WO2013183656A1 - Conjugué de ligand de liaison à un récepteur couplé à une protéine g et d'une molécule d'acide nucléique - Google Patents

Conjugué de ligand de liaison à un récepteur couplé à une protéine g et d'une molécule d'acide nucléique Download PDF

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WO2013183656A1
WO2013183656A1 PCT/JP2013/065512 JP2013065512W WO2013183656A1 WO 2013183656 A1 WO2013183656 A1 WO 2013183656A1 JP 2013065512 W JP2013065512 W JP 2013065512W WO 2013183656 A1 WO2013183656 A1 WO 2013183656A1
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formula
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nucleic acid
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龍 永田
雅則 戸邊
拓士 中川
慶介 柿口
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Sumitomo Pharma Co Ltd
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Sumitomo Dainippon Pharma Co Ltd
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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
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to a conjugate of a G protein coupled receptor (GPCR) ligand and a nucleic acid molecule.
  • GPCR G protein coupled receptor
  • the present invention provides a novel compound containing a conjugate of a GPCR ligand and a nucleic acid molecule, and a pharmaceutical composition containing the novel compound as an active ingredient.
  • the present invention provides methods for making these compounds and methods for introducing nucleic acid molecules into cells using these compounds for the treatment of various diseases associated with cells with GPCRs. .
  • Nucleic acid drugs use nucleic acid molecules such as antisense oligonucleotides, siRNAs, and micro RNAs (miRNAs) as drugs, and are attracting attention as next-generation drugs next to low-molecular drugs and antibody drugs.
  • An antisense oligonucleotide is an oligonucleotide having a structure complementary to a partial sequence region of mRNA encoding a target protein. Antisense oligonucleotides can inhibit translation into proteins by binding to the corresponding mRNA, and in addition, the activation of RNase H degrades the corresponding mRNA and suppresses mRNA expression.
  • Non-patent Document 1 Potential control of gene expression using oligonucleotides and application to therapy is expected.
  • siRNA is a double-stranded RNA that is converted into a single strand in the process of RNA interference, and then incorporated into RISC (RNA-induced silencing complex) to target RNA that has at least one highly complementary binding site. Recognize sequence-specifically. As a result, the target RNA is cleaved / degraded by the nuclease in RISC. Therefore, siRNA is expected as a new type of pharmaceutical that can suppress the expression of genes that cause diseases (Non-patent Document 2).
  • RISC RNA-induced silencing complex
  • miRNA is single-stranded RNA, and is incorporated into RISC in the process of RNA interference like siRNA, and shows specific gene expression suppression. miRNA has been reported to be associated with various diseases including cancer, such as involvement in suppression or promotion of cancer, and the possibility of treatment using miRNA is expected (Non-patent Documents 3 and 4). .
  • An antisense oligonucleotide having a complementary structure to a partial sequence region of miRNA has been reported to inhibit the function of endogenous miRNA upon intravenous administration to mice, and an antisense oligonucleotide targeting miRNA is a nucleic acid. Expected to be a pharmaceutical product (Non-Patent Document 5).
  • nucleic acid molecules as described above are considered to provide solutions to diseases that have been considered difficult so far, and more disease-related gene sequences are being identified.
  • Clinical trials of therapeutic methods using nucleic acid molecules for are currently underway.
  • nucleic acid molecules as therapeutic agents, pharmacology has solved problems such as stability in serum, delivery to appropriate organs or cells, and uptake into cells. There is still a need for nucleic acid molecules with properties.
  • Non-Patent Document 6 Endocytosis via receptors expressed on cell membranes is known as one of the methods for taking up nucleic acid molecules into cells.
  • Non-patent Document 7 A specific ligand for a membrane receptor expressed on the cell membrane is conjugated to the nucleic acid molecule.
  • conjugates reported so far are limited to liver diseases because sugar chain receptors are highly expressed in the liver, and folate receptors are limited to cancer because they are expressed in cancer cells.
  • Deliverying nucleic acid molecules for limited diseases Accordingly, development of a technique for incorporating intracellular nucleic acid molecules with great versatility is strongly desired.
  • GPCRs are a member of the cell membrane receptor superfamily. These receptors are biologically important and various GPCRs are involved in many diseases.
  • Non-patent Documents 8 and 9 For example, hypertension, myocardial infarction, arrhythmia, atherosclerosis, renal failure, diabetes, asthma, chronic obstructive pulmonary disease, rhinitis, inflammatory disease, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, pain, depression It is involved in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, multiple sclerosis, and cancer, but it is also considered to be involved in other than these diseases (Non-patent Documents 8 and 9).
  • Non-Patent Documents 10 and 11 Most GPCRs are stimulated by binding to GPCR-specific agonists, then phosphorylated by GPCR kinase, undergo endocytosis with GPCR-specific agonists through binding of arrestins, and are taken up into cells.
  • the present invention provides a transporter for effectively incorporating a nucleic acid drug into a diseased cell, more specifically, a novel compound (conjugate) of a nucleic acid molecule and a transporter (hereinafter referred to as “the compound of the present invention”).
  • the present inventors have focused on GPCRs that are specifically expressed in diseases. Therefore, when a compound in which a compound capable of becoming a GPCR ligand and a nucleic acid molecule are bound with a linker is produced and allowed to act on a cell, the target nucleic acid molecule is introduced into the target cell, and the present invention is achieved. It came to be completed. It was also confirmed that the nucleic acid molecule portion in the compound functions. Furthermore, it was confirmed that GPCR agonists function effectively in GPCR ligands.
  • the linker is represented by the following formula (I):
  • L is from a phosphate group, a phosphorothioate group, a boranophosphate group, a phosphoroselenate group, a boranophosphate ester group, a hydrogen phosphonate group, a phosphoramidate group, an alkylphosphonate group, an arylphosphonate group, and a phosphotriester group.
  • R 1 is — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 —, — (CH 2 CH 2 O) n4 —CH 2 CH 2 —, —CH 2 CH 2 — S—S—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — and —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 — are selected from the group consisting of Divalent groups (wherein — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 — and — (CH 2 CH 2 O) n4 —CH 2 CH 2 — are substitutable.
  • R 3 is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 4 is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 3 and R 4 may be bonded to each other to form a (C3-C8) member ring
  • W 1 represents —NH—, —O—, —NH—C (O) — (CH 2 ) m1 — and —NH—C (O) — (CH 2 ) m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 )
  • R 5 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 2 represents — (CH 2 ) m3 —, — (CH 2 ) m4 —NH —
  • — (CH 2 ) m5 —O— represents a divalent group selected from the group consisting of m3, m4 and m5 each independently represents an integer of 1 to 20
  • R 6 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 3 represents — (CH 2 ) m6 —, — (CH 2 ) m7 —NH -And- (CH 2 ) m8- represents a divalent group selected from the group consisting of -O-, and m6, m7 and m8 each independently represents an integer of 1 to 20)
  • R 5 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 4 represents — (CH 2 ) m9 —, — (CH 2 ) m10 — NH— and — (CH 2 ) m11 —O— represents a divalent group selected from the group consisting of m9, m10 and m11 each independently represents an integer of 1 to 20, and formula (IIe) :
  • R 6 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 5 represents — (CH 2 ) m12 —, — (CH 2 ) m13 — NH— and — (CH 2 ) m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20)
  • W 6 is a divalent group selected from the group consisting of — (CH 2 ) m15 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m16 — (the divalent group is a substitutable position)
  • W 8 is, - (CH 2) m20 - and -CH 2 CH 2 - (OCH 2 CH 2) m21 - divalent group (the bivalent group selected from the group consisting of the substitutable position Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m20 and m21 Each independently represents an integer of 1 to 20, R 7 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R 8 and R 9 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 7 and R 8 may be bonded to each other to form a (C3-C8) member ring), and IIh):
  • W 9 is a divalent group selected from the group consisting of — (CH 2 ) m22 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m23 — (the divalent group is a substitutable position)
  • R 10 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and
  • R 11 and R 12 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 10 and R 11 may be bonded to each other to form a (C3-C8) member ring)
  • a 1 represents (C3-C8) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, (C1-C9) heteroarylene, —O—. (C1-C9) heteroarylene, (C1-C9) heteroarylene-O-, (C3-C7) heterocyclylene, formula (IVa):
  • cycloalkylene may be substituted with 1 to 10 identical or different (C1-C6) alkyl groups, and (C6-C10) arylene. , -O- (C6-C10) arylene, (C6-C10) arylene-O-, (C1-C9) heteroarylene, -O- (C1-C9) heteroarylene, (C1-C9) heteroarylene-O- , (C3-C7) heterocyclylene, the arylene, heteroarylene and heterocyclylene moieties in formula (IVa), formula (IVb), formula (IVc) and formula (IVd) are halogen atoms, (C1-C6) alkyl Group, (C1-C6) alkoxy group, (C1-C6) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group And it may be substituted with 1-6 substituents selected from the group consisting of a trifluoromethoxy group
  • L is from a phosphate group, a phosphorothioate group, a boranophosphate group, a phosphoroselenate group, a boranophosphate ester group, a hydrogen phosphonate group, a phosphoramidate group, an alkylphosphonate group, an arylphosphonate group, and a phosphotriester group.
  • R 1 is — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 —, — (CH 2 CH A divalent group selected from the group consisting of 2 O) n4 —CH 2 CH 2 — and —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 —, where — (CH 2 ) n1 —, — (CH 2 ) n2 —O— (CH 2 ) n3 — and — (CH 2 CH 2 O) n4 —CH 2 CH 2 — are each independently a substitutable position of 1 to 3 N1 and n2 (which may be substituted with the same or different 1 to 5 substituents selected from the group consisting of (C1-C6) alkyl groups and hydroxyl groups
  • N3 and n4 each independently represents an integer of 3 to 20,
  • R 2 represents — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 —, —CH 2 CH 2 — (OCH 2 CH 2 ) n8 —, —C (O) —CH 2 CH 2 — (OCH 2 CH 2 ) n9 —, —CH 2 CH 2 — (OCH 2 CH 2 ) n10 —C (O) —, —C (O) —CH 2 CH 2 — (OCH 2 CH 2 ) n11 —NH—C (O) —, —C (O) — (CH 2 ) n12 —, —O— (CH 2 ) n13 — and — (CH 2 ) n14 — (OCH 2 CH 2 ) n15 —O—
  • R 3 is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 4 is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 3 and R 4 may be bonded to each other to form a (C3-C8) member ring
  • W 1 represents —NH—, —O—, —NH—C (O) — (CH 2 ) m1 — and —NH—C (O) — (CH 2 ) m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 )
  • R 5 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 2 represents — (CH 2 ) m3 —, — (CH 2 ) m4 —NH —
  • — (CH 2 ) m5 —O— represents a divalent group selected from the group consisting of m3, m4 and m5 each independently represents an integer of 1 to 20
  • R 6 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 3 represents — (CH 2 ) m6 —, — (CH 2 ) m7 —NH -And- (CH 2 ) m8- represents a divalent group selected from the group consisting of -O-, and m6, m7 and m8 each independently represents an integer of 1 to 20)
  • R 5 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 4 represents — (CH 2 ) m9 —, — (CH 2 ) m10 — NH— and — (CH 2 ) m11 —O— represents a divalent group selected from the group consisting of m9, m10 and m11 each independently represents an integer of 1 to 20, and formula (IIe) :
  • R 6 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 5 represents — (CH 2 ) m12 —, — (CH 2 ) m13 — NH— and — (CH 2 ) m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20)
  • W 6 is a divalent group selected from the group consisting of — (CH 2 ) m15 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m16 — (the divalent group is a substitutable position)
  • W 8 is, - (CH 2) m20 - and -CH 2 CH 2 - (OCH 2 CH 2) m21 - divalent group (the bivalent group selected from the group consisting of the substitutable position Each independently represents a (C1-C6) alkyl group, a hydroxyl group and an amino group, which may be substituted with the same or different 1 to 10 substituents selected from the group consisting of an alkyl group, m20 and m21 Each independently represents an integer of 1 to 20, R 7 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and R 8 and R 9 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 7 and R 8 may be bonded to each other to form a (C3-C8) member ring), and IIh):
  • W 9 is a divalent group selected from the group consisting of — (CH 2 ) m22 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m23 — (the divalent group is a substitutable position)
  • R 10 represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group, and
  • R 11 and R 12 are each independently (C1-C6) alkyl group or (C3-C8) cycloalkyl group, or R 10 and R 11 may be bonded to each other to form a (C3-C8) member ring)
  • a 1 represents (C3-C8) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, (C1-C9) heteroarylene, —O—. (C1-C9) heteroarylene, (C1-C9) heteroarylene-O-, (C3-C7) heterocyclylene, formula (IVa):
  • cycloalkylene may be substituted with 1 to 10 identical or different (C1-C6) alkyl groups, and (C6-C10) arylene. , -O- (C6-C10) arylene, (C6-C10) arylene-O-, (C1-C9) heteroarylene, -O- (C1-C9) heteroarylene, (C1-C9) heteroarylene-O- , (C3-C7) heterocyclylene, the arylene, heteroarylene and heterocyclylene moieties in formula (IVa), formula (IVb), formula (IVc) and formula (IVd) are halogen atoms, (C1-C6) alkyl Group, (C1-C6) alkoxy group, (C1-C6) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group And it may be substituted with 1-6 substituents selected from the group consisting of a trifluoromethoxy group
  • Z 1 and Z 2 each independently represents an oxygen atom or a sulfur atom
  • [5] The conjugate according to any one of [1] to [4], wherein the nucleic acid molecule is a single-stranded or double-stranded nucleic acid molecule.
  • [6] The conjugate according to any one of [1] to [5], wherein the nucleic acid molecule is a nucleic acid molecule having 7 to 100 bases.
  • nucleic acid molecule is a nucleic acid molecule that interacts with mRNA or a nucleic acid molecule that induces RNA interference.
  • nucleic acid molecule is selected from the group consisting of siRNA, miRNA, antisense oligonucleotide, and antagonistMir.
  • GPCR ligand is a non-peptide ligand structure that binds to GPCR.
  • the GPCR ligand is a non-peptide ligand structure that binds to a ⁇ 2 receptor or a 5-HT 4 receptor.
  • the GPCR ligand is of formula (VIa):
  • the GPCR ligand is of formula (VIa):
  • the GPCR ligand is of formula (VIIa):
  • the GPCR ligand is of formula (VIIa):
  • a novel compound comprising a conjugate of a GPCR ligand and a nucleic acid molecule that has an ability to efficiently incorporate into cells by binding to the GPCR is provided.
  • GPCR ligand G protein-coupled receptor binding ligand
  • GPCR G protein coupled receptor
  • Preferred GPCR ligands in the present invention include non-peptide small molecule agonists.
  • GPCR ligands include non-peptide ligand structures for GPCRs that are known to be internalized by agonists.
  • Preferred examples include non-peptidic ligand structures that bind to ⁇ 2 receptor or 5-HT 4 receptor, and more preferably formula (VIa):
  • a non-peptidic ligand structure that binds to the ⁇ 2 receptor represented by formula (VIIa):
  • non-peptidic ligand structure that binds to the 5-HT 4 receptor represented by the formula (VIa), (VIb), (VIe) or (VIf) described above is particularly preferred.
  • Most preferred are non-peptide ligand structures that bind to the ⁇ 2 receptor represented by the formula (VIa), formula (VIb) or formula (VIf) described above.
  • a non-peptidic ligand structure that binds to the ⁇ 2 receptor represented by formula (VIIa):
  • Non-peptidic ligand structures that bind to the ⁇ 2 receptor are particularly preferable.
  • nucleic acid molecule means a molecule (oligonucleotide) obtained by polymerizing a nucleotide structure composed of a nucleobase, a pentose group and a phosphate group as a basic unit. This includes modified nucleic acid molecules, and any of the nucleobases, pentose groups or phosphate groups may be modified.
  • the nucleic acid molecule may be DNA, RNA, or a chimera (a nucleic acid containing DNA and RNA in a single-stranded nucleic acid), and may be single-stranded or double-stranded. Examples of the length of the nucleic acid molecule include about 7 to 100 bases, preferably about 15 to 100 bases.
  • a double strand it may be a double-stranded DNA, a double-stranded RNA, a double-stranded chimera, an RNA / DNA hybrid, an RNA / chimeric hybrid, a DNA / chimeric hybrid, or a chimeric / chimeric hybrid.
  • nucleic acid molecules include nucleic acid molecules that interact with mRNA (eg, antisense oligonucleotides, antigomir (or antiMir) or exon skip nucleic acid molecules), or nucleic acid molecules that induce RNA interference (eg, siRNA). , MiRNA or shRNA).
  • Examples of the “antisense oligonucleotide” include a nucleic acid molecule of about 7 to 100 bases having a direct or indirect interaction with a partial sequence region of mRNA encoding a target protein, preferably 7 to 30 bases. A nucleic acid molecule of about 15 to 20 bases is more preferable. In another embodiment, the “antisense oligonucleotide” includes a nucleic acid molecule of about 15 to 100 bases having a complementary structure to the partial sequence region of mRNA encoding the target protein, preferably 15 to 30 bases. A nucleic acid molecule of about 15 to 20 bases is more preferable.
  • RNA includes a double-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases involved in RNA interference that suppresses gene expression specifically for the target mRNA sequence, and preferably 15 to 30.
  • RNA double-stranded nucleic acid molecule having about a base
  • a double-stranded nucleic acid molecule having about 21 to 23 bases is more preferable.
  • RNA includes a single-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases having a function of regulating the expression of other genes, preferably a single-stranded nucleic acid molecule of about 15 to 30 bases. A nucleic acid molecule, and more preferably a single-stranded nucleic acid molecule of about 21 to 25 bases. Further, the miRNA may be pri-miRNA or pre-miRNA which is a precursor of miRNA.
  • “AntagMir (or antiMir)” includes a single-stranded nucleic acid molecule (eg, RNA) of about 7 to 100 bases having a complementary structure to the partial sequence region of miRNA, and preferably about 7 to 30 bases.
  • RNA single-stranded nucleic acid molecule
  • a single-stranded nucleic acid molecule is mentioned, More preferably, a single-stranded nucleic acid molecule of about 21 to 25 bases is mentioned.
  • “antagoMir (or antiMir)” includes a single-stranded nucleic acid molecule (eg, RNA) of about 15 to 100 bases having a complementary structure to a partial sequence region of miRNA, preferably 15 Examples thereof include single-stranded nucleic acid molecules of about 30 to 30 bases, and more preferably single-stranded nucleic acid molecules of about 21 to 25 bases.
  • RNA single-stranded nucleic acid molecule
  • the “complementary structure” may not be a structure that maintains perfect complementarity, but may be a structure that partially maintains complementarity.
  • the nucleic acid molecule may have 5 bases or less at the 5 'or 3' end, preferably 2 bases, and have an additional base that does not form a base pair.
  • the additional base may be DNA or RNA.
  • Such additional base sequences include, for example, ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, tttt- Examples of the sequence include 3 ′ and uuu-3 ′, but are not limited thereto.
  • Nucleic acid base includes, for example, a purine base or a pyrimidine base. Specific examples thereof include adenine, guanine, thymine, cytosine, uracil and the like.
  • Modified nucleobases include, for example, modified purine bases and modified pyrimidine bases.
  • modified purine base include hypoxanthine, xanthine, isoguanine, 2-position substituted adenine derivative and 2-position substituted purine derivative, 2-position substituted with amino group, alkylamino, mercapto group, alkylthio group, etc.
  • 2-substituted alkyladenine derivatives substituted with alkyl groups such as methyl groups and 2-position alkyl-substituted hypoxanthine derivatives 2-position modified guanine derivatives modified with 2-position nitrogen with alkyl groups such as methyl groups
  • 6-positions such as methyl 6-position alkyl-substituted purine derivatives and 2-position amino 6-position alkyl-substituted purine derivatives modified with an alkyl group
  • the 6-position oxygen with an alkyl such as a methyl group 6-modified guanine derivative and 6-position modified hypoxanthine derivative modified with a group
  • 7-position modified guanine derivative modified in position 7 with methyl group 8-position alkyl group, halogen, amino group
  • modified pyrimidine base examples include, for example, 2-thiocytosine derivatives and 2-thiouracil derivatives, 3-position alkyl-substituted uracil derivatives substituted with an alkyl group such as a methyl group, 3-position alkyl-substituted cytosine derivatives, 4-thiouracil Derivatives: 4-position modified cytosine derivative in which 4-position nitrogen is modified with a substituent such as acetyl group, 5-position is halogen, trifluoromethyl group, alkyl group, amino group, alkylamino group, alkylaminomethyl group, arylamino group, 5-substituted uracil derivatives, 5-substituted 2-thiouracil derivatives, 5-substituted cytosine derivatives, 5-substituted pyrimidine derivatives, 5-uracil modified with substituents such as alkoxy, propynyl, methoxycarbonylmethyl, and acetic acid oxy
  • Further modified pyrimidine bases include, for example, dihydrouracil derivatives, 3-deaza-5-aza-cytosine derivatives, 6-azauracil derivatives, 6-azacytosine derivatives, 6-azothymine derivatives, 6-azapyrimidine derivatives, triazole derivatives, 3-azole derivatives, Also included are pyrrole derivatives such as nitropyrrole, 2-pyridinone derivatives and the like.
  • Other modified nucleobases include J. et al. Org. Chem. , 2011, 76, 7295, Angew. Chem. Int. Ed. , 1991, 30, 613.
  • pentose group examples include a ribose group and a 2-deoxyribose group.
  • modified pentose group examples include modification and conversion of the hydroxyl group at the 2-position of the ribose group, that is, modification of the 2-position of the 2-deoxyribose group.
  • substituent are selected from the group consisting of a hydrogen atom; a hydroxyl group; a halogen atom; a cyano group, an amino group optionally substituted with a (C1-C6) alkyl group, and a (C1-C6) alkoxy group.
  • (C1-C6) alkoxy group optionally substituted by one or two or more groups which are the same or different; cyano group; azido group; mercapto group; (C1-C6) thioalkoxy group; (C1-C6) An amino group optionally substituted with an alkyl group; (C1-C6) an aminooxy group optionally substituted with an alkyl group; an aminooxy group; a (C1-C6) alkyl group; a (C1-C6) alkenyl group; (C1-C6) alkynyl group; (C1-C6) alkylcarbonylamino group and the like.
  • Preferred examples of the substituent at the 2-position include a hydrogen atom, a hydroxyl group, a fluorine atom, a (C1-C6) alkoxy group, a 2-cyanoethoxy group, and a 2-methoxyethoxy group.
  • a locked nucleic acid (LNA) in which a hydroxyl group at the 2-position of the ribose group, an amino group, a mercapto group, and the like are bonded to the carbon at the 4-position of the ribose group by a methylene bridge, a hydroxyl group at the 2-position of the ribose group
  • a locked nucleic acid (ENA) in which an amino group, a mercapto group, etc.
  • BNA bridged nucleic acids
  • nucleic acid in which the pentose group is substituted with a morpholinyl group morpholino nucleic acid (PMO)
  • PMO morpholino nucleic acid
  • PNA peptide nucleic acid
  • PNA pentose group and a phosphate group are substituted with units such as N- (2-aminoethyl) glycine, and a chain structure in which the pentose group is opened
  • substituted nucleic acids unlocked nucleic acids (UNA)).
  • modified phosphate group examples include phosphorothioate group, phosphorodithioate group, boranophosphate group, phosphoroselenate group, boranophosphate ester group, hydrogen phosphonate group, phosphoramidate group, alkylphosphonate group Arylphosphonate groups and phosphotriester groups. Also included herein are compounds in which the linking oxygen of the phosphate group is replaced with sulfur (bridged phosphorothioate), nitrogen (bridged phosphoramidate) or carbon (bridged methylenephosphonate).
  • the linker in the present invention is not particularly limited as long as it is a part connecting the GPCR ligand and the nucleic acid molecule and has a structure having biocompatibility.
  • it contains triazolylene and an optionally substituted polyalkylene glycol chain or an optionally substituted alkylene chain, and the alkylene chain further comprises one or more —O—, —S—, —NH.
  • conjugate which is the compound of the present invention means a compound in which the GPCR ligand and the nucleic acid molecule are bound, and specifically includes the linker between them.
  • polyalkylene glycol chain means a polyether chain having a structure in which alkylene glycol is polymerized. Specific examples thereof include a polyethylene glycol chain and a polypropylene glycol chain.
  • alkylene chain means a linear alkylene chain, preferably a linear alkylene chain having 1 to 20 carbon atoms. Specific examples thereof include methylene chain, ethylene chain, propylene chain, butylene chain, pentylene chain, hexylene chain, heptylene chain, octylene chain, nonylene chain, decanylene chain, dodecanylene chain, tetradecanylene chain, hexadecanylene chain, icosylene chain, and the like. Can be mentioned.
  • substituent in the “optionally substituted polyalkylene glycol chain and optionally substituted alkylene chain” include a (C1-C6) alkyl group or hydroxyl group optionally substituted with 1 to 3 hydroxyl groups. Can be mentioned. Preferably, a (C1-C6) alkyl group is used.
  • “may be blocked by a divalent group or inserted at the end” means a structure in which a divalent group is inserted at any position or at the end of a polyalkylene glycol chain or an alkylene chain. .
  • L is covalently bonded to the pentose group of a nucleic acid molecule
  • B 6 represents a nucleobase
  • Y 2 represents a substituent at the 2-position of the ribose group
  • N 2 represents a single-stranded or double-stranded oligonucleotide
  • (C1-C6) alkyl means a linear or branched alkyl group having 1 to 6 carbon atoms. Specific examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl and the like.
  • (C3-C8) cycloalkyl means a saturated monocyclic cycloalkyl having 3 to 8 carbon atoms. Specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
  • (C1-C6) alkoxy means straight or branched alkoxy having 1 to 6 carbon atoms. Specific examples thereof include methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy and the like.
  • (C1-C6) alkoxy” in “(C1-C6) alkoxycarbonyl” has the same meaning.
  • (C3-C8) cycloalkylene means a divalent saturated monocyclic cycloalkylene having 3 to 8 carbon atoms. Specific examples thereof include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene and the like.
  • (C6-C10) arylene means a bivalent monocyclic or bicyclic aromatic ring having 6 to 10 carbon atoms. Specific examples thereof include phenylene, 1-naphthylene, 2-naphthylene and the like.
  • (C6-C10) arylene is a bicyclic aromatic ring, one in which one ring is saturated or partially saturated is also included. Specific examples thereof include 1,2,3,4-tetrahydronaphthylene or 2,3-dihydro-1H-indenylene.
  • (C1-C9) heteroarylene is a monocyclic or bicyclic ring having 1 to 9 carbon atoms and containing 1 to 4 nitrogen atoms, oxygen atoms and / or sulfur atoms.
  • pyridylene pyridazylene
  • pyrazinylene pyrimidinylene
  • triazinylene pyrrolylene
  • pyrazolylene triazolylene
  • tetrazolylene imidazolylene, furylene, thienylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, thiadiazolylene, oxadiazolylene, etc.
  • Preferred heteroarylenes include pyridylene, pyridazylene, pyrazinylene, pyrimidinylene, pyrrolylene, pyrazolylene, furylene, thienylene, indolenylene, indazolylene, quinolylene, isoquinolylene.
  • (C3-C7) heterocyclylene is a monocyclic 5 having 3 to 7 carbon atoms and containing 1 to 3 nitrogen atoms, oxygen atoms and / or sulfur atoms.
  • a monovalent 6-membered divalent ring containing 1 to 3 nitrogen, oxygen and / or sulfur atoms such as a valent heterocyclo ring, piperidinylene, morpholinylene, thiomorpholinylene, piperazinylene, tetrahydropyranylene or dioxanylene
  • a monocyclic 7-membered divalent heterocyclo ring containing 1 to 3 nitrogen atoms, oxygen atoms and / or sulfur atoms such as azepanylene, homopiperazinylene or oxepanylene.
  • Preferable heterocyclylene includes pyrrolidinylene, piperidinylene and the like.
  • triazolylene means a monocyclic 5-membered divalent heterocyclo ring containing three nitrogen atoms, and specific examples thereof include 1,2,3-triazolylene or 1,2 , 4-triazolylene. Preferred triazolylene includes 1,2,3-triazolylene.
  • halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
  • (C3-C8) membered ring means a divalent saturated monocyclic cycloalkane having 3 to 8 carbon atoms. Specific examples thereof include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and the like.
  • the compound of the present invention is a conjugate comprising a linker between a GPCR ligand and a nucleic acid molecule, wherein the linker is triazolylene and an optionally substituted polyalkylene glycol chain or an optionally substituted alkylene chain.
  • the alkylene chain is one or more —O—, —S—, —NH—, ⁇ N—, —N ⁇ , —SO 2 —, —C ( ⁇ O) —, (C6-C10
  • the linker is represented by the following formula (I):
  • R 1 is preferably — (CH 2 ) n1 —, —CH 2 CH 2 —SS—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — and —CH 2 CH 2
  • R 30 R 31 N—C (O) — and R 32 OC (O) — is a substitutable position from the group consisting of R 30 R 31 N—C (O) — and R 32 OC (O) —. It may be substituted with 1 to 3 identical or different substituents selected.
  • R 30, R 31 and R 32 each independently represent a hydrogen atom or a (C1-C6) alkyl group.) Can be mentioned.
  • R 1 is preferably a divalent group selected from the group consisting of — (CH 2 ) n1 — and —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 —.
  • a group (wherein n1 represents an integer of 3 to 10, and — (CH 2 ) n1 — represents a substitutable position selected from the group consisting of a (C1-C4) alkyl group, a hydroxyl group and a hydroxymethyl group. Or may be substituted with 1 to 5 different substituents). More preferred is a divalent group represented by — (CH 2 ) n1 — (where n1 represents an integer of 3 to 10).
  • R 2 is preferably — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 —, — (CH 2 ) n14 — (OCH 2 CH 2 ) n15 —O— ( CH 2) n16 - and - (CH 2) n17 -NH- C (O) - (CH 2 CH 2 O) n18 -CH 2 CH 2 -NH-C (O) - (CH 2) n19 - group consisting of (Wherein n5, n6, n7, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20, and the divalent group can be substituted) Each independently at 1 to 3 (C1-C6) alkyl group optionally substituted with 1 to 3 hydroxyl groups and 1 to 10 substituents selected from the group consisting of hydroxyl groups.
  • a valent group here, n5, n6, n7, n14, n15, n16, n17, n18 and n19 each independently represents an integer of 0 to 20, and the divalent group is a substitutable position; Each independently may be substituted with 1 to 3 (C1-C4) alkyl groups).
  • R 2 is preferably — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 — and — (CH 2 ) n14 — (OCH 2 CH 2 ).
  • a divalent group selected from the group consisting of n15 —O— (CH 2 ) n16 — (wherein n5, n6, n7, n14, n15 and n16 each independently represents an integer of 0 to 12, The divalent group is independently substituted with 1 to 5 identical or different substituents selected from the group consisting of a (C1-C4) alkyl group, a hydroxyl group and a hydroxymethyl group at substitutable positions. May be included).
  • R 3 is preferably a (C1-C6) alkyl group.
  • R 4 is preferably a hydrogen atom or a (C1-C6) alkyl group.
  • a (C3-C8) member ring formed by combining R 3 and R 4 with each other can be mentioned.
  • R 5 , R 5 ′ , R 6 , R 6 ′ , R 9 and R 12 are preferably each independently a hydrogen atom or a (C1-C6) alkyl group.
  • R 7 and R 10 are preferably each independently a hydrogen atom or a (C1-C6) alkyl group.
  • R 8 and R 11 are preferably each independently a (C1-C6) alkyl group.
  • Q 1 is preferably a divalent group selected from the group consisting of the formula (IIa), formula (IIb), formula (IIc), formula (IIe) and formula (IIf) described above. More preferably, a divalent group selected from the group consisting of the formula (IIb), the formula (IIc) and the formula (IIe) described above can be used, and more preferably the formula (IIc) and the formula (IIe) described above.
  • (CH 2 ) p1 and (CH 2 ) p3 described above may preferably be blocked or inserted at one end with one or more —O— or —SO 2 —NH—.
  • examples thereof include a divalent group selected from the group consisting of the above-described formula (IIIa) and formula (IIIb).
  • Q 2 preferably, (CH 2 ) p1 and (CH 2 ) p3 described above may be blocked or inserted at one end with one or more —O—.
  • a 1 is preferably (C3-C7) cycloalkylene, (C6-C10) arylene, —O— (C6-C10) arylene, (C6-C10) arylene-O—, and (C3-C7) heterogene.
  • Cyclylene a divalent group selected from the group consisting of formula (IVa), formula (IVb), formula (IVc) and formula (IVd) as described above (wherein (C3-C7) cycloalkylene and (C3-C7) ) Heterocyclylene may be substituted with 1 to 4 identical or different (C1-C4) alkyl groups, (C6-C10) arylene, —O— (C6-C10) arylene and (C6-C10) The arylene moiety in) arylene-O— is a halogen atom, a (C1-C4) alkyl group, a (C1-C4) alkoxy group, or a (C1-C4) alkoxy group.
  • sulfonyl group may be substituted with 1 to 4 substituents selected from the group consisting of a sulfonyl group, a carboxyl group, a cyano group, a hydroxyl group, a trifluoromethyl group, and a trifluoromethoxy group.
  • (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O- the above-described formula (IVa), formula (IVb), formula (IVc) and formula A divalent group selected from the group consisting of (IVd) (wherein (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O-, the arylene moiety is a halogen atom, 1 to 4 selected from the group consisting of (C1-C4) alkyl group, (C1-C4) alkoxy group, (C1-C4) alkoxycarbonyl group, carboxyl group, cyano group, hydroxyl group, trifluoromethyl group and trifluoromethoxy group.
  • a divalent group selected from the group consisting of (C6-C10) arylene (preferably phenylene), —O— (C6-C10) arylene (preferably —O-phenylene) and the above-described formula (IVc).
  • (C6-C10) arylene and -O- (C6-C10) arylene are halogen atoms, (C1-C4) alkyl groups, (C1-C4) alkoxy groups, (C1-C4)) And may be substituted with 1 to 4 substituents selected from the group consisting of an alkoxycarbonyl group, a carboxyl group, a cyano group, a hydroxyl group, a trifluoromethyl group, and a trifluoromethoxy group.
  • W 1 examples include —NH—C (O) — (CH 2 ) m1 —, —NH—C (O) — (CH 2 ) m2 —O—, —NH—, or —O—.
  • W 2 , W 3 , W 4 and W 5 are preferably each independently, — (CH 2 ) m3 —, — (CH 2 ) m6 —, — (CH 2 ) m9 — or — (CH 2 ) M12- .
  • W 6 , W 8 and W 9 are preferably each independently, — (CH 2 ) m15 —, — (CH 2 ) m20 — or — (CH 2 ) m22 — (wherein the divalent group Each independently may be substituted at the substitutable position with the same or different 1 to 10 substituents selected from the group consisting of (C1-C6) alkyl groups, hydroxyl groups and amino groups. Can be mentioned.
  • W 7 is preferably a single bond or a divalent group selected from the group consisting of —C (O) —NH— (CH 2 ) m17 — and —O— (CH 2 ) m19 — (wherein The divalent group is independently selected from the group consisting of C (O) —NH— (C1-C6) alkyl group and NH—C (O) — (C1-C6) alkyl at substitutable positions. And may be substituted with 1 to 10 substituents which may be the same or different.
  • Z 1 and Z 2 are preferably oxygen atoms.
  • N1 to n4 are each independently and preferably an integer of 3 to 10
  • n5 to n16 are each independently and preferably an integer of 0 to 10.
  • M1 to m23 are each independently preferably an integer of 1 to 10.
  • P1 to p4 are preferably each independently an integer of 1 to 10.
  • A is 0 or 1, and b is preferably 1.
  • R 1 , R 2 , Q 1 , Q 2 , A 1 , L, a and b and the GPCR ligand in formula (I) respectively represent:
  • R 1 is — (CH 2 ) n1 — (where n1 represents an integer of 3 to 10) or —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 —.
  • R 2 represents — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 — or — (CH 2 ) n14 — (OCH 2 CH 2 ) n15 —O— (CH 2 ) n16 -(Wherein n5, n6, n7, n14, n15 and n16 each independently represents an integer of 0 to 10);
  • Q 1 is represented by formula (IIa):
  • R 3 is (represents C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 4 is a hydrogen atom, (C1-C6) alkyl or (C3-C8) cycloalkyl group
  • R 3 and R 4 may be bonded to each other to form a (C3-C8) member ring
  • W 1 represents —NH—, —O—, —NH—C (O) — (CH 2 ) m1 — and —NH—C (O) — (CH 2 ) m2 —O— represents a divalent group selected from the group consisting of m1 and m2, each independently representing an integer of 1 to 20 )
  • R 5 represents a hydrogen atom or a (C1-C4) alkyl group
  • W 2 represents a divalent group represented by — (CH 2 ) m3 —
  • m3 represents an integer of 1 to 10.
  • R 6 represents a hydrogen atom or a (C1-C4) alkyl group
  • W 3 represents a divalent group represented by — (CH 2 ) m6 —
  • m6 represents an integer of 1 to 10.
  • R 6 ′ represents a hydrogen atom, a (C1-C6) alkyl group or a (C3-C8) cycloalkyl group
  • W 5 represents — (CH 2 ) m12 —, — (CH 2 ) m13 — NH— and — (CH 2 ) m14 —O— represents a divalent group selected from the group consisting of m12, m13 and m14 each independently represents an integer of 1 to 20) or formula (IIf) :
  • W 6 is a divalent group selected from the group consisting of — (CH 2 ) m15 — and —CH 2 CH 2 — (OCH 2 CH 2 ) m16 — (the divalent group is a substitutable position)
  • Q 2 represents a divalent group represented by the above formula (IIIa) or (IIIb);
  • a 1 is (C6-C10) arylene, —O— (C6-C10) arylene and (C6-C10) arylene-O—, the above-mentioned formula (IVa), formula (IVb), formula (IVc) and formula A divalent group selected from the group consisting of (IVd) (wherein (C6-C10) arylene, -O- (C6-C10) arylene and (C6-C10) arylene-O-, the arylene moiety is a halogen atom, 1 to 4 selected from the group consisting of (C1-C4) alkyl
  • L represents the above-described formula (V) (wherein Z 1 and Z 2 each independently represents an oxygen atom or a sulfur atom); a represents 0 or 1; b represents 0 or 1;
  • the GPCR ligand represents formula (VIa), formula (VIb), formula (VIc) or formula (VId) as described above.
  • R 1 , R 2 , Q 2 , A 1 , L, a and b and the GPCR ligand in formula (I) each represent:
  • R 1 is — (CH 2 ) n1 — (where n1 represents an integer of 3 to 10) or —CH 2 CH 2 —O—C (O) —NH— (CH 2 ) 6 —.
  • R 2 represents — (CH 2 ) n5 —, — (CH 2 ) n6 —O— (CH 2 ) n7 — or — (CH 2 ) n14 — (OCH 2 CH 2 ) n15 —O— (CH 2 ) n16 -In which n5, n6, n7, n14, n15 and n16 each independently represents an integer of 3 to 10;
  • Q 2 represents a divalent group represented by the above formula (IIIa) or (IIIb);
  • a 1 is a divalent group selected from the group consisting of phenylene, —O-phenylene, phenylene-O—, formula (IVa), formula (IVb), formula (IVc) and formula (IVd) described above (herein , Phenylene, —O-phenylene and (phenylene in —O— include a halogen atom, a (C1-C4) alkyl group, a (C1-C4) al
  • R 1 is — (CH 2 ) n1 —, —CH 2 CH 2 —S—S—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — or —CH 2 CH 2 —O—C
  • R 1 is — (CH 2 ) n1 —, —CH 2 CH 2 —S—S—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — or —CH 2 CH 2 —O—C
  • a divalent group which is (O) —NH— (CH 2 ) 6 — (where n1 represents an integer of 3 to 10, and —CH 2 CH 2 —S—S—CH 2 CH 2 —C (O) —NH— (CH 2 ) 6 — is a substitutable position and 1 to 3
  • R 30 R 31 N—C (O) — wherein R 30 , R 31 and R 32 are each independently Represents a hydrogen atom or a (C1-C6) alkyl
  • Q 1 represents a divalent group of formula (IIb), formula (IIc) or formula (IIe) as described above;
  • Q 2 represents the above-described formula (IIIa) or (IIIb) (wherein p1, p2, p3 and p4 each independently represents an integer of 1 to 20, and (CH 2 ) p1 and ( CH 2 ) p3 represents a divalent group that may be blocked or inserted at the end with a divalent group selected from the group consisting of one or more —O— and —SO 2 —NH—;
  • a 1 is a divalent group selected from the group consisting of phenylene, —O-phenylene, phenylene-O— and the above-described formula (IVc) (wherein phenylene, —O-phenylene and phenylene in (phenylene-O—) The moiety consists of a halogen atom, (C1-C4) alkyl group, (C1-C4) alkoxy group
  • L represents the above-described formula (V) (wherein Z 1 and Z 2 represent an oxygen atom); a represents 0 or 1; b represents 0 or 1;
  • the GPCR ligand represents a structure represented by the above formula (VIa), formula (VIb), formula (VIe), formula (VIf), formula (VIIa), formula (VIIb) or formula (VIIc).
  • Each of the above compound groups limited by one or a combination of any of these exemplifications is also an embodiment of a preferred compound of the present invention.
  • the number of substituents in the group defined as “optionally substituted” is not particularly limited as long as it is substitutable unless otherwise defined, and is one or more.
  • the description of each group also applies when the group is a part of another group or a substituent.
  • the compounds of the present invention may form pharmaceutically acceptable salts, such as salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid; formic acid, Salts with organic carboxylic acids such as acetic acid, fumaric acid, maleic acid, oxalic acid, citric acid, malic acid, tartaric acid, aspartic acid, glutamic acid; methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydroxybenzenesulfonic acid , Salts with sulfonic acids such as dihydroxybenzene sulfonic acid; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; ammonium salt; triethylamine salt, pyridine salt, picoline salt, Ethanolamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine and Salt
  • the compound of the present invention and a pharmaceutically acceptable salt thereof may be a hydrate or a solvate such as an ethanolate, and these hydrates and / or solvates are also compounds according to the present invention. And pharmaceutically acceptable salts thereof.
  • the compound of the present invention when the compound of the present invention has a stereoisomer, a tautomer and / or an optical isomer, the compound of the present invention includes a mixture of these isomers and an isolated isomer.
  • the compound of the present invention and a pharmaceutically acceptable salt thereof may be a crystal, and may be a single crystal form or a crystal form mixture.
  • compound (I) may be a deuterium converter.
  • the production method of the compound of the present invention is not particularly limited.
  • the compound of the present invention in which the linker is represented by the formula (I) can be produced by the following production methods 1 and 2 and the like.
  • the raw material compound can be produced by the production method shown below and the like, and can be easily obtained as a commercial product unless otherwise specified, or can be produced according to a method known per se or a method analogous thereto.
  • the compound obtained at each step in the following reaction formula can be used in the next reaction as a reaction solution or as a crude product.
  • the compound used by the following manufacturing method may form a salt in the range which does not interfere with reaction.
  • the compound can be isolated from the reaction mixture according to a conventional method, and can be easily purified by usual separation means such as recrystallization, distillation, chromatography and the like.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof is a novel compound, and can be produced, for example, by the method described below, the examples described later and a method analogous thereto.
  • R 1 , R 2 , Q 1 , A 1 , L, p 1 , p 2, a, b, *, and ** have the same meanings as [2] or [3] above).
  • Step 1 Compound (I-1) is produced by reacting compound (1-1) with compound (1-2).
  • the reaction can be carried out according to the reaction conditions of a conventional Huisgen azide-alkene cyclization reaction using a catalytic amount of a copper salt (Lallana et al., Angew. Chem. Int. Ed. (2011) 50: 8794-8804).
  • a copper salt include divalent copper salts such as copper sulfate, and monovalent copper salts such as copper bromide, copper iodide, and copper trifluoromethylsulfonate. Additives and ligands may be used.
  • Examples of the additive include reducing agents such as sodium ascorbate and tris (2-carboxyethyl) phosphine (TCEP).
  • Examples of the ligand include tris (benzyltriazolylmethyl) amine (TBTA), Examples include tris (hydroxypropyltriazolylmethyl) amine (THPTA), bathophenanthroline disulfonate (BPDS), and the like.
  • Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents.
  • the reaction temperature can usually be selected from the range of ⁇ 20 to 100 ° C., preferably 0 ° C. to room temperature.
  • R 1 , R 2 , Q 1 , A 1 , L, p3, p4, a, b, *, and ** have the same meanings as [2] or [3] above).
  • Step 1 Compound (I-2) is produced from compound (2-1) and compound (2-2) by the same method as in Step 1 described in Production Method 1.
  • the starting compound (1-2) used in Production Method 1 can be produced, for example, according to the following method.
  • R 1 , Q 1 , L, p2, a and ** have the same meanings as the above [2] or [3], and X 1 is, for example, a chlorine atom, a bromine atom, alkoxycarbonyloxy, phenoxy, Activating group or hydroxyl group of a carboxylic acid derivative such as p-nitrophenoxy or succinimidyloxy)
  • Step 1 Compound (1-2) is synthesized by reacting compound (3-1) with compound (3-2).
  • X 1 is an activating group of a carboxylic acid such as chlorine atom, bromine atom, alkoxycarbonyloxy, phenoxy, p-nitrophenoxy or succinimidyloxy
  • the reaction can be carried out in the presence or absence of a base.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide).
  • DMSO diisopropylethylamine
  • acetonitrile acetonitrile
  • ketone solvents acetone, etc.
  • water including buffer solution
  • alcohol solvents methanol, ethanol, etc.
  • the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably potassium carbonate, diiso
  • the amount of the base used is usually selected from the range of 1 to 5 equivalents relative to compound (3-2).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably in the range of ⁇ 78 to 50 ° C.
  • the reaction is performed in the presence of a condensing agent.
  • a condensing agent examples include carbodiimide (dicyclohexylcarbodiimide, 1-ethyl-3- (dimethylaminopropyl) carbodiimide, etc.), phosphonium salt (BOP, etc.), guanidinium salt (HBTU, etc.), etc.
  • the solvent examples include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, acetonitrile, etc.), ketone solvents (acetone, etc.), or mixed solvents thereof.
  • the reaction temperature is usually selected from the range of 0 to 100 ° C., preferably 0 to 50 ° C.
  • X 1 is a hydroxyl group
  • the hydroxyl group is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy, and then compound (3-2) Compound (1-2) is produced by reacting with.
  • X 1 is converted from a hydroxyl group to a chlorine atom or a bromine atom
  • thionyl chloride, oxalyl chloride, phosphorus trichloride, phosphorus pentachloride, phosphorus oxychloride, dichlorotriphenylphosphorane, carbon tetrachloride-triphenylphosphine, odor Thionyl chloride, dibromotriphenylphosphorane and the like are used.
  • the solvent include an aprotic solvent (such as toluene), a halogen-based solvent (such as methylene chloride and chloroform), or a mixed solvent thereof.
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 100 ° C.
  • X 1 When X 1 is converted from a hydroxyl group to alkoxycarbonyloxy, it is carried out by reacting, for example, an alkyl halide formate such as ethyl chloroformate, isopropyl chloroformate, or isobutyl chloroformate in the presence of a base.
  • an alkyl halide formate such as ethyl chloroformate, isopropyl chloroformate, or isobutyl chloroformate
  • the amount of the halogenated alkyl formate used is usually selected from the range of 1 to 5 equivalents relative to the carboxylic acid.
  • solvent examples include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), or a mixed solvent thereof. Is mentioned.
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- And diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like, preferably N-methylmorpholine, triethylamine, diisopropyl Ethylamine is mentioned.
  • the amount of the base used is usually selected from the range of 1 to 10 equivalents relative to the carboxylic acid.
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 100 ° C., preferably in the range of ⁇ 78 ° C. to room temperature.
  • X 1 is converted from a hydroxyl group to succinimidyloxy by reacting N-hydroxysuccinimide in the presence of a condensing agent.
  • the condensing agent include carbodiimide (dicyclohexylcarbodiimide, 1-ethyl-3- (dimethylaminopropyl) carbodiimide, etc.), phosphonium salt (BOP, etc.), guanidinium salt (HBTU, etc.), etc.
  • N-hydroxybenzotriazole, etc. and bases (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4] .3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like.
  • the amount of N-hydroxysuccinimide and condensing agent used is usually selected from the range of 1 to 5 equivalents relative to the carboxylic acid.
  • the solvent examples include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, acetonitrile, etc.), ketone solvents (acetone, etc.), or mixed solvents thereof.
  • the reaction temperature is usually selected from the range of 0 to 100 ° C., preferably 0 to 50 ° C.
  • the compound (3-2) is synthesized according to an ordinary nucleic acid synthesis method such as a phosphoramidite method using amidite reagent (Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, New York (2000)).
  • the starting compound (2-2) used in Production Method 2 can be produced, for example, according to the following method.
  • R 1 , Q 1 , L, p4, a and ** are as defined in [2] or [3] above, P 11 is a protecting group for carboxylic acid, and X 2 is a chlorine atom.
  • OMs methanesulfonyloxy group
  • OTs p-toluenesulfonyloxy group
  • Tf trifluoromethanesulfonyloxy group
  • OTf trifluoromethanesulfonyloxy group
  • Step 1 Compound (4-2) is synthesized by reacting compound (4-1) with an azidation reagent in the presence or absence of a base.
  • examples of the azide reagent include metal azide compounds such as sodium azide, organic azide compounds such as trimethylsilyl azide, tetrabutylammonium azide And quaternary ammonium azide compounds.
  • the amount of the azidation reagent used is usually selected from the range of 1 to 10 equivalents relative to the compound (4-1).
  • the solvent examples include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), Water, alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof can be used. If necessary, for example, a phase transfer catalyst such as tetrabutylammonium chloride may be added as an additive.
  • the reaction temperature can usually be selected from the range of 0 ° C. to 150 ° C., and preferably in the range of room temperature to 100 ° C.
  • examples of the azidation reagent include diphenyl phosphate azide, and the amount used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1).
  • examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO). , NMP, acetonitrile, HMPT, etc.), ketone solvents (acetone, etc.) or mixed solvents thereof.
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), organic bases ( N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1 , 4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably 1,4-diazabicyclo [5.4.0] undec-7-ene.
  • the amount of the base used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably 0 to 100 ° C.
  • X 2 is a hydroxyl group
  • X 2 When X 2 is converted from a hydroxyl group to a chlorine atom, a bromine atom or an iodine atom, it is carried out by reacting a halogenating agent in the presence or absence of an acid.
  • a halogenating agent include chlorinating agents such as thionyl chloride, oxalyl chloride, phosphorus trichloride, phosphorus pentachloride, phosphorus oxychloride, carbon tetrachloride-triphenylphosphine, N-chlorosuccinimide-triphenylphosphine, Brominating agents such as thionyl, hydrobromic acid, phosphorus tribromide, carbon tetrabromide-triphenylphosphine, N-bromosuccinimide-triphenylphosphine, bromine-triphenylphosphine, hydroiodic acid, potassium iodide, And an iodinating agent
  • Examples of the acid used include hydrochloric acid, sulfuric acid, phosphoric acid and the like.
  • Examples of the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents. (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), water, or a mixed solvent thereof may be mentioned, and no solvent may be used. Moreover, you may add a base as needed.
  • the base used include organic bases (imidazole, N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 100 ° C., preferably 0 to 100 ° C.
  • the reaction is performed by reacting a sulfonylating agent in the presence of a base.
  • a sulfonylating agent include methanesulfonyl chloride, tosyl chloride, trifluorosulfonic acid anhydride, and the like.
  • the amount of the sulfonylating agent used is usually selected from the range of 1 to 5 equivalents relative to the compound wherein X 2 is a hydroxyl group.
  • solvent examples include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), ketone solvents (acetone, etc.) or a mixed solvent thereof. It is done.
  • ether solvents diethyl ether, DME, 1,4-dioxane, etc.
  • halogen solvents methylene chloride, chloroform, etc.
  • ketone solvents acetone, etc.
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), organic bases ( N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-ene, 1 , 4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably N-methylmorpholine, triethylamine, diisopropylethylamine.
  • the amount of the base used is usually selected from the range of 1 to 10 equivalents relative to compound (4-1).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 100 ° C., preferably in the range of ⁇ 78 ° C. to
  • Step 2 For example, by using the general deprotection conditions for protecting groups of carboxylic acids shown in the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)), compound (4-2) can be used. Compound (4-3) is produced.
  • Step 3 Compound (2-2) is produced from compound (4-3) and compound (3-2) by the same method as in the case where X 1 in Step 1 of the production method described in (1-2) is a hydroxyl group. Is done. Specifically, a method using a condensing agent or a hydroxyl group of the carboxylic acid of the compound (4-3) is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy. Thereafter, compound (2-2) is produced by a method of reacting with compound (3-2).
  • the compound of the following formula (3-1a) in which a is 1 can be produced, for example, according to the following method.
  • R 3 , R 4 and p2 have the same meanings as [2] above, W 11 has the same meaning as W 1 in [2] above, and P 12 represents a hydrogen atom or a general amino group or hydroxyl group.
  • X 3 is a protecting group, for example, a chlorine atom, a bromine atom, an activation group of a carboxylic acid such as alkoxycarbonyloxy or succinimidyloxy, or a hydroxyl group
  • X 4 is phenoxy, p-nitrophenoxy or Activating group such as alkoxy.
  • Step 1 Compound (5-3) is produced from compound (5-1) and compound (5-2) by the same method as in Step 1 described in the production method for (1-2).
  • P 12 is a protecting group, for example, the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)) By using the general deprotection conditions shown in, once the P 12, Convert to hydrogen atom. Subsequently, in the presence of a base, for example, an aryl halide formate such as phenyl chloroformate or chloroformate (p-nitrophenyl), an alkyl formate such as ethyl chloroformate, isopropyl chloroformate or isobutyl chloroformate is reacted. To produce compound (3-1a).
  • a base for example, an aryl halide formate such as phenyl chloroformate or chloroformate (p-nitrophenyl)
  • an alkyl formate such as ethyl chloroformate, isopropyl chloroformate or isobutyl chloroformate is reacted.
  • a base for example, an
  • the amount of aryl halide formate or alkyl halide formate to be used is usually selected from the range of 1 to 5 equivalents relative to compound (5-3).
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic solvents (toluene, etc.), halogen solvents (methylene chloride, chloroform, etc.), or a mixed solvent thereof. Is mentioned.
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- And diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine and the like, preferably N-methylmorpholine, triethylamine, diisopropyl Ethylamine is mentioned.
  • the amount of the base used is usually selected from the range of 1 to 10 equivalents relative to the carboxylic acid.
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 100 ° C., preferably in the range of ⁇ 78 ° C. to room temperature.
  • the compound of the following formula (3-1b) in which a is 1 can be produced, for example, according to the following method.
  • X 1 is synonymous with the description of the production method of (1-2)
  • X 3 is synonymous with the description of the production method of (3-1a)
  • R 22 is a carboxylic acid or a protected carboxylic acid, a hydrogen atom Or a protective group for a hydroxyl group or an amine
  • X 5 is a chlorine atom, a bromine atom, an iodine atom, OMs, OTs, OTf, or a hydroxyl group.
  • Step 1 Compound (6-1) is produced from compound (5-2) and hydrazine by the same method as in Step 1 described in the production method of (1-2).
  • Step 2 Compound (6-4) is synthesized by reacting compound (6-2) with compound (6-3).
  • X 5 is, for example, a chlorine atom, a bromine atom, an iodine atom, OMs, OTs, or OTf
  • the reaction is performed in the presence of a base.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide).
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably cesium carbonate, potassium carbonate, hydrogenated Sodium is mentioned.
  • the amount of the base used is usually selected from the range of 1 to 5 equivalents relative to compound (6-2).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably in the range of room temperature to 100 ° C.
  • an azo compound such as diethyl azodicarboxylate or diisopropyl azodicarboxylate and a phosphorus reagent such as triphenylphosphine are used according to the Mitsunobu method (eg Synthesis, 1 (1981)).
  • the compound (6-4) can be produced.
  • the reaction solvent include an inert solvent such as THF, and examples of the reaction temperature include a range of 0 ° C. to the boiling point of the solvent.
  • Step 3 Compound (6-5) is synthesized by reacting compound (6-4) with compound (6-1) in the presence or absence of an acid.
  • the acid used include acetic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid and the like, and preferably acetic acid.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide).
  • reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably 0 to 100 ° C.
  • R 22 is a protecting group, for example, by using general deprotection conditions shown in the literature (Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.)), R 22 is once Convert to carboxylic acid or hydrogen atom. Subsequently, W 21 is — (CH 2 ) m3 — or — (CH 2 ) m6 — (wherein m3 and m6 each independently represents an integer of 1 to 20), and R 22 is In the case of a carboxylic acid, the compound (6-5) is converted to the compound (3-1b) by converting the hydroxyl group by the method described in Step 1 of the production of the raw material compound (1-2) as necessary. can do.
  • W 21 is — (CH 2 ) m4 —NH—, — (CH 2 ) m5 —O—, — (CH 2 ) m7 —NH— or — (CH 2 ) m8 —O— (where m4, m5 and m7 each independently represents an integer of 1 to 20, and when R 22 is a hydrogen atom, the compound (3-1) is produced by the same method as in Step 2 of the production method of (3-1a). 6-5) is converted to compound (3-1b).
  • the compound of the following formula (3-1c) in which a is 1 can be produced, for example, according to the following method.
  • X 1 has the same meaning as described in the production method of (1-2), R 24 is a carboxylic acid or a protected carboxylic acid, a hydrogen atom, a hydroxyl group or an amine protecting group, and X 6 is Chlorine atom, bromine atom, iodine atom, OMs, OTs or OTf.
  • Step 1 Compound (7-1) is synthesized by reacting compound (6-4) ′ with hydroxylamine.
  • the amount of hydroxylamine to be used is usually selected from the range of 1 to 10 equivalents relative to compound (6-4) ′.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide, DMSO, NMP, acetonitrile, HMPT, etc.), Water, alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof can be used. If necessary, for example, sodium acetate may be added as an additive.
  • the reaction temperature can usually be selected from the range of 0 ° C. to 150 ° C., and preferably in the range of room temperature to 100 ° C.
  • Compound (7-3) is synthesized by reacting compound (7-1) with compound (7-2) in the presence of a base.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide). , DMSO, NMP, acetonitrile, etc.), ketone solvents (acetone, etc.) or a mixed solvent thereof.
  • Examples of the base used include alkali metal carbonates (cesium carbonate, potassium carbonate, sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, etc.), alkali metal hydrides (sodium hydride, potassium hydride, etc.), alkali hydroxides Metal (potassium hydroxide, sodium hydroxide, etc.), organic base (N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5- Diazabicyclo [4.3.0] non-5-ene, 1,4-diazabicyclo [5.4.0] undec-7-ene, pyridine, etc.), preferably cesium carbonate, potassium carbonate, hydrogenated Sodium is mentioned.
  • the amount of the base to be used is usually selected from the range of 1 to 5 equivalents relative to compound (7-1).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C.,
  • the compound of the following formula (3-1d) in which a is 1 can be produced, for example, according to the following method.
  • Compound (8-3) is synthesized by reacting compound (8-1) with compound (8-2) in the presence or absence of an acid.
  • the acid used include acetic acid, hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, boron trifluoride diethyl ether complex and the like, and preferably acetic acid.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide).
  • reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably 0 to 50 ° C.
  • Step 2 Compound (8-4) is produced from compound (8-3) and compound (3-2) in the same manner as in the case where X 1 in Step 1 of the production method of (1-2) is a hydroxyl group. Is done. Specifically, the method using a condensing agent or the hydroxyl group of the carboxylic acid of the compound (8-3) is converted into an activating group such as a chlorine atom, a bromine atom, alkoxycarbonyloxy or succinimidyloxy. Thereafter, compound (8-4) is produced by a method of reacting with compound (3-2).
  • Step 3 Compound (1-3d) is produced by reacting compound (8-4) with compound (8-5).
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, acetonitrile, etc.), water ( Buffer solution), alcohol solvents (methanol, ethanol, etc.), or a mixed solvent thereof.
  • the reaction temperature can usually be selected from the range of 0 to 100 ° C., preferably in the range of room temperature to 80 ° C.
  • R 2 represents —C (CH 3 ) 2 —CH 2 —NH—C (O) — (CH 2 CH 2 O) 4 —CH 2 CH 2 —NH—C (O) — ( A compound that is CH 2 ) 5 — is synthesized by the methods of published patent (Yamazaki et al., WO2010 / 053115), Reference Example 35 and Example 34.
  • Step 1 Compound (9-3) is produced by reacting compound (9-1), which can be synthesized by a known method, with compound (9-2).
  • the reaction is synthesized by reacting compound (9-1) with compound (9-2) in the presence or absence of a buffer solution.
  • the buffer solution used include borate buffer solution, phosphate buffer solution, acetate buffer solution, citrate buffer solution, and preferably borate buffer solution.
  • the solvent include ether solvents (THF, diethyl ether, DME, 1,4-dioxane, etc.), halogen solvents (methylene chloride, chloroform, etc.), aprotic polar solvents (DMF, N, N-dimethylacetamide).
  • the reaction temperature can usually be selected from the range of ⁇ 78 to 150 ° C., preferably 0 ° C. to room temperature (see Reference Example 37, Reference Example 53, and Example 44).
  • raw material compounds of the compound of the present invention are known compounds or can be synthesized from known compounds by combining known synthesis methods.
  • a protection or deprotection technique can be used as necessary. Suitable protecting groups, protecting methods, and deprotecting methods are described in detail in the aforementioned Protective Groups in Organic Synthesis 3rd Edition (John Wiley & Sons, Inc.) and the like.
  • the compound of the present invention or an intermediate for producing the compound can be purified by methods known to those skilled in the art.
  • it can be purified by column chromatography (for example, silica gel column chromatography or ion exchange column chromatography) or recrystallization.
  • recrystallization solvent alcohol solvents such as methanol, ethanol or 2-propanol, ether solvents such as diethyl ether, ester solvents such as ethyl acetate, aromatic hydrocarbon solvents such as benzene or toluene, acetone, etc.
  • a ketone solvent such as hexane, a hydrocarbon solvent such as hexane, an aprotic solvent such as dimethylformamide or acetonitrile, water, or a mixed solvent thereof can be used.
  • the methods described in Experimental Chemistry Course (edited by the Chemical Society of Japan, Maruzen) Vol. 1 etc. can be used.
  • the administration route of the compound of the present invention is roughly classified into oral administration and parenteral administration.
  • the dose of the compound varies depending on the type of compound, administration form, administration method, patient symptom / age, etc., but is usually 0.005 to 150 mg / kg / day, preferably 0.05 to 20 mg / kg / day. And can be administered in one or several divided doses.
  • the compound of the present invention is usually administered as a pharmaceutical in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier.
  • a pharmaceutical carrier include oral preparations such as tablets and capsules, ointments, intravesical injections and the like.
  • External preparations such as liquid preparations for external use, patches, inhalants, nasal drops, injections such as intradermal injections, subcutaneous injections or intracavitary injections such as intraperitoneal and intraarticular cavity.
  • These pharmaceutical compositions are prepared according to conventional methods.
  • the pharmaceutical carrier a substance that is commonly used in the pharmaceutical field and does not react with the compound according to the present invention is used.
  • Specific examples of pharmaceutical carriers used for tablet and capsule production include lactose, corn starch, sucrose, mannitol, calcium sulfate, excipients such as crystalline cellulose, croscarmellose sodium, modified starch, carmellose calcium, Crospovidone, disintegrants such as low substituted hydroxypropylcellulose, binders such as methylcellulose, gelatin, gum arabic, ethylcellulose, hydroxypropylcellulose, povidone, light anhydrous silicic acid, magnesium stearate, talc, sucrose fatty acid ester And lubricants such as hydrogenated oil.
  • the tablet may be coated by a well-known method using a coating agent such as carnauba wax, hydroxypropylmethylcellulose, macrogol, cellulose acetate phthalate, hydroxypropylmethylcellulose acetate phthalate, sucrose, titanium oxide, sorbitan fatty acid ester, calcium phosphate.
  • a coating agent such as carnauba wax, hydroxypropylmethylcellulose, macrogol, cellulose acetate phthalate, hydroxypropylmethylcellulose acetate phthalate, sucrose, titanium oxide, sorbitan fatty acid ester, calcium phosphate.
  • the base of the patch include polymer bases such as polyvinyl pyrrolidone, polyisobutylene, vinyl acetate copolymer, acrylic copolymer, glycerin, propylene glycol, polyethylene glycol, triethyl citrate, citric acid Plasticizers such as acetyltriethyl, diethyl phthalate, diethyl sebacate, dibutyl sebacate, and acetylated monoglycerides.
  • polymer bases such as polyvinyl pyrrolidone, polyisobutylene, vinyl acetate copolymer, acrylic copolymer, glycerin, propylene glycol, polyethylene glycol, triethyl citrate, citric acid Plasticizers such as acetyltriethyl, diethyl phthalate, diethyl sebacate, dibutyl sebacate, and acetylated monoglycerides.
  • the dermal absorption enhancer may be any pharmacologically acceptable one, for example, alcohols such as ethanol and diethylene glycol; polar solvents such as dodecylpyrrolidone; urea; ethyl laurate; azone; olive oil Etc.
  • alcohols such as ethanol and diethylene glycol
  • polar solvents such as dodecylpyrrolidone
  • urea urea
  • ethyl laurate azone
  • olive oil Etc olive oil
  • inorganic fillers such as kaolin, bentonite, zinc oxide, titanium oxide; agarose, carrageenan, alginic acid or salts thereof, tragacanth, acacia gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, carboxyvinyl polymer, gelatin, corn starch
  • Viscosity modifiers such as polymers such as xanthan gum, dextrin and polyvinyl alcohol; anti-aging agents; pH regulators; humectants such as glycerin and propylene glycol may be added. Further, a surfactant may be added.
  • fatty acid alkali salts such as potassium laurate, potassium palmitate and potassium myristate; sodium lauryl sulfate, sodium cetyl sulfate, castor oil sulfate (funnel oil) Ionic surfactants such as sulfate esters such as sorbitan stearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate (so-called Span); polysorbate 20, polysorbate 40, polysorbate 60, Polysorbate 65, polysorbate 80, polysorbate 85, polyoxyethylene sorbitan fatty acid ester (so-called Tween); polyoxyethylene hydrogenated castor oil (so-called HCO); polyoxyethylene lauryl ester Polyoxyethylene alkyl ethers such as tellurium, polyoxyethylene cetyl ether and polyoxyethylene oleyl ether; polyethylene glycol fatty acid esters such as polyethylene glycol monolaurate and polyethylene glycol
  • An inhalant can be produced by converting the compound of the present invention into a powder or liquid form and blending it into an inhalation spray or carrier and filling the inhalation container such as a metered dose inhaler or a dry powder inhaler. it can. It may be a spray, an aerosol, or a spray.
  • the inhalable propellant conventionally known ones can be widely used.
  • CFCs such as 1,1,1,2-tetrafluoroethane
  • CFC alternatives such as HFA-227 and HFA-134a
  • examples thereof include hydrocarbon gas, diethyl ether, nitrogen gas, carbon dioxide gas and the like.
  • the carrier conventionally known ones can be widely used, and examples thereof include sugars, sugar alcohols, amino acids and the like.
  • preservatives benzalkonium chloride, parabens, etc.
  • coloring agents buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride, concentrated glycerin, etc.)
  • buffering agents sodium phosphate, sodium acetate, etc.
  • isotonic agents sodium chloride, concentrated glycerin, etc.
  • a sticking agent such as carboxyvinyl polymer
  • an antiseptic such as benzalkonium chloride, paraben
  • an absorption accelerator and the like as necessary.
  • lubricants stearic acid and its salts, etc.
  • binders starch, dextrin, etc.
  • excipients lactose, cellulose, etc.
  • coloring agents absorption enhancers, etc.
  • the nasal drops can take various forms such as a drop type, a coating type, and a spray type.
  • a spray type manual pump type nasal drops with a mechanism for ejecting liquid by manually moving the pump attached to the container, compressed gas (air or oxygen, nitrogen, carbonic acid, or mixed gas), etc.
  • compressed gas air or oxygen, nitrogen, carbonic acid, or mixed gas
  • an aerosol type nasal spray having a mechanism in which a liquid agent is automatically ejected by moving a valve attached to the container and filling the container with the propellant is automatically included.
  • the compound of the present invention is dissolved in distilled water for injection and, if necessary, a solution to which a solubilizing agent, buffer, pH adjuster, tonicity agent, soothing agent, preservative and the like are added.
  • a solubilizing agent, buffer, pH adjuster, tonicity agent, soothing agent, preservative and the like May be prepared by suspending the compound in distilled water for injection or vegetable oil, and in this case, a base, a suspending agent, a thickening agent, etc. may be added as necessary.
  • a base, a suspending agent, a thickening agent, etc. may be added as necessary.
  • dissolves a powder or a lyophilized product at the time of use may be sufficient, and an excipient
  • the content of the compound according to the present invention in the pharmaceutical composition varies depending on the dosage form, but is usually 0.0025 to 20% by weight in the total composition. These pharmaceutical compositions may also contain other therapeutically effective substances.
  • the administration timing of the compound of the present invention and the concomitant drug is not particularly limited, and the compounding ratio of the compound of the present invention and the concomitant drug can be appropriately set depending on the administration subject, administration method, disease, combination and the like.
  • the content of the concomitant drug in the concomitant drug of the present invention varies depending on the form of the preparation, but is usually 0.0025 to 20% by weight in the total composition.
  • Compound identification includes elemental analysis values, mass spectrum, high performance liquid chromatography / mass spectrometer (LCMS), time-of-flight mass spectrometer (TOF-MS), IR spectrum, NMR spectrum, high performance liquid chromatography (HPLC), etc. It went by.
  • LCMS high performance liquid chromatography / mass spectrometer
  • TOF-MS time-of-flight mass spectrometer
  • IR spectrum IR spectrum
  • NMR spectrum high performance liquid chromatography
  • HPLC high performance liquid chromatography
  • nucleic acid sequences include 3′- for the 3 ′ end, 5′- for the 5 ′ end, A, G, C, U, and T for adenine, guanine, cytosine, uracil, and thymine, respectively, as nucleobases.
  • the ribonucleotides a, g, c and t are deoxyribonucleotides having adenine, guanine, cytosine and thymine as nucleobases, respectively.
  • a (M), G (M), C (M), and U (M) represent 2′-O-methylated adenine, guanine, cytosine, and uracil, respectively, and A (F), G (F ), C (F) and U (F) mean ribonucleotides having a nucleobase of adenine, guanine, cytosine and uracil fluorinated at the 2 ′ position, respectively. Furthermore, ⁇ means a phosphorothioate group. “Room temperature” in the following examples usually indicates about 10 ° C. to about 35 ° C. The ratio shown in the mixed solvent is a volume ratio unless otherwise specified. Unless otherwise indicated, “%” indicates “% by weight”.
  • the title compound was obtained from 10-undecylic acid by reacting and treating in the same manner as described in Reference Example 1.
  • the reaction mixture was cooled to 0 ° C., diluted with water (205 mL), and neutralized with 1N aqueous hydrochloric acid (201 mL). Further, water (205 mL) and ethyl acetate (774 mL) were added for liquid separation. The organic layer was washed with saturated brine (194 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was crystallized by adding a mixed solution of diethyl ether / hexane to give the title compound (29.7 g) as a white solid.
  • Reference Example 15 Nucleic acid sequence: 3′-ttCGUGUCCUCUCUUCUCGUA-5 ′ (SEQ ID NO: 1) (1) Synthesis of oligonucleotides All oligonucleotides were synthesized by an AKTA oligopilot synthesizer.
  • RNA phosphoramidite with standard protecting group (5'-O-dimethoxytrityl-N 6 -benzoyl-2'- Triisopropylsilyloxymethyl-adenosine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N 4 -acetyl-2′-triisopropylsilyloxymethyl-cytidine -3'-ON, N'-diisopropyl-2-cyanoethyl phosphoramidite, 5'-O-dimethoxytrityl-N 2 -isobutyryl-2'-triisopropylsilyloxymethyl-guanosine-3'-ON , N'-Diisopropyl-2-cyanoethyl phosphoro
  • phosphoramidites were used at a concentration of 0.1M in acetonitrile. A 15 minute ligation / reuse time was used for RNA and a 5 minute ligation / reuse time was used for DNA.
  • phosphoramidite activator 5-benzylmercaptotetrazole (0.25M, manufactured by Wako Pure Chemical Industries, Ltd.) was used, and iodine / water / pyridine was used for the oxidation of phosphorous acid to phosphoric acid.
  • polystyrene solid carrier an aminated nonporous polystyrene solid carrier (Custom Primer Support C6 amino 80 manufactured by GE Healthcare) was used.
  • RNA phosphoramidite namely 5′-O-dimethoxytrityl-N 6 -benzoyl-2′-triisopropylsilyloxymethyladenosine-3′-ON, N′-diisopropyl -2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N 4 -acetyl-2′-triisopropylsilyloxymethylcytidine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl-N 2 -isobutyryl-2′-triisopropylsilyloxymethylguanosine-3′-ON, N′-diisopropyl-2-cyanoethyl phosphoramidite, 5′-O-dimethoxytrityl- 2'-
  • RNA DNA
  • DNA DNA
  • 5 ′ end modification 5 ′ end modification.
  • phosphoramidite activator 5-benzylmercaptotetrazole (0.25M, manufactured by Wako Pure Chemical Industries, Ltd.) was used, and iodine / water / pyridine was used for the oxidation of phosphorous acid to phosphoric acid.
  • Aqueous solution (0.47 mM, 560 ⁇ L) was mixed in order.
  • An aqueous solution (10 mM, 888 ⁇ L) of the compound of Reference Example 18 was added to the well mixed solution, and the reaction was performed at 25 ° C. for 2 hours. After the reaction was completed, HPLC analysis was performed using a C18 reverse phase column (X-bridge Nippon Waters) to confirm the reaction.
  • the mixture was prepared to be a 10 mM Tris-HCl buffer (pH 8.0) solution of 20 mM sodium chloride at a final concentration.
  • the solution was heated at 70 ° C. for 10 minutes, and then the temperature was decreased by 1 ° C. per minute to 30 ° C. for annealing. Annealing was confirmed by 19% non-denaturing polyacrylamide gel electrophoresis, and the compound of Example 1 was obtained.
  • Example 2-17 In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXIII):
  • Example 18-21 In the same manner as in Example 1, using the corresponding starting material compound, the following formula (XXIV):
  • the compound of Reference Example 53 was dissolved in deionized RNase-free water so as to be 0.50 mM.
  • a DMF solution (70 ⁇ L) of the compound of Reference Example 37 prepared to 10 mM was added to a mixed solution of 560 ⁇ L of this aqueous solution and 770 ⁇ L of 0.5 M borate buffer (pH 8.0), and the mixture was stirred at 25 ° C. for 1 hour.
  • Test Example 1 cAMP Assay of ⁇ 2 Receptor Expressing HEK293 Cells
  • HEK293 cell line was seeded at 6 ⁇ well plate at 3 ⁇ 10 5 cells / 2 mL / well and cultured overnight.
  • antibiotic-free 10% FBS / DMEM was used as the medium.
  • the beta 2 receptor was inserted into the mammalian expression vector such as pcDNA3.1 and pCI, were transfected with LipofectamineLTX (overnight culture at 37 ° C.). The cells were detached with trypsin, suspended in 2 mL of FBS ( ⁇ ) / DMEM, and centrifuged at 1000 rpm for 2 minutes.
  • the supernatant was removed, and the cells were suspended in 1 ⁇ 10 6 cells / mL with assay buffer (0.5 mM IBMX / 0.1% BSA / DMEM). To 384-well Low Volume Black Round Bottom PS NBS Microplate (Corning, Cat # 3676), 5 ⁇ L / well of the evaluation compound prepared to twice the final concentration was added, and 5 ⁇ L / well of the cell suspension was further added. After standing at 37 ° C. for 0.5 hour, the cAMP concentration produced by the cells was quantified using cAMP HTRF Kit (Cisbio, Cat # 62AM6PEJ).
  • HTRF homogeneous time-resolved fluorescence
  • Test Example 2 Receptor internalization assay 6-well plate using the beta 2 receptor expressing HEK293 cells with GFP2 crowded seeded HEK293 cell lines 3x10 5 cells / 2 mL / well , and cultured overnight.
  • antibiotic-free 10% FBS (0.22 ⁇ m-filtered) / DMEM was used.
  • those that combines GFP2 the beta 2 receptor is a mammalian expression vector pcDNA3.1 and pCI or various viral expression vector pAxcwit2, pDON-5 DNA, pLVSIN -CMV Neo, inserted like pFastBacMam, virus created Infected (37 ° C., 4-6 hours).
  • Emax value the concentration of a beta 2 agonist Salmeterol is inherent to the maximum as 100%, was expressed at a ratio of concentrations indicating the maximum internalization of each compound thereto.
  • the EC 50 value was calculated as the concentration of the compound that resulted in internalization of 50% of the Emax value of each compound.
  • the EC 50 value of Salmeterol was 1.7 nM.
  • the compounds of the present invention showed internalization in beta 2 receptor expressing HEK293 cells with GFP2.
  • Test Example 3 Knockdown experiments method HEK293 cell line beta 2 receptor expression plasmid using beta 2 receptor expressing HEK293 cells are transfected with LipofectamineLTX (overnight culture at 37 ° C.). Cells are detached with trypsin and suspended in 10% FBS / DMEM. Cells are seeded on a 12-well plate at 1.5 ⁇ 10 5 cells / 2 mL / well and cultured for 6 hours. Add compound and incubate for 24 hours. The culture fluid of the cultured cells is removed, and total RNA is prepared using the QuickGene-800 automatic nucleic acid extraction system (FUJIFILM) and RNA cultured cell kit S (FUJIFILM) according to the instructions.
  • FUJIFILM QuickGene-800 automatic nucleic acid extraction system
  • FUJIFILM RNA cultured cell kit S
  • RNA is used as a template, and a reverse transcriptase (RT) reaction is carried out according to the instructions of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
  • RT reverse transcriptase
  • Power SYBR Green PCR Master Mix Applied Biosystems
  • quantitative PCR is performed by the Applied Biosystems 7900HT real-time PCR system according to the instructions.
  • Step 1 50 ° C., 2 min
  • Step 2 95 ° C., 10 min
  • Step 3 95 ° C., 15 sec
  • Step 4 95 ° C., 15 sec
  • Step 5 60 ° C., 15 sec
  • Step 6 95 ° C., 15 sec.
  • PCR primers are peptidylprolylomerase B (cyclophilin B) (hereinafter referred to as PPIB), PPIB Forward Primer: 5'-gctagataggcaagcatgtgtgtgtt-3 ', PPIBrevert' Ribosomal protein, large, P0 (hereinafter referred to as RPLP0) is used as an internal standard gene, and PCR primers are RPLP0 Forward Primer: 5′-aggtgtcgacatggcatcatcact-3 ′, RPLP0 Reverse Primet 5 To do.
  • PPIB peptidylprolylomerase B
  • RPLP0 PPIB Revert' Ribosomal protein, large
  • PCR primers are RPLP0 Forward Primer: 5′-aggtgtcgacatggcatcatcact-3 ′, RPLP0 Reverse Primet 5 To do.
  • Test Example 4 Intracellular Uptake Assay Using Fluorescent Labeled Compound HEK293 cell line was seeded at 6 ⁇ well plate at 3 ⁇ 10 5 cells / 2 mL / well and cultured overnight at 37 ° C. in a 5% CO 2 incubator. As the medium, antibiotic-free 10% FBS / DMEM was used. Human beta 2 receptor was inserted into the mammalian expression vector, such as PTran3.1, were transfected with LipofectamineLTX (overnight culture at 37 ° C.).
  • Test Example 5 cAMP assay of 5-HT 4 receptor-expressing HEK293 cells
  • HEK293 cell line was seeded on a 6-well plate at 1.5 ⁇ 10 5 cells / 2 mL / well and cultured overnight.
  • antibiotic-free 10% FBS / DMEM was used as the medium.
  • 5-HT 4 receptor was inserted into the pcDNA3.1 mammalian expression vector, were transfected with Trans-IT LT1 reagent (cultured overnight at 37 ° C.). The cells were detached with trypsin, suspended in 2 mL of FBS ( ⁇ ) / DMEM, and centrifuged at 1000 rpm for 2 minutes.
  • HTRF homogeneous time-resolved fluorescence
  • the novel compound comprising a conjugate of an antisense oligonucleotide, siRNA, and miRNA represented by a miRNA and a ligand that binds to GPCR in the present invention has an efficient intracellular uptake ability via GPCR,
  • the functional expression of oligonucleotides can be shown and contributes greatly to the industrial fields such as research and medical fields.

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Abstract

L'invention concerne un transporteur pour l'incorporation efficace d'un médicament d'acide nucléique dans des cellules malades. Plus particulièrement, l'invention concerne un nouveau composé (conjugué) chimique d'une molécule d'acide nucléique et du transporteur. Le conjugué comprend un lieur entre un ligand de liaison à un récepteur couplé à une protéine G et une molécule d'acide nucléique. Le lieur comprend le triazolylène et une chaîne polyalkylène glycol qui peut être substituée, ou une chaîne alkylène qui peut être substituée (la chaîne alkylène peut avoir un ou plusieurs groupes divalents insérés de sorte qu'il ou ils interrompent la chaîne, ou à l'extrémité de celle-ci, choisi dans le groupe consistant en -O-, -S-, -NH-, =N-, -N=, -SO2-, -C(=O)-, arylène en (C6-C10), hétéroarylène en (C2-C9), cycloalkylène en (C3-C8) et hétérocyclène en (C3-C7)).
PCT/JP2013/065512 2012-06-04 2013-06-04 Conjugué de ligand de liaison à un récepteur couplé à une protéine g et d'une molécule d'acide nucléique Ceased WO2013183656A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
JP2016503394A (ja) * 2012-10-26 2016-02-04 エヌライフ、セラピューティックス、ソシエダッド、リミターダＮｌｉｆｅ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｓ．Ｌ． 細胞型へのオリゴヌクレオチド分子の選択的送達のための組成物および方法
CN105646285A (zh) * 2014-12-02 2016-06-08 上海医药工业研究院 一种维兰特罗中间体及其制备方法和应用
EP3136099A1 (fr) * 2015-08-31 2017-03-01 Sysmex Corporation Méthode d'analyse de sang ainsi que solution colorante et analyseur de sang utilisé pour celle-ci
CN106699576A (zh) * 2016-12-23 2017-05-24 常州瑞明药业有限公司 盐酸甲氧那明的合成方法
CN108976216A (zh) * 2018-09-07 2018-12-11 江苏工程职业技术学院 一种普卡必利的制备方法

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WO2009082606A2 (fr) * 2007-12-04 2009-07-02 Alnylam Pharmaceuticals, Inc. Conjugués du folate
JP2011505425A (ja) * 2007-12-04 2011-02-24 アルニラム ファーマスーティカルズ インコーポレイテッド オリゴヌクレオチドの送達剤としての糖質コンジュゲート

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WO2009082606A2 (fr) * 2007-12-04 2009-07-02 Alnylam Pharmaceuticals, Inc. Conjugués du folate
JP2011505425A (ja) * 2007-12-04 2011-02-24 アルニラム ファーマスーティカルズ インコーポレイテッド オリゴヌクレオチドの送達剤としての糖質コンジュゲート

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016503394A (ja) * 2012-10-26 2016-02-04 エヌライフ、セラピューティックス、ソシエダッド、リミターダＮｌｉｆｅ Ｔｈｅｒａｐｅｕｔｉｃｓ， Ｓ．Ｌ． 細胞型へのオリゴヌクレオチド分子の選択的送達のための組成物および方法
CN105646285A (zh) * 2014-12-02 2016-06-08 上海医药工业研究院 一种维兰特罗中间体及其制备方法和应用
CN105646285B (zh) * 2014-12-02 2017-11-24 上海医药工业研究院 一种维兰特罗中间体及其制备方法和应用
EP3136099A1 (fr) * 2015-08-31 2017-03-01 Sysmex Corporation Méthode d'analyse de sang ainsi que solution colorante et analyseur de sang utilisé pour celle-ci
US10295553B2 (en) 2015-08-31 2019-05-21 Sysmex Corporation Blood analyzing method as well as stain solution and blood analyzer used for the same
CN106699576A (zh) * 2016-12-23 2017-05-24 常州瑞明药业有限公司 盐酸甲氧那明的合成方法
CN108976216A (zh) * 2018-09-07 2018-12-11 江苏工程职业技术学院 一种普卡必利的制备方法
CN108976216B (zh) * 2018-09-07 2021-02-12 江苏工程职业技术学院 一种普卡必利的制备方法

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