WO2008016139A1 - Fluorescent oleamide derivative and use thereof - Google Patents

Fluorescent oleamide derivative and use thereof Download PDF

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Publication number
WO2008016139A1
WO2008016139A1 PCT/JP2007/065264 JP2007065264W WO2008016139A1 WO 2008016139 A1 WO2008016139 A1 WO 2008016139A1 JP 2007065264 W JP2007065264 W JP 2007065264W WO 2008016139 A1 WO2008016139 A1 WO 2008016139A1
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Prior art keywords
oleamide
fluorescent
linked
group
formula
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PCT/JP2007/065264
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French (fr)
Japanese (ja)
Inventor
Yasuyuki Kita
Hiroshi Nojima
Yusuke Ohba
Takashi Okamoto
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Osaka University
Nard Institute, Ltd.
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Publication of WO2008016139A1 publication Critical patent/WO2008016139A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/50—Compounds containing any of the groups, X being a hetero atom, Y being any atom
    • C07C311/51—Y being a hydrogen or a carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D303/46—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals by amide or nitrile radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a fluorescent oleamide derivative and use thereof, for example, a method of observing fluorescence of a cell using the fluorescent oleamide derivative, a gap binding inhibitor containing the fluorescent ureamide, and a cell of an oleamide derivative. It relates to tools for research on internal mechanisms, pharmaceuticals, food and drink, and supplements.
  • a gap bond is a connexin hexamer connexon linked between adjacent cells.
  • the conduit formed by this connection penetrates the cell membrane, and ions and low molecular weight proteins are transferred from cell to cell in the conduit. This mechanism is considered essential for maintaining homeostasis of proliferation of epidermal cells.
  • Connexin is a generic name for a family of membrane-bound proteins that constitute gap junctions, and more than 20 subtypes have been discovered due to differences in molecular weight. For example, a molecular weight of 26 kDa is called connexin 26 (connexin 26), and a 43 kDa molecular weight is called connexin 43.
  • oleamide an amide derivative of oleic acid
  • Olamide is an endogenous fatty acid primary amide that has been shown to accumulate in cerebrospinal fluid in the state of sleep disturbance and sleep loss during sleep recovery.
  • Olamide is a compound with very interesting activity known to have various effects, such as inducing physiological sleep in animals when administered by intraperitoneal or intravenous injection.
  • Olamide and its derivatives are also known as substances that inhibit gap bonding (Non-patent Document 3 below).
  • the present inventors have identified an oleamide derivative that specifically inhibits connexin 26 and exhibits good cancer metastasis-inhibiting activity, and one of them is referred to as “MI-18”. (Patent Document 2 below).
  • the present inventors have also conducted research on multimeric oleamide derivatives and their use in cancer treatment and the like! (Patent Document 3 below).
  • Non-Patent Document 4 For studying the mechanism of gap binding inhibition of oleamide, for example, the fluidity of bulk membranes and perturbations at membrane protein interfaces have been studied (Non-Patent Document 4 below). Olamide acts on the higher order structure of membrane-bound proteins in bulk membranes and interacts directly with gap-bound proteins. However, the mechanism of its action has not been clarified yet, and the mode of distribution to the cells has not been understood at all!
  • a method of introducing a fluorescent group into the amide group (peptide group) (fluorescence introduction method) and observing fluorescence is used.
  • Typical examples of the fluorescent group used at this time include a dansyl group and an NBD group.
  • conversion of the amide group part has a great influence on its biological activity. For example, the ability to inhibit gap binding is lost by introducing fluorescence.
  • Patent Document 1 Japanese Patent Laid-Open No. 2001-17184
  • Patent Document 2 International Publication WO2004 / 060398
  • Patent Document 3 International Publication WO2006 / 049157
  • Non-patent literature l Ito, A. et al .: J. Clin. Invest., 105: 1189— 1197, 2000.
  • Non-Patent Document 2 Cravatt et al. Science 1995, 1506-1509; Lerner et al. Proc. Natl. Acad. Sci. USA 1994, 91, 9505-9508; Cravatt et al J. Am. Chem. Soc. 1996, 118, 580-590
  • Non-Patent Document 3 Boger, D. et al .: Proc. Nat. Acad. Sci. USA., 95: 4810—4815. 1998.
  • Non-Patent Document 4 Lars Bastiaase et al. J. Membrane Biol. 1993, 136, 135-145 Disclosure of the Invention
  • an object of the present invention is to provide a fluorescent oleamide derivative in which the original biological activity of oleamide is not impaired.
  • Another object of the present invention is to provide a method for observing fluorescence of cells using the fluorescent oleamide derivative, a gap binding inhibitor containing the fluorescent oleamide, and an oleamide derivative. It is to provide tools for studying intracellular action mechanisms, pharmaceuticals, foods and drinks, and supplements.
  • the compound of the present invention is a fluorescent oleamide derivative represented by the following formula (I), a tautomer or stereoisomer thereof, or a salt thereof.
  • L 1 is a single bond, -0- or -S-
  • L 2 is a single bond or a linear or branched alkylene group
  • R 1 and R 2 are each a hydrogen atom or an arbitrary substituent, and may be the same or different.
  • R is a fluorescent group.
  • the compound of the present invention is a dimer type compound of the fluorescent olamide derivative of the above formula (I) represented by the following formula (IV), a tautomer or stereoisomer thereof, or a salt thereof. Always. [Chemical 8]
  • L 2 , R 1 , R 2 and R are the same as those in the formula (I), and each L 1 may be the same or different from each other R 1 Can be the same or different
  • R 3 is a hydrogen atom or an optional substituent
  • 1 is a positive integer.
  • the compound of the present invention is a multimeric compound of the fluorescent oleamide derivative of the formula (I) represented by the following formula (V), a tautomer or stereoisomer thereof, or a salt thereof. Also good.
  • L, L, R and R are the same as the formula (I), and each L may be the same or different.
  • R 4 is a hydrogen atom or an optional substituent
  • n, h and k are each a positive integer.
  • the method for observing fluorescence of cells according to the present invention includes a fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof.
  • the product of the present invention comprises a fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof, It is used as a gap binding inhibitor, a tool for studying the intracellular action mechanism of an oleamide derivative, a pharmaceutical, a food or drink, or a supplement.
  • the fluorescent oleamide derivative represented by the above formula (1), (IV) or (V) can emit fluorescence without significantly impairing the original biological activity by introduction of the fluorescent group. others Therefore, the compound of the present invention, that is, the fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof, is a method for observing fluorescence of a cell, It is useful as a tool for studying the intracellular action mechanism of oleamide derivatives.
  • the compound of the present invention can be used as a gap binding inhibitor, a pharmaceutical, a food or drink, or a supplement due to its biological activity. Furthermore, the use of the compound of the present invention is not limited to these, and can be used for all uses.
  • FIG. 1 is a graph showing the GJIC score of each test substance (oleamide, dansyl compound (compounds la to lc), and Dansyl oleamide in a comparative example) in dye transfer assembly.
  • Fig. 2a is a diagram showing the state after the dye transfer assembly with each test substance (olamide, Dansyl form (compounds la to lc)), Fig. 2a) is a control diagram, Fig. 2b) Fig. 2c) is a diagram of the compound lc (C10 linked dansyl oleamide). In the original image, cells that have been dyed are displayed in green.
  • FIG. 3 is a diagram showing the results of introducing an oleamide derivative having an NBD group (compounds 2a to 2c and 4c) into HeLa_Cx43 cells or HeLa cells.
  • Figures 3a) and 3b) show the results of introducing compounds 2a-2c and 4c into HeLa_Cx43 cells
  • Figures 3c) and 3d) show the results of introduction into HeLa cells.
  • Figures 3a) and 3c) show the measurement results by DIC
  • Figures 3b) and 3d) show the measurement results using Cy2-filter. In the original drawing, the fluorescent spots in b) and d) are displayed in green.
  • FIG. 4 is a graph showing the results of introducing oleamide derivatives having dansyl groups (compounds la to lc and 3c) into HeLa_Cx43 cells or HeLa cells.
  • FIGS. 4a) and 4b) show the results of introducing compounds la to lc and 3c into HeLa_Cx43 cells
  • FIGS. 4c) and 4d) show the results of introduction into HeLa cells.
  • Figures 4a) and 4c) show the measurement results by DIC
  • Figures 4b) and 4d) show the measurement results using the DAH-filter.
  • FIG. 5 shows the results of double staining of Tigl cells with C10-linked oleamide, which is a fluorescent oleamide derivative of the present invention, and mitotracker.
  • a) is the result of staining with MitoTracker TM Red using a rhodamine filter (the fluorescent part is shown in red in the original figure)
  • b) is the result of observing the localization of C10-linled NBD oleamide using the FITC filter ( Original drawing In Fig. 1, the fluorescent part is displayed in green)
  • c) is the result of the merge of a) and b) (Merge) (the Merge part is displayed in yellow in the original figure)
  • d) is a DIC-filter. It is an observation result.
  • FIG. 6 shows the results of double staining of Tigl cells with C10-linked oleamide and FM TM 4-64, which are fluorescent oleamide derivatives of the present invention.
  • a) is the result of staining with FM TM 4_64 (fluorescent sites are displayed in red in the original image)
  • b) is the result of observing the localization of C10-linled NBD oleamide (in the original image, the fluorescent sites are displayed in green)
  • c ) Is the result of the merge of a) and b) (Merge is displayed in yellow in the original figure)
  • d) is the observation result using the DIC-filter.
  • the bar length is 10 ⁇ m.
  • L 2 is, for example, a single bond or a carbon atoms;! Ku linear if the - 12 there is preferably fixture L 2 be branched alkylene group , Single bond, methylene group or 2 carbon atoms
  • R is not particularly limited and may be any fluorescent group.
  • a dansyl group represented by the following formula ( ⁇ ), an NBD group represented by the following formula (III), or a fluorescent group such as naphthalimide is preferable.
  • using fluorescent groups with different excitation wavelengths according to the purpose is more suitable for use in cell fluorescence observation methods, intracellular action mechanism research tools, and the like.
  • R 1 and R 2 are a hydrogen atom or an arbitrary substituent as described above. An alkyl group is preferred. In the formula (I), R 1 and R 2 are more preferably a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.
  • R 3 in the above formula (IV) and R 4 in the above formula (V) are a hydrogen atom or an arbitrary substituent as described above.
  • the alkyl group may be an alkyl group, and the alkyl group preferably has 1 to 6 carbon atoms, for example.
  • R 3 or R 4 may have another fluorescent group R as represented by the following formula (K).
  • the definitions of L 2 , R 2 and R in the following formula (IX) are the same as described above, and in the formula (IV) or (V), each L 2 is the same or different.
  • Each R 2 may be the same or different, and each R may be the same or different.
  • 1 is not particularly limited, but is, for example, 3 to 8, preferably 3 to 5, particularly preferably 3.
  • m is not particularly limited, but is, for example, 3 to 8, preferably 3 to 5, and particularly preferably 3.
  • h is not particularly limited, it is, for example, 0 to 2, preferably 0 to; 1, particularly preferably 1.
  • k is not particularly limited, but is preferably 3 to 4, for example. Preferably it is 3.
  • halogen refers to any halogen element, and examples thereof include fluorine, chlorine, bromine and iodine.
  • the alkyl group is not particularly limited, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group.
  • alkylene group include polymethylene groups such as ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, and otatamethylene group.
  • the salt of the compound represented by the formula (1), (IV) or (V) may be an acid addition salt or a base addition salt.
  • the acid forming the acid addition salt may be an inorganic acid or an organic acid.
  • the inorganic acid is not particularly limited, and examples thereof include sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid and the like.
  • the organic acid is not particularly limited, and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citrate, benzoic acid, and acetic acid.
  • the base forming the base addition salt may be an inorganic base or an organic base.
  • the inorganic base include, but are not limited to, ammonium hydroxide, alkali metal hydroxide, alkaline earth metal hydroxide, carbonate, bicarbonate, and the like. More specifically, for example, Sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like are possible.
  • the organic base is not particularly limited, and for example, ethanolamine, triethylamine, tris (hydroxymethyl) aminomethane and the like are possible.
  • a method for producing a salt of the compound represented by the formula (1), (IV) or (V) is not particularly limited.
  • the compound represented by the formula 0), (IV) or (V) Acids and bases such as those described above can be obtained by known methods. It can be produced by a method such as adding appropriately.
  • a fluorescent oleamide derivative represented by any one of the following formulas la to lc, 2a to 2c, 3a to 3c, and 4a to 4c, It is a tautomer or stereoisomer, or a salt thereof.
  • the compound of the present invention may be produced by any method, for example, chemically synthesized, or if it is found that it exists in nature, it may be isolated and purified from a natural product. good. Alternatively, a substance obtained from a natural product is used as a raw material and subjected to a treatment such as a reaction. A compound may be produced. If necessary, the compound of the present invention may be produced using a biological material such as a microorganism. When the compound of the present invention is produced by chemical synthesis, the method is not particularly limited. For example, a compound of the following formulas ( ⁇ ), ( ⁇ ) and (vm) is used as a raw material, and an amine and a halide are synthesized. It is preferable to produce by the production method of the present invention including the condensation reaction step.
  • the compound of the present invention can be easily synthesized from commercially available raw materials.
  • the reaction conditions are not particularly limited.
  • the reaction solvent in the condensation reaction is not particularly limited, and examples thereof include ethers such as jetyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and t-butylmethyl ether, halogenated solvents such as dichloromethane and chloroform, or dimethylformamide, Examples include dimethyl sulfoxide, acetonitrile, acetone, toluene, and benzene.
  • the reaction temperature and reaction time are also not particularly limited, and can be appropriately selected according to, for example, the compound to be reacted and the solvent.
  • the reaction temperature is, for example, 0 to 130 ° C, preferably 0 to 30 ° C, particularly preferably 0 to 25 ° C.
  • the reaction time is, for example, !! to 24 hours, preferably 3 to 12 hours, particularly preferably 6 to 12 hours.
  • the compound of the formula (I) can be synthesized, for example, according to the following schemes 1 to 4.
  • the following formula ( ⁇ ) shows the case where L 1 is a single bond in the formula (I).
  • the method for producing the compounds of the formulas (IV) and (V) is not particularly limited. It is possible to produce a multimer-type oleamide derivative described in International Publication No. WO2006 / 049157 (Patent Document 3) combined with a condensation reaction between an amine and a halide. For example, after producing a multimeric oleamide derivative, a fluorescent group may be introduced by a condensation reaction between an amine and a halide, or after producing a fluorescent ureamide, it may be converted into a multimeric oleamide.
  • the compound of the present invention is useful for a method for observing fluorescence of a cell and a tool for studying the intracellular action mechanism of an oleamide derivative.
  • the compounds of the present invention can also be used as gap binding inhibitors, pharmaceuticals, foods and drinks or supplements due to their biological activity.
  • a pharmaceutical such as an anticancer agent (anticancer agent), a supplement having an anticancer activity or a cancer preventing effect, a functional food (edible composition) It can be used as a raw material.
  • the use of the compound of the present invention is not limited to these, and can be used for any application.
  • the compound of the present invention when used for drug development, as one aspect thereof, the compound of the present invention may be used as a lead compound in the drug development process.
  • the compound of the present invention can be administered to humans or animals) as it is or as a pharmaceutical composition together with a conventional pharmaceutical carrier.
  • the dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary.
  • Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are, for example, starch, lactose, sucrose, trenorose, mannitol, force noboxymethylenorescenellose, corn starch, inorganic salts Etc. are manufactured according to a conventional method.
  • the compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately set.
  • the ability to use binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances, etc. as appropriate is reduced.
  • the dosage is adjusted according to the patient's age, weight, degree of disease, etc. For example, it is administered by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection or the like.
  • This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent.
  • this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use.
  • an isotonic agent, stabilizer, preservative, and soothing agent may be added as necessary.
  • the compounding amount of the compound of the present invention in these preparations is not particularly limited and can be arbitrarily set. Examples of other parenteral preparations include coating solutions for external use, ointments and the like, suppositories for rectal administration, etc., and these are also produced according to conventional methods.
  • DDS drug delivery system
  • the compound of the present invention may be encapsulated in a carrier such as ribosome and administered in the body! /.
  • a carrier such as ribosome
  • the compound of the present invention can be efficiently transported to the target site.
  • the compound of the present invention can be used in foods and drinks (edible compositions) such as supplements and functional foods. That is, the compound of the present invention is added to foods and drinks as raw materials for various beverages and various processed foods, and if necessary, pellets, tablets, excipients such as dextrin, lactose, starch, flavorings, pigments, etc. Can be processed into granules, etc., or coated with gelatin and molded into capsules for use as health food or health food
  • Nuclear magnetic resonance (NMR) spectra were measured at 0 to 25 ° C by JEOL equipment JNM—EX270 type (2 70MHz, 67 ⁇ 8) ⁇ ), JEOL JNM—EX300 type (300MHz, 75.3MHz) (trade name) ( 1 H measurement was performed using 270 or 300 MHz). Chemical shifts are expressed in parts per million (ppm). Tetramethylsilane (TMS) was used as the internal standard Oppm. Coupling constants (J) are shown in hertz, and the abbreviations s, d, t, q, m and br are singlet, doublet, triplet, quadruple, respectively. quartet, multiplet and wide line ( broad).
  • Mass spectrometry (MS or LRMS) was measured by the FAB method using an instrument JMS-D300 (trade name) manufactured by JEOL.
  • High resolution mass spectrometry (HRMS) was measured by the FAB method using an instrument JMS-600 (trade name) manufactured by JEOL.
  • the gap binding inhibitory activity was measured by dye transfer assay, and the results were recorded and photographed.
  • the specific assembly method was the same as the method described in WO2006 / 049157.
  • FIG. 1 is a graph showing the GJIC score of each test substance (olamide, Dansyl form (compounds la to lc) and comparative example Dansyl oleamide).
  • FIG. 2 is a diagram showing the state after the dye transfer assay by each test substance, FIG. 2a) is a control diagram, and FIG. 2b) is an oleamide diagram.
  • Figure 2c) is a diagram of the compound lc (C10 linked dansyl oleamide).
  • the fluorescent oleamide derivative synthesized in this example was introduced into HeLa cells, HeLa_Cx43 (HeLa C x43) cells, cancer cells such as MCF-7 cells, and normal cells such as TIG1 cells, and fluorescence was observed. Was recorded and photographed.
  • the specific introduction method is almost the same as the method described in WO2006 / 049157.
  • each cell was individually suspended in DMEM containing 10% FCS. And all the cells were cultured on the cover glass in a petri dish.
  • 40 M of a fluorescent oleamide derivative was first added to the cell culture, incubated for 2 hours, and then washed 3 times with a phosphate buffer. After adding 1 mL of 4% formaldehyde solution (diluted with phosphate buffer, not containing methanol), allowed to stand for 5 minutes, it was washed again with phosphate buffer three times. After the cover glass was sufficiently drained, it was sealed on the preparation. afterwards, Cells were observed.
  • FIG. 3 is a diagram showing the result of introducing an oleamide derivative (compounds 2a to 2c and 4c) having an NBD group into HeLa_Cx43 cells or HeLa cells.
  • Figures 3a) and 3b) show the results of introducing compounds 2a-2c and 4c into HeLa_Cx43 cells, and Figures 3c) and 3d) show the results of introduction into HeLa cells.
  • Figures 3a) and 3c) show the measurement results by DIC
  • Figures 3b) and 3d) show the measurement results using Cy2-filter.
  • FIG. 3 is a diagram showing the result of introducing an oleamide derivative (compounds 2a to 2c and 4c) having an NBD group into HeLa_Cx43 cells or HeLa cells.
  • Figures 3a) and 3b) show the results of introducing compounds 2a-2c and 4c into HeLa_Cx43 cells
  • Figures 3c) and 3d) show the results of introduction into HeLa cells.
  • FIG. 4 is a diagram showing the results of introducing oleamide derivatives having dansyl groups (compounds la to lc and 3c) into HeLa_Cx43 cells or HeLa cells.
  • Figures 4a) and 4b) show the results of introducing the compounds la to lc and 3c into HeLa_Cx43 cells
  • Figures 4c) and 4d) show the results of introduction into HeLa cells.
  • Figures 4a) and 4c) show the results of measurement by DIC
  • Figures 4b) and 4d) show the results of measurement using DAPI-filter.
  • the compound of this example can be introduced into both HeLa_Cx43 cells and HeLa, and the fluorescence of the cells could be observed. Contrary to expectations, oleamide was observed not only in the cell membrane but also in the cytoplasm other than the cell nucleus.
  • Figs. 5 and 6 show the results of double staining of the fluorescent oleamide of the present invention with another fluorescent dye.
  • Fig. 5 shows the result of double staining of Tigl cells with C10-linked NBD ol eamide, a fluorescent oligomer of the present invention, and the fluorescent dye MitoTracker TM Red.
  • FIG. 6 shows the results of double staining with linked NBD oleamide and fluorescent dye FM TM 4-64.
  • MitoTracker TM Red and FM TM 4-64 are both purchased from Molecular Probes Inc. MitoTracker Red (Chloromethyl-X-rosamine) selectively stains cell mitochondria.
  • FM TM 4-64 N- (3-triethylammoniumpropyi) -4- (6- (4- (diethylaminophenyl) hexatnenyl) pyridinium dibromide) is stained in the process of endocytosis.
  • All fluorescent oleamide derivatives in this example introduced fluorescence related to cell types (various cancer cells such as HeLa cells, HeLa_Cx43 cells, MCF-7 cells, and normal cells such as TIG1 cells). It can.
  • C10-linked dansyl MI-18 (compound 3c) and C10-linked NBD MI-18 (compound 4c) do not inhibit gap binding in HeLa_Cx43 cells, but Cn-linked dansyl or NBD oleamide (compound la ⁇ Lc and 2a ⁇ 2c) have the property of inhibiting gap junction formation in HeLa-Cx43 cells.
  • the ability to inhibit gap formation can be changed by changing the olefin moiety of oleamide.
  • fluorescent oleamides in which a fluorescent part and an oleamide part were connected with carbon chains of various lengths were synthesized.
  • Cx43 connexin43
  • the present invention is the first and innovative example in which fluorescence is introduced into oleamide and its analogs without significantly impairing biological activity.
  • the compound of this example is a very excellent compound that can introduce fluorescence into various living cells and can be observed under a microscope.
  • HeLa_Cx43 introduced connexin 43 into HeLa cells Fluorescence could be observed in cancer cells such as HeLa cells and MCF-7 cells, and normal cells such as TIG1 cells. This suggests that the expression of gap bond formation inhibitory activity of oleamide via Cx43 does not directly act on connexin43. In other words, it can be inferred that oleamide changes the fluidity of the membrane taken into the membrane in any cell, thereby changing the interaction between the cell membrane and connexin and inhibiting the formation of gap junctions. By further applying the compound of the present invention, there is a high possibility that the previously unknown actions of oleamide and its derivatives can be discovered.
  • the compound of the present invention can emit fluorescence without significantly impairing the original biological activity by introduction of a fluorescent group. Therefore, the compound of the present invention is useful for a method for observing fluorescence of cells and a tool for studying the intracellular action mechanism of an oleamide derivative. Moreover, the compound of this invention can be used also as a gap coupling
  • the compound of the present invention when the compound of the present invention has a cancer metastasis inhibitory activity, an antitumor activity, etc., a pharmaceutical such as an anticancer agent (anticancer agent), a supplement having an anticancer effect or a cancer preventive effect, a functional food (edible food) It can be used as a raw material for the composition. Furthermore, the use of the compound of the present invention is not limited to these, and can be used for any application.

Abstract

An oleamide can emit fluorescence without largely deteriorating its biological activity by introducing a fluorescent group into the oleamide via a carbon chain, as shown in the formula (I). The fluorescent group (R) may be a dansyl group of the formula (II) or an NBD group of the formula (III). The compound is useful for any application such as a method for fluorescence observation of a cell, a gap binding inhibitor, a tool for the study of the intracellular action mechanism of an oleamide derivative, a pharmaceutical product, a food, a beverage and a supplement.

Description

明 細 書  Specification
蛍光性ォレアミド誘導体とその利用  Fluorescent oleamide derivatives and their use
技術分野  Technical field
[0001] 本発明は、蛍光性ォレアミド誘導体とその利用に関し、例えば、前記蛍光性ォレア ミド誘導体を用いた細胞の蛍光観察方法、および前記蛍光性ォレアミドを含む gap結 合阻害剤、ォレアミド誘導体の細胞内作用メカニズム研究用ツール、医薬品、飲食 品、サプリメントに関する。  [0001] The present invention relates to a fluorescent oleamide derivative and use thereof, for example, a method of observing fluorescence of a cell using the fluorescent oleamide derivative, a gap binding inhibitor containing the fluorescent ureamide, and a cell of an oleamide derivative. It relates to tools for research on internal mechanisms, pharmaceuticals, food and drink, and supplements.
背景技術  Background art
[0002] gap (ギャップ）結合とは、コネキシン（connexin) 6量体のコネクソンが隣接細胞間で 連結したものである。この連結によって形成された導管は細胞膜を貫通し、その導管 内をイオンや低分子量タンパク質が細胞から細胞へと受け渡される。この機構は、上 皮性細胞の増殖の恒常性維持などのために必須であると考えられている。コネキシ ンは、 gap (ギャップ)結合を構成する膜結合タンパク質ファミリーの総称であり、その 分子量の違いによって、 20を超えるサブタイプが発見されている。例えば、分子量が 26kDaのものはコネキシン 26 (connexin26)、 43kDaのものはコネキシン 43 (connexin43 )と称されている。本発明者らは、以前、癌の転移機序を遺伝子レベルで解明する基 礎研究を行っている中で、コネキシン 26が癌転移に密接に関わっていること、より具 体的には、コネキシン 26の機能を抑制することにより癌の転移が抑制されることを見 出して!/、る (下記の特許文献 1および非特許文献 1)。  [0002] A gap bond is a connexin hexamer connexon linked between adjacent cells. The conduit formed by this connection penetrates the cell membrane, and ions and low molecular weight proteins are transferred from cell to cell in the conduit. This mechanism is considered essential for maintaining homeostasis of proliferation of epidermal cells. Connexin is a generic name for a family of membrane-bound proteins that constitute gap junctions, and more than 20 subtypes have been discovered due to differences in molecular weight. For example, a molecular weight of 26 kDa is called connexin 26 (connexin 26), and a 43 kDa molecular weight is called connexin 43. The present inventors have previously conducted basic research to elucidate the mechanism of cancer metastasis at the gene level, and that connexin 26 is closely related to cancer metastasis, more specifically, connexin. It has been found that suppression of the function of 26 suppresses cancer metastasis! (Patent Document 1 and Non-Patent Document 1 below).
[0003] 一方、ォレイン酸のアミド誘導体であるォレアミドは、睡眠回復における睡眠妨害お よび睡眠消失の状態下において、脳脊髄液中に蓄積することが示された内在性の脂 肪酸 1級アミドである (下記の非特許文献 2)。ォレアミドは、腹腔内または静脈内注射 により投与したとき、動物における生理学的睡眠を誘導するなど、様々な作用を有す ることが知られている非常に興味深い活性を有する化合物である。  [0003] On the other hand, oleamide, an amide derivative of oleic acid, is an endogenous fatty acid primary amide that has been shown to accumulate in cerebrospinal fluid in the state of sleep disturbance and sleep loss during sleep recovery. (The following non-patent document 2). Olamide is a compound with very interesting activity known to have various effects, such as inducing physiological sleep in animals when administered by intraperitoneal or intravenous injection.
[0004] ォレアミドおよびその誘導体は、 gap結合を阻害する物質としても知られている（下 記の非特許文献 3)。本発明者らは、ォレアミド誘導体のうち、コネキシン 26を特異的 に阻害し、良好な癌転移抑制活性を示すものを同定し、その 1つについて「MI-18」と 命名した（下記の特許文献 2)。さらに、本発明者らは、多量体型ォレアミド誘導体と、 その癌治療等への利用につ!/、ての研究も行って!/、る（下記の特許文献 3)。 [0004] Olamide and its derivatives are also known as substances that inhibit gap bonding (Non-patent Document 3 below). The present inventors have identified an oleamide derivative that specifically inhibits connexin 26 and exhibits good cancer metastasis-inhibiting activity, and one of them is referred to as “MI-18”. (Patent Document 2 below). Furthermore, the present inventors have also conducted research on multimeric oleamide derivatives and their use in cancer treatment and the like! (Patent Document 3 below).
[0005] ォレアミドの gap結合阻害メカニズム研究としては、例えば、 bulk膜の流動性、膜ータ ンパク質界面における摂動が検討されている（下記の非特許文献 4)。ォレアミドは、 b ulk膜において膜結合タンパク質の高次構造に作用し、 gap結合タンパク質と直接相 互作用する。し力もながら、その作用機序は未だ明らかになっておらず、その細胞へ の分布様式等も全く分かって!/、なかった。  [0005] For studying the mechanism of gap binding inhibition of oleamide, for example, the fluidity of bulk membranes and perturbations at membrane protein interfaces have been studied (Non-Patent Document 4 below). Olamide acts on the higher order structure of membrane-bound proteins in bulk membranes and interacts directly with gap-bound proteins. However, the mechanism of its action has not been clarified yet, and the mode of distribution to the cells has not been understood at all!
[0006] アミドゃペプチドの生物活性メカニズムを研究するには、アミド基 (ペプチド基）に蛍 光基を導入し (蛍光導入法）、蛍光を観察する方法が用いられる。このときに用いられ る蛍光基として代表的なものに、例えば、 Dansyl基、 NBD基等がある。し力、し、ォレア ミドの場合、アミド基部分を変換すると、その生物活性に多大な影響を与え、例えば、 蛍光導入により、 gap結合阻害能は消失してしまう。  [0006] In order to study the biological activity mechanism of an amide peptide, a method of introducing a fluorescent group into the amide group (peptide group) (fluorescence introduction method) and observing fluorescence is used. Typical examples of the fluorescent group used at this time include a Dansyl group and an NBD group. In the case of oleamide, conversion of the amide group part has a great influence on its biological activity. For example, the ability to inhibit gap binding is lost by introducing fluorescence.
[0007] 特許文献 1：特開 2001— 17184号公報  [0007] Patent Document 1: Japanese Patent Laid-Open No. 2001-17184
特許文献 2：国際公開 WO2004/060398号公報  Patent Document 2: International Publication WO2004 / 060398
特許文献 3：国際公開 WO2006/049157号公報  Patent Document 3: International Publication WO2006 / 049157
非特許文献 l : Ito, A. et al. ： J. Clin. Invest. , 105 : 1189— 1197, 2000.  Non-patent literature l: Ito, A. et al .: J. Clin. Invest., 105: 1189— 1197, 2000.
非特許文献 2 : Cravatt et al. Science 1995， 1506-1509; Lerner et al. Proc. Natl. Acad. Sci. USA 1994， 91， 9505—9508; Cravatt et al J. Am. Chem. Soc. 1996， 118， 580-590  Non-Patent Document 2: Cravatt et al. Science 1995, 1506-1509; Lerner et al. Proc. Natl. Acad. Sci. USA 1994, 91, 9505-9508; Cravatt et al J. Am. Chem. Soc. 1996, 118, 580-590
非特許文献 3 : Boger, D.し et al. ： Proc. Nat. Acad. Sci. USA. , 95 : 4810 —4815 . 1998.  Non-Patent Document 3: Boger, D. et al .: Proc. Nat. Acad. Sci. USA., 95: 4810—4815. 1998.
非特許文献 4 : Lars Bastiaase et al. J. Membrane Biol. 1993， 136， 135-145 発明の開示  Non-Patent Document 4: Lars Bastiaase et al. J. Membrane Biol. 1993, 136, 135-145 Disclosure of the Invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0008] したがって、本発明の目的は、ォレアミド本来の生物活性が損なわれていない蛍光 性ォレアミド誘導体を提供することである。 [0008] Accordingly, an object of the present invention is to provide a fluorescent oleamide derivative in which the original biological activity of oleamide is not impaired.
[0009] さらに、本発明の他の目的は、前記蛍光性ォレアミド誘導体を用いた細胞の蛍光 観察方法、および前記蛍光性ォレアミドを含む gap結合阻害剤、ォレアミド誘導体の 細胞内作用メカニズム研究用ツール、医薬品、飲食品、サプリメントを提供することで ある。 [0009] Furthermore, another object of the present invention is to provide a method for observing fluorescence of cells using the fluorescent oleamide derivative, a gap binding inhibitor containing the fluorescent oleamide, and an oleamide derivative. It is to provide tools for studying intracellular action mechanisms, pharmaceuticals, foods and drinks, and supplements.
課題を解決するための手段  Means for solving the problem
[0010] 前述の通り、ォレアミドのアミド基に直接蛍光基を導入すると、ォレアミド本来の生物 活性が大きく損なわれてしまう。本発明者らは、この課題を解決するために鋭意研究 を重ねた。その結果、ォレアミドのアミド基に炭素鎖を結合させ、その炭素鎖を介して 蛍光基を導入することにより、ォレアミド本来の生物活性が大きく損なわれることが防 げることを見出した。 [0010] As described above, when a fluorescent group is directly introduced into the amide group of oleamide, the original biological activity of oleamide is greatly impaired. The present inventors have intensively studied to solve this problem. As a result, it has been found that by binding a carbon chain to the amide group of oleamide and introducing a fluorescent group through the carbon chain, the original biological activity of oleamide can be prevented from being greatly impaired.
[0011] すなわち、本発明の化合物は、下記式 (I)で表される蛍光性ォレアミド誘導体、その 互変異体もしくは立体異性体、またはそれらの塩である。  That is, the compound of the present invention is a fluorescent oleamide derivative represented by the following formula (I), a tautomer or stereoisomer thereof, or a salt thereof.
[化 7]  [Chemical 7]
Figure imgf000006_0001
Figure imgf000006_0001
前記式 (I)中、 In the formula (I),
L1は、単結合、 -0- または -S- であり、 L 1 is a single bond, -0- or -S-
L2は、単結合または直鎖もしくは分枝アルキレン基であり、 L 2 is a single bond or a linear or branched alkylene group,
R1および R2は、それぞれ水素原子または任意の置換基であり、同一でも異なってい ても良ぐ R 1 and R 2 are each a hydrogen atom or an arbitrary substituent, and may be the same or different.
Rは蛍光基である。  R is a fluorescent group.
また、本発明の化合物は、下記式 (IV)で表される、前記式 (I)の蛍光性ォレアミド誘 導体の二量体型化合物、その互変異体もしくは立体異性体、またはそれらの塩であ つても い。 [化 8] Further, the compound of the present invention is a dimer type compound of the fluorescent olamide derivative of the above formula (I) represented by the following formula (IV), a tautomer or stereoisomer thereof, or a salt thereof. Always. [Chemical 8]
Figure imgf000007_0001
Figure imgf000007_0001
(IV) 前記式 (IV)中、 、 L2、 R1, R2および Rは、それぞれ前記式 (I)と同じであり、各 L1は同 一でも異なっていても良ぐ各 R1は同一でも異なっていても良ぐ (IV) In the formula (IV), L 2 , R 1 , R 2 and R are the same as those in the formula (I), and each L 1 may be the same or different from each other R 1 Can be the same or different
R3は水素原子または任意の置換基であり、 R 3 is a hydrogen atom or an optional substituent,
1は正の整数である。  1 is a positive integer.
さらに、本発明の化合物は、下記式 (V)で表される、前記式 (I)の蛍光性ォレアミド誘 導体の多量体型化合物、その互変異体もしくは立体異性体、またはそれらの塩であ つても良い。  Furthermore, the compound of the present invention is a multimeric compound of the fluorescent oleamide derivative of the formula (I) represented by the following formula (V), a tautomer or stereoisomer thereof, or a salt thereof. Also good.
[化 9] [Chemical 9]
Figure imgf000008_0001
Figure imgf000008_0001
前記式 (V)中、 L、 L、 Rおよび Rは、それぞれ前記式 (I)と同じであり、各 Lは同一で も異なっていても良ぐ In the formula (V), L, L, R and R are the same as the formula (I), and each L may be the same or different.
R4は水素原子または任意の置換基であり、 R 4 is a hydrogen atom or an optional substituent,
m、 hおよび kはそれぞれ正の整数である。  m, h and k are each a positive integer.
[0014] また、本発明による細胞の蛍光観察方法は、前記式 (1)、（IV)もしくは (V)で表される 蛍光性ォレアミド誘導体、その互変異体もしくは立体異性体、またはそれらの塩を細 胞に導入する工程を含む。 [0014] In addition, the method for observing fluorescence of cells according to the present invention includes a fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof. A step of introducing the cell into the cell.
[0015] さらに、本発明の製品は、前記式 (1)、（IV)もしくは (V)で表される蛍光性ォレアミド誘 導体、その互変異体もしくは立体異性体、またはそれらの塩を含み、 gap結合阻害剤 、ォレアミド誘導体の細胞内作用メカニズム研究用ツール、医薬品、飲食品またはサ プリメントとして用いられる。 [0015] Further, the product of the present invention comprises a fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof, It is used as a gap binding inhibitor, a tool for studying the intracellular action mechanism of an oleamide derivative, a pharmaceutical, a food or drink, or a supplement.
発明の効果  The invention's effect
[0016] 前記式 (1)、（IV)もしくは (V)で表される蛍光性ォレアミド誘導体は、蛍光基の導入に より本来の生物活性が大きく損なわれることがなぐ蛍光を発することができる。このた め、本発明の化合物すなわち前記式 (1)、（IV)もしくは (V)で表される蛍光性ォレアミド 誘導体、その互変異体もしくは立体異性体、またはそれらの塩は、細胞の蛍光観察 方法、およびォレアミド誘導体の細胞内作用メカニズム研究用ツールに有用である。 また、本発明の化合物は、その生物活性により、 gap結合阻害剤、医薬品、飲食品ま たはサプリメントとしても用いること力 Sできる。さらに、本発明の化合物の用途はこれら に限定されず、あらゆる用途に使用可能である。 [0016] The fluorescent oleamide derivative represented by the above formula (1), (IV) or (V) can emit fluorescence without significantly impairing the original biological activity by introduction of the fluorescent group. others Therefore, the compound of the present invention, that is, the fluorescent oleamide derivative represented by the formula (1), (IV) or (V), a tautomer or stereoisomer thereof, or a salt thereof, is a method for observing fluorescence of a cell, It is useful as a tool for studying the intracellular action mechanism of oleamide derivatives. In addition, the compound of the present invention can be used as a gap binding inhibitor, a pharmaceutical, a food or drink, or a supplement due to its biological activity. Furthermore, the use of the compound of the present invention is not limited to these, and can be used for all uses.
図面の簡単な説明 Brief Description of Drawings
[図 1]色素 トランスファーアツセィにおける各被検物質 (ォレアミド、 Dansyl体（化合 物 la〜lc)および比較例の Dansyl oleamide)の GJICスコアを示すグラフである。  FIG. 1 is a graph showing the GJIC score of each test substance (oleamide, Dansyl compound (compounds la to lc), and Dansyl oleamide in a comparative example) in dye transfer assembly.
[図 2]各被検物質 (ォレアミド、 Dansyl体（化合物 la〜lc) )による前記色素 トランスフ ァーアツセィ後の状態を示す図であり、図 2a)は対照（control)の図であり、図 2b)はォ レアミドの図であり、図 2c)は化合物 lc (C10 linked dansyl oleamide)の図である。 原図では、色素が移入された細胞は緑色で表示される。 [Fig. 2] Fig. 2a is a diagram showing the state after the dye transfer assembly with each test substance (olamide, Dansyl form (compounds la to lc)), Fig. 2a) is a control diagram, Fig. 2b) Fig. 2c) is a diagram of the compound lc (C10 linked dansyl oleamide). In the original image, cells that have been dyed are displayed in green.
[図 3]NBD基を有するォレアミド誘導体（化合物 2a〜2cおよび 4c)を HeLa_Cx43細胞 または HeLa細胞に導入した結果を示す図である。図 3a)および図 3b)は、化合物 2a〜 2cおよび 4cを HeLa_Cx43細胞に導入した結果を示し、図 3c)および図 3d)は、 HeLa細 胞に導入した結果を示す。また、図 3a)および図 3c)は、 DICによる測定結果を示し、 図 3b)および図 3d)は、 Cy2-filterを用いた測定結果を示す。原図では、 b)、 d)の蛍光 箇所は緑色で表示される。  FIG. 3 is a diagram showing the results of introducing an oleamide derivative having an NBD group (compounds 2a to 2c and 4c) into HeLa_Cx43 cells or HeLa cells. Figures 3a) and 3b) show the results of introducing compounds 2a-2c and 4c into HeLa_Cx43 cells, and Figures 3c) and 3d) show the results of introduction into HeLa cells. Figures 3a) and 3c) show the measurement results by DIC, and Figures 3b) and 3d) show the measurement results using Cy2-filter. In the original drawing, the fluorescent spots in b) and d) are displayed in green.
[図 4]ダンシル基を有するォレアミド誘導体（化合物 la〜lcおよび 3c)を HeLa_Cx43細 胞または HeLa細胞に導入した結果を示す図である。図 4a)および図 4b)は、化合物 la 〜lcおよび 3cを HeLa_Cx43細胞に導入した結果を示し、図 4c)および図 4d)は、 HeLa 細胞に導入した結果を示す。また、図 4a)および図 4c)は、 DICによる測定結果を示し 、図 4b)および図 4d)は、 DAH-filterを用いた測定結果を示す。  FIG. 4 is a graph showing the results of introducing oleamide derivatives having dansyl groups (compounds la to lc and 3c) into HeLa_Cx43 cells or HeLa cells. FIGS. 4a) and 4b) show the results of introducing compounds la to lc and 3c into HeLa_Cx43 cells, and FIGS. 4c) and 4d) show the results of introduction into HeLa cells. Figures 4a) and 4c) show the measurement results by DIC, and Figures 4b) and 4d) show the measurement results using the DAH-filter.
[図 5]Tigl細胞を、本発明の蛍光性ォレアミド誘導体である C10-linked oleamideおよ びミトトラッカーで二重染色した結果を示す図である。 a)は、ローダミン一フィルターを 用いて MitoTracker™ Redで染色した結果 (原図では蛍光箇所は赤色で表示）、 b)は 、 FITC フィルターを用いて C10-linled NBD oleamideの局在を観察した結果（原図 では蛍光箇所は緑色で表示）、 c)は、 a)と b)との重ね合わせ (Merge)の結果 (原図で は Mergeの箇所は黄色で表示）、 d)は、 DIC—フィルターを用いた観察結果である。 FIG. 5 shows the results of double staining of Tigl cells with C10-linked oleamide, which is a fluorescent oleamide derivative of the present invention, and mitotracker. a) is the result of staining with MitoTracker ™ Red using a rhodamine filter (the fluorescent part is shown in red in the original figure), and b) is the result of observing the localization of C10-linled NBD oleamide using the FITC filter ( Original drawing In Fig. 1, the fluorescent part is displayed in green), c) is the result of the merge of a) and b) (Merge) (the Merge part is displayed in yellow in the original figure), and d) is a DIC-filter. It is an observation result.
[図 6]Tigl細胞を、本発明の蛍光性ォレアミド誘導体である C10-linked oleamideおよ び FM™ 4-64で二重染色した結果を示す図である。 a)は、 FM™ 4_64で染色した結果 (原図では蛍光箇所は赤色で表示）、 b)は、 C10-linled NBD oleamideの局在を観察 した結果 (原図では蛍光箇所は緑色で表示）、 c)は、 a)と b)との重ね合わせ (Merge) の結果（原図では Mergeの箇所は黄色で表示）、 d)は、 DIC—フィルターを用いた観 察結果である。バーの長さは 10 μ m。  FIG. 6 shows the results of double staining of Tigl cells with C10-linked oleamide and FM ™ 4-64, which are fluorescent oleamide derivatives of the present invention. a) is the result of staining with FM ™ 4_64 (fluorescent sites are displayed in red in the original image), b) is the result of observing the localization of C10-linled NBD oleamide (in the original image, the fluorescent sites are displayed in green), c ) Is the result of the merge of a) and b) (Merge is displayed in yellow in the original figure), and d) is the observation result using the DIC-filter. The bar length is 10 μm.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 以下、本発明の好ましい実施形態について説明する力 本発明は、下記の実施形 態によって何ら限定されるものではなぐその要旨を変更することなぐ様々に改変し て実施することができるものである。 [0018] In the following, the ability to explain preferred embodiments of the present invention The present invention is not limited in any way by the following embodiments, and can be implemented with various modifications without changing its gist. It is.
[0019] まず、本発明の化合物の好ましい形態について説明する。 [0019] First, preferred forms of the compound of the present invention will be described.
前記式 (1)、（IV)または (V)中、 L2は、例えば、単結合または炭素数;!〜 12の直鎖もし くは分枝アルキレン基であることが好ましぐ L2が、単結合、メチレン基または炭素数 2Formula (1), in (IV) or (V), L 2 is, for example, a single bond or a carbon atoms;! Ku linear if the - 12 there is preferably fixture L 2 be branched alkylene group , Single bond, methylene group or 2 carbon atoms
〜8のポリメチレン基であることがより好まし!/、。 More preferred is a polymethylene group of ~ 8!
[0020] 前記式 (1)、（IV)または (V)中、 Rは特に限定されず、どのような蛍光基であっても良い[0020] In the formula (1), (IV) or (V), R is not particularly limited and may be any fluorescent group.
1S 例えば、下記式 (Π)で表されるダンシル基、下記式 (III)で表される NBD基、または ナフタリミド等の蛍光基であることが好ましい。例えば、励起波長が異なる蛍光基を目 的に応じて使い分けると、細胞の蛍光観察方法、細胞内作用メカニズム研究用ツー ル等に用いる際により好適である。 1S For example, a dansyl group represented by the following formula (Π), an NBD group represented by the following formula (III), or a fluorescent group such as naphthalimide is preferable. For example, using fluorescent groups with different excitation wavelengths according to the purpose is more suitable for use in cell fluorescence observation methods, intracellular action mechanism research tools, and the like.
Figure imgf000011_0001
Figure imgf000011_0001
[0021] 前記式 (1)、（IV)または (V)中、 R1および R2は、前述の通り水素原子または任意の置換 基であるが、例えば、それぞれ水素原子または直鎖もしくは分枝アルキル基であるこ とが好ましい。また、前記式 (I)中、 R1および R2が、それぞれ水素原子または炭素数 1 〜6の直鎖もしくは分枝アルキル基であることがより好ましい。 [0021] In the formula (1), (IV) or (V), R 1 and R 2 are a hydrogen atom or an arbitrary substituent as described above. An alkyl group is preferred. In the formula (I), R 1 and R 2 are more preferably a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.
[0022] 前記式 (IV)中の R3および前記式 (V)中の R4は、前述の通り水素原子または任意の置 換基であるが、例えば、それぞれ水素原子または直鎖もしくは分枝アルキル基であつ ても良く、前記アルキル基の炭素数は、例えば 1〜6が好ましい。また、前記 R3または R4は、下記式 (K)で表されるようにもう一つの蛍光基 Rを有していても良い。この場合 において、下記式 (IX)中の L2、 R2および Rの定義は、前述と同じであり、前記式 (IV)ま たは (V)中、各 L2は同一でも異なっていても良ぐ各 R2は同一でも異なっていても良く 、各 Rは同一でも異なっていても良い。 [0022] R 3 in the above formula (IV) and R 4 in the above formula (V) are a hydrogen atom or an arbitrary substituent as described above. The alkyl group may be an alkyl group, and the alkyl group preferably has 1 to 6 carbon atoms, for example. R 3 or R 4 may have another fluorescent group R as represented by the following formula (K). In this case, the definitions of L 2 , R 2 and R in the following formula (IX) are the same as described above, and in the formula (IV) or (V), each L 2 is the same or different. Each R 2 may be the same or different, and each R may be the same or different.
[化 11]
Figure imgf000011_0002
[Chemical 11]
Figure imgf000011_0002
(IX) さらに、前記式 (IV)中、 1は、特に限定されないが、例えば 3〜8、好ましくは 3〜5、 特に好ましくは 3である。前記式 (V)中、 mは、特に限定されないが、例えば 3〜8、好 ましくは 3〜5、特に好ましくは 3である。 hは、特に限定されないが、例えば 0〜2、好 ましくは 0〜； 1、特に好ましくは 1である。 kは、特に限定されないが、例えば 3〜4、好 ましくは 3である。 (IX) Further, in the formula (IV), 1 is not particularly limited, but is, for example, 3 to 8, preferably 3 to 5, particularly preferably 3. In the formula (V), m is not particularly limited, but is, for example, 3 to 8, preferably 3 to 5, and particularly preferably 3. Although h is not particularly limited, it is, for example, 0 to 2, preferably 0 to; 1, particularly preferably 1. k is not particularly limited, but is preferably 3 to 4, for example. Preferably it is 3.
[0024] なお、本発明で「ハロゲン」とは、任意のハロゲン元素を指すが、例えば、フッ素、塩 素、臭素およびヨウ素が挙げられる。また、アルキル基としては、特に限定されないが 、例えば、メチル基、ェチル基、 n-プロピル基、イソプロピル基、 n-ブチル基、イソブ チル基、 sec-ブチル基および tert-ブチル基等が挙げられる。アルキレン基としては、 例えば、エチレン基、トリメチレン基、テトラメチレン基、ペンタメチレン基、へキサメチ レン基、ヘプタメチレン基、オタタメチレン基等のポリメチレン基が挙げられる。  In the present invention, “halogen” refers to any halogen element, and examples thereof include fluorine, chlorine, bromine and iodine. The alkyl group is not particularly limited, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. . Examples of the alkylene group include polymethylene groups such as ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, and otatamethylene group.
[0025] また、前記式 0)、（IV)または (V)により表される化合物またはその塩力 互変異性体 または立体異性体（例：幾何異性体および配座異性体）を有するときは、それらの分 離された各異性体および混合物も本発明の範囲に含まれる。そして、前記式 (1)、 (IV) または (V)で表される化合物またはその塩が、その構造に不斉炭素を有するときは、 それらの光学活性体およびラセミ混合物も本発明の範囲に含まれる。さらに、前記式 (1)、（IV)または (V)により表される化合物またはその塩は、例えば、適宜な溶媒から再 結晶する等の方法により、結晶化させて用いることもできる。  [0025] In addition, when it has a compound represented by the above formula 0), (IV) or (V), or its salt power tautomer or stereoisomer (eg, geometric isomer and conformer) These separated isomers and mixtures are also included in the scope of the present invention. When the compound represented by the formula (1), (IV) or (V) or a salt thereof has an asymmetric carbon in its structure, those optically active substances and racemic mixtures are also within the scope of the present invention. included. Furthermore, the compound represented by the formula (1), (IV) or (V) or a salt thereof can be used after being crystallized, for example, by recrystallization from an appropriate solvent.
[0026] 前記式 (1)、（IV)または (V)により表される化合物の塩は、酸付加塩でも塩基付加塩で も良い。前記酸付加塩を形成する酸は、無機酸でも有機酸でも良い。無機酸として は、特に限定されないが、例えば、硫酸、リン酸、塩酸、臭化水素酸、ヨウ化水素酸 等が可能である。有機酸も特に限定されないが、例えば、 p—トルエンスルホン酸、メ タンスルホン酸、シユウ酸、 p—ブロモベンゼンスルホン酸、炭酸、コハク酸、クェン酸 、安息香酸、酢酸等が可能である。前記塩基付加塩を形成する塩基は、無機塩基で も有機塩基でも良い。無機塩基としては、特に限定されないが、例えば、水酸化アン モニゥム、アルカリ金属水酸化物、アルカリ土類金属水酸化物、炭酸塩、炭酸水素塩 等が可能であり、より具体的には、例えば、水酸化ナトリウム、水酸化カリウム、炭酸力 リウム、炭酸ナトリウム、炭酸水素ナトリウム、炭酸水素カリウム、水酸化カルシウム、炭 酸カルシウム等が可能である。有機塩基も特に限定されないが、例えば、エタノール ァミン、トリェチルァミン、トリス（ヒドロキシメチル）ァミノメタン等が可能である。前記式 ( 1)、（IV)または (V)で表される化合物の塩の製造方法も特に限定されず、例えば、前記 式 0)、（IV)または (V)で表される化合物に、前記のような酸や塩基を公知の方法により 適宜付加させる等の方法で製造することができる。 [0026] The salt of the compound represented by the formula (1), (IV) or (V) may be an acid addition salt or a base addition salt. The acid forming the acid addition salt may be an inorganic acid or an organic acid. The inorganic acid is not particularly limited, and examples thereof include sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid and the like. The organic acid is not particularly limited, and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citrate, benzoic acid, and acetic acid. The base forming the base addition salt may be an inorganic base or an organic base. Examples of the inorganic base include, but are not limited to, ammonium hydroxide, alkali metal hydroxide, alkaline earth metal hydroxide, carbonate, bicarbonate, and the like. More specifically, for example, Sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like are possible. The organic base is not particularly limited, and for example, ethanolamine, triethylamine, tris (hydroxymethyl) aminomethane and the like are possible. A method for producing a salt of the compound represented by the formula (1), (IV) or (V) is not particularly limited. For example, the compound represented by the formula 0), (IV) or (V) Acids and bases such as those described above can be obtained by known methods. It can be produced by a method such as adding appropriately.
本発明の化合物中、生物活性等の観点から特に好ましいのは、例えば、下記式 la 〜lc、 2a〜2c、 3a〜3cおよび 4a〜4cのうちいずれかで表される蛍光性ォレアミド誘導 体、その互変異体もしくは立体異性体、またはそれらの塩である。 Among the compounds of the present invention, particularly preferred from the viewpoint of biological activity and the like, for example, a fluorescent oleamide derivative represented by any one of the following formulas la to lc, 2a to 2c, 3a to 3c, and 4a to 4c, It is a tautomer or stereoisomer, or a salt thereof.
Figure imgf000014_0001
Figure imgf000014_0001
1a (C2-linked dansyl oleamide, n = 0)  1a (C2-linked dansyl oleamide, n = 0)
1b (C4-linked dansyl oleamide, n = 2)  1b (C4-linked dansyl oleamide, n = 2)
1c (C10-linked dansyl oleamide, n = 8)  1c (C10-linked dansyl oleamide, n = 8)
Figure imgf000014_0002
Figure imgf000014_0002
2a (C2-linked NBD oleamide, n = 0) 2a (C2-linked NBD oleamide, n = 0)
2b (C4-linked NBD oleamide, n = 2)  2b (C4-linked NBD oleamide, n = 2)
2c (C10-linked NBD oleamide, n = 8)  2c (C10-linked NBD oleamide, n = 8)
Figure imgf000014_0003
Figure imgf000014_0003
3a (C2-linked dansyl MI-18, n = 0)  3a (C2-linked dansyl MI-18, n = 0)
3b (C4-linked dansyl MI-18, n = 2)  3b (C4-linked dansyl MI-18, n = 2)
3c (C10-linked dansyl MI-18, n = 8)  3c (C10-linked dansyl MI-18, n = 8)
Figure imgf000014_0004
Figure imgf000014_0004
4a (C2-linked NBD MI-18, n = 0)  4a (C2-linked NBD MI-18, n = 0)
4b (C4-linked NBD MI-18, n = 2)  4b (C4-linked NBD MI-18, n = 2)
4c (C10-linked NBD MI-18, n = 8)  4c (C10-linked NBD MI-18, n = 8)
[0028] 次に、本発明の化合物の製造方法について説明する。 [0028] Next, a method for producing the compound of the present invention will be described.
[0029] 本発明の化合物はどのような方法によって製造しても良ぐ例えば化学的に合成し ても良いし、もし天然に存在することがわかれば、天然物から単離 ·精製しても良い。 あるいは、天然物から得られた物質を原材料として反応等の処理を施し、本発明の 化合物を製造しても良い。また、必要であれば、微生物などの生物材料を使用して 本発明の化合物を生産しても良い。本発明の化合物を化学的合成により製造する場 合、その方法は特に限定されないが、例えば、下記式 (νι)、（νπ)および (vm)の化合 物を原料として用い、ァミンとハロゲン化物との縮合反応工程を含む本発明の製造方 法により製造することが好ましレ、。 [0029] The compound of the present invention may be produced by any method, for example, chemically synthesized, or if it is found that it exists in nature, it may be isolated and purified from a natural product. good. Alternatively, a substance obtained from a natural product is used as a raw material and subjected to a treatment such as a reaction. A compound may be produced. If necessary, the compound of the present invention may be produced using a biological material such as a microorganism. When the compound of the present invention is produced by chemical synthesis, the method is not particularly limited. For example, a compound of the following formulas (νι), (νπ) and (vm) is used as a raw material, and an amine and a halide are synthesized. It is preferable to produce by the production method of the present invention including the condensation reaction step.
[化 13]  [Chemical 13]
Figure imgf000015_0001
Figure imgf000015_0001
(VI) (VII) (VIII) 前記式 (VI)、（VII)および (vm)中、
Figure imgf000015_0002
じ、 R1, R2および Rは、それぞれ前記式 0)、 (IV) または (V)と同じであり、 X1および X2は、それぞれハロゲンであり、同一でも異なってい ても良い。 (VI) (VII) (VIII) In the above formulas (VI), (VII) and (vm),
Figure imgf000015_0002
R 1 , R 2 and R are the same as those in the above formula 0), (IV) or (V), and X 1 and X 2 are each halogen and may be the same or different.
[0030] 前記本発明の製造方法によれば、本発明の化合物を、市販の原料から簡便に合 成することも可能である。反応条件は特に限定されず、例えば、ァミンとハロゲン化物 との縮合反応は、同様の反応における公知の反応条件を適宜応用することも可能で ある。前記縮合反応における反応溶媒は、特に限定されないが、例えば、ジェチル エーテル、テトラヒドロフラン、ジォキサン、ジメトキシェタン、 t ブチルメチルエーテ ル等のエーテル、ジクロロメタン、クロ口ホルム等のハロゲン化溶媒、またはジメチルホ ルムアミド、ジメチルスルホキシド、ァセトニトリル、アセトン、トルエン、ベンゼン等が挙 げられる。反応温度および反応時間も特に限定されず、例えば、反応させる化合物 および溶媒に応じて適宜選択することができる。前記反応温度は、例えば 0〜； 130°C 、好ましくは 0〜30°C、特に好ましくは 0〜25°Cである。前記反応時間は、例えば；!〜 24時間、好ましくは 3〜； 12時間、特に好ましくは 6〜； 12時間である。  [0030] According to the production method of the present invention, the compound of the present invention can be easily synthesized from commercially available raw materials. The reaction conditions are not particularly limited. For example, for the condensation reaction between an amine and a halide, known reaction conditions in the same reaction can be appropriately applied. The reaction solvent in the condensation reaction is not particularly limited, and examples thereof include ethers such as jetyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and t-butylmethyl ether, halogenated solvents such as dichloromethane and chloroform, or dimethylformamide, Examples include dimethyl sulfoxide, acetonitrile, acetone, toluene, and benzene. The reaction temperature and reaction time are also not particularly limited, and can be appropriately selected according to, for example, the compound to be reacted and the solvent. The reaction temperature is, for example, 0 to 130 ° C, preferably 0 to 30 ° C, particularly preferably 0 to 25 ° C. The reaction time is, for example, !! to 24 hours, preferably 3 to 12 hours, particularly preferably 6 to 12 hours.
[0031] 前記式 (I)の化合物は、例えば、下記スキーム 1ないし 4に従って合成することができ る。なお、下記式 (Γ )は、前記式 (I)において L1が単結合の場合を示す。 [化 14] [0031] The compound of the formula (I) can be synthesized, for example, according to the following schemes 1 to 4. The following formula (Γ) shows the case where L 1 is a single bond in the formula (I). [Chemical 14]
R1HNへ L2へ NHR2 R 1 To HN To L 2 NHR 2
Figure imgf000016_0001
Figure imgf000016_0001
H,H,
Figure imgf000016_0002
Figure imgf000016_0002
(1，） スキーム 1 (1,) Scheme 1
[化 15]
Figure imgf000016_0003
[Chemical 15]
Figure imgf000016_0003
Figure imgf000016_0004
Figure imgf000016_0004
(1，） ム 2 O 1 O (1,) Mu 2 O 1 O
H3C、 H 3 C,
OCH3 H 、 OCH 3 H,
■OCH  ■ OCH
Figure imgf000017_0001
Figure imgf000017_0002
スキーム 3
Figure imgf000017_0001
Figure imgf000017_0002
Scheme 3
[化 17]
Figure imgf000017_0003
Figure imgf000017_0004
[Chemical 17]
Figure imgf000017_0003
Figure imgf000017_0004
(I)  (I)
スキーム 4 また、前記式 (IV)および (V)の化合物の製造方法は特に限定されないが、例えば、 国際公開 WO2006/049157号公報（前記特許文献 3)に記載されている多量体型ォ レアミド誘導体の製造方法に、ァミンとハロゲン化物との縮合反応を組み合わせて製 造すること力 Sできる。例えば、多量体型ォレアミド誘導体を製造した後に、ァミンとハロ ゲン化物との縮合反応により蛍光基を導入しても良いし、蛍光性ォレアミドを製造し た後に、多量体型ォレアミドに変換しても良い。 Scheme 4 In addition, the method for producing the compounds of the formulas (IV) and (V) is not particularly limited. It is possible to produce a multimer-type oleamide derivative described in International Publication No. WO2006 / 049157 (Patent Document 3) combined with a condensation reaction between an amine and a halide. For example, after producing a multimeric oleamide derivative, a fluorescent group may be introduced by a condensation reaction between an amine and a halide, or after producing a fluorescent ureamide, it may be converted into a multimeric oleamide.
[0033] 次に、本発明の化合物の用途について説明する。 [0033] Next, uses of the compound of the present invention will be described.
[0034] 前述の通り、本発明の化合物は、細胞の蛍光観察方法、およびォレアミド誘導体の 細胞内作用メカニズム研究用ツールに有用である。また、本発明の化合物は、その 生物活性により、 gap結合阻害剤、医薬品、飲食品またはサプリメントとしても用いるこ とができる。例えば、本発明の化合物が癌の転移抑制活性、抗腫瘍活性等を有する 場合は、抗癌剤 (制癌剤）などの医薬、さらには抗癌作用または癌予防効果をもつサ プリメント、機能性食品（食用組成物）の原材料などに利用可能である。さらに、本発 明の化合物の用途はこれらに限定されず、あらゆる用途に使用可能である。  [0034] As described above, the compound of the present invention is useful for a method for observing fluorescence of a cell and a tool for studying the intracellular action mechanism of an oleamide derivative. The compounds of the present invention can also be used as gap binding inhibitors, pharmaceuticals, foods and drinks or supplements due to their biological activity. For example, when the compound of the present invention has a cancer metastasis inhibitory activity, an antitumor activity, etc., a pharmaceutical such as an anticancer agent (anticancer agent), a supplement having an anticancer activity or a cancer preventing effect, a functional food (edible composition) It can be used as a raw material. Furthermore, the use of the compound of the present invention is not limited to these, and can be used for any application.
[0035] 本発明の化合物を医薬開発に利用する場合、その一態様として、本発明の化合物 を医薬品開発過程におけるリード化合物として利用するものであってもよい。  [0035] When the compound of the present invention is used for drug development, as one aspect thereof, the compound of the present invention may be used as a lead compound in the drug development process.
[0036] 本発明の化合物を医薬品（医薬用組成物）に用いる場合の一例について説明する 。本発明の化合物は、これをそのまま、あるいは慣用の医薬製剤担体とともに医薬用 組成物となし、ヒトほたは動物）に投与することができる。医薬用組成物の剤形として は特に制限されるものではなく必要に応じて適宜選択すればよいが、例えば、錠剤、 カプセル剤、顆粒剤、細粒剤、散剤等の経口剤、注射剤、坐剤、塗布剤等の非経口 剤が挙げられる。  [0036] An example of using the compound of the present invention for a pharmaceutical product (pharmaceutical composition) will be described. The compound of the present invention can be administered to humans or animals) as it is or as a pharmaceutical composition together with a conventional pharmaceutical carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be appropriately selected as necessary. For example, tablets, capsules, granules, fine granules, powders and other oral preparations, injections, Examples include parenteral preparations such as suppositories and coating agents.
[0037] 錠剤、カプセル剤、顆粒剤、細粒剤、散剤等の経口剤は、例えば、デンプン、乳糖 、白糖、トレノヽロース、マンニット、力ノレボキシメチノレセノレロース、コーンスターチ、無機 塩類等を用いて常法に従って製造される。これらの製剤中の本発明の化合物の配合 量は特に限定されるものではなく適宜設定できる。この種の製剤には、結合剤、崩壊 剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を適宜に使用す ること力 Sでさる。  [0037] Oral preparations such as tablets, capsules, granules, fine granules, powders and the like are, for example, starch, lactose, sucrose, trenorose, mannitol, force noboxymethylenorescenellose, corn starch, inorganic salts Etc. are manufactured according to a conventional method. The compounding amount of the compound of the present invention in these preparations is not particularly limited and can be appropriately set. For this type of preparation, the ability to use binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances, etc. as appropriate is reduced.
[0038] 非経口剤の場合、患者の年齢、体重、疾患の程度などに応じて用量を調節し、例 えば、静注、点滴静注、皮下注射、筋肉注射などによって投与する。この非経口剤は 常法に従って製造され、希釈剤として一般に注射用蒸留水、生理食塩水等を用いる ことができる。さらに必要に応じて、殺菌剤、防腐剤、安定剤を加えてもよい。また、こ の非経口剤は安定性の点から、バイアル等に充填後冷凍し、通常の凍結乾燥処理 により水分を除き、使用直前に凍結乾燥物から液剤を再調製することもできる。さらに 必要に応じて、等張化剤、安定剤、防腐剤、無痛化剤を加えてもよい。これら製剤中 の本発明の化合物の配合量は特に限定されるものではなく任意に設定できる。その 他の非経口剤の例として、外用液剤、軟膏等の塗布剤、直腸内投与のための坐剤 等が挙げられ、これらも常法に従って製造される。 [0038] In the case of parenteral agents, the dosage is adjusted according to the patient's age, weight, degree of disease, etc. For example, it is administered by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection or the like. This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent. Furthermore, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and the liquid preparation can be re-prepared from the freeze-dried product immediately before use. Furthermore, an isotonic agent, stabilizer, preservative, and soothing agent may be added as necessary. The compounding amount of the compound of the present invention in these preparations is not particularly limited and can be arbitrarily set. Examples of other parenteral preparations include coating solutions for external use, ointments and the like, suppositories for rectal administration, etc., and these are also produced according to conventional methods.
[0039] なお、公知の DDS (ドラッグ ·デリバリー ·システム）を利用し、例えば、本発明の化合 物をリボソームなどの運搬体に封入して体内投与してもよ!/、。このとき標的部位 (癌細 胞等）を特異的に認識する運搬体などを利用すれば、標的部位に本発明の化合物 を効率よく運ぶことができ効果的である。  [0039] It should be noted that a known DDS (drug delivery system) may be used, for example, the compound of the present invention may be encapsulated in a carrier such as ribosome and administered in the body! /. At this time, if a carrier that specifically recognizes the target site (cancer cell or the like) is used, the compound of the present invention can be efficiently transported to the target site.
[0040] また前述したように、本発明の化合物は、サプリメント、機能性食品などの飲食品（ 食用組成物）に利用することができる。すなわち、各種飲料や各種加工食品の原材 料として本発明の化合物を飲食品に添加したり、必要に応じてデキストリン、乳糖、澱 粉等の賦形剤や香料、色素等とともにペレット、錠剤、顆粒等に加工したり、またゼラ チン等で被覆してカプセルに成形加工して健康食品や保健食品等として利用できる [0040] As described above, the compound of the present invention can be used in foods and drinks (edible compositions) such as supplements and functional foods. That is, the compound of the present invention is added to foods and drinks as raw materials for various beverages and various processed foods, and if necessary, pellets, tablets, excipients such as dextrin, lactose, starch, flavorings, pigments, etc. Can be processed into granules, etc., or coated with gelatin and molded into capsules for use as health food or health food
〇 Yes
実施例  Example
[0041] 次に、本発明の実施例について説明する。  Next, examples of the present invention will be described.
[0042] (測定条件等）  [0042] (Measurement conditions, etc.)
核磁気共鳴（NMR)スペクトルは、 0〜25°Cで JEOL社製の機器 JNM— EX270型 (2 70MHz, 67·8ΜΗζ)、 JEOL JNM— EX300型（300MHz，75·3MHz) (商品名）（1H 測定時 270もしくは 300MHz)を用いて測定した。ケミカルシフトは百万分率（ppm)で 表している。内部標準 Oppmには、テトラメチルシラン (TMS)を用いた。結合定数 (J) は、ヘルツで示しており、略号 s、 d、 t、 q、 mおよび brは、それぞれ、一重線 (singlet)、 二重線 (doublet)、三重線 (triplet)、四重線 (quartet)、多重線 (multiplet)および広幅線（ broad)を表す。質量分析（MSあるいは LRMS)は、 JEOL社製の機器 JMS— D300型 ( 商品名）を用い、 FAB法により測定した。高分解能質量分析 (HRMS)は、 JEOL社製 の機器 JMS— 600型（商品名）を用い、 FAB法により測定した。カラムクロマトグラフィ 一分離の固定相には、 Fuji Silysia Chemical silicagel BW— 300を用いた。 全ての化学物質は、試薬級であり、 TCI社力、ら NBDクロリド、和光化学力、らォレイン 酸、その他の試薬はアルドリッチ社から購入した。 Nuclear magnetic resonance (NMR) spectra were measured at 0 to 25 ° C by JEOL equipment JNM—EX270 type (2 70MHz, 67 · 8) ζ), JEOL JNM—EX300 type (300MHz, 75.3MHz) (trade name) ( 1 H measurement was performed using 270 or 300 MHz). Chemical shifts are expressed in parts per million (ppm). Tetramethylsilane (TMS) was used as the internal standard Oppm. Coupling constants (J) are shown in hertz, and the abbreviations s, d, t, q, m and br are singlet, doublet, triplet, quadruple, respectively. Quartet, multiplet and wide line ( broad). Mass spectrometry (MS or LRMS) was measured by the FAB method using an instrument JMS-D300 (trade name) manufactured by JEOL. High resolution mass spectrometry (HRMS) was measured by the FAB method using an instrument JMS-600 (trade name) manufactured by JEOL. Column chromatography Fuji Silysia Chemical silicagel BW-300 was used as the stationary phase for one separation. All chemicals were reagent grade, and TCI, et al. NBD chloride, Wako Chemical, laoleic acid, and other reagents were purchased from Aldrich.
(1.合成）  (1. Synthesis)
下記スキーム 5ないし 8により、 Dansyl化ォレアミド誘導体（Dansyl体） la〜lc、 NBD 化ォレアミド誘導体（NBD体） 2a〜2c、蛍光基を導入した MI-18 (蛍光ラベル化 MI-18 ) 3cおよび 4cを合成した。また、比較例として、リンカ一なしの蛍光ォレアミドである下 記 lzのダンシルォレアミド（Dansyl oleamide)を合成した。  According to the following schemes 5 to 8, Dansyl-modified amide derivatives (Dansyl-form) la to lc, NBD-formamide derivatives (NBD-form) 2a to 2c, MI-18 (fluorescence-labeled MI-18) 3c and 4c with a fluorescent group introduced Was synthesized. Further, as a comparative example, the following lz dansyl oleamide, which is a fluorescent oleamide without a linker, was synthesized.
[化 18] [Chemical 18]
Dansyl-CIDansyl-CI
Figure imgf000020_0001
Figure imgf000020_0001
5a (n = 0, 84%)  5a (n = 0, 84%)
5b (n = 2, 90%)  5b (n = 2, 90%)
5c (n = 8, 83%)  5c (n = 8, 83%)
Figure imgf000020_0002
Figure imgf000020_0002
1a; C2-hnked dansyl oleamide (n = 0, 66%)  1a; C2-hnked dansyl oleamide (n = 0, 66%)
1 b; C4-linked dansyl oleamide (n = 2, 91 %)  1 b; C4-linked dansyl oleamide (n = 2, 91%)
1c; C10-linked dansyl oleamide (n = 8, 87%) スキーム 5
Figure imgf000021_0001
1c; C10-linked dansyl oleamide (n = 8, 87%) Scheme 5
Figure imgf000021_0001
6a (n = 0, 34%)  6a (n = 0, 34%)
6b (n = 2, 34%)  6b (n = 2, 34%)
6c (n = 8, 90%)  6c (n = 8, 90%)
Figure imgf000021_0002
Figure imgf000021_0002
2a; C2-linked NBD oleamide (n = 0, 80%)  2a; C2-linked NBD oleamide (n = 0, 80%)
2b; C4-linked NBD oleamide (n = 2, 26%)  2b; C4-linked NBD oleamide (n = 2, 26%)
2c; C10-linked NBD oleamide (n = 8, 20%) スキーム 6  2c; C10-linked NBD oleamide (n = 8, 20%) Scheme 6
[化 20]
Figure imgf000021_0003
Figure imgf000021_0004
[Chemical 20]
Figure imgf000021_0003
Figure imgf000021_0004
Figure imgf000021_0005
Figure imgf000021_0005
3c; C10-Nnked dansyl MI-18 (n = 8)  3c; C10-Nnked dansyl MI-18 (n = 8)
スキーム 7 [化 21] Scheme 7 [Chemical 21]
Figure imgf000022_0001
Figure imgf000022_0001
4c; ClO-linked NBD I-18 (n = 8) スキーム 8  4c; ClO-linked NBD I-18 (n = 8) Scheme 8
[化 22] [Chemical 22]
Figure imgf000022_0002
Figure imgf000022_0002
1 z (dansyl oleamide) 比較例のダンシルォレアミド（Dansyl oleamide)、即ち、 N-(5_dimethylamino)naphth yl-sulfonyl-(Z)-9-octadecenanoylamide (N— (5—ジメチルァミノ）ナフチルスルホニ ルー（Z)— 9ーォクタデカノィルアミド)は、下記に示すように、直接ォレアミドに蛍光 基を導入して合成した。  1 z (dansyl oleamide) Dansyl oleamide of comparative example, ie, N- (5_dimethylamino) naphth yl-sulfonyl- (Z) -9-octadecenanoylamide (N— (5-dimethylamino) naphthylsulfonyl Z) -9-octadecanolamide) was synthesized by directly introducing a fluorescent group into oleamide as shown below.
[化 23]
Figure imgf000022_0003
詳細には、窒素雰囲気下 0。Cで、水素化ナトリウム (60% in oil, 21 mg， 0.53 mmol) のテトラヒドロフラン溶液（2.5 mL)に、ォレインアミド（100 mg， 0.36 mmol)のテトラヒド 口フラン溶液 (1 mL)をゆっくりとキヤ二ユレーシヨンした。室温で 30分撹拌後、 0 °Cで、 ダンシルクロリド（94 mg， 0.36 mmol)をゆっくりとカロえ、室温で 3時間撹拌した。 TLCで 反応終了を確認後、水を加えジクロロメタンで抽出した。得られた有機層を無水硫酸 ナトリウムで乾燥後、減圧濃縮した。得られた粗生成物をシリカゲルクロマトグラフィー (へキサン:酢酸ェチル = 4: 1)を用いて精製し、 Dansyl oleamide (32 mg， 18%)を得た 。以下に、この化合物の機器分析データを示す。 [Chemical 23]
Figure imgf000022_0003
Specifically, 0 under nitrogen atmosphere. C, slowly add tetrahydrofuran solution (1 mL) of oleinamide (100 mg, 0.36 mmol) to tetrahydrofuran solution (2.5 mL) of sodium hydride (60% in oil, 21 mg, 0.53 mmol). did. After stirring at room temperature for 30 minutes, dansyl chloride (94 mg, 0.36 mmol) was slowly added at 0 ° C and stirred at room temperature for 3 hours. After confirming the completion of the reaction by TLC, water was added and extracted with dichloromethane. The obtained organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained crude product was subjected to silica gel chromatography. Purification was performed using (hexane: ethyl acetate = 4: 1) to obtain Dansyl oleamide (32 mg, 18%). The instrumental analysis data of this compound is shown below.
(化合物 lz)  (Compound lz)
:H NMR (CDC1， 300MHz); σ 0.87 (3H， t， J = 6.2 Hz), 1.11—1.62 (10H, m)， 1.91—2 : H NMR (CDC1, 300MHz); σ 0.87 (3H, t, J = 6.2 Hz), 1.11—1.62 (10H, m), 1.91—2
.05 (4H， m)， 2.21 (2H， t， J = 7.5 Hz), 2.90 (6H， s)， 5.29-5.34 (2H， m)， 7.19 (1H， d， J = 7.2 Hz), 7.59 (2H， m)， 8.21 (1H， d， J = 9.0 Hz), 8.50 (2H， m)， 8.62 (1H， d， J = 8 .4 Hz). .05 (4H, m), 2.21 (2H, t, J = 7.5 Hz), 2.90 (6H, s), 5.29-5.34 (2H, m), 7.19 (1H, d, J = 7.2 Hz), 7.59 ( 2H, m), 8.21 (1H, d, J = 9.0 Hz), 8.50 (2H, m), 8.62 (1H, d, J = 8.4 Hz).
[0045] 次に、前記スキーム 5ないし 8についてさらに詳しく説明する。  Next, the schemes 5 to 8 will be described in more detail.
(1 - 1. Dansyl体の合成）  (1-1. Synthesis of Dansyl body)
前記スキーム 5に従い、まずダンシルクロリド（Dansy卜 C1)とポリメチレンジァミンとの 縮合反応により化合物 5a〜5cを合成し、さらに、化合物 5a〜5cとォレイン酸クロリドと の縮合反応により Dansyl体 la〜lcを合成した。以下、この合成についてさらに詳しく 説明する。  In accordance with Scheme 5 above, compounds 5a to 5c were first synthesized by a condensation reaction of dansyl chloride (Dansy 卜 C1) and polymethylenediamine, and then a Dansyl compound la was synthesized by a condensation reaction of compounds 5a to 5c and oleic acid chloride. ~ Lc was synthesized. Hereinafter, this synthesis will be described in more detail.
[0046] 1 -{(5-dimethylamino)naphthyl-sulfonyiamino}- 10-decanamine  [0046] 1-{(5-dimethylamino) naphthyl-sulfonyiamino}-10-decanamine
1 - { (5—ジメチルァミノ）ナフチル一スルホニルァミノ }— 10—デカンァミン (化合物 5 c)  1-{(5-Dimethylamino) naphthyl monosulfonylamino} — 10-decanamine (Compound 5 c)
[0047] 窒素雰囲気下 0°Cで、 1 , 10—デカンジァミン (228mg， 1.5mmol)のジクロロメタン溶 液 (3mL)に、ダンシルクロリド (135mg， 0.5mmol)のジクロロメタン溶液 (2mL)を発熱しな いようにゆっくりと加え、室温で 6時間撹拌した。 TLCで反応終了を確認後、 10質量 %水酸化ナトリウム水溶液を加え、ジクロロメタンで抽出した。得られた有機層を無水 硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗生成物をシリカゲルクロマトダラ フィー (ジクロロメタン:メタノール = 2: 1)を用いて精製し、 1— { (5 ジメチルァミノ）ナフ チルースルホニルァミノ } 10—デカンァミン (168mg， 83%)を得た。以下に、この化 合物の機器分析データを示す。  [0047] Dosyl chloride (135 mg, 0.5 mmol) in dichloromethane (2 mL) is not exothermic to 1,10-decandiamine (228 mg, 1.5 mmol) in dichloromethane (3 mL) at 0 ° C under a nitrogen atmosphere. And then stirred at room temperature for 6 hours. After confirming the completion of the reaction by TLC, a 10 mass% aqueous sodium hydroxide solution was added, and the mixture was extracted with dichloromethane. The obtained organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was purified using silica gel chromatography (dichloromethane: methanol = 2: 1) to give 1-{(5 dimethylamino) naphthylsulfonylsulfonyl} 10-decanamine (168 mg, 83%). It was. The instrumental analysis data for this compound is shown below.
[0048] (化合物 5c)  [0048] (Compound 5c)
'HNMRCCDCl， 270MHz); δ 1·00_1·52(16Η， m)， 2·67(2Η， t， J=6.8Hz), 2.85-2 'HNMRCCDCl, 270MHz); δ 1 · 00_1 · 52 (16Η, m), 2 · 67 (2Η, t, J = 6.8Hz), 2.85-2
• 90(2H， m)， 2.85(6H, s)， 7.20(1H, d， J=7.3 Hz), 7.53(2H, m)， 8.26(2H, m)， 8.54(1H, d， J=8.9Hz). [0049] l-{(5-dimethylamino)naphthyl-sulfonylamino}-4-butanamine • 90 (2H, m), 2.85 (6H, s), 7.20 (1H, d, J = 7.3 Hz), 7.53 (2H, m), 8.26 (2H, m), 8.54 (1H, d, J = 8.9 Hz). [0049] l-{(5-dimethylamino) naphthyl-sulfonylamino} -4-butanamine
1一 { (5—ジメチルァミノ）ナフチルースルホニルァミノ } 5—ブタンァミン (化合物 5b) 1 1 {(5-Dimethylamino) naphthylsulfonylsulfonyl 5- 5-butanamine (Compound 5b)
[0050] 1— { (5 ジメチルァミノ）ナフチル—スルホニルァミノ }— 10 デカンァミンと同様 の手法を用いて、 1 , 4 ブタンジァミン (132mg， 1.5mmol) とダンシルクロリド （135m g， 0.5mmol) から、 1 { (5 ジメチルァミノ）ナフチルースルホニルァミノ } 5—ブ タンアミン （145mg， 90%) を得た。以下に、この化合物の機器分析データを示す。 [0050] 1— {(5 Dimethylamino) naphthyl-sulfonylamamino} — 10 Using the same procedure as for decanamine, 1, 4 butanediamin (132 mg, 1.5 mmol) and dansyl chloride (135 mg, 0.5 mmol), 1 {(5 Dimethylamino) naphthylsulfonylsulfonylamino 5-butanamine (145 mg, 90%) was obtained. The instrumental analysis data of this compound is shown below.
[0051] (化合物 5b)  [0051] (Compound 5b)
:HNMR (CDCl， 270MHz); δ 1.40- 1·52(2Η， m)， 2·65(2Η， t， J=5.4Hz), 2.82 : HNMR (CDCl, 270MHz); δ 1.40- 1 · 52 (2Η, m), 2.65 (2Η, t, J = 5.4Hz), 2.82
-2.9Κ2Η, m)， 2·82(6Η， s)， 7· 17(1Η， d， J=7.3Hz), 7.48- 7·56(2Η， m)， 8.22(1 H， d， J=8.6Hz), 8.33(2H, d， J=8.9Hz), 8.52(1H, d， J=8.6Hz). -2.9Κ2Η, m), 2.82 (6Η, s), 7 · 17 (1Η, d, J = 7.3Hz), 7.48-7 · 56 (2Η, m), 8.22 (1 H, d, J = 8.6Hz), 8.33 (2H, d, J = 8.9Hz), 8.52 (1H, d, J = 8.6Hz).
[0052] l-{(5-dimethylamino)naphthyl-sulfonylamino}-2-ethanamine  [0052] l-{(5-dimethylamino) naphthyl-sulfonylamino} -2-ethanamine
l—{ (5—ジメチルァミノ）ナフチルースルホニルァミノ }ー 2—エタンァミン (化合物 5a) l— {(5-Dimethylamino) naphthylsulfonylsulfonyl}-2-Ethanamine (Compound 5a)
[0053] 1— { (5 ジメチルァミノ）ナフチル—スルホニルァミノ }— 10 デカンァミンと同様 の手法を用いて、 1 , 2 エタンジァミン (90mg， 1.5mmol)とダンシルクロリド (135mg， 0 .5mmol)力、ら、 1— { ( 5 ジメチルァミノ）ナフチル -スルホニルァミノ } 2 ェタンアミ ン (123mg， 84%)を得た。以下に、この化合物の機器分析データを示す。 [0053] 1 — {(5 Dimethylamino) naphthyl-sulfonylamamino} — 10 Using the same procedure as for decanamine, 1,2 ethanediamin (90 mg, 1.5 mmol) and dansyl chloride (135 mg, 0.5 mmol) force, etc. 1-{(5 dimethylamino) naphthyl-sulfonylamino} 2 ethaneamine (123 mg, 84%) was obtained. The instrumental analysis data of this compound is shown below.
[0054] (化合物 5a)  [0054] (Compound 5a)
:HNMR (CDCl， 270MHz); δ 2·72(2Η， t， J=6.5Hz), 2·89- 3·01(2Η， m)， 2.89 : HNMR (CDCl, 270MHz); δ 2 · 72 (2Η, t, J = 6.5Hz), 2 · 89-3 · 01 (2Η, m), 2.89
(6Η， s)， 7·20(1Η， d， J=7.3Hz), 7·55(2Η， m)， 8·28(2Η， m)， 8·54(1Η， d， J=8. 6Hz). (6Η, s), 7 · 20 (1Η, d, J = 7.3Hz), 7 · 55 (2Η, m), 8 · 28 (2Η, m), 8 · 54 (1Η, d, J = 8. 6Hz).
[0055] 1 -{(5-dimethylamino)naphthyl-sulfonylamino}- 10-{(Z)-9-octadecenanoylamino}-deca ne  [0055] 1-{(5-dimethylamino) naphthyl-sulfonylamino} -10-{(Z) -9-octadecenanoylamino} -deca ne
l—{ (5 ジメチルァミノ）ナフチルースルホニルァミノ }— 10— { (Z)—9 ォクタデカ ノィルアミノ}デカン（化合物 lc ; C10-linked dansyl oleamide)  l— {(5 dimethylamino) naphthylsulfonylsulfonyl} — 10— {(Z) —9 Octadecanolamino} decane (compound lc; C10-linked dansyl oleamide)
[0056] 窒素雰囲気下、ォレイン酸 (25mg， 0.089mmol)の無水メチレンクロリド溶液 (1.7mL) に氷冷下、ォキザリルクロリド (32 ,1 L， 0.36mmol)を滴下して、室温で 4時間攪拌した。 溶媒を減圧留去した後、ジクロロメタン (lmL)に溶解し、氷冷した 1—{ (5 ジメチルァ ミノ）ナフチル スルホニルァミノ } - 10 -デカンァミン (30mg， 0.074mmol)のジクロ口 メタン溶液 (0.5mL)にゆっくり加えて、室温で 3時間攪拌した。 TLCで反応終了を確認 後、ジクロロメタンで抽出した。得られた有機層を飽和食塩水で洗浄し、無水硫酸ナト リウムで乾燥後、減圧濃縮した。得られた粗生成物をシリカゲルクロマトグラフィー (へ キサン:酢酸ェチル = 1 : 1)を用いて精製し、 1 - { (5 ジメチルァミノ）ナフチル—スル ホニルアミノ}— 10— { (Z)— 9 ォクタデカノィルァミノ }デカン (42mg， 87%)を得た。 以下に、この化合物の機器分析データを示す。 [0056] Under a nitrogen atmosphere, oxalyl chloride (32, 1 L, 0.36 mmol) was added dropwise to a solution of oleic acid (25 mg, 0.089 mmol) in anhydrous methylene chloride (1.7 mL) under ice-cooling. Stir for hours. The solvent was distilled off under reduced pressure, then dissolved in dichloromethane (lmL) and ice-cooled 1-{(5 dimethylamino) naphthylsulfonylamino}-10 -decanamine (30 mg, 0.074 mmol) in dichloromethane. Slowly added to methane solution (0.5 mL) and stirred at room temperature for 3 hours. After confirming the completion of the reaction by TLC, extraction with dichloromethane was performed. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified using silica gel chromatography (hexane: ethyl acetate = 1: 1) to give 1-{(5 dimethylamino) naphthyl-sulfonylamino} — 10— {(Z) — 9 octa Decanoylamino} decane (42 mg, 87%) was obtained. The instrumental analysis data of this compound is shown below.
[0057] (ィ匕合物 lc ; C10-linked dansyl oleamide) [0057] (I compound lc; C10-linked dansyl oleamide)
:HNMR (CDCl， 300MHz); δ 0·88(3Η， t， J=6.2Hz), 1.11— 1·58(38Η， m)， 1.9 : HNMR (CDCl, 300MHz); δ 0 · 88 (3Η, t, J = 6.2Hz), 1.11— 1 · 58 (38Η, m), 1.9
9-2.18(6H, m)， 2.84(6H, s)， 2·84— 2·90(2Η， m)， 3.19— 3·26(2Η， m)， 4.57(1H, br-s), 5.34(2H, br— s)， 7.22(1H, d， J=8.4Hz), 7.51- 7·60(2Η， m)， 8.24-8.30(2 H， m)， 8.54(1H, d， J=8.4Hz). 9-2.18 (6H, m), 2.84 (6H, s), 2.84—2.90 (2Η, m), 3.19—3.26 (2Η, m), 4.57 (1H, br-s), 5.34 (2H, br—s), 7.22 (1H, d, J = 8.4Hz), 7.51-760 (2mm, m), 8.24-8.30 (2H, m), 8.54 (1H, d, J = 8.4 Hz).
LRMS(FAB) m/z 670(MH+). HRMS(FAB) calcd for C H O N S, 670.4981; fo und, 670.5003. LRMS (FAB) m / z 670 (MH + ). HRMS (FAB) calcd for CHONS, 670.4981; fo und, 670.5003.
[0058] l-{(5-dimethylamino)naphthyl-sulfonylamino}-4-{(Z)-9-octadecenanoylamino}-butan e  [0058] l-{(5-dimethylamino) naphthyl-sulfonylamino} -4-{(Z) -9-octadecenanoylamino} -butan e
l - { (5—ジメチルァミノ）ナフチルースルホニルァミノ }ー4 { (Z) 9ーォクタデカノ ィルァミノ }ブタン (化合物 lb ; C4-linked dansyl oleamide)  l-{(5-Dimethylamino) naphthylsulfonylsulfonyl} -4 {(Z) 9-octadecanylamino} butane (compound lb; C4-linked dansyl oleamide)
[0059] l—{ (5 ジメチルァミノ）ナフチル—スルホニルァミノ }— 10— { (Z)—9 ォクタデ カノィルァミノ }デカンと同様の手法を用いて、 1 { (5—ジメチルァミノ）ナフチルース ノレホニルァミノ } 5 ブタンァミン (48mg， 0.150mmol)とォレイン酸 (50mg， 0.177mm ol)、ォキザリノレクロリド (64 0.72mmol)力、ら、 1 { (5 ジメチルァミノ）ナフチルー スルホニルァミノ } -4 - { (Z) 9ーォクタデカノィルァミノ }ブタン (80mg， 91%)を得た 。以下に、この化合物の機器分析データを示す。  [0059] l- {(5 Dimethylamino) naphthyl-sulfonylamino} — 10— {(Z) -9 Octadecanoylamino} Using the same method as decane, 1 {(5-Dimethylamino) naphthylose norephonylamino} 5 Butanamine ( 48 mg, 0.150 mmol) and oleic acid (50 mg, 0.177 mmol), oxalinorechloride (64 0.72 mmol) force, et al, 1 {(5 dimethylamino) naphthylsulfonylsulfonylamino} -4-{(Z) 9- Octadecanolamino} butane (80 mg, 91%) was obtained. The instrumental analysis data of this compound is shown below.
[0060] (化合物 lb ; C4-linked dansyl oleamide)  [0060] (Compound lb; C4-linked dansyl oleamide)
:HNMR (CDCl， 270MHz); δ 0·88(3Η， t， J=7.0Hz), 1·26_1·59(26Η， m)， 1.9 : HNMR (CDCl, 270MHz); δ 0 · 88 (3Η, t, J = 7.0Hz), 1 · 26_1 · 59 (26Η, m), 1.9
9-2.18(6H, m)， 2.90(6H, s)， 2·89_2·91(2Η， m)， 3· 12_3· 15(2Η， m)， 4.88(1H, br-s), 5.34(2H, br-s), 7.2 (1H， d， J=7.6Hz), 7.50— 7·60(2Η， m)， 8.22-8.30(2 H， m)， 8.55(1H, d， J=8.4Hz). LRMS(FAB) m/z 586(MH+). HRMS(FAB) calcd for C H O N S, 586.4042; fo und, 586.4033. 9-2.18 (6H, m), 2.90 (6H, s), 2.89_2 · 91 (2Η, m), 3 / 12_3 · 15 (2Η, m), 4.88 (1H, br-s), 5.34 (2H , br-s), 7.2 (1H, d, J = 7.6Hz), 7.50—7.60 (2mm, m), 8.22-8.30 (2H, m), 8.55 (1H, d, J = 8.4Hz) . LRMS (FAB) m / z 586 (MH + ). HRMS (FAB) calcd for CHONS, 586.4042; fo und, 586.4033.
[0061] l-{(5-dimethylamino)naphthyl-sulfonylamino}-2-{(Z)-9-octadecenanoylamino}-ethan e  [0061] l-{(5-dimethylamino) naphthyl-sulfonylamino} -2-{(Z) -9-octadecenanoylamino} -ethan e
l—{ (5 ジメチルァミノ）ナフチルースルホニルァミノ } 2— { (Z)— 9ーォクタデカノ ィルァミノ }ェタン (化合物 la ; C2-linked dansyl oleamide)  l— {(5 Dimethylamino) naphthylsulfonylsulfonyl} 2— {(Z) — 9-octadecanylamino} ethane (compound la; C2-linked dansyl oleamide)
[0062] l—{ (5 ジメチルァミノ）ナフチル一スルホニルァミノ }— 10— { (Z)—9 ォクタデ カノィルァミノ }デカンと同様の手法を用いて、 1 { (5—ジメチルァミノ）ナフチルース ノレホニルァミノ }— 2—エタンァミン (44mg， 0.150mmol)とォレイン酸 (50mg， 0.177mm ol)、ォキザリノレクロリド (64 0.72mmol) 力、ら、 1—{ (5 ジメチルァミノ）ナフチル —スルホニルアミノ}— 2— { (Z)— 9 ォクタデカノィルァミノ }ェタン (54mg， 66%)を得 た。以下に、この化合物の機器分析データを示す。  [0062] l— {(5 Dimethylamino) naphthyl monosulfonylamino} — 10— {(Z) -9 Octadecanoylamino} Using a method similar to decane, 1 {(5-Dimethylamino) naphthylose norephonylamino} — 2— Ethanamine (44 mg, 0.150 mmol) and oleic acid (50 mg, 0.177 mmol), oxalinorechloride (64 0.72 mmol) force, 1— {(5 dimethylamino) naphthyl —sulfonylamino} — 2— {(Z ) —9 Octadecanolamino} ethane (54 mg, 66%) was obtained. The instrumental analysis data of this compound is shown below.
[0063] (化合物 la ; C2- linked dansyl oleamide)  [0063] (Compound la; C2- linked dansyl oleamide)
:HNMR (CDCl， 270MHz); δ 0·80(3Η， t， J=7.3Hz), 1.19— 1·49(22Η， m)， 1.8 : HNMR (CDCl, 270MHz); δ 0 · 80 (3Η, t, J = 7.3Hz), 1.19—1 · 49 (22Η, m), 1.8
9-1.98(6H, m)， 2.83(6H, s)， 2·94_2·98(2Η， m)， 3.19- 3·25(2Η， m)， 5.26(3H, br-s), 5.63(1H, br-s), 7.19(1H, d， J=7.3Hz), 7.48- 7·57(2Η， m)， 8.15-8.18(2 H， m)， 8.50(1H, d， J=8.6Hz). 9-1.98 (6H, m), 2.83 (6H, s), 2.94_2 · 98 (2Η, m), 3.19-325 (2Η, m), 5.26 (3H, br-s), 5.63 (1H , br-s), 7.19 (1H, d, J = 7.3Hz), 7.48-7.57 (2mm, m), 8.15-8.18 (2H, m), 8.50 (1H, d, J = 8.6Hz) .
LRMS(FAB) m/z 558(MH+). HRMS(FAB) calcd for C H O N S, 558.3729; fo und, 558.3735. LRMS (FAB) m / z 558 (MH + ). HRMS (FAB) calcd for CHONS, 558.3729; fo und, 558.3735.
[0064] (1 2· NBD体の合成） [0064] (1 2 · NBD synthesis)
前記スキーム 6に従い、まずォレイン酸クロリドとポリメチレンジァミンとの縮合反応に より化合物 6a〜6cを合成し、さらに、化合物 6a〜6cと NBDクロリド（NBD-C1)との縮合 反応により NBD体 2a〜2cを合成した。以下、この合成についてさらに詳しく説明する In accordance with Scheme 6 above, compounds 6a to 6c are first synthesized by a condensation reaction of oleic acid chloride and polymethylenediamine, and further NBD compounds are obtained by a condensation reaction of compounds 6a to 6c and NBD chloride (NBD-C1). 2a-2c were synthesized. Hereinafter, this synthesis will be described in more detail.
〇 Yes
[0065] l_{(Z)_9_octadecenanoylamino}_lO_decanamine  [0065] l _ {(Z) _9_octadecenanoylamino} _lO_decanamine
1 - { (Z) 9ーォクタデカノィルァミノ } 10 デカンァミン（化合物 6c)  1-{(Z) 9-octadecanolamino} 10 Decanamine (Compound 6c)
[0066] 窒素雰囲気下、ォレイン酸 (70mg， 0.25mmol)の無水メチレンクロリド溶液 (ImL)に氷 冷下、ォキザリルクロリド (87 L， 0.99mmol)を滴下して、室温で 4時間攪拌した。溶 媒を減圧留去した後、ジクロロメタン (1.5mL)に溶解し、氷冷した 1 , 10—デカンジアミ ン (128mg， 0.74mmol)のジクロロメタン溶液 (lmUにゆっくり加えて、室温で 6時間攪拌 した。 TLCで反応終了を確認後、ジクロロメタンで抽出した。得られた有機層を飽和 食塩水で洗浄し、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗生成物を シリカゲルクロマトグラフィー (メタノール:ジクロロメタン = 1 :2)を用いて精製し、 1 - { (Z )— 9 ォクタデカノィルァミノ }— 10—デカンァミン (90mg， 83%)を得た。以下に、こ の化合物の機器分析データを示す。 [0066] Under a nitrogen atmosphere, oxalyl chloride (87 L, 0.99 mmol) was added dropwise to an anhydrous methylene chloride solution (ImL) of oleic acid (70 mg, 0.25 mmol) under ice cooling, and the mixture was stirred at room temperature for 4 hours. . Melting The solvent was distilled off under reduced pressure, and the residue was dissolved in dichloromethane (1.5 mL) and slowly added to ice-cooled 1,10-decanediamine (128 mg, 0.74 mmol) in dichloromethane (lmU) and stirred at room temperature for 6 hours. The organic layer obtained was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the resulting crude product was chromatographed on silica gel (methanol: dichloromethane). = 1: 2) to obtain 1-{(Z) — 9 Octadecanolamine} — 10-decanamine (90 mg, 83%), the following is the analytical data of this compound Indicates.
[0067] (化合物 6c) [0067] (Compound 6c)
:HNMR (CDCl， 270MHz); δ 0·87(3Η， t， J=6.8Hz), 1·28_1·62(42Η， m)， 2.1 : HNMR (CDCl, 270MHz); δ 0 · 87 (3Η, t, J = 6.8Hz), 1 · 28_1 · 62 (42Η, m), 2.1
5(2Η， t， J=6.9Hz)， 2·60_2·80(2Η， m)， 3·23(2Η， dd， J=12.7, 6·2Ηζ)， 5·34(3Η， br). 5 (2Η, t, J = 6.9Hz), 2 · 60_2 · 80 (2Η, m), 3 · 23 (2Η, dd, J = 12.7, 6 · 2Ηζ), 5 · 34 (3Η, br).
[0068] l-{(Z)-9-octadecenanoylamino}-4-butanamine  [0068] l-{(Z) -9-octadecenanoylamino} -4-butanamine
1 - { (Z) 9ーォクタデカノィルァミノ }ー4 ブタンァミン（化合物 6b)  1-{(Z) 9-octadecanolamino} -4 Butanamine (Compound 6b)
[0069] 1 { (Z) 9 ォクタデカノイノレアミノ } 10 デカンァミンと同様の手法を用レ、て、 1 ,4—ブタンジァミン （66mg， 0.74mmol) とォレイン酸 (70mg， 0.25mmol)、ォキザリ ルクロリド （87 0.99mmol) 力、ら、 1—{ (Z)—9 ォクタデカノィルァミノ }—4— ブタンァミン (30mg， 34%)を得た。以下に、この化合物の機器分析データを示す。  [0069] 1 {(Z) 9 Octadecano-inoreamino} 10 1,4-Butadiamine (66 mg, 0.74 mmol), oleic acid (70 mg, 0.25 mmol), oxali Luclide (87 0.99 mmol) force, et al. 1- {(Z) -9 Octadecanoylamino} -4-butanamine (30 mg, 34%) was obtained. The instrumental analysis data of this compound is shown below.
[0070] (化合物 6b)  [0070] (Compound 6b)
:HNMR (CDCl， 270MHz); δ 0·80(3Η， t， J=6.8Hz), 1·20_1·95(32Η， m)， 2.0 : HNMR (CDCl, 270MHz); δ 0 · 80 (3Η, t, J = 6.8Hz), 1 · 20_1 · 95 (32Η, m), 2.0
9(2Η， t， J=8.1Hz), 2·77(2Η， t， J=5.9Hz), 3.16 (2H， t， J=5.6Hz), 3.35(1H, b r— s)， 5.27(3H, br-s). 9 (2Η, t, J = 8.1Hz), 2.77 (2Η, t, J = 5.9Hz), 3.16 (2H, t, J = 5.6Hz), 3.35 (1H, br—s), 5.27 (3H , br-s).
[0071] l_{(Z)_9_octadecenanoylamino}_2_ethanamine  [0071] l _ {(Z) _9_octadecenanoylamino} _2_ethanamine
1 - { (Z) 9ーォクタデカノィルァミノ } 2—エタンァミン (化合物 6a)  1-{(Z) 9-octadecanolamino} 2-Ethanamine (Compound 6a)
[0072] 1 { (Z) 9 ォクタデカノイノレアミノ } 10—デカンァミンと同様の手法を用レ、て、 1 ,2—エタンジァミン （45mg， 0.74mmol) とォレイン酸 (70mg， 0.25mmol)、ォキザリ ルクロリド (87 L， 0.99mmol)力、ら、 1—{ (Z)—9 ォクタデカノィルァミノ }—2—エタ ンァミン (27mg， 34%)を得た。以下に、この化合物の機器分析データを示す。  [0072] 1 {(Z) 9 Octadecanoinoreamino} Using a method similar to 10-decanamine, 1,2-ethanediamine (45 mg, 0.74 mmol) and oleic acid (70 mg, 0.25 mmol), Oxalyl chloride (87 L, 0.99 mmol) force, 1- {(Z) -9 octadecanoylamino} -2-ethanamine (27 mg, 34%) was obtained. The instrumental analysis data of this compound is shown below.
[0073] (化合物 6a) HNMR (CDC1， 270MHz); δ 0·80(3Η， t， J=6.8Hz), 1.19-1.90 (28H, m)， 2. [0073] (Compound 6a) HNMR (CDC1, 270MHz); δ 0 · 80 (3Η, t, J = 6.8Hz), 1.19-1.90 (28H, m), 2.
3  Three
14(2H， t， J=8.1Hz), 2.77(2H, t， J=5.4Hz), 3.24(2H, t， J=5.9Hz), 3.35(1H, b r— s)， 5.27(3H, br-s).  14 (2H, t, J = 8.1Hz), 2.77 (2H, t, J = 5.4Hz), 3.24 (2H, t, J = 5.9Hz), 3.35 (1H, br—s), 5.27 (3H, br -s).
[0074] 10-(7-nitro-2, l,3-benzoxadiazol-4-yl)amino-l-{(Z)-9-octadecenanoylamino}-decan e  [0074] 10- (7-nitro-2, l, 3-benzoxadiazol-4-yl) amino-l-{(Z) -9-octadecenanoylamino} -decan e
10— (7 ニトロ一 2, 1 , 3—ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { (Z)— 9 ーォクタデセノィルァミノ }—デカン (化合物 2c ; C10-linked NBD oleamide)  10- (7 Nitro-1, 2, 1-, 3-Benzoxadiazol, 4-yl) amino 1- {(Z)-9-octadecenoylamino} -decane (compound 2c; C10-linked NBD oleamide)
[0075] 窒素雰囲気下、 0°Cで、 1 { (Z)— 9ーォクタデカノィルァミノ }—10 デカンアミン( 50mg， O. l lmmol)の無水ァセトニトリル溶液 (0.25mUに、トリェチルァミン (24 0.1 7mmol)を加えた後、 NBD-C1の無水ァセトニトリル溶液 (0.5M in CH CN, 0.34mL, [0075] 1 {(Z) -9-octadecanolamino} -10 decaneamine (50 mg, O. l lmmol) in anhydrous acetononitrile (0.25 mU, triethylamine (24 0.17 mmol), and then NBD-C1 in anhydrous acetonitrile (0.5M in CH CN, 0.34mL,
3  Three
0.17mmol)を 5分かけてゆっくり加え、室温で 24時間撹拌した。 TLCで反応終了を確 認後、水を加え、ジクロロメタンで抽出した。得られた有機層を水と飽和食塩水で洗 浄し、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗生成物をシリカゲルク 口マトグラフィー (酢酸ェチル：へキサン = 1 : 1) を用いて精製し、 10— (7 二トロー 2 , 1 , 3—ベンゾキサジァゾ一ルー 4 ィル）アミノー 1— { (Z)— 9—ォクタデセノィルァ ミノ }—デカン (14mg， 20%)を得た。以下に、この化合物の機器分析データを示す。  0.17 mmol) was slowly added over 5 minutes and stirred at room temperature for 24 hours. After confirming the completion of the reaction by TLC, water was added and the mixture was extracted with dichloromethane. The obtained organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified using silica gel chromatography (ethyl acetate: hexane = 1: 1) to give 10- (7 Nitroen 2,1,3-benzoxaziazol 4-yl) amino-1 — {(Z) — 9-octadecenoylamino} -decane (14 mg, 20%) was obtained. The instrumental analysis data of this compound is shown below.
[0076] (化合物 2c ; C10-linked NBD oleamide) [0076] (Compound 2c; C10-linked NBD oleamide)
:H NMR(CDC1 , 270MHz); δ 0·81(3Η， t， J=6.8Hz), 1· 19— 1·76(42Η， m)， 1.9 : H NMR (CDC1, 270MHz); δ 0 · 81 (3Η, t, J = 6.8Hz), 1 · 19— 1 · 76 (42Η, m), 1.9
3  Three
2- 1·98(4Η， m)， 2·09(2Η， t， J=7.8Hz)， 3.16- 3· 18(2Η， m)， 3.41- 3·43(2Η， m)， 2- 1.98 (4 mm, m), 2.09 (2 mm, t, J = 7.8 Hz), 3.16- 3.18 (2 mm, m), 3.41- 3.43 (2 mm, m),
5.23- 5·27(3Η， m)， 6· 11(1Η， d， J=8.9Hz), 6·27(1Η， br-s), 8·43(1Η， d， J=8.95.23- 5 · 27 (3Η, m), 6 · 11 (1Η, d, J = 8.9Hz), 6 · 27 (1Η, br-s), 8 · 43 (1Η, d, J = 8.9
Hz) Hz)
LRMS(FAB) m/z 600(MH+). HRMS(FAB) calcd for C H O N , 600.4489; fou LRMS (FAB) m / z 600 (MH + ). HRMS (FAB) calcd for CHON, 600.4489; fou
34 58 4 5  34 58 4 5
nd, 600.4515.  nd, 600.4515.
[0077] 4-(7-nitro-2, l,3-benzoxadiazol-4-yl)amino-l-{(Z)-9-octadecenanoylamino}-butane 4— (7 ニトロ一 2, 1 , 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { (Z)— 9— ォクタデセノィルァミノ }—ブタン (化合物 2b ; C4-linked NBD oleamide)  [0077] 4- (7-nitro-2, l, 3-benzoxadiazol-4-yl) amino-l-{(Z) -9-octadecenanoylamino} -butane 4— (7 nitro 1, 2, 3 benzoxa Diazol 4-yl) amino 1- {(Z)-9- Octadecenoylamino} -butane (compound 2b; C4-linked NBD oleamide)
[0078] 10— (7 ニトロ一 2, 1 , 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { (Z)— 9ーォクタデセノィルァミノ }—デカンと同様の手法を用いて、 1— { (Ζ)—9 ォクタデ カノィルァミノ }— 4—ブタンァミン (26mg， 0.074mmol)と NBD- C1(0.5M in DMF, 0.1 8mL， 0.089mmol)、トリエチノレアミン (14 O. lOmmol)から、 4— (7 ニトロ一 2, 1 , 3—ベンゾキサジァゾール一 4—ィル）ァミノ一 1— { (Z)— 9—ォクタデセノィルァミノ } ブタン (10mg， 26%)を得た。以下に、この化合物の機器分析データを示す。 [0078] 10— (7 Nitro 1, 2, 3 Benzoxadiazol 4 4-yl) amino 1— {(Z) — 9-octadecenoylamino} — 1— {(Ζ) —9 Canoylamino} — From 4-butanamine (26 mg, 0.074 mmol), NBD-C1 (0.5 M in DMF, 0.1 8 mL, 0.089 mmol), triethinoreamine (14 O. lOmmol), 4— (7 Nitro 1, 2, 1 , 3-Benzoxadiazol 4-yl) amino 1-{(Z) -9-octadecenoylamino} butane (10 mg, 26%) was obtained. The instrumental analysis data of this compound is shown below.
[0079] (化合物 2b ; C4-linked NBD oleamide) [0079] (Compound 2b; C4-linked NBD oleamide)
:HNMR (CDC1， 270MHz); δ 0·87(3Η， t， J=6.8Hz), 1·25_1·87(26Η， m)， 1.9 : HNMR (CDC1, 270MHz); δ 0 · 87 (3Η, t, J = 6.8Hz), 1 · 25_1 · 87 (26Η, m), 1.9
3  Three
8-2.0K4H, m)， 2.20(2H, t， J=7.2Hz), 3.20- 3·35(2Η， m)， 3·45_3·62(2Η， m)， 8-2.0K4H, m), 2.20 (2H, t, J = 7.2Hz), 3.20-35 (2Η, m), 3.45_3 · 62 (2Η, m),
5.30- 5·34(3Η， m)， 5.60(1H, br— s)， 6.2(1H, d， J=8.4Hz), 8.49(1H, d， J=8.7H z) 5.30-5.34 (3mm, m), 5.60 (1H, br—s), 6.2 (1H, d, J = 8.4Hz), 8.49 (1H, d, J = 8.7H z)
LRMS(FAB) m/z 516(MH+). HRMS(FAB) calcd for C H O N , 516.3549; fou LRMS (FAB) m / z 516 (MH + ) .HRMS (FAB) calcd for CHON, 516.3549; fou
28 46 4 5  28 46 4 5
nd, 516.3561.  nd, 516.3561.
[0080] 2-(7-nitro-2,l,3-benzoxadiazol-4-yl)amino-l-{(Z)-9-octadecenanoylamino}-ethane 2— (7 ニトロ一 2, 1 , 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { (Z)— 9— ォクタデセノィルァミノ }ーェタン (化合物 2a; C2-linked NBD oleamide)  [0080] 2- (7-nitro-2, l, 3-benzoxadiazol-4-yl) amino-l-{(Z) -9-octadecenanoylamino} -ethane 2— (7 nitro 1, 2, 3 benzoxa Diazol 4-yl) amino- 1— {(Z) — 9— Octadecenoylamino} -ethan (compound 2a; C2-linked NBD oleamide)
[0081] 10— (7 ニトロ一 2, 1 , 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { (Z)— 9ーォクタデセノィルァミノ }—デカンと同様の手法を用いて、 1— { (Ζ)—9 ォクタデ カノィルァミノ }— 2—エタンァミン (30mg， 0.092mmol)と NBD- C1(0.5M in DMF, 0.2 2mL， 0.11010101)、トリェチノレァミン(18〃 0.13mmol) から、 2— (7 二卜口— 2, 1 , 3—ベンゾキサジァゾール一 4—ィル）ァミノ一 1— { (Z)— 9—ォクタデセノィルァミノ } ーェタン (36mg， 80%)を得た。以下に、この化合物の機器分析データを示す。 [0081] 10— (7 Nitro-1, 2, 1, Benzoxadiazol, 4 yl) amino 1— {(Z) — 9-octadecenoylamino} —The same procedure as decane 1- {(Ζ) -9 Octadecanolamino} — 2-Ethanamine (30 mg, 0.092 mmol) and NBD-C1 (0.5 M in DMF, 0.2 2 mL, 0.110101 0 1), Toretinoreamine (18 〃 0.13 mmol) to 2— (7 Nittoguchi— 2, 1, 3—Benzoxadiazol 4- 4-yl) amino 1— {(Z) — 9-octadecenoylamino} ethane ( 36 mg, 80%). The instrumental analysis data of this compound is shown below.
[0082] (化合物 2a; C2-linked NBD oleamide)  [0082] (Compound 2a; C2-linked NBD oleamide)
:HNMR (CDC1， 270MHz); δ 0·81(3Η， t， J=7.0Hz), 1.19— 1·56(26Η， m)， 1.9 : HNMR (CDC1, 270MHz); δ 0 · 81 (3Η, t, J = 7.0Hz), 1.19— 1 · 56 (26Η, m), 1.9
3  Three
2-1.98(4H, m)， 2.20(2H, t， J=7.0Hz), 3·55— 3·64(4Η， m)， 5·23— 5·26(2Η， m)， 5.91(1H, br-s), 6.08(1H, d， J=8.6Hz), 7.79 (1H， br— s)， 8.41(1H, d， J=8.4Hz )  2-1.98 (4H, m), 2.20 (2H, t, J = 7.0Hz), 3 · 55— 3 · 64 (4Η, m), 5 · 23— 5 · 26 (2Η, m), 5.91 (1H , br-s), 6.08 (1H, d, J = 8.6Hz), 7.79 (1H, br—s), 8.41 (1H, d, J = 8.4Hz)
LRMS(FAB) m/z 488(MH+). HRMS(FAB) calcd for C H O N , 488.3237; fou LRMS (FAB) m / z 488 (MH + ). HRMS (FAB) calcd for CHON, 488.3237; fou
26 42 4 5  26 42 4 5
nd, 488.3249.  nd, 488.3249.
[0083] (1 3·蛍光ラベル化 MI-18) 前記スキーム 7に従!/、Dansyl化 MI-18 (化合物 3c)を合成し、前記スキーム 8に従レヽ NBD化 MI-18 (化合物 4c)を合成した。以下、これらの合成についてさらに詳しく説明 する。 [0083] (1 3 · Fluorescent labeling MI-18) According to Scheme 7, Dansylated MI-18 (Compound 3c) was synthesized, and according to Scheme 8, NBD MI-18 (Compound 4c) was synthesized. Hereinafter, these syntheses will be described in more detail.
[0084] 10-{(5-dimethylamino)naphthyl-sulfonylamino}-l-{8-[(2b， 3Rノ- 3_octyloxiranyl]octa noylamino}-aecane  [0084] 10-{(5-dimethylamino) naphthyl-sulfonylamino} -l- {8-[(2b, 3R-3_octyloxiranyl] octa noylamino} -aecane
10—（（5 ジメチルァミノ）ナフチルースルホニルァミノ） 1 { 8— [ (2S*, 3R*] 3 ォクチルォキシラニル}オタタノィルァミノ） デカン（化合物 3c ; C10-linked da nsyl MI-18)  10-((5 dimethylamino) naphthylsulfonylsulfonyl) 1 {8— [(2S *, 3R *] 3 octyloxylanyl} otatanylamino) Decane (compound 3c; C10-linked da nsyl MI- 18)
[0085] 窒素雰囲気下、 0°Cで、ォレイン酸メチル (l.Og, 3.37mmol)の無水メチレンクロリド溶 液 (34mL)に、 mCPBA(872mg， 5.05mmol)を加え、室温で 12時間撹拌した。 TLCで反 応終了を確認後、水を加え、ジクロロメタンで抽出した。得られた有機層を水と飽和 食塩水で洗浄し、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗生成物を シリカゲルクロマトグラフィー (酢酸ェチル：へキサン = 10: 1)を用いて精製し、メチル— 8 - [ (2S*, 3R*)— 3 ォクチルォキシラン一 2 ィル]オタタノエイト (828mg， 79%)を得 た。  [0085] Under a nitrogen atmosphere, mCPBA (872 mg, 5.05 mmol) was added to an anhydrous methylene chloride solution (34 mL) of methyl oleate (l.Og, 3.37 mmol) at 0 ° C, and the mixture was stirred at room temperature for 12 hours. . After confirming the completion of the reaction by TLC, water was added and the mixture was extracted with dichloromethane. The obtained organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified using silica gel chromatography (ethyl acetate: hexane = 10: 1) and methyl-8-[(2S *, 3R *)-3 octyloxysilane-2-yl] Otata Eight (828 mg, 79%) was obtained.
[0086] 上記により得られたメチル 8— [ (2S*， 3R*)— 3 ォクチルォキシラン一 2 ィル]ォ クタノエイト (38mg， 0.12mmol)の水 エタノール溶液 (v/v=l/l , 1.2mL)に、水酸化リ チウム (3mg， O. mmol)を加え、 50°Cで 3時間攪拌した。 TLCで反応終了を確認後、 1 0%塩酸水溶液を加え、酢酸ェチルで抽出した。得られた有機層を飽和食塩水で洗 浄し、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗成生物 (36mg， 0.12m mol)を、ジクロロメタン (0.8mL)に溶解し、氷冷下、ォキザリルクロリド (38 μ L， 0.43mmo 1)を滴下して、室温で 4時間攪拌した。溶媒を減圧留去した後、ジクロロメタン (0.4mL) に溶解し、氷冷した 1—{ (5—ジメチルァミノ）ナフチル—スルホニルァミノ }— 10—デ カンアミン (32mg， 0.08mmol)のジクロロメタン溶液 (0.4mUにゆっくり加えて、室温で 3 時間攪拌した。 TLCで反応終了を確認後、ジクロロメタンで抽出した。得られた有機 層を飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた粗 生成物をシリカゲルクロマトグラフィー (へキサン:酢酸ェチル = 1 : 1)を用いて精製し 10 — (7 ニトロ一 2, 1 , 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— { 8— [ (2S*, 3R*]— 3 ォクチルォキシラニル}オタタノィルァミノ） デカン (27mg， 33%)を得た。 以下に、この化合物の機器分析データを示す。 [0086] Methyl 8-[(2S *, 3R *) — 3-octyloxysilane-2-yl] octanoate (38mg, 0.12mmol) obtained in the above solution in water (v / v = l / l , 1.2 mL) was added lithium hydroxide (3 mg, O. mmol), and the mixture was stirred at 50 ° C. for 3 hours. After confirming the completion of the reaction by TLC, 10% aqueous hydrochloric acid solution was added, and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product (36 mg, 0.12 mmol) was dissolved in dichloromethane (0.8 mL), and oxalyl chloride (38 μL, 0.43 mmo 1) was added dropwise under ice cooling, and at room temperature for 4 hours. Stir. The solvent was distilled off under reduced pressure, and the residue was dissolved in dichloromethane (0.4 mL) and ice-cooled 1-{(5-dimethylamino) naphthyl-sulfonylamino} -10-decanamine (32 mg, 0.08 mmol) in dichloromethane (0.4 After slowly adding to mU and stirring at room temperature for 3 hours, the completion of the reaction was confirmed by TLC, followed by extraction with dichloromethane, and the resulting organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product was purified using silica gel chromatography (hexane: ethyl acetate = 1: 1). 10 — (7 Nitro-1,2,1,3 Benzoxadiazole, 4-yl) amino-1- — {8— [(2S *, 3R *] — 3 octyloxylanyl} otatanylamino) decane (27 mg, 33%). The instrumental analysis data of this compound is shown below.
[0087] (化合物 3c ; C10-linked dansyl MI-18) [0087] (Compound 3c; C10-linked dansyl MI-18)
:HNMR (CDC1， 270MHz); δ 0·79(3Η， t， J=7.0Hz), 1· 16_1·77(42Η， m)， 2.0 : HNMR (CDC1, 270MHz); δ 0 · 79 (3Η, t, J = 7.0Hz), 1 · 16_1 · 77 (42Η, m), 2.0
3— 2· 12(4Η， m)， 2.83(2H, br— s)， 3.13- 3·21(2Η， m)， 3.41-3.462H, m)， 5.34 (1 H， br-s), 6.11(1H, d， J=8.6Hz), 6.34(1H, br— s)， 8.43(1H, d， J=8.4Hz) LRMS(FAB) m/z 616(MH+). HRMS(FAB) calcd for C H O N , 616.4448; fou nd, 616.4460. 3—2 · 12 (4Η, m), 2.83 (2H, br—s), 3.13-3.21 (2Η, m), 3.41-3.462H, m), 5.34 (1 H, br-s), 6.11 (1H, d, J = 8.6Hz), 6.34 (1H, br—s), 8.43 (1H, d, J = 8.4Hz) LRMS (FAB) m / z 616 (MH + ). HRMS (FAB) calcd for CHON, 616.4448; fou nd, 616.4460.
[0088] 10-(7-nitro-2,l,3-benzoxadiazol-4-yl)amino-l-{8-[(2b， 3R*)-3-octyloxiranyl]octan oylamino}-decane  [0088] 10- (7-nitro-2, l, 3-benzoxadiazol-4-yl) amino-l- {8-[(2b, 3R *)-3-octyloxiranyl] octan oylamino} -decane
10— (7 ニトロ一 2, 1, 3 ベンゾキサジァゾール一 4 ィル）ァミノ一 1— {8— [ (2 S*， 3R*]— 3 ォクチルォキシラニル}オタタノィルァミノ）一デカン（化合物 4c ; C10- linked NBD MI-18)  10— (7 Nitro-1, 2, 1, 3 Benzoxadiazole, 4-yl) amino 1— {8— [(2 S *, 3R *] — 3 octyloxylanyl} otatanylamino ) One decane (compound 4c; C10- linked NBD MI-18)
[0089] 窒素雰囲気下、 0°Cで、 10—（7 二トロー 2, 1, 3—ベンゾキサジァゾ一ルー 4ーィ ノレ）ァミノ一 1 KZ)— 9 ォクタデセノィルァミノ }—デカン（16 mg， 0.026 mmol)の 無水メチレンクロリド溶液（0.26 mL)に、 mCPBA (9 mg， 0.052 mmol)を加え、室温で 12時間撹拌した。 TLCで反応終了を確認後、ハイポ水を加え、ジクロロメタンで抽出 した。得られた有機層を水と飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥後、減 圧濃縮した。得られた粗生成物をシリカゲルクロマトグラフィー（酢酸ェチル：へキサン = 1: 1)を用いて精製し、 10— (7 ニトロ 2, 1 , 3 ベンゾキサジァゾール— 4 ィ ル）アミノー 1— { 8— [ (2S*， 3R*] - 3—ォクチルォキシラニル}オタタノィルァミノ） - デカン（14 mg， 83%)を得た。以下に、この化合物の機器分析データを示す。  [0089] 10— (7 Nitrole 2, 1, 3-benzoxaziazol 4-1-nor) amino-1 KZ) —9 Octadecenoylamino} —Decan (0 ° C under nitrogen atmosphere) MCPBA (9 mg, 0.052 mmol) was added to an anhydrous methylene chloride solution (0.26 mL) of 16 mg, 0.026 mmol), and the mixture was stirred at room temperature for 12 hours. After confirming the completion of the reaction by TLC, hypo water was added and extracted with dichloromethane. The obtained organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude product was purified using silica gel chromatography (ethyl acetate: hexane = 1: 1) to give 10— (7 nitro 2,1,3 benzoxazazole-4-yl) amino 1—. {8 — [(2S *, 3R *]-3-octyloxylanyl} otatanylamino) -decane (14 mg, 83%) was obtained. The instrumental analysis data of this compound is shown below.
[0090] (化合物 4c ; C10-linked NBD MI-18)  [0090] (Compound 4c; C10-linked NBD MI-18)
:HNMR (CDC1， 270MHz); δ 0·81(3Η， t， J=6.7Hz), 1.14— 1·49(42Η， m)， 2.0 : HNMR (CDC1, 270MHz); δ 0 · 81 (3Η, t, J = 6.7Hz), 1.14—1 · 49 (42Η, m), 2.0
8(2H， t， J=7.6Hz)， 2.82(6H, s)， 2·82_2·85(2Η， m)， 3.14- 3· 16(2Η， m)， 4.53(1 H， br-s), 5.34(1H, br-s), 7.13(1H, br), 7.47- 7·53(2Η， m)， 8.17- 8·22(2Η， m) ， 8.49(1H, br). 8 (2H, t, J = 7.6Hz), 2.82 (6H, s), 2.82_285 (2Η, m), 3.14- 3 · 16 (2Η, m), 4.53 (1 H, br-s) , 5.34 (1H, br-s), 7.13 (1H, br), 7.47-7 · 53 (2Η, m), 8.17-8 · 22 (2Η, m), 8.49 (1H, br).
LRMS(FAB) m/z 686(MH+). HRMS(FAB) calcd for C H O N S, 686.4930; fo und, 686.4951. LRMS (FAB) m / z 686 (MH + ). HRMS (FAB) calcd for CHONS, 686.4930; fo und, 686.4951.
[0091] (2.色素 トランスファーアツセィによる蛍光性ォレアミド誘導体の gap結合阻害活 性測定）  [0091] (2. Measurement of gap binding inhibitory activity of fluorescent oleamide derivatives by dye transfer assembly)
本実施例で合成した蛍光性ォレアミド誘導体について、色素 トランスファーアツセ ィ (Dye transfer assay)により、 gap結合阻害活性測定を測定し、その結果を記録お よび写真撮影した。具体的なアツセィ方法については、 WO2006/049157号公報に記 載の方法と同様に行った。  For the fluorescent oleamide derivative synthesized in this example, the gap binding inhibitory activity was measured by dye transfer assay, and the results were recorded and photographed. The specific assembly method was the same as the method described in WO2006 / 049157.
図 1のグラフおよび図 2に、前記色素 トランスファーアツセィの結果を示す。図 1は 、各被検物質（ォレアミド、 Dansyl体（化合物 la〜lc)および比較例の Dansyl oleamide )の GJICスコアを示すグラフである。また、図 2は、各被検物質による前記色素 トラ ンスファーアツセィ後の状態を示す図であり、図 2a)は対照（control)の図であり、図 2 b)はォレアミドの図であり、図 2c)は化合物 lc (C10 linked dansyl oleamide)の図で ある。  The graph of FIG. 1 and FIG. 2 show the results of the dye transfer assembly. FIG. 1 is a graph showing the GJIC score of each test substance (olamide, Dansyl form (compounds la to lc) and comparative example Dansyl oleamide). FIG. 2 is a diagram showing the state after the dye transfer assay by each test substance, FIG. 2a) is a control diagram, and FIG. 2b) is an oleamide diagram. Figure 2c) is a diagram of the compound lc (C10 linked dansyl oleamide).
[0092] 図 1および 2から分かる通り、 Dansyl oleamideは Cx43阻害活性を消失したのに対 し、 Cn linked Dansyl oleamide (化合物 la〜lc)は、 oleamideと類似の Cx43阻害活 性を示した。中でも、化合物 lc (C10 linked dansyl oleamide)が特に高い活性を示 した。  [0092] As can be seen from FIGS. 1 and 2, Dansyl oleamide lost Cx43 inhibitory activity, whereas Cn linked Dansyl oleamide (compounds la to lc) showed Cx43 inhibitory activity similar to oleamide. Among them, the compound lc (C10 linked dansyl oleamide) showed particularly high activity.
[0093] (3.種々の細胞内への蛍光性ォレアミド誘導体の導入）  [0093] (3. Introduction of fluorescent oleamide derivatives into various cells)
本実施例で合成した蛍光性ォレアミド誘導体を、 HeLa細胞、 HeLa_Cx43 (HeLa C x43)細胞、 MCF-7細胞等の癌細胞、および TIG1細胞等の正常細胞に導入して蛍光 を観察し、その結果を記録および写真撮影した。  The fluorescent oleamide derivative synthesized in this example was introduced into HeLa cells, HeLa_Cx43 (HeLa C x43) cells, cancer cells such as MCF-7 cells, and normal cells such as TIG1 cells, and fluorescence was observed. Was recorded and photographed.
具体的な導入方法は、 WO2006/049157号公報に記載の方法とほぼ同様であり、 観察の前日、おのおのの細胞をそれぞれ別個に、 10%FCS含有 DMEM中に懸濁 させた。そして、いずれの細胞も、シャーレ内のカバーガラス上に培養した。蛍光観 察当日、まず細胞培養液に蛍光性ォレアミド誘導体を 40 M添加し、 2時間インキュ ペートした後、リン酸緩衝液で 3回洗浄した。 4%ホルムアルデヒド溶液 (リン酸緩衝液 で希釈、メタノール不含)を lmL加え、 5分静置した後、再びリン酸緩衝液で 3回洗浄 した。カバーガラスの水分を十分に切った後、プレパラート上にシールした。その後、 細胞を観察した。 The specific introduction method is almost the same as the method described in WO2006 / 049157. On the day before the observation, each cell was individually suspended in DMEM containing 10% FCS. And all the cells were cultured on the cover glass in a petri dish. On the day of the fluorescence observation, 40 M of a fluorescent oleamide derivative was first added to the cell culture, incubated for 2 hours, and then washed 3 times with a phosphate buffer. After adding 1 mL of 4% formaldehyde solution (diluted with phosphate buffer, not containing methanol), allowed to stand for 5 minutes, it was washed again with phosphate buffer three times. After the cover glass was sufficiently drained, it was sealed on the preparation. afterwards, Cells were observed.
図 3および 4に、その結果を示す。図 3は、 NBD基を有するォレアミド誘導体 (化合 物 2a〜2cおよび 4c)を HeLa_Cx43細胞または HeLa細胞に導入した結果を示す図で ある。図 3a)および図 3b)は、化合物 2a〜2cおよび 4cを HeLa_Cx43細胞に導入した結 果を示し、図 3c)および図 3d)は、 HeLa細胞に導入した結果を示す。また、図 3a)およ び図 3c)は、 DICによる測定結果を示し、図 3b)および図 3d)は、 Cy2-filterを用いた測 定結果を示す。図 4は、ダンシル基を有するォレアミド誘導体 (化合物 la〜lcおよび 3 c)を HeLa_Cx43細胞または HeLa細胞に導入した結果を示す図である。図 4a)および 図 4b)は、化合物 la〜lcおよび 3cを HeLa_Cx43細胞に導入した結果を示し、図 4c)お よび図 4d)は、 HeLa細胞に導入した結果を示す。また、図 4a)および図 4c)は、 DICに よる測定結果を示し、図 4b)および図 4d)は、 DAPI-filterを用いた測定結果を示す。  Figures 3 and 4 show the results. FIG. 3 is a diagram showing the result of introducing an oleamide derivative (compounds 2a to 2c and 4c) having an NBD group into HeLa_Cx43 cells or HeLa cells. Figures 3a) and 3b) show the results of introducing compounds 2a-2c and 4c into HeLa_Cx43 cells, and Figures 3c) and 3d) show the results of introduction into HeLa cells. Figures 3a) and 3c) show the measurement results by DIC, and Figures 3b) and 3d) show the measurement results using Cy2-filter. FIG. 4 is a diagram showing the results of introducing oleamide derivatives having dansyl groups (compounds la to lc and 3c) into HeLa_Cx43 cells or HeLa cells. Figures 4a) and 4b) show the results of introducing the compounds la to lc and 3c into HeLa_Cx43 cells, and Figures 4c) and 4d) show the results of introduction into HeLa cells. Figures 4a) and 4c) show the results of measurement by DIC, and Figures 4b) and 4d) show the results of measurement using DAPI-filter.
[0094] 図 3および図 4から分かる通り、本実施例の化合物は、 HeLa_Cx43細胞および HeLa のいずれに対しても導入することが可能であり、細胞の蛍光を観察することができた。 予想に反して、ォレアミドが細胞膜以外にも細胞の核以外の細胞質部分でも観察さ れることを明らかにした。  [0094] As can be seen from Figs. 3 and 4, the compound of this example can be introduced into both HeLa_Cx43 cells and HeLa, and the fluorescence of the cells could be observed. Contrary to expectations, oleamide was observed not only in the cell membrane but also in the cytoplasm other than the cell nucleus.
[0095] さらに、図 5および図 6に、本発明の蛍光ォレアミドと他の蛍光色素との二重染色の 結果を示す。図 5は、 Tigl細胞を、本発明の蛍光ォレアミドである C 10-linked NBD ol eamideおよび蛍光色素ミトトラッカー（MitoTracker™ Red)で二重染色した結果、図 6 は、 Tigl細胞を、 C 10-linked NBD oleamideおよび蛍光色素 FM™ 4-64で二重染色し た結果を示す図である。 MitoTracker™ Redおよび FM™ 4-64は、いずれも Molecular Probes Inc.社より購入し 7こ。 MitoTracker Red (Chloromethyl—X—rosamine)によって 、細胞のミトコンドリアが選択的に染色される。一方、 FM™ 4-64 (N-(3-triethylammon iumpropyi)-4-(6-(4-(diethylamino phenyl)hexatnenyl)pyridinium dibromide) (ュ、エノ ドサイト一シスの過程を染色する。  [0095] Further, Figs. 5 and 6 show the results of double staining of the fluorescent oleamide of the present invention with another fluorescent dye. Fig. 5 shows the result of double staining of Tigl cells with C10-linked NBD ol eamide, a fluorescent oligomer of the present invention, and the fluorescent dye MitoTracker ™ Red. FIG. 6 shows the results of double staining with linked NBD oleamide and fluorescent dye FM ™ 4-64. MitoTracker ™ Red and FM ™ 4-64 are both purchased from Molecular Probes Inc. MitoTracker Red (Chloromethyl-X-rosamine) selectively stains cell mitochondria. On the other hand, FM ™ 4-64 (N- (3-triethylammoniumpropyi) -4- (6- (4- (diethylaminophenyl) hexatnenyl) pyridinium dibromide)) is stained in the process of endocytosis.
図 5に示すように、 MitoTracker™ Redと二重染色した場合、 C lO-linked NBD oleam ideと MitoTracker™ Redとはほとんど mergeしない（局在が重ならない）。また、二種の 蛍光は互いに干渉しない。これに対して、 FM™ 4-64と二重染色した場合、図 6に示 すように、 C 10-linked NBD oleamideと FM™ 4-64とはきれいに mergeする（局在がよく 重なる）。この結果は、膜に入り込んだ蛍光ォレアミドが、エンドサイト一シスによって 細胞に取り込まれてレ、ることを示して!/、る。 As shown in Figure 5, when double-staining with MitoTracker ™ Red, ClO-linked NBD oleamide and MitoTracker ™ Red hardly merge (localization does not overlap). Also, the two types of fluorescence do not interfere with each other. In contrast, when double-stained with FM ™ 4-64, as shown in Fig. 6, C 10-linked NBD oleamide and FM ™ 4-64 merge together cleanly (localization is good) Overlap). This result indicates that the fluorescent olamide that has entered the membrane is taken up by the cells by endocytosis!
[0096] (4.結果） [0096] (4. Results)
本実施例の化合物について、以下の機能的性質を確認することができた。  The following functional properties could be confirmed for the compound of this example.
[1] 蛍光部として励起光の異なるダンシル基、 NBD基を導入することで、用途に合わ せて二種類を使レヽ分けること力 Sできる。  [1] By introducing dansyl group and NBD group with different excitation light as the fluorescent part, it is possible to use two types according to the application.
[2] 従来技術では、ォレアミドのアミド部分を変換すると、ォレアミドの gap結合阻害能 は半分以下に低下する。し力、しながら、本実施例の化合物では、様々な長さの炭素 鎖でォレアミド部と蛍光部を繋ぐことで、蛍光部によってォレアミドの生物活性が損わ れることなぐ connexin43(Cx43)を介した gap結合阻害作用を示す。  [2] In the prior art, when the amide part of oleamide is converted, the gap bond inhibitory ability of oleamide decreases to less than half. However, in the compound of this example, the oleamide part and the fluorescent part are connected by carbon chains of various lengths, so that the biological activity of the oleamide is not impaired by the fluorescent part via connexin43 (Cx43). The gap binding inhibitory action is shown.
[3] 生細胞に導入し、生きたまま蛍光を観察できる。  [3] Introduce into living cells and observe fluorescence alive.
[4] 本実施例におけるすべての蛍光性ォレアミド誘導体は、細胞の種類 (HeLa細胞 、 HeLa_Cx43細胞、 MCF-7細胞等の種々の癌細胞、 TIG1細胞等の正常細胞)に関 係なぐ蛍光を導入できる。しかしながら、 C10-linked dansyl MI-18 (化合物 3c)およ び C10-linked NBD MI-18 (化合物 4c)は、 HeLa_Cx43細胞における gap結合阻害 能はなぐ一方で Cn-linked dansyl or NBD oleamide (化合物 la〜lcおよび 2a〜2 c)は、 HeLa-Cx43細胞における gap結合形成を阻害するという特性を有している。す なわち、ォレアミドのォレフイン部の変換によって gap結合形成阻害能を変化させるこ とが可能となる。  [4] All fluorescent oleamide derivatives in this example introduced fluorescence related to cell types (various cancer cells such as HeLa cells, HeLa_Cx43 cells, MCF-7 cells, and normal cells such as TIG1 cells). it can. However, C10-linked dansyl MI-18 (compound 3c) and C10-linked NBD MI-18 (compound 4c) do not inhibit gap binding in HeLa_Cx43 cells, but Cn-linked dansyl or NBD oleamide (compound la ~ Lc and 2a ~ 2c) have the property of inhibiting gap junction formation in HeLa-Cx43 cells. In other words, the ability to inhibit gap formation can be changed by changing the olefin moiety of oleamide.
[5] 蛍光性ォレアミドと異なる波長の蛍光色素を併用することで、二重染色が可能で あり、その汎用性は高い。  [5] By using fluorescent dyes with fluorescent dyes of different wavelengths, double staining is possible, and its versatility is high.
[0097] このように、本実施例では、種々の長さの炭素鎖で蛍光部とォレアミド部をつないだ 蛍光性ォレアミドを合成した。それにより、ォレアミドの作用の一つとして知られる conn exin43 (Cx43)を介した gap結合形成阻害能を損なうことなぐォレアミドに蛍光を導入 できた。ォレアミドおよびその類縁体に対して生物活性を大きく損なうことなく蛍光を 導入した例はこれまでになぐ本発明が初めてであり革新的である。さらに、本実施 例の化合物は、種々の生細胞中に蛍光を導入し、顕微鏡下観察することができる非 常に優れた化合物である。その際、 HeLa細胞に connexin 43を導入した HeLa_Cx43 細胞のみでなぐ HeLa細胞および MCF-7細胞等の癌細胞、および TIG1細胞といつ た正常細胞においても蛍光が観察できた。このことは、ォレアミドの Cx43を介した gap 結合形成阻害活性の発現が、 connexin43に直接作用しているのではないことを示唆 していると推測される。すなわち、ォレアミドはいずれの細胞においても膜に取り込ま れるカ 膜の流動性を変化させることで、細胞膜とコネキシンとの相互作用を変化さ せ、 gap結合の形成を阻害すると類推できる。本発明の化合物をさらに応用すること で、ォレアミドおよびその誘導体のこれまで未知であった作用等が発見できる可能性 が高い。 [0097] Thus, in this example, fluorescent oleamides in which a fluorescent part and an oleamide part were connected with carbon chains of various lengths were synthesized. As a result, it was possible to introduce fluorescence into oleamide without impairing the ability to inhibit gap bond formation via connexin43 (Cx43), which is known as one of the actions of oleamide. The present invention is the first and innovative example in which fluorescence is introduced into oleamide and its analogs without significantly impairing biological activity. Further, the compound of this example is a very excellent compound that can introduce fluorescence into various living cells and can be observed under a microscope. At that time, HeLa_Cx43 introduced connexin 43 into HeLa cells Fluorescence could be observed in cancer cells such as HeLa cells and MCF-7 cells, and normal cells such as TIG1 cells. This suggests that the expression of gap bond formation inhibitory activity of oleamide via Cx43 does not directly act on connexin43. In other words, it can be inferred that oleamide changes the fluidity of the membrane taken into the membrane in any cell, thereby changing the interaction between the cell membrane and connexin and inhibiting the formation of gap junctions. By further applying the compound of the present invention, there is a high possibility that the previously unknown actions of oleamide and its derivatives can be discovered.
産業上の利用可能性 Industrial applicability
以上説明した通り、本発明の化合物は、蛍光基の導入により本来の生物活性が大 きく損なわれることがなぐ蛍光を発すること力 Sできる。このため、本発明の化合物は、 細胞の蛍光観察方法、およびォレアミド誘導体の細胞内作用メカニズム研究用ツー ルに有用である。また、本発明の化合物は、その生物活性により、 gap結合阻害剤、 医薬品、飲食品またはサプリメントとしても用いることができる。例えば、本発明の化 合物が癌の転移抑制活性、抗腫瘍活性等を有する場合は、抗癌剤 (制癌剤）などの 医薬、さらには抗癌作用または癌予防効果をもつサプリメント、機能性食品（食用組 成物）の原材料などに利用可能である。さらに、本発明の化合物の用途はこれらに限 定されず、あらゆる用途に使用可能である。  As described above, the compound of the present invention can emit fluorescence without significantly impairing the original biological activity by introduction of a fluorescent group. Therefore, the compound of the present invention is useful for a method for observing fluorescence of cells and a tool for studying the intracellular action mechanism of an oleamide derivative. Moreover, the compound of this invention can be used also as a gap coupling | bonding inhibitor, a pharmaceutical, food-drinks, or a supplement by the biological activity. For example, when the compound of the present invention has a cancer metastasis inhibitory activity, an antitumor activity, etc., a pharmaceutical such as an anticancer agent (anticancer agent), a supplement having an anticancer effect or a cancer preventive effect, a functional food (edible food) It can be used as a raw material for the composition. Furthermore, the use of the compound of the present invention is not limited to these, and can be used for any application.

Claims

請求の範囲 The scope of the claims
下記式 (I)で表される蛍光性ォレアミド誘導体、その互変異体もしくは立体異性体、 またはそれらの塩。  A fluorescent oleamide derivative represented by the following formula (I), a tautomer or stereoisomer thereof, or a salt thereof.
[化 1]  [Chemical 1]
Figure imgf000036_0001
Figure imgf000036_0001
前記式 (I)中、 In the formula (I),
L1は、単結合、 -0- または -S- であり、 L 1 is a single bond, -0- or -S-
L2は、単結合または直鎖もしくは分枝アルキレン基であり、 L 2 is a single bond or a linear or branched alkylene group,
R1および R2は、それぞれ水素原子または任意の置換基であり、同一でも異なってい ても良ぐ R 1 and R 2 are each a hydrogen atom or an arbitrary substituent, and may be the same or different.
Rは蛍光基である。  R is a fluorescent group.
[2] 前記式 (I)中、 L2が、単結合または炭素数 1〜； 12の直鎖もしくは分枝アルキレン基で ある請求項 1記載の蛍光性ォレアミド誘導体、その互変異体もしくは立体異性体、ま たはそれらの塩。 [2] The fluorescent oleamide derivative according to claim 1, wherein L 2 is a single bond or a linear or branched alkylene group having 1 to 12 carbon atoms in the formula (I), or a tautomer or stereoisomer thereof. Body or their salts.
[3] 前記式 (I)中、 Rが、下記式 (Π)で表されるダンシル基（5— (ジメチルァミノ）—ナフタ レン 1ースルホニル基）、下記式 (III)で表される NBD基 (7 二トロべンズー 2 ォキ サー 1 , 3—ジァゾール一 4—ィル基)、またはナフタリミド基 (6—ァミノ一 1 , 3—ジォキ ソ— 1H, 3H ベンゾデイソキノリル基)などの蛍光基である請求項 1または 2に記載 の蛍光性ォレアミド誘導体、その互変異体もしくは立体異性体、またはそれらの塩。  [3] In the formula (I), R is a dansyl group (5- (dimethylamino) -naphthalene 1-sulfonyl group) represented by the following formula (Π), an NBD group represented by the following formula (III) ( 7 Fluorobenzene such as 2-trobens-2-oxer 1, 3-diazol-4-yl group) or naphthalimido group (6-amino-1,3-dioxo-1H, 3H benzodeisoquinolyl group) The fluorescent oleamide derivative according to claim 1 or 2, its tautomer or stereoisomer, or a salt thereof.
[化 2] [Chemical 2]
Figure imgf000037_0001
Figure imgf000037_0001
[4] 前記式 (I)中、 R1および R2が、それぞれ水素原子または直鎖もしくは分枝アルキル基 である請求項 1〜3のいずれかに記載の蛍光性ォレアミド誘導体、その互変異体もし くは立体異性体、またはそれらの塩。 [4] The fluorescent oleamide derivative according to any one of claims 1 to 3, wherein R 1 and R 2 in the formula (I) are each a hydrogen atom or a linear or branched alkyl group, or a tautomer thereof Or a stereoisomer or a salt thereof.
[5] 下記式 la〜lc、 2a〜2c、 3a〜3cおよび 4a〜4cのうちいずれかで表される請求項 1に 記載の蛍光性ォレアミド誘導体、その互変異体もしくは立体異性体、またはそれらの  [5] The fluorescent oleamide derivative according to claim 1, represented by any one of the following formulas la to lc, 2a to 2c, 3a to 3c, and 4a to 4c, or a tautomer or stereoisomer thereof, or of
[化 3] [Chemical 3]
Figure imgf000038_0001
Figure imgf000038_0001
1a (C2-linked dansyl oleamide, n = 0)  1a (C2-linked dansyl oleamide, n = 0)
1b (C4-linked dansyl oleamide, n = 2)  1b (C4-linked dansyl oleamide, n = 2)
1c (C10-linked dansyl oleamide, n = 8)  1c (C10-linked dansyl oleamide, n = 8)
Figure imgf000038_0002
Figure imgf000038_0002
2a (C2-linked NBD oleamide, n = 0) 2a (C2-linked NBD oleamide, n = 0)
2b (C4-linked NBD oleamide, n = 2)  2b (C4-linked NBD oleamide, n = 2)
2c (C10-linked NBD oleamide, n = 8)  2c (C10-linked NBD oleamide, n = 8)
Figure imgf000038_0003
Figure imgf000038_0003
3a (C2-linked dansyl MI-18, n = 0)  3a (C2-linked dansyl MI-18, n = 0)
3b (C4-linked dansyl MI-18, n = 2)  3b (C4-linked dansyl MI-18, n = 2)
3c (C10-linked dansyl MI-18, n = 8)  3c (C10-linked dansyl MI-18, n = 8)
Figure imgf000038_0004
Figure imgf000038_0004
4a (C2-linked NBD MI-18, n = 0)  4a (C2-linked NBD MI-18, n = 0)
4b (C4-linked NBD MI-18, n = 2)  4b (C4-linked NBD MI-18, n = 2)
4c (C10-linked NBD MI-18, n = 8)  4c (C10-linked NBD MI-18, n = 8)
[6] 下記式 (IV)で表される、請求項;!〜 5のいずれかに記載の蛍光性ォレアミド誘導体 の二量体型化合物、その互変異体もしくは立体異性体、またはそれらの塩。 [6] The dimer compound of the fluorescent oleamide derivative according to any one of claims 5 to 6 represented by the following formula (IV), a tautomer or stereoisomer thereof, or a salt thereof.
[化 4]
Figure imgf000039_0001
[Chemical 4]
Figure imgf000039_0001
(IV) 前記式 (IV)中、 、 L2、 R1, R2および Rは、それぞれ前記式 (I)と同じであり、各 L1は同 一でも異なっていても良ぐ各 R1は同一でも異なっていても良ぐ (IV) In the formula (IV), L 2 , R 1 , R 2 and R are the same as those in the formula (I), and each L 1 may be the same or different from each other R 1 Can be the same or different
R3は水素原子または任意の置換基であり、 R 3 is a hydrogen atom or an optional substituent,
1は正の整数である。  1 is a positive integer.
[7] 下記式 (V)で表される、請求項;!〜 5のいずれかに記載の蛍光性ォレアミド誘導体 の多量体型化合物、その互変異体もしくは立体異性体、またはそれらの塩。  [7] A multimeric compound of the fluorescent oleamide derivative according to any one of claims 5 to 6 represented by the following formula (V), a tautomer or stereoisomer thereof, or a salt thereof.
[化 5] [Chemical 5]
Figure imgf000040_0001
Figure imgf000040_0001
(V) 前記式 (V)中、
Figure imgf000040_0002
R2および Rは、それぞれ前記式 (I)と同じであり、各 L1は同一で も異なっていても良ぐ (V) In the formula (V),
Figure imgf000040_0002
R 2 and R are the same as in the above formula (I), and each L 1 may be the same or different.
R4は水素原子または任意の置換基であり、 R 4 is a hydrogen atom or an optional substituent,
m、 hおよび kはそれぞれ正の整数である。  m, h and k are each a positive integer.
下記式 (νι)、（νπ)および (vm)の化合物を原料として用い、ァミンとハロゲン化物との 縮合反応工程を含む、請求項;!〜 7のいずれかに記載の蛍光性ォレアミド誘導体、 その互変異体もしくは立体異性体、またはそれらの塩の製造方法。  The fluorescent oleamide derivative according to any one of Claims 1 to 7, which comprises a condensation reaction step of an amine and a halide, using compounds of the following formulas (νι), (νπ) and (vm) as raw materials: A method for producing a tautomer or stereoisomer, or a salt thereof.
[化 6] [Chemical 6]
Figure imgf000040_0003
Figure imgf000040_0003
(VI) (VI I) (VIII) 前記式 (vi)、（vii)および (vm)中、
Figure imgf000041_0001
R2および Rは、それぞれ前記式 0)、 (ιν) または (v)と同じであり、 X1および X2は、それぞれハロゲンであり、同一でも異なってい ても良い。 (VI) (VI I) (VIII) In the formulas (vi), (vii) and (vm),
Figure imgf000041_0001
R 2 and R are the same as those in the above formulas 0), (ιν) or (v), and X 1 and X 2 are each halogen and may be the same or different.
[9] 請求項;!〜 7のいずれかに記載の蛍光性ォレアミド誘導体、その互変異体もしくは 立体異性体、またはそれらの塩を細胞に導入する工程を含む、細胞の蛍光観察方 法。  [9] A method for observing fluorescence of a cell, comprising the step of introducing the fluorescent oleamide derivative according to any one of claims! To 7 or a tautomer or stereoisomer thereof, or a salt thereof into the cell.
[10] 請求項;!〜 7のいずれかに記載の蛍光性ォレアミド誘導体、その互変異体もしくは 立体異性体、またはそれらの塩を含み、 gap結合阻害剤、ォレアミド誘導体の細胞内 作用メカニズム研究用ツール、医薬品、飲食品またはサプリメントとして用いられる製 [10] A method for studying the intracellular action mechanism of a gap binding inhibitor or an oleamide derivative, comprising the fluorescent oleamide derivative according to any one of claims 1 to 7 or a tautomer or stereoisomer thereof, or a salt thereof. Made as a tool, medicine, food or drink or supplement
PP
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PCT/JP2007/065264 2006-08-04 2007-08-03 Fluorescent oleamide derivative and use thereof WO2008016139A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011173859A (en) * 2010-01-28 2011-09-08 Mie Univ Diagnostic composition and method of analysis using new indocyanine compound
US9056131B2 (en) 2010-01-28 2015-06-16 National University Corporation Mie University Indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition
US9844606B2 (en) 2010-01-28 2017-12-19 National University Corporation Mie University Indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition
US10086090B2 (en) 2010-01-28 2018-10-02 National University Corporation Mie University Indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition
US10350310B2 (en) 2010-01-28 2019-07-16 National University Corporation Mie University Indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition
US11344632B2 (en) 2010-01-28 2022-05-31 National University Corporation Mie University Indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition

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