WO2010026743A1 - Reagent for measuring hypoxic environment - Google Patents
Reagent for measuring hypoxic environment Download PDFInfo
- Publication number
- WO2010026743A1 WO2010026743A1 PCT/JP2009/004313 JP2009004313W WO2010026743A1 WO 2010026743 A1 WO2010026743 A1 WO 2010026743A1 JP 2009004313 W JP2009004313 W JP 2009004313W WO 2010026743 A1 WO2010026743 A1 WO 2010026743A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- group
- compound
- hypoxic environment
- compound according
- measuring
- Prior art date
Links
- 206010021143 Hypoxia Diseases 0.000 title claims abstract description 52
- 230000001146 hypoxic effect Effects 0.000 title claims abstract description 51
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 16
- 150000001875 compounds Chemical class 0.000 claims abstract description 77
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 15
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 12
- 125000002947 alkylene group Chemical group 0.000 claims abstract description 11
- 125000004453 alkoxycarbonyl group Chemical group 0.000 claims abstract description 6
- 125000001140 1,4-phenylene group Chemical group [H]C1=C([H])C([*:2])=C([H])C([H])=C1[*:1] 0.000 claims abstract description 5
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 claims abstract description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 26
- 239000001301 oxygen Substances 0.000 claims description 26
- 229910052760 oxygen Inorganic materials 0.000 claims description 26
- -1 p-dimethylaminophenyl group Chemical group 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 17
- 125000000816 ethylene group Chemical group [H]C([H])([*:1])C([H])([H])[*:2] 0.000 claims description 13
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 13
- 125000004432 carbon atom Chemical group C* 0.000 claims description 11
- 125000001424 substituent group Chemical group 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 125000004400 (C1-C12) alkyl group Chemical group 0.000 claims description 7
- 125000004642 (C1-C12) alkoxy group Chemical group 0.000 claims description 4
- 101150053185 P450 gene Proteins 0.000 claims description 4
- 125000005843 halogen group Chemical group 0.000 claims description 4
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 125000003545 alkoxy group Chemical group 0.000 abstract description 2
- 125000003282 alkyl amino group Chemical group 0.000 abstract 1
- 229910052736 halogen Inorganic materials 0.000 abstract 1
- 150000002367 halogens Chemical class 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- 239000000203 mixture Substances 0.000 description 15
- 150000003839 salts Chemical class 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 14
- 210000001853 liver microsome Anatomy 0.000 description 13
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 10
- 238000005259 measurement Methods 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 description 9
- 102100023897 NADPH-cytochrome P450 reductase Human genes 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000005160 1H NMR spectroscopy Methods 0.000 description 7
- 239000012300 argon atmosphere Substances 0.000 description 7
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 description 7
- 238000002189 fluorescence spectrum Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 6
- 239000006184 cosolvent Substances 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- 239000008057 potassium phosphate buffer Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical class CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 229940125904 compound 1 Drugs 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 230000035699 permeability Effects 0.000 description 4
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 4
- 238000011894 semi-preparative HPLC Methods 0.000 description 4
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 description 4
- VTVWTPGLLAELLI-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzenesulfonyl chloride Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(S(Cl)(=O)=O)C=C1 VTVWTPGLLAELLI-UHFFFAOYSA-N 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 125000003668 acetyloxy group Chemical group [H]C([H])([H])C(=O)O[*] 0.000 description 3
- 229940126214 compound 3 Drugs 0.000 description 3
- 238000002059 diagnostic imaging Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- NJYVEMPWNAYQQN-UHFFFAOYSA-N 5-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C21OC(=O)C1=CC(C(=O)O)=CC=C21 NJYVEMPWNAYQQN-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 108010010349 Cytochrome Reductases Proteins 0.000 description 2
- 102000015793 Cytochrome Reductases Human genes 0.000 description 2
- 102100025287 Cytochrome b Human genes 0.000 description 2
- 108010075028 Cytochromes b Proteins 0.000 description 2
- 108090000371 Esterases Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101001112118 Homo sapiens NADPH-cytochrome P450 reductase Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 102000004960 NAD(P)H dehydrogenase (quinone) Human genes 0.000 description 2
- 108020000284 NAD(P)H dehydrogenase (quinone) Proteins 0.000 description 2
- 102100022365 NAD(P)H dehydrogenase [quinone] 1 Human genes 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010066657 azoreductase Proteins 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 229940125782 compound 2 Drugs 0.000 description 2
- UKJLNMAFNRKWGR-UHFFFAOYSA-N cyclohexatrienamine Chemical group NC1=CC=C=C[CH]1 UKJLNMAFNRKWGR-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 2
- 102000047583 human POR Human genes 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- 210000001589 microsome Anatomy 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 125000004817 pentamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 description 2
- 125000000843 phenylene group Chemical group C1(=C(C=CC=C1)*)* 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000012453 solvate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 2
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
- 125000002030 1,2-phenylene group Chemical group [H]C1=C([H])C([*:1])=C([*:2])C([H])=C1[H] 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
- UBWNIXUTEFITDF-OCOZRVBESA-N CC(C)(C)OC(N(CC1)CCN1S(c(cc1)ccc1/N=N/c(cc1)ccc1N(C)C)(=O)=O)=O Chemical compound CC(C)(C)OC(N(CC1)CCN1S(c(cc1)ccc1/N=N/c(cc1)ccc1N(C)C)(=O)=O)=O UBWNIXUTEFITDF-OCOZRVBESA-N 0.000 description 1
- PQKZXFZOLZSBDM-JQOKOOLQSA-N CN(C)C(CC1)=CC=C1/N=N/c(cc1)ccc1S(NCCCCCCNC(c(cc1C(O)=O)ccc1C(c(c(O1)c2)ccc2O)=C(C=C2)C1=CC2=O)=O)(=O)=O Chemical compound CN(C)C(CC1)=CC=C1/N=N/c(cc1)ccc1S(NCCCCCCNC(c(cc1C(O)=O)ccc1C(c(c(O1)c2)ccc2O)=C(C=C2)C1=CC2=O)=O)(=O)=O PQKZXFZOLZSBDM-JQOKOOLQSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- LFZAGIJXANFPFN-UHFFFAOYSA-N N-[3-[4-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)piperidin-1-yl]-1-thiophen-2-ylpropyl]acetamide Chemical compound C(C)(C)C1=NN=C(N1C1CCN(CC1)CCC(C=1SC=CC=1)NC(C)=O)C LFZAGIJXANFPFN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229940123934 Reductase inhibitor Drugs 0.000 description 1
- 108091007187 Reductases Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 125000005227 alkyl sulfonate group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- RSJLWBUYLGJOBD-UHFFFAOYSA-M diphenyliodanium;chloride Chemical compound [Cl-].C=1C=CC=CC=1[I+]C1=CC=CC=C1 RSJLWBUYLGJOBD-UHFFFAOYSA-M 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000001839 endoscopy Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 125000001634 furandiyl group Chemical group O1C(=C(C=C1)*)* 0.000 description 1
- 150000002333 glycines Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000031852 maintenance of location in cell Effects 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- RRVXZLSGEQVYGY-UHFFFAOYSA-N tert-butyl n-(1-aminohexyl)carbamate Chemical compound CCCCCC(N)NC(=O)OC(C)(C)C RRVXZLSGEQVYGY-UHFFFAOYSA-N 0.000 description 1
- AOCSUUGBCMTKJH-UHFFFAOYSA-N tert-butyl n-(2-aminoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCN AOCSUUGBCMTKJH-UHFFFAOYSA-N 0.000 description 1
- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 description 1
- 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
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
- C07D311/82—Xanthenes
- C07D311/90—Xanthenes with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
Definitions
- the present invention relates to a compound useful as a low oxygen environment measurement reagent or a salt thereof.
- the present invention also relates to a low oxygen environment measuring reagent containing the above compound or a salt thereof.
- hypoxic microenvironment in vivo has been suggested to be associated with various diseases, so detecting tissues and cells in hypoxic conditions is useful for early diagnosis of diseases and determination of treatment strategies.
- detecting tissues and cells in hypoxic conditions is useful for early diagnosis of diseases and determination of treatment strategies.
- several proposals have been made as methods capable of measuring a hypoxic environment. For example, PET tracers and immunostaining have been proposed, but these methods have problems in terms of safety and simplicity.
- a method for measuring a trace component in a living body or a specific environment using a fluorescent probe is simple and high safety can be expected. Therefore, development of a method for measuring a hypoxic microenvironment using a fluorescent probe is required.
- hitherto few fluorescent probes capable of measuring a hypoxic microenvironment have been provided, and there have been few proposals for fluorescent probes using small molecules.
- Some of the reported fluorescent probes have many problems in terms of specificity and excitation wavelength (for example, JP 2007-77036 A, ChemBioChem., 9, pp.426-432, 2008). .
- An object of the present invention is to provide a compound useful for measurement of a hypoxic environment. More specifically, it is an object of the present invention to provide a compound capable of imaging a tissue in a living body or a hypoxic environment of a cell with fluorescence safely and easily.
- the present inventors have reduced the substantially non-fluorescent compound represented by the following general formula (I) in a low oxygen environment, and emits strong fluorescence. And by using these compounds, it has been found that a hypoxic microenvironment in cells and tissues in a living body can be efficiently imaged.
- the present invention has been completed based on these findings.
- Y 1 represents an alkylene group having 1 to 6 carbon atoms
- X 1 represents a single bond, —CO—, or —SO 2 —
- X 2 represents —OY 2 —N (R 14 ) —
- Y 2 represents an alkylene group having 1 to 6 carbon atoms
- R 14 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
- m represents 0 or 1
- pC 6 H 4 - represents a p- phenylene group
- Ar represents an aryldiyl group
- R 13 represents a group represented by showing a monoalkylamino group or a dialkylamino group
- R 2 is an optionally substituted group good C 1-12 alkyl group, which may have a substituent C 1-12 alkoxy group, a carboxy group, a sulfo group, or an optionally substituted C 1-12 alkoxycarbonyl group
- the above compound wherein R 2 is a C 1-12 alkyl group or a carboxy group; the above compound wherein R 2 is a methyl group or a carboxy group; R 3 , R 4 , R 5 ,
- R 6 , R 7 and R 8 are hydrogen atoms; the above compound wherein Ar—R 13 is a p-dimethylaminophenyl group; the above compound wherein m is 0; X 1 is —SO 2
- Y 1 is an ethylene group; the above compound wherein R 11 and R 12 are bonded to each other to form an ethylene group; and Y 1 is an ethylene group, and R 11 and There is provided the above compound wherein R 12 is bonded to each other to form an ethylene group.
- a reagent for measuring a low oxygen environment comprising the compound represented by the above general formula (I) is provided by the present invention, and preferably in the presence of an enzyme capable of reductively cleaving an azo bond.
- an enzyme capable of reductively cleaving an azo bond In the presence of NADPH-cytochrome) P450 reductase in the presence of NADPH-cytochrome P450 reductase. ) Is provided.
- an enzyme substrate having the ability to reductively cleave an azo bond comprising a compound represented by the general formula (I), particularly NADPH-cytochrome ⁇ ⁇ ⁇ P450 reductase substrate, is provided by the present invention.
- a method for measuring a low oxygen environment comprising the following steps: (A) introducing the compound represented by the general formula (I) into the low oxygen environment; and (B) the above steps ( fluorescent compound formed by a) or a group bound to X 1 and an azo group cleavable in (the general formula (I) - (X 2) m - group bonded is p- aminophenyl to X 1 through a
- a method comprising the step of measuring the fluorescence of the underlying compound).
- the step (A) is performed in the presence of an enzyme having the ability to reductively cleave the azo bond in a hypoxic environment, such as NADPH-cytochrome P450 reductase, azoreductase, DT diaphorase and the like.
- an enzyme having the ability to reductively cleave the azo bond in a hypoxic environment such as NADPH-cytochrome P450 reductase, azoreductase, DT diaphorase and the like.
- the compound of the present invention is useful as a reagent for the measurement of a hypoxic environment, particularly a hypoxic microenvironment.
- a hypoxic environment particularly a hypoxic microenvironment.
- tissues and cells in a hypoxic environment such as solid tumors on the surface of mucous membranes, skin or organs can be safely and simply Can be imaged.
- FIG. 3 shows the results of a reaction between DBCF-PIP (3 ⁇ M) and rat liver microsomes in the presence of NADPH or NADH under normal and hypoxic conditions.
- ⁇ indicates the results in a hypoxic environment
- ⁇ indicates the results in a normal oxygen environment
- (A) indicates the results of 50 ⁇ M NADPH
- (B) indicates the results of 250 ⁇ M NADH.
- FIG. 2 is a diagram showing an absorption spectrum (A) of DBCF-PIP and a fluorescence spectrum (B) when rat liver microsomes are reacted with DBCF-PIP under the respective conditions of DBCF-PIP, hypoxic environment and normal oxygen environment.
- DBCF-PIP is the fluorescence spectrum of DBCF-PIP
- MS-aerobic is the fluorescence spectrum of rat liver microsomes and DBCF-PIP in a normal oxygen environment
- MS-hypoxic is the rat in a hypoxic environment.
- the fluorescence spectrum at the time of making liver microsome and DBCF-PIP react is shown. It is the figure which showed the result of having added the NADPH-cytochrome P450 reductase inhibitor in the hypoxic environment and reacting DBCF-PIP with the rat liver microsome.
- FIG. 3 (A) shows the results of changes over time
- FIG. 3 (B) shows the measurement results after 1 hour of reaction.
- FIG. 4 is a graph showing the results of reaction with rat liver microsomes in a hypoxic environment for the four compounds obtained in Examples 1 to 4.
- an “alkyl group” or an alkyl part of a substituent containing an alkyl part means an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof.
- the “alkylene group” may be linear or branched, and may include a cyclic structure. For example, ethylene group, propylene group, cyclopentane-1,2-diyl group, cyclohexane-1,2-diyl group, cyclohexane-1,3-diyl group, cyclohexane-1,4-diyl group, etc. Also good.
- the “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
- C 1-12 alkyl group represented by R 2 C 1-12 alkoxy group, or the C 1-12 alkoxycarbonyl group has a substituent
- the kind of substituents, number, and substitution position is not particularly limited, for example, ,
- a halogen atom, a hydroxyl group, an amino group, a carboxy group, an alkoxycarbonyl group, a sulfo group, an alkyl sulfonate group, or the like may be used as a substituent.
- Examples of the aryldiyl group represented by Ar include aromatic hydrocarbon diyl groups such as a phenylene group, a naphthalenediyl group, an anthracenediyl group, and a phenanthrenediyl group, as well as a pyridinediyl group, a quinolinediyl group, a furandiyl group, and a thiophenediyl group. Or a heteroaromatic diyl group.
- R 11 and R 12 are preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom. It is also preferred that R 11 and R 12 are combined to form an ethylene group.
- Y 1 is preferably a methylene group, an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group.
- R 11 and R 12 are combined to form an ethylene group, Y 1 is preferably an ethylene group.
- X 1 is preferably —CO— or —SO 2 —, more preferably —SO 2 —.
- the X 2 Y 2 is a methylene group, an ethylene group, or propylene group, it is preferred that R 14 is a hydrogen atom or a methyl group.
- m represents 0 or 1, but is preferably 0. In this case, it means that X 2 does not exist and becomes a single bond.
- the aryldiyl group represented by Ar is preferably a phenylene group, more preferably an o-phenylene group or a p-phenylene group, and particularly preferably a p-phenylene group.
- R 13 is preferably a monomethylamino group or a dimethylamino group, and more preferably a dimethylamino group.
- R 2 is preferably a C 1-12 alkyl group or a carboxy group, more preferably a C 1-6 alkyl group or a carboxy group, and even more preferably a methyl group or a carboxy group.
- the C 1-12 alkyl group optionally having a substituent represented by R 2 in the above general formula (I), substituted The water-solubility of the compound of the present invention by appropriately selecting a C 1-12 alkoxy group which may have a group or a substituent of a C 1-12 alkoxycarbonyl group which may have a substituent, It can be used as a cell membrane permeation type and non-membrane permeation type probe.
- the compound of the present invention having one or two, preferably three or more, sulfo groups and carboxy groups as the substituents is highly water-soluble and exhibits non-membrane permeability and cannot be taken into cells. It can be suitably used for detection of extracellular hypoxic environment.
- the compound of the present invention in which R 2 is an acetoxymethoxycarbonyl group has high lipid solubility and exhibits cell membrane permeability and is taken into cells (the acetoxymethoxycarbonyl group is hydrolyzed by esterase present in the cells).
- R 2 in the above general formula (I) may be converted to a compound represented by a carboxy group, resulting in a decrease in fat solubility and intracellular retention). In some cases, it can be suitably used.
- the 6-position hydroxyl group of the xanthene ring moiety of the above general formula (I) can be acetylated and converted to an acetoxy group to enhance the lipid solubility of the compound of the present invention and impart cell membrane permeability (the acetoxy group).
- the group is hydrolyzed by the esterase present in the cell, it is converted into the compound represented by the above general formula (I)).
- the compound of the present invention is a fluorescein-like compound in which R 2 in the above general formula (I) is a carboxy group
- the fluorescein-like compound takes a lactone form and the carbonyl group at the 3-position of the xanthene ring moiety is a hydroxyl group
- one or both of the hydroxyl groups at the 3rd and 6th positions of the xanthene ring moiety are acetylated to convert them into acetoxy groups, thereby increasing the fat solubility of the compound of the present invention.
- Cell membrane permeability can be imparted.
- R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are preferably hydrogen atoms.
- Preferred compounds as the compound represented by the general formula (I) are: (a) Ar—R 13 is a p-dimethylaminophenyl group; (b) m is 0; (c) X 1 is —SO 2 —; (d) Y 1 is an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group; or (e) a case where R 11 and R 12 bonded to each other is an ethylene group, more preferably a case where two or more of the above (a) to (e) are included, and particularly preferably the above (a) to (e ).
- the compound represented by the general formula (I) may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.
- the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.
- the compound represented by the general formula (I) may exist in the form of a salt, and the scope of the present invention includes a salt form. Examples of the salt include acid addition salts and base addition salts.
- mineral salts such as hydrochloride and sulfate
- organic acid salts such as maleate and p-toluenesulfonate
- sodium salt and potassium in addition to metal salts such as salts, organic amine salts such as ammonium salts and triethylamine salts, salts of amino acids such as glycine salts may be used.
- the compound represented by the general formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.
- the compound of the present invention represented by the general formula (I) is generally an amine compound represented by the general formula (I) in which the terminal carbonyl group is replaced with a hydrogen atom in the group represented by the general formula (A). It can be easily produced by reacting a compound represented by R 1 with a carboxy group. Since the production method of a representative compound of the compound represented by the general formula (I) of the present invention is specifically shown in the examples of the present specification, those skilled in the art will be based on the specific description of the examples. The compound of the present invention can be easily produced by appropriately selecting starting materials and reaction reagents, and appropriately changing or modifying the reaction conditions and steps as necessary.
- the target product can be efficiently produced by carrying out the reaction while protecting specific functional groups as necessary in the reaction step.
- Organic synthesis Protective Groups, Organic Synthesis, TWGreene, John Wiley & Sons, Inc., 1981
- Organic synthesis and the like can be selected by those skilled in the art.
- isolation and purification of the product in the above production method can be performed by appropriately combining methods used in ordinary organic synthesis, for example, filtration, extraction, washing, drying, concentration, crystallization, various chromatography and the like.
- the production intermediate in the above step can be subjected to the next reaction without particular purification.
- the salt of the compound of the present invention when the salt of each compound is obtained in the above production method, it may be purified as it is.
- the free form compound is obtained, the free form compound is obtained. May be dissolved or suspended in a suitable solvent, acid or base may be added to form a salt, and purification may be performed as necessary.
- the compound of the present invention represented by the general formula (I) When the compound of the present invention represented by the general formula (I) is placed in a hypoxic environment under mild conditions, for example, physiological conditions, the azo group in the general formula (A) is cleaved, and the general formula (A group or bound to X 1 in I) - (X 2) m - is a group bound to X 1 via a has the property of giving the compound a p- aminophenyl group.
- the compound represented by the general formula (I) is substantially non-fluorescent, while the above compound produced by cleavage of the azo group has a property of emitting high intensity fluorescence.
- hypoxic environment examples include a hypoxic environment of about 0% to 5% (38 mmHg) in solid cancer. It is not limited to this state.
- examples of the hypoxic environment include cancer tissue, cancer cells, or ischemic tissue.
- cancer tissue for example, a solid cancer generated on the mucosal surface, skin, or organ surface is preferable. It is.
- a tissue in a hypoxic environment can be identified using a reagent of the present invention by means such as endoscopy, and the presence of cancer can be detected at an early stage.
- resistance to radiation increases when the intratumoral oxygen partial pressure pO 2 is less than 10 mmHg, and the reagent of the present invention is also useful for identifying tumors having such radiation resistance.
- the compound (reagent) of the present invention When the compound (reagent) of the present invention is introduced (applied) into a hypoxic environment in a biological sample (tissue or cell), it has the ability to reductively cleave the azo bond in the hypoxic environment present in the tissue or cell.
- a compound in which the azo group is cleaved is generated by the enzyme having it, and generates fluorescence.
- Enzymes that have the ability to reductively cleave the azo bond in such a hypoxic environment include NADPH-cytochrome P450 reductase (present in various tissue microsomes), cytochrome-b 5 reductase (present in various tissue microsomes).
- DT diaphorase present in the cytosol of various tissues
- azoreductase derived from microorganisms
- NADPH-cytochrome P450 reductase is preferable because it exists in a wide range of tissues and cells.
- the enzyme may be derived from a sample depending on the sample for measuring the hypoxic environment or the test conditions, or may be added from the outside. When added from the outside, it may be obtained by a genetic recombination technique.
- the method for measuring a hypoxic environment of the present invention generally comprises (A) a step of introducing the compound represented by the above general formula (I) into a low oxygen environment, and (B) the step (A).
- a step of measuring fluorescence A step of measuring fluorescence.
- Measurement of the fluorescence of a compound with an azo group cleaved can be performed by a normal method, such as a method of measuring a fluorescence spectrum in vitro or a method of measuring a fluorescence spectrum in vivo using a bioimaging method. Can do. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method.
- DBTG-PIP has the property of being taken up into cells, and other probes can be easily converted into a structure that can be taken up into cells by protecting the hydroxyl group of the xanthene skeleton with an acetyl group. Is possible. Therefore, a hypoxic environment localized in individual cells and tissues can be measured with high sensitivity by a bioimaging technique.
- the compound represented by the above general formula (I) may be used as it is, but if necessary, it is composed of additives usually used for the preparation of the reagent. You may use as a thing.
- additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible.
- These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.
- the compound of the present invention can be the main component of the diagnostic imaging composition by preparing it as a pharmacologically or pharmaceutically acceptable formulation (formulation) as necessary.
- a diagnostic imaging composition based on the compound of the present invention is provided.
- applications of the above reagents and compositions to diagnostic imaging are also included.
- Example 1 DBCF-PIP synthesis (1) Compound 1
- N-Boc-piperazine (366 mg, 2 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 334 ⁇ L of triethylamine and dabsyl chloride (332 mg, 1 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, the residue was purified on silica gel to obtain 327 mg of the target compound 1 (yield 69%, orange powder).
- N-Boc-ethylenediamine (345.8 mg, 2.16 mmol) was dissolved in 40 mL of anhydrous dichloromethane, 334 ⁇ L of triethylamine and dabsyl chloride (330.4 mg, 1.02 mmol) were added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by silica gel column chromatography to obtain 392.5 mg of the target compound 2 (yield 85.8%, dark red solid).
- N-Boc-hexanediamine (216 mg, 1 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 167 ⁇ L of triethylamine and dabsyl chloride (241 mg, 0.74 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere.
- Example 5 Reaction between DBCF-PIP and rat liver microsomes
- reaction between DBCF-PIP (3 ⁇ M) and rat liver microsomes was performed under normal or hypoxic conditions. It was. Measurement was performed at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent. A hypoxic environment was created by bubbling 100% argon for 30 minutes. The results are shown in Figure 1.
- DMSO dimethyl sulfoxide
- (1) shows the results in a hypoxic environment
- ⁇ shows the results in a normal oxygen environment
- (A) shows the results in the presence of 50 ⁇ M NADPH
- (B) shows the results in the presence of 250 ⁇ M NADH.
- Liver microsomes contain cytochrome reductases, and those using NADPH as an electron donor are NADPH-cytochrome P450 reductase, and those using NADH as an electron donor are cytochrome-b 5 reductases.
- the compounds of the present invention have been shown to have excellent properties as substrates for cytochrome reductases in liver microsomes, particularly NADPH-cytochrome P450 reductase, under anaerobic (hypoxic) conditions.
- Fig. 2 (A) shows the absorption spectrum (3 ⁇ M) of DBCF-PIP
- Fig. 2 (B) shows the fluorescence spectrum when reacted with rat liver microsomes under the conditions of DBCF-PIP and hypoxic / normal oxygen environment.
- the sample was prepared with potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and reacted with rat liver microsomes in the presence of 250 ⁇ M NADH for 3 hours, measured at an excitation wavelength of 490 nm. Went.
- DMSO dimethyl sulfoxide
- the absorption spectrum of DBCF-PIP showed a spectrum shape in which dabsyl and fluorescein were superimposed, and the fluorescence intensity was very small (FIG. 2B, fluorescence quantum yield 0.01). A slight increase in fluorescence was observed even in a normal oxygen environment, but a very large increase in fluorescence of 10 times or more that in a normal oxygen environment was observed in a low oxygen environment.
- Diphenyliodonium chloride an inhibitor of NADPH-cytochrome-P450-reductase
- a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm).
- FIG. 3 (A) shows the results of changes over time
- FIG. 3 (B) shows the measurement results after 1 hour of reaction. In a hypoxic environment, the increase in fluorescence was suppressed depending on the concentration of the inhibitor (FIG. 3 (A)).
- FIG. 4 shows the results of reaction with DBCF-PIP (3 ⁇ M) using purified human NADPH-cytochrome-P450-reductase (5 ⁇ g).
- a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm). While a large increase in fluorescence was observed in the hypoxic environment, no substantial increase in fluorescence was observed in the normal oxygen environment.
- DMSO dimethyl sulfoxide
- Example 6 The four compounds (3 ⁇ M) obtained in Examples 1 to 4 were reacted with rat liver microsomes (50 ⁇ L, 5-fold dilution) in a hypoxic environment in the same manner as in Example 5.
- a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and measurement was performed in the presence of 50 ⁇ M NADPH (excitation wavelength 490 nm). It was confirmed that all four compounds of the present invention obtained in Examples 1 to 4 fluoresce in a hypoxic environment.
- DMSO dimethyl sulfoxide
Landscapes
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pyrane Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Disclosed is a compound represented by formula (I), which can be used for fluorescently measuring a hypoxic environment conveniently and with high sensitivity (in formula (I), R1 represents -CO-N(R11)-Y1-N(R12)-X1-(X2)m-p-C6H4-N=N-Ar-R13 [wherein R11 and R12 independently represent H or an alkyl (including an alkylene formed by the binding between R11 and R12); Y1 represents an alkylene; X1 represents a single bond, -CO-, or -SO2-; X2 represents -O-Y2-N(R14)- (wherein Y2 represents an alkylene; and R14 represents H or an alkyl); m represents a number of 0 or 1; p-C6H4- represents a p-phenylene; Ar represents an aryldiyl; and R13 represents an alkylamino]; R2 represents an alkyl, an alkoxy, a carboxy, a sulfo or an alkoxycarbonyl; and R3 to R8 independently represent H or a halogen).
Description
本発明は、低酸素環境測定試薬として有用な化合物又はその塩に関するものである。また、本発明は上記化合物又はその塩を含む低酸素環境測定試薬に関する。
The present invention relates to a compound useful as a low oxygen environment measurement reagent or a salt thereof. The present invention also relates to a low oxygen environment measuring reagent containing the above compound or a salt thereof.
生体内における低酸素微小環境は様々な疾患との関連が示唆されていることから、低酸素状態にある組織や細胞などを検出することは疾患の早期診断や治療方針の決定などに有用である。低酸素環境を測定可能な方法として現在までにいくつかの提案がなされている。例えば、PETトレーサーや免疫染色などが提案されているが、これらの方法は安全性や簡便性から問題を有している。
The hypoxic microenvironment in vivo has been suggested to be associated with various diseases, so detecting tissues and cells in hypoxic conditions is useful for early diagnosis of diseases and determination of treatment strategies. . To date, several proposals have been made as methods capable of measuring a hypoxic environment. For example, PET tracers and immunostaining have been proposed, but these methods have problems in terms of safety and simplicity.
一般に生体内微量成分や特定の環境を蛍光プローブを用いて測定する方法は簡便で高い安全性が期待できることから、蛍光プローブを用いて低酸素微小環境を測定する方法の開発が求められている。しかしながら、従来、低酸素微小環境を測定可能な蛍光プローブはほとんど提供されておらず、特に小分子を用いた蛍光プローブの提案はほとんどなされていない。報告されているいくつかの蛍光プローブも特異性や励起波長などの点で多くの問題を有している（例えば特開2007-77036号公報、ChemBioChem., 9, pp.426-432, 2008）。
Generally, a method for measuring a trace component in a living body or a specific environment using a fluorescent probe is simple and high safety can be expected. Therefore, development of a method for measuring a hypoxic microenvironment using a fluorescent probe is required. However, hitherto, few fluorescent probes capable of measuring a hypoxic microenvironment have been provided, and there have been few proposals for fluorescent probes using small molecules. Some of the reported fluorescent probes have many problems in terms of specificity and excitation wavelength (for example, JP 2007-77036 A, ChemBioChem., 9, pp.426-432, 2008). .
本発明の課題は、低酸素環境の測定のために有用な化合物を提供することにある。より具体的には、安全かつ簡便に生体内組織や細胞の低酸素環境を蛍光によりイメージングできる化合物を提供することが本発明の課題である。
An object of the present invention is to provide a compound useful for measurement of a hypoxic environment. More specifically, it is an object of the present invention to provide a compound capable of imaging a tissue in a living body or a hypoxic environment of a cell with fluorescence safely and easily.
本発明者らは上記の課題を解決すべく鋭意努力した結果、下記の一般式(I)で表される実質的に非蛍光性の化合物が低酸素環境下において還元され、強い蛍光を発する化合物を与えること、及びこれらの化合物を用いることにより生体内の細胞や組織などにおける低酸素微小環境を効率的にイメージングできることを見出した。本発明はこれらの知見を基にして完成されたものである。
As a result of diligent efforts to solve the above problems, the present inventors have reduced the substantially non-fluorescent compound represented by the following general formula (I) in a low oxygen environment, and emits strong fluorescence. And by using these compounds, it has been found that a hypoxic microenvironment in cells and tissues in a living body can be efficiently imaged. The present invention has been completed based on these findings.
すなわち本発明は、下記の式(I)：
〔式中、R1は下記の一般式(A)：
-CO-N(R11)-Y1-N(R12)-X1-(X2)m-p-C6H4-N=N-Ar-R13
[式中、R11及びR12はそれぞれ独立に水素原子又は炭素数1ないし6個のアルキル基を示し、R11及びR12は互いに結合して炭素数2ないし6個のアルキレン基となってもよく、Y1は炭素数1ないし6個のアルキレン基を示し、X1は単結合、-CO-、又は-SO2-を示し、X2は-O-Y2-N(R14)-（式中、Y2は炭素数1ないし6個のアルキレン基を示し、R14は水素原子又は炭素数1ないし6個のアルキル基を示す）を示し、mは0又は1を示し、p-C6H4-はp-フェニレン基を示し、Arはアリールジイル基を示し、R13はモノアルキルアミノ基又はジアルキルアミノ基を示す]で表される基を示し、R2は置換基を有していてもよいC1-12アルキル基、置換基を有していてもよいC1-12アルコキシ基、カルボキシ基、スルホ基、又は置換基を有していてもよいC1-12アルコキシカルボニル基を示し；R3、R4、R5、R6、R7、及びR8はそれぞれ独立に水素原子又はハロゲン原子を示す〕で表される化合物が提供される。 That is, the present invention provides the following formula (I):
[Wherein R 1 represents the following general formula (A):
-CO-N (R 11 ) -Y 1 -N (R 12 ) -X 1- (X 2 ) m -pC 6 H 4 -N = N-Ar-R 13
[Wherein, R 11 and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R 11 and R 12 are bonded to each other to form an alkylene group having 2 to 6 carbon atoms. Y 1 represents an alkylene group having 1 to 6 carbon atoms, X 1 represents a single bond, —CO—, or —SO 2 —, and X 2 represents —OY 2 —N (R 14 ) — ( In the formula, Y 2 represents an alkylene group having 1 to 6 carbon atoms, R 14 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), m represents 0 or 1, and pC 6 H 4 - represents a p- phenylene group, Ar represents an aryldiyl group, R 13 represents a group represented by showing a monoalkylamino group or a dialkylamino group], R 2 is an optionally substituted group good C 1-12 alkyl group, which may have a substituent C 1-12 alkoxy group, a carboxy group, a sulfo group, or an optionally substituted C 1-12 alkoxycarbonyl group R 3, R 4, R 5 , R 6, R 7, and R 8 compounds represented by each independently represent a hydrogen atom or a halogen atom] is provided.
-CO-N(R11)-Y1-N(R12)-X1-(X2)m-p-C6H4-N=N-Ar-R13
[式中、R11及びR12はそれぞれ独立に水素原子又は炭素数1ないし6個のアルキル基を示し、R11及びR12は互いに結合して炭素数2ないし6個のアルキレン基となってもよく、Y1は炭素数1ないし6個のアルキレン基を示し、X1は単結合、-CO-、又は-SO2-を示し、X2は-O-Y2-N(R14)-（式中、Y2は炭素数1ないし6個のアルキレン基を示し、R14は水素原子又は炭素数1ないし6個のアルキル基を示す）を示し、mは0又は1を示し、p-C6H4-はp-フェニレン基を示し、Arはアリールジイル基を示し、R13はモノアルキルアミノ基又はジアルキルアミノ基を示す]で表される基を示し、R2は置換基を有していてもよいC1-12アルキル基、置換基を有していてもよいC1-12アルコキシ基、カルボキシ基、スルホ基、又は置換基を有していてもよいC1-12アルコキシカルボニル基を示し；R3、R4、R5、R6、R7、及びR8はそれぞれ独立に水素原子又はハロゲン原子を示す〕で表される化合物が提供される。 That is, the present invention provides the following formula (I):
-CO-N (R 11 ) -Y 1 -N (R 12 ) -X 1- (X 2 ) m -pC 6 H 4 -N = N-Ar-R 13
[Wherein, R 11 and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R 11 and R 12 are bonded to each other to form an alkylene group having 2 to 6 carbon atoms. Y 1 represents an alkylene group having 1 to 6 carbon atoms, X 1 represents a single bond, —CO—, or —SO 2 —, and X 2 represents —OY 2 —N (R 14 ) — ( In the formula, Y 2 represents an alkylene group having 1 to 6 carbon atoms, R 14 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), m represents 0 or 1, and pC 6 H 4 - represents a p- phenylene group, Ar represents an aryldiyl group, R 13 represents a group represented by showing a monoalkylamino group or a dialkylamino group], R 2 is an optionally substituted group good C 1-12 alkyl group, which may have a substituent C 1-12 alkoxy group, a carboxy group, a sulfo group, or an optionally substituted C 1-12 alkoxycarbonyl group R 3, R 4, R 5 , R 6, R 7, and R 8 compounds represented by each independently represent a hydrogen atom or a halogen atom] is provided.
上記発明の好ましい態様によれば、R2がC1-12アルキル基又はカルボキシ基である上記の化合物；R2がメチル基又はカルボキシ基である上記の化合物；R3、R4、R5、R6、R7、及びR8が水素原子である上記の化合物；Ar-R13がp-ジメチルアミノフェニル基である上記の化合物；mが0である上記の化合物；X1が-SO2-である上記の化合物；Y1がエチレン基である上記の化合物；R11及びR12が互いに結合してエチレン基を形成する上記の化合物；及びY1がエチレン基であり、かつR11及びR12が互いに結合してエチレン基を形成する上記の化合物が提供される。
According to a preferred embodiment of the above invention, the above compound wherein R 2 is a C 1-12 alkyl group or a carboxy group; the above compound wherein R 2 is a methyl group or a carboxy group; R 3 , R 4 , R 5 , The above compound wherein R 6 , R 7 and R 8 are hydrogen atoms; the above compound wherein Ar—R 13 is a p-dimethylaminophenyl group; the above compound wherein m is 0; X 1 is —SO 2 The above compound wherein Y 1 is an ethylene group; the above compound wherein R 11 and R 12 are bonded to each other to form an ethylene group; and Y 1 is an ethylene group, and R 11 and There is provided the above compound wherein R 12 is bonded to each other to form an ethylene group.
別の観点からは、上記の一般式(I)で表される化合物を含む低酸素環境測定用試薬が本発明により提供され、好ましくはアゾ結合を還元的に開裂する能力を有する酵素の存在下において低酸素環境を測定するために用いる上記の一般式(I)で表される化合物、特に好ましくはNADPH-チトクローム P450 レダクターゼの存在下において低酸素環境を測定するために用いる上記の一般式(I)で表される化合物が提供される。また、一般式(I)で表される化合物からなるアゾ結合を還元的に開裂する能力を有する酵素基質、特にNADPH-チトクローム P450 レダクターゼ基質が本発明により提供される。
From another viewpoint, a reagent for measuring a low oxygen environment comprising the compound represented by the above general formula (I) is provided by the present invention, and preferably in the presence of an enzyme capable of reductively cleaving an azo bond. In the presence of NADPH-cytochrome) P450 reductase in the presence of NADPH-cytochrome P450 reductase. ) Is provided. In addition, an enzyme substrate having the ability to reductively cleave an azo bond comprising a compound represented by the general formula (I), particularly NADPH-cytochrome ク ロ ー P450 reductase substrate, is provided by the present invention.
さらに、本発明により、低酸素環境の測定方法であって、下記の工程：(A)上記一般式(I)で表される化合物を低酸素環境に導入する工程、及び(B)上記工程(A)により生成した蛍光性化合物（上記一般式(I)においてアゾ基が開裂してX1に結合する基又は-(X2)m-を介してX1に結合する基がp-アミノフェニル基となった化合物）の蛍光を測定する工程を含む方法が提供される。好ましくは、上記工程(A)は低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素、例えばNADPH-チトクローム P450 レダクターゼ、アゾレダクターゼ、DTジアホラーゼ等の存在下に行われる。
Further, according to the present invention, there is provided a method for measuring a low oxygen environment, comprising the following steps: (A) introducing the compound represented by the general formula (I) into the low oxygen environment; and (B) the above steps ( fluorescent compound formed by a) or a group bound to X 1 and an azo group cleavable in (the general formula (I) - (X 2) m - group bonded is p- aminophenyl to X 1 through a There is provided a method comprising the step of measuring the fluorescence of the underlying compound). Preferably, the step (A) is performed in the presence of an enzyme having the ability to reductively cleave the azo bond in a hypoxic environment, such as NADPH-cytochrome P450 reductase, azoreductase, DT diaphorase and the like.
本発明の化合物は低酸素環境、特に低酸素微小環境の測定用試薬として有用であり、例えば、粘膜や皮膚あるいは臓器表面に生じた固形癌など低酸素環境にある組織や細胞を安全かつ簡便にイメージングすることができる。
The compound of the present invention is useful as a reagent for the measurement of a hypoxic environment, particularly a hypoxic microenvironment. For example, tissues and cells in a hypoxic environment such as solid tumors on the surface of mucous membranes, skin or organs can be safely and simply Can be imaged.
本明細書において、「アルキル基」又はアルキル部分を含む置換基（例えばアルコキシ基など）のアルキル部分は、直鎖、分枝鎖、環状、又はそれらの組み合わせからなるアルキル基を意味している。
本明細書において、「アルキレン基」は直鎖状又は分枝鎖状のいずれであってもよく、環状構造を含むものであってもよい。例えば、エチレン基、プロピレン基などや、シクロペンタン-1,2-ジイル基、シクロヘキサン-1,2-ジイル基、シクロヘキサン-1,3-ジイル基、シクロヘキサン-1,4-ジイル基などであってもよい。
本明細書において、「ハロゲン原子」はフッ素原子、塩素原子、臭素原子、ヨウ素原子のいずれであってもよい。 In the present specification, an “alkyl group” or an alkyl part of a substituent containing an alkyl part (such as an alkoxy group) means an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof.
In the present specification, the “alkylene group” may be linear or branched, and may include a cyclic structure. For example, ethylene group, propylene group, cyclopentane-1,2-diyl group, cyclohexane-1,2-diyl group, cyclohexane-1,3-diyl group, cyclohexane-1,4-diyl group, etc. Also good.
In the present specification, the “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
本明細書において、「アルキレン基」は直鎖状又は分枝鎖状のいずれであってもよく、環状構造を含むものであってもよい。例えば、エチレン基、プロピレン基などや、シクロペンタン-1,2-ジイル基、シクロヘキサン-1,2-ジイル基、シクロヘキサン-1,3-ジイル基、シクロヘキサン-1,4-ジイル基などであってもよい。
本明細書において、「ハロゲン原子」はフッ素原子、塩素原子、臭素原子、ヨウ素原子のいずれであってもよい。 In the present specification, an “alkyl group” or an alkyl part of a substituent containing an alkyl part (such as an alkoxy group) means an alkyl group composed of a straight chain, a branched chain, a ring, or a combination thereof.
In the present specification, the “alkylene group” may be linear or branched, and may include a cyclic structure. For example, ethylene group, propylene group, cyclopentane-1,2-diyl group, cyclohexane-1,2-diyl group, cyclohexane-1,3-diyl group, cyclohexane-1,4-diyl group, etc. Also good.
In the present specification, the “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
R2が示すC1-12アルキル基、C1-12アルコキシ基、又はC1-12アルコキシカルボニル基が置換基を有する場合、置換基の種類、個数、及び置換位置は特に限定されないが、例えば、ハロゲン原子、水酸基、アミノ基、カルボキシ基、アルコキシカルボニル基、スルホ基、又はアルキルスルホネート基などを置換基として有していてもよい。
Arが示すアリールジイル基としては、例えば、フェニレン基、ナフタレンジイル基、アントラセンジイル基、フェナントレンジイル基などの芳香族炭化水素ジイル基のほか、ピリジンジイル基、キノリンジイル基、フランジイル基、チオフェンジイル基などのヘテロ芳香族ジイル基であってもよい。 C 1-12 alkyl group represented by R 2, C 1-12 alkoxy group, or the C 1-12 alkoxycarbonyl group has a substituent, the kind of substituents, number, and substitution position is not particularly limited, for example, , A halogen atom, a hydroxyl group, an amino group, a carboxy group, an alkoxycarbonyl group, a sulfo group, an alkyl sulfonate group, or the like may be used as a substituent.
Examples of the aryldiyl group represented by Ar include aromatic hydrocarbon diyl groups such as a phenylene group, a naphthalenediyl group, an anthracenediyl group, and a phenanthrenediyl group, as well as a pyridinediyl group, a quinolinediyl group, a furandiyl group, and a thiophenediyl group. Or a heteroaromatic diyl group.
Arが示すアリールジイル基としては、例えば、フェニレン基、ナフタレンジイル基、アントラセンジイル基、フェナントレンジイル基などの芳香族炭化水素ジイル基のほか、ピリジンジイル基、キノリンジイル基、フランジイル基、チオフェンジイル基などのヘテロ芳香族ジイル基であってもよい。 C 1-12 alkyl group represented by R 2, C 1-12 alkoxy group, or the C 1-12 alkoxycarbonyl group has a substituent, the kind of substituents, number, and substitution position is not particularly limited, for example, , A halogen atom, a hydroxyl group, an amino group, a carboxy group, an alkoxycarbonyl group, a sulfo group, an alkyl sulfonate group, or the like may be used as a substituent.
Examples of the aryldiyl group represented by Ar include aromatic hydrocarbon diyl groups such as a phenylene group, a naphthalenediyl group, an anthracenediyl group, and a phenanthrenediyl group, as well as a pyridinediyl group, a quinolinediyl group, a furandiyl group, and a thiophenediyl group. Or a heteroaromatic diyl group.
R13がジアルキルアミノ基を示す場合、2個のアルキル基は同一でも異なっていてもよい。
R11及びR12としては水素原子、メチル基、又はエチル基が好ましく、共に水素原子であることがより好ましい。R11及びR12が結合してエチレン基となることも好ましい。
Y1としてはメチレン基、エチレン基、プロピレン基、ブチレン基、ペンタメチレン基、又はヘキサメチレン基が好ましい。R11及びR12が結合してエチレン基となる場合にはY1がエチレン基であることが好ましい。 When R 13 represents a dialkylamino group, the two alkyl groups may be the same or different.
R 11 and R 12 are preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom. It is also preferred that R 11 and R 12 are combined to form an ethylene group.
Y 1 is preferably a methylene group, an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group. When R 11 and R 12 are combined to form an ethylene group, Y 1 is preferably an ethylene group.
R11及びR12としては水素原子、メチル基、又はエチル基が好ましく、共に水素原子であることがより好ましい。R11及びR12が結合してエチレン基となることも好ましい。
Y1としてはメチレン基、エチレン基、プロピレン基、ブチレン基、ペンタメチレン基、又はヘキサメチレン基が好ましい。R11及びR12が結合してエチレン基となる場合にはY1がエチレン基であることが好ましい。 When R 13 represents a dialkylamino group, the two alkyl groups may be the same or different.
R 11 and R 12 are preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom. It is also preferred that R 11 and R 12 are combined to form an ethylene group.
Y 1 is preferably a methylene group, an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group. When R 11 and R 12 are combined to form an ethylene group, Y 1 is preferably an ethylene group.
X1としては-CO-又は-SO2-が好ましく、-SO2-がより好ましい。
X2においてY2がメチレン基、エチレン基、又はプロピレン基であることが好ましく、R14が水素原子又はメチル基であることが好ましい。mは0又は1を示すが0であることが好ましく、この場合はX2が存在せずに単結合となることを意味する。
Arが示すアリールジイル基としてはフェニレン基が好ましく、o-フェニレン基又はp-フェニレン基がより好ましく、p-フェニレン基が特に好ましい。 X 1 is preferably —CO— or —SO 2 —, more preferably —SO 2 —.
Preferably the X 2 Y 2 is a methylene group, an ethylene group, or propylene group, it is preferred that R 14 is a hydrogen atom or a methyl group. m represents 0 or 1, but is preferably 0. In this case, it means that X 2 does not exist and becomes a single bond.
The aryldiyl group represented by Ar is preferably a phenylene group, more preferably an o-phenylene group or a p-phenylene group, and particularly preferably a p-phenylene group.
X2においてY2がメチレン基、エチレン基、又はプロピレン基であることが好ましく、R14が水素原子又はメチル基であることが好ましい。mは0又は1を示すが0であることが好ましく、この場合はX2が存在せずに単結合となることを意味する。
Arが示すアリールジイル基としてはフェニレン基が好ましく、o-フェニレン基又はp-フェニレン基がより好ましく、p-フェニレン基が特に好ましい。 X 1 is preferably —CO— or —SO 2 —, more preferably —SO 2 —.
Preferably the X 2 Y 2 is a methylene group, an ethylene group, or propylene group, it is preferred that R 14 is a hydrogen atom or a methyl group. m represents 0 or 1, but is preferably 0. In this case, it means that X 2 does not exist and becomes a single bond.
The aryldiyl group represented by Ar is preferably a phenylene group, more preferably an o-phenylene group or a p-phenylene group, and particularly preferably a p-phenylene group.
R13としてはモノメチルアミノ基又はジメチルアミノ基が好ましく、ジメチルアミノ基がより好ましい。
R2としてはC1-12アルキル基又はカルボキシ基が好ましく、C1-6アルキル基又はカルボキシ基がより好ましく、メチル基又はカルボキシ基がさらに好ましい。また、本発明の化合物を細胞、生体組織又はイン・ビボで使用する場合には、上記一般式(I)のR2が示す置換基を有していてもよいC1-12アルキル基、置換基を有していてもよいC1-12アルコキシ基又は置換基を有していてもよいC1-12アルコキシカルボニル基の置換基を適宜選択して本発明の化合物の水溶性を調節し、細胞膜透過型及び非膜透過型のプローブとして使用することができる。 R 13 is preferably a monomethylamino group or a dimethylamino group, and more preferably a dimethylamino group.
R 2 is preferably a C 1-12 alkyl group or a carboxy group, more preferably a C 1-6 alkyl group or a carboxy group, and even more preferably a methyl group or a carboxy group. In addition, when the compound of the present invention is used in cells, living tissues, or in vivo, the C 1-12 alkyl group optionally having a substituent represented by R 2 in the above general formula (I), substituted The water-solubility of the compound of the present invention by appropriately selecting a C 1-12 alkoxy group which may have a group or a substituent of a C 1-12 alkoxycarbonyl group which may have a substituent, It can be used as a cell membrane permeation type and non-membrane permeation type probe.
R2としてはC1-12アルキル基又はカルボキシ基が好ましく、C1-6アルキル基又はカルボキシ基がより好ましく、メチル基又はカルボキシ基がさらに好ましい。また、本発明の化合物を細胞、生体組織又はイン・ビボで使用する場合には、上記一般式(I)のR2が示す置換基を有していてもよいC1-12アルキル基、置換基を有していてもよいC1-12アルコキシ基又は置換基を有していてもよいC1-12アルコキシカルボニル基の置換基を適宜選択して本発明の化合物の水溶性を調節し、細胞膜透過型及び非膜透過型のプローブとして使用することができる。 R 13 is preferably a monomethylamino group or a dimethylamino group, and more preferably a dimethylamino group.
R 2 is preferably a C 1-12 alkyl group or a carboxy group, more preferably a C 1-6 alkyl group or a carboxy group, and even more preferably a methyl group or a carboxy group. In addition, when the compound of the present invention is used in cells, living tissues, or in vivo, the C 1-12 alkyl group optionally having a substituent represented by R 2 in the above general formula (I), substituted The water-solubility of the compound of the present invention by appropriately selecting a C 1-12 alkoxy group which may have a group or a substituent of a C 1-12 alkoxycarbonyl group which may have a substituent, It can be used as a cell membrane permeation type and non-membrane permeation type probe.
例えば、前記置換基としてスルホ基及びカルボキシ基を1つ又は2つ、好ましくは3つ以上有する本発明の化合物は、水溶性が非常に高く非膜透過性を示して細胞内に取り込まれないため、細胞外の低酸素環境の検出に好適に用いることができる。また、例えば、R2がアセトキシメトキシカルボニル基である本発明の化合物は、脂溶性が高まり細胞膜透過性を示して細胞内に取り込まれ（該アセトキシメトキシカルボニル基は細胞内に存在するエステラーゼにより加水分解されると、上記一般式(I)のR2がカルボキシ基で示される化合物に変換されて脂溶性が低下し、細胞内滞留性を示す場合がある）、細胞内の低酸素環境の検出に好適に用いることができる場合がある。
For example, the compound of the present invention having one or two, preferably three or more, sulfo groups and carboxy groups as the substituents is highly water-soluble and exhibits non-membrane permeability and cannot be taken into cells. It can be suitably used for detection of extracellular hypoxic environment. In addition, for example, the compound of the present invention in which R 2 is an acetoxymethoxycarbonyl group has high lipid solubility and exhibits cell membrane permeability and is taken into cells (the acetoxymethoxycarbonyl group is hydrolyzed by esterase present in the cells). In this case, R 2 in the above general formula (I) may be converted to a compound represented by a carboxy group, resulting in a decrease in fat solubility and intracellular retention). In some cases, it can be suitably used.
なお、上記一般式(I)のキサンテン環部位の6位の水酸基をアセチル化してアセトキシ基に変換して、本発明の化合物の脂溶性を高めて細胞膜透過性を付与することもできる（該アセトキシ基は細胞内に存在するエステラーゼにより加水分解されると、上記一般式(I)で示される化合物に変換される）。また、本発明の化合物が上記一般式(I)の R2がカルボキシ基であるフルオレセイン様化合物の場合には、該フルオレセイン様化合物がラクトンフォームをとってキサンテン環部位の3位のカルボニル基が水酸基となる互変異性体を形成するが、この場合にはキサンテン環部位の3位及び6位の水酸基の一方又は両方をアセチル化してアセトキシ基に変換して、本発明の化合物の脂溶性を高めて細胞膜透過性を付与することができる。
R3、R4、R5、R6、R7、及びR8は水素原子であることが好ましい。 In addition, the 6-position hydroxyl group of the xanthene ring moiety of the above general formula (I) can be acetylated and converted to an acetoxy group to enhance the lipid solubility of the compound of the present invention and impart cell membrane permeability (the acetoxy group). When the group is hydrolyzed by the esterase present in the cell, it is converted into the compound represented by the above general formula (I)). Further, when the compound of the present invention is a fluorescein-like compound in which R 2 in the above general formula (I) is a carboxy group, the fluorescein-like compound takes a lactone form and the carbonyl group at the 3-position of the xanthene ring moiety is a hydroxyl group In this case, one or both of the hydroxyl groups at the 3rd and 6th positions of the xanthene ring moiety are acetylated to convert them into acetoxy groups, thereby increasing the fat solubility of the compound of the present invention. Cell membrane permeability can be imparted.
R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are preferably hydrogen atoms.
R3、R4、R5、R6、R7、及びR8は水素原子であることが好ましい。 In addition, the 6-position hydroxyl group of the xanthene ring moiety of the above general formula (I) can be acetylated and converted to an acetoxy group to enhance the lipid solubility of the compound of the present invention and impart cell membrane permeability (the acetoxy group). When the group is hydrolyzed by the esterase present in the cell, it is converted into the compound represented by the above general formula (I)). Further, when the compound of the present invention is a fluorescein-like compound in which R 2 in the above general formula (I) is a carboxy group, the fluorescein-like compound takes a lactone form and the carbonyl group at the 3-position of the xanthene ring moiety is a hydroxyl group In this case, one or both of the hydroxyl groups at the 3rd and 6th positions of the xanthene ring moiety are acetylated to convert them into acetoxy groups, thereby increasing the fat solubility of the compound of the present invention. Cell membrane permeability can be imparted.
R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are preferably hydrogen atoms.
一般式(I)で表される化合物として好ましい化合物は、
(a)Ar-R13がp-ジメチルアミノフェニル基であり；
(b)mが0であり；
(c)X1が-SO2-であり；
(d)Y1がエチレン基、プロピレン基、ブチレン基、ペンタメチレン基、若しくはヘキサメチレン基であり；又は
(e)互いに結合したR11及びR12がエチレン基である
場合であり、さらに好ましくは上記(a)ないし(e)の2以上を含む場合であり、特に好ましくは上記(a)ないし(e)の全てを含む場合である。 Preferred compounds as the compound represented by the general formula (I) are:
(a) Ar—R 13 is a p-dimethylaminophenyl group;
(b) m is 0;
(c) X 1 is —SO 2 —;
(d) Y 1 is an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group; or
(e) a case where R 11 and R 12 bonded to each other is an ethylene group, more preferably a case where two or more of the above (a) to (e) are included, and particularly preferably the above (a) to (e ).
(a)Ar-R13がp-ジメチルアミノフェニル基であり；
(b)mが0であり；
(c)X1が-SO2-であり；
(d)Y1がエチレン基、プロピレン基、ブチレン基、ペンタメチレン基、若しくはヘキサメチレン基であり；又は
(e)互いに結合したR11及びR12がエチレン基である
場合であり、さらに好ましくは上記(a)ないし(e)の2以上を含む場合であり、特に好ましくは上記(a)ないし(e)の全てを含む場合である。 Preferred compounds as the compound represented by the general formula (I) are:
(a) Ar—R 13 is a p-dimethylaminophenyl group;
(b) m is 0;
(c) X 1 is —SO 2 —;
(d) Y 1 is an ethylene group, a propylene group, a butylene group, a pentamethylene group, or a hexamethylene group; or
(e) a case where R 11 and R 12 bonded to each other is an ethylene group, more preferably a case where two or more of the above (a) to (e) are included, and particularly preferably the above (a) to (e ).
一般式(I)で表される化合物は水和物又は溶媒和物として存在する場合もあるが、これらの物質はいずれも本発明の範囲に包含される。溶媒和物を形成する溶媒の種類は特に限定されないが、例えば、エタノール、アセトン、イソプロパノールなどの溶媒を例示することができる。また、上記一般式(I)で表される化合物は塩の形態で存在する場合もあり、本発明の範囲には塩の形態も包含される。塩としては酸付加塩又は塩基付加塩などを挙げることができ、例えば、塩酸塩、硫酸塩などの鉱酸塩、マレイン酸塩、p-トルエンスルホン酸塩などの有機酸塩、ナトリウム塩、カリウム塩などの金属塩、アンモニウム塩、トリエチルアミン塩などの有機アミン塩のほか、グリシン塩などのアミノ酸の塩であってもよい。
The compound represented by the general formula (I) may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated. The compound represented by the general formula (I) may exist in the form of a salt, and the scope of the present invention includes a salt form. Examples of the salt include acid addition salts and base addition salts. For example, mineral salts such as hydrochloride and sulfate, organic acid salts such as maleate and p-toluenesulfonate, sodium salt and potassium In addition to metal salts such as salts, organic amine salts such as ammonium salts and triethylamine salts, salts of amino acids such as glycine salts may be used.
一般式(I)で表される化合物は、置換基の種類に応じて1個または2個以上の不斉炭素を有する場合があり、光学異性体又はジアステレオ異性体などの立体異性体が存在する場合がある。純粋な形態の立体異性体、立体異性体の任意の混合物、ラセミ体などはいずれも本発明の範囲に包含される。
The compound represented by the general formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.
一般式(I)で表される本発明の化合物は、一般的には、一般式(A)で表される基において末端のカルボニル基を水素原子で置き換えたアミン化合物を一般式(I)で表される化合物においてR1がカルボキシ基である化合物に対して反応させることにより容易に製造することができる。本発明の一般式(I)で表される化合物の代表的化合物の製造方法が本明細書の実施例に具体的に示されているので、当業者は実施例の具体的説明を基にして、出発原料及び反応試薬を適宜選択し、必要に応じて反応条件や工程を適宜変更ないし修飾することにより、本発明の化合物を容易に製造することが可能である。
The compound of the present invention represented by the general formula (I) is generally an amine compound represented by the general formula (I) in which the terminal carbonyl group is replaced with a hydrogen atom in the group represented by the general formula (A). It can be easily produced by reacting a compound represented by R 1 with a carboxy group. Since the production method of a representative compound of the compound represented by the general formula (I) of the present invention is specifically shown in the examples of the present specification, those skilled in the art will be based on the specific description of the examples. The compound of the present invention can be easily produced by appropriately selecting starting materials and reaction reagents, and appropriately changing or modifying the reaction conditions and steps as necessary.
なお、反応工程において特定の官能基を必要に応じて保護して反応を行うことにより、目的物を効率的に製造することができる場合があるが、保護基については、プロテクティブ・グループス・イン・オーガニック・シンセシス (Protective Groups in Organic Synthesis, T.W.Greene, John Wiley & Sons, Inc., 1981)などに詳しく説明されており、当業者は適宜の保護基を選択することが可能である。
In some cases, the target product can be efficiently produced by carrying out the reaction while protecting specific functional groups as necessary in the reaction step. Organic synthesis (Protective Groups, Organic Synthesis, TWGreene, John Wiley & Sons, Inc., 1981) and the like can be selected by those skilled in the art.
また、上記製造法における生成物の単離、精製は通常の有機合成で用いられる方法、例えば濾過、抽出、洗浄、乾燥、濃縮、結晶化、各種クロマトグラフィー等を適宜組み合わせ行うことができる。また、上記工程における製造中間体は、特に精製することなく次の反応に供することも可能である。本発明の化合物の塩を製造する場合には、上記製造法においてそれぞれの化合物の塩が得られる場合はそのまま精製すればよればよく、遊離形態の化合物が得られる場合には、遊離形態の化合物を適当な溶媒に溶解又は懸濁した後、酸あるいは塩基を加えて塩を形成させ、必要に応じて精製を行えばよい。
In addition, isolation and purification of the product in the above production method can be performed by appropriately combining methods used in ordinary organic synthesis, for example, filtration, extraction, washing, drying, concentration, crystallization, various chromatography and the like. In addition, the production intermediate in the above step can be subjected to the next reaction without particular purification. In the case of producing the salt of the compound of the present invention, when the salt of each compound is obtained in the above production method, it may be purified as it is. When the free form compound is obtained, the free form compound is obtained. May be dissolved or suspended in a suitable solvent, acid or base may be added to form a salt, and purification may be performed as necessary.
上記一般式(I)で表される本発明の化合物は、緩和な条件下、例えば生理的条件下において低酸素環境に置かれると一般式(A)におけるアゾ基が開裂し、上記一般式(I)においてX1に結合する基又は-(X2)m-を介してX1に結合する基がp-アミノフェニル基となった化合物を与える性質を有している。一般式(I)で表される化合物は実質的に非蛍光性であり、一方、アゾ基が開裂して生成する上記化合物は高強度の蛍光を発する性質を有している。従って、上記一般式(I)で表される化合物又はその塩を低酸素環境に導入し、アゾ基が開裂した化合物の蛍光を測定することによって、低酸素環境を高感度に測定することが可能である。
When the compound of the present invention represented by the general formula (I) is placed in a hypoxic environment under mild conditions, for example, physiological conditions, the azo group in the general formula (A) is cleaved, and the general formula (A group or bound to X 1 in I) - (X 2) m - is a group bound to X 1 via a has the property of giving the compound a p- aminophenyl group. The compound represented by the general formula (I) is substantially non-fluorescent, while the above compound produced by cleavage of the azo group has a property of emitting high intensity fluorescence. Therefore, it is possible to measure the hypoxic environment with high sensitivity by introducing the compound represented by the above general formula (I) or a salt thereof into the hypoxic environment and measuring the fluorescence of the compound in which the azo group is cleaved. It is.
本発明の試薬により測定可能な低酸素環境としては、例えば、固形癌における0%～5%（38mmHg）程度の低酸素環境などを挙げることができるが、本発明により測定可能な低酸素環境はこの状態に限定されることはない。低酸素環境としては、例えば、癌組織、癌細胞、又は虚血組織などを挙げることができ、癌組織としては、例えば、粘膜表面、皮膚、又は臓器表面などに生じた固形癌が好ましい測定対象である。例えば、内視鏡検査などの手段により本発明の試薬を用いて低酸素環境の組織を特定することができ、癌の存在を早期に発見することができる。また、腫瘍内酸素分圧pO2が10mmHgを下回ると放射線に対する抵抗が増大することが知られており、本発明の試薬は、このような放射線耐性を有する腫瘍の識別にも有用である。
Examples of the hypoxic environment that can be measured by the reagent of the present invention include a hypoxic environment of about 0% to 5% (38 mmHg) in solid cancer. It is not limited to this state. Examples of the hypoxic environment include cancer tissue, cancer cells, or ischemic tissue. As the cancer tissue, for example, a solid cancer generated on the mucosal surface, skin, or organ surface is preferable. It is. For example, a tissue in a hypoxic environment can be identified using a reagent of the present invention by means such as endoscopy, and the presence of cancer can be detected at an early stage. In addition, it is known that resistance to radiation increases when the intratumoral oxygen partial pressure pO 2 is less than 10 mmHg, and the reagent of the present invention is also useful for identifying tumors having such radiation resistance.
本発明の化合物（試薬）を生物試料（組織や細胞）における低酸素環境に導入（適用）した場合、該組織や細胞中に存在する低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素によりアゾ基が開裂した化合物が生成され蛍光を生じる。このような低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素としては、NADPH-チトクローム P450 レダクターゼ（各種組織のミクロゾームに存在）、チトクローム-b5 レダクターゼ（各種組織のミクロゾームに存在）、DTジアホラーゼ（各種組織のサイトゾルに存在）、アゾレダクターゼ（微生物由来）等をあげることができる。これらのうち、NADPH-チトクローム P450 レダクターゼが広範な組織や細胞に存在するので好ましい。前記酵素は、低酸素環境を測定する試料や試験条件により、試料由来のものが使用されてもよいし、外部から加えてもよい。外部から加える場合には、遺伝子組み換え技術により得られたものであってもよい。
When the compound (reagent) of the present invention is introduced (applied) into a hypoxic environment in a biological sample (tissue or cell), it has the ability to reductively cleave the azo bond in the hypoxic environment present in the tissue or cell. A compound in which the azo group is cleaved is generated by the enzyme having it, and generates fluorescence. Enzymes that have the ability to reductively cleave the azo bond in such a hypoxic environment include NADPH-cytochrome P450 reductase (present in various tissue microsomes), cytochrome-b 5 reductase (present in various tissue microsomes). DT diaphorase (present in the cytosol of various tissues), azoreductase (derived from microorganisms), and the like. Of these, NADPH-cytochrome P450 reductase is preferable because it exists in a wide range of tissues and cells. The enzyme may be derived from a sample depending on the sample for measuring the hypoxic environment or the test conditions, or may be added from the outside. When added from the outside, it may be obtained by a genetic recombination technique.
本明細書において用いられる「測定」という用語は、定量、定性、又は診断などの目的で行われる測定、検査、検出などを含めて、最も広義に解釈しなければならない。本発明の低酸素環境の測定方法は、一般的には、(A)上記一般式(I)で表される化合物を低酸素環境に導入する工程、及び(B) 上記工程(A)で生成したアゾ基が開裂した化合物（上記一般式(I)においてX1に結合する基又は-(X2)m-を介してX1に結合する基がp-アミノフェニル基となった化合物）の蛍光を測定する工程を含んでいる。
As used herein, the term “measurement” should be interpreted in the broadest sense, including measurements, tests, detections, etc. performed for purposes such as quantification, qualitative or diagnostic. The method for measuring a hypoxic environment of the present invention generally comprises (A) a step of introducing the compound represented by the above general formula (I) into a low oxygen environment, and (B) the step (A). Of the azo group cleaved (a compound in which the group bonded to X 1 in the above general formula (I) or the group bonded to X 1 through — (X 2 ) m — is a p-aminophenyl group) A step of measuring fluorescence.
アゾ基が開裂した化合物の蛍光の測定は通常の方法で行うことができ、インビトロで蛍光スペクトルを測定する方法や、バイオイメージングの手法を用いてインビボで蛍光スペクトルを測定する方法などを採用することができる。例えば、定量を行う場合には、常法に従って予め検量線を作成しておくことが望ましい。本発明の試薬の中でもDBTG-PIPは細胞内に取り込まれる性質を有しており、また他のプローブもキサンテン骨格の水酸基をアセチル基で保護する事により容易に細胞内に取り込まれる構造へと変換が可能である。そのため個々の細胞や組織内に局在する低酸素環境をバイオイメージング手法により高感度に測定できる。
Measurement of the fluorescence of a compound with an azo group cleaved can be performed by a normal method, such as a method of measuring a fluorescence spectrum in vitro or a method of measuring a fluorescence spectrum in vivo using a bioimaging method. Can do. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method. Among the reagents of the present invention, DBTG-PIP has the property of being taken up into cells, and other probes can be easily converted into a structure that can be taken up into cells by protecting the hydroxyl group of the xanthene skeleton with an acetyl group. Is possible. Therefore, a hypoxic environment localized in individual cells and tissues can be measured with high sensitivity by a bioimaging technique.
本発明の低酸素環境測定用試薬としては、上記一般式(I)で表される化合物をそのまま用いてもよいが、必要に応じて、試薬の調製に通常用いられる添加剤を配合して組成物として用いてもよい。例えば、生理的環境で試薬を用いるための添加剤として、溶解補助剤、pH調節剤、緩衝剤、等張化剤などの添加剤を用いることができ、これらの配合量は当業者に適宜選択可能である。これらの組成物は、粉末形態の混合物、凍結乾燥物、顆粒剤、錠剤、液剤など適宜の形態の組成物として提供される。
As the reagent for measuring a low oxygen environment of the present invention, the compound represented by the above general formula (I) may be used as it is, but if necessary, it is composed of additives usually used for the preparation of the reagent. You may use as a thing. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amount of these additives is appropriately selected by those skilled in the art. Is possible. These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.
上記の説明を基にすれば、必要に応じて薬物学的又は薬剤学的に許容される処方（製剤）形態として調製することにより、本発明の化合物が画像診断用組成物の主成分となりうること及び本発明の化合物を主成分とする画像診断用組成物が提供されることが当然に理解されよう。本発明の範囲には、上記した本発明の化合物のほか、上記試薬や上記組成物の画像診断への適用も包含される。
Based on the above explanation, the compound of the present invention can be the main component of the diagnostic imaging composition by preparing it as a pharmacologically or pharmaceutically acceptable formulation (formulation) as necessary. Of course, it will be understood that a diagnostic imaging composition based on the compound of the present invention is provided. Within the scope of the present invention, in addition to the compounds of the present invention described above, applications of the above reagents and compositions to diagnostic imaging are also included.
以下、実施例により本発明をさらに具体的に説明するが、本発明の範囲は下記の実施例に限定されることはない。
例１：DBCF-PIPの合成
(1)化合物1
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1: DBCF-PIP synthesis
(1)Compound 1
例１：DBCF-PIPの合成
(1)化合物1
Example 1: DBCF-PIP synthesis
(1)
N-Boc-ピペラジン(366 mg, 2 mmol)を20 mLの無水ジクロロメタンに溶解し、334μLのトリエチルアミン、塩化ダブシル（332 mg, 1 mmol）を加えアルゴン雰囲気下室温で5時間攪拌した。溶媒を減圧留去後、シリカゲルで精製を行い、目的の化合物1を327 mg得た（収率69%、オレンジ色粉末）。
1H-NMR (300 MHz, CDCl3) δ:1.40 (s, 9H), 3.01 (t, 4H, J = 5.13 Hz), 3.13 (s, 6H), 3.51 (t, 4H, J = 5.13 Hz), 6.76 (d, 2H, J = 8.99 Hz), 7.82-7.95 (m, 6H)
LRMS (ESI+) 474 (M+H)+ N-Boc-piperazine (366 mg, 2 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 334 μL of triethylamine and dabsyl chloride (332 mg, 1 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, the residue was purified on silica gel to obtain 327 mg of the target compound 1 (yield 69%, orange powder).
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.40 (s, 9H), 3.01 (t, 4H, J = 5.13 Hz), 3.13 (s, 6H), 3.51 (t, 4H, J = 5.13 Hz) , 6.76 (d, 2H, J = 8.99 Hz), 7.82-7.95 (m, 6H)
LRMS (ESI +) 474 (M + H) +
1H-NMR (300 MHz, CDCl3) δ:1.40 (s, 9H), 3.01 (t, 4H, J = 5.13 Hz), 3.13 (s, 6H), 3.51 (t, 4H, J = 5.13 Hz), 6.76 (d, 2H, J = 8.99 Hz), 7.82-7.95 (m, 6H)
LRMS (ESI+) 474 (M+H)+ N-Boc-piperazine (366 mg, 2 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 334 μL of triethylamine and dabsyl chloride (332 mg, 1 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, the residue was purified on silica gel to obtain 327 mg of the target compound 1 (yield 69%, orange powder).
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.40 (s, 9H), 3.01 (t, 4H, J = 5.13 Hz), 3.13 (s, 6H), 3.51 (t, 4H, J = 5.13 Hz) , 6.76 (d, 2H, J = 8.99 Hz), 7.82-7.95 (m, 6H)
LRMS (ESI +) 474 (M + H) +
(2)DBCF-PIP
(2) DBCF-PIP
化合物1（47.4 mg, 0.1 mmol）を10 mLのジクロロメタンに溶解し、この溶液を氷冷下トリフルオロ酢酸20 mL中に滴下した。1時間後、トルエンを加えてトリフルオロ酢酸を減圧留去した。残渣を5 mLの脱水N,N-ジメチルホルムアミドに溶解し、HOBT・H2O（28.5 mg, 0.16 mmol）、HBTU（60.6 mg, 0.16 mmol）、N,N-ジイソプロピルエチルアミン（132 mL, 0.8 mmol）、5-カルボキシフルオレセイン（30 mg, 0.08 mmol）を加え、アルゴン雰囲気下、室温で一晩攪拌する。溶媒を減圧留去後、セミ分取HPLCで精製を行い、目的物23 mgを得た（収率39%、暗赤色粉末）。
1H-NMR (300 MHz, CDCl3) δ: 2.39 (br, 8H), 3.16 (s, 6H), 6.37 (d, 2H, J = 2.20 Hz), 6.57 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 6.79 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
13C-NMR (100 MHz, CDCl3) δ:9.8, 40.3, 46.7, 103.4, 111.1, 112.4, 113.5, 123.3, 124.2, 125.3, 126.4, 128.2, 129.8, 130.2, 132.7, 134.8, 136.4, 138.6, 144.2, 153.4, 154.4, 154.5, 156.6, 160.8, 168.9, 169.0
HRMS (ESI+) Calcd for [M+H]+, 732.2128, Found, 732.2122 (-0.54 mmu) Compound 1 (47.4 mg, 0.1 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 20 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 5 mL of dehydrated N, N-dimethylformamide, and HOBT · H 2 O (28.5 mg, 0.16 mmol), HBTU (60.6 mg, 0.16 mmol), N, N-diisopropylethylamine (132 mL, 0.8 mmol) ), 5-carboxyfluorescein (30 mg, 0.08 mmol) is added and stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by semi-preparative HPLC to obtain 23 mg of the desired product (yield 39%, dark red powder).
1 H-NMR (300 MHz, CDCl 3 ) δ: 2.39 (br, 8H), 3.16 (s, 6H), 6.37 (d, 2H, J = 2.20 Hz), 6.57 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 6.79 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
13 C-NMR (100 MHz, CDCl 3 ) δ: 9.8, 40.3, 46.7, 103.4, 111.1, 112.4, 113.5, 123.3, 124.2, 125.3, 126.4, 128.2, 129.8, 130.2, 132.7, 134.8, 136.4, 138.6, 144.2 , 153.4, 154.4, 154.5, 156.6, 160.8, 168.9, 169.0
HRMS (ESI +) Calcd for [M + H] +, 732.2128, Found, 732.2122 (-0.54 mmu)
1H-NMR (300 MHz, CDCl3) δ: 2.39 (br, 8H), 3.16 (s, 6H), 6.37 (d, 2H, J = 2.20 Hz), 6.57 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 6.79 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
13C-NMR (100 MHz, CDCl3) δ:9.8, 40.3, 46.7, 103.4, 111.1, 112.4, 113.5, 123.3, 124.2, 125.3, 126.4, 128.2, 129.8, 130.2, 132.7, 134.8, 136.4, 138.6, 144.2, 153.4, 154.4, 154.5, 156.6, 160.8, 168.9, 169.0
HRMS (ESI+) Calcd for [M+H]+, 732.2128, Found, 732.2122 (-0.54 mmu) Compound 1 (47.4 mg, 0.1 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 20 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 5 mL of dehydrated N, N-dimethylformamide, and HOBT · H 2 O (28.5 mg, 0.16 mmol), HBTU (60.6 mg, 0.16 mmol), N, N-diisopropylethylamine (132 mL, 0.8 mmol) ), 5-carboxyfluorescein (30 mg, 0.08 mmol) is added and stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by semi-preparative HPLC to obtain 23 mg of the desired product (yield 39%, dark red powder).
1 H-NMR (300 MHz, CDCl 3 ) δ: 2.39 (br, 8H), 3.16 (s, 6H), 6.37 (d, 2H, J = 2.20 Hz), 6.57 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 6.79 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
13 C-NMR (100 MHz, CDCl 3 ) δ: 9.8, 40.3, 46.7, 103.4, 111.1, 112.4, 113.5, 123.3, 124.2, 125.3, 126.4, 128.2, 129.8, 130.2, 132.7, 134.8, 136.4, 138.6, 144.2 , 153.4, 154.4, 154.5, 156.6, 160.8, 168.9, 169.0
HRMS (ESI +) Calcd for [M + H] +, 732.2128, Found, 732.2122 (-0.54 mmu)
例２：DBCF-Etの合成
(1)化合物2の合成
Example 2: Synthesis of DBCF-Et
(1) Synthesis of compound 2
(1)化合物2の合成
(1) Synthesis of compound 2
N-Boc-エチレンジアミン(345.8 mg, 2.16 mmol)を40 mLの無水ジクロロメタンに溶解し、334μLのトリエチルアミン、塩化ダブシル（330.4 mg, 1.02 mmol）を加えてアルゴン雰囲気下室温で一晩攪拌した。溶媒を減圧留去後、シリカゲルカラムクロマトグラフィーで精製を行い、目的の化合物2を392.5 mg得た（収率85.8%、暗赤色固体）。
1H-NMR (300 MHz, CDCl3) δ:1.43 (s, 9H), 3.07-3.13 (m, 8H), 3.21-3.27 (m, 2H), 6.75 (d, 2H, J = 9.35 Hz), 7.89-7.93 (m, 6H)
LRMS (ESI+) 470 (M+Na+) N-Boc-ethylenediamine (345.8 mg, 2.16 mmol) was dissolved in 40 mL of anhydrous dichloromethane, 334 μL of triethylamine and dabsyl chloride (330.4 mg, 1.02 mmol) were added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by silica gel column chromatography to obtain 392.5 mg of the target compound 2 (yield 85.8%, dark red solid).
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.43 (s, 9H), 3.07-3.13 (m, 8H), 3.21-3.27 (m, 2H), 6.75 (d, 2H, J = 9.35 Hz), 7.89-7.93 (m, 6H)
LRMS (ESI +) 470 (M + Na + )
1H-NMR (300 MHz, CDCl3) δ:1.43 (s, 9H), 3.07-3.13 (m, 8H), 3.21-3.27 (m, 2H), 6.75 (d, 2H, J = 9.35 Hz), 7.89-7.93 (m, 6H)
LRMS (ESI+) 470 (M+Na+) N-Boc-ethylenediamine (345.8 mg, 2.16 mmol) was dissolved in 40 mL of anhydrous dichloromethane, 334 μL of triethylamine and dabsyl chloride (330.4 mg, 1.02 mmol) were added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After the solvent was distilled off under reduced pressure, purification was performed by silica gel column chromatography to obtain 392.5 mg of the target compound 2 (yield 85.8%, dark red solid).
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.43 (s, 9H), 3.07-3.13 (m, 8H), 3.21-3.27 (m, 2H), 6.75 (d, 2H, J = 9.35 Hz), 7.89-7.93 (m, 6H)
LRMS (ESI +) 470 (M + Na + )
(2)DBCF-Et
(2) DBCF-Et
化合物2（268.2 mg, 0.6 mmol）を20 mLのジクロロメタンに溶解し、この溶液を氷冷下トリフルオロ酢酸60 mL中に滴下した。1時間後、トルエンを加えトリフルオロ酢酸を減圧留去した。残渣を15 mLの脱水N,N-ジメチルホルムアミドに溶解させ、HOBT・H2O（149.8 mg, 0.84 mmol）、HBTU（320.3 mg, 0.84 mmol）、N,N-ジイソプロピルエチルアミン（612 μL, 4.0 mmol）、5-カルボキシフルオレセイン（153.8 mg, 0.41 mmol）を加え、アルゴン雰囲気下、室温で一晩攪拌した。溶媒を減圧留去後、シリカゲルカラムクロマトグラフィー、セミ分取HPLCで精製を行い、目的物20.5 mgを得た（収率7.0%、暗赤色粉末）
1H-NMR (400 MHz, CD3OD) δ: 3.01-3.40 (m, 8H), 3.41 (t, 2H, J = 5.6 Hz), 6.48-6.70 (m, 8H), 7.15 (d, J = 7.8 Hz), 7.65-7.99 (m, 7H), 8.31 (s, 1H)
13C-NMR (100 MHz, CD3OD) δ: 40.5, 41.2, 43.3, 103.5, 111.8, 113.0, 114.8, 122.9, 123.1, 125.8, 126.5, 127.2, 129.0, 129.1, 130.7, 130.9, 135.1, 137.6, 141.4, 144.4, 154.9, 155.1, 156.1, 163.0, 168.6, 170.0
HRMS (ESI+) Calcd for [M+H]+, 706.1971, Found, 706.1989 (+1.74 mmu ) Compound 2 (268.2 mg, 0.6 mmol) was dissolved in 20 mL of dichloromethane, and this solution was added dropwise to 60 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 15 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (149.8 mg, 0.84 mmol), HBTU (320.3 mg, 0.84 mmol), N, N-diisopropylethylamine (612 μL, 4.0 mmol) ), 5-carboxyfluorescein (153.8 mg, 0.41 mmol) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After evaporating the solvent under reduced pressure, purification was performed by silica gel column chromatography and semi-preparative HPLC to obtain 20.5 mg of the desired product (yield 7.0%, dark red powder)
1 H-NMR (400 MHz, CD 3 OD) δ: 3.01-3.40 (m, 8H), 3.41 (t, 2H, J = 5.6 Hz), 6.48-6.70 (m, 8H), 7.15 (d, J = 7.8 Hz), 7.65-7.99 (m, 7H), 8.31 (s, 1H)
13 C-NMR (100 MHz, CD 3 OD) δ: 40.5, 41.2, 43.3, 103.5, 111.8, 113.0, 114.8, 122.9, 123.1, 125.8, 126.5, 127.2, 129.0, 129.1, 130.7, 130.9, 135.1, 137.6, 141.4, 144.4, 154.9, 155.1, 156.1, 163.0, 168.6, 170.0
HRMS (ESI +) Calcd for [M + H] + , 706.1971, Found, 706.1989 (+1.74 mmu)
1H-NMR (400 MHz, CD3OD) δ: 3.01-3.40 (m, 8H), 3.41 (t, 2H, J = 5.6 Hz), 6.48-6.70 (m, 8H), 7.15 (d, J = 7.8 Hz), 7.65-7.99 (m, 7H), 8.31 (s, 1H)
13C-NMR (100 MHz, CD3OD) δ: 40.5, 41.2, 43.3, 103.5, 111.8, 113.0, 114.8, 122.9, 123.1, 125.8, 126.5, 127.2, 129.0, 129.1, 130.7, 130.9, 135.1, 137.6, 141.4, 144.4, 154.9, 155.1, 156.1, 163.0, 168.6, 170.0
HRMS (ESI+) Calcd for [M+H]+, 706.1971, Found, 706.1989 (+1.74 mmu ) Compound 2 (268.2 mg, 0.6 mmol) was dissolved in 20 mL of dichloromethane, and this solution was added dropwise to 60 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 15 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (149.8 mg, 0.84 mmol), HBTU (320.3 mg, 0.84 mmol), N, N-diisopropylethylamine (612 μL, 4.0 mmol) ), 5-carboxyfluorescein (153.8 mg, 0.41 mmol) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After evaporating the solvent under reduced pressure, purification was performed by silica gel column chromatography and semi-preparative HPLC to obtain 20.5 mg of the desired product (yield 7.0%, dark red powder)
1 H-NMR (400 MHz, CD 3 OD) δ: 3.01-3.40 (m, 8H), 3.41 (t, 2H, J = 5.6 Hz), 6.48-6.70 (m, 8H), 7.15 (d, J = 7.8 Hz), 7.65-7.99 (m, 7H), 8.31 (s, 1H)
13 C-NMR (100 MHz, CD 3 OD) δ: 40.5, 41.2, 43.3, 103.5, 111.8, 113.0, 114.8, 122.9, 123.1, 125.8, 126.5, 127.2, 129.0, 129.1, 130.7, 130.9, 135.1, 137.6, 141.4, 144.4, 154.9, 155.1, 156.1, 163.0, 168.6, 170.0
HRMS (ESI +) Calcd for [M + H] + , 706.1971, Found, 706.1989 (+1.74 mmu)
例３：DBCF-Hexの合成
(1)化合物3
Example 3: Synthesis of DBCF-Hex
(1) Compound 3
(1)化合物3
(1) Compound 3
N-Boc-ヘキサンジアミン(216 mg, 1 mmol)を20 mLの無水ジクロロメタンに溶解し、167μLのトリエチルアミン、塩化ダブシル（241 mg, 0.74 mmol）を加えアルゴン雰囲気下室温で5時間攪拌した。溶媒を減圧留去後、シリカゲルで精製を行い、目的の化合物3を297 mg得た（収率79%、オレンジ色粉末）
1H-NMR (300 MHz, CDCl3) δ:1.26-1.63 (m, 18H), 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
LRMS (ESI+) 504 (M+H)+ N-Boc-hexanediamine (216 mg, 1 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 167 μL of triethylamine and dabsyl chloride (241 mg, 0.74 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, purification with silica gel yielded 297 mg of the target compound 3 (yield 79%, orange powder)
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.26-1.63 (m, 18H), 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H )
LRMS (ESI +) 504 (M + H) +
1H-NMR (300 MHz, CDCl3) δ:1.26-1.63 (m, 18H), 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H)
LRMS (ESI+) 504 (M+H)+ N-Boc-hexanediamine (216 mg, 1 mmol) was dissolved in 20 mL of anhydrous dichloromethane, 167 μL of triethylamine and dabsyl chloride (241 mg, 0.74 mmol) were added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. After evaporating the solvent under reduced pressure, purification with silica gel yielded 297 mg of the target compound 3 (yield 79%, orange powder)
1 H-NMR (300 MHz, CDCl 3 ) δ: 1.26-1.63 (m, 18H), 2.91-3.10 (m, 10H), 6.74 (d, 2H, J = 9.17 Hz), 7.87-7.97 (m, 6H )
LRMS (ESI +) 504 (M + H) +
(2)DBCF-Hex
(2) DBCF-Hex
化合物3（85.6 mg, 0.17 mmol）を10 mLのジクロロメタンに溶解し、この溶液を氷冷下トリフルオロ酢酸20 mL中に滴下した。1時間後、トルエンを加えトリフルオロ酢酸を減圧留去した。残渣を5 mLの脱水N,N-ジメチルホルムアミドに溶解し、HOBT・H2O（72 mg, 0.40 mmol）、HBTU（152 mg, 0.40 mmol）、N,N-ジイソプロピルエチルアミ330μL、5-カルボキシフルオレセイン（75.2 mg, 0.20 mmol）を加え、アルゴン雰囲気下、室温で一晩攪拌した。溶媒を減圧留去後、セミ分取HPLCで精製を行い、目的物21.7 mgを得た（収率16.9%、暗赤色粉末）
1H-NMR (300 MHz, CD3OD) δ: 1.27-1.64 (m, 8H), 2.90 (t, 2H, J = 6.97 Hz), 3.16 (s, 6H), 3.38 (t, 2H, J = 9.97 Hz), 6.74 (m, 2H), 6.85-6.90 (m, 5H), 7.33 (d, J = 8.07 Hz), 7.79-7.90 (m, 6H), 8.17 (dd, 1H, J = 7.90, 1.56 Hz), 8.53 (s, 1H)
13C-NMR (100 MHz, CD3OD) δ:27.0, 27.1, 30.2, 30.4, 41.0, 41.0, 43.9, 103.5, 113.3, 113.9, 116.3, 122.6, 127.1, 127.7, 128.1, 129.2, 129.9, 130.0, 131.5, 134.4, 138.1, 141.4, 141.5, 143.7, 154.5, 155.7, 156.4, 165.4, 168.1, 169.3
HRMS (ESI-) Calcd for [M-H]-, 760.2441, Found, 760.2480 (+3.89 mmu ) Compound 3 (85.6 mg, 0.17 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 20 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 5 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (72 mg, 0.40 mmol), HBTU (152 mg, 0.40 mmol), N, N-diisopropylethylamino 330 μL, 5-carboxyl Fluorescein (75.2 mg, 0.20 mmol) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After distilling off the solvent under reduced pressure, purification was performed by semi-preparative HPLC to obtain 21.7 mg of the desired product (yield 16.9%, dark red powder)
1 H-NMR (300 MHz, CD 3 OD) δ: 1.27-1.64 (m, 8H), 2.90 (t, 2H, J = 6.97 Hz), 3.16 (s, 6H), 3.38 (t, 2H, J = 9.97 Hz), 6.74 (m, 2H), 6.85-6.90 (m, 5H), 7.33 (d, J = 8.07 Hz), 7.79-7.90 (m, 6H), 8.17 (dd, 1H, J = 7.90, 1.56 Hz), 8.53 (s, 1H)
13 C-NMR (100 MHz, CD 3 OD) δ: 27.0, 27.1, 30.2, 30.4, 41.0, 41.0, 43.9, 103.5, 113.3, 113.9, 116.3, 122.6, 127.1, 127.7, 128.1, 129.2, 129.9, 130.0, 131.5, 134.4, 138.1, 141.4, 141.5, 143.7, 154.5, 155.7, 156.4, 165.4, 168.1, 169.3
HRMS (ESI-) Calcd for [MH] - , 760.2441, Found, 760.2480 (+3.89 mmu)
1H-NMR (300 MHz, CD3OD) δ: 1.27-1.64 (m, 8H), 2.90 (t, 2H, J = 6.97 Hz), 3.16 (s, 6H), 3.38 (t, 2H, J = 9.97 Hz), 6.74 (m, 2H), 6.85-6.90 (m, 5H), 7.33 (d, J = 8.07 Hz), 7.79-7.90 (m, 6H), 8.17 (dd, 1H, J = 7.90, 1.56 Hz), 8.53 (s, 1H)
13C-NMR (100 MHz, CD3OD) δ:27.0, 27.1, 30.2, 30.4, 41.0, 41.0, 43.9, 103.5, 113.3, 113.9, 116.3, 122.6, 127.1, 127.7, 128.1, 129.2, 129.9, 130.0, 131.5, 134.4, 138.1, 141.4, 141.5, 143.7, 154.5, 155.7, 156.4, 165.4, 168.1, 169.3
HRMS (ESI-) Calcd for [M-H]-, 760.2441, Found, 760.2480 (+3.89 mmu ) Compound 3 (85.6 mg, 0.17 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 20 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 5 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (72 mg, 0.40 mmol), HBTU (152 mg, 0.40 mmol), N, N-diisopropylethylamino 330 μL, 5-carboxyl Fluorescein (75.2 mg, 0.20 mmol) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. After distilling off the solvent under reduced pressure, purification was performed by semi-preparative HPLC to obtain 21.7 mg of the desired product (yield 16.9%, dark red powder)
1 H-NMR (300 MHz, CD 3 OD) δ: 1.27-1.64 (m, 8H), 2.90 (t, 2H, J = 6.97 Hz), 3.16 (s, 6H), 3.38 (t, 2H, J = 9.97 Hz), 6.74 (m, 2H), 6.85-6.90 (m, 5H), 7.33 (d, J = 8.07 Hz), 7.79-7.90 (m, 6H), 8.17 (dd, 1H, J = 7.90, 1.56 Hz), 8.53 (s, 1H)
13 C-NMR (100 MHz, CD 3 OD) δ: 27.0, 27.1, 30.2, 30.4, 41.0, 41.0, 43.9, 103.5, 113.3, 113.9, 116.3, 122.6, 127.1, 127.7, 128.1, 129.2, 129.9, 130.0, 131.5, 134.4, 138.1, 141.4, 141.5, 143.7, 154.5, 155.7, 156.4, 165.4, 168.1, 169.3
HRMS (ESI-) Calcd for [MH] - , 760.2441, Found, 760.2480 (+3.89 mmu)
例４：DBTG-PIPの合成
Example 4: Synthesis of DBTG-PIP
化合物1（94.6 mg, 0.2 mmol）を10 mLのジクロロメタンに溶解し、この溶液を氷冷下トリフルオロ酢酸30 mL中に滴下した。1時間後、トルエンを加えトリフルオロ酢酸を減圧留去した。残渣を7 mLの脱水N,N-ジメチルホルムアミドに溶解し、HOBT・H2O（72 mg, 0.40 mmol）、HBTU（152 mg, 0.40 mmol）、N,N-ジイソプロピルエチルアミン330μL、2-Me-4-COOH TokyoGreen（9-[1-(4-カルボキシ-2-メチルフェニル)]-6-ヒドロキシ-3H-キサンテン-3-オン, 文献:Org. Lett., 8，pp.5963-5966, 2006; 62 mg, 0.20 mmol）（TokyoGreenは登録商標）を加え、アルゴン雰囲気下、室温で一晩攪拌した。反応溶液に水を加え、ジクロロメタンで抽出を行い、有機層を減圧留去した。残渣をシリカゲルカラムクロマトグラフィー、セミ分取HPLCにより精製し、目的物67.4 mgを得た。（収率48%、暗赤色粉末）
1H-NMR (300 MHz, DMSO-d6) δ: 1.97 (s, 3H), 3.07 (br, 10H), 3.66 (br, 4H), 6.82-6.95 (m, 6H), 7.14 (d, 2H, J = 9.17 Hz), 7.30-7.41 (m, 2H), 7.47 (s, 1H), 7.80-7.96 (m, 6H)
13C-NMR (100 MHz, CD3OD) δ:19.7, 40.5, 40.6, 42.8, 103.7, 113.0, 117.7, 121.9, 123.4, 125.9, 127.1, 127.3, 129.2, 129.9, 130.1, 130.5, 134.0, 136.4, 138.2, 138.7, 144.5, 155.2, 156.9, 161.0, 171.5, 173.7
HRMS (ESI-) Calcd for [M+Na]+, 724.2205, Found, 724.2202 (+0.29 mmu ) Compound 1 (94.6 mg, 0.2 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 30 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 7 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (72 mg, 0.40 mmol), HBTU (152 mg, 0.40 mmol), N, N-diisopropylethylamine 330 μL, 2-Me- 4-COOH TokyoGreen (9- [1- (4-Carboxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3-one, Literature: Org. Lett., 8, pp.5963-5966, 2006 62 mg, 0.20 mmol) (TokyoGreen is a registered trademark) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. Water was added to the reaction solution, extraction was performed with dichloromethane, and the organic layer was distilled off under reduced pressure. The residue was purified by silica gel column chromatography and semi-preparative HPLC to obtain 67.4 mg of the desired product. (Yield 48%, dark red powder)
1 H-NMR (300 MHz, DMSO-d 6 ) δ: 1.97 (s, 3H), 3.07 (br, 10H), 3.66 (br, 4H), 6.82-6.95 (m, 6H), 7.14 (d, 2H , J = 9.17 Hz), 7.30-7.41 (m, 2H), 7.47 (s, 1H), 7.80-7.96 (m, 6H)
13 C-NMR (100 MHz, CD 3 OD) δ: 19.7, 40.5, 40.6, 42.8, 103.7, 113.0, 117.7, 121.9, 123.4, 125.9, 127.1, 127.3, 129.2, 129.9, 130.1, 130.5, 134.0, 136.4, 138.2, 138.7, 144.5, 155.2, 156.9, 161.0, 171.5, 173.7
HRMS (ESI-) Calcd for [M + Na] + , 724.2205, Found, 724.2202 (+0.29 mmu)
1H-NMR (300 MHz, DMSO-d6) δ: 1.97 (s, 3H), 3.07 (br, 10H), 3.66 (br, 4H), 6.82-6.95 (m, 6H), 7.14 (d, 2H, J = 9.17 Hz), 7.30-7.41 (m, 2H), 7.47 (s, 1H), 7.80-7.96 (m, 6H)
13C-NMR (100 MHz, CD3OD) δ:19.7, 40.5, 40.6, 42.8, 103.7, 113.0, 117.7, 121.9, 123.4, 125.9, 127.1, 127.3, 129.2, 129.9, 130.1, 130.5, 134.0, 136.4, 138.2, 138.7, 144.5, 155.2, 156.9, 161.0, 171.5, 173.7
HRMS (ESI-) Calcd for [M+Na]+, 724.2205, Found, 724.2202 (+0.29 mmu ) Compound 1 (94.6 mg, 0.2 mmol) was dissolved in 10 mL of dichloromethane, and this solution was added dropwise to 30 mL of trifluoroacetic acid under ice cooling. After 1 hour, toluene was added and trifluoroacetic acid was distilled off under reduced pressure. The residue was dissolved in 7 mL of dehydrated N, N-dimethylformamide, HOBT · H 2 O (72 mg, 0.40 mmol), HBTU (152 mg, 0.40 mmol), N, N-diisopropylethylamine 330 μL, 2-Me- 4-COOH TokyoGreen (9- [1- (4-Carboxy-2-methylphenyl)]-6-hydroxy-3H-xanthen-3-one, Literature: Org. Lett., 8, pp.5963-5966, 2006 62 mg, 0.20 mmol) (TokyoGreen is a registered trademark) was added, and the mixture was stirred overnight at room temperature under an argon atmosphere. Water was added to the reaction solution, extraction was performed with dichloromethane, and the organic layer was distilled off under reduced pressure. The residue was purified by silica gel column chromatography and semi-preparative HPLC to obtain 67.4 mg of the desired product. (Yield 48%, dark red powder)
1 H-NMR (300 MHz, DMSO-d 6 ) δ: 1.97 (s, 3H), 3.07 (br, 10H), 3.66 (br, 4H), 6.82-6.95 (m, 6H), 7.14 (d, 2H , J = 9.17 Hz), 7.30-7.41 (m, 2H), 7.47 (s, 1H), 7.80-7.96 (m, 6H)
13 C-NMR (100 MHz, CD 3 OD) δ: 19.7, 40.5, 40.6, 42.8, 103.7, 113.0, 117.7, 121.9, 123.4, 125.9, 127.1, 127.3, 129.2, 129.9, 130.1, 130.5, 134.0, 136.4, 138.2, 138.7, 144.5, 155.2, 156.9, 161.0, 171.5, 173.7
HRMS (ESI-) Calcd for [M + Na] + , 724.2205, Found, 724.2202 (+0.29 mmu)
例５：DBCF-PIPとラット肝ミクロゾームとの反応
NADPHまたはNADHの存在下で、通常酸素環境下又は低酸素環境下それぞれの条件下においてDBCF-PIP(3μM)とラット肝ミクロゾームとの反応を行った。励起波長490 nm、蛍光波長510 nmで測定を行い、試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製した。低酸素環境は100%アルゴンを30分間バブリングして作成した。結果を図1に示す。■は低酸素環境下、●は通常酸素環境下の結果を示し、(A)は50μM NADPH、(B)は250μM NADH存在下の結果を示す。何れの場合においても通常酸素環境下では殆ど蛍光強度の上昇が認められないのに対し、低酸素環境下では蛍光強度の大きな上昇が認められた。肝ミクロゾームは、チトクロムレダクターゼ類を含んでおり、NADPHを電子供与体とするものはNADPH-チトクローム P450 レダクターゼ、NADHを電子供与体とするものはチトクローム-b5 レダクターゼである。本発明の化合物は嫌気的（低酸素）条件下で肝ミクロゾーム中のチトクロムレダクターゼ類、特にNADPH-チトクローム P450 レダクターゼの基質として優れた性質を有することが示された。 Example 5: Reaction between DBCF-PIP and rat liver microsomes In the presence of NADPH or NADH, reaction between DBCF-PIP (3μM) and rat liver microsomes was performed under normal or hypoxic conditions. It was. Measurement was performed at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent. A hypoxic environment was created by bubbling 100% argon for 30 minutes. The results are shown in Figure 1. (1) shows the results in a hypoxic environment, ● shows the results in a normal oxygen environment, (A) shows the results in the presence of 50 μM NADPH, and (B) shows the results in the presence of 250 μM NADH. In any case, almost no increase in fluorescence intensity was observed in a normal oxygen environment, whereas a large increase in fluorescence intensity was observed in a low oxygen environment. Liver microsomes contain cytochrome reductases, and those using NADPH as an electron donor are NADPH-cytochrome P450 reductase, and those using NADH as an electron donor are cytochrome-b 5 reductases. The compounds of the present invention have been shown to have excellent properties as substrates for cytochrome reductases in liver microsomes, particularly NADPH-cytochrome P450 reductase, under anaerobic (hypoxic) conditions.
NADPHまたはNADHの存在下で、通常酸素環境下又は低酸素環境下それぞれの条件下においてDBCF-PIP(3μM)とラット肝ミクロゾームとの反応を行った。励起波長490 nm、蛍光波長510 nmで測定を行い、試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製した。低酸素環境は100%アルゴンを30分間バブリングして作成した。結果を図1に示す。■は低酸素環境下、●は通常酸素環境下の結果を示し、(A)は50μM NADPH、(B)は250μM NADH存在下の結果を示す。何れの場合においても通常酸素環境下では殆ど蛍光強度の上昇が認められないのに対し、低酸素環境下では蛍光強度の大きな上昇が認められた。肝ミクロゾームは、チトクロムレダクターゼ類を含んでおり、NADPHを電子供与体とするものはNADPH-チトクローム P450 レダクターゼ、NADHを電子供与体とするものはチトクローム-b5 レダクターゼである。本発明の化合物は嫌気的（低酸素）条件下で肝ミクロゾーム中のチトクロムレダクターゼ類、特にNADPH-チトクローム P450 レダクターゼの基質として優れた性質を有することが示された。 Example 5: Reaction between DBCF-PIP and rat liver microsomes In the presence of NADPH or NADH, reaction between DBCF-PIP (3μM) and rat liver microsomes was performed under normal or hypoxic conditions. It was. Measurement was performed at an excitation wavelength of 490 nm and a fluorescence wavelength of 510 nm, and a sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent. A hypoxic environment was created by bubbling 100% argon for 30 minutes. The results are shown in Figure 1. (1) shows the results in a hypoxic environment, ● shows the results in a normal oxygen environment, (A) shows the results in the presence of 50 μM NADPH, and (B) shows the results in the presence of 250 μM NADH. In any case, almost no increase in fluorescence intensity was observed in a normal oxygen environment, whereas a large increase in fluorescence intensity was observed in a low oxygen environment. Liver microsomes contain cytochrome reductases, and those using NADPH as an electron donor are NADPH-cytochrome P450 reductase, and those using NADH as an electron donor are cytochrome-b 5 reductases. The compounds of the present invention have been shown to have excellent properties as substrates for cytochrome reductases in liver microsomes, particularly NADPH-cytochrome P450 reductase, under anaerobic (hypoxic) conditions.
図2(A)にDBCF-PIPの吸収スペクトル(3μM)を示し、図2(B)にDBCF-PIP及び低酸素環境・通常酸素環境それぞれの条件でラット肝ミクロゾームと反応させた際の蛍光スペクトルを示す。試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製し、250μM NADHの存在下でラット肝ミクロゾームとの反応を3時間行い、励起波長490 nmで測定を行った。DBCF-PIPの吸収スペクトルはダブシルとフルオレセインを重ね合わせたスペクトル形状を示し、蛍光強度は非常に小さく抑えられていた（図2B、蛍光量子収率0.01）。通常酸素環境でも若干の蛍光上昇が認められたが、低酸素環境では通常酸素環境の10倍以上の非常に大きな蛍光上昇が認められた。
Fig. 2 (A) shows the absorption spectrum (3μM) of DBCF-PIP, and Fig. 2 (B) shows the fluorescence spectrum when reacted with rat liver microsomes under the conditions of DBCF-PIP and hypoxic / normal oxygen environment. Indicates. The sample was prepared with potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and reacted with rat liver microsomes in the presence of 250 μM NADH for 3 hours, measured at an excitation wavelength of 490 nm. Went. The absorption spectrum of DBCF-PIP showed a spectrum shape in which dabsyl and fluorescein were superimposed, and the fluorescence intensity was very small (FIG. 2B, fluorescence quantum yield 0.01). A slight increase in fluorescence was observed even in a normal oxygen environment, but a very large increase in fluorescence of 10 times or more that in a normal oxygen environment was observed in a low oxygen environment.
低酸素環境下、NADPH-チトクローム P450 レダクターゼの阻害剤であるジフェニルヨードニウムクロリドを様々な濃度で加えた。試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製し、50μMのNADPHの存在下で測定を行った(励起波長490 nm)。図3(A)は経時変化の結果、図3(B)は1時間の反応後の測定結果を示す。低酸素環境において阻害剤の濃度依存的に蛍光の上昇が抑制された（図3(A)）。また、通常酸素環境での実質的な蛍光上昇はなかった（図3(B)）。以上の結果は、本発明の化合物DBCF-PIPは、通常酸素環境では分解されないが、低酸素環境においてはNADPH-チトクローム P450レダクターゼにより分解され、蛍光強度が上昇したことを示している。
Diphenyliodonium chloride, an inhibitor of NADPH-cytochrome-P450-reductase, was added at various concentrations in a hypoxic environment. A sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 μM NADPH (excitation wavelength 490 nm). FIG. 3 (A) shows the results of changes over time, and FIG. 3 (B) shows the measurement results after 1 hour of reaction. In a hypoxic environment, the increase in fluorescence was suppressed depending on the concentration of the inhibitor (FIG. 3 (A)). In addition, there was no substantial increase in fluorescence in a normal oxygen environment (FIG. 3 (B)). The above results indicate that the compound DBCF-PIP of the present invention is not decomposed in a normal oxygen environment, but was decomposed by NADPH-cytochrome P450 reductase in a low oxygen environment, and the fluorescence intensity increased.
精製したヒトNADPH-チトクローム P450 レダクターゼ(5μg)を用いてDBCF-PIP(3μM)と反応させた結果を図4に示す。試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製し、50μMのNADPHの存在下で測定を行った(励起波長490 nm)。低酸素環境において大きな蛍光上昇が観測される一方、通常酸素環境では実質的な蛍光上昇は観察されなかった。
FIG. 4 shows the results of reaction with DBCF-PIP (3 μM) using purified human NADPH-cytochrome-P450-reductase (5 μg). A sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent and measured in the presence of 50 μM NADPH (excitation wavelength 490 nm). While a large increase in fluorescence was observed in the hypoxic environment, no substantial increase in fluorescence was observed in the normal oxygen environment.
例６
例1～4で得た4種の化合物(3μM)について例５と同様に低酸素環境下においてラット肝ミクロゾーム(50μL、5倍希釈)との反応を行った。試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製し、50μMのNADPHの存在下で測定を行った(励起波長490 nm)。例1～4で得た4種の本発明の化合物はいずれも低酸素環境下において蛍光を発することが確認された。 Example 6
The four compounds (3 μM) obtained in Examples 1 to 4 were reacted with rat liver microsomes (50 μL, 5-fold dilution) in a hypoxic environment in the same manner as in Example 5. A sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and measurement was performed in the presence of 50 μM NADPH (excitation wavelength 490 nm). It was confirmed that all four compounds of the present invention obtained in Examples 1 to 4 fluoresce in a hypoxic environment.
例1～4で得た4種の化合物(3μM)について例５と同様に低酸素環境下においてラット肝ミクロゾーム(50μL、5倍希釈)との反応を行った。試料は0.1% ジメチルスルホキシド(DMSO)を共溶媒として含むリン酸カリウム緩衝液(pH 7.4)で調製し、50μMのNADPHの存在下で測定を行った(励起波長490 nm)。例1～4で得た4種の本発明の化合物はいずれも低酸素環境下において蛍光を発することが確認された。 Example 6
The four compounds (3 μM) obtained in Examples 1 to 4 were reacted with rat liver microsomes (50 μL, 5-fold dilution) in a hypoxic environment in the same manner as in Example 5. A sample was prepared with a potassium phosphate buffer (pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) as a co-solvent, and measurement was performed in the presence of 50 μM NADPH (excitation wavelength 490 nm). It was confirmed that all four compounds of the present invention obtained in Examples 1 to 4 fluoresce in a hypoxic environment.
Claims (13)
- 下記の式(I)：
-CO-N(R11)-Y1-N(R12)-X1-(X2)m-p-C6H4-N=N-Ar-R13
[式中、R11及びR12はそれぞれ独立に水素原子又は炭素数1ないし6個のアルキル基を示し、R11及びR12は互いに結合して炭素数2ないし6個のアルキレン基となってもよく、Y1は炭素数1ないし6個のアルキレン基を示し、X1は単結合、-CO-、又は-SO2-を示し、X2は-O-Y2-N(R14)-（式中、Y2は炭素数1ないし6個のアルキレン基を示し、R14は水素原子又は炭素数1ないし6個のアルキル基を示す）を示し、mは0又は1を示し、p-C6H4-はp-フェニレン基を示し、Arはアリールジイル基を示し、R13はモノアルキルアミノ基又はジアルキルアミノ基を示す]で表される基を示し、R2は置換基を有していてもよいC1-12アルキル基、置換基を有していてもよいC1-12アルコキシ基、カルボキシ基、スルホ基、又は置換基を有していてもよいC1-12アルコキシカルボニル基を示し；R3、R4、R5、R6、R7、及びR8はそれぞれ独立に水素原子又はハロゲン原子を示す〕で表される化合物。 Formula (I) below:
-CO-N (R 11 ) -Y 1 -N (R 12 ) -X 1- (X 2 ) m -pC 6 H 4 -N = N-Ar-R 13
[Wherein, R 11 and R 12 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and R 11 and R 12 are bonded to each other to form an alkylene group having 2 to 6 carbon atoms. Y 1 represents an alkylene group having 1 to 6 carbon atoms, X 1 represents a single bond, —CO—, or —SO 2 —, and X 2 represents —OY 2 —N (R 14 ) — ( In the formula, Y 2 represents an alkylene group having 1 to 6 carbon atoms, R 14 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms), m represents 0 or 1, and pC 6 H 4 - represents a p- phenylene group, Ar represents an aryldiyl group, R 13 represents a group represented by showing a monoalkylamino group or a dialkylamino group], R 2 is an optionally substituted group good C 1-12 alkyl group, which may have a substituent C 1-12 alkoxy group, a carboxy group, a sulfo group, or an optionally substituted C 1-12 alkoxycarbonyl group R 3, R 4, R 5 , R 6, R 7, and R 8 of the compound represented by each independently represent a hydrogen atom or a halogen atom]. - R2がC1-12アルキル基又はカルボキシ基である請求項１に記載の化合物。 The compound according to claim 1, wherein R 2 is a C 1-12 alkyl group or a carboxy group.
- R3、R4、R5、R6、R7、及びR8が水素原子である請求項１又は２に記載の化合物。 The compound according to claim 1 or 2, wherein R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are hydrogen atoms.
- Ar-R13がp-ジメチルアミノフェニル基である請求項１ないし３のいずれか１項に記載の化合物。 The compound according to any one of claims 1 to 3, wherein Ar-R 13 is a p-dimethylaminophenyl group.
- mが0である請求項４に記載の化合物。 The compound according to claim 4, wherein m is 0.
- X1が-SO2-である請求項４又は５に記載の化合物。 The compound according to claim 4 or 5, wherein X 1 is -SO 2- .
- Y1が炭素数1ないし6個の直鎖アルキレン基である請求項４ないし６のいずれか１項に記載の化合物。 The compound according to any one of claims 4 to 6, wherein Y 1 is a linear alkylene group having 1 to 6 carbon atoms.
- R11及びR12が互いに結合してエチレン基を形成する請求項４ないし７のいずれか１項に記載の化合物。 The compound according to any one of claims 4 to 7, wherein R 11 and R 12 are bonded to each other to form an ethylene group.
- 上記の一般式(I)で表される化合物を含む低酸素環境測定用試薬。 A reagent for measuring a low oxygen environment, comprising the compound represented by the general formula (I).
- 低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素の存在下において低酸素環境を測定するために用いる請求項１ないし８のいずれか１項に記載の化合物。 The compound according to any one of claims 1 to 8, which is used for measuring a hypoxic environment in the presence of an enzyme having an ability to reductively cleave an azo bond in a hypoxic environment.
- 低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素がNADPH-チトクローム P450 レダクターゼである請求項１０に記載の化合物。 The compound according to claim 10, wherein the enzyme capable of reductively cleaving an azo bond in a hypoxic environment is NADPH-cytochrome チ P450 reductase.
- 低酸素環境の測定方法であって、下記の工程：
(A)上記一般式(I)で表される化合物を低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素の存在下において低酸素環境に導入する工程、及び
(B)上記工程(A)により生成した蛍光性化合物の蛍光を測定する工程
を含む方法。 A method for measuring a hypoxic environment comprising the following steps:
(A) introducing the compound represented by the general formula (I) into a hypoxic environment in the presence of an enzyme having an ability to reductively cleave the azo bond in the hypoxic environment; and
(B) A method comprising a step of measuring the fluorescence of the fluorescent compound produced by the step (A). - 低酸素環境下でアゾ結合を還元的に開裂する能力を有する酵素がNADPH-チトクローム P450 レダクターゼである請求項１２に記載の方法。 The method according to claim 12, wherein the enzyme capable of reductively cleaving an azo bond in a hypoxic environment is NADPH-cytochrome ク ロ ー P450 reductase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010529181A JP5360609B2 (en) | 2008-09-03 | 2009-09-02 | Reagent for low oxygen environment measurement |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008225389 | 2008-09-03 | ||
| JP2008-225389 | 2008-09-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2010026743A1 true WO2010026743A1 (en) | 2010-03-11 |
Family
ID=41796927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2009/004313 WO2010026743A1 (en) | 2008-09-03 | 2009-09-02 | Reagent for measuring hypoxic environment |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP5360609B2 (en) |
| WO (1) | WO2010026743A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012111818A1 (en) * | 2011-02-18 | 2012-08-23 | 国立大学法人 東京大学 | Fluorescent probe |
| WO2012111817A1 (en) * | 2011-02-18 | 2012-08-23 | 国立大学法人 東京大学 | Fluorescent probe |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62120378A (en) * | 1985-11-19 | 1987-06-01 | ザ ジヨ−ンズ ホプキンズ ユニバ−シテイ− | Protain label and medicine release system |
| JP2007077036A (en) * | 2005-09-12 | 2007-03-29 | Kyoto Univ | New compound selectively increasing fluorescence intensity at target part and composition for image diagnosis |
| WO2007076379A2 (en) * | 2005-12-20 | 2007-07-05 | Gilead Colorado, Inc. | 4,7-dihydrothieno[2,3-b]pyridine compounds and pharmaceutical compositions |
| WO2007092963A1 (en) * | 2006-02-09 | 2007-08-16 | Spectrum Pharmaceuticals, Inc. | Bladder cancer treatment involving determination of tumor enzyme level |
-
2009
- 2009-09-02 WO PCT/JP2009/004313 patent/WO2010026743A1/en active Application Filing
- 2009-09-02 JP JP2010529181A patent/JP5360609B2/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS62120378A (en) * | 1985-11-19 | 1987-06-01 | ザ ジヨ−ンズ ホプキンズ ユニバ−シテイ− | Protain label and medicine release system |
| JP2007077036A (en) * | 2005-09-12 | 2007-03-29 | Kyoto Univ | New compound selectively increasing fluorescence intensity at target part and composition for image diagnosis |
| WO2007076379A2 (en) * | 2005-12-20 | 2007-07-05 | Gilead Colorado, Inc. | 4,7-dihydrothieno[2,3-b]pyridine compounds and pharmaceutical compositions |
| WO2007092963A1 (en) * | 2006-02-09 | 2007-08-16 | Spectrum Pharmaceuticals, Inc. | Bladder cancer treatment involving determination of tumor enzyme level |
Non-Patent Citations (1)
| Title |
|---|
| SHEN, H. ET AL.: "Poly(ethylene glycol) carbodiimide coupling reagents for the biological and chemical functionalization of water-soluble nanoparticles", ACSNANO, vol. 3, no. 4, 2009, pages 915 - 923 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012111818A1 (en) * | 2011-02-18 | 2012-08-23 | 国立大学法人 東京大学 | Fluorescent probe |
| WO2012111817A1 (en) * | 2011-02-18 | 2012-08-23 | 国立大学法人 東京大学 | Fluorescent probe |
| US9170266B2 (en) | 2011-02-18 | 2015-10-27 | The University Of Tokyo | Fluorescent probe |
| JP2016033134A (en) * | 2011-02-18 | 2016-03-10 | 国立大学法人 東京大学 | Fluorescent probe |
| US9329184B2 (en) | 2011-02-18 | 2016-05-03 | The University Of Tokyo | Fluorescent probe |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5360609B2 (en) | 2013-12-04 |
| JPWO2010026743A1 (en) | 2012-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9714260B2 (en) | Asymmetrical Si rhodamine and rhodol synthesis | |
| US9329184B2 (en) | Fluorescent probe | |
| JP5228190B2 (en) | Peroxynitrite fluorescent probe | |
| JP4713343B2 (en) | Fluorescent probe | |
| JP5588962B2 (en) | Fluorescent probe for protease measurement | |
| US20050123478A1 (en) | Agent for measurement of singlet oxygen | |
| US8394850B2 (en) | Fluorescent probe specific to hydrogen peroxide | |
| JP4373608B2 (en) | Reagent for singlet oxygen measurement | |
| US9170266B2 (en) | Fluorescent probe | |
| JP5360609B2 (en) | Reagent for low oxygen environment measurement | |
| EP2183230B1 (en) | Novel fluorescent derivatives of polyamine, method for preparing same and applications thereof as diagnosis tools in the treatment of cancerous tumours | |
| JP2018145126A (en) | Fluorescent probe for detection of carboxypeptidase activity | |
| WO2010070232A1 (en) | Novel rare earth element cryptates including a tetraazatriphenylene unit | |
| JP2010180191A (en) | Photogenesis substrate of luciferase | |
| WO2017090631A1 (en) | Fluorescent probe for detecting extracellular metabolite and screening method employing said fluorescent probe | |
| JP2005022985A (en) | Zinc fluorescent probe | |
| JPWO2007013201A1 (en) | Zinc fluorescent probe | |
| JP2004101389A (en) | Probe for measuring aluminum ion and/or ferric ion | |
| US20040044228A1 (en) | Fluorescent probe for magnesium ion determination | |
| Wang et al. | Structure–reactivity relationship of probes based on the H 2 S-mediated reductive cleavage of the C [double bond, length as m-dash] C bond | |
| WO2023219136A1 (en) | Fluorescent probe for detecting peptide-linked hydrolase | |
| US20150276751A1 (en) | Reagent for imaging intracellular acetylation | |
| JP2021127305A (en) | Compound having sulfonylaniline skeleton or salt thereof or organic fluorescence material having the same | |
| TWI454455B (en) | Salicylic acid derivates with fluorophores and method of making and using the same | |
| JP2009275006A (en) | Reagent for measuring hypoxic environment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09811273 Country of ref document: EP Kind code of ref document: A1 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2010529181 Country of ref document: JP |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 09811273 Country of ref document: EP Kind code of ref document: A1 |