WO2010059241A2 - Traitement de la sclérose latérale amyotrophique - Google Patents

Traitement de la sclérose latérale amyotrophique Download PDF

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Publication number
WO2010059241A2
WO2010059241A2 PCT/US2009/006237 US2009006237W WO2010059241A2 WO 2010059241 A2 WO2010059241 A2 WO 2010059241A2 US 2009006237 W US2009006237 W US 2009006237W WO 2010059241 A2 WO2010059241 A2 WO 2010059241A2
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Prior art keywords
compound
formula
formulae
independently selected
optionally substituted
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PCT/US2009/006237
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English (en)
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WO2010059241A3 (fr
Inventor
Donald R. Kirsch
Radhia Benmohamed
Anthony C. Arvanites
Richard I. Morimoto
Tian Chen
Richard B. Silverman
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Cambria Pharmaceuticals, Inc.
Northwestern University
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Priority to AU2009318098A priority Critical patent/AU2009318098B2/en
Priority to CA2744016A priority patent/CA2744016C/fr
Priority to EP09827890.6A priority patent/EP2367798B1/fr
Priority to ES09827890.6T priority patent/ES2668556T3/es
Priority to US13/129,854 priority patent/US10167263B2/en
Application filed by Cambria Pharmaceuticals, Inc., Northwestern University filed Critical Cambria Pharmaceuticals, Inc.
Publication of WO2010059241A2 publication Critical patent/WO2010059241A2/fr
Publication of WO2010059241A3 publication Critical patent/WO2010059241A3/fr
Priority to IL212992A priority patent/IL212992A0/en
Priority to US13/354,156 priority patent/US9428467B2/en
Priority to US14/103,728 priority patent/US9145424B2/en
Priority to US14/867,624 priority patent/US9809556B2/en
Priority to US15/683,131 priority patent/US10526289B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/18—One oxygen or sulfur atom
    • C07D231/20—One oxygen atom attached in position 3 or 5
    • C07D231/22—One oxygen atom attached in position 3 or 5 with aryl radicals attached to ring nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/412—Detecting or monitoring sepsis
    • 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
    • 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
    • C07D401/12—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 chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • ALS Amyotrophic lateral sclerosis
  • motor neuron death a fatal neurodegenerative disorder caused by motor neuron death (Rowland et al, N. Engl. J. Med, 2001, 334, 1688-1700) and characterized in part by the presence of abnormal aggregates of insoluble protein in selectively vulnerable populations of neurons and glia.
  • ALS an orphan disease, is estimated to afflict about 87,000 people worldwide, but its prevalence would be much higher were it not for the fact that ALS patients survive for only 3 to 5 years on average after diagnosis. Approximately 10% of ALS cases are familial, with the rest of the cases being sporadic (Rowland et al, N. Engl. J. Med., 2001, 334, 1688-1700).
  • SODl Cu/Zn superoxide dismutase
  • Rodent models in mutant SODl are often used as a disease model because of its phenotypic and pathologic resemblance to sporadic and familial human ALS (Dal Canto et al, Brain Res., 1995, 676, 25-40; Wong, et al, Neuron, 1995, 14, 1105-1116; Bruijin et al, Science, 1998, 281, 1851-1854; Bruijn et al, Neuron, 1997, 18, 327-338; Wang et al, Hum. MoI Genet., 2003, 12, 2753-2764; Wang et al, Neurobiol Dis., 2002, 10, 128-138; Jonsson et al, Brain, 2004, 127, 73-88).
  • Riluzole which decreases glutamate excitotoxicity, is the only FDA approved ALS drug (Jimonet et al, J. Med. Chem., 1999, 42, 2828-2843.). However, it can only extend median survival life for 2 to 3 months, suggesting mechanisms other than glutamate-mediated excitotoxicity should be considered during ALS drug development (Miller et al, ALS and Other Motor Neuron Disorders, 2003, 4, 191-206; Taylor et al., Neurology, 2006, 67, 20-27).
  • Imbalances in protein homeostasis which can be caused by cell stress or expression of certain mutant proteins, can result in the appearance of alternative conformational states that are able to self-associate to form protein aggregates and inclusion bodies.
  • aberrant protein aggregation has been reported as a common feature (Bruijin et al., Science, 1998, 281, 1851-1854; Leigh et al, Cytoskeletalpathology in Motor Neuron Diseases, in: Rowland, L. P.
  • the present invention encompasses the recognition that there exists a need new compounds and methods for treating patients with amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases characterized by the presence of aberrant protein aggregates.
  • ALS amyotrophic lateral sclerosis
  • the present invention relates to the identification of provided compounds and pharmaceutical compositions thereof to treat neurodegenerative diseases.
  • the present invention provides methods of treating amyotrophic lateral sclerosis (ALS) with provided compounds.
  • ALS amyotrophic lateral sclerosis
  • provided compounds may be useful in the treatment of ALS or other neurodegenerative diseases where abnormal protein aggregation has been implicated, as they may prevent the aggregation of protein in a cell or limit the toxicity of such aggregates.
  • the invention provides compounds of the formula:
  • the invention provides methods for treating a subject with amyotrophic lateral sclerosis (ALS) or other neurodegenerative disease (e.g., Alzheimer's Disease, prion disease, or Huntington's Disease) by administering a therapeutically effective amount of an inventive compound, or a pharmaceutical composition thereof.
  • the compound is of one of the classes described herein or is a species described herein.
  • the invention provides methods for treating a subject with ALS or other neurodegenerative disease by administering both an inventive compound or pharmaceutical composition thereof, and a second therapeutic agent or pharmaceutical composition thereof.
  • the two compounds and/or compositions may be administered as a combination composition comprising both compounds. Alternatively, the two compounds may be administered separately (e.g., as two different compositions) either simultaneously or sequentially.
  • the present invention also provides methods of identifying compounds that protect against the cytotoxic effects of abnormal protein aggregation. Additionally, the present invention provides methods of identifying compounds that prevent, inhibit, or reverse protein aggregation.
  • the inventive assay involves the use of PC 12 cells expressing the protein SODl.
  • SODl is labeled with a detectable moiety (e.g., a fluorescent moiety). Assays may be high-throughput assays.
  • the present invention provides methods of inhibiting or reversing abnormal protein aggregation (e.g., SODl protein aggregates). Inhibiting or reversing abnormal protein aggregation may occur in vivo (e.g., in a subject as described herein) or in vitro (e.g., in a cell).
  • the invention provides methods of protecting cells from the cytotoxic effects of aggregated protein (e.g., SODl) using an inventive compound. Protection of cells may occur in vivo or in vitro. In some embodiments, protection occurs in vitro and the cells are PC 12 cells.
  • the invention provides methods of modulating proteasome activity in vivo or in vitro using a provided compound. In some embodiments, protection occurs in vitro, and the cells used are PC12 cells or HeLa cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are in an organism (e.g., a mammal).
  • the present invention contemplates provided compounds for use in medicine.
  • Aliphatic means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-20 aliphatic carbon atoms.
  • aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • cycloaliphatic refers to a monocyclic C 3 -C 6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • Alkyl refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • Alkylene refers to a bivalent alkyl group.
  • An "alkylene chain” is a polymethylene group, i.e., -(CH 2 ),,-, wherein n is a positive integer, preferably from 1 to 20, 1 to 12, 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • alkenyl refers to unsaturated aliphatic groups analogous in possible substitution to the alkyls described above, but that contain at least one double bond.
  • alkenylene refers to a bivalent alkenyl group.
  • a substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • Alkynyl refers to unsaturated aliphatic groups analogous in possible substitution to the alkyls described above, but that contain at least one triple bond.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal ⁇ e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically-engineered animal, and/or a clone.
  • the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). In some embodiments, use of the term “about” in reference to dosages means ⁇ 5 mg/kg/day.
  • Aryl The term “aryl” used alone or as part of a larger moiety as in “aralkyl,”
  • aryl refers to monocyclic and bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • Arylalkyl refers to an aryl substituted alkyl group, wherein the terms “aryl” and “alkyl” are defined herein, and wherein the aryl group is attached to the alkyl group, which in turn is attached to the parent molecule.
  • An exemplary arylalkyl group includes benzyl.
  • Characteristic portion As used herein, the phrase a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. Each such continuous stretch generally will contain at least two amino acids. Furthermore, those of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids are required to be characteristic of a protein. In general, a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact protein.
  • Characteristic sequence is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.
  • an "effective amount” is an amount that achieves, or is expected to achieve, a desired result, and can be administered in one dose or in multiple doses.
  • Compositions may be considered to contain an effective amount if they include a dose that is effective in the context of a dosing regimen, even if the composition as a single dose is not expected to be effective.
  • Heteroaryl used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy,” refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein one or more ring in the system is aromatic, one or more ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
  • heteroaryl and “heteroar-,” as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4 ⁇ )-one.
  • heteroaryl group may be mono- or bicyclic.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.
  • heteroarylkyl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • Heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl)).
  • Heterocycle As used herein, the terms “heterocycle,” “heterocyclyl,”
  • heterocyclic radical and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10— membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • the nitrogen may be N (as in 3,4— dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or 4 NR (as in iV-substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • Intraperitoneal The phrases "intraperitoneal administration" and
  • administered intraperitonealy as used herein have their art-understood meaning referring to administration of a compound or composition into the peritoneum of a subject.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism ⁇ e.g., animal, plant, and/or microbe).
  • m vivo refers to events that occur within an organism (e.g., animal, plant, and/or microbe).
  • Lower alkyl refers to a C 1-4 straight or branched alkyl group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.
  • Lower haloalkyl refers to a C 1-6 straight or branched alkyl group that is substituted with one or more halogen atoms.
  • substituents of the invention may contain “optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • suitable monovalent substituents on R° are independently halogen, -(CH 2 )o- 2 R*, -(haloR * ), - ⁇ CH 2 )o_ 2 OH, -(CH 2 )( ⁇ 2 OR*, - (CH 2 )o- 2 CH(OR*) 2 ; -O(haloR*), -CN, -N 3 , - ⁇ CH 2 )o- 2 C(0)R*, -(CH 2 ) ⁇ 2 C(O)OH, -(CH 2 )o_ 2 C(O)OR*, -(CH 2 )o- 2 SR # , -(CH 2 )o- 2 SH, -(CH 2 )o- 2 NH 2 , -(CH 2 ) ⁇ 2 NHR*, -(CH 2 )o_ 2 NR* 2 , - NO
  • suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: - O(CR 2 ) 2 - 3 O-, wherein each independent occurrence of R * is selected from hydrogen, aliphatic which may be substituted as defined below, or an unsubstituted 5— 6— membered saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R * substituents on the aliphatic group of R * include halogen, -R*, -(haloR * ), -OH, -OR*, -O(haloR'), -CN, -C(O)OH, -C(O)OR*, - NH 2 , -NHR*, -NR* 2 , or -NO 2 , wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently C 1 ⁇ aliphatic, - CH 2 Ph, -0(CH 2 )o_iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • substituents on a substitutable nitrogen of an "optionally substituted" group include -C(O)R 1 , -C(O)OR 1 , -C(O)C(O)R f , - C(O)CH 2 C(O)R 1 , -S(O) 2 R ⁇ -S(O) 2 NR ⁇ , -C(S)NR ⁇ , -C(NH)NR ⁇ , or -N(R ⁇ S(O) 2 R*; wherein each R* is independently hydrogen, Ci_ 6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated,
  • suitable substituents on the aliphatic group of R ⁇ are independently halogen, -R*, -(haloR'), -OH, -OR', -O(haloR'), -CN, -C(O)OH, - C(O)OR*, -NH 2 , -NHR", -NR # 2 , or -NO 2 , wherein each R # is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci_ 4 aliphatic, -CH 2 Ph, -0(CH 2 )o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • oral administration and “administered orally” as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.
  • Parenteral administration and “administered parenterally” as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.
  • Partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • patient refers to any organism to which a provided compound is administered in accordance with the present invention e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects include animals ⁇ e.g., mammals such as mice, rats, rabbits, non- human primates, and humans; insects; worms; etc.).
  • a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition (e.g., a neurodegenerative disease, a disease, disorder or condition associated with protein aggregation, ALS, etc.).
  • a disease, disorder, and/or condition e.g., a neurodegenerative disease, a disease, disorder or condition associated with protein aggregation, ALS, etc.
  • compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Prodrug A general, a "prodrug,” as that term is used herein and as is understood in the art, is an entity that, when administered to an organism, is metabolized in the body to deliver a therapeutic agent of interest.
  • Various forms of "prodrugs” are known in the art. For examples of such prodrug derivatives, see: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder, et al.
  • Protecting group The term "protecting group,” as used herein, is well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
  • Suitable amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-d ⁇ -t- buty l-[9-( 10,10-dioxo- 10,10,10,10-tetrahydrothioxanthy l)]methy 1 carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2— trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1 -(I -adamanty I)-I- methylethyl carbamate (Adpoc), l,l-dimethyl-2-hal
  • suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids.
  • suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, triisopropylsilyl, and the like.
  • suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4— dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2- yl.
  • suitable alkenyl groups include allyl.
  • suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl.
  • suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4- dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p- cyanobenzyl), and 2- and 4—picolyl.
  • suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4— methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), r-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2— trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (
  • the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4— methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1— methoxy ethylidene ortho
  • Protein refers to a polypeptide ⁇ i.e., a string of at least two amino acids linked to one another by peptide bonds).
  • proteins include only naturally-occurring amino acids.
  • proteins include one or more non-naturally-occurring amino acids ⁇ e.g., moieties that form one or more peptide bonds with adjacent amino acids).
  • one or more residues in a protein chain contains a non-amino-acid moiety ⁇ e.g., a glycan, etc).
  • a protein includes more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • proteins contain L-amino acids, D-amino acids, or both; in some embodiments, proteins contain one or more amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • the term "peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • each R 1 is independently -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -CN, -NO 2 - NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, - N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, -C(O)N(R) 2 , -OC(O)N(R) 2 , or an optionally substituted 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms
  • X is N or C; each R 5 is independently -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -CN, - NO 2 -NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, - C(O)N(R) 2 , -OC(O)N(R) 2 , or a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or ary
  • R 0 is R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, N(R) 2 , -NRC(O)R, -NRC(O)(CO)R, - NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -
  • L is a valence bond or a bivalent saturated or partially unsaturated Cj.io hydrocarbon chain, wherein 1-4 methylene units of L are optionally and independently replaced by -0-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -S(O)-, -S(O) 2 -, -OSO 2 O-, - N(R)C(O)-, -C(O)NR-, -N(R)C(O)O-, -OC(O)NR-, -N(R)C(O)NR-, -N(R 6 )-, or -Cy-, and where
  • R 6 is -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -C(O)R, -C(O)OR, -OC(O)R, - OC(O)OR, -C(O)N(R) 2 , -OC(O)N(R) 2 , a suitable amino protecting group, or an optionally substituted 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur;
  • Ring A is a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein Ring A is optionally substituted with m occurrences of R 4 ; m is 0-5; and each R 4 is independently -R, -OR, -SR, -CN, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -NO 2 , - NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C
  • the term "provided compound” may encompass prodrugs and/or esters of compounds of formula I. As discussed herein, provided compounds may be provided in salt form. In particular, in some embodiments, a provided compound is provided as a pharmaceutically acceptable salt of a compound of formula I.
  • Provided compounds may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and trans- isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. AU such isomers, as well as mixtures thereof, are intended to be included in this invention. That is, in some embodiments, the present invention provides isolated single isomers. In some embodiments, the present invention provides mixtures of two or more isomers. In certain embodiments, the present invention relates to a provided compound represented by any of the structures outlined herein, wherein the compound is provided as a single stereoisomer.
  • acid or base addition salt also comprise the hydrates and the solvent addition forms which the compounds are able to form. Examples of such forms are, e.g., hydrates, alcoholates and the like.
  • Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof, wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound.
  • the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants, which are in themselves known, but are not mentioned here.
  • stereochemically isomeric forms refers to different compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which are not interchangeable.
  • chemical compositions may be provided as pure preparations of individual stereochemically isomeric forms of a compound; in some embodiments, chemical compositions may be provided that are or include mixtures of two or more stereochemically isomeric forms of the compound. In certain embodiments, such mixtures contain equal amounts of different stereochemically isomeric forms; in certain embodiments, such mixtures contain different amounts of at least two different stereochemically isomeric forms.
  • a chemical composition may contain all diastereomers and/or enantiomers of the compound. In some embodiments, a chemical composition may contain less than all diastereomers and/or enantiomers of a compound.
  • the present invention encompasses all stereochemically isomeric forms of relevant compounds, whether in pure form or in admixture with one another. If a particular enantiomer of a compound of the present invention is desired, it may be prepared, for example, by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, diastereomeric salts are formed with an appropriate optically-active acid, and resolved, for example, by fractional crystallization.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and/or chemical phenomena.
  • Susceptible to An individual who is "susceptible to" a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • Tautomeric forms The phrase "tautomeric forms," as used herein, is used to describe different isomeric forms of organic compounds that are capable of facile interconversion. Tautomers may be characterized by the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond. In some embodiments, tautomers may result from prototropic tautomerism ⁇ i.e., the relocation of a proton). In some embodiments, tautomers may result from valence tautomerism (i.e., the rapid reorganization of bonding electrons). All such tautomeric forms are intended to be included within the scope of the present invention.
  • tautomeric forms of a compound exist in mobile equilibrium with each other, so that attempts to prepare the separate substances results in the formation of a mixture.
  • tautomeric forms of a compound are separable and isolatable compounds.
  • chemical compositions may be provided that are or include pure preparations of a single tautomeric form of a compound.
  • chemical compositions may be provided as mixtures of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain embodiments, such mixtures contain different amounts of at least two different tautomeric forms of a compound.
  • chemical compositions may contain all tautomeric forms of a compound. In some embodiments of the invention, chemical compositions may contain less than all tautomeric forms of a compound. In some embodiments of the invention, chemical compositions may contain one or more tautomeric forms of a compound in amounts that vary over time as a result of interconversion. Unless otherwise indicated, the present invention encompasses all tautomeric forms of relevant compounds, whether in pure form or in admixture with one another.
  • Therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • therapeutically effective amount means an amount of a substance ⁇ e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • a composition may be considered to include a therapeutically effective amount of a provided compound when it includes an amount that is effective when administered as part of a dosing regimen even if a single dose (i.e., only the amount in the composition) alone is not expected to be effective.
  • Treat refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition; in some embodiments, treatment prevents one or more symptoms of features of the disease, disorder, or condition.
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • Systemic The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” as used herein have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient's system.
  • Unsaturated means that a moiety has one or more units of unsaturation.
  • FIG. 1 Proteasome inhibition is selectively toxic to PC 12 cells expressing mutant G93A SODl. Cell survival was determined using the viability stain calcein-AM. At 24 hours, treatment with 100 nM MG 132 shows no toxicity against any of the cell lines although large numbers of aggregates are seen in the G85R SODl cell lines (panel A). Treatment with 100 nM MG 132 is selectively toxic to the G93A SODl cell line after 48 hours (panel B). Washing of the cells at 24 hours to remove the compound does not reverse toxicity to the G93A SODl cell line suggesting that an irreversible toxic event, potentially related the aggregation of G93A SODl, has been triggered prior to wash out (panel C).
  • Figure 2 Radicicol protects PC12 cells expressing mutant G93A SODl from the toxic effects of proteasome inhibitor MG 132.
  • FIG. 3 Mutant but not wild type SODl aggregates in cells treated with the proteasome inhibitor MG 132. Fluorescence micrographs of PC 12 cells expressing YFP tagged wild type (WT), G93A mutant (G93A), and G85R mutant (G85R) SODl proteins. The micrographs show the effects of treating cells with 200 nM MG132 for 24 hours (untreated cells are shown in insets on left). The wild type SODl expressing cells are unaffected while cells expressing mutant SODl show large perinuclear aggregates.
  • FIG. 4 Radicicol decreases mutant SODl aggregation induced by proteasome inhibitor MGl 32. Fluorescence micrographs of PC 12 cells expressing YFP tagged G93A SODl (left) or G85R SODl (right) proteins. The cells were untreated, treated with 200 nM MG132 to induce protein aggregation, or co-treated with MG132 and radicicol for 24 hours. Without radicicol treatment, cells show large perinuclear aggregates. The aggregates are reduced in radicicol-treated cells. While the behavior of the two cell lines is generally similar, G85R SODl cells show 'brighter' aggregates and more contrast between the aggregates and the cytoplasm.
  • FIG. 5 Automated detection of mutant SODl aggregates. Left: compound microscope fluorescence micrographs of the same G85R SODl cells using GFP filter set to image the SODl-YFP aggregates (top) and the TRITC filter set to image iT-WGA plasma membrane (bottom). Right: Aggregate detection from the Cellomics Arrayscan 3.5 using spot detector software to image cells with Image-iT in channel 1 and SODl-YFP aggregates in channel 2. Data are expressed as spot count (aggregates) per object (cell).
  • HeLa hse-luc cells were treated with arylsulfanyl pyrazolones (l ⁇ M- lOO ⁇ M), celestrol (l ⁇ M-5 ⁇ M), or CdC12 (lO ⁇ M-lOO ⁇ M), for 8 h and activation of the heat shock response determined by HSP70 promoter activity.
  • Figure 7 Arylsulfanyl pyrazolones prevent MG132-induced accumulation of
  • Ubi-YFP HeLa cells were co-transfected with Ubi-YFP and CMV-CFP plasmids, treated with 1 ⁇ M MGl 32 and 25 ⁇ M arylsulfanyl pyrazolones and Ubi-YFP fluorescence intensity normalized to CFP.
  • Figure 10 Plasma standard curve for CMB-087229.
  • Figure 14 Stuctures of TC-I-165 metabolite candidates (sulfoxide metabolite candidate TC-II-68 and sulfone metabolite candidate TC-II-70).
  • Figure 16 HPLC trace of TC-I-165 [5 min NADPH (tube 2)] and a new peak with a retention time of 5.900 minutes.
  • Figure 17 HPLC trace of TC-I- 165 [60 min NADPH (tube 1 )] and a new peak with a retention time of 5.917 minutes.
  • Figure 18 HPLC trace of TC-II-68 [10 ⁇ M standard solution (tube I)] and a new peak with a retention time of 5.933 minutes.
  • Figure 19 HPLC trace of TC-II-70 [10 ⁇ M standard solution (tube 2)] and a new peak with a retention time of 11.000 minutes.
  • Figure 20 Structures of compounds used in the rat liver microsomal stability study of TC-II-70.
  • Figure 21 NADPH dependent rat liver microsomal stability study of TC-II-
  • Figure 25 Preliminary SAR of selected arylsulfanylpyrazolones.
  • Imbalances in protein homeostasis are often associated with protein misfolding and/or protein conformational changes that lead to protein aggregation and formation of protein inclusion bodies.
  • Many neurodegenerative diseases including the polyglutamine (polyQ)-repeat diseases, Alzheimer's disease, Parkinson's disease, prion diseases, and ALS, are characterized by the appearance of damaged and aggregated proteins, including huntingtin, polyQ proteins, amyloid A prion (PrP and Sup35) fibrils, and mutant SODl (Taylor et al, Science 2002, 296(5575), 1991-1995; Ross, C.A., Neuron. 1997, 19(6), 1147-1150; Perutz, M.F., Brain Res. Bull.
  • Affected proteins could include ubiquitin, components of the proteasome, components of the cytoskeleton, transcription factors (TBP (i.e., TATA binding protein), EYA (i.e., Eyes Absent protein), CBP (i.e., CREB binding protein), and molecular chaperones Hsc-70, Hsp-70, Hdj-1, and Hdj-2 (Davies et al, Cell 1997, 90(3), 537-548; Ross, C.A., Neuron.
  • TBP i.e., TATA binding protein
  • EYA i.e., Eyes Absent protein
  • CBP i.e., CREB binding protein
  • Hsc-70, Hsp-70, Hdj-1, and Hdj-2 Daavies et al, Cell 1997, 90(3), 537-548; Ross, C.A., Neuron.
  • Molecular chaperones are a large and diverse protein family which includes Hspl04, Hsp90, Hsp70, dnaJ (Hsp40), immunophilins (Cyp40, FKBP), Hsp60 (chaperonins), the small heat shock proteins, and components of the steroid aporeceptor complex (p23, Hip, Hop, Bagl) (Gething, M.J., Nature 1997, 388(6640) 329- 331; Bakau, B., Amsterdam: Harwood Academic Publishers. 1999, 690).
  • molecular chaperones can suppress the toxicity of mutant huntingtin, ⁇ -synuclein, and SODl (Sakahira et al., Proc. Natl. Acad. ScL U S A. 2002, 99 Suppl 4, 6412-6418; Stenoien et al., Hum. MoI. Genet. 1999, 8(5), 731-741; Warrick et al., Nat. Genet. 1999, 23(4), 425-428; Carmichael et al, Proc. Natl. Acad. ScL U S A. 2000, 97(17), 9701-9705; Takeuchi et al, Brain Res.
  • the chaperone system is a highly appealing therapeutic target, because multiple small molecular weight modulators of chaperone activity have already been identified, two of which are active in a mouse model of ALS (Westerheide et al, J. Biol. Chem. 2005, 280(39), 33097-33100; Kieran et al, Nat. Med. 2004, 10(4), 402-405; and Traynor et al, Neurology 2006, 67(1), 20-27). Accordingly, recent analyses identified protein folding/misfolding and protein aggregation as a relevant therapeutic target for neurodegenerative diseases (Pasinelli et al, Nat. Rev. Neurosci. 2006, 7(9), 710-723; Lansbury et al, Nature 2006, 443(7113), 774-9; Rubinsztein et al, Nature 2006, 443(7113), 780-786).
  • the present invention provides compounds and methods for treating patients with amyotrophic lateral sclerosis (ALS) or other neurodegenerative diseases characterized by the presence of aberrant protein aggregates.
  • ALS amyotrophic lateral sclerosis
  • compounds and methods of the invention are useful in inhibiting or reversing abnormal protein aggregation or reducing the toxicity of protein aggregation (e.g., SODl).
  • provided compounds are useful in modulating proteosome function.
  • the invention provides methods for treating a subject with ALS or other neurodegenerative disease including the step of administering to the subject a therapeutically effective amount of an inventive compound or a pharmaceutical composition thereof.
  • the subject is a mammal.
  • the present invention provides compounds of the formula:
  • each R 1 is independently -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -CN, -NO 2 - NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, - N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, -C(O)N(R) 2 , -OC(O)N(R) 2 , or an optionally substituted 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently
  • X is N or C; each R 5 is independently -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -CN, - NO 2 -NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, - C(O)N(R) 2 , -OC(O)N(R) 2 , or a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or ary
  • R 0 is R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, N(R) 2 , -NRC(O)R, -NRC(O)(CO)R, - NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -
  • L is a valence bond or a bivalent saturated or partially unsaturated C] -I0 hydrocarbon chain, wherein 1-4 methylene units of L are optionally and independently replaced by -0-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -S(O)-, -S(O) 2 -, -OSO 2 O-, - N(R)C(O)-, -C(O)NR-, -N(R)C(O)O-, -OC(O)NR-, -N(R)C(O)NR-, -N(R 6 )-, or -Cy-, and wherein L is
  • Ring A is a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein Ring A is optionally substituted with m occurrences of R 4 ; m is 0-5; and each R 4 is independently -R, -OR, -SR, -CN, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -NO 2 , - NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C
  • n is O. In certain embodiments, n is 1.
  • p is 1. In certain embodiments, p is 2.
  • One such exemplary pair of tautomers is as shown below:
  • each R 1 is independently -R, -OR, -SR, -S(O)R, -
  • each R , 1 1 is independently -R, -OR, -SR, -S(O)R, -SO 2 R,
  • -OSO 2 R N(R) 2 , -NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, - N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, -OC(O)OR, -C(O)N(R) 2 , or - OC(O)N(R) 2 .
  • At least one R 1 is optionally substituted 3-8 membered saturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 3-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 5-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • At least one R 1 is an optionally substituted 3-8 membered partially unsaturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 3-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • At least one R 1 is an optionally substituted 5-6 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 5-6 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R is an optionally substituted 5 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 6 membered aryl ring having 1-3 nitrogens. In some embodiments, at least one R 1 is an optionally substituted phenyl. In certain embodiments, at least one R 1 is unsubstituted phenyl.
  • At least one R 1 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 8-10 membered saturated bicyclic carbocycle.
  • At least one R 1 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 8-10 membered partially unsaturated carbocycle. [0115] In some embodiments, at least one R 1 is an optionally substituted 9-10 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • At least one R 1 is an optionally substituted 9-10 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 9 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is an optionally substituted 10 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 1 is optionally substituted naphthyl.
  • two R 1 are taken together to form " ⁇ , wherein
  • X is N or C. In certain embodiments wherein X is N, one R 5 group is present. In certain embodiments wherein X is C, either one or two R 5 groups may be present. In certain embodiments, two R are taken together to form any one of the following formulae:
  • each R 5 is independently -R, -OR, -SR, -S(O)R, -SO 2 R,
  • At least one R 5 is a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein R 5 is
  • R 2 is independently -R, -OR, -SR, -
  • R 2 is -R, -OR, -SR, -N(R) 2 , or a suitable amino protecting group
  • R 2 is halogen or R.
  • R 2 is hydrogen, methyl, ethyl, propyl, or butyl.
  • R 2 is hydrogen.
  • R 2 is methyl.
  • R 2 is an optionally substituted 3-8 membered saturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 3-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 5-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R is an optionally substituted 3-8 membered partially unsaturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 2 is an optionally substituted 3-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 2 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 2 is an optionally substituted 5-6 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 5-6 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 5 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 6 membered aryl ring having 1-3 nitrogens. In some embodiments, R is an optionally substituted phenyl. In certain embodiments, R 2 is unsubstituted phenyl.
  • R 2 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 8-10 membered saturated bicyclic carbocycle.
  • R 2 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 8-10 membered partially unsaturated carbocycle.
  • R is an optionally substituted 9-10 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 9-10 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 9 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 2 is an optionally substituted 10 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is optionally substituted naphthyl.
  • R 2 heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazblyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxaliny
  • R 3 is independently -R, -OR, -SR, -
  • R 3 is-R, -OR, -SR, -N(R) 2 , or a suitable amino protecting group
  • R 3 is halogen or R.
  • R 3 is hydrogen, methyl, ethyl, propyl, or butyl.
  • R 3 is hydrogen.
  • R 3 is methyl.
  • R 3 is an optionally substituted 3-8 membered saturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 3-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 5-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 3 is an optionally substituted 3-8 membered partially unsaturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 3-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 3 is an optionally substituted 5-6 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 5-6 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 5 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 6 membered aryl ring having 1-3 nitrogens. In some embodiments, R 3 is an optionally substituted phenyl. In certain embodiments, R 3 is unsubstituted phenyl.
  • R 3 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 8-10 membered saturated bicyclic carbocycle.
  • R 3 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 8-10 membered partially unsaturated carbocycle.
  • R 3 is an optionally substituted 9-10 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 9-10 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is an optionally substituted 9 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 10 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 3 is optionally substituted naphthyl.
  • R 3 heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,
  • R 3 is of any one of the following formulae:
  • R 0 is -R -OR, -SR, -S(O)R, -SO 2 R, -
  • R 0 is R, wherein R is hydrogen, optionally substituted
  • Ci- 6 aliphatic optionally substituted Ci -6 heteroaliphatic, or optionally substituted phenyl.
  • R 0 is methyl, ethyl, propyl, or butyl.
  • R 0 is m v_ y 5 wherein R 4 , m, and Ring A are
  • R 0 is ' m v_y ? wherein R 4 , m, and Ring A are as defined above and L is not a valence bond.
  • L is a valence bond or a bivalent saturated or partially unsaturated CM O hydrocarbon chain, wherein 1-4 methylene units of L are optionally and independently replaced by -0-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, - S(O)-, -S(O) 2 -, -OSO 2 O-, -N(R)C(O)-, -C(O)NR-, -N(R)C(O)O-, -OC(O)NR-, - N(R)C(O)NR-, -N(R 6 )-, or -Cy-, and wherein L is optionally substituted with 1-4 R groups; wherein: each -Cy- is independently a bivalent optionally substituted saturated, partially unsaturated, or aromatic monocyclic or bicyclic ring selected from a 6-10 membered
  • R 6 is -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -C(O)R, -C(O)OR, -OC(O)R, - OC(O)OR, -C(O)N(R) 2 , -OC(O)N(R) 2 , a suitable amino protecting group, or an optionally substituted 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an optionally substituted 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • L a bivalent saturated or partially unsaturated Cj.io hydrocarbon chain, wherein 1-4 methylene units of L are optionally and independently replaced by -0-, -N(R 6 )-, -S-, -C(O)-, -OC(O)-, -C(O)O-, -OC(O)O-, -S(O)-, -S(O) 2 -, - OSO 2 O-, -N(R)C(O)-, -C(O)NR-, -N(R)C(O)O-, -OC(O)NR-, -N(R)C(O)NR-, and wherein L is optionally substituted with 1-4 R groups.
  • L a bivalent saturated or partially unsaturated C 2-7 hydrocarbon chain, wherein 1-3 methylene units of L are optionally and independently replaced by -N(R 6 )-, -N(R)C(O)-, -C(O)N(R)-, -0-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -S(O)- or -SO 2 -, and wherein L is optionally substituted with 1-4 R groups.
  • L is a bivalent saturated C 2-7 hydrocarbon chain, wherein one or more methylene units of L is replaced by -N(R 6 )-, -N(R)C(O)-, -C(O)N(R)-.
  • L is a bivalent saturated C 2-7 hydrocarbon chain, wherein one or more methylene units of L is replaced by -0-, -C(O)-, -OC(O)-, or -C(O)O-.
  • L is a bivalent saturated C 2-7 hydrocarbon chain, wherein one or more methylene units of L is replaced by -S-, -S(O)- or -SO 2 -.
  • L is of any of the following formulae: [0143] In certain embodiments, L is of any of the following formulae:
  • L is of any of the following formulae:
  • L is a bivalent saturated Ci -10 hydrocarbon chain, wherein one or more methylene unit is independently replaced by -Cy-, and wherein one or more -Cy- is independently a bivalent optionally substituted saturated monocyclic ring. In some embodiments, one or more -Cy- is independently a bivalent optionally substituted partially unsaturated monocyclic ring. In some embodiments, one or more -Cy- is independently a bivalent optionally substituted aromatic monocyclic ring. In certain embodiments, -Cy- is optionally substituted phenylene.
  • one or more -Cy- is independently a bivalent optionally substituted saturated bicyclic ring. In some embodiments, one or more -Cy- is independently a bivalent optionally substituted partially unsaturated bicyclic ring. In some embodiments, one or more -Cy- is independently a bivalent optionally substituted aromatic bicyclic ring. In certain embodiments, -Cy- is optionally substituted naphthylene.
  • one or more -Cy- is independently an optionally substituted 6-10 membered arylene. In some embodiments, one or more -Cy- is independently an optionally substituted a 5-10 membered heteroarylene having 1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted a 5-6 membered heteroarylene having 1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 5 membered heteroarylene having 1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 6 membered heteroarylene having 1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur.
  • Exemplary optionally substituted -Cy- heteroarylene groups include thienylene, furanylene, pyrrolylene, imidazolylene, pyrazolylene, triazolylene, tetrazolylene, oxazolylene, isoxazolylene, oxadiazolylene, thiazolylene, isothiazolylene, thiadiazolylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, indolizinylene, purinylene, naphthyridinylene, pteridinylene, indolylene, isoindolylene, benzothienylene, benzofuranylene, dibenzofuranylene, indazolylene, benzimidazolylene, benzthiazolylene, quinolylene, isoquinolylene, cinnolinylene, phthalazinylene, quinazolinylene, quinoxalin
  • -Cy- is selected from the group consisting of tetrahydropyranylene, tetrahydrofuranylene, morpholinylene, thiomorpholinylene, piperidinylene, piperazinylene, pyrrolidinylene, tetrahydrothiophenylene, and tetrahydrothiopyranylene, wherein each ring is optionally substituted.
  • one or more -Cy- is independently an optionally substituted 3-8 membered carbocyclylene. In some embodiments, one or more -Cy- is independently an optionally substituted 3-6 membered carbocyclylene. In some embodiments, one or more -Cy- is independently an optionally substituted cyclopropylene, cyclopentylene, or cyclohexylene.
  • one or more -Cy- is independently an optionally substituted 3-10 membered heterocyclylene having 1-4 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 5-7 membered heterocyclylene having 1-3 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 3 membered heterocyclylene having 1 heteroatom independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 5 membered heterocyclylene having 1-2 heteroatoms independently selected from oxygen, nitrogen, or sulfur. In some embodiments, one or more -Cy- is independently an optionally substituted 6 membered heterocyclylene having 1-3 heteroatoms independently selected from oxygen, nitrogen, or sulfur.
  • Exemplary -Cy- saturated 3-8 membered optionally substituted heterocyclenes include oxiranylene, oxetanylene, tetrahydrofuranylene, tetrahydropyranylene, oxepaneylene, aziridineylene, azetidineylene, pyrrolidinylene, piperidinylene, azepanylene, thiiranylene, thietanylene, tetrahydrothiophenylene, tetrahydrothiopyranylene, thiepanylene, dioxolanylene, oxathiolanylene, oxazolidinylene, imidazolidinylene, thiazolidinylene, dithiolanylene, dioxanylene, mo ⁇ holinylene, oxathianylene, piperazinylene, thiomorpholinylene, dithianylene, dioxepanylene, oxazepanylene,
  • R 6 is -R, -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -C(O)R, -
  • R 6 is a suitable amino protecting group.
  • R 6 is -SO 2 R.
  • R 6 is R.
  • R 6 is optionally substituted arylalkyl.
  • R 6 is an optionally substituted 3-8 membered saturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 3-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 5-6 membered saturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is an optionally substituted 3-8 membered partially unsaturated monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 3-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0156] In some embodiments, R 6 is an optionally substituted 5-6 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is an optionally substituted 5-6 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 5 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 6 membered aryl ring having 1-3 nitrogens. In some embodiments, R 6 is an optionally substituted phenyl. In certain embodiments, R 6 is unsubstituted phenyl.
  • R 6 is an optionally substituted 8-10 membered saturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 8-10 membered saturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R is an optionally substituted 8-10 membered saturated bicyclic carbocycle.
  • R 6 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 8-10 membered partially unsaturated bicyclic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R 6 is an optionally substituted 8-10 membered partially unsaturated carbocycle.
  • R is an optionally substituted 9-10 membered aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is an optionally substituted 9-10 membered aryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is an optionally substituted 9 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is an optionally substituted 10 membered aryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 6 is optionally substituted naphthyl.
  • R 6 heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,
  • L is a bivalent saturated C 2-7 hydrocarbon chain wherein two methylene units are replaced by -N(R 6 )-, -N(R)C(O)-, -C(O)N(R)-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -S(O)- or -SO 2 -.
  • L is a bivalent saturated C 2- 7 hydrocarbon chain wherein two methylene units are replaced by -0-, -S-, -S(O)-, or -SO 2 -.
  • L is a bivalent saturated C 2 hydrocarbon chain wherein at least one methylene unit is replaced by -N(R 6 )-, -N(R)C(O)-, -C(O)N(R)-, -0-, -C(O)-, -OC(O)-, -C(O)O-, -S-, -S(O)- or -SO 2 -.
  • L is a bivalent saturated C 2 hydrocarbon chain wherein at least one methylene unit is replaced by -N(R 6 )-, -O-, -S-, - S(O)-, Or-SO 2 -.
  • L is of any of the following formulae:
  • Ring A is a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein Ring A is optionally substituted with m occurrences of R 4 , wherein m is 0-5. In some embodiments, m is O, 1, 2, or 3.
  • Ring A is phenyl substituted with 1-5 R 4 groups. In certain embodiments, Ring A is unsubstituted phenyl.
  • Ring A is naphthyl substituted with 1-5 R 4 groups. In certain embodiments, Ring A is unsubstituted naphthyl.
  • Ring A is a 5-6 membered monocyclic saturated, partially unsaturated or aromatic heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-5 R 4 groups.
  • Ring A is a 5 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-2 R 4 groups.
  • Ring A is a 6 membered monocyclic heteroaryl ring having 1-2 nitrogens independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-2 R 4 groups.
  • Ring A is an 8-10 membered bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-5 R 4 groups. In some embodiments, Ring A is an 8 membered bicyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-3 R 4 groups. In some embodiments, Ring A is a 9 membered bicyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-3 R 4 groups. In some embodiments, Ring A is a 10 membered bicyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and optionally substituted with 1-3 R 4 groups. In some embodiments, Ring A is an 8-10 membered bicyclic ring comprised of 0-2 aromatic rings and optionally substituted with 1-5 R 4 groups.
  • Exemplary Ring A heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quino
  • each R 4 is independently -R, -OR, -
  • R 4 is independently -OR, -SR, -S(O)R, -SO 2 R, -OSO 2 R, -N(R) 2 , -CN, -NO 2 , -NRC(O)R, -NRC(O)(CO)R, - NRC(O)N(R) 2 , -NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -C(O)OR, - OC(O)R, -OC(O)OR, -C(O)N(R) 2 , -OC(O)N(R) 2 , or a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • at least one R 4 is independently -OR, -SR, -S(O)
  • m is 1 and R 4 is -OMe. In certain embodiments, m is 1 and R 4 is -NO 2 . In certain embodiments, m is 1 and R 4 is -NR 2 . In certain embodiments, m is 1 and R 4 is - NMe 2 .
  • At least one R 4 is independently a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring optionally containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • At least one R 4 is independently a 3-6 membered saturated monocyclic ring optionally containing 0-1 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, at least one R 4 is independently a 3 membered saturated monocyclic ring optionally containing 0-1 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, at least one R 4 is cyclopropyl. In certain embodiments, two R 4 are cyclopropyl.
  • At least one R 4 is optionally substituted phenyl. In certain embodiments, at least one R 4 is unsubstituted phenyl. In some embodiments, at least one R 4 is independently a 5-6 membered aryl monocyclic ring containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • R 4 is of any of the following formulae:
  • each R 4 is R, wherein R is hydrogen, halogen, or optionally substituted C 1-20 aliphatic. In certain embodiments, each R 4 is independently Ci -6 aliphatic. In certain embodiments, each R 4 is independently selected from the group consisting of methyl, ethyl, propyl, or butyl. In certain embodiments, m is 1 and R 4 is methyl, ethyl, propyl, or butyl. In certain embodiments, m is 1 and R 4 is C H aliphatic. In some embodiments, m is 2 and each R 4 is methyl. In some embodiments, m is 2 and each R 4 is -CF 3 .
  • each R 4 is independently selected from the group consisting of fluorine, chlorine, bromine, and iodine.
  • m is 1 and R 4 is fluorine.
  • m is 1 and R is chlorine.
  • m is 1 and R 4 is bromine.
  • m is 2 and each R 4 is fluorine.
  • m is 2 and each R 4 is chlorine.
  • m is 2 and each R 4 is bromine.
  • m is 2, wherein one R 4 is fluorine and one R 4 is chlorine.
  • m is 3 and each R 4 is independently methyl or chlorine.
  • Ring A is of any one of the formulae:
  • R 4 is as defined above and herein.
  • Ring A is of any one of the formulae:
  • Ring A is of either one of the formulae:
  • Ring A is of the formula:
  • Ring A is of any one of the formulae:
  • R is as defined above and herein.
  • Ring A is of the formula:
  • Ring A is of any one of the formulae:
  • R is as defined above and herein.
  • Ring A is of either of the formulae:
  • Ring A is of any one of the formulae:
  • R is as defined above and herein.
  • a provided compound is of either of the formulae:
  • a provided compound is of either of the formulae:
  • L is not a bivalent saturated C 2 hydrocarbon chain wherein one methylene unit is replaced with -S(O)-.
  • L is a bivalent saturated C 2 hydrocarbon chain wherein one methylene unit is replaced with - S-, -O-, or -SO 2 -.
  • R 1 , R 2 , or R 3 is hydrogen. In certain embodiments, wherein compounds are of formula la(i-a) or lb(i-a), at least two of R 1 , R 2 , and/or R 3 are hydrogen. In certain embodiments, wherein compounds are of formula la(i-a) or lb(i-a), R 1 , R 2 , and R 3 are all hydrogen. [0191] In certain embodiments, a provided compound is of the formula:
  • R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R 3 , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae: [0199] In some embodiments, a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • R 1 , R 2 , R 3 , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R . 1 , R r>2 , r R>3 , and R are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R , R , R , and R are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae: [0212] In some embodiments, a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R 3 , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • R 4 is halogen or Cj-C 6 alkyl.
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R 3 , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae: [0228] In some embodiments, a provided compound is of the formula:
  • R , R , R , and R are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R 3 , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is the formula:
  • a provided compound is the formula:
  • R, R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • a provided compound is the formula:
  • a provided compound is the formula:
  • R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R 3 , R 4 , R 6 and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R, R 1 , R 2 , R 3 , R 4 , and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • R 1 , R 2 , R , and R 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • R 1 , R T ⁇ 2 2 , R ⁇ > 3 3 , and ] R r» 4 4 are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae: [0247] In some embodiments, a provided compound is of either of the formulae:
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein each R 5 is independently -R.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein each R 5 is independently -OR, -SR, -S(O)R, - SO 2 R, -OSO 2 R, -N(R) 2 , -CN, -NO 2 , -NRC(O)R, -NRC(O)(CO)R, -NRC(O)N(R) 2 , - NRC(O)OR, -N(R)S(O)R, -N(R)SO 2 R, -N(R)SO 2 OR, -C(O)R, -C(O)OR, -OC(O)R, - OC(O)OR, -C(O)N(R) 2 , or
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein at least one R 5 is a 3-8 membered saturated, partially unsaturated, or aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein R is optionally substituted with 1-5 R groups.
  • at least one R 5 is a 5-6 membered aryl monocyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein R 5 is optionally substituted with 1-5 R groups.
  • at least one R 5 is phenyl optionally substituted with 1-5 R groups.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein at least one R is a 5-6 membered heteroaryl monocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein R 5 is optionally substituted with 1-5 R groups.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 is an 8-10 membered saturated, partially unsaturated, or aryl bicyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein R 5 is optionally substituted with 1-5 R groups.
  • R 5 heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quino
  • a provided compound is of formula lb(i-b) or lb(i-c :) above, wherein > RR 55 is of the formula:
  • R is as defined and described above and herein.
  • a provided compound is of formula llbb((ii-b) or lb(i-c) above, wherein R 5 is of any of the formulae:
  • R is as def fiinneedd aanndd ddeessccrriibbeedd aabboovvee aanndd hheerreeiinn.
  • R is as defined and described above and herein.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 is of any of the formulae:
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 is of any of the formulae:
  • R is as defined and described above and herein.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 of the formula:
  • R is as defined and described above and herein.
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 of any of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of formula lb(i-b) or lb(i-c) above, wherein R 5 of any of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • R, R 3 , R 4 , L, and m are as defined and described above and herein.
  • a provided compound is of any one of the formulae:
  • a provided compound is of any one of the formulae:
  • L is selected from the group consisting Of -CH 2 O-, -CH 2 S-, -CH 2 S(O)-, or -CH 2 SO 2 -.
  • a provided compound is of the formula:
  • a provided compound is of the formula:
  • R, R 3 , R 4 , and m are as defined and described above and herein, and wherein L is selected from the group consisting Of -CH 2 O-, -CH 2 S-, -CH 2 S(O)-, or -CH 2 SO 2 -.
  • Exemplary compounds of the present invention having an EC50 of ⁇ 20 ⁇ M in assays for protection of mutant SODl -induced cytotoxicity are as shown below in Table 1 :
  • ALS amyotrophic lateral sclerosis
  • the present invention provides methods of treating ALS comprising administering a therapeutically effective amount of a provided compound or analog thereof, or pharmaceutical composition thereof, to a subject with ALS.
  • the inventive pyrazolone may be administered to a subject in need thereof using any method of administration known in the medical arts.
  • treatment may be administered orally or parenterally.
  • treatment is administered once a day.
  • the compound is administered two, three, four, or five times a day.
  • the compound is administered every other day.
  • the compound is administered every two days.
  • the compound is administered every three days.
  • the compound is administered every four days.
  • the compound is administered every five days.
  • the compound is administered every six days. In some embodiments, the compound is administered once a week. In some embodiments, the compound is administered at intervals as instructed by a physician for the duration of the life of the patient being treated. In certain embodiments, treatment is administered as many times a day as necessary to provide a therapeutically effective amount of a provided compound to treat a subject with ALS.
  • the subject with ALS is a mammal. In some embodiments, the subject with ALS is a rodent, such as a rat or mouse, for example, a mouse model of ALS. In certain embodiments, the subject with ALS is a human.
  • the efficacy of the inventive treatment may be evaluated and followed using any method known in the medical arts.
  • the treatment of ALS may be evaluated, for example, by physical examination, laboratory testing, imaging studies, electrophysiological studies, etc.
  • the present invention provides a method of inhibiting or reversing abnormal protein aggregation comprising contacting in vitro or in vivo a compound of the instant invention with a cell in a therapeutically effective amount to inhibit or reverse abnormal protein aggregation.
  • inhibition of abnormal protein aggregation occurs in vivo in a subject with ALS or another neurodegenerative disease characterized by aberrant protein aggregation ⁇ e.g., Huntington's disease, prion disease, or Alzheimer's disease).
  • the subject is a mammal.
  • the subject is a mouse or rat.
  • the subject is a human.
  • contact occurs in vitro, and the cell is derived from a mammalian cell line. In certain embodiments, contact occurs in vitro, and the cell is derived from a PC 12 cell line. In certain embodiments, PC 12 cells may additionally contain a detectable moiety to measure the extent of inhibition of aggregation. In certain embodiments, a detectable moiety is associated with a protein ⁇ e.g., SODl). In certain embodiments, the detectable moiety is a flourescent moiety ⁇ e.g., a YFP tag).
  • the detectable moiety is a phosphorescent moiety, a radiolabel, or any other detectable moiety known in the art, and may be detected using any of the methods known in the art.
  • the detectable moiety may be detected using a high content microscopy system to allow for high-throughput screening.
  • the detectable moiety allows for the measurement of cell viability. Assays for the identification of compounds that protect against protein aggregate-induced cytotoxicity
  • the present invention also provides assays for the identification of compounds that protect against protein aggregate-induced cytotoxicity.
  • the assays are cell protection assays that are used to identify compounds that protect cells from the cytotoxic effects of aberrant protein aggregation.
  • the assays are protein aggregation inhibition assays that are used to identify compounds that inhibit protein aggregation in a cell or in vitro.
  • the inventive assay may screen at least 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000 compounds in parallel.
  • the present invention provides a method of identifying compounds that protect against protein aggregate-induced cytotoxicity comprising contacting a cell expressing SODl or another protein susceptible to aggregation with a test compound, incubating the cell with the test compound under suitable conditions for an amount of time sufficient to observe a protective effect against protein aggregate-induced cytotoxicity, and then measuring viability in the cells treated with the test compound.
  • the extent of protein aggregation- induced cytotoxicity is measured by determining the level of a detectable moiety (e.g., a fluorescent moiety) in the cell.
  • the expressed protein is a mutant SODl protein. In some embodiments, the expressed protein is an SODl protein associated with a detectable moiety. In certain embodiments, the expressed protein is a fluoresently tagged mutant SODl protein, and the flourescent moiety is a YFP tag. In some embodiments, the detectable moiety is a phosphorescent moiety, epitope, or radiolabel. In some embodiments, the detectable moiety is any suitable detectable moiety known to those or ordinary skill in the art and may be detected using any method known in the art. In some embodiments, the detectable moiety is a fluorescent tag (e.g., a YFP tag) that can be detected with a high content microscopy system.
  • a fluorescent tag e.g., a YFP tag
  • the high content microscopy system detects cell viability and facilitates high-throughput screening of a plurality of compounds.
  • Cells may be pre-treated with an agent that modulates the expression of a protein of interest (e.g., SODl) in the assay.
  • the agent may, for instance, induce the expression of a gene responsible for the protein of interest (e.g., doxycycline-inducible promoter).
  • cells may also be treated with an agent that modulates proteasome activity.
  • the agent may be a proteasome inhibitor (e.g., MG132).
  • cell viability of cells pre-treated with an agent described herein is measured using methods described above.
  • the time of incubation of a cell with a test compound ranges from approximately 1 minute to approximately 1 week. In some embodiments, the time of incubation ranges from approximately 5 minutes to approximately 1 week. In some embodiments, the time of incubation ranges from approximately 30 minutes to approximately 2 days. In some embodiments, the time of incubation ranges from approximately 30 minutes to approximately 1 day. In some embodiments, the time of incubation ranges from approximately 1 hour to approximately 1 day. In some embodiments, the time of incubation ranges from approximately 1 hour to approximately 18 hours. In some embodiments, the time of incubation ranges from approximately 1 hour to approximately 12 hours. In some embodiments, the time of incubation ranges from approximately 1 hour to approximately 6 hours.
  • the time of incubation ranges from approximately 1 hour to approximately 3 hours. In some embodiments, the time of incubation is approximately 6 hours. In some embodiments, the time of incubation is approximately 12 hours. In some embodiments, the time of incubation is approximately 18 hours. In some embodiments, the time of incubation is approximately 24 hours.
  • the temperature during incubation of a cell with a test compound ranges from approximately 20 0 C to approximately 45 0 C. In certain embodiments, the temperature ranges from approximately 20 °C to approximately 40 0 C. In certain embodiments, the temperature ranges from approximately 25 0 C to approximately 40 0 C. In certain embodiments, the temperature ranges from approximately 30 0 C to approximately 40 0 C. In certain embodiments, the temperature is approximately 30 0 C. In certain embodiments, the temperature is approximately 37 0 C.
  • the present invention provides a method of identifying compounds that inhibit aberrant protein aggregation comprising contacting a cell expressing SODl or other protein susceptible to aggregation with a test compound, incubating the cell with the test compound under suitable conditions, and then measuring the extent of protein aggregation in the cells treated with the test compound as compared to a control.
  • the extent of inhibition of protein aggregation is measured by staining the protein aggregates with a detectable stain (e.g., Image-iT plasma membrane dye).
  • the detectable stain is detected using a scanning device (e.g., Cellomics Arrayscan).
  • the protein aggregates are detected using any method of detecting protein aggregates known in the art.
  • Compounds identified using the above-mentioned assays may be further examined using biological assays to guide structure-activity relationship (SAR) analyses of the identified compounds.
  • biological assays and SAR analyses are known to those of skill in the art.
  • the present invention provides a method of protecting cells against the cytotoxic effects of aggregated SODl protein comprising contacting in vitro or in vivo a compound of the invention with a cell in a therapeutically effective amount to protect the cell from the effects of aggregated SODl.
  • protection of a cell occurs in vivo in a subject with ALS.
  • the subject is a mammal.
  • the subject is a mouse or rat.
  • the subject is a human.
  • SODl occurs in vitro.
  • protection occurs in vitro in a cell culture.
  • compounds of the invention are contacted with a cell line in vitro and the cell line is a mammalian cell line.
  • the cell line is the PC 12 cell line.
  • cells are associate with a detectable moiety such as those described above.
  • cells contain a protein labeled with a detectable moiety.
  • the protein labeled with a detectable moiety is SODl and the detectable moiety is a fluoresent moiety.
  • the flourescent moiety e.g., a YFP tag
  • the detectable moiety is a phosphorescent moiety, an epitope, radiolabel, or any other detectable moiety known in the art, and may be detected using any of the methods known in the art.
  • the detectable moiety allows for the measurement of cell viability.
  • the present invention provides a method of measuring changes in ubiquitin proteasome activity comprising contacting in vitro or in vivo a compound of the instant invention with a cell in a therapeutically effective amount to effect a change in ubiquitin proteasome activity.
  • modulation of proteasome activity with an inventive compound occurs in vivo in a subject with ALS.
  • the subject is a mammal.
  • the subject is a mouse or rat.
  • the subject is a human.
  • contact occurs in vitro by contacting a test compound with a cell, incubating the test compound with the cell using methods described above, and measuring the extent of inhibition of protein aggregation in the cell.
  • the cell is derived from a mammalian cell line.
  • the cell is derived from a PC 12 cell line or a HeLa cell line.
  • the cells contain a detectable moiety to measure the extent to which proteasome activity is inhibited.
  • cells contain a protein labeled with a detectable moiety.
  • the protein is SODl and the detectable moiety is a fluoresent moiety.
  • the detectable moiety is a flourescent moiety (e.g., a Ubi-YFP tag). In some embodiments, the detectable moiety is a Ubi-YFP tag, which is detectable by fluorescence microscopy. In some embodiments, the detectable moiety is a phosphorescent moiety, an epitope, a radiolabel, or any other detectable moiety known in the art, and may be detected using any of the methods known in the art. In some embodiments, the detectable moiety may be detected using a high content microscopy system to allow for high-throughput screening. In certain embodiments, the detectable moiety allows for the measurement of cell viability.
  • a flourescent moiety e.g., a Ubi-YFP tag
  • the detectable moiety is a Ubi-YFP tag, which is detectable by fluorescence microscopy.
  • the detectable moiety is a phosphorescent moiety, an epitope, a radiolabel, or any other detectable
  • the present invention provides pharmaceutically compositions, which comprise a therapeutically effective amount of one or more of a provided compound, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents for use in treating ALS or any other diseases, disorders, or conditions.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions
  • compositions of provided compounds include conventional nontoxic salts or quaternary ammonium salts of a compound, e.g., from nontoxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • provided compounds may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutical ly-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutical ly-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. See, for example, Berge et al., supra.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
  • Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.
  • an aforementioned formulation renders orally bioavailable a compound of the present invention.
  • Methods of preparing formulations or compositions comprising provided compounds include a step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, g
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Tablets may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent.
  • Tablets, and other solid dosage forms of pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Dissolving or dispersing the compound in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the compound across the skin. Either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux. [0314] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
  • a compound or pharmaceutical preparation is administered orally. In other embodiments, the compound or pharmaceutical preparation is administered intravenously. Alternative routs of administration include sublingual, intramuscular, and transdermal administrations.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingual Iy.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved.
  • one or more provided compounds, or pharmaceutical compositions thereof is provided to a synucleinopathic subject chronically.
  • Chronic treatments include any form of repeated administration for an extended period of time, such as repeated administrations for one or more months, between a month and a year, one or more years, or longer.
  • chronic treatment involves administering one or more provided compounds, or pharmaceutical compositions thereof, repeatedly over the life of the subject.
  • Preferred chronic treatments involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month.
  • a suitable dose such as a daily dose of one or more provided compounds, or pharmaceutical compositions thereof, will be that amount of the one or more provided compound that is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • doses of the compounds of this invention for a patient when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day.
  • the daily dosage will range from 0.001 to 50 mg of compound per kg of body weight, and even more preferably from 0.01 to 10 mg of compound per kg of body weight.
  • the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.
  • the effective daily dose of one or more provided compounds may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a provided compound While it is possible for a provided compound to be administered alone, it is preferable to administer a provided compound as a pharmaceutical formulation (composition) as described above.
  • Provided compounds may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • provided compounds for treating neurological conditions or diseases can be formulated or administered using methods that help the compounds cross the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the vertebrate brain (and CNS) has a unique capillary system unlike that in any other organ in the body.
  • the unique capillary system has morphologic characteristics which make up the blood-brain barrier (BBB).
  • the blood-brain barrier acts as a system-wide cellular membrane that separates the brain interstitial space from the blood.
  • the unique morphologic characteristics of the brain capillaries that make up the BBB are: (a) epithelial-like high resistance tight junctions which literally cement all endothelia of brain capillaries together, and (b) scanty pinocytosis or transendothelial channels, which are abundant in endothelia of peripheral organs. Due to the unique characteristics of the blood-brain barrier, hydrophilic drugs and peptides that readily gain access to other tissues in the body are barred from entry into the brain or their rates of entry and/or accumulation in the brain are very low.
  • provided compounds that cross the BBB are particularly useful for treating synucleinopathies.
  • provided compounds that cross the BBB are particularly useful for treating amyotrophic lateral sclerosis (ALS). Therefore it will be appreciated by a person of ordinary skill in the art that some of the compounds of the invention might readily cross the BBB.
  • the compounds of the invention can be modified, for example, by the addition of various substitutuents that would make them less hydrophilic and allow them to more readily cross the BBB.
  • Another approach to increasing the permeability of the BBB to drugs involves the intra-arterial infusion of hypertonic substances which transiently open the blood-brain barrier to allow passage of hydrophilic drugs.
  • hypertonic substances are potentially toxic and may damage the blood-brain barrier.
  • Antibodies are another method for delivery of compositions of the invention.
  • an antibody that is reactive with a transferrin receptor present on a brain capillary endothelial cell can be conjugated to a neuropharmaceutical agent to produce an antibody-neuropharmaceutical agent conjugate (US 5,004,697, incorporated herein in its entirety by reference).
  • the method is conducted under conditions whereby the antibody binds to the transferrin receptor on the brain capillary endothelial cell and the neuropharmaceutical agent is transferred across the blood brain barrier in a pharmaceutically active form.
  • the uptake or transport of antibodies into the brain can also be greatly increased by cationizing the antibodies to form cationized antibodies having an isoelectric point of between about 8.0 to 11.0 (US 5,527,527, incorporated herein in its entirety by reference).
  • a ligand-neuropharmaceutical agent fusion protein is another method useful for delivery of compositions to a host (US 5,977,307, incorporated herein in its entirety by reference).
  • the ligand is reactive with a brain capillary endothelial cell receptor.
  • the method is conducted under conditions whereby the ligand binds to the receptor on a brain capillary endothelial cell and the neuropharmaceutical agent is transferred across the blood brain barrier in a pharmaceutically active form.
  • a ligand- neuropharmaceutical agent fusion protein which has both ligand binding and neuropharmaceutical characteristics, can be produced as a contiguous protein by using genetic engineering techniques.
  • Gene constructs can be prepared comprising DNA encoding the ligand fused to DNA encoding the protein, polypeptide or peptide to be delivered across the blood brain barrier.
  • the ligand coding sequence and the agent coding sequence are inserted in the expression vectors in a suitable manner for proper expression of the desired fusion protein.
  • the gene fusion is expressed as a contiguous protein molecule containing both a ligand portion and a neuropharmaceutical agent portion.
  • the permeability of the blood brain barrier can be increased by administering a blood brain barrier agonist, for example bradykinin (US 5,112,596, incorporated herein in its entirety by reference), or polypeptides called receptor mediated permeabilizers (RMP) (US 5,268,164, incorporated herein in its entirety by reference).
  • a blood brain barrier agonist for example bradykinin (US 5,112,596, incorporated herein in its entirety by reference), or polypeptides called receptor mediated permeabilizers (RMP) (US 5,268,164, incorporated herein in its entirety by reference).
  • Exogenous molecules can be administered to the host's bloodstream parenterally by subcutaneous, intravenous or intramuscular injection or by absorption through a bodily tissue, such as the digestive tract, the respiratory system or the skin.
  • the form in which the molecule is administered depends, at least in part, on the route by which it is administered.
  • the administration of the exogenous molecule to the host's bloodstream and the intravenous injection of the agonist of blood-brain barrier permeability can occur simultaneously or sequentially in time.
  • a therapeutic drug can be administered orally in tablet form while the intravenous administration of an agonist of blood-brain barrier permeability is given later (e.g., between 30 minutes later and several hours later). This allows time for the drug to be absorbed in the gastrointestinal tract and taken up by the bloodstream before the agonist is given to increase the permeability of the blood-brain barrier to the drug.
  • an agonist of blood-brain barrier permeability e.g., bradykinin
  • co-administration is used herein to mean that the agonist of blood- brain barrier and the exogenous molecule will be administered at times that will achieve significant concentrations in the blood for producing the simultaneous effects of increasing the permeability of the blood-brain barrier and allowing the maximum passage of the exogenous molecule from the blood to the cells of the central nervous system.
  • a provided compound can be formulated as a prodrug with a fatty acid carrier (and optionally with another neuroactive drug).
  • the prodrug is stable in the environment of both the stomach and the bloodstream and may be delivered by ingestion.
  • the prodrug passes readily through the blood brain barrier.
  • the prodrug preferably has a brain penetration index of at least two times the brain penetration index of the drug alone.
  • the prodrug which preferably is inactive, is hydrolyzed into the fatty acid carrier and a provided compound or analog thereof (and optionally another drug).
  • the carrier preferably is a normal component of the central nervous system and is inactive and harmless. The compound and/or drug, once released from the fatty acid carrier, is active.
  • the fatty acid carrier is a partially-saturated straight chain molecule having between about 16 and 26 carbon atoms, and more preferably 20 and 24 carbon atoms.
  • Examples of fatty acid carriers are provided in U.S. Patents. 4,939,174; 4,933,324; 5,994,932; 6,107,499; 6,258,836; and 6,407,137, the disclosures of which are incorporated herein by reference in their entirety.
  • Administration of agents of the present invention may be for either prophylactic or therapeutic purposes.
  • the agent is provided in advance of disease symptoms.
  • the prophylactic administration of the agent serves to prevent or reduce the rate of onset of symptoms of ALS.
  • the agent is provided at (or shortly after) the onset of the appearance of symptoms of actual disease.
  • the therapeutic administration of the agent serves to reduce the severity and duration of the disease.
  • Cultured cells are utilized to conduct high throughput assays for compounds that protect against mutant SODl -induced cytotoxicity. Two assays are used: first, in the cytotoxicity protection assay, compounds are screened for their ability to protect cells from the cytotoxic effects of aggregated mutant SODl, irrespective of mechanism of drug action. Second, in the protein aggregation assay, compounds are screened for their ability to reduce aggregation of mutant SODl.
  • the high throughput cytotoxicity protection assay is the primary screen and compounds active in the primary screen (and their analogs) move forward into the secondary screen for protein aggregation.
  • the high throughput cytotoxicity protection assay was carried out in PC 12 cells that express mutant G93A SODl as a YFP fusion protein from a doxycycline-inducible promoter.
  • cytotoxicity of protein aggregates in ALS is due at least in part to inhibition of the proteasome (Bruijin et al. Annu. Rev. Neurosci. 2004, 27, 723-729; Cleveland et al Nat. Rev. Neurosci. 2001, 2(11), 806). This idea was tested by examining the sensitivity of PC 12 cells to SODl aggregates in the presence and absence of proteasome inhibitor MG132.
  • PC12 cells expressing no SODl, wild type SODl, G85R SODl or G93A SODl were grown with or without MG132 ( Figure 1).
  • Cells expressing no SODl, wild type SODl and G85R SODl were relatively insensitive to MGl 32, with an IC 50 of approximately 400 nM.
  • cells expressing G93A SODl were approximately 5- fold more sensitive to MG132 (IC 50 ⁇ 75 nM). In these cells, protein aggregation was detected after 24 h and loss of cell viability was detected at approximately 48 h.
  • Compounds that are active in the above assay could theoretically protect against mutant SODl-induced cytotoxicity through a number of mechanisms, including the following: 1) compounds could nonspecifically block or reverse protein aggregation via chaperone induction, as observed for radicicol and geldanamycin; 2) compounds could block or reverse the aggregation of a specific aggregated protein form; 3) compounds could interfere with an event downstream of protein aggregation that plays a critical role in mutant SODl-induced cytotoxicity (e.g., proteasome function); and 4) compounds could act directly on SODl in a manner that prevents mutant SODl aggregation. These possibilities were tested using an assay that directly measures protein aggregation.
  • the protein aggregation assay is based on G85R SODl; this broadens the scope of the screening strategy, and should eliminate compounds with highly specific activity (i.e., G93A SODl limited) against protein aggregation.
  • Arrayscan ® high content microscopy system was used for screening and quantification. Initial experiments indicated that G85R SODl aggregates were more readily recognized by the high content microscopy system and its computer algorithm. Because the most robust high content assays measure events on a per cell basis, it was necessary to select a fluorescent stain that marks whole cells to be used with a compatible stain that marks intracellular structures. On the basis of pilot experiments with a number of vital dyes, an Image-iT conjugated wheat germ agglutinin (WGA) dye from Molecular Probes was selected and a computer algorithm for detecting WGA was developed. As shown in Figure 5, WGA provided an excellent cellular marker that did not interfere with detection of YFP-tagged SODl.
  • WGA Image-iT conjugated wheat germ agglutinin
  • Mutant SODl-induced cytotoxicity protection assay automated high throughput screening protocol.
  • PC12 cells were maintained in Dulbecco's Modified Eagle's Medium
  • Mutant SODl aggregation assay automated high content screening protocol.
  • PC 12 cells were maintained in Dulbecco's Modified Eagle's Medium
  • Test compounds were incubated with the cells for 24h prior to the addition of MGl 32.
  • CMB-3350 was used as a positive control at 5 ⁇ M
  • DMSO as a negative control
  • test compounds were applied at 512M.
  • MG132 was then added at 1 ⁇ M final concentration and the cells were grown for an additional 24 hours.
  • cell culture medium was then removed and the cells were washed once with HBSS then incubated in HBSS containing 5 ⁇ g/ml of the Image-iT WGA plasma membrane dye (Molecular Probes) for 15min at 37°C. Cells were then washed with PBS and imaged using the Cellomics Arrayscan ® .
  • Arrayscan images were recorded using a 2Ox objective and the XF93-TRITC filter set in channel 1 for the Image-iT WGA plasma membrane dye or the XF93-FITC filter set for SODl-YFP and analyzed using Cellomics spot detector software. Software parameters were set to maximally differentiate cells from background and maximize recognition of SODl aggregates without recognizing background cytoplasmic staining as a spot. Data were recorded as spot count (aggregate)/object (cell).
  • a library was screened using the cytotoxicity protection assay as the primary screen and the protein aggregation assay as the secondary screen.
  • the overall screen produced an average Z 1 factor of 0.5 and an average Z factor of 0.6.
  • 195 primary actives were recovered (0.38% primary hit rate).
  • All primary actives were retested in dose response curves yielding 68 confirmed actives (0.13% confirmed hit rate). These actives were then tested for auto-fluorescence and inhibition of protein synthesis, potential causes of artifactual positives. Confirmed primary actives were assayed in the high content protein aggregation screen and grouped into compounds that were positive or negative in the secondary screen.
  • Cheminformatic analysis of these compounds identified 17 chemotypes of structurally-related active and inactive compounds plus 15 singleton hits. Most active compounds were also active in the secondary high content assay, two of the confirmed primary actives were not.
  • Initial biological analysis focused on the arylsulfanyl pyrazolone lead series. The compound collection includes riluzole, which tested negative indicating that the actives tested here differ from the sole clinically approved drug for ALS.
  • the high throughput cytotoxicity protection assay and the high content protein aggregation assay only score compounds as active or inactive.
  • Two quantitative metrics were therefore developed to evaluate compound activity for SAR development: potency (EDs 0 ), which is the concentration producing half maximal cell viability, and efficacy, which is defined as the maximum viability produced by a compound at its optimum dosage. Cytotoxicity is measured by determining the concentration that reduces PC 12 cell viability by 50% and is reported as the cytotoxicity IC 50 ( Figure 2).
  • Compounds were also tested for nonspecific inhibition of protein synthesis, inhibition of protein aggregation (i.e., SODl-YFP fusion) and autofluorescence (to ensure the detection method was not detecting false positives).
  • CMB-3319 & CMB-3350 are the C-5 substituted pyrazoles containing an arylsulfanyl group.
  • CMB-3299 is similar to CMB-3319, but has a methyl substitution at C2 rather than a chlorine group on the arylsulfanyl ring system.
  • HeLa cells stably transfected with a luciferase reporter plasmid under control of the HSP70 promoter, were plated in a 96 well plate at a density of 7500 cells/100 ⁇ l. Following a 16 h incubation at 37°C/5% CO 2 , compounds were added and cells incubated for an additional 8 h. Cells were lysed by the addition of 100 ⁇ l BrightGlo reagent (Promega) according to manufacturer's instructions and luciferase activity measured by quantifying luminescence signal. Cells were treated with test compounds at concentrations ranging from 10 - 100 ⁇ M celastrol or CdCl 2 serve as positive controls while DMSO is a negative control.
  • HeLa cells (20,000/chamber) were plated on 8 chambered Tissue-tek coverglass pretreated with 1 mg/ml poly-D-lysine and allowed to adhere for 18 h at 37 °C/5% CO 2 .
  • Cells were transfected with 0.2 ⁇ g each CMV-CFP and Ubi-YFP plasmids using Lipofectamine 2000 according to manufacturer's instructions.
  • Cells were allowed to express CFP and Ubi-YFP for 18 h followed by a 6 h incubation with test compounds (25 ⁇ M).
  • MG 132 (1 ⁇ M) was added for a final 18 h incubation and Ubi-YFP degradation assessed by fluorescence microscopy. Fluorescence intensity of CFP and YFP was calculated on a cell- by-cell basis from captured images using AxioVision software (Zeiss). YFP fluorescence was normalized to CFP fluorescence.
  • Nonspecific cytotoxicity was determined using PC 12 and other cell lines in conventional dose response assays employing calcein-AM fluorescence as the indicator of cell viability.
  • compounds could score a false positive by selectively reducing the synthesis of aggregated proteins (i.e., SODl-YFP fusion).
  • Reduction in SODl protein concentration was determined using a modification of the high content assay in which YFP fluorescence is measured on a plate reader prior to determining number of cells per well with the Arrayscan. Specific YFP activity, which is equivalent to SODl content per cell, was then calculated. Expanded assays for aggregated protein spectrum.
  • TDP-43 is an aggregated protein that accumulates in neural cells in patients with sporadic ALS (Neumann et al.
  • Active compounds are tested for ability to prevent TDP- 43 aggregation and/or reduce its associated cytotoxicity.
  • Compounds that prevent TDP-43 - associated cytotoxicity and/or aggregation are expected to have greater potential for efficacy in sporadic ALS patients.
  • the in vitro ADMET approach is based on the use of a suite of chemical tests (compound integrity, compound solubility, compound aggregation, lipophilicity, pKa) and biological assays (Caco-2 and/or PAMPA assays, cytochrome P450 metabolism and inhibition, cardiac risk, and cytotoxicity) that assess the absorption, distribution, metabolism, excretion, and toxic effects of test compounds.
  • the output from ADMET assays is used to identify and select for compounds with low predicted toxicity and desirable predicted pharmacological properties during SAR-based optimization.
  • Compounds optimized by this approach are selected for analysis in the mouse model of ALS.
  • the following suite of assays are performed:
  • Cytotoxicity Cytotoxicity is determined in cultured cells.
  • Compound Aggregation of screening hits is measured using dynamic light scattering.
  • Solubility Because compounds are stored in DMSO, it is necessary to determine aqueous solubility of initial hit compounds and their analogs. High solubility in aqueous solution is necessary for high GI absorption, bioavailability, and for chemical formulation. Compounds should be soluble in aqueous solution at >10 ⁇ g/ml. Solubility of > 50 ⁇ g/ml is preferred.
  • GI absorption and optimal bioavailability Passive permeability is typically assessed in the PAMPA artificial membrane assay (Kansy et al, J. Med. Chem. 2002, 45(8), 1712-1722).
  • Cell-based permeability determinations using the Caco-2 cell assay are more resource intensive but more predictive of active transport or efflux in vivo (Artursson et al., Adv. Drug. Deliv. Rev. 2001, 46(1-3), 27-43). Caco-2 assays are performed as needed during SAR development.
  • Lipophilicity and pKa Lipophilicity is determined via octanol-water partition at pH 7.4 (Hitzel et al, Pharm. Res. 2000, 17(11), 1389-1395) and pKa is determined by capillary electrophoresis and photodiode array detection (Ishihama et al, J. Pharm. ScL 2002. 91(4), 933-942).
  • Metabolism microsome, S9 fraction, CYP450: Liver Cytochrome P450
  • CYP450 enzymes are the major route for xenobiotic metabolism and microsomal and hepatocyte stability is the best predictor of pharmacokinetic half-life. Cross-species comparisons of metabolism in liver microsomes can predict potential issues with liver toxicity in humans. Selected active compounds are tested in liver microsomes from efficacy species (mouse), toxicity species (rat, dog, monkey), and humans for metabolic stability.
  • hERG ion channel inhibition is implicated in greater than 90% of reported cases of cardiac toxicity, and is a common cause of after market drug failures and withdrawals.
  • Information on hERG inhibition as well as inhibition of five major human CYP450s (1A2, 2C9. 2 C19, 3A4, 2D6) allows for accurate prediction of potential drug- associated cardiac risk.
  • Plasma stability Plasma stability of active compounds is assessed in efficacy species (mouse), toxicity species (rat, dog, monkey), and human cells.
  • Plasma protein binding Plasma protein binding is examined in efficacy species (mouse), toxicity species (rat, dog, monkey), and human cells using ultrafiltration methodology.
  • the compounds identified were evaluated in the transgenic G93A ALS mouse model.
  • ADME data was used to determine optimal route of administration.
  • LD 50 and maximally-tolerated dose were determined, and that information was used to design and execute pharmacokinetic studies to assess brain bioavailability, and to guide choice of dosing regimen (i.e., frequency, dose, route).
  • Three-dose basic efficacy studies were performed on compounds demonstrating acceptable tolerability and bioavailability. For compounds that demonstrate efficacy, a more complete preclinical study is performed and the efficacies of the test compound and previously characterized neuroprotective agents are compared. This may include further dose optimization, phenotype assessment, comparison with compounds of established efficacy, and analysis of brain tissue in treated animals.
  • LD50 for each test compound was determined in wild type mice by increasing the dose b.i.d. one-fold each injection.
  • the route of administration (p.o. by gavage or i.p.) and starting dose was based on solubility and other output from ADME studies.
  • Initial pharmacokinetic (pK) studies were conducted by giving animals a single dose, sacrificing them after 30 min, 1 h, 2 h, 4 h, 6 h, or 12 h, and dissecting brains and spinal cords and determining drug concentration in the target tissue.
  • Drug steady-state levels were determined in animals that have been dosed for 1 week prior to sacrifice. These data are used to optimize the dosing regimen for efficacy studies.
  • Drug doses should achieve a desirable drug concentration in the brain and spinal cord of treated mice. Working doses at least 10-fold lower than the acute tolerability dose are preferable.
  • Efficacy studies Efficacy is measured using endpoints that indicate neuroprotective function. These include reversal of degenerative lesions in the brain and neuronal tissues, improved motor function, increased body weight, and prolonged survival. Some mice cohorts are sacrificed at a predetermined time point, while others are sacrificed when they reach criteria for euthanasia.
  • Weight loss is a sensitive measure of disease progression in transgenic G93A SODl mice and of toxicity in transgenic and wild type mice.
  • Motor/behavioral Quantitative methods of testing motor function are used including Rotarod and analysis of open field behavior. Decline of motor function is a sensitive measure of disease onset and progression.
  • mice are euthanized at 120 days for isolation and analysis of spinal cord tissue. For this purpose, mice are deeply anesthetized and perfused transcardially with 4% buffered paraformaldehyde at the desired time point. These and other studies are performed in a blinded manner, to avoid bias in interpretation of the results. Brains are weighed, serially sectioned at 50 ⁇ m and stained for quantitative morphology (cresyl violet), to assess protein aggregates, and to label ventral neurons. Remaining tissue samples/sections are stored for future use. Stereology is used to quantify ventral horn atrophy, neuronal atrophy, neuronal loss, and protein aggregate load.
  • L3-L5 spinal cord segments are used for gross spinal cord areas and neuronal analysis. Gross areas of the spinal cord sections are quantified from each experimental cohort using NIH Image. From the same sections, the ventral horn is delineated by a line from the central canal laterally and circumscribing the belly of gray matter. Absolute neuronal counts of Nissl- positive neurons are performed in the ventral horns in the lumbar spinal cord. Only those neurons with nuclei are counted. All counts are performed with the experimenter (JM) blinded to treatment conditions. Counts are performed using Image J (NIH) and manually verified and the data represent the average neuronal number from the sections used.
  • NIH Image J
  • TC-I-53 4-Benzylidene-3-(hydroxymethyI)-l/T-pyrazol-5(4H)-one (TC-I-53): To a suspension of ethanol (2 mL) and TC-I-49 (367 mg, 2 mmol) was added an ethanolic solution of hydrazine (2 N, 1 mL). The resulting suspension was refluxed for 1 h, during which time all of the TC-I-49 dissolved and a new precipitate formed. The resulting mixture was cooled in an ice-bath for 20 min. The white precipitate was filtered and washed with cold ethanol to give TC-I-53 (77 mg, 28%).
  • Benzenethiol (61 ⁇ L, 0.60 mmol) was added to a suspension of TC-I-82 (0.13 g, 0.50 mmol) and anhydrous potassium carbonate (0.42 g, 3.0 mmol) in DMF (2 ml) at room temperature. The reaction temperature was gently brought to 70 °C. After the resulting suspension was stirred for 18 h, the reaction mixture was quenched with HCl (0.25 N), and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over Na 2 SO 4 , and evaporated to dryness.
  • Reagents and conditions (a) TBHP, vanadyl acetylacetonate, CH 2 Cl 2 , room temp, overnight; (b) NH 2 NH 2 , EtOH, room temp, overnight.
  • TC-I-164 (3.00 g, 11.0 mmol) was mixed with t- butyl hydrogen peroxide (70 wt% in water, 1.5 mL, 10.50 mmol) in CH 2 Cl 2 (50 mL) at room temperature, and vanadyl acetylacetonate (0.1% mol) was added slowly. Extra t- butyl hydrogen peroxide (6 mL, 41. 94 mmol) was added to the reaction mixture after 2 h. The resulting suspension was stirred overnight at room temperature.
  • Ethyl 4-(4-chlorophenoxy)-3-oxobutanoate (TC-II-48): A solution of 4- chlorophenol (6.4 g, 50 mmol) in THF (25 mL) was treated with NaH (60% in mineral oil, 2 g, 50 mmol) at 0 0 C. In another flask, a solution of ethyl 4-chloroacetoacetate (10.21 mL, 75 mmol) in THF (25 mL) was treated NaH (60% in mineral oil, 3.5 g, 75 mmol) at -20 0 C. The resulting yellowish suspension was slowly added to the solution of sodium 4- chlorophenoxide, which was kept at 0 0 C.
  • Ethyl 4-(3,5-dichlorophenoxy)-3-oxobutanoate (TC-IV-12): EtOAc (0.9 mL. 9.19 mmol) was added to a solution of LiHMDS (1 ⁇ in THF, 21 mL, 21 mmol) at 0 0 C and stirred. After 60 min, a THF solution of the Wienreb amide TC-IV-10 (2.4 g, 9.19 mmol) was added at -78 0 C. After the resulting solution was stirred at -78 0 C for 8 h, the reaction mixture was quenched with diluted HCl (0.25 ⁇ ), and the aqueous layer was extracted with Et 2 O.
  • TC-IV-120 3,5-Dibromophenol (1.0 g, 3.97 mmol), phenylboronic acid (2 g,
  • TC-IV-150 (30 mg, 0.068 mmol) and phenol (60 mg, 0.64 mmol) were added to a solution of HBr in water (48%, 0.75 mL). After the resulting suspension was stirred at 100 0 C for 8 h, the reaction mixture was partitioned between water and EtOAc, and the aqueous layer was extracted with EtOAc. The combined organic layer was evaporated to dryness and purified by flash column chromatography (ethyl acetate) to give TC-IV-154 (10 mg, 57%) as a white solid.
  • Reagents and conditions (a) K 2 CO 3 , M-BuOH, reflux, 72 h; (b) K 2 CO 3 , acentone, 50 0 C, overnight; (c) EtOAc, LiHMDS, THF -78 0 C, 16 h; (d) NH 2 NH 2 , EtOH, rt, overnight.
  • l-(3,5-Difluorophenyl)piperazine (TC-IV-174): Bis(2-chloroethyl)amine hydrochloride (2.07 g, 11.60 mmol) was added to a solution of 3,5-difluoroaniline (1.50 g, 11.62 mmol) in n-BuOH (20 mL). After the resulting solution was refluxed for 48 h, anhydrous potassium carbonate (1.61 g, 11.62 mmol) was added. After being refluxed for another 24 h, the reaction mixture was cooled, partitioned between water and CHCl 3 , and the aqueous layer was extracted with CHCl 3 .
  • R is cyclic aliphatic.
  • Reagents and conditions (a) K 2 CO 3 , M-BuOH, reflux, 48 0 C; (b) K 2 CO 3 , , acetone, 50 0 C, overnight; (c) EtOAc, LiHMDS, THF, -78 °C, 16h; (d) NH 2 NH 2 , EtOH, rt, overnight. Table 2. H NMR data for selected compounds.
  • CMB-003319 In vitro ADME study ofCMB-003319. CMB-003319 was evaluated for its in vitro ADME properties. The study showed that CMB-003319 has a medium metabolic potential, medium CaCO-2 permeability, poor plasma stability, and medium aqueous- solubility.
  • TC-I-165 Rat liver microsomal stability study of TC-I-165.
  • TC-I-165 was applied for an NADPH dependent rat liver microsomal stability study by HPLC. Preliminary results showed that TC-I-165 has poor microsomal stability like CMB-003319.
  • the test (TC-I- 165), positive control (minaprine), and internal standard (haloperidol) are shown in Figure 11.
  • TC-I-165 was used in a NADPH dependent rat liver microsomal metabolite study by HPLC.
  • TC-II-68 and TC-II-70 were proposed as possible metabolites and prepared before the study. Results showed that only TC-II-68 was a direct metabolite.
  • TC-II-125 was prepared as a double 15 N isotope labeled TC-I-165.
  • TC-I-165, TC-II-125, and TC-II-68 were sent for NADPH dependent rat liver microsomal metabolite determination. The results showed that during the first hour the only direct metabolite from TC-I-165 was TC-II-68, with no nitrogen loss on the pyrazolone ring.
  • Test agents (15 ⁇ M) were incubated in Eppendorf tubes with mouse microsomes (pooled male rat liver microsomes (Sprague Dawley), BD Science) at 37 0 C. Each reaction mixture was carried out in PBS (pH 7.4), which contained 1 mg/mL microsomal protein with 1.3 mM NADP + , 3.3 mM glucose-6-phosphate, 0.4 LVmL glucose- 6-phosphate dehydrogenase, and 3.3 mM magnesium chloride. A control using minaprine was run to test the activity of the microsomal activity. After 0 and 20 minutes incubation, 40 ⁇ L of acetonitrile was added to quench the reactions.
  • reaction mixture was vortexed and incubated in an ice bath for 2 h.
  • Haloperidol 100 ⁇ L, 100 ⁇ M was added to every test tube as an internal standard.
  • the samples were diluted in water and centrifuged to remove the precipitated protein.
  • the supernatant solutions were loaded for solid phase extraction. Extraction fractions were evaporated and reconstituted for HPLC analysis.
  • TC-I-165 was used for a NADPH dependent rat liver microsomal metabolites study, and the corresponding sulfoxide, TC-II-68, and sulfone, TC-II-70, also were prepared ( Figure 14).
  • TC-II-68 was a metabolite from TC-I-165 after 60 minutes of NADPH dependent rat liver microsome incubation.
  • TC-II-70 is not a metabolite. Only one new peak was observed from the microsomal residue of TC-I-165. Using the same HPLC program (25% acetonitrile, isocratic, 30 minutes), the retention time of the new peak was approximately identical to the one from TC-II-68. The retention time of TC-II-70 was well seperated from that of the new peak (see Figures 15-20).
  • TC-II-70 was used in a NADPH dependent rat liver microsomal stability study.
  • the test (TC-II-70), positive control (minaprine), negative control (warfarin), and internal standard (haloperidol) compounds in this experiment are listed in Figure 21.
  • the experiment was carried out mostly according to the general procedure from the microsomal stability study of TC-I-165. After 0, 20, and 40 minutes incubation, 100 ⁇ L acetonitrile was added to quench reactions. The reaction mixture was vortexed and incubated in an ice bath for 2 h.
  • Haloperidol 50 ⁇ L, 100 ⁇ M was added to every test tube as an internal standard. The samples were centrifuged to remove the precipitated protein. The supernatant solutions were loaded directly for HPLC analysis.
  • TC-II-70 is more stable in a NADPH dependent rat liver microsomal stability study compare with its sulfide, TC-I-165 ( Figures 22-25). Table 5 shows results of microsomal stability data processing.
  • BBB blood brain barrier
  • CMB-087229 BBB penetration experiment was carried out for CMB-087229.
  • CMB-003319 was used as the internal standard due to its structural similarity to CMB-003299.
  • CMB-087229 was formulated in 100 ⁇ L DMSO in 2mL phosphate buffered saline.
  • Blood concentration of CMB-087229 (MTD of 75 mg/kd IP) 1 hour following a single lmg IP dose was approximately 10 nM.
  • Brain concentration of CMB-087229 1 hour following a single lmg IP dose was approximately 120 nM. After four hours, concentration in the brain was 194 ⁇ M. At 3 hours, plasma concentration measured 342 ⁇ M. At 6 hours, plasma concentration measured 347 ⁇ M. At 12 hours, plasma concentration measured 248 ⁇ M. At 24 hours, plasma concentration measured 1.68 ⁇ M.
  • CMB-087229 reaches approximately 55% of the blood level in the brain. Brain uptake procedure
  • CMB-087229 Mice Brain and Plasma Data
  • CMB-087229 (1 mg/kg) was administrated to mice (Tg6799 colony with B6/SJL background) by intraperitoneal injection in a 60% PBS and 40% dimethyl sulfoxide solution. At 10 minutes after compound administration the mice were sacrificed. Blood was harvested and plasma was transferred to flash freeze in liquid nitrogen after centrifugation. Mice were perfused. The brain was homogenized in 100% acetonitrile.
  • CMB-003319 (0.5 ⁇ g, 1.73 nmol) was added as an internal standard. The homogenates were centrifuged at 12000 * g for 12 min, and the supernatant was evaporated.
  • the means are not intended to be limited to the means disclosed herein for performing the recited function, but are intended to cover in scope any means, known now or later developed, for performing the recited function.

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Abstract

La présente invention concerne l'identification de composés et de leurs compositions pharmaceutiques destinés au traitement de sujets souffrant de sclérose latérale amyotrophique (SLA) et d'autres maladies neurodégénératives. La présente invention concerne également des méthodes de synthèse des composés concernés.
PCT/US2009/006237 2008-11-20 2009-11-20 Traitement de la sclérose latérale amyotrophique WO2010059241A2 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
AU2009318098A AU2009318098B2 (en) 2008-11-20 2009-11-20 Treatment of amyotrophic lateral sclerosis
CA2744016A CA2744016C (fr) 2008-11-20 2009-11-20 Traitement de la sclerose laterale amyotrophique
EP09827890.6A EP2367798B1 (fr) 2008-11-20 2009-11-20 Dérivés pyrazolone utiles dans le traitement de la sclérose latérale amyotrophique
ES09827890.6T ES2668556T3 (es) 2008-11-20 2009-11-20 Derivados de pirazolona útiles en el tratamiento de esclerosis lateral amiotrófica
US13/129,854 US10167263B2 (en) 2008-11-20 2009-11-20 Treatment of amyotrophic lateral sclerosis
IL212992A IL212992A0 (en) 2008-11-20 2011-05-19 Treatment of amyotrophic lateral sclerosis
US13/354,156 US9428467B2 (en) 2008-11-20 2012-01-19 Selective calcium channel antagonists
US14/103,728 US9145424B2 (en) 2008-11-20 2013-12-11 Treatment of amyotrophic lateral sclerosis
US14/867,624 US9809556B2 (en) 2008-11-20 2015-09-28 Treatment of amyotrophic lateral sclerosis
US15/683,131 US10526289B2 (en) 2008-11-20 2017-08-22 Substituted pyrazolone compounds for use in treatment of amyotrophic lateral sclerosis

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US61/116,571 2008-11-20

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US13/354,156 Continuation-In-Part US9428467B2 (en) 2008-11-20 2012-01-19 Selective calcium channel antagonists
US14/103,728 Continuation-In-Part US9145424B2 (en) 2008-11-20 2013-12-11 Treatment of amyotrophic lateral sclerosis

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US20150203456A1 (en) * 2009-05-05 2015-07-23 Northwestern University Pyrimidine-2,4,6-triones for use in the treatment of amyotrophic lateral sclerosis
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WO2016109559A3 (fr) * 2014-12-29 2016-08-25 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Inhibiteurs à petite molécule de lactate déshydrogénase et procédés pour les utiliser
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US20170349552A1 (en) * 2008-11-20 2017-12-07 Northwestern University Treatment of Amyotrophic Lateral Sclerosis
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US8940798B2 (en) 2011-11-04 2015-01-27 Centre National De La Recherche Scientifique (Cnrs) Compounds useful for treating and/or preventing disease-associated bone loss
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