STRUCTURAL FORMULA II COMPOUND, ITS USE AND PHARMACEUTICAL COMPOSITION

专利摘要:
modulators of methyl modifying enzymes, their compositions and use. provided herein are agents having structural formula (ii), defined below, for the modulation of histone methyl modifying enzymes, their compositions and use, for example, as anti-cancer agents.
公开号:BR112015018508B1
申请号:R112015018508-8
申请日:2014-02-11
公开日:2021-08-24
发明作者:Brian K. Albrecht；James Edmund Audia；Andrew S. Cook；Les A. Dakin；Martin Duplessis；Victor S. Gehling；Jean-Christophe Harmange；Christopher G. Nasveschuk；Rischi G. Vaswani
申请人:Constellation Pharmaceuticals, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] This order claims the priority of International OrderNo. PCT/US2013/025639, filed February 11, 2013. International Application PCT/US2013/025639 claims priority to United States Interim Applications Nos. Serial 61/597,695, filed February 10, 2012 and 61/667,821, filed July 3, 2012. The entire contents of the aforementioned applications are hereby incorporated by reference. FUNDAMENTALS OF THE INVENTION
[0002] Eukaryotic chromatin is composed of macromolecular complexes called nucleosomes. A nucleosome has 147 base pairs of DNA wrapped around a protein octamer having two subunits each of the histone proteins H2A, H2B, H3 and H4. Histone proteins are subject to post-translational modifications which in turn affect chromatin structure and gene expression. One type of post-translational modification found in histones is the methylation of lysine and arginine residues. Histone methylation plays a critical role in regulating gene expression in eukaryotes. Methylation affects chromatin structure and has been associated with both activation and repression of transcription (Zhang and Reinberg, Genes Dev. 15:23432360, 2001). Enzymes that catalyze the attachment and removal of methyl groups from histones are implicated in gene silencing, embryonic development, cell proliferation, and other processes. SUMMARY OF THE INVENTION
[0003] The present disclosure encompasses the recognition that methyl-modifying enzymes are an attractive target for modulation, given their role in regulating various biological processes. It has now been found that the compounds of this invention, and their pharmaceutically acceptable compositions, are effective as agents that stimulate the activity of histone methyl modifying enzymes, including histone methylases and methylases demethylases. Such compounds have the general formula II:

[0004] or a pharmaceutically acceptable salt thereof, wherein each variable is as defined herein.
[001] The compounds of the present invention, and their pharmaceutically acceptable compositions, are useful for the treatment of a variety of diseases, disorders or conditions associated with a methyl-modifying enzyme. Such diseases, disorders or conditions include those described herein.
[002] The compounds provided by this invention are also useful for the study of methyl-modifying enzymes in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by methyl-modifying enzymes and the comparative evaluation of novel methyl-modifying enzyme modulators. BRIEF DESCRIPTION OF THE DRAWINGS
[003] Figure 1. Exemplary compounds of Formula II. DETAILED DESCRIPTION OF CERTAIN MODALITIES 1. General Description of the Compounds of the Invention
[004] In certain embodiments, the present invention provides a compound of Formula II:

[0005] or a pharmaceutically acceptable salt thereof, wherein:
[0006] A is CH or N;
[0007] R1a is selected from C1-C2 alkyl and -O-(C1-C2 alkyl), wherein R1a is optionally substituted with one or more fluorine;
[0008] R4a is selected from -(C1-C4 alkylene)-O-(C1-C3 alkyl), 1-substituted-pipieridin-4-yl, C3-C6 cycloalkyl optionally substituted with one or more fluorine, and tetrahydropyranyl ; and
[0009] R13 is selected from hydrogen, halo, phenyl, pyridinyl and -O-(C1-C4 alkyl).2. Compounds and Definitions
[005] The definitions of specific functional groups and thermochemicals are described in more detail below. For purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., back cover, and specific functional groups are generally defined as described herein. Additionally, general principles of organic chemistry, as well as specific functional components and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; whose entire contents of each are hereby incorporated by reference.
[006] Unless otherwise noted, structures depicted herein are also intended to include all isomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) of the structure; for example, the R and S configurations for each asymmetric center, the Z and E double bond isomers, and the Z and E conformational isomers. Therefore, the individual stereochemical isomers as well as the enantiomeric, diastereomeric and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise mentioned, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise noted, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a carbon enriched with 13C or 14C, are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
[007] Where a particular enantiomer is preferred, it may, in some embodiments, be provided substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched". "Optically enriched", as used herein, means that the compound is prepared from a significantly greater proportion of one enantiomer. In certain embodiments, the compound is composed of at least about 90% by weight of a preferred enantiomer. In other embodiments, the compound is composed of at least about 95%, 98% or 99% by weight of a preferred enantiomer. Preferred enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).
[008] A wavy bond (—) at a chiral center with a chemical structure is used to indicate compounds of the invention that are optically pure, but whose optical rotation has not been determined. A straight bond at a chiral center indicates a racemic mixture although, as mentioned above, the invention also includes all possible isomeric forms of the racemate.
[009] The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine (fluoro, -F), chlorine (chlorine, -Cl), bromine (bromine, -Br) and iodine (iodine , -I).
[0010] The term "alkylene" refers to a bivalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., (CH2)n, where n is a positive integer, preferably from 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below with respect to a substituted aliphatic group.
[0011] The term "alkyl", as used herein, refers to a straight or branched chain saturated monovalent hydrocarbon radical derived from an aliphatic component containing between one and six carbon atoms by removal of a single hydrogen atom . In some embodiments, alkyl contains 1 to 5 carbon atoms. In another embodiment, alkyl contains 1 to 4 carbon atoms. In most other embodiments, alkyl contains 1 to 3 carbon atoms. In yet another embodiment, alkyl contains 1 to 2 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n- pentyl, neopentyl, n-hexyl, sec-hexyl, and more.
[0012] As described herein, compounds of the invention may contain "optionally substituted" components. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated component are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in a given structure may be substituted with more than one substituent selected from a specified group, the substituent can be the same or different at each position. The combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically viable compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions that allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed here.
Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH2)o-4R°; -(CH2)o—4OR°; -O-(CH2)o-4C(O)OR°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)0-4O(CH2)0-1Ph which may be substituted with R°; -CH=CHPh, which can be substituted with R°; -NO2; -CN; -N3; -(CH2)0-4N(R°)2; -(CH2)0-4N(R°)C(O)R°; -N(R°)C(S)R°; -(CH2)0-4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)0-4N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)R°; -C(S)R°; -(CH2)0-4C(O)OR°; -(CH2)0-4C(O)SR°; -(CH2)0-4C(O)OSiR°3; -(CH2)0 -4OC(O)R°; -OC(O)(CH2)0-4SR-; -SC(S)SR°; -(CH2)0-4SC(O)R°; -(CH2)0-4C(O)NR°2; -C(S)NR°2; -C(S)S R°; -SC(S)SR°; -(CH2)0-4OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)0-4SSRC°; -(CH2)0-4S(O)2R°; -(CH2)0-4S(O)2OR°; -(CH2)0-4OS(O)2R°; -S(O)2NR°2; -(CH2)0-4S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -P(O)2R°; -P(O)R°2; - OP(O)R°2; -OP(O)(OR°)2; -SiR°3; -(C1-4 alkylene straight or branched)O-N(R°)2; or -(C1-4 straight or branched alkylene)C(O)ON(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -O(CH2) 0-1Ph, or a 5- to 6-membered saturated or partially saturated aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, despite the above definition, two independent occurrences of Ro, taken together with the its intervening atoms form a 3- to 12-membered saturated or partially unsaturated aryl mono- or bicyclic ring having from 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.
Suitable substituents on a replaceable nitrogen of an "optionally substituted" group include -Ri, -NR4 , -C(O)Rt, -C(O)ORt -C(O)C(O)Rt, -C (O)CH2C(O)Rt, -S(O)2Rt, -S(O)2NRi2, -C(S)NRi2, -C(NH)NRi2, or -N(Ri)S(O)2Ri; wherein each Ri is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted OPh, or an unsubstituted 5- to 6-membered saturated or partially unsaturated aryl ring having 0 to 4 independently selected nitrogen heteroatoms , oxygen or sulfur, or, despite the above definition, two independent occurrences of Ri, taken together with its intervening atom form an unsubstituted 3- to 12-membered mono- or bicyclic saturated or partially unsaturated aryl ring independently selected from nitrogen, oxygen or sulfur.
Suitable substituents on the aliphatic group of Ri are independently halogen, -R*, -(haloR*), -OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C( O) OR*, -NH2, -NHR*, -NR*2, or -NO2, where each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C1- 4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5- to 6-membered saturated or partially saturated aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As used herein, the term "inhibitor" is defined as a compound that binds and/or inhibits an S-adenosylmethionine (SAM) target using the enzyme with measurable affinity. In certain embodiments, an inhibitor has an IC50 and/or binding constant of less than about 50 µM, less than about 1 µM, less than about 500 nM, less than about 100 nM, or less than about 10 nM.
[0017] The terms "measurable affinity" and "formally measurable inhibit", as used herein, mean a measurable change in the activity of at least one SAM using the enzyme between a sample comprising a supplied compound, or its composition, and at least a SAM-dependent enzyme, and an equivalent sample comprising at least one SAM-dependent enzyme, in the absence of said compound, or its composition. Description of Exemplary Compounds
[0018] In some embodiments of Formula II, R1a is selected from -OCH3, -CH3, -OCHF2 and -CH2CH3.
In some embodiments of Formula II, R4a is selected from -CH2OCH3, -CH(CH3)OCH3, 4,4-difluorocyclohexyl, cyclopropyl, tetrahydropyran-4-yl, 1-(t-butoxycarbonyl)-piperidin-4 -yl, 1-(isobutoxycarbonyl)-piperidin-4-yl, 1-(isopropoxycarbonyl)-piperidin-4-yl, 1-(2-fluoroethyl)-piperidin-4-yl, 1-(2,2-difluoroethyl) -piperidin-4-yl, 1-(2,2,2-trifluoroethyl)-piperidin-4-yl, 1-(2-hydroxyisobutyl)-piperidin-4-yl, 1-(hydroxyisopropylcarbonyl)-piperidin-4-yl , 1-(ethoxycarbonylmethyl)-piperidin-4-yl, 1-(isopropylcarbonyl)-piperidin-4-yl, 1-methylpiperidin-4-yl, 1-(methylsulfonyl)-piperidin-4-yl, 1-(ethylsulfonyl) -piperidin-4-yl, 1-(isopropylsulfonyl)-piperidin-4-yl, 1-(phenyl)-piperidin-4-yl, 1-(oxetan-3-yl)piperidin-4-yl, 1-(pyridin -2-yl)-piperidin-4-yl and 1-(pyrimidin-2-yl)-piperidin-4-yl.
[0020] In some embodiments of Formula II, R13 is selected from hydrogen, chlorine, fluorine, OCH(CH3)2, phenyl and pyridin-2-yl.
[0021] Exemplary compounds of Formula II are shown in Figure 1. In some cases, two (or more) of the compounds in Figure 1 having one or more wavy bonds will have the exact structure. Since the wavy bond represents a chiral center of indeterminate optical rotation, such compounds will be understood to be separate and distinct optical isomers. Figure 1 is noted to indicate sets of two or more compounds that have the same depicted structure but are of different stereochemistry.4. Use, Formulation and Administration Pharmaceutically acceptable compositions
According to another embodiment, the invention provides a composition comprising a compound of this invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle. The amount of compound in the compositions of this invention is such that it is effective to measurably modulate a methyl histone modifying enzyme, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the amount of compound in the compositions of this invention is such that it is effective to measurably modulate a methyl histone modifying enzyme, or a mutant thereof, in a biological sample or in a patient.
In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such a composition. In some embodiments, a composition of this invention is formulated for oral administration to a patient.
The term "patient", as used herein, means an animal, preferably a mammal, and more preferably a human.
The term "pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering substances such as phosphates , glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.
[0026] A "pharmaceutically acceptable derivative" means any salt, ester, salt of an ester or other non-toxic derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of this invention or an inhibitory active metabolite or its residue.
The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or through an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention can be an aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixed oils are conventionally employed as a solvent or suspending medium.
[0028] For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.
The pharmaceutically acceptable compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutically acceptable compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutically acceptable compositions of this invention can also be administered topically, especially when the target of treatment includes areas or organs easily accessible by topical application, including diseases of the eyes, skin or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs.
Topical application to the lower intestinal tract can be made in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically transdermal patches can also be used.
[0033] For topical applications, the provided pharmaceutically acceptable compositions may be formulated into a suitable ointment containing the active component in suspension or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions provided may be formulated in a suitable lotion or cream containing the active components in suspension or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
[0034] For ophthalmic use, the pharmaceutically acceptable compositions provided may be formulated as micronized isotonic suspensions, sterile saline with pH adjusted or, preferably, as solutions in sterile saline with adjusted pH isotonic, with or without a preservative such as benzylalkonium chloride . Alternatively, for ophthalmic use, pharmaceutically acceptable compositions can be formulated in an ointment such as petrolatum.
The pharmaceutically acceptable compositions of the present invention can also be administered by aerosol or nasal inhalation. Such compositions are prepared in accordance with techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons and/or other solubilizing agents or conventional dispersion.
More preferably, the pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of this invention are administered with food.
The amount of compounds of the present invention that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host treated and the particular mode of administration. Preferably, the compositions provided should be formulated so that a dosage of between 0.01 to 100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.
[0038] It is also to be understood that a specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, excretion rate, drug combination, and the attending physician's assessment and severity of the particular illness being treated. The amount of a compound of the present invention in the composition will also depend on the particular compound in the composition. Use of Pharmaceutically Acceptable Compounds and Compositions
[0039] The compounds and compositions described herein are generally useful for modulating the activity of one or more enzymes involved in epigenetic regulation.
[0040] Epigenetics is the study of hereditary changes in gene expression caused by different mechanisms that alter the underlying DNA sequence. Molecular mechanisms that play a role in epigenetic regulation include DNA methylation and chromatin/histone modifications. Histone methylation, in particular, is fundamental in many epigenetic phenomena.
[0041] Chromatin, the organized set of nuclear DNA and histone proteins, is the basis for a multitude of vital nuclear processes including the regulation of transcription, replication, DNA damage repair and progression through the cell cycle. Several factors, such as chromatin modifying enzymes, have been identified which play an important role in maintaining the dynamic balance of chromatin (Margueron, et al. (2005) Curr. Opin. Genet. Dev. 15:163-176) .
[0042] Histones are the major components of the protein dechromatin. They act as coils around which the DNA revolves, and they play a role in gene regulation. There are a total of six histone classes (H1, H2A, H2B, H3, H4, and H5) organized into two superclasses: core histones (H2A, H2B, H3 and H4) and binding histones (H1 and H5). The basic unit of chromatin is the nucleosome, which consists of about 147 base pairs of DNA around the histone octamer, which consists of two copies each of the core histones H2A, H2B, H3 and H4 (Luger, et al. al. (1997) Nature 389:251-260).
Histones, in particular the amino-terminal residues of histones H3 and H4 and the amino- and carboxyl-terminal residues of histones H2A, H2B and H1, are susceptible to a variety of post-translational modifications including acetylation, methylation, phosphorylation, ribosylation , sumoylation, ubiquitination, citrullination, deimination and biotinylation. The nucleus of histones H2A and H3 can also be modified. Histone modifications are an integral part of several biological processes such as gene regulation, DNA repair and chromosome condensation.
[0044] The present disclosure provides compounds and compositions for the modulating activity of the methyl histone modifying enzyme. Histone methyl modifying enzymes are fundamental regulators of cellular and developmental processes. Histone methyl modifying enzymes can be characterized as histone methyl transferases or histone demethylases. Histone demethylase enzymes have modules that mediate binding to methylated residues. For example, multiple demethylases contain a Tudor domain (eg JMJD2C/GASC1) or a PHD domain (eg JARID1C/SMCX, PHF8).
The lysine specificities of many histone methyltransferases have been characterized. For example, SET7/9, SMYD3, and MLL1-5 are specific for H3K4. SUV39H1, DIM-5, and G9a are specific for H3K9. SET8 is specific for H4K20.
[0046] DOT1 is an example of a non-SET domain containing histone methylase. DOT1 H3 methylates in lysine 79.
[0047] Equally histone methylases have been shown to deregulate transcriptional activity, chromatin structure, and gene silencing, demethylases have also been discovered which affect gene expression. LSD1 was the first histone lysine demethylase to be characterized. This enzyme has FAD-dependent homology to amine oxidases and acts as a co-repressor of neuronal gene transcription (Shi et al., Cell 119:941-953, 2004). Additional demethylases that define separate demethylase families have been discovered, including the JHDM1 (or KDM2), JHDM2 (or KDM3), JMJD2 (or KDM4), JARID (or KDM5), JMJD3 (or KDM6), and JMJD6 (Lan) families. et al., Curr. Opin. Cell Biol. 20(3):316-325, 2008).
[0048] Demethylases act on specific lysine residues within substrate sequences and discriminate between the degree of methylation present in a given residue. For example, LSD1 removes mono or dimethyl groups from H3K4. Members of the JARID1A-D family remove trimethyl groups from H3K4. UTX and JMJD3 demethylate H3K27, neutralizing effects of EZH2 methylase activity. The substrate specificities of other demethylases have been characterized (see Shi, Nat. Rev. 8:829-833, 2007).
[0049] A class of histone methylases is characterized by the presence of a SET domain, named after the domain-sharing proteins, Su(var)3-9, the zest enhancer [E(Z)] and trithorax. A SET domain includes about 130 amino acids. Methylase families containing the SET domain include the SUV39H1, SET1, SET2, EZH2, RIZ1, SMYD3, SUV4-20H1, SET7/9 and PR-SET7/SET8 families (reviewed in Dillon et al., Genome Biol. 6:227 , 2005). Members of a family typically include similar sequence motifs in proximity to and within the SET domain. The human genome encodes an additional 50 histone protein methylases containing the SET domain, any of which can be used in an assay described herein.
EZH2 is an example of a methylase containing the human SET domain. EZH2 associates with EED (Ectodermal Embryonic Development) and SUZ12 (Zest 12 homolog suppressor) to form a complex known as PRC2 (Polycomb Group 2 Repressive Complex) having the ability to tri-methylate histone H3 to lysine 27 ( Cao and Zhang, Mol. Cell 15:57-67, 2004). PRC2 complexes can also include the RBAP46 and RBAP48 subunits.
[0051] The oncogenic activities of EZH2 have been demonstrated by several studies. In cell lineage experiments, overexpression of EZH2 induces cell invasion, growth in soft agar, and the motility although unexpected of EZH2 inhibits cell proliferation and cell invasion (Kleer et al., 2003, Proc. Nat. Acad. Sci. USA 100:11606-11611; Varambally et al., (2002), "The polycomb group protein EZH2 is involved in progression of prostate cancer," Nature 419, 624-629). EZH2 has been shown to repress the expression of several tumor suppressors, including E-cadherin, DAB2IP and RUNX3 among others. In xenograft models, the unexpected EZH2 inhibits the growth of tumors and metastases. Recently, EZH2 down-modulation in murine models has been shown to block prostate cancer metastasis (Min et al., “An oncogene-tumor suppressor cascade drives metastatic prostate cancer by coordinately activating Ras and nuclear factor-kappaB,” Nat Med . 2010 Mar; 16(3):286-94). EZH2 overexpression is associated with aggressiveness of certain cancers such as breast cancer (Kleer et al., Proc. Nat. Acad. Sci. USA 100:11606-11611, 2003). Recent studies also suggest that the prostate cancer-specific oncogenic fusion gene TMPRSS2-ERG induces repressive epigenetic programs through direct activation of EZH2 (Yu et al., “An Integrated Network of Androgen Receptor, Polycomb, and TMPRSS2-ERG Gene Fusions in Prostate Cancer Progression,” Cancer Cell. 2010 May 18;17(5):443-454).
[0052] In some embodiments, the compounds of the present invention modulate the activity of one or more enzymes involved in epigenetic regulation. In some embodiments, compounds of the present invention modulate the activity of a methyl histone modifying enzyme, or a mutant thereof. In some embodiments, compounds of the present invention modulate EZH2 activity. In certain embodiments, compounds of the present invention down-regulate or suppress EZH2 activity. In some embodiments, compounds of the present invention are antagonists of EZH2 activity.
In some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with a histone methyl modifying enzyme. Accordingly, in some embodiments, the present invention provides a method of modulating a disease and/or disorder associated with a histone methyl modifying enzyme. In some embodiments, the present invention provides a method of treating an individual suffering from a disease and/or disorder associated with a histone methyl modifying enzyme which comprises the step of administering a compound or composition of Formula II.
In some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with overexpression of EZH2. In some embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with EZH2 overexpression comprising the step of administering a compound or composition of Formula II. In some embodiments, the above method additionally comprises the preliminary step of determining whether the individual overexpresses EZH2.
In some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with cell proliferation. In some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with cell cycle dysregulation or DNA repair. In some embodiments, the compounds and compositions of the present invention are useful in treating cancer. Exemplary types of cancer include breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme cancer, bladder cancer, melanoma, bronchial cancer, lymphoma, and liver cancer.
The study of EZH2 knockouts, missense and frameshift mutations suggest that EZH2 functions as a tumor suppressor in blood disorders such as myelodysplastic syndromes (MDS) and myeloid neoplasms (Ernst et al. , Nat Genet. 2010 Aug; 42(8):722-6; Nikoloski et al., Nat Genet. 2010 Aug; 42(8):665-7). Accordingly, in some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with the presence of a mutant form of EZH2. In some embodiments, the compounds and compositions of the present invention are useful in treating diseases and/or disorders associated with the presence of Y641N EZH2. In certain embodiments, the disease or disorder associated with the presence of a mutant form of EZH2 is human B-cell lymphoma. In certain embodiments, the disease and/or disorder associated with the presence of Y641N EZH2 is follicular lymphoma or diffuse large B-cell lymphoma. In some embodiments, the compounds or compositions of the present invention are useful in treating blood disorders such as myelodysplastic syndromes, leukemia, anemia and cytopenia. Sneeringer et al., “Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas,” Proceedings of the National Academy of Sciences, PNAS Early Edition published ahead of print on November 15, 2010.
[0057] In some embodiments, the present invention provides a method of reducing EZH2 activity in an individual comprising the step of administering a compound or composition of Formula II. In some embodiments, the present invention provides a method of reducing large-type EZH2 activity in a subject comprising the step of administering a compound or composition of Formula II. In some embodiments, the present invention provides a method of reducing the activity of a mutant form of EZH2 in a subject comprising the step of administering a compound or composition of Formula II. In certain embodiments, the present invention provides a method of reducing the activity of a mutant form of EZH2 in a subject comprising the step of administering a compound or composition of Formula II, wherein the mutant form of EZH2 is Y641N EZH2. In certain embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with EZH2 which comprises the step of administering a compound or composition of Formula II. In some embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with EZH2 of the large type which comprises the step of administering a compound or composition of Formula II. In some embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with a mutant form of EZH2 which comprises the step of administering a compound or composition of Formula II. In certain embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with a mutant form of EZH2 which comprises the step of administering a compound or composition of Formula II, wherein the form mutant of EZH2 is Y641N EZH2. In certain embodiments, the above method further comprises the preliminary step of determining whether the individual expresses a mutant form of EZH2, such as Y641N EZH2. In certain embodiments, the present invention provides a method of reducing the activity of a mutant form of EZH2, such as EZH2 Y641N, in a subject in need thereof, comprising the step of administering a compound or composition of Formula II. In some embodiments, the present invention provides a method of treating a subject suffering from a disease and/or disorder associated with a mutant form of EZH2 which comprises the step of administering a compound or composition of Formula II. In certain embodiments, the above method further comprises the preliminary step of determining whether the individual expresses a mutant form of EZH2, such as Y641N EZH2. In some embodiments, this determination is made by determining whether the individual has increased levels of specific trimethylation of histone H3 Lys-27 (H3K27me3), compared to an individual known not to express a mutant form of EZH2. EQUIVALENTS
[0058] The following representative examples are intended to help illustrate the invention, and are not intended, and should not be construed, to limit the scope of the invention. Indeed, various modifications of the invention and many other embodiments thereof, in addition to those presented and described herein, will become apparent to those skilled in the art from the full contents of this document, including the following examples and references to scientific and scientific literature. patent cited herein. It should further be noted that the contents of these cited references are hereby incorporated by reference to help illustrate the state of the art.
It will be appreciated that for the compound preparations described herein, when reverse phase HPLC is used to purify a compound, a compound may exist as an acid addition salt. In some embodiments, a compound can exist as a salt of formic acid or mono-, di-, or tri-trifluoroacetic acid.
[0060] It will further be appreciated that the present invention contemplates the individual compounds described herein. When the individual compounds exemplified are isolated and/or characterized as a salt, for example, as a salt of trifluoroacetic acid, the present invention contemplates a free base of the salt, as well as other pharmaceutically acceptable salts of the free base.
[0061] The following examples contain important additional information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and its equivalents.
The procedures for the preparation of the compounds exemplified below, as well as the additional compounds/intermediates in the synthesis schemes, can be seen in International Application No. PCT/US2013/025639, the contents of which are incorporated herein by reference. EXAMPLES
[0063] As described in the Examples below, in certain exemplary embodiments, the compounds are prepared according to the following general procedures. It will be appreciated that while the synthetic methods and schemes represent the synthesis of certain compounds of the present invention, the following methods and other methods known to a person of practical skill in the art can be applied to all compounds and subclasses and species of each of these compounds as described herein.
[0064] Unless otherwise mentioned, all solvents, chemicals and reagents were commercially obtained and used without purification. 1H NMR spectra were obtained in CDCl3, d6-DMSO, CD3OD, or d6-acetone at 25 oC at 300 MHz on an OXFORD (Varian) with chemical shift (δ, ppm) recorded against TMS as an internal standard. HPLC-MS chromatograms and spectra were obtained with the Shimadzu LC-MS-2020 system. Chiral analysis and purification were obtained with Yilite P270.Example 1. Synthesis of Compounds 327 and 346 and Related Compounds and Intermediates. The title compounds of this and other Related Compounds were prepared according to the following general scheme. In addition, where indicated, modifications of this scheme are disclosed for the synthesis of further additional Related Compounds of the invention and their intermediates. -90% Stage 1

Step 1: 4-(((tert-butylsulfinyl)imino)methyl)piperidine-1-carboxylate (S,E)-tert-butyl:(S)-2-methylpropane-2-sulfinamide

To a round bottom flask charged with a magnetic stir bar were added (S)-2-methylpropane-2-sulfinamide (20.46 g, 169 mmol), tert-butyl 4-formylpiperidine-1-carboxylate ( 30 g, 141 mmol), DCM (300 ml), and Ti(OEt) 4 (59.0 ml, 281 mmol). The solution was stirred at room temperature for 3 h before it was rapidly cooled with brine (80 ml). The solution was stirred for 30 minutes before filtering. The filter cake was washed with DCM and the filtrate was placed in a separatory funnel and washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue solidified with the title compound (29 g, 92 mmol, 65.1% yield) m/z 217.
[0066] The intermediate shown in the following table was prepared according to the general procedure outlined in Step 1 using the appropriate starting materials and modifications. Name Structure m/z(S,E)-2-methyl-N-((tetra- hydro-2H-pyran-4-yl)methylene)propane-2-sulfinamide,2
Step 2: tert-butyl 4-((S)-1-((R or S)-1,1-dimethylethylsulfinamido)ethyl)piperidine-1-carboxylate:

To a round bottom flask loaded with a magnetic stir bar were added (S,E)-tert-butyl 4-((tert-butylsulfinylimino)methyl)piperidine-1-carboxylate (36.4 g, 115 mmol) ), DCM (400 ml), and the solution was cooled to 0 °C in an ice bath with stirring. To this solution was added MeMgBr (77 ml, 230 mmol) (3M in diethyl ether) and the reaction stirred for 4 h while warming to room temperature. The reaction was carefully quenched by addition of saturated aqueous NH4Cl. The solid was broken up by the addition of 1N HCl. The layers were separated and the aqueous phase was extracted with DCM. The combined organic phase was dried over Na 2 SO 4 , filtered, and concentrated in vacuo to give the title compound (29 g, >9:1 dr) which is used without further purification in the next step.
[0068] The intermediate shown in the following table was prepared according to the general procedure outlined in Step 2 using the appropriate starting materials and modifications.Name Structure m/z(S)-2-methyl-N-((R or S) -1-(tetrahydro-2H-pyran-4-yl)ethyl)propane-2-sulfinamide ° y / / HN-S* 'b 234
Step 3: (R or S)-tert-butyl 4-(1-aminoethyl)piperidine-1-carboxylate:

To a 1 L round bottom flask charged with a magnetic stir bar was added tert- 4-((S)-1-((S)-1,1-dimethylethylsulfinamido)ethyl)piperidine-1-carboxylate. Crude butyl (29 g) was taken up in MeOH (200 ml) before addition of a 4 N solution of HCl in 1,4-dioxane (24.06 ml, 96 mmol). The resulting solution was then stirred at room temperature for 1 h at rt. Methanol was then removed in vacuo to give viscous oil which was treated with saturated aqueous NaHCO 3 (~500 ml) and extracted with ethyl acetate (2 x 500 ml). This organic phase was combined, dried over MgSO4, filtered, and the solvent was then removed in vacuo to give the title compound (22 g) which was used without further purification.
[0070] The intermediate shown in the following table was prepared according to the general procedure outlined in Step 3 using the appropriate starting materials. Name Structure m/z(R or S)-1-(tetrahydro-2H-pyran-4 -yl)ethanamine ° yz NH2 130
Step 4: Methyl 2-(2-bromophenyl)-3-oxobutanoate:

A round bottom flask was charged with a magnetic stir bar and methyl 2-(2-bromophenyl)acetate (25 g, 109 mmol) and THF (50 ml). This solution was cooled to -78°C before the dropwise addition of a 1 M solution of LiHMDS in THF (218 ml, 218 mmol). The reaction was stirred for 30 min at -78 °C before the addition of 1-(1H-imidazol-1-yl)ethanone (14.42 g, 131 mmol), dissolved in a mixture of THF:DMF (112 ml of THF, 24 ml DMF). The solution was stirred for 1 h before quenching with saturated aqueous NH 4 Cl (~250 ml) and diluting with EtOAc. The layers were separated and the aqueous phase was extracted with EtOAc (~2 x 250 ml). The combined organic extract was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was purified by chromatography on silica gel using an eluent of ethyl acetate/hexanes (10:1) to give methyl 2-(2-bromophenyl)-3-oxobutanoate (32.5 g, 102 mmol). , 93% yield).
The intermediates shown in the following table were prepared according to the general procedure outlined in Step 4 using the appropriate starting materials. — Methyl 3042-(2-bromo-4-methoxyphenyl)-3-oxobutanoate oZ CHO o — Methyl 3022-(2-bromo-4-fluorophenyl)-3-oxobutanoate r°Y 1 289
Step 5: 4-(1-(3-(2-Bromophenyl)-4-methoxy-4-oxobut-2-en-2-ylamino)ethyl)piperidine-1-carboxylate (R or S, E and Z) -tert-butyl:

To a round bottom flask were added (R or S)-tert-butyl 4-(1-aminoethyl)piperidine-1-carboxylate (9.35 g, 40.9 mmol), EtOH (75 ml) , and methyl 2-(2-bromophenyl)-3-oxobutanoate (7.40 g, 27.3 mmol) (from Step 4). To this solution was added AcOH (1.563 ml, 27.3 mmol) and the reaction was heated overnight at 85 °C before cooling to room temperature and concentration. The crude residue was purified by silica gel chromatography (330 g, 100% hexanes to 25% EA in hexanes) to give the title compound (6.45 g, 13.40 mmol, 49.1% yield ).
The intermediates shown in the following table were prepared according to the general procedure outlined in Step 5 using the appropriate starting materials.

Step 6: 1-(1-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-2-methyl-1H-
(R or S)-methyl indole-3-carboxylate:
[0075] A 250 ml round bottom flask was charged with a magnetic stir bar, 4-(1-(3-(2-bromophenyl)-4-methoxy-4-oxobut-2-en-2-ylamino)ethyl (R or S,Z)-tert-butyl)piperidine-1-carboxylate (3.33 g, 6.92 mmol), RuPhos Pre-catalyst II (Methanesulfonate(2-dicyclohexylphosphino-2',6'-di- i-propoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl) palladium(II)) (0.463 g, 0.553 mmol), dicyclohexyl(2',6'-diisopropoxybiphenyl- 2-yl)phosphine (0.387 g, 0.830 mmol), anhydrous 1,4-dioxane (27.7 ml, 6.92 mmol), and sodium methoxide (0.561 g, 10.38 mmol). The reaction mixture was purged and again supplied with nitrogen and heated to 100 °C with stirring overnight before being allowed to cool to rt. The reaction was diluted with ethyl acetate (~100 ml) and the mixture was filtered through a bed of diatomaceous earth. The filtrate was pre-absorbed onto silica gel (~30 g) and purified by chromatography on silica gel (120 g) using ethyl acetate/hexanes (1:1) as eluent to give the title compound (2 .01 g, 4.77 mmol, 68.9% yield).
[0076] The intermediates shown in the following table were prepared according to the general procedure outlined in Step 6 using the appropriate starting materials.
Step 7: (R or S)-2-methyl-1-(1-(tetrahydro-2H-pyran-4-i)ethyl)-1H-indole-3-carboxylic acid:

[0077] A 1 L round bottom flask was charged with a magnetic stir bar, 2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3- (R or S)-methyl carboxylate (11.60 g, 38.5 mmol), ethanol (96 ml, 38.5 mmol), and 6N aqueous NaOH (64.1 ml, 385 mmol). The flask was fitted with a reflux condenser and heated to reflux for 6 h before being allowed to cool to rt. Volatiles were removed in vacuo and the resulting mixture was poured into 10% HCl (~300 ml). A precipitate formed which was collected by vacuum filtration using a Buchner funnel. The filter cake was rinsed with an additional portion of water (~200 ml), collected, and dried under vacuum to provide the title compound (10.87 g, 35.9 mmol, 93% yield) as an off-white solid. .
[0078] The intermediates shown in the following table were prepared according to the general procedure outlined in Step 7 using the appropriate starting materials.
Step 8: 4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-2-methyl-1H-indole-1- (R or S)-tert-butyl yl)ethyl)piperidine-1-carboxylate (Compound 327).

A 250 ml round bottom flask was charged with a magnetic stir bar, acid (R or S)-1-(1-(1-(tert-butoxycarbonyl)piperidin-4-yl)ethyl)-2- methyl-1H-indole-3-carboxylic (1.950 g, 5.05 mmol), 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one hydrochloride (2.065 g, 10.09 mmol) , DMF (25.2 ml, 5.05 mmol), Hunig's base (3.52 ml, 20.18 mmol). The reaction mixture was cooled to 0 °C and COMU (2.16 g, 5.05 mmol) was added. The reaction was allowed to stir overnight to room temperature. The reaction mixture was diluted with water and extracted with EtOAc. The combined organic extract was washed with brine, dried over MgSO4, filtered and conc. in vacuo to give the crude material which was purified by chromatography on silica gel (120 g) using MeOH/ethyl acetate (1:5) as eluant to give the title compound (1.86 g, 3.29 mmol, 65.3% yield). LCMS 537 (M + 1) + 1H NMR (400 MHz, DMSO-d6) δ = 11.83 - 11.71 (m, 1H), 7.80 (br.s., 1H), 7.73 (d, J = 7.6 Hz, 1 H), 7.62 (d, J = 7.8 Hz, 1 H), 7.06 (td, J = 7.1, 14.4 Hz, 2 H ), 6.21 (s, 1H), 4.32 (br.s., 2H), 4.16 (br.s., 1H), 4.02 (br.s., 1H) , 3.85 (s, 3H), 3.75 (br.s., 1H), 2.70 (br.s., 1H), 2.58 (s, 3H), 2.37 (br.s., 1 H), 2.21 (s, 3 H), 1.90 (d, J = 12.9 Hz, 1 H), 1.53 (d, J = 6.9 Hz, 3H), 1.35 (s, 10H), 1.21 (br.s., 1H), 0.89 (d, J = 8.7Hz, 1H), 0.67 (d, J = 11.8 Hz, 1H).
The compounds shown in the following table were prepared according to the general procedure outlined in Step 8 using the appropriate starting materials. The structures of the compounds are shown in Figure 1.


Step 9: (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-) hydrochloride -(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (Compound 326).

A 250 ml round bottom flask was charged with a magnetic stir bar, 4-(1-(3-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-) (R or S)-tert-butyl 3-yl)methylcarbamoyl)-2-methyl-1H-indol-1-yl)ethyl)piperidine-1-carboxylate (Compound 327) (1.850 g, 3.45 mmol), MeOH (13.79 ml, 3.45 mmol), and HCl (2.59 ml, 10.34 mmol) (4N in dioxane). The reaction was allowed to stir at rt for 6 h before being conc. in vacuo to give the title compound (1.65 g, 3.14 mmol, 91% yield). LCMS 437 (M+1)+.
The compound shown in the following table was prepared according to the general procedure outlined in Step 9 using the appropriate starting materials. The structure of this compound is shown in Figure 1. Compound Number Name 1H NMR m/z376 (R or S)-1-(1-(1-(azetidin-3-yl)piperidin-4-yl)ethyl) hydrochloride - N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide (400 MHz, DMSO-d6) δ 12.27 -12.10 (m, 1H), 11.96 - 11.72 (m, 1H), 9.80 (br.s., 1H), 9.19 (br.s., 2H), 7.89 - 7.67 (m, 2H), 7.62 (d, J = 7.6Hz, 1H), 7.09 (quin, J = 6.6Hz, 2H ), 5.99 (s, 1H), 4.59 - 4.36 (m, 3H), 4.24 - 3.95 (m, 2H), 3.48 (d, J = 13. 2Hz, 1H), 3.17 (d, J = 12.0Hz, 1H), 2.87 (br.s., 1H), 2.70 (br.s., 2H), 2.58 (s, 3H), 2.34 - 2.25 (m, 3H), 2.19 - 2.10 (m, 3H), 1.75 (d, J = 12.3 Hz , 1 H), 1.57 (d, J = 6.7 Hz, 3 H), 1.47 (d, J = 12.7 Hz, 2 H), 1.33 - 1.21 (m, 2 H), 0.85 (d, J = 13.6 Hz, 1 H) 476
Step 10: 4-(1-(3-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methylcarbamoyl)-2-methyl-1H-indole-1- (R or S)-isopropyl yl)ethyl)piperidine-1-carboxylate (Compound 346).

[0083] A 250 ml round bottom flask was charged with a magnetic stir bar, (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine) hydrochloride -3-yl)methyl)-2-methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (0.467 g, 0.987 mmol), DMF (2.468 ml, 0.987 mmol) , THF (2.468 ml, 0.987 mmol), and N-ethyl-N-isopropylpropan-2-amine (0.638 g, 4.94 mmol). The reaction was cooled to 0 °C and isopropyl carbonchloridate (0.160 ml, 1.086 mmol) was added dropwise via syringe. The reaction was allowed to stir for 2 h at rt and was then treated with 5 N LiOH for 1 h to remove any acylated pyridone. This material was extracted with ethyl acetate, washed with brine, dried over MgSO4 and filtered and conc. in vacuo. The resulting material was purified by chromatography on silica gel (50 g) using ethyl acetate/MeOH (5:1) as eluent to give pure title compound as a pale yellow solid (0.300 g, 0.545 mmol, 55, 2% yield). LCMS 523 (M+1)+; 1H NMR (DMSO-d6, 400 MHz) δ 11.59 (br.s., 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.69 (t, J = 4 0.9Hz, 1H), 7.62 (d, J = 7.8Hz, 1H), 7.13 - 7.01 (m, 2H), 6.15 (s, 1H), 4 .78 - 4.67 (m, 1H), 4.32 (d, J = 4.9Hz, 2H), 4.23 - 4.12 (m, 1H), 4.12 - 4, 02 (m, 1H), 3.84 (s, 3H), 3.82 - 3.74 (m, 1H), 2.79 - 2.66 (m, 1H), 2.58 ( s, 3H), 2.46 - 2.34 (m, 2H), 2.20 (s, 3H), 1.96 - 1.88 (m, 1H), 1.58 - 1, 46 (m, 4H), 1.15 (d, J = 6.0Hz, 6H), 0.95 - 0.89 (m, 1H), 0.74 - 0.65 (m, 1 H).
The compounds shown in the following table were prepared according to the general procedure outlined in Step 10 using the appropriate starting materials. The structures of the compounds are shown in Figure 1.





Example 2. Synthesis of (R or S)-1-(1-(1-isopropylpiperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1,2-di- hydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide (Compound 358)

[0085] A 25 ml vial was charged with a magnetic stir bar, (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-hydrochloride) yl)methyl)-2-methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide, THF (2.114 ml, 0.211 mmol), propan-2-one (0.061 g, 1.057 mmol), and sodium triacetoxyborohydride (0.224 g, 1.057 mmol). The reaction was allowed to stir at rt for 12 h. The reaction was quenched inversely in saturated aqueous NaHCO 3 , extracted with ethyl acetate and conc. in vacuo. The resulting material was treated with 10 ml of 7N ammonia in MeOH and conc in vacuo to yield material which was purified by chromatography on silica gel (10g) using DCM/MeOH/NH4OH (90:1:0.1 ) as eluent to provide 33 mg (0.065 mmol, 31.0% yield) of the title compound as a white solid. LCMS 479 (M+1)+; 1H NMR (DMSO-d6, 400 MHz)
δ 11.59 (s, 1H), 7.64 - 7.82 (m, 2H), 7.59 (d, 1H), 6.95 - 7.17 (m, 2H), 6. 15 (s, 1H), 4.32 (d, 2H), 4.04 - 4.24 (m, 1H), 3.84 □, 3H), 2.77 - 2.93 (□, 2H), 2.68 (□, 1H), 2.60 (□, 3H), 2.20 (□, 2H), 0.91 (□, 6H), 0.71 - 0.67 (□, 2H).
The compounds shown in the following table were prepared according to the general procedure outlined in this Example using the appropriate starting materials. The structures of the compounds are shown in Figure 1.





Example 3. Synthesis of (R or S)-1-(1-(1-(2-fluoroethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1 ,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide (Compound 356):

[0088] A 25 ml vial was charged with a magnetic stir bar, (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-hydrochloride) yl)methyl)-2-methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (0.062 g, 0.131 mmol), K2CO3 (0.072 g, 0.524 mmol), MeCN ( 0.655 ml, 0.131 mmol), DMF (0.262 ml, 0.131 mmol) and 1-bromo-2-fluoroethane (0.020 ml, 0.262 mmol). The reaction was capped and heated to 82 °C with stirring for 4 h. The reaction was allowed to cool to rt, filtered, and the filtrate was pre-absorbed onto silica gel (12 g). The material was purified by chromatography on SiO2 (25 g) using DCM/MeOH/Et3N (85:15:0.5) as eluent to give the title compound as an off-white solid (30 mg, 0.059 mmol, 45, 1% yield). LCMS 483 (M+1)+ ; 1H NMR (DMSO-d6, 400 MHz) δ 11.59 (s, 1H), 7.75 - 7.68 (m, 2H), 7.60 (d, 1H) 7.09 - 7.03 ( m, 2H), 6.15 (s, 1H) 4.53 - 4.51 (m, 1H), 4.42 - 4.39 (m, 1H), 4.32 (d, 2H), 4. 24 - 4.2 (m, 1H), 3.84 (s, 3H), 2.98 (br.d., 1H), 2.70 - 2.49 (m, 4H), 2.60 (s , 3H), 2.20 (s, 3H), 2.01 (dt, 1H), 1.92 - 1.90 (m, 1H), 1.75 - 1.71 (m, 1H), 1. 54 (d, 3H), 1.38 - 1.36 (m, 1H), 1.02 - 0.98 (m, 1H), 0.7 - 0.66 (br.d., 1H).
The compounds shown in the following table were prepared according to the general procedure outlined in this Example using the appropriate starting materials. The composite structures are shown in Figure 1.


Example 4. Synthesis of (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1 -(1-(pyrimidin-2-yl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (Compound 361).

To a reusable vial were added 2-chloropyrimidine (185 mg, 1.611 mmol), (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-di-hydrochloride) hydrochloride hydropyridin-3-yl)methyl)-2-methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (508 mg, 1.074 mmol), and EtOH (8 ml). To this solution was added Et3N (449 µl, 3.22 mmol). The vial was sealed and heated to 100°C overnight. The solution was allowed to cool to room temperature and concentrated in vacuo. The raw residue was
[0011] purified by chromatography on silica gel (10g) using DCM/MeOH/NH4OH (90:1:0.1) as eluent to provide 33 mg (0.065 mmol, 31.0% yield) of the title compound as a white solid. LCMS 479 (M+1)+; 1H NMR (DMSO-d6, 400 MHz)
d 11.59 (s., 1H), 7.64 - 7.82 (m, 2H), 7.59 (d, 1H), 6.95 - 7.17 (m, 2H), 6 1.15 (s, 1H), 4.32 (d, 2H), 4.04 - 4.24 (m, 1H), 3.84 (s, 3H), 2.77 - 2.93 (m, 2H ), 2.68 (d, 1H), 2.60 (s, 3H), 2.20 (s, 3H), 2.08 - 2.15 (m, 1H), 1.92 (d, 1H) , 1.83 (br.s., 1H), 1.54 (d, 3H), 1.27 - 1.43 (m, 2H), 0.91 (t, 6H), 0.71-0, 67 (m, 2H).
The compounds shown in the following table were prepared according to the general procedure outlined in this Example using the appropriate starting materials. The composite structures are shown in Figure 1.
Example 5. Synthesis of (R or S)-1-(1-(1-(2-hydroxyethyl)piperidin-4-yl)ethyl)-N-((4-methoxy-6-methyl-2-oxo-1 ,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide (Compound 347).

[0092] In a sealed tube loaded with a magnetic stir bar was added (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl )-2-methyl-1-(1-(piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (0.1 g, 0.229 mmol), DCM (3 ml) was added and the reaction cooled to 0°C. To the cooled reaction mixture was added oxirane which was condensed in the reaction flask (~1 ml). The reaction was allowed to stir at rt for 4 h and was then conc. in vacuo to give the crude material which was purified by chromatography on silica gel (12 g) using ethyl acetate/MeOH (4:1) as eluant to give the title compound as a white solid (50 mg). LCMS 481 (M + 1)+ ;1H NMR (DMSO-d6, 400 MHz) δ 11.58 (s, 1H), 7.77 - 7.65 (m, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.11 - 6.99 (m, 2H), 6.14 (s, 1H), 4.54 - 4.44 (m, 1H), 4. 31 (d, J = 5.1Hz, 3H), 4.13 (dd, J = 7.1, 10.3Hz, 1H), 3.83 (s, 3H), 3.42 (q , J = 6.0Hz, 2H), 2.93 (br.s., 1H), 2.71 - 2.56 (m, 4H), 2.31 (br.s., 2H) ), 2.19 (s, 3H), 2.03 - 1.83 (m, 2H), 1.64(br.s., 1H), 1.53 (d, J = 6.9 Hz, 3H), 1.32 (d, J = 11.1 Hz, 1H), 1.02 (d,J = 10.3 Hz, 1H), 0.65 (d, J = 11, 8Hz, 1H).
The compounds shown in the following table were prepared according to the general procedure outlined in this Example using the appropriate starting materials. The structures of the compounds are shown in Figure 1.

Example 6. Synthesis of (R or S)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-6-phenyl- 1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxamide (Compound 374).

[0094] A 25 ml reaction tube was loaded with a magnetic stir bar, phenyl boronic acid (72.6 mg, 0.596 mmol), K3PO4 (103 mg, 0.447 mmol), X-Phos pre-catalyst (Chlorine(2 -dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)) (4.92 mg, 5.96 μmol), and the bottle was sealed. The flask was evacuated/refilled with nitrogen (3x) before the addition of 6-chloro-2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole- Methyl 3-carboxylate (Compound 314) (100 mg, 0.298 mmol) as a solution in 1,4-dioxane (1 ml). The flask was then heated to 100°C overnight with stirring. The flask was then allowed to cool to room temperature and the reaction concentrated in vacuo. The crude residue was purified by chromatography on SiO2 (10g) using an eluent of ethyl acetate/hexanes (4:1) to give the title compound as a white solid (106 mg, 0.281 mmol, 94% yield) . ). LCMS 514 (M+1)+ ; 1H NMR 1H NMR (400 MHz, DMSO-d6) δ = 11.59 (s, 1H), 7.98 - 7.84 (m, 2H), 7.75 - 7.67 (m, 3H) ), 7.47 (t, J = 7.8 Hz, 2 H), 7.39 (d, J = 8.5 Hz, 1 H), 7.35 - 7.27 (m, 1 H), 6.15 (s, 1 H), 4.35 (d, J = 4.9 Hz, 2 H), 4.25 - 4.12 (m, 1 H), 3.93 (d, J = 8 .5Hz, 1H), 3.86 - 3.77 (m, 3H), 3.67 (d, J = 8.5Hz, 1H), 3.39 - 3.32 (m, 1 H), 3.10 - 3.00 (m, 1H), 2.62 (s, 3H), 2.21 (s, 3H), 1.85 (d, J = 10.0Hz, 1H), 1.63 - 1.49 (m, 4H), 1.45 - 1.33 (m, 1H), 1.20 - 0.99 (m, 1H), 0.66 ( d, J = 12.0 Hz, 1 H). Example 7. Synthesis of (R or S)-2-(4-(1-(3-((4-methoxy-6-methyl-2-oxo-) acid) 1,2-dihydropyridin-3-yl)methylcarbamoyl)-2-methyl-1H-indol-1-yl)ethyl)piperidin-1-yl)acetic (Compound 364).
To a round bottom flask was charged with a magnetic stir bar was added (R or S)-ethyl-2-(4-(1-(3-((4-methoxy-6-methyl-2-oxo-1) ,2-dihydropyridin-3-yl)methylcarbamoyl)-2-methyl-1H-indol-1-yl)ethyl)piperidin-1-yl)acetate (Compound 363) (69mg, 0.132mmol), THF (1 .5 ml), MeOH (1.5 ml), and water (0.75 ml). To this solution was added lithium hydroxide monohydrate (5.54 mg, 0.132 mmol) and the reaction stirred at room temperature for 1 h. The organics were removed under reduced pressure and the resulting aqueous solution purified by reverse phase HPLC (water/MeCN) 0^95% to give the title compound (66 mg, 0.108 mmol, 82% yield). LCMS 514 (M + 1) + 1H NMR (400 MHz, DMSO-d6) δ =11.67 (s, 1H), 9.65 (s, 1H), 7.84 - 7.68 (m, 2H), 7.63 (d, J = 7.4Hz, 1H), 7.14 - 7.03 (m, 2H), 6.18 (s, 1H), 4.33 (d, J = 3.6Hz, 2H), 4.27 -4.15 (m, 1H), 4.04 (br.s., 2H), 3.85 (s, 3H), 3. 57 (s, 1H), 3.35 - 3.23 (m, 1H), 3.14 - 2.99 (m, 1H), 2.86 - 2.74 (m, 1H), 2.62 (s, 3H), 2.21 (s, 3H), 2.18 - 2.08 (m, 1H), 1.75 (s, 1H), 1.60 - 1, 49 (m, 4H), 1.46 - 1.33 (m, 1H), 0.92 - 0.81 (m, 1H). Example 8. Synthesis of 2-methyl-6-(pyridine- (R or S)-methyl 3-yl)-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxylate. This intermediate was used as an alternative starting material in Step 7 presented in Example 1 for the synthesis of other compounds of the invention. 2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl) (R or S)-methyl -6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-3-carboxylate

To a round bottom flask were added Pd(OAc)2 (10.03 mg, 0.045 mmol), potassium acetate (219 mg, 2.233 mmol), 4,4.4',4',5.5 ,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (567 mg, 2.233 mmol), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos) (85 mg, 0.179 mmol), and the vial was sealed. To this vessel was added (R or S)-methyl 6-chloro-2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indole-3-carboxylate ( Step 6) (500 mg, 1.489 mmol) dissolved in dioxane (3.4 ml) and the reaction evacuated/supplied again with N2 (3*) before heating to 100 °C overnight. The reaction was then allowed to cool to rt and was diluted with EtOAc. The reaction was filtered through diatomaceous earth and the filtrate concentrated to provide the title compound which was used in subsequent reactions without further purification. LCMS 428 (M + 1)+.2-methyl-6-(pyridin-3-yl)-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-1H-indol-3- (R or S)-methyl carboxylate:

To a reusable vial were added K2CO3 (206 mg, 1.488 mmol), PdCl2(dppf)-CH2Cl2 adduct (60.8 mg, 0.074 mmol), and the vial was sealed. This flask was evacuated/supplied again with N2 (3X) before the addition of 2-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)ethyl)-6-(4,4,5 ,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-3-carboxylate (R or S)-methyl (318 mg, 0.744 mmol) dissolved in 1,4-dioxane (4 ml) , 3-bromopyridine (71.7 µl, 0.744 mmol), and water (400 µl). The reaction was evacuated/supplied again with N2 (3X) before heating to 100°C. The solution was cooled to room temperature and diluted with EtOAc. The solution was filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (10 g, EtOAc/hex (1:1)) to give the title compound (101 mg, 0.267 mmol, 35.9% yield). LCMS 379 (M+1)+.Example 9. Other Alkyl Carboxylate Intermediates. The following alkyl carboxylate intermediates were synthesized in a manner analogous to that shown in Step 2 of Example 1 using an appropriate starting material and reagent.

Example 10. Other Compounds of the Invention Produced from Carboxylic Acid Intermediates. The following compounds were synthesized in a manner analogous to that shown in Step 4 of Example 1 using an appropriate starting material. The structures of these compounds are shown in Figure 1.






Example 11. Synthesis of methyl 1-(1-(1,4-dioxan-2-yl)ethyl)-2-methyl-1H-indole-3-carboxylate. The title compound was used as an alternative alkyl carboxylate starting material in Step 3 of Example 1. Step 1: 1-(1,4-dioxan-2-yl)ethanone:

To a solution of benzoic peroxide (20 g, 141 mmol) in 200 ml of 1,4-dioxane at room temperature under nitrogen atmosphere was added biacetyl (24.3 g, 282 mmol). After addition, the mixture was heated to reflux and stirred for 24 hours. The reaction mixture was cooled to 0 °C. The pH was adjusted to around 9 by progressive addition of 2N sodium hydroxide below 0 °C, extracted with 2-methoxy-2-methylpropane (10 ml x 3), and concentrated to provide 1-(1,4 -dioxan-2-yl)ethanone (13 g, 36%) as a yellow oil which was used directly in the next step without purification. Step 2: 1-(1,4-dioxan-2-yl)ethanamine:

To a solution of 1-(1,4-dioxan-2-yl)ethanone (12g, 92.2mmol) in 1,2-dichloroethane (100ml) was added (4-methoxyphenyl)methanamine (25g , 184.4 mmol) at room temperature. The mixture was allowed to stir for 3 hours, then sodium triacetoxyborohydride (39 g, 184.4 mmol) was added. The resulting mixture was allowed to stir for 48 hours at room temperature. The reaction mixture was quenched by the addition of water, extracted with dichloromethane (100 ml x 3). The combined organic phase was dried over anhydrous sodium sulfate, then filtered. The filtrate was concentrated and purified by column chromatography on silica gel (eluate: dichloromethane/methanol 100:1 50:1 ^20:1) to give 1-(1,4-dioxan-2-yl)-N -(4-methoxybenzyl)ethanamine (16.4 g, 71%) as a yellow solid. LCMS (M + H+) m/z: calc. 251.15, observed 251.9. To a solution of 1-(1,4-dioxan-2-yl)-N-(4-methoxybenzyl)ethanamine (5 g, 19.9 mmol) in anhydrous methanol (100 ml) was added 10% palladium on carbon ( 240 mg, 2 mmol), then purged with hydrogen (30 psi), the mixture was allowed to stir overnight at room temperature. The reaction mixture was filtered, and the filtrate was concentrated to provide the title compound (2.5 g, 96%) as a brown solid.
The amine intermediates shown in the following table were prepared according to the general procedure outlined above using the appropriate starting materials and modifications.

Step 3: (E)-methyl 3-((1-(1,4-dioxan-2-yl)ethyl)imino)-2-(2-bromophenyl)butanoate:

To a solution of 1-(1,4-dioxan-2-yl)ethanamine (2.5 g, 19 mmol) in methanol (100 ml) was added 2-(2-bromophenyl)-3-oxobutanoate. methyl (5.4 g, 20 mmol) and acetic acid (1.8 g, 30 mmol). The resulting reaction system was heated to reflux and allowed to stir overnight. The reaction mixture was concentrated and purified by column chromatography on silica gel (eluted: dichloromethane/methanol 50:1^20:1^5:1) to give the title compound (1 g, 14%) as a solid. Brown. LCMS (M + H+) m/z: calc. 383.07, observed 384.9.
[00101] The imino-bromo intermediates shown in the following table were prepared according to the general procedure outlined above using the appropriate starting materials (eg one of the amines shown in the table in Step 2 of this example) and modifications.

Step 4: Methyl 1-(1-(1,4-dioxan-2-yl)ethyl)-2-methyl-1H-indole-3-carboxylate:

[00102] To a solution of (E)-methyl 3-((1-(1,4-dioxan-2-yl)ethyl)imino)-2-(2-bromophenyl)butanoate (400mg, 1.1 mmol) in dioxane (3 ml) were added Chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropylbiphenyl][2-(2-aminoethyl)phenyl]Pd(π) ( 160 mg, 0.2 mmol), 2-Dicyclohexyphosphine-2',6'-diisopropoxybiphenyl (93 mg, 0.2 mmol) and sodium tert-butoxide (192 mg, 2 mmol). The resulting reaction mixture was heated to 120 °C with stirring for 30 min in a microwave oven. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate (25 ml x 3). The combined organic phase was dried over anhydrous sodium sulfate, then filtered. The filtrate was concentrated and purified by column chromatography on silica gel (eluted: petroleum ether / acetic ester 10:1^5:1^2:1) to give the title compound (282 mg, 89%) as a solid. yellow. LCMS (M + H+) m/z: calc. 303.15, observed 303.9.
[00103] The compound shown in the following table was prepared according to the general procedure outlined above using the appropriate starting materials (eg one of the imino-bromo intermediates shown in the table in Step 3 of this example) and modifications.

These alkyl carboxylates were also used as starting material in Step 3 of Example 1 in the synthesis of certain compounds of the invention. Example 12. Chiral Separation of Compound 219 to provide Compounds 223 and 224. N-((4- methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(1-methoxypropan-2-yl)-2-methyl-1H-indole-3-carboxamide (200 mg) (Compound 219) was subjected to chiral chromatography by means of supercritical fluid chromatography (SFC) (A:C2H6OH,B:NHS^H2O. A:B = 55:45 column AD) to provide the separated enantiomers 223 (peak 1) and 224 (Peak 2) (60 mg each) LCMS 398 (M + 1) + 1H NMR (400 MHz, CD3OD) δ 7.69 (d, J=7.2 Hz, 1H), 7.53 (d, J = 7.6Hz, 1H), 7.12 (m, 2H), 6.26 (s,1H), 4.80 (m,1H), 4.52 (s,2H), 3 .99 (m, 4H), 3.75 (m, 1H), 3.20 (s, 3H), 2.62 (s, 3H), 2.31 (s, 3H), 1.59 (d, J = 7.2 Hz, 3H). The optical rotation of each enantiomer has not been determined.
[00105] The compounds shown in the following table were prepared according to the general chiral chromatography procedure outlined above. The optical rotation of the separated enantiomers has not been determined, but the elution peak (“Peak 1” or “Peak 2”) is indicated. The structures of each compound are shown in Figure 1.


Example 1. Synthesis of tert-butyl 1-(2,3-dihydro-1H-inden-1-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate. The title compound as starting material in Step 3 of Example 36 in the synthesis of certain compounds of the invention. tert-butyl 2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate:

[00106] To a 500 ml round bottom flask containing N-acetyl-N-(3-bromopyridin-2-yl)acetamide (14'815 g'57'6 mmol) was added copper(I) iodide ( 1'098 g'5'76mmol)' L-proline (1'327 g'11'53mmol)' cesium carbonate (28'2 g' 86 mmol)' then t-butyl acetoacetate (11'47 ml 69/2 mmol) and dioxane (100 ml). The reaction was vac/purged with 3X N2 then fitted with a septum and N2 inlet and heated overnight at 70°C. Inorganic solids were removed by filtration through celite and the cake was washed with 100 ml of EtOAc. This solution was concentrated and the residue was partitioned between 250 ml of brine and 250 ml of EtOAc. The aq layer. was further extracted with EtOAc (2 x 250 ml) and the combined organic layer was dried by Na2SO4'filtered' concentrated and purified by bi CC using 1:1 EtOAc:Hex as eluent to give (2'7 g'20'2 %) of tert-butyl 2-methyl-1H-pyrrolo[2'3-b]pyridine-3-carboxylate. LRMS (M + H+) m/z: calc. 233'28; found 233'1.1-(2,3-dihydro-1H-inden-1-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate tert-butyl:

[00107] A solution of tert-butyl ethyl 2-methyl-1H-pyrrolo[2'3-b]pyridine-3-carboxylate (100 mg' 0'74 mmol)' 2'3-dihydro-1H- inden-1-ol (176 mg, 0.74 mmol), PPh3 (195 mg, 1.49 mmol) was stirred in dry THF (10 ml) at 0 °C under a nitrogen atmosphere. To this mixture was added DIAD dropwise (150 mg, 1.48 mmol) over a period of 5 min, and the reaction was stirred at room temperature for 16 hours. The mixture was washed with brine, dried and concentrated to provide the crude product. The crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate = 5:1) to provide tert-butyl-1-(2,3-dihydro-1H-inden-1-yl) - 2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (150 mg, 60%).
[00108] The compound shown in the following table was prepared according to the general procedure outlined above using the appropriate starting materials and modifications.

[00109] Each of the above alkyl carboxylates was used as starting material in Step 3 of Example 1 in the synthesis of certain compounds of the invention. Example 13. Synthesis of isolated N-((4-methoxy-6-methyl-) diastereomers 2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R or 2S, 3R or 3S)-3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2 ,3-b]pyridine-3-carboxamide (Compounds 261, 266, 267 and 302). Step 1: 2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b] tert-butyl pyridine-3-carboxylate:

[00110] To a solution of tert-butyl 2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (5.0 g, 21.53 mmol) in CH3CN (50 ml) was added Cs2CO3 (21.0g, 64.58mmol), potassium iodide (3.57g, 21.53mmol). The mixture was stirred at 27°C for 30 minutes. Then 3-chlorobutan-2-one (2.75 g, 25.83 mmol) was added and the mixture was stirred at 70°C for 12 hours. The mixture was filtered and the filtrate was concentrated. The residue was purified by column (Eluate: Petroleum ether: Ethyl acetate = 50:1) to give 2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b] tert-butyl pyridine-3-carboxylate as a yellow-green oil. (3.23 g, 50% yield) LCMS (M + H+) m/z: calc 303.37; observed 302.9. 1H NMR (400 MHz, CDCh): at 8.32 - 8.30 (m, 1H), 8.25 - 8.23 (m, 1H), 7.17 - 7.14 (m, 1H), 5 .50 - 5.44 (m, 1H), 2.71 (s, 3H), 1.96 (s, 3H), 1.65 - 1.67 (d, 3H), 1.64 (s, 9H ).Step 2: 1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate tert-butyl:

[00111] To tert-butyl 2-methyl-1-(3-oxobutan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylate solution (3.1 g, 10.25 mmol) in methanol (30 ml) was added sodium borohydride (0.30 g, 8.2 mmol) at 0 °C. After 30 minutes, another batch of sodium borohydride (0.30 g, 8.2 mmol) was added at 0 °C. After the reaction was completed around 2 h later, water (30 ml) was added dropwise very carefully to quench the reaction. The mixture was extracted with CH2Cl2. The extraction was dried over Na2SO4, filtered and concentrated in vacuo to give tert-butyl 1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate as a yellow solid. (3.0 g, iield 96%) LCMS (M + H+) m/z: calc 305.38; observed 304.9. 1H NMR (400 MHz, CDCl3): at 8.31 - 8.29 (m, 1H), 8.13 - 8.12 (m, 1H), 7.11 - 7.07 (m, 1H), 4 .46 - 4.43 (m, 1H), 4.12 (m, 1H), 2.73 (s, 3H), 1.58 (s, 9H), 1.51 - 1.49 (d, 3H ), 0.92 - 0.91 (d, 3H). Step 3: Tert-butyl-1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine- 3-carboxylate:

[00112] To dry THF (20 ml) was added NaH (60% in mineral oil, 2.37 g, 59.14 mmol). Then the mixture was stirred at 27°C for 20 minutes, then tert-butyl 1-(3-hydroxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (3 .0 g, 9.86 mmol) was added. The mixture was stirred at 27°C for 1 hour, then added in CH 3 I (13.99 g, 98.6 mmol). The mixture was stirred for 12 hours at 27°C and then cooled to 0°C. NH4CI sat. was added and extracted with CH2Cl2. The extraction was dried over sodium sulfate, filtered and concentrated to give tert-butyl-1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate as a yellow oil. (3.2 g, 100% yield) LCMS (M + H+) m/z: calc. 319.41; observed 318.9. Step 4: 1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid:

To pre-cooled solution of 1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylate of tert-butyl (3.0 g, 9.42 mmol) in CH 2 Cl 2 (20 ml) was added trifluoroacetic acid (20 ml) dropwise. The solution was stirred at 27°C for 1.5 hours. The solvent was removed under vacuum at 27°C. The residue was used for the next step without being purified. LCMS (M + H+) m/z: calc 263.30; found 262.9. Step 5: N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3-methoxybutan-2-yl) -2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide:

[00114] To a solution of 1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (2.4 g, 9.15 mmol) in DMF (30 ml) was added TEA (4.2 g, 41.50 mmol), 3-(aminomethyl)-4-methoxy-6-methylpyridin-2(1H)-one hydrochloride (2.1 g, 12 .81 mmol). After stirring for 10 minutes at 27°C, the mixture was cooled and HATU added (5.56 g, 14.64 mmol). The mixture was stirred at 27°C for 72 hours and 30% SM remained. Then the mixture was heated to 80°C for 5 hours. The solution was diluted with brine (100 ml) and extracted with CH2Cl2 (100 ml*3). The extractions were combined and dried over Na2SO4. The solvent was evaporated under vacuum and the residue was purified by flash column (Eluent: dichloromethane: methanol = 95:5) to give N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridine) -3-yl)methyl)-1-(3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide. (3.6g, 95% yield). Step 6: Separation of N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-( 3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide: Isomers (Compounds 261, 266, 267, and 302):

[00115] Mixture of isomers from Step 5, N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-(3-methoxybutan-2- il)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide, was purified by prep-HPLC (Condition: Column: SHIMADZU LC-8A, 250*50mm *10um; Mobile phase A: water with 0.2% formic acid; Mobile phase B: MeCN; Column temperature: 30°C; Gradient: B in A 10~50%) to provide a major isomer pair (Compound 261 and Compound 266 combined) (1.0 g, 98.8%) purity) and a minor isomer pair (Compound 267 and Compound 302 combined) (180 mg, 63 % purity). The resulting isomer pairs were individually separated by SFC (Condition: Column: Chiralpak AD 250*30 mm *5 um; Mobile phase A: Supercritical CO2; Mobile phase B: IPA+NHa^O; Gradient: B/A: 75: 25) to provide the following individual isolated compounds:
[00116] Compound 261, N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R or 2S, 3R or 3S)- 3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (Main Isomer Pair; Peak 1): 1H NMR (400 MHz, CDCl3): δ 8.173 - 8.157 (m, 1H), 8.140 - 8.116 (m, 1H), 7.582 - 7.555 (m, 1H), 6.968 - 6.936 (m, 1H), 5.927 (s, 1H), 4.707 - 4.609 (m, 2H), 4.348 (s, 1H), 3.892 (s, 3H), 2.869 (s, 3H), 2.788 (s, 3H), 2.173 (s, 3H), 1.644 - 1.627 (d, 3H), 1.263 - 1.249 (d, 3H).
[00117] Compound 266, N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R or 2S, 3R or 3S)- 3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (Main Isomer Pair; Peak 2): 1H NMR (400 MHz, CDCl3): δ 8.179 - 8.163 (m, 1H), 8.143 - 8.120 (m, 1H), 7.558 - 7.531 (m, 1H), 6.986 - 6.954 (m, 1H), 5.931 (s, 1H), 4.702 - 4.605 (m, 2H), 3.897 (s, 3H), 2.892 (s, 3H), 2.789 (s, 3H), 2.189 (s, 3H), 1.647 - 1.629 (d, 3H), 1.267 - 1.252 (d, 3H).
[00118] Compound 267, N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R or 2S, 3R or 3S)- 3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (Secondary Isomer Pair; Peak 1): 1H NMR (400 MHz, CDCl3): δ 8.174 - 8.162 (d, 1H), 8.111 - 8.094 (d, 1H), 7.551 - 7.526 (m, 1H), 6.993 - 6.961 (m, 1H), 5.935 (s, 1H), 4.683 - 4.579 (m, 2H), 3.887 (s, 3H), 3.442 (s, 3H), 2.753 (s, 3H), 2.194 (s, 3H), 1.695 - 1.678 (d, 3H), 0.781 - 0.768 (d, 3H).
[00119] Compound 302, N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-1-((2R or 2S, 3R or 3S)- 3-methoxybutan-2-yl)-2-methyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (Secondary Isomer Pair; Peak 2): 1H NMR (400 MHz, CDCl3): δ 8.177 - 8.166 (d, 1H), 8.122 - 8.104 (d, 1H), 7.587 - 7.562 (m, 1H), 6.984 - 6.952 (m, 1H), 5.933 (s, 1H), 4.698 - 4.591 (m, 2H), 4.426 (s, 2H), 3.983 (s, 3H), 3.448 (s, 3H), 2.764 (s, 3H), 2.180 (s, 3H), 1.701 - 1.684 (d, 3H), 0.786 - 0.772 (d, 3H). Example 14. Synthesis of (±)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-phenylethyl)- 1H-pyrrolo[2,3-c]pyridine-3-carboxamideStep 1: 1-(3-methoxyphenyl)ethanol:

To a stirred solution of 3-Amino-4-picoline (7 g, 64.8 mmol) in anhydrous THF (200 ml), sec-BuLi (150 ml, 1.3M in cyclohexane, 194 mmol) was added by dropping over 20 minutes at -78°C. The solution was warmed to room temperature and stirred for 3 hours. Ethyl acetate (2.3 g, 25.9 mmol) was added dropwise into the reaction at -78°C and the mixture was stirred at the same temperature for 2 hours. Methanol (50 ml) was added dropwise into the reaction over 10 minutes. The mixture was warmed to room temperature and stirred for 1 hour. A half-saturated NH4Cl (250 ml) was added. The mixture was extracted with EA. The combined organic layers were washed with brine, dried and concentrated to provide the crude product. The crude product was purified by silica gel bichromatography (petroleum ether / ethyl acetate = 10:1) to give 2-methyl-1H-pyrrolo[2,3-c]pyridine (2.5 g, 73.5 %). Step 2: 2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone:

[00121] To a stirred solution of 2-methyl-1H-pyrrolo[2,3-c]pyridine (2.5 g, 18.9 mmol) and aluminum chloride (5 g, 37.8 mmol) in DCM ( 100 ml), trichloroacetyl chloride (4.1 g, 22.7 mmol) was added dropwise in a reaction over 0.5 hour at room temperature. After stirring for 2 hours, the reaction was cooled to 0°C and quenched with water (100 ml). The resulting precipitate was isolated by filtration to provide 2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone which was used for the next step without further purification. 100% yield is assumed. (5.24 g). Step 3: Methyl 2-methyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate:

[00122] A mixture of 2,2,2-trichloro-1-(2-methyl-1H-pyrrolo[2,3-c]pyridin-3-yl)ethanone (5.24 g, 18.9 mmol) and KOH (1.2 g, 20.9 mmol) in MeOH (100 ml) was stirred at room temperature for 16 hours. The reaction mixture was concentrated to remove MeOH, the residue was partitioned between EA and Water. The organic layer was washed with brine, dried and concentrated to provide methyl 2-methyl-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (3 g, 83%). Step 4: 2-methyl-1 -(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate:

Methyl carboxylate (550 mg, 2.89 mmol) and sodium hydride (200 mg, 4.34 mmol) in N,N-dimethylformamide (3.0 ml) was stirred at room temperature for 0.5 hour , and then (1-bromoethyl)benzene (589 mg, 3.18 mmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was poured into saturated NH4Cl and extracted with ethyl acetate. The organic layers were combined and concentrated to provide a residue. The residue was purified by chromatography (petroleum ether / ethyl acetate = 5:1) to give methyl 2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (800 mg, 94%). Step 5: 2-Methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylic acid:

[00124] To a mixture of methyl 2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylate (800 mg, 2.72 mmol) and KOH (1, 5 g, 27.2 mmol) in (15 ml) and water (5 ml) was refluxed for 2 hours. The mixture was adjusted to pH 2 by 10% HCl and extracted with EA. The combined organic layers were washed with brine, dried and concentrated to provide the crude product. The crude product was used in the next step without further purification. 100% yield. (760 mg). Step 6: (±)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1- phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxamide (Compound 203):

[00125] A mixture of 2-methyl-1-(1-phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxylic acid (280 mg, 1.0 mmol) was added to HATU (456 mg, 1.2 mmol), TEA (1 g, 10 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (182 mg, 1.2 mmol) in anhydrous dichloromethane (30 ml), was stirred at room temperature for 16 hours. To the reaction mixture, water (10 ml) was added, extracted with dichloromethane (30 ml x 2). The organic layers were combined and concentrated to provide a residue. The residue was recrystallized from MeCN to give the compound N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1- phenylethyl)-1H-pyrrolo[2,3-c]pyridine-3-carboxamide as an off-white solid (80mg, 21.6%). LRMS (M + H+) m/z: calc 414.21; found 414. 1H NMR (400 MHz, Methanol-d4) δ: 8.84 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 8.03 (d, J = 6, 8Hz, 1H), 7.44-7.37 (m, 5H), 6.09 (s, 1H), 6.01-5.99 (m, 1H), 4.49 (s, 2H), 2.73 (s, 3H), 2.38 (s, 3H), 2.22 (s, 3H), 2.06 (d, J = 7.2 Hz, 3H).
[00126] The compounds shown in the following table were prepared according to the general procedure outlined in this example using the appropriate starting materials and modifications. The structures are shown in Figure 1.
Example 15. General Procedures for Synthesizing Other Compounds of the Invention General Procedure A: Indol Alkylation

[00127] To a cooled (0 oC) solution of NH indole ester (1 equivalent) in N,N-dimethylformamide (volume to constitute 0.4M concentration) was added sodium hydride (60% w/w, 1 ,1 equivalent with respect to indole). The resulting mixture was stirred for 15 minutes. Then RX (2 equivalents) was added and the reaction was allowed to warm to room temperature. The reaction was kept at room temperature for 12 hours. The reaction mixture was poured into saturated ammonium chloride solution (100 ml) with stirring. The mixture was extracted with ethyl acetate (200 ml x 2) and the combined organic phase was washed with brine, dried over magnesium sulfate, filtered and concentrated to give the crude product which was purified by column chromatography (silica gel, ether of petroleum/ethyl acetate = 20:1) to provide the desired alkylated indole ester product. General Procedure B: Alkylated indole ester saponification

[00128] To a solution of alkylated indole ester (1 equivalent) in tetrahydrofuran:methanol:water (2.5:5:1, volume to make concentration 0.05M) was added lithium hydroxide (4 equivalents). The resulting reaction mixture was stirred at 60°C for 48 hours. The mixture was concentrated in vacuo. Then the residue was diluted with water (40 ml) and slowly acidified with 1N hydrogen chloride to pH = 4 to 5. The mixture was extracted with ethyl acetate (100 ml x 3). The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated to provide the crude indole acid, which was used in the next step without further purification. General Procedure C: Amide Bond Formation

[00129] To a solution of indole acid (1 equivalent) in dichloromethane (volume to constitute concentration 0.05 M) were added 1-hydroxybenzotriazole (1.5 equivalent), 1-(3-dimethylaminopropyl)-hydrochloride. 3-ethylcarbodiimide (1.5 equiv.) and triethylamine (3 equiv.). The resulting mixture was stirred at room temperature for 30 minutes. Then pyridone amine (1.2 equiv.) was added and the resulting mixture was stirred at room temperature for 16 hours. Water (50 ml) was added to the mixture. The mixture was extracted with dichloromethane (100 ml x 2). The organic layer was concentrated in vacuo to furnish the crude product which was purified by column chromatography (silica gel, dichloromethane / methanol = 20:1) to furnish the target compound. General Procedure D: Chiral chromatography
[00130] The separation of the chiral compounds was performed by means of HPLC or normal phase SFC (supercritical carbon dioxide fluid chromatography). Separated compounds were typically >95% ee. The absolute configuration of the chiral centers has not been determined. General Procedure L: Sulfonylation

To a solution of chiral amine (1 equiv.) in dichloromethane (volume to make 0.1 M concentration) was added triethylamine (4 equiv.) at 18°C under N2. The reaction was cooled to 0°C and methanesulfonyl chloride (1.5 equiv.) was added. The reaction was stirred at 0 °C for 1 h. Then the mixture was concentrated in vacuo and methanol and potassium carbonate were added and the reaction was stirred for a further 1 h. The mixture was filtered and the crude product was purified by preparative HPLC.
[00132] The table below lists the compounds of the invention and the above general methods that were used in their synthesis. The structures of these compounds are shown in Figure 1.
Example 16. IC50 measurements for Inhibitors using EZH2.
[00133] EZH2 Assay: Assays were performed by mixing rPRC2 together with biotinylated oligonucleosome substrates in the presence of the radiolabeled enzyme cofactor, S-adenosyl-L-methionine (3H SAM) (Perkin Elmer) and monitoring the enzymatically mediated transfer of tritiated methyl groups from 3 H SAM to histone lysine residues. The amount of resulting tritiated methyl histone product was measured by first capturing the biotinylated oligonucleosomes in streptavidin (SAV) coated on FlashPlates (Perkin Elmer), followed by a wash step to remove unreacted 3H SAM, and then counting on a counter of TopCount NXT 384 well plate scintillation (Perkin Elmer). The final assay conditions for EZH2 were as follows: 50 mM Tris buffer pH 8.5, 1 mM DTT, 69 µM Brij-35 detergent, 5.0 mM MgCl2, 0.1 mg/ml BSA, 0.2 µM 3H SAM, 0.2 µM biotinylated moligonucleosomes, 3.6 µM peptide H3K27me3 and 2 nM EZH2.
Compound IC50 measurements were obtained as follows: Compounds were first dissolved in 100% DMSO as 10 mM stock solutions. Ten point dose response curves were generated by distributing varying amounts of the 10 mM compound solution into 10 wells of the 384 well plate (Echo; Labcyte), pure DMSO was then used to backfill the wells to ensure that all reservoirs had the same amount of DMSO. A 12.5 µl volume of HMT enzyme, H3K27me3 peptide and oligonucleosome substrate in assay buffer was added to each well of the assay plate using a Multidrop Combi (ThermoFisher). Compounds were pre-incubated with the enzyme for 20 min, followed by initiation of the methyltransferase reaction by adding 12.5 μl of 3H SAM in assay buffer (final volume = 25 μl). The final concentrations of compounds ranged from a standard concentration above 80 µM to 0.16 µM in ten steps of 2-fold dilution. Reactions were carried out for 60 minutes and quenched with 20 µl per reservoir of 1.96 mM SAH, 50 mM Tris pH 8.5, 200 mM EDTA. Stopped reactions were transferred to SAV-coated Flashplates (Perkin Elmer), incubated for 120 minutes, washed with a plate washer, and then read into TopCount NXT (1.0 min/well) to measure the amount of methyl histone product formed during the reaction. The amount of methyl histone product was compared to the amount of product formed in the 0% and 100% inhibition control reservoirs which allow calculation of % inhibition in the presence of the individual compounds at various concentrations. The IC50s were calculated using a 4-parameter adapted nonlinear curve fitting software package (XLFIT, part of the database package, ActivityBase (IDBS)), where the four parameters were the IC50. The ascent slope, pre-transition baseline (0 % INH), and post-transition reference value (100 % INH); with the last two parameters being set to zero and 100%, respectively, by default.
Assay for Y641N EZH2 was performed as above using reconstituted H3K27Me2 oligonucleosomes as substrate.
[00136] Table 2 shows the activity of selected compounds of the present invention in the EZH2 and Y641N EZH2 activity inhibition assay. IC50 values are reported as follows: "A" indicates an IC50 value of less than 100 nM; "B" indicates an IC50 value of 100 nM to 1 µM; "C" indicates an IC50 value greater than 1 μM and less than 10 μM for each enzyme; "D" indicates an IC50 value of more than 10 µM for each enzyme; and “*(X µM)” indicates that no inhibition was observed at the highest concentration (ie, X pM) of compound tested.Table 2. IC50 values for compounds of Formula I against mutant enzymes EZH2 and Y641N EZH2.



Example 17. EC50 measurements for inhibitors in HeLa cell assays.
[00137] H3K27me3 MSD Hela Assay. Trypsin-treated HeLa cells were counted and diluted in 10% DMEM (Life Technologies, Cat. #10569) to 5000 cells/75 µl. Seventy-five µl of cells were placed in each well of a 96-well flat-bottom plate and incubated at 37°C for 4 hours. Twenty-five µl of test compound (at various concentrations) was added to the cells and incubation continued at 37°C for 96 hours. The medium was then removed and the cells rinsed once with ice-cold PBS. Forty µl ice-cold MSD AT buffer (10 mM HEPES, pH 7.9, 5 mM MgCl 2 , 0.25 M sucrose, Benzonase (1:10000), 1% Triton X100 supplemented with fresh 1x and 1 mM protease inhibitor cocktail of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)) were added to each well and the plates placed on ice for 30 minutes. Ten µl of 5 M NaCl was then added to each well and incubation on ice continued for a further 15 minutes. The material in each well was suspended by pipetting up and down and then transferred to a new 96-well plate. Empty wells were rinsed with 150 µl ice-cold 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, supplemented with fresh cocktail of 1x protease inhibitor and 1 mM AEBSF ("NO salt NO detergent buffer) and transferred to the respective wells on the new plate.Three hundred μl of NO salt NO detergent buffer was then added to each lysate well and the plates frozen at -80°C.
[00138] On the same day, an appropriate number of standard MSD binding 96 well plates were coated with 30 µl/well of total H3 capture antibody (Millipore, Cat # MAB3422) at a concentration of 1 µg/ml in PBS. The antibody solution was evenly distributed by first gently tapping the sides of the plates and then shaking the plates for a few minutes at 1000 rpm. Antibody coated plates were stored at 4°C overnight.
[00139] The next day the lysates are thawed at room temperature. Antibody coated MSD plates are washed 3X with TBS-T (Tris-buffered saline (Fisher Scientific, Cat # BP2471-1) + 0.2% Tween-20). One hundred and fifty μl of 5% Blocker A in TBS-T is added to each well. Reservoirs are covered and shaken on a shaker at RT for one hour. Blocker A step is repeated a second time. After removing the blockers, 25 µl of cell lysate is transferred into each antibody coated well. Plates are shaken for 2 hours at RT, lysate removed and plates again washed with Blocker A in TBS-T. Twenty-five μl of freshly prepared appropriate antibody mix (including both primary and secondary antibodies) is added to each well and the plates shaken for one hour at RT. The mixture of antibodies used was one (or both) of those indicated in the table below:

[00140] Both H3 antibodies were obtained from Cell Signaling (Cat #s 4499 and 9733). Goat anti-rabbit antibody was obtained from Meso-Scale Discovery (Cat #R32AB-1).
The antibody mixture was then removed and the wells washed with Blocker A. One hundred and fifty µl of freshly prepared 1X MSD Reading Buffer (Meso-Scale Discovery; Cat # R927C-2) was then added to each well and the plates read on a plate reader on an MSD Sector 2400 Plate Reader.
[00142] Data were analyzed using the Assay Assistant model (Constellation Pharmaceuticals In-house product) and Activity Base (IDBS Ltd, Surrey, UK). Data files were imported from Assay Assistant and assay conditions were specified. A single Analysis ID was created and the data files exported to Activity Base. An analysis model was created in Activity Base to measure dose-dependent inhibition of the H3K27me3 tag and cell viability, respectively. The reading of the DMSO reservoirs was used to normalize the data. The resulting curves were fitted using Activity Base Model 205 software (IDBS Ltd, Surrey, UK). Data were quality checked, validated and integrated in excel format using SARview (IDBS Ltd, Surrey, UK).
[00143] H3K27me3 Alpha Hela Assay (AlphaLISA). Ten different doses of each test compound (in a 3-fold dilution series) were plated onto tissue culture treated plates from 384 duplicate wells (Catalog # 781080; Greiner Bio One; Monroe, North Carolina). HeLa cells grown in the culture were trypsinized and counted using a Countess® cell counter (Catalog # C10281; Life Technologies, Grand Island, NY). Cells were diluted to 67,000 cells per ml in 10% DMEM (Catalog # 10569-010 Life Technologies, Grand Island, NY) and 15 µl (1000 cells) were plated into each well using the Biotek MicroFloTM Select Dispenser (BioTek Instruments, Inc. Vermont, USA), of the 384 reservoir plate. Plates were incubated at 37°C/5% CO 2 for 72 hours. One of the duplicate plates was processed by the HeLa assay and the other for viability.
[00144] To the plate processed by AlphaLISA was added 5 µl per well of Cell-Histone Lysis (1X) buffer (Catalog # AL009F1 Perkin Elmer; Waltham, MA) and the plate was incubated at RT for 30 minutes on a plate shaker with low speed (Model# 4625-Q Thermo Scientific; Waltham, MA). Then, 10 µl per well of Histone Extraction buffer (catalog # AL009F2; Perkin Elmer; Waltham, MA) was added and the plate further incubated at RT for 20 min on a low speed plate shaker. 10 μl per well of a 5X mixture of anti-K27me3 acceptor globules plus anti-Histone antibody treated with Biotin H3 (C-ter) (diluted in 3 nM final) was then added to each well (Catalog #AL118 Perkin Elmer; Waltham , MA). Dilution of acceptor and then anti-Histone H3 cells was with 1X Histone Detection Buffer (Catalog # AL009F3 Perkin Elmer; Waltham, MA) which was produced diluted from the 10X stock provided. The plate was sealed with an aluminum plate sealer and incubated at 23°C for 60 min. Then 10 μl of 5X Streptavidin Donor bead solution (Catalog #6760002 Perkin Elmer; Waltham, MA) (20 μg/mL final with 1X Histone Detection Buffer) was added to the plate sealed with aluminum plate sealer and incubated at 23°C. 0°C for 30 min. Plates were then read using an EnVision-Alpha Reader (model #2104 Perkin Elmer; Waltham, MA).
[00145] Cell viability was tested by adding 15 µl of Cell Titer Glo ((Catalog #G7571 Promega Madison, WI) to each cell well with media. Plates were incubated at RT for 15 to 20 minutes on a shaker plates were then read using an EnVision-Alpha Reader (model #2104 Perkin Elmer; Waltham, MA).
Data from both trials were analyzed using the standard Assay Assistant (Constellation Pharmaceuticals In-house product) and Activity Base (IDBS Ltd, Surrey, UK). Data files were imported from Assay Assistant and assay conditions were specified. The unique Analysis ID was created and the data files exported from the Activity Base. An analysis model was created in Activity Base to measure dose-dependent inhibition of the H3K27me3 tag and cell viability, respectively. The complete reading of the DMSO reservoirs was used to normalize the data. The resulting curves were fitted using Activity Base Model 205 software (IDBS Ltd, Surrey, UK). Data were quality checked, validated and integrated in excel format using SARview (IDBS Ltd, Surrey, UK).
[00147] Table 3 shows the activity of selected compounds of this invention in the two different HeLa cell assays described above. EC50 values are reported as follows: "A" indicates an EC50 value of less than 400 nM; "B" indicates an EC50 value of 400 nM to 2 µM; "C" indicates an EC50 value greater than 2 μM and less than 10 μM for each enzyme; "D" indicates an EC50 value greater than 10 µM for each enzyme; and “*(X µM)” indicates that no inhibition was observed at the highest concentration (ie, X µM) of the compound tested. Table 3. EC50 Values for Selected Compounds of the Invention in HeLa Cells Expressing the H3k27 EZH2 Mutant .


Example 18. Tumor Growth Inhibition Assay
[00148] The antitumor efficacy of Compound 362 and 365 in the subcutaneous Karpas422 human lymphoma xenograft model in female CB-17 SCID mice was as follows. 18 to 22 gNumber of animals: 50 mice plus reservesAnimal supplier: Shanghai SLAC Laboratory Animal Co., LTD.Cell Culture
[00149] Karpas422 tumor cells were maintained in vitro as suspension culture in RPMI1640 medium supplemented with heat-inactive fetal calf serum at 37 °C in an atmosphere of 5% CO2 with air. Tumor cells were routinely subcultured twice a week. Cells that developed in an exponential growth phase were harvested and counted for tumor inoculation. Tumor inoculation
[00150] Each mouse was inoculated subcutaneously on the right flank with the Karpas422 tumor cells (5 x 106) in 0.2 ml PBS with Matrigel (1:1) for tumor development. 23 days after tumor inoculation was like day 0 after the start of treatment, when the mean tumor size reached approximately 300 mm3. Each group consisted of 10 tumor-bearing mice. Tumor Measurements
[00151] Tumor size was measured three times a week in two dimensions using a caliper, and volume was expressed in mm3 using the formula: V = 0.536 ax b2, where a and b are the long and short diameters of the tumor, respectively. Tumor size was then used for calculations of T/C values. The T/C value (in percent) is an indication of antitumor efficacy; T and C are the mean volumes of the treated and control groups, respectively, on a given day.
[00152] The TGI was calculated for each group using the formula: TGI (%) = [1-(Ti-T0)/ (Vi-V0)] x100; Ti is the mean tumor volume of a treatment group on a given day, T0 is the mean tumor volume of the treatment group on the day of start of treatment, Vi is the mean tumor volume of the vehicle control group on the same day. day with Ti, and V0 is the mean tumor volume of the vehicle group on the day of initiation of treatment. Experimental End Stage and Sample Collection1) Plasma, tumor and muscle in the ZPE-6438 group were collected on day 16 after the start of treatment within 6 h after dosing. Plasma, tumor and muscle in vehicle, groups CPI-524369, CPI-524416 and CPI591780 were collected on day 25 after treatment initiation at 1 h after dosing. 2) All blood was taken from each animal with EDTA-K2 as anticoagulant. The plasma was divided into two parts. The first part was for PK analysis; the second part has been frozen for backup. 3) The tumor was divided into three parts. The first part was instantly frozen for PK; the second part was snap frozen for PD analysis; the third part has been frozen for backup. 4) The muscle was divided into two parts. The first part was instantly frozen for PK; the second part was frozen for backup. Tumor Growth Inhibition Analysis Table 4. Tumor growth inhibition calculation for Compounds 362 and 365 in Karpass422 xenograft model calculated based on day 25 or day tumor volume measurements day 16 after starting treatment
Grade A. Mean ± SEM. B. Tumor Growth Inhibition is calculated by dividing the mean tumor volume of the group versus the treated group by the mean tumor volume of the group versus the control group (T/C). For a test article to be considered to have antitumor activity, the T/C must be 0.5 or less. d. Statistically significant difference (one-way ANOVA) vs vehicle: ***p < 0.001.

权利要求:
Claims (12)
[0001]
1. Compound having the structural Formula (II):
[0002]
2. Compound according to claim 1, characterized in that R1a is selected from -OCH3, -CH3, -OCHF2 and -CH2CH3.
[0003]
3. Compound according to claim 1 or 2, characterized in that R4a is selected from 4,4-difluorocyclohexyl, cyclopropyl, tetrahydropyran-4-yl, 1-(2-fluoroethyl)-piperidin-4-yl , 1-(2,2-difluoroethyl)-piperidin-4-yl and 1-(2,2,2-trifluoroethyl)-piperidin-4-yl.
[0004]
4. Compound according to any one of claims 1 to 3, characterized in that R13 is selected from hydrogen, chlorine, fluorine, -OCH(CH3)2, phenyl and pyridin-2-yl.
[0005]
5. Compound according to claim 1, characterized in that the compound is N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)- 2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0006]
6. Compound according to claim 5, characterized in that the compound is RN-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)- 2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0007]
7. A compound according to claim 1, characterized in that the compound is 1-(1-(1-(2,2-difluoroethyl)piperidin-4-yl)ethyl)-N-((4-methoxy- 6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0008]
8. Compound according to claim 7, characterized in that the compound is R-1-(1-(1-(2,2-difluoroethyl)piperidin-4-yl)ethyl)-N-((4 -methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1H-indole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0009]
9. Pharmaceutical composition, characterized in that it comprises a compound, as defined in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
[0010]
10. Use of a compound as defined in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, characterized in that it is for the preparation of a medicament for the treatment of a disease or disorder associated with cell proliferation .
[0011]
11. Use according to claim 10, characterized in that the disease is cancer.
[0012]
12. Use according to claim 11, characterized in that the cancer is selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial cancer , lymphoma and liver cancer.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-08-20| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-15| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-07-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-08-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/02/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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