US20080287484A1 - Neurotrophin antagonist compositions - Google Patents

Neurotrophin antagonist compositions Download PDF

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US20080287484A1
US20080287484A1 US11/905,406 US90540607A US2008287484A1 US 20080287484 A1 US20080287484 A1 US 20080287484A1 US 90540607 A US90540607 A US 90540607A US 2008287484 A1 US2008287484 A1 US 2008287484A1
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loweralkyl
isoquinoline
dioxo
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nitro
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Ashok Tehim
Xiannong Chen
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Priority claimed from GBGB9710904.5A external-priority patent/GB9710904D0/en
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Priority to US11/905,406 priority Critical patent/US20080287484A1/en
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Priority to US12/453,032 priority patent/US20090215815A1/en
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/06—Ring systems of three rings
    • C07D221/14—Aza-phenalenes, e.g. 1,8-naphthalimide
    • 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
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • 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
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the present invention relates to neurotrophin antagonists.
  • the present invention relates to compositions comprising an effective amount of a compound which inhibits or reduces undesirable neurotrophin activity, and a pharmaceutically acceptable carrier.
  • neurotrophins A family of structurally and functionally related neurotrophic factors exist which are collectively known as neurotrophins.
  • the family of neurotrophins includes the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6).
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4 neurotrophin-4
  • NT-5 neurotrophin-5
  • NT-6 neurotrophin-6
  • the neurotrophins exhibit similar structural conformations, including three surface ⁇ -hairpin loops, a ⁇ -strand, an internal reverse turn region, and N- and C-termini. With respect to sequence similarities, the neurotrophins share approximately 50% amino acid identity.
  • the neurotrophins are also functionally similar in that they each exhibit low affinity binding to a receptor known as the “p75 nerve growth factor receptor” or p75 NGFR .
  • Each neurotrophin also exhibits binding to a receptor of the tyrosine kinase (trk) family which is of higher affinity than the binding to the p75 receptor. This interaction is believed to be related to neuron survival, but is also involved with neuron differentiation including process formation.
  • trk tyrosine kinase
  • the trk receptor-neurotrophin interaction has been found to be more selective than neurotrophin interaction with the p75 NGFR receptor.
  • NGF binds only a trk receptor known as the TrkA receptor, while BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor.
  • TrkA receptor a trk receptor known as the TrkA receptor
  • BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor.
  • NT-3 is less selective and, although it binds primarily with a TrkC receptor, it also exhibits some binding to the TrkA and TrkB receptors (Ibanez et al., EMBO J. 1993, 12:2281).
  • the neurotrophins function primarily to promote survival of certain classes of peripheral and central neurons both during development and following neuronal damage.
  • NGF in particular, is involved with the development of neurons in the peripheral nervous system and supports neuronal survival, as well as enhancing and maintaining the differentiated state of neurons.
  • the neurotrophins may also support inappropriate neurite outgrowth thereby facilitating the progression of a disease condition.
  • neurotrophins promote the undesirable sprouting of hippocampal “mossy fibres”. Such inappropriate sprouting of mossy fibres is a common accompaniment of epilepsy in humans. It is also postulated that the pain experienced by patients suffering from some chronic pain syndromes may be associated with sprouting of sensory pain fibers responsive to NGF in particular into the spinal cord.
  • pathological states such as Alzheimer's disease, aberrant process growth, known as dystrophic neurite formation, is a strong correlate of disease severity.
  • neurotrophins are essential for the normal development and growth of neurons, they may be detrimental under certain circumstances.
  • ligands capable of inhibiting or reducing selected neurotrophin-mediated activities would be desirable therapeutically to treat neurodegenerative diseases and conditions including neuropathic pain and to repair nervous system injury.
  • compositions capable of inhibiting, or at least reducing, undesirable neurotrophin-mediated activity.
  • composition which comprises a carrier and an effective amount of a compound of Formula I:
  • R 1 is selected from alkyl; aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;
  • R 4 is phenyl optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and halo; phenyl optionally monosubstituted or disubstituted with a substituent selected from amino, loweralkoxy, hydroxy and loweralkyl; NHCH 2 CH 2 OX wherein X represents an in vivo hydrolyzable ester, and loweralkyl-(R 5 )(R 6 ) wherein one of R 5 and R 6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R 2 and R 3 are independently selected from H, NO 2 , halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR 7 wherein Z is selected from C and S and R 7 is selected from H, loweralkylamino and aryla
  • a method for inhibiting a neurotrophin-mediated activity comprising the step of exposing neurons to a composition as described above.
  • a further aspect of the present invention provides a method for inhibiting neurotrophin-mediated activity in a mammal comprising the step of administering a composition as described above to said mammal.
  • alkyl as used herein means straight and branched chain alkyl radicals containing from one to eight carbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyl, pentyl, hexyl, heptyl, octyl and the like.
  • loweralkyl as used herein means straight and branched chain alkyl radicals containing from one to four carbon atoms and includes methyl, ethyl, propyl, isopropyl tert-butyl and the like.
  • alkoxy as used herein means straight and branched chain alkoxy radicals containing from one to eight carbon atoms and includes methoxy, ethoxy, tert-butoxy and the like.
  • loweralkoxy as used herein means straight and branched chain alkoxy radicals containing from one to four carbon atoms and includes methoxy, ethoxy, tert-butoxy and the like.
  • aryl as used herein means a 5 or 6 membered aromatic or heteroaromatic ring containing 1, 2 or 3 heteroatoms independently selected from O, N and S, and includes phenyl, pyridyl, thienyl, furanyl, pyrrolo, imidazolo and the like.
  • heterocycle means a five or six membered, non-aromatic ring optionally containing one or more double bonds and one or two heteroatoms selected from O, S, and N, and includes dihydropyran, tetrahydropyran, tetrahydrofuranyl, azacyclohexane, azacyclohexene, dihydrothiapyran, tetrahydrothiapyran, morpholino and the like.
  • halo as used herein means halide and includes fluoro, chloro, bromo and iodo.
  • hydrolyzable esters or amides are those readily hydrolyzable esters or amides of compounds of Formula I, which are known and used in the pharmaceutical industry and include ⁇ -acyloxyalkyl and esters of C 3-20 -fatty acids.
  • neurotrophin refers to neurotrophic factors that are structurally homologous to NGF, i.e. include three surface ⁇ -hairpin loops, a ⁇ -strand, an internal reverse turn region, and N- and C-termini, and which promote at least one of neuron survival and neuron differentiation, as determined using assays of conventional design such as the in vitro assay exemplified herein and described by Riopelle et al. in Can. J. of Phys. and Pharm., 1982, 60:707.
  • Mammalian nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6) are examples of neurotrophins.
  • Neurotrophin-mediated activity is a biological activity that is normally promoted, either directly or indirectly, in the presence of a neurotrophin.
  • Neurotrophin-mediated activities include, for example, neurotrophin binding to the p75 NGFR receptor or neurotrophin binding to one of the trk receptors, neuron survival, neuron differentiation including neuron process formation and neurite outgrowth, and biochemical changes such as enzyme induction.
  • a biological activity that is mediated by a particular neurotrophin, e.g. NGF is referred to herein by reference to that neurotrophin, e.g. NGF-mediated activity.
  • conventional in vitro and in vivo assays can be used.
  • a receptor binding assay such as the assay described herein in Example 1, can be used to assess the extent to which a compound inhibits neurotrophin/receptor binding. Inhibition of neurite survival and outgrowth can be determined using the in vitro assay described by Riopelle et al. in the Can. J. of Phys. and Pharm, 1982, 60:707, illustrated herein in Example 2.
  • the present invention relates to compositions comprising an effective amount of a compound of Formula I, or pharmaceutically acceptable salts or in vivo hydrolyzable esters or amides thereof (hereinafter referred to as a compound of Formula I), which inhibits neurotrophin-mediated activity, and a pharmaceutically acceptable carrier.
  • a compound of Formula I or pharmaceutically acceptable salts or in vivo hydrolyzable esters or amides thereof (hereinafter referred to as a compound of Formula I), which inhibits neurotrophin-mediated activity, and a pharmaceutically acceptable carrier.
  • compounds of Formula I include those in which R 1 is selected from allyl; aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;
  • R 4 is phenyl optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and halo; phenyl optionally monosubstituted or disubstituted with a substituent selected from amino, loweralkoxy, hydroxy and loweralkyl; NHCH 2 CH 2 OX wherein X represents an in vivo hydrolyzable ester, and loweralkyl-(R 5 )(R 6 ) wherein one of R 5 and R 6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R 2 and R 3 are independently selected from H, NO 2 , halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR 7 wherein Z is selected from C and S and R 7 is selected from H, loweralkylamino and aryla
  • compounds of Formula I include those in which R 1 is selected from aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and hydroxyloweralkyl; benzimidaz-2-yl; NHCH 2 CH 2 OX wherein X represents an in vivo hydrolyzable ester; and loweralkyl-(R 5 )(R 6 ) wherein one of R 5 and R 6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R 2 and R 3 are independently selected from H, NO 2 , di(loweralkyl)amino, and phenyl-S—; and pharmaceutically acceptable salts thereof.
  • compounds of Formula I include those in which R 1 is selected from amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and hydroxyloweralkyl; NHCH 2 CH 2 OX wherein X represents an in vivo hydrolyzable ester; and loweralkyl-(R 5 )(R 6 ) wherein one of R 5 and R 6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R 2 and R 3 are independently selected from H and NO 2 ; and pharmaceutically acceptable salts thereof.
  • compounds of Formula I include:
  • compounds of Formula I include:
  • compounds of Formula I include:
  • compounds of Formula I include:
  • the compounds of the present invention can be prepared by techniques well known in the art
  • Compounds of formula I wherein R 1 , R 2 and R 3 are as defined above can be prepared by reacting a 1,8-naphthalic anhydride of Formula A with a primary amine of Formula B in a suitable solvent such as toluene, methanol, ethanol, propanol or acetone and at temperatures in the range of 0° C. to the boiling point of the solvent used.
  • a suitable solvent such as toluene, methanol, ethanol, propanol or acetone
  • Acid addition salts of the compounds of Formula I are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, sulphuric or phosphoric acids and organic acids e.g. succinic, maleic, acetic or fumaric acid.
  • Other non-pharmaceutically acceptable salts e.g. oxalates may be used for example in the isolation of the compound of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
  • solvates and hydrates of the invention are also included within the scope of the invention.
  • the conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether.
  • the free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.
  • base e.g. sodium carbonate or potassium hydroxide
  • In vivo hydrolyzable esters or amides of certain compounds of Formula I can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform.
  • Suitable bases include triethylamine or pyridine.
  • compounds of Formula I having a free carboxy group may be esterified using standard conditions which may include activation followed by treatment with the desired alcohol in the presence of a suitable base.
  • compositions of the present invention are useful to inhibit or reduce undesirable neurotrophin activity both in vitro and in vivo.
  • a composition comprising an effective amount of a compound of Formula I and a suitable carrier.
  • suitable carrier is meant a carrier which admixes with the compound of Formula I to yield a composition suitable for the application for which it is to be used.
  • effective amount is meant an amount of the compound sufficient to inhibit an undesired neurotrophin-mediated activity to a measurable extent, preferably by about 20%, more preferably by about 40%, most preferably by about 50%, as determined using assays of conventional design such as those described herein in the specific examples.
  • the present composition has use as a media supplement to prevent undesirable neurotrophin-mediated activity of neuron cells in vitro.
  • primary sensory neurons require NGF for survival in cell culture; however, NGF also influences neuron differentiation, notably process formation and outgrowth, which are undesirable for the use of primary sensory neurons in cell culture.
  • NGF is added to the cell culture media along with the compound of Formula I.
  • the compound is first combined with a carrier which will not adversely affect the growth of the cells in culture.
  • Such carriers will include, for example, physiologically acceptable fluids such as water or any other fluid suitable for addition to the cell culture.
  • the compound can be combined with media suitable for culturing neuronal cells prior to being added to the cell culture.
  • concentration of the compound in the cell culture will be in the range of from about 1-500 ⁇ M, and preferably from about 1-100 ⁇ M.
  • concentration of compound for use in preventing neuron differentiation in cell culture will, of course, vary depending on the extent of inhibition desired as well as the type of neuronal cells involved.
  • compositions for in vivo administration e.g. for treating neurological conditions such as epilepsy or Alzheimer's disease, or for treating chronic pain
  • Such compositions comprise a therapeutically effective amount of the compound of Formula I together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means acceptable for use in the pharmaceutical and veterinary arts, i.e. non-toxic and not adversely affecting the activity of the compound.
  • therapeutically effective amount means an amount of the compound sufficient to reduce undesirable neurotrophin-mediated activity, as determined using assays of conventional design, in an inflicted individual without causing adverse effects.
  • compositions for in viva administration include conventional carriers used in formulating drugs, such as diluents, excipients and the like. Reference may be made to “Remington's Pharmaceutical Sciences”, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, for guidance on drug formulations generally. As will be appreciated, the pharmaceutical carriers used to prepare compositions in accordance with the present invention will depend on the dosage form to be used to treat the inflicted individual.
  • a compound of Formula I is formulated for administration by injection intraventricularly, and is accordingly provided as an aqueous solution in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the compound may be administered in distilled water or, more desirably, in saline or 5% dextrose solution.
  • Water solubility of the compound of the invention may be enhanced, if desired, by incorporating into the composition a solubility enhancer, such as acetyltrimethylammonium bromide or chloride.
  • Lyoprotectants such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
  • a pharmaceutical composition appropriate for treatment For use in treating individuals with a neurological condition, precise dosage sizes of a pharmaceutical composition appropriate for treatment are established in appropriately controlled trials, and will correspond to an amount of a compound of Formula I that reduces undesirable neurotrophin-mediated activity without causing intolerable side effects to the individual being treated. It is anticipated that an effective treatment regimen for patients will involve the intraventricular administration of dosages which achieve a level of the compound in the spinal fluid of the individual being treated of about 1-500 ⁇ M. It will be appreciated, of course, that the dosage sizes required to attain this in vivo concentration will vary according to the route of administration, the frequency of administration, on the individual being treated and on the neurological condition being treated.
  • NGF was labelled using the Lactoperoxidase labelling method (Sutter et al., J. Biol. Chem., 1979) and the labelled NGF was separated from radiolabelling agents and free iodide using a PD-10 Sephadex G-25 column.
  • PC12 cells were grown in RPMI with 10% heat inactivated donor horse serum and 5% fetal calf serum. Cells were harvested for binding by washing off the media with calcium-magnesium free balanced salt solution (Gey's solution) and incubated in 5 ml Gey's solution at 37° C. for 15 minutes. Cells were pelleted by centrifugation and suspended in Hepes-Krebs Ringer buffer (HKR) (10 mM Hepes pH7.35, containing 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 . 1 mg/ml BSA and 1.0 mg/ml glucose) at a concentration of 4 ⁇ 10 6 /ml and kept on ice.
  • HLR Hepes-Krebs Ringer buffer
  • the reaction was performed in a 96 well plate. Suspended cells (150 ul, 10 6 cells) were added to 125 I-NGF (final concentration of 0.5 mM) and the competing compound of Formula I in a final volume of 300 ul of HKR buffer. The plates were incubated with shaking for 2 hr at 4° C. At the end of the incubation, 100 ul aliquots of the reaction sample were added to 400 ul microcentrifuge tubes containing 200 ul of 10% glycerol in HKR buffer. The tubes were centrifuged for 1 minute at ⁇ 5000 rpm and the tip containing the cell pellet was cut off.
  • Radioactivity bound to the cells was determined by measuring the 125 I-NGF associated with each pellet in a gamma counter. Specific binding is calculated as the difference between the amount of 125 I-NGF bound in the absence (total) and presence (NSB) of 50 nM unlabeled NGF. TrkA binding is determined similarly except 10 nM BDNF is added to all reactions. Table 1 summarizes the values obtained from this experiment for the inhibition of binding of NGF to P75 and TrkA by compounds of Formula I.
  • the cell suspension was then preplated on a 100-mm Flacon culture dish and incubated for 45-60 min at 37° C. in a 5% CO, humidified atmosphere. Cells enriched in neurons were decanted for the bioassay, since non-neuronal cells of DRG preferentially stick to the culture substrate.
  • the inside wells of 96-well Falcon microculture plates were coated with polylysine (0.1 mg/mL) (Sigma) for 4 h at 37° C. (the outside wells were filled with distilled water to provide humidity) and, following a rinse with tissue culture media, 100 mL of neuron-rich cell suspension was added to each well at 10 5 cells/mL.
  • a rinse with tissue culture media 100 mL of neuron-rich cell suspension was added to each well at 10 5 cells/mL.
  • Ninety (90) mL of NGF solution (prepared in tissue culture media) was then added to each well to a final concentration of 0.25 ng/mL NGF per well.
  • Ten (10) mL of test compound solution i.e.
  • tissue culture media admixed with a compound of Formula I was then added to test wells in duplicate to yield wells containing compound concentrations ranging from 0 ⁇ M-100 ⁇ M.
  • 10 mL of Ham's F12 medium was added to duplicate NGF-containing wells. The plates were covered and incubated in the dark for 24-30 hrs. at 37° C. in a 5% CO 2 humidified atmosphere.
  • the bioassays were read using a Leitz Diavert microscope with phase optics. To afford adequate optics, the meniscus effect of each well was removed by filling the well with a balanced salt solution until a flat, air-filled interface was achieved at the top of the well. At least 100 neurons per well were counted, and the assay was scored as the ratio of cells bearing neurites greater than one cell diameter to total viable (phase bright) cells. These results are summarized in Table 2.
  • Compound A N- ⁇ 5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione ⁇ -2-aminoethanol
  • Nerve ligation injury was performed according to the method described by Kim and Chung (Pain 50: 355-363, 1992). Rats were anesthetized with halothane and the vertebrae over the L4 to S2 region were exposed. L5 and L6 spinal nerves were exposed, carefully isolated, tightly ligated with 4-0 silk suture distal to the DRG. After ensuring homeostatic stability, the wounds were sutured, and the animals were allowed to recover in the cages. Sham-operated rats were prepared in an identical fashion except that the L5/L6 nerve roots are not ligated.
  • test compounds were injected through indwelling i.th. catheters. While under anesthesia, PE-10 tubing (8 cm) was inserted through an incision made in the atlanto-occipitil membrane to the level of the lumbar enlargement of the rat and secured. Drug injections were made in a volume of 5 ⁇ l of 50% aqueous DMSO followed by a 9 ⁇ l saline flush.
  • Thermal hyperalgesia was determined by focusing a radiant heat source onto the plantar surface of the affected paw of nerve-injured or sham-operated rats. Paw withdrawal latencies are determined by a photodetection device which halts the stimulus and the timer after a maximum cut-off of 40 sec to prevent tissue damage. The withdrawal latency of sham-operated rats were compared to those of ligated rats to measure the degree of hyperalgesia.
  • Acute nociception was determined by using the nociceptive warm water tail-flick reflex. This test was performed by placing the tail of the nerve-injured or sham-operated rats in a heated water bath maintained at 55° C. The latency until tail withdrawal (rapid flick) from the bath was determined and compared among treatments. A 15 second cut-off was employed to avoid tissue damage.
  • Compound A and morphine were tested in the nerve-ligated injury model of neuropathic pain using 3 routes of administration: intraopertoneally (i.p.), intra-thecally (i.th.) and intracerebroventrically (i.c.v.). The compounds were evaluated for three endpoints: tactile allodynia, thermal hyperalgesia and acute nociception. Compound A is not active when given i.c.v. The results for i.p and i.th. administration are shown in Table 3.

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Abstract

A pharmaceutical composition comprising a compound of Formula I
Figure US20080287484A1-20081120-C00001
wherein R1 is selected from, inter alia, alkyl, aryl-loweralkyl, heterocycle-loweralkyl, loweralkyl-carbonate; optionally substituted amino; benzimidaz-2-yl; optionally substituted phenyl; loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester; and R2 and R3 are independently selected from H, NO2, halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR7 wherein Z is selected from C and S and R7 is selected from H, loweralkylamino and arylamino; or pharmaceutically acceptable salts or certain in vivo hydrolyzable esters or amides thereof, in an amount effective to inhibit neurotrophin-mediated activity, and a suitable carrier, is described.
The compositions are useful to inhibit undesirable neurotrophin-mediated activity such as the neurite outgrowth that occurs in some neurodegenerative disease states.

Description

    RELATED APPLICATIONS
  • This application is a continuation of U.S. Ser. No. 09/592,015, filed Jun. 12, 2000, which is a continuation of U.S. Ser. No. 09/440,505, filed Nov. 15, 1999, which is a continuation of U.S. Ser. No. 09/292,458, filed Apr. 15, 1999, which is a continuation of the U.S. designation of international application PCT/CA97/00779, filed Oct. 20, 1997, which claims priority to GB9710904.5, filed May 27, 1997, and GB9621902.7, filed Oct. 21, 1996, the entire teachings of which are incorporated herein by reference.
    • FIELD OF THE INVENTION
  • The present invention relates to neurotrophin antagonists. In particular, the present invention relates to compositions comprising an effective amount of a compound which inhibits or reduces undesirable neurotrophin activity, and a pharmaceutically acceptable carrier.
    • BACKGROUND OF THE INVENTION
  • A family of structurally and functionally related neurotrophic factors exist which are collectively known as neurotrophins. The family of neurotrophins includes the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6).
  • The neurotrophins exhibit similar structural conformations, including three surface β-hairpin loops, a β-strand, an internal reverse turn region, and N- and C-termini. With respect to sequence similarities, the neurotrophins share approximately 50% amino acid identity. The neurotrophins are also functionally similar in that they each exhibit low affinity binding to a receptor known as the “p75 nerve growth factor receptor” or p75NGFR. Each neurotrophin also exhibits binding to a receptor of the tyrosine kinase (trk) family which is of higher affinity than the binding to the p75 receptor. This interaction is believed to be related to neuron survival, but is also involved with neuron differentiation including process formation. The trk receptor-neurotrophin interaction has been found to be more selective than neurotrophin interaction with the p75NGFR receptor. In particular, NGF binds only a trk receptor known as the TrkA receptor, while BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor. NT-3 is less selective and, although it binds primarily with a TrkC receptor, it also exhibits some binding to the TrkA and TrkB receptors (Ibanez et al., EMBO J. 1993, 12:2281).
  • The neurotrophins function primarily to promote survival of certain classes of peripheral and central neurons both during development and following neuronal damage. NGF, in particular, is involved with the development of neurons in the peripheral nervous system and supports neuronal survival, as well as enhancing and maintaining the differentiated state of neurons. However, in some neurological disease states, the neurotrophins may also support inappropriate neurite outgrowth thereby facilitating the progression of a disease condition. For example, neurotrophins promote the undesirable sprouting of hippocampal “mossy fibres”. Such inappropriate sprouting of mossy fibres is a common accompaniment of epilepsy in humans. It is also postulated that the pain experienced by patients suffering from some chronic pain syndromes may be associated with sprouting of sensory pain fibers responsive to NGF in particular into the spinal cord. In other pathological states, such as Alzheimer's disease, aberrant process growth, known as dystrophic neurite formation, is a strong correlate of disease severity.
  • Thus, although the neurotrophins are essential for the normal development and growth of neurons, they may be detrimental under certain circumstances. In such instances, ligands capable of inhibiting or reducing selected neurotrophin-mediated activities would be desirable therapeutically to treat neurodegenerative diseases and conditions including neuropathic pain and to repair nervous system injury.
    • SUMMARY OF THE INVENTION
  • It is an object of the present invention to provide compositions capable of inhibiting, or at least reducing, undesirable neurotrophin-mediated activity.
  • In an aspect of the present invention, a composition is provided which comprises a carrier and an effective amount of a compound of Formula I:
  • Figure US20080287484A1-20081120-C00002
  • wherein
    R1 is selected from alkyl; aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;
  • Figure US20080287484A1-20081120-C00003
  • wherein R4 is phenyl optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and halo; phenyl optionally monosubstituted or disubstituted with a substituent selected from amino, loweralkoxy, hydroxy and loweralkyl; NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester, and loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R2 and R3 are independently selected from H, NO2, halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR7 wherein Z is selected from C and S and R7 is selected from H, loweralkylamino and arylamino;
    and pharmaceutically acceptable salts thereof.
  • In a further aspect of the present invention, there is provided a method for inhibiting a neurotrophin-mediated activity comprising the step of exposing neurons to a composition as described above.
  • A further aspect of the present invention provides a method for inhibiting neurotrophin-mediated activity in a mammal comprising the step of administering a composition as described above to said mammal.
  • These and other aspects of the present invention will be described in greater detail hereinbelow.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The term “alkyl” as used herein means straight and branched chain alkyl radicals containing from one to eight carbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyl, pentyl, hexyl, heptyl, octyl and the like.
  • The term “loweralkyl” as used herein means straight and branched chain alkyl radicals containing from one to four carbon atoms and includes methyl, ethyl, propyl, isopropyl tert-butyl and the like.
  • The term “alkoxy” as used herein means straight and branched chain alkoxy radicals containing from one to eight carbon atoms and includes methoxy, ethoxy, tert-butoxy and the like.
  • The term “loweralkoxy” as used herein means straight and branched chain alkoxy radicals containing from one to four carbon atoms and includes methoxy, ethoxy, tert-butoxy and the like.
  • The term “aryl” as used herein means a 5 or 6 membered aromatic or heteroaromatic ring containing 1, 2 or 3 heteroatoms independently selected from O, N and S, and includes phenyl, pyridyl, thienyl, furanyl, pyrrolo, imidazolo and the like.
  • The term “heterocycle” as used herein means a five or six membered, non-aromatic ring optionally containing one or more double bonds and one or two heteroatoms selected from O, S, and N, and includes dihydropyran, tetrahydropyran, tetrahydrofuranyl, azacyclohexane, azacyclohexene, dihydrothiapyran, tetrahydrothiapyran, morpholino and the like.
  • The term “halo” as used herein means halide and includes fluoro, chloro, bromo and iodo.
  • As used herein, in vivo hydrolyzable esters or amides are those readily hydrolyzable esters or amides of compounds of Formula I, which are known and used in the pharmaceutical industry and include α-acyloxyalkyl and esters of C3-20-fatty acids.
  • As it is used herein, the term “neurotrophin” refers to neurotrophic factors that are structurally homologous to NGF, i.e. include three surface β-hairpin loops, a β-strand, an internal reverse turn region, and N- and C-termini, and which promote at least one of neuron survival and neuron differentiation, as determined using assays of conventional design such as the in vitro assay exemplified herein and described by Riopelle et al. in Can. J. of Phys. and Pharm., 1982, 60:707. Mammalian nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6) are examples of neurotrophins.
  • “Neurotrophin-mediated activity” is a biological activity that is normally promoted, either directly or indirectly, in the presence of a neurotrophin. Neurotrophin-mediated activities include, for example, neurotrophin binding to the p75NGFR receptor or neurotrophin binding to one of the trk receptors, neuron survival, neuron differentiation including neuron process formation and neurite outgrowth, and biochemical changes such as enzyme induction. A biological activity that is mediated by a particular neurotrophin, e.g. NGF, is referred to herein by reference to that neurotrophin, e.g. NGF-mediated activity. To determine the ability of a compound to inhibit a neurotrophin-mediated activity, conventional in vitro and in vivo assays can be used. For example, a receptor binding assay, such as the assay described herein in Example 1, can be used to assess the extent to which a compound inhibits neurotrophin/receptor binding. Inhibition of neurite survival and outgrowth can be determined using the in vitro assay described by Riopelle et al. in the Can. J. of Phys. and Pharm, 1982, 60:707, illustrated herein in Example 2.
  • The present invention relates to compositions comprising an effective amount of a compound of Formula I, or pharmaceutically acceptable salts or in vivo hydrolyzable esters or amides thereof (hereinafter referred to as a compound of Formula I), which inhibits neurotrophin-mediated activity, and a pharmaceutically acceptable carrier.
  • In embodiments of the invention, compounds of Formula I include those in which R1 is selected from allyl; aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;
  • Figure US20080287484A1-20081120-C00004
  • wherein R4 is phenyl optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and halo; phenyl optionally monosubstituted or disubstituted with a substituent selected from amino, loweralkoxy, hydroxy and loweralkyl; NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester, and loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R2 and R3 are independently selected from H, NO2, halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR7 wherein Z is selected from C and S and R7 is selected from H, loweralkylamino and arylamino;
    and pharmaceutically acceptable salts thereof.
  • In another embodiment of the invention, compounds of Formula I include those in which R1 is selected from aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and hydroxyloweralkyl; benzimidaz-2-yl; NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester; and loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R2 and R3 are independently selected from H, NO2, di(loweralkyl)amino, and phenyl-S—; and pharmaceutically acceptable salts thereof.
  • In a further embodiment of the invention, compounds of Formula I include those in which R1 is selected from amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and hydroxyloweralkyl; NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester; and loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R2 and R3 are independently selected from H and NO2; and pharmaceutically acceptable salts thereof.
  • In a specific embodiment of the invention, compounds of Formula I include:
    • N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;
    • N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic acid;
    • N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • N-Furfuryl-1,8-naphthalimide; —(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;
    • N-(Pyrid-2-ylethyl)-1,8-naphthalimide;
    • 1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline;
    • 2-{2-(4-Methylphenylsulphonanido)phenyl}-6-(N,N-dimethylamino) naphthalimide;
    • 1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydro-benzo[i]isoquinoline;
    • N-Octyl-5-nitronaphthalimide;
    • 5-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;
    • 1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline;
    • 6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide;
    • N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • Naphthalicacid-N-aminoimide;
    • 2-{2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl}naphthalimide;
    • 3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;
    • 1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 6-Nitro-2-(pyrid-3-methyl)naphthalimide;
    • 3-Amino-7,4-bis(ethyl-1,3-dioxo)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 2-(2-Aminophenyl)naphthalimide;
    • 1,3-Dioxo-2-{4,5-dimethyl-2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 3-Methyl-3-(1,3-dioxo-5-nitro(1H,3H)benz[de]isoquinolyl)butyric acid methylester;
    • 1,3-Dioxo-N-methyltetrahydrofurfur-2-yl-5-nitro-1,2,3,4-tetrahydro-[i]isoquinoline;
    • N-(4-Ethoxyphenyl)-5-nitronaphthalimide;
    • 6-Nitro-2-(furfuryl)naphthalimide;
    • Ethyl-5-nitro-1,3-dioxo-1H-benz[de]isoquinoline-2-3H-acetate;
    • Naphthalicacid-N,N′-diimide;
    • 2-(2-Hydroxyphenyl)naphthalimide;
    • 5-Amino-N-butylnaphthalimide;
    • 1,3-Dioxo-5-nitro-n-propylmorpholino-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 6-Nitro-2-(pyrid-2-ylethyl)naphthalimide;
    • N-Methylnaphthalimide;
    • N-(Pyrid-2-ylmethyl)naphthalimide;
    • N-(3,5-Dimethylphenyl)-1,8-naphthalimide;
    • 6-Bromo-N-dimethylamino-1,3-dioxo-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;
    • N-(1,3-Dioxo-6-phenylmercapto-1,2,3,4-tetrahydrobenzo[i]isoquinoline)-aminoethanol; and
    • N-Anilino-1,8-naphthalimide.
  • In a preferred embodiment of the invention, compounds of Formula I include:
    • N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;
    • N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic acid;
    • N-Acetoxy-1,3-dioxo-1,2,3,1-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • N-Furfuryl-1,8-naphthalimide;
    • 6-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;
    • N-(Pyrid-2-ylethyl)-1,8-naphthalimide;
    • 1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline;
    • 2-{2-(4-Methylphenylsulphonamido)phenyl}-6-(N,N-dimethylamino) naphthalimide;
    • 1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydro-benzo[i]isoquinoline;
    • N-Octyl-5-nitronaphthalimide;
    • 5-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;
    • 1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline;
    • 6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide;
    • N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • Naphthalicacid-N-aminoimide;
    • 2-{2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl}naphthalimide;
    • 3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;
    • 1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 6-Nitro-2-(pyrid-3-methyl)naphthalimide;
    • 3-Amino-7,4-bis(ethyl-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline; and
    • 2-(2-Aminophenyl)naphthalimide.
  • In a more preferred embodiment of the invention, compounds of Formula I include:
    • N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)dione}-2-aminoethanol;
    • N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic acid;
    • N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • N-Furfuryl-1,8-naphthalimide;
    • 6-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;
    • N-(Pyrid-2-ylethyl)-1,8-naphthalimide; and
    • 1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline.
  • In a most preferred embodiment of the invention, compounds of Formula I include:
    • N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;
    • N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic acid;
    • N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline; and
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol.
  • Another embodiment of the invention includes an in vivo hydrolyzable ester or amide of a compound selected from the group consisting of:
    • N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic acid;
    • N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;
    • Naphthalicacid-N-aminoimide;
    • 3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;
    • 1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 3-Amino-7,4-bis(ethyl-1,3-dioxo)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;
    • 2-(2-Aminophenyl)naphthalimide; and
    • 2-(2-Hydroxyphenyl)naphthalimide.
  • The compounds of the present invention can be prepared by techniques well known in the art Compounds of formula I wherein R1, R2 and R3 are as defined above can be prepared by reacting a 1,8-naphthalic anhydride of Formula A with a primary amine of Formula B in a suitable solvent such as toluene, methanol, ethanol, propanol or acetone and at temperatures in the range of 0° C. to the boiling point of the solvent used. Both reagent A and reagent B are commercially available or can be prepared using procedures known to one skilled ii the art.
  • Figure US20080287484A1-20081120-C00005
  • Acid addition salts of the compounds of Formula I are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, sulphuric or phosphoric acids and organic acids e.g. succinic, maleic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates may be used for example in the isolation of the compound of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.
  • The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.
  • In vivo hydrolyzable esters or amides of certain compounds of Formula I can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of Formula I having a free carboxy group may be esterified using standard conditions which may include activation followed by treatment with the desired alcohol in the presence of a suitable base.
  • The compositions of the present invention are useful to inhibit or reduce undesirable neurotrophin activity both in vitro and in vivo. Thus, in an aspect of the invention, a composition comprising an effective amount of a compound of Formula I and a suitable carrier is provided. By “suitable carrier” is meant a carrier which admixes with the compound of Formula I to yield a composition suitable for the application for which it is to be used. By “effective amount” is meant an amount of the compound sufficient to inhibit an undesired neurotrophin-mediated activity to a measurable extent, preferably by about 20%, more preferably by about 40%, most preferably by about 50%, as determined using assays of conventional design such as those described herein in the specific examples.
  • The present composition has use as a media supplement to prevent undesirable neurotrophin-mediated activity of neuron cells in vitro. For example, primary sensory neurons require NGF for survival in cell culture; however, NGF also influences neuron differentiation, notably process formation and outgrowth, which are undesirable for the use of primary sensory neurons in cell culture. Thus, to preserve neuron survival in vitro while inhibiting cell differentiation, NGF is added to the cell culture media along with the compound of Formula I. For addition to the cell culture, the compound is first combined with a carrier which will not adversely affect the growth of the cells in culture. Such carriers will include, for example, physiologically acceptable fluids such as water or any other fluid suitable for addition to the cell culture. Alternatively, the compound can be combined with media suitable for culturing neuronal cells prior to being added to the cell culture. To be effective to prevent neuron differentiation, the concentration of the compound in the cell culture will be in the range of from about 1-500 μM, and preferably from about 1-100 μM. The optimal concentration of compound for use in preventing neuron differentiation in cell culture will, of course, vary depending on the extent of inhibition desired as well as the type of neuronal cells involved.
  • Compositions for in vivo administration, e.g. for treating neurological conditions such as epilepsy or Alzheimer's disease, or for treating chronic pain, are also contemplated. Such compositions comprise a therapeutically effective amount of the compound of Formula I together with a pharmaceutically acceptable carrier. In this context, the term “pharmaceutically acceptable” means acceptable for use in the pharmaceutical and veterinary arts, i.e. non-toxic and not adversely affecting the activity of the compound. The term “therapeutically effective amount” means an amount of the compound sufficient to reduce undesirable neurotrophin-mediated activity, as determined using assays of conventional design, in an inflicted individual without causing adverse effects.
  • Pharmaceutically acceptable carriers useful to prepare compositions for in viva administration include conventional carriers used in formulating drugs, such as diluents, excipients and the like. Reference may be made to “Remington's Pharmaceutical Sciences”, 17th Ed., Mack Publishing Company, Easton, Pa., 1985, for guidance on drug formulations generally. As will be appreciated, the pharmaceutical carriers used to prepare compositions in accordance with the present invention will depend on the dosage form to be used to treat the inflicted individual.
  • According to one embodiment of the invention, a compound of Formula I is formulated for administration by injection intraventricularly, and is accordingly provided as an aqueous solution in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compound may be administered in distilled water or, more desirably, in saline or 5% dextrose solution. Water solubility of the compound of the invention may be enhanced, if desired, by incorporating into the composition a solubility enhancer, such as acetyltrimethylammonium bromide or chloride. Lyoprotectants, such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
  • For use in treating individuals with a neurological condition, precise dosage sizes of a pharmaceutical composition appropriate for treatment are established in appropriately controlled trials, and will correspond to an amount of a compound of Formula I that reduces undesirable neurotrophin-mediated activity without causing intolerable side effects to the individual being treated. It is anticipated that an effective treatment regimen for patients will involve the intraventricular administration of dosages which achieve a level of the compound in the spinal fluid of the individual being treated of about 1-500 μM. It will be appreciated, of course, that the dosage sizes required to attain this in vivo concentration will vary according to the route of administration, the frequency of administration, on the individual being treated and on the neurological condition being treated.
  • Specific embodiments of the present invention are described in more detail in the following examples which are not to be construed as limiting.
    • EXAMPLE 1 Binding of [125I]NGF to PC12 Cells in the Presence and Absence of BDNF
  • The ability of the compounds of Formula I to antagonize NGF interaction with the p75 and trkA receptors was determined as follows.
    • (A) Iodination of NGF
  • NGF was labelled using the Lactoperoxidase labelling method (Sutter et al., J. Biol. Chem., 1979) and the labelled NGF was separated from radiolabelling agents and free iodide using a PD-10 Sephadex G-25 column.
    • (B) Cell Culture and Cell Preparation
  • PC12 cells were grown in RPMI with 10% heat inactivated donor horse serum and 5% fetal calf serum. Cells were harvested for binding by washing off the media with calcium-magnesium free balanced salt solution (Gey's solution) and incubated in 5 ml Gey's solution at 37° C. for 15 minutes. Cells were pelleted by centrifugation and suspended in Hepes-Krebs Ringer buffer (HKR) (10 mM Hepes pH7.35, containing 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4. 1 mg/ml BSA and 1.0 mg/ml glucose) at a concentration of 4×106/ml and kept on ice.
    • (C) NGF Binding
  • The reaction was performed in a 96 well plate. Suspended cells (150 ul, 106 cells) were added to 125I-NGF (final concentration of 0.5 mM) and the competing compound of Formula I in a final volume of 300 ul of HKR buffer. The plates were incubated with shaking for 2 hr at 4° C. At the end of the incubation, 100 ul aliquots of the reaction sample were added to 400 ul microcentrifuge tubes containing 200 ul of 10% glycerol in HKR buffer. The tubes were centrifuged for 1 minute at ˜5000 rpm and the tip containing the cell pellet was cut off. Radioactivity bound to the cells was determined by measuring the 125I-NGF associated with each pellet in a gamma counter. Specific binding is calculated as the difference between the amount of 125I-NGF bound in the absence (total) and presence (NSB) of 50 nM unlabeled NGF. TrkA binding is determined similarly except 10 nM BDNF is added to all reactions. Table 1 summarizes the values obtained from this experiment for the inhibition of binding of NGF to P75 and TrkA by compounds of Formula I.
  • TABLE 1
    %
    Inhibition
    Compound at 100 uM
    N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]iso- 59
    quinoline
    N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic 58
    acid
    N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline 57
    N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)-aminoethanol 44
    N-Furfuryl-1,8-naphthalimide 36
    6-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide 36
    N-(Pyrid-2-ylethyl)-1,8-naphthalimide 35
    1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro- 33
    benzo[i]isoquinoline
    2-{2-(4-methylphenylsulphonamido)phenyl}-6-(N,N-dimethylamino)- 29
    naphthalimide
    1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4- 29
    tetrahydrobenzo[i]isoquinoline
    N-Octyl-5-nitronaphthalimide 29
    5-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo[i]- 28
    isoquinoline
    1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline 28
    6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide 28
    N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol 27
    Naphthalicacid-N-aminoimide 27
    2-[2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl]naphthalimide 26
    3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene 25
    1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline 24
    6-Nitro-2-(pyrid-3-methyl)naphthalimide 23
    3-Amino-7,4-bis(ethyl-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline 23
    2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline 23
    2-(2-Aminophenyl)naphthalimide 20
    1,3-Dioxo-2-[4,5-dimethyl-2-(4-methylphenylsulphonamido)phenyl]- 19
    1,2,3,4-tetrahydrobenzo[i]isoquinoline
    N-(4-Ethoxyphenyl)-5-nitronaphthalimide 18
    1,3-Dioxo-N-methyltetrahydrofurfur-2-yl-5-nitro-1,2,3,4-tetrahydro- 18
    [i]isoquinoline
    3-Methyl-3-(1,3-dioxo-5-nitro(1H,3H)benz[de]isoquinolyl)butyric acid 17
    methylester
    6-Nitro-2-(furfuryl)naphthalimide 17
    Ethyl-5-nitro-1,3-dioxo-1H-benz[de]isoquinoline-2-3H-acetate 16
    Naphthalicacid-N,N′-diimide 13
    2-(2-Hydroxyphenyl)naphthalimide 13
    5-Amino-N-butylnaphthalimide 13
    1,3-Dioxo-5-nitro-n-propylmorpholino-1,2,3,4-tetrahydrobenzo[i]- 11
    isoquinoline
    6-Nitro-2-(pyrid-2-ylethyl)naphthalimide 11
    N-Methylnaphthalimide 11
    N-(Pyrid-2-ylmethyl)naphthalimide 10
    N-(3,5-Dimethylphenyl)-1,8-naphthalimide 9
    6-Bromo-N-dimethylamino-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]- 5
    isoquinoline
    N-(1,3-Dioxo-6-phenylmercapto-1,2,3,4-tetrahydrobenzo[i]iso- 3
    quinoline)aminoethanol
    N-Anilino-1,8-naphthalimide 1
    • EXAMPLE 2 Inhibition of Neurite Outgrowth
  • The ability of N-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol (Compound A) to inhibit neurite outgrowth was determined using the following assay.
  • Eight-day chick embryo dorsal root ganglia (DRG) were freed of meninges and removed aseptically. The DRG were kept at 4° C. at all times. Ganglia from six embryos (40-50 per embryo) were washed in Ca2+- and Mg2+-free Gey's balanced salt solution (Gibco) and exposed to 0.01% trypsin (Worthington) in the same solution for 10 nm u at 37° C. A half-volume of phosphate-buffered Gey's balanced salt solution was added for a further 5 min at 37° C. and the reaction was then terminated with one-third volume of Ham's F12 medium (Gibco) containing 5% fetal calf serum (FCS, Gibco). The ganglia were then triturated using a 5 mL narrow-tip pipette to a single cell suspension. Following filtration through 37-mm nylon mesh (Small Parts Inc., Miami, Fla.) in a Millipore chamber to remove clumps, the cell suspension was washed through a 500-ml FCS undercut (700×g for 5 min at 4° C.) and resuspended in 4 mL of Ham's F12 medium plus 5% FCS. The cell suspension was then preplated on a 100-mm Flacon culture dish and incubated for 45-60 min at 37° C. in a 5% CO, humidified atmosphere. Cells enriched in neurons were decanted for the bioassay, since non-neuronal cells of DRG preferentially stick to the culture substrate.
  • The inside wells of 96-well Falcon microculture plates were coated with polylysine (0.1 mg/mL) (Sigma) for 4 h at 37° C. (the outside wells were filled with distilled water to provide humidity) and, following a rinse with tissue culture media, 100 mL of neuron-rich cell suspension was added to each well at 105 cells/mL. Ninety (90) mL of NGF solution (prepared in tissue culture media) was then added to each well to a final concentration of 0.25 ng/mL NGF per well. Ten (10) mL of test compound solution, i.e. tissue culture media admixed with a compound of Formula I, was then added to test wells in duplicate to yield wells containing compound concentrations ranging from 0 μM-100 μM. For control assays, 10 mL of Ham's F12 medium was added to duplicate NGF-containing wells. The plates were covered and incubated in the dark for 24-30 hrs. at 37° C. in a 5% CO2 humidified atmosphere.
  • The bioassays were read using a Leitz Diavert microscope with phase optics. To afford adequate optics, the meniscus effect of each well was removed by filling the well with a balanced salt solution until a flat, air-filled interface was achieved at the top of the well. At least 100 neurons per well were counted, and the assay was scored as the ratio of cells bearing neurites greater than one cell diameter to total viable (phase bright) cells. These results are summarized in Table 2.
  • TABLE 2
    Group Ratio Std Dev SEM
    NGF Control 1.000 0.16 0.06
     5 μM Compound A 1.1 0.42 0.15
     50 μM Compound A 0.16 0.15 0.05
    100 μM Compound A 0.016 0.02 0.01
    • EXAMPLE 3 Animal Models of Neuropathic Pain
  • For pain related to nerve injury, the compound N-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol (Compound A) was tested in nerve-ligated rats for activity against tactile allodynia, thermal hyperalgesia and in direct production of thermal antinociception. The nerve-ligation model is commonly accepted as representing aspects of neuropathic pain reported by humans. Sham operated rats served as appropriate controls for the neuropathic experiments.
    • Nerve Ligation Injury:
  • Nerve ligation injury was performed according to the method described by Kim and Chung (Pain 50: 355-363, 1992). Rats were anesthetized with halothane and the vertebrae over the L4 to S2 region were exposed. L5 and L6 spinal nerves were exposed, carefully isolated, tightly ligated with 4-0 silk suture distal to the DRG. After ensuring homeostatic stability, the wounds were sutured, and the animals were allowed to recover in the cages. Sham-operated rats were prepared in an identical fashion except that the L5/L6 nerve roots are not ligated.
    • Intrathecal Catheter Placement:
  • The test compounds were injected through indwelling i.th. catheters. While under anesthesia, PE-10 tubing (8 cm) was inserted through an incision made in the atlanto-occipitil membrane to the level of the lumbar enlargement of the rat and secured. Drug injections were made in a volume of 5 μl of 50% aqueous DMSO followed by a 9 μl saline flush.
    • Endpoints: (A) Evaluation of Tactile Allodynia:
  • Mechanical alloydynia was determined in the manner described by Chaplan et al. (J. Neurosci. Meth. 53:55-63, 1994). The paw withdrawal threshold was determined in response to probing with calibrated von Frey filaments. The rats were kept in suspended cages with mesh floors and the von Frey filaments were applied perpendicularly to the plantar surface of the paw of the rat until it buckled slightly, and held for 3-6 sec. A positive response was indicated by a sharp withdrawal of the paw. The 50% paw withdrawal threshold was determined by the non-parametric method of Dixon (Ann. rev. Pharmacol. Toxicol. 20: 441-462, 1980).
    • (B) Evaluation of Thermal Hyperalgesia:
  • Thermal hyperalgesia was determined by focusing a radiant heat source onto the plantar surface of the affected paw of nerve-injured or sham-operated rats. Paw withdrawal latencies are determined by a photodetection device which halts the stimulus and the timer after a maximum cut-off of 40 sec to prevent tissue damage. The withdrawal latency of sham-operated rats were compared to those of ligated rats to measure the degree of hyperalgesia.
    • (C) Evaluation of Acute Nociceptive Responses:
  • Acute nociception was determined by using the nociceptive warm water tail-flick reflex. This test was performed by placing the tail of the nerve-injured or sham-operated rats in a heated water bath maintained at 55° C. The latency until tail withdrawal (rapid flick) from the bath was determined and compared among treatments. A 15 second cut-off was employed to avoid tissue damage.
  • Compound A and morphine were tested in the nerve-ligated injury model of neuropathic pain using 3 routes of administration: intraopertoneally (i.p.), intra-thecally (i.th.) and intracerebroventrically (i.c.v.). The compounds were evaluated for three endpoints: tactile allodynia, thermal hyperalgesia and acute nociception. Compound A is not active when given i.c.v. The results for i.p and i.th. administration are shown in Table 3.
  • TABLE 3
    Summary of the A50 doses with 95% Confidence Limits
    (C.L.) for Compound A and Morphine in L5/L6 Nerve Ligated
    and Sham-Operated Rats in Models of Tactile
    Allodynia, Acute Nociception and Thermal Hyperalgesia
    Compound A Morphine
    Treatment A50 (95% C.L.) A50 (95% C.L.)
    Allodynia-Ligated i.th 34.6 μg >100 μg
    i.p. 38 mg/kg 7.1 mg/kg
    Tail Flick-Ligated i.th 78 μg 2.3 μg
    i.p. 27.8 mg/kg na*
    Tail Flick-Sham i.th 34 μg 1.45 μg
    i.p. 25.7 mg/kg na
    Hot Plate-Ligated i.th 14.8 μg na
    i.p. 18.6 mg/kg na
    Hot Plate-Sham i.th 48.1 μg na
    i.p. 34.9 mg/kg na
    *na = not available
    • EXAMPLE 4 Preparation OF N-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol (Compound A)
  • 3-Nitro-1,8-naphthalic anhydride (1 eq) and 2-hydroxyethylhydrazine (1 eq) are dissolved in toluene and heated to reflux. The reaction is monitored by tlc and halted when all of the starting materials are consumed. The solvent is removed under reduced pressure and the product purified, if necessary, by recrystallization or silica gel chromatography.
  • Other compounds of Formula I can be prepared in an analogous manner, or are available commercially from Ryan Scientific Inc., Isle of Palms, S.C., U.S.

Claims (13)

1. A pharmaceutical composition comprising a compound of Formula I,
Figure US20080287484A1-20081120-C00006
wherein
R1 is selected from alkyl; aryl-loweralkyl; heterocycle-loweralkyl; loweralkyl-carbonate; amino optionally monosubstituted or disubstituted with a substituent selected from loweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;
Figure US20080287484A1-20081120-C00007
wherein R4 is phenyl optionally monosubstituted or disubstituted with a substituent selected from loweralkyl and halo; phenyl optionally monosubstituted or disubstituted with a substituent selected from amino, loweralkoxy, hydroxy and loweralkyl;
NHCH2CH2OX wherein X represents an in vivo hydrolyzable ester, and loweralkyl-(R5)(R6) wherein one of R5 and R6 is selected from H and loweralkyl and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and
R2 and R3 are independently selected from H, NO2, halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR7 wherein Z is selected from C and S and R7 is selected from H, loweralkylamino and arylamino;
and pharmaceutically acceptable salts thereof, in an amount effective to inhibit neurotrophin-mediated activity, and a suitable carrier.
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
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