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WO2015042685A1 - Inhibiteurs d'agrégats d'arn associés à la répétition de polynucléotides et leurs utilisations - Google Patents

Inhibiteurs d'agrégats d'arn associés à la répétition de polynucléotides et leurs utilisations Download PDF

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Publication number
WO2015042685A1
WO2015042685A1 PCT/CA2013/050730 CA2013050730W WO2015042685A1 WO 2015042685 A1 WO2015042685 A1 WO 2015042685A1 CA 2013050730 W CA2013050730 W CA 2013050730W WO 2015042685 A1 WO2015042685 A1 WO 2015042685A1
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compound
substituted
alkyl
aryl
optionally substituted
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PCT/CA2013/050730
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Pascal Chartrand
Emmanuelle QUERIDO
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Valorisation-Recherche
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Priority to CA2923503A priority Critical patent/CA2923503A1/fr
Priority to PCT/CA2013/050730 priority patent/WO2015042685A1/fr
Priority to EP13894694.2A priority patent/EP3049393A4/fr
Priority to US15/024,169 priority patent/US10092574B2/en
Publication of WO2015042685A1 publication Critical patent/WO2015042685A1/fr

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/16Amides, e.g. hydroxamic acids
    • A61K31/18Sulfonamides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4151,2-Diazoles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4151,2-Diazoles
    • A61K31/41551,2-Diazoles non condensed and containing further heterocyclic rings
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445Non condensed piperidines, e.g. piperocaine
    • A61K31/451Non condensed piperidines, e.g. piperocaine having a carbocyclic group directly attached to the heterocyclic ring, e.g. glutethimide, meperidine, loperamide, phencyclidine, piminodine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445Non condensed piperidines, e.g. piperocaine
    • A61K31/4523Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/53751,4-Oxazines, e.g. morpholine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/53751,4-Oxazines, e.g. morpholine
    • A61K31/53771,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/63Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00Drugs for disorders of the muscular or neuromuscular system
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/30Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/45Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups at least one of the singly-bound nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfonamides
    • C07C311/46Y being a hydrogen or a carbon atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D231/00Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10Heterocyclic 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
    • C07D231/14Heterocyclic 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
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D409/00Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00Systems containing only non-condensed rings
    • C07C2601/06Systems containing only non-condensed rings with a five-membered ring
    • C07C2601/08Systems containing only non-condensed rings with a five-membered ring the ring being saturated
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00Systems containing at least three condensed rings
    • C07C2603/56Ring systems containing bridged rings
    • C07C2603/58Ring systems containing bridged rings containing three rings
    • C07C2603/70Ring systems containing bridged rings containing three rings containing only six-membered rings
    • C07C2603/74Adamantanes

Definitions

  • the present invention generally relates to polynucleotide repeat disorders such as trinucleotide repeat disorders, and more particularly to compounds and uses thereof such as for the treatment of diseases associated with the presence of polynucleotide repeats, such as myotonic dystrophy.
  • Polynucleotide repeat disorders are a set of genetic disorders caused by polynucleotide repeat expansions (typically trinucleotide repeat expansions).
  • the expanded polynucleotide/trinucleotide repeats have been shown to cause the retention of transcripts in the nucleus, where it accumulates in numerous foci (RNA aggregates).
  • RNA aggregates or foci have been identified in different pathologies, for example Myotonic Dystrophy type 1 (DM1 ) (Davis et al., 1997) and type 2 (DM2) (Liquori et al., 2001 ), Fragile X-associated tremor/ataxia syndrome (FXTAS) (Tassone et al., 2004), Spinocerebellar ataxia type 8 (SCA8) (Daughters et al., 2009) and Huntington's disease-like 2 (HDL2) (Rudnicki et al., 2007). All these diseases are characterized by microsatellite expansions of CNG or CCTG repeats in specific genes, leading to the accumulation of their transcripts as nuclear RNA foci (Ranum and Cooper, 2006).
  • DM1 Myotonic Dystrophy type 1
  • DM2 type 2
  • FXTAS Fragile X-associated tremor/ataxia syndrome
  • SCA8 Spinocer
  • Myotonic dystrophy is a chronic, slowly progressing, highly variable, inherited multisystemic disease.
  • Type 1 also known as Steinert disease
  • Type 2 also known as proximal myotonic myopathy (PROMM)
  • PROMM proximal myotonic myopathy
  • DM1 is the most common adult form of muscular dystrophy with a prevalence of up to 1 in 7,000 worldwide.
  • DM1 is highly prevalent in the Saguenay region of Quebec where its carrier rate reaches 1 in 550, making DM1 a Canadian-specific disease.
  • FIG. 1 It is a multisystemic disorder (FIG. 1 ), caused by an expansion of CUG trinucleotide repeats in the 3' untranslated region (UTR) of the protein kinase DMPK mRNA (Brook et al. 1992, Buxton et al. 1992, Fu et al. 1992, Mahadevan et al. 1992).
  • the expanded CUG repeats have been shown to cause the retention of this transcript in the nucleus, where it accumulates in numerous foci (FIG. 2; Taneja et al. 1995, Davis et al. 1997).
  • the present invention relates to compounds, as well as to uses thereof, such as to treat a polynucleotide repeat disorder (such as a trinucleotide repeat disorder, such as myotonic dystrophy).
  • a polynucleotide repeat disorder such as a trinucleotide repeat disorder, such as myotonic dystrophy.
  • the present invention provides method for treating a polynucleotide repeat disorder, said method comprising administering to a subject in need thereof an effective amount of a compound of formula (1 ), (2), or (3):
  • R 1 is:
  • aryl, heteroaryl, arylmethyl, or heteroarylmethyl said aryl, heteroaryl, arylmethyl, and heteroarylmethyl being optionally substituted on the aryl ring or on the methyl group by alkyl, alkenyl, or alkynyl, and said heteroaryl and heteroarylmethyl comprising only oxygen and/or nitrogen atoms as heteroatom(s), or
  • alkyl alkenyl or alkynyl, said alkyl, alkenyl and alkynyl being substituted by cycloalkyl, cycloalkenyl or cycloalkynyl,
  • R 2 and R 3 are independently:
  • alkyl alkenyl, or alkynyl
  • cycloalkyl cycloalkenyl, cycloalkynyl
  • R 2 and R 3 are not a hydrogen atom or a Ci-C 2 alkyl
  • R 2 and R 3 together with the nitrogen atom to which they are attached, form a 6- or more membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen atom,
  • R 4 and R 5 are independently: a hydrogen atom
  • alkyi alkenyl, or alkynyl
  • cycloalkyl cycloalkenyl, cycloalkynyl
  • R 4 and R 5 are not a hydrogen atom or a Ci-C 2 alkyi
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form a 6 membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen or oxygen atom.
  • R 6 is H, halogen, or CrC 8 alkyi optionally substituted with one or more halogen atoms,
  • R 7 is Ci-C 8 alkyi optionally substituted with one or more halogen atoms
  • R 8 and R 9 are independently alkyi, alkenyl, or alkynyl, said alkyi, alkenyl, and alkynyl being substituted by optionally substituted aryl or optionally substituted thioaryl, or
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5- or more membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen atom, and substituted with optionally substituted aryl,
  • R 7 is C-i alkyi
  • R 8 and R 9 are independently alkyi, alkenyl, or alkynyl, said alkyi, alkenyl, and alkynyl being substituted by aryl or thioaryl,
  • R 1 is substituted aryl, such as aryl substituted with one to three alkyi groups, preferably methyl groups.
  • the substituted aryl is phenyl substituted with two methyl groups, such as:
  • R 1 is an arylmethyl, preferably:
  • the arylmethyl such as benzyl
  • the alkyl group substitutes the methyl group of the arylmethyl.
  • R 1 is:
  • R 1 is an unsubstituted or substituted heteroaryl.
  • the heteroaryl comprises one or two oxygen atoms such as: :
  • the heteroaryl comprises one or two nitrogen atoms, preferably one nitrogen atom, such as:
  • the heteroaryl may be substituted, for example with one to three alkyl groups, preferably methyl groups.
  • the substituted heteroaryl is 1 -phenyl-1 H-pyrazole substituted with one methyl group, such as:
  • R 1 is an alkyl substituted cycloalkyl, preferably Ci -6 alkyl substituted with C 5 - 6 cycloalkyl. In a further embodiment R 1 is:
  • one of R 2 and R 3 is hydrogen or alkyl (preferably C1-C3 alkyl), and the other is alkyl comprising 3 or more carbon atoms, aralkyi (preferably phenyl-alkyl) or cycloalkyi (preferably cyclohexyl or adamantyl).
  • R 2 and R 3 are C 3-6 alkyl, preferably propyl.
  • R 2 is H and R 3 is a C 3 -C 6 alkyl, in a further embodiment, R 3 is a C 4 alkyl, such as n-butyl, ferf-butyl, or sec-butyl.
  • R 2 is H and R 3 is a C3-C12 cycloalkyi.
  • R 3 is adamantyl, such as adamant-1-yl:
  • R 2 is H and R 3 is an arylalkyl, such as benzyl.
  • R 2 is alkyl and R 3 is alkyl comprising more than 3 carbon atoms.
  • R 2 and R 3 form a substituted or unsubstituted 6- to 12-membered aliphatic heterocycle, which optionally comprises one or more additional nitrogen atom.
  • said heterocycle is 6-membered.
  • said heterocycle is 6- membered heterocycloalkyl comprising 0 or 1 additional nitrogen atom.
  • the substituents are one to three Ci -6 alkyl, preferably methyl.
  • R 2 and R 3 form:
  • R 1 is substituted aryl, such as aryl substituted with one to three alkyl groups.
  • the aryl is phenyl.
  • the alkyl groups are methyl. In embodiments, there are two methyl groups.
  • R 1 is:
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form a 6-membered, optionally substituted, aliphatic heterocycle, optionally comprising one or more additional nitrogen or oxygen atom.
  • the heterocycle comprises one further oxygen atom.
  • the heterocycle is substituted with one to three alkyl groups, preferably two methyl groups.
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form:
  • R 6 is H or a halogen, in a further embodiment the halogen is CI.
  • R 7 is a C-
  • R7 is methyl, ethyl, propyl, or -CF 3 , preferably methyl, propyl or -CF 3 , more preferably propyl.
  • R 8 and R 9 are independently alkyls, such as Ci -3 alkyls, preferably methyl groups, substituted by aryl or thioaryl, preferably phenyl and/or 2-thienyl.
  • NR 8 R 9 is:
  • R 7 when R 7 is not a C-i alkyl, R 8 and R 9 , together with the nitrogen atom to which they are attached, join to form a substituted 5- or more membered aliphatic heterocycle.
  • the aliphatic heterocycle may be pyrrolidinyl or piperidinyl.
  • the heterocycle is substituted with an optionally substituted aryl, preferably optionally substituted phenyl. Substituents for the aryl include C ⁇ e alkyl and alkyloxy.
  • NR 8 R 9 is:
  • the compound is:
  • the present invention provides the compound defined above for treating a polynucleotide repeat disorder in a subject. In another aspect, the present invention provides the compound defined above for the manufacture of a medicament for treating a polynucleotide repeat disorder in a subject.
  • the present invention provides the use of the compound defined above for treating a polynucleotide repeat disorder in a subject. In another aspect, the present invention provides the use of the compound defined above for the manufacture of a medicament for treating a polynucleotide repeat disorder in a subject.
  • the polynucleotide repeat disorder is myotonic dystrophy, in a further embodiment myotonic dystrophy type 1 (Steinert's disease).
  • the present invention provides a compound, wherein said compound is the compound defined above.
  • the present invention provides a composition comprising the above- mentioned compounds and a pharmaceutically r embodiments of
  • the present invention provides a method for reducing the formation of RNA foci or aggregates in a cell, said method comprising contacting said cell with the compound defined above.
  • the method is in vitro.
  • the method is in vivo.
  • the RNA is a mutated mRNA comprising polynucleotide repeats, such as trinucleotide (or triplet) repeats.
  • the present invention provides a method for determining whether a test compound may be useful for treating a polynucleotide repeat disorder, said method comprising contacting said test compound with a cell expressing a reporter construct, said reporter construct comprising a first domain encoding a reporter transcript and a second domain comprising a plurality of polynucleotide repeats located downstream of said first domain, and determining the aggregation of said reporter transcript in said cell, wherein a decrease in the aggregation of said reporter transcript in said cell in the presence of said test compound, relative to the aggregation in the absence of said test compound, is indicative that said test compound may be useful for treating a polynucleotide repeat disorder.
  • said second domain of said reporter construct comprises at least 100 polynucleotide repeats, in a further embodiment, at least 200 polynucleotide repeats, in a further embodiment, at least 300 polynucleotide repeats, in a further embodiment, 100-1500 polynucleotide repeats, in further embodiments, 100, 200, 300 or 1250 polynucleotide repeats.
  • the polynucleotide repeat is a trinucleotide repeat.
  • the decrease in the aggregation of said reporter transcript in said cell is determined by quantifying the number of reporter transcript foci in the nucleus of said cell.
  • the above-mentioned reporter transcript is a luciferase or beta- galactosidase transcript.
  • the above-mentioned reporter construct is under inducible expression. In an embodiment, the above-mentioned reporter construct is operably linked to a tetracycline-responsive element (TRE).
  • TRE tetracycline-responsive element
  • the above-mentioned cell further expresses the tetracycline-responsive transcriptional activator (tTA) from the strong immediate early promoter of cytomegalovirus (PCMV)-
  • tTA tetracycline-responsive transcriptional activator
  • PCMV cytomegalovirus
  • the above-mentioned expression is induced by culturing said cell in the absence of tetracycline (Tc), or a derivative thereof.
  • the tetracycline (Tc) derivative is doxycycline (Dox).
  • the above-mentioned method comprises:
  • Tc tetracycline
  • Tc tetracycline
  • FIG. 1 shows the symptoms of myotonic dystrophy
  • FIG. 2 shows DM1 patient fibroblasts with a 2000 CUG-triplet repeat expansion in the 3'UTR of the DMPK mRNA. This mutant mRNA forms nuclear foci which can be detected by fluorescent in situ hybridisation (FISH) (arrows);
  • FISH fluorescent in situ hybridisation
  • FIG. 3 shows the structure of two compounds (compounds #15 and 32) identified in the first screening experiments
  • FIG. 4 shows that compounds #15 and 32 reduce the formation of mutant DMPK mRNA foci in DM1 patient myoblasts.
  • Myoblast cells with 1250 CTG-repeats in the 3'UTR of the DMPK gene were treated with compounds 15, 32 or DMSO for 7 days.
  • the formation of (CUG)n RNA foci was detected by FISH.
  • the surface occupied by CUG-repeat RNA foci in the nucleus of the DM1 myoblasts was quantified with the ImarisTM program. Each data point represents one nucleus and the median value is shown on the graph. ***p ⁇ 0,001 ;
  • FIG. 5 shows a structure-activity relationship (SAR) analysis of analogues of compounds 15 and 32.
  • a screen of 49 analogues of compounds 15 and 32 was performed in C2C12 myoblasts expressing a LacZ-145CUG reporter mRNA. Sixteen analogues increase the beta-galactosidase activity in the reporter cell line (table).
  • EC 50 assays of the analogue compounds show that they increase the expression of the beta-galactosidase expressed from the LacZ-145CUG mRNA, but not from the LacZ-5CUG mRNA.
  • the cells were treated with 0.2, 1 , 2, 5 and 25 ⁇ concentrations of each compound for 24 hrs.
  • the EC 50 and maximal induction of beta-galactosidase (Max) were measured for each compound; and
  • FIG. 6A and B shows the structure of analogues of compounds #15 (Fig. 6A) and 32 (Fig.
  • FIG. 6C shows further analogs (no. 102, 195, 196, 213, 214, 215, 216, 217, 218, 219, 220, 221 ) of these compounds;
  • FIG. 7 shows the effect of compounds #15 and 32 and representative analogues thereof on the levels of the CUGBP1 alternative splicing factor.
  • A Western blot on the CUGBP1 protein in normal human myoblasts (WT) and DM1 patient myoblasts treated with compound 15 (and its analogues 8 and 12), or compound 32 (and its analogue 43) at a concentration of 20 ⁇ for 7 days.
  • B Quantification of the bands in panel (A) acquired with the ChemiDocTM MP imaging system (Bio-Rad). GAPDH is used as a loading control;
  • FIG. 8 shows the effect of compound 15 on the alternative splicing of three transcripts mis- spliced in DM1 .
  • Normal human myoblasts (WT) or myoblasts from a DM1 patient were treated with compound 15 at 20 ⁇ or DMSO during 7 days.
  • FIG. 1 shows the effect of compound 15 on the alternative splicing of three transcripts mis- spliced in DM1 .
  • FIG. 10 shows the effect of compounds #15 and 32 and representative analogues thereof on the alternative splicing of the endogenous MBNL2, MBNL1 and Sercal transcripts.
  • Myoblasts from a DM1 patient were treated with DMSO, compound 15 (or analogs 8, 12, 19, 21 , 22, 23, 24, 27, 28, 29, 31 , 32n, 34), or compound 32 (or analogs 43, 46) at 20 ⁇ concentration during 7 days. Correction of mis-splicing is reported in %, where 0% means no correction, while 100% reflects complete splicing correction.
  • Toxicity of compounds is indicated by the ratio of viable cell number (where 1 corresponds to no toxicity), and the percentage of dead cells (%). Toxicity was measured by treating the cells for 7 days with 20 ⁇ of compound or DMSO. All the values were obtained from the average of 3 independent experiments;
  • FIG. 11 shows the effect of analogs of compounds 15 and 32 on the alternative splicing of the endogenous MBNL2, MBNL1 , Sercal and TNNT2 transcripts.
  • Myoblasts from a DM1 patient were treated with DMSO, analogues of compound 15 (8, 32n), or compound 32 (and analogues 102, 195, 196, 213, 214, 215, 216, 217, 218, 219, 220, 221 ) at 20 ⁇ concentration during 7 days. Correction of mis-splicing is reported in %, where 0% means no correction, while 100% reflects complete splicing correction. All the values were obtained from the average of 3 independent experiments;
  • FIGs. 12A and 12B shows a dose-response effect of compound 32 on the splicing of exon 7 of MBNL2 mRNA (FIG. 12A) and exon 7 of MBNL1 mRNA (FIG. 12B) in normal (M908) or DM1 myoblasts (DM1 ).
  • UNT untreated cells. All the values were obtained from the average of 3 independent experiments;
  • FIG. 13 shows the effect of various analogues of compounds 15 and 32 on the alternative splicing of the exon 2 of hnRNPA2/B1 mRNA, which is not mis-spliced in DM1 myoblasts.
  • M908 normal myoblasts
  • DM1 DM1 patient myoblasts. All the values were obtained from the average of 3 independent experiments;
  • FIGs. 14A-D shows a dose-response assay of compounds 32 (FIGs. 14A-B) and 196 (FIGs. 14C-D) on the ratio of viable cell number and the percentage (%) of cell death.
  • Both normal (M908) and DM1 myoblasts (DM1 ) were treated for 7 days at concentrations varying from 5 to 60 ⁇ .
  • RNA foci reduce the formation of mutant DMPK mRNA foci in DM1 patient myoblasts, have the capacity to correct the stabilization of the CUGBP1 alternative splicing factor in DM1 myoblasts, and correct the mis-splicing of specific mRNAs in DM1 cells.
  • These compounds may thus be useful for the treatment of diseases associated with the accumulation of RNA foci (RNA aggregates), such as expanded polynucleotide (e.g, trinucleotide) repeat disorders.
  • the present invention provides method for treating a polynucleotide repeat disorder, said method comprising administering to a subject in need thereof an effective amount of a compound of formula (1 ), (2), or (3):
  • R 1 is:
  • aryl, heteroaryl, arylmethyl, or heteroarylmethyl said aryl, heteroaryl, arylmethyl, and heteroarylmethyl being optionally substituted on the aryl ring or on the methyl group by alkyl, alkenyl, or alkynyl, and said heteroaryl and heteroarylmethyl comprising only oxygen and/or nitrogen atoms as heteroatom(s), or
  • alkyl alkenyl or alkynyl, said alkyl, alkenyl and alkynyl being substituted by cycloalkyl, cycloalkenyl or cycloalkynyl,
  • R 2 and R 3 are:
  • alkyl alkenyl, or alkynyl
  • cycloalkyl cycloalkenyl, cycloalkynyl
  • R 2 and R 3 are not a hydrogen atom or a C-
  • R 2 and R 3 together with the nitrogen atom to which they are attached, form a 6- or more membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen atom,
  • R 4 and R 5 are:
  • alkyl alkenyl, or alkynyl
  • cycloalkyl cycloalkenyl, cycloalkynyl
  • R 4 and R 5 are not a hydrogen atom or a Ci-C 2 alkyl
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form a 6 membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen or oxygen atom,
  • R 6 is H, halogen, or C-
  • R 7 is C-
  • R 8 and R 9 are independently alkyl, alkenyl, or alkynyl, said alkyl, alkenyl, and alkynyl being substituted by optionally substituted aryl or optionally substituted thioaryl, or
  • R 8 and R 9 together with the nitrogen atom to which they are attached, form a 5- or more membered, optionally substituted, aromatic or aliphatic heterocycle, optionally comprising one or more additional nitrogen atom, and substituted with optionally substituted aryl,
  • R 8 and R 9 are independently alkyl, alkenyl, or alkynyl, said alkyl, alkenyl, and alkynyl being substituted by aryl or thioaryl,
  • alkyl or the prefix “alk” refers to an optionally substituted straight or branched chain saturated hydrocarbon group.
  • straight or branched chain alkyl groups include, but are not limited to, methyl, trifluoromethyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl,
  • a substituted alkyl can be substituted with one or more (e.g., 2, 3, 4, 5, 6, or 7) substituent groups such as -halogen, -NH 2 , -NH(C Ci 2 alkyl), -N(C Ci 2 alkyl) 2 , -OH, -0-(C Ci 2 alkyl), or C 6 -Ci 0 aryl groups, such as phenyl or naphthyl groups, or any other substituent group described herein.
  • the alkyl group contains 1 -12 carbons, in further embodiments 1 -8, 1 -6 or 1 -3 carbons.
  • aryl refers to an optionally substituted monocyclic or polycyclic structure wherein all rings are aromatic, either fused together (e.g. naphthalene) or linked together (e.g. biphenyl) and formed by carbon atoms.
  • exemplary aryl groups include phenyl, naphthyl, and biphenyl. Where an aryl group is substituted, substituents can include any substituent groups described herein.
  • the aryl comprises from 6 to 15 carbons (C 6 -Ci 5 aryl).
  • heteroaryl or “heteroaromatic” refers to an aryl where one or more carbon atom has been replaced by an heteroatom, such as N, O, or S. In an embodiment, the heteroaryl is 5-6 membered.
  • arylalkyl refers to an aryl group attached to the parent molecular group through an alkyl group.
  • the "arylalkyl” is a "C 7 -C 14 arylalkyl” having the formula -(C x -alkyl)-(C y -aryl) wherein (x+y) is an integer between 7 and 14 and x is at least 1 .
  • substituents can include any substituent group described herein.
  • arylalkenyl and “arylalkynyl” refer to an aryl group attached to the parent molecular group through an alkenyl and alkynyl group, respectively.
  • cycloalkyi refers to an optionally substituted, aliphatic, saturated or unsaturated monocyclic or polycyclic (e.g., bicyclic or tricyclic) hydrocarbon ring system. Polycyclic cycloalkyls may be linear, fused, bridged, or spirocyclic. In an embodiment, the cycloalkyi contains 3-12 carbon atoms (C 3 -C 12 cycloalkyi).
  • cycloalkylalkyl refers to a cycloalkyi group attached to the parent molecular group through an alkyl group.
  • cycloalkylalkenyl and “cycloalkylalkynyl” refer to a cycloalkyi group attached to the parent molecular group through an alkenyl and alkynyl group, respectively.
  • alkenyl refers to an optionally substituted unsaturated, straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond.
  • the alkenyl comprises from 2 to 8 carbon atoms "C 2 -C 8 alkenyl", in a further embodiment from 2 to 6 or 2 to 4 carbon atoms.
  • alkynyl refers to an optionally substituted unsaturated, straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond.
  • the alkynyl comprises from 2 to 8 carbon atoms "C 2 -C 8 alkynyl", in a further embodiment from 2 to 6, or 2 to 4 carbon atoms.
  • halogen refers to -F, -CI, -Br, or -I.
  • heterocycle or “heterocyclyl” is an optionally substituted aromatic or aliphatic monocyclic or bicyclic ring system that includes one or more carbon atoms and heteroatoms (e.g., 1 , 2, 3, or 4 heteroatoms), such as oxygen, nitrogen, and sulfur.
  • 3- to 9-membered heterocycles include, but are not limited to, aziridinyl, oxiranyl, thiiranyl, azirinyl, diaziridinyl, diazirinyl, oxaziridinyl, azetidinyl, azetidinonyl, oxetanyl, thietanyl, diazinanyl, piperidinyl, tetrahydropyridinyl, piperazinyl, morpholinyl, azepinyl or any partially or fully saturated derivatives thereof, diazepinyl or any partially or fully saturated derivatives thereof, pyrrolyl, oxazinyl, thiazinyl, diazinyl, triazinyl, tetrazinyl, imidazolyl, benzimidazolyl, tetrazolyl, indolyl, isoquinolinyl, quinolinyl,
  • heterocyclylalkyl refers to a heterocyclic group attached to the parent molecular group through an optionally substituted alkyl group.
  • aromatic refers to a cyclic ring system having (4n +2) ⁇ electrons in conjugation, where n is 1 , 2, or 3.
  • any group described herein may be substituted or unsubstituted. When substituted, they may be with any desired substituent or substituents that do not adversely affect the desired activity of the compound.
  • substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as substituents such as: halogen (chloro, iodo, bromo, or fluoro); Ci -6 alkyl; C 2-6 alkenyl; C 2-6 alkynyl; hydroxyl; Ci -6 alkoxyl; amino (primary, secondary, or tertiary); nitro; thiol; thioether; imine; cyano; amido; carbamoyl; phosphonato; phosphine; a phosphorus (V) containing group; carboxyl; thiocarbonyl; sulfonyl; sulfonamide; ketone; aldehyde; ester; oxo; haloalkyl (e.
  • substituent groups includes benzyloxy; -N(CH 3 ) 2 ; O-alkyl (0-CH 3 ); O-aryl; aryl; aryl-lower alkyl; - C0 2 CH 3 ; -OCH 2 CH 3 ; methoxy; -CONH 2 ; -OCH 2 CONH 2 ; -S0 2 NH 2 ; -OCHF 2 ; -CF 3 ; and -OCF 3 .
  • a substituted group may have 1 , 2, 3, 4, 5, 6, 7, or 8 substituent groups. These substituent groups may optionally be further substituted with a substituent listed herein. Substituents may also be optionally substituted by a fused-ring structure or bridge, for example -OCH 2 0-. In other embodiments, these substituents are not further substituted.
  • salt(s) includes but are not limited to salts of acidic or basic groups that may be present in compounds used in the present compositions. Exemplary pharmaceutically acceptable salts are described in Berge et al., J. Pharm. Sci. 1977; 66:1 -19 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and CG. Wermuth, Wiley-VCH, 2008). Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,
  • compounds of the invention that include ionizable hydrogens can be combined with various inorganic and organic bases to form salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • R 1 is substituted aryl, such as aryl substituted with one to three alkyl groups, preferably methyl groups.
  • the substituted aryl is phenyl substituted with two methyl groups, such as:
  • R 1 is an arylmethyl. In embodiments, R 1 is an unsubstituted arylmethyl. In embodiments, the aryl group in the arylmethyl is phenyl. In a further embodiment, R 1 is:
  • R 1 is an arylmethyl substituted by an alkyl group, preferably a C 1-3 alkyl group, such as ethyl.
  • the alkyl group substitutes the methyl group of the arylmethyl.
  • R 1 is:
  • R 1 is unsubstituted or substituted heteroaryl.
  • the heteroaryl group is unsubstituted.
  • the heteroaryl has an aryl ring fused to a 5-membered ring comprising one or two heteroatoms, preferably two oxygen atoms.
  • R 1 is:
  • the heteroaryl has two aryl rings fused to a 6-membered ring comprising one or two heteroatoms, preferably two oxygen atoms.
  • R 1 is:
  • R 1 is a heteroaryl comprising one or two heteroatoms.
  • the heteroaryl is unsubstituted.
  • the heteroaryl is 6-membered..
  • the heteroaryl has one heteroatom.
  • the heteroatom is a nitrogen atom.
  • R 1 is:
  • the heteroaryl is substituted, for example with one to three alkyl groups, preferably methyl groups.
  • the heteroaryl comprises a 6-membered ring linked to a 5-membered ring.
  • the 5-membered ring comprises one or two heteroatoms, preferably two nitrogen atoms.
  • the substituted heteroaryl is 1 -phenyl-1 H-pyrazole substituted with one methyl group, such as:
  • R 1 is alkyl substituted with cycloalkyl. In a further embodiment R1 is otherwise unsubstituted alkyl substituted with unsubstituted cycloalkyl.
  • the alkyl group in the alkylcycloalkyl is a C 1-6 alkyl, such as a alkyl.
  • the alkyl is an ethyl group.
  • the cycloalkyl is a 5-membered cycloalkyl.
  • R 1 is:
  • R 1 is not:
  • one of R 2 and R 3 is hydrogen or alkyl (preferably C C 3 alkyl), and the other is alkyl comprising 3 or more carbon atoms, aralkyi (preferably phenyl-alkyl) or cycloalkyl (preferably cyclohexyl or adamantyl).
  • R 2 is H and R 3 is a C 3 -C 6 alkyl. In an embodiment, R 3 is linear. In another embodiment, R 3 is branched. In a further embodiment, R 3 is a C 4 alkyl. In a further embodiment, NR 2 R 3 is:
  • NR R is:
  • NR 2 R 3 is:
  • NR 2 R 3 is:
  • R 2 and R 3 are C 3-6 alkyi, preferably propyl.
  • R 2 is H and R 3 is a C 3 -C 12 cycloalkyl, in a further embodiment a Cio cycloalkyl.
  • R 3 is adamantly, and NR 2 R 3 is for example adamant-1 -yl:
  • R 2 is H and R 3 is a C7-C14 arylalkyl, such as a C7-C1 0 arylalkyl, in a further embodiment an unsubstituted arylalkyl.
  • R 3 is:
  • R 2 and R 3 together with the nitrogen atom to which they are attached form an optionally substituted 6- to 9-membered non heterocycle.
  • the heterocycle comprises zero or one additional heteroatom, in a further embodiment the additional heteroatom is nitrogen.
  • the heterocycle is a 6-membered aliphatic heterocycle.
  • the heterocycle is substituted, in a further embodiment substituted with an alkyi, such as a C-
  • the substituent is methyl.
  • 2 R 3 is:
  • NR 2 R 3 is: In an embodiment, NR 2 R 3 is:
  • NR 2 R 3 is not:
  • the compound is a compound of formula (1 b):
  • R 2 and R 3 are as defined above, or a pharmaceutically acceptable salt thereof.
  • the compound is: 26
  • the compound is:
  • the compound is:
  • the compound is:
  • the compound is:
  • the compound is:
  • the compound is:
  • the compound is:
  • the compound is: or a pharmaceutically acceptable salt thereof.
  • the compound is:
  • R 1 is a substituted or unsubstituted aryl, in a further embodiment a substituted aryl.
  • the aryl is substituted with an alkyl, for example one or three alkyl, in a further embodiment a Ci-C 6 or C1-C4 alkyl, such as a methyl.
  • the substituted aryl is phenyl substituted with two methyl groups, such as:
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form a 6-membered, optionally substituted, aliphatic heterocycle, optionally comprising one or more additional nitrogen or oxygen atom.
  • the heterocycle comprises one further oxygen atom.
  • the heterocycle is substituted with one to three alkyl groups, preferably two alkyl groups, preferably methyl groups.
  • R 4 and R 5 together with the nitrogen atom to which they are attached, form:
  • the compound is:
  • R 6 is H or a halogen, in a further embodiment the halogen is CI.
  • R 7 is a C 3 -C 8 alkyl, in a further embodiment a C 3 -C 6 alkyl.
  • the alkyl is substituted with one or more halogen atoms, for examples one to three such atoms, for example three fluoride atoms.
  • R 8 and R 9 are independently alkyls, such as Ci -3 alkyls, preferably methyl groups, substituted by optionally substituted aryl or optionally substituted thioaryl. In embodiment, substituted preferably monocyclic aryl or thioaryl, for example phenyl and/or 2-thienyl, which are preferably unsubstituted.
  • NR 8 R 9 is:
  • R 7 is not a Ci alkyl
  • R 8 and R 9 together with the nitrogen atom to which they are attached, join to form a 5- or more membered substituted aliphatic heterocycle.
  • the aliphatic heterocycle may be substituted pyrrolidinyl or piperidinyl:
  • Z 5 is a substituted or unsubstituted aryl.
  • Z 5 is a substituted aryl, in a further embodiment the substituent is alkyloxy, such as a methoxy (-0-CH 3 ).
  • NR 8 R 9 is:
  • the compound is: 36
  • the compound is:
  • the compound is:
  • the present invention provides a compound of formula 1 , 1 a, 1 b, 2, 2a or (3) as defined above.
  • the compound is:
  • the above-mentioned compound reduces the formation of mRNA foci or aggregates in a cell.
  • the above-defined compounds can contain one or more asymmetric carbon atoms which give rise to enantiomers.
  • the compounds can be prepared as racemates or can be made from enantiomeric intermediates. Both racemates and enantiomers form part of the present invention.
  • the present invention also provides prodrugs of the compounds of the invention.
  • Prodrugs include derivatives of the compounds of the invention that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound of the invention.
  • Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, and biohydrolyzable phosphate analogues.
  • prodrugs of the compounds of the invention with carboxyl functional groups are the lower alkyl esters of the carboxylic acid.
  • the carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.
  • Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery 6 th ed. (Donald J. Abraham ed., 2001 , Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).
  • Biohydrolyzable moieties of a compounds of the invention 1 do not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) are biologically inactive but are converted in vivo to the biologically active compound.
  • biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.
  • biohydrolyzable amides include, but are not limited to, lower alkyl amides, a-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
  • biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines.
  • the compounds of formula 1 , 1 a and 1 b may be synthesized, for example, based on the methods disclosed in PCT publication No. WO 99/36398.
  • a compound described herein is formulated in a pharmaceutical composition, in association with one or more carrier(s).
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a compound of the invention is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents may be used.
  • Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, polyethylene glycol 300, water, ethanol, polysorbate 20, wetting or emulsifying agents, or pH buffering agents.
  • excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, polyethylene glycol 300, water, ethanol, polysorbate 20, wetting or emulsifying agents, or pH buffering agents.
  • treatment is an approach for obtaining beneficial or desired therapeutic results, such as clinical results.
  • beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions (e.g., for myotonic dystrophy, one or more of the symptoms depicted in FIG. 1 , such as muscle weakness, muscle wasting); diminishment of extent of disease, disorder, or condition; stabilization (i.e., not worsening) of state of disease, disorder, or condition; prevention of spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total).
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • polynucleotide repeat disorders refers to disorders characterized based upon the presence of unstable and abnormal expansions of DNA-repeats, which in turn typically results in abnormal expression of one or more genes.
  • the most common types of repeats are trinucleotide repeats (often CAG, but also CCG, CTG, CGG, GCC or GAA). Accumulation of RNA foci (or RNA aggregates) is a hallmark of polynucleotide repeat disorders.
  • polynucleotide repeat disorders include Huntington's disease (HD), Huntington's disease-like 2 (HDL2), spinocerebellar ataxias (types 1-8, 12 and 17), fragile X syndrome, fragile X-E syndrome, fragile X-associated tremor/ataxia syndrome, myotonic dystrophy (type 1 or type 2), juvenile myoclonic epilepsy, Dentatorubral-pallidoluysian atrophy (DRPLA), Friedreich's ataxia (FRDA), spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease), chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD, associated with an hexanucleotide (GGGGCC) repeat expansion).
  • the trinucleotide repeat disorder is myotonic dystrophy, in a further embodiment myotonic dystrophy type 1 (DM1 ) (Steinert's disease).
  • the present invention provides a method for inhibiting the accumulation of RNA foci (RNA aggregates) in a cell, said method comprising contacting said cell with one or more compounds of formula 1 , 1 a, 1 b, 2 and/or 3 defined above, or a composition defined above.
  • the method is an in vitro method.
  • the above-mentioned method is an in vivo method.
  • the present invention provides a method for treating a disease associated with accumulation of RNA foci (RNA aggregates), said method comprising administering to a subject in need thereof an effective amount of one or more compounds of formula 1 , 1 a, 1 b, 2 and/or 3 defined above, or a composition defined above.
  • Diseases associated with accumulation of RNA foci include, for example, the polynucleotide repeat disorders noted above.
  • the patient or subject is an animal, including, but not limited to, a human, mammal, e.g., cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit, mouse or guinea pig, or other animal such as a chicken, turkey, or quail.
  • a human mammal
  • mammal e.g., cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit, mouse or guinea pig
  • the patient or subject is a human.
  • compositions which comprise an effective amount of one or more of the above-defined compounds, can be administered by any convenient route, for example by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, intestinal mucosa, etc.), and can be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer a compound. In certain embodiments, more than one compound is administered to a patient.
  • Methods of administration include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically to the ears, nose, eyes, or skin.
  • the preferred mode of administration is left to the discretion of the practitioner.
  • This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, formulating with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant.
  • the compounds can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • the compounds can be delivered in a vesicle, in particular a liposome.
  • the compounds can be delivered in a controlled-release system.
  • the present compositions may include an effective amount of one or more of the above-defined compounds and a pharmaceutically acceptable carrier.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • compositions which are acidic in nature are capable of forming base salts with various pharmacologically or cosmetically acceptable cations.
  • examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.
  • the compounds of the invention are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compounds for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the compositions may also include a solubilizing agent.
  • Compositions for intravenous administration may optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions for oral delivery may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example.
  • Orally administered compositions may contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation.
  • sweetening agents such as fructose, aspartame or saccharin
  • flavoring agents such as peppermint, oil of wintergreen, or cherry
  • coloring agents such as peppermint, oil of wintergreen, or cherry
  • preserving agents to provide a pharmaceutically palatable preparation.
  • the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time.
  • Oral compositions can include standard carriers such as mann
  • the amount of the compound that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and may be determined for a given case. Suitable effective dosage ranges for intravenous administration are generally about 0.01 to about 5 g, preferably about 0.01 to about 1 g of the compound per kilogram body weight. In specific embodiments, the i.v.
  • a suitable dose range for i.v. administration may be obtained using doses of about 1 to about 2000 mg, without adjustment for a patient's body weight or body surface area.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 10 mg/kg body weight.
  • Suppositories generally contain 0.5% to 20% by weight of one or more compounds of the invention alone or in combination with another therapeutic agent.
  • Oral compositions can contain about 10% to about 95% by weight of one or more compounds of the invention alone or in combination with another therapeutic agent.
  • suitable dose ranges for oral administration are generally about 0.1 to about 200 mg, preferably about 0.5 to about 100 mg, and more preferably about 1 to about 50 mg of a compound of the invention per kilogram body weight or their equivalent doses expressed per square meter of body surface area.
  • the oral dose is about 0.25 to about 75 mg/kg, about 1.0 to about 50 mg/kg, about 2.0 to about 25 mg/kg, about 2.5 to about 15 mg/kg, or about 5.0 to about 20 mg/kg (or the equivalent doses expressed per square meter of body surface area).
  • a suitable dose range for oral administration from about 10 to about 4000 mg, without adjustment for a patient's body weight or body surface area.
  • Other effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.
  • kits comprising one or more containers containing one or more of the above-defined compounds.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the kit may also contain one or more agents useful for treating the polynucleotide repeat disorder to be administered in combination with one or more of the above-defined compounds.
  • Example 1 Materials and Methods
  • C2C12 murine myoblast cells and Phoenix retrovirus packaging cells were cultivated in DMEM supplemented with 10% FBS.
  • Phoenix cells were transfected with the desired retroviral vector by the Calcium-Phosphate method and, 48 hours after transfection, retrovirus-containing supernatant was supplemented with polybrene and deposited on cells to infect.
  • C2C12 Tet-Off cell lines were generated by infecting cells with the retrovirus pTet-Off IN (Clontech) and selecting for Doxycycline (Dox)-regulated clones.
  • Cell lines expressing the LacZ-5CUG, LacZ-145CUG, LUC- 5CUG or LUC-200CUG transcripts were generated by infecting C2C12 Tet-Off cells with the desired reporter construct in the pRevTRE plasmid (Clontech) and selecting for stable clones with inducible expression.
  • the 3'UTR of human DMPK containing 5 CTG repeats or 200 CTG repeats was cloned downstream of the luciferase or beta-galactosidase open reading frames.
  • the C2C12 cell line(s) are cultivated in media containing 1 ng/ml Dox to repress transcription of the reporter constructs.
  • the 20mM compound stock in 100% DMSO is pipetted directly into the media, Dox is also added to 0.1 ng/ml, and the tube is mixed well by inversion and gentle vortexing, and immediately overlayed on the cells. Pure DMSO is used for the control cells. The final concentration of DMSO is never higher than 0.5 %.
  • Stocks of compound are prepared in 100% ultra-pure tissue culture tested DMSO (SIGMA D2650), aliquoted in single use volumes to avoid freezing-thawing, and kept at -20°C. Beta-galactosidase expression is determined with the Fluorescein di(Beta-D-galactopyranoside) FDG assay.
  • the lysis buffer is prepared as follows (for 50 ml): 15 ⁇ Triton X-100, 5ml HEPES 100mM pH 7.8, and 400 ⁇ MgS0 4 8mM.
  • the FDG substrate (Invitrogen F1 179) stock is prepared at 20mM in a solution of dH20/DMSO/EtOH 8:1 :1 .
  • Cells are washed in 1X PBS, then lysed in 90 ⁇ of lysis buffer, and 10 ⁇ of 1 mM FDG (diluted in dH20) is added for a final concentration of 100 ⁇ .
  • the plate is incubated at room temp for 2 hrs (protected from light) and read in fluorescence at 490nm/515nm.
  • Human myoblasts were obtained from the Myosix institute associated with the Association Francaise cor les Myopathies (AFM). Five primary myoblast lines were obtained as controls, and one line from a DM1 patient with a 3.8 Kb CTG triplet amplification corresponding to approximately 1250 CTG repeats.
  • the human myoblasts are cultivated in HAM-F10 media (SIGMA N6635) with 15% FBS, 1 % chick embryo extract, and 390 ng/ml of dexamethasone.
  • 2 X 10 5 cells were plated in 100mm plates (for CUGBP1 western blot or RT-PCR alternative splicing assay) or 2 X 10 4 cells/well are seeded in 12-well plates containing 12mm round glass coverslips (for FISH to detect RNA foci).
  • media is replaced with fresh media containing the compounds at the desired concentration in this manner: the media is put in a screw-cap tube and heated at 37°C.
  • the 20mM compound stock in 100% DMSO is pipetted directly into the media and the tube is mixed well by inversion and gentle vortexing, and immediately overlayed on the cells. Pure DMSO is used for the control cells. Final concentrations on myoblasts are never higher than 0.5 %. This procedure is repeated twice during the seven days of treatment, the media is thus replaced with fresh media containing compound every 2-3 days. Cells are collected or fixed for FISH after one week.
  • CUGBP1 was detected with the 3B1 -3D1 1 monoclonal antibody (mAb) (SIGMA C51 12) and GAPDH was detected with the 6C5 mAb (Millipore MAB374).
  • the enhanced chemiluminescence signal (ECL) was acquired and quantified with the ChemiDocTM MP imaging system (Bio-Rad).
  • ChemiDocTM MP imaging system Bio-Rad.
  • total RNA was collected with TrizolTM (Invitrogen), and 1 ⁇ g was used for reverse transcription with random primers with the RevertAidTM H Minus M-MuL RT (Fermentas) according to manufacturer's instructions.
  • PCR amplification was performed with primers 5'-ACAAGTGACAACACCGTAACCG-3' (SEQ ID NO:1 ) and 5'-TTTGGTAAAGGATGAAGAGCACC-3' (SEQ ID NO:2).
  • SERCA1 was amplified with primers 5'-ATGATCTTCAAGCTCCGGGC-3' (SEQ ID NO:3) and 5'-CAGCTCTGCCTGAAGATGTG-3' (SEQ ID NO:4).
  • TNNT2 cardiac Troponin T
  • PCR was carried out with primers 5'- AT AG AAG AG GTG GTG G AAG AGT AC-3' (SEQ ID NO:5) and 5'-
  • GTCTCAGCCTCTGCTTCAGCATCC-3' (SEQ ID NO:6).
  • the alternative splicing of the hnRNPA2/B1 transcript was detected with the primers 5'-CTGAAGCGACTGAGTCCGCG-3' (SEQ ID NO:7) and 5'-ACAGTCTGTAAGCTTTCCCC-3' (SEQ ID NO:8).
  • PCR was performed with Taq using standard conditions but the number of amplification cycles was optimized to be in the quantitative range for each gene.
  • the products of PCR amplification were resolved on 3.5 % agarose gels containing GelredTM (Biotium) and images were acquired and quantified with the ChemiDocTM MP imaging system (Bio-Rad). Detection and Quantification of CUG-triplet repeat RNA foci
  • FISH fluorescent in situ hybridization
  • the threshold value for the CUGn RNA foci was determined by obtaining the average value for background nucleoplasm ⁇ fluorescence when WT human myoblasts are hybridized with the (CAG)io-Cy3 probe (this background fluorescence signal is a combination of non-specific hybridization of the probe, autofluorescence of the cells and camera background).
  • the MetaMorphTM program then calculates the surface area covered in each nucleus for all the foci in the Cy3 image that have a) pixels with a fluorescence value above the threshold, and b) a size larger than 3 pixels (to remove single pixel background). For each treatment condition, 60 to 80 cells were analyzed and all the data points were presented, with the median value indicated. Each experiment was repeated at least three times.
  • a high-throughput screen (HTS) of chemical compounds was performed in order to identify and validate drug candidates that relieve the nuclear retention of CUGn mRNAs and dislocate nuclear foci formation in a DM1 cell culture model.
  • a reporter mRNA containing a luciferase ORF with the DMPK 3' untranslated region containing 200 CUG repeats was used as a model system for DM1 .
  • fluorescent in situ hybridization (FISH) was used to visualize the disruption of the nuclear CUG-rich RNA foci. From these screens, two chemical compounds which: 1 ) increase nuclear export and translation of reporter mRNAs containing expanded CUG triplet repeats, and 2) decrease the number and intensity of nuclear CUGn RNA foci, were identified.
  • compound 15 and 32 have the structure depicted in FIG. 3.
  • Compound 15 has a para- sulfonamide structure
  • compound 32 is a benzyl methylthiophene amide. Both are small molecules which fit the Lipinski's "rule of five" for drug-like properties (C.A. Lipinski; F. Lombardo; B.W. Dominy and P.J. Feeney (2001 ). Adv Drug Del Rev 46: 3-26) and should thus demonstrate good ADME (absorption, distribution, metabolism and excretion) profiles.
  • the dose-response effect of candidate drugs 15 and 32 on the nuclear export of luciferase-CUG200 mRNA was assessed in the C2C12 myoblast cell line.
  • the amount of CUG repeat RNA that get exported and properly translated into luciferase in response to treatment with varying concentrations of target drugs was measured in order to determine the dose-response curves.
  • DM1 myoblasts with approximately 1250 CTG triplets in the DMPK gene were treated for 7 days with 15 ⁇ of compound 15 or 5 ⁇ of compound 32, compared to the control (DMSO only).
  • FISH fluorescent in situ hybridization
  • Example 5 Structure-activity relationship (SAR) analysis of compounds 15 and 32 A screen of small-molecules structurally similar to compounds 15 and 32 isolated in our original high-throughput screen was performed. 49 compounds that are structural analogues of compound 15 or 32 were chosen. These are shown in the tables below:
  • MS mass spectra
  • ESI electrospray ion-mass spectroscopy
  • MeMgBr methylmagnesium bromide
  • MgS0 4 magnesium sulfate
  • NaHCOa sodium bicarbonate
  • Na 2 S0 4 sodium sulfate
  • NMR nuclear magnetic resonance spectrosc
  • Ph phenyl
  • TFA-NHS trifluoroacetic /V-hydroxysuccinimide
  • TMEDA ⁇ /, ⁇ /, ⁇ /', ⁇ /'-tetramethylethylenediamine
  • T3P Propylphosphonic anhydride
  • Step 1 Thionyl chloride (0.90 ml_, 12 mmol, 5.0 eq.) was added dropwise to a stirred suspension of 5-methyl-1 -phenyl- • //-/-pyrazole-4-carboxylic acid (0.5 g, 2.5 mmol; prepared according to Dycman et al. WO 2004/099156) in DCM (5.0 mL) at 0°C. The reaction was subsequently warmed to rt and stirred for an additional 6 hours before being concentrated to dryness under high vacuum. The residue was used directly in the next step.
  • Step 2 DIPEA (2.0 eq.) was added to a solution of the product from Step 1 , an appropriate secondary amine (1 .2 eq.) and DMAP (0.1 eq.) in DCM (0.2 M). The reaction was stirred at rt for 3 hours before being concentrated to dryness and the residue purified directly by flash chromatography on silica gel eluting with an increasing proportion of EtOAc in hexanes.
  • yVjyV-Dibenzyl-S-methyl-l -phenyl-iH-pyrazole ⁇ -carboxamide Compound 214)
  • Step 1 Dipropylamine (1.9 ml_, 14 mmol, 3.0 eq.) was added dropwise to a suspension of 4-nitrobenzenesulfonyl chloride (1 .0 g, 4.5 mmol) in DCM (15 mL) at 0°C. After 10 minutes at 0°C, the reaction was stirred at rt for 2 hours before being partitioned between water and DCM. The layers were separated and the aqueous layer was extracted with DCM (2X). The combined organic layers were dried (MgS0 4 ), filtered and concentrated under reduced pressure and the residue was purified by flash chromatography on silica gel eluting with an increasing proportion of EtOAc in hexanes.
  • Step 2 A saturated solution of NH 4 CI (0.5 mL) and iron powder (0.59 g, 10 mmol, 10 eq.) were added to a solution of the product from Step 1 (0.30 g, 1 .0 mmol) in EtOH (4.0 mL). The mixture was heated at 65°C for 18 hours before being cooled to rt and partitioned between EtOAc and water. The layers were separated and the aqueous layer extracted with EtOAc (3X). The combined organics layers were dried (MgS0 4 ), filtered, concentrated and dried under suction and high vacuum to afford 4-amino-/V,/V-dipropylbenzenesulfonamide which was used directly without further purification.
  • Step 3 The product from Step 2 (42 mg, 0.16 mmol, 1 .2 eq.) was added to a solution of 5- methyl-1 -phenyl- • //-/-pyrazole-4-carbonyl chloride (30 mg, 0.14 mmol) and DIPEA (48 ⁇ _, 0.27 mmol, 2.0 eq.) in DCM (0.68 mL). The reaction was stirred at rt for 18 hours before being concentrated to dryness. The residue was purified by flash chromatography on silica gel eluting with an increasing proportion of EtOAc in hexanes to afford the title compound as a yellow solid.
  • Step 1 Benzylamine (3.8 mL, 35 mmol, 1.05 eq.) and acetic acid (9.6 mL, 170 mmol, 5 eq.) were added to a solution of 2-thiophenecarboxaldehyde (3.1 mL, 34 mmol) in DCE (1 10 mL) with stirring at rt for 18 hours.
  • Sodium triacetoxyborohydride (8.95 g, 42.3 mmol, 1.2 eq.) was then added with continued stirring at rt for an additional hour.
  • the reaction vessel contents were then cooled to 0°C and a saturated solution of NaHC0 3 was added slowly to pH 8.
  • Step 2 DIPEA (96 ⁇ _, 0.55 mmol, 3.0 eq.) was added to a suspension of 1 -phenyl-5- (trifluoromethyl)-i/-/-pyrazole-4-carboxylic acid (50 mg, 0.20 mmol, 1.05 eq.), the product from Step 1 (38 mg, 0.19 mmol) and HATU (81 mg, 0.21 mmol, 1 .2 eq.) in EtOAc (2.0 ml_). The mixture was stirred for 18 hours before being partitioned between EtOAc and a saturated solution of NaHC0 3 . The layers were separated and the aqueous layer extracted with EtOAc (3X). The combined organic layers were dried (MgS0 4 ), filtered and concentrated under reduced pressure. The product was isolated as a colorless solid by flash chromatography on silica gel eluting with an increasing proportion of EtOAc in hexanes.
  • Example 7 Effect of disruption of CUG-rich RNA foci on expression of splicing factors and mRNA splicing
  • RNA toxicity in DM1 is dysfunction of two classes of splicing regulators: the CUG binding protein 1 (CUGBP1/CELF1 ) and the muscleblind protein 1 (MBNL1 ).
  • MBNL1 recognizes YGCY motifs in RNA and binds with high affinity to CUG repeats (Goers et al. 2010).
  • MBNL1 has been shown to co-localize in vivo with the nuclear foci of CUG-triplet repeat transcripts (Fardei et al. 2001 ), suggesting that the sequestration of this factor in the nucleus may lead to DM1 pathogenesis.
  • Expanded CUG-repeat RNA is believed to increase CUGBP1 levels and activity in DM1 cells via activation of protein kinase C (PKC), which phosphorylates and stabilizes CUGBP1 protein levels (Kuyumcu-Martinez et al. 2007).
  • PKC protein kinase C
  • CUGBP1 was found to bind CUG-repeat RNA but does not co-localize in vivo with the nuclear DMPK expanded-repeat foci (Fardei et al. 2001 ). It was next tested whether treatment of DM1 patient myoblasts with compounds 15 and 32 and representative analogues thereof could correct the increased CUGBP1 protein levels observed in these cells (FIG. 7).
  • DM1 cells have a level of CUGBP1 about 2.5-times higher than WT cells (lane 1 compared to lanes 3 and 4).
  • the myoblast cells were treated for 7 days at a 20 ⁇ concentration of the compounds or DMSO.
  • As a control it was verified that the treatment of WT cells with compound 15 does not change the levels of CUGBP1 (lane 2 compared to lane 1 ).
  • Treatment of the DM1 myoblasts with compound 15 reduces CUGBP1 levels by 85% (lane 7 compared to lanes 3 and 4).
  • Compounds 32 and 43 also reduce CUGBP1 levels by 30% and 45%, respectively (lanes 8 and 9 compared to lanes 3 and 4).
  • the drug candidates have the capacity to correct the stabilization of the CUGBP1 alternative splicing factor in DM1 myoblasts.
  • the abnormal increase in CUGBP1 levels has been linked to the severe muscle wasting which debilitates DM1 patients in advanced stages of the disease (Orengo et al. 2008).
  • Treatment with compound 15 restores inclusion of exon 22 to 17% of total transcripts.
  • the SERCA1 exon 22+ variant is the adult form, and the SERCA1 exon 22- is the neonatal form (Kimura et al. 2005). Mis-splicing of SERCA1 is thought to be involved in muscle wasting in DM1 .
  • MBNL2 is a muscleblind splicing regulator that is ubiquitously expressed.
  • FIG. 8D it is shown that treatment with compound 15 reduces the inclusion of exon 7 from 40% to 25%. Mis- splicing of MBNL2 is thought to result in the mis-splicing of several transcripts in the brain in DM1 (Charizanis et al. 2012).
  • MBNL2 exon 7 mis-splicing The data depicted in FIG. 9 shows that in DM1 cells which expressed 60% of total transcripts with the foetal form (exon 7+), treatment with the compounds reduces inclusion of exon 7 to about 45% of total transcripts. Overall, these results show that the compounds can correct some of the molecular defects in DM1 patient cells.
  • Example 8 Further testing of compounds in the alternative splicing assay
  • DM1 patient myoblasts were treated for 7 days with a 20 ⁇ concentration of each compound.
  • Total RNA was extracted, and used in RT-PCR reactions to quantify the alternative splicing of exon 7 of MBNL2 and MBNL1 mRNAs, and exon 22 of Sercal mRNA, which are all mis- spliced to re-express a foetal or neonatal form in DM1 cells (Charizanis et al. 2012, Kimura et al. 2005).
  • analogue 31 which exhibits cellular toxicity
  • all the analogues partially corrected the mis-splicing of MBNL2 and MBNL1 mRNA (FIG. 10).
  • analogues 102, 196, and 214 were able to correct at least 3 mis-splicing of the pre-mRNAs tested.
  • analogue 196 which is a "chimera" of compounds 15 and 32
  • a dose- response assay was performed with the alternative splicing assay using compound 32.
  • Normal (M908) and patient (DM1 ) myoblasts were treated for 7 days with compound 32 at concentrations varying from 5 to 60 ⁇ .
  • Total RNA was extracted, and used in RT-PCR reactions to quantify the alternative splicing of the endogenous MBNL2 and MBNL1 mRNAs.
  • FIGs. 12A and 12B a clear dose-response effect can be observed on the splicing of MBNL2 (FIG. 12A) and MBNL1 (FIG. 12B) mRNAs upon treatment of DM1 myoblasts, as the splicing pattern of both mRNAs shift toward the pattern observed in normal myoblasts as the concentration of compound 32 increases.
  • the cellular toxicity of compounds 15 and 32, and their analogs was also measured using a validated cytotoxicity assay.
  • Normal or DM1 myoblasts were treated with 20 ⁇ of compound or DMSO.
  • the ratio of viable cells was measured with a High Content Screening microscope in live cells treated with Hoescht 33342 (labels all cells) and propidium iodide (labels dead cells). The total number of cells was counted and the number of dead cells was subtracted to obtain the number of viable cells, which is presented as a ratio (drug-treated versus DMSO-treated cells).
  • the percentage of dead cells was also measured using propidium iodide. As shown in FIG. 10, while some compounds like 15 and 31 show some toxicity, other compounds like 32 and 46 had little toxicity on the cells at this concentration.
  • the cellular toxicity of the compounds was also measured in a dose-response assay.
  • normal (M908) and patient (DM1 ) myoblasts were treated for 7 days with compound 32 or 196 at concentrations varying from 5 to 60 ⁇ .
  • Cell number and cell death was quantified by High Content Screening microscope, as specified above.
  • Little cytotoxicity was observed at concentration of 20 ⁇ and below (FIGs. 14A-D).
  • the main phenotype observed was a cytostatic effect at higher concentrations, as cell death remains below 10% at concentrations lower than 60 ⁇ .
  • Huntington's disease-like 2 is associated with CUG repeat-containing RNA foci. Ann. Neurol. 61 , 272-282.

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Abstract

La présente invention concerne des composés qui inhibent la formation et/ou l'accumulation d'agrégats d'ARN, tels que ceux dus à des répétitions de polynucléotides (par exemple des répétitions de trinucléotides). La présente invention concerne également des utilisations de tels composés, telles que pour l'inhibition de la formation et de l'accumulation de tels agrégats d'ARN, ainsi que pour le traitement d'affections associées à la répétition de polynucléotides (par exemple des affections associées à la répétition de trinucléotides), telles que la dystrophie myotonique (par exemple DM1). De tels composés comprennent des composés de formule 1, 1a, 1b, 2, 2a et (3) décrits dans la description.
PCT/CA2013/050730 2012-09-26 2013-09-25 Inhibiteurs d'agrégats d'arn associés à la répétition de polynucléotides et leurs utilisations WO2015042685A1 (fr)

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CA2923503A CA2923503A1 (fr) 2013-09-25 2013-09-25 Inhibiteurs d'agregats d'arn associes a la repetition de polynucleotides et leurs utilisations
PCT/CA2013/050730 WO2015042685A1 (fr) 2013-09-25 2013-09-25 Inhibiteurs d'agrégats d'arn associés à la répétition de polynucléotides et leurs utilisations
EP13894694.2A EP3049393A4 (fr) 2013-09-25 2013-09-25 Inhibiteurs d'agrégats d'arn associés à la répétition de polynucléotides et leurs utilisations
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EP3740482A1 (fr) * 2018-01-17 2020-11-25 Migal Galilee Research Institute Ltd. Nouveaux inhibiteurs de la voie métabolique de méthionine
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US11647748B2 (en) 2018-01-17 2023-05-16 Migal—Galilee Research Institute Ltd. Sulphonamides as methionine metabolic pathway inhibitors

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