WO2018203559A1 - Pharmaceutical compositions for polyglutamine diseases - Google Patents

Pharmaceutical compositions for polyglutamine diseases Download PDF

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WO2018203559A1
WO2018203559A1 PCT/JP2018/017448 JP2018017448W WO2018203559A1 WO 2018203559 A1 WO2018203559 A1 WO 2018203559A1 JP 2018017448 W JP2018017448 W JP 2018017448W WO 2018203559 A1 WO2018203559 A1 WO 2018203559A1
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group
halogen atom
compound
polyglutamine
pharmaceutical composition
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PCT/JP2018/017448
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French (fr)
Japanese (ja)
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萩原正敏
奥野友紀子
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic 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
    • A61K31/415—1,2-Diazoles
    • A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic 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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic 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
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present disclosure relates to pharmaceutical compositions relating to polyglutamine diseases, their use, and methods for preventing, improving, suppressing progression and / or treating polyglutamine diseases using them.
  • the present disclosure also relates to a method for screening a compound for polyglutamine disease.
  • Huntington's disease In hereditary neurological diseases such as Huntington's disease, which are summarized as polyglutamine diseases, CAG repeats in the causative gene are overextended, and a protein with polyglutamine overextension is generated using this CAG repeat as a template. Accumulate in and form aggregates. This polyglutamine aggregate causes triggering, cell damage, and subsequent neuronal loss. Huntington's disease is the second most common neurological disease after Alzheimer's disease and Parkinson's disease, but the main Huntington's disease drugs currently on the market are symptomatic treatments and drugs that can still be fundamentally treated. I can't say there is.
  • Non-Patent Document 1 discloses a reporter protein for forming a polyglutamine aggregate in which 150 repeats of Huntington gene exon 1 and a fluorescent protein are fused.
  • the present disclosure in one aspect, provides a pharmaceutical composition relating to polyglutamine disease, their use, and a method for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease using them. In another aspect, the present disclosure provides a method for screening a compound relating to polyglutamine disease.
  • the present disclosure provides an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a receptor tyrosine kinase (c-Met) inhibitor compound encoded by the c-met gene, and VEGFR and c-Met.
  • VEGFR intravascular epidermal growth factor receptor
  • c-Met receptor tyrosine kinase
  • the present invention relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease, which contains at least one selected from the group consisting of both inhibitory compounds as an active ingredient.
  • the present disclosure provides a method for screening a candidate substance for an active ingredient of a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease, Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell; Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell; Comparing signal (A) and signal (B); and
  • the present invention relates to a screening method comprising selecting, as a candidate substance, a test substance that reduces signal (A) over signal (B) based on the comparison.
  • FIG. 1 is a schematic diagram of an expression system of a fusion protein of polyglutamine and a reporter protein.
  • FIG. 2 is a schematic diagram of an example of a screening scheme.
  • FIG. 3 shows the expression induction of the assay system used for screening, and the Z ′ factor.
  • FIG. 4 is a graph showing the concentration dependency of compound 4 inhibition of polyglutamine aggregate formation.
  • FIG. 5 is a graph showing the concentration dependence of reporter protein expression level reduction by compound 4.
  • the present disclosure discloses a pharmaceutical composition of a pharmaceutical composition for polyglutamine disease (hereinafter, also referred to as “the pharmaceutical composition according to the present disclosure”).
  • the pharmaceutical composition for polyglutamine disease refers to a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease in one or more embodiments.
  • polyglutamine disease is an overextension of a CAG repeat in a causative gene, generation of a protein having polyglutamine overextension using the CAG repeat as a template, or a nerve cell of this protein It refers to a disease caused by at least one of accumulation in the body and aggregate formation.
  • Polyglutamine disease includes, in one or more embodiments, Huntington's disease, hereditary spinocerebellar degeneration, or bulbar spinal muscular atrophy.
  • an inhibitor of intravascular epidermal growth factor receptor (VEGFR), an inhibitor of c-Met, or both VEGFR and c-Met
  • VEGFR intravascular epidermal growth factor receptor
  • a substance having inhibitory activity against VEGFR2 a substance having inhibitory activity against both VEGFR and c-Met, or a substance having inhibitory activity against both VEGFR2 and c-Met
  • a substance that suppresses the formation of polyglutamine aggregates a plurality of known VEGER inhibitors and substances having inhibitory activity against both VEGFR and c-Met were selected by the screening method according to the present disclosure described later.
  • VEGFR inhibitor that is an active ingredient includes a VEGFR inhibitor having a structure represented by the following formula (I).
  • W, Z and R 1 in the following formula (I) are arbitrary groups.
  • R 1 is a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom
  • W is
  • R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom
  • X 1 is a C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom or an imino group
  • X 2 and Y are each independently, C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom, or an imino group, or, X 2 and Y together with the atom marked with a form an aromatic ring or a heteroaromatic ring, said ring being unsubstitute
  • Examples thereof include a compound or a pharmaceutically acceptable salt thereof having one or more substituents which are oxy groups.
  • a bond with a wavy line indicates a bond portion with the compound or group represented by the formula (I), (II) or (III).
  • the VEGFR inhibitor in the present disclosure is a control in which the substance is not added to the activity of VEGFR when the substance is added in a known VEGFR inhibition assay system in at least one of in vitro and in vivo. Compared to, for example, it refers to a compound that can be inhibited to 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In the assay system, the amount of substance added is 0.01-10 ⁇ M in one or more embodiments.
  • the c-Met inhibitory substance in the present disclosure refers to the activity of c-Met when the substance is added in a known c-Met inhibition assay system in at least one of in vitro and in vivo. Compared with the control to which the substance is not added, it refers to a compound that can be inhibited to, for example, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In the assay system, the amount of substance added is 0.01-10 ⁇ M in one or more embodiments.
  • the inhibitor of both VEGFR and C-Met refers to the activity of VEGFR in the known VEGFR inhibition assay system in at least one of in vitro and in vivo in the one or a plurality of embodiments when the substance is added. Compared with a control to which the substance is not added, for example, it can be inhibited to 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, and at least one of in vitro and in vivo
  • the activity of c-Met when the substance is added is, for example, 60% or less, 50% or less, 40% or less, 30% or less compared to a control without the substance. , 20% or less, or a compound that can be inhibited to 10% or less.
  • the amount of substance added is 0.01-10 ⁇ M in one or more embodiments.
  • the alkyl group or alkylene group includes a linear, branched, or cyclic alkyl group or alkylene group.
  • the “C 1-4 alkyl group” is a linear, branched or cyclic alkyl group having 1 to 4 carbon atoms in one or a plurality of embodiments.
  • the linear or branched alkyl group having 1 to 4 carbon atoms includes a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a sec-butyl group. And tert-butyl group.
  • Examples of the cyclic alkyl group having 3 to 4 carbon atoms include a cyclopropyl group and a cyclobutyl group in one or more embodiments.
  • Heterocycle in one or more embodiments, contains 1-2 heteroatoms in the atoms constituting the ring, may contain double bonds in the ring, and is non-aromatic. Or an aromatic ring. “Heteroaromatic ring” means an aromatic heterocycle. “Hetero atom” means, in one or more embodiments, a sulfur atom, an oxygen atom or a nitrogen atom.
  • the “pharmaceutically acceptable salt” includes a pharmacologically and / or pharmaceutically acceptable salt, for example, an inorganic acid salt, an organic acid salt, an inorganic basic salt, an organic basic salt, an acidic amino acid salt. Or a basic amino acid salt etc. are mentioned.
  • Preferable examples of the inorganic acid salt include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferable examples of the organic acid salt include, for example, acetate, succinate, Examples thereof include fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.
  • Preferred examples of the inorganic base salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt.
  • Preferable examples of the organic base salt include diethylamine salt, diethanolamine salt, meglumine salt, N, N′-dibenzylethylenediamine salt and the like.
  • Preferred examples of the acidic amino acid salt include aspartate and glutamate.
  • Preferable examples of the basic amino acid salt include arginine salt, lysine salt, ornithine salt and the like.
  • the “salt of a compound” may include a hydrate that can be formed by absorbing moisture when the compound is left in the air. Further, in the present disclosure, the “salt of a compound” may include a solvate that can be formed by absorbing a certain kind of other solvent.
  • R 1 in the above formula (I) includes, in one or more embodiments, a halogen atom, or a C 1-4 alkyl group substituted with a halogen atom, and in one or more embodiments, fluorine atom Or a trifluoromethyl group.
  • W in the above formula (I) is one or more embodiments, Is mentioned.
  • R 2 and R 3 each independently represents a hydrogen atom or a halogen atom.
  • X 1 is an imino group in one or a plurality of embodiments
  • X 2 and Y are each independently an imino group or a group in one or a plurality of embodiments.
  • W in the above formula (I) has an inhibitory activity against c-Met in addition to the inhibitory activity against VEGFR.
  • R 2 , R 3 , X 2 and Y are the same as above.
  • Z in the above formula (I) is, in one or more embodiments, Is mentioned.
  • R 4 and R 5 in one or more embodiments, are each independently a halogen atom or an amino group, or R 4 and R 5 , together with atoms marked with b and c, Is mentioned.
  • R 6 and R 7 are each independently a methyl group, or Is mentioned.
  • the formula (I) is the following formula (III) in one or more embodiments.
  • W is R 1 -R 3 , R 6 and R 7 , X 1 , X 2 and Y are the same as in formula (I). ]
  • the formula (I) is represented by the following formula (IV) in one or more embodiments. [In formula (IV), R 1 -R 5 , X 2 and Y are the same as in formula (I)]
  • VEGFR inhibitor which is an active ingredient
  • the following compound 1-5 may be mentioned.
  • One or more embodiments of a substance having both VEGFR and c-Met inhibitory activity include the following compound 2-4.
  • the active ingredient of the pharmaceutical composition according to the present disclosure may include the following compound 6-11.
  • the above-mentioned compound that is an active ingredient of the pharmaceutical composition according to the present disclosure is, in one or a plurality of embodiments, a mixture or a single isomer. It has been released.
  • cis-trans isomers may be mentioned.
  • prodrug includes those that are easily hydrolyzed in vivo and regenerate the above compound.
  • carboxyl group is an alkoxycarbonyl group.
  • a compound that becomes an alkylthiocarbonyl group, or a compound that becomes an alkylaminocarbonyl group is an alkoxycarbonyl group.
  • a compound having an amino group a compound in which the amino group is substituted with an alkanoyl group to become an alkanoylamino group, a compound in which the amino group is substituted with an alkoxycarbonyl group to become an alkoxycarbonylamino group, an acyloxymethylamino group, Or a compound that has become hydroxylamine.
  • a compound having a hydroxyl group a compound in which the hydroxyl group is substituted with the acyl group to become an acyloxy group, a compound that has become a phosphate ester, or a compound that has become an acyloxymethyloxy group can be given.
  • alkyl moiety of the group used for forming a prodrug examples include an alkyl group described later, and the alkyl group may be substituted (for example, with an alkoxy group having 1 to 6 carbon atoms).
  • the alkyl group may be substituted (for example, with an alkoxy group having 1 to 6 carbon atoms).
  • lower alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, methoxymethoxycarbonyl, ethoxymethoxy, etc.
  • Examples include lower (eg, having 1 to 6 carbon atoms) alkoxycarbonyl substituted with an alkoxy group such as carbonyl, 2-methoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, and pivaloyloxymethoxycarbonyl.
  • the pharmaceutical composition of the present disclosure contains the inhibitor as an active ingredient, and further includes a pharmaceutically acceptable carrier, preservative, diluent, excipient, or other pharmaceutical agent. May contain acceptable components.
  • the “pharmaceutical composition” may be a dosage form suitable for an administration form by applying a well-known formulation technique in one or a plurality of embodiments.
  • the dosage form include, but are not limited to, oral administration in a dosage form such as a tablet, capsule, granule, powder, pill, troche, syrup, and liquid.
  • parenteral administration in dosage forms such as injections, solutions, aerosols, suppositories, patches, patches, lotions, liniments, ointments, eye drops and the like can be mentioned.
  • These preparations can be produced by known methods using additives such as, but not limited to, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
  • excipient examples include, but are not limited to, starch such as starch, potato starch, and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like.
  • coating agent examples include, but are not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, paraffin, and the like.
  • binder include, but are not limited to, polyvinyl pyrrolidone, macrogol and the same compound as the excipient.
  • disintegrant examples include, but are not limited to, compounds similar to the excipients and chemically modified starch and celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.
  • stabilizer examples include, but are not limited to, paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenol, cresol Mention may be made of such phenols; thimerosal; dehydroacetic acid; and sorbic acid.
  • flavoring agent examples include, but are not limited to, sweeteners, acidulants, and fragrances that are commonly used.
  • the solvent is not limited to these, but ethanol, phenol, chlorocresol, purified water, distilled water and the like can be used, and a surfactant or an emulsifier can also be used as necessary.
  • a surfactant or an emulsifier include, but are not limited to, polysorbate 80, polyoxyl 40 stearate, lauromacrogol, and the like.
  • the method of using the pharmaceutical composition according to the present disclosure may vary depending on symptoms, age, administration method, and the like.
  • the method of use is not limited to these, but intermittently or continuously, orally, transdermally, submucosally, subcutaneously, so that the in vivo concentration of the compound as the active ingredient is between 100 nM and 1 mM.
  • Administration can be intramuscular, intravascular, intracerebral, or intraperitoneal.
  • the lower limit is 0.01 mg (preferably converted into the compound represented by the above general formula (I) per day for a subject (adult if human)) 0.1 mg), and as an upper limit, 2000 mg (preferably 500 mg, more preferably 100 mg) can be divided into one or several doses and administered according to symptoms.
  • the lower limit is 0.001 mg (preferably 0.01 mg) and the upper limit is 500 mg (preferably 50 mg) per day for a subject (adult if human). Is divided into one or several times and administered according to symptoms.
  • the present disclosure is a method for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease, the method comprising administering a pharmaceutical composition according to the present disclosure to a subject.
  • administration of the pharmaceutical composition according to the present disclosure can be based on the above-described method of using the pharmaceutical composition. Examples of the subject include humans and non-human animals.
  • the present disclosure relates to the use of the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease.
  • the present compound or the pharmaceutical thereof is an active ingredient for producing a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease. It relates to the use of top acceptable salts.
  • the present disclosure relates to a method for screening a substance that affects the formation of a polyglutamine aggregate.
  • the screening method of this aspect is: Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell; Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell; Comparing signal (A) and signal (B); and Selecting a test substance that reduces the signal (A) over the signal (B) as a candidate substance based on the comparison.
  • the assay cell in the screening method according to the present disclosure has a gene capable of expressing polyglutamine fused with a reporter protein.
  • the assay cell can be prepared by introducing a gene expression vector constructed and adapted to express a polyglutamine mRNA fused with a reporter protein into the cell.
  • the cell used for the production of the assay cell that is, the cell into which the vector is introduced is not particularly limited, and in one or more embodiments, is a mammalian cell.
  • the mammalian cell is a human, bovine, cat, monkey, dog, hamster, mink, mouse, pig, rabbit, rat cell.
  • the type of cell is not particularly limited, and in one or a plurality of embodiments, a nerve cell or a cultured cell thereof can be mentioned.
  • the vector may be a type that performs transient expression or a type that performs stable expression.
  • the promoter of the gene expression vector a promoter known in the art or developed in the future that can induce protein expression in the assay cell can be used.
  • the reporter protein include a photoprotein in one or a plurality of embodiments, and examples include a fluorescent protein.
  • a gene capable of expressing polyglutamine fused with a reporter protein those described in the Examples can be used.
  • the virtual cell body region refers to a predetermined region including the periphery of the cell nucleus.
  • the reporter signal in this region By measuring the reporter signal in this region by image analysis, polyglutamine aggregates can be detected with high sensitivity.
  • calculating the average luminance in the polyglutamine protein existing region from the total luminance of the signal in the polyglutamine protein existing region of the virtual cell body region and the area of the existing region Is mentioned.
  • the virtual cell body region can be set, for example, as a region including a region in which the peripheral region of the nucleus is expanded with a certain number of pixels.
  • cell nuclei in mammalian cell images are defined as a region stained by the DNA stain DAPI. Only one cell nucleus exists in a cell, and the cell body is regarded as a space surrounding it.
  • each cell nucleus region is defined in the captured image of the DAPI-stained region, and the region having a certain number of pixels is defined as the cytoplasm attached to the cell nucleus.
  • a reporter fluorescent region having a certain luminance or higher existing in the identified cytoplasmic region is defined as a polyglutamine protein existing region belonging to the nucleus / cell. The total luminance in the existing region and the area of the identified existing region are calculated, and the average luminance in the polyglutamine protein existing region is calculated.
  • the aggregate is defined as a region having a high reporter-derived fluorescence value on the image
  • the polyglutamine protein existing region identified in the above method is identified relatively narrowly as compared with the case where it is not formed. Therefore, when aggregates are significantly formed in the virtual cell body region, the difference between the average luminance in the polyglutamine protein-existing region and the presence or absence of aggregate formation is larger than the total difference, and the measurement is derived from the size of the aggregate. Variations in results are offset, thus increasing measurement accuracy.
  • the present disclosure may relate to one or more of the following embodiments;
  • [1] Containing as an active ingredient at least one selected from the group consisting of an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a c-Met inhibitor compound, and both VEGFR and c-Met inhibitor compounds
  • the inhibitory compound is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
  • R 1 is a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom
  • W is
  • R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom
  • X 1 is a C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom or an imino group
  • X 2 and Y are each independently, C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom, or an imino group, or, X 2 and Y together with the atom marked with a form an aromatic ring or a heteroaromatic ring, said ring being unsubstituted or hydrogen atom, halogen atom, C 1-4 alkyl A C 1-4 alky
  • W is R 1 -R 3 , X 1 , X 2 and Y are the same as in formula (I), R 6 and R 7 is a hydrogen atom, a halogen atom, C 1-4 alkyl groups, C 1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring.
  • R 6 and R 7 is a hydrogen atom, a halogen atom, C 1-4 alkyl groups, C 1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring.
  • a pharmaceutical composition for preventing, ameliorating, inhibiting progression and / or treating polyglutamine disease comprising as an active ingredient at least one selected from the group consisting of the following compounds 1-11.
  • a method of prevention, improvement, progression suppression, and / or treatment of polyglutamine disease comprising administering to a subject the pharmaceutical composition according to any one of [1] to [6] Method.
  • Intravascular epidermal growth factor receptor (VEGFR) inhibitory compound Intravascular epidermal growth factor receptor (VEGFR) inhibitory compound, c-Met inhibitory compound for producing a pharmaceutical composition for prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease, And at least one use selected from the group consisting of both VEGFR and c-Met inhibitor compounds.
  • VEGFR vascular epidermal growth factor receptor
  • a screening method for a candidate substance of an active ingredient of a pharmaceutical composition for prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell; Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell; Comparing signal (A) and signal (B); and A screening method comprising selecting, as a candidate substance, a test substance that reduces the signal (A) over the signal (B) based on the comparison.
  • Experimental Example 1 Screening for a substance that affects the formation of polyglutamine aggregates
  • a reporter as shown in FIG. 1 was fused to a host neuron (Neuro2a cell).
  • Cells into which a polyglutamine expression system was introduced (hereinafter referred to as “polyQ cells”) were used (Matsumoto et al. Molecular Cell, 2011; 44: 279-289). Screening was performed using polyQ cells according to the scheme shown in FIG.
  • the concentration at which the relative average luminance fluorescence of polyglutamine per visual field was 50% of that of DMSO control was measured as IC50.
  • the virtual cell body region was acquired as a 20x objective lens equipped with a cell image analyzer ArrayScan VTi, and each cell nucleus region was defined in the captured image of the DAPI stained region, and was defined as a region having a width of 15 pixels. .
  • compound 1-5 was a compound known as a VEGFR inhibitor.
  • Compound 2-4 was a compound known as a VEGFR2 inhibitor and a c-MET inhibitor.
  • Compound 8 was a known compound as a MAPK / ERK-kinase (MEK) inhibitor.
  • Experimental Example 2 Inhibition of polyglutamine aggregate formation by Compound 4 4 mM in a culture solution of polyQ cells cultured for 1 day after seeding (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 ⁇ g / ml streptomycin) dbcAMP and 10 ⁇ g / ml Dox were added to induce expression of the 24h reporter. Thereafter, the medium was replaced with a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 ⁇ g / ml streptomycin) excluding 10 ⁇ g / ml Dox, and then compound 4 (10 nM-10 ⁇ M) was added and cultured for 2 days. After fixing with 4% PFA and nuclear staining with DAPI, the emission luminance of the aggregates in the virtual cell body region was measured with a cell image analyzer. The result is shown in FIG.
  • Compound 4 inhibited the formation of polyglutamine aggregates in an induced concentration-dependent manner after reporter expression.
  • the addition of compound 4 shows an inhibitory effect on aggregates even when added after the transient expression of the reporter, and the action point of this drug is the false action due to the suppression of the tet-on promoter used during reporter construction. It was also shown not to be positive.
  • Experimental Example 3 Relationship between Compound 4 and Reporter Protein Mass 4 mM in a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 ⁇ g / ml streptomycin) cultured for 1 day after seeding. dbcAMP and 10 ⁇ g / ml Dox were added to induce expression of the 24h reporter. Thereafter, the medium was replaced with a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 ⁇ g / ml streptomycin) excluding 10 ⁇ g / ml Dox, and then compound 4 (10 nM-10 ⁇ M) was added and cultured for 2 days. .
  • Compound 4 decreased the expression level of the reporter protein and the RNA level in a concentration-dependent manner. Together with the results in FIG. 4, it is suggested that the present compound promotes the degradation of reporter protein or RNA.

Abstract

Provided are: pharmaceutical compositions for polyglutamine diseases and the uses thereof; methods for prevention, amelioration, progress-suppression and/or treatment of polyglutamine diseases using such pharmaceutical compositions; and a method for screening for a compound for polyglutamine diseases. One embodiment of the present invention pertains to a pharmaceutical composition that is for the prevention, amelioration, progress-suppression, and/or treatment of polyglutamine diseases, and that contains, as an active ingredient, at least one selected from the group consisting of vascular endothelial growth factor receptor (VEGFR) inhibitor compounds, compounds for inhibiting receptor tyrosine kinase (c-Met) encoded by c-Met gene, and compounds for inhibiting both VEGFR and c-Met. Another embodiment of the present invention pertains to a method for screening for a substance that has an effect on the formation of a polyglutamine aggregate, the method comprising a step for measuring, as an indicator of polyglutamine aggregates formed in a culture solution in which cells having introduced therein an expression system that expresses polyglutamine fused with a reporter protein have been cultured, a signal of the reporter protein present in a putative somatic region including a cell nucleus.

Description

ポリグルタミン病に関する医薬組成物Pharmaceutical composition for polyglutamine disease
　本開示は、ポリグルタミン病に関する医薬組成物、それらの使用、それらを用いたポリグルタミン病の予防、改善、進行抑制及び／又は治療方法に関する。本開示はまた、ポリグルタミン病に関する化合物のスクリーニング方法に関する。 The present disclosure relates to pharmaceutical compositions relating to polyglutamine diseases, their use, and methods for preventing, improving, suppressing progression and / or treating polyglutamine diseases using them. The present disclosure also relates to a method for screening a compound for polyglutamine disease.
　ハンチントン病等のポリグルタミン病と総括される遺伝性神経疾患においては、原因遺伝子内のCAGリピートが過伸長し、それを鋳型とするポリグルタミン過伸長を有する蛋白質が生成され、この蛋白質が神経細胞内で蓄積して凝集体を形成する。このポリグルタミン凝集体がトリガー、細胞障害と、それに続く神経細胞の欠落が生じる。ハンチントン病は神経疾患としてはアルツハイマー病、パーキンソン病に次いで頻度の高い疾病であるが、現在のところ上市されている主なハンチントン病薬は対症療法薬であり、まだ根本的な治療が可能な薬があるとはいえない。 In hereditary neurological diseases such as Huntington's disease, which are summarized as polyglutamine diseases, CAG repeats in the causative gene are overextended, and a protein with polyglutamine overextension is generated using this CAG repeat as a template. Accumulate in and form aggregates. This polyglutamine aggregate causes triggering, cell damage, and subsequent neuronal loss. Huntington's disease is the second most common neurological disease after Alzheimer's disease and Parkinson's disease, but the main Huntington's disease drugs currently on the market are symptomatic treatments and drugs that can still be fundamentally treated. I can't say there is.
　非特許文献１は、ハンチントン遺伝子エキソン１の１５０リピートと蛍光蛋白質とを融合したポリグルタミン凝集体形成のレポーター蛋白質を開示する。 Non-Patent Document 1 discloses a reporter protein for forming a polyglutamine aggregate in which 150 repeats of Huntington gene exon 1 and a fluorescent protein are fused.
　本開示は、一態様において、ポリグルタミン病に関する医薬組成物、それらの使用、それらを用いたポリグルタミン病の予防、改善、進行抑制及び／又は治療方法を提供する。本開示は、その他の態様において、ポリグルタミン病に関する化合物のスクリーニング方法を提供する。 The present disclosure, in one aspect, provides a pharmaceutical composition relating to polyglutamine disease, their use, and a method for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease using them. In another aspect, the present disclosure provides a method for screening a compound relating to polyglutamine disease.
　本開示は、一態様において、血管内上皮細胞増殖因子受容体（VEGFR）阻害化合物、c-met遺伝子にコードされた受容体型チロシンキナーゼ(c-Met)阻害化合物、及びVEGFRとc-Metとの両方の阻害化合物からなる群から選択される少なくとも１種類を有効成分として含有する、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物に関する。 In one aspect, the present disclosure provides an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a receptor tyrosine kinase (c-Met) inhibitor compound encoded by the c-met gene, and VEGFR and c-Met. The present invention relates to a pharmaceutical composition for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease, which contains at least one selected from the group consisting of both inhibitory compounds as an active ingredient.
　本開示は、その他の態様において、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物の有効成分の候補物質のスクリーニング方法であって、
　レポーター蛋白質と融合したポリグルタミンを発現するアッセイ細胞をテスト物質と接触させて培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ａ）を測定すること、
　前記アッセイ細胞を該テスト物質と接触させることなく培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ｂ）を測定すること、
　シグナル（Ａ）とシグナル（Ｂ）とを比較すること、及び、
　前記比較に基づいてシグナル（Ｂ）よりもシグナル（Ａ）を低減させるテスト物質を候補物質として選択することを含む、スクリーニング方法に関する。 In another aspect, the present disclosure provides a method for screening a candidate substance for an active ingredient of a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease,
Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell;
Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell;
Comparing signal (A) and signal (B); and
The present invention relates to a screening method comprising selecting, as a candidate substance, a test substance that reduces signal (A) over signal (B) based on the comparison.
図１は、ポリグルタミンとレポーター蛋白質との融合蛋白質の発現系の概略図である。FIG. 1 is a schematic diagram of an expression system of a fusion protein of polyglutamine and a reporter protein. 図２は、スクリーニングスキームの一例の概略図である。FIG. 2 is a schematic diagram of an example of a screening scheme. 図３は、スクリーニングに用いられるアッセイ系の発現誘導の様子、及びZ' factorを示す。FIG. 3 shows the expression induction of the assay system used for screening, and the Z ′ factor. 図４は、化合物４によるポリグルタミン凝集体形成阻害の濃度依存性を示すグラフである。FIG. 4 is a graph showing the concentration dependency of compound 4 inhibition of polyglutamine aggregate formation. 図５は、化合物４によるレポーター蛋白質発現量低下の濃度依存性を示すグラフである。FIG. 5 is a graph showing the concentration dependence of reporter protein expression level reduction by compound 4.
　本開示は、レポーター蛋白質と融合したポリグルタミンの凝集体の測定方法として、仮想細胞領域内のシグナルを測定する方法を採用すると、アッセイ感度が向上し、ポリグルタミン凝集体の形成に関わる物質を簡便にスクリーニングできる、という新しい知見に基づく。 In this disclosure, if a method of measuring a signal in a virtual cell region is used as a method for measuring a polyglutamine aggregate fused with a reporter protein, assay sensitivity is improved, and a substance involved in the formation of a polyglutamine aggregate is simplified. Based on the new knowledge that screening is possible.
　［ポリグルタミン病のための医薬組成物］
　本開示は、一態様において、ポリグルタミン病のための医薬組成物の医薬組成物（以下、「本開示に係る医薬組成物」ともいう。）を開示する。
　本開示において、ポリグルタミン病のための医薬組成物とは、一又は複数の実施形態において、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物をいう。
　本開示において、ポリグルタミン病とは、一又は複数の実施形態において、原因遺伝子内のCAGリピートの過伸長、それを鋳型とするポリグルタミン過伸長を有する蛋白質の生成、又は、この蛋白質の神経細胞内での蓄積及び凝集体形成の少なくとも１つに起因する疾病をいう。ポリグルタミン病として、一又は複数の実施形態において、ハンチントン病、遺伝性脊髄小脳変性症、又は、球脊髄性筋萎縮症が挙げられる。 [Pharmaceutical composition for polyglutamine disease]
In one aspect, the present disclosure discloses a pharmaceutical composition of a pharmaceutical composition for polyglutamine disease (hereinafter, also referred to as “the pharmaceutical composition according to the present disclosure”).
In the present disclosure, the pharmaceutical composition for polyglutamine disease refers to a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease in one or more embodiments.
In this disclosure, in one or a plurality of embodiments, polyglutamine disease is an overextension of a CAG repeat in a causative gene, generation of a protein having polyglutamine overextension using the CAG repeat as a template, or a nerve cell of this protein It refers to a disease caused by at least one of accumulation in the body and aggregate formation. Polyglutamine disease includes, in one or more embodiments, Huntington's disease, hereditary spinocerebellar degeneration, or bulbar spinal muscular atrophy.
　［有効成分］
　本開示に係る医薬組成物の有効成分として、一又は複数の実施形態において、血管内上皮細胞増殖因子受容体（VEGFR）の阻害物質、c-Metの阻害物質、又はVEGFRとc-Metの両方の阻害物質が挙げられ、好ましくは、VEGFR2に対する阻害活性を有する物質、VEGFRとc-Metの両方に対する阻害活性を有する物質又はVEGFR2とc-Metの両方に対する阻害活性を有する物質が挙げられる。後述する本開示に係るスクリーニング方法により、ポリグルタミン凝集体の形成を抑制する物質として、複数の公知のVEGER阻害物質及びVEGFRとc-Metの両方に対する阻害活性を有する物質が選択された。 [Active ingredients]
As an active ingredient of a pharmaceutical composition according to the present disclosure, in one or more embodiments, an inhibitor of intravascular epidermal growth factor receptor (VEGFR), an inhibitor of c-Met, or both VEGFR and c-Met Preferably, a substance having inhibitory activity against VEGFR2, a substance having inhibitory activity against both VEGFR and c-Met, or a substance having inhibitory activity against both VEGFR2 and c-Met can be mentioned. As a substance that suppresses the formation of polyglutamine aggregates, a plurality of known VEGER inhibitors and substances having inhibitory activity against both VEGFR and c-Met were selected by the screening method according to the present disclosure described later.
　有効成分であるVEGFR阻害物質の一又は複数の実施形態として、下記式（Ｉ）で表わされる構造を有するVEGFR阻害物質が挙げられる。ここで、下記式（Ｉ）におけるＷ、Ｚ、Ｒ1は任意の基とする。
Figure JPOXMLDOC01-appb-C000010
 One or a plurality of embodiments of a VEGFR inhibitor that is an active ingredient includes a VEGFR inhibitor having a structure represented by the following formula (I). Here, W, Z and R 1 in the following formula (I) are arbitrary groups.
Figure JPOXMLDOC01-appb-C000010
　上記式（Ｉ）で表わされる構造を有するVEGFR阻害物質のその他の一又は複数の実施形態として、式（Ｉ）において、
　Ｒ1は、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
　Ｗは、
Figure JPOXMLDOC01-appb-C000011
 であり、
　Ｒ2及びＲ3は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
　Ｘ1は、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であり、
　Ｘ2及びＹは、それぞれ独立して、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であるか、あるいは、
　Ｘ2及びＹは、ａで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有し、
　Ｚは、
Figure JPOXMLDOC01-appb-C000012
 であり、
　Ｒ4及びＲ5は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基、アミノ基、アジド基、シアノ基、ニトロ基、水酸基であるか、あるいは、
　Ｒ4及びＲ5は、ｂ及びｃで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有する、化合物又はその製薬上許容される塩が挙げられる。
　なお、本開示において波線を付した結合手は、式（Ｉ）、（ＩＩ）又は（ＩＩＩ）で表される化合物又は基との結合部分を示す。 As one or more embodiments of the VEGFR inhibitor having the structure represented by the above formula (I), in the formula (I),
R 1 is a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
W is
Figure JPOXMLDOC01-appb-C000011
 And
R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
X 1 is a C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom or an imino group,
X 2 and Y are each independently, C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom, or an imino group, or,
X 2 and Y together with the atom marked with a form an aromatic ring or a heteroaromatic ring, said ring being unsubstituted or hydrogen atom, halogen atom, C 1-4 alkyl A C 1-4 alkyl group, a C 1-4 alkyloxy group substituted by a group, a halogen atom or an alicyclic ring or a heterocyclic ring, or a C 1-4 alkyloxy group substituted by a halogen atom, an alicyclic ring or a heterocyclic ring Have one or more substituents,
Z is
Figure JPOXMLDOC01-appb-C000012
 And
R 4 and R 5 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom, an amino group, an azide group, a cyano group, or a nitro group. A hydroxyl group, or
R 4 and R 5 together with the atoms marked with b and c form an aromatic ring or a heteroaromatic ring, which ring is unsubstituted or is a hydrogen atom, halogen atom, C 1 -4 alkyl group, a halogen atom or an aliphatic ring or a C 1-4 alkyl group substituted with a heterocycle, C 1-4 alkyl group, or a halogen atom or an aliphatic ring or a C 1-4 alkyl substituted with a heterocycle Examples thereof include a compound or a pharmaceutically acceptable salt thereof having one or more substituents which are oxy groups.
In the present disclosure, a bond with a wavy line indicates a bond portion with the compound or group represented by the formula (I), (II) or (III).
　本開示においてVEGFR阻害物質とは、一又は複数の実施形態において、インビトロ及びインビボの少なくとも一方における公知のVEGFR阻害アッセイ系において、前記物質を加えた場合のVEGFRの活性を、前記物質を加えないコントロールと比べて、例えば６０％以下、５０％以下、４０％以下、３０％以下、２０％以下、又は１０％以下にまで阻害できる化合物をいう。前記アッセイ系において、添加する物質の量は、一又は複数の実施形態において、０．０１－１０μＭである。
　本開示においてc-Met阻害物質とは、一又は複数の実施形態において、インビトロ及びインビボの少なくとも一方における公知のc-Met阻害アッセイ系において、前記物質を加えた場合のc-Metの活性を、前記物質を加えないコントロールと比べて、例えば６０％以下、５０％以下、４０％以下、３０％以下、２０％以下、又は１０％以下にまで阻害できる化合物をいう。前記アッセイ系において、添加する物質の量は、一又は複数の実施形態において、０．０１－１０μＭである。
　本開示においてVEGFR及びC-Metの両方の阻害物質とは、一又は複数の実施形態において、インビトロ及びインビボの少なくとも一方における公知のVEGFR阻害アッセイ系において、前記物質を加えた場合のVEGFRの活性を、前記物質を加えないコントロールと比べて、例えば６０％以下、５０％以下、４０％以下、３０％以下、２０％以下、又は１０％以下にまで阻害でき、かつ、インビトロ及びインビボの少なくとも一方における公知のc-Met阻害アッセイ系において、前記物質を加えた場合のc-Metの活性を、前記物質を加えないコントロールと比べて、例えば６０％以下、５０％以下、４０％以下、３０％以下、２０％以下、又は１０％以下にまで阻害できる化合物をいう。前記アッセイ系において、添加する物質の量は、一又は複数の実施形態において、０．０１－１０μＭである。 In one or a plurality of embodiments, the VEGFR inhibitor in the present disclosure is a control in which the substance is not added to the activity of VEGFR when the substance is added in a known VEGFR inhibition assay system in at least one of in vitro and in vivo. Compared to, for example, it refers to a compound that can be inhibited to 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In the assay system, the amount of substance added is 0.01-10 μM in one or more embodiments.
In one or more embodiments, the c-Met inhibitory substance in the present disclosure refers to the activity of c-Met when the substance is added in a known c-Met inhibition assay system in at least one of in vitro and in vivo. Compared with the control to which the substance is not added, it refers to a compound that can be inhibited to, for example, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In the assay system, the amount of substance added is 0.01-10 μM in one or more embodiments.
In the present disclosure, the inhibitor of both VEGFR and C-Met refers to the activity of VEGFR in the known VEGFR inhibition assay system in at least one of in vitro and in vivo in the one or a plurality of embodiments when the substance is added. Compared with a control to which the substance is not added, for example, it can be inhibited to 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, and at least one of in vitro and in vivo In a known c-Met inhibition assay system, the activity of c-Met when the substance is added is, for example, 60% or less, 50% or less, 40% or less, 30% or less compared to a control without the substance. , 20% or less, or a compound that can be inhibited to 10% or less. In the assay system, the amount of substance added is 0.01-10 μM in one or more embodiments.
　アルキル基又はアルキレン基としては、一又は複数の実施形態において、直鎖若しくは分枝若しくは環状のアルキル基又はアルキレン基が挙げられる。「Ｃ1-4アルキル基」とは、一又は複数の実施形態において、炭素数１－４個の直鎖若しくは分枝若しくは環状のアルキル基である。炭素数１－４個の直鎖又は分枝のアルキル基としては、一又は複数の実施形態において、メチル基、エチル基、プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基が挙げられる。炭素数３－４個の環状のアルキル基としては、一又は複数の実施形態において、シクロプロピル基、シクロブチル基などが挙げられる。 In one or a plurality of embodiments, the alkyl group or alkylene group includes a linear, branched, or cyclic alkyl group or alkylene group. The “C 1-4 alkyl group” is a linear, branched or cyclic alkyl group having 1 to 4 carbon atoms in one or a plurality of embodiments. In one or more embodiments, the linear or branched alkyl group having 1 to 4 carbon atoms includes a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a sec-butyl group. And tert-butyl group. Examples of the cyclic alkyl group having 3 to 4 carbon atoms include a cyclopropyl group and a cyclobutyl group in one or more embodiments.
　ハロゲン原子としては、一又は複数の実施形態において、フッ素原子、塩素原子、臭素原子、ヨウ素原子が挙げられる。
　「複素環」とは、一又は複数の実施形態において、環を構成する原子中に１－２個のヘテロ原子を含有し、環中に二重結合を含んでいてもよく、非芳香族性の環又は芳香族性の環を意味する。「複素芳香環」とは、芳香族性の複素環を意味する。「ヘテロ原子」とは、一又は複数の実施形態において、硫黄原子、酸素原子又は窒素原子を意味する。 As a halogen atom, in one or some embodiment, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom are mentioned.
“Heterocycle”, in one or more embodiments, contains 1-2 heteroatoms in the atoms constituting the ring, may contain double bonds in the ring, and is non-aromatic. Or an aromatic ring. “Heteroaromatic ring” means an aromatic heterocycle. “Hetero atom” means, in one or more embodiments, a sulfur atom, an oxygen atom or a nitrogen atom.
　本開示において「製薬上許容される塩」とは、薬理上及び／又は医薬上許容される塩を含有し、例えば、無機酸塩、有機酸塩、無機塩基塩、有機塩基塩、酸性アミノ酸塩又は塩基性アミノ酸塩などが挙げられる。 In the present disclosure, the “pharmaceutically acceptable salt” includes a pharmacologically and / or pharmaceutically acceptable salt, for example, an inorganic acid salt, an organic acid salt, an inorganic basic salt, an organic basic salt, an acidic amino acid salt. Or a basic amino acid salt etc. are mentioned.
　前記無機酸塩の好ましい例としては、例えば塩酸塩、臭化水素酸塩、硫酸塩、硝酸塩、リン酸塩などが挙げられ、有機酸塩の好ましい例としては、例えば酢酸塩、コハク酸塩、フマル酸塩、マレイン酸塩、酒石酸塩、クエン酸塩、乳酸塩、ステアリン酸塩、安息香酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩などが挙げられる。 Preferable examples of the inorganic acid salt include hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferable examples of the organic acid salt include, for example, acetate, succinate, Examples thereof include fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.
　前記無機塩基塩の好ましい例としては、例えばナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩、マグネシウム塩などのアルカリ土類金属塩、アルミニウム塩、アンモニウム塩などが挙げられる。前記有機塩基塩の好ましい例としては、例えばジエチルアミン塩、ジエタノールアミン塩、メグルミン塩、Ｎ，Ｎ'－ジベンジルエチレンジアミン塩などが挙げられる。 Preferred examples of the inorganic base salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferable examples of the organic base salt include diethylamine salt, diethanolamine salt, meglumine salt, N, N′-dibenzylethylenediamine salt and the like.
　前記酸性アミノ酸塩の好ましい例としては、例えばアスパラギン酸塩、グルタミン酸塩などが挙げられる。前記塩基性アミノ酸塩の好ましい例としては、例えばアルギニン塩、リジン塩、オルニチン塩などが挙げられる。 Preferred examples of the acidic amino acid salt include aspartate and glutamate. Preferable examples of the basic amino acid salt include arginine salt, lysine salt, ornithine salt and the like.
　本開示において「化合物の塩」には、化合物が大気中に放置されることにより、水分を吸収して形成されうる水和物が包含され得る。また、本開示において「化合物の塩」には、化合物が他のある種の溶媒を吸収して形成されうる溶媒和物も包含され得る。 In the present disclosure, the “salt of a compound” may include a hydrate that can be formed by absorbing moisture when the compound is left in the air. Further, in the present disclosure, the “salt of a compound” may include a solvate that can be formed by absorbing a certain kind of other solvent.
　上記式（Ｉ）のＲ1は、一又は複数の実施形態において、ハロゲン原子、又はハロゲン原子で置換されたＣ1-4アルキル基が挙げられ、限定されない一又は複数の実施形態において、フッ素原子、又はトリフルオロメチル基が挙げられる。 R 1 in the above formula (I) includes, in one or more embodiments, a halogen atom, or a C 1-4 alkyl group substituted with a halogen atom, and in one or more embodiments, fluorine atom Or a trifluoromethyl group.
　上記式（Ｉ）のＷは、一又は複数の実施形態において、
Figure JPOXMLDOC01-appb-C000013
 が挙げられる。
　Ｒ2及びＲ3は、一又は複数の実施形態において、それぞれ独立して、水素原子又はハロゲン原子が挙げられる。Ｘ1は、一又は複数の実施形態において、イミノ基であり、Ｘ2及びＹは、一又は複数の実施形態において、それぞれ独立して、イミノ基若しくは
Figure JPOXMLDOC01-appb-C000014
 あるいは、Ｘ2及びＹは、ａで印をつけた原子と共に、
Figure JPOXMLDOC01-appb-C000015
 が挙げられる。 W in the above formula (I) is one or more embodiments,
Figure JPOXMLDOC01-appb-C000013
 Is mentioned.
In one or a plurality of embodiments, R 2 and R 3 each independently represents a hydrogen atom or a halogen atom. X 1 is an imino group in one or a plurality of embodiments, and X 2 and Y are each independently an imino group or a group in one or a plurality of embodiments.
Figure JPOXMLDOC01-appb-C000014
 Alternatively, X 2 and Y, together with the atoms marked with a,
Figure JPOXMLDOC01-appb-C000015
 Is mentioned.
　上記式（Ｉ）のＷは、VEGFRに対する阻害活性に加えて、c-Metに対する阻害活性を有する点から、一又は複数の実施形態において、
Figure JPOXMLDOC01-appb-C000016
 が挙げられる。
　Ｒ2、Ｒ3、Ｘ2及びＹは、上記と同様である。 In the one or more embodiments, W in the above formula (I) has an inhibitory activity against c-Met in addition to the inhibitory activity against VEGFR.
Figure JPOXMLDOC01-appb-C000016
 Is mentioned.
R 2 , R 3 , X 2 and Y are the same as above.
　上記式（Ｉ）のＺは、一又は複数の実施形態において、
Figure JPOXMLDOC01-appb-C000017
 が挙げられる。Ｒ4及びＲ5は、一又は複数の実施形態において、それぞれ独立して、ハロゲン原子若しくはアミノ基、あるいは、Ｒ4及びＲ5は、ｂ及びｃで印をつけた原子と共に、
Figure JPOXMLDOC01-appb-C000018
 が挙げられる。Ｒ6及びＲ7は、一又は複数の実施形態において、それぞれ独立して、メチル基、又は、
Figure JPOXMLDOC01-appb-C000019
 が挙げられる。 Z in the above formula (I) is, in one or more embodiments,
Figure JPOXMLDOC01-appb-C000017
 Is mentioned. R 4 and R 5 , in one or more embodiments, are each independently a halogen atom or an amino group, or R 4 and R 5 , together with atoms marked with b and c,
Figure JPOXMLDOC01-appb-C000018
 Is mentioned. In one or more embodiments, R 6 and R 7 are each independently a methyl group, or
Figure JPOXMLDOC01-appb-C000019
 Is mentioned.
　上記式（Ｉ）は、一又は複数の実施形態において、下記式（ＩＩ）である。
Figure JPOXMLDOC01-appb-C000020
 　［式（ＩＩ）において、
　Ｗは、
Figure JPOXMLDOC01-appb-C000021
 であり、Ｒ1－Ｒ5、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様である。］ The above formula (I) is the following formula (II) in one or more embodiments.
Figure JPOXMLDOC01-appb-C000020
 [In the formula (II),
W is
Figure JPOXMLDOC01-appb-C000021
 R 1 -R 5 , X 1 , X 2 and Y are the same as those in formula (I). ]
　上記式（Ｉ）は、一又は複数の実施形態において、下記式（ＩＩＩ）である。
Figure JPOXMLDOC01-appb-C000022
 　［式（ＩＩＩ）において、
　Ｗは、
Figure JPOXMLDOC01-appb-C000023
 　であり、Ｒ1－Ｒ3、Ｒ6及びＲ7、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様である。］ The formula (I) is the following formula (III) in one or more embodiments.
Figure JPOXMLDOC01-appb-C000022
 [In Formula (III),
W is
Figure JPOXMLDOC01-appb-C000023
 R 1 -R 3 , R 6 and R 7 , X 1 , X 2 and Y are the same as in formula (I). ]
　上記式（Ｉ）は、一又は複数の実施形態において、下記式（ＩＶ）である。
Figure JPOXMLDOC01-appb-C000024
 　［式（ＩＶ）において、Ｒ1－Ｒ5、Ｘ2及びＹは式（Ｉ）と同様である］ The formula (I) is represented by the following formula (IV) in one or more embodiments.
Figure JPOXMLDOC01-appb-C000024
 [In formula (IV), R 1 -R 5 , X 2 and Y are the same as in formula (I)]
　有効成分であるVEGFR阻害物質の一又は複数の実施形態として、下記化合物１－５が挙げられる。VEGFR及びc-Metの両方の阻害活性を有する物質の一又は複数の実施形態として、下記化合物２－４が挙げられる。
Figure JPOXMLDOC01-appb-C000025
 As one or a plurality of embodiments of the VEGFR inhibitor which is an active ingredient, the following compound 1-5 may be mentioned. One or more embodiments of a substance having both VEGFR and c-Met inhibitory activity include the following compound 2-4.
Figure JPOXMLDOC01-appb-C000025
　本開示に係る医薬組成物の有効成分として、その他の一又は複数の実施形態において、下記化合物６－１１も挙げられる。
Figure JPOXMLDOC01-appb-C000026
 In one or more other embodiments, the active ingredient of the pharmaceutical composition according to the present disclosure may include the following compound 6-11.
Figure JPOXMLDOC01-appb-C000026
　本開示に係る医薬組成物の有効成分である上記化合物は、不斉炭素原子が存在する場合及び／又は立体異性体が存在する場合、一又は複数の実施形態において、各異性体の混合物又は単離されたものである。立体異性体の特に限定されない一又は複数の実施形態において、シス－トランス異性体が挙げられる。 In the case where an asymmetric carbon atom is present and / or a stereoisomer is present, the above-mentioned compound that is an active ingredient of the pharmaceutical composition according to the present disclosure is, in one or a plurality of embodiments, a mixture or a single isomer. It has been released. In one or more embodiments of the stereoisomer that are not particularly limited, cis-trans isomers may be mentioned.
　本開示に係る医薬組成物の有効成分である上記化合物は、プロドラッグの形態で医薬組成物に含有されていてもよい。「プロドラッグ」は、一又は複数の実施形態において、生体内で容易に加水分解され、上記化合物を再生するものが挙げられ、例えばカルボキシル基を有する化合物であればそのカルボキシル基がアルコキシカルボニル基となった化合物、アルキルチオカルボニル基となった化合物、又はアルキルアミノカルボニル基となった化合物が挙げられる。また、例えばアミノ基を有する化合物であれば、そのアミノ基がアルカノイル基で置換されアルカノイルアミノ基となった化合物、アルコキシカルボニル基により置換されアルコキシカルボニルアミノ基となった化合物、アシロキシメチルアミノ基となった化合物、又はヒドロキシルアミンとなった化合物が挙げられる。また例えば水酸基を有する化合物であれば、その水酸基が前記アシル基により置換されてアシロキシ基となった化合物、リン酸エステルとなった化合物、又はアシロキシメチルオキシ基となった化合物が挙げられる。これらのプロドラッグ化に用いる基のアルキル部分としては後述するアルキル基が挙げられ、そのアルキル基は置換（例えば炭素原子数１～６のアルコキシ基等により）されていてもよい。一又は複数の実施形態において、例えばカルボキシル基がアルコキシカルボニル基となった化合物を例にとれば、メトキシカルボニル、エトキシカルボニルなどの低級（例えば炭素数１～６）アルコキシカルボニル、メトキシメトキシカルボニル、エトキシメトキシカルボニル、２－メトキシエトキシカルボニル、２－メトキシエトキシメトキシカルボニル、ピバロイロキシメトキシカルボニルなどのアルコキシ基により置換された低級（例えば炭素数１～６）アルコキシカルボニルが挙げられる。 The above-mentioned compound that is an active ingredient of the pharmaceutical composition according to the present disclosure may be contained in the pharmaceutical composition in the form of a prodrug. In one or a plurality of embodiments, “prodrug” includes those that are easily hydrolyzed in vivo and regenerate the above compound. For example, in the case of a compound having a carboxyl group, the carboxyl group is an alkoxycarbonyl group. Or a compound that becomes an alkylthiocarbonyl group, or a compound that becomes an alkylaminocarbonyl group. Further, for example, in the case of a compound having an amino group, a compound in which the amino group is substituted with an alkanoyl group to become an alkanoylamino group, a compound in which the amino group is substituted with an alkoxycarbonyl group to become an alkoxycarbonylamino group, an acyloxymethylamino group, Or a compound that has become hydroxylamine. In addition, for example, in the case of a compound having a hydroxyl group, a compound in which the hydroxyl group is substituted with the acyl group to become an acyloxy group, a compound that has become a phosphate ester, or a compound that has become an acyloxymethyloxy group can be given. Examples of the alkyl moiety of the group used for forming a prodrug include an alkyl group described later, and the alkyl group may be substituted (for example, with an alkoxy group having 1 to 6 carbon atoms). In one or a plurality of embodiments, for example, when a compound in which a carboxyl group is an alkoxycarbonyl group is taken as an example, lower (eg, having 1 to 6 carbon atoms) alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, methoxymethoxycarbonyl, ethoxymethoxy, etc. Examples include lower (eg, having 1 to 6 carbon atoms) alkoxycarbonyl substituted with an alkoxy group such as carbonyl, 2-methoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, and pivaloyloxymethoxycarbonyl.
　本開示の医薬組成物は、一又は複数の実施形態において、上記阻害物質を有効成分として含有し、さらに、医薬的に許容される担体、防腐剤、希釈剤、賦形剤又はその他の医薬的に許容される成分を含んでよい。 In one or a plurality of embodiments, the pharmaceutical composition of the present disclosure contains the inhibitor as an active ingredient, and further includes a pharmaceutically acceptable carrier, preservative, diluent, excipient, or other pharmaceutical agent. May contain acceptable components.
　本開示において「医薬組成物」は、一又は複数の実施形態において、周知の製剤技術を適用し、投与形態に適した剤形とすることができる。その投与形態としては、これらに限定されないが、例えば、錠剤、カプセル剤、顆粒剤、散剤、丸剤、トローチ剤、シロップ剤、液剤等の剤形による経口投与が挙げられる。或いは、注射剤、液剤、エアゾール剤、座剤、貼付剤、パップ剤、ローション剤、リニメント剤、軟膏剤、点眼剤等の剤形による非経口投与を挙げることができる。これらの製剤は、これらに限定されないが、賦形剤、滑沢剤、結合剤、崩壊剤、安定化剤、矯味矯臭剤、希釈剤などの添加剤を用いて周知の方法で製造されうる。 In the present disclosure, the “pharmaceutical composition” may be a dosage form suitable for an administration form by applying a well-known formulation technique in one or a plurality of embodiments. Examples of the dosage form include, but are not limited to, oral administration in a dosage form such as a tablet, capsule, granule, powder, pill, troche, syrup, and liquid. Alternatively, parenteral administration in dosage forms such as injections, solutions, aerosols, suppositories, patches, patches, lotions, liniments, ointments, eye drops and the like can be mentioned. These preparations can be produced by known methods using additives such as, but not limited to, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
　前記賦形剤としては、これらに限定されないがデンプン、バレイショデンプン、トウモロコシデンプン等のデンプン、乳糖、結晶セルロース、リン酸水素カルシウム等を挙げることができる。前記コーティング剤としては、これらに限定されないが、エチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、セラック、タルク、カルナウバロウ、パラフィン等を挙げることができる。前記結合剤としては、これらに限定されないが、ポリビニルピロリドン、マクロゴール及び前記賦形剤と同様の化合物を挙げることができる。前記崩壊剤としては、これらに限定されないが、前記賦形剤と同様の化合物及びクロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドンのような化学修飾されたデンプン・セルロース類を挙げることができる。前記安定化剤としては、これらに限定されないが、メチルパラベン、プロピルパラベンのようなパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコールのようなアルコール類；塩化ベンザルコニウム；フェノール、クレゾールのようなフェエノール類；チメロサール；デヒドロ酢酸；及びソルビン酸を挙げることができる。前記矯味矯臭剤としては、これらに限定されないが、通常使用される、甘味料、酸味料、香料等を挙げることができる。 Examples of the excipient include, but are not limited to, starch such as starch, potato starch, and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like. Examples of the coating agent include, but are not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, paraffin, and the like. Examples of the binder include, but are not limited to, polyvinyl pyrrolidone, macrogol and the same compound as the excipient. Examples of the disintegrant include, but are not limited to, compounds similar to the excipients and chemically modified starch and celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone. . Examples of the stabilizer include, but are not limited to, paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenol, cresol Mention may be made of such phenols; thimerosal; dehydroacetic acid; and sorbic acid. Examples of the flavoring agent include, but are not limited to, sweeteners, acidulants, and fragrances that are commonly used.
　また、液剤の製造には、溶媒として、これらに限定されないが、エタノール、フェノール、クロロクレゾール、精製水、蒸留水等を使用することができ、必要に応じて界面活性剤又は乳化剤等も使用できる。前記界面活性剤又は乳化剤としては、これらに限定されないが、ポリソルベート８０、ステアリン酸ポリオキシル４０、ラウロマクロゴール等を挙げることができる。 In the production of the liquid agent, the solvent is not limited to these, but ethanol, phenol, chlorocresol, purified water, distilled water and the like can be used, and a surfactant or an emulsifier can also be used as necessary. . Examples of the surfactant or emulsifier include, but are not limited to, polysorbate 80, polyoxyl 40 stearate, lauromacrogol, and the like.
　本開示に係る医薬組成物の使用方法は、症状、年齢、投与方法等により異なりうる。使用方法は、これらに限定されないが、有効成分である上記化合物の体内濃度が１００ｎＭ～１ｍＭの間のいずれかになるように、間欠的若しくは持続的に、経口、経皮、粘膜下、皮下、筋肉内、血管内、脳内、又は腹腔内に投与することができる。限定されない実施形態として、経口投与の場合、対象（ヒトであれば成人）に対して１日あたり、前記一般式（Ｉ）で表される化合物に換算して、下限として０．０１ｍｇ（好ましくは０．１ｍｇ）、上限として、２０００ｍｇ（好ましくは５００ｍｇ、より好ましくは１００ｍｇ）を１回又は数回に分けて、症状に応じて投与することが挙げられる。限定されない実施形態として、静脈内投与の場合には、対象（ヒトであれば成人）に対して１日当たり、下限として０．００１ｍｇ（好ましくは０．０１ｍｇ）、上限として、５００ｍｇ（好ましくは５０ｍｇ）を１回又は数回に分けて、症状に応じて投与することが挙げられる。 The method of using the pharmaceutical composition according to the present disclosure may vary depending on symptoms, age, administration method, and the like. The method of use is not limited to these, but intermittently or continuously, orally, transdermally, submucosally, subcutaneously, so that the in vivo concentration of the compound as the active ingredient is between 100 nM and 1 mM. Administration can be intramuscular, intravascular, intracerebral, or intraperitoneal. As a non-limiting embodiment, in the case of oral administration, the lower limit is 0.01 mg (preferably converted into the compound represented by the above general formula (I) per day for a subject (adult if human)) 0.1 mg), and as an upper limit, 2000 mg (preferably 500 mg, more preferably 100 mg) can be divided into one or several doses and administered according to symptoms. As a non-limiting embodiment, in the case of intravenous administration, the lower limit is 0.001 mg (preferably 0.01 mg) and the upper limit is 500 mg (preferably 50 mg) per day for a subject (adult if human). Is divided into one or several times and administered according to symptoms.
　［ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療］
　本開示は、一又は複数の実施形態において、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療の方法であって、本開示に係る医薬組成物を対象に投与することを含む方法に関する。本開示に係る医薬組成物の投与は、一又は複数の実施形態において、前述の医薬組成物の使用方法に準じることができる。対象としては、ヒト、ヒト以外の動物が挙げられる。 [Prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease]
In one or a plurality of embodiments, the present disclosure is a method for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease, the method comprising administering a pharmaceutical composition according to the present disclosure to a subject. About. In one or a plurality of embodiments, administration of the pharmaceutical composition according to the present disclosure can be based on the above-described method of using the pharmaceutical composition. Examples of the subject include humans and non-human animals.
　本開示は、一又は複数の実施形態において、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための有効成分である上記化合物又はその製薬上許容される塩の使用に関する。
　本開示は、その他の一又は複数の実施形態において、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物を製造するための有効成分である上記化合物又はその製薬上許容される塩の使用に関する。 In one or a plurality of embodiments, the present disclosure relates to the use of the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease.
In one or a plurality of other embodiments of the present disclosure, the present compound or the pharmaceutical thereof is an active ingredient for producing a pharmaceutical composition for prevention, amelioration, progression inhibition, and / or treatment of polyglutamine disease. It relates to the use of top acceptable salts.
　［スクリーニング方法］
　本開示は、一態様において、ポリグルタミン凝集体の形成に影響を与える物質のスクリーニング方法に関する。本態様のスクリーニング方法は、
　レポーター蛋白質と融合したポリグルタミンを発現するアッセイ細胞をテスト物質と接触させて培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ａ）を測定すること、
　前記アッセイ細胞を該テスト物質と接触させることなく培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ｂ）を測定すること、
　シグナル（Ａ）とシグナル（Ｂ）とを比較すること、及び、
　前記比較に基づいてシグナル（Ｂ）よりもシグナル（Ａ）を低減させるテスト物質を候補物質として選択することを含む。 [Screening method]
In one aspect, the present disclosure relates to a method for screening a substance that affects the formation of a polyglutamine aggregate. The screening method of this aspect is:
Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell;
Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell;
Comparing signal (A) and signal (B); and
Selecting a test substance that reduces the signal (A) over the signal (B) as a candidate substance based on the comparison.
　本開示にかかるスクリーニング方法におけるアッセイ細胞は、レポーター蛋白質と融合したポリグルタミンを発現可能な遺伝子を有する。アッセイ細胞は、一又は複数の実施形態において、レポーター蛋白質と融合したポリグルタミンのｍＲＮＡが発現されうるように構成及び適合された遺伝子発現ベクターを細胞に導入することで作製できる。アッセイ細胞の作製に使用する、すなわち、前記ベクターの導入対象となる細胞は、特に制限されず、一又は複数の実施形態において、哺乳類細胞である。哺乳類細胞は、一又は複数の実施形態において、ヒト、ウシ、ネコ、サル、イヌ、ハムスター、ミンク、マウス、ブタ、ウサギ、ラットの細胞である。細胞の種類は、特に制限されないが、一又は複数の実施形態において、神経細胞又はその培養細胞が挙げられる。前記ベクターは、一過性発現を行うタイプであってもよく、安定発現を行うタイプであってもよい。また、遺伝子発現ベクターのプロモーターは、アッセイ細胞内でタンパク質の発現を誘導できる、従前知られた又は今後開発されるプロモーターを使用できる。前記レポーター蛋白質としては、一又は複数の実施形態において、発光蛋白質が挙げられ、例えば、蛍光蛋白質が挙げられる。
　レポーター蛋白質と融合したポリグルタミンを発現可能な遺伝子としては、実施例に記載のものが使用できる。 The assay cell in the screening method according to the present disclosure has a gene capable of expressing polyglutamine fused with a reporter protein. In one or a plurality of embodiments, the assay cell can be prepared by introducing a gene expression vector constructed and adapted to express a polyglutamine mRNA fused with a reporter protein into the cell. The cell used for the production of the assay cell, that is, the cell into which the vector is introduced is not particularly limited, and in one or more embodiments, is a mammalian cell. In one or more embodiments, the mammalian cell is a human, bovine, cat, monkey, dog, hamster, mink, mouse, pig, rabbit, rat cell. The type of cell is not particularly limited, and in one or a plurality of embodiments, a nerve cell or a cultured cell thereof can be mentioned. The vector may be a type that performs transient expression or a type that performs stable expression. Further, as the promoter of the gene expression vector, a promoter known in the art or developed in the future that can induce protein expression in the assay cell can be used. Examples of the reporter protein include a photoprotein in one or a plurality of embodiments, and examples include a fluorescent protein.
As a gene capable of expressing polyglutamine fused with a reporter protein, those described in the Examples can be used.
　本開示において、仮想細胞体領域とは、細胞核の周りを含む所定の領域をいう。この領域にあるレポーターシグナルを画像分析で測定することにより、ポリグルタミン凝集体を感度よく検出できる。
　本態様のスクリーニング方法の一又は複数の実施形態として、仮想細胞体領域のポリグルタミンタンパク存在領域におけるシグナルの輝度総計と、存在領域の面積とから、ポリグルタミンタンパク存在領域内平均輝度を算出することが挙げられる。
　仮想細胞体領域の設定は、例えば、核の周辺域を一定の画素数幅で拡張した領域を含む領域として行うことができる。一般に、哺乳細胞の画像における細胞核はDNA染色剤DAPIによって染色される一領域として定義される。細胞核は細胞に一つのみ存在し、細胞体はそれを囲む空間として捉えられる。例えば、DAPI染色領域の撮像画像において各細胞核領域を定義し、それより一定の画素数の幅を持つ領域までをその細胞核に付属する細胞質として定義する。この同定された細胞質領域の中に存在する一定輝度以上のレポーター蛍光領域を当該核/細胞に帰属するポリグルタミンタンパク存在領域とする。この存在領域内における輝度総計と、同定された存在領域の面積を計算し、ポリグルタミンタンパク存在領域内平均輝度を算出する。凝集体は画像上でレポーター由来蛍光値の高い領域として定義されるため、上記方法において同定されるポリグルタミンタンパク存在領域は形成されない場合と比べ相対的に狭く同定される。したがって、凝集体が仮想細胞体領域に有意に形成された場合、ポリグルタミンタンパク存在領域内平均輝度の凝集体形成有無に対する差は、総計の差よりも大きく、さらに凝集体の大小に由来する測定結果のバラつきが相殺され、したがって測定精度が上昇する。 In the present disclosure, the virtual cell body region refers to a predetermined region including the periphery of the cell nucleus. By measuring the reporter signal in this region by image analysis, polyglutamine aggregates can be detected with high sensitivity.
As one or a plurality of embodiments of the screening method of this aspect, calculating the average luminance in the polyglutamine protein existing region from the total luminance of the signal in the polyglutamine protein existing region of the virtual cell body region and the area of the existing region Is mentioned.
The virtual cell body region can be set, for example, as a region including a region in which the peripheral region of the nucleus is expanded with a certain number of pixels. In general, cell nuclei in mammalian cell images are defined as a region stained by the DNA stain DAPI. Only one cell nucleus exists in a cell, and the cell body is regarded as a space surrounding it. For example, each cell nucleus region is defined in the captured image of the DAPI-stained region, and the region having a certain number of pixels is defined as the cytoplasm attached to the cell nucleus. A reporter fluorescent region having a certain luminance or higher existing in the identified cytoplasmic region is defined as a polyglutamine protein existing region belonging to the nucleus / cell. The total luminance in the existing region and the area of the identified existing region are calculated, and the average luminance in the polyglutamine protein existing region is calculated. Since the aggregate is defined as a region having a high reporter-derived fluorescence value on the image, the polyglutamine protein existing region identified in the above method is identified relatively narrowly as compared with the case where it is not formed. Therefore, when aggregates are significantly formed in the virtual cell body region, the difference between the average luminance in the polyglutamine protein-existing region and the presence or absence of aggregate formation is larger than the total difference, and the measurement is derived from the size of the aggregate. Variations in results are offset, thus increasing measurement accuracy.
　すなわち、本開示は以下の一又は複数の実施形態に関しうる；
［１］　血管内上皮細胞増殖因子受容体（VEGFR）阻害化合物、c-Met阻害化合物、及びVEGFRとc-Metとの両方の阻害化合物からなる群から選択される少なくとも一種類を有効成分として含有する、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物。
［２］　前記阻害化合物が下記式（Ｉ）で表わされる化合物又はその製薬上許容される塩である、［１］記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000027
 　［式（Ｉ）において、
　Ｒ1は、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
　Ｗは、
Figure JPOXMLDOC01-appb-C000028
 であり、
　Ｒ2及びＲ3は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
　Ｘ1は、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であり、
　Ｘ2及びＹは、それぞれ独立して、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であるか、あるいは、
　Ｘ2及びＹは、ａで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有し、
　Ｚは、
Figure JPOXMLDOC01-appb-C000029
 であり、
　Ｒ4及びＲ5は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基、アミノ基、アジド基、シアノ基、ニトロ基、水酸基であるか、あるいは、
　Ｒ4及びＲ5は、ｂ及びｃで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有する。］
［３］　前記式（Ｉ）で表わされる化合物が、下記式（ＩＩ）で表わされる化合物である、［２］記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000030
 　［式（ＩＩ）において、
　Ｗは、
Figure JPOXMLDOC01-appb-C000031
 であり、Ｒ1－Ｒ5、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様である。］
［４］　前記式（Ｉ）で表わされる化合物が、下記式（ＩＩＩ）で表わされる化合物である、［２］記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000032
 　［式（ＩＩＩ）において、
　Ｗは、
Figure JPOXMLDOC01-appb-C000033
 であり、Ｒ1－Ｒ3、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様であり、
　Ｒ6及びＲ7は、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基である。］
［５］　前記式（Ｉ）で表わされる化合物が、下記式（ＩＶ）で表わされる化合物である、［２］記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000034
 　［式（ＩＶ）において、Ｒ1－Ｒ5、Ｘ2及びＹは式（Ｉ）と同様である］
［６］　下記化合物１－１１からなる群から選択される少なくとも１種類を有効成分として含有する、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物。
Figure JPOXMLDOC01-appb-C000035
 ［７］　前記ポリグルタミン病が、ハンチントン病、遺伝性脊髄小脳変性症、又は、球脊髄性筋萎縮症である、［１］から［６］のいずれかに記載の医薬組成物。
［８］　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療の方法であって、［１］から［６］のいずれかに記載の医薬組成物を対象に投与することを含む、方法。［９］　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物を製造するための血管内上皮細胞増殖因子受容体（VEGFR）阻害化合物、c-Met阻害化合物、及びVEGFRとc-Metとの両方の阻害化合物からなる群から選択される少なくとも一種類の使用。
［１０］　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物の有効成分の候補物質のスクリーニング方法であって、
　レポーター蛋白質と融合したポリグルタミンを発現するアッセイ細胞をテスト物質と接触させて培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ａ）を測定すること、
　前記アッセイ細胞を該テスト物質と接触させることなく培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ｂ）を測定すること、
　シグナル（Ａ）とシグナル（Ｂ）とを比較すること、及び、
　前記比較に基づいてシグナル（Ｂ）よりもシグナル（Ａ）を低減させるテスト物質を候補物質として選択することを含む、スクリーニング方法。 That is, the present disclosure may relate to one or more of the following embodiments;
[1] Containing as an active ingredient at least one selected from the group consisting of an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a c-Met inhibitor compound, and both VEGFR and c-Met inhibitor compounds A pharmaceutical composition for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease.
[2] The pharmaceutical composition according to [1], wherein the inhibitory compound is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000027
 [In Formula (I),
R 1 is a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
W is
Figure JPOXMLDOC01-appb-C000028
 And
R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
X 1 is a C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom or an imino group,
X 2 and Y are each independently, C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom, or an imino group, or,
X 2 and Y together with the atom marked with a form an aromatic ring or a heteroaromatic ring, said ring being unsubstituted or hydrogen atom, halogen atom, C 1-4 alkyl A C 1-4 alkyl group, a C 1-4 alkyloxy group substituted by a group, a halogen atom or an alicyclic ring or a heterocyclic ring, or a C 1-4 alkyloxy group substituted by a halogen atom, an alicyclic ring or a heterocyclic ring Have one or more substituents,
Z is
Figure JPOXMLDOC01-appb-C000029
 And
R 4 and R 5 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom, an amino group, an azide group, a cyano group, or a nitro group. A hydroxyl group, or
R 4 and R 5 together with the atoms marked with b and c form an aromatic ring or a heteroaromatic ring, which ring is unsubstituted or is a hydrogen atom, halogen atom, C 1 -4 alkyl group, a halogen atom or an aliphatic ring or a C 1-4 alkyl group substituted with a heterocycle, C 1-4 alkyl group, or a halogen atom or an aliphatic ring or a C 1-4 alkyl substituted with a heterocycle It has one or more substituents that are oxy groups. ]
[3] The pharmaceutical composition according to [2], wherein the compound represented by the formula (I) is a compound represented by the following formula (II).
Figure JPOXMLDOC01-appb-C000030
 [In the formula (II),
W is
Figure JPOXMLDOC01-appb-C000031
 R 1 -R 5 , X 1 , X 2 and Y are the same as those in formula (I). ]
[4] The pharmaceutical composition according to [2], wherein the compound represented by the formula (I) is a compound represented by the following formula (III).
Figure JPOXMLDOC01-appb-C000032
 [In Formula (III),
W is
Figure JPOXMLDOC01-appb-C000033
 R 1 -R 3 , X 1 , X 2 and Y are the same as in formula (I),
R 6 and R 7 is a hydrogen atom, a halogen atom, C 1-4 alkyl groups, C 1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring. ]
[5] The pharmaceutical composition according to [2], wherein the compound represented by the formula (I) is a compound represented by the following formula (IV).
Figure JPOXMLDOC01-appb-C000034
 [In formula (IV), R 1 -R 5 , X 2 and Y are the same as in formula (I)]
[6] A pharmaceutical composition for preventing, ameliorating, inhibiting progression and / or treating polyglutamine disease, comprising as an active ingredient at least one selected from the group consisting of the following compounds 1-11.
Figure JPOXMLDOC01-appb-C000035
 [7] The pharmaceutical composition according to any one of [1] to [6], wherein the polyglutamine disease is Huntington's disease, hereditary spinocerebellar degeneration, or bulbar spinal muscular atrophy.
[8] A method of prevention, improvement, progression suppression, and / or treatment of polyglutamine disease, comprising administering to a subject the pharmaceutical composition according to any one of [1] to [6] Method. [9] Intravascular epidermal growth factor receptor (VEGFR) inhibitory compound, c-Met inhibitory compound for producing a pharmaceutical composition for prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease, And at least one use selected from the group consisting of both VEGFR and c-Met inhibitor compounds.
[10] A screening method for a candidate substance of an active ingredient of a pharmaceutical composition for prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease,
Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell;
Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell;
Comparing signal (A) and signal (B); and
A screening method comprising selecting, as a candidate substance, a test substance that reduces the signal (A) over the signal (B) based on the comparison.
　以下、実施例により本開示をさらに詳細に説明するが、これらは例示的なものであって、本開示はこれら実施例に制限されるものではない。なお、本開示中に引用された文献は、すべて本開示の一部として組み入れられる。 Hereinafter, the present disclosure will be described in more detail by way of examples. However, these examples are illustrative, and the present disclosure is not limited to these examples. It should be noted that all documents cited in this disclosure are incorporated as part of this disclosure.
　実験例１：ポリグルタミン凝集体の形成に影響を与える物質のスクリーニング
　細胞内でポリグルタミン凝集体を形成させる系として、宿主の神経細胞（Neuro2a細胞）に図１に記載のようなレポーターが融合したポリグルタミンの発現系が導入された細胞（以下、「ポリQ細胞」という）を利用した（Matsumoto et al. Molecular Cell, 2011;44:279-289）。
　ポリQ細胞を用い、図２に示すスキームでスクリーニングを行った。まず、ポリQ細胞を播種して1日培養したあと（高グルコースDMEM培地、10% FBS、100u/mL ペニシリン、100μg/ml ストレプトマイシン）、4mM dibutylil cyclic AMP (dbcAMP)を添加して分化誘導するとともに、10μg/ml Doxicyclin (DOX)を添加してレポーターが融合したポリグルタミンの発現誘導を行い、さらに、テスト化合物を添加する（10μM、コントロールは0.1% DMSO）。この後2日間培養した細胞を4% パラホルムアルデヒド（PFA）、4℃、30分の処理で固定し、DAPIで核染色をした後、細胞イメージアナライザーArrayScan VTiで測定した。測定は、核の周辺領域と仮想細胞体領域と設定し、該領域にあるポリグルタミン凝集体（Venus）の輝度を測定した。仮想細胞体領域を設定してその領域内の凝集体の輝度を測定し、平均化することで、図３に示すように、発現誘導（dbcAMP+DOX）の有無に応じた凝集体平均輝度の差が顕著になり、アッセイ系の最適度を表す指標であるZ' factorが0.73という良好な数値を示した。
　また、この系において、1視野あたりのポリグルタミンの相対平均輝度蛍光がDMSOコントロールの50％となる濃度をIC50として測定した。
　なお、仮想細胞体領域は、細胞イメージアナライザーArrayScan VTi搭載20倍対物レンズで画像を取得し、DAPI染色領域の撮像画像において各細胞核領域を定義し、それより15ピクセルの幅を持つ領域として定義した。 Experimental Example 1: Screening for a substance that affects the formation of polyglutamine aggregates As a system for forming polyglutamine aggregates in cells, a reporter as shown in FIG. 1 was fused to a host neuron (Neuro2a cell). Cells into which a polyglutamine expression system was introduced (hereinafter referred to as “polyQ cells”) were used (Matsumoto et al. Molecular Cell, 2011; 44: 279-289).
Screening was performed using polyQ cells according to the scheme shown in FIG. First, after seeding poly Q cells and culturing for 1 day (high glucose DMEM medium, 10% FBS, 100u / mL penicillin, 100μg / ml streptomycin), 4mM dibutylil cyclic AMP (dbcAMP) is added to induce differentiation Then, 10 μg / ml Doxicyclin (DOX) is added to induce expression of polyglutamine fused with the reporter, and a test compound is added (10 μM, control is 0.1% DMSO). Thereafter, the cells cultured for 2 days were fixed by treatment with 4% paraformaldehyde (PFA) at 4 ° C. for 30 minutes, subjected to nuclear staining with DAPI, and then measured with a cell image analyzer ArrayScan VTi. The measurement was performed by setting the peripheral region of the nucleus and the virtual cell body region, and measuring the luminance of the polyglutamine aggregate (Venus) in the region. By setting the virtual cell body region and measuring and averaging the brightness of the aggregates in the region, as shown in FIG. 3, the aggregate average brightness according to the presence or absence of expression induction (dbcAMP + DOX) The difference became prominent, and the Z ′ factor, which is an index representing the optimality of the assay system, showed a favorable value of 0.73.
In this system, the concentration at which the relative average luminance fluorescence of polyglutamine per visual field was 50% of that of DMSO control was measured as IC50.
In addition, the virtual cell body region was acquired as a 20x objective lens equipped with a cell image analyzer ArrayScan VTi, and each cell nucleus region was defined in the captured image of the DAPI stained region, and was defined as a region having a width of 15 pixels. .
　上記のスクリーニングシステムと、京都大学医学研究支援センター保有の機能既知化合物コレクション（2520化合物）とを用いてスクリーニングを行った。その結果、下記化合物１－１１を選出した。これらの化合物について、上記スクリーニング系において1視野におけるポリグルタミン凝集体の相対平均輝度蛍光がDMSOコントロールの50％となる濃度をIC50として測定した（n=2）。その結果を下記表１に示す。
Figure JPOXMLDOC01-appb-C000036
 Screening was performed using the above screening system and a collection of known compounds (2520 compounds) possessed by the Kyoto University Medical Research Support Center. As a result, the following compound 1-11 was selected. With respect to these compounds, the concentration at which the relative average luminance fluorescence of polyglutamine aggregates in one visual field in the above screening system was 50% of DMSO control was measured as IC50 (n = 2). The results are shown in Table 1 below.
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-T000037
Figure JPOXMLDOC01-appb-T000037
　上記化合物のうち、化合物１－５は、VEGFRインヒビターとして既知の化合物であった。また、化合物２－４は、VEGFR2インヒビター及びc-METインヒビターとして既知の化合物であった。化合物８は、MAPK/ERK kinase（MEK）インヒビターとして既知の化合物であった。 Of the above compounds, compound 1-5 was a compound known as a VEGFR inhibitor. Compound 2-4 was a compound known as a VEGFR2 inhibitor and a c-MET inhibitor. Compound 8 was a known compound as a MAPK / ERK-kinase (MEK) inhibitor.
　実験例２：化合物４のポリグルタミン凝集体形成抑制能
　播種後1日培養したポリQ細胞の培養液（高グルコースDMEM培地、10％FBS、100u/mL ペニシリン、100μg/mlストレプトマイシン）に、4 mM dbcAMP、10μg/ml Doxを添加して24hレポーターを発現誘導した。その後10μg/ml Doxを除いた培養液（高グルコースDMEM培地、10％FBS、100u/mL ペニシリン、100μg/mlストレプトマイシン）に置換し、ついで化合物４（10nM－10μM）を添加して2日間培養後、4% PFAで固定し、DAPIで核染色をした後、仮想細胞体領域内の凝集体の発光輝度を細胞イメージアナライザーで測定した。その結果を図４に示す。 Experimental Example 2: Inhibition of polyglutamine aggregate formation by Compound 4 4 mM in a culture solution of polyQ cells cultured for 1 day after seeding (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) dbcAMP and 10 μg / ml Dox were added to induce expression of the 24h reporter. Thereafter, the medium was replaced with a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) excluding 10 μg / ml Dox, and then compound 4 (10 nM-10 μM) was added and cultured for 2 days. After fixing with 4% PFA and nuclear staining with DAPI, the emission luminance of the aggregates in the virtual cell body region was measured with a cell image analyzer. The result is shown in FIG.
　図４に示すとおり、化合物４は、レポーター発現後に誘導濃度依存的にポリグルタミン凝集体の形成を阻害した。本実験では化合物４の添加はレポーターの一過的発現後に添加しても凝集体抑制効果が見られており、本薬剤の作用点はレポーター構築時に利用しているtet-onプロモーターの抑制による偽陽性でないことも示された。 As shown in FIG. 4, Compound 4 inhibited the formation of polyglutamine aggregates in an induced concentration-dependent manner after reporter expression. In this experiment, the addition of compound 4 shows an inhibitory effect on aggregates even when added after the transient expression of the reporter, and the action point of this drug is the false action due to the suppression of the tet-on promoter used during reporter construction. It was also shown not to be positive.
　実験例３：化合物４とレポーター蛋白質量との関連性
　播種後１日培養したポリQ細胞の培養液（高グルコースDMEM培地、10％FBS、100u/mL ペニシリン、100μg/mlストレプトマイシン）に、4 mM dbcAMP、10μg/ml Doxを添加して24hレポーターを発現誘導した。その後10μg/ml Doxを除いた培養液（高グルコースDMEM培地、10％FBS、100u/mL ペニシリン、100μg/mlストレプトマイシン）に置換し、ついで化合物４（10nM－10μM）を添加して2日間培養した。その後、細胞層タンパク及びRNAを回収し、ウェスタンブロッティングでレポーター蛋白質(Venus)量の測定、及びリバーストランスクリプション ポリメラーゼチェインリアクション(RT-PCR)法によりレポーター遺伝子発現量を測定した。その結果を図５に示す。 Experimental Example 3: Relationship between Compound 4 and Reporter Protein Mass 4 mM in a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) cultured for 1 day after seeding. dbcAMP and 10 μg / ml Dox were added to induce expression of the 24h reporter. Thereafter, the medium was replaced with a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) excluding 10 μg / ml Dox, and then compound 4 (10 nM-10 μM) was added and cultured for 2 days. . Thereafter, cell layer proteins and RNA were collected, and the amount of reporter protein (Venus) was measured by Western blotting, and the expression level of the reporter gene was measured by the reverse transcription polymerase chain reaction (RT-PCR) method. The result is shown in FIG.
　図５に示すとおり、化合物４は濃度依存的にレポーター蛋白質の発現量、及びRNA量を減少させていた。図４の結果と合わせ、本化合物によりレポータータンパク質ないしRNAの分解促進が起こっていることが示唆される。 As shown in FIG. 5, Compound 4 decreased the expression level of the reporter protein and the RNA level in a concentration-dependent manner. Together with the results in FIG. 4, it is suggested that the present compound promotes the degradation of reporter protein or RNA.

Claims (10)

  1. 　血管内上皮細胞増殖因子受容体（VEGFR）阻害化合物、c-met遺伝子にコードされた受容体型チロシンキナーゼ(c-Met)阻害化合物、並びにVEGFR及びc-Metの両方の阻害化合物からなる群から選択される少なくとも１種類を有効成分として含有する、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物。 Selected from the group consisting of an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a receptor tyrosine kinase (c-Met) inhibitor compound encoded by the c-met gene, and both VEGFR and c-Met inhibitor compounds A pharmaceutical composition for preventing, ameliorating, inhibiting progression and / or treating polyglutamine disease, comprising at least one of the above as an active ingredient.
  2. 　前記阻害化合物が、下記式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000001
     で表わされる化合物又はその製薬上許容される塩である、請求項１記載の医薬組成物
    　［式（Ｉ）において、
    　Ｒ1は、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
    　Ｗは、
    Figure JPOXMLDOC01-appb-C000002
     であり、
    　Ｒ2及びＲ3は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基であり、
    　Ｘ1は、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であり、
    　Ｘ2及びＹは、それぞれ独立して、Ｃ1-4アルキレン基、ハロゲン原子で置換されたＣ1-4アルキレン基、又はイミノ基であるか、あるいは、
    　Ｘ2及びＹは、ａで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有し、
    　Ｚは、
    Figure JPOXMLDOC01-appb-C000003
     であり、
    　Ｒ4及びＲ5は、それぞれ独立して、水素原子、ハロゲン原子、Ｃ1-4アルキル基、又はハロゲン原子で置換されたＣ1-4アルキル基、アミノ基、アジド基、シアノ基、ニトロ基、水酸基であるか、あるいは、
    　Ｒ4及びＲ5は、ｂ及びｃで印をつけた原子と共に、芳香族環又は複素芳香族環を形成し、前記環は、無置換であるか、あるいは、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基、Ｃ1-4アルキルオキシ基、又はハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキルオキシ基である置換基を１つ以上有する。］。     The inhibitor compound is represented by the following formula (I):
    Figure JPOXMLDOC01-appb-C000001
     Or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 1, wherein the compound is represented by the formula (I):
    R 1 is a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
    W is
    Figure JPOXMLDOC01-appb-C000002
     And
    R 2 and R 3 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom;
    X 1 is a C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom or an imino group,
    X 2 and Y are each independently, C 1-4 alkylene group, C 1-4 alkylene group substituted with a halogen atom, or an imino group, or,
    X 2 and Y together with the atom marked with a form an aromatic ring or a heteroaromatic ring, said ring being unsubstituted or hydrogen atom, halogen atom, C 1-4 alkyl A C 1-4 alkyl group, a C 1-4 alkyloxy group substituted by a group, a halogen atom or an alicyclic ring or a heterocyclic ring, or a C 1-4 alkyloxy group substituted by a halogen atom, an alicyclic ring or a heterocyclic ring Have one or more substituents,
    Z is
    Figure JPOXMLDOC01-appb-C000003
     And
    R 4 and R 5 are each independently a hydrogen atom, a halogen atom, a C 1-4 alkyl group, or a C 1-4 alkyl group substituted with a halogen atom, an amino group, an azide group, a cyano group, or a nitro group. A hydroxyl group, or
    R 4 and R 5 together with the atoms marked with b and c form an aromatic ring or a heteroaromatic ring, which ring is unsubstituted or is a hydrogen atom, halogen atom, C 1 -4 alkyl group, a halogen atom or an aliphatic ring or a C 1-4 alkyl group substituted with a heterocycle, C 1-4 alkyl group, or a halogen atom or an aliphatic ring or a C 1-4 alkyl substituted with a heterocycle It has one or more substituents that are oxy groups. ].
  3. 　前記式（Ｉ）で表わされる化合物が、下記式（ＩＩ）：
    Figure JPOXMLDOC01-appb-C000004
     で表わされる化合物である、請求項２記載の医薬組成物
    　［式（ＩＩ）において、
    　Ｗは、
    Figure JPOXMLDOC01-appb-C000005
     であり、Ｒ1－Ｒ5、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様である。］。     The compound represented by the formula (I) is represented by the following formula (II):
    Figure JPOXMLDOC01-appb-C000004
     A pharmaceutical composition according to claim 2, which is a compound represented by the formula (II):
    W is
    Figure JPOXMLDOC01-appb-C000005
     R 1 -R 5 , X 1 , X 2 and Y are the same as those in formula (I). ].
  4. 　前記式（Ｉ）で表わされる化合物が、下記式（ＩＩＩ）：
    Figure JPOXMLDOC01-appb-C000006
     で表わされる化合物である、請求項２記載の医薬組成物
    　［式（ＩＩＩ）において、
    　Ｗは、
    Figure JPOXMLDOC01-appb-C000007
     であり、Ｒ1－Ｒ3、Ｘ1、Ｘ2及びＹは式（Ｉ）と同様であり、
    　Ｒ6及びＲ7は、水素原子、ハロゲン原子、Ｃ1-4アルキル基、ハロゲン原子若しくは脂肪環若しくは複素環で置換されたＣ1-4アルキル基である。］。     The compound represented by the formula (I) is represented by the following formula (III):
    Figure JPOXMLDOC01-appb-C000006
     The pharmaceutical composition according to claim 2, which is a compound represented by the formula (III):
    W is
    Figure JPOXMLDOC01-appb-C000007
     R 1 -R 3 , X 1 , X 2 and Y are the same as in formula (I),
    R 6 and R 7 is a hydrogen atom, a halogen atom, C 1-4 alkyl groups, C 1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring. ].
  5. 　前記式（Ｉ）で表わされる化合物が、下記式（ＩＶ）：
    Figure JPOXMLDOC01-appb-C000008
     で表わされる化合物である、請求項２記載の医薬組成物
    　［式（ＩＶ）において、Ｒ1－Ｒ5、Ｘ2及びＹは式（Ｉ）と同様である］。     The compound represented by the formula (I) is represented by the following formula (IV):
    Figure JPOXMLDOC01-appb-C000008
     The pharmaceutical composition according to claim 2, wherein R 1 -R 5 , X 2 and Y are the same as in formula (I).
  6. 　下記化合物１－１１、すなわち、
    Figure JPOXMLDOC01-appb-C000009
     からなる群から選択される少なくとも１種類を有効成分として含有する、ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物。     The following compound 1-11, that is,
    Figure JPOXMLDOC01-appb-C000009
     A pharmaceutical composition for preventing, ameliorating, suppressing progression and / or treating polyglutamine disease, comprising as an active ingredient at least one selected from the group consisting of:
  7. 　前記ポリグルタミン病が、ハンチントン病、遺伝性脊髄小脳変性症、又は、球脊髄性筋萎縮症である、請求項１から６のいずれかに記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 6, wherein the polyglutamine disease is Huntington's disease, hereditary spinocerebellar degeneration, or bulbar spinal muscular atrophy.
  8. 　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療の方法であって、請求項１から６のいずれかに記載の医薬組成物を対象に投与することを含む、方法。 A method for prevention, improvement, progression inhibition, and / or treatment of polyglutamine disease, comprising administering the pharmaceutical composition according to any one of claims 1 to 6 to a subject.
  9. 　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物を製造するための血管内上皮細胞増殖因子受容体（VEGFR）阻害化合物、c-met遺伝子にコードされた受容体型チロシンキナーゼ(c-Met)阻害化合物、並びにVEGFR及びc-Metの両方の阻害化合物からなる群から選択される少なくとも１種類の使用。 Intravascular epidermal growth factor receptor (VEGFR) inhibitory compound for producing a pharmaceutical composition for preventing, improving, inhibiting progression and / or treating polyglutamine disease, receptor encoded by c-met gene At least one use selected from the group consisting of a body tyrosine kinase (c-Met) inhibitory compound, and both VEGFR and c-Met inhibitory compounds.
  10. 　ポリグルタミン病の予防、改善、進行抑制、及び／又は、治療のための医薬組成物の有効成分の候補物質のスクリーニング方法であって、
    　レポーター蛋白質と融合したポリグルタミンを発現するアッセイ細胞をテスト物質と接触させて培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ａ）を測定すること、
    　前記アッセイ細胞を該テスト物質と接触させることなく培養し、該アッセイ細胞の仮想細胞体領域にあるレポーターシグナル（Ｂ）を測定すること、
    　シグナル（Ａ）とシグナル（Ｂ）とを比較すること、及び、
    　前記比較に基づいてシグナル（Ｂ）よりもシグナル（Ａ）を低減させるテスト物質を候補物質として選択することを含む、スクリーニング方法。     A method for screening a candidate substance for an active ingredient of a pharmaceutical composition for prevention, improvement, progression inhibition and / or treatment of polyglutamine disease, comprising:
    Culturing an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, and measuring a reporter signal (A) in a virtual cell body region of the assay cell;
    Culturing the assay cell without contacting with the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell;
    Comparing signal (A) and signal (B); and
    A screening method comprising selecting, as a candidate substance, a test substance that reduces the signal (A) over the signal (B) based on the comparison.
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