JP7217460B2 - A novel compound and a reagent for detecting lipid droplets containing the compound - Google Patents
A novel compound and a reagent for detecting lipid droplets containing the compound Download PDFInfo
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- JP7217460B2 JP7217460B2 JP2019090840A JP2019090840A JP7217460B2 JP 7217460 B2 JP7217460 B2 JP 7217460B2 JP 2019090840 A JP2019090840 A JP 2019090840A JP 2019090840 A JP2019090840 A JP 2019090840A JP 7217460 B2 JP7217460 B2 JP 7217460B2
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- carbon atoms
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- lipid droplets
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- 150000001875 compounds Chemical class 0.000 title claims description 86
- 150000002632 lipids Chemical class 0.000 title claims description 70
- 239000003153 chemical reaction reagent Substances 0.000 title description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 79
- 239000007850 fluorescent dye Substances 0.000 claims description 50
- 125000000217 alkyl group Chemical group 0.000 claims description 26
- 125000003545 alkoxy group Chemical group 0.000 claims description 24
- 125000005843 halogen group Chemical group 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 125000001188 haloalkyl group Chemical group 0.000 claims description 19
- 125000005055 alkyl alkoxy group Chemical group 0.000 claims description 18
- 150000003839 salts Chemical class 0.000 claims description 15
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 14
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 13
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 9
- NAWXUBYGYWOOIX-SFHVURJKSA-N (2s)-2-[[4-[2-(2,4-diaminoquinazolin-6-yl)ethyl]benzoyl]amino]-4-methylidenepentanedioic acid Chemical compound C1=CC2=NC(N)=NC(N)=C2C=C1CCC1=CC=C(C(=O)N[C@@H](CC(=C)C(O)=O)C(O)=O)C=C1 NAWXUBYGYWOOIX-SFHVURJKSA-N 0.000 claims description 8
- LFZXBUFQMSGPMQ-UHFFFAOYSA-N 2,4-bis(trifluoromethyl)quinolin-7-amine Chemical compound FC(F)(F)C1=CC(C(F)(F)F)=NC2=CC(N)=CC=C21 LFZXBUFQMSGPMQ-UHFFFAOYSA-N 0.000 claims description 7
- 230000005284 excitation Effects 0.000 claims description 6
- WZCQRUWWHSTZEM-UHFFFAOYSA-N 1,3-phenylenediamine Chemical compound NC1=CC=CC(N)=C1 WZCQRUWWHSTZEM-UHFFFAOYSA-N 0.000 claims description 5
- 229940018564 m-phenylenediamine Drugs 0.000 claims description 5
- RZAUIOKDXQWSQE-UHFFFAOYSA-N quinolin-7-amine Chemical compound C1=CC=NC2=CC(N)=CC=C21 RZAUIOKDXQWSQE-UHFFFAOYSA-N 0.000 claims description 5
- QAMFBRUWYYMMGJ-UHFFFAOYSA-N hexafluoroacetylacetone Chemical compound FC(F)(F)C(=O)CC(=O)C(F)(F)F QAMFBRUWYYMMGJ-UHFFFAOYSA-N 0.000 claims description 4
- 230000001678 irradiating effect Effects 0.000 claims description 4
- KDYVCOSVYOSHOL-UHFFFAOYSA-N quinolin-7-ylmethanamine Natural products C1=CC=NC2=CC(C)=CC=C21 KDYVCOSVYOSHOL-UHFFFAOYSA-N 0.000 claims description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- -1 2-methyl-cyclopropyl group Chemical group 0.000 description 77
- 210000004027 cell Anatomy 0.000 description 48
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 36
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 36
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 28
- 239000002904 solvent Substances 0.000 description 18
- 239000003054 catalyst Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- QARVLSVVCXYDNA-UHFFFAOYSA-N bromobenzene Chemical compound BrC1=CC=CC=C1 QARVLSVVCXYDNA-UHFFFAOYSA-N 0.000 description 12
- 239000012442 inert solvent Substances 0.000 description 11
- KZPYGQFFRCFCPP-UHFFFAOYSA-N 1,1'-bis(diphenylphosphino)ferrocene Chemical compound [Fe+2].C1=CC=C[C-]1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=C[C-]1P(C=1C=CC=CC=1)C1=CC=CC=C1 KZPYGQFFRCFCPP-UHFFFAOYSA-N 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- 238000000862 absorption spectrum Methods 0.000 description 8
- 150000003248 quinolines Chemical class 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 7
- 239000002585 base Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- LPNYRYFBWFDTMA-UHFFFAOYSA-N potassium tert-butoxide Chemical compound [K+].CC(C)(C)[O-] LPNYRYFBWFDTMA-UHFFFAOYSA-N 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 239000012299 nitrogen atmosphere Substances 0.000 description 6
- YJVFFLUZDVXJQI-UHFFFAOYSA-L palladium(ii) acetate Chemical compound [Pd+2].CC([O-])=O.CC([O-])=O YJVFFLUZDVXJQI-UHFFFAOYSA-L 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 5
- 238000001953 recrystallisation Methods 0.000 description 5
- 238000010992 reflux Methods 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
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- 230000015572 biosynthetic process Effects 0.000 description 4
- 229960001701 chloroform Drugs 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- GUJOJGAPFQRJSV-UHFFFAOYSA-N dialuminum;dioxosilane;oxygen(2-);hydrate Chemical compound O.[O-2].[O-2].[O-2].[Al+3].[Al+3].O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O GUJOJGAPFQRJSV-UHFFFAOYSA-N 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000012046 mixed solvent Substances 0.000 description 4
- 239000002798 polar solvent Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 125000001424 substituent group Chemical group 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- BWHDROKFUHTORW-UHFFFAOYSA-N tritert-butylphosphane Chemical compound CC(C)(C)P(C(C)(C)C)C(C)(C)C BWHDROKFUHTORW-UHFFFAOYSA-N 0.000 description 4
- XHCAGOVGSDHHNP-UHFFFAOYSA-N 1-bromo-4-tert-butylbenzene Chemical compound CC(C)(C)C1=CC=C(Br)C=C1 XHCAGOVGSDHHNP-UHFFFAOYSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 230000008033 biological extinction Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
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- MUJIDPITZJWBSW-UHFFFAOYSA-N palladium(2+) Chemical compound [Pd+2] MUJIDPITZJWBSW-UHFFFAOYSA-N 0.000 description 3
- LXNAVEXFUKBNMK-UHFFFAOYSA-N palladium(II) acetate Substances [Pd].CC(O)=O.CC(O)=O LXNAVEXFUKBNMK-UHFFFAOYSA-N 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- QJPJQTDYNZXKQF-UHFFFAOYSA-N 4-bromoanisole Chemical compound COC1=CC=C(Br)C=C1 QJPJQTDYNZXKQF-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- NPWZMUWHIPTQLO-UHFFFAOYSA-N N-phenyl-2,4-bis(trifluoromethyl)quinolin-7-amine Chemical compound C=1C2=NC(C(F)(F)F)=CC(C(F)(F)F)=C2C=CC=1NC1=CC=CC=C1 NPWZMUWHIPTQLO-UHFFFAOYSA-N 0.000 description 2
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- MUALRAIOVNYAIW-UHFFFAOYSA-N binap Chemical group C1=CC=CC=C1P(C=1C(=C2C=CC=CC2=CC=1)C=1C2=CC=CC=C2C=CC=1P(C=1C=CC=CC=1)C=1C=CC=CC=1)C1=CC=CC=C1 MUALRAIOVNYAIW-UHFFFAOYSA-N 0.000 description 2
- GZUXJHMPEANEGY-UHFFFAOYSA-N bromomethane Chemical compound BrC GZUXJHMPEANEGY-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
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- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical compound ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 description 2
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- 238000001816 cooling Methods 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 description 2
- 125000003963 dichloro group Chemical group Cl* 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
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- 238000002372 labelling Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
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- 230000007935 neutral effect Effects 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 229910052763 palladium Inorganic materials 0.000 description 2
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- 229910052700 potassium Inorganic materials 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 2
- COIOYMYWGDAQPM-UHFFFAOYSA-N tris(2-methylphenyl)phosphane Chemical compound CC1=CC=CC=C1P(C=1C(=CC=CC=1)C)C1=CC=CC=C1C COIOYMYWGDAQPM-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- CYPYTURSJDMMMP-WVCUSYJESA-N (1e,4e)-1,5-diphenylpenta-1,4-dien-3-one;palladium Chemical compound [Pd].[Pd].C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1.C=1C=CC=CC=1\C=C\C(=O)\C=C\C1=CC=CC=C1 CYPYTURSJDMMMP-WVCUSYJESA-N 0.000 description 1
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- JIRHAGAOHOYLNO-UHFFFAOYSA-N (3-cyclopentyloxy-4-methoxyphenyl)methanol Chemical compound COC1=CC=C(CO)C=C1OC1CCCC1 JIRHAGAOHOYLNO-UHFFFAOYSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
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- QFMZQPDHXULLKC-UHFFFAOYSA-N 1,2-bis(diphenylphosphino)ethane Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)CCP(C=1C=CC=CC=1)C1=CC=CC=C1 QFMZQPDHXULLKC-UHFFFAOYSA-N 0.000 description 1
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- 125000006433 1-ethyl cyclopropyl group Chemical group [H]C([H])([H])C([H])([H])C1(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000006438 1-i-propyl cyclopropyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C1(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000006432 1-methyl cyclopropyl group Chemical group [H]C([H])([H])C1(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000006439 1-n-propyl cyclopropyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C1(*)C([H])([H])C1([H])[H] 0.000 description 1
- KHGHGZPESHUYCR-UHFFFAOYSA-N 1h-phosphindole Chemical group C1=CC=C2PC=CC2=C1 KHGHGZPESHUYCR-UHFFFAOYSA-N 0.000 description 1
- IQHSSYROJYPFDV-UHFFFAOYSA-N 2-bromo-1,3-dichloro-5-(trifluoromethyl)benzene Chemical group FC(F)(F)C1=CC(Cl)=C(Br)C(Cl)=C1 IQHSSYROJYPFDV-UHFFFAOYSA-N 0.000 description 1
- SDTMFDGELKWGFT-UHFFFAOYSA-N 2-methylpropan-2-olate Chemical compound CC(C)(C)[O-] SDTMFDGELKWGFT-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
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Images
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Quinoline Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
特許法第３０条第２項適用 第４８回 複素環化学討論会実行委員会、「第４８回複素環化学討論会講演要旨集」第６８ページ、平成３０年８月２０日Application of
本発明は、新規化合物及び該化合物を含む脂肪滴検出用試薬に関する。特に、低極性環境中でのみ特異的に蛍光発光し、且つ、細胞の蛍光イメージングにおける特異的集積性を示す、新規化合物及び該化合物を含む脂肪滴検出用試薬に関する。 The present invention relates to a novel compound and a lipid droplet detection reagent containing the compound. In particular, it relates to a novel compound and a reagent for detecting lipid droplets containing the compound, which specifically emits fluorescence only in a low-polarity environment and shows specific accumulation in fluorescence imaging of cells.
現在まで様々な骨格をもつ有機蛍光分子が開発されており、細胞内小器官（オルガネラ）の蛍光イメージングプローブとして利用されている。また近年、高脂血症や肥満のような生活習慣病患者数の増加が社会問題となっている中で、生体内脂質分子への関心が高まっている。脂肪酸は細胞膜を構成する必須の成分であるが、細胞内ではコレステロールやトリグリセリドのような中性脂質分子が蓄積した「脂肪滴」と呼ばれるオルガネラの役割も注目されている。例えば受精卵の発育に脂肪滴が関連していることが最近報告された他（非特許文献１参照）、ＨＣＶ（Ｃ型肝炎ウィルス）のウィルス粒子形成にも関与していることなども知られている（非特許文献２参照）。
以上の背景から脂肪滴を検出する蛍光プローブは生物学的研究において有用であり、「ナイルレッド」と呼ばれる赤色蛍光分子（下記式（ｉ）参照）が古くから利用されている（非特許文献３参照）。また最近ナイルレッドに変わるＬｉｐｉＤｙｅ（登録商標）と呼ばれるベンゾホスホール骨格をもつ新規蛍光分子（非特許文献４参照）が市販されている（下記式（ii）参照）。
From the above background, a fluorescent probe that detects lipid droplets is useful in biological research, and a red fluorescent molecule called "Nile Red" (see formula (i) below) has been used for a long time (Non-Patent
また、これまでに新規二環性プッシュ―プル型蛍光分子として、“ＴＦＭＡＱ”と呼ばれる２，４－ジトリフルオロ－７－アミノキノリン誘導体が合成され、独特な蛍光発光特性を示すことが報告されている（非特許文献５参照）。
また、蛍光分子は一般的に溶媒極性の影響を受けてスペクトルが変化する「ソルバトクロミズム」を示すことが知られている。
A 2,4-ditrifluoro-7-aminoquinoline derivative called "TFMAQ" has been synthesized as a novel bicyclic push-pull fluorescent molecule, and has been reported to exhibit unique fluorescence emission properties. (See Non-Patent Document 5).
It is also known that fluorescent molecules generally exhibit "solvatochromism" in which the spectrum changes under the influence of solvent polarity.
従来の強発光性の蛍光プローブを用いた細胞イメージングにおいては、脂肪滴以外の非特異的な蛍光染色画像が得られるという問題があった。またＬｉｐｉＤｙｅ（登録商標）は合成ステップが６工程以上あるため、製造コストも高価であるという問題があった。
そこで、本発明の目的は、これら蛍光分子に代わる、特異性の高い新規脂肪滴蛍光染色プローブを提供することにある。
また、本発明の目的は、特異性の高い新規脂肪滴蛍光染色プローブに用いる新規キノリン誘導体及びより少ない工程を含む新規キノリン誘導体の製造方法を提供することにある。
In cell imaging using conventional fluorescent probes with strong luminescence, there was a problem that non-specific fluorescent staining images other than lipid droplets were obtained. Moreover, since LipiDye (registered trademark) has six or more synthetic steps, there is a problem that the production cost is high.
Therefore, an object of the present invention is to provide novel lipid droplet fluorescent staining probes with high specificity that can replace these fluorescent molecules.
Another object of the present invention is to provide a novel quinoline derivative used for a highly specific novel lipid droplet fluorescent staining probe and a method for producing a novel quinoline derivative involving fewer steps.
本発明者らは、上記目的を達成するため鋭意検討を重ねた結果、特定のキノリン環骨格を有する化合物の８位に芳香環を導入した新規キノリン誘導体又はその塩が、細胞内の脂肪滴の蛍光プローブとして使用できるとことを見出し、本発明を完成させた。 The present inventors have made intensive studies to achieve the above objects, and as a result, a novel quinoline derivative or a salt thereof, in which an aromatic ring is introduced at the 8-position of a compound having a specific quinoline ring skeleton, is effective for intracellular lipid droplets. They found that it can be used as a fluorescent probe, and completed the present invention.
すなわち、本発明は以下の［１］乃至［１０］の発明に関する。
［１］下記式（１）：
Ｒ１は、水素原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、炭素原子数１乃至１０のアルキルアルコキシ基、又は置換基を有してもよいフェニル基を表し、
Ｒ２は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｍが２以上の整数を表すとき、各々のＲ２は互いに同一であっても又は互いに相異なってもよく、
Ｒ３は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、
ｍ及びｎは、夫々独立して、０乃至５の整数を表す。）
で表される化合物又はその塩。
［２］前記式（１）で表される化合物が、下記式（２）：
で表される化合物である、［１］に記載の化合物又はその塩。
［３］前記Ｒ４が、水素原子、ｔ－ブチル基又はメトキシ基を表す、［２］に記載の化合物又はその塩。
［４］下記式（３）：
Ｒ２は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｍが２以上の整数を表すとき、各々のＲ２は互いに同一であっても又は互いに相異なってもよく、
Ｒ３は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、
ｍ及びｎは、夫々独立して、０乃至５の整数を表す。）
で表される化合物の製造方法であって、
ｍ－フェニレンジアミンと、ヘキサフルオロアセチルアセトンとを反応させて２，４－ジトリフルオロメチル－７－アミノキノリンを製造する工程Ａ、
該７－アミノキノリンを、下記式（１０）：
［５］前記式（１０）で表される化合物が下記式（１２）
［６］下記式（５）：
ル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、ｍ及びｎは、夫々独立して、０乃至５の整数である。）で表される化合物の製造方法であって、
ｍ－フェニレンジアミンと、ヘキサフルオロアセチルアセトンとを反応させて７－アミノキノリンを製造する工程Ａ、
該７－アミノキノリンを、下記式（１０）
該式（４）で表される化合物を、下記式（１４）：
Ｒ５－Ｘ （１４）
（式中、Ｘは、ハロゲン原子を表し、Ｒ５は、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルキルアルコキシ基、炭素原子数１乃至１０のアルコキシ基、又は置換基を有してもよいフェニル基を表す。）で表される化合物と反応させて、下記式（６）
［８］（ａ）［７］に記載の蛍光プローブを、脂肪滴を含む細胞又は脂肪滴と接触させる工程、及び（ｂ）該細胞又は脂肪滴に対し励起光を照射して蛍光を生じさせる工程を含む
脂肪滴の検出方法
［９］さらに、（ｃ）蛍光プローブの蛍光を測定する工程を含む、［８］に記載の方法。［１０］前記（ａ）工程の前に、（ｄ）［７］に記載の蛍光プローブを、生きている生物個体内（ヒトを除く）に投与する工程を含む、［８］又は［９］に記載の方法。
That is, the present invention relates to the following inventions [1] to [10].
[1] Formula (1) below:
R 1 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, an alkylalkoxy group having 1 to 10 carbon atoms, or substituted represents a phenyl group which may have a group,
R 2 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group, and when m represents an integer of 2 or more, each R 2 may be the same or different from each other,
R 3 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group and n represents an integer of 2 or more, each R 3 may be the same or different from each other,
m and n each independently represent an integer of 0 to 5; )
A compound represented by or a salt thereof.
[2] The compound represented by the above formula (1) is represented by the following formula (2):
The compound or its salt according to [1], which is a compound represented by
[3] The compound or salt thereof according to [2], wherein R 4 represents a hydrogen atom, a t-butyl group or a methoxy group.
[4] Formula (3) below:
R 2 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group, and when m represents an integer of 2 or more, each R 2 may be the same or different from each other,
R 3 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group and n represents an integer of 2 or more, each R 3 may be the same or different from each other,
m and n each independently represent an integer of 0 to 5; )
A method for producing a compound represented by
Step A of reacting m-phenylenediamine with hexafluoroacetylacetone to produce 2,4-ditrifluoromethyl-7-aminoquinoline;
The 7-aminoquinoline is represented by the following formula (10):
[5] The compound represented by the above formula (10) is represented by the following formula (12)
[6] Formula (5) below:
Step A of producing 7-aminoquinoline by reacting m-phenylenediamine with hexafluoroacetylacetone;
The 7-aminoquinoline is represented by the following formula (10)
The compound represented by the formula (4) is represented by the following formula (14):
R 5 -X (14)
(wherein X represents a halogen atom, R 5 represents an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkylalkoxy group having 1 to 10 carbon atoms, represents an alkoxy group of 1 to 10, or a phenyl group which may have a substituent.) to react with a compound represented by the following formula (6)
[8] (a) The fluorescent probe according to [7], the step of contacting with cells or lipid droplets containing lipid droplets, and (b) irradiating the cells or lipid droplets with excitation light to generate fluorescence The method according to [8], further comprising the step of (c) measuring the fluorescence of the fluorescent probe. [10] [8] or [9], including the step of administering (d) the fluorescent probe according to [7] into a living organism (excluding humans) before step (a) The method described in .
本発明によれば、特定キノリン誘導体のキノリン環の８位に芳香環を導入することで、蛍光団共役系の延長及び分子内電荷移動状態を変化させた新規キノリン誘導体を提供できる。
また、本発明よれば、低コストに抑えられた上記キノリン誘導体の３又は４ステップの製造方法を提供できる。
また、本発明によれば、高極性のジメチルスルホキシド（ＤＭＳＯ）溶液中ではほとんど蛍光発光を示さないが（蛍光量子収率１％以下）、低極性溶媒中では４８０－５６０ｎｍ付近に３０－５０％程度の蛍光量子収率で緑色蛍光発光することが可能な、低極性環境中でのみ特異的に蛍光発光する性質（Ｓ／Ｎ比２０倍以上）を有する新規キノリン誘導体を提供できる。
そして、本発明によれば、該新規キノリン誘導体を利用して、中性脂質が集積した脂肪滴部位に対して、特異的集積性を示す蛍光発光が観察される細胞の蛍光イメージングに使用する汎用性の高い蛍光プローブを提供できる。
According to the present invention, by introducing an aromatic ring at the 8-position of the quinoline ring of a specific quinoline derivative, it is possible to provide a novel quinoline derivative in which the fluorophore conjugated system is extended and the intramolecular charge transfer state is changed.
Moreover, according to the present invention, it is possible to provide a 3- or 4-step production method of the quinoline derivative at low cost.
In addition, according to the present invention, in a highly polar dimethyl sulfoxide (DMSO) solution, it hardly exhibits fluorescence emission (fluorescence quantum yield of 1% or less), but in a low polar solvent, 30-50% fluorescence is emitted near 480-560 nm. It is possible to provide a novel quinoline derivative that can emit green fluorescence with a certain level of fluorescence quantum yield and has the property of specifically emitting fluorescence only in a low-polarity environment (S/N ratio of 20 times or more).
Then, according to the present invention, the novel quinoline derivative is used for fluorescence imaging of cells in which fluorescence emission showing specific accumulation is observed for lipid droplet sites where neutral lipids are accumulated. It is possible to provide a highly efficient fluorescent probe.
本発明の化合物は、下記式（１）：
Ｒ１は、水素原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、炭素原子数１乃至１０のアルキルアルコキシ基、又は置換基を有してもよいフェニル基を表し、
Ｒ２は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｍが２以上の整数を表すとき、各々のＲ２は互いに同一であっても又は互いに相異なってもよく、
Ｒ３は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、
ｍ及びｎは、夫々独立して、０乃至５の整数を表す。）
で表される新規化合物又はその塩である。
The compounds of the present invention have the following formula (1):
R 1 is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, an alkylalkoxy group having 1 to 10 carbon atoms, or substituted represents a phenyl group which may have a group,
R 2 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group, and when m represents an integer of 2 or more, each R 2 may be the same or different from each other,
R 3 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group and n represents an integer of 2 or more, each R 3 may be the same or different from each other,
m and n each independently represent an integer of 0 to 5; )
It is a novel compound represented by or a salt thereof.
炭素原子数１乃至１０のアルキル基としては、炭素原子数１乃至１０のアルキル基としては、メチル基、エチル基、ｎ－プロピル基、i－プロピル基、シクロプロピル基、ｎ－ブチル基、ｉ－ブチル基、ｓ－ブチル基、t－ブチル基、シクロブチル基、１－メチル－シクロプロピル基、２－メチル－シクロプロピル基、ｎ－ペンチル基、１－メチル－ｎ－ブチル基、２－メチル－ｎ－ブチル基、３－メチル－ｎ－ブチル基、１,１－ジメチル－ｎ－プロピル基、１,２－ジメチル－ｎ－プロピル基、２,２－ジメチル－ｎ－プロピル基、１－エチル－ｎ－プロピル基、シクロペンチル基、１－メチル－シクロブチル基、２－メチル－シクロブチル基、３－メチル－シクロブチル基、１,２－ジメチル－シクロプロピル基、２,３－ジメチル－シクロプロピル基、１－エチル－シクロプロピル基、２－エチル－シクロプロピル基、ｎ－ヘキシル基、１－メチル－ｎ－ペンチル基、２－メチル－ｎ－ペンチル基、３－メチル－ｎ－ペンチル基、４－メチル－ｎ－ペンチル基、１,１－ジメチル－ｎ－ブチル基、１,２－ジメチル－ｎ－ブチル基、１,３－ジメチル－ｎ－ブチル基、２,２－ジメチル－ｎ－ブチル基、２,３－ジメチル－ｎ－ブチル基、３,３－ジメチル－ｎ－ブチル基、１－エチル－ｎ－ブチル基、２－エチル－ｎ－ブチル基、１,１,２－トリメチル－ｎ－プロピル基、１,２,２－トリメチル－ｎ－プロピル基、１－エチル－１－メチル－ｎ－プロピル基、１－エチル－２－メチル－ｎ－プロピル基、シクロヘキシル基、１－メチル－シクロペンチル基、２－メチル－シクロペンチル基、３－メチル－シクロペンチル基、１－エチル－シクロブチル基、２－エチル－シクロブチル基、３－エチル－シクロブチル基、１,２－ジメチル－シクロブチル基、１,３－ジメチル－シクロブチル基、２,２－ジメチル－シクロブチル基、２,３－ジメチル－シクロブチル基、２,４－ジメチル－シクロブチル基、３,３－ジメチル－シクロブチル基、１－ｎ－プロピル－シクロプロピル基、２－ｎ－プロピル－シクロプロピル基、１－i－プロピル－シクロプロピル基、２－ｉ－プロピル－シクロプロピル基、１,２,２－トリメチル－シクロプロピル基、１,２,３－トリメチル－シクロプロピル基、２,２,３－トリメチル－シクロプロピル基、１－エチル－２－メチル－シクロプロピル基、２－エチル－１－メチル－シクロプロピル基、２－エチル－２－メチル－シクロプロピル基及び２－エチル－３－メチル－シク
ロプロピル基等が挙げられる。
Examples of alkyl groups having 1 to 10 carbon atoms include methyl group, ethyl group, n-propyl group, i-propyl group, cyclopropyl group, n-butyl group, i -butyl group, s-butyl group, t-butyl group, cyclobutyl group, 1-methyl-cyclopropyl group, 2-methyl-cyclopropyl group, n-pentyl group, 1-methyl-n-butyl group, 2-methyl -n-butyl group, 3-methyl-n-butyl group, 1,1-dimethyl-n-propyl group, 1,2-dimethyl-n-propyl group, 2,2-dimethyl-n-propyl group, 1- ethyl-n-propyl group, cyclopentyl group, 1-methyl-cyclobutyl group, 2-methyl-cyclobutyl group, 3-methyl-cyclobutyl group, 1,2-dimethyl-cyclopropyl group, 2,3-dimethyl-cyclopropyl group , 1-ethyl-cyclopropyl group, 2-ethyl-cyclopropyl group, n-hexyl group, 1-methyl-n-pentyl group, 2-methyl-n-pentyl group, 3-methyl-n-pentyl group, 4 -methyl-n-pentyl group, 1,1-dimethyl-n-butyl group, 1,2-dimethyl-n-butyl group, 1,3-dimethyl-n-butyl group, 2,2-dimethyl-n-butyl group, 2,3-dimethyl-n-butyl group, 3,3-dimethyl-n-butyl group, 1-ethyl-n-butyl group, 2-ethyl-n-butyl group, 1,1,2-trimethyl- n-propyl group, 1,2,2-trimethyl-n-propyl group, 1-ethyl-1-methyl-n-propyl group, 1-ethyl-2-methyl-n-propyl group, cyclohexyl group, 1-methyl -cyclopentyl group, 2-methyl-cyclopentyl group, 3-methyl-cyclopentyl group, 1-ethyl-cyclobutyl group, 2-ethyl-cyclobutyl group, 3-ethyl-cyclobutyl group, 1,2-dimethyl-cyclobutyl group, 1, 3-dimethyl-cyclobutyl group, 2,2-dimethyl-cyclobutyl group, 2,3-dimethyl-cyclobutyl group, 2,4-dimethyl-cyclobutyl group, 3,3-dimethyl-cyclobutyl group, 1-n-propyl-cyclo propyl group, 2-n-propyl-cyclopropyl group, 1-i-propyl-cyclopropyl group, 2-i-propyl-cyclopropyl group, 1,2,2-trimethyl-cyclopropyl group, 1,2,3 -trimethyl-cyclopropyl group, 2,2,3-trimethyl-cyclopropyl group, 1-ethyl-2-methyl-cyclopropyl group, 2-ethyl-1- Examples include methyl-cyclopropyl group, 2-ethyl-2-methyl-cyclopropyl group and 2-ethyl-3-methyl-cyclopropyl group.
炭素原子数１乃至１０のハロアルキル基としては上述の炭素原子数１乃至１０のアルキル基において、フッ素原子、塩素原子、臭素原子及びヨウ素原子からなる群から選択される少なくとも一種のハロゲン原子で置換されたアルキル基を挙げることができる。 The haloalkyl group having 1 to 10 carbon atoms is the above alkyl group having 1 to 10 carbon atoms, which is substituted with at least one halogen atom selected from the group consisting of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. and alkyl groups.
炭素原子数１乃至１０のアルコキシ基としては、炭素原子数１乃至１０のアルコキシ基としてはメトキシ基、エトキシ基、ｎ－プロポキシ基、ｉ－プロポキシ基、ｎ－ブトキシ基、ｉ－ブトキシ基、ｓ－ブトキシ基、ｔ－ブトキシ基、ｎ－ペントキシ基、１－メチル－ｎ－ブトキシ基、２－メチル－ｎ－ブトキシ基、３－メチル－ｎ－ブトキシ基、１，１－ジメチル－ｎ－プロポキシ基、１，２－ジメチル－ｎ－プロポキシ基、２，２－ジメチル－ｎ－プロポキシ基、１－エチル－ｎ－プロポキシ基、ｎ－ヘキシルオキシ基、１－メチル－ｎ－ペンチルオキシ基、２－メチル－ｎ－ペンチルオキシ基、３－メチル－ｎ－ペンチルオキシ基、４－メチル－ｎ－ペンチルオキシ基、１，１－ジメチル－ｎ－ブトキシ基、１，２－ジメチル－ｎ－ブトキシ基、１，３－ジメチル－ｎ－ブトキシ基、２，２－ジメチル－ｎ－ブトキシ基、２，３－ジメチル－ｎ－ブトキシ基、３，３－ジメチル－ｎ－ブトキシ基、１－エチル－ｎ－ブトキシ基、２－エチル－ｎ－ブトキシ基、１，１，２－トリメチル－ｎ－プロポキシ基、１，２，２，－トリメチル－ｎ－プロポキシ基、１－エチル－１－メチル－ｎ－プロポキシ基、及び１－エチル－２－メチル－ｎ－プロポキシ基等が挙げられる。 Examples of alkoxy groups having 1 to 10 carbon atoms include methoxy group, ethoxy group, n-propoxy group, i-propoxy group, n-butoxy group, i-butoxy group, s -butoxy, t-butoxy, n-pentoxy, 1-methyl-n-butoxy, 2-methyl-n-butoxy, 3-methyl-n-butoxy, 1,1-dimethyl-n-propoxy group, 1,2-dimethyl-n-propoxy group, 2,2-dimethyl-n-propoxy group, 1-ethyl-n-propoxy group, n-hexyloxy group, 1-methyl-n-pentyloxy group, 2 -methyl-n-pentyloxy group, 3-methyl-n-pentyloxy group, 4-methyl-n-pentyloxy group, 1,1-dimethyl-n-butoxy group, 1,2-dimethyl-n-butoxy group , 1,3-dimethyl-n-butoxy group, 2,2-dimethyl-n-butoxy group, 2,3-dimethyl-n-butoxy group, 3,3-dimethyl-n-butoxy group, 1-ethyl-n -butoxy group, 2-ethyl-n-butoxy group, 1,1,2-trimethyl-n-propoxy group, 1,2,2,-trimethyl-n-propoxy group, 1-ethyl-1-methyl-n- Propoxy group, 1-ethyl-2-methyl-n-propoxy group, and the like.
炭素原子数１乃至１０のアルキルアルコキシ基としては、上述のアルキル基において、上述のアルコキシ基で置換された炭素原子数１乃至１０のアルキルアルコキシ基を挙げることができる。 Examples of the alkylalkoxy group having 1 to 10 carbon atoms include alkylalkoxy groups having 1 to 10 carbon atoms in which the above alkyl group is substituted with the above alkoxy group.
置換基を有するフェニル基の置換基としては、ハロゲン原子、ヒドロキシ基、炭素原子数１～１０のアルキル基又はアルコキシル基が挙げられ、好ましくは、炭素原子数１～５のアルキル基若しくはアルコキシル基が挙げられる。 Substituents of the phenyl group having a substituent include a halogen atom, a hydroxy group, an alkyl group having 1 to 10 carbon atoms, or an alkoxyl group, preferably an alkyl group having 1 to 5 carbon atoms or an alkoxyl group. mentioned.
上記ハロゲン原子としてはフッ素原子、塩素原子、臭素原子及びヨウ素原子が挙げられる。 The halogen atoms include fluorine, chlorine, bromine and iodine atoms.
本発明の化合物の好ましい例は、下記式（２）で表される位化合物である。
＜式（１）で表される化合物又はその塩の製造＞
また、本発明は、式（１）で表される化合物又はその塩の製造方法に関し、下記の製造方法により式（１）で表される化合物を得ることができる。
（工程Ａ）
触媒としては、コンベス キノリン反応に使用可能な酸触媒、例えば、メタンスルホン酸等の脂肪族スルホン酸、ｐ－トルエンスルホン酸、ベンゼンスルホン酸およびキシレンスルホン酸等の芳香族スルホン酸を包含するスルホン酸；硫酸、発煙硫酸、リン酸等の鉱酸；ギ酸、酢酸、プロピオン酸、トリフルオロ酢酸等のカルボン酸；三フッ化ホウ素－テトラヒドロフラン（ＴＨＦ）錯体、塩化アルミニウム、塩化亜鉛等のルイス酸；モンモリロナイトＫ－１０、硫酸化ジルコニア等の固体酸等が挙げられ、好ましくはモンモリロナイトＫ１０が挙げられる。
不活性溶媒としては、反応に関与しないものであればよく、ヘキサン、ヘプタン等の飽和炭化水素系、ベンゼン、トルエン等の芳香族炭化水素系、ジエチルエーテル、ジイソプロピルエーテル、ｔ－ブチルメチルエーテル、テトラヒドロフラン、ジオキサン等のエーテル系、ジクロロメタン、トリクロロメタン、ジクロロエタン等のハロゲン系、酢酸メチル、酢酸エチル、酢酸プロピル、酢酸ブチル等のエステル系又はこれらの混合溶媒を挙げることができる、特に芳香族炭化水素系、例えばトルエンが好ましい。
例えば、不活性溶媒としてトルエン中、モンモリロナイトＫ１０触媒の存在下、好ましくは８０乃至１００の温度で２０分乃至３時間反応を行う。
<Production of compound represented by formula (1) or salt thereof>
The present invention also relates to a method for producing the compound represented by formula (1) or a salt thereof, and the compound represented by formula (1) can be obtained by the following production method.
(Step A)
Examples of the catalyst include acid catalysts that can be used in the Convesquinoline reaction, sulfonic acids including aliphatic sulfonic acids such as methanesulfonic acid, and aromatic sulfonic acids such as p-toluenesulfonic acid, benzenesulfonic acid and xylenesulfonic acid. Mineral acids such as sulfuric acid, fuming sulfuric acid and phosphoric acid; Carboxylic acids such as formic acid, acetic acid, propionic acid and trifluoroacetic acid; Lewis acids such as boron trifluoride-tetrahydrofuran (THF) complex, aluminum chloride and zinc chloride; Montmorillonite Examples include solid acids such as K-10 and sulfated zirconia, and preferably montmorillonite K10.
Any inert solvent may be used as long as it does not participate in the reaction, and includes saturated hydrocarbons such as hexane and heptane, aromatic hydrocarbons such as benzene and toluene, diethyl ether, diisopropyl ether, t-butyl methyl ether, and tetrahydrofuran. , ethers such as dioxane; halogens such as dichloromethane, trichloromethane and dichloroethane; esters such as methyl acetate, ethyl acetate, propyl acetate and butyl acetate; , for example toluene, is preferred.
For example, the reaction is carried out in toluene as an inert solvent in the presence of a montmorillonite K10 catalyst at a temperature of preferably 80 to 100 for 20 minutes to 3 hours.
（工程Ｂ１）
式（１０）で表される化合物としては、ブロモベンゼンが好ましい。
(Step B1)
Bromobenzene is preferred as the compound represented by formula (10).
（工程Ｂ２）
４）（式中、Ｘ及びＲ５は上記と同じ意味を表す。）で表される化合物と反応させて、式（６）で表される化合物を得る。
式（１４）で表される化合物としては、ヨウ化メチル、臭化メチル、塩化メチル、臭化エチル等のハロアルキル類又はブロモベンゼンが好ましい。
(Step B2)
4) (wherein X and R5 have the same meanings as above) to obtain a compound represented by formula (6).
As the compound represented by formula (14), haloalkyls such as methyl iodide, methyl bromide, methyl chloride and ethyl bromide, or bromobenzene are preferred.
この工程Ｂ１及びＢ２は、例えば、バックワルド・ハートウィッグアミノ化反応、ゴールドバーグ アミノ化反応及びジョルダン・ウルマン・ゴルトベルク反応等の公知の方法に準じて行うことができ、例えば、触媒、塩基及びリガンドの存在下において、不活性溶媒中で、室温乃至溶媒の還流温度の温度範囲にて行われる。 These steps B1 and B2 can be carried out, for example, according to known methods such as Buchwald-Hartwig amination reaction, Goldberg amination reaction and Jordan-Ullmann-Goldberg reaction. in the presence of and in an inert solvent at a temperature range from room temperature to the reflux temperature of the solvent.
触媒としては、例えばパラジウム触媒又は銅触媒を使用できる。
パラジウム触媒としては、例えば、ジクロロビス（トリフェニルホスフィン）パラジウム、テトラキストリフェニルホスフィンパラジウム（０）、トリス（ジベンジリデンアセトン）ジパラジウム、酢酸パラジウム（ＩＩ）またはジクロロビス（ジフェニルホスフィンフェロセニル）パラジウム（ＩＩ）などの触媒が挙げられ、工程Ｂ１にはジクロロビス（ジフェニルホスフィンフェロセニル）パラジウム（ＩＩ）が好ましく、工程Ｂ２には酢酸パラジウム（ＩＩ）が好ましい。
As a catalyst, for example a palladium catalyst or a copper catalyst can be used.
Palladium catalysts include, for example, dichlorobis(triphenylphosphine)palladium, tetrakistriphenylphosphinepalladium(0), tris(dibenzylideneacetone)dipalladium, palladium(II) acetate or dichlorobis(diphenylphosphineferrocenyl)palladium(II ), preferably dichlorobis(diphenylphosphineferrocenyl)palladium(II) for step B1 and palladium(II) acetate for step B2.
また、リガンドとしては、例えば、トリ（ｏ－トリル）ホスフィン、トリシクロヘキシルホスフィン、トリｔｅｒｔ－ブチルホスフィン、トリフェニルホスフィン、トリス（２－メチルフェノキシ）ホスフィン、トリフェノキシホスフィン、トリメトキシホスフィン、４，５－ビス（ジフェニルホスフィノ）－９，９’－ジメチルキサンテン、１，２－ビス（ジフェニルホスフィノ）エタン、２，２’－ビス（ジフェニルホスフィノ）－１，１’－ビナフチル（ＢＩＮＡＰ）および１，１’－ビス（ジフェニルホスファニル）フェロセンが挙げられ、工程Ｂ１には１，１’－ビス（ジフェニルホスファニル）フェロセンが好ましく、工程Ｂ２にはトリｔｅｒｔ－ブチルホスフィンが好ましい。 Examples of ligands include tri(o-tolyl)phosphine, tricyclohexylphosphine, tritert-butylphosphine, triphenylphosphine, tris(2-methylphenoxy)phosphine, triphenoxyphosphine, trimethoxyphosphine, 4,5 -bis(diphenylphosphino)-9,9'-dimethylxanthene, 1,2-bis(diphenylphosphino)ethane, 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP) and 1,1′-bis(diphenylphosphanyl)ferrocene is preferred for step B1 and tri-tert-butylphosphine is preferred for step B2.
銅触媒としては、ヨウ化銅（Ｉ）、臭化銅（Ｉ）、塩化銅（Ｉ）、酢酸銅（ＩＩ）、硫酸銅（ＩＩ）等を挙げることができる。また、リガンドとしては、例えばトランス－Ｎ，Ｎ‘－ジメチルシクロヘキサン－１，２－ジアミン等が挙げられる。 Copper catalysts include copper (I) iodide, copper (I) bromide, copper (I) chloride, copper (II) acetate, and copper (II) sulfate. Examples of ligands include trans-N,N'-dimethylcyclohexane-1,2-diamine.
塩基としては、例えば、酢酸カリウム、炭酸セシウム、炭酸カリウムもしくは炭酸ナトリウム、水酸化バリウム、リン酸カリウム、カリウムｔｅｒｔ－ブトキシド、フッ化カルシウム、フッ化カリウムまたはリン酸三カリウムが挙げられ、工程Ｂ１及びＢ２にはカリウムｔｅｒｔ－ブトキシドが好ましい。 Bases include, for example, potassium acetate, cesium carbonate, potassium carbonate or sodium carbonate, barium hydroxide, potassium phosphate, potassium tert-butoxide, calcium fluoride, potassium fluoride or tripotassium phosphate. Potassium tert-butoxide is preferred for B2.
不活性溶媒としては、上記に挙げた不活性溶媒を挙げることができ、特に芳香族炭化水素系、例えばトルエンが好ましい。
反応温度は室温乃至の溶媒の還流温度、好ましくは８０℃乃至溶媒の還流温度である。反応時間は反応温度によって異なるが、５分～４８時間、好ましくは４～１２時間である。
Examples of the inert solvent include the inert solvents listed above, and aromatic hydrocarbons such as toluene are particularly preferred.
The reaction temperature is from room temperature to the reflux temperature of the solvent, preferably from 80° C. to the reflux temperature of the solvent. Although the reaction time varies depending on the reaction temperature, it is 5 minutes to 48 hours, preferably 4 to 12 hours.
また、２，４－ジトリフルオロメチル－７－アミノキノリンに、１当量以上、例えば２乃至５当量の式（１０）で表される化合物を反応させることで、式（４）で表される化合物と式（６）で表される化合物との混合物を得ることができる。この混合物は、各々単離して又は混合物の状態で、次の工程Ｃ１及びＣ２の原料として使用できる。また混合物は、例えば、シリカゲルクロマトグラフ等の公知の方法により単離することができる。 Further, by reacting 2,4-ditrifluoromethyl-7-aminoquinoline with 1 equivalent or more, for example, 2 to 5 equivalents of the compound represented by formula (10), the compound represented by formula (4) is obtained. and a compound of formula (6) can be obtained. This mixture can be used as a starting material for the subsequent steps C1 and C2, either individually or in the form of a mixture. Also, the mixture can be isolated by a known method such as silica gel chromatography.
（工程Ｃ１及びＣ２）
式（１１）で表される化合物として、ブロモベンゼン、４－ｔｅｒｔ－ブチル－１－ブロモベンゼン、４－メトキシ－１－ブロモベンゼンが好ましい。
反応は、クロスカップリング反応等の公知の方法に準じて行うことができ、例えば、不活性溶媒中、触媒、塩基及びリガンドの存在下において室温乃至溶媒の還流温度の温度範囲にて行われる。
(Steps C1 and C2)
Bromobenzene, 4-tert-butyl-1-bromobenzene, and 4-methoxy-1-bromobenzene are preferred as the compound represented by formula (11).
The reaction can be carried out according to a known method such as a cross-coupling reaction, and is carried out, for example, in an inert solvent in the presence of a catalyst, a base and a ligand at a temperature ranging from room temperature to the reflux temperature of the solvent.
不活性溶媒、触媒、リガンド、塩基及び反応条件は上記に例示したものを使用できる
例えば、不活性溶媒としてトルエン、触媒として酢酸１，１－ビス（ジフェニルホスフィノ）フェロセンパラジウム（ＩＩ）ジクロリド、塩基としてカリウムｔｅｒｔ－ブトキシドを用いることができる。
また、例えば、不活性溶媒としてトルエン、触媒として酢酸パラジウム（ＩＩ）、リガンドとして１，１’－ビス（ジフェニルホスフィノ）フェロセン、塩基としてカリウムｔｅｒｔ－ブトキシドを用いることができる。
Inert solvent, catalyst, ligand, base and reaction conditions can be those exemplified above. For example, toluene as inert solvent, 1,1-bis(diphenylphosphino)ferrocene palladium (II) dichloride acetate as catalyst, base Potassium tert-butoxide can be used as the
Alternatively, for example, toluene can be used as an inert solvent, palladium(II) acetate as a catalyst, 1,1′-bis(diphenylphosphino)ferrocene as a ligand, and potassium tert-butoxide as a base.
また、反応温度及び反応時間は、使用する式（１１）で表されル化合物により適宜調整でき、例えば、８０℃～乃至不活性溶媒の還流温度（例えばトルエンの場合は約１１０℃）で３０分乃至２４時間、好ましくは２時間乃至１８時間、例えば３時間乃至１２時間である。 In addition, the reaction temperature and reaction time can be appropriately adjusted depending on the compound represented by formula (11) to be used. to 24 hours, preferably 2 to 18 hours, such as 3 to 12 hours.
また、各工程において必要に応じて精製を行うことができる。精製方法としてはクロマトグラフィー、再結晶、蒸留、昇華、再沈殿、吸着、分液処理等が挙げられる。精製剤の具体例としてはシリカゲル、ＮＨ２シリカゲル、アルミナ、活性炭等が挙げられる。 Moreover, refinement|purification can be performed as needed in each process. Purification methods include chromatography, recrystallization, distillation, sublimation, reprecipitation, adsorption, liquid separation treatment, and the like. Specific examples of purification agents include silica gel, NH2 silica gel, alumina, activated carbon, and the like.
＜脂肪滴の検出用蛍光プローブ＞
本発明は、上記の本発明の式（１）で表される化合物又はその塩を含有する、細胞内の脂肪滴の検出用蛍光プローブである。
該蛍光プローブは、式（１）で表される化合物又はその塩を１種又は２種以上を含有しても良く、また脂肪滴の検出に用いられる公知の化合物を含んでいても良い。
脂肪滴の検出に用いられる公知の化合物としては、例えばナイルレッド、ＢＯＤＩＰＹ４９３／５０３、ＬＤ４５０、リピッドグリーン及びＳＦ４４が挙げられる。
<Fluorescent probe for detecting lipid droplets>
The present invention is a fluorescent probe for detecting intracellular lipid droplets, which contains the compound represented by formula (1) of the present invention or a salt thereof.
The fluorescent probe may contain one or more compounds represented by formula (1) or salts thereof, and may contain known compounds used for detecting lipid droplets.
Known compounds used for detecting lipid droplets include, for example, Nile Red, BODIPY493/503, LD450, Lipid Green and SF44.
本発明の式（１）で表される化合物を含む蛍光プローブは、溶媒の極性の影響を受けてスペクトルが変化するソルバトクロミズムを示し、溶媒の極性が増大するとともに蛍光発光が低下する。そして、低極性溶媒中では、例えばＳ／Ｎ比が１０倍以上、好ましくは２
０倍以上で、強く蛍光発光するので、蛍光プローブは、ソルバトクロミックの蛍光プローブとして有用である。
A fluorescent probe containing the compound represented by the formula (1) of the present invention exhibits solvatochromism, in which the spectrum changes under the influence of the polarity of the solvent, and fluorescence emission decreases as the polarity of the solvent increases. And, in a low polar solvent, for example, the S/N ratio is 10 times or more, preferably 2
Fluorescent probes are useful as solvatochromic fluorescent probes because they emit strong fluorescence at 0-fold or higher.
本発明の蛍光プローブは、周囲の環境によって吸収極大波長及び蛍光極大波長が変化する化合物であるから、蛍光させようとするものに併せた波長の光を照射することにより所望の蛍光を得ることができる。例えば、本発明の蛍光プローブにあわせた波長の光を照射した場合には、極性溶媒雰囲気下では本発明の蛍光プローブはほとんど励起されず蛍光しない一方、脂肪滴中のように非極性溶媒雰囲気下では本発明の蛍光プローブは蛍光する。
また、本発明の蛍光プローブは、細胞膜及び脂肪滴内部に存在しやすく、特に非極性の親油性（疎水性）部分である脂肪滴内部に存在しやすい。このことから、本発明の蛍光プローブにあわせた波長の光を照射した場合には、脂肪滴内部の本発明の蛍光プローブがより強く蛍光発色するのに対し、細胞内の脂肪滴以外に存在する蛍光プローブは蛍光発色しないので、本発明の蛍光プローブは細胞内の脂肪滴の検出に非常に有用である。
つまり、本発明の蛍光プローブを使用した場合には、細胞中の脂肪滴を必要に応じて蛍光発光させることが可能である。このため、本発明の蛍光プローブは、細胞中に脂肪滴ができた場合の挙動や、脂肪滴を含む細胞又は脂肪滴がどのように変化するのか解析が可能である。
Since the fluorescent probe of the present invention is a compound whose absorption maximum wavelength and fluorescence maximum wavelength change depending on the surrounding environment, it is possible to obtain desired fluorescence by irradiating with light having a wavelength that matches what is to be fluoresced. can. For example, when irradiated with light of a wavelength that matches the fluorescent probe of the present invention, the fluorescent probe of the present invention is hardly excited and does not fluoresce in a polar solvent atmosphere, while in a nonpolar solvent atmosphere like in lipid droplets The fluorescent probe of the present invention then fluoresces.
In addition, the fluorescent probe of the present invention tends to exist inside cell membranes and lipid droplets, and particularly tends to exist inside lipid droplets, which are non-polar lipophilic (hydrophobic) moieties. From this, when irradiated with light having a wavelength that matches the fluorescent probe of the present invention, the fluorescent probe of the present invention inside the lipid droplets is more strongly fluorescent, whereas other than the intracellular lipid droplets present Since the fluorescent probe does not develop fluorescent color, the fluorescent probe of the present invention is very useful for detecting intracellular lipid droplets.
That is, when the fluorescent probe of the present invention is used, lipid droplets in cells can be caused to fluoresce as necessary. Therefore, the fluorescent probe of the present invention can analyze the behavior when lipid droplets are formed in cells, and how cells or lipid droplets containing lipid droplets change.
＜脂肪滴の検出方法＞
また本発明は、（ａ）本発明の蛍光プローブを、脂肪滴を含む細胞又は脂肪滴と接触させる工程、及び（ｂ）該細胞に対し励起光を照射して蛍光を生じさせる工程を含む脂肪滴の検出方法である。
さらに、本発明の脂肪滴の検出方法は、（ｃ）蛍光プローブの蛍光を測定する工程を含むことができる。
<Method for detecting lipid droplets>
The present invention also provides (a) the fluorescent probe of the present invention, the step of contacting with cells or lipid droplets containing lipid droplets, and (b) lipid comprising the step of irradiating the cells with excitation light to generate fluorescence Drop detection method.
Furthermore, the method for detecting lipid droplets of the present invention can include (c) the step of measuring the fluorescence of a fluorescent probe.
本発明の式（１）で表される化合物又はその塩の添加量は、使用する細胞や脂肪滴の割合などによっても変化し得るが、例えば、０．０１～１００μＭ、好ましくは０．１～１０μＭの終濃度で脂肪滴を含む細胞に添加することができる。
本発明の化合物を溶媒に溶解させてから脂肪滴を含む細胞に添加する場合、該溶媒として、例えば、ジメチルスルホキシド（ＤＭＳＯ）及び生理食塩水等の極性溶媒を用いることができ、好ましくはジメチルスルホキシドである。
The added amount of the compound represented by the formula (1) of the present invention or a salt thereof may vary depending on the ratio of cells and lipid droplets used, for example, 0.01 to 100 μM, preferably 0.1 to A final concentration of 10 μM can be added to cells containing lipid droplets.
When the compound of the present invention is dissolved in a solvent and then added to cells containing lipid droplets, the solvent may be, for example, a polar solvent such as dimethylsulfoxide (DMSO) and saline, preferably dimethylsulfoxide is.
本発明の式（１）で表される化合物を添加する細胞としては、脂肪滴を含む細胞であれば特に制限はなく、例えば３Ｔ３－Ｌ１細胞、単離脂肪細胞が挙げられる。また、脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞中に人為的に脂肪滴を形成させた細胞を用いてもよい。脂肪滴を含まない細胞又は脂肪滴の含有量が少ない細胞としては、ＨｅＬａ細胞、ＵＥＥＴ－１２細胞、ＮＩＨ３Ｔ３細胞などが挙げられる。また、脂肪滴を形成させる方法としては、例えば、オレイン酸を細胞に添加する方法、インスリン、ＩＢＭＸ，ＤＥＸのカクテルを細胞に添加することにより脂肪滴を誘導する方法が挙げられる。 The cells to which the compound represented by formula (1) of the present invention is added are not particularly limited as long as they contain lipid droplets, and examples thereof include 3T3-L1 cells and isolated adipocytes. Alternatively, cells in which lipid droplets are artificially formed in cells containing no lipid droplets or cells with a low lipid droplet content may be used. Cells containing no lipid droplets or cells with a low content of lipid droplets include HeLa cells, UEET-12 cells, NIH3T3 cells, and the like. In addition, methods for forming lipid droplets include, for example, a method of adding oleic acid to cells, and a method of inducing lipid droplets by adding a cocktail of insulin, IBMX, and DEX to cells.
本発明の脂肪滴の検出方法は、脂肪滴を含む細胞又は脂肪滴に蛍光プローブを接触させて、脂肪滴を蛍光プローブで標識し、該標識された脂肪滴に蛍光プローブにあわせた励起光を照射して蛍光させ、生じた蛍光を観察する。 The method for detecting lipid droplets of the present invention involves contacting a fluorescent probe to cells or lipid droplets containing lipid droplets, labeling the lipid droplets with a fluorescent probe, and applying excitation light to the labeled lipid droplets in accordance with the fluorescent probe. Illuminate to fluoresce, and observe the resulting fluorescence.
脂肪滴の標識は、蛍光プローブを脂肪滴に取り込ませること、あるいは、細胞内に取り込ませた蛍光プローブを脂肪滴に取り込ませることで行うことができる。
蛍光プローブで標識された脂肪滴に、式（１）で表される化合物を励起する励起光を照射し、蛍光顕微鏡やフローサイトメーター等のインビトロ蛍光イメージング装置を用いたインビトロ蛍光イメージングによって蛍光プローブからの蛍光を観察することができる。例えば、波長範囲が例えば３００～８００ｎｍ、例えば３００～５００、例えば３５０～
４５０ｎｍ、例えば３８０～４８０ｎｍの励起光を照射して式（１）で表される化合物を励起し、例えば４００～８００ｎｍ、例えば４５０～６００ｎｍ波長範囲の蛍光を観察する。
Labeling of lipid droplets can be performed by incorporating a fluorescent probe into the lipid droplets, or by incorporating into the lipid droplets a fluorescent probe that has been incorporated into cells.
The lipid droplets labeled with a fluorescent probe are irradiated with excitation light that excites the compound represented by formula (1), and from the fluorescent probe by in vitro fluorescence imaging using an in vitro fluorescence imaging device such as a fluorescence microscope or a flow cytometer. fluorescence can be observed. For example, if the wavelength range is eg 300-800 nm, eg 300-500, eg 350-
Excitation light of 450 nm, eg, 380 to 480 nm is irradiated to excite the compound represented by formula (1), and fluorescence in the wavelength range of, eg, 400 to 800 nm, eg, 450 to 600 nm is observed.
本発明において、生体は、蛍光観察を行うことができる動物であれば特に限定は無い。好ましくは哺乳動物である。特に好ましくは、マウス、ラット等のげっ歯類の哺乳動物である。なお、マウス等の生体内への蛍光プローブで標識された標的細胞の移植は、後述するとおりに行うことができる。 In the present invention, the living body is not particularly limited as long as it is an animal capable of fluorescence observation. Mammals are preferred. Particularly preferred are rodent mammals such as mice and rats. In addition, transplantation of target cells labeled with a fluorescent probe into a living body such as a mouse can be performed as described later.
本発明の蛍光プローブを用いることで、インビトロ及びインビボのいずれにおいても、蛍光プローブで標識された標的細胞を観察することができる。 By using the fluorescent probe of the present invention, target cells labeled with the fluorescent probe can be observed both in vitro and in vivo.
また、本発明の脂肪滴の検出方法は、（ｄ）本発明の蛍光プローブを、生きている生物個体内に投与する工程を含むことができる。
これにより、生きている生物個体内に存在する脂肪滴を含む細胞をも検出することができる。つまり、本発明の蛍光プローブは、生体内の脂肪滴を含む細胞又は該細胞から構成される脂肪組織を検出するための試薬として非常に有用である。生体内の脂肪組織としては、皮下脂肪、内臓脂肪、異所性脂肪（例えば、筋肉、肝臓、心臓、膵臓、腎臓などの臓器に蓄積する脂肪）などが挙げられる。
Moreover, the method for detecting lipid droplets of the present invention can include (d) the step of administering the fluorescent probe of the present invention into a living organism.
This makes it possible to detect even cells containing lipid droplets present in living organisms. That is, the fluorescent probe of the present invention is very useful as a reagent for detecting cells containing lipid droplets in vivo or adipose tissue composed of such cells. Fat tissue in vivo includes subcutaneous fat, visceral fat, and ectopic fat (eg, fat accumulated in organs such as muscle, liver, heart, pancreas, and kidney).
例えば、倒立共焦点顕微鏡等を用いた生体分子イメージング手法等により本発明の蛍光プローブの蛍光シグナルを観測することにより、生きた状態で生体内の脂肪組織を検出することができる。
本発明の化合物の投与形態としては、例えば、静脈内投与、皮下投与、筋肉内投与が挙げられる。
また、本発明の化合物の投与量は、投与対象となる動物、投与形態によっても異なるが、例えば、０．０１～１．０μＭ／ｋｇ体重、好ましくは０．０５～０．５μＭ／ｋｇ体重の範囲で該化合物を投与することができる。
本発明の化合物を溶媒に溶解させてから生体内に投与する場合、該溶媒としては、例えば、ＤＭＳＯなどを用いることが出来る。
投与対象となる生物個体としては、特に限定されず、例えば、哺乳動物（マウス、ヒト、ブタ、イヌ、ウサギなど）を含む脊椎動物や無脊椎動物が挙げられる。また、投与対象にはヒトが含まれていても含まれていなくてもよい。
For example, by observing the fluorescence signal of the fluorescent probe of the present invention by a biomolecular imaging technique using an inverted confocal microscope or the like, it is possible to detect adipose tissue in vivo in a living state.
Modes of administration of the compounds of the present invention include, for example, intravenous administration, subcutaneous administration, and intramuscular administration.
The dose of the compound of the present invention varies depending on the animal to be administered and the mode of administration. The compound can be administered in a range.
When the compound of the present invention is dissolved in a solvent and then administered in vivo, examples of the solvent that can be used include DMSO.
The organism to be administered is not particularly limited, and examples thereof include vertebrates and invertebrates including mammals (mouse, human, pig, dog, rabbit, etc.). In addition, administration subjects may or may not include humans.
本発明の蛍光プローブは、多数（６個、２４個、９６個、３８４個等）のウェル（穴）を有するプレート、洗浄液、固定液、使用説明書等を含有した脂肪滴検出用アッセイキットとして提供することもできる。 The fluorescent probe of the present invention is a plate having a large number (6, 24, 96, 384, etc.) wells (holes), a washing solution, a fixative, a lipid droplet detection assay kit containing instructions for use, etc. can also be provided.
以下、実施例を挙げて、本発明をより具体的に説明するが、本発明は下記の実施例に限定されるものではない。
なお、実施例において、試料の合成及び調製に用いた試薬は下記のメーカーから購入した。
（１）１，１’－ビス（ジフェニルホスフィノ）フェロセンパラジウム（ＩＩ）ジクロリド：アークファーム（Ａｒｋ Ｐｈａｒｍ）社
（２）カリウムｔｅｒｔ－ブトキシド：ナカライテクス株式会社
（３）ブロモベンゼン：ナカライテクス株式会社
（４）酢酸パラジウム（ＩＩ）：東京化成工業株式会社
（５）１－ブロモ－４－ｔｅｒｔ－ブチルベンゼン：東京化成工業株式会社
（６）４－ブロモ兄ソール：ナカライテクス株式会社
（７）トルエン：富士フィルム和光純薬社
（８）ジメチルスルホキシド（ＤＭＳＯ）：富士フィルム和光純薬社
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
In the examples, reagents used for synthesizing and preparing samples were purchased from the following manufacturers.
(1) 1,1′-Bis(diphenylphosphino)ferrocene palladium (II) dichloride: Ark Pharm Co., Ltd. (2) Potassium tert-butoxide: Nacalai Techs Co., Ltd. (3) Bromobenzene: Nacalai Techs Co., Ltd. (4) palladium acetate (II): Tokyo Chemical Industry Co., Ltd. (5) 1-bromo-4-tert-butylbenzene: Tokyo Chemical Industry Co., Ltd. (6) 4-bromo brother sole: Nacalai Techs Co., Ltd. (7) toluene : Fuji Film Wako Pure Chemical Co., Ltd. (8) Dimethyl sulfoxide (DMSO): Fuji Film Wako Pure Chemical Co., Ltd.
実施例において、試料の調製及び物性の分析に用いた装置は下記の通りである。
（１）赤外分光光度計（ＩＲ）
装置：日本分光社製 ４２０ＦＴ－ＩＲ
（２）核磁気共鳴装置（ＮＭＲ）
装置：ブルカーバイオスピン社製ＡＶＡＮＣＥ ３００
（３）高分解能質量分析装置（ＨＲＭＳ（ＥＳＩ））
装置：日本電子データム社製 ＪＭＰ－Ｔ１００ＬＰ
（４）紫外可視吸収スペクトル
装置：日本分光社製 Ｖ７６０
（５）分光蛍光光度計・光量子収率測定
装置：ＪＡＳＣＯ ＦＰ８５００
（６）共焦点レーザー顕微鏡
装置：株式会社ニコン社製 ＡＩ ＨＤ２５
（７）プレートリーダー
装置：Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ
In the examples, the equipment used for sample preparation and physical property analysis is as follows.
(1) Infrared spectrophotometer (IR)
Apparatus: 420FT-IR manufactured by JASCO Corporation
(2) Nuclear Magnetic Resonance (NMR)
Apparatus: AVANCE 300 manufactured by Bruker Biospin
(3) High resolution mass spectrometer (HRMS (ESI))
Apparatus: JMP-T100LP manufactured by JEOL Datum Co., Ltd.
(4) UV-visible absorption spectrometer: V760 manufactured by JASCO Corporation
(5) Spectrofluorometer/photon yield measuring device: JASCO FP8500
(6) Confocal laser microscope device: AI HD25 manufactured by Nikon Corporation
(7) Plate reader device: Thermo Fisher
下記スキームに従って、２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ）、Ｎ－フェニル－２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ－Ｐｈ）、Ｎ，８－ジフェニル－２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ－８Ｐｈ）、８－（４－（ターシャリブチル）フェニル－Ｎ－フェニル－２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ－８Ｐｈ－ｔ－Ｂｕ）、８－（４－メトキシフェニル）－Ｎ－フェニルー２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ－８Ｐｈ－ＯＭｅ）を合成した。
実施例１：ＴＦＭＡＱ－８Ｐｈの合成
ｍ－フェニレンジアミン１．０ｇ（９．２６ｍｍｏｌ）及びモンモリロナイトＫ１０ １．５ｇを溶解したトルエン３０ｍＬ溶液に、ヘキサフルオロアセトン２．３５ｇ（１１．３５ｍｍｏｌ）を添加し、９０℃で１時間反応させた。反応溶液を濾過し、濾液を減圧下で約１０ｍＬに濃縮した。冷却後、得られた沈殿物を濾過により集めて、２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ）を黄色の固体２．５ｇ（８．９３ｍｍｏｌ、収率９６％）で得た。
また、酢酸エチル／ヘキサン混合溶媒で再結晶することで黄色単結晶を得た。
Example 1: Synthesis of TFMAQ-8Ph
To a solution of 1.0 g (9.26 mmol) of m-phenylenediamine and 1.5 g of montmorillonite K10 dissolved in 30 mL of toluene, 2.35 g (11.35 mmol) of hexafluoroacetone was added and reacted at 90° C. for 1 hour. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to about 10 mL. After cooling, the resulting precipitate was collected by filtration to give 2,4-bis(trifluoromethyl)quinolin-7-amine (TFMAQ) as a yellow solid 2.5 g (8.93 mmol, 96% yield). Obtained.
Moreover, a yellow single crystal was obtained by recrystallization with an ethyl acetate/hexane mixed solvent.
次いで、窒素置換したシュレンク管に、得られたＴＦＭＡＱ１．０ｇ（３．６ｍｍｏｌ）、１，１’－ビス（ジフェニルホスフィノ）フェロセン（ｄｐｐｆ）０．３ｇ（０．５４ｍｍｏｌ）、ジクロロ［１，１’－ビス（ジフェニルホスフィノ）フェロセン］パラジウム（ＩＩ）ジクロロメタン付加物０．１５ｇ（０．１８ｍｍｏｌ）、カリウムｔｅｒｔ
－ブトキシド０．４４ｇ（４．０ｍｍｏｌ）及び凍結脱気したトルエン５ｍＬを加えて溶解させた。この溶液に、ブロモベンゼン０．７５ｍＬ（７．１ｍｍｏｌ）を添加し、窒素雰囲気下、１００℃で６時間反応させた。この反応溶液に水を加え、酢酸エチル５０ｍＬで３回抽出した。合わせた有機層を硫酸マグネシウムで乾燥させ、減圧下で蒸発させた。残留物を展開溶媒（ヘキサン：酢酸エチル＝１００：１）を用いてシリカゲルクロマトグラフィーによって精製し、Ｎ－フェニル－２，４－ビス（トリフルオロメチル）キノリン－７－アミン（ＴＦＭＡＱ－Ｐｈ）を、淡黄色固体０．９１ｇ（２．５６ｍｍｏｌ、収率７２％）で得た。
また、エタノール／ヘキサン（１０：３）混合溶媒（４℃）で再結晶することで黄色板状結晶を得、またエタノール／ヘキサン（１：１０）混合溶媒（２０℃）で再結晶することで黄色ブロック状結晶を得た。
Then, 1.0 g (3.6 mmol) of the obtained TFMAQ, 0.3 g (0.54 mmol) of 1,1′-bis(diphenylphosphino)ferrocene (dppf), dichloro[1,1 0.15 g (0.18 mmol) of '-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct, potassium tert
0.44 g (4.0 mmol) of -butoxide and 5 mL of freeze-degassed toluene were added and dissolved. 0.75 mL (7.1 mmol) of bromobenzene was added to this solution and reacted at 100° C. for 6 hours under nitrogen atmosphere. Water was added to this reaction solution, and the mixture was extracted with 50 mL of ethyl acetate three times. The combined organic layers were dried over magnesium sulphate and evaporated under reduced pressure. The residue was purified by silica gel chromatography using a developing solvent (hexane:ethyl acetate=100:1) to give N-phenyl-2,4-bis(trifluoromethyl)quinolin-7-amine (TFMAQ-Ph). , as a pale yellow solid, 0.91 g (2.56 mmol, 72% yield).
In addition, by recrystallization with ethanol/hexane (10:3) mixed solvent (4°C), yellow plate-like crystals were obtained, and by recrystallization with ethanol/hexane (1:10) mixed solvent (20°C), Yellow blocky crystals were obtained.
得られたＴＦＭＡＱ－Ｐｈ２６ｍｇ（０．０７３ｍｍｏｌ）、ジクロロ［１，１’－ビス（フィフェニルホスフィノ）フェロセン］パラジウム（ＩＩ）（１８．３ｍｏｌ％）、カリウムｔ－ブトキシド３４．９ｍｇ（０．３１ｍｍｏｌ）をシュレンク管に入れた。脱気したシュレンク管にトルエンを加え、窒素雰囲気下でブロモベンゼン０．０３０ｍＬ（０．２８ｍｍｏｌ）を加えて、１１０℃で加熱した。３時間後加熱を止め、反応溶液をセライト濾過し、濾液に水を加えジエチルエーテルで３回抽出した。合わせた有機層を硫酸マグネシウムで乾燥させ、展開溶媒（ヘキサン：酢酸エチル＝３０：１）を用いてシリカゲルクロマトグラフィーによって精製した。ＴＦＭＡＱ－８Ｐｈの淡黄色固体２７．７ｍｇ（０．０６４ ｍｍｏｌ）を８７．７％で得た。
また、ヘキサン、酢酸エチル混合溶媒で再結晶することで淡黄色針状結晶を得た。
IR (KBr) 3380.6, 2924.5, 1597.7, 1499.4, 1269.9, 1210.1, 1139.7, 896.7, 749.2, 704.9 cm-1.
1H-NMR (CDCl3, 600 MHz):δ8.06 (dd, J= 1.9, 9.4 Hz, 1H), 7.83 (d, J= 9.5 Hz, 1H), 7.74 (s, 1H), 7.55 (t, J= 7.5 Hz, 2H), 7.48 - 7.44 (m, 3H), 7.35 (t, J= 7.9 Hz, 2H), 7.15 (d, J= 7.7 Hz, 2H), 7.12 (t, J= 7.4 Hz, 1H), 6.13 (s, 1H) ppm.
13C-NMR (CDCl3, 150 MHz):148.2, 147.3(q, J=35.3 Hz),144.2, 140.9, 136.1(q, J=31.9 Hz), 134.3, 131.3, 130.1, 129.8, 129.0, 128.2, 126.3, 126.1, 124.7, 124.2, 123.9, 122.4, 122.1, 121.7, 121.1, 120.2, 118.4, 110.6 ppm.
ESI-HRMS: Calcd. for [C23H14F6N2] 433.1139 [M+H]+, Found 433.1111.
Obtained TFMAQ-Ph 26mg (0.073mmol), dichloro[1,1'-bis(fiphenylphosphino)ferrocene]palladium (II) (18.3mol%), potassium t-butoxide 34.9mg (0.31mmol) ) was placed in the Schlenk tube. Toluene was added to the degassed Schlenk tube, 0.030 mL (0.28 mmol) of bromobenzene was added under a nitrogen atmosphere, and the mixture was heated at 110°C. After 3 hours, the heating was stopped, the reaction solution was filtered through Celite, water was added to the filtrate, and the mixture was extracted with diethyl ether three times. The combined organic layers were dried over magnesium sulfate and purified by silica gel chromatography using a developing solvent (hexane:ethyl acetate=30:1). 27.7 mg (0.064 mmol) of pale yellow solid of TFMAQ-8Ph was obtained with 87.7%.
Further, by recrystallization with a mixed solvent of hexane and ethyl acetate, pale yellow needle crystals were obtained.
IR (KBr) 3380.6, 2924.5, 1597.7, 1499.4, 1269.9, 1210.1, 1139.7, 896.7, 749.2, 704.9 cm -1 .
1 H-NMR (CDCl 3 , 600 MHz): δ 8.06 (dd, J = 1.9, 9.4 Hz, 1H), 7.83 (d, J = 9.5 Hz, 1H), 7.74 (s, 1H), 7.55 (t , J= 7.5 Hz, 2H), 7.48 - 7.44 (m, 3H), 7.35 (t, J= 7.9 Hz, 2H), 7.15 (d, J= 7.7 Hz, 2H), 7.12 (t, J= 7.4 Hz , 1H), 6.13 (s, 1H) ppm.
13 C-NMR (CDCl 3 , 150 MHz): 148.2, 147.3 (q, J=35.3 Hz), 144.2, 140.9, 136.1 (q, J=31.9 Hz), 134.3, 131.3, 130.1, 129.8, 129.0, 128.2, 126.3, 126.1, 124.7, 124.2, 123.9, 122.4, 122.1, 121.7, 121.1, 120.2, 118.4, 110.6 ppm.
ESI-HRMS: Calcd . for [ C23H14F6N2 ] 433.1139 [M+H] + , Found 433.1111 .
実施例２：ＴＦＭＡＱ－８Ｐｈ-ｔ-Ｂｕの合成
ＭＡＱ－８Ｐｈ－ｔ－Ｂｕの淡黄色固体８２．３ｍｇ（０．１７ｍｍｏｌ、収率３０．１％）で得た。
また、ＤＭＳＯに溶解させ凍結乾燥させることにより黄緑色針状結晶を得た。
IR (KBr) 3389.3, 2966.0, 1592.9, 1501.3, 1436.7, 1274.7, 1133.0, 1021.1, 876.5, 679.8 cm-1.
1H-NMR (CDCl3, 600 MHz) :δ8.04 (dd, J= 1.6, 9.4 Hz, 1H), 7.81 (d, J= 9.4 Hz, 1H), 7.73 (s, 1H), 7.55 (dd, J= 1.6, 6.7 Hz, 2H), 7.39 (d, J= 8.3 Hz, 2H), 7.35 (t, J= 7.9 Hz, 2H), 7.17 (d, J= 7.7 Hz, 2H), 7.12 (t, J= 7.4 Hz, 1H), 6.22 (s, 1H), 1.41 (s, 9H)ppm.
13C-NMR (CDCl3, 150 MHz):δ150.9, 148.2, 147.2 (q, J= 35.3 Hz), 144.4, 141.1, 136.0 (q, J= 32.0 Hz), 131.0, 130.9, 129.8, 125.9, 124.7, 124.1, 123.7, 121.8, 121.1, 120.3, 118.5, 110.6, 34.9, 31.5 ppm.
ESI-HRMS: Calcd. for [C27H22F6N2] 489.1765 [M+H]+, Found 489.1801.
Example 2: Synthesis of TFMAQ-8Ph-t-Bu
Obtained 82.3 mg (0.17 mmol, 30.1% yield) of pale yellow solid MAQ-8Ph-t-Bu.
Further, yellow-green needle crystals were obtained by dissolving in DMSO and freeze-drying.
IR (KBr) 3389.3, 2966.0, 1592.9, 1501.3, 1436.7, 1274.7, 1133.0, 1021.1, 876.5, 679.8 cm -1 .
1 H-NMR (CDCl 3 , 600 MHz): 8.04 (dd, J= 1.6, 9.4 Hz, 1H), 7.81 (d, J= 9.4 Hz, 1H), 7.73 (s, 1H), 7.55 (dd , J= 1.6, 6.7 Hz, 2H), 7.39 (d, J= 8.3 Hz, 2H), 7.35 (t, J= 7.9 Hz, 2H), 7.17 (d, J= 7.7 Hz, 2H), 7.12 (t , J= 7.4 Hz, 1H), 6.22 (s, 1H), 1.41 (s, 9H)ppm.
13 C-NMR (CDCl 3 , 150 MHz): δ 150.9, 148.2, 147.2 (q, J = 35.3 Hz), 144.4, 141.1, 136.0 (q, J = 32.0 Hz), 131.0, 130.9, 129.8, 125.9, 124.7, 124.1, 123.7, 121.8, 121.1, 120.3, 118.5, 110.6, 34.9, 31.5 ppm.
ESI - HRMS: Calcd . for [ C27H22F6N2 ] 489.1765 [M+H] + , Found 489.1801.
実施例３：ＴＦＭＡＱ－８Ｐｈ-ＯＭｅの合成
また、ヘキサン／メタノールで溶解後、０℃に冷却することで黄色結晶を得た。
IR (KBr) 3398.0, 2924.5, 1592.9, 1435.7, 1278.6, 1251.6, 1180.2, 1128.2, 874.6, 691.4 cm-1.
δ1H NMR (600 MHz, CDCl3) :δ 8.03 (dd, J= 1.9, 9.5 Hz, 1H), 7.82 (d, J= 9.4 Hz,
1H), 7.72 (s, 1H), 7.37 (d, J= 8.8 Hz, 2H), 7.34 (t, J= 8.0 Hz, 2H), 7.15 (d, J= 8.6 Hz, 2H), 7.11 (t, J= 8.4 Hz, 1H), 7.08 (d, J= 8.7 Hz, 2H), 6.17 (s, 1H), 3.90 (s, 3H) ppm.
13C-NMR (CDCl3, 150 MHz):δ159.4, 148.3, 147.2 (q, J= 35.6 Hz), 144.3, 141.1, 136.1 (q, J= 32.1 Hz), 132.5, 129.8, 127.9, 126.1, 124.6, 124.0, 123.7, 122.4, 122.1, 121.5, 121.1, 120.3, 118.5, 114.5, 110.6, 55.4.
ESI-HRMS: Calcd. for [C24H16F6N2O] 485.1065 [M+Na]+, Found 485.1096
Example 3: Synthesis of TFMAQ-8Ph-OMe
Further, yellow crystals were obtained by cooling to 0° C. after dissolving in hexane/methanol.
IR (KBr) 3398.0, 2924.5, 1592.9, 1435.7, 1278.6, 1251.6, 1180.2, 1128.2, 874.6, 691.4 cm -1 .
δ 1 H NMR (600 MHz, CDCl 3 ): δ 8.03 (dd, J= 1.9, 9.5 Hz, 1H), 7.82 (d, J= 9.4 Hz,
1H), 7.72 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 7.11 (t, J= 8.4 Hz, 1H), 7.08 (d, J= 8.7 Hz, 2H), 6.17 (s, 1H), 3.90 (s, 3H) ppm.
13 C-NMR (CDCl 3 , 150 MHz): δ 159.4, 148.3, 147.2 (q, J = 35.6 Hz), 144.3, 141.1, 136.1 (q, J = 32.1 Hz), 132.5, 129.8, 127.9, 126.1, 124.6, 124.0, 123.7, 122.4, 122.1, 121.5, 121.1, 120.3, 118.5, 114.5, 110.6, 55.4.
ESI - HRMS: Calcd . for [ C24H16F6N2O ] 485.1065 [M+Na] + , Found 485.1096
実施例４：スペクトル測定
実施例１で合成したＴＦＭＡＱ－８Ｐｈを、ｎ－ヘキサン、クロロホルム、酢酸エチル又はジメチルスルホキシドに溶解して得られた溶液５０μｍｏｌ／Ｌの紫外可視吸収スペクトルと蛍光スペクトルを測定した結果を図１に示す。
図１中、左軸はモル吸光係数を表し、右軸は正規化蛍光強度を表す。
Example 4: Spectral measurement The ultraviolet-visible absorption spectrum and fluorescence spectrum of a 50 µmol/L solution obtained by dissolving TFMAQ-8Ph synthesized in Example 1 in n-hexane, chloroform, ethyl acetate, or dimethylsulfoxide were measured. The results are shown in FIG.
In FIG. 1, the left axis represents the molar extinction coefficient and the right axis represents the normalized fluorescence intensity.
実施例５：スペクトル測定
実施例２で合成したＴＦＭＡＱ－８Ｐｈ－ｔ－Ｂｕを、ｎ－ヘキサン、クロロホルム、酢酸エチル又はジメチルスルホキシドに溶解して得られた溶液５０μｍｏｌ／Ｌの紫外可視吸収スペクトルと蛍光スペクトルを測定した結果を図２に示す。
図２中、左軸はモル吸光係数を表し、右軸は正規化蛍光強度を表す。
Example 5: Spectral measurement UV-visible absorption spectrum and fluorescence of 50 µmol/L solution obtained by dissolving TFMAQ-8Ph-t-Bu synthesized in Example 2 in n-hexane, chloroform, ethyl acetate or dimethylsulfoxide FIG. 2 shows the result of measuring the spectrum.
In FIG. 2, the left axis represents the molar extinction coefficient and the right axis represents the normalized fluorescence intensity.
実施例６：スペクトル測定
実施例３で合成したＴＦＭＡＱ－８Ｐｈ－ｔ－ＯＭｅを、ｎ－ヘキサン、クロロホルム、酢酸エチル又はジメチルスルホキシドに溶解して得られた溶液５０μｍｏｌ／Ｌの紫外可視吸収スペクトルと蛍光スペクトルを測定した結果を図３に示す。
図３中、左軸はモル吸光係数を表し、右軸は正規化蛍光強度を表す。
Example 6: Spectral measurement UV-visible absorption spectrum and fluorescence of 50 μmol/L solution obtained by dissolving TFMAQ-8Ph-t-OMe synthesized in Example 3 in n-hexane, chloroform, ethyl acetate or dimethylsulfoxide FIG. 3 shows the result of measuring the spectrum.
In FIG. 3, the left axis represents the molar extinction coefficient and the right axis represents the normalized fluorescence intensity.
実施例７：
実施例４乃至実施例６の紫外可視吸収スペクトルと蛍光スペクトルの測定結果から得られた、ＴＦＭＡＱ-８Ｐｈ, ＴＦＭＡＱ-８Ｐｈ-ｔ-Bu, ＴＦＭＡＱ-８Ｐｈ-ＯＭｅの各溶媒における吸収極大（λａｂ
ｍａｘ）、蛍光極大（λｆｌ
ｍａｘ）及び蛍光光量子収率（φｆｌ）を下記表１に示す。なお、表１中のｎ．ｄ．は検出できなかったことを表す。
Absorption maxima (λ ab max ), fluorescence maximum (λ fl max ) and fluorescence photon yield (φ fl ) are shown in Table 1 below. In addition, n. d. indicates that it could not be detected.
表１より、本発明の化合物は、溶媒の極性が増加するに従って、吸収極大及び蛍光極大が長波長側にシフト（レッドシフト）し、蛍光光量子収率が低下し、ＤＭＳＯ溶媒中では蛍光光量子収率（φｆｌ）が１未満と低く蛍光発色が観察されないことが明らかである。
したがって、本発明の化合物は、脂肪滴検出用の蛍光プローブとして非常に有用なことが理解できる。
From Table 1, as the polarity of the solvent increases, the compound of the present invention shifts the absorption maximum and fluorescence maximum to the longer wavelength side (red shift), decreases the fluorescence photon yield, and decreases the fluorescence photon yield in DMSO solvent. It is clear that the ratio (φ fl ) is as low as less than 1 and no fluorescence is observed.
Therefore, it can be understood that the compound of the present invention is very useful as a fluorescent probe for detecting lipid droplets.
実施例８：ＴＦＭＡＱ－８Ｐｈの脂肪細胞への取り込み
脂肪組織に分化する３Ｔ３－Ｌ１細胞を用いて、実施例１で合成したＴＦＭＡＱ－８Ｐｈの取り込みについて検討を行った。３Ｔ３－Ｌ１細胞にイソブチルメチルキサンチン、インスリン、デキサメタゾンを添加することで細胞内にトリグリセリドやコレステロールなどから成る脂肪滴を含む脂肪細胞を形成した。脂肪細胞に分化した３Ｔ３－Ｌ１細胞の培養液中にＴＦＭＡＱ－８Ｐｈの１μＭ ＤＭＳＯ溶液を添加し、５％ＣＯ２雰囲気下、３７℃で３０分間インキュベートを行い、共焦点レーザー顕微鏡を用いて明視野観察（倍率：４０倍）及び蛍光観察（倍率：４０倍）を行った。
実施例２で合成したＴＦＭＡＱ－８Ｐｈ－ｔ－Ｂｕ及び実施例３で合成したＴＦＭＡＱ－８Ｐｈ－ＯＭｅについても同様の操作を行った。
ＴＦＭＡＱ－８Ｐｈ、ＴＦＭＡＱ－８Ｐｈ－ｔ－Ｂｕ又はＴＦＭＡＱ－８Ｐｈ－ＯＭｅを添加した３Ｔ３－Ｌ１細胞の明視野観察画像及び蛍光観察画像を図４乃至図６に示す。
図４乃至図６より、本発明の蛍光プローブは、細胞内の脂肪滴を特異的に検出できることが示された。
Example 8 Uptake of TFMAQ-8Ph into Adipocytes Uptake of TFMAQ-8Ph synthesized in Example 1 was examined using 3T3-L1 cells that differentiate into adipose tissue. Addition of isobutylmethylxanthine, insulin, and dexamethasone to 3T3-L1 cells formed adipocytes containing lipid droplets composed of triglycerides, cholesterol, and the like in the cells. A 1 μM DMSO solution of TFMAQ-8Ph was added to the culture medium of adipocyte-differentiated 3T3-L1 cells, incubated at 37° C. for 30 min in a 5% CO 2 atmosphere, and bright field images were obtained using a confocal laser microscope. Observation (magnification: 40 times) and fluorescence observation (magnification: 40 times) were performed.
TFMAQ-8Ph-t-Bu synthesized in Example 2 and TFMAQ-8Ph-OMe synthesized in Example 3 were subjected to the same operation.
Bright field observation images and fluorescence observation images of 3T3-L1 cells added with TFMAQ-8Ph, TFMAQ-8Ph-t-Bu or TFMAQ-8Ph-OMe are shown in FIGS. 4 to 6. FIG.
4 to 6 show that the fluorescent probe of the present invention can specifically detect intracellular lipid droplets.
実施例９：ＭＴＳアッセイ法による細胞増殖阻害試験
９６ウェルプレートに１ウェルあたりＨｅＬａ細胞１．０×１０４ｃｅｌｌを播種し、ＣＯ２インキュベーター中３７℃で２４時間インキュベートした。化合物を各濃度（１，１０，５０，１００μＭ）加え、２時間インキュベートした後に各ウェルをＤＭＥＭ培地で洗浄した。ウェルにＤＭＥＭ培地とＣｅｌｌＴｉｔｅｒ ９６（登録商標）を加えてＣＯ２インキュベーター中３７℃で４時間インキュベートした後に、４９０ｎｍの吸光度を測定することで細胞生存率を算出した（ｎ＝３）。化合物濃度と細胞生存率の結果を図７に示す。
図７より、本発明の化合物を添加した細胞の生存率は高く、本発明の化合物は細胞毒性を有さないことが期待できる。
Example 9 Cell Growth Inhibition Test by MTS Assay 1.0×10 4 HeLa cells were seeded per well in a 96-well plate and incubated at 37° C. in a CO 2 incubator for 24 hours. A compound was added at each concentration (1, 10, 50, 100 μM), incubated for 2 hours, and then each well was washed with DMEM medium. Cell viability was calculated by measuring absorbance at 490 nm after adding DMEM medium and CellTiter 96® to the wells and incubating for 4 hours at 37° C. in a CO 2 incubator (n=3). The results of compound concentration and cell viability are shown in FIG.
From FIG. 7, it can be expected that the survival rate of the cells added with the compound of the present invention is high, and that the compound of the present invention has no cytotoxicity.
実施例１０：光安定性試験
１ｃｍ角の石英セルに実施例１乃至３で製造したＴＦＭＡＱ－８Ｐｈ、ＴＦＭＡＱ－８Ｐｈ－ｔ－Ｂｕ及びＴＦＭＡＱ－８Ｐｈ－ＯＭｅ、並びにＬｉｐｉＤｙｅ（登録商標）、ナイルレッドの２０μＭメタノール溶液を２ｍＬ加えて、溶液に高圧水銀ランプ（１０００Ｗ、ウシオ電機株式会社製）を６０分間照射した。光照射開始後、任意の時間毎に溶液のＵＶ吸収スペクトルを測定し、吸光度の減少率を算出することで光安定性を評価した。各化合物の光照射時間に対する正規化吸光度を図８に示す。
図８より、本発明の化合物は従来のナイルレッドよりも優れた光安定性を有し、またＬｉｐｉＤｙｅ（登録商標）と同程度の光安定性を有することが示された。
Example 10: Photostability test TFMAQ-8Ph, TFMAQ-8Ph-t-Bu and TFMAQ-8Ph-OMe produced in Examples 1 to 3, LipiDye (registered trademark), and Nile Red were added to a 1 cm square quartz cell. 2 mL of a 20 μM methanol solution was added, and the solution was irradiated with a high-pressure mercury lamp (1000 W, manufactured by Ushio Inc.) for 60 minutes. After the start of light irradiation, the UV absorption spectrum of the solution was measured at arbitrary time intervals, and the photostability was evaluated by calculating the rate of decrease in absorbance. FIG. 8 shows the normalized absorbance of each compound with respect to the irradiation time.
FIG. 8 shows that the compounds of the present invention have better photostability than conventional Nile Red and comparable photostability to LipiDye®.
Claims (9)
Ｒ１は、水素原子を表し、
Ｒ２は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｍが２以上の整数を表すとき、各々のＲ２は互いに同一であっても又は互いに相異なってもよく、
Ｒ３は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、
ｍ及びｎは、夫々独立して、０乃至５の整数を表す。）
で表される化合物又はその塩。 Formula (1) below:
R 1 represents a hydrogen atom ,
R 2 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group, and when m represents an integer of 2 or more, each R 2 may be the same or different from each other,
R 3 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group and n represents an integer of 2 or more, each R 3 may be the same or different from each other,
m and n each independently represent an integer of 0 to 5; )
A compound represented by or a salt thereof.
で表される化合物である、請求項１に記載の化合物又はその塩。 The compound represented by the formula (1) is represented by the following formula (2):
The compound or its salt according to claim 1, which is a compound represented by:
Ｒ２は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｍが２以上の整数を表すとき、各々のＲ２は互いに同一であっても又は互いに相異なってもよく、
Ｒ３は、ヒドロキシ基、ハロゲン原子、炭素原子数１乃至１０のアルキル基、炭素原子数１乃至１０のハロアルキル基、炭素原子数１乃至１０のアルコキシ基、又は炭素原子数１乃至１０のアルキルアルコキシ基を表し、ｎが２以上の整数を表すとき、各々のＲ３は互いに同一であっても又は互いに相異なってもよく、
ｍ及びｎは、夫々独立して、０乃至５の整数を表す。）
で表される化合物の製造方法であって、
ｍ－フェニレンジアミンと、ヘキサフルオロアセチルアセトンとを反応させて２，４－ジトリフルオロメチル－７－アミノキノリンを製造する工程Ａ、
該７－アミノキノリンを、下記式（１０）：
Ｘは、ハロゲン原子を表し、
Ｒ２及びｍは、上記と同じ意味を表す。）
で表される化合物と反応させて、下記式（４）：
で表される化合物を製造する工程Ｂ１、及び
該式（４）で表される化合物を、下記式（１１）：
で表される化合物と反応させて、上記式（３）で表される化合物を製造する工程Ｃ１、
を含む方法。 Formula (3) below:
R 2 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group, and when m represents an integer of 2 or more, each R 2 may be the same or different from each other,
R 3 is a hydroxy group, a halogen atom, an alkyl group having 1 to 10 carbon atoms, a haloalkyl group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, or an alkylalkoxy group having 1 to 10 carbon atoms represents a group and n represents an integer of 2 or more, each R 3 may be the same or different from each other,
m and n each independently represent an integer of 0 to 5; )
A method for producing a compound represented by
Step A of reacting m-phenylenediamine with hexafluoroacetylacetone to produce 2,4-ditrifluoromethyl-7-aminoquinoline;
The 7-aminoquinoline is represented by the following formula (10):
X represents a halogen atom,
R 2 and m have the same meanings as above. )
By reacting with a compound represented by the following formula (4):
Step B1 for producing a compound represented by and the compound represented by the formula (4) in the following formula (11):
Step C1 for producing a compound represented by the above formula (3) by reacting with a compound represented by
method including.
Ｘは、ハロゲン原子を表し、
Ｒ４は、水素原子、炭素原子数１乃至４のアルキル基又は炭素原子数１乃至４のアルコキシ基を表す。）で表される化合物である、
請求項４に記載の方法。 The compound represented by the formula (10) is the following formula (12)
X represents a halogen atom,
R 4 represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms or an alkoxy group having 1 to 4 carbon atoms. ) is a compound represented by
5. The method of claim 4.
（ｂ）該細胞又は脂肪滴に対し励起光を照射して蛍光を生じさせる工程
を含む脂肪滴の検出方法。 (A) the fluorescent probe according to claim 6 , the step of contacting with cells or lipid droplets containing lipid droplets, and (b) the step of irradiating the cells or lipid droplets with excitation light to generate fluorescence. Method for detecting lipid droplets.
内（ヒトを除く）に投与する工程を含む、請求項７又は請求項８に記載の方法。 9. The method according to claim 7 or 8 , comprising the step of administering (d) the fluorescent probe according to claim 6 into a living organism (excluding humans) before the step (a). Method.
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|---|---|---|---|---|
| US20110263861A1 (en) | 2009-11-19 | 2011-10-27 | Kyushu University, National University Corporation | Quinoline compound |
| WO2014142320A1 (en) | 2013-03-15 | 2014-09-18 | 独立行政法人理化学研究所 | Novel compound, and reagent for detecting lipid droplets and/or adipose tissue containing said compound |
| CN104529893A (en) | 2014-12-30 | 2015-04-22 | 中国科学技术大学 | Novel quinoline dye capable of being used as Golgi apparatus organelle probe |
| JP2018145422A (en) | 2017-03-02 | 2018-09-20 | 国立大学法人名古屋大学 | Condensed-ring thiophene compound and oil droplet dyeing agent prepared therewith |
-
2019
- 2019-05-13 JP JP2019090840A patent/JP7217460B2/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110263861A1 (en) | 2009-11-19 | 2011-10-27 | Kyushu University, National University Corporation | Quinoline compound |
| WO2014142320A1 (en) | 2013-03-15 | 2014-09-18 | 独立行政法人理化学研究所 | Novel compound, and reagent for detecting lipid droplets and/or adipose tissue containing said compound |
| CN104529893A (en) | 2014-12-30 | 2015-04-22 | 中国科学技术大学 | Novel quinoline dye capable of being used as Golgi apparatus organelle probe |
| JP2018145422A (en) | 2017-03-02 | 2018-09-20 | 国立大学法人名古屋大学 | Condensed-ring thiophene compound and oil droplet dyeing agent prepared therewith |
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