EA043327B1 - CANCER TREATMENT METHOD - Google Patents

Description

Related applications

This application claims priority and benefit under U.S. Patent Application No.

62/064948, filed October 16, 2014, and No. 62/065590, filed October 17, 2014, the contents of each of which are incorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION

Disease-associated chromatin-modifying enzymes (eg, EZH2) play a role in diseases such as proliferative disorders, metabolic disorders, and blood disorders. Thus, there is a need to develop small molecules capable of modulating EZH2 activity.

Brief description of the invention

The present invention provides a method of treating or alleviating a symptom of a disorder, for example, immunosurveillance evasion, a malfunction of the immune system induced by a cancer cell, a weakened immune response, a decreased intensity of inflammation, a decreased level of major histocompatibility complex (MHC) expression, or a cancer characterized by abnormal, abnormal regulated or increased activity and/or expression of enhancer of Zeste homolog-2 (EZH2) protein in a cell or subject in need thereof by contacting the cell with a therapeutically effective amount of an EZH2 inhibitor or administering it to the subject.

In one aspect, the method relates to preventing a cancer cell from escaping immunosurveillance, the method comprising the step of contacting the cell with a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to treating and/or correcting a dysfunction of the immune system induced by a cancer cell in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to enhancing, enhancing or inducing an immune response in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to increasing the expression level of a major histocompatibility complex (MHC) in a cancer cell, the method comprising the step of contacting the cancer cell with a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to increasing the intensity of inflammation in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to treating a cancer or cell proliferation disorder in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

In one aspect, the method relates to determining the likelihood of an EZH2 inhibitor treatment being effective for a subject in need thereof, the method comprising the steps of (1) obtaining a biological sample from the subject and (2) analyzing the biological sample for downregulation of the major complex expression level. histocompatibility (MHC) compared to the control sample. A treatment providing an EZH2 inhibitor will be more likely to be effective for a subject if the biological sample has a reduced level of MHC expression compared to the control sample.

In one aspect, the method relates to determining the likelihood of an EZH2 inhibitor treatment being effective for a subject in need thereof, the method comprising analyzing a biological sample obtained from the subject for a reduction in the level of major histocompatibility complex (MHC) expression in the sample compared to a control sample. A treatment providing an EZH2 inhibitor will be more likely to be effective for a subject if the biological sample has a reduced level of MHC expression compared to the control sample.

In one aspect, the method relates to screening the effectiveness of an EZH2 inhibitor treatment for a subject in need thereof, the method comprising (1) obtaining first and second biological samples from the subject; (2) contacting a second biological sample with an EZH2 inhibitor; (3) analyzing the first and second biological samples for major histocompatibility complex (MHC) expression; and (4) comparing MHC expression in the first biological sample with MHC expression in the second biological sample. A treatment providing an EZH2 inhibitor will be more likely to be effective for a subject if the second biological sample has an increased level of MHC expression compared to the expression in the first biological sample.

In embodiments of the above aspects, the cell or subject exhibits abnormal, misregulated, or increased EZH2 activity.

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In embodiments according to the above aspects, the cell or subject is characterized by the chromosomal translocation t(x; 18)(p 11.2;q11.2).

In embodiments according to the above aspects, the translocation results in the formation of an SS18-SSX fusion gene.

In embodiments of the above aspects, the cell or subject has reduced function or expression level of INI1 (also referred to herein as BAF47, SNF5 or SMARCB1).

In embodiments according to the above aspects, the cell or subject has reduced function or expression level of INI1.

In embodiments according to the above aspects, the EZH2 inhibitor is Compound A (also referred to herein as E7438, or EPZ-6438, or tazemetostat), characterized by the following formula:

and its pharmaceutically acceptable salts or stereoisomers.

In embodiments according to the above aspects, the EZH2 inhibitor is one or more of

and their pharmaceutically acceptable salts or stereoisomers.

In embodiments according to the above aspects, the method further includes administering a chemotherapeutic compound, for example, a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

In embodiments according to the above aspects, the cancer cell is located in the body of a subject, such as a human.

In embodiments according to the above aspects, immune evasion, dysfunction of the immune system induced by a cancer cell, an immune response requiring enhancement, inflammation, or cancer is characterized by a reduced level of expression of one or more of MHC, β2-microglobulin, tumor necrosis factor (TNF) receptor ), low molecule polypeptide 2 (LMP2), small molecule polypeptide 7 (LMP7), transporter associated with antigen processing (TAP), and TAP-associated glycoprotein (tapasin) in a cancer cell compared to a non-cancerous cell. MHC may be a human leukocyte antigen (HLA), for example, HLA-A, HLA-B, HLA-C, HLA-DM alpha, HLA-DM beta, HLA-DO alpha, HLA-DO beta 1, HLA-DP alpha 1, HLA-DP beta 1, HLA-DR alpha, HLA-DR beta 1, HLA-DR beta 3, HLA-DR beta 4, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L .

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Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be understood by one skilled in the art to which this invention relates. As used herein, the singular number also includes the plural unless the context clearly requires otherwise. Unless specifically stated or obvious from the context, singular forms used herein are understood to mean singular or plural. Unless specifically stated or obvious from the context, the term or as used herein should be understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, the term should be approximately understood to be within the range of normal deviation in a given area, for example, within 2 standard deviations of the mean. Approximate can be understood as being within 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05 or 0.01% of the specified value. Unless the context clearly requires otherwise, all numerical values provided herein are modified by the term approximately.

Although methods and materials similar or equivalent to those described herein may 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. The references provided in this document are not considered to be prior art for the claimed invention. In the event of a conflict, this description, including definitions, will control. Moreover, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Any of the above aspects and embodiments may be combined with any other aspect or embodiment.

Other features and advantages of the present invention will be apparent from the following detailed description and the accompanying claims.

Description of the figures

In fig. 1 shows Western blot results of cell lysates demonstrating expression of SS18-SSX1 and down-regulation of INI1 (also referred to herein as BAF47, SNF5 or SMARCB1) in the HS-SY-II cell line.

In fig. Figure 2A shows the results of Western blot analysis of isolated histones, demonstrating the levels of H3K27 trimethylation (H3K27me3) and H3K27 dimethylation (H3K27me2) in various cell lines. In fig. 2B-2D are a series of graphs showing the quantitative ratio of H3K27me3/total H3 (B), H3K27me2/total H3 (C), or H3K27me3/H3K27me2 (D) in various cell lines. These quantitative data were derived from calculations based on protein bands obtained from Western blot analysis.

In fig. 3A-3D are a series of graphs demonstrating that HS-SY-II cells are highly sensitive to EZH2 inhibitors, whereas SW982 cells are not. The HS-SY-II cell line shown in FIG. 3A, and the SW982 cell line shown in FIG. 3B were treated with Compound E. The HS-SY-II cell line shown in FIG. 3C, and the SW982 cell line shown in FIG. 3D were treated with Compound A (also referred to herein as E7438 or EPZ-6438). Each cell type was pretreated with a compound (Compound E or Compound A) for 7 days at specified concentrations and subcultured and treated for an additional 7 days. Cell viability was determined using the CellTiter-Glo® luminescence-based cell viability assay.

In fig. 4A-4F demonstrate that reduction in INI1 levels confers sensitivity to EZH2 inhibitor (EZH2i) in soft tissue sarcoma cell lines. (A) shows Western blot analysis of cell lysates demonstrating INI1 expression in various tumor cell lines. Chondrosarcoma tumor cell lines were characterized by down-regulation of INI1 (eg, in cell lines b and c). (B) and (C) are graphs demonstrating that cell line b (Panel B) and cell line c (Panel C) are sensitive to EZH2 inhibitors. (D) shows Western blot results of cell lysates demonstrating expression of INI1 and SS18 in various cell lines. (E) is a graph demonstrating that SSX-SS18-positive cells are sensitive to EZH2 inhibitors. (F) is a graph showing that 33X-3318 negative cells are not sensitive to EZH2 inhibitors.

In fig. 5A and 5B are a series of graphs demonstrating that HS-SY-II cells are highly sensitive to Compound A (FIG. 5A), whereas SW982 cells are not (FIG. 5B). Each cell type was treated with compound A at specific concentrations. Cells were subcultured on day 7 and treated for an additional 7 days. Cell viability was determined using the CellTiter-Glo® luminescence-based cell viability assay.

In fig. 6A shows reduced H3K27me3/total H3 ratios (ratio to control) in HS-SY-II cells and SW982 cells after treatment with Compound A. Cells were treated with Compound

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And within 96 hours the histones were extracted. Changes in histone abundance as a marker were analyzed using enzyme-linked immunosorbent assay (ELISA). Changes in histone marker abundance were comparable between HS-SY-II and SW982, indicating that the changes were independent of the presence of the SS18-SSX fusion protein. In fig. 6B shows the concentration (IC ₅₀ ) of the compound required to reduce the H3K27me3/total H3 ratio by 50%.

In fig. 7A and 7B show pharmacokinetic (PK) values and pharmacodynamic (PD) changes in the HS-SY-II xenograft model, respectively. In fig. 7A shows the plasma concentrations of Compound A. In this case, Compound A was given to mice orally twice daily for 7 days. Peripheral blood samples were collected from mice treated with Compound A approximately 5 minutes before and 3 hours after the last dose. An analysis was carried out regarding the concentration of Compound A in blood plasma. Concentrations are shown graphically (n=5). Each bar represents the mean plasma concentration in each group. In fig. 7B shows the inhibitory effects of Compound A in mice on H3K27me3 in HS-SY-II xenograft. In this case, Compound A was given to mice orally twice daily for 7 days. Indicators of H3K27me3 in the tumor are presented in graphical form (n=5). Each bar represents the mean ±SEM of the trimethylation level in each group. In table 2 and 3 below present the results of statistical analyzes regarding the data shown in FIG. 7B.

The results of statistical analyzes confirm the dose-dependent change.

In fig. 8 shows changes in the expression of putative PD markers after treatment with Compound A in HS-SY-II and SW982 in vitro experiments. Here, each cell type was treated with Compound A or EPZ-011989 (which is also an EZH2 inhibitor and is also referred to herein as Compound C); concentration and time period (days) are shown. Changes in gene expression were analyzed by RT-PCR. Gene expression levels were normalized to GAPDH levels. Bars are shown as ratios to 0 μM treated controls. In table 4 below presents the results of statistical analysis regarding the data shown in FIG. 8. Asterisks indicate significant changes compared to levels in 0 µM treated groups.

In fig. 9 shows changes in the expression of putative PD markers after treatment with Compound A in an in vivo HS-SY-II experiment. In this case, Compound A was given to mice orally twice daily for 7 days. Tumor samples were collected approximately 3 hours after the last dose. Changes in gene expression were analyzed by RT-PCR. Gene expression levels were normalized to GAPDH levels. Bars are shown as ratios relative to data from the vehicle group.

In fig. 10A-10C show the average tumor volumes for nude mice bearing HS-SY-II xenografts that were administered either vehicle (orally or iv), Compound A (orally), doxorubicin (iv), or the Compound A/doxorubicin combination. in certain doses for 28 days. Tumor volumes were measured twice a week. Two independent studies were carried out. In fig. 10A and 10B show the results of the first study, and FIG. Figure 1C shows the results of the second study. Tumors from animals from the second study were removed on day 28 (3 hours after the last dose) and analyzed for H3K27me3 using ELISA (Fig. 10D) or immunohistochemistry (IHC) for the proliferation marker Ki67 (Fig. 10E).

In fig. 11A-11D show the mean tumor volumes and percentage survival for nude mice bearing two different patient-derived xenografts (PDX) tumors consistent with synovial sarcoma and treated with either vehicle (orally) or Compound A (orally ), or doxorubicin (iv) in certain doses for 35 days. In fig. 11A and 11B show data obtained for PDX mice from a 57 year old male subject with high grade spindle cell sarcoma (CTG-0771). In fig. 11C and 11D show data obtained for PDX mice from a 16 year old female subject (CTG0331). Tumor volumes were measured twice a week. Dosing was stopped on day 35 and animals were observed until day 60, with measurements taken twice per week. If the tumor volume of a given animal exceeded 2000 mm ³ , the mouse was euthanized (analyzed based on a graph according to the Kaplan-Meier method).

In fig. 12A and 12B show a dataset obtained from PDX tumors consistent with synovial sarcoma from subsets of mice that were removed at day 35 and analyzed by RNA-seq analysis. In fig. 12A shows data obtained for PDX mice from a 57 year old male subject; in fig. 12B shows data from mice carrying PDX from a 16 year old female subject. Shown are the results of HLA gene expression in tumors from mice treated with either vehicle or Compound A BID at a concentration of 400 mg/kg. The figure indicates low expression level and high expression level.

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Detailed Description of the Invention

The present invention provides a method of treating or alleviating a symptom of a disorder, for example, immunosurveillance evasion, a malfunction of the immune system induced by a cancer cell, a weakened immune response, a decreased intensity of inflammation, a decreased level of major histocompatibility complex (MHC) expression, or a cancer characterized by abnormal, abnormal regulated or increased activity or expression of enhancer of Zeste homolog-2 (EZH2) protein in a cell or subject in need thereof by contacting the cell with a therapeutically effective amount of an EZH2 inhibitor or administering it to the subject. EZH2 is an enzymatic subunit of transcriptional repression complex 2 (PRC2), a complex that catalyzes the methylation of histone 3 at lysine 27 (H3K27). Histone 3 lysine 27 (H3K27) methylation is an epigenetic marker of transcriptional repression that is causally associated with a number of cancers, including hematologic malignancies and solid cancers. In human cancers, several molecular mechanisms have been reported to lead to increased levels of H3K27 trimethylation.

The present invention is based in part on the discovery that immunosurveillance evasion or disorders characterized by or induced by malfunction of the immune system induced by a cancer cell, a weakened immune response, decreased inflammation, and decreased MHC expression can be effectively treated with EZH2 inhibitors.

In some embodiments, cells, subjects, tumors, or tumor cells characterized by abnormal, misregulated, or increased EZH2 activity are sensitive to the EZH2 inhibitors of the present invention.

Human synovial sarcoma accounts for 8-10% of all malignant soft tissue tumors and most often occurs in the extremities of young people. A recurrent chromosomal translocation, t(x;18)(p11.2;q11.2), which results in the fusion of the SS18 gene on chromosome 18 with one of three closely related genes on the X chromosome SSX1, SSX2 and, less commonly, SSX4, results in formation of an in-frame fusion protein in which the C-terminal eight amino acids of SS18 are replaced by the C-terminal 78 amino acids of SSX. This results in the expression of the oncogenic SS18-SSX fusion protein, which binds to the SWI/SNF complex, displacing both wild-type SS18 and the tumor suppressor INI1, which are then degraded. This leads to abnormal gene expression and ultimately the development of cancer.

The present invention provides methods for preventing a cancer cell from escaping immunosurveillance, comprising the step of contacting the cell with a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for treating or correcting a dysfunction of the immune system induced by a cancer cell in a subject in need thereof, which includes the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for enhancing, enhancing, or inducing an immune response in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for increasing the level of major histocompatibility complex (MHC) expression or activity in a cancer cell, comprising the step of contacting the cancer cell with a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for increasing the intensity of inflammation in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for treating cancer or a cell proliferation disorder in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an EZH2 inhibitor.

The present invention may also provide methods for determining the likelihood of an EZH2 inhibitor treatment being effective for a subject in need thereof, comprising the steps of (1) obtaining a biological sample from the subject and (2) analyzing the biological sample for downregulation of major histocompatibility complex (MHC) expression. ) compared to the control sample. A treatment providing an EZH2 inhibitor will be more likely to be effective for a subject if the biological sample has a reduced level of MHC expression compared to the control sample.

The present invention may also provide methods for determining the likelihood of an EZH2 inhibitor treatment being effective for a subject in need thereof, comprising analyzing a biological sample obtained from the subject for a reduction in the level of major histocompatibility complex (MHC) expression in the sample compared to a control sample. An EZH2 inhibitor treatment will be more likely to be effective for a subject if the biological sample has reduced expression levels

MHC compared to the control sample.

The present invention may also provide methods for screening the effectiveness of an EZH2 inhibitor treatment for a subject in need thereof, comprising (1) obtaining first and second biological samples from the subject; (2) contacting a second biological sample with an EZH2 inhibitor; (3) analyzing the first and second biological samples for major histocompatibility complex (MHC) expression; and (4) comparing MHC expression in the first biological sample with MHC expression in the second biological sample. A treatment providing an EZH2 inhibitor will be more likely to be effective for a subject if the second biological sample has an increased level of MHC expression compared to the expression in the first biological sample.

In embodiments of the present invention, a cell or subject exhibits abnormal, misregulated, or increased EZH2 activity.

In embodiments of the present invention, the cell or subject is characterized by the chromosomal translocation t(x;18)(p11.2;q11.2).

In embodiments of the present invention, the translocation results in the formation of an SS18-SSX fusion gene.

In embodiments of the present invention, the cell or subject has reduced function or expression level of INI1 (also referred to herein as BAF47, SNF5 or SMARCB1). In embodiments of the present invention, the cell or subject has reduced function or expression level of INI1. In some embodiments, the cell or subject is suspected or determined to be INI1 negative or INI1 deficient.

In embodiments of the present invention, the EZH2 inhibitor is Compound A having the following formula:

and pharmaceutically acceptable salts thereof.

In embodiments of the present invention, the EZH2 inhibitor is one or more of

and pharmaceutically acceptable salts thereof.

In embodiments of the present invention, the method further comprises administering a chemotherapeutic compound, for example, a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

According to embodiments of the present invention, the cancer cell is located in the body of a subject, such as a human.

According to embodiments of the present invention, immunosurveillance evasion, malfunction of the immune system induced by a cancer cell, an immune response requiring enhancement, inflammation, or cancer is characterized by a reduced level of expression of one or more of MHC, β2-microglobulin, tumor necrosis factor (TNF) receptor, low molecule polypeptide 2 (LMP2), low molecule polypeptide 7 (LMP7), transporter associated with antigen processing (TAP), and TAP-associated glycoprotein (tapasin) in a cancer cell compared to a non-cancerous cell. MHC may be a human leukocyte antigen (HLA), for example, HLA-A, HLA-B, HLA-C, HLA-DM alpha, HLA-DM beta, HLA-DO alpha, HLA-DO beta 1, HLA-DP alpha 1, HLA-DP beta 1, HLA-DR alpha, HLA-DR beta 1, HLA-DR beta 3, HLA-DR beta 4, HLA-E, HLA-F, HLA-G, HLA-K or HLA-L .

Accordingly, the present invention may provide methods for treating immunosurveillance evasion, immune system dysfunction induced by a cancer cell, a weakened immune response, decreased inflammation, decreased MHC expression in a cell or subject in need thereof by contacting the cell with a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof, or by administering it to a subject. The present invention may further provide for the use of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof for the preparation of a medicament useful for the treatment of immunosurveillance evasion, cancer cell-induced dysfunction of the immune system, weakened immune response, reduced inflammation, reduced MHC expression level.

In embodiments, the disorders are characterized by abnormal, misregulated, or increased EZH2 activity or expression. Abnormal EZH2 activity as used herein refers to the mislocalization of EZH2 in the cell or the misbinding of EZH2 to/within the protein complex. In embodiments, abnormal EZH2 activity results from loss of INI1 regulatory function, which in turn can occur with a number of genetic alterations, examples of some of which are discussed in more detail herein. In some embodiments, abnormal, misregulated, or increased EZH2 activity or expression results in or is associated with increased levels of H3K27 trimethylation.

In embodiments, the disorder is characterized by chromosomal translocation t(x;18)(p11.2;q11.2). This translocation results in the formation of the SS18-SSX fusion gene.

In embodiments, the subject in need of treatment has abnormal, dysregulated, or increased EZH2 activity.

In embodiments, the subject in need of treatment has decreased INI1 function or expression level, or both. In embodiments, the subject has no detectable INI1 function or expression, or both.

According to embodiments of the present invention, synovial sarcoma is characterized by an SSX1 fusion. In another embodiment, the synovial sarcoma is characterized by an SSX2 fusion. In embodiments, synovial sarcoma is characterized by an SSX4 fusion. In embodiments, the subject in need of treatment, the treatment regimen, dose, and frequency of administration are selected according to the type of SSX fusion that is being detected. In embodiments, the EZH2 inhibitor to be administered is also selected according to an SSX fusion associated with cancer.

The present invention may also provide methods for treating cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, wherein the subject in need of treatment has a t( x;18)(p11.2;q11.2) or SS18-SSX fusion gene. The present invention further provides the use of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof for the preparation of a medicament useful for the treatment of cancer.

In embodiments, the method includes the step of determining the presence of a t(x;18)(p11.2;q11.2) chromosomal translocation or SS18-SSX fusion gene in a sample from the subject prior to the administration step.

Determining the presence of chromosomal translocation t(x;18)(p11.2;q11.2) in a sample can be carried out using any method known in the art. For example, it can be determined by karyotyping and RT-PCR of SS18-SSX transcripts or by FISH (fluorescence in situ hybridization). The SS18-SSX fusion gene can be detected using any method known in the art. For example, it can be detected by RT-PCT, immunohistochemical staining assay, or fluorescence in situ hybridization (FISH).

Treatable cancer can be assessed using DNA cytometry, flow cytometry or imaging cytometry. A cancer to be treated may be defined as having 10, 20, 30, 40, 50, 60, 70, 80, or 90% of cells in the DNA synthesis phase of cell division (eg, the S phase of cell division). The cancer to be treated can be defined as having a low proportion of cells in S phase or a high proportion of cells in S phase.

According to aspects of the present invention, the synovial sarcoma is a monophasic synovial sarcoma. According to other aspects of the present invention, the synovial sarcoma is a biphasic synovial sarcoma.

The present invention also provides methods that include the steps of detecting the presence of a t(x;18)(p11.2;q11.2) chromosomal translocation or SS18-SSX fusion gene in a sample from a subject and treating the subject by administering a therapeutically effective amount of the compound or pharmaceutically an acceptable salt, solvate or polymorph.

The present invention further provides methods that include selecting a treatment regimen comprising administering to a candidate subject a therapeutically effective amount of a compound or a pharmaceutically acceptable salt or solvate thereof. The treatment regimen may also include surgery, chemotherapy, radiation therapy, immunotherapy, or any combination thereof.

In embodiments, a method of the present invention comprises administering to a subject a therapeutically effective amount of an EZH2 inhibitor, surgery, chemotherapy, radiation therapy, acupuncture, immunotherapy, or any combination thereof. Chemotherapy (usually doxorubicin and/or ifosfamide) may be recommended in the treatment of synovial sarcoma, particularly in advanced or metastatic disease.

The present invention further provides a method of treating cancer or cell proliferation disorder associated with abnormal, misregulated or increased EZH2 activity in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof. For example, the cancer is synovial sarcoma. For example, the cancer is epithelioid sarcoma, extraskeletal myxoid chondrosarcoma, malignant rhabdoid tumor, or atypical chordoma.

For example, an EZH2 inhibitor that can be used herein includes Compound A, B, C, D, or E. Compound A is also referred to herein as E7438 or EPZ6438.

As used herein, a subject in need of treatment is a subject with cancer associated with abnormal, misregulated or increased EZH2 activity or a subject with cancer mediated by the t(x;18)(p11.2;q11.2) chromosomal translocation. For example, the subject in need of treatment has synovial sarcoma.

In embodiments, the subject in need of treatment has received at least one prior therapy to treat a disorder associated with abnormal, misregulated, or increased EZH2 activity.

In embodiments, the subject has refractory cancer at the time of most recent therapy. Refractory cancer means any cancer described in this document, including synovial sarcoma, that does not respond to treatment. The cancer may be resistant at the beginning of treatment or may become resistant during treatment. Refractory cancer is also called resistant cancer. In embodiments, the subject in need of treatment experiences a recurrence of cancer after being in remission for the most recent therapy. In embodiments, the subject has received and failed all known effective therapies for the synovial sarcoma from which the subject suffers.

In embodiments, the subject is concurrently treated with another therapy to treat a cancer mediated by the t(x;18)(p11.2;q11.2) chromosomal translocation, such as synovial sarcoma.

Subject includes a mammal. The mammal can be, for example, any mammal, such as a human, primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or pig. Preferably the mammal is a human.

In embodiments, the subject has an increased level of histone H3 Lys27 trimethylation (H3-K27me3). In embodiments, an abnormal, dysregulated, or upregulated EZH2 or chromosomal translocation t(x;18)(p11.2;q11.2) is associated with increased levels of H3-K27me trimethylation.

As used herein, a subject sample refers to any suitable sample containing cells or cell components obtained from or originating from a subject. For example, in embodiments, the sample contains cancer cells. In embodiments, the sample is a biopsy obtained, for example, from soft tissue (for example,

- 8 043327 tavs). In embodiments, the sample is a biopsy obtained from, or in addition to, tissue other than soft tissue. For example, in embodiments, the sample is a cancer biopsy, such as from a tumor consisting of cancer cells. Cells in a sample can be isolated from other components of the sample according to methods well known to those skilled in the art. For example, in embodiments, the sample is a tissue, organ, or biological fluid, such as whole blood, blood plasma, serum, urine, saliva, genital secretions, cerebrospinal fluid, sweat, or waste products.

As used herein, monotherapy refers to the administration of a single active or therapeutic compound to a subject in need thereof. Preferably, monotherapy will comprise administration of a therapeutically effective amount of a single active compound. For example, cancer monotherapy involves administering one compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need of treatment of cancer associated with abnormal, misregulated or increased EZH2 activity. In one aspect, the one active compound is a compound of the present invention or a pharmaceutically acceptable salt, solvate, or polymorph thereof. In a certain embodiment, the one active compound is tazemetostat.

As used herein, treat or treat describes the management and care of a patient to combat a disease, condition or disorder associated with abnormal, misregulated or increased activity of EZH2, and includes the administration of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof for the purpose of relieving symptoms or complications of a disease, condition or disorder or eliminating a disease, condition or disorder.

A compound of the present invention, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, may also be used to prevent a disease, condition, or disorder associated with abnormal, misregulated, or increased EZH2 activity. As used herein, warning or warning describes reducing the likelihood of or eliminating the occurrence of symptoms or complications of a disease, condition or disorder.

As used herein, the term alleviate describes the process by which the severity of a sign or symptom of a disorder is reduced. It is important to note that a sign or symptom can be relieved without eliminating it. In a preferred embodiment, administration of the pharmaceutical compositions of the present invention results in resolution of the sign or symptom even though resolution is not required. Effective dosages are expected to reduce the severity of the sign or symptom. For example, a sign or symptom of a disorder, such as cancer, that can occur in multiple sites is alleviated if the severity of the cancer is reduced within at least one of the multiple sites.

As used herein, the term symptom is defined as an indicator of a disease, disorder, lesion or malfunction of some kind in the body. Symptoms are felt and noted by the individual experiencing the symptom and cannot be noted by others. Others are defined as non-medical specialists.

As used herein, the term symptom is also defined as an indicator of some disorder in the body. But signs are defined as factors that can be detected by a doctor, nurse or other medical professional.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in tumor size or volume, or a reduction in tumor growth rate, or regrowth, or any combination of the above. A decrease in tumor size or volume can also be called tumor regression. Preferably, after treatment, the size of the tumor is reduced by 5% or more relative to its size before treatment; more preferably, the tumor size is reduced by 10% or more; more preferably reduced by 20% or more; more preferably reduced by 30% or more; more preferably reduced by 40% or more; even more preferably, it is reduced by 50% or more, and most preferably, it is reduced by more than 75% or more. The size or volume of the tumor can be determined using any reproducible measurement methods. The size or volume of the tumor can be measured as the diameter of the tumor.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity or expression with the compounds described herein may result in a reduction in the number of tumors. Preferably, after treatment, the number of tumors is reduced by 5% or more relative to the number before treatment; more preferably, the number of tumors is reduced by 10% or more; more preferably reduced by 20% or more; more preferably reduced by 30% or more; more preferably, reduced by 40% or more; even more

- 9 043327 is preferably reduced by 50% or more and most preferably reduced by more than 75%. The number of tumors can be determined using any reproducible quantification methods. The number of tumors can be determined by counting tumors visible to the naked eye or at a given magnification.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in increased median survival time or decreased mortality rates or both, in a population of treated subjects compared with a population of untreated subjects or subjects receiving vehicle alone. In a certain embodiment, the population of subjects being treated is treated with a drug or combination of drugs that is not a compound of the present invention. Preferably, the average survival time is increased by more than 10, 20 or 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 a population can be determined using any reproducible methods. The increase in population mean survival time can be determined, for example, by calculating the population mean survival time after initiation of treatment with the active compound. The increase in population mean survival time can be determined, for example, by calculating the population mean survival time after completion of the first cycle of treatment with the active compound.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in the mortality rate of the treated population , compared to a population receiving vehicle alone. Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in the mortality rate of the treated population , compared to the untreated population. Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in the mortality rate of the treated population , compared to a population receiving monotherapy with a drug that is not a compound of the present invention or a pharmaceutically acceptable salt or solvate 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%. A reduction in the mortality rate of a population of subjects receiving treatment can be determined using any reproducible methods. The reduction in the mortality rate of a population can be determined, for example, by calculating for the population the average number of disease-related deaths per unit time after initiation of treatment with the active compound. The reduction in population mortality may also be determined, for example, by calculating for the population the average number of disease-related deaths per unit time after completion of the first cycle of treatment with the active compound.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in the rate of tumor growth. Preferably, after treatment, the tumor growth rate is reduced by at least 5% relative to pre-treatment; more preferably, the tumor growth rate is reduced by at least 10%; more preferably reduced by at least 20%; more preferably reduced by at least 30%; more preferably reduced by at least 40%; more preferably reduced by at least 50%; even more preferably, it is reduced by at least 50%, and most preferably, it is reduced by at least 75%. The rate of tumor growth can be determined using any reproducible detection methods. The tumor growth rate can be determined according to the change in tumor diameter per unit time.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may 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

- 10 043327 preferably less than 75%. Tumor regrowth can be determined using any reproducible detection methods. Tumor regrowth is determined, for example, by determining the increase in tumor diameter following previous tumor shrinkage following treatment. Reduced tumor regrowth is determined by the failure of tumors to recur after treatment is stopped.

Treatment or prevention of cancer(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in a reduction in the number or proportion of cells with abnormal appearance or morphology. Preferably, after treatment, the number of cells with abnormal morphology is reduced by at least 5% relative to its pre-treatment value; more preferably reduced by at least 10%; more preferably reduced by at least 20%; more preferably reduced by at least 30%; more preferably reduced by at least 40%; more preferably reduced by at least 50%; even more preferably, it is reduced by at least 50%, and most preferably, it is reduced by at least 75%. Abnormal cell appearance or morphology can be determined using any reproducible detection methods. Abnormal cell morphology can be determined by microscopy, such as using an inverted tissue culture microscope. Abnormal cell morphology may be expressed as nuclear pleomorphism.

Treatment of cancer(s) or cell proliferation disorder(s) associated with abnormal, misregulated or increased EZH2 activity with the compounds described herein may result in cell death, and preferably cell death results to reduce the number of cells in the population by at least 10%. More preferably, cell death means a decrease of at least 20%; more preferably a reduction of at least 30%; more preferably a reduction of at least 40%; more preferably a reduction of at least 50%; most preferably a reduction of at least 75%. The number of cells in a population can be determined using any reproducible methods. The number of cells in a population can be determined using fluorescence-excited cell sorting (FACS), immunofluorescence microscopy, and light microscopy. Methods for determining cell death are described in Li et al., Proc. Natl. Acad. Sci. U S A. 100(5): 2674-8, 2003. In one aspect, cell death occurs as a result of apoptosis.

As used herein, the term selective means the tendency to occur with greater frequency in one population than in another population. The populations being compared may be populations of cells. Preferably, the compound of the present invention, or a pharmaceutically acceptable salt, solvate or polymorph thereof, is selective against a tumor or pre-tumor cell and not against a normal cell. Preferably, a compound of the present invention, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, acts selectively to modulate one molecular target (e.g., a methyltransferase target protein) but does 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 EZH2. Preferably, an event occurs selectively in population A relative to population B if it occurs more than twice as often in population A compared to population B. An event occurs selectively if it occurs more than five times as often in population A. An event occurs selectively , if it occurs more than ten times more often in population A; more preferably more than fifty times; even more preferably more than 100 times and most preferably more than 1000 times more often in population A compared to population B. For example, cell death in cancer cells would be considered selective if it occurs more than twice as often in cancer cells compared to normal cells.

Administration of a compound of the present invention, or a pharmaceutically acceptable salt, solvate or polymorph thereof, to a cell or subject in need thereof may result in modulation (ie inhibition) of EZH2 activity.

Administration of a compound of the present invention, or a pharmaceutically acceptable salt, solvate or polymorph thereof, to a cell or subject in need thereof results in modulation (ie inhibition) of EZH2.

Activation refers to bringing a composition of the present invention (eg, a protein or nucleic acid) into a state suitable for performing the desired biological function. The composition of the present invention, capable of being activated, can also be in an inactivated state. The activated composition of the present invention may have an inhibitory or stimulatory biological function, or both.

Enhancement refers to an increase in the desired biological activity of a composition of the present invention (eg, a protein or nucleic acid). The increase may occur due to

- 11 043327 increasing the concentration of the composition of the present invention.

A compound (i.e., EZH2 inhibitor) that can be used in any of the methods described herein may be characterized by the following Formula I:

or be in the form of a pharmaceutically acceptable salt, solvate or polymorph thereof;

where R ⁷⁰¹ represents H, F, OR ⁷⁰⁷ , NHR ⁷⁰⁷ , -(CΞC)-(CH2)n7-R ⁷⁰⁸ , phenyl, 5- or 6-membered heteroaryl, C3-8cycloalkyl or 4-7-membered heterocycloalkyl containing 1-3 heteroatoms, wherein each of phenyl, 5- or 6-membered heteroaryl, _C3-8 cycloalkyl or 4-7 membered heterocycloalkyl is independently optionally substituted with one or more groups selected from halogen, _C1-3 alkyl, OH, O-C ₁ . ₆ alkyl, NH-C ₁ . ₆ alkyl and C _1-3 alkyl substituted with C ₃ . ₈ cycloalkyl or 4-7 membered heterocycloalkyl containing 1-3 heteroatoms, where each of O-C ₁ . ₆ alkyl and NH-C ₁ . The ₆ alkyl is optionally substituted with hydroxyl, O-C ₁ . ₃ alkyl or NH-C ₁ . ₃ alkyl, with each of OC ₁ . ₃ alkyl and NH-C ₁ . ₃ alkyl is optionally further substituted with OC ₁ . ₃ alkyl or NH-C ₁ . ₃ alkyl;

R ⁷⁰² and R ⁷⁰³ are each independently H, halogen, C _1-4 alkyl, C ₁ . ₆ alkoxy or C ₆ C ₁₀ aryloxy, each optionally substituted with one or more halogens;

R ⁷⁰⁴ and R ⁷⁰⁵ are each independently C _1-4 alkyl;

R ⁷⁰⁶ is cyclohexyl substituted with N(C ₁ . ₄ alkyl) ₂ , where one or both of the C _1-4 alkyls are substituted with C ₁ . ₆ alkoxy; or R ⁷⁰⁶ is tetrahydropyranyl;

R ⁷⁰⁷ is C _1-4 alkyl, optionally substituted with one or more groups selected from hydroxyl, C ₁ . ₄ alkoxy, amino, mono- or di-C1. ₄ alkylamino, C _3-8 cycloalkyl and 4-7 membered heterocycloalkyl containing 1-3 heteroatoms, where each of C ₃ . ₈ cycloalkyl or 4-7 membered heterocycloalkyl is independently further optionally substituted with C ₁ . ₃ alkyl;

R ⁷⁰⁸ is C _1-4 alkyl, optionally substituted with one or more groups selected from OH, halogen and C ₁ . ₄ alkoxy, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms, or OC ₁ . ₆ alkylα, where the 4-7 membered heterocycloalkyl may be optionally further substituted with OH or C ₁ . ₆ alkyl; and n ₇ is 0, 1 or 2.

For example, R ⁷⁰⁶ is cyclohexyl substituted with N(C ₁ . ₄ alkyl) ₂ , where one C _1-4 alkyl is unsubstituted and the other is substituted with methoxy.

For example, R ⁷⁰⁶ is

For example, the compound is represented by formula II:

For example, R ⁷⁰² toxyl.

For example R ⁷⁰⁴

For example, R ⁷⁰¹ is methyl or isopropyl, and R ⁷⁰³ is methyl or substituted with OCH ₃ or morpholine.

represents methyl.

represents OR ⁷⁰⁷ , and R ⁷⁰⁷ represents C _1-3 alkyl, optionally sub- 12 043327

For example, R ⁷⁰¹ is H or F.

For example, R ⁷⁰¹ is tetrahydropyranyl, phenyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl or pyrazolyl, each of which is optionally substituted with methyl, methoxy, ethyl, substituted morpholine, or -OCH2CH2OCH3.

For example, R ⁷⁰⁸ is morpholine, piperidine, piperazine, pyrrolidine, diazepane or azetidine, each of which is optionally substituted with OH or C _1-6 alkyl.

For example, R ⁷⁰⁸ is morpholine.

For example, R ⁷⁰⁸ is piperazine substituted with C _1-6 alkyl.

For example, R ⁷⁰⁸ represents methyl, tert-butyl or C(CH ₃ )2OH.

A compound (i.e., EZH2 inhibitor) that can be used in any of the methods described herein may be characterized by the following formula III:

or is a pharmaceutically acceptable salt thereof.

In this formula, R ⁸⁰¹ represents C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C3-8cycloalkyl, 4-7 membered heterocycloalkyl containing 1-3 heteroatoms, phenyl or 5 or 6 membered heteroaryl, each of which is substituted with O- C1-6alkyl-Rx or NH-C1-6alkyl-Rx, wherein Rx is hydroxyl, O-C1-3alkyl or NH-C1-3alkyl, and wherein Rx is optionally further substituted with O-C1-3alkyl or NH-C1-3alkyl , except when Rx is hydroxyl; or R ⁸⁰¹ is phenyl substituted by -Q2-T2, where Q2 is a bond or C1-C ₃ αlkyl as a linker, optionally substituted with halogen, cyano, hydroxyl or C1-C ₆ alkoxy, and T2 is optionally substituted with 4-12 -membered heterocycloalkyl; and R ⁸⁰¹ is optionally further substituted;

each of R ⁸⁰² and r ⁸⁰³ independently represents H, halogen, C _1-4 alkyl, C _1-6 alkoxy or C 6 C ₁₀ aryloxy, each optionally substituted with one or more halogens;

R ⁸⁰⁴ and R ⁸⁰⁵ are each independently C _1-4 alkyl; And

R ⁸⁰⁶ represents -Q _x -T _x where Q _x represents a bond or C _1-4 alkyl as a linker, T _x represents H, optionally substituted C _1-4 alkyl, optionally substituted C ₃ C ₈ cycloalkyl or optionally substituted 4-14 membered heterocycloalkyl.

For example, each of Q _x and Q2 independently represents a bond or methyl as a linker, and each of T _x and T2 independently represents tetrahydropyranyl, piperidinyl substituted with 1, 2, or 3 C _1-4 alkyl groups, or cyclohexyl substituted N(C _1-4 alkyl) ₂ where one or both of the C _1-4 alkyls are optionally substituted with C _1-6 αlkoxy;

For example, R ⁸⁰⁶ is cyclohexyl substituted with N(C _1-4 alkyl) ₂ or R ⁸⁰⁶ is tetrahydropyranyl.

For example, R ⁸⁰⁶ is

For example, R ⁸⁰¹ is phenyl or 5- or 6-membered heteroaryl substituted with O-C _1-6 alkyl-R _x , or R ⁸⁰¹ is substituted with CH2-tetrahydropyranyl.

For example, a compound of the present invention is represented by formula IVa or IVb:

- 13 043327

or

(IVb) where Z' represents CH or N and R ⁸⁰⁷ represents C ₂ . ₃ alkyl-R _x .

For example, R ⁸⁰⁷ is -CH2CH2OH, CH ₂ CH ₂ OCH ₃ or -CH ₂ CH ₂ OCH ₂ CH ₂ OCH ₃ .

For example, R ⁸⁰² is methyl or isopropyl, and R ⁸⁰³ is methyl or methoxyl.

For example, R ⁸⁰⁴ is methyl.

The compound of the present invention may be characterized by the following formula (V):

or be a pharmaceutically acceptable salt or ester thereof.

In this formula, R2, R4 and _R12 are each independently _C1-6 alkyl;

R6 is a C ₆ -C ₁₀ aryl or 5- or 6-membered heteroaryl, each of which is optionally substituted by one or more -Q2-T2, where Q2 is a bond or a C ₁ -C ₃ alkyl as a linker, optionally substituted with halogen , cyano, hydroxyl or C1-C ₆ alkoxy, and T ₂ represents H, halogen, cyano, -ORa, -NR _a R _b , -(NR _a R _b R _c ) ⁺ A, -C(O)Ra, -C(O)ORa, -C(O)NRaRb, -NRbC(O)Ra, -NRbC(O)ORa, -S(O)2Ra, -S(O)2NRaRb or RS2, in which each of Ra, Rb and Rc are independently H or RS3, A ^is a pharmaceutically acceptable anion, RS2 and R _S3 are each independently C1- _C6 alkyl, _C3 - _C8 cycloalkyl, _C6 - _C10 aryl, 4-12 -membered heterocycloalkyl or 5- or 6-membered heteroaryl, or Ra and R _b together with the N atom to which they are attached form a 4-12 membered heterocycloalkyl ring with or without 1 additional heteroatom, and each of R _S2 , R _S3 and the 4-12 membered heterocycloalkyl ring formed by Ra and _Rb are optionally substituted by one or more -Q ₃ -T ₃ where Q ₃ is a bond or O ₁ -C ₃ alkyl as a linker, each optionally substituted halogen, cyano, hydroxyl or C ₁ -C ₆ alkoxy, and T ₃ is selected from the group consisting of halogen, cyano, C 1 -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12- membered heterocycloalkyl, 5- or 6-membered heteroaryl, _ORd , COORd, _S (O) _2Rd , _-NRdRe , and -C(O) _NRdRe , wherein each of Rd and Re is independently H or C1- _C6 alkyl, or _-Q3 - _T3 is oxo; or any two adjacent -Q ₂ -T ₂ , together with the atoms to which they are attached, form a 5- or 6-membered ring, optionally containing 1-4 heteroatoms selected from N, O and S, and optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, COOH, C(O)O-C ₁ -C ₆ alkyl, cyano, C1-C ₆ alkoxyl, amino, mono-C ₁ -C ₆ alkylamino, di-C1- C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12 membered heterocycloalkyl and 5- or 6-membered heteroaryl;

R ₇ is -Q4-T4, in which Q4 is a bond, a C1- _C4 alkyl as a linker, or a _C2 - _C4 alkenyl as a linker, each linker being optionally substituted with halogen, cyano, hydroxyl, or C1-C ₆ alkoxy, and T ₄ is H, halogen, cyano, NRfRg, -ORf, -C(O)Rf, -C(O)ORf, -C(O)NRfRg, C(O)NRfORg, -NRfC(O )Rg, -S(O)2Rf or RS4, in which Rf and Rg are each independently H or _RS5 , _RS4 and _RS5 are each independently C1- _C6 alkyl, _C2 - _C6 alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12 membered heterocycloalkyl or 5- or 6-membered heteroaryl, and each of R _S4 and R _S5 is optionally substituted by one or more - Q ₅ -T ₅ , where Q5 represents a bond, C(O), C(O)NR _k , NR _k C(O), S(O) ₂ or C ₁ -C ₃ alkyl as a linker, R _k represents is H or C ₁ -C ₆ alkyl, and T ₅ is H, halogen, C1-C ₆ alkyl, hydroxyl, cyano, C1-C ₆ alkoxy, amino, mono-C1-C ₆ alkylamino, di-C1-C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12 membered heterocycloalkyl, 5- or 6-membered heteroaryl or S(O)qRq in which q is 0, 1 or 2, and

- 14 043327

Rq 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, 4-12 membered heterocycloalkyl or 5- or 6-membered heteroaryl, and T ₅ is optionally substituted with one or several substituents selected from the group consisting of halogen, 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, 4-12 membered heterocycloalkyl and 5- or 6-membered heteroaryl, except when T ₅ represents H, halogen, hydroxyl or cyano; or -Q5-T5 is oxo; And

R ₈ is H, halogen, hydroxyl, COOH, cyano, _RS6 , ORS6 or COORS6, in which RS6 is C1- _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 -C ₈ cycloalkyl, 4-12 membered heterocycloalkyl, amino, mono-C1-C ₆ alkylamino or di-C ₁ -C ₆ αalkylamino, and RS6 is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, COOH, C(O)O-C ₁ -C ₆ alkyl, cyano, C1-C ₆ alkoxyl, amino, mono-C1-C ₆ alkylamino and di-C1-C ₆ alkylamino; or R ₇ and R ₈ , together with the N atom to which they are attached, form a 4-11 membered heterocycloalkyl ring with or without 2 additional heteroatoms, and wherein the 4-11 membered heterocycloalkyl ring formed by R ₇ and R ₈ , optionally substituted by one or more -Q ₆ -T ₆ , where Q ₆ represents a bond, C(O), C(O)NRm, NRmC(O), S(O) ₂ or C1-C ₃ alkyl as a linker Rm is H or C1- _C6 alkyl, and _T6 is H, halogen, C1- _C6 alkyl, hydroxyl, cyano, C1- _C6 alkoxy, amino, mono-C1- _C6 alkylamino, di-C1 -C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12 membered heterocycloalkyl, 5- or 6-membered heteroaryl or S(O)pRp, in which p is 0, 1 or 2 and Rp is is C1- _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C8 cycloalkyl, _C6 - _C10 aryl, 4-12 membered heterocycloalkyl or 5- or 6-membered heteroaryl, and T ₆ is optionally substituted with one or more substituents selected from the group consisting of halogen, C1- _C6 alkyl, hydroxyl, cyano, C1- _C6 alkoxyl, amino, mono-C1- _C6 alkylamino, di-C1- _C6 alkylamino, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12 membered heterocycloalkyl and 5- or 6-membered heteroaryl, except when T ₆ represents H, halogen, hydroxyl or cyano; or -Q ₆ -T ₆ is oxo.

For example, R ₆ is C ₆ -C ₁₀ aryl or 5- or 6-membered heteroaryl, each of which is optionally independently substituted with one or more -Q ₂ -T ₂ where Q ₂ represents a bond or C ₁ -C ₃ alkyl as a linker, and T ₂ represents H, halogen, cyano, -ORa, -NRaRb, -(NR _a R _b R _c ) ⁺ A', -C(O)NR _a R _b , NR _b C(O) R _a , -S(O) ₂ R _a or R _S2 , in which Ra and R _b are each independently H or R _S3 , R _S2 and R _S3 are each independently C1-C ₆ alkyl, or Ra and Rb together with the N atom to which they are attached form a 4-7 membered heterocycloalkyl ring with or without 1 additional heteroatom, and each of _RS2 , _RS3 and the 4-7 membered heterocycloalkyl ring formed by R _a and R _b , optionally independently substituted by one or more -Q ₃ -T ₃ where Q ₃ represents a bond or C ₁ -C ₃ alkyl as a linker, and T ₃ is selected from the group consisting of halogen, C ₁ -C ₆ alkyl, 4 -7-membered heterocycloalkyl, ORd, -S(O) ₂ Rd and -NRdRe, wherein each of Rd and Re independently represents H or C1-C ₆ alkyl or -Q ₃ -T ₃ represents oxo; or any two adjacent -Q ₂ -T ₂ , together with the atoms to which they are attached, form a 5- or 6-membered ring, optionally containing 1-4 heteroatoms selected from N, O and S.

For example, the compound of the present invention is represented by formula (VI):

or is a pharmaceutically acceptable salt thereof, wherein Q ₂ is a bond or methyl as a linker, T ₂ is H, halogen, -ORa, -NRaRb, -(NR _a R _b R _c ) ⁺ A, or -S( O) ₂ NR _a R _b , R ₇ is piperidinyl, tetrahydropyran, cyclopentyl or cyclohexyl, each optionally substituted with one -Q ₅ -T ₅ , and R ₈ is ethyl.

The compound of the present invention may be characterized by the following formula (VIa):

Ra I

where each of R _a and R _b independently represents H or R _S3 , R _S3 represents C ₁

- 15 043327

C ₆ alkyl, C ₃ -C ₈ cycloalkyl, C6-C1oaryl, 4-12-membered heterocycloalkyl or 5- or 6-membered heteroaryl, or R _a and R _b together with the N atom to which they are attached form 4-12 -membered heterocycloalkyl ring with or without 1 additional heteroatom, and each of R _S3 and the 4 to 12 membered heterocycloalkyl ring formed by R _a and R _b is optionally substituted with one or more -Q ₃ -T ₃ , where Q ₃ is bond or C ₁ -C ₃ alkyl as a linker, each optionally substituted with halogen, cyano, hydroxyl or C ₁ -C ₆ alkoxy, and T ₃ selected from the group consisting of halogen, cyano, C 1 -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-12-membered heterocycloalkyl, 5- or 6-membered heteroaryl, OR _d , COOR _d , S(O) ₂ Rd, -NRdRe and -C(O)NR _d R _e , wherein each of R _d and R _e independently represents H or C1-C ₆ alkyl, or -Q ₃ -T ₃ represents oxo;

R7 is -Q4-T4, in which _Q4 is a bond, a C1- _C4 alkyl as a linker, or a _C2 - _C4 alkenyl as a linker, each linker being optionally substituted with halogen, cyano, hydroxyl, or C1-C ₆ alkoxy, and T ₄ is H, halogen, cyano, NRfRg, -OR _f , -C(O)R _f , -C(O)OR _f , -C(O)NR _f Rg, C(O)NRfORg , -NRfC(O)Rg, -S(O)2R _f or RS4, in which Rf and Rg are each independently H or _RS5 , RS4 and _RS5 are each independently C1- _C6 alkyl, _C2 -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-7 membered heterocycloalkyl or 5- or 6-membered heteroaryl, and each of RS4 and _RS5 is optionally substituted with one or several -Q ₅ -T ₅ , where Q ₅ represents a bond, C(O), C(O)NR _k , NR _k C(O), S(O) ₂ or C1-C ₃ alkyl as a linker, R _k is H or C1- _C6 alkyl, and _T5 is H, halogen, C1- _C6 alkyl, hydroxyl, cyano, C1- _C6 alkoxy, amino, mono-C1- _C6 alkylamino, di-C1- C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-7 membered heterocycloalkyl, 5- or 6-membered heteroaryl or S(O) _q R _q in which q is 0, 1 or 2, and R _q represents C1- _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C3 - _C8 cycloalkyl, _C6 - _C10 aryl, 4-7 membered heterocycloalkyl or 5 or 6 member heteroaryl , and T ₅ is optionally substituted with one or more substituents selected from the group consisting of halogen, C1- _C6 alkyl, hydroxyl, cyano, C1- _C6 alkoxyl, amino, mono-C1- _C6 alkylamino, di-C1-C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C6-C10 aryl, 4-7 membered heterocycloalkyl and 5 or 6 membered heteroaryl, except when T ₅ represents H, halogen, hydroxyl or cyano; or -Q ₅ -T ₅ is oxo; with the proviso that R ₇ is not H; And

R ₈ is H, halogen, hydroxyl, COOH, cyano, _RS6 , OR _S6 or COOR _S6 , in which _RS6 is C1- _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, amino , mono-C1-C ₆ alkylamino or di-C1-C ₆ alkylamino, and R _S6 is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, COOH, C(O)O-C ₁ -C ₆ alkyl, cyano, C1-C ₆ alkoxyl, amino, mono-C1-C ₆ alkylamino and di-C1-C ₆ alkylamino; or R7 and _R8 together with the N atom to which they are attached form a 4-11 membered heterocycloalkyl ring which contains no or 2 additional heteroatoms and is optionally substituted by one or more -Q ₆ -T ₆ where Q ₆ is bond, C(O), C(O)NR _m , NR _m C(O), S(O) ₂ or C1-C ₃ alkyl as a linker, Rm is H or C1-C ₆ alkyl, and T ₆ represents H, halogen, C1- _C6 alkyl, hydroxyl, cyano, C1- _C6 alkoxyl, amino, mono-C1- _C6 alkylamino, di-C1- _C6 alkylamino, _C3 - _C8 cycloalkyl, _C6 - C ₁₀ aryl, 4-7 membered heterocycloalkyl, 5- or 6-membered heteroaryl or S(O)pRp, in which p is 0, 1 or 2, and Rp is C1- _C6 alkyl, _C2 - _C6 alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₀ aryl, 4-7 membered heterocycloalkyl or 5- or 6-membered heteroaryl, and T ₆ is optionally substituted with one or more substituents selected from the group consisting of halogen, C ₁ -C ₆ alkyl, hydroxyl, cyano, C1-C ₆ alkoxyl, amino, mono-C1-C ₆ alkylamino, di-C1-C ₆ alkylamino, C ₃ -C ₈ cycloalkyl, C6-C10aryl, 4- 7-membered heterocycloalkyl and 5- or 6-membered heteroaryl, except when T ₆ represents H, halogen, hydroxyl or cyano; or -Q ₆ -T ₆ is oxo.

For example, R _a and R _b , together with the N atom to which they are attached, form a 4-7 membered heterocycloalkyl ring with or without 1 additional heteroatom at the N atom, and the ring is optionally substituted with one or more -Q3-T3 where heterocycloalkyl is azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl or morpholinyl.

For example, R ₇ is C ₃ -C ₈ cycloalkyl or 4-7 membered heterocycloalkyl, each optionally substituted with one or more -Q ₅ -T ₅ .

For example, R ₇ is piperidinyl, tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl or cycloheptyl, each optionally substituted with one or more -Q ₅ -T ₅ .

For example, R ₈ is H or C1- _C6 alkyl, which is optionally substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, COOH, C(O)OC1- _C6 alkyl, cyano, C1- _C6 alkoxyl, amino, mono-C1-C ₆ alkylamino and di-C1-C ₆ alkylamino.

In embodiments, a compound that can be used in any of the methods provided herein is:

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(compound A, also referred to herein as E7438 or EPZ-6438) or a pharmaceutically acceptable salt thereof.

In embodiments, a compound that can be used in any of the methods provided herein is:

and pharmaceutically acceptable salts thereof.

Alternatively, the EZH2 inhibitor is selected from the group consisting of compounds B and C, stereoisomers thereof, and pharmaceutically acceptable salts thereof.

In embodiments, a compound that can be used in any of the methods presented herein is compound F

or pharmaceutically acceptable salts thereof.

In embodiments, compounds suitable for use in the method of the present invention include compounds of formula (VII)

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(VII) wherein V ¹ is N or CR ⁷ , V ² is N or CR ² , provided that if V ¹ is N, V ² is N, X and Z are independently selected from the group consisting of from hydrogen, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, unsubstituted or substituted (C3-C8)cycloalkyl, unsubstituted or substituted (C3-C8)cycloalkyl-(C1-C8)alkyl or -(C2C8)alkenyl, unsubstituted or substituted (C5-C8)cycloalkenyl, unsubstituted or substituted (C5-C8)cycloalkenyl-(C1-C8)alkyl or -(C2-C8)alkenyl, (C6-C10)bicycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted heterocycloalkyl-(C1-C8)alkyl or -(C2-C8)alkenyl, unsubstituted or substituted aryl, unsubstituted or substituted aryl-(C1-C8)alkyl or -(C2-C8)alkenyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted heteroaryl-(C1-C8)alkyl or -(C2-C8)alkenyl, halogen, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -CONR ^a NR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O )NR ^a R ^b , -NR ^a NR ^a C(O)ORa, -OR ^a , -OC(O)R ^a and -OC (O)NR ^a R ^b ;

Y represents H or halogen;

R ¹ represents (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )kynyl, unsubstituted or substituted (C ₃ -C ₈ )cycloalkyl, unsubstituted or substituted (C ₃ - C ₈ )cycloalkyl-(C ₁ -C ₈ )alkyl or -(C ₂ -C ₈ )alkenyl, unsubstituted or substituted (C ₅ -C ₈ )cycloalkenyl, unsubstituted or substituted (C ₅ -C ₈ )cycloalkenyl-(C ₁ -C ₈ )alkyl or -(C ₂ -C ₈ )alkenyl, unsubstituted or substituted (C ₆ -C ₁₀ )bicycloalkyl, unsubstituted or substituted heterocycloalkyl or -(C ₂ -C ₈ )alkenyl, unsubstituted or substituted heterocycloalkyl-( C ₁ -C ₈ )alkyl, unsubstituted or substituted aryl, unsubstituted or substituted aryl-(C ₁ -C ₈ )alkyl or -(C ₂ -C ₈ )alkenyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted heteroaryl-(C ₁ -C ₈ )alkyl or -(C ₂ -C ₈ )alkenyl, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -CONR ^a NR ^a R ^b ;

R ² represents hydrogen, (C ₁ -C ₈ )alkyl, trifluoromethyl, alkoxy or halogen, wherein said (C ₁ -C ₈ )alkyl is optionally substituted by one or two groups selected from amino and (C ₁ -C ₃ )alkylamino ;

R ⁷ represents hydrogen, (C ₁ -C ₃ )alkyl or alkoxy;

R ³ is hydrogen, (C ₁ -C ₈ )alkyl, cyano, trifluoromethyl, -NR ^a R ^b or halogen;

R ⁶ is selected from the group consisting of hydrogen, halogen, (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, unsubstituted or substituted (C ₃ -C ₈ )cycloalkyl , unsubstituted or substituted (C ₃ -C ₈ )cycloalkyl-(C ₁ -C ₈ )alkyl, unsubstituted or substituted (C ₅ -C ₈ )cycloalkenyl, unsubstituted or substituted (C ₅ -C ₈ )cycloalkenyl-(C ₁ - C ₈ )alkyl, (C ₆ -C ₁₀ )bicycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted heterocycloalkyl-(C ₁ -C ₈ )alkyl, unsubstituted or substituted aryl, unsubstituted or substituted aryl-(C ₁ -C ₈ ) alkyl, unsubstituted or substituted heteroaryl, unsubstituted or substituted heteroaryl-(C ₁ -C ₈ )alkyl, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -CONR ^a NR ^a R ^b , -SR ^a , - SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O) OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O)NR ^a R ^b , NR ^a NR ^a C(O)OR ^a , -OR ^a , -OC(O)R ^a , -OC(O)NR ^a R ^b ;

where any (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, cycloalkyl, cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with 1, 2 or 3 groups, independently selected from the group consisting of -O(C ₁ -C ₆ )alkyl(R ^c )1_2, -S(C1-C6)alkyl(R ^c )1_ ₂ , -(C ₁ -C ₆ )alkyl(R ^c )1_2, -(C1-C8)alkyl-heterocycloalkyl, (C3-C8)cycloalkylheterocycloalkyl, halogen, (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (C1-C6)haloalkyl, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a , OC (O)NR ^a R ^b , heterocycloalkyl, aryl, heteroaryl, aryl(C1- _C4 )alkyl and heteroaryl( _C1 - _C4 )alkyl;

wherein any aryl or heteroaryl moiety of said aryl, heteroaryl, aryl(C ₁ -C ₄ )alkyl or heteroaryl(C ₁ -C ₄ )alkyl is optionally substituted with 1, 2 or 3 groups independently selected from the group consisting of halogen, (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₅ -C ₈ )cycloalkenyl, (C ₁ -C ₆ )haloalkyl, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO ₂ NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , NR ^a C(O)NR ^a R ^b , - NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a and

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-OC(O)NR ^a R ^b ;

R ^a and R ^b are each independently hydrogen, (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, (C ₃ -C ₈ )cycloalkyl, (C ₅ -C ₈ )cycloalkenyl, (C ₆ -C ₁₀ )bicycloalkyl, heterocycloalkyl, aryl or heteroaryl, where the specified (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl , cycloalkyl, cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with 1, 2 or 3 groups independently selected from halogen, hydroxyl, (C ₁ -C ₄ )alkoxy, amino, (C ₁ -C ₄ )alkylamino, ( (C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl)amino, -CO ₂ H, -CO ₂ (QC ₄ )alkyl, -CONH2, -CONH(C ₁ -C ₄ )alkyl, - CoN((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl), -SO ₂ (C ₁ -C ₄ )alkyl, SO2NH2, -SO ₂ NH(C ₁ -C ₄ )alkyl and SO ₂ N((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl);

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 5-8 membered saturated or unsaturated ring, optionally containing an additional heteroatom selected from oxygen, nitrogen and sulfur, wherein said ring is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₄ )alkyl, (Ci-C ₄ )haloalkyl, amino, (C ₁ -C ₄ )alkylamino, ((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl)amino, hydroxyl, oxo, (C ₁ -C ₄ )alkoxy and (C ₁ -C ₄ )alkoxy (C ₁ -C ₄ )alkyl, wherein said ring is optionally fused with (C ₃ -C ₈ ) a cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring;

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 6-10 membered bridged bicyclic ring system, optionally fused to a (C ₃ C ₈ )cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring;

each R ^c is independently (C1-C4)alkylamino, -NR ^a SO2R ^b , -SOR ^a , -SO ₂ R ^a , -NR ^a C(O)OR ^a , -NR ^a R ^b , or -CO2R ^a ;

or its salt.

The subgroups of compounds covered by the general structure of formula (I) are as follows.

Subgroup A of formula (VII) X and Z are selected from the group consisting of (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -NR ^a R ^b and -OR ^a ;

Y is H or F;

R ¹ is selected from the group consisting of (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

R ² represents hydrogen, (C ₁ -C ₈ )alkyl, trifluoromethyl, alkoxy or halogen, wherein said (C ₁ -C ₈ )alkyl is optionally substituted by one or two groups selected from amino and (C ₁ -C ₃ )alkylamino ;

R ⁷ represents hydrogen, (C ₁ -C ₃ )alkyl or alkoxy;

R ³ is selected from the group consisting of hydrogen, (C ₁ -C ₈ )alkyl, cyano, trifluoromethyl, -NR ^a R ^b and halogen;

R ⁶ is selected from the group consisting of hydrogen, halogen, cyano, trifluoromethyl, amino, (C1-C8)alkyl, (C3-C8)cycloalkyl, aryl, heteroaryl, acylamino, (C2-C8)alkynyl, arylalkynyl, heteroarylalkynyl, - SO2R ^a , -SO2NR ^a R ^b and -NR ^a SO ₂ R ^b ;

where any (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₂ -C ₈ )alkynyl, arylalkynyl, heteroarylalkynyl group is optionally substituted with 1, 2 or 3 groups independently selected from -O(C ₁ -C ₆ )alkyl(R ^c )1.2, -S(C1-C6)alkyl(R ^c )1. ₂ , -(C ₁ -C ₆ )alkyl(R ^c ) ₁ . ₂ , -(C ₁ -C ₈ )alkyl-heterocycloalkyl, (C ₃ -C ₈ )cycloalkyl-heterocycloalkyl, halogen, (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₅ -C ₈ )cycloalkenyl, (C ₁ -C ₆ )haloalkyl, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, - NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a , -OC(O)NR ^a R ^b , heterocycloalkyl, aryl, heteroaryl, aryl(C ₁ -C ₄ )alkyl and heteroaryl (C ₁ C ₄ )alkyl;

R ^a and R ^b are each independently hydrogen, (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C2-C8)alkynyl, (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl , (C6-C10)bicycloalkyl, heterocycloalkyl, aryl or heteroaryl, where the specified (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, cycloalkyl, cycloalkenyl, bicycloalkyl, heterocycloalkyl , the aryl or heteroaryl group is optionally substituted with 1, 2 or 3 groups independently selected from halogen, hydroxyl, (C ₁ -C ₄ )alkoxy, amino, (C ₁ -C ₄ )alkylamino, ((C ₁ -C ₄ )alkyl )((C ₁ -C ₄ )alkyl) amino, -CO ₂ H, -CO ₂ (C ₁ C ₄ )alkyl, -CONH2, -CONH(C ₁ -C ₄ )alkyl, -CON((C ₁ - C ₄ )alkyl)((C ₁ -C ₄ )alkyl), -SO ₂ (C ₁ -C ₄ )alkyl, SO2NH2, -SO ₂ NH(C ₁ -C ₄ )alkyl and -SO ₂ N((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl);

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 5-8 membered saturated or unsaturated ring, optionally containing an additional heteroatom selected from oxygen, nitrogen and sulfur, wherein said ring is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )haloalkyl, amino, (C ₁ -C ₄ )alkylamino, ((C ₁ -C ₄ )alkyl)((C ₁ - C ₄ )alkyl)amino, hydroxyl, oxo, (C ₁ -C ₄ )alkoxy and (C ₁ -C ₄ )alkoxy (C ₁ -C ₄ )alkyl, wherein said ring is optionally fused with a (C3-C8)cycloalkyl , heterocycloalkyl, aryl or heteroaryl ring;

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 6-10-19 043327-membered bridged bicyclic ring system, optionally fused to a (C ₃ -C 8 )cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring. The aryl or heteroaryl group in this particular subgroup A is independently selected from the group consisting of furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, oxadiazole, thiadiazole, triazole, tetrazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine, quinoline, cinnoline, quinazoline, quinoxaline and naphthyridine or other aryl or heteroaryl group such as the following:

where in (1) A represents O, NH or S; B represents CH or N, and C represents hydrogen or C ₁ -C8 alkyl; or

where in (2) D represents N or C, optionally substituted with hydrogen or C1- _C8 alkyl;

or

where in (3) E represents NH or CH ₂ ; F represents O or CO; and G is NH or CH ₂ ; or

where in (4) J represents O, S or CO; or

where in (5) Q represents CH or N;

M represents CH or N, and

L/(5) represents hydrogen, halogen, amino, cyano, (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, -COR ^a , -CO2R ^a , -CONRaRb, CONR ^a NR ^a R ^b , -SO2R ^a , -SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a SO2R ^b , NR ^a SO ₂ NR ^a R ^b , -NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O)NR ^a R ^b or -OR ^a , where any (C ₁ -C ₈ )alkyl or (C ₃ -C ₈ )cycloalkyl group optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₅ -C ₈ )cycloalkenyl, (C1-C6)haloalkyl, cyano, -COR ^a , -CO2R ^a , CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R b , ^{-NR a} C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a and -OC(O) NR ^a R ^b ; where R ^a and R ^b are defined above; or

where in (6) L/(6) represents NH or CH ₂ ; or

where in 7 M/(7) represents hydrogen, halogen, amino, cyano, (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -CONR ^a NR ^a R ^b , -SO2R ^a , -SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , - NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O)NR ^a R ^b or -OR ^a , where any (C ₁ -C ₈ )αalkyl α, (C _{The 3} -C ₈ cycloalkyl or heterocycloalkyl group is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₅ -C ₈ )cycloalkenyl, (C ₁ -C ₆ )haloalkyl, cyano, -CORa, -CO ₂ R ^a , -CONR ^a R ^b , -SR ^a , -SOR ^a , -SO ₂ R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , NR ^a SO2NR ^a R ^b , -OR ^a ,

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-OC(O)R ^a and -OC(O)NR ^a R ^b ; where R ^a and R ^b are defined above; or

where in (8) P represents CH ₂ , NH, O or S; Q/(8) represents CH or N; and n is 0-2; or

R

where in (9) S/(9) and T/(9) are C, or S/(9) is C, and T/(9) is N, or S/(9) is N, and T/(9) represents C;

R is hydrogen, amino, methyl, trifluoromethyl or halogen;

U represents hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, (C ₁ -C ₈ )alkyl, (C3-C ₈ )cycloalkyl, -COR ^a , -CO2R ^a , CONR ^a R ^b , -SO2R ^a , - SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a SO2R ^b , -NR ^a SO ₂ NR ^a R ^b , -NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -OR ^a or 4-(1H-pyrazol-4-yl), wherein any (C ₁ -C ₈ )alkyl or (C ₃ -C ₈ )cycloalkyl group is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C5-C ₈ )cycloalkenyl, (C1-C6)haloalkyl, cyano, -COR ^a , -CO2R ^a , CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a and -OC(O)NR ^a R ^b ; where R ^a and R ^b are defined above.

Subgroup B of formula (VII) X and Z are independently selected from the group consisting of (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -NR ^a R ^b and -OR ^a ;

Y represents H;

R ¹ represents (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl or heterocycloalkyl;

R ² represents hydrogen, (C ₁ -C ₃ )alkyl or halogen, wherein said (C ₁ -C ₃ )alkyl is optionally substituted by one or two groups selected from amino and (C ₁ -C ₃ )alkylamino;

R ⁷ represents hydrogen, (C ₁ -C ₃ )alkyl or alkoxy;

R ³ represents hydrogen, (C ₁ -C ₈ )alkyl or halogen;

R ⁶ represents hydrogen, halogen, cyano, trifluoromethyl, amino, (C ₁ -C ₈ )alkyl, (C3-C8)cycloalkyl, aryl, heteroaryl, acylamino; (C2-C8)alkynyl, arylalkynyl, heteroarylalkynyl, -SO2R ^a , -SO2NR ^a R ^b or -NR ^a SO2R ^b ;

wherein any (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C ₂ -C ₈ )alkynyl, arylalkynyl or heteroarylalkynyl group is optionally substituted with 1, 2 or 3 groups independently selected from halogen, (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C5-C ₈ )cycloalkenyl, (C ₁ -C ₆ )haloalkyl, cyano, -COR ^a , -CO2R ^a , CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C( O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a , -OC(O)NR ^a R ^b , heterocycloalkyl, aryl, heteroaryl, aryl(C1 -C ₄ )alkyl and heteroaryl(C ₁ -C ₄ )alkyl;

R ^a and R ^b are each independently hydrogen, (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, (C ₃ -C ₈ )cycloalkyl, (C5 -C ₈ )cycloalkenyl, (C ₆ -C ₁₀ )bicycloalkyl, heterocycloalkyl, aryl or heteroaryl, where said (C ₁ -C ₈ )alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, a cycloalkyl, cycloalkenyl, bicycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with 1, 2 or 3 groups independently selected from halogen, hydroxyl, (C ₁ -C ₄ )alkoxy, amino, (C ₁ -C ₄ )alkylamino, (( C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl)amino, -CO ₂ H, -CO ₂ (C ₁ C ₄ )alkyl, -CONH2, -CONH(C ₁ -C ₄ )alkyl, -CON((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl), -SO ₂ (C ₁ -C ₄ )alkyl, SO ₂ NH ₂ , -SO ₂ NH(C ₁ -C ₄ )alkyl and -SO ₂ N((C ₁ -C ₄ )alkyl)((C ₁ -C ₄ )alkyl);

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 5-8 membered saturated or unsaturated ring, optionally containing an additional heteroatom selected from oxygen, nitrogen and sulfur, wherein said ring is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )haloalkyl, amino, (C ₁ -C ₄ )alkylamino, ((C ₁ -C ₄ )alkyl)((C ₁ - C ₄ )alkyl)amino, hydroxyl, oxo, (C ₁ -C ₄ )alkoxy and (C ₁ -C ₄ )alkoxy (C ₁ -C ₄ )alkyl, wherein said ring is optionally fused with (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring;

or R ^a and R ^b , taken together with the nitrogen atom to which they are attached, represent a 6-10 membered bridged bicyclic ring system, optionally fused to a (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring. Aryl and heteroaryl for the purposes of this definition are selected from the group consisting of furan, thiophene, pyrrole, oxazol, thiazole, imidazole, pyrazole, oxadiazole, thiadiazole, triazole, tetrazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, phenyl, pyridine , pyridazine, pyrimidine, pyrazine, triazine, tetrazine, quinoline, cinnoline, quinazoline, quinoxaline and naphthyridine, or a compound of another aryl or heteroaryl group, such as the following:

where in (1) A represents O, NH or S; B represents CH or N, and C represents hydrogen or C1- _C8 alkyl; or

where in (2) D represents N or C, optionally substituted with hydrogen or C1- _C8 alkyl; or

where in (3) E represents NH or CH2; F represents O or CO; and G represents NH or CH2; or

where in (4) J represents O, S or CO; or where in (5) Q represents CH or N;

M is CH or N, and L/(5) is hydrogen, halogen, amino, cyano, (C1-C8)alkyl, (C3-C8)cycloalkyl, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -CONR ^a NR ^a R ^b , -SO2R ^a , -SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , - NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O)NR ^a R ^b or -OR ^a , where any (C1-C8)alkyl, (C3-C8 )cycloalkyl, the group is optionally substituted with 1, 2 or 3 groups independently selected from (C1-C6)alkyl, (C3-C8)cycloalkyl, (C5-C8)cycloalkenyl, (C1C6)haloalkyl, cyano, -COR ^a , -CO2R ^a , CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O) NR ^a R ^b , -NR ^a C(O)OR ^a , NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a and -OC (O)NR ^a R ^b , where R ^a and R ^b are defined above; or

where in (6) L/(6) represents NH or CH2; or

where in (7) M/(7) represents hydrogen, halogen, amino, cyano, (C ₁ -C ₈ )αalkyl, (C ₃ -C ₈ )cycloalkyl, heterocycloalkyl, -CORa, -CO ₂ R ^a , - CONRaRb, -CONR ^a NR ^a R ^b , -SO2R ^a , -SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , - NR ^a NR ^a R ^b , -NR ^a NR ^a C(O)R ^b , -NR ^a NR ^a C(O)NR ^a R ^b or -OR ^a , where any (C ₁ -C ₈ )alkyl, (C _{The 3} -C ₈ cycloalkyl, heterocycloalkyl group is optionally substituted with 1, 2 or 3 groups independently selected from (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C5 -C ₈ )cycloalkenyl, (C ₁ -C ₆ )haloalkyl, cyano, -COR ^a , -CO2R ^a , -CONR ^a R ^b , -SR ^a , -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC( O)R ^a , -OC(O)NR ^a R ^b ; where R ^a and R ^b are defined above; or

- 22 043327 where in (8) P represents CH ₂ , NH, O or S; Q/(8) represents CH or N; and n equals

0-2; or

where in (9) S/(9) and T/(9) are C, or S/(9) is C, and T/(9) is N, or S/(9) is N, and T/(9) represents C;

R represents hydrogen, amino, methyl, trifluoromethyl, halogen;

U represents hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, (C ₁ -C ₈ )alkyl, (C ₃ -C ₈ )cycloalkyl, -COR ^a , -CO2R ^a , CONRaRb, -SO2R ^a , -SO2NR ^a R ^b , -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a SO2R ^b , NR ^a SO ₂ NR ^a R ^b , -NR ^a NR ^a R ^b , -NR ^a NR ^a C(O )R ^b , -OR ^a or 4-(1H-pyrazol-4-yl), wherein any (C ₁ -C ₈ )alkyl or (C ₃ -C ₈ )cycloalkyl group is optionally substituted with 1, 2 or 3 groups, regardless selected from (C ₁ -C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, (C5-C ₈ )cycloalkenyl, (C1-C6)haloalkylα, cyano, -CORa, -CO^a, -CONRaRb, -SOR ^a , -SO2R ^a , -SO2NR ^a R ^b , nitro, -NR ^a R ^b , -NR ^a C(O)R ^b , -NR ^a C(O)NR ^a R ^b , -NR ^a C(O)OR ^a , -NR ^a SO2R ^b , -NR ^a SO2NR ^a R ^b , -OR ^a , -OC(O)R ^a and -OC(O)NR ^a R ^b , where Ra and Rb are defined above.

In embodiments, the EZH2 inhibitor is Compound G or GSK126

or stereoisomers thereof, or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, an EZH2 inhibitor that can be used in any of the methods provided herein is Compound H

or stereoisomers thereof, or pharmaceutically acceptable salts and solvates thereof.

In some embodiments, the EZH2 inhibitor, which can be used in any of the methods presented herein, is any of the Ga-Gc compounds

- 23 043327

or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

In some embodiments, an EZH2 inhibitor that can be used in any of the methods provided herein is CPI-1205 or GSK343.

In one embodiment, a compound disclosed herein (eg, an EZH2 inhibitor) is a compound itself, i.e. free base or naked molecule. In another embodiment, the compound is a salt thereof, for example, a mono-HCl or tri-HCl salt, a mono-HBr or tri-HBr salt of the bare molecule.

The compounds described herein can be synthesized according to any method known in the art. For example, compounds characterized by formula (VII) can be synthesized according to the method described in WO 2011/140325; WO 2011/140324 and WO 2012/005805, each of which is incorporated herein by reference in its entirety.

As used herein, alkyl, C1, _C2 , _C3 , _C4 , _C5 or C6alkyl or C1-C6alkyl is intended to include straight chain (linear) saturated aliphatic hydrocarbon groups of C1, _C2 , _C3 , _C4 , C ₅ or C6 atoms and branched saturated aliphatic hydrocarbon groups of C ₃ , C ₄ , C ₅ or C6 atoms. For example, C1-C6alkyl is intended to include C1, _C2 , _C3 , _C4 , _C5 and C6alkyl groups. Examples of alkyl include moieties containing one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, npentyl, s-pentyl, or n-hexyl.

In embodiments, the straight or branched chain alkyl contains six or fewer carbon atoms (e.g., C1-C6 in the case of a straight chain, C ₃ -C ₆ in the case of a branched chain), and in another embodiment, the straight or branched chain alkyl contains four or fewer carbon atoms.

As used herein, the term cycloalkyl refers to a saturated or unsaturated non-aromatic hydrocarbon single or multi-ring system (eg, fused, bridged or spiro ring) with 3-30 carbon atoms (eg, C ₃ -C ₁₀ ). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl. The term heterocycloalkyl refers to a saturated or unsaturated non-aromatic 3-8 membered monocyclic, 7-12 membered bicyclic (fused, bridged or spiro ring) or 11-14 membered tricyclic ring system (fused, bridged or spiro ring) with one or more heteroatoms ( such as O, N, S or Se) unless otherwise noted. 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, oxetanyl, tietanyl, 1,2 ,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl, 2-oxa-5azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6- azaspiro[3.3]heptanyl, 2,6diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, etc.

The term optionally substituted alkyl refers to unsubstituted alkyl or alkyl with designated substituents replacing one or more hydrogen atoms on one or more carbon atoms of the hydrocarbon backbone. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl then, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfones up to , nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An arylalkyl or aralkyl moiety is an alkyl substituted with aryl (eg, phenylmethyl (benzyl)). An alkylaryl moiety is aryl substituted with alkyl (eg, methylphenyl).

The alkyl linker used herein is intended to include straight chain saturated divalent aliphatic hydrocarbon groups of C1, _C2 , _C3 , _C4 , _C5 or _C6 atoms and branched saturated aliphatic hydrocarbon groups of _C3 , C ₄ , C ₅ or C ₆ atoms. For example, the C1- _C6 alkyl linker is intended to include C1, _C2 , _C3 , _C4 ,

- 24 043327

C ₅ and C ₆ alkyl linker groups. Examples of alkyl as a linker include moieties with one to six carbon atoms, such as, but not limited to, methyl (-CH2-), ethyl (-CH2CH2-), n-propyl (-CH2CH2CH2-), isopropyl (-CHCH3CH2-), n- butyl (-CH2CH2CH2CH2-), s-butyl (-CHCH ₃ CH ₂ CH ₂ -), isobutyl (-C(CH ₃ ) ₂ CH ₂ -), n-pentyl (-CH2CH2CH2CH2CH2-), s-pentyl (-CHCH ₃ CH ₂ CH ₂ CH ₂ -) or n-hexyl (-CH2CH2CH2CH2CH2CH2-).

Alkenyl includes unsaturated aliphatic groups similar in length and possible substitution to alkyls described above, but which contain at least one double bond. For example, the term alkenyl includes straight chain alkenyl groups (eg, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl) and branched alkenyl groups. In embodiments, a straight or branched chain alkenyl group contains six or fewer carbon atoms in its backbone (eg, C ₂ -C ₆ in the case of a straight chain, C ₃ C ₆ in the case of a branched chain). The term C ₂ -C ₆ includes alkenyl groups containing two to six carbon atoms. The term C ₃ -C ₆ includes alkenyl groups containing three to six carbon atoms.

The term optionally substituted alkenyl refers to unsubstituted alkenyl or alkenyl with designated substituents replacing one or more hydrogen atoms on one or more carbon atoms of the hydrocarbon backbone. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl then, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfones up to , nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Alkynyl includes unsaturated aliphatic groups similar in length and possible substitution to alkyls described above, but which contain at least one triple bond. For example, alkynyl includes straight chain alkynyl groups (eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, nonynyl, decynyl) and branched alkynyl groups. In embodiments, a straight or branched chain alkynyl group contains six or fewer carbon atoms in its backbone (eg, C ₂ -C ₆ in the case of a straight chain, C ₃ -C ₆ in the case of a branched chain). The term C ₂ -C ₆ includes alkynyl groups containing two to six carbon atoms. The term C ₃ -C ₆ includes alkynyl groups containing three to six carbon atoms.

The term optionally substituted alkynyl refers to unsubstituted alkynyl or alkynyl with designated substituents replacing one or more hydrogen atoms on one or more carbon atoms of the hydrocarbon backbone. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonyl then, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfones up to , nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl) include both unsubstituted moieties and moieties with one or more designated substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6tetramethylpiperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

Aryl includes groups with a certain degree of aromatization, including conjugated or multicyclic systems with at least one aromatic ring, and not containing any heteroatom in the ring structure. Examples include phenyl, benzyl, 1,2,3,4tetrahydronaphthalenyl and the like.

Heteroaryl groups are aryl groups as defined above except those containing one to four heteroatoms in the ring structure, and may also be referred to as aryl heterocycles or heteroaromatic compounds. As used herein, the term heteroaryl is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic aromatic heterocyclic ring that consists of carbon atoms and one or more heteroatoms, for example 1, or 1-2, or 1-3, or 1-4, or 1-5, or 1-6 heteroatoms, or, for example, 1, 2, 3, 4, 5 or 6 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (ie N or NR, where R represents H or other substituents, as defined). Heteroatoms,

- 25 043327 representing nitrogen and sulfur, may optionally be oxidized (that is, N^O and S(O)p, where p = 1 or 2). It should be noted that the total number of S and O atoms in an aromatic heterocycle is no more than 1.

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

In addition, the terms aryl and heteroaryl include multicyclic aryl and heteroaryl groups, e.g. tricyclic, bicyclic, e.g. naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

In the case of multicyclic aromatic rings, only one of the rings is required to be aromatic (eg, 2,3-dihydroindole), although all of the rings may be aromatic (eg, quinoline). The second ring can also be fused or bridged.

A cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring may be substituted at one or more positions on the ring (e.g., a ring-forming carbon atom or a heteroatom such as N) with substituents such 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, aminocarbonyl, alk ylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido , heterocyclyl, alkylaryl or an aromatic or heteroaromatic moiety. Aryl and heteroaryl groups can be fused to or bridged with alicyclic or heterocyclic rings that are non-aromatic to form a multicyclic system (eg, tetralin, methylenedioxyphenyl).

As used herein, a carbocycle or carbocyclic ring is intended to include any stable monocyclic, bicyclic or tricyclic ring with a specified number of carbon atoms, each of which may be saturated, unsaturated or aromatic. The carbocycle includes cycloalkyl and aryl. For example, the C ₃ C ₁₄ carbocycle is intended to include a monocyclic, bicyclic or tricyclic ring 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, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, and tetrahydronaphthyl. Bridged rings are also included in the definition of carbocycle, including, for example, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane, and [2.2.2]bicyclooctane. A bridging ring occurs when one or more carbon atoms link two carbon atoms that are not adjacent. In one embodiment, the bridging rings represent one or two carbon atoms. It should be noted that a bridge always converts a monocyclic ring to a tricyclic ring. If the ring is bridged, the substituents listed for the ring may also be present in the bridge. Fused (eg naphthyl, tetrahydronaphthyl) rings and spiro rings are also included.

As used herein, a heterocycle or heterocyclic group includes any ring structure (saturated, unsaturated, or aromatic) that contains at least one heteroatom in the ring (eg, N, O, or S). The heterocycle includes heterocycloalkyl and heteroaryl. Examples of heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine and tetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl , chromanil, chromenil, cinnolinil, decahydroquinolinyl, 2H, 6H-1,5,2dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isathionyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-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, phenoxatinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4piperidonyl , piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl ,

- 26 043327 quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1 ,3,4thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4 -triazolyl and xanthenyl.

As used herein, the term substituted means that any one or more hydrogen atoms on the designated atom are replaced by one selected from the indicated groups, provided that the normal valence of the designated atom is not exceeded and that the substitution results in a stable compound. If the substituent is oxo or keto (i.e. =O), then 2 hydrogen atoms are replaced per atom. Keto substituents are absent on aromatic moieties. Ring double bonds as used herein are double bonds that form between two adjacent ring atoms (eg, C=C, C=N, or N=N). A stable compound and a stable structure are meant to mean a compound that is sufficiently stable to withstand isolation to a suitable degree of purity from the reaction mixture and formulation into an effective therapeutic agent.

If the bond to a substituent is shown to cross a bond connecting two atoms in the ring, then that substituent can be bonded to any atom in the ring. When a substituent is listed without specifying the atom through which such substituent is attached to the rest of the compound of a given formula, then such substituent may be linked through any atom in such formula. Combinations of substituents or variables (or both) are permitted, but only if such combinations result in stable compounds.

When any variable (such as R1) appears more than once in any constituent or formula of a compound, its definition in each occurrence is independent of its definition in each other occurrence. Thus, for example, if a group is shown to be substituted by 0-2 R1 moieties, then the group may optionally be substituted by up to two R1 moieties, and R1 is selected in each case regardless of the definition of R1. Combinations of substituents or variables (or both) are also permitted, but only if such combinations result in stable compounds.

The term hydroxy or hydroxyl includes groups with -OH or ^-O- .

As used herein, halogen or halide refers to fluorine, chlorine, bromine and iodine. The term perhalogenated generally refers to a moiety in which all of the hydrogen atoms are replaced by halogen atoms. The term haloalkyl or haloalkoxy refers to alkyl or alkoxy substituted with one or more halogen atoms.

The term carbonyl includes compounds and moieties that contain a carbon atom attached by 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, etc.

The term carboxyl refers to -COOH or its C1-C6 alkyl ester.

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

Aroyl includes moieties with an aryl or heteroaromatic moiety attached to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthylcarboxy, etc.

Alkoxyalkyl, alkylaminoalkyl and thioalkoxyalkyl include alkyl groups described above in which one or more carbon atoms of the hydrocarbon skeleton are replaced by oxygen, nitrogen or sulfur atoms.

The term alkoxy or alkoxyl includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently bonded to an oxygen atom. Examples of alkoxy groups or alkoxy 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 may be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonato, phosphinato, amino ( including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido , nitro, trifluoromethyl, cyano,

- 27 043327 azido, heterocyclyl, alkylaryl or aromatic or heteroaromatic fragments. Examples of halogen substituted with alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

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

The term ester includes compounds or moieties that contain a carbon atom or heteroatom bonded to an oxygen atom that is bonded to a carbon atom of a carbonyl group. The term ester includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.

The term thioalkyl includes compounds or moieties that contain an alkyl group attached via a sulfur atom. Thioalkyl groups may be substituted with groups such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl yl, alkoxyl, amino (in including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro , trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl or aromatic or heteroaromatic moieties.

The term thiocarbonyl or thiocarboxy includes compounds and moieties that contain a carbon atom attached by a double bond to a sulfur atom.

The term thioethers includes moieties that contain a sulfur atom bonded to two carbon atoms or heteroatoms. Examples of thioethers include, but are not limited to, alkthioalkyls, alkthioalkenyls, and alkthioalkynyls. The term alkthioalkyl includes moieties with an alkyl, alkenyl or alkynyl group bonded to a sulfur atom that is bonded to the alkyl group. Likewise, the term alkthioalkenyls refers to moieties in which an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom that is covalently bonded to the alkenyl group; and alkthioalkynyls refers to moieties in which an alkyl, alkenyl or alkynyl group is linked to a sulfur atom that is covalently bonded to the alkynyl group.

As used herein, amine or amino refers to unsubstituted or substituted -NH2. Alkylamino includes groups of compounds in which the nitrogen atom -NH2 is bonded to at least one alkyl group. Examples of alkylamino groups include benzylamino, methylamino, ethylamino, phenethylamino, etc. Dialkylamino includes groups in which the nitrogen atom -NH2 is bonded 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 atom is bonded to at least one or two aryl groups, respectively. Aminoaryl and aminoaryloxy refer to aryl and aryloxy substituted with amino.

Alkylarylamino, alkylaminoaryl or arylaminoalkyl refers to an amino group that is bonded to at least one alkyl group and at least one aryl group. Alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bonded to a nitrogen atom that is also bonded to the alkyl group. Acylamino includes groups in which a nitrogen atom is bonded to an acyl group. Examples of acylamino include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido groups.

The term amide or aminocarboxy includes compounds or moieties that contain a nitrogen atom that is bonded to a carbon atom of a carbonyl or thiocarbonyl group. The term includes alkaminocarboxyl groups, which include alkyl, alkenyl or alkynyl groups bonded to an amino group that is bonded to the carbon atom of the carbonyl or thiocarbonyl group. It also includes arylaminocarboxyl groups, which include aryl or heteroaryl moieties linked to an amino group that is linked to the carbon atom of the carbonyl or thiocarbonyl group. The terms alkylaminocarboxy, alkenylaminocarboxy, alkynylaminocarboxy and arylaminocarboxy include those moieties in which the alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bonded to a nitrogen atom, which in turn is bonded to a carbon atom of the carbonyl group. Amides may be substituted with substituents such as straight chain alkyl, branched alkyl, cycloalkyl, aryl, heteroaryl or heterocycle. Substituents on amide groups can be further substituted.

In the present specification, in some cases, for convenience, the structural formula of a compound is a specific isomer, but the present invention includes all isomers, such as geometric isomers, optical isomers based on an asymmetric carbon atom, stereoisomers, tautomers, and the like, it is understood that that not all isomers can be characterized by the same level of activity. In addition, crystal polymorphism may be present in the compounds represented by the formula. It should be noted that any crystalline form, mixture of crystalline forms, or anhydride or hydrate thereof is included within the scope of the present invention.

Isomerism means compounds that have the same molecular formula but differ in the bonding sequence of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of atoms in space are called stereoisomers. Stereoisomers that are characterized by non-mirror images of each other are called diastereoisomers, and stereoisomers that are characterized by non-overlapping mirror images of each other are called enantiomers or sometimes optical isomers. A mixture containing equal amounts of forms of individual enantiomers with opposite chirality is called a racemic mixture.

A carbon atom bonded to four unequal substituents is called a chiral center.

Chiral isomer means a compound with at least one chiral center. Compounds that have more than one chiral center may exist, either as a single diastereomer or as a mixture of diastereomers, which is called a diastereomeric mixture. When a single chiral center is present, the stereoisomer can be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the spatial arrangement of substituents attached to the chiral center. The substituents attached to the chiral center in question are arranged according to the Cahn, Ingold and Prelog sequence rule. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 ( London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

Geometric isomer refers to diastereomers that owe their existence to hindered rotation around double bonds or a cycloalkyl linker (eg 1,3cyclobutyl). These configurations are distinguished in their names by the prefixes cis and trans or Z- and E, which indicate that the groups are on one side or the other of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.

It should be understood that the compounds disclosed in this invention may be represented as various chiral isomers or geometric isomers. It should also be understood that where compounds have chiral isomeric or geometric isomeric forms, all isomeric forms are intended to be included within the scope of the present invention, and the names of the compounds do not exclude any isomeric forms, it being understood that not all isomers may have the same level of activity.

Moreover, the structures and other compounds discussed herein include all of their atropic isomers, it being understood that not all atropic isomers may have the same level of activity. Atropoisomers are a type of stereoisomer in which the atoms of the two isomers are arranged differently in space. Atropoisomers owe their existence to limited rotation caused by the slowing of rotation of large groups around a central bond. Such atropisomers usually exist as a mixture, but recent advances in chromatographic techniques have made it possible in some cases to separate mixtures of two atropisomers.

A tautomer is one or more structural isomers that exist in equilibrium and easily transform from one isomeric form to another. This transformation results in the formal migration of a hydrogen atom, accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerization is possible, chemical equilibrium of the tautomers was achieved. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The model in which tautomers are interconvertible upon tautomerization is called tautomerism.

Of the different possible types of tautomerism, two are usually observed. In keto-enol tautomerism, a simultaneous displacement of electrons and a hydrogen atom occurs. Ring-chain tautomerism occurs when the aldehyde group (-CHO) on a chain sugar molecule reacts with one of the hydroxyl groups (-OH) on the same molecule to give it the cyclic (ring-shaped) shape that characterizes glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide-imide tautomerism in heterocyclic rings (for example, in nucleic bases such as guanine, thymine and cytosine), imine-enamine and enamine-enamine. An example of a keto-enol equilibrium between pyridin-2(1H)-ones and the corresponding pyridin-2-ols is shown below.

oh he

pyridin-2(1/-/)-one pyridin-2-ol

- 29 043327

In the connections described in this document,

ABOUT

in each case of its use should be interpreted as

It should be understood that the compounds of the present invention may be shown as various tautomers. It should also be understood that if compounds have tautomeric forms, then all tautomeric forms are included within the scope of the present invention, and the names of the compounds do not exclude any tautomeric form. Certain tautomers, as will be appreciated, may exhibit higher levels of activity than others.

The term crystalline polymorphs, polymorphs or crystalline forms refers to crystal structures in which a compound (or its salt or solvate) can crystallize to form different types of crystal lattice, each having the same elemental composition. Different crystalline forms are typically characterized by different X-ray diffraction patterns, different infrared spectrum, melting point values, density values, crystal shapes, optical and electrical properties, stability and solubility. Solvent recrystallization, crystallization rate, storage temperature, and other factors may cause the predominance of one crystalline form. Crystalline polymorphs of compounds can be obtained by crystallization under different conditions.

Compounds of any formula disclosed herein include the compounds themselves, as well as their salts and solvates thereof, as applicable. A salt, for example, can be formed between an anion and a positively charged group (eg, an amino group) on an 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, naphthalene sulfonate and acetate (eg trifluoroacetate). The term pharmaceutically acceptable anion refers to an anion suitable to form a pharmaceutically acceptable salt. Likewise, a salt can also be formed between a cation and a negatively charged group (eg, a carboxylate) on an aryl or heteroaryl-substituted benzene compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, as well as ammonium cation such as tetramethylammonium ion. Aryl- or heteroaryl-substituted benzene compounds also include salts containing quaternary nitrogen atoms.

In addition, the compounds of the present invention, such as salts of the compounds, may exist in either hydrated or non-hydrated (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.

Solvate means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds tend to hold a fixed molar ratio of solvent molecules in a crystalline solid state, thus forming a solvate. If the solvent is water, the solvate is a hydrate; and if the solvent is an alcohol, the solvate formed is an alcoholate. Hydrates are formed by combining one or more molecules of water with one molecule of a substance in which water retains its molecular state as H2O.

The term bioisostere refers to a compound resulting from the exchange of an atom or groups of atoms for another, generally similar, atom or group of atoms. The goal of bioisosteric substitution is to create a new compound with biological properties similar to the original compound. Bioisosteric substitution can be physicochemical or topological. Examples of carboxylic acid bioisosteres include, but are not limited to, acyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, for example, Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.

The present invention includes all isotopes of atoms found in the present compounds. Isotopes include those atoms that have 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.

The present invention provides methods for synthesizing compounds of any formula disclosed herein. The present invention also provides in detail methods for synthesizing various disclosed compounds of the present invention in accordance with the following schemes shown in the examples.

Throughout this specification, when compositions are described as having, including, or with

- 30 043327 containing specific components, it is assumed that these compositions also consist essentially of, or consist of, the specified components. Likewise, when methods or methods are described as being characterized by, including or involving specific method steps, the methods also substantially include or involve the listed method steps. In addition, it should be understood that the order of the steps or the order in which certain actions are carried out does not matter, provided that the present invention remains executable. Moreover, two or more stages or actions can be carried out simultaneously.

A wide range of functional groups can be tolerated in the synthesis methods of the present invention, and therefore a variety of substituted starting materials can be used. In the methods, the desired final compound is generally obtained at or near the end of the overall process, although in some cases additional conversion of the compound to its pharmaceutically acceptable salt, solvate or polymorph may be required.

The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates using standard synthetic methods and procedures that are either known to those skilled in the art or will be apparent to those skilled in the art. in light of the ideas presented in this document. Standard synthetic methods and procedures for preparing organic molecules, as well as methods for converting and manipulating functional groups, can be found in the relevant scientific literature or standard textbooks in the field. Although not limited to one or more sources, classic texts such as Smith, M.W., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, ^5th edition, John Wiley & Sons: New York, 2001; Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994) and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), incorporated herein by reference, are suitable and generally accepted references for organic synthesis known to those skilled in the art. The following descriptions of synthetic methods are intended to illustrate, and not to limit, general methods for preparing the compounds of the present invention.

The compounds of the present invention can be easily prepared using various methods known to those skilled in the art. The compounds of the present invention with any formula disclosed herein can be prepared in accordance with the procedures illustrated in Schemes 1-10 below from commercially available starting materials or starting materials that can be prepared using procedures known in the literature. The Z and R groups (such as R ₂ , R ₃ , R 4 , R ₆ , R ₇ , R ₈ and R ₁₂ ) in Schemes 1-10 are as defined in any formula disclosed herein unless otherwise noted .

One skilled in the art will appreciate that within the reaction sequences and synthesis schemes described herein, the order of certain steps may be changed, such as the introduction and removal of protecting groups.

One skilled in the art will appreciate that certain groups may require protection from reaction conditions through the use of protecting groups. Protecting groups can also be used to differentiate similar functional groups in molecules. A list of protecting groups and methods for introducing and removing these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

in the case of a hydroxyl fragment - TBS, benzyl, THP, Ac;

in the case of carboxylic acids - benzyl ester, methyl ester, ethyl ester, allyl ester;

in the case of amines - Cbz, BOC, DMB;

in the case of diols - Ac (x2) TBS (x2) or acetonides taken together;

in the case of thiols - Ac;

in the case of benzimidazoles - SEM, benzyl, RMB, DMB;

in the case of aldehydes, dialkyl acetals such as dimethoxyacetal or diethyl acetal.

In the reaction schemes described herein, multiple stereoisomers can be obtained. If a specific stereoisomer is not specified, this means that all possible stereoisomers that can be obtained from the reaction are meant. One skilled in the art will appreciate that reactions can be optimized to produce one preferred isomer, or new schemes can be developed to produce one isomer. If mixtures are obtained, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

The following abbreviations are used throughout this specification and are defined below:

Acetyl;

- 31 043327

AcOH acetic acid;

aq. water;

BID or b.i.d. twice a day (twice a day);

BOC tert-butoxycarbonyl;

Cbz benzyloxycarbonyl;

CDCl ₃ deuterated chloroform;

CH ₂ Cl ₂ dichloromethane;

DCM dichloromethane;

DMB 2,4-dimethoxybenzyl;

MF N,N-dimethylformamide;

DMSO dimethyl sulfoxide;

EA or EtOAc ethyl acetate;

EDC or EDCI N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide;

ESI-electrospray negative ion mode;

ESI+ electrospray positive ion mode;

EtOH ethanol;

h clock;

H ₂ O water;

HOBt 1 -hydroxybenzotriazole;

HCl hydrogen chloride or hydrochloric acid;

HPLC high performance liquid chromatography;

K ₂ CO ₃ potassium carbonate;

LC/MS or LC-MS liquid chromatography with mass spectrometry;

M molar;

MeCN acetonitrile;

min. minutes;

Na ₂ CO ₃ sodium carbonate;

Na ₂ SO ₄ sodium sulfate;

NaHCO ₃ sodium bicarbonate;

NaHMDS sodium hexamethyldisilazide;

NaOH sodium hydroxide;

NaHCO3 sodium bicarbonate;

Na2SO4 sodium sulfate;

NMR nuclear magnetic resonance;

Pd(OH)2 palladium dihydroxide;

RMV para-methoxybenzyl;

p.o. by mouth (oral administration);

ppm parts per million;

preparative HPLC preparative high performance liquid chromatography;

PYBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate;

Rt or RT room temperature;

TVME tert-butyl methyl ether;

TFA trifluoroacetic acid;

THF tetrahydrofuran;

THP tetrahydropyran.

An effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof is not significantly cytotoxic to normal cells. A therapeutically effective amount of a compound is not significantly cytotoxic to normal cells unless administration of the compound in a therapeutically effective amount induces cell death in more than 10% of normal cells. A therapeutically effective amount of a compound does not significantly affect the viability of normal cells unless administration of a therapeutically effective amount of the compound does not induce cell death in more than 10% of normal cells. In one aspect, cell death occurs as a result of apoptosis.

Contacting a cell with a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof can selectively induce or activate cell death in cancer cells. Administration to a subject in need of a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof can selectively induce or activate cell death in cancer cells. Contacting a cell with a compound of the present invention or a pharmaceutically acceptable salt, solvate or polymorph thereof can selectively induce cell death in one or more cells affected by the cell proliferation disorder. It is preferable to administer to a subject in need a compound of the present invention or a pharmaceutically acceptable salt, solvate thereof

- 32 043327 or polymorph selectively induces cell death in one or more cells affected by a disorder of cell proliferation.

One skilled in the art may refer to the general reference texts for detailed descriptions of known techniques discussed herein or similar techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual ( ^3rd edition), 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 edition (1990). Of course, these texts may also be referred to when making or using an aspect of the present invention.

The present invention may also provide pharmaceutical compositions containing a compound of the present invention, for example, compound A, B, C or D, in combination with at least one pharmaceutically acceptable excipient or carrier.

A pharmaceutical composition is a composition containing the compounds of the present invention in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition may be in bulk or in unit dosage form. The unit dosage form is any of various forms, including, for example, a capsule, an intravenous bag, a tablet, a single-cylinder aerosol inhaler pump, or a vial. The amount of active ingredient in a unit dose of the composition is an effective amount and will vary depending on the particular treatment intended. One skilled in the art will appreciate that it is sometimes necessary to make customary changes in dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. Various routes are provided, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for topical or transdermal administration of a compound of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalers. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any desired preservatives, buffers or propellants.

As used herein, the phrase pharmaceutically acceptable refers to those compounds, materials, compositions, carriers, or dosage forms that have been medically evaluated for use in contact with tissues of humans and animals without causing undue toxicity, irritation, allergic reaction, or other problem or complication. in accordance with a reasonable benefit/risk ratio.

Pharmaceutically acceptable excipient means an excipient suitable for the preparation of a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes an excipient acceptable for veterinary and veterinary uses. the field of pharmaceuticals in relation to humans. As used herein and the appended claims, a pharmaceutically acceptable excipient includes one or more such excipients.

The pharmaceutical composition of the present invention is formulated so that it is compatible with its intended route of administration. Example routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous use may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as citrates or phosphates; and tonicity adjusters such as sodium chloride or dextrose. The pH value can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. The drug for parenteral administration may be contained in ampoules, single-use syringes, or multi-dose vials made of glass or plastic.

The compound or pharmaceutical composition of the present invention can be administered to a subject using a variety of well-known methods used today for chemotherapy treatment. For example, for the treatment of cancer(s), the compound of the present invention can be administered by injection directly into tumors, administered by injection into the bloodstream or body cavities, or taken orally or applied through the skin using patches. The dose chosen should be sufficient to provide effective treatment, but not so high as to cause unacceptable side effects. The stage of the disease condition (eg, cancer, precancerous condition, etc.) and the patient's health should be carefully monitored during and for a reasonable period of time after treatment.

As used herein, the term therapeutically effective amount refers to an amount of a pharmaceutical agent to treat, alleviate, or prevent an identified disease or condition or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected using any analytical method known from the prior art. The exact effective amount for a subject will depend on the subject's body weight, size and health status, the nature and severity of the condition, and the therapeutic agent selected for administration. Therapeutically effective amounts for a particular situation may be determined by routine experimentation within the skill and discretion of the clinician. In a preferred aspect, the disease or condition being treated is cancer. In another aspect, the disease or condition being treated is a disorder associated with cell proliferation.

For any compound, a therapeutically effective amount can be determined initially or by assays in cell culture, such as tumor cells, or in animal models, typically rats, mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Such data can then be used to determine suitable doses and routes of administration for humans.

In embodiments of the present invention, EZH2 inhibitors are administered at a dose of 100-1600 mg/kg. In embodiments, the dose is 100 mg/kg, or 200 mg/kg, or 400 mg/kg, or 800 mg/kg, or 1600 mg/kg. In certain embodiments, the dosage is administered once daily or twice daily. In embodiments, the EZH2 inhibitor is Compound A, and the dose is 100 mg/kg, or 200 mg/kg, or 400 mg/kg, or 800 mg/kg, or 1600 mg/kg twice daily. In a preferred embodiment, the EZH2 inhibitor is Compound A and the dose is 800 mg/kg twice daily.

To ensure sufficient levels of the active agent(s) or to maintain the desired effect, the dosage and route of administration are adjusted. Factors that may be taken into account include the severity of the disease, the general health of the subject, the age, weight and sex of the subject, diet, timing and frequency of administration, drug interaction(s), sensitivity reactions and tolerability/response to therapy. Long-acting pharmaceutical compositions can be administered every 3-4 days, every week or every two weeks depending on the half-life and clearance rate of the particular composition.

Pharmaceutical compositions containing the active compounds of the present invention can be prepared in a manner generally known, for example, by conventional methods of mixing, dissolving, granulating, drageeing, trituration, emulsifying, encapsulating, encapsulating or lyophilizing. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and/or excipients (or both) to help convert the active compounds into drugs that can be used for pharmaceutical purposes. Of course, the appropriate composition depends on the chosen route of administration.

Suitable pharmaceutical compositions for use in injectable form include sterile aqueous solutions (in the case of water-soluble) or dispersions and sterile powders for the preparation of customized sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and it must be fluid to such an extent that it can be administered through a syringe. It must be stable under the conditions of manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium and contain, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol and polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using an emulsifier such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an absorption delaying agent, for example, aluminum monostearate and gelatin.

Sterile injection solutions can be prepared by introducing the active compound in the required amount into an appropriate solvent with one or a combination of the above

- 34 043327 ingredients, followed by filter sterilization if necessary. Typically, dispersions are prepared by introducing the active compound into a sterile carrier medium containing the starting dispersion medium and other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, production methods include vacuum drying and lyophilization, thereby obtaining a powder of the active ingredient and any desired ingredient from a pre-sterilized solution thereof.

Compositions for oral administration generally include an inert diluent or an edible pharmaceutically acceptable carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of administering a therapeutic agent for oral administration, the active compound can be included together with excipients and administered in the form of tablets, lozenges or capsules. Compositions for oral use can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is administered orally and rinsed and spit or swallowed.

Pharmaceutically compatible binders or excipients may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; a filler such as starch or lactose; a tablet disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavor.

For administration by inhalation, the compounds are delivered in the form of a spray aerosol from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be done via the transmucosal or transdermal route. For transmucosal or transdermal administration, the formulation uses wetting agents appropriate to the barrier through which it must pass. Such wetting agents are generally known in the art and include, for example, in the case of transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved using a nasal spray or suppositories. In the case of transdermal administration, the active compounds are formulated in the form of ointments, balms, gels or creams, generally known in the art.

The active compounds can be formulated together with pharmaceutically acceptable carriers that will protect the compound from rapid removal from the body, for example, in the form of a controlled release formulation, including implants and delivery systems in microencapsulated form. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to one skilled in the art. Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal. Suspensions of liposomes (including liposomes containing monoclonal antibodies against viral antigens that target infected cells) can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to one skilled in the art, such as those described in US Pat. No. 4,522,811.

Particularly preferred is the formulation of the composition for oral or parenteral use in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable for unit dosage administration to the subject being treated; each unit containing a predetermined amount of active compound, calculated to obtain the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification for unit dosage forms of the present invention is determined by and depends directly on the individual characteristics of the active compound and the particular therapeutic effect to be achieved.

In the context of therapeutic use, the dosages of the pharmaceutical compositions used in accordance with the present invention will vary depending on the agent, age, weight and clinical condition of the patient receiving treatment and the experience and judgment of the clinician or practitioner prescribing the therapy, among other factors influencing to choose the dosage. Generally, the dose should be sufficient to produce the result of slowing, and preferably regression, tumor growth, and preferably the result of complete regression of the cancer. Dosages may range from about 0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects, dosages may range from about 1 mg/kg per day to about 1000 mg/kg per day. In one aspect, the dose will range from about 0.1 mg/day to about 50 g/day, from about

- 35 043327 specifically 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 up to approximately 1 g/day, in single, divided or continuous doses (each dose can be adjusted according to the patient's weight in kg, body area in m ² and age in years). An effective amount of a pharmaceutical agent is an amount that produces an objectively detectable improvement, as determined by a clinician or other qualified observer. For example, regression of a patient's tumor may be determined in relation to tumor diameter. A decrease in tumor diameter indicates regression. Regression is also indicated by the failure of tumors to recur after treatment is stopped. As used herein, the term effective dosage refers to the amount of active compound to produce the desired biological effect in a subject or cell.

The pharmaceutical compositions may be contained in a container, package or dispenser along with instructions for administration.

The compounds of the present invention may further form salts.

Pharmaceutically acceptable salts as used herein refer to salts of the compounds of the present invention wherein the parent compound is modified by forming acidic or basic salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of inorganic or organic acids with basic moieties such as amines, alkalis, or organic salts with acidic moieties such as carboxylic acids and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts formed from the parent compound, for example, non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, salts derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonate, carbonic acid, citric, ethylenediaminetetraacetic, ethionic, 1,2-ethanesulfonic , fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycolylarsanilic, hexylresorcinolic, hydrabamine, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, almond, methanesulfonic, on psilovic, nitrogenous, oxalic, pamoic , pantothenic, phenylacetic, orthophosphoric, polygalacturonic, propionic, salicylic, stearic, weak acetic, succinic, sulfamic, sulfanyl, sulfuric, tannic, tartaric, toluenesulfonic and commonly occurring amino acids, such as glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include those of caproic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2 ,2,2]-octa-2-en-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid and the like. The present invention also covers salts formed when an acid proton present in the parent compound is replaced either by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or is reacted with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

The compounds or pharmaceutically acceptable salts, solvates or polymorphs thereof are administered by the oral, nasal, transdermal, pulmonary, inhalation, buccal, sublingual, intraperitoneal, subcutaneous, intramuscular, intravenous, rectal, intrapleural, intrathecal and parenteral routes. In one embodiment, the compound is administered orally. One skilled in the art will appreciate the advantages of certain routes of administration.

The dosage regimen using the compounds is selected according to a number of factors, including 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 liver function, and the specific compound or salt thereof used. The average physician or veterinarian can easily determine and prescribe the effective amount of drug required to prevent, counteract, or stop the progression of the condition.

Methods for formulation and administration of the compounds disclosed herein can be found in Remington: the Science and Practice of Pharmacy, ^19th edition, Mack Publishing Co., Easton, PA (1995). In one embodiment, the compounds described herein and their pharmaceutically acceptable salts are used as pharmaceuticals in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid excipients or diluents and sterile aqueous or organic solutions. In such pharmaceutical compositions, the compounds will be present in quantities sufficient to provide the desired dosage amount within the range described herein.

For the purposes of this document, all percentages and ratios are by weight, unless

- 36 043327 not otherwise stated. Other features and advantages of the present invention become apparent from various examples. The examples presented illustrate various components and methodology useful in practicing the present invention. The examples do not limit the claims. Based on the present disclosure, one skilled in the art will be able to identify and use other components and methodology useful in practicing the present invention.

In the synthesis schemes described herein, compounds may be depicted in one specific configuration for the purpose of simplicity. Such specific configurations should not be construed as limiting the present invention to any particular isomer, tautomer, regioisomer or stereoisomer, nor as excluding mixtures of isomers, tautomers, regioisomers or stereoisomers.

Additional compounds suitable for the methods of the present invention, as well as pharmaceutical compositions based thereon and their routes of administration, are described in WO12/142504 and WO12/142513, the contents of each of which are incorporated herein in their entirety by reference.

Post-translational modification of histone proteins and ATP-dependent chromatin remodeling are key processes for regulating the fidelity of normal gene expression. In cancer, proteins involved in these processes, such as the PRC2 and SWI/SNF complexes, are often genetically altered. It is important to note that these two complexes under normal conditions compete with each other to bind and influence chromatin; in this case, genetic changes can provide an imbalance in this antagonism. For example, INI1 is a subunit of the SWI/SNF complex that is absent in almost all rhabdoid tumors, resulting in oncogenic dependence in the PRC2-EZH2 methyltransferase, and rhabdoid tumor models are sensitive to small molecule inhibitors of EZH2. In synovial sarcoma, another INI1-deficient tumor type, a recurrent chromosomal translocation results in the fusion of the SS18 gene (a subunit of the chromatin remodeling complex SWI/SNF) on chromosome 18 with one of three related genes on the X chromosome, SSX1, SSX2, and less commonly SSX4. This results in the expression of the oncogenic SS18-SSX fusion protein, which binds to the SWI/SNF complex, displacing both wild-type SS18 and the tumor suppressor INI1, which are then degraded. This leads to abnormal gene expression and ultimately the development of cancer.

Described herein are data showing that Compound A (also referred to herein as E7438 or EPZ-6438), which is an early-stage clinically tested, selective, and orally bioavailable small molecule inhibitor of EZH2 enzymatic activity, induces antiproliferative activity. in preclinical models of synovial sarcoma, both as a single agent and in combination with chemotherapy. The compound induces dose-dependent inhibition of cell growth and cell death, particularly in a synovial sarcoma cell line (which is positive for the SS18-SSX1 fusion) in vitro. Treatment of mice with either a cell line-based xenograft model or two patient-derived xenograft (PDX) models resulted in dose-dependent inhibition of tumor growth with a corresponding reduction in the levels of trimethylation of the EZH2-specific substrate, lysine 27 in histone H3. These data demonstrate the dependence of SS18-SSX1 positive synovial sarcoma varieties on EZH2 enzymatic activity in xenograft models and indicate the potential utility of EZH2-targeting drugs in a given genetically defined cancer(s) with a corresponding biomarker.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and separately identified as being included herein. using the link. Reference to publications and patent documents does not imply as an admission that any such reference relates to prior art, nor does it constitute any admission as to their contents or date. From the present invention, having been described by means of the written description, one skilled in the art will understand that the present invention may be practiced in various embodiments and that the foregoing description and examples are provided for purposes of illustration and not limitation of the appended claims.

Examples

Example 1. Protein levels of EZH2, INI1, SS18-SSX1 and β-actin load control in different cell lines.

Human synovial sarcoma HS-SY-II and SW982 cells, human rhabdomyosarcoma RD cells, human rhabdoid tumor G401 cells, and human embryonic kidney cells HEK293 were lysed using 1x cell lysis buffer (#9803, Cell Signaling Technology, Danvers, MA). containing 1x protease inhibitor cocktail (Thermo Scientific, Rockford, IL). The samples were subjected to ultrasound and clarified by centrifugation at 10,000xg for 10 min at 4°C. The protein content of the lysates was determined using a BCA protein assay kit (Thermo Scientific). The sample solution was prepared by mixing 2× loading buffer (β-ME for sample processing with Tris SDS, Cosmo bio, Tokyo, Japan) and water with the cell lysate and incubated for 5 min at 95°C. Western blot analysis was performed as follows. Sample solutions were separated on a 2-15% gradient polyacrylamide gel for SS18 and EZH2 or on a 4-20% gradient polyacrylamide gel for INI1 and β-actin under reducing conditions and transferred to nitrocellulose membranes (GE Healthcare, Waukesha, WI) . Blots were blocked for 1 h at room temperature using the following blocking solutions: 1x Block Ace solution (Yukijirushi, Sapporo, Japan) for EZH2, INI1, and SS18, and TBS containing 0.5% Tween 20 and 5% skim milk. in the case of p-actin. Blots were incubated with primary antibodies overnight at 4°C under the following dilution conditions: anti-EZH2 antibody (07-689, Millipore, Billerica, MA) at a 1:1000 dilution in TBS containing 0.5% Tween 20 and 0.1x Block Ace solution, anti-INI1 antibody (#8745, Cell Signaling Technology) diluted 1:1000 in TBS containing 0.5% Tween 20 and 0.1x Block Ace solution, anti-SS18 antibody (sc-365170, Santa Cruz, Santa Cruz Cruz, California) at a 1:500 dilution in TBS containing 0.5% Tween 20 and 0.1x Block Ace solution, and anti-β-actin antibody (A5441, Sigma-Aldrich, St. Louis, MO) at a 1:2000 dilution in TBS containing 0.5% Tween 20 and 5% skim milk. After washing with TBS containing 0.5% Tween 20, the blots were further incubated with horseradish peroxidase conjugated to anti-rabbit IgG (Cell Signaling Technology) or anti-mouse IgG (Cell Signaling Technology) at room temperature for 60 min under the following dilution conditions: a 1:1000 dilution of anti-rabbit IgG in TBS containing 0.5% Tween 20 and 0.1x Block Ace solution, in the case of EZH2, INI1 and SS18, and a 1:2000 dilution of anti-mouse IgG in TBS containing 0.5% Tween 20 and 5% skim milk for β-actin. After thorough washing with TBS containing 0.5% Tween 20, blots were developed using Immobilon chemiluminescent Western blotting substrate HRP (Millipore). Immunoreactive bands were visualized by chemiluminescence using a LAS-3000 Luminescence Image Analyzer (Fuji Film, Tokyo, Japan). Exemplary images of the Western blot result are shown in FIG. 1. Expression of SS18-SSX1 was confirmed in HS-SY-II cells. Down-regulation of INI1 was also observed in this cell line. However, another synovial sarcoma line, SW982, did not express the fusion protein and had similar INI1 expression to RD and HEK293 cells.

Example 2: Levels of trimethylation and dimethylation of H3K27 in various cell lines.

Human synovial sarcoma cells HS-SY-II and SW982, human rhabdomyosarcoma RD cells, human rhabdoid tumor G401 cells, human diffuse large B-cell lymphoma cells WSU-DLCL2 and OCI-LY19, and human embryonic kidney cells HEK293 were suspended in 500 μl of lysis buffer ( 10 mmol/L MgCl ₂ , 10 mmol/L Tris-HCl, 25 mmol/L KCl, 1% Triton X-100, 8.6% sucrose and 1x protease inhibitor mixture).

After 5 min of incubation on ice, nuclei were collected by centrifugation at 600 × g for 5 min at 4°C and washed once with ice-cold PBS. After centrifugation at 600xg for 5 min at 4°C, the pellet was resuspended in 100 μl of 0.2 mol/l glacial sulfuric acid for 1 h by vortexing several times during the incubation. The supernatant was clarified by centrifugation at 10,000xg for 10 min at 4°C, and 1 ml of ice-cold acetone was added to the collected supernatant. Histones were precipitated at -20°C for 1 h, the pellet was isolated by centrifugation at 10,000xg for 10 min at 4°C and resuspended in 100 μl of water. The amount of extracted histones was determined using a BCA protein assay kit (Pierce). The sample solution was prepared by mixing 2x loading buffer (β-ME for sample processing with Tris SDS, Cosmo bio) and water with the cell lysate and incubated for 5 min at 95°C. Western blot analysis was performed as follows. Sample solutions were separated on a 15–25% gradient polyacrylamide gel under reducing conditions and transferred to nitrocellulose membranes (GE Healthcare, Waukesha, WI). Blots were blocked for 1 h at room temperature using the following blocking solutions: 1x Block Ace solution for H3K27me3 and H3K27me2 and TBS containing 0.5% Tween 20 and 5% skim milk for total histone H3. Blots were incubated with primary antibodies overnight at 4°C under the following dilution conditions: anti-H3K27me3 antibody (#9733, Cell Signaling Technology) and anti-H3K27me2 antibody (#9728, Cell Signaling Technology) at a dilution of 1:1000 in TBS containing 0 .5% Tween 20 and 0.1x Block Ace solution, and anti-total histone H3 antibody (ab1791, Abcam, Cambridge, MA) diluted 1:2000 in TBS containing 0.5% Tween 20 and 5% skim milk. After washing with TBS containing 0.5% Tween 20, the blots were further incubated with horseradish peroxidase conjugated to anti-rabbit IgG antibody (Cell Signaling Technology) at room temperature for 60 min at a 1:1000 dilution in TBS containing 0 .5% Tween 20 and 0.1x Block Ace solution, in the case of H3K27me3 and H3K27me2, or diluted 1:2000 in TBS containing 0.5% Tween 20 and 5% skim milk, in the case of general

- 38 043327 histone H3. After thorough washing with TBS containing 0.5% Tween 20, blots were developed using Immobilon chemiluminescent Western blotting substrate HRP (Millipore). Immunoreactive bands were visualized by chemiluminescence using a LAS-3000 Luminescence Image Analyzer (Fuji Film, Tokyo, Japan). Exemplary images of the Western blot result are shown in FIG. 2A. Signals from protein bands were quantified using Multi Gauge software version 3.0 (Fuji Film). A series of graphs are shown showing the quantitative ratio of H3K27me3/total H3 (Fig. 2B), H3K27me2/total H3 (Fig. 2C), or H3K27me3/H3K27me2 (Fig. 2D) in various cell lines. In contrast to the high levels of H3K27me3/H3K27me2 observed in WSU-DLCL2 with the EZH2 mutation at Y646, HS-SY-II was not characterized by high levels of H3K27me3/H3K27me2.

Example 3. Analysis of long-term proliferation of cell lines attached to the substrate. Protocol.

Inoculate into 96-well plates for analysis within 7 days. To obtain each substrate-attached cell line, cells were seeded in a volume of 100 μl in 96-well plates subject to three treatments, either in the evening (with compound treatment the next day) or in the morning (with compound treatment in the evening) to ensure that cells were able to attach to the plates before performing compound processing.

Divide into 6-well plates for analysis within 7-14 days. 2 ml of cells were seeded into each well of a 6-well plate at the proper density. The proper density was calculated from the 96-well plate growth curve by multiplying the 96-well density by 30. The factor 30 was derived from the 6-well plate growth area divided by the 96-well plate growth area (9.5 ^cm2 /0. 32 cm ² =29.7, rounded to 30), which corresponds to a 30-fold increase in surface area in a 6-well plate compared to a 96-well plate.

Day 0: Treat with compound or DMSO by removing the media and adding back either 100 µl (for a 96-well plate) or 2 ml (for a 6-well plate) of properly diluted compound/DMSO media. Table 1 includes an example of a 96-well plate design. The plates were incubated for 96 hours.

Table 1

A 1 2 3 4 5 6 7 8 9 10 eleven 12 IN Raw 10 2.5 0.63 0.16 0.04 0.01 0.0024 0.0006 DMSO WITH Raw 10 2.5 0.63 0.16 0.04 0.01 0.0024 0.0006 DMSO D Raw 10 2.5 0.63 0.16 0.04 0.01 0.0024 0.0006 DMSO E Compound Breedings ___ ___ ___ ___ — > F G N

Read one 96-well plate using CellTiter-Glo® to obtain Day 0 readings (medium change is not necessary for this plate).

- 39 043327

Day 4: Read one 96-well plate using CellTiter-Glo® to obtain readings on Day 4 (media change before reading is not necessary for this plate). Replace media in 96-well plates and 6-well plates with fresh media containing compound on day 4.

Day 7: Read the last 96-well plate using CellTiter-Glo®.

Count individual wells of 6-well plates and subseed cells at the original seeding density into 96-well plates as described above.

Repeat the steps performed on days 0-7 for days 7-14 of the proliferation assay.

Specifically, approximately 12 hours before compound treatment, HS-SY-II or SW982 cells were seeded in 6-well plates at a density of 24,000 cells/well and 7,500 cells/well, respectively. On day 0, cells were treated with either DMSO or compound, starting at 10 μmol/L and decreasing to fourfold dilutions. On day 7, cells in 6-well plates were trypsinized, counted using a TC10 automated cell count (Bio-Rad, Richmond, CA), and subcultured in triplicate into 96-well plates at a density of 800 cells/well and 250 cells. /well for HS-SY-II and SW982, respectively. Cells were allowed to adhere to the plate overnight and treated with either DMSO or compound as on day 0. Cell viability was determined on days 0, 7, 11, and 14 using the CellTiter-Glo® Luminescence Cell Viability Assay Kit (Promega). using the Envision 2103 Multifunction Reader (Perkin-Elmer, Wellesley, MA). The compound/medium was replaced with new ones on days 4 and 11. The ratios of the measured values on days 7, 11 and 14 and the measured values on day 7 were used to construct a graph showing proliferation from day 7 to day 14 and to calculate IC ₅₀ values on day 14 Representative graphs showing the results of experiments using Compound E and Compound A (also referred to herein as E7438 and EPZ-6438) are shown in FIG. 3A, 3B and 3C, 3D respectively. HS-SY-II cells were highly sensitive to Compound E and Compound A by day 14 of the long-term proliferation assay, whereas SW982 cells were not.

Example 4 Treatments with Compound A in vitro and in vivo.

In vitro, HS-SY-II cells were highly sensitive to Compound A and exhibited dose-dependent inhibition of cell proliferation. However, SW982 cells were insensitive to compound A (Fig. 5A and 5B).

A decrease in H3K27me3 levels was observed in both cell lines following EZH2 inhibitor treatment (Figure 6A), and _IC50 values were comparable to each other (Figure 6B). This suggests that changes in histone abundance resulting from EZH2 inhibitor treatment were independent of the presence of the SS18-SSX fusion protein.

PK values and PD changes were analyzed in the HS-SY-II mouse xenograft model. Concentrations of Compound A in blood plasma were determined 5 minutes before the last dose and 3 hours after it. A dose-dependent effect was observed (Fig. 7A). At the same time, a dose-dependent decrease in H3K27me3 levels in tumor tissues was also observed (Fig. 7B). In table 2 and 3 present the results of statistical analyzes relative to the data shown in FIG. 7B.

- 40 043327

table 2

Number of families 1 Number of comparisons per family 3 Alpha 0.05 Dunnett's multiple comparison test Mean difference 95% CI for difference Is it significant? Summary Medium carrier compared to E7438, 125 mg/kg 0.3 0.16810.4319 Yes Medium carrier compared to E7438, 250 mg/kg 0.45 0.31010.5899 Yes Medium carrier compared to E7438, 500 mg/kg 0.696 0.56410.8279 Yes Test Details Average 1 Average 2 Mean difference SE differences nl n2 q DE Medium carrier compared to E7438, 125 mg/kg 1 0.7 0.3 0.05052 5 5 5.93 8 15 Medium carrier compared to E7438, 250 mg/kg 1 0.55 0.45 0.05359 5 4 8.39 8 15 Medium carrier compared to E7438, 500 mg/kg 1 0.304 0.696 0.05052 5 5 13.7 8 15

Table 3

Ultimate Test for Linear Trend Incline -0.1119 R-square 0.9104 P-values <0.0001 Summary of P-values **** Is the linear trend significant? (P<0.05) Yes

Changes in gene expression resulting from treatment with Compound A or EPZ-011989 (i.e. Compound C) were analyzed in HS-SY-II and SW982 cultures in vitro. The PRC2 complex has been reported to be recruited by the SS18-SSX fusion protein through TLE1 and to repress ATF2 target genes (EGR1, ATF3, MEIS2 and CDKN2A) (Cancer Cell 21, 333-347, 2012). Here, the expression levels of EGR1, ATF3 and CDKN2A were studied. A dose- and time-dependent increase in CDKN2A expression was observed in HS-SY-II, whereas in SW982 the CDKN2A locus was known to be subject to homosytic deletion (Fig. 8). The expression of ATF3 and EGR1 was increased by treatment with Compound A and EPZ-011989 in HS-SY-II but not in SW982. Thus, compound A induced changes in the expression of genes involved in the pathogenesis of synovial sarcoma.

In table 4 presents the results of statistical analysis relative to the data shown in FIG. 8.

- 41 043327

Table 4

0 µM compared to day 2 day 4 day 7 SW982 E7438 ATF3 0.1 µM ns ns ns 1 µM ns ns ns EGR1 0.1 µM ns ns ns 1 µM ns ns ns EPZ011989 ATF3 0.1 µM ns ns ns 1 µM ns ns ns EGR1 0.1 µM * * * ns 1 µM ns ns * HS-SY-II E7438 ATF3 0.1 µM ns ns ns 1 µM ns * ** EGR1 0.1 µM ns ns ns 1 µM ns ns * * CDKN2A 0.1 µM ns ns ns 1 µM * * * * * * * * * * EPZ01198g ATF3 0.1 µM ns * * ns 1 µM * * * * EGR1 0.1 µM ns ** ns 1 µM ns * * * * CDKN2A 0.1 µM ns ns 1 µM ***

Asterisks indicate significant changes compared to levels in 0 μM treated groups.

Gene expression changes resulting from Compound A treatment were also analyzed in the HS-SY-II xenograft model. CDKN2A expression was significantly increased in mice treated with 500 mg/kg Compound A (Fig. 9). In this case, compound A also induced changes in the expression of genes involved in the pathogenesis of synovial sarcoma.

In fig. 10A-1C show average in vivo tumor volumes for athymic nude mice bearing HS-SY-II xenografts. In the first study, mice were administered either vehicle (orally for 28 days or iv on day 1 and day 22) or compound A (orally: 125 mg/kg, 250 mg/kg or 500 mg/kg for 28 days). , or doxorubicin (iv: 10 mg/kg, day 1 and day 22); while tumor volumes were measured twice a week (Figs. 10A and 10B). In the second study, mice were treated with either vehicle (orally for 28 days), Compound A (orally: 250 mg/kg or 500 mg/kg for 28 days), or doxorubicin (iv: 10 mg/kg on day 1 and day 22), or a combination of doxorubicin (iv: 10 mg/kg on day 1 and day 22) and compound A (oral: 250 mg/kg for 28 days) (Fig. 10C). Tumors from animals from the second study were removed on day 28 (3 hours after the last dose) and analyzed for H3K27me3 using ELISA (Fig. 10D) or IHC for the proliferation marker Ki67 (Fig. 10E).

Despite sensitivity to Compound A in 2D cell culture, xenografts based on the HS-SY-II cell line were characterized by a dose-dependent increase in tumor volume in a murine xenograft model; at the same time, the mechanism underlying this observation was studied.

In fig. 11A and 11C show average in vivo tumor volumes for athymic nude mice bearing one of two different PDX tumors consistent with synovial sarcoma. In fig. 11B and 11D show percentage survival rates for PDX mice. Mice were treated with either vehicle (orally for 35 days), Compound A (orally: 125 mg/kg, 250 mg/kg, or 500/400 mg/kg for 35 days), or doxorubicin (iv: 3 mg/kg). kg, once a week for 3 weeks). In fig. 11A and 11B show data obtained for PDX mice from a 57 year old male subject with high grade spindle cell sarcoma. In fig. 11C and 11D show data obtained for PDX mice from a 16 year old female subject. In contrast to xenografts based on the HS-SY-II cell line, PDX in mice exhibited a dose-dependent reduction in tumor volume in vivo.

In fig. 12A and 12B show a dataset obtained from PDX tumors consistent with synovial sarcoma from subsets of mice that were removed at day 35 and analyzed by RNA-seq analysis. In fig. 12A shows data obtained for PDX mice from a 57 year old male subject; in fig. 2B shows data obtained for PDX mice from a 16-year-old female subject. Expression of HLA, Fas and KLRD1 genes was increased in mice,

- 42 043327 treated with 400 mg/kg Compound A, compared to expression in mice treated with vehicle. In the figure, low expression level is indicated and high expression level is indicated.

Materials and methods

Cell culture.

HS-SY-II (RCB2231, RIKEN Bioresource Center) and SW982 (NTV-93, ATCC) were grown in RPMI1640 with 10% FBS under conditions of 37°C, 5% CO ₂ . HS-SY-II cells had an SS18-SSX1 fusion, whereas SW982 cells had wild-type SS18.

Changes in H3K27 methylation induced by compound A.

HS-SY-II and SW982 cells were treated with either DMSO or Compound A, starting at 40 nmol/L and decreasing to fourfold dilutions, for 96 h. Cells were washed with ice-cold PBS, collected using a cell scraper, and lysed with 100 µl of nuclear extraction buffer (10 mM Tris-HCl, 10 mM _MdCl2 , 25 mM KC1, 1% Triton X-100, 8.6% sucrose and 1x Halt protease inhibitor cocktail (18 61281, Thermo Scientific). Nuclei were collected by centrifugation at 600 g for 5 min at 4°C and washed once with ice-cold PBS. The supernatant was removed and the histones were extracted for one hour with 100 μl of 0.4 N ice-cold sulfuric acid. Extracts were clarified by centrifugation at 10,000 g for 10 min at 4°C and transferred to a new microcentrifuge tube containing 1 ml of ice-cold acetone.Histones were precipitated at -2.0°C overnight, the sediment was isolated by centrifugation at 10,000 g for 10 min and resuspended in 100 µL of water Histones were quantified using the BCA Protein Assay Kit (23225, Pierce). Immulon 4HBX plates (3855, Thermo Scientific) were coated with diluted histones overnight and ELISA was performed. Briefly, after blocking with PBS containing 0.05% Tween 20 and 2% bovine serum albumin (BSA), plates were incubated with anti-HK27te3 antibody (#9733, Cell Signaling Technology) or anti-total histone H3 antibody (AB1791, Abeam). . The plates were then incubated with horseradish peroxidase conjugated to anti-rabbit IgG (#7074, Cell Signaling Technology), followed by incubation with TMB substrate (TMBS-0100-01, BioFx Laboratories). The intensity of the developed color in the wells was then measured using a plate spectrophotometer (SpectraMax 250, Molecular Devices) at 450 nm (reference wavelength 650 nm). H3K27te3 levels were adjusted by the level of total histone H3 and expressed as fold change relative to the DMSO control.

In fig. 6A shows a representative graph. The range of 1C ₅ o values (two independent experiments) shown in Fig. 6B was determined using GraphPad Prism (GraphPad Software). Changes in histone marker abundance were comparable between HS-SY-II and SW982, with changes independent of the presence of the SS18-SSX fusion protein.

PK values and PD changes in the HS-SY-II xenograft model.

HS-SY-II cells were harvested at mid-log growth phase and resuspended in Hanks' balanced salt solution with 50% Matrigel (BD Biosciences). Balb/C nude mice (Charles River Laboratories, Japan) were injected subcutaneously with 1x10 ⁷ cells (0.1 ml cell suspension) into the right flank. Mice with tumors of approximately 200 mm ³ (31 days after injection) were divided into treatment groups based on similar mean tumor volumes. Compound A or vehicle (0.5% MS + 0.1% Tween-80 in water) was administered orally by gavage at specified doses twice daily for 7 days. Each dose was delivered in a volume of 0.2 ml/20 g mouse (10 ml/kg), and adjusted to the last recorded weight for individual animals. Peripheral blood samples were collected from mice treated with Compound A approximately 5 minutes before and 3 hours after the last dose. After obtaining blood plasma samples by centrifugation, the concentrations of Compound A in the blood plasma were analyzed using the liquid chromatography tandem mass spectrometry (LC/MS/MS) method. To quantify compound A, a UPLC system (Acquity, Waters) equipped with a mass spectrometer (Quattro premier, MICROMASS) was used. Concentrations are shown graphically (n=5) in FIG. 7A. Each bar represents the mean plasma concentration in each group.

Tumor samples were collected from mice used in the analysis of PK values and PD changes approximately 3 h after the last dose. Tumor samples were stored using RNAlater reagent (AM7020, Life technologies). Total RNA extraction and reverse transcription were performed using the RNeasy Mini Kit (74104, Qiagen) and the cDNA High Efficiency Reverse Transcription Kit (4368814, Life technologies) according to the manufacturer's instructions. cDNA samples were used in real-time PCR as described above. Expression levels were adjusted for GAPDH and expressed as fold change relative to vehicle control (Fig. 9).

Regarding FIG. 10D, tumors were flash frozen in liquid nitrogen. Frozen tumor samples were cut into 20 mg pieces and placed in 500 μl of ice-cold nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl ₂ , 25 mM KCl, 1% Triton X-100, 8.6% sucrose and 1x Halt protease inhibitor mixture (1861281, Thermo Scientific)) and homogenized using a manual microhomogenizer. Nuclei were collected by centrifugation at 600 g for 5 min at 4°C and washed once with ice-cold PBS. The supernatant was removed and histones were extracted for one hour with 100 μl of 0.4 N. glacial sulfuric acid. The extracts were clarified by centrifugation at 10,000 g for 10 min at 4°C and transferred to a new microcentrifuge tube containing 1 ml of ice-cold acetone. Histones were precipitated at -20°C overnight, the sediment was isolated by centrifugation at 10,000 g for 10 min and resuspended in 100 μl of water. The amount of histones was determined using the BCA Protein Assay Kit (23225, Pierce). Immulon 4HBX plates (3855, Thermo Scientific) were coated with diluted histones overnight and ELISA was performed. Briefly, after blocking with PBS containing 0.05% Tween 20 and 2% bovine serum albumin (BSA), plates were incubated with anti-H3K27me3 antibody (#9733, Cell Signaling Technology) or anti-total histone H3 antibody (AB1791, Abeam). . The plates were then incubated with horseradish peroxidase conjugated to anti-rabbit IgG (#7074, Cell Signaling Technology), followed by incubation with TMB substrate (TMBS-0100-01, BioFx Laboratories). The intensity of the developed color in the wells was then measured using a plate spectrophotometer (SpectraMax 250, Molecular Devices) at 450 nm (reference wavelength 650 nm). H3K27me3 levels were adjusted by total histone H3 levels and expressed as fold change relative to vehicle control. The values are shown graphically (n=6) in FIG. 10D. Each bar represents the mean value in each group. A significant reduction in H3K27me3 levels was observed in tumors obtained from mice treated with Compound A. Histones were extracted and quantified, and H3K27me3 levels were analyzed as described above. The levels of H3K2 7 trimethylation in the tumor are shown graphically (n=5) in Fig. 7B. Each bar represents the mean + SEM of the trimethylation level in each group.

Changes in gene expression in vitro after treatment with compound A.

HS-SY-II and SW982 cells were treated with EZH2 inhibitors (compound A or EPZ-011989) and harvested at specific time points. Total RNA was isolated and cDNA was synthesized using the TaqMan Gene Expression Cells-to-CT kit (4399002, Life technologies) in accordance with the manufacturer's protocol. The expression of ATF3, EGR1, CDKN2A and GAPDH was analyzed using the TaqMan gene expression assay kit (Life technologies, 4331182) and TaqMan probes (Hs00231069_m1, Hs00152928_m1, Hs00233365_m1 and Hs99999905_m1, respectively). Expression levels were adjusted for GAPDH and expressed as fold change relative to the DMSO control (Fig. 8).

Changes in gene expression in vivo after treatment with compound A.

Tumor samples were collected from mice used in the analysis of PK values and PD changes approximately 3 h after the last dose. Tumor samples were stored using RNAlater reagent (AM7020, Life technologies). Total RNA extraction and reverse transcription were performed using the RNeasy Mini Kit (74104, Qiagen) and the cDNA High Efficiency Reverse Transcription Kit (4368814, Life technologies) according to the manufacturer's instructions. cDNA samples were used in real-time PCR as described above. Expression levels were adjusted for GAPDH and expressed as fold change relative to vehicle control (Figure 9). Additionally or alternatively, total RNA samples were isolated from frozen tumor samples. The TruSeq™ RNA Sample Kit (Illumina) was used to generate a cDNA library for paired-end sequencing on the Illumina HiSeq (details are described in the Illumina TruSeq RNA Sample Kit manual). Using the Standard Cluster Generation Kit v5, cDNA libraries were bound to the surface of the flow cell. Paired-end reads from ~50 million clusters per sample were obtained using the TruSeq SBS kit on Illumina HiSeq.

Processing of RNA sequencing data.

Raw reads obtained from sequencing in FASTQ format were aligned to the human hg19 genome as well as the mmlO mouse genome. Transcriptome quantification was performed using RSEM v1.1.13 using the transcriptome from the USCS KnownGene database as a reference. Expression calculations in RSEM were performed with parameters optimized for 50x50 paired-end Illumina sequencing.

Analysis of transcriptomic data obtained by PEC sequencing for biological interpretation.

Gene group abundance analysis (GSEA) was performed to compare two samples treated with Compound A or DMSO for both PDX models in 5 replicate simulations using desktop GSEA software with Java support (version 2.0.13, available online: iviviv.broadinstitute .org/gsea/index.jsp). The gene level count data obtained from RSEM, after normalization, was used for input into GSEA. Permutation was performed on a group of genes rather than a phenotype to ensure a small sample size. For comparison, the KEGG pathway as well as the transcription factor binding motif gene group from MSigDB version 4.0, available online

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Claims (5)

(rvrvrv.broadinstitute.org/gsea/msigdb/index.jsp) was used for all GSEA analyses. Regarding FIG. 10E, selected tumor samples were fixed in formalin and embedded in paraffin. Paraffin sections were obtained, deparaffinized, and processed for IHC following the general procedure according to the polymer-based system method (Envision, DAKO, Japan). Deparaffinized sections were processed for antigen retrieval by autoclaving for 20 min at 121°C at 2 atmospheres of target retrieval solution (DAKO, diluted to a final concentration of 10%) and treated with 3% hydrogen peroxide for 5 min to block activity of endogenous peroxidase. A commercially available primary antibody was allowed to react with Ki-67 for approximately one hour (mouse monoclonal antibody to human Ki67, DAKO, Japan, diluted 1:50) at room temperature. The following steps were performed using the Envision system following the manufacturer's instructions, including secondary antibody treatment for 30 min at room temperature; in this case, 3,3'-diaminobenzidine was used as a chromogen and hematoxylin as a contrast dye. For Figure 10E, microscopic slides were digitized using an Aperio XT™ scanner. Digitized microscopic slides were analyzed using Aperio Image Scope™ software and the percentage of positive cells was quantified. Quantification of IHC-stained Ki67 showed no change in cell proliferation across treatment groups. Each bar represents the mean value in each group. (No significant change in the percentage of Ki67-positive cells was observed across all dose groups). Methods for sequencing RNA from synovial sarcoma samples. Patient-derived xenograft (PDX) models CTG-0331 and CTG-0771 were short-passage established Champions TumorGraft™ models of human synovial sarcoma. Female immunocompromised mice (Taconic; NCr deficient) were implanted on one side in the flank with tumor fragments (5 mm x 5 mm x 5 mm) extracted from donor animals, each implanted after a certain number of passages (CTG-0331 and CTG-0771 for P4). When tumors reached a volume of approximately 100,300 mm ³ , animals were assigned based on tumor volume into treatment and control groups and dosed. Vehicle (0.5% NaCMC and 0.1% Tween 80 in water) or Compound A (500 mg/kg for CTG-0331 and 500 mg/kg tapered to 400 mg/kg on day 17 for CTG -0771) were administered orally BID at a dose volume of 10 μl/g for 35 days. The size of the tumors was measured twice a week. On day 35, at least 5 mice with the highest tumor burden were euthanized to select tumors 3 hours after the last dose and the latter were snap frozen. Frozen tumor samples (30 mg) from the vehicle and highest dose of Compound A groups were transported for expression analysis, RNA processing, and RNA sequence analysis. The entire disclosure of each patent document and scientific article associated with this document is incorporated by reference for all purposes. The present invention may be embodied in other specific forms without deviating from the idea or essential characteristics. Therefore, the foregoing embodiments should be considered as illustrative rather than limiting of the present invention described herein. Accordingly, the scope of the present invention is given by the appended claims and not by the foregoing description, and all modifications that come within the meaning and scope of the claims are intended to be included herein. CLAIM 1. A method of treating cancer, comprising contacting a human cancer cell with an amount of an EZH2 inhibitor effective to increase the expression of a major histocompatibility complex (MHC) in the cancer cell, where the cancer cell contains a t(x;18)(p11.2;q11) chromosomal translocation. 2), which leads to the formation of the SS18-SSX fusion gene. 2. A method of treating cancer, comprising contacting a human cancer cell with an amount of an EZH2 inhibitor effective to increase the expression of a major histocompatibility complex (MHC) in the cancer cell, wherein the function and/or expression of INI1 in the cancer cell is reduced. 3. A method of treating cancer comprising contacting a human cancer cell with an amount of an EZH2 inhibitor effective to increase major histocompatibility complex (MHC) expression in the cancer cell, wherein the cancer cell contains aberrant, misregulated or increased EZH2 activity. 4. The method of claim 2, wherein the cancer cell has reduced INI1 function and expression. 5. Method according to any one of claims 1 to 4, wherein the EZH2 inhibitor is compound A having the following formula: -