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WO2017136699A1 - Inhibiteurs d'arginine méthyltransférase et leurs utilisations - Google Patents

Inhibiteurs d'arginine méthyltransférase et leurs utilisations Download PDF

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WO2017136699A1
WO2017136699A1 PCT/US2017/016472 US2017016472W WO2017136699A1 WO 2017136699 A1 WO2017136699 A1 WO 2017136699A1 US 2017016472 W US2017016472 W US 2017016472W WO 2017136699 A1 WO2017136699 A1 WO 2017136699A1
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disorder
cell
cancer
rmt
compound
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PCT/US2017/016472
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Lorna Helen Mitchell
Kerren Kalai SWINGER
Gideon Shapiro
Paula Ann Boriack-Sjodin
Richard Chesworth
Kenneth W. Duncan
Kevin Wayne Kuntz
Oscar Miguel Moradei
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Epizyme, Inc
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Publication of WO2017136699A1 publication Critical patent/WO2017136699A1/fr

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    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
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    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
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    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/08—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
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    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D403/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing aromatic rings
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    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • 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
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    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
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    • C07D—HETEROCYCLIC COMPOUNDS
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Definitions

  • Epigenetic regulation involves heritable modification of genetic material without changing its nucleotide sequence.
  • epigenetic regulation is mediated by selective and reversible modification (e.g. , methylation) of DNA and proteins (e.g. , histones) that control the conformational transition between transcriptionally active and inactive states of chromatin.
  • methylation e.g. , methylation
  • proteins e.g. , histones
  • methyltransferases e.g., arginine methyltransferases
  • many of which are associated with specific genetic alterations that can cause human disease are associated with specific genetic alterations that can cause human disease.
  • methyltransferases play a role in diseases such as proliferative disorders, autoimmune disorders, muscular disorders, vascular disorders, metabolic disorders, and neurological disorders.
  • diseases such as proliferative disorders, autoimmune disorders, muscular disorders, vascular disorders, metabolic disorders, and neurological disorders.
  • small molecules that are capable of inhibiting the activity of arginine methyltransferases.
  • Arginine methyltransferases are attractive targets for modulation given their role in the regulation of diverse biological processes. It has now been found that compounds described herein, and pharmaceutically acceptable salts and compositions thereof, are effective as inhibitors of arginine methyltransferases. Such compounds are listed in Table 1, infra.
  • compounds described herein inhibit activity of an arginine methyltransferase (RMT) (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8).
  • RMT arginine methyltransferase
  • methods of inhibiting an arginine methyltransferase comprise contacting the arginine methyltransferase with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof.
  • the RMT may be purified or crude, and may be present in a cell, tissue, or a subject. Thus, such methods encompass inhibition of RMT activity both in vitro and in vivo.
  • the RMT is wild-type.
  • the RMT is overexpressed.
  • the RMT is a mutant. In certain embodiments, the RMT is in a cell. In some embodiments, the RMT is expressed at normal levels in a subject, but the subject would benefit from RMT inhibition (e.g., because the subject has one or more mutations in an RMT substrate that causes an increase in methylation of the substrate with normal levels of RMT). In some embodiments, the RMT is in a subject known or identified as having abnormal RMT activity (e.g. , overexpression).
  • methods of modulating gene expression in a cell comprise contacting a cell with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the cell in culture in vitro.
  • cell is in an animal, e.g. , a human.
  • methods of modulating transcription in a cell comprise contacting a cell with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the cell in culture in vitro.
  • the cell is in an animal, e.g. , a human.
  • methods of treating an RMT-mediated disorder are provided which comprise administering to a subject suffering from an RMT-mediated disorder an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the RMT-mediated disorder is a proliferative disorder.
  • compounds described herein are useful for treating cancer.
  • compounds described herein are useful for treating breast cancer, prostate cancer, lung cancer, colon cancer, bladder cancer, or leukemia.
  • the RMT-mediated disorder is a muscular disorder.
  • the RMT-mediated disorder is an autoimmune disorder.
  • the RMT-mediated disorder is a neurological disorder.
  • the RMT-mediated disorder is a vascular disorder. In certain embodiments, the RMT-mediated disorder is a metabolic disorder. [0010] Compounds described herein are also useful for the study of arginine
  • methyltransferases in biological and pathological phenomena, the study of intracellular signal transduction pathways mediated by arginine methyltransferases, and the comparative evaluation of new RMT inhibitors.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g. , enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19 F with 18 F, or the replacement of a carbon by a 13 C- or C-enriched carbon are within the scope of the disclosure.
  • Such compounds are useful, for example, as analytical tools or probes in biological assays.
  • “Pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response, and the like, and are
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds describe herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts 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, oxalic 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.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, 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
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (Ci_ 4 alkyl) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, quaternary salts.
  • a "subject" to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or other non-human animals, for example, non-human mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.
  • humans e.g., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)
  • non-human mammals e.g., primates (e.g., cyn
  • the non-human animal is a mammal.
  • the non-human animal may be a male or female at any stage of development.
  • a non-human animal may be a transgenic animal.
  • Treating encompasses an action that occurs while a subject is suffering from a condition which reduces the severity of the condition or retards or slows the progression of the condition ("therapeutic treatment”).
  • Treating also encompasses an action that occurs before a subject begins to suffer from the condition and which inhibits or reduces the severity of the condition (“prophylactic treatment”).
  • an "effective amount" of a compound refers to an amount sufficient to elicit the desired biological response, e.g. , treat the condition.
  • the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject.
  • An effective amount encompasses therapeutic and prophylactic treatment.
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term "therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • a prophylactically effective amount of a compound is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • methyltransferase represents transferase class enzymes that are able to transfer a methyl group from a donor molecule to an acceptor molecule, e.g. , an amino acid residue of a protein or a nucleic base of a DNA molecule.
  • Methytransferases typically use a reactive methyl group bound to sulfur in S-adenosyl methionine (SAM) as the methyl donor.
  • SAM S-adenosyl methionine
  • a methyltransferase described herein is a protein methyltransferase.
  • a methyltransferase described herein is a histone methyltransferase.
  • Histone methyltransf erases are histone-modifying enzymes, (including histone-lysine N-methyltransferase and histone-arginine N-methyltransferase), that catalyze the transfer of one or more methyl groups to lysine and arginine residues of histone proteins.
  • a methyltransferase described herein is a histone-arginine N-methyltransferase.
  • RMT arginine methyltransferase
  • a provided compound inhibits an RMT (e.g. , PRMTl , PRMT3, CARMl, PRMT6, and/or PRMT8). In certain embodiments, a provided compound inhibits wild-type PRMTl, PRMT3, CARMl , PRMT6, and/or PRMT8. In certain embodiments, a provided compound inhibits a mutant RMT. In certain embodiments, a provided compound inhibits PRMTl , PRMT3, CARMl, PRMT6, and/or PRMT8, e.g. , as measured in an assay described herein. In certain embodiments, the RMT is from a human.
  • a provided compound inhibits an RMT (e.g., PRMTl, PRMT3, CARMl , PRMT6, and/or PRMT8) at an IC 50 less than or equal to 10 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g., PRMTl , PRMT3, CARMl , PRMT6, and/or PRMT8) at an IC5 0 less than or equal to 1 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g. , PRMTl, PRMT3, CARMl , PRMT6, and/or PRMT8) at an IC5 0 less than or equal to 0.1 ⁇ .
  • RMT e.g., PRMTl, PRMT3, CARMl , PRMT6, and/or PRMT8
  • a provided compound inhibits an RMT (e.g., PRMTl, PRMT3, CARMl , PRMT6, and/or PRMT8) at an IC50 less than or equal to 0.01 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g. , PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC 30 less than or equal to 10 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g., PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC 30 less than or equal to 12 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g., PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8) at an IC50 less than or equal to 0.01 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g. , PRMTl, PRMT3, CARM1, PRMT6,
  • a provided compound inhibits PRMTl in a cell at an EC 30 less than or equal to 12 ⁇ . In certain embodiments, a provided compound inhibits PRMTl in a cell at an EC 30 less than or equal to 3 ⁇ . In certain embodiments, a provided compound inhibits an RMT (e.g. , PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC 30 less than or equal to 1 ⁇ .
  • RMT e.g. , PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8
  • a provided compound inhibits an RMT (e.g., PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8) in a cell at an EC 30 less than or equal to 0.1 ⁇ . In certain embodiments, a provided compound inhibits cell proliferation at an EC 50 less than or equal to 10 ⁇ . In certain embodiments, a provided compound inhibits cell proliferation at an EC5 0 less than or equal to 1 ⁇ . In certain embodiments, a provided compound inhibits cell proliferation at an EC5 0 less than or equal to 0.1 ⁇ .
  • RMT e.g., PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8
  • the RMT can be wild- type, or any mutant or variant.
  • compositions comprising a compound described herein, e.g. , a compound of Table 1, or a pharmaceutically acceptable salt thereof, as described herein, and optionally a pharmaceutically acceptable excipient.
  • a compound described herein, or salts thereof may be present in various forms, such as amorphous, hydrates, solvates, or polymorphs.
  • a provided composition comprises two or more compounds described herein.
  • a compound described herein, or a pharmaceutically acceptable salt thereof is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a
  • the effective amount is an amount effective for inhibiting an RMT (e.g. , PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, the effective amount is an amount effective for treating an RMT- mediated disorder (e.g., a PRMTl -, PRMT3-, CARM1-, PRMT6-, and/or PRMT8 -mediated disorder). In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is an amount effective to prevent an RMT-mediated disorder.
  • compositions agents include any and all solvents, diluents, or other liquid vehicles, dispersions, suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as suited to the particular dosage form desired.
  • solvents diluents, or other liquid vehicles, dispersions, suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like.
  • compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing a compound described herein (the "active ingredient") into association with a carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions of the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition described herein is sterilized.
  • compositions used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
  • Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
  • Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
  • crospovidone cross-linked poly(vinyl-pyrrolidone)
  • sodium carboxymethyl starch sodium starch glycolate
  • Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cell
  • Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
  • methylcellulose methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
  • Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT),
  • the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic sa
  • Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
  • Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, camauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myri state, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea
  • Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
  • Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may comprise 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 (e.g., cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • 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,
  • the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • solubilizing agents such as CremophorTM, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active ingredient 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, 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 and g
  • Solid compositions of a similar type can 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 and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can 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 which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type can 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 active ingredient can be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g. , tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can 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.
  • opacifying agents include polymeric substances and waxes.
  • Dosage forms for topical and/or transdermal administration of a provided compound may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
  • the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and/or any desired preservatives and/or buffers as can be required.
  • the present disclosure encompasses the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body.
  • Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium.
  • the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
  • Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self propelling
  • solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure.
  • the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension.
  • Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation for buccal
  • formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a provided pharmaceutical composition can be prepared, packaged, and/or sold in a formulation for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein.
  • Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this disclosure.
  • compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
  • compositions provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of provided compositions will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease, disorder, or condition being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the compounds and compositions provided herein can be administered by any route, including enteral (e.g. , oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g. , oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal
  • topical as by powders, ointments, creams, and/or drops
  • mucosal
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g. , its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g. , whether the subject is able to tolerate oral administration).
  • the exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like.
  • the desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks.
  • the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.
  • a compound described herein may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • a compound described herein is administered one or more times per day, for multiple days. In some embodiments, the dosing regimen is continued for days, weeks, months, or years.
  • dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a compound or composition, as described herein can be administered in combination with one or more additional therapeutically active agents.
  • a compound or composition provided herein is administered in combination with one or more additional therapeutically active agents that improve its bioavailability, reduce and/or modify its metabolism, inhibit its excretion, and/or modify its distribution within the body.
  • the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects.
  • the compound or composition can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents.
  • the additional therapeutically active agent is a a compound of Table 1.
  • the additional therapeutically active agent is not a a compound of Table 1.
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions.
  • the particular combination to employ in a regimen will take into account compatibility of a provided compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved.
  • additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized
  • Exemplary additional therapeutically active agents include, but are not limited to, small organic molecules such as drug compounds (e.g. , compounds approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • drug compounds e.g. , compounds approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)
  • CFR Code of Federal Regulations
  • peptides e.g., compounds approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulation
  • an additional therapeutically active agent is prednisolone, dexamethasone, doxorubicin, vincristine, mafosfamide, cisplatin, carboplatin, Ara-C, rituximab, azacitadine, panobinostat, vorinostat, everolimus, rapamycin, ATRA (all- trans retinoic acid), daunorubicin, decitabine, Vidaza, mitoxantrone, or IBET-151.
  • kits e.g. , pharmaceutical packs
  • the kits provided may comprise a provided pharmaceutical composition or compound and a container (e.g. , a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g. , a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a provided pharmaceutical composition or compound.
  • a provided pharmaceutical composition or compound provided in the container and the second container are combined to form one unit dosage form.
  • a provided kits further includes instructions for use.
  • RMT e.g. , PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8.
  • methods of treating an RMT-mediated disorder in a subject comprise administering an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof), to a subject in need of treatment.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the subject is suffering from a RMT-mediated disorder.
  • the subject is susceptible to a RMT-mediated disorder.
  • RMT-mediated disorder means any disease, disorder, or other pathological condition in which an RMT (e.g. , PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) is known to play a role. Accordingly, in some embodiments, the present disclosure relates to treating or lessening the severity of one or more diseases in which an RMT is known to play a role.
  • the present disclosure provides a method of inhibiting an RMT comprising contacting the RMT with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof.
  • the RMT may be purified or crude, and may be present in a cell, tissue, or subject.
  • such methods encompass both inhibition of in vitro and in vivo RMT activity.
  • the method is an in vitro method, e.g.,
  • Ill such as an assay method. It will be understood by one of ordinary skill in the art that inhibition of an RMT does not necessarily require that all of the RMT be occupied by an inhibitor at once.
  • Exemplary levels of inhibition of an RMT include at least 10% inhibition, about 10% to about 25% inhibition, about 25% to about 50% inhibition, about 50% to about 75% inhibition, at least 50% inhibition, at least 75% inhibition, about 80% inhibition, about 90% inhibition, and greater than 90% inhibition.
  • a method of inhibiting RMT activity in a subject in need thereof comprising administering to the subject an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • a method of modulating gene expression in a cell which comprises contacting a cell with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof.
  • the cell is in culture in vitro.
  • the cell is in an animal, e.g. , a human.
  • the cell is in a subject in need of treatment.
  • a method of modulating transcription in a cell which comprises contacting a cell with an effective amount of a compound of Table 1, or a pharmaceutically acceptable salt thereof.
  • the cell is in culture in vitro.
  • the cell is in an animal, e.g. , a human.
  • the cell is in a subject in need of treatment.
  • a method is provided of selecting a therapy for a subject having a disease associated with an RMT-mediated disorder or mutation comprising the steps of determining the presence of an RMT-mediated disorder or gene mutation in an RMT gene (e.g., a PRMTl, PRMT3, CARM1, PRMT6, and/or PRMT8 gene) or and selecting, based on the presence of an RMT-mediated disorder a gene mutation in the RMT gene a therapy that includes the administration of a provided compound.
  • the disease is cancer.
  • a method of treatment for a subject in need thereof comprising the steps of determining the presence of an RMT-mediated disorder or a gene mutation in the RMT gene and treating the subject in need thereof, based on the presence of a RMT-mediated disorder or gene mutation in the RMT gene with a therapy that includes the administration of a provided compound.
  • the subject is a cancer patient.
  • a compound provided herein is useful in treating a proliferative disorder, such as cancer.
  • PRMTs protein arginine methylation by PRMTs is a modification that has been implicated in signal transduction, gene transcription, DNA repair and mRNA splicing, among others; and overexpression of PRMTs within these pathways is often associated with various cancers.
  • compounds which inhibit the action of PRMTs, as provided herein, are effective in the treatment of cancer.
  • compounds provided herein are effective in treating cancer through the inhibition of PRMTl .
  • PRMTl overexpression has been observed in various human cancers, including, but not limited to, breast cancer, prostate cancer, lung cancer, colon cancer, bladder cancer, and leukemia.
  • PRMTl specifically deposits an asymmetric dimethylarginine (aDMA) mark on histone H4 at arginine 3
  • H4R3me2a H4R3me2a
  • this mark is associated with transcription activation.
  • the methylation status of H4R3 positively correlates with increasing tumor grade and can be used to predict the risk of prostate cancer recurrence (Seligson et al, Nature 2005 435, 1262- 1266).
  • inhibitors of PRMTl as described herein, are useful in treating cancers associated with the methylation status of H4R3, e.g., prostate cancer.
  • TDRD3 methylarginine effector molecule
  • H4R3me2a mark the methylarginine effector molecule
  • overexpression of TDRD3 is linked to poor prognosis for the survival of patients with breast cancer (Nagahata et al, Cancer Sci. 2004 95, 218-225).
  • inhibitors of PRMTl as described herein, are useful in treating cancers associated with overexpression of TDRD3, e.g., breast cancer, as inhibition of PRMTl leads to a decrease in methylation of H4R3, thereby preventing the association of overexpressed TDRD3 with H4R3me2a.
  • PRMTl is known to have non-histone substrates.
  • PRMTl when localized to the cytoplasm, methylates proteins that are involved in signal transduction pathways, e.g., the estrogen receptor (ER).
  • ER the estrogen receptor
  • the expression status of ER in breast cancer is critical for prognosis of the disease, and both genomic and non-genomic ER pathways have been implicated in the pathogenesis of breast cancer.
  • SRC a proto-oncogene tyrosine- protein kinase
  • FAK focal adhesion kinase
  • PRMTl -mediated ERa methylation is required for the activation of the SRC-PI3K-FAK cascade and AKT, coordinating cell proliferation and survival.
  • hypermethylation of ERa in breast cancer is thought to cause hyperactivation of this signaling pathway, providing a selective survival advantage to tumor cells (Le Romancer et al, Mol. Cell 2008 31, 212- 221 ; Le Romancer et al, Steroids 2010 75, 560-564).
  • inhibitors of PRMTl, as described herein are useful in treating cancers associated with ERa methylation, e.g., breast cancer.
  • PRMTl has been shown to be involved in the regulation of leukemia development.
  • SRC-associated in mitosis 68 kDa protein is a well-characterized PRMTl substrate, and when either SAM68 or PRMTl is fused directly to the myeloid/lymphoid leukemia (MLL) gene, these fusion proteins can activate MLL oncogenic properties, implying that the methylation of SAM68 by PRMTl is a critical signal for the development of leukemia (Cheung et al, Nature Cell Biol. 2007 9, 1208-1215). Accordingly, in some embodiments, inhibitors of PRMTl, as described herein, are useful in treating cancers associated with SAM68 methylation, e.g., leukemia.
  • MLL myeloid/lymphoid leukemia
  • PRMTl is implicated in leukemia development through its interaction with AE9a, a splice isoform of AML1-ETO (Shia et al, Blood 2012 119:4953-62). Knockdown of PRMTl affects expression of certain AE9a-activated genes and suppresses AE9a's self-renewal capability. It has also been shown that AE9a recruits PRMTl to AE9a activated gene promoters, which leads to increased H4 Arg3 methylation, H3 Lys9/14 acetylation, and transcription activated.
  • inhibitors of PRMTl are useful in treating cancers associated with AML1-ETO, e.g., leukemia.
  • the inhibition of PRMTl e.g., by compounds described herein, is beneficial in the treatment of cancer.
  • compounds provided herein are effective in treating cancer through the inhibition of PRMT3.
  • the DAL1 tumor suppressor protein has been shown to interact with PRMT3 and inhibits its methyltransferase activity (Singh et al, Oncogene 2004 23, 7761-7771).
  • Epigenetic downregulation of DAL1 has been reported in several cancers (e.g., meningiomas and breast cancer), thus PRMT3 is expected to display increased activity, and cancers that display DAL1 silencing may, in some aspects, be good targets for PRMT3 inhibitors, e.g., those described herein.
  • the inhibition of PRMT3, e.g., by compounds described herein is beneficial in the treatment of cancer.
  • compounds provided herein are effective in treating cancer through the inhibition of PRMT4, also known as CARMl .
  • PRMT4 levels have been shown to be elevated in castration-resistant prostate cancer (CRPC), as well as in aggressive breast tumors (Hong et al, Cancer 2004 101, 83-89; Majumder et al, Prostate 2006 66, 1292-1301).
  • inhibitors of PRMT4, as described herein are useful in treating cancers associated with PRMT4 overexpression.
  • PRMT4 has also been shown to affect ERa-dependent breast cancer cell differentiation and proliferation (Al-Dhaheri et al, Cancer Res.
  • PRMT4 inhibitors as described herein, are useful in treating ERa-dependent breast cancer by inhibiting cell differentiation and proliferation .
  • PRMT4 has been shown to be recruited to the promoter of E2F1 (which encodes a cell cycle regulator) as a transcriptional co-activator (Frietze et al, Cancer Res. 2008 68, 301-306).
  • E2F1 which encodes a cell cycle regulator
  • PRMT4- mediated upregulation of E2F1 expression may contribute to cancer progression and chemoresistance as increased abundance of E2F1 triggers invasion and metastasis by activating growth receptor signaling pathways, which in turn promote an antiapoptotic tumor environment (Engelmann and Piitzer, Cancer Res 2012 72; 571).
  • the inhibition of PRMT4, e.g., by compounds provided herein is useful in treating cancers associated with E2F1 upregulation.
  • the inhibition of PRMT4, e.g., by compounds described herein is beneficial in the treatment of cancer.
  • compounds provided herein are effective in treating cancer through the inhibition of PRMT6.
  • PRMT6 has been reported to be
  • the inhibition of PRMT6, by compounds provided herein, is useful in treating cancers associated with PRMT6
  • PRMT6 is primarily thought to function as a transcriptional repressor, although it has also been reported that PRMT6 functions as a co-activator of nuclear receptors.
  • PRMT6 suppresses the expression of thrombospondin 1 (TSP1 ; also known as THBS 1; a potent natural inhibitor of angiogenesis and endothelial cell migration) and p21 (a natural inhibitor of cyclin dependent kinase), thereby contributing to cancer development and progression (Michaud-Levesque and Richard, J. Biol. Chem. 2009 284, 21338-21346; Kleinschmidt et al., PLoS ONE 2012 7, e41446).
  • TSP1 thrombospondin 1
  • p21 a natural inhibitor of cyclin dependent kinase
  • the inhibition of PRMT6, by compounds provided herein is useful in treating cancer by preventing the repression of THBsl and/or p21.
  • the inhibition of PRMT6, e.g., by compounds described herein is beneficial in the treatment of cancer.
  • compounds provided herein are effective in treating cancer through the inhibition of PRMT8. For example, deep-sequencing efforts of cancer genomes (e.g., COSMIC) have revealed that of all the PRMTs, PRMT8 is reported to be the most mutated.
  • PRMT8 is thought to contribute to the initiation or progression of cancer.
  • the inhibition of PRMT8, e.g., by compounds described herein, is beneficial in the treatment of cancer.
  • compounds described herein are useful for treating a cancer including, but not limited to, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma;
  • angiosarcoma e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma
  • bronchus cancer carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypere
  • cervical cancer e.g.
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis CML
  • chronic neutrophilic leukemia CML
  • hypereosinophilic syndrome HES
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • NF neurofibromatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), mela
  • a compound provided herein is useful in treating diseases associated with increased levels of circulating asymmetric dimethylarginine (aDMA), e.g., cardiovascular disease, diabetes, kidney failure, renal disease, pulmonary disease, etc.
  • aDMA asymmetric dimethylarginine
  • Circulating aDMA is produced by the proteolysis of asymmetrically dimethylated proteins.
  • PRMTs which mediate aDMA methylation include, e.g. , PRMTl, PRMT3, PRMT4, PRMT6, and PRMT8.
  • aDMA levels are directly involved in various diseases as aDMA is an endogenous competitive inhibitor of nitric oxide synthase (NOS), thereby reducing the production of nitric oxide (NO) (Vallance et al, J. Cardiovasc. Pharmacol. 1992 20(Suppl. 12):S60-2).
  • NO nitric oxide synthase
  • NO nitric oxide
  • PRMTl is a major enzyme that generates aDMA
  • cardiovascular diseases Boger et al, Ann. Med. 2006 38: 126-36
  • other pathophysiological conditions such as diabetes mellitus (Sydow et al, Vase. Med. 2005 10(Suppl. l): S35-43), kidney failure (Vallance et al, Lancet 1992 339:572-5), and chronic pulmonary diseases (Zakrzewicz et al, BMC Pulm. Med. 2009 9:5).
  • PRMTl and PRMT3 are increased in coronary heart disease (Chen et al, Basic Res. Cardiol.
  • aDMA elevation is seen in patients with renal failure, due to impaired clearance of this metabolite from the circulation (Jacobi et al, Am. J. Nephrol. 2008 28:224-37).
  • circulating aDMA levels is observed in many pathophysiological situations.
  • the inhibition of PRMTs e.g. , by compounds described herein, results in the decrease of circulating aDMA, which is beneficial in the treatment of diseases associated with increased levels of circulating aDMA, e.g., cardiovascular disease, diabetes, kidney failure, renal disease, pulmonary disease, etc.
  • a compound described herein is useful for treating or preventing vascular diseases.
  • a compound provided herein is useful in treating metabolic disorders.
  • PRMTl has been shown to enhance mRNA levels of FoxOl target genes in gluconeogenesis, which results in increased hepatic glucose production, and knockdown of PRMT promotes inhibition of FoxOl activity and thus inhibition of hepatic gluconeogenesis (Choi et al, Hepatology 2012 56: 1546-56).
  • genetic haploinsufficiency of Prmtl has been shown to reduce blood glucose levels in mouse models.
  • the inhibition of PRMT1 e.g., by compounds described herein, is beneficial in the treating of metabolic disorders, such as diabetes.
  • a provided compound is useful in treating type I diabetes.
  • a provided compound is useful in treating type II diabetes.
  • a compound provided herein is useful in treating muscular dystrophies.
  • PRMT1 methylate the nuclear poly(A)-binding protein (PABPNl) in a region located near its C-terminus (Perreault et al, J. Biol Chem. 2007 282:7552-62).
  • PABPNl nuclear poly(A)-binding protein
  • This domain is involved in the aggregation of the PABPNl protein, and abnormal aggregation of this protein is involved in the disease oculopharyngeal muscular dystrophy (Davies et al, Int. J. Biochem. Cell Biol. 2006 38: 1457-62).
  • the inhibition of PRMTs e.g.
  • muscular dystrophies e.g. , oculopharyngeal muscular dystrophy
  • PABPNl oculopharyngeal muscular dystrophy
  • CARM1 is also the most abundant PRMT expressed in skeletal muscle cells, and has been found to selectively control the pathways modulating glycogen metabolism, and associated AMPK (AMP-activated protein kinase) and p38 MAPK (mitogen-activated protein kinase) expression. See, e.g., Wang et al, Biochem (2012) 444:323-331.
  • inhibitors of CARMl, as described herein are useful in treating metabolic disorders, e.g., for example skeletal muscle metabolic disorders, e.g., glycogen and glucose metabolic disorders.
  • Exemplary skeletal muscle metabolic disorders include, but are not limited to, Acid Maltase Deficiency (Glycogenosis type 2; Pompe disease), Debrancher deficiency (Glycogenosis type 3), Phosphorylase deficiency (McArdle's; GSD 5), X-linked syndrome (GSD9D), Autosomal recessive syndrome (GSD9B), Tarui's disease (Glycogen storage disease VII; GSD 7), Phosphoglycerate Mutase deficiency (Glycogen storage disease X; GSDX; GSD 10), Lactate dehydrogenase A deficiency (GSD 11), Branching enzyme deficiency (GSD 4), Aldolase A (muscle) deficiency, ⁇ -Enolase deficiency, Triosephosphate isomerase (TIM) deficiency, Lafora's disease (Progressive myoclonic epilepsy 2), Glycogen storage disease (Mus
  • Glycogenin Deficiency (GSD 15).
  • a compound provided herein is useful in treating autoimmune disease.
  • PRMT inhibitors may be valuable for the treatment of autoimmune diseases, e.g. , rheumatoid arthritis.
  • PRMTs are known to modify and regulate several critical immunomodulatory proteins. For example, post-translational modifications (e.g. , arginine methylation), within T cell receptor signaling cascades allow T lymphocytes to initiate a rapid and appropriate immune response to pathogens.
  • Co-engagement of the CD28 costimulatory receptor with the T cell receptor elevates PRMT activity and cellular protein arginine methylation, including methylation of the guanine nucleotide exchange factor Vavl (Blanchet et al, J. Exp. Med. 2005 202:371-377).
  • PRMT inhibitors are thus expected to diminish methylation of the guanine exchange factor Vavl, resulting in diminished IL-2 production.
  • siRNA directed against PRMT5 was shown to both inhibit NFAT-driven promoter activity and IL-2 secretion (Richard et al, Biochem J. 2005 388:379-386).
  • PRMT1 is known to cooperate with PRMT4 to enhance NFkB p65-driven transcription and facilitate the transcription of p65 target genes like TNFa (Covic et al, Embo. J. 2005 24:85-96).
  • PRMTl and/or PRMT4 inhibitors are useful in treating autoimmune disease by decreasing the transcription of p65 target genes like TNFa. These examples demonstrate an important role for arginine methylation in inflammation.
  • the inhibition of PRMTs e.g., by compounds described herein, is beneficial in the treatment of autoimmune diseases.
  • a compound provided herein is useful in treating neurological disorders, such as amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • TLS/FUS a gene involved in ALS, TLS/FUS, often contains mutated arginines in certain familial forms of this disease (Kwiatkowski et al, Science 2009 323: 1205-8). These mutants are retained in the cytoplasm, which is similar to reports documenting the role arginine methylation plays in nuclear-cytoplasmic shuffling (Shen et al, Genes Dev. 1998 12:679-91).
  • PRMT e.g., PRMTl
  • TLS/FUS is methylated on at least 20 arginine residues
  • SAM S-adenosylmethionine
  • SAH S-adenosylhomocysteine
  • bicine Tween20
  • dimethylsulfoxide DMSO
  • bovine skin gelatin BSG
  • Tris(2- carboxyethyl)phosphine hydrochloride solution TCEP
  • 3 H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
  • 384-well streptavidin Flashplates were purchased from PerkinElmer.
  • Recombinant baculovirus were generated according to Bac- to-Bac kit instructions (Life Technologies). Protein over-expression was accomplished by infecting exponentially growing High Five insect cell culture at 1.5X10 6 cell/ml with 1 : 100 ratio of virus. Infections were carried out at 27°C for 48 hours, harvested by centrifugation, and stored at -80°C for purification.
  • DMSO DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of PRMTl, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
  • the final concentrations of the components were as follows: PRMTl was 0.5 nM, 3 H-SAM was 200 nM, non-radiolabeled SAM was 1.5 uM, peptide was 20 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%.
  • the assays were stopped by the addition of non- radiolabeled SAM (lOul) to a final concentration of 300 uM, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable.
  • dpm disintegrations per minute
  • cmpd signal in assay well
  • min and max are the respective minimum and maximum signal controls.
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • SAM S-adenosylmethionine
  • SAH S-adenosylhomocysteine
  • bicine Tween20
  • dimethylsulfoxide DMSO
  • bovine skin gelatin BSG
  • 3 H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
  • 384-well streptavidin Flashplates were purchased from PerkinElmer.
  • Flag-PRMT6-His (SEQ ID NO.: 7)
  • DMSO DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of PRMT6, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
  • the final concentrations of the components were as follows: PRMT6 was 1 nM, 3 H-SAM was 200 nM, non-radiolabeled SAM was 250 nM, peptide was 75 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%.
  • the assays were stopped by the addition of non- radiolabeled SAM (lOul) to a final concentration of 400 uM, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable.
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • SAM S-adenosylmethionine
  • SAH S-adenosylhomocysteine
  • bicine Tween20
  • dimethylsulfoxide DMSO
  • bovine skin gelatin BSG
  • isopropyl- ⁇ -D-thiogalactopyranoside IPTG
  • Tris(2-carboxyethyl)phosphine hydrochloride solution TCEP
  • 3 H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
  • 384-well streptavidin Flashplates were purchased from PerkinElmer.
  • E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl 2 method were transformed with the PRMT8 construct and ampicillin selection. Protein over-expression was accomplished by growing the PRMT8 expressing E. coli clone and inducing expression with 0.3 mM IPTG at 16°C. The culture was grown for 12 hours, harvested by centrifugation, and stored at -80°C for purification.
  • GST was removed by reloading the cleaved protein sample onto glutathione sepharose column and PRMT8 was collected in the flow-through fractions.
  • PRMT8 was purified further by ceramic hydroxyapatite chromatography. The column was washed with 50 mM phosphate buffer, 100 mM NaCl, 5% glycerol, 5 mM ⁇ -mercaptoethanol, pH 7.8 and PRMT8 was eluted by 100 mM phosphate in the same buffer. Protein was concentrated and buffer was exchanged to 50 mM Tris, 300 mM NaCl, 10% glycerol, 5 mM ⁇ - mercaptoethanol, pH 7.8 by ultrafiltration. The purity of recovered protein was 89%.
  • DMSO DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of PRMT8, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
  • the final concentrations of the components were as follows: PRMT8 was 1.5 nM, 3 H-SAM was 50 nM, non-radiolabeled SAM was 550 nM, peptide was 150 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%.
  • the assays were stopped by the addition of non- radiolabeled SAM (lOul) to a final concentration of 400 uM, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable.
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • SAM S-adenosylmethionine
  • SAH S-adenosylhomocysteine
  • bicine Tween20
  • dimethylsulfoxide DMSO
  • bovine skin gelatin BSG
  • IPTG isopropyl- -D-thiogalactopyranoside
  • TCEP Tris(2-carboxyethyl)phosphine hydrochloride solution
  • E. coli (BL21(DE3) Gold, Stratagene) made competent by the CaCl 2 method were transformed with the PRMT3 construct and ampicillin selection. Protein over-expression was accomplished by growing the PRMT3 expressing E. coli clone and inducing expression with 0.3 mM IPTG at 16°C. The culture was grown for 12 hours, harvested by centrifugation, and stored at -80°C for purification.
  • DMSO DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of PRMT3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
  • the final concentrations of the components were as follows: PRMT3 was 0.5 nM, 3 H-SAM was 100 nM, non-radiolabeled SAM was 1.8 uM, peptide was 330 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%.
  • the assays were stopped by the addition of potassium chloride (lOul) to a final concentration of 100 mM. 50ul of the reaction in the 384- well polypropylene plate was then transferred to a 384-well Flashplate and the biotinylated peptides were allowed to bind to the streptavidin surface for at least 1 hour before being washed once with 0.
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • SAM S-adenosylmethionine
  • SAH S-adenosylhomocysteine
  • bicine Tween20
  • dimethylsulfoxide DMSO
  • bovine skin gelatin BSG
  • 3 H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
  • 384-well streptavidin Flashplates were purchased from PerkinElmer.
  • DMSO DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of CARMl, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
  • the final concentrations of the components were as follows: CARMl was 0.25 nM, 3 H-SAM was 30 nM, peptide was 250 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%.
  • the assays were stopped by the addition of non-radiolabeled SAM (lOul) to a final concentration of 300 uM, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable. 50ul of the reaction in the 384-well
  • polypropylene plate was then transferred to a 384-well Flashplate and the biotinylated peptides were allowed to bind to the streptavidin surface for at least 1 hour before being washed once with 0. l%Tween20 in a Biotek ELx405 plate washer.
  • the plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3 H-labeled peptide bound to the Flashplate surface, measured as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
  • top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit.
  • the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit.
  • Y is the % inhibition and X is the compound concentration.
  • RKO adherent cells were purchased from ATCC (American Type Culture
  • DMEM/Glutamax medium penicillin-streptomycin, heat inactivated fetal bovine serum, 0.05% trypsin and D-PBS were purchased from Life
  • Imaging blocking buffer 800CW goat anti-rabbit IgG (H+L) antibody, and Licor Odyssey infrared scanner were purchased from Licor Biosciences, Lincoln, NE, USA.
  • Mono-methyl arginine antibody was purchased from Cell Signaling Technology, Danvers, MA, USA.
  • Methanol was purchased from VWR, Franklin, MA, USA.
  • 10% Tween 20 was purchased from KPL, Inc., Gaithersburg, Maryland, USA.
  • DRAQ5 was purchased from Biostatus Limited, Leicestershire, UK.
  • RKO adherent cells were maintained in growth medium (DMEM/Glutamax medium supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin) and cultured at 37 °C under 5% CO2.
  • growth medium DMEM/Glutamax medium supplemented with 10% v/v heat inactivated fetal bovine serum and 100 units/mL penicillin-streptomycin
  • Each plate included fourteen control wells of DMSO only treatment (minimum activation) as well as fourteen control wells for maximum activation treated with 20 ⁇ of a reference compound. The average of the ratio values for each control type was calculated and used to determine the percent activation for each test well in the plate. Reference compound was serially diluted three-fold in DMSO for a total of nine test concentrations, beginning at 20 ⁇ . Percent activation was determined and EC 30 curves were generated using triplicate wells per concentration of compound.

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Abstract

L'invention concerne divers composés, et des sels pharmaceutiquement acceptables de ceux-ci, ainsi que des compositions pharmaceutiques de ceux-ci, utiles pour inhiber l'activité de l'arginine méthyltransférase. L'invention concerne également des procédés d'utilisation desdits composés pour traiter des troubles médiés par l'arginine méthyltransférase.
PCT/US2017/016472 2016-02-05 2017-02-03 Inhibiteurs d'arginine méthyltransférase et leurs utilisations WO2017136699A1 (fr)

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US10118918B2 (en) 2012-12-21 2018-11-06 Epizyme, Inc. PRMT5 inhibitors and uses thereof
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WO2019054944A1 (fr) * 2017-09-12 2019-03-21 Agency For Science, Technology And Research Composés utiles en tant qu'inhibiteurs d'isoprénylcystéine carboxyl méthyltranférase
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WO2019196720A1 (fr) * 2018-04-08 2019-10-17 中国科学院上海药物研究所 Inhibiteur d'arginine méthyltransférase, composition pharmaceutique de celui-ci et son utilisation
CN110845474A (zh) * 2019-11-07 2020-02-28 四川大学 一种靶向i型prmt的化合物及其制备方法和应用
US10632103B2 (en) 2013-03-14 2020-04-28 Epizyme, Inc. Arginine methyltransferase inhibitors and uses thereof
US10653693B2 (en) 2014-08-04 2020-05-19 Epizyme, Inc. PRMT5 inhibitors and uses thereof
US11028083B2 (en) 2018-03-01 2021-06-08 Board Of Regents, The University Of Texas System Ethanediamine-heterocycle derivatives as inhibitors of protein arginine methyltransferases
CN113533727A (zh) * 2020-04-21 2021-10-22 上海市第一人民医院 精氨酸甲基转移酶3在乳腺癌诊疗中的应用
US11834455B2 (en) 2013-03-15 2023-12-05 Epizyme, Inc. Carm1 inhibitors and uses thereof
WO2025021997A1 (fr) 2023-07-27 2025-01-30 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Nouveaux inhibiteurs de map4k1

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