WO2014152261A1 - Composés 7-déazapurine substitués - Google Patents

Composés 7-déazapurine substitués Download PDF

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Publication number
WO2014152261A1
WO2014152261A1 PCT/US2014/027134 US2014027134W WO2014152261A1 WO 2014152261 A1 WO2014152261 A1 WO 2014152261A1 US 2014027134 W US2014027134 W US 2014027134W WO 2014152261 A1 WO2014152261 A1 WO 2014152261A1
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Prior art keywords
compound
cancer
subject
cell
leukemia
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PCT/US2014/027134
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English (en)
Inventor
Richard Chesworth
Kevin Wayne Kuntz
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Epizyme, Inc.
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Publication of WO2014152261A1 publication Critical patent/WO2014152261A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • DOT1L Disease-associated chromatin-modifying enzymes
  • the invention provides compounds useful for modulating the aberrant action of epigenetic enzymes.
  • the present invention also provides pharmaceutically acceptable salts, esters, and/or N-oxides, of these compounds.
  • the present invention features a substituted 7-deazapurine compound of Formula I) below or an N-oxide, a pharmaceutically acceptable salt or ester thereof.
  • Rj is halo, cyano, amino, C ⁇ -C(, alkyl, C 2 -C6 alkenyl, C2-C 6 alkynyl, or C 3 -Cg cycloalkyl, in which, each of the C1-C6 alkyl, C 2 -C6 alkenyl, C2-C 6 alkynyl, and C 3 - C8 cycloalkyl, independently, is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, Cj -C6 alkoxyl, and amino.
  • One subset of the compounds of Formula (I) includes those of Formula (IA):
  • the compounds of Formulae (I), (IA), and (IB) can include one or more of the following features:
  • Ri is halo
  • Ri is F, CI, Br, or I.
  • Ri is alkyl
  • Ri is methyl, ethyl, or isopropyl.
  • R is haloalkyl
  • R is CF 3 .
  • R is C 3 -C 8 eye loalkyl.
  • Ri is cyclopropyl or cyclobutyl.
  • Ri is C 2 -C 6 alkenyl or C 2 -C 6 alkynyl.
  • Ri is ethynyl, vinyl, isopropenyl, or 1 -propenyl.
  • Ri is amino
  • Ri is NH2, mono-alkylamino or di-alkylamino.
  • Ri is cyano
  • the present invention features a compound that is selected from compounds listed in Table 1 , N-oxides and pharmaceutically acceptable salts thereof.
  • the invention also relates to a pharmaceutical composition of a compound of any of the Formulae described herein and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of N-oxide or a salt of a compound of any of the Formulae described herein and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a solvate (e.g., a hydrate) of a compound of any of the Formulae described herein and a pharmaceutically acceptable carrier.
  • a solvate e.g., a hydrate
  • the present invention provides methods of treating or preventing cancer.
  • the present invention provides methods of treating cancer.
  • the present invention also provides methods of preventing cancer.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae described herein.
  • the cancer can be a hematological cancer.
  • the cancer is leukemia. More preferably, the cancer is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • the present invention provides methods of treating a disease or disorder mediated by translocation of a gene on chromosome 1 l q23.
  • the present invention also provides methods of preventing a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae described herein.
  • the present invention provides methods of treating a disease or disorder in which DOT1 -mediated protein methylation plays a part or a disease or disorder mediated by DOT1- mediated protein methylation.
  • the present invention also provides methods of preventing a disease or disorder in which DOT1 -mediated protein methylation plays a part or a disease or disorder mediated by DOT 1 -mediated protein methylation.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae described herein.
  • the present invention provides methods of inhibiting DOT1L activity in a cell.
  • the method includes contacting the cell with an effective amount of one or more of the compound of any of the Formulae described herein.
  • Still another aspect of the invention relates to a method of reducing the level of Histone H3 Lysine residue 79 (H3-K79) methylation in a cell.
  • the method includes contacting a cell with a compound of the present invention.
  • Such method can be used to ameliorate any condition which is caused by or potentiated by the activity of DOT1 through H3-K79 methylation.
  • the present invention relates to use of the compounds disclosed herein in preparation of a medicament for treating or preventing cancer.
  • the use includes a compound of any of the Formulae described herein for administration to a subject in need thereof in a therapeutically effective amount.
  • the cancer can be a hematological cancer.
  • the cancer is leukemia. More preferably, the cancer is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • the present invention provides use of the compounds disclosed herein in preparation of a medicament for treating or preventing a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the use includes a compound of any of the Formulae described herein for administration to a subject in need thereof in a therapeutically effective amount.
  • the present invention provides use of the compounds disclosed herein in preparation of a medicament for treating or preventing a disease or disorder in which DOTl -mediated protein methylation plays a part or a disease or disorder mediated by DOTl -mediated protein methylation.
  • the use includes a compound of any of the Formulae described herein for administration to a subject in need thereof in a therapeutically effective amount.
  • the present invention provides use of the compounds disclosed herein for inhibiting DOT1L activity in a cell.
  • the use includes contacting the cell with an effective amount of one or more of the compound of any of the Formulae described herein.
  • Still another aspect of the invention relates to a use of the compounds disclosed herein for reducing the level of Histone H3 Lysine residue 79 (H3-K79) methylation in a cell.
  • the use includes contacting a cell with a compound of the present invention.
  • Such use can ameliorate any condition which is caused by or potentiated by the activity of DOTl through H3-K79 methylation.
  • the present invention provides a method for treating or alleviating a symptom of leukemia comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein.
  • the leukemia is characterized by a chromosomal rearrangement.
  • the chromosomal rearrangement is chimeric fusion of mixed lineage leukemia gene (MLL) or partial tandem duplication of the MLL gene (MLL-PTD).
  • the subject has an increased level of HOXA9, Fms-like tyrosine kinase 3 (FLT3), MEIS1 , and/or DOT1L.
  • the present invention provides a method for treating or alleviating a symptom of leukemia comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein, wherein the subject has an increased level of HOXA9, FLT3, MEIS1 , and/or DOT1L.
  • the present invention provides a method for treating or alleviating a symptom of leukemia in a subject comprising: obtaining a sample from the subject; detecting the level of HOXA9, FLT3, MEIS1 , and/or DOT1L, wherein an increased level of HOXA9, FLT3, MEIS1 , and/or DOT1L indicates the subject is responsive to a compound described herein; and administering to the subject a therapeutically effective amount of said compound when said subject is responsive to said compound.
  • the present invention provides a method for treating or alleviating a symptom of leukemia in a subject comprising: obtaining a sample from the subject; detecting the presence of a genetic lesion of MLL in the sample; and administering to the subject a therapeutically effective amount of a compound described herein when said genetic lesion is present in the sample.
  • the genetic lesion is chimeric fusion of MLL or MLL-PTD.
  • the sample is selected from bone marrow, peripheral blood cells, blood, plasma, serum, urine, saliva, a cell, or a tumor tissue.
  • the present invention provides a method for treating a disorder mediated by translocation, deletion and/or duplication of a gene on chromosome 1 lq23, comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein.
  • the invention features a method of selecting a therapy for a subject having leukemia.
  • the method includes the steps of: detecting the presence of a partial tandem duplication of the MLL gene (MLL-PTD) in a sample from the subject; and selecting, based on the presence of the MLL-PTD, a therapy for treating leukemia.
  • the therapy includes administering to the subject a therapeutically effective amount of a compound described herein.
  • the method further includes administrating to the subject a therapeutically effective amount of a compound described herein.
  • the leukemia is characterized by partial tandem duplication of the MLL gene.
  • a method of treatment for a subject in need thereof, the method comprising the steps of: detecting presence of a partial tandem duplication of the MLL gene (MLL-PTD) in a sample from the subject; and treating the subject based on the presence of MLL-PTD with a therapy that includes administrating to the subject a therapeutically effective amount of a compound described herein.
  • the subject in need thereof has leukemia that is is characterized by partial tandem duplication of the MLL gene.
  • the invention features a method of selecting a therapy for a subject having leukemia.
  • the method includes the steps of: detecting the level of HOXA9, FLT3, MEISl , and/or DOTIL in a sample from the subject; and selecting, based on the presence of the increased level of HOXA9, FLT3, MEISl , and/or DOTIL a therapy for treating leukemia.
  • the therapy includes administering to the subject a therapeutically effective amount of a compound described herein.
  • the method further includes administrating to the subject a therapeutically effective amount of a compound described herein.
  • the leukemia is characterized by partial tandem duplication of the MLL gene.
  • the leukemia is characterized by overexpression of HOXA9, FLT3, MEISl and/or DOTIL.
  • a method of treatment for a subject in need thereof, the method comprising the steps of: detecting the the level of HOXA9, FLT3, MEISl, and/or DOTIL in a sample from the subject; and treating the subject based on the presence of the increased level of HOXA9, FLT3, MEISl , and/or DOTIL with a therapy that includes administrating to the subject a therapeutically effective amount of a compound described herein.
  • the subject in need thereof has leukemia that is characterized by partial tandem duplication of the MLL gene.
  • the subject in need thereof has leukemia that is characterized by overexpression of HOXA9, FLT3, MEISl and/or DOTIL.
  • the invention provides methods of synthesizing the foregoing compounds.
  • a therapeutically effective amount of one or more of the compounds can be formulated with a pharmaceutically acceptable carrier for administration to a mammal, particularly humans, for use in modulating an epigenetic enzyme.
  • the compounds of the present invention are useful for treating, preventing, or reducing the risk of cancer or for the manufacture of a medicament for treating, preventing, or reducing the risk of cancer.
  • the compounds or the formulations can be administered, for example, via oral, parenteral, otic, ophthalmic, nasal, or topical routes, to provide an effective amount of the compound to the mammal.
  • the present invention provides a family of compounds that can be used to selectively modulate the aberrant action of an epigenetic enzyme. Further, the compounds can be used to treat or prevent a disease state in a mammal caused or mediated by aberrant action of an epigenetic enzyme.
  • the present invention includes pharmaceutically acceptable salts, esters, tautomers, and N-oxides of these compounds.
  • the present invention provides novel substituted 7-deazapurine compounds, synthetic methods for making the compounds, pharmaceutical compositions containing them and various uses of the compounds.
  • the present invention provides the compounds of Formula (I) below or an N-oxide, a pharmaceuticall acceptable salt or ester thereof.
  • Ri is halo, cyano, amino, C ⁇ -Ce alkyl, C 2 -C6 alkenyl, C 2 -C6 alkynyl, or C3-C8 cycloalkyl, in which, each of the C1-C6 alkyl, C 2 -C6 alkenyl, C 2 -C 6 alkynyl, and C3-C8 cycloalkyl, independently, is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxyl, carboxyl, cyano, C1-C6 alkoxyl, and amino.
  • R ⁇ is halo
  • R ⁇ is F, CI, Br, or I.
  • R ⁇ is alkyl
  • R ⁇ is methyl, ethyl, or isopropyl.
  • Ri is haloalkyl
  • R ⁇ is CF 3 .
  • R ⁇ is C3-C8 cycloalkyl.
  • R ⁇ is cyclopropyl or cyclobutyl.
  • R] is C 2 -C6 alkenyl or C 2 -C6 alkynyl.
  • R ⁇ is ethynyl, vinyl, isopropenyl, or 1 -propenyl
  • Ri is amino
  • Rj is NH 2 , mono-alkylamino or di-alkylamino.
  • Ri is cyano
  • One subset of the compounds of Formula (I) includes those compounds of Formula (IA):
  • Another subset of the compounds of Formula (I) includes those compounds of Formulf
  • the present invention features a compound is selected from compounds listed in Table 1 , N-oxides, pharmaceutically acceptable salts polymorphs, solvates (e.g., hydrates), and stereoisomers thereof.
  • This invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of any of the Formulae described herein and a pharmaceutically acceptable carrier.
  • One aspect of the present invention is based in part upon the discovery that DOT1 L inliibitors can effectively treat leukemia that is characterized by partial tandem duplication of the MLL gene.
  • Another aspect of the present invention is based in part upon the discovery that DOT1L inhibitors can effectively treat leukemia that is characterized by overexpression of HOXA9, FLT3, MEIS1 and/or DOT1L.
  • tumors or tumor cells having increased mRNA or protein level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L are sensitive to the DOT1L inhibitors of the present invention.
  • the present invention provides methods of treating or alleviating a symptom of leukemia in a subject by administering a therapeutically effective amount of a DOT1L inhibitor described herein to the subject, particular leukemia associated with overexpression of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOTlL.
  • the compounds of the present invention inhibit the histone methyltransferase activity of DOTIL or a mutant thereof.
  • methylation regulation by DOTIL involves in tumor formation, particular tumors bearing an increased mRNA, protein and/or activity (function) level of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL
  • the compounds described herein are suitable candidates for treating cancers, i.e., to decrease methylation or restore methylation in a cancer cell to roughly its level in counterpart normal cells.
  • the present invention features a method for treating or alleviating a symptom of cancer.
  • the method includes administering to a subject in need thereof, a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, solvate, or stereoisomeror thereof.
  • the present invention provides methods for the treatment of a cancer mediated by DOT1 (e.g., DOT1 L)-mediated protein methylation in a subject in need thereof by administering to a subject in need of such treatment, a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof.
  • DOT1 e.g., DOT1 L
  • the present invention further provides the use of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, for the preparation of a medicament useful for the treatment of a cancer mediated by DOTlL-mediated protein methylation.
  • the present invention provides methods for the treatment of a cancer the course of which is influenced by modulating the methylation status of histones or other proteins, wherein said methylation status is mediated at least in part by the activity of DOTIL.
  • Modulation of the methylation status of histones can in turn influence the level of expression of target genes activated by methylation, and/or target genes suppressed by methylation.
  • the method includes administering to a subject in need of such treatment, a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, solvate, or stereoisomeror thereof.
  • the present invention also provides methods of protecting against or preventing a cancer in which DOTlL-mediated protein methylation plays a part in a subject in need thereof by administering a therapeutically effective amount of compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to a subject in need of such treatment.
  • the present invention also provides the use of compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, solvate, or stereoisomeror thereof, for the preparation of a medicament useful for the prevention of a cell proliferative disorder.
  • the cancer is a cancer selected from the group consisting of brain and CNS cancer, kidney cancer, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, prostate cancer, or a hematological cancer.
  • the hematological cancer is leukemia or lymphoma.
  • the cancer is leukemia.
  • the present invention further provides the use of a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, metabolite, polymorph or solvate thereof in the treatment of leukemia, or, for the preparation of a medicament useful for the treatment of such leukemia.
  • the leukemia can be acute or chronic leukemia.
  • the leukemia is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • exemplary leukemia that may be treated is mixed linage leukemia (MLL).
  • MLL mixed linage leukemia
  • the MLL that can be treated by the compound of the present invention is chimeric fusion of MLL, partial tandem duplication of the MLL gene (MLL-PTD) or non-rearranged .. . MLL.
  • MLL Mixed lineage leukemia
  • AML adult acute myeloid leukemias
  • MLL disease A universal hallmark of MLL disease is a chromosomal translocation affecting the MLL gene on chromosome 1 lq23 (Hess, 2004; Krivtsov and Armstrong, 2007).
  • the MLL gene encodes for a SET-domain histone methyltransferase that catalyzes the methyiation of lysine 4 of histone H3 (H3K4) at specific gene loci (Milne et al. (2002) Mol Cell 10, 1 107-1 1 17; Nakamura et al. (2002), Mol Cell 10, 11 19-1128).
  • H3K4 histone H3
  • fusion partners are capable of interacting directly, or indirectly, with another histone methyltransferase, DOT1L (Bitoun et al. (2007) Hum Mol Genet 16, 92-106; Mohan et al. (2010) Genes Dev. 24, 574-589; Mueller et al. (2007) Blood 1 10, 4445-4454; Mueller et al. (2009) PLoS Biol 7, el 000249; Okada et al. (2005) Cell 121, 167-178; Park et al. (2010) Protein J 29, 213-223; Yokoyama et al. (2010) Cancer Cell 17, 198-212; Zhang et al. (2006) J Biol Chem 281 , 18059-18068).
  • DOT1L histone methyltransferase
  • translocation products retain gene-specific recognition elements within the remainder of the MLL protein, but also gain the ability to recruit DOT1L, to these locations (Monroe et al. (2010) Exp Hematol. 2010 Sepl 8. [Epub ahead of print] Pubmed PMID: 20854876; Mueller et al, 2007; Mueller et al, 2009; Okada et al., 2005).
  • DOT1L catalyzes the methylation of H3K79, a chromatin modification associated with actively transcribed genes (Feng et al. (2002) Curr Biol 12, 1052-1058; Steger et al. (2008) Mol Cell Biol 28, 2825-2839).
  • DOT1L is not genetically altered in the disease per se, its mislocated enzymatic activity is a direct consequence of the chromo'somal translocation affecting MLL patients; thus, DOT1L has been proposed to be a catalytic driver of leukemogenesis in this disease (Krivtsov et al, 2008; Monroe et al, 2010; Okada et al, 2005; Yokoyama et al. (2010) Cancer Cell 77, 198-212).
  • Such genetic lesions include chromosomal rearrangements, such as translocations, deletions, and/or duplications of the MLL gene.
  • MLL has been categorized or characterized as having a chimeric fusion of MLL, partial tandem duplication of the MLL gene (MLL-PTD), or nonrearranged MLL.
  • Chromosomal rearrangements or translocations can be identified by methods known in the art. For example, chromosomal rearrangements resulting in chimeric fusions can be detected by probe-based assays, such as FISH (fluorescence in situ hybridization) or sequence amplification by PCR.
  • FISH fluorescence in situ hybridization
  • MLL-PTD can be identified by DNA sequencing.
  • MLL chimeric fusions can be identified by FISH.
  • Diagnosis of MLL can be performed by detection of rearrangements of the MLL gene, or increased mRNA, protein, and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1 L, as further described herein.
  • Compounds of the present invention can selectively inhibit proliferation of tumor or tumor cells characterized with an increased mRNA, protein and/or activity (function) level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1 L.
  • the present invention provides methods for treating or alleviating a symptom of leukemia characterized with an increased mRNA, protein and/or activity (function) level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L by a compound of the present invention, or a pharmaceutically acceptable salt, ester, prodrug, metabolite, polymorph or solvate thereof.
  • exemplary leukemia that may be treated is mixed linage leukemia (MLL).
  • MLL that can be treated by the compound of the present invention is chimeric fusion of MLL, partial tandem duplication of MLL (MLL-PTD) or nonrearranged MLL.
  • the present invention also provides methods for treating or alleviating a symptom of leukemia characterized by the presence of a genetic lesion of MLL.
  • this method comprises obtaining sample from the subject; detecting the presence of a genetic lesion of MLL in the sample; and when the genetic lesion is present in the sample, administering to the subject a therapeutically effective amount of a DOT1L inhibitor (i.e. , a compound listed in Table 1 or an N-oxide, pharmaceutically acceptable salt, polymorph, solvate (e.g., hydrate), and stereoisomer thereof).
  • a DOT1L inhibitor i.e. , a compound listed in Table 1 or an N-oxide, pharmaceutically acceptable salt, polymorph, solvate (e.g., hydrate), and stereoisomer thereof.
  • the genetic lesion is chimeric fusion of MLL or MLL-PTD.
  • the present invention also provides methods for treating a disorder medicated by translocation, deletion and/or duplication of a gene on chromosome 1 lq23, comprising administering to a subject in need thereof a therapeutically effective amount of a compound listed in Table 1, or an N-oxide, pharmaceutically acceptable salt, polymorph, solvate (e.g., hydrate), and stereoisomer thereof.
  • the present invention provides personalized medicine, treatment and/or cancer management for a subject by genetic screening of increased gene expression (mRNA or protein), and/or increased function or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L in the subject.
  • the present invention provides methods for treating, preventing or alleviating a symptom of cancer or a precancerous condition by determining responsiveness of the subject to a DOT1L inhibitor and when the subject is responsive to the DOT1L inhibitor, administering to the subject a therapeutically effective amount of the DOT1L inhibitor, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, solvate, or stereoisomeror thereof.
  • the responsiveness is determined by obtaining a sample from the subject and detecting increased mRNA or protein, and/or increased activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, and the presence of such gain of expression and/or function indicates that the subject is responsive to the DOT1L inhibitor.
  • a therapeutically effective amount of a DOT1L inhibitor for example, any compound from Table 1, can be administered.
  • the therapeutically effective amount of a DOT1L inhibitor can be determined by one of ordinary skill in the art.
  • responsiveness is interchangeable with terms “responsive”, “sensitive”, and “sensitivity”, and it is meant that a subject is showing therapeutic responses when administered an DOT1L inhibitor, e.g. , tumor cells or tumor tissues of the subject undergo apoptosis and/or necrosis, and/or display reduced growing, dividing, or proliferation.
  • DOT1L inhibitor e.g., tumor cells or tumor tissues of the subject undergo apoptosis and/or necrosis, and/or display reduced growing, dividing, or proliferation.
  • a "subject” is interchangeable with a "subject in need thereof, both of which refer to a subject having a disorder in which DOTlL-mediated protein methylation plays a part, or a subject having an increased risk of developing such disorder relative to the population at large.
  • a subject in need thereof may be a subject having a disorder associated with DOT1L.
  • a subject in need thereof can have a precancerous condition.
  • a subject in need thereof has cancer.
  • a subject in need thereof can have cancer associated with DOT1L.
  • a subject in need thereof can have cancer associated with increased expression (mRNA or protein) and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L.
  • a subject in need thereof has one or more cancers selected from the group consisting of brain and central nervous system (CNS) cancer, head and neck cancer, kidney cancer, ovarian cancer, pancreatic cancer, leukemia, lung cancer, lymphoma, myeloma, sarcoma, breast cancer, prostate cancer and a hematological cancer.
  • CNS central nervous system
  • a subject in need thereof has a hematologic cancer, wherein the hematologic cancer is leukemia or lymphoma.
  • hematologic cancer is leukemia or lymphoma.
  • MLL myeloma
  • lymphoma including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childliood lymphomas, and lymphomas of lymphocytic and cutaneous origin
  • leukemia including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia
  • myeloid neoplasms and mast cell neoplasms can include multiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childliood lymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia (including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lympho
  • a "subject" includes a mammal.
  • the mammal can be e.g., a human or appropriate non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig.
  • the subject can also be a bird or fowl.
  • the mammal is a human.
  • a subject can be male or female.
  • a subject in need thereof can be one who has been previously diagnosed or identified as having cancer or a precancerous condition.
  • a subject in need thereof can also be one who is having (suffering from) cancer or a precancerous condition.
  • a subject in need thereof can be one who is having an increased risk of developing such disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large).
  • a subject in need thereof has already undergone, is undergoing or will undergo, at least one therapeutic intervention for the cancer or precancerous condition.
  • a subject in need thereof may have refractory cancer on most recent therapy.
  • Refractory cancer means cancer that does not respond to treatment.
  • the cancer may be resistant at the beginning of treatment or it may become resistant during treatment.
  • Refractory cancer is also called resistant cancer.
  • the subject in need thereof has cancer recurrence following remission on most recent therapy.
  • the subject in need thereof received and failed all known effective therapies for cancer treatment.
  • the subject in need thereof received at least one prior therapy.
  • a subject in need thereof may have a secondary cancer as a result of a previous therapy.
  • Secondary cancer means cancer that arises due to or as a result from previous carcinogenic therapies, such as chemotherapy.
  • the secondary cancer is a hematologic cancer, such as leukemia.
  • a subject in need thereof may have increased mRNA, protein, and/or activity level of at least of at least one signaling component downstream of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL.
  • at least one signaling component downstream of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL.
  • downstream components are readily known in the art, and can include other transcription factors, or signaling proteins.
  • the term "increase in activity” refers to increased or a gain of function of a gene product/protein compared to the wild type. In one aspect of the present invention, increased activity can be caused by increased mRNA and/or increased protein levels.
  • Increased mRNA levels can be caused by gene amplification and increased transcription, for example. Increased protein levels can be caused by increased stability, inhibition of degradation pathways, or increased transcription. Alternatively, increased activity levels can be caused by a gain of function mutation resulting from a point mutation (e.g., a substitution, a missense mutation, or a nonsense mutation), an insertion, and/or a deletion, or a rearrangement in a polypeptide selected from the group consisting of HOXA9, FLT3, MEIS l and DOTIL, or a nucleic acid sequence encoding a polypeptide selected from the group consisting of HOXA9, FLT3, MEIS l and DOTI L.
  • the mutations referred herein are somatic mutations.
  • wild-type refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or "wild-type” form of the gene.
  • an increase in mRNA or protein expression and/or activity levels can be detected using any suitable method available in the art.
  • an increase in activity level can be detected by measuring the biological function of a gene product, such as the histone methyltransferase activity of DOT1L (i.e., methylation of histone substrates such as H3K79 by immunoblot); transcriptional activity of HOXA9 or MEIS1 (i.e., expression levels of HOXA9 or MEIS1 target genes by RT-PCR); or phosphorylation activity of FLT3 (i.e., phosphorylation status of FLT3 targets by immunoblot or radioimmunoassay).
  • a gene product such as the histone methyltransferase activity of DOT1L (i.e., methylation of histone substrates such as H3K79 by immunoblot); transcriptional activity of HOXA9 or MEIS1 (i.e., expression levels of HOXA9 or MEIS1 target genes by RT
  • a gain of function mutation can be determined by detecting any alternation in a nucleic acid sequence encoding a protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L.
  • a nucleic acid sequence encoding HOXA9, FLT3, MEIS1 and DOT1L having a gain of function mutation can be detected by whole-genome resequencing or target region resequencing (the latter also known as targeted resequencing) using suitably selected sources of DNA and polymerase chain reaction (PCR) primers in accordance with methods well known in the art.
  • PCR polymerase chain reaction
  • the method typically and generally entails the steps of genomic DNA purification, PCR amplification to amplify the region of interest, cycle sequencing, sequencing reaction cleanup, capillary electrophoresis, and/or data analysis.
  • the method may include the use of microarray-based targeted region genomic DNA capture and/or sequencing. Kits, reagents, and methods for selecting appropriate PCR primers and performing resequencing are commercially available, for example, from Applied Biosystems, Agilent, and NimbleGen (Roche Diagnostics GmbH).
  • Detection of mRNA expression can be detected by methods known in the art, such as Northern blot, nucleic acid PCR, and quantitative RT-PCR. Detection of polypeptide expression (i.e., wild-type or mutant) can be carried out with any suitable immunoassay in the art, such as Western blot analysis.
  • sample means any biological sample derived from the subject, includes but is not limited to, cells, tissues samples, body fluids (including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen), tumor cells, and tumor tissues.
  • body fluids including, but not limited to, mucus, blood, plasma, serum, urine, saliva, and semen
  • tumor cells and tumor tissues.
  • the sample is selected from bone marrow, peripheral blood cells, blood, plasma and serum. Samples can be provided by the subject under treatment or testing. Alternatively samples can be obtained by the physician according to routine practice in the art.
  • the present invention also provides methods for diagnosing leukemia in a subject by obtaining a sample from the subject and detecting an increased mRNA, protein and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL, and the presence of such increased mRNA, protein and/or activity level indicates that the subject has or is at risk for developing leukemia compared to a subject without such increased mRNA, protein and/or activity level, or a subject that does not have leukemia.
  • the present invention also provides methods for determining predisposition of a subject to leukemia by obtaining a sample from the subject and detecting an increased mRNA, protein and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL, and the presence of such increased mRNA, protein and/or activity level indicates that the subject is predisposed to (i.e., having higher risk of) developing leukemia compared to a subject without such increased mRNA, protein and/or activity level.
  • predisposed as used herein in relation to cancer or a precancerous condition is to be understood to mean the increased probability (e.g., at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more increase in probability) that a subject with an increased mRNA, protein and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL, will suffer leukemia, as compared to the probability that another subject not having an increased mRNA, protein and/or activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEISl and DOTIL, will suffer leukemia, under circumstances where other risk factors (e.g. , chemical/environment, food, and smoking history, etc.) for having leukemia between the subjects are the same.
  • risk factors e.g. , chemical/environment, food, and smoking history, etc.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post- measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion.
  • the present invention provides methods of cancer management in a subject by determining predisposition of the subject to a cancer or a precancerous condition periodically.
  • the methods comprise steps of obtaining a sample from the subject and detecting increased rnRNA or protein, and/or increased activity level of at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L, and the presence of such gain of expression and/or function indicates that the subject is predisposed to developing the cancer or the precancerous condition compared to a subject without such gain of mRNA or protein expression and/or function of the at least one protein selected from the group consisting of HOXA9, FLT3, MEIS1 and DOT1L.
  • Any compound (e.g., DOT1L inhibitor) of the present invention can be used for the methods described above.
  • cell proliferative disorder refers to conditions in which unregulated or abnormal growth, or both, of cells can lead to the development of an unwanted condition or disease, which may or may not be cancerous.
  • Exemplary cell proliferative disorders of the invention encompass a variety of conditions wherein cell division is deregulated.
  • Exemplary cell proliferative disorder include, but are not limited to, neoplasms, benign tumors, malignant tumors, pre-cancerous conditions, in situ tumors, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells.
  • the term "rapidly dividing cell” as used herein is defined as any cell that divides at a rate that exceeds or is greater than what is expected or observed among neighboring or juxtaposed cells within the same tissue.
  • a cell proliferative disorder includes a precancer or a precancerous condition.
  • a cell proliferative disorder includes cancer.
  • the methods provided herein are used to treat or alleviate a symptom of cancer.
  • the term “cancer” includes solid tumors, as well as, hematologic tumors and/or malignancies.
  • a "precancer cell” or “precancerous cell” is a cell manifesting a cell proliferative disorder that is a precancer or a precancerous condition.
  • a “cancer cell” or “cancerous cell” is a cell manifesting a cell proliferative disorder that is a cancer. Any reproducible means of measurement may be used to identify cancer cells or precancerous cells. Cancer cells or precancerous cells can be identified by histological typing or grading of a tissue sample (e.g., a biopsy sample). Cancer cells or precancerous cells can be identified through the use of appropriate molecular markers.
  • non-cancerous conditions or disorders include, but are not limited to, rheumatoid arthritis; inflammation; autoimmune disease; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout, other arthritic conditions; sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic renal disease; irritable bowel syndrome; pyresis; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute and chronic pain; allergic rhinitis; allergic conjunctivitis; chronic
  • Exemplary cancers include, but are not limited to, adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extraliepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma,
  • medulloblastoma supratentorial primitive neuroectodeimal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal
  • a "cell proliferative disorder of the hematologic system” is a cell proliferative disorder involving cells of the hematologic system.
  • a cell proliferative disorder of the hematologic system can include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic myelocytic leukemia, agnogenic myeloid metaplasia, and essential thrombocythemia.
  • a cell proliferative disorder of the hematologic system can include hyperplasia, dysplasia, and metaplasia of cells of the hematologic system.
  • compositions of the present invention may be used to treat a cancer selected from the group consisting of a hematologic cancer of the present invention or a hematologic cell proliferative disorder of the present invention.
  • a hematologic cancer of the present invention can include multiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia (including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms and mast cell neoplasms.
  • lymphoma including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin
  • leukemia including childhood leukemia, hairy-cell leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic
  • a "cell proliferative disorder of the lung” is a cell proliferative disorder involving cells of the lung.
  • Cell proliferative disorders of the lung can include all forms of cell proliferative disorders affecting lung cells.
  • Cell proliferative disorders of the lung can include lung cancer, a precancer or precancerous condition of the lung, benign growths or lesions of the lung, and malignant growths or lesions of the lung, and metastatic lesions in tissue and organs in the body other than the lung.
  • compositions of the present invention may be used to treat lung cancer or cell proliferative disorders of the lung.
  • Lung cancer can include all forms of cancer of the lung.
  • Lung cancer can include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors.
  • Lung cancer can include small cell lung cancer ("SCLC"), non-small cell lung cancer ("NSCLC”), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, adenosquamous cell carcinoma, and mesothelioma.
  • Lung cancer can include "scar carcinoma,” bronchioalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma.
  • Lung cancer can include lung neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).
  • Cell proliferative disorders of the lung can include all forms of cell proliferative disorders affecting lung cells.
  • Cell proliferative disorders of the lung can include lung cancer, precancerous conditions of the lung.
  • Cell proliferative disorders of the lung can include hyperplasia, metaplasia, and dysplasia of the lung.
  • Cell proliferative disorders of the lung can include asbestos-induced hyperplasia, squamous metaplasia, and benign reactive mesothelial metaplasia.
  • Cell proliferative disorders of the lung can include replacement of columnar epithelium with stratified squamous epithelium, and mucosal dysplasia.
  • Prior lung diseases that may predispose individuals to development of cell proliferative disorders of the lung can include chronic interstitial lung disease, necrotizing pulmonary disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathic pulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, and Hodgkin's disease.
  • a "cell proliferative disorder of the colon” is a cell proliferative disorder involving cells of the colon.
  • the cell proliferative disorder of the colon is colon cancer.
  • compositions of the present invention may be used to treat colon cancer or cell proliferative disorders of the ' colon.
  • Colon cancer can include all forms of cancer of the colon.
  • Colon cancer can include sporadic and hereditary colon cancers.
  • Colon cancer can include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors.
  • Colon cancer can include adenocarcinoma, squamous cell carcinoma, and adenosquamous cell carcinoma.
  • Colon cancer can be associated with a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot 's syndrome and juvenile polyposis.
  • Colon cancer can be caused by a hereditary syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Koz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.
  • Cell proliferative disorders of the colon can include all forms of cell proliferative disorders affecting colon cells.
  • Cell proliferative disorders of the colon can include colon cancer, precancerous conditions of the colon, adenomatous polyps of the colon and metachronous lesions of the colon.
  • a cell proliferative disorder of the colon can include adenoma.
  • Cell proliferative disorders of the colon can be characterized by hyperplasia, metaplasia, and dysplasia of the colon.
  • Prior colon diseases that may predispose individuals to development of cell proliferative disorders of the colon can include prior colon cancer.
  • Current disease that may predispose individuals to development of cell proliferative disorders of the colon can include Crohn's disease and ulcerative colitis.
  • a cell proliferative disorder of the colon can be associated with a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.
  • An individual can have an elevated risk of developing a cell proliferative disorder of the colon due to the presence of a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.
  • a "cell proliferative disorder of the pancreas” is a cell proliferative disorder involving cells of the pancreas.
  • Cell proliferative disorders of the pancreas can include all forms of cell proliferative disorders affecting pancreatic cells.
  • Cell proliferative disorders of the pancreas can include pancreas cancer, a precancer or precancerous condition of the pancreas, hyperplasia of the pancreas, and dysaplasia of the pancreas, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, and metastatic lesions in tissue and organs in the body other than the pancreas.
  • Pancreatic cancer includes all forms of cancer of the pancreas.
  • Pancreatic cancer can include ductal adenocarcinoma,
  • pancreatic cancer can also include pancreatic neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).
  • a "cell proliferative disorder of the prostate” is a cell proliferative disorder involving cells of the prostate.
  • Cell proliferative disorders of the prostate can include all forms of cell proliferative disorders affecting prostate cells.
  • Cell proliferative disorders of the prostate can include prostate cancer, a precancer or precancerous condition of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, and metastatic lesions in tissue and organs in the body other than the prostate.
  • Cell proliferative disorders of the prostate can include hyperplasia, metaplasia, and dysplasia of the prostate.
  • a "cell proliferative disorder of the skin” is a cell proliferative disorder involving cells of the skin.
  • Cell proliferative disorders of the skin can include all forms of cell proliferative disorders affecting skin cells.
  • Cell proliferative disorders of the skin can include a precancer or precancerous condition of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissue and organs in the body other than the skin.
  • Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of the skin.
  • a "cell proliferative disorder of the ovary” is a cell proliferative disorder involving cells of the ovary.
  • Cell proliferative disorders of the ovary can include all forms of cell proliferative disorders affecting cells of the ovary.
  • Cell proliferative disorders of the ovary can include a precancer or precancerous condition of the ovary, benign growths or lesions of the ovary, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissue and organs in the body other than the ovary.
  • Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of cells of the ovary.
  • a "cell proliferative disorder of the breast” is a cell proliferative disorder involving cells of the breast.
  • Cell proliferative disorders of the breast can include all forms of cell proliferative disorders affecting breast cells.
  • Cell proliferative disorders of the breast can include breast cancer, a precancer or precancerous condition of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, and metastatic lesions in tissue and organs in the body other than the breast.
  • Cell proliferative disorders of the breast can include hyperplasia, metaplasia, and dysplasia of the breast.
  • a cell proliferative disorder of the breast can be a precancerous condition of the breast.
  • Compositions of the present invention may be used to treat a precancerous condition of the breast.
  • a precancerous condition of the breast can include atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0 growth or lesion of the breast ⁇ e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).
  • a precancerous condition of the breast can be staged according to the TNM classification scheme as accepted by the American Joint Committee on Cancer (AJCC), where the primary tumor (T) has been assigned a stage of TO or Tis; and where the regional lymph nodes (N) have been assigned a stage of NO; and where distant metastasis (M) has been assigned a stage of MO.
  • AJCC American Joint Committee on Cancer
  • the cell proliferative disorder of the breast can be breast cancer.
  • compositions of the present invention may be used to treat breast cancer.
  • Breast cancer includes all forms of cancer of the breast.
  • Breast cancer can include primary epithelial breast cancers.
  • Breast cancer can include cancers in which the breast is involved by other tumors such as lymphoma, sarcoma or melanoma.
  • Breast cancer can include carcinoma of the breast, ductal carcinoma of the breast, lobular carcinoma of the breast, undifferentiated carcinoma of the breast, cystosarcoma phyllodes of the breast, angiosarcoma of the breast, and primary lymphoma of the breast.
  • Breast cancer can include Stage I, II, IIIA, IIIB, IIIC and IV breast cancer.
  • Ductal carcinoma of the breast can include invasive carcinoma, invasive carcinoma in situ with predominant intraductal component, inflammatory breast cancer, and a ductal carcinoma of the breast with a histologic type selected from the group consisting of comedo, mucinous (colloid), medullary, medullary with lymphcytic infiltrate, papillary, scirrhous, and tubular.
  • Lobular carcinoma of the breast can include invasive lobular carcinoma with predominant in situ component, invasive lobular carcinoma, and infiltrating lobular carcinoma.
  • Breast cancer can include Paget's disease, Paget' s disease with intraductal carcinoma, and Paget's disease with invasive ductal carcinoma.
  • Breast cancer can include breast neoplasms having histologic and ultrastructual heterogeneity (e.g., mixed cell types).
  • compound of the present invention may be used to treat breast cancer.
  • a breast cancer that is to be treated can include familial breast cancer.
  • a breast cancer that is to be treated can include sporadic breast cancer.
  • a breast cancer that is to be treated can arise in a male subject.
  • a breast cancer that is to be treated can arise in a female subject.
  • a breast cancer that is to be treated can arise in a premenopausal female subject or a postmenopausal female subject.
  • a breast cancer that is to be treated can arise in a subject equal to or older than 30 years old, or a subject younger than 30 years old.
  • a breast cancer that is to be treated has arisen in a subject equal to or older than 50 years old, or a subject younger than 50 years old.
  • a breast cancer that is to be treated can arise in a subject equal to or older than 70 years old, or a subject younger than 70 years old.
  • a breast cancer that is to be treated can be typed to identify a familial or spontaneous mutation in BRCA1 , BRCA2, or p53.
  • a breast cancer that is to be treated can be typed as having a HER2/neu gene amplification, as overexpressing HER2/neu, or as having a low, intermediate or high level of HER2/neu expression.
  • a breast cancer that is to be treated can be typed for a marker selected from the group consisting of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.
  • ER estrogen receptor
  • PR progesterone receptor
  • Ki-67 human epidermal growth factor receptor-2
  • Ki-67 Ki-67
  • CA15-3 CA 27-29
  • CA 27-29 CA 27-29
  • c-Met c-Met
  • a breast cancer that is to be treated can be typed as ER -negative or ER-positive.
  • ER-typing of a breast cancer may be performed by any reproducible means. ER-typing of a breast cancer may be performed as set forth in Onkologie 27: 175-179 (2004).
  • a breast cancer that is to be treated can be typed as PR-unknown, PR-rich, or PR-poor.
  • a breast cancer that is to be treated can be typed as PR-negative or PR-positive.
  • a breast cancer that is to be treated can be typed as receptor positive or receptor negative.
  • a breast cancer that is to be treated can be typed as being associated with elevated blood levels of CA 15-3, or CA 27-29, or both.
  • a breast cancer that is to be treated can include a localized tumor of the breast.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with a negative sentinel lymph node (SLN) biopsy.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with a positive sentinel lymph node (SLN) biopsy.
  • a breast cancer that is to be treated can include a tumor of the breast that is associated with one or more positive axillary lymph nodes, where the axillary lymph nodes have been staged by any applicable method.
  • a breast cancer that is to be treated can include a tumor of the breast that has been typed as having nodal negative status (e.g., node-negative) or nodal positive status (e.g., node-positive).
  • a breast cancer that is to be treated can include a tumor of the breast that has metastasized to other locations in the body.
  • a breast cancer that is to be treated can be classified as having metastasized to a location selected from the group consisting of bone, lung, liver, or brain.
  • a breast cancer that is to be treated can be classified according to a characteristic selected from the group consisting of metastatic, localized, regional, local-regional, locally advanced, distant, multicentric, bilateral, ipsilateral, contralateral, newly diagnosed, recurrent, and inoperable.
  • a compound of the present invention may be used to treat or prevent a cell proliferative disorder of the breast, or to treat or prevent breast cancer, in a subject having an increased risk of developing breast cancer relative to the population at large.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject with a family history or personal history of breast cancer.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject having a germ- line or spontaneous mutation in BRCA1 or BRCA2, or both.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female subject with a family history of breast cancer and a genn-line or spontaneous mutation in BRCA1 or BRCA2, or both.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a female who is greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, greater than 80 years old, or greater than 90 years old.
  • a subject with an increased risk of developing breast cancer relative to the population at large is a subject with atypical hyperplasia of the breast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, or a stage 0 growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).
  • DCIS ductal carcinoma in situ
  • LCIS lobular carcinoma in situ
  • lobular neoplasia or a stage 0 growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).
  • a breast cancer that is to be treated can histologically graded according to the Scarff- Bloom-Richardson system, wherein a breast tumor has been assigned a mitosis count score of 1 , 2, or 3; a nuclear pleiomorphism score of 1 , 2, or 3; a tubule formation score of 1 , 2, or 3; and a total Scarff-Bloom-Richardson score of between 3 and 9.
  • a breast cancer that is to be treated can be assigned a tumor grade according to the International Consensus Panel on the Treatment of Breast Cancer selected from the group consisting of grade 1 , grade 1 -2, grade 2, grade 2-3, or grade 3.
  • a cancer that is to be treated can be staged according to the American Joint
  • TNM classification system where the tumor (T) has been assigned a stage of TX, Tl , Tlmic, Tl a, Ti b, Tic, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N) have been assigned a stage of NX, NO, Nl , N2, N2a, N2b, N3, N3a, N3b, or N3c; and where distant metastasis (M) can be assigned a stage of MX, MO, or Ml .
  • a cancer that is to be treated can be staged according to an American Joint Committee on Cancer (AJCC) classification as Stage I, Stage IIA, Stage IEB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV.
  • AJCC American Joint Committee on Cancer
  • a cancer that is to be treated can be assigned a grade according to an AJCC classification as Grade GX (e.g., grade cannot be assessed), Grade 1 , Grade 2, Grade 3 or Grade 4.
  • a cancer that is to be treated can be staged according to an AJCC pathologic classification (pN) of pNX, pNO, PNO (I-), PNO (I+), PNO (mol-), PNO (mol+), PN1 , PNl (mi), PNl a, PNl b, PNl c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.
  • pN AJCC pathologic classification
  • a cancer that is to be treated can include a tumor that has been determined to be less than or equal to about 2 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be from about 2 to about 5 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be greater than or equal to about 3 centimeters in diameter.
  • a cancer that is to be treated can include a tumor that has been determined to be greater than 5 centimeters in diameter.
  • a cancer that is to be treated can be classified by microscopic appearance as well differentiated, moderately differentiated, poorly differentiated, or undifferentiated.
  • a cancer that is to be treated can be classified by microscopic appearance with respect to mitosis count (e.g., amount of cell division) or nuclear pleiomorphism (e.g., change in cells).
  • a cancer that is to be treated can be classified by microscopic appearance as being associated with areas of necrosis (e.g., areas of dying or degenerating cells).
  • a cancer that is to be treated can be classified as having an abnormal karyotype, having an abnormal number of chromosomes, or having one or more chromosomes that are abnormal in appearance.
  • a cancer that is to be treated can be classified as being aneuploid, triploid, tetraploid, or as having an altered ploidy.
  • a cancer that is to be treated can be classified as having a chromosomal translocation, or a deletion or duplication of an entire chromosome, or a region of deletion, duplication or amplification of a portion of a chromosome.
  • a cancer that is to be treated can be evaluated by DNA cytometry, flow cytometry, or image cytometry.
  • a cancer that is to be treated can be typed as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division (e.g., in S phase of cell division).
  • a cancer that is to be treated can be typed as having a low S-phase fraction or a high S-phase fraction.
  • a "normal cell” is a cell that cannot be classified as part of a "cell proliferative disorder”.
  • a normal cell lacks unregulated or abnormal growth, or both, that can lead to the development of an unwanted condition or disease.
  • a normal cell possesses normally functioning cell cycle checkpoint control mechanisms.
  • contacting a cell refers to a condition in which a compound or other composition of matter is in direct contact with a cell, or is close enough to induce a desired biological effect in a cell.
  • candidate compound refers to a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, that has been or will be tested in one or more in vitro or in vivo biological assays, in order to determine if that compound is likely to elicit a desired biological or medical response in a cell, tissue, system, animal or human that is being sought by a researcher or clinician.
  • a candidate compound is a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof.
  • the biological or medical response can be the treatment of cancer.
  • the biological or medical response can be treatment or prevention of a cell proliferative disorder.
  • In vitro or in vivo biological assays can include, but are not limited to, enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.
  • an in vitro biological assay that can be used includes the steps of (1) mixing a histone substrate (e.g., an isolated histone sample for a histone or modified histone of interest, or an isolated oligonucleosome substrate) with recombinant DOTIL enzyme (e.g., recombinant protein containing amino acids 1 -416); (2) adding a candidate compound of the invention to this mixture; (3) adding non-radioactive and ⁇ -labeled S-Adenosyl methionine (SAM) to start the reaction; (4) adding excessive amount of non-radioactive SAM to stop the reaction; (4) washing off the free non-incorporated H-SAM; and (5) detecting the quantity of 3 H-labeled histone substrate by any methods known in the art (e.g., by a PerkinElmer TopCount platereader).
  • a histone substrate e.g., an isolated histone sample for a histone or modified histone of interest, or an isolated oligonucleosome substrate
  • an in vitro cell viability assay that can be used includes the steps of (1) culturing cells (e.g., EOL-1 cells) in the presence of increasing concentration of a candidate compound; (2) determining viable cell number every 3-4 days by methods known in the art (e.g. , using the Millipore Guava Viacount assay); (3) plotting concentration-dependence growth curves; and optionally (4) calculating IC50 values from the concentration-dependence growth curves using methods known in the art (e.g., using GraphPad Prism Software).
  • culturing cells e.g., EOL-1 cells
  • determining viable cell number every 3-4 days e.g. , using the Millipore Guava Viacount assay
  • concentration-dependence growth curves e.g., using GraphPad Prism Software
  • a histone methylation assay that can be used includes the steps of (1) culturing cells (e.g., EOL-1 cells) in the presence of a candidate compound; (2) harvesting the cells; (3) extracting histone proteins, using methods known in the art (e.g., sulfuric acid precipitation); (4) fractionating histone extracts by SDS-PAGE electrophoresis and transferring to a filter; (5) probing the filter with antibodies specific to a protein or methylated-protein of interest (e.g., H3K79me2-specific antibody and total histone IB- specific antibody); and (6) detecting the signal of the antibodies using methods known in the art (e.g. , Li-cor Odyssey infrared imager).
  • a gene expression assay that can be used includes the steps of (1) culturing cells (e.g., EOL-1 , Molml 3, MV411 , LOUCY, SemK2, Reh, HL60, BV173, or Jurkat cells) in the presence or absence of a candidate compound; (2) harvesting the cells; (3) extracting the RNA using methods known in the art (e.g., Qiagen RNeasy Kit); (4) synthesizing cDNA from the extracted RNA (e.g., Applied Biosystems reverse transcriptase kit); (5) preparing qPCR reactions using, for example, primers and probes (e.g., predesigned labeled primer and probe sets for HOXA9, MEIS1 , FLT3, DOT1L, and P2-microglobulin from Applied Biosystems), synthesized sample cDNA, and qPCR master mix reagent (e.g., Applied Biosystems Taqman universal PCR master mix); (6) running
  • monotherapy refers to the administration of a single active or therapeutic compound to a subject in need thereof.
  • monotherapy will involve administration of a therapeutically effective amount of a single active compound.
  • cancer monotherapy with one of the compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, to a subject in need of treatment of cancer.
  • the single active compound is a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof.
  • treating describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder.
  • a compound of the present invention can also be used to prevent a disease, condition or disorder.
  • preventing or “prevent” describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder.
  • the term "alleviate” is meant to describe a process by which the severity of a sign or symptom of a disorder is decreased.
  • a sign or symptom can be alleviated without being eliminated.
  • the administration of phannaceutical compositions of the invention leads to the elimination of a sign or symptom, however, elimination is not required.
  • Effective dosages are expected to decrease the severity of a sign or symptom.
  • a sign or symptom of a disorder such as cancer, which can occur in multiple locations, is alleviated if the severity of the cancer is decreased within at least one of multiple locations.
  • severity is meant to describe the potential of cancer to transform from a precancerous, or benign, state into a malignant state.
  • severity is meant to describe a cancer stage, for example, according to the TNM system (accepted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other art-recognized methods.
  • TNM system accepted by the International Union against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)
  • UNM system International Union against Cancer
  • AJCC American Joint Committee on Cancer
  • Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (spread of cancer into lymph nodes).
  • Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary with each type of cancer. Severity also describes a histologic grade, also called differentiation, which refers to how much the tumor cells resemble normal cells of the same tissue type (see, National Cancer Institute, www.cancer.gov). Furthermore, severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).
  • severity describes the degree to which a tumor has secreted growth factors, degraded the extracellular matrix, become vascularized, lost adhesion to juxtaposed tissues, or metastasized. Moreover, severity describes the number of locations to which a primary tumor has metastasized. Finally, severity includes the difficulty of treating tumors of varying types and locations. For example, inoperable tumors, those cancers which have greater access to multiple body systems (hematological and immunological tumors), and those which are the most resistant to traditional treatments are considered most severe.
  • symptom is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. Others are defined as non-health-care professionals.
  • signs are also defined as an indication that something is not right in the body. But signs are defined as things that can be seen by a doctor, nurse, or other health care professional.
  • Cancer is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.
  • pancreas cancers for example, do not usually grow large enough to be felt from the outside of the body. Some pancreatic cancers do not cause symptoms until they begin to grow around nearby nerves (this causes a backache). Others grow around the bile duct, which blocks the flow of bile and leads to a yellowing of the skin known as jaundice. By the time a pancreatic cancer causes these signs or symptoms, it has usually reached an advanced stage.
  • a cancer may also cause symptoms such as fever, fatigue, or weight loss. This may be because cancer cells use up much of the body's energy supply or release substances that change the body's metabolism. Or the cancer may cause the immune system to react in ways that produce these symptoms.
  • cancer cells release substances into the bloodstream that cause symptoms not usually thought to result from cancers.
  • some cancers of the pancreas can release substances which cause blood clots to develop in veins of the legs.
  • Some lung cancers make hormone-like substances that affect blood calcium levels, affecting nerves and muscles and causing weakness and dizziness
  • Cancer presents several general signs or symptoms that occur when a variety of subtypes of cancer cells are present. Most people with cancer will lose weight at some time with their disease. An unexplained (unintentional) weight loss of 10 pounds or more may be the first sign of cancer, particularly cancers of the pancreas, stomach, esophagus, or lung.
  • Fever is very common with cancer, but is more often seen in advanced disease. Almost all patients with cancer will have fever at some time, especially if the cancer or its treatment affects the immune system and makes it harder for the body to fight infection. Less often, fever may be an early sign of cancer, such as with leukemia or lymphoma.
  • Fatigue may be an important symptom as cancer progresses. It may happen early, though, in cancers such as with leukemia, or if the cancer is causing an ongoing loss of blood, as in some colon or stomach cancers.
  • cancer subtypes present specific signs or symptoms. Changes in bowel habits or bladder function could indicate cancer. Long-term constipation, diarrhea, or a change in the size of the stool may be a sign of colon cancer. Pain with urination, blood in the urine, or a change in bladder function (such as more frequent or less frequent urination) could be related to bladder or prostate cancer.
  • Changes in skin condition or appearance of a new skin condition could indicate cancer.
  • Skin cancers may bleed and look like sores that do not heal.
  • a long-lasting sore in the mouth could be an oral cancer, especially in patients who smoke, chew tobacco, or frequently drink alcohol. Sores on the penis or vagina may either be signs of infection or an early cancer.
  • Unusual bleeding or discharge could indicate cancer. Unusual bleeding can happen in either early or advanced cancer. Blood in the sputum (phlegm) may be a sign of lung cancer. Blood in the stool (or a dark or black stool) could be a sign of colon or rectal cancer. Cancer of the cervix or the endometrium (lining of the uterus) can cause vaginal bleeding. Blood in the urine may be a sign of bladder or kidney cancer. A bloody discharge from the nipple may be a sign of breast cancer.
  • a thickening or lump in the breast or in other parts of the body could indicate the presence of a cancer. Many cancers can be felt through the skin, mostly in the breast, testicle, lymph nodes (glands), and the soft tissues of the body. A lump or thickening may be an early or late sign of cancer. Any lump or thickening could be indicative of cancer, especially if the formation is new or has grown in size.
  • Indigestion or trouble swallowing could indicate cancer. While these symptoms commonly have other causes, indigestion or swallowing problems may be a sign of cancer of the esophagus, stomach, or pharynx (throat).
  • Recent changes in a wart or mole could be indicative of cancer. Any wart, mole, or freckle that changes in color, size, or shape, or loses its definite borders indicates the potential development of cancer.
  • the skin lesion may be a melanoma.
  • a persistent cough or hoarseness could be indicative of cancer.
  • a cough that does not go away may be a sign of lung cancer.
  • Hoarseness can be a sign of cancer of the larynx (voice box) or thyroid.
  • Treating cancer can result in a reduction in size of a tumor.
  • a reduction in size of a tumor may also be referred to as "tumor regression".
  • tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.
  • Treating cancer can result in a reduction in tumor volume.
  • tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Tumor volume may be measured by any reproducible means of measurement.
  • Treating cancer results in a decrease in number of tumors.
  • tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • Number of tumors may be measured by any reproducible means of measurement.
  • the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification.
  • the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.
  • Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site.
  • the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • the number of metastatic lesions may be measured by any reproducible means of measurement.
  • the number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof.
  • the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%.
  • a decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means.
  • a decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound.
  • a decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in tumor growth rate.
  • tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, 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, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.
  • Treating cancer can result in a decrease in tumor regrowth.
  • tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%.
  • Tumor regrowth may be measured by any reproducible means of
  • Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation.
  • the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the rate of cellular proliferation may be measured by any reproducible means of measurement.
  • the rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
  • Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells.
  • the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the proportion of proliferating cells may be measured by any reproducible means of measurement.
  • the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample.
  • the proportion of proliferating cells can be equivalent to the mitotic index.
  • Treating or preventing a cell proliferative disorder can result in a decrease in size of an area or zone of cellular proliferation.
  • size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; 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, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • Size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement.
  • the size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.
  • Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology.
  • the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; 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, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement.
  • An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope.
  • An abnormal cellular morphology can take the form of nuclear pleiomorphism.
  • the term "selectively" means tending to occur at a higher frequency in one population than in another population.
  • the compared populations can be cell populations.
  • a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof acts selectively on a cancer or precancerous cell but not on a normal cell.
  • a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof acts selectively to modulate one molecular target (e.g., a target-protein
  • the invention also provides a method for selectively inhibiting the activity of an enzyme, such as a protein methyltransferase.
  • an event occurs selectively in population A relative to population B if it occurs greater than two times more frequently in population A as compared to population B.
  • An event occurs selectively if it occurs greater than five times more frequently in population A.
  • An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B.
  • cell death would be said to occur selectively in cancer cells if it occurred greater than twice as frequently in cancer cells as compared to normal cells.
  • a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof can modulate the activity of a molecular target (e.g., a target protein methyltransferase). Modulating refers to stimulating or inhibiting an activity of a molecular target.
  • a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 2-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • a compound of the present invention modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target by at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • the activity of a molecular target may be measured by any reproducible means.
  • the activity of a molecular target may be measured in vitro or in vivo.
  • the activity of a molecular target may be measured in vitro by an enzymatic activity assay or a DNA binding assay, or the activity of a molecular target may be measured in vivo by assaying for expression of a reporter gene.
  • a compound of the present invention does not significantly modulate the activity of a molecular target if the addition of the compound does not stimulate or inhibit the activity of the molecular target by greater than 10% relative to the activity of the molecular target under the same conditions but lacking only the presence of said compound.
  • the term "isozyme selective" means preferential inhibition or stimulation of a first isoform of an enzyme in comparison to a second isoform of an enzyme (e.g., preferential inhibition or stimulation of a protein methyltransferase isozyme alpha in comparison to a protein methyltransferase isozyme beta).
  • a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof demonstrates a minimum of a fourfold differential, preferably a tenfold differential, more preferably a fifty fold differential, in the dosage required to achieve a biological effect.
  • a compound of the present invention demonstrates this differential across the range of inhibition, and the differential is exemplified at the IC50, i.e., a 50% inhibition, for a molecular target of interest.
  • Administering a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to a cell or a subject in need thereof can result in modulation (i.e., stimulation or inhibition) of an activity of a protein
  • the present invention provides methods to assess biological activity of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof or methods of identifying a test compound as a modulator (e.g., an inhibitor) of DOT1L.
  • DOT1L polypeptides and nucleic acids can be used to screen for compounds that bind to and/or modulate (e.g., increase or decrease) one or more biological activities of DOT1L, including but not limited to H3K79 HMTase activity, SAM binding activity, histone and/or nucleosome binding activity, AF10 binding activity, AF10-MLL or other MLL fusion protein binding activity, and/or any other biological activity of interest.
  • a DOT1L polypeptide can be a functional fragment of a full-length DOT1 L polypeptide or functional equivalent thereof, and may comprise any DOT1 domain of interest, including but not limited to the catalytic domain, the SAM binding domain and/or the positively charged domain, the AF10 interaction domain and/or a nuclear export signal.
  • Methods of assessing DOT1L binding to histones, nucleosomes, nucleic acids or polypeptides can be carried out using standard techniques that will be apparent to those skilled in the art (see the Exemplification for exemplary methods). Such methods include yeast and mammalian two-hybrid assays and co-immunoprecipitation techniques.
  • a compound that modulates DOT1L H3K79 HMTase activity can be verified by: contacting a DOT1L polypeptide with a histone or peptide substrate comprising H3 in the presence of a test compound; detecting the level of H3K79 methylation of the histone or peptide substrate under conditions sufficient to provide H3K79 methylation, wherein an elevation or reduction in H3K79 methylation in the presence of the test compound as compared with the level of histone H3K79 methylation in the absence of the test compound indicates that the test compound modulates DOT1L H3K79 HMTase activity.
  • the screening methods of the invention can be carried out in a cell-based or cell-free system.
  • the assay can be performed in a whole animal (including transgenic non-human animals).
  • the DOT1L polypeptide (or any other polypeptide used in the assay) can be added directly to the cell or can be produced from a nucleic acid in the cell.
  • the nucleic acid can be endogenous to the cell or can be foreign (e.g., a genetically modified cell).
  • immunological reagents e.g., antibodies and antigens
  • Fluorescence can be utilized in the measurement of enzymatic activity in some assays.
  • fluorescence refers to a process through which a molecule emits a photon as a result of absorbing an incoming photon of higher energy by the same molecule. Specific methods for assessing the biological activity of the disclosed compounds are described in the examples.
  • Administering a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to a cell or a subject in need thereof results in modulation ⁇ i.e., stimulation or inhibition) of an activity of an intracellular target (e.g. , substrate).
  • an intracellular target e.g. , substrate
  • intracellular targets can be modulated with the compounds of the present invention, including, but not limited to, protein methyltrasferase.
  • Activating refers to placing a composition of matter (e.g., protein or nucleic acid) in a state suitable for carrying out a desired biological function.
  • a composition of matter capable of being activated also has an unactivated state.
  • An activated composition of matter may have an inhibitory or stimulatory biological function, or both.
  • Elevation refers to an increase in a desired biological activity of a composition of matter (e.g., a protein or a nucleic acid). Elevation may occur through an increase in concentration of a composition of matter.
  • a composition of matter e.g., a protein or a nucleic acid
  • a cell cycle checkpoint pathway refers to a biochemical pathway that is involved in modulation of a cell cycle checkpoint.
  • a cell cycle checkpoint pathway may have stimulatory or inhibitory effects, or both, on one or more functions comprising a cell cycle checkpoint.
  • a cell cycle checkpoint pathway is comprised of at least two compositions of matter, preferably proteins, both of which contribute to modulation of a cell cycle checkpoint.
  • a cell cycle checkpoint pathway may be activated through an activation of one or more members of the cell cycle checkpoint pathway.
  • a cell cycle checkpoint pathway is a biochemical signaling pathway.
  • cell cycle checkpoint regulator refers to a composition of matter that can function, at least in part, in modulation of a cell cycle checkpoint.
  • a cell cycle checkpoint regulator may have stimulatory or inhibitory effects, or both, on one or more functions comprising a cell cycle checkpoint.
  • a cell cycle checkpoint regulator can be a protein or not a protein.
  • Treating cancer or a cell proliferative disorder can result in cell death, and preferably, cell death results in a decrease of at least 10% in number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%.
  • Number of cells in a population may be measured by any reproducible means. A number of cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence microscopy and light microscopy. Methods of measuring cell death are as shown in Li et al, Proc Natl Acad Sci U S A. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis.
  • an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof is not significantly cytotoxic to normal cells.
  • a therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells.
  • a therapeutically effective amount of a compound does not significantly affect the viability of normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. In an aspect, cell death occurs by apoptosis.
  • administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, induces cell death selectively in one or more cells affected by a cell proliferative disorder.
  • the present invention relates to a method of treating or preventing cancer by administering a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, to a subject in need thereof, where administration of the compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, results in one or more of the following: accumulation of cells in Gl and/or S phase of the cell cycle, cytotoxicity via cell death in cancer cells without a significant amount of cell death in normal cells, antitumor activity in animals with a therapeutic index of at least 2, and activation of a cell cycle checkpoint.
  • therapeutic index is the maximum tolerated dose divided by the efficacious dose.
  • the present disclosure also provides a composition comprising a compound described herein, a N-oxide or a pharmaceutically acceptable salt thereof, and one or more therapeutic agents.
  • the present invention provides for the administration of a compound described herein, a N-oxide or a pharmaceutically acceptable salt thereof, and one or more therapeutic agents as a co- formulation or separate formulations, wherein the administration of formulations is simultaneous, sequential, or in alternation.
  • the one or more therapeutic agents can be an agent that is recognized in the art as being useful to treat the disease or condition being treated by the composition of the present invention.
  • the one or more therapeutic agents can be an agent that is not recognized in the art as being useful to treat the disease or condition being treated by the composition of the present invention.
  • the other therapeutic agents can be an agent that imparts a beneficial attribute to the composition of the present invention ⁇ e.g., an agent that affects the viscosity of the composition).
  • the beneficial attribute to the composition of the present invention includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of a compound of Formula (I) and one or more therapeutic agents.
  • the one or more therapeutic agents can be anticancer agents or chemotherapeutic agents.
  • the one or more therapeutic agents can be selected from Ara-C, Daunorubicin, Decitabine, Vidaza, Mitoxantrone, JQ1 , IBET151, Panobinostat, Vorinostat, Quizartinib, Midostaurin, Tranylcypromine, LSD 1 inhibitor II, Navitoclax, or functional analogs, derivatives, produgs, and metabolites thereof.
  • the therapeutic agent is Ara-C or Daunorubicin or functional analogs, derivatives, produgs, and metabolites thereof.
  • the therapeutic agents aretopoisomerase inhibitors (e.g., Mitoxantrone), liypomethylating agents ⁇ e.g., Decitabine or Vidaza), Bromodomain inhibitors ⁇ e.g., IBET-151 ), HDAC inhibitors ⁇ e.g., Panobinostat), Bcl-2 inhibitors ⁇ e.g., Navitoclax) or FLT inhibitors ⁇ e.g., Quizartinib).
  • Mitoxantrone liypomethylating agents
  • liypomethylating agents e.g., Decitabine or Vidaza
  • Bromodomain inhibitors ⁇ e.g., IBET-151
  • HDAC inhibitors e.g., Panobinostat
  • Bcl-2 inhibitors e.g., Navitoclax
  • FLT inhibitors e.g., Quizartinib
  • the other therapeutic agent is an anticancer agent
  • the anticancer agent is a compound that affects histone modifications, such as an HDAC inhibitor.
  • an anticancer agent is selected from the group consisting of chemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG,
  • Clofarabine ClolarTM, Cytoxan®, daunorubicin hydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L-PAM, Lysodren, Matulane®, mithracin, Mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®, Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®, Platinol®, prolifeprospan 20 with
  • Thalomid®, Tykerb®, Velcade® and ZevalinTM corticosteroids, (such as dexamethasone sodium phosphate, Deltasone® and Delta-Cortef®); hormonal therapies (such as Arimidex®, Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®, Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, PlenaxisTM and Zoladex®); and radiopharmaceuticals (such as Iodotope®, Metastron®, Pliosphocol® and Samarium SM- 153).
  • corticosteroids such as dexamethasone sodium phosphate, Deltasone® and Delta-Cortef®
  • hormonal therapies such as Arimidex®, Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®
  • an alkylating agent selected from the group including an antibiotic; an anti-metabolite; a detoxifying agent; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor;
  • VEGF VEGFR inhibitor a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting drug, a topoisomerase poison drug, an inhibitor of a molecular target or enzyme (e.g., a kinase or a protein methyltransferase), a cytidine analogue drug or any chemotherapeutic, anti-neoplastic or anti-proliferative agent listed in
  • alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin (Zanosar).
  • cyclophosphamide Cytoxan; Neosar
  • chlorambucil Leukeran
  • melphalan Alkeran
  • antibiotics include, but are not limited to, doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane);
  • daunorubicin (Cerubidine); daunorubicin liposomal (DaunoXome); dactinomycin
  • Exemplary anti-metabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine
  • Exemplary detoxifying agents include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).
  • interferons include, but are not limited to, interferon alfa-2b (Intron A) or interferon alfa-2a (Roferon-A).
  • Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab
  • cetuxan cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodinel 31 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In- 11 1 ; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris) ordenosumab.
  • Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.
  • Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.
  • Histone Deacetylase Inhibitors include, but are not limited to, vorinostat (Zolinza).
  • Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;
  • Nolvadex Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron; Lupron Depot; Eiigard; Viadur) ; f lvestrant (Faslodex); Ietrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot) ; exemestane (Aromasin) ; goserelin (Zoladex) ; bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo- Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone
  • Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan
  • Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afnitor) or temsirolimus Torisel); rapamune, ridaforolimus; or AP23573.
  • Exemplary multi-kinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.
  • Exemplary serine/threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril/easudil hydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS-032 (BMS-387032); Pkc412; bryostatin; KAI-9803;SF1 126; VX-680; Azdl 152; Arry- 142886 (AZD-6244); SCIO-469; GW681323; CC-401 ; CEP-1347 or PD 332991.
  • Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib
  • VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.
  • microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastin, nocodazole, epothilones and navelbine.
  • Exemplary topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.
  • Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.
  • Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine
  • the other therapeutic agent is a chemotherapeutic agent or a cytokine such as G-CSF (granulocyte colony stimulating factor).
  • G-CSF granulocyte colony stimulating factor
  • the other therapeutic agents can be standard chemotherapy combinations such as, but not restricted to, CMF (cyclophosphamide, methotrexate and 5- fluorouracil), CAF (cyclophosphamide, adriamycin and 5 -fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP), Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molar ratio of 1 :0.4: 1), Camptothecin-1 1 (CPT-11 , Irinotecan or CamptosarTM), CHOP (cyclophosphamide,
  • CMF cyclopho
  • the other therapeutic agents can be an inhibitor of an enzyme, such as a receptor or non-receptor kinase.
  • Receptor and non-receptor kinases are, for example, tyrosine kinases or serine/threonine kinases.
  • Kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides, or antibodies.
  • Exemplary kinase inhibitors include, but are not limited to, Bevacizumab (targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux (targets Erbl),
  • Exemplary serine/threonine kinase inhibitors include, but are not limited to,
  • Rapamune targets mTOR/FRAPl
  • Deforolimus targets mTOR
  • Certican Everolimus targets mTOR/FRAPl
  • AP23573 targets mTOR/FRAPl
  • Eril/Fasudil hydrochloride targets RHO
  • Flavopiridol targets CDK
  • Seliciclib/CYC202/Roscovitrine targets CDK
  • SNS-032/BMS-387032 targets CDK
  • Ruboxistaurin targets PKC
  • Pkc412 targets PKC
  • Bryostatin targets PKC
  • KAI-9803 targets PKC
  • SFl 126 targets PI3K
  • VX-680 targets Aurora kinase
  • Azdl 152 targets Aurora kinase
  • Arry-142886/AZD-6244 targets
  • MAP/MEK MAP/MEK
  • SCIO-469 targets MAP/MEK
  • GW681323 targets MAP/MEK
  • CC-401 targets INK
  • CEP-1347 targets INK
  • PD 332991 targets CDK
  • Other examples of the other therapeutic agents and combination therapy can be found in, e.g., co-owned US Provisional Application No. 61/ 61/785,446 with the title "Combination Therapy For Treating Cancer” filed on March 14, 2013 (Attorney Docket No. 41478-520P01US), the contents of which are hereby incorporated by reference in its entirety.
  • “combination therapy” or “co-therapy” includes the administration of a compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph or solvate thereof, and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • “Combination therapy” may be, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
  • Combination therapy is intended to embrace administration of these therapeutic agents in a sequential manner, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents concurrently, or in a substantially simultaneous manner. Simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes.
  • a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally.
  • all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.
  • the sequence in which the therapeutic agents are administered is not narrowly critical. Therapeutic agents may also be administered in alternation.
  • the combination therapies featured in the present invention can result in a synergistic effect in the treatment of a disease or cancer.
  • a "synergistic effect” is defined as where the efficacy of a combination of therapeutic agents is greater than the sum of the effects of any of the agents given alone.
  • a synergistic effect may also be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents.
  • the synergistic effect may include, but is not limited to, an effect of treating cancer by reducing tumor size, inhibiting tumor growth, or increasing survival of the subject.
  • the synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.
  • Combination therapy also embraces the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non- drug therapies (e.g., surgery or radiation treatment).
  • the combination therapy further comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • the compounds described herein, N-oxides, pharmaceutically acceptable salts thereof, and/or the compositions described herein may be administered in combination with radiation therapy.
  • Radiation therapy can also be administered in combination with a composition of the present invention and another chemotherapeutic agent described herein as part of a multiple agent therapy.
  • the compounds of the instant invention can also be utilized to treat or prevent neurologic diseases or disorders in monotherapy or combination therapy.
  • Neurologic diseases or disorders that may be treated with the compounds of this invention include epilepsy, schizophrenia, bipolar disorder or other psychological and/or psychiatric disorders, neuropathies, skeletal muscle atrophy, and neurodegenerative diseases, e.g., a
  • neurodegenerative disease exemplary neurodegenerative diseases include: Alzheimer's, Amyotrophic Lateral Sclerosis (ALS), and Parkinson's disease. Another class of ALS and ALS.
  • ALS Amyotrophic Lateral Sclerosis
  • Parkinson's disease Another class of ALS
  • Neurodegenerative diseases includes diseases caused at least in part by aggregation of poly- glutamine.
  • Diseases of this class include: Huntington's Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy's Disease) Dentatorubropallidoluysian Atrophy (DRPLA), Spinocerebellar Ataxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease (MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7 (SCA7), and Spinocerebellar Ataxia 12 (SCA12).
  • SCA1 Spinalbulbar Muscular Atrophy
  • SCA2 Spinocerebellar Ataxia 2
  • MJD Machado-Joseph Disease
  • SCA6 Spinocerebellar Ataxia 6
  • SCA7 Spinocerebellar Ataxia 7
  • SCA12 Spinocer
  • Any other disease in which epigenetic methylation, which is mediated by DOT1 , plays a role may be treatable or preventable using compounds and methods described herein.
  • the compound suitable for the method of the invention e.g., a DOT1L inhibitor
  • a DOT1L inhibitor is a compound of Formula (I) described herein, or a N-oxide, pharmaceutically acceptable salt, polymorph, solvate (e.g., hydrate), or stereoisomer thereof:
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a compound of any of the Formulae disclosed herein and a
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a salt of a compound of any of the Formulae disclosed herein and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a hydrate of a compound of any of the Formulae disclosed herein and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a compound selected from Table 1 and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a salt of a compound selected from Table 1 and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of an N-oxide of a compound selected from Table 1 and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of an N-oxide of salt of a compound selected from Table 1 and a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition of a therapeutically effective amount of a hydrate of a compound selected from Table 1 and a pharmaceutically acceptable carrier.
  • the present invention provides methods of treating or preventing cancer.
  • the present invention provides methods of treating cancer.
  • the present invention also provides methods of preventing cancer.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae disclosed herein.
  • the cancer can be a hematological cancer.
  • the cancer is leukemia. More preferably, the cancer is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • the present invention provides methods of treating or preventing a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the present invention provides metliods of treating a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the present invention also provides methods of preventing a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae disclosed herein.
  • the present invention provides metliods of treating or preventing a disease or disorder in which DOTlL-mediated protein methylation plays a part or a disease or disorder mediated by DOT 1 L- mediated protein methylation.
  • the present invention provides methods of treating a disease or disorder in which DOTl L-mediated protein methylation plays a part or a disease or disorder mediated by DOTlL-mediated protein methylation.
  • the present invention also provides methods of preventing a disease or disorder in which DOT1L- mediated protein methylation plays a part or a disease or disorder mediated by DOT1 remediated protein methylation.
  • the method includes administering to a subject in need thereof a therapeutically effective amount of the compound of any of the Formulae disclosed herein.
  • the present invention provides methods of inhibiting DOT1L activity in a cell.
  • the method includes contacting the cell with an effective amount of one or more of the compound of any of the Formulae disclosed herein.
  • Still another aspect of the invention relates to a method of reducing the level of Histone H3 Lysine residue 79 (H3-K79) methylation in a cell.
  • the method includes contacting a cell with a compound of the present invention.
  • Such method can be used to ameliorate any condition which is caused by or potentiated by the activity of DOT1L through H3-K79 methylation.
  • the present invention relates to use of the compounds disclosed herein in preparation of a medicament for treating or preventing cancer.
  • the use includes a compound of any of the Formulae disclosed herein for administration to a subject in need thereof in a
  • the cancer can be a hematological cancer.
  • the cancer is leukemia. More preferably, the cancer is acute myeloid leukemia, acute lymphocytic leukemia or mixed lineage leukemia.
  • the present invention provides use of the compounds disclosed herein in preparation of a medicament for treating or preventing a disease or disorder mediated by translocation of a gene on chromosome 1 lq23.
  • the use includes a compound of any of the Formulae disclosed herein for administration to a subject in need thereof in a therapeutically effective amount.
  • the present invention provides use of the compounds disclosed herein in preparation of a medicament for treating or preventing a disease or disorder in which DOTlL-mediated protein methylation plays a part or a disease or disorder mediated by DOTlL-mediated protein methylation.
  • the use includes a compound of any of the Formulae disclosed herein for administration to a subject in need thereof in a therapeutically effective amount.
  • the present invention provides use of the compounds disclosed herein for inhibiting DOT1L activity in a cell.
  • the use includes contacting the cell with an effective amount of one or more of the compound of any of the Formulae disclosed herein.
  • Still another aspect of the invention relates to a use of the compounds disclosed herein for reducing the level of Histone H3 Lysine residue 79 (H3-K79) methylation in a cell.
  • the use includes contacting a cell with a compound of the present invention.
  • Such use can ameliorate any condition which is caused by or potentiated by the activity of DOT 1 L through H3-K79 methylation.
  • the invention provides methods of synthesizing the foregoing compounds.
  • a therapeutically effective amount of one or more of the compounds can be formulated with a pharmaceutically acceptable carrier for administration to a mammal, particularly humans, for use in modulating an epigenetic enzyme.
  • the compounds of the present invention are useful for treating, preventing, or reducing the risk of cancer or for the manufacture of a medicament for treating, preventing, or reducing the risk of cancer.
  • the compounds or the formulations can be administered, for example, via oral, parenteral, otic, ophthalmic, nasal, or topical routes, to provide an effective amount of the compound to the mammal.
  • Representative compounds of the present invention include compounds listed i 1.
  • alkyl As used herein, "alkyl”, “d, C 2 , C 3 , C 4> C 5 or C 6 alkyl” or “C,-C 6 alkyl” is intended to include Ci, C 2 , C3, C 4 , C5 or C(, straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C 4 , C5 or branched saturated aliphatic hydrocarbon groups.
  • C1 -C6 alkyl is intended to include Ci , C2, C3, C4, C5 and C , alkyl groups.
  • alkyl examples include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.
  • a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C -Ce for straight chain, C3-C6 for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms.
  • cycloalkyl refers to a saturated or unsaturated nonaromatic hydrocarbon mono-or multi-ring system having 3 to 30 carbon atoms (e.g., C3-C10).
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl.
  • heterocycloalkyl refers to a saturated or unsaturated nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1 -14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se).
  • heterocycloalkyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.
  • optionally substituted alkyl refers to unsubstituted alkyl or alkyl having designated substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
  • aryloxycarbonyloxy carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
  • aminocarbonyl alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (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.
  • arylalkyl or an “aralkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • alkylaryl moiety is an aryl substituted with an alkyl (e.g., methylphenyl).
  • alkyl linker is intended to include Ci, C 2 , C3, C 4 , C5 or Cs straight chain (linear) saturated divalent aliphatic hydrocarbon groups and C3, C 4 , C or C3 ⁇ 4 branched saturated aliphatic hydrocarbon groups.
  • C 1 -C6 alkyl linker is intended to include C i , C2, C3, C4, C5 and Cfr alkyl linker groups.
  • alkyl linker examples include, moieties having from one to six carbon atoms, such as, but not limited to, methyl (-(3 ⁇ 4-), ethyl (-CH2CH2-), n-propyl (-CH 2 CH 2 CH 2 -), i-propyl (-CHCH3CH2-), n-butyl (- CH2CH2CH2CH2-), s-butyl (-CHCH3CH2CH2-), i-butyl (-C(CH 3 ) 2 CH 2 -), n-pentyl (- CH2CH2CH2CH2CH2-), s-pentyl (-CHCH3CH2CH2CH2-) or n-hexyl (- CH2CH2CH2CH2CH2-).
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
  • alkenyl includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups.
  • a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain).
  • C 2 -C6 includes alkenyl groups containing two to six carbon atoms.
  • C3-C6 includes alkenyl groups containing three to six carbon atoms.
  • alkenyl refers to unsubstituted alkenyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
  • aminocarbonyl alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
  • Alkynyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
  • alkynyl includes straight chain alkynyl groups (e.g. , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups.
  • a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C 2 -C6 for straight chain, C3-C6 for branched chain).
  • C2-C6 includes alkynyl groups containing two to six carbon atoms.
  • C3-C6 includes alkynyl groups containing three to six carbon atoms.
  • optionally substituted alkynyl refers to unsubstituted alkynyl or alkynyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
  • aminocarbonyl alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (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,
  • optionally substituted moieties include both the unsubstituted moieties and the moieties having one or more of the designated substituents.
  • Aryl includes groups with aromaticity, including “conjugated,” or multicyclic systems with at least one aromatic ring and do not contain any heteroatom in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, etc.
  • Heteroaryl groups are aryl groups, as defined above, except having from one to four heteroatoms in the ring structure, and may also be referred to as “aryl heterocycles” or “heteroaromatics.”
  • heteroaryl is intended to include a stable 5- or 6-membered monocyclic or 7-, 8-, 9-, 10-, 1 1 - or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g.
  • heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • the nitrogen atom may be substituted or unsubstituted (i. e. , N or NR wherein R is H or other substituents, as defined).
  • heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
  • aryl and heteroaryl include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole,
  • benzothiazole benzoimidazole, benzothiophene, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.
  • the aryl or heteroaryl aromatic ring can be substituted at one or more ring positions with such substituents as 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, alkylthiocarbonyl, 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, sulfam
  • Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methyl enedioxyphenyl such as benzo[d][l ,3]dioxole-5-yl).
  • alicyclic or heterocyclic rings which are not aromatic so as to form a multicyclic system (e.g., tetralin, methyl enedioxyphenyl such as benzo[d][l ,3]dioxole-5-yl).
  • Carbocycle or “carbocyclic ring” is intended to include any stable monocyclic, bicyclic or tricyclic ring having the specified number of carbons, any of which may be saturated, unsaturated, or aromatic.
  • a C3-C14 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.
  • 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 bridged ring occurs when one or more carbon atoms link two non-adjacent carbon atoms.
  • bridge rings are one or two carbon atoms. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro rings are also included.
  • heterocycle includes any ring structure (saturated or partially unsaturated) which contains at least one ring heteroatom (e.g., N, O or S).
  • heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine and tetrahydrofuran.
  • heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
  • benzisothiazolyl benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l ,5,2-dithiazinyl,
  • substituted means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.
  • 2 hydrogen atoms on the atom are replaced.
  • Keto substituents are not present on aromatic moieties.
  • “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • hydroxy or "hydroxyl” includes groups with an -OH or -O " .
  • halo or halogen refers to fluoro, chloro, bromo and iodo.
  • perhalogenated generally refers to a moiety wherein all hydrogen atoms are replaced by halogen atoms.
  • haloalkyl or “haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or more halogen atoms, e.g., -CX a 3 ⁇ 4, in which X is halo, a and b each are an integer, a is not 0, and the sum of a and b is 3.
  • carbonyl includes compounds and moieties which contain a carbon connected with a double bond to an oxygen atom.
  • moieties containing a carbonyl include, but are not limited to, aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.
  • carboxyl refers to -COOH or its C r C 6 alkyl ester.
  • Acyl includes moieties that contain the acyl radical (R-C(O)-) or a carbonyl group.
  • substituted acyl includes acyl groups where one or more of the hydrogen atoms are replaced by, for example, 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, aryl
  • Aroyl includes moieties with an aryl or heteroaromatic moiety bound to a carbonyl group. Examples of aroyl groups include phenylcarboxy, naphthyl carboxy, etc. [0277] "Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkyl groups, as described above, wherein oxygen, nitrogen, or sulfur atoms replace one or more hydrocarbon backbone carbon atoms.
  • alkoxy or "alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.
  • alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups.
  • substituted alkoxy groups include halogenated alkoxy groups.
  • the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
  • aryloxycarbonyloxy carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
  • halogen substituted alkoxy groups include 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 moieties.
  • ether or "alkoxy” includes compounds or moieties which contain an oxygen bonded to two carbon atoms or heteroatoms.
  • alkoxyalkyl refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to an alkyl group.
  • esters includes compounds or moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group.
  • ester includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
  • thioalkyl includes compounds or moieties which contain an alkyl group connected with a sulfur atom.
  • the thioalkyl groups can be substituted with groups such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
  • arylcarbonylamino, carbamoyl and ureido amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxyiate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifiuoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
  • thiocarbonyl or "thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.
  • thioether includes moieties which contain a sulfur atom bonded to two carbon atoms or heteroatoms.
  • thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls, and alkthioalkynyls.
  • alkthioalkyls include moieties with an alkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group.
  • alkthioalkenyls refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkenyl group
  • alkthioalkynyls refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.
  • amine refers to unsubstituted or substituted -N3 ⁇ 4.
  • Alkylamino includes groups of compounds wherein nitrogen of -N3 ⁇ 4 is bound to at least one alkyl group. Examples of alkylamino groups include benzylamino, methylamino, ethylamino, phenethylamino, etc.
  • Dialkylamino includes groups wherein the nitrogen of - N3 ⁇ 4 is bound to at least two additional alkyl groups. Examples of dialkylamino groups include, but are not limited to, dimethylamino and diethylamino.
  • Arylamino and
  • diarylamino include groups wherein the nitrogen is bound 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 which is bound to at least one alkyl group and at least one aryl group.
  • Alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.
  • Acylamino includes groups wherein nitrogen is bound to an acyl group. Examples of acylamino include, but are not limited to, alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.
  • amide or "aminocarboxy” includes compounds or moieties that contain a nitrogen atom that is bound to the carbon of a carbonyl or a thiocarbonyl group.
  • alkaminocarboxy groups that include alkyl, alkenyl or alkynyl groups bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group.
  • arylaminocarboxy groups that include aryl or heteroaryl moieties bound to an amino group that is bound to the carbon of a carbonyl or thiocarbonyl group.
  • alkylaminocarboxy alkenylaminocarboxy
  • alkynylaminocarboxy and
  • arylaminocarboxy include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group.
  • Amides can be substituted with substituents such as straight chain alkyl, branched alkyl, cycloalkyl, aryl, heteroaryl or heterocycle. Substituents on amide groups may be further substituted.
  • N- oxides can be converted to N- oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the present invention.
  • an oxidizing agent e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides
  • mCPBA 3-chloroperoxybenzoic acid
  • hydrogen peroxides e.g., hydrogen peroxides
  • all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N->0 or N + -0 " ).
  • the nitrogens in the compounds of the present invention can be converted to N-hydroxy or N-alkoxy compounds.
  • N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m-CPBA.
  • nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., N-OH) and N-alkoxy (i.e. , N-OR, wherein R is substituted or unsubstituted Q-C e alkyl, Ci-Ce alkenyl, Ci-Ce alkynyl, 3-14-membered carbocycle or 3-14- membered heterocycle) derivatives.
  • N-OH N-hydroxy
  • N-alkoxy i.e. , N-OR, wherein R is substituted or unsubstituted Q-C e alkyl, Ci-Ce alkenyl, Ci-Ce alkynyl, 3-14-membered carbocycle or 3-14- membered heterocycle
  • the structural formula of the compound represents a certain isomer for convenience in some cases, but the present invention includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon,
  • a crystal polymorphism may be present for the compounds represented by the formula. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present invention. Furthermore, so-called metabolite which is produced by degradation of the present compound in vivo is included in the scope of the present invention.
  • stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed
  • enantiomers or sometimes optical isomers.
  • a mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture.”
  • chiral center A carbon atom bonded to four nonidentical substituents is termed a "chiral center.”
  • Chiral isomer means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture.” When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.
  • Gaometric isomer means the diastereomers that owe their existence to hindered rotation about double bonds or a cycloalkyl linker (e.g., 1,3-cylcobutyl).
  • atropic isomers are a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to a restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as a mixture, however as a result of recent advances in chromatography techniques, it has been possible to separate mixtures of two atropic isomers in select cases.
  • Tautomer is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion 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, a chemical equilibrium of the tautomers will be reached.
  • the exact ratio of the tautomers depends on several factors, including temperature, solvent and pH.
  • the concept of tautomers that are interconvertable by tautomerizations is called tautomerism.
  • keto-enol tautomerism a simultaneous shift of electrons and a hydrogen atom occurs.
  • Ring- chain tautomerism arises as a result of the aldehyde group (-CHO) in a sugar chain molecule reacting with one of the hydroxy groups (-OH) in the same molecule to give it a cyclic (ring- shaped) form as exhibited by glucose.
  • tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide- imidic acid tautomerism in heterocyclic rings (e.g., in nucleobases such as guanine, thymine and cytosine), amine-enamine and enamine-enamine.
  • Benzimidazoles also exhibit tautomerism, when the benzimidazole contains one or more substituents in the 4, 5, 6 or 7 positions, the possibility of different isomers arises.
  • 2,5-dimethyl-lH- benzo[d]imidazole can exist in equilibrium with its isomer 2,6-dimethyl-lH- benzo[d]imidazole via tautomerization.
  • crystal polymorphs means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.
  • Compounds of the invention may be crystalline, semi-crystalline, non-crystalline, amorphous, mesomorphous, etc.
  • the compounds of any of the Formulae disclosed herein include the compounds themselves, as well as their N-oxides, salts, their solvates, and their prodrugs, if applicable.
  • a salt for example, can be formed between an anion and a positively charged group (e.g., amino) on the compound or inhibitor (e.g., a substituted nucleoside compound such as a substituted 7-deazapurine compound).
  • Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate.
  • a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on the compound or inhibitor (e.g., a substituted nucleoside compound such as a substituted 7-deazapurine compound).
  • Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion.
  • the compound or inhibitor e.g., a substituted nucleoside compound such as a substituted 7-deazapurine compound
  • examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active substituted nucleoside compound such as a substituted 7-deazapurine.
  • the compounds of the present invention can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules.
  • hydrates include hemihydrates, monohydrates, dihydrates, trihydrates, etc.
  • solvates include ethanol solvates, acetone solvates, etc.
  • Solvate means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as 3 ⁇ 40. A hemihydrate is formed by the combination of one molecule of water with more than one molecule of the substance in which the water retains its molecular state as 3 ⁇ 40.
  • analog refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group).
  • an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
  • the term “derivative” refers to compounds that have a common core structure, and are substituted with various groups as described herein. For example, all of the compounds represented by Formula (I) are substituted 7-deazapurine compounds, and have Formula (I) as a common core.
  • bioisostere refers to a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms.
  • the objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound.
  • the bioisosteric replacement may be physicochemically or topologically based.
  • Examples of carboxyiic acid bioisosteres include, but are not limited to, acyl sulfonimides, tetrazoles, sulfonates and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.
  • the present invention is intended to include all isotopes of atoms occurring in the present compounds.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium
  • isotopes of carbon include C-13 and C-14.
  • the present invention also provides methods for the synthesis of the compounds of any of the Formulae disclosed herein.
  • the present invention also provides detailed methods for the synthesis of various disclosed compounds of the present invention according to the schemes and the Examples described in WO2012/075381 , WO2012/075492,
  • compositions are described as having, including, or comprising specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components.
  • methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps.
  • order of steps or order for performing certain actions is immaterial so long as the invention remains operable.
  • two or more steps or actions can be conducted simultaneously.
  • the synthetic processes of the invention can tolerate a wide variety of functional groups, therefore various substituted starting materials can be used.
  • the processes generally provide the desired final compound at or near the end of the overall process, although it may be desirable in certain instances to further convert the compound to a pharmaceutically acceptable salt, ester, or prodrug thereof.
  • Preferred protecting groups include, but are not limited to:
  • aldehydes di-alkyl acetals such as dimethoxy acetal or diethyl acetyl.
  • DIPEA ⁇ , ⁇ -diisopropylethylamine (Hunig's base)
  • the invention provides methods for making the compounds of the invention.
  • 5'-Amino-7-deazapurine-ribose intermediates (B-V) can be synthesized as depicted in Scheme 1 above.
  • a suitable protected 7-deazapurine-ribose intermediate containing a 6- chloro substituent (B-I )may be converted into the corresponding 6-amino derivative (B-II) via treatment with the appropriate amine (including ammonia) in the presence of a base such as Et 3 N, K 2 C0 3 or Hunig's base in solvent such as MeCN or DMF, THF, iPrOH or a mixture thereof.
  • the reaction may be heated to 100 °C (if the temperature required is greater than the boiling point of one or more of the components in the mixture, the reaction may be performed in a sealed tube).
  • the R groups in the scheme may represent alkyl protecting groups (e.g. 2, 4 dimethoxybenzyl).
  • the 6-amino product (B-II) may be transformed into the 5 '-azido intermediate (B-III) by converting the 5 '-hydroxyl group into a leaving group such as MsO by treatment with MsCl in the presence of a base such as Et 3 N, pyridine or K2C0 3 in an inert solvent such as (3 ⁇ 4(3 ⁇ 4, THF, MeCN, DMF or a mixture thereof.
  • the 5 '-leaving group is then displaced with azide anion from an azide source such as NaN 3 in an inert solvent such as DMF.
  • an azide source such as NaN 3 in an inert solvent such as DMF.
  • (B-II) may be directly transformed into (B-III) by treatment with DPPA, Ph 3 P and DIAD in a solvent such as THF.
  • the azido group of (B-III) may be reduced to the primary amine (B-IV) by reduction with 3 ⁇ 4 in the presence of a metal catalyst (e.g. Pd/C, Pt(3 ⁇ 4) or by a Staudinger reaction with a phosphine such as Ph 3 P or PMe 3 .
  • a metal catalyst e.g. Pd/C, Pt(3 ⁇ 4) or by a Staudinger reaction with a phosphine such as Ph 3 P or PMe 3 .
  • the primary amine (B-IV) may be converted into the secondary amine (B-V) by treatment with the appropriate ketone or aldehyde in the presence of a suitable reducing agent such as NaBH(OAc) 3 or NaCNBH 3 . Additional reagents such as Ti(OiPr)4 may be added.
  • a suitable reducing agent such as NaBH(OAc) 3 or NaCNBH 3 .
  • Additional reagents such as Ti(OiPr)4 may be added.
  • the 5 '-hydroxy intermediate (B-II) may be treated with the sulfonamide (B-VI), DEAD and Ph 3 P in an inert solvent such as THF.
  • the resultant sulfonamide product may then be treated with benzenethiol in the presence of a base such as K2C0 3 , Cs2C0 3 to give the secondary amine (B-V).
  • These reaction sequences may also be applied to lyxose derivatives starting from (B
  • reaction sequences may be employed for 2'-deoxy, or 3'-deoxy, or substituted ribose or lyxose (B-VIII) above to obtain 5 '-amino 7-deazapurine-ribose/lyxose intermediates.
  • An alternative method for introduction of a 6-NH 2 group is via treating (B-IX) derivatives with NaN 3 to produce a 6-azido intermediate followed by reduction to the NH 2 moiety (B-X) with a trialkyl phosphine such as PMe 3 or PPh 3 .
  • Cyclobutanes of formulae (G-VIII), (G-XV) and (G-XXI) may be synthesized as depicted in Scheme 1.
  • the alkenyl esters (G-I) may be subjected to a [2+2] cycloaddition with trichloroacetyl chloride in the presence of Zn/Cu couple in an inert solvent such as Et 2 0, DME, THF or a mixture thereof.
  • the [2+2] cycloaddition reaction may be performed using Zn dust under sonication conditions.
  • the dichlorides (G-II) are reduced via treatment with Zn powder in the presence of a proton donor such as NH 4 C1 in a solvent such as MeOH.
  • the cyclobutanones (G-IV) (which include (G-III)) may be further elaborated by treatment with a phosphonate (G-V) to give the , ⁇ unsaturated esters (G-VI).
  • the acid (VI) is converted to the Weinreb amide (G-VII) under standard conditions (e.g. iso-butyl chloroformate, Hunig's base, ⁇ , ⁇ -dimethyl hydroxylamine).
  • the double bond may then be reduced via hydrogenation using H 2 in the presence of a metal catalyst such as Pd/C, Pt0 2 or Pd(OH) 2 to give the cyclobutane intermediates (G-VIII).
  • the cyclobutanones may be treated with the Wittig reagent (G-X) to give the cyclobutane enol ether (G-XI) which upon deprotection gives the corresponding acid (G-XII).
  • the cyclobutanones (G-IX) may also be treated with the stabilized phosphonate (G- V) in the presence of a base such as KOtBu, LDA, NaHMDS, KHMDS or LiHMDS or with Et3N in the presence of LiCl in an inert solvent to give the ⁇ , ⁇ unsaturated ester (G-XIII) which can be reduced to the (G-XIV) by treatment with H 2 in the presence of a metal catalyst such as Pd/C, Pd(OH) 2 or Pt0 2 in an inert solvent.
  • the acid functionality of (G-XIV) may be converted into the corresponding Weinreb amide by treatment with ⁇ , ⁇ - dimethylhydroxylamine in presence of a suitable coupling agent such as iso- butylchloroformate and a base such as Hunig's base to give (G-XV).
  • a suitable coupling agent such as iso- butylchloroformate and a base such as Hunig's base
  • the cyclobutanones (G-XVI) may also be treated with N,0-dimethylhydroxylamine in presence of a suitable coupling agent such as iso-butylchloroformate and a base such as Hunig's base to give the corresponding Weinreb amide (G-XVII) which upon reductive amination with an ammonia equivalent followed by deprotection as needed gives the amine (G-XVIII).
  • a suitable coupling agent such as iso-butylchloroformate
  • a base such as Hunig's base
  • Suitable ammonia equivalents include benzhydryl amine, N3 ⁇ 4, NH4CI, BnN3 ⁇ 4, PMB-NH 2 , 2,4 DMB-NH 2 which may be treated with the ketone (G-XVII) and a suitable reducing agent such as NaCN(BH3) or Na(OAc) 3 BH in the presence of an acid if required such as HC1 or AcOH.
  • a suitable reducing agent such as NaCN(BH3) or Na(OAc) 3 BH in the presence of an acid if required such as HC1 or AcOH.
  • Protecting groups on the reductive amination products may be removed by methods known to those of ordinary skill in the art.
  • the ketone (G- XVII) can be treated with hydroxyl amine to form the corresponding oxime which then can be reduced with 3 ⁇ 4 in the presence of a metal catalyst such as Pd/C, PtC>2 or Pd(OH)2 to give the intermediate (G-XVIII).
  • a metal catalyst such as Pd/C, PtC>2 or Pd(OH)2
  • the cyclobutane (G-IV) may converted into the amine (G-XXI) via a multi-step sequence involving treating (G-IV) with the phosphorane (G-V) to produce the enol ether (G- XIX).
  • Suitable ammonia equivalents include benzhydryl amine, NH 3 , NH 4 C1, BnNH 2 , PMB-NH 2 , 2,4 DMB-NH 2 .
  • Suitable reducing agents for the reductive amination include NaCN(BH 3 ) or Na(OAc) 3 BH used in the presence of an acid if required such as HC1 or AcOH. Protecting groups on the reductive amination products may be removed by methods known to those of ordinary skill in the art.
  • ketones (K-VI), (K-VII) and (K-VIII) and the aldehydes (K-XII), (K-XIII) and (K-XIV) are converted into the corresponding benzimidazoles (K-IX) and (K-XV), ureas (K-X) and (K-XVI) and amides (K-XI) and (K-XVII) via reductive amination with (K-V).
  • the reductive amination can be performed with a suitable reducing agent such as NaCN(BI3 ⁇ 4) or Na(OAc)3BH in the presence of an acid if required such as HC1 or AcOH or a Lewis acid/dehydrating agent such as Ti(OiPr)4 or MgSC .
  • a suitable reducing agent such as NaCN(BI3 ⁇ 4) or Na(OAc)3BH
  • an acid if required such as HC1 or AcOH or a Lewis acid/dehydrating agent such as Ti(OiPr)4 or MgSC .
  • compounds of the invention may be synthesized by routes depicted in the following schemes.
  • scheme 1A compound (I) may be protected with a protecting group such as TBS under standard conditions (e.g. TBS-C1, Et 3 N, aprotic solvent such as CH 2 C1 2 or THF) to give the corresponding protected compound, which may then be brominated using a reagent such as NBS in a solvent such as DMF to give compound (II).
  • the alcohol protecting group may be removed at this point under standard conditions and the resulting primary alcohol transformed into compound (III) by a Mitsunobu reaction using Phthalimide, PPI13, DIAD in a solvent such as THF.
  • Compound (III) may be treated with NaN 3 in an aprotic solvent such as DMF at a temperature of RT to 80 °C to give the azido compound (IV).
  • the azide may be reduced with a reagent such as PMe3 in a solvent such as THF to give compound (V).
  • the phthalimido group may be converted into the corresponding primary amine by treatment with hydrazine in a solvent such as EtOH at a temperature of RT to 78 °C to give the intermediate (VI).
  • the amino and CF 3 analogs may be synthesized as depicted in scheme 3A.
  • the primary alcohol of compound (I) may be protected with a suitable protecting group such as acetate under standard conditions.
  • the resultant acetate is then subjected to nitrating conditions such as HN0 3 , H 2 S0 4 in a solvent such as Ac 2 0, CH 2 C1 2 or H 2 0 to give the nitro compound (XII).
  • the alcohol protecting group may be removed at this point under standard conditions and the resulting primary alcohol transformed into compound (XIII) by a
  • the amino of compound (XIV) group may be transformed into a CF 3 group by initially forming a diazo intermediate by treatment with HN0 2 or tBuONO in the presence of CuBr at a temperature of -10 °C to RT, followed by treatment with CF 3 -I or the compound (XV).
  • the resultant CF 3 containing compound (XVI) may be transformed into the amino containing compound (XVII) by first treating with NaN 3 in a solvent such as DMF at a temperature of RT to 60°C to produce the azido intermediate, followed by reduction of azido functionality with a reagent such as PMe3 in a solvent such as THF.
  • the phthalimido group may be converted into the corresponding primary amine by treatment with hydrazine in a solvent such as EtOH at a temperature of RT to 78 °C to give the intermediate (XVIII).
  • the cyclopropyl group may be introduced via cyclopropanation of the intermediate produced by treating the compound (XIX) with vinyl boronic acid in the presence of K2CO 3 , Pd(PPli 3 )4 in a solvent mixture of water/dioxane at a temperature of RT to 100°C.
  • the resulting vinyl compound is the treated with di-iodomethane in the presence of ZnEt2 in solvent such as CH2CI2 at a temperature of 0-40°C to give the cyclopropyl containing compound (XX).
  • the alkynyl compound (XXI) may be produced by first treating the halo compound (XIX) with TMS-acetylene in the presence of a Pd catalyst such as Pd(PPh 3 ) 4 or Pd2(dba)3, Cul, a tertiary basic amine such as Et 3 N in a solvent such as DMF at a temperature of RT to 100 °C.
  • a Pd catalyst such as Pd(PPh 3 ) 4 or Pd2(dba)3, Cul
  • Et 3 N a tertiary basic amine
  • the silyl group may then be removed by treatment with TBAF or K2CO 3 in a solvent such as MeOH to give the compound (XXI).
  • the cyano compound (XXII) may be produced by treating compound (XIX) with a cyano source such as CuCN or Zn(CN) 2 .
  • a Pd catalyst such as Pd(PPh 3 ) 4 or Pd 2 (dba) 3 may also be added.
  • DMF may be used as a solvent for the reaction at a temperature of RT to 120 °C or alternatively the reaction may be enhanced by the use of microwave irradiation.
  • the Me containing compound (XXVI) may be produced by treating the compound (XIX) with SnMe 4 (Stille type coupling) or 2,4,6-trimethyl-boroxine (Suzuki coupling).
  • suitable reaction conditions include a Pd catalyst such as Pd(PPh 3 ) 4 or Pd 2 (dba) 3 in an aprotic solvent such as THF or DMF at a temperature of RT to 100 °C.
  • a Pd catalyst such as Pd(PPh 3 ) 4 or Pd 2 (dba) 3 in an aprotic solvent such as THF or DMF at a temperature of RT to 100 °C.
  • AsPh 3 may also be added to reaction mixture.
  • Pd(PPh 3 ) 4 maybe used in a solvent mixture of water/dioxane at a temperature of RT to 100°C.
  • the elaborated compounds (XXVII) may be coupled with the cyclcobutanone (XXVIII) via a reductive amination to give the compound (XXIX).
  • the reaction may be run using Na(OAc)3BH or NaCNBE ⁇ as the reducing agent in a solvent such as 1,2 DCE at a temperature of 0 to 80 °C.
  • the resulting compound (XXIX) can undergo a further reductive amination to give the amine (XXX).
  • dimethoxypropane is used in the presence of Na(OAc)3NH or NaCNB3 ⁇ 4 as the reducing agent in a solvent such as 1,2 DCE at a temperature of 0 to 80 °C.
  • AcOH may be added to the mixture.
  • the protecting groups may be removed under standard conditions.
  • the reductive amination to produce (XXIX) usually produces a mixture of cis and trans isomers around the cyclobutane ring. These isomers may be separated using HPLC, chiral HPLC, SFC crystallization or a combination of these techniques. These separation techniques may be employed at any stage after the reductive amination with the cyclobutanone (XXVIII).
  • X F, CI, Br, I, Me, Et, iPr, CF 3 , NH 2 , cyclopropyl, CN
  • compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present invention also consist essentially of, or consist of, the recited components, and that the processes of the present invention also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions are immaterial so long as the invention remains operable.
  • Compounds suitable for the methods of the invention can be characterized using a variety of assays known to those skilled in the art to determine whether the compounds have biological activity.
  • the molecules can be characterized by conventional assays, including but not limited to those assays described below, to determine whether they have a predicted activity, binding activity and/or binding specificity.
  • high-throughput screening can be used to speed up analysis using such assays. As a result, it can be possible to rapidly screen the molecules described herein for activity, using techniques known in the art. General methodologies for performing high- throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker; and U.S. Patent No. 5,763,263. High-throughput assays can use one or more different assay techniques including, but not limited to, those described herein.
  • measurements of inhibition of cytochrome P450 enzymes and phase II metabolizing enzyme activity can also be measured either using recombinant human enzyme systems or more complex systems like human liver microsomes. Further, compounds can be assessed as substrates of these metabolic enzyme activities as well. These activities are useful in determining the potential of a compound to cause drug-drug interactions or generate metabolites that retain or have no useful antimicrobial activity.
  • solubility and Caco-2 assays are a cell line from human epithelium that allows measurement of drug uptake and passage through a Caco-2 cell monolayer often growing within wells of a 24-well microtiter plate equipped with a 1 micron membrane. Free drug concentrations can be measured on the basolateral side of the monolayer, assessing the amount of drug that can pass through the intestinal monolayer. Appropriate controls to ensure monolayer integrity and tightness of gap junctions are needed. Using this same system one can get an estimate of P-glycoprotein mediated efflux.
  • P-glycoprotein is a pump that localizes to the apical membrane of cells, forming polarized monolayers. This pump can abrogate the active or passive uptake across the Caco-2 cell membrane, resulting in less drug passing through the intestinal epithelial layer. These results are often done in conjunction with solubility measurements and both of these factors are known to contribute to oral bioavailability in mammals. Measurements of oral bioavailability in animals and ultimately in man using traditional pharmacokinetic experiments will determine the absolute oral bioavailability.
  • Experimental results can also be used to build models that help predict physical- chemical parameters that contribute to drug-like properties. When such a model is verified, experimental methodology can be reduced, with increased reliance on the model predictability.
  • the present invention also provides pharmaceutical compositions comprising a compound of any of the Formulae disclosed herein in combination with at least one pharmaceutically acceptable excipient or carrier.
  • a "pharmaceutical composition” is a formulation containing the compounds of the present invention in a form suitable for administration to a subject.
  • the pharmaceutical composition is in bulk or in unit dosage form.
  • the unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler or a vial.
  • the quantity of active ingredient ⁇ e.g. , a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.
  • active ingredient ⁇ e.g. , a formulation of the disclosed compound or salt, hydrate, solvate or isomer thereof
  • the dosage will also depend on the route of administration.
  • routes of administration A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
  • the phrase "pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a "pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral ⁇ e.g., inhalation), transdermal (topical), and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • a compound or pharmaceutical composition of the invention can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment.
  • a compound of the invention may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches.
  • the dose chosen should be sufficient to constitute effective treatment but not as high as to cause unacceptable side effects.
  • the state of the disease condition ⁇ e.g., cancer, precancer, and the like
  • the health of the patient should preferably be closely monitored during and for a reasonable period after treatment.
  • terapéuticaally effective amount refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect.
  • the effect can be detected by any assay method known in the art.
  • the precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic selected for administration.
  • Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.
  • the disease or condition to be treated is cancer.
  • the disease or condition to be treated is a cell proliferative disorder.
  • the therapeutically effective amount can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug interaction(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • compositions containing active compounds of the present invention may be manufactured in a manner that is generally known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • Phannaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and/or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol and sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a 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 sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as macrocrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a 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
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g. , a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g. , a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,81 1.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • the dosages of the pharmaceutical compositions used in accordance with the invention vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the cancer. Dosages can range from about 0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects, dosages can range from about 1 mg/kg per day to about 1000 mg/kg per day.
  • the dose will be in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, or continuous doses (which dose may be adjusted for the patient's weight in kg, body surface area in m 2 , and age in years).
  • An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, regression of a tumor in a patient may be measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Regression is also indicated by failure of tumors to reoccur after treatment has stopped.
  • the term "dosage effective manner" refers to amount of an active compound to produce the desired biological effect in a subject or cell.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • pharmaceutically acceptable salts refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.
  • the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric,
  • compositions include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like.
  • the present invention also encompasses salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • a metal ion e.g. , an alkali metal ion, an alkaline earth ion, or an aluminum ion
  • organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • the compounds of the present invention can also be prepared as esters, for example, pharmaceutically acceptable esters.
  • a carboxylic acid function group in a compound can be converted to its corresponding ester, e.g., a methyl, ethyl or other ester.
  • an alcohol group in a compound can be converted to its corresponding ester, e.g., acetate, propionate or other ester.
  • the compounds of the present invention can also be prepared as prodrugs, for example, pharmaceutically acceptable prodrugs.
  • prodrug and “prodrug” are used interchangeably herein and refer to any compound which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of
  • the compounds of the present invention can be delivered in prodrug form.
  • the present invention is intended to cover prodrugs of the presently claimed compounds, methods of delivering the same and compositions containing the same.
  • Prodrugs are intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when such prodrug is administered to a subject.
  • Prodrugs in the present invention are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • Prodrugs include compounds of the present invention wherein a hydroxy, amino, sulfhydryl, carboxy or carbonyl group is bonded to any group that may be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl group, respectively.
  • prodrugs include, but are not limited to, esters (e.g., acetate,
  • dialkylaminoacetates formates, phosphates, sulfates and benzoate derivatives
  • carbamates e.g., ⁇ , ⁇ -dimethylaminocarbonyl
  • esters e.g., ethyl esters, morpholinoethanol esters
  • N-acyl derivatives e.g., N-acetyl
  • N-Mannich bases Schiff bases and enaminones of amino functional groups
  • oximes acetals, ketals and enol esters of ketone and aldehyde functional groups in compounds of the invention, and the like
  • Bundegaard, H. Design of Prodrugs, pi -92, Elesevier, New York-Oxford (1985).
  • the compounds, or pharmaceutically acceptable salts, esters or prodrugs thereof are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally and parenterally.
  • the compound is administered orally.
  • One skilled in the art will recognize the advantages of certain routes of administration.
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.
  • the compounds described herein, and the pharmaceutically acceptable salts thereof are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent.
  • suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.
  • TBSC1 (2.78 g, 18.42 mmol) was added to a suspension of [(3aR,4R,6R,6aR)-6- ⁇ 4- chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl ⁇ -2,2-dim ethyl -tetrahydro-2H-furo[3,4-d][l,3]dioxol- 4-yl]methanol (5.00 g, 15.35 mmol) and imidazole (2.51 g, 36.84 mmol) in DCM (80 ml) at RT and stirred overnight. The reaction mixture was washed with 0.5 N HC1 (100 ml), dried over Na 2 S0 4 , filtered and evaporated in vacuo. Purification by silica gel column
  • DIAD (2.85 ml, 14.46 mmol) was added slowly to a solution of [(3aR,4R,6R,6aR)-6- ⁇ 5-bromo-4-chloro-7H ⁇ yrrolo[2,3-d]pyrimidin-7-yl ⁇ -2,2-dimethyl-tetrahydro-2H-furo[3,4 d][l,3]dioxol-4-yl]met anol (3.90 g, 9.64 mmol), pht alimide (1.42 g, 9.64 mmol) and PPh 3 (3.79 g, 14.46 mmol) in THF (50 ml) at 0 °C and stirred whilst allowing to warm to RT over 8 h.
  • EOL-1 Human leukemia cell line EOL-1 (Catalog # ACC-386) is purchased from DSMZ and are grown in Roswell Park Memorial Institute medium (RPMI) with 10% Fetal Bovine Serum (FBS). Cells are kept in log growth as outlined in the technical data sheet provided by the vendor.
  • RPMI Roswell Park Memorial Institute medium
  • FBS Fetal Bovine Serum
  • THP-1, RS4;1 1 , and MV4-1 1 are obtained from ATCC, MOLM-13 cells are obtained from DSMZ. All lines are grown in RPMI 1640 containing 10% FBS and maintained using the vendors recommended cell densities and environmental conditions. Media is supplemented with non essential amino acids and L- Glutamine. THP-1 cells are also supplemented with 0.05 mM ⁇ -Mercaptoethanol.
  • Purified histones are quantitated using the BCA protein assay (Pierce) with a BSA standard curve. 400 ng of isolated histones are fractionated by SDS-PAGE on a 4-20% gel and transferred to nitrocellulose membranes. Membranes are incubated with various primary and secondary antibodies and imaged on the Licor imaging system (Odyssey).
  • the H3K79- Me2 rabbit polyclonal is purchased from Abeam.
  • Other rabbit polyclonal antibodies including H3K4-Me3, H3K9-Me3, H3K27-Me2, and H3K27-Me3 are purchased from Cell Signaling Technologies (CST).
  • a mouse monoclonal total H3 antibody is used as a loading control (CST). Fluorescently labeled secondary antibodies are purchased from Odyssey.
  • Exponentially growing cells e.g., EOL-1 , THP-1, MV4-11 and MOLM-13 cells
  • EOL-1 EOL-1 , THP-1, MV4-11 and MOLM-13 cells
  • 96-well plates a density of 3 ⁇ 10 4 viable cells/well. Each treatment is seeded in triplicate with a final well volume of 150 ⁇ . Cells are incubated with increasing concentrations of DOT1L inhibitor up to50 ⁇ . Viable cell number is determined every 3 - 4 days for 1 1 days using the Guava Viacount assay (Millipore # 4000-0040) and analyzed on a Guava EasyCyte Plus instrument according to the manufacturer's protocol.
  • Frozen pellets are allowed to thaw briefly on ice and then lysed by a 5 minute incubation on ice with 250 ⁇ nuclear extraction buffer (10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 25 mM KC1, 1% Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet 1836153001). Nuclei are collected by centrifugation at 600 g for 5 minutes at 4°C and washed once in Tris/EDTA buffer (pH 7.4). Supernatant is removed and histones extracted for one hour with 60 ⁇ 0.4 N cold sulfuric acid.
  • Extracts are clarified by centrifugation at 10,000 g for 10 minutes at 4°C and transferred to a fresh microcentrifuge tube containing 600 ⁇ ice cold acetone. Histones are precipitated at -20° C for 2 hours, pelleted by centrifugation at 10,000 g for 10 minutes and resuspended in 60 ⁇ distilled water (DI water). Total protein of the acid extracts is assessed using a bicinchoninic acid (BCA) protein quantification assay with a bovine serum albumin (BSA) standard (Pierce Biotechnology).
  • BCA bicinchoninic acid
  • BSA bovine serum albumin
  • EOL-1 cells For immunoblot analysis of the H3K79me2 inhibition by DOT 1L inhibitor, exponentially growing cells (e.g., EOL-1 cells) are seeded at 2x 10 ⁇ cells/mL and incubated in the presence of increasing concentrations of DOT1 L inhibitor for 4 days. Following incubation, cells (2-3 ⁇ 10 ⁇ ) are harvested and histones extracted as described. Histones (400 ng) are fractionated on a 10-20% Tris HC1 gels (Bio-Rad) with Tris-Glycine SDS running buffer (Pnvitrogen) under denaturing conditions and transferred to a nitrocellulose filter.
  • Tris HC1 gels Bio-Rad
  • Tris-Glycine SDS running buffer Pnvitrogen
  • the filter is incubated for 1 hour in blocking buffer (Odyssey blocking buffer, Li-cor, 927-40000) at RT and then incubated overnight at 4°C in blocking buffer containing a antibody specific for H3K79me2 (1 :5000 dilution, abeam ab3594). Filters are washed 3 times for 5 minutes with wash buffer (PBST) and incubated with infrared tagged secondary antibody (Alexa Flour 680 goat anti-rabbit IgG (1 :20,000), Invitrogen A-21076) at RT for 1 hour.
  • blocking buffer Odyssey blocking buffer, Li-cor, 927-40000
  • Filters are washed in PBST and reprobed for 1 hour at RT with the appropriate total histone antibody control (mouse anti-histone H3 (1 :20,000), CST 3638, or mouse anti-histone H4 (1 : 10,000), CST 2935). Filters are washed again in PBST and incubated with infrared tagged secondary antibody (IRDye 800Cw donkey-anti-mouse IgG (1 :20,000), Li- Cor 926-32212) at RT for 1 hour. After a final wash in PBST, filters are scanned using the Odyssey infared imager (Li- cor). Signal intensities specific for each methyl-specific antibody is quantified using Odyssey software and normalized to that of the appropriate total histone control signal on the same filter by dividing the methyl-specific antibody signal intensity by the total histone control signal intensity.
  • Exponentially growing cells e.g., EOL-1 cells
  • EOL-1 cells Exponentially growing cells
  • Cells are plated in a 12 well plate at 2x 10 ⁇ cells/mL.
  • Cells are incubated in the presence of increasing concentrations of a test compound up to 10 ⁇ .
  • On day 4 cells are maintained in log phase culture by reseeding at 5 ⁇ 10 ⁇ cells/mL and compound is replenished.
  • cells are washed twice with PBS and pelleted by centrifugation at 200 X g.
  • Cell pellets are lysed in 300 ⁇ buffer (Qiagen) and total RNA is isolated using the RNeasy total RNA isolation kit (Qiagen 74106).
  • RNA (1 ⁇ g) was reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems 4368813). RNA isolation and cDNA synthesis are carried out according to the manufacturer's protocol. Predesigned labeled primer and probe sets for HOXA9 (Hs00365956), MEIS1 (HsOO 180020) and FLT3 (Hs00975659) are purchased from Applied Biosystems. Quantitative real-time PCR (qPCR) reactions contained 50 ng cDNA, IX labeled primer and probe set, and IX Taqman universal PCR master mix (Applied Biosystems 4368813). RNA isolation and cDNA synthesis are carried out according to the manufacturer's protocol. Predesigned labeled primer and probe sets for HOXA9 (Hs00365956), MEIS1 (HsOO 180020) and FLT3 (Hs00975659) are purchased from Applied Biosystems. Quantitative real-time PCR (qPCR) reactions contained 50 ng cDNA,
  • Biosystems 4304437 Samples are run on a 7900 HT Fast Real Time PCR machine (Applied Biosystems 4351405) with cycling conditions of 2 min 50°C, 10 min 95°C, 40 cycles at 15 sec 95°C and 1 min 60°C.
  • Target gene cycle numbers are normalized to the house keeping gene p2-microglobulin (Applied Biosystems 4333766) to get a ACT value.
  • Test compounds are serially diluted 3 fold in DMSO for 10 points and 1 ⁇ is plated in a 384 well microtiter plate.
  • Positive control (100% inliibition standard) is 2.5 uM final concentration of S-adenosyl-L-homocysteine and negative control (0% inhibition standard) contained 1 ⁇ of DMSO.
  • Compound is then incubated for 30 minutes with 40 ⁇ per well of D0T1L(1 -416) (0.25 nM final concentration in assay buffer: 20 mM TRIS, pH 8.0, 10 mM NaCl, 0.002% Tween20, 0.005% Bovine Skin Gelatin, 100 mM KC1, and 0.5 mM DTT). 10 ⁇ per well of substrate mix (same assay buffer with 200 nM S-[methyl- H]-adenosyl-L methionine, 600 nM of unlabeled S-[methyl- H]-adenosyl-L methionine, and 20 nM oligonucleosome) is added to initiate the reaction.
  • substrate mix standard assay buffer with 200 nM S-[methyl- H]-adenosyl-L methionine, 600 nM of unlabeled S-[methyl- H]-adenosyl-L methionine, and 20 nM
  • Reaction is incubated for 120 minutes at room temperature and quenched with 10 ul per well of 100 uM S-methyl-adenosyl -L methionine.
  • substrate from 50 ul of reaction is immobilized on a 384 well Streptavidin coated Flashplate (Perkin Elmer) (also coated with 0.2% polyethyleneimine) and read on a Top Count scintillation counter (Perkin Elmer).
  • EOL-1 cells a leukemia cell line characterized by MLL PTD
  • EOL-1 cells are plated in 96-well plates at a density of 3x10 4 viable cells/well.
  • Cells are incubated with increasing concentrations of DOT1L inhibitor between the 0.003 ⁇ - 50 ⁇ .
  • the number of viable cells is determined every 3-4 days for 11 days.
  • Cells are maintained in log phase by reseeding and replenishing growth media and the indicated concentration of DOT1L inhibitor on each day of cell counts (Day 0, Day 4, Day 7, and Day 1 1). Total cell number is expressed as split-adjusted viable cells per well.
  • DMSO-treated cells are used as a control.
  • Example 3A Effect of DOT1L Inhibition on Cell Growth and Viability
  • MV4-1 1 suspension cells were maintained in growth medium (IMDM supplemented with 10% v/v heat inactivated fetal bovine serum and cultured at 37 °C under 5% CO2.
  • cells were incubated in assay medium (IMDM supplemented with 10% v/v heat inactivated fetal bovine serum) and 100 units/mL penicillin-streptomycin) at 37 °C under 5% C0 2 .
  • assay medium IMDM supplemented with 10% v/v heat inactivated fetal bovine serum
  • penicillin-streptomycin 100 units/mL penicillin-streptomycin
  • polypropylene 384-well plate with an assigned container number was prepared by performing triplicate nine-point 3 -fold serial dilutions in DMSO, beginning at 10 mM (final top concentration of compound in the assay was 20 ⁇ and the final concentration of DMSO was 0.2%). A 100 nL aliquot from the compound stock plate was added to its respective well in the cell plate. The 100% inhibition control consisted of cells treated with 200 nM final concentration of staurosporine and the 0% inhibition control consisted of DMSO treated cells. After addition of the compounds, assay plates were incubated for 7 days at 37 °C, 5% C0 2 , relative humidity > 90%.
  • Cell viability was measured by quantitation of ATP present in the cell cultures, by adding 35 ⁇ of Cell Titer Glo ® reagent to the cell plates. Luminescence was read in the SpectraMax M5 instrument. The concentration inhibiting cell viability by 50% was determined using a 4-parametric fit of the normalized dose response curves.
  • Compound 7 was determined to have an IC 5 o of 0.003 ⁇ .
  • Inhibition of methylation of H3K79 is assessed after 4 days of treatment of DOT1L compounds in exponentially growing cells (e.g., EOL-1 cells).
  • H3K79 methylation status after treatment with a test compound is first examined by immunoblot. Following treatment, cells are harvested and histones are extracted. Western blot analysis is performed using antibodies specific for H3K79me2 and total histone 3 (as a control). Signal intensities specific to H3K79me2 is quantified and normalized to that of the total histone 3 signal.
  • IC50 and inhibition constants of the DOT1L inhibitors described herein can be determined from concentration-dependence growth curves, as described in Example 2.
  • concentration-dependence curves at day 1 1 for a test compound and a control compound are plotted on a single log graph and compared.
  • the inhibition constant (Ki) can also be determined for the DOT1 L inhibitors described herein. For example, Ki values for a test compound and a control compound can be compared. The test compound having the lowest Ki, which indicates that a very low concentration (e.g., 0.08 nM) of the test compound is required to decrease the maximal rate of the reaction to half of the uninhibited value, in the presence of a low substrate
  • Example 5A Determination of IC50
  • Test compounds were serially diluted 3 fold in DMSO for 10 points and 1 ⁇ was plated in a 384 well microtiter plate. Positive control (100% inhibition standard) was 50 uM final concentration of S-adenosyl-L-homocysteine and negative control (0% inhibition standard) contained 1 ⁇ of DMSO. The test compound was then incubated for 30 minutes with 40 uL per well of D0T1L(1-416) (0.25 nM final concentration in assay buffer: 20 mM TRIS, pH 8.0, 10 mM NaCl, 0.002% Tween20, 0.005% Bovine Skin Gelatin, 100 mM KC1, and 0.5 mM DTT).
  • substrate from 50 ⁇ of reaction was immobilized on a 384 well Streptavidin coated Flashplate (Perkin Elmer) (also coated with 0.2% polyethyleneimine) and read on a Top Count scintillation counter (Perkin Elmer).
  • Compound 7 was determined to have an IC5 0 of 0.47 nM.
  • Example 6 Gene Overexpression in Leukemias
  • HOXA9 expression levels are assessed in a panel of leukemia cells lines, demonstrating that HOXA9 is often overexpressed in various hematologic cancers.
  • the leukemia cells assessed include MolM13 (acute monocytic leukemia cell line), MV41 1 (acute myelocytic leukemia), LOUCY (T-cell acute lymphoblastic leukemia), EOL-1 (eosinophilic leukemia) SemK2 (B-cell acute lymphoblastic leukemia), Reh (acute lymphoblastic leukemia), HL60 (promyelocytic leukemia) and BV173 (pre-B-cell leukemia).
  • Molml 3, MV411 , and SemK2 cell lines are characterized as having MLL fusions.
  • LOUCY and Reh cell lines are characterized as having non-MLL chromosomal rearrangements.
  • EOL- 1 cells are characterized as having MLL-PTD.
  • HOXA9 is determined by quantitative real-time PCR. HOXA9 expression level is normalized to the lowest HOXA9 expresser.
  • Jurkat cells an immortalized T-lymphocyte cell line, are used as control.
  • HL60, BV173, and Reh cell lines had very low overexpression of HOXA9.
  • SemK2, EOL-1 , LOUCY, MV41 1, and Molml3 had extremely high overexpression of HOXA9, with at least a 2000-fold increase over the lowest expresser.
  • Overexpression of other cancer-associated genes, such as FLT3, MEISl , or DOTIL can be determined using similar methods as those described herein.
  • Leukemia cell lines can be treated with DOTIL inhibitors and the expression of select genes can be examined to assess the effects of DOTIL inhibition on cancer-associated gene overexpression.
  • Leukemia cell lines are treated with increasing concentrations of candidate DOT1 L inhibitors up to 10 ⁇ for 6 days.
  • For the vehicle control, cells are treated with DM SO. Cells are then harvested, RNA is extracted, and cDNA is prepared as described in Example 1.
  • Expression levels of select genes are determined by quantitative real-time PCR. Expression of HOXA9, FLT3 and/or MEISl can be reduced in a dose-dependent manner after treatment with a test compound. As expected, DOTI L gene expression is not reduced after treatment of DOTIL inhibitor and can be considered useful for control purposes.

Abstract

La présente invention concerne des composés 7-déazapurine substitués. La présente invention concerne également des compositions pharmaceutiques contenant ces composés et des méthodes de traitement de troubles dans lesquels la méthylation de protéines médiée par DOT 1 joue un rôle, par exemple le cancer, par administration de ces composés et compositions pharmaceutiques à des sujets en ayant besoin.
PCT/US2014/027134 2013-03-14 2014-03-14 Composés 7-déazapurine substitués WO2014152261A1 (fr)

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WO2012075381A1 (fr) * 2010-12-03 2012-06-07 Epizyme, Inc. Composés purines et 7-déazapurines substituées en tant que modulateurs d'enzymes épigéniques
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CN113024620A (zh) * 2021-03-11 2021-06-25 沈阳药科大学 一种嘌呤衍生物及其制备方法和用途
CN113024620B (zh) * 2021-03-11 2023-02-28 沈阳药科大学 一种嘌呤衍生物及其制备方法和用途

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