JP7413932B2 - Fluorescent labeling agents and fluorescent dyes - Google Patents
Fluorescent labeling agents and fluorescent dyes Download PDFInfo
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- JP7413932B2 JP7413932B2 JP2020099441A JP2020099441A JP7413932B2 JP 7413932 B2 JP7413932 B2 JP 7413932B2 JP 2020099441 A JP2020099441 A JP 2020099441A JP 2020099441 A JP2020099441 A JP 2020099441A JP 7413932 B2 JP7413932 B2 JP 7413932B2
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 125000006606 n-butoxy group Chemical group 0.000 description 1
- 125000006608 n-octyloxy group Chemical group 0.000 description 1
- 125000005186 naphthyloxy group Chemical group C1(=CC=CC2=CC=CC=C12)O* 0.000 description 1
- 125000005484 neopentoxy group Chemical group 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 125000003261 o-tolyl group Chemical group [H]C1=C([H])C(*)=C(C([H])=C1[H])C([H])([H])[H] 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000001037 p-tolyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)C([H])([H])[H] 0.000 description 1
- 125000002255 pentenyl group Chemical group C(=CCCC)* 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 125000000951 phenoxy group Chemical group [H]C1=C([H])C([H])=C(O*)C([H])=C1[H] 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000008105 phosphatidylcholines Chemical class 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 229940067605 phosphatidylethanolamines Drugs 0.000 description 1
- 150000008106 phosphatidylserines Chemical class 0.000 description 1
- 239000001007 phthalocyanine dye Substances 0.000 description 1
- 125000000587 piperidin-1-yl group Chemical group [H]C1([H])N(*)C([H])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000004309 pyranyl group Chemical group O1C(C=CC=C1)* 0.000 description 1
- 125000005412 pyrazyl group Chemical group 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 125000003548 sec-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000005017 substituted alkenyl group Chemical group 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- 125000005346 substituted cycloalkyl group Chemical group 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- HXJUTPCZVOIRIF-UHFFFAOYSA-N sulfolane Chemical compound O=S1(=O)CCCC1 HXJUTPCZVOIRIF-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 229910052714 tellurium Inorganic materials 0.000 description 1
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001973 tert-pentyl group Chemical group [H]C([H])([H])C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 229910052718 tin Inorganic materials 0.000 description 1
- 125000004784 trichloromethoxy group Chemical group ClC(O*)(Cl)Cl 0.000 description 1
- 125000003866 trichloromethyl group Chemical group ClC(Cl)(Cl)* 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910052726 zirconium Inorganic materials 0.000 description 1
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Description
本発明は、蛍光標識剤と、それに用いられる蛍光色素に関する。 The present invention relates to a fluorescent labeling agent and a fluorescent dye used therefor.
バイオイメージングは、生体内分子や細胞、生体組織の動態及び機能を可視化する技術であり、生体内分子・細胞機能の解明や創薬の研究等、生物学、医学の研究領域で幅広く活用されている。中でも蛍光バイオイメージングは、現象の動的な観察、多色観察、高感度観察が可能な手法である。 Bioimaging is a technology that visualizes the dynamics and functions of biological molecules, cells, and biological tissues, and is widely used in biological and medical research fields, such as elucidation of biological molecular and cellular functions and drug discovery research. There is. Among them, fluorescence bioimaging is a method that allows dynamic observation of phenomena, multicolor observation, and high-sensitivity observation.
蛍光バイオイメージングは、標的物質と特異的に結合する、あるいは、標的部位に集積する蛍光色素を用い、その蛍光色素に光を照射した際に色素が発する蛍光を検出することにより、標的を可視化する手法である。 Fluorescent bioimaging uses a fluorescent dye that specifically binds to the target substance or accumulates at the target site, and visualizes the target by detecting the fluorescence emitted by the dye when the fluorescent dye is irradiated with light. It is a method.
蛍光色素の細胞集積性を高める手段として、例えば特許文献１では、蛍光分子にリン脂質構造を導入した蛍光色素が開発されている。これにより、蛍光色素が細胞膜上に良好に固定化され、高い蛍光標識能を示す。 As a means for increasing the cell accumulation of fluorescent dyes, for example, in Patent Document 1, a fluorescent dye in which a phospholipid structure is introduced into a fluorescent molecule has been developed. This allows the fluorescent dye to be well immobilized on the cell membrane and exhibits high fluorescent labeling ability.
特許文献１、特許文献２および特許文献３に記載の細胞膜集積型蛍光色素は吸収・蛍光波長を可視域に持つ。しかしながら、可視域の吸収波長を持つ蛍光色素では、タイムラプスイメージング中に励起光によって細胞損傷を引き起こすため、長期観察のための蛍光色素として適切でない。また可視光は、生体内物質や生体中の水による、光の吸収・散乱が大きいため、生体組織下や臓器内部のイメージングに適用できない等の制約があった。 The cell membrane-integrated fluorescent dyes described in Patent Document 1, Patent Document 2, and Patent Document 3 have absorption and fluorescence wavelengths in the visible range. However, fluorescent dyes with absorption wavelengths in the visible range cause cell damage due to excitation light during time-lapse imaging, and are therefore not suitable as fluorescent dyes for long-term observation. In addition, visible light has limitations such as its inability to be applied to imaging underneath living tissues and inside organs because of the large amount of light absorption and scattering caused by substances in living bodies and water in living bodies.
上述の課題は、近赤外領域の光を用いることで解決できる。近赤外蛍光色素は、シアニンを基本骨格としたものが広く利用されている。しかし、シアニン骨格は光照射により生じる活性酸素により分解しやすいため、蛍光観察中に色素が退色してしまうという課題がある。 The above-mentioned problems can be solved by using light in the near-infrared region. Near-infrared fluorescent dyes with cyanine as their basic skeleton are widely used. However, since the cyanine skeleton is easily decomposed by active oxygen generated by light irradiation, there is a problem that the dye fades during fluorescence observation.
本発明が解決しようとする課題は、リン脂質への集積性が高く、イン・ビトロ（ｉｎ ｖｉｔｒｏ）およびイン・ビボ（ｉｎ ｖｉｖｏ）イメージングに用いる蛍光標識剤に適した蛍光強度を有する蛍光色素を提供することである。 The problem to be solved by the present invention is to develop a fluorescent dye that has a high ability to accumulate in phospholipids and has a fluorescence intensity suitable for a fluorescent labeling agent used in in vitro and in vivo imaging. It is to provide.
本発明者らは、前記諸問題を解決するために鋭意検討を重ねた結果、上記課題を解決するための優れた蛍光色素を見出し、本発明をなしたものである。 The present inventors have made intensive studies to solve the above problems, and as a result, have discovered an excellent fluorescent dye to solve the above problems, and have accomplished the present invention.
すなわち、本発明は、一般式（１）で表される蛍光色素を含む蛍光標識剤に関する。
一般式（１）
（Ｒ１～Ｒ１６は、それぞれ独立に、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換のアルケニル基、置換もしくは無置換の複素環基、－ＡＢ、－ＳＯ３Ｍ２、－ＣＯＯＭ３、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１～Ｒ１６は、隣接する置換基同士が互いに連結して、環を形成してもよい。Ａは、１６族元素を表す。Ｂは、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換の複素環基を表す。Ｍ２、Ｍ３はそれぞれ独立に１価のカチオンを表す。
Ｒ１７は、それぞれ独立に、水酸基、ハロゲン元素、置換または無置換のアルコキシ基、置換または無置換のアリールオキシ基、－ＯＰ（＝Ｏ）Ｒ１８Ｒ１９、－ＯＣ（＝Ｏ）Ｒ２０、－ＯＳ（＝Ｏ）２Ｒ２１、－ＯＳｉＲ２２Ｒ２３Ｒ２４、－Z－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１８及びＲ１９は、それぞれ独立に、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｒ２０は、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２１は、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２２～Ｒ２４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。
Ｍ1は、２価～５価の金属原子を表し、Ｍ1が２価の金属原子である場合はｎは０であり、Ｍ1が３価の金属原子である場合はｎは１であり、Ｍ1が４価の金属原子である場合はｎは２である。
ただし、Ｒ１～Ｒ１７の少なくとも１つは－Z－Ｌ１－Ｃ（＝Ｏ）－Ｘである。
Ｚは直接結合、－Ｏ－、－ＯＰ（＝Ｏ）Ｌ２－、－ＯＣ（＝Ｏ）－、－ＯＳ（＝Ｏ）２－、－ＯＳｉＬ３Ｌ４－、－Ｃ（＝Ｏ）－、－Ｃ（＝Ｏ）ＮＨ－を表す。Ｌ１は、直接結合、置換もしくは無置換のアルキレン基、置換もしくは無置換のアリーレン基、置換もしくは無置換の複素環基を表す。Ｘはリン脂質の残基を表す。Ｌ２は、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｌ３、Ｌ４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。）
That is, the present invention relates to a fluorescent labeling agent containing a fluorescent dye represented by general formula (1).
General formula (1)
(R 1 to R 16 each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkenyl group, Represents an unsubstituted heterocyclic group, -AB, -SO 3 M 2 , -COOM 3 , -Z-L 1 -C(=O)-X. R 1 to R 16 indicate that adjacent substituents are mutually They may be linked to form a ring.A represents a group 16 element.B is a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group. represents a substituted or unsubstituted heterocyclic group. M 2 and M 3 each independently represents a monovalent cation.
R 17 is each independently a hydroxyl group, a halogen element, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)R 18 R 19 , -OC(=O)R 20 , -OS(=O) 2 R 21 , -OSiR 22 R 23 R 24 , -Z-L 1 -C(=O)-X. R 18 and R 19 are each independently a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted Or represents an unsubstituted heterocyclic group. R 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 22 to R 24 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group.
M 1 represents a divalent to pentavalent metal atom; when M 1 is a divalent metal atom, n is 0; when M 1 is a trivalent metal atom, n is 1; , n is 2 when M 1 is a tetravalent metal atom.
However, at least one of R 1 to R 17 is -Z-L 1 -C(=O)-X.
Z is a direct bond, -O-, -OP(=O)L 2 -, -OC(=O)-, -OS(=O) 2 -, -OSiL 3 L 4 -, -C(=O)- , -C(=O)NH-. L 1 represents a direct bond, a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heterocyclic group. X represents a phospholipid residue. L2 is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heterocyclic group represents. L 3 and L 4 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. )
また、本発明は、リン脂質集積型蛍光標識剤である、前記蛍光標識剤に関する。 The present invention also relates to the fluorescent labeling agent, which is a phospholipid-accumulating fluorescent labeling agent.
また、本発明は、一般式（１）のＲ１７が－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘである、前記蛍光標識剤に関する。 The present invention also relates to the fluorescent labeling agent, wherein R 17 in general formula (1) is -Z-L 1 -C(=O)-X.
また、本発明は、一般式（２）で表される化合物に関する。
一般式（２）
（Ｒ１～Ｒ１６は、それぞれ独立に、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換のアルケニル基、置換もしくは無置換の複素環基、－ＡＢ、－ＳＯ３Ｍ２、－ＣＯＯＭ３、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１～Ｒ１６は、隣接する置換基同士が互いに連結して、環を形成してもよい。Ａは、１６族元素を表す。Ｂは、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換の複素環基を表す。Ｍ２、Ｍ３はそれぞれ独立に１価のカチオンを表す。
Ｒ１７は、それぞれ独立に、水酸基、ハロゲン元素、置換または無置換のアルコキシ基、置換または無置換のアリールオキシ基、－ＯＰ（＝Ｏ）Ｒ１８Ｒ１９、－ＯＣ（＝Ｏ）Ｒ２０、－ＯＳ（＝Ｏ）２Ｒ２１、－ＯＳｉＲ２２Ｒ２３Ｒ２４、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１８及びＲ１９は、それぞれ独立に、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｒ２０は、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２１は、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２２～Ｒ２４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。
Ｍ1は、２価～５価の金属原子を表し、Ｍ1が２価の金属原子である場合はｎは０であり、Ｍ1が３価の金属原子である場合はｎは１であり、Ｍ1が４価の金属原子である場合はｎは２である。
ただし、Ｒ１～Ｒ１７の少なくとも１つは－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘである。
Ｚは直接結合、－Ｏ－、－ＯＰ（＝Ｏ）Ｌ２－、－ＯＣ（＝Ｏ）－、－ＯＳ（＝Ｏ）２－、－ＯＳｉＬ３Ｌ４－、－Ｃ（＝Ｏ）－、－Ｃ（＝Ｏ）ＮＨ－を表す。Ｌ１は、直接結合、置換もしくは無置換のアルキレン基、置換もしくは無置換のアリーレン基、置換もしくは無置換の複素環基を表す。Ｘはリン脂質の残基を表す。Ｌ２は、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｌ３、Ｌ４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。）
The present invention also relates to a compound represented by general formula (2).
General formula (2)
(R 1 to R 16 each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkenyl group, Represents an unsubstituted heterocyclic group, -AB, -SO 3 M 2 , -COOM 3 , -Z-L 1 -C(=O)-X. R 1 to R 16 indicate that adjacent substituents are mutually They may be linked to form a ring.A represents a group 16 element.B is a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group. represents a substituted or unsubstituted heterocyclic group. M 2 and M 3 each independently represent a monovalent cation.
R 17 is each independently a hydroxyl group, a halogen element, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)R 18 R 19 , -OC(=O)R 20 , -OS(=O) 2 R 21 , -OSiR 22 R 23 R 24 , -Z-L 1 -C(=O)-X. R 18 and R 19 are each independently a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted Or represents an unsubstituted heterocyclic group. R 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 22 to R 24 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group.
M 1 represents a divalent to pentavalent metal atom; when M 1 is a divalent metal atom, n is 0; when M 1 is a trivalent metal atom, n is 1; , n is 2 when M 1 is a tetravalent metal atom.
However, at least one of R 1 to R 17 is -ZL 1 -C(=O)-X.
Z is a direct bond, -O-, -OP(=O)L 2 -, -OC(=O)-, -OS(=O) 2 -, -OSiL 3 L 4 -, -C(=O)- , -C(=O)NH-. L 1 represents a direct bond, a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heterocyclic group. X represents a phospholipid residue. L2 is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heterocyclic group represents. L 3 and L 4 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. )
本発明によって、リン脂質への集積性が高く、イン・ビトロ（ｉｎ ｖｉｔｒｏ）およびイン・ビボ（ｉｎ ｖｉｖｏ）イメージングに用いる蛍光標識剤に適した蛍光強度を有する蛍光色素を提供することが可能となった。 According to the present invention, it is possible to provide a fluorescent dye that has a high ability to accumulate in phospholipids and has a fluorescence intensity suitable for a fluorescent labeling agent used for in vitro and in vivo imaging. became.
以下、本発明を詳細に説明する。
本発明の蛍光標識剤は、一般式（１）で表される化合物を含む。一般式（１）で表され
る化合物は、フタロシアニン色素骨格を有する蛍光色素である。
一般式（１）
The present invention will be explained in detail below.
The fluorescent labeling agent of the present invention includes a compound represented by general formula (1). The compound represented by general formula (1) is a fluorescent dye having a phthalocyanine dye skeleton.
General formula (1)
アルキル基としては、直鎖状または分岐鎖状のアルキル基が挙げられる。具体例としては、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基、ドデシル基、オクタデシル基、イソプロピル基、イソブチル基、イソペンチル基、２－エチルヘキシル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ｓｅｃ－ペンチル基、ｔｅｒｔ－ペンチル基、ｔｅｒｔ－オクチル基、ネオペンチル基等を挙げることができる。アルキル基の炭素数は１～３０の範囲内であることが好ましい。 Examples of the alkyl group include linear or branched alkyl groups. Specific examples include methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, dodecyl group, octadecyl group, isopropyl group, isobutyl group, isopentyl group, Examples include 2-ethylhexyl group, sec-butyl group, tert-butyl group, sec-pentyl group, tert-pentyl group, tert-octyl group, and neopentyl group. The number of carbon atoms in the alkyl group is preferably within the range of 1 to 30.
上記アルキル基における置換基としては、フッ素、塩素、臭素等のハロゲン原子、水酸基、アミノ基、ニトロ基、ホルミル基、シアノ基、カルボキシル基等の他、上述したアルキル基、後述するアリール基、シクロアルキル基、複素環基が挙げられる。また、構造の一部が、アミド結合（－ＮＨＣＯ－）やエステル結合（－ＣＯＯ－）、エーテル結合（－Ｏ－）、ウレア結合（－ＮＨＣＯＮＨ－）、ウレタン結合（－ＮＨＣＯＯ－）で置換されている場合、その置換部分も「置換基」として含めるものとする。 Substituents for the above alkyl groups include halogen atoms such as fluorine, chlorine, and bromine, hydroxyl groups, amino groups, nitro groups, formyl groups, cyano groups, carboxyl groups, etc., as well as the alkyl groups mentioned above, aryl groups mentioned below, cyclo Examples include alkyl groups and heterocyclic groups. In addition, part of the structure is substituted with an amide bond (-NHCO-), an ester bond (-COO-), an ether bond (-O-), a urea bond (-NHCONH-), or a urethane bond (-NHCOO-). If so, the substituted moiety shall also be included as a "substituent."
したがって、置換アルキル基としては、上記の置換基で置換されたアルキル基を意味する。一つ又は二つ以上の置換基で置換されたものであっても良い。例えば、ハロゲン原子で置換されたアルキル基の具体例としては、トリフルオロメチル基、２，２，２－トリフルオロエチル基、－（ＣＦ２）４ＣＦ３、－（ＣＦ２）５ＣＦ３、－（ＣＦ２）６ＣＦ３、－（ＣＦ２）７ＣＦ３、－（ＣＦ２）８ＣＦ３、トリクロロメチル基２，２－ジブロモエチル基等を挙げることができる。 Therefore, the substituted alkyl group means an alkyl group substituted with the above-mentioned substituents. It may be substituted with one or more substituents. For example, specific examples of alkyl groups substituted with halogen atoms include trifluoromethyl group, 2,2,2-trifluoroethyl group, -(CF 2 ) 4 CF 3 , -(CF 2 ) 5 CF 3 , -(CF 2 ) 6 CF 3 , -(CF 2 ) 7 CF 3 , -(CF 2 ) 8 CF 3 , trichloromethyl group, 2,2-dibromoethyl group, and the like.
また、アミド結合で置換されたアルキル基の具体例としては、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ（－ＣＨ３）－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ（ＣＨ２－ＣＨ３）－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ３、－(ＣＨ２)５－ＮＨＣＯ－（ＣＨ２）１１－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－Ｃ(－ＮＨＣＯ－ＣＨ２－ＣＨ３) ３等を挙げることができる。アミド結合で置換されたアルキル基の炭素数は、２～３０の範囲内であることが好ましい。 Specific examples of alkyl groups substituted with amide bonds include -CH 2 -CH 2 -CH 2 -NHCO-CH 2 -CH 3 , -CH 2 -CH(-CH 3 )-CH 2 -NHCO- CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -NHCO-CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -CH 2 -NHCO -CH 2 -CH (CH 2 -CH 3 ) -CH 2 -CH 2 -CH 2 -CH 3 , -(CH 2 ) 5 -NHCO-(CH 2 ) 11 -CH 3 , -CH 2 -CH 2 -CH 2 -C(-NHCO-CH 2 -CH 3 ) 3 etc. can be mentioned. The number of carbon atoms in the alkyl group substituted with an amide bond is preferably within the range of 2 to 30.
また、エステル結合で置換されたアルキル基の具体例としては、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＯＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ（－ＣＨ３）－ＣＨ２－ＣＯＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＯＣＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ２－ＣＯＯ－ＣＨ２－ＣＨ（ＣＨ２－ＣＨ３）－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ３、－(ＣＨ２)５－ＣＯＯ－（ＣＨ２）１１－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ－(ＣＯＯ－ＣＨ２－ＣＨ３) ２等を挙げることができる。エステル結合で置換されたアルキル基の炭素数は、２～３０の範囲内であることが好ましい。 Specific examples of alkyl groups substituted with ester bonds include -CH 2 -CH 2 -CH 2 -COO-CH 2 -CH 3 , -CH 2 -CH(-CH 3 )-CH 2 -COO- CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -OCO-CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -CH 2 -COO-CH 2 -CH (CH 2 -CH 3 ) -CH 2 -CH 2 -CH 2 -CH 3 , -(CH 2 ) 5 -COO-(CH 2 ) 11 -CH 3 , -CH 2 -CH 2 -CH 2 -CH-(COO-CH 2 -CH 3 ) 2 etc. can be mentioned. The number of carbon atoms in the alkyl group substituted with an ester bond is preferably within the range of 2 to 30.
また、エーテル結合で置換されたアルキル基の具体例としては、－ＣＨ２－Ｏ－ＣＨ３、－ＣＨ２－ＣＨ２－Ｏ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－Ｏ－ＣＨ２－ＣＨ３、－（ＣＨ２－ＣＨ２－Ｏ）ｎ－ＣＨ３（ここでｎは１から８の整数である）、－（ＣＨ２－ＣＨ２－ＣＨ２－Ｏ）ｍ－ＣＨ３（ここでｍは１から５の整数である）、－ＣＨ２－ＣＨ（ＣＨ３）－Ｏ－ＣＨ２－ＣＨ３－、－ＣＨ２－ＣＨ－（ＯＣＨ３）２等を挙げることができるが、これらに限定されるものではない。エーテル結合で置換されたアルキル基の炭素数は、２～３０の範囲内であることが好ましい。 Specific examples of alkyl groups substituted with ether bonds include -CH 2 -O-CH 3 , -CH 2 -CH 2 -O-CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 - O-CH 2 -CH 3 , -(CH 2 -CH 2 -O) n -CH 3 (where n is an integer from 1 to 8), -(CH 2 -CH 2 -CH 2 -O) m -CH 3 (where m is an integer from 1 to 5), -CH 2 -CH(CH 3 )-O-CH 2 -CH 3 -, -CH 2 -CH-(OCH 3 ) 2 , etc. However, it is not limited to these. The number of carbon atoms in the alkyl group substituted with an ether bond is preferably within the range of 2 to 30.
また、ウレア結合（－ＮＨＣＯＮＨ－）で置換されたアルキル基の具体例としては、－ＣＨ２－ＮＨＣＯＮＨ－ＣＨ３、－ＣＨ２－ＣＨ２－ＮＨＣＯＮＨ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯＮＨ－ＣＨ２－ＣＨ３、－（ＣＨ２－ＣＨ２－ＮＨＣＯＮＨ）ｎ－ＣＨ３（ここでｎは１から８の整数である）、－（ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯＮＨ）ｍ－ＣＨ３（ここでｍは１から５の整数である）、－ＣＨ２－ＣＨ（ＣＨ３）－ＮＨＣＯＮＨ－ＣＨ２－ＣＨ３－、－ＣＨ２－ＣＨ－（ＮＨＣＯＮＨＣＨ３）２等を挙げることができるが、これらに限定されるものではない。エーテル結合で置換されたアルキル基の炭素数は、２～３０の範囲内であることが好ましい。 Further, specific examples of alkyl groups substituted with a urea bond (-NHCONH-) include -CH 2 -NHCONH-CH 3 , -CH 2 -CH 2 -NHCONH-CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -NHCONH-CH 2 -CH 3 , -(CH 2 -CH 2 -NHCONH) n -CH 3 (where n is an integer from 1 to 8), -(CH 2 -CH 2 -CH 2 -NHCONH) m -CH 3 (where m is an integer from 1 to 5), -CH 2 -CH(CH 3 )-NHCONH-CH 2 -CH 3 -, -CH 2 -CH-(NHCONHCH 3 ) 2 , etc., but are not limited to these. The number of carbon atoms in the alkyl group substituted with an ether bond is preferably within the range of 2 to 30.
また、ウレタン結合で置換されたアルキル基の具体例としては、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ（－ＣＨ３）－ＣＨ２－ＮＨＣＯＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯＯ－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯＯ－ＣＨ２－ＣＨ（ＣＨ２－ＣＨ３）－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ３、－(ＣＨ２)５－ＮＨＣＯＯ－（ＣＨ２）１１－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ－(ＮＨＣＯＯ－ＣＨ２－ＣＨ３) ２等を挙げることができる。エステル結合で置換されたアルキル基の炭素数は、２～３０の範囲内であることが好ましい。 Specific examples of alkyl groups substituted with urethane bonds include -CH 2 -CH 2 -CH 2 -NHCOO-CH 2 -CH 3 , -CH 2 -CH(-CH 3 )-CH 2 -NHCOO- CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -NHCOO-CH 2 -CH 3 , -CH 2 -CH 2 -CH 2 -CH 2 -NHCOO -CH 2 -CH (CH 2 -CH 3 ) -CH 2 -CH 2 -CH 2 -CH 3 , -(CH 2 ) 5 -NHCOO-(CH 2 ) 11 -CH 3 , -CH 2 -CH 2 -CH 2 -CH-(NHCOO-CH 2 -CH 3 ) 2 etc. can be mentioned. The number of carbon atoms in the alkyl group substituted with an ester bond is preferably within the range of 2 to 30.
また、アミド結合（－ＮＨＣＯ－）、エステル結合（－ＣＯＯ－）、およびエーテル結合（－Ｏ－）、ウレア結合（－ＮＨＣＯＮＨ－）、ウレタン結合（－ＮＨＣＯＯ－）のうち２種以上の置換基で置換されたアルキル基の具体例としては、－ＣＨ２－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ２－Ｏ－ＣＨ２－ＣＨ（ＣＨ２－ＣＨ３）－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ３、－ＣＨ２－ＣＨ２－ＣＯＯ－ＣＨ２－ＣＨ２－Ｏ－ＣＨ２－ＣＨ２－ＮＨＣＯＯ－ＣＨ２－ＣＨ（ＣＨ２－ＣＨ３）－ＣＨ２－ＣＨ２－ＣＨ２－ＣＨ３を挙げることができる。アミド結合（－ＮＨＣＯ－）、エステル結合（－ＣＯＯ－）、エーテル結合（－Ｏ－）、ウレア結合（－ＮＨＣＯＮＨ－）、およびウレタン結合（－ＮＨＣＯＯ－）のうち２種以上の置換基で置換されたアルキル基の炭素数は、３～３０の範囲内であることが好ましい。 In addition, two or more substituents among amide bond (-NHCO-), ester bond (-COO-), ether bond (-O-), urea bond (-NHCONH-), and urethane bond (-NHCOO-) Specific examples of alkyl groups substituted with -CH 2 -CH 2 -NHCO-CH 2 -CH 2 -O-CH 2 -CH(CH 2 -CH 3 )-CH 2 -CH 2 -CH 2 - CH 3 , -CH 2 -CH 2 -COO-CH 2 -CH 2 -O-CH 2 -CH 2 -NHCOO-CH 2 -CH (CH 2 -CH 3 ) -CH 2 -CH 2 -CH 2 -CH 3 can be mentioned. Substituted with two or more substituents among amide bond (-NHCO-), ester bond (-COO-), ether bond (-O-), urea bond (-NHCONH-), and urethane bond (-NHCOO-) The number of carbon atoms in the alkyl group is preferably within the range of 3 to 30.
シクロアルキル基としては、シクロペンチル基、シクロへキシル基、２，５－ジメチルシクロペンチル基、４－ｔｅｒｔ－プチルシクロヘキシル基等が挙げられる。また、シクロアルキル基の炭素数は５～１２の範囲内であることが好ましい。置換シクロアルキル基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。 Examples of the cycloalkyl group include a cyclopentyl group, a cyclohexyl group, a 2,5-dimethylcyclopentyl group, and a 4-tert-butylcyclohexyl group. Further, the number of carbon atoms in the cycloalkyl group is preferably within the range of 5 to 12. Examples of the substituent for the substituted cycloalkyl group include the same substituents as those for the alkyl group described above.
アルケニル基としては、直鎖状または分岐鎖状のアルケニル基が挙げられる。アルケニル基はその構造中に一つの二重結合を一般的に指すが、本明細書においては複数の二重結合を有するものもアルケニル基に含めるものとする。具体例としては、ビニル基、１－プロペニル基、アリル基、２－ブテニル基、３－ブテニル基、イソプロペニル基、イソブテニル基、１－ペンテニル基、２－ペンテニル基、３－ペンテニル基、４－ペンテニル基、１－ヘキセニル基、２－ヘキセニル基、３－ヘキセニル基、４－ヘキセニル基、１，３－ブタジエニル基等を挙げることができる。アルケニル基の炭素数は２～１８の範囲内であることが好ましい。置換アルケニル基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。 Examples of the alkenyl group include linear or branched alkenyl groups. An alkenyl group generally refers to one double bond in its structure, but in this specification, alkenyl groups also include those having multiple double bonds. Specific examples include vinyl group, 1-propenyl group, allyl group, 2-butenyl group, 3-butenyl group, isopropenyl group, isobutenyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4- Examples include pentenyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, and 1,3-butadienyl group. The number of carbon atoms in the alkenyl group is preferably within the range of 2 to 18. Examples of the substituent for the substituted alkenyl group include the same substituents as those for the alkyl group described above.
アリール基としては、単環または縮合多環のアリール基が挙げられる。例えば、フェニル基、１－ナフチル基、２－ナフチル基、ｐ－ビフェニル基、ｍ－ビフェニル基、２－アントリル基、９－アントリル基、２－フェナントリル基、３－フェナントリル基、９－フェナントリル基、２－フルオレニル基、３－フルオレニル基、９－フルオレニル基、１－ピレニル基、２－ピレニル基、３－ペリレニル基、ｏ－トリル基、ｍ－トリル基、ｐ－トリル基、４－メチルビフェニル基、ターフェニル基、４－メチル－１－ナフチル基、４－ｔｅｒｔ－ブチル－１－ナフチル基、４－ナフチル－１－ナフチル基、６－フェニル－２－ナフチル基、１０－フェニル－９－アントリル基、スピロフルオレニル基、２－ベンゾシクロブテニル基等が挙げられる。アリール基の炭素数は６～１８の範囲内であることが好ましい。
置換アリール基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。
Examples of the aryl group include monocyclic or condensed polycyclic aryl groups. For example, phenyl group, 1-naphthyl group, 2-naphthyl group, p-biphenyl group, m-biphenyl group, 2-anthryl group, 9-anthryl group, 2-phenanthryl group, 3-phenanthryl group, 9-phenanthryl group, 2-fluorenyl group, 3-fluorenyl group, 9-fluorenyl group, 1-pyrenyl group, 2-pyrenyl group, 3-perylenyl group, o-tolyl group, m-tolyl group, p-tolyl group, 4-methylbiphenyl group , terphenyl group, 4-methyl-1-naphthyl group, 4-tert-butyl-1-naphthyl group, 4-naphthyl-1-naphthyl group, 6-phenyl-2-naphthyl group, 10-phenyl-9-anthryl group group, spirofluorenyl group, 2-benzocyclobutenyl group, and the like. The number of carbon atoms in the aryl group is preferably within the range of 6 to 18.
Examples of the substituent for the substituted aryl group include the same substituents as those for the alkyl group described above.
複素環基としては、脂肪族複素環基や芳香族複素環基が挙げられる。具体例としては、ピリジル基、ピラジル基、ピペリジノ基、ピラニル基、モルホリノ基、アクリジニル基等が挙げられる。また、下記構造式で表される基も挙げられる。複素環基の炭素数は、４～１２であることが好ましい。環員数は、５～１３であることが好ましい。 Examples of the heterocyclic group include an aliphatic heterocyclic group and an aromatic heterocyclic group. Specific examples include pyridyl group, pyrazyl group, piperidino group, pyranyl group, morpholino group, acridinyl group, and the like. Also included are groups represented by the following structural formulas. The number of carbon atoms in the heterocyclic group is preferably 4 to 12. The number of ring members is preferably 5 to 13.
置換複素環基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。例えば、複素環基３－メチルピリジル基、Ｎ－メチルピペリジル基、Ｎ－メチルピローリル基等が挙げられる。 Examples of the substituent of the substituted heterocyclic group include the same substituents as those of the alkyl group described above. Examples include heterocyclic groups such as 3-methylpyridyl group, N-methylpiperidyl group, and N-methylpyrrolyl group.
アルコキシ基としては、直鎖状または分岐鎖状のアルコキシル基が挙げられる。具体例としては、メトキシ基、エトキシ基、プロポキシ基、イソプロポキシ基、ｎ－ブトキシ基、イソブトキシ基、ｔｅｒｔ－ブトキシ基、ネオペンチルオキシ基、２，３－ジメチル－３－ペンチルオキシ基、ｎ－へキシルオキシ基、ｎ－オクチルオキシ基、ステアリルオキシ基、２－エチルへキシルオキシ基等が挙げられる。アルコキシル基の炭素数は１～６の範囲内であることが好ましい。置換アルコキシル基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。 Examples of the alkoxy group include linear or branched alkoxyl groups. Specific examples include methoxy group, ethoxy group, propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, tert-butoxy group, neopentyloxy group, 2,3-dimethyl-3-pentyloxy group, n- Examples include hexyloxy group, n-octyloxy group, stearyloxy group, and 2-ethylhexyloxy group. The number of carbon atoms in the alkoxyl group is preferably within the range of 1 to 6. Examples of the substituent for the substituted alkoxyl group include the same substituents as those for the alkyl group described above.
置換アルコキシ基の置換基としては、上述したアルキル基における置換基と同じ置換基が挙げられる。具体例としては、トリクロロメトキシ基、トリフルオロメトキシ基、２，２，２－トリフルオロエトキシ基、２，２，３，３－テトラフルオロプロポキシ基、２，２－ビス（トリフルオロメチル）プロポキシ基、２－エトキシエトキシ基、２－ブトキシエトキシ基、２－ニトロプロポキシ基、ベンジルオキシ基等が挙げられる。 Examples of the substituent for the substituted alkoxy group include the same substituents as those for the alkyl group described above. Specific examples include trichloromethoxy group, trifluoromethoxy group, 2,2,2-trifluoroethoxy group, 2,2,3,3-tetrafluoropropoxy group, and 2,2-bis(trifluoromethyl)propoxy group. , 2-ethoxyethoxy group, 2-butoxyethoxy group, 2-nitropropoxy group, benzyloxy group and the like.
アリールオキシ基としては、単環または縮合多環のアリールオキシ基が挙げられる。具体例としては、フェノキシ基、ｐ－メチルフェノキシ基、ナフチルオキシ基、アンスリルオキシ基等が挙げられる。アリールオキシ基は、単環のアリールオキシ基が好ましい。また、炭素数６～１２のアリールオキシ基が好ましい。 Examples of the aryloxy group include monocyclic or condensed polycyclic aryloxy groups. Specific examples include phenoxy group, p-methylphenoxy group, naphthyloxy group, and anthryloxy group. The aryloxy group is preferably a monocyclic aryloxy group. Furthermore, an aryloxy group having 6 to 12 carbon atoms is preferred.
置換アリールオキシ基の置換基としては、上述したアリール基における置換基と同じ置換基が挙げられる。例えば、ｐ－ニトロフェノキシ基、ｐ－メトキシフェノキシ基、２，４－ジクロロフェノキシ基、ペンタフルオロフェノキシ基、２－メチル－４－クロロフェノキシ基等が挙げられる。 Examples of the substituent for the substituted aryloxy group include the same substituents as those for the aryl group described above. Examples include p-nitrophenoxy group, p-methoxyphenoxy group, 2,4-dichlorophenoxy group, pentafluorophenoxy group, and 2-methyl-4-chlorophenoxy group.
アルキレン基としては、アルキル基から一つの水素原子を除いた二価の基が挙げられる。
置換もしくは無置換のアルキレン基の具体例としては、－ＣＨ２－ＣＨ２－、－ＣＨ２－ＣＨ２－ＣＨ２－ＮＨＣＯ－ＣＨ２－ＣＨ２－、－ＣＨ２－ＣＨ２－ＣＨ２－ＯＣＯ－ＣＨ２－ＣＨ２－、－ＣＨ２－ＣＨ２－ＣＨ２－Ｏ－ＣＨ２－ＣＨ２－等が挙げられる。
Examples of the alkylene group include divalent groups obtained by removing one hydrogen atom from an alkyl group.
Specific examples of substituted or unsubstituted alkylene groups include -CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -NHCO-CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 - Examples include OCO-CH 2 -CH 2 -, -CH 2 -CH 2 -CH 2 -O-CH 2 -CH 2 -, and the like.
アリーレン基としては、アリール基から一つの水素原子を除いた二価の基が挙げられる。置換もしくは無置換のアリーレン基の具体例としては、下記構造式で表される基が挙げられる。 Examples of the arylene group include divalent groups obtained by removing one hydrogen atom from an aryl group. Specific examples of substituted or unsubstituted arylene groups include groups represented by the following structural formula.
第１６族元素としては、酸素、硫黄、セレン、テルル等が挙げられる。この内、酸素、硫黄、セレンが好ましく、合成の容易さや安定性の点で酸素、硫黄がより好ましい。 Examples of Group 16 elements include oxygen, sulfur, selenium, tellurium, and the like. Among these, oxygen, sulfur, and selenium are preferred, and oxygen and sulfur are more preferred in terms of ease of synthesis and stability.
Ｍ１の２価の金属原子としては、Ｍg、Ｃu、Ｚn等が挙げられる。３価の金属原子としては、Ａｌ、Ｇａ、Ｉｎ等が挙げられる。４価の金属原子としては、Ｓｉ、Ｍｎ、Ｓｎ、Ｃｒ、Ｚｒ等が挙げられる。蛍光強度の観点からは、Ｍｇ、Ａｌ、Ｓｉ、Ｚｎが好ましく、耐光性の観点からは、Ａｌ、Ｓｉがより好ましい。 Examples of the divalent metal atom of M1 include Mg, Cu, and Zn. Examples of trivalent metal atoms include Al, Ga, and In. Examples of the tetravalent metal atoms include Si, Mn, Sn, Cr, and Zr. From the viewpoint of fluorescence intensity, Mg, Al, Si, and Zn are preferable, and from the viewpoint of light resistance, Al and Si are more preferable.
リン脂質としては、特に限定されないが、ホスファチジルエタノールアミン類、ホスファチジルコリン類、ホスファチジルセリン類、スフィンゴミエリン類、セラミドホスホリルエタノールアミン類、１，２－ジミリストイル－１，２－デオキシホスファチジルコリン類、又はプラスマロゲン類などを採用できる。
これらのリン脂質における脂肪酸残基は特に限定されない。例えば、炭素数１２～２０個程度の飽和又は不飽和の脂肪酸残基を１個又は２個有するリン脂質を用いることができ、具体的には、カプリン酸、ラウリン酸、ミリスチン酸、ペンタデシル酸、パルミチン酸、パルミトレイン酸、ステアリン酸、オレイン酸、リノール酸等の脂肪酸由来のアシル基を１個又は２個以上有するリン脂質を用いることができる。
Examples of phospholipids include, but are not limited to, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, sphingomyelins, ceramidephosphorylethanolamines, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholines, or plasmalogens. etc. can be adopted.
The fatty acid residues in these phospholipids are not particularly limited. For example, phospholipids having one or two saturated or unsaturated fatty acid residues having about 12 to 20 carbon atoms can be used, and specifically, capric acid, lauric acid, myristic acid, pentadecyl acid, Phospholipids having one or more acyl groups derived from fatty acids such as palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid can be used.
リン脂質の残基とは、リン脂質の親水性末端にあるアミノ基または第4級アンモニウム基から水素原子または置換基を除いた残基を表す。 A phospholipid residue refers to a residue obtained by removing a hydrogen atom or a substituent from an amino group or a quaternary ammonium group at the hydrophilic end of a phospholipid.
一般式（１）において、Ｒ１～Ｒ１７の少なくとも１つは－Z－Ｌ１－Ｃ（＝Ｏ）－Ｘであり、Ｒ１７が－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘであることが好ましい。 In general formula (1), at least one of R 1 to R 17 is -Z-L 1 -C(=O)-X, and R 17 is -Z-L 1 -C(=O)-X It is preferable that there be.
本発明の蛍光標識剤は、生化学研究から医療診断までの幅広い分野におけるバイオイメージング用蛍光標識として応用可能であり、例えば、遺伝子診断分野、免疫診断分野、医療開発分野、再生医療分野、環境試験分野、バイオテクノロジー分野、蛍光検査等における蛍光標識等として使用することができる。 The fluorescent labeling agent of the present invention can be applied as a fluorescent label for bioimaging in a wide range of fields from biochemical research to medical diagnosis, such as genetic diagnosis, immunodiagnosis, medical development, regenerative medicine, and environmental testing. It can be used as a fluorescent label in the fields of biotechnology, fluorescence testing, etc.
なかでも、本発明の蛍光標識剤は、リン脂質集積型蛍光標識剤として好適に使用できる。リン脂質集積型蛍光標識剤は、細胞膜の染色、エクソソームの追跡、ドラッグデリバリーシステム（ＤＤＳ）のためのリポソームイメージングなどにおける蛍光標識剤として好適に使用できる。 Among these, the fluorescent labeling agent of the present invention can be suitably used as a phospholipid-accumulating fluorescent labeling agent. The phospholipid-accumulating fluorescent labeling agent can be suitably used as a fluorescent labeling agent in cell membrane staining, exosome tracking, liposome imaging for drug delivery systems (DDS), and the like.
本発明の蛍光色素の濃度は特に限定されないが、例えば、細胞を扱う場合、細胞の機能障害や増殖阻害等への影響から、濃度は低い方が好ましく、本発明の蛍光色素の濃度は１００μＭ以下であることが好ましい。 The concentration of the fluorescent dye of the present invention is not particularly limited, but for example, when handling cells, a lower concentration is preferable due to the influence on cell dysfunction and growth inhibition, and the concentration of the fluorescent dye of the present invention is 100 μM or less. It is preferable that
本発明における蛍光標識剤は、本発明の蛍光色素を含有していれば、必要に応じてその他の成分を含有していてもよい。その他の成分としては、例えば、水や有機溶剤、両親媒性物質等が挙げられる。 The fluorescent labeling agent of the present invention may contain other components as necessary, as long as it contains the fluorescent dye of the present invention. Examples of other components include water, organic solvents, amphiphilic substances, and the like.
本発明の蛍光色素のうち、フタロシアニンの合成方法としては特に限定されないが、例えば、フタロニトリル誘導体を原料として公知の方法でアルミニウムフタロシアニンを合成した後、対応する軸成分とジメチルスルホキシド溶媒中で加熱撹拌することで得ることができる。 Among the fluorescent dyes of the present invention, the method for synthesizing phthalocyanine is not particularly limited, but for example, aluminum phthalocyanine is synthesized by a known method using a phthalonitrile derivative as a raw material, and then heated and stirred in a dimethyl sulfoxide solvent with the corresponding shaft component. You can get it by doing.
原料であるフタロニトリル誘導体が非対称の構造である場合、得られるフタロシアニンは置換基の位置が異なる異性体の混合物として得られる。本明細書においては、異性体の構造のうち一例を示す。 When the raw material phthalonitrile derivative has an asymmetric structure, the resulting phthalocyanine is obtained as a mixture of isomers with different substituent positions. In this specification, one example of the structure of the isomer is shown.
本発明の蛍光色素の具体例としては、以下の蛍光色素が挙げられるが、本発明の蛍光色素はこれらに限定されない。 Specific examples of the fluorescent dye of the present invention include the following fluorescent dyes, but the fluorescent dye of the present invention is not limited thereto.
以下に、本発明を実施例に基づいて説明するが、本発明はこれによって限定されるものではない。なお、実施例中「部」とは、「質量部」を表す。 The present invention will be described below based on examples, but the present invention is not limited thereto. In addition, "parts" in the examples represent "parts by mass."
[製造例１]
＜化合物A－１の製造方法＞
スルホラン２００部、１，８－ジアザビシクロ［５，４，０］－７－ウンデセン（ＤＢＵ）１５．７部に１，３－ジイミノイソインドリン５部および四塩化ケイ素８．８部を加え、１６０～１７０℃で８時間加熱撹拌後、室温（２５℃）まで冷却した。メタノール２００部を加え、析出した沈澱を濾別して、メタノール：水（質量比４：１）混合溶液で洗浄後、乾燥して、収率６３．６％で表２に示す化合物Ａ－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝５７５．９９（理論値５７５．９７）に分子イオンピークが検出され、表２に示す化合物Ａ－１の構造を有することが同定された。
[Manufacturing example 1]
<Method for producing compound A-1>
5 parts of 1,3-diiminoisoindoline and 8.8 parts of silicon tetrachloride were added to 200 parts of sulfolane, 15.7 parts of 1,8-diazabicyclo[5,4,0]-7-undecene (DBU), and 160 parts of silicon tetrachloride was added. After heating and stirring at ~170°C for 8 hours, the mixture was cooled to room temperature (25°C). Add 200 parts of methanol, filter the precipitate, wash with methanol:water (4:1 mass ratio) mixed solution, and dry to obtain compound A-1 shown in Table 2 with a yield of 63.6%. Ta. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 575.99 (theoretical value 575.97), and the molecular ion peak of compound A-1 shown in Table 2 was detected. It was identified that it has a structure.
[製造例２～５]
＜化合物A－２～A－５の製造方法＞
化合物A－１の製造方法で使用した１，３－ジイミノイソインドリンを、表２に示すイソインドリン誘導体に変更した以外は、化合物A－１の製造と同様にして、表２に示す化合物A－２～A－５をそれぞれ製造した。尚、イソインドリン誘導体は、化合物A－１の製造における１，３－ジイミノイソインドリンと同モル量使用した。得られた化合物Ａ－２～Ａ－５の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表２に示した構造を有することが確認された。表２にマススペクトルの分析結果を示した。
[Manufacturing examples 2 to 5]
<Production method of compounds A-2 to A-5>
Compound A shown in Table 2 was produced in the same manner as in the production of Compound A-1, except that 1,3-diiminoisoindoline used in the production method of Compound A-1 was changed to the isoindoline derivative shown in Table 2. -2 to A-5 were produced respectively. The isoindoline derivative was used in the same molar amount as the 1,3-diiminoisoindoline used in the production of compound A-1. The structures of the obtained compounds A-2 to A-5 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 2. Table 2 shows the mass spectrum analysis results.
[製造例６]
＜化合物A－６の製造方法＞
キノリン５０部および無水塩化アルミニウム１部の溶液にアンモニアガスを導入し、フタロニトリルを５部添加した。１８０℃で７時間反応させた。これを室温まで冷却した後、メタノール２００部と１０％塩酸水溶液２００部を加え、析出した固体をろ取し、水２００部で洗浄を行った。８０℃で乾燥させ、収率８６．０％で表３に示す化合物Ａ－６を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝５７５．９９（理論値５７５．９７）に分子イオンピークが検出され、表３に示す化合物Ａ－６の構造を有することが同定された。
[Manufacturing example 6]
<Method for producing compound A-6>
Ammonia gas was introduced into a solution of 50 parts of quinoline and 1 part of anhydrous aluminum chloride, and 5 parts of phthalonitrile were added. The reaction was carried out at 180°C for 7 hours. After cooling this to room temperature, 200 parts of methanol and 200 parts of a 10% aqueous hydrochloric acid solution were added, and the precipitated solid was collected by filtration and washed with 200 parts of water. It was dried at 80° C. to obtain Compound A-6 shown in Table 3 with a yield of 86.0%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 575.99 (theoretical value 575.97), and the molecular ion peak of compound A-6 shown in Table 3 was detected. It was identified that it has a structure.
[製造例７～１０]
＜化合物A－７～A－１０の製造方法＞
化合物A－７の製造方法で使用したフタロニトリルを、表３に示すフタロニトリル誘導体に変更した以外は、化合物A－６の製造と同様にして、表３に示す化合物A－７～A－１０をそれぞれ製造した。尚、フタロニトリル誘導体は、化合物A－６の製造におけるフタロニトリルと同モル量使用した。得られた化合物Ａ－７～Ａ－１０の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表３に示した構造を有することが確認された。表３にマススペクトルの分析結果を示した。
[Manufacturing examples 7 to 10]
<Production method of compounds A-7 to A-10>
Compounds A-7 to A-10 shown in Table 3 were produced in the same manner as in the production of Compound A-6, except that the phthalonitrile used in the production method of Compound A-7 was changed to the phthalonitrile derivative shown in Table 3. were manufactured respectively. The phthalonitrile derivative was used in the same molar amount as the phthalonitrile used in the production of compound A-6. The structures of the obtained compounds A-7 to A-10 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 3. Table 3 shows the mass spectrum analysis results.
[製造例１１]
＜化合物Ｂ－１の製造方法＞
化合物Ａ－１を１部と３－アミノプロピルジメチルエトキシシラン０．６部をピリジンに溶解させ、１１５℃で３時間還流した。エバポレーターでピリジンを除去した後、エタノール１０部と水５０部の混合溶液を加え、析出した固体をろ取し、水５０部で洗浄を行った。８０℃で乾燥させ、収率５４．５％で表４に示す化合物B－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝８０６．１５（理論値８０６．１４）に分子イオンピークが検出され、表４に示す化合物B－１の構造を有することが同定された。
[Manufacture example 11]
<Method for producing compound B-1>
1 part of Compound A-1 and 0.6 part of 3-aminopropyldimethylethoxysilane were dissolved in pyridine, and the mixture was refluxed at 115°C for 3 hours. After removing pyridine using an evaporator, a mixed solution of 10 parts of ethanol and 50 parts of water was added, and the precipitated solid was collected by filtration and washed with 50 parts of water. It was dried at 80°C to obtain Compound B-1 shown in Table 4 with a yield of 54.5%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 806.15 (theoretical value 806.14), and the molecular ion peak of compound B-1 shown in Table 4 was detected. It was identified that it has a structure.
[製造例１２～２０]
＜化合物Ｂ－２～Ｂ－１０の製造方法＞
化合物Ｂ－１の製造方法で使用した化合物Ａ－１を、表４示す化合物Ａ－２～Ａ－１０に変更した以外は、化合物Ａ－１の製造と同様にして、表４に示す化合物Ｂ－２～Ｂ－１０をそれぞれ製造した。尚、化合物Ａ－２～Ａ－１０は、化合物Ｂ－１の製造における化合物Ａ－１と同モル量使用した。得られた化合物Ｂ－２～Ｂ－１０の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表４に示した構造を有することが確認された。表４にマススペクトルの分析結果を示した。
[Manufacturing examples 12 to 20]
<Method for producing compounds B-2 to B-10>
Compound B shown in Table 4 was produced in the same manner as in the production of Compound A-1, except that Compound A-1 used in the production method of Compound B-1 was changed to Compounds A-2 to A-10 shown in Table 4. -2 to B-10 were produced respectively. Note that Compounds A-2 to A-10 were used in the same molar amount as Compound A-1 in the production of Compound B-1. The structures of the obtained compounds B-2 to B-10 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 4. Table 4 shows the mass spectrum analysis results.
[製造例２１]
＜化合物Ｃ－１の製造方法＞
化合物Ａ－１を０．７部と４－アミノ酪酸０．４部をジメチルスルホキシド５０部に溶解させ、１，８－ジアザビシクロ［５．４．０］－７－ウンデセン０．３部を添加した後、９０℃で５時間反応させた。これを室温まで冷却した後、水１００部と食塩１０部を加え、析出した固体をろ取し、水５０部で洗浄を行った。８０℃で乾燥させ、収率４０．２％で表５に示す化合物Ｃ－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝７４５．８３（理論値７４５．８５）に分子イオンピークが検出され、表５に示す化合物Ｃ－１の構造を有することが同定された。
[Manufacturing example 21]
<Method for producing compound C-1>
0.7 parts of compound A-1 and 0.4 parts of 4-aminobutyric acid were dissolved in 50 parts of dimethyl sulfoxide, and 0.3 parts of 1,8-diazabicyclo[5.4.0]-7-undecene was added. Afterwards, the mixture was reacted at 90°C for 5 hours. After cooling this to room temperature, 100 parts of water and 10 parts of common salt were added, and the precipitated solid was collected by filtration and washed with 50 parts of water. It was dried at 80°C to obtain compound C-1 shown in Table 5 with a yield of 40.2%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 745.83 (theoretical value 745.85), and the molecular ion peak of compound C-1 shown in Table 5 was detected. It was identified that it has a structure.
[製造例２２～２４]
＜化合物Ｃ－２～Ｃ－４の製造方法＞
化合物Ｃ－１の製造方法で使用した化合物Ａ－１と４－アミノ酪酸を、表５に示す化合物Ａと軸配位子に変更した以外は、化合物Ｃ－１の製造と同様にして、表５に示す化合物Ｃ－２～Ｃ－４をそれぞれ製造した。尚、化合物Aおよび軸配位子は、化合物Ｃ－１の製造における化合物A－１および４－アミノ酪酸と同モル量使用した。得られた化合物Ｃ－２～Ｃ－４の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表５に示した構造を有することが確認された。表５にマススペクトルの分析結果を示した。
[Manufacturing examples 22 to 24]
<Production method of compounds C-2 to C-4>
In the same manner as in the production of compound C-1, except that compound A-1 and 4-aminobutyric acid used in the production method of compound C-1 were changed to compound A and the axial ligand shown in Table 5, Compounds C-2 to C-4 shown in No. 5 were prepared, respectively. Note that Compound A and the axial ligand were used in the same molar amounts as Compound A-1 and 4-aminobutyric acid in the production of Compound C-1. The structures of the obtained compounds C-2 to C-4 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 5. Table 5 shows the mass spectrum analysis results.
[製造例２５]
＜化合物Ｄ－１の製造方法＞
クロロホルム６部に化合物Ｂ－１を４部、無水コハク酸を１部溶解させた。これにトリエチルアミンを３部加え、室温で２時間攪拌した。エバポレーターでクロロホルムを除去した後、水５部で洗浄を行った。８０℃で乾燥させ、収率７２．８％で表６に示す化合物Ｄ－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝１００６．２８（理論値１００６．２８）に分子イオンピークが検出され、表６に示す化合物Ｄ－１の構造を有することが同定された。
[Manufacturing example 25]
<Method for producing compound D-1>
4 parts of compound B-1 and 1 part of succinic anhydride were dissolved in 6 parts of chloroform. Three parts of triethylamine was added to this, and the mixture was stirred at room temperature for 2 hours. After removing chloroform with an evaporator, washing was performed with 5 parts of water. It was dried at 80°C to obtain Compound D-1 shown in Table 6 with a yield of 72.8%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 1006.28 (theoretical value 1006.28), and the molecular ion peak of compound D-1 shown in Table 6 was detected. It was identified that it has a structure.
[製造例２６～３８]
＜化合物Ｄ－２～Ｄ～１４の製造方法＞
化合物Ｄ－１の製造方法で使用した化合物Ｂ－１を、化合物Ｂ－２～Ｂ－１０、化合物Ｃ－１～Ｃ－４のいずれかに変更した以外は、化合物Ｄ－１の製造と同様に、表６に示す化合物Ｄ－２～Ｄ－１４をそれぞれ製造した。尚、化合物Ｂ－２～Ｂ－１０、化合物Ｃ－１～Ｃ－４は、化合物Ｂ－１と同モル量使用した。得られた化合物Ｄ－２～Ｄ－１４の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表６に示した構造を有することが確認された。表６にマススペクトルの分析結果を示した。
[Manufacturing examples 26 to 38]
<Method for producing compounds D-2 to D-14>
Same as the production of compound D-1 except that compound B-1 used in the production method of compound D-1 was changed to one of compounds B-2 to B-10 and compounds C-1 to C-4. Then, compounds D-2 to D-14 shown in Table 6 were produced, respectively. Note that Compounds B-2 to B-10 and Compounds C-1 to C-4 were used in the same molar amounts as Compound B-1. The structures of the obtained compounds D-2 to D-14 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 6. Table 6 shows the mass spectrum analysis results.
[製造例３９]
＜化合物Ｅ－１の製造方法＞
化合物A－１を０．５部と（２－カルボキシエチル）フェニルホスフィン酸０．２９部をジメチルスルホキシド２０部に溶解させ、８０℃で８時間反応させた。これを室温まで冷却した後、水５０部と食塩１０部を加え、析出した固体をろ取し、水５０部で洗浄を行った。８０℃で乾燥させ、収率７４．４％で０．４６部の表７に示す化合物Ｅ－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝９６７．９４（理論値９６７．９２）に分子イオンピークが検出され、表７に示す化合物Ｅ－１の構造を有することが同定された。
[Manufacture example 39]
<Method for producing compound E-1>
0.5 parts of Compound A-1 and 0.29 parts of (2-carboxyethyl)phenylphosphinic acid were dissolved in 20 parts of dimethyl sulfoxide, and the mixture was reacted at 80°C for 8 hours. After cooling this to room temperature, 50 parts of water and 10 parts of common salt were added, and the precipitated solid was collected by filtration and washed with 50 parts of water. It was dried at 80° C. to obtain 0.46 parts of Compound E-1 shown in Table 7 with a yield of 74.4%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 967.94 (theoretical value 967.92), and the molecular ion peak of compound E-1 shown in Table 7 was detected. It was identified that it has a structure.
[製造例３９～４２]
＜化合物Ｅ－２～Ｅ－４の製造方法＞
化合物Ｅ－１の製造方法で使用した化合物A－１と（２－カルボキシエチル）フェニルホスフィン酸を、表７に示す化合物Aと軸配位子に変更した以外は、化合物Ｅ－１の製造と同様にして、表７に示す化合物Ｅ－２～Ｅ－４をそれぞれ製造した。尚、化合物Aおよび軸配位子は、化合物Ｅ－１の製造における化合物Ｅ－１および（２－カルボキシエチル）フェニルホスフィン酸と同モル量使用した。得られた化合物Ｆ－２～Ｆ－４の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表７に示した構造を有することが確認された。表７にマススペクトルの分析結果を示した。
[Manufacturing examples 39 to 42]
<Method for producing compounds E-2 to E-4>
The process for producing Compound E-1 was performed with the exception that Compound A-1 and (2-carboxyethyl)phenylphosphinic acid used in the production method for Compound E-1 were changed to Compound A and the axial ligand shown in Table 7. Compounds E-2 to E-4 shown in Table 7 were produced in the same manner. Note that Compound A and the axial ligand were used in the same molar amounts as Compound E-1 and (2-carboxyethyl)phenylphosphinic acid in the production of Compound E-1. The structures of the obtained compounds F-2 to F-4 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 7. Table 7 shows the mass spectrum analysis results.
[製造例４３]
＜化合物Ｆ－１の製造方法＞
脱水N，N－ジメチルホルムアミド７０００部に化合物D－1を４７部、Ｎ－ヒドロキシコハク酸イミドを１６部、１－エチル－３－（３－ジメチルアミノプロピル)カルボジイミド塩酸塩を３１部溶解させ、窒素雰囲気下、室温で３時間攪拌した。水を１００００部加え、析出した固体をろ取し、水５０部で洗浄を行った。８０℃で乾燥させ、収率５１．４％で表８に示す化合物Ｆ－１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝１２００．４１（理論値１２００．４３）に分子イオンピークが検出され、表８に示す化合物Ｆ－１の構造を有することが同定された。
[Manufacturing example 43]
<Method for producing compound F-1>
47 parts of compound D-1, 16 parts of N-hydroxysuccinimide, and 31 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in 7000 parts of dehydrated N,N-dimethylformamide, The mixture was stirred at room temperature for 3 hours under a nitrogen atmosphere. 10,000 parts of water was added, and the precipitated solid was collected by filtration and washed with 50 parts of water. It was dried at 80°C to obtain compound F-1 shown in Table 8 with a yield of 51.4%. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 1200.41 (theoretical value 1200.43), and the molecular ion peak of compound F-1 shown in Table 8 was detected. It was identified that it has a structure.
[製造例４４～６０]
＜化合物Ｆ－２～Ｆ－１８の製造方法＞
化合物Ｆ－１の製造方法で使用した化合物Ｄ－１を、化合物Ｄ－２～Ｄ－１４、Ｅ－１～Ｅ－４のいずれかに変更した以外は、化合物Ｆ－１の製造と同様に、表８に示す化合物Ｆ－２～Ｆ－１８をそれぞれ製造した。尚、化合物Ｄ－２～Ｄ－１４、Ｅ－１～Ｅ－４は、化合物Ｄ－１と同モル量使用した。得られた化合物Ｆ－２～Ｆ－１８の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表８に示した構造を有することが確認された。表８にマススペクトルの分析結果を示した。
[Manufacturing examples 44 to 60]
<Production method of compounds F-2 to F-18>
The same process as in the production of compound F-1 except that compound D-1 used in the production method of compound F-1 was changed to one of compounds D-2 to D-14 and E-1 to E-4. , Compounds F-2 to F-18 shown in Table 8 were produced, respectively. Note that Compounds D-2 to D-14 and E-1 to E-4 were used in the same molar amount as Compound D-1. The structures of the obtained compounds F-2 to F-18 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 8. Table 8 shows the mass spectrum analysis results.
［実施例１］
＜蛍光色素１の製造方法＞
クロロホルム３０００部に化合物Ｆ－１を１０部溶解させた。これに１，２－ジパルミトイル－ｓｎ－グリセロ－３－ホスホエタノールアミン（DPPE）１１．５部とジイソプロピルアミノエタノール１部を溶解させたクロロホルム４０００部を少しずつ加えた。室温で３日攪拌した。エバポレーターでクロロホルムを留去した後、粗製物を中圧分取液体クロマトグラフ（バイオタージ製フラッシュ自動精製システム、Ｉｓｏｒｅｌａ Ｏｎｅ）を用いて逆相シリカゲルカラムクロマトグラフィーにより精製し、収率７５．８％で表１に示す蛍光色素１を得た。質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）により分析した結果ｍ／ｚ＝２３５４．２１（理論値２３５４．２０）に分子イオンピークが検出され、表１に示す蛍光色素１の構造を有することが同定された。
[Example 1]
<Method for producing fluorescent dye 1>
10 parts of compound F-1 was dissolved in 3000 parts of chloroform. To this was added little by little 4000 parts of chloroform in which 11.5 parts of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1 part of diisopropylaminoethanol were dissolved. The mixture was stirred at room temperature for 3 days. After chloroform was distilled off using an evaporator, the crude product was purified by reverse phase silica gel column chromatography using a medium pressure preparative liquid chromatograph (Biotage flash automatic purification system, Isorela One), yield 75.8%. Fluorescent dye 1 shown in Table 1 was obtained. As a result of analysis using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), a molecular ion peak was detected at m/z = 2354.21 (theoretical value 2354.20), and the structure of fluorescent dye 1 shown in Table 1 was detected. was identified as having
[実施例２～２１]
＜蛍光色素２～２１の製造方法＞
蛍光色素１の製造方法で使用した化合物Ｆ－１とDPPEを、表９に示す化合物Ｆ－２～Ｆ－１８とリン脂質に変更し、蛍光色素１の製造と同様にして、表１に示す蛍光色素２～２１をそれぞれ製造した。化合物Ｆ－１～Ｆ－１８は、蛍光色素１の製造における化合物Ｆ－１と同モル量使用した。リン脂質は、蛍光色素１の製造におけるDPPEと同モル量使用した。得られた蛍光色素２～２１の構造は、質量分析装置（ＴＯＦ－ＭＳ：ブルカー・ダルトニクス社製 ａｕｔｏｆｌｅｘＩＩ）によって同定し、表１に示した構造を有することが確認された。表９にマススペクトルの分析結果を示した。
[Examples 2 to 21]
<Production method of fluorescent dyes 2 to 21>
The compounds F-1 and DPPE used in the production method of fluorescent dye 1 were changed to compounds F-2 to F-18 and phospholipids shown in Table 9, and the production methods shown in Table 1 were carried out in the same manner as in the production of fluorescent dye 1. Fluorescent dyes 2 to 21 were each produced. Compounds F-1 to F-18 were used in the same molar amount as compound F-1 in the production of fluorescent dye 1. The phospholipid was used in the same molar amount as DPPE in the production of fluorescent dye 1. The structures of the obtained fluorescent dyes 2 to 21 were identified using a mass spectrometer (TOF-MS: autoflex II manufactured by Bruker Daltonics), and it was confirmed that they had the structures shown in Table 1. Table 9 shows the mass spectrum analysis results.
[実施例２２]
＜蛍光標識剤１の作製＞
ジメチルスルホキシド１０部に蛍光色素１を０．０００４７部溶解した。０．２μｍナイロン製メンブレンフィルターでろ過した後、ＲＰＭＩ１６４０培地で１００倍希釈し、蛍光色素１を含む蛍光標識剤１を作製した。
[Example 22]
<Preparation of fluorescent labeling agent 1>
0.00047 parts of fluorescent dye 1 was dissolved in 10 parts of dimethyl sulfoxide. After filtering through a 0.2 μm nylon membrane filter, the mixture was diluted 100 times with RPMI1640 medium to prepare fluorescent labeling agent 1 containing fluorescent dye 1.
[実施例２３～４２]
＜蛍光標識剤２～２１の作製＞
蛍光標識剤１の作製で使用した蛍光色素１を蛍光色素２～２１に変更し、蛍光標識剤２～２１をそれぞれ作製した。ただし、蛍光色素２～２１は蛍光色素１と同モル量使用した。
[Examples 23-42]
<Preparation of fluorescent labeling agents 2 to 21>
Fluorescent dye 1 used in the production of fluorescent labeling agent 1 was changed to fluorescent dyes 2 to 21 to produce fluorescent labeling agents 2 to 21, respectively. However, fluorescent dyes 2 to 21 were used in the same molar amount as fluorescent dye 1.
[比較例１～１０]
＜蛍光標識剤２２～３１の作製＞
蛍光標識剤１の作製で使用した蛍光色素１を化合物Ａ－１～Ａ－１０に変更し、比較例１～１０である蛍光標識剤２２～３１をそれぞれ作製した。ただし、化合物１～１０は蛍光色素１と同モル量使用した。
[Comparative Examples 1 to 10]
<Preparation of fluorescent labeling agents 22 to 31>
Fluorescent dye 1 used in the production of fluorescent labeling agent 1 was changed to compounds A-1 to A-10, and fluorescent labeling agents 22 to 31, which are Comparative Examples 1 to 10, were respectively produced. However, Compounds 1 to 10 were used in the same molar amount as Fluorescent Dye 1.
＜蛍光標識剤の細胞毒性評価＞
ヒト類上皮がん細胞Ａ４３１を９６ウェルプレートに播種（１×１０４ｃｅｌｌ／ｗｅｌｌ）し、１０％Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（ＦＢＳ）および１％ペニシリン―ストレプトマイシンを含ませたＲＰＭＩ１６４０培地を用いて、インキュベーター（３７℃、５％ＣＯ２含有Ａｉｒ、加湿環境）内で２４時間培養した。その後、培地を取り除き、実施例２２～４２、比較例１～１０にて作製した蛍光標識剤と、リファレンスとして１％ジメチルスルホキシドを含むＲＰＭＩ１６４０培地（ＤＭＳＯ培地溶液）をそれぞれ添加した。これをインキュベーター内に１時間静置した後、ＲＰＭＩ１６４０培地で洗浄した。各ｗｅｌｌにセルカウンティングキット－８（同仁化学製）を１０μＬずつ添加し、インキュベーター（３７℃、５％ＣＯ２含有Ａｉｒ、加湿環境）内で１時間静置した。プレートリーダー（ＴＥＣＡＮ社製、ＳＰＡＲＫ）を用いて、４５０ｎｍにおける吸光度を測定した。リファレンスの吸光度を１として各々の蛍光標識剤の吸光度の相対値を算出し、下記の基準に基づいて評価した。評価が〇であれば細胞毒性を示さないといえる。尚、蛍光標識剤の吸光度の相対値を算出際には、測定した吸光度からセルカウンティングキット－８（同仁化学製）添加前の吸光度を差し引いた値を用いた。
〇：０．９以上
×：０．９未満
<Cytotoxicity evaluation of fluorescent labeling agents>
Human epithelioid cancer cells A431 were seeded in a 96-well plate (1×10 4 cells/well), and incubated in an incubator ( The cells were cultured for 24 hours at 37° C. in a humidified air containing 5% CO 2 environment. Thereafter, the medium was removed, and the fluorescent labeling agents prepared in Examples 22 to 42 and Comparative Examples 1 to 10 and RPMI1640 medium (DMSO medium solution) containing 1% dimethyl sulfoxide as a reference were added. After leaving this in an incubator for 1 hour, it was washed with RPMI1640 medium. 10 μL of Cell Counting Kit-8 (manufactured by Dojindo Chemical Co., Ltd.) was added to each well, and the mixture was left standing in an incubator (37° C., 5% CO 2 -containing air, humidified environment) for 1 hour. The absorbance at 450 nm was measured using a plate reader (TECAN, SPARK). The relative value of the absorbance of each fluorescent labeling agent was calculated using the absorbance of the reference as 1, and evaluated based on the following criteria. If the evaluation is ○, it can be said that the substance does not exhibit cytotoxicity. In addition, when calculating the relative value of the absorbance of the fluorescent labeling agent, the value obtained by subtracting the absorbance before addition of Cell Counting Kit-8 (manufactured by Dojindo Chemical Co., Ltd.) from the measured absorbance was used.
〇: 0.9 or more ×: less than 0.9
以下、評価結果を表１０中の「細胞毒性」に示す。 The evaluation results are shown in "Cytotoxicity" in Table 10 below.
＜蛍光標識剤の蛍光強度評価＞
ヒト類上皮がん細胞Ａ４３１を９６ウェルプレートに播種（１×１０４ｃｅｌｌ／ｗｅｌｌ）し、１０％Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（ＦＢＳ）および１％ペニシリン―ストレプトマイシンを含ませたＲＰＭＩ１６４０培地を用いて、インキュベーター（３７℃、５％ＣＯ２含有Ａｉｒ、加湿環境）内で２４時間培養した。その後、培地を取り除き、実施例２２～４２、比較例１～１０にて作製した蛍光標識剤を添加し、インキュベーター内に１時間静置した後、ＲＰＭＩ１６４０培地で洗浄した。プレートリーダー（ＴＥＣＡＮ社製、ＳＰＡＲＫ、励起波長６７０ｎｍ（バンド幅：４０ｎｍ）、測定波長７４０ｎｍ（バンド幅：４０ｎｍ））を用いて、表１７に示す蛍光波長の範囲内での蛍光強度評価を行い、下記の基準に基づいて評価した。実施例２２～４２は、比較例１～１０に比べ蛍光強度が高く、ＤＰＰＥ基の導入によって細胞集積能が向上していることが確認された。
３：１０００以上
２：２００以上～１０００未満
１：２００未満
<Evaluation of fluorescence intensity of fluorescent labeling agent>
Human epithelioid cancer cells A431 were seeded in a 96-well plate (1×10 4 cells/well), and incubated in an incubator ( The cells were cultured for 24 hours at 37° C. in a humidified air containing 5% CO 2 environment. Thereafter, the medium was removed, the fluorescent labels prepared in Examples 22 to 42 and Comparative Examples 1 to 10 were added, and the cells were left standing in an incubator for 1 hour, and then washed with RPMI1640 medium. Using a plate reader (manufactured by TECAN, SPARK, excitation wavelength 670 nm (bandwidth: 40 nm), measurement wavelength 740 nm (bandwidth: 40 nm)), the fluorescence intensity was evaluated within the range of fluorescence wavelengths shown in Table 17. Evaluation was made based on the following criteria. Examples 22 to 42 had higher fluorescence intensity than Comparative Examples 1 to 10, and it was confirmed that the introduction of the DPPE group improved cell accumulation ability.
3: 1000 or more 2: 200 or more - less than 1000 1: less than 200
図１に蛍光標識剤１、及び２２の蛍光強度評価結果を示す。 FIG. 1 shows the fluorescence intensity evaluation results of fluorescent labeling agents 1 and 22.
以下、評価結果を表１０中の「蛍光強度」に示す。 The evaluation results are shown in "Fluorescence intensity" in Table 10 below.
＜細胞の視認性評価＞
ヒト類上皮がん細胞Ａ４３１を９６ウェルプレートに播種（１×１０４ｃｅｌｌ／ｗｅｌｌ）し、１０％Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ（ＦＢＳ）および１％ペニシリン―ストレプトマイシンを含ませたＲＰＭＩ１６４０培地を用いて、インキュベーター（３７℃、５％ＣＯ２含有Ａｉｒ、加湿環境）内で２４時間培養した。その後、培地を取り除き、実施例２２～４２、比較例１～１０にて作製した蛍光標識剤を添加し、インキュベーター内に１時間静置した後、ＲＰＭＩ１６４０培地で洗浄した。適切な波長の励起フィルターおよび蛍光フィルターを設置した蛍光顕微鏡（キーエンス社製、ＢＺ－Ｘ８００、励起波長７１０ｎｍ（バンド幅：７５ｎｍ）、測定波長８１０ｎｍ（バンド幅：９０ｎｍ））を用いて、細胞の暗視野像と蛍光像を観察し、下記の基準に基づいて評価した。蛍光顕微鏡観察の結果から、実施例２２～４２は比較例１～１０に比べ明瞭に細胞標識できていることから、ＤＰＰＥ基の導入によって細胞集積能が向上していることが確認された。
〇：明瞭
×：不明瞭
<Evaluation of cell visibility>
Human epithelioid cancer cells A431 were seeded in a 96-well plate (1×10 4 cells/well), and incubated in an incubator ( The cells were cultured for 24 hours at 37° C. in a humidified air containing 5% CO 2 environment. Thereafter, the medium was removed, the fluorescent labels prepared in Examples 22 to 42 and Comparative Examples 1 to 10 were added, and the cells were left standing in an incubator for 1 hour, and then washed with RPMI1640 medium. Using a fluorescence microscope (manufactured by Keyence Corporation, BZ-X800, excitation wavelength 710 nm (bandwidth: 75 nm), measurement wavelength 810 nm (bandwidth: 90 nm)) equipped with excitation filters and fluorescence filters of appropriate wavelengths, dark cells were detected. Visual field images and fluorescence images were observed and evaluated based on the following criteria. The results of fluorescence microscopy showed that Examples 22 to 42 were more clearly labeled with cells than Comparative Examples 1 to 10, confirming that the cell accumulation ability was improved by the introduction of the DPPE group.
〇: Clear ×: Unclear
図２に蛍光標識剤１で標識した細胞、図３に蛍光標識剤２２で標識した細胞の視認性評価結果を示す（倍率：１０倍、蛍光取り込み時間：２秒）。 FIG. 2 shows the visibility evaluation results of cells labeled with fluorescent labeling agent 1, and FIG. 3 shows the visibility evaluation results of cells labeled with fluorescent labeling agent 22 (magnification: 10 times, fluorescence capture time: 2 seconds).
以下、評価結果を表１０中の「視認性」に示す。 The evaluation results are shown in "Visibility" in Table 10 below.
Claims (4)
一般式（１）
（Ｒ１～Ｒ１６は、それぞれ独立に、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換のアルケニル基、置換もしくは無置換の複素環基、－ＡＢ、－ＳＯ３Ｍ２、－ＣＯＯＭ３、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ａは、１６族元素を表す。Ｂは、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換の複素環基を表す。Ｍ２、Ｍ３はそれぞれ独立に１価のカチオンを表す。
Ｒ１～Ｒ１６は、隣接する置換基同士が互いに連結して、環を形成してもよい。
Ｒ１７は、それぞれ独立に、水酸基、ハロゲン元素、置換または無置換のアルコキシ基、置換または無置換のアリールオキシ基、－ＯＰ（＝Ｏ）Ｒ１８Ｒ１９、－ＯＣ（＝Ｏ）Ｒ２０、－ＯＳ（＝Ｏ）２Ｒ２１、－ＯＳｉＲ２２Ｒ２３Ｒ２４、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１８及びＲ１９は、それぞれ独立に、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｒ２０は、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２１は、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２２～Ｒ２４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。
Ｍ1は、２価～５価の金属原子を表し、Ｍ1が２価の金属原子である場合はｎは０であり、Ｍ1が３価の金属原子である場合はｎは１であり、Ｍ1が４価の金属原子である場合はｎは２である。
ただし、Ｒ１～Ｒ１７の少なくとも１つは－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘである。
Ｚは直接結合、－Ｏ－、－ＯＰ（＝Ｏ）Ｌ２－、－ＯＣ（＝Ｏ）－、－ＯＳ（＝Ｏ）２－、－ＯＳｉＬ３Ｌ４－、－Ｃ（＝Ｏ）－、－Ｃ（＝Ｏ）ＮＨ－を表す。Ｌ１は、直接結合、置換もしくは無置換のアルキレン基、置換もしくは無置換のアリーレン基、置換もしくは無置換の複素環基を表す。Ｘはリン脂質の残基を表す。Ｌ２は、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｌ３、Ｌ４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。） A fluorescent labeling agent containing a fluorescent dye represented by general formula (1).
General formula (1)
(R 1 to R 16 each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkenyl group, Represents an unsubstituted heterocyclic group, -AB, -SO 3 M 2 , -COOM 3 , -Z-L 1 -C(=O)-X. A represents a group 16 element. B is a hydrogen atom , represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group. M 2 and M 3 are each independently a monovalent cation represents.
Adjacent substituents of R 1 to R 16 may be linked to each other to form a ring.
R 17 is each independently a hydroxyl group, a halogen element, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)R 18 R 19 , -OC(=O)R 20 , -OS(=O) 2 R 21 , -OSiR 22 R 23 R 24 , -Z-L 1 -C(=O)-X. R 18 and R 19 are each independently a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted Or represents an unsubstituted heterocyclic group. R 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 22 to R 24 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group.
M 1 represents a divalent to pentavalent metal atom; when M 1 is a divalent metal atom, n is 0; when M 1 is a trivalent metal atom, n is 1; , n is 2 when M 1 is a tetravalent metal atom.
However, at least one of R 1 to R 17 is -ZL 1 -C(=O)-X.
Z is a direct bond, -O-, -OP(=O)L 2 -, -OC(=O)-, -OS(=O) 2 -, -OSiL 3 L 4 -, -C(=O)- , -C(=O)NH-. L 1 represents a direct bond, a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heterocyclic group. X represents a phospholipid residue. L2 is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heterocyclic group represents. L 3 and L 4 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. )
一般式（２）
（Ｒ１～Ｒ１６は、それぞれ独立に、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換のアルケニル基、置換もしくは無置換の複素環基、－ＡＢ、－ＳＯ３Ｍ２、－ＣＯＯＭ３、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ａは、１６族元素を表す。Ｂは、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のシクロアルキル基、置換もしくは無置換の複素環基を表す。Ｍ２、Ｍ３はそれぞれ独立に１価のカチオンを表す。
Ｒ１～Ｒ１６は、隣接する置換基同士が互いに連結して、環を形成してもよい。
Ｒ１７は、それぞれ独立に、水酸基、ハロゲン元素、置換または無置換のアルコキシ基、置換または無置換のアリールオキシ基、－ＯＰ（＝Ｏ）Ｒ１８Ｒ１９、－ＯＣ（＝Ｏ）Ｒ２０、－ＯＳ（＝Ｏ）２Ｒ２１、－ＯＳｉＲ２２Ｒ２３Ｒ２４、－Ｚ－Ｌ１－Ｃ（＝Ｏ）－Ｘを表す。Ｒ１８及びＲ１９は、それぞれ独立に、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｒ２０は、水素原子、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２１は、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。Ｒ２２～Ｒ２４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。
Ｍ1は、２価～５価の金属原子を表し、Ｍ1が２価の金属原子である場合はｎは０であり、Ｍ1が３価の金属原子である場合はｎは１であり、Ｍ1が４価の金属原子である場合はｎは２である。
ただし、Ｒ１～Ｒ１７の少なくとも１つは－Ｚ－（Ｌ１）n－Ｃ（＝Ｏ）－Ｘである。
Ｚは直接結合、－Ｏ－、－ＯＰ（＝Ｏ）Ｌ２－、－ＯＣ（＝Ｏ）－、－ＯＳ（＝Ｏ）２－、－ＯＳｉＬ３Ｌ４－、－Ｃ（＝Ｏ）－、－Ｃ（＝Ｏ）ＮＨ－を表す。Ｌ１は、直接結合、置換もしくは無置換のアルキレン基、置換もしくは無置換のアリーレン基、置換もしくは無置換の複素環基を表す。Ｘはリン脂質の残基を表す。Ｌ２は、水素原子、水酸基、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換のアルコキシ基、置換もしくは無置換のアリールオキシ基、置換もしくは無置換の複素環基を表す。Ｌ３、Ｌ４は、それぞれ独立に、置換もしくは無置換のアルキル基、置換もしくは無置換のアリール基、置換もしくは無置換の複素環基を表す。）
A compound represented by general formula (2).
General formula (2)
(R 1 to R 16 each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkenyl group, Represents an unsubstituted heterocyclic group, -AB, -SO 3 M 2 , -COOM 3 , -Z-L 1 -C(=O)-X. A represents a group 16 element. B is a hydrogen atom , represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclic group. M 2 and M 3 are each independently a monovalent cation represents.
Adjacent substituents of R 1 to R 16 may be linked to each other to form a ring.
R 17 is each independently a hydroxyl group, a halogen element, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, -OP(=O)R 18 R 19 , -OC(=O)R 20 , -OS(=O) 2 R 21 , -OSiR 22 R 23 R 24 , -Z-L 1 -C(=O)-X. R 18 and R 19 are each independently a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted Or represents an unsubstituted heterocyclic group. R 20 represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 21 represents a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. R 22 to R 24 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group.
M 1 represents a divalent to pentavalent metal atom; when M 1 is a divalent metal atom, n is 0; when M 1 is a trivalent metal atom, n is 1; , n is 2 when M 1 is a tetravalent metal atom.
However, at least one of R 1 to R 17 is -Z-(L 1 ) n -C(=O)-X.
Z is a direct bond, -O-, -OP(=O)L 2 -, -OC(=O)-, -OS(=O) 2 -, -OSiL 3 L 4 -, -C(=O)- , -C(=O)NH-. L 1 represents a direct bond, a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heterocyclic group. X represents a phospholipid residue. L2 is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkoxy group, a substituted or unsubstituted aryloxy group, a substituted or unsubstituted heterocyclic group represents. L 3 and L 4 each independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. )
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