US20210087143A1 - Agents for differentiating stem cells and treating cancer - Google Patents

Agents for differentiating stem cells and treating cancer Download PDF

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US20210087143A1
US20210087143A1 US16/498,640 US201816498640A US2021087143A1 US 20210087143 A1 US20210087143 A1 US 20210087143A1 US 201816498640 A US201816498640 A US 201816498640A US 2021087143 A1 US2021087143 A1 US 2021087143A1
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stem cells
optionally substituted
alkyl
cancer
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Cynthia Bamdad
Scott Moe
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Minerva Biotechnologies Corp
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Minerva Biotechnologies Corp
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Definitions

  • This invention generally relates to methods and compositions for the treatment of cancers that are characterized by the function of the compounds to differentiate stem cells.
  • human stem cells cultured under standard conditions, are not in a truly pluripotent state. Rather they have undergone some differentiation and have made certain cell fate decisions as evidenced by the accumulation of various methylation marks.
  • human cultured stem cells When comparing human cultured stem cells to cells of mouse embryos it was determined that the human cultured stem cells look and behave more like mouse stem cells from the epiblast portion of the embryo, which has begun to differentiate, rather than the truly pluripotent stem cells of the inner cell mass.
  • researchers dubbed the true pluripotent stem cells of the inner cell mass ‘na ⁇ ve’ and the more differentiated cells ‘primed’.
  • na ⁇ ve stem cells have two active X chromosomes whereas primed state stem cells have already inactivated one X chromosome by methylation. Additionally, it is now known that na ⁇ ve state stem cells have far less methylation marks, which essentially are early differentiation decisions, also known as cell fate decisions, which limit the types of mature cells that the stem cells can become.
  • a drug screen in which agents are screened for their ability to preferentially inhibit pluripotency of na ⁇ ve stem cells more than primed stem cells.
  • Agents that are screened may be antibodies or antibody like molecules, polyclonal, monoclonal, antibody fragment fusion proteins, antibody mimics, peptides or peptide mimics, small molecules or natural products.
  • agents that inhibit cancer growth, inhibit the growth of metastatic cancer cells, or inhibit the metastatic potential of cancer cells wherein the agents were identified by their ability to induce differentiation or inhibit pluripotency of na ⁇ ve stem cells and their relative inability to induce differentiation or inhibit pluripotency of primed stem cells.
  • the agents that are disclosed are disclosed for use as an anti-cancer or anti-metastasis therapeutic for the treatment or prevention of cancers.
  • novel anti-cancer or anti-metastasis drug targets are identified by identifying genes that are upregulated in na ⁇ ve stem cells but not in primed stem cells.
  • novel anti-cancer or anti-metastasis drug targets are identified by identifying microRNAs that are upregulated in na ⁇ ve stem cells but not in primed stem cells.
  • the invention is directed to a method for identifying an agent for the treatment or prevention of cancer or metastatic cancer comprising the steps of
  • the stem cell may be na ⁇ ve state stem cell.
  • the stem cell may be na ⁇ ve state or primed state stem cell, wherein the effect of the agent on na ⁇ ve state stem cell is compared to the effect on primed state stem cell, wherein if the agent has a greater effect on the na ⁇ ve state stem cell compared with primed state stem cell, then the agent is determined to be an anti-cancer agent.
  • the agent may be a polyclonal antibody, monoclonal antibody, antibody like molecule, antibody fragment fusion protein, antibody mimic, peptide, peptide mimic, small molecule or natural product.
  • the stem cell may be human.
  • the stem cell may be maintained in a na ⁇ ve state by culturing in a medium comprising NME7 AB or NME7-X1.
  • the cancer may be breast, ovarian, melanoma, prostate, colon, lung or pancreatic.
  • the cancer may be MUC1 positive or MUC1* positive cancer.
  • the cancer may be NME7 AB or NME7-X1 positive cancer.
  • the agent may not be generally cytotoxic.
  • the agent may not be cytotoxic to fibroblasts or fibroblast progenitor cells.
  • the invention is directed to a method for preventing or treating cancer comprising administering to the subject the agent obtained by the method according to above.
  • the cancer may be breast, ovarian, melanoma, prostate, colon, lung or pancreatic.
  • the cancer may be a MUC1 positive or MUC1* positive cancer.
  • the cancer may be an NME7 AB or NME7-X1 positive cancer.
  • the invention is directed to a method for preventing metastasis of cancer comprising administering to the subject the agent obtained by the method according to above.
  • the invention is directed to a method of inhibiting cancer growth, migration or invasiveness comprising administering to the subject the agent obtained by the method according to above.
  • the invention is directed to a method of inhibiting the growth of metastatic cancer cells comprising administering to the subject the agent obtained by the method according to above.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis target for drug discovery comprising identifying a gene or gene product that is upregulated in na ⁇ ve state stem cells compared to primed state stem cells.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis target for drug discovery comprising identifying a gene or gene product that is downregulated in na ⁇ ve state stem cells compared to primed state stem cells.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis agent comprising (i) identifying gene or gene product that is downregulated in na ⁇ ve state stem cells compared to primed state stem cells; (ii) contacting the na ⁇ ve stem cells with an agent; and (iii) identifying an agent that increases expression or activity of the downregulated gene or gene product in na ⁇ ve state stem cells.
  • the down-regulated gene may be a gene that is upregulated when stem cells initiate differentiation.
  • the down-regulated gene may be fibronectin, vimentin, or NF1.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis agent comprising (i) identifying gene or gene product that is upregulated in na ⁇ ve state stem cells compared to primed state stem cells; (ii) contacting the na ⁇ ve stem cells with an agent; and (iii) identifying an agent that inhibits expression or activity of the upregulated gene or gene product in na ⁇ ve state stem cells.
  • the upregulated gene may be E-cadherin, CXCR4, ⁇ -catenin, AXIN2, MUC1, NME7, or NME7-X1.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis agent comprising (i) identifying gene or gene product that is upregulated in na ⁇ ve state stem cells compared to fibroblast cells; (ii) contacting the na ⁇ ve stem cells with an agent; and (iii) identifying an agent that inhibits expression or activity of the upregulated gene or gene product in na ⁇ ve state stem cells.
  • the upregulated gene may be E-cadherin, CXCR4, ⁇ -catenin, AXIN2, MUC1, NME7, or NME7-X1.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis agent comprising (i) identifying gene or gene product that is downregulated in na ⁇ ve state stem cells compared to fibroblast cells; (ii) contacting the na ⁇ ve stem cells with an agent; and (iii) identifying an agent that increases expression or activity of the downregulated gene or gene product in na ⁇ ve state stem cells.
  • the down-regulated gene may be a gene that is upregulated when stem cells initiate differentiation.
  • the down-regulated gene may be fibronectin, vimentin, NF1, or microRNA-145.
  • the down-regulated gene may be a superenhancer target gene, such as HES3, GNAS, VLDLR, EXT1, FBXL17, RHOC or GREB1L.
  • the invention is directed to a method of identifying anti-cancer or anti-metastasis agent comprising (i) identifying microRNA that is upregulated in na ⁇ ve state stem cells compared to primed stem cells or fibroblast cells; (ii) contacting the na ⁇ ve stem cells with an agent; and (iii) identifying an agent that inhibits expression or activity of the upregulated microRNA in na ⁇ ve state stem cells.
  • the invention is directed to the compounds of Formulae 1 to 17.
  • the invention is directed to a method of treating cancer in a subject, comprising administering to the subject a compound of Formula 1 to 17 or as set forth in FIG. 18A-18E , or as drawn out in the present specification at or about pages 48-64.
  • the cancer may be a MUC1 positive, or MUC1* positive, or a MUC1 negative cancer.
  • the cancer may be an NME7 AB or NME7-X1 positive cancer.
  • the invention is directed to a method for preventing or treating cancer or cancer metastasis comprising the steps of: (i) analyzing a cancerous sample from the patient and determining that it is MUC1* positive, NME7 AB positive or NME7-X1 positive; and
  • the analyzing step may be carried out by PCR.
  • the cancerous sample may express mRNA level of MUC1 gene, NME7 gene or NME7-X1 gene that is at least 0.5% of the mRNA expression level of EEF1A1 gene, it is determined to be MUC1* positive, NME7 AB positive or NME7-X1 positive.
  • the analyzing step may be carried out by immunohistochemistry.
  • the cancerous sample when the cancerous sample may be contacted with an antibody that binds to the PSMGFR peptide or the N-10 peptide and stains the tissue with a pathologist's standard score 1-4 (“+ ⁇ ++++”), it is determined to be MUC1* positive.
  • the cancerous sample may be contacted with an antibody that binds to the B3 peptide of NME7 and stains the tissue with a pathologist's standard score 1-4 (“+ ⁇ ++++”), it is determined to be NME7 AB positive or NME7-X1 positive.
  • the invention is directed to a method of identifying an agent for the prevention or treatment of an inflammatory disease or condition, comprising the steps of (i) exposing stem cells to an agent, and (ii) identifying an agent that inhibits stem cell pluripotency or growth, or induces stem cell differentiation, wherein the agent or its analog is an agent for treating inflammatory disease or condition.
  • the inflammatory disease or condition may be rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, osteoarthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves' disease, gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease, angina, cancer metastasis, small artery disease, or mitochondrial disease.
  • COPD chronic obstructive pulmonary diseases
  • the invention is directed to a method of treating an inflammatory disease or condition comprising administering to a person in need thereof, an agent that when contacted with stem cells, inhibits stem pluripotency or growth or induces stem cell differentiation.
  • the inflammatory disease or condition may be rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, osteoarthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves'
  • FIG. 1 shows the chemical structures of a set of small molecules that were tested for their ability to inhibit pluripotency, growth or induce differentiation of na ⁇ ve state or primed state stem cells.
  • FIG. 2 is a Table that summarizes the results of testing small molecules, an anti-MUC1* Fab “E6”, a MUC1* extracellular domain peptide “FLR” and anti-NME7 antibodies #56 and #61.
  • FIG. 3A-3L shows photographs at 10 ⁇ magnification of human primed state stem cells, grown in stem cell media with growth factor FGF, over a layer of MEFs and treated for 3 days with in the presence of a test agent.
  • FIG. 3A shows photograph of primed stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 3B shows photograph of primed stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR
  • FIG. 3C shows photograph of control primed stem cells
  • FIG. 3D shows photograph of primed stem cells cultured in 0.2% DMSO as control for small molecules in 0.2% DMSO
  • FIG. 3E shows photograph of primed stem cells cultured in the presence of MN0642, FIG.
  • FIG. 3F shows photograph of primed stem cells cultured in the presence of MN1130
  • FIG. 3G shows photograph of primed stem cells cultured in the presence of MN0572
  • FIG. 3H shows photograph of primed stem cells cultured in the presence of MN0947
  • FIG. 3I shows photograph of primed stem cells cultured in the presence of MN0129
  • FIG. 3J shows photograph of primed stem cells cultured in the presence of MN0676
  • FIG. 3K shows photograph of primed stem cells cultured in the presence of MN0992
  • FIG. 3L shows photograph of primed stem cells cultured in the presence of MN0402.
  • FIG. 4A-4L shows photographs at 20 ⁇ magnification of human primed state stem cells, grown in stem cell media with growth factor FGF, over a layer of MEFs and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 4A shows photograph of primed stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 4B shows photograph of primed stem cells cultured in presence of a MUC1* ecd peptide extracellular domain peptide, also known as FLR,
  • FIG. 4C shows photograph of control primed stem cells, FIG.
  • FIG. 4D shows photograph of primed stem cells cultured in 0.2% DMSO as control for small molecules in 0.2% DMSO
  • FIG. 4E shows photograph of primed stem cells cultured in the presence of MN0642
  • FIG. 4F shows photograph of primed stem cells cultured in the presence of MN1130
  • FIG. 4G shows photograph of primed stem cells cultured in the presence of MN0572
  • FIG. 4H shows photograph of primed stem cells cultured in the presence of MN0947
  • FIG. 4I shows photograph of primed stem cells cultured in the presence of MN0129
  • FIG. 4J shows photograph of primed stem cells cultured in the presence of MN0676
  • FIG. 4K shows photograph of primed stem cells cultured in the presence of MN0992
  • FIG. 3L shows photograph of primed stem cells cultured in the presence of MN0402.
  • FIG. 5A-5L shows photographs at 10 ⁇ magnification of human primed state stem cells, grown in stem cell media without growth factor FGF, over a layer of MEFs and treated for 3 days with in the presence of a test agent.
  • FIG. 5A shows photograph of primed stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 5B shows photograph of primed stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR
  • FIG. 5C shows photograph of primed stem cells cultured in presence of an anti-NME7 polyclonal antibody #56
  • FIG. 5D shows photograph of primed stem cells cultured in presence of an anti-NME7 polyclonal antibody #61,
  • FIG. 5A shows photograph of primed stem cells cultured in presence of an anti-MUC1* Fab, named E6
  • FIG. 5B shows photograph of primed stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR
  • FIG. 5C shows
  • FIG. 5E shows photograph of primed stem cells cultured in the presence of MN0642
  • FIG. 5F shows photograph of primed stem cells cultured in the presence of MN1130
  • FIG. 5G shows photograph of primed stem cells cultured in the presence of MN0572
  • FIG. 5H shows photograph of primed stem cells cultured in the presence of MN0947
  • FIG. 5I shows photograph of primed stem cells cultured in the presence of MN0129
  • FIG. 5J shows photograph of primed stem cells cultured in the presence of MN0676
  • FIG. 5K shows photograph of primed stem cells cultured in the presence of MN0992
  • FIG. 5L shows photograph of primed stem cells cultured in the presence of MN0402.
  • FIG. 6A-6L shows photographs at 20 ⁇ magnification of human primed state stem cells, grown in stem cell media without growth factor FGF, over a layer of MEFs and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 6A shows photograph of primed stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 6B shows photograph of primed stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR,
  • FIG. 6C shows photograph of primed stem cells cultured in presence of an anti-NME7 polyclonal antibody #56, FIG.
  • FIG. 6D shows photograph of primed stem cells cultured in presence of an anti-NME7 polyclonal antibody #61
  • FIG. 6E shows photograph of primed stem cells cultured in the presence of MN0642
  • FIG. 6F shows photograph of primed stem cells cultured in the presence of MN1130
  • FIG. 6G shows photograph of primed stem cells cultured in the presence of MN0572
  • FIG. 6H shows photograph of primed stem cells cultured in the presence of MN0947
  • FIG. 6I shows photograph of primed stem cells cultured in the presence of MN0129
  • FIG. 6J shows photograph of primed stem cells cultured in the presence of MN0676
  • FIG. 6K shows photograph of primed stem cells cultured in the presence of MN0992
  • FIG. 6L shows photograph of primed stem cells cultured in the presence of MN0402.
  • FIG. 7A-7L shows photographs at 10 ⁇ magnification of human na ⁇ ve state stem cells, grown in stem cell media with growth factor NME7 AB , over a MUC1* antibody, C3, surface and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 7A shows photograph of na ⁇ ve stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 7B shows photograph of na ⁇ ve stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR,
  • FIG. 7C shows photograph of control na ⁇ ve stem cells, FIG.
  • FIG. 7D shows photograph of na ⁇ ve stem cells cultured in 0.2% DMSO as control for small molecules in 0.2% DMSO
  • FIG. 7E shows photograph of na ⁇ ve stem cells cultured in the presence of MN0642
  • FIG. 7F shows photograph of na ⁇ ve stem cells cultured in the presence of MN1130
  • FIG. 7G shows photograph of na ⁇ ve stem cells cultured in the presence of MN0572
  • FIG. 7H shows photograph of na ⁇ ve stem cells cultured in the presence of MN0947
  • FIG. 7I shows photograph of na ⁇ ve stem cells cultured in the presence of MN0129
  • FIG. 7J shows photograph of na ⁇ ve stem cells cultured in the presence of MN0676
  • FIG. 7K shows photograph of na ⁇ ve stem cells cultured in the presence of MN0992
  • FIG. 7L shows photograph of na ⁇ ve stem cells cultured in the presence of MN0402.
  • FIG. 8A-8L shows photographs at 20 ⁇ magnification of human na ⁇ ve state stem cells, grown in stem cell media with growth factor NME7 AB , over a MUC1* antibody, C3, surface and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 8A shows photograph of na ⁇ ve stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 8B shows photograph of na ⁇ ve stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR,
  • FIG. 8C shows photograph of control na ⁇ ve stem cells, FIG.
  • FIG. 8D shows photograph of na ⁇ ve stem cells cultured in 0.2% DMSO as control for small molecules in 0.2% DMSO
  • FIG. 8E shows photograph of na ⁇ ve stem cells cultured in the presence of MN0642
  • FIG. 8F shows photograph of na ⁇ ve stem cells cultured in the presence of MN1130
  • FIG. 8G shows photograph of na ⁇ ve stem cells cultured in the presence of MN0572
  • FIG. 8H shows photograph of na ⁇ ve stem cells cultured in the presence of MN0947
  • FIG. 8I shows photograph of na ⁇ ve stem cells cultured in the presence of MN0129
  • FIG. 8J shows photograph of na ⁇ ve stem cells cultured in the presence of MN0676
  • FIG. 8K shows photograph of na ⁇ ve stem cells cultured in the presence of MN0992
  • FIG. 8L shows photograph of na ⁇ ve stem cells cultured in the presence of MN0402.
  • FIG. 9A-9L shows photographs at 10 ⁇ magnification of human na ⁇ ve state stem cells, grown in stem cell media without growth factor NME7 AB , over a MUC1* antibody, C3, surface and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 9A shows photograph of na ⁇ ve stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 9B shows photograph of na ⁇ ve stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR, FIG.
  • FIG. 9C shows photograph of na ⁇ ve stem cells cultured in presence of an anti-NME7 polyclonal antibody #56
  • FIG. 9D shows photograph of na ⁇ ve stem cells cultured in presence of an anti-NME7 polyclonal antibody #61
  • FIG. 9E shows photograph of na ⁇ ve stem cells cultured in the presence of MN0642
  • FIG. 9F shows photograph of na ⁇ ve stem cells cultured in the presence of MN1130
  • FIG. 9G shows photograph of na ⁇ ve stem cells cultured in the presence of MN0572
  • FIG. 9H shows photograph of na ⁇ ve stem cells cultured in the presence of MN0947
  • FIG. 9I shows photograph of na ⁇ ve stem cells cultured in the presence of MN0129
  • FIG. 9J shows photograph of na ⁇ ve stem cells cultured in the presence of MN0676
  • FIG. 9K shows photograph of na ⁇ ve stem cells cultured in the presence of MN0992
  • FIG. 9L shows photograph of na ⁇ ve stem cells cultured in the presence of MN0402.
  • FIG. 10A-10L shows photographs at 20 ⁇ magnification of human na ⁇ ve state stem cells, grown in stem cell media without NME7 AB , over a MUC1* antibody, C3, surface and treated for 3 days with in the presence of a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 10A shows photograph of na ⁇ ve stem cells cultured in presence of an anti-MUC1* Fab, named E6,
  • FIG. 10B shows photograph of na ⁇ ve stem cells cultured in presence of a MUC1* extracellular domain peptide, FLR,
  • FIG. 10C shows photograph of na ⁇ ve stem cells cultured in presence of an anti-NME7 polyclonal antibody #56, FIG.
  • FIG. 10D shows photograph of na ⁇ ve stem cells cultured in presence of an anti-NME7 polyclonal antibody #61
  • FIG. 10E shows photograph of na ⁇ ve stem cells cultured in the presence of MN0642
  • FIG. 10F shows photograph of na ⁇ ve stem cells cultured in the presence of MN1130
  • FIG. 10G shows photograph of na ⁇ ve stem cells cultured in the presence of MN0572
  • FIG. 10H shows photograph of na ⁇ ve stem cells cultured in the presence of MN0947
  • FIG. 10I shows photograph of na ⁇ ve stem cells cultured in the presence of MN0129
  • FIG. 10J shows photograph of na ⁇ ve stem cells cultured in the presence of MN0676
  • FIG. 10K shows photograph of na ⁇ ve stem cells cultured in the presence of MN0992
  • FIG. 10L shows photograph of na ⁇ ve stem cells cultured in the presence of MN0402.
  • FIG. 11A-11F shows photographs at 4 ⁇ magnification of human primed state stem cells, previously grown in bFGF over MEFs, but cultured in the absence of bFGF during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 11A shows photograph of primed stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 11B shows photograph of primed stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 11C shows photograph of primed stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 11D shows photograph of primed stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 11E shows photograph of primed stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 11 F shows photograph of primed stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 12A-12F shows photographs at 20 ⁇ magnification of human primed state stem cells, previously grown in bFGF over MEFs, but cultured in the absence of bFGF during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 12A shows photograph of primed stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 12B shows photograph of primed stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 12C shows photograph of primed stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 12D shows photograph of primed stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 12E shows photograph of primed stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 12F shows photograph of primed stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 13A-13F shows photographs at 4 ⁇ magnification of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 13A shows photograph of na ⁇ ve stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 13B shows photograph of na ⁇ ve stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 13C shows photograph of na ⁇ ve stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 13D shows photograph of na ⁇ ve stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 13E shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 13F shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 14A-14F shows photographs at 20 ⁇ magnification of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 14A shows photograph of na ⁇ ve stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 14B shows photograph of na ⁇ ve stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 14C shows photograph of na ⁇ ve stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 14D shows photograph of na ⁇ ve stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 14E shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 14F shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 15A-15F shows photographs at 4 ⁇ magnification of human na ⁇ ve state stem cells, previously grown in NME1 dimers over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 15A shows photograph of na ⁇ ve stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 15B shows photograph of na ⁇ ve stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 15C shows photograph of na ⁇ ve stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 15D shows photograph of na ⁇ ve stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 15E shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 15F shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 16A-16F shows photographs at 20 ⁇ magnification of human na ⁇ ve state stem cells, previously grown in NME1 dimers over a MUC1* antibody surface, C3, but cultured in the absence of NME1 dimers during the experiment, and treated for 3 days with a test agent. Dotted lines indicate areas where stem cell pluripotency or growth is inhibited or differentiation is induced.
  • FIG. 16A shows photograph of na ⁇ ve stem cells cultured in presence of a control scrambled sequence siRNA
  • FIG. 16B shows photograph of na ⁇ ve stem cells cultured in presence of a BRD4 specific siRNA
  • FIG. 16C shows photograph of na ⁇ ve stem cells cultured in presence of a JMJD6 specific siRNA
  • FIG. 16D shows photograph of na ⁇ ve stem cells cultured in presence of an inactive stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1 ⁇
  • FIG. 16E shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1 aka JQ1+ at 500 nM
  • FIG. 16F shows photograph of na ⁇ ve stem cells cultured in presence of the active stereoisomer of purported BRD4 inhibitor JQ1+ at 1 uM.
  • FIG. 17 shows chemical structures of some compounds previously reported to inhibit cancer cell migration as well as some that the inventors previously disclosed.
  • FIG. 18A-18E shows summary of biological data for compounds of the invention and various other previously known chemical compounds.
  • FIG. 19A-19P shows photographs of human stem cells cultured for 3 days with either control media or a small molecule that had been previously reported to inhibit cancer cell migration, which is a characteristic of cancer metastasis.
  • the cells were na ⁇ ve state stem cells, previously grown in the growth factor NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment.
  • the cells were primed state stem cells, previously grown in the growth factor FGF over a layer of inactivated MEFs, but cultured in the absence of FGF during the experiment.
  • FIG. 20 is a bar graph showing the measured percent inhibition of cancer cell migration.
  • the cancer cell line used was T47D breast cancer cell line. Multi-well plate was coated with collagen and cells were plated using Platypus system that restricts cells from entering center of wells until cells have attached. The percent area that remains free of cells at 126 hrs was measured using Image J and graphed.
  • the agents that were tested were: an anti-MUC1* Fab “E6”, which has been shown to inhibit proliferation of virtually all MUC1* positive cells tested, in vitro and in vivo; JQ1, a BRD4 inhibitor reported to inhibit cancer cell migration and proliferation in vitro and in vivo; small molecules reported by others to inhibit migration of a range of cancer cells; and novel small molecules of the invention.
  • FIG. 21A-21P shows representative photographs of the cancer cell migration assay at 126 hours, wherein the cancer cells were treated with a panel of agents. Small molecules were dosed at 6 uM final concentration unless otherwise indicated. The “+” or “ ⁇ ” indicates the score each agents received in the na ⁇ ve/primed stem cell assay. For example +++/ ⁇ indicates the compound profoundly inhibited the pluripotency and proliferation of na ⁇ ve stem cells but had no effect on primed stem cells.
  • FIG. 21A cells were treated with control PBS.
  • FIG. 21B-21D cells were treated with anti-MUC1* Fab E6.
  • FIG. 21E-211 shows cells treated with control amount of DMSO at time zero.
  • FIG. 21F-21G cells were treated with JQ1.
  • FIG. 21A-21P shows representative photographs of the cancer cell migration assay at 126 hours, wherein the cancer cells were treated with a panel of agents. Small molecules were dosed at 6 uM final concentration unless otherwise indicated.
  • 21H-21M shows cells treated with control amount of DMSO at 126 hours.
  • FIG. 21J shows cells treated with novel molecule MN1194.
  • FIG. 21K shows cells treated with novel molecule MN1186.
  • FIG. 21L shows cells treated with novel molecule MN1137.
  • FIG. 21N shows cells treated with novel molecule MN1193.
  • FIG. 21O shows cells treated with novel molecule MN1203.
  • FIG. 21P shows cells treated with novel molecule MN1184.
  • FIG. 22A-22X shows the results of cancer cell migration assays in which novel compounds of the invention that inhibited na ⁇ ve stem cell pluripotency or proliferation were tested for their ability to inhibit cancer cell invasion or migration.
  • FIG. 22A-22U shows photographs of a migration, invasion assay performed on T47D breast cancer cells in the presence of novel compounds of the invention or the control, DMSO alone, at 120 hours.
  • FIG. 22V is a graph showing the measured inhibition of cancer cell migration at time 0, 24 hours or 48 hours for a number of compounds.
  • FIG. 22W is a graph showing the inhibitory effect of the small molecules as a function of concentration, where units are uM.
  • FIG. 22X is a graph showing how IC50's of the small molecules of the invention were measured and calculated.
  • FIG. 23A-23D shows photographs of human fibroblasts in culture, treated only with 0.2% DMSO as a control.
  • FIG. 24A-24F shows photographs of the effect of JQ1+ ( FIG. 24A-24C ) versus the effect of the inactive enantiomer JQ1 ⁇ ( FIG. 24D-24F ) on human na ⁇ ve state stem cells ( FIG. 24A, 24D ), human primed state stem cells ( FIG. 24B, 24E ), or human fibroblasts ( FIG. 24C, 24F ).
  • FIG. 25A-25F show photographs of the effect of JQ1 compared to previously known cancer cell migration inhibitors, versus compounds of the invention, on the growth of human fibroblast progenitor cells.
  • FIG. 26A-26H show photographs of stem cell control experiments and a previously known compound, Dorsomorphin.
  • FIG. 26A-26B show primed state stem cells culture in same concentration of DMSO that the compounds were dissolved in.
  • FIG. 26E-26F show na ⁇ ve state stem cells culture in same concentration of DMSO that the compounds were dissolved in.
  • FIG. 26 C- 26 D show the effect of Dorsomorphin on primed state stem cells.
  • FIG. 26G-26H show the effect of Dorsomorphin on na ⁇ ve state stem cells.
  • FIG. 27A-27F show photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIG. 27G-27L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIG. 28A-28L show photographs of control experiments carried out on different human stem cell lines.
  • FIG. 28A, 28B, 28E, 28F show photographs of a female induced pluripotent stem cell line, iPS 9X, that is in the na ⁇ ve state as evidenced by documentation that the second X chromosome has been re-activated.
  • FIG. 28C, 28D, 28G, 28H are human embryonic stem cell line, HES-3, growing in bFGF which keeps stem cells in primed state.
  • FIG. 28I-28L shows photographs of human fibroblasts, BJ line available from the ATCC.
  • FIG. 29A-29F shows photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIG. 29G-29L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIG. 29M-29R show photographs of human fibroblast cells treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the morphology or proliferation of the cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on morphology or proliferation of the cells.
  • FIG. 30A-30F shows photographs of control experiments on stem cell lines that were used in the next series of drug screening experiments.
  • FIGS. 31-35 A-F show photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 31-35 G-L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 31-35 M-R show photographs of human fibroblast cells treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM, unless otherwise indicated.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the morphology or proliferation of the cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on morphology or proliferation of the cells.
  • FIG. 36 A 1 - 36 L 4 shows photographs of a cancer cell migration, invasion assay performed on T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 37 shows measured IC50 curves for each of the compounds for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 38 A 1 - 38 R 4 shows photographs of a cancer cell migration, invasion assay performed on T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 39 shows measured IC50 curves for each of the compounds for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 40 A 1 - 40 R 4 shows photographs of a cancer cell migration, invasion assay performed on T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 41 shows measured IC50 curves for each of the compounds for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 42 A 1 - 42 R 4 shows photographs of a cancer cell migration, invasion assay performed on T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 122 hours.
  • FIG. 43 shows measured IC50 curves for each of the compounds for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 122 hours.
  • FIG. 44 A 1 - 44 R 4 shows photographs of a cancer cell migration, invasion assay performed on T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 124 hours.
  • FIG. 45 shows measured IC50 curves for each of the compounds for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 124 hours.
  • FIG. 46A-46F shows photographs of the control stem cells and fibroblast cells treated with the same concentration of DMSO as is in the test compounds.
  • FIGS. 46A-46C are 10 ⁇ magnification photographs.
  • FIGS. 46D-46F are 20 ⁇ magnification photographs.
  • FIGS. 46A and 46D are photographs of na ⁇ ve state stem cells.
  • FIGS. 46B and 46E are photographs of primed state stem cells.
  • FIGS. 46C and 46F are photographs of human fibroblast cells.
  • FIGS. 47-49 A-F show photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 47-49 G-L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 47-49 M-R show photographs of human fibroblast cells treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the morphology or proliferation of the cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on morphology or proliferation of the cells.
  • FIG. 50A-50F shows photographs of the control stem cells and fibroblast cells for the next set of experiments, where the cells were treated with the same concentration of DMSO as is in the test compounds.
  • FIGS. 50A-50C are 10 ⁇ magnification photographs.
  • FIGS. 50D-50F are 20 ⁇ magnification photographs.
  • FIGS. 50A and 50D are photographs of na ⁇ ve state stem cells.
  • FIGS. 50B and 50E are photographs of primed state stem cells.
  • FIGS. 50C and 50F are photographs of human fibroblast cells.
  • FIGS. 51-54 A-F show photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 51-54 G-L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 51-54 M-R show photographs of human fibroblast cells treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the morphology or proliferation of the cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on morphology or proliferation of the cells.
  • FIG. 55A-55F shows photographs of the control stem cells and fibroblast cells for the next set of experiments, where the cells were treated with the same concentration of DMSO as is in the test compounds.
  • FIGS. 55A-55C are 10 ⁇ magnification photographs.
  • FIGS. 55D-55F are 20 ⁇ magnification photographs.
  • FIGS. 55A and 55D are photographs of na ⁇ ve state stem cells.
  • FIGS. 55B and 55E are photographs of primed state stem cells.
  • FIGS. 55C and 55F are photographs of human fibroblast cells.
  • FIGS. 56-64 A-F show photographs of human na ⁇ ve state stem cells, previously grown in NME7 AB over a MUC1* antibody surface, C3, but cultured in the absence of NME7 AB during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Inhibition of proliferation can be seen as holes, or blank areas, in the layer of stem cells.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 56-64 G-L show photographs of human primed state stem cells, previously grown in FGF over a layer of MEFs, but cultured in the absence of FGF during the experiment, and treated for a brief 24 hours with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the pluripotency or proliferation of the stem cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on pluripotency or proliferation.
  • Primed state stem cells grow in defined colonies rather than a uniform layer like na ⁇ ve stem cells.
  • Inhibition of proliferation can be seen as a reduction in the colony size.
  • Inhibition of pluripotency which is also induction of differentiation, is seen as increase in cell size with a decrease in the size of the nucleus, elongation and flattening of cells or rounding up of cells and floating off the plate.
  • FIGS. 56-64 M-R show photographs of human fibroblast cells treated for 3 days with a small molecule drug candidate at a final concentration of 6 uM.
  • a score of ⁇ , or +, ++, +++, or ++++ is given, wherein “ ⁇ ” indicates that at the indicated concentration the drug candidate did not have an obvious effect on the morphology or proliferation of the cells.
  • a “+” indicates a mild effect and “++++” indicates a profound effect on morphology or proliferation of the cells.
  • FIG. 65 A-L shows photographs of a cancer cell migration assay in which the effect of novel compound 1420 is tested for its ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 66 A 1 - 66 R 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 67 A 1 - 67 R 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 68 A 1 - 68 H 3 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 69 A 1 - 69 K 3 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 70 A 1 - 70 I 2 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIGS. 71-75 show measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 76 A 1 - 76 L 3 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 77 A 1 - 77 R 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 78 A 1 - 78 T 3 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of T47D breast cancer cells, 120 hours after single addition of the compound at the indicated concentrations.
  • FIGS. 79-80 show measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of T47D breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 81 A 1 - 81 J 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of a Herceptin resistant breast cancer cell line, BT474-resistant, aka BT-Res2, 120 hours after single addition of the compound at the indicated concentrations.
  • FIG. 82 shows measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of a Herceptin resistant breast cancer cell line, BT474-resistant, aka BT-Res2, over a range of concentrations, or the control, DMSO alone, at 120 hours.
  • FIG. 83 A 1 - 83 F 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of HCT-MUC1*, which is an engineered cell line, where MUC1-negative HCT-116 colon cancer cells were stably transfected with the growth factor receptor MUC1*.
  • Compounds of the invention were added once over a range of concentrations and images were taken at 72 hours post addition of compound.
  • FIG. 84 shows measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of HCT-MUC1* cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 72 hours.
  • FIG. 85 A 1 - 85 J 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of BT20s, a triple negative breast cancer cell line. Compounds of the invention were added once over a range of concentrations and images were taken at 72 hours post addition of compound.
  • FIG. 86 shows measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of BT20s, triple negative breast cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 72 hours.
  • FIG. 87 A 1 - 87 J 4 shows photographs of a cancer cell migration assay in which the effect of compounds of the invention are tested for their ability to inhibit the migration of MUC1-negative HCT-116 colon cancer cells.
  • Compounds of the invention were added once over a range of concentrations and images were taken at 72 hours post addition of compound.
  • FIG. 88 shows measured IC50 curves for compounds of the invention for the ability to inhibit cancer cell migration or invasion of HCT-116 colon cancer cells in the presence of compounds of the invention, over a range of concentrations, or the control, DMSO alone, at 72 hours.
  • FIGS. 89A-89H show graphs of RT-PCR measurement of na ⁇ ve state stem cells treated for 72 hours with compounds of the invention at the indicated concentrations, wherein the genes that are measured are AXIN2, a surrogate for beta-catenin, plus HES3, GNAS, VLDLR, EXT1, FBXL17, RHOC, and GREB1L, which are all super-enhancer target genes that are critical for induction of differentiation.
  • FIGS. 90A-90C show graphs of RT-PCR measurement of cancer cells treated for 72 hours with compounds of the invention at the indicated concentrations, wherein the genes that are measured are AXIN2, a surrogate for beta-catenin, which is suppressed as differentiation is induced, plus NME7 AB and NME7-X1, which are metastatic growth factors.
  • FIGS. 91A-91C show graphs of RT-PCR measurement of na ⁇ ve state stem cells treated for 72 hours with compounds of the invention at the indicated concentrations, wherein the gene that is measured is micro-RNA-145, which is a harbinger of stem cell differentiation.
  • FIGS. 92A-92C show graphs of RT-PCR measurement of T47D cancer cells treated for 72 hours with compounds of the invention at the indicated concentrations, wherein the gene that is measured is micro-RNA-145, which is a harbinger of stem cell differentiation
  • “about” or “substantially” generally provides a leeway from being limited to an exact number.
  • “about” or “substantially” indicates that the polypeptide is not to be limited to the recited number of amino acids. A few amino acids add to or subtracted from the N-terminus or C-terminus may be included so long as the functional activity such as its binding activity is present.
  • administration “in combination with” one or more further therapeutic agents include simultaneous (concurrent) and consecutive administration in any order.
  • carriers include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • pharmaceutically acceptable carrier is an aqueous pH buffered solution.
  • Examples of pharmaceutically acceptable carriers include without limitation buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material 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 (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • MUC1 Growth Factor Receptor is a functional definition meaning that portion of the MUC1 receptor that interacts with an activating ligand, such as a growth factor or a modifying enzyme such as a cleavage enzyme, to promote cell proliferation.
  • the MGFR region of MUC1 is that extracellular portion that is closest to the cell surface and is defined by most or all by the primary sequence of MGFR (PSMGFR).
  • PSMGFR primary sequence of MGFR
  • the MGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc.
  • Results of the invention are consistent with a mechanism in which this portion is made accessible to the ligand upon MUC1 cleavage at a site associated with tumorigenesis that causes release of the some or all of the IBR from the cell.
  • MGFR is also known as MUC1*.
  • PSMGFR MUC1 Growth Factor Receptor
  • FLR FLR
  • PSMGFR Primary Sequence of the MUC1 Growth Factor Receptor
  • the PSMGFR is defined as SEQ ID NO:3 listed below in Table 1, and all functional variants and fragments thereof having any integer value of amino acid substitutions up to 20 (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and/or any integer value of amino acid additions or deletions up to 20 at its N-terminus and/or C-terminus.
  • a “functional variant or fragment” in the above context refers to such variant or fragment having the ability to specifically bind to, or otherwise specifically interact with, ligands that specifically bind to, or otherwise specifically interact with, the peptide of SEQ ID NO:3.
  • nat-PSMGFR a functional variant of the PSMGFR peptide of SEQ NO:3
  • var-PSMGFR SEQ ID NO:11
  • var-PSMGFR differs from nat-PSMGFR by including an -SPY- sequence instead of the native -SRY- (see bold text in sequence listings).
  • Var-PSMGFR may have enhanced conformational stability, when compared to the native form, which may be important for certain applications such as for antibody production.
  • the PSMGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation, etc.
  • PSMGFR is an acronym for Primary Sequence of MUC1 Growth Factor Receptor as set forth as GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:3).
  • N-10 PSMGFR N-15 PSMGFR
  • N-20 PSMGFR refers to the number of amino acid residues that have been deleted at the N-terminal end of PSMGFR.
  • C-number as in “C-10 PSMGFR”, “C-15 PSMGFR”, or “C-20 PSMGFR” refers to the number of amino acid residues that have been deleted at the C-terminal end of PSMGFR.
  • the extracellular domain of MUC1* refers to the extracellular portion of a MUC1 protein that is devoid of the tandem repeat domain.
  • MUC1* is a cleavage product wherein the MUC1* portion consists of a short extracellular domain devoid of tandem repeats, a transmembrane domain and a cytoplasmic tail.
  • the precise location of cleavage of MUC1 is not known perhaps because it appears that it can be cleaved by more than one enzyme.
  • the extracellular domain of MUC1* will include most of the PSMGFR sequence but may have an additional 10-20 N-terminal amino acids.
  • NME family proteins or “NME family member proteins”, numbered 1-10, are proteins grouped together because they all have at least one NDPK (nucleotide diphosphate kinase) domain. In some cases, the NDPK domain is not functional in terms of being able to catalyze the conversion of ATP to ADP.
  • NME proteins were formerly known as NM23 proteins, numbered H1 and H2. Recently, as many as ten (10) NME family members have been identified. Herein, the terms NM23 and NME are interchangeable.
  • NME1, NME2, LAMES, NME6, NME7, NME8 and NME9 are used to refer to the native protein as well as NME variants.
  • NME7 as used in the specification can mean the native protein or a variant, such as NME7-AB that has superior commercial applicability because variations allow high yield expression of the soluble, properly folded protein in E. coli .
  • NME7-AB consists primarily of the NME7 A and B domains but is devoid of most of the DM10 domain (SEQ ID NO:12), which is at the N-terminus of the native protein.
  • NME1 as referred to herein is interchangeable with “NM23-H1”. It is also intended that the invention not be limited by the exact sequence of the NME proteins.
  • the mutant NME1-S120G also called NM23-S120G, are used interchangeably throughout the application.
  • the S120G mutants and the P96S mutant are preferred because of their preference for dimer formation, but may be referred to herein as NM23 dimers, NME1 dimers, or dimeric NME1, or dimeric NM23.
  • NME7 as referred to herein is intended to mean native NME7 having a molecular weight of about 42 kDa.
  • a “family of NME7” refers to full length NME7 as well as naturally occurring or artificially created cleaved form having a molecular weight about 30 kDa, 33 kDa, or a cleaved form having a molecular weight of about 25 kDa, a variant devoid or partially devoid of the DM10 leader sequence (SEQ ID NO:12), which is NME7 about amino acids 1-95 of NME7 represented by SEQ ID NO:5, such as NME7b, NME7-X1, NME7-AB or a recombinant NME7 protein, or variants thereof whose sequence may be altered to allow for efficient expression or that increase yield, solubility or other characteristics that make the NME7 more effective or commercially more viable.
  • the “family of NME7” may also include “NME7-AB-like” protein, which is a protein in the range of 30 to 33 kDa that is expressed in cancer cells.
  • an agent that “induces differentiation, or inhibits stem cell pluripotency or growth of the stem cell” refers to a protein, small molecule or nucleic acid that alone or in combination causes the stem cells either in the prime state or in the na ⁇ ve state, to differentiate or inhibit stem cell pluripotency or growth of the stem cell.
  • agents include SMAD inhibitors and dorsomorphin.
  • an agent that “inhibits expression or activity of an up regulated gene in the na ⁇ ve state” with reference to primed stem cell refers to a protein, small molecule or nucleic acid that alone or in combination causes the inhibition of the normally upregulated gene in na ⁇ ve stem cells.
  • agents include siRNA, anti-sense nucleic acids and small molecules.
  • an agent that “increases expression or activity of down regulated gene in the na ⁇ ve state” with reference to primed cell refers to a protein, small molecule or nucleic acid that alone or in combination causes the upregulation of the normally down regulated gene in na ⁇ ve stem cells.
  • agents include genes coding for proteins that are indicative of differentiation such as vimentin, fibronectin and NF1 and also microRNAs such as miR-145.
  • an agent that “inhibits expression or activity of an up regulated gene in the na ⁇ ve state” with reference to fibroblasts refers to a protein, small molecule or nucleic acid that alone or in combination causes the inhibition of the normally upregulated gene in na ⁇ ve stem cells.
  • agents include anti-sense nucleic acids or siRNA specific for pluripotency genes OCT4, SOX2, KLF4 or c-Myc, and genes that encode vimentin, fibronectin, NF1 or the gene products themselves.
  • an agent that “increases expression or activity of down regulated gene in the na ⁇ ve state” with reference to fibroblasts refers to a protein, small molecule or nucleic acid that alone or in combination causes the upregulation of the normally down regulated gene in na ⁇ ve stem cells.
  • agents include nucleic acids that encode the downregulated genes or the proteins themselves, and agents that induce differentiation such as SMAD inhibitors, dorsomorphin and the like.
  • an “an agent that promotes pluripotency” or “reverts somatic cells to a stem-like or cancer-like state” refers to a protein, small molecule or nucleic acid that alone or in combination induces expression of or suppresses expression of certain genes such that the genetic signature shifts to one that more closely resembles stem cells or cancer cells.
  • NME1 dimers examples include but are not limited to NME1 dimers, NME7, NME7-X1, NME7-AB, 2i, 5i, nucleic acids such as siRNA that suppress expression of MBD3, CHD4, BRD4, or JMJD6, microbial NME proteins that have high sequence homology to human NME1, NME2, NME5, NME6, NME7, NME8, or NME9, preferably with the regions that house NDPK domains.
  • small molecule in reference to an agent being referred to as a “small molecule”, it may be a synthetic chemical or chemically based molecule having a molecular weight between 50 Da and 2000 Da, more preferably between 150 Da and 1000 Da, still more preferably between 200 Da and 750 Da.
  • cancer may include but is not limited to: biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; colon cancer, rectal cancer; sarcom
  • cancer treatment may include but is not limited to: chemotherapy, radiotherapy, adjuvant therapy, or any combination of the aforementioned methods. Aspects of treatment that may vary include, but are not limited to: dosages, timing of administration, or duration or therapy; and may or may not be combined with other treatments, which may also vary in dosage, timing, or duration.
  • Another treatment for cancer is surgery, which can be utilized either alone or in combination with any of the aforementioned treatment methods.
  • One of ordinary skill in the medical arts may determine an appropriate treatment.
  • inflammatory disease refers to disease or conditions characterized by an immune response that involves non-specific immune responses in particular areas.
  • disease or condition may include without limitation, rheumatoid arthritis, inflammatory bowel syndrome, Crohn's disease, osteoarthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post transplantation late and chronic solid organ rejection, multiple sclerosis, systemic lupus erythematosus, Sjogren syndrome, Hashimoto thyroiditis, polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis, pulmonary fibrosis, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury, post-angioplasty restenosis, chronic obstructive pulmonary diseases (COPD), Graves' disease, gastrointestinal allergy, conjunctivitis, atherosclerosis, coronary artery disease
  • COPD chronic
  • tissue sample refers to any body tissue or body fluid sample obtained from a subject.
  • body fluids for example lymph, saliva, blood, urine, milk and breast secretions, and the like. Blood is preferred in certain embodiments.
  • Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods including, but not limited to: tissue biopsy, including punch biopsy and cell scraping, needle biopsy, and collection of blood or other bodily fluids by aspiration or other methods.
  • a “subject”, as used herein, refers to any mammal (preferably, a human), and preferably a mammal that has a disease that may be treated by administering the inventive composition to a site within the subject. Examples include a human, non-human primate, cow, horse, pig, sheep, goat, dog, or cat. Generally, the invention is directed toward use with humans.
  • a “MUC1-positive cancer” or a “MUC1*-positive cancer” refers to a cancer that is characterized by the aberrant expression of MUC1, wherein aberrant may refer to the overexpression of the MUC1 gene or gene product, or the loss of the normal expression pattern of MUC1 or MUC1* which, in the healthy state, is restricted to the apical border of the cell or the luminal edge of a duct or an increase in the amount of MUC1 that is cleaved and shed from the cell surface.
  • nucleotide symbols other than a, g, c, t they follow the convention set forth in WIPO Standard ST.25, Appendix 2, Table 1, wherein k represents t or g; n represents a, c, t or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t and y represents c or t.
  • MUC1 Receptor (Mucin 1 precursor, Genbank Accession number: P15941) (SEQ ID NO: 2) GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWG IALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPT YHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL describes a truncated MUC1 receptor isoform having nat-PSMGFR at its N-terminus and including the transmembrane and cytoplasmic sequences of a full-length MUC1 receptor.
  • GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA describes the extracellular domain of Native Primary Sequence of the MUC1 Growth Factor Receptor (nat-PSMGFR-an example of “PSMGFR”).
  • SEQ ID NO: 4 QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA describes N-10 peptide of PSMGFR in which ten amino acids at the N-terminus has been removed.
  • AIFGKTKIQNAVHCTDLPEDGLLEVQYFF describes B3, which is NME7B peptide 3 (B domain).
  • GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA describes the extracellular domain of “SPY” functional variant of the native Primary Sequence of the MUC1 Growth Factor Receptor having enhanced stability (var-PSMGFR-An example of “PSMGFR”).
  • stem cells and cancer cells have a lot in common.
  • researchers are now discovering that many of the markers of undifferentiated stem cells are in fact also markers of cancer cells.
  • many of the molecular markers that were once considered markers of cancer are now being redefined as stem cell markers.
  • CXCR4 which was previously identified as a marker of metastatic cancer, is a marker of na ⁇ ve stem cells.
  • Cancer cells have also been characterized as undergoing epithelial to mesenchymal transition (EMT), where epithelial cells are terminally differentiated and mesenchymal cells are less differentiated and stem-like cell (Mani et al., 2008).
  • EMT epithelial to mesenchymal transition
  • Pathologists use the appearance of the degree of differentiation to classify cancer stage, with early cancers classified as moderately differentiated and aggressive or metastatic cancers being classified as poorly differentiated.
  • NME7 AB a growth factor that binds to and activates growth, survival and self-renewal functions of MUC1* (Carter et al., 2016).
  • Human stem cells can be maintained in a pluripotent state by culturing in a minimal media containing NME7 AB as the only growth factor.
  • Stem cells cultured in NME7 AB are maintained in the earliest state called na ⁇ ve.
  • NME7 AB is in every cell of Day 3 human morula, where all the cells are in the earliest na ⁇ ve state. By Day 5 of the human blastocyst, NME7 AB is confined to the inner cell mass, where the cells are na ⁇ ve by definition. NME7 AB should be turned off after Day 5 of a human blastocyst except that it is found in testis. However, we found that NME7, in truncated forms corresponding to NME7 AB and NME7-X1, are expressed in aggressive and metastatic cancers (WO2015/023694).
  • NME7 AB to regular cancer cells made them transition to more metastatic cancer cells that formed tumors in animals from as few as 50 implanted cancer cells, whereas non-metastatic cancer cells require 4-6M implanted cells to form a tumor. Additionally, injecting the animals with NME7 AB caused the engrafted cancer cells to metastasize. These data further establish a functional link, at the molecular level, between stem cells and cancer cells and more particularly between aggressive or metastatic cancers and na ⁇ ve stem cells.
  • agents that inhibit stem pluripotency or growth, or induce stem cell differentiation are agents that, when administered to a patient, are effective anti-cancer agents for the prevention or treatment of cancers.
  • na ⁇ ve stem cells are similar in many ways to aggressive or metastatic cancer cells. These results imply that the pathways that promote pluripotency in na ⁇ ve stem cells are the same pathways that promote metastasis in cancer cells.
  • agents that inhibit na ⁇ ve stem pluripotency or growth, or induce stem cell differentiation are agents that, when administered to a patient, are effective anti-cancer agents for the prevention or treatment of metastatic cancers.
  • na ⁇ ve stem cells The vast differences between na ⁇ ve stem cells and primed stem cells suggest that these two distinct types of stem cells grow pluripotently and resist differentiation by different pathways. Therefore, drug candidates that inhibit the pluripotency or proliferation of na ⁇ ve stem cells, but not of primed state stem cells, or have a milder effect on primed state stem cells, are drug candidates that would be most effective in the treatment or prevention of aggressive or metastatic cancers.
  • stem cells are cultured in the presence of an agent that may be a drug candidate, it is observed that the agent inhibits stem cell pluripotency, or growth, or induces stem cell differentiation and said agent is administered to a patient for the prevention or treatment of cancers.
  • the stem cells are human.
  • the stem cells are in the na ⁇ ve state.
  • the stem cells are maintained in the na ⁇ ve state by culturing in NME1 dimers, NME7, NME7 AB , NME7-X1 or by other methods reported to maintain stem cells in a more na ⁇ ve state (Silva et al., 2008; Hanna et al., 2010; Gafni et al., 2013; Theunissen et al., 2014; Ware et al., 2014).
  • the agent is observed to inhibit pluripotency, or growth, or induce differentiation of na ⁇ ve stem cells, but not primed state stem cells, or the agent has a lesser effect on primed state stem cells and the agent is administered to a patient at risk of developing or has been diagnosed with metastatic cancer. Because we have found that all pluripotent stem cells are MUC1* positive, and na ⁇ ve stem cells express NME7 AB , agents identified as described above will be most effective for the treatment of MUC1* positive, or NME7 AB positive, or NME7-X1 positive cancers.
  • cancer miration and “invasion”, as used herein are synonymous and are characteristic of metastatic cancer cells.
  • Migration assay refers to coating a surface with an extracellular matrix protein such as collagen, fibronectin or the like, plating cancer cells onto that surface, but either removing them from an area or restricting them from being plated onto an area, and then photographing the cells as they move into the restricted space or, in the presence of an effective inhibitory agent, are inhibited from moving into the restricted space.
  • Migration assays in which cells are removed from an area are called scratch assays or wound assays and those that restrict cells from being plated in an area, herein is called a platypus assay.
  • Metastatic cancer as used herein includes cancers that have infiltrated or invaded neighboring tissues, or that have moved into lymph nodes, or have moved into organs other than the organ of original cancer.
  • metastatic cancer includes those cancers that are known to readily become metastatic. For example, melanoma that are of a certain depth of skin are statistically going to metastasize within a predictable period of time. Another example is pancreatic cancer, which is known to metastasize, especially to the liver, within a predictable period of time.
  • Grade 1 means the tumor cells look the most like normal cells, called well-differentiated. Well-differentiated cancer cells are slow growing. Grade 2 means the tumor are moderately differentiated and so are faster growing. Grade 3 means the tumor cells look very abnormal and look poorly differentiated, which are the fastest growing cancer cells.
  • TNM TNM system of scoring tumors based on analysis of biopsied tissues and other diagnostic techniques.
  • T stands for extension into adjacent tissues
  • N stands for involvement of lymph nodes
  • M metastasis to distal organs.
  • the T score ranges from 0-4 where zero indicates no evidence of tumor and 4 relates to large tumor that has extended into adjacent tissues.
  • the N score ranges from 0-3, where NO means no evidence of lymph node involvement, N1 means cancer has spread to nearby lymph nodes or a small number of nodes.
  • N2 and N3 indicates tumor has spread to greater number of lymph nodes and/or to more distant nodes.
  • the M score is either 0 or 1, where M0 means no evidence of metastasis and M1 means cancer has spread to distant organs or organs other than the organ of origin.
  • these therapeutics are for the prevention or treatment of cancers that are MUC1-positive, MUC1*-positive, NME7-positive, NME7 AB positive or NME7-X1-positive.
  • MUC1-positive, MUC1*-positive, NME7-positive, NME7 AB positive or NME7-X1-positive we have determined that the signaling pathways that control the growth and pluripotency of na ⁇ ve stem cells are different from those that control the growth and pluripotency of primed stem cells. Further, we discovered that the same pathways that mediate growth or pluripotency of na ⁇ ve stem cells also mediate the growth and metastatic potential of cancer cells.
  • agents that inhibit stem cell pluripotency or growth, or induce stem cell differentiation are agents that inhibit cancer cell proliferation and when administered to a patient, are effective agents for the prevention or treatment of cancers.
  • Agents that inhibit na ⁇ ve stem cell pluripotency or growth, or induce na ⁇ ve stem cell differentiation are agents that inhibit cancer cell migration, which is a characteristic of metastatic cancers, and when administered to a patient, would be effective anti-cancer agents for the prevention or treatment of aggressive or metastatic cancers.
  • Agents that inhibit pluripotency or growth, or induce stem cell differentiation of na ⁇ ve stem cells but not primed stem cells, or have a far lesser effect on primed stem cells are effective agents for the prevention or treatment of aggressive or metastatic cancers.
  • na ⁇ ve stem cells grow stem cells in na ⁇ ve state; 2) contact stem cells with drug candidates; 3) identify drug candidates that inhibit pluripotency or growth, or induce differentiation of na ⁇ ve stem cells; and 4) conclude that drug candidates that inhibit pluripotency or growth, or induce differentiation of na ⁇ ve stem cells are anti-cancer agents for the treatment or prevention of aggressive cancers or cancer metastasis.
  • na ⁇ ve stem cells in na ⁇ ve state and, optionally, in parallel grow stem cells in primed state 1) grow stem cells in na ⁇ ve state and, optionally, in parallel grow stem cells in primed state; 2) contact both populations of stem cells with drug candidates; 3) identify drug candidates that inhibit pluripotency or growth, or induce differentiation of na ⁇ ve stem cells, but, optionally, not primed stem cells or have a far lesser effect on primed stem cells; and 4) conclude that drug candidates that inhibit pluripotency or growth, or induce differentiation of na ⁇ ve stem cells, but, optionally not primed stem cells, or have a far lesser effect on primed stem cells, are anti-cancer agents for the treatment or prevention of cancer metastasis.
  • Agents screened in these ways to assess their potential as anti-cancer or anti-metastasis agents may be of any form including but not limited to small molecules, natural products, antibodies, antibody fragments, libraries or antibodies or antibody fragments, peptides, peptide mimics, nucleic acids, anti-sense nucleic acids, DNA, RNA, coding or non-coding, inhibitory RNAs, bacteria and microbes.
  • the stem cells are of human origin.
  • the stem cells are of primate origin.
  • the stem cells are of mammal origin.
  • the stem cells are of rodent origin.
  • novel anti-cancer or anti-metastasis drug targets are identified by identifying genes that are upregulated in na ⁇ ve stem cells but not in primed stem cells.
  • novel anti-cancer or anti-metastasis drug targets are identified by identifying microRNAs that are upregulated in na ⁇ ve stem cells but not in primed stem cells.
  • WO2009/042815 discloses that in a direct binding assay a series of carboline molecules inhibited the interaction between the extracellular domain of MUC1* and NME proteins, especially NME1 dimers and NME7 AB .
  • NME1 dimers and NME7 AB we also previously showed that the same series of carbolines that inhibited MUC1*-NME interaction also inhibited cancer cell growth.
  • JQ1 is a small molecule that reportedly inhibits BRD4 and has been shown to inhibit cancer cell migration and cancer cell proliferation, but has not been reported to have any effect on stem cells.
  • the stem cell screening assay was performed in both the presence and absence of the stem cell growth factors: NME7 AB for growing na ⁇ ve stem cells or FGF for growing primed stem cells. If the cognate growth factor was present, then the biological or small molecule would have to compete away the growth factor to get an effect. Therefore, we expected to see more of an effect when the growth factor, FGF for primed stem cells or NME7 AB or NME1 dimers for na ⁇ ve stem cells, was absent. The results are summarized in the table of FIG. 2 .
  • the effect of the compounds on stem cells was visually determined and compounds were ranked 0-4, with 4 being the greatest effect and 0 being no observable effect.
  • the major effect that was observed was a change from pluripotent stem cell morphology, which is a cobblestone pattern of small round cells with a large nucleus to cytoplasm ratio, to that of differentiating stem cells, which are elongated, large and flattened cells with a smaller nucleus to cytoplasm ratio. Some compounds also severely inhibited growth of the stem cells.
  • the compounds were added to a final concentration of 6 uM to either na ⁇ ve state stem cells or primed state stem cells.
  • the na ⁇ ve state stem cells were maintained in a na ⁇ ve state by culturing in a media containing NME7 AB or NME1 dimers.
  • other methods such as 2i and 5i (Silva et al., 2008, Nichols and Smith, 2009, Theunissen et al., 2014)] can be used to maintain stem cells in a more na ⁇ ve state.
  • primed state stem cells were cultured in bFGF over a layer of MEFs, although it is known that any bFGF containing media will maintain stem cells in the primed state.
  • JQI has an inhibitory effect on na ⁇ ve stem cell growth but not primed stem cell growth.
  • previous studies have shown JQ1 has anti-inflammatory effects (Belkina et al, 2013; Meng et al, 2014). Therefore, the compounds identified in this study should also have anti-inflammatory effects and be useful in the treatment of inflammation in obesity, asthma, chronic peptic ulcer, tuberculosis, rheumatoid arthritis, chronic periodontitis, ulcerative colitis and Crohn's disease, chronic sinusitis, Chronic active hepatitis etc.
  • FIG. 2 is a summary of how those drug candidates performed in the na ⁇ ve versus primed stem cell drug in which a confirmed drug hit is one in which the compound induced differentiation of the na ⁇ ve stem cells but had no effect or a lesser effect on the FGF-grown primed stem cells.
  • FIGS. 3-10 show photographs of stem cells that were treated with the small molecules, the Fab, the MUC1* extracellular domain peptide “FLR” or the small molecules.
  • FIGS. 7-10 show that several agents induced differentiation of na ⁇ ve state stem cells. Differentiating portions are indicated by dashed lines. Specifically, at these concentrations, the anti-MUC1* E6 Fab, the FLR peptide, anti-NME7 #61, MN572, MN0642 and MN1130 all induced differentiation of na ⁇ ve state stem cells and are predicted to be potent inhibitors of cancer and inhibitors of metastatic cancers. They could be administered to patients for the prevention or treatment of cancers or metastatic cancers. The E6 Fab has been shown to inhibit the growth of all MUC1* positive cancer cells.
  • anti-MUC1* E6 Fab was shown to robustly inhibit MUC1* positive tumor growth in animals.
  • Compound MN0642 similarly has been shown to inhibit the growth of cancer cells in vitro.
  • the FLR (PSMGFR) peptide and anti-NME7 #61 have been shown to inhibit the transition of regular cancer cells to metastatic cancer cells.
  • a small molecule that bears no resemblance to compounds of the invention but that were reported to inhibit cancer growth or migration were tested and found to inhibit pluripotency, or growth or induce differentiation of stem cells, particularly na ⁇ ve stem cells.
  • a small molecule that bears no resemblance to carbolines, JQ1(+) ( FIG. 1 ) reportedly inhibits inflammation (Belkina et al., 2013), cancer pluripotency (Fillippakopoulos et al., 2010) and cancer cell migration (Tang et al., 2013).
  • JQ1(+) reportedly inhibits BRD4 and its inactive enantiomer, JQ1( ⁇ ), has no effect (Fillippakopoulos et al., 2010).
  • BRD4 has been reported to be a regulator of NME7, a regulator of oncogene c-Myc and a component of super-enhancers that overexpress a selected few genes in cancer cells and in stem cells. At this time, it is not entirely clear which of these purported functions of BRD4 are correct.
  • Primed state stem cells were treated for 3 days with JQ1(+), inactive stereoisomer JQ1( ⁇ ), BRD4 specific siRNA, or JMJD6 specific siRNA.
  • JQ1(+) may have a modest effect on the size of primed stem cell colonies ( FIG. 11 ), and also appeared to cause some abnormal morphology ( FIG. 12 ).
  • JQ1(+) dramatically induced differentiation of na ⁇ ve state stem cells and inhibited their growth ( FIGS. 14 E-F, 15 E-F and 16 E-F). Whether the na ⁇ ve stem cells were cultured in NME7 AB ( FIG. 13-14 ) or NME1 dimers ( FIG. 15-16 ), JQ1(+) inhibited na ⁇ ve stem cell pluripotency and growth and induced differentiation.
  • JQ1 (+) is a known inhibitor of inflammation, cancer cell migration and cancer cell proliferation, these results show that agents that are effective treatments for inflammation or the prevention or treatment of cancers, also inhibit na ⁇ ve stem cell pluripotency or growth or induce stem cell differentiation. Therefore, the agents that inhibit na ⁇ ve stem cell pluripotency or growth or induce stem cell differentiation are also effective treatments for inflammation or the prevention or treatment of cancers.
  • FIG. 19A-19P shows photographs of control stem cells or stem cells to which was added known anti-migration compounds Dexamethasone and SU11274.
  • Potent cancer cell migration is characteristic of cancer cell invasion of other tissues and of metastasis.
  • Typical migration assays involve coating a cell culture plate with fibronectin, collagen or the like, plating cancer cells and making a scar across through the cells and measuring the time it takes for the cancer cells to invade the void space.
  • An alternative approach that gives more reliable data is the Platypus System which is a special multi-well cell culture plate with a juxtaposed set of plugs that block off a circle in the center of each well. Cancer cells are plated while the plugs are in place, then they are removed after the cells attach to the plate surface. Drug candidates are added to each well and photographs are then taken as a function of time to track the inhibitory effect of the drug candidates on cancer cell migration or invasion.
  • FIG. 20 A bar graph summarizing the results of such a cancer cell migration assay is shown in FIG. 20 .
  • the effects of known anti-migration compounds are compared to the anti-MUC1* Fab E6 and the first few small molecule leads.
  • the results of the cancer cell migration assay are shown in FIG. 21 .
  • Photographs of the cancer cell migration assay and bar graphs summarizing their activities are shown in FIG. 22 .
  • the effect of two novel small molecules MN1186 and MN1194, compared to the known anti-migration molecule SU11274, is shown in FIG. 22A-22U .
  • FIG. 22A-22U A bar graph summarizing the results of such a cancer cell migration assay.
  • FIG. 22V is a graph showing the measured inhibition of cancer cell migration at time 0, 24 hours or 48 hours for a number of compounds.
  • FIG. 22W is a graph showing the inhibitory effect of the small molecules as a function of its concentration.
  • FIG. 22X is a graph showing how IC50's of the small molecules of the invention were measured and calculated.
  • All human pluripotent stem cells are MUC1* positive.
  • Na ⁇ ve state stem cells also express the primitive growth factor NME7 AB which is an activating ligand of MUC1*.
  • the breast cancer cell line T47D was derived from a metastatic breast cancer patient.
  • T47D cells express the highest levels of MUC1* of any commercially available cell line.
  • NME7 AB and an alternative splice isoform NME7-X1 which are both growth factors that activate the MUC1* growth factor receptor.
  • Compound hits are first identified in the stem cell drug screening assay for their ability to inhibit stem cell pluripotency or proliferation. We then test the hits for their ability to inhibit cancer cell migration, invasion, which is a characteristic of metastatic cancers, and then finally we test the hits for their ability to inhibit cancer cell proliferation. The result is that compounds that inhibit stem cell pluripotency and/or proliferation also inhibited cancer cell migration, invasiveness and/or proliferation. These studies showed that compounds of the invention inhibit migration and/or invasion of a wide range of cancer cells.
  • Compounds of the invention were shown to inhibit migration, invasion and/or proliferation of DU145 (MUC1* + /NME7AB +++ /NME7-X1 +++ ) prostate cancer cells, and SK-OV-3 (MUC1* + ) ovarian cancer cells, A549 (MUC1* LO ) lung cancer cells, PC-3 (MUC1* ⁇ /NME7 AB +++ /NME7-X1 +++ ) prostate cancer cells, CHL-1 (MUC1* + /NME7 + ) melanoma cells, OV-90 (MUC1* ⁇ ) ovarian cancer cells, CAPAN-2 (MUC1* + ) pancreatic cancer cells, ZR-75-1 (MUC1* ++ ) breast cancer cells, as well as others.
  • Small molecule inhibition of cancer cell migration or proliferation studies were also performed using previously reported inhibitors of cancer cell migration or invasion, such as the BRD4 inhibitor JQ1+ and its inactive enantiomer JQ1 ⁇ , c-Met inhibitor SU11274, and others shown in FIG. 17 .
  • Some of these compounds inhibited cancer cell migration or invasion to some degree, however most also inhibited the growth of fibroblast cells, which are a surrogate for normal healthy cells, which implies they could have toxic side effects on patients.
  • FIG. 18A-18E The biological testing data for compounds of the invention are shown in FIG. 18A-18E .
  • fibroblasts are more differentiated than stem cells but are able to self-replicate for defined periods of time.
  • fibroblasts as a surrogate for normal cells. Since fibroblasts do not change morphology, the readout of this assay was only what effect the compounds had on proliferation.
  • FIG. 23A-23D shows photographs of human fibroblasts in culture, treated only with 0.2% DMSO as a control.
  • FIG. 24A-24F shows photographs of the effect of JQ1+ ( FIG. 24A-24C ) versus the effect of the inactive enantiomer JQ1 ⁇ , both at 500 nM final concentration, ( FIG. 24D-24F ) on human na ⁇ ve state stem cells ( FIG. 24A, 24D ), human primed state stem cells ( FIG.
  • FIG. 24B, 24E shows photographs of the effect of previously known cancer cell migration inhibitors JQ1 and SU11274 versus the original hits that led to the derivatives that are now compounds of this invention, on the growth of human fibroblast progenitor cells.
  • most of the novel compounds of the invention have little or no effect on the growth of fibroblast cells.
  • novel compounds of the invention inhibit pluripotency, proliferation and/or migration of both stem cells and cancer cells by inducing maturation, also known as differentiation.
  • RT-PCR measurements of na ⁇ ve stem cells that have been treated with compounds of the invention showed that the compounds of the invention induced upregulation of markers of differentiation.
  • the genes whose expression increased as a result of treatment with the compounds of the invention, in a concentration dependent manner, are fibronectin and vimentin, which both increase as stem cells initiate differentiation and NF1, which is one of the first genes to increase when stem cells begin to differentiate down the neural lineage.
  • fibronectin, vimentin or NF1 expression increases in response to treatment with compounds of the invention shows that the compounds induce differentiation and terminally differentiated cells do not self-replicate.
  • compounds of the invention that induce markers of differentiation are useful for the treatment of cancers, because cancer cells, by definition, have de-differentiated, which allows them to continually self-replicate.
  • E-cadherin which is upregulated in cancers, was down regulated when the cancer cells were treated with compounds of the invention.
  • JQ1+ and SU11274 did not cause upregulation of markers of differentiation, i.e. induce differentiation of the stem cells.
  • novel compounds of the invention induced differentiation of cancer cells. Expression of metastatic marker E-cadherin was reduced and expression of differentiation markers fibronectin, vimentin and NF1 were increased.
  • Novel compounds of the invention are highly specific. They specifically inhibit pluripotency and/or proliferation of stem cells and cancer cells. Novel compounds of the invention are most effective against cancers that are MUC1* positive and/or NME7 AB or NME7-X1 positive. Although we discovered that NME1 dimers, NME7 AB and NME7-X1 are all activating ligands of the MUC1* growth factor receptor and they bind to its extracellular domain, we have developed ample evidence that both NME7 AB and NME7-X1 have other binding partners and can exert oncogenic effects, independent of MUC1*.
  • NME7 AB is the natural growth factor that makes the earliest na ⁇ ve stem cells grow. NME7 AB alone is sufficient for the growth and pluripotency of na ⁇ ve human stem cells. In human Day 3 blastocysts, all cells are positive for NME7 AB . By Day 5, the NME7 AB cells are restricted to the inner cell mass, which by definition contains na ⁇ ve state stem cells. Although NME7 AB is expressed in all na ⁇ ve stem cells, it reportedly is not expressed in adult tissues except in testis. However, we have found it in every metastatic cancer we have examined. We have shown that both na ⁇ ve stem cells and cancer cells secrete NME7 AB and NME7-X1.
  • both NME7 AB and NME7-X1 bind to the extracellular domain of MUC1* and activate pluripotency and growth via ligand-induced dimerization of the MUC1* extracellular domain.
  • Numerous immunohistochemistry studies we have performed show that both NME7 AB and NME7-X1 are overexpressed in cancer cells and the increase in expression correlates to tumor stage.
  • PCR experiments show that the compounds of the invention cause a decrease in the expression of NME7 AB and NME7-X1 in cancer cells.
  • FIGS. 26-35 show photographs of the effects of the compounds on either na ⁇ ve state stem cells, primed state stem cell or fibroblasts. Compounds that inhibit stem cell pluripotency, especially na ⁇ ve state pluripotency but do not affect more mature cells like fibroblasts are predicted to be effective anti-cancer therapeutics. As can be seen in the tabulated data of FIG.
  • FIGS. 36-45 show photographs, graphs and IC50 curves that quantify the effect of these new compounds on cancer cell migration.
  • MN1413 inhibited na ⁇ ve stem cell pluripotency and proliferation by 100% or a score of ‘4’, while having no effect on the more mature primed state stem cells and also having no effect on fibroblast cells, which are a surrogate for normal cells.
  • MN1413 inhibited cancer cell migration by 83% with an IC50 of 10 nM, and inhibited cancer cell proliferation by about 50%.
  • MN1423 inhibited na ⁇ ve stem cell pluripotency and proliferation by 100%, or score of ‘4’, but had no effect on primed state stem cells or fibroblasts.
  • MN1423 inhibited cancer cell migration by 84% with an IC50 of 12 nM and inhibited cancer cell proliferation by 50%.
  • MN1428 also inhibited na ⁇ ve stem cell pluripotency and proliferation by 100%, or score of ‘4’, but had no effect on primed state stem cells or fibroblasts.
  • MN1428 inhibited cancer cell migration by 79% with an IC50 of 7 nM. The results of the stem cell drug screening of these compounds are shown in FIGS. 46-64 .
  • FIGS. 65-88 show photographs and graphs showing the effects of these compounds on cancer cell migration or invasion and graphs indicating the IC50 of each compound.
  • the compounds of the invention inhibited tumor cell migration and invasion and such activity was independent of whether the cancer cells were positive or negative for the common cancer growth factor receptor, MUC1*. Recall that 100% of na ⁇ ve stem cells are MUC1* positive. Most cancers are MUC1* positive as well.
  • MUC1* cancer cell lines including T47D breast cancer cells, BT20 triple negative breast cancer cells, BT474-Res2 chemo resistant HER2 positive breast cancer cells, SKOV3 ovarian cancer cells, DU145 prostate cancer cells and Capan2 pancreatic cancer cells, as well as many others.
  • compounds of the invention have also been shown to inhibit migration of some MUC1* negative prostate cancer cells. For example, compounds of the invention inhibited migration and proliferation of PC3 prostate cancer cells and HCT-116 MUC1* negative colon cancer cells.
  • the genes that are super-upregulated in primed state stem cells, but not in na ⁇ ve stem cells include LIN7A, VLDLR, GNAS, ZIC5, HES3, BDNF, FBXL17, RHOC, KLHL4, GREB1L, EXT1, FEZF1, SULF1, BRD2, CDH9, and LRRTM2.
  • BRD2 which itself regulates expression of 1,450 other genes through its interaction with chromatin
  • HES3 which regulates basic helix-loop-helix transcription factors
  • GNAS which mediates the activity of a host of factors that are critical for differentiation.
  • MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells. Cell. 137(4), p 647-658, 15 May 2009. DOI:10.1016/j.cell.2009.02.038; and Smagghe et al PLoS ONE 2013).
  • Sachdeva and Mo Cancer Res: 70(1); 378-87, 2010
  • MiR-145 directly suppresses the tumor metastasis gene MUC1, and by extension MUC1*, which then suppresses expression of activated ⁇ -catenin.
  • FIG. 90 shows that compounds of the invention suppressed expression of MUC1* ligands NME7 AB and NME7-X1, which we have shown induce cancer metastasis in vitro and in animals.
  • Compounds of the invention also increased expression of miR-145 which has been shown to induce differentiation and suppress tumor cell invasiveness and migration.
  • FIG. 91A-91C shows a graph of RT-PCR measurement of na ⁇ ve state stem cells treated with compounds MN1413, MN1423 and 1428. As can be seen, these compounds increased expression of miR-145.
  • FIG. 92A-92C shows a graph of RT-PCR measurement of T47D cancer cells treated with compounds MN1413, MN1423 and 1428.
  • these compounds increased expression of miR-145 in cancer cells also.
  • compounds of the invention inhibit tumor cell migration and invasiveness by inducing expression of genes that are critical for differentiation, some of which are super-enhancer target genes, and miR-145, while decreasing expression of ⁇ -catenin, MUC1 and its growth factor NME7 AB .
  • Novel compounds of the invention are powerful agents for the treatment or prevention of cancers and metastatic cancers. The novel compounds of the invention will be most effective for the treatment of cancers that are MUC1* positive and/or NME7 AB or NME7-X1 positive.
  • a biological sample from a patient is tested for the presence of MUC1*, NME7 AB or NME7-X1, and upon finding that the patient's cancer is positive for MUC1*, NME7 AB or NME7-X1, a compound of the invention is administered to the patient in an amount suitable to prevent or treat the cancer.
  • the patient sample is subjected to a test, such as PCR, to determine the amount of nucleic acid that encodes MUC1, NME7 or NME7-X1.
  • the patient's cancer is considered to be MUC1* positive, NME7 AB positive or NME7-X1 positive if expression of those genes is comparable to, or higher than, their expression in human pluripotent stem cells.
  • the patient's cancer is considered to be MUC1* positive, NME7 AB positive or NME7-X1 positive if expression of those genes is equal to or greater than 0.5% of EEF1A1 expression in those cells.
  • the patient's cancer is considered to be MUC1* positive if the patient's tissue specimen is contacted with an antibody that binds to the PSMGFR peptide or the N-10 peptide and stains the tissue with a pathologist's standard score 1-4 (“+ ⁇ ++++”).
  • the patient's cancer is considered to be NME7 AB positive or NME7-X1 positive if the patient's tissue specimen is contacted with an antibody that binds to the B3 peptide of NME7 and stains the tissue with a pathologist's standard score 1-4 (“+ ⁇ ++++”).
  • FIGS. 18A-18E A Table summarizing the below exemplified compounds is set forth in FIGS. 18A-18E .
  • Described herein are compounds for use in the treatment or prevention of cancer or cancer metastasis.
  • the following definitions apply:
  • an “alkyl” substituent group or an alkyl moiety in a substituent group may be linear or branched, or be or include one or more cycloalkyl groups.
  • Suitable alkyl groups include but are not limited to C1-C9 alkyl groups, C1-C6 alkyl groups, C1-C4 alkyl groups, and C1-C3 alkyl groups.
  • alkyl groups/moieties include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2,4,4-trimethylpentyl, 2-methylcyclopentyl, cyclopentylmethyl and cycloalkyl groups/moieties as exemplified below. All alkyl groups, unless otherwise stated, may be substituted or unsubstituted.
  • Alkyl refers to alkyl groups that do not contain heteroatoms. Thus the phrase includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.
  • the phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: —CH(CH 3 ) 2 , —H(CH 3 )(CH 2 CH 3 ), —CH(CH 2 CH 3 ) 2 , —C(CH 3 ) 3 , —C(CH 2 CH 3 ) 3 , —CH 2 CH(CH 3 ) 2 , CH 2 CH(CH 3 )(CH 2 CH 3 ), —CH 2 CH(CH 2 CH 3 ) 2 , —CH 2 C(CH 3 ) 3 , —CH 2 C(CH 2 CH 3 ) 3 , —CH(CH 3 ), —CH(CH 3 )(CH 2 CH 3 ), —CH 2 CH 2 CH(CH 3 ) 2 , —CH 2 CH 2 CH(CH 3 )(CH 2 CH 3 ), —CH 2 CH 2 CH(CH 3 ) 2 , —CH 2 CH 2 CH(CH 3 ) 2
  • Halogen or “halo” refers to chloro, bromo, fluoro, and iodo groups.
  • haloalkyl refers to an alkyl radical substituted with one or more halogen atoms.
  • haloalkoxy refers to an alkoxy radical substituted with one or more halogen atoms.
  • haloalkyl substituent group or a haloalkyl moiety in a substituent group refers to an alkyl group or moiety in which one or more, e.g. one, two, three, four or five, hydrogen atoms are replaced independently by halogen atoms, i.e. by fluorine, chlorine, bromine or iodine atoms.
  • Suitable haloalkyl groups include but are not limited to halo (C1-C3)alkyl, and halo(C1-C)alkyl. Examples of haloalkyl groups/moieties include fluoromethyl, difluoromethyl, trifluoromethyl and 2,2,2-trifluoroethyl.
  • a “cycloalkyl” substituent group or a cycloalkyl moiety in a substituent group refers to a saturated hydrocarbyl ring containing, for example, from 3 to 8 carbon atoms, examples of which include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Unless stated otherwise, a cycloalkyl substituent group or moiety may include monocyclic, bicyclic (e.g. fused or spiro) and polycyclic hydrocarbyl rings.
  • a “cycloalkyl” substituent group or a cycloalkyl moiety in a substituent group includes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • heteroalkyl substituent group or a heteroalkyl moiety in a substituent group refers to an alkyl group or moiety in which from 1 to 4 secondary or tertiary carbon atoms, including any secondary or tertiary carbon atoms through which the group or moiety is attached to the rest of the molecule, are replaced independently by heteroatoms selected from nitrogen, oxygen and sulphur in the case of secondary carbon atoms, or by nitrogen in the case of tertiary carbon atoms.
  • heteroalkyl groups/moieties include methoxy, methylamino, methylsulphanyl, ethoxy, ethylamino, dimethylamino, ethylsulphanyl, propyloxy, methoxyethyl, propylamino, methylethylamino, propylsulphanyl, methylsulphanylethyl, tetrahydropyranyloxy, N-methylpyrrolidinyl, and heterocycloalkyl groups/moieties as exemplified below.
  • heterocycloalkyl substituent group or a heterocycloalkyl moiety in a substituent group refers to a cycloalkyl group or moiety in which from 1 to 4 secondary or tertiary carbon atoms, including any secondary or tertiary carbon atoms through which the group or moiety is attached to the rest of the molecule, are replaced independently by heteroatoms selected from nitrogen, oxygen and sulphur in the case of secondary carbon atoms, or by nitrogen in the case of tertiary carbon atoms.
  • heterocycloalkyl groups/moieties examples include tetrahydrofuranyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl and thiomorpholinyl.
  • alkenyl substituent group or an alkenyl moiety in a substituent group refers to an unsaturated alkyl group or moiety having one or more carbon-carbon double bonds.
  • Suitable “alkenyl” group include but are not limited to C1-C9 alkenyl, C1-C6 alkenyl, C1-C4 alkenyl, and C1-C3 alkenyl.
  • alkenyl groups/moieties examples include ethenyl, propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 1-hexenyl, 1,3-butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, 1,4-hexadienyl and cycloalkenyl groups/moieties as exemplified below.
  • a “cycloalkenyl” substituent group or a cycloalkenyl moiety in a substituent group refers to an unsaturated hydrocarbyl ring having one or more carbon-carbon double bonds and containing, for example, from 3 to 8 carbon atoms, examples of which include cyclopent-1-en-1-yl, cyclohex-1-en-1-yl and cyclohex-1,3-dien-1-yl.
  • a cycloalkenyl substituent group or moiety may include monocyclic, bicyclic (e.g. fused or spiro) and polycyclic hydrocarbyl rings.
  • heteroalkenyl substituent group or a heteroalkenyl moiety in a substituent group refers to an alkenyl group or moiety in which from 1 to 4 secondary or tertiary carbon atoms, including any secondary or tertiary carbon atoms through which the group or moiety is attached to the rest of the molecule, are replaced independently by heteroatoms selected from nitrogen, oxygen and sulphur in the case of secondary carbon atoms, or by nitrogen in the case of tertiary carbon atoms.
  • heteroalkenyl groups/moieties include ethenyloxy, ethenylamino, ethenylsulphanyl, ethenyloxyethyl and heterocycloalkenyl groups/moieties as exemplified below.
  • heterocycloalkenyl substituent group or a heterocycloalkenyl moiety in a substituent group refers to a cycloalkenyl group or moiety in which from 1 to 4 secondary or tertiary carbon atoms, including any secondary or tertiary carbon atoms through which the group or moiety is attached to the rest of the molecule, are replaced independently by heteroatoms selected from nitrogen, oxygen and sulphur in the case of secondary carbon atoms, or by nitrogen in the case of tertiary carbon atoms.
  • heterocycloalkenyl groups/moieties include dihydropyranyl and dihydrofuranyl.
  • alkynyl substituent group or an alkynyl moiety in a substituent group refers to an unsaturated alkyl group or moiety having one or more carbon-carbon triple bonds.
  • alkynyl groups/moieties include ethynyl, propargyl, but-1-ynyl and but-2-ynyl.
  • heteroalkynyl substituent group or a heteroalkynyl moiety in a substituent group refers to an alkynyl group or moiety in which from 1 to 4 secondary or tertiary carbon atoms, including any secondary or tertiary carbon atoms through which the group or moiety is attached to the rest of the molecule, are replaced independently by heteroatoms selected from nitrogen, oxygen and sulphur in the case of secondary carbon atoms, or by nitrogen in the case of tertiary carbon atoms.
  • heteroalkynyl groups/moieties include ethynyloxy and propargylamino.
  • heteroaryl substituent group or a heteroaryl moiety in a substituent group includes monocyclic aromatic and polycyclic fused ring aromatic groups in which from 1 to 4 ring atoms are independently selected from nitrogen, oxygen and sulphur, with the remainder of the ring atoms being carbon.
  • heteroaryl groups/moieties include the following:
  • arylalkyl arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl
  • the last mentioned moiety contains the atom by which the group is attached to the rest of the molecule.
  • An example of an arylalkyl group is benzyl.
  • An example of cycloalkylalkyl is cyclopropylmethyl.
  • hetero is used in relation to a combination of moieties referred to as one group, for example “hetero(arylalkyl)”, any or all of the moieties within the combination may be a hetero moiety.
  • hetero(arylalkyl) encompasses heteroaryl-alkyl, aryl-heteroalkyl and heteroaryl-heteroalkyl.
  • hetero(arylalkyl) groups/moieties include pyridinylmethyl, phenoxy, N-anilinyl and pyridinyloxyethyl.
  • a group may be substituted, the group may be substituted by, for example, one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H
  • the invention discloses compounds of Formula 1:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C3-C4 cycloalkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH 3 , —OC 2 H 5 , —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be C2-C4 alkyl, or C3-C4 cycloalkyl.
  • R1 can be methyl
  • R1 can be ethyl, isopropyl, cyclopropyl, or isobutyl.
  • R1 can be ethyl, isopropyl, or cyclopropyl.
  • R2 can be H, halogen or methyl.
  • R2 can be H, F, Cl, or Me.
  • R2 is H.
  • R1 is ethyl, isopropyl, cyclopropyl, or isobutyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isopropyl, cyclopropyl, or isobutyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —NH 2 , —CN, —CHO, —COOH, or —CONH 2 .
  • R1 is ethyl, or isopropyl, or cyclopropyl and R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, or C1-C6 alkyl.
  • the invention discloses compounds of Formula 2:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is H, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • Z1 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —C3-C7 cycloalkyl-CH 2 —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—; —C3-C7 cycloalkyl-CH 2 NH(CO)—, —C3-C7 cycloalkyl-CH 2 NCH3(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —
  • Z3 is —OH, —OCH3, —O—C1-C6 alkyl, —O—CH2C6H5, —NH 2 , —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —C1-C6 alkyl;
  • R3 is H, optionally substituted C1-C9 alkyl, C2-C6 alkenyl; optionally substituted C6-C12 aryl, optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; or an optionally substituted C3-C7 cycloalkyl; —(CH 2 ) n —NH(CO)O—(C1-C6 alkyl); —CH 2 O(CH 2 ) p —NH(CO)O—(C1-C6) alkyl; —(CH 2 ) p —NHCO—(CH 2 ) m —NH(CO)O—C1-C6 alkyl); —NH(CO)O-tert-butyl; —O-tert-butyl; or -tert-butyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be H, C1-C4 alkyl (e.g. methyl, ethyl, isopropyl, isobutyl), phenyl, phenyl substituted with halogen, methylcarboxy, methoxy, ethoxy, methyl; heteroaryl, pyridyl, benzyl or alpha-methylbenzyl.
  • C1-C4 alkyl e.g. methyl, ethyl, isopropyl, isobutyl
  • phenyl e.g. methyl, ethyl, isopropyl, isobutyl
  • phenyl phenyl substituted with halogen, methylcarboxy, methoxy, ethoxy, methyl
  • heteroaryl pyridyl, benzyl or alpha-methylbenzyl.
  • R1 can be H or C2-C4 alkyl.
  • R1 is H.
  • R2 can be H, halogen, methyl or methoxy.
  • Z1 can be a bond, —NH—, —CH 2 —, —(CH2)2-, —(CH2)3-, —CH ⁇ CH—, substituted phenyl, —CH2NH(CO)O—, —(CH2)2NH(CO)O—, —(CH2)3NH(CO)O—, —(CH2)4NH(CO)O—, —(CH2)5NH(CO)O—, —CH2NH(CO)—, —CH(CH3)NH(CO)O—, —CH2NH(CO)NH—, —CH2NH(CO)CH2NH(CO)O—, —CH2O(CH2)2NH(CO)O— or -cyclohexyl-CH2NH(CO)O—.
  • Z3 can be —OH, —OCH3, —O—C1-C6 alkyl, —NH2, —N(C1-C6 alkyl)2, or —C1-C6 alkyl.
  • R3 can be ethyl, butyl, isobutyl, pentyl, 2,4,4-trimethylpentyl, heptyl, octyl, phenyl, phenyl substituted with methyl, ethyl, halogen, ethoxy or methoxy.
  • R1 is isobutyl
  • R3 is —NH(CO)O-tert-butyl
  • R2 can be hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • Z1 is cyclohexylmethyl
  • R3 is —NH(CO)O-tert-butyl
  • R2 can be hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • Z1 is C1-C5 alkyl
  • R3 is —NH(CO)O-tert-butyl or —NH(CO)CH2-isopropyl
  • R2 can be hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R3 is —NH(CO)O-tert-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isobutyl, isopropyl, benzyl, Z1 is (CH2) 4-9 -, R3 is —NH(CO)O-tert-butyl, R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • Z1 is (CH2) 4-9 -
  • R3 is —NH(CO)O-tert-butyl
  • R2 can be hydrogen
  • R1 is a phenyl ring substituted with hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • Z1 cyclohexylmethyl or a C3-C7 cycloalkyl-CH2- group R3 is —NH(CO)O-tert-butyl, R1 is isobutyl, R2 is halogen, methyl, or methoxy.
  • the invention discloses compounds of Formula 3:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; an optionally substituted unsubstituted C3-C8 cycloalkyl; or optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • G1 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —C3-C7 cycloalkyl-, —C3-C7 cycloalkyl-CH 2 —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) p NH(CO)—, —(CH 2 ) p NH(CO)O—, —(CH 2 ) p NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-NCH3(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-CH 2 NCH3(CO)O—,
  • Z3 is —OH, —OCH3, —O—C1-C6 alkyl, —O—CH2C6H5, —NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —C1-C6 alkyl;
  • R5 is H, methyl, or optionally substituted C1-C6 alkyl
  • R4 is H, optionally substituted C1-C9 alkyl such as but not limited to tert-butyl; optionally substituted C2-C6 alkenyl; optionally substituted C6-C12 aryl such as but not limited to optionally substituted naphthyl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; an optionally substituted C3-C7 cycloalkyl; —(CH 2 ) p —NH(CO)O—(C1-C6 alkyl); —CH 2 O(CH 2 ) p —NH(CO)O—(C1-C6) alkyl; —(CH 2 ) p —NHCO—(CH 2 ) n —NH(CO)O—C1-C6 alkyl); —NH(CO
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be hydrogen, C1-C4 alkyl (e.g. methyl, ethyl, isopropyl, isobutyl), benzyl, heteroaryl such as pyridyl, phenyl, and phenyl substituted with halogen, trifluoromethyl, methoxy, cyano or dialkylamino.
  • C1-C4 alkyl e.g. methyl, ethyl, isopropyl, isobutyl
  • benzyl heteroaryl such as pyridyl, phenyl, and phenyl substituted with halogen, trifluoromethyl, methoxy, cyano or dialkylamino.
  • R1 can be H or C1-C4 alkyl.
  • R1 is H.
  • R2 can be hydrogen, halogen, methyl or methoxy.
  • R2 is H.
  • Z2 can be O, NH, —CH 2 —, —(CH2)2-, —(CH2)3-, —(CH2)4-, —(CH2)5-, —CH(CH3)-, —CH2NH(CO)CH2-, —CH2O(CH2)2-, -cyclohexyl-CH2- or a bond.
  • Z2 is O.
  • Z3 can be —OH, —OCH3, —O—C1-C6 alkyl, —NH2, —N(C1-C6 alkyl)2, or —C1-C6 alkyl.
  • G1 is —(CH2)-, —(CH2)2-, —(CH2)3-, —(CH2)4-, —(CH2)5-, —CH2OCH2CH2-, —CH(CH3)-, —CH2NHCOCH2- or -cyclohexyl-CH2-.
  • G1 is -cyclohexyl-CH2-.
  • R5 can be hydrogen, methyl or 2-phenylethyl.
  • R5 is methyl
  • R4 can be optionally substituted phenyl, naphthyl, benzyl, substituted isopropyl or t-butyl.
  • R4 can be C4 alkyl, e.g. t-butyl.
  • Z2 and R4 taken together are —O-C1-C4 alkyl, such as —O—C4 alkyl, e.g. —O-t-butyl.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen and R4 is tert-butyl
  • G1 has no oxygens
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is tert-butyl
  • G1 is cyclohexylmethyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen or CH2
  • R4 is tert-butyl or isopropyl
  • G1 is C1-C5 alkylene
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is tert-butyl
  • R2 can be hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isobutyl, isopropyl, or benzyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is tert-butyl
  • G1 is (CH2) 4-9 -
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is tert-butyl
  • G1 is (CH2) 4-9 -
  • R2 is hydrogen
  • R1 is a phenyl ring substituted with hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is tert-butyl
  • R1 is isobutyl
  • R2 is halogen, methyl, or methoxy
  • G1 is cyclohexylmethyl or C3-C7 cycloalkyl-CH2- group.
  • the invention discloses compounds of Formula 4:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • G1 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —C3-C7 cycloalkyl-, —C3-C7 cycloalkyl-CH2-, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-NH(
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —; —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) p NH(CO)—, —(CH 2 ) p NH(CO)O—, —(CH 2 ) p NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-NH(CO)NH—, or —N(CH 2 CH 2 C 6 H 5 )—;
  • Z3 is —OH, —OCH3, —O—C1-C6 alkyl, —OCH2C6H5, —NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —C1-C6 alkyl;
  • R5 is H, methyl, or optionally substituted C1-C6 alkyl
  • X is H, C1-C3 alkyl, or C1-C3 arylalkyl
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be hydrogen, methyl, ethyl, isopropyl, isobutyl, benzyl, heteroaryl such as pyridyl, phenyl and phenyl substituted with halogen, methyl, trifluoromethyl, methoxy, cyano, or dialkylamino.
  • R1 can be H or C1-C4 alkyl.
  • R1 is H
  • R2 can be hydrogen, halogen, methyl or methoxy.
  • R is H.
  • G1 can be —(CH2)-, —(CH2)2-, —(CH2)3-, —(CH2)4-, —(CH2)5-, —CH2OCH2CH2-, —CH(CH3)-, —CH2NHCOCH2-, —CH2O(CH2)2-, -cyclohexyl-CH2- or a bond.
  • G1 is -cyclohexyl-CH2-.
  • Z2 can be O, NH, —CH2- or a bond.
  • Z2 is O.
  • Z3 can be —OH, —OCH3, —O—C1-C6 alkyl, —NH2, —N(C1-C6 alkyl)2, or —C1-C6 alkyl.
  • Z3 can be C1-C4 alkyl.
  • Z3 is methyl
  • R5 can be hydrogen or methyl.
  • R5 is methyl
  • X can be hydrogen or methyl.
  • X is methyl
  • R1 is isobutyl
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G1 is a chain spanning 4-9 bond lengths and has no oxygen atoms
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G1 is cyclohexylmethyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • X is methyl or hydrogen
  • Z2 is oxygen or CH 2
  • G1 is C1-5 methylene group
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G1 is a linker of 4-9 bond lengths
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isobutyl, isopropyl, benzyl
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G1 is (CH2) 4-9 -
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G1 is (CH2) 4-9 -
  • R2 is hydrogen
  • R1 is a phenyl ring substituted with hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • R1 is isobutyl
  • R2 is halogen, methyl, or methoxy
  • G1 is cyclohexylmethyl or C3-C7 cycloalkyl-CH 2 - group.
  • the invention discloses compounds of Formula 5:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted arylalkenyl; an optionally substituted C3-C8 cycloalkyl; or optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • G2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—; —C3-C7 cycloalkyl-such as but not limited to -cyclohexyl-, or —N(CH 2 CH 2 C 6 H 5 )—;
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 — or —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) p NH(CO)—, —(CH 2 ) p NH(CO)O—, —(CH 2 ) p NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-NH(CO)NH—, or —N(CH 2 CH 2 C 6 H 5 )—;
  • Z3 is —OH, —OCH3, —O—C1-C6 alkyl, —OCH2C6H5, —NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —C1-C6 alkyl;
  • R5 is H, methyl, or optionally substituted C1-C6 alkyl
  • X is H, C1-C3 alkyl, or C1-C3 arylalkyl
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be hydrogen, C1-C4 alkyl (e.g. methyl, ethyl, isopropyl, isobutyl), benzyl, heteroaryl such as pyridyl, phenyl, phenyl substituted with halogen, trifluoromethyl, methyl, methoxy, cyano, or dialkylamino.
  • C1-C4 alkyl e.g. methyl, ethyl, isopropyl, isobutyl
  • heteroaryl such as pyridyl, phenyl, phenyl substituted with halogen, trifluoromethyl, methyl, methoxy, cyano, or dialkylamino.
  • R1 can be H or C1-C4 alkyl.
  • R1 is H.
  • R2 can be hydrogen, halogen, methyl or methoxy.
  • R2 is H.
  • G2 can be a bond, —CH2-, —(CH2)2-, —(CH2)3-, —(CH2)4-, —CH2OCH2-, —CH(CH3)-, —CH2NHCO— or -cyclohexyl-.
  • G2 is cyclohexyl
  • Z2 is O, CH2 or NH.
  • Z2 is O.
  • Z3 can be —OH, —OCH3, —O—C1-C6 alkyl, —NH2, —N(C1-C6 alkyl)2, or —C1-C6 alkyl.
  • Z3 is C1-C4 alkyl.
  • Z3 is methyl
  • R5 can be hydrogen or methyl.
  • R5 is methyl
  • X can be hydrogen or methyl.
  • X is methyl
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R5 is hydrogen
  • X is methyl
  • G2 has no oxygens
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G2 is cyclohexyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • Z2 is oxygen or CH 2
  • R5 is hydrogen or methyl
  • X is methyl
  • G2 is a bond or —(CH2) 1-4 -
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as methoxy or ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl
  • Z2 is oxygen
  • R5 is hydrogen
  • X is methyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isobutyl, isopropyl, benzyl, Z2 is oxygen, R5 is hydrogen, X is methyl, G2 is —(CH2) 2-5 , R2 can be hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • G2 is —(CH2) 2-5
  • R2 is hydrogen
  • R1 is a phenyl ring substituted with hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R5 is hydrogen
  • X is methyl
  • Z2 is oxygen
  • R1 is isobutyl
  • R2 is halogen, methyl, or methoxy
  • G2 is cyclohexyl or C3-C7 cycloalkyl-CH2- group.
  • the invention discloses compounds of Formula 6:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • R5 is H, methyl, or optionally substituted C1-C6 alkyl
  • X is H, C1-C3 alkyl, or C1-C3 arylalkyl
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —(CH 2 ) n —; —CH ⁇ CH—, —CO—, —SO—, —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—; —C3-C7 cycloalkyl-NH(CO)—, —C3-C7 cycloalkyl-CH 2 NH(CO)O—, —C3-C7 cycloalkyl-NH(CO)NH—, or —N(CH 2 CH 2 C 6 H 5 )—;
  • Z3 is —OH, —OCH3, —O—C1-C6 alkyl, —OCH2C6H5, —NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, —C1-C6 alkyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be isopropyl or isobutyl.
  • R1 is H.
  • R2 can be H, halogen or methyl.
  • R2 is H.
  • R5 can be H.
  • X can be methyl
  • Z2 can be O.
  • Z3 can be —OH, —OCH3, —O—C1-C6 alkyl, —NH2, —N(C1-C6 alkyl)2, or —C1-C6 alkyl.
  • Z3 can be C1-C4 alkyl.
  • Z3 is methyl
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • X is hydrogen
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isopropyl
  • R5 is hydrogen
  • Z2 is oxygen
  • X is hydrogen
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen or methyl
  • Z2 is oxygen
  • X is hydrogen
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen
  • Z2 is —CH2- or oxygen
  • X is hydrogen or CH3
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • the invention discloses compounds of Formula 7:
  • R1 is H, optionally substituted C1-C6 alkyl; C3-C4 cycloalkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • R8 is H, optionally substituted C1-C6 alkyl; C3-C4 cycloalkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be C2-C4 alkyl, or C3-C4 cycloalkyl.
  • R1 can be ethyl, isopropyl or isobutyl.
  • R1 can be ethyl or isopropyl.
  • R2 can be H, halogen or methyl.
  • R2 can be H, F, Cl, or Me.
  • R2 is H.
  • R8 is H.
  • R8 is Me.
  • R1 is ethyl, isopropyl, or isobutyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is ethyl, isopropyl, or isobutyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —NH 2 , —CN, —CHO, —COOH, or —CONH 2 .
  • R1 is ethyl, or isopropyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, or C1-C6 alkyl.
  • the invention discloses compounds of Formula 8:
  • X is O, NH, S, or CH2;
  • Y is O, N—R1, N—CH2-R1, CH—R1, or CH—CH2-R1;
  • R0 is H, or C1-C5 alkyl
  • R1 is H, C1-5 alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • R2 is H, or optionally substituted aryl
  • R3 is H or C1-3 alkyl
  • n 0 or 1
  • n 0 or 1
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —OCH3, —OC2H5, —O—C1-C4 alkyl, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • X may be O.
  • X may be CH2.
  • Y may be O, N—R1, or CH—R1. In some embodiments, Y may be N—R1. Alternatively, Y may be CH—R1.
  • R0 is H or methyl.
  • R1 is H, optionally substituted aryl, or optionally substituted heteroaryl; and R2 is H.
  • R1 may be H, and R2 is optionally substituted aryl.
  • aryl may refer to phenyl or substituted phenyl.
  • Substituted aryl or phenyl may refer to aryl or phenyl substituted with one or more (e.g. 1-3 or 1-2) selected from halogen, methoxyl, methyl, amino, and nitro.
  • the term “optionally substituted heteroaryl” may refer to optionally substituted pyridyl, thiazoyl, imidazolyl, or pyrimidinyl.
  • the heteroaryls may be substituted with one or more (e.g. 1-3 or 1-2) selected from halogen, methoxy, methyl, amino and nitro.
  • R1 is methyl, phenyl, 4-pyridyl, 3-pyridyl, 2-pyridyl, 4-aminophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4-pyridyl, 3-pyridyl, 2-pyridyl, 4-pyrimidinyl, 4-nitrophenyl, 2-thiazolyl, 4-(2-methyl)pyridyl, 2-imidazolyl, 4-imidazolyl, or 1-imidazolyl
  • R3 is H or methyl.
  • X is O or NH;
  • R1 is phenyl, phenyl substituted with halogen, amino, methoxy, or nitro (such as 4-aminophenyl, 4-fluorophenyl, 4-methoxyphenyl, and 4-nitrophenyl), pyridyl (such as 4-pyridyl, 3-pyridyl, 2-pyridyl), pyrimidinyl (such as 4-pyrimidinyl), 2-thiazolyl, 4-(2-methyl)pyridyl, 4-pyridylmethyl, 2-imidazolyl, 4-imidazolyl, or 1-imidazolyl;
  • R2 is H;
  • R3 is H;
  • the compounds of Formula 8 may be selected from MN1420, MN1427, MN1428, MN1429, MN1430, MN1432, MN1433, MN1434, MN1435, MN1436, MN1437, MN1438, MN1439, MN1440, MN1441, MN1442, MN1444, MN1445, MN1447, MN1448, MN1449, MN1450, MN1451, MN1452, MN1453, MN1454, MN1455, MN1456, MN1457, MN1458, MN1459, MN1460, or MN1461.
  • Y is N—R1;
  • X is NH;
  • R0 is H or CH3;
  • R1 is phenyl, 2-pyridyl, or 3-pyridyl;
  • R2 is H;
  • R3 is H;
  • m is 1; and
  • n is 1.
  • Y is CH—R1;
  • X is NH;
  • R0 is CH3;
  • R1 is 4-pyridyl or 2-pyridyl;
  • R2 is H;
  • R3 is H;
  • n is 1; and
  • m is 1.
  • Y is CH—R1;
  • X is O;
  • R0 is CH3;
  • R1 is phenyl, 4-pyridyl, H, t-Bu-CON(CH3)-CH2-, 3-pyridyl, 4-pyrimidinyl, 2-pyrimidinyl, 4-nitrophenyl, 2-thiazolyl, 3-fluorophenyl, 4-methoxyphenyl, 4-(2-methyl)pyridyl, 4-pyridylmethyl, 4-pyridyl, 2-imidazolyl, 4-imidazolyl, 1-imidazolyl, or 4-aminophenyl.
  • the invention discloses compounds of Formula 9:
  • Q is heteroaryl
  • R0 is H or C1-4 alkyl
  • X is O, NH, CH2;
  • R5 is H or CH3
  • n 1, 2, or 3.
  • Q may be a monocyclic or bicyclic heteroaryl.
  • Q may be a monocyclic or bicyclic heteroaryl containing 1-2 nitrogen atoms.
  • Q may be pyridine, isoquinoline, indole, or azaindole.
  • R0 may be H or CH3.
  • R0 may be CH3.
  • X is O.
  • R5 is H.
  • the compounds of Formula 9 may be selected from MN1462, MN1463, MN1465, MN1468, MN1467, and MN1466.
  • the invention discloses compounds of Formula 10:
  • R0 is H or C1-4 alkyl
  • X is O, NH, or CH2;
  • R5 is H or C1-4 alkyl
  • G is NH, —CH ⁇ CH—, O or S;
  • n 1 or 2.
  • heterocyclic moiety is connected at either position 2 or 3.
  • R0 is H or CH3.
  • R0 may be CH3.
  • X is O.
  • R5 is H or CH3.
  • R5 may be H.
  • G is NH or —CH ⁇ CH—.
  • the compounds of Formula 10 may be selected from MN1462, MN1463, and MN1465.
  • the invention discloses compounds of Formula 11:
  • R0 is H or C1-4 alkyl
  • X is O, CH2, or NH
  • R4 is H, CH3, OH, NH2
  • R5 is H or C1-4 alkyl
  • n 1-3.
  • R0 is H or CH3.
  • R0 is CH3.
  • X is O.
  • R5 is H or CH3.
  • R5 may be H.
  • R4 is H.
  • the compounds of Formula 11 may be selected from MN1468, MN1467, and MN1466.
  • the invention discloses compounds of Formula 12:
  • R0 is H or C1-4 alkyl
  • X is O, NH or CH2;
  • Y is N or CH.
  • R0 is H or CH3.
  • R0 is CH3.
  • X is O.
  • Y is CH or N.
  • the compounds of Formula 12 may be selected from MN1431, and MN1464.
  • the invention discloses compounds of Formula 13:
  • R0 is H or C1-4 alkyl
  • X is O, NH or CH2.
  • R0 is H or CH3.
  • R0 is CH3.
  • X is O.
  • R0 is CH3 and X is O.
  • the compound of Formula 13 is compound MN1434.
  • the invention discloses compounds of Formula 14:
  • R0 is H or C1-4 alkyl
  • X is O, NH or CH2.
  • R0 is H or CH3.
  • R0 is CH3.
  • X is O.
  • R0 is CH3; and X is O.
  • the compound of Formula 14 is compound MN1460.
  • the invention discloses compounds of Formula 15:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • R5 is H, methyl, ethyl, C1-C6 alkyl, C1-C3 arylalkyl, or 2-phenylethyl;
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —CH 2 —, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 —, —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—;
  • R4 is H, optionally substituted C1-C9 alkyl such as but not limited to tert-butyl; optionally substituted C2-C6 alkenyl; optionally substituted C6-C12 aryl such as but not limited to optionally substituted phenyl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; —O-tert-butyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be isopropyl or isobutyl.
  • R1 can be H.
  • R2 can be H, halogen or methyl.
  • R5 can be H or CH 3 .
  • R5 is CH 3 .
  • R4 is t-butyl
  • Z2 can be O.
  • Z2 can be —NH—.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isopropyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is t-butyl
  • R1 is H
  • R5 is CH 3
  • R1 may be H
  • R5 may be CH3
  • R2 may be H, halogen or methyl
  • Z2 may be —O— or —NH—
  • R4 may be C4 alkyl (such as t-butyl).
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen or methyl
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen
  • Z2 is —CH2- or oxygen
  • R4 is t-butyl. or CH3
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • the invention discloses compounds of Formula 16:
  • G3 is CH or N
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • R5 is methyl, ethyl, C1-C6 alkyl, C1-C3 arylalkyl, or 2-phenylethyl;
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —CH 2 —, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 —, —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH 2 NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—;
  • R4 is H, optionally substituted C1-C9 alkyl such as but not limited to tert-butyl; optionally substituted C2-C6 alkenyl; optionally substituted C6-C12 aryl such as but not limited to optionally substituted phenyl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; —O-tert-butyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • G3 can be H or N.
  • R1 can be a C1-4 alkyl, such as but not limited to methyl, ethyl, propyl, butyl, and cyclopropyl.
  • R1 can be isopropyl or isobutyl.
  • R1 can be methyl
  • R1 can be ethyl
  • R1 can be cyclopropyl
  • R1 can be H.
  • R2 can be H, halogen or methyl.
  • R5 can be H or CH3.
  • R5 can be CH3.
  • R5 can be ethyl
  • R4 is t-butyl
  • Z2 can be —O— or —NH—.
  • Z2 can be O.
  • Z2 can be —NH—.
  • R5 is methyl; Z2 is —O—; and R4 is t-butyl.
  • R5 is methyl; Z2 is —NH—; and R4 is t-butyl.
  • R5 is H; Z2 is —O—; and R4 is t-butyl.
  • R1 is also C1-3 alkyl; and/or R2 is H or methyl.
  • R1 is C1-4 alkyl; R2 is H, halogen or methyl; R5 is methyl; Z2 is —O—; R4 is t-butyl.
  • G3 may be CH.
  • G3 is N
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • G3 is N
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen or methyl
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • G3 is N
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen
  • Z2 is —CH2- or oxygen
  • R4 is t-butyl. or CH3
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • the invention discloses compounds of Formula 17:
  • R1 is H, optionally substituted C1-C6 alkyl; optionally substituted C2-C6 alkenyl; optionally substituted C1-C6 alkoxy; optionally substituted C6-C12 aryl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; optionally substituted C2-C15 heteroarylalkyl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkenyl; optionally substituted C3-C8 cycloalkyl; or an optionally substituted C4-C8 cycloalkylalkyl;
  • R2 is hydrogen, C1-C6 alkoxy such as but not limited to methoxy or ethoxy, trifluoromethyl, halogen, methylcarboxy, ethylcarboxy, optionally substituted C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H;
  • R5 is H, methyl, ethyl, C1-C6 alkyl, C1-C3 arylalkyl, or 2-phenylethyl;
  • Z2 is a bond, —NH—, —O—, —S—, —CH(CH 3 )—, —CH 2 —, —(CH 2 ) n —, —CH ⁇ CH—, —CO—, —SO—, —SO 2 —, —C( ⁇ NH)—, —CH 2 NH(CO)—, —CH 2 NH(CO)O—, —CH2NH(CO)NH—; —(CH 2 ) n NH(CO)—, —(CH 2 ) n NH(CO)O—, —(CH 2 ) m NH(CO)NH—;
  • R4 is H, optionally substituted C1-C9 alkyl such as but not limited to tert-butyl; optionally substituted C2-C6 alkenyl; optionally substituted C6-C12 aryl such as but not limited to optionally substituted phenyl; optionally substituted C1-C9 heteroaryl with 1 to 4 ring atoms independently selected from N, S, and O; optionally substituted C7-C15 arylalkyl such as but not limited to benzyl or alpha-methylbenzyl; —O-tert-butyl;
  • substituted means substituted with one or more independently selected from halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, methoxy, ethoxy, C1-C6 alkoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 can be isopropyl or isobutyl.
  • R1 can be H.
  • R2 can be H, halogen or methyl.
  • R2 can be H.
  • R5 can be H or CH3.
  • R5 can be CH3.
  • R4 is t-butyl
  • Z2 can be —O— or —NH—.
  • Z2 can be O.
  • Z2 can be —NH—.
  • R5 is methyl; Z2 is —O—; and R4 is t-butyl.
  • R5 is methyl; Z2 is —NH—; and R4 is t-butyl.
  • R1 is isobutyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isopropyl
  • R5 is hydrogen
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen or methyl
  • Z2 is oxygen
  • R4 is t-butyl
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • R1 is isobutyl or isopropyl
  • R5 is hydrogen
  • Z2 is —CH2- or oxygen
  • R4 is t-butyl. or CH3
  • R2 is hydrogen, halogen, trifluoromethyl, methylcarboxy, ethylcarboxy, C1-C6 alkoxy such as but not limited to methoxy and ethoxy, C1-C6 alkyl, —OH, —SH, —NH 2 , —N 3 , —CN, —NO 2 , —CHO, —COOH, —CONH 2 , —C( ⁇ NH)NH 2 , or —SO 3 H.
  • Coupling Methods F-H The basic secondary nitrogen of the tetrahydro-beta-carboline was then acylated with a carboxylic acid (in the presence of coupling agents), an acid chloride in the presence of a base, or with an isocyanate to generate ureas.
  • Samples were typically prepared in methylene chloride, at a concentration of 1 mg/mL, injecting 1 uL for each acquisition.
  • Mass spectroscopy experiments were performed by Dr. Tun-Li Shen of Brown University (Providence, R.I.). pH measurements were determined either by using either Hydracid Papers 1-6 (Micro Essential Laboratory—Brookly, N.Y.) or with a Fisher Scientific pH meter, model number AB15. Controlled additions of reagents were performed using a Hamilton 10 mL gas tight syringe attached to a KD Scientific, model 100 syringe pump.
  • HPLC High performance liquid chromatograph
  • Tryptamine (1.6 g, 10 mmol) was dissolved in 1,1,1,3,3,3-hexafluoro-2-isopropanol (16 mL) and added to isovaleraldehyde (1.3 mL; 12 mmol) by syringe.
  • the reaction was heated to reflux for 18.5 hrs and stirred under an inert atmosphere of nitrogen.
  • the solvent was evaporated and azeotroped with CHCl 3 (3 ⁇ 50 mL) under vacuum.
  • Hexane (16 mL) was added and the mixture was sonicated in a bath for 10 min and then stirred overnight. The mixture was filtered, yielding a solid (1.9 g).
  • Tryptamine (8.0 g, 50 mmol) was dissolved in CH 2 Cl 2 (400 mL) and placed under an inert atmosphere of argon for 20 min.
  • Isovaleraldehyde (5.36 mL, 50.0 mmol) was added to the solution and the reaction was placed in a ⁇ 80° C. ice bath for 20 minutes.
  • TFA 38.3 mL was added drop-wise over 15 minutes.
  • the reaction was removed from the water bath, allowed to warm to room temperature, and stirred for 20 hrs.
  • the solvent was evaporated, yielding a black oil.
  • the oil was dissolved in CH 2 Cl 2 (250 mL) and 1N NaOH was added and shaken.
  • TFA salt olive-colored powder
  • the TFA salt was recrystallized from refluxing acetonitrile
  • This intermediate was used in the synthesis of the following compounds: MN1132, MN1133, MN1137, MN1138, MN1157, MN1186, MN1189, MN1190, MN1194, MN1195, MN1197, MN1203, MN1206, MN1207, MN1208, MN1209, MN1212, MN1213, MN1214, MN1220, MN1221, MN1222, MN1223, MN1224, MN1225, MN1226, MN1231, MN1232, MN1246.
  • the solution was diluted with EtOAc (100 mL), washed with 1N HCl (3 ⁇ 25 mL), sat. NaHCO 3 (3 ⁇ 50 mL), and sat. NaCl (25 mL).
  • the organic layer was dried (anhyd. Na 2 SO 4 ), filtered, and evaporated under vacuum.
  • the resulting oil was dissolved in CH 2 Cl 2 (5 mL), and the solvent was removed under vacuum.
  • the oily residue was washed with hexanes (3 mL) top remove any hexane-soluable impurities.
  • This material was further purified by silica gel chromatography: 5 fractions (200 mL each) consisting of 0%, 5%, 10%, 15%, and 20% EtOAc in hexane. Fractions containing product were combined, the solvent was removed under vacuum resulting in an oil. The oil was dissolved in CH 2 Cl 2 ( ⁇ 1 mL) and was slowly evaporated in an ice bath, yielding a white solid.
  • This method was used in the synthesis of the following compounds: MN0477, MN0642, MN0908, MN1132, MN1133, MN1135, MN1137, MN1138, MN1152, MN1156, MN1157, MN1188, MN1193, MN1197, MN1203, MN1206, MN1207, MN1208, MN1209, MN1210, MN1211, MN1212, MN1213, MN1214, MN1216, MN1217, MN1218, MN1219.
  • MN1462 MN1463, MN1464, MN1465, MN1466, MN1467, MN1468, MN1469, MN1470, and MN1471.
  • N-(4-tert-butylbenzyl)-2-(1H-indol-3-yl)ethanamine To a solution of tryptamine (1.5 g, 9.4 mmol) was in abs. EtOH (15 mL) was added 4-t-butylbenzaldehyde (2.0 mL, 12 mmol). The reaction was stirred for 1 h before cooling to OC and then adding NaBH4 (750 mg, 19 mmol). The solution was stirred for 1 h at 0 C. The solution was concentrated in vacuo and then dried under high vacuum. The reaction was then quenched with 1N HCl ( ⁇ 20 mL), then EtOAc (100 mL) was added to form a precipitate.
  • 6-Fluoro-1-isobutyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (246 mg, 1.00 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC-HCl) (192 mg, 1.00 mmol), 4-dimethylaminopyridine (DMAP) (12 mg, 0.10 mmol), hydroxybenzotriazole (HOBT) (51 mg, 0.33 mmol), and 5-(tert-butoxycarbonylamino)pentanoic acid (217 mg, 1.00 mmol) were all dissolved in acetonitrile (1.25 mL), dimethylformamide (DMF) (5 mL), and diisopropylethylamine (DIEA) (200 ⁇ L, 1.20 mmol).
  • DMAP 1-ethyl-3-(3-dimethylaminopropyl)carbod
  • 6-Fluoro-1-isopropyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 232 mg, 1.00 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl EDC-HCl
  • 4-dimethylaminopyridine DMAP
  • HOBT hydroxybenzotriazole
  • boc-glycine (175 mg, 1.00 mmol) were all dissolved in acetonitrile (1.25 mL), dimethylformamide (DMF) (5 mL), and diisopropylethylamine (DIEA) (200 ⁇ L, 1.20 mmol).
  • 6-Fluoro-1-isopropyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 232 mg, 1.00 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl EDC-HCl
  • 4-dimethylaminopyridine DMAP
  • HOBT hydroxybenzotriazole
  • boc-valeric acid 217 mg, 1.00 mmol
  • acetonitrile (1.25 mL)
  • DMF dimethylformamide
  • DIEA diisopropylethylamine
  • 6-Fluoro-1-isopropyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 232 mg, 1.00 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl EDC-HCl
  • 4-dimethylaminopyridine DMAP
  • HOBT hydroxybenzotriazole
  • boc-tranexamic acid (257 mg, 1.00 mmol) were all dissolved in acetonitrile (1.25 mL), dimethylformamide (DMF) (5 mL), and diisopropylethylamine (DIEA) (200 ⁇ L, 1.20 mmol).
  • the reaction was stirred for 17 hours at RT.
  • the reaction mixture was diluted with EtOAc (100 mL), washed with sat. NaCl (2 ⁇ 50 mL), 1M citric acid (3 ⁇ 25 mL), sat. NaHCO 3 (3 ⁇ 25 mL), and sat. NaCl (50 mL).
  • the organic layer was dried (anhyd. Na 2 SO 4 ), filtered, and evaporated under vacuum. This material was further purified by silica gel (25-30 g) chromatography using: 3 fractions (200 mL) consisting of 20%, 25%, and 35% EtOAc in hexane.
  • 6-Fluoro-1-isobutyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 151 mg, 0.614 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl EDC-HCl
  • 4-dimethylaminopyridine DMAP
  • HOBT hydroxybenzotriazole
  • trans-4-(Boc-methylaminomethyl)cyclohexane carboxylic acid 167 mg, 0.614 mmol
  • acetonitrile 768 ⁇ L
  • dimethylformamide DMF
  • DIEA diisopropylethylamine
  • 6-Fluoro-1-isopropyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole 65 mg, 0.280 mmol
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl EDC-HCl
  • 4-dimethylaminopyridine DMAP
  • HOBT hydroxybenzotriazole
  • trans-4-(Boc-methylaminomethyl)cyclohexane carboxylic acid 76 mg, 0.280 mmol
  • acetonitrile 350 ⁇ L
  • dimethylformamide (DMF) (1.40 mL)
  • DIEA diisopropylethylamine
  • Boc-N-methy-tranexamic acid (176 mg, 0.65 mmol) and fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (TFFH) (198 mg, 0.75 mmol) were dissolved in 1,2-dichloroethane (DCE) (2.25 mL) and diisopropylethylamine (DIEA) (372 uL, 2.25 mmol). This was stirred at room temperature for 30 minutes before the addition of methyl 1-isobutyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (143 mg, 0.5 mmol).
  • DCE 1,2-dichloroethane
  • DIEA diisopropylethylamine
  • the reaction was refluxed at 80° C. for 1 hour before adding a solution of Boc-N-methy-tranexamic acid (176 mg, 0.65 mmol), fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (198 mg, 0.75 mmol), diisopropylethylamine (372 uL, 2.25 mmol), and 1,2-dichloroethane (2.25 mL). This was refluxed at 80° C. for 1.5 hours before being azeotroped with toluene (3 ⁇ 50 mL). The crude product was purified by silica gel chromatography. Product was recovered as a solid (141 mg, 52%).
  • Ethyl 2-(2,3,4,9-tetrahydro-1H-indeno[2,1-c]pyridin-1-yl)acetate were all dissolved in acetonitrile (525 ⁇ L), dimethylformamide (DMF) (2.1 mL), and diisopropylethylamine (DIEA) (83 ⁇ L, 0.50 mmol).
  • 6-Chlorotryptamine 500 mg, 2.57 mmol was dissolved in CH 2 Cl 2 (21 mL).
  • Isobutyraldehyde (234 uL, 2.57 mmol) was added via syringe to the solution and the mixture was placed in a dry ice propanol bath for 5 min.
  • TFA (1.97 mL, 25.7 mmol) was added to the reaction mixture dropwise over 6 min and then was removed from the ice bath and allowed to warm to RT.
  • Methyl 1-isobutyl-2,3,4,9-tetrahydro-1H-indeno[2,1-c]pyridine-3-carboxylate 143 mg, 0.50 mmol
  • trans-4-(Boc-methylaminomethyl)cyclohexanecarboxylic acid 352 mg, 1.30 mmol
  • Tetramethylfluoroformamidinium hexafluorophosphate THFH
  • reaction mixture was azeotroped with toluene (3 ⁇ 50 mL).
  • This material was further purified by silica gel (25-30 g) chromatography using: 5 fractions (200 mL) consisting of CH 2 Cl 2 , 6% EtOAc in CH 2 Cl 2 , 10% EtOAc in CH 2 Cl 2 , 15% EtOAc in CH 2 Cl 2 , and 20% EtOAc in CH 2 Cl 2 . Fractions containing product were combined, and the solvent was evaporated under vacuum, yielding a solid (190 mg).
  • 6-Chlorotryptamine (389 mg, 2.00 mmol) was dissolved in a solution of 10% water in MeOH (2 mL).
  • Propionaldehyde (216 uL mL, 3.00 mmol) was added via syringe followed by conc. H 2 SO 4 (1.4 mL) slowly via syringe.
  • the reaction was refluxed for 17 hrs.
  • the reaction was cooled to room temperature and then made basic with ammonium hydroxide to give a solid.
  • the solution was triturated with hexane (2 ⁇ 15 mL) and Et 2 O (2 ⁇ 20 mL). The result was filtered, and the filtrate was evaporated.
  • the resulting solid was dissolved in EtOAc (20 mL) and filtered.
  • 6-Methyltryptamine (360.7 mg, 2.07 mmol) was dissolved in CH 2 Cl 2 (16 mL). The mixture was stirred while propionaldehyde (180 uL, 2.48 mmol) was added via syringe, causing the solution to become clear. The reaction mixture was cooled in a dry ice/2-propanol bath for 5 min and then 10% TFA solution in CH 2 Cl 2 (4.76 mL) was added dropwise via syringe over 8 min. The reaction was stirred for 17 hrs and was allowed to warm slowly to RT. The mixture was concentrated and dried under vacuum, resulting in a brown solid. The result was triturated with ACN (2 ⁇ 2 mL) and EtOAc (2 mL).
  • the aqueous layer was extracted with EtOAc (2 ⁇ 50 mL) and the aqueous layers were combined.
  • the EtOAc layer was washed with sat. NaCl (20 mL), dried (anhyd. Na 2 SO 4 ), filtered, and evaporated under vacuum.
  • the reaction was stirred for 17 hours at RT.
  • the reaction mixture was diluted with EtOAc (100 mL), washed with sat. NaCl (2 ⁇ 50 mL), 1M citric acid (3 ⁇ 25 mL), sat. NaHCO 3 (3 ⁇ 25 mL), and sat. NaCl (50 mL).
  • the organic layer was dried (anhyd. Na 2 SO 4 ), filtered, and evaporated under vacuum.
  • Benzyl 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (306 mg, 1.00 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC-HCl) (192 mg, 1.00 mmol), 4-dimethylaminopyridine (DMAP) (12 mg, 0.1 mmol), hydroxybenzotriazole (HOBT) (51 mg, 0.33 mmol), and trans-4-(Boc-methylaminomethyl)cyclohexane carboxylic acid (271 mg, 1.00 mmol) were all dissolved in acetonitrile (1.25 mL), dimethylformamide (DMF) (5 mL), and diisopropylethylamine (DIEA) (200 uL, 1.20 mmol).
  • DMAP 1-ethyl-3-(3-dimethylaminopropyl)car
  • ⁇ -Methyltryptamine 174 mg, 1.00 mmol was dissolved in hexafluoro-2-propanol (HFIP) (1.6 mL).
  • Paraformaldehyde (30 mg, 1.0 mmol) was dissolved in HFIP (1.0 mL) and added to the former solution dropwise in 250 uL portions.
  • Lanthanum (III) trifluoromethanesulfonate (La(OTf) 3 ) (80 mg, 0.136 mmol) was heated using a heat gun to 200+° C. under vacuum. Argon was back-filled into the tube and L-1,2,3,4-tetrahydronorharman-3-carboxylic acid methyl ester.HCl (520 mg, 1.95 mmol) was added. The solids were dissolved in 2N NH 3 in EtOH (12 mL). The reaction mixture was capped and heated to 60° C. for 48 hr. The mixture was cooled to RT, filtered with a 0.45 um syringe, and dried under vacuum.

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