Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the estrogen receptor (ER) plays important roles in various cancers, including breast cancers. A variety of treatments have been developed to target the estrogen receptor and/or its activities.
• estrogen receptors include those coded for by both wild-type and mutant versions (e.g., those containing activating mutations) of the gene encoding Estrogen Receptor-alpha (ERa), Estrogen Receptor 1 (ESR1).
• Selective estrogen receptor modulators (SERMs) or degraders (SERDs) are a particularly useful or promising tools for such therapy.
• the estrogen receptor is a tripartite protein comprising two distinct transcriptional activation functions (AF1 and AF2). Complete anti-estrogen activity requires inactivation of both AF1 and AF2.
• Activating mutations in the gene that codes for estrogen receptor 1 allows for activation of both AF1 and AF2 even in the absence of estrogen.
• the present disclosure provides a method of treating a patient or subject suffering from a cancer associated with the estrogen receptor (ER) comprising administration of an estrogen receptor antagonist and a CDK4/6 inhibitor.
• ER estrogen receptor
• the present disclosure provides a method of treating a patient or subject suffering from a cancer associated with the estrogen receptor (ER) comprising administration of an estrogen receptor antagonist and a PIK3CA inhibitor.
• ER estrogen receptor
• the present disclosure provides a method of treating a patient or subject suffering from a cancer associated with the estrogen receptor (ER) comprising administration of an estrogen receptor antagonist and an mTOR inhibitor.
• ER estrogen receptor
• the present disclosure provides a method of treating a patient or subject suffering from a cancer, wherein the cancer has metastasized to the brain, bones, lungs, or liver, comprising administrating to the patient or subject Compound 1 :
• the present disclosure provides an assay system for assessing compounds or compositions for inhibition of estrogen receptor activating function 1 (AF1) and/or activating function 2 (AF2).
• AF1 estrogen receptor activating function 1
• AF2 activating function 2
• the present disclosure provides a method for treating a patient suffering from a cancer, the method comprising oral administration of Compound 1.
• FIG. 1A is a scatter plot measuring percent of estrogenic response as a function of Log[M] for certain estrogen receptor antagonist compounds where no estrogen has been added.
• FIG. IB is a scatter plot measuring percent of estrogenic response as a function of Log[M] for certain estrogen receptor antagonist compounds where estrogen has been added.
• FIG. 2 is a chart measuring degradation of the estrogen receptor protein across multiple cell lines for multiple estrogen receptor antagonists.
• FIGs. 3A-3C are scatter plots illustrating the decreased percent of estrogen response for Compound 1 in combination with various CDK4/6 inhibitors.
• FIGs. 4A-4B are scatter plots illustrating the decreased percent of estrogen proliferation to MCF-7 cells when treated with Compound 1 in combination with a PIK3CA inhibitor.
• FIGs. 5A-5F are scatter plots for cell lines illustrating dose response of Compound 1 for AF1 inhibition with most common ESR1 mutations.
• FIG. 6 is a scatter plot illustrating change in tumor volume for varying doses of Compound 1.
• FIGs. 7A-7D are scatter plots measuring drug exposure (ng/ml) over time for mouse FIG. 7 A), rat (FIG. 7B), dog (FIG. 7C), and monkey (FIG. 7D).
• FIGs. 8A-8D are scatter plots illustrating the decreased in estrogen concentration across different cell lines.
• FIG. 9 is a bar graph illustrating that mutant ERs increase ligand-independent alkaline phosphatase activity (AP) in Ishikawa endometrial cancer cells.
• FIG. 10 is a bar graph illustrating that activation domain 1 (AF1) of the ER is required for AP activity.
• FIG. 11 is a reproduction of Figure 1A of Patel & Bihani Pharm & Therap 186:1, 2018, illustrating that mammals express two major isoforms of the ER, known as ERa and ERP, each of which is a member of the nuclear hormone receptor family.
• Aromatase inhibitors prevent ER signaling by inhibiting synthesis of estradiol
• SERMs prevent ER signaling by binding to ER and causing an inactive complex
• SERDs prevent ER signaling by causing degradation of ER.
• FIG. 12 is a reproduction of Figure 3 of Hewitt & Korach Endocrine Rev. 39:664-674 (June 12, 2018), illustrating variations in the basic mechanism of E2 response.
• the present disclosure provides, among other things, methods of treating patients or subjects suffering from a cancer related to the estrogen receptor, and mutations of the estrogen receptor, comprising administering an estrogen receptor antagonist.
• the estrogen receptor antagonist is Compound 1 :
• Administration typically refers to the administration of a composition to a subject or system, for example to achieve delivery of an agent that is, or is included in or otherwise delivered by, the composition.
• agent refers to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.).
• entity e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof
• phenomenon e.g., heat, electric current or field, magnetic force or field, etc.
• Antagonist may refer to an agent, or condition whose presence, level, degree, type, or form is associated with a decreased level or activity of a target.
• An antagonist may include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity.
• an antagonist may be a“direct antagonist” in that it binds directly to its target; in some embodiments, an antagonist may be an “indirect antagonist” in that it exerts its influence by means other than binding directly to its target; e.g., by interacting with a regulator of the target, so that the level or activity of the target is altered).
• an“antagonist” may be referred to as an“inhibitor”.
• Two events or entities are“associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
• a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc
• two or more entities are physically“associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
• two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
• Biological Sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein.
• a source of interest comprises an organism, such as an animal or human.
• a biological sample is or comprises biological tissue or fluid.
• a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc.
• a biological sample is or comprises cells obtained from an individual.
• obtained cells are or include cells from an individual from whom the sample is obtained.
• a sample is a“primary sample” obtained directly from a source of interest by any appropriate means.
• a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g, blood, lymph, feces etc.), etc.
• biopsy e.g., fine needle aspiration or tissue biopsy
• body fluid e.g, blood, lymph, feces etc.
• the term“sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample.
• Such a“processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
• Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
• the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
• “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
• combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
• Dosage form or unit dosage form may be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject.
• each such unit contains a predetermined quantity of active agent.
• such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
• 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 administration of multiple dosage forms.
• Dosing regimen or therapeutic regimen may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
• a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
• a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
• individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
• all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
• Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect.
• suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• oral administration and“administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.
• Parenteral The phrases“parenteral administration” and“administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.
• a patient refers to any organism to which a provided composition is or may be administered, e.g., for 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).
• a patient is a human.
• a patient or a subject is suffering from or susceptible to one or more disorders or conditions.
• a patient or subject displays one or more symptoms of a disorder or condition.
• a patient or subject has been diagnosed with one or more disorders or conditions.
• a patient or a subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
• composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers.
• the active agent is present in unit dose amounts appropriate for administration in a therapeutic regimen to a relevant subject (e.g., in amounts that have been demonstrated to show a statistically significant probability of achieving a predetermined therapeutic effect when administered), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.).
• comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
• composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion 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.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com 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;
• compositions that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
• Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences , 66: 1-19 (1977).
• pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group 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.
• nontoxic acid addition salts which are salts of an amino group 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.
• 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, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate
• Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
• pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
• Therapeutic agent in general refers to any agent that elicits a desired pharmacological effect when administered to an organism.
• an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population.
• the appropriate population may be a population of model organisms.
• an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc.
• a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• a“therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans.
• a“therapeutic agent” is an agent for which a medical prescription is required for administration to humans.
• Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
• Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
• treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
• therapeutically effective amount refers to an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
• a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
• the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
• the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder and/or condition.
• a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
• the estrogen receptor (“ER”) is involved in a variety of biological processes, relating, for example, to development of the female reproductive system, maintenance of bone mass, protection of cardiovascular and/or central nervous system components, etc (see, for example, Pearce & Jordan Crit. Rev. Onc/Hem 50:3, 2004; Heldring Phys. Rev. 87:905, 2007).
• ERa and ERP are a member of the nuclear hormone receptor family and has a structural organization as depicted in Figure 1A of Patel & Bihani Pharm & Therap 186: 1, 2018, reproduced in FIG. 11.
• Aromatase inhibitors prevent ER signaling by inhibiting synthesis of estradiol
• SERMs prevent ER signaling by binding to ER and causing an inactive complex
• SERDs prevent ER signaling by causing degradation of ER.
• ER“domains” labeled A-F
• Domain A/B found at the amino-terminal end of the ER protein, is the largest domain and includes one of the two so-called“transcriptional activation function” elements, AFl; AF2 is found in the E domain, which also includes the ligand-binding domain and element(s) that are believed to participate in ER dimerization and nuclear localization (see, for example, Hewitt & Korach Endocrine Rev 39:664, 2018).
• binding of ligand to the ligand-binding domain triggers a structural re-organization of alpha-helices within the E domain, and this reorganization may contribute to activity of AF2 (e.g., in interacting with certain mediator components).
• the ER’s C domain includes its DNA binding domain, which mediates interaction with so-called“estrogen responsive element(s)” (ERE(s)) operatively associated with genes whose transcription is regulated by the ER.
• ERP estrogen responsive element
• ERa and ERP regulate expression of different ERE- associated genes, and show different cellular and tissue distribution patterns.
• DNA binding by the ER appears to be mediated by two zinc finger structures within the C domain, although additional element(s) may contribute (see, for example, Hewitt & Korach Endocrine Rev 39:664, 2018).
• the ER’s C domain may also participate in or otherwise contribute to ER dimerization.
• the ER’s D domain is also called the“hinge region” and includes amino acid element(s) that may participate in ER dimerization and/or nuclear localization.
• the ER’s F domain may play a role in ER protein stability. This domain appears to be characteristic of estrogen receptors, as compared with other nuclear receptor family members, and may contribute to responsiveness to certain therapies (e.g., tamoxifen) (see, for example, Arao et al, J Bio. Chem 293:22, 8495).
• the ER In the presence of a natural ligand (e.g., 17P-estradiol), the ER undergoes a conformational change, homodimerizes, and localizes to the nucleus, where it binds to EREs and regulates transcription of its target genes (see, for example, Pawlak et al. ; Kumar & Chambor; Hall & McDonnell ); this series of events has been described as the“genomic” mechanism of ER gene regulation. Other mechanisms, such as “tethered”, “non-genomic”, and “ligand- independent”, have also been described, as depicted in Figure 3 of Hewitt & Korach, reproduced in FIG. 12.
• a natural ligand e.g., 17P-estradiol
• E2 response mechanisms As illustrated in FIG. 12 and reported by Hewitt & Korach, variations in the basic mechanism of E2 response.
• the genomic mechanism involves interaction between ER and ERE DNA motifs.
• the tethered mechanism involves indirect interaction between ER and other transcriptional regulators, such as the API DNA motif that binds the FOS/JUN dimer.
• ER is“tethered” to the DNA via, in this example, FOS/JUN binding to its API DNA motif.
• Nongenomic signaling is so-called because it initiates a signal from extracellular E2 that leads to rapid signal cascades in the cytoplasm, and thus the response does not involve interaction with genomic features.
• Ligand-independent signaling involves transduction of extracellular growth factor (GF) activation of cell membrane GF receptor (GFR), which initiates signaling cascades, such as MAPK. The signal is received by the ER, activating its transcriptional modulation of target genes, despite lacking E2 ligand.
• GF extracellular growth factor
• GFR cell membrane GF receptor
• ER has been implicated in a variety of cancers.
• ER + tumors In many tumors that express the estrogen receptor (i.e., ER + tumors), active ERa signaling has been demonstrated to drive cell proliferation (although ERp signaling has been reported to be able to achieve tumor suppressor effects; see, for example, Nilsson & Gustafson Clin. Pharmacol. Ther. 89:44, 2011).
• tumors e.g., breast tumors
• therapies targeting the ER are standard of care for many patients with ER + tumors (see, for example, Cardoso et al Annals One.
• Some ER-targeting agents are designed and/or documented to reduce levels of estrogen (i.e., 17b estradiol) production.
• Some ER-targeting agents are designed and/or documented to bind directly to the ER; in some cases, such agents compete with estrogen for binding to the ER and/or interfere with the allosteric changes that estrogen binding would naturally produce. Often, the term“antiestrogen” is used to refer to agents that bind to the ER, and sometimes is specifically used to indicate those agents that compete with estrogen for ER binding.
• SERM selective estrogen receptor modulator
• SERM selective estrogen receptor degrader
• Tamoxifen which has been an important breast cancer therapeutic, credited with having“saved the lives of half a million women around the world” (see“Bringing the Investigational Breast Cancer Drug Endoxifen From Bench to Bedside with NCI Support, available at https://www.cancer.gov/news-events/caneer-currents- bi og/2017/endoxifen-breast-cancer-NCI-support (last accessed July 7, 2019), but known to be less effective in women with low CYP2D6 activity, and also susceptible to development of resistance.
• Endoxifen the active metabolite of Tamoxifen, originally developed to address Tamoxifen’s failure in women with low CYP2D6 activity, which reduces their ability to convert Tamoxifen into Endoxifen. (see Cancer Currents Blog, National Cancer Institute, Aug 31, 2017).
• ARN-810 (Brilanestrant; GDC-810), which has been described as “a novel, potent, non-steroidal, orally bioavailable, selective ER antagonist/ER degrader that induces tumor regression in tamoxifen-sensitive and resistant ER+ BC xenograft models” (see Dickler et al. Cancer Res. 75(15 Suppl): Abstract nr CT231, 2015), and which was carried into Phase II clinical trials for treatment of patients with ER+ bereast cancer who had failed other hormonal agents, but whose further development may subsequently have been dropped (see, for example, Biospace April 27, 2017).
• AZD9496 which has been described as“an oral nonsteroidal, small-molecule inhibitor of estrogen receptor alpha (ERa) and a potent and selective antagonist and degrader of ERa” (see Hamilton et al Clin Cancer Res 1 :3519, 2018); AZD9496 has been reported to“antagonize] and degrad[e] ER with anti-tumour activity in both endocrine-sensitive and endocrine-resistant models”, and has been described as“comparable to fulvestrant in antagonising ER and circumventing endocrine resistance” (see, Nardone et al. Br. J. Cancer 120:331, 2019).
• Fulvestrant was the first SERD to earn FDA approval, and has been approved for treatment of certain ER+ cancers, including in combination with palbociclib or abemaciclib. Fulvestrant is a“selective estrogen receptor degrader that binds, blocks and degrades the estrogen receptor (ER), leading to complete inhibition of estrogen signaling through the ER” (see Nathan & Schmid Oncol Ther 5: 17, 2017). Fulvestrant has achieved significant clinical success, and is often considered to be the“gold standard” against which ER-targeted therapies are compared. However, Fulvestrant is administered by injection rather than orally, and in fact requires administration of 500 mg via intramuscular injection once per month (after initial dosing).
• Fulvestrant success stems from its ability to function as a complete estrogen receptor antagonist (a“CERAN”) that (1) inhibits both AF1 and AF2, so that it can inhibit AF1 activity that remains present in constitutively active ER mutants; (2) promotes ER degradation; and (3) lacks the partial ER agonist activity observed with certain other agents (see, for example, FIG 1 A which, among other things, documents that each of ARN- 810, AZD-9496, and Endoxifen increases ER activity in the absence of added estrogen, even while reducing ER activity when estrogen is there).
• a“CERAN” complete estrogen receptor antagonist
• fulvestrant As compared, for example, with therapies that limit estrogen production (e.g., anastrozole) or with partial antagonists (e.g., tamoxifen), fulvestrant exhibits superior activity and is the preferred treatment option for patients with hormone receptor-positive locally advanced or metastatic breast cancer. See Robertson, el al ., The Lancet , 388(10063):2997-3005 (Dec. 17, 2016). Without wishing to be bound by any particular theory, it is proposed that Fulvestrant’s ability to inhibit both AFl and AF2 may be attributable to its recruitment of co-repressors to the ER complex.
• therapies that limit estrogen production e.g., anastrozole
• partial antagonists e.g., tamoxifen
• ARN-810 ARN-810
• AZD9496 tamoxifen
• tamoxifen tamoxifen
• Fulvestrant matches Fulvestrant’ s CERAN attributes, and furthermore offers further valuable properties, including, for example that it (i) is orally bioavailable and has a long half-life; and (ii) shows good blood brain barrier penetration.
• the present disclosure demonstrates that Compound 1 is uniquely useful in certain contexts, including for (a) treatment of cancers associated with ER mutations, including ligand-independent/constitutive mutations; (b) treatment of cancers with CNS (e.g., brain) metastases or tumors; (c) use in combination with certain other agents, including certain agents demonstrated or proposed to be useful with Fulvestrant.
• CNS e.g., brain
• the present disclosure reports a discovery that Compound 1 exhibits complete estrogen receptor antagonism, and furthermore that such antagonism is comparable to Fulvestrant in various assays.
• the present disclosure demonstrates that Compound 1 is characterized by an ability to inhibit AFl and AF2, and therefore is properly described as a“complete estrogen receptor antagonist.”
• Compound 1 may be particularly useful or effective for treatment of diseases, disorders, or conditions (e.g., cancers) associated with presence of one or more ER mutants, specifically including ligand-independent ER mutants.
• the present disclosure provides methods of treatment in which Compound 1 is administered to a subject who expresses (e.g., in relevant cells or tissues) one or more ERs, and particular one or more ligand-independent ERs; in some embodiments, such subject(s) has been demonstrated to express such mutant ER(s) prior to the administration.
• a mutant ER protein is detected; in some embodiments, a nucleic acid encoding a mutant protein (e.g., a mutant ESR1 gene) is detected.
• Compound 1 to inhibit both AF1 and AF2 allows Compound 1 to function as a CERAN despite an activating mutation of the estrogen receptor (e.g., ESR1).
• ESR1 an activating mutation of the estrogen receptor
• the present disclosure provides methods of treating a subject (or a population of subjects) suffering from a cancer, wherein the subject is carrying an ESR1 mutation (and/or is expressing a mutant ER protein), the method comprising administering to the subject Compound 1 :
• useful ER antagonist agents are ones with CERAN activity as described herein.
• One aspect of the present disclosure is an insight that conventional strategies for assessing or characterizing ER antagonist (and/or potential antagonist) agents were insufficient at least in that they typically did not distinguish between SERDs and CERANs. In particular, most such conventional strategies did not assess an agent’s ability to specifically impact AF1.
• particularly useful ER antagonist agents are those that can inhibit ligand-independent ER activity; in some embodiments including activity observed with constitutive ER variant(s) such as, for example, AF2 deletions or truncations and/or LBD mutants (e.g., D538G and Y537S).
• constitutive ER variant(s) such as, for example, AF2 deletions or truncations and/or LBD mutants (e.g., D538G and Y537S).
• particularly useful ER antagonist agents are characterized by each of: a. Inhibition of AFl (e.g., inhibition of at least one, and preferably all known, constitutive ER variants)
• particularly useful ER antagonist agents are further characterized by one or more of:
• activity of ER antagonist agent(s) may be assessed relative to that of one or more of ARN-810, AZD9496, Endoxifen, Fulvestrant, RAD 1901, Tamoxifen, and/or Compound 1; in some such embodiments, comparison is contemporaneous, or alternatively, in some embodiments, it may be with a historical record or future result.
• the present disclosure further offers an insight that the unique ability of Compound 1 to function as an inhibitor of both AF1 and AF2 makes it particularly attractive for use in certain combination therapies.
• Compound 1 an estrogen receptor antagonist, and indeed a complete estrogen receptor antagonist, in combination with a secondary agent can substantially eliminate cell proliferation.
• the present disclosure encompasses the recognition that a combination of certain agents can beneficially be used to completely antagonize the estrogen receptor by inactivating both AF1 and AF2.
• the present disclosure provides a method of treating a subject suffering from a cancer comprising administering a compound that is an inhibitor of activating function 2 and a secondary agent that is an inhibitor of activating function 1.
• the compound is an estrogen receptor antagonist selected from AZD9496, RAD- 1901, ARN-810, endoxifen, Fulvestrant, and Compound 1.
• the compound is selected from fulvestrant and Compound 1
• the present disclosure provides a method of treating a patient or subject suffering from a cancer, the method comprising administering Compound 1 and a secondary agent selected from a CDK2, CDK4, CDK6, or CDK7 inhibitor.
• the secondary agent is a CDK2 inhibitor.
• the secondary agent is a CDK4 inhibitor.
• the secondary agent is a CDK6 inhibitor.
• the secondary agent is a CDK7 inhibitor.
• the secondary agent is a CDK4/6 inhibitor (i.e., inhibits one or both of CDK4 and CDK6).
• the secondary agent is a CDK2/4/6 inhibitor (i.e., inhibits one or more of CDK2, CDK4 and CDK6).
• the secondary agent is a CDK4/6 inhibitor selected from palbocociclib, ribociclib, abemaciclib, lerociclib, trilaciclib, and SHR6390.
• the CDK 4/6 inhibitor is palbocociclib.
• the CDK4/6 inhibitor is ribociclib.
• the CDK4/6 inhibitor is abemaciclib.
• the CDK4/6 inhibitor is lerociclib.
• the CDK4/6 inhibitor is trilaciclib.
• the CDK 4/6 inhibitor is SHR6390.
• the present disclosure provides a method of treating a patient or subject suffering from a cancer, the method comprising administering Compound 1 and a secondary agent, wherein the secondary agent is a PIK3CA inhibitor.
• the PIK3CA inhibitor is selected from alpelisib, taselisib, and LY3023414.
• the PIK3CA inhibitor is alpelisib.
• the PIK3CA inhibitor is taselisib.
• the PIK3CA inhibitor is LY3023414.
• the present disclosure provides a method of treating a patient or subject suffering from a cancer, the method comprising administering Compound 1 and a secondary agent, wherein the secondary agent is an mTOR inhibitor.
• the mTOR inhibitor is selected from sirolimus, temsirolimus, everolimus, and LY3023414.
• the mTOR inhibitor is sirolimus.
• the mTOR inhibitor is temsirolimus.
• the mTOR inhibitor is everolimus.
• the mTOR inhibitor is LY3023414.
• the present disclosure encompasses the recognition that certain disorders or conditions, e.g., cancer, can be effectively treated using amounts of active compound that is less than other compounds of similar activity.
• Compound 1 was found to reduce tumor volume more efficiently than other CERANs e.g., fulvestrant.
• Compound 1 is suitable for oral administration, which is a benefit over other CERANs, e.g., fulvestrant, which must be administered parenterally.
• the present disclosure provides a method of treating a patient or subject suffering from a cancer, the method comprising administering a composition comprising Compound 1.
• the composition comprises Compound 1 and a pharmaceutically acceptable excipient, carrier, or diluent.
• Said composition may be administered orally, parenterally, by inhalation or nasal spray, topically (e.g., as by powders, ointments, or drops), rectally, buccally, intravaginally, intraperitoneally, intracistemally or via an implanted reservoir, depending on the severity of the condition being treated.
• the compositions are administered orally, intraperitoneally or intravenously.
• provided compounds are administered orally or parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
• compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
• the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
• Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
• additional substances other than inert diluents e.g., lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
• 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.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• 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 starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol
• Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings (i.e. buffering agents) and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
• Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• the oral compositions can also include adj
• compositions described herein may be administered in the form of suppositories for rectal or vaginal administration.
• suppositories for rectal or vaginal administration.
• suitable non-irritating excipients or carriers that are solid at room temperature but liquid at body (e.g. rectal or vaginal) temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.
• suitable non-irritating excipients or carriers that are solid at room temperature but liquid at body (e.g. rectal or vaginal) temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.
• Such materials include cocoa butter, a suppository wax (e.g., beeswax) and polyethylene glycols.
• the composition is administered orally. In some embodiments, the composition is administered in an amount that is 30 mg/kg or less of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 10 mg/kg or less of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg/kg of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 3 mg/kg or less of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 1 mg/kg or less of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 0.9, 0.8. 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1, mg/kg of the weight of the patient or subject. In some embodiments, the composition is administered in an amount that is 0.1 mg/kg of the weight of the patient or subject.
• Compound 1 is administered as a unit dosage form. In some embodiments, Compound 1 is administered in the form of a capsule. In some embodiments, Compound is administered in the form of a tablet. In some embodiments, Compound 1 is administered as a suspension. In some embodiments, Compound 1 is administered as a solution.
• Compound 1 is administered as a daily dose (QD). In some embodiments, Compound 1 is administered as a twice daily dose (BID). In some embodiments, Compound 1 is administered every other day (QOD). In some embodiments, Compound 1 is administered as a weekly dose (QW). In some embodiments, Compound 1 is administered as a monthly dose (Q4W).
• FIGs. 7A-7D drug exposure (ng/ml) over time is high and even for mouse (FIG. 7A), rat (FIG. 7B), dog (FIG. 7C), and monkey (FIG. 7D) subjects.
• Compound 1 advantageously can be used to treat metastasized cancers, e.g., cancers that have spread to the brain, bones, lungs, liver, or the central nervous system.
• metastasized cancers e.g., cancers that have spread to the brain, bones, lungs, liver, or the central nervous system.
• Compound 1 when administered in a single oral 300 mg/kg dose, is able to penetrate the blood brain barrier.
• Other estrogen receptor antagonists e.g., fulvestrant, are unable to penetrate the blood-brain barrier in analogous quantities.
• the present disclosure provides a method of treating a patient or subject suffering from a cancer that has metastasized to the brain, bones, lungs, liver or the central nervous system, comprising administering Compound 1 :
• the cancer includes one or more CNS tumors (e.g., metastases); in some embodiments, the cancer has metastasized to the brain, bones, lungs, or liver. In some embodiments, the cancer has metastasized to the central nervous system.
• CNS tumors e.g., metastases
• the cancer has metastasized to the brain, bones, lungs, or liver. In some embodiments, the cancer has metastasized to the central nervous system.
• aq. aqueous
• ACN acetonitrile
• CSA camphorsulfonic acid
• d day or days
• DCM di chi orom ethane
• DEA diethylamine
• DHP dihydropyran
• DMF N,N-dimethylformamide
• DIPEA N,N- diisopropylethylamine
• DMAP 4-dimethylaminopyridine
• DMSO dimethyl sulphoxide
• EA ethyl acetate
• ee enantiomeric excess
• Step 1 Preparation of 1-propionylazeti din-3 -one
• the compound 3-azetidinone hydrochloride (10.000 g, 93.0 mmol, 1.0 equiv.), anhydrous 1,2-dichloroethane (200 mL) and diisopropylethylamine (38.9 mL, 223 mmol, 2.4 equiv.) were added to a round bottom flask (500 mL) to provide a light yellow suspension.
• the suspension was sonicated for 1 h and then cooled to -10 °C (dry-ice/MeOH) for 10 min.
• Lithium aluminum hydride (10.397 g, 273.9 mmol, 3.0 equiv.) was suspended into THF (200 mL) and cooled in an ice bath.
• Trifluoromethanesulfonic anhydride (5.0 mL, 29.7 mmol, 1.3 equiv.) was added dropwise to a 0 °C solution of 2-fluoro-2-methylpropanol (2.090 g, 22.7 mmol, 1.0 equiv.) and 2,6 lutidine (3.40 mL, 29.4 mmol, 1.3 equiv.) in DCM (25 mL) over 30 minutes. After 2 hours, the red solution had turned light brown. TLC (20:80 EA:Hex, KMnCh stain) indicated that the starting material was not present. The reaction mixture was washed with 1M HC1 solution (2 x 20 mL) and sat.
• reaction solution was diluted in DCM, filtered, and washed with saturated Na2CCh solution.
• the aqueous layer was extracted with DCM and the combined organic layers were dried over Na2SC>4.
• the solution was filtered and concentrated.
• the residue was dissolved into acetonitrile (2 mL) and filtered through a syringe filter before purification via prep LC (40 to 90% ACNiFEO over 18 min, followed by isocratic 90% ACN for 7 min).
• the present Example describes assessment of various compounds (ARN-810, AZD9496, Compound 1, Endoxifen, and Fulvestrant) on ERa protein level in a variety of cell lines.
• 90,000-500,000 cells per well were previously plated into each well of a 12-well dish and incubated in phenol red-free media containing 5% charcoal dextran stripped fetal bovine serum (stripped FBS) (HyClone) for at least 24 hours.
• Cells were treated with 300 nM antiestrogen for 4 hours in serum-free medium and lysates subsequently lysed with RIPA buffer supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific).
• the present Example describes assays that assess impact of tested compounds on human MCF-7 cells, which are a human ER + breast cancer cell line. Specifically, 1000 MCF-7 cells (Cheryl Walker, Baylor College of Medicine) per well were plated into a 96-well plate in phenol red-free media (ThermoFisher Scientific) containing 5% stripped FBS. At least 4 hours later cells were treated with antiestrogens and media was diluted to 2.5% stripped FBS in the presence of 100 pM E2 for 6-8 days. Proliferation was measured using CyQuant fluorescent DNA-binding dye kit (ThermoFisher Scientific) using 1 :200 GR dye and reading fluorescence at 485 nm excitation and 538 nm.
• the present Example describes studies in which certain estrogen receptor constructs were transfected into Ishikawa cells, which are a human endometrial cancer cell line, endogenous alkaline phosphatase is assayed. 15,000 Ishikawa cells per well were plated into a 96-well plate in phenol-red media containing 5% stripped FBS. At the time of plating, cells in each well were transiently transfected with 75-100 ng of an estrogen receptor construct (or empty vector, pSG5) using Lipofectamine LTX (ThermoFisher Scientific). Approximately 4 hours later, cells were treated with indicated amount of anti-estrogen (in the absence of E2), or 500 pM E2 (FIG.
• Compound 1 has ER Antagonist and not Agonist. Activity
• Wild type ER HEGO
• empty vector pSG5
• indicated LBD mutant ER was transiently transfected into Ishikawa cells as described above. Only the empty vector was treated with 500 pM 17/5-estradiol (E2). 72 hours later, cells were assayed for AP activity. Results are presented in FIG. 9. Bars represent mean absorbance at 405 nm from triplicate wells, +s.e.m. As can be seen, various of the tested ER mutants were observed to be“activating mutants” in that they showed more activity than did the wild type ER when ligand was not present.
• AFlWild type ER HEGO, AA 1-595
• empty vector pSG5
• indicated ER missing the activation domain 2 (“AF2”) AA 1-282
• activation domain 1 AF1
• Bars represent mean absorbance at 405 nm from quadruplicate wells, + s.e.m.
• FI AF1 is required for ligand-independent ER activity that is observed when the ER is truncated (AAF2), even in the presence of the activating Y537S mutation (AAF1/Y5372).
• FIGs. 5A-5F each of FIG. 5 A to 5F reports a particular cell line, as indicated in each figure. Points represent mean AP activity normalized to vehicle, +/- s.e.m. from duplicate wells. Dose response curves of Compound 1 and Fulvestrant were fit using the least squares fit method and pICso (-Log ICso) were calculated using a variable slope sigmoid dose-response model.
• Line represents the normalized AP activity of the endogenous receptor (transfected with empty vector (pSG5)).
• pSG5 empty vector
• Line represents AP activity of the endogenous receptor (transfected with empty vector (pSG5)).
• Endoxifen RAD- 1901, ARN-810 (GDC-0810) or AZD-9496 can inhibit activity of the ligand-independent ER variants as Compound 1 and Fulvestrant do.

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