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WO2022118966A1 - Agent for promoting nucleic acid molecule uptake into cells, pharmaceutical composition, and novel compound - Google Patents

Agent for promoting nucleic acid molecule uptake into cells, pharmaceutical composition, and novel compound Download PDF

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Publication number
WO2022118966A1
WO2022118966A1 PCT/JP2021/044526 JP2021044526W WO2022118966A1 WO 2022118966 A1 WO2022118966 A1 WO 2022118966A1 JP 2021044526 W JP2021044526 W JP 2021044526W WO 2022118966 A1 WO2022118966 A1 WO 2022118966A1
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channel
groups
alkoxycarbonyl
substituted
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Japanese (ja)
Inventor
泰生 森
龍 永田
正仁 下條
聡 小比賀
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Kyoto University NUC
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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • 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
    • A61K31/495—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
    • 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
    • A61K31/495—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 or sparfloxacin
    • 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
    • A61K31/658—Medicinal preparations containing organic active ingredients o-phenolic cannabinoids, e.g. cannabidiol, cannabigerolic acid, cannabichromene or tetrahydrocannabinol
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • 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
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to an agent for promoting the uptake of extracellular nucleic acid molecules into cells, a pharmaceutical composition, a novel compound, an agent for enhancing autophagy of cells, and an agent for enhancing the activity of nucleic acid molecules. Etc.
  • Nucleic acid drugs are being researched and developed as new drugs following small molecule drugs and antibody drugs.
  • Antibodies are well known as drugs used in molecular targeted therapies, but most of them target epitopes on the cell surface.
  • nucleic acid drugs are theoretically considered to be able to target intracellular nucleic acids such as cytoplasm and nucleus.
  • the Transient Receptor Potential channel (also referred to as a TRP channel in the present specification) basically functions as a cation channel in the cell membrane. Twenty-eight subtypes of TRP channels are known, many of which take up extracellular calcium ions into the cell when activated. By taking calcium ions into the cell, extracellular environmental changes are transmitted into the cell.
  • PPZ1 and PPZ2 having the structures shown below are known as compounds that activate TRPC channels (Non-Patent Document 1).
  • PPZ1 and PPZ2 activate TRPC3 channel, TRPC6 channel, and TRPC7 channel (also abbreviated as TRPC3, TRPC6, and TRPC7, respectively in the present specification) among TRP channels.
  • Cannabidiol is also known to activate TRPV1 channels, TRPV2 channels and TRPA1 channels (Non-Patent Document 2).
  • GSK1702934A (1,3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazole -2-one) is also known to activate TRPC3 and TRPC6 channels (Non-Patent Document 3).
  • One issue is to provide an agent for promoting the uptake of nucleic acid molecules into cells.
  • One issue is the provision of new compounds.
  • One issue is to provide a pharmaceutical composition or the like containing a substance that promotes the uptake of nucleic acid molecules into cells.
  • One of the challenges is to provide a drug for enhancing cell autophagy.
  • One issue is to provide an agent for enhancing the activity of a nucleic acid molecule.
  • the present inventors activate at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • TRPC6 channel preferably TRPC6 channel
  • a substance for example, a compound represented by the formula (1) described later
  • TRPC6 channel activating effect and the like.
  • Item 1 Nucleic acid intracellularly containing a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof.
  • the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is the compound represented by the formula (1), cannabidiol, and 1,3-dihydro-1.
  • the agent according to Item 1 which is at least one compound.
  • A indicates a carbon atom or a nitrogen atom.
  • B represents a carbon atom or a nitrogen atom.
  • R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • n represents an integer from 0 to 5 and represents R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group.
  • n represents an integer from 0 to 5 and represents p indicates 0 or 1 and represents q indicates 0 or 1.
  • Item 3 The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A is a carbon atom or a nitrogen atom. Show, B represents a carbon atom or a nitrogen atom.
  • R1 represents a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and when there are a plurality of them, May be the same or different, m represents an integer from 0 to 4 and represents R2 is a fluorine atom; a chlorine atom; a bromine atom; a trifluoromethyl group; a trichloromethyl group; a tribromomethyl group; a C1-C4 alkyl group; a C2-C5 acyloxy group; a C2-C5 alkoxycarbonyl group; a C1-C3 alkyl.
  • An amide group which may be substituted with one or two groups selected from the group consisting of a group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group.
  • Piperidinylcarbonyl group optionally substituted with one or more groups; or substituted with one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • n represents an integer from 0 to 4 and represents p indicates 0 or 1 and represents q indicates 0 or 1, Item 2.
  • Item 4. The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom.
  • B represents a carbon atom or a nitrogen atom.
  • R 1 represents a fluorine atom, a chlorine atom, a C1 to C4 alkyl group, a C1 to C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group, and when there are a plurality of them.
  • m indicates an integer from 0 to 3 and represents R2 is a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 acyloxy group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group.
  • An amide group which may be substituted with one or two groups selected from the group consisting of; one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom. B represents a carbon atom or a nitrogen atom.
  • R1 represents a chlorine atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, or a trichloromethyl group, and when there are a plurality of them, they may be the same or different.
  • m indicates an integer of 0 to 2 and represents R2 is selected from the group consisting of a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. May be substituted with one or two groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. It shows a good piperidinylcarbonyl group and may be the same or different if there are more than one.
  • n represents an integer from 0 to 3 and represents p indicates 1, q indicates 1.
  • Item 6. Item 1 or 2, wherein the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is one or more of the compounds shown below. The listed agent.
  • Item 7. Item 6. The agent according to any one of Items 1 to 6, wherein at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is TRPC6 channel.
  • Item 6. The agent according to any one of Items 1 to 8, wherein the cell is a target cell of a nucleic acid molecule.
  • Item 10. Item 6. The agent according to any one of Items 1 to 9, wherein the cells are small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells or nerve cells.
  • Item 11. Item 8. The agent according to any one of Items 1 to 10, which is used for the prevention or treatment of a disease for which administration of a nucleic acid molecule is expected.
  • the agent described in the section. Item 13. A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier. And (a) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or (b) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof.
  • the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the following formula (1), cannabidiol, and 1,3-dihydro-. Select from the group consisting of 1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one.
  • Item 3 The pharmaceutical composition according to Item 13, which is at least one compound thereof. [During the ceremony, A indicates a carbon atom or a nitrogen atom.
  • R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • m represents an integer from 0 to 5 and represents R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • n represents an integer from 0 to 5 and represents p indicates 0 or 1 and represents q indicates 0 or 1.
  • Nucleic acid molecule consists of a group consisting of small interfering RNA (siRNA), micro RNA, antogomir, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme, decoy nucleic acid, splice switching nucleic acid, mRNA, and plasmid.
  • siRNA small interfering RNA
  • micro RNA micro RNA
  • antogomir small hairpin RNA
  • shRNA small hairpin RNA
  • gRNA guide RNA
  • antisense nucleic acid ribozyme
  • decoy nucleic acid splice switching nucleic acid
  • mRNA splice switching nucleic acid
  • plasmid plasmid.
  • R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • m represents an integer from 0 to 5 and represents R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • R 3 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • A indicates a carbon atom or a nitrogen atom.
  • B represents a carbon atom or a nitrogen atom.
  • R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • m indicates an integer from 0 to 5 and represents R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • n represents an integer from 0 to 5 and represents p indicates 0 or 1 and represents q indicates 0 or 1.
  • a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel acts to promote the uptake of extracellular nucleic acid molecule into the cell and enhance the activity of the nucleic acid molecule. Therefore, it can be used as an intracellular nucleic acid molecule uptake promoter, a cell autophagy enhancer, a nucleic acid molecule activity enhancer, and the like.
  • the compound represented by the formula (1), (1-1), or (1-2) is at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • FIG. 1 is a graph showing relative amounts of TRPC6 channel, TRPC3 channel, TRPC7 channel and SRRM4 gene expression in various cells (Test Example 2).
  • FIG. 2 shows the transition of the fluorescence intensity (intracellular Ca 2+ concentration) measured from STC-1 cells in the presence (right figure) and in the absence (left figure) of the compound (60 ⁇ M) of Example 2 (test).
  • Example 3 shows the amount of Ca 2+ influx into the cell.
  • FIG. 3 shows the fluorescence intensity (nucleic acid uptake into cells) measured from STC-1 cells in the presence (right figure) and in the absence (left figure) of the compound (60 ⁇ M) of Example 2 (test).
  • Example 3 shows the amount of nucleic acid taken up into cells.
  • FIG. 4 is a photograph of the fluorescence wavelength derived from the fluorescent dye-conjugated antisense nucleic acid (Test Example 3). The left figure is a photograph immediately after the compound addition of Example 2, and the right figure is a photograph 10 minutes after the compound addition of Example 2.
  • FIG. 5 shows the transition of the fluorescence intensity (intracellular Ca 2+ concentration) measured in the presence of cannabidiol (6 ⁇ M) (Test Example 3).
  • FIG. 6 shows the transition of the fluorescence intensity (amount of nucleic acid incorporated into cells) measured in the presence of cannabidiol (6 ⁇ M) (Test Example 3).
  • FIG. 7 is a photograph of the fluorescence wavelength derived from the fluorescent dye-conjugated antisense nucleic acid in the presence of cannabidiol (6 ⁇ M) (Test Example 3).
  • the left figure is a photograph immediately after the addition of cannabidiol, and the right figure is a photograph 10 minutes after the addition of cannabidiol.
  • FIG. 8 is a graph showing the relative amount of SRRM4 mRNA after 6 hours (left figure) and 24 hours (right figure) of compound addition of Example 2 measured in Test Example 4.
  • FIG. 7 is a photograph of the fluorescence wavelength derived from the fluorescent dye-conjugated antisense nucleic acid in the presence of cannabidiol (6 ⁇ M) (Test Example 3).
  • the left figure is a photograph immediately after the addition of cannabidiol
  • the right figure is a photograph 10 minutes after the addition of cannabidiol.
  • FIG. 8 is a graph showing the relative amount of SRRM4
  • FIG. 9 is a graph showing the relative amount of MALAT-1 expression by the compound of Example 2 in A549 cells (Test Example 5).
  • FIG. 10 is a graph showing the effect of antisense nucleic acid on enhancing MALAT-1 cleavage activity 24 hours (upper figure) and 48 hours (lower figure) after the addition of the compound of Example 2 in A549 cells (Test Example). 6).
  • FIG. 11 is a graph showing the effect of the compound of Example 2 on the proliferation of A549 cells (Test Example 7). The upper figure shows the relative number of cells, and the lower figure shows the ratio of the number of living cells.
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Fluorine atoms, chlorine atoms, and bromine atoms are preferable, and fluorine atoms and chlorine atoms are more preferable.
  • alkyl group examples include C1 to C8 alkyl groups including linear, branched or cyclic structures, preferably C1 to C6 alkyl groups, and more preferably C1 to C4 alkyl.
  • a group particularly preferably a C1-C3 alkyl group.
  • the linear or branched alkyl group includes a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 1-butyl group, a 2-butyl group, an isobutyl group, and a tert-butyl.
  • Examples include a group, an n-pentyl group, a neopentyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, and the like, and examples of the alkyl group containing a cyclic structure include a cyclopropyl group, a cyclopropylmethyl group, a cyclobutyl group, and a cyclo. Examples thereof include a butylmethyl group, a cyclopentyl group, a cyclopentylmethyl group, a cyclohexyl group, a cyclohexylmethyl group, and a cyclohexylethyl group.
  • Preferred examples include a methyl group, an ethyl group, a 2-propyl group, a t-butyl group, a cyclopropyl group and the like.
  • alkyl group substituted with a halogen atom examples include the alkyl group comprising a linear, branched or cyclic structure substituted with a halogen atom.
  • the number of substituents contained in the alkyl group is 1 to the maximum number that can be substituted, and preferably 3 to the maximum number that can be substituted.
  • the halogen atoms may be the same or different, but they are preferably the same.
  • C1-C4 alkyl groups substituted with the maximum number of replaceable halogen atoms can be mentioned, preferably C1-C3 alkyl groups substituted with the maximum number of replaceable halogen atoms, more preferably substitutable.
  • alkyl groups substituted with halogen atoms include perfluoro-n-butyl group, perfluoro-t-butyl group, perfluoro-n-propyl group, perfluoro-i-propyl group, pentylfluoroethyl group, Trifluoromethyl group, perchloro-n-butyl group, perchloro-t-butyl group, perchloro-n-propyl group, perchloro-i-propyl group, pentachloroethyl group, trichloromethyl group, perbromo-n-butyl group, perbromo -T-butyl group, perbromo-n-propyl group, perbromo-i-propyl group, pentabromoethyl group, tribromomethyl group, periodo
  • Preferred examples of the alkyl group substituted with the halogen atom are a pentylfluoroethyl group, a trifluoromethyl group, a pentachloroethyl group, a trichloromethyl group, a pentabromoethyl group, a tribromomethyl group, a pentaiodoethyl group and a triiode.
  • Examples thereof include a methyl group, a trifluoromethyl group, a trichloromethyl group and a tribromomethyl group are more preferable, and a trifluoromethyl group and a trichloromethyl group are even more preferable.
  • alkoxy group examples include C1 to C8 alkoxy groups including linear, branched or cyclic structures, preferably C1 to C6 alkoxy groups, and more preferably C1 to C4 alkoxy. Groups, particularly preferably C1-C3 alkoxy groups.
  • the linear or branched alkoxy group includes a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, a 1-butoxy group, a 2-butoxy group, an isobutoxy group, and a tert-butoxy.
  • Examples thereof include a group, an n-pentyloxy group, a neopentyloxy group, an n-hexyloxy group, an isohexyloxy group, a 3-methylpentyloxy group and the like.
  • Examples of the alkoxy group containing a cyclic structure include a cyclopropoxy group, a cyclopropylmethoxy group, a cyclobutyloxy group, a cyclobutylmethoxy group, a cyclopentyloxy group, a cyclopentylmethoxy group, a cyclohexyloxy group, a cyclohexylmethoxy group and a cyclohexylethoxy group.
  • Preferred examples include a methoxy group, an ethoxy group, a 2-propoxy group, a tert-butoxy group, a cyclopropoxy group and the like.
  • examples of the "acyloxy group” include C2-C9 acyloxy groups containing a linear, branched or cyclic structure, preferably C2-C7 acyloxy groups, and more preferably C2-C5 acyloxy groups.
  • the linear or branched acyloxy group includes a methoxycarbonyl group, an ethoxycarbonyl group, a 1-propoxycarbonyl group, a 2-propoxycarbonyl group, a 1-butoxycarbonyl group, a 2-butoxycarbonyl group, and the like.
  • Examples thereof include an isobutoxycarbonyl group, a tert-butoxycarbonyl group, an n-pentyloxycarbonyl group, a neopentyloxycarbonyl group, an n-hexyloxycarbonyl group, an isohexyloxycarbonyl group and a 3-methylpentyloxycarbonyl group.
  • Examples of the acyloxy group containing a cyclic structure include a cyclopropoxycarbonyl group, a cyclopropylmethoxycarbonyl group, a cyclobutyloxycarbonyl group, a cyclobutylmethoxycarbonyl group, a cyclopentyloxycarbonyl group, a cyclopentylmethoxycarbonyl group, a cyclohexyloxycarbonyl group and a cyclohexylmethoxy.
  • Examples thereof include a carbonyl group and a cyclohexylethoxycarbonyl group.
  • Preferred examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, a 2-propoxycarbonyl group, a tert-butoxycarbonyl group, a cyclopropoxycarbonyl group and the like.
  • the "alkoxycarbonyl group” includes a carbonyl group bonded to the alkoxy group, and examples thereof include a carbonyl group bonded to a C1 to C8 alkoxy group including a linear, branched or cyclic structure. It is preferably a C2-C7 alkoxycarbonyl group, more preferably a C2-C5 alkoxycarbonyl group, and particularly preferably a C2-C4 alkoxycarbonyl group.
  • the linear or branched alkoxy group constituting the alkoxycarbonyl group includes a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, a 1-butoxy group, a 2-butoxy group, an isobutoxy group, and a tert-. Examples thereof include a butoxy group, an n-pentyloxy group, a neopentyloxy group, an n-hexyloxy group, an isohexyloxy group, a 3-methylpentyloxy group and the like.
  • Examples of the alkoxy group containing a cyclic structure constituting the alkoxycarbonyl group include a cyclopropoxy group, a cyclopropylmethoxy group, a cyclobutyloxy group, a cyclobutylmethoxy group, a cyclopentyroxy group, a cyclopentylmethoxy group, a cyclohexyloxy group, and a cyclohexylmethoxy group. , Cyclohexylethoxy group and the like.
  • Preferred examples of the alkoxy group constituting the alkoxycarbonyl group include a methoxy group, an ethoxy group, a 2-propoxy group, a tert-butoxy group, a cyclopropoxy group and the like.
  • the "amide group which may be substituted with one or two groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group” is an amide group, a mono or a di-alkylamide group, a mono or a di-. It includes an alkoxycarbonylamide group, an alkyl group and an amide group di-substituted with an alkoxycarbonyl group, preferably an amide group, a dialkylamide group, and more preferably a dialkylamide group.
  • the substituents may be the same or different.
  • Examples of the monoalkylamide group include an amide group mono-substituted with the alkyl group, preferably an amide group mono-substituted with a C1 to C4 alkyl group, and more preferably an amide mono-substituted with a C1 to C3 alkyl group.
  • Groups are mentioned, and particularly preferably, a methylamide group and an ethylamide group are mentioned.
  • the dialkylamide group include an amide group di-substituted with the alkyl group, preferably an amide group di-substituted with a C1 to C4 alkyl group, and more preferably an amide group di-substituted with a C1 to C3 alkyl group.
  • a dimethylamide group particularly preferred are a dimethylamide group, a diethylamide group and an ethylmethylamide group.
  • the monoalkoxycarbonylamide group include an amide group mono-substituted with the alkoxycarbonyl group, preferably an amide group mono-substituted with a C2-C5 alkoxycarbonyl group, and more preferably a mono-alkoxycarbonyl group with a C2-C4 alkoxycarbonyl group.
  • Substituted amide groups particularly preferably methoxycarbonylamides and ethoxycarbonylamide groups can be mentioned.
  • dialkoxycarbonylamide group examples include an amide group di-substituted with the alkoxycarbonyl group, preferably an amide group di-substituted with a C2-C5 alkoxycarbonyl group, and more preferably a C2-C4 alkoxycarbonyl group.
  • Substituted amide groups particularly preferably di (methoxycarbonyl) amides, di (ethoxycarbonyl) amide groups can be mentioned.
  • Examples of the amide group substituted with the alkyl group and the alkoxycarbonyl group include the amide group di-substituted with the alkyl group and the alkoxycarbonyl group, preferably the C1-C4 alkyl group and the C2-C5 alkoxycarbonyl group.
  • Substituted amide groups more preferably di-substituted amide groups with C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups, particularly preferably methylmethoxycarbonylamide groups, methylethoxycarbonylamide groups, ethylmethoxycarbonylamide groups, Ethylethoxycarbonylamide groups can be mentioned.
  • the number of substituents of the piperidinylcarbonyl group in "a piperidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group” is It is 0 to 5, preferably 0 to 4, more preferably 0 to 3, even more preferably 0 to 2, and particularly preferably 0 or 1.
  • the substituents they may be the same or different.
  • the piperidinylcarbonyl group has a substituent, at least one substituent is on the carbon atom at the 3-position of the piperidine ring (specifically, the 3-position when the nitrogen atom constituting the piperidine ring is at the 1-position).
  • substituents include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1 to C4 alkyl group, a C2-C5 alkoxycarbonyl group, and more preferably a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Particularly preferably, a methyl group, an ethyl group, a methoxycarbonyl group and an ethoxycarbonyl group can be mentioned.
  • the number of substituents of the pyrrolidinylcarbonyl group in "a pyrrolidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group” is It is 0 to 4, preferably 0 to 3, more preferably 0 to 2, and particularly preferably 0 or 1.
  • the substituents they may be the same or different.
  • the pyrrolidinylcarbonyl group has a substituent, at least one substituent is on the carbon atom at the 3-position of the pyrrolidine ring (specifically, the 3-position when the nitrogen atom constituting the pyrrolidine ring is at the 1-position).
  • substituents include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1 to C4 alkyl group, a C2-C5 alkoxycarbonyl group, and more preferably a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Particularly preferably, a methyl group, an ethyl group, a methoxycarbonyl group and an ethoxycarbonyl group can be mentioned.
  • the agent of the present invention is an agent for promoting the uptake of nucleic acid molecules into cells (also referred to as “nucleic acid molecule uptake promoter” in the present specification), and an agent for enhancing cell autophagy (the present invention).
  • nucleic acid molecule uptake promoter also referred to as “nucleic acid molecule uptake promoter” in the present specification
  • autophagy enhancer an agent for enhancing nucleic acid molecular activity
  • nucleic acid molecule activity enhancer also referred to as “nucleic acid molecule activity enhancer” in the present specification.
  • Nucleic acid molecule uptake promoter The agent for promoting the uptake of nucleic acid molecules into cells of the present invention activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • -[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one at least one selected from the group.
  • promoting means that the amount of nucleic acid molecule taken up into the cell increases when the accelerator is applied as compared with the case where the accelerator is not applied.
  • the degree of increase can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more).
  • the accelerator of the present invention typically increases the uptake of nucleic acid molecules administered to a living body and the uptake of nucleic acid molecules added to cells outside the body (eg, cells in a container). ..
  • the nucleic acid molecule activity enhancer of the present invention is (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and is pharmaceutically acceptable thereof.
  • a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel and is pharmaceutically acceptable thereof.
  • cannavidiol By containing the following salts, or prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,,).
  • the activity of the nucleic acid molecule is enhanced in vivo or in vitro.
  • enhancing the activity of a nucleic acid molecule means enhancing the function expected to be exerted by the nucleic acid molecule.
  • the target RNA Enhancement of cleavage activity of (mRNA, non-coding RNA), enhancement of expression of target protein in case of plasmid, mRNA, antagomir, etc., enhancement of modification of splicing of target mRNA in case of splice switching oligonucleotide, etc., decoy nucleic acid, etc.
  • enhancing means that the activity of the nucleic acid molecule is enhanced when the nucleic acid molecule activity enhancer is applied as compared with the case where the nucleic acid molecule activity enhancer is not applied.
  • the degree of enhancement can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more).
  • the nucleic acid molecule activity enhancer of the present invention typically enhances the activity of a nucleic acid molecule administered to a living body and enhances the activity of a nucleic acid molecule added to an in vitro cell (for example, a cell in a container). Let me.
  • the autophagy enhancer of the present invention is a pharmaceutically acceptable substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • a) TRPC3 channel, TRPC6 channel, and TRPC7 channel By containing salts, or prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1-[1-[(5,6,7,, At least one compound selected from the group consisting of 8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, pharmaceutically acceptable thereof.
  • the autophagy of cells is enhanced in vivo or in vitro. Therefore, the autophagy enhancer is useful for the prevention or treatment of diseases caused by the accumulation of abnormal proteins such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and Brion's disease without relying on nucleic acid molecules.
  • autophagy refers to a phenomenon in which intracellular proteins are degraded.
  • autophagy activity is achieved by quantifying an increase or decrease in the amount of conversion from LC3-i to LC3-ii, which correlates with autophagy activity. Can be evaluated for induction and inhibition of.
  • LC3-i is decomposed by Atg4 (cysteine protease) and becomes LC3-ii when LC3-i binds to phosphatidylethanolamine
  • the amount of LC3-ii shows a positive correlation with autophagosome formation. It is used to evaluate the induction and suppression of autophagy.
  • “enhancement” means that the autophagy of the cell enhances the autophagy when the autophagy enhancer is applied as compared with the case where the autophagy enhancer is not applied.
  • the degree of enhancement can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more).
  • the autophagy enhancer of the present invention typically enhances the activity of a nucleic acid molecule administered to a living body and enhances the activity of a nucleic acid molecule added to an in vitro cell (for example, a cell in a container). .. It has been reported that enhancement of autophagy enhances the knockdown (KD) effect of antisense nucleic acid (J. Ochaba et al., A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity, Nucleic Acids). Research, 47, 11284-11303 (2019)).
  • nucleic acid molecule uptake promoter the nucleic acid molecule activity enhancer, and the autophagy enhancer may be collectively referred to as "the agent of the present invention” or simply "the agent”.
  • the agent of the present invention When the agent of the present invention is applied to cells in the presence of calcium ions, the amount of nucleic acid molecule uptake in cells increases. Therefore, the agent of the present invention is preferably used in the presence of calcium ions. Since calcium ions are present inside and outside cells in the in vivo environment, it is not always necessary to add calcium ions, but when the agent of the present invention is applied to cells outside the living body, it must be in the presence of calcium ions. Is preferable.
  • the agent of the present invention can be added to cells in a culture medium containing calcium ions.
  • the agent of the present invention since it has an action of promoting the uptake of nucleic acid molecule into cells, an action of enhancing autophagy, an action of enhancing the activity of nucleic acid molecule, etc., it may contain a nucleic acid molecule or a nucleic acid molecule (for example, It is preferably used in combination with a nucleic acid drug).
  • the agent of the present invention may be administered or added before, after, or at the same time as the administration or addition of the nucleic acid molecule.
  • cells expected to take up nucleic acid molecules are suitable.
  • cells targeted for nucleic acid molecules preferably cells targeted for nucleic acid molecules (typically nucleic acid drugs) administered to a living body, and uptake of nucleic acid molecules into cells in vitro are tested and evaluated. It is a cell to be equalized.
  • the cell has at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. Having such a channel is advantageous for increasing the uptake of nucleic acid molecules, enhancing autophagy, or enhancing the activity of nucleic acid molecules. Therefore, the agents of the present invention allow cells having at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel to take up nucleic acid molecules, enhance autophagy, or nucleic acid molecules. It is useful for enhancing the activity of.
  • TRPC6 channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • the agent of the present invention allows cells having at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel to take up a nucleic acid drug (nucleic acid molecule) and enhance the activity of the nucleic acid molecule. It is useful for drug delivery systems (eg, drug delivery systems for delivering nucleic acid molecules to cells having at least one channel selected from the group consisting of TRPC3 channels, TRPC6 channels, and TRPC7 channels).
  • the cells include small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells, nerve cells and the like, preferably small cell lung cancer cells, non-small cell lung cancer cells, muscle cells and the like. It is a nerve cell. Further, the cells are preferably human cells.
  • nucleic acid molecules examples include small interfering RNA (siRNA), microRNA, antogomir, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme, decoy nucleic acid, splice switching nucleic acid, mRNA, plasmid and the like. These are preferably antisense nucleic acids, splice switching nucleic acids and the like. Nucleic acid molecules can be used alone or in combination of two or more. Specific examples of nucleic acid molecules include Viltepso, Vyondys53, Spinraza, Exondys51 and the like. Nucleic acid molecules can be made based on known sequence information.
  • the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is preferably at least one channel selected from the group consisting of TRPC3 channel and TRPC6 channel, more preferably. May include substances that can activate TRPC6 channels and promote the intracellular influx of extracellular calcium ions.
  • the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the following formula (1) (in the present specification, "Compound (1)). ”), Cannabidiol, 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cycloheptane [b] thien-2-yl) carbonyl] -4-piperidinyl ] -2H-benzimidazol-2-one (also referred to as GSK1702934A) and the like can be mentioned.
  • A indicates a carbon atom or a nitrogen atom.
  • B represents a carbon atom or a nitrogen atom.
  • R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • m indicates an integer from 0 to 5 and represents R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • n represents an integer from 0 to 5 and represents p indicates 0 or 1 and represents q indicates 0 or 1.
  • the formula (1) may be the formula (1-1) or the formula (1-2).
  • the compound (1) is a compound represented by the following formula (1-1) (also referred to as “compound (1-1)” in the present specification) or a compound represented by the following formula (1-2) (present). In the specification, it may also be referred to as “compound (1-2)”. ”).
  • Compound (1-1) and compound (1-2) are novel compounds.
  • [In equation (1-1), R 1 , R 2 , m, and n are the same as in equation (1). ]
  • A indicates a carbon atom or a nitrogen atom.
  • B represents a carbon atom or a nitrogen atom.
  • R 3 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
  • R2 and n are the same as in the equation (1). ]
  • A indicates a carbon atom or a nitrogen atom
  • B indicates a carbon atom or a nitrogen atom.
  • a and B do not show carbon atoms at the same time.
  • a and B are both carbon atoms
  • A is a carbon atom and B is a nitrogen atom
  • A is a nitrogen atom and B is a carbon atom
  • a and B are both nitrogen atoms. That is, the ring containing A and B is a benzene ring, a pyridine ring, or a pyridazine ring in the formula (1), and a pyridine ring or a pyridazine ring in the formula (1-2).
  • both A and B are carbon atoms
  • A is a carbon atom and B is a nitrogen atom
  • A is a nitrogen atom and B is a carbon atom.
  • both B are carbon atoms or A is a nitrogen atom and B is a carbon atom.
  • A is a carbon atom and B is a nitrogen atom
  • A is a nitrogen atom and B is a carbon atom
  • A is a nitrogen atom and B is a carbon atom. It is more preferably an atom.
  • the carbon atom may have a substituent R1 .
  • R1 When there are a plurality of R1 , they may be the same or different.
  • R1 a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group is preferable, and a fluorine atom, Chlorine atoms, C1-C4 alkyl groups, C1-C4 alkoxy groups, C2-C4 alkoxycarbonyl groups, trifluoromethyl groups, trichloromethyl groups, or tribromomethyl groups are more preferred, chlorine atoms, C1-C3 alkyl groups, C1.
  • R 1 is located at the ortho position with respect to the carbon atom directly bonded to the nitrogen atom constituting (NH) p among the carbon atoms constituting the benzene ring. It is preferably bonded to at least one of the two carbon atoms.
  • R 1 bonded to the carbon atom located at the ortho position include an alkoxy group or an alkoxycarbonyl group, preferably a C2-C5 alkoxycarbonyl group, more preferably a C2-C4 alkoxycarbonyl group, and a methoxycarbonyl group or an ethoxy.
  • a carbonyl group is more preferred, and an ethoxycarbonyl group is particularly preferred.
  • the ring to which R 1 is bonded is a benzene ring, and among the carbon atoms constituting the benzene ring, R 1 is located at the meta position with respect to the carbon atom directly bonded to the nitrogen atom constituting (NH) p .
  • it is preferably a halogen atom, more preferably a chlorine atom or a fluorine atom, and particularly preferably a fluorine atom.
  • R 1 is a carbon atom directly bonded to a nitrogen atom constituting (NH) p among the carbon atoms constituting the pyridine ring or the pyridazine ring. It is preferable to bond to a carbon atom located between A and A.
  • R 1 may be, for example, a halogen atom, an alkyl group, an alkoxy group, or an alkoxycarbonyl group, and may be a fluorine atom, a chlorine atom, or a C1-C4 alkyl.
  • Groups, C1-C4 alkoxy groups, or C2-C5 alkoxycarbonyl groups are preferred, C1-C4 alkoxy groups are more preferred, and methoxy or ethoxy groups are particularly preferred.
  • R3 may be a halogen atom , an alkyl group, an alkoxy group, or an alkoxycarbonyl group, preferably a fluorine atom, a chlorine atom, a C1 to C4 alkyl group, a C1 to C4 alkoxy group, or a C2 to C5 alkoxycarbonyl group.
  • C1 to C4 alkoxy groups are more preferable, and methoxy groups or ethoxy groups are particularly preferable.
  • an integer of 0 to 4 is more preferable, 0, 1, or 2 is more preferable, 0 or 1 is further preferable, and 1 is particularly preferable.
  • Partial structure represented by the following in equation (1) And the partial structure represented by the following in the equation (1-1). [In both substructures, * indicates the side bonded to the nitrogen atom constituting the substructure (NH) p, and the other symbols have the same meanings as described above. ]
  • R represents an alkyl group, preferably a C1 to C4 alkyl group, more preferably a C1 to C2 alkyl group, Hal is a halogen atom, preferably a chlorine atom or fluorine. Atoms are shown.
  • the partial structure the following structure is more preferable.
  • R2 includes fluorine atom; chlorine atom; bromine atom; trifluoromethyl group; trichloromethyl group; tribromomethyl group; C1-C4 alkyl group; C2-C5 acyloxy group; C2-C5 alkoxycarbonyl group; C1-C3.
  • An amide group that may be substituted with one or two groups selected from the group consisting of an alkyl group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group.
  • a piperidinyl carbonyl group that may be substituted with one or more groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • Substitutable pyrrolidinylcarbonyl groups are preferred, preferably fluorine atoms; chlorine atoms; trifluoromethyl groups; trichloromethyl groups; C1-C4 alkyl groups; C2-C4 acyloxy groups; C2-C4 alkoxycarbonyl groups; C1-C3.
  • An amide group that may be substituted with one or two groups selected from the group consisting of an alkyl group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group.
  • a piperidinyl carbonyl group that may be substituted with one or more groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • Substitutable pyrrolidinylcarbonyl groups are more preferred: fluorine atoms; chlorine atoms; trifluoromethyl groups; trichloromethyl groups; C1-C4 alkyl groups; C2-C4 alkoxycarbonyl groups; C1-C3 alkyl groups and C2-.
  • An amide group that may be substituted with one or two groups selected from the group consisting of C4 alkoxycarbonyl groups; or one selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • a piperidinylcarbonyl group which may be substituted with two or more groups is more preferable, and a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a methyl group; an ethyl group; a methoxycarbonyl group; an ethoxycarbonyl group; an amide group; C1.
  • the piperidinyl carbonyl group obtained is more preferable.
  • R 2 is one of two carbon atoms located in the ortho position with respect to the carbon atom directly bonded to the nitrogen atom constituting the piperazine ring among the carbon atoms constituting the benzene ring to which R 2 is bonded. Alternatively, it is preferably bonded to two atoms, and more preferably bonded to one ortho-position carbon atom. R 2 may be bonded to a carbon atom located at the para position. R2 is (a) bonded to only one of the two ortho-position carbon atoms, and (b) one of the two ortho-position carbon atoms and the para-position carbon atom.
  • the atom is bonded to (c) one of the two carbon atoms at the ortho position and one of the two carbon atoms at the meta position.
  • R2 is either (a) bonded to only one of the two ortho-position carbon atoms, or (b) one of the ortho-position two carbon atoms and the para-position carbon. It is more preferable that it is bonded to an atom.
  • the R 2 bonded to the carbon atom located at the ortho position is preferably a halogen atom, a trihalogenomethyl group, or a C1-C3 alkyl group, preferably a fluorine atom, a chlorine atom, a bromine atom, a trifluoromethyl group, a methyl group, or a methyl group.
  • An ethyl group is more preferable, and a chlorine atom, a trifluoromethyl group, or a methyl group is particularly preferable.
  • R 2 bonded to the carbon atom located at the para position is substituted with one or two groups selected from the group consisting of, for example, a halogen atom, an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group.
  • a halogen atom an alkyl group
  • an acyloxy group an alkoxycarbonyl group
  • an alkyl group and an alkoxycarbonyl group May be an amide group; a piperidinyl carbonyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group; or a group consisting of an alkyl group and an alkoxycarbonyl group.
  • It may be a pyrrolidinylcarbonyl group which may be substituted with one or more groups selected from, chlorine atom; bromine atom; fluorine atom; C1-C3 alkyl group; C2-C4 acyloxy group; C2. ⁇ C4 alkoxycarbonyl group; an amide group which may be substituted with one or two groups selected from the group consisting of C1 to C3 alkyl groups and C2 to C4 alkoxycarbonyl groups; C1 to C3 alkyl groups and C2 to C4.
  • a piperidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of alkoxycarbonyl groups; or selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups.
  • a pyrrolidinylcarbonyl group which may be substituted with one or more groups is preferred and is selected from a chlorine atom; a methyl group; an ethyl group; a methoxycarbonyl group; an ethoxycarbonyl group; an amide group; a C1-C3 alkyl group.
  • a carbonyl group is more preferred.
  • n an integer of 0 to 4, an integer of 0 to 3, is more preferable, 0, 1, or 2 is more preferable, 1 or 2 is more preferable, and 2 is particularly preferable.
  • Examples of the compound (1), the compound (1-1), or the compound (1-2) include the compounds shown in the following (a) to (m).
  • compound (1), the compound (1-1), or the compound (1-2) may be referred to as the above (a) to (e), (g), (h), (j), or (k). It may be the compound shown. Furthermore, compound (1), compound (1-1), or compound (1-2) is the compound represented by the above (a) to (d), (g), (h), or (k). May be.
  • Examples of compound (1) or compound (1-1) include 2- (4- (2-chloro-4-piperidin-1-carbonyl) phenyl) piperazine-1-yl) -N- (2-ethoxyphenyl). ) Acetamide is particularly preferred.
  • the compound (1), the compound (1-1), and the compound (1-2) can be produced by a known production method for the known compounds included therein.
  • the new compound can be produced, for example, by appropriately changing or combining the production method 1 or 2, a method similar thereto, a known method, or the like described in detail below.
  • the compound used as the raw material compound may be used as a salt, respectively.
  • the method shown below is merely an example, and can be appropriately produced by another method based on the knowledge of a person who is proficient in organic synthesis.
  • the compound represented by the formula (1A) can be produced by the synthetic team represented by the following reaction step formula-1. That is, the compound represented by the formula (1A) can be produced from the compound represented by the formula (2), the compound represented by the formula (3) and the compound represented by the formula (6).
  • X is a halogen atom
  • D is a protecting group
  • A, B, R 1 , m, R 2 , and n are the same as above.
  • Examples of X include a chlorine atom, a fluorine atom, a bromine atom, and an iodine atom, and a bromine atom is preferable.
  • Examples of D include a protecting group such as a halogen atom, a tert-butyl group, a phenyl group, a methyl group and an ethyl group, and a tert-butoxy group is preferable.
  • a protecting group such as a halogen atom, a tert-butyl group, a phenyl group, a methyl group and an ethyl group, and a tert-butoxy group is preferable.
  • R 2 is preferably an alkoxycarbonyl group and a halogen atom. It is more preferable that an alkoxycarbonyl group is bonded to the carbon atom at the para position. It is more preferable that the alkoxycarbonyl group is bonded to the carbon atom at the para position and the halogen atom is bonded to the carbon atom at the ortho position.
  • Step 1 that is, the step of reacting the compound represented by the formula (2) with the compound represented by the formula (3) (which may be a hydrochloride) to synthesize the compound represented by the formula (4) is
  • the compound represented by the formula (3) for example, if it is 2- (4- (2-chlorophenyl) piperazine-1-yl) acetic acid (CAS No. 119357-76-5), a commercially available product can be used, and the hydrochloric acid thereof can be used. Salt (CAS No.
  • 856843-13-5) can be produced by the method described in Journal of the American Chemical Society 1955, 77, 1, 40-42, and other compounds can also be commercially available and can be produced by a known production method. It can be manufactured, or it can be manufactured by appropriately changing a known manufacturing method.
  • inert solvent in this reaction examples include ether solvents such as diethyl ether, tetrahydrofuran (THF), dioxane and dimethoxymethane, aromatic hydrocarbon solvents such as toluene, benzene and xylene, dichloromethane, chloroform, dichloroethane and tetrachloride.
  • ether solvents such as diethyl ether, tetrahydrofuran (THF), dioxane and dimethoxymethane
  • aromatic hydrocarbon solvents such as toluene, benzene and xylene
  • dichloromethane chloroform
  • dichloroethane and tetrachloride examples include a halogenated hydrocarbon solvent such as carbon, a ketone solvent such as acetone, an aproton solvent such as dimethyl sulfoxide, N, N-dimethylformamide (DMF) and acetonitrile, and pyr
  • Examples of the base include metal hydrides such as sodium hydride and potassium hydride, metal hydroxides such as potassium hydroxide and sodium hydroxide, and metals such as potassium carbonate, potassium hydrogen carbonate, sodium carbonate, sodium hydrogen carbonate and cesium carbonate.
  • metal hydrides such as sodium hydride and potassium hydride
  • metal hydroxides such as potassium hydroxide and sodium hydroxide
  • metals such as potassium carbonate, potassium hydrogen carbonate, sodium carbonate, sodium hydrogen carbonate and cesium carbonate.
  • alkylamines such as carbonates, triethylamine and ethyldiisopropylamine
  • metal alkoxides such as sodium methoxydo and potassium t-butoxide.
  • the amount of the compound represented by the formula (2) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (3). It is mol, more preferably 0.9 to 1.5 mol.
  • the amount of the base used is usually 1 mol or more, preferably 1 to 5 times mol, and more preferably 1 to 2 times mol with respect to 1 mol of the compound represented by the formula (3).
  • the reaction temperature is usually ⁇ 20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C.
  • the reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.
  • Step 2 that is, the step of converting the compound represented by the formula (4) into the compound represented by the formula (5) is possible, for example, by treating the compound represented by the formula (4) with an acidic organic solvent. be.
  • Examples of the acidic organic solvent in this reaction include hydrochloric acid-containing 1,4-dioxane and the like. Two or more kinds of these solvents may be mixed and used in an appropriate ratio. The amount of the acidic organic solvent used may be such that the compound represented by the formula (4) can be oxidized.
  • Step 3 that is, the step of reacting the compound represented by the formula (5) with the compound represented by the formula (6) to synthesize the compound represented by the formula (1A) is, for example, in a base and condensation in an inert solvent. It can be done in the presence of the agent.
  • the details of the inert solvent and the base are the same as described above.
  • condensing agent examples include 1- [dimethylamino (dimethylimino) methyl] -1H-1,2,3-triazolo [4,5-b] pyridine 3-oxide hexafluorophosphart (in the present specification). Also referred to as "HATU”) and the like.
  • the amount of the compound represented by the formula (6) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (5). It is mol, more preferably 0.9 to 1.5 mol.
  • the amount of the condensing agent to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 mol, more preferably 0, with respect to 1 mol of the compound represented by the formula (5). It is 9 to 1.5 mol.
  • the reaction temperature is usually ⁇ 20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C.
  • the reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.
  • the corresponding carboxylic acid is obtained by treating this compound, for example, in an inert solvent in the presence of a base. It may be converted into a body. Then, an appropriate substituent is introduced into this carboxylic acid body. For example, in an inert solvent, in the presence of a condensing agent, and if necessary, in the presence of a base, the reaction with a compound corresponding to the substituent (for example, piperidin, amine, etc.) is carried out to form the corresponding amide group or pi as R2 .
  • a compound represented by the formula (1A) having a peridinylcarbonyl group can also be obtained.
  • the details of the inert solvent, base and condensing agent are the same as described above.
  • the amount of the compound corresponding to the substituent such as piperidin or amine is usually 0.5 mol or more with respect to 1 mol of the compound represented by the formula (1A) in which R2 is an alkoxycarbonyl group. It is 0.8 mol or more, preferably 0.9 to 10 mol, and more preferably 0.9 to 8 mol.
  • the reaction temperature is usually ⁇ 20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C.
  • the reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.
  • the compound represented by the formula (1A) can be produced by the synthetic scheme represented by the following reaction step formula-2. That is, the compound represented by the formula (1A) can be produced from the compound represented by the formula (6), the compound represented by the formula (7) and the compound represented by the formula (3).
  • Step 1 that is, the step of reacting the compound represented by the formula (6) with the compound represented by the formula (7) to synthesize the compound represented by the formula (8) is, for example, the presence of a base in an inert solvent. Can be done below. The details of the inert solvent and the base are the same as described above.
  • the amount of the compound represented by the formula (7) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (6). It is mol, more preferably 0.9 to 1.5 mol.
  • the reaction temperature is usually ⁇ 20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably ⁇ 10 ° C. to 40 ° C.
  • the reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.
  • Step 2 that is, the step of reacting the compound represented by the formula (8) with the compound represented by the formula (3) to synthesize the compound represented by the formula (1A) is, for example, the presence of a base in an inert solvent. Can be done below. The details of the inert solvent and the base are the same as described above.
  • the amount of the compound represented by the formula (3) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (8). It is mol, more preferably 0.9 to 1.5 mol.
  • the reaction temperature is usually ⁇ 20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably ⁇ 10 ° C. to 40 ° C.
  • the reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.
  • Cannabidiol is represented by the following chemical structural formula 2-[(1R, 6R) -3-methyl-6-(1-methylethenyl) -2-cyclohexen-1-yl] -5-pentyl-1,3- It is Benzenediol, which was found by the present inventors to have a non-selective TRP channel activating effect and also a TRPC6 channel activating effect. Cannabidiol can be produced by known methods or obtained from nature.
  • GSK1702934A (1,3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2 -one) is a known compound having the following structure and has a TRPC3 channel activating effect.
  • the method for producing GSK1702934A is known, and for example, it can be produced by the method described in Non-Patent Document 3.
  • the GSK1702934A can also be a commercially available product.
  • the compound represented by the formula (1) according to the present invention (the compound represented by the formula (1) includes the compound represented by the formula (1-1) and the compound represented by the formula (1-2). ) And its intermediate compounds can be produced by the above-mentioned production method, and the starting material compound, the compound represented by the formula (1) according to the present invention, and the intermediate compound thereof are described herein.
  • Known or known techniques eg, B.R.
  • the starting material compound and the intermediate compound shown in each of the above reaction formulas are subjected to the reaction, if necessary, the functional groups are protected with an appropriate protecting group by a known method, and after the reaction is completed, the functional groups are protected.
  • the protecting group can be deprotected by a known method.
  • Each of the target compounds obtained according to the above reaction formula can be isolated and purified. For example, after cooling the reaction mixture, isolation procedures such as filtration, concentration, and extraction are performed to separate the crude reaction product, and then the crude reaction product is subjected to general column chromatography, recrystallization, etc. By subjecting to a purification procedure, it can be isolated and purified from the reaction mixture.
  • a solvent is used in the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and the starting material compound, intermediate compound, etc. shown in each of the above reaction formulas. Contains compounds in the form of added solvates (eg, hydrates, ethanol solvates, etc.).
  • alkali metal salts such as sodium salt and potassium salt
  • alkaline earth metal salts such as calcium salt and magnesium salt
  • inorganic metal salts such as zinc salt
  • triethylamine, triethanolamine and trihydroxy the compound represented by the formula (1), cannavidiol, and 1,3-dihydro-1-[ 1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-A pharmaceutical
  • Organic base salts such as methylaminomethane and amino acids
  • inorganic acid salts such as hydrochlorides, hydrobromates, sulfates, phosphates and nitrates; acetates, carbonates, propionates, succinates and lactates.
  • These salts can be produced according to a conventional method.
  • Various isomers can be isolated by a known separation method.
  • a racemic compound can be derived into a sterically pure isomer by a general optical resolution method (for example, optical resolution by crystallization, direct optical resolution by chromatography, etc.).
  • the optically active compound can also be produced by using an appropriate optically active raw material.
  • the starting material compound, the intermediate compound, and the target compound represented in each of the above reaction process formulas can be used in an appropriate salt form.
  • one or more atoms can be replaced with one or more isotope atoms.
  • isotope atoms include deuterium (2H), tritium (3H), 13C, 14N, 18O and the like.
  • [Pharmaceutical composition] in the present invention, (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof. And a pharmaceutical composition containing a pharmaceutically acceptable carrier, or a compound represented by (b) formula (1), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,,). At least one compound selected from the group consisting of 7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceuticals.
  • compositions containing an acceptable salt, or a prodrug thereof, and a pharmaceutically acceptable carrier include an acceptable salt, or a prodrug thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention comprises a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a compound represented by the formula (1), cannabidiol, and 1 , 3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one ,
  • the pharmaceutically acceptable salt thereof, or their prodrugs are formulated in the form of conventional pharmaceutical compositions.
  • the pharmaceutical composition of the present invention comprises (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof.
  • Prodrugs or compounds represented by formula (1) (b), benzimidazole, and 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b) ] Thien-2-yl) carbonyl] -4-piperidinyl] -2H-Benzimidazole-2-one prepared using at least one compound selected from the group and a pharmaceutically acceptable carrier.
  • the carrier include commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants and other diluents or excipients.
  • a prodrug refers to a compound that is converted into the compound of the present invention by a reaction in vivo (for example, an enzymatic reaction or a reaction with gastric acid).
  • a reaction in vivo for example, an enzymatic reaction or a reaction with gastric acid.
  • the carboxyl is converted into an ester.
  • the ester include methyl ester, ethyl ester, 1-propyl ester, 2-propyl ester, pivaloyloxymethyl ester, acetyloxymethyl ester, cyclohexylacetyloxymethyl ester, 1-methylcyclohexylcarbonyloxymethyl ester and ethyloxy.
  • Examples thereof include carbonyloxy-1-ethyl ester and cyclohexyloxycarbonyloxy-1-ethyl ester.
  • the pharmaceutical composition of the present invention can be selected from various forms according to the purpose of treatment, and typical examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules and capsules. Examples include suppositories, injections (liquids, suspensions, etc.), ointments, inhalants and the like.
  • carrier used for molding tablets known carriers can be widely used, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, water, ethanol, etc. Binders such as propanol, simple syrup, glucose solution, starch solution, gelatin solution, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, carboxymethyl cellulose, cellac, sodium alginate, dried starch, agar powder, laminaran powder, calcium carbonate, sodium hydrogen carbonate.
  • excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, water, ethanol, etc.
  • Binders such as propanol, simple syrup, glucose solution, starch solution, gelatin solution, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, carboxymethyl cellulose, cellac, sodium alginate
  • Polyoxyethylene sorbitan fatty acid esters sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrants such as lactose, absorption promoters such as quaternary ammonium base, sodium lauryl sulfate, stearic acid, cocoa butter, hydrogenated oil, etc.
  • Disintegration inhibitors moisturizers such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearic acid, sodium borate, lubricants such as polyethylene glycol and the like. Be done.
  • the tablet can be a tablet with a normal lock skin, for example, a sugar coating, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet, or a multi-layer tablet, if necessary.
  • a normal lock skin for example, a sugar coating, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet, or a multi-layer tablet, if necessary.
  • Known carriers can be widely used for molding pills, for example, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, talc, gum arabic powder, tragant powder, gelatin. , Binders such as ethanol, disintegrants such as laminarane and agar, and the like.
  • the carrier used for molding the suppository known ones can be widely used, and examples thereof include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glyceride and the like.
  • the liquid, emulsion and suspension are preferably sterilized and isotonic with blood.
  • emulsions and suspending agents known ones can be widely used, for example, water, ethanol, propylene glycol, polyoxylated isostearyl alcohol, ethoxylated iso. Examples thereof include stearyl alcohol and polyoxyethylene sorbitan fatty acid esters.
  • a sufficient amount of salt, glycerin, glucose, etc. can be contained in the pharmaceutical preparation to prepare an isotonic solution, and usual solubilizing agents, buffers, and pain-relieving agents can be contained.
  • Agents and the like can be contained, and if necessary, colorants, preservatives, flavors, flavoring agents, sweeteners and the like and other pharmaceutical products can be contained.
  • the ointment has a form such as a paste, a cream, or a gel, and when preparing these forms, for example, white petrolatum, paraffin, glycerin, a cellulose derivative, polyethylene glycol, silicone, bentonite, etc. are used as a diluent. can.
  • white petrolatum, paraffin, glycerin, a cellulose derivative, polyethylene glycol, silicone, bentonite, etc. are used as a diluent. can.
  • the inhalant is a preparation intended to be applied to the bronchi or lungs by inhaling the active ingredient as an aerosol, and includes a powder inhalant, an inhalation solution, an inhalation aerosol and the like.
  • a powder inhalant is a preparation that is inhaled as an aerosol of solid powder particles, and is usually produced by making the active ingredient into fine particles and mixing them with an additive such as lactose to make them homogeneous. can.
  • the inhalation solution refers to a liquid inhalation agent applied by a nebulizer or the like, and can usually be produced by adding a solvent, an appropriate tonicity agent, a pH adjuster, or the like to the active ingredient and mixing them.
  • the inhalation aerosol agent is a fixed-quantity spray-type inhalant that sprays a certain amount of the active ingredient together with the propellant filled in the container.
  • Inhalation aerosols are usually prepared by adding a solvent, an appropriate dispersant, a stabilizer, etc. to the active ingredient to make a solution or suspension, filling a pressure-resistant container with a liquid propellant, and installing a metering valve. It can be manufactured by.
  • the pharmaceutical composition of the present invention may contain a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent, and other pharmaceutical products, if necessary.
  • the amount of the prodrug is not particularly limited and may be appropriately selected from a wide range, but is usually 0.5 to 90% by mass, preferably 1 to 85% by mass, preferably 1 to 80% by mass in the pharmaceutical composition. %.
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited, and the pharmaceutical composition is administered by a method according to various pharmaceutical forms, the age, sex, disease state, and other conditions of the patient.
  • the pharmaceutical composition is administered by a method according to various pharmaceutical forms, the age, sex, disease state, and other conditions of the patient.
  • tablets, pills, liquids, suspensions, emulsions, granules and capsules they are orally administered.
  • it may be administered intravenously alone or mixed with a normal replacement fluid such as glucose or amino acid, or if necessary, intramuscularly, intradermally, subcutaneously, intraperitoneally, etc.
  • a normal replacement fluid such as glucose or amino acid
  • intramuscularly intradermally
  • subcutaneously intraperitoneally, etc.
  • nasally In the case of suppositories, it is administered intrarectally.
  • inhalants it is administered nasally.
  • the dose of the pharmaceutical composition of the present invention may be selected in consideration of usage, age, sex, degree of disease, and other conditions of the patient, and is selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • a substance that activates at least one channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof is usually used.
  • the amount is 0.01 to 100 mg, preferably 0.1 to 50 mg per 1 kg of body weight per day, divided into 1 to several times per day, or 2 days, 3 days, 4 days, 5 days. , 6 days, 1 week, 2 weeks or 4 weeks at intervals. Since the dose varies depending on various conditions, a dose smaller than the above range may be sufficient, or a dose exceeding the above range may be required.
  • the pharmaceutical composition of the present invention can be used as a concomitant drug in combination with other drugs.
  • the pharmaceutical composition of the present invention is preferably formulated in combination with a drug containing a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof.
  • the pharmaceutical composition of the present invention may be used in combination with an anticancer agent.
  • anticancer agents include antitumor antibiotics such as doxorubicin, idarvidin and mitomycin C, microtube inhibitors such as docetaxel and bincristine, platinum preparations such as carboplatin, cisplatin and oxaliplatin, and histone deacetylases such as bolinostat.
  • HDAC HDAC
  • kinase inhibitors such as snitinib, imatinib, gefetinib, errotinib, afatinib, dasatinib, tramethinib
  • topoisomerase inhibitors such as irinotecan and etopocid
  • carcinurin inhibitors such as cyclosporin and tachlorimus
  • cyclophosphamide cyclophosphamide
  • Alkylating agents such as iosphamide and dacarbazine
  • metabolic antagonists such as pentostatin, fludalabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine and enocitabine
  • molecular targeting agents such as rituximab, setuccimab and trusszumab
  • proteasome inhibitors such as voltezomib.
  • hormonal therapeutic agents such as tamoxifen, bicardamide, anti-PD-1 antibodies such as nibolumab, penbrolizumab, pidirisumab, atezolizumab, avelumab, or anti-PD-L1 antibodies such as durvalumab, BMS-936559, anti-CTLA such as ipilimumab or tremellimumab. 4 Inhibitors can be mentioned.
  • agent or pharmaceutical composition of the present invention can promote the uptake of nucleic acid molecules into cells, it is desirable to use them in combination with nucleic acid molecules. Further, the agent or pharmaceutical composition of the present invention is preferably used for diseases that are expected to be prevented or treated by administration of nucleic acid molecules.
  • Diseases to be prevented or treated include diseases for which administration of nucleic acid molecules is expected, such as lung cancer, prostate cancer, breast cancer, pulmonary fibrosis, renal fibrosis, muscular dystrophy, amyotrophic lateral sclerosis, and Huntington's disease. Examples include muscular atrophy and Parkinson's disease. Preferred diseases are lung cancer and prostate cancer.
  • the agent or pharmaceutical composition of the present invention is (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and a pharmaceutically acceptable salt thereof.
  • a pharmaceutically acceptable salt thereof Or to contain prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,7,8-).
  • the cells include small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells, nerve cells and the like, preferably small cell lung cancer cells, non-small cell lung cancer cells, muscle cells, etc. Prostate cancer cells and nerve cells. Further, the cells are preferably human cells.
  • the present invention is a substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel, and a pharmaceutically acceptable salt thereof, for a subject receiving nucleic acid molecule administration. , Or an effective amount of their prodrug, or (b) the compound represented by formula (1), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,7).
  • the present invention is pharmaceutically acceptable as a substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel for a subject to be administered with a nucleic acid molecule.
  • a salt or a prodrug thereof or (b) the compound represented by formula (1), benzimidazole, and 1,3-dihydro-1- [1-[(5,6). , 7,8-Tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or their prodrugs It may include methods of enhancing cell autophagy, including administration of an effective amount of.
  • the present invention is a pharmaceutically acceptable substance for a subject receiving nucleic acid molecule administration, which activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • a) TRPC3 channel, TRPC6 channel, and TRPC7 channel activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel.
  • 6,7,8-Tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or their pros It may include methods of enhancing the activity of a nucleic acid molecule, including administering an effective amount of the drug.
  • the present invention activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel for a subject to be prevented or treated for a disease for which administration of a nucleic acid molecule is expected. Administering an effective amount of the substance, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or (b) the compound represented by formula (1), benzimidazole, and 1,3-dihydro-1-.
  • the present invention relates to (1) an agent for promoting the uptake of nucleic acid molecules into cells, (2) an autophagy enhancer, (3) an agent for enhancing nucleic acid molecule activity, or (4) prevention of diseases in which administration of nucleic acid molecules is expected.
  • prodrugs or compounds represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1-[1-[(5,6,7,8-tetrahydro-4H-cyclohepta] b] Thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or the use of prodrugs thereof may be included.
  • Bromoacetic acid bromide (0.87 mL, 9.99 mmol) is added dropwise at 0 ° C. to a solution of 2-ethoxyaniline (1.37 g, 9.99 mmol) and triethylamine (1.462 mL, 10.49 mmol) in dichloromethane (20 mL). Then, the reaction mixture was stirred at 0 ° C. for 1 hour to obtain a reaction solution containing 2-bromo-N- (2-ethoxyphenyl) acetamide. The obtained reaction solution was used as it was for the reactions of Examples 8 and 9.
  • Bromoacetic acid bromide (0.26 mL, 3 mmol) was added dropwise at 0 ° C. to a solution of 2-aminobenzoic acid methyl ester (453 mg, 3 mmol) and triethylamine (1.254 mL, 9 mmol) in dichloromethane (15 mL) to give the reaction mixture. The mixture was stirred at 0 ° C. for 1 hour.
  • TRPC6-expressing HEK293 cells Influx of Ca 2+ into HEK293 cells transiently expressing TRPC channels (1) Introduction and expression of TRPC channels into HEK293 cells The TRPC6 gene was introduced into HEK293 cells (human fetal kidney-derived cells) by the lipofection method. TRPC6 channels were expressed on the cell membrane. That is, HEK293 cells in which TRPC6 channels were expressed on the cell membrane by introducing TRPC6 plasmid DNA (pCI-neo (promega)) into HEK293 cells using a lipofection agent and then culturing at 37 ° C for 24 to 48 hours. (TRPC6-expressing HEK293 cells) were prepared. Specifically, the method described in Non-Patent Document 1 (S.
  • TRPC3 HEK293 cells (TRPC3-expressing HEK293 cells) in which the TRPC3 channel was expressed on the cell membrane and HEK293 cells (TRPC7-expressing HEK293 cells) in which the TRPC7 channel was expressed on the cell membrane were generated in the same manner.
  • TRPC3, TRPC6 or TRPC7-expressed HEK293 cultured cells were peeled off with trypsin, re-spread on a cover glass, and then cultured again at 37 ° C. for 3 hours.
  • the cover glass was transferred to 500 ⁇ l of a medium containing the calcium indicator Fura-2 AM (final concentration 1 ⁇ M), cultured at 37 ° C. for 30 minutes, and incorporated into cells.
  • Ca 2+ -containing solution and Ca 2+ free solution were prepared as extracellular solutions.
  • Ca 2+ -containing solution (2 mM CaCl 2 , 132 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM glucose, 5 mM HEPES (pH 7.4))
  • Ca 2+ free solution (132 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM glucose, 5 mM HEPES (pH 7.4))
  • Test Example 1-1 Ca into HEK293 cells expressing TRPC6 channel 2+ Effect of the compounds of the Examples on the influx of
  • the change in calcium ion concentration was measured according to the measuring method described in Test Example 1.
  • the compounds of Examples 1 to 3, the compounds of Examples 5 to 11 and PPZ2 were used as the test compounds, and the addition amount thereof was 30 ⁇ M.
  • 0.1% DMSO was used instead of the test compound as a control.
  • the amount of Ca 2+ increase in TRPC6 channel-expressing HEK293 cells was obtained.
  • the test compound having a larger Ca 2+ increase than the control using DMSO was used as the substance that activates the TRPC6 channel.
  • the amount of increase in Ca 2+ of PPZ2 was set to 100%, and the amount of increase in Ca 2+ of other test compounds was calculated as a relative value. The results are shown in Table 1.
  • Fura-2 AM has high cell membrane permeability due to the presence of AM group (acetoxymethyl group), is easily taken up into cells, and the AM group is hydrolyzed inside the cell to become Fura-2. , Ca 2+ and chelate formation become possible, and the loss of the AM group makes it difficult for the cell to leak out of the cell.
  • AM group acetoxymethyl group
  • PPZ2 is known to activate the TRPC6 channel and allow Ca 2+ to flow into the cells, and in this study as well, the addition of 30 ⁇ M PPZ2 increased the Ca 2+ concentration of 80 nM-120 nM.
  • the compounds of Examples 1 to 3 and Examples 5 to 11 also increased the intracellular Ca 2+ concentration more than DMSO, confirming that these compounds activate the TRPC6 channel. Since the compound of Example 4 has a similar chemical structure to the compound of Example 2, the compound of Example 3, and the like, it is presumed that the TRPC6 channel is activated in the same manner as these compounds.
  • Test Example 1-2 Effect of the compound of Example 2 on the influx of Ca 2+ into HEK293 cells expressing TRPC3 channel, TRPC6 channel or TRPC7 channel TRPC3-expressing HEK293 cells (TRPC3 / HEK293), TRPC6 prepared by the method described in Test Example 1.
  • TRPC3 / HEK293 TRPC3-expressing HEK293 cells
  • TRPC6 prepared by the method described in Test Example 1.
  • Each cell was used in the same manner as in Test Example 1-1 except that the expressed HEK293 cells (TRPC6 / HEK293) and TRPC7 expressed HEK293 cells (TRPC7 / HEK293) were used and the compound of Example 2 (30 ⁇ M) was used as the test compound.
  • the amount of Ca 2+ increase inward was calculated. The results are shown in Table 2.
  • the compound of Test Example 2 has not only the TRPC6 channel activating effect but also the TRPC3 channel activating effect and the TRPC7 channel activating effect.
  • Test Example 1-3 Action of Cannabidiol on the influx of Ca 2+ into HEK293 cells expressing TRPC6 channel
  • Cannabidiol (6 ⁇ M; 2-[(1R, 6R) -3-methyl-6-(1-methylethenyl) -2-cyclohexen- 1-yl] -5-pentyl-1,3-Benzenediol) was used.
  • the same measurement method as described in Test Example 1 was used except for the following protocol.
  • the Ca 2+ increase by Cannabidiol was measured and found to be 83 nM.
  • Cannabidiol was known to activate TRPV1 and TRPA1 channels, but this study newly confirmed that it activates TRPC6 channels.
  • TRPC6-expressing cells were placed in a measurement chamber containing a Ca 2+ -containing solution. After 30 seconds: The Ca 2+ -containing solution was replaced with a Ca 2+ free solution. After 3 minutes: The test compound Cannabidiol (6 ⁇ M) was added to the Ca 2+ free solution. 7 minutes later: The Ca 2+ free solution was replaced with a Ca 2+ -containing solution containing the test compound Cannabidiol (6 ⁇ M). 15 minutes later: Measurement is completed.
  • Test Example 2 TRPC6 channel, TRPC3 channel, TRPC7 channel and SRRM4 gene expressing cells in various cells including STC1 and A549 cells (NCI-N417 (human small cell lung cancer), STC1 (human small cell lung cancer), H1650 (human non-small cell lung cancer) ), A549 (human non-small cell lung cancer), DU145 (human prostate cancer), VCaP (human prostate cancer)) after extracting total RNA using RNeasy mini kit (Qiagen), 20 ⁇ L of reaction solution (SuperScript VILO) Transcription reaction to cDNA using total RNA (1 ⁇ g) in master mix (ThermoFisher): 4 ⁇ L, H 2 O: 16 ⁇ L (priming at 25 ° C for 10 minutes, reverse transcription at 42 ° C for 60 minutes, no Activation was performed at 85 ° C.
  • NCI-N417 human small cell lung cancer
  • STC1 human small cell lung cancer
  • H1650 human non-small cell lung cancer
  • hTRPC3 forward 5'-cagggtgaaaccacccagt-3'(SEQ ID NO: 1); hTRPC3 reverse: 5 '-cctaggtccttccagccaga-3' (SEQ ID NO: 2); hTRPC6 forward: 5'-tgaaagctttggacctctgc-3' (SEQ ID NO: 3); hTRPC6 reverse: 5'-tcataaaggccacaaacacca-3' (SEQ ID NO: 4); hTRPC7 forward: 5'- tacgtgctgcacttgtggaa-3'(SEQ ID NO: 5); hTRPC7 reverse: 5'-atgaagcgt
  • the expression of the TRPC3 channel gene was confirmed in DU145, H165, STC1 and VCaP cells, the expression of the TRPC6 channel gene was confirmed in A549, DU145, H165, STC1 and VCaP cells, and the expression of the TRPC7 channel gene was confirmed in VCaP cells.
  • the expression of the SRRM4 gene targeted by the antisense nucleic acid L21 (AmNA-7174, L21) used in Test Example 4 was confirmed in N417, STC1 and VCaP cells.
  • Test Example 3 Uptake of Ca 2+ into STC-1 cells and uptake test of fluorescent dye-conjugated antisense nucleic acid by the compound of Example 2 STC-1 cells (1.0 ⁇ 10 5 cell / well, RPMI medium- High glucose) was sown. The next day, the medium was removed, 500 ⁇ L of a 2 mM Ca 2+ solution containing Fluo-4 (final concentration 1 ⁇ M) was added, and the cells were cultured for 30 minutes and incorporated into cells. After washing with 500 ⁇ L of PBS, 500 ⁇ L of 2 mM (or 0 mM) Ca 2+ solution was added.
  • a 10 ⁇ M fluorescent dye-conjugated antisense nucleic acid (L26-alexa647, final concentration 10 nM) was added, and the mixture was lightly stirred.
  • 30 mM Example 2 compound (final concentration 60 ⁇ M) or 3 mM Cannabidiol (1 ⁇ L, final concentration 6 ⁇ M) was added as test compound (Cannabidiol activates TRPC6 channel (see Test Example 1-3)). ..
  • DMSO was added as a control instead of the test compound.
  • the fluorescence of Fluo-4 (BZ-9000, Keyence, lens x 20) with a BZ-X filter GFP OP-87763 (absorption wavelength: 525-575 nm) was used.
  • FIGS. 2 and 3 By monitoring the maximum fluorescence wavelength: 518 nm), the intracellular Ca 2+ concentration transition is observed, and a fluorescence microscope equipped with a BZ-X filter Cy5 OP-87766 (absorption wavelength: 700-775 nm) (BZ-9000, The intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid was observed by monitoring the fluorescence of Alexa647 (maximum fluorescence wavelength: 668 nm) using Keyence (lens using ⁇ 20). The monitored intracellular Ca 2+ concentration and the intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid are shown in FIGS. 2 and 3, respectively. Further, FIG.
  • FIG. 4 shows fluorescence micrographs of Alexa647-derived fluorescence immediately after the compound addition of Example 2 (left figure) and 10 minutes later (right figure).
  • each chart line represents the transition of the target substance (Ca 2+ concentration in FIG. 2 and nucleic acid uptake amount in FIG. 3) in one cell.
  • the graph contains about 20 charts, which represent the amount of substance of interest in about 20 cells. The same is true for other similar graphs.
  • the left figure of FIG. 4 is a fluorescence micrograph immediately after the compound addition of Example 2, and the right figure is a fluorescence micrograph 10 minutes after the compound addition of Example 2.
  • FIGS. 5 and 6 The intracellular Ca 2+ concentration of Cannabidiol and the intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid are shown in FIGS. 5 and 6, respectively. Further, FIG. 7 shows fluorescence micrographs of Alexa647-derived fluorescence immediately after the addition of cannabidiol (left figure) and 10 minutes later (right figure).
  • Test Example 4 Enhancement of SRRM4 gene knockdown by antisense nucleic acid of the compound of Example 2 in STC-1 cells
  • STC-1 cells (2.0 ⁇ 10 5 cells / well, RPMI medium-high glucose) were seeded on a 6-well plate. The next day, the medium was changed from RPMI-high glucose to DMEM-high glucose, and the cells were cultured for another 24 hours.
  • Add 1 ⁇ L each of 100 ⁇ M negative control antisense nucleic acid (AmNA-NEG # 26, L26) or antisense nucleic acid targeting the SRRM4 gene (AmNA-7174, L21) stir lightly, and allow to stand for about 10 minutes (final). Concentration 100 nM).
  • L26 is a gapmer-type oligonucleic acid that does not have the knock-down activity of the SRRM4 gene and has a non-selective control sequence
  • L21 is a gapmer-type antisense oligo that has the knock-down activity of the SRRM4 gene. It is a nucleic acid. 2 ⁇ L of 30 mM test compound was added, and the cells were cultured for 6 hours or 24 hours (final concentration 30 ⁇ M). Extraction of Total RNA and measurement of the knockdown effect of SRRM4 gene were performed according to the qRT-PCR method described in M. Shimojo et al., Scientific Reports (2019) 9:7618.
  • hSRRM4 forward 5'-tgacaaagacttgacaccacc-3'(SEQ ID NO: 7); hSRRM4 reverse: 5'-acctgcgtcgcttgtgttt-3' (SEQ ID NO: 8); ⁇ actin forward: 5'- ggccgtcttccctccatcg-3'(SEQ ID NO: 9); ⁇ actin reverse: 5'-ccagttggtgacgatgccgtgc-3' (SEQ ID NO: 10).
  • the results are shown in FIG.
  • the left figure of FIG. 8 shows the total RNA amount 6 hours after the administration of the test compound, and the right figure shows the total RNA amount 24 hours after the administration of the test compound.
  • the compound of Example 2 induced cleavage of the SRRM4 gene by antisense nucleic acid.
  • Test Example 5 Increased expression of MALAT-1 by the compound of Example 2 in A549 cells
  • A549 cells (5.0 ⁇ 10 3 cells / well, DMEM medium) were seeded on 96-well plates and cultured for 1 day. The medium was removed and washed with 120 ⁇ L of PBS solution, and 50 ⁇ L of DMEM medium was added thereto. Further, 50 ⁇ L of DMEM medium containing DMSO (final concentration 0.1%) or the compound of Example 2 (final concentration 3,10,30 ⁇ M) was added, and the cells were cultured for 24 hours.
  • RNA is extracted using SuperPrep® Cell Lysis & RT Kit for qPCR (TOYOBO) according to the instructions for use and transcribed into cDNA (priming at 25 ° C for 10 minutes for reverse transcription). It was performed at 25 ° C for 40 minutes and for inactivation at 85 ° C for 5 minutes). The obtained cDNA was retained at 4 ° C. Gene amplification was performed using StepOnePlus TM Real-Time PCR Systems (Thermo Fisher Scientific).
  • hGAPDH forward 5'-gagtcaacggatttggtcgt-3'(SEQ ID NO: 11); hGAPDH reverse: 5'-gacaagcttcccgttctcag-3' (SEQ ID NO: 12), hMALAT1 forward: 5' -aagcaaggtctccccacaag -3'(SEQ ID NO: 13); hMALAT1reverse: 5'-gcccacaaggatccaagcta-3' (SEQ ID NO: 14).
  • MALAT-1 Metal Absorption Associated Lung Adenocarcinoma Transcript 1
  • lncRNA long non-coding RNA
  • autophagy activation for example, LncRNA expression profile during autophagy and Malat1 function in macrophages, Z. Ma. et al., P ONE 14: e0221104 (2019)
  • the compound of Example 2 increased the expression of MALAT-1 in A549 cells, it is considered to activate autophagy.
  • Test Example 6 Enhancement of MALAT-1 knockdown by antisense nucleic acid of the compound of Example 2 in A549 cells
  • A549 cells (5.0 ⁇ 10 3 cells / well, DMEM medium) were seeded on 96-well plates and cultured for 1 day. The medium was removed and washed with 120 ⁇ L of PBS solution, and 50 ⁇ L of DMEM medium was added thereto. Add 50 ⁇ L of DMEM medium containing antisense nucleic acid targeting MALAT-1 (final concentration 80 nM) and DMSO (final concentration 0.1%) or test compound (final concentration 3,10,30 ⁇ M) for 24 hours or It was cultured for 48 hours.
  • ASO antisense nucleic acid targeting MALAT-1
  • (L) represents LNA.
  • ASO for MALAT-1 (SEQ ID NO: 15) Name: MALAT-1-5714-LNA (15) Array (5' ⁇ 3'): T (L) ⁇ A (L) ⁇ G (L) ⁇ t ⁇ t ⁇ g ⁇ g ⁇ c ⁇ a ⁇ t ⁇ c ⁇ a ⁇ A (L) ⁇ G (L) ) ⁇ g
  • ASO for negative control SEQ ID NO: 16) Name: NEG # L11 Array (5' ⁇ 3'): G (L) ⁇ A (L) ⁇ G (L) ⁇ c ⁇ a ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ c ⁇ G (L) ⁇ T (L) ) ⁇ g Ex
  • the compound of Example 2 enhanced the cleavage activity of long non-coding RNA (MALAT-1) by antisense nucleic acid in a time-dependent manner.
  • MALAT-1 long non-coding RNA
  • activation of TRPC3 channel, TRPC6 channel, or TRPC7 channel promotes intracellular uptake of extracellular nucleic acid molecules via endosomes, followed by endosome nucleic acid molecules generated by autophagy activation. It was considered that the escape of endosomes was promoted and the nucleic acid molecule activity exerted in the cytoplasm was enhanced.
  • Test Example 7 Effect of the compound of Example 2 on the proliferation of A549 cells
  • A549 cells (3.0 ⁇ 10 3 cells / well, DMEM medium) were inoculated on 96-well plates and cultured for 1 day.
  • 50 ⁇ L of DMEM medium containing DMSO (final concentration 0.1%) or test compound (final concentration 0.1,1,3,10,30 ⁇ M) was placed in each well and cultured for 2 days.
  • Hoechst 33342 and Propidium Iodide (PI) (dojindo) were diluted with phosphate buffered saline (PBS), dropped onto the medium, and cultured for 30 minutes.
  • PBS phosphate buffered saline
  • the compound of Example 2 having an action of activating TRPC3 channel, TRPC6 channel, and TRPC7 channel does not have an action of promoting the growth of cancer cells, and suppresses it at a concentration of 10 ⁇ M or more. Therefore, it can be used as a concomitant drug for nucleic acid drugs in cancer treatment.
  • the present invention can be used in fields where intracellular uptake of nucleic acid molecules is expected, such as pharmaceuticals and nucleic acid molecule uptake tests.

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Abstract

A purpose of the present invention is to provide an agent for promoting the uptake of nucleic acid molecules into cells.　The present invention relates to, inter alia, an agent for promoting the uptake of nucleic acid molecules into cells, the agent containing a substance that activates at least one channel selected from the group consisting of TRPC3 channels, TRPC6 channels, and TRPC7 channels, or a pharmaceutically acceptable salt thereof, or a prodrug of these.

Description

細胞内への核酸分子取り込み促進剤、医薬組成物、及び新規化合物Nucleic acid molecule uptake promoter, pharmaceutical composition, and novel compound into cells

　本発明は、細胞外の核酸分子を細胞内への取り込みを促進するための剤、医薬組成物、新規化合物、細胞のオートファジーを増強するための剤、核酸分子の活性を増強するための剤等に関する。 The present invention relates to an agent for promoting the uptake of extracellular nucleic acid molecules into cells, a pharmaceutical composition, a novel compound, an agent for enhancing autophagy of cells, and an agent for enhancing the activity of nucleic acid molecules. Etc.

　核酸医薬は、低分子医薬、抗体医薬に続く、新たな医薬として研究及び開発されている。分子標的治療に使用される医薬としては抗体が良く知られているが、その多くは細胞表面のエピトープを標的にするものである。これに対し、核酸医薬では、理論上は細胞質、核といった細胞内の核酸等を標的にできると考えられている。しかし、実際には、細胞外の核酸分子を、対象とする細胞の内部へ取り込めるケースは少ないという問題があった。 Nucleic acid drugs are being researched and developed as new drugs following small molecule drugs and antibody drugs. Antibodies are well known as drugs used in molecular targeted therapies, but most of them target epitopes on the cell surface. On the other hand, nucleic acid drugs are theoretically considered to be able to target intracellular nucleic acids such as cytoplasm and nucleus. However, in reality, there is a problem that there are few cases where extracellular nucleic acid molecules can be taken into the inside of a target cell.

　Transient Receptor Potentialチャネル（本明細書中、TRPチャネルとも称する。）は基本的に細胞膜において陽イオンチャネルとして機能する。TRPチャネルとしては28のサブタイプが知られており、その多くは活性化されると細胞外のカルシウムイオンを細胞内へ取り込む。細胞内へカルシウムイオンを取り込むことによって細胞外の環境変化が細胞内に伝達される。TRPチャネルのうちTRPCチャネルを活性化する化合物として下に示される構造を有するPPZ1及びPPZ2が知られている（非特許文献１）。PPZ1及びPPZ2はTRPチャネルのうち、TRPC3チャネル、TRPC6チャネル、TRPC7チャネル（本明細書中、それぞれ、TRPC3、TRPC6、TRPC7とも略称する。）を活性化する。また、カンナビジオール（Cannabidiol）もTRPV1チャネル、TRPV2チャネル及びTRPA1チャネルを活性化することが知られている（非特許文献２）。さらに、GSK1702934A（1,3-Dihydro-1-[1-[(5,6,7,8-tetrahydro-4H-cyclohepta[b]thien-2-yl)carbonyl]-4-piperidinyl]-2H-benzimidazol-2-one）もTRPC3チャネル及びTRPC6チャネルを活性化することが知られている（非特許文献３）。 The Transient Receptor Potential channel (also referred to as a TRP channel in the present specification) basically functions as a cation channel in the cell membrane. Twenty-eight subtypes of TRP channels are known, many of which take up extracellular calcium ions into the cell when activated. By taking calcium ions into the cell, extracellular environmental changes are transmitted into the cell. Among the TRP channels, PPZ1 and PPZ2 having the structures shown below are known as compounds that activate TRPC channels (Non-Patent Document 1). PPZ1 and PPZ2 activate TRPC3 channel, TRPC6 channel, and TRPC7 channel (also abbreviated as TRPC3, TRPC6, and TRPC7, respectively in the present specification) among TRP channels. Cannabidiol is also known to activate TRPV1 channels, TRPV2 channels and TRPA1 channels (Non-Patent Document 2). In addition, GSK1702934A (1,3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazole -2-one) is also known to activate TRPC3 and TRPC6 channels (Non-Patent Document 3).

Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008

S. Sawamura et al., Mol. Pharmacol., 89, 348-363 (2016)S. Sawamura et al., Mol. Pharmacol., 89, 348-363 (2016) N. Qin et al., J. Neurosci. 28, 6231-6238 (2008)N. Qin et al., J. Neurosci. 28, 6231-6238 (2008) B. Doleschal et al., Cardiovascular Res., 106, 163-173 (2015)B. Doleschal et al., Cardiovascular Res., 106, 163-173 (2015)

　細胞内への核酸分子取り込み促進するための剤の提供を一つの課題とする。新規化合物等の提供を一つの課題とする。核酸分子の細胞内への取り込みを促進する物質を含む医薬組成物等の提供を一つの課題とする。細胞のオートファジーを増強するための剤の提供を一つの課題とする。核酸分子の活性を増強するための剤の提供を一つの課題とする。 One issue is to provide an agent for promoting the uptake of nucleic acid molecules into cells. One issue is the provision of new compounds. One issue is to provide a pharmaceutical composition or the like containing a substance that promotes the uptake of nucleic acid molecules into cells. One of the challenges is to provide a drug for enhancing cell autophagy. One issue is to provide an agent for enhancing the activity of a nucleic acid molecule.

　本発明者らは、上記課題を解決すべく、鋭意検討した結果、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネル（好適にはTRPC6チャネル）を活性化する物質（例えば後述の式（１）で表される化合物）等が、核酸分子の細胞内への取り込みを促進すること、細胞のオートファジーを増強すること、核酸分子の活性を増強すること、カンナビジオールがTRPC6チャネル活性化作用を有すること、等を見出し、本発明を完成した。代表的な本発明は以下の通りである。 As a result of diligent studies to solve the above problems, the present inventors activate at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. A substance (for example, a compound represented by the formula (1) described later) promotes the uptake of nucleic acid molecules into cells, enhances cell autophagy, enhances the activity of nucleic acid molecules, and cannavi. The present invention was completed by finding that the diol has a TRPC6 channel activating effect and the like. Typical inventions are as follows.

　項１．
TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有し、細胞内への核酸分子の取り込みを促進するための、オートファジーを増強するための、又は、核酸分子活性を増強するための剤。
　項２．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物である、項１に記載の剤。

Figure JPOXMLDOC01-appb-C000009
［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］
　項３．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、フッ素原子、塩素原子、臭素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ５アシルオキシ基、Ｃ２～Ｃ５アルコキシカルボニル基、又はトリハロゲノメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～４の整数を示し、
Ｒ２は、フッ素原子；塩素原子；臭素原子；トリフルオロメチル基；トリクロロメチル基；トリブロモメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ５アシルオキシ基；Ｃ２～Ｃ５アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～４の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す、
項２に記載の剤。
　項４．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、フッ素原子、塩素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、トリクロロメチル基、又はトリブロモメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～３の整数を示し、
Ｒ２は、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アシルオキシ基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～３の整数を示し、
ｐは、１を示し、
ｑは、１を示す、
項２又は３に記載の剤。
　項５．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、塩素原子、Ｃ１～Ｃ３アルキル基、Ｃ１～Ｃ３アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、又はトリクロロメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～２の整数を示し、
Ｒ２は、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～３の整数を示し、
ｐは、１を示し、
ｑは、１を示す、
項２～４のいずれか一項に記載の剤。
　項６．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、以下に示された化合物の１種又は２種以上である、項１又は２に記載の剤。
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
 　項７．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルがTRPC6チャネルである、項１～６のいずれか一項に記載の剤。
　項８．
前記細胞が、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを発現する細胞である、項１～７のいずれか一項に記載の剤。
　項９．
前記細胞が、核酸分子の標的細胞である、項１～８のいずれか一項に記載の剤。
　項１０．
前記細胞が、小細胞肺がん細胞、非小細胞肺がん細胞、前立腺がん細胞、乳がん細胞、筋細胞又は神経細胞である、項１～９のいずれか一項に記載の剤。
　項１１．
核酸分子の投与が期待される疾患の予防又は治療用である項１～１０のいずれか一項に記載の剤。
　項１２．
肺がん、前立腺がん、乳がん、肺線維症、腎線維症、筋ジストロフィー、筋萎縮性側索硬化症、ハンチントン病、筋萎縮症又はパーキンソン病の予防又は治療用である項１～１１のいずれか一項に記載の剤。
　項１３．
TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグ、及び薬学的に許容される担体を含有し、（ａ）核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグをさらに含有する、或いは（ｂ）核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグを含有する薬剤とともに組み合わせて処方される、
医薬組成物。
　項１４．
前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、下記式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物である、項１３に記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000012
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］
　項１５．
核酸分子が、small interfering RNA(siRNA)、micro RNA、antagomir、small hairpin RNA(shRNA)、guide RNA(gRNA)、アンチセンス核酸、リボザイム、デコイ核酸、スプライススィッチング核酸、mRNA、及びプラスミドからなる群から選択される少なくとも１種である、項１３又は１４に記載の医薬組成物。
　項１６．
式（１－１）
Figure JPOXMLDOC01-appb-C000013
 ［式中、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示す。］
で表わされる化合物、
式（１－２）
Figure JPOXMLDOC01-appb-C000014
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ａ及びＢは、同時に炭素原子を示さず、
Ｒ３は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示す。］
で表わされる化合物、
それらの塩、又はそれらのプロドラッグ。
　項１７．
式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有し、細胞内への核酸分子の取り込みを促進するための、オートファジーを増強するための、又は核酸分子活性を増強するための剤。
Figure JPOXMLDOC01-appb-C000015
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］ Item 1.
Nucleic acid intracellularly containing a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof. An agent for promoting the uptake of molecules, enhancing autophagy, or enhancing nucleic acid molecule activity.
Item 2.
The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is the compound represented by the formula (1), cannabidiol, and 1,3-dihydro-1. -[1-[(5,6,7,8-tetrahydro-4H-cycloheptane [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one selected from the group Item 2. The agent according to Item 1, which is at least one compound.
Figure JPOXMLDOC01-appb-C000009
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m represents an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ]
Item 3.
The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A is a carbon atom or a nitrogen atom. Show,
B represents a carbon atom or a nitrogen atom.
R1 represents a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and when there are a plurality of them, May be the same or different,
m represents an integer from 0 to 4 and represents
R2 is a fluorine atom; a chlorine atom; a bromine atom; a trifluoromethyl group; a trichloromethyl group; a tribromomethyl group; a C1-C4 alkyl group; a C2-C5 acyloxy group; a C2-C5 alkoxycarbonyl group; a C1-C3 alkyl. An amide group which may be substituted with one or two groups selected from the group consisting of a group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Piperidinylcarbonyl group optionally substituted with one or more groups; or substituted with one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Indicates a pyrrolidinylcarbonyl group which may be the same or different if there are more than one.
n represents an integer from 0 to 4 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1,
Item 2. The agent according to Item 2.
Item 4.
The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a fluorine atom, a chlorine atom, a C1 to C4 alkyl group, a C1 to C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group, and when there are a plurality of them. May be the same or different,
m indicates an integer from 0 to 3 and represents
R2 is a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 acyloxy group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. An amide group which may be substituted with one or two groups selected from the group consisting of; one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Piperidinylcarbonyl group optionally substituted with; or pyrrolidinylcarbonyl optionally substituted with one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Indicates a group, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 3 and represents
p indicates 1,
q indicates 1.
Item 2. The agent according to Item 2 or 3.
Item 5.
The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R1 represents a chlorine atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, or a trichloromethyl group, and when there are a plurality of them, they may be the same or different.
m indicates an integer of 0 to 2 and represents
R2 is selected from the group consisting of a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. May be substituted with one or two groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. It shows a good piperidinylcarbonyl group and may be the same or different if there are more than one.
n represents an integer from 0 to 3 and represents
p indicates 1,
q indicates 1.
The agent according to any one of Items 2 to 4.
Item 6.
Item 1 or 2, wherein the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is one or more of the compounds shown below. The listed agent.
Figure JPOXMLDOC01-appb-C000010
Figure JPOXMLDOC01-appb-C000011
 Item 7.
Item 6. The agent according to any one of Items 1 to 6, wherein at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is TRPC6 channel.
Item 8.
Item 6. The agent according to any one of Items 1 to 7, wherein the cell expresses at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel.
Item 9.
Item 6. The agent according to any one of Items 1 to 8, wherein the cell is a target cell of a nucleic acid molecule.
Item 10.
Item 6. The agent according to any one of Items 1 to 9, wherein the cells are small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells or nerve cells.
Item 11.
Item 8. The agent according to any one of Items 1 to 10, which is used for the prevention or treatment of a disease for which administration of a nucleic acid molecule is expected.
Item 12.
Any one of Items 1 to 11 for the prevention or treatment of lung cancer, prostate cancer, breast cancer, pulmonary fibrosis, renal fibrosis, muscular dystrophy, amyotrophic lateral sclerosis, Huntington's disease, muscle atrophy or Parkinson's disease. The agent described in the section.
Item 13.
A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier. And (a) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or (b) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof. Prescribed in combination with drugs containing
Pharmaceutical composition.
Item 14.
The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the following formula (1), cannabidiol, and 1,3-dihydro-. Select from the group consisting of 1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one. Item 3. The pharmaceutical composition according to Item 13, which is at least one compound thereof.
Figure JPOXMLDOC01-appb-C000012
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m represents an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ]
Item 15.
Nucleic acid molecule consists of a group consisting of small interfering RNA (siRNA), micro RNA, antogomir, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme, decoy nucleic acid, splice switching nucleic acid, mRNA, and plasmid. Item 6. The pharmaceutical composition according to Item 13 or 14, which is at least one selected.
Item 16.
Equation (1-1)
Figure JPOXMLDOC01-appb-C000013
 [During the ceremony,
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m represents an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer of 0 to 5. ]
Compound represented by,
Equation (1-2)
Figure JPOXMLDOC01-appb-C000014
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
A and B do not show carbon atoms at the same time,
R 3 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer of 0 to 5. ]
Compound represented by,
Their salts, or their prodrugs.
Item 17.
The compound represented by the formula (1), benzimidazole, and 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl). ] -4-Piperidinyl] -Contains at least one compound selected from the group consisting of -2H-benzimidazol-2-one, a pharmaceutically acceptable salt thereof, or a prodrug thereof and into the cell. An agent for promoting the uptake of nucleic acid molecules, enhancing autophagy, or enhancing nucleic acid molecule activity.
Figure JPOXMLDOC01-appb-C000015
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m indicates an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ]

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質は、細胞外の核酸分子を細胞内へ取り込むことを促進し核酸分子の活性を増強する作用を有するため、細胞内への核酸分子取り込み促進剤、細胞のオートファジー増強剤、核酸分子活性増強剤等として使用できる。式（１）、（１－１）、又は（１－２）で表わされる化合物はTRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネル（好適にはTRPC6チャネル）を活性化して、細胞外のカルシウムイオンを細胞内へ流入させるため、細胞内への核酸分子取り込み促進剤、細胞のオートファジー増強剤、核酸分子活性増強剤等として使用できる。 A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel acts to promote the uptake of extracellular nucleic acid molecule into the cell and enhance the activity of the nucleic acid molecule. Therefore, it can be used as an intracellular nucleic acid molecule uptake promoter, a cell autophagy enhancer, a nucleic acid molecule activity enhancer, and the like. The compound represented by the formula (1), (1-1), or (1-2) is at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. Can be used as an intracellular nucleic acid molecule uptake promoter, a cell autophagy enhancer, a nucleic acid molecule activity enhancer, and the like because it activates and causes extracellular calcium ions to flow into the cell.

図１は、種々の細胞におけるTRPC6チャネル、TRPC3チャネル、TRPC7チャネル及びSRRM4遺伝子発現相対量（試験例２）を示すグラフである。FIG. 1 is a graph showing relative amounts of TRPC6 channel, TRPC3 channel, TRPC7 channel and SRRM4 gene expression in various cells (Test Example 2). 図２は、実施例２の化合物（60μM）存在下（右図）及び非存在下（左図）でSTC-1細胞から測定された蛍光強度（細胞内Ca２＋濃度）の推移を示す（試験例３）。下図は、細胞内へのCa２＋流入量を示す。FIG. 2 shows the transition of the fluorescence intensity (intracellular Ca 2+ concentration) measured from STC-1 cells in the presence (right figure) and in the absence (left figure) of the compound (60 μM) of Example 2 (test). Example 3). The figure below shows the amount of Ca 2+ influx into the cell. 図３は、実施例２の化合物（60μM）存在下（右図）及び非存在下（左図）でSTC-1細胞から測定された蛍光強度（細胞内への核酸取り込み量）を示す（試験例３）。下図は、細胞内への核酸取り込み量を示す。FIG. 3 shows the fluorescence intensity (nucleic acid uptake into cells) measured from STC-1 cells in the presence (right figure) and in the absence (left figure) of the compound (60 μM) of Example 2 (test). Example 3). The figure below shows the amount of nucleic acid taken up into cells. 図４は、蛍光色素共役アンチセンス核酸に由来する蛍光波長を撮影した写真である（試験例３）。左図は実施例２の化合物添加直後の写真であり、右図は実施例２の化合物添加１０分後の写真である。FIG. 4 is a photograph of the fluorescence wavelength derived from the fluorescent dye-conjugated antisense nucleic acid (Test Example 3). The left figure is a photograph immediately after the compound addition of Example 2, and the right figure is a photograph 10 minutes after the compound addition of Example 2. 図５は、カンナビジオール（6μM）存在下で測定された蛍光強度（細胞内Ca２＋濃度）の推移を示す（試験例３）。FIG. 5 shows the transition of the fluorescence intensity (intracellular Ca 2+ concentration) measured in the presence of cannabidiol (6 μM) (Test Example 3). 図６は、カンナビジオール（6μM）存在下で測定された蛍光強度（細胞内への核酸取り込み量）の推移を示す（試験例３）。左図は、2 mMCa2+溶液存在下を表わし、右図は、Ca2+非含有溶液存在下を表わす。FIG. 6 shows the transition of the fluorescence intensity (amount of nucleic acid incorporated into cells) measured in the presence of cannabidiol (6 μM) (Test Example 3). The figure on the left shows the presence of a 2 mM Ca 2+ solution, and the figure on the right shows the presence of a solution containing no Ca 2+ . 図７は、カンナビジオール（6μM）存在下で蛍光色素共役アンチセンス核酸に由来する蛍光波長を撮影した写真である（試験例３）。左図はカンナビジオール添加直後の写真であり、右図はカンナビジオール添加１０分後の写真である。FIG. 7 is a photograph of the fluorescence wavelength derived from the fluorescent dye-conjugated antisense nucleic acid in the presence of cannabidiol (6 μM) (Test Example 3). The left figure is a photograph immediately after the addition of cannabidiol, and the right figure is a photograph 10 minutes after the addition of cannabidiol. 図８は、試験例４で測定された実施例２の化合物添加６時間後（左図）及び２４時間後（右図）のSRRM4 mRNAの相対量を表わすグラフである。FIG. 8 is a graph showing the relative amount of SRRM4 mRNA after 6 hours (left figure) and 24 hours (right figure) of compound addition of Example 2 measured in Test Example 4. 図９は、A549細胞における実施例２の化合物によるMALAT-1発現相対量を表わすグラフである（試験例５）。FIG. 9 is a graph showing the relative amount of MALAT-1 expression by the compound of Example 2 in A549 cells (Test Example 5). 図１０は、A549細胞における実施例２の化合物の添加24時間後（上図）及び48時間後（下図）の、アンチセンス核酸によるMALAT-1切断活性の増強効果を表すグラフである（試験例６）。FIG. 10 is a graph showing the effect of antisense nucleic acid on enhancing MALAT-1 cleavage activity 24 hours (upper figure) and 48 hours (lower figure) after the addition of the compound of Example 2 in A549 cells (Test Example). 6). 図１１は、実施例２の化合物がA549細胞の増殖に与える影響を表すグラフである（試験例７）。上図は相対細胞数を表し、下図は生細胞数の割合を表す。FIG. 11 is a graph showing the effect of the compound of Example 2 on the proliferation of A549 cells (Test Example 7). The upper figure shows the relative number of cells, and the lower figure shows the ratio of the number of living cells.

　本発明において「ハロゲン原子」としては、フッ素原子、塩素原子、臭素原子およびヨウ素原子が挙げられる。好ましくはフッ素原子、塩素原子、及び臭素原子、より好ましくはフッ素原子、及び塩素原子が挙げられる。 Examples of the "halogen atom" in the present invention include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Fluorine atoms, chlorine atoms, and bromine atoms are preferable, and fluorine atoms and chlorine atoms are more preferable.

　本発明において「アルキル基」としては、例えば、直鎖状、分枝状、又は環状構造を含む、Ｃ１～Ｃ８アルキル基が挙げられ、好ましくはＣ１～Ｃ６アルキル基、より好ましくはＣ１～Ｃ４アルキル基、特に好ましくはＣ１～Ｃ３アルキル基である。具体的には、直鎖状または分枝状のアルキル基としては、メチル基、エチル基、１－プロピル基、２－プロピル基、１－ブチル基、２－ブチル基、イソブチル基、ｔｅｒｔ－ブチル基、ｎ－ペンチル基、ネオペンチル基、ｎ－ヘキシル基、イソヘキシル基、３－メチルペンチル基等が挙げられ、環状構造を含むアルキル基としては、シクロプロピル基、シクロプロピルメチル基、シクロブチル基、シクロブチルメチル基、シクロペンチル基、シクロペンチルメチル基、シクロヘキシル基、シクロヘキシルメチル基、シクロヘキシルエチル基等が挙げられる。好ましくは、メチル基、エチル基、２－プロピル基、ｔ－ブチル基、シクロプロピル基等が挙げられる。 Examples of the "alkyl group" in the present invention include C1 to C8 alkyl groups including linear, branched or cyclic structures, preferably C1 to C6 alkyl groups, and more preferably C1 to C4 alkyl. A group, particularly preferably a C1-C3 alkyl group. Specifically, the linear or branched alkyl group includes a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 1-butyl group, a 2-butyl group, an isobutyl group, and a tert-butyl. Examples include a group, an n-pentyl group, a neopentyl group, an n-hexyl group, an isohexyl group, a 3-methylpentyl group, and the like, and examples of the alkyl group containing a cyclic structure include a cyclopropyl group, a cyclopropylmethyl group, a cyclobutyl group, and a cyclo. Examples thereof include a butylmethyl group, a cyclopentyl group, a cyclopentylmethyl group, a cyclohexyl group, a cyclohexylmethyl group, and a cyclohexylethyl group. Preferred examples include a methyl group, an ethyl group, a 2-propyl group, a t-butyl group, a cyclopropyl group and the like.

　本発明において「ハロゲン原子で置換されたアルキル基」としては、例えば、ハロゲン原子で置換された、直鎖状、分枝状、又は環状構造を含む、前記アルキル基が挙げられる。アルキル基が有する置換基の数は１～置換可能な最大の数であり、好ましくは３～置換可能な最大の数である。当該ハロゲン原子が複数あるときは同一でも異なってもよいが、同一であることが好ましい。例えば、置換可能な最大の数のハロゲン原子で置換されたＣ１～Ｃ４アルキル基が挙げられ、好ましくは置換可能な最大の数のハロゲン原子で置換されたＣ１～Ｃ３アルキル基、より好ましくは置換可能な最大の数のハロゲン原子で置換されたＣ１～Ｃ２アルキル基、特に好ましくはトリハロゲノメチル基である。ハロゲン原子で置換されたアルキル基の例としては、パーフルオロ－ｎ－ブチル基、パーフルオロ－ｔ－ブチル基、パーフルオロ－ｎ－プロピル基、パーフルオロ－ｉ－プロピル基、ペンチルフルオロエチル基、トリフルオロメチル基、パークロロ－ｎ－ブチル基、パークロロ－ｔ－ブチル基、パークロロ－ｎ－プロピル基、パークロロ－ｉ－プロピル基、ペンタクロロエチル基、トリクロロメチル基、パーブロモ－ｎ－ブチル基、パーブロモ－ｔ－ブチル基、パーブロモ－ｎ－プロピル基、パーブロモ－ｉ－プロピル基、ペンタブロモエチル基、トリブロモメチル基、パーヨード－ｎ－ブチル基、パーヨード－ｔ－ブチル基、パーヨード－ｎ－プロピル基、パーヨード－ｉ－プロピル基、ペンタヨードエチル基、トリヨードメチル基が挙げられる。ハロゲン原子で置換されたアルキル基の好ましい例としては、ペンチルフルオロエチル基、トリフルオロメチル基、ペンタクロロエチル基、トリクロロメチル基、ペンタブロモエチル基、トリブロモメチル基、ペンタヨードエチル基、トリヨードメチル基が挙げられ、トリフルオロメチル基、トリクロロメチル基、トリブロモメチル基がより好ましく、トリフルオロメチル基、トリクロロメチル基がより一層好ましい。 Examples of the "alkyl group substituted with a halogen atom" in the present invention include the alkyl group comprising a linear, branched or cyclic structure substituted with a halogen atom. The number of substituents contained in the alkyl group is 1 to the maximum number that can be substituted, and preferably 3 to the maximum number that can be substituted. When there are a plurality of the halogen atoms, they may be the same or different, but they are preferably the same. For example, C1-C4 alkyl groups substituted with the maximum number of replaceable halogen atoms can be mentioned, preferably C1-C3 alkyl groups substituted with the maximum number of replaceable halogen atoms, more preferably substitutable. A C1-C2 alkyl group substituted with the largest number of halogen atoms, particularly preferably a trihalogenomethyl group. Examples of alkyl groups substituted with halogen atoms include perfluoro-n-butyl group, perfluoro-t-butyl group, perfluoro-n-propyl group, perfluoro-i-propyl group, pentylfluoroethyl group, Trifluoromethyl group, perchloro-n-butyl group, perchloro-t-butyl group, perchloro-n-propyl group, perchloro-i-propyl group, pentachloroethyl group, trichloromethyl group, perbromo-n-butyl group, perbromo -T-butyl group, perbromo-n-propyl group, perbromo-i-propyl group, pentabromoethyl group, tribromomethyl group, periodo-n-butyl group, periodo-t-butyl group, periodo-n-propyl group , Periodo-i-propyl group, pentaiodoethyl group, triiodomethyl group and the like. Preferred examples of the alkyl group substituted with the halogen atom are a pentylfluoroethyl group, a trifluoromethyl group, a pentachloroethyl group, a trichloromethyl group, a pentabromoethyl group, a tribromomethyl group, a pentaiodoethyl group and a triiode. Examples thereof include a methyl group, a trifluoromethyl group, a trichloromethyl group and a tribromomethyl group are more preferable, and a trifluoromethyl group and a trichloromethyl group are even more preferable.

　本発明において「アルコキシ基」としては、例えば、直鎖状、分枝状、又は環状構造を含む、Ｃ１～Ｃ８アルコキシ基が挙げられ、好ましくはＣ１～Ｃ６アルコキシ基、より好ましくはＣ１～Ｃ４アルコキシ基、特に好ましくはＣ１～Ｃ３アルコキシ基である。具体的には、直鎖状または分枝状のアルコキシ基としては、メトキシ基、エトキシ基、１－プロポキシ基、２－プロポキシ基、１－ブトキシ基、２－ブトキシ基、イソブトキシ基、ｔｅｒｔ－ブトキシ基、ｎ－ペンチルオキシ基、ネオペンチルオキシ基、ｎ－ヘキシルオキシ基、イソヘキシルオキシ基、３－メチルペンチルオキシ基等が挙げられる。環状構造を含むアルコキシ基としては、シクロプロポキシ基、シクロプロピルメトキシ基、シクロブチロキシ基、シクロブチルメトキシ基、シクロペンチロキシ基、シクロペンチルメトキシ基、シクロヘキシロキシ基、シクロヘキシルメトキシ基、シクロヘキシルエトキシ基等が挙げられる。好ましくは、メトキシ基、エトキシ基、２－プロポキシ基、ｔｅｒｔ－ブトキシ基、シクロプロポキシ基等が挙げられる。 Examples of the "alkoxy group" in the present invention include C1 to C8 alkoxy groups including linear, branched or cyclic structures, preferably C1 to C6 alkoxy groups, and more preferably C1 to C4 alkoxy. Groups, particularly preferably C1-C3 alkoxy groups. Specifically, the linear or branched alkoxy group includes a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, a 1-butoxy group, a 2-butoxy group, an isobutoxy group, and a tert-butoxy. Examples thereof include a group, an n-pentyloxy group, a neopentyloxy group, an n-hexyloxy group, an isohexyloxy group, a 3-methylpentyloxy group and the like. Examples of the alkoxy group containing a cyclic structure include a cyclopropoxy group, a cyclopropylmethoxy group, a cyclobutyloxy group, a cyclobutylmethoxy group, a cyclopentyloxy group, a cyclopentylmethoxy group, a cyclohexyloxy group, a cyclohexylmethoxy group and a cyclohexylethoxy group. Can be mentioned. Preferred examples include a methoxy group, an ethoxy group, a 2-propoxy group, a tert-butoxy group, a cyclopropoxy group and the like.

　本発明において「アシルオキシ基」としては、例えば、直鎖状、分枝状、又は環状構造を含む、Ｃ２～Ｃ９アシルオキシ基が挙げられ、好ましくはＣ２～Ｃ７アシルオキシ基、より好ましくはＣ２～Ｃ５アシルオキシ基、特に好ましくはＣ２～Ｃ４アシルオキシ基である。具体的には、直鎖状または分枝状のアシルオキシ基としては、メトキシカルボニル基、エトキシカルボニル基、１－プロポキシカルボニル基、２－プロポキシカルボニル基、１－ブトキシカルボニル基、２－ブトキシカルボニル基、イソブトキシカルボニル基、ｔｅｒｔ－ブトキシカルボニル基、ｎ－ペンチルオキシカルボニル基、ネオペンチルオキシカルボニル基、ｎ－ヘキシルオキシカルボニル基、イソヘキシルオキシカルボニル基、３－メチルペンチルオキシカルボニル基等が挙げられる。環状構造を含むアシルオキシ基としては、シクロプロポキシカルボニル基、シクロプロピルメトキシカルボニル基、シクロブチロキシカルボニル基、シクロブチルメトキシカルボニル基、シクロペンチロキシカルボニル基、シクロペンチルメトキシカルボニル基、シクロヘキシロキシカルボニル基、シクロヘキシルメトキシカルボニル基、シクロヘキシルエトキシカルボニル基等が挙げられる。好ましくは、メトキシカルボニル基、エトキシカルボニル基、２－プロポキシカルボニル基、ｔｅｒｔ－ブトキシカルボニル基、シクロプロポキシカルボニル基等が挙げられる。 In the present invention, examples of the "acyloxy group" include C2-C9 acyloxy groups containing a linear, branched or cyclic structure, preferably C2-C7 acyloxy groups, and more preferably C2-C5 acyloxy groups. A group, particularly preferably a C2-C4 acyloxy group. Specifically, the linear or branched acyloxy group includes a methoxycarbonyl group, an ethoxycarbonyl group, a 1-propoxycarbonyl group, a 2-propoxycarbonyl group, a 1-butoxycarbonyl group, a 2-butoxycarbonyl group, and the like. Examples thereof include an isobutoxycarbonyl group, a tert-butoxycarbonyl group, an n-pentyloxycarbonyl group, a neopentyloxycarbonyl group, an n-hexyloxycarbonyl group, an isohexyloxycarbonyl group and a 3-methylpentyloxycarbonyl group. Examples of the acyloxy group containing a cyclic structure include a cyclopropoxycarbonyl group, a cyclopropylmethoxycarbonyl group, a cyclobutyloxycarbonyl group, a cyclobutylmethoxycarbonyl group, a cyclopentyloxycarbonyl group, a cyclopentylmethoxycarbonyl group, a cyclohexyloxycarbonyl group and a cyclohexylmethoxy. Examples thereof include a carbonyl group and a cyclohexylethoxycarbonyl group. Preferred examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, a 2-propoxycarbonyl group, a tert-butoxycarbonyl group, a cyclopropoxycarbonyl group and the like.

　本発明において「アルコキシカルボニル基」としては、前記アルコキシ基と結合したカルボニル基が挙げられ、例えば直鎖状、分枝状、又は環状構造を含む、Ｃ１～Ｃ８アルコキシ基と結合したカルボニル基が挙げられ、好ましくはＣ２～Ｃ７アルコキシカルボニル基、より好ましくはＣ２～Ｃ５アルコキシカルボニル基、特に好ましくはＣ２～Ｃ４アルコキシカルボニル基である。アルコキシカルボニル基を構成する直鎖状または分枝状のアルコキシ基としては、メトキシ基、エトキシ基、１－プロポキシ基、２－プロポキシ基、１－ブトキシ基、２－ブトキシ基、イソブトキシ基、ｔｅｒｔ－ブトキシ基、ｎ－ペンチルオキシ基、ネオペンチルオキシ基、ｎ－ヘキシルオキシ基、イソヘキシルオキシ基、３－メチルペンチルオキシ基等が挙げられる。アルコキシカルボニル基を構成する環状構造を含むアルコキシ基としては、シクロプロポキシ基、シクロプロピルメトキシ基、シクロブチロキシ基、シクロブチルメトキシ基、シクロペンチロキシ基、シクロペンチルメトキシ基、シクロヘキシロキシ基、シクロヘキシルメトキシ基、シクロヘキシルエトキシ基等が挙げられる。アルコキシカルボニル基を構成するアルコキシ基としては、好ましくは、メトキシ基、エトキシ基、２－プロポキシ基、ｔｅｒｔ－ブトキシ基、シクロプロポキシ基等が挙げられる。 In the present invention, the "alkoxycarbonyl group" includes a carbonyl group bonded to the alkoxy group, and examples thereof include a carbonyl group bonded to a C1 to C8 alkoxy group including a linear, branched or cyclic structure. It is preferably a C2-C7 alkoxycarbonyl group, more preferably a C2-C5 alkoxycarbonyl group, and particularly preferably a C2-C4 alkoxycarbonyl group. The linear or branched alkoxy group constituting the alkoxycarbonyl group includes a methoxy group, an ethoxy group, a 1-propoxy group, a 2-propoxy group, a 1-butoxy group, a 2-butoxy group, an isobutoxy group, and a tert-. Examples thereof include a butoxy group, an n-pentyloxy group, a neopentyloxy group, an n-hexyloxy group, an isohexyloxy group, a 3-methylpentyloxy group and the like. Examples of the alkoxy group containing a cyclic structure constituting the alkoxycarbonyl group include a cyclopropoxy group, a cyclopropylmethoxy group, a cyclobutyloxy group, a cyclobutylmethoxy group, a cyclopentyroxy group, a cyclopentylmethoxy group, a cyclohexyloxy group, and a cyclohexylmethoxy group. , Cyclohexylethoxy group and the like. Preferred examples of the alkoxy group constituting the alkoxycarbonyl group include a methoxy group, an ethoxy group, a 2-propoxy group, a tert-butoxy group, a cyclopropoxy group and the like.

　本発明において「アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基」は、アミド基、モノ又はジ－アルキルアミド基、モノ又はジ－アルコキシカルボニルアミド基、アルキル基及びアルコキシカルボニル基でジ置換されたアミド基を包含し、好ましくはアミド基、ジアルキルアミド基、より好ましくはジアルキルアミド基である。アミド基が２つの基で置換される場合、置換基は同一でも異なってもよい。モノアルキルアミド基としては、前記アルキル基でモノ置換されたアミド基が挙げられ、好ましくはＣ１～Ｃ４アルキル基でモノ置換されたアミド基、より好ましくはＣ１～Ｃ３アルキル基でモノ置換されたアミド基が挙げられ、特に好ましくはメチルアミド基、エチルアミド基が挙げられる。ジアルキルアミド基としては、前記アルキル基でジ置換されたアミド基が挙げられ、好ましくはＣ１～Ｃ４アルキル基でジ置換されたアミド基、より好ましくはＣ１～Ｃ３アルキル基でジ置換されたアミド基、特に好ましくはジメチルアミド基、ジエチルアミド基、エチルメチルアミド基が挙げられる。モノアルコキシカルボニルアミド基としては、前記アルコキシカルボニル基でモノ置換されたアミド基が挙げられ、好ましくはＣ２～Ｃ５アルコキシカルボニル基でモノ置換されたアミド基、より好ましくはＣ２～Ｃ４アルコキシカルボニル基でモノ置換されたアミド基、特に好ましくはメトキシカルボニルアミド、エトキシカルボニルアミド基が挙げられる。ジアルコキシカルボニルアミド基としては、前記アルコキシカルボニル基でジ置換されたアミド基が挙げられ、好ましくはＣ２～Ｃ５アルコキシカルボニル基でジ置換されたアミド基、より好ましくはＣ２～Ｃ４アルコキシカルボニル基でジ置換されたアミド基、特に好ましくはジ（メトキシカルボニル）アミド、ジ（エトキシカルボニル）アミド基が挙げられる。アルキル基及びアルコキシカルボニル基で置換されたアミド基としては、前記アルキル基と前記アルコキシカルボニル基でジ置換されたアミド基が挙げられ、好ましくはＣ１～Ｃ４アルキル基とＣ２～Ｃ５アルコキシカルボニル基でジ置換されたアミド基、より好ましくはＣ１～Ｃ３アルキル基とＣ２～Ｃ４アルコキシカルボニル基でジ置換されたアミド基、特に好ましくはメチルメトキシカルボニルアミド基、メチルエトキシカルボニルアミド基、エチルメトキシカルボニルアミド基、エチルエトキシカルボニルアミド基が挙げられる。 In the present invention, the "amide group which may be substituted with one or two groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group" is an amide group, a mono or a di-alkylamide group, a mono or a di-. It includes an alkoxycarbonylamide group, an alkyl group and an amide group di-substituted with an alkoxycarbonyl group, preferably an amide group, a dialkylamide group, and more preferably a dialkylamide group. When the amide group is substituted with two groups, the substituents may be the same or different. Examples of the monoalkylamide group include an amide group mono-substituted with the alkyl group, preferably an amide group mono-substituted with a C1 to C4 alkyl group, and more preferably an amide mono-substituted with a C1 to C3 alkyl group. Groups are mentioned, and particularly preferably, a methylamide group and an ethylamide group are mentioned. Examples of the dialkylamide group include an amide group di-substituted with the alkyl group, preferably an amide group di-substituted with a C1 to C4 alkyl group, and more preferably an amide group di-substituted with a C1 to C3 alkyl group. Particularly preferred are a dimethylamide group, a diethylamide group and an ethylmethylamide group. Examples of the monoalkoxycarbonylamide group include an amide group mono-substituted with the alkoxycarbonyl group, preferably an amide group mono-substituted with a C2-C5 alkoxycarbonyl group, and more preferably a mono-alkoxycarbonyl group with a C2-C4 alkoxycarbonyl group. Substituted amide groups, particularly preferably methoxycarbonylamides and ethoxycarbonylamide groups can be mentioned. Examples of the dialkoxycarbonylamide group include an amide group di-substituted with the alkoxycarbonyl group, preferably an amide group di-substituted with a C2-C5 alkoxycarbonyl group, and more preferably a C2-C4 alkoxycarbonyl group. Substituted amide groups, particularly preferably di (methoxycarbonyl) amides, di (ethoxycarbonyl) amide groups can be mentioned. Examples of the amide group substituted with the alkyl group and the alkoxycarbonyl group include the amide group di-substituted with the alkyl group and the alkoxycarbonyl group, preferably the C1-C4 alkyl group and the C2-C5 alkoxycarbonyl group. Substituted amide groups, more preferably di-substituted amide groups with C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups, particularly preferably methylmethoxycarbonylamide groups, methylethoxycarbonylamide groups, ethylmethoxycarbonylamide groups, Ethylethoxycarbonylamide groups can be mentioned.

　本発明において「アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基」においてピペリジニルカルボニル基が有する置換基の数は０～５であり、好ましくは０～４、より好ましくは０～３、より一層好ましくは０～２、特に好ましくは０又は１である。当該置換基が複数あるときは同一でも異なってもよい。ピペリジニルカルボニル基が置換基を有する場合、少なくとも１個の置換基がピペリジン環の３位（詳細には、ピペリジン環を構成する窒素原子を１位とした場合の３位）の炭素原子に結合していることが好ましい。置換基としては、例えば前記のアルキル基及び前記のアルコキシカルボニル基が挙げられ、好ましくはＣ１～Ｃ４アルキル基、Ｃ２～Ｃ５アルコキシカルボニル基、より好ましくはＣ１～Ｃ３アルキル基、Ｃ２～Ｃ４アルコキシカルボニル基、特に好ましくはメチル基、エチル基、メトキシカルボニル基、エトキシカルボニル基が挙げられる。 In the present invention, the number of substituents of the piperidinylcarbonyl group in "a piperidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group" is It is 0 to 5, preferably 0 to 4, more preferably 0 to 3, even more preferably 0 to 2, and particularly preferably 0 or 1. When there are a plurality of the substituents, they may be the same or different. When the piperidinylcarbonyl group has a substituent, at least one substituent is on the carbon atom at the 3-position of the piperidine ring (specifically, the 3-position when the nitrogen atom constituting the piperidine ring is at the 1-position). It is preferable that they are bonded. Examples of the substituent include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1 to C4 alkyl group, a C2-C5 alkoxycarbonyl group, and more preferably a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Particularly preferably, a methyl group, an ethyl group, a methoxycarbonyl group and an ethoxycarbonyl group can be mentioned.

　本発明において「アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基」においてピロリジニルカルボニル基が有する置換基の数は０～４であり、好ましくは０～３、より好ましくは０～２、特に好ましくは０又は１である。当該置換基が複数あるときは同一でも異なってもよい。ピロリジニルカルボニル基が置換基を有する場合、少なくとも１個の置換基がピロリジン環の３位（詳細には、ピロリジン環を構成する窒素原子を１位とした場合の３位）の炭素原子に結合していることが好ましい。置換基としては、例えば前記のアルキル基及び前記のアルコキシカルボニル基が挙げられ、好ましくはＣ１～Ｃ４アルキル基、Ｃ２～Ｃ５アルコキシカルボニル基、より好ましくはＣ１～Ｃ３アルキル基、Ｃ２～Ｃ４アルコキシカルボニル基、特に好ましくはメチル基、エチル基、メトキシカルボニル基、エトキシカルボニル基が挙げられる。 In the present invention, the number of substituents of the pyrrolidinylcarbonyl group in "a pyrrolidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group" is It is 0 to 4, preferably 0 to 3, more preferably 0 to 2, and particularly preferably 0 or 1. When there are a plurality of the substituents, they may be the same or different. When the pyrrolidinylcarbonyl group has a substituent, at least one substituent is on the carbon atom at the 3-position of the pyrrolidine ring (specifically, the 3-position when the nitrogen atom constituting the pyrrolidine ring is at the 1-position). It is preferable that they are bonded. Examples of the substituent include the above-mentioned alkyl group and the above-mentioned alkoxycarbonyl group, preferably a C1 to C4 alkyl group, a C2-C5 alkoxycarbonyl group, and more preferably a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Particularly preferably, a methyl group, an ethyl group, a methoxycarbonyl group and an ethoxycarbonyl group can be mentioned.

　本発明の剤は、細胞内への核酸分子の取り込みを促進するための剤（本明細書中、「核酸分子取り込み促進剤」とも称する。）、細胞のオートファジーを増強するための剤（本明細書中、「オートファジー増強剤」とも称する。）、又は、核酸分子活性を増強するための剤（本明細書中、「核酸分子活性増強剤」とも称する。）である。 The agent of the present invention is an agent for promoting the uptake of nucleic acid molecules into cells (also referred to as "nucleic acid molecule uptake promoter" in the present specification), and an agent for enhancing cell autophagy (the present invention). In the specification, it is also referred to as "autophagy enhancer") or an agent for enhancing nucleic acid molecular activity (also referred to as "nucleic acid molecule activity enhancer" in the present specification).

核酸分子取り込み促進剤
　本発明の細胞内への核酸分子取り込み促進するための剤は、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、生体内又は生体外において細胞外の核酸分子が細胞内へ取り込まれることを促進する。ここで、促進する、とは、細胞内への核酸分子の取り込み量が、促進剤が適用されない場合と比較して、促進剤が適用された場合に増加することをいう。増加の程度は、例えば1.2倍以上、1.3倍以上、1.4倍以上、1.5倍以上（好ましくは2倍以上、より好ましくは2.5倍以上）とできる。本発明の促進剤は、代表的には、生体に投与された核酸分子の取り込み量を増加させる、及び生体外の細胞（例えば容器中の細胞）に添加された核酸分子の取り込み量を増加させる。 Nucleic acid molecule uptake promoter The agent for promoting the uptake of nucleic acid molecules into cells of the present invention activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel. By containing a substance, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a compound represented by the formula (1) (b), cannavidiol, and 1,3-dihydro-1- [1]. -[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one at least one selected from the group. The inclusion of a compound of a species, a pharmaceutically acceptable salt thereof, or a prodrug thereof facilitates the uptake of extracellular nucleic acid molecules into cells in vivo or in vitro. Here, "promoting" means that the amount of nucleic acid molecule taken up into the cell increases when the accelerator is applied as compared with the case where the accelerator is not applied. The degree of increase can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more). The accelerator of the present invention typically increases the uptake of nucleic acid molecules administered to a living body and the uptake of nucleic acid molecules added to cells outside the body (eg, cells in a container). ..

核酸分子活性増強剤
　本発明の核酸分子活性増強剤は、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、生体内又は生体外において核酸分子の活性を増強する。ここで、「核酸分子の活性を増強する」とは、核酸分子が発揮することを期待されている機能を増強することをいい、例えばアンチセンス核酸、siRNA, miRNA, shRNA等であれば標的RNA (mRNA, non-coding RNA)の切断活性の増強、plasmid, mRNA, antagomir等であれば標的タンパク質の発現の増強、splice switching oligonucleotide等であれば標的mRNAのsplicingの修正の増強、デコイ核酸等であれば転写活性の低減、guide RNA等であればゲノム編集活性の増強、リボザイム等であれば酵素活性の増強をいう。また、増強する、とは、核酸分子の活性が、核酸分子活性増強剤が適用されない場合と比較して、核酸分子活性増強剤が適用された場合に活性が増強することをいう。増強の程度は、例えば1.2倍以上、1.3倍以上、1.4倍以上、1.5倍以上（好ましくは2倍以上、より好ましくは2.5倍以上）とできる。本発明の核酸分子活性増強剤は、代表的には、生体に投与された核酸分子の活性を増強させる、及び生体外の細胞（例えば容器中の細胞）に添加された核酸分子の活性を増強させる。 Nucleic Acid Molecular Activity Enhancer The nucleic acid molecule activity enhancer of the present invention is (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and is pharmaceutically acceptable thereof. By containing the following salts, or prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,,). At least one compound selected from the group consisting of 7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceuticals. By containing an acceptable salt or a prodrug thereof, the activity of the nucleic acid molecule is enhanced in vivo or in vitro. Here, "enhancing the activity of a nucleic acid molecule" means enhancing the function expected to be exerted by the nucleic acid molecule. For example, in the case of antisense nucleic acid, siRNA, miRNA, shRNA, etc., the target RNA. Enhancement of cleavage activity of (mRNA, non-coding RNA), enhancement of expression of target protein in case of plasmid, mRNA, antagomir, etc., enhancement of modification of splicing of target mRNA in case of splice switching oligonucleotide, etc., decoy nucleic acid, etc. If there is, it means reduction of transcriptional activity, if it is guide RNA etc., it means enhancement of genome editing activity, and if it is ribozyme etc., it means enhancement of enzyme activity. Further, "enhancing" means that the activity of the nucleic acid molecule is enhanced when the nucleic acid molecule activity enhancer is applied as compared with the case where the nucleic acid molecule activity enhancer is not applied. The degree of enhancement can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more). The nucleic acid molecule activity enhancer of the present invention typically enhances the activity of a nucleic acid molecule administered to a living body and enhances the activity of a nucleic acid molecule added to an in vitro cell (for example, a cell in a container). Let me.

オートファジー増強剤
　本発明のオートファジー増強剤は、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有することによって、生体内又は生体外において細胞のオートファジー（Autophagy）を増強する。このため、オートファジー増強剤は、核酸分子に依拠することなく、アルツハイマー病、パーキンソン病、ハンチントン病、ブリオン病等の異常タンパク質の蓄積に起因する疾患の予防又は治療に有用である。ここで、オートファジーとは、細胞内のタンパク質が分解される現象をいい、例えば、オートファジー活性と相関するLC3-iからLC3-iiへの変換量の増減を定量することで、オートファジー活性の誘導及び抑制を評価することができる。なお、LC3-iはAtg4（システインプロテアーゼ）で分解されて生じ、LC3-iがホスファチジルエタノールアミンと結合するとLC3-iiになり、LC3-iiの量はオートファゴソーム形成と正の相関を示すため、オートファジーの誘導及び抑制の評価に利用されている。また、増強する、とは、細胞のオートファジーが、オートファジー増強剤が適用されない場合と比較して、オートファジー増強剤が適用された場合に、オートファジーを増強することをいう。増強の程度は、例えば1.2倍以上、1.3倍以上、1.4倍以上、1.5倍以上（好ましくは2倍以上、より好ましくは2.5倍以上）とできる。本発明のオートファジー増強剤は、代表的には、生体に投与された核酸分子の活性を増強させる、及び生体外の細胞（例えば容器中の細胞）に添加された核酸分子の活性を増強させる。なお、オートファジーの増強がアンチセンス核酸のノックダウン（KD）効果を増強することが報告されている（J. Ochaba et al., A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity, Nucleic Acids Research, 47, 11284-11303 (2019)）。 Autophagy Enhancer The autophagy enhancer of the present invention is a pharmaceutically acceptable substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel. By containing salts, or prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1-[1-[(5,6,7,, At least one compound selected from the group consisting of 8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, pharmaceutically acceptable thereof. By containing the following salts, or prodrugs thereof, the autophagy of cells is enhanced in vivo or in vitro. Therefore, the autophagy enhancer is useful for the prevention or treatment of diseases caused by the accumulation of abnormal proteins such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and Brion's disease without relying on nucleic acid molecules. Here, autophagy refers to a phenomenon in which intracellular proteins are degraded. For example, autophagy activity is achieved by quantifying an increase or decrease in the amount of conversion from LC3-i to LC3-ii, which correlates with autophagy activity. Can be evaluated for induction and inhibition of. Since LC3-i is decomposed by Atg4 (cysteine protease) and becomes LC3-ii when LC3-i binds to phosphatidylethanolamine, the amount of LC3-ii shows a positive correlation with autophagosome formation. It is used to evaluate the induction and suppression of autophagy. Further, "enhancement" means that the autophagy of the cell enhances the autophagy when the autophagy enhancer is applied as compared with the case where the autophagy enhancer is not applied. The degree of enhancement can be, for example, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more (preferably 2 times or more, more preferably 2.5 times or more). The autophagy enhancer of the present invention typically enhances the activity of a nucleic acid molecule administered to a living body and enhances the activity of a nucleic acid molecule added to an in vitro cell (for example, a cell in a container). .. It has been reported that enhancement of autophagy enhances the knockdown (KD) effect of antisense nucleic acid (J. Ochaba et al., A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity, Nucleic Acids). Research, 47, 11284-11303 (2019)).

　本明細書中、細胞内への核酸分子取り込み促進剤、核酸分子活性増強剤、及びオートファジー増強剤を総称して「本発明の剤」又は単に「剤」と称することがある。 In the present specification, the nucleic acid molecule uptake promoter, the nucleic acid molecule activity enhancer, and the autophagy enhancer may be collectively referred to as "the agent of the present invention" or simply "the agent".

　本発明の剤は、カルシウムイオンの存在下で細胞に適用されると、細胞の核酸分子取り込み量が増大する。このため、本発明の剤はカルシウムイオンの存在下で使用されることが好ましい。生体内の環境では細胞内外にカルシウムイオンが存在するため、カルシウムイオンの追加は必ずしも必要ではないが、生体外の細胞に促進本発明の剤を適用するときは、カルシウムイオンの存在下であることが好ましい。例えば、カルシウムイオンを含有する培養液中の細胞に本発明の剤を添加することができる。 When the agent of the present invention is applied to cells in the presence of calcium ions, the amount of nucleic acid molecule uptake in cells increases. Therefore, the agent of the present invention is preferably used in the presence of calcium ions. Since calcium ions are present inside and outside cells in the in vivo environment, it is not always necessary to add calcium ions, but when the agent of the present invention is applied to cells outside the living body, it must be in the presence of calcium ions. Is preferable. For example, the agent of the present invention can be added to cells in a culture medium containing calcium ions.

　本発明の剤は、細胞内への核酸分子取り込みを促進する作用、オートファジーを増強する作用、又は核酸分子の活性を増強する作用等を有するため、核酸分子を含有すること又は核酸分子（例えば核酸薬剤）と組み合わせて使用されることが好ましい。本発明の剤は、核酸分子と組み合わせて使用される場合、核酸分子の投与又は添加の前、後、又は同時に、投与又は添加されてよい。 Since the agent of the present invention has an action of promoting the uptake of nucleic acid molecule into cells, an action of enhancing autophagy, an action of enhancing the activity of nucleic acid molecule, etc., it may contain a nucleic acid molecule or a nucleic acid molecule (for example, It is preferably used in combination with a nucleic acid drug). When used in combination with a nucleic acid molecule, the agent of the present invention may be administered or added before, after, or at the same time as the administration or addition of the nucleic acid molecule.

　本発明の剤の適用対象となる細胞としては、核酸分子の取り込みが期待される細胞が好適である。例えば、核酸分子の標的となる細胞、好適には、生体に投与される核酸分子（代表的には核酸薬剤）の標的となる細胞及び生体外で細胞内への核酸分子の取り込みが試験、評価等される細胞である。 As the cells to which the agent of the present invention is applied, cells expected to take up nucleic acid molecules are suitable. For example, cells targeted for nucleic acid molecules, preferably cells targeted for nucleic acid molecules (typically nucleic acid drugs) administered to a living body, and uptake of nucleic acid molecules into cells in vitro are tested and evaluated. It is a cell to be equalized.

　細胞はTRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネル（好ましくはTRPC6チャネル）を有していることが好ましい。細胞がそのようなチャネルを有していると核酸分子の取り込み量の増大、オートファジーの増強、又は核酸分子の活性の増強に有利である。したがって、本発明の剤は、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを有する細胞に核酸分子を取り込ませること、オートファジーを増強すること、又は核酸分子の活性を増強すること等に有用である。本発明の剤は、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを有する細胞に核酸薬剤（核酸分子）を取り込ませ、核酸分子の活性を増強させるため、ドラッグデリバリーシステム（例えば、核酸分子をTRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを有する細胞に送達するためのドラッグデリバリーシステム）に有用である。 It is preferable that the cell has at least one channel (preferably TRPC6 channel) selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. Having such a channel is advantageous for increasing the uptake of nucleic acid molecules, enhancing autophagy, or enhancing the activity of nucleic acid molecules. Therefore, the agents of the present invention allow cells having at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel to take up nucleic acid molecules, enhance autophagy, or nucleic acid molecules. It is useful for enhancing the activity of. The agent of the present invention allows cells having at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel to take up a nucleic acid drug (nucleic acid molecule) and enhance the activity of the nucleic acid molecule. It is useful for drug delivery systems (eg, drug delivery systems for delivering nucleic acid molecules to cells having at least one channel selected from the group consisting of TRPC3 channels, TRPC6 channels, and TRPC7 channels).

　細胞としては、例えば小細胞肺がん細胞、非小細胞肺がん細胞、前立腺がん細胞、乳がん細胞、筋細胞又は神経細胞等が挙げられ、好ましくは小細胞肺がん細胞、非小細胞肺がん細胞、筋細胞及び神経細胞である。また、細胞はヒトの細胞が好ましい。 Examples of the cells include small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells, nerve cells and the like, preferably small cell lung cancer cells, non-small cell lung cancer cells, muscle cells and the like. It is a nerve cell. Further, the cells are preferably human cells.

　核酸分子としては、例えばsmall interfering RNA(siRNA)、micro RNA、antagomir、small hairpin RNA(shRNA)、guide RNA(gRNA)、アンチセンス核酸、リボザイム、デコイ核酸、スプライススィッチング核酸、mRNA、プラスミド等が挙げられ、好ましくはアンチセンス核酸及びスプライススィッチング核酸等である。核酸分子は１種単独でも２種以上でも使用できる。核酸分子の具体例としては、Viltepso、Vyondys53、Spinraza、Exondys51等が挙げられる。核酸分子は、公知の配列情報に基づいて作製することができる。 Examples of nucleic acid molecules include small interfering RNA (siRNA), microRNA, antogomir, small hairpin RNA (shRNA), guide RNA (gRNA), antisense nucleic acid, ribozyme, decoy nucleic acid, splice switching nucleic acid, mRNA, plasmid and the like. These are preferably antisense nucleic acids, splice switching nucleic acids and the like. Nucleic acid molecules can be used alone or in combination of two or more. Specific examples of nucleic acid molecules include Viltepso, Vyondys53, Spinraza, Exondys51 and the like. Nucleic acid molecules can be made based on known sequence information.

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質は、好ましくはTRPC3チャネル及びTRPC6チャネルからなる群から選択される少なくとも１種のチャネル、さらに好ましくはTRPC6チャネルを活性化し、細胞外のカルシウムイオンの細胞内への流入を促進することができる物質を包含しうる。 The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is preferably at least one channel selected from the group consisting of TRPC3 channel and TRPC6 channel, more preferably. May include substances that can activate TRPC6 channels and promote the intracellular influx of extracellular calcium ions.

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質としては、下記式（１）で表される化合物（本明細書中、「化合物（１）」とも称する。）、カンナビジオール、1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン（GSK1702934Aとも称する。）等が挙げられる。

Figure JPOXMLDOC01-appb-C000016
［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］ The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the following formula (1) (in the present specification, "Compound (1)). ”), Cannabidiol, 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cycloheptane [b] thien-2-yl) carbonyl] -4-piperidinyl ] -2H-benzimidazol-2-one (also referred to as GSK1702934A) and the like can be mentioned.
Figure JPOXMLDOC01-appb-C000016
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m indicates an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ]

　式（１）は、式（１－１）又は式（１－２）であってもよい。化合物（１）は下記式（１－１）で表わされる化合物（本明細書中、「化合物（１－１）」とも称する。」）又は下記式（１－２）で表される化合物（本明細書中、「化合物（１－２）」とも称する。」）であってよい。化合物（１－１）及び化合物（１－２）は新規な化合物である。

Figure JPOXMLDOC01-appb-C000017
［式（１－１）中、Ｒ１、Ｒ２、ｍ、及びｎは、式（１）と同じ。］
Figure JPOXMLDOC01-appb-C000018
 ［式（１－２）中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ａ及びＢは、同時に炭素原子を示さず、
Ｒ３は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
Ｒ２、及びｎは、式（１）と同じ。］ The formula (1) may be the formula (1-1) or the formula (1-2). The compound (1) is a compound represented by the following formula (1-1) (also referred to as “compound (1-1)” in the present specification) or a compound represented by the following formula (1-2) (present). In the specification, it may also be referred to as “compound (1-2)”. ”). Compound (1-1) and compound (1-2) are novel compounds.
Figure JPOXMLDOC01-appb-C000017
 [In equation (1-1), R 1 , R 2 , m, and n are the same as in equation (1). ]
Figure JPOXMLDOC01-appb-C000018
 [In equation (1-2),
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
A and B do not show carbon atoms at the same time,
R 3 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
R2 and n are the same as in the equation (1). ]

　化合物（１）及び（１－１）から以下に示される化合物が除かれてもよい。

Figure JPOXMLDOC01-appb-C000019
The compounds shown below may be excluded from the compounds (1) and (1-1).
Figure JPOXMLDOC01-appb-C000019

　化合物（１）及び（１－１）から以下に示される化合物が除かれてもよい。

Figure JPOXMLDOC01-appb-C000020
The compounds shown below may be excluded from the compounds (1) and (1-1).
Figure JPOXMLDOC01-appb-C000020

　Ａは、炭素原子又は窒素原子を示し、Ｂは炭素原子又は窒素原子を示す。但し、式（１－２）においては、Ａ及びＢは、同時に炭素原子を示さない。
　Ａ及びＢの組み合わせとしては、Ａ及びＢがともに炭素原子、Ａが炭素原子及びＢが窒素原子、Ａが窒素原子及びＢが炭素原子、Ａ及びＢがともに窒素原子のいずれかである。つまりＡ及びＢを含有する環は、式（１）において、ベンゼン環、ピリジン環、またはピリダジン環であり、式（１－２）において、ピリジン環又はピリダジン環である。
　式（１）において、Ａ及びＢの組み合わせとしては、Ａ及びＢがともに炭素原子、Ａが炭素原子及びＢが窒素原子、又はＡが窒素原子及びＢが炭素原子であることが好ましく、Ａ及びＢがともに炭素原子又はＡが窒素原子及びＢが炭素原子であることがより好ましい。
　式（１－２）において、Ａ及びＢの組み合わせとしては、Ａが炭素原子及びＢが窒素原子、又はＡが窒素原子及びＢが炭素原子であることが好ましく、Ａが窒素原子及びＢが炭素原子であることがより好ましい。
　Ａ及びＢのいずれか又は両方が炭素原子であるときは、当該炭素原子は置換基Ｒ１を有してもよい。 A indicates a carbon atom or a nitrogen atom, and B indicates a carbon atom or a nitrogen atom. However, in the formula (1-2), A and B do not show carbon atoms at the same time.
As a combination of A and B, A and B are both carbon atoms, A is a carbon atom and B is a nitrogen atom, A is a nitrogen atom and B is a carbon atom, and A and B are both nitrogen atoms. That is, the ring containing A and B is a benzene ring, a pyridine ring, or a pyridazine ring in the formula (1), and a pyridine ring or a pyridazine ring in the formula (1-2).
In the formula (1), as the combination of A and B, it is preferable that both A and B are carbon atoms, A is a carbon atom and B is a nitrogen atom, or A is a nitrogen atom and B is a carbon atom. It is more preferable that both B are carbon atoms or A is a nitrogen atom and B is a carbon atom.
In the formula (1-2), as the combination of A and B, it is preferable that A is a carbon atom and B is a nitrogen atom, or A is a nitrogen atom and B is a carbon atom, and A is a nitrogen atom and B is a carbon atom. It is more preferably an atom.
When either or both of A and B are carbon atoms, the carbon atom may have a substituent R1 .

　Ｒ１が複数あるときは同一であっても異なってもよい。Ｒ１としては、フッ素原子、塩素原子、臭素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ５アシルオキシ基、Ｃ２～Ｃ５アルコキシカルボニル基、又はトリハロゲノメチル基が好ましく、フッ素原子、塩素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、トリクロロメチル基、又はトリブロモメチル基がより好ましく、塩素原子、Ｃ１～Ｃ３アルキル基、Ｃ１～Ｃ３アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、又はトリクロロメチル基がさらに好ましく、塩素原子、メトキシ基、エトキシ基、メトキシカルボニル基、又はエトキシカルボニル基が特に好ましい。 When there are a plurality of R1 , they may be the same or different. As R1 , a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group is preferable, and a fluorine atom, Chlorine atoms, C1-C4 alkyl groups, C1-C4 alkoxy groups, C2-C4 alkoxycarbonyl groups, trifluoromethyl groups, trichloromethyl groups, or tribromomethyl groups are more preferred, chlorine atoms, C1-C3 alkyl groups, C1. -C3 alkoxy group, C2-C4 alkoxycarbonyl group, trifluoromethyl group, or trichloromethyl group is more preferable, and chlorine atom, methoxy group, ethoxy group, methoxycarbonyl group, or ethoxycarbonyl group is particularly preferable.

　Ｒ１は、Ｒ１が結合する環がベンゼン環であるときは、ベンゼン環を構成する炭素原子のうち、（ＮＨ）ｐを構成する窒素原子と直接結合する炭素原子に対してオルト位に位置する２個の炭素原子のうちの少なくとも１個に結合していることが好ましい。オルト位に位置する炭素原子に結合するＲ１としては、例えばアルコキシ基又はアルコキシカルボニル基が挙げられ、Ｃ２～Ｃ５アルコキシカルボニル基が好ましく、Ｃ２～Ｃ４アルコキシカルボニル基がより好ましく、メトキシカルボニル基又はエトキシカルボニル基がさらに好ましく、エトキシカルボニル基が特に好ましい。 When the ring to which R 1 is bonded is a benzene ring, R 1 is located at the ortho position with respect to the carbon atom directly bonded to the nitrogen atom constituting (NH) p among the carbon atoms constituting the benzene ring. It is preferably bonded to at least one of the two carbon atoms. Examples of R 1 bonded to the carbon atom located at the ortho position include an alkoxy group or an alkoxycarbonyl group, preferably a C2-C5 alkoxycarbonyl group, more preferably a C2-C4 alkoxycarbonyl group, and a methoxycarbonyl group or an ethoxy. A carbonyl group is more preferred, and an ethoxycarbonyl group is particularly preferred.

　Ｒ１は、Ｒ１が結合する環がベンゼン環であって、ベンゼン環を構成する炭素原子のうち、（ＮＨ）ｐを構成する窒素原子と直接結合する炭素原子に対してメタ位に位置する２個の炭素原子のいずれか又は両方に結合している場合、ハロゲン原子であることが好ましく、塩素原子又はフッ素原子であることがより好ましく、フッ素原子が特に好ましい。 In R 1 , the ring to which R 1 is bonded is a benzene ring, and among the carbon atoms constituting the benzene ring, R 1 is located at the meta position with respect to the carbon atom directly bonded to the nitrogen atom constituting (NH) p . When bonded to either or both of the two carbon atoms, it is preferably a halogen atom, more preferably a chlorine atom or a fluorine atom, and particularly preferably a fluorine atom.

　Ｒ１は、Ａ及びＢを含む環がピリジン環又はピリダジン環であるときは、ピリジン環又はピリダジン環を構成する炭素原子のうち、（ＮＨ）ｐを構成する窒素原子と直接結合する炭素原子とＡとの間に位置する炭素原子に結合することが好ましい。
　Ｒ１は、Ａ及びＢを含む環がピリジン環又はピリダジン環であるときは、例えばハロゲン原子、アルキル基、アルコキシ基、又はアルコキシカルボニル基であってよく、フッ素原子、塩素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、又はＣ２～Ｃ５アルコキシカルボニル基が好ましく、Ｃ１～Ｃ４アルコキシ基がより好ましく、メトキシ基又はエトキシ基が特に好ましい。 When the ring containing A and B is a pyridine ring or a pyridazine ring, R 1 is a carbon atom directly bonded to a nitrogen atom constituting (NH) p among the carbon atoms constituting the pyridine ring or the pyridazine ring. It is preferable to bond to a carbon atom located between A and A.
When the ring containing A and B is a pyridine ring or a pyridazine ring, R 1 may be, for example, a halogen atom, an alkyl group, an alkoxy group, or an alkoxycarbonyl group, and may be a fluorine atom, a chlorine atom, or a C1-C4 alkyl. Groups, C1-C4 alkoxy groups, or C2-C5 alkoxycarbonyl groups are preferred, C1-C4 alkoxy groups are more preferred, and methoxy or ethoxy groups are particularly preferred.

　Ｒ３は、ハロゲン原子、アルキル基、アルコキシ基、又はアルコキシカルボニル基であってよく、フッ素原子、塩素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、又はＣ２～Ｃ５アルコキシカルボニル基が好ましく、Ｃ１～Ｃ４アルコキシ基がより好ましく、メトキシ基又はエトキシ基が特に好ましい。 R3 may be a halogen atom , an alkyl group, an alkoxy group, or an alkoxycarbonyl group, preferably a fluorine atom, a chlorine atom, a C1 to C4 alkyl group, a C1 to C4 alkoxy group, or a C2 to C5 alkoxycarbonyl group. C1 to C4 alkoxy groups are more preferable, and methoxy groups or ethoxy groups are particularly preferable.

　ｍとしては、０～４の整数が好ましく、０～３の整数がより好ましく、０、１、又は２がさらに好ましく、０又は１がさらに好ましく、１が特に好ましい。 As m, an integer of 0 to 4, an integer of 0 to 3, is more preferable, 0, 1, or 2 is more preferable, 0 or 1 is further preferable, and 1 is particularly preferable.

　式（１）における下記に表わされる部分構造

Figure JPOXMLDOC01-appb-C000021
、及び式（１－１）における下記に表わされる部分構造
Figure JPOXMLDOC01-appb-C000022
 ［両部分構造において、＊は部分構造（ＮＨ）ｐを構成する窒素原子と結合する側を示し、他の符号は前記と同意義を示す。］
としては、下記の構造（下記の構造中、Ｒはアルキル基、好適にはＣ１～Ｃ４アルキル基、より好適にはＣ１～Ｃ２アルキル基を示し、Ｈａｌはハロゲン原子、好適には塩素原子又はフッ素原子を示す。）が好ましい。
Figure JPOXMLDOC01-appb-C000023
 　前記部分構造としては、下記の構造がより好ましい。
Figure JPOXMLDOC01-appb-C000024
 Partial structure represented by the following in equation (1)
Figure JPOXMLDOC01-appb-C000021
 , And the partial structure represented by the following in the equation (1-1).
Figure JPOXMLDOC01-appb-C000022
 [In both substructures, * indicates the side bonded to the nitrogen atom constituting the substructure (NH) p, and the other symbols have the same meanings as described above. ]
In the following structure (in the following structure, R represents an alkyl group, preferably a C1 to C4 alkyl group, more preferably a C1 to C2 alkyl group, Hal is a halogen atom, preferably a chlorine atom or fluorine. Atoms are shown.) Are preferred.
Figure JPOXMLDOC01-appb-C000023
 As the partial structure, the following structure is more preferable.
Figure JPOXMLDOC01-appb-C000024

Ｒ２が複数あるときは同一であっても異なってもよい。Ｒ２としては、フッ素原子；塩素原子；臭素原子；トリフルオロメチル基；トリクロロメチル基；トリブロモメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ５アシルオキシ基；Ｃ２～Ｃ５アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基が好ましく、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アシルオキシ基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基がより好ましく、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基がさらに好ましく、塩素原子；トリフルオロメチル基；トリクロロメチル基；メチル基；エチル基；メトキシカルボニル基；エトキシカルボニル基；アミド基；Ｃ１～Ｃ３アルキル基から選択される１種又は２種の基で置換されたアミド基；ピペリジニルカルボニル基；或いはメトキシカルボニル基及びエトキシカルボニル基から選択される１種又は２種以上の基で置換されたピペリジニルカルボニル基がさらに好ましい。 When there are a plurality of R 2 , they may be the same or different. R2 includes fluorine atom; chlorine atom; bromine atom; trifluoromethyl group; trichloromethyl group; tribromomethyl group; C1-C4 alkyl group; C2-C5 acyloxy group; C2-C5 alkoxycarbonyl group; C1-C3. An amide group that may be substituted with one or two groups selected from the group consisting of an alkyl group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. A piperidinyl carbonyl group that may be substituted with one or more groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Substitutable pyrrolidinylcarbonyl groups are preferred, preferably fluorine atoms; chlorine atoms; trifluoromethyl groups; trichloromethyl groups; C1-C4 alkyl groups; C2-C4 acyloxy groups; C2-C4 alkoxycarbonyl groups; C1-C3. An amide group that may be substituted with one or two groups selected from the group consisting of an alkyl group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. A piperidinyl carbonyl group that may be substituted with one or more groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Substitutable pyrrolidinylcarbonyl groups are more preferred: fluorine atoms; chlorine atoms; trifluoromethyl groups; trichloromethyl groups; C1-C4 alkyl groups; C2-C4 alkoxycarbonyl groups; C1-C3 alkyl groups and C2-. An amide group that may be substituted with one or two groups selected from the group consisting of C4 alkoxycarbonyl groups; or one selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. A piperidinylcarbonyl group which may be substituted with two or more groups is more preferable, and a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a methyl group; an ethyl group; a methoxycarbonyl group; an ethoxycarbonyl group; an amide group; C1. An amide group substituted with one or two groups selected from the C3 alkyl group; a piperidinylcarbonyl group; or substituted with one or more groups selected from a methoxycarbonyl group and an ethoxycarbonyl group. The piperidinyl carbonyl group obtained is more preferable.

　Ｒ２は、Ｒ２の結合するベンゼン環を構成する炭素原子のうち、ピペラジン環を構成する窒素原子と直接結合する炭素原子に対してオルト位に位置する２個の炭素原子のうちの１個又は２個に結合していることが好ましく、１個のオルト位炭素原子に結合していることがより好ましい。Ｒ２は、パラ位に位置する炭素原子に結合してもよい。Ｒ２は、（ａ）オルト位の２個の炭素原子のうちの１個のみに結合していること、（ｂ）オルト位の２個の炭素原子のうちの１個とパラ位の炭素原子に結合していること、及び（ｃ）オルト位の２個の炭素原子のうちの１個とメタ位の２個の炭素原子のうちの１個に結合していることが好ましい。Ｒ２は、（ａ）オルト位の２個の炭素原子のうちの１個のみに結合していること、又は（ｂ）オルト位の２個の炭素原子のうちの１個とパラ位の炭素原子に結合していることがより好ましい。 R 2 is one of two carbon atoms located in the ortho position with respect to the carbon atom directly bonded to the nitrogen atom constituting the piperazine ring among the carbon atoms constituting the benzene ring to which R 2 is bonded. Alternatively, it is preferably bonded to two atoms, and more preferably bonded to one ortho-position carbon atom. R 2 may be bonded to a carbon atom located at the para position. R2 is (a) bonded to only one of the two ortho-position carbon atoms, and (b) one of the two ortho-position carbon atoms and the para-position carbon atom. It is preferable that the atom is bonded to (c) one of the two carbon atoms at the ortho position and one of the two carbon atoms at the meta position. R2 is either (a) bonded to only one of the two ortho-position carbon atoms, or (b) one of the ortho-position two carbon atoms and the para-position carbon. It is more preferable that it is bonded to an atom.

　オルト位に位置する炭素原子に結合するＲ２としては、ハロゲン原子、トリハロゲノメチル基、又はＣ１～Ｃ３アルキル基が好ましく、フッ素原子、塩素原子、臭素原子、トリフルオロメチル基、メチル基、又はエチル基がより好ましく、塩素原子、トリフルオロメチル基、又はメチル基が特に好ましい。 The R 2 bonded to the carbon atom located at the ortho position is preferably a halogen atom, a trihalogenomethyl group, or a C1-C3 alkyl group, preferably a fluorine atom, a chlorine atom, a bromine atom, a trifluoromethyl group, a methyl group, or a methyl group. An ethyl group is more preferable, and a chlorine atom, a trifluoromethyl group, or a methyl group is particularly preferable.

　パラ位に位置する炭素原子に結合するＲ２は、例えばハロゲン原子、アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基であってよく、塩素原子；臭素原子；フッ素原子；Ｃ１～Ｃ３アルキル基；Ｃ２～Ｃ４アシルオキシ基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基が好ましく、塩素原子；メチル基；エチル基；メトキシカルボニル基；エトキシカルボニル基；アミド基；Ｃ１～Ｃ３アルキル基から選択される１種又は２種の基で置換されたアミド基；ピペリジニルカルボニル基；或いはメトキシカルボニル基及びエトキシカルボニル基から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基がさらに好ましい。 R 2 bonded to the carbon atom located at the para position is substituted with one or two groups selected from the group consisting of, for example, a halogen atom, an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. May be an amide group; a piperidinyl carbonyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group and an alkoxycarbonyl group; or a group consisting of an alkyl group and an alkoxycarbonyl group. It may be a pyrrolidinylcarbonyl group which may be substituted with one or more groups selected from, chlorine atom; bromine atom; fluorine atom; C1-C3 alkyl group; C2-C4 acyloxy group; C2. ~ C4 alkoxycarbonyl group; an amide group which may be substituted with one or two groups selected from the group consisting of C1 to C3 alkyl groups and C2 to C4 alkoxycarbonyl groups; C1 to C3 alkyl groups and C2 to C4. A piperidinylcarbonyl group that may be substituted with one or more groups selected from the group consisting of alkoxycarbonyl groups; or selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. A pyrrolidinylcarbonyl group which may be substituted with one or more groups is preferred and is selected from a chlorine atom; a methyl group; an ethyl group; a methoxycarbonyl group; an ethoxycarbonyl group; an amide group; a C1-C3 alkyl group. An amide group substituted with one or two groups; a piperidinylcarbonyl group; or a piperidinyl optionally substituted with one or more groups selected from a methoxycarbonyl group and an ethoxycarbonyl group. A carbonyl group is more preferred.

　ｎとしては、０～４の整数が好ましく、０～３の整数がより好ましく、０、１、又は２がさらに好ましく、１又は２がさらに好ましく、２が特に好ましい。 As n, an integer of 0 to 4, an integer of 0 to 3, is more preferable, 0, 1, or 2 is more preferable, 1 or 2 is more preferable, and 2 is particularly preferable.

　式（１）、（１－１）及び（１－２）において下記に表わされる部分構造

Figure JPOXMLDOC01-appb-C000025
［ここで、＊はピペラジン環を構成する窒素原子と結合する側を示す。］
としては、下記の構造が好ましい。
Figure JPOXMLDOC01-appb-C000026
 Substructures represented below in equations (1), (1-1) and (1-2)
Figure JPOXMLDOC01-appb-C000025
 [Here, * indicates the side bonded to the nitrogen atom constituting the piperazine ring. ]
The following structure is preferable.
Figure JPOXMLDOC01-appb-C000026

　ｐとしては、１が好ましい。 As p, 1 is preferable.

　ｑとしては、１が好ましい。 As q, 1 is preferable.

　化合物（１）、化合物（１－１）、又は化合物（１－２）としては、以下の（ａ）～（ｍ）に示す化合物を例示できる。
化合物（ａ）　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル
化合物（ｂ）　２－（４－（２－クロロ－４－ピぺリジン－１－カルボニル）フェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド
化合物（ｃ）　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）－Ｎ，Ｎ－ジメチルベンズアミド
化合物（ｄ）　１－（３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）ベンゾイル）ピぺリジン－４－カルボン酸メチルエステル
化合物（ｅ）　Ｎ－（４－クロロ－２－メトキシフェニル）－２－（４－（２－クロロフェニル）ピペラジン－１－イル）アセトアミド
化合物（ｆ）　２－（２－（４－（２－クロロフェニル）ピペラジン－１－イル）アセトアミド）安息香酸メチルエステル
化合物（ｇ）　２－（２－（４－（２－トリフルオロメチル）フェニル）ピペラジン－１－イル）アセトアミド）安息香酸メチルエステル
化合物（ｈ）　２－（４－（２，５－ジクロロフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド
化合物（ｉ）　２－（４－（２，４－ジクロロフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド
化合物（ｊ）　２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（３－メトキシピリジン－４－イル）アセトアミド
化合物（ｋ）　２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（２－メトキシピリジン－３－イル）アセトアミド
化合物（ｌ）　（４－（５－クロロ－２－メチルフェニル）ピペラジン－１－イル）（３－フルオロフェニル）メタノン
化合物（ｍ）　２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド

Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028
 Examples of the compound (1), the compound (1-1), or the compound (1-2) include the compounds shown in the following (a) to (m).
Compound (a) 3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) benzoic acid methyl ester compound (b) 2- (4-( 2-Chloro-4-piperidin-1-carbonyl) phenyl) piperazin-1-yl) -N- (2-ethoxyphenyl) acetamide compound (c) 3-chloro-4- (4- (2-((((() 2-ethoxyphenyl) amino) -2-oxoethyl) piperazin-1-yl) -N, N-dimethylbenzamide compound (d) 1- (3-chloro-4- (4- (2-((2-ethoxyphenyl)) ) Amino) -2-oxoethyl) piperazin-1-yl) benzoyl) piperidine-4-carboxylic acid methyl ester compound (e) N- (4-chloro-2-methoxyphenyl) -2- (4- (2) -Chlorophenyl) piperazin-1-yl) acetamide compound (f) 2- (2- (4- (2-chlorophenyl) piperazin-1-yl) acetamide) benzoic acid methyl ester compound (g) 2- (2- (4) -(2-Trifluoromethyl) phenyl) piperazin-1-yl) acetamide) benzoic acid methyl ester compound (h) 2- (4- (2,5-dichlorophenyl) piperazin-1-yl) -N- (2-) Ethoxyphenyl) acetamide compound (i) 2- (4- (2,4-dichlorophenyl) piperazin-1-yl) -N- (2-ethoxyphenyl) acetamide compound (j) 2- (4- (2,3-) Dimethylphenyl) piperazin-1-yl) -N- (3-methoxypyridine-4-yl) acetamide compound (k) 2- (4- (2,3-dimethylphenyl) piperazin-1-yl) -N- ( 2-methoxypyridin-3-yl) acetamide compound (l) (4- (5-chloro-2-methylphenyl) piperazin-1-yl) (3-fluorophenyl) methanone compound (m) 2- (4- (4-( 2,3-Dimethylphenyl) piperazin-1-yl) -N- (2-ethoxyphenyl) acetamide
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000028

　また、化合物（１）、化合物（１－１）、又は化合物（１－２）は、上記の（ａ）～（ｅ）、（ｇ）、（ｈ）、（ｊ）、又は（ｋ）として示される化合物であってよい。さらにまた、化合物（１）、化合物（１－１）、又は化合物（１－２）は、上記の（ａ）～（ｄ）、（ｇ）、（ｈ）、又は（ｋ）として示される化合物であってよい。 Further, the compound (1), the compound (1-1), or the compound (1-2) may be referred to as the above (a) to (e), (g), (h), (j), or (k). It may be the compound shown. Furthermore, compound (1), compound (1-1), or compound (1-2) is the compound represented by the above (a) to (d), (g), (h), or (k). May be.

　化合物（１）又は化合物（１－１）としては、２－（４－（２－クロロ－４－ピぺリジン－１－カルボニル）フェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミドが特に好ましい。 Examples of compound (1) or compound (1-1) include 2- (4- (2-chloro-4-piperidin-1-carbonyl) phenyl) piperazine-1-yl) -N- (2-ethoxyphenyl). ) Acetamide is particularly preferred.

　化合物（１）、化合物（１－１）、及び化合物（１－２）は、これに包含される公知化合物については公知製造法により製造できる。新規化合物については、例えば以下に詳述する製造法１又は２、これらに準ずる方法、公知の方法等を適宜変更、組み合わせること等によって製造することができる。原料化合物として用いられる化合物は、それぞれ塩として用いてもよい。なお、以下に示す方法は単なる例示であり、有機合成に習熟している者の知識に基づき、適宜、他の方法で製造することもできる。 The compound (1), the compound (1-1), and the compound (1-2) can be produced by a known production method for the known compounds included therein. The new compound can be produced, for example, by appropriately changing or combining the production method 1 or 2, a method similar thereto, a known method, or the like described in detail below. The compound used as the raw material compound may be used as a salt, respectively. The method shown below is merely an example, and can be appropriately produced by another method based on the knowledge of a person who is proficient in organic synthesis.

　製造における各反応において、必要に応じて、官能基を保護することができる。保護基並びにその保護および脱保護の技術は、公知の方法、例えば、T.W.Greene and P.G.M.Wuts,“Protective Groups in Organic Synthesis”,3rd Ed.,John Wiley and Sons,inc.,New York(1999)に記載の方法を適宜適用できる。 In each reaction in manufacturing, functional groups can be protected as needed. Protecting groups and their protection and deprotection techniques are described in known methods such as T.W.Greene and P.G.M.Wuts, “Protective Groups in Organic Synthesis”, 3rd Ed., John Wiley and Sons, inc., New York (1999). The described method can be applied as appropriate.

［製造法１］
　一実施形態において、式（１Ａ）で表される化合物は、下記の反応工程式－１で示される合成スキ－ムで製造することができる。すなわち、式（１Ａ）で表される化合物は、式（２）で表される化合物と式（３）で表される化合物と式（６）で表わされる化合物から製造することができる。

Figure JPOXMLDOC01-appb-C000029
［式中、Ｘはハロゲン原子であり、Ｄは保護基であり、Ａ、Ｂ、Ｒ１、ｍ、Ｒ２、及びｎは前記と同じ。］ [Manufacturing method 1]
In one embodiment, the compound represented by the formula (1A) can be produced by the synthetic team represented by the following reaction step formula-1. That is, the compound represented by the formula (1A) can be produced from the compound represented by the formula (2), the compound represented by the formula (3) and the compound represented by the formula (6).
Figure JPOXMLDOC01-appb-C000029
 [In the formula, X is a halogen atom, D is a protecting group, and A, B, R 1 , m, R 2 , and n are the same as above. ]

　Ｘとしては、例えば、塩素原子、フッ素原子、臭素原子、ヨウ素原子を挙げることができ、臭素原子が好ましい。 Examples of X include a chlorine atom, a fluorine atom, a bromine atom, and an iodine atom, and a bromine atom is preferable.

　Ｄとしては、ハロゲン原子、ｔｅｒｔ－ブチル基、フェニル基、メチル基、エチル基等の保護基を挙げることができ、ｔｅｒｔ－ブトキシ基が好ましい。 Examples of D include a protecting group such as a halogen atom, a tert-butyl group, a phenyl group, a methyl group and an ethyl group, and a tert-butoxy group is preferable.

　製造法１において、Ｒ２は、アルコキシカルボニル基及びハロゲン原子であることが好ましい。パラ位の炭素原子にアルコキシカルボニル基が結合していることがより好ましい。パラ位の炭素原子にアルコキシカルボニル基が結合し、且つ、オルト位の炭素原子にハロゲン原子が結合していることがより好ましい。 In the production method 1, R 2 is preferably an alkoxycarbonyl group and a halogen atom. It is more preferable that an alkoxycarbonyl group is bonded to the carbon atom at the para position. It is more preferable that the alkoxycarbonyl group is bonded to the carbon atom at the para position and the halogen atom is bonded to the carbon atom at the ortho position.

　工程１、すなわち式（２）で表される化合物と式（３）で表される化合物（塩酸塩であってもよい）を反応させて式（４）で表わされる化合物を合成する工程は、例えば不活性溶媒中、塩基の存在下で行うことができる。式（３）で表わされる化合物としては、例えば2－（4－（2－クロロフェニル）ピペラジン－1－イル）酢酸（CAS No.119357-76-5）であれば市販品を使用でき、その塩酸塩（CAS No. 856843-13-5）はJournal of the American Chemical Society  1955, 77, 1, 40-42に記載の方法により製造でき、その他の化合物も市販品を使用でき、公知の製造法により製造でき、又は公知の製造法を適宜変更することにより製造できる。 Step 1, that is, the step of reacting the compound represented by the formula (2) with the compound represented by the formula (3) (which may be a hydrochloride) to synthesize the compound represented by the formula (4) is For example, it can be carried out in an inert solvent in the presence of a base. As the compound represented by the formula (3), for example, if it is 2- (4- (2-chlorophenyl) piperazine-1-yl) acetic acid (CAS No. 119357-76-5), a commercially available product can be used, and the hydrochloric acid thereof can be used. Salt (CAS No. 856843-13-5) can be produced by the method described in Journal of the American Chemical Society 1955, 77, 1, 40-42, and other compounds can also be commercially available and can be produced by a known production method. It can be manufactured, or it can be manufactured by appropriately changing a known manufacturing method.

　この反応における、不活性溶媒としては、ジエチルエーテル、テトラヒドロフラン（ＴＨＦ）、ジオキサン、ジメトキシメタン等のエーテル系溶媒、トルエン、ベンゼン、キシレン等の芳香族炭化水素系溶媒、ジクロロメタン、クロロホルム、ジクロロエタン、四塩化炭素等のハロゲン化炭化水素系溶媒、アセトン等のケトン系溶媒、ジメチルスルホキシド、Ｎ、Ｎ－ジメチルホルムアミド（ＤＭＦ）、アセトニトリル等の非プロトン溶媒、ピリジン等が挙げられる。これらの溶媒は、２種類以上を適宜の割合で混合して用いてもよい。 Examples of the inert solvent in this reaction include ether solvents such as diethyl ether, tetrahydrofuran (THF), dioxane and dimethoxymethane, aromatic hydrocarbon solvents such as toluene, benzene and xylene, dichloromethane, chloroform, dichloroethane and tetrachloride. Examples thereof include a halogenated hydrocarbon solvent such as carbon, a ketone solvent such as acetone, an aproton solvent such as dimethyl sulfoxide, N, N-dimethylformamide (DMF) and acetonitrile, and pyridine. Two or more kinds of these solvents may be mixed and used in an appropriate ratio.

　塩基としては、例えば水素化ナトリウム、水素化カリウム等の金属ヒドリド、水酸化カリウム、水酸化ナトリウム等の金属水酸化物、炭酸カリウム、炭酸水素カリウム、炭酸ナトリウム、炭酸水素ナトリウム、炭酸セシウム等の金属炭酸塩、トリエチルアミン、エチルジイソプロピルアミン等のアルキルアミン類、ナトリウムメトキシド、カリウムｔ－ブトキシド等の金属アルコキシドが挙げられる。 Examples of the base include metal hydrides such as sodium hydride and potassium hydride, metal hydroxides such as potassium hydroxide and sodium hydroxide, and metals such as potassium carbonate, potassium hydrogen carbonate, sodium carbonate, sodium hydrogen carbonate and cesium carbonate. Examples thereof include alkylamines such as carbonates, triethylamine and ethyldiisopropylamine, and metal alkoxides such as sodium methoxydo and potassium t-butoxide.

　式（２）で表される化合物の使用量は、式（３）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～２モル、より好ましくは０．９～１．５モルである。 The amount of the compound represented by the formula (2) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (3). It is mol, more preferably 0.9 to 1.5 mol.

　塩基の使用量は、式（３）で表される化合物１モルに対して、通常１モル以上、好ましくは１～５倍モル、より好ましくは１～２倍モルである。 The amount of the base used is usually 1 mol or more, preferably 1 to 5 times mol, and more preferably 1 to 2 times mol with respect to 1 mol of the compound represented by the formula (3).

　反応温度は、通常－２０℃～溶媒の沸点より１０℃高い温度、好ましくは０℃～４０℃である。反応時間は、通常１０分～４８時間、好ましくは１０分～２４時間、より好ましくは３０分～１８時間である。 The reaction temperature is usually −20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.

　工程２、すなわち式（４）で表される化合物を式（５）で表される化合物へ変換する工程は、例えば式（４）で表される化合物を酸性有機溶媒で処理することにより可能である。 Step 2, that is, the step of converting the compound represented by the formula (4) into the compound represented by the formula (5) is possible, for example, by treating the compound represented by the formula (4) with an acidic organic solvent. be.

　この反応における、酸性有機溶媒としては、塩酸含有１，４－ジオキサン等が挙げられる。これらの溶媒は、２種類以上を適宜の割合で混合して用いてもよい。酸性有機溶媒の使用量は式（４）で表わされる化合物を酸化できる程度の量であればよい。 Examples of the acidic organic solvent in this reaction include hydrochloric acid-containing 1,4-dioxane and the like. Two or more kinds of these solvents may be mixed and used in an appropriate ratio. The amount of the acidic organic solvent used may be such that the compound represented by the formula (4) can be oxidized.

　工程３、すなわち式（５）で表される化合物と式（６）で表される化合物を反応させて式（１Ａ）で表わされる化合物を合成する工程は、例えば不活性溶媒中、塩基及び縮合剤の存在下で行うことができる。不活性溶媒及び塩基の詳細は前記と同様である。 Step 3, that is, the step of reacting the compound represented by the formula (5) with the compound represented by the formula (6) to synthesize the compound represented by the formula (1A) is, for example, in a base and condensation in an inert solvent. It can be done in the presence of the agent. The details of the inert solvent and the base are the same as described above.

　縮合剤としては、例えば１‐［ジメチルアミノ（ジメチルイミニオ）メチル］‐１Ｈ‐１，２，３‐トリアゾロ［４，５‐ｂ］ピリジン３‐オキシド・ヘキサフルオロホスファート（本明細書中、「ＨＡＴＵ」とも称する。）等が挙げられる。 Examples of the condensing agent include 1- [dimethylamino (dimethylimino) methyl] -1H-1,2,3-triazolo [4,5-b] pyridine 3-oxide hexafluorophosphart (in the present specification). Also referred to as "HATU") and the like.

　式（６）で表される化合物の使用量は、式（５）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～２モル、より好ましくは０．９～１．５モルである。 The amount of the compound represented by the formula (6) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (5). It is mol, more preferably 0.9 to 1.5 mol.

　縮合剤の使用量は、式（５）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～２モル、より好ましくは０．９～１．５モルである。 The amount of the condensing agent to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 mol, more preferably 0, with respect to 1 mol of the compound represented by the formula (5). It is 9 to 1.5 mol.

　反応温度は、通常－２０℃～溶媒の沸点より１０℃高い温度、好ましくは０℃～４０℃である。反応時間は、通常１０分～４８時間、好ましくは１０分～２４時間、より好ましくは３０分～１８時間である。 The reaction temperature is usually −20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.

　工程３によって、Ｒ２がアルコキシカルボニル基である式（１Ａ）で表される化合物を得た場合、この化合物を、例えば不活性溶媒中、塩基の存在下で処理することによって、対応するカルボン酸体に変換してもよい。次いで、このカルボン酸体に適当な置換基を導入する。例えば不活性溶媒中、縮合剤の存在下、必要に応じてさらに塩基の存在下で、置換基に対応する化合物（例えばピペリジン、アミン等）と反応させて、Ｒ２として対応するアミド基又はピペリジニルカルボニル基を有する式（１Ａ）で表される化合物を得ることもできる。不活性溶媒、塩基及び縮合剤の詳細は前記と同様である。 When the compound represented by the formula (1A) in which R 2 is an alkoxycarbonyl group is obtained by step 3, the corresponding carboxylic acid is obtained by treating this compound, for example, in an inert solvent in the presence of a base. It may be converted into a body. Then, an appropriate substituent is introduced into this carboxylic acid body. For example, in an inert solvent, in the presence of a condensing agent, and if necessary, in the presence of a base, the reaction with a compound corresponding to the substituent (for example, piperidin, amine, etc.) is carried out to form the corresponding amide group or pi as R2 . A compound represented by the formula (1A) having a peridinylcarbonyl group can also be obtained. The details of the inert solvent, base and condensing agent are the same as described above.

　この場合、ピペリジン、アミン等の置換基に対応する化合物の使用量は、Ｒ２がアルコキシカルボニル基である式（１Ａ）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～１０モル、より好ましくは０．９～８モルである。反応温度は、通常－２０℃～溶媒の沸点より１０℃高い温度、好ましくは０℃～４０℃である。反応時間は、通常１０分～４８時間、好ましくは１０分～２４時間、より好ましくは３０分～１８時間である。 In this case, the amount of the compound corresponding to the substituent such as piperidin or amine is usually 0.5 mol or more with respect to 1 mol of the compound represented by the formula (1A) in which R2 is an alkoxycarbonyl group. It is 0.8 mol or more, preferably 0.9 to 10 mol, and more preferably 0.9 to 8 mol. The reaction temperature is usually −20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably 0 ° C. to 40 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.

［製造法２］
　一実施形態において、式（１Ａ）で表される化合物は、下記の反応工程式－２で示される合成スキームで製造することができる。すなわち、式（１Ａ）で表される化合物は、式（６）で表される化合物と式（７）で表される化合物と式（３）で表される化合物から製造することができる。 [Manufacturing method 2]
In one embodiment, the compound represented by the formula (1A) can be produced by the synthetic scheme represented by the following reaction step formula-2. That is, the compound represented by the formula (1A) can be produced from the compound represented by the formula (6), the compound represented by the formula (7) and the compound represented by the formula (3).

Figure JPOXMLDOC01-appb-C000030
［式中、Ｘ、Ｄ、Ａ、Ｂ、Ｒ１、ｍ、Ｒ２、及びｎは前記と同じ。］
Figure JPOXMLDOC01-appb-C000030
 [In the formula, X, D, A, B, R 1 , m, R 2 , and n are the same as described above. ]

　工程１、すなわち式（６）で表される化合物と式（７）で表される化合物を反応させて式（８）で表わされる化合物を合成する工程は、例えば不活性溶媒中、塩基の存在下で行うことができる。不活性溶媒及び塩基の詳細は前記と同様である。 Step 1, that is, the step of reacting the compound represented by the formula (6) with the compound represented by the formula (7) to synthesize the compound represented by the formula (8) is, for example, the presence of a base in an inert solvent. Can be done below. The details of the inert solvent and the base are the same as described above.

　式（７）で表される化合物の使用量は、式（６）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～２モル、より好ましくは０．９～１．５モルである。 The amount of the compound represented by the formula (7) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (6). It is mol, more preferably 0.9 to 1.5 mol.

　反応温度は、通常－２０℃～溶媒の沸点より１０℃高い温度、好ましくは－１０℃～４０℃である。反応時間は、通常１０分～４８時間、好ましくは１０分～２４時間、より好ましくは３０分～１８時間である。 The reaction temperature is usually −20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably −10 ° C. to 40 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.

　工程２、すなわち式（８）で表される化合物と式（３）で表される化合物を反応させて式（１Ａ）で表わされる化合物を合成する工程は、例えば不活性溶媒中、塩基の存在下で行うことができる。不活性溶媒及び塩基の詳細は前記と同様である。 Step 2, that is, the step of reacting the compound represented by the formula (8) with the compound represented by the formula (3) to synthesize the compound represented by the formula (1A) is, for example, the presence of a base in an inert solvent. Can be done below. The details of the inert solvent and the base are the same as described above.

　式（３）で表される化合物の使用量は、式（８）で表される化合物１モルに対して、通常、０．５モル以上、０．８モル以上、好ましくは０．９～２モル、より好ましくは０．９～１．５モルである。 The amount of the compound represented by the formula (3) to be used is usually 0.5 mol or more, 0.8 mol or more, preferably 0.9 to 2 with respect to 1 mol of the compound represented by the formula (8). It is mol, more preferably 0.9 to 1.5 mol.

　反応温度は、通常－２０℃～溶媒の沸点より１０℃高い温度、好ましくは－１０℃～４０℃である。反応時間は、通常１０分～４８時間、好ましくは１０分～２４時間、より好ましくは３０分～１８時間である。 The reaction temperature is usually −20 ° C. to 10 ° C. higher than the boiling point of the solvent, preferably −10 ° C. to 40 ° C. The reaction time is usually 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and more preferably 30 minutes to 18 hours.

　カンナビジオールは、下の化学構造式で示される2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-Benzenediolであり、本発明者らによって非選択的なTRPチャネル活性化作用を有し、TRPC6チャネル活性化作用も有することが見出された。カンナビジオールは公知の方法により製造するか、天然から得ることができる。

Figure JPOXMLDOC01-appb-C000031
Cannabidiol is represented by the following chemical structural formula 2-[(1R, 6R) -3-methyl-6-(1-methylethenyl) -2-cyclohexen-1-yl] -5-pentyl-1,3- It is Benzenediol, which was found by the present inventors to have a non-selective TRP channel activating effect and also a TRPC6 channel activating effect. Cannabidiol can be produced by known methods or obtained from nature.
Figure JPOXMLDOC01-appb-C000031

　GSK1702934A（1,3-Dihydro-1-[1-[(5,6,7,8-tetrahydro-4H-cyclohepta[b]thien-2-yl)carbonyl]-4-piperidinyl]-2H-benzimidazol-2-one）は下記の構造を有する公知の化合物であり、TRPC3チャネル活性化作用を有する。GSK1702934Aの製造方法は公知であり、例えば非特許文献３に記載の方法で製造できる。またGSK1702934Aは市販品を使用することもできる。

Figure JPOXMLDOC01-appb-C000032
GSK1702934A (1,3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2 -one) is a known compound having the following structure and has a TRPC3 channel activating effect. The method for producing GSK1702934A is known, and for example, it can be produced by the method described in Non-Patent Document 3. The GSK1702934A can also be a commercially available product.
Figure JPOXMLDOC01-appb-C000032

　本発明による式（１）で表される化合物（式（１）で表される化合物には式（１－１）で表される化合物及び式（１－２）で表される化合物が包含される。）及びその中間体化合物は、上記の製造法により製造することができ、また、出発原料化合物、本発明による式（１）で表される化合物、及びその中間体化合物は、本明細書の実施例等に記載された合成方法に基づき本出願時に既知または公知の技術（例えば、B. R. Kiran et al., SYNTHESIS, EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITIES OF SUBSTITUTED 1,2-BENZOXAZOLONE AND 3-CHLORO-1,2-BENZOXAZOLE DERIVATIVES, International Journal of Pharmaceutical Sciences and Research, 2015; 6: 2918-2925.）を参酌して製造することもできる。 The compound represented by the formula (1) according to the present invention (the compound represented by the formula (1) includes the compound represented by the formula (1-1) and the compound represented by the formula (1-2). ) And its intermediate compounds can be produced by the above-mentioned production method, and the starting material compound, the compound represented by the formula (1) according to the present invention, and the intermediate compound thereof are described herein. Known or known techniques (eg, B.R. Kiran et al., SYNTHESIS, EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITIES OF SUBSTITUTED 1,2-BENZOXAZOLONE AND 3-CHLORO-1,2-BENZOXAZOLE DERIVATIVES, International Journal of Pharmaceutical Sciences and Research, 2015; 6: 2918-2925.) Can also be taken into consideration when manufacturing.

　上記反応式のそれぞれに示された出発原料化合物及び中間体化合物は、反応に供する前に、必要に応じて、公知の方法を用いて官能基を適当な保護基で保護し、反応終了後、公知の方法で当該保護基を脱保護することができる。 The starting material compound and the intermediate compound shown in each of the above reaction formulas are subjected to the reaction, if necessary, the functional groups are protected with an appropriate protecting group by a known method, and after the reaction is completed, the functional groups are protected. The protecting group can be deprotected by a known method.

　上記の反応式に従って得られる目的化合物のそれぞれを単離および精製することができる。例えば、反応混合物を冷却した後に、粗反応生成物を分離するために濾過、濃縮、抽出等の単離手順を行い、その後粗反応生成物を、カラムクロマトグラフィー、再結晶化等の一般的な精製手順に供することにより、反応混合物から単離および精製することができる。 Each of the target compounds obtained according to the above reaction formula can be isolated and purified. For example, after cooling the reaction mixture, isolation procedures such as filtration, concentration, and extraction are performed to separate the crude reaction product, and then the crude reaction product is subjected to general column chromatography, recrystallization, etc. By subjecting to a purification procedure, it can be isolated and purified from the reaction mixture.

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、及び上記反応式のそれぞれに示された出発原料化合物、中間体化合物等には、溶媒が付加した溶媒和物（例えば、水和物、エタノール和物等）の形態である化合物が含まれる。 A solvent is used in the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and the starting material compound, intermediate compound, etc. shown in each of the above reaction formulas. Contains compounds in the form of added solvates (eg, hydrates, ethanol solvates, etc.).

　上記各反応工程式において得られる中間体化合物、出発原料化合物、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、式（１）で表される化合物、カンナビジオール、並びに1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンに、二重結合、環、縮合環における異性体（Ｅ、Ｚ、シス、トランス体）、不斉炭素の存在等による異性体（Ｒ、Ｓ体、α、β体、エナンチオマー、ジアステレオマー）、旋光性を有する光学活性体（Ｄ、Ｌ、ｄ、ｌ体）、クロマトグラフ分離による極性体（高極性体、低極性体）、平衡化合物、回転異性体、これらの任意の割合の混合物、ラセミ混合物は幾何異性体、立体異性体、光学異性体などの異性体が存在する場合には、全ての異性体が包含される。例えば、光学異性体は、公知の各種の分割法（例えば、結晶化による光学分割、クロマトグラフィーによる直接光学分割する方法等）を利用して分離することができる。 A substance that activates at least one channel selected from the group consisting of the intermediate compound, the starting material compound, the TRPC3 channel, the TRPC6 channel, and the TRPC7 channel obtained in each of the above reaction step formulas, represented by the formula (1). Compounds, cannavidiol, and 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl]- 2H-benzimidazol-2-one with isomers (E, Z, cis, trans) in double bonds, rings, fused rings, isomers due to the presence of asymmetric carbon (R, S, α, β) (Body, enantiomer, diastereomeric), optically active substance (D, L, d, l-form) with diversion, polar body (high polar body, low polar body) by chromatograph separation, equilibrium compound, rotational isomer, The mixture of these arbitrary proportions and the racemic mixture include all isomers when isomers such as geometric isomers, steric isomers and optical isomers are present. For example, optical isomers can be separated by using various known splitting methods (for example, optical resolution by crystallization, direct optical resolution by chromatography, etc.).

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、式（１）で表される化合物、カンナビジオール、並びに1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンの薬学的に許容される塩としては、特に制限されず、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩；亜鉛塩等の無機金属塩；トリエチルアミン、トリエタノールアミン、トリヒドロキシメチルアミノメタン、アミノ酸等の有機塩基塩；塩酸塩、臭化水素酸塩、硫酸塩、リン酸塩、硝酸塩等の無機酸塩；酢酸塩、炭酸塩、プロピオン酸塩、コハク酸塩、乳酸塩、リンゴ酸塩、酒石酸塩、クエン酸塩、マレイン酸塩、フマル酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩、ベンゼンスルホン酸塩、アスコルビン酸塩等の有機酸塩等が挙げられる。これらの塩は常法に従って製造することができる。 A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, the compound represented by the formula (1), cannavidiol, and 1,3-dihydro-1-[ 1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-A pharmaceutically acceptable salt of benzimidazol-2-one The above is not particularly limited, and for example, alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic metal salts such as zinc salt; triethylamine, triethanolamine and trihydroxy. Organic base salts such as methylaminomethane and amino acids; inorganic acid salts such as hydrochlorides, hydrobromates, sulfates, phosphates and nitrates; acetates, carbonates, propionates, succinates and lactates. , Maltate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, benzenesulfonate, organic acid salts such as ascorbate and the like. These salts can be produced according to a conventional method.

　各種の異性体は公知の分離法により単離できる。例えば、ラセミ化合物は一般的な光学分割法（例えば、結晶化による光学分割、クロマトグラフィーによる直接光学分割する方法等）により、立体的に純粋な異性体に導くことができる。また、光学活性な化合物は適当な光学活性な原料を用いることにより製造することもできる。 Various isomers can be isolated by a known separation method. For example, a racemic compound can be derived into a sterically pure isomer by a general optical resolution method (for example, optical resolution by crystallization, direct optical resolution by chromatography, etc.). The optically active compound can also be produced by using an appropriate optically active raw material.

　上記反応工程式のそれぞれに表される出発原料化合物、中間体化合物、及び目的化合物を適切な塩形態で使用することができる。 The starting material compound, the intermediate compound, and the target compound represented in each of the above reaction process formulas can be used in an appropriate salt form.

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質等では、１つまたは複数の原子を１つまたは複数の同位体原子で置換することができる。同位体原子の例としては重水素（２Ｈ）、三重水素（３Ｈ）、１３Ｃ、１４Ｎ、１８Ｏ等が挙げられる。 In a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, one or more atoms can be replaced with one or more isotope atoms. .. Examples of isotope atoms include deuterium (2H), tritium (3H), 13C, 14N, 18O and the like.

［医薬組成物］
　本発明には、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグ、及び薬学的に許容される担体を含有する医薬組成物、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグ、及び薬学的に許容される担体を含有する医薬組成物が包含される。TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンが細胞の拡散分子取り込み量を増大させる機能を有するため、本発明の医薬組成物は、核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグをさらに含有すること、及び、核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグを含有する薬剤とともに組み合わせて処方されることが好ましい。 [Pharmaceutical composition]
In the present invention, (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof. And a pharmaceutical composition containing a pharmaceutically acceptable carrier, or a compound represented by (b) formula (1), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,,). At least one compound selected from the group consisting of 7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceuticals. Included are pharmaceutical compositions containing an acceptable salt, or a prodrug thereof, and a pharmaceutically acceptable carrier. A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a compound represented by the formula (1), cannavidiol, and 1,3-dihydro-1-[ 1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one increases the amount of diffuse molecule uptake in cells Due to their ability to increase, the pharmaceutical compositions of the invention may further contain nucleic acid molecules, pharmaceutically acceptable salts thereof, or prodrugs thereof, and nucleic acid molecules, pharmaceutically acceptable thereof. It is preferably formulated in combination with agents containing salts or prodrugs thereof.

　本発明の医薬組成物は、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、又はそれらのプロドラッグを通常の医薬組成物の形態に製剤したものである。本発明の医薬組成物は、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグと、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物と、薬学的に許容される担体とを用いて調製される。当該担体としては、通常使用される充填剤、増量剤、結合剤、付湿剤、崩壊剤、表面活性剤、滑沢剤等の希釈剤あるいは賦形剤が挙げられる。 The pharmaceutical composition of the present invention comprises a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a compound represented by the formula (1), cannabidiol, and 1 , 3-Dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one , The pharmaceutically acceptable salt thereof, or their prodrugs are formulated in the form of conventional pharmaceutical compositions. The pharmaceutical composition of the present invention comprises (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. Prodrugs or compounds represented by formula (1) (b), benzimidazole, and 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b) ] Thien-2-yl) carbonyl] -4-piperidinyl] -2H-Benzimidazole-2-one prepared using at least one compound selected from the group and a pharmaceutically acceptable carrier. To. Examples of the carrier include commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants and other diluents or excipients.

　本明細書において、プロドラッグは、生体内における反応（例えば酵素反応、胃酸による反応）によって本発明の化合物に変換される化合物をいう。例えば、本発明の化合物がカルボキシルを有する場合、該カルボキシルがエステルに変換された化合物である。該エステルとしては、メチルエステル、エチルエステル、１－プロピルエステル、２－プロピルエステル、ピバロイルオキシメチルエステル、アセチルオキシメチルエステル、シクロヘキシルアセチルオキシメチルエステル、１－メチルシクロヘキシルカルボニルオキシメチルエステル、エチルオキシカルボニルオキシ－１－エチルエステル、シクロヘキシルオキシカルボニルオキシ－１－エチルエステルなどが挙げられる。 In the present specification, a prodrug refers to a compound that is converted into the compound of the present invention by a reaction in vivo (for example, an enzymatic reaction or a reaction with gastric acid). For example, when the compound of the present invention has a carboxyl, the carboxyl is converted into an ester. Examples of the ester include methyl ester, ethyl ester, 1-propyl ester, 2-propyl ester, pivaloyloxymethyl ester, acetyloxymethyl ester, cyclohexylacetyloxymethyl ester, 1-methylcyclohexylcarbonyloxymethyl ester and ethyloxy. Examples thereof include carbonyloxy-1-ethyl ester and cyclohexyloxycarbonyloxy-1-ethyl ester.

　本発明の医薬組成物としては、治療目的に応じて種々の形態の中から選択でき、その代表的なものとして錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、坐剤、注射剤（液剤、懸濁剤等）、軟膏剤、吸入剤等が挙げられる。 The pharmaceutical composition of the present invention can be selected from various forms according to the purpose of treatment, and typical examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules and capsules. Examples include suppositories, injections (liquids, suspensions, etc.), ointments, inhalants and the like.

　錠剤の成形に使用する担体としては、公知のものを広く使用でき、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、尿素、デンプン、炭酸カルシウム、カオリン、結晶セルロース等の賦形剤、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン溶液、メチルセルロース、リン酸カリウム、ポリビニルピロリドン、カルボキシメチルセルロース、セラック等の結合剤、アルギン酸ナトリウム、乾燥デンプン、寒天末、ラミナラン末、炭酸カルシウム、炭酸水素ナトリウム、ポリオキシエチレンソルビタン脂肪酸エステル類、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、デンプン、乳糖等の崩壊剤、第４級アンモニウム塩基、ラウリル硫酸ナトリウム等の吸収促進剤、ステアリン、カカオバター、水素添加油等の崩壊抑制剤、グリセリン、デンプン等の保湿剤、デンプン、乳糖、カオリン、ベントナイト、コロイド状ケイ酸等の吸着剤、精製タルク、ステアリン酸塩、ホウ酸末、ポリエチレングリコール等の滑沢剤等が挙げられる。 As the carrier used for molding tablets, known carriers can be widely used, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, water, ethanol, etc. Binders such as propanol, simple syrup, glucose solution, starch solution, gelatin solution, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, carboxymethyl cellulose, cellac, sodium alginate, dried starch, agar powder, laminaran powder, calcium carbonate, sodium hydrogen carbonate. , Polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrants such as lactose, absorption promoters such as quaternary ammonium base, sodium lauryl sulfate, stearic acid, cocoa butter, hydrogenated oil, etc. Disintegration inhibitors, moisturizers such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearic acid, sodium borate, lubricants such as polyethylene glycol and the like. Be done.

　さらに、錠剤は、必要に応じて通常の錠皮を施した錠剤、例えば、糖衣剤、ゼラチン被包錠、腸溶被錠、フィルムコーティング錠あるいは二重錠、多層錠とすることができる。 Further, the tablet can be a tablet with a normal lock skin, for example, a sugar coating, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet, or a multi-layer tablet, if necessary.

　丸剤の成形に使用する担体としては、公知のものを広く使用でき、例えば、ブドウ糖、乳糖、デンプン、カカオ脂、硬化植物油、カオリン、タルク等の賦形剤、アラビアゴム末、トラガント末、ゼラチン、エタノール等の結合剤、ラミナラン、寒天等の崩壊剤等が挙げられる。 Known carriers can be widely used for molding pills, for example, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, talc, gum arabic powder, tragant powder, gelatin. , Binders such as ethanol, disintegrants such as laminarane and agar, and the like.

　坐剤の成形に使用する担体としては、公知のものを広く使用でき、例えば、ポリエチレングリコール、カカオ脂、高級アルコール、高級アルコールのエステル類、ゼラチン、半合成グリセライド等が挙げられる。 As the carrier used for molding the suppository, known ones can be widely used, and examples thereof include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glyceride and the like.

　注射剤として調製される場合は、液剤、乳剤および懸濁剤は殺菌され、かつ血液と等張であるのが好ましい。これらの液剤、乳剤および懸濁剤の調製の際に使用する希釈剤としては、公知のものを広く使用することができ、例えば、水、エタノール、プロピレングリコール、ポリオキシ化イソステアリルアルコール、エトキシ化イソステアリルアルコール、ポリオキシエチレンソルビタン脂肪酸エステル類等が挙げられる。なお、注射剤の場合、等張性の溶液を調製するのに十分な量の食塩、グリセリン、ブドウ糖等を医薬製剤中に含有させることができ、また通常の溶解補助剤、緩衝剤、無痛化剤等を含有させることができ、更に必要に応じて着色剤、保存剤、香料、風味剤、甘味剤等や他の医薬品を含有させることができる。 When prepared as an injection, the liquid, emulsion and suspension are preferably sterilized and isotonic with blood. As the diluent used in the preparation of these liquids, emulsions and suspending agents, known ones can be widely used, for example, water, ethanol, propylene glycol, polyoxylated isostearyl alcohol, ethoxylated iso. Examples thereof include stearyl alcohol and polyoxyethylene sorbitan fatty acid esters. In the case of an injection, a sufficient amount of salt, glycerin, glucose, etc. can be contained in the pharmaceutical preparation to prepare an isotonic solution, and usual solubilizing agents, buffers, and pain-relieving agents can be contained. Agents and the like can be contained, and if necessary, colorants, preservatives, flavors, flavoring agents, sweeteners and the like and other pharmaceutical products can be contained.

　軟膏剤は、ペースト、クリーム、またはゲルなどの形態があり、これらの形態に調製するに際しては、希釈剤として、例えば、白色ワセリン、パラフィン、グリセリン、セルロース誘導体、ポリエチレングリコール、シリコーン、ベントナイト等を使用できる。 The ointment has a form such as a paste, a cream, or a gel, and when preparing these forms, for example, white petrolatum, paraffin, glycerin, a cellulose derivative, polyethylene glycol, silicone, bentonite, etc. are used as a diluent. can.

　吸入剤は、有効成分をエアゾールとして吸入させることにより、気管支または肺に適用することを目的とした製剤であり、粉末吸入剤、吸入液剤、吸入エアゾール剤等が含まれる。粉末吸入剤は、粉末状固体粒子のエアゾールとして吸入する製剤をいい、通常、有効成分を微細な粒子とし、必要に応じて乳糖等の添加剤と混和して均質とすることにより製造することができる。吸入液剤は、ネブライザ等により適用する液状の吸入剤をいい、通常、有効成分に溶剤および適切な等張化剤、ｐＨ調節剤等を加え、混和することにより製造できる。吸入エアゾール剤は、容器に充填した噴射剤と共に、一定量の有効成分を噴霧する定量噴霧式吸入剤である。吸入エアゾール剤は、通常、有効成分に溶剤および適切な分散剤、安定化剤等を加えて、溶液または懸濁液とし、液状の噴射剤と共に耐圧性の容器に充填し、定量バルブを装着することにより製造することができる。 The inhalant is a preparation intended to be applied to the bronchi or lungs by inhaling the active ingredient as an aerosol, and includes a powder inhalant, an inhalation solution, an inhalation aerosol and the like. A powder inhalant is a preparation that is inhaled as an aerosol of solid powder particles, and is usually produced by making the active ingredient into fine particles and mixing them with an additive such as lactose to make them homogeneous. can. The inhalation solution refers to a liquid inhalation agent applied by a nebulizer or the like, and can usually be produced by adding a solvent, an appropriate tonicity agent, a pH adjuster, or the like to the active ingredient and mixing them. The inhalation aerosol agent is a fixed-quantity spray-type inhalant that sprays a certain amount of the active ingredient together with the propellant filled in the container. Inhalation aerosols are usually prepared by adding a solvent, an appropriate dispersant, a stabilizer, etc. to the active ingredient to make a solution or suspension, filling a pressure-resistant container with a liquid propellant, and installing a metering valve. It can be manufactured by.

　本発明の医薬組成物は、必要に応じて着色剤、保存剤、香料、風味剤、甘味剤などや他の医薬品を含有してもよい。 The pharmaceutical composition of the present invention may contain a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent, and other pharmaceutical products, if necessary.

　本発明の医薬組成物中に含有されるTRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグの量は、特に限定されず広い範囲内から適宜選択することができるが、医薬組成物中に、通常、０．５～９０質量％、１～８５質量％、好ましくは１～８０質量％である。 A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel contained in the pharmaceutical composition of the present invention, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. The amount of the prodrug is not particularly limited and may be appropriately selected from a wide range, but is usually 0.5 to 90% by mass, preferably 1 to 85% by mass, preferably 1 to 80% by mass in the pharmaceutical composition. %.

　本発明の医薬組成物の投与方法は特に制限されず、各種製剤形態、患者の年齢、性別、疾患の状態、その他の条件に応じた方法で投与される。例えば、錠剤、丸剤、液剤、懸濁剤、乳剤、顆粒剤およびカプセル剤の場合には経口投与される。また、注射剤の場合には、単独であるいはブドウ糖、アミノ酸等の通常の補液と混合して静脈内に投与したり、更には必要に応じて単独で筋肉内、皮内、皮下、腹腔内等に投与することができる。坐剤の場合には、直腸内に投与される。吸入剤の場合には経鼻投与される。 The administration method of the pharmaceutical composition of the present invention is not particularly limited, and the pharmaceutical composition is administered by a method according to various pharmaceutical forms, the age, sex, disease state, and other conditions of the patient. For example, in the case of tablets, pills, liquids, suspensions, emulsions, granules and capsules, they are orally administered. In the case of an injection, it may be administered intravenously alone or mixed with a normal replacement fluid such as glucose or amino acid, or if necessary, intramuscularly, intradermally, subcutaneously, intraperitoneally, etc. Can be administered to. In the case of suppositories, it is administered intrarectally. In the case of inhalants, it is administered nasally.

　本発明の医薬組成物の投与量は、用法、患者の年齢、性別、疾患の程度、その他の条件を考慮して選択すればよく、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグ、或いは、核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグが、通常、１日あたり体重１ｋｇに対して０．０１～１００ｍｇ、好ましくは０．１～５０ｍｇとなる量で、１日あたり１回～数回に分けて、あるいは２日、３日、４日、５日、６日、１週間、２週間または４週間に１回の間隔で投与される。投与量は、種々の条件で変動するので、上記範囲より少ない投与量で充分な場合もあるし、また上記範囲を超えた投与量が必要な場合もある。 The dose of the pharmaceutical composition of the present invention may be selected in consideration of usage, age, sex, degree of disease, and other conditions of the patient, and is selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. A substance that activates at least one channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof is usually used. The amount is 0.01 to 100 mg, preferably 0.1 to 50 mg per 1 kg of body weight per day, divided into 1 to several times per day, or 2 days, 3 days, 4 days, 5 days. , 6 days, 1 week, 2 weeks or 4 weeks at intervals. Since the dose varies depending on various conditions, a dose smaller than the above range may be sufficient, or a dose exceeding the above range may be required.

　また、本発明の医薬組成物は、他の薬剤と組み合わせた併用剤とすることもできる。本発明の医薬組成物は、核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグを含有する薬剤とともに組み合わせて処方されることが好ましい。また、本発明の医薬組成物は、抗がん剤と併用してもよい。抗がん剤としては、ドキソルビシン、イダルビジン、マイトマイシンＣ等の抗腫瘍性抗生物質、ドセタキセル、ビンクリスチン等の微小管阻害剤、カルボプラチン、シスプラチン、オキサリプラチン等のプラチナ製剤、ボリノスタット等のヒストン脱アセチル化酵素（HDAC）阻害剤、スニチニブ、イマチニブ、ゲフェチニブ、エルロチニブ、アファチニブ、ダサチニブ、トラメチニブ等のキナーゼ阻害剤、イリノテカン、エトポシド等のトポイソメラーゼ阻害剤、シクロスポリン、タクロリムス等のカルシニューリン阻害薬、シクロホスファミド、ベンダムスチン、イオスファミド、ダカルバジン等のアルキル化薬、ペントスタチン、フルダラビン、クラドリビン、メソトレキセート、５－フルオロウラシル、６－メルカプトプリン、エノシタビン等の代謝拮抗薬、リツキシマブ、セツキシマブ、トラスツズマブ等の分子標的薬、ボルテゾミブ等のプロテアソーム阻害剤、タモキシフェン、ビカルダミド等のホルモン療法薬、ニボルマブ、ペンブロリズマブ、ピジリズマブ等の抗ＰＤ－１抗体、アテゾリズマブ、アベルマブ、又はデュルバルマブ、ＢＭＳ－９３６５５９等の抗ＰＤ－Ｌ１抗体、イピリムマブ又はトレメリムマブ等の抗ＣＴＬＡ－４抗体を挙げることができる。 Further, the pharmaceutical composition of the present invention can be used as a concomitant drug in combination with other drugs. The pharmaceutical composition of the present invention is preferably formulated in combination with a drug containing a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof. In addition, the pharmaceutical composition of the present invention may be used in combination with an anticancer agent. Examples of anticancer agents include antitumor antibiotics such as doxorubicin, idarvidin and mitomycin C, microtube inhibitors such as docetaxel and bincristine, platinum preparations such as carboplatin, cisplatin and oxaliplatin, and histone deacetylases such as bolinostat. (HDAC) Inhibitors, kinase inhibitors such as snitinib, imatinib, gefetinib, errotinib, afatinib, dasatinib, tramethinib, topoisomerase inhibitors such as irinotecan and etopocid, carcinurin inhibitors such as cyclosporin and tachlorimus, cyclophosphamide, cyclophosphamide Alkylating agents such as iosphamide and dacarbazine, metabolic antagonists such as pentostatin, fludalabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine and enocitabine, molecular targeting agents such as rituximab, setuccimab and trusszumab, and proteasome inhibitors such as voltezomib. Drugs, hormonal therapeutic agents such as tamoxifen, bicardamide, anti-PD-1 antibodies such as nibolumab, penbrolizumab, pidirisumab, atezolizumab, avelumab, or anti-PD-L1 antibodies such as durvalumab, BMS-936559, anti-CTLA such as ipilimumab or tremellimumab. 4 Inhibitors can be mentioned.

　本発明の剤又は医薬組成物は、核酸分子の細胞内への取り込みを促進できるため、核酸分子と併用されることが望ましい。また本発明の剤又は医薬組成物は、核酸分子の投与により予防又は治療が期待される疾患に対して好ましく使用される。 Since the agent or pharmaceutical composition of the present invention can promote the uptake of nucleic acid molecules into cells, it is desirable to use them in combination with nucleic acid molecules. Further, the agent or pharmaceutical composition of the present invention is preferably used for diseases that are expected to be prevented or treated by administration of nucleic acid molecules.

　予防又は治療対象となる疾患としては、核酸分子の投与が期待される疾患、例えば肺がん、前立腺がん、乳がん、肺線維症、腎線維症、筋ジストロフィー、筋萎縮性側索硬化症、ハンチントン病、筋萎縮症、パーキンソン病等が挙げられる。好ましい疾患は、肺がん、前立腺がんである。 Diseases to be prevented or treated include diseases for which administration of nucleic acid molecules is expected, such as lung cancer, prostate cancer, breast cancer, pulmonary fibrosis, renal fibrosis, muscular dystrophy, amyotrophic lateral sclerosis, and Huntington's disease. Examples include muscular atrophy and Parkinson's disease. Preferred diseases are lung cancer and prostate cancer.

　また、本発明の剤又は医薬組成物は、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有するため、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有するため、TRPチャネルを多く発現している細胞膜を有する細胞の核酸分子の取り込みの促進、オートファジーの増強、核酸分子の活性の増強に特に有用である。細胞としては、例えば小細胞肺がん細胞、非小細胞肺がん細胞、前立腺がん細胞、乳がん細胞、筋細胞又は神経細胞等が挙げられ、好ましくは小細胞肺がん細胞、非小細胞肺がん細胞、筋細胞、前立腺がん細胞及び神経細胞である。また、細胞はヒトの細胞が好ましい。 In addition, the agent or pharmaceutical composition of the present invention is (a) a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, and a pharmaceutically acceptable salt thereof. , Or to contain prodrugs thereof, or the compound represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,7,8-). At least one compound selected from the group consisting of tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, pharmaceutically acceptable thereof. Since it contains salts or prodrugs thereof, it is particularly useful for promoting the uptake of nucleic acid molecules, enhancing autophagy, and enhancing the activity of nucleic acid molecules in cells having a cell membrane expressing a large amount of TRP channels. Examples of the cells include small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells, nerve cells and the like, preferably small cell lung cancer cells, non-small cell lung cancer cells, muscle cells, etc. Prostate cancer cells and nerve cells. Further, the cells are preferably human cells.

　本発明は、核酸分子投与を受ける対象に、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与すること、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与することを含む、細胞内への核酸分子の取り込みを促進する方法を包含しうる。また、本発明は、核酸分子投与を受ける対象に、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与すること、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与することを含む、細胞のオートファジーを増強する方法を包含しうる。さらにまた、本発明は、核酸分子投与を受ける対象に、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与すること、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与することを含む、核酸分子の活性を増強する方法を包含しうる。 The present invention is a substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel, and a pharmaceutically acceptable salt thereof, for a subject receiving nucleic acid molecule administration. , Or an effective amount of their prodrug, or (b) the compound represented by formula (1), cannavidiol, and 1,3-dihydro-1- [1-[(5,6,7). , 8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or the efficacy of their prodrugs It may include methods of facilitating the uptake of nucleic acid molecules into cells, including administration of an amount. Further, the present invention is pharmaceutically acceptable as a substance that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel for a subject to be administered with a nucleic acid molecule. Or administer an effective amount of a salt or a prodrug thereof, or (b) the compound represented by formula (1), benzimidazole, and 1,3-dihydro-1- [1-[(5,6). , 7,8-Tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or their prodrugs It may include methods of enhancing cell autophagy, including administration of an effective amount of. Furthermore, the present invention is a pharmaceutically acceptable substance for a subject receiving nucleic acid molecule administration, which activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel. To administer an effective amount of the salt to be obtained, or a prodrug thereof, or (b) the compound represented by the formula (1), benzimidazole, and 1,3-dihydro-1- [1-[(5,,). 6,7,8-Tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or their pros It may include methods of enhancing the activity of a nucleic acid molecule, including administering an effective amount of the drug.

　本発明は、核酸分子の投与が期待される疾患の予防又は治療を受ける対象に、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグの有効量を投与すること、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、またはそれらのプロドラッグの有効量を投与することを含む、核酸分子の投与が期待される疾患の予防又は治療方法を包含しうる。 The present invention activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel for a subject to be prevented or treated for a disease for which administration of a nucleic acid molecule is expected. Administering an effective amount of the substance, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or (b) the compound represented by formula (1), benzimidazole, and 1,3-dihydro-1-. [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable Can include prophylactic or therapeutic methods of disease for which administration of benzimidazole is expected, including administration of effective amounts of the salts, or prodrugs thereof.

　本発明は、（１）細胞内への核酸分子取り込み促進剤、（２）オートファジー増強剤、（３）核酸分子活性増強剤、又は（４）核酸分子の投与が期待される疾患の予防又は治療剤の製造のための、（ａ）TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、またはそれらのプロドラッグの使用、或いは（ｂ）式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オン、その薬学的に許容される塩、またはそれらのプロドラッグの使用を包含しうる。 The present invention relates to (1) an agent for promoting the uptake of nucleic acid molecules into cells, (2) an autophagy enhancer, (3) an agent for enhancing nucleic acid molecule activity, or (4) prevention of diseases in which administration of nucleic acid molecules is expected. A substance, a pharmaceutically acceptable salt thereof, or a substance thereof that activates at least one channel selected from the group consisting of (a) TRPC3 channel, TRPC6 channel, and TRPC7 channel for the production of a therapeutic agent. Use of prodrugs, or compounds represented by formula (1) (b), cannavidiol, and 1,3-dihydro-1-[1-[(5,6,7,8-tetrahydro-4H-cyclohepta] b] Thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one, its pharmaceutically acceptable salts, or the use of prodrugs thereof may be included.

　以下、実施例等によって本発明の一実施態様を更に詳細に説明するが、本発明はこれらに限定されない。 Hereinafter, one embodiment of the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited thereto.

参考例１Reference example 1
２－（４－（２－クロロフェニル）ピペラジン－１－イル）酢酸ｔｅｒｔブチルエステル2- (4- (2-Chlorophenyl) piperazine-1-yl) tert-butyl acetate

Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033

　2－クロロフェニルピペラジン（１．９６６ｇ、１０mmol；C1102（東京化成社））、2－ブロモ酢酸tertブチルエステル（１．９５ｇ、１０mmol）、トリエチルアミン（２．０９mＬ、１５mmol）のＴＨＦ（４０ｍＬ）溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、2－（4－（2－クロロフェニル）ピペラジン－1－イル）ブロモ酢酸tertブチルエステル（２．７４ｇ）を得た。収率８８％。 A solution of 2-chlorophenylpiperazine (1.966 g, 10 mmol; C1102 (Tokyo Kasei Co., Ltd.)), 2-bromoacetic acid tert-butyl ester (1.95 g, 10 mmol) and triethylamine (2.09 mL, 15 mmol) in THF (40 mL) at room temperature. The mixture was stirred overnight, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 2- (4- (2-chlorophenyl) piperazine-1-yl) bromoacetic acid tert-butyl ester (2.74 g). Yield 88%.

参考例２Reference example 2
２－（４－（２－クロロフェニル）ピペラジン－１－イル）酢酸　塩酸塩2- (4- (2-Chlorophenyl) piperazine-1-yl) acetic acid hydrochloride

Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034

　参考例１で得た2－（4－（2－クロロフェニル）ピペラジン－1－イル）ブロモ酢酸tertブチルエステルを過剰の塩酸及び酢酸エチルで処理し、2－（4－（2－クロロフェニル）ピペラジン－1－イル）酢酸　塩酸塩を得た。 The 2- (4- (2-chlorophenyl) piperazine-1-yl) bromoacetic acid tert-butyl ester obtained in Reference Example 1 was treated with excess hydrochloric acid and ethyl acetate, and 2- (4- (2-chlorophenyl) piperazine- 1-Il) Acetic acid hydrochloride was obtained.

参考例３Reference example 3
２－ブロモ－Ｎ－（２－エトキシフェニル）アセトアミド（CAS349120-97-4）2-Bromo-N- (2-ethoxyphenyl) acetamide (CAS349120-97-4)

Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000035

　２－エトキシアニリン（１．３７ｇ、９．９９mmol）とトリエチルアミン（１．４６２ｍL、１０．４９mmol）のジクロロメタン（２０ｍL）の溶液に、ブロモ酢酸ブロミド（０．８７ｍL、９．９９mmol）を０℃で滴下し、反応混合物を０℃で１時間撹拌し、２－ブロモ－Ｎ－（２－エトキシフェニル）アセトアミドを含む反応液を得た。得られた反応溶液をそのまま実施例８及び９の反応に用いた。 Bromoacetic acid bromide (0.87 mL, 9.99 mmol) is added dropwise at 0 ° C. to a solution of 2-ethoxyaniline (1.37 g, 9.99 mmol) and triethylamine (1.462 mL, 10.49 mmol) in dichloromethane (20 mL). Then, the reaction mixture was stirred at 0 ° C. for 1 hour to obtain a reaction solution containing 2-bromo-N- (2-ethoxyphenyl) acetamide. The obtained reaction solution was used as it was for the reactions of Examples 8 and 9.

参考例４Reference example 4
４－（４－（２－（ｔｅｒｔ－ブトキシ）－２－オキソエチル）ピペラジン－１－イル）－３－クロロ安息香酸メチルエステル4- (4- (2- (tert-butoxy) -2-oxoethyl) piperazine-1-yl) -3-chlorobenzoic acid methyl ester

Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000036

　３－クロロ－４－（ピペラジン－１－イル）安息香酸メチルエステル（２．５４ｇ、９．９７mmol；ST-0611（Combi-Blocks社））、2－ブロモ酢酸tertブチルエステル（２．０４２ｇ、１０．４７mmol）、トリエチルアミン（２．０８５mＬ、１４．９６mmol）のＤＭＦ（４０ｍＬ）溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、４－（４－（２－（ｔｅｒｔ－ブトキシ）－２－オキソエチル）ピペラジン－１－イル）－３－クロロ安息香酸メチルエステル（３．８１８ｇ）を得た。収率１００％。
LC-MS: r.t. 1.96 min., m/z 369.1 (M++1)。 3-Chloro-4- (piperazin-1-yl) benzoic acid methyl ester (2.54 g, 9.97 mmol; ST-0611 (Combi-Blocks)), 2-bromoacetic acid tert-butyl ester (2.042 g, 10) A solution of .47 mmol) and triethylamine (2.085 mL, 14.96 mmol) in DMF (40 mL) was stirred overnight at room temperature, saturated aqueous ammonium chloride was added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and 4- (4- (2- (tert-butoxy) -2-oxoethyl) piperazine-1-yl) -3-chlorobenzoic acid methyl ester (3). .818 g) was obtained. Yield 100%.
LC-MS: rt 1.96 min., m / z 369.1 (M + + 1).

参考例５Reference example 5
２－（４－（２－（クロロ－４－（メトキシカルボニル）フェニル）ピペラジン－１－イル）酢酸　塩酸塩2- (4- (2- (Chloro-4- (methoxycarbonyl) phenyl) piperazine-1-yl) acetic acid hydrochloride

Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000037

　４－（４－（２－（ｔｅｒｔ－ブトキシ）－２－オキソエチル）ピペラジン－１－イル）－３－クロロ安息香酸メチルエステル（１．３７９ｇ、３．１５mmol）を４Ｎ塩酸－１，４－ジオキサン（１３ｍＬ）で処理し、溶媒を留去、乾燥し２－（４－（２－（クロロ－４－（メトキシカルボニル）フェニル）ピペラジン－１－イル）酢酸　塩酸塩（１．１ｇ）を得た。収率８０％。
LC-MS: r.t. 1.44 min., m/z 313.0 (M+ + 1)。 4- (4- (2- (tert-butoxy) -2-oxoethyl) piperazine-1-yl) -3-chlorobenzoic acid methyl ester (1.379 g, 3.15 mmol) with 4N hydrochloric acid-1,4-dioxane The treatment was carried out with (13 mL), the solvent was distilled off, and the mixture was dried to obtain 2- (4- (2- (chloro-4- (methoxycarbonyl) phenyl) piperazine-1-yl) acetate hydrochloride (1.1 g). . Yield 80%.
LC-MS: rt 1.44 min., m / z 313.0 (M + + 1).

実施例１Example 1
３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル（化合物（ａ））3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) benzoic acid methyl ester (Compound (a))

Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000038

　２－（４－（２－（クロロ－４－（メトキシカルボニル）フェニル）ピペラジン－１－イル）酢酸　塩酸塩（３４９．２ｍｇ，１．０mmol）、２－エトキシアニリン（１６５ｍｇ、１．２mmol）、ＨＡＴＵ（４５６ｍｇ、１．２mmol）とトリエチルアミン（４１０μＬ、３．０mmol）のＤＭＦ（５ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル（１１３．３ｍｇ、Ｒｆ＝０．４５（１：１ヘキサン／酢酸エチル））を得た。収率２６％。
１H NMR (CDCl３, 400 MHz) δ9.85 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 8.05 (d, 1 H, J = 1.6 Hz), 7.91 (dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (dd, 1 H, J = 2.0, 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 8.0 Hz), 6.87 (dd, 1 H, J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.90 (s, 3 H), 3.20 - 3.30 (br, 4 H), 3.25 (s, 2 H), 2.80 - 2.90 (bt, 4H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 2.57 min., m/z 432.2 (M+ + 1)。 2- (4- (2- (Chloro-4- (methoxycarbonyl) phenyl) piperazine-1-yl) acetate hydrochloride (349.2 mg, 1.0 mmol), 2-ethoxyaniline (165 mg, 1.2 mmol), A solution of HATU (456 mg, 1.2 mmol) and DMF (5 mL) of triethylamine (410 μL, 3.0 mmol) was stirred overnight at room temperature, saturated aqueous ammonium chloride was added, and the organic layer was extracted with magnesium sulfate. After drying, the solvent was distilled off under reduced pressure, the residue was purified by normal phase preparative liquid chromatography, and 3-chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine. -1-yl) Methyl benzoate (113.3 mg, Rf = 0.45 (1: 1 hexane / ethyl acetate)) was obtained. The yield was 26%.
1 H NMR (CDCl 3 , 400 MHz) δ9.85 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 8.05 (d, 1 H, J = 1.6 Hz), 7.91 ( dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (dd, 1 H, J = 2.0, 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 8.0 Hz), 6.87 (dd, 1 H, J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.90 (s, 3 H), 3.20 --3.30 (br, 4 H), 3.25 (s, 2 H), 2.80 --2.90 (bt, 4H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: rt 2.57 min., m / z 432.2 (M +) +1).

実施例２Example 2
２－（４－（２－クロロ－４－ピぺリジン－１－カルボニル）フェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド（化合物（ｂ））2- (4- (2-Chloro-4-piperidin-1-carbonyl) phenyl) piperazine-1-yl) -N- (2-ethoxyphenyl) acetamide (compound (b))

Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000039

実施例２－１
３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸 Example 2-1
3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) benzoic acid

Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000040

　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル（２００ｍｇ、０．４６３mmol）を５：２ジメトキシエタン／１Ｎ水酸化ナトリウム（７ｍＬ）で処理し、１Ｎ塩酸で中和し酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥、溶媒を減圧留去し、３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸（１６４ｍｇ）を得た。収率８５％。
１H NMR (CDCl３, 400 MHz) δ9.83 (bs, 1 H), 8.43 (dd, 1 H, J = 1.6, 8.0 Hz), 7.39 (d, 1 H, J = 8.0 Hz), 7.08 (d, 1 H, J = 2.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.99 (dd, 1 H, J = 1.6, 8.0 Hz), 6.96 (dt, 1 H, J = 2.0, 8.0 Hz), 6.87 (dd, 1 H, J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.62 - 3.78 (br, 2 H), 3.25 (s, 2 H), 3.11- 3.21 (br, 4H), 2.76 - 2.88 (br, 4 H), 1.63 - 1.73 (br, 1 H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 1.97 min., m/z 418.2 (M+ + 1)。 3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) benzoic acid methyl ester (200 mg, 0.463 mmol) 5: 2 dimethoxyethane / It was treated with 1N sodium hydroxide (7 mL), neutralized with 1N hydrochloric acid and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and 3-chloro-4- (4-(2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) benzoic acid ( 164 mg) was obtained. Yield 85%.
1 H NMR (CDCl 3 , 400 MHz) δ9.83 (bs, 1 H), 8.43 (dd, 1 H, J = 1.6, 8.0 Hz), 7.39 (d, 1 H, J = 8.0 Hz), 7.08 ( d, 1 H, J = 2.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.99 (dd, 1 H, J = 1.6, 8.0 Hz), 6.96 (dt, 1 H, J = 2.0, 8.0 Hz), 6.87 (dd, 1 H, J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.62 --3.78 (br, 2 H), 3.25 (s, 2 H) ), 3.11-3.21 (br, 4H), 2.76 --2.88 (br, 4 H), 1.63 --1.73 (br, 1 H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: rt 1.97 min., m / z 418.2 (M + + 1).

実施例２－２
２－（４－（２－クロロ－４－ピぺリジン－１－カルボニル）フェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド Example 2-2
2- (4- (2-Chloro-4-piperidin-1-carbonyl) phenyl) piperazine-1-yl) -N- (2-ethoxyphenyl) acetamide

Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000041

　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸（１６４ｍｇ，０．３９２mmol）、ピぺリジン（２０１ｍｇ、２．３５５mmol）とＨＡＴＵ（２２４ｍｇ、０．５８９mmol）のＤＭＦ（２ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル（１７８．６ｍｇ）を得た。収率９４％。
１H NMR (CDCl３, 400 MHz) δ9.87 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.53 (d, 1 H, J = 2.0 Hz), 7.29 (dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.2, 7.6 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.52 - 3.75 (br, 2 H), 3.31 - 3.52 (br, 2 H), 3.25 (s, 2 H), 3.12 - 3.22 (br, 4H), 2.80 - 2.88 (brt, 4 H), 1.65 - 1.73 (br, 4 H), 1.54 - 1.65 (br, 2 H), 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 2.31 min., m/z 485.1 (M+ + 1)。 3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazin-1-yl) benzoic acid (164 mg, 0.392 mmol), piperidine (201 mg, 2. A solution of DMF (2 mL) of 355 mmol) and HATU (224 mg, 0.589 mmol) was stirred overnight at room temperature, saturated aqueous ammonium chloride was added and extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to perform 3-chloro-4- (4- (2-((2-ethoxyphenyl) amino)). -2-oxoethyl) piperazine-1-yl) benzoic acid methyl ester (178.6 mg) was obtained. Yield 94%.
1 H NMR (CDCl 3 , 400 MHz) δ9.87 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.53 (d, 1 H, J = 2.0 Hz), 7.29 ( dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.2, 7.6 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.52 --3.75 (br, 2 H), 3.31 --3.52 (br, 2 H), 3.25 (s, 2 H), 3.12 --3.22 (br, 4H), 2.80 --2.88 (brt, 4 H), 1.65 --1.73 (br, 4 H), 1.54 --1.65 (br, 2 H) , 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: rt 2.31 min., m / z 485.1 (M + + 1).

実施例３Example 3
３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）－Ｎ，Ｎ－ジメチルベンズアミド（化合物（ｃ））3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) -N, N-dimethylbenzamide (Compound (c))

Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000042

　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸（２０．９ｍｇ，０．０５mmol）、ジメチルアミン（１１．３ｍｇ、０．２５mmol）、ＨＡＴＵ（２８．５ｍｇ、０．０７５mmol）とトリエチルアミン（２０．９μＬ、０．１５mmol）のＤＭＦ（１ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）－Ｎ，Ｎ－ジメチルベンズアミド（２２．２ｍｇ、Ｒｆ＝０．３３（１：２ヘキサン／酢酸エチル））を得た。収率１００％。
１H NMR (CDCl３, 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 2.0, 8.0 Hz), 7.46 (d, 1 H, J = 2.0 Hz), 7.33 (dd, 1 H, J = 2.0, 8.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 8.0 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.12 - 3.22 (br, 4H), 3.08 (bs, 3 H), 3.04 (bs, 3 H), 2.81 - 2.87 (br, 4 H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 1.93 min., m/z 445.2 (M+ + 1)。 3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazine-1-yl) Benzoic acid (20.9 mg, 0.05 mmol), dimethylamine (11.3 mg) , 0.25 mmol), a solution of HATU (28.5 mg, 0.075 mmol) and triethylamine (20.9 μL, 0.15 mmol) in DMF (1 mL) overnight at room temperature, saturated ammonium chloride water added, ethyl acetate. Extracted with. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to perform 3-chloro-4- (4- (2-((2-ethoxyphenyl) amino)). -2-oxoethyl) piperazin-1-yl) -N, N-dimethylbenzamide (22.2 mg, Rf = 0.33 (1: 2 hexane / ethyl acetate)) was obtained. Yield 100%.
1 H NMR (CDCl 3 , 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 2.0, 8.0 Hz), 7.46 (d, 1 H, J = 2.0 Hz), 7.33 ( dd, 1 H, J = 2.0, 8.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 8.0 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.12 --3.22 (br, 4H) , 3.08 (bs, 3 H), 3.04 (bs, 3 H), 2.81 --2.87 (br, 4 H), 1.50 (t, 3 H, J = 6.8 Hz), LC-MS: rt 1.93 min., m / z 445.2 (M + + 1).

実施例４Example 4
１－（３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）ベンゾイル）ピぺリジン－４－カルボン酸メチルエステル（化合物（ｄ））1- (3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazin-1-yl) benzoyl) piperidine-4-carboxylic acid methyl ester (compound (compound) d))

Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000043

　３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸（１０５ｍｇ，０．２５１mmol）、ピぺリジン－４－カルボン酸メチルエステル（４３．２ｍｇ、０．３０２mmol）とＨＡＴＵ（１４３ｍｇ、０．３７７mmol）のＤＭＦ（２ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、１－（３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）ベンゾイル）ピぺリジン－４－カルボン酸メチルエステル（１１７ｍｇ）を得た。収率８６％。
１H NMR (CDCl３, 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.43 (d, 1 H, J = 2.0 Hz), 7.30 (dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 7.6 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.71 (s, 3 H), 3.25 (s, 2 H), 3.52 - 3.75 (br, 2 H), 3.31 - 3.52 (br, 2 H), 3.25 (s, 2 H), 3.12 - 3.22 (br, 4H), 3.0 - 3.12 (br, 1 H), 2.80 - 2.88 (brt, 4 H), 2.60 (tt, 1 H, J = 4.0, 6.8 Hz), 1.85 - 2.10 (br, 2 H), 1.65 - 1.80 (br, 2 H), 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 2.26 min., m/z 543.2 (M+ + 1)。 3-Chloro-4- (4- (2-((2-ethoxyphenyl) amino) -2-oxoethyl) piperazin-1-yl) benzoic acid (105 mg, 0.251 mmol), piperazine-4-carboxylic acid A solution of methyl ester (43.2 mg, 0.302 mmol) and HATU (143 mg, 0.377 mmol) in DMF (2 mL) was stirred overnight at room temperature, saturated ammonium chloride water was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to perform 1- (3-chloro-4- (4- (2-((2-ethoxyphenyl)). ) Amino) -2-oxoethyl) piperazin-1-yl) benzoyl) piperidine-4-carboxylic acid methyl ester (117 mg) was obtained. Yield 86%.
1 H NMR (CDCl 3 , 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.43 (d, 1 H, J = 2.0 Hz), 7.30 ( dd, 1 H, J = 2.0, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 7.03 (d, 1 H, J = 8.0 Hz), 6.96 (dt, 1 H, J = 1.6, 7.6 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.71 (s, 3 H), 3.25 (s, 2 H), 3.52 --3.75 (br, 2 H), 3.31 --3.52 (br, 2 H), 3.25 (s, 2 H), 3.12 --3.22 (br, 4H), 3.0 --3.12 (br, 1 H), 2.80 --2.88 (brt, 4 H), 2.60 (tt, 1 H, J = 4.0, 6.8 Hz), 1.85 ―― 2.10 (br, 2 H), 1.65 ―― 1.80 (br, 2 H), 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: rt 2.26 min., m / z 543.2 (M + + 1).

実施例５Example 5
Ｎ－（４－クロロ－２－メトキシフェニル）－２－（４－（２－クロロフェニル）ピペラジン－１－イル）アセトアミド（化合物（ｅ））N- (4-Chloro-2-methoxyphenyl) -2- (4- (2-chlorophenyl) piperazine-1-yl) acetamide (Compound (e))

Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000044

　２－（４－（２－クロロフェニル）ピペラジン－１－イル）酢酸（２５．５ｍｇ，０．１mmol）、４－クロロ－２－メトキシアニリン　塩酸塩（２３．３ｍｇ、０．１２mmol）、ＨＡＴＵ（５７．１ｍｇ、０．１５mmol）とトリエチルアミン（６９．８μＬ、０．５mmol）のＤＭＦ（１ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、Ｎ－（４－クロロ－２－メトキシフェニル）－２－（４－（２－クロロフェニル）ピペラジン－１－イル）アセトアミド（２１．３ｍｇ、Ｒｆ＝０．２（１：２ヘキサン／酢酸エチル））を得た。収率５４％。
１H NMR (CDCl３, 400 MHz) δ9.78 (bs, 1 H), 8.34 (dd, 1 H, J = 1.6, 8.0 Hz), 7.37 (d, 1 H, J = 8.0 Hz), 7.29 (dd, 1 H, J = 2.0, 8.4 Hz), 7.06 (d, 1 H, J = 8.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.96 (dt, 1 H, J = 1.2, 7.6 Hz), 6.87 (s, 1 H), 3.90 (s, 3 H), 3.25 (s, 2 H), 3.08 - 3.20 (br, 4H), 2.76 - 2.88 (br, 4 H), LC-MS: r.t. 2.38 min., m/z 394.1 (M+)。 2- (4- (2-Chlorophenyl) piperazin-1-yl) acetic acid (25.5 mg, 0.1 mmol), 4-chloro-2-methoxyaniline hydrochloride (23.3 mg, 0.12 mmol), HATU (57) A solution of DMF (1 mL) of .1 mg, 0.15 mmol) and triethylamine (69.8 μL, 0.5 mmol) was stirred overnight at room temperature, saturated aqueous ammonium chloride was added, and the mixture was extracted with ethyl acetate. The organic layer was dried with magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to purify N- (4-chloro-2-methoxyphenyl) -2- (4- (2- (2-). A chlorophenyl) piperazin-1-yl) acetamide (21.3 mg, Rf = 0.2 (1: 2 hexane / ethyl acetate)) was obtained. Yield 54%.
1 H NMR (CDCl 3 , 400 MHz) δ9.78 (bs, 1 H), 8.34 (dd, 1 H, J = 1.6, 8.0 Hz), 7.37 (d, 1 H, J = 8.0 Hz), 7.29 ( dd, 1 H, J = 2.0, 8.4 Hz), 7.06 (d, 1 H, J = 8.0 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.96 (dt, 1 H, J = 1.2, 7.6 Hz), 6.87 (s, 1 H), 3.90 (s, 3 H), 3.25 (s, 2 H), 3.08 --3.20 (br, 4H), 2.76 --2.88 (br, 4 H), LC -MS: rt 2.38 min., m / z 394.1 (M + ).

実施例６Example 6
２－（２－（４－（２－クロロフェニル）ピペラジン－１－イル）アセトアミド）安息香酸メチルエステル（化合物（ｆ））2- (2- (4- (2-Chlorophenyl) piperazine-1-yl) acetamide) Methyl benzoate (Compound (f))

Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000045

　２－（４－（２－クロロフェニル）ピペラジン－１－イル）酢酸（１３．２ｍｇ，０．０５２mmol）、２－アミノ安息香酸メチルエステル（２３．５ｍｇ、０．１５５mmol）、トリエチルアミン（３６．１μL、０．２５９mmol）とＨＡＴＵ（３９．４ｍｇ、０．１０４mmol）のＤＭＦ（０．５ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、３－クロロ－４－（４－（２－（（２－エトキシフェニル）アミノ）－２－オキソエチル）ピペラジン－１－イル）安息香酸メチルエステル（２．０ｍｇ）を得た。収率１０％。
１H NMR (CDCl３, 400 MHz) δ12.10 (bs, 1 H), 8.80 (d, 1 H, J = 8.8 Hz), 8.04 (d, 1 H, J = 8.4 Hz), 7.55 (t, 1 H, J = 8.4 Hz), 7.38 (d, 1 H, J = 8.0 Hz), 7.25 (t, 1 H, J = 8.0 Hz), 7.12 (d, 1 H, J = 8.0 Hz), 7.10 (t, 1 H, J = 8.0 Hz), 6.99 (t, 1 H. J = 8.0 Hz), 3.92 (s, 3 H), 3.29 (s, 2 H), 3.20 - 3.28 (br, 4H), 2.80 - 2.88 (br, 4 H), LC-MS: r.t. 2.89 min., m/z 388.1 (M+ + 1)。 2- (4- (2-Chlorophenyl) piperazin-1-yl) acetic acid (13.2 mg, 0.052 mmol), 2-aminobenzoic acid methyl ester (23.5 mg, 0.155 mmol), triethylamine (36.1 μL,) A solution of 0.259 mmol) and HATU (39.4 mg, 0.104 mmol) in DMF (0.5 mL) was stirred overnight at room temperature, saturated aqueous ammonium chloride was added and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to perform 3-chloro-4- (4- (2-((2-ethoxyphenyl) amino)). -2-oxoethyl) piperazine-1-yl) benzoic acid methyl ester (2.0 mg) was obtained. Yield 10%.
1 H NMR (CDCl 3 , 400 MHz) δ12.10 (bs, 1 H), 8.80 (d, 1 H, J = 8.8 Hz), 8.04 (d, 1 H, J = 8.4 Hz), 7.55 (t, 1 H, J = 8.4 Hz), 7.38 (d, 1 H, J = 8.0 Hz), 7.25 (t, 1 H, J = 8.0 Hz), 7.12 (d, 1 H, J = 8.0 Hz), 7.10 ( t, 1 H, J = 8.0 Hz), 6.99 (t, 1 H. J = 8.0 Hz), 3.92 (s, 3 H), 3.29 (s, 2 H), 3.20 --3.28 (br, 4H), 2.80 --2.88 (br, 4 H), LC-MS: rt 2.89 min., m / z 388.1 (M + + 1).

実施例７Example 7
２－（２－（４－（２－トリフルオロメチル）フェニル）ピペラジン－１－イル）アセトアミド）安息香酸メチルエステル（化合物（ｇ））2- (2- (4- (2-trifluoromethyl) phenyl) piperazin-1-yl) acetamide) Methyl benzoate (compound (g))

Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000046

　２－アミノ安息香酸メチルエステル（４５３ｍｇ、３mmol）とトリエチルアミン（１．２５４ｍL、９mmol）のジクロロメタン（１５ｍL）の溶液に、ブロモ酢酸ブロミド（０．２６ｍL、３mmol）を０℃で滴下し、反応混合物を０℃で１時間撹拌した。続いて、１－（２－（トリフルオロメチル）フェニル）ピペラジン（６９１ｍｇ、３mmol）のDMF（１０ｍL）の溶液を0℃で滴下し、室温で終夜撹拌して、飽和塩化アンモニウム水を加え酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、２－（２－（４－（２－トリフルオロメチル）フェニル）ピペラジン－１－イル）アセトアミド）安息香酸メチルエステル（１５７ｍｇ）を得た。収率１２．４％。
１H NMR (CDCl３, 400 MHz) δ12.14 (bs, 1 H), 8.81 (d, 1 H, J = 8.0 Hz), 8.04 (dd, 1 H, J = 1.6, 8.0 Hz), 7.64 (d, 1 H, J = 8.0 Hz), 7.55 (dt, 1 H, J = 1.6, 8.4 Hz), 7.53 (d, 1 H, J = 7.6 Hz), 7.48 (d, 1 H, J = 7.6 Hz), 7.24 (t, 1 H, J = 7.6 Hz), 7.10 (t, 1 H, J = 8.0 Hz), 3.97 (s, 3 H), 3.28 (s, 2 H), 3.08 - 3.16 (br, 4H), 2.76 - 2.83 (br, 4 H), LC-MS: r.t. 3.04 min., m/z 422.2 (M+ + 1)。 Bromoacetic acid bromide (0.26 mL, 3 mmol) was added dropwise at 0 ° C. to a solution of 2-aminobenzoic acid methyl ester (453 mg, 3 mmol) and triethylamine (1.254 mL, 9 mmol) in dichloromethane (15 mL) to give the reaction mixture. The mixture was stirred at 0 ° C. for 1 hour. Subsequently, a solution of 1- (2- (trifluoromethyl) phenyl) piperazine (691 mg, 3 mmol) in DMF (10 mL) was added dropwise at 0 ° C., stirred overnight at room temperature, saturated aqueous ammonium chloride water was added, and ethyl acetate was added. Extracted with. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by normal phase preparative liquid chromatography to 2-(2- (4- (2-trifluoromethyl) phenyl) piperazine-1-. Il) acetamide) Methyl benzoate (157 mg) was obtained. Yield 12.4%.
1 H NMR (CDCl 3 , 400 MHz) δ12.14 (bs, 1 H), 8.81 (d, 1 H, J = 8.0 Hz), 8.04 (dd, 1 H, J = 1.6, 8.0 Hz), 7.64 ( d, 1 H, J = 8.0 Hz), 7.55 (dt, 1 H, J = 1.6, 8.4 Hz), 7.53 (d, 1 H, J = 7.6 Hz), 7.48 (d, 1 H, J = 7.6 Hz) ), 7.24 (t, 1 H, J = 7.6 Hz), 7.10 (t, 1 H, J = 8.0 Hz), 3.97 (s, 3 H), 3.28 (s, 2 H), 3.08 --3.16 (br, 4H), 2.76 --2.83 (br, 4 H), LC-MS: rt 3.04 min., m / z 422.2 (M + + 1).

実施例８Example 8
２－（４－（２，５－ジクロロフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド（化合物（ｈ））2- (4- (2,5-dichlorophenyl) piperazine-1-yl) -N- (2-ethoxyphenyl) acetamide (compound (h))

Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000047

　１－（２，５－ジクロロフェニル）ピペラジン塩酸塩（80 ｍｇ、0.3 mmol）とトリエチルアミン（104μL、0.75 mmol）のDMF（2 ｍL）の溶液に２－ブロモ－N－（２－エトキシフェニル）アセトアミド（64.5 ｍｇ、0.25 mmol相当）のジクロロメタン溶液（64.5 ｍｇ、0.25 mmol相当）を0℃で滴下し、室温で終夜撹拌して、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、２－（４－（２，５－ジクロロフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド（54.1 ｍｇ）を得た。収率５３％。
１H NMR (CDCl３, 400 MHz)δ9.85 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.29 (d, 1 H, J = 8.0 Hz), 7.29 (dd, 1 H, J = 2.0, 8.4 Hz), 7.04 (dt, 1 H, J = 2.0, 7.6 Hz), 6.98 - 7.00 (m, 2 H), 6.96 (dt, 1 H, J = 1.2, 8.0 Hz), 6.88 (dd, 1 H. J = 1.6, 8.0 Hz), 4.12 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.08 - 3.18 (br, 4 H), 2.78 - 2.88 (brt, 4 H), 1.53 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 2.85 min., m/z 408.1 (M+ + 1)。 2-bromo-N- (2-ethoxyphenyl) acetamide (2-bromo-N- (2-ethoxyphenyl) acetamide in a solution of 1- (2,5-dichlorophenyl) piperazine hydrochloride (80 mg, 0.3 mmol) and triethylamine (104 μL, 0.75 mmol) in DMF (2 mL). A solution of nitrogen (64.5 mg, equivalent to 0.25 mmol) (64.5 mg, equivalent to 0.25 mmol) was added dropwise at 0 ° C., the mixture was stirred overnight at room temperature, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, the residue was purified by normal phase preparative liquid chromatography, and 2- (4- (2,5-dichlorophenyl) piperazine-1-yl) -N- (2-ethoxyphenyl) acetamide (54.1 mg) was obtained. Yield 53%.
1 H NMR (CDCl 3 , 400 MHz) δ9.85 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.29 (d, 1 H, J = 8.0 Hz), 7.29 ( dd, 1 H, J = 2.0, 8.4 Hz), 7.04 (dt, 1 H, J = 2.0, 7.6 Hz), 6.98 --7.00 (m, 2 H), 6.96 (dt, 1 H, J = 1.2, 8.0 Hz), 6.88 (dd, 1 H. J = 1.6, 8.0 Hz), 4.12 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.08 --3.18 (br, 4 H), 2.78 --2.88 (brt, 4 H), 1.53 (t, 3 H, J = 6.8 Hz), LC-MS: rt 2.85 min., m / z 408.1 (M + + 1).

実施例９Example 9
２－（４－（２，４－ジクロロフェニル）ピペラジン－１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド（化合物（ｉ））2- (4- (2,4-dichlorophenyl) piperazine-1-yl) -N- (2-ethoxyphenyl) acetamide (Compound (i))

Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000048

　１－（２，４－ジクロロフェニル）ピペラジン（69.3 ｍｇ、0.3 mmol）とトリエチルアミン（52μL、0.375 mmol）のDMF（2 ｍL）の溶液に2－ブロモ－N－（２－エトキシフェニル）アセトアミド（64.5 ｍｇ、0.25 mmol相当）のジクロロメタン溶液を0℃で滴下し、室温で終夜撹拌して、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥し、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、２－（４－（２，４－ジクロロフェニル）ピペラジンー１－イル）－Ｎ－（２－エトキシフェニル）アセトアミド（55.7 ｍｇ）を得た。収率５４．６％。１H NMR (CDCl３, 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.39 (d, 1 H, J = 2.0 Hz), 7.21 (dd, 1 H, J = 2.4, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.97 (dt, 1 H, J = 1.6, 8.0 Hz), 6.95 (d, 1 H, J = 8.4 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.10 - 3.21 (br, 4 H), 2.78 - 2.88 (brt, 4 H), 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: r.t. 2.86 min., m/z 408.1 (M+ + 1)。 2-bromo-N- (2-ethoxyphenyl) acetamide (64.5 mg) in a solution of 1- (2,4-dichlorophenyl) piperazine (69.3 mg, 0.3 mmol) and triethylamine (52 μL, 0.375 mmol) in DMF (2 mL). , 0.25 mmol) was added dropwise at 0 ° C., the mixture was stirred overnight at room temperature, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, the residue was purified by normal phase preparative liquid chromatography, and 2- (4- (2,4-dichlorophenyl) piperazin-1-yl) -N- ( 2-ethoxyphenyl) acetamide (55.7 mg) was obtained. Yield 54.6%. 1 H NMR (CDCl 3 , 400 MHz) δ9.86 (bs, 1 H), 8.44 (dd, 1 H, J = 1.6, 8.0 Hz), 7.39 (d, 1 H, J = 2.0 Hz), 7.21 ( dd, 1 H, J = 2.4, 8.4 Hz), 7.03 (dt, 1 H, J = 2.0, 7.6 Hz), 6.97 (dt, 1 H, J = 1.6, 8.0 Hz), 6.95 (d, 1 H, J = 8.4 Hz), 6.87 (dd, 1 H. J = 1.6, 8.0 Hz), 4.11 (q, 2 H, J = 6.8 Hz), 3.25 (s, 2 H), 3.10 --3.21 (br, 4 H) ), 2.78 --2.88 (brt, 4 H), 1.51 (t, 3 H, J = 6.8 Hz), LC-MS: rt 2.86 min., m / z 408.1 (M + + 1).

実施例１０Example 10
２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（３－メトキシピリジン－４－イル）アセトアミド（化合物（ｊ））2- (4- (2,3-Dimethylphenyl) piperazine-1-yl) -N- (3-methoxypyridin-4-yl) acetamide (Compound (j))

Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000049

　２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）酢酸　塩酸塩（０．３ｍｍｏｌ）、３－メトキシピリジン－４－アミン（０．３ mmol）、ＨＡＴＵ（０．４mmol）とジイソプロピルエチルアミン（０．８mmol）のＤＭＦ（１ｍＬ）の溶液を室温で終夜撹拌し、飽和塩化アンモニウム水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（３－メトキシピリジン－４－イル）アセトアミド（１１．２ｍｇ、Ｒｆ＝０．３３（３：１ヘキサン／酢酸エチル））を得た。収率５％。
１H NMR (CDCl３, 400 MHz)δ10.05 (bs, 1 H), 8.33 (d, 1 H, J = 5.2 Hz), 8.23 (bd, 2 H), 7.12 (t, 1 H, J = 8.0 Hz), 6.94 (d, 2 H, J = 8.0 Hz), , 4.01 (s, 3 H), 3.26 (s, 2 H), 2.95 - 3.02 (br, 4H), 2.78 - 2.84 (br, 4 H), 2.28 (s, 3 H), 2.23 (s, 3 H), LC-MS: r.t. 1.54 min., m/z 355.0 (M+ + 1)。 2- (4- (2,3-dimethylphenyl) piperazine-1-yl) acetate hydrochloride (0.3 mmol), 3-methoxypyridin-4-amine (0.3 mmol), HATU (0.4 mmol) A solution of DMF (1 mL) of diisopropylethylamine (0.8 mmol) was stirred overnight at room temperature, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was dried with magnesium sulfate, the solvent was distilled off under reduced pressure, the residue was purified by normal phase preparative liquid chromatography, and 2- (4- (2,3-dimethylphenyl) piperazin-1-yl) -N- (3-methoxypyridin-4-yl) acetamide (11.2 mg, Rf = 0.33 (3: 1 hexane / ethyl acetate)) was obtained. Yield 5%.
1 H NMR (CDCl 3 , 400 MHz) δ 10.05 (bs, 1 H), 8.33 (d, 1 H, J = 5.2 Hz), 8.23 (bd, 2 H), 7.12 (t, 1 H, J = 8.0 Hz), 6.94 (d, 2 H, J = 8.0 Hz), 4.01 (s, 3 H), 3.26 (s, 2 H), 2.95 --3.02 (br, 4H), 2.78 --2.84 (br, 4) H), 2.28 (s, 3 H), 2.23 (s, 3 H), LC-MS: rt 1.54 min., m / z 355.0 (M + + 1).

実施例１１Example 11
２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（２－メトキシピリジン－３－イル）アセトアミド（化合物（ｋ））2- (4- (2,3-Dimethylphenyl) piperazine-1-yl) -N- (2-methoxypyridin-3-yl) acetamide (Compound (k))

Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000050

　２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）酢酸　塩酸塩（０．３ｍｍｏｌ）、２－メトキシピリジン－３－アミン（０．３ mmol）、ＨＡＴＵ（０．４mmol）とジイソプロピルエチルアミン（０．８mmol）のＤＭＦ（１ｍＬ）の溶液を室温で終夜撹拌し、飽和重曹水を加え、酢酸エチルで抽出した。有機層を硫酸マグネシウムで乾燥、溶媒を減圧留去し、残渣を順相分取液体クロマトグラフィーで精製し、２－（４－（２，３－ジメチルフェニル）ピペラジン－１－イル）－Ｎ－（２－メトキシピリジン－３－イル）アセトアミド（８５．３ｍｇ、Ｒｆ＝０．３８（３：１ヘキサン／酢酸エチル））を得た。収率８０％。
１H NMR (CDCl３, 400 MHz) δ9.78 (bs, 1 H), 8.60 (dd, 1 H, J = 7.6, 1.6 Hz), 7.86 (dd, 1 H, J = 5.2, 1.6 Hz), 7.12 (t, 1 H, J = 8.0 Hz), 6.96 (d, 1 H, J = 8.0 Hz), 6.94 (d, 1 H, J = 8.0 Hz), 6.91 (dd, 1 H, J = 7.6, 5.2 Hz), 4.04 (s, 3 H), 3.24 (s, 2 H), 2.97 - 3.01 (br, 4H), 2.78 - 2.84 (br, 4 H), 2.28 (s, 3 H), 2.23 (s, 3 H), LC-MS: r.t. 2.02 min., m/z 354.9 (M++ 1)。 2- (4- (2,3-dimethylphenyl) piperazine-1-yl) acetate hydrochloride (0.3 mmol), 2-methoxypyridin-3-amine (0.3 mmol), HATU (0.4 mmol) A solution of DMF (1 mL) of diisopropylethylamine (0.8 mmol) was stirred overnight at room temperature, saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate. The organic layer was dried with magnesium sulfate, the solvent was distilled off under reduced pressure, the residue was purified by normal phase preparative liquid chromatography, and 2- (4- (2,3-dimethylphenyl) piperazin-1-yl) -N- (2-methoxypyridin-3-yl) acetamide (85.3 mg, Rf = 0.38 (3: 1 hexane / ethyl acetate)) was obtained. Yield 80%.
1 H NMR (CDCl 3 , 400 MHz) δ9.78 (bs, 1 H), 8.60 (dd, 1 H, J = 7.6, 1.6 Hz), 7.86 (dd, 1 H, J = 5.2, 1.6 Hz), 7.12 (t, 1 H, J = 8.0 Hz), 6.96 (d, 1 H, J = 8.0 Hz), 6.94 (d, 1 H, J = 8.0 Hz), 6.91 (dd, 1 H, J = 7.6, 5.2 Hz), 4.04 (s, 3 H), 3.24 (s, 2 H), 2.97 --3.01 (br, 4H), 2.78 --2.84 (br, 4 H), 2.28 (s, 3 H), 2.23 (s) , 3 H), LC-MS: rt 2.02 min., m / z 354.9 (M + + 1).

試験例１
TRPCチャネルを一過的に発現したHEK293細胞内へのCa ２＋ の流入
（１）TRPCチャネルのHEK293細胞への導入および発現
　HEK293細胞（ヒト胎児腎由来細胞）にTRPC6遺伝子をリポフェクション法で導入し、細胞膜にTRPC6チャネルを発現させた。すなわち、TRPC6のプラスミドDNA（pCI-neo (promega社)）を、リポフェクション剤を用いてHEK293細胞へ導入後、37℃で24時間～48時間培養することで、TRPC6チャネルが細胞膜に発現したHEK293細胞（TRPC6発現HEK293細胞）を作製した。具体的には、非特許文献１（S. Sawamura et al., Mol. Pharmacol., 89, 348-363 (2016)）に記載された方法に従った。TRPC3遺伝子またはTRPC7遺伝子を使用し、同様にして、TRPC3チャネルが細胞膜に発現したHEK293細胞（TRPC3発現HEK293細胞）及びTRPC7チャネルが細胞膜に発現したHEK293細胞（TRPC7発現HEK293細胞）を作製した。 Test Example 1
Influx of Ca 2+ into HEK293 cells transiently expressing TRPC channels (1) Introduction and expression of TRPC channels into HEK293 cells The TRPC6 gene was introduced into HEK293 cells (human fetal kidney-derived cells) by the lipofection method. TRPC6 channels were expressed on the cell membrane. That is, HEK293 cells in which TRPC6 channels were expressed on the cell membrane by introducing TRPC6 plasmid DNA (pCI-neo (promega)) into HEK293 cells using a lipofection agent and then culturing at 37 ° C for 24 to 48 hours. (TRPC6-expressing HEK293 cells) were prepared. Specifically, the method described in Non-Patent Document 1 (S. Sawamura et al., Mol. Pharmacol., 89, 348-363 (2016)) was followed. Using the TRPC3 gene or TRPC7 gene, HEK293 cells (TRPC3-expressing HEK293 cells) in which the TRPC3 channel was expressed on the cell membrane and HEK293 cells (TRPC7-expressing HEK293 cells) in which the TRPC7 channel was expressed on the cell membrane were generated in the same manner.

（２）TRPC発現HEK293細胞内におけるカルシウムイオン濃度変化の測定方法　TRPC3、TRPC6またはTRPC7発現させたHEK293培養細胞をトリプシンで剥がし、カバーガラスに撒き直したのち、再び37℃で3時間培養した。カルシウム指示薬Fura-2 AM(終濃度1μM)が入った培地500μlにカバーガラスを移し替え、30分間37℃で培養し細胞に取り込ませた。細胞外溶液にはCa２＋含有溶液およびCa２＋フリー溶液を用意した。
Ca２＋含有溶液：(2 mM CaCl2, 132 mM NaCl, 4 mM KCl, 1mM MgCl2, 5 mMグルコース, 5 mM HEPES (pH7.4))
Ca２＋フリー溶液：(132 mM NaCl, 4 mM KCl, 1mM MgCl2, 5 mMグルコース, 5 mM HEPES (pH7.4)) (2) Method for measuring changes in calcium ion concentration in TRPC-expressed HEK293 cells TRPC3, TRPC6 or TRPC7-expressed HEK293 cultured cells were peeled off with trypsin, re-spread on a cover glass, and then cultured again at 37 ° C. for 3 hours. The cover glass was transferred to 500 μl of a medium containing the calcium indicator Fura-2 AM (final concentration 1 μM), cultured at 37 ° C. for 30 minutes, and incorporated into cells. Ca 2+ -containing solution and Ca 2+ free solution were prepared as extracellular solutions.
Ca 2+ -containing solution: (2 mM CaCl 2 , 132 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM glucose, 5 mM HEPES (pH 7.4))
Ca 2+ free solution: (132 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM glucose, 5 mM HEPES (pH 7.4))

　Ca2+含有溶液に細胞を入れた後、室温で、Fura-2の蛍光強度（510 nm）をAQUACOSMOS（Hamamatsu Photonics社）で経時的に測定しつつ以下の操作を行った。０分：Ca２＋含有溶液を入れた測定用チャンバーにTRPC3発現細胞、TRPC6発現細胞またはTRPC7発現細胞を入れた。
２分後：Ca２＋含有溶液に所定量の試験化合物を添加した。
１０分後：測定終了 After putting the cells in the Ca 2+ -containing solution, the following operation was performed while measuring the fluorescence intensity (510 nm) of Fura-2 over time with AQUACOSMOS (Hamamatsu Photonics) at room temperature. 0 min: TRPC3-expressing cells, TRPC6-expressing cells or TRPC7-expressing cells were placed in a measurement chamber containing a Ca 2+ -containing solution.
After 2 minutes: A predetermined amount of the test compound was added to the Ca 2+ -containing solution.
10 minutes later: Measurement finished

試験例１－１Test Example 1-1
TRPC6チャネルを発現したHEK293細胞内へのCaCa into HEK293 cells expressing TRPC6 channel ２＋2+ の流入に対する実施例の化合物の作用Effect of the compounds of the Examples on the influx of

　試験例１に記載の測定方法に従ってカルシウムイオン濃度変化を測定した。試験化合物には実施例1～3の化合物、実施例5～11の化合物及びPPZ2を使用し、その添加量は30μMであった。また、試験化合物に代えて0.1％のDMSOを使用し、コントロールとした。Fura-2の蛍光強度を経時的に測定することによって、TRPC6チャネル発現HEK293細胞内におけるCa２＋増加量（試験化合物添加後に測定されたCa２＋濃度の最大値から初期の（試験化合物添加前の）Ca２＋濃度を控除した値）を求めた。DMSOを使用したコントロールよりもCa２＋増加量が大きかった試験化合物を、TRPC6チャネルを活性化する物質とした。PPZ2のCa２＋増加量を100％とし、他の試験化合物のCa２＋増加量を相対値として算出した。結果を表１に示す。 The change in calcium ion concentration was measured according to the measuring method described in Test Example 1. The compounds of Examples 1 to 3, the compounds of Examples 5 to 11 and PPZ2 were used as the test compounds, and the addition amount thereof was 30 μM. In addition, 0.1% DMSO was used instead of the test compound as a control. By measuring the fluorescence intensity of Fura-2 over time, the amount of Ca 2+ increase in TRPC6 channel-expressing HEK293 cells (from the maximum value of Ca 2+ concentration measured after the addition of the test compound to the initial value (before the addition of the test compound)). The value obtained by subtracting the Ca 2+ concentration) was obtained. The test compound having a larger Ca 2+ increase than the control using DMSO was used as the substance that activates the TRPC6 channel. The amount of increase in Ca 2+ of PPZ2 was set to 100%, and the amount of increase in Ca 2+ of other test compounds was calculated as a relative value. The results are shown in Table 1.

　なお、本試験では、TRPCチャネル活性化作用を有する物質が細胞に適用されると、TRPCチャネルが開口し、細胞外溶液中のCa２＋が細胞内に多量に流入する現象が観察される。また、Fura-2 AMは、AM基（アセトキシメチル基）の存在により細胞膜透過性が高く、細胞内に容易に取り込まれ、細胞内でAM基が加水分解を受けることでFura-2となる結果、Ca２＋とキレート形成が可能となるとともに、AM基を失ったことで細胞外へ漏れ出しにくくなる。 In this test, when a substance having a TRPC channel activating effect is applied to cells, the TRPC channel opens and a large amount of Ca 2+ in the extracellular solution flows into the cells. In addition, Fura-2 AM has high cell membrane permeability due to the presence of AM group (acetoxymethyl group), is easily taken up into cells, and the AM group is hydrolyzed inside the cell to become Fura-2. , Ca 2+ and chelate formation become possible, and the loss of the AM group makes it difficult for the cell to leak out of the cell.

Figure JPOXMLDOC01-appb-T000051
Figure JPOXMLDOC01-appb-T000051

　PPZ2はTRPC6チャネルを活性化させ細胞内へCa２＋を流入させることが知られており、本試験でも30μMのPPZ2の添加により、80nM－120nMのCa２＋濃度の増加が見られた。実施例1～3及び実施例5～11の化合物も、DMSOよりも、細胞内のCa２＋濃度を増加させたことから、これら化合物はTRPC6チャネルを活性化することが確認された。実施例4の化合物は、実施例2の化合物、実施例3の化合物等と化学構造が近似しているため、これら化合物と同様にTRPC6チャネルを活性化すると推測される。 PPZ2 is known to activate the TRPC6 channel and allow Ca 2+ to flow into the cells, and in this study as well, the addition of 30 μM PPZ2 increased the Ca 2+ concentration of 80 nM-120 nM. The compounds of Examples 1 to 3 and Examples 5 to 11 also increased the intracellular Ca 2+ concentration more than DMSO, confirming that these compounds activate the TRPC6 channel. Since the compound of Example 4 has a similar chemical structure to the compound of Example 2, the compound of Example 3, and the like, it is presumed that the TRPC6 channel is activated in the same manner as these compounds.

試験例１－２
TRPC3チャネル、TRPC6チャネル又はTRPC7チャネルを発現したHEK293細胞内へのCa ２＋ の流入に対する実施例２の化合物の作用
　試験例１に記載の方法で作製されたTRPC3発現HEK293細胞（TRPC3/HEK293）、TRPC6発現HEK293細胞（TRPC6/HEK293）、TRPC7発現HEK293細胞（TRPC7/HEK293）を使用し、試験化合物として実施例２の化合物（30μM）を使用した以外は試験例１－１と同様にして、各細胞内へのCa２＋増加量を求めた。結果を表２に示す。 Test Example 1-2
Effect of the compound of Example 2 on the influx of Ca 2+ into HEK293 cells expressing TRPC3 channel, TRPC6 channel or TRPC7 channel TRPC3-expressing HEK293 cells (TRPC3 / HEK293), TRPC6 prepared by the method described in Test Example 1. Each cell was used in the same manner as in Test Example 1-1 except that the expressed HEK293 cells (TRPC6 / HEK293) and TRPC7 expressed HEK293 cells (TRPC7 / HEK293) were used and the compound of Example 2 (30 μM) was used as the test compound. The amount of Ca 2+ increase inward was calculated. The results are shown in Table 2.

Figure JPOXMLDOC01-appb-T000052
Figure JPOXMLDOC01-appb-T000052

　試験例２の化合物は、TRPC6チャネル活性化作用だけでなく、TRPC3チャネル活性化作用及びTRPC7チャネル活性化作用を有することが確認された。 It was confirmed that the compound of Test Example 2 has not only the TRPC6 channel activating effect but also the TRPC3 channel activating effect and the TRPC7 channel activating effect.

試験例１－３
TRPC6チャネルを発現したHEK293細胞内へのCa ２＋ の流入に対するCannabidiolの作用
　試験化合物としてCannabidiol（6μM；2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-Benzenediol）を使用した。本試験では、以下のプロトコルを除き、試験例１に記載の測定方法と同様にした。CannabidiolによるCa２＋増加量を測定したところ、83nMであった。CannabidiolはTRPV1チャネル及びTRPA1チャネルを活性化することが知られていたが、本試験によりTRPC6チャネルを活性化することが新たに確認された。 Test Example 1-3
Action of Cannabidiol on the influx of Ca 2+ into HEK293 cells expressing TRPC6 channel As a test compound, Cannabidiol (6 μM; 2-[(1R, 6R) -3-methyl-6-(1-methylethenyl) -2-cyclohexen- 1-yl] -5-pentyl-1,3-Benzenediol) was used. In this test, the same measurement method as described in Test Example 1 was used except for the following protocol. The Ca 2+ increase by Cannabidiol was measured and found to be 83 nM. Cannabidiol was known to activate TRPV1 and TRPA1 channels, but this study newly confirmed that it activates TRPC6 channels.

０分：Ca２＋含有溶液を入れた測定用チャンバーにTRPC6発現細胞を入れた。
３０秒後：Ca２＋含有溶液をCa2+フリー溶液に置き換えた。
３分後：Ca２＋フリー溶液に試験化合物Cannabidiol（6μM）を添加した。
７分後：Ca２＋フリー溶液を、試験化合物Cannabidiol（6μM）を含むCa２＋含有溶液に置き換えた。
１５分後：測定終了。 0 min: TRPC6-expressing cells were placed in a measurement chamber containing a Ca 2+ -containing solution.
After 30 seconds: The Ca 2+ -containing solution was replaced with a Ca 2+ free solution.
After 3 minutes: The test compound Cannabidiol (6 μM) was added to the Ca 2+ free solution.
7 minutes later: The Ca 2+ free solution was replaced with a Ca 2+ -containing solution containing the test compound Cannabidiol (6 μM).
15 minutes later: Measurement is completed.

　また、Cannabidiolを使用した試験では、細胞外溶液としてCa２＋フリー溶液を使用すると、試験化合物の非選択的な作用により小胞体からCa２＋が細胞質へ排出され、一過性に細胞内のCa２＋濃度が上昇した後、速やかに低下した。続いて、細胞外溶液としてCa２＋含有溶液を適用すると、試験化合物のTRPC6活性化作用により、TRPC6チャネルが開口し、細胞外溶液中のCa２＋が細胞内に流入した。 In the test using Cannabidiol, when Ca 2+ free solution was used as the extracellular solution, Ca 2+ was excreted from the endoplasmic reticulum to the cytoplasm by the non-selective action of the test compound, and the intracellular Ca 2+ was transiently performed. After the concentration increased, it decreased rapidly. Subsequently, when a Ca 2+ -containing solution was applied as an extracellular solution, the TRPC6 channel was opened by the TRPC6 activating action of the test compound, and Ca 2+ in the extracellular solution flowed into the cell.

試験例２
STC1及びA549細胞を含む種々の細胞におけるTRPC6チャネル、TRPC3チャネル、TRPC7チャネル及びSRRM4遺伝子発現
　各細胞（NCI-N417 (ヒト小細胞肺がん), STC1 (ヒト小細胞肺がん), H1650 (ヒト非小細胞肺がん), A549 (ヒト非小細胞肺がん), DU145 (ヒト前立腺がん), VCaP (ヒト前立腺がん)）よりtotal RNAをRNeasy mini kit (Qiagen)を用いて抽出後、20μLの反応液（SuperScript VILO master mix (ThermoFisher): 4 μL , H2O: 16μL）中、total RNA (1 μg)を用いてcDNAへの転写反応（プライミングに25℃で10分間、逆転写に42℃で60分間、不活性化に85℃で5分間）を行った。
　遺伝子増幅はStepOnePlusTM Real-Time PCR Systems (Thermo Fisher Scientific)を用いて行った。
　各TRPCチャネルのmRNA及びSRRM4 mRNAの発現解析は、下記の配列特異的プライマーを用いたqRT-PCRにて行った；hTRPC3 forward: 5’-cagggtgaaaaccacccagt- 3’（配列番号１）；hTRPC3 reverse: 5’-cctaggtccttccagccaga-3’（配列番号２）；hTRPC6 forward: 5’-tgaaagctttggacctctgc-3’（配列番号３）；hTRPC6 reverse: 5’-tcataaaggccacaaacacca-3’（配列番号４）；hTRPC7 forward: 5’-tacgtgctgcacttgtggaa-3’（配列番号５）；hTRPC7 reverse: 5’-atgaagcgtgctgtgaagga-3’（配列番号６）；hSRRM4 forward: 5’-tgacaaagacttgacaccacc-3’（配列番号７）；hSRRM4 reverse: 5’-acctgcgtcgcttgtgttt-3’（配列番号８）。
　cDNA(逆転写の1/20量)を用い、Fast SYBR Green Master Mix (10 μL)を含む 反応液（20 μL）中、プライマー（それぞれ、500 pmol）を使用し、qPCR反応 (初期活性化に95℃で20秒、続く40サイクルの増幅には3秒間95℃で加熱した後、60℃で30秒加熱)を行った。蛍光強度は１サイクルごとに、使用説明書にしたがって測定した。相対mRNA量は標準曲線法に従って定量し、値はβ-actinのCt値で補正した。結果を図１に示す。なお、図１中、４種の棒グラフは左から順にTRPC3、TRPC6、TRPC7、SRRM4を示す。また、図１中、縦軸は、VCaP細胞における各遺伝子の発現量を100とした場合の相対値（RQ）を示す。 Test Example 2
TRPC6 channel, TRPC3 channel, TRPC7 channel and SRRM4 gene expressing cells in various cells including STC1 and A549 cells (NCI-N417 (human small cell lung cancer), STC1 (human small cell lung cancer), H1650 (human non-small cell lung cancer) ), A549 (human non-small cell lung cancer), DU145 (human prostate cancer), VCaP (human prostate cancer)) after extracting total RNA using RNeasy mini kit (Qiagen), 20 μL of reaction solution (SuperScript VILO) Transcription reaction to cDNA using total RNA (1 μg) in master mix (ThermoFisher): 4 μL, H 2 O: 16 μL (priming at 25 ° C for 10 minutes, reverse transcription at 42 ° C for 60 minutes, no Activation was performed at 85 ° C. for 5 minutes).
Gene amplification was performed using StepOnePlus TM Real-Time PCR Systems (Thermo Fisher Scientific).
Expression analysis of mRNA and SRRM4 mRNA of each TRPC channel was performed by qRT-PCR using the following sequence-specific primers; hTRPC3 forward: 5'-cagggtgaaaaccacccagt-3'(SEQ ID NO: 1); hTRPC3 reverse: 5 '-cctaggtccttccagccaga-3' (SEQ ID NO: 2); hTRPC6 forward: 5'-tgaaagctttggacctctgc-3' (SEQ ID NO: 3); hTRPC6 reverse: 5'-tcataaaggccacaaacacca-3' (SEQ ID NO: 4); hTRPC7 forward: 5'- tacgtgctgcacttgtggaa-3'(SEQ ID NO: 5); hTRPC7 reverse: 5'-atgaagcgtgctgtgaagga-3' (SEQ ID NO: 6); hSRRM4 forward: 5'-tgacaaagacttgacaccacc-3'(SEQ ID NO: 7); hSRRM4 reverse: 5'-acctgcgtcgctt 3'(SEQ ID NO: 8).
QPCR reaction (for initial activation) using cDNA (1/20 amount of reverse transcription), primer (500 pmol each) in reaction solution (20 μL) containing Fast SYBR Green Master Mix (10 μL) After heating at 95 ° C for 20 seconds at 95 ° C, followed by 40 cycles of amplification at 95 ° C for 3 seconds, heating at 60 ° C for 30 seconds) was performed. The fluorescence intensity was measured every cycle according to the instruction manual. The amount of relative mRNA was quantified according to the standard curve method, and the value was corrected by the Ct value of β-actin. The results are shown in FIG. In FIG. 1, the four types of bar graphs show TRPC3, TRPC6, TRPC7, and SRRM4 in order from the left. Further, in FIG. 1, the vertical axis shows a relative value (RQ) when the expression level of each gene in VCaP cells is 100.

　TRPC3チャネル遺伝子の発現はDU145、H165、STC1、及びVCaP細胞で、TRPC6チャネル遺伝子の発現はA549、DU145、H165、STC1、及びVCaP細胞で、TRPC7チャネル遺伝子の発現はVCaP細胞で確認できた。また、試験例４で用いたアンチセンス核酸L21（AmNA-7174、L21）の標的となるSRRM4遺伝子の発現はN417、STC1、及びVCaP細胞で確認できた。 The expression of the TRPC3 channel gene was confirmed in DU145, H165, STC1 and VCaP cells, the expression of the TRPC6 channel gene was confirmed in A549, DU145, H165, STC1 and VCaP cells, and the expression of the TRPC7 channel gene was confirmed in VCaP cells. In addition, the expression of the SRRM4 gene targeted by the antisense nucleic acid L21 (AmNA-7174, L21) used in Test Example 4 was confirmed in N417, STC1 and VCaP cells.

試験例３
実施例２の化合物によるSTC-1細胞へのCa ２＋ の取り込み及び蛍光色素共役アンチセンス核酸の取り込み試験
　24well(Poly-L coating)にSTC-1細胞 (1.0×105cell/well、RPMI培地-high glucose) を播種した。
　翌日培地を抜き、Fluo-4(終濃度1μM)の入った2 mM Ca2+溶液500μLを加え30分間培養し細胞に取り込ませた。
　500μLのPBSで洗浄後、500μLの2 mM（または0 mM）Ca2+溶液を投入した。　10μMの蛍光色素共役アンチセンス核酸(L26-alexa647、終濃度10nM）をいれ、軽く撹拌した。
　試験化合物として30 mMの実施例２の化合物(終濃度60μM)または3 mＭのCannabidiol (1μL, 終濃度6μM)を加えた（CannabidiolはTRPC6チャネルを活性化する。（試験例１－３参照））。また、これとは別に、コントロールとして試験化合物に代えてDMSOを加えた。
　直後に、BZ-Xフィルタ GFP OP-87763（吸収波長：525-575 nm）を備えた蛍光顕微鏡（BZ－9000、キーエンス社、レンズは×20を使用）を使用してFluo-4の蛍光（最大蛍光波長：518 nm）をモニターすることで細胞内のCa２＋濃度推移を観察し、BZ-Xフィルタ Cy5 OP-87766（吸収波長：700-775 nm）を備えた蛍光顕微鏡（BZ－9000、キーエンス社、レンズは×20を使用）を使用してAlexa647の蛍光（最大蛍光波長：668nm）をモニターすることで蛍光色素共役アンチセンス核酸の細胞内取り込み量を観察した。
　モニターされた細胞内Ca２＋濃度と蛍光色素共役アンチセンス核酸の細胞内取り込み量を、各々、図２と図３に示す。さらに、実施例２の化合物添加の直後（左図）と10分後（右図）のAlexa647由来の蛍光を撮影した蛍光顕微鏡写真を図４に示す。図２及び図３中の左上及び右上のグラフにおいて、各チャート線は、一つの細胞内の対象物質（図２ではCa２＋濃度であり、図３では核酸取り込み量）の推移を表す。当該グラフには約20個のチャートが含まれ、これにより約20個の細胞内の対象物質量を表す。他の類似のグラフについても同様である。図４の左図は実施例２の化合物添加直後の蛍光顕微鏡写真であり、右図は実施例２の化合物添加１０分後の蛍光顕微鏡写真である。
　Cannabidiolの細胞内Ca２＋濃度と蛍光色素共役アンチセンス核酸の細胞内取り込み量を、各々、図５と図６に示す。さらに、Cannabidiol添加の直後（左図）と10分後（右図）のAlexa647由来の蛍光を撮影した蛍光顕微鏡写真を図７に示す。 Test Example 3
Uptake of Ca 2+ into STC-1 cells and uptake test of fluorescent dye-conjugated antisense nucleic acid by the compound of Example 2 STC-1 cells (1.0 × 10 5 cell / well, RPMI medium- High glucose) was sown.
The next day, the medium was removed, 500 μL of a 2 mM Ca 2+ solution containing Fluo-4 (final concentration 1 μM) was added, and the cells were cultured for 30 minutes and incorporated into cells.
After washing with 500 μL of PBS, 500 μL of 2 mM (or 0 mM) Ca 2+ solution was added. A 10 μM fluorescent dye-conjugated antisense nucleic acid (L26-alexa647, final concentration 10 nM) was added, and the mixture was lightly stirred.
30 mM Example 2 compound (final concentration 60 μM) or 3 mM Cannabidiol (1 μL, final concentration 6 μM) was added as test compound (Cannabidiol activates TRPC6 channel (see Test Example 1-3)). .. Separately, DMSO was added as a control instead of the test compound.
Immediately afterwards, the fluorescence of Fluo-4 (BZ-9000, Keyence, lens x 20) with a BZ-X filter GFP OP-87763 (absorption wavelength: 525-575 nm) was used. By monitoring the maximum fluorescence wavelength: 518 nm), the intracellular Ca 2+ concentration transition is observed, and a fluorescence microscope equipped with a BZ-X filter Cy5 OP-87766 (absorption wavelength: 700-775 nm) (BZ-9000, The intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid was observed by monitoring the fluorescence of Alexa647 (maximum fluorescence wavelength: 668 nm) using Keyence (lens using × 20).
The monitored intracellular Ca 2+ concentration and the intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid are shown in FIGS. 2 and 3, respectively. Further, FIG. 4 shows fluorescence micrographs of Alexa647-derived fluorescence immediately after the compound addition of Example 2 (left figure) and 10 minutes later (right figure). In the upper left and upper right graphs in FIGS. 2 and 3, each chart line represents the transition of the target substance (Ca 2+ concentration in FIG. 2 and nucleic acid uptake amount in FIG. 3) in one cell. The graph contains about 20 charts, which represent the amount of substance of interest in about 20 cells. The same is true for other similar graphs. The left figure of FIG. 4 is a fluorescence micrograph immediately after the compound addition of Example 2, and the right figure is a fluorescence micrograph 10 minutes after the compound addition of Example 2.
The intracellular Ca 2+ concentration of Cannabidiol and the intracellular uptake of the fluorescent dye-conjugated antisense nucleic acid are shown in FIGS. 5 and 6, respectively. Further, FIG. 7 shows fluorescence micrographs of Alexa647-derived fluorescence immediately after the addition of cannabidiol (left figure) and 10 minutes later (right figure).

　図４及び図７の右図（試験化合物添加１０分後の蛍光顕微鏡写真）では、左図（試験化合物添加直後の蛍光顕微鏡写真）に比べて、蛍光色素共役アンチセンス核酸に由来する明るい蛍光が多く確認され、核酸分子が細胞内へ取り込まれていることが確認された。試験例２においてTRPC3チャネル及びTRPC6チャネルを有することが確認されたSTC-1細胞において、試験例１－１及び１－３においてTRPC6活性化作用を有することが確認された実施例２の化合物及びカンナビジオールは細胞外Ca2+を細胞内に流入させるだけでなく、核酸分子の細胞内取り込みを誘導した。 In the right figure of FIGS. 4 and 7 (fluorescence micrograph 10 minutes after the addition of the test compound), the bright fluorescence derived from the fluorescent dye-conjugated antisense nucleic acid is higher than that of the left figure (fluorescence micrograph immediately after the addition of the test compound). Many were confirmed, and it was confirmed that the nucleic acid molecule was taken up into the cell. In STC-1 cells confirmed to have TRPC3 channel and TRPC6 channel in Test Example 2, the compound of Example 2 and cannavi confirmed to have a TRPC6 activating effect in Test Examples 1-1 and 1-3. The diol not only allowed extracellular Ca 2+ to flow into the cell, but also induced the intracellular uptake of nucleic acid molecules.

試験例４
STC-1細胞における実施例２の化合物の、アンチセンス核酸によるSRRM4遺伝子ノックダウンの増強作用
　6wellプレートにSTC-1細胞（2.0×105cell/well、RPMI培地-high glucose）を播種した。翌日培地をRPMI-high glucoseからDMEM-high glucoseに変更し、さらに24時間培養した。
　100μMの陰性対照のアンチセンス核酸（AmNA-NEG#26、L26)またはSRRM4遺伝子を標的とするアンチセンス核酸（AmNA-7174、L21）をそれぞれ1μL加え軽く撹拌し、約10分静置した（終濃度100 nM）。なお、L26はSRRM4遺伝子のknock-down活性を有さず、非選択的なコントロール配列を持ったギャップマー型オリゴ核酸であり、L21はSRRM4遺伝子のknock-down活性を有するギャップマー型アンチセンスオリゴ核酸である。
　30 mMの試験化合物を2μL加え、6時間または24時間培養した（終濃度30μM）。
　Total RNAの抽出とSRRM4遺伝子のノックダウン効果の測定はM. Shimojo et al., Scientific Reports (2019) 9:7618に記載のqRT-PCR法に従って行った。
　各種プライマーとしては、以下のものを用いた；hSRRM4 forward: 5’-tgacaaagacttgacaccacc-3’（配列番号７）；hSRRM4 reverse: 5’-acctgcgtcgcttgtgttt-3’（配列番号８）；βactin forward: 5’-ggccgtcttcccctccatcg-3’（配列番号９）；βactin reverse: 5’-ccagttggtgacgatgccgtgc-3’（配列番号１０）。
　結果を図８に示す。図８左図は試験化合物投与６時間後、右図は試験化合物投与２４時間後のTotal RNA量を示す。 Test Example 4
Enhancement of SRRM4 gene knockdown by antisense nucleic acid of the compound of Example 2 in STC-1 cells STC-1 cells (2.0 × 10 5 cells / well, RPMI medium-high glucose) were seeded on a 6-well plate. The next day, the medium was changed from RPMI-high glucose to DMEM-high glucose, and the cells were cultured for another 24 hours.
Add 1 μL each of 100 μM negative control antisense nucleic acid (AmNA-NEG # 26, L26) or antisense nucleic acid targeting the SRRM4 gene (AmNA-7174, L21), stir lightly, and allow to stand for about 10 minutes (final). Concentration 100 nM). L26 is a gapmer-type oligonucleic acid that does not have the knock-down activity of the SRRM4 gene and has a non-selective control sequence, and L21 is a gapmer-type antisense oligo that has the knock-down activity of the SRRM4 gene. It is a nucleic acid.
2 μL of 30 mM test compound was added, and the cells were cultured for 6 hours or 24 hours (final concentration 30 μM).
Extraction of Total RNA and measurement of the knockdown effect of SRRM4 gene were performed according to the qRT-PCR method described in M. Shimojo et al., Scientific Reports (2019) 9:7618.
The following primers were used as various primers; hSRRM4 forward: 5'-tgacaaagacttgacaccacc-3'(SEQ ID NO: 7); hSRRM4 reverse: 5'-acctgcgtcgcttgtgttt-3' (SEQ ID NO: 8); βactin forward: 5'- ggccgtcttcccctccatcg-3'(SEQ ID NO: 9); βactin reverse: 5'-ccagttggtgacgatgccgtgc-3' (SEQ ID NO: 10).
The results are shown in FIG. The left figure of FIG. 8 shows the total RNA amount 6 hours after the administration of the test compound, and the right figure shows the total RNA amount 24 hours after the administration of the test compound.

　STC-1細胞において、実施例２の化合物は、アンチセンス核酸によるSRRM4遺伝子の切断を誘導した。 In STC-1 cells, the compound of Example 2 induced cleavage of the SRRM4 gene by antisense nucleic acid.

試験例５
A549細胞における実施例２の化合物によるMALAT-1の発現上昇
　96well プレートにA549細胞（5.0×103cells/well、DMEM培地）を播種し、1日培養した。
　培地を抜きPBS溶液120μLで洗浄し、そこにDMEM培地を50μLずつ加えた。
　さらにDMSO（終濃度0.1％）または実施例２の化合物（終濃度3,10,30μM)の入ったDMEM培地を50μLずつ加え、24時間培養した。
　qRT-PCR: トータルRNAはSuperPrep（登録商標） Cell Lysis & RT Kit for qPCR (TOYOBO)を用いて使用説明書にしたがって抽出し、cDNAへの転写反応（プライミングに25℃で10分間、逆転写に25℃で40分間、不活性化に85℃で5分間）を行った。得られたcDNAは4℃で保持した。
　遺伝子増幅はStepOnePlusTMReal-Time PCR Systems (Thermo Fisher Scientific)を用いて行った。QPCR用のプライマーとして、下記のものを用いた；hGAPDH forward: 5’-gagtcaacggatttggtcgt-3’（配列番号１１）;hGAPDH reverse: 5’-gacaagcttcccgttctcag-3’（配列番号１２）, hMALAT1 forward: 5’-aagcaaggtctccccacaag -3’（配列番号１３）;hMALAT1reverse: 5’-gcccacaaggatccaagcta-3’（配列番号１４）。
　PowerUp SYBR Green Master Mix 反応液（20μL）中、プライマー（それぞれ、20 pmol）を使用し、cDNA(逆転写の1/20量)を用いqPCR反応 (初期活性化に95℃で20秒、続く40サイクルの増幅には3秒間95℃で加熱した後60℃で30秒間加熱)を行い、蛍光強度は１サイクルごとに、使用説明書にしたがって測定した。
　相対mRNA量は標準曲線法に従って定量し、値はhGAPDHのCt値で補正した。結果を図９に示す。 Test Example 5
Increased expression of MALAT-1 by the compound of Example 2 in A549 cells A549 cells (5.0 × 10 3 cells / well, DMEM medium) were seeded on 96-well plates and cultured for 1 day.
The medium was removed and washed with 120 μL of PBS solution, and 50 μL of DMEM medium was added thereto.
Further, 50 μL of DMEM medium containing DMSO (final concentration 0.1%) or the compound of Example 2 (final concentration 3,10,30 μM) was added, and the cells were cultured for 24 hours.
qRT-PCR: Total RNA is extracted using SuperPrep® Cell Lysis & RT Kit for qPCR (TOYOBO) according to the instructions for use and transcribed into cDNA (priming at 25 ° C for 10 minutes for reverse transcription). It was performed at 25 ° C for 40 minutes and for inactivation at 85 ° C for 5 minutes). The obtained cDNA was retained at 4 ° C.
Gene amplification was performed using StepOnePlus TM Real-Time PCR Systems (Thermo Fisher Scientific). The following primers were used for QPCR; hGAPDH forward: 5'-gagtcaacggatttggtcgt-3'(SEQ ID NO: 11); hGAPDH reverse: 5'-gacaagcttcccgttctcag-3' (SEQ ID NO: 12), hMALAT1 forward: 5' -aagcaaggtctccccacaag -3'(SEQ ID NO: 13); hMALAT1reverse: 5'-gcccacaaggatccaagcta-3' (SEQ ID NO: 14).
In PowerUp SYBR Green Master Mix reaction solution (20 μL), use primers (20 pmol each) and use cDNA (1/20 amount of reverse transcription) for qPCR reaction (initial activation at 95 ° C for 20 seconds, followed by 40). The cycle was amplified by heating at 95 ° C for 3 seconds and then at 60 ° C for 30 seconds), and the fluorescence intensity was measured for each cycle according to the instruction manual.
The amount of relative mRNA was quantified according to the standard curve method, and the value was corrected by the Ct value of hGAPDH. The results are shown in FIG.

　MALAT-1（Metastasis Associated Lung Adenocarcinoma Transcript 1）はオートファジー活性化作用と正の相関を示すlong non-coding RNA（lncRNA）である（例えば、LncRNA expression profile during autophagy and Malat1 function in macrophages, Z. Ma et al., P ONE 14: e0221104 (2019)を参照）。実施例２の化合物は、A549細胞においてMALAT-1の発現を上昇させたことから、オートファジーを活性化させると考えられる。 MALAT-1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is a long non-coding RNA (lncRNA) that shows a positive correlation with autophagy activation (for example, LncRNA expression profile during autophagy and Malat1 function in macrophages, Z. Ma. et al., P ONE 14: e0221104 (2019)). Since the compound of Example 2 increased the expression of MALAT-1 in A549 cells, it is considered to activate autophagy.

試験例６
A549細胞における実施例２の化合物の、アンチセンス核酸によるMALAT-1ノックダウンの増強作用
　96well プレートにA549細胞（5.0×103cells/well、DMEM培地）を播種し、1日培養した。
　培地を抜きPBS溶液120μLで洗浄し、そこにDMEM培地を50μLずつ加えた。
　MALAT-1を標的とするアンチセンス核酸（終濃度80nM）と、DMSO（終濃度0.1％）または試験化合物（終濃度3,10,30μM)とが入ったDMEM培地を50μLずつ加え、24時間または48時間培養した。MALAT-1を標的とするアンチセンス核酸（ASO）としては以下の配列のものを用いた。なお、以下の配列中、^はP=S、(L)はLNAを表す。
 
MALAT-1用ASO（配列番号１５）
名称：MALAT-1-5714-LNA(15)
配列（5'→3'）：T(L)^A(L)^G(L)^t^t^g^g^c^a^t^c^a^A(L)^G(L)^g 

ネガティブコントロール用ASO（配列番号１６）
名称：NEG#L11
配列（5'→3'）：G(L)^A(L)^G(L)^c^a^a^a^g^c^c^a^t^G(L)^T(L)^g 

　Total RNAの抽出は試験例５と同様にして行った。
　MALAT-1は試験例５と同じものを使用し、MALAT-1の発現上昇効果は試験例５と同様に、QPCR法にて測定した。
　MALAT-1とGAPDHのprimerは試験例５と同じものを用いた。
　相対mRNA量はGAPDHのCt値で補正した。
　結果を図１０に示す。 Test Example 6
Enhancement of MALAT-1 knockdown by antisense nucleic acid of the compound of Example 2 in A549 cells A549 cells (5.0 × 10 3 cells / well, DMEM medium) were seeded on 96-well plates and cultured for 1 day.
The medium was removed and washed with 120 μL of PBS solution, and 50 μL of DMEM medium was added thereto.
Add 50 μL of DMEM medium containing antisense nucleic acid targeting MALAT-1 (final concentration 80 nM) and DMSO (final concentration 0.1%) or test compound (final concentration 3,10,30 μM) for 24 hours or It was cultured for 48 hours. As the antisense nucleic acid (ASO) targeting MALAT-1, the one having the following sequence was used. In the following sequence, ^ represents P = S and (L) represents LNA.

ASO for MALAT-1 (SEQ ID NO: 15)
Name: MALAT-1-5714-LNA (15)
Array (5'→ 3'): T (L) ^ A (L) ^ G (L) ^ t ^ t ^ g ^ g ^ c ^ a ^ t ^ c ^ a ^ A (L) ^ G (L) ) ^ g

ASO for negative control (SEQ ID NO: 16)
Name: NEG # L11
Array (5'→ 3'): G (L) ^ A (L) ^ G (L) ^ c ^ a ^ a ^ a ^ g ^ c ^ c ^ a ^ t ^ G (L) ^ T (L) ) ^ g

Extraction of Total RNA was carried out in the same manner as in Test Example 5.
The same MALAT-1 as in Test Example 5 was used, and the effect of increasing the expression of MALAT-1 was measured by the QPCR method in the same manner as in Test Example 5.
The same MALAT-1 and GAPDH primers as in Test Example 5 were used.
The amount of relative mRNA was corrected by the Ct value of GAPDH.
The results are shown in FIG.

　A549細胞において、実施例２の化合物は、アンチセンス核酸によるlong non-coding RNA（MALAT-1）の切断活性を時間依存的に増強した。以上の試験より、TRPC3チャネル、TRPC6チャネル、またはTRPC7チャネルの活性化によって、細胞外の核酸分子のエンドソームを介した細胞内への取り込みが促進され、続く、オートファジー活性化によって生じるエンドソーム内核酸分子のエンドソーム脱出が促進され、細胞質で発揮される核酸分子活性が増強されると考えられた。 In A549 cells, the compound of Example 2 enhanced the cleavage activity of long non-coding RNA (MALAT-1) by antisense nucleic acid in a time-dependent manner. From the above tests, activation of TRPC3 channel, TRPC6 channel, or TRPC7 channel promotes intracellular uptake of extracellular nucleic acid molecules via endosomes, followed by endosome nucleic acid molecules generated by autophagy activation. It was considered that the escape of endosomes was promoted and the nucleic acid molecule activity exerted in the cytoplasm was enhanced.

試験例７
実施例２の化合物のA549細胞の増殖に対する影響
　96wellプレートにA549細胞（3.0×103cells/well、DMEM培地）を播種し、1日培養した。
　各wellにDMSO（終濃度0.1％）または試験化合物(終濃度0.1,1,3,10,30μM)の入ったDMEM培地を50μLずつ入れ、2日間培養した。
　Hoechst33342とPropidium Iodide (PI)(dojindo)をリン酸緩衝食塩水(PBS)で希釈し培地上に滴下、30分間培養した。(終濃度1μg/mL)
　ImageXpress Pico (MOLECULAR DEVICES)を用い、Hoechst33342による461 nmの蛍光とPIによる620 nmの蛍光の強度を測定することにより、それぞれ、全細胞数と生細胞数の相対値を計測した。
　結果を図１１に示す。図１１中、上図は相対細胞数を表し、下図は生細胞数の割合を表す。 Test Example 7
Effect of the compound of Example 2 on the proliferation of A549 cells A549 cells (3.0 × 10 3 cells / well, DMEM medium) were inoculated on 96-well plates and cultured for 1 day.
50 μL of DMEM medium containing DMSO (final concentration 0.1%) or test compound (final concentration 0.1,1,3,10,30 μM) was placed in each well and cultured for 2 days.
Hoechst 33342 and Propidium Iodide (PI) (dojindo) were diluted with phosphate buffered saline (PBS), dropped onto the medium, and cultured for 30 minutes. (Final concentration 1 μg / mL)
Using ImageXpress Pico (MOLECULAR DEVICES), the relative values of total cell count and viable cell count were measured by measuring the intensity of 461 nm fluorescence by Hoechst 33342 and 620 nm fluorescence by PI, respectively.
The results are shown in FIG. In FIG. 11, the upper figure shows the relative number of cells, and the lower figure shows the ratio of the number of living cells.

　TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルを活性化する作用を有する実施例２の化合物はがん細胞の増殖を亢進する作用を有さず、10μM以上の濃度では抑制する。このため、がん治療における核酸医薬の併用剤として利用することができる。 The compound of Example 2 having an action of activating TRPC3 channel, TRPC6 channel, and TRPC7 channel does not have an action of promoting the growth of cancer cells, and suppresses it at a concentration of 10 μM or more. Therefore, it can be used as a concomitant drug for nucleic acid drugs in cancer treatment.

　本発明は、核酸分子の細胞内取り込みが期待される分野、例えば医薬、核酸分子取り込み試験等の分野で利用できる。 The present invention can be used in fields where intracellular uptake of nucleic acid molecules is expected, such as pharmaceuticals and nucleic acid molecule uptake tests.

Claims (17)

TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグを含有し、細胞内への核酸分子の取り込みを促進するための、オートファジーを増強するための、又は、核酸分子活性を増強するための剤。 Nucleic acid intracellularly containing a substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof. An agent for promoting the uptake of molecules, enhancing autophagy, or enhancing nucleic acid molecule activity. 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物である、請求項１に記載の剤。
Figure JPOXMLDOC01-appb-C000001
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］ The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is the compound represented by the formula (1), cannabidiol, and 1,3-dihydro-1. -[1-[(5,6,7,8-tetrahydro-4H-cycloheptane [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one selected from the group The agent according to claim 1, which is at least one compound.
Figure JPOXMLDOC01-appb-C000001
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m indicates an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ] 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、フッ素原子、塩素原子、臭素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ５アシルオキシ基、Ｃ２～Ｃ５アルコキシカルボニル基、又はトリハロゲノメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～４の整数を示し、
Ｒ２は、フッ素原子；塩素原子；臭素原子；トリフルオロメチル基；トリクロロメチル基；トリブロモメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ５アシルオキシ基；Ｃ２～Ｃ５アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～４の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す、
請求項２に記載の剤。 The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A is a carbon atom or a nitrogen atom. Show,
B represents a carbon atom or a nitrogen atom.
R1 represents a fluorine atom, a chlorine atom, a bromine atom, a C1-C4 alkyl group, a C1-C4 alkoxy group, a C2-C5 acyloxy group, a C2-C5 alkoxycarbonyl group, or a trihalogenomethyl group, and when there are a plurality of them, May be the same or different,
m indicates an integer from 0 to 4 and represents
R2 is a fluorine atom; a chlorine atom; a bromine atom; a trifluoromethyl group; a trichloromethyl group; a tribromomethyl group; a C1-C4 alkyl group; a C2-C5 acyloxy group; a C2-C5 alkoxycarbonyl group; a C1-C3 alkyl. An amide group which may be substituted with one or two groups selected from the group consisting of a group and a C2-C4 alkoxycarbonyl group; selected from the group consisting of a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. Piperidinylcarbonyl group optionally substituted with one or more groups; or substituted with one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Indicates a pyrrolidinylcarbonyl group which may be the same or different if there are more than one.
n represents an integer from 0 to 4 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1,
The agent according to claim 2. 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、フッ素原子、塩素原子、Ｃ１～Ｃ４アルキル基、Ｃ１～Ｃ４アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、トリクロロメチル基、又はトリブロモメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～３の整数を示し、
Ｒ２は、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アシルオキシ基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～３の整数を示し、
ｐは、１を示し、
ｑは、１を示す、
請求項２又は３に記載の剤。 The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a fluorine atom, a chlorine atom, a C1 to C4 alkyl group, a C1 to C4 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, a trichloromethyl group, or a tribromomethyl group, and when there are a plurality of them. May be the same or different,
m indicates an integer from 0 to 3 and represents
R2 is a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 acyloxy group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. An amide group which may be substituted with one or two groups selected from the group consisting of; one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Piperidinylcarbonyl group optionally substituted with; or pyrrolidinylcarbonyl optionally substituted with one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. Indicates a group, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 3 and represents
p indicates 1,
q indicates 1.
The agent according to claim 2 or 3. 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、前記式（１）で表わされる化合物であって、Ａは、窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、塩素原子、Ｃ１～Ｃ３アルキル基、Ｃ１～Ｃ３アルコキシ基、Ｃ２～Ｃ４アルコキシカルボニル基、トリフルオロメチル基、又はトリクロロメチル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～２の整数を示し、
Ｒ２は、フッ素原子；塩素原子；トリフルオロメチル基；トリクロロメチル基；Ｃ１～Ｃ４アルキル基；Ｃ２～Ｃ４アルコキシカルボニル基；Ｃ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；或いはＣ１～Ｃ３アルキル基及びＣ２～Ｃ４アルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～３の整数を示し、
ｐは、１を示し、
ｑは、１を示す、
請求項２～４のいずれか一項に記載の剤。 The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the above formula (1), and A represents a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R1 represents a chlorine atom, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C2-C4 alkoxycarbonyl group, a trifluoromethyl group, or a trichloromethyl group, and when there are a plurality of them, they may be the same or different.
m indicates an integer of 0 to 2 and represents
R2 is selected from the group consisting of a fluorine atom; a chlorine atom; a trifluoromethyl group; a trichloromethyl group; a C1-C4 alkyl group; a C2-C4 alkoxycarbonyl group; a C1-C3 alkyl group and a C2-C4 alkoxycarbonyl group. May be substituted with one or two groups; or one or more groups selected from the group consisting of C1-C3 alkyl groups and C2-C4 alkoxycarbonyl groups. It shows a good piperidinylcarbonyl group and may be the same or different if there are more than one.
n represents an integer from 0 to 3 and represents
p indicates 1,
q indicates 1.
The agent according to any one of claims 2 to 4. 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、以下に示された化合物の１種又は２種以上である、請求項１又は２に記載の剤。
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000003
 Claim 1 or 2 wherein the substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is one or more of the compounds shown below. The agent described in.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000003
 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルがTRPC6チャネルである、請求項１～６のいずれか一項に記載の剤。 The agent according to any one of claims 1 to 6, wherein at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is TRPC6 channel. 前記細胞が、TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを発現する細胞である、請求項１～７のいずれか一項に記載の剤。 The agent according to any one of claims 1 to 7, wherein the cell expresses at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel. 前記細胞が、核酸分子の標的細胞である、請求項１～８のいずれか一項に記載の剤。 The agent according to any one of claims 1 to 8, wherein the cell is a target cell of a nucleic acid molecule. 前記細胞が、小細胞肺がん細胞、非小細胞肺がん細胞、前立腺がん細胞、乳がん細胞、筋細胞又は神経細胞である、請求項１～９のいずれか一項に記載の剤。 The agent according to any one of claims 1 to 9, wherein the cells are small cell lung cancer cells, non-small cell lung cancer cells, prostate cancer cells, breast cancer cells, muscle cells or nerve cells. 核酸分子の投与が期待される疾患の予防又は治療用である請求項１～１０のいずれか一項に記載の剤。 The agent according to any one of claims 1 to 10, which is for the prevention or treatment of a disease for which administration of a nucleic acid molecule is expected. 肺がん、前立腺がん、乳がん、肺線維症、腎線維症、筋ジストロフィー、筋萎縮性側索硬化症、ハンチントン病、筋萎縮症又はパーキンソン病の予防又は治療用である請求項１～１１のいずれか一項に記載の剤。 Any of claims 1 to 11 for the prevention or treatment of lung cancer, prostate cancer, breast cancer, pulmonary fibrosis, renal fibrosis, muscular dystrophy, amyotrophic lateral sclerosis, Huntington's disease, muscle atrophy or Parkinson's disease. The agent described in paragraph 1. TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質、その薬学的に許容される塩、又はそれらのプロドラッグ、及び薬学的に許容される担体を含有し、（ａ）核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグをさらに含有する、或いは（ｂ）核酸分子、その薬学的に許容される塩、又はそれらのプロドラッグを含有する薬剤とともに組み合わせて処方される、
医薬組成物。 A substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel, a pharmaceutically acceptable salt thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier. And (a) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof, or (b) a nucleic acid molecule, a pharmaceutically acceptable salt thereof, or a prodrug thereof. Prescribed in combination with drugs containing
Pharmaceutical composition. 前記TRPC3チャネル、TRPC6チャネル、及びTRPC7チャネルからなる群から選択される少なくとも１種のチャネルを活性化する物質が、下記式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物である、請求項１３に記載の医薬組成物。
Figure JPOXMLDOC01-appb-C000004
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］ The substance that activates at least one channel selected from the group consisting of TRPC3 channel, TRPC6 channel, and TRPC7 channel is a compound represented by the following formula (1), cannabidiol, and 1,3-dihydro-. Select from the group consisting of 1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl] -4-piperidinyl] -2H-benzimidazol-2-one. The pharmaceutical composition according to claim 13, which is at least one compound thereof.
Figure JPOXMLDOC01-appb-C000004
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m represents an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ] 核酸分子が、small interfering RNA(siRNA)、micro RNA、antagomir、small hairpin RNA(shRNA)、guide RNA(gRNA)、アンチセンス核酸、リボザイム、デコイ核酸、スプライススィッチング核酸、mRNA、及びプラスミドからなる群から選択される少なくとも１種である、請求項１３又は１４に記載の医薬組成物。 Nucleic acid molecule consists of a group consisting of small interfering RNA (siRNA), microRNA, antagomir, smallhairpinRNA (shRNA), guideRNA (gRNA), antisense nucleic acid, ribozyme, decoy nucleic acid, splice switching nucleic acid, mRNA, and plasmid. The pharmaceutical composition according to claim 13 or 14, which is at least one selected. 式（１－１）
Figure JPOXMLDOC01-appb-C000005
 ［式中、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示す。］
で表わされる化合物、
式（１－２）
Figure JPOXMLDOC01-appb-C000006
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ａ及びＢは、同時に炭素原子を示さず、
Ｒ３は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示す。］
で表わされる化合物、
それらの塩、又はそれらのプロドラッグ。 Equation (1-1)
Figure JPOXMLDOC01-appb-C000005
 [During the ceremony,
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m indicates an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer of 0 to 5. ]
Compound represented by,
Equation (1-2)
Figure JPOXMLDOC01-appb-C000006
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
A and B do not show carbon atoms at the same time,
R 3 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer of 0 to 5. ]
Compound represented by,
Their salts, or their prodrugs. 式（１）で表される化合物、カンナビジオール、及び1,3-ジヒドロ-1-[1-[(5,6,7,8-テトラヒドロ-4H-シクロヘプタ[b]チエン-2-イル)カルボニル]-4-ピペリジニル]-2H-ベンズイミダゾール-2-オンからなる群から選択される少なくとも１種の化合物、その薬学的に許容される塩、又はそれらのプロドラッグを含有し、細胞内への核酸分子の取り込みを促進するための、オートファジーを増強するための、又は核酸分子活性を増強するための剤。
Figure JPOXMLDOC01-appb-C000007
 ［式中、
Ａは、炭素原子又は窒素原子を示し、
Ｂは、炭素原子又は窒素原子を示し、
Ｒ１は、ハロゲン原子、アルキル基、アルコキシ基、アシルオキシ基、アルコキシカルボニル基、又はハロゲン原子で置換されたアルキル基を示し、複数あるときは同一又は異なってよく、
ｍは、０～５の整数を示し、
Ｒ２は、ハロゲン原子；トリハロゲノメチル基；アルキル基；アシルオキシ基；アルコキシカルボニル基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種の基で置換されてもよいアミド基；アルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピペリジニルカルボニル基；或いはアルキル基及びアルコキシカルボニル基からなる群から選択される１種又は２種以上の基で置換されてもよいピロリジニルカルボニル基を示し、複数あるときは同一又は異なってよく、
ｎは、０～５の整数を示し、
ｐは、０又は１を示し、
ｑは、０又は１を示す。］ The compound represented by the formula (1), benzimidazole, and 1,3-dihydro-1- [1-[(5,6,7,8-tetrahydro-4H-cyclohepta [b] thien-2-yl) carbonyl). ] -4-Piperidinyl] -Contains at least one compound selected from the group consisting of -2H-benzimidazol-2-one, a pharmaceutically acceptable salt thereof, or a prodrug thereof and into the cell. An agent for promoting the uptake of nucleic acid molecules, enhancing autophagy, or enhancing nucleic acid molecule activity.
Figure JPOXMLDOC01-appb-C000007
 [During the ceremony,
A indicates a carbon atom or a nitrogen atom.
B represents a carbon atom or a nitrogen atom.
R 1 represents a halogen atom, an alkyl group, an alkoxy group, an acyloxy group, an alkoxycarbonyl group, or an alkyl group substituted with a halogen atom, and when there are a plurality of them, they may be the same or different.
m represents an integer from 0 to 5 and represents
R 2 is an amide group which may be substituted with one or two groups selected from the group consisting of a halogen atom; a trihalogenomethyl group; an alkyl group; an acyloxy group; an alkoxycarbonyl group; an alkyl group and an alkoxycarbonyl group. One selected from the group consisting of an alkyl group and an alkoxycarbonyl group or a piperidinylcarbonyl group which may be substituted with two or more groups; or one selected from the group consisting of an alkyl group and an alkoxycarbonyl group. Alternatively, it indicates a pyrrolidinylcarbonyl group which may be substituted with two or more groups, and when there are multiple groups, they may be the same or different.
n represents an integer from 0 to 5 and represents
p indicates 0 or 1 and represents
q indicates 0 or 1. ]
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