WO2020045570A1 - Self-assembly for activating immunity - Google Patents

Self-assembly for activating immunity Download PDF

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WO2020045570A1
WO2020045570A1 PCT/JP2019/033920 JP2019033920W WO2020045570A1 WO 2020045570 A1 WO2020045570 A1 WO 2020045570A1 JP 2019033920 W JP2019033920 W JP 2019033920W WO 2020045570 A1 WO2020045570 A1 WO 2020045570A1
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self
assembly
compound
formula
shows
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PCT/JP2019/033920
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French (fr)
Japanese (ja)
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志成 上杉
ブー・フエ・ティ
大樹 吉田
尚孝 野田
ジン・シュウユウ
晶 山▲崎▼
徹 島根
石井　健
仰 日置
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国立大学法人京都大学
国立大学法人大阪大学
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Priority to JP2020539590A priority Critical patent/JPWO2020045570A1/en
Publication of WO2020045570A1 publication Critical patent/WO2020045570A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link

Definitions

  • the present invention relates to a novel compound forming a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
  • Self-assembly which is the spontaneous self-assembly of molecules, plays an important role in the formation of biological hierarchical structures in living organisms.
  • DNA, lipid membranes, cytoskeletal fibers, and the like are examples of natural self-assemblies.
  • a single compound molecule has a single function
  • a self-assembly has complex physical or chemical properties and functions depending on the surrounding environment (Non-Patent Document 1). Therefore, the synthesis of a new self-assembly has attracted attention as one of new methods for drug development that regulates the function and biological activity of biological systems.
  • Non-Patent Document 2 synthetic oligonucleotides containing unmethylated CG sequences (CpG- ODN) is known (Non-Patent Document 2). As described above, several self-assemblies that mimic the natural self-assembly structure have been reported, but most of them use peptides as constituent units of the self-assembly.
  • Non-Patent Documents 3 to 10 Although some examples of self-assembly of non-peptide synthetic chemical molecules are also known, their biological activities are mainly related to cytotoxicity and cell death (Non-Patent Documents 3 to 10). Therefore, new and useful self-assembly of synthetic chemical molecules that exhibit biological activity is desired.
  • One of the problems to be solved by the present invention is to provide a novel compound which forms a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, a small molecule compound having a partial structure in which a specific chemical modification has been made to bile acid forms a new self-assembly, and the self-assembly concerned Showed that they exhibited an immunostimulatory effect, and thus completed the present invention.
  • the self-assembled substance formed by linking the compounds of the formula (I) can exhibit a biological activity, for example, an immunostimulatory action, and can be useful as an adjuvant.
  • FIG. 1 shows the hydrodynamic diameter (nm) (vertical axis) of a compound (Hit @ compounds) that formed a self-assembly in the compound library.
  • FIG. 2 shows the hydrodynamic radii (average R h (nm)) (vertical axis) of self-assemblies of the compounds of Example 1, Reference Example 2 and Reference Example 3 in DMEM solutions at various concentrations ( ⁇ M).
  • FIG. 3 shows the IL-6 production amount (ng / mL) (vertical axis) of the self-assembly of the compound of Example 1 in DMEM solutions at various concentrations ( ⁇ M).
  • FIG. 1 shows the hydrodynamic diameter (nm) (vertical axis) of a compound (Hit @ compounds) that formed a self-assembly in the compound library.
  • FIG. 2 shows the hydrodynamic radii (average R h (nm)) (vertical axis) of self-assemblies of the compounds of Example 1, Reference
  • FIG. 4 shows the IL-6 production amount (ng / mL) (vertical axis) of the compounds of Example 1 and Reference Examples 1 to 3 in a 30 ⁇ M @DMEM solution.
  • DCA shows the result for deoxycholic acid.
  • NC is a negative control of 1% (v / v) DMSO, and LPS is a positive control.
  • FIG. 5 shows the relative value (vertical axis) of the amount of IL-6 produced by the self-assembly of each example compound in a 30 ⁇ M @DMEM solution when the negative control (DMSO) is set to 1.
  • FIG. 6 shows the particle size distribution (%) of the self-assembly of the compound of Example 1 in a 30 ⁇ M @DMEM solution (right side).
  • FIG. 9 shows the results of an IL-6 ⁇ ELISA assay (ng / mL) using FcR ⁇ / MyD88 double knockout BMDC (right) and wild-type BMDC (WT) (left).
  • FIG. 12 shows IL-6 production (pg / mL) of wild-type (WT) and TLR7 knockout (Tlr7 ⁇ / ⁇ ) mice.
  • the bar on the left side of each test substance shows the result of the wild type, and the bar on the right side shows the result of the TLR7 knockout mouse.
  • FIG. 18 shows the production amount (ng / mL) of the anti-OVA antibody (IgE). Each bar in each test substance shows the results at 1, 10 and 100 ⁇ g from the left, respectively.
  • FIG. 20A shows the relationship between the number of days after influenza infection (horizontal axis) and the weight loss rate (%) (vertical axis).
  • ⁇ indicates a self-assembly of the compound of Example 1
  • ⁇ indicates a self-assembly of the compound of Example 7
  • ⁇ indicates a self-assembly of the compound of Example 10
  • ⁇ indicates DCA
  • ⁇ indicates 1% DMSO
  • ⁇ indicates an aluminum salt.
  • FIG. 20B shows the relationship between the number of days after influenza infection (horizontal axis) and the survival rate (%) (vertical axis).
  • ⁇ indicates a self-assembly of the compound of Example 1
  • ⁇ indicates a self-assembly of the compound of Example 7
  • ⁇ indicates data when DCA
  • ⁇ indicates 1% DMSO
  • FIG. 21A shows the number of days after influenza SV injection (horizontal axis) and weight loss rate (%) (vertical axis) for each dose ( ⁇ : 1 ⁇ g, ⁇ : 10 ⁇ g, ⁇ : 100 ⁇ g) of the self-assembly of the compound of Example 10. Shows the relationship.
  • ⁇ indicates data when 1% DMSO was used, and ⁇ indicates data when aluminum salt was used.
  • FIG. 23 shows the results of a SEAP assay for null and no TLR9 using HEK293 cells.
  • FIG. 24 shows the results of a SEAP assay for TLR2 and TLR4 using HEK293 cells without an agonist (null).
  • FIG. 25 shows the results of the IL-6 quantitative PCR assay (qPCR).
  • Item 2 The self-assembly according to Item 1, wherein R x is the same as R y .
  • R x is of the formula (II ′): Is a group represented by R y has the formula (III ′): Item 3.
  • Item 4 The self-assembly according to any one of Items 1 to 3, wherein R 3 and R 4 are each independently C 1-3 alkyl.
  • Item 3 The self-assembly according to Item 1 or 2, wherein R x and R y are both H.
  • Item 6 The self-assembly according to any one of Items 1 to 5, wherein n is an integer of 5 to 10.
  • Item 7 The self-assembly according to any one of Items 1 to 6, which forms an aggregate having a hydrodynamic size of 40 nm to 12,000 nm.
  • Item 8 An immunostimulator comprising the self-assembly according to any one of Items 1 to 7.
  • Item 9 The immunostimulant according to Item 8, which is an adjuvant.
  • Item 10 A vaccine composition comprising the immunostimulating agent according to Item 9 and an antigen.
  • Item 11 The vaccine composition according to Item 10, for treating and / or preventing a disease selected from the group consisting of cancer, infectious disease, allergic disease, autoimmune disease, hypertension and Alzheimer's disease.
  • R x is H or formula (II): Is a group represented by R y is H or formula (III): Is a group represented by R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl; Wavy lines indicate binding sites, n is an integer of 1 to 12]
  • C 1-12 alkyl refers to a linear or branched saturated hydrocarbon group having 1 to 12 carbon atoms.
  • “C 1-12 alkyl” includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 2,2-dimethylpentyl , 2,2,3-trimethylbutyl, n-octyl, 3,4-dimethylhexyl, 2,2,4-trimethylpentyl, n
  • C 1-6 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl and the like.
  • C 1-12 haloalkyl refers to a C 1-12 alkyl in which one or more optional hydrogen atoms have been replaced with the same or different halogen.
  • C 1-12 haloalkyl includes difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2-chloroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 1,1 -Difluoropropyl, 3,3,3-trifluoropropyl, 2,2,2-trifluoro-1-methyl-ethyl, 1,1-difluoro-2-methylpropyl, 4-chlorobutyl, 5-fluoropentyl and the like No.
  • halo or “halogen” includes fluoro, chloro, bromo, iodo and the like.
  • self-assembly refers to an assembly formed by self-assembling, defined as the spontaneous self-assembly of a molecule, and comprising the molecule as a constituent unit, and two or more molecules. Is a micellar, fibrous, crystalline, colloidal, or particulate aggregate formed by non-covalent bonding.
  • non-covalent bond include a hydrophobic bond, ⁇ - ⁇ stacking, a hydrogen bond, a van der Waals bond, and an arbitrary combination thereof.
  • the molecules constituting the self-assembly include small molecule compounds having a partial structure in which bile acid is chemically modified, and preferably a compound of the formula (I).
  • the self-assembly may have a plurality of hydrophobic pockets, and through this hydrophobic pocket, various substances such as other molecules and antigens can be incorporated into the assembly structure.
  • a self-assembly may also be an aggregate of compounds of formula (I) having different structures based on the differences in R x , R y and R 1 -R 4 , and compounds other than compounds of formula (I) Mixed together to form an aggregate.
  • the structure and properties of the self-assembly can be appropriately controlled by chemical modification of a molecule serving as a structural unit.
  • Self-assemblies may exhibit immunostimulatory effects through interaction with membrane proteins.
  • the hydrodynamic size of the self-assembly is not particularly limited and may depend on the surrounding environment such as the concentration in the medium, the concentration in the blood and the concentration in the tissue, but is, for example, 40 to 12,000 nm, and is preferably It is 45 to 5,000 nm, more preferably 100 to 1,000 nm, further preferably 140 to 800 nm, and particularly preferably 150 to 500 nm.
  • the term "hydrodynamic size" refers to the hydrodynamic diameter, which may be measured, for example, using dynamic light scattering (DLS).
  • the self-assembly may also have an increased immunostimulatory effect as its concentration in the medium, blood or tissue increases.
  • immunostimulatory effect refers to endogenous or exogenous agonist activity for a pattern recognition receptor on the cell surface.
  • agonist activity antigen presenting cells such as macrophages and dendritic cells can be activated to produce cytokines such as interleukins.
  • the immunostimulatory activity is preferably an agonist activity for a Toll-like receptor (TLR), more preferably a TLR7 agonist activity or a TLR9 agonist activity, and particularly preferably a TLR7 agonist activity.
  • TLR Toll-like receptor
  • Immunostimulatory effects can also be increased as the hydrodynamic size of the self-assembly increases.
  • TLR7 stimulates the production of interferon and the like by sensing single-stranded RNA or synthetic small molecule ligand derived from virus. Therefore, TLR7 agonist has an immunostimulatory effect, for example, various diseases such as infectious diseases and autoimmune diseases. May be useful as therapeutics for Since TLR9 is commonly found in bacteria and viruses and plays a role in stimulating innate and adaptive immune responses to unmethylated DNA, TLR9 agonists can improve the immunogenicity of antigens used in vaccine compositions.
  • immunostimulant refers to a substance or composition that exhibits an immunostimulatory effect, and can be used for immunotherapy of various diseases.
  • the immunostimulant may contain a self-assembly.
  • an immunostimulant is used in combination with an antigen or a vaccine composition containing the antigen, immunogenicity to the antigen can be obtained or enhanced, and the duration of immunity can be prolonged.
  • the immunostimulant may be administered alone, may be used to be administered separately, sequentially or simultaneously at an interval from the antigen or vaccine composition comprising the antigen, or May be used as a component of the product. Examples of the immunostimulant include an adjuvant.
  • the amount of the self-assembly contained in the immunostimulant is not particularly limited as long as it is an immunologically acceptable amount, but is, for example, 0.1 to 20 w / w%, preferably 0.2 to 20 w / w%. It is 15 w / w%, more preferably 0.3 to 10 w / w%, particularly preferably 0.5 to 5 w / w%.
  • antigen refers to a substance that provokes an immune response, such as a peptide or protein.
  • Antigens include cancer antigens, viral antigens, bacterial antigens, fungal antigens, protozoal or parasite antigens, allergy-related antigens, and disease-related antigens.
  • the term “vaccine composition” refers to a composition containing an antigen, and can be used for immunotherapy of various diseases.
  • the vaccine composition may be administered alone, may be used so that it is administered separately, sequentially or simultaneously at an interval from the immunostimulant, or a vaccine containing the immunostimulant. It may be used as a composition.
  • the vaccine composition may also include a pharmaceutically acceptable carrier.
  • Such pharmaceutically acceptable carriers include additives commonly used in the art, such as excipients, isotonic agents, buffers, pH adjusters, suspending agents, preservatives, and the like.
  • the vaccine composition may be administered by administration routes commonly used in the art.
  • administration route examples include oral administration; and parenteral administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intrathecal administration, and transdermal administration.
  • parenteral administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intrathecal administration, and transdermal administration.
  • the amount of each component contained in the vaccine composition is not particularly limited as long as it is an immunologically acceptable amount usually used in the art.
  • disease includes cancer (eg, leukemia, lymphoma, neurological tumor, melanoma, breast cancer, lung cancer, head and neck cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, vaginal cancer , Testicular cancer, prostate cancer, penile cancer, bone tumor, vascular tumor, lip cancer, nasopharyngeal cancer, pharyngeal cancer, esophageal cancer, rectal cancer, gallbladder cancer, bile duct cancer, laryngeal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor Thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma, etc., infectious diseases (eg, influenza, human immunodeficiency syndrome (HIV), hepatitis such as viral hepatitis, viral infections such as Ebola hemorrhagic fever; pertussis, diphtheria, tetanus, tubercul
  • infectious diseases
  • -Bacterial infections caused by H. pylori, pneumococci, etc . fungal infections such as Candida; protozoal or parasite infections such as malaria; allergic diseases (eg, atopic dermatitis, allergic nose) , Asthma, etc.), autoimmune diseases (including, for example, diabetes mellitus (type 1 diabetes and type 2 diabetes), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.), hypertension, Alzheimer's disease.
  • allergic diseases eg, atopic dermatitis, allergic nose
  • Asthma Asthma
  • autoimmune diseases including, for example, diabetes mellitus (type 1 diabetes and type 2 diabetes), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.
  • hypertension Alzheimer's disease.
  • R x and R y are both H.
  • R 1 and R 2 are preferably the same.
  • R 1 and R 2 are preferably H, C 1-3 alkyl or C 1-3 haloalkyl, more preferably H or isopropyl. Particularly preferably, R 1 and R 2 are both H.
  • R 3 and R 4 are preferably the same.
  • R 3 and R 4 are preferably C 1-3 alkyl or C 1-3 haloalkyl, more preferably C 1-3 alkyl, and particularly preferably methyl or ethyl.
  • ⁇ n is preferably from 2 to 12, more preferably from 5 to 10, and particularly preferably from 5 to 8.
  • the compound of formula (I) preferably has the formula (I-1): [Wherein, R x , R y and n have the same meanings as in formula (I)] It is a compound shown by these.
  • R a -COOH is a bile acid, such as a primary bile acid or a secondary bile acid, preferably deoxycholic acid
  • R b is C 1-12 alkyl, preferably C 2-10 alkyl, more preferably C 5-10 alkyl, particularly preferably C 5-8 alkyl
  • R c is H, C 1-6 alkyl or C 1-6 haloalkyl
  • the compounds of formula (I) can be obtained by subjecting compounds (a), (b), (c) and (d) to a condensation reaction in a solvent.
  • the condensation reaction may be performed by mixing the compounds (b) and (c) to obtain an imine intermediate, and then adding the compounds (a) and (d) to the mixture.
  • the solvent include alcohols such as methanol and ethanol, and aprotic polar solvents such as DMF, and ethanol is preferable.
  • the reaction temperature is from room temperature to 60 ° C.
  • the compound of formula (I) can also be prepared when R x and R y are both H, for example according to the following method.
  • the compound of formula (I) can be obtained by subjecting compound (a), compound (b) and the dehydrating condensing agent to a condensation reaction in a solvent in the presence of a base as appropriate.
  • the dehydrating condensing agent include carbodiimides such as WSCI ⁇ HCl and N, N′-dicyclohexylcarbodiimide.
  • the base include DMAP.
  • the solvent include a non-polar solvent such as dichloromethane.
  • the reaction temperature is from room temperature to 40 ° C.
  • the self-assembly of the compound of the formula (I) can be produced from the compound of the formula (I) produced as described above, for example, according to the following method. After dissolving the compound of formula (I) in an aprotic polar solvent (eg, DMSO), the solution is added to a tissue culture medium (eg, DMEM) containing an additive (eg, serum) as appropriate. Mix. The mixed solution is allowed to stand to obtain a self-assembly of the compound of the formula (I).
  • an aprotic polar solvent eg, DMSO
  • DMEM tissue culture medium
  • an additive eg, serum
  • the formation of the self-assembly can be confirmed by mixing the self-assembly with the environment-sensitive fluorescent probe in an aqueous solvent (for example, water) and measuring the fluorescence intensity of the mixture.
  • aqueous solvent for example, water
  • Environmentally sensitive fluorescent probes are essentially non-fluorescent in aqueous solvents, but show fluorescence in less polar solvents or in hydrophobic environments.
  • Hydrophobic environments include, for example, self-assembled hydrophobic pockets.
  • Examples of the environmentally sensitive fluorescent probe include 8-anilino-1-naphthalenesulfonic acid (ANS) and Nile Red.
  • Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 ⁇ l, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours.
  • the EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (10/1).
  • the silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (1.3 mg, 0.0011 mmol).
  • WSCI ⁇ HCl 230 mg, 1.2 mmol
  • DMAP 122.17 mg, 1.0 mmol
  • extraction was performed three times with chloroform.
  • the organic layer was washed with a 2M aqueous NaOH solution, 1M hydrochloric acid and saturated saline, and the solvent was distilled off under reduced pressure.
  • the crude product was purified by column chromatography, the solution was concentrated under reduced pressure, and dried under vacuum to obtain the title compound (160 mg, 0.19 mmol).
  • Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 ⁇ l, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours.
  • the EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (20/1).
  • the silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (25 mg, 0.046 mmol).
  • Each self-assembly was produced from the above example compound and reference example compound according to the following method.
  • Each compound was dissolved in DMSO to prepare a 10 mM stock solution. After preparing a 3 mM DMSO solution (20 ⁇ l) of each compound from the stock solution, each solution was added to DMEM (2 ml) containing 10% FBS and 1% penicillin-streptomycin solution at room temperature, and mixed by pipetting. The mixed solution was allowed to stand for 1 hour to obtain a self-assembly of each compound.
  • mice MyD88-deficient mice (Oriental Yeast Co., Ltd.) and C57BL / 6 (CLEA Japan) background FcR ⁇ -deficient mice (RIKEN) were used.
  • cell RAW-Blue TM cells (InvivoGen) expressed the secreted fetal alkaline phosphatase (SEAP) reporter gene by NF- ⁇ B and AP-1 activation.
  • SEAP secreted fetal alkaline phosphatase
  • Bone marrow-derived dendritic cells were prepared according to the method described in Ishikawa et al., Cell Host & Microbe, April 17, 2013, 13, 477-488.
  • bone marrow cells were cultured in RPMI1640 medium supplemented with 10% (v / v) FBS and ⁇ -mercaptoethanol together with GM-CSF released from MGM-5 at 37 ° C. for 10 days, Bone marrow-derived dendritic cells were prepared.
  • RAW 264.7 cells American Type Culture Collection
  • DMEM DMEM containing 10% FBS and 1% penicillin-streptomycin solution.
  • Trehalose-6,6′-dimicolate (reagent) Trehalose-6,6′-dimicolate (TDM; Sigma) was dissolved at 1 mg / mL in a chloroform: methanol solution (2: 1 (v / v)) and diluted with 2-propanol to a final concentration of 50 ⁇ g / mL. After coating on a 96-well plate at 20 ⁇ L / well, the solvent was distilled off. The amount of TDM coated was 1 ⁇ g / well.
  • Lipopolysaccharide (LPS; L4516; Sigma) and curdlan (InvivoGen) were diluted with RPMI or DMEM containing 5% or 10% FBS and used for immunostimulation property measurement.
  • FIG. 1 shows the hydrodynamic diameter of a compound that formed a self-assembly in the compound library.
  • Test Example 1-2 Measurement of hydrodynamic size of self-assembly (2) For each example compound, a self-assembly was formed according to the method of Test Example 1-1. Samples were prepared at various concentrations in DMEM medium containing 10% FBS (Biowest) and 1% penicillin-streptomycin (Nacalai Tesque Inc.) at 25 ° C., and 100 ⁇ L in a 96-well UV plate (Corning Inc.). / Well.
  • the hydrodynamic radius of the self-assembly was measured using a DynaPro Plate reader II (Wyatt Technology Corp .; 165 °; automatic decay mode at 25 ° C .; laser wavelength 830 nm; scattering angle 58 °; data acquisition time 10 seconds / well; 10 acquisitions / measurements).
  • Hydrodynamic diameter converted from the self-assembly of 30 [mu] M DMEM solution measured hydrodynamic radius in (R h) and the R h values of the Example compounds are as follows.
  • FIG. 2 shows the results of the Rh values of the self-assembly of the compound of Example 1 and the compound of Reference Example in a DMEM solution.
  • the compound of Reference Example 1 did not form a self-assembly.
  • Immunostimulation properties (1) The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. 20,000 RAW 264.7 cells / well were seeded in 96-well tissue culture plates in DMEM containing 10% FBS at 37 ° C. for 24 hours. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% (v / v) DMSO and 100 pg / mL LPS were used as negative and positive controls, respectively.
  • FIG. 4 shows the result of comparison between the self-assembly of the compound of Example 1 and the compound of Reference Example.
  • FIG. 5 shows the results of the self-assembly of each example compound.
  • the particle size distribution of the self-assembly of the compound of Example 1 in a 30 ⁇ M DMEM solution is shown in FIG.
  • Immunostimulation properties (3) The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. After seeding BMDCs at 1 ⁇ 10 6 cells / well in RPMI 1640 containing 5% FBS in 96-well tissue culture plates, compounds of Examples 1, 7, 7 and 10 at 5, 10, 30, 50 and 100 ⁇ M respectively Of a self-assembled RPMI solution (containing 5% FBS) was added.
  • the mouse IL-6 concentration in the supernatant was measured using a Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex (Bio-Rad Laboratories, Inc.).
  • Deoxycholic acid (DCA) and DMSO (1%) were used as negative controls, and LPS (20 ng) and R848 (2 ⁇ g) were used as positive controls.
  • DCA Deoxycholic acid
  • DMSO DMSO
  • LPS (20 ng) and R848 (2 ⁇ g) were used as positive controls.
  • the result is shown in FIG.
  • the results show that the immunostimulatory effect induced by the self-assembly is TLR7-dependent, as is the positive control R848 (TLR7 agonist).
  • the test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 ⁇ M, respectively, were added to a 96-well plate, and 1 ⁇ 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA.
  • FIG. 13 shows the results.
  • Test examples to evaluate the agonistic activity on self-assembling of IL-6 production using T57-9 of T57-9 using C57BL / 6J (WT) or Tlr 9 ⁇ / ⁇ bone marrow-derived dendritic cells The test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 ⁇ M, respectively, were added to a 96-well plate, and 1 ⁇ 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA.
  • FIG. 14 shows the results.
  • Example 7 compound 1, 10 and 100 ⁇ g
  • Example 10 compound 1, 10 and 100 ⁇ g
  • deoxycholic acid 1, 10 and 100 ⁇ g
  • Deoxycholic acid and PBS were used as a negative control
  • aluminum salt Al salt
  • a 96-well plate was coated with 10 ⁇ g / mL OVA at 4 ° C. overnight. After washing, the plates were incubated with blocking buffer for 1 hour at room temperature. Plates were washed and incubated with diluted serum for 2 hours. The plates were then washed and horseradish peroxidase-conjugated anti-mouse total IgG, IgG1 or IgG2c antibodies (Southern Biotech) were each added to the plates. One hour later, the plate was washed and TMB Microwell Peroxidase Substrate System (KPL) was added to the wells. After 20 min incubation, the reaction was stopped by adding 2N H 2 SO 4.
  • KPL TMB Microwell Peroxidase Substrate System
  • the production amounts of anti-OVA total IgG, IgG1 and IgG2c are shown in FIGS. 15 to 17, respectively.
  • Anti-OVA total IgE was measured using a DS mouse IgE ELISA (OVA) kit (DS Pharma Biomedical).
  • FIG. 18 shows the amount of anti-OVA IgE produced.
  • mice were treated on day 0 and day 14 with influenza SV (1 ⁇ g), DMSO (final concentration: 1%), Example 1 compound (1, 10 and 100 ⁇ g), Example 7 compound (1, 10 And 100 ⁇ g) or a self-assembly of the compound of Example 10 (1, 10 and 100 ⁇ g), 100 ⁇ l / shot of a PBS solution containing either deoxycholic acid (1, 10 and 100 ⁇ g) or an aluminum salt (500 ⁇ g) subcutaneously.
  • mice were infected intranasally with 10 LD50 of clinically isolated A / Puerto Rico / 8/1934 influenza (H1N1) virus.
  • Anti-HA IgG1 and IgG2c in sera collected on day 21 were measured by ELISA.
  • the production amounts of anti-HA IgG1 and IgG2c are shown in FIGS. 19A and 19B. Mice were followed for body weight and survival 21 days after influenza infection on day 34.
  • Influenza SV (1 ⁇ g), self-assembly of DMSO (final concentration: 1%), Example 1 compound (100 ⁇ g), Example 7 compound (100 ⁇ g) or Example 10 compound (100 ⁇ g), deoxycholic acid (100 ⁇ g) Changes in body weight and viability for each mouse using either subcutaneous immunization, or with aluminum salt (500 ⁇ g) are shown in FIGS. 20A and 20B.
  • FIGS. 21A and 21B changes in body weight and survival rate are shown.
  • TLR293 cells In order to elucidate the SEAP assay signal pathway using HEK293 cells , several major TLRs were used using human embryonic kidney cells (HEK293) stably transfected with an NF- ⁇ B-responsive SEAP reporter gene.
  • HEK cells expressing TLR2, TLR4 or TLR9 are seeded at 3 ⁇ 10 4 cells / well in 96-well tissue culture plates in DMEM (100 ⁇ l) containing 10% FBS and self-assembled with the compound of Example 1 at 37 ° C. Stimulation was for 24 or 48 hours.
  • SEAP in the culture supernatant was stained with an alkaline phosphatase detection reagent QUANTI-Blue (InvivoGen) for 3 hours or 6 hours, and optical density (630 nm) was measured with a Multiskan microplate reader (Thermo Fisher Scientific Inc.). OD value) was measured.
  • DMSO was used as a negative control (NC)
  • oligodeoxynucleotide ODN1826; tlrl-1826; InvivoGen
  • TLR2 agonist Pam3 Pam3CSK4; InvivoGen
  • LPS Self-assembled SEAP responses were elevated in TLR9.
  • IL-6 Quantitative PCR assay RAW264.7 cells were seeded 24 hours 6 ⁇ 10 5 cells / well in DMEM containing 10% FBS at six-well tissue culture plates at 37 ° C.. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% DMSO and 100 ng / mL LPS were used as negative and positive controls, respectively. After 4 hours, the cells were washed twice with PBS. Cells were lysed with ISOGENE (1 mL / well; Nippon Gene Co., Ltd.) for 5 minutes. Then, chloroform (200 ⁇ L) was added.
  • the mixture was mixed for 15 minutes by inverting the test tube and then centrifuged at 14,000 rpm for 15 minutes at 4 ° C.
  • the upper 400 ⁇ L of the aqueous layer was collected in another 1.5 mL test tube and mixed with 500 ⁇ L of isopropanol.
  • the test tube was centrifuged again at 14,000 rpm, and the supernatant was removed.
  • the residue was washed twice with ethanol (700 ⁇ L), air-dried and redissolved in TE buffer (pH 5.2).
  • PrimeScript (TM) II 1 st strand cDNA Synthesis Kit Takara Bio Inc.
  • reverse transcribed total mRNA samples (2 [mu] g)
  • Quantitative real-time PCR was performed using the Fast CYBR Green Master Mix kit (Thermo Fisher Scientific Inc.). All assays were performed in triplicate and TNF- ⁇ and ⁇ -actin were used as internal standards. All primers for specific target genes are shown below: The results are shown in FIG.
  • a self-assembly obtained by linking the compounds of the formula (I) can be useful as an adjuvant, and is expected to be used as a vaccine composition for treating and / or preventing various diseases.

Abstract

Provided are: a novel compound that forms a self-assembly; the self-assembly; production methods for the compound and the self-assembly; and pharmaceutical use of the compound and the self-assembly. The compound is represented by formula (I) [in the formula, Rx represents H or a group represented by formula (II), Ry represents H or a group represented by formula (III), R1, R2, R3, and R4 each independently represent H, C1-6 alkyl, or C1-6 haloalkyl, a wavy line represents a binding site, and n represents an integer of 1-12]. The self-assembly is formed by the composition. The production methods are for the compound and the self-assembly, and the pharmaceutical use are for the compound and the self-assembly.

Description

免疫を賦活化する自己集合体Self-assembly that activates immunity
　本発明は、自己集合体を形成する新規化合物、その自己集合体並びにこれらの製法及び医薬用途に関する。 (4) The present invention relates to a novel compound forming a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
　生体における生物学的階層構造の形成において、分子の自発的な自己組織化である自己集合は重要な役割を担っている。例えば、ＤＮＡ、脂質膜や細胞骨格線維等は天然の自己集合体の一例である。一般に、単一の化合物分子は単一の機能を有するのに対し、自己集合体は周囲環境に応じて複雑な物理的又は化学的な特性や機能を有する（非特許文献１）。そのため、新たな自己集合体の合成は、生物系の機能や生物活性を調節する医薬品開発の新たな手法の一つとして注目されている。 自己 Self-assembly, which is the spontaneous self-assembly of molecules, plays an important role in the formation of biological hierarchical structures in living organisms. For example, DNA, lipid membranes, cytoskeletal fibers, and the like are examples of natural self-assemblies. Generally, a single compound molecule has a single function, whereas a self-assembly has complex physical or chemical properties and functions depending on the surrounding environment (Non-Patent Document 1). Therefore, the synthesis of a new self-assembly has attracted attention as one of new methods for drug development that regulates the function and biological activity of biological systems.
　生物活性のうち免疫賦活作用に関して、１９２０年代にアルミニウム化合物を用いたアジュバントが開発されて以来、種々の免疫賦活剤がジフテリアやインフルエンザ等の感染症等の疾患のワクチンに用いられている。これらの免疫賦活剤には、トール様受容体（ＴＬＲ）等のパターン認識受容体に作用することにより免疫細胞を活性化するものがあり、例えば非メチル化ＣＧ配列を含む合成オリゴヌクレオチド（ＣｐＧ－ＯＤＮ）等が知られている（非特許文献２）。このように、天然の自己集合構造を模した自己集合体がいくつか報告されているが、そのほとんどがペプチドを自己集合体の構成単位としている。非ペプチド合成化学分子の自己集合体の例もいくつか知られているが、それらの生物活性は主に細胞毒性や細胞死に関するものである（非特許文献３～１０）。したがって、生物活性を示す新規かつ有用なさらなる合成化学分子の自己集合体が望まれている。 Regarding immunostimulatory activity among biological activities, since an adjuvant using an aluminum compound was developed in the 1920s, various immunostimulants have been used in vaccines for diseases such as infectious diseases such as diphtheria and influenza. Some of these immunostimulants activate immune cells by acting on pattern recognition receptors such as Toll-like receptor (TLR). For example, synthetic oligonucleotides containing unmethylated CG sequences (CpG- ODN) is known (Non-Patent Document 2). As described above, several self-assemblies that mimic the natural self-assembly structure have been reported, but most of them use peptides as constituent units of the self-assembly. Although some examples of self-assembly of non-peptide synthetic chemical molecules are also known, their biological activities are mainly related to cytotoxicity and cell death (Non-Patent Documents 3 to 10). Therefore, new and useful self-assembly of synthetic chemical molecules that exhibit biological activity is desired.
　本発明が解決しようとする課題の一つは、自己集合体を形成する新規化合物、その自己集合体並びにこれらの製法及び医薬用途を提供することである。 課題 One of the problems to be solved by the present invention is to provide a novel compound which forms a self-assembly, the self-assembly, a method for producing the same, and a pharmaceutical use.
　本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、胆汁酸に特定の化学修飾がなされた部分構造をもつ小分子化合物が新たな自己集合体を形成し、当該自己集合体が免疫賦活作用を示すことを見出し、本発明を完成した。 The present inventors have conducted intensive studies to solve the above problems, and as a result, a small molecule compound having a partial structure in which a specific chemical modification has been made to bile acid forms a new self-assembly, and the self-assembly concerned Showed that they exhibited an immunostimulatory effect, and thus completed the present invention.
　ある態様において、式（Ｉ）：
Figure JPOXMLDOC01-appb-C000009
 ［式中、Ｒｘは、Ｈ又は式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000010
 で示される基であり、
　Ｒｙは、Ｈ又は式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000011
 で示される基であり、
　Ｒ１、Ｒ２、Ｒ３及びＲ４は、それぞれ独立して、Ｈ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
　波線は結合部位を示し、
　ｎは１～１２の整数である］
で示される化合物が非共有結合により連結してなる自己集合体が提供される。 In some embodiments, formula (I):
Figure JPOXMLDOC01-appb-C000009
 Wherein R x is H or formula (II):
Figure JPOXMLDOC01-appb-C000010
 Is a group represented by
R y is H or formula (III):
Figure JPOXMLDOC01-appb-C000011
 Is a group represented by
R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl;
Wavy lines indicate binding sites,
n is an integer of 1 to 12]
And a self-assembly obtained by linking the compounds represented by the following formulas by a non-covalent bond.
　別の態様において、式（Ｉ）の化合物が連結してなる自己集合体の医薬用途が提供される。 In another aspect, there is provided a pharmaceutical use of a self-assembly obtained by linking the compounds of the formula (I).
　さらに別の態様において、式（Ｉ）の化合物が提供される。 In yet another aspect, there is provided a compound of formula (I).
　さらに別の態様において、式（Ｉ）の化合物が連結してなる自己集合体の製造方法が提供される。 In still another aspect, a method for producing a self-assembly obtained by linking the compounds of the formula (I) is provided.
　式（Ｉ）の化合物が連結してなる自己集合体は、生物活性、例えば免疫賦活作用を示すことができ、アジュバントとして有用であり得る。 (5) The self-assembled substance formed by linking the compounds of the formula (I) can exhibit a biological activity, for example, an immunostimulatory action, and can be useful as an adjuvant.
図１は化合物ライブラリーのうち自己集合体を形成した化合物（Hit compounds）の流体力学的直径（ｎｍ）（縦軸）を示す。FIG. 1 shows the hydrodynamic diameter (nm) (vertical axis) of a compound (Hit @ compounds) that formed a self-assembly in the compound library. 図２は種々の濃度（μＭ）のＤＭＥＭ溶液中の実施例１化合物、参考例２化合物及び参考例３化合物の自己集合体の流体力学的半径（平均Ｒｈ（ｎｍ））（縦軸）を示す。FIG. 2 shows the hydrodynamic radii (average R h (nm)) (vertical axis) of self-assemblies of the compounds of Example 1, Reference Example 2 and Reference Example 3 in DMEM solutions at various concentrations (μM). Show. 図３は種々の濃度（μＭ）のＤＭＥＭ溶液中の実施例１化合物の自己集合体のＩＬ－６産生量（ｎｇ／ｍＬ）（縦軸）を示す。FIG. 3 shows the IL-6 production amount (ng / mL) (vertical axis) of the self-assembly of the compound of Example 1 in DMEM solutions at various concentrations (μM). 図４は３０μＭ ＤＭＥＭ溶液中の実施例１及び参考例１～３化合物のＩＬ－６産生量（ｎｇ／ｍＬ）（縦軸）を示す。ＤＣＡはデオキシコール酸の結果を示す。ＮＣは陰性対照である１％（ｖ／ｖ）ＤＭＳＯ、ＬＰＳは陽性対照である。FIG. 4 shows the IL-6 production amount (ng / mL) (vertical axis) of the compounds of Example 1 and Reference Examples 1 to 3 in a 30 μM @DMEM solution. DCA shows the result for deoxycholic acid. NC is a negative control of 1% (v / v) DMSO, and LPS is a positive control. 図５は３０μＭ ＤＭＥＭ溶液中の各実施例化合物の自己集合体のＩＬ－６産生量について陰性対照（ＤＭＳＯ）を１とした場合の相対値（縦軸）を示す。FIG. 5 shows the relative value (vertical axis) of the amount of IL-6 produced by the self-assembly of each example compound in a 30 μM @DMEM solution when the negative control (DMSO) is set to 1. 図６は３０μＭ ＤＭＥＭ溶液中の実施例１化合物の自己集合体の粒度分布（％）を示す（右側）。左側の分布は血清由来の粒度分布（％）を示す。FIG. 6 shows the particle size distribution (%) of the self-assembly of the compound of Example 1 in a 30 μM @DMEM solution (right side). The distribution on the left shows the particle size distribution (%) derived from serum. 図７は実施例１化合物の自己集合体の各濃度について左から遠心分離前及び後のＩＬ－６産生量（ｎｇ／ｍＬ）を示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 7 shows the amount of IL-6 produced (ng / mL) before and after centrifugation from the left for each concentration of the self-assembly of the compound of Example 1. Error bars indicate standard deviation (n = 3). 図８はＢＭＤＣにおけるＩＬ－６産生量（ｐｇ／ｍＬ）を示す。各試験物質における各プロットは、左からそれぞれ５、１０、３０、５０及び１００μＭでの結果を示す。エラーバーは標準偏差（ｎ＝１～２）を示す。FIG. 8 shows the amount of IL-6 produced in BMDC (pg / mL). Each plot for each test substance shows the results at 5, 10, 30, 50 and 100 μM, respectively, from the left. Error bars indicate standard deviation (n = 1 to 2). 図９はＦｃＲγ／ＭｙＤ８８ダブルノックアウトＢＭＤＣ（右）及び野生型ＢＭＤＣ（ＷＴ）（左）を用いたＩＬ－６ ＥＬＩＳＡアッセイ結果（ｎｇ／ｍＬ）を示す。実施例１化合物の自己集合体は３０μＭ ＤＭＥＭ溶液、ＬＰＳは１００ｎｇ／ｍＬ、カードランは１００ｎｇ／ｍＬ、ＴＤＭは１μg／ウェルにて用いた。データは平均値±標準偏差を示す。エラーバーは標準偏差（ｎ＝２）を示す。FIG. 9 shows the results of an IL-6Δ ELISA assay (ng / mL) using FcRγ / MyD88 double knockout BMDC (right) and wild-type BMDC (WT) (left). The self-assembly of the compound of Example 1 was used in a 30 μM DMEM solution, LPS was used at 100 ng / mL, curdlan was used at 100 ng / mL, and TDM was used at 1 μg / well. Data show mean ± standard deviation. Error bars indicate standard deviation (n = 2). 図１０は野生型（左）及びＦｃＲγノックアウトＢＭＤＣ（右）を用いた実施例１化合物の自己集合体（３０μＭ ＤＭＥＭ溶液）のＩＬ－６産生量（ｎｇ／ｍＬ）を示す。データは平均値±標準偏差を示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 10 shows the amount of IL-6 produced (ng / mL) of the self-assembly of the compound of Example 1 (30 μM in DMEM) using wild-type (left) and FcRγ knockout BMDC (right). Data show mean ± standard deviation. Error bars indicate standard deviation (n = 3). 図１１は野生型（左）及びＭｙＤ８８ノックアウトＢＭＤＣ（右）を実施例１化合物の自己集合体（３０μＭ ＤＭＥＭ溶液）のＩＬ－６産生量（ｐｇ／ｍＬ）を示す。データは平均値±標準偏差を示す。エラーバーは標準偏差（ｎ＝２）を示す。FIG. 11 shows the amount of IL-6 production (pg / mL) of the wild type (left) and the self-assembly of the compound of Example 1 (30 μM in DMEM) with MyD88 knockout BMDC (right). Data show mean ± standard deviation. Error bars indicate standard deviation (n = 2). 図１２は野生型（ＷＴ）及びＴＬＲ７ノックアウト（Ｔｌｒ７－／－）マウスのＩＬ－６産生量（ｐｇ／ｍＬ）を示す。各試験物質における左側のバーは野生型の結果を示し、右側のバーはＴＬＲ７ノックアウトマウスの結果を示す。FIG. 12 shows IL-6 production (pg / mL) of wild-type (WT) and TLR7 knockout (Tlr7 − / − ) mice. The bar on the left side of each test substance shows the result of the wild type, and the bar on the right side shows the result of the TLR7 knockout mouse. 図１３は野生型（ＷＴ）及びＴＬＲ７ノックアウト（ＫＯ）マウスのＩＬ－６産生量（ｎｇ／ｍＬ）を示す。エラーバーは標準偏差（ｎ＝１～３）を示す。FIG. 13 shows IL-6 production (ng / mL) of wild-type (WT) and TLR7 knockout (KO) mice. Error bars indicate standard deviation (n = 1 to 3). 図１４は野生型（ＷＴ）及びＴＬＲ９ノックアウト（ＫＯ）マウスのＩＬ－６産生量（ｎｇ／ｍＬ）を示す。エラーバーは標準偏差（ｎ＝１～３）を示す。FIG. 14 shows the amount of IL-6 produced (ng / mL) in wild type (WT) and TLR9 knockout (KO) mice. Error bars indicate standard deviation (n = 1 to 3). 図１５は抗ＯＶＡ総抗体（ＩｇＧ）価（１０ｎ）を示す。各試験物質における各プロットは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝４）を示す。ＤＣＡ（１００μｇ）に対する有意差として、＊はｐ＜０．０５を示し、＊＊はｐ＜０．０１を示し、＊＊＊はｐ＜０．００１を示す。FIG. 15 shows anti-OVA total antibody (IgG) titers (10 n ). Each plot for each test substance shows the results at 1, 10, and 100 μg, respectively, from the left. Error bars indicate standard deviation (n = 4). * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001 as a significant difference with respect to DCA (100 μg). 図１６は抗ＯＶＡ抗体（ＩｇＧ１）価（１０ｎ）を示す。各試験物質における各プロットは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝４）を示す。ＤＣＡ（１００μｇ）に対する有意差として、＊はｐ＜０．０５を示し、＊＊はｐ＜０．０１を示し、＊＊＊はｐ＜０．００１を示す。FIG. 16 shows the anti-OVA antibody (IgG1) titer (10 n ). Each plot for each test substance shows the results at 1, 10, and 100 μg, respectively, from the left. Error bars indicate standard deviation (n = 4). * Indicates p <0.05, ** indicates p <0.01, and *** indicates p <0.001 as a significant difference with respect to DCA (100 μg). 図１７は抗ＯＶＡ抗体（ＩｇＧ２ｃ）価（１０ｎ）を示す。各試験物質における各プロットは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝４）を示す。ＤＣＡ（１００μｇ）に対する有意差として、＊はｐ＜０．０５を示す。FIG. 17 shows the anti-OVA antibody (IgG2c) titer (10 n ). Each plot for each test substance shows the results at 1, 10, and 100 μg, respectively, from the left. Error bars indicate standard deviation (n = 4). * Indicates p <0.05 as a significant difference with respect to DCA (100 μg). 図１８は抗ＯＶＡ抗体（ＩｇＥ）の産生量（ｎｇ／ｍＬ）を示す。各試験物質における各バーは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝４）を示す。FIG. 18 shows the production amount (ng / mL) of the anti-OVA antibody (IgE). Each bar in each test substance shows the results at 1, 10 and 100 μg from the left, respectively. Error bars indicate standard deviation (n = 4). 図１９Ａは抗ＨＡ抗体（ＩｇＧ１）価（１０ｎ）を示す。各試験物質における各バーは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 19A shows the anti-HA antibody (IgG1) titer (10 n ). Each bar in each test substance shows the results at 1, 10 and 100 μg from the left, respectively. Error bars indicate standard deviation (n = 3). 図１９Ｂは抗ＨＡ抗体（ＩｇＧ２ｃ）価（１０ｎ）を示す。各試験物質における各バーは、左からそれぞれ１、１０及び１００μｇでの結果を示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 19B shows the anti-HA antibody (IgG2c) titer (10 n ). Each bar in each test substance shows the results at 1, 10 and 100 μg from the left, respectively. Error bars indicate standard deviation (n = 3). 図２０Ａはインフルエンザ感染後の日数（横軸）と体重減少率（％）（縦軸）の関係を示す。●は実施例１化合物の自己集合体、■は実施例７化合物の自己集合体、▲は実施例１０化合物の自己集合体、◆はＤＣＡ、×は１％ＤＭＳＯ、▼はアルミニウム塩をそれぞれ用いた場合のデータを示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 20A shows the relationship between the number of days after influenza infection (horizontal axis) and the weight loss rate (%) (vertical axis). ● indicates a self-assembly of the compound of Example 1, ■ indicates a self-assembly of the compound of Example 7, ▲ indicates a self-assembly of the compound of Example 10, ◆ indicates DCA, × indicates 1% DMSO, and ▼ indicates an aluminum salt. Here is the data when there is Error bars indicate standard deviation (n = 3). 図２０Ｂはインフルエンザ感染後の日数（横軸）と生存率（％）（縦軸）の関係を示す。●は実施例１化合物の自己集合体、■は実施例７化合物の自己集合体、◆はＤＣＡ、×は１％ＤＭＳＯ、▼はアルミニウム塩をそれぞれ用いた場合のデータを示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 20B shows the relationship between the number of days after influenza infection (horizontal axis) and the survival rate (%) (vertical axis). ● indicates a self-assembly of the compound of Example 1, ■ indicates a self-assembly of the compound of Example 7, ◆ indicates data when DCA, × indicates 1% DMSO, and ▼ indicates data when an aluminum salt was used. Error bars indicate standard deviation (n = 3). 図２１Ａは実施例１０化合物の自己集合体の各投与量（●：１μｇ、▲：１０μｇ、■：１００μｇ）に対するインフルエンザＳＶ注射後の日数（横軸）と体重減少率（％）（縦軸）の関係を示す。×は１％ＤＭＳＯ、▼はアルミニウム塩をそれぞれ用いた場合のデータを示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 21A shows the number of days after influenza SV injection (horizontal axis) and weight loss rate (%) (vertical axis) for each dose (●: 1 μg, ▲: 10 μg, Δ: 100 μg) of the self-assembly of the compound of Example 10. Shows the relationship. × indicates data when 1% DMSO was used, and ▼ indicates data when aluminum salt was used. Error bars indicate standard deviation (n = 3). 図２１Ｂは実施例１０化合物の自己集合体の各投与量（●：１μｇ、▲：１０μｇ、■：１００μｇ）に対するインフルエンザＳＶ注射後の日数（横軸）と生存率（％）（縦軸）の関係を示す。×は１％ＤＭＳＯ、▼はアルミニウム塩をそれぞれ用いた場合のデータを示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 21B shows the number of days (horizontal axis) and the survival rate (%) (vertical axis) after influenza SV injection for each dose (●: 1 μg, ▲: 10 μg, Δ: 100 μg) of the self-assembly of the compound of Example 10. Show the relationship. × indicates data when 1% DMSO was used, and ▼ indicates data when aluminum salt was used. Error bars indicate standard deviation (n = 3). 図２２Ａはインフルエンザ感染後の日数（横軸）と体重減少率（％）（縦軸）の関係についての実施例１０化合物の自己集合体（▲）と対照（◆：ＤＣＡ）の比較を示す。エラーバーは標準偏差（ｎ＝３）を示す。＊はｐ＜０．０５を示す。FIG. 22A shows a comparison between the self-assembly of the compound of Example 10 (▲) and the control (◆: DCA) regarding the relationship between the number of days after influenza infection (horizontal axis) and the weight loss rate (%) (vertical axis). Error bars indicate standard deviation (n = 3). * Indicates p <0.05. 図２２Ｂはインフルエンザ感染後の日数（横軸）と生存率（％）（縦軸）の関係についての実施例１０化合物の自己集合体（▲）と対照（◆：ＤＣＡ）の比較を示す。エラーバーは標準偏差（ｎ＝３）を示す。FIG. 22B shows a comparison between the self-assembly of the compound of Example 10 (▲) and the control (◆: DCA) regarding the relationship between the number of days after influenza infection (horizontal axis) and the survival rate (%) (vertical axis). Error bars indicate standard deviation (n = 3). 図２３はＨＥＫ２９３細胞を用いた、アゴニストなし（ｎｕｌｌ）及びＴＬＲ９に対するＳＥＡＰアッセイ結果を示す。横軸の濃度はＤＭＥＭ溶液中の実施例１化合物の自己集合体の濃度（μＭ）を示す。エラーバーは標準偏差（ｎ＝２）を示す。FIG. 23 shows the results of a SEAP assay for null and no TLR9 using HEK293 cells. The concentration on the horizontal axis indicates the concentration (μM) of the self-assembly of the compound of Example 1 in the DMEM solution. Error bars indicate standard deviation (n = 2). 図２４はＨＥＫ２９３細胞を用いた、アゴニストなし（ｎｕｌｌ）、ＴＬＲ２及びＴＬＲ４に対するＳＥＡＰアッセイ結果を示す。横軸の濃度はＤＭＥＭ溶液中の実施例１化合物の自己集合体の濃度（μＭ）を示す。データは平均値±標準偏差を示す。エラーバーは標準偏差（ｎ＝２）を示す。FIG. 24 shows the results of a SEAP assay for TLR2 and TLR4 using HEK293 cells without an agonist (null). The concentration on the horizontal axis indicates the concentration (μM) of the self-assembly of the compound of Example 1 in the DMEM solution. Data show mean ± standard deviation. Error bars indicate standard deviation (n = 2). 図２５はＩＬ－６定量的ＰＣＲアッセイ結果（ｑＰＣＲ）を示す。FIG. 25 shows the results of the IL-6 quantitative PCR assay (qPCR).
　本発明の具体的態様を以下に例示する。
［項１］
　式（Ｉ）：
Figure JPOXMLDOC01-appb-C000012
 ［式中、Ｒｘは、Ｈ又は式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000013
 で示される基であり、
　Ｒｙは、Ｈ又は式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000014
 で示される基であり、
　Ｒ１、Ｒ２、Ｒ３及びＲ４は、それぞれ独立して、Ｈ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
　波線は結合部位を示し、
　ｎは１～１２の整数である］
で示される化合物が非共有結合により連結してなる、自己集合体。 Specific embodiments of the present invention will be exemplified below.
[Item 1]
Formula (I):
Figure JPOXMLDOC01-appb-C000012
 Wherein R x is H or formula (II):
Figure JPOXMLDOC01-appb-C000013
 Is a group represented by
R y is H or formula (III):
Figure JPOXMLDOC01-appb-C000014
 Is a group represented by
R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl;
Wavy lines indicate binding sites,
n is an integer of 1 to 12]
A self-assembly comprising a compound represented by the formula (1) linked by a non-covalent bond.
［項２］
　ＲｘがＲｙと同一である、項１に記載の自己集合体。 [Item 2]
Item 2. The self-assembly according to Item 1, wherein R x is the same as R y .
［項３］
　Ｒｘが式（ＩＩ’）：
Figure JPOXMLDOC01-appb-C000015
 で示される基であり、
　Ｒｙが式（ＩＩＩ’）：
Figure JPOXMLDOC01-appb-C000016
 で示される基である、項１又は２に記載の自己集合体。 [Item 3]
R x is of the formula (II ′):
Figure JPOXMLDOC01-appb-C000015
 Is a group represented by
R y has the formula (III ′):
Figure JPOXMLDOC01-appb-C000016
 Item 3. The self-assembly according to Item 1 or 2, which is a group represented by the formula:
［項４］
　Ｒ３及びＲ４がそれぞれ独立してＣ１－３アルキルである、項１～３のいずれかに記載の自己集合体。 [Item 4]
Item 4. The self-assembly according to any one of Items 1 to 3, wherein R 3 and R 4 are each independently C 1-3 alkyl.
［項５］
　Ｒｘ及びＲｙがともにＨである、項１又は２に記載の自己集合体。 [Item 5]
Item 3. The self-assembly according to Item 1 or 2, wherein R x and R y are both H.
［項６］
　ｎが５～１０の整数である、項１～５のいずれかに記載の自己集合体。 [Item 6]
Item 6. The self-assembly according to any one of Items 1 to 5, wherein n is an integer of 5 to 10.
［項７］
　流体力学的サイズ４０ｎｍ～１２，０００ｎｍを有する凝集体を形成する、項１～６のいずれかに記載の自己集合体。 [Item 7]
Item 7. The self-assembly according to any one of Items 1 to 6, which forms an aggregate having a hydrodynamic size of 40 nm to 12,000 nm.
［項８］
　項１～７のいずれかに記載の自己集合体を含む、免疫賦活剤。 [Item 8]
Item 8. An immunostimulator comprising the self-assembly according to any one of Items 1 to 7.
［項９］
　アジュバントである、項８に記載の免疫賦活剤。 [Item 9]
Item 9. The immunostimulant according to Item 8, which is an adjuvant.
［項１０］
　項９に記載の免疫賦活剤及び抗原を含む、ワクチン組成物。 [Item 10]
Item 10. A vaccine composition comprising the immunostimulating agent according to Item 9 and an antigen.
［項１１］
　がん、感染症、アレルギー疾患、自己免疫疾患、高血圧症及びアルツハイマー病からなる群から選択される疾患の治療及び／又は予防のための、項１０に記載のワクチン組成物。 [Item 11]
Item 11. The vaccine composition according to Item 10, for treating and / or preventing a disease selected from the group consisting of cancer, infectious disease, allergic disease, autoimmune disease, hypertension and Alzheimer's disease.
［項１２］
　式（Ｉ）：
Figure JPOXMLDOC01-appb-C000017
 ［式中、Ｒｘは、Ｈ又は式（ＩＩ）：
Figure JPOXMLDOC01-appb-C000018
 で示される基であり、
　Ｒｙは、Ｈ又は式（ＩＩＩ）：
Figure JPOXMLDOC01-appb-C000019
 で示される基であり、
　Ｒ１、Ｒ２、Ｒ３及びＲ４は、それぞれ独立して、Ｈ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
　波線は結合部位を示し、
　ｎは１～１２の整数である］
で示される化合物。 [Item 12]
Formula (I):
Figure JPOXMLDOC01-appb-C000017
 Wherein R x is H or formula (II):
Figure JPOXMLDOC01-appb-C000018
 Is a group represented by
R y is H or formula (III):
Figure JPOXMLDOC01-appb-C000019
 Is a group represented by
R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl;
Wavy lines indicate binding sites,
n is an integer of 1 to 12]
A compound represented by the formula:
　用語「Ｃ１－１２アルキル」とは、炭素数１～１２の直鎖状又は分枝鎖状の飽和炭化水素基をいう。「Ｃ１－１２アルキル」としては、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、ネオペンチル、１，１－ジメチルプロピル、１－エチルプロピル、ｎ－ヘキシル、イソヘキシル、３，３－ジメチルブチル、２，２－ジメチルブチル、１，１－ジメチルブチル、２－エチルブチル、ｎ－ヘプチル、２－メチルヘキシル、２，２－ジメチルペンチル、２，２，３－トリメチルブチル、ｎ－オクチル、３，４－ジメチルヘキシル、２，２，４－トリメチルペンチル、ｎ－ノニル、ｎ－デカニル、ｎ－ウンデカニル、ｎ－ドデカニル等が挙げられる。「Ｃ１－６アルキル」としては、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ｎ－ペンチル、イソペンチル、ネオペンチル、１，１－ジメチルプロピル、１－エチルプロピル、ｎ－ヘキシル、イソヘキシル、３，３－ジメチルブチル、２，２－ジメチルブチル、１，１－ジメチルブチル、２－エチルブチル等が挙げられる。 The term “C 1-12 alkyl” refers to a linear or branched saturated hydrocarbon group having 1 to 12 carbon atoms. “C 1-12 alkyl” includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 2,2-dimethylpentyl , 2,2,3-trimethylbutyl, n-octyl, 3,4-dimethylhexyl, 2,2,4-trimethylpentyl, n-nonyl, n-decanyl, n-undecanyl, n-dodecanyl and the like. “C 1-6 alkyl” includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 1,1-dimethylpropyl, -Ethylpropyl, n-hexyl, isohexyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2-ethylbutyl and the like.
　用語「Ｃ１－１２ハロアルキル」とは、１つ以上の任意の水素原子が同一又は異なるハロゲンで置き換えられたＣ１－１２アルキルをいう。「Ｃ１－１２ハロアルキル」としては、ジフルオロメチル、トリフルオロメチル、２－フルオロエチル、２－クロロエチル、２，２－ジフルオロエチル、２，２，２－トリフルオロエチル、ペンタフルオロエチル、１，１－ジフルオロプロピル、３，３，３－トリフルオロプロピル、２，２，２－トリフルオロ－１－メチル－エチル、１，１－ジフルオロ－２－メチルプロピル、４－クロロブチル、５－フルオロペンチル等が挙げられる。 The term “C 1-12 haloalkyl” refers to a C 1-12 alkyl in which one or more optional hydrogen atoms have been replaced with the same or different halogen. “C 1-12 haloalkyl” includes difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2-chloroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 1,1 -Difluoropropyl, 3,3,3-trifluoropropyl, 2,2,2-trifluoro-1-methyl-ethyl, 1,1-difluoro-2-methylpropyl, 4-chlorobutyl, 5-fluoropentyl and the like No.
　用語「ハロ」又は「ハロゲン」としては、フルオロ、クロロ、ブロモ、ヨード等が挙げられる。 The term “halo” or “halogen” includes fluoro, chloro, bromo, iodo and the like.
　用語「自己集合体」とは、分子の自発的な自己組織化として定義される自己集合（self-assembling）により形成される、当該分子を構成単位とする集合体をいい、２つ以上の分子が非共有結合により連結して形成される、ミセル状、繊維状、結晶状、コロイド状又は粒子状の凝集体である。ここで、非共有結合としては、疎水結合、π－πスタッキング、水素結合、ファンデルワールス結合及びこれらの任意の組合せが挙げられる。自己集合体を構成する分子としては、胆汁酸に特定の化学修飾がなされた部分構造をもつ小分子化合物が挙げられ、好ましくは式（Ｉ）の化合物である。自己集合体は、複数の疎水性ポケットを有していてもよく、この疎水性ポケットを介して他の分子及び抗原等の種々の物質を集合体構造中に取り込むことができる。自己集合体はまた、Ｒｘ、Ｒｙ及びＲ１～Ｒ４の相違に基づく異なる構造を有する式（Ｉ）の化合物の集合体であってもよく、また式（Ｉ）の化合物以外の化合物が混入して集合体を形成したものも包含される。自己集合体はまた、構成単位となる分子の化学修飾により、その構造及び特性を適宜コントロールすることができる。自己集合体は、膜タンパク質との相互作用を通じて免疫賦活作用を示してもよい。自己集合体の流体力学的サイズは、特に限定されるものではなく、培地中濃度、血中濃度及び組織中濃度等の周囲環境に依存しうるが、例えば４０～１２，０００ｎｍであり、好ましくは４５～５，０００ｎｍであり、より好ましくは１００～１，０００ｎｍであり、さらに好ましくは１４０～８００ｎｍであり、特に好ましくは１５０～５００ｎｍである。ここで、用語「流体力学的サイズ」は、流体力学的直径をいい、例えば動的光散乱法（ＤＬＳ）を用いて測定してもよい。自己集合体はまた、その培地中濃度、血中濃度又は組織中濃度が上昇するに従い、免疫賦活作用が上昇しうる。 The term "self-assembly" refers to an assembly formed by self-assembling, defined as the spontaneous self-assembly of a molecule, and comprising the molecule as a constituent unit, and two or more molecules. Is a micellar, fibrous, crystalline, colloidal, or particulate aggregate formed by non-covalent bonding. Here, examples of the non-covalent bond include a hydrophobic bond, π-π stacking, a hydrogen bond, a van der Waals bond, and an arbitrary combination thereof. Examples of the molecules constituting the self-assembly include small molecule compounds having a partial structure in which bile acid is chemically modified, and preferably a compound of the formula (I). The self-assembly may have a plurality of hydrophobic pockets, and through this hydrophobic pocket, various substances such as other molecules and antigens can be incorporated into the assembly structure. A self-assembly may also be an aggregate of compounds of formula (I) having different structures based on the differences in R x , R y and R 1 -R 4 , and compounds other than compounds of formula (I) Mixed together to form an aggregate. The structure and properties of the self-assembly can be appropriately controlled by chemical modification of a molecule serving as a structural unit. Self-assemblies may exhibit immunostimulatory effects through interaction with membrane proteins. The hydrodynamic size of the self-assembly is not particularly limited and may depend on the surrounding environment such as the concentration in the medium, the concentration in the blood and the concentration in the tissue, but is, for example, 40 to 12,000 nm, and is preferably It is 45 to 5,000 nm, more preferably 100 to 1,000 nm, further preferably 140 to 800 nm, and particularly preferably 150 to 500 nm. Here, the term "hydrodynamic size" refers to the hydrodynamic diameter, which may be measured, for example, using dynamic light scattering (DLS). The self-assembly may also have an increased immunostimulatory effect as its concentration in the medium, blood or tissue increases.
　用語「免疫賦活作用」とは、細胞表面にあるパターン認識受容体に対する内因性又は外因性アゴニスト活性をいう。当該アゴニスト活性によりマクロファージや樹状細胞等の抗原提示細胞が活性化されてインターロイキン等のサイトカインが産生されうる。免疫賦活作用は、好ましくはトール様受容体（ＴＬＲ）に対するアゴニスト活性であり、より好ましくはＴＬＲ７アゴニスト活性又はＴＬＲ９アゴニスト活性であり、特に好ましくはＴＬＲ７アゴニスト活性である。免疫賦活作用はまた、自己集合体の流体力学的サイズが大きくなると、増大しうる。ＴＬＲ７はウイルス由来の一本鎖ＲＮＡ又は合成低分子リガンドを感知することにより、インターフェロン等の産生を促すため、ＴＬＲ７アゴニストは免疫賦活作用を示し、例えば、感染症及び自己免疫疾患等の種々の疾患の治療薬として有用でありうる。ＴＬＲ９は一般にバクテリア及びウイルスに見られ、非メチル化ＤＮＡに対する自然免疫応答及び適応免疫応答を刺激する役割を担うため、ＴＬＲ９アゴニストはワクチン組成物に用いられる抗原の免疫原性を向上しうる。 The term "immunostimulatory effect" refers to endogenous or exogenous agonist activity for a pattern recognition receptor on the cell surface. By the agonist activity, antigen presenting cells such as macrophages and dendritic cells can be activated to produce cytokines such as interleukins. The immunostimulatory activity is preferably an agonist activity for a Toll-like receptor (TLR), more preferably a TLR7 agonist activity or a TLR9 agonist activity, and particularly preferably a TLR7 agonist activity. Immunostimulatory effects can also be increased as the hydrodynamic size of the self-assembly increases. TLR7 stimulates the production of interferon and the like by sensing single-stranded RNA or synthetic small molecule ligand derived from virus. Therefore, TLR7 agonist has an immunostimulatory effect, for example, various diseases such as infectious diseases and autoimmune diseases. May be useful as therapeutics for Since TLR9 is commonly found in bacteria and viruses and plays a role in stimulating innate and adaptive immune responses to unmethylated DNA, TLR9 agonists can improve the immunogenicity of antigens used in vaccine compositions.
　用語「免疫賦活剤」とは、免疫賦活作用を示す物質又は組成物をいい、種々の疾患の免疫療法に用いうる。免疫賦活剤は、自己集合体を含有してもよい。免疫賦活剤を、抗原又は抗原を含むワクチン組成物と併用した場合、その抗原に対する免疫原性が獲得又は増強され、免疫持続期間が延長されうる。免疫賦活剤は、それのみで投与してもよく、抗原又は抗原を含むワクチン組成物と一定間隔を空けて別々に、連続して又は同時に投与されるように用いてもよく、或いは、ワクチン組成物の含有成分として用いてもよい。免疫賦活剤としては、例えばアジュバントが挙げられる。免疫賦活剤に含有される自己集合体の量は、免疫学的に許容される量であれば特に制限されるものではないが、例えば０．１～２０ｗ／ｗ％、好ましくは０．２～１５ｗ／ｗ％、より好ましくは０．３～１０ｗ／ｗ％、特に好ましくは０．５～５ｗ／ｗ％である。 The term “immunostimulant” refers to a substance or composition that exhibits an immunostimulatory effect, and can be used for immunotherapy of various diseases. The immunostimulant may contain a self-assembly. When an immunostimulant is used in combination with an antigen or a vaccine composition containing the antigen, immunogenicity to the antigen can be obtained or enhanced, and the duration of immunity can be prolonged. The immunostimulant may be administered alone, may be used to be administered separately, sequentially or simultaneously at an interval from the antigen or vaccine composition comprising the antigen, or May be used as a component of the product. Examples of the immunostimulant include an adjuvant. The amount of the self-assembly contained in the immunostimulant is not particularly limited as long as it is an immunologically acceptable amount, but is, for example, 0.1 to 20 w / w%, preferably 0.2 to 20 w / w%. It is 15 w / w%, more preferably 0.3 to 10 w / w%, particularly preferably 0.5 to 5 w / w%.
　用語「抗原」とは、免疫応答を生じさせる物質をいい、例えばペプチド又はタンパク質が挙げられる。抗原としては、がん抗原、ウイルス抗原、細菌性抗原、真菌性抗原、原虫性又は寄生虫性抗原、アレルギー関連抗原、疾患関連抗原が挙げられる。 The term "antigen" refers to a substance that provokes an immune response, such as a peptide or protein. Antigens include cancer antigens, viral antigens, bacterial antigens, fungal antigens, protozoal or parasite antigens, allergy-related antigens, and disease-related antigens.
　用語「ワクチン組成物」とは、抗原を含む組成物をいい、種々の疾患の免疫療法に用いうる。ワクチン組成物は、それのみで投与してもよく、免疫賦活剤と一定間隔を空けて別々に、連続して又は同時に投与されるように用いてもよく、或いは、免疫賦活剤を含有するワクチン組成物として用いてもよい。ワクチン組成物はまた、医薬的に許容される担体を含んでもよい。当該医薬的に許容される担体としては、当分野で通常用いられる賦形剤、等張化剤、緩衝剤、ｐＨ調節剤、懸濁化剤、保存剤等の添加物が挙げられる。ワクチン組成物は、当分野で通常用いられる投与経路で投与してもよい。当該投与経路としては、経口投与；又は静脈内注射、動脈内注射、皮下注射、皮内注射、筋肉内注射、髄腔内投与、経皮投与等の非経口投与が挙げられる。ワクチン組成物に含有される各成分の量は、当分野で通常用いられる、免疫学的に許容される量であれば、特に制限されない。 The term “vaccine composition” refers to a composition containing an antigen, and can be used for immunotherapy of various diseases. The vaccine composition may be administered alone, may be used so that it is administered separately, sequentially or simultaneously at an interval from the immunostimulant, or a vaccine containing the immunostimulant. It may be used as a composition. The vaccine composition may also include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers include additives commonly used in the art, such as excipients, isotonic agents, buffers, pH adjusters, suspending agents, preservatives, and the like. The vaccine composition may be administered by administration routes commonly used in the art. Examples of the administration route include oral administration; and parenteral administration such as intravenous injection, intraarterial injection, subcutaneous injection, intradermal injection, intramuscular injection, intrathecal administration, and transdermal administration. The amount of each component contained in the vaccine composition is not particularly limited as long as it is an immunologically acceptable amount usually used in the art.
　用語「疾患」としては、がん（例えば白血病、リンパ腫、神経性腫瘍、メラノーマ、乳癌、肺癌、頭頸部癌、胃癌、結腸癌、肝癌、膵癌、子宮頸癌、子宮癌、卵巣癌、腟癌、精巣癌、前立腺癌、陰茎癌、骨腫瘍、血管腫瘍、***癌、上咽頭癌、咽頭癌、食道癌、直腸癌、胆嚢癌、胆管癌、喉頭癌、肺癌、膀胱癌、腎臓癌、脳腫瘍、甲状腺癌、ホジキン病、非ホジキンリンパ腫等）、感染症（例えばインフルエンザ、ヒト免疫不全症候群（ＨＩＶ）、ウイルス性肝炎等の肝炎、エボラ出血熱等のウイルス感染症；百日咳、ジフテリア、破傷風、結核、ヘリコバクター・ピロリ、肺炎球菌等による細菌感染症；カンジダ等の真菌感染症；マラリア等の原虫性又は寄生虫性感染症等）、アレルギー疾患（例えばアトピー性皮膚炎、アレルギー性鼻炎、喘息等）、自己免疫疾患（例えば糖尿病（１型糖尿病及び２型糖尿病を含む）、多発性硬化症、関節リウマチ、全身性エリテマトーデス、乾癬等）、高血圧症、アルツハイマー病が挙げられる。 The term “disease” includes cancer (eg, leukemia, lymphoma, neurological tumor, melanoma, breast cancer, lung cancer, head and neck cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, cervical cancer, uterine cancer, ovarian cancer, vaginal cancer , Testicular cancer, prostate cancer, penile cancer, bone tumor, vascular tumor, lip cancer, nasopharyngeal cancer, pharyngeal cancer, esophageal cancer, rectal cancer, gallbladder cancer, bile duct cancer, laryngeal cancer, lung cancer, bladder cancer, kidney cancer, brain tumor Thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma, etc., infectious diseases (eg, influenza, human immunodeficiency syndrome (HIV), hepatitis such as viral hepatitis, viral infections such as Ebola hemorrhagic fever; pertussis, diphtheria, tetanus, tuberculosis, Helicobacter. -Bacterial infections caused by H. pylori, pneumococci, etc .; fungal infections such as Candida; protozoal or parasite infections such as malaria; allergic diseases (eg, atopic dermatitis, allergic nose) , Asthma, etc.), autoimmune diseases (including, for example, diabetes mellitus (type 1 diabetes and type 2 diabetes), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.), hypertension, Alzheimer's disease.
　ある態様において、Ｒｘ及びＲｙはともにＨである。 In some embodiments, R x and R y are both H.
　Ｒ１及びＲ２は、好ましくは同一である。Ｒ１及びＲ２は、好ましくはＨ、Ｃ１－３アルキル又はＣ１－３ハロアルキルであり、より好ましくはＨ又はイソプロピルである。特に好ましくは、Ｒ１及びＲ２はともにＨである。 R 1 and R 2 are preferably the same. R 1 and R 2 are preferably H, C 1-3 alkyl or C 1-3 haloalkyl, more preferably H or isopropyl. Particularly preferably, R 1 and R 2 are both H.
　Ｒ３及びＲ４は、好ましくは同一である。Ｒ３及びＲ４は、好ましくはＣ１－３アルキル又はＣ１－３ハロアルキルであり、より好ましくはＣ１－３アルキルであり、特に好ましくはメチル又はエチルである。 R 3 and R 4 are preferably the same. R 3 and R 4 are preferably C 1-3 alkyl or C 1-3 haloalkyl, more preferably C 1-3 alkyl, and particularly preferably methyl or ethyl.
　ｎは、好ましくは２～１２であり、より好ましくは５～１０であり、特に好ましくは５～８である。 Δn is preferably from 2 to 12, more preferably from 5 to 10, and particularly preferably from 5 to 8.
　式（Ｉ）の化合物は、好ましくは式（Ｉ－１）：
Figure JPOXMLDOC01-appb-C000020
 ［式中、Ｒｘ、Ｒｙ及びｎは式（Ｉ）におけるものと同義である］
で示される化合物である。 The compound of formula (I) preferably has the formula (I-1):
Figure JPOXMLDOC01-appb-C000020
 [Wherein, R x , R y and n have the same meanings as in formula (I)]
It is a compound shown by these.
　本明細書において、以下の略語を用いることがある。
Ｅｘ．：実施例化合物、又は試験例においては当該実施例化合物の自己集合体を示す場合もある。
ＲＥｘ．：参考例
ＡＮＳ：８－アニリノ－１－ナフタレンスルホン酸
ＢＭＤＣ：骨髄由来樹状細胞
ＤＭＡＰ：Ｎ，Ｎ－ジメチル－４－アミノピリジン
ＤＭＥＭ：ダルベッコ改変イーグル培地
ＤＭＳＯ：ジメチルスルホキシド
Ｅｔ：エチル
ＥｔＯＨ：エタノール
ＦＢＳ：ウシ胎児血清
ＧＭ－ＣＳＦ：顆粒球単球コロニー刺激因子
ｉＰｒ：イソプロピル
ＬＰＳ：リポ多糖
ＭＧＭ－５：
ＯＶＡ：オボアルブミン
ＰＢＳ：リン酸緩衝生理食塩水
ＲＰＭＩ：ロズウェルパーク記念研究所培地
ＴＤＭ：トレハロース－６，６’－ジミコレート
ＴＬＣ：薄層クロマトグラフィー
ＷＳＣＩ・ＨＣｌ：１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩
ＷＴ：野生型 In this specification, the following abbreviations may be used.
Ex. : In some cases, a self-assembly of an example compound or a test example is shown in the test examples.
REx. Reference Example ANS: 8-anilino-1-naphthalenesulfonic acid BMDC: bone marrow-derived dendritic cells DMAP: N, N-dimethyl-4-aminopyridine DMEM: Dulbecco's modified Eagle's medium DMSO: dimethyl sulfoxide Et: ethyl EtOH: ethanol FBS : Fetal calf serum GM-CSF: granulocyte monocyte colony stimulating factor iPr: isopropyl LPS: lipopolysaccharide MGM-5:
OVA: Ovalbumin PBS: Phosphate buffered saline RPMI: Roswell Park Memorial Laboratory Medium TDM: Trehalose-6,6′-dimicolate TLC: Thin-layer chromatography WSCI.HCl: 1-ethyl-3- (3-dimethyl Aminopropyl) carbodiimide hydrochloride WT: wild type
［一般製法１］
　式（Ｉ）の化合物は、例えば以下のスキームで示されるウギ反応に従い製造することができる。
Figure JPOXMLDOC01-appb-C000021
 ［スキーム中、Ｒａ－ＣＯＯＨは胆汁酸、例えば一次胆汁酸又は二次胆汁酸、好ましくはデオキシコール酸であり、
　ＲｂはＣ１－１２アルキルであり、好ましくはＣ２－１０アルキルであり、より好ましくはＣ５－１０アルキルであり、特に好ましくはＣ５－８アルキルであり、
　ＲｃはＨ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
　ＲｄはＣＨ２Ｃ（＝Ｏ）Ｏ－Ｒ３又はＣＨ２Ｃ（＝Ｏ）Ｏ－Ｒ４であり、
　Ｒ３及びＲ４は前記定義されるとおりである］
　化合物（ａ）、（ｂ）、（ｃ）及び（ｄ）を溶媒中で縮合反応させることにより式（Ｉ）の化合物を得ることができる。縮合反応は、化合物（ｂ）及び（ｃ）を混合してイミン中間体を得た後、当該混合物に化合物（ａ）及び（ｄ）を添加して行ってもよい。溶媒としては、メタノール又はエタノール等のアルコール、ＤＭＦ等の非プロトン性極性溶媒が挙げられ、好ましくはエタノールである。反応温度は、室温から６０℃である。 [General manufacturing method 1]
The compound of the formula (I) can be produced, for example, according to the Ugi reaction shown in the following scheme.
Figure JPOXMLDOC01-appb-C000021
 [In the scheme, R a -COOH is a bile acid, such as a primary bile acid or a secondary bile acid, preferably deoxycholic acid;
R b is C 1-12 alkyl, preferably C 2-10 alkyl, more preferably C 5-10 alkyl, particularly preferably C 5-8 alkyl,
R c is H, C 1-6 alkyl or C 1-6 haloalkyl;
R d is CH 2 C (＝ O) OR 3 or CH 2 C (＝ O) OR 4 ,
R 3 and R 4 are as defined above.
The compounds of formula (I) can be obtained by subjecting compounds (a), (b), (c) and (d) to a condensation reaction in a solvent. The condensation reaction may be performed by mixing the compounds (b) and (c) to obtain an imine intermediate, and then adding the compounds (a) and (d) to the mixture. Examples of the solvent include alcohols such as methanol and ethanol, and aprotic polar solvents such as DMF, and ethanol is preferable. The reaction temperature is from room temperature to 60 ° C.
［一般製法２］
　式（Ｉ）の化合物はまた、Ｒｘ及びＲｙがともにＨである場合、例えば以下の方法に従い製造することができる。
　化合物（ａ）、化合物（ｂ）及び脱水縮合剤を、溶媒中、適宜塩基存在下にて縮合反応させることにより式（Ｉ）の化合物を得ることができる。脱水縮合剤としては、ＷＳＣＩ・ＨＣｌ、Ｎ，Ｎ’－ジシクロヘキシルカルボジイミド等のカルボジイミドが挙げられる。塩基としては、ＤＭＡＰが挙げられる。溶媒としては、ジクロロメタン等の非極性溶媒が挙げられる。反応温度は、室温から４０℃である。 [General manufacturing method 2]
The compound of formula (I) can also be prepared when R x and R y are both H, for example according to the following method.
The compound of formula (I) can be obtained by subjecting compound (a), compound (b) and the dehydrating condensing agent to a condensation reaction in a solvent in the presence of a base as appropriate. Examples of the dehydrating condensing agent include carbodiimides such as WSCI · HCl and N, N′-dicyclohexylcarbodiimide. Examples of the base include DMAP. Examples of the solvent include a non-polar solvent such as dichloromethane. The reaction temperature is from room temperature to 40 ° C.
［自己集合体の製造］
　式（Ｉ）の化合物の自己集合体は、上記のようにして製造した式（Ｉ）の化合物から、例えば以下の方法に従い製造することができる。
　式（Ｉ）の化合物を非プロトン性極性溶媒（例えば、ＤＭＳＯ）に溶解させた後、当該溶液を、適宜、添加物（例えば、血清）を含む組織培養用培地（例えば、ＤＭＥＭ）に加えて混和する。混和した溶液を静置させて、式（Ｉ）の化合物の自己集合体を得る。 [Manufacture of self-assembly]
The self-assembly of the compound of the formula (I) can be produced from the compound of the formula (I) produced as described above, for example, according to the following method.
After dissolving the compound of formula (I) in an aprotic polar solvent (eg, DMSO), the solution is added to a tissue culture medium (eg, DMEM) containing an additive (eg, serum) as appropriate. Mix. The mixed solution is allowed to stand to obtain a self-assembly of the compound of the formula (I).
　自己集合体の生成は、自己集合体と環境感受性蛍光プローブとを水性溶媒（例えば水）中で混合し、当該混合物の蛍光強度を測定することにより確認することができる。環境感受性蛍光プローブは、本質的には水性溶媒中で非蛍光性であるが、低極性溶媒中又は疎水性環境中では蛍光性を示す。疎水性環境としては、例えば自己集合体の疎水性ポケットが挙げられる。環境感受性蛍光プローブとしては、例えば８－アニリノ－１－ナフタレンスルホン酸（ＡＮＳ）、ナイルレッドが挙げられる。 生成 The formation of the self-assembly can be confirmed by mixing the self-assembly with the environment-sensitive fluorescent probe in an aqueous solvent (for example, water) and measuring the fluorescence intensity of the mixture. Environmentally sensitive fluorescent probes are essentially non-fluorescent in aqueous solvents, but show fluorescence in less polar solvents or in hydrophobic environments. Hydrophobic environments include, for example, self-assembled hydrophobic pockets. Examples of the environmentally sensitive fluorescent probe include 8-anilino-1-naphthalenesulfonic acid (ANS) and Nile Red.
　以下に実施例及び試験例を挙げて本発明をより詳細に説明するが、本発明はこれらに限定されるものではない。 本 The present invention will be described in more detail with reference to examples and test examples below, but the present invention is not limited to these.
［実施例１］ジエチル 6,12-ビス(4-((3R,5R,8R,9S,10S,12S,13R,14S,17S)-3,12-ジヒドロキシ-10,13-ジメチルヘキサデカヒドロ-1H-シクロペンタ[a]フェナントレン-17-イル)ペンタノイル)-4,14-ジオキソ-3,6,12,15-テトラアザヘプタデカンジオエートの製造
　ホルムアルデヒド（3.6 mg, 0.12 mmol）、カダベリン（0.06 mmol）及びEtOH（1 ml）をガラス管に充填した。混合物を40℃にて15分間撹拌した。デオキシコール酸（0.12 mmol）及びイソシアノ酢酸エチル（14.1μl, 0.12 mmol）を反応混合物に添加し、12時間撹拌した。窒素雰囲気下EtOHを留去し、粗製物をジクロロメタンに溶解させた。1M塩酸、2M NaOH水溶液及び飽和食塩水で洗浄し、有機層を分取用TLCに充填し、クロロホルム／メタノール（10/1）で展開させた。生成物を含むシリカを掻き取り、アセトニトリル／EtOH溶液の入った平底ガラス瓶に入れた。撹拌棒で粉末化し、ろ過した。溶液を減圧濃縮し、真空乾燥して標題化合物（1.3 mg, 0.0011 mmol）を得た。 Example 1 Diethyl 6,12-bis (4-((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17S) -3,12-dihydroxy-10,13-dimethylhexadecahydro- Preparation of 1H-cyclopenta [a] phenanthrene-17-yl) pentanoyl) -4,14-dioxo-3,6,12,15-tetraazaheptadecandioate formaldehyde (3.6 mg, 0.12 mmol), cadaverine (0.06 mmol) ) And EtOH (1 ml) were filled into glass tubes. The mixture was stirred at 40 ° C. for 15 minutes. Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 μl, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours. The EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (10/1). The silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (1.3 mg, 0.0011 mmol).
　実施例１の方法に従い、以下の実施例化合物を製造した。
Figure JPOXMLDOC01-appb-T000022
According to the method of Example 1, the following example compounds were produced.
Figure JPOXMLDOC01-appb-T000022
［実施例１０］(4R,4'R)-N,N'-(ペンタン-1,5-ジイル)ビス(4-((3R,5S,8R,9S,10S,12S,13R,17R)-3,12-ジヒドロキシ-10,13-ジメチルヘキサデカヒドロ-1H-シクロペンタ[a]フェナントレン-17-イル)ペンタアミド)の製造
Figure JPOXMLDOC01-appb-C000023
 　デオキシコール酸（382 mg, 0.97 mmol）、カダベリン（5 mmol）及びジクロロメタン（5 ml）をガラス管に充填し、撹拌した。混合液へWSCI・HCl（230 mg, 1.2 mmol）及びDMAP（122.17 mg, 1.0 mmol）を加えて室温で3時間撹拌した。水を加えて反応を停止させたのちに、クロロホルムで三回抽出を行った。有機層を2M NaOH水溶液、1M 塩酸及び飽和食塩水で洗浄し、減圧下で溶媒を留去した。粗生成物をカラムクロマトグラフィーで精製し、溶液を減圧濃縮し、真空乾燥して標題化合物（160 mg, 0.19 mmol）を得た。 Example 10 (4R, 4'R) -N, N '-(pentane-1,5-diyl) bis (4-((3R, 5S, 8R, 9S, 10S, 12S, 13R, 17R)- Preparation of 3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthren-17-yl) pentaamide)
Figure JPOXMLDOC01-appb-C000023
 Deoxycholic acid (382 mg, 0.97 mmol), cadaverine (5 mmol) and dichloromethane (5 ml) were charged into a glass tube and stirred. WSCI · HCl (230 mg, 1.2 mmol) and DMAP (122.17 mg, 1.0 mmol) were added to the mixture, and the mixture was stirred at room temperature for 3 hours. After water was added to stop the reaction, extraction was performed three times with chloroform. The organic layer was washed with a 2M aqueous NaOH solution, 1M hydrochloric acid and saturated saline, and the solvent was distilled off under reduced pressure. The crude product was purified by column chromatography, the solution was concentrated under reduced pressure, and dried under vacuum to obtain the title compound (160 mg, 0.19 mmol).
　各実施例化合物の物性データを以下に示す。
Figure JPOXMLDOC01-appb-T000024

Figure JPOXMLDOC01-appb-I000025


Figure JPOXMLDOC01-appb-I000026


Figure JPOXMLDOC01-appb-I000027

The physical property data of each example compound is shown below.
Figure JPOXMLDOC01-appb-T000024

Figure JPOXMLDOC01-appb-I000025


Figure JPOXMLDOC01-appb-I000026


Figure JPOXMLDOC01-appb-I000027
［参考例１］エチル N-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-ジヒドロキシ-10,13-ジメチルヘキサデカヒドロ-1H-シクロペンタ[a]フェナントレン-17-イル)ペンタノイル)-N-メチルグリシルグリシネートの製造
Figure JPOXMLDOC01-appb-C000028
 　ホルムアルデヒド（3.6 mg, 0.12 mmol）、メチルアミン（0.12 mmol）及びEtOH（1 ml）をガラス管に充填した。混合物を40℃にて15分間撹拌した。デオキシコール酸（0.12 mmol）及びイソシアノ酢酸エチル（14.1μl, 0.12 mmol）を反応混合物に添加し、12時間撹拌した。窒素雰囲気下EtOHを留去し、粗製物をジクロロメタンに溶解させた。1M塩酸、2M NaOH水溶液及び飽和食塩水で洗浄し、有機層を分取用TLCに充填し、クロロホルム／メタノール（20/1）で展開させた。生成物を含むシリカを掻き取り、アセトニトリル／EtOH溶液の入った平底ガラス瓶に入れた。撹拌棒で粉末化し、ろ過した。溶液を減圧濃縮し、真空乾燥して標題化合物（25 mg, 0.046 mmol）を得た。 Reference Example 1 Ethyl N-((R) -4-((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,12-dihydroxy-10,13-dimethylhexadecahydro Preparation of -1H-cyclopenta [a] phenanthrene-17-yl) pentanoyl) -N-methylglycylglycinate
Figure JPOXMLDOC01-appb-C000028
 Formaldehyde (3.6 mg, 0.12 mmol), methylamine (0.12 mmol) and EtOH (1 ml) were charged into a glass tube. The mixture was stirred at 40 ° C. for 15 minutes. Deoxycholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 μl, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours. The EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (20/1). The silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (25 mg, 0.046 mmol).
［参考例２］ジエチル 6,12-ビス(4-((3R,5R,8R,9S,10S,13R,14S,17S)-3-ヒドロキシ-10,13-ジメチルヘキサデカヒドロ-1H-シクロペンタ[a]フェナントレン-17-イル)ペンタノイル)-4,14-ジオキソ-3,6,12,15-テトラアザヘプタデカンジオエートの製造
Figure JPOXMLDOC01-appb-C000029
 　ホルムアルデヒド（3.6 mg, 0.12 mmol）、カダベリン（0.06 mmol）及びEtOH（1 ml）をガラス管に充填した。混合物を40℃にて15分間撹拌した。リトコール酸（0.12 mmol）及びイソシアノ酢酸エチル（14.1μl, 0.12 mmol）を反応混合物に添加し、12時間撹拌した。窒素雰囲気下EtOHを留去し、粗製物をジクロロメタンに溶解させた。1M塩酸、2M NaOH水溶液及び飽和食塩水で洗浄し、有機層を分取用TLCに充填し、クロロホルム／メタノール（20/1）で展開させた。生成物を含むシリカを掻き取り、アセトニトリル／EtOH溶液の入った平底ガラス瓶に入れた。撹拌棒で粉末化し、ろ過した。溶液を減圧濃縮し、真空乾燥して標題化合物（1.7 mg, 0.0015 mmol）を得た。 Reference Example 2 Diethyl 6,12-bis (4-((3R, 5R, 8R, 9S, 10S, 13R, 14S, 17S) -3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [ a) Preparation of phenanthrene-17-yl) pentanoyl) -4,14-dioxo-3,6,12,15-tetraazaheptadecandioate
Figure JPOXMLDOC01-appb-C000029
 Formaldehyde (3.6 mg, 0.12 mmol), cadaverine (0.06 mmol) and EtOH (1 ml) were charged into a glass tube. The mixture was stirred at 40 ° C. for 15 minutes. Lithocholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 μl, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours. The EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1 M hydrochloric acid, 2 M NaOH aqueous solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (20/1). The silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (1.7 mg, 0.0015 mmol).
［参考例３］ジエチル 4,14-ジオキソ-6,12-ビス((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-トリヒドロキシ-10,13-ジメチルヘキサデカヒドロ-1H-シクロペンタ[a]フェナントレン-17-イル)ペンタノイル)-3,6,12,15-テトラアザヘプタデカンジオエートの製造
Figure JPOXMLDOC01-appb-C000030
 　ホルムアルデヒド（3.6 mg, 0.12 mmol）、カダベリン（0.06 mmol）及びEtOH（1 ml）をガラス管に充填した。混合物を40℃にて15分間撹拌した。コール酸（0.12 mmol）及びイソシアノ酢酸エチル（14.1μl, 0.12 mmol）を反応混合物に添加し、12時間撹拌した。窒素雰囲気下EtOHを留去し、粗製物をジクロロメタンに溶解させた。1M塩酸、2M NaOH水溶液及び飽和食塩水で洗浄し、有機層を分取用TLCに充填し、クロロホルム／メタノール（20/3）で展開させた。生成物を含むシリカを掻き取り、アセトニトリル／EtOH溶液の入った平底ガラス瓶に入れた。撹拌棒で粉末化し、ろ過した。溶液を減圧濃縮し、真空乾燥して標題化合物（0.4 mg, 0.00034 mmol）を得た。 Reference Example 3 Diethyl 4,14-dioxo-6,12-bis ((R) -4-((3R, 5S, 7R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,7) Preparation of (12,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta [a] phenanthrene-17-yl) pentanoyl) -3,6,12,15-tetraazaheptadecandioate
Figure JPOXMLDOC01-appb-C000030
 Formaldehyde (3.6 mg, 0.12 mmol), cadaverine (0.06 mmol) and EtOH (1 ml) were charged into a glass tube. The mixture was stirred at 40 ° C. for 15 minutes. Cholic acid (0.12 mmol) and ethyl isocyanoacetate (14.1 μl, 0.12 mmol) were added to the reaction mixture and stirred for 12 hours. The EtOH was distilled off under a nitrogen atmosphere and the crude was dissolved in dichloromethane. After washing with 1M hydrochloric acid, 2M aqueous NaOH solution and saturated saline, the organic layer was filled in a preparative TLC and developed with chloroform / methanol (20/3). The silica containing the product was scraped and placed in a flat-bottomed glass bottle containing the acetonitrile / EtOH solution. Powdered with a stir bar and filtered. The solution was concentrated under reduced pressure and dried under vacuum to obtain the title compound (0.4 mg, 0.00034 mmol).
　各参考例化合物の物性データを以下に示す。
Figure JPOXMLDOC01-appb-T000031
Physical property data of each reference example compound is shown below.
Figure JPOXMLDOC01-appb-T000031
　上記実施例化合物及び参考例化合物から以下の方法に従い、それぞれの自己集合体を製造した。
　それぞれの化合物をＤＭＳＯに溶解させ、１０ｍＭのストック溶液を調製した。当該ストック溶液から各化合物の３ｍＭ ＤＭＳＯ溶液（２０μｌ）を調製した後、それぞれの溶液を室温にて１０％ＦＢＳ及び１％ペニシリン－ストレプトマイシン溶液を含むＤＭＥＭ（２ｍｌ）に加え、ピペッティングにより混和した。混和した溶液を１時間静置させ、各化合物の自己集合体を得た。 Each self-assembly was produced from the above example compound and reference example compound according to the following method.
Each compound was dissolved in DMSO to prepare a 10 mM stock solution. After preparing a 3 mM DMSO solution (20 μl) of each compound from the stock solution, each solution was added to DMEM (2 ml) containing 10% FBS and 1% penicillin-streptomycin solution at room temperature, and mixed by pipetting. The mixed solution was allowed to stand for 1 hour to obtain a self-assembly of each compound.
［試験例］
　試験例では、特に明記しない場合、以下の動物、細胞及び試薬を用いた。
（マウス）
　ＭｙＤ８８欠損マウス（オリエンタル酵母工業株式会社）及びＣ５７ＢＬ／６（日本クレア株式会社）バックグラウンドのＦｃＲγ欠損マウス（理化学研究所）を用いた。
（細胞）
　ＲＡＷ－Ｂｌｕｅ（商標）細胞（InvivoGen社）に、ＮＦ－κＢ及びＡＰ－１活性化により分泌型胎児性アルカリホスファターゼ（ＳＥＡＰ）レポーター遺伝子を発現させた。
　骨髄由来樹状細胞は、Ishikawa et al., Cell Host & Microbe, April 17, 2013, 13, 477-488に記載の方法に従い調製した。具体的には、１０％（ｖ／ｖ）ＦＢＳ及びβ－メルカプトエタノールを補充したＲＰＭＩ１６４０培地にて骨髄細胞を、ＭＧＭ－５から放出されるＧＭ－ＣＳＦとともに、３７℃にて１０日間培養し、骨髄由来樹状細胞を調製した。
　１０％ＦＢＳ及び１％ペニシリン－ストレプトマイシン溶液を含むＤＭＥＭにてＲＡＷ２６４．７細胞（American Type Culture Collection社）を培養した。
（試薬）
　トレハロース－６，６’－ジミコレート（ＴＤＭ；シグマ社）を１ｍｇ／ｍＬにてクロロホルム：メタノール溶液（２：１（ｖ／ｖ））に溶解し、２－プロパノールで最終濃度５０μｇ／ｍＬまで希釈し、９６ウェルプレートに２０μＬ／ウェルにてコーティングした後、溶媒を留去した。コーティングしたＴＤＭの量は１μｇ／ウェルとした。
　リポ多糖（ＬＰＳ；L4516；シグマ社）及びカードラン（InvivoGen社）を５％又は１０％ＦＢＳを含むＲＰＭＩ又はＤＭＥＭで希釈し、免疫刺激特性測定に用いた。 [Test example]
In the test examples, the following animals, cells and reagents were used unless otherwise specified.
(mouse)
MyD88-deficient mice (Oriental Yeast Co., Ltd.) and C57BL / 6 (CLEA Japan) background FcRγ-deficient mice (RIKEN) were used.
(cell)
RAW-Blue ™ cells (InvivoGen) expressed the secreted fetal alkaline phosphatase (SEAP) reporter gene by NF-κB and AP-1 activation.
Bone marrow-derived dendritic cells were prepared according to the method described in Ishikawa et al., Cell Host & Microbe, April 17, 2013, 13, 477-488. Specifically, bone marrow cells were cultured in RPMI1640 medium supplemented with 10% (v / v) FBS and β-mercaptoethanol together with GM-CSF released from MGM-5 at 37 ° C. for 10 days, Bone marrow-derived dendritic cells were prepared.
RAW 264.7 cells (American Type Culture Collection) were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin solution.
(reagent)
Trehalose-6,6′-dimicolate (TDM; Sigma) was dissolved at 1 mg / mL in a chloroform: methanol solution (2: 1 (v / v)) and diluted with 2-propanol to a final concentration of 50 μg / mL. After coating on a 96-well plate at 20 μL / well, the solvent was distilled off. The amount of TDM coated was 1 μg / well.
Lipopolysaccharide (LPS; L4516; Sigma) and curdlan (InvivoGen) were diluted with RPMI or DMEM containing 5% or 10% FBS and used for immunostimulation property measurement.
［試験例１－１］自己集合体の流体力学的サイズ測定（１）
　上記のように製造した自己集合体の流体力学的直径を、キュムラント相関関数を用いた動的光散乱光度計（ELS-Z2；165°；オートアジャストモード；大塚電子株式会社）により測定した。
　具体的には、上記実施例１化合物を含む化合物ライブラリーの試験化合物のＰＢＳ（pH 7.4；Thermo Fisher Scientific社）溶液（５０μＭ）を、１％（ｖ／ｖ）最終濃度のＤＭＳＯを含むように、Coster Ｖ底ポリプロピレン９６ウェルプレートにて調製した。各サンプルをピペッティングにより混合し、室温にて１５分間インキュベーションした。インキュベーションの後、各サンプルに蛍光試薬（１μＬ）を加え、ピペッティングにより混合した。さらに１５分間インキュベーションした後、マイクロプレートリーダーにより蛍光強度を記録した。蛍光試薬として、ＡＮＳの１．５ｍＭ ＰＢＳ溶液（励起波長＝365 nm、発光波長＝450 nm）またはナイルレッドの０．３ｍＭ ＤＭＳＯ溶液（励起波長＝550 nm、発光波長＝610 nm）のいずれかを用いた。全サンプルについて、５０μＭ ＰＢＳ溶液を２５℃にて調製し、流体力学的直径を測定した。
　化合物ライブラリーのうち自己集合体を形成した化合物の流体力学的直径を図１に示す。 [Test Example 1-1] Measurement of hydrodynamic size of self-assembly (1)
The hydrodynamic diameter of the self-assembly produced as described above was measured by a dynamic light scattering photometer (ELS-Z2; 165 °; auto-adjust mode; Otsuka Electronics Co., Ltd.) using a cumulant correlation function.
Specifically, a PBS (pH 7.4; Thermo Fisher Scientific) solution (50 μM) of a test compound in the compound library containing the compound of Example 1 was added so as to contain 1% (v / v) final concentration of DMSO. And Coster V-bottom polypropylene 96-well plate. Each sample was mixed by pipetting and incubated for 15 minutes at room temperature. After incubation, a fluorescent reagent (1 μL) was added to each sample and mixed by pipetting. After a further 15 minute incubation, the fluorescence intensity was recorded with a microplate reader. As a fluorescent reagent, either a 1.5 mM PBS solution of ANS (excitation wavelength = 365 nm, emission wavelength = 450 nm) or a 0.3 mM DMSO solution of Nile Red (excitation wavelength = 550 nm, emission wavelength = 610 nm) was used. Using. For all samples, a 50 μM PBS solution was prepared at 25 ° C. and the hydrodynamic diameter was measured.
FIG. 1 shows the hydrodynamic diameter of a compound that formed a self-assembly in the compound library.
［試験例１－２］自己集合体の流体力学的サイズ測定（２）
　各実施例化合物について、試験例１－１の方法に従い、自己集合体を形成させた。サンプルは１０％ＦＢＳ（Biowest社）及び１％ペニシリン－ストレプトマイシン（Nacalai Tesque Inc.社）を含むＤＭＥＭ培地中２５℃にて種々の濃度で調製し、９６ウェルＵＶプレート（Corning Inc.社）に１００μＬ／ウェルにて移した。自己集合体の流体力学的半径を、DynaPro Plate reader II（Wyatt Technology Corp.社；165°；25℃での自動減衰モード；レーザー波長830 nm；散乱角度58°；データ取得時間10秒／ウェル；10取得／測定）を用いて測定した。
　各実施例化合物の自己集合体の３０μＭ ＤＭＥＭ溶液中で測定した流体力学的半径（Ｒｈ）及び当該Ｒｈ値から換算した流体力学的直径は以下のとおりである。
Figure JPOXMLDOC01-appb-T000032
　また、実施例１化合物及び参考例化合物の自己集合体のＤＭＥＭ溶液中のＲｈ値の結果を図２に示す。なお、参考例１化合物は自己集合体を形成しなかった。 [Test Example 1-2] Measurement of hydrodynamic size of self-assembly (2)
For each example compound, a self-assembly was formed according to the method of Test Example 1-1. Samples were prepared at various concentrations in DMEM medium containing 10% FBS (Biowest) and 1% penicillin-streptomycin (Nacalai Tesque Inc.) at 25 ° C., and 100 μL in a 96-well UV plate (Corning Inc.). / Well. The hydrodynamic radius of the self-assembly was measured using a DynaPro Plate reader II (Wyatt Technology Corp .; 165 °; automatic decay mode at 25 ° C .; laser wavelength 830 nm; scattering angle 58 °; data acquisition time 10 seconds / well; 10 acquisitions / measurements).
Hydrodynamic diameter converted from the self-assembly of 30 [mu] M DMEM solution measured hydrodynamic radius in (R h) and the R h values of the Example compounds are as follows.
Figure JPOXMLDOC01-appb-T000032
FIG. 2 shows the results of the Rh values of the self-assembly of the compound of Example 1 and the compound of Reference Example in a DMEM solution. The compound of Reference Example 1 did not form a self-assembly.
［試験例１－３］自己集合体の流体力学的サイズ測定（３）
　実施例１０化合物について、試験例１－１の方法に従い、自己集合体を形成させた。自己集合体の流体力学的直径を、ゼータサイザーナノS（英国マルバーン・パナリティカル社製）を用いて測定し（auto-attenuationモード、散乱角173度、レーザー波長633 nm）、粒子からの散乱光より得られた自己相関関数をキュムラント法で解析することにより得た。サンプルは蒸留水中２５℃にて種々の濃度で調製し、４０μＬ小容量キュベット（英国マルバーン・パナリティカル社製）を用いて測定した。実施例１０の蒸留水中各濃度における自己集合体の流体力学的直径（Diameter）は以下のとおりである。
Figure JPOXMLDOC01-appb-T000033
 [Test Example 1-3] Measurement of hydrodynamic size of self-assembly (3)
For the compound of Example 10, a self-assembly was formed according to the method of Test Example 1-1. The hydrodynamic diameter of the self-assembly was measured using Zetasizer Nano S (manufactured by Malvern Panalytical, UK) (auto-attenuation mode, scattering angle 173 degrees, laser wavelength 633 nm), and the scattered light from the particles The obtained autocorrelation function was obtained by analyzing by the cumulant method. Samples were prepared at various concentrations at 25 ° C. in distilled water and measured using a 40 μL small-volume cuvette (manufactured by Malvern Panalytical Co., UK). The hydrodynamic diameter (Diameter) of the self-assembly at each concentration in the distilled water of Example 10 is as follows.
Figure JPOXMLDOC01-appb-T000033
［試験例２－１］免疫刺激特性（１）
　自己集合体の免疫刺激特性を、ＩＬ－６産生量を指標としてアッセイした。９６ウェル組織培養プレートにて１０％ＦＢＳを含むＤＭＥＭ中で２０，０００ＲＡＷ２６４．７細胞／ウェルを３７℃にて２４時間播種した。次いで、細胞培地を試験化合物のＤＭＥＭ溶液（１０％ＦＢＳ及び最終濃度１％のＤＭＳＯを含む）で置き換えた。１％（ｖ／ｖ）ＤＭＳＯ及び100 pg/mLのＬＰＳをそれぞれ陰性及び陽性対照として用いた。２４時間後、試験化合物を含む細胞培地を回収し、２０，０００ｒｐｍ（４℃）にて遠心分離して微粒子を除去し、－３０℃にて貯蔵するか、又はそのままアッセイした。アッセイは、製造業者のプロトコールに従い、ＩＬ－６ ＥＬＩＳＡキット（Ｒ＆Ｄシステムズ社）を用いて三重又は二重に行った。
　ＤＭＥＭ溶液（０、１、３、１０、１５、２０、３０及び５０μＭ）中の実施例１化合物の自己集合体の結果を図３に示す。実施例１化合物と参考例化合物の自己集合体の比較結果を図４に示す。各実施例化合物の自己集合体の結果を図５に示す。実施例１化合物の自己集合体の３０μＭ ＤＭＥＭ溶液中での粒度分布を図６に示す。 [Test Example 2-1] Immunostimulation properties (1)
The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. 20,000 RAW 264.7 cells / well were seeded in 96-well tissue culture plates in DMEM containing 10% FBS at 37 ° C. for 24 hours. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% (v / v) DMSO and 100 pg / mL LPS were used as negative and positive controls, respectively. Twenty-four hours later, the cell culture medium containing the test compound was collected, centrifuged at 20,000 rpm (4 ° C.) to remove microparticles, and stored at −30 ° C. or assayed as is. Assays were performed in triplicate or duplicate using an IL-6 ELISA kit (R & D Systems) according to the manufacturer's protocol.
The results of self-assembly of the compound of Example 1 in DMEM solution (0, 1, 3, 10, 15, 20, 30, and 50 μM) are shown in FIG. FIG. 4 shows the result of comparison between the self-assembly of the compound of Example 1 and the compound of Reference Example. FIG. 5 shows the results of the self-assembly of each example compound. The particle size distribution of the self-assembly of the compound of Example 1 in a 30 μM DMEM solution is shown in FIG.
［試験例２－２］免疫刺激特性（２）
　自己集合体のＩＬ－６産生量を、遠心分離（１４，０００ｒｐｍ）の前後で比較した。実施例１化合物の自己集合体のＤＭＥＭ溶液（０、３、１０、３０及び５０μＭ）について、遠心分離前のサンプルではＩＬ－６産生が見られたのに対し、遠心分離後の溶液から微粒子を除去すると免疫応答が消失した。結果を図７に示す。 [Test Example 2-2] Immunostimulation properties (2)
Self-assembled IL-6 production was compared before and after centrifugation (14,000 rpm). In a DMEM solution (0, 3, 10, 30, and 50 μM) of a self-assembled compound of Example 1, IL-6 production was observed in the sample before centrifugation, whereas fine particles were removed from the solution after centrifugation. Removal eliminated the immune response. FIG. 7 shows the results.
［試験例２－３］免疫刺激特性（３）
　自己集合体の免疫刺激特性を、ＩＬ－６産生量を指標としてアッセイした。９６ウェル組織培養プレートに５％FBSを含むＲＰＭＩ１６４０中で１×１０６細胞／ウェルのＢＭＤＣを播種後、それぞれ５、１０、３０、５０及び１００μＭの実施例１、実施例７及び実施例１０化合物の自己集合体のRPMI溶液（５％ＦＢＳを含む）を添加した。０．３７ｐｇ／ｍＬのＬＰＳ（SIGMA社）、Ｋ３　ＣｐＧ（ＴＬＲ９アゴニスト；Gene Design社；２００ｎｇ）、Ｄ３５　ＣｐＧ（ＴＬＲ９アゴニスト；Gene Design社；２００ｎｇ）、Ｒ８４８（ＴＬＲ７アゴニスト；Enzo Life Sciences；２μｇ）、ｐｏｌｙ ＩＣ（ＴＬＲ３アゴニスト；SIGMA社；１μｇ）及びＤＭＳＯ（１％）をそれぞれ対照として用いた。２４時間後、試験化合物を含む細胞培地を回収し、－３０℃にて貯蔵するか、又はそのままアッセイした。アッセイは、製造業者のプロトコールに従い、ＩＬ－６ ＥＬＩＳＡキット（Ｒ＆Ｄシステムズ社）を用いて行った。結果を図８に示す。 [Test Example 2-3] Immunostimulation properties (3)
The immunostimulatory properties of the self-assembly were assayed using the amount of IL-6 produced as an index. After seeding BMDCs at 1 × 10 6 cells / well in RPMI 1640 containing 5% FBS in 96-well tissue culture plates, compounds of Examples 1, 7, 7 and 10 at 5, 10, 30, 50 and 100 μM respectively Of a self-assembled RPMI solution (containing 5% FBS) was added. 0.37 pg / mL LPS (SIGMA), K3 CpG (TLR9 agonist; Gene Design, 200 ng), D35 CpG (TLR9 agonist; Gene Design, 200 ng), R848 (TLR7 agonist; Enzo Life Sciences; 2 μg), Poly IC (TLR3 agonist; SIGMA; 1 μg) and DMSO (1%) were used as controls, respectively. Twenty-four hours later, the cell culture medium containing the test compound was collected and stored at −30 ° C. or assayed directly. The assay was performed using an IL-6 ELISA kit (R & D Systems) according to the manufacturer's protocol. FIG. 8 shows the results.
［試験例３－１］ＦｃＲγ／ＭｙＤ８８ダブルノックアウト骨髄由来樹状細胞を用いたＩＬ－６産生量測定
　自己集合体が標的する免疫受容体を評価するために、ＦｃＲγ／ＭｙＤ８８ダブルノックアウト骨髄由来樹状細胞（ＢＭＤＣ）を用いてＩＬ－６産生量を測定した。ＦｃＲγ及びＭｙＤ８８はそれぞれＣ型レクチン受容体（ＣＬＲ）及びＴＬＲの下流シグナルタンパク質である。
　実施例１化合物の自己集合体のＤＭＳＯ溶液を５％又は１０％ＲＰＭＩで希釈し、９６ウェルプレートに１００μｌ／ウェルにて加えた。１×１０５ＢＭＤＣを２４時間３７℃にて自己集合体とともに又はなしで培養した。上清中のＩＬ－６濃度をＥＬＩＳＡ（ＢＤバイオサイエンス社）で測定した。陰性対照としてＤＭＳＯを用い、陽性対照としてＬＰＳ、カードラン及びＴＤＭ（シグマ社）を用いた。結果を図９に示す。
　次に、ＦｃＲγノックアウト細胞及びＭｙＤ８８ノックアウト細胞を用いたアッセイを行った。陰性対照としてＤＭＳＯを用いた。結果を図１０及び図１１に示す（図中、ＬＰＳ及びＴＤＭは陽性対照である）。
　結果は、自己集合体が少なくともＴＬＲファミリーメンバーとの相互作用を通じて免疫刺激作用を奏することを示す。 [Test Example 3-1] Measurement of IL-6 production using FcRγ / MyD88 double knockout bone marrow-derived dendritic cells In order to evaluate the immunoreceptor targeted by the self-assembly, FcRγ / MyD88 double knockout bone marrow-derived dendrites were used. The amount of IL-6 produced was measured using cells (BMDC). FcRγ and MyD88 are C-type lectin receptor (CLR) and TLR downstream signal proteins, respectively.
A DMSO solution of a self-assembly of the compound of Example 1 was diluted with 5% or 10% RPMI and added to a 96-well plate at 100 μl / well. 1 × 10 5 BMDCs were cultured for 24 hours at 37 ° C. with or without self-assembly. The concentration of IL-6 in the supernatant was measured by ELISA (BD Bioscience). DMSO was used as a negative control, and LPS, curdlan and TDM (Sigma) were used as positive controls. FIG. 9 shows the results.
Next, an assay was performed using FcRγ knockout cells and MyD88 knockout cells. DMSO was used as a negative control. The results are shown in FIGS. 10 and 11 (in the figures, LPS and TDM are positive controls).
The results indicate that self-assembly exerts an immunostimulatory effect at least through interaction with TLR family members.
［試験例３－２］Ｃ５７ＢＬ／６Ｊ（ＷＴ）又はＴｌｒ７ － ／ － 骨髄由来樹状細胞を用いたＩＬ－６産生量測定
　自己集合体のＴＬＲ－７に対するアゴニスト活性を評価するために、Ｃ５７ＢＬ／６Ｊ（ＷＴ）又はＴｌｒ７－／－骨髄由来樹状細胞（ＢＭＤＣ）を用いてＩＬ－６産生量を測定した。
　Ｃ５７ＢＬ／６Ｊ（ＷＴ）、Ｔｌｒ７＋／－、Ｔｌｒ７－／－、Ｔｌｒ９＋／－又はＴｌｒ９－／－マウスの脛骨及び大腿骨から骨髄細胞を単離し、これらの細胞を２０ｎｇ／ｍＬのＧＭ－ＣＳＦ(PeproTech社)と共に７日間培養することによってＢＭＤＣを調製した。
　５％ＦＢＳの存在下、それぞれ１０、３０及び１００μＭの実施例１、実施例７及び実施例１０化合物の自己集合体のＲＰＭＩ１６４０溶液９６ウェルプレートに１００μｌ／ウェルにて加えた。１×１０６細胞／ウェルのＢＭＤＣを２４時間３７℃にて自己集合体とともに又はなしで培養した。上清中のマウスのＩＬ－６濃度をBio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex(Bio-Rad Laboratories, Inc.)を用いて測定した。陰性対照としてデオキシコール酸（ＤＣＡ）及びＤＭＳＯ（１％）を用い、陽性対照としてＬＰＳ（２０ｎｇ）及びＲ８４８（２μｇ）を用いた。結果を図１２に示す。結果は、自己集合体により誘導される免疫刺激作用が、陽性対照であるＲ８４８（ＴＬＲ７アゴニスト）と同様にＴＬＲ７依存性であることを示す。 [Test Example 3-2] To evaluate the agonistic activity of C57BL / 6J (WT) or Tlr7 − / − bone marrow-derived dendritic cells on TLR-7 in self-assembly for measuring the amount of IL-6 production , C57BL / J- IL-6 production was measured using 6J (WT) or Tlr7 − / − bone marrow-derived dendritic cells (BMDC).
C57BL / 6J (WT), Tlr7 + / -, Tlr7 - / -, Tlr9 + / - or TLR9 - / - mouse tibia and femur of isolated bone marrow cells single, GM-CSF of the cells 20 ng / mL BMDC were prepared by culturing with (PeproTech) for 7 days.
RPMI1640 solutions of self-assembled compounds of Examples 1, 7, and 10 at 10, 30, and 100 μM, respectively, were added at 100 μl / well in the presence of 5% FBS. 1 × 10 6 cells / well of BMDC were cultured for 24 hours at 37 ° C. with or without self-assembly. The mouse IL-6 concentration in the supernatant was measured using a Bio-Plex Pro Mouse Cytokine Grp I Panel 23-Plex (Bio-Rad Laboratories, Inc.). Deoxycholic acid (DCA) and DMSO (1%) were used as negative controls, and LPS (20 ng) and R848 (2 μg) were used as positive controls. The result is shown in FIG. The results show that the immunostimulatory effect induced by the self-assembly is TLR7-dependent, as is the positive control R848 (TLR7 agonist).
［試験例３－３］Ｃ５７ＢＬ／６Ｊ（ＷＴ）又はＴｌｒ７ － ／ － 骨髄由来樹状細胞を用いたＩＬ－６産生量測定
　自己集合体のＴＬＲ－７に対するアゴニスト活性を評価するために、試験例３－２と同様に試験を行った。具体的には、それぞれ１０、３０、５０及び１００μＭの実施例１、実施例７及び実施例１０化合物の自己集合体の溶液を９６ウェルプレートに加え、５％ＦＢＳの存在下、１×１０６細胞／ウェルのＢＭＤＣを２４時間自己集合体とともに又はなしで培養した。上清中のマウスのＩＬ－６濃度をＥＬＩＳＡによって測定した。１％（ｖ／ｖ）ＤＭＳＯを陰性対照、またＬＰＳ（２０ｎｇ）、Ｒ８４８（２μｇ）、Ｋ３　ＣｐＧ（２００ｎｇ）、ｃ－ｄｉ－ＡＭＰ（STINGアゴニスト；ヤマサ醤油株式会社；２μｇ）、Ｄ３５　ＣｐＧ（２００ｎｇ）をそれぞれ陽性対照として用いた。結果を図１３に示す。 [Test Example 3-3] Test Examples to evaluate the agonistic activity on self-assembling of IL-6 production using T57-7 for TLR-7 using dendritic cells derived from bone marrow derived from C57BL / 6J (WT) or Tlr7 - / - The test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 μM, respectively, were added to a 96-well plate, and 1 × 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA. 1% (v / v) DMSO was used as a negative control, and LPS (20 ng), R848 (2 μg), K3 CpG (200 ng), c-di-AMP (STING agonist; Yamasa Shoyu Co., Ltd .; 2 μg), D35 CpG (200 ng) ) Were used as positive controls. FIG. 13 shows the results.
［試験例３－４］Ｃ５７ＢＬ／６Ｊ（ＷＴ）又はＴｌｒ９ － ／ － 骨髄由来樹状細胞を用いたＩＬ－６産生量測定
　自己集合体のＴＬＲ－９に対するアゴニスト活性を評価するために、試験例３－２と同様に試験を行った。具体的には、それぞれ１０、３０、５０及び１００μＭの実施例１、実施例７及び実施例１０化合物の自己集合体の溶液を９６ウェルプレートに加え、５％ＦＢＳの存在下、１×１０６細胞／ウェルのＢＭＤＣを２４時間自己集合体とともに又はなしで培養した。上清中のマウスのＩＬ－６濃度をＥＬＩＳＡによって測定した。１％（ｖ／ｖ）ＤＭＳＯ及びを陰性対照、またＬＰＳ（２０ｎｇ）、Ｒ８４８（２μｇ）、Ｋ３　ＣｐＧ（２００ｎｇ）、ｃ－ｄｉ－ＡＭＰ（２μｇ）、Ｄ３５　ＣｐＧ（２００ｎｇ）をそれぞれ陽性対照として用いた。結果を図１４に示す。 [Test Example 3-4] Test examples to evaluate the agonistic activity on self-assembling of IL-6 production using T57-9 of T57-9 using C57BL / 6J (WT) or Tlr 9 − / − bone marrow-derived dendritic cells The test was performed in the same manner as in 3-2. Specifically, solutions of the self-assembly of the compounds of Examples 1, 7 and 10 at 10, 30, 50 and 100 μM, respectively, were added to a 96-well plate, and 1 × 10 6 in the presence of 5% FBS. Cells / well of BMDC were cultured with or without self-assembly for 24 hours. The IL-6 concentration of the mice in the supernatant was measured by ELISA. 1% (v / v) DMSO and a negative control, LPS (20 ng), R848 (2 μg), K3 CpG (200 ng), c-di-AMP (2 μg) and D35 CpG (200 ng) were used as positive controls, respectively. Was. FIG. 14 shows the results.
［試験例４］マウスモデルにおけるＯＶＡ抗原特異的抗体価の測定
　自己集合体のアジュバント活性を評価するために、野生型（C57BL/6JJcl）マウス（クレア社）を用いて、ＯＶＡ特異的抗体反応による抗ＯＶＡ総ＩｇＧ、ＩｇＧ１、ＩｇＧ２ｃ及びＩｇＥ力価を測定した。
　マウスを、第０日目及び第１４日目において、１０μｇ ＯＶＡ（関東化学株式会社；ロット番号：01103-31）と、ＤＭＳＯ（最終濃度：１％）、実施例１化合物（１、１０及び１００μｇ）、実施例７化合物（１、１０及び１００μｇ）若しくは実施例１０化合物（１、１０及び１００μｇ）の自己集合体、デオキシコール酸（１、１０及び１００μｇ）又は５００μｇ ミョウバンのいずれかを含む、１００μｌ／ショットのＰＢＳ溶液で皮下免疫化した（それぞれの群について、ｎ＝４マウス）。陰性対照として、デオキシコール酸及びＰＢＳを用い、陽性対照として、アルミニウム塩（Ａｌｕｍ）を用いた。最初の免疫化から２１日後に、血清を回収し、ＯＶＡ特異的抗体反応に用いた。
　血清中の抗原特異的総ＩｇＧ、ＩｇＧ１及びＩｇＧ２ｃを、ＥＬＩＳＡによって測定した。具体的には、９６ウェルプレートを、１０μｇ／ｍＬ ＯＶＡで、４℃で一晩コーティングした。洗浄後、プレートをブロッキング緩衝液と共に、室温で１時間インキュベートした。プレートを洗浄し、希釈血清と共に２時間インキュベートした。次いで、プレートを洗浄し、西洋ワサビペルオキシダーゼ結合抗マウス総ＩｇＧ、ＩｇＧ１又はＩｇＧ２ｃ抗体（Southern Biotech社）をそれぞれ、プレートに加えた。１時間後、プレートを洗浄し、TMB Microwell Peroxidase Substrate System（KPL）をウェルに加えた。２０分間インキュベートした後、２Ｎ Ｈ２ＳＯ４を加えて反応を停止させた。抗体価は、１００倍希釈した血清を５倍ずつ7段階希釈して、それぞれの吸光度をプロットして標準曲線を描き、ＯＤ＝０．２に対応する希釈値の逆数をその検体の抗体価とした。抗ＯＶＡ 総ＩｇＧ、ＩｇＧ１及びＩｇＧ２ｃの産生量をそれぞれ図１５～１７に示す。
　ＤＳマウスＩｇＥ ＥＬＩＳＡ（ＯＶＡ）キット（ＤＳファーマバイオメディカル社）を用いて、抗ＯＶＡ 総ＩｇＥを測定した。抗ＯＶＡ ＩｇＥの産生量を図１８に示す。 [Test Example 4] Measurement of OVA antigen-specific antibody titer in mouse model In order to evaluate the adjuvant activity of the self-assembly, a wild-type (C57BL / 6JJcl) mouse (CLEA) was used to evaluate the OVA-specific antibody reaction. Anti-OVA total IgG, IgG1, IgG2c and IgE titers were measured.
On day 0 and day 14, mice were challenged with 10 μg OVA (Kanto Chemical Co., Ltd .; lot number: 01103-31), DMSO (final concentration: 1%), Example 1 compound (1, 10 and 100 μg). 100 μl containing self-assembly of Example 7 compound (1, 10 and 100 μg) or Example 10 compound (1, 10 and 100 μg), deoxycholic acid (1, 10 and 100 μg) or 500 μg alum / Shot was immunized subcutaneously with PBS solution (n = 4 mice for each group). Deoxycholic acid and PBS were used as a negative control, and aluminum salt (Alum) was used as a positive control. Twenty-one days after the first immunization, serum was collected and used for OVA-specific antibody reactions.
Antigen-specific total IgG, IgG1 and IgG2c in serum were measured by ELISA. Specifically, a 96-well plate was coated with 10 μg / mL OVA at 4 ° C. overnight. After washing, the plates were incubated with blocking buffer for 1 hour at room temperature. Plates were washed and incubated with diluted serum for 2 hours. The plates were then washed and horseradish peroxidase-conjugated anti-mouse total IgG, IgG1 or IgG2c antibodies (Southern Biotech) were each added to the plates. One hour later, the plate was washed and TMB Microwell Peroxidase Substrate System (KPL) was added to the wells. After 20 min incubation, the reaction was stopped by adding 2N H 2 SO 4. The antibody titer was obtained by diluting 100-fold diluted serum five-fold in seven steps, plotting the absorbance of each, drawing a standard curve, and calculating the reciprocal of the dilution value corresponding to OD = 0.2 as the antibody titer of the sample. did. The production amounts of anti-OVA total IgG, IgG1 and IgG2c are shown in FIGS. 15 to 17, respectively.
Anti-OVA total IgE was measured using a DS mouse IgE ELISA (OVA) kit (DS Pharma Biomedical). FIG. 18 shows the amount of anti-OVA IgE produced.
［試験例５］ウイルス感染モデルにおける、自己集合体により免疫賦活化されたインフルエンザスプリットワクチン（ＳＶ）の有効性
　インフルエンザＳＶ（A/New Caledonia/20/1999 (H1N1)）に対する自己集合体のアジュバント活性を評価するために、野生型（C57BL/6JJcl）マウス（クレア社）を用いて、インフルエンザウイルス特異的抗体反応による抗ＨＡ－ＩｇＧ１及びＩｇＧ２力価を測定した。
　マウスを、第０日目及び第１４日目において、インフルエンザＳＶ（１μｇ）と、ＤＭＳＯ（最終濃度：１％）、実施例１化合物（１、１０及び１００μｇ）、実施例７化合物（１、１０及び１００μｇ）若しくは実施例１０化合物（１、１０及び１００μｇ）の自己集合体、デオキシコール酸（１、１０及び１００μｇ）又はアルミニウム塩（５００μｇ）のいずれかを含む、１００μｌ／ショットのＰＢＳ溶液で皮下免疫化した（それぞれの群について、ｎ＝３マウス）。陰性対照として、デオキシコール酸及びＰＢＳを用い、陽性対照として、アルミニウム塩を用いた。第２１日目において、血清を回収し、第３４日目においてマウスに、１０ ＬＤ５０の臨床的に単離したA/Puerto Rico/8/1934インフルエンザ（Ｈ１Ｎ１）ウイルスを鼻腔内感染させた。
　第２１日目に回収した血清中の、抗ＨＡ ＩｇＧ１及びＩｇＧ２ｃをＥＬＩＳＡによって測定した。抗ＨＡ ＩｇＧ１及びＩｇＧ２ｃの産生量を、図１９Ａ及びＢに示す。
　第３４日目のインフルエンザ感染後２１日間、マウスの体重及び生存率を追跡した。インフルエンザＳＶ（１μｇ）と、ＤＭＳＯ（最終濃度：１％）、実施例１化合物（１００μｇ）、実施例７化合物（１００μｇ）若しくは実施例１０化合物（１００μｇ）の自己集合体、デオキシコール酸（１００μｇ）又はアルミニウム塩（５００μｇ）のいずれかを皮下免疫化に用いたそれぞれのマウスについて、体重及び生存率の変化を、図２０Ａ及びＢに示す。
　インフルエンザＳＶ（１μｇ）と、ＤＭＳＯ（最終濃度：１％）、実施例１０化合物の自己集合体（１、１０又は１００μｇ）又はアルミニウム塩（５００μｇ）のいずれかを皮下免疫化に用いたそれぞれのマウスについて、体重及び生存率の変化を、図２１Ａ及びＢに示す。
　インフルエンザＳＶ（１μｇ）と、実施例１０化合物（１００μｇ）又は陰性対照としてデオキシコール酸（１００μｇ）のいずれかを皮下免疫化に用いたそれぞれのマウスについて、体重及び生存率の変化を、図２２Ａ及びＢに示す。 [Test Example 5] Effectiveness of influenza split vaccine (SV) immunostimulated by self-assembly in virus infection model Adjuvant activity of self-assembly against influenza SV (A / New Caledonia / 20/1999 (H1N1)) In order to evaluate, anti-HA-IgG1 and IgG2 titers by influenza virus-specific antibody reaction were measured using wild-type (C57BL / 6JJcl) mice (CLEA).
Mice were treated on day 0 and day 14 with influenza SV (1 μg), DMSO (final concentration: 1%), Example 1 compound (1, 10 and 100 μg), Example 7 compound (1, 10 And 100 μg) or a self-assembly of the compound of Example 10 (1, 10 and 100 μg), 100 μl / shot of a PBS solution containing either deoxycholic acid (1, 10 and 100 μg) or an aluminum salt (500 μg) subcutaneously. Immunized (n = 3 mice for each group). Deoxycholic acid and PBS were used as a negative control, and an aluminum salt was used as a positive control. On day 21, serum was collected and on day 34, mice were infected intranasally with 10 LD50 of clinically isolated A / Puerto Rico / 8/1934 influenza (H1N1) virus.
Anti-HA IgG1 and IgG2c in sera collected on day 21 were measured by ELISA. The production amounts of anti-HA IgG1 and IgG2c are shown in FIGS. 19A and 19B.
Mice were followed for body weight and survival 21 days after influenza infection on day 34. Influenza SV (1 μg), self-assembly of DMSO (final concentration: 1%), Example 1 compound (100 μg), Example 7 compound (100 μg) or Example 10 compound (100 μg), deoxycholic acid (100 μg) Changes in body weight and viability for each mouse using either subcutaneous immunization, or with aluminum salt (500 μg) are shown in FIGS. 20A and 20B.
Each mouse used for subcutaneous immunization with influenza SV (1 μg), DMSO (final concentration: 1%), self-assembly of the compound of Example 10 (1, 10 or 100 μg) or aluminum salt (500 μg) For FIGS. 21A and 21B, changes in body weight and survival rate are shown.
Changes in body weight and viability for each mouse using subcutaneous immunization with influenza SV (1 μg) and either Example 10 compound (100 μg) or deoxycholic acid (100 μg) as a negative control are shown in FIG. 22A and FIG. B.
［試験例６］ＨＥＫ２９３細胞を用いたＳＥＡＰアッセイ
　シグナル経路を解明するために、ＮＦ－κＢ応答ＳＥＡＰレポーター遺伝子で安定にトランスフェクトしたヒト胎児腎臓細胞（ＨＥＫ２９３）を用いて、いくつかの主なＴＬＲについて試験した。
　ＴＬＲ２、ＴＬＲ４又はＴＬＲ９発現ＨＥＫ細胞を９６ウェル組織培養プレートにて１０％ＦＢＳを含むＤＭＥＭ（１００μｌ）中で３×１０４細胞／ウェルにて播種し、実施例１化合物の自己集合体で３７℃２４時間又は４８時間刺激させた。培養液の上清中のＳＥＡＰをアルカリホスファターゼ検出試薬QUANTI-Blue（InvivoGen社）で３時間又は６時間かけて染色し、Multiskanマイクロプレートリーダー（Thermo Fisher Scientific Inc.）により630 nmにて光学濃度（ＯＤ値）を測定した。
　結果を図２３及び図２４に示す。陰性対照（ＮＣ）としてＤＭＳＯを用い、陽性対照として図２３ではオリゴデオキシヌクレオチド（ODN1826；tlrl-1826；InvivoGen社）を、図２４ではＴＬＲ２アゴニストＰａｍ３（Pam3CSK4；InvivoGen社）及びＬＰＳを用いた。自己集合体によるＳＥＡＰ応答はＴＬＲ９で上昇した。 [Test Example 6] In order to elucidate the SEAP assay signal pathway using HEK293 cells , several major TLRs were used using human embryonic kidney cells (HEK293) stably transfected with an NF-κB-responsive SEAP reporter gene. Was tested.
HEK cells expressing TLR2, TLR4 or TLR9 are seeded at 3 × 10 4 cells / well in 96-well tissue culture plates in DMEM (100 μl) containing 10% FBS and self-assembled with the compound of Example 1 at 37 ° C. Stimulation was for 24 or 48 hours. SEAP in the culture supernatant was stained with an alkaline phosphatase detection reagent QUANTI-Blue (InvivoGen) for 3 hours or 6 hours, and optical density (630 nm) was measured with a Multiskan microplate reader (Thermo Fisher Scientific Inc.). OD value) was measured.
The results are shown in FIGS. DMSO was used as a negative control (NC), and oligodeoxynucleotide (ODN1826; tlrl-1826; InvivoGen) was used in FIG. 23 as a positive control, and TLR2 agonist Pam3 (Pam3CSK4; InvivoGen) and LPS were used in FIG. Self-assembled SEAP responses were elevated in TLR9.
［試験例７］ＩＬ－６定量的ＰＣＲアッセイ
　ＲＡＷ２６４．７細胞について、６ウェル組織培養プレートにて１０％ＦＢＳを含むＤＭＥＭ中で６×１０５細胞／ウェルを３７℃にて２４時間播種した。次いで、細胞培地を試験化合物のＤＭＥＭ溶液（１０％ＦＢＳ及び最終濃度１％のＤＭＳＯを含む）で置き換えた。１％ＤＭＳＯ及び100 ng/mLのＬＰＳをそれぞれ陰性及び陽性対照として用いた。４時間後、細胞をＰＢＳで２回洗浄した。細胞をISOGENE（１ｍＬ／ウェル；Nippon Gene Co., Ltd.）で５分間溶解させた。次いで、クロロホルム（２００μＬ）を加えた。試験管を逆さにすることにより混合物を１５分間混合した後、１４，０００ｒｐｍで４℃にて１５分間遠心分離した。水層の上部４００μＬを別の１．５ｍＬ試験管に回収し、イソプロパノール５００μＬと混合した。試験管を１４，０００ｒｐｍにて再度遠心分離し、上清を除去した。残渣をエタノール（７００μＬ）で２回洗浄し、空気乾燥し、ＴＥ緩衝液に再溶解した（ｐＨ５．２）。PrimeScript（商標）II 1st strand cDNA Synthesis Kit（タカラバイオ株式会社）により全ｍＲＮＡサンプル（２μｇ）を逆転写し、ｃＤＮＡの合成に用いた。定量的リアルタイムＰＣＲをFast CYBR Green Master Mixキット（Thermo Fisher Scientific Inc.）を用いて行った。全アッセイは三重に行い、ＴＮＦ－α及びβ－アクチンを内部標準として用いた。特異的な標的遺伝子についての全プライマーを以下に示す：
Figure JPOXMLDOC01-appb-T000034

　結果を図２５に示す。 For Test Example 7] IL-6 Quantitative PCR assay RAW264.7 cells were seeded 24 hours 6 × 10 5 cells / well in DMEM containing 10% FBS at six-well tissue culture plates at 37 ° C.. The cell culture medium was then replaced with a DMEM solution of the test compound (containing 10% FBS and 1% final concentration of DMS0). 1% DMSO and 100 ng / mL LPS were used as negative and positive controls, respectively. After 4 hours, the cells were washed twice with PBS. Cells were lysed with ISOGENE (1 mL / well; Nippon Gene Co., Ltd.) for 5 minutes. Then, chloroform (200 μL) was added. The mixture was mixed for 15 minutes by inverting the test tube and then centrifuged at 14,000 rpm for 15 minutes at 4 ° C. The upper 400 μL of the aqueous layer was collected in another 1.5 mL test tube and mixed with 500 μL of isopropanol. The test tube was centrifuged again at 14,000 rpm, and the supernatant was removed. The residue was washed twice with ethanol (700 μL), air-dried and redissolved in TE buffer (pH 5.2). PrimeScript (TM) II 1 st strand cDNA Synthesis Kit ( Takara Bio Inc.) by reverse transcribed total mRNA samples (2 [mu] g), were used for the synthesis of cDNA. Quantitative real-time PCR was performed using the Fast CYBR Green Master Mix kit (Thermo Fisher Scientific Inc.). All assays were performed in triplicate and TNF-α and β-actin were used as internal standards. All primers for specific target genes are shown below:
Figure JPOXMLDOC01-appb-T000034

The results are shown in FIG.
　式（Ｉ）の化合物が連結してなる自己集合体はアジュバントとして有用であり得、種々の疾患の治療及び／又は予防のためのワクチン組成物としての利用が期待される。 (5) A self-assembly obtained by linking the compounds of the formula (I) can be useful as an adjuvant, and is expected to be used as a vaccine composition for treating and / or preventing various diseases.

Claims (12)

  1. 　式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000001
     ［式中、Ｒｘは、Ｈ又は式（ＩＩ）：
    Figure JPOXMLDOC01-appb-C000002
     で示される基であり、
    　Ｒｙは、Ｈ又は式（ＩＩＩ）：
    Figure JPOXMLDOC01-appb-C000003
     で示される基であり、
    　Ｒ１、Ｒ２、Ｒ３及びＲ４は、それぞれ独立して、Ｈ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
    　波線は結合部位を示し、
    　ｎは１～１２の整数である］
    で示される化合物が非共有結合により連結してなる、自己集合体。     Formula (I):
    Figure JPOXMLDOC01-appb-C000001
     Wherein R x is H or formula (II):
    Figure JPOXMLDOC01-appb-C000002
     Is a group represented by
    R y is H or formula (III):
    Figure JPOXMLDOC01-appb-C000003
     Is a group represented by
    R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl;
    Wavy lines indicate binding sites,
    n is an integer of 1 to 12]
    A self-assembly comprising a compound represented by the formula (1) linked by a non-covalent bond.
  2. 　ＲｘがＲｙと同一である、請求項１に記載の自己集合体。 2. The self-assembly according to claim 1, wherein Rx is the same as Ry .
  3. 　Ｒｘが式（ＩＩ’）：
    Figure JPOXMLDOC01-appb-C000004
     で示される基であり、
    　Ｒｙが式（ＩＩＩ’）：
    Figure JPOXMLDOC01-appb-C000005
     で示される基である、請求項１又は２に記載の自己集合体。     R x is of the formula (II ′):
    Figure JPOXMLDOC01-appb-C000004
     Is a group represented by
    R y has the formula (III ′):
    Figure JPOXMLDOC01-appb-C000005
     The self-assembly according to claim 1, which is a group represented by the formula:
  4. 　Ｒ３及びＲ４がそれぞれ独立してＣ１－３アルキルである、請求項１～３のいずれかに記載の自己集合体。 4. The self-assembly according to claim 1, wherein R 3 and R 4 are each independently C 1-3 alkyl.
  5. 　Ｒｘ及びＲｙがともにＨである、請求項１又は２に記載の自己集合体。 The self-assembly according to claim 1, wherein R x and R y are both H.
  6. 　ｎが５～１０の整数である、請求項１～５のいずれかに記載の自己集合体。 The self-assembly according to any one of claims 1 to 5, wherein n is an integer of 5 to 10.
  7. 　流体力学的サイズ４０ｎｍ～１２，０００ｎｍを有する凝集体を形成する、請求項１～６のいずれかに記載の自己集合体。 The self-assembly according to any one of claims 1 to 6, which forms an aggregate having a hydrodynamic size of 40 nm to 12,000 nm.
  8. 　請求項１～７のいずれかに記載の自己集合体を含む、免疫賦活剤。 (8) An immunostimulator comprising the self-assembly according to any one of (1) to (7).
  9. 　アジュバントである、請求項８に記載の免疫賦活剤。 The immunostimulator according to claim 8, which is an adjuvant.
  10. 　請求項９に記載の免疫賦活剤及び抗原を含む、ワクチン組成物。 ワ ク チ ン A vaccine composition comprising the immunostimulant according to claim 9 and an antigen.
  11. 　がん、感染症、アレルギー疾患、自己免疫疾患、高血圧症及びアルツハイマー病からなる群から選択される疾患の治療及び／又は予防のための、請求項１０に記載のワクチン組成物。 11. The vaccine composition according to claim 10, for treating and / or preventing a disease selected from the group consisting of cancer, infectious disease, allergic disease, autoimmune disease, hypertension and Alzheimer's disease.
  12. 　式（Ｉ）：
    Figure JPOXMLDOC01-appb-C000006
     ［式中、Ｒｘは、Ｈ又は式（ＩＩ）：
    Figure JPOXMLDOC01-appb-C000007
     で示される基であり、
    　Ｒｙは、Ｈ又は式（ＩＩＩ）：
    Figure JPOXMLDOC01-appb-C000008
     で示される基であり、
    　Ｒ１、Ｒ２、Ｒ３及びＲ４は、それぞれ独立して、Ｈ、Ｃ１－６アルキル又はＣ１－６ハロアルキルであり、
    　波線は結合部位を示し、
    　ｎは１～１２の整数である］
    で示される化合物。     Formula (I):
    Figure JPOXMLDOC01-appb-C000006
     Wherein R x is H or formula (II):
    Figure JPOXMLDOC01-appb-C000007
     Is a group represented by
    R y is H or formula (III):
    Figure JPOXMLDOC01-appb-C000008
     Is a group represented by
    R 1 , R 2 , R 3 and R 4 are each independently H, C 1-6 alkyl or C 1-6 haloalkyl;
    Wavy lines indicate binding sites,
    n is an integer of 1 to 12]
    A compound represented by the formula:
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