US20070219244A1 - Histone deacetylase inhibitors as therapeutics for neurological diseases - Google Patents

Histone deacetylase inhibitors as therapeutics for neurological diseases Download PDF

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US20070219244A1
US20070219244A1 US11/595,779 US59577906A US2007219244A1 US 20070219244 A1 US20070219244 A1 US 20070219244A1 US 59577906 A US59577906 A US 59577906A US 2007219244 A1 US2007219244 A1 US 2007219244A1
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alkyl
aryl
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heteroaryl
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Kai Jenssen
David Herman
Joel Gottesfeld
Ryan Burnett
C. Chou
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Scripps Research Institute
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Assigned to SCRIPPS RESEARCH INSTITUTE, THE reassignment SCRIPPS RESEARCH INSTITUTE, THE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JENSSEN, KAI, BURNETT, RYAN, CHOU, C. JAMES, GOTTESFELD, JOEL M., HERMAN, DAVID M.
Publication of US20070219244A1 publication Critical patent/US20070219244A1/en
Priority to US12/773,032 priority patent/US20110021562A1/en
Priority to US13/862,727 priority patent/US8835502B2/en
Priority to US14/480,899 priority patent/US9957225B2/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: SCRIPPS RESEARCH INSTITUTE
Assigned to NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR reassignment NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: THE SCRIPPS RESEARCH INSTITUTE
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Definitions

  • the invention relates to histone deacetylase (HDAC) inhibitors and their uses as therapeutics.
  • HDAC histone deacetylase
  • FRDA Friedreich's ataxia
  • Frataxin insufficiency leads to progressive spinocerebellar neurodegeneration resulting in gait and hand in-coordination, slurred speech, muscle weakness and sensory loss with extraneural scoliosis, cardiomyopathy and diabetes.
  • an affected individual is confined to a wheelchair and in later stages, become completely incapacitated.
  • Most affected individuals die in early adulthood of heart disease.
  • antioxidant- and iron-chelator-based strategies have been used to treat FRDA, these strategies only treat the symptoms of the disease and not the cause, i.e. frataxin deficiency. Therefore, there is a need to develop molecules that could restore frataxin protein expression for the treatment of a neurological condition such as FRDA.
  • Triplet repeat expansion in genomic DNA is associated with many other neurodegenerative and neuromuscular diseases including, without limitation, myotonic dystrophy, spinal muscular atrophy, fragile X syndrome, Huntington's disease, spinocerebellar ataxias, amyotrophic lateral sclerosis, Kennedy's disease, spinal and bulbar muscular atrophy and Alzheimer's disease. Triplet repeat expansion may cause disease by altering gene expression.
  • the invention provides small molecules that could be used to treat a neurological disease such as FRDA.
  • the invention provides small molecule inhibitors that are effective in restoring the normal function of a gene, e.g. restoring transcription of frataxin mRNA.
  • the present invention involves the discovery that lymphocytes from FRDA patients that have been incubated with histone deacetylase (HDAC) inhibitors show elevated levels of acetylated histones.
  • HDAC histone deacetylase
  • the invention concerns the discovery that the HDAC inhibitor BML-2 10 and other novel HDAC inhibitors have the effect of increasing frataxin mRNA in lymphocytes from FRDA patients.
  • the invention is directed to pharmaceutical compositions of HDAC inhibitors and their use as therapeutics for chronic and acute neurological diseases such as, for example, Friedreich's ataxia.
  • the invention is also directed to novel HDAC inhibitors, as well as novel methods for their synthesis.
  • the invention provides a compound of formula Ia: wherein:
  • the compounds of formula Ia are HDAC inhibitors.
  • the invention provides methods for preparing compounds of formula I: wherein:
  • compounds of formula I may be prepared by contacting a compound of formula V: with one or more coupling agents and a compound of formula VI: R 2 —NH(R b ) (VI) to provide the compound of formula I.
  • the compound of formula V may be prepared by contacting a compound of formula III: with a compound of formula IV: R 1 —NH(R a ) (IV) to provide the compound of formula V.
  • the compound of formula III may be prepared by contacting a compound of formula II: with a dehydrating agent to provide the compound of formula III.
  • the invention provides pharmaceutical compositions that include a compound of formula I in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be suitable for oral administration.
  • Pharmaceutical compositions suitable for oral administration can be in the form of a tablet, capsule, or elixir.
  • the pharmaceutical compositions can also be suitable for parenteral administration such as by intravenous, intraperitoneal or subcutaneous administration.
  • the pharmaceutical composition can also be in the form of a sustained-release formulation.
  • the pharmaceutical compositions can include an amount of the compound of formula I that is effective to increase frataxin mRNA levels in a cell.
  • the cell can be a mammalian cell.
  • the mammalian cell can be a human cell such as a lymphocyte, cardiomyocyte or neuronal cell.
  • the invention also provides an article of manufacture that includes the compound of formula I contained within packaging materials that have a label indicating that the compound of formula I can be used for treating Friedreich's ataxia.
  • the invention provides a method of treating, or preventing or delaying the onset of, a neurodegenerative or neuromuscular condition in a mammal such as a human.
  • the method involves administering to the mammal a compound of formula I in an amount effective to alter the level of histone acetylation in the mammal.
  • the compound of formula I may be administered orally or parenterally.
  • the method may also include identifying the mammal as one suffering from, or at risk for, the neurodegenerative or neuromuscular condition.
  • the neurodegenerative condition may be Huntington's disease, spinocerebellar ataxia, Friedreich's ataxia, Fragile X syndrome, Kennedy's disease, spinal and bulbar muscular atrophy, amyotrophic lateral sclerosis and Alzheimer's disease.
  • the neuromuscular condition may be spinal muscular atrophy or myotonic dystrophy.
  • the invention provides a method of treating, or preventing or delaying the onset of, Friedreich's ataxia in a mammal. This method involves administering to the mammal a compound of formula I in an amount effective to increase frataxin mRNA in the mammal. The method may include identifying the mammal as one suffering from or at risk for Friedreich's ataxia.
  • a mammal suffering from or at risk for Friedreich's ataxia may be identified by determining the length, extent or number of expansion of a GAA triplet repeat in intron 1 of the frataxin gene.
  • the mammal may also be identified by determining the level of frataxin mRNA or protein.
  • radicals, substituents, and ranges are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
  • Alkyl, alkoxy, alkenyl, and the like denote both straight and branched groups.
  • alkyl refers to a linear or branched hydrocarbon radical that is optionally unsaturated and optionally substituted with functional groups as described herein.
  • the alkyl group can contain from 1 to about 20 carbon atoms.
  • alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, 3-pentyl, hexyl, heptyl, or octyl.
  • alkyl is preferably (C 1 -C 8 )alkyl.
  • alkyl is preferably (C 1 -C 4 )alkyl.
  • alkyl group is an alkenyl group.
  • Alkenyl groups can be, for example, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 7-octenyl, and branched isomers thereof.
  • alkoxy refers to an optionally substituted alkyl group that is substituted with an oxygen radical.
  • Alkoxy can be, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexyloxy.
  • aryl refers to a monovalent aromatic hydrocarbon radical of 6-18 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Aryl groups are typically made up of 6-10 carbon atoms and additionally can possess optional substituents as described herein.
  • Typical aryl groups include, but are not limited to, radicals derived from benzene, naphthalene, anthracene, biphenyl, and the like.
  • heteroaryl refers to a monocyclic, bicyclic, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one (typically one to about three) nitrogen, oxygen, or sulfur atoms in an aromatic ring. Heteroaryl groups can possess optional substituents as described herein.
  • heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, ⁇ -carbolinyl, carbazolyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,
  • heteroaryl denotes a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms independently selected from non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl.
  • heteroaryl denotes an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • optionally substituted group refers to the substitution of a group in which one or more hydrogen atoms are each independently replaced with a non-hydrogen substituent.
  • Groups that are optionally substituted are typically substituted with one to five substituents. In other embodiments, optionally substituted groups are substituted with one to three substituents.
  • Typical substituents include, but are not limited to, —X, —R, —O ⁇ , ⁇ O —OR, —S ⁇ , —SR, —S( ⁇ O)R, —S( ⁇ O) 2 R, —S( ⁇ O) 2 O ⁇ , —S( ⁇ O) 2 OH, —OS( ⁇ O) 2 OR, —S( ⁇ O) 2 NR, —NR 2 , —N + R 3 , ⁇ NR, —N ⁇ C ⁇ O, —NCS, —NO, —NO 2 , ⁇ N 2 , —N 3 , NC( ⁇ O)R, —CX 3 , —C(O)O ⁇ , —C( ⁇ O)R, —C(O)OR, —C( ⁇ O)X, —C( ⁇ O)NRR, —C(S)R, —C(S)OR, —C(O)SR, —C(S)
  • any of the above groups that contain one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible.
  • the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
  • nitrogen protecting group refers to any group which, when bound to a nitrogen group, can serve to prevent undesired reactions from occurring at this group and which can be removed by conventional chemical or enzymatic steps to reestablish the free nitrogen (e.g, a —NH— group or a —N ⁇ group) at a later stage.
  • the hydroxyl, carboxyl, amino, and amido groups of the compounds described herein can include optional protecting groups. Suitable protecting groups are known to those skilled in the art. A large number of protecting groups and corresponding chemical cleavage reactions that can be used in conjunction with the compounds of the invention are described in Protective Groups in Organic Synthesis, Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991, ISBN 0-471-62301-6), which is incorporated herein by reference in its entirety. Included therein are hydroxyl protecting groups, carboxylic acid protecting groups, and amide-forming groups.
  • a “base” refers to any molecule, ion, or other entity that acts as a proton acceptor.
  • a base can be an organic compound or ion with an unshared electron pair. Typical bases include mono-, di-, and tri-alkyl substituted amines.
  • a base can also be an inorganic compound or ion, such as a metal oxide or metal hydroxide. Bases used in organic synthesis are well known to those of skill in the art. Many bases are disclosed in, for example, the Aldrich Handbook of Fine Chemicals, 2003-2004 (Milwaukee, Wis.).
  • solvent refers to a substance, usually a liquid, capable of dissolving another substance, e.g., a solid substance, semi-solid substance, or a liquid.
  • Typical solvents include water and organic solvents. It is appreciated by those of skill in the art that the solvent should not chemically react with any of the starting materials or reagents present in the reaction mixture, to any significant degree, under the reaction conditions employed.
  • solvent system refers to a medium that includes one or more solvents.
  • a solvent system can be homogeneous (miscible solvents) or heterogeneous (e.g, an organic/aqueous system).
  • reflux refers to the process of boiling a liquid solvent system in a vessel, for example, a vessel attached to a condenser, so that the vapors of the solvent system continuously condense for reboiling.
  • purifying refers to the process of ridding a substrate (e.g., crystals, an amorphous solid, a liquid, or an oil) of impurities. Suitable methods of purifying include, for example, filtering, washing, recrystallizing and drying, distilling, and chromatography.
  • isolated and purified refer to substances that are substantially free of other agents, for example, at least about 90%, at least about 95%, , at least about 98%, or, at least about 99% pure by weight.
  • anhydrous refers to a substance that contains less than about 10 wt. % water, less than about 1 wt. % water, less than about 0.5 wt. % water, less than about 0.1 wt. % water, or less than about 0.01 wt. % water.
  • Anhydrous conditions refer to reaction conditions that have less than about 2 wt. % water, less than about 1 wt. % water, less than about 0.5 wt. % water, less than about 0.1 wt. % water, or less than about 0.01 wt. % water present.
  • contacting refers to the act of touching, making contact, or of bringing into immediate proximity.
  • Compounds are typically contacted by forming a solution in a suitable solvent system.
  • the numerical ranges given herein are those amounts that provide functional results in the composition.
  • ranges are generally introduced with the term “about” to indicate a certain flexibility in the range.
  • the term “about” can refer to +/ ⁇ one integer from a given number or the upper or lower limit of range.
  • the term “about” can refer to +/ ⁇ two integers from a given number or the upper or lower limit of range.
  • the term “about” can also refer to +/ ⁇ 20% of a given number or numerical range.
  • the term “about” can refer to +/ ⁇ 10%, or +/ ⁇ 5% of a given number or numerical range.
  • the term “about refers to +/ ⁇ 1%.
  • the term “about” refers to exactly the given number or numerical range.
  • FIG. 1 are bar graphs illustrating the histone modifications on frataxin gene chromatin.
  • Chromatin immunoprecipitation (ChIP) experiments were performed with the FRDA cell line (GM15850) and the normal cell line (GM15851) using antibodies to the acetylated forms of human histones H3 and H4 (acetylated at the lysine residues indicated).
  • Primer pairs for the frataxin promoter (Pro), and regions immediately upstream (Up) and downstream (Down) of the GAA repeats in the first intron of the frataxin gene were used.
  • Relative recovery as determined by real-time PCR, is expressed in relation to GAPDH, and the recovery on the Up GAA region for each antibody is set to a value of 100.
  • FIG. 2 are bar graphs illustrating the effects of histone deacetylase inhibitors on acetylation and frataxin mRNA in FRDA cells.
  • A Effects of histone deacetylase inhibitors on the levels of H3 and H4 acetylation in an FRDA lymphoid cell line (15850B). Cells were either untreated or treated with the indicated compounds for 12 hours prior to isolation of acid soluble nuclear proteins, SDS-PAGE and western blotting with antibodies to total histone H4/H3 or acetylated H4/H3. The fold changes in normalized ratio of AcH4 or AcH3 to total H4 or H3 are shown in the bar graph.
  • B Frataxin mRNA levels were determined by quantitative RT-PCR. All values are normalized to GAPDH mRNA levels, which were unaffected by the HDAC inhibitors. Each of the HDAC inhibitors was tested at the IC 50 value reported by the commercial supplier, as indicated. Error bars are s.e.m.
  • FIG. 3 is an autoradiogram showing that HDAC inhibitors increase frataxin protein in the FRDA lymphoid cell line.
  • Cells were incubated with the indicated concentrations of HDAC inhibitors for 4 days prior to western blot analysis with antibody to human frataxin or actin. Equivalent amounts of total cell extract protein were loaded in each lane.
  • the fold changes in frataxin protein compared to untreated control cells (denoted “Ctrl” in the figure), normalized to the actin signals, are 1.6 (2.5 ⁇ M 4c /BML-210), 3.4 (5 ⁇ M 4c), and 3.5 (2.5 ⁇ M 4b).
  • FIG. 4 are bar graphs showing that HDAC inhibitors increase frataxin mRNA in primary lymphocytes from FRDA patients. Frataxin mRNA levels were determined by qRT-PCR, relative to that of GAPDH, in lymphocytes from an unaffected individual A (normal range of repeats) and his/her FRDA sibling (affected S, with frataxin alleles containing and 906 and 88 GAA repeats) (A); in lymphocytes from carrier C and affected AC (801 and 597 repeats) (B); and in lymphocytes from carrier D, and affected J (550 and 530 repeats) and M (1030 and 650 repeats) (C).
  • FIG. 5 are bar graphs illustrating the effects of HDAC inhibitors on histone acetylation at the frataxin gene.
  • HDAC inhibitor 4b increases histone acetylation at particular H3 and H4 lysines on the frataxin gene.
  • FRDA cells were treated with 4b (5 ⁇ M for 96 h) prior to ChIP with the indicated antibodies, and PCR was performed with primers for the region upstream of the GAA repeats. Data are shown for both untreated cell lines and the FRDA cells treated with 4b. Recovery is expressed as percent of GAPDH, and all values are normalized to those for GM15851 cells.
  • SAHA and TSA do not affect histone acetylation on the frataxin gene.
  • FRDA cells were incubated for 96 hours with 2.5 ⁇ M SAHA or 0.1 ⁇ M TSA and processed for ChIP as in (A). Recovery is expressed relative to untreated GM15850 cells, normalized for GAPDH. Error bars are s.e.m.
  • the invention provides small molecules that could be used to treat a neurological condition such as FRDA.
  • the invention concerns the discovery that lymphocytes from FRDA patients that have been incubated with histone deacetylase (HDAC) inhibitors show elevated levels of acetylated histones.
  • HDAC histone deacetylase
  • the invention concerns the discovery that the HDAC inhibitors, suberoylanilide orthoaminoanilide (SAOA, BML-210) and pimeloylanilide orthoaminoanilide (PAOA), as well as novel derivatives of SAOA and PAOA, have the effect of increasing frataxin mRNA and protein expression in lymphocytes from FRDA patients.
  • the invention provides pharmaceutical compositions of HDAC inhibitors and their use as therapeutics for chronic and acute neurological diseases such as, for example, Friedreich's ataxia.
  • the invention also provides novel HDAC inhibitors, as well as novel methods for their synthesis.
  • FRDA DNA abnormality found in 98% of FRDA patients is the unstable hyper-expansion of a GAA triplet repeat in the first intron of the frataxin gene that results in a defect in transcription of the frataxin gene (see Campuzano et al. (1996) Science 271: 1423-7).
  • FRDA patients have a marked deficiency of frataxin mRNA, and longer GAA triplet repeats also cause a more profound frataxin deficiency.
  • FRDA is typical of triplet repeat diseases: normal alleles have 6-34 repeats while FRDA patient alleles have 66-1700 repeats. Longer GAA triplet repeats are associated with earlier onset and increased severity of the disease.
  • the invention provides for histone deacetylase (HDAC) inhibitors that can restore gene function in a neurological disease that is associated with expansion of a triplet repeat such as FRDA.
  • HDAC histone deacetylase
  • a HDAC of the invention can increase frataxin mRNA and protein in lymphocytes from FRDA patients.
  • a “histone deacetylase inhibitor” is a small molecule that binds to one or more histone deacetylase (HDAC) to modulate the levels of acetylation of histones, non-histone chromosomal proteins, and other cellular proteins.
  • An HDAC inhibitor of the invention may interact with a HDAC to modulate the level of acetylation of cellular targets.
  • a histone deacetylase may be any polypeptide having features characteristics of polypeptides that catalyze the removal of the acetyl group (deacetylation) from acetylated target proteins.
  • HDAC histone deacetylase
  • a HDAC may be a polypeptide that represses gene transcription by deacetylating the ⁇ -amino groups of conserved lysine residues located at the N-termini of histones, e.g. H3, H4, H2A and H2B, that form the nucleosome.
  • HDACs may also deacetylate other proteins such as p53, E2F, ⁇ -tubulin and Myo D. See Annemieke et al. (2003) Biochem. J. 370: 737. HDAC may also be localized to the nucleus or one that may be found in both the nucleus and cytoplasm.
  • An HDAC inhibitor of the invention may interact with any HDAC.
  • an HDAC inhibitor of the invention may interact with HDAC from one of the three known classes of HDAC.
  • An HDAC inhibitor of the invention may interact with an HDAC of the class I or class II family of HDAC.
  • Class I HDACs are those that most closely resemble the yeast transcriptional regulator RPD3. Examples of class I HDACs include HDACs 1, 2, 3 and 8, as well as any HDAC that has a deacetylase domain exhibiting from 45% to 93% identity in amino acid sequence to HDACs 1, 2, 3 and 8.
  • Class II HDACs are those that most closely resemble the yeast HDA1 enzyme, and examples of class II HDACs include HDACs 4, 5, 6, 7, 9 and 10.
  • HDAC inhibitor of the invention may also interact with the NAD + -dependent family of HDACs, which most closely resemble the yeast SIR2 protein.
  • An HDAC inhibitor of the invention may also interact with HDACs that do not fall into one of the above classes, see e.g. Gao et al. (2002) J. Biol. Chem. 277: 25748.
  • HDAC inhibitors of the invention include SAOA and PAOA, derivatives of SAOA and PAOA described herein and salts thereof.
  • HDAC inhibitors of the invention include compounds of formula I: wherein:
  • the alkyl, aryl or heteroaryl substitutent may be other than carboxyl, carboxy ester, or carboxamide.
  • R 1 can be aryl. In another embodiment, R 1 can be heteroaryl. In other embodiments, R 1 can be phenyl, 2-aminophenyl, 3-aminophenyl, 4-aminophenyl, 2-methoxyphenyl, 3-methoxyphenyl, or 4-methoxyphenyl. In other embodiments R 1 can be 2-methylphenyl, 3-methylphenyl, or 4-methylphenyl. In other embodiments, R 1 can be 2,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 2,4-diaminophenyl, 3,5-diaminophenyl, or 3,4,5-triaminophenyl. In still other embodiments, R 1 can be 2-pyridinyl, 3-quinolinyl, or 8-quinolinyl.
  • R 2 can be aryl. In another embodiment, R 2 can be heteroaryl. In certain embodiments, R 2 can be phenyl, 2-aminophenyl, 3-aminophenyl, 4-aminophenyl, 2-methoxyphenyl, 3-methoxyphenyl, or 4-methoxyphenyl. In other embodiments R 1 can be 2-methylphenyl, 3-methylphenyl, or 4-methylphenyl. In other embodiments, R2 can be 2,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl, 2,4-diaminophenyl, 3,5-diaminophenyl, or 3,4,5-triaminophenyl. In yet another embodiment, R 2 can be 2-pyridinyl, 3-quinolinyl, or 8-quinolinyl.
  • R 1 and R 2 can be the same. In other embodiments, R 1 and R 2 are not the same.
  • R a is H. In another embodiment, R b is H. In yet another embodiment, R a is a nitrogen protecting group. In yet another embodiment, R b can be a nitrogen protecting group.
  • n is about 3 to about 6. In another embodiment, n is 5. In yet another embodiment, n is 6.
  • R 1 can be substituted with one or more substituents.
  • R 1 can be substituted with one to about five, or one to about three, substituents.
  • R 1 can be substituted with two amino groups.
  • R 1 can be substituted with two methoxy groups.
  • R 2 can be substituted with one or more substituents.
  • R 2 can be substituted with one to about five, or one to about three, substituents.
  • R 2 can be substituted with two amino groups.
  • R 2 can be substituted with two methoxy groups.
  • compounds of formula I may be prepared by contacting a compound of formula V: with one or more coupling agents and a compound of formula VI: R 2 —NH(R b ) (VI) to provide the compound of formula I.
  • the coupling agents may be 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt).
  • EDC 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride
  • HOAt 1-hydroxy-7-azabenzotriazole
  • the coupling of compounds of formula V and VI may be carried out in the presence of one or more basic compounds. Suitable basic compounds or “bases” include alkyl amines.
  • the alkyl amine may be tri-alkyl substituted amines, for example, triethylamine or diisopropylethylamine.
  • Hindered amines such as 2,6-lutidine and 2,4,6-collidine may also be used in certain embodiments of the invention.
  • the coupling of compounds of formula V and VI may be carried out in the presence of a solvent system.
  • Typical solvent systems may be one solvent or more than one solvent.
  • the solvent system may be one or more organic solvents.
  • Two component solvent systems include two solvents that are miscible with one another.
  • the solvent system may dissolve the compounds of formula V and VI to a degree that allows the reaction to proceed to the formation of the compound of formula I.
  • Suitable solvents include dimethylformamide, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone (NMP), tetrahydrofurane (THF), 1,4-dioxane, dichloromethane, and any other suitable non-protic solvent.
  • the compound of formula I may be isolated and purified. Purification techniques that may be used include precipitation, filtration, recrystallization, and other forms of chromatography including, but not limited to GC, HPLC, reverse phase chromatography, gel plate, thin layer chromatography and the like.
  • the invention also provides a method of preparing the compound of formula V by contacting a compound of formula III: with a compound of formula IV: R 1 —NH(R a ) (IV) to provide the compound of formula V.
  • the compounds of formula III and IV may be contacted in the presence of a solvent system.
  • the solvent system may include one or more organic solvents. Suitable solvents include ether, tetrahydrofuran, and dioxane. In one embodiment, the solvent is tetrahydrofuran.
  • the invention also provides a method of preparing the compound of formula III by contacting a compound of formula II: with a dehydrating agent to provide the compound of formula III.
  • the dehydrating agent may be a carboxylic anhydride.
  • the carboxylic anhydride is acetic anhydride.
  • Other alkyl or aryl carboxylic anhydrides may also be employed in the reaction.
  • the formation of compounds of formula III are typically carried out under anhydrous conditions. Anhydrous conditions may be achieved by suitable drying of reactants, reagents, and equipment.
  • the compound of formula II and the dehydrating agent may be heated to facilitate the formation of the compound of formula III.
  • the temperature of the reaction may be increased, for example, to about 35° C., to about 40° C., to about 50° C., to about 70° C., or to about 100° C.
  • the temperature of the reaction may also be determined by the temperature at which the solvent system achieves reflux. In such cases, the reaction may be to the reflux temperature of the solvent system employed.
  • the synthetic protocol for preparing a compound of formula V from a compound of formula II may be illustrated as shown below in Scheme 1:
  • the compound of formula III can be isolated and purified.
  • the compound of formula III can be converted to the compound of formula V directly without purification.
  • HDAC inhibitors of the invention may be prepared as described herein or using any other applicable techniques of organic synthesis known in the art. Many applicable techniques not described herein are well known in the art. However, many of the known techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Volumes 1-6; as well as March, J., Advanced Organic Chemistry, 3 rd Ed. (John Wiley & Sons, New York, 1985), and Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modern Organic Chemistry, in 9 Volumes, Barry M. Trost, Ed.-in-Chief (Pergamon Press, New York, 1993 printing).
  • reaction conditions such as temperature, reaction time, solvents, work-up procedures, and the like, will be those common in the art for the particular reaction to be performed.
  • the cited reference material, together with material cited therein, contains detailed descriptions of such conditions.
  • the temperatures will be about ⁇ 100° C. to about 200° C.
  • solvents will be aprotic or protic depending on the conditions required
  • reaction times will be 1 minute to 10 days. Reaction times are adjusted to achieve desired conversions.
  • Work-up of reactions can include removal of solvent to provide crude products, precipitation and filtration, and/or quenching of any unreacted reagents followed by partition between a water / organic layer system (extraction) and separation of the layer containing the product.
  • protecting group may refer to any group which, when bound to a hydroxyl, nitrogen, or other heteroatom, prevents undesired reactions from occurring at this group and which can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl group.
  • removable protecting group employed is not critical and preferred removable hydroxyl and nitrogen protecting groups include conventional substituents such as, for example, allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, methyl methoxy, silyl ethers (e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)) and any other group that can be introduced chemically onto a hydroxyl or nitrogen functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
  • silyl ethers e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), or t-butyldimethylsilyl (TBS)
  • TMS trimethylsilyl
  • TDPS t-buty
  • Suitable hydroxyl protecting groups are known to those skilled in the art and disclosed in more detail in T. W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981, (“Greene”) and the references cited therein, and Kocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York, 1994), both of which are incorporated herein by reference in its entirety.
  • Protecting groups are available, commonly known and used, and are optionally used to prevent side reactions with the protected group during synthetic procedures, i.e. routes or methods to prepare the compounds by the methods of the invention. For the most part the decision as to which groups to protect, when to do so, and the nature of the chemical protecting group will be dependent upon the chemistry of the reaction to be protected against (e.g., acidic, basic, oxidative, reductive or other conditions) and the intended direction of the synthesis.
  • protecting groups do not need to be, and generally are not, the same if the compound is substituted with multiple protecting groups.
  • protecting groups will be used to protect functional groups such as carboxyl, hydroxyl, thio, or amino groups and to thus prevent side reactions or to otherwise facilitate the synthetic efficiency.
  • the order of deprotection to yield free, deprotected groups is dependent upon the intended direction of the synthesis and the reaction conditions to be encountered, and may occur in any order as determined by the artisan.
  • protecting groups for —OH groups include “ether- or ester-forming groups”.
  • Ether- or ester-forming groups are capable of functioning as chemical protecting groups in the synthetic schemes set forth herein.
  • some hydroxyl and thio protecting groups are neither ether- nor ester-forming groups, as will be understood by those skilled in the art.
  • carboxylic acid protecting groups and other protecting groups for acids see Greene as set forth below.
  • Such groups include by way of example and not limitation, esters, amides, hydrazides, and the like.
  • Ester- and Amide-forming groups include: (1) carboxyl ester/amide-forming groups, and (2) sulfur ester-forming groups, such as sulfonate, sulfate, and sulfinate.
  • a protecting group typically is bound to any acidic group such as, by way of example and not limitation, a —CO 2 H group, thereby resulting in —CO 2 R a where R a is as defined herein.
  • Examples of protecting groups include:
  • Heterocycle or aryl radicals optionally can be polycyclic or monocyclic. Examples include phenyl, spiryl, 2- and 3-pyrrolyl, 2- and 3-thienyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 3- and 4-isoxazolyl, 2-, 4- and 5-thiazolyl, 3-, 4- and 5-isothiazolyl, 3- and 4-pyrazolyl, 1-, 2-, 3- and 4-pyridinyl, and 1-, 2-, 4- and 5-pyrimidinyl; and
  • Such groups include 2-, 3- and 4-alkoxyphenyl (C 1 -C 12 alkyl), 2-, 3- and 4-methoxyphenyl, 2-, 3- and 4-ethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-diethoxyphenyl, 2- and 3-carboethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-5-hydroxyphenyl, 2- and 3-ethoxy-6-hydroxyphenyl, 2-, 3- and 4-O-acetylphenyl, 2-, 3- and 4-dimethylaminophenyl, 2-, 3- and 4-methylmercaptophenyl, 2-, 3- and 4-halophenyl (including 2-, 3- and 4-fluorophenyl and 2-, 3- and 4-chlorophenyl), 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dimethylphenyl, 2,3-, 2,4-, 2,5-, 2,
  • protecting groups are ester moieties that, for example, can be bonded via an oxygen of the compound of the invention to —C(O)—O—PG′ wherein PG′ is —CH 2 —C(O)—N(R 1 ) 2 , —CH 2 —S(O)(R 1 ), —CH 2 —S(O) 2 (R 1 ), —CH 2 —O—C(O)—CH 2 —C 6 H 5 , 3-cholesteryl, 3-pyridyl, N-ethylmorpholino, —CH 2 —O—C(O)—C 6 H 5 , —CH 2 —O—C(O)—CH 2 CH 3 , —CH 2 —O—C(O)—C(CH 3 ) 3 , —CH 2 —CCl 3 , —C 6 H 5 , —NH—CH 2 —C(O)O—CH 2 CH 3 , —N(CH 3 )—CH 2 —
  • esters can be synthesized by reacting the compound herein having a free hydroxyl (or acid group) with the corresponding halide (chloride or acyl chloride and the like) and N,N-dicyclohexyl-N-morpholine carboxamidine (or another base such as DBU, triethylamine, CsCO 3 , N,N-dimethylaniline and the like) in DMF (or other solvent such as acetonitrile or N-methylpyrrolidone). Coupling reagents can be used to facilitate linkage of the compound and the protecting group.
  • Other esters can be synthesized by the methods described by Greene, or by other methods well known to those of skill in the art.
  • Protecting groups also includes “double ester” forming pro-functionalities such as —CH 2 OC(O)OCH 3 , —CH 2 SCOCH 3 , —CH 2 OCON(CH 3 ) 2 , dihydro-furan-2-one-5-yl, or alkyl- or aryl-acyloxyalkyl groups (linked to oxygen of the acidic group) (see U.S. Pat. No. 4,968,788).
  • Another example is the pivaloyloxymethyl group.
  • alkylacyloxymethyl esters and their derivatives including: 2-(adamantine-1-carboxylate)-ethyl, —CH(CH 2 CH 2 OCH 3 )OC(O)C(CH 3 ) 3 , —CH 2 OC(O)C 10 H 15 , —CH 2 O C(O)C(CH 3 ) 3 , —CH(CH 2 OCH 3 )OC(O)C(CH 3 ) 3 , —CH(CH(CH 3 ) 2 )OC(O)C(CH 3 ) 3 , —CH 2 OC(O)CH 2 CH(CH 3 ) 2 , —CH 2 O C(O)C 6 H 11 , —CH 2 OC(O)C 6 H 5 , —CH 2 OC(O)C 10 H 15 , —CH 2 OC(O)CH 2 CH 3 , —CH 2 OC(O)CH(CH 3 ) 2 , —CH 2 OC(O)C 10 H 15
  • One or more acidic hydroxyls can be protected. If more than one acidic hydroxyl is protected then the same or a different protecting group can be employed, e.g., the esters may be different or the same, or a mixed amidate and ester may be used.
  • Typical nitrogen and hydroxy protecting groups described in Greene include substituted methyl and alkyl ethers, substituted benzyl ethers, silyl ethers, esters including sulfonic acid esters, carbonates, sulfates, and sulfonates.
  • substituted methyl and alkyl ethers substituted benzyl ethers, silyl ethers, esters including sulfonic acid esters, carbonates, sulfates, and sulfonates.
  • HDAC inhibitors of the invention may be used prophylactically or as a treatment for various neurodegenerative or neuromuscular conditions. More specifically, a HDAC inhibitor of the invention may be used to delay or prevent the onset of a neurodegenerative or neuromuscular condition, as well as to treat a mammal suffering from a neurodegenerative or neuromuscular condition.
  • neurodegenerative conditions include, without limitation, fragile X syndrome, Friedreich's ataxia, Huntington's disease, spinocerebellar ataxias, amyotrophic lateral sclerosis, Kennedy's disease, spinal and bulbar muscular atrophy and Alzheimer's disease.
  • neuromuscular conditions include spinal muscular atrophy and myotonic dystrophy.
  • Mammals, e.g. humans, to which HDAC inhibitors may be administered include those suffering from the conditions discussed above as well as those who are at risk for developing the above conditions.
  • a mammal at risk for developing a neurodegenerative condition may be identified in numerous ways, including, for example, first determining (1) the length, extent or number of repeats of particular nucleic acid sequences in the individual's genome; the degree of acetylation of core histones; or the expression level of a particular mRNA or protein, and then (2) comparing it with that of a normal individual.
  • An individual at risk for developing a neurodegenerative or neuromuscular condition is one who has an aberrant number of repeat of a particular nucleic aid sequence, degree of acetylation of core histones or expression of a particular gene.
  • a mammal at risk for developing Friedreich's ataxia may be identified by determining the length, extent or number of repeats of a GAA triplet in the first intron of the dataxin gene.
  • a mammal would be at risk for Friedreich's ataxia if the above analysis indicates that there are more than 34 repeats of the GAA triplet, for example, if the mammal has more than 66 repeats of the GAA triplet.
  • a mammal at risk for Friedreich's ataxia could also be identified by determining the levels of frataxin mRNA or protein expressed in the mammal.
  • a mammal would be at risk for Friedreich's ataxia if the levels of frataxin mRNA or protein is lower than the level normally observed in a healthy individual such as for example, an unaffected sibling.
  • the amount of HDAC inhibitor to be administered to the mammal may be any amount appropriate to restore the level of histone acetylation, or the level of mRNA or protein expression, in the afflicted mammal to that typical of a healthy individual such as an unaffected sibling.
  • the amount of the HDAC inhibitor to be administered may be an effective dose or an appropriate fraction thereof. Such amounts will depend on individual patient parameters including age, physical condition, size, weight, the condition being treated, the severity of the condition, and any concurrent treatment. For example, the effective dose range that is necessary to prevent or delay the onset of the neurodegenerative condition may be significantly lower than the effective dose range for inhibiting the progression of the condition being treated.
  • Factors that determine appropriate dosages are well known to those of ordinary skill in the art and may be addressed with routine experimentation. For example, determination of the physicochemical, toxicological and pharmacokinetic properties may be made using standard chemical and biological assays and through the use of mathematical modeling techniques known in the chemical, pharmacological and toxicological arts. The therapeutic utility and dosing regimen may be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and/or pharmacodynamic models. The precise amount of HDAC inhibitor administered to a patient will be the responsibility of the attendant physician. However, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • HDAC inhibitors of the invention may be administered in numerous ways.
  • HDAC inhibitors of the invention may be administered orally, rectally, topically, or by intramuscular, intraperitoneal subcutaneous or intravenous injection.
  • the inhibitors are administered orally or by injection.
  • Other routes include intrathecal administration directly into spinal fluid and direct introduction onto, in the vicinity of or within the target cells. The route of administration may depend on the condition being treated and its severity.
  • HDAC inhibitors of the invention may be administered orally or by injection at a dose of from 0.1 to 30 mg per kg weight of the mammal, preferably 2 to 15 mg/kg weight of the mammal.
  • the dose range for adult humans is generally from 8 to 2,400 mg/day and preferably 35 to 1,050 mg/day.
  • HDAC inhibitors may be administered neat or, preferably, as pharmaceutical compositions.
  • Pharmaceutical compositions of the invention include an appropriate amount of the HDAC inhibitor in combination with an appropriate carrier as well as other useful ingredients.
  • HDAC inhibitors of the invention include the compounds of formula I, and wherein applicable, acceptable salts thereof.
  • Acceptable salts include, but are not limited to, those prepared from the following acids: alkyl, alkenyl, aryl, alkylaryl and alkenylaryl mono-, di- and tricarboxylic acids of 1 to 20 carbon atoms, optionally substituted by 1 to 4 hydroxyls; alkyl, alkenyl, aryl, alkylaryl and alkenylaryl mono-, di- and trisulfonic acids of 1 to 20 carbon atoms, optionally substituted by 1 to 4 hydroxyls; and mineral acids.
  • Examples include hydrochloric; hydrobromic; sulphuric; nitric; phosphoric; maleic; acetic; salicyclic; p-toluenesulfonic; tartaric; citric; methanesulphonic; formic; malonic; succinic; naphthalene-2-sulphonic; and benzenesulphonic acid.
  • pharmaceutically-acceptable salts may be prepared as amine salts, ammonium salts, or alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. These are formed from alkaline metal or alkaline earth metal bases or from amine compounds.
  • analogs of the foregoing compounds that act as functional equivalents also are intended to be embraced as equivalents and within the scope of the invention.
  • compositions of HDAC inhibitors suitable for oral administration may be in the form of (1) discrete units such as capsules, cachets, tablets or lozenges each containing a predetermined amount of the HDAC inhibitor; (2) a powder or granules; (3) a bolus, electuary or paste; (4) a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or (4) an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • compositions suitable for topical administration in the mouth for example buccally or sublingually, include lozenges.
  • Compositions suitable for parenteral administration include aqueous and non-aqueous sterile suspensions or injection solutions.
  • Compositions suitable for rectal administration may be presented as a suppository.
  • compositions of HDAC inhibitors may be formulated using a solid or liquid carrier.
  • the solid or liquid carrier would be compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • HDAC inhibitor is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the carrier is a finely divided solid in admixture with the finely divided active ingredient.
  • the powders and tablets may contain up to 99% of the active ingredient.
  • Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • a solid carrier may include one or more substances that may act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents.
  • a suitable carrier may also be an encapsulating material.
  • liquid carriers may be used.
  • the HDAC inhibitor is dissolved or suspended in a pharmaceutically acceptable liquid carrier.
  • suitable examples of liquid carriers for oral and parenteral administration include (1) water, (2) alcohols, e.g. monohydric alcohols and polyhydric alcohols such as glycols, and their derivatives, and (3) oils, e.g. fractionated coconut oil and arachis oil.
  • the carrier may also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Liquid carriers for pressurized compositions include halogenated hydrocarbon or other pharmaceutically acceptable propellent.
  • the liquid carrier may contain other suitable pharmaceutical additives such as solubilizers; emulsifiers; buffers; preservatives; sweeteners; flavoring agents; suspending agents; thickening agents; colors; viscosity regulators; stabilizers; osmo-regulators; cellulose derivatives such as sodium carboxymethyl cellulose; anti-oxidants; and bacteriostats.
  • suitable pharmaceutical additives such as solubilizers; emulsifiers; buffers; preservatives; sweeteners; flavoring agents; suspending agents; thickening agents; colors; viscosity regulators; stabilizers; osmo-regulators; cellulose derivatives such as sodium carboxymethyl cellulose; anti-oxidants; and bacteriostats.
  • Other carriers include those used for formulating lozenges such as sucrose, acacia, tragacanth, gelatin and glycerin as well as those used in formulating suppositories such as cocoa butter or polyethylene glycol.
  • solutions of the HDAC inhibitor may be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the composition suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium as described above.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the HDAC inhibitor in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the HDAC inhibitor, plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • compositions of the invention may be in unit-dose or multi-dose form or in a form that allows for slow or controlled release of the HDAC inhibitor.
  • Each unit-dose may be in the form of a tablet, capsule or packaged composition such as, for example, a packeted powder, vial, ampoule, prefilled syringe or sachet containing liquids.
  • the unit-dose form also may be the appropriate number of any such compositions in package form.
  • Pharmaceutical compositions in multi-dose form may be in packaged in containers such as sealed ampoules and vials.
  • the HDAC inhibitor may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Epstein Barr virus transformed lymphoblast cell lines GM15850 from a FRDA patient (alleles with 650 and 1030 GAA repeats in the frataxin gene, from the Coriell Cell Repository, Camden, N.J.), and GM15851 from an unaffected sibling (normal range of repeats), were propagated in RPMI 1640 medium with 2 mM L-glutamine and 15% fetal bovine serum at 37° C. in 5% CO 2 . Cell growth and morphology were monitored by phase contrast microscopy, and viability by trypan blue exclusion. HDAC inhibitors were dissolved in DMSO and added to the culture medium at the concentrations indicated in the table and figure captions, for the indicated times.
  • the final DMSO concentration in the culture medium did not exceed 0.5% (v/v). All control samples were treated with the same concentration of DMSO lacking compounds.
  • the suppliers of the HDAC inhibitors were: valproic acid (VPA), Calbiochem (San Diego, Calif.); trichostatin A (TSA), suberoyl bis-hydroxamic acid (SBHA), suberoylanilide hydroxamic acid (SAHA), and BML-210, Bio-Mol (Plymouth Meeting, Pa.); each tested at the IC 50 value reported by the supplier, as indicated in the figures.
  • Quantitative real-time RT-PCR was performed using iScript One-Step RT-PCR kit with SYBR green (BioRad). Statistical analysis was performed on three independent quantitative RT-PCR experiments for each RNA sample, and error bars shown in the figures represent standard errors of the mean.
  • Protein levels in HDAC inhibitor-treated and untreated cells were monitored by western blotting with antibodies to histones H3 and H4 (Upstate Biotechnology) or with antibodies to the acetylated versions of these proteins. Histones were purified by acid extraction as described in the protocols provided by Upstate Biotechnology. Antibodies to human frataxin were from Mitoscience (Eugene, Oreg.) and anti-actin antibodies were from Santa Cruz Biotechnology (CA). Total cell extracts were used for frataxin and actin western blots. Signals were detected by chemiluminescence after probing the blot with HRP-conjugated secondary antibody (Supersignal West, Pierce). To quantify the relative levels of proteins, autoradiograms (within the linear response range of X-ray film) were converted into digital images and the signals quantified using Molecular Dynamics ImageQuant software.
  • Each of the histone deacetylase inhibitors was assayed with the BioMol AK500 kit to determine IC 50 values. Samples were processed as described by BioMol and read with a 96-well fluorescence plate reader. A semi-logarithmic plot of the data was analyzed with Kaleidagraph software to obtain the IC 50 value.
  • the Friedreich's Ataxia Research Alliance (Arlington, Va.) recruited a series of families with affected individuals and siblings or parents for anonymous blood donation (with a Human Subjects Protocol approved by the Scripps Clinic Human Subjects Committee and by NINDS, with appropriate informed consent). Blood was collected in heparinized Vacutainer tubes (#364680, BD Biosciences) and lymphocytes were isolated by density centrifugation using Ficoll-Paque PLUS (Amersham Biosciences), according to the manufacturer. Lymphocytes were maintained in the same culture medium and conditions as the established cell lines, and HDAC inhibitor treatment was as described above. Cells were treated with HDAC inhibitors after 16 h, and RNA isolated after subsequent 48 h incubation. Under these culture conditions, no increases in cell number were observed.
  • the synthetic scheme is as shown below.
  • the RP-HPLC system was set to a flow rate of 5 mL/min in 10% acetonitrile/water/0.1% TFA—100% acetonitrile/0.1% TFA for 0-60 minutes and then 100% acetonitrile/0.1% TFA—10% acetonitrile/water/0.1% TFA for 60-75 minutes. All MALDI-ToF spectra were measured on Voyager System 1089 from Applied Biosystems; ⁇ -cyanohydroxy-cinnamic (CHCA) acid was used as matrix. For flash column chromatography, silica gel (mesh 60-200) purchased from J. T. Baker was used. TLC plates were purchased from J. T. Baker (Si250F).
  • CHCA cyano-4-hydroxycinnamic acid
  • DCM diichloromethane
  • DIPEA diisopropylethylamine
  • DMSO dimethylsulfoxide
  • DMF dimethylformamide
  • EDC ⁇ HCl N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • HOBt 1-hydroxy-1H-benzotriazole hydrate
  • MeOH methanol
  • room temperature r.t
  • THF tetrahydrofurane
  • a solution of adipic acid (5.00 g, 34.0 mmol) in acetic anhydride (10 mL) was heated under reflux for 1 hour. After cooling to room temperature, the solvent was removed in vacuo. The crude yellow oil was used without any further purification for the next step.
  • Aniline (3.00 mL, 28.7 mmol) was added to a stirred solution of the produced anhydride in anhydrous THF (10 mL). After stirring at room temperature for 1 hour, the solvent was removed and the residue was recrystallized from water/acetonitrile.
  • Pimelic acid 160 mg, 1.00 mmol was refluxed in 5 mL acetic anhydride for 1 hour. The solvent was removed to complete dryness. The residue was diluted in 10 mL dry THF, then P1 (200 mg, 0.96 mmol) in 5 mL dry THF was added drop-wise. The reaction was stirred overnight and then diluted with water. The resulting colorless solid was collected by filtration and recrystallized from ethanol.
  • Boc protecting groups were cleaved by adding a mixture of DCM/TFA (5 mL, 3:2) and stirring for 2 hours at room temperature. After the solvent was removed, the product was lyophilized. If necessary the product can be further purified by preparative HPLC.
  • Pimelic acid 200 mg, 1.25 mmol was refluxed in 10 mL acetic anhydride for 30 minutes. The solvent was removed to complete dryness. The residue was diluted in 5 mL dry THF, then P2 (208 mg, 1.00 mmol) in 5 mL dry THF was added dropwise. The reaction was stirred overnight and then the solvent was evaporated. The residue was taken up in 100 mL ethylacetate and then washed two times with 50 mL with water and two times with saturated NaCl. After drying over MgSO 4 the solvent was evaporated. The crude residue was purified by flashcolumn chromatography on silica gel (DCM/MeOH 40:1).
  • the synthesis method was the same as that for 100b, with the exception that p-toluidine was used in place of o-toluidine.
  • the cell line from the FRDA patient has a markedly lower level (13 ⁇ 6%, range of 20 determinations (Burnett et al. P.N.A.S. 103: 11497-502 (2006)) of frataxin mRNA compared to the cell line from the unaffected sibling, as determined by quantitative real time/reverse transcriptase PCR (qRT-PCR, see below).
  • Primers that interrogate the chromatin regions upstream or downstream of the GAA ⁇ TTC repeats in the first intron of the frataxin gene, as well as the promoter element, were used in the ChIP experiments, with the levels of immunoprecipitated DNA quantified by real-time PCR ( FIG. 1A ).
  • GAPDH glyceraldehyde-3-phosphodehydrogenase
  • H3-K9 is highly trimethylated in the FRDA cell line compared to the normal cell line ( FIG. 1B ).
  • trimethylation of H3-K9 is a hallmark of heterochromatin (Elgin & Grewal, Curr. Biol. 13:R895-8 (2003)).
  • heterochromatin Elgin & Grewal, Curr. Biol. 13:R895-8 (2003).
  • the histone postsynthetic modification states within the coding region of inactive frataxin alleles are consistent with a chromatin-mediated mechanism as the cause of gene silencing in FRDA (Saveliev et al. Nature 422:909-13 (2003)).
  • results in FIG. 2A indicate that all of the HDAC inhibitors increased the levels of total acetylated histones in the FRDA cell line when used at their reported IC 50 value for HDAC inhibition. Cyclic peptide inhibitors were also tested, but were found to be highly cytotoxic to the lymphoid cells, and thus not pursued. Each of the HDAC inhibitors was also tested for effects on frataxin mRNA levels in the FRDA cell line by qRT-PCR (at the IC 50 values), and only BML-210 increased the level of frataxin mRNA ⁇ 2-fold ( FIG. 2B ). The levels of GAPDH mRNA were not changed by the HDAC inhibitors and were used for normalization in all qRT-PCR experiments.
  • BML-210 is not cytotoxic to the lymphoid cell lines (determined by trypan blue exclusion) and does not markedly affect cell growth rates.
  • the structurally related compound SAHA had no effect on frataxin transcription and SBHA had a negative effect (50% decrease), even though both compounds were more effective HDAC inhibitors than BML-210 ( FIG. 2A ).
  • the compounds were tested for their effects on frataxin mRNA levels in the FRDA cell line by qRT-PCR and for their activity as HDAC inhibitors in a HeLa nuclear extract (Table 4).
  • the IC 50 values representing the general HDAC inhibitory activities of these compounds, range from 14 ⁇ M (compound 16b) to >1000 ⁇ M (compound 14b, Table 4).
  • the same IC 50 values were obtained with an extract from FRDA lymphoid cells for several of the compounds (not shown). For activation of transcription, each of the compounds was tested at a concentration of 5 ⁇ M in culture medium for 96 hours.
  • the quinoline derivatives of pimeloylanilide are also highly active (compounds 11b and 12b).
  • the symmetric diamino compound 16b N 1 ,N 7 -bis(2-aminophenyl) heptanediamide, is the most effective compound in the FRDA cell line (3.1-fold increase in frataxin mRNA at 5 ⁇ M and 3.5-fold increase at 10 ⁇ M).
  • 16b exhibits an IC 50 value of 14 ⁇ M in a HeLa nuclear extract HDAC inhibition assay, compared to an IC 50 of 87 ⁇ M for 4c and 78 ⁇ M for 4b.
  • HDAC Inhibitors Increase Frataxin Protein Levels
  • HDAC Inhibitors Increase Frataxin mRNA in Primary Lymphocytes from FRDA Patients
  • Frataxin protein deficiency in the human disease affects non-proliferating cell types (neuronal cells and cardiomyocytes). While these human cells are not readily available for experimentation, primary lymphocytes can be obtained from donor blood, and lymphocytes that are not treated with cytokines do not divide in culture under the conditions of our experiments. We thus tested the effect of HDAC inhibitors on frataxin mRNA levels in primary lymphocytes obtained from FRDA patients and carrier or normal relatives of these patients (under an approved Human Subjects Protocol, with appropriate informed consent).
  • Lymphocytes were isolated by Ficoll gradient centrifugation, and cells were incubated in culture for 16 h prior to the addition of 2.5 or 5 ⁇ M 4b or 4c to the culture medium; cells were harvested and RNA purified after an additional 48 hours in culture. Similar to the established cell lines, the HDAC inhibitors did not affect viability of primary lymphocytes over this time period. Lymphocytes from affected individual S had 33 ⁇ 2% of the level of frataxin mRNA compared to lymphocytes from his/her homozygous normal sibling A ( FIG. 4A ).
  • the relative levels of frataxin mRNA increased by 1.8-fold (80% increase) with 5 ⁇ M 4c, and by 2.3-fold (130% increase) with 5 ⁇ M 4b in FRDA lymphocytes.
  • frataxin mRNA was nearly doubled in the carrier, suggesting that the inactive frataxin allele has been nearly completely re-activated. While differences in the fold-increases in frataxin mRNA are observed with 4b in primary lymphocytes from different donors (compare FIGS. 4 A-C), this compound consistently increases frataxin mRNA in FRDA and carrier lymphocytes obtained from 12 out of 12 families, and in each instance the frataxin mRNA level in the FRDA lymphocytes is increased to approximately that of untreated lymphocytes from a carrier relative. We have thus obtained a level of gene activation that represents a therapeutically useful increase in frataxin mRNA. We note that the HDAC inhibitors are more effective in primary lymphocytes than in the FRDA cell line, and this difference may be related to the more severe silencing of the frataxin gene observed in the FRDA cell line.
  • HDAC Inhibitors act Directly on the Frataxin Gene
  • Campuzano, V. et al Friedreich's ataxia autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423-7 (1996).
  • HDAC Histone deacetylase

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EP1951656B1 (de) 2015-08-05
US20130210918A1 (en) 2013-08-15
EP1951656A1 (de) 2008-08-06
US20150080472A1 (en) 2015-03-19
US8835502B2 (en) 2014-09-16
JP2009515887A (ja) 2009-04-16

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