JPWO2018203559A1 - Pharmaceutical composition for polyglutamine disease - Google Patents

Abstract

Provided are a pharmaceutical composition for polyglutamine disease, use thereof, a method for preventing, ameliorating, suppressing the progression of and/or treating polyglutamine disease using the same, and a method for screening a compound for polyglutamine disease. In one aspect, from 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 It relates to a pharmaceutical composition for preventing, ameliorating, suppressing the progression and/or treating polyglutamine disease, which comprises at least one selected from the group consisting of as an active ingredient. In another embodiment, as an index of the polyglutamine aggregate formed in the culture medium in which the cell into which the expression system expressing the polyglutamine fused with the reporter protein has been introduced, is introduced, the polyglutamine aggregate in the virtual cell body region including the cell nucleus is The present invention relates to a method for screening a substance that affects the formation of polyglutamine aggregates, which comprises measuring the signal of a reporter protein.

Description

  The present disclosure relates to a pharmaceutical composition for polyglutamine disease, use thereof, and a method for preventing, ameliorating, suppressing the progression of and / or treating polyglutamine disease using the same. The present disclosure also relates to methods for screening compounds for polyglutamine disease.

  In inherited neurological diseases such as Huntington's disease and polyglutamine diseases, CAG repeats in the causative gene are overstretched, and a protein with polyglutamine overstretching is produced using the CAG repeat as a template. It accumulates within and forms aggregates. This polyglutamine aggregate triggers 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 drug currently on the market is a symptomatic drug, and a drug that can still be fundamentally treated. It cannot be said that there is.

  Non-Patent Document 1 discloses a reporter protein for polyglutamine aggregate formation, which is obtained by fusing 150 repeats of exon 1 of Huntington gene and a fluorescent protein.

Matsumoto, G et al .: Serine 403 Phosphorylation of p62 / SQSTM1 Regulates Selective Autophagic Clearance of Ubiquitinated Proteins. Molecular Cell 44, 279289, October 21, 2011

  The present disclosure provides, in one aspect, a pharmaceutical composition for polyglutamine disease, a use thereof, and a method for preventing, ameliorating, suppressing the progression of and / or treating polyglutamine disease using the same. The present disclosure provides, in another aspect, a method for screening a compound for polyglutamine disease.

  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 the progression and / or treating polyglutamine disease, which comprises, as an active ingredient, at least one selected from the group consisting of both inhibitory compounds.

The present disclosure provides, in another aspect, a method for screening a candidate substance for an active ingredient of a pharmaceutical composition for the prevention, amelioration, inhibition of progression, and / or treatment of polyglutamine disease,
Assaying an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance and culturing, and measuring a reporter signal (A) in a virtual cell body region of the assay cell,
Culturing the assay cell without contacting the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell,
Comparing signal (A) with signal (B), and
The present invention relates to a screening method, which comprises selecting, as a candidate substance, a test substance that reduces the signal (A) more than the 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 state of the assay system used for screening and Z'factor. FIG. 4 is a graph showing the concentration dependence of the inhibition of polyglutamine aggregate formation by Compound 4. FIG. 5 is a graph showing the concentration dependence of the decrease in the reporter protein expression level by Compound 4.

  The present disclosure adopts a method of measuring a signal in a virtual cell region as a method for measuring an aggregate of polyglutamine fused with a reporter protein, which improves assay sensitivity and simplifies substances involved in the formation of polyglutamine aggregate. Based on the new knowledge that it can be screened.

[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 “pharmaceutical composition according to the present disclosure”).
In the present disclosure, the pharmaceutical composition for polyglutamine disease refers to, in one or more embodiments, a pharmaceutical composition for prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease.
In the present disclosure, polyglutamine disease, in one or more embodiments, overgrowth of CAG repeats in the causative gene, production of a protein having polyglutamine overgrowth using it as a template, or nerve cells of this protein It refers to a disease caused by at least one of accumulation and aggregation in the body. Polyglutamine disease, in one or more embodiments, includes Huntington's disease, hereditary spinocerebellar degeneration, or bulbospinal muscular atrophy.

[Active ingredient]
As an active ingredient of the 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. And a substance having an inhibitory activity against VEGFR2, a substance having an inhibitory activity against both VEGFR and c-Met, or a substance having an inhibitory activity against both VEGFR2 and c-Met. By the screening method according to the present disclosure described below, a plurality of known VEGER inhibitors and substances having an inhibitory activity against both VEGFR and c-Met were selected as substances that suppress the formation of polyglutamine aggregates.

As one or more embodiments of the VEGFR inhibitor which is an active ingredient, a VEGFR inhibitor having a structure represented by the following formula (I) can be mentioned. Here, W, Z, and R ¹ in the following formula (I) are arbitrary groups.

As another 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
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 a hydrogen atom, halogen atom, C _1-4 alkyl group, C _1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring, with C _1-4 alkyl group, or a halogen atom or an aliphatic ring or a C _1-4 alkyl group substituted by a heterocyclic Have one or more substituents,
Z is
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 azido group, a cyano group, a nitro group. , Is a hydroxyl group, or
R ⁴ and R ⁵ together with the atoms marked b and c form an aromatic or heteroaromatic ring, said ring being unsubstituted or a hydrogen atom, a halogen atom, C _{1 -4} alkyl group, C _1-4 alkyl group substituted with a halogen atom or an alicyclic ring or heterocyclic ring, C _1-4 alkyloxy group, or C _1-4 alkyl substituted with a halogen atom or an alicyclic ring or heterocyclic ring Examples thereof include a compound or a pharmaceutically acceptable salt thereof, which has one or more substituents which are oxy groups.
In the present disclosure, a wavy-lined bond represents a bond with a compound or group represented by formula (I), (II) or (III).

The VEGFR inhibitor in the present disclosure, in one or a plurality of embodiments, in the known VEGFR inhibition assay system in at least one of in vitro and in vivo, the activity of VEGFR when the substance is added, the control without adding the substance In comparison with, for example, a compound capable of inhibiting up 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 the present disclosure, the c-Met inhibitor, in one or more embodiments, in a known c-Met inhibition assay system in at least one of in vitro and in vivo, the activity of c-Met when the substance is added, It refers to a compound that can inhibit up to, for example, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less compared to a control to which the above substance is not added. 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, in one or more embodiments, in the known VEGFR inhibition assay system in at least one of in vitro and in vivo, the activity of VEGFR when the substance is added. , Compared to a control without the addition of the substance, for example, can be inhibited to 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less, and in 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. , A compound that can inhibit up to 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.

Examples of the alkyl group or alkylene group include a linear, branched, or cyclic alkyl group or alkylene group in one or more embodiments. 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. As the linear or branched alkyl group having 1 to 4 carbon atoms, in one or more embodiments, a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group. , 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.

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom in one or a plurality of embodiments.
The “heterocycle”, in one or more embodiments, contains 1-2 heteroatoms in the atoms constituting the ring, may contain a double bond in the ring, and is non-aromatic. Or a ring of aromaticity. “Heteroaromatic ring” means an aromatic heterocycle. “Heteroatom” means, in one or more embodiments, a sulfur atom, oxygen atom or nitrogen atom.

  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. Alternatively, basic amino acid salts and the like can be mentioned.

  Preferred examples of the inorganic acid salt include, for example, hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like, and preferred examples of the organic acid salt include, for example, acetate, succinate, Examples include fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate and the like.

  Preferable 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, ammonium salt and the like. Preferred 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, glutamate and the like. Preferred examples of the basic amino acid salt include arginine salt, lysine salt, ornithine salt and the like.

  In the present disclosure, the “compound salt” may include a hydrate that can be formed by absorbing water when the compound is left in the air. Further, in the present disclosure, “a salt of a compound” may also include a solvate which may be formed by absorbing a certain other solvent by the compound.

R ^{1 in the} above formula (I) includes, in one or a plurality of embodiments, a halogen atom or a C _1-4 alkyl group substituted with a halogen atom, and is not limited, and in one or a plurality of embodiments, a fluorine atom. , Or a trifluoromethyl group.

W in the above formula (I) is, in one or more embodiments,
Is mentioned.
In one or a plurality of embodiments, R ² and R ³ each independently include 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 independently an imino group in one or a plurality of embodiments.
Alternatively, X ² and Y are, along with the atom marked a,
Is mentioned.

W of the above formula (I) has an inhibitory activity against c-Met in addition to an inhibitory activity against VEGFR, and in one or a plurality of embodiments,
Is mentioned.
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 are, in one or more embodiments, each independently, a halogen atom or an amino group, or R ⁴ and R ⁵ together with the atoms marked b and c,
Is mentioned. R ⁶ and R ⁷ are, in one or more embodiments, each independently a methyl group, or
Is mentioned.

The above formula (I) is, in one or more embodiments, the following formula (II).
[In the formula (II),
W is
And R ¹ -R ⁵ , X ¹ , X ² and Y are the same as in formula (I). ]

The above formula (I) is, in one or more embodiments, the following formula (III).
[In Formula (III),
W is
And R ¹ -R ³ , R ⁶ and R ⁷ , X ¹ , X ² and Y are the same as in formula (I). ]

The above formula (I) is, in one or more embodiments, the following formula (IV).
[In the formula (IV), R ¹ -R ⁵ , X ² and Y are the same as those in the formula (I)]

The following compound 1-5 is mentioned as one or some embodiment of the VEGFR inhibitor which is an active ingredient. One or more embodiments of the substance having both VEGFR and c-Met inhibitory activity include the following compound 2-4.

In one or more other embodiments, the active ingredients of the pharmaceutical composition according to the present disclosure also include the following compounds 6-11.

  In the case where an asymmetric carbon atom is present and / or a stereoisomer is present, the above-mentioned compound which is an active ingredient of the pharmaceutical composition according to the present disclosure is, in one or more embodiments, a mixture or a single isomer of each isomer. It has been separated. In one or more non-limiting embodiments of stereoisomers, cis-trans isomers may be mentioned.

  The above compound, which 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. The “prodrug” includes, in one or more embodiments, those that are easily hydrolyzed in vivo to regenerate the above compound. For example, in the case of a compound having a carboxyl group, the carboxyl group is an alkoxycarbonyl group. And a compound having an alkylthiocarbonyl group or a compound having 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 substituted with an alkoxycarbonyl group to become an alkoxycarbonylamino group, and an acyloxymethylamino group And a compound converted to hydroxylamine. Further, for example, in the case of a compound having a hydroxyl group, a compound in which the hydroxyl group is substituted with the above acyl group to be an acyloxy group, a compound to be a phosphoric acid ester, or a compound to be an acyloxymethyloxy group can be mentioned. Examples of the alkyl moiety of the group used for prodrug formation include the alkyl groups described below, 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, a lower (for example, 1 to 6 carbon atoms) alkoxycarbonyl such as methoxycarbonyl or ethoxycarbonyl, methoxymethoxycarbonyl, ethoxymethoxy. Examples thereof include lower (for example, 1 to 6 carbon atoms) alkoxycarbonyl substituted with an alkoxy group such as carbonyl, 2-methoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl and pivaloyloxymethoxycarbonyl.

  In one or a plurality of embodiments, the pharmaceutical composition of the present disclosure contains the above inhibitor as an active ingredient, and further comprises a pharmaceutically acceptable carrier, preservative, diluent, excipient or other pharmaceutical agent. It may contain a component acceptable to the.

  In the present disclosure, the “pharmaceutical composition” can be made into a dosage form suitable for an administration form by applying well-known formulation techniques in one or a plurality of embodiments. The dosage form is not limited to these, but includes oral administration in the form of tablets, capsules, granules, powders, pills, troches, syrups, liquids, and the like. Alternatively, parenteral administration in the form of injections, solutions, aerosols, suppositories, patches, poultices, lotions, liniments, ointments, eye drops and the like can be mentioned. These preparations can be manufactured by a known method using additives such as, but not limited to, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.

  Examples of the excipient include, but are not limited to, starch such as starch, potato starch, 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, hydroxypropylmethyl cellulose, shellac, talc, carnauba wax, and paraffin. The binder may include, but is not limited to, polyvinylpyrrolidone, macrogol and compounds similar to the excipients above. The disintegrants include, but are not limited to, compounds similar to the excipients and chemically modified starch / celluloses such as croscarmellose sodium, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone. .. Examples of the stabilizer include, but are not limited to, paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; phenol and cresol. Such as phenols; thimerosal; dehydroacetic acid; and sorbic acid. The flavoring agents include, but are not limited to, commonly used sweeteners, acidulants, flavors and the like.

  Further, in the production of the liquid agent, as a solvent, but not limited to, ethanol, phenol, chlorocresol, purified water, distilled water or the like can be used, and if necessary, a surfactant or an emulsifier can also be used. .. The surfactants or emulsifiers 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 oral or transdermal, submucosal, subcutaneous, intermittently or continuously, so that the concentration of the compound as an active ingredient in the body is between 100 nM and 1 mM. It can be administered intramuscularly, intravascularly, intracerebrally, or intraperitoneally. As a non-limiting embodiment, in the case of oral administration, the lower limit of 0.01 mg (preferably, the compound represented by the general formula (I) per day for a subject (an adult for a human) is calculated. 0.1 mg), and as an upper limit, 2000 mg (preferably 500 mg, more preferably 100 mg) may be administered once or in several divided doses depending on the 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 (an adult for a human). May be administered once or in several divided doses depending on the symptoms.

[Prevention, amelioration, suppression of progression and / or treatment of polyglutamine disease]
The present disclosure, in one or more embodiments, is a method for preventing, ameliorating, inhibiting the progression, and / or treating polyglutamine disease, which comprises administering a pharmaceutical composition according to the present disclosure to a subject. Regarding The administration of the pharmaceutical composition according to the present disclosure can be according to the method of using the pharmaceutical composition described above in one or more embodiments. Targets include humans and non-human animals.

The present disclosure relates, in one or more embodiments, to the use of the above compound or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention, amelioration, inhibition of progression, and / or treatment of polyglutamine disease.
In one or more other embodiments, the present disclosure provides the above compound or a pharmaceutical compound thereof, which is an active ingredient for producing a pharmaceutical composition for preventing, ameliorating, suppressing the progression of, and / or treating polyglutamine disease. Regarding the use of acceptable salts above.

[Screening method]
In one aspect, the present disclosure relates to a method for screening a substance that affects the formation of polyglutamine aggregates. The screening method of this embodiment is
Assaying an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance and culturing, and measuring a reporter signal (A) in a virtual cell body region of the assay cell,
Culturing the assay cell without contacting the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell,
Comparing signal (A) with signal (B), and
Selecting a test substance that reduces signal (A) over signal (B) based on the comparison as a candidate substance.

The assay cell in the screening method according to the present disclosure has a gene capable of expressing polyglutamine fused with a reporter protein. Assay cells, in one or more embodiments, can be made by introducing into a cell a gene expression vector constructed and adapted to express polyglutamine mRNA fused to a reporter protein. The cells used for producing the assay cells, that is, the cells into which the vector is introduced are not particularly limited, and in one or a plurality of embodiments, mammalian cells. The mammalian cell is, in one or more embodiments, a human, bovine, cat, monkey, dog, hamster, mink, mouse, pig, rabbit, rat cell. The type of cell is not particularly limited, but 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 previously known or later developed promoter capable of inducing the expression of the protein in the assay cell can be used. Examples of the reporter protein include a photoprotein in one or a plurality of embodiments, and examples thereof include a fluorescent protein.
As a gene capable of expressing polyglutamine fused with a reporter protein, those described in Examples can be used.

In the present disclosure, the virtual cell body region refers to a predetermined region including around 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 the present aspect, to calculate the average brightness in the polyglutamine protein existing region from the total brightness 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 by a constant number of pixels. Generally, the cell nucleus in an image of a mammalian cell is defined as a region stained with the DNA stain DAPI. There is only one cell nucleus in the cell, and the cell body is regarded as the space surrounding it. For example, each cell nucleus region is defined in the captured image of the DAPI-stained region, and regions up to a region having a certain number of pixels are defined as the cytoplasm attached to the cell nucleus. A reporter fluorescence region having a certain brightness or higher existing in the identified cytoplasmic region is defined as a polyglutamine protein existing region belonging to the nucleus / cell. The total brightness in the existing area and the area of the identified existing area are calculated, and the average brightness in the polyglutamine protein existing area is calculated. Since the aggregate is defined on the image as a region having a high fluorescence value derived from the reporter, 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 the aggregates were significantly formed in the virtual cell body region, the difference in the average brightness in the polyglutamine protein presence region with respect to the presence or absence of the aggregates was larger than the total difference, and the measurement was derived from the size of the aggregates. Variations in the results are canceled out, thus increasing the measurement accuracy.

That is, the present disclosure may relate to one or more of the following embodiments;
[1] At least one selected from the group consisting of an intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a c-Met inhibitor compound, and an inhibitor compound of both VEGFR and c-Met as an active ingredient A pharmaceutical composition for the prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease.
[2] The pharmaceutical composition according to [1], wherein the inhibitor compound is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
[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
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 a hydrogen atom, halogen atom, C _1-4 alkyl group, C _1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring, with C _1-4 alkyl group, or a halogen atom or an aliphatic ring or a C _1-4 alkyl group substituted by a heterocyclic Have one or more substituents,
Z is
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 azido group, a cyano group, a nitro group. , Is a hydroxyl group, or
R ⁴ and R ⁵ together with the atoms marked b and c form an aromatic or heteroaromatic ring, said ring being unsubstituted or a hydrogen atom, a halogen atom, C _{1 -4} alkyl group, C _1-4 alkyl group substituted with a halogen atom or an alicyclic ring or heterocyclic ring, C _1-4 alkyloxy group, or C _1-4 alkyl substituted with a halogen atom or an alicyclic ring or heterocyclic ring It has one or more substituents which 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).
[In the formula (II),
W is
And R ¹ -R ⁵ , X ¹ , X ² and Y are the same as 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).
[In Formula (III),
W is
And R ¹ -R ³ , X ¹ , X ² and Y are the same as those in formula (I),
R ⁶ and R ⁷ are a hydrogen atom, a halogen atom, a C _1-4 alkyl group, a halogen atom or a C _1-4 alkyl group substituted with an alicyclic ring or a 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).
[In the formula (IV), R ¹ -R ⁵ , X ² and Y are the same as those in the formula (I)]
[6] A pharmaceutical composition for the prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease, which comprises at least one selected from the group consisting of the following compounds 1-11 as an active ingredient.
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the polyglutamine disease is Huntington's disease, hereditary spinocerebellar degeneration, or bulbospinal muscular atrophy.
[8] A method for preventing, ameliorating, suppressing the progression, and / or treating polyglutamine disease, which comprises administering the pharmaceutical composition according to any one of [1] to [6] to a subject. Method. [9] An intravascular epidermal growth factor receptor (VEGFR) inhibitor compound, a c-Met inhibitor compound, for producing a pharmaceutical composition for preventing, ameliorating, suppressing the progression and / or treating polyglutamine disease, And at least one use selected from the group consisting of compounds that inhibit both VEGFR and c-Met.
[10] A method for screening a candidate substance for an active ingredient of a pharmaceutical composition for the prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease, the method comprising:
Assaying an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance, culturing, 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) with 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.

  Hereinafter, the present disclosure will be described in more detail with reference to Examples, but these are exemplary and the present disclosure is not limited to these Examples. It should be noted that all references cited in this disclosure are incorporated as part of this disclosure.

Experimental Example 1: Screening for substances that affect the formation of polyglutamine aggregates As a system for forming polyglutamine aggregates in cells, a host neuron (Neuro2a cell) was fused with a reporter as shown in FIG. 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 poly Q cells according to the scheme shown in FIG. First, poly-Q cells were seeded and cultured for 1 day (high glucose DMEM medium, 10% FBS, 100u / mL penicillin, 100 μg / ml streptomycin), and 4 mM dibutylil cyclic AMP (dbcAMP) was added to induce differentiation. , 10 μg / ml Doxicyclin (DOX) is added to induce expression of polyglutamine fused with a reporter, and a test compound is further added (10 μM, control 0.1% DMSO). After that, cells cultured for 2 days were fixed by treatment with 4% paraformaldehyde (PFA) at 4 ° C. for 30 minutes, nuclear staining was performed with DAPI, and then the cells were measured with a cell image analyzer ArrayScan VTi. In the measurement, the peripheral region of the nucleus and the virtual cell body region were set, and the brightness of the polyglutamine aggregate (Venus) in the region was measured. By setting the virtual cell body area, measuring the brightness of the aggregates in the area, and averaging, the average brightness of the aggregates according to the presence or absence of expression induction (dbcAMP + DOX) is measured as shown in FIG. The difference became remarkable, and the Z'factor, which is an index showing the optimality of the assay system, was a good 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.
Note that the virtual cell body region was obtained by acquiring an image with a 20x objective lens equipped with a cell image analyzer ArrayScan VTi, and defining each cell nucleus region in the captured image of the DAPI stained region, and defining it as a region having a width of 15 pixels. ..

Screening was performed using the screening system described above and a collection of compounds with known functions (2520 compounds) owned by the Kyoto University Medical Research Support Center. As a result, the following compound 1-11 was selected. For these compounds, the concentration at which the relative average luminance fluorescence of the polyglutamine aggregate in one visual field in the above screening system was 50% of the DMSO control was measured as IC50 (n = 2). The results are shown in Table 1 below.

  Of the above compounds, compounds 1-5 were known compounds as VEGFR inhibitors. Compound 2-4 was a compound known as a VEGFR2 inhibitor and a c-MET inhibitor. Compound 8 was a compound known as a MAPK / ERK kinase (MEK) inhibitor.

Experimental Example 2: Polyglutamine aggregate formation-inhibiting ability of compound 4 4 mM was added to a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) of poly Q cells cultured for 1 day after seeding. Expression of the 24h reporter was induced by adding dbcAMP and 10 µg / ml Dox. Then, the medium was replaced with 10 μg / ml Dox-free medium (high glucose DMEM medium, 10% FBS, 100u / mL penicillin, 100 μg / ml streptomycin), and then compound 4 (10 nM-10 μM) was added, followed by incubation for 2 days. After fixing with 4% PFA and nuclear staining with DAPI, the emission brightness of aggregates in the virtual cell body region was measured with a cell image analyzer. The result is shown in FIG.

  As shown in FIG. 4, Compound 4 inhibited the formation of polyglutamine aggregates after reporter expression in an induced concentration-dependent manner. In this experiment, addition of compound 4 showed an inhibitory effect on aggregates even after the transient expression of the reporter, and the action point of this drug is pseudo by the suppression of the tet-on promoter used in the construction of the reporter. It was also shown not to be positive.

Experimental Example 3: Relationship between Compound 4 and Reporter Protein Mass 4 mM was added to a culture solution (high glucose DMEM medium, 10% FBS, 100 u / mL penicillin, 100 μg / ml streptomycin) of poly-Q cells cultured for 1 day after seeding. Expression of the 24h reporter was induced by adding dbcAMP and 10 µg / ml Dox. After that, the medium was replaced with a culture medium (high glucose DMEM medium, 10% FBS, 100u / mL penicillin, 100 µg / ml streptomycin) from which 10 µg / ml Dox was removed, 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 amount of reporter gene expression was measured by the reverse transcription polymerase chain reaction (RT-PCR) method. The result is shown in FIG.

  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 of FIG. 4, it is suggested that this compound promotes the degradation of the reporter protein or RNA.

Claims (10)

  Select from the group consisting of intravascular epidermal growth factor receptor (VEGFR) inhibitor compounds, receptor tyrosine kinase (c-Met) inhibitor compounds encoded by the c-met gene, and both VEGFR and c-Met inhibitor compounds A pharmaceutical composition for preventing, ameliorating, suppressing the progression, and / or treating polyglutamine disease, which comprises at least one of the above-mentioned compounds as an active ingredient. The inhibitor compound has the following formula (I):
The pharmaceutical composition according to claim 1, which is a compound represented by: or a pharmaceutically acceptable salt thereof [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
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 a hydrogen atom, halogen atom, C _1-4 alkyl group, C _1-4 alkyl group substituted by a halogen atom or an aliphatic ring or heterocyclic ring, with C _1-4 alkyl group, or a halogen atom or an aliphatic ring or a C _1-4 alkyl group substituted by a heterocyclic Have one or more substituents,
Z is
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 azido group, a cyano group, a nitro group. , Is a hydroxyl group, or
R ⁴ and R ⁵ together with the atoms marked b and c form an aromatic or heteroaromatic ring, said ring being unsubstituted or a hydrogen atom, a halogen atom, C _{1 -4} alkyl group, C _1-4 alkyl group substituted with a halogen atom or an alicyclic ring or heterocyclic ring, C _1-4 alkyloxy group, or C _1-4 alkyl substituted with a halogen atom or an alicyclic ring or heterocyclic ring It has one or more substituents which are oxy groups. ]. The compound represented by the formula (I) has the following formula (II):
The pharmaceutical composition according to claim 2, which is a compound represented by the formula [in the formula (II):
W is
And R ¹ -R ⁵ , X ¹ , X ² and Y are the same as in formula (I). ]. The compound represented by the formula (I) has the following formula (III):
The pharmaceutical composition according to claim 2, which is a compound represented by the formula [in the formula (III),
W is
And R ¹ -R ³ , X ¹ , X ² and Y are the same as those in formula (I),
R ⁶ and R ⁷ are a hydrogen atom, a halogen atom, a C _1-4 alkyl group, a halogen atom or a C _1-4 alkyl group substituted with an alicyclic ring or a heterocyclic ring. ]. The compound represented by the formula (I) has the following formula (IV):
The pharmaceutical composition according to claim 2, which is a compound represented by the formula [In formula (IV), R ¹ -R ⁵ , X ² and Y are the same as in formula (I)]. Compound 1-11 below, that is,
A pharmaceutical composition for the prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease, which comprises at least one selected from the group consisting of as an active ingredient.   The pharmaceutical composition according to any one of claims 1 to 6, wherein the polyglutamine disease is Huntington's disease, hereditary spinocerebellar degeneration, or bulbospinal muscular atrophy.   A method for preventing, ameliorating, suppressing the progression of, and / or treating polyglutamine disease, which comprises administering to a subject the pharmaceutical composition according to any one of claims 1 to 6.   Intravascular epidermal growth factor receptor (VEGFR) inhibitory compound for producing a pharmaceutical composition for the prevention, amelioration, progression inhibition and / or treatment of polyglutamine disease, receptor encoded by c-met gene At least one use selected from the group consisting of somatic tyrosine kinase (c-Met) inhibitory compounds, and inhibitors of both VEGFR and c-Met. A method for screening a candidate substance for an active ingredient of a pharmaceutical composition for prevention, amelioration, suppression of progression, and / or treatment of polyglutamine disease,
Assaying an assay cell expressing polyglutamine fused with a reporter protein in contact with a test substance and culturing, and measuring a reporter signal (A) in a virtual cell body region of the assay cell,
Culturing the assay cell without contacting the test substance, and measuring a reporter signal (B) in a virtual cell body region of the assay cell,
Comparing signal (A) with signal (B), and
A screening method comprising selecting, as a candidate substance, a test substance that reduces the signal (A) more than the signal (B) based on the comparison.