US20060275912A1 - Fluorescent probes - Google Patents
Fluorescent probes Download PDFInfo
- Publication number
- US20060275912A1 US20060275912A1 US10/547,305 US54730506A US2006275912A1 US 20060275912 A1 US20060275912 A1 US 20060275912A1 US 54730506 A US54730506 A US 54730506A US 2006275912 A1 US2006275912 A1 US 2006275912A1
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- alkyl group
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- alkyl
- Prior art date
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- 239000007850 fluorescent dye Substances 0.000 title description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 89
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims abstract description 84
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims abstract description 63
- 238000005259 measurement Methods 0.000 claims abstract description 41
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 33
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 24
- 125000003277 amino group Chemical group 0.000 claims abstract description 23
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 17
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 12
- 125000003118 aryl group Chemical group 0.000 claims abstract description 11
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims abstract description 7
- 125000004454 (C1-C6) alkoxycarbonyl group Chemical group 0.000 claims abstract description 7
- 125000000168 pyrrolyl group Chemical group 0.000 claims abstract description 7
- 125000001544 thienyl group Chemical group 0.000 claims abstract description 7
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims abstract description 7
- 150000003839 salts Chemical class 0.000 claims description 26
- -1 2-carboxy-1-ethyl groups Chemical group 0.000 claims description 18
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 6
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 6
- 125000006239 protecting group Chemical group 0.000 claims description 5
- 239000000126 substance Substances 0.000 abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 13
- 229910052760 oxygen Inorganic materials 0.000 description 13
- 239000001301 oxygen Substances 0.000 description 13
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 11
- 239000011541 reaction mixture Substances 0.000 description 11
- 125000001424 substituent group Chemical group 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 230000005284 excitation Effects 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- 238000005160 1H NMR spectroscopy Methods 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000007935 neutral effect Effects 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 0 [1*]C.[2*]C.[3*]C.[4*]C.[5*]C1=C2C(C3=CC=CC=C3)=C3C([8*])=C([9*])C([10*])=N3B(F)(F)N2C([7*])=C1[6*] Chemical compound [1*]C.[2*]C.[3*]C.[4*]C.[5*]C1=C2C(C3=CC=CC=C3)=C3C([8*])=C([9*])C([10*])=N3B(F)(F)N2C([7*])=C1[6*] 0.000 description 6
- 238000006862 quantum yield reaction Methods 0.000 description 6
- 239000007832 Na2SO4 Substances 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 229910052786 argon Inorganic materials 0.000 description 5
- 125000003710 aryl alkyl group Chemical group 0.000 description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 150000002467 indacenes Chemical class 0.000 description 5
- 239000012074 organic phase Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 description 5
- DYUUGILMVYJEHY-UHFFFAOYSA-N 1-$l^{1}-oxidanyl-4,4,5,5-tetramethyl-3-oxido-2-phenylimidazol-3-ium Chemical compound CC1(C)C(C)(C)N([O])C(C=2C=CC=CC=2)=[N+]1[O-] DYUUGILMVYJEHY-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 4
- 239000006184 cosolvent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N tetrahydropyridine hydrochloride Natural products C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 125000006702 (C1-C18) alkyl group Chemical group 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium on carbon Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 238000000695 excitation spectrum Methods 0.000 description 3
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 3
- 150000003852 triazoles Chemical class 0.000 description 3
- HZNVUJQVZSTENZ-UHFFFAOYSA-N 2,3-dichloro-5,6-dicyano-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(C#N)=C(C#N)C1=O HZNVUJQVZSTENZ-UHFFFAOYSA-N 0.000 description 2
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 2
- QSSNLQPWTLQYTB-UHFFFAOYSA-N 4-(1-hydroxy-4,4,5,5-tetramethyl-3-oxidoimidazol-3-ium-2-yl)benzoic acid Chemical compound CC1(C)C(C)(C)N(O)C(C=2C=CC(=CC=2)C(O)=O)=[N+]1[O-] QSSNLQPWTLQYTB-UHFFFAOYSA-N 0.000 description 2
- IIOCECYTBZBBAL-UHFFFAOYSA-N 4-amino-3-nitrobenzaldehyde Chemical compound NC1=CC=C(C=O)C=C1[N+]([O-])=O IIOCECYTBZBBAL-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 125000005227 alkyl sulfonate group Chemical group 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 230000005587 bubbling Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
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- 125000000524 functional group Chemical group 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
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- HEKYHUQPSNIMJD-UHFFFAOYSA-N methyl 3-(2,4-dimethyl-1h-pyrrol-3-yl)propanoate Chemical compound COC(=O)CCC=1C(C)=CNC=1C HEKYHUQPSNIMJD-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- CNCOWQRMRJIFOH-UHFFFAOYSA-N n-(4-formyl-2-nitrophenyl)acetamide Chemical compound CC(=O)NC1=CC=C(C=O)C=C1[N+]([O-])=O CNCOWQRMRJIFOH-UHFFFAOYSA-N 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 125000000389 2-pyrrolyl group Chemical group [H]N1C([*])=C([H])C([H])=C1[H] 0.000 description 1
- 125000000175 2-thienyl group Chemical group S1C([*])=C([H])C([H])=C1[H] 0.000 description 1
- SKLUWKYNZNXSLX-UHFFFAOYSA-N 4-Acetamidobenzaldehyde Chemical compound CC(=O)NC1=CC=C(C=O)C=C1 SKLUWKYNZNXSLX-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
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- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 229910001413 alkali metal ion Inorganic materials 0.000 description 1
- 125000005103 alkyl silyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
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- KCXNIAMRWFWMOX-UHFFFAOYSA-N benzyl 4-(3-methoxy-3-oxopropyl)-3,5-dimethyl-1h-pyrrole-2-carboxylate Chemical compound COC(=O)CCC1=C(C)NC(C(=O)OCC=2C=CC=CC=2)=C1C KCXNIAMRWFWMOX-UHFFFAOYSA-N 0.000 description 1
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- 125000003754 ethoxycarbonyl group Chemical group C(=O)(OCC)* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
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- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 238000005558 fluorometry Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
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- 239000011707 mineral Substances 0.000 description 1
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- LFMTUFVYMCDPGY-UHFFFAOYSA-N n,n-diethylethanamine oxide Chemical compound CC[N+]([O-])(CC)CC LFMTUFVYMCDPGY-UHFFFAOYSA-N 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
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- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
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- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
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- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- FIQMHBFVRAXMOP-UHFFFAOYSA-N triphenylphosphane oxide Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=O)C1=CC=CC=C1 FIQMHBFVRAXMOP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/145555—Hetero-N
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/17—Nitrogen containing
- Y10T436/177692—Oxides of nitrogen
Definitions
- Diaminorhodamine derivatives that can efficiently react with nitrogen monoxide under a neutral condition, and give a triazole derivative having superior fluorescence intensity have been proposed (U.S. Pat. No. 6,201,134).
- the fluorescence of the diaminorhodamine derivatives shifts to a longer wavelength side compared with the aforementioned fluorescein derivatives, thus the fluorescence does not substantially overlaps the autofluorescence of cells, and the fluorescence does not attenuate even in an acidic region. Therefore, the fluorescence of the diaminorhodamine derivatives does not damage living tissues and cells, and enable measurement of nitrogen monoxide in a fluorescent region of a longer wavelength than in the autofluorescent region of cells.
- the inventors of the present invention conducted various researches to provide a fluorescent probe that specifically and efficiently captures nitrogen monoxide to emit fluorescence. As a result, they successfully provided an agent for nitrogen monoxide measurement that can efficiently react with nitrogen monoxide under a neutral condition to give a fluorescent substance having superior fluorescence intensity by using fluorescence chromophores of indacene derivatives (Japanese Patent Application No. 2002-80230).
- the inventors of the present invention further conducted researches, and as a result, they found that an agent for nitrogen monoxide measurement further having superior water-solubility was successfully provided by introducing a substituent such as a carboxy-substituted alkyl group into the fluorescence chromophore of the indacene derivatives, and that an fluorescence intensity of the fluorescent triazole derivative, produced by trapping of nitrogen monoxide by the agent, was subjected to almost no influence by pH fluctuation.
- the present invention was achieved on the basis of these findings.
- R 1 and R 2 represent amino groups that substitute at adjacent positions on the benzene ring, wherein one of the amino groups may have one alkyl group which may be substituted;
- R 3 and R 4 independently represent hydrogen atom, a C 1-6 alkyl group, or a C 1-6 alkoxy group,
- R 5 and R 8 independently represent a C 1-6 alkyl group which may be substituted,
- R 6 and R 9 independently represent a carboxy-substituted C 1-6 alkyl group, an alkoxycarbonyl-substituted C 1-6 alkyl group, a sulfo-substituted C 1-6 alkyl group, or an alkyl sulfonate-substituted C 1-6 alkyl group, and
- R 7 and R 10 independently represent a C 1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C 1-6 alkoxycarbonyl group which
- the aforementioned compound or a salt thereof wherein R 6 and R 9 are 2-carboxy-1-ethyl groups, and R 5 , R 7 , R 8 , and R 10 are methyl groups is provided.
- the present invention also provides an agent for measurement of nitrogen monoxide comprising the aforementioned compound or a salt thereof.
- the present invention provides a compound represented by the following formula (II): wherein, R 11 and R 12 combine together to represent a group represented by —N ⁇ N—NR 30 — which forms a ring structure at adjacent positions on the benzene ring wherein R 30 represents hydrogen atom, or an alkyl group which may be substituted, or R 11 and R 12 represent a combination of an amino group (which may have an alkyl group which may be substituted or a protective group for amino group) and nitro group that substitute at adjacent positions on the benzene ring; R 13 and R 14 independently represent hydrogen atom, a C 1-6 alkyl group, or a C 1-6 alkoxy group, R 15 and R 18 independently represent a C 1-6 alkyl group which may be substituted, R 16 and R 19 independently represent a carboxy-substituted C 1-6 alkyl group, an alkoxycarbonyl-substituted C 1-6 alkyl group, a sulfo-substituted
- FIG. 1 shows the results of the measurement of changes of excitation and fluorescence spectra of Compound (7) after addition of NOC 13.
- (A) represent the excitation spectrum (Em: 535 nm)
- (B) represents the fluorescence spectrum (Ex: 520 nm).
- the curves indicate the results for the NOC 13 concentrations of 5.0 ⁇ M, 2.0 ⁇ M, 1.0 ⁇ M, 0.5 ⁇ M, and 0 ⁇ M in the order that the fluorescence intensities shown by the curves decrease from the highest.
- FIG. 2 shows the results of HPLC measurement of the reaction product obtained by adding NOC 13 to Compound (7).
- FIG. 3 shows the results of measurement of fluorescence intensity of Compounds (7) and (8) at various pH values.
- FIG. 4 shows changes of fluorescence intensity observed when a solution of NO in chloroform was added to a solution of Compound (9) in chloroform.
- the alkyl group may be a linear, branched, or cyclic alkyl group, or a combination thereof, unless otherwise specifically mentioned.
- An alkyl moiety of other substituents containing the alkyl moiety e.g. alkoxy group
- the type, number, and substitution position of the substituent are not particularly limited.
- the functional group may have, for example, a halogen atom (any of fluorine atom, chlorine atom, bromine atom, and iodine atom), hydroxy group, amino group, carboxy group, sulfo group, an alkyl sulfonate group, or the like as the substituent.
- a halogen atom any of fluorine atom, chlorine atom, bromine atom, and iodine atom
- the carboxy-substituted C 1-6 alkyl group represented by R 6 or R 9 is preferably a monocarboxy-substituted C 1-6 alkyl group.
- the carbon number of the C 1-6 alkyl moiety of the carboxy-substituted C 1-6 alkyl group is preferably 1 to 4, more preferably 2 or 3, and most preferably 2. It is most preferred that R 6 and R 9 are 2-carboxy-1-ethyl groups.
- Examples of the alkoxycarbonyl-substituted C 1-6 alkyl group represented by R 6 or R 9 include a C 1-6 alkyl ester of the aforementioned carboxy-substituted C 1-6 alkyl group.
- Preferred examples include an ethoxycarbonyl-substituted C 1-6 alkyl group, and the like.
- a monosulfo-substituted C 1-6 alkyl group is preferred.
- a monoalkyl sulfonate-substituted C 1-6 alkyl group is preferred.
- alkyl sulfonate group in the alkyl sulfonate-substituted C 1-6 alkyl group a C 1-6 alkyl sulfonate (C 1-6 alkyl-O—SO 2 —) is preferred.
- R 6 and R 9 are monocarboxy-substituted C 1-6 alkyl groups, especially when R 6 and R 9 are 2-carboxy-1-ethyl groups, superior effects are obtained in that water-solubility of the compounds remarkably increases, and fluorescence intensity of the compounds represented by the formula (II) produced by a reaction with nitrogen monoxide is not subjected to influence of pH fluctuation.
- a phenyl group is preferred.
- the phenyl group has a substituent, sulfo group, a sulfonate group, and the like are preferred as the substituent, and a sulfo group is particularly preferred.
- the C 1-6 alkoxycarbonyl group represented by R 7 or R 10 ethoxycarbonyl group is preferred.
- the substituent which exists on the vinyl group represented by R 7 or R 10 include a phenyl group, monoaminophenyl group, and a diaminophenyl group (for example, 3,4-diaminophenyl group).
- R 6 and R 9 are 2-carboxy-1-ethyl groups.
- R 5 , R 7 , R 8 , and R 10 are C 1-6 alkyl groups which may be substituted.
- R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 in the formula (II) should be understood in the same manner as R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 .
- R 1 and R 2 represent amino groups that substitute at adjacent positions on the benzene ring. Both of R 1 and R 2 may be unsubstituted amino groups. Alternatively, either of R 1 and R 2 may be substituted with one alkyl group, and the alkyl group may have one or more substituents. Examples of the alkyl group which substitutes on the amino group include, for example, a linear or branched C 1-18 alkyl group (preferably a C 1-6 alkyl group). Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group and the like can be used.
- alkyl group which has a substituent examples include, for example, a C 1-6 alkyl group substituted with a substituted or unsubstituted aryl group (aralkyl group) and the like.
- aryl-substituted alkyl group for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
- R 11 and R 12 combine together to represent a —N ⁇ N—NR 30 — group which forms a ring structure at adjacent positions on the benzene ring.
- R 30 represents hydrogen atom, or an alkyl group which may be substituted. Examples of this alkyl group include a linear or branched C 1-18 alkyl group (preferably a C 1-6 alkyl group), and examples of the alkyl group which has a substituent include, for example, a substituted or unsubstituted aralkyl group.
- the aralkyl group for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
- R 11 and R 12 also represent a combination of an amino group (which may have one substituent) and nitro group that substitute at adjacent positions on the benzene ring, wherein either of R 11 and R 12 represents an amino group, and the other represents nitro group.
- the amino group represented by either R 11 or R 12 may be unsubstituted, or the group may have one alkyl group, for example, a C 1-18 alkyl group, preferably a C 1-6 alkyl group. Further, the alkyl group may be substituted, and for example, a substituted or unsubstituted aralkyl group or the like may substitute on the amino group.
- the amino group may have a protective group for amino group, for example, an acyl group such as acetyl group, trifluoroacetyl group, and benzoyl group; an alkylsilyl group such as trimethylsilyl groups, or the like.
- An aralkyl group such as benzyl group may also be used as the protective group.
- the compounds of the present invention represented by the formulas (I) and (II) may form a salt.
- Type of the salt is not particularly limited, and the salt may be an acid addition salt or base addition salt.
- the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, citrate, p-toluenesulfonate, and oxalate.
- Examples of the base addition salt include metal salts such as sodium salt, potassium salt, and calcium salt, ammonium salts, and organic amine salts such as methylamine salt, and triethylamine salt.
- the compounds may form a salt with an amino acid such as glycine.
- salts of the compounds of the present invention are not limited to these specific examples.
- the compounds of the present invention represented by the formula (I) or (II) may have one or more asymmetric carbon. Any of optical isomers in an optically pure form, arbitrary mixtures of optical isomers, racemates, diastereoisomers in a pure form, mixtures of diastereoisomers, and the like based on one or more asymmetric carbon atoms fall within the scope of the present invention.
- the compounds of the present invention may exist as hydrates or solvates, and it should be understood that these substances also fall within the scope of the present invention.
- the compounds represented by the aforementioned formula (II) are useful as synthetic intermediates of the compounds represented by the formula (I).
- the compounds represented by the formula (II) the compounds wherein R 11 and R 12 combine together to represent the —N ⁇ N—NR 30 — group, which forms a ring structure at adjacent positions on the phenyl ring, can be prepared by reacting a compound represented by the aforementioned formula (I) with nitrogen monoxide. These compounds have strong fluorescent property as described later, and are useful for measurement of nitrogen monoxide.
- the compounds represented by the formula (I) of the present invention have a property that they efficiently react with nitrogen monoxide under a neutral condition and provide compounds of the formula (II) wherein R 11 and R 12 combine together to form the group —N ⁇ N—NR 30 — which forms a ring structure at adjacent positions on the benzene ring in a good yield.
- the compounds represented by the formula (I), per se, emit almost no fluorescence when irradiated with excitation light of around 485 nm under a neutral condition, whereas the compounds of the above formula (II) have the property of emitting extremely strong fluorescence under the same condition.
- nitrogen monoxide in living tissues or cells can be measured by introducing the compound represented by the formula (I) into a living tissue or a cell to allow the compound to react with nitrogen monoxide to form the fluorescent compound of the above formula (II), and measuring the fluorescence of the compound.
- the compounds of the formula (I) of the present invention have superior reactivity with nitrogen monoxide, and thus have an outstanding characteristic that they enable measurement of nitrogen monoxide with high sensitivity and accuracy.
- the term “measurement” used in the specification should be construed in its broadest sense, which includes various measurement purposes such as, for example, detection, quantification, qualitative analysis and the like.
- the above reaction can preferably be carried out under a neutral condition, for example, in a range of from pH 6.0 to 8.0, preferably in a range of from pH 6.5 to 7.8, and more preferably in a range of from pH 6.8 to 7.6.
- a neutral condition for example, in a range of from pH 6.0 to 8.0, preferably in a range of from pH 6.5 to 7.8, and more preferably in a range of from pH 6.8 to 7.6.
- the measurement of nitrogen monoxide using the compounds of the present invention is not limited to the measurements under the neutral range or weakly acidic range.
- measurement can also be performed under a strongly acidic condition such as in gastric mucosal cells.
- the compounds represented by the formula (II) of the present invention have an extremely superior characteristic that fluorescence intensity does not change in a wide range of pH, and thus they enable accurate measurement without being influenced by pH fluctuation.
- the measurement of fluorescence can be carried out according to a known fluorometry method (see, for example, Wiersma, J. H., Anal. Lett., 3, pp. 123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp. 761-775, 1971; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; and Misko, T. P., Anal. Biochem., 214, pp. 11-16, 1993, and the like).
- the nitrogen monoxide measurement according to the present invention for example, irradiation with light of about 520 nm as excitation light, and measurement of fluorescence of about 535 nm may preferably be performed.
- the compounds represented by the formula (II) of the present invention have a superior characteristic that they give sufficient fluorescence intensity even with an excitation light having a long wavelength, and therefore they can reduce damages on the living body, tissues, cells, and the like. Further, by using the light having such a wavelength, efficient cut off can be obtained by using a fluorescence filter provided on an ordinary fluorescence microscope, and measurement with high sensitivity can be achieved without using an unordinary filter.
- the aforementioned measurement of nitrogen monoxide may be carried out in the presence of an oxygen source.
- an oxygen source for example, oxygen, ozone, oxide compounds or the like can be used.
- oxygen dissolved oxygen can generally be used, and if desired, oxygen gas may be introduced into the reaction system, or an agent that can generate oxygen (e.g., hydrogen peroxide) may be added.
- the oxide compounds are not particularly limited so long as they have an oxide bond that can easily be cleaved, e.g., N—O, S—O, or P—O.
- PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Maeda, H., et al., J. Leuk. Biol., 56, pp. 588-592, 1994; and Akaike, T., et al., Biochemistry, 32, pp. 827-832, 1993
- carboxy-PTIO which has carboxyl group introduced at the para-position of the phenyl group of PTIO and the like
- triphenylphosphine oxide triethylamine oxide or the like
- PTIO and derivatives thereof are particularly preferred compounds, and they can be readily obtained by those skilled in the art (listed in, for example, Organic Chemicals Catalog, 32, 1994, Tokyo Kasei Co., Ltd.).
- the oxide compounds, per se, may be used as a reaction agent, or those encapsulated in liposomes or other may also be used.
- An amount of the oxygen source is not particularly limited. A preferable amount may be at least 1 ⁇ mol or more, preferably 10 to 30 ⁇ mol, and more preferably about 10 to 20 ⁇ mol, based on nitrogen monoxide to be measured.
- the oxygen source For the measurement of a sample from a living body, from about 10 to 20 ⁇ mol of the oxygen source may preferably be added to the samples.
- a required amount of the oxygen source is generally supplied by dissolved oxygen. If the amount of oxygen source is extremely small, measuring sensitivity may sometimes be lowered, and if an extremely large amount of oxygen source exists, emission of fluorescence may be disadvantageously affected. Therefore, it is preferred that an amount of nitrogen monoxide to be measured is predicted by a preliminary experiment or a known method so that the oxygen source within an appropriate concentration range can be added.
- the reaction can be carried out at a temperature of from 10 to 25° C.
- a mixed acid was prepared by adding fuming nitric acid (1 mL), which was added 3 times as divided portions, to concentrated sulfuric acid (6 mL) under ice cooling, and added portionwise with 4-acetamidobenzaldehyde (1.91 g, 11.7 mmol). After the addition, the reaction mixture was immediately poured onto ice, and the solids were collected by filtration. The solids were sufficiently washed with cold water, dried, purified by silica gel column chromatography (developing solvent: CH 2 Cl 2 ), and then recrystallized from water to obtain the target compound as pale yellow needlelike solid (1.57 g, yield: 64%).
- Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate (3.1 g, 9.8 mmol) was dissolved in acetone (100 mL). This solution was added with 10% Pd—C, and stirred at room temperature under hydrogen gas. When the starting material disappeared, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure. The residue was added with trifluoroacetic acid (10 mL), and heated at 40° C. for 10 minutes under an argon flow. The reaction mixture was added with chloroform, and washed once with water, and the aqueous layer was extracted twice with chloroform.
- 6-(4-Amino-3-nitrophenyl)-4,4′-bis(2-methoxycarbonylethyl)-3,3,5,5′-tetramethylpyrromethene (213 mg, 0.42 mmol) was dissolved in dichloromethane (20 mL). The solution was added with diisopropylethylamine (DIEA, 1 mL, 5.7 mmol) under an argon flow, and stirred at room temperature for 10 minutes. The solution was further added with boron trifluoride diethyl etherate (1 mL, 7.9 mmol), and stirred for 40 minutes. A little after the addition, fluorescence appeared.
- DIEA diisopropylethylamine
- the reaction mixture was washed once with water and twice with aqueous 2 N NaOH, and the aqueous layer and the NaOH layer were combined, and extracted 3 times with dichloromethane.
- the fluorescence characteristics of Compound (7) and Compound (8) were measured.
- the quantum yield was measured by using F-4500 (Hitachi), and the other spectra were measured by using LS50B (Perkin Elmer). The measurement was performed at 20° C. for a solution of a sample dissolved in 0.1 M sodium phosphate buffer (pH 7.4) using dimethyl sulfoxide of less than 0.2% as a cosolvent.
- the quantum yield was calculated relative to that of fluorescein, which was 0.85 in aqueous 0.1 M NaOH. The results are shown in Table 1.
- NOC 13 which is a nitrogen monoxide generating agent, was added to Compound (7), and changes of excitation and fluorescence emission spectra were measured.
- the compound (7) at 5 ⁇ M (cosolvent: 0.1% DMSO) and NOC 13 were incubated at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4), and then excitation and fluorescence spectra were measured. The measurement was performed with slit widths of Ex/Em 2.5/2.5 nm, excitation wavelength of 520 nm, and emission wavelength of 535 nm. The results are shown in FIG. 1 . Increase of fluorescence depending on the concentration of added NOC 13 was observed.
- FIG. 2 The results obtained by incubating Compound (7) at 5 ⁇ M (cosolvent: 0.1% DMSO) and NOC 13 (20 ⁇ M) at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4) and then analyzing the product by HPLC are shown in FIG. 2 .
- a solution of DAMBO-P Me at 1 ⁇ M in chloroform was added with 5 ⁇ L/minute of a solution of NO in chloroform (prepared by bubbling argon gas in chloroform for 10 minutes and then bubbling NO gas in the same for 3 minutes), and the measurement was performed for 10 minutes.
- FIG. 4 shows that measurement of NO in an organic solvent is also possible by using p-DAMBO-P Me . From the result, it can be understood that the compound of the present invention has cell membrane permeability, and achieves efficient detection of NO even in a liposoluble environment such as biomembranes.
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Abstract
A compound represented by the formula (I) wherein R1 and R2 represent amino groups that substitute at adjacent positions on the benzene ring, wherein one of the amino groups may have one alkyl group; R3 and R4 represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R5 and R8 independently represent a C1-6 alkyl group, R6 and R9 represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R7 and R10 represent a C1-6 alkyl group, an aryl group, a C1-6 alkoxycarbonyl group, a vinyl group, a thienyl group, or a pyrrolyl group, and an agent for measurement of nitrogen monoxide containing the compound. Said compound can give a fluorescent substance that is free from change in fluorescence intensity in a wide pH range.
Description
- The present invention relates to a fluorescent probe. More specifically, the present invention relates to a fluorescent probe that captures nitrogen monoxide to emit fluorescence.
- Recently, it has been reported that particular fluorescein derivatives that per se have almost no fluorescent property react with nitrogen monoxide under a neutral condition to provide a triazole compound having a strong fluorescence intensity, and the triazole derivative can emit intense fluorescence at wavelength of around 515 nm under excitation light at a long wavelength of around 495 nm (U.S. Pat. No. 5,874,590). When these fluorescein derivatives are used as an agent for nitrogen monoxide measurement, the excitation light can be easily cut off with a fluorescence filter provided on an ordinary fluorescence microscope, and intracellular nitrogen monoxide concentration can be conveniently measured by measuring fluorescence in individual cells.
- Diaminorhodamine derivatives that can efficiently react with nitrogen monoxide under a neutral condition, and give a triazole derivative having superior fluorescence intensity have been proposed (U.S. Pat. No. 6,201,134). The fluorescence of the diaminorhodamine derivatives shifts to a longer wavelength side compared with the aforementioned fluorescein derivatives, thus the fluorescence does not substantially overlaps the autofluorescence of cells, and the fluorescence does not attenuate even in an acidic region. Therefore, the fluorescence of the diaminorhodamine derivatives does not damage living tissues and cells, and enable measurement of nitrogen monoxide in a fluorescent region of a longer wavelength than in the autofluorescent region of cells.
- Indacene derivatives, which are known to have an ion capturing moiety useful for measurement of alkali metal ions or cations (Japanese Patent Laid-Open Publication (KOKAI) Nos. 10-338695 and 11-5796). However, no attempt has been reported for measurement of nitrogen monoxide by using the fluorescence chromophore of the indacene derivatives.
- The inventors of the present invention conducted various researches to provide a fluorescent probe that specifically and efficiently captures nitrogen monoxide to emit fluorescence. As a result, they successfully provided an agent for nitrogen monoxide measurement that can efficiently react with nitrogen monoxide under a neutral condition to give a fluorescent substance having superior fluorescence intensity by using fluorescence chromophores of indacene derivatives (Japanese Patent Application No. 2002-80230).
- The inventors of the present invention further conducted researches, and as a result, they found that an agent for nitrogen monoxide measurement further having superior water-solubility was successfully provided by introducing a substituent such as a carboxy-substituted alkyl group into the fluorescence chromophore of the indacene derivatives, and that an fluorescence intensity of the fluorescent triazole derivative, produced by trapping of nitrogen monoxide by the agent, was subjected to almost no influence by pH fluctuation. The present invention was achieved on the basis of these findings.
- The present invention thus provides a compound represented by the following formula (I):
wherein, R1 and R2 represent amino groups that substitute at adjacent positions on the benzene ring, wherein one of the amino groups may have one alkyl group which may be substituted; R3 and R4 independently represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R5 and R8 independently represent a C1-6 alkyl group which may be substituted, R6 and R9 independently represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R7 and R10 independently represent a C1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C1-6 alkoxycarbonyl group which may be substituted, a vinyl group which may be substituted, a thienyl group which may be substituted, or a pyrrolyl group which may be substituted, or a salt thereof. - According to a preferred embodiment of the present invention, the aforementioned compound or a salt thereof wherein R6 and R9 are 2-carboxy-1-ethyl groups, and R5, R7, R8, and R10 are methyl groups is provided. The present invention also provides an agent for measurement of nitrogen monoxide comprising the aforementioned compound or a salt thereof.
- From another aspect, the present invention provides a compound represented by the following formula (II):
wherein, R11 and R12 combine together to represent a group represented by —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring wherein R30 represents hydrogen atom, or an alkyl group which may be substituted, or R11 and R12 represent a combination of an amino group (which may have an alkyl group which may be substituted or a protective group for amino group) and nitro group that substitute at adjacent positions on the benzene ring; R13 and R14 independently represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R15 and R18 independently represent a C1-6 alkyl group which may be substituted, R16 and R19 independently represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R17 and R20 independently represent a C1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C1-6 alkoxycarbonyl group which may be substituted, a vinyl group which may be substituted, a thienyl group which may be substituted, or a pyrrolyl group which may be substituted, or a salt thereof. According to a preferred embodiment of the aforementioned invention, the compound or a salt thereof wherein R16 and R19 are 2-carboxy-1-ethyl groups, and R15, R17, R18, and R20 are methyl groups is provided. - From a further aspect, the present invention provides a method for measurement of nitrogen monoxide, which comprises the steps of (a) reacting a compound represented by the aforementioned formula (I) with nitrogen monoxide; and (b) detecting a compound represented by the aforementioned formula (II) produced in the aforementioned step (a) wherein R11 and R12 combine together to form a group represented by —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring wherein R30 represents hydrogen atom, or an alkyl group which may be substituted.
-
FIG. 1 shows the results of the measurement of changes of excitation and fluorescence spectra of Compound (7) after addition ofNOC 13. In the drawing, (A) represent the excitation spectrum (Em: 535 nm), and (B) represents the fluorescence spectrum (Ex: 520 nm). The curves indicate the results for theNOC 13 concentrations of 5.0 μM, 2.0 μM, 1.0 μM, 0.5 μM, and 0 μM in the order that the fluorescence intensities shown by the curves decrease from the highest. -
FIG. 2 shows the results of HPLC measurement of the reaction product obtained by addingNOC 13 to Compound (7). -
FIG. 3 shows the results of measurement of fluorescence intensity of Compounds (7) and (8) at various pH values. -
FIG. 4 shows changes of fluorescence intensity observed when a solution of NO in chloroform was added to a solution of Compound (9) in chloroform. - In the specification, the alkyl group may be a linear, branched, or cyclic alkyl group, or a combination thereof, unless otherwise specifically mentioned. An alkyl moiety of other substituents containing the alkyl moiety (e.g. alkoxy group) should be understood in the same manner. Further, when “which may be substituted” is referred to for a certain functional group, the type, number, and substitution position of the substituent are not particularly limited. The functional group may have, for example, a halogen atom (any of fluorine atom, chlorine atom, bromine atom, and iodine atom), hydroxy group, amino group, carboxy group, sulfo group, an alkyl sulfonate group, or the like as the substituent. Further, when the aryl group is referred to in the specification, the group may be either a monocyclic or polycyclic aryl group. Phenyl group can be preferably used. The aromatic ring should be understood in the same manner.
- In the formula (I), when R3 and/or R4 represent a C1-6 alkyl group, or a C1-6 alkoxy group, it is preferred that these groups bind at the 2- and 6-position on the benzene ring. When these groups exist, the quantum yield and reaction rate may be improved, and thus detection sensitivity may sometimes be increased. As the alkyl group represented by R3 or R4, methyl group is preferred, and methoxy group is preferred as the alkoxy group. It is also preferred that both R3 and R4 are hydrogen atoms. R13 and R14 in the formula (II) should be understood in the same manner.
- The carboxy-substituted C1-6 alkyl group represented by R6 or R9 is preferably a monocarboxy-substituted C1-6 alkyl group. The carbon number of the C1-6 alkyl moiety of the carboxy-substituted C1-6 alkyl group is preferably 1 to 4, more preferably 2 or 3, and most preferably 2. It is most preferred that R6 and R9 are 2-carboxy-1-ethyl groups. Examples of the alkoxycarbonyl-substituted C1-6 alkyl group represented by R6 or R9 include a C1-6 alkyl ester of the aforementioned carboxy-substituted C1-6 alkyl group. Preferred examples include an ethoxycarbonyl-substituted C1-6 alkyl group, and the like. As the sulfo-substituted C1-6 alkyl group represented by R6 or R9, a monosulfo-substituted C1-6 alkyl group is preferred. As the alkyl sulfonate-substituted C1-6 alkyl group represented by R6 or R9, a monoalkyl sulfonate-substituted C1-6 alkyl group is preferred. As the alkyl sulfonate group in the alkyl sulfonate-substituted C1-6 alkyl group, a C1-6 alkyl sulfonate (C1-6 alkyl-O—SO2—) is preferred. When R6 and R9 are monocarboxy-substituted C1-6 alkyl groups, especially when R6 and R9 are 2-carboxy-1-ethyl groups, superior effects are obtained in that water-solubility of the compounds remarkably increases, and fluorescence intensity of the compounds represented by the formula (II) produced by a reaction with nitrogen monoxide is not subjected to influence of pH fluctuation.
- As the aryl group represented by R7 or R10, a phenyl group is preferred. When the phenyl group has a substituent, sulfo group, a sulfonate group, and the like are preferred as the substituent, and a sulfo group is particularly preferred. As the C1-6 alkoxycarbonyl group represented by R7 or R10, ethoxycarbonyl group is preferred. Examples of the substituent which exists on the vinyl group represented by R7 or R10 include a phenyl group, monoaminophenyl group, and a diaminophenyl group (for example, 3,4-diaminophenyl group). As the thienyl group or pyrrolyl group represented by R7 or R10, 2-thienyl group, or 2-pyrrolyl group is preferred, respectively. When R7 and R10 are groups other than alkyl group, fluorescence wavelength of the compounds may sometimes shift to a longer wavelength side.
- In the formula (I), it is more preferred that R6 and R9 are 2-carboxy-1-ethyl groups. Further, in the formula (I), it is preferred that R5, R7, R8, and R10 are C1-6 alkyl groups which may be substituted. For example, the compounds wherein R5, R7, R8, and R10 are methyl groups are preferred embodiments of the present invention. R15, R16, R17, R18, R19, and R20 in the formula (II) should be understood in the same manner as R5, R6, R7, R8, R9, and R10.
- In the aforementioned formula (I), R1 and R2 represent amino groups that substitute at adjacent positions on the benzene ring. Both of R1 and R2 may be unsubstituted amino groups. Alternatively, either of R1 and R2 may be substituted with one alkyl group, and the alkyl group may have one or more substituents. Examples of the alkyl group which substitutes on the amino group include, for example, a linear or branched C1-18 alkyl group (preferably a C1-6 alkyl group). Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group and the like can be used. Examples of the alkyl group which has a substituent include, for example, a C1-6 alkyl group substituted with a substituted or unsubstituted aryl group (aralkyl group) and the like. As the aryl-substituted alkyl group, for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
- In the aforementioned formula (II), R11 and R12 combine together to represent a —N═N—NR30— group which forms a ring structure at adjacent positions on the benzene ring. R30 represents hydrogen atom, or an alkyl group which may be substituted. Examples of this alkyl group include a linear or branched C1-18 alkyl group (preferably a C1-6 alkyl group), and examples of the alkyl group which has a substituent include, for example, a substituted or unsubstituted aralkyl group. As the aralkyl group, for example, benzyl group, phenethyl group, paramethoxybenzyl group, paraethoxycarbonylbenzyl group, paracarboxybenzyl group, and the like can be used.
- Further, R11 and R12 also represent a combination of an amino group (which may have one substituent) and nitro group that substitute at adjacent positions on the benzene ring, wherein either of R11 and R12 represents an amino group, and the other represents nitro group. The amino group represented by either R11 or R12 may be unsubstituted, or the group may have one alkyl group, for example, a C1-18 alkyl group, preferably a C1-6 alkyl group. Further, the alkyl group may be substituted, and for example, a substituted or unsubstituted aralkyl group or the like may substitute on the amino group. Moreover, the amino group may have a protective group for amino group, for example, an acyl group such as acetyl group, trifluoroacetyl group, and benzoyl group; an alkylsilyl group such as trimethylsilyl groups, or the like. An aralkyl group such as benzyl group may also be used as the protective group.
- The compounds of the present invention represented by the formulas (I) and (II) may form a salt. Type of the salt is not particularly limited, and the salt may be an acid addition salt or base addition salt. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as methanesulfonate, citrate, p-toluenesulfonate, and oxalate. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, and calcium salt, ammonium salts, and organic amine salts such as methylamine salt, and triethylamine salt. In addition, the compounds may form a salt with an amino acid such as glycine. However, salts of the compounds of the present invention are not limited to these specific examples.
- The compounds of the present invention represented by the formula (I) or (II) may have one or more asymmetric carbon. Any of optical isomers in an optically pure form, arbitrary mixtures of optical isomers, racemates, diastereoisomers in a pure form, mixtures of diastereoisomers, and the like based on one or more asymmetric carbon atoms fall within the scope of the present invention. The compounds of the present invention may exist as hydrates or solvates, and it should be understood that these substances also fall within the scope of the present invention.
- Among the compounds represented by the aforementioned formula (I), and the compounds represented by the aforementioned formula (II) wherein R11 and R12 represent a combination of an amino group and nitro group that substitute at adjacent positions on the benzene ring, those wherein R3 and R4 are hydrogen atoms, R6 and R9 are 2-carboxy-1-ethyl groups, and R5, R7, R8, and R10 are methyl groups, and those wherein R13 and R14 are hydrogen atoms, R16 and R19 are 2-carboxy-1-ethyl groups, and R15, R17, R18, and R20 are methyl groups, which are typical compounds, are specifically mentioned in the examples of the specification as for preparation examples thereof. Therefore, it will be understood that the compounds represented by the aforementioned formula (II) are useful as synthetic intermediates of the compounds represented by the formula (I). Among the compounds represented by the formula (II), the compounds wherein R11 and R12 combine together to represent the —N═N—NR30— group, which forms a ring structure at adjacent positions on the phenyl ring, can be prepared by reacting a compound represented by the aforementioned formula (I) with nitrogen monoxide. These compounds have strong fluorescent property as described later, and are useful for measurement of nitrogen monoxide.
- It will be understood by those skilled in the art that the compounds falling within the scope of the formula (I) or (II) can be easily produced by referring to the specific explanations of the examples of the specification. Further, synthetic methods of the indacene structure are mentioned in, for example, Japanese Patent Laid-Open Publication (KOKAI) Nos. 10-338695 and 11-5796, as well as in New J. Chem., 25, pp. 289-292, 2001; Tetrahedron Letters, 42, pp. 6711-6713, 2001; Angew. Chem. Int. Ed., 40 pp. 385-387, 2001; Japanese Patent Application No. 2002-80230, and the like, and therefore, those skilled in the art can prepare the compounds of the present invention still more easily by referring to these publications.
- The compounds represented by the formula (I) of the present invention have a property that they efficiently react with nitrogen monoxide under a neutral condition and provide compounds of the formula (II) wherein R11 and R12 combine together to form the group —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring in a good yield. The compounds represented by the formula (I), per se, emit almost no fluorescence when irradiated with excitation light of around 485 nm under a neutral condition, whereas the compounds of the above formula (II) have the property of emitting extremely strong fluorescence under the same condition. Therefore, nitrogen monoxide in living tissues or cells can be measured by introducing the compound represented by the formula (I) into a living tissue or a cell to allow the compound to react with nitrogen monoxide to form the fluorescent compound of the above formula (II), and measuring the fluorescence of the compound. In particular, the compounds of the formula (I) of the present invention have superior reactivity with nitrogen monoxide, and thus have an outstanding characteristic that they enable measurement of nitrogen monoxide with high sensitivity and accuracy.
- The method for measurement of nitrogen monoxide provided by the present invention comprises the steps of reacting a compound represented by the above formula (I) with nitrogen monoxide to form a compound of formula (II), and measuring fluorescence of the compound of the formula (II) wherein R11 and R12 combine together to represent the —N=N—NR30— group which forms a ring structure at adjacent positions on the benzene ring. The term “measurement” used in the specification should be construed in its broadest sense, which includes various measurement purposes such as, for example, detection, quantification, qualitative analysis and the like. The above reaction can preferably be carried out under a neutral condition, for example, in a range of from pH 6.0 to 8.0, preferably in a range of from pH 6.5 to 7.8, and more preferably in a range of from pH 6.8 to 7.6. However, the measurement of nitrogen monoxide using the compounds of the present invention is not limited to the measurements under the neutral range or weakly acidic range. For example, measurement can also be performed under a strongly acidic condition such as in gastric mucosal cells. In particular, the compounds represented by the formula (II) of the present invention have an extremely superior characteristic that fluorescence intensity does not change in a wide range of pH, and thus they enable accurate measurement without being influenced by pH fluctuation.
- The measurement of fluorescence can be carried out according to a known fluorometry method (see, for example, Wiersma, J. H., Anal. Lett., 3, pp. 123-132, 1970; Sawicki, C. R., Anal. Lett., 4, pp. 761-775, 1971; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; Damiani, P. and Burini, G., Talanta, 8, pp. 649-652, 1986; and Misko, T. P., Anal. Biochem., 214, pp. 11-16, 1993, and the like). For the nitrogen monoxide measurement according to the present invention, for example, irradiation with light of about 520 nm as excitation light, and measurement of fluorescence of about 535 nm may preferably be performed. The compounds represented by the formula (II) of the present invention have a superior characteristic that they give sufficient fluorescence intensity even with an excitation light having a long wavelength, and therefore they can reduce damages on the living body, tissues, cells, and the like. Further, by using the light having such a wavelength, efficient cut off can be obtained by using a fluorescence filter provided on an ordinary fluorescence microscope, and measurement with high sensitivity can be achieved without using an unordinary filter.
- Where particularly high sensitive measurement is required, the aforementioned measurement of nitrogen monoxide may be carried out in the presence of an oxygen source. As the oxygen source, for example, oxygen, ozone, oxide compounds or the like can be used. As the oxygen, dissolved oxygen can generally be used, and if desired, oxygen gas may be introduced into the reaction system, or an agent that can generate oxygen (e.g., hydrogen peroxide) may be added. The oxide compounds are not particularly limited so long as they have an oxide bond that can easily be cleaved, e.g., N—O, S—O, or P—O. For example, PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, Maeda, H., et al., J. Leuk. Biol., 56, pp. 588-592, 1994; and Akaike, T., et al., Biochemistry, 32, pp. 827-832, 1993) or derivatives thereof (carboxy-PTIO which has carboxyl group introduced at the para-position of the phenyl group of PTIO and the like), triphenylphosphine oxide, triethylamine oxide or the like can be used.
- Among the oxide compounds mentioned above, PTIO and derivatives thereof (e.g., carboxy-PTIO) are particularly preferred compounds, and they can be readily obtained by those skilled in the art (listed in, for example, Organic Chemicals Catalog, 32, 1994, Tokyo Kasei Co., Ltd.). The oxide compounds, per se, may be used as a reaction agent, or those encapsulated in liposomes or other may also be used. An amount of the oxygen source is not particularly limited. A preferable amount may be at least 1 μmol or more, preferably 10 to 30 μmol, and more preferably about 10 to 20 μmol, based on nitrogen monoxide to be measured. For the measurement of a sample from a living body, from about 10 to 20 μmol of the oxygen source may preferably be added to the samples. A required amount of the oxygen source is generally supplied by dissolved oxygen. If the amount of oxygen source is extremely small, measuring sensitivity may sometimes be lowered, and if an extremely large amount of oxygen source exists, emission of fluorescence may be disadvantageously affected. Therefore, it is preferred that an amount of nitrogen monoxide to be measured is predicted by a preliminary experiment or a known method so that the oxygen source within an appropriate concentration range can be added. The reaction can be carried out at a temperature of from 10 to 25° C. In addition, the method for measurement of nitrogen monoxide by using a fluorescent probe is described in detail in Tetsuo Nagano et al., “Kagaku to Kyoiku (Chemistry and Education)”, 47, pp. 665-669, 1999, and the like, and therefore those skilled in the art can measure nitrogen monoxide with high sensitivity by using the compounds of the present invention by referring to the aforementioned publication.
- The present invention will be more specifically explained with reference to the examples. However, the scope of the present invention is not limited to the following examples.
- (1) 4-Acetamido-3-nitrobenzaldehyde
- A mixed acid was prepared by adding fuming nitric acid (1 mL), which was added 3 times as divided portions, to concentrated sulfuric acid (6 mL) under ice cooling, and added portionwise with 4-acetamidobenzaldehyde (1.91 g, 11.7 mmol). After the addition, the reaction mixture was immediately poured onto ice, and the solids were collected by filtration. The solids were sufficiently washed with cold water, dried, purified by silica gel column chromatography (developing solvent: CH2Cl2), and then recrystallized from water to obtain the target compound as pale yellow needlelike solid (1.57 g, yield: 64%).
- 1H-NMR (300 MHz, CDCl3)
- 2.36 (3H, s), 8.16 (1H, dd, J=8.79, 1.65 Hz), 8.74 (1H, d, J=1.65 Hz), 9.04 (1H, d, J=8.79 Hz), 9.99 (1H, S), 10.63 (1H, S)
- MS (El): 208 (M+)
- m.p.: 156° C.
(2) 4-Amino-3-nitrobenzaldehyde - 4-Acetamido-3-nitrobenzaldehyde (2.08 g, 10 mmol) was dissolved in methanol (200 mL). The solution was added with aqueous 2 N HCl (50 mL), and refluxed by heating at 80° C. for 8 hours under an argon flow. The reaction mixture was concentrated under reduced pressure to remove the methanol, and the residue was made basic with aqueous 2 N NaOH, and extracted 5 times with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain the target compound as yellow solid (1.62 g, yield: 98%).
- 1H-NMR (300 MHz, CDCl3)
- 6.91 (1H, d, J=8.58 Hz), 7.92 (1H, dd, J=8.58, 2.01 Hz), 8.63 (1H, d, J=2.01 Hz), 9.82 (1H, s)
- MS (El): 166 (M+)
(3) 3-(2-Methoxycarbonylethyl)-2,4-dimethylpyrrole - Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate (3.1 g, 9.8 mmol) was dissolved in acetone (100 mL). This solution was added with 10% Pd—C, and stirred at room temperature under hydrogen gas. When the starting material disappeared, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure. The residue was added with trifluoroacetic acid (10 mL), and heated at 40° C. for 10 minutes under an argon flow. The reaction mixture was added with chloroform, and washed once with water, and the aqueous layer was extracted twice with chloroform. The organic phases were combined, washed once with aqueous Na2CO3 and once with water, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: CH2Cl2) to obtain the target compound as brown liquid (1.6 g, yield: 90%).
- 1H-NMR (300 MHz, CDCl3)
- 2.03 (3H, d, J=0.90 Hz), 2.18 (3H, s), 2.45 (2H, m), 2.72 (2H, m), 3.67 (3H, s), 6.38 (1H, d, J=0.90 Hz), 7.53 (1H, s)
- MS (El): 181 (M+)
(4) 6-(4-Amino-3-nitrophenyl)-4,4′-bis(2-methoxycarbonylethyl)-3,3′,5,5′-tetramethylpyrromethene - 4-Amino-3-nitrobenzaldehyde (760 mg, 4.58 mmol) and 3-(2-methoxycarbonyl-ethyl)-2,4-dimethylpyrrole (1.6 g, 9.16 mmol) were dissolved in dichloromethane (250 mL). This solution was added with several drops of trifluoroacetatic acid under an argon flow, and stirred overnight at room temperature with shielding from light. After disappearance of the starting materials was confirmed by thin layer chromatography (developing solvent: dichloromethane, silica gel), the reaction mixture was added with 120 mL of a solution of dichlorodicyanoparabenzoquinone (DDQ, 1.07 g, 4.58 mmol) in dichloromethane. The reaction mixture was stirred for 15 minutes, then washed once with water, dried over anhydrous MgSO4, and concentrated under reduced pressure. The residue was purified by alumina column chromatography (developing solvent: methanol/dichloromethane=1/20) to obtain the target compound (yield: 76%).
- 1H-NMR (300 MHz, CDCl3)
- 1.37 (6H, s), 2.33 (6H, S), 2.36 (4H, dd, J=8.43, 7.14 Hz), 2.63 (4H, dd, J=8.43, 7.14 Hz), 3.65 (6H, S), 6.32 (2H, S), 6.92 (1H, d, J=8.61Hz), 7.26 (1H, dd, J=8.61, 2.01 Hz), 8.06 (1H, d, J=2.01 Hz)
- MS (El): 508 (M+)
(5) 8-(4-Amino-3-nitrophenyl)-4,4-difluoro-2,6-bis(2-methoxycarbonylethyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene - 6-(4-Amino-3-nitrophenyl)-4,4′-bis(2-methoxycarbonylethyl)-3,3,5,5′-tetramethylpyrromethene (213 mg, 0.42 mmol) was dissolved in dichloromethane (20 mL). The solution was added with diisopropylethylamine (DIEA, 1 mL, 5.7 mmol) under an argon flow, and stirred at room temperature for 10 minutes. The solution was further added with boron trifluoride diethyl etherate (1 mL, 7.9 mmol), and stirred for 40 minutes. A little after the addition, fluorescence appeared. After completion of the reaction, the reaction mixture was washed once with water and twice with aqueous 2 N NaOH, and the aqueous layer and the NaOH layer were combined, and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure, and the residue was purified by alumina column chromatography (developing solvent: methanol/dichloromethane=1/20) to obtain the target compound as orange solid (72 mg, yield: 31%).
- 1H-NMR (300 MHz, CDCl3)
- 1.46 (6H, s), 2.37 (4H, dd, J=8.25, 7.32 Hz), 2.54 (6H, S), 2.65 (4H, dd, J=8.25, 7.32 Hz), 3.66 (6H, S), 6.32 (2H, S), 6.99 (1H, d, J=8.40 Hz), 7.24 (1H, dd, J=8.40, 1.83 Hz), 8.06 (1H, d, J=1.83 Hz)
- MS (El): 556 (M+)
(6) 8-(4-Amino-3-nitrophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene - 8-(4-Amino-3-nitrophenyl)-4,4-difluoro-2,6-bis(2-methoxycarbonylethyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (182.5 mg, 0.33 mmol) was dissolved in methanol (100 mL), and this solution was added with aqueous 0.2 N NaOH (10 mL), and refluxed by heating at 80° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the methanol, made acidic with aqueous 2 N HCl, and extracted 3 times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: methanol/dichloromethane=1/10) to obtain the target compound (87.1 mg, yield: 50%).
- 1H-NMR (300 MHz, CD3OD)
- 1.44 (6H, s), 2.26 (4H, dd, J=7.86, 7.32 Hz), 2.40 (6H, S), 2.57 (4H, dd, J=7.86, 7.32 Hz), 7.07 (1H, d, J=8.61 Hz), 7.17 (1H, dd, J=8.61, 2.01 Hz), 7.88 (1H, d, J=2.01 Hz)
- MS (FAB): 528 (M+)
(7) 8-(3,4-Diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (p-DAMBO-PH) - 8-(4-Amino-3-nitrophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetrame thyl-4-bora-3a,4a-diaza-s-indacene (87.1 mg, 0.16 mmol) was dissolved in methanol (100 mL), and this solution was added with 10% Pd—C, and stirred at room temperature under hydrogen gas. After disappearance of the starting material was confirmed, the reaction mixture was filtered, and the filtrate was distilled under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: H2O/acetonitrile=1/10) to obtain the target compound as brown solid (31.5 mg, yield: 38%).
- 1H-NMR (300 MHz, CD3OD)
- 1.51 (6H, s), 2.28 (4H, dd, J=8.40, 7.14 Hz), 2.47 (6H, S), 2.64 (4H, dd, J=8.40, 7.14 Hz), 6.46 (1H, dd, J=7.86, 2.01 Hz), 6.59 (1H, d, J=2.01 Hz), 6.83 (1H, d, J=7.86 Hz)
- MS (FAB): 498 (M+)
(8) 8-(5-Benzotriazolyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (p-DAMBO-PH-T) - p-DAMBO-PH (17.4 mg, 0.035 mmol) was added with aqueous 1 N HCl (20 mL), and this solution was added portionwise with aqueous NaNO2 (2.5 mg, 0.036 mmol) with stirring under ice cooling. After completion of the addition, the reaction mixture was returned to room temperature, and stirred for 15 minutes. The solution was extracted 3 times with ethyl acetate. The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: H2O/acetonitrile=1/10) to obtain the target compound as dark reddish brown solid (14.6 mg, yield: 82%).
- 1H-NMR (300 MHz, CD3OD)
- 1.24 (6H, s), 2.30 (4H, dd, J=8.25, 7.14 Hz), 2.51 (6H, S), 2.63 (4H, dd, J=8.25, 7.14 Hz), 7.43 (1H, d, J=8.43 Hz), 7.89 (1H, s), 8.08 (1H, d, J=8.43 Hz)
- MS (FAB): 509 (M+)
(9) 8-(3,4-Diaminophenyl)-4,4-difluoro-2,6-bis(2-methoxycarbonylethyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (p-DAMBO-PMe) - 8-(4-Amino-3-nitrophenyl)-4,4-difluoro-2,6-bis(2-methoxycarbonylethyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (131 mg, 0.24 mmol) was dissolved in dichloromethane (50 mL), and further added with methanol (100 ml). This solution was added with 10% Pd—C, and stirred overnight at room temperature under hydrogen gas. After disappearance of the starting materials was confirmed by alumina TLC (developing solvent: dichloromethane), the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by alumina column chromatography (developing solvent: dichloromethane, 85 mg, yield: 68%), and recrystallized from hexane/chloroform.
- 1H-NMR (300 MHz, CDCl3)
- 1.44 (6H, s), 2.35 (4H, dd, J=8.61, 7.32 Hz), 2.53 (6H, S), 2.64 (4H, dd, J=8.61, 7.32 Hz), 3.46 (2H, S), 3.54 (2H, S), 3.66 (6H, S), 6.55 (1H, d, J=6.39 Hz), 6.56 (s, 1H), 6.79 (1H, d, J=6.39 Hz)
- MS (ESl): 549 ([M+Na]+)
- (a) Fluorescence Spectrum
- The fluorescence characteristics of Compound (7) and Compound (8) were measured. The quantum yield was measured by using F-4500 (Hitachi), and the other spectra were measured by using LS50B (Perkin Elmer). The measurement was performed at 20° C. for a solution of a sample dissolved in 0.1 M sodium phosphate buffer (pH 7.4) using dimethyl sulfoxide of less than 0.2% as a cosolvent. The quantum yield was calculated relative to that of fluorescein, which was 0.85 in aqueous 0.1 M NaOH. The results are shown in Table 1. When similar quantum yield measurements were performed for a compound of the formula (I) wherein R6 and R9 were hydrogen atoms (p-DAMBO) and a compound of the formula (II) wherein R16 and R19 were hydrogen atoms (p-DAMBO-T) as controls, the quantum yields were 0.001 and 0.40, respectively. Accordingly, the compounds of the present invention were found to successfully give a large quantum yield. Moreover, the Stokes shift of the compounds of the present invention was 16 nm, which was larger than those of p-DAMBO and p-DAMBO-T, namely, a large Stokes shift almost comparable to that of fluorescein was obtained (both the Stokes shifts of p-DAMBO and p-DAMBO-T were 9 nm).
TABLE 1 Extinction Extinction coefficient Emission Relative maximum (×104 M−1 maximum quantum Compound (nm) cm−1) (nm) yield p-DAMBO-PH 519 5.8 535 0.002 p-DAMBO-PH-T 521 5.6 537 0.74
(b) Reaction with Nitrogen Monoxide -
NOC 13, which is a nitrogen monoxide generating agent, was added to Compound (7), and changes of excitation and fluorescence emission spectra were measured. The compound (7) at 5 μM (cosolvent: 0.1% DMSO) andNOC 13 were incubated at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4), and then excitation and fluorescence spectra were measured. The measurement was performed with slit widths of Ex/Em=2.5/2.5 nm, excitation wavelength of 520 nm, and emission wavelength of 535 nm. The results are shown inFIG. 1 . Increase of fluorescence depending on the concentration of addedNOC 13 was observed. The results obtained by incubating Compound (7) at 5 μM (cosolvent: 0.1% DMSO) and NOC 13 (20 μM) at 37° C. for 1 hour in 0.1 M sodium phosphate buffer (pH 7.4) and then analyzing the product by HPLC are shown inFIG. 2 . The HPLC conditions were as follows: column: Inertsil ODS-3 (4.6×250 mm), eluent: aqueous 0.1% H3PO4/acetonitrile=55/45 (v/v), detection: UV-vis (520 nm)/emission (520/535 nm), flow rate: 1 mL/min, and sample volume: 5 μL. Occurrence of extremely rapid reaction of Compound (7) and nitrogen monoxide was observed. - (c) The Effect of pH on the Fluorescence Intensity
- Compounds (7) and (8) were dissolved in 0.1 M sodium phosphate buffer (cosolvent: 0.1% DMSO) adjusted to various pH values, and fluorescence was measured in the same manner as those in the aforementioned (b). The results are shown in
FIG. 3 . Almost no change in the fluorescence intensity of Compound (8) was observed in an extremely wide pH range of from 3 to 13. Therefore, according to the measurement of nitrogen monoxide by using the compound of the present invention, extremely high accuracy of the measurement is achieved for a biosample with pH fluctuation. (d) Change in fluorescence caused by a reaction of Compound (9) (p-DAMBO-PMe) with NO in an organic solvent was measured. A solution of DAMBO-PMe at 1 μM in chloroform was added with 5 μL/minute of a solution of NO in chloroform (prepared by bubbling argon gas in chloroform for 10 minutes and then bubbling NO gas in the same for 3 minutes), and the measurement was performed for 10 minutes. The measurement was performed with slit widths of Ex/Em=5.0/0 nm, excitation wavelength of 520 nm, and emission wavelength of 535 nm. The results are shown inFIG. 4 .FIG. 4 shows that measurement of NO in an organic solvent is also possible by using p-DAMBO-PMe. From the result, it can be understood that the compound of the present invention has cell membrane permeability, and achieves efficient detection of NO even in a liposoluble environment such as biomembranes.
Claims (10)
1. A compound represented by the following formula (I):
wherein, R1 and R2 represent amino groups that substitute at adjacent positions on the benzene ring, wherein one of the amino groups may have one alkyl group which may be substituted; R3 and R4 independently represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R5 and R8 independently represent a C1-6 alkyl group which may be substituted, R6 and R9 independently represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R7 and R10 independently represent a C1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C1-6 alkoxycarbonyl group which may be substituted, a vinyl group which may be substituted, a thienyl group which may be substituted, or a pyrrolyl group which may be substituted, or a salt thereof.
2. The compound or a salt thereof according to claim 1 , wherein R6 and R9 are 2-carboxy-1-ethyl groups, and R5, R7, R8, and R10 are methyl groups.
3. The compound or a salt thereof according to claim 1 , wherein R6 and R9 are 2-methoxycarbonyl-1-ethyl groups, and R5, R7, R8, and R10 are methyl groups.
4. An agent for measurement of nitrogen monoxide comprising the compound or a salt thereof according to claim 1 .
5. A compound represented by the following formula (II):
wherein, R11 and R12 combine together to represent a group represented by —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring wherein R30 represents hydrogen atom, or an alkyl group which may be substituted, or R11 and R12 represent a combination of an amino group (which may have an alkyl group which may be substituted or a protective group for amino group) and nitro group that substitute at adjacent positions on the benzene ring; R13 and R14 independently represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R15 and R18 independently represent a C1-6 alkyl group which may be substituted, R16 and R19 independently represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R17 and R20 independently represent a C1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C1-6 alkoxycarbonyl group which may be substituted, a vinyl group which may be substituted, a thienyl group which may be substituted, or a pyrrolyl group which may be substituted, or a salt thereof.
6. The compound or a salt thereof according to claim 5 , wherein R16 and R19 are 2-carboxy-1-ethyl groups, and R15, R17, R18, and R20 are methyl groups.
7. The compound or a salt thereof according to claim 5 , wherein R16 and R19 are 2-methoxycarboxy-1-ethyl groups, and R15, R17, R18, and R20 are methyl groups.
8. A method for measurement of nitrogen monoxide, which comprises the steps of:
(a) reacting a compound represented by the formula (I) according to claim 1 with nitrogen monoxide; and
(b) detecting a compound represented by the formula (II) according to the following formula (II):
wherein, R11 and R12 combine together to represent a group represented by —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring wherein R30 represents hydrogen atom, or an alkyl group which may be substituted, or R11 and R12 represent a combination of an amino group (which may have an alkyl group which may be substituted or a protective group for amino group) and nitro group that substitute at adjacent positions on the benzene ring; R13 and R14 independently represent hydrogen atom, a C1-6 alkyl group, or a C1-6 alkoxy group, R15 and R18 independently represent a C1-6 alkyl group which may be substituted, R16 and R19 independently represent a carboxy-substituted C1-6 alkyl group, an alkoxycarbonyl-substituted C1-6 alkyl group, a sulfo-substituted C1-6 alkyl group, or an alkyl sulfonate-substituted C1-6 alkyl group, and R 17 and R20 independently represent a C1-6 alkyl group which may be substituted, an aryl group which may be substituted, a C1-6 alkoxycarbonyl group which may be substituted, a vinyl group which may be substituted, a thienyl group which may be substituted, or a pyrrolyl group which may be substituted, or a salt thereof, produced in the aforementioned step (a) wherein R11 and R12 combine together to form a group represented by —N═N—NR30— which forms a ring structure at adjacent positions on the benzene ring wherein R30 represents hydrogen atom, or an alkyl group which may be substituted.
9. An agent for measurement of nitrogen monoxide comprising the compound or a salt thereof according to claim 2 .
10. An agent for measurement of nitrogen monoxide comprising the compound or a salt thereof according to claim 3.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/688,625 US20100120160A1 (en) | 2003-02-28 | 2010-01-15 | Fluorescent Probe |
US13/425,802 US8673957B2 (en) | 2003-02-28 | 2012-03-21 | Fluorescent probe |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003-052256 | 2003-02-28 | ||
JP2003052256 | 2003-02-28 | ||
PCT/JP2004/002407 WO2004076466A1 (en) | 2003-02-28 | 2004-02-27 | Fluorescent probes |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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US12/688,625 Continuation US20100120160A1 (en) | 2003-02-28 | 2010-01-15 | Fluorescent Probe |
Publications (1)
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US20060275912A1 true US20060275912A1 (en) | 2006-12-07 |
Family
ID=32923393
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
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US10/547,305 Abandoned US20060275912A1 (en) | 2003-02-28 | 2004-02-27 | Fluorescent probes |
US12/688,625 Abandoned US20100120160A1 (en) | 2003-02-28 | 2010-01-15 | Fluorescent Probe |
US13/425,802 Expired - Fee Related US8673957B2 (en) | 2003-02-28 | 2012-03-21 | Fluorescent probe |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
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US12/688,625 Abandoned US20100120160A1 (en) | 2003-02-28 | 2010-01-15 | Fluorescent Probe |
US13/425,802 Expired - Fee Related US8673957B2 (en) | 2003-02-28 | 2012-03-21 | Fluorescent probe |
Country Status (4)
Country | Link |
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US (3) | US20060275912A1 (en) |
EP (1) | EP1604994A4 (en) |
JP (1) | JP4522365B2 (en) |
WO (1) | WO2004076466A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070298507A1 (en) * | 2004-02-23 | 2007-12-27 | Tetsuo Nagano | Fluorescent Probe |
US20090258434A1 (en) * | 2005-11-14 | 2009-10-15 | The University Of Tokyo | Fluorescent probe for peroxynitrite |
US8258171B2 (en) | 2006-11-15 | 2012-09-04 | The University Of Tokyo | pH-sensitive fluorescent probe |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2006306752A (en) * | 2005-04-27 | 2006-11-09 | Kanagawa Acad Of Sci & Technol | Chemiluminescent compound and marker comprising the same |
JP2007238489A (en) * | 2006-03-07 | 2007-09-20 | Kanagawa Acad Of Sci & Technol | Chemiluminescent compound and labeling agent composed of the same |
US20100231125A1 (en) * | 2009-03-12 | 2010-09-16 | Sheng Li | Organic light emitting device to emit in near infrared |
KR101397530B1 (en) | 2012-08-10 | 2014-05-20 | 한국과학기술원 | Facile “one pot” route to the novel benzazulene-type dye class: asymmetric, derivatizable, 5-7-6 fused ring puckered half BODIPY design |
CN103194214A (en) * | 2013-04-11 | 2013-07-10 | 武汉大学 | Fluorescent probe for nitrogen monoxide detection |
CN104194773B (en) * | 2014-08-26 | 2016-08-17 | 武汉大学 | A kind of cell membrane targeting nitric oxide fluorescent probe and its preparation method and application |
CN106220664B (en) * | 2016-08-10 | 2018-06-22 | 浙江大学 | A kind of fluorescence probe for detecting autophagy stream and its preparation and application |
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US5248782A (en) * | 1990-12-18 | 1993-09-28 | Molecular Probes, Inc. | Long wavelength heteroaryl-substituted dipyrrometheneboron difluoride dyes |
US5874590A (en) * | 1997-02-19 | 1999-02-23 | Tetsuo Nagano | Diaminofluorescein derivative |
US5981746A (en) * | 1997-05-30 | 1999-11-09 | Avl Medical Instruments | Luminescence indicator |
US6201134B1 (en) * | 1997-07-02 | 2001-03-13 | Tetsuo Nagano | Diaminorhodamine derivatives |
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Publication number | Priority date | Publication date | Assignee | Title |
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JP3967943B2 (en) * | 2002-03-22 | 2007-08-29 | 哲雄 長野 | Fluorescent probe |
FR2882056B1 (en) * | 2005-02-15 | 2007-06-01 | Centre Nat Rech Scient | UNSATURATED BOROCARBON DIPYRROMETHENES-BORE |
US8178669B2 (en) * | 2005-11-14 | 2012-05-15 | The University Of Tokyo | Fluorescent probe for peroxynitrite |
EP2098529B1 (en) * | 2006-11-15 | 2014-06-18 | The University of Tokyo | Ph-sensitive fluorescent probe |
-
2004
- 2004-02-27 EP EP04715541A patent/EP1604994A4/en not_active Withdrawn
- 2004-02-27 US US10/547,305 patent/US20060275912A1/en not_active Abandoned
- 2004-02-27 WO PCT/JP2004/002407 patent/WO2004076466A1/en active Application Filing
- 2004-02-27 JP JP2005502965A patent/JP4522365B2/en not_active Expired - Fee Related
-
2010
- 2010-01-15 US US12/688,625 patent/US20100120160A1/en not_active Abandoned
-
2012
- 2012-03-21 US US13/425,802 patent/US8673957B2/en not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US5248782A (en) * | 1990-12-18 | 1993-09-28 | Molecular Probes, Inc. | Long wavelength heteroaryl-substituted dipyrrometheneboron difluoride dyes |
US5874590A (en) * | 1997-02-19 | 1999-02-23 | Tetsuo Nagano | Diaminofluorescein derivative |
US5981746A (en) * | 1997-05-30 | 1999-11-09 | Avl Medical Instruments | Luminescence indicator |
US6001999A (en) * | 1997-05-30 | 1999-12-14 | Avl Medical Instruments | Luminescence indicator including ionophoric moiety |
US6201134B1 (en) * | 1997-07-02 | 2001-03-13 | Tetsuo Nagano | Diaminorhodamine derivatives |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070298507A1 (en) * | 2004-02-23 | 2007-12-27 | Tetsuo Nagano | Fluorescent Probe |
US7939330B2 (en) | 2004-02-23 | 2011-05-10 | Tetsuo Nagano | Fluorescent probe |
US20090258434A1 (en) * | 2005-11-14 | 2009-10-15 | The University Of Tokyo | Fluorescent probe for peroxynitrite |
US8178669B2 (en) | 2005-11-14 | 2012-05-15 | The University Of Tokyo | Fluorescent probe for peroxynitrite |
US8258171B2 (en) | 2006-11-15 | 2012-09-04 | The University Of Tokyo | pH-sensitive fluorescent probe |
Also Published As
Publication number | Publication date |
---|---|
EP1604994A1 (en) | 2005-12-14 |
EP1604994A4 (en) | 2009-03-25 |
JP4522365B2 (en) | 2010-08-11 |
WO2004076466A1 (en) | 2004-09-10 |
US8673957B2 (en) | 2014-03-18 |
US20120178174A1 (en) | 2012-07-12 |
US20100120160A1 (en) | 2010-05-13 |
JPWO2004076466A1 (en) | 2006-06-01 |
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