituted or, when preceded by "halo", substituted by only one or more halogens and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 3-6 membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0091] Small molecule: As used herein, the term "small molecule" refers to an organic and / or inorganic compound of low molecular weight. Generally, a "small molecule" is a molecule having a size of less than about 5 kilodaltons (kD). In some embodiments, the small molecule is less than about 4 kD, 3 kD, about 2 kD, or less than about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, the small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, the small molecule is not a polymer.

[0092] In some embodiments, the small molecule does not contain a polymer moiety. In some embodiments, the small molecule is not and / or does not contain a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, the small molecule is not and / or does not contain a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, the small molecule is not and / or does not contain a polysaccharide; for example, in some embodiments, the small molecule is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, the small molecule is not a lipid.

[0093] In some embodiments, the small molecule is a modulator (e.g., an inhibitor or activator). In some embodiments, the small molecule is biologically active. In some embodiments, the small molecule is detectable (e.g., contains at least one detectable moiety). In some embodiments, the small molecule is a therapeutic agent.

[0094] Upon reading the present disclosure, those skilled in the art will understand that the specific small molecule compounds described herein can be provided and / or utilized in any of various forms, such as crystalline forms (e.g., polymorphs, solvates, etc.), salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, and the like.

[0095] Those skilled in the art will understand that a specific small molecule compound can have a structure in which it can exist in one or more stereoisomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of a racemic mixture.

[0096] Those skilled in the art will understand that a specific small molecule compound can have a structure in which it can exist in one or more tautomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual tautomers or in a form that interconverts between tautomeric forms.

[0097] Those skilled in the art will understand that a specific small molecule compound can have a structure that allows for isotope substitution (e.g., for H, 2H or 3H; for 12C, 11C, 13C or 14C; for 14N, 13N or 15N; for 16O, 17O or 18O; for XXC, 36Cl; for XXF, 18F; for XXXI, 131I, etc.). In some embodiments, such small molecules can be utilized in accordance with the present disclosure in one or more isotopically modified forms, or mixtures thereof.

[0098] In some embodiments, reference to a particular small molecule compound may relate to a particular form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in salt form (e.g., an acid addition or base addition salt form, depending on the compound), and in some such embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0099] In some embodiments, if a small molecule compound is a compound that exists in nature or is a compound found in nature, that compound may be provided and / or utilized in accordance with the present disclosure in a form that is different from the form in which it exists or is found in nature. One of ordinary skill in the art will understand that in some embodiments, a preparation of a particular small molecule compound containing an absolute or relative amount of the compound or form that is different (e.g., with respect to another component of the preparation, including a different form of the compound) from the absolute or relative amount of the compound or form present in a reference preparation of interest (e.g., a primary sample from a source of interest such as a biological or environmental source). Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound may be considered a compound in a form different from a racemic mixture of the compound, a particular salt of a small molecule compound may be considered a compound in a form different from another salt form of the compound, a preparation containing only the form of a compound containing one stereoisomer ((Z) or (E)) of a double bond may be considered a compound in a form different from one containing the other stereoisomer ((E) or (Z)) of the double bond, and a preparation in which one or more atoms are isotopes different from those present in the reference preparation may be considered a different form.

[0100] Therapeutic agent: As used herein, the term "therapeutic agent" generally refers to any agent that, when administered to an organism, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it shows a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms. In some embodiments, the appropriate population can be defined by various criteria such as a particular age group, gender, genetic background, existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to reduce, ameliorate, relieve, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features 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 available for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

[0101] Treat: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely reduce, ameliorate, relieve, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment can be administered to a subject that does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to a subject that exhibits only early signs of a disease, disorder, and / or condition, for example, for the purpose of reducing the risk of developing the medical conditions associated with the disease, disorder, and / or condition.

[0102] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, the 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 in a subject who has or is susceptible to having the disease, disorder, and / or condition. As will be understood by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, the target cell or tissue, etc. For example, the effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that reduces, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, the therapeutically effective amount is administered as a single dose, and in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0103] The symbols used herein

Chemical formula

[0104] Compounds referred to herein can be enriched with one or more isotopes, such as deuterium ( 2 H or D), in any or all atoms that exceed the naturally occurring isotope ratio, as will be understood by those skilled in the art.

[0105] The compounds of the present invention, or pharmaceutically acceptable salts thereof, may be in either the (R) or (S) configuration, or may contain chiral centers that can include mixtures thereof, unless otherwise specified. Accordingly, this application includes, where applicable, the stereoisomers of the compounds described herein, individually or in any proportion mixed. Stereoisomers may include, but are not limited to, enantiomers, diastereomers, racemic mixtures, and combinations thereof. Such stereoisomers can be prepared and isolated using conventional techniques, either by reacting enantiomeric starting materials or by separating the isomers of the compounds of this application.

[0106] Estrogen receptor-related diseases The estrogen receptor ("ER") is involved in various biological processes related to, for example, the development of the female reproductive system, maintenance of bone mass, and protection of cardiovascular and / or central nervous system components (see, for example, Pearce & Jordan Crit. Rev. Onc / Hem 50:3, 2004, Heldring Phys. Rev. 87:905, 2007). ER is involved in various cancers. In many tumors that express the estrogen receptor (i.e., ER + tumors), activation of ERα signaling has been demonstrated to cause cell proliferation (however, ERβ signaling has been reported to be able to achieve a tumor-suppressive effect; see, for example, Nilsson & Gustafson Clin. Pharmacol. Ther. 89:44, 2011). Typically, only 1% of tumors (e.g., breast tumors) with cells positively stained for ER are classified as "ER + ". Therapies targeting ER are ER +This is the standard treatment for many patients with tumors (see, for example, 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 breast cancer patients, the recommended therapy typically involves tumor resection, followed by ER-targeted chemotherapy (discussed below). For advanced breast cancer, including metastatic breast cancer, ER-targeted chemotherapy is the mainstay.

[0107] Given the importance of ER signaling in many cancers, as well as certain cardiovascular, inflammatory, and neurodegenerative diseases, significant efforts have been devoted to the development of therapeutic agents and modalities that target the ER. The terms used to describe ER-targeted agents have some fluidity / flexibility, but various agents with different mechanisms have been developed and / or studied.

[0108] Some ER-targeted agents are designed and / or demonstrated to reduce the level of estrogen (i.e., 17β-estradiol) production.

[0109] Some ER-targeted agents are designed and / or demonstrated to bind directly to the ER, and in some cases, such agents compete with estrogen for binding to the ER and / or prevent the allosteric changes that estrogen binding naturally induces. In many cases, the term "anti-estrogen" is used to refer to agents that bind to the ER, and in some instances, it is specifically used to denote agents that compete with estrogen for ER binding.

[0110] The term "selective estrogen receptor modulator" ("SERM") refers to compounds that are designed and / or demonstrated to alter some aspects of ER activity. In some descriptions, "SERM" refers to a particular class of anti-estrogens, however, other descriptions more generally use the term "SERM" to refer to compounds that specifically affect some characteristics of ER (especially ERα) expression and / or activity.

[0111] The term "selective estrogen receptor degrader" ("SERD") refers to compounds that are designed and / or demonstrated to induce or enhance the degradation of ER. Often, a compound may be called a SERD if the presence of the compound correlates with a reduction in the level of ER. In some descriptions, compounds are classified as either SERMs or SERDs, and other descriptions refer to SERDs as a particular class or species of compounds that are SERMs.

[0112] Regardless of the mechanism of action of a particular agent, previous clinical experience has revealed that incomplete effects (e.g., within individual patients and / or across patient populations) and / or the development of resistance remain problems.

[0113] 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 the 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 the ER, and other mutations, including deletions and / or fusions that remove the ligand-binding domain, may have a similar effect (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 by circulating tumor DNA.

[0114] Brain metastases occur in approximately 10-16% of breast cancer patients and are the second most common cause of brain metastases after lung cancer. Leone, Exp Hematol Oncol 4, 33(2015) doi:10.1186 / s40164-015-0028-8. The prognosis is poor, and the overall survival from diagnosis ranges from several months to several years. Frisk, et al., Breast Cancer Res.Treat. 166:887-896(2017). In particular, in the case of hormone-positive diseases, such as ER-positive breast cancer, the incidence of breast cancer brain metastases (BCBM) is 14%, and the median overall survival after the onset of brain metastases is 9-10 months. Brosnan & Anders, Ann Transl Med., 2016;6(9):163. There is no FDA-approved treatment for breast cancer brain metastases, only surgery and radiotherapy, including whole brain radiotherapy.

[0115] The challenge in treating breast cancer brain metastases is to provide therapies that can cross the blood-brain barrier. As pointed out by Brosnan & Anders, the BBB selectively regulates what enters the brain and exists to protect the brain from toxic substances, including chemotherapeutic agents and targeted drugs. In particular, "in addition to the intrinsic drug efflux pumps, the unpredictable nature and heterogeneity in the permeability of the BBB make it difficult to effectively deliver a sufficient amount of drug to brain metastases to achieve apoptosis." Brosnan & Anders, Ann Transl Med., 2016;6(9):163.

[0116] The present disclosure establishes a specific class of compounds that are complete estrogen receptor antagonists capable of crossing the blood-brain barrier, thereby providing a practicable mode of treatment for breast cancer brain metastases.

[0117] Estrogen receptor antagonist Substantial investment has been and continues to be made in pursuing effective therapies targeting ER (see, for example, Patel & Bihani Pharmacol.& Therap. 186:1,2018).

[0118] Among the most advanced compounds in clinical development are the following: a. Tamoxifen, an important breast cancer treatment drug, has been evaluated as "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 on July 7, 2019)), but it is known to be less effective and more likely to develop resistance in women with low CYP2D6 activity). b. Endoxifen, an active metabolite of tamoxifen, was originally developed to address the problems of tamoxifen in women with low CYP2D6 activity and reduces the ability to convert tamoxifen to endoxifen. (See Cancer Currents Blog, National Cancer Institute, August 31, 2017) c. ARN-810 (brivanestrant, GDC-810), 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), which has failed in other hormonal agents but has been brought into a Phase II clinical trial for the treatment of ER+ breast cancer patients in whom further growth may have declined (see, for example, Biospace April 27, 2017) d. AZD9496 is described as an "oral non-steroidal 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 an "antagonist and degrader of ER with antitumor activity in both endocrine-sensitive and endocrine-resistant models" and is described as "comparable to fulvestrant in antagonizing ER and circumventing endocrine resistance" (see Nardone et al.Br.Cancer 120:331,2019). RAD-1901 (elacestrant) is described as a "novel non-steroidal oral SERD that demonstrated single-agent activity in heavily pre-treated 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, in preclinical studies, it has been reported that "elacestrant significantly inhibited the growth of xenograft models with ESR1 mutations, including those with the Y537S or D538G mutations and models that were insensitive to fulvestrant and tamoxifen" (see Patel et al.Cancer Res 79:Abstract nr P6-20-08,2019). f. Fulvestrant (Faslodex®) is the first SERD to receive FDA approval 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 via the ER" (see Nathan & Schmid Oncol Ther 5:17, 2017). Fulvestrant has had clinically significant 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 actually requires a 500 mg dose by intramuscular injection once monthly (after the initial dose). Also, certain retrospective analyses have provided hope that fulvestrant may have some utility in the treatment of patients with ER variants, but confirmation of activity has not been obtained (see, for example, Fribbens et al. J Clin Oncol. 34:2961, 2916, Spoerke et al. Nat Commun 7:11579, 2016).

Chem.

[0119] Fulvestrant continues to be the gold standard for the treatment of ER-related diseases and disorders for patients who have developed resistance to currently approved therapies such as tamoxifen and endoxifen. This disclosure is based on the understanding that the success of fulvestrant results from its ability to (1) inhibit both AF1 and AF2, and thus inhibit the AF1 activity that persists in constitutively active ER variants, (2) promote ER degradation, and (3) function as a complete estrogen receptor antagonist (“CERAN”) lacking the partial ER agonist activity observed with certain other agents. 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.

[0120] Nevertheless, this disclosure further understands that many other compounds, including, for example, ARN-810, AZD9496, tamoxifen, and others, are at least partially only ER antagonists, specifically inhibiting the activation of AF2 but not AF1, and are thus less effective than fulvestrant. However, it is well known that fulvestrant further has a number of deficiencies, including poor oral bioavailability and the inability to cross the blood-brain barrier.

[0121] This disclosure encompasses the insight that the treatment of ER-related brain metastases involves the administration of certain classes of complete estrogen receptor antagonists that are orally bioavailable and can also cross the blood-brain barrier. Accordingly, in some embodiments, this disclosure provides a method of treating cancer, the method comprising administering to a subject afflicted with ER-related cancer a composition that delivers a complete estrogen receptor antagonist to the subject's brain, wherein the subject has been determined to have or is suspected of having brain metastases.

[0122] Estrogen receptor antagonist In some embodiments, this disclosure teaches the particular utility of compound(s) that are complete estrogen receptor antagonists. In some embodiments, a “complete estrogen receptor antagonist,” as the term is used herein, is characterized by the complete antagonism of the estrogen receptor that has no residual estrogen receptor agonist activity. For example, a complete estrogen antagonist is understood to be an agent (e.g., a small molecule compound) that does not exhibit ER antagonism and ER agonism in one or more of an ERα protein level assay, an MCF-7 cell line assay, an Ishikawa cell line assay (measuring certain variants including wild-type ER and certain variants lacking the aAF1 and / or AF2 domains), and a rodent uterine weight gain assay. See generally WO2017 / 059139 and US9,018,244. Alternatively or additionally, in some embodiments, a complete estrogen receptor antagonist has three characteristics: (1) inhibits both activation function 1 (AF1) and activation function 2 (AF2) because complete anti-estrogen activity requires inactivation of both AF1 and AF2, (2) promotes ER degradation, and (3) lacks the partial ER agonist activity observed with certain other agents. Without being bound by theory, it is understood that complete inhibition of both AF1 and AF2 is required for complete estrogen receptor activity, and activating mutations in the gene encoding estrogen receptor 1 can enable activation of both AF1 and AF2 even in the absence of estrogen.

[0123] Given the importance of ER signaling in many cancers, and certain cardiovascular, inflammatory, and neurodegenerative diseases, significant efforts are being made to develop therapeutics and modalities that target the ER. While the terminology used to describe ER-targeting agents has some fluidity / flexibility, various agents with different mechanisms have been developed and / or studied.

[0124] Currently, fulvestrant is the only approved therapy with each of these characteristics. However, as described above, fulvestrant has a number of drawbacks, including poor oral bioavailability and inability to cross the blood-brain barrier, and is completely ineffective for the treatment of brain metastases associated with ER-related diseases or disorders.

[0125] Certain structural features of exemplary compounds This disclosure encompasses the insight that a particular class of compounds can fully antagonize the estrogen receptor (i.e., are complete estrogen receptor antagonists) and at the same time can cross the blood-brain barrier and are suitable for the treatment of brain metastases associated with ER-related diseases or disorders. For example, among other things, this disclosure reports certain estrogen receptor antagonists that exhibit complete estrogen receptor antagonism comparable to fulvestrant in various assays.

[0126] Suitable complete estrogen receptor antagonists include those reported in WO2012 / 084711, WO2014 / 191726, WO2016 / 097072, WO2017 / 059139, and WO2019 / 245974, each of which is incorporated herein by reference. For example, in some embodiments, the complete estrogen receptor antagonist is GDC-9545, SAR439859, AZD9833, and compounds of Formula I

Chemical formula

Chemical formula

[0127] In some embodiments, the complete estrogen receptor antagonist is SAR439859.

Chemical formula

[0128] In some embodiments, the complete estrogen receptor antagonist is AZD9833.

[0129] In some embodiments, the complete estrogen receptor antagonist is GDC-9545.

[0130] In some embodiments, the estrogen receptor antagonist is [Chemistry] is.

[0131] In some embodiments, the estrogen receptor antagonist is [Chemistry] is.

[0132] In some embodiments, the complete estrogen receptor antagonist is a compound of formula I [Chemistry] or a pharmaceutically acceptable salt thereof, wherein R a , R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , X, and Y are described in the classes and subclasses herein.

[0133] In some embodiments, generally as described above, X is -NH-, -CH2-, or -O-. In some embodiments, X is -NH- or -O-. In some embodiments, X is -CH2- or -O-. In some embodiments, X is -NH- or -CH2-. In some embodiments, X is -NH-. In some embodiments, X is -CH2-. In some embodiments, X is -O-.

[0134] In some embodiments, generally as described above, R a is hydrogen or halo. In some embodiments, R a is hydrogen. In some embodiments, R a is halo. In some embodiments, R はis fluoro, bromo, or chloro. In some embodiments, R は is fluoro.

[0135] In some embodiments, generally as described above, R 1 , R 2 , R 3 , and R 4 are each independently selected from hydrogen and halo. In some embodiments, R 1 , R 2 , R 3 , and R 4 are each hydrogen. In some embodiments, R 1 , R 2 , and R 3 are each hydrogen and R 4 is halo. In some embodiments, R 1 , R 2 , and R 3 are each hydrogen and R 4 is fluoro. In some embodiments, R 2 , R 3 , and R 4 are each hydrogen and R 1 is halo. In some embodiments, R 2 , R 3 , and R 4 are each hydrogen and R 1 is fluoro. In some embodiments, R 1 , R 3 , and R 4 are each hydrogen and R 2 is halo. In some embodiments, R 1 , R 3 , and R 4 are each hydrogen and R 2 is fluoro. In some embodiments, R 1 , R 2 , and R 4 are each hydrogen and R 3 is halo. In some embodiments, R 1 , R 2 , and R 4 are each hydrogen and R 3is fluoro. In some embodiments, R 1 and R 2 are each hydrogen, and R 3 and R 4 are each halo. In some embodiments, R 1 and R 2 are each hydrogen, and R 3 and R 4 are each fluoro. In some embodiments, R 3 and R 4 is hydrogen, and R 1 and R 2 are halo. In some embodiments, R 3 and R 4 are hydrogen, and R 1 and R 2 are fluoro.

[0136] In some embodiments, R 1 and R 2 are each hydrogen, R 3 and R 4 are each hydrogen or halo, and when one of R 3 or R 4 is halo, the other of R 3 or R 4 is hydrogen. In some embodiments, R 1 and R 2 are each hydrogen, R 3 and R 4 are each hydrogen or fluoro, and when one of R 3 or R 4 is fluoro, the other of R 3 or R 4 is hydrogen.

[0137] In some embodiments, generally as described above, Y is

Chemical formula

Chemical formula

Chemical formula

[0138] In some embodiments, generally as described above, R 5 is hydrogen, or an optionally substituted group selected from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C1-C6 heteroalkyl. In some embodiments, R 5 is an optionally substituted group selected from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, and C1-C6 heteroalkyl. In some embodiments, R 5 is an optionally substituted group selected from C1-C6 alkyl, C1-C6 haloalkyl, and C2-C6 alkenyl. In some embodiments, R 5 is an optionally substituted group selected from C1-C6 alkyl and C1-C6 haloalkyl.

[0139] In some embodiments, R 5 is optionally substituted C1-C6 alkyl. In some embodiments, R 5 is unsubstituted C1-C6 alkyl. In some embodiments, R 5 is methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R 5 is methyl, ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl. In some embodiments, R 5 is n-propyl.

[0140] In some embodiments, R 5 is optionally substituted C1-C6 haloalkyl. In some embodiments, R 5is -CH2-halo, -CH2-CH2-halo, -CH2-CH2-CH2-halo, or -CH2-CH2-CH2-CH2-halo. In some embodiments, R 5 is -CH2-fluoro, -CH2-CH2-fluoro, -CH2-CH2-CH2-fluoro, or -CH2-CH2-CH2-CH2-fluoro. In some embodiments, R 5 is -CH2-F or -CH2-CH2-CH2-F. In some embodiments, R 5 is -CH2-F. In some embodiments, R 5 is -CH2-CH2-CH2-F.

[0141] In some embodiments, Y is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0142] In some embodiments, Y is

Chemical formula

[0143] In some embodiments, generally as described above, R 6 is a group optionally substituted with hydrogen, or C1-C6 alkyl and C1-C6 haloalkyl. In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is a group optionally substituted with C1-C6 alkyl and C1-C6 haloalkyl.

[0144] In some embodiments, R 6 is optionally substituted C1-C6 alkyl. In some embodiments, R 6 is C1-C6 alkyl substituted with one or more groups selected from halogen and -(CH2)0-4OR°. In some embodiments, R 6 is C1-C6 alkyl substituted with one or more groups selected from halogen and -OR°. In some embodiments, R 6 is C1-C6 alkyl substituted with one or more groups selected from halogen and -OH. In some embodiments, R 6 is [Chemical formula] is as follows.

[0145] In some embodiments, R 6 is C1-C6 haloalkyl. In some embodiments, R 6 is C1-C4 haloalkyl. In some embodiments, R 6 is -CH2-C(CH3)2-F or -CH2-CF3.

[0146] In some embodiments, generally as described above, R 7 and R 8is independently selected from hydrogen and optionally substituted C1-C6 alkyl. In some embodiments, R 7 and R 8 one of which is hydrogen and R 7 and R 8 the other of which is C1-C6 alkyl. In some embodiments, R 7 and R 8 one of which is hydrogen and R 7 and R 8 the other of which is methyl. In some embodiments, R 7 is hydrogen and R 8 is R-methyl (i.e., methyl having the stereochemical orientation shown as R). In some embodiments, R 7 is hydrogen and R 8 is S-methyl (i.e., methyl having the stereochemical orientation shown as S).

[0147] Thus, in some embodiments, the complete estrogen receptor antagonist is a compound of formula (I), and the complete estrogen receptor antagonist is

Chemical formula

[0148] In some embodiments, the complete estrogen receptor antagonist is Compound 1

Chemical formula

[0149] In some embodiments, the complete estrogen receptor antagonist is the free base form of Compound 1

Chemical formula

[0150] In some embodiments, the complete estrogen receptor antagonist is Compound 2 [Chemical Structure] as follows.

[0151] In some embodiments, the complete estrogen receptor antagonist is Compound 3 [Chemical Structure] as follows.

[0152] In some embodiments, the complete estrogen receptor antagonist is Compound 4 [Chemical Structure] as follows.

[0153] In some embodiments, the complete estrogen receptor antagonist is Compound 5 [Chemical Structure] as follows.

[0154] In some embodiments, the complete estrogen receptor antagonist is Compound 5a: [Chemical Structure] as follows.

[0155] In some embodiments, the estrogen receptor antagonist is Compound 5b: [Chemical Structure] as follows.

[0156] In some embodiments, the complete estrogen receptor antagonist is (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, (1R,3R)-1-(2,6-difluoro-4-((1-propylazetidin-3-yl)oxy)phenyl)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9,-tetrahydro-1H-pyrido[3,4-b]indole, (1R,3R)-1-(2,6-difluoro-4-(2-(3-(fluoromethyl)azetidin-1-yl)ethoxy)phenyl)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9,-tetrahydro-1H-pyrido[3,4-b]indole, (1R,3R)-1-(2,6-difluoro-4-((1-(3-fluoropropyl)azetidin-3-yl)oxy)phenyl)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9,-tetrahydro-1H-pyrido[3,4-b]indole, and a compound selected from 3-((1R,3R)-1-(2,6-difluoro-4-((1-(3-fluoropropyl)azetidin-3-yl)amino)phenyl)-3-methyl-1,3,4,9,-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol.

[0157] In some embodiments, the complete estrogen receptor antagonist is a free base. In some embodiments, the complete estrogen receptor antagonist is in the form of a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically acceptable salt is as described herein. In some embodiments, the pharmaceutically acceptable salt is selected from succinate (e.g., succinate form), tartrate (e.g., tartrate form), and fumarate (e.g., fumarate form).

[0158] In some embodiments, the compounds described herein can optionally be labeled with a radioactive label, for example, an isotope of a particular atom. These radiolabeled compounds are useful for the detection and visualization of certain ER + tumors (e.g., via positron emission tomography (PET)). Exemplary radiolabels include 11 C, 18 F, 15 O, 13 N, and 131 I, but are not limited thereto.

[0159] In particular, it should be understood that certain features of the compounds described herein enable them to be combinations of complete estrogen antagonists, be orally bioavailable, and be able to cross the blood-brain barrier. However, similar compounds lack these three properties. For example, AZD9496, a selective estrogen receptor degrader (SERD), has been found to be orally bioavailable, but fails to fully antagonize the estrogen receptor due to the presence of at least some residual agonist activity. Without being bound by theory, it should be noted that AZD9496 lacks the heterocyclic moiety (e.g., the azetidinyl moiety) present in the compounds described herein. In particular, without being bound by theory, the combination of a heterocyclic moiety (e.g., the azetidinyl moiety) and pyrido[3,4-b]indole is theorized to provide compounds with certain desired properties.

[0160] Also, the absence of halogen substitution on the phenyl bridge is theorized to contribute to the ability of certain compounds to cross the blood-brain barrier.

[0161] Evaluation of ER Antagonists In particular, the present disclosure teaches that useful ER antagonist agents have CERAN activity as described herein.

[0162] One aspect of the present disclosure is the insight that conventional strategies for evaluating or characterizing ER antagonist (and / or potential antagonist) agents were insufficient in that they typically did not distinguish between SERD and CERAN. In particular, most such conventional strategies did not evaluate the ability of agents to specifically affect AF1.

[0163] In particular, the present disclosure teaches that particularly useful ER antagonist agents are those that can inhibit ligand-independent ER activity and, in some embodiments, include activities observed in constitutive ER variant(s) such as, for example, AF2 deletion or truncation and / or LBD variants (e.g., D538G and Y537S).

[0164] Furthermore, the present disclosure teaches that particularly useful ER antagonist agents a. inhibit AF1 (e.g., inhibit at least one, preferably all known constitutive ER variants) b. inhibit AF2 (e.g., inhibit ligand-dependent ER activity) c. promote ER degradation, respectively.

[0165] Furthermore, in some embodiments, particularly useful ER antagonist agents a. have oral bioavailability and a long half-life. b. cross the blood-brain barrier, and are further characterized by one or more of the above.

[0166] In certain embodiments, the activity of the ER antagonist agent(s) may be evaluated relative to the activity of one or more of ARN-810, AZD9496, endoxifen, fulvestrant, RAD1901, tamoxifen, and / or a compound of Formula I, and in some such embodiments, the comparison may be made simultaneously or, in some embodiments, compared to historical records or future results.

[0167] Method of Use This disclosure encompasses the insight that certain complete estrogen receptor antagonists have several uses, including the treatment, detection, and / or prognostic evaluation of certain tumors, including the treatment of ER-related disorders (such as ER-related cancers such as breast cancer including metastatic brain tumors).

[0168] Method of treatment For example, in some embodiments, this disclosure provides a method of treating a particular condition in a subject having an ER-related disease, disorder, or condition. For example, in some embodiments, this disclosure provides a method of treating an ER-related disorder in a subject who has been determined or suspected to have a brain metastasis. In some embodiments, the subject has developed a brain metastasis related to an ER-related cancer, such as breast cancer, or a mutation in the estrogen receptor.

[0169] In some embodiments, this disclosure provides a method of treating metastatic breast cancer in a subject, comprising administering to the subject a complete estrogen receptor antagonist, where the subject has been previously treated with a selective estrogen receptor modulator. In some embodiments, the selective estrogen receptor modulator is selected from tamoxifen, endoxifen, raloxifene, toremifene, lasofoxifene, and ospemifene.

[0170] In some embodiments, this disclosure provides a method of treating cancer in a subject afflicted with an ER-related cancer, comprising administering to the subject a composition that delivers a complete estrogen receptor antagonist to the subject's brain, where the subject has been determined or suspected to have a brain metastasis.

[0171] In some embodiments, this disclosure provides a method of treating a subject's cancer with a complete estrogen receptor antagonist, comprising administering to the subject a complete estrogen receptor antagonist, where the subject has been determined or suspected to have a brain metastasis.

[0172] In some embodiments, the present disclosure provides a method of treating ER-related cancer by administering to a population of subjects afflicted with brain metastases a composition comprising a complete estrogen receptor antagonist, whereby the brain metastases are on average reduced or eliminated.

[0173] In some embodiments, the ER-related disorder is cancer. In some embodiments, the ER-related disorder is breast cancer. In some embodiments, the subject afflicted with the ER-related disorder has developed brain metastases.

[0174] In some embodiments, the present disclosure provides a method of preventing metastatic spread of cancer to the brain of a subject, the method comprising administering compound 1:

Chemical formula

[0175] Detection method The present disclosure further encompasses the insight that a radiolabeled version of certain compounds described herein, such as any of the compounds described herein, is useful for the detection of certain ER-related diseases, disorders, and conditions. For example, an ER-related tumor can be detected, e.g., via PET, using a compound described herein in which a non-radioactive fluorine atom is replaced with 18 F. In some embodiments, the present disclosure provides a method of detecting an ER-related disorder in a subject, wherein the subject has been determined or is suspected of having brain metastases. That is, in some embodiments, the present disclosure provides a method of detecting ER-related metastases in the brain.

[0176] Characterization method The present disclosure further encompasses the insight that the effectiveness of certain compounds described herein is evaluated by in vitro and in vivo model assays. For example, the specific compounds described herein are characterized according to any of the model assays described herein, including, for example, ERα protein level assays, MCF-7 cell line assays, Ishikawa cell line assays (measuring specific mutants including wild-type ER and mutants lacking the AF1 and / or AF2 domains), and rodent uterine weight gain assays. Further, in some embodiments, the compounds described herein exhibit complete estrogen receptor antagonism when characterized by any reference assay, as the term is defined herein.

[0177] Administration The present disclosure provides a method of treating a subject suffering from an ER-related disorder, wherein the subject has developed or is suspected of developing brain metastases, the method comprising administering a composition comprising a complete estrogen receptor antagonist. In some embodiments, the composition comprises a complete estrogen receptor antagonist and a pharmaceutically acceptable excipient, carrier, or diluent. The foregoing composition may be administered orally, parenterally, by inhalation or nasal spray, topically (e.g., by powder, ointment, or instillation), rectally, intraorally, intravaginally, intraperitoneally, intracapsularly, or via an implanted reservoir, depending on the severity of the condition being treated. Preferably, the composition is administered orally, intraperitoneally, or intravenously. In certain embodiments, the compounds provided are administered orally or parenterally at dosage levels 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.

[0178] 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, the active compound may be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also, as is normal practice, contain additional substances other than inert diluents, for example lubricants such as magnesium stearate and microcrystalline cellulose and other tablet excipients. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0179] 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 pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and / or i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. The active compound may also be in microencapsulated form using one or more of the excipients described above.

[0180] Solid compositions of the same kind may also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose, i.e., milk sugar, and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coating agents and shell agents such as enteric coatings (i.e., buffering agents) and other coatings well known in pharmaceutical formulation technology. These may optionally contain opacifying agents and may also be compositions that release only or preferentially the active ingredient(s) in a particular part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0181] 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, 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 oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

[0182] Alternatively, the pharmaceutically 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., rectal or vaginal), and thus dissolve in the rectal or vaginal cavity to release the active compound. Such materials include cocoa butter, suppository wax (e.g., beeswax), and polyethylene glycol.

[0183] One of ordinary skill in the art will readily understand how the therapeutically effective dose determined for an animal is converted to the corresponding human equivalent dose. Thus, one of ordinary skill in the art will understand that, for example, by using the tables provided by Nair & Jacob, J. Basic Clin. Pharm., 7(2):27-31(2016), suitable doses in humans can be determined using specific provided data regarding an animal (e.g., a mouse).

[0184] The present disclosure provides a dosing regimen in which the compounds reported herein are administered according to levels and / or regimens corresponding to those exemplified herein for Compound 1 (see, e.g., Example 4). That is, the dose (i.e., the composition optionally including additional pharmaceutically acceptable excipients) refers to a specific ratio of the weight of the compound per kilogram of the subject. For example, a dose of 3 mg / kg refers to a composition optionally including a pharmaceutically acceptable excipient, and the compound is administered to the subject in an amount of 3 mg per kilogram of the subject's body weight. It is understood that the weight of the compound is determined according to the weight of the free base of the compound (e.g., if the compound is a salt, the corresponding weight of the free base of the compound is used to determine the amount of the compound in the dose). Thus, in some embodiments, a human subject is provided with a dose corresponding to 3 mg / kg to 30 mg / kg in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / kg in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 3 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 5 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 10 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 15 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 20 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 25 mg / kg or more in a mouse. In some embodiments, a human subject is provided with a dose corresponding to 30 mg / kg or more in a mouse.

[0185] In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a unit dosage form. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered in the form of a capsule. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered in the form of a tablet. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a suspension. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a solution.

[0186] In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a once-daily (QD) dosage. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a twice-daily (BID) dosage. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered every other day (QOD). In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a weekly (QW) dosage. In some embodiments, a composition comprising a complete estrogen receptor antagonist is administered as a monthly (Q4W) dosage.

[0187] The present disclosure also encompasses the recognition that Compound 1 can be advantageously used to treat metastatic cancer, such as cancer that has spread to the brain, bone, lung, liver, or central nervous system. As exemplified in the table below, Compound 1 can cross the blood-brain barrier when administered at a single oral dose of 300 mg / kg. Other estrogen receptor antagonists, such as fulvestrant, cannot cross the blood-brain barrier in similar amounts. [Table 1]

[0188] Combination Therapy This disclosure encompasses the recognition that certain combinations of agents can be beneficially used to fully antagonize estrogen receptors. Accordingly, in some embodiments, this disclosure provides methods of treating a subject afflicted with an ER-related disorder (e.g., cancer or breast cancer) that include administering a complete estrogen receptor antagonist and an anti-cancer agent. For example, in some embodiments, the anti-cancer agent is a CDK4 / 6 inhibitor, a PI3KCA inhibitor, or an mTOR inhibitor.

[0189] In some embodiments, this disclosure provides methods of treating a patient or subject afflicted with cancer, the method including administering a complete estrogen receptor antagonist, wherein the anti-cancer agent is a CDK4 / 6 inhibitor (i.e., inhibits one or both of CDK4 and CDK6). In some embodiments, the secondary agent is a CDK4 / 6 inhibitor selected from palbociclib, ribociclib, abemaciclib, lerociclib, trilaciclib, and SHR6390. In some embodiments, the CDK4 / 6 inhibitor is palbociclib. 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 lerociclib. In some embodiments, the CDK4 / 6 inhibitor is trilaciclib. In some embodiments, the CDK4 / 6 inhibitor is SHR6390.

[0190] In some embodiments, this disclosure provides methods of treating a patient or subject afflicted with cancer, the method including administering a complete estrogen receptor antagonist and an anti-cancer agent, wherein the secondary 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.

[0191] In some embodiments, the present disclosure provides a method of treating a patient or subject suffering from cancer, the method comprising administering a complete estrogen receptor antagonist and an anti-cancer agent, the anti-cancer 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.

[0192] It is understood that the administration of the complete estrogen receptor antagonist and the anti-cancer agent described herein can be administered simultaneously or separately. For example, in some embodiments, the complete estrogen receptor antagonist and the anti-cancer agent are administered simultaneously. In some embodiments, the anti-cancer agent is administered prior to the administration of the complete estrogen receptor antagonist. In some embodiments, the anti-cancer agent is administered after the administration of the complete estrogen receptor antagonist. Exemplary embodiments Embodiment 1. A method of treating cancer, comprising: administering to a subject suffering from ER-related cancer a composition that delivers a complete estrogen receptor antagonist to the subject's brain, wherein the subject has been determined or is suspected to have brain metastases. Embodiment 2. The method of Embodiment 1, wherein the complete estrogen receptor antagonist is GDC-9545. Embodiment 3. The method of Embodiment 1, wherein the complete estrogen receptor antagonist is SAR439859. Embodiment 4. The method of Embodiment 1, wherein the complete estrogen receptor antagonist is AZD9833. Embodiment 5. The complete estrogen antagonist is a compound of Formula I

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0193] Illustration The examples provided in this specification demonstrate and support certain aspects of the present disclosure, but are not intended to limit any of the claims. Unless specifically presented in the past tense, including in an example is not intended to imply that the described operations have been completed or even executed. The following non-limiting examples are provided to further illustrate certain teachings provided by the present disclosure. Those skilled in the art will understand that, in light of this application, various changes can be made in the specific embodiments illustrated in these examples without departing from the spirit and scope of the teachings of the present invention.

[0194] In the following examples, the following abbreviations can be used. (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 (dimethyl sulfoxide); EA (ethyl acetate); ee (enantiomeric excess); 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 (minute or minutes); NMR (nuclear magnetic resonance); Pd / C (palladium on carbon); PPh3O (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).

[0195] Example 1: Synthesis of Compound 1 The complete synthesis of Compound 1 includes PCT Publication No. WO 2017 / 059139 (Compound B, or (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), which is incorporated herein by reference and repeated below.

[0196] Preparation of 4-((1-propylazetidin-3-yl)oxy)benzaldehyde

Chemical formula

Chemical formula

[0197] Step 2.1 - Preparation of 1-propylazetidin-3-ol

Chemical Structure

[0198] (R)-1-(1H-Indol-3-yl)-N-((R)-1-phenylethyl)propan-2-amine Preparation:

Chemical formula

[0199] 1 1H 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. LCMS: ES+ [M+H] 279.0.

[0200] Preparation of (2R)-1-(1H-indol-3-yl)propan-2-amine

Chemical Structure

[0201] 1 1H 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). LCMS: ES+ [M+H]+ 174.9.

[0202] Preparation of 2-fluoro-2-methylpropanol

Chemical Structure

[0203] Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate

Chemical formula

[0204] 11H NMR (300 MHz, CDCl3) δ 1.46 (d, J = 20.4 Hz, 6H), 4.41 (d, J = 18.6 Hz, 2H). 19 19F NMR (282 MHz, CDCl3) δ -147.1, -74.5.

[0205] Preparation of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine: [Chemical Structure] Compound 2-fluoro-2-methylpropyl trifluoromethanesulfonate (9.587 g, 42.8 mmol, 1.1 eq) (solution in DCM, 16 wt% 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 h. After 3 h, LCMS indicated that a small amount of indoleamine starting material was still present. TLC (10% MeOH / DCM) indicated that the triflate (Rf = 0.54) had been consumed. NMR of the unused triflate SM (286-30) indicated that the triflate did not decompose overnight, so another 0.1 eq (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 indicated that the reaction was complete and TLC (10% MeOH / DCM) showed one spot (Rf = 0.24) (TLC using 50% EA / Hex, one streak spot Rf ≤ 0.12, another spot at Rf = 0). EtOAc (50 mL) was added and the solution was washed with NaHCO3 (2 × 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 product (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-tailed peak. The 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.

[0206] 11H 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 19F NMR (282 MHz, CDCl3) δ -144.2. m / z: ES+ [M + H] 249.0.

[0207] 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). A molecular sieve was added and the solution was stirred at 80 °C for 8 h under N2 in the dark. 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), filtered through a syringe filter, and then purified via preparative LC (18 min with 40 - 90% ACN:H2O followed by 7 min with 90% ACN of uniform concentration). The 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)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole as a white powder.

[0208] Example 2: Synthesis of Compounds 2 - 4 The specific synthetic methods for each of Compounds 2, 3, and 4 can be found in WO2016 / 097072, which is incorporated herein by reference.

[0209] Example 3: Synthesis of Compounds 5 to 5b Specific synthetic methods for each of Compounds 5, 5a, and 5b can be found in WO2019 / 245974, which is incorporated herein by reference.

[0210] Example 4: In Vivo Evaluation of Compound 1 Model Development Intracranial Tumor Implantation For intracranial inoculation of 8 NMRI nude mice, patient-derived tumors with subcutaneous ST941 tumors were used.

[0211] Treatment was carried out according to Minerva imaging SOP18.1.2. Fresh tissue and single cell suspensions from intracranial injections.

[0212] Briefly, subcutaneous ST941 PDX tumors grown in NMRI nude mice were harvested. The tumors were cut into small pieces and enzymatically digested to obtain a single cell suspension. The digestion was stopped, filtered through a 100 μm filter, washed with PBS, and resuspended in PBS. The viability of the tumor cells was checked by trypan blue staining, and the final concentration was 20 million viable cells / mL. The cells were maintained on ice until inoculation.

[0213] The mice were anesthetized with hypnorm / midazolam (1 mL / 100 g body weight) and placed in a stereotaxic frame to fix the head. The scalp was incised longitudinally to expose the skull. Using a microdrill, a hole was made in the skull 1.5 mm to the right of the sagittal suture and 1.0 mm behind the coronal suture. 10 μl of the cell suspension (200,000 cells) was injected at a depth of 2 - 2.5 mm at a rate of 60 nl / sec using a 100 μl syringe with a 25-gauge needle placed on a microinfusion pump. The needle was left in place for 3 minutes and then withdrawn. Bupivacain (0.2 mg / 100 g body weight) and Lidocain (1 mg / 100 g body weight) were administered to the incision site for local anesthesia, and the skin was closed with sutures.

[0214] The mice were marked for identification, returned to the cage, and monitored here until they fully recovered from anesthesia.

[0215] MR Imaging, Tumor Monitoring and Euthanasia Tumor growth was monitored by T2-weighted MR imaging. The first scan was performed 1 week after inoculation and then twice a week thereafter. Body weight and scoring were performed at least twice a week. When a weight loss of ≥10% was observed, daily scoring of the animals was performed.

[0216] When the humane endpoints were met, the mice were euthanized by cervical dislocation.

[0217] Efficacy Intracranial Tumor Implantation For intracranial inoculation of 54 thymus-deficient nude mice, tumors from parental origin with subcutaneous ST941 tumors were used.

[0218] The treatment was carried out according to Minerva Imaging SOP18.1.2. Fresh tissue and single cell suspension from intracranial injection. See the above brief description for reference.

[0219] MR Imaging for Inclusion Based on MR imaging, the mice were continuously enrolled in the study. The first MR imaging session was started 1 - 3 weeks after transplantation according to the model establishment results. MR imaging was then performed twice a week until inclusion.

[0220] When the tumor volume reached 2 - 5 mm 3 the mice were enrolled in the study. Inclusion was on the day after the actual MR imaging. The first 40 mice that met the inclusion criteria were randomized into 5 groups. See Table 1. The mice were randomized so that all groups had the same mean tumor volume at the time of enrollment. [Table 2]

[0221] Estrogen supplementation and depletion Animals were given 17β - estradiol in the drinking water starting 2 days before intracranial inoculation. When the animals were incorporated into the study, estrogen was depleted and administration was started 2 days later.

[0222] Therapy, body weight monitoring and MR imaging Mice were treated according to Table 1 (above). Therapy was given by forced oral administration between 9:00 and 10:00 am daily. The first dose was administered 2 days after incorporation.

[0223] During therapy, body weight and visual assessments were monitored daily. Animals were scored three times a week according to the following table and more frequently if a 5% weight loss was evident.

Table 3

[0224] Tumor growth was monitored by MR on days 5, 10, 15, 20 and 25 compared to the day of incorporation.

[0225] Post - therapy monitoring and euthanasia Body weight monitoring and scoring were performed three times a week for 1 week after treatment and daily if a 5% weight loss was observed. Mice were euthanized by cervical dislocation when humane endpoints were met.

[0226] Tissue preservation The brain with tumor tissue was excised, fixed in 10% neutral buffered formalin for 48 hours (fixation ratio should be at least 1:20), and transferred to 70% ethanol. Samples were stored at 4 °C until shipment.

[0227] Figure 1A shows the volume (mm by MRI at the time of incorporation in ST941 brain metastases for each mouse within each group A - E3 ) is a scatter plot for measuring

[0228] Figure 1B is a scatter plot for measuring body weight (g) by MRI at the time of incorporation in ST941 brain metastases for each mouse within each of groups A to E.

[0229] Figure 2 is a scatter plot for measuring the average tumor volume for each of groups A to E over time.

[0230] Figure 3A is a scatter plot for measuring the average body weight for each of groups A to E over time.

[0231] Figure 3B is a scatter plot for measuring the rate of change in the average body weight for each of groups A to E over time.

[0232] Figure 4 is a Kaplan-Meier plot showing the proportion of mice surviving within each of groups A to E over time. The tick marks indicate censored data reflecting that the mice were enrolled on different days.

[0233] Example 5: Assay of Estrogen Receptor Protein Level This example describes the evaluation of various compounds (ARN-810, AZD9496, Compound 1, endoxifen, and fulvestrant) at the ERα protein level 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 plate and incubated for at least 24 hours in phenol red-free medium containing 5% charcoal dextran-treated fetal bovine serum (treated FBS) (HyClone). Cells were treated with 300 nM anti-estrogen in serum-free medium for 4 hours, and subsequently, the lysate was lysed with RIPA buffer (ThermoFisher Scientific) supplemented with protease and phosphatase inhibitors. Total protein extracts were separated on a 10% SDS-PAGE TGX gel and transferred to a nitrocellulose membrane (BioRad). The blot was incubated with either a mouse monoclonal anti-ERα, D12 (#sc-8005, SantaCruz Biotechnology) or SP1 (#MA5-14501, ThermoFisher Scientific). A β-actin monoclonal antibody (#MA5-15739 or #MA5-16410 (ThermoFisher Scientific) or #sc-47778 (SantaCruz Biotechnology)) was used as a loading control. The blot was incubated with an appropriate secondary antibody conjugated to horseradish peroxidase (ThermoFisher Scientific). Signals were detected with Super Signal Femto chemiluminescent reagent (ThermoFisher Scientific). The results are shown in Figure 6. As shown in the figure, all compounds except endoxifen showed significant ability to reduce ER protein levels in most cell lines, and Compound 1 and fulvestrant were the most effective in reducing ER protein levels and showed comparable activity in this regard.

[0234] Example 6: Cell Proliferation Assay This example describes human ER +An assay is described for evaluating the effect of a test compound on the human MCF-7 cell line, which is a breast cancer cell line. Specifically, 1000 MCF-7 cells per well (Cheryl Walker, Baylor College of Medicine) were plated in a 96-well plate in phenol red-free medium (ThermoFisher Scientific) containing 5% treated FBS. After at least 4 hours, the cells were treated with anti-estrogen, and the medium was diluted to 2.5% treated FBS in the presence of 100 pM E2 for 6 - 8 days. Proliferation was measured using the CyQuant fluorescent DNA binding dye kit (ThermoFisher Scientific) with a 1:200 GR dye and fluorescence readings at 485 nm excitation and 538 nm.

[0235] Example 7: Estrogen Receptor Antagonism Analysis Transient Transfection of Estrogen Receptors and Variants In this example, a study is described in which a specific estrogen receptor construct was transfected into Ishikawa cells, which are a human endometrial cancer cell line, and endogenous alkaline phosphatase was assayed. 15,000 Ishikawa cells per well were plated in a 96-well plate in phenol red medium containing 5% treated FBS. At the time of plating, the cells in each well were transiently transfected with 75 - 100 ng of the estrogen receptor construct (or empty vector, pSG5) using Lipofectamine LTX (ThermoFisher Scientific). After approximately 4 hours, the cells were treated with the indicated amount of anti-estrogen (in the absence of E2) or 500 pM E2 (Figure 13), and the medium was diluted to 2.5% treated FBS. The cells were incubated for 3 days, the medium was removed, and the plates were frozen at -80°C. The thawed plates were incubated with p-nitrophenyl phosphate (ThermoFisher Scientific), which is a chromogenic substrate for AP and thus reveals the AP activity level. After 40 - 80 minutes at 40°C, the absorbance was read at 405 nm.

[0236] Compound 1 has ER antagonist activity and no agonist activity and is active. 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 17-β-estradiol (E2) (antagonist mode). The results are shown in Figure 5A (agonist mode) and Figure 5B (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.

[0237] Certain mutant ERs increase ligand-independent ER activity Wild-type ER (HEGO), empty vector (pSG5), or the indicated LBD mutant ERs were transiently transfected into Ishikawa cells as described above. Only the empty vector was treated with 500 pM 17-β-estradiol (E2). After 72 hours, the cells were assayed for AP activity. The results are shown in Figure 13. Bars represent the mean absorbance + standard error at 405 nm from triplicate wells. As shown in the figure, various tested ER mutants were observed to be "activated mutants" in that they showed more activity than wild-type ER in the absence of ligand.

[0238] Activation function 1 (AF1) is required for ligand-independent activity observed in certain ER mutants. The AF1 wild-type ER (HEGO, AA1-595), empty vector (pSG5), or ER lacking activation domain 2 ("AF2") (AA1-282) or lacking activation domain 1 ("AF1") (AA178-595, with or without the Y537S mutation) was transiently transfected into Ishikawa cells as described above. After 72 hours, the cells were assayed for AP activity. Bars represent the mean absorbance at 405 nm + standard error from quadruplicate wells. As shown in the figure, F1AF1 is required for ligand-independent ER activity observed when the ER is truncated (ΔAF2), even in the presence of the activating Y537S mutation (ΔAF1 / Y5372).

[0239] 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 assayed for activity in the presence of compound 1 or fulvestrant. The results are shown in FIGS. 9A-9F. Points represent the mean AP activity + / − standard error normalized to vehicle from duplicate wells. The dose-response curves for compound 1 and fulvestrant were fitted using the least squares fitting method, and pIC 50 (-Log IC 50 ) was calculated using a variable slope sigmoid dose-response model. The line represents the normalized AP activity of the endogenous receptor (transfected with the empty vector (pSG5)). As shown in the figure, compound 1 inhibited the activity of each ligand-independent ER mutant with an IC50 comparable to that of fulvestrant.

[0240] A particular clinical candidate cannot inhibit the activity of ligand-independent ER mutants The wild-type ER or the expressed ER variants were transiently transfected into Ishikawa cells as described above and assayed for activity in the presence of compound 1 or fulvestrant, compared to endoxifen, RAD-1901, ARN-810 (GDC-0810), or AZD-9496 (results are shown in FIGS. 12A - 12B, where compound 1 and fulvestrant are compared to endoxifen and RAD-1901 in FIGS. 12A and 12B, or to ARN-810 (GDC-0810) and AZD-9496 in FIGS. 12C - 12D. Points represent the mean absorbance + standard error at 405 nm from triplicate wells. Lines represent the AP activity of the endogenous receptor (transfected with empty vector (pSG5)). As shown, none of endoxifen, RAD-1901, ARN-810 (GDC-0810), or AZD-9496 were able to inhibit the activity of the ligand-independent ER variant as compound 1 and fulvestrant do.

[0241] Example 8: Xenograft Analysis of Compound 1 in ST941 PDX Brain Metastases This example describes the effect of compound 1 on tumors derived from a patient-derived xenograft (PDX) model ST941 directly transplanted into the mouse brain.

[0242] In particular, this example describes the effect of compound 1 on estrogen receptor (ER)-positive tumors directly transplanted into the brains of mice using patient-derived human breast cancer cells containing an activating mutation of the estrogen receptor, the Y537SESR1 mutation.

[0243] Comparative regimens included ovariectomy + vehicle, fulvestrant, tamoxifen, compound 1, and combinations of ribociclib and compound 1 monotherapy versus vehicle in the ST941 intracranial breast cancer brain metastasis model.

[0244] Protocol and Materials In Vitro Treatments For intracranial inoculation of 64 thymus-deficient nude mice, tumors derived from the parental line with subcutaneous ST941 tumors were used. The procedure was carried out according to the following method. Subcutaneous ST941 PDX tumors grown in NMRI nude mice were harvested. The tumors were cut into small pieces, enzymatically digested to obtain a single-cell suspension. The digestion was stopped, filtered through a 100-μm filter, washed with PBS, and resuspended in PBS. The viability of the tumor cells was checked by trypan blue staining, and the cells were suspended according to the following table. The cells were maintained on ice until inoculation.

Table 4

[0245] In Vivo Treatment

[0246] Eight female NMRI nude mice (ordered by age, approximately 6 weeks old within a one-week time frame) were used for model development.

[0247] Tumor Transplantation and Estrogen Administration Tumors were prepared and transplanted by the following method.

[0248] The animals were anesthetized with hypnorm / midazolam (1 mL / 100 g body weight) and placed in a stereotaxic frame to fix the head. The scalp was incised longitudinally to expose the skull. Using a microdrill, a hole was made in the skull 1.5 mm to the right of the sagittal suture and 1.0 mm behind the coronal suture. 10 μl of the cell suspension (200,000 cells) was injected at a rate of 60 nl / second to a depth of 2 - 2.5 mm using a 100-μl syringe with a 25-gauge needle placed on a microinfusion pump. The needle was left in place for 3 minutes and then withdrawn. Bupivacain (0.2 mg / 100 g body weight) and Lidocain (1 mg / 100 g body weight) were administered to the incision site for local anesthesia, and the skin was closed with sutures.

[0249] The animals were chipped for identification, returned to their cages, and monitored here until fully recovered from anesthesia.

[0250] Mice were tracked by MR imaging and registered when the tumor volume reached 2 - 5 mm 3 Incorporation was performed either 1 or 2 days after MR imaging when an appropriate tumor size was confirmed. Mice were randomized so that all groups had approximately equal tumor volumes at the time of registration. MR imaging was performed according to the following method:

[0251] The first MR imaging session was started 2 weeks after transplantation. Once the tumors were established, MR imaging was performed weekly or twice a week. Animals were registered when the tumor volume reached 2 - 5 mm3. Incorporation was on the day after the actual MR imaging. Animals that met the registration criteria were stratified into 6 groups according to Table 8 - 1. Animals were stratified so that all groups had the same mean tumor volume at the time of registration.

[0252] Estrogen supplement was provided via 17β - estradiol in the drinking water starting 2 days before intracranial inoculation. Once a sufficient tumor volume was reached, estrogen supplement was discontinued for each animal. Administration to each mouse was started 2 days after registration.

[0253] Table 8 - 1 summarizes the treatments applied to different groups.

Table 5

[0254] Animals were treated according to Table 8 - 1. Therapy by forced oral administration was performed between 9:00 am and 10:00 am every day. The first dose was administered 2 days after incorporation.

[0255] Compound 1 was dissolved in DMSO to form a clear solution, thereby preparing a Compound 1 formulation. This solution was transferred to 0.5% CMC in Millipore water such that the final concentration of DMSO was less than 5% v / v. During the addition of the DMSO solution, Compound 1 precipitated to form a finely divided suspension within the vehicle. This suspension settled over time. Prior to administering the test substance, sonication and stirring were performed.

[0256] Ribociclib was formulated in an amount of 15 mg / mL. 100 mg of ribociclib was suspended in 6.66 mL of vehicle to reach a final concentration of 15 mg / mL.

[0257] The combined formulation of Compound 1 and ribociclib was prepared by mixing 1:1 mL of the solutions prior to administration.

[0258] Ovariectomy

[0259] The animals in Group B had their estrogen depleted by ovariectomy on the day of incorporation. Ovariectomy was performed according to the following method:

[0260] The animals were anesthetized (in ambient air supplemented with 100% O2 at a ratio of approximately 4:1 with sevoflurane at 2 - 4%) and placed on a heating pad for recovery. Carprofen (5 mg / kg) was administered subcutaneously before the surgery and daily for 3 days after the surgery.

[0261] The area around the incision was disinfected with iodine. A 1 cm incision was made along the midline, and the muscle layer was separated from the skin using curved scissors. A small incision 1 cm outside the midline was made through the muscle to reach the abdominal cavity. The white adipose tissue surrounding the ovaries was removed using forceps. The proximal blood vessels and uterine horns were ligated using monofilament suture thread. The ovaries were removed using small scissors, and the remaining tissue was returned into the abdominal cavity.

[0262] The muscle layer wound and skin layer wound were separately closed with absorbable sutures. This procedure was repeated on the opposite side.

[0263] Therapy, body weight monitoring, and MR imaging

[0264] During therapy, body weight and visual evaluation were monitored daily. Animals were scored three times a week according to the following table and more frequently if a 10% weight loss was evident.

Table 6

[0265] Tumor growth was monitored by MR on days 5, 10, 15, 20, and 25 compared to the implantation day.

[0266] When humane endpoints were met, animals were euthanized by cervical dislocation.

[0267] Tissue preservation

[0268] When animals reached the humane endpoints, the following sampling was performed. · Blood (plasma sample) · Four hours after the last administration, whole blood was collected by cardiac puncture and transferred to an EDTA tube. The sample was centrifuged at 2000 × g for 10 minutes at 4°C. Plasma was transferred to 2 mL round-bottom Eppendorf tubes and stored at -80°C. · The brain with tumor tissue was excised. · Four brains from each group were stored in 10% neutral buffered formalin for 48 hours (fixation ratio should be at least 1:20) and transferred to 70% ethanol. Samples were stored at 4°C until shipment. · Four brains from each group were snap-frozen and stored at -80°C until shipment.

[0269] Results The results of the xenograft studies described herein are provided in FIGS. 20A-27. These results show that mice provided with Compound 1 alone and in combination with ribocyclib showed improved survival rates compared to other therapies including fulvestrant. For example, nearly all mice administered Compound 1 survived throughout the 100-day study period, while at least half of the mice administered fulvestrant died within less than 30 days (see FIGS. 21, 24, and 25). Thus, the present disclosure encompasses the insight that Compound 1 exhibits improved properties over fulvestrant as a complete estrogen receptor antagonist. Tamoxifen was the only other tested therapy that slowed the change in tumor volume, but not to the same extent as Compound 1 (see FIG. 22B). However, animals treated with tamoxifen (Group D) began to show an increase in tumor volume after 100 days (see, e.g., FIGS. 22A, 22B, 24, 25), and the survival probability decreased after two members of Group D died (see FIG. 21). The present disclosure also encompasses the insight that Compound 1 exhibits improved properties over tamoxifen in its ability to treat brain metastases. While Compound 1 is a complete estrogen receptor antagonist, tamoxifen, as described above, is a partial estrogen receptor agonist, among other reasons, which makes Compound 1 superior to tamoxifen.

[0270] FIG. 20A is a scatter plot measuring the body weight of each animal at time 0.

[0271] FIG. 20B is a scatter plot measuring the tumor burden of each animal at time 0.

[0272] FIG. 21 is a Kaplan-Meier plot showing the percentage of surviving mice in each group over time. The tick marks indicate censored data reflecting that the mice were enrolled on different days. “Cmpd1” refers to “Compound 1” described herein.

[0273] Figure 22A is a scatter plot measuring the average tumor volume over time for each group. "Cmpd1" refers to "Compound 1" described herein.

[0274] Figure 22B is a scatter plot measuring the rate of change in tumor volume over time for each group. "Cmpd1" refers to "Compound 1" described herein.

[0275] Figure 23A is a waterfall plot measuring the rate of change in tumor volume for each group.

[0276] Figure 23B is a zoomed-in view of the waterfall plot of Figure 23A showing the decrease in tumor volume size for a particular group.

[0277] Figure 24 is a scatter plot measuring the average tumor volume of individual animals in each group over time.

[0278] Figure 25 is a scatter plot measuring the rate of change in tumor volume over time for individual animals in each group.

[0279] Figure 26 is a scatter plot measuring the body weight of animals over time for each group.

[0280] Figure 27 is a scatter plot measuring the rate of change in the change in body weight of animals over time for each group.

[0281] The above is a description of specific non-limiting embodiments of the subject matter described herein. Therefore, it should be understood that the embodiments described herein merely exemplify the subject matter reported internally. References to the details of the exemplified embodiments are not intended to limit the claims that list features regarded as essential in themselves.

[0282] The claimed systems and methods are intended to embrace variations and adaptations developed using information from the embodiments described herein. Adaptations, modifications, or both of the systems and methods described herein may be made by those of ordinary skill in the relevant art.

[0283] Where a system is described as having, including, or comprising a particular component, or a method is described as having, including, or comprising a particular step, it is also contemplated that there are systems embraced by the subject matter of the invention that consist essentially of, or consist of, the recited components, and that there are methods embraced by the subject matter of the invention that consist essentially of, or consist of, the recited process steps.

[0284] It should be understood that the order of steps or the order of performance of particular operations is not critical so long as any embodiment of the subject matter described herein is operable. Further, two or more steps or operations may be performed concurrently. The present invention provides, for example, the following items. (Item 1) A method for treating cancer, comprising administering to a subject suffering from ER-related cancer a composition that delivers a complete estrogen receptor antagonist to the subject's brain, wherein the subject has been determined to have or is suspected of having brain metastases. (Item 2) The method according to Item 1, wherein the complete estrogen receptor antagonist is GDC-9545. (Item 3) The method according to Item 1, wherein the complete estrogen receptor antagonist is SAR439859. (Item 4) The method according to Item 1, wherein the complete estrogen receptor antagonist is AZD9833. (Item 5) The complete estrogen antagonist is a compound of formula I, or a pharmaceutically acceptable salt thereof,

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Claims

1. A pharmaceutical composition for the treatment of ER-related cancer in a subject, wherein the subject has been determined or suspected to have brain metastases, and the pharmaceutical composition is 【Chemical 74】 or a pharmaceutically acceptable salt thereof, the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the treatment further comprises administering a second anti-cancer agent to the subject.

3. The pharmaceutical composition according to claim 2, wherein the second anti-cancer agent is a CDK4 / 6 inhibitor, a PI3KCA inhibitor, or an mTOR inhibitor.

4. The pharmaceutical composition according to claim 3, wherein the second anti-cancer agent is a CDK4 / 6 inhibitor.

5. The pharmaceutical composition according to claim 4, wherein the CDK4 / 6 inhibitor is selected from palbociclib, ribociclib, abemaciclib, lerociclib, and trilaciclib.

6. The pharmaceutical composition according to claim 5, wherein the CDK4 / 6 inhibitor is selected from ribociclib, palbociclib, and abemaciclib.

7. The pharmaceutical composition according to claim 6, wherein the CDK4 / 6 inhibitor is ribociclib.

8. The pharmaceutical composition according to claim 3, wherein the second anti-cancer agent is a PIK3CA inhibitor.

9. The pharmaceutical composition according to claim 8, wherein the PIK3CA inhibitor is selected from alpelisib and taselisib.

10. The pharmaceutical composition according to claim 3, wherein the anti-cancer agent is an mTOR inhibitor.

11. The pharmaceutical composition according to claim 10, wherein the mTOR inhibitor is selected from sirolimus, temsirolimus, and everolimus.

12. The pharmaceutical composition according to claim 1, wherein the subject has been previously treated with a selective estrogen receptor modulator.

13. The pharmaceutical composition according to claim 12, wherein the selective estrogen receptor modulator is an estrogen receptor agonist or a partial estrogen receptor agonist.

14. The pharmaceutical composition according to claim 13, wherein the estrogen receptor agonist or partial estrogen receptor agonist is tamoxifen, raloxifene, or toremifene.

15. For the treatment of ER-related cancer, 【Chemical 74】 or a pharmaceutical composition comprising a pharmaceutically acceptable salt thereof, wherein the treatment is The pharmaceutical composition, which is administered to a population of subjects suffering from brain metastases, such that, on average, the brain metastases are reduced or eliminated. [

16. ] A pharmaceutical composition for preventing metastatic spread of ER-related cancer to the brain of a subject, wherein the pharmaceutical composition comprises Compound 1: 【Chemical 74】 or a pharmaceutically acceptable salt thereof.

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