USOO963 O961 B2

(12) United States Patent (10) Patent No.: US 9,630,961 B2 Chesworth et al. (45) Date of Patent: *Apr. 25, 2017

(54) ARGINNE METHYLTRANSFERASE (56) References Cited INHIBITORS AND USES THEREOF U.S. PATENT DOCUMENTS Applicant: Epizyme, Inc., Cambridge, MA (US) (71) 5,011,849 A 4, 1991 Gassner et al. 5,204,482 A 4, 1993 Gassner et al. (72) Inventors: Richard Chesworth, Concord, MA 5,932,737 A 8, 1999 Itoh et al. (US); Lorna Helen Mitchell, 6,566,376 B1 5/2003 Baxter et al. Cambridge, MA (US); Gideon Shapiro, 6,914, 160 B1 7/2005 Armour et al. Gainesville, FL (US); Oscar Miguel 7,485,722 B2 2/2009 Egle et al. 7,629,294 B2 12/2009 Gebauer et al. Moradei, Burlington, MA (US) 7,632,855 B2 12/2009 Barrillalonso et al. 7,759,336 B2 7/2010 Habashita et al. (73) Assignee: Epizyme, Inc., Cambridge, MA (US) 8,063,071 B2 11/2011 Kerns et al. 8,097,708 B2 1/2012 Sugimoto et al. (*) Notice: Subject to any disclaimer, the term of this 8,133,904 B2 3/2012 McElroy et al. patent is extended or adjusted under 35 8,153,625 B2 4/2012 Habashita et al. 8.338,437 B2 12/2012 Wahhab et al. U.S.C. 154(b) by 0 days. 8,906,900 B2 12/2014 Duncan et al. 8,940,726 B2 1/2015 Duncan et al. This patent is Subject to a terminal dis 8,952,026 B2 2/2015 Mitchell et al. claimer. 8,993,555 B2 3/2015 Duncan et al. 9,023,883 B2 5, 2015 Kuntz et al. (21) Appl. No.: 14/808,270 9,045.455 B2 6, 2015 Mitchell et al. 9,120,757 B2 9/2015 Chesworth et al. (22) Filed: Jul. 24, 2015 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2016/O185772 A1 Jun. 30, 2016 DE 10149370 A1 4/2003 EP 1571 146 A1 9, 2005 Related U.S. Application Data (Continued) (63) Continuation of application No. 14/212.713, filed on OTHER PUBLICATIONS Mar. 14, 2014, now Pat. No. 9,133,189. Ito, Nobuyuki. Cancer Science 94(1), (2003) 3-8.* Database Registry Chemical Abstracts Service, Columbus, Ohio, (60) Provisional application No. 61/781,046, filed on Mar. Accession No. RN 1179596-03-2, Entered STN; Sep. 3, 2009.* 14, 2013. International Search Report and Written Opinion for International Application No. PCT/US2014/029009 mailed May 28, 2014. (51) Int. C. (Continued) CO7D 47L/04 (2006.01) CO7D 409/04 (2006.01) CO7D 403/04 (2006.01) Primary Examiner — Samantha Shterengarts CO7D 413/04 (2006.01) (74) Attorney, Agent, or Firm — Wolf, Greenfield & CO7D 23L/2 (2006.01) Sacks, P.C. CO7D 407/04 (2006.01) CO7D 4II/04 (2006.01) CO7D 405/04 (2006.01) (57) ABSTRACT CO7D 417/04 (2006.01) Described herein are compounds of Formula (I), pharma A6 IK 3L/45 (2006.01) ceutically acceptable salts thereof, and pharmaceutical com A6 IK 3/4I55 (2006.01) positions thereof. Compounds described herein are useful A6 IK 3/428 (2006.01) for inhibiting arginine methyltransferase activity. Methods A6 IK 3/437 (2006.01) of using the compounds for treating arginine methyltrans A6 IK 3/42 (2006.01) ferase-mediated disorders are also described. (52) U.S. C. CPC ...... C07D 471/04 (2013.01); A61K 31/415 (2013.01); A61K 31/4155 (2013.01); A61 K 3 1/42 (2013.01); A61K 31/428 (2013.01); HN R3 A61K 31/437 (2013.01); C07D 231/12 Q /N/ (2013.01); C07D 403/04 (2013.01); C07D 405/04 (2013.01); C07D 407/04 (2013.01); C07D 409/04 (2013.01); C07D 411/04 (2013.01); C07D 413/04 (2013.01); C07D 41704 (2013.01) (58) Field of Classification Search None See application file for complete search history. 24 Claims, No Drawings US 9,630,961 B2 Page 2

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INHIBITORS AND USES THEREOF In certain embodiments, methods of inhibiting an arginine methyltransferase are provided which comprise contacting RELATED APPLICATIONS the arginine methyltransferase with an effective amount of a compound of Formula (I), or a pharmaceutically acceptable The present application is a continuation of and claims salt thereof. The RMT may be purified or crude, and may be priority under 35 U.S.C. S 120 to U.S. patent application, present in a cell, tissue, or a Subject. Thus, such methods U.S. Ser. No. 14/212,713, filed Mar. 14, 2014, now U.S. Pat. encompass inhibition of RMT activity both in vitro and in No. 9,133,189, which claims priority under 35 U.S.C. S 119 vivo. In certain embodiments, the RMT is wild-type. In (e) to U.S. Provisional Patent Application, U.S. Ser. No. 10 certain embodiments, the RMT is overexpressed. In certain 61/781,046, filed Mar. 14, 2013, the entire contents of each embodiments, the RMT is a mutant. In certain embodiments, of which is incorporated herein by reference. the RMT is in a cell. In some embodiments, the RMT is expressed at normal levels in a subject, but the subject would BACKGROUND OF THE INVENTION benefit from RMT inhibition (e.g., because the subject has 15 one or more mutations in an RMT Substrate that causes an Epigenetic regulation of gene expression is an important increase in methylation of the substrate with normal levels biological determinant of protein production and cellular of RMT). In some embodiments, the RMT is in a subject differentiation and plays a significant pathogenic role in a known or identified as having abnormal RMT activity (e.g., number of human diseases. overexpression). Epigenetic regulation involves heritable modification of In certain embodiments, methods of modulating gene genetic material without changing its nucleotide sequence. expression in a cell are provided which comprise contacting Typically, epigenetic regulation is mediated by selective and a cell with an effective amount of a compound of Formula reversible modification (e.g., methylation) of DNA and (I), or a pharmaceutically acceptable salt thereof, or a proteins (e.g., histones) that control the conformational pharmaceutical composition thereof. In certain embodi transition between transcriptionally active and inactive 25 ments, the cell in culture in vitro. In certain embodiments, states of chromatin. These covalent modifications can be cell is in an animal, e.g., a human. controlled by enzymes such as methyltransferases (e.g., In certain embodiments, methods of modulating transcrip arginine methyltransferases), many of which are associated tion in a cell are provided which comprise contacting a cell with specific genetic alterations that can cause human dis with an effective amount of a compound of Formula (I), or CaSC. 30 a pharmaceutically acceptable salt thereof, or a pharmaceu Disease-associated chromatin-modifying enzymes (e.g., tical composition thereof. In certain embodiments, the cell in arginine methyltransferases) play a role in diseases such as culture in vitro. In certain embodiments, the cell is in an proliferative disorders, autoimmune disorders, muscular dis animal, e.g., a human. orders, vascular disorders, metabolic disorders, and neuro In some embodiments, methods of treating an RMT logical disorders. Thus, there is a need for the development 35 mediated disorder (e.g., a PRMT1-, PRMT3-, CARM1-, of small molecules that are capable of inhibiting the activity PRMT6-, or PRMT8-mediated disorder) are provided which of arginine methyltransferases. comprise administering to a subject Suffering from an RMT mediated disorder an effective amount of a compound DETAILED DESCRIPTION OF CERTAIN described herein (e.g., a compound of Formula (I)), or a EMBODIMENTS 40 pharmaceutically acceptable salt thereof, or a pharmaceuti cal composition thereof. In certain embodiments, the RMT Arginine methyltransferases are attractive targets for mediated disorder is a proliferative disorder. In certain modulation given their role in the regulation of diverse embodiments, compounds described herein are useful for biological processes. It has now been found that compounds treating cancer. In certain embodiments, compounds described herein, and pharmaceutically acceptable salts and 45 described herein are useful for treating breast cancer, pros compositions thereof, are effective as inhibitors of arginine tate cancer, lung cancer, colon cancer, bladder cancer, or methyltransferases. Such compounds have the general For leukemia. In certain embodiments, the RMT-mediated dis mula (I): order is a muscular disorder. In certain embodiments, the RMT-mediated disorder is an autoimmune disorder. In cer 50 tain embodiments, the RMT-mediated disorder is a neuro logical disorder. In certain embodiments, the RMT-mediated disorder is a vascular disorder. In certain embodiments, the RMT-mediated disorder is a metabolic disorder. Compounds described herein are also useful for the study 55 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. or a pharmaceutically acceptable salt thereof, wherein X, Y, This application refers to various issued patent, published Z, Q, R, and R are as defined herein. 60 patent applications, journal articles, and other publications, In some embodiments, pharmaceutical compositions are all of which are incorporated herein by reference. provided which comprise a compound described herein Definitions of specific functional groups and chemical (e.g., a compound of Formula (I)), or a pharmaceutically terms are described in more detail below. The chemical acceptable salt thereof, and optionally a pharmaceutically elements are identified in accordance with the Periodic Table acceptable excipient. 65 of the Elements, CAS version, Handbook of Chemistry and In certain embodiments, compounds described herein Physics, 75" Ed., inside cover, and specific functional inhibit activity of an arginine methyltransferase (RMT) groups are generally defined as described therein. Addition US 9,630,961 B2 3 4 ally, general principles of organic chemistry, as well as Co. C1-6 C1-s: C-4 C1-3. C1-2, C2-6. C2-s: C2-4 C2-3. C3-6. specific functional moieties and reactivity, are described in Cs-s: C-4 C4-6. Cals, and CS-6 alkyl. Thomas Sorrell, Organic Chemistry, University Science “Alkyl refers to a radical of a straight-chain or branched Books, Sausalito, 1999; Smith and March, March's saturated hydrocarbon group having from 1 to 20 carbon Advanced Organic Chemistry, 5" Edition, John Wiley & atoms (“Co alkyl). In some embodiments, an alkyl group Sons, Inc., New York, 2001: Larock, Comprehensive has 1 to 10 carbon atoms (“Clio alkyl). In some embodi Organic Transformations, VCH Publishers, Inc., New York, ments, an alkyl group has 1 to 9 carbon atoms (“Co alkyl). 1989; and Carruthers. Some Modern Methods of Organic In some embodiments, an alkyl group has 1 to 8 carbon Synthesis, 3" Edition, Cambridge University Press, Cam atoms (“Cs alkyl). In some embodiments, an alkyl group bridge, 1987. 10 has 1 to 7 carbon atoms (“C, alkyl). In some embodi Compounds described herein can comprise one or more ments, an alkyl group has 1 to 6 carbonatoms ("Ce alkyl). asymmetric centers, and thus can exist in various isomeric In some embodiments, an alkyl group has 1 to 5 carbon forms, e.g., enantiomers and/or diastereomers. For example, atoms (“Cs alkyl). In some embodiments, an alkyl group the compounds described herein can be in the form of an has 1 to 4 carbon atoms ("C. alkyl). In some embodi individual enantiomer, diastereomer or geometric isomer, or 15 ments, an alkyl group has 1 to 3 carbonatoms (“Cs alkyl). can be in the form of a mixture of stereoisomers, including In some embodiments, an alkyl group has 1 to 2 carbon racemic mixtures and mixtures enriched in one or more atoms ("C. alkyl). In some embodiments, an alkyl group Stereoisomer. Isomers can be isolated from mixtures by has 1 carbon atom ("Ce alkyl). In some embodiments, an methods known to those skilled in the art, including chiral alkyl group has 2 to 6 carbon atoms ("Ce alkyl). Examples high pressure liquid chromatography (HPLC) and the for of C alkyl groups include methyl (C), ethyl (C), n-pro mation and crystallization of chiral salts; or preferred iso pyl (C), isopropyl (C), n-butyl (C), tert-butyl (C), sec mers can be prepared by asymmetric syntheses. See, for butyl (C), iso-butyl (C), n-pentyl (Cs), 3-pentanyl (Cs), example, Jacques et al., Enantiomers, Racemates and Reso amyl (Cs), neopentyl (Cs), 3-methyl-2-butanyl (Cs), tertiary lutions (Wiley Interscience, New York, 1981); Wilen et al., amyl (Cs), and n-hexyl (C). Additional examples of alkyl Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Car 25 groups include n-heptyl (C7), n-octyl (Cs) and the like. In bon Compounds (McGraw-Hill, NY. 1962); and Wilen, certain embodiments, each instance of an alkyl group is Tables of Resolving Agents and Optical Resolutions p. 268 independently optionally Substituted, e.g., unsubstituted (an (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, “unsubstituted alkyl) or substituted (a “substituted alkyl) Ind. 1972). The present disclosure additionally encompasses with one or more substituents. In certain embodiments, the compounds described herein as individual isomers Substan 30 alkyl group is unsubstituted Co alkyl (e.g., —CH). In tially free of other isomers, and alternatively, as mixtures of certain embodiments, the alkyl group is Substituted Co various isomers. alkyl. It is to be understood that the compounds of the present In some embodiments, an alkyl group is substituted with invention may be depicted as different tautomers. It should one or more halogens. “Perhaloalkyl is a substituted alkyl also be understood that when compounds have tautomeric 35 group as defined herein wherein all of the hydrogen atoms forms, all tautomeric forms are intended to be included in are independently replaced by a halogen, e.g., fluoro, bromo, the scope of the present invention, and the naming of any chloro, or iodo. In some embodiments, the alkyl moiety has compound described herein does not exclude any tautomer 1 to 8 carbon atoms (“Cs perhaloalkyl). In some embodi form. ments, the alkyl moiety has 1 to 6 carbon atoms ("Ce

N'-((3-(1H-indazol-4-yl)-1H-pyrazol-4- yl)methyl)-N'-methylethane-1,2-diamine

55 Unless otherwise stated, structures depicted herein are perhaloalkyl). In some embodiments, the alkyl moiety has also meant to include compounds that differ only in the 1 to 4 carbon atoms ("Ca perhaloalkyl). In some embodi presence of one or more isotopically enriched atoms. For ments, the alkyl moiety has 1 to 3 carbon atoms ("C. example, compounds having the present structures except perhaloalkyl). In some embodiments, the alkyl moiety has for the replacement of hydrogen by deuterium or tritium, 60 1 to 2 carbon atoms (“C, perhaloalkyl). In some embodi replacement of F with F, or the replacement of a carbon ments, all of the hydrogen atoms are replaced with fluoro. In by a C- or 'C-enriched carbon are within the scope of the Some embodiments, all of the hydrogen atoms are replaced disclosure. Such compounds are useful, for example, as with chloro. Examples of perhaloalkyl groups include analytical tools or probes in biological assays. —CF, CFCF, —CFCFCF —CC1, —CFC1, When a range of values is listed, it is intended to encom 65 —CFC1, and the like. pass each value and Sub-range within the range. For example As used herein, “alkenyl refers to a radical of a straight "Ce alkyl is intended to encompass, C, C, C, C, Cs, chain or branched hydrocarbon group having from 2 to 20 US 9,630,961 B2 5 6 carbon atoms and one or more carbon-carbon double bonds “Carbocyclyl or “carbocyclic” refers to a radical of a (e.g., 1, 2, 3, or 4 double bonds), and optionally one or more non-aromatic cyclic hydrocarbon group having from 3 to 10 triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("Coalkenyl). ring carbon atoms (“Clio carbocyclyl) and Zero heteroa In certain embodiments, alkenyl does not comprise triple toms in the non-aromatic ring system. In some embodi bonds. In some embodiments, an alkenyl group has 2 to 10 ments, a carbocyclyl group has 3 to 8 ring carbon atoms carbon atoms ("Coalkenyl). In some embodiments, an (“Cs carbocyclyl). In some embodiments, a carbocyclyl alkenyl group has 2 to 9 carbon atoms (“Coalkenyl). In group has 3 to 6 ring carbon atoms (“C. carbocyclyl). In Some embodiments, an alkenyl group has 2 to 8 carbon Some embodiments, a carbocyclyl group has 3 to 6 ring atoms (“Cs alkenyl). In some embodiments, an alkenyl carbon atoms ("C. carbocyclyl). In some embodiments, a 10 carbocyclyl group has 5 to 10 ring carbon atoms (“Cso group has 2 to 7 carbon atoms ("C-7 alkenyl). In some carbocyclyl). Exemplary C. carbocyclyl groups include, embodiments, an alkenyl group has 2 to 6 carbon atoms without limitation, cyclopropyl (C), cyclopropenyl (C), ("Ce alkenyl). In some embodiments, an alkenyl group cyclobutyl (C), cyclobutenyl (C), cyclopentyl (Cs), cyclo has 2 to 5 carbon atoms (“Cs alkenyl). In some embodi pentenyl (Cs), cyclohexyl (C), cyclohexenyl (C), cyclo ments, an alkenyl group has 2 to 4 carbon atoms ("C. 15 hexadienyl (C), and the like. Exemplary Cs carbocyclyl alkenyl). In some embodiments, an alkenyl group has 2 to groups include, without limitation, the aforementioned C. 3 carbon atoms ("C- alkenyl). In some embodiments, an carbocyclyl groups as well as cycloheptyl (C7), cyclohep alkenyl group has 2 carbon atoms ("Calkenyl). The one or tenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7). more carbon-carbon double bonds can be internal (such as in cyclooctyl (Cs), cyclooctenyl (Cs), bicyclo2.2.1]heptanyl 2-butenyl) or terminal (such as in 1-butenyl). Examples of (C7), bicyclo[2.2.2]octanyl (Cs), and the like. Exemplary Calkenyl groups include ethenyl (C), 1-propenyl (C), C. carbocyclyl groups include, without limitation, the 2-propenyl (C), 1-butenyl (C), 2-butenyl (C), butadienyl aforementioned Cls carbocyclyl groups as well as (C), and the like. Examples of Calkenyl groups include cyclononyl (C), cyclononenyl (C), cyclodecyl (C), the aforementioned Calkenyl groups as well as pentenyl cyclodecenyl (Co), octahydro-1H-indenyl (Co), decahy (Cs), pentadienyl (Cs), hexenyl (C), and the like. Additional 25 dronaphthalenyl (Co), Spiro4.5 decanyl (Co), and the like. examples of alkenyl include heptenyl (C7), octenyl (Cs), As the foregoing examples illustrate, in certain embodi octatrienyl (Cs), and the like. In certain embodiments, each ments, the carbocyclyl group is either monocyclic ("mono instance of an alkenyl group is independently optionally cyclic carbocyclyl) or contain a fused, bridged or spiro ring substituted, e.g., unsubstituted (an “unsubstituted alkenyl) system such as a bicyclic system (“bicyclic carbocyclyl) or substituted (a “substituted alkenyl) with one or more 30 and can be saturated or can be partially unsaturated. "Car Substituents. In certain embodiments, the alkenyl group is bocyclyl also includes ring systems wherein the carbocy unsubstituted Co alkenyl. In certain embodiments, the clyl ring, as defined above, is fused with one or more aryl or alkenyl group is substituted Coalkenyl. heteroaryl groups wherein the point of attachment is on the As used herein, “alkynyl refers to a radical of a straight carbocyclyl ring, and in Such instances, the number of chain or branched hydrocarbon group having from 2 to 20 35 carbons continue to designate the number of carbons in the carbon atoms and one or more carbon-carbon triple bonds carbocyclic ring system. In certain embodiments, each (e.g., 1, 2, 3, or 4 triple bonds), and optionally one or more instance of a carbocyclyl group is independently optionally double bonds (e.g., 1, 2, 3, or 4 double bonds) ("Co Substituted, e.g., unsubstituted (an "unsubstituted carbocy alkynyl). In certain embodiments, alkynyl does not com clyl) or substituted (a “substituted carbocyclyl) with one prise double bonds. In some embodiments, an alkynyl group 40 or more Substituents. In certain embodiments, the carbocy has 2 to 10 carbon atoms (“Clio alkynyl). In some embodi clyl group is unsubstituted Co carbocyclyl. In certain ments, an alkynyl group has 2 to 9 carbon atoms ("C- embodiments, the carbocyclyl group is a substituted Co alkynyl). In some embodiments, an alkynyl group has 2 to carbocyclyl. 8 carbon atoms ("Cs alkynyl). In some embodiments, an In some embodiments, “carbocyclyl is a monocyclic, alkynyl group has 2 to 7 carbon atoms ("C-7 alkynyl). In 45 saturated carbocyclyl group having from 3 to 10 ring carbon Some embodiments, an alkynyl group has 2 to 6 carbon atoms (“Clio cycloalkyl). In some embodiments, a atoms ("C. alkynyl). In some embodiments, an alkynyl cycloalkyl group has 3 to 8 ring carbon atoms ("Cs group has 2 to 5 carbon atoms (“Cs alkynyl). In some cycloalkyl). In some embodiments, a cycloalkyl group has embodiments, an alkynyl group has 2 to 4 carbon atoms 3 to 6 ring carbon atoms (“C cycloalkyl). In some (“C alkynyl). In some embodiments, an alkynyl group 50 embodiments, a cycloalkyl group has 5 to 6 ring carbon has 2 to 3 carbon atoms (“C- alkynyl). In some embodi atoms (“Cs cycloalkyl). In some embodiments, a ments, an alkynyl group has 2 carbon atoms ("C. alkynyl). cycloalkyl group has 5 to 10 ring carbon atoms (“Cso The one or more carbon-carbon triple bonds can be internal cycloalkyl). Examples of Cse cycloalkyl groups include (such as in 2-butynyl) or terminal (such as in 1-butynyl). cyclopentyl (Cs) and cyclohexyl (Cs). Examples of C. Examples of Calkynyl groups include, without limitation, 55 cycloalkyl groups include the aforementioned Cs. ethynyl (C), 1-propynyl (C), 2-propynyl (C), 1-butynyl cycloalkyl groups as well as cyclopropyl (C) and cyclobu (C), 2-butynyl (C), and the like. Examples of Calkenyl tyl (C). Examples of Css cycloalkyl groups include the groups include the aforementioned C. alkynyl groups as aforementioned C. cycloalkyl groups as well as cyclohep well as pentynyl (Cs), hexynyl (C), and the like. Additional tyl (C7) and cyclooctyl (Cs). In certain embodiments, each examples of alkynyl include heptynyl (C7), octynyl (Cs), 60 instance of a cycloalkyl group is independently unsubsti and the like. In certain embodiments, each instance of an tuted (an “unsubstituted cycloalkyl) or substituted (a “sub alkynyl group is independently optionally Substituted, e.g., stituted cycloalkyl) with one or more substituents. In cer unsubstituted (an "unsubstituted alkynyl) or substituted (a tain embodiments, the cycloalkyl group is unsubstituted “substituted alkynyl) with one or more substituents. In Co cycloalkyl. In certain embodiments, the cycloalkyl certain embodiments, the alkynyl group is unsubstituted 65 group is Substituted Co cycloalkyl. Co alkynyl. In certain embodiments, the alkynyl group is “Heterocyclyl or "heterocyclic” refers to a radical of a 3 Substituted Co alkynyl. to 10-membered non-aromatic ring system having ring car US 9,630,961 B2 7 8 bon atoms and 1 to 4 ring heteroatoms, wherein each heterocyclyl groups containing two heteroatoms include, heteroatom is independently selected from nitrogen, oxygen, without limitation, triazinanyl. Exemplary 7-membered het and sulfur (3-10 membered heterocyclyl). In heterocyclyl erocyclyl groups containing one heteroatom include, with groups that contain one or more nitrogen atoms, the point of out limitation, azepanyl, oxepanyl and thiepanyl. Exemplary attachment can be a carbon or nitrogen atom, as Valency 5 8-membered heterocyclyl groups containing one heteroatom permits. A heterocyclyl group can either be monocyclic include, without limitation, azocanyl, oxecanyl, and thioca ("monocyclic heterocyclyl) or a fused, bridged or spiro ring nyl. Exemplary 5-membered heterocyclyl groups fused to a system such as a bicyclic system (“bicyclic heterocyclyl), Caryl ring (also referred to herein as a 5,6-bicyclic het and can be saturated or can be partially unsaturated. Het erocyclic ring) include, without limitation, indolinyl, isoin erocyclyl bicyclic ring systems can include one or more 10 dolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzo heteroatoms in one or both rings. “Heterocyclyl also xazolinonyl, and the like. Exemplary 6-membered includes ring systems wherein the heterocyclyl ring, as heterocyclyl groups fused to an aryl ring (also referred to defined above, is fused with one or more carbocyclyl groups herein as a 6,6-bicyclic heterocyclic ring) include, without wherein the point of attachment is either on the carbocyclyl limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, or heterocyclyl ring, or ring systems wherein the heterocy 15 and the like. clyl ring, as defined above, is fused with one or more aryl or "Aryl refers to a radical of a monocyclic or polycyclic heteroaryl groups, wherein the point of attachment is on the (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., heterocyclyl ring, and in Such instances, the number of ring having 6, 10, or 14 it electrons shared in a cyclic array) members continue to designate the number of ring members having 6-14 ring carbon atoms and Zero heteroatoms pro in the heterocyclyl ring system. In certain embodiments, vided in the aromatic ring system ("Caryl'). In some each instance of heterocyclyl is independently optionally embodiments, an aryl group has six ring carbon atoms (“C Substituted, e.g., unsubstituted (an "unsubstituted heterocy aryl'; e.g., phenyl). In some embodiments, an aryl group has clyl) or substituted (a “substituted heterocyclyl) with one ten ring carbon atoms (“Co aryl”; e.g., naphthyl such as or more Substituents. In certain embodiments, the heterocy 1-naphthyl and 2-maphthyl). In some embodiments, an aryl clyl group is unsubstituted 3-10 membered heterocyclyl. In 25 group has fourteen ring carbon atoms ("C. aryl', e.g., certain embodiments, the heterocyclyl group is Substituted anthracy1). “Aryl also includes ring systems wherein the 3-10 membered heterocyclyl. aryl ring, as defined above, is fused with one or more In some embodiments, a heterocyclyl group is a 5-10 carbocyclyl or heterocyclyl groups wherein the radical or membered non-aromatic ring system having ring carbon point of attachment is on the aryl ring, and in Such instances, atoms and 1-4 ring heteroatoms, wherein each heteroatom is 30 the number of carbon atoms continue to designate the independently selected from nitrogen, oxygen, and Sulfur number of carbon atoms in the aryl ring system. In certain (“5-10 membered heterocyclyl”). In some embodiments, a embodiments, each instance of an aryl group is indepen heterocyclyl group is a 5-8 membered non-aromatic ring dently optionally Substituted, e.g., unsubstituted (an "unsub system having ring carbon atoms and 1-4 ring heteroatoms, stituted aryl') or substituted (a “substituted aryl') with one wherein each heteroatom is independently selected from 35 or more Substituents. In certain embodiments, the aryl group nitrogen, oxygen, and Sulfur ("5-8 membered heterocy is unsubstituted Caryl. In certain embodiments, the aryl clyl). In some embodiments, a heterocyclyl group is a 5-6 group is Substituted Caryl. membered non-aromatic ring system having ring carbon “Heteroaryl” refers to a radical of a 5-10 membered atoms and 1-4 ring heteroatoms, wherein each heteroatom is monocyclic or bicyclic 4n+2 aromatic ring system (e.g., independently selected from nitrogen, oxygen, and Sulfur 40 having 6 or 10 t electrons shared in a cyclic array) having (“5-6 membered heterocyclyl). In some embodiments, the ring carbon atoms and 1-4 ring heteroatoms provided in the 5-6 membered heterocyclyl has 1-3 ring heteroatoms aromatic ring system, wherein each heteroatom is indepen selected from nitrogen, oxygen, and Sulfur. In some embodi dently selected from nitrogen, oxygen and sulfur (“5-10 ments, the 5-6 membered heterocyclyl has 1-2 ring heteroa membered heteroaryl'). In heteroaryl groups that contain toms selected from nitrogen, oxygen, and Sulfur. In some 45 one or more nitrogen atoms, the point of attachment can be embodiments, the 5-6 membered heterocyclyl has one ring a carbon or nitrogen atom, as Valency permits. Heteroaryl heteroatom selected from nitrogen, oxygen, and Sulfur. bicyclic ring systems can include one or more heteroatoms Exemplary 3-membered heterocyclyl groups containing in one or both rings. “Heteroaryl” includes ring systems one heteroatom include, without limitation, azirdinyl, oxi wherein the heteroaryl ring, as defined above, is fused with ranyl, and thiorenyl. Exemplary 4-membered heterocyclyl 50 one or more carbocyclyl or heterocyclyl groups wherein the groups containing one heteroatom include, without limita point of attachment is on the heteroaryl ring, and in Such tion, aZetidinyl, oxetanyl, and thietanyl. Exemplary 5-mem instances, the number of ring members continue to designate bered heterocyclyl groups containing one heteroatom the number of ring members in the heteroaryl ring system. include, without limitation, tetrahydrofuranyl, dihydrofura “Heteroaryl also includes ring systems wherein the het nyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, 55 eroaryl ring, as defined above, is fused with one or more aryl dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-mem groups wherein the point of attachment is either on the aryl bered heterocyclyl groups containing two heteroatoms or heteroaryl ring, and in Such instances, the number of ring include, without limitation, dioxolanyl, oxasulfuranyl, dis members designates the number of ring members in the ulfuranyl, and oxazolidin-2-one. Exemplary 5-membered fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl heterocyclyl groups containing three heteroatoms include, 60 groups wherein one ring does not contain a heteroatom (e.g., without limitation, triazolinyl, oxadiazolinyl, and thiadiaz indolyl, quinolinyl, carbazolyl, and the like) the point of olinyl. Exemplary 6-membered heterocyclyl groups contain attachment can be on either ring, e.g., either the ring bearing ing one heteroatom include, without limitation, piperidinyl, a heteroatom (e.g., 2-indolyl) or the ring that does not tetrahydropyranyl, dihydropyridinyl, and thianyl. Exem contain a heteroatom (e.g., 5-indolyl). plary 6-membered heterocyclyl groups containing two het 65 In some embodiments, a heteroaryl group is a 5-10 eroatoms include, without limitation, piperazinyl, morpholi membered aromatic ring system having ring carbon atoms nyl, dithianyl, and dioxanyl. Exemplary 6-membered and 1-4 ring heteroatoms provided in the aromatic ring US 9,630,961 B2 10 system, wherein each heteroatom is independently selected -continued from nitrogen, oxygen, and sulfur (“5-10 membered het 21 eroaryl'). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms Sa and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected O from nitrogen, oxygen, and sulfur (“5-8 membered het eroaryl'). In some embodiments, a heteroaryl group is a 5-6 / / membered aromatic ring system having ring carbon atoms 2 10 NSas and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and Sulfur ("5-6 membered het No1S-1~21 eroaryl'). In some embodiments, the 5-6 membered het 21NCC)NNN-V/ NaNs'CN eroaryl has 1-3 ring heteroatoms selected from nitrogen, 15 21 e oxygen, and Sulfur. In some embodiments, the 5-6 mem O, S, bered heteroaryl has 1-2 ring heteroatoms selected from C N n 1S nitrogen, oxygen, and Sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from Sl-O N nitrogen, oxygen, and Sulfur. In certain embodiments, each instance of a heteroaryl group is independently optionally 7 u/ 21 / substituted, e.g., unsubstituted (“unsubstituted heteroaryl') or substituted (“substituted heteroaryl') with one or more Substituents. In certain embodiments, the heteroaryl group is N, unsubstituted 5-14 membered heteroaryl. In certain embodi 25 ) ments, the heteroaryl group is substituted 5-14 membered heteroaryl. Exemplary 5-membered heteroaryl groups containing one y y heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups con 30 e taining two heteroatoms include, without limitation, imida S Zolyl pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothi S. o M azolyl. Exemplary 5-membered heteroaryl groups C. N N containing three heteroatoms include, without limitation, 35 21 N21 triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-mem S, bered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-mem C N N bered heteroaryl groups containing one heteroatom include, e e without limitation, pyridinyl. Exemplary 6-membered het 40 N eroaryl groups containing two heteroatoms include, without N s / N S/ N-N limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exem plary 6-membered heteroaryl groups containing three or four e 21 e 21 N2 heteroatoms include, without limitation, triazinyl and tet N ss N 45 / 2O7 razinyl, respectively. Exemplary 7-membered heteroaryl N s-N-/ groups containing one heteroatom include, without limita tion, azepinyl, oxepinyl, and thiepinyl. Exemplary 6,6- e N N bicyclic heteroaryl groups include, without limitation, naph N Y. N Y / 2 thyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, N % o? 50 YN quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, 21 N2 any one of the following formulae: N, NH, O. N21N C. s s 55 N O S, N ry 2 AR 2N/ N S O N H e e e N 21 2N 60 N, NH, O, S. N 1S OOOOOOS. S. s21N N C eS N N N N2 S, 65 S. 2 ). Cy CyO C) N2N/N2 H N s N 2Ns/ N y 4. O

US 9,630,961 B2 13 14 -continued -continued CC)Sl-N CI)N N CySl-N CXCC)e 2N. e 2N N21N2N.C) -N. N N 1- s re s 2N N, CC)2NO CO,% s/ CO4. s/ ls, Ns S. NN/ N 10 1- N, r- N, and 4N2N N N4 s/ Na2 s/ Na2 s/ N--/ Nsu-NN/ s -N-N/ N N 15 In any of the monocyclic or bicyclic heteroaryl groups, the point of attachment can be any carbon or nitrogen atom, as 4Ns/ 2Ns/ 4Ns/ Valency permits. N “Partially unsaturated refers to a group that includes at least one double or triple bond. The term “partially unsatu N 21 s/ 4. s/ S N/ rated' is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic 21 N N N N groups (e.g., aryl or heteroaryl groups) as herein defined. Likewise, “saturated’ refers to a group that does not contain N1SNCCC). 4. S Cy4. S 25 a double or triple bond, i.e., contains all single bonds. In some embodiments, alkyl, alkenyl, alkynyl, carbocy N N N N clyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or N4CC) s' Na2NsCy Na2NsCy “unsubstituted alkyl, “substituted' or “unsubstituted alk N 30 enyl, “substituted' or “unsubstituted alkynyl, “substituted or “unsubstituted carbocyclyl, “substituted' or “unsubsti y Syr tuted” heterocyclyl, “substituted” or “unsubstituted” aryl or 2 y Nu- N-N? “substituted' or “unsubstituted heteroaryl group). In gen eral, the term “substituted, whether preceded by the term 35 “optionally or not, means that at least one hydrogen present y ry N21 N2 on a group (e.g., a carbon or nitrogen atom) is replaced with C) sus. ls-- a permissible Substituent, e.g., a Substituent which upon Substitution results in a stable compound, e.g., a compound 21 N2 21 2N N which does not spontaneously undergo transformation Such 40 as by rearrangement, cyclization, elimination, or other reac Nn N/ N s^ ls-- tion. Unless otherwise indicated, a “substituted’ group has a N N substituent at one or more substitutable positions of the 21 e 2 e N21 e group, and when more than one position in any given N, ZN ZN structure is substituted, the substituent is either the same or Nsu-N, N-N, Nsu-N, 45 different at each position. The term “substituted' is contem N plated to include substitution with all permissible substitu ents of organic compounds, including any of the Substituents 1- N, 21 N2\ N, N2 y s described herein that results in the formation of a stable ls-- Ns-N, N-- compound. The present disclosure contemplates any and all N N 50 Such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitro gen may have hydrogen Substituents and/or any Suitable N&CC). NN/ C).N-N M N-N?C.) substituent as described herein which satisfy the valencies of N the heteroatoms and results in the formation of a stable 55 moiety. s s N, Exemplary carbon atom Substituents include, but are not pyrry limited to, halogen, —CN, - NO. —N, -SOH, ls-- s-N? N- M - SOH, OH, OR", ON(R), N(R), N N N(R),"X, N(OR)R, SH, SR', SSR, rSlyN, r-, N, 1 N, 60 C(=O)R", COH, CHO, C(OR), COR", OC(=O)R*, OCOR', C(=O)N(R), OC N-N M N-N M N-N / (—O)N(R), NRC(=O)R', NRCO.R.", N NRC(=O)N(R), C(-NR)R, C(=NR) 1- N, 21 N2\ N, N2 N s OR, OC( NR)R*, OC( NR)OR*, 65 C(-NR)N(R), OC(-NR)N(R), NRC ls -NN/ Ns-N-N/N Nu (—NR)N(R), C(=O)NRSOR, NRSOR, - SON(R) - SOR", SOOR", OSOR",

US 9,630,961 B2 17 18 Co perhaloalkyl, Coalkenyl, Co alkynyl, Cao car carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluene bocyclyl. 3-14 membered heterocyclyl, C-aryl, and 5-14 sulfonyl)ethyl carbamate, 2-(1,3-dithianyl)methyl carbam membered heteroaryl, or two R groups attached to a ate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- nitrogen atom are joined to form a 3-14 membered hetero dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl cyclyl or 5-14 membered heteroaryl ring, wherein each 5 carbamate (Peoc), 2-triphenylphosphonioisopropyl carbam alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and ate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro heteroaryl is independently substituted with 0, 1, 2, 3, 4, or p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl car 5 R* groups, and wherein R", R', R* and R* are as bamate, 5-benzisoxazolylmethyl carbamate, defined above. 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), In certain embodiments, the Substituent present on a 10 m-nitrophenyl carbamate, 3.5-dimethoxybenzyl carbamate, nitrogen atom is a nitrogen protecting group (also referred to o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl car as an amino protecting group). Nitrogen protecting groups bamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl include, but are not limited to, —OH, - OR', —N(R), carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbam -C(=O)R", C(=O)N(R), CO.R.", -SOR", ate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopen C(-NR)R", C(-NR)OR', C(-NR)N 15 tyl carbamate, cyclopropylmethyl carbamate, p-decyloxy (R), -SON(R), -SOR-, -SOOR-, -SOR", benzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N. C(=S)N(R), C(=O)SR, C(=S)SR, Co N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3- alkyl (e.g., aralkyl, heteroaralkyl). Coalkenyl, C2-io alky (N,N-dimethylcarboxamido)propyl carbamate, 1,1- nyl, Co carbocyclyl, 3-14 membered heterocyclyl, Ca dimethylpropynyl carbamate, di(2-pyridyl)methyl aryl, and 5-14 membered heteroaryl groups, wherein each carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbam alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, ate, isoborynl carbamate, isobutyl carbamate, isonicotinyl aryl, and heteroaryl is independently substituted with 0, 1, 2, carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 3, 4, or 5 R' groups, and wherein R', R. R., and R* are 1-methylcyclobutyl carbamate, 1-methylcyclohexyl car as defined herein. Nitrogen protecting groups are well bamate, 1-methyl-1-cyclopropylmethyl carbamate, known in the art and include those described in detail in 25 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, Protecting Groups in Organic Synthesis, T. W. Greene and 1-methyl-1-(p-phenylaZophenyl)ethyl carbamate, 1-methyl P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, 1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl car incorporated herein by reference. bamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, Amide nitrogen protecting groups (e.g., —C(=O)R“) 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium) include, but are not limited to, formamide, acetamide, chlo 30 benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate. roacetamide, trichloroacetamide, trifluoroacetamide, pheny Sulfonamide nitrogen protecting groups (e.g., —S(=O) lacetamide, 3-phenylpropanamide, picolinamide, 3-pyridyl R“) include, but are not limited to, p-toluenesulfonamide carboxamide, N-benzoylphenylalanyl derivative, (Ts), benzenesulfonamide, 2.3.6.-trimethyl-4-methoxyben benzamide, p-phenylbenzamide, o-nitophenylacetamide, Zenesulfonamide (Mitr), 2,4,6-trimethoxybenzenesulfona o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzy 35 mide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide loxyacylamino)acetamide, 3-(p-hydroxyphenyl)propana (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide mide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrop (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimeth henoxy)propanamide, 2-methyl-2-(o-phenylaZophenoxy) ylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylben propanamide, 4-chlorobutanamide, 3-methyl-3- Zenesulfonamide (iMds), 2.2.5.7.8-pentamethylchroman-6- nitrobutanamide, o-nitrocinnamide, N-acetylmethionine, 40 Sulfonamide (Pmc), methanesulfonamide (Ms), o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide. B-trimethylsilylethanesulfonamide (SES), 9-anthracenesul Carbamate nitrogen protecting groups (e.g., —C(=O) fonamide, 4-(4,8-dimethoxynaphthylmethyl)benzenesulfo OR') include, but are not limited to, methyl carbamate, namide (DNMBS), benzylsulfonamide, trifluoromethylsul ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), fonamide, and phenacylsulfonamide. 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo) fluo 45 Other nitrogen protecting groups include, but are not roenylmethyl carbamate, 2,7-di-t-butyl-9-(10,10-dioxo-10, limited to, phenothiazinyl-(10)-acyl derivative, N'-p-tolu 10,10,10-tetrahydrothioxanthyl)methyl carbamate (DBD enesulfonylaminoacyl derivative, N'-phenylaminothioacyl Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2.2.2- derivative, N-benzoylphenylalanyl derivative, N-acetylme trichloroethyl carbamate (Troc), 2-trimethylsilylethyl thionine derivative, 4,5-diphenyl-3-oxazolin-2-one, carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-ada 50 N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenyl mantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2- maleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyld haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl car isilylazacyclopentane adduct (STABASE), 5-substituted bamate (DB-t-BOC), 1,1-dimethyl-2.2.2-trichloroethyl 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl car 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted bamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl car 55 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, bamate (t-Bumeoc), 2-(2- and 4'-pyridyl)ethyl carbamate N-2-(trimethylsilyl)ethoxymethylamine (SEM), N-3-ac (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, etoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-py t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), roolin-3-yl)amine, quaternary ammonium salts, N-ben vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropy Zylamine, N-di(4-methoxyphenyl)methylamine, N-5- lallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-ni 60 dibenzosuberylamine, N-triphenylmethylamine (Tr), N-(4- trocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hy methoxyphenyl)diphenylmethylamine (MMTr), N-9- droxypiperidinyl carbamate, alkyldithio carbamate, benzyl phenylfluorenylamine (PhF), N-2,7-dichloro-9- carbamate (Cbz), p-methoxybenzyl carbamate (MoZ), p-ni fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), tobenzyl carbamate, p-bromobenzyl carbamate, p-chlo N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyl robenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-meth 65 eneamine, N-benzylideneamine, N-p-methoxybenzylide ylsulfinylbenzyl carbamate (MSZ), 9-anthrylmethyl neamine, N-diphenylmethyleneamine, N-(2-pyridyl)mesi carbamate, diphenylmethyl carbamate, 2-methylthioethyl tylmethyleneamine, N—(N',N'-dimethylaminomethylene) US 9,630,961 B2 19 20 amine, N,N'-isopropylidenediamine, N-p- ylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsi nitrobenzylideneamine, N-Salicylideneamine, N-5- lyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthex chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl) ylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl phenylmethyleneamine. N-cyclohexylideneamine, N-(5.5- (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl derivative, N-diphenylborinic acid derivative, N-phenyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, (pentaacylchromium- or tungsten)acylamine, N-copper dichloroacetate, trichloroacetate, trifluoroacetate, methoxy chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, acetate, triphenylmethoxyacetate, phenoxyacetate, p-chloro amine N-oxide, diphenylphosphinamide (Dpp), dimethylth phenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (le iophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), 10 Vulinate), 4.4-(ethylenedithio)pentanoate dialkyl phosphoramidates, dibenzyl phosphoramidate, (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, diphenyl phosphoramidate, benzenesulfenamide, o-ni 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2.4.6- trobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfena trimethylbenzoate (mesitoate), methyl carbonate, 9-fluore mide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxy nylmethyl carbonate (Fmoc), ethyl carbonate, 2.2.2-trichlo benzenesulfenamide, triphenylmethylsulfenamide, and 15 roethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate 3-nitropyridinesulfenamide (Npys). (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(tri In certain embodiments, the Substituent present on an phenylphosphonio)ethyl carbonate (Peoc), isobutyl carbon oxygen atom is an oxygen protecting group (also referred to ate, vinyl carbonate, allyl carbonate, t-butyl carbonate as a hydroxyl protecting group). Oxygen protecting groups (BOC), p-nitrophenyl carbonate, benzyl carbonate, include, but are not limited to, R", N(R'), C(=O) p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbon SR', C(=O)R, COR, C(=O)N(R), ate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, C(-NR)R", C(-NR)OR, C(-NR)N S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, (R), S(=O)R', SOR", Si(R), -P(R), methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, - P(R) - P(=O).R", P(=O)(R') - P(=O) 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, (OR), P(=O)N(R), and P(=O)(NR), 25 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, wherein R", R', and R* are as defined herein. Oxygen 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxym protecting groups are well known in the art and include those ethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2.6- described in detail in Protecting Groups in Organic Synthe dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4- sis, T. W. Greene and P. G. M. Wuts, 3' edition, John Wiley bis(1,1-dimethylpropyl)phenoxyacetate, & Sons, 1999, incorporated herein by reference. 30 chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2- Exemplary oxygen protecting groups include, but are not methyl-2-butenoate, o-(methoxyacyl)benzoate, C.-naph limited to, methyl, methoxylmethyl (MOM), methylthiom thoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiami ethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) date, alkyl N-phenylcarbamate, borate, methoxymethyl (SMOM), benzyloxymethyl (BOM), dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, p-methoxybenzyloxymethyl (PMBM), (4-methoxyphe 35 Sulfate, methanesulfonate (mesylate), benzylsulfonate, and noxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxym tosylate (Ts). ethyl, 4-pentenyloxymethyl (POM), siloxymethyl, In certain embodiments, the Substituent present on a 2-methoxyethoxymethyl (MEM), 2.2.2-trichloroethoxym Sulfur atom is a Sulfur protecting group (also referred to as ethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl) a thiol protecting group). Sulfur protecting groups include, ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bro 40 but are not limited to, R', N(R), —C(=O)SR", motetrahydropyranyl, tetrahydrothiopyranyl. C(=O)R, COR', C(=O)N(R), C(-NR) 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl R*, C(—NR)OR, C(-NR)N(R), S(=O) (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetra R", SOR', Si(R) P(R) P(R), hydrothiopyranyl S.S.-dioxide, 1-(2-chloro-4-methyl)phe - P(=O).R.", P(=O)(R') - P(=O)(OR), nyl-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tet 45 - P(=O)N(R), and P(=O)(NR), wherein R*, rahydrofuranyl, tetrahydrothiofuranyl, 2.3.3a,4,5,6,7,7a R”, and R* are as defined herein. Sulfur protecting groups octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, are well known in the art and include those described in 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1- detail in Protecting Groups in Organic Synthesis, T. W. methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-ben Greene and P. G. M. Wuts, 3' edition, John Wiley & Sons, Zyloxy-2-fluoroethyl, 2.2.2-trichloroethyl 2-trimethylsilyl 50 1999, incorporated herein by reference. ethyl 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, These and other exemplary substituents are described in p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), more detail in the Detailed Description, Examples, and p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, claims. The present disclosure is not intended to be limited p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cya in any manner by the above exemplary listing of Substitu nobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2- 55 entS. picolyl N-oxido, diphenylmethyl, p.p'-dinitrobenzhydryl, "Pharmaceutically acceptable salt” refers to those salts 5-dibenzosuberyl, triphenylmethyl, C.-naphthyldiphenylm which are, within the scope of Sound medical judgment, ethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphe Suitable for use in contact with the tissues of humans and nyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4-bro other animals without undue toxicity, irritation, allergic mophenacyloxyphenyl)diphenylmethyl, 4,4',4'-tris(4,5- 60 response, and the like, and are commensurate with a rea dichlorophthalimidophenyl)methyl, 4,4',4'-tris sonable benefit/risk ratio. Pharmaceutically acceptable salts (levulinoyloxyphenyl)methyl, 4,4',4'-tris are well known in the art. For example, Berge et al. describe (benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4,4'- pharmaceutically acceptable salts in detail in J. Pharmaceu dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1- tical Sciences (1977) 66:1-19. Pharmaceutically acceptable pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9- 65 salts of the compounds describe herein include those derived phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, from Suitable inorganic and organic acids and bases. benzisothiazolyl S.S.-dioxido, trimethylsilyl (TMS), trieth Examples of pharmaceutically acceptable, nontoxic acid US 9,630,961 B2 21 22 addition salts are salts of an amino group formed with the condition. The term “therapeutically effective amount inorganic acids such as hydrochloric acid, hydrobromic acid, can encompass an amount that improves overall therapy, phosphoric acid, Sulfuric acid and perchloric acid or with reduces or avoids symptoms or causes of the condition, or organic acids such as acetic acid, oxalic acid, maleic acid, enhances the therapeutic efficacy of another therapeutic tartaric acid, citric acid, Succinic acid, or malonic acid or by agent. using other methods used in the art such as ion exchange. A “prophylactically effective amount of a compound is Other pharmaceutically acceptable salts include adipate, an amount Sufficient to prevent a condition, or one or more alginate, ascorbate, aspartate, benzenesulfonate, benzoate, symptoms associated with the condition or prevent its recur bisulfate, borate, butyrate, camphorate, camphorsulfonate, rence. A prophylactically effective amount of a compound citrate, cyclopentanepropionate, digluconate, dodecylsul 10 fate, ethanesulfonate, formate, fumarate, glucoheptonate, means an amount of a therapeutic agent, alone or in com glycerophosphate, gluconate, hemisulfate, heptanoate, bination with other agents, which provides a prophylactic hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lacto benefit in the prevention of the condition. The term “pro bionate, lactate, laurate, lauryl Sulfate, malate, maleate, phylactically effective amount can encompass an amount malonate, methanesulfonate, 2-naphthalenesulfonate, nico 15 that improves overall prophylaxis or enhances the prophy tinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, lactic efficacy of another prophylactic agent. persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, As used herein, the term “methyltransferase' represents propionate, Stearate. Succinate, Sulfate, tartrate, thiocyanate, transferase class enzymes that are able to transfer a methyl p-toluenesulfonate, undecanoate, Valerate salts, and the like. group from a donor molecule to an acceptor molecule, e.g., Salts derived from appropriate bases include alkali metal, an amino acid residue of a protein or a nucleic base of a alkaline earth metal, ammonium and N(Calkyl) salts. DNA molecule. Methyltransferases typically use a reactive Representative alkali or alkaline earth metal salts include methyl group bound to Sulfur in S-adenosyl methionine Sodium, lithium, potassium, calcium, magnesium, and the (SAM) as the methyl donor. In some embodiments, a like. Further pharmaceutically acceptable salts include, methyltransferase described herein is a protein methyltrans when appropriate, quaternary salts. 25 ferase. In some embodiments, a methyltransferase described A “subject’ to which administration is contemplated herein is a histone methyltransferase. Histone methyltrans includes, but is not limited to, humans (e.g., a male or female ferases (HMT) are histone-modifying enzymes, (including of any age group, e.g., a pediatric Subject (e.g., infant, child, histone-lysine N-methyltransferase and histone-arginine adolescent) or adult Subject (e.g., young adult, middle-aged N-methyltransferase), that catalyze the transfer of one or adult or senior adult)) and/or other non-human animals, for 30 more methyl groups to lysine and arginine residues of example, non-human mammals (e.g., primates (e.g., cyno histone proteins. In certain embodiments, a methyltrans molgus monkeys, rhesus monkeys); commercially relevant ferase described herein is a histone-arginine N-methyltrans mammals such as cattle, pigs, horses, sheep, goats, cats, ferase. and/or dogs), birds (e.g., commercially relevant birds Such As generally described above, provided herein are com as chickens, ducks, geese, and/or turkeys), rodents (e.g., rats 35 pounds useful as arginine methyltransferase (RMT) inhibi and/or mice), reptiles, amphibians, and fish. In certain tors. In some embodiments, the present disclosure provides embodiments, the non-human animal is a mammal. The a compound of Formula (I): non-human animal may be a male or female at any stage of development. A non-human animal may be a transgenic animal. 40 “Condition,” “disease,” and “disorder are used inter changeably herein. “Treat,” “treating and “treatment' encompasses an action that occurs while a subject is Suffering from a condition which reduces the severity of the condition or 45 retards or slows the progression of the condition (“thera peutic treatment”). “Treat,” “treating and “treatment” 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'). 50 or a pharmaceutically acceptable salt thereof, An “effective amount of a compound refers to an amount wherein Sufficient to elicit the desired biological response, e.g., treat X is N, Z is NR, and Y is CR; or the condition. As will be appreciated by those of ordinary X is NR, Z is N, and Y is CR; or skill in this art, the effective amount of a compound X is CR, Z is NR, and Y is N; or described herein may vary depending on Such factors as the 55 X is CR, Z is N, and Y is NR; desired biological endpoint, the pharmacokinetics of the Q is an optionally substituted, monocyclic or bicyclic compound, the condition being treated, the mode of admin heteroaryl having 1-4 heteroatoms independently selected istration, and the age and health of the subject. An effective from nitrogen, oxygen, and Sulfur, wherein Q is not pyridine; amount encompasses therapeutic and prophylactic treat R is hydrogen, C. alkyl, or Cs a cycloalkyl; ment. 60 R" is hydrogen, optionally substituted C. alkyl, option A “therapeutically effective amount of a compound is an ally substituted C- alkenyl, optionally substituted C amount sufficient to provide a therapeutic benefit in the alkynyl, optionally substituted C-7 cycloalkyl, optionally treatment of a condition or to delay or minimize one or more substituted 4- to 7-membered heterocyclyl; or optionally symptoms associated with the condition. A therapeutically Substituted C. alkyl-Cy; effective amount of a compound means an amount of 65 Cy is optionally substituted C-7 cycloalkyl, optionally therapeutic agent, alone or in combination with other thera substituted 4- to 7-membered heterocyclyl, optionally sub pies, which provides a therapeutic benefit in the treatment of stituted aryl, or optionally substituted heteroaryl; and US 9,630,961 B2 23 24 R is hydrogen, halo, —CN, optionally substituted Ca or a pharmaceutically acceptable salt thereof, wherein Q, R, alkyl, or optionally substituted C. cycloalkyl. R. R. and R are as described herein. In certain embodiments, a provided compound is of As defined generally above, Q is optionally substituted Formula (II): heteroaryl. In some embodiments, Q is an optionally Sub stituted 5- to 10-membered heteroaryl having 1-4 heteroa toms independently selected from nitrogen, oxygen, and II Sulfur. In some embodiments, Q is an optionally Substituted HN-R 5- to 8-membered heteroaryl having 1-4 heteroatoms inde pendently selected from nitrogen, oxygen, and Sulfur. In 10 Some embodiments, Q is an optionally Substituted 5- to N 6-membered heteroaryl having 1-4 heteroatoms indepen Q Ye dently selected from nitrogen, oxygen, and Sulfur. In some ( \ embodiments, Q is an optionally substituted 5- to 6-mem YN R5 bered heteroaryl having 1-3 heteroatoms independently 15 selected from nitrogen, oxygen, and Sulfur. In some embodi ments, Q is an optionally substituted 5- to 6-membered heteroaryl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and Sulfur. In some embodiments, Q or a pharmaceutically acceptable salt thereof, wherein Q, R, is an optionally substituted 5- to 6-membered heteroaryl R. R. and R are as described herein. having 1 heteroatom selected from nitrogen, oxygen, and In certain embodiments, a provided compound is of sulfur. Formula (III): In some embodiments, Q is an optionally substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and Sulfur. In III 25 some embodiments, Q is an optionally substituted 9- to HN-R 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and Sulfur. In Q N Some embodiments, Q is an optionally Substituted 9-mem bered bicyclic heteroaryl having 1-4 heteroatoms indepen Ye 30 dently selected from nitrogen, oxygen, and Sulfur. In some NN 2 embodiments, Q is an optionally substituted 9-membered R4 1 n N R bicyclic heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and Sulfur. In some embodi or a pharmaceutically acceptable salt thereof, wherein Q, R, ments, Q is an optionally substituted 9-membered bicyclic 35 heteroaryl having 1-2 heteroatoms independently selected R. R. and R are as described herein. from nitrogen, oxygen, and Sulfur. In some embodiments, Q In certain embodiments, a provided compound is of is an optionally substituted 9-membered bicyclic heteroaryl Formula (IV): having 1 heteroatom selected from nitrogen, oxygen, and Sulfur. In some embodiments, Q is an optionally Substituted IV 40 10-membered bicyclic heteroaryl having 1-4 heteroatoms HN-R independently selected from nitrogen, oxygen, and Sulfur. In Some embodiments, Q is an optionally Substituted 10-mem bered bicyclic heteroaryl having 1-3 heteroatoms indepen Q N dently selected from nitrogen, oxygen, and Sulfur. In some V 45 embodiments, Q is an optionally substituted 10-membered K. R bicyclic heteroaryl having 1-2 heteroatoms independently ( \, selected from nitrogen, oxygen, and Sulfur. In some embodi R5 N1 ments, Q is an optionally substituted 10-membered bicyclic heteroaryl having 1 heteroatom selected from nitrogen, 50 oxygen, and Sulfur. In certain embodiments, Q is Substituted. In certain or a pharmaceutically acceptable salt thereof, wherein Q, R, embodiments, Q is unsubstituted. R. R. and R are as described herein. In certain embodiments, Q is substituted pyrrolyl. In In certain embodiments, a provided compound is of certain embodiments, Q is unsubstituted pyrrolyl. In certain Formula (V): 55 embodiments, Q is substituted furanyl. In certain embodi ments, Q is unsubstituted furanyl. In certain embodiments, Q is substituted thienyl. In certain embodiments, Q is V unsubstituted thienyl. In certain embodiments, Q is substi HN-R tuted imidazolyl. In certain embodiments, Q is unsubstituted 60 imidazolyl. In certain embodiments, Q is substituted pyra Q N Zolyl. In certain embodiments, Q is unsubstituted pyrazolyl. V In certain embodiments, Q is Substituted oxazolyl. In certain o R embodiments, Q is unsubstituted oxazolyl. In certain N embodiments, Q is substituted thiazolyl. In certain embodi R5 N- NR4 65 ments, Q is unsubstituted thiazolyl. In certain embodiments, Q is substituted isothiazolyl. In certain embodiments, Q is unsubstituted isothiazolyl. In certain embodiments, Q is US 9,630,961 B2 25 26 substituted triazolyl. In certain embodiments, Q is unsub embodiments, Q is Substituted quinazolinyl. In certain stituted triazolyl. In certain embodiments, Q is substituted embodiments, Q is unsubstituted quinazolinyl. In certain oxadiazolyl. In certain embodiments, Q is unsubstituted embodiments, Q is substituted phthalazinyl. In certain thiadiazolyl. In certain embodiments, Q is substituted oxadi embodiments, Q is unsubstituted phthalazinyl. azolyl. In certain embodiments, Q is unsubstituted thiadiaz In some embodiments, Q is substituted with one or more olyl. In certain embodiments, Q is substituted tetrazolyl. In R" groups, wherein each R is independently selected from certain embodiments, Q is unsubstituted tetrazolyl. the group consisting of halo. —CN, NO, optionally In certain embodiments, Q is substituted pyridyl. In substituted alkyl, optionally substituted alkenyl, optionally certain embodiments, Q is unsubstituted pyridyl. In certain substituted alkynyl, optionally substituted carbocyclyl, embodiments, Q is substituted pyrimidyl. In certain embodi 10 optionally substituted aryl, optionally substituted heterocy ments, Q is unsubstituted pyrimidyl. In certain embodi clyl, optionally substituted heteroaryl, -OR", N(R), ments, Q is Substituted pyrazinyl. In certain embodiments, Q - SR", C(=O)R, C(O)CR', C(O)SR', C(O)N is unsubstituted pyrazinyl. In certain embodiments, Q is (R), C(O)N(R)N(R), OC(O)R', OC(O)N(R), Substituted pyridazinyl. In certain embodiments, Q is unsub NRC(O)R', NRC(O)N(R), NRC(O)N(R)N stituted pyridazinyl. In certain embodiments, Q is Substi 15 (R), NRC(O)OR', SC(O)R, C(-NR)R', tuted triazinyl. In certain embodiments, Q is unsubstituted C(—NNR)R', C(—NOR")R', C(-NR)N(R), triazinyl. NRC(-NR)R’, C(-S)R, C(-S)N(R), In certain embodiments, Q is substituted indolyl. In NRC(=S)R', S(O)R', OS(O).R', SOR, certain embodiments, Q is unsubstituted indolyl. In certain - NRSOR', or -SON(R); wherein each R is inde embodiments, Q is substituted isoindolyl. In certain embodi pendently selected from the group consisting of hydrogen, ments, Q is unsubstituted isoindolyl. In certain embodi optionally substituted alkyl, optionally substituted alkenyl, ments, Q is substituted indazolyl. In certain embodiments, Q optionally substituted alkynyl, optionally substituted carbo is unsubstituted indazolyl. In certain embodiments, Q is cyclyl, optionally Substituted heterocyclyl, optionally Sub substituted benzotriazolyl. In certain embodiments, Q is stituted aryl, optionally substituted heteroaryl, an oxygen unsubstituted benzotriazolyl. In certain embodiments, Q is 25 protecting group when attached to an oxygen atom, and a substituted benzothiophenyl. In certain embodiments, Q is Sulfur protecting group when attached to a Sulfur atom; and unsubstituted benzothiophenyl. In certain embodiments, Q each R is independently selected from the group consisting is substituted isobenzothiophenyl. In certain embodiments, of hydrogen, optionally Substituted alkyl, optionally Substi Q is unsubstituted isobenzothiophenyl. In certain embodi tuted alkenyl, optionally substituted alkynyl, optionally Sub ments, Q is substituted benzofuranyl. In certain embodi 30 stituted carbocyclyl, optionally substituted heterocyclyl, ments, Q is unsubstituted benzofuranyl. In certain embodi optionally substituted aryl, optionally substituted heteroaryl, ments, Q is substituted benzoisofuranyl. In certain and a nitrogen protecting group, or two R groups are taken embodiments, Q is unsubstituted benzoisofuranyl. In certain together with their intervening atoms to form an optionally embodiments, Q is substituted benzimidazolyl. In certain substituted heterocyclic ring. In some embodiments, R is embodiments, Q is unsubstituted benzimidazolyl. In certain 35 halo. In certain embodiments, R is fluoro. In certain embodiments, Q is substituted benzoxazolyl. In certain embodiments, R is chloro. In some embodiments, R is embodiments, Q is unsubstituted benzoxazolyl. In certain optionally substituted alkyl, optionally substituted alkenyl, embodiments, Q is substituted benzoxadiazolyl. In certain optionally Substituted alkynyl, or optionally Substituted car embodiments, Q is unsubstituted benzoxadiazolyl. In certain bocyclyl. In certain embodiments, R is optionally substi embodiments, Q is substituted benzisoxazolyl. In certain 40 tuted C. alkyl, optionally substituted Calkenyl, option embodiments, Q is unsubstituted benzisoxazolyl. In certain ally substituted C- alkynyl, or optionally substituted C embodiments, Q is substituted benzthiazolyl. In certain carbocyclyl. In certain embodiments, R is optionally sub embodiments, Q is unsubstituted benzthiazolyl. In certain stituted C. alkyl. In certain embodiments, R is substituted embodiments, Q is substituted benzisothiazolyl. In certain C. alkyl. In certain embodiments, R is —CF. In certain embodiments, Q is unsubstituted benzisothiazolyl. In certain 45 embodiments, R is —CHF. In certain embodiments, R is embodiments, Q is substituted benzthiadiazolyl. In certain —Calkyl-carbocyclyl. In certain embodiments, R is embodiments, Q is unsubstituted benzthiadiazolyl. In certain —CH-cyclopropyl or —CH-cyclobutyl. In certain embodiments, Q is substituted indolizinyl. In certain embodiments, R is unsubstituted C. alkyl. In certain embodiments, Q is unsubstituted indolizinyl. In certain embodiments, R is methyl, ethyl, propyl, butyl, or pentyl. In embodiments, Q is substituted purinyl. In certain embodi 50 certain embodiments, R is isopropyl, isobutyl, or isoamyl. ments, Q is unsubstituted purinyl. In certain embodiments, In certain embodiments, R is isobutyl. In some embodi Q is substituted pyrrolopyridinyl. In certain embodiments, Q ments, R is —CN. In some embodiments, R is optionally is unsubstituted pyrrolopyridinyl. In certain embodiments, Q Substituted carbocyclyl, optionally Substituted aryl, option is substituted triazolopyridinyl. In certain embodiments, Q is ally substituted heterocyclyl, or optionally substituted het unsubstituted triazolopyridinyl. 55 eroaryl. In some embodiments, R is cyclopropyl or In certain embodiments, Q is substituted naphthyridinyl. cyclobutyl. In some embodiments, R is —OR, N(R), In certain embodiments, Q is unsubstituted naphthyridinyl. - SR", C(=O)R', C(O)CR, C(O)SR, C(O)N In certain embodiments, Q is substituted pteridinyl. In (R), C(O)N(R)N(R), OC(O)R', OC(O)N(R), certain embodiments, Q is unsubstituted pteridinyl. In cer NRC(O)N(R), NRC(O)N(R)N(R), NRC(O) tain embodiments, Q is substituted quinolinyl. In certain 60 OR', SC(O)R, C(-NR)R, C(-NNR)R', embodiments, Q is unsubstituted quinolinyl. In certain C(-NOR")R', C(—NR)N(R), NRC(—NR) embodiments, Q is Substituted isoquinolinyl. In certain R’, C(-S)R, C(-S)N(R), NRC(—S)R', embodiments, Q is unsubstituted isoquinolinyl. In certain - S(O)R, OS(O).R., SOR", or -SON(R). In embodiments, Q is substituted cinnolinyl. In certain embodi certain embodiments, R is N(R). In certain embodi ments, Q is unsubstituted cinnolinyl. In certain embodi 65 ments, R is NHR'. In certain embodiments, R is ments, Q is Substituted quinoxalinyl. In certain embodi NHR, wherein R is optionally substituted C. alkyl. In ments, Q is unsubstituted quinoxalinyl. In certain certain embodiments, R is NHR, wherein R is unsub US 9,630,961 B2 27 28 stituted C, alkyl. In certain embodiment, R is NHR'. In certain embodiments, Q is of Formula (q-2): wherein R is substituted C. alkyl. In certain embodi ments, R is NH-benzyl. In certain embodiments, R is —N(R), wherein each R is independently optionally (q-2) substituted C. alkyl. In certain embodiments, R is v.5% VNSy 4 92S N3 —N(R), wherein each R’ is independently unsubstituted C, alkyl. In certain embodiments, R is N(CH3)R’. V. ( )Oywn-1/ wherein each R is independently optionally substituted C. N27 Nvi alkyl. In certain embodiments, R is N(CH3)R’, wherein each R is independently unsubstituted C. alkyl. In certain 10 In certain embodiments, Q is of Formula (q-3): embodiments, R is N(CHCH)R’, wherein each R is independently optionally Substituted C. alkyl. In certain embodiments, R is N(CHCH)R’, wherein each R is (q-3) independently unsubstituted C. alkyl. In certain embodi orm ments, R is N(R), wherein each R is independently 15 v52V3 Sy2S selected from the group consisting of methyl, ethyl, isopro V2. pyl, isobutyl, isoamyl, and benzyl. In some embodiments, A Su) R is N(R), wherein each R is the same. In some V& 2S, embodiments, R is N(R), wherein each R is different. In certain embodiments, R is NH. In certain embodi In certain embodiments, Q is of Formula (q-4): ments, R is —OR". In certain embodiments, R is —OH. In certain embodiments, R is —OR", wherein R is optionally substituted alkyl, optionally substituted alkenyl, optionally (q-4) substituted alkynyl, or optionally substituted carbocyclyl. In 25 certain embodiments, R is —O-isobutylenyl. In certain 4 embodiments, R is —OR", wherein R is optionally sub A vas W9 a\ stituted C, alkyl. In certain embodiments, R is —OR", ( )O wherein R is unsubstituted C alkyl. In certain embodi ments, R is —O-propyl. —O-isopropyl. —O-isobutyl, or 30 -O-isoamyl. In certain embodiments, R is —OR", As used herein, each instance of V, V, V, V, V, V, V, V, and V may independently be O, S, N, NR, C, or wherein R is substituted C. alkyl. In certain embodi CR, as valency permits, wherein at least one of V, V, V, ments, R is —O Cealkyl-O-C-alkyl. In certain V, V, V, V, V, and V is O, S, N, NRY, and wherein: embodiments, R is –OCHCHOCH O 35 each instance of R is independently selected from the —OCHCHCHOCH. In certain embodiments, R is group consisting of halo. —CN. —NO, optionally Substi —O—Calkyl-carbocyclyl. In certain embodiments, R is tuted alkyl, optionally substituted alkenyl, optionally sub —O—CH2-cyclobutyl or —O—CH2-cyclopropyl. In cer stituted alkynyl, optionally Substituted carbocyclyl, option tain embodiments, R is —O—Calkyl-heterocyclyl. In ally substituted aryl, optionally substituted heterocyclyl, certain embodiments, R is —O CH-tetrahydropyranyl or 40 optionally substituted heteroaryl, -OR", N(R) - SR", —O CH2-oxetanyl. In certain embodiments, R is —OR", C(=O)R, C(O)OR, C(O)SR, C(O)N(R), wherein R is optionally substituted heterocyclyl. In certain C(O)N(R)N(R), OC(O)R', OC(O)N(R), NRC(O)R, NRC(O)N(R), NRC(O)N(R)N embodiments, R is —O-tetrahydropyranyl or —O— oxeta (R), NRC(O)OR', SC(O)R, C(-NR)R', nyl. In certain embodiments, R is —OR", wherein R is C(NNR)R, C(—NOR")R', C(-NR)N(R), optionally substituted aryl. In certain embodiments, R is 45 NRC(-NR)R’, C(-S)R, C(-S)N(R), -O-phenyl. In certain embodiments, R is —OR", wherein NRC(=S)R', S(O)R, OS(O).R., SOR, R" is optionally substituted heteroaryl. NRSOR", or SON(R); and each instance of RY is independently selected from the group consisting of hydrogen, optionally Substituted alkyl, 50 optionally substituted alkenyl, optionally substituted alky In some embodiments, Q is unsubstituted. In some nyl, optionally Substituted carbocyclyl, optionally Substi embodiments, Q is substituted with one R group. In some tuted heterocyclyl, optionally substituted aryl, optionally embodiments, Q is substituted with two R groups. In some substituted heteroaryl, -C(=O)R', -C(=O)CR', embodiments, Q is substituted with three R groups. In some C(=O)SR', C(=O)N(R) C(-NR)R', embodiments, Q is substituted with four R groups. 55 C(-NNR)R, C(-NOR)R, C(-NR)N(R), In certain embodiments, Q is of Formula (q-1): C(=S)R, C(=S)N(R), S(=O)R', SOR, —SO.N(R), and a nitrogen protecting group. In certain embodiments, V' is O, S, N or NR'. In certain embodiments, V is N or NRY. In certain embodiments, V' sas 92S 60 is O. In certain embodiments, V is S. In certain embodi V V N 2 V ments, only one of V, V, V, V, V, V, V7, V, and V V. / is selected from the group consisting of O, S, N, and NR'. 2 Nyl In certain embodiments, only one of V, V, V, V, V, V, V, V, and V is selected from the group consisting of N Y." 65 and NRY. In certain embodiments, only one of V, V, V, V, V, V, V, V, and V is O. In certain embodiments, only one of V, V, V, V, V, V, V7, V, and V is S. In US 9,630,961 B2 29 certain embodiments, only two of V, V, V, V, V, V, V7, V, and V are each independently selected from the group consisting of O, S, N, and NR. In certain embodi ments, only two of V, V, V, V, V, V, V, V, and V are each independently selected from the group consisting of 5 N and NR. In certain embodiments, only two of V, V, V, V, V, V, V, V, and V are each independently selected from the group consisting of O, N and NRY. In certain embodiments, only two of V, V, V, V, V, V, V, V, and V are each independently selected from the group 10 In compounds of Formula (q-6), V, V, V, V, V, consisting of S, N and NRY. In certain embodiments, only and V' are each independently selected from the group three of V, V, V, V, V, V, V7, V, and V are each consisting of N or CR, wherein at least one of V, V, independently selected from the group consisting of O, S, N, V, V, V, and V' is N. In certain embodiments, only and NRY. In certain embodiments, only three of V, V, V, 15 one of V, V, V'7, V, V, and V* is N. In certain V, V, V, V, V, and V are each independently selected embodiments, only two of V'', V, V7, V, V, and V? from the group consisting of N and NR'. In certain embodi are N. In certain embodiments, only three of V, V, V7, ments, only three of V, V, V, V, V, V, V7, V, and V V, V, and V2 are N. are each independently selected from the group consisting of O, N and NRY. In certain embodiments, only three of V, V, In certain embodiments, Q has one of the following V, V, V, V, V, V, and V are each independently Structures: selected from the group consisting of S, N and NR'. In certain embodiments, only four of V, V, V, V, V, V, V7, V, and V are each independently selected from the N1 v (Ri)0-3 N group consisting of O, S, N, and NR. In certain embodi 25 3. (R)-3-H / ments, only four of V, V, V, V, V, V, V7, V, and V are each independently selected from the group consisting of N and NRY. In certain embodiments, only four of V, V, V, V, V, V, V, V, and V are each independently selected from the group consisting of O, N and NR'. In 30 N 1S N 21 S certain embodiments, only four of V, V, V, V, V, V, - (Ra) (R9) - - A V, V, and V are each independently selected from the 2 N ŽS(R) group consisting of S, N and NR'. In certain embodiments, only five of V, V, V, V, V, V, V7, V, and V are each independently selected from the group consisting of O, S, N, 35 and NRY. In certain embodiments, only five of V, V, V, V, V, V, V, V, and V are each independently selected N N from the group consisting of N and NRY. In certain embodiments, Q is an optionally substituted 2NMN(Ra) 5-membered heteroaryl ring. In certain embodiments, Q is of 40 Formula (c-5):

45

50 As used herein, each instance of V'', V'', V'', V', and V may independently be O, S, N, NRY, C, or CR, as valency permits, wherein RY and R are as defined herein, and wherein at least one of V'', V'', V'', V', and V' is O, 55 S, N, or NRY. In certain embodiments, only one of V'', V'', V, V, and V* is selected from the group consisting of O, S, N, and NR'. In certain embodiments, only two of V', V'', V'', V', and V* are selected from the group consist ing of O, S, N, and NR'. In certain embodiments, only three 60 of V'', V'', V'', V', and V* are selected from the group consisting of O, S, N, and NR. In certain embodiments, only four of V, V, V, V, and V* are selected from the group consisting of O, S, N, and NR'. In certain embodiments, Q is an optionally substituted 65 6-membered heteroaryl ring. In certain embodiments, Q is of Formula (c-6): US 9,630,961 B2 31 32 -continued In some embodiments, R is (R)0-3 XY),

As defined generally above, R is hydrogen, C. alkyl, or In some embodiments, R is Cs a cycloalkyl. In certain embodiments, R is hydrogen. In 10 certain embodiments, R is C, alkyl. In certain embodi ments, R is methyl. In certain embodiments, R is ethyl. In certain embodiments, R is propyl or butyl. In certain embodiments, R is cyclopropyl. In certain embodiment, R N.21 Xc21 O is cyclobutyl. 15 As defined generally above, R is hydrogen, optionally Substituted C. alkyl, optionally Substituted C- alkenyl, optionally Substituted C- alkynyl, optionally Substituted 2N. C-7 cycloalkyl, optionally Substituted 4- to 7-membered Xr heterocyclyl; or optionally substituted C. alkyl-Cy, wherein Cy is optionally substituted C-7 cycloalkyl, option As defined generally above, R is hydrogen, halo, —CN, ally substituted 4- to 7-membered heterocyclyl, optionally optionally Substituted C. alkyl, or optionally Substituted substituted aryl, or optionally substituted heteroaryl. In C. cycloalkyl. In certain embodiments, R is hydrogen. In certain embodiments, R is hydrogen. In certain embodi certain embodiments, R is halo. In certain embodiments, R ments, R is optionally substituted C. alkyl. In certain 25 is chloro. In certain embodiments, R is fluoro. In certain embodiments, R is unsubstituted C. alkyl. In certain embodiments, R is —CN. In certain embodiments, R is embodiments, R is methyl, ethyl, or isopropyl. In certain optionally substituted C. alkyl. In certain embodiments, R embodiments, R is substituted C. alkyl. In certain is unsubstituted C. alkyl. In certain embodiments, R is embodiments, R is methoxyethyl. In certain embodiments, 30 methyl. In certain embodiments, R is ethyl. In certain R" is hydroxyethyl or propane-1,2-diol. In certain embodi embodiments, R is propyl or butyl. In certain embodiments, ments, R is optionally substituted C-7 cycloalkyl. In certain R is C, alkyl substituted with one or more fluoro groups. embodiments, R is unsubstituted C-7 cycloalkyl. In certain In certain embodiments, R is CF. In certain embodi embodiments, R is cyclopropyl, cyclobutyl, cyclopentyl, or ments, R is —CHF. In certain embodiments, R is option cyclohexyl. In certain embodiments, R is optionally sub 35 ally substituted C. cycloalkyl. In certain embodiments, R stituted 4- to 7-membered heterocyclyl. In certain embodi is cyclopropyl or cyclobutyl. ments, R is optionally substituted 4- to 7-membered het As defined generally above, R is optionally substituted erocyclyl having 1-2 heteroatoms independently selected C. alkyl or optionally Substituted C. cycloalkyl. In cer from nitrogen, oxygen, and Sulfur. In certain embodiments, tain embodiments, R is optionally Substituted C. alkyl. In R" is oxetane, tetrahydrofuran, or tetrahydropyran. In certain 40 certain embodiments, R is unsubstituted C alkyl. In embodiments, R is optionally substituted C. alkyl-Cy, certain embodiments, R is methyl. In certain embodiments, wherein Cy is optionally substituted C-7 cycloalkyl, option R is ethyl. In certain embodiments, R is isopropyl. In ally substituted 4- to 7-membered heterocyclyl, optionally certain embodiments, R is propyl or butyl. In certain substituted aryl, or optionally substituted heteroaryl. In some embodiments, R is substituted Calkyl. In certain embodi embodiments, Cy is optionally substituted C-7 cycloalkyl. 45 ments, R is C alkyl substituted with hydroxyl or alkoxy. In some embodiments, Cy is optionally Substituted 4- to In certain embodiments, R is hydroxyethyl or methoxy 7-membered heterocyclyl having 1-2 heteroatoms indepen ethyl. In certain embodiments, R is optionally substituted dently selected from nitrogen, oxygen, and Sulfur. In some C. cycloalkyl. In certain embodiments, R is unsubstituted embodiments, Cy is oxetane, tetrahydrofuran, or tetrahydro C. cycloalkyl. In certain embodiments, R is cyclopropyl. pyran. In some embodiments, Cy is optionally substituted 50 In certain embodiments, R is cyclobutyl. aryl. In some embodiments, Cy is optionally Substituted As defined generally above, each R" is independently phenyl. In some embodiments, Cy is unsubstituted phenyl. selected from the group consisting of hydrogen, optionally In some embodiments, Cy is optionally substituted het substituted alkyl, optionally substituted alkenyl, optionally eroaryl having 1-3 heteroatoms independently selected from substituted alkynyl, optionally substituted carbocyclyl, nitrogen, oxygen, and Sulfur. In some embodiments, Cy is 55 optionally substituted heterocyclyl, optionally substituted optionally substituted 5- to 6-membered heteroaryl having aryl, optionally Substituted heteroaryl, an oxygen protecting 1-3 heteroatoms independently selected from nitrogen, oxy group when attached to an oxygen atom, and a Sulfur gen, and Sulfur. In some embodiments, Cy is pyridyl. In protecting group when attached to a Sulfur atom. In some some embodiments, R is embodiments, R is hydrogen. In some embodiments, R is 60 optionally substituted alkyl. In some embodiments, R is optionally substituted alkyl substituted with a Cy group to form optionally substituted alkyl-Cy, wherein Cy is described herein. In some embodiments, R is optionally substituted alkenyl or optionally substituted alkynyl. In 65 some embodiments, R is optionally substituted carbocy clyl, optionally substituted heterocyclyl, optionally substi tuted aryl, or optionally substituted heteroaryl. In some US 9,630,961 B2 33 34 embodiments, R is an oxygen protecting group when TABLE 1-continued attached to an oxygenatom. In some embodiments, R is not an oxygen protecting group. In some embodiments, R is Exemplary Compounds Sulfur protecting group when attached to an Sulfur atom. In Some embodiments, R is not a sulfur protecting group. 5 Cmpd LC-MS As defined generally above, each R is independently mz selected from the group consisting of hydrogen, optionally No Structure (M + H) substituted alkyl, optionally substituted alkenyl, optionally 232.28 substituted alkynyl, optionally substituted carbocyclyl, 10 optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl, and a nitrogen protecting group, or two R' groups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring. In some embodiments, R is hydrogen. In 15 some embodiments, R is optionally substituted alkyl. In some embodiments, R is optionally alkyl substituted with a Cy group to form optionally substituted alkyl-Cy, wherein 236.34 Cy is described herein. In some embodiments, R is option ally substituted alkenyl or optionally substituted alkynyl. In some embodiments, R is optionally substituted carbocy clyl, optionally substituted heterocyclyl, optionally substi tuted aryl, or optionally substituted heteroaryl. In some embodiments, R is a nitrogen protecting group. In some embodiments, R is not a nitrogen protecting group. In some 25 embodiments, two R' groups are taken together with their 2212 intervening atoms to form an optionally Substituted hetero cyclic ring. In certain embodiments, a provided compound is a com 30 pound listed in Table 1, or a pharmaceutically acceptable salt thereof.

TABLE 1. 35 2212 Exemplary Compounds

Cmpd LC-MS mz No Structure (M + H) 40 1 S 301.2

O 235.1 45 1N1a ON NH 2 N2NO 247.2 50 1. n-1N 288.1 55 ON/ NH / N HN 3 S 237.1 le

60 -N

65 US 9,630,961 B2 35 36 TABLE 1-continued TABLE 1-continued Exemplary Compounds Exemplary Compounds Cmpd LC-MS Cmpd LC-MS 5 mz mz No Structure (M + H) No Structure (M + H) 249.4 10 2SO.O

10

15 11 / 263.2 297.1 2- 2O N \ S. W 1N1 NH2 N 25 N HN

12 N-N 2212 30 18 OH 265.1

35 13 2 NH 271-2

N N 40 HN le 19 NH2 285.4 e N 45 S NN

NH2 s HN 14 221.3 50 N2 / N 7 55 2O 21 ty 272.2 N-4 N1s N 15 Na 235.4 HN le N 60 1. / 1N-1 NH2 -N N N HN 65 US 9,630,961 B2 37 38 TABLE 1-continued TABLE 1-continued Exemplary Compounds Exemplary Compounds Cmpd LC-MS Cmpd LC-MS 5 mz mz No Structure (M + H) No Structure (M + H) 26 263.4

21 271.1 10

15

27 271.3

NH2

22 262.3

25 -N

NH2

30 28 273.1

23 277.1 s N 35 \ 7 1N1 NH2 N N 40 HN

29 e 22O2 HN / 24 3.11.1 45 / 1N1 NH2 N N HN

50 30 271.3 N /

55 2 N f N-NH. 25 249.3 60 In certain embodiments, a provided compound inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In certain embodiments, a provided compound inhibits wild-type PRMT1, PRMT3, CARM1, PRMT6, and/ or PRMT8. In certain embodiments, a provided compound 65 inhibits a mutant RMT. In certain embodiments, a provided compound inhibits PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8, e.g., as measured in an assay described US 9,630,961 B2 39 40 herein. In certain embodiments, the RMT is from a human. ers, lubricants, and the like, as Suited to the particular dosage In certain embodiments, a provided compound inhibits an form desired. General considerations in formulation and/or RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or manufacture of pharmaceutical compositions agents can be PRMT8) at an ICs less than or equal to 10 uM. In certain found, for example, in Remington's Pharmaceutical Sci embodiments, a provided compound inhibits an RMT (e.g., 5 ences, Sixteenth Edition, E. W. Martin (Mack Publishing PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8) at an Co., Easton, Pa., 1980), and Remington. The Science and ICs less than or equal to 1 LM. In certain embodiments, a Practice of Pharmacy, 21st Edition (Lippincott Williams & provided compound inhibits an RMT (e.g., PRMT1, Wilkins, 2005). PRMT3, CARM1, PRMT6, and/or PRMT8) at an ICs less Pharmaceutical compositions described herein can be than or equal to 0.1 LM. In certain embodiments, a provided 10 prepared by any method known in the art of pharmacology. compound inhibits an RMT (e.g., PRMT1, PRMT3, In general. Such preparatory methods include the steps of CARM1, PRMT6, and/or PRMT8) at an IC50 less than or bringing a compound described herein (the “active ingredi equal to 0.01 uM. In certain embodiments, a provided ent') into association with a carrier and/or one or more other compound inhibits an RMT (e.g., PRMT1, PRMT3, accessory ingredients, and then, if necessary and/or desir CARM1, PRMT6, and/or PRMT8) in a cell at an EC less 15 able, shaping and/or packaging the product into a desired than or equal to 10 uM. In certain embodiments, a provided single- or multi-dose unit. compound inhibits an RMT (e.g., PRMT1, PRMT3, Pharmaceutical compositions can be prepared, packaged, CARM1, PRMT6, and/or PRMT8) in a cell at an EC less and/or sold in bulk, as a single unit dose, and/or as a plurality than or equal to 12 LM. In certain embodiments, a provided of single unit doses. As used herein, a “unit dose' is discrete compound inhibits an RMT (e.g., PRMT1, PRMT3, amount of the pharmaceutical composition comprising a CARM1, PRMT6, and/or PRMT8) in a cell at an EC less predetermined amount of the active ingredient. The amount than or equal to 3 LM. In certain embodiments, a provided of the active ingredient is generally equal to the dosage of compound inhibits PRMT1 in a cell at an EC less than or the active ingredient which would be administered to a equal to 12 LM. In certain embodiments, a provided com Subject and/or a convenient fraction of Such a dosage Such pound inhibits PRMT1 in a cell at an EC less than or equal 25 as, for example, one-half or one-third of Such a dosage. to 3 LM. In certain embodiments, a provided compound Relative amounts of the active ingredient, the pharma inhibits an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, ceutically acceptable excipient, and/or any additional ingre and/or PRMT8) in a cell at an EC less than or equal to 1 dients in a pharmaceutical composition of the present dis LM. In certain embodiments, a provided compound inhibits closure will vary, depending upon the identity, size, and/or an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, and/or 30 condition of the Subject treated and further depending upon PRMT8) in a cell at an EC less than or equal to 0.1 uM. the route by which the composition is to be administered. By In certain embodiments, a provided compound inhibits cell way of example, the composition may comprise between proliferation at an ECs less than or equal to 10 LM. In 0.1% and 100% (w/w) active ingredient. certain embodiments, a provided compound inhibits cell In some embodiments, a pharmaceutical composition proliferation at an ECso less than or equal to 1 LM. In certain 35 described herein is sterilized. embodiments, a provided compound inhibits cell prolifera Pharmaceutically acceptable excipients used in the manu tion at an ECso less than or equal to 0.1 uM. facture of provided pharmaceutical compositions include It will be understood by one of ordinary skill in the art that inert diluents, dispersing and/or granulating agents, Surface the RMT can be wild-type, or any mutant or variant. active agents and/or emulsifiers, disintegrating agents, bind The present disclosure provides pharmaceutical compo 40 ing agents, preservatives, buffering agents, lubricating sitions comprising a compound described herein, e.g., a agents, and/or oils. Excipients such as cocoa butter and compound of Formula (I), or a pharmaceutically acceptable Suppository waxes, coloring agents, coating agents, Sweet salt thereof, as described herein, and optionally a pharma ening, flavoring, and perfuming agents may also be present ceutically acceptable excipient. It will be understood by one in the composition. of ordinary skill in the art that the compounds described 45 Exemplary diluents include calcium carbonate, sodium herein, or salts thereof, may be present in various forms, carbonate, calcium phosphate, dicalcium phosphate, cal Such as amorphous, hydrates, Solvates, or polymorphs. In cium sulfate, calcium hydrogen phosphate, sodium phos certain embodiments, a provided composition comprises phate lactose, Sucrose, cellulose, microcrystalline cellulose, two or more compounds described herein. In certain kaolin, mannitol, Sorbitol, inositol, Sodium chloride, dry embodiments, a compound described herein, or a pharma 50 starch, cornstarch, powdered Sugar, and mixtures thereof. ceutically acceptable salt thereof, is provided in an effective Exemplary granulating and/or dispersing agents include amount in the pharmaceutical composition. In certain potato starch, corn Starch, tapioca Starch, sodium starch embodiments, the effective amount is a therapeutically glycolate, clays, alginic acid, guar gum, citrus pulp, agar, effective amount. In certain embodiments, the effective bentonite, cellulose and wood products, natural sponge, amount is an amount effective for inhibiting an RMT (e.g., 55 cation-exchange resins, calcium carbonate, silicates, sodium PRMT1, PRMT3, CARM1, PRMT6, and/or PRMT8). In carbonate, cross-linked poly(Vinyl-pyrrolidone) (crospovi certain embodiments, the effective amount is an amount done), Sodium carboxymethyl starch (sodium starch glyco effective for treating an RMT-mediated disorder (e.g., a late), carboxymethyl cellulose, cross-linked sodium car PRMT1-, PRMT3-, CARM1-, PRMT6-, and/or PRMT8 boxymethyl cellulose (croscarmellose), methylcellulose, mediated disorder). In certain embodiments, the effective 60 pregelatinized starch (starch 1500), microcrystalline starch, amount is a prophylactically effective amount. In certain water insoluble starch, calcium carboxymethyl cellulose, embodiments, the effective amount is an amount effective to magnesium aluminum silicate (Veegum), sodium lauryl Sul prevent an RMT-mediated disorder. fate, quaternary ammonium compounds, and mixtures Pharmaceutically acceptable excipients include any and thereof. all solvents, diluents, or other liquid vehicles, dispersions, 65 Exemplary Surface active agents and/or emulsifiers Suspension aids, Surface active agents, isotonic agents, include natural emulsifiers (e.g., acacia, agar, alginic acid, thickening or emulsifying agents, preservatives, Solid bind Sodium alginate, tragacanth, chondruX, cholesterol, Xanthan, US 9,630,961 B2 41 42 pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, Exemplary antifungal preservatives include butyl para and lecithin), colloidal clays (e.g., bentonite (aluminum ben, methyl paraben, ethyl paraben, propyl paraben, benzoic silicate) and Veegum (magnesium aluminum silicate)), long acid, hydroxybenzoic acid, potassium benzoate, potassium chain amino acid derivatives, high molecular weight alco Sorbate, Sodium benzoate, sodium propionate, and Sorbic hols (e.g., Stearyl alcohol, cetyl alcohol, oleyl alcohol, 5 acid. triacetin monostearate, ethylene glycol distearate, glyceryl Exemplary alcohol preservatives include ethanol, poly monostearate, and propylene glycol monostearate, polyvinyl ethylene glycol, phenol, phenolic compounds, bisphenol, alcohol), carbomers (e.g., carboxy polymethylene, poly chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. acrylic acid, acrylic acid polymer, and carboxyvinyl poly Exemplary acidic preservatives include Vitamin A, vitamin mer), carrageenan, cellulosic derivatives (e.g., carboxym 10 C. vitamin E, beta-carotene, citric acid, acetic acid, dehy ethylcellulose sodium, powdered cellulose, hydroxymethyl droacetic acid, ascorbic acid, Sorbic acid, and phytic acid. cellulose, hydroxypropyl cellulose, hydroxypropyl methyl Other preservatives include tocopherol, tocopherol cellulose, methylcellulose), Sorbitan fatty acid esters (e.g., acetate, deteroxime mesylate, cetrimide, butylated hydroxy polyoxyethylene sorbitan monolaurate (Tween 20), poly anisol (BHA), butylated hydroxytoluened (BHT), ethylene oxyethylene sorbitan (Tween 60), polyoxyethylene sorbitan 15 diamine, sodium lauryl sulfate (SLS), sodium lauryl ether monooleate (Tween 80), sorbitan monopalmitate (Span 40), sulfate (SLES), sodium bisulfite, sodium metabisulfite, sorbitan monostearate (Span 60, sorbitan tristearate (Span potassium sulfite, potassium metabisulfite, Glydant Plus, 65), glyceryl monooleate, sorbitan monooleate (Span 80)), Phenonip, methylparaben, Germall 115, Germaben II, polyoxyethylene esters (e.g., polyoxyethylene monostearate Neolone, Kathon, and Euxyl. In certain embodiments, the (Myril 45), polyoxyethylene hydrogenated castor oil, poly preservative is an anti-oxidant. In other embodiments, the ethoxylated castor oil, polyoxymethylene Stearate, and Solu preservative is a chelating agent. tol), Sucrose fatty acid esters, polyethylene glycol fatty acid Exemplary buffering agents include citrate buffer solu esters (e.g., CremophorTM), polyoxyethylene ethers, (e.g., tions, acetate buffer solutions, phosphate buffer solutions, polyoxyethylene lauryl ether (Brij 30)), poly(vinyl-pyrroli ammonium chloride, calcium carbonate, calcium chloride, done), diethylene glycol monolaurate, triethanolamine 25 calcium citrate, calcium glubionate, calcium gluceptate, oleate, sodium oleate, potassium oleate, ethyl oleate, oleic calcium gluconate, D-gluconic acid, calcium glycerophos acid, ethyl laurate, sodium lauryl sulfate, Pluronic F68, phate, calcium lactate, propanoic acid, calcium levulinate, Poloxamer 188, cetrimonium bromide, cetylpyridinium pentanoic acid, dibasic calcium phosphate, phosphoric acid, chloride, benzalkonium chloride, docusate Sodium, and/or tribasic calcium phosphate, calcium hydroxide phosphate, mixtures thereof. 30 potassium acetate, potassium chloride, potassium gluconate, Exemplary binding agents include starch (e.g., cornstarch potassium mixtures, dibasic potassium phosphate, monoba and Starch paste), gelatin, Sugars (e.g., Sucrose, glucose, sic potassium phosphate, potassium phosphate mixtures, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), Sodium acetate, Sodium bicarbonate, sodium chloride, natural and Synthetic gums (e.g., acacia, Sodium alginate, Sodium citrate, sodium lactate, dibasic sodium phosphate, extract of Irish moss, panwar gum, ghatti gum, mucilage of 35 monobasic sodium phosphate, Sodium phosphate mixtures, isapol husks, carboxymethylcellulose, methylcellulose, eth tromethamine, magnesium hydroxide, aluminum hydroxide, ylcellulose, hydroxyethylcellulose, hydroxypropyl cellu alginic acid, pyrogen-free water, isotonic Saline, Ringer's lose, hydroxypropyl methylcellulose, microcrystalline cel solution, ethyl alcohol, and mixtures thereof. lulose, cellulose acetate, poly(Vinyl-pyrrolidone), Exemplary lubricating agents include magnesium Stear magnesium aluminum silicate (Veegum), and larch araboga 40 ate, calcium Stearate, Stearic acid, silica, talc, malt, glyceryl lactan), alginates, polyethylene oxide, polyethylene glycol, behanate, hydrogenated vegetable oils, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, Sodium benzoate, sodium acetate, Sodium chloride, leucine, waxes, water, alcohol, and/or mixtures thereof. magnesium lauryl Sulfate, Sodium lauryl Sulfate, and mix Exemplary preservatives include antioxidants, chelating tures thereof. agents, antimicrobial preservatives, antifungal preserva 45 Exemplary natural oils include almond, apricot kernel, tives, alcohol preservatives, acidic preservatives, and other avocado, babassu, bergamot, black current seed, borage, preservatives. cade, camomile, canola, caraway, carnauba, castor, cinna Exemplary antioxidants include alpha tocopherol, ascor mon, cocoa butter, coconut, cod liver, coffee, corn, cotton bic acid, acorbyl palmitate, butylated hydroxyanisole, buty seed, emu, eucalyptus, evening primrose, fish, flaxseed, lated hydroxytoluene, monothioglycerol, potassium meta 50 geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl bisulfite, propionic acid, propyl gallate, sodium ascorbate, myristate, jojoba, kukui nut, lavandin, lavender, lemon, sodium bisulfite, sodium metabisulfite, and sodium sulfite. litsea cubeba, macademia nut, mallow, mango seed, mead Exemplary chelating agents include ethylenediaminetet owfoam seed, mink, nutmeg, olive, orange, orange roughy, raacetic acid (EDTA) and salts and hydrates thereof (e.g., palm, palm kernel, peach kernel, peanut, poppy seed, pump Sodium edetate, disodium edetate, trisodium edetate, cal 55 kin seed, rapeseed, rice bran, rosemary, safflower, Sandal cium disodium edetate, dipotassium edetate, and the like), wood, Sasquana, Savoury, Sea buckthorn, sesame, shea but citric acid and salts and hydrates thereof (e.g., citric acid ter, silicone, soybean, Sunflower, tea tree, thistle, tsubaki, monohydrate), fumaric acid and salts and hydrates thereof, Vetiver, walnut, and wheat germ oils. Exemplary synthetic malic acid and salts and hydrates thereof, phosphoric acid oils include, but are not limited to, butyl Stearate, caprylic and salts and hydrates thereof, and tartaric acid and salts and 60 triglyceride, capric triglyceride, cyclomethicone, diethyl hydrates thereof. Exemplary antimicrobial preservatives sebacate, dimethicone 360, isopropyl myristate, mineral oil, include benzalkonium chloride, benzethonium chloride, octyldodecanol, oleyl alcohol, silicone oil, and mixtures benzyl alcohol, bronopol, cetrimide, cetylpyridinium chlo thereof. ride, chlorhexidine, chlorobutanol, chlorocresol, chloroxyle Liquid dosage forms for oral and parenteral administra nol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, 65 tion include pharmaceutically acceptable emulsions, micro phenol, phenoxyethanol, phenylethyl alcohol, phenylmercu emulsions, Solutions, Suspensions, syrups and elixirs. In ric nitrate, propylene glycol, and thimerosal. addition to the active ingredients, the liquid dosage forms US 9,630,961 B2 43 44 may comprise inert diluents commonly used in the art Such example, cetyl alcohol and glycerol monostearate, h) absor as, for example, water or other solvents, solubilizing agents bents such as kaolin and bentonite clay, and i) lubricants and emulsifiers such as ethyl alcohol, isopropyl alcohol, Such as talc, calcium Stearate, magnesium Stearate, Solid ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl ben polyethylene glycols, Sodium lauryl Sulfate, and mixtures Zoate, propylene glycol, 1,3-butylene glycol, dimethylfor thereof. In the case of capsules, tablets and pills, the dosage mamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, form may comprise buffering agents. castor, and sesame oils), glycerol, tetrahydrofurfuryl alco Solid compositions of a similar type can be employed as hol, polyethylene glycols and fatty acid esters of Sorbitan, fillers in soft and hard-filled gelatin capsules using Such and mixtures thereof. Besides inert diluents, the oral com excipients as lactose or milk Sugar as well as high molecular positions can include adjuvants such as wetting agents, 10 emulsifying and Suspending agents, Sweetening, flavoring, weight polyethylene glycols and the like. The Solid dosage and perfuming agents. In certain embodiments for parenteral forms of tablets, dragees, capsules, pills, and granules can be administration, the compounds described herein are mixed prepared with coatings and shells such as enteric coatings with solubilizing agents such as CremophorTM. alcohols, and other coatings well known in the pharmaceutical for oils, modified oils, glycols, polysorbates, cyclodextrins, 15 mulating art. They may optionally comprise opacifying polymers, and mixtures thereof. agents and can be of a composition that they release the Injectable preparations, for example, Sterile injectable active ingredient(s) only, or preferentially, in a certain part aqueous or oleaginous Suspensions can be formulated of the intestinal tract, optionally, in a delayed manner. according to the known art using Suitable dispersing or Examples of embedding compositions which can be used wetting agents and Suspending agents. The sterile injectable include polymeric Substances and waxes. Solid composi preparation can be a sterile injectable solution, Suspension or tions of a similar type can be employed as fillers in Soft and emulsion in a nontoxic parenterally acceptable diluent or hard-filled gelatin capsules using Such excipients as lactose Solvent, for example, as a solution in 1,3-butanediol. Among or milk Sugar as well as high molecular weight polyethylene the acceptable vehicles and solvents that can be employed glycols and the like. are water, Ringer's solution, U.S.P. and isotonic sodium 25 The active ingredient can be in micro-encapsulated form chloride solution. In addition, sterile, fixed oils are conven with one or more excipients as noted above. The solid tionally employed as a solvent or Suspending medium. For dosage forms of tablets, dragees, capsules, pills, and gran this purpose any bland fixed oil can be employed including ules can be prepared with coatings and shells such as enteric synthetic mono- or diglycerides. In addition, fatty acids Such coatings, release controlling coatings and other coatings as oleic acid are used in the preparation of injectables. 30 well known in the pharmaceutical formulating art. In Such The injectable formulations can be sterilized, for example, Solid dosage forms the active ingredient can be admixed by filtration through a bacterial-retaining filter, or by incor with at least one inert diluent such as sucrose, lactose, or porating sterilizing agents in the form of sterile Solid com starch. Such dosage forms may comprise, as is normal positions which can be dissolved or dispersed in sterile practice, additional Substances other than inert diluents, e.g., water or other sterile injectable medium prior to use. 35 tableting lubricants and other tableting aids Such a magne In order to prolong the effect of a drug, it is often desirable sium Stearate and microcrystalline cellulose. In the case of to slow the absorption of the drug from subcutaneous or capsules, tablets, and pills, the dosage forms may comprise intramuscular injection. This can be accomplished by the use buffering agents. They may optionally comprise opacifying of a liquid Suspension of crystalline or amorphous material agents and can be of a composition that they release the with poor water solubility. The rate of absorption of the drug 40 active ingredient(s) only, or preferentially, in a certain part then depends upon its rate of dissolution which, in turn, may of the intestinal tract, optionally, in a delayed manner. depend upon crystal size and crystalline form. Alternatively, Examples of embedding compositions which can be used delayed absorption of a parenterally administered drug form include polymeric Substances and waxes. is accomplished by dissolving or Suspending the drug in an Dosage forms for topical and/or transdermal administra oil vehicle. 45 tion of a provided compound may include ointments, pastes, Compositions for rectal or vaginal administration are creams, lotions, gels, powders, solutions, sprays, inhalants typically Suppositories which can be prepared by mixing the and/or patches. Generally, the active ingredient is admixed compounds described herein with Suitable non-irritating under Sterile conditions with a pharmaceutically acceptable excipients or carriers such as cocoa butter, polyethylene carrier and/or any desired preservatives and/or buffers as can glycol or a Suppository wax which are solid at ambient 50 be required. Additionally, the present disclosure encom temperature but liquid at body temperature and therefore passes the use of transdermal patches, which often have the melt in the rectum or vaginal cavity and release the active added advantage of providing controlled delivery of an ingredient. active ingredient to the body. Such dosage forms can be Solid dosage forms for oral administration include cap prepared, for example, by dissolving and/or dispensing the Sules, tablets, pills, powders, and granules. In such solid 55 active ingredient in the proper medium. Alternatively or dosage forms, the active ingredient is mixed with at least one additionally, the rate can be controlled by either providing a inert, pharmaceutically acceptable excipient or carrier Such rate controlling membrane and/or by dispersing the active as sodium citrate or dicalcium phosphate and/or a) fillers or ingredient in a polymer matrix and/or gel. extenders such as starches, lactose. Sucrose, glucose, man Formulations suitable for topical administration include, nitol, and silicic acid, b) binders such as, for example, 60 but are not limited to, liquid and/or semi liquid preparations carboxymethylcellulose, alginates, gelatin, polyvinylpyrro Such as liniments, lotions, oil in water and/or water in oil lidinone. Sucrose, and acacia, c) humectants such as glyc emulsions such as creams, ointments and/or pastes, and/or erol, d) disintegrating agents such as agar, calcium carbon Solutions and/or Suspensions. Topically-administrable for ate, potato or tapioca starch, alginic acid, certain silicates, mulations may, for example, comprise from about 1% to and Sodium carbonate, e) solution retarding agents such as 65 about 10% (w/w) active ingredient, although the concentra paraffin, f) absorption accelerators such as quaternary tion of the active ingredient can be as high as the Solubility ammonium compounds, g) wetting agents such as, for limit of the active ingredient in the solvent. Formulations for US 9,630,961 B2 45 46 topical administration may further comprise one or more of formulations for buccal administration may comprise a the additional ingredients described herein. powder and/or an aerosolized and/or atomized solution A provided pharmaceutical composition can be prepared, and/or Suspension comprising the active ingredient. Such packaged, and/or sold in a formulation Suitable for pulmo powdered, aerosolized, and/or aerosolized formulations, nary administration via the buccal cavity. Such a formulation when dispersed, may have an average particle and/or droplet may comprise dry particles which comprise the active size in the range from about 0.1 to about 200 nanometers, ingredient and which have a diameter in the range from and may further comprise one or more of the additional about 0.5 to about 7 nanometers or from about 1 to about 6 ingredients described herein. nanometers. Such compositions are conveniently in the form A provided pharmaceutical composition can be prepared, of dry powders for administration using a device comprising 10 packaged, and/or sold in a formulation for ophthalmic a dry powder reservoir to which a stream of propellant can administration. Such formulations may, for example, be in be directed to disperse the powder and/or using a self the form of eye drops including, for example, a 0.1/1.0% propelling solvent/powder dispensing container Such as a (w/w) solution and/or Suspension of the active ingredient in device comprising the active ingredient dissolved and/or an aqueous or oily liquid carrier. Such drops may further Suspended in a low-boiling propellant in a sealed container. 15 comprise buffering agents, salts, and/or one or more other of Such powders comprise particles wherein at least 98% of the the additional ingredients described herein. Other opthalmi particles by weight have a diameter greater than 0.5 nano cally-administrable formulations which are useful include meters and at least 95% of the particles by number have a those which comprise the active ingredient in microcrystal diameter less than 7 nanometers. Alternatively, at least 95% line form and/or in a liposomal preparation. Ear drops and/or of the particles by weight have a diameter greater than 1 eye drops are contemplated as being within the scope of this nanometer and at least 90% of the particles by number have disclosure. a diameter less than 6 nanometers. Dry powder composi Although the descriptions of pharmaceutical composi tions may include a solid fine powder diluent Such as Sugar tions provided herein are principally directed to pharmaceu and are conveniently provided in a unit dose form. tical compositions which are Suitable for administration to Low boiling propellants generally include liquid propel 25 humans, it will be understood by the skilled artisan that such lants having a boiling point of below 65° F. at atmospheric compositions are generally Suitable for administration to pressure. Generally the propellant may constitute 50 to animals of all sorts. Modification of pharmaceutical com 99.9% (w/w) of the composition, and the active ingredient positions suitable for administration to humans in order to may constitute 0.1 to 20% (w/w) of the composition. The render the compositions Suitable for administration to vari propellant may further comprise additional ingredients such 30 ous animals is well understood, and the ordinarily skilled as a liquid non-ionic and/or solid anionic Surfactant and/or veterinary pharmacologist can design and/or perform Such a solid diluent (which may have a particle size of the same modification with ordinary experimentation. order as particles comprising the active ingredient). Compounds provided herein are typically formulated in Pharmaceutical compositions formulated for pulmonary dosage unit form for ease of administration and uniformity delivery may provide the active ingredient in the form of 35 of dosage. It will be understood, however, that the total daily droplets of a solution and/or Suspension. Such formulations usage of provided compositions will be decided by the can be prepared, packaged, and/or sold as aqueous and/or attending physician within the scope of Sound medical dilute alcoholic Solutions and/or suspensions, optionally judgment. The specific therapeutically effective dose level sterile, comprising the active ingredient, and may conve for any particular subject or organism will depend upon a niently be administered using any nebulization and/or atomi 40 variety of factors including the disease, disorder, or condi Zation device. Such formulations may further comprise one tion being treated and the severity of the disorder; the or more additional ingredients including, but not limited to, activity of the specific active ingredient employed; the a flavoring agent such as Saccharin sodium, a volatile oil, a specific composition employed; the age, body weight, gen buffering agent, a Surface active agent, and/or a preservative eral health, sex and diet of the subject; the time of admin such as methylhydroxybenzoate. The droplets provided by 45 istration, route of administration, and rate of excretion of the this route of administration may have an average diameter in specific active ingredient employed; the duration of the the range from about 0.1 to about 200 nanometers. treatment; drugs used in combination or coincidental with Formulations described herein as being useful for pulmo the specific active ingredient employed; and like factors well nary delivery are useful for intranasal delivery of a phar known in the medical arts. maceutical composition. Another formulation Suitable for 50 The compounds and compositions provided herein can be intranasal administration is a coarse powder comprising the administered by any route, including enteral (e.g., oral), active ingredient and having an average particle from about parenteral, intravenous, intramuscular, intra-arterial, 0.2 to 500 micrometers. Such a formulation is administered intramedullary, intrathecal. Subcutaneous, intraventricular, by rapid inhalation through the nasal passage from a con transdermal, interdermal, rectal, intravaginal, intraperito tainer of the powder held close to the nares. 55 neal, topical (as by powders, ointments, creams, and/or Formulations for nasal administration may, for example, drops), mucosal, nasal, bucal, Sublingual; by intratracheal comprise from about as little as 0.1% (w/w) and as much as instillation, bronchial instillation, and/or inhalation; and/or 100% (w/w) of the active ingredient, and may comprise one as an oral spray, nasal spray, and/or aerosol. Specifically or more of the additional ingredients described herein. A contemplated routes are oral administration, intravenous provided pharmaceutical composition can be prepared, 60 administration (e.g., systemic intravenous injection), packaged, and/or sold in a formulation for buccal adminis regional administration via blood and/or lymph Supply, tration. Such formulations may, for example, be in the form and/or direct administration to an affected site. In general the of tablets and/or lozenges made using conventional meth most appropriate route of administration will depend upon a ods, and may contain, for example, 0.1 to 20% (w/w) active variety of factors including the nature of the agent (e.g., its ingredient, the balance comprising an orally dissolvable 65 stability in the environment of the gastrointestinal tract), and/or degradable composition and, optionally, one or more and/or the condition of the subject (e.g., whether the subject of the additional ingredients described herein. Alternately, is able to tolerate oral administration). US 9,630,961 B2 47 48 The exact amount of a compound required to achieve an achieved. In general, it is expected that additional therapeu effective amount will vary from subject to subject, depend tically active agents utilized in combination be utilized at ing, for example, on species, age, and general condition of levels that do not exceed the levels at which they are utilized a subject, severity of the side effects or disorder, identity of individually. In some embodiments, the levels utilized in the particular compound(s), mode of administration, and the 5 combination will be lower than those utilized individually. like. The desired dosage can be delivered three times a day, Exemplary additional therapeutically active agents two times a day, once a day, every other day, every third day, include, but are not limited to, Small organic molecules Such every week, every two weeks, every three weeks, or every as drug compounds (e.g., compounds approved by the U.S. four weeks. In certain embodiments, the desired dosage can Food and Drug Administration as provided in the Code of be delivered using multiple administrations (e.g., two, three, 10 Federal Regulations (CFR)), peptides, proteins, carbohy four, five, six, seven, eight, nine, ten, eleven, twelve, thir drates, monosaccharides, oligosaccharides, polysaccharides, teen, fourteen, or more administrations). nucleoproteins, mucoproteins, lipoproteins, synthetic poly In certain embodiments, an effective amount of a com peptides or proteins, Small molecules linked to proteins, pound for administration one or more times a day to a 70 kg glycoproteins, steroids, nucleic acids, DNAS, RNAS, nucleo adult human may comprise about 0.0001 mg to about 3000 15 tides, nucleosides, oligonucleotides, antisense oligonucle mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to otides, lipids, hormones, vitamins, and cells. In certain about 1000 mg, about 0.001 mg to about 1000 mg, about embodiments, an additional therapeutically active agent is 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg. prednisolone, dexamethasone, doxorubicin, Vincristine, about 1 mg to about 1000 mg, about 1 mg to about 100 mg. mafosfamide, cisplatin, carboplatin, Ara-C, rituximab, about 10 mg to about 1000 mg, or about 100 mg to about aZacitadine, panobinostat, Vorinostat, everolimus, rapamy 1000 mg. of a compound per unit dosage form. cin, ATRA (all-trans retinoic acid), daunorubicin, decitabine, In certain embodiments, a compound described herein Vidaza, mitoxantrone, or IBET-151. may be administered at dosage levels sufficient to deliver Also encompassed by the present disclosure are kits (e.g., from about 0.001 mg/kg to about 1000 mg/kg, from about pharmaceutical packs). The kits provided may comprise a 0.01 mg/kg to about mg/kg, from about 0.1 mg/kg to about 25 provided pharmaceutical composition or compound and a 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from container (e.g., a vial, ampule, bottle, Syringe, and/or dis about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg penser package, or other Suitable container). In some to about 10 mg/kg, or from about 1 mg/kg to about 25 embodiments, provided kits may optionally further include mg/kg, of Subject body weight per day, one or more times a a second container comprising a pharmaceutical excipient day, to obtain the desired therapeutic effect. 30 for dilution or Suspension of a provided pharmaceutical In some embodiments, a compound described herein is composition or compound. In some embodiments, a pro administered one or more times per day, for multiple days. vided pharmaceutical composition or compound provided in In some embodiments, the dosing regimen is continued for the container and the second container are combined to form days, weeks, months, or years. one unit dosage form. In some embodiments, a provided kits It will be appreciated that dose ranges as described herein 35 further includes instructions for use. provide guidance for the administration of provided phar Compounds and compositions described herein are gen maceutical compositions to an adult. The amount to be erally useful for the inhibition of RMT (e.g., PRMT1, administered to, for example, a child or an adolescent can be PRMT3, CARM1, PRMT6, and/or PRMT8). In some determined by a medical practitioner or person skilled in the embodiments, methods of treating an RMT-mediated disor art and can be lower or the same as that administered to an 40 der in a Subject are provided which comprise administering adult. an effective amount of a compound described herein (e.g., a It will be also appreciated that a compound or composi compound of Formula (I)), or a pharmaceutically acceptable tion, as described herein, can be administered in combina salt thereof), to a subject in need of treatment. In certain tion with one or more additional therapeutically active embodiments, the effective amount is a therapeutically agents. In certain embodiments, a compound or composition 45 effective amount. In certain embodiments, the effective provided herein is administered in combination with one or amount is a prophylactically effective amount. In certain more additional therapeutically active agents that improve embodiments, the subject is suffering from a RMT-mediated its bioavailability, reduce and/or modify its metabolism, disorder. In certain embodiments, the subject is susceptible inhibit its excretion, and/or modify its distribution within the to a RMT-mediated disorder. body. It will also be appreciated that the therapy employed 50 As used herein, the term “RMT-mediated disorder” means may achieve a desired effect for the same disorder, and/or it any disease, disorder, or other pathological condition in may achieve different effects. which an RMT (e.g., PRMT1, PRMT3, CARM1, PRMT6, The compound or composition can be administered con and/or PRMT8) is known to play a role. Accordingly, in currently with, prior to, or Subsequent to, one or more Some embodiments, the present disclosure relates to treating additional therapeutically active agents. In certain embodi 55 or lessening the severity of one or more diseases in which an ments, the additional therapeutically active agent is a com RMT is known to play a role. pound of Formula (I). In certain embodiments, the additional In some embodiments, the present disclosure provides a therapeutically active agent is not a compound of Formula method of inhibiting an RMT comprising contacting the (I). In general, each agent will be administered at a dose RMT with an effective amount of a compound described and/or on a time schedule determined for that agent. In will 60 herein (e.g., a compound of Formula (I)), or a pharmaceu further be appreciated that the additional therapeutically tically acceptable salt thereof. The RMT may be purified or active agent utilized in this combination can be administered crude, and may be present in a cell, tissue, or subject. Thus, together in a single composition or administered separately such methods encompass both inhibition of in vitro and in in different compositions. The particular combination to vivo RMT activity. In certain embodiments, the method is an employ in a regimen will take into account compatibility of 65 in vitro method, e.g., Such as an assay method. It will be a provided compound with the additional therapeutically understood by one of ordinary skill in the art that inhibition active agent and/or the desired therapeutic effect to be of an RMT does not necessarily require that all of the RMT US 9,630,961 B2 49 50 be occupied by an inhibitor at once. Exemplary levels of the methylation status of H4R3 positively correlates with inhibition of an RMT (e.g., PRMT1, PRMT3, CARM1, increasing tumor grade and can be used to predict the risk of PRMT6, and/or PRMT8) include at least 10% inhibition, prostate cancer recurrence (Seligson et al., Nature 2005 435, about 10% to about 25% inhibition, about 25% to about 50% 1262–1266). Thus, in some embodiments, inhibitors of inhibition, about 50% to about 75% inhibition, at least 50% PRMT1, as described herein, are useful in treating cancers inhibition, at least 75% inhibition, about 80% inhibition, associated with the methylation status of H4R3, e.g., pros about 90% inhibition, and greater than 90% inhibition. tate cancer. Additionally, the methylarginine effector mol In some embodiments, provided is a method of inhibiting ecule TDRD3 interacts with the H4R3me2a mark, and RMT activity in a subject in need thereof (e.g., a subject overexpression of TDRD3 is linked to poor prognosis for the diagnosed as having an RMT-mediated disorder) comprising 10 administering to the Subject an effective amount of a com Survival of patients with breast cancer (Nagahata et al., pound described herein (e.g., a compound of Formula (I)), or Cancer Sci. 200495,218-225). Thus, in some embodiments, a pharmaceutically acceptable salt thereof, or a pharmaceu inhibitors of PRMT1, as described herein, are useful in tical composition thereof. treating cancers associated with overexpression of TDRD3. In certain embodiments, provided is a method of modu 15 e.g., breast cancer, as inhibition of PRMT1 leads to a lating gene expression in a cell which comprises contacting decrease in methylation of H4R3, thereby preventing the a cell with an effective amount of a compound of Formula association of overexpressed TDRD3 with H4R3me2a. In (I), or a pharmaceutically acceptable salt thereof. In certain other examples, PRMT1 is known to have non-histone embodiments, the cell is in culture in vitro. In certain substrates. For example, PRMT1, when localized to the embodiments, the cell is in an animal, e.g., a human. In cytoplasm, methylates proteins that are involved in signal certain embodiments, the cell is in a Subject in need of transduction pathways, e.g., the estrogen receptor (ER). The treatment. expression status of ER in breast cancer is critical for In certain embodiments, provided is a method of modu prognosis of the disease, and both genomic and non-ge lating transcription in a cell which comprises contacting a nomic ER pathways have been implicated in the pathogen cell with an effective amount of a compound of Formula (I), 25 esis of breast cancer. For example, it has been shown that or a pharmaceutically acceptable salt thereof. In certain PRMT1 methylates ERC, and that ERC. methylation is embodiments, the cell is in culture in vitro. In certain required for the assembly of ERC. with SRC (a proto embodiments, the cell is in an animal, e.g., a human. In oncogene tyrosine-protein kinase) and focal adhesion kinase certain embodiments, the cell is in a Subject in need of (FAK). Further, the silencing of endogenous PRMT1 treatment. 30 resulted in the inability of estrogen to activate AKT. These In certain embodiments, a method is provided of selecting results suggested that PRMT1-mediated ERC. methylation is a therapy for a subject having a disease associated with an required for the activation of the SRC-PI3K-FAK cascade RMT-mediated disorder or mutation comprising the steps of and AKT, coordinating cell proliferation and survival. Thus, determining the presence of an RMT-mediated disorder or hypermethylation of ERC. in breast cancer is thought to gene mutation in an RMT gene (e.g., a PRMT1, PRMT3, 35 cause hyperactivation of this signaling pathway, providing a CARM1, PRMT6, and/or PRMT8 gene) or and selecting, selective Survival advantage to tumor cells (Le Romancer et based on the presence of an RMT-mediated disorder a gene al., Mol. Cell 2008 31, 212-221. Le Romancer et al., Steroids mutation in the RMT gene a therapy that includes the 2010 75, 560-564). Accordingly, in some embodiments, administration of a provided compound. In certain embodi inhibitors of PRMT1, as described herein, are useful in ments, the disease is cancer. 40 treating cancers associated with ERC. methylation, e.g., In certain embodiments, a method of treatment is pro breast cancer. In yet another example, PRMT1 has been vided for a subject in need thereof comprising the steps of shown to be involved in the regulation of leukemia devel determining the presence of an RMT-mediated disorder or a opment. For example, SRC-associated in mitosis 68 kDa gene mutation in the RMT gene and treating the Subject in protein (SAM68; also known as KHDRBS1) is a well need thereof, based on the presence of a RMT-mediated 45 characterized PRMT1 substrate, and when either SAM68 or disorder or gene mutation in the RMT gene with a therapy PRMT1 is fused directly to the myeloid/lymphoid leukemia that includes the administration of a provided compound. In (MLL) gene, these fusion proteins can activate MLL onco certain embodiments, the Subject is a cancer patient. genic properties, implying that the methylation of SAM68 In some embodiments, a compound provided herein is by PRMT1 is a critical signal for the development of useful in treating a proliferative disorder, Such as cancer. For 50 leukemia (Cheung et al., Nature Cell Biol. 2007 9, 1208 example, while not being bound to any particular mecha 1215). Accordingly, in some embodiments, inhibitors of nism, protein arginine methylation by PRMTs is a modifi PRMT1, as described herein, are useful in treating cancers cation that has been implicated in signal transduction, gene associated with SAM68 methylation, e.g., leukemia. In still transcription, DNA repair and mRNA splicing, among oth another example, PRMT1 is implicated in leukemia devel ers; and overexpression of PRMTs within these pathways is 55 opment through its interaction with AE9a, a splice isoform often associated with various cancers. Thus, compounds of AML1-ETO (Shia et al., Blood 2012 119:4953-62). which inhibit the action of PRMTs, as provided herein, are Knockdown of PRMT1 affects expression of certain AE9a effective in the treatment of cancer. activated genes and Suppresses AE9a's self-renewal capa In some embodiments, compounds provided herein are bility. It has also been shown that AE9a recruits PRMT1 to effective in treating cancer through the inhibition of PRMT1. 60 AE9a activated gene promoters, which leads to increased H4 For example, PRMT1 overexpression has been observed in Arg3 methylation, H3 Lys9/14 acetylation, and transcription various human cancers, including, but not limited to, breast activated. Accordingly, in Some embodiments, inhibitors of cancer, prostate cancer, lung cancer, colon cancer, bladder PRMT1, as described herein, are useful in treating cancers cancer, and leukemia. In one example, PRMT1 specifically associated with AML1-ETO, e.g., leukemia. Thus, without deposits an asymmetric dimethylarginine (al)MA) mark on 65 being bound by any particular mechanism, the inhibition of histone H4 at arginine 3 (H4R3me2a), and this mark is PRMT1, e.g., by compounds described herein, is beneficial associated with transcription activation. In prostate cancer, in the treatment of cancer. US 9,630,961 B2 51 52 In some embodiments, compounds provided herein are bound by any particular mechanism, the inhibition of effective in treating cancer through the inhibition of PRMT3. PRMT6, e.g., by compounds described herein, is beneficial In one example, the DAL1 tumor Suppressor protein has in the treatment of cancer. been shown to interact with PRMT3 and inhibits its meth In some embodiments, compounds provided herein are yltransferase activity (Singh et al., Oncogene 2004 23, effective in treating cancer through the inhibition of PRMT8. 7761-7771). Epigenetic downregulation of DAL1 has been For example, deep-sequencing efforts of cancer genomes reported in several cancers (e.g., meningiomas and breast (e.g., COSMIC) have revealed that of all the PRMTs, cancer), thus PRMT3 is expected to display increased activ PRMT8 is reported to be the most mutated. Of 106 ity, and cancers that display DAL1 silencing may, in some sequenced genomes, 15 carry mutations in the PRMT8 10 coding region, and nine of these result in an amino acid aspects, be good targets for PRMT3 inhibitors, e.g., those change (Forbes et al., Nucleic Acids Res. 2011 39, D945 described herein. Thus, without being bound by any par D950). Because of its high rate of mutation in cancer, ticular mechanism, the inhibition of PRMT3, e.g., by com PRMT8 is thought to contribute to the initiation or progres pounds described herein, is beneficial in the treatment of sion of cancer. Thus, without being bound by any particular CaCC. 15 mechanism, the inhibition of PRMT8, e.g., by compounds In some embodiments, compounds provided herein are described herein, is beneficial in the treatment of cancer. effective in treating cancer through the inhibition of PRMT4, In some embodiments, compounds described herein are also known as CARM1. For example, PRMT4 levels have useful for treating a cancer including, but not limited to, been shown to be elevated in castration-resistant prostate acoustic neuroma, adenocarcinoma, adrenal gland cancer, cancer (CRPC), as well as in aggressive breast tumors (Hong anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymp et al., Cancer 2004 101, 83-89; Majumder et al., Prostate hangioendotheliosarcoma, hemangiosarcoma), appendix 2006 66, 1292-1301). Thus, in some embodiments, inhibi cancer, benign monoclonal gammopathy, biliary cancer tors of PRMT4, as described herein, are useful in treating (e.g., cholangiocarcinoma), bladder cancer, breast cancer cancers associated with PRMT4 overexpression. PRMT4 (e.g., adenocarcinoma of the breast, papillary carcinoma of has also been shown to affect ERC.-dependent breast cancer 25 the breast, mammary cancer, medullary carcinoma of the cell differentiation and proliferation (Al-Dhaheri et al., Can breast), brain cancer (e.g., meningioma; glioma, e.g., astro cer Res. 2011 71,2118-2128), thus in some aspects PRMT4 cytoma, oligodendroglioma; medulloblastoma), bronchus inhibitors, as described herein, are useful in treating ERC.- cancer, carcinoid tumor, cervical cancer (e.g., cervical dependent breast cancer by inhibiting cell differentiation and adenocarcinoma), choriocarcinoma, chordoma, craniophar proliferation. In another example, PRMT4 has been shown 30 yngioma, colorectal cancer (e.g., colon cancer, rectal cancer, to be recruited to the promoter of E2F1 (which encodes a colorectal adenocarcinoma), epithelial carcinoma, cell cycle regulator) as a transcriptional co-activator (Frietze ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, et al., Cancer Res. 2008 68, 301-306). Thus, PRMT4 multiple idiopathic hemorrhagic sarcoma), endometrial can mediated upregulation of E2F1 expression may contribute to cer (e.g., uterine cancer, uterine sarcoma), esophageal cancer cancer progression and chemoresistance as increased abun 35 (e.g., adenocarcinoma of the esophagus, Barrett's adenoca dance of E2F1 triggers invasion and metastasis by activating rinoma), Ewing sarcoma, eye cancer (e.g., intraocular mela growth receptor signaling pathways, which in turn promote noma, retinoblastoma), familiar hypereosinophilia, gall an antiapoptotic tumor environment (Engelmann and Pitzer, bladder cancer, gastric cancer (e.g., stomach adenocarci Cancer Res 2012 72: 571). Accordingly, in some embodi noma), gastrointestinal stromal tumor (GIST), head and ments, the inhibition of PRMT4, e.g., by compounds pro 40 neck cancer (e.g., head and neck Squamous cell carcinoma, vided herein, is useful in treating cancers associated with oral cancer (e.g., oral squamous cell carcinoma (OSCC), E2F1 upregulation. Thus, without being bound by any throat cancer (e.g., laryngeal cancer, pharyngeal cancer, particular mechanism, the inhibition of PRMT4, e.g., by nasopharyngeal cancer, oropharyngeal cancer)), hematopoi compounds described herein, is beneficial in the treatment of etic cancers (e.g., leukemia Such as acute lymphocytic CaCC. 45 leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute In some embodiments, compounds provided herein are myelocytic leukemia (AML) (e.g., B-cell AML, T-cell effective in treating cancer through the inhibition of PRMT6. AML), chronic myelocytic leukemia (CML) (e.g., B-cell For example, PRMT6 has been reported to be overexpressed CML, T-cell CML), and chronic lymphocytic leukemia in a number of cancers, e.g., bladder and lung cancer (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as (Yoshimatsu et al., Int. J. Cancer 2011 128,562-573). Thus, 50 Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and in some embodiments, the inhibition of PRMT6, by com non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as pounds provided herein, is useful in treating cancers asso diffuse large cell lymphoma (DLCL) (e.g., diffuse large ciated with PRMT6 overexpression. In some aspects, B-cell lymphoma (DLBCL)), follicular lymphoma, chronic PRMT6 is primarily thought to function as a transcriptional lymphocytic leukemia/small lymphocytic lymphoma (CLL/ repressor, although it has also been reported that PRMT6 55 SLL), mantle cell lymphoma (MCL), marginal Zone B-cell functions as a co-activator of nuclear receptors. For lymphomas (e.g., mucosa-associated lymphoid tissue example, as a transcriptional repressor, PRMT6 Suppresses (MALT) lymphomas, nodal marginal Zone B-cell lym the expression of thrombospondin 1 (TSP1; also known as phoma, splenic marginal Zone B-cell lymphoma), primary THEBS1; a potent natural inhibitor of angiogenesis and mediastinal B-cell lymphoma, Burkitt lymphoma, lympho endothelial cell migration) and p21 (a natural inhibitor of 60 plasmacytic lymphoma (e.g., “Waldenström's macroglobu cyclin dependent kinase), thereby contributing to cancer linemia), hairy cell leukemia (HCL), immunoblastic large development and progression (Michaud-Levesque and Rich cell lymphoma, precursor B-lymphoblastic lymphoma and ard, J. Biol. Chem. 2009 284, 21338-21346; Kleinschmidt et primary central nervous system (CNS) lymphoma; and al., PLoS ONE 2012 7, e41446). Accordingly, in some T-cell NHL such as precursor T-lymphoblastic lymphoma/ embodiments, the inhibition of PRMT6, by compounds 65 leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cuta provided herein, is useful in treating cancer by preventing neous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, the repression of THEBs1 and/or p21. Thus, without being Sezary syndrome), angioimmunoblastic T-cell lymphoma, US 9,630,961 B2 53 54 extranodal natural killer T-cell lymphoma, enteropathy type the expression of PRMT1 and PRMT3 are increased in T-cell lymphoma, Subcutaneous panniculitis-like T-cell lym coronary heart disease (Chen et al., Basic Res. Cardiol. 2006 phoma, anaplastic large cell lymphoma); a mixture of one or 101:346-53). In another example, aloMA elevation is seen in more leukemia/lymphoma as described above; and multiple patients with renal failure, due to impaired clearance of this myeloma (MM)), heavy chain disease (e.g., alpha chain 5 metabolite from the circulation (Jacobi et al., Am. J. Neph disease, gamma chain disease, mu chain disease), heman rol. 2008 28:224–37). Thus, circulating alDMA levels is gioblastoma, inflammatory myofibroblastic tumors, immu observed in many pathophysiological situations. Accord nocytic amyloidosis, kidney cancer (e.g., nephroblastoma ingly, without being bound by any particular mechanism, the a.k.a. Wilms tumor, renal cell carcinoma), liver cancer (e.g., inhibition of PRMTs, e.g., by compounds described herein, hepatocellular cancer (HCC), malignant hepatoma), lung 10 results in the decrease of circulating aloMA, which is cancer (e.g., bronchogenic carcinoma, Small cell lung cancer beneficial in the treatment of diseases associated with (SCLC), non-small cell lung cancer (NSCLC), adenocarci increased levels of circulating aloMA, e.g., cardiovascular noma of the lung), leiomyosarcoma (LMS), mastocytosis disease, diabetes, kidney failure, renal disease, pulmonary (e.g., systemic mastocytosis), myelodysplastic syndrome disease, etc. In certain embodiments, a compound described (MDS), mesothelioma, myeloproliferative disorder (MPD) 15 herein is useful for treating or preventing vascular diseases. (e.g., polycythemia Vera (PV), essential thrombocytosis In some embodiments, a compound provided herein is (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelo useful in treating metabolic disorders. For example, PRMT1 fibrosis (MF), chronic idiopathic myelofibrosis, chronic has been shown to enhance mRNA levels of FoxO1 target myelocytic leukemia (CML), chronic neutrophilic leukemia genes in gluconeogenesis, which results in increased hepatic (CNL), hypereosinophilic syndrome (HES)), neuroblas glucose production, and knockdown of PRMT promotes toma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or inhibition of FoxO1 activity and thus inhibition of hepatic type 2, Schwannomatosis), neuroendocrine cancer (e.g., gas gluconeogenesis (Choi et al., Hepatology 2012 56:1546-56). troenteropancreatic neuroendoctrine tumor (GEP-NET), Additionally, genetic haploinsufficiency of Prmt1 has been carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cys shown to reduce blood glucose levels in mouse models. tadenocarcinoma, ovarian embryonal carcinoma, ovarian 25 Thus, without being bound by any particular mechanism, the adenocarcinoma), papillary adenocarcinoma, pancreatic inhibition of PRMT1, e.g., by compounds described herein, cancer (e.g., pancreatic andenocarcinoma, intraductal pap is beneficial in the treating of metabolic disorders, such as illary mucinous neoplasm (IPMN), Islet cell tumors), penile diabetes. In some embodiments, a provided compound is cancer (e.g., Paget’s disease of the penis and scrotum), useful in treating type I diabetes. In some embodiments, a pinealoma, primitive neuroectodermal tumor (PNT), pros 30 provided compound is useful in treating type II diabetes. tate cancer (e.g., prostate adenocarcinoma), rectal cancer, In some embodiments, a compound provided herein is rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., useful in treating muscular dystrophies. For example, squamous cell carcinoma (SCC), keratoacanthoma (KA), PRMT1, as well as PRMT3 and PRMT6, methylate the melanoma, basal cell carcinoma (BCC)), Small bowel cancer nuclear poly(A)-binding protein (PABPN1) in a region (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant 35 located near its C-terminus (Perreault et al., J. Biol. Chem. fibrous histiocytoma (MFH), liposarcoma, malignant 2007 282:7552–62). This domain is involved in the aggre peripheral nerve sheath tumor (MPNST), chondrosarcoma, gation of the PABPN1 protein, and abnormal aggregation of fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, this protein is involved in the disease oculopharyngeal Sweat gland carcinoma, synovioma, testicular cancer (e.g., muscular dystrophy (Davies et al., Int. J. Biochem. Cell. seminoma, testicular embryonal carcinoma), thyroid cancer 40 Biol. 2006 38:1457-62). Thus, without being bound by any (e.g., papillary carcinoma of the thyroid, papillary thyroid particular mechanism, the inhibition of PRMTs, e.g., by carcinoma (PTC), medullary thyroid cancer), urethral can compounds described herein, is beneficial in the treatment of cer, vaginal cancer and Vulvar cancer (e.g., Paget’s disease muscular dystrophies, e.g., oculopharyngeal muscular dys of the Vulva). trophy, by decreasing the amount of methylation of In some embodiments, a compound provided herein is 45 PABPN1, thereby decreasing the amount of PABPN1 aggre useful in treating diseases associated with increased levels of gation. circulating asymmetric dimethylarginine (al)MA), e.g., car CARM1 is also the most abundant PRMT expressed in diovascular disease, diabetes, kidney failure, renal disease, skeletal muscle cells, and has been found to selectively pulmonary disease, etc. Circulating a MA is produced by control the pathways modulating glycogen metabolism, and the proteolysis of asymmetrically dimethylated proteins. 50 associated AMPK (AMP-activated protein kinase) and p38 PRMTs which mediate alMA methylation include, e.g., MAPK (mitogen-activated protein kinase) expression. See, PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8... alDMA e.g., Wang et al., Biochem (2012) 444:323-331. Thus, in levels are directly involved in various diseases as aloMA is some embodiments, inhibitors of CARM1, as described an endogenous competitive inhibitor of nitric oxide synthase herein, are useful in treating metabolic disorders, e.g., for (NOS), thereby reducing the production of nitric oxide (NO) 55 example skeletal muscle metabolic disorders, e.g., glycogen (Vallance et al., J. Cardiovasc. Pharmacol. 1992 200Suppl. and glucose metabolic disorders. Exemplary skeletal muscle 12):S60-2). NO functions as a potent vasodilator in endothe metabolic disorders include, but are not limited to, Acid lial vessels, and as such inhibiting its production has major Maltase Deficiency (Glycogenosis type 2: Pompe disease), consequences on the cardiovascular system. For example, Debrancher deficiency (Glycogenosis type 3), Phosphory since PRMT1 is a major enzyme that generates aloMA, the 60 lase deficiency (McArdles; GSD 5), X-linked syndrome dysregulation of its activity is likely to regulate cardiovas (GSD9D), Autosomal recessive syndrome (GSD9B), Tarui's cular diseases (Boger et al., Ann. Med. 2006 38:126-36), and disease (Glycogen storage disease VII; GSD 7), Phospho other pathophysiological conditions such as diabetes melli glycerate Mutase deficiency (Glycogen storage disease X: tus (Sydow et al., Vasc. Med. 2005 10(Suppl. 1):S35-43), GSDX; GSD 10), Lactate dehydrogenase A deficiency (GSD kidney failure (Vallance et al., Lancet 1992 339:572-5), and 65 11), Branching enzyme deficiency (GSD 4), Aldolase A chronic pulmonary diseases (Zakrzewicz et al., BMC Pulm. (muscle) deficiency, B-Enolase deficiency, Triosephosphate Med. 2009 9:5). Additionally, it has been demonstrated that isomerase (TIM) deficiency, Lafora's disease (Progressive US 9,630,961 B2 55 56 myoclonic epilepsy 2). Glycogen storage disease (Muscle, -continued Type 0, Phosphoglucomutase 1 Deficiency (GSD 14)), and R3 Glycogenin Deficiency (GSD 15). NN Boc XIV = Q-B(OR)2 In some embodiments, a compound provided herein is I /N/ - - -> useful in treating autoimmune disease. For example, several 5 N Suzuki coupling lines of evidence strongly suggest that PRMT inhibitors may \ be valuable for the treatment of autoimmune diseases, e.g., X Y rheumatoid arthritis. PRMTs are known to modify and NSO), Z 2 k regulate several critical immunomodulatory proteins. For XIII example, post-translational modifications (e.g., arginine 10 Boc methylation), within T cell receptor signaling cascades allow T lymphocytes to initiate a rapid and appropriate immune \ NR3 response to pathogens. Co-engagement of the CD28 Q /N/ costimulatory receptor with the T cell receptor elevates PRMT activity and cellular protein arginine methylation, 15 including methylation of the guanine nucleotide exchange factor Vav1 (Blanchet et al., J. Exp. Med. 2005 202:371 377). PRMT inhibitors are thus expected to diminish meth ylation of the guanine exchange factor Vav 1, resulting in 1. optional functional group diminished IL-2 production. In agreement, siRNA directed modification/substitution steps against PRMT5 was shown to both inhibit NFAT-driven 2. Boc deprotection promoter activity and IL-2 secretion (Richard et al., Bio chem J. 2005 388:379-386). In another example, PRMT1 is NNR3 known to cooperate with PRMT4 to enhance NFkB p65 25 driven transcription and facilitate the transcription of p85 target genes like TNFO. (Covic et al., Embo. J. 2005 24:85 96). Thus, in some embodiments, PRMT1 and/or PRMT4 inhibitors, e.g., those described herein, are useful in treating autoimmune disease by decreasing the transcription of p65 30 target genes like TNFC. These examples demonstrate an important role for arginine methylation in inflammation. Scheme 1 shows a general synthesis route to pyrazole Thus, without being bound by any particular mechanism, the compounds of formula I, wherein Q is either the same as Q inhibition of PRMTs, e.g., by compounds described herein, 35 as or is precursor of Q which after functional group modi is beneficial in the treatment of autoimmune diseases. fication or substitution yields Q wherein Q, R. R. X, Y and In some embodiments, a compound provided herein is Z are as defined above. In the first step iodopyrazole useful in treating neurological disorders, such as amyo carboxaldehydes of general formula XI are allowed to react trophic lateral sclerosis (ALS). For example, a gene with mono-Boc protected ethylenediamines XII under involved in ALS, TLS/FUS, often contains mutated argin 40 reductive amination conditions (e.g. sodium cyanoborohy ines in certain familial forms of this disease (Kwiatkowski dride and catalytic acid such as acetic acid) in an appropriate et al., Science 2009 323:1205-8). These mutants are retained Solvent such as methanol to give intermediates of general in the cytoplasm, which is similar to reports documenting formula XIII. Suzuki reaction of the latter with boronic acids the role arginine methylation plays in nuclear-cytoplasmic or boronic esters of general formula XIV in the presence of shuffling (Shen et al., Genes Dev. 1998 12:679-91). This 45 a palladium catalyst (e.g. PdCl2(dppf)) and a base (e.g. implicates PRMT, e.g., PRMT1, function in this disease, as potassium carbonate) in an organic solvent (e.g. toluene) at it was demonstrated that TLS/FUS is methylated on at least elevated temperature yields intermediates of general for 20 arginine residues (Rappsilber et al., Anal. Chem. 2003 mula XV. In a subsequent optional step or steps, functional 75:3107-14). Thus, in some embodiments, the inhibition of groups on Q' may be modified to yield certain embodiments PRMTs, e.g., by compounds provided herein, are useful in O of final compounds of formula I. For example compounds treating ALS by decreasing the amount of TLS/FUS arginine wherein Q is substituted with an alkoxy group can be methylation. synthesized by alkylating intermediates of formula XV where Q' has a corresponding OH group by alkylation reaction with a suitable alkylbromide or iodide using an Scheme 1 55 appropriate base (e.g. potassium carbonate) in a suitable organic solvent (e.g. tetrahydrofuran). In a final deprotection R3 step the N-Boc protecting can be removed by treating XV with an acid (e.g. HCl) in a suitable organic solvent (e.g. NNBoc ethanol) to give compounds of formula I. H N. ~ 60 In certain embodiments, iodopyrazole carboxaldehydes of I CHO \ general formula XI may be prepared from suitable known R pyrazole compound intermediates by established synthetic ) ( XII chemistry methods. Standard methods include direct iodi X O Y reductive nation of a pyrazole 3-carboxylate and Sandmeyer reaction NS Z 2 amination 65 of a 3-amino pyrazole 4-carboxylate. In certain embodi XI ments, iodopyrazole carboxaldehydes are derived from iodopyrazole carboxylates by reduction to a hydroxymethyl US 9,630,961 B2 57 group followed by oxidation to carboxaldehyde. The mono -continued Boc protected ethylenediamines XII can be synthesized by R3 standard methods known in the literature for derivatizing or preparing ethylenediamines. For example intermediates of N Q /N/ H formula XII may be prepared by treatment of the corre- 5 N sponding unprotected diamine precursors with Boc-O and V purifying the mixture of mono and dibocylated products. Y \ R Heteroaromatic boronic acids or esters of general formula N XIV are either commercially available or can be prepared by YN R5 standard method for example by boronylation of the corre 10 R4 sponding commercially available heteroaryl bromides. II In certain embodiments, pyrazole compounds of general formula II are prepared from iodopyrazole carboxaldehydes of general formula XXI as depicted in Scheme 2. In cases 15 where R is hydrogen compounds of general formula II are Scheme 3 equivalent to compounds of general formula III which are tautomers. In certain embodiments R' is a protecting group HN COEt COEt NaNO such as tetrahydropyranyl (THP) which maybe cleaved to NC RNHNH f \ H2SO4 hydrogen under acidic conditions in the final Boc-deprotec -e- N -e- tion step. In certain embodiments, iodopyrazole carboxal \ N R5 KI dehydes of general formula XXI can be prepared as depicted OEt in Scheme 3. Rs R4 I COEt I CHH 25 Scheme 2 Y \ LiOH / \ NN N R5 THF NN N R5 HO XII R4 R4 -- 30 reductive amination Tipt

35 I CHO I CHOH IBX N| \ - DMSO N/ \ 40 YN R5 YN R5 XIV N Hip \ Suzuki coupling XXI

45 In certain embodiments, iodopyrazole carboxaldehydes of general formula XXI are prepared as depicted in Scheme 4 XXII which also provides iodopyrazole carboxyaldehydes of gen eral formula XXXI. Thus, alkylation of intermediates of general formula XXX gives a mixture of pyrazole nitrogen 50 alkylated isomers which are separated by chromatography to give pure isomers XXI and XXXI. In certain embodiments, pyrazole compounds of general formula III can be prepared from iodopyrazole carboxaldehydes of general formula V 55 XXXI as depicted in Scheme 5.

Scheme 4

XXIII I COEt I COEt f \ DHP f \ reduction 1. optional functional group NN 5 80 id N e.g. LlLiAlH4 modification substitution steps R YN R5 2. Boc deprotection 65 H US 9,630,961 B2 59 60 -continued XLI may be a selected protecting group Such as tetrahydro I COOH I CHO pyranyl (THP) which can be cleaved to hydrogen under acidic conditions in the final Boc-deprotection step. Nf \ He-oxidation NW \ N R5 e.g. IBX N N R s 5 Scheme 6 THP THP I CHO

" 10 W \N reductiveXII I CHO I CHO R5 N1 amination I CHO s RS R4' S -- N1 R5 RX YN1 R5 XLI R41N R5 R4 alkylation H 15 R3 conditions XXXI XXI NaH, THF XXX NN Boc I ~ XIV V Suzuki coupling 2O W \ R N SchClele 5 R5 N / I CHO R4' - A - 25 XLII N reductive R41 Né R5 amination XXXI R3 i NN NNBoe 30 Q /N/ Boc I /N/ XIV N N -e-Suzuki coupling / \ R N NN 2 R5 R41 YN R5 35 XXXII R4' XLIII

NN Boc Q /N/ 40 1. optional functional group N modification substitution steps o V 2. Boc deprotectionp R R41 NNYN 2 R5 XXXIII 45 3 R

1.. optionalOptional functionalTunctional group N-H modification substitution steps Q ~ 2. Boc deprotection \ 50 W \ R R3 N R5 N1 NSH Q /N/ R4 N IV k 55 R4 -NaN R5 In certain embodiments, iodopyrazole carboxaldehydes of III general formula XLI and LI can be prepared as depicted in Scheme 7. In certain embodiments, an R" group of iodopy In certain embodiments, pyrazole compounds of general razole carboxaldehydes is introduced by alkylation of inter formula IV are prepared from iodopyrazole carboxaldehydes mediates of formula XLVII. This reaction can give a mixture of general formula XLI as depicted in Scheme 6. In certain of intermediate compounds of formulas XLI and LI which embodiments where R is hydrogen compounds of general may be separated by chromatography. The THP protected formula IV are equivalent to compounds of general formula 65 intermediates of formula XLVI can be used to prepare V which are tautomers. In certain embodiments where R' in compounds of formula IV where R–H as also depicted in compounds of formula IV is hydrogen, R' in intermediate Scheme 7. US 9,630,961 B2

Scheme 7 COEt I COEt I COEt I CHOH f \ iodination W \ DHP W \ LiAlH4 ) ( R5 N1 e.g. NIS R5 N1 H R5 - R51N1 DMF/K2CO H H THP THP XLII XLIII XLIV XLV

oxidation e.g. MnO2

I CHO I CHO I CHO

I CHO

o / \, 4 / \ -- W \ -- R5 N1 - A - R5 N1 -'. R5 N1 N N alkylation R5 N1 n R4 R4 conditions H THP NaH, THF LI XLI XLVII XLVI

Aeps as in Scheme 6 R3

Q -U/h N

} (N \, R5 N1

H IV = V (R is H)

In certain embodiments, pyrazole compounds of general -continued formula V are prepared from iodopyrazole carboxaldehydes R3 of general formula LI as depicted in Scheme 8. NN Q /N/ Boc 45 N Scheme 8 o k I CHO R5 NY N N R4 XII LIII N reductive 50 R5 NY N R4 amination LI 1. optional functional group modification substitution steps 2. Boc deprotection 55 R3

NN H R3 Q N/N/ NN Boc 60 V I /N/ XIV R' N -e-Suzuki coupling R5 N- NR4 R V R5 N YNN R4 65 LII In certain embodiments, phenylboronic acids or esters of general formula XIV are commercially available. In certain US 9,630,961 B2 63 64 embodiments phenyl boronic acids or esters of general heated using conventional hotplate apparatus or heating formula XIV can be prepared from commercially available mantle or microwave irradiation equipment. Reactions were or known corresponding heteroaryl bromide precursors of conducted with or without stirring, under atmospheric or general formula LX by standard methods. One such method elevated pressure in either open or closed vessels. Reaction to prepare pinacol boranes of general formula XIVa is 5 progress was monitored using conventional techniques such as TLC, HPLC, UPLC, or LCMS using instrumentation and depicted in Scheme 9. methods described below. Reactions were quenched and crude compounds isolated using conventional methods as Scheme 9 described in the specific examples provided. Solvent 10 removal was carried out with or without heating, under Q atmospheric or reduced pressure, using either a rotary or O O Y centrifugal evaporator. Compound purification was carried N B-B M o/ NO out as needed using a variety of traditional methods includ M M ing, but not limited to, preparative chromatography under O O 15 acidic, neutral, or basic conditions using either normal phase Q - Br Ho Pd catalyst or reverse phase HPLC or flash columns or Prep-TLC plates. LX XIVa. Compound purity and mass confirmations were conducted using standard HPLC and/or UPLC and/or MS spectrom eters and/or LCMS and/or GC equipment (e.g., including, but not limited to the following instrumentation: Waters Examples Alliance 2695 with 2996 PDA detector connected with ZQ detector and ESI source: Shimadzu LDMS-2020; Waters In order that the invention described herein may be more Acquity H Class with PDA detector connected with SQ fully understood, the following examples are set forth. It detector and ESI source: Agilent 1100 Series with PDA should be understood that these examples are for illustrative 25 detector; Waters Alliance 2695 with 2998 PDA detector; AB purposes only and are not to be construed as limiting this SCIEX API 2000 with ESI source: Agilent 7890 GC). invention in any manner. Exemplified compounds were dissolved in either MeOH or Synthetic Methods MeCN to a concentration of approximately 1 mg/mL and General methods and experimental procedures for pre analyzed by injection of 0.5-10 uL into an appropriate paring and characterizing compounds of the present inven LCMS system using the methods provided in the following tion are set forth below. Wherever needed, reactions were table:

MS MS Heat Detector Mobile Phase Mobile Phase Flow Rate Block Voltage Method Column A. B (mL/min) Gradient Profile Temp ( C.) (kV) A. Shim-pack Water/0.05% ACN/O.05% 1 5% to 100% B in 2.0 250 1.5 XR-ODS TFA TFA minutes, 100% B for 1.1 2.21 Im minutes, 100% to 5% B in 3.0 x 50 mm 0.2 minutes, then stop B Gemini-NX WateriO.04% ACN 1 5% to 100% B in 2.0 200 0.75 3 Im C18 Ammonia minutes, 100% B for 1.1 110A minutes, 100% to 5% B in 0.1 minutes, then stop C Shim-pack Water/0.05% ACN/O.05% 1 5% to 100% B in 2.0 250 O.85 XR-ODS FA FA minutes, 100% B for 1.1 1.6 m minutes, 100% to 5% B in 2.0 x 50 mm 0.1 minutes, then stop D Shim-pack Water/0.05% ACN/O.05% 1 5% to 100% B in 2.0 250 O.9S XR-ODS TFA TFA minutes, 100% B for 1.1 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop E Waters Water O.05% ACNOOS90 O.9 5% to 100% B in 2.0 250 1.5 XSelect C18 FA FA minutes, 100% B for 1.2 3.5 m minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop F Shim-pack Water/0.05% ACN/O.05% 1 5% to 80% B in 3.25 200 O.9S XR-ODS TFA TFA minutes, 80% B for 1.35 2.2 In minutes, 80% to 5% B in 0.3 3.0 x 50 mm minutes, then stop G Shim-pack Water/0.05% ACN/O.05% 1 5% to 70% B in 2SO 200 O.9S XR-ODS TFA TFA minutes, 70% B for 0.70 2.2 In minutes, 70% to 5% B in 0.1 3.0 x 50 mm minutes, then stop H Shim-pack Water/0.05% ACN/O.05% 1 5% to 100% B in 2.20 250 O.9S XR-ODS TFA TFA minutes, 100% B for 1.00 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop I Shim-pack Water/0.05% ACN/O.05% 1 5% to 100% B in 1.20 250 O.9S XR-ODS minutes, 100% B for 1.00 2.2 In TFA TFA minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop US 9,630,961 B2 65 66 -continued

MS MS Heat Detector Mobile Phase Mobi e Phase Flow Rate Block Voltage Method Column A. B (mL/min) Gradient Profile Temp ( C.) (kV) Shim-pack Water,0.05% ACNOOS90 5% to 70% B in 3.20 250 XR-ODS TFA TFA minutes, 70% B for 0.75 2.2 In minutes, 70% to 5% B in 3.0 x 50 mm 0.35 minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 80% B in 3.00 250 XR-ODS TFA TFA minutes, 80% B for 0.8 2.2 In minutes, 80% to 5% B in 0.1 3.0 x 50 mm minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 100% B in 3.00 250 XR-ODS TFA TFA minutes, 100% B for 0.8 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 100% B in 2.20 250 XR-ODS TFA TFA minutes, 100% B for 1.00 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 80% B in 2.20 250 XR-ODS TFA TFA minutes, 80% B for 1.0 2.2 In minutes, 80% to 5% B in 0.1 3.0 x 50 mm minutes, then stop Zorbax Water,0.05% ACNOOS90 5% to 70% B in 8.00 250 Eclipse Plus TFA TFA minutes, 70% B for 2.0 C18 minutes, then stop 4.6 x 100 mm Shim-pack Water,0.05% ACNOOS90 5% to 65% B in 3.00 250 XR-ODS TFA TFA minutes, 65% B for 0.80 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 60% B in 2.50 250 XR-ODS TFA TFA minutes, 60% B for 0.7 2.2 In minutes, 60% to 5% B in 0.1 3.0 x 50 mm minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to SO% B in 2.50 250 XR-ODS TFA TFA minutes, 50% B for 0.7 2.2 In minutes, 50% to 5% B in 0.1 3.0 x 50 mm minutes, then stop XBridge Water,0.05% ACNOOS90 5% to 95% B in 2.20 250 TFA TFA minutes, 95% B for 1.00 minutes, 95% to 5% B in 0.1 minutes, then stop Water,0.05% ACNOOS90 0.7 5% to 100% B in 2.0 250 O.85 FA FA minutes, 100% B for 1.1 minutes, 100% to 5% B in 0.1 minutes, then stop Water,0.05% ACNOOS90 5% to 40% B in 2.50 250 TFA TFA minutes, 40% B for 0.7 minutes, 40% to 5% B in 0.1 minutes, then stop Water,0.05% ACNOOS90 5% to 60% B in 4.20 1.OS TFA TFA minutes, 60% B for 1.0 minutes, 60% to 5% B in 0.1 minutes, then stop Shim-pack Water,0.05% ACNOOS90 5% to 100% B in 2.20 XR-ODS TFA TFA minutes, 100% B for 1.00 minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop Shim-pack Water,0.05% ACNOOS90 0.7 5% to 100% B in 2.0 O.85 XR-ODS FA FA minutes, 100% B for 1.1 1.6 m minutes, 100% to 5% B in 2.0 x 50 mm 0.1 minutes, then stop Ecliplis Plus Water,0.05% ACN 5% to 100% B in 2.0 250 C18 3.5 m TFA minutes, 100% B for 1.0 4.6 x 50 mm minutes, 100% to 5% B in 0.1 minutes, then stop Ecliplis Plus Waterf10 ACNS90 5% to 100% B in 2.0 250 1.1 C18 3.5 m mM Water minutes, 100% B for 1.0 4.6 x 50 mm ammonium minutes, 100% to 5% B in carbonate 0.1 minutes, then stop A1 Shim-pack Water,0.05% ACN 5% to 100% B in 2.0 250 XR-ODS TFA minutes, 100% B for 1.0 2.2 In minutes, 100% to 5% B in 3.0 x 50 mm 0.1 minutes, then stop A2 Ecliplis Plus Waterf ACN 5% to 100% B in 2.0 250 C18 3.5 m 10 mM minutes, 100% B for 1.4 US 9,630,961 B2 67 68 -continued

MS MS Heat Detector Mobile Phase Mobile Phase Flow Rate Block Voltage Method Column A. B (mL/min) Gradient Profile Temp ( C.) (kV) 4.6 x 50 mm ammonium minutes, 100% to 5% B in acetate 0.1 minutes, then stop A3 Acquity Waterf ACNO.1% 0.55 5% B at 0.01 min up to 0.4 BEH C18 5 mM FA min, 35% B at 0.8 min, 55% 1.7 m ammonium B at 1.2 min, 100% B in 1.3 2.1 x 50 mm acetate? minutes, at 2.5 min up to O.1% FA 3.30 min, 5% B at 3.31 min up to 4.0 min, then stop

15 Compound structure confirmations were carried out using Intermediate Synthesis standard 300 or 400 MHz NMR spectrometers with NOe’s conducted whenever necessary. Synthesis of intermediate tert-butyl (2-(((3-iodo-1- The following abbreviations are used herein: (tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl) 2O (methyl)amino)ethyl)carbamate

Abbreviation Meaning

ACN acetonitrile atn. atmosphere 25 O DCM dichloromethane DHP dihydropyran 5-( N DIBAL. disobutyl aluminum hydride / / O DIEA diisopropyl ethylamine I N DMF dimethyl formamide 30 \ DMF-DMA dimethyl formamide dimethyl acetal DMSO dimethyl sulfoxide ( \ dppf 1,1'-bis(diphenylphosphino)ferrocene YN EA ethyl acetate ESI electrospray ionization 35 O EtOH ethanol FA formic acid GC gas chromatography l hour Hex hexanes 40 HMDS hexamethyl disilazide Step 1: tert-butyl (2-(((3-iodo-1-(tetrahydro-2H HPLC high performance liquid chromatography pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino) PA isopropanol ethyl)carbamate LCMS liquid chromatography/mass spectrometry MeOH methanol 45 min minutes NBS N-bromo succinimide NCS N-chloro Succinimide O NIS N-iodo Succinimide NMR nuclear magnetic resonance 50 5-( N NOe nuclear Overhauser effect /-/ O Prep. preparative PTSA para-toluene Sulfonic acid I v Rf retardation factor rt room temperature ( ) N RT retention time Sat. Saturated SGC silica gel chromatography O TBAF tetrabutyl ammonium fluoride 60 TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbal TLC thin layer chromatography dehyde (3.2 g, 10.45 mmol. 1.00 equiv), tert-butyl N-2- UPLC ultra performance liquid chromatography 65 (methylamino)ethylcarbamate (2.2 g, 12.63 mmol. 1.21 equiv) and NaBH(OAc) (6.65 g, 31.38 mmol. 3.00 equiv) in dichloroethane (30 mL) was stirred for 2 h at room US 9,630,961 B2 69 70 temperature. The reaction was quenched with 50 mL of MHz, CDC1): 88.18 (s, 1H), 4.38-4.29 (m, 2H), 1.41-1.33 saturated aqueous sodium bicarbonate solution. The result (m, 3H) ppm. LCMS (method B, ESI): RT=1.36 min, ing mixture was extracted with 3x200 mL of dichlorometh m/z=267.0 M+H". ane. The combined organic layers was dried over anhydrous Sodium Sulfate and concentrated under vacuum. The residue 5 Step 2: Ethyl 3-iodo-1-(tetrahydro-2H-pyran-2-yl)- was purified on a silica gel column eluted with 30-100% 1H-pyrazole-4-carboxylate ethyl acetate in petroleum ether to give 4.05 g (83%) of tert-butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate as a light yellow oil. "H NMR (300 MHz, CDC1): 8 7.48 (s, 1H), 10 O N-1 5.35-5.30 (m. 1H), 4.13-4.03 (m, 1H), 3.71-3.63 (m. 1H), 3.36 (s. 2H), 3.26-3.25 (m, 2H), 2.52-2.49 (m, 2H), 2.21 (s, 3H), 2.09-201 (m, 3H), 1.68-1.58 (m, 3H), 1.44 (s, 9H) ppm. LCMS (method C, ESI): RT=0.58 min, m/z 465.0 M+H". 15 Synthesis of intermediate tert-butyl (2-(((3-iodo-1- (tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl) (methyl)amino)ethyl)(methyl)carbamate A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (10.8 g. 40.60 mmol. 1.00 equiv), 3,4-dihydro-2H-pyran (10 g, 118.88 mmol. 2.93 equiv) and TsOH (780 mg. 4.53 mmol. 0.11 equiv) in THF (100 mL) was stirred for 2 h at 60° C. f. 25 The reaction mixture was cooled to room temperature and quenched by the addition of 100 mL of saturated sodium bicarbonate solution. The resulting solution was extracted // { with 2x80 mL of dichloromethane. The combined organic \ 30 layers was dried over anhydrous sodium Sulfate and con centrated under vacuum. The residue was purified on a silica gel column eluted with ethyl acetate/petroleum ether (1:20) to give 13 g (91%) of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyra Zole-4-carboxylate as a yellow oil. "H NMR (400 MHz, 35 CDC1): 8 8.04 (s, 1H), 5.40-5.38 (m. 1H), 4.34-4.29 (m, 2H), 408-4.05 (m, 1H), 3.73-3.70 (m, 1H), 2.07-1.98 (m, 3H), 1.69-1.62 (m, 3H), 1.39-1.32 (m, 3H) ppm. LCMS (method C, ESI): RT=1.53 min, m/z 351.0 M+H".

Step 1: Ethyl 3-iodo-1H-pyrazole-4-carboxylate 40 Step 3: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H pyrazole-4-carboxylic acid

45

50 To a stirred solution of ethyl 3-amino-1H-pyrazole-4- carboxylate (10g, 64.45 mmol. 1.00 equiv) in 50% sulfuric acid (90 mL) at 5° C. was added dropwise a solution of NaNO (7.4g, 107.25 mmol. 1.66 equiv) in water (15 mL). 55 The reaction was stirred at 5° C. for another 30 min. A To a solution of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole solution of KI (32.1 g, 193.37 mmol. 3.00 equiv) in water 4-carboxylate (85g, 242.75 mmol. 1.00 equiv) in THF (300 (15 mL) was added dropwise at 5° C. The reaction was mL) and methanol (300 mL) was added a solution of LiOH allowed to stir at 5° C. for 1 h and then quenched by the (17.5 g, 730.69 mmol. 3.01 equiv) in water (400 mL). The addition of 50 mL of water. The precipitate was collected by 60 resulting solution was stirred at room temperature overnight filtration and then dissolved in 150 mL of ethyl acetate. The and then concentrated under vacuum to remove the organic resulting solution was washed sequentially with 1x100 mL solvent. The resulting solution was diluted with 400 mL of of saturated NaSO solution, 1x100 mL of saturated sodium HO and then acidified to pH 6.0 with 1M hydrochloric acid. bicarbonate solution and 1x100 mL of brine. The organic The mixture was extracted with 3x800 mL of dichlorometh layer was dried over anhydrous Sodium sulfate and concen 65 ane. The combined organic layers was washed with 3x1000 trated under vacuum to give 10.8 g (63%) of ethyl 3-iodo mL of brine, dried over anhydrous sodium sulfate and 1H-pyrazole-4-carboxylate as a yellow solid. "H NMR (300 concentrated under vacuum to give 75 g (96%) of 3-iodo US 9,630,961 B2 71 72 1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid as an off-white quenched by the addition of 100 mL of saturated NHCl solid. LCMS (method D, ESI): RT=1.23 min, m/z 323.0 solution. The resulting mixture was extracted with 3x400 M+H". mL of dichloromethane. The combined organic layers was dried over anhydrous sodium Sulfate and concentrated under Step 4: (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H vacuum. The residue was purified on a silica gel column pyrazol-4-yl)methanol eluted with ethyl acetate/petroleum ether (1:20) to give 13.48 g (90%) of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-car baldehyde as a golden oil. 1H NMR (300 MHz, DMSO-d6): & 9.69 (s, 1H), 8.57 (s, 1H), 5.49 (dd, J=2.7 Hz, 9.9 Hz, 1H), OH 10 3.95-3.91 (m. 1H), 3.68-3.62 (m. 1H), 2.11-2.01 (m, 3H), 1.69-1.62 (m, 3H) ppm. LCMS (method A, ESI): RT=1.35 min, m/z 3.07.0 M+H". Step 6: tert-Butyl (2-(((3-iodo-1-(tetrahydro-2H 15 pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino) ethyl)(methyl)carbamate

To a solution of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-car boxylic acid (28 g. 86.93 mmol. 1.00 equiv) in anhydrous THF (300 mL) maintained under nitrogen at 5°C. was added a 1M solution of BH in THF (300 mL) dropwise with stirring. The reaction was stirred overnight at room tem 25 perature and then quenched by the addition of 300 mL of saturated NHCl solution. The resulting mixture was extracted with 3x1000 mL of dichloromethane. The com \ f. bined organic layers was dried over anhydrous sodium 30 Sulfate and concentrated under vacuum. The residue was K. purified on a silica gel column eluted with ethyl acetate/ \ petroleum ether (1:1) to give 12.67 g (47%) of (3-iodo-1- (oxan-2-yl)-1H-pyrazol-4-yl)methanol as a white solid. "H NMR (400 MHz, DMSO-d6): 8 7.73 (s, 1H), 5.37-5.34 (m, 35 1H), 4.92 (s, 1H), 4.20 (d. J=3.6 Hz, 2H), 3.89-3.88 (m. 1H), 3.65-3.57 (m. 1H), 2.09-2.00 (m, 1H), 1.99-190 (m, 2H), 1.69-1.61 (m. 1H), 1.49-1.46 (m. 2H) ppm. LCMS (method A, ESI): RT=1.16 min, m/z 3.09.0 M+H". Step 5: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H 40 pyrazole-4-carbaldehyde

45

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbal 50 dehyde (21.5 g., 70.24 mmol. 1.00 equiv), tert-butyl N-methyl-N-(2-(methylamino)ethyl)carbamate (20 g, 106.23 mmol, 1.51 equiv) and NaBH(OAc) (29.8g., 137.98 mmol. 1.96 equiv) in dichloroethane (300 mL) was stirred for 1 h at room temperature. The reaction was diluted with 55 300 mL of dichloromethane and then washed with 3x300 Into a 250-mL 3-necked round-bottom flask purged and. mL of brine. The organic layer was dried over anhydrous To a stirred solution of oxalyl chloride (18.576 g. 146.35 Sodium sulfate and concentrated under vacuum. The residue mmol. 3.01 equiv) in anhydrous dichloromethane (300 mL) was purified on a silica gel column eluted with 0-7% maintained under nitrogen at -78° C. was added DMSO methanol in dichloromethane to give 31 g (92%) of tert (15.138 g., 193.75 mmol. 3.98 equiv) dropwise. The reaction 60 butyl (2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyra mixture was stirred at -65° C. for 30 min. A solution of Zol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate as (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol (15.0 g, a yellow oil. "H NMR (300 MHz, CDC1): 8 7.62 (s, 1H), 48.68 mmol. 1.00 equiv) in dichloromethane (100 mL) was 5.34-5.30 (m. 1H), 406-4.02 (m, 1H), 3.68-3.62 (m. 1H), then added dropwise at -65° C. and the reaction was stirred 3.42-3.38 (m, 4H), 2.85 (s, 4H), 2.62-2.53 (m, 2H), 2.47 for another 60 min at -65°C. Triethylamine (40.6 mL) was 65 2.46 (m, 2H), 2.13-1.97 (m, 3H), 1.74-1.69 (m, 3H), 1.46 (s, added dropwise at -65° C. and the reaction was stirred for 9H) ppm. LCMS (method A, ESI): RT=1.17 min, m/z 479.0 30 min at -65° C. The reaction was warmed to 0° C. then M+H".

US 9,630,961 B2 75 76 umn, 5um, 19x150 mm; mobile phase, water with 10 mmol Step 1: 1-isobutyl-4-(4,4,5,5-tetramethyl-1,3,2-di NHHCO, and CHCN (18% CHCN up to 58% in 10 min, oxaborolan-2-yl)-1H-pyrazole up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm to afford 40 mg (20%) of tert-butyl 2-(((3- (imidazol-2-alpyridin-6-yl)-1-(tetrahydro-2H-pyran-2-yl)- 1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate as a colorless oil. LCMS (method A, ESI): RT=1.04 min, m/Z-455.0 M+H". S/A- Step 2: Compound 21 N'-((3-(imidazo[1,2-alpyri 10 din-6-yl)-1H-pyrazol-4-yl)methyl)-N'-methylethane 1.2-diamine

15

A mixture of 4-(tetramethyl-1,3,2-dioxaborolan-2-yl)- 1H-pyrazole (1 g, 5.15 mmol. 1.00 equiv), 1-bromo-2- methylpropane (1.05 g, 7.66 mmol. 1.49 equiv) and CsCO NNNA (3.36 g, 10.31 mmol. 2.00 equiv) in acetonitrile (60 mL) was stirred at 80° C. for 4 h. The reaction was cooled to room 25 temperature and the solid material was removed by filtra A solution of tert-butyl 2-(((3-(imidazo[1,2-alpyridin-6- tion. The filtrate was diluted with ethyl acetate (30 mL) and yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl) then washed with brine (40 mL). The organic layer was dried (methyl)amino)ethyl)carbamate (40 mL, 1.00 equiv) in tet over anhydrous sodium sulfate and concentrated under rahydrofuran (3 mL) and 12N hydrochloric acid (2 mL) was vacuum to give 1.14 g (88%) of 1-iso-butyl-4-(4.4.5.5- stirred at room temperature overnight. The pH of the mixture 30 tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole as a col was adjusted to 7 by addition of triethylamine. The resulting orless oil. H-NMR (300 MHz, CDC1): 8 7.78 (s, 1H), 7.65 mixture was concentrated under vacuum and the residue (s, 1H), 3.91 (d. J=7.2 Hz, 2H), 2.24-2.19 (m. 1H), 1.32 (s, purified by Prep-HPLC under the following conditions (1 ii 12H), 0.90 (d. J–7.2 Hz, 6H) ppm. LCMS (method D, ESI): Pre-HPLC-005 (Waters)): Column, XBridge Shield RP18 RT=1.51 min, m/z 251.0 M+H". OBD Column, 5um, 19x150 mm; mobile phase, water with 35 10 mmol NHHCO, and CHCN (18% CHCN up to 58% Step 2: (R/S) tert-butyl 2-(((2-iso-butyl-1-(tetra in 10 min, up to 95% in 1 min, down to 18% in 2 min); hydro-2H-pyran-2-yl)-3,4'-bi(1H-pyrazol)-4-yl) Detector, UV 254/220 nm to give 5 mg (21%) of N'-((3- methyl)(methyl)amino)ethyl)carbamate (imidazo 1,2-alpyridin-6-yl)-1H-pyrazol-4-yl)methyl)-N- methylethane-1,2-diamine as a colorless oil. H-NMR (300 40 MHz, CDOD): 8 8.94 (s, 1H), 7.93 (s, 1H), 7.74-7.72 (m, 2H), 7.65-7.62 (m, 2H), 3.59 (s. 2H), 2.87-2.82 (m, 2H), 2.58-2.54 (m, 2H), 2.27 (s, 3H) ppm. LCMS (method A2, N NHBOc ESI): RT=1.32 min, m/z 271.0 M+H". C N 45 // \ Compound 23

50 N'-((2-isobutyl-3,4'-bi(1H-pyrazol)-4-yl)methyl)- THP N'-methylethane-1,2-diamine A mixture of tert-butyl N-2-(3-iodo-1-(oxan-2-yl)-1H pyrazol-4-yl)methyl(methyl)amino)ethylcarbamate (800 55 mg, 1.72 mmol. 1.00 equiv), 1-iso-butyl-4-(4.4.5.5-tetram ethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (860 mg, 3.44 mmol. 2.00 equiv), Pd(dppf)Cl. (251 mg, 0.34 mmol, 0.20 equiv) and potassium carbonate (712 mg, 5.15 mmol. 2.99 equiv) in 1,4-dioxane (50 mL) and water (10 mL) was stirred 60 at 90° C. for 12 h. The reaction was cooled to room o N temperature and diluted with HO (20 mL) and the resulting \ mixture was extracted with 2x50 mL of ethyl acetate. The combined organic layers was washed with 2x100 mL of ( ) brine, dried over anhydrous sodium Sulfate and concentrated 65 under vacuum. The residue was purified on a silica gel column eluted with 0-30% of ethyl acetate in petroleum ether to give 400 mg (50%) of tert-butyl 2-(((2-iso-butyl

US 9,630,961 B2 79 80 carbonate solution. The precipitate was removed by filtra Step 2: (R/S) tert-butyl 5-(4-(((2-(tert-butoxycarbo tion. The filtrate which contained the crude product was nylamino)ethyl)(methyl)amino)methyl)-1-(tetra purified by Pre-HPLC with the following conditions (1 ii hydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)-1H-inda Pre-HPLC-005 (Waters)): Column, XBridge Shield RP18 Zole-1-carboxylate OBD Column, 5um, 19x150 mm; mobile phase, water with 5 10 mmol NHHCO, and CHCN (18% CHCN up to 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm to give 23 mg (37%) of N'-((1'- benzyl-3,4'-bi(1H-pyrazol)-4-yl)methyl)-N'-methylethane 1,2-diamine as a white solid. 'H-NMR (300 MHz, CDOD): 10 88.04 (s, 1H), 7.88 (s, 1H), 7.60 (s, 1H), 7.41-7.29 (m, 5H), 5.41 (s. 2H), 3.52 (s. 2H), 2.77-2.73 (m, 2H), 2.51-2.47 (m, 2H), 2.22 (s, 3H) ppm. LCMS (method A1, ESI): RT=1.04 Boc min, m/z. 311.0 M+H". 15 Compound 27 A mixture of (R/S) tert-butyl N-2-(3-iodo-1-(oxan-2- N'-((3-(1H-indazol-5-yl)-1H-pyrazol-4-yl)methyl)- yl)-1H-pyrazol-4-yl)methyl(methyl)amino)ethylcarbam N'-methylethane-1,2-diamine ate (200 mg, 0.43 mmol. 1.00 equiv), KPO3H2O (439 mg, 1.65 mmol. 3.83 equiv), 1-(tert-butoxycarbonyl)-1H-inda Zol-5-ylboronic acid (300 mg, 1.14 mmol. 2.66 equiv) and Pd(dppf)Cl.CHCl (135 mg) in ethylene glycol dimethyl 25 ether (20 mL) and water (1 mL) was stirred under nitrogen at 85°C. for 3 h. The resulting mixture was cooled to room temperature and concentrated under vacuum. The residue was dissolved in 5 mL of methanol and 5 mL of DMSO and then purified by Prep-HPLC with the following conditions 30 (1H-Pre-HPLC-005 (Waters)): Column, XBridge Shield RP18 OBD Column, 5 um, 19x150 mm; mobile phase, water with 10 mmol NHHCO, and CHCN (18% CHCN up to 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm to give 50 mg (21%) of 35 (R/S) tert-butyl 5-(4-(((2-(tert-butoxycarbonylamino)ethyl) (methyl)amino)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H pyrazol-3-yl)-1H-indazole-1-carboxylate as a colorless oil. LCMS (method A, ESI): RT=1.34 min, m/z 555.1 M+H". 40 Step 3: N'-((3-(1H-indazol-5-yl)-1H-pyrazol-4-yl) methyl)-N'-methylethane-1,2-diamine (Compound Step 1: 27) 1-(tert-butoxycarbonyl)-1H-indazol-5-ylboronic acid 45

OH N 50 Boc s 2 OH HN N-NH A solution of (1H-indazol-5-yl)boronic acid (300 mg. 1.85 mmol. 1.00 equiv), triethylamine (280 mg), BocO 55 A solution of (R/S) tert-butyl 5-(4-(((2-(tert-butoxycarbo (804 mg, 3.68 mmol. 1.99 equiv) and 4-dimethylaminopyri nylamino)ethyl)(methyl)amino)methyl)-1-(tetrahydro-2H dine (23 mg, 0.19 mmol, 0.10 equiv) in acetonitrile (15 mL) pyran-2-yl)-1H-pyrazol-3-yl)-1H-indazole-1-carboxylate was stirred at 25° C. overnight. Water (20 mL) was then (50 mg, 0.09 mmol. 1.00 equiv) in tetrahydrofuran (10 mL) added and the resulting mixture was extracted with 3x60 mL and 12N hydrochloric acid (2 mL) was stirred overnight at of ethyl acetate. The combined organic extracts was washed 60 25° C. The resulting mixture was concentrated under with 2x3 mL of water and 3x3 mL of brine. The organic vacuum to remove most of the THF. The pH value of the layer was dried over anhydrous Sodium sulfate and concen solution was adjusted to 9 with 10% sodium carbonate trated under vacuum. The residue was purified on a silica gel Solution. The resulting mixture was concentrated under column eluted with 0-5% of methanol in dichloromethane to vacuum. The residue was dissolved in 5 mL of methanol and give 320 mg (68%) of 1-(tert-butoxycarbonyl)-1H-indazol 65 then purified by Pre-HPLC with the following conditions 5-ylboronic acid as a white solid. LCMS (method A, ESI): (1H-Pre-HPLC-005 (Waters)): Column, XBridge Shield RT=1.34 min, m/z =263.0 M+H". RP18 OBD Column, 5 m, 19x150 mm; mobile phase, water US 9,630,961 B2 81 82 with 10 mmol NHHCO, and CHCN (18% CHCN up to Predicted Translations: 58% in 10 min, up to 95% in 1 min, down to 18% in 2 min); Detector, UV 254/220 nm to give 7 mg (29%) of (2-amino ethyl)(3-(1H-indazol-5-yl)-1H-pyrazol-4-yl)methyl) GST-tagged PRMT1 (SEQ ID NO. : 4) methylamine as a white solid. 'H-NMR (300 MHz, 5 MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL CDOD): 8 8.14 (s, 1H), 8.10 (s, 1H), 7.72-7.64 (m, 3H), 3.63 (s. 2H), 3.10-3.09 (m, 2H), 2.86-2.77 (m, 2H), 2.24 (s, EFPNLPYYIDGDWKLTOSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL 3H) ppm. LCMS (method A1, ESI): RT=0.78 min, DIRYGWSRIAYSKDFETLKWDFLSKLPEMLKMFEDRLCHKTYLNGDHWTH m/Z-271.0 M+H". Biological Methods 10 PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPOIDKYLKSSKYIA PRMT1 Biochemical Assay WPLOGWOATFGGGDHPPKSDENLYFOGGNSDYKDDDDKMAAAEAANCIME General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine NFVATLANGMSLOPPLEEVSCGOAESSEKPNAEDMTSKDYYPDSYAHFGI (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2-carboxyethyl)phos 15 HEEMLKDEWRTLTYRNSMFHNRHLFKDKWWLDWGSGTGILCMFAAKAGAR phine hydrochloride solution (TCEP) were purchased from KWIGIECSSISDYAWKIWKANKLDHWWTIIKGKWEEWELPWEKWDIIISE Sigma-Aldrich at the highest level of purity possible. H-SAM was purchase from American Radiolabeled WMGYCLFYESMLNTVLYARDKWLAPDGLIFPDRATLYWTAIEDROYKDYK Chemicals with a specific activity of 80 Ci/mmol. 384-well 20 IHWWENVYGFDMSCIKDVAIKEPLVDVVDPKOLVTNACLIKEVDIYTVKV streptavidin Flashplates were purchased from PerkinElmer. Substrates. EDLTFTSPFCLOWKRNDYWHALWAYFNIEFTRCHKRTGFSTSPESPYTHW Peptide representative of human histone H4 residues KOTWFYMEDYLTVKTGEEIFGTIGMRPNAKNNRDLDFTIDLDFKGOLCEL 36-50 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21 Century Biochemi- 25 SCSTDYRMR cals. The peptide was purified by high-performance liquid General Procedure for PRMT1 Enzyme Assays on Pep chromatography (HPLC) to greater than 95% purity and tide Substrates. confirmed by liquid chromatography mass spectrometry The assays were all performed in a buffer consisting of 20 (LC-MS). The sequence was Biot-Ahx-RLARRGGVKRIS mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and GLI-amide (SEQ ID NO.: 1). 30 0.002% Tween 20, prepared on the day of use. Compounds Molecular Biology: in 100% DMSO (1 ul) were spotted into a polypropylene Full-length human PRMT1 isoform 1 (NM 001536.5) 384-well V-bottom plates (Greiner) using a Platemate Plus transcript clone was amplified from an HEK 293 cDNA outfitted with a 384-channel head (Thermo Scientific). library, incorporating flanking 5' sequence encoding a FLAG DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows 35 A-H for the maximum signal control and 1 ul of SAH, a tag (DYKDDDDK) (SEQ ID NO.:2) fused directly to Met known product and inhibitor of PRMT1, was added to 1 of PRMT1. The amplified gene was subcloned into pFast columns 11, 12, 23, 24, rows I-P for the minimum signal BacI (Life Technologies) modified to encode an N-terminal control. A cocktail (40 ul) containing the PRMT1 enzyme GST tag and a TEV cleavage sequence (MSPILGYWKIK was added by Multidrop Combi (Thermo-Fisher). The com GLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK- 40 pounds were allowed to incubate with PRMT1 for 30 min at KFELGLEFPNLPYYI DGDVKLTQSMAIIRYIADKHN room temperature, then a cocktail (10 ul) containing SAM MLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK and peptide was added to initiate the reaction (final vol VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM ume=51 ul). The final concentrations of the components LYDALDVVLYMDPMCLDAFPKL VCFKKRIEAIPQID were as follows: PRMT1 was 0.5 nM, H-SAM was 200 KYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLY. 45 nM, non-radiolabeled SAM was 1.5uM, peptide was 20 nM, FQGGNS) (SEQ ID NO.:3) fused to Asp of the Flag tag of SAH in the minimum signal control wells was 1 mM, and PRMT1. the DMSO concentration was 2%. The assays were stopped Protein Expression. by the addition of non-radiolabeled SAM (10 ul) to a final Recombinant baculovirus were generated according to concentration of 300 uM, which dilutes the H-SAM to a Bac-to-Bac kit instructions (Life Technologies). Protein 50 level where its incorporation into the peptide substrate is no over-expression was accomplished by infecting exponen longer detectable. 50 ul of the reaction in the 384-well tially growing High Five insect cell culture at 1.5x10' polypropylene plate was then transferred to a 384-well cell/ml with 1:100 ratio of virus. Infections were carried out Flashplate and the biotinylated peptides were allowed to at 27° C. for 48 hours, harvested by centrifugation, and bind to the streptavidin surface for at least 1 hour before 55 being washed once with 0.1% Tween20 in a Biotek ELX405 stored at -80° C. for purification. plate washer. The plates were then read in a PerkinElmer Protein Purification. TopCount plate reader to measure the quantity of H-labeled Expressed full-length human GST-tagged PRMT1 protein peptide bound to the Flashplate Surface, measured as disin was purified from cell paste by glutathione Sepharose affinity tegrations per minute (dpm) or alternatively, referred to as chromatography after equilibration of the resin with 50 mM 60 counts per minute (cpm). phosphate buffer, 200 mM. NaCl, 5% glycerol, 5 mM %. Inhibition Calculation (3-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT1 was eluted with 50 mM Tris, 2 mM glutathione, pH 7.8, dialysed in buffer A and concentrated to 1 mg/mL. The dpmend - dipmni, % inch = 100- (TE) x 100 purity of recovered protein was 73%. Reference: Wasilko, 65 dpm - dipmrain D. J. and S. E. Lee: “TIPS: titerless infected-cells preser vation and scale-up' Bioprocess J., 5 (2006), pp. 29-32. US 9,630,961 B2 83 84 Where dpm disintegrations per minute, cmpd-signal in Predicted Translations: assay well, and min and max are the respective minimum and maximum signal controls. Four-Parameter ICs Fit Flag-PRMT6-His (SEO ID NO. : 7) MDYKDDDDKSOPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRE (Top - Bottom) Y = Bottom ( -- ( X Coefficient RDOLYYECYSDWSWHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAG Cso TGILSIFCAOAGARRVYAVEASAIWOOAREVWRFNGLEDRVHVLPGPVET 10 Where top and bottom are the normally allowed to float, WELPEQVDAIVSEWMGYGLLHESMLSSWLHARTKWLKEGGLLLPASAELF but may be fixed at 100 or 0 respectively in a 3-parameter IAPISDOMLEWRLGFWSOVKOHYGVDMSCLEGFATRCLMGHSEIWVOGLS fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition 15 GEDVLARPORFAOLELSRAGLEOELEAGWGGRFRCSCYGSAPMHGFAIWF and X is the compound concentration. PRMT6 Biochemical Assay OWTFPGGESEKPLVLSTSPFHPATHWKOALLYLNEPVOVEODTDVSGEIT General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine LLPSRDNPRRLRVLLRYKVGDOEEKTKDFAMEDHHHHHH (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), sodium butyrate and Tris(2- General Procedure for PRMT6 Enzyme Assays on Pep carboxyethyl)phosphine hydrochloride solution (TCEP) tide Substrates. were purchased from Sigma-Aldrich at the highest level of The assays were all performed in a buffer consisting of 20 purity possible. H-SAM was purchase from American mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and Radiolabeled Chemicals with a specific activity of 80 25 0.002% Tween 20, prepared on the day of use. Compounds Ci/mmol. 384-well streptavidin Flashplates were purchased in 100% DMSO (1 ul) were spotted into a polypropylene from PerkinElmer. 384-well V-bottom plates (Greiner) using a Platemate Plus Substrates. outfitted with a 384-channel head (Thermo Scientific). Peptide representative of human histone H4 residues DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows 36-50 was synthesized with an N-terminal linker-affinity tag 30 motif and a C-terminal amide cap by 21 Century Biochemi A-H for the maximum signal control and 1 ul of SAH, a cals. The peptide was purified by high-performance liquid known product and inhibitor of PRMT6, was added to chromatography (HPLC) to greater than 95% purity and columns 11, 12, 23, 24, rows I-P for the minimum signal confirmed by liquid chromatography mass spectrometry control. A cocktail (40 ul) containing the PRMT6 enzyme (LC-MS). The sequence was Biot-Ahx-RLARRGGVKRIS 35 was added by Multidrop Combi (Thermo-Fisher). The com GLI-amide and contained a monomethylated lysine at posi pounds were allowed to incubate with PRMT6 for 30 min at tion 44 (SEQ ID NO.:5). room temperature, then a cocktail (10 ul) containing SAM Molecular Biology: and peptide was added to initiate the reaction (final vol Full-length human PRMT6 (NM_018137.2) transcript ume=51 ul). The final concentrations of the components clone was amplified from an HEK 293 cDNA library, 40 were as follows: PRMT6 was 1 nM., H-SAM was 200 nM, incorporating a flanking 5' sequence encoding a FLAG tag non-radiolabeled SAM was 250 nM, peptide was 75 nM. (MDYKDDDDK) (SEQID NO.: 6) fused directly to Ser 2 of SAH in the minimum signal control wells was 1 mM, and PRMT6 and a 3' sequence encoding a hexa His sequence the DMSO concentration was 2%. The assays were stopped (HHHHHH) fused directly to Asp 375. The amplified gene by the addition of non-radiolabeled SAM (10 ul) to a final was subcloned into pFastEacMam (Viva Biotech). 45 concentration of 400 uM, which dilutes the H-SAM to a Protein Expression. level where its incorporation into the peptide substrate is no Recombinant baculovirus were generated according to longer detectable. 50 ul of the reaction in the 384-well Bac-to-Bac kit instructions (Life Technologies). Protein polypropylene plate was then transferred to a 384-well over-expression was accomplished by infecting exponen Flashplate and the biotinylated peptides were allowed to tially growing HEK 293F cell culture at 1.3x10 cell/ml with 50 bind to the streptavidin surface for at least 1 hour before virus (MOI-10) in the presence of 8 mM sodium butyrate. being washed once with 0.1% Tween20 in a Biotek ELX405 Infections were carried out at 37° C. for 48 hours, harvested by centrifugation, and stored at -80° C. for purification. plate washer. The plates were then read in a PerkinElmer Protein Purification. TopCount plate reader to measure the quantity of H-labeled Expressed full-length human Flag- and His-tagged 55 peptide bound to the Flashplate Surface, measured as disin PRMT6 protein was purified from cell paste by NiNTA tegrations per minute (dpm) or alternatively, referred to as agarose affinity chromatography after equilibration of the counts per minute (cpm). resin with buffer containing 50 mM Tris, 300 mM. NaCl, %. Inhibition Calculation 10% glycerol, pH 7.8 (Buffer Ni-A). Column was washed with 20 mMimidazole in the same buffer and Flag-PRMT6 60 His was eluted with 150 mM imidazole. Pooled fractions dpmend - dipm, were dialysed against buffer Ni-A and further purified by % inch=100- dpm - dipmii. )x100 anti-flag M2 affinity chromatography. Flag-PRMT6-His was eluted with 200 ug/ml FLAG peptide in the same buffer. Pooled fractions were dialysed in 20 mM Tris, 150 mM 65 Where dpm disintegrations per minute, cmpd-signal in NaCl, 10% glycerol and 5 mM f-mercaptoethanol, pH 7.8. assay well, and min and max are the respective minimum The purity of recovered protein was 95%. and maximum signal controls. US 9,630,961 B2 85 86 Four-Parameter ICs Fit phosphate buffer, 100 mM. NaCl, 5% glycerol, 5 mM (3-mercaptoethanol, pH 7.8 and PRMT8 was eluted by 100 mM phosphate in the same buffer. Protein was concentrated Y = Bottom (Top - Bottom) and buffer was exchanged to 50 mM Tris, 300 mM NaCl, ( -- ( X Coefficient 10% glycerol, 5 mM f-mercaptoethanol, pH 7.8 by ultra IC50 filtration. The purity of recovered protein was 89%. Predicted Translations: Where top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter 10 GST-tagged PRMT8 fit. The Hill Coefficient normally allowed to float but may (SEQ ID NO. : 10) also be fixed at 1 in a 3-parameter fit. Y is the % inhibition MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL and X is the compound concentration. PRMT8 Biochemical Assay EFPNLPYYIDGDWKLTOSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL General Materials. 15 DIRYGWSRIAYSKDFETLKWDFLSKLPEMLKMFEDRLCHKTYLNGDHWTH S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPOIDKYLKSSKYIA bovine skin gelatin (BSG), isopropyl-B-D-thiogalactopyra WPLOGWOATFGGGDHPPKSDLVPRGSPEFMGMKHSSRCLLLRRKMAENAA noside (IPTG), and Tris(2-carboxyethyl)phosphine hydro chloride solution (TCEP) were purchased from Sigma ESTEVNSPPSOPPOPVVPAKPVOCWHHVSTOPSCPGRGKMSKLLNPEEMT Aldrich at the highest level of purity possible. H-SAM was SRDYYFDSYAHFGIHEEMLKDEWRTLTYRNSMYHNKHWFKDKWWLDWGSG purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well streptavidin Flash TGILSMFAAKAGAKKWFGIECSSISDYSEKIIKANHLDNIITIFKGKWEE plates were purchased from PerkinElmer. WELPWEKWDIIISEWMGYCLFYESMLNTWIFARDKWLKPGGLMFPDRAAL Substrates. 25 Peptide representative of human histone H4 residues YVVAIEDROYKDFKIHWWENVYGFDMTCIRDWAMKEPLVDIVDPKOVVTN 31-45 was synthesized with an N-terminal linker-affinity tag motif and a C-terminal amide cap by 21 Century Biochemi ACLIKEVDIYTVKTEELSFTSAFCLOIORNDYWHALVTYFNIEFTKCHKK cals. The peptide was purified by high-performance liquid MGFSTAPDAPYTHWKOTWFYLEDYLTVRRGEEIYGTISMKPNAKNVRDLD chromatography (HPLC) to greater than 95% purity and 30 confirmed by liquid chromatography mass spectrometry FTVDLDFKGOLCETSVSNDYKMR (LC-MS). The sequence was Biot-Ahx-KPAIRRLARRG GVKR-amide (SEQ ID NO.:8). General Procedure for PRMT8 Enzyme Assays on Pep Molecular Biology: tide Substrates. Full-length human PRMT8 (NM 019854.4) isoform 1 35 The assays were all performed in a buffer consisting of 20 transcript clone was amplified from an HEK 293 cDNA mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and library and subcloned into pGEX-4T-1 (GE Life Sciences). 0.002% Tween 20, prepared on the day of use. Compounds The resulting construct encodes an N-terminal GST tag and in 100% DMSO (1 ul) were spotted into a polypropylene a thrombin cleavage sequence (MSPILGYWKIKGLVOP 384-well V-bottom plates (Greiner) using a Platemate Plus TRLLLEYLEEKYEEHLYERDEGDKWRNKKFEL 40 outfitted with a 384-channel head (Thermo Scientific). GLEFPNLPYYI DGDVKLTQSMAIIRYIADKHNMLG DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows GCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK A-H for the maximum signal control and 1 ul of SAH, a VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM known product and inhibitor of PRMT8, was added to LYDALDVVLYMDPMCLDAFPKL VCFKKRIEAIPQID columns 11, 12, 23, 24, rows I-P for the minimum signal KYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRG 45 control. A cocktail (40 ul) containing the PRMT8 enzyme SPEF) (SEQID NO.:9) fused directly to Met 1 of PRMT8. was added by Multidrop Combi (Thermo-Fisher). The com Protein Expression. pounds were allowed to incubate with PRMT8 for 30 min at E. coli (BL21 (DE3) Gold, Stratagene) made competent by room temperature, then a cocktail (10 ul) containing the CaCl method were transformed with the PRMT8 con H-SAM and peptide was added to initiate the reaction (final struct and amplicillin selection. Protein over-expression was 50 volume=51 ul). The final concentrations of the components accomplished by growing the PRMT8 expressing E. coli were as follows: PRMT8 was 1.5 nM, H-SAM was 50 nM, clone and inducing expression with 0.3 mM IPTG at 16°C. non-radiolabeled SAM was 550 nM, peptide was 150 nM, The culture was grown for 12 hours, harvested by centrifu SAH in the minimum signal control wells was 1 mM, and gation, and stored at -80° C. for purification. the DMSO concentration was 2%. The assays were stopped Protein Purification. 55 by the addition of non-radiolabeled SAM (10 ul) to a final Expressed full-length human GST-tagged PRMT8 protein concentration of 400 uM, which dilutes the H-SAM to a was purified from cell paste by glutathione Sepharose affinity level where its incorporation into the peptide substrate is no chromatography after the resin was equilibrated with 50 mM longer detectable. 50 ul of the reaction in the 384-well phosphate buffer, 200 mM. NaCl, 5% glycerol, 5 mM polypropylene plate was then transferred to a 384-well (3-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT8 60 Flashplate and the biotinylated peptides were allowed to was eluted with 50 mM Tris, 2 mM glutathione, pH 7.8. bind to the streptavidin surface for at least 1 hour before Pooled fractions were cleaved by thrombin (10 U) and being washed once with 0.1% Tween20 in a Biotek ELX405 dialysed in buffer A. GST was removed by reloading the plate washer. The plates were then read in a PerkinElmer cleaved protein sample onto glutathione Sepharose column TopCount plate reader to measure the quantity of H-labeled and PRMT8 was collected in the flow-through fractions. 65 peptide bound to the Flashplate Surface, measured as disin PRMT8 was purified further by ceramic hydroxyapatite tegrations per minute (dpm) or alternatively, referred to as chromatography. The column was washed with 50 mM counts per minute (cpm). US 9,630,961 B2 88 %. Inhibition Calculation Protein Purification. Expressed full-length human GST-tagged PRMT3 protein was purified from cell paste by glutathione Sepharose affinity don - dom. chromatography after equilibration of the resin with 50 mM % inch = 100 -(TE)xdpm - dpmni, 100 phosphate buffer, 200 mM. NaCl, 5% glycerol, 1 mM EDTA, 5 mM (3-mercaptoethanol, pH6.5 (Buffer A). GST-tagged PRMT3 was eluted with 50 mM Tris, 2 mM glutathione, pH Where dpm disintegrations per minute, cmpd-signal in 7.1 and 50 mM Tris, 20 mM glutathione, pH 7.1. Pooled assay well, and min and max are the respective minimum fractions were dialysed in 20 mM Tris, 50 mM. NaCl, 5% and maximum signal controls. 10 glycerol, 1 mM EDTA, 1 mM DTT, pH7.5 (Buffer B) and Four-Parameter ICs Fit applied to a Q Sepharose Fast Flow column. GST-tagged PRMT3 was eluted by 500 mM. NaCl in buffer B. Pooled fractions were dialyzed in 25 mM phosphate buffer, 100 mM Y = Bottom- (TopTop - Bottom) NaCl, 5% glycerol, 2 mM DTT, pH 6.8 (Buffer C) and 15 -- - - loaded on to a ceramic hydroxyapatite column. GST-tagged ( ( CsoX cer PRMT3 eluted with 25-400 mM phosphate in buffer C. Protein was concentrated and buffer was exchanged to 20 mM Tris, 150 mM. NaCl, 5% glycerol, 5 mM B-mercap Where top and bottom are the normally allowed to float, toethanol, pH7.8 by ultrafiltration. The purity of recovered but may be fixed at 100 or 0 respectively in a 3-parameter protein was 70%. fit. The Hill Coefficient normally allowed to float but may Predicted Translations: also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration. PRMT3 Biochemical Assay GST-tagged PRMT3 25 (SEQ ID NO. : 13) General Materials. MSPILGYWKIKGLVOPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), EFPNLPYYIDGDWKLTOSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL bovine skin gelatin (BSG), isopropyl-B-D-thiogalactopyra DIRYGWSRIAYSKDFETLKWDFLSKLPEMLKMFEDRLCHKTYLNGDHWTH noside (IPTG), and Tris(2-carboxyethyl)phosphine hydro chloride solution (TCEP) were purchased from Sigma 30 PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPOIDKYLKSSKYIA Aldrich at the highest level of purity possible. H-SAM was WPLOGWOATFGGGDHPPKSDLWPRGSCSLASGATGGRGAVENEEDLPELS purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol. 384-well streptavidin Flash DSGDEAAWEDEDDADLPHGKOOTPCLFCNRLFTSAEETFSHCKSEHOFNI plates were purchased from PerkinElmer. 35 DSMWHKHGLEFYGYIKLINFIRLKNPTWEYMNSIYNPWPWEKEEYLKPWL Substrates. Peptide containing the classic RMT substrate motif was EDDLLL.OFDVEDLYEPVSVPFSYPNGLSENTSVVEKLKHMEARALSAEAA synthesized with an N-terminal linker-affinity tag motif and LARAREDLOKMKOFAODFWMHTDVRTCSSSTSVIADLOEDEDGVYFSSYG a C-terminal amide cap by 21 Century Biochemicals. The peptide was purified by high-performance liquid chroma 40 HYGIHEEMLKDKIRTESYRDFIYONPHIFKDKVVLDVGCGTGILSMFAAK tography (HPLC) to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS). The AGAKKVLGVDOSEILYOAMDIIRLNKLEDTITLIKGKIEEWHLPVEKVDV sequence was Biot-Ahx-GGRGGFGGRGGFGGRGGFG IISEWMGYFLLFESMLDSWLYAKNKYLAKGGSWYPDICTISLWAWSDWNK amide (SEQ ID NO.11). HADRIAFWDDWYGFKMSCMKKAWIPEAWWEWLDPKTLISEPCGIKHIDCH Molecular Biology: 45 Full-length human PRMT3 (NM_005788.3) isoform 1 TTSISDLEFSSDFTLKITRTSMCTAIAGYPDIYFEKNCHNRVVFSTGPOS transcript clone was amplified from an HEK 293 cDNA TKTHWKOTVFLLEKPFSVKAGEALKGKVTVHKNKKDPRSLTVTLTLNNST library and subcloned into pGEX-KG (GE Life Sciences). The resulting construct encodes an N-terminal GST tag and QTYGLQ a thrombin cleavage sequence (MSPILGYWKIKGLVOP 50 TRLLLEYLEEKYEEHLYERDEGDKWRNKKFEL General Procedure for PRMT3 Enzyme Assays on Pep tide Substrates. GLEFPNLPYYI DGDVKLTQSMAIIRYIADKHNMLG GCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLK The assays were all performed in a buffer consisting of 20 VDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and 55 0.002% Tween 20, prepared on the day of use. Compounds LYDALDVVLYMDPMCLDAFPKL VCFKKRIEAIPQID in 100% DMSO (1 ul) were spotted into a polypropylene KYLKSSKYIAWPLQGWQATFGGGDHPPKS 384-well V-bottom plates (Greiner) using a Platemate Plus DLVPRGS) (SEQ ID NO. 12) fused directly to Cys 2 of outfitted with a 384-channel head (Thermo Scientific). PRMT3. DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows Protein Expression. 60 A-H for the maximum signal control and 1 ul of SAH, a E. coli (BL21 (DE3) Gold, Stratagene) made competent by known product and inhibitor of PRMT3, was added to the CaCl method were transformed with the PRMT3 con columns 11, 12, 23, 24, rows I-P for the minimum signal struct and amplicillin selection. Protein over-expression was control. A cocktail (40 ul) containing the PRMT3 enzyme accomplished by growing the PRMT3 expressing E. coli was added by Multidrop Combi (Thermo-Fisher). The com clone and inducing expression with 0.3 mM IPTG at 16°C. 65 pounds were allowed to incubate with PRMT3 for 30 min at The culture was grown for 12 hours, harvested by centrifu room temperature, then a cocktail (10 ul) containing SAM gation, and stored at -80° C. for purification. and peptide was added to initiate the reaction (final vol US 9,630,961 B2 89 90 ume=51 ul). The final concentrations of the components The gene sequence encoding isoform 1 containing a deletion were as follows: PRMT3 was 0.5 nM, H-SAM was 100 of amino acids 539-561 was amplified subsequently and nM, non-radiolabeled SAM was 1.8 uM, peptide was 330 subcloned into pastBacMam (Viva Biotech). nM. SAH in the minimum signal control wells was 1 mM, Protein Expression. and the DMSO concentration was 2%. The assays were Recombinant baculovirus were generated according to stopped by the addition of potassium chloride (10 ul) to a Bac-to-Bac kit instructions (Life Technologies). Protein final concentration of 100 mM. 50 ul of the reaction in the over-expression was accomplished by infecting exponen 384-well polypropylene plate was then transferred to a tially growing HEK293F cell culture at 1.3x10 cell/ml with 384-well Flashplate and the biotinylated peptides were virus (MOI-10) in the presence of 8 mM sodium butyrate. allowed to bind to the streptavidin surface for at least 1 hour 10 Infections were carried out at 37° C. for 48 hours, harvested before being washed once with 0.1% Tween20 in a Biotek by centrifugation, and stored at -80° C. for purification. ELX405 plate washer. The plates were then read in a Protein Purification. PerkinElmer TopCount plate reader to measure the quantity Expressed full-length human Flag- and His-tagged of H-labeled peptide bound to the Flashplate surface, 15 CARM1 protein was purified from cell paste by anti-flag M2 measured as disintegrations per minute (dpm) or alterna affinity chromatography with resin equilibrated with buffer tively, referred to as counts per minute (cpm). containing 20 mM Tris, 150 mM. NaCl, 5% glycerol, pH 7.8. %. Inhibition Calculation Column was washed with 500 mM NaCl in buffer A and Flag-CARM1-His was eluted with 200 ug/ml FLAG peptide in buffer A. Pooled fractions were dialyzed in 20 mM Tris, dpmend - dipmnin 150 mM. NaCl, 5% glycerol and 1 mM DTT, pH 7.8. The % inch=100- dpm - dipmrain )x100 purity of recovered protein was 94. Predicted Translations: Where dpm disintegrations per minute, cmpd-signal in 25 assay well, and min and max are the respective minimum Flag-CARM1-His and maximum signal controls. (SEQ ID NO. : 16) Four-Parameter ICs Fit MDYKDDDDKAAAAAAWGPGAGGAGSAWPGGAGPCATWSWFPGARLLTIGD ANGEIORHAEOOALRLEVRAGPDSAGIALYSHEDWCVFKCSVSRETECSR 30 Y = Bottom- (TopTop - Bottom) VGKOSFIITLGCNSVLIOFATPNDFCSFYNILKTCRGHTLERSVFSERTE X Hill Coefficient ( 1 ( Cso ) ESSAVOYFOFYGYLSOOONMMODYVRTGTYORAILONHTDFKDKIVLDVG CGSGILSFFAAOAGARKIYAVEASTMAOHAEWLVKSNNLTDRIVVIPGKV

Where top and bottom are the normally allowed to float, 35 EEVSLPEOVDIIISEPMGYMLFNERMLESYLHAKKYLKPSGNMFPTIGDV but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may HLAPFTDEOLYMEOFTKANFWYOPSFHGVDLSALRGAAVDEYFROPVVDT also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration. FDIRILMAKSWKYTWNFLEAKEGDLHRIEIPFKFHMLHSGLWHGLAFWFD CARM1 Biochemical Assay 40 WAFIGSIMTVWLSTAPTEPLTHWYOVRCLFOSPLFAKAGDTLSGTCLLIA General Materials. S-adenosylmethionine (SAM), S-adenosylhomocysteine NKROSYDISIVAOVDOTGSKSSNLLDLKNPFFRYTGTTPSPPPGSHYTSP (SAH), bicine, Tween20, dimethylsulfoxide (DMSO), SENMWNTGSTYNLSSGMAWAGMPTAYDLSSWIASGSSWGHNNLIPLGSSG bovine skin gelatin (BSG), sodium butyrate and Tris(2- carboxyethyl)phosphine hydrochloride solution (TCEP) 45 AOGSGGGSTSAHYAVNSOFTMGGPAISMASPMSIPTNTMHYGSEGHHHHH were purchased from Sigma-Aldrich at the highest level of purity possible. H-SAM was purchase from American H Radiolabeled Chemicals with a specific activity of 80 General Procedure for CARM1 Enzyme Assays on Pep Ci/mmol. 384-well streptavidin Flashplates were purchased tide Substrates. from PerkinElmer. 50 The assays were all performed in a buffer consisting of 20 Substrates. mM Bicine (pH=7.6), 1 mM TCEP, 0.005% BSG, and Peptide representative of human histone H3 residues 0.002% Tween 20, prepared on the day of use. Compounds 16-30 was synthesized with an N-terminal linker-affinity tag in 100% DMSO (1 ul) were spotted into a polypropylene motif and a C-terminal amide cap by 21 Century Biochemi 384-well V-bottom plates (Greiner) using a Platemate Plus cals. The peptide was purified by high-performance liquid 55 outfitted with a 384-channel head (Thermo Scientific). chromatography (HPLC) to greater than 95% purity and DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows confirmed by liquid chromatography mass spectrometry A-H for the maximum signal control and 1 ul of SAH, a (LC-MS). The sequence was Biot-AhX-PRKQLAT known product and inhibitor of CARM1, was added to KAARKSAP-amide and contained a monomethylated argi columns 11, 12, 23, 24, rows I-P for the minimum signal nine at position 26 (SEQ ID NO.14). 60 control. A cocktail (40 ul) containing the CARM1 enzyme Molecular Biology: was added by Multidrop Combi (Thermo-Fisher). The com Human CARM1 (PRMT4) (NM 199141.1) transcript pounds were allowed to incubate with CARM1 for 30 min clone was amplified from an HEK 293 cDNA library, at room temperature, then a cocktail (10 ul) containing incorporating a flanking 5' sequence encoding a FLAG tag H-SAM and peptide was added to initiate the reaction (final (MDYKDDDDK) (SEQID NO.: 6) fused directly to Ala 2 of 65 volume=51 ul). The final concentrations of the components CARM1 and 3' sequence encoding a hexa His sequence were as follows: CARM1 was 0.25 nM, H-SAM was 30 (EGHHHHHH) (SEQID NO.15) fused directly to Ser 608. nM, peptide was 250 nM, SAH in the minimum signal US 9,630,961 B2 91 92 control wells was 1 mM, and the DMSO concentration was TABLE 2-continued 2%. The assays were stopped by the addition of non radiolabeled SAM (10 ul) to a final concentration of 300 uM, Biochemical IC which dilutes the H-SAM to a level where its incorporation Cmpd No. PRMT1 PRMT6 PRMT8 PRMT3 CARM1 into the peptide substrate is no longer detectable. 50 ul of the 5 reaction in the 384-well polypropylene plate was then trans 29 C C D ferred to a 384-well Flashplate and the biotinylated peptides 30 B A. D were allowed to bind to the streptavidin surface for at least "—" indicates no data provided. For Table 2, “A” indicates an IC50s 0.100 LM, “B” indicates an ICso of 0.101-1.00 M, 1 hour before being washed once with 0.1% Tween20 in a “C” indicates an ICso of 1.01-3.00 LM, “D” indicates an IC50 of 3.01-10 M, and ICs > Biotek ELX405 plate washer. The plates were then read in a 10 10.01 um. PerkinElmer TopCount plate reader to measure the quantity RKO Methylation Assay of H-labeled peptide bound to the Flashplate surface, RKO adherent cells were purchased from ATCC (Ameri measured as disintegrations per minute (dpm) or alterna can Type Culture Collection), Manassas, Va., USA. DMEM/ tively, referred to as counts per minute (cpm). 15 Glutamax medium, penicillin-Streptomycin, heat inactivated %. Inhibition Calculation fetal bovine serum, 0.05% trypsin and D-PBS were pur chased from Life Technologies, Grand Island, N.Y., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG dpmend - dipm, (H+L) antibody, and Licor Odyssey infrared scanner were dpm - dipm purchased from Licor Biosciences, Lincoln, Nebr., USA. % inch=100- rain )x100 Mono-methyl arginine antibody was purchased from Cell Signaling Technology, Danvers, Mass., USA. Methanol was purchased from VWR, Franklin, Mass., USA. 10% Tween Where dpm disintegrations per minute, cmpd-signal in 20 was purchased from KPL, Inc., Gaithersburg, Md., USA. assay well, and min and max are the respective minimum DRAO5 was purchased from Biostatus Limited, Leicester and maximum signal controls. 25 shire, UK. Four-Parameter ICs Fit 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 peni Y = Bottom (Top - Bottom) cillin-streptomycin) and cultured at 37° C. under 5% CO. ( -- ( X Coefficient 30 Cell Treatment, in Cell Western (ICW) for Detection of IC50 Mono-Methyl Arginine and DNA Content. RKO cells were seeded in assay medium at a concentra tion of 20,000 cells per mL to a poly-D-lysine coated 384 Where top and bottom are the normally allowed to float, well culture plate (BD Biosciences 356697) with 50 uL per but may be fixed at 100 or 0 respectively in a 3-parameter well. Compound (100 mL) from a 96-well source plate was fit. The Hill Coefficient normally allowed to float but may 35 added directly to 384 well cell plate. Plates were incubated also be fixed at 1 in a 3-parameter fit. Y is the % inhibition at 37° C., 5% CO, for 72 hours. After three days of and X is the compound concentration. incubation, plates were brought to room temperature outside of the incubator for ten minutes and blotted on paper towels to remove cell media. 50 uL of ice cold 100% methanol was TABLE 2 40 added directly to each well and incubated for 30 min at room Biochemical ICso temperature. After 30 min, plates were transferred to a Biotek EL406 plate washer and washed 2 times with 100 uL Cmpd No. PRMT1 PRMT6 PRMT8 PRMT3 CARM1 per well of wash buffer (IX PBS). Next 60 uL per well of Odyssey blocking buffer (Odyssey Buffer with 0.1% Tween 45 20 (v/v)) were added to each plate and incubated 1 hour at room temperature. Blocking buffer was removed and 20 LL per well of primary antibody was added (mono-methyl arginine diluted 1:200 in Odyssey buffer with 0.1% Tween 20 (v/v)) and plates were incubated overnight (16 hours) at 4° C. Plates were washed 5 times with 100 uL per well of 50 wash buffer. Next 20 uL per well of secondary antibody was 1 added (1:200 800CW goat anti-rabbit IgG (H+L) antibody, 1:1000 DRAQ5 (Biostatus limited) in Odyssey buffer with 0.1% Tween 20 (v/v)) and incubated for 1 hour at room s temperature. The plates were washed 5 times with 100 L 55 per well wash buffer then 2 times with 100 uL per well of water. Plates were allowed to dry at room temperature then imaged on the Licor Odyssey machine which measures integrated intensity at 700 nm and 800 nm wavelengths. Both 700 and 800 channels were scanned. 60 Calculations: First, the ratio for each well was determined by: 2 3 24 s monomethyl Arginine 800 nm value 2 7 65 ( DRAO5 700 nm value ) 2 8 p US 9,630,961 B2 93 94 Each plate included fourteen control wells of DMSO only TABLE 3-continued treatment (minimum activation) as well as fourteen control wells for maximum activation treated with 20 uM of a In Cell Western reference compound. The average of the ratio values for each control type was calculated and used to determine the Cmpd Cmpd percent activation for each test well in the plate. Reference No. EC3o No. ECso compound was serially diluted three-fold in DMSO for a 9 B 21 B total of nine test concentrations, beginning at 20 uM. Percent 12 C 22 B activation was determined and ECso curves were generated 13 B 23 A. using triplicate wells per concentration of compound. 14 C 24 A. 10 15 C 25 C 16 C 26 C (Individual Test Sample Ratio)- 17 A. 27 A. (Minimum Activation Ratio) 18 C 28 C Percent Activation = 100 ------(Maximum Activation Ratio) - 3: 100 For Table 3, “A” indicates an EC3oz 3.00 uM, “B” indicates an ECo of 3.01-12.0 LM, and (Minimum Activation Ratio) “C" indicates an ECo a 12.01 uM. 15

TABLE 3 Other Embodiments In Cell Western The foregoing has been a description of certain non limiting embodiments of the invention. Those of ordinary Cmpd Cmpd skill in the art will appreciate that various changes and No. EC3o No. ECso modifications to this description may be made without 4 C 19 B departing from the spirit or scope of the present invention, 8 C 2O C as defined in the following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 17

<21 Os SEQ ID NO 1 &211s LENGTH: 16 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221s NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is Acp

<4 OOs SEQUENCE: 1 Xaa Arg Lieu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser Gly Lieu. Ile 1. 5 1O 15

SEO ID NO 2 LENGTH: 8 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Polypeptide

<4 OOs SEQUENCE: 2 Asp Tyr Lys Asp Asp Asp Asp Llys 1. 5

SEO ID NO 3 LENGTH: 23 O TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Polypeptide

<4 OOs SEQUENCE: 3 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Lieu Val Glin Pro 1. 5 1O 15

Thr Arg Lieu. Lieu. Lieu. Glu Tyr Lieu. Glu Glu Lys Tyr Glu Glu. His Lieu. US 9,630,961 B2 95 - Continued

2O 25 3O Tyr Glu Arg Asp Glu Gly Asp Llys Trp Arg Asn Llys Llys Phe Glu Lieu. 35 4 O 45 Gly Lieu. Glu Phe Pro Asn Lieu Pro Tyr Tyr Ile Asp Gly Asp Wall Lys SO 55 6 O Lieu. Thr Glin Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Llys His Asn 65 70 7s 8O Met Lieu. Gly Gly Cys Pro Llys Glu Arg Ala Glu Ile Ser Met Lieu. Glu 85 90 95 Gly Ala Val Lieu. Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 1OO 105 11 O Lys Asp Phe Glu Thir Lieu Lys Val Asp Phe Lieu. Ser Llys Lieu Pro Glu 115 12 O 125 Met Lieu Lys Met Phe Glu Asp Arg Lieu. Cys His Llys Thr Tyr Lieu. Asn 13 O 135 14 O Gly Asp His Val Thr His Pro Asp Phe Met Lieu. Tyr Asp Ala Lieu. Asp 145 150 155 160 Val Val Lieu. Tyr Met Asp Pro Met Cys Lieu. Asp Ala Phe Pro Llys Lieu 1.65 17O 17s Val Cys Phe Llys Lys Arg Ile Glu Ala Ile Pro Glin Ile Asp Llys Tyr 18O 185 19 O Lieu Lys Ser Ser Llys Tyr Ile Ala Trp Pro Lieu. Glin Gly Trp Glin Ala 195 2OO 2O5 Thr Phe Gly Gly Gly Asp His Pro Pro Llys Ser Asp Glu Asn Lieu. Tyr 210 215 220 Phe Glin Gly Gly Asn Ser 225 23 O

<210s, SEQ ID NO 4 &211s LENGTH: 609 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 4 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Lieu Val Glin Pro 1. 5 1O 15 Thir Arg Lieu. Lieu. Lieu. Glu Tyr Lieu. Glu Glu Lys Tyr Glu Glu. His Lieu. 2O 25 3O Tyr Glu Arg Asp Glu Gly Asp Llys Trp Arg Asn Llys Llys Phe Glu Lieu. 35 4 O 45 Gly Lieu. Glu Phe Pro Asn Lieu Pro Tyr Tyr Ile Asp Gly Asp Wall Lys SO 55 6 O Lieu. Thr Glin Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Llys His Asn 65 70 7s 8O

Met Lieu. Gly Gly Cys Pro Llys Glu Arg Ala Glu Ile Ser Met Lieu. Glu 85 90 95

Gly Ala Val Lieu. Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 1OO 105 11 O

Lys Asp Phe Glu Thir Lieu Lys Val Asp Phe Lieu. Ser Llys Lieu Pro Glu 115 12 O 125 Met Lieu Lys Met Phe Glu Asp Arg Lieu. Cys His Llys Thr Tyr Lieu. Asn 13 O 135 14 O Gly Asp His Val Thr His Pro Asp Phe Met Lieu. Tyr Asp Ala Lieu. Asp US 9,630,961 B2 97 - Continued

145 150 155 160 Val Val Lieu. Tyr Met Asp Pro Met Cys Lieu. Asp Ala Phe Pro Llys Lieu 1.65 17O 17s Val Cys Phe Llys Lys Arg Ile Glu Ala Ile Pro Glin Ile Asp Llys Tyr 18O 185 19 O Lieu Lys Ser Ser Llys Tyr Ile Ala Trp Pro Lieu. Glin Gly Trp Glin Ala 195 2OO 2O5 Thr Phe Gly Gly Gly Asp His Pro Pro Llys Ser Asp Glu Asn Lieu. Tyr 21 O 215 22O Phe Glin Gly Gly Asn. Ser Asp Tyr Lys Asp Asp Asp Asp Llys Met Ala 225 23 O 235 24 O Ala Ala Glu Ala Ala Asn. Cys Ile Met Glu Asn. Phe Val Ala Thir Lieu 245 250 255 Ala Asn Gly Met Ser Lieu Gln Pro Pro Leu Glu Glu Val Ser Cys Gly 26 O 265 27 O Glin Ala Glu Ser Ser Glu Lys Pro Asn Ala Glu Asp Met Thir Ser Lys 27s 28O 285 Asp Tyr Tyr Phe Asp Ser Tyr Ala His Phe Gly Ile His Glu Glu Met 29 O 295 3 OO Lieu Lys Asp Glu Val Arg Thr Lieu. Thir Tyr Arg Asn. Ser Met Phe His 3. OS 310 315 32O Asn Arg His Lieu. Phe Lys Asp Llys Val Val Lieu. Asp Val Gly Ser Gly 3.25 330 335 Thr Gly Ile Lieu. Cys Met Phe Ala Ala Lys Ala Gly Ala Arg Llys Val 34 O 345 35 O Ile Gly Ile Glu. Cys Ser Ser Ile Ser Asp Tyr Ala Val Lys Ile Val 355 360 365 Lys Ala Asn Llys Lieu. Asp His Val Val Thir Ile Ile Llys Gly Llys Val 37 O 375 38O Glu Glu Val Glu Lieu Pro Val Glu Lys Val Asp Ile Ile Ile Ser Glu 385 390 395 4 OO Trp Met Gly Tyr Cys Lieu Phe Tyr Glu Ser Met Lieu. Asn Thr Val Lieu. 4 OS 41O 415 Tyr Ala Arg Asp Llys Trp Lieu Ala Pro Asp Gly Lieu. Ile Phe Pro Asp 42O 425 43 O Arg Ala Thir Lieu. Tyr Val Thr Ala Ile Glu Asp Arg Glin Tyr Lys Asp 435 44 O 445 Tyr Lys Ile His Trp Trp Glu Asn Val Tyr Gly Phe Asp Met Ser Cys 450 45.5 460 Ile Lys Asp Wall Ala Ile Llys Glu Pro Lieu Val Asp Val Val Asp Pro 465 470 47s 48O Lys Glin Lieu Val Thr Asn Ala Cys Lieu. Ile Lys Glu Val Asp Ile Tyr 485 490 495

Thr Val Llys Val Glu Asp Lieu. Thr Phe Thr Ser Pro Phe Cys Lieu. Glin SOO 505 51O Val Lys Arg Asn Asp Tyr Val His Ala Lieu Val Ala Tyr Phe Asn. Ile 515 52O 525 Glu Phe Thr Arg Cys His Lys Arg Thr Gly Phe Ser Thr Ser Pro Glu 53 O 535 54 O Ser Pro Tyr Thr His Trp Lys Glin Thr Val Phe Tyr Met Glu Asp Tyr 5.45 550 555 560

Lieu. Thr Val Lys Thr Gly Glu Glu Ile Phe Gly Thr Ile Gly Met Arg 565 st O sts US 9,630,961 B2 99 100 - Continued

Pro Asn Ala Lys Asn. Asn Arg Asp Lieu. Asp Phe Thir Ile Asp Lieu. Asp 585 59 O Phe Lys Gly Glin Lieu. Cys Glu Lieu Ser Cys Ser Thr Asp Tyr Arg Met 595 6OO 605 Arg

<210s, SEQ ID NO 5 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is Acp <4 OOs, SEQUENCE: 5 Xaa Arg Lieu Ala Arg Arg Gly Gly Wall Lys Arg Ile Ser Gly Lieu. Ile 1. 5 15

SEQ ID NO 6 LENGTH: 9 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polypeptide

SEQUENCE: 6 Met Asp Tyr Lys Asp Asp Asp Asp Llys 1. 5

SEO ID NO 7 LENGTH: 389 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 7

Met Asp Tyr Lys Asp Asp Asp Asp Ser Glin Pro Arg 1. 5 1O 15

Lell Ser Gly Gly Gly Gly Glu Gly Gly Glu Gly Thir Glu Glu Glu 25

Asp Ala Glu Arg Glu Ala Ala Luell Glu Arg Pro Arg Arg Thir 35 4 O 45

Arg Arg Asp Glin Lell Tyr Glu Ser Asp Wall Ser Wall 55 6 O

His Glu Met Ile Ala Asp Arg Wall Arg Thir Asp Ala Tyr Arg Luell 65 70 7s 8O

Gly Luell Arg Asn Trp Ala Ala Luell Arg Gly Thir Wall Luell Asp 85 90 95

Wall Ala Gly Thir Gly Ile Luell Ser Ile Phe Ala Glin Ala Gly 1OO 105 11 O

Ala Arg Arg Wall Ala Wall Glu Ala Ser Ala Ile Trp Glin Glin Ala 115 12 O 125

Arg Glu Wall Wall Arg Phe Asn Gly Luell Glu Asp Arg Wall His Wall Luell 13 O 135 14 O

Pro Gly Pro Wall Glu Thir Wall Glu Luell Pro Glu Glin Wall Asp Ala Ile 145 150 155 160 US 9,630,961 B2 101 102 - Continued Val Ser Glu Trp Met Gly Tyr Gly Lieu. Lieu. His Glu Ser Met Leu Ser 1.65 17O 17s Ser Val Lieu. His Ala Arg Thr Lys Trp Lieu Lys Glu Gly Gly Lieu. Lieu. 18O 185 19 O Lieu Pro Ala Ser Ala Glu Lieu. Phe Ile Ala Pro Ile Ser Asp Gln Met 195 2OO 2O5 Lieu. Glu Trp Arg Lieu. Gly Phe Trp Ser Glin Val Lys Gln His Tyr Gly 21 O 215 22O Val Asp Met Ser Cys Lieu. Glu Gly Phe Ala Thr Arg Cys Lieu Met Gly 225 23 O 235 24 O His Ser Glu Ile Val Val Glin Gly Lieu. Ser Gly Glu Asp Wall Lieu Ala 245 250 255 Arg Pro Glin Arg Phe Ala Glin Lieu. Glu Lieu. Ser Arg Ala Gly Lieu. Glu 26 O 265 27 O Glin Glu Lieu. Glu Ala Gly Val Gly Gly Arg Phe Arg Cys Ser Cys Tyr 27s 28O 285 Gly Ser Ala Pro Met His Gly Phe Ala Ile Trp Phe Glin Val Thr Phe 29 O 295 3 OO Pro Gly Gly Glu Ser Glu Lys Pro Leu Val Lieu Ser Thr Ser Pro Phe 3. OS 310 315 32O His Pro Ala Thr His Trp Llys Glin Ala Lieu. Lieu. Tyr Lieu. Asn. Glu Pro 3.25 330 335 Val Glin Val Glu Glin Asp Thr Asp Val Ser Gly Glu Ile Thr Lieu. Lieu 34 O 345 35. O Pro Ser Arg Asp ASn Pro Arg Arg Lieu. Arg Val Lieu Lieu. Arg Tyr Lys 355 360 365 Val Gly Asp Glin Glu Glu Lys Thir Lys Asp Phe Ala Met Glu Asp His 37 O 375 38O

His His His His His 385

<210s, SEQ ID NO 8 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is Acp <4 OOs, SEQUENCE: 8 Xaa Llys Pro Ala Ile Arg Arg Lieu Ala Arg Arg Gly Gly Wall Lys Arg 1. 5 1O 15

<210s, SEQ ID NO 9 &211s LENGTH: 229 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 9 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Lieu Val Glin Pro 1. 5 1O 15

Thir Arg Lieu. Lieu. Lieu. Glu Tyr Lieu. Glu Glu Lys Tyr Glu Glu. His Lieu. 2O 25 3O

Tyr Glu Arg Asp Glu Gly Asp Llys Trp Arg Asn Llys Llys Phe Glu Lieu. US 9,630,961 B2 103 104 - Continued

35 4 O 45

Gly Luell Glu Phe Pro Asn Lell Pro Ile Asp Gly Asp Wall SO 55 6 O

Lell Thir Glin Ser Met Ala Ile Ile Arg Ile Ala Asp His Asn 65 70

Met Luell Gly Gly Cys Pro Glu Arg Ala Glu Ile Ser Met Luell Glu 85 90 95

Gly Ala Wall Luell Asp Ile Gly Wall Ser Arg Ile Ala Ser 105 11 O

Asp Phe Glu Thir Lell Wall Asp Phe Luell Ser Lys Luell Pro Glu 115 12 O 125

Met Luell Met Phe Glu Asp Arg Luell His Lys Thir Luell Asn 13 O 135 14 O

Gly Asp His Wall Thir His Pro Asp Phe Met Luell Tyr Asp Ala Luell Asp 145 150 155 160

Wall Wall Luell Tyr Met Asp Pro Met Luell Asp Ala Phe Pro Lys Luell 1.65 17O

Wall Phe Lys Ile Glu Ala Ile Pro Glin Ile Asp 18O 185 19 O

Lell Ser Ser Tyr Ile Ala Trp Pro Luell Glin Gly Trp Glin Ala 195

Thir Phe Gly Gly Gly Asp His Pro Pro Ser Asp Lell Wall Pro Arg 21 O 215 22O

Gly Ser Pro Glu Phe 225

SEQ ID NO 10 LENGTH: 623 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 10

Met Ser Pro Ile Lell Gly Tyr Trp Ile Gly Lell Wall Glin Pro 1. 5 1O 15

Thir Arg Luell Luell Lell Glu Tyr Luell Glu Glu Tyr Glu Glu His Luell 25 3O

Glu Arg Asp Glu Gly Asp Lys Trp Arg ASn Lys Phe Glu Luell 35 4 O 45

Gly Luell Glu Phe Pro Asn Lell Pro Ile Asp Gly Asp Wall SO 55 6 O

Lell Thir Glin Ser Met Ala Ile Ile Arg Ile Ala Asp His 65 70

Met Luell Gly Gly Cys Pro Glu Arg Ala Glu Ile Ser Met Luell Glu 85 90 95

Gly Ala Wall Luell Asp Ile Gly Wall Ser Arg Ile Ala Ser 1OO 105 11 O

Asp Phe Glu Thir Lell Wall Asp Phe Luell Ser Lys Luell Pro Glu 115 12 O 125

Met Luell Met Phe Glu Asp Arg Luell His Lys Thir Luell Asn 13 O 135 14 O

Gly Asp His Wall Thir His Pro Asp Phe Met Luell Tyr Asp Ala Luell Asp 145 150 155 160

Wall Wall Luell Met Asp Pro Met Luell Asp Ala Phe Pro Luell US 9,630,961 B2 105 106 - Continued

1.65 17O 17s

Wall Phe Lys Arg Ile Glu Ala Ile Pro Glin Ile Asp 18O 185 19 O

Lell Ser Ser Ile Ala Trp Pro Luell Glin Gly Trp Glin Ala 195

Thir Phe Gly Gly Gly Asp His Pro Pro Ser Asp Lell Wall Pro Arg 21 O 215

Gly Ser Pro Glu Phe Met Gly Met His Ser Ser Arg Luell Luell 225 23 O 235 24 O

Lell Arg Arg Met Ala Glu Asn Ala Ala Glu Ser Thir Glu Wall Asn 245 250 255

Ser Pro Pro Ser Glin Pro Pro Glin Pro Wall Wall Pro Ala Lys Pro Wall 26 O 265 27 O

Glin Wall His His Wall Ser Thir Glin Pro Ser Pro Gly Arg Gly 285

Met Ser Lell Lell Asn Pro Glu Glu Met Thir Ser Arg Asp 29 O 295 3 OO

Tyr Phe Asp Ser Ala His Phe Gly Ile His Glu Glu Met Luell Lys 3. OS 310 315

Asp Glu Wall Arg Thir Lell Thir Arg Asn Ser Met His Asn 3.25 330 335

His Wall Phe Lys Asp Wall Wall Luell Asp Wall Gly Ser Gly Thir Gly 34 O 345 35. O

Ile Luell Ser Met Phe Ala Ala Lys Ala Gly Ala Lys Wall Phe Gly 355 360 365

Ile Glu Ser Ser Ile Ser Asp Ser Glu Lys Ile Ile Ala 37 O 375

Asn His Luell Asp Asn Ile Ile Thir Ile Phe Lys Gly Wall Glu Glu 385 390 395 4 OO

Wall Glu Luell Pro Wall Glu Wall Asp Ile Ile Ile Ser Glu Trp Met 4 OS 415

Gly Luell Phe Glu Ser Met Luell ASn Thir Wall Ile Phe Ala 425 43 O

Arg Asp Lys Trp Lell Pro Gly Gly Luell Met Phe Pro Asp Arg Ala 435 44 O 445

Ala Luell Wall Wall Ala Ile Glu Asp Arg Glin Tyr Asp Phe 450 45.5 460

Ile His Trp Trp Glu Asn Wall Gly Phe Asp Met Thir Ile Arg 465 470 48O

Asp Wall Ala Met Lys Glu Pro Luell Wall Asp Ile Wall Asp Pro Lys Glin 485 490 495

Wall Wall Thir Asn Ala Cys Lell Ile Lys Glu Wall Asp Ile Tyr Thir Wall SOO 505 51O

Thir Glu Glu Lell Ser Phe Thir Ser Ala Phe Cys Lell Glin Ile Glin 515 52O 525

Arg Asn Asp Wall His Ala Luell Wall Thir Phe Asn Ile Glu Phe 53 O 535 54 O

Thir His Lys Met Gly Phe Ser Thir Ala Pro Asp Ala Pro 5.45 550 555 560

Thir His Trp Lys Glin Thir Wall Phe Tyr Luell Glu Asp Luell Thir 565 st O sts

Wall Arg Arg Gly Glu Glu Ile Gly Thir Ile Ser Met Lys Pro Asn 58O 585 59 O US 9,630,961 B2 107 108 - Continued

Ala Lys Asn Val Arg Asp Lieu. Asp Phe Thr Val Asp Lieu. Asp Phe Lys 595 605 Gly Glin Lieu. Cys Glu Thir Ser Val Ser Asn Asp Tyr Lys Met Arg 610 615

<210s, SEQ ID NO 11 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is Acp

<4 OOs, SEQUENCE: 11 Xaa Gly Gly Arg Gly Gly Phe Gly Gly Arg Gly Gly Phe Gly Gly Arg 1. 5 15 Gly Gly Phe Gly

SEQ ID NO 12 LENGTH: 226 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 12

Met Ser Pro Ile Lell Gly Tyr Trp Ile Gly Lell Wall Glin Pro 1. 5 1O 15

Thir Arg Luell Luell Lell Glu Tyr Luell Glu Glu Lys Tyr Glu Glu His Luell 25 3O

Glu Arg Asp Glu Gly Asp Lys Trp Arg ASn Lys Phe Glu Luell 35 4 O 45

Gly Luell Glu Phe Pro Asn Lell Pro Ile Asp Gly Asp Wall SO 55 6 O

Lell Thir Glin Ser Met Ala Ile Ile Arg Ile Ala Asp His Asn 65 70

Met Luell Gly Gly Cys Pro Glu Arg Ala Glu Ile Ser Met Luell Glu 85 90 95

Gly Ala Wall Luell Asp Ile Gly Wall Ser Arg Ile Ala Ser 105 11 O

Asp Phe Glu Thir Lell Wall Asp Phe Luell Ser Lys Luell Pro Glu 115 12 O 125

Met Luell Met Phe Glu Asp Arg Luell His Lys Thir Luell Asn 13 O 135 14 O

Gly Asp His Wall Thir His Pro Asp Phe Met Luell Tyr Asp Ala Luell Asp 145 150 155 160

Wall Wall Luell Met Asp Pro Met Luell Asp Ala Phe Pro Lys Luell 1.65 17O 17s

Wall Phe Lys Ile Glu Ala Ile Pro Glin Ile Asp 18O 185 19 O

Lell Ser Ser Tyr Ile Ala Trp Pro Luell Glin Gly Trp Glin Ala 195

Thir Phe Gly Gly Gly Asp His Pro Pro Ser Asp Lell Wall Pro Arg 21 O 215 22O US 9,630,961 B2 109 110 - Continued Gly Ser 225

<210s, SEQ ID NO 13 &211s LENGTH: 756 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 13 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Lieu Val Glin Pro 1. 5 1O 15 Thir Arg Lieu. Lieu. Lieu. Glu Tyr Lieu. Glu Glu Lys Tyr Glu Glu. His Lieu. 2O 25 3O Tyr Glu Arg Asp Glu Gly Asp Llys Trp Arg Asn Llys Llys Phe Glu Lieu. 35 4 O 45 Gly Lieu. Glu Phe Pro Asn Lieu Pro Tyr Tyr Ile Asp Gly Asp Wall Lys SO 55 6 O Lieu. Thr Glin Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Llys His Asn 65 70 7s 8O Met Lieu. Gly Gly Cys Pro Llys Glu Arg Ala Glu Ile Ser Met Lieu. Glu 85 90 95 Gly Ala Val Lieu. Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 1OO 105 11 O Lys Asp Phe Glu Thir Lieu Lys Val Asp Phe Lieu. Ser Llys Lieu Pro Glu 115 12 O 125 Met Lieu Lys Met Phe Glu Asp Arg Lieu. Cys His Llys Thr Tyr Lieu. Asn 13 O 135 14 O Gly Asp His Val Thr His Pro Asp Phe Met Lieu. Tyr Asp Ala Lieu. Asp 145 150 155 160 Val Val Lieu. Tyr Met Asp Pro Met Cys Lieu. Asp Ala Phe Pro Llys Lieu 1.65 17O 17s Val Cys Phe Llys Lys Arg Ile Glu Ala Ile Pro Glin Ile Asp Llys Tyr 18O 185 19 O Lieu Lys Ser Ser Llys Tyr Ile Ala Trp Pro Lieu. Glin Gly Trp Glin Ala 195 2OO 2O5 Thr Phe Gly Gly Gly Asp His Pro Pro Llys Ser Asp Leu Val Pro Arg 21 O 215 22O Gly Ser Cys Ser Lieu Ala Ser Gly Ala Thr Gly Gly Arg Gly Ala Val 225 23 O 235 24 O Glu Asn. Glu Glu Asp Lieu Pro Glu Lieu. Ser Asp Ser Gly Asp Glu Ala 245 250 255 Ala Trp Glu Asp Glu Asp Asp Ala Asp Lieu Pro His Gly Lys Glin Glin 26 O 265 27 O Thr Pro Cys Lieu Phe Cys Asn Arg Lieu Phe Thr Ser Ala Glu Glu Thr 27s 28O 285 Phe Ser His Cys Lys Ser Glu. His Glin Phe Asn Ile Asp Ser Met Val 29 O 295 3 OO His Llys His Gly Lieu. Glu Phe Tyr Gly Tyr Ile Llys Lieu. Ile Asin Phe 3. OS 310 315 32O

Ile Arg Lieu Lys Asn Pro Thr Val Glu Tyr Met Asn Ser Ile Tyr Asn 3.25 330 335

Pro Val Pro Trp Glu Lys Glu Glu Tyr Lieu Lys Pro Val Lieu. Glu Asp 34 O 345 35. O US 9,630,961 B2 111 112 - Continued

Asp Luell Luell Luell Glin Phe Asp Wall Glu Asp Luell Tyr Glu Pro Wall Ser 355 360 365

Wall Pro Phe Ser Pro Asn Gly Luell Ser Glu Asn Thir Ser Wall Wall 37 O 375 38O

Glu Luell His Met Glu Ala Arg Ala Luell Ser Ala Glu Ala Ala 385 390 395 4 OO

Lell Ala Arg Ala Arg Glu Asp Luell Glin Lys Met Glin Phe Ala Glin 4 OS 415

Asp Phe Wall Met His Thir Asp Wall Arg Thir Ser Ser Ser Thir Ser 42O 425 43 O

Wall Ile Ala Asp Lell Glin Glu Asp Glu Asp Gly Wall Tyr Phe Ser Ser 435 44 O 445

Gly His Gly Ile His Glu Glu Met Luell Lys Asp Ile Arg 450 45.5 460

Thir Glu Ser Arg Asp Phe Ile Glin ASn Pro His Ile Phe Lys 465 470

Asp Wall Wall Lell Asp Wall Gly Gly Thir Gly Ile Luell Ser Met 485 490 495

Phe Ala Ala Lys Ala Gly Ala Lys Wall Luell Gly Wall Asp Glin Ser SOO 505

Glu Ile Luell Glin Ala Met Asp Ile Ile Arg Lell Asn Luell Glu 515 52O 525

Asp Thir Ile Thir Lell Ile Lys Gly Ile Glu Glu Wall His Luell Pro 53 O 535 54 O

Wall Glu Wall Asp Wall Ile Ile Ser Glu Trp Met Gly Phe Lieu 5.45 550 555 560

Lell Phe Glu Ser Met Lell Asp Ser Wall Luell Ala Asn Lys 565 st O sts

Lell Ala Gly Gly Ser Wall Pro Asp Ile Thir Ile Ser Luell 585 59 O

Wall Ala Wall Ser Asp Wall Asn Lys His Ala Asp Arg Ile Ala Phe Trp 595 605

Asp Asp Wall Gly Phe Lys Met Ser Met Lys Ala Wall Ile 610 615

Pro Glu Ala Wall Wall Glu Wall Luell Asp Pro Lys Thir Lell Ile Ser Glu 625 630 635 64 O

Pro Gly Ile Lys His Ile Asp His Thir Thir Ser Ile Ser Asp 645 650 655

Lell Glu Phe Ser Ser Asp Phe Thir Luell Ile Thir Arg Thir Ser Met 660 665 67 O

Thir Ala Ile Ala Gly Phe Asp Ile Phe Glu Asn 675 685

His Asn Arg Wall Wall Phe Ser Thir Gly Pro Glin Ser Thir Thir His 69 O. 695 7 OO

Trp Glin Thir Wall Phe Lell Luell Glu Pro Phe Ser Wall Ala 7 Os 71O 71s 72O

Gly Glu Ala Luell Lys Gly Wall Thir Wall His Asn Lys Asp 72 73 O 73

Pro Arg Ser Luell Thir Wall Thir Luell Thir Luell ASn Asn Ser Thir Glin Thir 740 74. 7 O

Gly Luell Glin 7ss US 9,630,961 B2 113 114 - Continued

<210s, SEQ ID NO 14 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa is Acp <4 OOs, SEQUENCE: 14 Xaa Pro Arg Lys Glin Lieu Ala Thir Lys Ala Ala Arg Llys Ser Ala Pro 1. 5 1O 15

<210s, SEQ ID NO 15 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 15 Glu Gly His His His His His His 1. 5

<210s, SEQ ID NO 16 &211s LENGTH: 6O1 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 16 Met Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ala Ala Ala Ala Ala Val 1. 5 1O 15 Gly Pro Gly Ala Gly Gly Ala Gly Ser Ala Val Pro Gly Gly Ala Gly 2O 25 3O Pro Cys Ala Thr Val Ser Val Phe Pro Gly Ala Arg Lieu. Lieu. Thir Ile 35 4 O 45 Gly Asp Ala Asn Gly Glu Ile Glin Arg His Ala Glu Glin Glin Ala Lieu. SO 55 6 O Arg Lieu. Glu Val Arg Ala Gly Pro Asp Ser Ala Gly Ile Ala Lieu. Tyr 65 70 7s 8O Ser His Glu Asp Val Cys Val Phe Lys Cys Ser Val Ser Arg Glu Thr 85 90 95 Glu Cys Ser Arg Val Gly Lys Glin Ser Phe Ile Ile Thr Lieu. Gly Cys 1OO 105 11 O Asn Ser Val Lieu. Ile Glin Phe Ala Thr Pro Asn Asp Phe Cys Ser Phe 115 12 O 125 Tyr Asn. Ile Lieu Lys Thr Cys Arg Gly His Thr Lieu. Glu Arg Ser Val 13 O 135 14 O

Phe Ser Glu Arg Thr Glu Glu Ser Ser Ala Val Glin Tyr Phe Glin Phe 145 150 155 160

Tyr Gly Tyr Lieu Ser Glin Glin Glin Asn Met Met Glin Asp Tyr Val Arg 1.65 17O 17s Thr Gly Thr Tyr Glin Arg Ala Ile Leu Glin Asn His Thr Asp Phe Lys 18O 185 19 O

Asp Llys Ile Val Lieu. Asp Val Gly Cys Gly Ser Gly Ile Lieu. Ser Phe 195 2OO 2O5

Phe Ala Ala Glin Ala Gly Ala Arg Lys Ile Tyr Ala Val Glu Ala Ser US 9,630,961 B2 115 116 - Continued

21 O 215 22O

Thir Met Ala Glin His Ala Glu Wall Luell Wall Lys Ser Asn Asn Luell Thir 225 23 O 235 24 O

Asp Arg Ile Wall Wall Ile Pro Gly Wall Glu Glu Wall Ser Luell Pro 245 250 255

Glu Glin Wall Asp Ile Ile Ile Ser Glu Pro Met Gly Met Luell Phe 26 O 265 27 O

Asn Glu Arg Met Lell Glu Ser Tyr Luell His Ala Lys Luell 285

Pro Ser Gly Asn Met Phe Pro Thir Ile Gly Asp Wall His Luell Ala Pro 29 O 295 3 OO

Phe Thir Asp Glu Glin Lell Met Glu Glin Phe Thir Ala Asn Phe 3. OS 310 315

Trp Glin Pro Ser Phe His Gly Wall Asp Luell Ser Ala Luell Arg Gly 3.25 330 335

Ala Ala Wall Asp Glu Phe Arg Glin Pro Wall Wall Asp Thir Phe Asp 34 O 345 35. O

Ile Arg Ile Luell Met Ala Ser Wall Thir Wall Asn Phe Luell 355 360 365

Glu Ala Glu Gly Asp Lell His Arg Ile Glu Ile Pro Phe Phe 37 O 375

His Met Luell His Ser Gly Lell Wall His Gly Luell Ala Phe Trp Phe Asp 385 390 395 4 OO

Wall Ala Phe Ile Gly Ser Ile Met Thir Wall Trp Lell Ser Thir Ala Pro 405 410 415

Thir Glu Pro Luell Thir His Trp Glin Wall Arg Lell Phe Glin Ser 425 43 O

Pro Luell Phe Ala Ala Gly Asp Thir Luell Ser Gly Thir Luell Luell 435 44 O 445

Ile Ala Asn Arg Glin Ser Asp Ile Ser Ile Wall Ala Glin Wall 450 45.5 460

Asp Glin Thir Ser Lys Ser Ser Asn Luell Luell Asp Lell Asn Pro 465 470

Phe Phe Arg Thir Gly Thir Thir Pro Ser Pro Pro Pro Gly Ser His 485 490 495

Thir Ser Pro Ser Glu Asn Met Trp Asn Thir Gly Ser Thir Asn SOO 505 51O

Lell Ser Ser Gly Met Ala Wall Ala Gly Met Pro Thir Ala Asp Luell 515 525

Ser Ser Wall Ile Ala Ser Gly Ser Ser Wall Gly His Asn Asn Luell Ile 53 O 535 54 O

Pro Luell Gly Ser Ser Gly Ala Glin Gly Ser Gly Gly Gly Ser Thir Ser 5.45 550 555 560

Ala His Ala Wall Asn Ser Glin Phe Thir Met Gly Gly Pro Ala Ile 565 st O sts

Ser Met Ala Ser Pro Met Ser Ile Pro Thir ASn Thir Met His Gly 58O 585 59 O

Ser Glu Gly His His His His His His 595 6OO

<210s, SEQ ID NO 17 &211s LENGTH: 6 212. TYPE : PRT &213s ORGANISM: Artificial Sequence US 9,630,961 B2

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Polypeptide

<4 OOs, SEQUENCE: 17

His His His His His His 1. 5

10 What is claimed is: 1. A compoundp of Formula (I):(I) HN-R, (III)

15 A (I) Q N HN-R, V / o R

Y Na 2 R4 YN R5 2O or a pharmaceutically acceptable salt thereof. 4. The compound of claim 1, wherein the compound is of Formula (IV): or a pharmaceutically acceptable salt thereof, 25 wherein: (IV) HN-R,

X is N, Z is NR, and Y is CR; or 30 N X is NR, Z is N, and Y is CR; or Q Ye X is CR, Z is NR", and Y is N; or / \ X is CR, Z is N, and Y is NR; R5 - R is optionally substituted C alkyl or optionally sub 35 stituted C. cycloalkyl, Q is an optionally Substituted, monocyclic or bicyclic heteroaryl having 1-4 heteroatoms independently or a pharmaceutically acceptable salt thereof. Selected from nitrogen, oxygen, and Sulfur, wherein Q 5. The compound of claim 1, wherein the compound is of is not pyridine; 40 Formula (V): R is hydrogen, C. alkyl, or Csa cycloalkyl; R" is hydrogen, C. alkyl, or Cs a cycloalkyl; and (V) R is hydrogen, halo. —CN, optionally substituted Ca HN-R, alkyl, or optionally Substituted C. cycloalkyl. 45 Q N V o R 2. The compound of claim 1, wherein the compound is of Formula (II): 50 R5 N-N R4

(II) or a pharmaceutically acceptable salt thereof. HN-R, 6. The compound of claim 1, wherein Q is an optionally 55 substituted 8- to 10-membered heteroaryl having 1-4 het N eroatoms independently selected from nitrogen, oxygen, and Q Ye sulfur. 7. The compound of claim 6, wherein Q is an optionally NY \ substituted 9-membered heteroaryl having 1-3 heteroatoms YN R5 60 independently selected from nitrogen, oxygen, and Sulfur. 8. The compound of claim 1, wherein Q is an optionally substituted 5- to 6-membered heteroaryl having 1-3 heteroa toms independently selected from nitrogen, oxygen, and sulfur. or a pharmaceutically acceptable salt thereof. 65 9. The compound of claim 8, wherein Q is an optionally 3. The compound of claim 1, wherein the compound is of substituted 5-membered heteroaryl having 1-3 heteroatoms Formula (III): independently selected from nitrogen, oxygen, and Sulfur. US 9,630,961 B2 119 120

10. The compound of claim 1, wherein Q is an unsubsti -continued tuted, monocyclic or bicyclic heteroaryl having 1-4 heteroa toms independently selected from nitrogen, oxygen, and sulfur. 11. The compound of claim 1, wherein Q is substituted, monocyclic or bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and Sulfur. 12. The compound of claim 1, wherein Q is substituted or unsubstituted pyrrolyl, substituted or unsubstituted furanyl, substituted or unsubstituted thienyl, substituted or unsubsti tuted imidazolyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted oxazolyl, substituted or unsub stituted thiazolyl, substituted or unsubstituted isothiazolyl, substituted or unsubstituted triazolyl, substituted or unsub stituted thiadiazolyl, substituted or unsubstituted thiadiaz olyl, substituted or unsubstituted tetrazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted pyrim idyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted tri azinyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoindolyl, substituted or unsubstituted inda Zolyl, substituted or unsubstituted benzotriazolyl, substi tuted or unsubstituted benzothiophenyl, substituted or 25 wherein: unsubstituted isobenzothiophenyl, substituted or unsubsti each R is independently selected from the group consist tuted benzofuranyl, substituted or unsubstituted benzoiso ing of halo. —CN, NO, optionally substituted alkyl, furanyl, substituted or unsubstituted benzimidazolyl, substi optionally substituted alkenyl, optionally substituted tuted or unsubstituted benzoxazolyl, substituted or alkynyl, optionally Substituted carbocyclyl, optionally unsubstituted benzoxadiazolyl, substituted or unsubstituted 30 benzisoxazolyl, substituted or unsubstituted benzthiazolyl, substituted aryl, optionally substituted heterocyclyl, substituted or unsubstituted benzisothiazolyl, substituted or optionally substituted heteroaryl, -OR", N(R). unsubstituted benzthiadiazolyl, substituted or unsubstituted - SR", C(=O)R', C(O)OR, C(O)SR', indolizinyl, substituted or unsubstituted purinyl, substituted C(O)N(R), C(O)N(R)N(R), OC(O)R, or unsubstituted pyrrolopyridinyl, substituted or unsubsti 35 OC(O)N(R), NRC(O)R, NRC(O)N(R), tuted triazolopyridinyl, substituted or unsubstituted naph NRC(O)N(R)N(R), NRC(O)OR', SC(O) thyridinyl, substituted or unsubstituted pteridinyl, substi R", C(-NR)R', C(-NNR)R', C(-NOR") tuted or unsubstituted quinolinyl, substituted or R", C(-NR)N(R), NR°C( NR)R’, unsubstituted isoquinolinyl. Substituted or unsubstituted cin C(—S)R', C(—S)N(R), NRC(=S)R', nolinyl. Substituted or unsubstituted quinoxalinyl. Substi 40 S(O)R, OS(O).R', SOR', NRSO.R.", tuted or unsubstituted quinazolinyl, or Substituted or unsub and -SON(R): stituted phthalazinyl. each R is independently selected from the group con 13. The compound of claim 1, wherein Q is selected from sisting of hydrogen, optionally Substituted alkyl, the group consisting of: optionally substituted alkenyl, optionally substituted 45 alkynyl, optionally Substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally Substituted heteroaryl, an oxygen protecting N-(R)03, N N 1S N s group when attached to an oxygen atom, and a Sulfur / (R)-it N | --(R'). protecting group when attached to a Sulfur atom; and 2 50 each R is independently selected from the group con sisting of hydrogen, optionally Substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally Substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, 21 S e-y (R)-3 55 optionally Substituted heteroaryl, and a nitrogen pro (Ra) - X tecting group, or two R' groups are taken together with Sn SR), () s their intervening atoms to form an optionally substi tuted heterocyclic ring. 14. The compound of claim 1, wherein R is hydrogen or 60 methyl. N X (R)0-3 15. The compound of claim 1, wherein R is hydrogen. (R)- > / 16. The compound of claim 1, wherein R is methyl. 21 MY(Ra), S 17. The compound of claim 1, wherein R is ethyl. 18. The compound of claim 1, wherein R is hydroxyethyl 65 or methoxyethyl. 19. The compound of claim 1, wherein R is cyclopropyl or isopropyl. US 9,630,961 B2 121 122 20. The compound of claim 1, wherein R is hydrogen. -continued 21. A pharmaceutical composition comprising a com pound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. 5 22. A pharmaceutical composition comprising a com pound selected from the group consisting of:

S 10 O

-N-n 15 ON s NH N2-y O N, 2O

1. N N ON NH US 9,630,961 B2 123 124 -continued -continued

US 9,630,961 B2 125 126 -continued -continued

10 M N HN e 15 HN / / 1--"NH2, N HN 2O Y M

H 25 1 N

N MY N f N-N. / N HN le '' and pharmaceutically acceptable salts thereof, and a phar maceutically acceptable excipient. 23. A kit or packaged pharmaceutical comprising a com -N pound claim 1 or a pharmaceutically acceptable Salt thereof, and instructions for use thereof. 35 24. A method of inhibiting an arginine methyl transferase (RMT) comprising contacting a cell with an effective NH2 amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof. k k k k k