WO2010024911A1 - Procédés d’augmentation de la neurogenèse - Google Patents

Procédés d’augmentation de la neurogenèse Download PDF

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WO2010024911A1
WO2010024911A1 PCT/US2009/004885 US2009004885W WO2010024911A1 WO 2010024911 A1 WO2010024911 A1 WO 2010024911A1 US 2009004885 W US2009004885 W US 2009004885W WO 2010024911 A1 WO2010024911 A1 WO 2010024911A1
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alkyl
compound
substituted
aryl
neural
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WO2010024911A8 (fr
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Stephen J. Klaus
Javorina Milosevic
Thomas B. Neff
Johannes Schwarz
Gail Walkinshaw
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Fibrogen, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4743—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to methods and compounds useful for increasing neurogenesis. Methods and compounds for increasing dopaminergic differentiation are also provided. Methods for treating neurological disorders by increasing neurogenesis are also provided.
  • Neurogenesis is the process by which neurons are created. Until recently, neurogenesis was thought to occur only in developing organisms, but evidence now supports the occurance of adult neurogenesis.
  • Neurogenesis is an important aspect of neuronal plasticity, enabling organisms to adapt to environmental changes and influencing learning and memory throughout life.
  • the process of adult neurogenesis includes neural stem cell division (i.e., cell proliferation), migration, maturation, and differentiation.
  • SVZ subventricular zone
  • SGZ subgranular zone
  • hippocampus subgranular zone
  • Regulation of neurogenesis occurs at all levels including the proliferation, differentiation, and fate determination of adult neural progenitor cells (NPCs), and the survival, maturation, and integration of newborn neurons. Furthermore, these cells may be required for certain forms of learning and memory.
  • Neurogenesis is required for the actions of antidepressants, and adult neurogenesis may counter the pathological effects of stress and chronic depression.
  • adult hippocampal neurogenesis may relieve symptoms in psychiatric and neurologic disorders such as addiction, epilepsy, and schizophrenia.
  • Eisch et al. (2008) J Neurosci 28:11785-11791; Warner-Schmidt and Duman (2006) Hippocampus 16:239-249.
  • Various strategies directed at providing neurogenesis have been proposed.
  • one strategy would be to transplant neural progenitor cells into the brain or spinal cord, such that the neural progenitor cells develop into specialized neural cells.
  • Another strategy would be to increase or enhance endogenous neurogenesis by delivering biologically active molecules to the brain or spinal cord in order to stimulate the proliferation, migration, and differentiation of endogenous neural progenitor cells.
  • the availability of effective therapies for increasing or enhancing neurogenesis is limited, and there exists a need for identifying compounds and methods for modulating neurogenesis.
  • the present invention meets this need by providing methods and compounds effective at increasing neurogenesis and increasing neural differentiation and, in particular, dopaminergic differentiation.
  • the present invention provides methods and compounds useful for increasing neurogenesis.
  • the present invention provides a method for increasing neurogenesis, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neurogenesis.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neurogenesis, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • increasing neurogenesis is associated with increasing neural cell differentiation, increasing dopaminergic differentiation of neural cells, and increasing neural cell proliferation.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • Subjects in need of such treatment include subjects having cognitive impairement, due to injury, to disease, or to other condition, or subjects having altered dopaminergic responses.
  • Additional subjects suitable for treatment with increased neurogenesis include subjects having psychiatric disorders, such as addiction or schizophrenia, or subjects having a neurogenerative disorder, including Parkinson's disease, dystonia, essential tremor, or a chronic neurological disorder such as epilepsy.
  • the present methods can be applied to treatment of subjects experiencing depression or stress.
  • the present invention provides methods for treating a subject with altered dopaminergic response, the method comprising administering to the subject a compound that inhibits HIF prolyl hydroxylase activity.
  • the invention also provides for use of a compound that inhibits HIF prolyl hydroxlase activity in manufacturing a medicament for treatment of a subject having altered dopaminergic response.
  • the present invention provides methods for increasing neurogenesis in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HEF prolyl hydroxylase activity.
  • the present invention provides methods for increasing dopaminergic neurogenesis in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention also encompasses the use of a compound that inhibits HIF prolyl hydroxylase activity in manufacturing a medicament for increasing dopaminergic neurogenesis.
  • the present invention provides methods of treating a neurological disorder by increasing neurogenesis in a subject in need, the method comprising administering a compound that inhibits HIF prolyl hydroxylase activity to the subject, thereby increasing neurogenesis and treating the disorder.
  • the methods increase dopaminergic neurogenesis, thereby treating the disorder.
  • the invention provides methods of treating a neurological disorder by increasing neurogenesis in a subject in need, the method comprising administering a compound that inhibits HIF prolyl hydroxylase activity to the subject, wherein the neurological disorder is a neurodegenerative disorder.
  • the invention provides methods of treating a neurological disorder by increasing neurogenesis in a subject in need, the method comprising administering a compound that inhibits HIF prolyl hydroxylase activity to the subject, wherein the neurological disorder is Parkinson's disease.
  • the invention provides methods of treating a neurological disorder by increasing neurogenesis in a subject in need, the method comprising administering a compound that inhibits HIF prolyl hydroxylase activity to the subject, wherein the neurological disorder is a chronic stress disorder.
  • the invention provides methods of treating a neurological disorder by increasing neurogenesis in a subject in need, the method comprising administering a compound that inhibits HIF prolyl hydroxylase activity to the subject, wherein the neurological disorder is a cognitive impairment.
  • the present invention further provides a method for increasing neural cell differentiation, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neural cell differentiation.
  • the present invention provides methods for increasing neural cell differentiation in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neural cell differentiation, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • the neural cell in these methods is a neural progenitor cell such as, for example, a mesencephalic neural progenitor cell or a cortical (i.e., frontal) neural progenitor cell.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • the present invention provides methods for increasing dopaminergic differentiation of neural cells, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing dopaminergic differentiation of the neural cell.
  • the present invention provides methods for increasing dopaminergic differentiation of neural cells in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention provides methods for increasing dopaminergic neurons (i.e., increasing the number of dopaminergic neurons), the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing dopaminergic neurons.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neural dopaminergic differentiation, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • the present invention provides a method for increasing neural cell proliferation, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neural cell proliferation.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neural cell proliferation, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • the neural cell in these methods is a neural progenitor cell such as, for example, a mesencephalic neural progenitor cell or a cortical (i.e., frontal) neural progenitor cell.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • the compounds, methods, and medicaments of the present invention can be used to treat subjects wherein increased neurogenesis would be beneficial, in particular wherein increased hippocampal neurogenesis would be beneficial.
  • Such subjects may include patients with chronic stress disorders or depression.
  • the compounds and methods can be used to treat subjects having or at risk for having a major depressive disorder, post-traumatic stress disorder (PTSD), or Cushing's disease.
  • the compounds and methods can be used to relieve symptoms in a subject having or at risk for having a psychiatric or neurologic disorder such as addiction, epilepsy, or schizophrenia.
  • the compounds and methods may be used to treat a subject with mild cognitive impairment (MCI) or dementia, e.g., age-related dementia.
  • MCI mild cognitive impairment
  • dementia e.g., age-related dementia.
  • the present invention provides methods for increasing neurogenesis in a subject having cognitive impairment, e.g., mild cognitive impairment, the method comprising administering to the subject a compound that inhibits HIF prolyl hydroxylase activity.
  • the invention provides for use of a compound that inhibits HIF prolyl hydroxylase activity in manufacturing a medicament for treatment of mild cognitive impairment.
  • Increased neurogenesis, particularly increased dopaminergic neurogenesis may also be beneficial in treatment and functional recovery in a subject having progressive neurological disorders including various neurodegenerative disorders, such as Parkinson's disease and essential tremor.
  • the present invention provides methods for increasing dopaminergic neurogenesis in a subject having a neurodegenerative disease, the method comprising administering to the subject a compound that inhibits
  • the present invention provides methods for increasing dopaminergic neurogenesis in a subject having Parkinson's disease, the method comprising administering to the subject a compound that inhibits HEF prolyl hydroxylase activity. In one embodiment, the present invention provides methods for increasing dopaminergic neurogenesis in a subject having essential tremor, the method comprising administering to the subject a compound that inhibits HEF prolyl hydroxylase activity. In another embodiment, the invention provides for use of a compound that inhibits HEF prolyl hydroxylase activity in manufacturing a medicament for treatment of neurodegnerative disease such as Parkinson's disease, essential tremor, and various types of kinetic tremors.
  • neurodegnerative disease such as Parkinson's disease, essential tremor, and various types of kinetic tremors.
  • a compound used in the present methods is a structural mimetic of 2- oxoglutarate.
  • the compound is a structural mimetic of 2-oxoglutarate that inhibits HEF prolyl hydroxylase activity competitively with respect to 2-oxoglutarate.
  • compounds used in the present methods and medicaments provided herein are structural mimetics of 2-oxoglutarate, wherein the compound inhibits the target HEF prolyl hydroxylase enzyme competitively with respect to 2-oxoglutarate and noncompetitively with respect to iron.
  • compounds for use in the present invention include cyclic carboxamides, wherein the cyclic group is a carbocycle or a heterocycle. Therefore, in certain embodiments, the compounds used are carbocyclic carboxamides or heterocyclic carboxamides.
  • carbocyclic carboxamides for use in the present invention are naphthalene carboxamides.
  • heterocyclic carboxamides for use in the present invention are isoquinoline carboxamides and pyrrolopyridazine carboxamides.
  • Heterocyclic carboxamides for use in the present invention include hydroxy isoquinoline carboxamides, oxo pyrrolopyridazine carboxamides, hydroxy pyrrolopyridazine carboxamides, and hydroxy oxo pyrrolopyridazine carboxamides.
  • compounds for use in the present invention include variously substituted 4- hydroxy-isoquinoline-3-carbonyl glycines, 2-oxo-pyrrolopyridazine-3-carbonyl glycines, 4-hydroxy- pyrrolopyridazine-3-carbonyl glycines, and 4-hydroxy-2-oxo-pyrrolopyridazine-3-carbonyl glycines.
  • the compound used in the present invention is selected from the group consisting of [(4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound A), [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound B), ⁇ [l-Cyano-6-(2,6- dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound C), or ⁇ [4- Hydroxy-2 -oxo-1 -(4-trifluoromethyl-benzyl)-l,2-dihydro-pyrrolo[l,2-b]pyridazine-3-carbonyl]-amino ⁇ - acetic acid (Compound D).
  • a compound for use in the present invention is a compound encompassed by one of Formulae I, Ia, Ib, Ic, and Id; Formula II; Formulae HI and IHa; Formulae IVA, IVB, IVC, and IVD; Formulae V, VA, VB, VC, and VD; Formula VI; Formula VII; Formula VIII; Formula DC; Formula
  • Figures IA, IB, 1C, ID, and IE set forth data showing the effect of compound of the present invention, DFO, CPX, CoCl 2 , and DMOG, respectively, on human neural progenitor cell viability.
  • Figure 2 sets for data showing compound of the present invention increased the number of human neural progenitor cells.
  • Figure 3 sets forth data showing compound of the present invention increased S-phase of cell cycle in human neural progenitor cells.
  • Figures 4A, 4B, and 4C set forth data showing compound of the present invention increased neural cell proliferation and neurogenesis in human neural progenitor cells.
  • Figure 5 sets forth data showing compound of the present invention increased neural cell proliferation and neurogenesis in human neural progenitor cells.
  • Figure 6 sets forth data showing compound of the present invention increased dopaminergic differentiation in human neural progenitor cells.
  • Figure 7 sets forth data showing compound of the present invention increased dopaminergic differentiation in human neural progenitor cells.
  • Figure 8 set forth data showing compound of the present invention increased neuron specific enolase and tyrosine hydroxylase protein levels in human neural progenitor cells.
  • Figures 9A and 9B set forth data showing compound of the present invention increased percent neurospheres with neurites in neural progenitor cells.
  • Figures 1OA and IB set forth data showing compound of the present invention increased the number of neurites in neural progenitor cells.
  • Figure 11 sets forth data showing compound of the present invention increased VEGF mRNA expression in neural progenitor cells.
  • Figures 12A and 12B set forth data showing compound of the present invention increased the number of new mature neurons and improved contextual fear conditioning in an animal model.
  • the present invention relates to the discovery that inhibiting hypoxia-inducible factor (HIF) hydroxylase is effective at increasing neurogenesis.
  • the present invention provides methods and compounds useful for increasing neurogenesis.
  • the present invention provides a method for increasing neurogenesis, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neurogenesis.
  • the present invention provides methods for increasing neurogenesis in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neurogenesis, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • increasing neurogenesis is associated with increasing neural cell differentiation, increasing dopaminergic differentiation of neural cells, and increasing neural cell proliferation.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • a compound that inhibits HIF prolyl hydroxylase activity is a cyclic carboxamide.
  • the compound for use in the present invention is selected from a carbocyclic carboxamide or a heterocyclic carboxamide.
  • a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme is a heterocyclic carbonyl glycine of Formula VI.
  • the present invention provides a method for increasing dopaminergic neurogenesis in subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention also provides for use of a compound that inhibits HIF prolyl hydroxylase activity in manufacturing a medicament for increasing dopaminergic neurogenesis.
  • Increased neurogenesis may also be beneficial in treatment and functional recovery in a subject having progressive neurological disorders including various neurodegenerative disorders, such as Parkinson's disease and essential tremor.
  • the present invention provides methods for increasing dopaminergic neurogenesis in a subject having a neurodegenerative disease, the method comprising administering to the subject a compound that inhibits
  • the present invention provides methods for increasing dopaminergic neurogenesis in a subject having Parkinson's disease, the method comprising administering to the subject a compound that inhibits HEF prolyl hydroxylase activity. In one embodiment, the present invention provides methods for increasing dopaminergic neurogenesis in a subject having essential tremor, the method comprising administering to the subject a compound that inhibits HEF prolyl hydroxylase activity. In another embodiment, the invention provides for use of a compound that inhibits HEF prolyl hydroxylase activity in manufacturing a medicament for treatment of neurodegnerative disease such as Parkinson's disease, essential tremor, and various types of kinetic tremors.
  • neurodegnerative disease such as Parkinson's disease, essential tremor, and various types of kinetic tremors.
  • the present methods and compounds are also useful for treating various conditions wherein increased neurogenesis would be beneficial, such as chronic stress disorders, e.g., post-traumatic stress disorder (PTSD); depression, Cushing's disease, addiction, epilepsy, and schizophrenia.
  • the present methods and compounds are also useful for treating mild cognitive impairment (MCI) or dementia, e.g., age-related dementia.
  • MCI mild cognitive impairment
  • the present invention provides methods for increasing neurogenesis in a subject having cognitive impairment, e.g., mild cognitive impairment, the method comprising administering to the subject a compound that inhibits HIF prolyl hydroxylase activity.
  • the invention provides for use of a compound that inhibits HIF prolyl hydroxylase activity in manufacturing a medicament for treatment of mild cognitive impairment.
  • Proliferating neural cells can be identified by their expression of neuron-specific class III ⁇ -tubulin (TUJl) and doublecortin (DCX).
  • TUJl is expressed in neurons of the peripheral and central nervous systems and contributes to microtubule stability in neuronal cell bodies and axons.
  • DCX is a microtubule-associated protein expressed almost exclusively in immature (e.g., developing) neurons, and thus is a marker for neurogenesis.
  • Neural precursors begin to express DCX shortly after exiting the cell cycle, and continue to express DCX as the cells mature into neurons. (Couillard-Despres et al. (2005) Eur J Neurosci 21:1-14.)
  • the present invention provides a method for increasing expression of TUJl in a neural cell, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing expression of TUJl in the neural cell.
  • the present invention provides a method for increasing expression of DCX in a neural cell, the method comprising administering to or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing expression of DCX in the neural cell.
  • Neural cell markers associated with neurogenesis include increased expression of neuron-specific enolase (NSE) or increased expression of microtubule-associated protein 2 (MAP2).
  • NSE neuron-specific enolase
  • MAP2 microtubule-associated protein 2
  • the present invention provides a method for increasing expression of MAP2 in a neural cell, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing expression of MAP2 in the neural cell.
  • the present invention also provides a method for increasing expression of neuron specific enolase (NSE) in a neural cell, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing expression of neuron specific enolase in the neural cell.
  • NSE neuron specific enolase
  • the present invention further provides a method for increasing neural cell differentiation, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neural cell differentiation.
  • the present invention provides methods for increasing neural cell differentiation in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neural cell differentiation, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • the neural cell in these methods is a neural progenitor cell such as, for example, a mesencephalic neural progenitor cell or a cortical (i.e., frontal) neural progenitor cell.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • the present invention provides a method for increasing dopaminergic differentiation of neural cells, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing dopaminergic differentiation of the neural cell.
  • the present invention provides methods for increasing dopaminergic differentiation of neural cells in a subject in need, the method comprising administering to the subject an effective amount of a compound that inhibits HIF prolyl hydroxylase activity.
  • the present invention provides a method for increasing dopaminergic neurons (i.e., increasing the number of dopaminergic neurons), the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing dopaminergic neurons.
  • Dopaminergic neurons are neurons whose primary neurotransmitter is dopamine. Dopaminergic neurons are present chiefly in the ventral tegmental area of the midbrain, substantia nigra pars compacta, and arcuate nucleus of the hypothalamus. Dopaminergic neurons are associated with numerous functions in the brain, including important roles in behavior and cognition, motor activity, motivation and reward, inhibition of prolactin production (involved in lactation), sleep, mood, attention, and learning.
  • Dopaminergic differentiation and identification of dopaminergic neurons can be determined by any measure known to those skilled in the art.
  • dopaminergic differentiation and dopaminergic neurons are identified by the presence of characteristic and identifying dopaminergic markers, such as, for example, tyrosine hydroxylase (TH) and microtubule-associated protein 2 (MAP2).
  • TH tyrosine hydroxylase
  • MAP2 microtubule-associated protein 2
  • Tyrosine hydroxylase is the enzyme responsible for catalyzing the conversion of the L-tyrosine to dihydrophenylalanine, a precursor for dopamine.
  • MAP2 is a microtubule associated protein found exclusively in nerve cells.
  • the present invention also provides a method for increasing expression of tyrosine hydroxylase in a neural cell, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing expression of tyrosine hydroxylase in the neural cell.
  • the present invention provides a method for increasing neural cell proliferation, the method comprising administering to a subject or contacting a neural cell with an effective amount of a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme, thereby increasing neural cell proliferation.
  • the present invention also provides compounds for use in manufacturing a medicament for increasing neural cell proliferation, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme.
  • the neural cell in these methods is a neural progenitor cell such as, for example, a mesencephalic neural progenitor cell or a cortical (i.e., frontal) neural progenitor cell.
  • the neural cell in these methods is a neural stem cell, a neural progenitor cell, or a neural precursor cell.
  • Neural cell proliferation can be identified by any measure known to one skilled in the art.
  • proliferating neural cells are identified by the presence of the neural cell marker neuron-specific class IQ ⁇ -tubulin (TUJl) and the cell proliferating marker Ki67.
  • TUJl is expressed in neurons of the peripheral and central nervous systems and contributes to microtubule stability in neuronal cell bodies and axons.
  • Ki67 is a cellular marker for proliferation, is present in all active phases of the cell cycle (Gl, S, G2, and mitosis), but is absent in resting cells (GO). Ki67 is used as a marker to determine the growth fraction of a given cell population. (See, e.g., Gerdes et al. (1983) Int J Cancer 31 : 13-20 and Scholzen and Gerdes (2000) J Cell Physiol 182:311-322.)
  • Methods and compounds of the present invention were also effective at increasing neurites in neural progenitor cells and effective at increasing VEGF expression in neural progenitor cells. Subjects
  • the present invention relates to methods for increasing neurogenesis.
  • the invention is applicable to a variety of different organisms, including, for example, vertebrates, large animals, and primates.
  • the subject is a mammalian subject, and in a most preferred embodiment, the subject is a human subject.
  • medical applications with humans are clearly foreseen, veterinary applications are also envisaged herein.
  • the present methods of treatment involve administration of an effective amount of a compound to a subject in need, wherein the compound inhibits the activity of a HIF prolyl hydroxylase enzyme, and wherein the subject would benefit from increased neurogenesis, neural cell differentiation, dopaminergic differentiation, or neural cell proliferation.
  • the subject has a neurodegenerative disease wherein increased neurogenesis would be beneficial.
  • the subject has Parkinson's disease, essential tremor (ET), or any type of kinetic tremor.
  • the subject is a subject with an altered dopaminergic response.
  • the subject has a chronic neurological disorder such as epilepsy.
  • the subject has a phycological or neurological impairment, such as chronic stress disorders, e.g., post-traumatic stress disorder (PTSD); depression, Cushing's disease, addiction, epilepsy, or schizophrenia.
  • PTSD post-traumatic stress disorder
  • depression depression
  • Cushing's disease addiction
  • epilepsy or schizophrenia
  • MCI mild cognitive impairment
  • dementia e.g., age-related dementia.
  • HIF ⁇ hypoxia-inducible factor alpha
  • HIF hypoxia-inducible factor prolyl hydroxylase enzymes
  • a compound that inhibits the activity of HIF prolyl hydroxylase enzyme refers to any compound that reduces or otherwise moldulates the activity of at least one HIF prolyl hydroxylase enzyme.
  • HIF prolyl hydroxylase refers to any enzyme that is capable of hydroxylating a proline residue within an alpha subunit of HIF.
  • HIF prolyl hydroxylases include protein members of the EGL-9 (EGLN) 2-oxoglutarate- and iron-dependent dioxygenase family described by Taylor (2001) Gene 275: 125-132; and characterized by Aravind and Koonin (2001) Genome Biol 2:RESEARCH0007; Epstein et al. (2001) Cell 107:43-54; and Bruick and McKnight (2001) Science 294: 1337-1340.
  • prolyl hydroxylase inhibitors for use in the methods of the present invention are defined by their ability to inhibit an activity of a 2-oxoglutarate dioxygenase enzyme, wherein the enzyme has specific activity toward hypoxia inducible factor.
  • Such compounds are defined herein as prolyl hydroxylase inhibitors (PHIs).
  • PHIs prolyl hydroxylase inhibitors
  • the PHIs for use in the invention are small molecule compounds.
  • a compound that inhibits the activity of a HIF prolyl hydroxylase enzyme refers to any compound that reduces or otherwise modulates the activity of at least one HIF prolyl hydroxylase enzyme.
  • a compound may additionally show inhibitory activity toward one or more other 2- oxoglutarate- and iron-dependent dioxygenase enzymes, e.g. factor inhibiting HIF (FIH; GenBank Accession No. AAL27308), procollagen prolyl 4-hydroxylase (CP4H), etc.
  • a "compound that inhibits HIF prolyl hydroxylase” suitable for use in the claimed methods can be any compound that inhibits HIF prolyl hydroxylase activity.
  • the compound that inhibits HIF prolyl hydroxylase activity can be a structural mimetic of 2-oxoglutarate.
  • the compound is a structural mimetic of 2-oxoglutarate that inhibits HIF prolyl hydroxylase activity competitively with respect to 2-oxoglutarate.
  • compounds used in the present methods and medicaments provided herein are structural mimetics of 2-oxoglutarate, wherein the compound inhibits the target HIF prolyl hydroxylase enzyme competitively with respect to 2- oxoglutarate and noncompetitively with respect to iron.
  • a compound suitable for use in the present invention is a cyclic carboxamide.
  • the compound is a carbocyclic carboxamide.
  • the carbocyclic group can be a single ring group, e.g., a benzene, or can contain multiple condensed rings, e.g., a napthalene.
  • a compound suitable for use in the present invention is a heterocyclic carboxamide.
  • the heterocycle can be a single ring, for example, a pyridine, a pyrimidine, or a pyridazine.
  • the specified heterocyclic structure is a multiple condensed ring, for example, a quinoline, a cinnoline, an isoquinoline, a pyrrolopyridine, a napthyridine, a ⁇ -carboline, a chromene (coumarin), or a thiochromene (thiocoumarin).
  • Carboxamide compounds particularly suitable for use in the present invention include carboxamides substituted at the amide to form a carbonyl glycine. Therefore, in certain embodiments, a compound for use in the present invention is a cyclic carbonyl glycine, and in particular, a carbocyclic carbonyl glycine or a heterocyclic carbonyl glycine. Specifically encompassed by the term "carbonyl glycine” are structural and functional analogs thereof, including, in particular, carbonyl glycineamides (wherein the carboxyl moiety on the glycine is replaced with carboxamide).
  • prodrugs thereof such as carbonyl glycine esters (wherein the carboxyl moiety is esterified with a substituent such as an alkyl, e.g., methyl).
  • carbonyl glycine esters wherein the carboxyl moiety is esterified with a substituent such as an alkyl, e.g., methyl.
  • specific substitution at the ⁇ carbon of the glycine of a suitable heterocyclic carbonyl glycine compound results in replacement of the glycine with a comparable amino acid selected from the group consisting of alanine, valine, leucine, and isoleucine.
  • a cyclic carboxamide for use in the present invention is a heterocyclic carboxamide and, more specifically, an isoquinoline carboxamide.
  • the carboxamide can be positioned on the isoquinoline at any stereochemically appropriate point on the heterocycle.
  • Isoquinoline carboxamides particularly suited for use in the present invention include isoquinoline-3-carboxamides.
  • isoquinoline carboxamides examples include [(4-hydroxy-7-phenylsulfanyl-isoquinoline-3- carbonyl)-amino] -acetic acid (Compound A); [(4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]- acetic acid (Compound B); ⁇ [l-cyano-6-(2,6-dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]- amino ⁇ -acetic acid (Compound C), and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., United States Patent No. 6,093,730 and International Publication Nos. WO 2004/108681 and WO 2007/090068.
  • an isoquinoline carboxamide according to the present invention is additionally substituted with a hydroxyl group; specifically a hydroxy isoquinoline carboxamide.
  • the hydroxyl can be positioned at any stereochemically appropriate point on the heterocycle.
  • isoquinoline carboxamides suitable for use in the present invention include 4-hydroxy-isoquinoline-3 -carboxamides. Examples of such compounds include Compound A, Compound B, Compound C, and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., United States Patent No. 6,093,730 and International Publication Nos. WO 2004/108681 and WO 2007/090068.
  • a compound of the invention is an isoquinoline carboxamide
  • the amide on the carboxamide moiety of the isoquinoline carboxamide is substituted to form a glycine
  • the compound for use in the present invention is an isoquinoline carbonyl glycine.
  • isoquinoline-3-carboxamides isoquinoline-3 -carbonyl glycines are specifically contemplated herein, as are more substituted examples thereof, including 4-hydroxy- isoquinoline-3-carbonyl glycines, etc.
  • Such compounds include Compound A, Compound B, Compound C, and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., United States Patent No. 6,093,730 and in International Publication Nos. WO 2004/108681 and WO 2007/090068.
  • a cyclic carboxamide according to the present invention is a heterocyclic carboxamide and, more specifically, a pyrrolopyridazine carboxamide.
  • the carboxamide can be positioned at any stereochemically appropriate point on the heterocycle.
  • Pyrrolopyridazine carboxamides suitable for use in the present invention include pyrrolopyridazine-3-carboxamides.
  • Examples of such pyrrolopyridazine carboxamides include ⁇ [4-hydroxy-2-oxo-l-(4-trifluoromethyl- benzyO-l ⁇ -dihydro-pyrrolofl ⁇ -blpyridazine-S-carbonylJ-aminoJ-acetic acid (Compound D) and other compounds readily identified by those skilled in the art, including those described and claimed in, e.g., International Application No. PCT/US09/54473.
  • a pyrrolopyridazine carboxamide according to the present invention is additionally substituted with an oxo group; specifically, an oxo-pyrrolopyridazine carboxamide (pyrrolopyridazinone carboxamide).
  • the oxo can be positioned at any stereochemically appropriate point on the heterocycle.
  • Pyrrolopyridazine carboxamides particularly suited for use in the present invention include 2-oxo- pyrrolopyridazine-3 -carboxamides. Examples of such compounds include Compound D and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., International Application No. PCT/US09/54473.
  • the pyrrolopyridazine carboxamide according to the present invention is additionally substituted with a hydroxyl group; specifically, a hydroxy pyrrolopyridazine carboxamide.
  • hydroxyl can be positioned at any stereochemically appropriate point on the heterocycle.
  • Pyrrolopyridazine carboxamides suitable for use in the present invention include 4-hydroxy- pyrrolopyridazine-3-carboxamides. Examples of such compounds include Compound D and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., International Application No. PCT/US09/54473.
  • a pyrrolopyridazine carboxamide according to the present invention is additionally substituted with a hydroxyl group and an oxo group; specifically, specifically, a hydroxy oxo-pyrrolopyridazine carboxamide (hydroxy pyrrolopyridazinone carboxamide).
  • the oxo and hydroxyl can be independently positioned at any stereochemically appropriate point on the heterocycle.
  • Pyrrolopyridazine carboxamides suitable for use in the present invention include 4-hydroxy-2-oxo- pyrrolopyridazine-3 -carboxamide.
  • Examples of such compounds include Compound D and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., International Application No. PCT/US09/54473.
  • a compound of the invention is a pyrrolopyridazine carboxamide
  • the amide on the carboxamide moiety of the pyrrolopyridazine carboxamide is substituted to form a glycine
  • the compound for use in the present invention is a pyrrolopyridazine carbonyl glycine.
  • pyrrolopyridazine -3-carboxamides are specifically contemplated herein, as are more substituted examples thereof, including 4-hydroxy-pyrrolopyridazine-3-carbonyl glycines, 2-oxo-pyrrolopyridazine-3-carbonyl glycines, and 4-hydroxy-2-oxo-pyrrolopyridazine-3 -carboxamides.
  • Examples of such include Compound D and other compounds easily identified by those skilled in the art, including those described and claimed in, e.g., International Application No. PCT/US09/54473.
  • a compound of the invention is a heterocyclic carbonyl glycine of formula VI.
  • R is an optionally substituted heterocyclic moiety.
  • such compounds include, but are not limited to, substituted 3-hydroxy-pyridine- 2-carbonyl-glycines, 4-hydroxy-pyridazine-3-carbonyl-glycines, 3-hydroxy-quinoline-2-carbonyl- glycines, 4-hydroxy-2-oxo-l,2-dihydro-quinoline-3 -carbonyl -glycines, 4-hydroxy-2-oxo-l,2-dihydro- naphthyridine-3-carbonyl-glycines, 8-hydroxy-6-oxo-4,6-dihydro-pyridopyrazine-7-carbonyl-glycines, 4- hydroxy-isoquinoline-3 -carbonyl -glycines, 4-hydroxy-cinnoline-3 -carbonyl-glycines, 7-hydroxy- thienopyridine-6-carbonyl-glycines, 4-hydroxy-thienopyridine-5-carbonyl-glycines, 7-hydroxy-hydroxy-hydroxy-
  • Compounds can be identified for use in the present embodiments by measuring inhibitory activity of the compound on a HIF prolyl hydroxylase enzyme, e.g., using an enzyme assay as described herein. More generally, compounds can be identified for use in the present embodiments by measuring HIF stabilization induced by the compound, e.g., using a cell-based assay as described herein.
  • the heterocyclic carbonyl glycine is a quinoline carboxamide, an isoquinoline carboxamide, a pyridine carboxamide, a cinnoline carboxamide, or a beta-carboline carboxamide.
  • Heterocyclic carbonyl glycines effectively stabilize HIF ⁇ .
  • a compound of the invention is a compound that inhibits prolyl hydroxylase activity (e.g., a prolyl hydroxylase inhibitor).
  • a compound of the invention is a compound that inhibits HIF prolyl hydroxylase activity.
  • PKIs Prolyl hydroxylase inhibitors specifically contemplated for use in the present methods are described, e.g., in Majamaa et al., supra; Kivirikko and Myllyharju (1998) Matrix Biol 16:357-368; Bickel et al. (1998) Hepatology 28:404-411; Friedman et al. (2000) Proc Natl Acad Sci USA 97:4736- 4741; Franklin (1991) Biochem Soc Trans 19):812 815; Franklin et al. (2001) Biochem J 353:333-338. Examples of compounds that may be used in the methods and medicaments provided herein can be found, e.g., in Majamaa et al. (1984) Eur.
  • a compound for use in the present methods is a heterocyclic carbonyl glycine, in particular, a heterocyclic carbonyl glycine of Formula VI.
  • the compound used in the present methods is a compound selected from the group consisting of the compounds of Formula I, Formula ⁇ , Formula HI, Formula IV, Formula V, and Formula VI.
  • Formula I includes, but is not limited to, compounds of
  • Formulae Ia, Ib, Ic, and Id include, but is not limited to, the compounds of Formula HIa.
  • Formula IV includes, but is not limited to, compounds of Formulae IVA, IVB, IVC, and IVD.
  • Formula V includes, but is not limited to, compounds of Formulae VA, VB, VC, and VD.
  • compounds suitable for use in the present invention include isoquinoline carboxamides.
  • isoquinoline carboxamides according to the present invention are isoquinoline- 3-carboxamides.
  • a compound for use in the methods of the present invention is [(4- Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound A), [(4-Hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound B), ⁇ [l-Cyano-6-(2,6- dimethyl-phenoxy)-4-hydroxy-isoquinoline-3-carbonyl]-amino ⁇ -acetic acid (Compound C), or ⁇ [4-
  • compounds used in the methods of the invention are heterocyclic carboxamides selected from a compound of the formula (I)
  • A is 1,2-arylidene, 1,3-arylidene, 1,4-arylidene; or (d-C 4 )-alkylene, optionally substituted by one or two halogen, cyano, nitro, trifluoromethyl, (d-C 6 )-alkyl, (Ci-C 6 )-hydroxyalkyl, (C 1 - C 6 )-alkoxy, -O-[CH 2 ] ⁇ -C f H (2f+ i.
  • B is -CO 2 H, -NH 2 , -NHSO 2 CF 3 , tetrazolyl, imidazolyl, 3-hydroxyisoxazolyl, -CONHCOR'",
  • R' is aryl, heteroaryl, (C 3 -C 7 )-cycloalkyl, or (d-C 4 )-alkyl, optionally monosubstituted by (C 6 -C 12 )-aryl, heteroaryl, OH, SH, (C 1 -C 4 )- alkyl, (C,-C 4 )-alkoxy, (C,-C 4 )-thioalkyl, (C 1 -C 4 )-sulfinyl, (C r C 4 )-sulfonyl, CF 3 , Cl, Br, F, I, N02, -COOH, (C 2 -C 5 )-alkoxycarbonyl, NH 2 , mono-(C 1 -C 4 -alkyl)-amino, di-(C r C 4 - alkyl)-amino, or (C r C 4
  • G is a radical of an alcohol G-OH in which G is selected from (d-C 20 )-alkyl radical, (C 3 - C 8 ) cycloalkyl radical, (C 2 -C 20 )-alkenyl radical, (C 3 -C 8 )-cycloalkenyl radical, retinyl radical, (C 2 -C 20 )-alkynyl radical, (C 4 -C 20 )-alkenynyl radical, where the alkenyl, cycloalkenyl, alkynyl, and alkenynyl radicals contain one or more multiple bonds; (C 6 - C 16 )-carbocyclic aryl radical, (C 7 -C 16 )-carbocyclic aralkyl radical, heteroaryl radical, or heteroaralkyl radical, wherein a heteroaryl radical or heteroaryl moiety of a heteroaralkyl radical contains 5 or 6 ring atoms; and wherein radicals defined
  • C 16 )-aralkylcarbamoyl N-((C,-Cio)-alkoxy-(Ci-Cio)-alkyl)-carbamoyl, N-((C 6 -Ci 2 )- aryloxy-(C]-Cio)alkyl)-carbamoyl, N-((C 7 -Ci 6 )-aralkyloxy-(Ci-Cio)-alkyl)-carbamoyl, N- (C 1 -C 10 )-alkyl-N-((C 1 -Cio)-alkoxy-(C,-Cio)-alkyl)-carbamoyl, N-(C,-Cio)-alkyl-N-((C 6 - C I6 )-aryloxy-(Ci-Cio)-alkyl)-carbamoyl, N-(Ci-Cio)-alkyl
  • E is a heteroaryl radical, a (C 3 -C 8 )-cycloalkyl radical, or a phenyl radical of the formula F
  • v is 0-6, w is 0 or 1, t is 0-3, and R 7 , R 8 , R 9 , R 10 , and R 1 ' are identical or different and are hydrogen, halogen, cyano, nitro, trifluoromethyl, (C,-C 6 )-alkyl, (C 3 -C 8 )-cycloalkyl, (C r C 6 )-alkoxy, -O-[CH 2 ] X - C f H (2f+1 .
  • R 1 , R 2 and R 3 are identical or different and are hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl, (d-C 20 )-alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -C 8 )cycloalkyl-
  • R x and R v are each independently selected from hydrogen, (C,-C 6 )-alkyl, (C 3 -C 7 )- cycloalkyl, aryl, or the substituent of an ⁇ -carbon of an oamino acid, to which the L- and D-amino acids belong, s is 1-5, T is OH, or NR*R**, and R*, R** and R*** are identical or different and are selected from hydrogen, (C 6 -C 12 )-aryl, (C 7 -C,,)-aralkyl, (Ci-C 8 )-alkyl, (C 3 -C 8 )-cycloalkyl, (+)-dehydroabietyl, (C 1 -C 8 )-alkoxy-(C 1 -C 8 )-alkyl, (C 7 -Ci 2 )-aralkoxy-(Ci-C 8 )- alkyl, (C 6 -C 12 )-
  • V is S, O, or NR k
  • R k is selected from hydrogen, (Ci-C 6 )-alkyl, aryl, or benzyl; where an aryl radical may be optionally substituted by 1 to 5 substituents as defined above; and R 24 , R 25 , R 26 , and R 27 in each case independently of each other have the meaning of R 1 , R 2 and R 3 ; f is l to 8; g is 0 or 1 to (2f+l); x is 0 to 3; and h is 3 to 7; including the physiologically active salts and prodrugs derived therefrom.
  • Additional compounds according to Formula (I) are substituted heterocyclic carboxyamides described in U.S. Patent No. 5,620,995; 3-hydroxypyridine-2-carboxamidoesters described in U.S. Patent No. 6,020,350; sulfonamidocarbonylpyridine-2-carboxamides described in U.S. Patent No. 5,607,954; and sulfonamidocarbonyl-pyridine-2-carboxamides and sulfonamidocarbonyl-pyridine-2-carboxamide esters described in U.S. Patent Nos. 5,610,172 and 5,620,996. All compounds listed in these patents, in particular, those compounds listed in the compound claims and the final products of the working examples, are hereby incorporated into the present application by reference herein.
  • compounds according the present invention are in some embodiments heterocyclic carboxamides; in particular, quinoline carboxamides.
  • compounds for use in the invention are quinoline-2 -carboxamides.
  • the compound is selected from a compound of the Formula Ia wherein
  • A is -CR 5 R 6 -, and R 5 and R 6 are each independently selected from the group consisting of hydrogen, (Ci-C 6 )-alkyl, (C 3 -C 7 )-cycloalkyl, aryl, or a substituent of the ⁇ -carbon atom of an ⁇ -amino acid, wherein the amino acid is a natural L-amino acid or its D-isomer;
  • G is a radical of an alcohol G-OH in which G is selected from the group consisting of (Ci-C 20 )-alkyl radical, (C 3 -C 8 ) cycloalkyl radical, (C 2 -C 20 )- alkenyl radical, (C 3 -C 8 )-cycloalkenyl radical, retinyl radical, (C 2 -C 20 )-alkynyl radical, (C 4 -C 20 )- alkenynyl radical;
  • R 4 is selected from the group consisting of hydrogen, (Ci-Ci O )-alkyl, (C 2 -C ]0 )-alkenyl, (C 2 -C 10 )-alkynyl, wherein alkenyl or alkynyl contains one or two C-C multiple bonds; unsubstituted fluoroalkyl radical of the formula -[CH 2 ] x -C f H (2f+ i. g) -F g , aryl, heteroaryl, and (C 7 -Ci i)-aralkyl;
  • R 1 , R 12 , R 13 , R 14 and R 15 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (Ci-C 20 )-alkyl, (C 3 -C 8 )- cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -C, 2 )-aryl, (C 7 -Ci 6 )-aralkyl, (C 7 -Ci 6 )-aralkenyl, (C 7 -Ci 6 )- aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C,-C 20 )-alkoxy, (C 2 -C 20 )-alkenyloxy, (C 2 -C 20 )- alkynyloxy, retinyloxy, (
  • gj F g , -OCF 2 Cl, and -OCF 2 -CHFCl x is 0 to 3; f is 1 to 8; and g is 0 or 1 to (2f+l); including the physiologically active salts, esters, and prodrugs derived therefrom.
  • the quinoline-2-carboxamide is selected from a compound of the Formula Ia wherein
  • A is -CHR 5 and R 5 is hydrogen or methyl;
  • B is -CO 2 H;
  • X is O;
  • R 4 is hydrogen; and R 1 , R 12 , R 13 , R 14 and R 15 are identical or different and are selected from the group consisting hydrogen, chloro, aryl, aryloxy, and substituted aryloxy, including the physiologically active salts, esters, and prodrugs derived therefrom.
  • compounds according to the present invention include isoquinoloine caarboxamides.
  • compounds for use in the invention are isoquinoline-3- carboxamides.
  • the isoquinoline-3-carboxamide is selected from a compound of the Formula Ib wherein
  • A is -CR 5 R 6 -, and R 5 and R 6 are each independently selected from the group consisting of hydrogen, (Ci-C 6 )-alkyl, (C 3 -C 7 )-cycloalkyl, aryl, or a substituent of the ot-carbon atom of an ⁇ -amino acid, wherein the amino acid is a natural L-amino acid or its D-isomer;
  • G is a radical of an alcohol G-OH in which G is selected from the group consisting of (Ci-C 20 )-alkyl radical, (C 3 -C 8 ) cycloalkyl radical, (C 2 -C 20 )- alkenyl radical, (C 3 -C 8 )-cycloalkenyl radical, retinyl radical, (C 2 -C 2 o)-alkynyl radical, (C 4 -C 20 )- alkenynyl radical;
  • X is O;
  • Q is O;
  • R 4 is selected from the group consisting of hydrogen, (Ci-Cio)-alkyl, (C 2 -Ci 0 )-alkenyl, (C 2 -Ci 0 )-alkynyl, wherein alkenyl or alkynyl contains one or two C-C multiple bonds; unsubstituted fluoroalkyl radical
  • R 3 , R 16 , R 17 , R 18 and R 19 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (C r C 2 o)-alkyl, (C 3 -C 8 )- cycloalkyl, (C 3 -C 8 )-cycloalkoxy, (C 6 -Ci 2 )-aryl, (C 7 -C 16 )-aralkyl, (C 7 -C 16 )-aralkenyl, (C 7 -C 16 )- aralkynyl, (C 2 -C 20 )-alkenyl, (C 2 -C 20 )-alkynyl, (C r C 20 )-alkoxy, (C 2 -C 20 )
  • the isoquinoline-3-carboxamide is selected from a compound of the Formula Ib wherein
  • A is -CHR 5 where R 5 is selected hydrogen or methyl
  • X is O
  • R 4 is hydrogen, (C 1 -C 3 )-alkyl, or substituted (C r C 3 )-alkyl;
  • R 3 is hydrogen, chloro, or cyano
  • R 16 , R 17 , R 18 and R 19 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, aryl, heteroaryl, substituted heteroaryl, -OR 70 , -SR 70 , -SOR 70 , and -SO 2 R 70 wherein R 70 is selected from the group consisting of alkyl, substituted alkyl, cyclcoalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; including the physiologically active salts, esters, and prodrugs derived therefrom.
  • isoquinoline-3-carboxamides for use in the present invention include those disclosed in International Publication No. WO 2004/108681 and as represented by Formula IV, IVA, IVB, IVC, IVD, VA, VB, VC and VD below.
  • heterocyclic carboxamides for use in the invention may be thienopyridine carboxamides.
  • the thienopyridine carboxamide is selected from a thienopyridine-5 -carboxamide or a thienopyridine-6-carboxamide.
  • thienopyridine carboxamide compounds for use in the present invention are as disclosed in International Publication No. WO 2006/094292, represented by Formula II
  • R 30 is selected from the group consisting of hydrogen, (Ci-C 6 )-alkyl, (C 3 -C 7 )-cycloalkyl, aryl, or a substituent of the ⁇ -carbon atom of an ⁇ -amino acid, wherein the amino acid is a natural L-amino acid or its D-isomer;
  • G is a radical of an alcohol G-OH in which G is selected from the group consisting of (Ci-C 2 o)-alkyl radical, (C 3 -C 8 ) cycloalkyl radical, (C 2 -C 20 )- alkenyl radical, (C 3 -C 8 )-cycloalkenyl radical, retinyl radical, (C 2 -C 20 )-alkynyl radical, (C 4 -C 20 )- alkenynyl radical;
  • R 31 is selected from the group consisting of hydrogen, (Ci-Cio)-alkyl, (C 2 -Ci 0 )-alkenyl, (C 2 -Ci 0 )- alkynyl, wherein alkenyl or alkynyl contains one or two C-C multiple bonds; unsubstituted fluoroalkyl radical of the formula -[CH 2
  • R 32 , R 33 , and R 34 are identical or different and are selected from the group consisting of hydrogen, hydroxyl, halogen, cyano, trifluoromethyl, nitro, carboxyl; (C r C 20 )-alkyl, (C 3 -C 8 )-cycloalkyl, (C 3 -
  • the compound is a compound of Formula II wherein
  • R 30 and R 31 are hydrogen
  • R 32 is selected from hydrogen, halo, aryl, substituted aryl, aryloxy, and substituted aryloxy;
  • R 34 is hydrogen, aryl, or substituted aryl; including the physiologically active salts, esters, and prodrugs derived therefrom.
  • cyclic carboxamides are particularly suited for use in the present invention.
  • use of other compounds that inhibit HIF prolyl hydroxylase activity is specifically contemplated.
  • Such compounds are have been identified and are well-known in the art.
  • compounds according to the invention can include phenanthrolines and iron chelators, etc.
  • the compound for use in the present invention is an iron chelator, e.g., a hydroxamate.
  • hydroxamates for use in the methods of the invention are selected from a compound of the formula (EI) or pharmaceutically acceptable salts thereof, wherein: a is an integer from 1 to 4; b is an integer from 0 to 4; c is an integer from 0 to 4;
  • Z is selected from the group consisting Of (C 3 -Ci 0 ) cycloalkyl, (C 3 -Ci 0 ) cycloalkyl independently substituted with one or more Y 1 , 3-10 membered heterocycloalkyl and 3-10 membered heterocycloalkyl independently substituted with one or more Y 1 ; (C 5 -C 20 ) aryl, (C 5 -C 20 ) aryl independently substituted with one or more Y 1 , 5-20 membered heteroaryl and 5-20 membered heteroaryl independently substituted with one or more Y 1 ;
  • Ar 1 is selected from the group consisting of (C 5 -C 20 ) aryl, (C 5 -C 20 ) aryl independently substituted with one or more Y 2 , 5-20 membered heteroaryl and 5-20 membered heteroaryl independently substituted with one or more Y 2 ; each Y 1 is independently selected from the group consisting of a lipophilic functional group, (C 5 - C 20 ) aryl, (C 6 -C 26 ) alkaryl, 5-20 membered heteroaryl and 6-26 membered alk-heteroaryl; each Y 2 is independently selected from the group consisting of -R', -OR', -OR", -SR', -SR", - NR'R', -NO 2 , -CN, -halogen, -trihalomethyl, trihalomethoxy, -C(O)R', -C(O)OR', - C(O)NR 1 R', -C(O)NR
  • R 35 and R 36 are each independently selected from the group consisting of hydrogen, (Ci-Cg) alkyl, (C 2 -C 8 ) alkenyl, (C 2 -C 8 ) alkynyl, (C 3 -Ci 0 ) cycloalkyl, (C 5 -C 20 ) aryl, (C 5 -C 20 ) substituted aryl, (C 6 -C 26 ) alkaryl, (C 6 -C 26 ) substituted alkaryl, 5-20 membered heteroaryl, 5-20 membered substituted heteroaryl, 6-26 membered alk-heteroaryl, and 6-26 membered substituted alk-heteroaryl; and
  • R 37 is independently selected from the group consisting of hydrogen, (Ci-C 8 ) alkyl, (C 2 -C 8 ) alkenyl, and (C 2 -C 8 ) alkynyl.
  • compounds according to the present invention include isoquinoloine caarboxamides.
  • compounds for use in the invention are isoquinoline-3- carboxamides.
  • the compounds used in the present invention are as disclosed in International Publication No. WO 2004/108681, represented by formula (IV):
  • R a is -COOH or -WR 8 ; provided that when R a is -COOH then p is zero and when R a is -WR 8 then p is one;
  • W is selected from the group consisting of oxygen, -S(O) n - and -NR 9 - where n is zero, one or two, R 9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic and R 8 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, or when W is -NR 9 - then R 8 and R 9 , together with the nitrogen atom to which they are bound, can be joined to form a heterocyclic or a substituted heterocyclic group, provided that when W is -S(O) n - and n is one or two, then R 8 is not hydrogen;
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl or aryl or, when X is -NR 7 -, then R 7 and R 8 , together with the nitrogen atom to which they are bound, can be joined to form a heterocyclic or substituted heterocyclic group;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxy, cyano, -S(O) n -N(R 6 )-R 6 where n is 0, 1 , or 2, -NR 6 C(O)NR 6 R 6 , -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, each R 6 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic provided that when X is -SO- or -SO 2 -, then R 6 is not hydrogen, and R 7 is selected from the group consisting of hydrogen, alkyl, aryl, or
  • R 4 and R 5 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl or aryl or, when X is -NR 7 -, then R 7 and R 8 , together with the nitrogen atom to which they are bound, can be joined to form a heterocyclic or substituted heterocyclic group;
  • R is selected from the group consisting of hydrogen, deuterium and methyl
  • R'" is selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, acyloxy, cycloalkoxy, substituted cycloalkoxy, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, aryl, -S(O) n -R 10 wherein R 10 is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl and n is zero, one or two; and pharmaceutically acceptable salts, esters and prodrugs thereof.
  • the compounds of formula (IV) are represented by formula (FVA):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R, R', R", R'" and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R", R'", WR 8 and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof.
  • the invention is directed to compounds represented by the formula (IVC):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R, R', R", R'", WR 8 and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof.
  • the invention is directed to compounds represented by the formula (IVD):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R, R', R", R'" and q are as defined above; and pharmaceutically acceptable salts, esters, prodrugs thereof.
  • the invention is directed to isoquinoline carboxamide compounds represented by the formulae (VA), (VB), (VC), (V), wherein said formulae are defined below.
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl or aryl;
  • R is selected from the group consisting of hydrogen and methyl
  • R' is selected from the group consisting of alkyl and substituted alkyl; or R and R' may be joined to form a cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic; R" is selected from the group consisting of hydrogen and alkyl or R" together with R' and the nitrogen pendent thereto forms a heterocyclic or substituted heterocyclic group; or pharmaceutically acceptable salts and/or prodrugs thereof.
  • Formula VB
  • W is selected from the group consisting of oxygen, -S(O) n - and -NR 9 - where n is zero, one or two,
  • R 9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic
  • R 8 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic;
  • R" is selected from hydrogen and alkyl
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl or aryl; R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxy, cyano, -XR 6 where X is oxygen, -S(O)
  • R 4 and R 5 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is * hydrogen, alkyl or aryl; or pharmaceutically acceptable salts and/or prodrugs thereof.
  • Formula VC Formula VC:
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl, or aryl;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxy, cyano, -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl, or aryl;
  • R 4 and R 5 are independently selected from the group consisting of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl, or aryl;
  • R is selected from the group consisting of hydrogen and methyl
  • R' is selected from the group consisting of alkyl and substituted alkyl; or R and R' can be joined to form cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic,
  • R" is selected from the group consisting of hydrogen and alkyl or R" together with R' and the nitrogen pendent thereto forms a heterocyclic or substituted heterocyclic group;
  • W is selected from the group consisting of oxygen, -S(O) n - and -NR 9 - where n is zero, one or two, R 9 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 8 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic; or pharmaceutically acceptable salts and/or prodrugs thereof.
  • R" is selected from hydrogen and alkyl
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, halo, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and -XR 6 where X is oxygen, -S(O) n - or -NR 7 - where n is zero, one or two, R 6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and R 7 is hydrogen, alkyl or aryl;
  • R 1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, halo, alkoxy, aryloxy, substituted aryloxy, substituted aryl, alkylthio, aminoacyl, aryl, substituted amino, heteroaryl, heteroaryloxy, -S(O) n -aryl, -S(O) n -substituted aryl, -S(O) n -heteroaryl, and -S(O) n -substituted heteroaryl, where n is zero, one or two.
  • R 1 is selected from the group consisting of: (3-methoxyphenyl)sulfanyl; (4- chlorophenyl)sulfanyl; (4-methylphenyl)sulfanyl; 2-fluorophenoxy; 2-methoxyphenoxy; (2- methoxyphenyl)sulfanyl 3-fluorophenoxy; 3-methoxyphenoxy; 4-(methylcarbonylamino)phenoxy; A- (methylsulfonamido)phenoxy; 4-fluorophenoxy; 4-methoxyphenoxy; 4-methoxyphenylsulfanyl; A- methylphenyl; bromo; cMoro; dimethylaminomethyl; ethoxy; ethylsulfanyl; hydrogen; isopropyl; methoxy; methoxymethyl; methyl; N,N-dimethylaminocarbonyl; naphth-2-yloxy; naphthylsulfanyl; phenoxy; pheny
  • R 2 is preferably selected from the group consisting of substituted amino, aryloxy, substituted aryloxy, alkoxy, substituted alkoxy, halo, hydrogen, alkyl, substituted alkyl, aryl, -S(O) n -aryl, -S(O) n -substituted aryl, -S(O) n -cycloalkyl, where n is zero, one or two, aminocarbonylamino, heteroaryloxy, and cycloalkyloxy.
  • R 2 is selected from the group consisting of: (4-methoxy)phenylsulfonylamino; 2,6-dimethylphenoxy; 3,4-difluorophenoxy; 3,5- difluorophenoxy; 3-chloro-4-fluorophenoxy; 3-methoxy-4-fluorophenoxy; 3-methoxy-5-fluorophenoxy;
  • R 3 is preferably selected from the group consisting of: substituted aryloxy, substituted alkoxy, alkoxy, substituted alkyl, alkyl, amino, cycloalkyloxy, hydrogen, halo, aryl, -S(O) n -aryl, -S(O) n -substituted aryl, -S(O) n -heteroaryl, and -S(O) n -substituted heteroaryl, where n is zero, one or two, aminocarbonylamino, and heteroaryloxy.
  • R 3 is selected from the group consisting of: amino; (4- methyl)phenylsulfonylaminophenoxy; 3,4-difluorophenoxy; 3,5-difluorophenoxy; 3-fluoro-5-methoxy- phenoxy; 3-chloro-4-fluorophenoxy; 4-CF 3 -O-phenoxy; 4-CF 3 -phenoxy; 4-chlorophenoxy; A- fluorophenoxy; 4-(4-fluorophenoxy)phenoxy; 4-methoxyphenoxy; benzyloxy; bromo; butoxy; CF 3 ; chloro; cyclohexyloxy; hydrogen; iodo; isopropoxy; phenoxy; phenyl; phenylsulfanyl; phenylsulfonyl; phenylsulfinyl; phenylurea; pyridin-1-ylsulfanyl; pyridin-3-yloxy; and pyri
  • R 2 and R 3 are joined to form an aryl group.
  • the aryl group is phenyl.
  • R 4 is preferably selected from the group consisting of: substituted arylthio, halo, hydrogen, substituted alkyl and aryl.
  • R 4 is selected from the group consisting of: 4-chlorophenyl sulfanyl; chloro; hydrogen; methoxymethyl; and phenyl.
  • R 5 is preferably hydrogen or aryl. More preferably R 5 is hydrogen or phenyl.
  • R is preferably selected from the group consisting of hydrogen, deuterium, aryl and alkyl. More preferably R is selected from the group consisting of phenyl, hydrogen, deuterium and methyl.
  • R' is selected from the group consisting of preferably hydrogen, deuterium, alkyl, substituted alkyl, and substituted amino. More preferably, R' is selected from the group consisting of: 4-aminobutyl; 4-hydroxybenzyl; benzyl; carboxylmethyl; deuterium; hydroxymethyl; imidazol-4-ylmethyl; isopropyl; methyl; and propyl.
  • R, R' and the carbon atom pendent thereto join to form a cycloalkyl and more preferably cyclopropyl.
  • R" is preferably hydrogen, alkyl or substituted alkyl. More preferably, R" is hydrogen, methyl or carboxylmethyl (-CH 2 C(O)OH). Alternatively, R', R" and the carbon atom and nitrogen atom respectively pendent thereto join to form a heterocyclic group and more preferably pyrrolidinyl.
  • R'" is selected from the group consisting of hydrogen, hydroxy, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, thiol, acyloxy and aryl.
  • R'" is selected from the group consisting of: hydroxy; benzyloxy; ethoxy; thiol; methoxy; methylcarbonyloxy; and phenyl.
  • WR 8 is preferably selected from the group consisting of amino, substituted amino, aminoacyl, hydroxy, and alkoxy. More preferably, WR 8 is selected from the group consisting of: amino; dimethylamino; hydroxy; methoxy; and methylcarbonylamino.
  • compounds according the present invention are in some embodiments heterocyclic carboxamides; in particular, pyrrolopyridazine carboxamides.
  • compounds for use in the invention are pyrrolopyridazine-3 -carboxamides.
  • the compound is a compound of formula VII:
  • R 52 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl
  • R 53 is selected from the group consisting of hydrogen, deuterium, alkyl, and substituted alkyl;
  • R 54 is selected from the group consisting of hydrogen, deuterium, alkyl, and substituted alkyl;
  • R 56 , R 57 and R 58 independently are selected from the group consisting of hydrogen, hydroxy, cyano, halo, nitro, acyl, amino, substituted amino, acylamino, sulfonyl, substituted sulfonyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, heterocyclyloxy, substituted heterocyclyloxy, carboxyl, carboxyl ester, carboxyl amide, oxycarbonylamino, aminocarbonyloxy, aminocarbonylamino, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroary
  • R 59 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
  • R 60 is -NR 61 R 62 or -OR 63 ;
  • R 61 and R 62 independently are selected from the group consisting hydrogen, alkyl, alkylene- cycloalkyl, C 3 -C 8 heterocyclic, aryl, and -C(O)(Ci-C 4 alkyl); or R 61 and R 62 taken together with the nitrogen to which they are attached form a 5- or 6- membered heterocyclic or substituted heterocyclic; and R 63 is selected from the group consisting of hydrogen and alkyl which is unsubstituted or substituted with one or more substituents independently selected from the group consisting of cycloalkyl, heterocyclic, aryl, and heteroaryl; or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, ester, tautomer or prodrug thereof.
  • the invention relates to compounds of Formula VII wherein R 52 and R 53 are hydrogen;
  • R 54 is selected from the group consisting of hydrogen and methyl
  • R 56 , R 57 , and R 58 independently are selected from the group consisting of hydrogen, halo, and aryl;
  • R 59 is selected from the group consisting of hydrogen, alkyl, -CH 2 -aryl, -CH 2 -substituted aryl, or -
  • R 60 is -OR 63 ; wherein R 63 is hydrogen or alkyl.
  • the invention relates to compounds of Formula VII wherein
  • R 52 , R 53 , and R 54 are hydrogen
  • R 56 and R 58 independently are selected from the group consisting of hydrogen and halo;
  • R 57 is selected from the group consisting of hydrogen, halo, and aryl
  • R 59 is selected from the group consisting of hydrogen, alkyl, -CH 2 -aryl, -CH 2 -substituted aryl, or ⁇ CH 2 -heteroaryl;
  • R 60 is -OR 63 ; wherein R 63 is hydrogen or alkyl.
  • hydroxy or "hydroxyl” refer to the group -OH.
  • halo or halogen refers to fluoro, chloro, bromo, and iodo.
  • cyano refers to the group -CN.
  • nitro refers to the group -NO 2 .
  • carboxyl refers to -COOH or salts thereof.
  • alkyl refers to saturated monovalent hydrocarbyl groups having from 1 to 10 carbon atoms; more particularly, from 1 to 5 carbon atoms; and, even more particularly, 1 to 3 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, /-butyl, n-pentyl, and the like.
  • cycloalkyl refers to a saturated or an unsaturated, but nonaromatic, cyclic alkyl groups of from 3 to 10, 3 to 8, or 3 to 6 carbon atoms having single or multiple cyclic rings including, by way of example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, cyclohexenyl, and the like.
  • cycloalkoxy refers to an -O-cycloalkyl group.
  • aryl refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-l,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the point of attachment is the aryl group.
  • Preferred aryls include phenyl and naphthyl.
  • heterocyclic or “heterocyclyl” refer to a saturated or unsaturated ring system having a single ring or multiple condensed rings, from 1 to 10 carbon atoms, and from 1 to 4 hetero atoms selected from the group consisting of nitrogen, sulfur, or oxygen within the ring.
  • heteroaryl refers to an aromatic heterocyclic group of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, and 1 to 4 heteroatoms within the ring selected from the group consisting of oxygen, nitrogen, and sulfur.
  • Such heteroaryl groups can have a single ring (e.g., pyridinyl, furyl, or thienyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), which condensed rings may or may not be aromatic provided the point of attachment is through a ring containing the heteroatom and that ring is aromatic.
  • the nitrogen can optionally be oxidized to provide for the N-oxide
  • the sulfur ring atoms can optionally be oxidized to provide for the sulfoxide and sulfone derivatives.
  • heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, furan, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, phthal
  • groups are exemplified by vinyl (ethen-1-yl), allyl, but-3-enyl, and the like.
  • alkynyl refers to acetylinic unsaturated monovalent hydrocarbyl groups having from 2 to 6, preferably from 2 to 3, carbon atoms and having at least 1, preferably from 1 to 2, sites of acetylenic (-C ⁇ €-) unsaturation. This group is exemplified by ethyn-1-yl, propyn-1-yl, propyn-2-yl, and the like.
  • alkoxy refers to the group “alkyl-O-,” which includes, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, f-butoxy, sec-butoxy, n-pentoxy, and the like.
  • alkenyloxy refers to the group “alkenyl-O-.”
  • alkynyloxy refers to the group “alkynyl-O-.”
  • aryloxy refers to the group aryl-O- that includes, by way of example, phenoxy, naphthoxy, and the like.
  • aralkyloxy refers to the group aralkyl-O- that includes, by way of example, benzyloxy, and the like.
  • aminoacyl or “amide”, or the prefixes “carbamoyl” or “carboxamide,” refer to the group -C(0)NR q R q where each R q is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, and heterocyclic; or where each R q is joined to form together with the nitrogen atom a heterocyclic wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • amino refers to the group -NH 2 .
  • alkylsulfanyl refers to the groups -S-alkyl where alkyl is as defined above.
  • sulfinyl refers to the group -S(O)-.
  • sulfonyl refers to the group -S(O) 2 -.
  • heterocyclyloxy refers to the group -O-heterocyclic.
  • cycloalkylene refers to divalent cycloalkyl groups as defined above.
  • cycloalkylthio or “cycloalkylsulfanyl” refer to the groups -S-cycloalkyl where cycloalkyl is as defined herein.
  • arylthio or “arylsulfanyl” refer to the group -S-aryl, where aryl is as defined herein.
  • heteroarylthio or “heteroarylsulfanyl” refer to the group -S-heteroaryl, where heteroaryl is as defined herein.
  • heterocyclicthio or “heterocyclicsulfanyl” refer to the group -S-heterocyclic, where heterocyclic is as defined herein.
  • alkyl alcohol refers to the group “alkyl-OH”. "Alkyl alcohol” is meant to include methanol, ethanol, 2-propanol, 2-butanol, butanol, etc.
  • acyl refers to the groups H-C(O)-, alkyl-C(O)-, alkenyl-C(O)-, alkynyl-C(O)-, cycloalkyl- C(O)-, aryl-C(O)-, heteroaryl-C(O)-, and heterocyclic-C(O)-, provided that a nitrogen atom of the heterocyclic is not bound to the -C(O)- group, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • acyloxy refers to the groups alkyl-C(O)O-, alkenyl-C(O)O-, alkynyl-C(O)O-, aryl-C(O)O-, cycloalkyl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O-, wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • alkenyl refers to a vinyl unsaturated monovalent hydrocarbyl group having from 2 to 6 carbon atoms, and preferably 2 to 4 carbon atoms, and having at least 1, and preferably from 1 to 2 sites of vinyl
  • alkynyl refers to acetylinic unsaturated monovalent hydrocarbyl groups having from 2 to 6, preferably from 2 to 3, carbon atoms and having at least 1, preferably from 1 to 2, sites of acetylenic (-C ⁇ C-) unsaturation. This group is exemplified by ethyn-1-yl, propyn-1-yl, propyn-2-yl, and the like.
  • acylamino refers to the groups -NR'C(O)alkyl, -NR 1 C(O)CyClOaIlCyI, -NR'C(O)alkenyl,
  • R 1 is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are defined herein.
  • carbonyloxyamino refers to the groups -NR u C(O)O-alkyl, -NR u C(O)O-alkenyl, -NR u C(0)0- alkynyl, -NR u C(O)O-cycloalkyl, -NR u C(O)O-aryl, -NR u C(O)O-heteroaryl, and -NR u C(O)O-heterocyclic, where R u is hydrogen or alkyl and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • oxycarbonylamino refers to the groups -NR u C(O)O-alkyl, -NR u C(O)O-alkenyl, -NR 11 C(O)O- alkynyl, -NR u C(O)O-cycloalkyl, -NR u C(O)O-aryl, -NR u C(O)O-heteroaryl, and -NR u C(O)O-heterocyclic, where R" is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • oxythiocarbonylamino refers to the groups -NR u C(S)O-alkyl,-NR u C(S)O-alkenyl, -NR u C(S)O-alkynyl, -NR u C(S)O-cycloalkyl, -NR u C(S)O-aryl, -NR u C(S)O-heteroaryl, and -NR U C(S)O- heterocyclic, where R u is hydrogen or alkyl, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • aminocarbonyloxy or the prefix “carbamoyloxy” refer to the groups -OC(O)NR V R V where each R v is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclic; or where each R v is joined to form, together with the nitrogen atom, a heterocyclic, and wherein alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, substituted heteroaryl, and heterocyclic are as defined herein.
  • aminocarbonylamino refers to the group -NR W C(O)N(R W ) 2 where each R w is independently selected from the group consisting of hydrogen and alkyl.
  • aminothiocarbonylamino refers to the group -NR W C(S)N(R W ) 2 where each R w is independently selected from the group consisting of hydrogen and alkyl.
  • aryloxyaryl refers to the group -aryl-O-aryl.
  • carboxyl ester refers to the groups -C(O)O-alkyl, -C(O)O-alkenyl, -C(O)O-alkynyl, -C(O)O- cycloalkyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-heteroaryl, -C(O)O-substituted heeteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic.
  • cycloalkylene refers to divalent cycloalkyl groups as defined above.
  • heteroaryloxy refers to the group -O-heteroaryl.
  • sulfonyl refers to the group -S(O) 2 -, and may be included in the groups -S(O) 2 H, -SO 2 -alkyl,
  • alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.
  • heterocyclyloxy refers to the group -O-heterocyclic.
  • arylthio or "arylsulfanyl” refer to the group -S-aryl.
  • heteroarylthio or “heteroarylsulfanyl” refer to the group -S-heteroaryl.
  • heterocyclicthio or “heterocyclicsulfanyl” refer to the group -S -heterocyclic. Conjugated terms refer to a linear arrangement of the separate substituents as each separate term is defined herein.
  • aralkyl refers to an aryl-alkyl group and includes, by way of example, benzyl; the term “aralkylcarbamoyl” refers to an aryl-alkyl-carbomoyl substituent wherein each term is as defined herein, etc.
  • impermissible substitution patterns e.g., methyl substituted with 5 fluoro groups or a hydroxyl group alpha to ethenylic or acetylenic unsaturation.
  • impermissible substitution patterns are well known to the skilled artisan.
  • pharmaceutically acceptable salt refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art, and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and, when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like.
  • stereoisomer or “stereoisomers” refer to compounds that differ in the chirality of one or more stereocenters.
  • Stereoisomers include enantiomers (compounds are non-superimposable mirror images) and diastereomers (compounds having more than one stereogenic center that are non-mirror images of each other and wherein one or more stereogenic center differs between the two stereoisomers).
  • the compounds of the invention can be present as a mixture of stereoisomers or as a single stereoisomer.
  • prodrug refers to compounds that include chemical groups which, in vivo, can be converted into the carboxylate group and/or can be split off from the amide N-atom and/or can be split off from the R atom to provide for the active drug, a pharmaceutically acceptable salt thereof, or a biologically active metabolite thereof.
  • Suitable groups are well known in the art and particularly include: for the carboxylic acid moiety, a prodrug selected from, e.g., esters including, but not limited to, those derived from alkyl alcohols, substituted alkyl alcohols, hydroxy substituted aryls and heteroaryls and the like; amides, particularly amides derived from amines of the Formula HNR 200 R 210 where R 200 and R 210 are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and the like; hydroxymethyl, aldehyde and derivatives thereof.
  • esters refers to compounds that include the group -COOR where R is alkyl, substituted alkyl, alkoxy, or substituted alkoxy.
  • a compound of the invention is one that stabilizes HIF ⁇ .
  • the ability of a compound to stabilize or activate HIFa can be measured, for example, by direct measurement of HIF ⁇ in a sample, indirect measurement of HIF ⁇ , e.g., by measuring a decrease in HDFa associated with the von Hippel Lindau protein (see, e.g., International
  • HIF responsive target genes or reporter constructs See, e.g., U.S. Patent No. 5,942,434. Measuring and comparing levels of HIF and/or HIF-responsive target proteins in the absence and presence of the compound will identify compounds that stabilize HIF ⁇ and/or activate HIF. Suitable compounds for use in the present methods may be identified and characterized using the assay described in International Publication No. WO 2005/118836, or in Example 10 of
  • a compound of the invention is one that inhibits HIF hydroxylase activity.
  • Assays for hydroxylase activity are standard in the art. Such assays can directly or indirectly measure hydroxylase activity.
  • an assay can measure hydroxylated residues, e.g., proline, asparagine, etc., present in the enzyme substrate, e.g., a target protein, a synthetic peptide mimetic, or a fragment thereof. (See, e.g., Palmerini et al. (1985) J Chromatogr 339:285-292.)
  • a reduction in hydroxylated residue, e.g., proline or asparagine, in the presence of a compound is indicative of a compound that inhibits hydroxylase activity.
  • assays can measure other products of the hydroxylation reaction, e.g., formation of succinate from 2-oxoglutarate.
  • assays can measure other products of the hydroxylation reaction, e.g., formation of succinate from 2-oxoglutarate.
  • Target protein may include HIF ⁇ or a fragment thereof, e.g., HIF(556-575).
  • Enzyme may include, e.g., HIF prolyl hydroxylase (see, e.g., GenBank Accession No. AAG33965, etc.) or HIF asparaginyl hydroxylase (see, e.g., GenBank Accession No. AAL27308, etc.), obtained from any source. Enzyme may also be present in a crude cell lysate or in a partially purified form. For example, procedures that measure HIF hydroxylase activity are described in Ivan et al.
  • compositions of the present invention can be delivered directly or in pharmaceutical compositions containing excipients, as is well known in the art.
  • the present methods of treatment involve administration of an effective amount of a compound of the present invention to a subject in need, wherein the subject would benefit from increased neurogenesis, neural cell differentiation, dopaminergic differentiation, or neural cell proliferation.
  • an effective amount, e.g., dose, of compound or drug can readily be determined by routine experimentation, as can an effective and convenient route of administration and an appropriate formulation.
  • Various formulations and drug delivery systems are available in the art. (See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Limbird, and Gilman, eds. (2001)
  • Suitable routes of administration may, for example, include oral, rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteral administration.
  • Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration.
  • Secondary routes of administration include intraperitoneal, intra-arterial, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration.
  • the indication to be treated, along with the physical, chemical, and biological properties of the drug, dictate the type of formulation and the route of administration to be used, as well as whether local or systemic delivery would be preferred.
  • the compounds of the present invention are administered orally.
  • the present invention provides for oral administration of [(4-Hydroxy-7- phenylsulfanyl-isoquinoline-3-carbonyl)-amino]-acetic acid (Compound A), [(4-Hydroxy-7-phenoxy- isoquinoline-3-carbonyl)-amino] -acetic acid (Compound B), ⁇ [l-Cyano-6-(2,6-dimethyl-phenoxy)-4- hydroxy-isoquinoline-3-carbonyl] -amino ⁇ -acetic acid (Compound C), or ⁇ [4-Hydroxy-2-oxo-l-(4- trifluoromethyl-benzyl)-l,2-dihydro-pyrrolo[l,2-b]pyridazine-3-carbonyl]-amino ⁇ -acetic acid.
  • Pharmaceutical dosage forms of a compound of the invention may be provided in an instant release, controlled release, sustained release, or target drug-delivery system.
  • Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, and lyophilized formulations.
  • special devices may be required for application or administration of the drug, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks.
  • Pharmaceutical dosage forms are often composed of the drug, an excipient(s), and a container/closure system.
  • One or multiple excipients also referred to as inactive ingredients, can be added to a compound of the invention to improve or facilitate manufacturing, stability, administration, and safety of the drug, and can provide a means to achieve a desired drug release profile. Therefore, the type of excipient(s) to be added to the drug can depend on various factors, such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing procedure.
  • Pharmaceutically acceptable excipients are available in the art, and include those listed in various pharmacopoeias. (See, e.g., USP, JP, EP, and BP,
  • compositions of the present invention can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.
  • the composition may be formulated in aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH, and a tonicity agent, such as, for example, sodium chloride or dextrose.
  • physiologically compatible buffers including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH
  • a tonicity agent such as, for example, sodium chloride or dextrose.
  • semisolid, liquid formulations, or patches may be preferred, possibly containing penetration enhancers.
  • penetration enhancers are generally known in the art.
  • the compounds can be formulated in liquid or solid dosage forms and as instant or controlled/sustained release formulations.
  • Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, and emulsions.
  • the compounds may also be formulated in rectal compositions, such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • Solid oral dosage forms can be obtained using excipients, which may include, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, antiadherants, cationic exchange resins, wetting agents, antioxidants, preservatives, coloring, and flavoring agents.
  • excipients may include, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, glidants, antiadherants, cationic exchange resins, wetting agents, antioxidants, preservatives, coloring, and flavoring agents.
  • excipients can be of synthetic or natural source.
  • excipients examples include cellulose derivatives, citric acid, dicalcium phosphate, gelatine, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrolidone, silicates, silicium dioxide, sodium benzoate, sorbitol, starches, stearic acid or a salt thereof, sugars (i.e. dextrose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes. Ethanol and water may serve as granulation aides.
  • coating of tablets with, for example, a taste-masking film, a stomach acid resistant film, or a release-retarding film is desirable.
  • Natural and synthetic polymers, in combination with colorants, sugars, and organic solvents or water, are often used to coat tablets, resulting in dragees.
  • the drug powder, suspension, or solution thereof can be delivered in a compatible hard or soft shell capsule.
  • the compounds of the present invention can be administered topically, such as through a skin patch, a semi-solid or a liquid formulation, for example a gel, a (micro)-emulsion, an ointment, a solution, a (nano/micro)-suspension, or a foam.
  • the penetration of the drug into the skin and underlying tissues can be regulated, for example, using penetration enhancers; the appropriate choice and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsifiers; by pH adjustment; and use of complexing agents.
  • Other techniques, such as iontophoresis may be used to regulate skin penetration of a compound of the invention. Transdermal or topical administration would be preferred, for example, in situations in which local delivery with minimal systemic exposure is desired.
  • the compounds for use according to the present invention are conveniently delivered in the form of a solution, suspension, emulsion, or semisolid aerosol from pressurized packs, or a nebuliser, usually with the use of a propellant, e.g., halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas.
  • a propellant e.g., halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas.
  • hydrocarbons like butane, isobutene, and pentane are useful.
  • the appropriate dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator may be formulated. These typically contain a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions formulated for parenteral administration by injection are usually sterile and, can be presented in unit dosage forms, e.g., in ampoules, syringes, injection pens, or in multi-dose containers, the latter usually containing a preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as buffers, tonicity agents, viscosity enhancing agents, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives.
  • the vehicle may contain water, a synthetic or vegetable oil, and/or organic co-solvents.
  • the parenteral formulation would be reconstituted or diluted prior to administration.
  • Depot formulations providing controlled or sustained release of a compound of the invention, may include injectable suspensions of nano/micro particles or nano/micro or non- micronized crystals.
  • Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof, can serve as controlled/sustained release matrices, in addition to others well known in the art.
  • Other depot delivery systems may be presented in form of implants and pumps requiring incision.
  • Suitable carriers for intravenous injection for the molecules of the invention are well-known in the art and include water-based solutions containing a base, such as, for example, sodium hydroxide, to form an ionized compound, sucrose or sodium chloride as a tonicity agent, for example, the buffer contains phosphate or histidine.
  • a base such as, for example, sodium hydroxide
  • sucrose or sodium chloride as a tonicity agent
  • the buffer contains phosphate or histidine.
  • Co-solvents such as, for example, polyethylene glycols, may be added.
  • These water-based systems are effective at dissolving compounds of the invention and produce low toxicity upon systemic administration.
  • the proportions of the components of a solution system may be varied considerably, without destroying solubility and toxicity characteristics.
  • the identity of the components may be varied.
  • low-toxicity surfactants such as polysorbates or poloxamers
  • polyethylene glycol or other co-solvents polyethylene glycol or other co-solvents
  • biocompatible polymers such as polyvinyl pyrrolidone may be added, and other sugars and polyols may substitute for dextrose.
  • composition useful for the present methods of treatment a therapeutically effective dose can be estimated initially using a variety of techniques well-known in the art. Initial doses used in animal studies may be based on effective concentrations established in cell culture assays. Dosage ranges appropriate for human subjects can be determined, for example, using data obtained from animal studies and cell culture assays.
  • a therapeutically effective dose or amount of a compound, agent, or drug of the present invention refers to an amount or dose of the compound, agent, or drug that results in amelioration of symptoms or a prolongation of survival in a subject.
  • Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ ED50. Agents that exhibit high therapeutic indices are preferred.
  • the effective amount or therapeutically effective amount is the amount of the compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician, e.g., treatment of cancer, including induction of anti-tumor effects, etc.
  • Dosages preferably fall within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending upon the dosage form employed and/or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be chosen according to methods known in the art, in view of the specifics of a subject's condition.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects, i.e., minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from, for example, in vitro data and animal experiments. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
  • effective doses for compounds of the invention include doses of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg,
  • effective treatment regimes for compounds of the invention include administration two or three times weekly.
  • the amount of agent or composition administered may be dependent on a variety of factors, including the sex, age, and weight of the subject being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient.
  • a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • hNPC mesencephalic and cortical human neural progenitor cell
  • MTT cell viability assay Human mesencephalic neural progenitor cell (hmNPC) and human cortical neural progenitor cell (frontal, hfNPCs) cultures were derived from tissue obtained from a 10-14-week-old human fetus. Two-midbrain-derived hNPC (hmNPC M 1 and hmNPC M2) and one frontal-derived hNPC (hfNPC F 1 ) were used in this MTT cell viability assay. The hNPCs were propagated in an undifferentiated state using methods previously described.
  • hNPCs were expanded in defined media (expansion media) (DMEM/F12) supplemented with epidermal growth factor
  • EGF fibroblast growth factor-2
  • FGF-2 fibroblast growth factor-2
  • hNPCs obtained and cultured as described above were seeded into 96-well culture plates at a density of 20,000 cells/well (in 100 ⁇ l medium), cultured for 24 hours in expansion media in 21% oxygen and then treated with various concentrations of Compound A (0, 5, 10, 20, and 30 ⁇ M, Figure IA), deferoxamine (DFO; 0, 10, 50, 100, and 200 ⁇ M, Figure IB), ciclopirox olamine (CPX; 0, 1, 5, 10, and 20 ⁇ M, Figure 1C), cobalt chloride (CoCl 2 ; 0, 10, 25, 50, and 100 ⁇ M, Figure ID), or dimethyloxallyl glycine (DMOG; 0, 50, 100, 500, and 1000 ⁇ M, Figure IE) in expansion media for 72 hours.
  • Compound A (0, 5, 10, 20, and 30 ⁇ M, Figure IA)
  • DFO deferoxamine
  • CPX ciclopirox olamine
  • CoCl 2 cobalt chloride
  • DMOG dimethylo
  • hmNPCs were cultured and expanded in 21% oxygen as described above in Example 1, and cell cycle phase distribution of the cells was determined as follows. DNA content of the cells, as reflected by the fluorescence signal of propidium iodide, was measured using a flow cytometer (Becton Dickinson,
  • Example 3 Increased Proliferation and Neurons in hmNPCs
  • hmNPCs were allowed to differentiate in vitro for 2 weeks.
  • Proliferating hNPCs were identified by double-immunostaining for TUJl and the proliferation marker Ki67 as follows.
  • Control and compound-treated hmNPCs (from at least 2 different tissue preparations) were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature, rinsed with PBS, counterstained with the DNA-binding dye 4'-6-Diamidino-2-phenylindole (DAPI, 2 ⁇ g/ml in PBS) for 15 minutes at room temperature, then twice rinsed in PBS followed by incubation in blocking buffer (10% FCS, 0,2% Triton-X 100 in PBS, pH 7.2) for 30 minutes at room temperature.
  • blocking buffer (10% FCS, 0,2% Triton-X 100 in PBS, pH 7.2
  • Neurogenesis e.g., increase in neural cell proliferation, increase in newly generated neurons, and increase in immature neurons
  • TUJl neuron-specific class UJ ⁇ -tubulin
  • DCX doublecortin
  • TUJl neuron-specific class UJ ⁇ -tubulin
  • DCX doublecortin
  • TUJl neuron-specific class UJ ⁇ -tubulin
  • DCX doublecortin
  • TUJl is expressed in neurons of the peripheral and central nervous systems and contributes to microtubule stability in neuronal cell bodies and axons.
  • DCX is a microtubule-associated protein expressed almost exclusively in immature (e.g., developing) neurons, and thus is a marker for neurogenesis.
  • Neural precursors begin to express DCX shortly after exiting the cell cycle, and continue to express DCX as the cells mature into neurons.
  • the number of hNPCs positive for TUJl + , Ki67 + , or both markers were determined in control and Compound A-treated cultures and normalized to the total number of DAPI-stained cells.
  • a representative human midbrain-derived NPC was immunostained for the neuronal marker microtubule- associated protein 2 (MAP2) and the dopaminergic marker tyrosine hydroxylase (TH) upon 1 week of differentiation in 21% oxygen in the presence or absence of 10 ⁇ M Compound A.
  • MAP2 neuronal marker microtubule- associated protein 2
  • TH dopaminergic marker tyrosine hydroxylase
  • administration of Compound A increased TH and MAP2 expression in neural cells.
  • TH is red
  • MAP2 is green
  • DAPI is blue.
  • Immunoblotting was performed as follows. Combined cytoplasmic and nuclear extracts of cultured hmNPCS were prepared in extraction buffer as described previously (Milosevic et al., 2007a). Protein concentrations were determined by the Bradford method, using bovine serum albumin as a standard. Denaturated proteins were resolved on a sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to a Hybond-ECL nitrocellulose membrane (GE Healthcare, Freiburg, Germany) by semidry blotting.
  • SDS sodium dodecyl sulfate
  • Membranes were stained with Ponceau S (Sigma) to confirm equal protein loading and transfer followed by blocking with 5% (wt/vol) nonfat dry milk in PBS-T (PBS, 0.1% (vol/vol) Tween 20) for • 2 hours at room temperature and subsequent incubation with desired primary antibody (diluted in PBS containing 5% non-fat milk and 0.1% Tween 20) overnight at 4 0 C with gentle agitation.
  • PBS-T PBS, 0.1% (vol/vol) Tween 20
  • the antibodies used were as follows: mouse monoclonal anti-actin (C4, ICN Biomedicals), rabbit polyclonal anti- tyrosine hydroxylase (anti-TH) antibody (Santa Cruz), mouse monoclonal anti-TUJl (Covance) mouse monoclonal anti-neuron specific enolase (NSE, Chemicon International, Hampshire, UK), horseradish peroxidase-coupled secondary antibodies (Pierce, Rockford, IL, USA). Chemiluminescence detection was performed by incubating the membranes with SuperSignal-Dura substrate (Pierce) followed by analyzing on a CCD cooling camera (Fuji LAS-1000plus). The chemiluminescence was quantified using AIDA, two-dimensional densitometry software (Raytest Isotopenmeb, Straubenhardt, Germany).
  • the numbers of immunopositive cells (TH + cells) expressed as the percentages of DAPI stained nuclei are counted and presented as mean ⁇ SEM.
  • FIG 8 shows mmunoblots representing alterations in the expression of neuronal (NSE) and dopaminergic (TH) markers obtained in hmNPCs following differentiation of NPC cultures in the presence of Compound A. As shown in Figure 8, immunoblots of both mature (as determined by neuron- specific enolase (NSE) reactivity) and dopaminergic neuron (TH) markers further confirmed their increased expression following treatment of NPC cultures with Compound A ( Figure 8).
  • NSE neuronal
  • TH dopaminergic neuron
  • Neural progenitor cells were obtained from Lonza (Poietic NHNP Cells PT-2599 NHNP Neural Progenitor cells). The cells were cultured in media (Maintenance Medium NPMM, Lonza) containing fibroblast growth factor (FGF) and epidermal growth factor (EGF).
  • media Maintenance Medium NPMM, Lonza
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • neural progenitor cells were cultured under growth conditions that support self-renewal and proliferation of non-differentiated cells, but which do not support spontaneous differentiation of the neural progenitor cells.
  • neural progenitor cells were cultured under growth conditions that support differentiation of neural progenitor cells into mature neural cells.
  • NPMM Maintenance Medium
  • EGF EGF
  • various concentrations of Compound B for three days, during which time neurospheres formed.
  • the cells were then re-suspended, counted, and equivalent numbers of cells were plated onto laminin-coated wells (R&D Systems) in the absence of Compound B, EGF, and FGF (differentiation phase).
  • the cells were then plated onto laminin coated slide chambers (60,000 cells per /well), and cultured in NPDMTM - Neural Progenitor Differentiation media (CC-3229, Lonza).
  • VEGF vascular endothelial cell growth factor
  • VEGF expression (Gene Expression Assay, 00173626 ml, Applied Biosystems).
  • Compound B increased VEGF expression in the neural progenitor cells compared to that observed in non-treated control cultures.
  • Example 7 Increased Neurogenesis in vivo
  • mice Male C57B1/6 mice (8-10 weeks old; Charles River Laboratories) were singly housed under standard conditions and provided with food and water ad libitum. Animals were randomly divided into 4 groups of 8 animals each, and groups were treated 3 times per week for 3 weeks (10 treatments) with either vehicle control, Compound A (60 mg/kg), Compound C (60 mg/kg), or Compound D (60 mg/kg) by oral delivery. On the day after the final dose, all groups received two intraperitoneal injections with 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg for each injection) with a two hour interval to label actively dividing cells.
  • NrdU 5-bromo-2-deoxyuridine
  • mice were deeply anaesthetized using isoflurane and tissues were perfused with saline followed by 4% cold paraformaldehyde by transcardiac perfusion. Brains were removed and post-fixed overnight in 4% paraformaldehyde at 4 0 C, trimmed to remove excess rostral and caudal brain tissue, and then cryoprotected in 30% sucrose. Brains were coronally embedded in TISSUE-TEK OCT (Optimal Cutting Temperature) compound (Sakura) and stored at -80 0 C. Brains from 4 animals per treatment group were serially sectioned into 35 um sections on a cryostat and stored in
  • Sections were blocked for 30 min in Tris Buffered Saline (TBS) supplemented with 0.1% TritonX-100 and 5% normal donkey serum; sections were incubated overnight with primary antibodies (BrdU 1 : 100, Serotec; NeuN 1 :500, Chemicon) diluted in blocking buffer at 4°C.
  • TBS Tris Buffered Saline
  • Primary antibodies BrdU 1 : 100, Serotec; NeuN 1 :500, Chemicon
  • mice treated with compounds and methods of the present invention had an increased percentage of new mature neurons relative to vehicle-treated controls. These results indicated that methods and compounds of the present invention are effective at increasing neurogenesis in vivo.
  • mice Female C57BL/6NCrl mice (28 day-old; Charles River Laboratories) were treated every other day for 3 weeks (11 treatments) with either vehicle or Compound A (60mg/kg) by intraperitoneal injection. Groups of animals were examined at either 1, 3 or 4 weeks after the final treatment to test for hippocampal-dependent contextual fear conditioning as described by Adamcio et al. (2008) BMC Biol 6:37. In brief, after a 120s period in which baseline freezing was assessed, mice received a 2s, 0.4mA foot shock. Contextual memory was assessed 72 h after the training by monitoring mice over a 2 min period for freezing in the same conditioning chamber.
  • mice treated with compound A showed a clear improvement in contextual memory in fear conditioning at both 3 and 4 weeks after treatment cessation.
  • the compounds and methods of the invention increased neurogenesis in the hippocampus and improved functional hippocampal-dependent contextual memory.

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Abstract

La présente invention concerne des procédés et des compositions permettant d’augmenter la neurogenèse. L’invention a également trait à des procédés et à des composés permettant d’augmenter la différenciation dopaminergique. L’invention concerne par ailleurs des méthodes de traitement de troubles neurologiques par augmentation de la neurogenèse.
PCT/US2009/004885 2008-08-26 2009-08-26 Procédés d’augmentation de la neurogenèse WO2010024911A1 (fr)

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