Combination Therapies for Treating Cancer

Abstract

To provide compositions comprising inhibitors of human histone methyltransferase EZH2 and one or more other therapeutic agents, particularly anticancer agents such as prednisone.SOLUTION: The present invention relates to compositions comprising inhibitors of human histone methyltransferase EZH2 and one or more other therapeutic agents, particularly to compositions comprising anticancer agents such as prednisone, and to methods of combination therapy for administering to subjects in need thereof for treatment of cancer.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] This application is a U.S. national stage application of an international application, No. PCT / US2013 / 036452, filed April 12, 2013, which claims priority to and the benefit of U.S. Provisional Patent Application No. 61 / 624,194, filed April 13, 2012, and U.S. Provisional Patent Application No. 61 / 785,169, filed March 14, 2013, the contents of each of which are incorporated herein by reference in their entirety. Incorporation by reference of sequence listing The contents of the text file named "EPIZ009001WO_ST2.5.txt", created on October 14, 2014, is 45KB in size and is incorporated herein by reference in its entirety.

[0002] The present invention relates to compositions comprising inhibitors of human histone methyltransferase EZH2, the catalytic subunit of the PRC2 complex that catalyzes the mono- to tri-methylation of lysine 27 on histone H3 (H3-K27), and one or more other therapeutic agents, particularly anti-cancer agents, and methods of combination therapy for treating cancer. [Background technology]

[0003] Combination therapy treatments for cancer are becoming more common, due in part to the recognized benefits of attacking the disease by multiple means. Although many effective combination therapy treatments have been identified over the past several decades, given the continuing high number of deaths that occur each year as a result of cancer, there is a continuing need to identify effective therapeutic regimens for use in anti-cancer treatment. Summary of the Invention [Means for solving the problem]

[0004] (Summary of the Invention) In one aspect, the invention features a composition that includes a compound of Formula (IIa) below and one or more other therapeutic agents or pharmaceutically acceptable salts or esters thereof:

[0005] [ka] The compounds of formula (IIa) may include one or more of the following features:

[0006] R a and R b are each independently H or C1-C6 alkyl.

[0007] R a and R b together with the N atom to which they are attached are a 4- to 7-membered heterocycloalkyl ring having 0 or 1 additional heteroatoms, a 4- to 12-membered (e.g., 4- to 7-membered) heterocycloalkyl ring optionally substituted with C-C alkyl and one or more -Q-T.

[0008] Q3 is a bond or an unsubstituted or substituted C1-C3 alkyl linker.

[0009] T3 is H, halo, 4- to 7-membered heterocycloalkyl, C1-C3 alkyl, OR d , COOR d , -S(O)2R d , or -NR d R e and R d and R e are each independently H or C1-C6 alkyl.

[0010] R7 is C1-C6 alkyl, C3-C8 cycloalkyl, or 4- to 12-membered (e.g., 4- to 7-membered) heterocycloalkyl, each optionally substituted with one or more -Q5-T5. For example, R7 is not H.

[0011] R7 is a 4- to 7-membered heterocycloalkyl optionally substituted with one or more -Q5-T5.

[0012] R7 is piperidinyl, tetrahydropyran, cyclopentyl, or cyclohexyl, each optionally substituted with one -Q5-T5.

[0013] T5 is H, halo, C1-C6 alkyl, C1-C6 alkoxyl, C3-C8 cycloalkyl, C6-C 10 aryl, or 4 to 12 membered (eg, 4 to 7 membered) heterocycloalkyl.

[0014] Q5 is a bond and T5 is C1-C6 alkyl, C3-C8 cycloalkyl, or 4- to 12-membered (eg, 4- to 7-membered) heterocycloalkyl.

[0015] Q5 is CO, S(O)2, or NHC(O); and T5 is C1-C6 alkyl, C1-C6 alkoxyl, C3-C8 cycloalkyl, or 4- to 12-membered (e.g., 4- to 7-membered) heterocycloalkyl.

[0016] Q5 is a C1-C3 alkyl linker and T5 is H or C6-C 10 It is aryl.

[0017] Q5 is a C1-C3 alkyl linker and T5 is a C3-C8 cycloalkyl, a 4- to 7-membered heterocycloalkyl, or S(O) q R q is.

[0018] R7 is cyclopentyl or cyclohexyl, each optionally substituted with one -Q5-T5.

[0019] Q5 is NHC(O) and T5 is C1-C6 alkyl or C1-C6 alkoxy.

[0020] R7 is isopropyl.

[0021] R2 and R4 are each independently H or amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, or C6-C 10 It is a C1-C6 alkyl optionally substituted with an aryl.

[0022] R8 is H, methyl, or ethyl.

[0023] R8 is methyl.

[0024] R8 is ethyl.

[0025] R8 is a 4 to 7 membered heterocycloalkyl, for example, tetrahydropyran.

[0026] The present invention features compositions including a compound selected from Table 1, or a pharmaceutically acceptable salt or ester thereof, and one or more other therapeutic agents.

[0027] The present invention relates to compound 44

[0028] [ka] or a pharmaceutically acceptable salt or ester thereof, and one or more other therapeutic agents.

[0029] In one embodiment of this and other aspects of the invention, the other therapeutic agent is an anti-cancer agent.

[0030] In one embodiment of this and other aspects of the invention, the other therapeutic agent is a glucocorticoid.

[0031] In one embodiment of this and other aspects of the invention, the other therapeutic agent is selected from prednisone, prednisolone, cyclophosphamide, vincristine, doxorubicin, mafosfamide, cisplatin, AraC, everolimus, decitabine, dexamethasone, and analogs, derivatives, or combinations thereof.

[0032] In one embodiment of this and other aspects of the invention, the other therapeutic agent is prednisone, or an analogue or derivative thereof.

[0033] The present invention also provides a pharmaceutical composition comprising a compound selected from the compounds of formula (IIa) disclosed herein or a pharmaceutically acceptable salt thereof and one or more therapeutic agents, and a pharmaceutically acceptable carrier.

[0034] The present invention also provides a compound selected from Table I, one or more other therapeutic agents, or a pharmaceutical Also provided is a pharmaceutical composition comprising an acceptable salt thereof and a pharmaceutically acceptable carrier.

[0035] The present invention also provides a pharmaceutical composition comprising a compound selected from the compounds of formula (IIa) disclosed herein or a pharmaceutically acceptable salt thereof, one or more other therapeutic agents, and a pharmaceutically acceptable carrier.

[0036] Another aspect of the present invention is a method for treating or preventing a disease by administering to a subject in need thereof a therapeutically effective amount of a composition comprising a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents. The diseases of the present invention can be affected, treated, or prevented by modulating the methylation status of histones or other proteins. For example, the disease is cancer, a precancerous condition, or a neurological disease. Preferably, the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma, or diffuse large B-cell lymphoma. Alternatively, the leukemia is chronic myeloid leukemia (CML). The precancerous condition is, for example, myelodysplastic syndrome (MDS, formerly known as preleukemia).

[0037] The subject of the present invention includes any human subject diagnosed with, suffering from, or at risk of developing cancer or a precancerous condition. The subject of the present invention includes any human subject expressing an EZH2 mutant. For example, the EZH2 mutant contains one or more mutations, where the mutation is a substitution, point mutation, nonsense mutation, missense mutation, deletion, or insertion. The EZH2 mutant of the present invention may contain a mutation in the substrate pocket domain defined by SEQ ID NO: 6. The EZH2 mutant may have a substitution at amino acid Y641. Preferably, the EZH2 mutant has one of the following mutations: a substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 with phenylalanine (F) (Y641F); a substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 with histidine (H) (Y641H); a substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 with asparagine (N) (Y641N); a substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 with serine (S) (Y641S); a substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 with cysteine ​​(C) (Y641C).

[0038] Other mutations in EZH2 include, but are not limited to, a substitution of the wild-type residue alanine (A) at amino acid position 677 of SEQ ID NO:1 with glycine (G) (A677G); a substitution of the wild-type residue alanine (A) at amino acid position 687 of SEQ ID NO:1 with valine (V) (A687V); a substitution of the wild-type residue valine (V) at amino acid position 674 of SEQ ID NO:1 with methionine (M) (V674M); a substitution of the wild-type residue valine (V) at amino acid position 685 of SEQ ID NO:1 with methionine (M) (V674M); Substitution of the wild-type residue arginine (R) with histidine (H) (R685H); substitution of the wild-type residue arginine (R) at amino acid position 685 of SEQ ID NO: 1 with cysteine ​​(C) (R685C); substitution of the wild-type residue asparagine (N) at amino acid position 322 of SEQ ID NO: 3 with serine (S) (N322S); substitution of the wild-type residue arginine (R) at amino acid position 288 of SEQ ID NO: 3 with glutamine (Q) (R288Q ... substitution of the wild-type residue threonine (T) at amino acid position 573 with isoleucine (I) (T573I); substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with glutamic acid (E) (D664E); substitution of the wild-type residue arginine (R) at amino acid position 458 of SEQ ID NO: 5 with glutamine (Q) (R458Q); substitution of the wild-type residue glutamic acid (E) at amino acid position 249 of SEQ ID NO: 3 with lysine (K) (E2 49K), a substitution of the wild-type residue arginine (R) at amino acid position 684 of SEQ ID NO: 3 with cysteine ​​(C) (R684C), a substitution of the wild-type residue arginine (R) at amino acid position 628 of SEQ ID NO: 11 with histidine (H) (R628H), a substitution of the wild-type residue glutamine (Q) at amino acid position 501 of SEQ ID NO: 5 with histidine (H) (Q501H), a substitution of the wild-type residue aspartic acid (D) at amino acid position 192 of SEQ ID NO: 3 with aspartic acid (D) (Q501H), a substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with valine (V) (D664V); a substitution of the wild-type residue valine (V) at amino acid position 704 of SEQ ID NO: 3 with leucine (L) (V704L); a substitution of the wild-type residue proline (P) at amino acid position 132 of SEQ ID NO: 3 with serine (S) (P132S); a substitution of the wild-type residue glutamic acid (E) at amino acid position 669 of SEQ ID NO: 11 with lysine (K) (E669K); a substitution of the wild-type residue alanine (A) at amino acid position 255 of SEQ ID NO: 3 with leucine (L) (V704L); substitution of wild-type residue glutamic acid (E) at amino acid position 726 of SEQ ID NO:3 with valine (V) (E726V); substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO:3 with tyrosine (Y) (C571Y); substitution of wild-type residue phenylalanine (F) at amino acid position 145 of SEQ ID NO:3 with cysteine ​​(C) (F145C); substitution of wild-type residue asparagine (N) at amino acid position 693 of SEQ ID NO:3 with threonine (T) (N693T); substitution of wild-type residue phenylalanine (F) at amino acid position 145 of SEQ ID NO:3 with cysteine ​​(C) (F145C). substitution of wild-type residue phenylalanine (F) with serine (S) (F145S), substitution of wild-type residue glutamine (Q) at amino acid position 109 of SEQ ID NO: 11 with histidine (H) (Q109H), substitution of wild-type residue phenylalanine (F) at amino acid position 622 of SEQ ID NO: 11 with cysteine ​​(C) (F622C), substitution of wild-type residue glycine (G) at amino acid position 135 of SEQ ID NO: 3 with arginine (R) (G135R), substitution of wild-type residue arginine (R) at amino acid position 168 of SEQ ID NO: 5 with glutamine (Q) (R168Q), substitution of wild-type residue arginine (R) at amino acid position 168 of SEQ ID NO: 3 with cysteine ​​(C) (F622C). substitution of the wild-type residue arginine (R) at amino acid position 310 of SEQ ID NO: 5 with cysteine ​​(C) (R310C); substitution of the wild-type residue arginine (R) at amino acid position 561 of SEQ ID NO: 3 with histidine (H) (R561H); substitution of the wild-type residue arginine (R) at amino acid position 634 of SEQ ID NO: 11 with histidine (H) (R634H); substitution of the wild-type residue glycine (G) at amino acid position 660 of SEQ ID NO: 3 with arginine (R) (G660R);Substitution of the wild-type residue tyrosine (Y) at amino acid position 181 of SEQ ID NO: 3 with cysteine ​​(C) (Y181C), substitution of the wild-type residue histidine (H) at amino acid position 297 of SEQ ID NO: 3 with arginine (R) (H297R), substitution of the wild-type residue cysteine ​​(C) at amino acid position 612 of SEQ ID NO: 11 with serine (S) (C612S), substitution of the wild-type residue histidine (H) at amino acid position 694 of SEQ ID NO: 3 with tyrosine (Y) (H694Y), substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with alanine (A) (D664A) ), a substitution of the wild-type residue isoleucine (I) at amino acid position 150 of SEQ ID NO:3 with threonine (T) (I150T), a substitution of the wild-type residue isoleucine (I) at amino acid position 264 of SEQ ID NO:3 with arginine (R) (I264R), a substitution of the wild-type residue proline (P) at amino acid position 636 of SEQ ID NO:3 with leucine (L) (P636L), a substitution of the wild-type residue isoleucine (I) at amino acid position 713 of SEQ ID NO:3 with threonine (T) (I713T), a substitution of the wild-type residue glutamine (Q) at amino acid position 501 of SEQ ID NO:5 with proline (P) (Q5 01P), a substitution of the wild type residue lysine (K) at amino acid position 243 of SEQ ID NO: 3 with glutamine (Q) (K243Q), a substitution of the wild type residue glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5 with aspartic acid (D) (E130D), a substitution of the wild type residue arginine (R) at amino acid position 509 of SEQ ID NO: 3 with glycine (G) (R509G), a substitution of the wild type residue arginine (R) at amino acid position 566 of SEQ ID NO: 3 with histidine (H) (R566H), a substitution of the wild type residue aspartic acid (D) at amino acid position 677 of SEQ ID NO: 3 with histidine (H) (R566H), substitution (D677H), substitution of the wild type residue lysine (K) at amino acid position 466 of SEQ ID NO: 5 with asparagine (N) (K466N), substitution of the wild type residue arginine (R) at amino acid position 78 of SEQ ID NO: 3 with histidine (H) (R78H), substitution of the wild type residue lysine (K) at amino acid position 1 of SEQ ID NO: 6 with methionine (M) (K6M), substitution of the wild type residue serine (S) at amino acid position 538 of SEQ ID NO: 3 with leucine (L) (S538L), substitution of the wild type residue leucine (L) at amino acid position 149 of SEQ ID NO: 3 with glutamine (Q) (L149Q),Substitution of the wild-type residue leucine (L) at amino acid position 252 of SEQ ID NO: 3 with valine (V) (L252V, ), a substitution of the wild-type residue leucine (L) at amino acid position 674 of SEQ ID NO:3 with valine (V) (L674V), a substitution of the wild-type residue alanine (A) at amino acid position 656 of SEQ ID NO:3 with valine (V) (A656V), a substitution of the wild-type residue alanine (A) at amino acid position 731 of SEQ ID NO:3 with aspartic acid (D) (Y731D), a substitution of the wild-type residue alanine (A) at amino acid position 345 of SEQ ID NO:3 with threonine (T) (A345T), a substitution of the wild-type residue alanine (A) at amino acid position 244 of SEQ ID NO:3 with aspartic acid (D) (Y244D), a substitution of the wild-type residue cysteine ​​(C) at amino acid position 576 of SEQ ID NO:3 with threonine (T) (Y244D), substitution of the wild-type residue asparagine (N) at amino acid position 640 of SEQ ID NO: 3 with lysine (K) (N640K); substitution of the wild-type residue asparagine (N) at amino acid position 675 of SEQ ID NO: 3 with lysine (K) (N675K); substitution of the wild-type residue aspartic acid (D) at amino acid position 579 of SEQ ID NO: 11 with tyrosine (Y) (D579Y); substitution of the wild-type residue asparagine (N) at amino acid position 693 of SEQ ID NO: 3 with isoleucine (I) (N693I); and substitution of the wild-type residue asparagine (N) at amino acid position 693 of SEQ ID NO: 3 with lysine (K) (N693K).

[0039] Other mutations in EZH2 may include a frameshift at amino acid position 730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 73, 656, 718, 374, 592, 505, 730, or 363 of SEQ ID NO: 3, 5, or 11, or the corresponding nucleotide position of a nucleic acid sequence encoding SEQ ID NO: 3, 5, or 11; a deletion of glutamic acid (E) and leucine (L) at amino acid positions 148 and 149 of SEQ ID NO: 3, 5, or 11, or a nonsense mutation at amino acid position 733, 25, 317, 62, 553, 328, 58, 207, 123, 63, 137, or 60 of SEQ ID NO: 3, 5, or 11.

[0040] A subject of the present invention may have resistance to any one or more other therapeutic agents or any compound described herein, for example, a subject may have resistance to an EZH inhibitor or prednisone.

[0041] The invention features a method for inhibiting cancer cell proliferation, comprising contacting the cancer cell with a composition comprising any compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more additional therapeutic agents. Inhibiting cancer cell proliferation includes slowing cancer cell proliferation, inducing cell death, reducing cancer cell viability, inhibiting or slowing tumor growth, or reducing tumor size.

[0042] The present invention features a method of combination therapy in which any compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents are administered simultaneously or sequentially. For example, any compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, may be administered followed by one or more other therapeutic agents. For example, any compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, may be administered followed by a composition comprising any compound of Formula (IIa), or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. The simultaneous or sequential administration of any compound of Formula (IIa) and / or each therapeutic agent may be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same or different routes.

[0043] The combination therapy methods featured in the present invention may result in a synergistic effect, where the effect of the combination of compounds or other therapeutic agents is greater than the sum of the effects resulting from the administration of either compound or other therapeutic agent as a single agent. A synergistic effect may be an effect that cannot be achieved by the administration of either compound or other therapeutic agent as a single agent. Synergistic effects include, but are not limited to, a reduction in tumor size. Synergistic effects include the effect of treating cancer by promoting the growth of a cancer cell, inhibiting tumor growth, or increasing the survival time of a subject. Synergistic effects may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or slowing cancer cell growth.

[0044] The composition of the present invention may be administered at a dose of 0.01 mg / kg per day to about 1000 mg / kg per day. Any compound of formula (IIa) or a pharmaceutically acceptable salt thereof may be administered at a dose of 0.01 mg / kg per day to about 1000 mg / kg per day. Any other therapeutic agent may be administered at a dose of 0.01 mg / kg per day to about 1000 mg / kg per day.

[0045] Unless otherwise defined, 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. As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0046] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0047] [Figure 1]FIG. 1 is a schematic diagram detailing in vitro treatment schedules for determining the effect of combination therapy on cell viability. (A) Administration of Compound 44 prior to administration of Compound 44 and components of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone). (B) Administration of Compound 44 prior to components of CHOP. (C) Administration of components of CHOP prior to administration of Compound 44 and components of CHOP. After 4 days, cell viability was determined. [Figure 2] Figure 1 shows four graphs measuring in vitro cancer cell viability after treatment with Compound 44 and CHOP components, alone or in combination. WSU-DLCL2 cells were treated as shown in Figure 1A. Cells were first treated with varying concentrations of Compound 44. Four days later, cells were treated with varying concentrations of Compound 44 and a combination of (A) mafosfamide (a cyclophosphamide metabolite), (B) doxorubicin, (C) vincristine, or (D) prednisolone (a prednisone metabolite). Control cells were treated with DMSO. Cell viability was normalized to the percentage of DMSO-treated cell viability for each Compound 44 concentration. [Figure 3] Figure 1 shows three graphs measuring cancer cell viability after various treatment schedules. WSU-DCLC2 cells were treated with increasing concentrations of prednisolone and Compound 44. Cells were treated with Compound 44 and prednisolone according to the treatment schedules in (A) Figure 1A, (B) Figure 1B, or (C) Figure 1C. Cell viability was normalized to DMSO / DMSO-treated samples. [Figure 4]These graphs show three measurements of cancer cell viability after treatment with prednisolone and Compound 44 in cell lines with different EZH2 mutations. Cells were treated with increasing concentrations of prednisolone and Compound 44 as described in Figure 1A. Cell viability was normalized to the DMSO / DMSO-treated sample. (A) WSU-DLCL2 cells express the Y641F mutation and are sensitive to EZH2 inhibitors. (B) RL cells express the Y641N mutation and are resistant to EZH2 inhibitors and prednisolone. (C) OCI-LY19 cells are wild-type at Y641 and show sensitivity to prednisolone but no increased sensitivity to Compound 44 or the combination of Compound 44 and prednisolone. [Figure 5] Figure 5 shows a panel of five graphs depicting the pharmacokinetic profile of in vivo coadministration of Compound 44 with a CHOP component. BALB / c mice were administered a single oral dose of Compound 44 and a CHOP component. Plasma Compound 44 concentrations (ng / mL) were measured at various time points, 0-24 hours post-administration. Cyclophosphamide was administered intraperitoneally at 30 mg / kg. Vincristine was administered intravenously at 0.375 mg / kg. Doxorubicin was administered intravenously at 2.475 mg / kg. Prednisolone was administered orally at 0.15 mg / kg. Compound 44 was administered orally at 225 mg / kg. [Figure 6]These graphs show the effect of Compound 44 and CHOP administration in a SUDHL6 xenograft model on tumor growth and survival. Athymic nude mice were subcutaneously injected with 1x107 SUDHL6 human lymphoma cells. After tumors reached approximately 120mm3 in size, Compound 44 was administered three times daily (TID) or twice daily (BID) for 28 days at the indicated doses. CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisone) was administered twice daily at 225mg / kg on days 1 and 8 (225mg / kg BID and CHOP). Control mice received no Compound 44 (vehicle) or CHOP alone (CHOP). Tumor volume was measured twice weekly. Animals were euthanized when tumor volumes reached 2000mm3 or 60 days after the first administration. (A) For tumor growth delay analysis, median tumor volumes were calculated for each treatment group by measuring tumors twice weekly for 60 days or until tumors reached 2000 mm. (B) Kaplan-Meier curves show mouse survival rates. [Figure 7]These graphs show the effects of Compound 44 and CHOP administration in a WSU-DLCL2 xenograft model on tumor growth and survival. SCID mice were subcutaneously injected with 1x10 WSU-DLCL2 human lymphoma cells. After tumors reached approximately 120 mm in size, Compound 44 was administered three times daily (TID), twice daily (BID), or once daily (QD) at the indicated doses for 28 days. CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisone) was administered twice daily at 225 mg / kg on days 1 and 22 to mice receiving 225 mg / kg BID and CHOP. Control mice received no Compound 44 (vehicle) or COP only (COP). Tumor volume was measured twice weekly. Animals were euthanized 28 days after the first administration. (A) Treatment efficacy was determined by measuring mean tumor volume over the course of treatment. (B) Compound 44 concentrations (ng / mL) were measured in plasma from mice 28 days before dosing (trough) and 3 hours after dosing (post-dose). (C) Compound 44 concentrations (ng / g) were measured in tumor tissue from mice 3 hours after dosing on day 28. [Figure 8] Western blot analysis and graph showing histone methylation status from tumor samples from the WSU-DLCL2 xenograft model. Tumors were harvested 28 days after injection, and histones were extracted. (A) Western blots were probed with an antibody that specifically recognizes trimethylated lysine 27 of histone H3 and total histone H3 protein (H3K27me3). (B) Tumor methylation status was determined by ELISA. H3K27 trimethylation was detected and normalized to total histone H3 levels. [Figure 9]These four graphs show the effect of administration of Compound 44 and COP in a SUDHL10 xenograft model on tumor growth and survival. SCID mice were subcutaneously injected with 1x107 SUDHL10 human lymphoma cells. After tumors reached approximately 120 mm3 in size, Compound 44 was administered twice daily (BID) at the indicated doses for 28 days. COP (cyclophosphamide, vincristine, and prednisone) was administered at 250 mg / kg twice daily on days 1 and 22 to mice receiving COP (250 mg / kg BID and COP). Control mice received no Compound 44 (vehicle) or COP alone (COP). Tumor volume was measured twice weekly. (A) Treatment efficacy was determined by measuring the mean tumor volume over the course of treatment. (B) Mean tumor weight was determined after mice were euthanized 28 days after the first dose. (C) For tumor growth delay analysis, the mean tumor volume and SEM were calculated for each treatment group by measuring tumors twice a week for 60 days or until tumors reached 2000 mm. (D) Kaplan-Meier curves show the survival rate of treated mice. [Figure 10] These four graphs show the pharmacokinetic and pharmacodynamic profiles of COP and Compound 44 in a SUDHL10 xenograft model. Tumor-bearing mice were euthanized 28 days after treatment, and tissues were harvested. (A) Compound 44 concentrations (ng / mL) were measured in plasma from mice by LC-MS / MS at 28 days before administration (trough) and after administration (post-administration). The methylation status of various tissues from tumor-bearing mice was determined by ELISA: (B) tumor, (C) spleen, and (D) bone marrow. H3K27 trimethylation was detected and normalized to total histone H3 levels. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention is based on the discovery that the combination of an EZH2 histone methyltransferase inhibitor with another anticancer agent can treat certain tumors with better results than those achieved by treating the tumor with the EZH2 histone methyltransferase inhibitor and the anticancer agent alone. Accordingly, the present invention provides compositions comprising an EZH2 histone methyltransferase inhibitor and one or more other therapeutic agents, and methods for their use to treat diseases, such as cancer, whose course can be affected by modulating the methylation status of histones or other proteins. In certain embodiments, the present invention features a composition comprising a compound of Formula (IIa) and prednisone. The present invention also includes methods for combination therapy comprising an EZH2 histone methyltransferase inhibitor and one or more therapeutic agents, such as a compound of Formula (IIa) and prednisone, for treating cancer, such as follicular lymphoma (FL) or diffuse large B-cell lymphoma (DCLBL). In particular, the methods of the present invention are useful for treating or preventing cancer or inhibiting cancer cell growth.

[0049] EZH2 is a histone methyltransferase, a catalytic subunit of the PRC2 complex, that catalyzes the mono- to tri-methylation of lysine 27 (H3-K27) on histone H3. Histone H3-K27 trimethylation is a mechanism for repressing the transcription of specific genes near the histone modification site. This trimethylation is known to be a cancer marker whose expression is altered in cancers such as prostate cancer (see, for example, U.S. Patent Application Publication No. 2003 / 0175736, the entire contents of which are incorporated herein by reference). Other studies have provided evidence for a functional link between deregulated EZH2 expression, transcriptional repression, and neoplastic transformation. Varambally et al. (2002) Nature 419(6907):624-9; Kleer et al. (2003) Proc Natl Acad Sci USA 100(20):11606-11.

[0050] One aspect of the present invention relates to a method for treating or alleviating symptoms of cancer or precancerous conditions in a subject by administering a therapeutically effective amount of an EZH2 inhibitor and one or more other therapeutic agents to the subject expressing an EZH2 mutant. The EZH2 mutant of the present invention refers to a mutant EZH2 polypeptide or a nucleic acid sequence encoding the mutant EZH2 polypeptide. In certain embodiments, the EZH2 mutant contains one or more mutations in its substrate pocket domain as defined in SEQ ID NO: 6. For example, the mutation may be a substitution, point mutation, nonsense mutation, missense mutation, deletion, or insertion.

[0051] Human EZH2 nucleic acids and polypeptides have been previously described, e.g., by Chen et al. (1996) Genomics 38:30-7 [746 amino acids]; Swiss-Prot accession number Q15910 [746 amino acids]; GenBank accession numbers NM_004456 and NP_004447 (isoform a [751 amino acids]); and GenBank accession numbers NM_152998 and NP_694543 (isoform b [707 amino acids]), each of which is incorporated by reference in its entirety.

[0052] [Table 1]

[0053] [Table 2-1] [Table 2-2]

[0054] [Table 3]

[0055] [Table 4-1] [Table 4-2]

[0056] [Table 5]

[0057] [Table 6]

[0058] [Table 7-1] [Table 7-2] A structural model of a partial EZH2 protein based on the A chain of nuclear receptor-binding SET domain protein 1 (NSD1) is provided below, which corresponds to amino acid residues 533-732 of the EZH2 sequence of SEQ ID NO:1.

[0059] [ka]

[0060] The corresponding amino acid sequence of this structural model is provided below: Substrate pocket domain Residues in the SET domain are italicized.

[0061] [ka]

[0062] The catalytic site of EZH2 is thought to be residues in a conserved domain of proteins known as the SET domain. The amino acid sequence of the SET domain of EZH2 is provided by the following partial sequence spanning amino acid residues 613-726 of Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1):

[0063] [ka] The underlined tyrosine (Y) residue in SEQ ID NO: 7 is Tyr641 (Y641) in Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1).

[0064] The SET domain of GenBank Accession No. NP_004447 (SEQ ID NO: 3) spans amino acid residues 618-731 and is identical to SEQ ID NO: 6. The tyrosine residue corresponding to Y641 in Swiss-Prot Accession No. Q15910, underlined in SEQ ID NO: 7, is Tyr646 (Y646) in GenBank Accession No. NP_004447 (SEQ ID NO: 3).

[0065] The SET domain of GenBank Accession No. NP_694543 (SEQ ID NO: 5) spans amino acid residues 574-687 and is identical to SEQ ID NO: 7. The tyrosine residue corresponding to Y641 in Swiss-Prot Accession No. Q15910, underlined in SEQ ID NO: 7, is Tyr602 (Y602) in GenBank Accession No. NP_694543 (SEQ ID NO: 5).

[0066] The nucleotide sequence encoding the SET domain of GenBank accession number NP_004447 is

[0067] [ka] (SEQ ID NO: 8) (wherein the codon encoding Y641 is underlined).

[0068] For purposes of this application, amino acid residue Y641 of human EZH2 should be understood to refer to the tyrosine residue that is or corresponds to Y641 in Swiss-Prot Accession No. Q15910.

[0069] [Table 8] Also, for purposes of this application, a Y641 mutant of human EZH2, or equivalently, a Y641 mutant of EZH2, should be understood to refer to human EZH2 in which the amino acid residue corresponding to Y641 in wild-type human EZH2 has been replaced with an amino acid residue other than tyrosine.

[0070] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from the amino acid sequence of wild-type human EZH2 only by substitution of a single amino acid residue corresponding to Y641 of wild-type human EZH2 with an amino acid residue other than tyrosine.

[0071] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to Y641 in wild-type human EZH2 with phenylalanine (F). The Y641 mutant of EZH2 according to this embodiment is referred to herein as the Y641F mutant, or equivalently, Y641F.

[0072] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to Y641 in wild-type human EZH2 with histidine (H). The Y641 mutant of EZH2 according to this embodiment is referred to herein as the Y641H mutant, or equivalently, Y641H.

[0073] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to Y641 in wild-type human EZH2 with asparagine (N). The Y641 mutant of EZH2 according to this embodiment is referred to herein as the Y641N mutant, or equivalently, Y641N.

[0074] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to Y641 in wild-type human EZH2 with serine (S). The Y641 mutant of EZH2 according to this embodiment is referred to herein as the Y641S mutant, or equivalently, Y641S.

[0075] In one embodiment, the amino acid sequence of the Y641 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to Y641 in wild-type human EZH2 with cysteine ​​(C). The Y641 mutant of EZH2 according to this embodiment is referred to herein as the Y641C mutant, or equivalently, Y641C.

[0076] In one embodiment, the amino acid sequence of the A677 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to A677 in wild-type human EZH2 with a non-alanine amino acid, preferably glycine (G). The A677 mutant of EZH2 according to this embodiment is referred to herein as the A677 mutant, preferably the A677G mutant, or equivalently, A677G.

[0077] In one embodiment, the amino acid sequence of the A687 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to A687 in wild-type human EZH2 with a non-alanine amino acid, preferably valine (V). The A687 mutant of EZH2 according to this embodiment is referred to herein as an A687 mutant, preferably an A687V mutant, or equivalently, A687V.

[0078] In one embodiment, the amino acid sequence of the R685 mutant of EZH2 differs from that of wild-type human EZH2 only by the substitution of a single amino acid residue corresponding to R685 in wild-type human EZH2 with a non-arginine amino acid, preferably histidine (H) or cysteine ​​(C). The R685 mutant of EZH2 according to this embodiment is referred to herein as an R685 mutant, preferably an R685C mutant or an R685H mutant, or equivalently, R685H or R685C.

[0079] In one embodiment, the amino acid sequence of the mutant of EZH2 differs from the amino acid sequence of wild-type human EZH2 at one or more amino acid residues in its substrate pocket domain as defined in SEQ ID NO: 6. A mutant of EZH2 according to this embodiment is referred to herein as an EZH2 mutant.

[0080] Other exemplary substitution amino acid mutations include substitutions at amino acid positions 677, 687, 674, 685, or 641 of SEQ ID NO:1, such as, but not limited to, a substitution of the wild-type residue alanine (A) at amino acid position 677 of SEQ ID NO:1 with glycine (G) (A677G); a substitution of the wild-type residue alanine (A) at amino acid position 687 of SEQ ID NO:1 with valine (V) (A687V); a substitution of the wild-type residue valine (V) at amino acid position 674 of SEQ ID NO:1 with methionine (M) (V674M); a substitution of the wild-type residue arginine (R) at amino acid position 685 of SEQ ID NO:1 with histidine (H) (R685H); a substitution of the wild-type residue arginine (R) at amino acid position 685 of SEQ ID NO:1 with histidine (H) (R685H); substitution of the wild-type residue arginine (R) at amino acid position 641 of SEQ ID NO:1 with cysteine ​​(C) (R685C); substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 with phenylalanine (F) (Y641F); substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 with histidine (H) (Y641H); substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 with asparagine (N) (Y641N); substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 with serine (S) (Y641S); or substitution of the wild-type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO:1 with cysteine ​​(C) (Y641C).

[0081] The mutations of the present invention also include a substitution of the wild type residue asparagine (N) at amino acid position 322 of SEQ ID NO: 3 with serine (S) (N322S), a substitution of the wild type residue arginine (R) at amino acid position 288 of SEQ ID NO: 3 with glutamine (Q) (R288Q ... asparagine (N) at amino acid position 322 of SEQ ID NO: 3 with serine (S) (N322S), a substitution of the wild type residue arginine (R) at amino acid position 288 of SEQ ID NO: 3 with glutamine (Q) (R288Q), a substitution of the wild type residue asparagine (N) at amino acid position 322 of SEQ ID NO: 3 with a substitution of the wild-type residue threonine (T) at amino acid position 573 with isoleucine (I) (T573I); a substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with glutamic acid (E) (D664E); a substitution of the wild-type residue arginine (R) at amino acid position 458 of SEQ ID NO: 5 with glutamine (Q) (R458Q); a substitution of the wild-type residue glutamic acid (E) at amino acid position 249 of SEQ ID NO: 3 with lysine (K) (E249K); a substitution of the wild-type residue arginine (R) at amino acid position 684 of SEQ ID NO: 3 with cysteine ​​(C) (R684C); a substitution of the wild-type residue arginine (R) at amino acid position 628 of SEQ ID NO: 11 with histidine (H) (R628H); a substitution of the wild-type residue glutamine (Q) at amino acid position 501 of SEQ ID NO: 5 with histidine (H) (Q501H); a substitution of the wild-type residue aspartic acid (D) at amino acid position 192 of SEQ ID NO: 3 with asparagine (N) (D192N); a substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with valine (V) (D664V); a substitution of the wild-type residue valine (V) at amino acid position 704 of SEQ ID NO: 3 with leucine (L) (V704L); Substitution of wild-type residue proline (P) at amino acid position 132 of SEQ ID NO: 3 with serine (S) (P132S), substitution of wild-type residue glutamic acid (E) at amino acid position 669 of SEQ ID NO: 11 with lysine (K) (E669K), substitution of wild-type residue alanine (A) at amino acid position 255 of SEQ ID NO: 3 with threonine (T) (A255T), substitution of wild-type residue glutamic acid (E) at amino acid position 726 of SEQ ID NO: 3 with valine (V) (E726V), substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO: 3 with tyrosine (Y) (C571Y), substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO: 3 with tyrosine (Y) (C571Y), substitution of wild-type residue alanine (A) at amino acid position 255 of SEQ ID NO: 3 with threonine (T) (A255T), substitution of wild-type residue glutamic acid (E) at amino acid position 726 of SEQ ID NO: 3 with valine (V) (E726V), substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO: 3 with tyrosine (Y) (C571Y), substitution of wild-type residue alanine (A) at amino acid position 255 of SEQ ID NO: 3 with threonine (T) (A255T), substitution of wild-type residue glutamic acid (E) at amino acid position 255 of SEQ ID NO: 3 with threonine (T) (A255V), substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO: 3 with a substitution of the wild-type residue phenylalanine (F) at amino acid position 145 with cysteine ​​(C) (F145C), a substitution of the wild-type residue asparagine (N) at amino acid position 693 of SEQ ID NO: 3 with threonine (T) (N693T), a substitution of the wild-type residue phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3 with serine (S) (F145S), a substitution of the wild-type residue glutamine (Q) at amino acid position 109 of SEQ ID NO: 11 with histidine (H) (Q109H), a substitution of the wild-type residue phenylalanine (F) at amino acid position 622 of SEQ ID NO: 11 with cysteine ​​(C) (F622C),a substitution of the wild-type residue glycine (G) at amino acid position 135 of SEQ ID NO: 3 with arginine (R) (G135R), a substitution of the wild-type residue arginine (R) at amino acid position 168 of SEQ ID NO: 5 with glutamine (Q) (R168Q), a substitution of the wild-type residue glycine (G) at amino acid position 159 of SEQ ID NO: 3 with arginine (R) (G159R), a substitution of the wild-type residue arginine (R) at amino acid position 310 of SEQ ID NO: 5 with cysteine ​​(C) (R310C), a substitution of the wild-type residue arginine (R) at amino acid position 561 of SEQ ID NO: 3 with histidine (H) (R561H), Substitution of the wild-type residue arginine (R) at amino acid position 634 of SEQ ID NO: 11 with histidine (H) (R634H), substitution of the wild-type residue glycine (G) at amino acid position 660 of SEQ ID NO: 3 with arginine (R) (G660R), substitution of the wild-type residue tyrosine (Y) at amino acid position 181 of SEQ ID NO: 3 with cysteine ​​(C) (Y181C), substitution of the wild-type residue histidine (H) at amino acid position 297 of SEQ ID NO: 3 with arginine (R) (H297R), substitution of the wild-type residue cysteine ​​(C) at amino acid position 612 of SEQ ID NO: 11 with serine (S) (C612S), a substitution of the wild-type residue histidine (H) at amino acid position 694 of SEQ ID NO:3 with tyrosine (Y) (H694Y); a substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO:3 with alanine (A) (D664A); a substitution of the wild-type residue isoleucine (I) at amino acid position 150 of SEQ ID NO:3 with threonine (T) (I150T); a substitution of the wild-type residue isoleucine (I) at amino acid position 264 of SEQ ID NO:3 with arginine (R) (I264R); a substitution of the wild-type residue proline (P) at amino acid position 636 of SEQ ID NO:3 with leucine (L) (P636L); a substitution of the wild-type residue isoleucine (I) at amino acid position 713 of SEQ ID NO:3 with threonine (T) (I713T); a substitution of the wild-type residue glutamine (Q) at amino acid position 501 of SEQ ID NO:5 with proline (P) (Q501P); a substitution of the wild-type residue lysine (K) at amino acid position 243 of SEQ ID NO:3 with glutamine (Q) (K243Q); a substitution of the wild-type residue glutamic acid (E) at amino acid position 130 of SEQ ID NO:5 with aspartic acid (D) (E130D); a substitution of the wild-type residue arginine (R) at amino acid position 509 of SEQ ID NO:3 with glycine (G) (R509G); Substitution of wild-type residue arginine (R) at amino acid position 566 of SEQ ID NO:3 with histidine (H) (R566H), substitution of wild-type residue aspartic acid (D) at amino acid position 677 of SEQ ID NO:3 with histidine (H) (D677H), substitution of wild-type residue lysine (K) at amino acid position 466 of SEQ ID NO:5 with asparagine (N) (K466N), substitution of wild-type residue arginine (R) at amino acid position 78 of SEQ ID NO:3 with histidine (H) (R78H), substitution of wild-type residue lysine (K) at amino acid position 1 of SEQ ID NO:6 with methionine (M) ( K6M), substitution of wild-type residue serine (S) at amino acid position 538 of SEQ ID NO: 3 with leucine (L) (S538L), substitution of wild-type residue leucine (L) at amino acid position 149 of SEQ ID NO: 3 with glutamine (Q) (L149Q), substitution of wild-type residue leucine (L) at amino acid position 252 of SEQ ID NO: 3 with valine (V) (L252V), substitution of wild-type residue leucine (L) at amino acid position 674 of SEQ ID NO: 3 with valine (V) (L674V), substitution of wild-type residue alanine (A) at amino acid position 656 of SEQ ID NO: 3 with valine (V) ( A656V), a substitution of the wild-type residue alanine (A) at amino acid position 731 of SEQ ID NO: 3 with aspartic acid (D) (Y731D), a substitution of the wild-type residue alanine (A) at amino acid position 345 of SEQ ID NO: 3 with threonine (T) (A345T), a substitution of the wild-type residue alanine (A) at amino acid position 244 of SEQ ID NO: 3 with aspartic acid (D) (Y244D), a substitution of the wild-type residue cysteine ​​(C) at amino acid position 576 of SEQ ID NO: 3 with tryptophan (W) (C576W), a substitution of the wild-type residue asparagine at amino acid position 640 of SEQ ID NO: 3 with tryptophan (W) (C576W), The wild-type residues may also include a substitution of aspartic acid (D) at amino acid position 679 of SEQ ID NO: 11 with tyrosine (Y) (D579Y), a substitution of aspartic acid (N) at amino acid position 693 of SEQ ID NO: 3 with isoleucine (I) (N693I), and a substitution of asparagine (N) at amino acid position 693 of SEQ ID NO: 3 with lysine (K) (N693K).

[0082] The mutation of the present invention may be a frameshift at amino acid position 730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 73, 656, 718, 374, 592, 505, 730, or 363 of SEQ ID NO: 3, 5, or 11, or the corresponding nucleotide position of a nucleic acid sequence encoding SEQ ID NO: 3, 5, or 11. The EZH2 mutation may be an insertion of glutamic acid (E) between amino acid positions 148 and 149 of SEQ ID NO: 3, 5, or 11. Another example of an EZH2 mutation is a deletion of glutamic acid (E) and leucine (L) between amino acid positions 148 and 149 of SEQ ID NO: 3, 5, or 11. The EZH2 mutant may further comprise a nonsense mutation at amino acid position 733, 25, 317, 62, 553, 328, 58, 207, 123, 63, 137 or 60 of SEQ ID NO: 3, 5 or 11.

[0083] Cells heterozygous for EZH2 are expected to exhibit a malignant phenotype due to the efficient formation of H3-K27me1 by the WT enzyme and the efficient subsequent transition of this precursor species to H3-K27me2, and particularly to H3-K27me3, by the mutant enzyme form.

[0084] Previous results indicate that the pathogenesis of follicular lymphoma and diffuse large B-cell lymphoma depends on the enzyme that performs H3-K27 mono-methylation and the enzymatic coupling of certain mutant forms of EZH2.For example, cells expressing Y641 mutant EZH2 may be more sensitive to small molecule EZH2 inhibitors than cells expressing WT EZH2.In ​​particular, cells expressing Y641 mutant EZH2 show reduced growth, division or proliferation, or even undergo apoptosis or necrosis after treatment with EZH2 inhibitors.In contrast, cells expressing WT EZH2 do not respond to the anti-proliferative effects of EZH2 inhibitors (U.S. Patent Application No. 61 / 381,684, the entire contents of which are incorporated herein by reference).

[0085] One aspect of the present invention is a method for treating or alleviating symptoms of cancer or a precancerous condition in a subject expressing an EZH2 mutant comprising a mutation in the substrate pocket domain as defined in SEQ ID NO:6 by administering to the subject a therapeutically effective amount of an EZH2 inhibitor described herein, e.g., a compound of Formula (IIa) in combination with another agent suitable for simultaneous, sequential, or staggered administration together.

[0086] Another aspect of the present invention is a method for inhibiting the conversion of H3-K27 to trimethylated H3-K27 in a subject. This inhibition may include inhibiting the conversion of unmethylated H3-K27 to monomethylated H3-K27, monomethylated H3-K27 to dimethylated H3-K27, dimethylated H3-K27 to trimethylated H3-K27, or any combination thereof, such as the conversion of monomethylated H3-K27 to dimethylated H3-K27 and dimethylated H3-K27 to trimethylated H3-K27, in a subject. As used herein, unmethylated H3-K27 refers to histone H3 that does not contain a methyl group covalently attached to the amino group of lysine 27. As used herein, monomethylated H3-K27 refers to histone H3 that contains a single methyl group covalently attached to the amino group of lysine 27. Monomethylated H3-K27 is also referred to herein as H3-K27me1. As used herein, di-methylated H3-K27 refers to histone H3 containing two methyl groups covalently attached to the amino group of lysine 27. Di-methylated H3-K27 is also referred to herein as H3-K27me2. As used herein, tri-methylated H3-K27 refers to histone H3 containing three methyl groups covalently attached to the amino group of lysine 27. Tri-methylated H3-K27 is also referred to herein as H3-K27me3.

[0087] Histone H3 is a 136 amino acid long protein, the sequence of which is known. See, for example, GenBank Accession No. CAB02546, the contents of which are incorporated herein by reference. As further disclosed herein, in addition to full-length histone H3, peptide fragments of histone H3 containing a lysine residue corresponding to K27 of full-length histone H3 can be used as substrates for EZH2 (as well as mutant forms of EZH2) to assess the conversion of H3-K27m1 to H3-K27m2 and H3-K27m2 to H3-K27m3. In one embodiment, such peptide fragments correspond to amino acid residues 21-44 of histone H3. Such peptide fragments have the amino acid sequence LATKAARKSAPATGGVKKPHRYRP (SEQ ID NO: 10).

[0088] The compositions of the present invention comprise a compound of formula (IIa) and one or more other therapeutic agents, or pharmaceutically acceptable salts thereof.The compound of formula (IIa) is suitable for administration as part of a combined therapy with one or more other therapeutic agents or treatment modalities, suitable for administration together, sequentially, or alternately.Other compounds of formula (IIa) suitable for the method of the present invention are described in U.S. Patent Application Publication No. 20120264734, the contents of which are incorporated herein by reference in their entirety.

[0089] The present invention relates to a compound of formula (IIa):

[0090] [ka] (In the formula, R7, R8, R a and R b is defined herein.) or a pharmaceutically acceptable salt or ester thereof.

[0091] The compounds of formula (IIa) may include one or more of the following features:

[0092] For example, R aand R b are each independently H or C1-C6 alkyl optionally substituted with one or more -Q3-T3.

[0093] For example, R a and R b One of them is H.

[0094] For example, R a and R b together with the N atom to which they are attached form a 4- to 7-membered heterocycloalkyl ring having 0 or 1 additional heteroatoms relative to the N atom (e.g., azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, etc.), which ring is optionally substituted with one or more -Q-T.

[0095] For example, R a and R b together with the N atom to which they are attached form azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahydrofuranyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl, which ring is optionally substituted with one or more -Q-T.

[0096] For example, one or more -Q3-T3 is oxo.

[0097] For example, Q3 is a bond or an unsubstituted or substituted C1-C3 alkyl linker.

[0098] For example, T3 can be H, halo, 4- to 7-membered heterocycloalkyl, C1-C3 alkyl, OR d , COORd , -S(O)2R d , or -NR d R e is.

[0099] For example, R d and R e are each independently H or C1-C6 alkyl.

[0100] For example, R7 is C3-C8 cycloalkyl or 4- to 7-membered heterocycloalkyl, each optionally substituted with one or more -Q5-T5.

[0101] For example, R7 is piperidinyl, tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl, or cycloheptyl, each optionally substituted with one or more -Q5-T5.

[0102] For example, R7 is cyclopentyl, cyclohexyl, or tetrahydro-2H-thiopyranyl, each optionally substituted with one or more -Q5-T5.

[0103] For example, Q5 is NHC(O) and T5 is each C1-C6 alkyl or C1-C6 alkoxy.

[0104] For example, one or more -Q5-T5 is oxo.

[0105] For example, R7 is 1-oxide-tetrahydro-2H-thiopyranyl or 1,1-dioxide-tetrahydro-2H-thiopyranyl.

[0106] For example, Q5 is a bond and T5 is amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino.

[0107] For example, Q5 is CO, S(O)2, or NHC(O), and T5 is C1-C6 alkyl, C1-C6 alkoxyl, C3-C8 cycloalkyl, or 4- to 7-membered heterocycloalkyl.

[0108] For example, R8 is H or C1-C6 alkyl optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, COOH, C(O)O—C1-C6 alkyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, and di-C1-C6 alkylamino.

[0109] For example, R8 is H, methyl, or ethyl.

[0110] In one embodiment, the compound of the present invention is compound 44

[0111] [ka] or a pharmaceutically acceptable salt thereof.

[0112] In one embodiment, the compound of the invention is the compound itself, i.e., the free base or "naked" molecule. In another embodiment, the compound is a salt thereof, e.g., a mono-HCl or tri-HCl salt, a mono-HBr or tri-HBr salt of the naked molecule.

[0113] Representative compounds of the present invention include those listed in Table 1. In the table below:

[0114] [ka] Each time it appears,

[0115] [ka] should be interpreted as follows.

[0116]

Table 9

[0117] As used herein, "alkyl," "C1, C2, C3, C4, C5, or C6 alkyl," or "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, or C6 straight-chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, or C6 branched-chain saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Examples of alkyl include, but are not limited to, moieties having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl, or n-hexyl.

[0118] In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and in other embodiments, a straight chain or branched chain alkyl has 4 or fewer carbon atoms.

[0119] As used herein, the term "cycloalkyl" refers to a group having 3 to 30 carbon atoms (e.g., C3-C 10cycloalkyl refers to a saturated or unsaturated non-aromatic hydrocarbon mono- or polycyclic (e.g., fused, bridged, or spiro) ring system having one or more heteroatoms (such as O, N, S, or Se). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl. The term "heterocycloalkyl," unless otherwise specified, refers to a saturated or unsaturated non-aromatic 3- to 8-membered monocyclic, 7- to 12-membered bicyclic (fused, bridged, or spiro) or 11- to 14-membered tricyclic (fused, bridged, or spiro) ring system having one or more heteroatoms (such as O, N, S, or Se). Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahydrofuranyl, oxiranyl, azetidinyl, oxetane, and the like. Examples include thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, and 1,4-dioxa-8-azaspiro[4.5]decanyl.

[0120] The term "optionally substituted alkyl" refers to an unsubstituted alkyl or an alkyl having specified substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbonyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moiety.

[0121] An "arylalkyl" or "aralkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). An "alkylaryl" moiety is an aryl substituted with an alkyl (e.g., methylphenyl).

[0122] As used herein, "alkyl linker" is intended to include C1, C2, C3, C4, C5, or C6 straight-chain (linear) saturated divalent aliphatic hydrocarbon groups and C3, C4, C5, or C6 branched-chain saturated aliphatic hydrocarbon groups. For example, a C1-C6 alkyl linker is intended to include C1, C2, C3, C4, C5, and C6 alkyl linker groups. Examples of alkyl linkers include, but are not limited to, moieties having 1 to 6 carbon atoms, such as methyl (-CH-), ethyl (-CHCH-), n-propyl (-CHCHCH-), i-propyl (-CHCHCH-), n-butyl (-CHCHCHCH-), s-butyl (-CHCHCHCH-), i-butyl (-C(CH)CH-), n-pentyl (-CHCHCHCHCHCH-), s-pentyl (-CHCHCHCHCH-), or n-hexyl (-CHCHCHCHCHCHCH-).

[0123] "Alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but containing at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl) and branched-chain alkenyl groups. In certain embodiments, a straight-chain or branched-chain alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term "C2-C6" includes alkenyl groups containing 2 to 6 carbon atoms. The term "C3-C6" includes alkenyl groups containing 3 to 6 carbon atoms.

[0124] The term "optionally substituted alkenyl" refers to an unsubstituted alkenyl or an alkenyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0125] "Alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but containing at least one triple bond. For example, "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl) and branched-chain alkynyl groups. In certain embodiments, a straight-chain or branched-chain alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term "C2-C6" includes alkynyl groups containing from 2 to 6 carbon atoms. The term "C3-C6" includes alkynyl groups containing from 3 to 6 carbon atoms.

[0126] The term "optionally substituted alkynyl" refers to unsubstituted alkynyl or alkynyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moiety.

[0127] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both unsubstituted moieties and moieties having one or more of the specified substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl-piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0128] "Aryl" includes groups that have aromatic character, such as "conjugated" or polycyclic ring systems that contain at least one aromatic ring but do not contain heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, and the like.

[0129] A "heteroaryl" group is an aryl group as defined above except that it contains one to four heteroatoms in the ring structure, and is sometimes referred to as an "aryl heterocycle" or "heteroaromatic." As used herein, the term "heteroaryl" is intended to include stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic aromatic heterocycles consisting of carbon atoms and one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, e.g., 1 or 1-2, or 1-3, or 1-4, or 1-5, or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or other substituent as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)). p (wherein p=1 or 2). It should be noted that the total number of S and O atoms in the aromatic heterocycle is not more than 1.

[0130] Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0131] Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0132] In the case of polycyclic aromatic rings, only one ring need be aromatic (e.g., 2,3-dihydroindole), but all rings may be aromatic (e.g., quinoline), and the second ring may be fused or bridged.

[0133] A cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring may contain at one or more ring positions (e.g., a ring carbon or a heteroatom such as N) such substituents as those described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aryl The aryl and heteroaryl groups may be substituted with aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Aryl and heteroaryl groups may also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic so as to form polycyclic systems (e.g., tetralin, methylenedioxyphenyl).

[0134] As used herein, "carbocycle" or "carbocyclic ring" is intended to include any stable monocyclic, bicyclic, or tricyclic ring having the specified number of carbons, any of which may be saturated, unsaturated, or aromatic. Carbocycles include cycloalkyl and aryl. For example, C3-C 14Carbocycle is intended to include monocyclic, bicyclic, or tricyclic rings having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cyclopentyl, cyclopentyl, cyclopentyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopent ... Carbocycles include phenyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, and tetrahydronaphthyl. Bridged rings are also included in the definition of carbocycle, such as [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane, and [2.2.2]bicyclooctane. A bridged ring occurs when one or more carbon atoms connect two non-adjacent carbon atoms. In one embodiment, the bridged ring is one or two carbon atoms. Note that a bridge always converts a monocycle into a tricycle. When rings are bridged, the substituents described for the ring may also be present on the bridge. Fused rings (e.g., naphthyl, tetrahydronaphthyl) and spirocycles are also included.

[0135] As used herein, a "heterocycle" or "heterocyclic group" includes any ring structure (saturated, unsaturated, or aromatic) containing at least one ring heteroatom (e.g., N, O, or S). Heterocycles include heterocycloalkyl and heteroaryl. Examples of heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, and tetrahydrofuran.

[0136] Examples of heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydroxybenzoyl, benzophenone ... Flo[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-o Oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazol 5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolidine Zolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,Examples include 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

[0137] As used herein, the term "substituted" means that any one or more hydrogen atoms on the designated atom are replaced with one selected from the indicated group, provided that the normal valence of the designated atom is not exceeded and that the substitution results in a stable compound. When the substituent is oxo or keto (i.e., =0), two hydrogen atoms on the atom are replaced. Keto No substituents are present on the aromatic moiety. A ring double bond, as used herein, is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N). "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0138] When a bond to a substituent is shown to cross the bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom to which such substituent is bonded to the remainder of the compound of a given formula, then such substituent may be bonded by any atom in such formula. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0139] When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R moieties, that group may also be optionally substituted with up to 2 R moieties, and each occurrence of R is selected independently of the definition of R. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0140] The term "hydroxy" or "hydroxyl" refers to the radicals -OH or -O - The group includes a group having the formula:

[0141] As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo. The term "perhalogenated" generally refers to a moiety in which all hydrogen atoms are replaced with halogen atoms. The terms "haloalkyl" or "haloalkoxyl" refer to an alkyl or alkoxyl substituted with one or more halogen atoms.

[0142] The term "carbonyl" includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom. Examples of carbonyl-containing moieties include, but are not limited to, aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, and the like.

[0143] The term "carboxyl" refers to -COOH or its C1-C6 alkyl esters.

[0144] "Acyl" includes moieties that contain the acyl radical (RC(O)-) or a carbonyl group. "Substituted acyl" includes acyl groups in which one or more hydrogen atoms are replaced by, for example, an alkyl group, an alkynyl group, a halogen, a hydroxyl, an alkylcarbonyloxy, an arylcarbonyloxy, an alkoxycarbonyloxy, an aryloxycarbonyloxy, a carboxylate, an alkylcarbonyl, an arylcarbonyl, an alkoxycarbonyl, an aminocarbonyl, an alkylaminocarbonyl, a dialkylaminocarbonyl, an alkylthiocarbonyl, an alkoxyl, a phosphate, a phosphonato, a phosphinato, an amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), an acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, a sulfhydryl, an alkylthio, an arylthio, a thiocarboxylate, a sulfate, an alkylsulfinyl, a sulfonato, a sulfamoyl, a sulfonamido, a nitro, a trifluoromethyl, a cyano, an azido, a heterocyclyl, an alkylaryl, or an aromatic or heteroaromatic moiety.

[0145] "Aroyl" includes moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthylcarboxy, and the like.

[0146] "Alkoxyalkyl," "alkylaminoalkyl," and "thioalkoxyalkyl" include alkyl groups as defined above, where oxygen, nitrogen, or sulfur atoms replace one or more hydrocarbon backbone carbon atoms.

[0147] The term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups or radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. Alkoxy groups can be optionally substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.

[0148] The term "ether" or "alkoxy" includes compounds or moieties that contain an oxygen bonded to two carbon atoms or heteroatoms. For example, this term includes "alkoxyalkyl," which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom that is covalently bonded to an alkyl group.

[0149] The term "ester" includes compounds or moieties which contain a carbon or heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term "ester" includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, and the like.

[0150] The term "thioalkyl" includes compounds or moieties which contain an alkyl group connected with a sulfur atom. Thioalkyl groups include alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxylic acid, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (such as alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heterocyclic alkyl groups. It may be substituted with groups such as heteroaromatic moieties.

[0151] The term "thiocarbonyl" or "thiocarboxy" includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.

[0152] The term "thioether" includes moieties containing a sulfur atom bonded to two carbon or heteroatoms. Examples of thioethers include, but are not limited to, alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term "alkthioalkyl" includes moieties having an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom bonded to an alkyl group. Similarly, the term "alkthioalkenyl" refers to a moiety in which an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom covalently bonded to an alkenyl group, and "alkthioalkynyl" refers to a moiety in which an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atom covalently bonded to an alkynyl group.

[0153] As used herein, "amine" or "amino" refers to unsubstituted or substituted -NH2. "Alkylamino" includes compounds in which the nitrogen of -NH2 is bound to at least one alkyl group. Examples of alkylamino groups include benzylamino, methylamino, ethylamino, and phenethylamino. "Dialkylamino" includes groups in which the nitrogen of -NH2 is bound to at least two additional alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino and diethylamino. "Arylamino" and "diarylamino" include groups in which the nitrogen is bound to at least one or two aryl groups, respectively. "Aminoaryl" and "aminoaryloxy" refer to aryl and aryloxy groups substituted with amino. "Alkylarylamino," "alkylaminoaryl," or "arylaminoalkyl" refers to an amino group bound to at least one alkyl group and at least one aryl group. "Alkaminoalkyl" refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom that is also bound to an alkyl group. "Acylamino" includes groups in which the nitrogen is bound to an acyl group. Examples of acylamino include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

[0154] The term "amide" or "aminocarboxy" includes compounds or moieties containing a nitrogen atom bonded to the carbon of a carbonyl or thiocarbonyl group. This term includes "alkaminocarboxy" groups, which include alkyl, alkenyl, or alkynyl groups bonded to an amino group bonded to the carbon of a carbonyl or thiocarbonyl group. It also includes "arylaminocarboxy" groups, which include aryl or heteroaryl moieties bonded to an amino group bonded to the carbon of a carbonyl or thiocarbonyl group. The terms "alkylaminocarboxy," "alkenylaminocarboxy," "alkynylaminocarboxy," and "arylaminocarboxy" include moieties in which alkyl, alkenyl, alkynyl, and aryl moieties are bonded to a nitrogen atom, which is similarly bonded to the carbon of a carbonyl group, respectively. Amides may be substituted with substituents such as linear alkyl, branched alkyl, cycloalkyl, aryl, heteroaryl, or heterocycle. The substituents on the amido group may be further substituted.

[0155] Nitrogen-containing compounds of the invention can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and / or hydrogen peroxide) to yield other compounds of the invention. Thus, all depicted and claimed nitrogen-containing compounds, where permitted by valency and structure, include compounds as depicted and their N-oxide derivatives (N→O or N + -O - In addition, in other examples, the nitrogen in the compounds of the present invention may be designated as an N-hydrogen group. The N-hydroxy compounds can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as m-CPBA. All depicted and claimed nitrogen-containing compounds are also considered to encompass both the compounds as depicted and their N-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, where R is substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3- to 14-membered carbocyclic or 3- to 14-membered heterocyclic) derivatives, where permitted by valency and structure.

[0156] In this specification, the structural formula of a compound may conveniently represent a specific isomer in some cases, but the present invention includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, and tautomers. Furthermore, the compounds represented by the formula may have crystalline polymorphism. It should be noted that any crystalline form, crystalline mixture, or anhydride or hydrate thereof is included within the scope of the present invention. Furthermore, so-called metabolites produced by in vivo decomposition of the compounds of the present invention are also included within the scope of the present invention.

[0157] "Isomers" means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers" or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture."

[0158] A carbon atom bonded to four non-identical substituents is termed a "chiral center."

[0159] "Chiral isomer" means a compound having at least one chiral center. Compounds with more than one chiral center may exist as individual diastereomers or as a mixture of diastereomers called a "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the Sequence Rule of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, p. 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, p. 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), p. 612; Cahn et al., Experientia 1956, 12, p. 81; Cahn, J. Chem. Educ. 1964, 41, p. 116).

[0160] "Geometric isomers" refers to diastereomers that owe their existence to restricted rotation about a double bond or a cycloalkyl linker (e.g., 1,3-cyclobutyl). These configurations are distinguished in their names by the prefixes cis and trans, or Z and E, indicating that the groups are on the same or opposite sides of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.

[0161] It is to be understood that the compounds of the present invention may be described as different chiral or geometric isomers, and it is also to be understood that when a compound has chiral or geometric isomeric forms, all isomeric forms are intended to be included within the scope of the present invention, and the name of the compound does not exclude any isomeric form.

[0162] Furthermore, the structures and other compounds discussed in this invention include all atropisomers thereof. "Atropisomers" are compounds in which the atoms of two isomers are arranged differently in space. Atropisomers are stereoisomeric forms of molecules that are confined to a single molecule. Atropisomers owe their existence to restricted rotation caused by the hindrance of rotation of large groups around a central bond. Such atropisomers typically exist as mixtures, although recent advances in chromatographic techniques have made it possible to separate mixtures of two atropisomers in selected cases.

[0163] A "tautomer" is one of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by the switching of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric sets in solution. In solutions where tautomerization is possible, a chemical equilibrium of tautomers is reached. The exact ratio of tautomers depends on several factors, such as temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism.

[0164] Of the various types of tautomerism that are possible, two are commonly observed: keto-enol tautomerism, in which a simultaneous shift of an electron and a hydrogen atom occurs; ring-chain tautomerism occurs when an aldehyde group (-CHO) in a sugar molecule reacts with one of the hydroxyl groups (-OH) in the same molecule, giving it a cyclic (ring-shaped) form, such as that exhibited by glucose.

[0165] Common tautomeric pairs are ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomers in heterocycles (e.g., in nucleobases such as guanine, thymine, and cytosine), imine-enamine, and enamine-enamine. An example of a keto-enol equilibrium is that between pyridin-2(1H)-one and the corresponding pyridin-2-ol, as shown below.

[0166] [ka]

[0167] It should be understood that the compounds of the invention may be described as different tautomeric forms, and it should also be understood that where the compounds have tautomeric forms, all tautomeric forms are intended to be included within the scope of the invention, and the name of the compound does not exclude any tautomeric form.

[0168] The terms "crystalline polymorph," "polymorph," or "crystalline form" refer to crystalline structures in which a compound (or a salt or solvate thereof) crystallizes in different crystalline packaging arrangements, all of which may have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystalline form to dominate. Crystalline polymorphs of a compound can be prepared by crystallization under different conditions.

[0169] The compounds of formula (IIa) disclosed herein include the compounds themselves, as well as, optionally, their salts, their esters, their solvates, and their prodrugs. For example, salts can be prepared by combining an anion with an aryl- or heteroaryl-substituted benzene compound. The salt can be formed between a cation and a negatively charged group (e.g., amino) on the aryl- or heteroaryl-substituted benzene compound. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, a salt can be formed between a cation and a negatively charged group (e.g., carboxylate) on the aryl- or heteroaryl-substituted benzene compound. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium ions, such as tetramethylammonium. The aryl- or heteroaryl-substituted benzene compounds also include salts thereof containing a quaternary nitrogen atom. It is understood that in the salt form, the ratio of the compound to the cation or anion of the salt can be 1:1 or any ratio other than 1:1, such as 3:1, 2:1, 1:2, or 1:3.

[0170] Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active aryl- or heteroaryl-substituted benzene compounds.

[0171] Furthermore, the compounds of the present invention, for example, salts of the compounds, can exist in hydrated or unhydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0172] "Solvate" refers to a solvent addition form containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. When the solvent is water, the solvate formed is a hydrate; when the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is formed by combining one or more molecules of water with one molecule of a substance in which the water retains its molecular state as HO.

[0173] As used herein, the term "analog" refers to a compound that is structurally similar to another but differs slightly in composition (such as the replacement of one atom with an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group with another). Thus, an analog is a compound that is similar or equivalent in function and appearance to the reference compound, but not in structure or origin.

[0174] The term "derivative," as defined herein, refers to compounds that share a common core structure and are substituted with various groups as described herein. For example, the compounds represented by formula (I) are all aryl- or heteroaryl-substituted benzene compounds and have formula (I) as a common core.

[0175] The term "bioisostere" refers to a compound resulting from the exchange of an atom or group of atoms with another broadly similar atom or group of atoms. The purpose of bioisosteric substitution is to create a new compound with biological properties similar to those of the parent compound. Bioisosteric substitution may be based on physicochemical or topology. Examples of carboxylic acid bioisosteres include, but are not limited to, acylsulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, pp. 3147-3176, 1996.

[0176] The present invention is intended to include all isotopes of atoms occurring in the parent compound. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.

[0177] Any compound of formula (IIa) of the present invention described herein may be an EZH2 inhibitor.

[0178] In certain embodiments of the present invention, an inhibitor of EZH2 "selectively inhibits" the histone methyltransferase activity of an EZH2 mutant if it inhibits the histone methyltransferase activity of an EZH2 mutant more efficiently than it inhibits the histone methyltransferase activity of wild-type EZH2. For example, in one embodiment, a selective inhibitor has an IC50 for an EZH2 mutant that is at least 40% lower than the IC50 for wild-type EZH2. In one embodiment, a selective inhibitor has an IC50 for an EZH2 mutant that is at least 50% lower than the IC50 for wild-type EZH2. In one embodiment, a selective inhibitor has an IC50 for an EZH2 mutant that is at least 60% lower than the IC50 for wild-type EZH2. In one embodiment, a selective inhibitor has an IC50 for an EZH2 mutant that is at least 70% lower than the IC50 for wild-type EZH2. In one embodiment, the selective inhibitor has an IC50 for the EZH2 mutant that is at least 80% lower than the IC50 for wild-type EZH2. In one embodiment, the selective inhibitor has an IC50 for the EZH2 mutant that is at least 90% lower than the IC50 for wild-type EZH2.

[0179] In one embodiment, a selective inhibitor of an EZH2 mutant exhibits essentially no inhibitory effect on wild-type EZH2.

[0180] In certain aspects of the present invention, the inhibitor inhibits the conversion of H3-K27me2 to H3-K27me3. In one embodiment, the inhibitor is said to inhibit H3-K27 trimethylation. Because the conversion of H3-K27me1 to H3-K27me2 precedes the conversion of H3-K27me2 to H3-K27me3, an inhibitor of the conversion of H3-K27me1 to H3-K27me2 naturally also inhibits the conversion of H3-K27me2 to H3-K27me3, i.e., the inhibitor inhibits H3-K27 trimethylation. The inhibitor can also inhibit the conversion of H3-K27me2 to H3-K27me3 without inhibiting the conversion of H3-K27me1 to H3-K27me2. This type of inhibition also results in the inhibition of H3-K27 trimethylation, despite not inhibiting the di-methylation of H3-K27.

[0181] In one embodiment, the inhibitor inhibits the conversion of H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3. Such an inhibitor can directly inhibit only the conversion of H3-K27me1 to H3-K27me2. Alternatively, such an inhibitor can directly inhibit both the conversion of H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3.

[0182] In certain embodiments of the present invention, the inhibitor compound inhibits histone methyltransferase activity. The inhibition of histone methyltransferase activity can be detected using any suitable method. This inhibition can be measured, for example, in terms of the rate of histone methyltransferase activity or as a product of histone methyltransferase activity.

[0183] Inhibition is measurable inhibition compared to a suitable control. In one embodiment, inhibition is at least 10% inhibition compared to a suitable control. That is, the rate of enzyme activity or amount of product with the inhibitor is 90% or less of the corresponding rate or amount without the inhibitor. In various other embodiments, inhibition is at least 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 95% inhibition compared to a suitable control. In one embodiment, inhibition is at least 99% inhibition compared to a suitable control. That is, the rate of enzyme activity or amount of product with the inhibitor is 1% or less of the corresponding rate or amount without the inhibitor.

[0184] The compositions of the present invention comprise a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents or pharmaceutically acceptable salts thereof. The present invention provides for the administration of a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents or pharmaceutically acceptable salts thereof, either as a co-formulation or as separate formulations, where the administration of the formulations is simultaneous, sequential, or alternating. In certain embodiments, the other therapeutic agent may be an agent recognized in the art as being useful for treating the disease or condition treated by the compositions of the present invention. In other embodiments, the other therapeutic agent may be an agent not recognized in the art as being useful for treating the disease or condition treated by the compositions of the present invention. In one aspect, the other therapeutic agent may be an agent that imparts beneficial properties to the compositions of the present invention (e.g., an agent that affects the viscosity of the composition). Beneficial properties for the compositions of the present invention include, but are not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of a compound of Formula (IIa) and one or more other therapeutic agents. For example, the one or more other therapeutic agents may be anti-cancer or chemotherapeutic agents. For example, one or more other therapeutic agents can be glucocorticoids.For example, one or more other therapeutic agents can be selected from prednisone, prednisolone, cyclophosphamide, vincristine, doxorubicin, mafosfamide, cisplatin, AraC, everolimus, decitabine, dexamethasone, or their functional analogs, derivatives, prodrugs, and metabolites.In another embodiment, the other therapeutic agent can be prednisone or its active metabolite, prednisolone.

[0185] The therapeutic agents listed below are for illustrative purposes and are not intended to be limiting. The present invention includes at least one other therapeutic agent selected from the list below. The present invention may include more than one other therapeutic agent, for example, 2, 3, 4, or 5 other therapeutic agents, so that the composition of the present invention can perform its intended function.

[0186] In one embodiment, the other therapeutic agent is an anti-cancer agent. In one embodiment, the anti-cancer agent is a compound that affects histone modifications, such as an HDAC inhibitor. In certain embodiments, the anti-cancer agent is a chemotherapeutic agent (e.g., 2CdA, 5-FU, 6-mercaptopurine, 6-TG, Abraxane™, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid, amethopterin, Ara-C, azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®, clofarabine, Clolar™, Cytoxan®, daunorubicin hydrochloride, DaunoXo®, or the like). me®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L -PAM, Lysodren, Matulane®, mithracin, mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®, nitrogen mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®, Platinol®, carmustine implant, Prolifeprospan 20, Sandostatin®, Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA, Trisenox®, Valstar®, Velban®, Vidaza™, vincristine sulfate, VM 26, Xeloda®, and Zanosar®; biologics (e.g., alpha interferon, Bacillus Calmette-Guerin, Bexxar®, Campath®, Ergamisol®, erlotinib, Herceptin®, interleukin-2, Iressa®, lenalidomide, Mylotarg®, Ontak®, Pegasys®, Revlimid®, Rituxan®, Tarceva™, Thalomid®, Tykerb®, Velcade®, and Zevalin™; corticosteroids (e.g., dexamethasone phosphate); sodium benzoate, DeltaSone®, and Delta-Cortef®; hormonal therapy agents (e.g., Arimidex®, Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®, Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™, and Zoladex®);and radiopharmaceuticals (e.g., Iodotope®, Metastron®, Phosphocol®, and Samarium SM-153);

[0187] In another embodiment, the other therapeutic agent is a chemotherapeutic agent (also called antineoplastic or antiproliferative agent) selected from the group including alkylating agents; antibiotics; antimetabolites; antidotes; interferons; polyclonal or monoclonal antibodies; EGFR inhibitors; HER2 inhibitors; histone deacetylase inhibitors; hormones; mitotic inhibitors; MTOR inhibitors; multikinase inhibitors; serine / threonine kinase inhibitors; tyrosine kinase inhibitors; VEGF / VEGFR inhibitors; taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule targeting agents, topoisomerase poisons, inhibitors of molecular targets or enzymes (e.g., kinases or protein methyltransferases), cytidine analogs, or any chemotherapeutic, anti-neoplastic, or anti-proliferative agent listed at www.cancer.org / docroot / cdg / cdg_0.asp.

[0188] Examples of alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).

[0189] Examples of antibiotics include, but are not limited to, doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal (DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

[0190] Examples of antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS) are examples.

[0191] Examples of antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0192] Examples of interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0193] Examples of polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab / iodine-131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris); ordenosumab.

[0194] Examples of EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.

[0195] Examples of HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0196] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0197] Examples of hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (Mega ce); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

[0198] Examples of antimitotic agents include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

[0199] Examples of MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; or AP23573.

[0200] Examples of VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.

[0201] Examples of microtubule targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0202] Examples of topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0203] Examples of taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0204] Examples of common chemotherapeutic, anti-neoplastic, and anti-proliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); and procarbazine (Matulane). ; Pegaspargase (Oncaspar); Denileukin diftitox (Ontak); Porfimer (Photofrin); Aldesleukin (Proleukin); Lenalidomide (Revlimid); Bexarotene (Targretin); Thalidomide (Thalomid); Temsirolimus (Torisel); Arsenic trioxide (Trisenox); Verteporfin (Visudyne); Mimosine (Leucenol); (1M Tegafur - 0.4M 5-chloro 1M potassium oxonate), or lovastatin.

[0205] In another embodiment, the other therapeutic agent is a chemotherapeutic agent or cytokine, for example, G-CSF (granulocyte colony-stimulating factor).

[0206] In yet another embodiment, the other therapeutic agent is selected from the group consisting of, but not limited to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil), CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP), Carboplatin, TS-1 (1:0.4:1 The chemotherapy agent may be a standard combination chemotherapy agent such as tegafur, gimestat, and otastat potassium in a molar ratio of 100 mg / kg / day, Camptothecin-11 (CPT-11, Irinotecan, or Camptosar™), CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, prednisone, or prednisolone), or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil, and prednisone).

[0207] In another embodiment, the other therapeutic agent may be an inhibitor of an enzyme, such as a receptor or non-receptor kinase. Receptor and non-receptor kinases are, for example, tyrosine kinases or serine / threonine kinases. The kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides, or antibodies.

[0208] Examples of kinase inhibitors include, but are not limited to, Bevacizumab (targets VEGF), BIBW2992 (targets EGFR and Erb2), Cetuximab / Erbitux (targets Erb1), Imatinib / Gleevic (targets Bcr-Abl), Trastuzumab (targets Erb2), Gefitinib / Iressa (targets EGFR), Ranibzumab (targets VEGF), Pegaptanib (targets VEGF), ), Erlotinib / Tarceva (targets Erb1), Nilotinib (targets Bcr-Abl), Lapatinib (targets Erb1 and Erb2 / Her2), GW-572016 / Lapatinib ditosylate (targets HER2 / Erb2), Panitumumab / Vectibix (targets EGFR), Vandetinib (targets RET / VEGFR), E7080 (targets multiple proteins including RET and VEGFR), Herce ptin (targets HER2 / Erb2), PKI-166 (targets EGFR), Canertinib / CI-1033 (targets EGFR), Sunitinib / SU-11464 / Sutent (targets EGFR and FLT3), Matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGF and FLT3), Vatalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), These include RANKL, RANKL-1, RANKL-2, RANKL-3, RANKL-4, RANKL-5, RANKL-6, RANKL-7, RANKL-8, RANKL-9, RANKL-10, RANKL-11, RANKL-12, RANKL-13, RANKL-24, RANKL-25, RANKL-36, RANKL-17, RANKL-26, RANKL-27, RANKL-28, RANKL-39, RANKL-39, RANKL-39, RANKL-40, RANKL-50, RANKL-14, RANKL-25, RANKL-26, RANKL-37, RANKL-28, RANKL-39, RANKL-40, RANKL-50, RANKL-29, RANKL-39, RANKL-25, RANKL-36, RANKL-26, RANKL-37, RANKL-27, RANKL-39, RANKL-40, RANKL-28, RANKL-39, RANKL-40, RANKL-29, RANKL-39, RANKL-40, RANKL-25, RANKL-39, RANKL-26, RANKL-39, RANKL-40, RANKL-25, RANKL-39 ...39, RANKL-39, RANKL-39,

[0209] Examples of serine / threonine kinase inhibitors include, but are not limited to, Rapamune (targets mTOR / FRAP1), Deforolimus (targets mTOR), Certican / Everolimus (targets mTOR / FRAP1), AP23573 (targets mTOR / FRAP1), Eril / Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK), Seliciclib / CYC202 / Roscovitrine (targets CDK), SNS-032 / BMS-387032 (targets CDK), Ruboxistaurin (targets PKC). ), Pkc412 (targets PKC), Bryostatin (targets PKC), KAI-9803 (targets PKC), SF1126 (targets PI3K), VX-680 (targets Aurora kinase), Azd1152 (targets Aurora kinase), Arry-142886 / AZD-6244 (targets MAP / MEK), SCIO-469 (targets MAP / MEK), GW681323 (targets MAP / MEK), CC-401 (targets JNK), CEP-1347 (targets JNK), and PD332991 (targets CDK).

[0210] Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); and alemtuzumab. (Campath); gemtuzumab (Mylotarg); temirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606; CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0211] The present invention provides a method for combination therapy in which a composition comprising a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents is administered to a subject in need of disease or cancer treatment. The combination therapy can also be administered to cancer cells to inhibit proliferation or induce cell death. In one embodiment, the compound of Formula (IIa) or a pharmaceutically acceptable salt thereof is administered before the administration of a composition of the present invention comprising a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents. In one embodiment, the compound of Formula (IIa) or a pharmaceutically acceptable salt thereof is administered before the administration of one or more therapeutic agents, such that the other therapeutic agents are administered in a single composition or in two or more compositions, e.g., simultaneously, sequentially, or staggered.

[0212] In one embodiment, the composition of the present invention comprises a compound of formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other anti-cancer agents, such as CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone or prednisolone) or R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, prednisone or prednisolone). In one embodiment, the composition of the present invention comprises a compound of formula (IIa) or a pharmaceutically acceptable salt thereof and prednisone or prednisolone. The method of the present invention includes a combination therapy of administering a compound of formula (IIa) or a pharmaceutically acceptable salt thereof and an anti-cancer agent, wherein the anti-cancer agent is CHOP, R-CHOP, prednisone, or prednisolone.

[0213] In certain embodiments, "combination therapy" is intended to encompass the administration of these therapeutic agents in a sequential manner, with each therapeutic agent being administered at a different time, and these therapeutic agents, or at least two therapeutic agents, being administered simultaneously or substantially simultaneously. Simultaneous administration can be achieved, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent, or multiple single capsules of each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, while other therapeutic agents of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally, or all therapeutic agents can be administered by intravenous injection. The therapeutic agents can also be administered alternately.

[0214] In certain embodiments of the present invention, the combination therapy described herein can produce a synergistic effect in the treatment of disease or cancer. "Synergistic effect" is defined as the effectiveness of the combination of therapeutic agents being greater than the sum of the effects of any agent administered alone. A synergistic effect may also be an effect that cannot be achieved by administering any compound or other therapeutic agent as a single agent. A synergistic effect may include, but is not limited to, the effect of treating cancer by reducing tumor size, inhibiting tumor growth, or increasing the survival time of a subject. A synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.

[0215] In certain embodiments of the present invention, "combined therapy" also includes the administration of the above-mentioned therapeutic agents in further combination with other bioactive ingredients and non-drug treatments (e.g., surgery or radiation treatment). When the combined therapy further includes a non-drug treatment, the non-drug treatment can be administered at any suitable time, as long as the beneficial effect resulting from the simultaneous action of the combination of the therapeutic agent and the non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is further achieved when the non-drug treatment is temporarily removed from the administration of the therapeutic agent, perhaps by several days or even weeks.

[0216] In another embodiment, a composition of the invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog, or derivative thereof, can be administered in conjunction with radiation therapy, which can also be administered as part of a multi-drug therapy, along with a composition of the invention and another chemotherapeutic agent described herein.

[0217] The present invention also provides pharmaceutical compositions comprising a compound of formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents disclosed herein, in admixture with a pharmaceutically suitable carrier or excipient, in a dose to treat or prevent a disease or condition described herein. In one embodiment, the present invention also provides pharmaceutical compositions comprising a compound or medicament of Table I, in admixture with a pharmaceutically suitable carrier or excipient, in a dose to treat or prevent a disease or condition described herein. In another aspect, the present invention also provides a pharmaceutical composition comprising compound 44, a pharmaceutically acceptable salt thereof, and one or more therapeutic agents, in an amount to treat or prevent a disease or condition described herein, in admixture with a pharmaceutically suitable carrier or excipient.

[0218] [ka] or a pharmaceutically acceptable salt thereof and one or more therapeutic agents. The pharmaceutical compositions of the present invention can also be administered simultaneously, sequentially, or alternately with other therapeutic agents or treatment modalities.

[0219] The mixture of the compositions of the present invention can be administered to patients as a simple mixture or in a suitable pharmaceutical composition.For example, one aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective dose of the EZH2 inhibitor of formula (IIa) or its pharmaceutically acceptable salt, hydrate, enantiomer or stereoisomer; one or more other therapeutic agents, and a pharmaceutically acceptable diluent or carrier.

[0220] A "pharmaceutical composition" is a formulation containing a compound of the present invention in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form may be in any of a variety of forms, such as a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The amount of active ingredient (e.g., a formulation of the disclosed compound or its salt, hydrate, solvate, or isomer) in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will understand that routine adjustments to the dosage may sometimes be necessary depending on the age and condition of the patient. The dosage also depends on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, and intranasal. Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the active compound is admixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that are required.

[0221] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, anions, cations, materials, compositions, carriers, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, as well as a reasonable benefit / risk ratio.

[0222] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and useful in preparing pharmaceutical compositions that are not biologically or otherwise undesirable, and is not intended to be a veterinary approved excipient. "Pharmaceutically acceptable excipient" as used herein and in the claims includes both one and more than one such excipient.

[0223] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. The parent preparation can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0224] The composition of the present invention can be administered to the subject by many well-known methods currently used for chemotherapy treatment.For example, for the treatment of cancer, the compound of the present invention can be directly injected into tumor, injected into bloodstream or body cavity, or taken orally, or applied through the skin using a patch.The selected dose should be sufficient to constitute effective treatment, but not so high as to cause unacceptable side effects.It should be preferable to closely monitor the state of disease state (for example, cancer, pre-cancer, etc.) and the health of the patient for a reasonable period after treatment.

[0225] As used herein, the term "therapeutically effective amount" refers to the amount of an agent that treats, alleviates, or prevents an identified disease or condition, or that exhibits a detectable therapeutic or inhibitory effect. This effect can be detected by any assay known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation that is within the skill and judgment of the physician. In a preferred embodiment, the disease or condition being treated is cancer. In another embodiment, the disease or condition being treated is a cell proliferation disorder.

[0226] In certain embodiments, the therapeutically effective amount of each agent used in combination is lower when used in combination compared to monotherapy using each agent alone, and such lower therapeutically effective amounts can provide for less toxicity of the treatment regimen.

[0227] For any compound, the therapeutically effective amount can be estimated initially, for example, in cell culture assays of neoplastic cells or in animal models, usually rats, mice, rabbits, dogs, or pigs.Animal models can also be used to determine suitable concentration ranges and administration routes.Such information can then be used to determine useful doses and routes for administration in humans.For example, ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 Therapeutic / prophylactic efficacy and toxicity, such as the dose lethal to 50% of the population, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 / ED 50 The therapeutic index can be expressed as a ratio. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0228] Dosage and administration are adjusted to provide sufficient level of active agent or maintain desired effect.Factors that can be considered include the severity of disease state, the general health of the patient, the age, weight and sex of the patient, the time and frequency of administration, drug combination, reaction sensitivity and the tolerance / response to treatment.Depending on the half-life and clearance rate of specific preparation, long-acting pharmaceutical composition can be administered every 3 to 4 days, every week or once every 2 weeks.

[0229] The pharmaceutical composition containing the active compound of the present invention can be prepared by generally known methods, for example, by conventional mixing, dissolving, granulating, sugar-coating, powdering, emulsifying, encapsulating, entrapping or lyophilization process.The pharmaceutical composition can be prepared by conventional methods using one or more pharmaceutically acceptable carriers, including excipients and / or auxiliary agents that facilitate the processing of active compound into pharmaceutical preparations.Of course, suitable preparation depends on the selected route of administration.

[0230] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0231] The required amount of active compound is incorporated into suitable solvent with one or combination of the components listed above, and then, if necessary, sterile filtering can be used to prepare sterile injection solution.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other necessary components from the above-listed ones.For the sterile powder that is used to prepare sterile injection solution, the method of preparation is vacuum drying and freeze-drying, which produces the powder of active compound and any other desired components from the solution that has been previously sterile filtered.

[0232] Oral compositions generally contain an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, where the compound in the liquid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients, or compounds of a similar nature: binders, such as microcrystalline cellulose, gum tragacanth, or gelatin; starch or It may contain excipients such as lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterot; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0233] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0234] Systemic administration may also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams that are generally known in the art.

[0235] The active compounds can be prepared with pharmaceutically acceptable carriers that protect the compounds against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations are clear to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0236] It is particularly advantageous to formulate oral or parenteral compositions into unit dosage form for ease of administration and uniformity of dosage.Unit dosage form as used herein refers to a physically separate unit that is suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect together with necessary pharmaceutical carrier.The specification of the unit dosage system of the present invention is directly determined depending on the unique characteristics of active compound and the specific therapeutic effect that is achieved.

[0237] In therapeutic applications, the dosage of an EZH2 inhibitor compound described herein, another therapeutic agent described herein, a composition comprising a compound of Formula (IIa) and one or more other therapeutic agents, or a pharmaceutical composition used in accordance with the present invention will vary depending on the agent, the age, weight, and clinical condition of the recipient patient, as well as the experience and judgment of the physician or practitioner administering the treatment, and other factors that may affect the selected dosage. Generally, the dosage should be sufficient to cause a slowing of tumor growth, preferably regression, and preferably complete regression of the cancer. Doses may range from about 0.01 mg / kg / day to about 5000 mg / kg / day. In a preferred embodiment, the dosage may range from about 1 mg / kg / day to about 1000 mg / kg / day. In one embodiment, the dose ranges from about 0.1 mg / day to about 50 g / day; from about 0.1 mg / day to about 25 g / day; from about 0.1 mg / day to about 10 g / day; from about 0.1 mg to about 3 g / day; or from about 0.1 mg to about 1 g / day (doses are expressed in kg for patient weight and m for body surface area) in single, divided, or continuous doses. 2 (These figures may be adjusted for age in years.) An effective amount of a drug is one that provides an objectively identifiable improvement as noted by a physician or other qualified observer. For example, tumor regression in a patient can be measured by reference to tumor diameter. A decrease in diameter indicates regression. Regression is also indicated by the failure of the tumor to recur after treatment has stopped. As used herein, the term "dosage-effective manner" refers to the amount of active compound that produces a desired biological effect in a subject or cell.

[0238] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0239] The compositions of the present invention can further form salts. The compositions of the present invention can form more than one salt per molecule, for example, mono-, di-, or tri-. All of these forms are encompassed within the scope of the claimed invention.

[0240] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present invention in which the parent compound is modified by making its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carboxylic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycollyarsanilic acid, hexylresorcylic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, and the like. and those derived from inorganic and organic acids selected from acetic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and commonly occurring amino acids such as glycine, alanine, phenylalanine, arginine, etc.

[0241] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, etc. The present invention also encompasses salts formed when an acidic proton present in any parent compound is replaced by a metal ion, e.g., an alkali metal ion, alkaline earth ion, or aluminum ion; or coordination forms with organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0242] It should be understood that any reference to a pharmaceutically acceptable salt also includes the solvent addition forms (solvates) or crystal forms (polymorphs), as defined herein, of the same salt.

[0243] The compositions of the present invention can also be prepared as esters, such as pharmaceutically acceptable esters. For example, a carboxylic acid functional group in a compound can be converted to its corresponding ester, such as a methyl, ethyl, or other ester. Also, an alcohol group in a compound can be converted to its corresponding ester, such as an acetate, propionate, or other ester.

[0244] The compositions of the present invention can also be prepared as prodrugs, e.g., pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein and refer to any compound that releases an active parent drug in vivo. Because prodrugs are known to enhance certain desirable properties of drugs (e.g., solubility, bioavailability, manufacturing, etc.), the compounds of the present invention can be delivered in prodrug form. Thus, the present invention is intended to encompass prodrugs of the compounds claimed in the present invention, methods of delivering the same, and compositions containing the same. "Prodrugs" are intended to include any covalently bonded carriers that release the active parent drug of the present invention in vivo when such prodrug is administered to a subject. Prodrugs of the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved to the parent compound by routine manipulation or in vivo. Prodrugs include compounds of the present invention in which a hydroxy, amino, sulfhydryl, carboxy, or carbonyl group is bonded to any group that can be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy, or free carbonyl group, respectively.

[0245] Examples of prodrugs include, but are not limited to, esters (e.g., acetate, dialkylaminoacetate, formate, phosphate, sulfate, and benzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in the compounds of the present invention, esters (e.g., ethyl ester, morpholinoethanol ester) of carboxyl functional groups, N-acyl derivatives (e.g., N-acetyl), N-Mannich bases, Schiff bases, and enantiomers of amino functional groups, oximes, acetals, ketals, and enol esters of ketone and aldehyde functional groups, etc. See Bundegaard, H., Design of Prodrugs, pp. 1-92, Elesevier, New York-Oxford (1985).

[0246] The composition or its pharmaceutically acceptable salt, ester or prodrug thereof can be administered orally, nasally, transdermally, pulmonary, inhalation, buccal, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally.In one embodiment, the compound is administered orally.Those skilled in the art will recognize the advantages of certain administration routes.

[0247] The dosage regimen using the compound is selected according to various factors, such as the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition being treated; the route of administration; the patient's renal and hepatic function; and the specific compound or its salt used. A physician or veterinarian of ordinary skill can easily determine and prescribe the effective amount of the drug required to prevent, counter, or stop the progression of the condition.

[0248] Techniques for formulating and administering the disclosed compounds of the present invention can be found in Remington: the Science and Practice of Pharmacy, 19th Edition, Mack Publishing Co., Easton, PA (1995). In some embodiments, the compounds described herein and their pharmaceutically acceptable salts are used in pharmaceutical preparations with pharmaceutically acceptable carriers or diluents. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compound is present in such pharmaceutical compositions in an amount sufficient to provide the desired dosage within the range described herein.

[0249] Unless otherwise indicated, all percentages and ratios used herein are by weight. Other features and advantages of the present invention will be apparent from the various examples. The examples provided are illustrative of various components and methods useful in practicing the invention. The examples do not limit the claimed invention. Based on this disclosure, one of ordinary skill in the art will be able to: Other components and methods useful in practicing the present invention may be identified and used.

[0250] The present invention provides compositions and methods for treating conditions and diseases whose course is influenced by modulating the methylation status of histones or other proteins, and wherein the methylation status is mediated at least in part by the activity of EZH2. Modulation of the methylation status of histones can in turn affect the expression levels of target genes activated by methylation and / or target genes repressed by methylation. The methods comprise administering to a subject in need of such treatment a therapeutically effective amount of a composition of the present invention or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof.

[0251] Based at least in part on the fact that aberrant histone methylation has been found to be associated with certain cancers and precancerous conditions, a method for treating cancer or a precancerous condition in a subject using an EZH2 mutant comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits methylation. In one embodiment, a method for treating cancer or a precancerous condition in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits the conversion of unmethylated H3-K27 to monomethylated H3-K27 (H3-K27me1). In one embodiment, a method for treating cancer or a precancerous condition in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits the conversion of monomethylated H3-K27 (K3-K27me1) to dimethylated H3-K27 (H3-K27me2). In one embodiment, a method for treating cancer or a precancerous condition in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits the conversion of H3-K27me2 to trimethylated H3-K27 (H3-K27me3). In one embodiment, a method for treating cancer or a precancerous condition in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibits both the conversion of H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3. It is important to note that disease-specific increases in methylation can occur at chromatin in important genomic loci in the absence of a global increase in histone or protein methylation at the cellular level. For example, aberrant hypermethylation at important disease-associated genes can occur in the setting of global histone or protein hypomethylation.

[0252] Methylation modulators can generally be used to modulate cell proliferation. For example, in some cases, excessive proliferation can be reduced using agents that decrease methylation, while insufficient proliferation can be stimulated using agents that increase methylation. Thus, diseases that can be treated include hyperproliferative diseases, such as benign cell proliferation and malignant cell proliferation (cancer).

[0253] The disorder in which EZH2-mediated protein methylation plays a role may be cancer, cell proliferation disorder, or precancerous condition. The present invention further provides the use of the composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, for the preparation of a medicament useful for treating cancer in a subject in need of such treatment. Examples of cancers that can be treated include lymphomas such as non-Hodgkin's lymphoma, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL); melanoma; and leukemias such as CML. Examples of precancerous conditions include myelodysplastic syndromes (MDS; formerly known as preleukemia).

[0254] In general, compounds that are methylation modulators can generally be used to modulate cell proliferation. For example, in some cases, excessive proliferation can be reduced using agents that decrease methylation, while insufficient proliferation can be reduced using agents that increase methylation. Thus, diseases that can be treated with the compounds of the present invention include hyperproliferative diseases such as benign and malignant cell proliferation.

[0255] As used herein, a "subject in need thereof" refers to a subject who has a disorder in which EZH2-mediated protein methylation plays a role, or a subject who is at a higher risk of developing such a disorder compared to the general population. A subject in need thereof may have a precancerous condition. Preferably, a subject in need thereof has cancer. A "subject" includes a mammal. A mammal may be, for example, any mammal, such as a human, a primate, a bird, a mouse, a rat, a chicken, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. Preferably, the mammal is a human.

[0256] The subject of the present invention includes any human subject who has been diagnosed with, has symptoms of, or is at risk of developing cancer or precancerous condition.The subject of the present invention includes any human subject who expresses EZH2 mutant.For example, the EZH2 mutant comprises one or more mutations, wherein the mutation is substitution, point mutation, nonsense mutation, missense mutation, deletion, or insertion, or any other EZH2 mutation described herein.

[0257] The subject in need thereof may have a refractory or resistant cancer. "Refractory or resistant cancer" means a cancer that does not respond to treatment. The cancer may be resistant at the start of treatment, or it may become resistant during treatment. In some embodiments, the subject in need thereof has cancer recurrence after remission for many recent treatments. In some embodiments, the subject in need thereof has undergone and failed all known effective therapies for cancer treatment. In some embodiments, the subject in need thereof has received at least one previous treatment. In certain embodiments, the previous treatment is a monotherapy. In certain embodiments, the previous treatment is a combination therapy.

[0258] In some embodiments, a subject in need thereof may have a secondary cancer as a result of a previous treatment. By "secondary cancer" is meant a cancer that arises from or results from a previous oncogenic treatment, such as chemotherapy.

[0259] The subject may also exhibit resistance to an EZH2 histone methyltransferase inhibitor or any other therapeutic agent.

[0260] The present invention also features a method for selecting a combination therapy for a subject with cancer. The method includes detecting one or more EZH2 mutations described herein in a sample from the subject; and selecting a combination therapy for treating the cancer based on the presence of one or more EZH2 mutations. In one embodiment, the treatment includes administering a composition of the present invention to the subject. In one embodiment, the method further includes administering a therapeutically effective amount of a composition of the present invention to the subject. EZH2 mutations can be detected using any suitable method known in the art. Further methods are described in U.S. Patent Application Publication No. 20130040906, the entire contents of which are incorporated herein by reference.

[0261] The methods and uses described herein may include detecting one or more EZH2 mutations described herein in a sample from a subject in need thereof before and / or after administering to the subject a composition of the invention (e.g., a composition comprising a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more therapeutic agents). The presence of one or more EZH2 mutations described herein in the tested sample indicates that the subject will respond to the combination therapy of the invention.

[0262] The present invention provides a method for the detection of one or more EZH2 mutations described herein in a subject using a genetic screening method. The present invention provides personalized medicine, treatment, and / or cancer management for a subject through combination therapy. For example, the present invention provides a method for treating or alleviating symptoms of cancer or precancerous conditions in a subject in need thereof by determining the subject's responsiveness to a combination therapy and, if the subject responds to the combination therapy, administering a composition of the present invention to the subject. Responsiveness is determined by obtaining a sample from the subject and detecting one or more EZH2 mutations described herein, where such one or more EZH2 mutations indicate that the subject will respond to the composition of the present invention. Once the subject's responsiveness has been determined, a therapeutically effective amount of a composition, such as a composition comprising a compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more therapeutic agents, can be administered. The therapeutically effective amount of the composition can be determined by one of ordinary skill in the art.

[0263] As used herein, the term "responsive" is interchangeable with the terms "responsive," "susceptible," and "sensitivity," and means that a subject exhibits a therapeutic response when administered a composition of the invention, e.g., the subject's tumor cells or tumor tissue undergo apoptosis and / or necrosis and / or exhibit reduced growth, division, or proliferation. The term also means that a subject has a higher probability, compared to the general population, of exhibiting a therapeutic response when administered a composition of the invention, e.g., the subject's tumor cells or tumor tissue undergo apoptosis and / or necrosis and / or exhibit reduced growth, division, or proliferation.

[0264] "Sample" refers to any biological sample derived from a subject, including, but not limited to, cell, tissue sample, body fluid (including, but not limited to, mucosa, blood, plasma, serum, urine, saliva, and semen), tumor cells, and tumor tissue. Preferably, the sample is selected from bone marrow, peripheral blood cells, blood, plasma, and serum. The sample may be provided by a subject under treatment or test. Alternatively, the sample may be obtained by a physician according to routine practice in the art.

[0265] As used herein, the term "cell proliferative disorder" refers to a condition in which unregulated or abnormal cell proliferation, or both, can lead to the development of an undesirable condition or disease, which may or may not be cancer. Examples of cell proliferative disorders of the present invention include various conditions in which cell division is deregulated. Examples of cell proliferative disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, precancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. As used herein, the term "rapidly dividing cells" is defined as any cell that divides at a rate that exceeds or is greater than that expected or observed among neighboring or juxtaposed cells within the same tissue. Cell proliferative disorders include precancers or precancerous conditions. Cell proliferative disorders include cancer. Preferably, the methods provided herein are used to treat or alleviate the symptoms of cancer. The term "cancer" includes solid tumors, as well as hematological tumors and / or malignant tumors. A "precancerous cell" or "precancerous cell" is a cell that exhibits a cell proliferative disorder that is a precancer or precancerous condition. A "cancer cell" or "cancerous cell" is a cell that exhibits a cell proliferative disorder that is cancer. Any reproducible means of measurement can be used to identify cancer or precancerous cells. Cancer or precancerous cells can be identified by histological typing or grading of a tissue sample (e.g., a biopsy sample). Cancer or precancerous cells can be identified by the use of appropriate molecular markers.

[0266] Examples of non-cancerous conditions or disorders include, but are not limited to, rheumatoid arthritis; inflammation; autoimmune diseases; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout, other joint conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammation Inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; liver fibrosis; acute and chronic kidney disease; irritable bowel syndrome; fever; restenosis; cerebral malaria; stroke and ischemic injury; neurotrauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute and chronic pain; allergic rhinitis; allergic conjunctivitis; chronic heart failure; acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter's syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; herniated condition, prolapse, or herniated disc syndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonary sarcoma; bone resorption diseases such as osteoporosis; graft-versus-host reaction; multiple sclerosis; lupus; fibromyalgia; AIDS and other viral diseases such as shingles, herpes simplex I or II, influenza virus, and cytomegalovirus; and diabetes.

[0267] Examples of cancer include, but are not limited to, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, and urinary bladder cancer. bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebellar astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, gastrointestinal tract, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelogenous leukemia Proliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Sézary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma tumor, eye cancer, islet cell tumor (pancreatic islet), Kaposi's sarcoma, kidney cancer, renal cancer, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell lymphoma cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell carcinoma of the cervix, oral cavity cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cavity cancer, oral cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,These include pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasmacytoma / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing's sarcoma family of tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary tracts, gestational trophoblastic tumor, urethral cancer, endometrial cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0268] A "cell proliferative disorder of the blood system" is a cell proliferative disorder involving cells of the blood system. Cell proliferative disorders of the blood system include lymphoma, leukemia, myeloid neoplasm, mast cell neoplasm, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic myeloid leukemia, primary myelofibrosis, and essential thrombocythemia. Cell proliferative disorders of the blood system may include hyperplasia, dysplasia, and metaplasia of cells of the blood system. Preferably, the compositions of the present invention can be used to treat a cancer selected from the group consisting of the blood cancers of the present invention or the blood cell proliferative disorders of the present invention. The blood cancers of the present invention may include multiple myeloma, lymphomas (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphoma, and lymphomas of lymphocytic and cutaneous origin), leukemias (such as childhood leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms, and mast cell neoplasms.

[0269] A "pulmonary cell proliferative disorder" is a cell proliferative disorder involving lung cells. A pulmonary cell proliferative disorder may include any form of cell proliferative disorder affecting lung cells. A pulmonary cell proliferative disorder may include lung cancer, precancer or precancerous conditions of the lung, benign lung growths or lesions, and malignant lung growths or lesions, as well as metastatic lesions in tissues and organs throughout the body other than the lung. Preferably, the compositions of the present invention can be used to treat lung cancer or a pulmonary cell proliferative disorder. Lung cancer may include any form of lung cancer. Lung cancer may include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer may include small cell lung cancer ("SCLC"), non-small cell lung cancer ("NSCLC"), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, adenosquamous carcinoma, and mesothelioma. Lung cancer may include "scar carcinoma," bronchoalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer may include lung neoplasms with histologic and ultrastructural heterogeneity (e.g., mixed cell types).

[0270] Pulmonary cell proliferative disorders may include any form of cell proliferative disorder affecting lung cells. Pulmonary cell proliferative disorders may include lung cancer and precancerous lung conditions. Pulmonary cell proliferative disorders may include pulmonary hyperplasia, metaplasia, and dysplasia. Pulmonary cell proliferative disorders may include asbestos-induced hyperplasia, squamous metaplasia, and benign reactive mesothelial metaplasia. Pulmonary cell proliferative disorders may include replacement of columnar epithelium with stratified squamous epithelium and mucosal dysplasia. Individuals exposed to inhaled harmful environmental factors such as tobacco smoke and asbestos may be at increased risk of developing pulmonary cell proliferative disorders. Previous lung diseases that may predispose individuals to developing pulmonary cell proliferative disorders may include chronic interstitial lung disease, necrotizing lung disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonia, tuberculosis, recurrent pneumonia, idiopathic pulmonary fibrosis, granulomas, asbestosis, fibrosing alveolitis, and Hodgkin's disease.

[0271] A "cell proliferative disorder of the colon" is a cell proliferative disorder involving colon cells. Preferably, the cell proliferative disorder of the colon is colon cancer. Preferably, the compositions of the present invention can be used to treat colon cancer or a cell proliferative disorder of the colon. Colon cancer may include all forms of colon cancer. Colon cancer may include sporadic colon cancer and hereditary colon cancer. Colon cancer may include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Colon cancer may include adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma. Colon cancer may be associated with a genetic syndrome selected from the group consisting of hereditary non-adenomatous colorectal cancer, familial adenomatous polyposis, Gardner syndrome, Peutz-Jeghers syndrome, Turcot syndrome, and juvenile polyposis. Colon cancer can be caused by a genetic syndrome selected from the group consisting of hereditary non-adenomatous colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis.

[0272] Cell proliferative disorders of the colon may include any form of cell proliferative disorder affecting colon cells. Cell proliferative disorders of the colon may include colon cancer, precancerous conditions of the colon, adenomatous polyps of the colon, and metachronous lesions of the colon. Cell proliferative disorders of the colon may include adenoma. Cell proliferative disorders of the colon may be characterized by colonic hyperplasia, metaplasia, and dysplasia. Previous colonic diseases that may predispose an individual to developing cell proliferative disorders of the colon include previous colon cancer. The current diseases that may predispose individuals to develop colon cell proliferation disorders may include Crohn's disease and ulcerative colitis.The colon cell proliferation disorder may be associated with a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.An individual may be at high risk of developing colon cell proliferation disorders due to the presence of a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.

[0273] A "pancreatic cell proliferative disorder" is a cell proliferative disorder involving pancreatic cells. Pancreatic cell proliferative disorders may include any form of cell proliferative disorder affecting pancreatic cells. Pancreatic cell proliferative disorders may include pancreatic cancer, precancer or precancerous conditions of the pancreas, pancreatic hyperplasia, pancreatic dysplasia, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, as well as metastatic lesions in tissues and organs throughout the body other than the pancreas. Pancreatic cancer may include any form of cancer of the pancreas. Pancreatic cancer may include ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary neoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serous cystadenoma. Pancreatic cancer can also include pancreatic neoplasms with histologic and ultrastructural heterogeneity (eg, mixed cell types).

[0274] A "cell proliferative disorder of the prostate" is a cell proliferative disorder involving cells of the prostate. Cell proliferative disorders of the prostate may include any form of cell proliferative disorder affecting prostate cells. Cell proliferative disorders of the prostate may include prostate cancer, precancer or precancerous conditions of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, as well as metastatic lesions in tissues and organs throughout the body other than the prostate. Cell proliferative disorders of the prostate may include hyperplasia, metaplasia, and dysplasia of the prostate.

[0275] A "cell proliferative disorder of the skin" is a cell proliferative disorder involving skin cells. A cell proliferative disorder of the skin may include any form of cell proliferative disorder affecting skin cells. A cell proliferative disorder of the skin may include precancer or precancerous conditions of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissues and organs throughout the body other than the skin. A cell proliferative disorder of the skin may include hyperplasia, metaplasia, and dysplasia of the skin.

[0276] A "cell proliferative disorder of the ovary" is a cell proliferative disorder involving cells of the ovary. Cell proliferative disorders of the ovary may include any form of cell proliferative disorder affecting cells of the ovary. Cell proliferative disorders of the ovary may include precancer or precancerous conditions of the ovary, benign growths or lesions of the ovary, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissues and organs throughout the body other than the ovaries. Cell proliferative disorders of the skin may include hyperplasia, metaplasia, and dysplasia of cells of the ovary.

[0277] A "cell proliferative disorder of the breast" is a cell proliferative disorder involving cells of the breast. Cell proliferative disorders of the breast may include any form of cell proliferative disorder affecting breast cells. Cell proliferative disorders of the breast may include breast cancer, precancer or precancerous conditions of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, as well as metastatic lesions in tissues and organs throughout the body other than the breast. Cell proliferative disorders of the breast may include hyperplasia, metaplasia, and dysplasia of the breast.

[0278] The cell proliferative disorder of the breast may be a precancerous condition of the breast. The compositions of the present invention can be used to treat precancerous conditions of the breast. Precancerous conditions of the breast may include atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasm, and stage 0 or grade 0 proliferations or lesions of the breast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ). Precancerous breast conditions are classified by the American Joint Committee on Cancer may be staged according to the TNM classification scheme accepted by the American Joint Committee on Cancer (AJCC), where the primary tumor (T) is assigned a stage of T0 or Tis; regional lymph nodes (N) are assigned a stage of N0; and distant metastases (M) are assigned a stage of M0.

[0279] The cell proliferative disorder of the breast may be breast cancer. Preferably, breast cancer can be treated using the compositions of the present invention. Breast cancer includes all forms of cancer of the breast. Breast cancer may include primary epithelial breast cancer. Breast cancer may include cancer in which the breast is invaded by other tumors, such as lymphoma, sarcoma, or melanoma. Breast cancer may include breast carcinoma, breast ductal carcinoma, breast lobular carcinoma, breast undifferentiated carcinoma, breast cystosarcoma phyllodes, breast angiosarcoma, and primary breast lymphoma. Breast cancer may include stage I, II, IIIA, IIIB, IIIC, and IV breast cancer. Breast ductal carcinoma may include invasive carcinoma, in situ invasive carcinoma with a predominant intraductal component, inflammatory breast cancer, and breast ductal carcinoma with a histology selected from the group consisting of acne, mucinous (colloid), medullary, medullary with lymphocytic infiltration, papillary, scirrhous, and ductal. Lobular carcinoma of the breast may include invasive lobular carcinoma with a predominant in situ component, invasive lobular carcinoma, and invasive lobular carcinoma. Breast cancer may include Paget's disease, Paget's disease with intraductal carcinoma, and Paget's disease with invasive ductal carcinoma. Breast cancer may include breast neoplasms with histologic and ultrastructural heterogeneity (e.g., mixed cell types).

[0280] Preferably, the compounds of the present invention, or pharmaceutically acceptable salts, prodrugs, metabolites, polymorphs, or solvates thereof, can be used to treat breast cancer. The breast cancer to be treated may include familial breast cancer. The breast cancer to be treated may include sporadic breast cancer. The breast cancer to be treated may also occur in male subjects. The breast cancer to be treated may occur in female subjects. The breast cancer to be treated may occur in premenopausal or postmenopausal female subjects. The breast cancer to be treated may occur in subjects aged 30 or older, or in subjects younger than 30 years. The breast cancer to be treated may occur in subjects aged 50 or older, or in subjects younger than 50 years. The breast cancer to be treated may occur in subjects aged 70 or older, or in subjects younger than 70 years.

[0281] The breast cancer to be treated can be classified to identify familial or spontaneous mutations in BRCA1, BRCA2, or p53. The breast cancer to be treated can be classified as having HER2 / neu gene amplification, overexpressing HER2 / neu, or having low, intermediate, or high levels of HER2 / neu expression. The breast cancer to be treated can be classified for a marker selected from the group consisting of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2, Ki-67, CA15-3, CA27-29, and c-Met. The breast cancer to be treated can be classified as ER-unknown, ER-rich, or ER-poor. The breast cancer to be treated can be classified as ER-negative or ER-positive. ER classification of breast cancer can be performed by any reproducible means. ER classification of breast cancer can be performed as described in Onkologie 27:175-179 (2004). The breast cancer to be treated can be classified as RP-unknown, PR-rich, or PR-poor.The breast cancer to be treated can be classified as PR-negative or PR-positive.The breast cancer to be treated can be classified as receptor-positive or receptor-negative.The breast cancer to be treated can be classified as associated with elevated blood levels of CA15-3, CA27-29, or both.

[0282] The breast cancer to be treated may include a localized tumor of the breast. The breast cancer to be treated may include a tumor of the breast associated with a negative sentinel lymph node (SLN) biopsy. The cancer may include a breast tumor associated with a positive sentinel lymph node (SLN) biopsy. The breast cancer to be treated may include a breast tumor associated with one or more positive axillary lymph nodes, where the axillary lymph nodes have been staged by any applicable method. The breast cancer to be treated has been classified as having node-negative status (e.g., node-negative) or node-positive status (e.g., node-positive). The breast cancer to be treated may include a breast tumor that has metastasized to another location in the body. The breast cancer to be treated may be classified as having metastasized to a location selected from the group consisting of bone, lung, liver, or brain. The breast cancer to be treated may be classified according to a characteristic selected from the group consisting of metastatic, localized, regional, localized, locally advanced, distant, multicentric, bilateral, ipsilateral, contralateral, newly diagnosed, recurrent, and inoperable.

[0283] The compound of the present invention, or its pharmaceutically acceptable salt, ester, prodrug, metabolite, polymorph or solvate, can be used to treat or prevent breast cell proliferation disorders, or treat or prevent breast cancer in subjects who are at a higher risk of developing breast cancer than the general population.The subjects who are at a higher risk of developing breast cancer than the general population are female subjects who have a family history or personal history of breast cancer.The subjects who are at a higher risk of developing breast cancer than the general population are female subjects who have germline or spontaneous mutations in BRCA1 or BRCA2, or both.The subjects who are at a higher risk of developing breast cancer than the general population are female subjects who have a family history of breast cancer and germline or spontaneous mutations in BRCA1 or BRCA2, or both.The subjects who are at a higher risk of developing breast cancer than the general population are women who are over 30 years old, over 40 years old, over 50 years old, over 60 years old, over 70 years old, over 80 years old, or over 90 years old. Subjects at increased risk of developing breast cancer compared to the general population are those with atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, and in The subject has lobular carcinoma in situ (LCIS), lobular neoplasia, or stage 0 growth or lesion of the breast (eg, stage 0 or grade 0 breast cancer, or carcinoma in situ).

[0284] The breast cancer to be treated can be histologically graded according to the Scarff-Bloom-Richardson system, where breast tumors are assigned a mitotic count score of 1, 2, or 3; a nuclear pleomorphism score of 1, 2, or 3; a duct formation score of 1, 2, or 3; and a total Scarff-Bloom-Richardson score of 3 to 9. The breast cancer to be treated can be assigned a tumor grade according to the International Consensus Panel on the Treatment of Breast Cancer selected from the group consisting of grade 1, grade 1-2, grade 2, grade 2-3, or grade 3.

[0285] The cancer to be treated can be staged according to the American Joint Committee on Cancer (AJCC) TNM classification system, in which tumors (T) are assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; regional lymph nodes (N) are assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, or N3c; and distant metastases (M) can be assigned a stage of MX, M0, or M1. The cancer to be treated can be staged according to the American Joint Committee on Cancer (AJCC) classification as stage I, stage IIA, stage IIB, stage IIIA, stage IIIB, stage IIIC, or stage IV. The cancer to be treated can be assigned a grade according to the AJCC classification as grade GX (e.g., grade cannot be assessed), grade 1, grade 2, grade 3, or grade 4. The treated cancers were classified as pNX, pN0, PN0(I-), PN0(I+), PN0(mol- ), PN0(mol+), PN1, PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

[0286] The cancer to be treated may include tumors determined to be about 2 centimeters or less in diameter. The cancer to be treated may include tumors determined to be about 2 to about 5 centimeters in diameter. The cancer to be treated may include tumors determined to be about 3 centimeters or more in diameter. The cancer to be treated may include tumors determined to be more than 5 centimeters in diameter. The cancer to be treated may be classified by microscopic appearance as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated. The cancer to be treated may be classified by microscopic appearance in terms of mitotic count (e.g., amount of cell division) or nuclear pleomorphism (e.g., changes in cells). The cancer to be treated may be classified by microscopic appearance as associated with areas of necrosis (e.g., areas of dead or degenerating cells). The cancer to be treated may be classified as having an abnormal karyotype, an abnormal chromosome number, or having one or more chromosomes that are abnormal in appearance. The cancer to be treated may be classified as being aneuploid, triploid, tetraploid, or having altered ploidy. The cancers to be treated can be classified as having a chromosomal translocation, or a deletion or duplication of an entire chromosome, or a region of deletion, duplication, or amplification of part of a chromosome.

[0287] The cancer to be treated can be evaluated by DNA cytometry, flow cytometry, or image cytometry.The cancer to be treated can be classified as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division (for example, in the S phase of cell division).The cancer to be treated can be classified as having a low S phase fraction or a high S phase fraction.

[0288] As used herein, a "normal cell" is a cell that cannot be classified as part of a "cell proliferation disorder." Normal cells lack unregulated or abnormal proliferation, or both, that can lead to the development of an undesirable condition or disease. Preferably, normal cells have normally functioning cell cycle checkpoint control mechanisms.

[0289] As used herein, "contacting a cell" refers to a condition in which the compound or other composition in question is in direct contact with the cell or is sufficiently close to induce a desired biological effect in the cell.

[0290] As used herein, "candidate compound" refers to a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, metabolite, polymorph, or solvate thereof, that has been or will be tested in one or more in vitro or in vivo biological assays to determine whether the compound has the potential to elicit a desired biological or medical response in cells, tissues, systems, animals, or humans as desired by a researcher or physician. A candidate compound is a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, metabolite, polymorph, or solvate thereof. The biological or medical response may be the treatment of cancer. The biological or medical response may be the treatment or prevention of a cell proliferation disorder. In vitro or in vivo biological assays include, but are not limited to, enzyme activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and assays described herein.

[0291] As used herein, "treating" or "treat" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes administering a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. This includes administration of solvates.

[0292] The compositions of the invention, or pharmaceutically acceptable salts, prodrugs, metabolites, polymorphs, or solvates thereof, can also be used to prevent a disease, condition, or disorder. As used herein, "preventing" or "prevent" describes reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder.

[0293] The term "alleviate" as used herein is meant to describe the process of reducing the severity of signs or symptoms of a disorder. Importantly, signs or symptoms can be alleviated without being eliminated. In a preferred embodiment, administration of the pharmaceutical composition of the present invention results in the elimination of signs or symptoms, but elimination is not necessary. An effective dose is expected to reduce the severity of signs or symptoms. For example, signs or symptoms of a disorder such as cancer that can occur in multiple locations are alleviated if the severity of the cancer is reduced in at least one of the multiple locations.

[0294] As used herein, the term "severity" refers to describing the potential of cancer to transform from a precancerous or benign state to a malignant state. Alternatively, or in addition, severity refers to describing the stage of cancer, for example, according to the TNM system (accepted by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other art-recognized methods. Cancer stage refers to the extent or severity of cancer based on factors such as the location of the primary tumor, tumor size, tumor number, and lymph node involvement (spread of cancer to lymph nodes). Alternatively, or in addition, severity refers to describing tumor grade according to art-recognized methods (see National Cancer Institute, www.cancer.gov). Tumor grade is a system used to classify cancer cells based on how abnormal they are under a microscope and how quickly they may grow and spread. When determining tumor grade, many factors are taken into account, such as the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary depending on the type of cancer. Severity also describes the histological grade, also called differentiation, which refers to how similar tumor cells are to normal cells of the same tissue type (see National Cancer Institute, www.cancer.gov). In addition, severity describes the nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of dividing tumor cells (see National Cancer Institute, www.cancer.gov).

[0295] In another embodiment of the present invention, the severity describes the extent to which a tumor secretes growth factors, degrades the extracellular matrix, becomes vascularized, loses adhesion to adjacent tissues, or metastasizes.Furthermore, the severity describes the number of locations to which the primary tumor has metastasized.Finally, the severity includes the difficulty of treating tumors of varying types and locations.For example, inoperable tumors, cancer tumors that have more access to multiple body systems (hematologic tumors and immunological tumors), and tumors that are most resistant to traditional treatments are considered to be the most severe.In these situations, an increase in the subject's life expectancy and / or reduction in pain, a reduction in the proportion of cancerous cells or limited cells to one system, and a reduction in the stage / tumor grade / histological grade / nuclear grade of cancer are considered to be alleviation of cancer signs or symptoms.

[0296] As used herein, the term "symptom" is defined as a manifestation of disease, illness, injury, or something not right with the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not be readily noticed by others, which are defined as non-medical professionals.

[0297] The term "symptom" as used herein is also defined as an indication that something is not right in the body, but a symptom is defined as something that can be seen by a doctor, nurse, or other medical professional.

[0298] Cancer is a group of diseases that can cause almost any sign or symptom. Signs and symptoms depend on where the cancer is, its size, and how much it affects nearby organs or structures. If the cancer spreads (metastasizes), symptoms may appear in different parts of the body.

[0299] The disorder in which EZH2-mediated protein methylation plays a role may be a neurological disease.Thus, the compounds of the present invention can also be used to treat neurological diseases, such as epilepsy, schizophrenia, bipolar disorder or other psychological and / or psychiatric disorders, neuropathy, skeletal muscle atrophy, and neurodegenerative diseases, such as neurodegenerative diseases.Examples of neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and Parkinson's disease.Another class of neurodegenerative diseases includes diseases that are at least partially caused by polyglutamine aggregation. Diseases in this class include Huntington's disease, spinal-bulbar muscular atrophy (SBMA or Kennedy disease), dentatorubral-pallidoluysian atrophy (DRPLA), spinocerebellar ataxia 1 (SCA1), spinocerebellar ataxia 2 (SCA2), Machado-Joseph disease (MJD; SCA3), spinocerebellar ataxia 6 (SCA6), spinocerebellar ataxia 7 (SCA7), and spinocerebellar ataxia 12 (SCA12).

[0300] Any other disease in which EZH2-mediated epigenetic methylation plays a role may be treatable or preventable using the compositions and methods described herein.

[0301] Cancer treatment can result in a reduction in tumor size. A reduction in tumor size can also be referred to as "tumor regression." Preferably, after treatment, tumor size is reduced by 5% or more compared to its size before treatment; more preferably, tumor size is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by 75% or more. Tumor size can be measured by any reproducible measurement means. Tumor size can be measured as the diameter of the tumor.

[0302] Cancer treatment can result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or more compared to its size before treatment; more preferably, tumor volume is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by 75% or more. Tumor volume can be measured by any reproducible measurement means.

[0303] Cancer treatment results in a reduction in tumor number. Preferably, after treatment, tumor number is reduced by 5% or more compared to the number before treatment; more preferably, tumor number is reduced by 10% or more; more preferably, by 20% or more; more preferably, by 30% or more; more preferably, by 40% or more; even more preferably, by 50% or more; and most preferably, by more than 75%. Tumor number can be measured by any reproducible measurement means. Tumor number can be measured by counting tumors visible to the naked eye or at a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0304] Cancer treatment results in a reduction in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. This can result in a reduction in the number of metastatic lesions by 5% or more after treatment compared to the number before treatment. Preferably, the number of metastatic lesions is reduced by 10% or more, more preferably by 20% or more, more preferably by 30% or more, more preferably by 40% or more, even more preferably by 50% or more, and most preferably by more than 75%. The number of metastatic lesions can be measured by any reproducible measurement means. The number of metastatic lesions can be measured by counting the number of metastatic lesions visible to the naked eye or under a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0305] Cancer treatment can result in an increase in the average survival time of the population of treated subjects compared with the population that receives only carrier.Preferably, average survival time increases by more than 30 days; more preferably, more than 60 days; more preferably, more than 90 days; and most preferably, more than 120 days.The increase in average survival time of a population can be measured by any reproducible means.For example, the increase in average survival time of a population can be measured by calculating the average length of survival after the start of treatment with active compound for the population.The increase in average survival time of a population can also be measured by calculating the average length of survival after the completion of the first round of treatment with active compound for the population.

[0306] Cancer treatment can cause the increase in the average survival time of the treated group of subjects compared with the group of untreated subjects.Preferably, the average survival time increases by more than 30 days; more preferably, more than 60 days; more preferably, more than 90 days; and most preferably, more than 120 days.The increase in the average survival time of the group can be measured by any reproducible means.For example, the increase in the average survival time of the group can be measured by calculating the average survival time of the group after the start of treatment with active compound.The increase in the average survival time of the group can also be measured by calculating the average survival time of the group after the completion of the first round of treatment with active compound.

[0307] Cancer treatment can result in an increase in the average survival time of the population of treated subjects compared with the population that receives a single drug therapy that is not the compound of the present invention or its pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative.Preferably, the average survival time increases by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.The increase in the average survival time of the population can be measured by any reproducible means.The increase in the average survival time of the population can be measured, for example, by calculating the average length of survival of the population after the start of treatment with active compound.The increase in the average survival time of the population can also be measured, for example, by calculating the average length of survival of the population after the completion of the first round of treatment with active compound.

[0308] The treatment of cancer can result in a reduction in the mortality rate of a population of treated subjects compared to a population that receives only carrier. The treatment of cancer can result in a reduction in the mortality rate of a population of treated subjects compared to a population that receives monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof. Preferably, the mortality rate is reduced by more than 2%; more preferably, more than 5%; more preferably, more than 10%; and most preferably, more than 25%. The reduction in the mortality rate of a population of treated subjects can be measured by any reproducible means. The reduction in the mortality rate of a population can be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the start of treatment with an active compound. The reduction in the mortality rate of a population can also be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the completion of the first round of treatment with an active compound.

[0309] Cancer treatment can cause tumor growth rate to decrease.Preferably, after treatment, tumor growth rate is reduced by at least 5% compared with the number before treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.Tumor growth rate can be measured by any reproducible measurement means.Tumor growth rate can be measured according to the change in tumor diameter per unit time.

[0310] Cancer treatment can result in a reduction in tumor regrowth.Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%.Tumor regrowth can be measured by any reproducible measurement method.Tumor regrowth is measured, for example, by measuring the increase in tumor diameter after previous tumor reduction by treatment.A reduction in tumor regrowth is indicated by the inability of tumors to recur after treatment is stopped.

[0311] Treatment or prevention of cell proliferation disorder can result in the reduction of cell proliferation rate.Preferably, after treatment, cell proliferation rate is reduced by at least 5%; more preferably, at least 10%; more preferably, at least 20%; more preferably, at least 30%; more preferably, at least 40%; more preferably, at least 50%; even more preferably, at least 50%; and most preferably, at least 75%.Cell proliferation rate can be measured by any reproducible measuring means.Cell proliferation rate is measured, for example, by measuring the number of dividing cells in tissue sample per unit time.

[0312] Treatment or prevention of cell proliferation disorder can result in a decrease in the proportion of proliferating cells.Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, at least 10%; more preferably, at least 20%; more preferably, at least 30%; more preferably, at least 40%; more preferably, at least 50%; even more preferably, at least 50%; and most preferably, at least 75%.The proportion of proliferating cells can be measured by any reproducible measurement means.Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells compared to the number of non-dividing cells in a tissue sample.The proportion of proliferating cells may be equal to the mitotic index.

[0313] Treatment or prevention of cell proliferation disorders can result in a reduction in the size of the area or zone of cell proliferation. Preferably, after treatment, the size of the area or zone of cell proliferation is reduced by at least 5% compared to its size before treatment; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The size of the area or zone of cell proliferation can be measured by any reproducible measurement means. The size of the area or zone of cell proliferation can be measured as the diameter or width of the area or zone of cell proliferation.

[0314] Treatment or prevention of a cell proliferation disorder can result in a decrease in the number or proportion of cells with an abnormal appearance or morphology. Preferably, after treatment, the number of cells with abnormal morphology is reduced by at least 5% compared to their size before treatment; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%. % reduction; more preferably, at least 40% reduction; more preferably, at least 50% reduction; even more preferably, at least 50% reduction; and most preferably, at least 75% reduction. Abnormal cell appearance or morphology can be measured by any reproducible measurement means. Abnormal cell morphology can be measured microscopically, for example, using an inverted tissue culture microscope. Abnormal cell morphology can also take the form of nuclear pleomorphism.

[0315] The term "selectively" as used herein means a tendency to occur more frequently in one population than in another. The populations being compared may be cell populations. Preferably, the compounds of the present invention, or pharmaceutically acceptable salts, prodrugs, metabolites, polymorphs, or solvates thereof, act selectively on cancer or precancerous cells but not on normal cells. Preferably, the compounds of the present invention, or pharmaceutically acceptable salts, prodrugs, metabolites, polymorphs, or solvates thereof, act selectively to modulate one molecular target (e.g., a target protein methyltransferase) but do not significantly modulate another molecular target (e.g., a non-target protein methyltransferase). The present invention also provides a method for selectively inhibiting the activity of an enzyme, such as a protein methyltransferase. Preferably, if an event occurs more than twice more frequently in population A compared to population B, the event occurs selectively in population A compared to population B. If an event occurs more than five times more frequently in population A, the event occurs selectively. An event is said to occur preferentially if it occurs more than 10 times more frequently in population A; more preferably, more than 50 times more frequently in population A compared to population B; even more preferably, more than 100 times; and most preferably, more than 1000 times. For example, cell death is said to occur preferentially in cancer cells if it occurs more than 2 times more frequently in cancer cells compared to normal cells.

[0316] Compositions of the present invention, such as compositions comprising any compound of Formula (IIa) or a pharmaceutically acceptable salt thereof and one or more other therapeutic agents, such as prednisone, can modulate the activity of a molecular target (e.g., a target protein methyltransferase). Modulating refers to stimulating or inhibiting the activity of a molecular target. Preferably, a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target at least two-fold compared to the activity of the molecular target under the same conditions but lacking the presence of the compound alone. More preferably, a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target at least five-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold compared to the activity of the molecular target under the same conditions but lacking the presence of the compound alone. The activity of molecular target can be measured by any reproducible means.The activity of molecular target can be measured in vitro or in vivo.For example, the activity of molecular target can be measured in vitro by enzyme activity assay or DNA binding assay, or the activity of molecular target can be measured in vivo by assaying for the expression of reporter gene.

[0317] A composition of the present invention does not significantly modulate the activity of a molecular target if the addition of the compound does not stimulate or inhibit the activity of the molecular target by more than 10% compared to the activity of the molecular target under the same conditions but in the absence of the compound alone.

[0318] As used herein, the term "isoenzyme selective" refers to the preferential inhibition or stimulation of an enzyme of a first isoform compared to an enzyme of a second isoform (e.g., a protein "preferential inhibition" refers to the preferential inhibition or stimulation of protein methyltransferase isozyme alpha relative to protein methyltransferase isozyme beta. Preferably, the compounds of the invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, exhibit a minimum of a 4-fold difference, preferably a 10-fold difference, and more preferably a 50-fold difference in the dose required to achieve a biological effect. Preferably, the compounds of the invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, exhibit this difference over the range of inhibition, which difference corresponds to the IC for the molecular target of interest. 50 , i.e., 50% inhibition.

[0319] Administration of the compositions of the invention to a cell or subject in need thereof can result in modulation (ie, stimulation or inhibition) of the activity of a protein methyltransferase in the subject.

[0320] Administration of a composition comprising a compound of the invention, e.g., any compound of Formula (IIa) or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents, such as prednisone, to a cell or subject in need thereof results in modulation (i.e., stimulation or inhibition) of the activity of an intracellular target (e.g., substrate). Several intracellular targets can be modulated using the compounds of the invention, including, but not limited to, protein methyltransferases.

[0321] Activation refers to placing a composition of matter (e.g., a protein or nucleic acid) in a state suitable for carrying out a desired biological function. A composition of matter that can be activated also has an inactivated state. An activated composition of matter may have an inhibitory or stimulatory biological function, or both.

[0322] Enhancement refers to an increase in a desired biological activity of a composition of matter (e.g., a protein or nucleic acid). Enhancement may occur by increasing the concentration of the composition of matter.

[0323] As used herein, "cell cycle checkpoint pathway" refers to a biochemical pathway involved in the modulation of cell cycle checkpoints. A cell cycle checkpoint pathway may have a stimulating or inhibitory effect, or both, on one or more functions, including cell cycle checkpoints. A cell cycle checkpoint pathway is composed of at least two components of substances, preferably proteins, both of which contribute to the modulation of cell cycle checkpoints. A cell cycle checkpoint pathway can be activated by the activation of one or more members of the cell cycle checkpoint pathway. Preferably, the cell cycle checkpoint pathway is a biochemical signaling pathway.

[0324] As used herein, "cell cycle checkpoint regulator" refers to a composition of matter that can function at least in part in modulating a cell cycle checkpoint. A cell cycle checkpoint regulator may have a stimulatory or inhibitory effect, or both, on one or more functions, including a cell cycle checkpoint. A cell cycle checkpoint regulator may or may not be a protein.

[0325] Treatment of cancer or a cell proliferation disorder can result in cell death, and preferably cell death results in at least a 10% reduction in the number of cells in a population. More preferably, cell death means at least a 20% reduction; more preferably at least a 30% reduction; more preferably at least a 40% reduction; more preferably at least a 50% reduction; and most preferably at least a 75% reduction. The number of cells in a population can be measured by any reproducible means. The number of cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence, or other methods. It can be measured by light microscopy and optical microscopy. Methods for measuring cell death are set forth in Li et al., Proc Natl Acad Sci USA. 100(5):2674-8, 2003. In one embodiment, cell death occurs by apoptosis.

[0326] Preferably, the effective amount of the composition of the present invention, or its pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate, is not significantly cytotoxic to normal cells.A therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if the administration of the compound at a therapeutically effective amount does not induce cell death in more than 10% of normal cells.A therapeutically effective amount of a compound is not significantly affected by the viability of normal cells if the administration of the compound at a therapeutically effective amount does not induce cell death in more than 10% of normal cells.In some embodiments, cell death occurs by apoptosis.

[0327] Contacting cells with a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, can selectively induce or activate cell death in cancer cells. Administering a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to a subject in need thereof can selectively induce or activate cell death in cancer cells. Contacting cells with a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, can selectively induce cell death in one or more cells affected by a cell proliferation disorder. Preferably, administering a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to a subject in need thereof, selectively induces cell death in one or more cells affected by a cell proliferation disorder.

[0328] The present invention relates to methods of treating or preventing cancer by administering a composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to a subject in need thereof, wherein administration of the composition of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, results in one or more of the following: prevention of cancer cell proliferation by accumulation of cells in one or more phases of the cell cycle (e.g., G1, G1 / S, G2 / M), or induction of cellular senescence, or promotion of tumor cell differentiation; promotion of cell death in cancer cells by cytotoxicity, necrosis, or apoptosis, without significant amounts of cell death in normal cells; anti-tumor activity in animals with a therapeutic index of at least 2. As used herein, "therapeutic index" is the maximum tolerated dose divided by the effective dose.

[0329] Those skilled in the art can refer to general reference textbooks for detailed descriptions of known techniques or equivalent techniques discussed herein. These textbooks include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, NY; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, NY; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th ed. (1990). Of course, these textbooks can also be referenced when making or using embodiments of the present invention.

[0330] [Example 1] Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]3-carboxamide

[0331] [ka]

[0332] Step 1: Synthesis of 5-bromo-2-methyl-3-nitrobenzoic acid

[0333] [ka]

[0334] To a stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in concentrated HSO (400 mL) was added 1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) in portions at room temperature, and the reaction mixture was stirred at room temperature for 5 h. The reaction mixture was poured onto ice-cold water, and the precipitated solid was filtered off, washed with water, and dried under reduced pressure to give the desired compound (140 g, 98%) as a solid. The isolated compound was taken directly to the next step. 1 HNMR (DMSO-d6, 400 MHz) δ 8. 31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).

[0335] Step 2: Synthesis of methyl 5-bromo-2-methyl-3-nitrobenzoate

[0336] [ka]

[0337] To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8 L) at room temperature, sodium carbonate (468 g, 4415 mmol) was added, followed by methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60° C. for 8 h. Upon completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate (1 L × 3). The combined filtrate was washed with water (3 L × 5), and the aqueous phase was back-extracted with ethyl acetate (1 L × 3). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give This afforded the title compound (290 g, 97% yield) as a solid. The isolated compound was taken directly to the next step. 1 H NMR (CDCl3,400MHz)δ8.17 (s, 1H), 7. 91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

[0338] Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

[0339] [ka]

[0340] To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5 L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5 L of water). The resulting mixture was stirred at 80° C., and iron powder (472 g, 8451 mmol) was added portionwise. The resulting reaction mixture was heated at 80° C. for 12 h. Upon completion, as determined by TLC, the reaction mixture was hot filtered onto Celite®, and the Celite bed was washed with methanol (5 L) followed by 30% MeOH in DCM (5 L). The combined filtrate was concentrated under reduced pressure, and the resulting residue was diluted with aqueous sodium bicarbonate solution (2 L) and extracted with ethyl acetate (5 L × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound (220 g, 85%) as a solid. This compound was taken directly to the next step. 1 HNMR(CDCl3,400MHz) δ 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s , 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

[0341] Step 4: Synthesis of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)benzoate

[0342] [ka]

[0343] To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (15 g, 61.5 mmol) and dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300 mL) was added acetic acid (22 g, 369 mmol). The reaction mixture was stirred at room temperature for 15 minutes. After that, the reaction mixture was cooled to 0 °C and sodium triacetoxyborohydride (39 g, 184 mmol) was added. The reaction mixture was stirred at room temperature overnight. Upon completion of the reaction, as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH of 7-8 was obtained. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate:hexane to give the desired compound (14 g, 69%) as a solid. 1 HNMR(DMSO-d6,400MHz) δ 7.01 (s, 1H), 6.98 (s, 1 H), 5.00 (d, 1H, J=7.6 Hz), 3.84-3.87 (m, 2H),3.79 (s, 3H), 3 .54-3.56 (m, 1H), 3.43 (t, 2H, J=12 Hz), 2.14(s, 3H), 1.81- 1.84 (m, 2H), 1.47-1.55 (m, 2H).

[0344] Step 5: Synthesis of methyl 5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate

[0345] [ka]

[0346] To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl)amino)benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0° C., and sodium triacetoxyborohydride (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction, as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH of 7-8 was obtained, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate:hexane to give the desired compound (14 g, 93%) as a viscous liquid. 1 H NMR (DMSO-d6,400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80(bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38(s, 3H), 1.52- 1.61 (m, 2H), 1.37-1.50 (m, 2H), 0.87 (t, 3H,J=6.8 Hz).

[0347] Step 6: Synthesis of 5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide

[0348] [ka]

[0349] To a stirred solution of 5-bromo-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (14 g, 39.4 mmol) in ethanol (100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol in 25 mL water), and the resulting mixture was stirred at 60 °C for 1 h. Upon completion of the reaction, as determined by TLC, the solvent was removed under reduced pressure, and the resulting residue was acidified with 1 N HCl until a pH of 7 was obtained, followed by the addition of aqueous citric acid solution until a pH of 5-6 was obtained. The aqueous layer was diluted with 10% M NaOH in DCM. Extraction with eOH (200 mL X3) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the corresponding acid (14 g, 100%).

[0350] The above acid (14 g, 40.9 mmol) was then dissolved in DMSO (70 mL), and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (12.4 g, 81.9 mmol) was added thereto. After the reaction mixture was stirred at room temperature for 15 minutes, PYBOP (31.9 g, 61.4 mmol) was added, and stirring was continued at room temperature overnight. Upon completion of the reaction, as determined by TLC, the reaction mixture was poured onto ice-cold water (700 mL), stirred for 30 minutes, and the precipitated solid was collected by filtration, washed with water (500 mL), and air-dried. The resulting solid was stirred with acetonitrile (75 mL × 2), filtered, and air-dried. The resulting solid was again stirred with 5% MeOH in DCM (100 mL), filtered, and thoroughly dried under reduced pressure to give the title compound (14 g, 74%) as a solid. 1 HNMR(DMSO-d6,400MHz) δ 11.47 (s, 1H), 8.23 ​​(t, 1H), 7.3 0 (s, 1H), 7.08 (s, 1H), 5.85 (s, 1H), 4.23(d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=10.4 Hz), 3.20-3.26 (m, 2H),3.00-3.07 (m, 1 H), 2.91-2.96 (m, 2H), 2.18 (s, 3H), 2.14(s, 3H), 2.10 (s, 3 H), 1.58-1.60 (m, 2H), 1.45-1.50 (m, 2H), 0.78(t, 3H, J=6.8 Hz).

[0351] Step 7: Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide

[0352] [ka]

[0353] To a stirred solution of 5-bromo-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino) 2-methylbenzamide (14 g, 29.5 mmol) in a dioxane / water mixture (70 mL / 14 mL) was added 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine (13.4 g, 44.2 mmol), followed by NaCO (11.2 g, 106.1 mmol). The solution was purged with argon for 15 min, after which Pd(PPh) (3.40 g, 2.94 mmol) was added, and the solution was purged again with argon for an additional 10 min. The reaction mixture was heated at 100 °C for 4 h. After completion (monitored by TLC), the reaction mixture was diluted with water and extracted with 10% MeOH / DCM. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with methanol:DCM to give the title compound (12 g, 71%) as a solid. Analytical data: LCMS: 573.35 (M+1) + HPLC: 99.5% (@254 nm) (Rt ;3.999;Method:Column:YMC ODS-A 150mm x 4.6mm x 5μ; Mobile phase: A; 0.05% in water TFA / B; 0.05% TFA in acetonitrile; injection volume: 10 μL, column temperature: 30 °C; flow rate: 1.4 mL / min; gradient: 5% B to 95% B in 8 min, 1.5 min hold, 9.51–12 min 5% B); 1 HNMR (DMSO-d6, 400MHz) δ 11.46 (s, 1H), 8.19 (t, 1H), 7.57 (d, 2H,J=7.2 Hz), 7.36-7. 39 (m, 3H), 7.21 (s, 1H), 5.85 (s, 1H), 4.28(d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57 (bs, 4H), 3.48(s, 2H), 3.24 (t, 2H, J=10.8Hz), 3.07-3.09(m,2H),3.01 (m, 1H),2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1.64-1.67(m, 2H), 1.51-1.53 ​​(m, 2H), 0.83 (t, 3H, J=6.4 Hz).

[0354] Step 8: Synthesis of N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide trihydrochloride

[0355] [ka]

[0356] N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H)-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide (12 g, 21.0 mmol) was dissolved in methanolic HCl (200 mL) and stirred at room temperature for 3 h. After 3 h of stirring, the reaction mixture was concentrated under reduced pressure. The resulting solid was stirred with ether (100 mL X2) to give the desired salt (11 g, 77%) as a solid. Analytical data for the tri-HCl salt: LCMS: 573.40 (M+1). + HPLC: 99.1% (@254 nm) (R t ;3.961;Method:Column:YMC ODS-A 150mm x 4.6mm x 5µ; Mobile phase: A; 0.05% TFA / B in water; 0.05% TFA in acetonitrile; Injection volume: 10µL, Column temperature: 30°C; Flow rate: 1.4mL / min; Gradient: 5% B to 95% B in 8min, 1.5min hold, 9.51-12min 5% B); 1 HNMR(D2O400MHz) δ 7.92 (bs, 1H,) 7.80 (s, 1H), 7.77 (d, 2H, J=8 Hz), 7.63(s, 1H), 7.61 (s, 1H), 6.30 (s, 1H), 4.48 (s, 2H), 4.42 (s, 2H), 4.09-4.11(m, 4H), 3.95-3.97 (m, 2H), 3.77 (t, 3H, J=10.4Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H), 2.35(s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1.76 (m, 2H), 1.04 (t, 3H,J=6.8 Hz).

[0357] [Example 2] Combination therapy of compound 44 and CHOP components Human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC575), OCI-Ly19 (DSMZ; ACC528), and RL (ATCC; CRL-2261) were obtained from the indicated sources and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were incubated at 37°C in a humidified incubator with 5% CO and 95% CO. Cultures were grown in tissue culture flasks in an air atmosphere.

[0358] The effect of combined treatment of Compound 44 with each individual CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisolone) component on cancer cell viability was investigated in vitro. The dosing schedule is shown in Figure 1A. WSU-DLCL2 human lymphoma cells were treated with increasing concentrations of Compound 44. After four days, increasing concentrations of Compound 44 in combination with each CHOP component were administered to the cells. After four days, cell viability was determined using Millipore Guava ViaCount Reagent and flow cytometry analysis. The percentage of viable cells in each sample was normalized to the percentage of viable cells in the DMSO-treated sample within each Compound 44 concentration group.

[0359] Cells treated with compound 44 and CHOP components alone showed decreased cell viability. Cells treated with compound 44 and mafosfamide (a cyclophosphamide metabolite) (Figure 2A) and doxorubicin (Figure 2B) did not show decreased cell viability at increasing concentrations of mafosfamide or doxorubicin. Combination treatment with compound 44 and vincristine (Figure 2C) showed decreased cell viability at the highest concentrations of compound 44. Importantly, combination treatment with compound 44 and prednisolone (a prednisone metabolite) (Figure 2D) showed a synergistic decrease in cell viability at the two highest doses of compound 44 and at all doses of prednisolone.

[0360] [Example 3] The synergistic effect of the combined treatment of compound 44 and prednisolone depends on the dosage regimen To investigate the role of timing of administration of compound 44 and prednisolone on cell viability, WSU-DLCL2 cells were treated with various dosing schedules as described in Figure 1. Cell viability was determined by staining with Millipore Guava ViaCount Reagent and then analyzed by flow cytometry.

[0361] Administration of compound 44 prior to co-administration of compound 44 and prednisolone resulted in a decrease in cell viability (Figure 3A). Cells were treated as described in Figure 1A. The effect of treatment with compound 44 alone was shown by normalizing cell viability to the DMSO / DMSO sample. Increasing concentrations of compound 44 resulted in a decrease in cell proliferation. Importantly, increasing concentrations of prednisolone in combination with compound 44 resulted in a further decrease in cell proliferation compared to treating cells with compound 44 or prednisolone independently as single agents. Thus, combined treatment with compound 44 and prednisolone produces a synergistic effect that reduces cancer cell viability when compound 44 is administered first.

[0362] Administration of compound 44 before administration of prednisolone resulted in a decrease in cell viability (Figure 3B). Cells were treated as described in Figure 1B. Cell viability was normalized to DMSO-treated samples for each compound 44 concentration group. Treatment with increasing concentrations of prednisolone as a single agent before or after administration of compound 44 also resulted in a further decrease in cell viability when compared to cells treated with prednisolone, thereby demonstrating a synergistic effect of the combined treatment of compound 44 and prednisolone.

[0363] Administration of prednisone prior to co-administration of Compound 44 and prednisolone did not reduce cell viability (Figure 3C). Cells were treated as described in Figure 1C. Cell viability was normalized to DMSO-treated samples for each Compound 44 concentration group. These results indicated that administration of prednisolone prior to treatment with a composition containing Compound 44 and prednisolone did not cause a synergistic effect on cell viability.

[0364] These data clearly demonstrate that the combination of Compound 44 and prednisolone reduces cancer cell viability or induces cancer cell death. In particular, the combination treatment in which cells are treated with Compound 44 before treatment with prednisolone or a combination of prednisolone and Compound 44 results in a synergistic effect on cell viability. The reduction in cell viability is greater than that induced by treatment with either prednisolone or Compound 44 as a single agent.

[0365] [Example 4] The synergistic effect of compound 44 with prednisolone depends on EZH2 mutations Different human lymphoma cancer cell lines carrying different EZH2 mutations were analyzed for their responsiveness to Compound 44 and prednisolone. Cells were treated first with Compound 44, followed 4 days later by a combination of Compound 44 and prednisolone, as shown in Figure 1A. Cell viability was assessed using Millipore Guava Cell viability was determined after 4 days using ViaCount reagent and flow cytometry analysis. The percentage of cell viability was normalized to that of DMSO-treated samples.

[0366] The OCI-LY19 cell line, a lymphoma cell line expressing wild-type (WT) EZH2, is resistant to treatment with an EZH2 inhibitor. Thus, treatment with increasing concentrations of compound 44 does not affect cell viability. Increasing concentrations of prednisolone have no additive or synergistic effect on cell viability (Figure 4A).

[0367] WSU-DLCL2 cells carrying the Y641F mutation are sensitive to treatment with EZH2 inhibitors, as evidenced by decreased cell viability at increasing concentrations of compound 44 when administered as a single agent. Furthermore, when treated in combination with prednisolone, the cells exhibit decreased cell viability (Figure 4B).

[0368] RL cells carry the Y641N mutation and are resistant to EZH2 inhibitor treatment. Administration of increasing concentrations of compound 44 alone did not result in a decrease in cell viability. However, coadministration of compound 44 with prednisolone resulted in a synergistic effect, with a greater decrease in cell viability than observed when compound 44 and prednisolone were administered as single agents (Figure 4C).

[0369] Taken together, these results suggest that combined treatment with compound 44 and prednisolone may have a synergistic effect in reducing cancer cell viability and increasing cancer cell death in cells expressing EZH2 mutants. Furthermore, cells that are resistant to either compound 44 or prednisolone when administered as single agents become sensitive to the combined treatment, resulting in reduced cell viability.

[0370] [Example 5] Pharmacokinetic analysis of in vivo co-administration of compound 44 with CHOP components Pharmacokinetic analysis of compound 44 in combination with each of the CHOP components (cyclophosphamide, vincristine, doxorubicin, and prednisolone) was performed to determine the absorption or distribution of compound 44 in vivo. Male BALB / c mice, 12 weeks old and weighing 20-40 g, were obtained from In Vivo, Bengaluru, India. Animals were administered one of the CHOP components as a single agent or in combination with compound 44. Cyclophosphamide was administered by intraperitoneal injection at 30 mg / kg. Vincristine was administered by intravenous injection at 0.375 mg / kg. Doxorubicin was administered by intravenous injection at 2.475 mg / kg. Prednisolone was administered by intravenous injection at 0.15 mg / kg. Compound 44 was administered orally at 225 mg / kg. Plasma samples were obtained at various time points over the course of 24 hours post-dose.

[0371] The extraction procedure for plasma test samples or spiked plasma calibration standards was identical: 25 μL of either the test sample or spiked calibration standard was added to individual pre-labeled microcentrifuge tubes. Then, 100 μL of IS (Glipizide, 500 ng / mL) prepared in ACN was added to the microcentrifuge tubes except for the blank sample, and acetonitrile was added and vortexed for 5 minutes. The samples were centrifuged at 15,000 rpm (20,600 g) for 10 minutes at 4°C. After centrifugation, 100 μL of supernatant was sampled from each centrifuge tube and transferred into an insert vial. These vials were loaded into the autosampler for LC / MS / MS analysis. Calibration standards were prepared by mixing 10 μL of analyte (compound 44, cyclophosphamide, doxorubicin, vincristine, and prednisolone) with 190 μL of blank mouse plasma.

[0372] Pharmacokinetic parameters were evaluated using the Noncompartmental Analysis module in WinNonlin® (version 5.2). The area under the concentration-time curve (AUC) was calculated by the linear trapezoidal rule. The AUC (AUC combo ) and the AUC of each CHOP component only (AUC single ) and the ratio (AUC combo / AUC single ) showed similar bioavailability when the CHOP component was administered alone or together with compound 44.

[0373] [Example 6] Analysis of the combination therapy of compound 44 and CHOP in a mouse xenograft model mouse Female Fox Chase SCID® mice (CB17 / Icr-Prkdc scid / IcrIcoCrl, Charles River Laboratories) or athymic nude mice (Crl:NU(Ncr)-Foxn1 nu Mice (Charles River Laboratories) were 8 weeks old on Day 1 of the study and had a body weight (BW) range of 16.0-21.1 g. Animals were provided ad libitum with water (reverse osmosis 1 ppm Cl) and an NIH31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. Mice were housed on illuminated Enrich-o'cobs™ bedding in static microisolators at 20-22°C (68-72°F) and 40-60% humidity with a 12-hour photoperiod. All procedures were performed in accordance with the Guide for Veterinary Care on restraint, animal husbandry, surgical procedures, food and drink regulations, and other regulations. Follow recommendations in the Care and Use of Laboratory Animals.

[0374] Tumor cell culture Human lymphoma cell lines were obtained from different sources (ATCC, DSMZ) and maintained at Piedmont as suspension cultures in RPMI 1640 medium containing 100 units / mL penicillin G sodium salt, 100 μg / mL streptomycin, and 25 μg / mL gentamicin. The medium was supplemented with 10% fetal bovine serum and 2 mM glutamine. Cells were grown in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.

[0375] In vivo tumor implantation Human lymphoma cell lines were harvested during mid-logarithmic growth and resuspended in PBS containing 50% Matrigel™ (BD Biosciences). 1x10 subcutaneously in the right flank 7 100 cells (0.2 mL of cell suspension) were received. The average volume was adjusted to the desired 80-120 mm 3 When tumors reached a size of 1 mm, they were measured in two dimensions with calipers to monitor growth. 3 The tumor size at

[0376]

number

[0377] Test substance The compound of formula (IIa) was stored at room temperature and protected from light. On each treatment day, fresh compound formulations were prepared by suspending the powder in 0.5% sodium carboxymethylcellulose (NaCMC) and 0.1% Tween® 80 in deionized water. A control group was treated with Compound 44 vehicle, 0.5% NaCMC, and 0.1% Tween® 80 in deionized water on the same schedule. The formulations were stored at 4°C away from light before administration.

[0378] Several chemotherapy agents were used in parallel with the Epizyme compound. Cyclophosphamide (Baxter, Lot No. 016591) was reconstituted to 20 mg / mL with sterile saline and stored at 4°C. Fresh dosing solutions were prepared for each dose by dilution with saline. Doxorubicin (Doxorubicin Meiji®, Meiji Pharmaceutical Co. Ltd., 1 mg / mL) was stored at 4°C and diluted with saline on each treatment day. Vincristine (Hospira, Inc., 1 mg / mL) was diluted with saline on each treatment day. Prednisone (Boehringer Ingelheim GmbH, 1 mg / mL) was diluted with PBS at the beginning of each 5-day dosing cycle.

[0379] Treatment Plan Mice were treated with compound doses ranging from 75-600 mg / kg and various daily doses by oral gavage or injection via intravenous, intraperitoneal, or subcutaneous routes on a TID (three times daily every 8 h), BID (twice daily every 12 h), or QD (once daily) schedule. Each dose was delivered in a 0.2 mL volume per 20 g mouse (10 mL / kg) and adjusted for the last recorded body weight of each individual animal. The maximum treatment length was 28 days.

[0380] Median tumor volume (MTV) and tumor growth inhibition (TGI) analysis Treatment efficacy was determined on the last treatment day. MTV(n), the median tumor volume relative to the number of animals (n), evaluable on the last day, was determined for each group. Percent tumor growth inhibition (%TGI) can be defined in several ways. First, the difference between the MTV(n) of a designated control group and the MTV(n) of a drug-treated group is expressed as a percentage of the MTV(n) of the control group:

[0381]

number

[0382] Another way to calculate %TGI is to take the change in tumor size from day 1 to day n, where n is the day of the last treatment.

[0383]

number

[0384] Tumor growth delay analysis Alternatively, mice were kept alive after the last treatment day for tumor growth delay analysis. Tumors were measured twice weekly with calipers and were then measured until the neoplasm reached 2000 mm 3 Each test animal was euthanized when it reached an endpoint volume of 0.01 or on the last pre-specified day of the study, whichever came first. The time to endpoint (TTE) for each mouse was calculated using the following formula:

[0385]

number

[0386] Treatment outcome was tumor growth delay (TGD), defined as the increase in median TTE in the treatment group compared to the control group, expressed in days:

[0387]

number

[0388]

number

[0389] toxicity Animals were weighed daily on days 1-5, then twice weekly until study completion. Mice were frequently examined for overt signs of any adverse treatment-related side effects, which were documented. Acceptable toxicity at the maximum tolerated dose (MTD) was defined as a group mean BW loss of less than 20% during the study and a mortality rate of 10% or less due to TR deaths. Deaths were classified as TR if they were due to treatment side effects as evidenced by clinical signs and / or necropsy, or due to unknown causes during the dosing period. Deaths were classified as NTR if there was evidence that the death was not related to treatment side effects. NTR deaths during the dosing interval are typically classified as NTRa (due to accident or human error) or NTRm (due to tumor dissemination confirmed by necropsy due to invasion and / or metastasis). Orally treated animals that die of unknown causes during the dosing period may be classified as NTRu if group performance does not support a TR classification and necropsy to rule out dosing error is not feasible.

[0390] sampling Mice were sampled in a pre-specified manner on several days during the study. Sampling included non-terminal bleeding (0.25 mL) from the mandibular vein without anesthesia and full-volume blood collection by terminal cardiac puncture under CO2 anesthesia. Blood samples were processed to plasma using K2-EDTA as an anticoagulant. Plasma samples were frozen at -80°C and stored prior to bioanalysis for compound levels.

[0391] Tumors were harvested from selected mice under RNAse-free conditions and bisected. 2-mm-thick sections from half of each tumor were fixed in formalin for 24 hours and transferred to 70% ethanol. The fixed tumor tissue was embedded in paraffin. The remaining tumor tissue from each animal was snap-frozen in liquid N2 and pulverized with a mortar and pestle.

[0392] Selected mice were sampled for surrogate tissues, including spleen, skin, bone marrow, and whiskers, each of which was isolated, fixed, and / or snap-frozen.

[0393] Statistical and Graphical Analysis All statistics and graph analyses were performed using Prism 3.03 (GraphPad) for Windows. Several analytical methods were applied. The central D29 tumor volume was compared using the Kruskal-Wallis test and the post hoc Dunn's multiple comparison test. These tests were performed three times.

[0394] Two-sided statistical analysis was performed at P = 0.05. Prism reports results as non-significant (ns) for P > 0.05, significant (indicated by " * ") for 0.01 < P < 0.05, very significant ( " ** ") for 0.001 < P < 0.01, and extremely significant ( " *** ") for P < 0.001.

[0395] To test the statistical significance between the control and treatment groups over the entire treatment time course, either a repeated measures ANOVA test followed by a Dunnett's multiple comparison post-test or a two-factor ANOVA test was used. Either was used.

[0396] For graph presentation, "box-and-whisker" plots were made to show the distribution of the individual tumor volumes for each group. The box represents the 25th to 75th percentile of the observations, the horizontal line corresponds to the median, and the "whiskers" show the maximum and minimum values. The central or mean (±SEM) tumor volume was graphed as a function of time on semi-logarithmic or linear plots. The change in group mean BW during the study was plotted as percent change from D1, ±SEM.

[0397] Scatter plots were made by group to show the TTE values. The TTE plots included NTR death, which was excluded from all other graph analyses. When an animal terminated the study due to tumor size, the final tumor volume recorded for the animal was included with the data used to calculate the central volume at subsequent time points. The percentage of animals remaining in each group during the study and time were presented in Kaplan-Meier survival plots.

[0398] Histone extraction For histone isolation, 60-90 mg of tumor tissue was homogenized in 1.5 ml of nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 1% Triton X-100, 8.6% sucrose + Roche protease inhibitor tablet 1836145) and incubated on ice for 5 minutes. Nuclei were collected by centrifugation at 600 g for 5 minutes at 4°C and washed once in PBS. The supernatant was removed, and histones were extracted with 0.4 N cold sulfuric acid for 1 hour, vortexing every 15 minutes. The extract was clarified by centrifugation at 10,000 g for 10 minutes at 4°C and transferred to a fresh microcentrifuge tube containing 10x the volume of ice-cold acetone. Histones were precipitated for 2 hours to overnight at -20°C, pelleted by centrifugation at 10,000 g for 10 minutes, and resuspended in water.

[0399] ELISA Histones were extracted from tumor samples as described above. Histones were prepared at equal concentrations in coating buffer (PBS + 0.05% BSA) to obtain 0.5 ng / μl samples. 100 μl of sample or standard was added in duplicate to two 96-well ELISA plates (Thermo Labsystems, Immulon 4HBX#3885). The plates were sealed and incubated overnight at 4°C. The following day, the plates were washed three times with 300 μl / well of PBST (PBS + 0.05% Tween 20; 10X PBST, KPL#51-14-02) on a BioTek plate washer. The plates were blocked with 300 μl / well of diluent (PBS + 2% BSA + 0.05% Tween 20), incubated for 2 hours at RT, and washed three times with PBST. All antibodies were diluted in diluent. 100 μl / well of anti-H3K27me3 (CST #9733, 50% glycerol stock 1:1,000) or anti-total H3 (Abcam ab1791, 50% glycerol 1:10,000) was added to each plate. Plates were incubated at RT for 90 minutes and washed three times with PBST. 100 μl / well of anti-Rb-IgG-HRP (Cell Signaling Technology, 7074) was added at 1:2,000 to the H3K27Me3 plate and at 1:6,000 to the H3 plate and incubated at RT for 90 minutes. Plates were washed four times with PBST. For detection, 100 μl / well of TMB substrate (BioFx Laboratories, #TMBS) was added, and plates were incubated in the dark at RT for 5 minutes. The reaction was stopped with 100 μl / well of 1N H2SO4. Absorbance at 450 nm was read on a SpectaMax M5 microplate reader.

[0400] Efficacy study in the SUDHL6 xenograft model The efficacy of treatment with Compound 44 and Compound 44 in combination with CHOP for tumor growth inhibition in vivo was determined in the SUDHL6 xenograft model. Comparison of tumor growth and survival rates demonstrated that administration of Compound 44 with CHOP inhibited or delayed tumor growth and increased survival time in tumor-bearing mice. 7 SUDHL6 human lymphoma cells were subcutaneously injected. Compound 44 was administered at the indicated concentrations (75 mg / kg, 150 mg / kg, or 225 mg / kg) once daily (QD), twice daily (BID), or three times daily (TID). Mice received CHOP on days 1 and 8. Tumor volumes were monitored twice weekly until the 60-day endpoint or when tumor volumes reached 200 mm. 3 The measurement was continued until either of the following was reached first.

[0401] Combination treatment with compound 44 and CHOP demonstrated tumor growth inhibition over the treatment course (28 days) compared with mice treated with CHOP or compound 44 alone (Figure 6A). Significantly, mice receiving combination treatment with compound 44 and CHOP showed durable regression of tumor size; 7 of 12 mice in the 225 mg / kg BID and CHOP combination group showed a complete response at day 60 (Figure 6A). Kaplan-Meier curves were determined to demonstrate mouse survival in the study. 57% of mice receiving combination treatment with compound 44 and CHOP survived for 60 days after injection (Figure 6B). No single-agent efficacy with compound 44 was observed over the 28-day treatment period, although high intragroup variability may mask the treatment effect of compound 44 as a single agent.

[0402] Efficacy study in WSU-DLCL2 xenograft model The efficacy of treatment with Compound 44 and Compound 44 in combination with CHOP for tumor growth inhibition in vivo was determined in a WSU-DLCL2 xenograft model. Comparison of tumor growth demonstrated that administration of Compound 44 and CHOP inhibited or delayed tumor growth in tumor-bearing mice. SCID mice were treated with 1x10 7WSU-DLCL2 human lymphoma cells were subcutaneously injected. Compound 44 was administered at the indicated concentrations (150 mg / kg, 225 mg / kg, 300 mg / kg, or 600 mg / kg) once daily (QD), twice daily (BID), or three times daily (TID). Mice received CHOP on days 1 and 22 (CHOP on days 1 and 8 was not tolerated in SCID mice). Tumor volume was measured twice weekly until the 28-day endpoint.

[0403] Compound 44 alone and in combination with CHOP treatment resulted in tumor growth inhibition (Figure 7A). Administration of compound 44 as a single agent at 150 mg / kg three times daily (TID) and 225 mg / kg twice daily (BID) resulted in significantly smaller tumors (by volume) compared to control vehicle-treated mice (p<0.05). Furthermore, combination treatment with compound 44 and CHOP showed statistically significant tumor growth inhibition compared to control vehicle-treated mice (p<0.001). Statistical analysis was performed using repeated measures ANOVA followed by Dunnett's post-hoc test. Tumor growth inhibition was also calculated, showing that treatment of tumors with compound 44 resulted in higher tumor growth inhibition at all doses (Table 2). Importantly, combination treatment with compound 44 and CHOP resulted in the highest tumor growth inhibition.

[0404] [Table 10]

[0405] To examine the absorption and distribution of the administered drug, pharmacokinetic analysis was performed to determine the concentration (ng / mL) of compound 44 in the plasma of tumor-bearing mice (Figure 7B). Plasma samples were obtained 5 minutes before the last dose ("trough") and 3 hours after the last dose ("post-dose") on day 28. Plasma samples were analyzed by LC-MS / MS to determine the concentration (ng / mL) of compound 44. Compound 44 levels were not significantly different between mice receiving 225 mg / kg of compound 44 twice daily (BID) as a single agent and mice receiving 225 mg / kg of compound 44 BID with CHOP at the trough time. However, 3 hours after administration of the last dose, compound 44 levels were significantly increased in mice receiving 225 mg / kg of compound 44 BID with CHOP compared to compound 44 as a single agent.

[0406] Pharmacokinetic analysis was also performed to determine the concentration (ng / g) of compound 44 in tumor tissue harvested from mice 3 hours after the last dose of treatment on day 28 (Figure 7C).

[0407] The efficacy of target inhibition in vivo was determined by analyzing histone methylation status in tumors 28 days after treatment. Western blot analysis (Figure 8A) showed that H3K27 methylation was inhibited by compound 44 at all doses and dosing schedules in WSU-DLCL2 tumors. Histones were extracted as described above. Protein concentrations of acid-extracted histones were determined by BCA assay (Pierce). 400-800 ng of each lysate was fractionated on a 10-20% Tris-Glycine gel (Biorad), transferred using iBlot (nitrocellulose transfer stack, program 3, 7 minutes), and probed with the following antibodies in Odyssey blocking buffer: rabbit anti-H3K27me3 (CST9733; 1:20,000 dilution) and mouse anti-total H3 (CST3638; 1:20,000 dilution). After primary Ab incubation, the membrane was probed with IRDye800CW donkey anti-mouse IgG (LiCOR #926-32212) and Alexa Fluor 680 goat anti-rabbit IgG (Invitrogen #A-21076) secondary Abs and imaged using the LiCOR Odyssey system. Signal from total histone H3 protein was used as a control.

[0408] Analysis of histone methylation in WSU-DLCL2 tumors by ELISA confirmed that histone methylation was inhibited by compound 44 at all doses and dosing schedules (Figure 8B). Tumors from mice receiving vehicle or CHOP did not show inhibition of methylation at H3K27. Histones were extracted from tumors 28 days after treatment and analyzed by ELISA as described above.

[0409] Efficacy study in the SUDHL10 xenograft model Compound 44 and Compound 44 in combination with COP (cyclophosphamide, Oncovin [vincristine], and prednisone) for tumor growth inhibition in vivo The efficacy of the treatment was determined in a SUDHL10 xenograft model. Comparison of tumor growth established that administration of Compound 44 and COP inhibited or delayed tumor growth in tumor-bearing mice. SCID mice were administered 1x10 7 SUDHL10 human lymphoma cells were subcutaneously injected. Compound 44 was administered twice daily (BID) or three times daily (TID) at the indicated concentrations (125 mg / kg, 250 mg / kg, or 500 mg / kg). Mice received COP on days 1 and 22 (CHOP on days 1 and 8 was not tolerated in SCID mice). Tumor volumes were measured twice weekly until the 28-day endpoint. Half of the cohort was euthanized after 28 days of treatment, while the other half was analyzed for tumor growth delay until the 60-day endpoint.

[0410] Figure 9A shows that administration of compound 44 alone demonstrated statistically significant tumor growth inhibition as a single agent at 250 mg / kg and 500 mg / kg delivered twice daily (BID) compared to control vehicle-treated mice (p<0.001). Furthermore, combination treatment of compound 44 with COP at 250 mg / kg twice daily (BID) demonstrated statistically significant inhibition of tumor growth compared to control vehicle-treated mice (p<0.001). Statistical analysis was performed using repeated measures ANOVA followed by Dunnett's post-hoc test. One mouse in the 500 mg / kg BID group was euthanized on day 15 due to poor physical condition. Mice receiving combination treatment with compound 44 and COP showed an 8% weight loss on day 25, and dosing was discontinued but resumed on day 27. On day 29, 1 / 16 mice and 2 / 15 mice were tumor-free in the 250 mg / kg and 500 mg / kg groups, respectively. Surprisingly, 10 / 14 mice receiving the combination treatment of compound 44 and COP were tumor-free on day 29.

[0411] Tumor growth inhibition was also calculated and showed that treatment of SUDHL10 xenograft tumors with compound 44, with or without COP, resulted in effective tumor growth inhibition at all doses (Table 3).

[0412] [Table 11]

[0413] Mice were euthanized on day 28, and tumors were weighed (FIG. 9B). Tumors from mice receiving single-agent administration of 125 mg / kg of compound 44 or COP were significantly smaller than control mice (vehicle). Tumors from mice receiving combination treatment with compound 44 and COP were significantly smaller than tumors from mice receiving compound 44 as a single agent at both 250 mg / kg and 500 mg / kg doses.

[0414] Half of the cohort was maintained until day 60 to determine efficacy of treatment by determining tumor growth delay (Figure 9C). Tumor growth in mice treated with compound 44 as a single agent at doses of 250 mg / kg and 500 mg / kg showed small tumors at day 28, while control and COP-treated mice showed large tumors that continued to grow after day 28. Surprisingly, mice receiving combination therapy showed tumor growth rates higher than all other treatment groups. Not only did the patients have smaller tumors than the control group, but they also demonstrated significant and durable tumor growth delay.

[0415] Figure 9D shows a Kaplan-Meier curve depicting the survival rate of mice treated with compound 44 alone or in combination with COP. Administration of compound 44 as a single agent at 125 mg / kg, 250 mg / kg, and 500 mg / kg increased the survival rate of mice compared with control mice or mice treated with COP alone. Significantly, administration of compound 44 in combination with COP improved the survival rate of mice compared with single agent administration.

[0416] Pharmacokinetic and pharmacodynamic studies were performed on SUDHL10 xenograft models. Figure 10A shows the pharmacokinetic analysis of compound 44 concentration (ng / mL) at plasma levels. Plasma samples were obtained from tumor-bearing mice on day 28, 5 minutes before the last dose ("trough") or 3 hours after the last dose ("post-dose"). Compound 44 levels were determined by LC-MS. Pharmacodynamic analysis was performed by ELISA to determine the proportion of trimethylated H3K27 in tumor samples. Pharmacodynamic analysis was also performed in other tissues of tumor-bearing mice, particularly the spleen and bone marrow, to demonstrate the effectiveness of compound 44 in inhibiting histone methylation in normal tissues of surrogate mice (Figures 10B and 10C).

[0417] [Example 7] Synergistic effects of combination therapy of compound 44 with anticancer drugs method Human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC575), SU-DHL-10 (DSMZ; ACC576), and Toledo (ATCC; CRL-2631) were obtained from the indicated sources and maintained in RPMI-1640 medium supplemented with 10%-20% heat-inactivated fetal bovine serum and 2 mM glutamine. Cells were cultured in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air. WSU-DLCL2 and SU-DHL-10 contain the Y641 EZH2 mutation, while the Toledo cell line contains WT EZH2.

[0418] The in vitro antiproliferative effects of compound 44 in combination with the following drugs were investigated: AraC, cisplatin, decitabine, dexamethasone, everolimus, prednisolone, and doxorubicin. WSU-DLCL2, SU-DHL-10, or Toledo human lymphoma cells were treated with increasing concentrations of compound 44 in flasks. Four days after treatment, WSU-DLCL2 or SU-DHL-10 cells were split to their initial seeding density, and each concentration of compound 44 was seeded in one row of a 96-well tissue culture plate. Six days later, Toledo cells were split to their initial seeding density, and each concentration of compound 44 was seeded in one row of a 96-well tissue culture plate. Increasing doses of drug were then added to the plate, with one dose per column forming a matrix of compound 44 and drug doses. After an additional 3 days of incubation, cell viability of WSU-DLCL2 or SU-DHL-10 was measured using Promega Cell Titer-Glo reagent followed by luminescence detection. After 5 days, viability of Toledo cells was measured using Promega Cell Titer-Glo reagent followed by luminescence detection.

[0419] Data analysis Synergistic effects were determined using the software package Calcusyn by Biosoft, which is based on the Chou-Talalay method for drug combinations using the median effect formula (Chou 2006). First, raw luminescence values ​​were converted to percent inhibition or fraction affected (Fa), calculated using DMSO-treated cells for maximum and minimum inhibition controls placed on each plate. The percent inhibition values ​​for each fixed ratio compound combination were entered into Calcusyn to determine the combination index value. Combinations less than 1 The index values ​​indicated a synergistic effect.

[0420] For test compounds that did not inhibit cell viability by 50%, synergy could not be determined using Calucusyn. Instead, data were reported as the IC50 fold shift from the IC50 of Compound 44. Alternatively, if Compound 44 had no effect on Toledo cells, the IC50 fold shift was reported for the drug instead of Compound 44.

[0421] result Treatment of WSU-DLCL2 and SU-DHL-10 cells with compound 44 and either AraC, cisplatin, doxorubicin, decitabine, or everolimus showed synergistic reduction in cell viability. The combination index values ​​for the combination of compound 44 and decitabine in WSU-DLCL2 and SU-DHL-10 cells were less than 0.1, indicating very strong synergy according to Chou-Talalay characterization (Chou 2006). The combination index values ​​for the combination of compound 44 and everolimus were less than 0.1 in WSU-DLCL2 cells and 0.1–0.3 in SU-DHL-10 cells, indicating very strong synergy and strong synergy, respectively. Combinations of compound 44 with either AraC, cisplatin, or doxorubicin in WSU-DLCL2 and SU-DHL-10 cells had combination index values ​​of 0.3–0.7, indicating synergism (Chou 2006). Combination of compound 44 with prednisolone enhanced the efficacy of compound 44 by 7-fold in WSU-DLCL2 cells and 3-fold in SU-DHL-10 cells at the highest prednisolone dose. Furthermore, combination of compound 44 with dexamethasone enhanced the efficacy of compound 44 by 17-fold in WSU-DLCL2 cells and 3-fold in SU-DHL-10 cells at the highest dexamethasone dose.

[0422] The Toledo cell line was not sensitive to compound 44, and no combination benefit was observed when compound 44 was combined with either AraC, cisplatin, doxorubicin, decitabine, everolimus, prednisolone, or dexamethasone. Thus, the combination benefit observed in these experiments appears to be dependent on the EZH2 mutation.

[0423] [Table 12]

[0424] Incorporation by Reference All publications and patent documents cited herein are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference. Citation of any publication or patent document is not intended as an admission that it is pertinent prior art, nor does it constitute any admission as to its contents or date. While the invention has been described herein by specification, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the following examples are intended to be illustrative and not limiting of the scope of the claims that follow.

[0425] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are, therefore, to be considered in all respects as illustrative rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0426] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) Formula (IIa): [ka] (In the formula, R a and R b are each independently H, or one or more -Q3-T3, where Q3 is a bond or an unsubstituted or substituted C1-C3 alkyl linker and T3 is H, halo, 4- to 7-membered heterocycloalkyl, C1-C3 alkyl, OR d , COOR d , -S(O)2R d , or -NR d R e and R d and R e are each independently H or C1-C6 alkyl, or -Q3T3 is oxo; R7 is isopropyl, or one or more -Q5-T5 (wherein Q5 is a bond, C(O), C(O)NR k , N.R. k C(O), S(O)2, or a C1-C3 alkyl linker; R k is H or C1-C6 alkyl, and T5 is H, halo, C1-C6 alkyl, hydroxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O) q R q (wherein q is 0, 1, or 2; R q is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, except when T5 is H, halo, hydroxyl, or cyano, T5 is halo, C1-C6 alkyl, hydroxyl, cyano, C1-C6 alkoxyl, amino, mono-C1-C6 alkylamino, di-C1-C6 alkylamino, C3-C8 cycloalkyl, C6-C 10optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; or -Q5-T5 is oxo; R8 is H, methyl, or ethyl. or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. (Item 2) A composition comprising any one of the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. (Section 3) A composition comprising compound 44, or a pharmaceutically acceptable salt thereof, and one or more therapeutic agents. (Section 4) 4. The composition according to any one of items 1 to 3, wherein the other therapeutic agent is an anticancer agent or a glucocorticoid. (Section 5) 4. The composition of any one of paragraphs 1 to 3, wherein the other therapeutic agent is selected from prednisone, prednisolone, cyclophosphamide, vincristine, doxorubicin, mafosfamide, cisplatin, AraC, everolimus, decitabine, dexamethasone, and analogs, derivatives, or combinations thereof. (Section 6) 4. The composition according to any one of paragraphs 1 to 3, wherein the other therapeutic agent is prednisone, or an analogue or derivative thereof. (Section 7) A pharmaceutical composition comprising a therapeutically effective amount of any of the above items 1 to 6 and a pharmaceutically acceptable carrier. (Section 8) A method for treating or preventing a disease, comprising administering a therapeutically effective amount of the composition according to item 1 to a subject in need thereof. (Section 9) 9. The method of paragraph 8 above, wherein the disease can be affected by modulating the methylation status of histones or other proteins. (Section 10) Item 9. The method according to item 8 above, wherein the disease is cancer or a precancerous condition. (Section 11) 10. The method of paragraph 9 above, wherein the methylation status is mediated at least in part by the activity of EZH2. (Section 12) A method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective dose of a compound of formula (IIa) and one or more other therapeutic agents, wherein formula (IIa) and the other therapeutic agents are administered simultaneously or sequentially. (Section 13) Item 13. The method according to item 12, wherein the compound of formula (IIa) is administered before the administration of the other therapeutic agent. (Section 14) A method for treating or preventing cancer, comprising administering a therapeutically effective dose of a compound of formula (IIa) or a pharmaceutically acceptable salt thereof to a subject in need thereof prior to administering a therapeutically effective dose of the composition described in item 1 above. (Section 15) The method according to item 8 or 12, wherein the composition according to item 1 is administered to a subject in need thereof at a dose of 0.01 mg / kg / day to about 1000 mg / kg / day. (Section 16) 15. The method according to item 12 or 14, wherein the compound of formula (IIa) is administered at a dose of 0.01 mg / kg / day to about 1000 mg / kg / day. (Section 17) 15. The method according to paragraph 12 or 14 above, wherein each of the one or more other therapeutic agents is administered at a dose of 0.01 mg / kg / day to about 1000 mg / kg / day. (Section 18) 15. The method of any one of paragraphs 8, 12 or 14, wherein the subject expresses an EZH2 mutant. (Section 19) The method according to item 18, wherein the EZH2 mutant has one or more mutations, and the mutations are substitutions, point mutations, nonsense mutations, missense mutations, deletions, or insertions. Law. (Section 20) 19. The method according to paragraph 18, wherein the EZH2 mutant has one or more mutations in its substrate pocket domain as defined in SEQ ID NO:6. (Section 21) 19. The method according to item 18, wherein the EZH2 mutant is a Y641 mutant. (Section 22) 22. The method according to claim 21, wherein the Y641 mutant is selected from Y641F, Y641H, Y641N and Y641S. (Section 23) 19. The method of claim 18, wherein the EZH2 mutant has a mutation at amino acid position 677, 687, 674, 685, or 641 of SEQ ID NO:1. (Section 24) The EZH2 mutants include a substitution of the wild-type residue alanine (A) at amino acid position 677 of SEQ ID NO:1 with glycine (G) (A677G); a substitution of the wild-type residue alanine (A) at amino acid position 687 of SEQ ID NO:1 with valine (V) (A687V); a substitution of the wild-type residue valine (V) at amino acid position 674 of SEQ ID NO:1 with methionine (M) (V674M); a substitution of the wild-type residue arginine (R) at amino acid position 685 of SEQ ID NO:1 with histidine (H) (R685H); a substitution of the wild-type residue arginine (R) at amino acid position 685 of SEQ ID NO:1 with cysteine ​​(C) (R68 5C); substitution of wild-type residue asparagine (N) at amino acid position 322 of SEQ ID NO: 3 with serine (S) (N322S), substitution of wild-type residue arginine (R) at amino acid position 288 of SEQ ID NO: 3 with glutamine (Q) (R288Q), substitution of wild-type residue threonine (T) at amino acid position 573 of SEQ ID NO: 3 with isoleucine (I) (T573I), substitution of wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with glutamic acid (E) (D664E), substitution of wild-type residue arginine (R) at amino acid position 458 of SEQ ID NO: 5 with glutamine (Q) (R4 58Q), substitution of wild-type residue glutamic acid (E) at amino acid position 249 of SEQ ID NO: 3 with lysine (K) (E249K), substitution of wild-type residue arginine (R) at amino acid position 684 of SEQ ID NO: 3 with cysteine ​​(C) (R684C), substitution of wild-type residue arginine (R) at amino acid position 628 of SEQ ID NO: 21 with histidine (H) (R628H), substitution of wild-type residue glutamine (Q) at amino acid position 501 of SEQ ID NO: 5 with histidine (H) (Q501H), substitution of wild-type residue aspartic acid (D) at amino acid position 192 of SEQ ID NO: 3 with asparagine (N). (D192N), a substitution of the wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with valine (V) (D664V), a substitution of the wild-type residue valine (V) at amino acid position 704 of SEQ ID NO: 3 with leucine (L) (V704L), a substitution of the wild-type residue proline (P) at amino acid position 132 of SEQ ID NO: 3 with serine (S) (P132S), a substitution of the wild-type residue glutamic acid (E) at amino acid position 669 of SEQ ID NO: 21 with lysine (K) (E669K), a substitution of the wild-type residue alanine (A) at amino acid position 255 of SEQ ID NO: 3 with threonine (T) (A255T),Substitution of wild-type residue glutamic acid (E) at amino acid position 726 of SEQ ID NO:3 with valine (V) (E726V), substitution of wild-type residue cysteine ​​(C) at amino acid position 571 of SEQ ID NO:3 with tyrosine (Y) (C571Y), substitution of wild-type residue phenylalanine (F) at amino acid position 145 of SEQ ID NO:3 with cysteine ​​(C) (F145C), substitution of wild-type residue asparagine (N) at amino acid position 693 of SEQ ID NO:3 with threonine (T) (N693T), substitution of wild-type residue phenylalanine (F) at amino acid position 145 of SEQ ID NO:3 with serine ( a substitution of wild-type residue glycine (G) at amino acid position 135 of SEQ ID NO:3 with arginine (R) (G135R); a substitution of wild-type residue arginine (R) at amino acid position 168 of SEQ ID NO:5 with glutamine (Q) (R168Q); a substitution of wild-type residue glycine (G) at amino acid position 168 of SEQ ID NO:3 with glutamine (Q) (R168Q); a substitution of wild-type residue glycine (G) at amino acid position 168 of SEQ ID NO:3 with arginine (R) (R168Q); substitution of glycine (G) with arginine (R) (G159R), substitution of wild-type residue arginine (R) at amino acid position 310 of SEQ ID NO: 5 with cysteine ​​(C) (R310C), substitution of wild-type residue arginine (R) at amino acid position 561 of SEQ ID NO: 3 with histidine (H) (R561H), substitution of wild-type residue arginine (R) at amino acid position 634 of SEQ ID NO: 21 with histidine (H) (R634H), substitution of wild-type residue glycine (G) at amino acid position 660 of SEQ ID NO: 3 with arginine (R) (G660R), substitution of wild-type residue glycine (G) at amino acid position 181 of SEQ ID NO: 3 with cysteine ​​(C) (R310C), substitution of wild-type residue tyrosine (Y) with cysteine ​​(C) (Y181C), substitution of wild-type residue histidine (H) at amino acid position 297 of SEQ ID NO: 3 with arginine (R) (H297R), substitution of wild-type residue cysteine ​​(C) at amino acid position 612 of SEQ ID NO: 21 with serine (S) (C612S), substitution of wild-type residue histidine (H) at amino acid position 694 of SEQ ID NO: 3 with tyrosine (Y) (H694Y), substitution of wild-type residue aspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 with alanine (A) (D664A), substitution of wild-type residue aspartic acid (D) at amino acid position 1 of SEQ ID NO: 3 with alanine (A) (D664A), substitution of wild-type residue cysteine ​​(C) at amino acid position 1 of SEQ ID NO: 3 with serine (S) (C612S). substitution of the wild type residue isoleucine (I) at amino acid position 264 of SEQ ID NO:3 with arginine (R) (I264R); substitution of the wild type residue proline (P) at amino acid position 636 of SEQ ID NO:3 with leucine (L) (P636L); substitution of the wild type residue isoleucine (I) at amino acid position 713 of SEQ ID NO:3 with threonine (T) (I713T); substitution of the wild type residue glutamine (Q) at amino acid position 501 of SEQ ID NO:5 with proline (P) (Q501P); a substitution of the wild-type residue lysine (K) at amino acid position 243 with glutamine (Q) (K243Q); a substitution of the wild-type residue glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5 with aspartic acid (D) (E130D); a substitution of the wild-type residue arginine (R) at amino acid position 509 of SEQ ID NO: 3 with glycine (G) (R509G); a substitution of the wild-type residue arginine (R) at amino acid position 566 of SEQ ID NO: 3 with histidine (H) (R566H); a substitution of the wild-type residue aspartic acid (D) at amino acid position 677 of SEQ ID NO: 3 with histidine (H) (D677H);a substitution of the wild type residue lysine (K) at amino acid position 466 of SEQ ID NO: 5 with asparagine (N) (K466N); a substitution of the wild type residue arginine (R) at amino acid position 78 of SEQ ID NO: 3 with histidine (H) (R78H); a substitution of the wild type residue lysine (K) at amino acid position 1 of SEQ ID NO: 6 with methionine (M) (K6M); a substitution of the wild type residue serine (S) at amino acid position 538 of SEQ ID NO: 3 with leucine (L) (S538L); a substitution of the wild type residue leucine (L) at amino acid position 149 of SEQ ID NO: 3 with methionine (M) (S538L). substitution of wild-type residue leucine (L) at amino acid position 252 of SEQ ID NO:3 with valine (V) (L252V); substitution of wild-type residue leucine (L) at amino acid position 674 of SEQ ID NO:3 with valine (V) (L674V); substitution of wild-type residue alanine (A) at amino acid position 656 of SEQ ID NO:3 with valine (V) (A656V); substitution of wild-type residue alanine (A) at amino acid position 731 of SEQ ID NO:3 with aspartic acid (D) (Y731D); a substitution of the wild-type residue alanine (A) at amino acid position 345 with threonine (T) (A345T); a substitution of the wild-type residue alanine (A) at amino acid position 244 of SEQ ID NO: 3 with aspartic acid (D) (Y244D); a substitution of the wild-type residue cysteine ​​(C) at amino acid position 576 of SEQ ID NO: 3 with tryptophan (W) (C576W); a substitution of the wild-type residue asparagine (N) at amino acid position 640 of SEQ ID NO: 3 with lysine (K) (N640K); 19. The method of claim 18, wherein the wild-type asparagine (N) residue at amino acid position 579 of SEQ ID NO: 21 is substituted with lysine (K) (N675K), the wild-type aspartic acid (D) residue at amino acid position 579 of SEQ ID NO: 21 is substituted with tyrosine (Y) (D579Y), the wild-type asparagine (N) residue at amino acid position 693 of SEQ ID NO: 3 is substituted with isoleucine (I) (N693I), and the wild-type asparagine (N) residue at amino acid position 693 of SEQ ID NO: 3 is substituted with lysine (K) (N693K). (Section 25) The EZH2 mutant is selected from the group consisting of amino acid positions 730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105 3, 656, 718, 374, 592, 505, 730, or 363, or a frameshift at the corresponding nucleotide position of the nucleic acid sequence encoding SEQ ID NO: 3, 5, or 21. (Section 26) 27. The method of claim 18, wherein the EZH2 mutant has a deletion of glutamic acid (E) and leucine (L) at amino acid positions 148 and 149 of SEQ ID NO: 3, 5, or 21. 19. The method of claim 18, wherein the EZH2 mutant has a nonsense mutation at amino acid position 733, 25, 317, 62, 553, 328, 58, 207, 123, 63, 137 or 60 of SEQ ID NO: 3, 5 or 21. (Section 28) 15. The method of any one of paragraphs 8, 12, or 14, wherein the subject has demonstrated tolerance to any one component of the composition of paragraph 1 when administered as a single agent. (Section 29) A method for inhibiting cancer cell proliferation, comprising contacting cancer cells with the composition described in item 1 above. (Section 30) A method of inhibiting cancer cell proliferation comprising contacting cancer cells with a compound of formula (IIa) and one or more other therapeutic agents, wherein formula (IIa) and the other therapeutic agents are delivered simultaneously or sequentially. (Section 31) 31. The method according to above item 30, wherein the compound of formula (IIa) is administered before the administration of the other therapeutic agent. (Section 32) A method for inhibiting cancer cell proliferation, comprising administering a therapeutically effective dose of a compound of formula (IIa) or a pharmaceutically acceptable salt thereof prior to administering a therapeutically effective dose of the composition described in item 1 above. (Section 33) 31. The method according to paragraph 12 or 30 above, wherein the other therapeutic agent is prednisone or prednisolone, or an analogue or derivative thereof. (Section 34) 33. The method of any one of paragraphs 12, 14, 29, 30, or 32, wherein the cancer is lymphoma, leukemia, or melanoma. (Section 35) 35. The method according to item 34, wherein the lymphoma is selected from the group consisting of non-Hodgkin's lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. (Section 36) 35. The method according to item 34 above, wherein the leukemia is chronic myeloid leukemia (CML). (Section 37) 35. The method according to paragraph 34 above, wherein the precancerous condition is myelodysplastic syndrome (MDS, formerly known as preleukemia).

Claims

1. A composition for treating follicular lymphoma, comprising a compound of formula (IIa): 【Chemical 1】 (where, R ａ and R ｂ are each independently H, or one or more -Q ３ -T ３ (In the formula, Q ３ is a bond, or unsubstituted or substituted C １ -C ３ an alkyl linker, and T ３ is H, halo, 4- to 7-membered heterocycloalkyl, C １ -C ３ Alkyl, OR ｄ , COOR ｄ , -S(O) ２ R ｄ , or -NR ｄ R ｅ where R ｄ and R ｅ are each independently H or C １ -C ６ alkyl, or -Q ３ T ３ is oxo. １ -C ６ is alkyl; R ７ is isopropyl, or one or more -Q ５ -T ５ (In the formula, Q ５ is a bond, C(O), C(O)NR ｋ , N.R. ｋ C(O), S(O) ２ , or C １ -C ３ is an alkyl linker, R ｋ is H or C １ -C ６ alkyl, and T ５ is H, halo, C １ -C ６ Alkyl, hydroxyl, cyano, C １ -C ６ Alkoxyl, Amino, Mono-C １ -C ６ Alkylamino, di-C １ -C ６ Alkylamino, C ３ -C ８ Cycloalkyl, C ６ -C １０ aryl, 4- to 12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O) ｑ R ｑ wherein q is 0, 1, or 2, and R ｑ is C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ aryl, 4- to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and T ５ T is H, halo, hydroxyl, or cyano, except when T is H, halo, hydroxyl, or cyano. ５ Halo, C １ -C ６ Alkyl, hydroxyl, cyano, C １ -C ６ Alkoxyl, Amino, Mono-C １ -C ６ Alkylamino, di-C １ -C ６ Alkylamino, C ３ -C ８ Cycloalkyl, C ６ -C １０ optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; or -Q ５ -T ５ is oxo. １ -C ６ Alkyl, C ３ -C ８ cycloalkyl, piperidinyl, tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl, or cyclohexyl, or a 4- to 12-membered heterocycloalkyl; and R ８ is H, methyl, or ethyl. or a pharmaceutically acceptable salt thereof Including; The compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to a subject simultaneously or sequentially with one or both of rituximab and lenalidomide. composition.

2. 2. The composition of claim 1, wherein the compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to the subject prior to administration of one or both of rituximab and lenalidomide.

3. The compound of formula (IIa) is compound 44: 【Chemistry 7】 or a pharmaceutically acceptable salt thereof.

4. R ７ But C １ -C ６ alkyl, 4- to 12-membered heterocycloalkyl, or C ３ -C ８ cycloalkyl, wherein said C １ -C ６ alkyl is isopropyl, said 4 to 12 membered heterocycloalkyl is selected from piperidinyl, tetrahydropyran, and tetrahydro-2H-thiopyranyl, and said C ３ -C ８ 3. The composition of claim 1 or 2, wherein cycloalkyl is selected from cyclopentyl and cyclohexyl.

5. The composition described in claim 1 or 2, characterized in that the compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to the subject together with both rituximab and lenalidomide.

6. The composition described in claim 3, characterized in that compound 44 or a pharmaceutically acceptable salt thereof is administered to the subject together with both rituximab and lenalidomide.

7. The composition of claim 1 or 2, wherein the compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to the subject together with rituximab.

8. The composition described in claim 3, characterized in that compound 44 or a pharmaceutically acceptable salt thereof is administered to the subject together with rituximab.

9. The composition described in claim 1 or 2, characterized in that the compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to the subject together with lenalidomide.

10. The composition described in claim 3, characterized in that compound 44 or a pharmaceutically acceptable salt thereof is administered to the subject together with lenalidomide.

11. 11. The composition of claim 3, 6, 8 or 10, wherein the compound is the hydrobromide salt of compound 44.

12. 4. The composition of claim 3, wherein compound 44 or a pharmaceutically acceptable salt thereof, rituximab, and lenalidomide are administered to the subject simultaneously.

13. The composition of claim 12, wherein the compound is the hydrobromide salt of compound 44.

14. 1. A composition for treating cancer, comprising a compound of formula (IIa): 【Chemistry 14】 (where, R ａ and R ｂ are each independently H, or one or more -Q ３ -T ３ (In the formula, Q ３ is a bond, or unsubstituted or substituted C １ -C ３ an alkyl linker, and T ３ is H, halo, 4- to 7-membered heterocycloalkyl, C １ -C ３ Alkyl, OR ｄ , COOR ｄ , -S(O) ２ R ｄ , or -NR ｄ R ｅ where R ｄ and R ｅ are each independently H or C １ -C ６ alkyl, or -Q ３ T ３ is oxo. １ -C ６ is alkyl; R ７ is isopropyl, or one or more -Q ５ -T ５ (In the formula, Q ５ is a bond, C(O), C(O)NR ｋ , N.R. ｋ C(O), S(O) ２ , or C １ -C ３ is an alkyl linker, R ｋ is H or C １ -C ６ alkyl, and T ５ is H, halo, C １ -C ６ Alkyl, hydroxyl, cyano, C １ -C ６ Alkoxyl, Amino, Mono-C １ - C ６ Alkylamino, di-C １ -C ６ Alkylamino, C ３ -C ８ Cycloalkyl, C ６ -C １０ aryl, 4- to 12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, or S(O) ｑ R ｑ wherein q is 0, 1, or 2, and R ｑ is C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C ８ Cycloalkyl, C ６ -C １０ aryl, 4- to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; and T ５ T is H, halo, hydroxyl, or cyano, except when T is H, halo, hydroxyl, or cyano. ５ Halo, C １ -C ６ Alkyl, hydroxyl, cyano, C １ -C ６ Alkoxyl, Amino, Mono-C １ -C ６ Alkylamino, di-C １ -C ６ Alkylamino, C ３ -C ８ Cycloalkyl, C ６ -C １０ optionally substituted with one or more substituents selected from the group consisting of aryl, 4- to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; or -Q ５ -T ５ is oxo. １ -C ６ Alkyl, C ３ -C ８ cycloalkyl, piperidinyl, tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl, or cyclohexyl, or a 4- to 12-membered heterocycloalkyl; and R ８ is H, methyl, or ethyl. or a pharmaceutically acceptable salt thereof Including; The compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to a subject simultaneously or sequentially with rituximab and lenalidomide. composition.

15. 15. The composition of claim 14, wherein the compound of formula (IIa) or a pharmaceutically acceptable salt thereof is administered to the subject prior to administration of rituximab and lenalidomide.

16. The compound of formula (IIa) is compound 44: 【Chemistry 20】 or a pharmaceutically acceptable salt thereof.

17. R ７ But C １ -C ６ alkyl, 4- to 12-membered heterocycloalkyl, or C ３ -C ８ cycloalkyl, wherein said C １ -C ６ alkyl is isopropyl, said 4 to 12 membered heterocycloalkyl is selected from piperidinyl, tetrahydropyran, and tetrahydro-2H-thiopyranyl, and said C ３ -C ８ 16. The composition of claim 14 or 15, wherein cycloalkyl is selected from cyclopentyl and cyclohexyl.

18. 17. The composition of claim 16, wherein the compound is the hydrobromide salt of compound 44.

19. The composition of any one of claims 14 to 18, wherein the cancer is lymphoma, leukemia, or melanoma.

20. 20. The composition of claim 19, wherein the cancer is lymphoma.

21. 21. The composition of any one of claims 16 or 18-20, wherein compound 44 or a pharmaceutically acceptable salt thereof, rituximab, and lenalidomide are administered to the subject simultaneously.

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