WO2013059944A1

WO2013059944A1 - Epigenetic regulators and uses thereof

Info

Publication number: WO2013059944A1
Application number: PCT/CA2012/050767
Authority: WO
Inventors: Andrew MUNGALL; Pierre Yulmin CHEUNG; Steven J.M. JONES; Oleksandr YAKOVENKO; Silvia THOENE
Original assignee: British Columbia Cancer Agency Branch
Priority date: 2011-10-28
Filing date: 2012-10-26
Publication date: 2013-05-02

Abstract

Protein chromobox homolog 2 (CBX2) is provided as a target for the development of therapeutics for the treatment of diseases associated with a hyperactive polycomb 2 complex (PRC2), such as lymphomas, lung cancer, breast cancer, prostate cancer and cancers of the neurological system. Compounds of general Formula (I) suitable for use as therapeutics for the treatment of diseases associated with a hyperactive PRC2, for example, diseases characterized by hypermethylation activity of EZH2, are provided. Formula (I)

Description

EPIGENETIC REGULATORS AND USES THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to the field of cancer therapeutics and, in particular, to compounds useful for the correction or normalization of de-regulated epigenetic pathways.

BACKGROUND OF THE INVENTION

[0002] Disruption of epigenetic mechanisms and pathways of gene regulation plays an important role in a spectrum of important disease states including cancer (Mai & Altucci, 2009, Int. J. Biochem. Cell Biol. 41 : 199-213). Poly comb-group (PcG) proteins are transcriptional repressors that function by modulating chromatin structure. These proteins reside in two main complexes, termed Poly comb repressive complexes 1 and 2 (PRCl and PRC2). PRC2 contains the protein EZH2, which catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), as well as EED and SUZ12. PRCl contains Ringl, Bmil, Mel- 18, and chromobox homolog (CBX) family proteins, including CBX2, with affinity for H3K27me3.

[0003] Interplay between PcG complexes and modified histones has been proposed to mediate stable transcriptional repression. Current models propose that PRC2 is recruited to specific genomic locations where it catalyzes H3K27me3, and the modified histones in turn recruit PRCl, which catalyzes H2Aubl and thereby impedes RNA polymerase II elongation. PRCl may also affect PRC2 function through as yet undefined mechanisms.

[0004] Genome- wide screening for somatic mutations in non-Hodgkin's lymphoma (NHL) has shown a high prevalence of mutations in genes/proteins involved in epigenetic regulation of gene expression. Recurrent somatic mutations in the protein EZH2 were found to be likely to contribute to development of NHL through increased activity of the PRC2 of which it is a constituent protein. In particular, NHL tumours with EZH2 mutations have been shown to have increased activity of the PRC2 in trimethylating histone H3 at residue Lysine 27 (H3K27) which leads to increased suppression of gene expression and tumourgenesis (Morin, et ah, 2010, Nat. Genet, 42(2): 181-185).

[0005] Overactivity and/or overexpression of EZH2 has been associated with a number of other cancers, including breast cancer, lung cancer, prostate cancer (in particular, late stage prostate cancer), multiple myeloma and cancers of the neurological system. In many cases, overactivity/overexpression of EZH2 has been associated with aggressive or drug resistant forms of these cancers.

[0006] Inhibitors of EZH2 or PRC2 have been proposed as therapeutics for the treatment of cancers. U.S. Patent Application Publication No. 2009/0137508 describes short interfering nucleic acid (siNA) molecules for modulating EZH2 expression and their use to treat diseases related to EZH2 expression, such as lung and prostate cancer.

[0007] U.S. Patent Application Publication No. 2011/0251216 describes micro RNA (miRNA) molecules and certain small molecules that target EZH2 expression in cancers such as prostate cancer, breast cancer and multiple myeloma. [0008] U.S. Patent Application Publication No. 2012/0071418 describes small molecule inhibitors of EZH2 that target the mutant form of EZH2 such that tri- methylation of lysine 27 on histone H3 (H3-K27), which is associated with certain types of cancers, is inhibited.

[0009] U.S. Patent Application Publication No. 2011/0237606 describes 3- deazaneplanocin derivatives that inhibit the function of PRC2 proteins and their use in the treatment of cancers, such as breast and prostate cancers.

[0010] U.S. Patent Application Publication No. 2005/0059682 describes methods for the treatment of conditions related to overexpression of EZH2, such as late stage prostate cancer, using a DNA methylation inhibitor and/or a histone deacetylase inhibitor, optionally in combination with an EZH2 antagonist.

[0011] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[0012] An object of the present invention is to provide epigenetic regulators and uses thereof. In accordance with one aspect of the present invention, there is provided a use of a compound of general Formula I or a pharmaceutically acceptable salt thereof for treating a disease characterized by increased methylation activity of EZH2 in a subject in need thereof:

I

[0013] wherein:

Ri, R₂, R-4 and Rs are each independently H or halo;

R3 is H, halo, C1-C4 alkyl or phenyl, and R9 is H or halo; or R3 and R9 taken together with the C atoms to which they are attached form phenyl;

R₅ is OR₇ or =0;

R₆ is X, CH₂X, C1-C4 alkyl, NH-NH₂, CH₂NRi₀, N ₀r piperidinyl;

R₇ is H or C1-C4 alkyl;

Rio is H, C1-C4 alkyl or CH₂-phenyl, and

X is halo. [0014] In accordance with another aspect of the invention, there is provided a use of a compound of general Formula I, as described above, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treatment of a disease characterized by increased methylation activity of EZH2. [0015] In accordance with another aspect of the invention, there is provided a method of treating a disease characterized by increased methylation activity of EZH2 in a subject comprising administering to the subject an effective amount of a compound of general Formula I, as described above, or a pharmaceutically acceptable salt thereof.

[0016] In accordance with another aspect of the invention, there is provided a method of screening for a candidate compound for treating a disease characterized by increased methylation activity of EZH2, the method comprising: (a) screening one or more compounds for the ability to modulate activity of chromobox homolog 2 (CBX2), wherein the modulation comprises an increase in binding affinity for mono- or di- methylated H3L27, a decrease in binding affinity for tri-methylated H3L27, or a combination thereof, and (b) selecting a compound having an increased binding affinity for mono- or di-methylated H3L27, a decrease in binding affinity for tri-methylated H3L27, or a combination thereof, wherein the compound selected in step (b) is a candidate compound for treating a disease characterized by increased methylation activity of EZH2.

BRIEF DESCRIPTION OF THE DRAWINGS [0017] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[0018] Figure 1 presents the results of an in vitro assay to determine cell viability of various lymphoma cell lines in the presence of Compound 1 (NSC112372); DoHH-2: wildtype for EZH2, MEF2B and MLL2; WSU-DLCL2: EZH2 mutation Y641F (wildtype for MEF2B and MLL2); DB: EZH2 mutation Y641N, the MEF2B mutation D83V and three mutations in MLL2. [0019] Figure 2 presents the results of xenograft experiments with the WSU-DLCL2 human diffuse large cell lymphoma cell line in which Compound 1 was injected into mice bearing the xenografted tumours. The top row shows the three treated tumours and the bottom row shows the three control tumours. [0020] Figure 3 presents a computer graphic illustrating the predicted interaction of Compound 1 with CBX2; (A) ribbon model with CBX2 shown in pale grey and the Histone H3 protein shown in mid-grey with the lysine 27 amino-acid side chain detailed, and (B) a surface based rendering of the model shown in (A).

[0021] Figure 4 presents the results from xenograft experiments using the WSU- DLCL2 human diffuse large cell lymphoma cell line in which mice were treated with vehicle, 1 mg/kg Compound 1 or 4 mg/kg Compound 1 (p = 0.0047 between control and 4 mg/kg groups).

[0022] Figure 5 presents the results of in vitro assays to determine cell viability of various lymphoma cell lines in the presence of Compound 3 (NSC2450) (cell lines as in Figure 1; SU-DHL-9: EZH2 wildtype, MEF2B wildtype, MLL2 indel).

[0023] Figure 6 presents the results from xenograft experiments using the WSU- DLCL2 human diffuse large cell lymphoma cell line in which mice were treated with vehicle or Compound 3 at 4 mg/kg for 5 days followed by 2 mg/kg for five days (p = 0.0125). [0024] Figure 7 presents the results of in vitro assays to determine cell viability of various breast cancer cell lines in the presence of Compound 1 or Compound 3.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to the identification of the protein chromobox homolog 2 (CBX2) as a target for the development of therapeutics for the treatment of diseases associated with a hyperactive polycomb 2 complex (PRC2), such as lymphomas, lung cancer, breast cancer, prostate cancer and cancers of the neurological system. [0026] As demonstrated herein, compounds predicted to interact with and modify the substrate specificity of CBX2 are capable of inhibiting the proliferation of cancer cells, such as lymphoma and breast cancer cells, and the growth of tumours in vivo. Certain embodiments of the invention thus relate to compounds that are predicted to interact with and modify the substrate specificity of CBX2 for the treatment of diseases associated with a hyperactive PRC2, for example, characterized by hypermethylation activity of EZH2.

[0027] Certain embodiments of the invention relate to CBX2 as a target for the development of therapies for treatment of Non-Hodgkin's lymphomas (NHL). Some embodiments relate to CBX2 as a target for the development of therapies for treatment of follicular lymphoma (FL). Other embodiments relate to CBX2 as a target for the development of therapies for treatment of the germinal centre B (GCB) subtype of diffuse large B-cell lymphoma (DLBCL).

[0028] Some embodiments of the invention relate to CBX2 as a target for the development of therapies for treatment of NHL, FL or GCB-DLBCL in which the histone methyltransferase protein EZH2 is mutated. Certain embodiments relate to CBX2 as a target for the development of therapies for treatment of NHL, FL or GCB- DLBCL in which EZH2 is mutated at amino acid position 641 (resulting in replacement of a single tyrosine residue at Tyr641 with an alternate amino acid) resulting in increased tri-methylation of lysine 27 residues in histone H3.

[0029] Some embodiments of the invention relate to CBX2 as a target for the development of therapies for treatment of NHL, FL or GCB-DLBCL in which the poly comb 2 complex (PRC2), of which EZH2 is a constituent, is hyperactive relative to normal state or wild-type. [0030] Some embodiments of the invention relate to CBX2 as a target for the development of therapies for treatment of cancers with hyperactive PRC2 complexes, including lung cancer, breast cancer and cancers of the neurological system.

Definitions [0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0033] The term "plurality" as used herein means more than one, for example, two or more, three or more, four or more, and the like.

[0034] The use of the word "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but it is also consistent with the meaning of "one or more," "at least one" and "one or more than one."

[0035] As used herein, the terms "comprising," "having," "including" and "containing," and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. The term "consisting essentially of when used herein in connection with a composition, use or method, denotes that additional elements and/or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term "consisting of when used herein in connection with a composition, use or method, excludes the presence of additional elements and/or method steps. A composition, use or method described herein as comprising certain elements and/or steps may also, in certain embodiments consist essentially of those elements and/or steps, and in other embodiments consist of those elements and/or steps, whether or not these embodiments are specifically referred to.

[0036] The terms "therapy" and "treatment," as used interchangeably herein, refer to an intervention performed with the intention of alleviating the symptoms associated with, preventing the development of, or altering the pathology of a disease. Thus, in certain embodiments, the terms therapy and treatment are used in the broadest sense, and in various embodiments may include one or more of the prevention (prophylaxis), moderation, reduction, and/or curing of a disease at various stages. Those in need of therapy/treatment thus may include those already having the disease as well as those prone to, or at risk of developing, the disease, disorder or condition and those in whom the disease is to be prevented.

[0037] The terms "subject" and "patient" as used herein refer to an animal in need of treatment.

[0038] The term "animal," as used herein, refers to both human and non-human animals.

[0039] Administration of the compounds of the invention "in combination with" one or more further therapeutic agents is intended to include simultaneous (concurrent) administration and consecutive administration. Consecutive administration is intended to encompass various orders of administration of the therapeutic agent(s) and the compound(s) of the invention to the subject with administration of the therapeutic agent(s) and the compound(s) being separated by a defined time period that may be short (for example in the order of minutes) or extended (for example in the order of days or weeks).

[0040] The term "C 1-C4 alkyl" refers to a straight chain or branched alkyl group of one to four carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl and tert-butyl (t-butyl).

[0041] The terms "halogen" and "halo" refer to fluorine, bromine, chlorine, and iodine atoms.

EPIGENETIC REGULATOR COMPOUNDS

[0042] Certain embodiments of the invention relate to compounds of general Formula I:

and pharmaceutically acceptable salts thereof, wherein: Ri, R₂, R4 and Rs are each independently H or halo;

R3 is H, halo, C₁-C4 alkyl or phenyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl;

R₅ is OR₇ or =0;

R₆ is X, CH₂X, C₁-C4 alkyl, NH-NH₂, CH₂NRi₀, N ₀r piperidinyl;

R₇ is H or C1-C4 alkyl;

Rio is H, C₁-C4 alkyl or CH₂-phenyl, and

X is halo. Some embodiments relate to compounds of general Formula I in which

Ri, R₂ and R4 are each independently H or halo;

R₅ is OH or =0;

R₆ is X, CH₂X, C₁-C4 alkyl, NH-NH₂, CH₂NRi₀ or piperidinyl;

R₈ is H; Rio is C2-C₃ alkyl or CH₂-phenyl, and X is halo.

[0045] Some embodiments relate to compounds of general Formula I in which

Ri, R₂ and R4 are each independently H or halo; R₃ is H, halo, C1-C4 alkyl or phenyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl;

R₅ is OH or =0;

R6 is CH₂X or piperidinyl;

R₈ is H; Rio is C2-C₃ alkyl or CH₂-phenyl, and

X is halo.

[0046] Some embodiments relate to compounds of general Formula I which have Formula II:

II [0047] and pharmaceutically acceptable salts thereof, wherein: Ri, R₂ and R4 are each independently H or halo; R3 is H, halo or phenyl; R₅ is OH or =0; R₆ is X, CH₂X or C C₄ alkyl, and X is halo.

[0048] Some embodiments relate to compounds of Formula II in which Ri, R₂ and R4 are each independently H or halo; R₃ is H, halo or phenyl;

R₅ is =0; R₆ is CH₂X, and X is halo.

[0049] Some embodiments relate to compounds of general Formula I which have Formula III:

[0050] and pharmaceutically acceptable salts thereof, wherein: Ri and R4 are each independently H or halo;

R3 is H, halo or C1-C4 alkyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl;

R₅ is OH or =0;

Re is NH-NH₂, CH₂NRio, or piperidinyl; Rio is C₂-C3 alkyl or CH₂-phenyl. [0051] Some embodiments relate to compounds of Formula III in which

Ri and R4 are each independently H or halo;

R3 is H, halo or C1-C4 alkyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl; R₅ is OH;

R6 is piperidinyl;

Rio is C2-C3 alkyl or CH₂-phenyl.

[0052] In certain embodiments of the invention in compounds of Formula I, II or III, each halo is CI or Br. [0053] In some embodiments in compounds of Formula I and II, X is Br.

[0054] Certain embodiments of the invention relate to compounds having the following structures:



 [0055] Certain embodiments of the invention relate to compounds 1, 2, 3, 4, 6, 8, 9, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27 and 28.

[0056] Compounds 1-28 above can be obtained/sourced from the NCI Developmental Therapeutics Program (DTP) open chemical repository at the National Cancer Institute (NCI)/N ational Institute of Health (NIH).

[0057] In certain embodiments, compounds of Formula I may possess a sufficiently acidic group, a sufficiently basic group, or both functional groups, and accordingly react with a number of organic and inorganic bases, or organic and inorganic acids, to form pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" as used herein, refers to a salt of a compound of Formula I, which is substantially nontoxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of general Formula I with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. [0058] Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulphonic acid, methanesulphonic acid, oxalic acid, p-bromophenylsulphonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such pharmaceutically acceptable salts are the sulphate, pyrosulphate, bisulphate, sulphite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxy benzoate, methoxybenzoate, phthalate, xylenesulphonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulphonate, propanesulphonate, naphthalene- 1 -sulfonate, napththalene-2- sulfonate, mandelate and the like. Pharmaceutically acceptable acid addition salts of particular interest are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulphonic acid.

[0059] Salts of amine groups may also comprise quarternary ammonium salts in which the amino nitrogen carries a suitable organic group such as an alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, or aralkyl moiety.

[0060] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing pharmaceutically acceptable salts thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0061] One skilled in the art will understand that the particular counterion forming a part of a pharmaceutically acceptable salt is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.

[0062] In some embodiments, the present invention further encompasses pharmaceutically acceptable solvates of a compound of Formula I. Many of the compounds of Formula I can combine with solvents such as water, methanol, ethanol and acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate and acetonitrilate.

[0063] Certain compounds of Formula I may have one or more asymmetric (chiral) centres and/or one or more unsaturated bonds. As a consequence, these compounds can be present as racemates, individual enantiomers, mixtures of enantiomers, individual diastereomers, mixtures of diastereomers, individual isomers and mixtures of isomers. Certain embodiments of the invention provide compounds of Formula I in an enantiomeric, diastereomeric or isomeric form, or as mixtures of enantiomers, diastereomers or isomers. [0064] In certain embodiments, the invention provides for prodrugs of the compounds of Formula I. The term "prodrug" as used herein refers to compound of Formula I that has undergone a chemical derivation such as substitution or addition of a further chemical group to change (for pharmaceutical use) one or more of its physico-chemical properties, and that yields the active compound per se by one or a series of metabolic transformations after administration to a subject. Physico-chemical properties that may be changed by conversion of the compound into a prodrug form include, for example, solubility, bioavailability, absorption, distribution, site specificity, stability, release characteristics, toxicity, and the like. Examples of chemical derivatives of compounds of Formula (I) that may be prepared in order to convert the compound into a prodrug include, but are not limited to, ester derivatives, ether derivatives, carbamate derivatives, amide derivatives, imine derivatives, and derivatization with an appropriate carrier moiety directly or via a linker group. Examples of prodrugs and methods of producing a prodrug of a given acting compound are well known to those skilled in the art and can be found, for example, in Krogsgaard-Larsen et al. {Textbook of Drug Design and Discovery, Taylor & Francis, New York, NY (April 2002)).

[0065] The preparation of salts, solvates and prodrugs can be carried out by methods known in the art. It will be appreciated that non-pharmaceutically acceptable salts, solvates or prodrugs also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts, solvates or prodrugs.

TESTING

[0066] Compounds of general Formula I may be tested for their ability to inhibit cancer cell proliferation and/or tumour growth in standard assays. Non-limiting examples of such assays are provided below. i) In vitro Testing

[0067] Initial determinations of the efficacy of compounds of general Formula I may be made using one or more standard in vitro assays. [0068] For example, the cytotoxicity of the compounds of general Formula I can be assayed in vitro using a suitable cell line, typically a cancer cell line. In general, cells of the selected test cell line are grown to an appropriate density and the candidate compound is added. After an appropriate incubation time (for example, about 48 to 72 hours), cell survival is assessed. Methods of determining cell survival are well known in the art and include, but are not limited to, the resazurin reduction test (see Fields & Lancaster (1993) Am. Biotechnol. Lab. 11 :48-50; O'Brien et al., (2000) Eur. J. Biochem. 267:5421-5426 and U.S. Patent No. 5,501,959), the sulforhodamine assay (Rubinstein et al, (1990) J. Natl. Cancer Inst. 82: 113-118) or the neutral red dye test (Kitano et al, (1991) Euro. J. Clin. Investg. 21 :53-58; West et al, (1992) J. Investigative Derm. 99:95-100). Cytotoxicity is determined by comparison of cell survival in the treated culture with cell survival in one or more control cultures, for example, untreated cultures and/or cultures pre-treated with a control compound (typically a known therapeutic). [0069] The ability of the compounds of general Formula I to inhibit proliferation of cancer cells in vitro can be assessed, for example, by culturing cells of a cancer cell line of interest in a suitable medium. After an appropriate incubation time, the cells can be treated with the compound of general Formula I and incubated for a further period of time. Cells are then counted and compared to an appropriate control. Suitable controls include, for example, cells treated with a standard chemotherapeutic and/or untreated cells.

[0070] Alternatively, the compounds of general Formula I can be tested in vitro by determining their ability to inhibit anchorage-independent growth of tumour cells. Anchorage-independent growth is known in the art to be a good indicator of tumourigenicity. In general, anchorage-independent growth is assessed by plating cells from a selected cancer cell-line onto soft agar and determining the number of colonies formed after an appropriate incubation period. Growth of cells treated with the compound of general Formula I can then be compared with that of control cells (as described above). [0071] A variety of cancer cell-lines suitable for testing the candidate compounds are known in the art and many are commercially available (for example, from the American Type Culture Collection, Manassas, VA).

[0072] If necessary, the toxicity of the compounds of general Formula I can also be initially assessed in vitro using standard techniques. For example, human primary fibroblasts can be treated in vitro with the compound of general Formula I and then tested at different time points following treatment for their viability using a standard viability assay, such as the assays described above, or the trypan-blue exclusion assay. Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluorocytometer cell sorter (FACS). ii) In vivo Testing

[0073] The ability of the compounds of general Formula I to inhibit tumour growth or proliferation in vivo can be determined in an appropriate animal model using standard techniques known in the art (see, for example, Enna, et al, Current Protocols in Pharmacology, J. Wiley & Sons, Inc., New York, NY).

[0074] In general, current animal models for screening anti-tumour compounds are xenograft models, in which a human tumour has been implanted into an animal. Examples of xenograft models of human cancer include, but are not limited to, human solid tumour xenografts, implanted by sub-cutaneous injection or implantation and used in tumour growth assays; human solid tumour isografts, implanted by fat pad injection and used in tumour growth assays; human solid tumour orthotopic xenografts, implanted directly into the relevant tissue and used in tumour growth assays; experimental models of lymphoma and leukaemia in mice, used in survival assays, and experimental models of lung metastasis in mice.

[0075] For example, the compounds of general Formula I can be tested in vivo on solid tumours using mice that are subcutaneously grafted bilaterally with 30 to 60 mg of a tumour fragment, or implanted with an appropriate number of cancer cells, on day 0. The animals bearing tumours are mixed before being subjected to the various treatments and controls. In the case of treatment of advanced tumours, tumours are allowed to develop to the desired size, animals having insufficiently developed tumours being eliminated. The selected animals are distributed at random to undergo the treatments and controls. Animals not bearing tumours may also be subjected to the same treatments as the tumour-bearing animals in order to be able to dissociate the toxic effect from the specific effect on the tumour. Chemotherapy generally begins from 3 to 22 days after grafting, depending on the type of tumour, and the animals are observed every day. The compound of general Formula I can be administered to the animals, for example, by i.p. injection or bolus infusion. The different animal groups are weighed about 3 or 4 times a week until the maximum weight loss is attained, after which the groups are weighed at least once a week until the end of the trial.

[0076] The tumours are measured after a pre-determined time period, or they can be monitored continuously by measuring about 2 or 3 times a week until the tumour reaches a pre-determined size and / or weight, or until the animal dies if this occurs before the tumour reaches the pre-determined size / weight. The animals are then sacrificed and the tissue histology, size and / or proliferation of the tumour assessed.

[0077] The effect of the compounds of general Formula I on tumour metastasis may be studied. Tumour cells are typically treated with the composition ex vivo and then injected into a suitable test animal. The spread of the tumour cells from the site of injection is then monitored over a suitable period of time.

[0078] In vivo toxic effects of the compounds may be evaluated by measuring their effect on animal body weight during treatment and by performing haematological profiles and liver enzyme analysis after the animal has been sacrificed. PHARMACEUTICAL COMPOSITIONS

[0079] Compounds of Formula I are typically formulated for therapeutic use. In certain embodiments, the present invention thus provides pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients.

[0080] Compounds of Formula I or pharmaceutical compositions comprising the compounds may be formulated for administration orally (including, for example, buccally or sublingually), topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. In the usual course of therapy, the active compound is incorporated into an acceptable vehicle and formulated into a form suitable for oral, rectal or parenteral administration, such as syrups, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositiories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injectables, or solutions. The term parenteral as used herein includes subcutaneous injections, intradermal, intraarticular, intravenous, intramuscular, intravascular, intrastemal, intrathecal injection or infusion techniques. [0081] Compositions intended for oral use may be prepared in either solid or fluid unit dosage forms. Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. An elixir is prepared by using a hydroalcoholic (for example, ethanol) vehicle with suitable sweeteners such as sugar and saccharin, together with an aromatic flavoring agent. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like. [0082] Solid formulations such as tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate: granulating and disintegrating agents for example, corn starch, or alginic acid: binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other conventional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, methylcellulose, and functionally similar materials. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0083] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil. [0084] Aqueous suspensions contain active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example hepta-decaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or Λ-propyl- /^hydroxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening agents, such as sucrose or saccharin. [0085] Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0086] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

[0087] Pharmaceutical compositions of the invention may also be in the form of oil- in-water emulsions. The oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0088] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally 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. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.

[0089] Compound(s) of Formula I may be administered, together or separately, in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

[0090] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philidelphia, PA (2000).

USES

[0091] Certain embodiments of the invention provide for the use of compounds of general Formula I for the treatment of diseases characterized by increased methylation activity of EZH2. [0092] Without being bound by any one theory, the compounds of general Formula I are believed to interact with the active site of CBX2 and modify the protein's substrate specificity such that it is less likely to bind to tri-methylated H3K27 and/or more likely to bind to mono- or di-methylated H3K27. Alteration of the activity of PRC 1 which contains CBX2 should mitigate the ability of cancer cells to continue to divide, particularly those which have a hyperactive PRC2.

[0093] In certain embodiments, the compounds can be used in the treatment of cancer and, in particular for the treatment of cancers characterized by increased methylation activity of EZH2. Overexpression of EZH2 has been associated with a number of cancers, including breast cancer, prostate cancer, ovarian cancer, endometrial cancer, lung cancer, multiple myeloma, cancers of the neurological system and lymphomas. Certain embodiments of the invention, therefore, relate to the use of compounds of general Formula I in the treatment of breast cancer, prostate cancer, ovarian cancer, endometrial cancer, lung cancer, multiple myeloma, cancers of the neurological system and lymphomas. Some embodiments provide for the use of the compounds of general Formula I in the treatment of aggressive, drug-resistant or refractory cancer which have been shown to be associated with overexpression of EZH2.

[0094] Certain embodiments of the invention relate to the use of compounds of general Formula I in the treatment of a cancer having a mutant form of EZH2. Some embodiments of the invention relate to the use of compounds of general Formula I in the treatment of a cancer having a mutant form of EZH2 in which the tyrosine at position 641 is replaced with an alternate amino acid (a Y641 mutant). Examples of such cancers, include, but are not limited to, lymphomas (such as non-Hodgkin's lymphoma (NHL), follicular lymphoma (FL) and germinal centre B subtype of diffuse large cell lymphoma (GCB-DLBCL)), prostate cancer, breast cancer, lung cancer and cancers of the neurological system.

[0095] Some embodiments relate to the use of compounds of general Formula I for treatment of NHL, FL or GCB-DLBCL in which a) the histone methyltransferase EZH2 is mutated, b) the histone methyltransferase protein EZH2 is mutated at amino acid position 641 (resulting in replacement of a single tyrosine residue at Tyr641 with an alternate amino acid) resulting in increased tri-methylation of lysine 27 residues in histone H3 or c) the polycomb 2 complex (PRC2), of which EZH2 is a constituent, is hyperactive relative to normal state or wild-type. [0096] Some embodiments relate to the use of compounds of general Formula I for treatment of cancers with hyperactive PRC2 complexes, including lung cancer, breast cancer and cancers of the neurological system.

[0097] Certain embodiments of the invention relate to the use of a compound of general Formula I for treatment of a subject having a cancer that expresses a Y641 mutant of EZH2. Accordingly, some embodiments of the invention relate to a method of treating cancer comprising the steps of performing an assay to detect a Y641 mutant of EZH2 in a sample comprising cancer cells from a subject having a cancer; and administering to a subject expressing a Y641 mutant of EZH2 a therapeutically effective amount of a compound of general Formula I, thereby treating the cancer. In some embodiments, the cancer is NHL, FL or GBC-DLBCL.

Combination Therapy

[0098] In certain embodiments of the invention relating to the use of a compound of general Formula I for the treatment of cancer, the compound may be used in combination with one or more chemotherapeutic agents.

[0099] Various chemotherapeutic agents are known in the art and include those that are specific for the treatment of a particular type of cancer as well as those that are applicable to a range of cancers, such as doxorubicin, capecitabine, mitoxantrone, irinotecan (CPT-11), cisplatin and gemcitabine. [00100] Chemotherapeutics typically used in the treatment of solid tumours include, for example, Gemicitabine (e.g. Gemzar®), Cyclophosphamide, Capecitabine (e.g. Xeloda®), Ifosfamide, Paclitaxel (e.g. Taxol®), Cisplatin, Docetaxel (e.g. Taxotere®), Carboplatin, Epi-doxorubicin (epirubicin), Doxorubicin (e.g. Adriamycin®) and 5- fluorouracil (5-FU). [00101] Chemotherapeutics typically used in the treatment of breast cancer include, for example, Capecitabine (e.g. Xeloda®), Cyclophosphamide, 5-fluorouracil (5-FU), Carboplatin, Paclitaxel (e.g. Taxol®), Cisplatin, Docetaxel (e.g. Taxotere®), Ifosfamide, Epi-doxorubicin (epirubicin), Doxorubicin (e.g. Adriamycin®), Trastuzumab (Herceptin®) and Tamoxifen. [00102] Chemotherapeutics typically used in the treatment of non-Hodgkin's lymphoma include, for example, Procarbazine (e.g. Matulane®), Cytarabine, Rituximab (e.g. Rituxan®) and Etoposide. [00103] Chemotherapeutics typically used in the treatment of prostate cancer include, for example, Goserelin Acetate (e.g. Zoladex®), Mitoxantrone (e.g. Novantrone®), Prednisone (e.g. Deltasone®), Liarozole, Nilutamide (e.g. Nilandron®), Flutamide (e.g. Eulexin®), Finasteride (e.g. Proscar®), Terazosin (e.g. Hytrin®), Doxazosin (e.g. Cardura®), Cyclophosphamide, Docetaxel (e.g. Taxotere®), Estramustine and Luteinizing hormone releasing hormone agonist.

[00104] Some embodiments of the invention relate to the use of compounds of general Formula I in combination with a therapeutic that targets EZH2 for the treatment of cancer. Examples of such compounds include, for example, those described in U.S. Patent Application Publication Nos. 2009/0137508, 2011/0251216, 2012/0071418 and 2011/0237606.

[00105] Certain embodiments of the invention relate to the use of compounds of general Formula I in combination with a chemotherapeutic for the treatment of a drug resistant tumours where drug resistance is the result of upregulation/over- activity of EZH2.

KITS

[00106] Certain embodiments of the invention provide for kits containing one or more compounds of Formula I, for example, therapeutic packs or kits. In those embodiments in which the compounds of Formula I are intended for use as part of a combination therapy, the kit may optionally contain the other therapeutic(s) that makes up the combination.

[00107] In certain embodiments, one or more of the components of the kit can be lyophilized and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized components. Individual components of the kit would typically be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. [00108] In certain embodiments, the compound(s) of Formula I are provided in the kit in the form of pharmaceutical compositions suitable for administration to a subject. In this case, if desired, the container may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the composition may be administered to the subject.

SCREENING METHODS

[00109] Certain embodiments of the invention relate to methods of screening for candidate therapeutics for treating a disease characterized by increased methylation activity of EZH2 by assessing the ability of a candidate compound to modulate activity of CBX2. Modulation of CBX2 activity comprises, for example, an increase in binding affinity for mono- or di-methylated H3-L27 and/or a decrease in binding affinity for tri- methylated H3-L27. Compounds that demonstrate an increased binding affinity for mono- or di-methylated H3-L27, a decreased binding affinity for tri-methylated H3- L27, or both, are then selected as candidate therapeutics. [00110] The ability of a candidate compound to modulate activity of CBX2 may be determined by computational methods, for example using in silico modelling, or they may be determined by standard in vitro or in vivo assays.

[00111] A number of computational methods for assessing binding affinities are available and would be known to those of skill in the art. Various in vitro and in vivo assays for assessing binding affinities are also known in the art (see, for example, Current Protocols in Protein Science, Coligan et al. (ed.), Wiley & Sons, New York, NY) and appropriate assays can be readily selected by the skilled worker.

[00112] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[00113] The following examples demonstrate the activity of Compound 1 and Compound 3. Compound 1 (2-bromo-l-(6,8-dichloro-2-phenylquinolin-4-yl)ethanone) and Compound 3 ([2-(3,4-dichlorophenyl)quinolin-4-yl]-piperidin-2-ylmethanol are part of the repository available from the National Cancer Institute and have been assigned numbers NSC112372 and NSC2450, respectively. Information available on these compounds from the PubChem database maintained by the National Center for Biotechnology Information indicates that Compound 1 was inactive when tested in the NCI In Vivo Anticancer Drug Screen against tumour model L1210 Leukemia (intraperitoneal) in CD2F1 (CDF1) mice, and Compound 3 showed activity in the NCI Yeast Anticancer Drug Screen using the rad50, mec2-l, sgslmgtl, cln2radl4, bub3 and mlhlradl8 strains.

EXAMPLE 1 : Effect of Compound 1 on Proliferation of Lymphoma Cell Lines

[00114] Compound 1 (NSC112372) was identified through computational modelling as a candidate to interact with the poly comb complex 1 protein constituent CBX2.

[00115] Compound 1 is expected to bind into the active pocket of the protein CBX2 (see Figure 3) and thus alter the specificity of the pocket, now possessing increased binding affinity to mono- and di-methylated forms of Lysine 27 of the Histone H3 protein and possibly also reducing the affinity to the tri-methylated form. As such, Compound 1, by interacting with CBX2 may diminish or reverse the normal effector (reader) function of the PRC1 complex, rendering it less sensitive (reduced binding) to histone tri-methylation and/or increased sensitivity (increased binding) to mono- and di-methylated forms of Lysine 27. Compound 1 was therefore expected to partially or wholly negate the pro-tumourigenic effects of somatic mutations in EZH2, for example.

[00116] Lymphoid malignancies with various mutations in the EZH2 gene have been shown to have increased activity of PRC2 in tri-methylating the histone H3 at the residue Lysine 27 (H3K27). Compound 1 was tested for its effect on the viability of three lymphoma cell lines in vitro using standard methods. The cell lines tested were: DoHH-2 (wildtype for EZH2, MEF2B and MLL2); WSU-DLCL2 (EZH2 mutation Y641F (wildtype for MEF2B and MLL2)) and DB (EZH2 mutation Y641N, the MEF2B mutation D83V and three mutations in MLL2 (two of which lead to a truncated protein after residue Q2736 and the third allele has a 1 base pair deletion at residue P480)).

[00117] The results are shown in Figure 1 and indicate that Compound 1 reduced the viability of all three cell lines in a dose dependent manner, but showed the highest activity against the cell-line WSU-DLCL2 which possesses the EZH2 Y641 mutation. EXAMPLE 2: Effect of Compound 1 on Tumour Growth in a Mouse Xenograft Model

[00118] Mouse xenografts of the WSU-DLCL2 cell line were established by subcutaneous injection of lxlO⁷ cells in 50 μΐ PBS into the flank of NOD/SCID/y^nu11 mice. [00119] Compound 1 was injected into mice bearing the WSU-DLCL2 xenografted tumours. The daily doses were lx l mg/kg, 2x 2 mg/kg, 2x 4 mg/kg, 2x 8 mg/kg and lx 16 mg/kg over 8 days dissolved in vehicle (50% DMSO/50% PEG-400). Control mice received the equivalent doses of vehicle only. At the end of the treatment the control tumours weighed 0.49 g, 0.60 g and 0.65 g. The tumours treated with Compound 1 weighed 0.35 g, 0.22 g and 0.22 g. The results are shown in Figure 2, in which the top row shows the three treated tumours and the bottom row shows the control tumours. EXAMPLE 3: Effect of Compound 1 on Tumour Growth in a Mouse Xenograft Model #2

[00120] WSU-DLCL2 tumour fragments were transplanted subcutaneously into the flank of male NSG mice. Mice were 9.0 - 9.3 weeks at transplantation and treatment was started when the mice were between 12.9 and 13.1 weeks. 8 mice were used per group and treatment was daily for ten days with evaluation at day 12. Tumour size determined by caliper every four days, tumour volume calculated by length x width²12.

[00121] The groups of mice were treated with vehicle (control), 1 mg/kg Compound 1 or 4 mg/kg Compound 1 by intraperitoneal injection. Vehicle was 50% DMSO/50% PEG-400.

[00122] The results are shown in Figure 4 and demonstrate that Compound 1 at both dosages was able to reduce tumour volume.

EXAMPLE 4: Effect of Compound 3 on Proliferation of Lymphoma Cell Lines

[00123] Compound 3 (NSC2540) was tested for its effect on the viability of four lymphoma cell lines in vitro using standard methods. The cell lines tested were DoHH- 2, WSU-DLCL2, DB and SU-DHL-9 (EZH2 wildtype, MEF2B wildtype, MLL2 indel).

[00124] The results are shown in Figure 5 and demonstrate that Compound 3 was able to decrease viability of the DoHH-2, WSU-DLCL2 and SU-DHL-9 cell lines.

EXAMPLE 5: Effect of Compound 3 on Tumour Growth in a Mouse Xenograft Model

[00125] WSU-DLCL2 tumour fragments were transplanted subcutaneously into the flank of male NSG mice. Mice were 5.3 - 9.4 weeks at transplantation and treatment was started when the mice were between 8.6 and 12.7 weeks. 8 mice were used in the control group and 6 mice were used in the Compound 3 treated group. Treatment was daily for ten days with evaluation at day 12. Tumour size determined by caliper every four days, tumour volume calculated by length x width² 12. [00126] The groups of mice were treated with vehicle (control) or Compound 1 by intraperitoneal injection at 4 mg/kg for the first 5 days and 2 mg/kg for the subsequent 5 days. Vehicle was 50% DMSO/50% PEG-400.

[00127] The results are shown in Figure 6 and demonstrate that Compound 3 was able to significantly reduce tumour volume.

EXAMPLE 6: Effect of Compounds 1 and 3 on Proliferation of Breast Cancer Cell Lines

[00128] Compounds 1 and 3 were tested for their effect on the viability of four breast cancer cell lines in vitro using standard methods. The cell lines tested were: MCF-7, MDA-MB-231, HCC202 (CRL-2316) and HCC1500 (CRL-2329).

[00129] The results are shown in Figure 7 and demonstrate that Compound 3 was able to decrease viability of the MDA-MB-231, HCC202 (CRL-2316) and HCC1500 (CRL- 2329) cell lines.

EXAMPLE 7: Identification of Further Compounds that Interact with CBX2 and Their Effect on Cancer Cell Viability

[00130] The compounds shown in Tables 1 and 2 were identified by on computer modelling techniques as compounds predicted to be able to bind into the active pocket of CBX2. The compounds can be obtained/sourced from the NCI Developmental Therapeutics Program (DTP) open chemical repository at the NCI/NIH and the NSC numbers for the compounds are provided.

[00131] The compounds were tested for their effect on cancer cell viability in vitro using standard methods. The results are provided in Tables 1 and 2.

Table 1 : NSC Numbers and Activity of Analogues of Compounds 1 and 2

Table 2: NSC Numbers and Activity of Analogues of Compound 3

[00132] The disclosures of all patents, patent applications, publications and database entries referenced in this specification are hereby specifically incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication and database entry were specifically and individually indicated to be incorporated by reference. [00133] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Use of a compound of general Formula I or a pharmaceutically acceptable salt thereof for treating a disease characterized by increased methylation activity of EZH2 in a subject in need thereof:

wherein:

R_1; R₂, R4 and Rs are each independently H or halo;

R3 is H, halo, C1-C4 alkyl or phenyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl;

R₅ is OR₇ or =0;

R₆ is X, CH₂X, C1-C4 alkyl, NH-NH₂, CH₂NRi₀, N or piperidinyl;

R₇ is H or C1-C4 alkyl;

Rio is H, C1-C4 alkyl or CH₂-phenyl, and

X is halo.

2. Use of a compound of general Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treatment of a disease characterized by increased methylation activity of EZH2:

I wherein:

Ri, R₂, R4 and Rs are each independently H or halo;

R₅ is OR₇ or =0;

R₆ is X, CH₂X, C1-C4 alkyl, NH-NH₂, CH₂NRi₀, N ₀r piperidinyl;

R₇ is H or C1-C4 alkyl;

Rio is H, C1-C4 alkyl or CH₂-phenyl, and

X is halo.

3. The use according to claim 1 or 2, wherein the disease is cancer.

4. The use according to claim 3, wherein the cancer is a lymphoma, breast cancer, prostate cancer, lung cancer, multiple myeloma or a cancer of the neurological system.

5. The use according to claim 4, wherein the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma or germinal centre B subtype of diffuse large cell lymphoma.

6. The use according to any one of claims 3-5, wherein the cancer expresses a Y641 mutant of EZH2.

7. The use according to any one of claims 3-6, wherein the compound administration in combination with a chemotherapeutic.

8 The use according to any one of claims 3-6, wherein the compound is for administration in combination with an EZH2 inhibitor.

9. The use according to any one of claims 1-8, wherein

Ri, R₂ and R4 are each independently H or halo;

R₅ is OH or =0;

R₆ is X, CH₂X, C1-C4 alkyl, NH-NH₂, CH₂NRi₀ or piperidinyl; R₈ is H;

Rio is C₂-C3 alkyl or CH₂-phenyl, and X is halo.

10. The use according to claim 9, wherein R6 is CH₂X or piperidinyl.

11. The use according to any one of claims 1-8, wherein the compound of general Formula I is a compound of Formula II or a pharmaceutically acceptable salt thereof:

II wherein: Ri, R₂ and R4 are each independently H or halo; R3 is H, halo or phenyl; R₅ is OH or =0;

R₆ is X, CH₂X or C C₄ alkyl, and X is halo.

12. The use according to claim 11, wherein R₅ is =0 and R6 is CH₂X.

13. The use according to any one of claims 1-8, wherein the compound of general Formula I is a compound of Formula III or a pharmaceutically acceptable salt thereof:

III wherein:

Ri and R4 are each independently H or halo;

R₃ is H, halo or C1-C4 alkyl, and R₉ is H or halo; or R₃ and R₉ taken together with the C atoms to which they are attached form phenyl;

R₅ is OH or =0;

Re is NH-NH₂, CH2NR1₀, or piperidinyl; Rio is C2-C3 alkyl or CH₂-phenyl.

14. The use according to claim 13, wherein R5 is OH and R6 is piperidinyl.

15. The use according to any one of claims 1-14, wherein each halo is CI or Br.

16. The use according to any one of claims 1-12, wherein X is Br.

17. The use according to any one of claims 1-8, wherein the compound of Formula I is selected from the group consisting of:

10 11 12

45

18. The use according to any one of claims 1-8, wherein the compound is selected from the group consisting of 1, 2, 3, 4, 6, 8, 9, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27 and 28.

19. The use according to claim 18, wherein the compound is Compound 1.

20. The use according to claim 18, wherein the compound is Compound 3.

21. A method of treating a disease characterized by increased methylation activity of EZH2 in a subject comprising administering to the subject an effective amount of a compound of general Formula I or a pharmaceutically acceptable salt thereof:

I wherein:

Ri, R₂, R4 and Rs are each independently H or halo;

R₅ is OR₇ or =0;

R₆ is X, CH₂X, C1-C4 alkyl, NH-NH₂, CH₂NRi₀, N ₀r piperidinyl;

R₇ is H or C1-C4 alkyl;

Rio is H, C1-C4 alkyl or CH₂-phenyl, and

X is halo.

22. The method according to claim 21, wherein the disease is cancer.

23. The method according to claim 22, wherein the cancer is a lymphoma, breast cancer, prostate cancer, lung cancer, multiple myeloma or a cancer of the neurological system.

24. The method according to claim 23, wherein the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma or germinal centre B subtype of diffuse large cell lymphoma.

25. The method according to any one of claims 22-24, wherein the cancer expresses a Y641 mutant of EZH2.

26. The method according to any one of claims 22-25, wherein the compound is administered in combination with a chemotherapeutic.

27. The method according to any one of claims 22-25, wherein the compound is administered in combination with an EZH2 inhibitor.

28. The method according to any one of claims 21-27, wherein

Ri, R₂ and R4 are each independently H or halo;

R₅ is OH or =0;

Rio is C₂-C3 alkyl or CH₂-phenyl, and X is halo.

29. The method according to claim 28, wherein R6 is CH₂X or piperidinyl.

30. The method according to any one of claims 21-27, wherein the compound of general Formula I is a compound of Formula II or a pharmaceutically acceptable salt thereof:

R₆ is X, CH₂X or C1-C4 alkyl, and X is halo.

31. The method according to claim 30, wherein R₅ is =0 and R6 is CH₂X.

32. The method according to any one of claims 21-27, wherein the compound of general Formula I is a compound of Formula III or a pharmaceutically acceptable salt thereof:

Ri and R4 are each independently H or halo;

R₅ is OH or =0;

Re is NH-NH₂, CH₂NRi₀, or piperidinyl; Rio is C₂-C3 alkyl or CH₂-phenyl. The method according to claim 32, wherein R₅ is OH and R6 is piperidinyl.

34. The method according to any one of claims 21-33, wherein each halo is CI or Br.

35. The method according to any one of claims 21-31, wherein X is Br.

36. The method according to any one of claims 21-27, wherein the compound of Formula I is selected from the group consisting of:

7 8 9

51

37. The method according to any one of claims 21-27, wherein the compound is selected from the group consisting of 1, 2, 3, 4, 6, 8, 9, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 27 and 28.

38. The method according to claim 37, wherein the compound is Compound 1.

39. The method according to claim 37, wherein the compound is Compound 3.

40. A method of screening for a candidate compound for treating a disease characterized by increased methylation activity of EZH2, the method comprising: (a) screening one or more compounds for the ability to modulate activity of chromobox homolog 2 (CBX2), wherein the modulation comprises an increase in binding affinity for mono- or di-methylated H3L27, a decrease in binding affinity for tri-methylated H3L27, or a combination thereof, and

(b) selecting a compound having an increased binding affinity for mono- or di- methylated H3L27, a decrease in binding affinity for tri-methylated H3L27, or a combination thereof, wherein the compound selected in step (b) is a candidate compound for treating a disease characterized by increased methylation activity of EZH2.

41. The method according to claim 40, wherein the screening in step (a) comprises in silico modelling.

42. The method according to claim 40, wherein the screening in step (a) comprises one or more in vitro assays.