WO2022196694A1 - 化合物、その製造方法、複合体および短波赤外蛍光剤 - Google Patents
化合物、その製造方法、複合体および短波赤外蛍光剤 Download PDFInfo
- Publication number
- WO2022196694A1 WO2022196694A1 PCT/JP2022/011674 JP2022011674W WO2022196694A1 WO 2022196694 A1 WO2022196694 A1 WO 2022196694A1 JP 2022011674 W JP2022011674 W JP 2022011674W WO 2022196694 A1 WO2022196694 A1 WO 2022196694A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- short
- wave infrared
- formula
- infrared fluorescent
- Prior art date
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 188
- 238000004519 manufacturing process Methods 0.000 title claims description 29
- 238000004132 cross linking Methods 0.000 claims abstract description 42
- 150000003839 salts Chemical group 0.000 claims abstract description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 164
- -1 phenylimino group Chemical group 0.000 claims description 22
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 14
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 9
- 125000000304 alkynyl group Chemical group 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- 125000000962 organic group Chemical group 0.000 claims description 6
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical group OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims description 6
- 239000000427 antigen Substances 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims description 5
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 claims description 3
- 238000003384 imaging method Methods 0.000 abstract description 31
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 70
- 238000005481 NMR spectroscopy Methods 0.000 description 67
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 51
- 206010006187 Breast cancer Diseases 0.000 description 43
- 208000026310 Breast neoplasm Diseases 0.000 description 43
- 239000000243 solution Substances 0.000 description 42
- 230000005284 excitation Effects 0.000 description 41
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 40
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 39
- 238000000799 fluorescence microscopy Methods 0.000 description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 34
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 description 31
- 238000002073 fluorescence micrograph Methods 0.000 description 30
- 239000007850 fluorescent dye Substances 0.000 description 30
- 229960004657 indocyanine green Drugs 0.000 description 29
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 27
- 239000007864 aqueous solution Substances 0.000 description 27
- 238000003786 synthesis reaction Methods 0.000 description 27
- 241000699670 Mus sp. Species 0.000 description 26
- 241000699666 Mus <mouse, genus> Species 0.000 description 24
- 210000004204 blood vessel Anatomy 0.000 description 24
- 239000007787 solid Substances 0.000 description 24
- 230000015572 biosynthetic process Effects 0.000 description 23
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 22
- 238000004458 analytical method Methods 0.000 description 22
- 238000004949 mass spectrometry Methods 0.000 description 22
- 239000012298 atmosphere Substances 0.000 description 21
- 238000004896 high resolution mass spectrometry Methods 0.000 description 21
- 238000004440 column chromatography Methods 0.000 description 20
- 239000000562 conjugate Substances 0.000 description 20
- 239000002872 contrast media Substances 0.000 description 20
- 230000003287 optical effect Effects 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 239000002904 solvent Substances 0.000 description 19
- 238000001917 fluorescence detection Methods 0.000 description 17
- 238000011580 nude mouse model Methods 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 17
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 16
- 229940098773 bovine serum albumin Drugs 0.000 description 16
- 239000011541 reaction mixture Substances 0.000 description 16
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 description 15
- 229940126657 Compound 17 Drugs 0.000 description 15
- 241000699660 Mus musculus Species 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 15
- 238000001727 in vivo Methods 0.000 description 15
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- 238000000034 method Methods 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 229960001612 trastuzumab emtansine Drugs 0.000 description 14
- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 13
- 201000011510 cancer Diseases 0.000 description 13
- 229940126543 compound 14 Drugs 0.000 description 13
- 229910052739 hydrogen Inorganic materials 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 210000001015 abdomen Anatomy 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 210000002216 heart Anatomy 0.000 description 11
- 210000003734 kidney Anatomy 0.000 description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 210000004185 liver Anatomy 0.000 description 10
- 210000000952 spleen Anatomy 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 9
- 229940125782 compound 2 Drugs 0.000 description 9
- 238000012632 fluorescent imaging Methods 0.000 description 9
- 239000001632 sodium acetate Substances 0.000 description 9
- 235000017281 sodium acetate Nutrition 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- 230000002490 cerebral effect Effects 0.000 description 8
- 229940125773 compound 10 Drugs 0.000 description 8
- 229940022353 herceptin Drugs 0.000 description 8
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- WJZSZXCWMATYFX-UHFFFAOYSA-N 1,1,2-trimethylbenzo[e]indole Chemical compound C1=CC=CC2=C(C(C(C)=N3)(C)C)C3=CC=C21 WJZSZXCWMATYFX-UHFFFAOYSA-N 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 7
- 239000000611 antibody drug conjugate Substances 0.000 description 7
- 229940049595 antibody-drug conjugate Drugs 0.000 description 7
- 229940126214 compound 3 Drugs 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000002523 gelfiltration Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 210000003462 vein Anatomy 0.000 description 7
- GHYOCDFICYLMRF-UTIIJYGPSA-N (2S,3R)-N-[(2S)-3-(cyclopenten-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]propanoyl]amino]propanamide Chemical compound C1(=CCCC1)C[C@@H](C(=O)[C@@]1(OC1)C)NC([C@H]([C@@H](C1=CC=C(C=C1)OC)O)NC([C@H](C)NC(CN1CCOCC1)=O)=O)=O GHYOCDFICYLMRF-UTIIJYGPSA-N 0.000 description 6
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- LNUFLCYMSVYYNW-ZPJMAFJPSA-N [(2r,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[(2r,3r,4s,5r,6r)-6-[[(3s,5s,8r,9s,10s,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-3-yl]oxy]-4,5-disulfo Chemical compound O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1[C@@H](COS(O)(=O)=O)O[C@H]([C@@H]([C@H]1OS(O)(=O)=O)OS(O)(=O)=O)O[C@@H]1C[C@@H]2CC[C@H]3[C@@H]4CC[C@@H]([C@]4(CC[C@@H]3[C@@]2(C)CC1)C)[C@H](C)CCCC(C)C)[C@H]1O[C@H](COS(O)(=O)=O)[C@@H](OS(O)(=O)=O)[C@H](OS(O)(=O)=O)[C@H]1OS(O)(=O)=O LNUFLCYMSVYYNW-ZPJMAFJPSA-N 0.000 description 6
- 125000003368 amide group Chemical group 0.000 description 6
- 239000006227 byproduct Substances 0.000 description 6
- 229940125797 compound 12 Drugs 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- GLGNXYJARSMNGJ-VKTIVEEGSA-N (1s,2s,3r,4r)-3-[[5-chloro-2-[(1-ethyl-6-methoxy-2-oxo-4,5-dihydro-3h-1-benzazepin-7-yl)amino]pyrimidin-4-yl]amino]bicyclo[2.2.1]hept-5-ene-2-carboxamide Chemical compound CCN1C(=O)CCCC2=C(OC)C(NC=3N=C(C(=CN=3)Cl)N[C@H]3[C@H]([C@@]4([H])C[C@@]3(C=C4)[H])C(N)=O)=CC=C21 GLGNXYJARSMNGJ-VKTIVEEGSA-N 0.000 description 5
- WWTBZEKOSBFBEM-SPWPXUSOSA-N (2s)-2-[[2-benzyl-3-[hydroxy-[(1r)-2-phenyl-1-(phenylmethoxycarbonylamino)ethyl]phosphoryl]propanoyl]amino]-3-(1h-indol-3-yl)propanoic acid Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)O)C(=O)C(CP(O)(=O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1C=CC=CC=1)CC1=CC=CC=C1 WWTBZEKOSBFBEM-SPWPXUSOSA-N 0.000 description 5
- IWZSHWBGHQBIML-ZGGLMWTQSA-N (3S,8S,10R,13S,14S,17S)-17-isoquinolin-7-yl-N,N,10,13-tetramethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-amine Chemical compound CN(C)[C@H]1CC[C@]2(C)C3CC[C@@]4(C)[C@@H](CC[C@@H]4c4ccc5ccncc5c4)[C@@H]3CC=C2C1 IWZSHWBGHQBIML-ZGGLMWTQSA-N 0.000 description 5
- UNILWMWFPHPYOR-KXEYIPSPSA-M 1-[6-[2-[3-[3-[3-[2-[2-[3-[[2-[2-[[(2r)-1-[[2-[[(2r)-1-[3-[2-[2-[3-[[2-(2-amino-2-oxoethoxy)acetyl]amino]propoxy]ethoxy]ethoxy]propylamino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-[(2r)-2,3-di(hexadecanoyloxy)propyl]sulfanyl-1-oxopropan-2-yl Chemical compound O=C1C(SCCC(=O)NCCCOCCOCCOCCCNC(=O)COCC(=O)N[C@@H](CSC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)C(=O)NCC(=O)N[C@H](CO)C(=O)NCCCOCCOCCOCCCNC(=O)COCC(N)=O)CC(=O)N1CCNC(=O)CCCCCN\1C2=CC=C(S([O-])(=O)=O)C=C2CC/1=C/C=C/C=C/C1=[N+](CC)C2=CC=C(S([O-])(=O)=O)C=C2C1 UNILWMWFPHPYOR-KXEYIPSPSA-M 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 5
- OPFJDXRVMFKJJO-ZHHKINOHSA-N N-{[3-(2-benzamido-4-methyl-1,3-thiazol-5-yl)-pyrazol-5-yl]carbonyl}-G-dR-G-dD-dD-dD-NH2 Chemical compound S1C(C=2NN=C(C=2)C(=O)NCC(=O)N[C@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(O)=O)C(N)=O)=C(C)N=C1NC(=O)C1=CC=CC=C1 OPFJDXRVMFKJJO-ZHHKINOHSA-N 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 239000002131 composite material Substances 0.000 description 5
- 229940125758 compound 15 Drugs 0.000 description 5
- 229940125810 compound 20 Drugs 0.000 description 5
- 229940126086 compound 21 Drugs 0.000 description 5
- 229940126208 compound 22 Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940082789 erbitux Drugs 0.000 description 5
- JAXFJECJQZDFJS-XHEPKHHKSA-N gtpl8555 Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)N[C@H](B1O[C@@]2(C)[C@H]3C[C@H](C3(C)C)C[C@H]2O1)CCC1=CC=C(F)C=C1 JAXFJECJQZDFJS-XHEPKHHKSA-N 0.000 description 5
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 4
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 description 4
- 239000007821 HATU Substances 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 229940126142 compound 16 Drugs 0.000 description 4
- 229940125898 compound 5 Drugs 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 4
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 4
- 150000002430 hydrocarbons Chemical group 0.000 description 4
- 210000003141 lower extremity Anatomy 0.000 description 4
- 210000001165 lymph node Anatomy 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 230000001936 parietal effect Effects 0.000 description 4
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 238000012984 biological imaging Methods 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 150000001993 dienes Chemical group 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 229960002725 isoflurane Drugs 0.000 description 3
- 238000004020 luminiscence type Methods 0.000 description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 150000004291 polyenes Chemical group 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 235000011152 sodium sulphate Nutrition 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- OIGKWPIMJCPGGD-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]propanoate Chemical compound [N-]=[N+]=NCCOCCOCCOCCOCCC(=O)ON1C(=O)CCC1=O OIGKWPIMJCPGGD-UHFFFAOYSA-N 0.000 description 2
- BATOPAZDIZEVQF-MQQKCMAXSA-N (E,E)-2,4-hexadienal Chemical compound C\C=C\C=C\C=O BATOPAZDIZEVQF-MQQKCMAXSA-N 0.000 description 2
- NJQOCRDPGFWEKA-UHFFFAOYSA-N 1-(2-aminoethyl)pyrrole-2,5-dione;hydrochloride Chemical compound Cl.NCCN1C(=O)C=CC1=O NJQOCRDPGFWEKA-UHFFFAOYSA-N 0.000 description 2
- PBLZLIFKVPJDCO-UHFFFAOYSA-N 12-aminododecanoic acid Chemical compound NCCCCCCCCCCCC(O)=O PBLZLIFKVPJDCO-UHFFFAOYSA-N 0.000 description 2
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 2
- KDJUNNVUQBKNAY-UHFFFAOYSA-N 4-[(2E)-2-[(2E,4E,6E)-7-[3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate Chemical compound CC1(C)\C(=C/C=C/C=C/C=C/C2=[N+](CCCCS([O-])(=O)=O)C3=C(C4=C(C=CC=C4)C=C3)C2(C)C)N(CCCCCC(=O)ON2C(=O)CCC2=O)C2=C1C1=C(C=CC=C1)C=C2 KDJUNNVUQBKNAY-UHFFFAOYSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- JJMDCOVWQOJGCB-UHFFFAOYSA-N 5-aminopentanoic acid Chemical compound [NH3+]CCCCC([O-])=O JJMDCOVWQOJGCB-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 2
- 229910000530 Gallium indium arsenide Inorganic materials 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 235000010724 Wisteria floribunda Nutrition 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- MHYFEEDKONKGEB-UHFFFAOYSA-N oxathiane 2,2-dioxide Chemical compound O=S1(=O)CCCCO1 MHYFEEDKONKGEB-UHFFFAOYSA-N 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 2
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 2
- BATOPAZDIZEVQF-UHFFFAOYSA-N sorbic aldehyde Natural products CC=CC=CC=O BATOPAZDIZEVQF-UHFFFAOYSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 150000003852 triazoles Chemical group 0.000 description 2
- ZDPHROOEEOARMN-UHFFFAOYSA-N undecanoic acid Chemical compound CCCCCCCCCCC(O)=O ZDPHROOEEOARMN-UHFFFAOYSA-N 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- VQTBINYMFPKLQD-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-(3-hydroxy-6-oxoxanthen-9-yl)benzoate Chemical compound C=12C=CC(=O)C=C2OC2=CC(O)=CC=C2C=1C1=CC=CC=C1C(=O)ON1C(=O)CCC1=O VQTBINYMFPKLQD-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- NBWRJAOOMGASJP-UHFFFAOYSA-N 2-(3,5-diphenyl-1h-tetrazol-1-ium-2-yl)-4,5-dimethyl-1,3-thiazole;bromide Chemical compound [Br-].S1C(C)=C(C)N=C1N1N(C=2C=CC=CC=2)N=C(C=2C=CC=CC=2)[NH2+]1 NBWRJAOOMGASJP-UHFFFAOYSA-N 0.000 description 1
- AJHPGXZOIAYYDW-UHFFFAOYSA-N 3-(2-cyanophenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)NC(C(O)=O)CC1=CC=CC=C1C#N AJHPGXZOIAYYDW-UHFFFAOYSA-N 0.000 description 1
- VAKXPQHQQNOUEZ-UHFFFAOYSA-N 3-[4-[[bis[[1-(3-hydroxypropyl)triazol-4-yl]methyl]amino]methyl]triazol-1-yl]propan-1-ol Chemical compound N1=NN(CCCO)C=C1CN(CC=1N=NN(CCCO)C=1)CC1=CN(CCCO)N=N1 VAKXPQHQQNOUEZ-UHFFFAOYSA-N 0.000 description 1
- XABCFXXGZPWJQP-UHFFFAOYSA-N 3-aminoadipic acid Chemical compound OC(=O)CC(N)CCC(O)=O XABCFXXGZPWJQP-UHFFFAOYSA-N 0.000 description 1
- IOBKAWMJICSVBL-UHFFFAOYSA-N 3-ethoxycarbonylpentan-3-ylphosphonic acid Chemical compound CCOC(=O)C(CC)(CC)P(O)(O)=O IOBKAWMJICSVBL-UHFFFAOYSA-N 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- NVRVNSHHLPQGCU-UHFFFAOYSA-N 6-bromohexanoic acid Chemical compound OC(=O)CCCCCBr NVRVNSHHLPQGCU-UHFFFAOYSA-N 0.000 description 1
- XDOLZJYETYVRKV-UHFFFAOYSA-N 7-Aminoheptanoic acid Chemical compound NCCCCCCC(O)=O XDOLZJYETYVRKV-UHFFFAOYSA-N 0.000 description 1
- UQXNEWQGGVUVQA-UHFFFAOYSA-N 8-aminooctanoic acid Chemical compound NCCCCCCCC(O)=O UQXNEWQGGVUVQA-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical class [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229940122530 Tubulin polymerization inhibitor Drugs 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 150000001299 aldehydes Chemical group 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000002583 angiography Methods 0.000 description 1
- SIPUZPBQZHNSDW-UHFFFAOYSA-N bis(2-methylpropyl)aluminum Chemical compound CC(C)C[Al]CC(C)C SIPUZPBQZHNSDW-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000001733 carboxylic acid esters Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 238000007449 liver function test Methods 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- PPNAOCWZXJOHFK-UHFFFAOYSA-N manganese(2+);oxygen(2-) Chemical class [O-2].[Mn+2] PPNAOCWZXJOHFK-UHFFFAOYSA-N 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- UIUXUFNYAYAMOE-UHFFFAOYSA-N methylsilane Chemical compound [SiH3]C UIUXUFNYAYAMOE-UHFFFAOYSA-N 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000001174 sulfone group Chemical group 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 235000019281 tert-butylhydroquinone Nutrition 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- 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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- 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
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/60—Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
Definitions
- the present invention relates to compounds, methods for producing the same, composites, and short-wave infrared fluorescent agents.
- ICG Indocyanine green
- An object of one aspect of the present invention is to provide a novel technique that enables bioimaging with short-wave infrared.
- n represents an integer of 3 to 5
- X represents a salt of a sulfonic acid group or a reactive cross-linking group for the molecular identifier.
- a complex according to one aspect of the present invention is a compound in which a molecular identifier and the compound are bound via a reactive cross-linking group residue in the compound of the present invention.
- a short-wave infrared fluorescent agent according to one aspect of the present invention contains one or both of the compound of the present invention and the complex of the present invention.
- a method for producing a compound represented by the following formula (1) comprises synthesizing a first compound represented by the following formula (1a) A first step, a second step of replacing the anilino group in formula (1a) with a structure represented by the following formula (1b), and a phenylimino group in the formula (1a) represented by the following formula (1c) and a third step of replacing the structure with
- n represents an integer of 3 to 5
- X represents a salt of a sulfonic acid group or a reactive cross-linking group for a molecular identifier.
- FIG. 4 is a graph showing the relationship between the concentration of Compound 4 and cell viability in Examples of the present invention.
- FIG. 4 is a diagram showing the relationship between the concentration of Compound 10 and cell viability in Examples of the present invention.
- FIG. 2 is a photograph showing a bright-field image and a short-wave infrared fluorescent image of the leg and abdomen of a hairless mouse using the short-wave infrared fluorescent agent 1 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of the leg and abdomen of a hairless mouse using the short-wave infrared fluorescent agent 2 in the example of the present invention as an optical contrast agent.
- FIG. 3 is a diagram showing a photograph representing an image
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescent image of the leg and abdomen of a hairless mouse using the short-wave infrared fluorescent agent 3 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescent image of the leg and abdomen of a hairless mouse using the short-wave infrared fluorescent agent 3 in the example of the present invention as an optical contrast agent.
- FIG. 10 is a photograph showing a visual field image and a short-wave infrared fluorescent image of the leg and abdomen of a hairless mouse using short-wave infrared fluorescent agent 4 as an optical contrast agent in an example of the present invention.
- FIG. 3 is a diagram showing a photograph representing an image
- FIG. 3 is a diagram showing fluorescence spectra of Complex 1 and Complex 2 in Examples of the present invention.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using short-wave infrared fluorescent agent 5 in an example of the present invention as an optical contrast agent. Photographs showing bright-field images and short-wave infrared fluorescence images of cancer tumors, heart, kidney, spleen, and liver of cancer-bearing mice using the short-wave infrared fluorescent agent 5 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a diagram showing; FIG.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using the short-wave infrared fluorescent agent 6 in the example of the present invention as an optical contrast agent. Photographs showing bright-field images and short-wave infrared fluorescence images of cancer tumors, heart, kidney, spleen, and liver of cancer-bearing mice using the short-wave infrared fluorescent agent 6 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a diagram showing; FIG.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using the short-wave infrared fluorescent agent 7 in the example of the present invention as an optical contrast agent. Photographs showing bright-field images and short-wave infrared fluorescence images of cancer tumor, heart, kidney, spleen, and liver of cancer-bearing mice using the short-wave infrared fluorescent agent 7 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using the short-wave infrared fluorescent agent 7 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a diagram showing; A photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using each of the short-wave infrared fluorescent agent 8 and the short-wave infrared fluorescent agent 9 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a diagram showing; A photograph showing a bright field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using each of the short-wave infrared fluorescent agent 10 and the short-wave infrared fluorescent agent 11 in the example of the present invention as an optical contrast agent.
- FIG. 4 is a diagram showing; A photograph showing a bright-field image and a short-wave infrared fluorescence image of a tumor site of a cancer-bearing mouse using each of the short-wave infrared fluorescent agent 10 and the short-wave infrared fluorescent agent 11 in the example of the present invention as an
- FIG. 4 is a diagram showing; Tumor areas of cancer-bearing mice injected with Kadcyla or not injected with Kadcyla, photographed using the short-wave infrared fluorescent agent 12 and the short-wave infrared fluorescent agent 13 in the example of the present invention as optical contrast agents.
- FIG. 2 is a photograph showing a bright-field image and a short-wave infrared fluorescence image of .
- the short-wave infrared fluorescent agent 13 in the example of the present invention as an optical contrast agent, a bright-field image and a short-wave infrared fluorescent image of the tumor site of a tumor-bearing mouse injected with Kadcyla were taken after a specific period of time.
- FIG. 3 is a diagram showing a representative photograph;
- n represents an integer of 3-5. n can be appropriately determined from the viewpoint that the compound of formula (1) emits short-wave infrared fluorescence and develops sufficient water solubility. In view of the above, n is preferably 3 or 4.
- short-wave infrared means electromagnetic waves having a wavelength of 900 nm or more and 1400 nm or less.
- the compound of formula (1) has a fluorescence intensity peak in the short-wave infrared wavelength region depending on its structure (for example, n). Therefore, the wavelength at which the fluorescence of the compound of formula (1) is detected may be appropriately determined within a range in which the compound of formula (1) emits sufficiently high intensity fluorescence in the short-wave infrared.
- the wavelength of the excitation light in the fluorescence of the compound of formula (1) may be the wavelength of the light that excites the compound of formula (1).
- the excitation wavelength can be appropriately determined in the range of 900 to 1100 nm, for example, according to the structure (eg, n) of the compound of formula (1) or the depth of the detection target site.
- X represents a salt of a sulfonic acid group or a reactive cross-linking group for the molecular identifier.
- the salt of the sulfonic acid group is not limited, and may be, for example, an alkali metal salt, more specifically a sodium salt.
- a reactive cross-linking group is a functional group used for bonding with a molecular identifier.
- the bond with the molecular identifier may be a bond suitable for the application of the compound of formula (1), and may be, for example, a covalent bond or a hydrogen bond.
- the bonding of the reactive cross-linking group to the molecular identifier may be in the form of bonding of the molecular identifier to the reactive cross-linking group, or the reactive cross-linking may be carried out with partial detachment of the reactive cross-linking group.
- a part of the group may be in a form that binds to the molecular identifier.
- the reactive cross-linking group is preferably a functional group that is subjected to cross-linking with the molecular identifier under mild conditions such as mixing at room temperature.
- the reactive cross-linking group is preferably one or more organic groups selected from the group consisting of N-hydroxysuccinimide ester groups, maleimide groups, alkynyl groups and azide groups.
- alkynyl group is not limited, it preferably has 5 or less carbon atoms, for example, from the viewpoint of the water solubility of the compound of formula (1).
- alkynyl groups include ethynyl and propynyl groups.
- the ester structure of the N-hydroxysuccinimide ester group may be any structure that can be bonded to a straight-chain hydrocarbon group or derived from an organic group containing a straight-chain hydrocarbon group. It's okay.
- the reactive cross-linking group may further have other molecular structures capable of constituting the compound of formula (1) within the range in which the effects of the present embodiment can be obtained.
- other reactive cross-linking groups can also be bonded to straight-chain hydrocarbon groups, or can be derived from organic groups containing straight-chain hydrocarbon groups (e.g., alkyl groups and amide groups). groups, etc.).
- the reactive cross-linking group described above can easily bond with a molecular identifier having an amino group, a sulfhydryl group, an azide group, or an alkynyl group. Therefore, it is preferable from the viewpoint of easily and accurately introducing a site that identifies a living tissue into the compound of formula (1).
- the reactive cross-linking group may be introduced into the compound of formula (1) by attachment of a linker substance used to attach the fluorescent substance to the amino acid.
- a reactive cross-linking group capable of binding to the amino group possessed by the molecular identifier can be introduced into the compound of formula (1).
- linker substances include 6-aminocaproic acid, 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 5-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 11-amino Included are undecanoic acid, 12-aminododecanoic acid, 2-aminobenzoic acid, 3-aminobenzoic acid and 4-aminobenzoic acid.
- a molecular identifier is a component that has a molecular structure that can identify a specific molecule.
- the molecular identifier may be a component containing a molecular structure capable of binding to a specific molecule, and may be of one type or more.
- Molecular identifiers also have sites available for bonding with reactive cross-linking groups. Examples of such moieties include amino groups, sulfhydryl groups, azide groups and alkynyl groups. Examples of molecular identifiers include peptides, proteins, nucleic acid derivatives, antibodies, fragments of such antibodies having antigen-binding ability, and cells.
- the compound of formula (1) is an organic dye that emits short-wave infrared fluorescence.
- organic dyes are expected to be applied to bioimaging as described above, and for this reason, compounds with various molecular skeletons have been investigated as short-wave infrared fluorescent agents (for example, "Jin T, ECS J. Solid State Sci. Technol, 8, R9-R13 (2019)”).
- short-wave infrared fluorescent agents for example, "Jin T, ECS J. Solid State Sci. Technol, 8, R9-R13 (2019)”.
- studies have been made on compounds having short-wave infrared fluorescence as a fluorescence characteristic peculiar to the molecular skeleton.
- the compound of formula (1) has a structure similar to that of ICG and a longer chain structure than ICG, its short-wave infrared fluorescence is similar to that of ICG in the near-infrared. It has an equivalent or higher luminous efficiency. Therefore, the compound of formula (1) is a unique compound that does not fit the above general trends, even though it has a larger molecular structure than ICG.
- the compound of formula (1) can be produced by a production method including the following three steps of the first to third steps.
- the meanings of the letters in the general formulas are the same as those of the compounds described above.
- the first step is to synthesize the first compound represented by the following formula (1a).
- n represents an integer of 3-5.
- the compound of formula (1a) can be synthesized by reacting a linear diene aldehyde in the presence of a phosphorus compound to appropriately extend the linear diene structure, and then reacting with aniline.
- the synthesis can proceed efficiently by appropriately adjusting the temperature.
- the terminal aldehyde of the linear diene structure in the intermediate product is replaced with another structure such as a carboxylic acid ester or an alcohol. can be temporarily replaced by
- the second step is to replace the anilino group in formula (1a) with a structure represented by formula (1b) below.
- the anilino group in the formula (1a) is converted to the compound represented by the following formula (1b1) in the presence of acetic anhydride and sodium acetate to form the compound of the formula (1a) or the phenylimino group in the formula (1a).
- Compounds of formula (1b1) are obtained by reacting 1,1,2-trimethyl-1H-benzo[e]indole with 1,4-butanesultone.
- the third step is to replace the phenylimino group in formula (1a) with a structure represented by formula (1c) below.
- the phenylimino group in formula (1a) can be replaced by the compound of formula (1a) or the formula A compound in which the phenylimino group in (1a) is replaced with the structure represented by formula (1b) can be used as a starting material to replace the structure represented by formula (1c).
- the phenylimino group in formula (1a) is first replaced with a structure represented by formula (1c1) below, and then reacted with a terminal carboxyl group.
- the structure of formula (1c) can be substituted by attaching a reactive bridging group or replacing the carboxyl group with a reactive bridging group.
- the replacement of the phenylimino group in formula (1a) with the structure represented by formula (1c1) can be performed, for example, by using the bromine salt of the compound of formula (1b1) in which the sulfonic acid group is replaced with a carboxyl group, as described above. It is possible to carry out in the same manner as the replacement of the anilino group in formula (1a) described above.
- the reaction of introducing a reactive cross-linking group to the carboxyl group after replacement with the structure represented by formula (1c1) may be performed by applying a known method according to the target reactive cross-linking group. .
- the order of the second step and the third step is determined from the viewpoint of the solubility of the raw material or the product or the reactivity of the raw material within the range where the desired compound of formula (1) can be produced. can be determined as appropriate.
- the second step and the third step are You can go all at once.
- the above-mentioned second step and third step can be carried out at once, for example, by reacting the compound of formula (1a) with two or more equivalents of the compound of formula (1b1) under the conditions of the second step. is possible.
- By performing the second step and the third step at once it is possible to more easily produce the compound of formula (1), and from the viewpoint of suppressing a decrease in yield due to an increase in the number of steps.
- the third step is carried out after the second step. is preferred.
- the target compound is the compound of formula (4), that is, when n in the above formula is 3 and X is a reactive cross-linking group
- the third step is performed after the second step. is preferred. In these cases, the third step may be carried out using the product of the second step as a raw material. Carrying out the third step after the second step in the preparation of the compound of formula (3) or the compound of formula (4) increases the yield of replacement of the phenylimino group in formula (1a) in the third step is preferable from the viewpoint of increasing the
- the third step is performed before the second step.
- the second step may be carried out using the product of the third step as a raw material. Carrying out the third step before the second step in the production of the compound of formula (5) is preferable from the viewpoint of increasing the yield in each replacement of the anilino group and the phenylimino group in formula (1a). be.
- the manufacturing method described above may further include steps other than the above-described first to third steps as long as the effects of the present embodiment can be obtained.
- the above production method may further include a step of purifying the product in each step.
- a purification step can be appropriately carried out by a known method such as washing with a solvent or column chromatography.
- the compounds of each formula can be synthesized using known techniques, but from the viewpoint of reactivity and productivity, it is preferable to appropriately select synthesis conditions.
- the synthesis reaction of the compound of formula (1a), the second step and the third step are accelerated by high temperature.
- the reaction temperature is raised, the target compound having the desired chain structure and the by-product having a shorter chain structure may be produced at approximately the same ratio.
- the target compound and by-products have similar physical properties such as polarity, and it can be easily expected that it would be difficult to separate the target compound from a mixture of these. are similar, and isolation of the target compound from the above mixture is difficult.
- the reaction rate will also be lowered, but it is possible to synthesize the target compound at a higher rate.
- the reaction rate is not high, but it is possible to efficiently obtain the target compound with high purity.
- the target compound can be obtained by appropriately adjusting synthesis conditions such as reaction temperature as necessary from the viewpoint of reactivity and productivity while using known techniques. It is possible to obtain with high efficiency.
- the carbon chain (straight polyene chain) in the compound of formula (1a) used is longer than in the synthesis of ICG.
- the third step prior to the second step.
- the preceding third step produces a product that is more soluble in the solvent, so that the subsequent second It becomes possible to carry out the process.
- the production of by-products is suppressed, making it possible to obtain substantially only the desired compound.
- a synthetic method with such a sequence of steps is preferable from the viewpoint of obtaining the target compound with high purity, although the reaction rate of the target compound is slightly low.
- the molecular identifier and the compound are bound via the reactive cross-linking group residue in the compound of the present embodiment.
- the meanings of the reactive cross-linking group and the molecular identifier are as described above in the description of the compounds of the embodiments of the present invention.
- the molecular identification agent is preferably a component that can bind to a specific site in a living body.
- the molecular identification agent is preferably an antibody or a fragment of the antibody having antigen-binding ability, among those exemplified above.
- a complex formed by binding such an antibody or fragment is useful as a fluorescent marker for specific biological tissues such as tumors.
- the complex can be used for detection of tumors and confirmation of pharmacological or therapeutic effects on tumors by short-wave infrared molecular imaging.
- the short-wave infrared fluorescent agent of an embodiment of the present invention is one or a composition containing the compound of formula (1) above and the complex above.
- one or more compounds of formula (1) may be used, and one or more complexes may be used.
- the compound of formula (1) or the complex containing it has the property of emitting short-wave infrared light. It can be used for fluorescence imaging with infrared.
- the short-wave infrared fluorescent agent of the present embodiment may further contain components other than the compound and complex of formula (1) described above within the range in which the effects of the present embodiment can be obtained.
- a short-wave infrared fluorescent agent can be used as a phosphate-buffered saline solution because it is usually used in living organisms.
- the stability of ICG and its conjugates in aqueous solution is enhanced in the presence of bovine serum albumin (BSA), and the fluorescence emission intensity of ICG and its conjugates is enhanced in the presence of BSA.
- BSA bovine serum albumin
- the short-wave infrared fluorescent agent in the embodiment of the present invention is from the viewpoint of increasing the stability of the compound and complex of the present embodiment in an aqueous solution, and the emission intensity of short-wave infrared fluorescence is preferable from the viewpoint of increasing
- the concentration of the compound or complex of the present embodiment in the short-wave infrared fluorescent agent may be any concentration that achieves sufficient fluorescence emission intensity according to the application of the short-wave infrared fluorescent agent.
- concentrations of compounds or conjugates of the present embodiments in short-wave infrared fluorescent agents form sufficiently distinct images in short-wave infrared biological or molecular imaging. From the viewpoint, it is preferably 0.1 ⁇ M or more, more preferably 1 ⁇ M or more, and even more preferably 10 ⁇ M or more.
- the concentration of the compound or complex of the present embodiment in the short-wave infrared fluorescent agent may be 100 ⁇ M or less, 10 ⁇ M or less, or 1 ⁇ M from the above viewpoint. may be:
- the content of BSA in the short-wave infrared fluorescent agent may be appropriately determined within the range in which the above effect of increasing the emission intensity of fluorescence can be obtained. you can
- any component that forms micelles with the short-wave infrared fluorescent agent is expected to have the same effect as the combined use of BSA.
- examples of other ingredients as well besides BSA include human serum albumin (HSA), phospholipids such as lecithin, and long chain fatty acids.
- the short-wave infrared fluorescent agent in the embodiment of the present invention functions as an excellent optical contrast agent in fluorescent imaging of a living body with short-wave infrared light having a wavelength region of 900 nm or more.
- the wavelength of excitation light and the detection wavelength of fluorescence can be appropriately determined from the ranges described above in the explanation of the compound.
- imaging of a plurality of biological tissues or biomolecules at different depths can be performed by a single administration of the short-wave infrared fluorescent agent to the living body. is possible.
- short-wave infrared fluorescent agents containing complexes in embodiments of the present invention can be easily prepared and used as optical contrast agents for molecular imaging.
- the short-wave infrared fluorescent agent in the embodiment of the present invention as an optical contrast agent, non-invasive imaging of blood vessels including intracerebral blood vessels, imaging of lymph nodes, and high sensitivity of cancer tumors using short-wave infrared light. detection is possible.
- the short-wave infrared fluorescent agent in the embodiment of the present invention can also be used for drug evaluation using an antibody-drug conjugate (ADC), for example, by conjugating a drug component to the molecular identification agent in the above-described conjugate. It is possible to use
- ADC antibody-drug conjugate
- administration of the short-wave infrared fluorescent agent when used for bioimaging can be performed by a known method suitable for the subject of imaging.
- administration of the short-wave infrared fluorescent agent may be direct administration to the part desired to be imaged, or administration into a blood vessel of a living body such as intravenous injection.
- the compound of formula (1) in the embodiment of the present invention is an analogue of ICG whose molecular structure is similar to ICG in clinical application.
- ICG is currently used as a pharmaceutical agent for human retinal angiography, liver function test (ICG test), and the like.
- the compound of formula (1) of the present embodiment has a molecular structure similar to that of ICG, and, like ICG, has substantially no cytotoxicity as shown in the examples below, and can be dissolved in an aqueous solution. can be used in any condition.
- the compound of formula (1) can be used in a form suitable for imaging with short-wave infrared, like conventionally used ICG, such as being able to bind to an antibody in the same manner as ICG. is possible.
- the compounds of formula (1) of this embodiment also have sufficiently high renal clearance to be substantially excreted in vitro.
- the compound of formula (1) which is an analogue of ICG, has high safety to living organisms.
- the compound of formula (1) has a sulfone group. Therefore, it is highly water soluble and dissolves in physiological buffers. Moreover, since it is an organic molecule with high water solubility, it is easily excreted from the body when it is introduced into a living body.
- the compound of formula (1) can be excited by light in the long wavelength region (900 nm or more) in the near infrared region. Moreover, in the state of an aqueous solution, it has sufficient short-wave infrared fluorescence intensity for fluorescence imaging of a living body. It is known that the short-wave infrared wavelength region is weakly absorbed and scattered by living tissue, and is weakly autofluorescent from living tissue. Therefore, the compound of formula (1) can realize fluorescence imaging at a deeper part than the part observable by fluorescence imaging of ICG in vivo.
- the excitation wavelength of the compound of formula (1) is on the longer wavelength side than that of ICG, the absorption of excitation light into living tissue is weakened more than in fluorescence imaging of ICG in a living body. Therefore, by applying the compound of formula (1) to fluorescence imaging of a living body, the influence of fluorescence imaging on living tissue can be further reduced.
- the compound of formula (1) when the compound of formula (1) has a reactive cross-linking group such as an N-hydroxysuccinimide ester group, it can be easily bound to a biomolecule such as an antibody, and can be used as a short-wave infrared fluorescent label. Complexes can be readily formed. Therefore, it is easy to develop optical contrast agents for molecular imaging, and noninvasive molecular imaging in the short-wave infrared region is possible.
- a reactive cross-linking group such as an N-hydroxysuccinimide ester group
- the compound of formula (1) can relatively shorten the exposure time of the excitation light. Therefore, by applying the compound of formula (1) to fluorescence imaging of a living body, the operation of detecting the fluorescence of the compound of formula (1) and the biological tissue thereby can be performed efficiently.
- the reactive cross-linking group in formula (1) may contain one or more organic groups selected from the group consisting of N-hydroxysuccinimide ester groups, maleimide groups, alkynyl groups and azide groups. This configuration is more effective from the viewpoint of obtaining a stable and high-yield complex when the reactive cross-linking group binds to a bio-derived molecular identification agent such as an antibody to form a complex.
- n in formula (1) may be 3 or 4. This configuration is much more effective from the viewpoint of exhibiting sufficient water solubility and sufficient fluorescence intensity of short-wave infrared rays.
- the compound of formula (1) may be a compound represented by any of formulas (2) to (9) described above. This configuration is suitable from the viewpoint of application to bioimaging and molecular imaging with short-wave infrared.
- the molecular identifier and the compound are bound via the residue of the reactive cross-linking group in the compound according to the embodiment of the present invention.
- the conjugates can be used for biological and molecular imaging in short wave infrared.
- the molecular identification agent may be an antibody or a fragment of the antibody having antigen-binding ability.
- the antibody or fragment thereof is an antibody or fragment thereof that specifically binds to an antigen contained in a living tissue that is the target of molecular imaging. This configuration is much more effective from the viewpoint of realizing molecular imaging in living tissue using short-wave infrared rays.
- the short-wave infrared fluorescent agent in the embodiment of the present invention contains one or both of the compound of the embodiment of the present invention and the complex of the embodiment of the present invention. Therefore, the short-wave infrared fluorescent agent can be used for bioimaging and molecular imaging by short-wave infrared.
- the method for producing a compound according to an embodiment of the present invention comprises a first step of synthesizing the first compound represented by the above formula (1a), and replacing the anilino group in the formula (1a) with the above formula (1b). and a third step of replacing the phenylimino group in formula (1a) with the structure represented by formula (1c) above. Therefore, the production method can produce a novel compound (compound of formula (1)) that enables bioimaging in short-wave infrared.
- n in the above formula is 3 and X is a salt of a sulfonic acid group, even if the second step and the third step are performed at once, good.
- This configuration is more effective from the viewpoint of easier production of the compound of formula (2) and from the viewpoint of suppressing a decrease in yield due to an increase in the number of steps.
- the third step may be performed after the second step.
- This configuration is more effective from the viewpoint of increasing the yield of substitution of the phenylimino group in formula (1a) in the third step in the production of the compound of formula (3) described above.
- the third step may be performed after the second step.
- This configuration is also more effective from the viewpoint of increasing the yield of substitution of the phenylimino group in formula (1a) in the third step in the production of the compound of formula (4).
- the third step may be performed before the second step.
- This configuration is more effective from the viewpoint of increasing the yield in each replacement of the anilino group and the phenylimino group in formula (1a) in the production of the compound of formula (5).
- Phosphoryl chloride (8 g, 52 mmol) was added dropwise to a mixed solution of dimethylformamide (10 mL, 130 mmol) and methanol (0.75 mL) at 0° C. with stirring under an inert atmosphere.
- the reaction temperature was raised to 50° C.
- 2,4-hexadienal 1 (2.5 g, 26 mmol) was added dropwise, the reaction mixture was stirred at 50° C. for about 4 hours, cooled to room temperature, and aqueous sodium perchlorate ( 5% aqueous solution) and extracted with dichloromethane (80 mL).
- the organic phase was filtered through sodium sulfate.
- MTT 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- ICG-C11-NHS can also be synthesized according to the synthesis scheme shown below. Other methods for synthesizing ICG-C11-NHS are specifically described below.
- short-wave infrared fluorescent agent 2 was prepared in the same manner as short-wave infrared fluorescent agent 1 except that compound 10 (ICG-C11) was used instead of compound 4.
- the excitation wavelength was 905 nm, and the fluorescence detection wavelength was 1000 nm.
- the excitation wavelength was 975 nm, and the fluorescence detection wavelength was 1100 nm.
- the excitation light intensity was 20-40 mW/cm 2 and the exposure time was 5-10 seconds.
- FIG. 3 shows a bright-field image of the lower limbs and abdomen of a hairless mouse and a photograph showing a short-wave infrared fluorescence image by short-wave infrared fluorescent agent 1.
- FIG. 4 shows a photograph showing a bright-field image and a short-wave infrared fluorescent image obtained by the short-wave infrared fluorescent agent 2 in the leg and abdomen of a hairless mouse.
- both short-wave infrared fluorescent agent 1 and short-wave infrared fluorescent agent 2 can form clear images of blood vessels in limbs and abdomen in short-wave infrared light. .
- the short-wave infrared fluorescent images obtained by the short-wave infrared fluorescent agent 1 and the short-wave infrared fluorescent agent 2 are clear with high fluorescent signal intensity and less blurring.
- the short-wave infrared fluorescent agent 1 and the short-wave infrared fluorescent agent 2 realize not only practical but also excellent fluorescence imaging of living organisms with short-wave infrared.
- the short-wave infrared fluorescent agent 2 was used instead of the short-wave infrared fluorescent agent 1
- the short-wave infrared fluorescence imaging of the cerebral blood vessels of hairless mice was performed in the same manner as the short-wave infrared fluorescence imaging using the short-wave infrared fluorescent agent 1. Fluorescence imaging was performed.
- the short-wave infrared fluorescent agent C1 is a fluorescent agent prepared in the same manner as the short-wave infrared fluorescent agent 1 except that ICG is used instead of compound 4.
- the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm.
- the excitation light intensity was 10 mW/cm 2 and the exposure time was 1 second.
- the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm.
- the excitation light intensity was 20-40 mW/cm 2 and the exposure time was 1-2.5 seconds.
- the excitation wavelength was 758 nm and the fluorescence detection wavelength was 900 nm.
- the excitation light intensity was 5 mW/cm 2 and the exposure time was 1 second.
- Fig. 5 shows a bright-field image of the parietal region of a hairless mouse and a photograph showing a short-wave infrared fluorescence image using short-wave infrared fluorescent agents C1, 1, and 2.
- the short-wave infrared fluorescent agents 1 and 2 compared with the short-wave infrared fluorescent agent C1 containing ICG, show images of brain vessels of hairless mice that can be observed from the outside with short-wave infrared light. , Clearly, and even the details of cerebral blood vessels can be formed.
- Short-wave infrared fluorescent agents 3 and 4 were prepared, respectively.
- compound 14 ICG-C9-NHS
- BSA bovine serum albumin
- compound 17 ICG-C11-NHS
- the preparation schemes of the short-wave infrared fluorescent agents 3 and 4 are shown below.
- BSA-bound compound 14 (ICG-C9-BSA) was obtained. Purification was performed using a gel filtration column (PD10, GE Healthcare) using physiological phosphate buffer (PBS) as an eluent. ICG-C9-BSA was dissolved in PBS to a concentration of 1 mg/mL. The resulting aqueous solution is used as the short-wave infrared fluorescent agent 3 .
- short-wave infrared fluorescent agent 4 was prepared in the same manner as short-wave infrared fluorescent agent 3, except that compound 17 was used instead of compound 14.
- FIG. 6 shows a photograph showing a bright-field image of the leg and abdomen of a hairless mouse and a short-wave infrared fluorescence image obtained by the short-wave infrared fluorescent agent 3. As shown in FIG.
- FIG. 7 shows a photograph showing a bright-field image of the leg and abdomen of a hairless mouse and a short-wave infrared fluorescence image obtained by the short-wave infrared fluorescent agent 4. As shown in FIG.
- the short-wave infrared fluorescent agent 3 and the short-wave infrared fluorescent agent 4 are similar to the short-wave infrared fluorescent agent 1 and the short-wave infrared fluorescent agent 2. A clear image of short-wave infrared light can be formed.
- Short-wave infrared fluorescence imaging of brain blood vessels of hairless mice was performed in the same manner as in Example 5 except that short-wave infrared fluorescent agent 3 or short-wave infrared fluorescent agent 4 was used instead of short-wave infrared fluorescent agent 1. .
- short-wave infrared fluorescence imaging of brain blood vessels in hairless mice was performed in the same manner as in Example 5, except that short-wave infrared fluorescent agent C2 was used instead of short-wave infrared fluorescent agent 1.
- Short-wave infrared fluorescent agent C2 is a fluorescent agent prepared in the same manner as short-wave infrared fluorescent agent 1 except that ICG-NHS is used instead of compound 14. ICG-NHS can be purchased from Goryo Kayaku Co., Ltd.
- the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm.
- the excitation light intensity was 10 mW/cm 2 and the exposure time was 7.5 seconds.
- the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm.
- the excitation light intensity was 20 to 40 mW/cm 2 and the exposure time was 1 second.
- the excitation wavelength was 758 nm and the fluorescence detection wavelength was 900 nm.
- the excitation light intensity was 5 mW/cm 2 and the exposure time was 20 seconds.
- Fig. 8 shows a bright-field image of the parietal region of a hairless mouse and a photograph showing a short-wave infrared fluorescence image using short-wave infrared fluorescent agents C2, 3, and 4.
- the short-wave infrared fluorescent agents 3 and 4 like the short-wave infrared fluorescent agents 1 and 2, clearly showed images of the cerebral blood vessels of hairless mice observable from the outside with short-wave infrared light. In addition, even the details of cerebral blood vessels can be formed.
- complex 2 (ICG-C11-Erbitux) in which compound 17 was modified with Erbitux was prepared in the same manner as complex 1, except that compound 17 was used instead of compound 14.
- Fluorescence spectra of aqueous solutions obtained by dissolving complexes 1 and 2 in PBS were measured.
- the concentration of the measured aqueous solution is 1 mg/mL.
- the wavelength of the excitation light is 785 nm for the complex 1 and 900 nm for the complex 2.
- Fluorescence spectra of complexes 1 and 2 are shown in FIG. As shown in FIG. 9, the fluorescence emission peak of complex 1 was at about 950 nm, and the fluorescence emission peak of complex 2 was at about 1100 nm.
- anti-HER2 antibody-tubulin polymerization inhibitor conjugate (Kadcyra (registered trademark of F Hoffmann La Roche)), which is an antibody-drug conjugate (ADC)
- ADC antibody-drug conjugate
- Chugai Compound 3 (ICG-C11-Kadcyla), in which compound 17 was modified with Kadcyla, was prepared in the same manner as the preparation of conjugate 2, except that the compound 17 was modified with Kadcyla.
- Conjugate 3 like Conjugate 2, is compound 17 (ICG-C11-NHS) bound to the antibody in the above antibody-drug conjugate via an amide group containing a carbonyl group. there is The excitation light wavelength and fluorescence peak of complex 3 are substantially the same as those of complex 2 .
- a short-wave infrared fluorescent agent 6 was prepared in the same manner as the short-wave infrared fluorescent agent 5 except that the composite 2 was used instead of the composite 1. Further, a short-wave infrared fluorescent agent 7 was prepared in the same manner as the short-wave infrared fluorescent agent 5 except that the composite 3 was used instead of the composite 1.
- the excitation wavelength was 905 nm and the fluorescence detection wavelength was 1000 nm.
- the excitation light intensity was 10 mW/cm 2 and the exposure time was 15 seconds.
- a photograph showing a bright-field image of a breast cancer tumor in nude mice in vivo and a short-wave infrared fluorescence image by short-wave infrared fluorescent agent 5 is shown in FIG. 11 shows bright-field images of breast cancer tumors, heart, kidney, spleen, and liver of nude mice ex vivo, and short-wave infrared fluorescence images obtained by short-wave infrared fluorescent agent 5.
- FIG. 11 shows bright-field images of breast cancer tumors, heart, kidney, spleen, and liver of nude mice ex vivo, and short-wave infrared fluorescence images obtained by short-wave infrared fluorescent agent 5.
- the excitation wavelength was 975 nm and the fluorescence detection wavelength was 1100 nm.
- the excitation light intensity was 20 to 40 mW/cm 2 and the exposure time was 15 to 30 seconds.
- a photograph showing a bright-field image of a breast cancer tumor in a nude mouse in vivo and a short-wave infrared fluorescence image by the short-wave infrared fluorescent agent 6 is shown in FIG. 13 shows bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo, and short-wave infrared fluorescence images obtained by the short-wave infrared fluorescent agent 6.
- FIG. 13 shows bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo, and short-wave infrared fluorescence images obtained by the short-wave infrared fluorescent agent 6.
- breast cancer tumors emit short-wave infrared fluorescence due to the accumulation of complex 1 or complex 2.
- both the short-wave infrared fluorescent agent 5 and the short-wave infrared fluorescent agent 6 can bind specifically to breast cancer tumors and can form images of breast cancer tumors by detection of short-wave infrared light. Recognize.
- Tumor-bearing mice were prepared in the same manner as the above-described breast cancer model mice except that KPL-4 cells (provided by Kawasaki Medical School, about 1.5 ⁇ 10 7 cells) were used as human breast cancer cells, and tumor cells were grown. Grows to several millimeters in size. Then, in vivo and ex vivo fluorescence imaging of breast cancer tumors in breast cancer model mice was performed in the same manner, except that the short-wave infrared fluorescent agent 7 was used instead of the short-wave infrared fluorescent agent 6 .
- FIG. 15 shows photographs showing bright-field images of breast cancer tumors, heart, kidney, spleen, and liver in nude mice ex vivo, and short-wave infrared fluorescence images obtained by short-wave infrared fluorescent agent 7 .
- the short-wave infrared fluorescent agent 7 like the short-wave infrared fluorescent agent 6, also specifically binds to breast cancer tumors, and can detect images of breast cancer tumors by detecting short-wave infrared light. can be formed.
- the fluorescence image was taken under conditions of an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more.
- the excitation light intensity was 20 mW/cm 2 and the exposure time was 5 seconds.
- Photographs representing bright-field and short-wave infrared fluorescence images of breast cancer tumors in nude mice in vivo for short-wave infrared fluorescent agent 8 and short-wave infrared fluorescent agent 9, respectively, are shown in FIG. From these results, it was shown that breast cancer tumors can be clearly detected even with the short-wave infrared fluorescent agents 8 and 9.
- the fluorescence image was taken under conditions of an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more.
- the excitation light intensity was 20 mW/cm 2 and the exposure times were 10 seconds (short-wave infrared fluorescent agent 10) and 30 seconds (short-wave infrared fluorescent agent 11).
- Photographs representing bright-field and short-wave infrared fluorescence images of breast cancer tumors in nude mice in vivo with short-wave infrared fluorescent agent 10 and short-wave infrared fluorescent agent 11, respectively, are shown in FIG. These results indicate that breast cancer tumors can be clearly detected even with the short-wave infrared fluorescent agents 10 and 11.
- the fluorescence image was taken under conditions of an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more.
- the excitation light intensity was 20 mW/cm 2 and the exposure times were 5 seconds (short-wave infrared fluorescent agent 12) and 10 seconds (short-wave infrared fluorescent agent 13).
- the fluorescence image was taken under conditions of an excitation wavelength of 905 nm and a fluorescence detection wavelength of 1000 nm or more.
- the excitation light intensity was 20 mW/cm 2 and the exposure time was 10 seconds.
- Photographs representing bright-field and short-wave infrared fluorescence images of breast cancer tumors in vivo in nude mice injected with Kadcyla in short-wave infrared fluorescent agent 13 after specific time lapses are shown in FIG.
- Kadcyla treatment induced apoptosis (cell death) of tumor cells and reduced tumor size. This result indicates that this short-wave infrared fluorescent agent is effective for confirming the effects of antibody-drug conjugates (ADC) at the level of animal experiments.
- the present invention can be used for short-wave infrared fluorescence imaging technology, and it is expected that the present invention will enable visualization of deep parts of the body with high resolution that far surpasses conventional technologies.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
本発明の実施形態の化合物は、下記式(1)で表される。
式(1)の化合物は、下記の第一から第三の工程の三工程を含む製造方法によって製造することが可能である。なお、以下の製造方法の説明において、一般式中の文字の意味は、前述の化合物のそれと同じである。
本発明の実施形態の複合体は、前述の本実施形態の化合物における反応性架橋基の残基を介して分子識別剤と当該化合物とが結合している。反応性架橋基および分子識別剤の意味は、本発明の実施形態の化合物の説明で前述したとおりである。分子イメージングへの適用の観点から、分子識別剤は、生体中の特定の部位に結合可能な成分であることが好ましい。このような観点から、分子識別剤は、先に例示した中でも、抗体、または、抗原結合能を有する当該抗体の断片であることが好ましい。このような抗体または断片と結合してなる複合体は、腫瘍などの特定の生体組織の蛍光マーカとして有用である。また、当該複合体は、短波赤外による分子イメージングによって腫瘍の検出および腫瘍に対する薬理効果または治療効果の確認に用いることが可能である。
本発明の実施形態の短波赤外蛍光剤は、前述の式(1)の化合物および前述の複合体の一方または含有する組成物である。本発明の実施形態の短波赤外蛍光剤における式(1)の化合物は一種でもそれ以上でもよく、複合体も一種でもそれ以上でもよい。本実施形態の短波赤外蛍光剤は、式(1)の化合物またはそれを含む上記複合体が短波赤外を発する性質を有することから、本発明の実施形態の短波赤外蛍光剤は、短波赤外による蛍光イメージングに使用することが可能である。
本発明の実施形態における短波赤外蛍光剤は、波長領域900nm以上の短波赤外光での生体の蛍光イメージングにおいて優れた光造影剤として機能する。生体の蛍光イメージングでは、化合物の説明で前述した範囲から、励起光の波長および蛍光の検出波長を適宜に決めることが可能である。また、励起波長および蛍光の検出波長の複数の異なる組み合わせを設定すると、短波赤外蛍光剤の生体への一回の投与によって、深さが異なる複数の生体組織または生体分子のイメージングを実施することが可能である。
本発明の実施形態における式(1)の化合物は、臨床応用されているICGにその分子構造が類似しているICGの類縁体である。ICGは、現在、ヒトの網膜の血管造影、肝機能検査(ICG検査)などの医薬品として使われている。本実施形態の式(1)の化合物は、ICGに類似の分子構造を有しており、ICGと同様に、後述の実施例で示すように細胞毒性を実質的に有さず、また水溶液の状態で使用し得る。さらには、式(1)の化合物は、ICGと同様の手法で抗体と結合させることができるなど、従来使用されているICGと同様に、短波赤外での造影に適した形態で使用することが可能である。
以上の説明から明らかなように、本発明の実施形態における化合物は、前述の式(1)で表される。よって、短波赤外での生体イメージングを可能とする新規な化合物が提供される。
下記実施例で使用する市販の溶剤および化学薬品は、シグマ アルドリッチ、東京化成工業株式会社、および富士フィルム和光純薬株式会社から購入した。
s=シングレット;d=ダブレット;t=トリプレット;q=クアルテット;m=多重項;dd=二重項-二重項;
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C9を合成した。
化合物1として、1,1,2-トリメチル-1H-ベンゾ[e]インドール(富士フィルム和光純薬)を用意した。
1,1,2-トリメチル-1H-ベンゾ[e]インドール(1)(2g、9.6mmol)と1,4-ブタンスルトン(2.6g、19.1mmol)の混合物を130℃で4時間撹拌した。得られた固体を真空濾過により収集し、アセトンで完全に洗浄し、真空下で乾燥させた。こうして、化合物2を淡青色の固体(2.85g、収率86%)として得た。化合物2の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C19H23NO3Sの[M-H]+に対する
計算値:344.1344
実測値:344.131
1H NMR(DMSO-d6,500MHz):δ=8.37(1H,d,J=8.6Hz),8.28(1H,d,J=9.2Hz),8.19-8.21(2H,m),7.77(1H,t,J=7.3Hz),7.72(1H,t,J=7.6Hz),4.60(2H,t,J=7.8Hz),2.94(3H,s),2.51-2.53(2H,m),2.03(2H,quintet,J=7.6Hz),1.76-1.80(2H,m),1.75(6H,s)
化合物3の合成スキームを以下に示す。
高分解能質量分析(m/z):分子式C19H18N2の[M+H]+に対する
計算値:275.1542
実測値:275.1534
1H NMR(CD3OD,500MHz):δ=8.30(2H,d,J=11.2Hz),7.65(2H,t,J=12.6Hz),7.42(4H,t,J=8.3Hz),7.31(4H,d,J=7.8Hz),7.22(2H,t,J=7.8Hz),6.49(7H,t,J=11.7Hz),6.27(2H,t,J=11.7Hz)
室温で、化合物2(0.5g、1.45mmol)の無水酢酸(10mL)溶液と化合物3(215mg、0.69mmol)の混合溶液に、撹拌しながら酢酸ナトリウム(200mg、2.42mmol)を加えた。不活性雰囲気下で酢酸(4mL)を滴下した。混合物を120℃で1時間撹拌し、室温に冷却した。反応混合物を30mLのジエチルエーテルに移し、得られた沈殿物を2-プロパノール:水(4:1、50mL)に溶解し、さらに2-プロパノール(300mL)で希釈し、再結晶するために一晩4℃で保存した。沈殿物を遠心分離により収集し、得られた残留物を、クロロホルム/メタノール(8:2)を溶出液としてカラムクロマトグラフィーによってさらに精製した。こうして、化合物4であるICG-C9を褐色の固体(270mg、収率23%)として得た。化合物4の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C45H49N2O6S2の[M]+に対する
計算値:777.3026
実測値:777.3005
1H NMR(DMSO-d6,500MHz):δ=8.22(2H,d,J=8.3Hz),7.89-8.05(6H,m),7.76-7.80(1H,m),7.74(2H,d,J=9.3Hz),7.59-7.66(3H,m),7.42-7.50(3H,m),6.59(2H,t,J=12.7Hz),6.42-6.51(2H,m),4.18(4H,t,J=6.3Hz),1.94-2.02(4H,m),1.90(12H,s),1.71-1.85(8H,m),1.02-1.06(2H,m)
HeLa細胞を96ウェルプレートに1ウェルあたり6×103個ずつ播種し、一晩培養した。0.1nM、1nM、10nMおよび100nMの各濃度の化合物4の水溶液を試験薬として調製し、各濃度の試験薬をウェルに添加し、HeLa細胞をさらに6、24、48時間培養した。各時間培養の後、ウェルにMTT(3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)試薬を加え、さらに2時間培養した。
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C11を合成した。
不活性雰囲気下、0℃で水素化ナトリウム(鉱油中60%、833mg、20.8mmol)のテトラヒドロフランの溶液に、撹拌しながらトリエチルホスホノ酢酸(5.2g、22.1mmol)を滴下した。混合物を0℃で30分間撹拌し、次に2,4-ヘキサジエナール1(2g、20.8mmol)を滴下して加えた。反応物を室温に温め、約2時間撹拌し、塩化アンモニウムでクエンチし、そして酢酸エチルで抽出した。有機相を硫酸ナトリウムで乾燥し、濾過し、そして真空中で濃縮して、残留物を得た。これをヘキサン/酢酸エチル(9.5:0.5)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物5を白色固体として得た(3.25g、収率94%)。化合物5の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C10H14O2の[M+H]+に対する
計算値:167.1066
実測値:167.1068
1H NMR(CDCl3,500MHz):δ=7.31(1H,dd,J=11.3,4.4Hz),6.53(1H,dd,J=10.7,4.4Hz),6.14-6.24(2H,m),5.90-5.98(1H,m),5.86(1H,d,J=15.1Hz),4.21(2H,m),1.34(3H,d,J=6.4Hz),1.30(3H,t,J=7.5Hz)
不活性雰囲気下、-60℃で化合物5のジクロロメタン溶液(2.5g、15mmol)を攪拌しながら、水素化ジイソブチルアルミニウム(ヘキサン中1.0M、45mL、45mmol)を滴下した。反応物を-60℃で30分間、次いで0℃で1時間撹拌した。水(10mL)をゆっくりと加え、続いて水酸化ナトリウム(15%水溶液10mL)を加えて反応をクエンチし、白色の沈殿物を得た。混合物を室温で1時間激しく撹拌し、硫酸マグネシウムを加え、混合物を、ジクロロメタンを含むセライトで濾過した。有機相を真空で濃縮して粗製物を得、これをヘキサン/酢酸エチル(8:2)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物6を白色固体として得た(1.65g、収率89%)。化合物6の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C8H10Oの[M+H-H2O]+に対する
計算値:107.0855
実測値:107.0860
1H NMR(CDCl3,500MHz):δ=6.20-6.28(2H,m),6.06-6.14(2H,m),5.71-5.83(2H,m),4.20(2H,d,J=5.8Hz),1.79(2H,d,J=6.8Hz),1.40(1H,s)
不活性雰囲気下、室温で、化合物6(1.5g、12.1mmol)のジクロロメタン溶液(30mL)溶液に、活性化酸化マンガン(7.4g、84.7mmol)を加え、反応混合物を一晩撹拌した。反応混合液を濾過し、濾液を濃縮してヘキサン/酢酸エチル(9:1)を溶出液としてカラムクロマトグラフィーによって精製された粗製物を得た。こうして、化合物7を黄色の固体として得た(1.08g、収率73%)。化合物7の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C8H10Oの[M+H]+に対する
計算値:123.0804
実測値:123.0804
1H NMR(CDCl3,500MHz):δ=9.58(1H,d,J=9.8Hz),7.12(1H,dd,J=11.3,3.9Hz),6.65(1H,dd,J=10.3,4.4Hz),6.34(1H,dd,J=10.7,4.0Hz),6.21(1H,m),6.14(1H,dd,J=7.8,7.3Hz),6.05(1H,m),1.87(3H,d,J=7.8Hz)
0℃でジメチルホルムアミド(3.2mL、42.5mmol)およびメタノール(0.4mL)の溶液に、撹拌しながら不活性雰囲気下で塩化ホスホリル(2.5g、16.4mmol)を滴下して加えた。反応温度を40℃に上げ、化合物7(1g、8.2mmol)のジメチルホルムアミド溶液(0.5mL)を滴下し、反応混合物を40℃で約4時間撹拌した。反応物を室温(室温)に冷却し、過塩素酸ナトリウム水溶液(20mL中1g)に加え、ジクロクロロメタン(25mL)で抽出した。有機相を硫酸ナトリウム層に通して濾過した。室温で攪拌したジクロクロロメタン相に、アニリン(1.5g、16.4mmol)を滴下し、一晩攪拌した。得られた沈殿物を濾過により収集し、ジクロロメタンで数回洗浄した。こうして、化合物8を緑色の固体として得た(1.25g、収率45%)。化合物8の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C21H20N2の[M+H]+に対する
計算値:301.1699
実測値:301.1699
1H NMR(CD3OD,500MHz):δ=8.21(2H,d,J=11.2Hz),7.52(2H,t,J=13.2Hz),7.4(4H,t,J=8.3Hz),7.367(1H,d,J=12.7Hz),7.27(4H,d,J=7.8Hz),7.18(2H,t,J=7.3Hz),6.47(2H,t,J=13.2Hz),6.26(2H,t,J=11.7Hz)
室温で化合物2(250mg、0.72mmol)および酢酸ナトリウム(178mg、2.17mmol)のエタノール(10mL)溶液に、当該溶液を撹拌しながら化合物8(244mg、0.72mmol)を加えた。次に無水酢酸(0.4mL、4.34mmol)を不活性雰囲気下で滴下した。反応を30分間続け、溶媒を真空で除去した。残留物をクロロホルム/メタノール(9:1)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物9を金褐色の固体(274mg、収率64%)として得た。化合物9の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C36H38N2O4Sの[M+H]+に対する
計算値:595.2625
実測値:595.2617
1H NMR(CD3OD,500MHz):δ=8.35(1H,d,J=7.3Hz),8.20-8.25(2H,m),8.11(1H,d,J=8.3Hz),7.86-7.94(2H,m),7.76(1H,t,J=7.3Hz),7.65(1H,t,J=7.8Hz),7.53-7.61(3H,m),7.28-7.36(3H,m),7.12(1H,d,J=14.6Hz),6.86(2H,q,J=14.1Hz),6.69(1H,t,J=11.2Hz),6.49(1H,t,J=13.6Hz),6.20(1H,t,J=11.7Hz),5.23(2H,t,J=13.7Hz),4.59(2H,t,J=7.3Hz),2.9(2H,t,J=7.3Hz),2.14(2H,quintet,J=7.8Hz),2.00(6H,s),1.89-1.98(2H,m)
室温で化合物2(29mg、0.084mmol)および酢酸ナトリウム(12mg、0.139mmol)のエタノール(3mL)溶液に、当該溶液を攪拌しながら無水酢酸(9μL、0.092mmol)を加えた。エタノール(3mL)に溶解した化合物9(50mg、0.084mmol)を、不活性雰囲気下で反応混合物に加えた。混合物を60℃で1時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(7.5:2.5)を使用するカラムクロマトグラフィーによって精製した。こうして、化合物10であるICG-C11を褐色の固体として得た(33mg、収率48%)。化合物10の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C47H51N2O6S2の[M]+に対する
計算値:803.3183
実測値:803.3221
1H NMR(DMSO-d6,500MHz):δ=8.21(2H,d,J=8.3Hz),8.01(4H,d,J=7.3Hz),7.81(2H,t,J=13.1Hz),7.71(2H,t,J=8.8Hz),7.61(2H,t,J=6.8Hz),7.47(2H,t,J=7.3Hz),7.34(2H,t,J=12.7Hz),7.01(1H,t,J=12.7Hz),6.48-6.57(4H,m),6.44(2H,d,J=13.6Hz),4.17(4H,t,J=6.8Hz),1.88(12H,s),1.75-1.81(12H,m)
化合物4に代えて化合物10を用いて試験薬を調製する以外は、化合物4の細胞毒性試験と同様にして、HeLa細胞の培養とウェルの細胞生存率とを行った。結果を図2に示す。上記試験結果から、化合物10は、化合物4と同様に、濃度1~100nMにおいて、6、24、48時間培養条件で細胞毒性を示さないことが明らかとなった。
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C9-NHSを合成した。
室温で化合物2(1g、2.89mmol)と酢酸ナトリウム(712mg、8.68mmol)とエタノール(25mL)の混合溶液に、当該溶液を撹拌しながら化合物3(900mg)を加えた。次に無水酢酸(1.7mL、17.37mmol)を不活性雰囲気下で滴下した。反応を30分間続け、溶媒を真空で除去し、残留物をクロロホルム/メタノール(9:1)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物11を金褐色の固体(1.12g、収率68%)として得た。化合物11の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C34H36N2OSの[M+H]+に対する
計算値:569.2468
実測値:569.2481
1H NMR(CD3OD,500MHz):δ=8.35(1H,d,J=8.3Hz),8.22(1H,dd,J=11.25,3.4Hz),8.18(1H,d,J=8.8Hz),8.11(1H,d,J=8.3Hz),8.00-8.04(1H,m),7.93(1H,d,J=8.7Hz),7.76(1H,t,J=7.3Hz),7.56-7.67(4H,m),7.31-7.36(3H,m),7.07(1H,d,J=15.1Hz),6.95(1H,dd,J=11.3Hz,3.0Hz),6.75(1H,dd,J=11.3,3.0Hz),6.33(1H,dd,J=11.7,2.5Hz),5.30(1H,dd,J=11.7Hz,2.0Hz),4.58(2H,t,J=7.8Hz),2.90(2H,t,J=7.3Hz),2.10-2.16(2H,m),2.00(6H,s),1.98(3H,s),1.95-1.97(2H,m)
化合物12の合成スキームを以下に示す。
高分解能質量分析(m/z):分子式C21H26NO2の[M]+に対する
計算値:324.1958
実測値:324.1949
1H NMR(DMSO-d6,500MHz):δ=8.38(1H,d,J=8.8Hz),8.29(1H,d,J=8.8Hz),8.22(1H,d,J=7.9Hz),8.15(1H,d,J=8.8Hz),7.78(1H,t,J=7.4Hz),7.73(1H,t,J=6.9Hz),4.57(2H,t,J=7.8Hz),2.93(3H,s),2.23(2H,t,J=7.3),1.87-1.93(2H,m),1.75(6H,s),1.54-1.60(2H,m),1.43-1.49(2H,m)
室温での化合物12(164mg、0.405mmol)と酢酸ナトリウム(55mg、0.673mmol)とエタノール(8mL)の混合溶液に、当該溶液を撹拌しながら無水酢酸(43μL、0.446mmol)を加えた。エタノール(10mL)に溶解した化合物11(230mg、0.405mmol)を、不活性雰囲気下で反応混合物に加えた。混合物を60℃で1時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(8:2)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物13を暗緑色の固体として得た(234mg、収率76%)。化合物13の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C47H52N2O5Sの[M+H]+に対する
計算値:757.3669
実測値:757.3666
1H NMR(CD3OD,500MHz):δ=8.20(2H,t,J=7.8Hz),7.85-8.00(7H,m),7.58-7.65(3H,m),7.52(1H,d,J=9.3Hz),7.34-7.48(4H,m),6.50-6.64(3H,m),6.42(1H,d,J=13.6Hz),6.25(1H,d,J=13.1Hz),4.24(2H,t,J=6.3Hz),4.16(2H,t,J=6.8Hz),3.61(1H,q,t,J=6.8Hz),2.92(2H,t,J=6.8Hz),2.29(2H,t,J=7.3Hz),1.99-2.07(4H,m),1.86(2H,quintet,J=7.3Hz),1.71(2H,quintet,J=7.3Hz),1.53(2H,quintet,J=7.3Hz)
不活性雰囲気下、化合物13(200mg、0.264mmol)およびN-ヒドロキシコハク酸イミド(76mg、0.660mmol)のクロロホルム溶液(10mL)に、当該溶液を撹拌しながらN,N’-ジシクロヘキシルカルボジイミド(136mg、0.660mmol)のクロロホルム(1mL)溶液を滴下した。反応混合物を室温で4時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(9:1)を溶出液とするカラムクロマトグラフィーによって精製した。こうして、化合物14であるICG-C9-NHSを暗緑色の固体(125mg、収率55%)として得た。化合物14の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C51H55N3O7Sの[M+H]+に対する
計算値:854.3833
実測値:854.3828
1H NMR(CD3OD,500MHz):δ=8.21(2H,t,J=7.3Hz),7.96(6H,m),7.62(3H,m),7.53(1H,d,J=8.8Hz),7.34-7.53(4H,m),6.50-6.65(3H,m),6.42(1H,d,J=13.6Hz),6.27(1H,d,J=13.1Hz),4.24(2H,t,J=6.3Hz),1.46(2H,t,J=7.3Hz),2.92(2H,t,J=7.3Hz),2.65-2.70(2H,m),2.00-2.07(4H,m),1.97(12H,s),1.82-1.92(6H,m),1.61-1.73(2H,m),1.39-1.45(2H,m)
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C11-NHSを合成した。
室温で化合物12(250mg、0.618mmol)と酢酸ナトリウム(101mg、1.236mmol)のエタノール(15mL)溶液に、当該溶液を撹拌しながら化合物8(208mg、0.618mmol)を加えた。次に無水酢酸(175μL、1.855mmol)を不活性雰囲気下で滴下した。反応を30分間続け、溶媒を真空で除去した。残留物をクロロホルム/メタノール(9:1)を溶出液とするカラムクロマトグラフィーによって精製した。こうして、化合物15を金褐色の固体(310mg、収率77%)として得た。化合物15の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C38H41N2O3の[M]+に対する
計算値:573.3111
実測値:573.3108
1H NMR(CD3OD,500MHz):δ=8.36(1H,d,J=8.3Hz),8.25(1H,dd,J=11.2,4.0Hz),8.19(1H,d,J=8.8Hz),8.12(1H,d,J=8.3Hz),7.89(2H,m),7.77(1H,t,J=7.3Hz),7.67(1H,t,J=7.3Hz),7.60(2H,j,J=7.4Hz),7.54(1H,t,J=7.8Hz),7.36(1H,dd,J=11.8,2.4Hz),7.30(2H,d,J=7.4Hz),7.04(1H,d,J=15.1Hz),6.79-6.90(2H,m),6.71(1H,dd,J=11.3,3.4Hz),6.50(1H,dd,J=11.3,3.0Hz),6.21(1H,dd,J=11.3,3.4Hz),5.23(1H,dd,J=11.3,3.0Hz),4.56(2H,t,J=7.3Hz),2.29(2H,t,J=7.3Hz)2.01(6H,s),1.92-1.97(5H,m),1.69(2H,quintet,J=6.8Hz),1.54(2H,quintet,J=7.3Hz)
不活性雰囲気下、0℃で化合物15(110mg、0.168mmol)とN-ヒドロキシコハク酸イミド(48mg、0.42mmol)のクロロホルム(10mL)溶液に、当該溶液を撹拌しながらN,N’-ジシクロヘキシルカルボジイミド(86mg、0.42mmol)のクロロホルム(1mL)溶液を滴下した。反応混合物を室温で2時間撹拌した。溶媒を真空で除去し、残留物を、クロロホルム/メタノール(9.5:0.5)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物16を褐色の固体として得た(88mg、収率70%)。化合物16の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C42H44N3O5の[M]+に対する
計算値:670.3275
実測値:670.3272
1H NMR(CD3OD,500MHz):δ=8.36(1H,d,J=8.3Hz),8.24(1H,t,J=11.7Hz),8.18(1H,d,J=9.3Hz),8.12(1H,d,J=7.8Hz),7.87-7.89(2H,m),7.77(1H,t,J=7.3Hz),7.67(1H,t,J=7.8Hz),7.54-7.61(4H,m),7.29-7.38(3H,m),7.04(1H,d,J=14.6Hz),6.78-6.90(2H,m),6.72(1H,t,J=13.6Hz),6.50(H,t,J=11.2Hz),6.21(1H,t,J=14.1Hz),5.24(1H,t,J=11.7Hz),4.57(2H,t,J=6.3Hz),2.76-2.84(4H,m),2.01(6H,s),1.89-1.97(4H,m),1.85(2H,quintet,J=6.8Hz),1.64(2H,quintet,J=5.0Hz)
室温で、化合物2(55mg、0.160mmol)および酢酸ナトリウム(13mg、0.160mmol)のエタノール(3mL)溶液に、当該溶液を撹拌しながら無水酢酸(15μL、0.160mmol)を加えた。エタノール(5mL)に溶解した化合物16(80mg、0.106mmol)を、不活性雰囲気下で反応混合物に加えた。混合物を60℃で1時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(9:1)を溶出液としてカラムクロマトグラフィーによって精製した。こうして、化合物17であるICG-C11-NHSを暗緑色の固体として得た(42mg、収率45%)。化合物17の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C53H57N3O7Sの[M+H]+に対する
計算値:880.3990
実測値:880.3985
1H NMR(DMSO-d6,500MHz):δ=8.25(1H,d,J=8.3Hz),8.18(1H,d,J=8.8Hz),8.06(2H,t,J=8.8Hz),7.99(2H,d,J=8.8Hz),7.92(1H,t,J=12.7Hz),7.80(1H,d,J=8.8Hz),7.71(1H,t,J=12.7Hz),7.65(1H,t,J=7.3Hz),7.60(2H,t,J=8.8Hz),7.51(1H,t,J=7.8Hz),7.37-7.44(2H,m),7.27(1H,t,J=12.7Hz),7.01(1H,t,J=13.1Hz),6.45-6.64(5H,m),6.22(1H,d,J=13.7Hz),4.25(2H,t,J=6.8Hz),4.09(2H,t,J=7.3Hz),2.80(4H,s),2.68(2H,t,J=6.8Hz),1.90(6H,s),1.88(6H,s),1.82-1.86(3H,m),1.68-1.79(7H,m),1.50(2H,quintet,J=7.3Hz),1.40(2H,quintet,J=6.8Hz)
なお、ICG-C11-NHSは、以下に示す合成スキームにしたがっても合成することが可能である。以下、ICG-C11-NHSの他の合成法を具体的に説明する。
室温で化合物2(175mg、0.505ミリモル)および酢酸ナトリウム(21mg、0.838ミリモル)のエタノール(10mL)溶液に、無水酢酸(53μL、0.555ミリモル)を加えた。次に、エタノール(10mL)に溶解した化合物15(330mg、0.505mmol)を、不活性雰囲気下で上記反応混合物に加え、混合物を60℃で約1.5時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(8:2)を溶出液とするカラムクロマトグラフィーによって精製した。こうして、化合物18を暗褐色の固体として得た(127mg、64%)。化合物18の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/z):分子式C49H54N2O5Sの[M+H]+に対する
計算値:783.3826
実測値:783.3817
1H NMR(DMSO-d6,500MHz):δ=8.25(1H,d,J=8.8Hz),8.19(1H,d,J=8.8Hz),8.06(2H,t,J=8.3Hz),8.00(2H,d,J=8.8Hz),7.92(1H,t,J=12.7Hz),7.80(1H,d,J=8.8Hz),7.71(1H,t,J=12.7Hz),7.65(1H,t,J=7.3Hz),7.60(2H,t,J=8.8Hz),7.51(1H,t,J=7.3Hz),7.37-7.44(2H,m),7.27(1H,t,J=12.7Hz),7.01(1H,t,J=12.7Hz),6.45-6.63(5H,m),6.22(1H,d,J=13.1Hz),4.24(2H,t,J=6.3Hz),4.09(2H,t,J=6.8Hz),2.14(2H,t,J=6.8Hz),1.90(6H,s),1.88(6H,s),1.82-1.84(2H,m),1.68-1.79(4H,m),1.55(2H,quintet,J=7.3Hz),1.40(2H,quintet,J=6.8Hz)
不活性雰囲気下0°Cで化合物18(53mg、0.0677mmol)およびN-ヒドロキシコハク酸イミド(20mg、0.169mmol)のクロロホルム(8mL)溶液に、当該溶液を攪拌しながら、N,N‘-ジシクロヘキシルカルボジイミド(35mg、0.169mmol)のクロロホルム(1mL)溶液を滴下し、反応混合物を室温で4時間撹拌した。溶媒を真空で除去し、残留物を、クロロホルム/メタノール(9:1)を溶出液とするカラムクロマトグラフィーによって精製した。こうして、ICG-C11-NHS(化合物17)を暗褐色の固体として得た(31mg、52%)。得られた化合物17の質量分析(MS)および核磁気共鳴(NMR)の分析結果は、前述の通りである。
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C9-マレイミドを合成した。
不活性雰囲気下、室温でジクロロメタン(10mL)中の化合物13(60mg、0.079ミリモル)の撹拌溶液に、1-[ビス(ジメチルアミノ)メチレン]-1H-1,2,3-トリアゾロ[4,5-b]ピリジニウム3-オキシドヘキサフルオロホスファート(HATU)(75mg、0.198ミリモル)、N、N-ジイソプロピルエチルアミン(DIPEA)(31mg、0.237ミリモル)、1-(2-アミノエチル)マレイミド塩酸塩(21mg、0.119mmol)を加えた。反応混合物を室温で約4~5時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(9:1)を使用するカラムクロマトグラフィーによって精製して、暗褐色の固体として化合物19を得た(45mg、64%)。化合物19の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析 (m/s): 分子式C53H58N4O6Sの[M+H]+に対する
計算値:879.4149
実測値:879.4121
1H NMR (DMSO-d6,500MHz):δ=8.56(1H,d,J=3.4Hz),8.36(1H,d,J=8.3Hz),8.22(2H,dd,J=17.5,8.3Hz),7.99-8.06(4H,m),7.85-7.88(2H,m),7.78(1H,d,J=8.8Hz),7.59-7.65(3H,m),7.43-7.51(3H,m),7.34-7.37(1H,m),6.46-6.64(4H,m),6.31(1H,d,J=13.2Hz),4.22(2H,t,J=6.3Hz),4.12(2H,t,J=6.8Hz),3.58(1H,s),3.38(2H,t,J=5.8Hz),3.15(2H,t,J=5.3Hz),1.98(2H,t,J=4.9Hz),1.90(6H,s),1.89(6H,s),1.84(2H,q,J=7.3Hz),1.77(2H,q,J=6.8Hz),1.69(2H,quintet,J=7.3Hz),1.51(2H,quintet,J=7.3Hz),1.34(2H,quintet,J=7.3Hz)
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C9-アルキンを合成した。
不活性雰囲気下、室温でCH2Cl2(10mL)中の化合物13(60mg、0.079ミリモル)の撹拌溶液に、HATU(75mg、0.198ミリモル)、DIPEA(31mg、0.238ミリモル)、プロパルギルアミン(9mg、0.159ミリモル)を加えた。反応混合物を室温で約4~5時間撹拌した。溶媒を真空で除去し、残留物をクロロホルム/メタノール(9:1)を使用するカラムクロマトグラフィーによって精製して、暗褐色の固体として化合物20を得た(43mg、68%)。化合物20の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/s):分子式C53H58N4O6Sの[M+H]+に対する
計算値:793.3907
実測値:793.3790
1H NMR(DMSO-d6,500MHz):δ=8.68(1H,d,J=4.3Hz),8.48(1H,d,J=8.3Hz),8.19-8.24(3H,m),8.00-8.06(4H,m),7.88(1H,t,J=13.2),7.78(1H,d,J=8.8Hz),7.60-7.65(3H,m),7.44-7.51(3H,m),6.50-6.64(3H,m),6.31(1H,d,J=13.6Hz),4.22(2H,t,J=6.3Hz),4.13(2H,t,J=6.8Hz),3.80-3.82(2H,m),3.06(1H,s),2.08(2H,t,J=7.3Hz),1.90(6H,s),1.89(6H,s),1.84(2H,q,J=6.8Hz),1.68-1.79(4H,m),1.56(2H,quintet,J=7.3Hz),1.38(2H,quintet,J=6.8Hz),1.22-1.25(2H,m)
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C11-マレイミドを合成した。
不活性雰囲気下、室温でジクロロメタン(10mL)中の化合物18(50mg、0.064ミリモル)の撹拌溶液に、HATU(60mg、0.160ミリモル)、DIPEA(25mg、0.192ミリモル)、1-(2-アミノエチル)マレイミド塩酸塩(17mg、0.10mmol)を加えた。反応混合物を室温で一晩撹拌した。溶媒を真空で除去し、残留物を、クロロホルム/メタノール(9:1)を使用するカラムクロマトグラフィーによって精製して、暗緑色の固体として化合物21を得た(32mg、55%)。化合物21の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/s):分子式C53H58N4O6Sの[M+H]+に対する
計算値:905.4306
実測値:905.4295
1H NMR(DMSO-d6,500MHz):δ=8.94(2H,s),8.24(1H,d,J=8.3Hz),8.18(1H,d,J=8.8Hz),8.05(2H,t,J=8.3Hz),7.97-8.00(2H,m),7.87-7.90(2H,m),7.79(1H,d,J=8.8Hz),7.57-7.75(5H,m),7.50(1H,t,J=7.3Hz),7.36-7.44(2H,m),7.28(1H,t,J=12.7Hz),6.98(2H,s),6.46-6.62(4H,m),6.25(1H,d,J=13.7Hz),4.23(2H,t,J=7.3Hz),4.09(2H,t,J=5.3Hz),3.38(2H,t,J=5.8Hz),3.11-3.15(2H,m),1.97(2H,t,J=7.3Hz),1.89(6H,s),1.87(6H,s),1.81-1.86(2H,m),1.76(2H,quintet,J=6.8Hz),1.68(2H,quintet,J=6.8Hz),1.50(2H,quintet,J=7.3Hz),1.32-1.37(2H,m)
以下に示す合成スキームにしたがって、本発明の化合物であるICG-C11-アルキンを合成した。
不活性雰囲気下、室温でジクロロメタン(10mL)中の化合物18(50mg、0.064ミリモル)の撹拌溶液に、HATU(61mg、0.160ミリモル)、DIPEA(25mg、0.192ミリモル)、プロパルギルアミン(7mg、0.128mmol)を加えた。反応混合物を室温で一晩撹拌した。溶媒を真空で除去し、残留物を、クロロホルム/メタノール(9:1)を使用するカラムクロマトグラフィーによって精製して、暗緑色の固体として化合物22得た(30mg、58%)。化合物22の質量分析(MS)および核磁気共鳴(NMR)の分析結果を以下に示す。
高分解能質量分析(m/s):分子式C53H58N4O6Sの[M+H]+に対する
計算値:920.4142
実測値:920.4130
1H NMR(DMSO-d6,500MHz):δ=8.17-8.24(3H,m),8.05(2H,t,J=7.8Hz),8.00(2H,d,J=8.3Hz),7.90(1H,t,J=13.2Hz),7.70-7.79(2H,m),7.58-7.66(3H,m),7.50(1H,t,J=7.3Hz),7.43(1H,t,J=7.3Hz),7.28(1H,t,J=13.2Hz),7.01(1H,t,J=12.7),6.46-6.62(4H,m),6.24(1H,d,J=13.2Hz),4.23(2H,t,J=6.8Hz),4.09(2H,t,J=5.3Hz),3.80-3.82(2H,m),3.06(1H,s),2.91(2H,s),2.08(2H,t,J=7.3Hz),1.89(6H,s),1.87(6H,s),1.81-1.84(2H,m),1.76(2H,quintet,J=7.3Hz),1.70(2H,quintet,J=6.8Hz),1.37(2H,quintet,J=6.8Hz)
[短波赤外蛍光剤1、2の調製]
化合物4(ICG-C9)の1mgを1mLのジメチルスルホキシド(富士フィルム和光純薬株式会社)に溶解する。次に、この溶液の0.1mLを重量比1%の牛血清アルブミン(シグマ アルドリッチ)0.9mLに加える。この溶液1mLを蒸留水で透析(スペクトラム透析、MWCO:50kDa)した。こうして、血管、およびリンパ節の造影用の蛍光プローブ水溶液(1mg/mL、1%牛血清アルブミン)を得た。当該水溶液を短波赤外蛍光剤1とする。
<マウスの下肢部および腹部の血管の造影>
ヘアレスマウス(オス5週齢、Hos:HR-1、日本エスエルシー株式会社)をイソフルラン(富士フィルム和光純薬株式会社)の麻酔下で、その尾静脈から0.2mLの短波赤外蛍光剤1を注入した。その後、ヘアレスマウスの下肢部および腹部の血管の短波赤外での蛍光イメージングを行った。
ヘアレスマウス(オス5週齢、Hos:HR-1、日本エスエルシー株式会社)をイソフルラン(富士フィルム和光純薬株式会社)の麻酔下で、その尾静脈から0.2mLの短波赤外蛍光剤1を注入した。その後、ヘアレスマウスの脳血管の短波赤外での蛍光イメージングを行った。蛍光画像の撮影は、浜松ホトニクス株式会社製、冷却InGaAs短波赤外カメラ(C10633-34、ペルチェ冷却+水冷 -70℃、暗電流132electrons/pixel/s)を使用した。
[短波赤外蛍光剤3、4の調製]
短波赤外蛍光剤3、4をそれぞれ調製した。短波赤外蛍光剤3は、化合物14(ICG-C9-NHS)が反応性架橋基の残基であるカルボニル基を含むアミド基を介して牛血清アルブミン(BSA)と結合しており、短波赤外蛍光剤4は、化合物17(ICG-C11-NHS)が当該アミド基を介してBSAと結合している。短波赤外蛍光剤3、4の調製スキームを以下に示す。
<マウスの下肢部および腹部の血管の造影>
短波赤外蛍光剤1に代えて短波赤外蛍光剤3または短波赤外蛍光剤4を用いる以外は実施例5と同様にして、ヘアレスマウスの下肢部および腹部の血管の短波赤外での蛍光イメージングを行った。
短波赤外蛍光剤1に代えて短波赤外蛍光剤3または短波赤外蛍光剤4を用いる以外は実施例5と同様にして、ヘアレスマウスの脳血管の短波赤外での蛍光イメージングを行った。
[複合体1~3の調製]
複合体1、2をそれぞれ調製した。複合体1は、化合物14(ICG-C9-NHS)が反応性架橋基の残基であるカルボニル基を含むアミド基を介して抗体(Erbitux)と結合しており、複合体2は、化合物17(ICG-C11-NHS)が当該アミド基を介して抗体と結合している。複合体1、2の調製スキームを以下に示す。
複合体1、2のそれぞれをPBSに溶解した水溶液の蛍光スペクトルを測定した。測定した水溶液の濃度は1mg/mLである。また、励起光の波長は、複合体1では785nm、複合体2では900nmである。複合体1、2の蛍光スペクトルを図9に示す。図9に示されるように、複合体1の蛍光の発光ピークは約950nmであり、複合体2の蛍光の発光ピークは約1100nmであった。
[短波赤外蛍光剤5~7の調製]
複合体1をPBSで希釈して濃度1mg/mLの複合体1の水溶液を得た。当該水溶液を短波赤外蛍光剤5とする。
<乳がんモデルマウスの作製>
5週令ヌードマウス(BALB/c Slc-nu/n、日本SLC)にヒト乳がん細胞(MDA-MB-468(ATCC)、約1.5×107個)をマウス下肢の第2乳房付近に移植した。こうして、乳がんモデルマウスとしての担がんマウスを作製した。当該マウスを2~3週間生育し、腫瘍のサイズを数ミリまで成長させた。
上記担がんマウスをイソフルラン(富士フィルム和光純薬株式会社)の麻酔下で、その尾静脈から0.2mLの短波赤外蛍光剤5を注入した、その後、担がんマウスの乳がん腫瘍の短波赤外での蛍光イメージングをin vivoで行った。その後、担がんマウスを解剖し、担がんマウスの乳がん腫瘍および臓器(心臓、腎臓、脾臓および肝臓)を取り出した。そして、取り出した乳がん腫瘍および各臓器の短波赤外での蛍光イメージングをex vivoで行った。
ヒト乳がん細胞にKPL-4細胞(川崎医大からの提供、約1.5×107個)を用いる以外は前述の乳がんモデルマウスの作製と同様にして、担がんマウスを作製し、腫瘍のサイズを数ミリまで成長させた。そして、短波赤外蛍光剤6に代えて短波赤外蛍光剤7を用いる以外は同様にして、in vivoおよびex vivoによる乳がんモデルマウスにおける乳がん腫瘍の蛍光イメージングを行った。in vivoでのヌードマウスにおける乳がん腫瘍の明視野画像と、短波赤外蛍光剤7による短波赤外蛍光画像とを表す写真を図14に示す。また、ex vivoでのヌードマウスにおける乳がん腫瘍、心臓、腎臓、脾臓および肝臓の明視野画像と、短波赤外蛍光剤7による短波赤外蛍光画像とを表す写真を図15に示す。
[複合体4、5の調製]
複合体4、5をそれぞれ調製した。複合体4は、化合物19(ICG-C9-マレイミド)が反応性架橋基の残基であるスクシンイミド基を介して抗体(Herceptin)と結合しており、複合体5は、化合物21(ICG-C11-マレイミド)が当該スクシンイミド基を介して当該抗体と結合している。複合体4、5の調製スキームを以下に示す。
[複合体6、7の調製]
複合体6、7をそれぞれ調製した。複合体6は、化合物20(ICG-C9-アルキン)が反応性架橋基の残基であるトリアゾール基を介して抗体(Herceptin)と結合しており、複合体7は、化合物22(ICG-C11-アルキン)が当該トリアゾール基を介して当該抗体と結合している。複合体6、7の調製スキームを以下に示す。
[短波赤外蛍光剤8、9の調製]
複合体4をPBSで希釈して濃度1mg/mLの複合体4の水溶液を得た。当該水溶液を短波赤外蛍光剤8とする。また、複合体5をPBSで希釈して濃度1mg/mLの複合体5の水溶液を得た。当該水溶液を短波赤外蛍光剤9とする。
HER2過剰発現乳がん細胞(KPL-4)を移植したヌードマウスに、0.2mLの短波赤外蛍光剤8(1mg/mLの濃度の複合体4)および0.2mLの短波赤外蛍光剤9(1mg/mLの濃度の複合体5)を尾静脈から注入し、3日後に、担がんマウスの乳がん腫瘍の短波赤外での蛍光イメージングをin vivoで行った。蛍光画像の撮影条件は、励起波長が905nmで蛍光検出波長は1000nm以上であった。励起光の強度は、20mW/cm2で、露光時間は5秒間であった。短波赤外蛍光剤8および短波赤外蛍光剤9のそれぞれにおけるin vivoでのヌードマウスにおける乳がん腫瘍の明視野画像と短波赤外蛍光画像とを表す写真を図16に示す。この結果から、短波赤外蛍光剤8、9によっても乳がん腫瘍が明瞭に検出できることが示された。
[短波赤外蛍光剤10、11の調製]
複合体6をPBSで希釈して濃度1mg/mLの複合体6の水溶液を得た。当該水溶液を短波赤外蛍光剤10とする。また、複合体7をPBSで希釈して濃度1mg/mLの複合体7の水溶液を得た。当該水溶液を短波赤外蛍光剤11とする。
HER2過剰発現乳がん細胞(KPL-4)を移植したヌードマウスに、0.2mLの短波赤外蛍光剤10(1mg/mLの濃度の複合体6)および0.2mLの短波赤外蛍光剤11(1mg/mLの濃度の複合体7)を尾静脈から注入し、3日後に担がんマウスの乳がん腫瘍の短波赤外での蛍光イメージングをin vivoで行った。蛍光画像の撮影条件は、励起波長が905nmで蛍光検出波長は1000nm以上であった。励起光の強度は、20mW/cm2で、露光時間は10秒間(短波赤外蛍光剤10)と30秒間(短波赤外蛍光剤11)であった。短波赤外蛍光剤10および短波赤外蛍光剤11のそれぞれにおけるin vivoでのヌードマウスにおける乳がん腫瘍の明視野画像と短波赤外蛍光画像とを表す写真を図17に示す。この結果から、短波赤外蛍光剤10、11によっても乳がん腫瘍が明瞭に検出できることが示された。
[複合体8、9の調製]
組み換えタンパク質であるアネキシンVの合成は発明者の論文(Setsuko Tsuboi and Takashi Jin, ChemBioChem 18, 2231-2235, 2017)で公表されている方法に従った。1mLのアネキシンVのPBS溶液(1mg/mL)に50μLの化合物14(ICG-C9-NHS)ないしは化合物17(ICG-C11-NHS)のジメチルスルホキシド溶液(1mg/mL)を加え、1時間反応させた。精製はゲル濾過カラムにより行い、複合体8(ICG-C9-アネキシンV)および複合体9(ICG-C11-アネキシンV)を得た。
[短波赤外蛍光剤12、13の調製]
複合体8をPBSで希釈して濃度0.5mg/mLの複合体8の水溶液を得た。当該水溶液を短波赤外蛍光剤12とする。また、複合体9をPBSで希釈して濃度0.5mg/mLの複合体9の水溶液を得た。当該水溶液を短波赤外蛍光剤13とする。
HER2過剰発現乳がん細胞(KPL-4)を移植したヌードマウスに、0.1mLのKadcyla(カドサイラ、2mg/mL)を尾静脈から注入し、3日後に腫瘍部分の短波赤外蛍光剤12(0.5mg/mLの濃度の複合体8)または短波赤外蛍光剤13(0.5mg/mLの濃度の複合体9)を注入した。翌日、担がんマウスの乳がん腫瘍の短波赤外での蛍光イメージングをin vivoで行った。蛍光画像の撮影条件は、励起波長が905nmで蛍光検出波長は1000nm以上であった。励起光の強度は、20mW/cm2で、露光時間は5秒間(短波赤外蛍光剤12)と10秒間(短波赤外蛍光剤13)であった。短波赤外蛍光剤12および短波赤外蛍光剤13のそれぞれについて、カドサイラを注入したヌードマウス(左)とカドサイラを注入していないヌードマウス(右)とにおけるin vivoでの乳がん腫瘍の明視野画像と短波赤外蛍光画像とを表す写真を図18に示す。
HER2過剰発現乳がん細胞(KPL-4)を移植したヌードマウスに、0.1mLのKadcyla(カドサイラ、2mg/mL)を尾静脈から注入し、3日後に腫瘍部分の短波赤外蛍光剤13(0.5mg/mLの濃度の複合体9)を注入した。その後、3日、5日、11日後に、担がんマウスの乳がん腫瘍の短波赤外での蛍光イメージングをin vivoで行った。蛍光画像の撮影条件は、励起波長が905nmで蛍光検出波長は1000nm以上であった。励起光の強度は、20mW/cm2で、露光時間は10秒間であった。短波赤外蛍光剤13におけるカドサイラを注入したヌードマウスのin vivoでの乳がん腫瘍の、特定の時間経過後における明視野画像と短波赤外蛍光画像とを表す写真を図19に示す。
Claims (12)
- 前記反応性架橋基は、N-ヒドロキシスクシンイミドエステル基、アルキニル基、マレイミド基およびアジド基からなる群から選ばれる一以上の有機基を含む、請求項1に記載の化合物。
- 前記nは、3または4である、請求項1または2に記載の化合物。
- 請求項1~4のいずれか一項の化合物における前記反応性架橋基の残基を介して分子識別剤と前記化合物とが結合している複合体。
- 前記分子識別剤は、抗体、または、抗原結合能を有する当該抗体の断片である、請求項5に記載の複合体。
- 請求項1~4のいずれか一項に記載の化合物、および、請求項5または6に記載の複合体、の一方または両方を含有する短波赤外蛍光剤。
- 前記nが3であり、前記Xがスルホン酸基の塩であり、前記第二の工程と前記第三の工程とを一度に行う、請求項8に記載の化合物の製造方法。
- 前記nが4であり、前記Xがスルホン酸基の塩であり、前記第二の工程の後に前記第三の工程を行う、請求項8に記載の化合物の製造方法。
- 前記nが3であり、前記Xが前記反応性架橋基であり、前記第二の工程の後に前記第三の工程を行う、請求項8に記載の化合物の製造方法。
- 前記nが4であり、前記Xが前記反応性架橋基であり、前記第二の工程の前に第三の工程を行う、請求項8に記載の化合物の製造方法。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/282,174 US20240174645A1 (en) | 2021-03-15 | 2022-03-15 | Compound, production method therefor, complex, and short wavelength infrared fluorescent agent |
JP2023507132A JPWO2022196694A1 (ja) | 2021-03-15 | 2022-03-15 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2021041717 | 2021-03-15 | ||
JP2021-041717 | 2021-03-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022196694A1 true WO2022196694A1 (ja) | 2022-09-22 |
Family
ID=83322323
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2022/011674 WO2022196694A1 (ja) | 2021-03-15 | 2022-03-15 | 化合物、その製造方法、複合体および短波赤外蛍光剤 |
Country Status (3)
Country | Link |
---|---|
US (1) | US20240174645A1 (ja) |
JP (1) | JPWO2022196694A1 (ja) |
WO (1) | WO2022196694A1 (ja) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009067690A (ja) * | 2007-09-11 | 2009-04-02 | Fujifilm Corp | メロシアニン色素を含む蛍光造影剤 |
US20140275526A1 (en) * | 2013-03-15 | 2014-09-18 | University Of Massachusetts | Sulfonate compounds |
JP2017503004A (ja) * | 2013-10-31 | 2017-01-26 | ベス・イスラエル・ディーコネス・メディカル・センター,インコーポレイテッド | 近赤外蛍光造影バイオイメージング剤及びその使用方法 |
WO2020041743A1 (en) * | 2018-08-23 | 2020-02-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Water soluble novel cyanine fluorophore with tunable properties between near ir and swir region for in vivo imaging |
WO2020229438A1 (en) * | 2019-05-13 | 2020-11-19 | Bracco Imaging Spa | Modified cyanine dyes and conjugates thereof |
-
2022
- 2022-03-15 WO PCT/JP2022/011674 patent/WO2022196694A1/ja active Application Filing
- 2022-03-15 JP JP2023507132A patent/JPWO2022196694A1/ja active Pending
- 2022-03-15 US US18/282,174 patent/US20240174645A1/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009067690A (ja) * | 2007-09-11 | 2009-04-02 | Fujifilm Corp | メロシアニン色素を含む蛍光造影剤 |
US20140275526A1 (en) * | 2013-03-15 | 2014-09-18 | University Of Massachusetts | Sulfonate compounds |
JP2017503004A (ja) * | 2013-10-31 | 2017-01-26 | ベス・イスラエル・ディーコネス・メディカル・センター,インコーポレイテッド | 近赤外蛍光造影バイオイメージング剤及びその使用方法 |
WO2020041743A1 (en) * | 2018-08-23 | 2020-02-27 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Water soluble novel cyanine fluorophore with tunable properties between near ir and swir region for in vivo imaging |
WO2020229438A1 (en) * | 2019-05-13 | 2020-11-19 | Bracco Imaging Spa | Modified cyanine dyes and conjugates thereof |
Also Published As
Publication number | Publication date |
---|---|
US20240174645A1 (en) | 2024-05-30 |
JPWO2022196694A1 (ja) | 2022-09-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1585791B1 (en) | Hydrophilic, thiol-reactive cyanine dyes and conjugates thereof with biomolecules for fluorescence diagnosis | |
JP7085633B2 (ja) | in vivo画像化のための非凝集性ヘプタメチンシアニンフルオロフォア | |
EP3496762A1 (en) | Near-ir light-cleavable conjugates and conjugate precursors | |
JP7344982B2 (ja) | 化合物及びこれを用いた蛍光標識生体物質 | |
François et al. | A functionalized heterobimetallic 99m Tc/Re complex as a potential dual-modality imaging probe: synthesis, photophysical properties, cytotoxicity and cellular imaging investigations | |
EP1104761B1 (en) | Fluorescent labelling reagents | |
US20230302160A1 (en) | Tumor contrast compound, preparation method therefor and application thereof in tumor diagnostic imaging | |
AU2019325658B2 (en) | Water soluble novel cyanine fluorophore with tunable properties between near IR and SWIR region for in vivo imaging | |
KR101663465B1 (ko) | 생체 분자 표지를 위한 시아닌 염료 및 그 제조방법 | |
US8916137B2 (en) | Monofunctional carbocyanine dyes for in vivo and in vitro imaging | |
US11242359B2 (en) | Biocompatible modular tetrazine platform | |
WO2022196694A1 (ja) | 化合物、その製造方法、複合体および短波赤外蛍光剤 | |
KR101842633B1 (ko) | 카텝신 b에 의해 활성화되는 암의 진단 또는 치료용 조성물, 이를 이용한 암의 근적외선 형광 이미징 및 광선 치료 | |
EP3943919B1 (en) | Fluorescence imaging of lipid droplets in cell and tissue with 3-(benzo[d]thiazol-2-yl)-2h-benzo[g]chromen-2-one derivatives and the corresponding benzo[d]oxazole and 1h-benzo[d]imidazole derivatives | |
CN108250125B (zh) | 一种肿瘤靶向探针化合物及其合成和应用 | |
JP2021520424A (ja) | アザシアニン色素及びその使用 | |
US11964965B2 (en) | Methods of manufacture and synthesis of fluorescent dye compounds and uses thereof | |
CN118159831A (zh) | 细胞和组织内脂滴的荧光成像试剂 | |
FR3064266A1 (fr) | Plateforme tetrazine modulaire biocompatible | |
JP2023039287A (ja) | 化合物、組成物、蛍光色素剤、キット、及び細胞、組織、又は器官の検出方法 | |
CN116947829A (zh) | 一种基于新吲哚菁绿ir820的荧光化合物及其制备和应用 |
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: 22771439 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2023507132 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 18282174 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 22771439 Country of ref document: EP Kind code of ref document: A1 |