JP2018090536A - Near-infrared fluorescent ratio type probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel near-infrared fluorescent pH probe.SOLUTION: The invention provides a compound represented by general formula (I), or a salt thereof.SELECTED DRAWING: None

Description

  The present invention relates to a novel near-infrared fluorescence ratio type probe and a method for measuring an acidic region in a cell using the same.

  The pH in the living body is strictly controlled and is involved in various life phenomena such as bone metabolism and cancer cell infiltration. Therefore, there is a need for a technique for measuring the pH of living animals.

  Currently, there are two in vivo pH measurement techniques. One is a method of directly inserting a pH electrode, which is simple, but only the pH of the portion where the electrode is inserted can be obtained, and it is difficult to obtain spatial information. The second is a technique using MRI. Spatial information can be obtained, but a long measurement time and a large amount of contrast medium are required due to poor sensitivity. The fluorescence imaging method has excellent spatial resolution, and is excellent in simplicity and time resolution due to high detection sensitivity.

  However, an excellent pH probe having an absorption / fluorescence wavelength in the near-infrared region suitable for in vivo fluorescence imaging has not been developed, and there are examples in which changes in pH are observed (Non-patent Document 1), but No method has been established for accurately quantifying pH.

Li, Y .; Wang, Y .; Yang, S .; Zhao, Y .; Yuan, L .; Zheng, J .; Yang, R., Hemicyanine-based high resolution ratiometric near-infrared fluorescent probe for monitoring pH changes in vivo. Anal. Chem. 2015, 87 (4), 2495-503.

  An object of the present invention is to provide a novel near-infrared fluorescent pH probe. In particular, an object of the present invention is to provide a near-infrared fluorescent pH probe capable of fluorescent imaging of a weakly acidic environment in vivo.

In in vivo imaging, a concentration difference is caused by the distribution of the fluorescent probe in the body. Therefore, when an off / on fluorescent probe is used, it is difficult to distinguish between a change in pH and a change in the distribution of the probe. Therefore, the present inventors have focused on the ratio imaging method as a useful technique.
The ratio imaging method is a method of obtaining an image as a ratio (ratio) of signals of two wavelengths using a fluorescent probe in which absorption or fluorescence changes in wavelength due to interaction with a target molecule, and corrects the concentration difference of the probe. can do.

In recent years, it has been reported that P-rhodamine (PR) obtained by substituting the 10-position Si element of Si rhodamine with a phosphorus element increases the absorption and fluorescence wavelengths by 50 to 70 nm. However, the near-infrared fluorescent dye PR has not been functionalized, and it has not been clarified whether P-rhodamine functions as a useful functional dye in ratio imaging.
The present inventors have so far found that Si rhodamine having piperazine functions as a pH probe. Therefore, it was examined whether a fluorescent probe having a P rhodamine skeleton and piperazine effectively functions as a pH probe in the near infrared region. When the 10-position Si element of Si rhodamine was replaced with a P element, the fluorescence quantum yield and pKa Was found to vary with the fluorescent dye skeleton. Furthermore, as a result of intensive studies, it was found that a near-infrared ratio type fluorescent probe can be provided by Prhodamine having a specific structure, and the present invention has been completed.

（式中、
Ｒ^１は、水素原子を示すか、又はベンゼン環上に存在する１ないし３個の同一又は異なる一価の置換基を示し、Ｒ^１は、同一であっても異なっていてもよく；
Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、一価の置換基を示し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^５及びＲ^６は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^７は、置換されていてもよいフェニル基を示し；
Ｒ^８及びＲ^９は、それぞれ独立に、水素原子又は炭素数１〜６個のアルキル基を示し、
Ｒ^８及びＲ^９は一緒になってＲ^８及びＲ^９が結合している窒素原子を含む４〜７員のヘテロシクリルを形成していてもよく、
Ｒ^８又はＲ^９、或いは、Ｒ^８及びＲ^９の両方は、夫々、Ｒ^３、Ｒ^５と一緒になって、Ｒ^８、Ｒ^９が結合している窒素原子を含む５〜７員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される１〜３個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数１〜６個のアルキル、炭素数２〜６個のアルケニル、又は炭素数２〜６個のアルキニル、炭素数６〜１０個のアラルキル基、炭素数６〜１０個のアルキル置換アルケニル基で置換されていてもよく；
Ｒ^１０は、水素原子、炭素数１〜６個のアルキル基又は炭素数１〜６個のフッ素化アルキル基を示す。）
で表される化合物又はその塩。
［２］Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、炭素数１〜６個のアルキル基、炭素数１〜６個のアルコキシ基又はハロゲン原子である、［１］に記載の化合物又はその塩。
［３］Ｒ^２ａ及びＲ^２ｂは、炭素数１〜６個のアルキル基である、［２］に記載の化合物又はその塩。
［４］Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、メチル基又はエチル基である、［１］〜［３］のいずれか１項に記載の化合物又はその塩。
［５］Ｒ^１０は、水素原子、メチル基、エチル基、イソプロピル基、トリフルオロメチル基又はトリフルオロエチル基である、［１］〜［４］のいずれか１項に記載の化合物又はその塩。
［６］Ｒ^８及びＲ^９は、それぞれ独立に、水素原子、メチル基又はエチル基である、［１］〜［５］のいずれか１項に記載の化合物又はその塩。
［７］Ｒ^１は、いずれも水素原子である、［１］〜［６］のいずれか１項に記載の化合物又はその塩。
［８］Ｒ^１の少なくとも１つは、カルボキシ基、カルボキシ基を有するアルキル基、エステル基、アルキルエステル基、アミノ基、アミド基、アルキルアミノ基、イソチオシアネート基、塩化スルホニル基、ハロアルキル基、ハロアセトアミド基、アジド基、又はアルキニル基から選ばれる［１］〜［６］のいずれか１項に記載の化合物又はその塩。
［９］Ｒ^１の少なくとも１つは、カルボキシ基、カルボキシル基を有するアルキル基、アミノ基、アミド基である、［８］に記載の化合物又はその塩。
［１０］以下の一般式（ＩＩ）：

（式中、
Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、一価の置換基を示し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^５及びＲ^６は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^７は、置換されていてもよいフェニル基を示し；
Ｒ^８及びＲ^９は、それぞれ独立に、水素原子又は炭素数１〜６個のアルキル基を示し、
Ｒ^８及びＲ^９は一緒になってＲ^８及びＲ^９が結合している窒素原子を含む４〜７員のヘテロシクリルを形成していてもよく、
Ｒ^８又はＲ^９、或いは、Ｒ^８及びＲ^９の両方は、夫々、Ｒ^３、Ｒ^５と一緒になって、Ｒ^８、Ｒ^９が結合している窒素原子を含む５〜７員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される１〜３個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数１〜６個のアルキル、炭素数２〜６個のアルケニル、又は炭素数２〜６個のアルキニル、炭素数６〜１０個のアラルキル基、炭素数６〜１０個のアルキル置換アルケニル基で置換されていてもよく；
Ｒ^１０は、水素原子、炭素数１〜６個のアルキル基又は炭素数１〜６個のフッ素化アルキル基を示し；
Ｘは、ラベル部位又は標的集積部位を導入することが可能な官能基がＴと結合した後の構造を示し；
Ｔは、架橋基を示し、該架橋基は、その片方または両方の端部に、ラベル部位又は標的集積部位を導入することが可能な官能基、ラベル部位又は標的集積部位と結合することができる官能基を有してもよく；
Ｒ^１’は、水素、又は同一又は異なる一価の置換基であり；
ｍは１〜２の整数であり、ｎは１〜２の整数であり、但し、ｍ＋ｎ＝３である；
で表される化合物又はその塩。
［１１］以下の一般式（ＩＩＩ）：

（式中、
Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、一価の置換基を示し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^５及びＲ^６は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^７は、置換されていてもよいフェニル基を示し；
Ｒ^８及びＲ^９は、それぞれ独立に、水素原子又は炭素数１〜６個のアルキル基を示し、
Ｒ^８及びＲ^９は一緒になってＲ^８及びＲ^９が結合している窒素原子を含む４〜７員のヘテロシクリルを形成していてもよく、
Ｒ^８又はＲ^９、或いは、Ｒ^８及びＲ^９の両方は、夫々、Ｒ^３、Ｒ^５と一緒になって、Ｒ^８、Ｒ^９が結合している窒素原子を含む５〜７員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される１〜３個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数１〜６個のアルキル、炭素数２〜６個のアルケニル、又は炭素数２〜６個のアルキニル、炭素数６〜１０個のアラルキル基、炭素数６〜１０個のアルキル置換アルケニル基で置換されていてもよく；
Ｒ^１０は、水素原子、炭素数１〜６個のアルキル基又は炭素数１〜６個のフッ素化アルキル基を示し；
Ｘは、ラベル部位又は標的集積部位を導入することが可能な官能基がＴと結合した後の構造を示し；
Ｔ’は、存在する場合は、架橋基がＳと結合した後の構造であり；
Ｓは。ラベル部位又は標的集積部位を示し；
Ｒ^１’は、水素、又は同一又は異なる一価の置換基であり；
ｍは１〜２の整数であり、ｎは１〜２の整数であり、但し、ｍ＋ｎ＝３である；
で表される化合物又はその塩。
［１２］−Ｘ−（Ｔ’）−Ｓは、以下の構造を有する、［１１］に記載の化合物又はその塩。

［１３］以下の一般式（ＩＶ）：

（式中、
Ｒ^２ａ及びＲ^２ｂは、それぞれ独立に、一価の置換基を示し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^５及びＲ^６は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^７は、置換されていてもよいフェニル基を示し；
Ｒ^８及びＲ^９は、それぞれ独立に、水素原子又は炭素数１〜６個のアルキル基を示し、
Ｒ^８及びＲ^９は一緒になってＲ^８及びＲ^９が結合している窒素原子を含む４〜７員のヘテロシクリルを形成していてもよく、
Ｒ^８又はＲ^９、或いは、Ｒ^８及びＲ^９の両方は、夫々、Ｒ^３、Ｒ^５と一緒になって、Ｒ^８、Ｒ^９が結合している窒素原子を含む５〜７員のヘテロシクリル又はヘテロアリールを形成していてもよく、環構成員として酸素原子、窒素原子及び硫黄原子からなる群から選択される１〜３個の更なるヘテロ原子を含有していてもよく、更に該ヘテロシクリル又はヘテロアリールは、炭素数１〜６個のアルキル、炭素数２〜６個のアルケニル、又は炭素数２〜６個のアルキニル、炭素数６〜１０個のアラルキル基、炭素数６〜１０個のアルキル置換アルケニル基で置換されていてもよく；
Ｒ^１０は、水素原子、炭素数１〜６個のアルキル基又は炭素数１〜６個のフッ素化アルキル基を示し；
Ｘは、生体高分子標識部位を導入することが可能な官能基がＴと結合した後の構造を示し；
Ｔ’は、存在する場合は、架橋基がＤｘと結合した後の構造であり；
Ｄｘは、デキストランを示し；
Ｒ^１’は、水素、又は同一又は異なる一価の置換基であり；
ｍは１〜２の整数であり、ｎは１〜２の整数であり、但し、ｍ＋ｎ＝３であり；
ｐは、デキストランとの結合当量を示す；
で表される化合物又はその塩。
［１４］［１］〜［１３］のいずれか１項に記載の化合物又はその塩を含む蛍光プローブ。
［１５］細胞内の酸性領域の測定方法であって、
（ａ）［１］〜［１４］に記載の化合物又はその塩を細胞内に導入する工程、及び
（ｂ）当該化合物又はその塩が細胞内で発する蛍光を測定する工程
を含む方法。
を、提供するものである。 That is, the present invention
[1] The following general formula (I):

(Where
R ¹ represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on the benzene ring, and R ¹ may be the same or different;
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a fluorinated alkyl group having 1 to 6 carbon atoms. )
Or a salt thereof.
[2] The compound or a salt thereof according to [1], wherein R ^2a and R ^2b are each independently an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom.
[3] The compound or a salt thereof according to [2], wherein R ^2a and R ^2b are an alkyl group having 1 to 6 carbon atoms.
[4] The compound or a salt thereof according to any one of [1] to [3], wherein R ^2a and R ^2b are each independently a methyl group or an ethyl group.
[5] The compound or a salt thereof according to any one of [1] to [4], wherein R ¹⁰ is a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a trifluoromethyl group, or a trifluoroethyl group. .
[6] The compound or a salt thereof according to any one of [1] to [5], wherein R ⁸ and R ⁹ are each independently a hydrogen atom, a methyl group, or an ethyl group.
[7] The compound or a salt thereof according to any one of [1] to [6], wherein R ¹ is a hydrogen atom.
[8] At least one of R ¹ is carboxy group, alkyl group having carboxy group, ester group, alkyl ester group, amino group, amide group, alkylamino group, isothiocyanate group, sulfonyl chloride group, haloalkyl group, halo The compound or a salt thereof according to any one of [1] to [6], which is selected from an acetamide group, an azide group, or an alkynyl group.
[9] The compound or salt thereof according to [8], wherein at least one of R ¹ is a carboxy group, an alkyl group having a carboxyl group, an amino group, or an amide group.
[10] The following general formula (II):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T;
T represents a bridging group, and the bridging group can be bonded to a functional group, a label site or a target accumulation site capable of introducing a label site or a target accumulation site at one or both ends thereof. May have a functional group;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer of 1 to 2 and n is an integer of 1 to 2, provided that m + n = 3;
Or a salt thereof.
[11] The following general formula (III):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T;
T ′, if present, is the structure after the bridging group is attached to S;
S. Indicates the label site or target accumulation site;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer of 1 to 2 and n is an integer of 1 to 2, provided that m + n = 3;
Or a salt thereof.
[12] The compound or salt thereof according to [11], wherein —X— (T ′) — S has the following structure.

[13] The following general formula (IV):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a biopolymer labeling site is bonded to T;
T ′, if present, is the structure after the bridging group is attached to Dx;
Dx represents dextran;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer from 1 to 2 and n is an integer from 1 to 2, provided that m + n = 3;
p represents the binding equivalent to dextran;
Or a salt thereof.
[14] A fluorescent probe comprising the compound or salt thereof according to any one of [1] to [13].
[15] A method for measuring an acidic region in a cell,
(A) introducing the compound or salt thereof according to [1] to [14] into a cell, and (b) measuring fluorescence emitted from the compound or salt thereof in the cell.
Is provided.

  INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a pH probe having a high fluorescence quantum yield and capable of performing fluorescence imaging in a weakly acidic environment of about 6.0 to 7.4, which is an in vivo pH. .

Pipeindo-PPhR chemical structure and photophysical properties absorption spectrum of pipenedo-PPhR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of absorption intensity ratio against pH of pipeindo-PPhR Fluorescence spectrum of pipenedo-PPhR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Excitation spectrum in pipenedo-PPhR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of fluorescence intensity ratio against pH of pipeindo-PPhR Chemical structure and photophysical properties of pEPPR Absorption spectrum of pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of absorption intensity ratio against pH of pEPPR Fluorescence spectrum of pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Excitation spectrum of pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of fluorescence intensity ratio against pH of pEPPR Chemical structure and photophysical properties of Me-pEPPR Absorption spectrum of Me-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of absorption intensity ratio against pH of Me-pEPPR Fluorescence spectrum of Me-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Excitation spectrum of Me-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of fluorescence intensity ratio against pH of Me-pEPPR Et-pEPPR chemical structure and photophysical properties Absorption spectrum of Et-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of absorption intensity ratio against pH of Et-pEPPR Fluorescence spectrum of Et-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Excitation spectrum of Et-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of fluorescence intensity ratio against pH of Et-pEPPR Chemical structure and photophysical properties of iPr-pEPPR Absorption spectrum of iPr-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of absorption intensity ratio against pH of iPr-pEPPR Fluorescence spectrum of iPr-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Excitation spectrum of iPr-pEPPR in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH Plot of fluorescence intensity ratio against pH of iPr-pEPPR The fluorescence ratio image of 2 micromol Me-pEPPR in 0.1M sodium phosphate buffer is shown. Plot of fluorescence intensity ratio against pH and pKa. The fluorescence ratio image of 2 micromol Me-pEPPR in 0.1M sodium phosphate buffer is shown. Plot of fluorescence intensity ratio against pH and pKa. The photophysical properties of probes (Dex-Me-pEPPR) labeled with various equivalents of Me-pEPPR on dextran are shown. Confocal fluorescence microscopic image of A549 cells loaded with Me-pEPPR or Dex-Me-pEPPR (DOL5.7) The fluorescence image of pH of the gastrointestinal tract of a mouse | mouth 2 hours after oral administration of 20 micromol Dex-Me-pEPPR (DOL = 5.7) (20 microliters) is shown. The gastrointestinal tract of 20 μM Dex-Me-pEPPR (DOL = 5.7) (20 μL × 2) after oral administration (upper) and then after administration of NaHCO ₃ aqueous solution (gastric antacid) (lower) The fluorescence image of pH is shown. Fluorescence ratio image 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to Colon 26 tumor model mice (when water is given to mice) Fluorescence ratio image 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to Colon 26 tumor model mice (when 200 mM NaHCO ₃ aqueous solution was given to mice) PH histograms of mice fed water (mock) and mice fed 200 mM NaHCO ₃ (NaHCO ₃ ) Average pH of mice fed water (mock) and mice fed 200 mM NaHCO ₃ (NaHCO ₃ ) Fluorescence ratio image 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to mice (when physiological saline is orally administered to mice for 1 week) Fluorescence ratio image 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to mice (when acetazolamide was orally administered to mice for 1 week) Mean pH of mice (mock) orally administered physiological saline for 1 week and mice (AZM) orally administered acetazolamide for 1 week

  In the present specification, the “alkyl group” or the alkyl part of a substituent containing an alkyl part (for example, an alkoxy group or the like) unless otherwise specified, for example, has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, More preferably, it means an alkyl group composed of a linear, branched, cyclic, or combination thereof having about 1 to 3 carbon atoms. More specifically, as the alkyl group, for example, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropyl A methyl group, n-pentyl group, n-hexyl group, etc. can be mentioned.

  In the present specification, the term “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom, a chlorine atom or a bromine atom.

One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as “embodiment 1”).

In the general formula (I), R ¹ represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on the benzene ring. R ¹ may be the same or different.
Here, R ¹ is introduced at three positions on the benzene ring other than the positions substituted by R ^2a and R ^2b . When R ¹ represents a monovalent substituent present on the benzene ring, it is preferable that about 1 to 2 substituents which are the same or different exist on the benzene ring. When R ¹ represents one or more monovalent substituents, the substituent can be substituted at any position on the benzene ring. Preferably, R ¹ represents a hydrogen atom (ie, all of R ¹ are hydrogen atoms) or when one substituent is present (ie, ^one of R ¹ is a monovalent substituent, otherwise Is a hydrogen atom).

Although the kind of monovalent substituent represented by R ¹ is not particularly limited, for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, carbon It is preferably selected from the group consisting of several to six alkoxy groups, hydroxyl groups, carboxy groups, sulfonyl groups, alkoxycarbonyl groups, halogen atoms, or amino groups. These monovalent substituents may further have one or more arbitrary substituents. For example, the alkyl group represented by R ¹ may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, and the like. For example, the alkyl group represented by R ¹ is a halogen atom. An alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group may be used. Further, for example, the amino group represented by R ¹ may be present one or two alkyl groups, an amino group represented by R ¹ may be a monoalkylamino group or a dialkylamino group. Furthermore, examples of the case where the alkoxy group represented by R ¹ has a substituent include a carboxy-substituted alkoxy group or an alkoxycarbonyl-substituted alkoxy group, and more specifically, a 4-carboxybutoxy group or 4-acetoxymethyloxy. A carbonyl butoxy group etc. can be mentioned.

In one preferred aspect, R ¹ is a monovalent substituent such as an alkyl group having 1 to 6 carbon atoms, and the substituent is present from the 3rd position to the 5th position on the benzene ring.

In one embodiment of the present invention, at least one of R ¹ is a functional group capable of introducing a label site or a target accumulation site (also referred to as “biopolymer labeling site”). The functional group capable of introducing a label site or a target accumulation site means a functional group capable of reacting with the label site or the target accumulation site, and examples thereof include a carboxyl group and an alkyl group having a carboxyl group. Ester group, alkyl ester group, amino group, amide group, alkylamino group, isothiocyanate group, sulfonyl chloride group, haloalkyl group, haloacetamide group, azide group, alkynyl group, etc. The alkyl group, amino group, and amide group are preferable.
In the case where at least one of R ¹ is a functional group capable of introducing a label site or a target accumulation site, the above monovalent substituent (the number of carbon atoms) even if other R ¹ is hydrogen An alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, A halogen atom or an amino group).

In one preferred aspect, at least one of R ¹ is a functional group capable of introducing a label site or a target accumulation site, and the functional group is present at the 3-position to the 5-position on the benzene ring.

In a preferred aspect of the present invention, at least one of R ¹ is a functional group capable of introducing a label site or a target accumulation site (particularly preferably, a carboxyl group, an alkyl group having a carboxyl group, an amino group, an amide group). And the other R ¹ is hydrogen.

In one preferred aspect, at least one of R ¹ is a carboxyl group, the other R ¹ is hydrogen, and the carboxyl group is at the 4-position on the benzene ring.

In general formula (I), R ^2a and R ^2b each independently represent a monovalent substituent.
Although the kind of monovalent substituent represented by R ^2a and R ^2b is not particularly limited, for example, as in R ¹ , for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, and the number of carbon atoms It is selected from the group consisting of 1 to 6 alkynyl groups, alkoxy groups having 1 to 6 carbon atoms, hydroxyl group, carboxy group, sulfonyl group, alkoxycarbonyl group, halogen atom, or amino group.
In one preferred embodiment of the present invention, R ^2a and R ^2b are each independently an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom.
Moreover, in one more preferable aspect of this invention, both R ^{< 2a>} and R ^<2b > are C1-C6 alkyl groups.
In one more preferred embodiment of the present invention, R ^2a and R ^2b are each independently a methyl group or an ethyl group.
While not intending to be bound by theory, in the P rhodamine skeleton, the carbon atom to which the benzene ring is attached is very susceptible to nucleophilic attack and may also be subjected to nucleophilic attack by water. However, when both R ^2a and R ^2b are alkyl groups having 1 to 6 carbon atoms, such nucleophilic attack can be prevented, and the stability of the probe in the solution can be improved.

In the general formula (I), R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
When R ³ or R ⁴ represents an alkyl group, the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, For example, the alkyl group represented by R ³ or R ⁴ may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like. R ³ and R ⁴ are each independently preferably a hydrogen atom or a halogen atom. When R ³ and R ⁴ are both hydrogen atoms, or R ³ and R ⁴ are both fluorine atoms or chlorine atoms. More preferred.

In general formula (I), R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom, and are the same as those described for R ³ and R ^4. . R ⁵ and R ⁶ are preferably both hydrogen atoms, both chlorine atoms, or both fluorine atoms.

In the general formula (I), R ⁷ represents an optionally substituted phenyl group. It is preferable that R ⁷ is an optionally substituted phenyl group because chemical stability against a nucleophilic species is increased.
Examples of the substituent include a phenyl group, a methyl group, an ethyl group, and a propyl group, and a phenyl group is preferable.

In general formula (I), R < ⁸ > and R < ⁹ > show a hydrogen atom or a C1-C6 alkyl group each independently.

R ⁸ and R ⁹ may together form a 4-7 membered heterocyclyl containing a nitrogen atom to which R ⁸ and R ⁹ are bonded. Examples of the heterocyclyl include azetidine and pyrrolidine, and these heterocyclyl may be substituted with a substituent such as alkyl having 1 to 6 carbon atoms.

In addition, R ⁸ or R ⁹ , or both R ⁸ and R ⁹ , together with R ³ and R ⁵ , respectively, are 5 to 7 members containing a nitrogen atom to which R ⁸ and R ⁹ are bonded. And may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur atoms as ring members, and The heterocyclyl or heteroaryl is alkyl having 1 to 6 carbon atoms, alkenyl having 2 to 6 carbon atoms, or alkynyl having 2 to 6 carbon atoms, aralkyl group having 6 to 10 carbon atoms (benzyl group, phenethyl group). Etc.), which may be substituted with an alkyl-substituted alkenyl group having 6 to 10 carbon atoms. Examples of the heterocyclyl or heteroaryl thus formed include, but are not limited to, pyrrolidine, piperidine, hexamethyleneimine, pyrrole, imidazole, pyrazole, oxazole, thiazole and the like.

In one preferred aspect of the present invention, R ⁸ and R ⁹ are each independently a hydrogen atom, a methyl group, or an ethyl group.
Although not intended to be bound by theory, from the viewpoint of obtaining an excellent ratio-type pH probe, it is preferable that the amino groups at the 3rd and 6th positions of the P rhodamine skeleton have an asymmetric structure. As the rhodamine skeleton has a symmetrical structure, the fluorescence quantum yield tends to be improved. The P-rhodamine skeleton of the compound of the present invention has a piperazine ring. By introducing a hydrogen atom, a methyl group or an ethyl group into the other amino group, it has excellent characteristics as a ratio type pH probe. It is possible to improve the fluorescence quantum yield.

In the general formula (I), R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a fluorinated alkyl group having 1 to 6 carbon atoms. R ¹⁰ is preferably a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a trifluoromethyl group, or a trifluoroethyl group, and more preferably a methyl group, an ethyl group, or an isopropyl group. By introducing these substituents as R ¹⁰ , it is possible to provide a pH probe having a weakly acidic pKa.

In another embodiment of the present invention, at least one of R ¹ in the general formula (I) is a functional group capable of introducing a label site or a target accumulation site, and the functional group is bonded to a crosslinking group. Or a salt thereof (hereinafter also referred to as “embodiment 2”).
Here, as a functional group capable of introducing a label site or a target accumulation site, a carbonyl group, an alkylcarbonyl group, an ester group, an alkyl ester group, an amino group, an alkylamino group, an amide group, an isothiocyanate group, a chloride group A sulfonyl group, a haloalkyl group, a haloacetamido group, an azide group, an alkynyl group, etc. are mentioned, and a carbonyl group and an alkylcarbonyl group are particularly preferable.

  Further, the crosslinking group may be any crosslinking group as long as it functions as a spacer for linking the functional group capable of introducing the label site or target accumulation site and the label site or target accumulation site. For example, substituted or unsubstituted hydrocarbon groups (alkanes, alkenes, alkynes, cycloalkanes, aromatic hydrocarbons, etc.), ethylene glycol groups, diethylene glycol groups, triethylene glycol groups, polyethylene glycol groups, alkyl cysteates and heterocyclic rings A group (for example, a piperidinyl group) and the like, but are not limited thereto. In addition, the crosslinking group has a functional group capable of introducing a label site or a target accumulation site, a functional group capable of binding to the label site or the target accumulation site at one or both ends thereof. Also good. Examples of such a functional group include an amino group, a carbonyl group, a carboxyl group, an amide group, a propargyl group, and the like.

One aspect of Embodiment 2 is a compound represented by the following general formula (II) or a salt thereof.

In general formula (II), R ^<2a> , R ^<2b> , R < ³ > -R < ¹⁰ > is as having described about general formula (I).

X represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T, and T is the above-described crosslinking group.
The bridging group may have a functional group capable of introducing a label site or a target accumulation site, a functional group capable of binding to the label site or the target accumulation site at one or both ends thereof. . Examples of such a functional group include an amino group, a carbonyl group, a carboxyl group, an amide group, a propargyl group, and the like.
R ¹ ′ is hydrogen or the same or different monovalent substituent defined as R ^{1 in the} general formula (I), and the details thereof are as described for the compound of the general formula (I).
R ¹ ′ is preferably hydrogen.

  In general formula (II), m is an integer of 1-2, n is an integer of 1-2, provided that m + n = 3.

In the general formula (II),-(XT) can be introduced at any position of the 3 to 5 positions of the benzene ring.
In a preferred aspect of the compound represented by the general formula (II), m is 1 and n is 2, in which case R ¹ ′ may be the same or different.

In another embodiment of the present invention, at least one of R ¹ in the general formula (I) or (II) is a functional group capable of introducing a label site or a target accumulation site, and the functional group is It is a compound or a salt thereof which is bonded to a crosslinking group, and in which the crosslinking group is further bonded to a label site or a target accumulation site.
In another embodiment of the present invention, at least one R ¹ in the general formula (I) or (II) is a functional group capable of introducing a label site or a target accumulation site, The group is a compound or a salt thereof bound to a label site or a target accumulation site without using a bridging group.
The two types of embodiments are collectively referred to as “embodiment 3” hereinafter.
The functional group and the crosslinking group capable of introducing the label site or the target accumulation site are as described in the second embodiment.

  Examples of the label site or target accumulation site include N-hydroxysuccinimide ester, Halo tag ligand (for example, 2- (2-((6-chlorohexyl) oxy) ethoxy) ethaneamino group), weakly basic amine, maleimide, iso Examples include a thiocyanate group, a sulfonyl chloride group, a haloalkyl group, a haloacetamide group, an azide group, an alkynyl group, a benzylguanine derivative, or a benzylcytosine derivative. The label site or target accumulation site also includes a polyethylene glycol group that may have a modifying group at one or both ends, and examples of the modifying group include an amino group, a carbonyl group, and a carboxyl group. A non-limiting example of a polyethylene glycol group having a modifying group is 3- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) propanoic acid.

  Further, a compound in which a label site or a target accumulation site is introduced into a part of a functional group capable of introducing a label site or a target accumulation site, that is, a functional group capable of introducing a label site or a target accumulation site And a compound having both a label site or a substituent introduced with a target accumulation site are also included in Embodiment 3.

One aspect of Embodiment 3 is a compound represented by the following general formula (III) or a salt thereof.

In general formula (III), R ^<2a> , R ^<2b> , R < ³ > -R < ¹⁰ > is as having described about general formula (I). X, R ¹ ′, m, and n are as described for the general formula (II).

In the general formula (II), T ′, when present, is a structure after the bridging group is bonded to S. Also, S is. Indicates the label site or target accumulation site;
R ¹ ′ is hydrogen or the same or different monovalent substituent.

In the general formula (III),-(X- (T ')-S) can be introduced at any of the 3 to 5 positions of the benzene ring.
In a preferred aspect represented by the general formula (III), m is 1 and n is 2, in which case R ¹ ′ may be the same or different.

In one preferable aspect of Embodiment 3, -S in general formula (III) is a succinimidyl ester body.
Moreover, in one preferable aspect of the compound of general formula (III),-(X- (T ')-S) has the following structure.

The compound of the general formula (III) can label a specific protein, sugar, antibody, etc., and can be localized in the cell, so that the pH in the cell can be measured in vivo. It is.
When a succinimidyl ester or a polyethylene glycol modified with a carboxyl group or the like is introduced into the molecule as a label site, it is labeled with dextran, which is a water-soluble polymer carrier that is a sugar polymer. It enables the fluorescent probe of the invention to respond to in vivo imaging.

上記の（１）〜（６）において、Ｒ^１、Ｒ^８、Ｒ^９及びＲ^１０は以下の通りである。
Ｒ^１＝Ｈ、ＣＯＯＨ、ＣＯＯＳｕ（Ｓｕ：Ｎ−スクシンイミド）、ＣＯＮＨＲ（Ｒ：炭素数１〜６のアルキル基）
Ｒ^８＝Ｅｔ、Ｍｅ、Ｈ
Ｒ^９＝Ｅｔ、Ｍｅ、Ｈ
Ｒ^１０＝Ｈ、Ｍｅ、Ｅｔ、^ｉＰｒ、ＣＨ_２ＣＦ_３、ＣＨ_２ＣＨ_２ＣＦ_３ Non-limiting examples of compounds of general formula (I), (II) or (III) of the present invention include the following compounds:

In the above (1) to (6), R ¹ , R ⁸ , R ⁹ and R ¹⁰ are as follows.
R ¹ = H, COOH, COOSu (Su: N-succinimide), CONHR (R: an alkyl group having 1 to 6 carbon atoms)
R ⁸ = Et, Me, H
R ⁹ = Et, Me, H
R ¹⁰ = H, Me, Et, ⁱ Pr, CH ₂ CF ₃ , CH ₂ CH ₂ CF ₃

Another embodiment of the present invention is a compound represented by the general formula (IV) or a salt thereof.

In general formula (IV), R ^<2a> , R ^<2b> , R < ³ > -R < ¹⁰ > is as having described about general formula (I). R ¹ ′, m, and n are as described for general formula (II).

In the general formula (IV), X represents a structure after a functional group capable of introducing a biopolymer labeling site is bonded to T, and T ′ is a crosslinking group bonded to Dx when present. It is the structure after doing.
Dx represents dextran.
p shows the bond equivalent with dextran, and p is usually 6 or less, Preferably it is 0.95-5.7.

  In general, since a water-soluble polymer carrier is not taken up by cells, it is known to increase the retention in blood and to be distributed in various tissues. By labeling with a certain dextran, the probe can easily reach the target tissue by being taken into a specific tissue or cell, and excellent in vivo imaging can be provided.

  The compounds of general formulas (I) to (VI) of the present invention can exist as acid addition salts or base addition salts. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, organic amine salts such as ammonium salt, and triethylamine salt. In addition to these, a salt with an amino acid such as glycine may be formed. The compounds of the present invention or salts thereof may exist as hydrates or solvates, and these substances are also within the scope of the present invention.

  The compounds of the general formulas (I) to (VI) of the present invention may have one or two or more asymmetric carbons depending on the type of substituent. In addition to stereoisomers such as diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates and the like are included in the scope of the present invention.

  The manufacturing method of the typical compound of the compound represented by general formula (I)-(IV) of this invention was shown concretely in the Example of this specification. Accordingly, those skilled in the art can appropriately select reaction raw materials, reaction conditions, reaction reagents, and the like based on these explanations, and modify or modify these methods as necessary, so that the general formula (I ) To (IV) can be produced.

  Another aspect of the present invention is a fluorescent probe comprising a compound of any one of the general formulas (I) to (IV) or a salt thereof.

Another aspect of the present invention is a method for measuring an acidic region in a cell,
(A) a step of introducing a fluorescent probe containing a compound of any one of the general formulas (I) to (IV) or a salt thereof into a cell, and (b) measuring fluorescence emitted from the compound or a salt thereof in the cell. It is a method including a process.

  In another embodiment of the present invention, (a) contacting a cell with a compound of any one of the general formulas (I) to (IV) or a salt thereof to form a contacted cell; and (b) the contact. Incubating the cells to allow the compound to enter the cells to form labeled cells; and (c) irradiating the labeled cells with an appropriate wavelength at which fluorescence is measured. And thereby monitoring the pH inside the cell, the method for monitoring the pH inside the living cell. Here, the cells include normal cells, cancer cells, nerve cells and the like.

Still another embodiment of the present invention provides (a) a step of administering a compound of general formula (IV) or a salt thereof to a subject, and (b) a suitable cell whose tissue is measured for fluorescence. Irradiating with a wavelength, and thereby monitoring the pH inside the cell, tissue or organ, for monitoring the pH inside the living body in vivo.
Examples of the administration method include oral administration and intravenous administration.
Examples of the cells include normal cells, cancer cells, nerve cells and the like. Examples of the tissue include normal muscle tissue, tumor tissue, adipose tissue and the like, and examples of the organ include stomach, digestive tract, pancreas, liver, kidney and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this.

[Synthesis Example 1]
According to Scheme 1 below, compound 1 (5- (2,6-dimethylphenyl) -1-methyl-10-phenyl-8- (piperazin-1-yl) -3,10-dihydro-2H-phosphinolino [3,2 -F] Indole-1-ium 10-oxide (pipeindo-PPhR)) was synthesized.

Scheme 1: Synthesis scheme of pipenodo-PPhR

(1) Synthesis of 1-Boc-4- (3-bromo-4-formylphenyl) piperazine ( 1 )

2-Bromo-4-fluorobenzaldehyde (4.76 g, 23 mmol), 1-Boc-piperazine (4.28 g, 23 mmol), K ₂ CO ₃ (4.04 g, 29 mmol) were dissolved in DMF (50 mL) and 100 ^o Stir at C for 31 h. The solution was cooled to room temperature, water was added, extracted with DCM and the organic phase was dried over anhydrous Na ₂ SO ₄ and then the solvent was removed under reduced pressure. The residue was purified by medium pressure prep (silica gel, 80/20 hexane / DCM → 50/50 hexane / DCM) to give 1-Boc-4- (3-bromo-4-formylphenyl) piperazine (4.41 g , 52% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 10.11 (1H, s, f), 7.81 (1H, d, J = 8.8 Hz, a), 6.98 (1H, d, J = 2.2 Hz, c), 6.82 (1H, dd, J = 8.8, 2.2 Hz, b), 3.59 (4H, t, J = 5.5 Hz, d), 3.39 (4H, t, J = 5.5 Hz, e), 1.49 (9H, s, g ).

(2) Synthesis of 1-Boc-4- (3-bromo-4-hydroxymethylphenyl) piperazine ( 2 )

1-Boc-4- (3-Bromo-4-formylphenyl) piperazine (2.57 g, 7.0 mmol) was dissolved in MeOH (50 mL), NaBH ₄ (290 mg, 7.7 mmol) was added and 1. was added at room temperature. Stir for 5 hours. Water was added thereto, extraction was performed with DCM, and the organic phase was dried over anhydrous Na ₂ SO _{4. Then} , the solvent was removed under reduced pressure, and 1-Boc-4- (3-bromo-4-hydroxymethylphenyl) piperazine ( 2.56 g, 99% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.32 (1H, d, J = 8.1 Hz, a), 7.08 (1H, d, J = 2.1 Hz, c), 6.85 (1H, dd, J = 8.1, 2.2 Hz, b), 4.66 (2H, d, J = 6.6 Hz, f), 3.56 (4H, t, J = 5.1 Hz, d), 3.13 (4H, t, J = 5.1 Hz, e), 2.02 ( 1H, t, J = 6.6 Hz, h), 1.48 (9H, s, g).
¹³ C NMR (75 MHz, CDCl ₃ ): δ 154.6, 151.8, 130.9, 130.0, 123.9, 120.1, 115.4, 80.1, 64.8, 48.9, 28.4.
HRMS: Calcd for [M + H] ⁺ , 371.0970, Found, 371.0922 (-4.8 mmu).

(3) Synthesis of N-methylindoline ( 3 ) Literature (Koide, Y .; Urano, Y .; Hanaoka, K .; Piao, W .; Kusakabe, M .; Saito, N .; Terai, T .; Okabe N. methylindoline according to J. Am. Chem. Soc. 2012, 134 (11), 5029-5031.), Nagano, T .; Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging. Was synthesized.

(4) Synthesis of tert-butyl 4- (3-bromo-4-((6-bromo-1-methylindoline-5-yl) methyl) phenyl) piperazine-1-carboxylate ( 4 )

1-Boc-4- (3-bromo-4-hydroxymethylphenyl) piperazine (1.94 g, 5.2 mmol) and N-methylindoline (1.11 g, 5.2 mmol) were dissolved in DCM (30 mL), BF3 · OEt ₂ (1.14 mL, 10 mmol) was added and stirred at room temperature for 3 hours. Water was added thereto, the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and then the solvent was removed under reduced pressure. The residue was purified by medium pressure prep (silica gel, DCM → 98/2 DCM / MeOH) and tert-butyl 4- (3-bromo-4-((6-bromo-1-methylindoline-5-yl) methyl ) Phenyl) piperazine-1-carboxylate (1.56 g, 53% yield) was obtained.
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.13 (1H, d, J = 2.1 Hz, e), 6.88 (1H, d, J = 8.7 Hz, c), 6.77 (1H, dd, J = 8.7, 2.1 Hz, d), 6.70 (1H, s, b), 6.65 (1H, s, a), 4.00 (2H, s, l), 3.56 (4H, t, J = 5.1 Hz, i), 3.30 (2H , t, J = 8.1 Hz, g), 3.10 (4H, t, J = 5.1 Hz, j), 2.82 (2H, t, J = 8.1 Hz, f), 1.48 (9H, s, k).
¹³ C NMR (75 MHz, CDCl ₃ ): δ 154.7, 153.1, 150.5, 131.2, 130.8, 130.2, 127.5, 126.1, 125.4, 123.1, 102.3, 115.6, 110.8, 80.0, 56.2, 49.1, 40.4, 36.0, 28.4, 28.3.
HRMS: Calcd for [M + H] ⁺ , 566.0841, Found, 566.0850 (+0.9 mmu).

(5) Synthesis of leuco Boppipe-indo-PPhR ( 5 )

tert-Butyl-4- (3-bromo-4-((6-bromo-1-methylindoline-5-yl) methyl) phenyl) piperazine-1-carboxylate (1.53 g, 2.7 mmol) substituted with Ar Under dissolved in THF (30 mL), s-BuLi (1M hexane solution) (5.7 mL, 5.7 mmol) was gradually added at −78 ^° C. and stirred for 30 minutes. A solution of dichlorophenylphosphine (441 μL, 3.2 mmol) dissolved in THF (5 mL) was added and stirred for 2.5 hours. After warming to 0 ^° C. and adding 30% aqueous hydrogen peroxide (5 mL, 44 mmol) and stirring for 1 hour, the solvent was removed under reduced pressure and the residue was collected at medium pressure (silica gel, 97/3 DCM / MeOH, then NH silica gel, 1/1 hexane / DCM → DCM) to give leuco Boppipe-indo-PPhR (228 mg, 16% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.69 (1H, dd, J = 2.2 Hz, J _HP = 13.2 Hz, h), 7.50-7.42 (2H, m, c), 7.37-7.22 (4H, m , a, b, f), 7.19 (1H, d, J _HP = 12.5 Hz, e), 7.06-6.97 (2H, m, d, g), 3.90-3.64 (2H, m, o), 3.58-3.55 (4H, m, m), 3.34-3.23 (2H, m, j), 3.19-3.16 (2H, m, l), 2.93 (2H, t, J = 8.1 Hz, i), 2.75 (3H, s, k), 1.48 (9H, s, n).
¹³ C NMR (75 MHz, CDCl ₃ ): δ 154.1, 152.0 (d, J _CP = 13.7 Hz), 149.5 (d, J _CP = 12.4 Hz), 134.5 (d, J _CP = 2.5 Hz), 134.0 (d , J _CP = 105 Hz), 132.4 (d, J _CP = 8.7 Hz), 130.8 (d, J _CP = 2.5 Hz), 130.5 (d, J _CP = 9.3 Hz), 130.1 (d, J _CP = 10.6 Hz ), 129.6 (d, J _CP = 100 Hz), 128.7 (d, J _CP = 11.2 Hz), 128.0 (d, J _CP = 11.8 Hz), 126.6 (d, J _CP = 101 Hz), 124.1 (d, J _CP = 12.4 Hz), 119.1 (d, J _CP = 1.9 Hz), 117.1 (d, J _CP = 7.5 Hz), 107.1 (d, J _CP = 8.1 Hz), 79.3, 55.3, 48.7, 35.6, 35.5, 28.9, 28.3, 27.9.
HRMS: Calcd for [M + H] ⁺ , 530.2573, Found, 530.2576 (+0.3 mmu).

(6) Synthesis of Boppipe-indo-PPhX ( 6 )

Leuco Boppipe-indo-PPhR (221 mg, 0.42 mmol) and chloranil (614 mg, 2.4 mmol) were dissolved in DCM (10 mL) and stirred at room temperature for 7 hours. The solvent was removed under reduced pressure and the residue was purified by medium pressure prep (NH silica gel, DCM) to give Boppipe-indo-PPhX (47 mg, 20% yield).
¹ H NMR (400 MHz, CDCl ₃ ): δ 8.32 (1H, dd, J = 8.8 Hz, J _HP = 5.9 Hz, f), 8.08 (1H, d, J _HP = 4.9 Hz, d), 7.61 (2H , dd, J = 7.3 Hz, J _HP = 12.7 Hz, c), 7.42-7.28 (4H, m, a, b, h), 7.05 (1H, dd, J = 9.0, 2.2 Hz, g), 6.79 ( 1H, d, J _HP = 13.7 Hz, e), 3.57-3.51 (6H, m, j, m), 3.40-3.35 (4H, m, l), 3.12-3.03 (2H, m, i), 2.86 ( 3H, s, k), 1.47 (9H, s, n).
¹³ C NMR (75 MHz, CDCl ₃ ): δ 180.0 (d, J _CP = 9.1 Hz), 156.0 (d, J _CP = 14.1 Hz), 154.5, 152.9 (d, J _CP = 12.4 Hz), 134.5 (d , J _CP = 96.8 Hz), 134.4 (d, J _CP = 98.5 Hz), 134.3, 131.4, 131.1 (d, J _CP = 9.9 Hz), 130.4 (d, J _CP = 9.9 Hz), 128.6 (d, J _CP = 12.4 Hz), 126.7 (d, J _CP = 6.6 Hz), 125.8 (d, J _CP = 6.6 Hz), 125.2 (d, J _CP = 10.8 Hz), 117.1, 114.7 (d, J _CP = 7.4 Hz ), 104.8 (d, J _CP = 7.4 Hz), 80.1, 54.3, 46.8, 42.9, 33.8, 28.3, 27.7.
HRMS: Calcd for [M + H] ⁺ , 544.2365, Found, 544.2367 (+0.2 mmu).

(7) Synthesis of pipenodo-PPhR ( 7 )

Boppipe-indo-PPhX (11 mg, 0.020 mmol) was dissolved in THF (5 mL) under Ar substitution, and (2,6-dimethylphenyl) magnesium bromide (1.0 M in THF) (2.0 mL, 2. mL). 0 mmol) was added in 5 portions and stirred for 5 hours. To this was added 2N hydrochloric acid (2 mL), and the organic solvent was removed under reduced pressure. The remaining solution was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to obtain pipeindo-PPhR (2.2 mg, 14% yield).
¹ H NMR (400 MHz, CD ₃ OD): δ 7.70 (1H, d, J _HP = 15.1 Hz, e), 7.66-7.51 (4H, m, a, c, h), 7.43 (1H, ddd, J = 7.6, 7.6 Hz, J _HP = 3.4 Hz, b), 7.32 (1H, t, J = 7.6 Hz, n), 7.22-7.16 (2H, m, o), 7.03 (1H, dd, J = 9.0, 2.2 Hz, g), 6.97 (1H, dd, J = 9.3 Hz, J _HP = 6.3 Hz, f), 6.70 (1H, d, J _HP = 4.4 Hz, d), 4.04 (2H, t, J = 6.8 Hz, j), 3.77 (4H, t, J = 5.1 Hz, l), 3.40 (3H, s, k), 3.28 (4H, t, J = 5.1 Hz, m), 3.00-2.93 (2H, m, i), 1.92 (3H, s, p), 1.90 (3H, s, q).
HPLC (A / B = 60/40 → 0/100, 30 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

[Synthesis Example 2]
According to Scheme 2 below, compound 2 (N- (10- (2,6-dimethylphenyl) -5-oxide-5-phenyl-7- (piperazin-1-yl) acridophosphine-3 (5H) -ylidene) -N-ethylethanaminium (pEPPR)) was synthesized.

Scheme 2: Synthesis scheme of pEPPR

(1) Synthesis of 3-bromo-N, N-diethylaniline ( 8 ) Literature (Lei, Z .; Li, X .; Li, Y .; Luo, X .; Zhou, M .; Yang, Y., 3-Bromo-N, N-diethylaniline according to Synthesis of Sterically Protected Xanthene Dyes with Bulky Groups at C-3 ′ and C-7 ′. J. Org. Chem. 2015, 80 (22), 11538-11543.) Was synthesized.

(2) Synthesis of tert-butyl 4- (3-bromo-4- (2-bromo-4- (diethylamino) benzyl) phenyl) piperazine-1-carboxylate ( 9 )

1-Boc-4- (3-bromo-4-hydroxymethylphenyl) piperazine (3.60 g, 9.7 mmol), 3-bromo-N, N-diethylaniline (2.21 g, 9.7 mmol) was added to DCM ( 80 mL), BF3 · OEt ₂ (2.10 mL, 19 mmol) was added, and the mixture was stirred at room temperature for 8 hours. Water was added thereto, the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and then the solvent was removed under reduced pressure. The residue was purified by medium pressure preparative (silica gel, 1/1 hexane / DCM → DCM) and tert-butyl 4 (3-bromo-4- (2-bromo-4- (diethylamino) benzyl) phenyl) piperazine- 1-carboxylate (4.28 g, 59% yield) was obtained.
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.10 (1H, dd, J = 2.2 Hz, f), 6.88-6.84 (2H, m, c, d), 6.80 (1H, d, J = 8.8 Hz, a), 6.70 (1H, dd, J = 8.8, 2.2 Hz, e), 6.48 (1H, dd, J = 8.8, 2.2 Hz, b), 3.96 (2H, s, l), 3.52 (4H, t, J = 4.4 Hz, j), 3.26 (4H, q, J = 7.0 Hz, g), 3.04 (4H, t, J = 4.4 Hz, i), 1.49 (9H, s, k), 1.11 (6H, t , J = 7.0 Hz, h).
¹³ C NMR (100 MHz, CDCl ₃ ): δ 154.3, 150.2, 147.1, 130.8, 130.6, 125.6, 125.1, 124.9, 119.9, 115.4, 115.0, 110.8, 79.5, 48.8, 44.0, 43.3, 39.7, 28.2, 12.3.
HRMS: Calcd for [M + H] ⁺ , 580.1174, Found, 580.1163 (-1.1 mmu).

ｔｅｒｔ−ブチル４ （３−ブロモ−４−（２−ブロモ−４−（ジエチルアミノ）ベンジル）フェニル）ピペラジン−１−カルボキシレート（５８０ｍｇ、１．０ｍｍｏｌ）をＡｒ置換下、ＴＨＦ（１５ｍＬ）に溶解し、−７８^ｏＣでｓ−ＢｕＬｉ（１Ｍヘキサン溶液）（２．０ｍＬ、２．０ｍｍｏｌ）を徐々に加え、１時間間攪拌した。ジクロロホスフィン（１６２μＬ、１．２ｍｍｏｌ）をＴＨＦ（３ｍＬ）に溶解したものを加え、２時間半攪拌した。０^ｏＣに温め、３０％過酸化水素水（２ｍＬ、１８ｍｍｏｌ）を加え、２時間攪拌した。これに水を加え、ＡｃＯＥｔで抽出して有機相を無水Ｎａ_２ＳＯ_４で乾燥させた後、溶媒を減圧除去し、残渣を中圧分取（ＮＨ ｓｉｌｉｃａ ｇｅｌ、１／２ヘキサン／ＤＣＭ→ＤＣＭ）で精製し、ｌｅｕｃｏ Ｂｏｃｐｉｐｅ−ｄｉＥｔ−ＰＰｈＲ（１６３ｍｇ、３０％収率）を得た。
¹H NMR (400 MHz, CDCl₃): δ 7.71 (1H, dd, J = 2.4 Hz, J_H-P = 13.2 Hz, i), 7.49-7.41 (3H, m, c, f), 7.35 (1H, t, J = 6.8 Hz, a), 7.29 (2H, ddd, J = 7.3, 7.3 Hz, J_H-P = 2.1 Hz, b), 7.23 (1H, dd, J = 8.3 Hz, J_H-P = 5.9 Hz, g), 7.18 (1H, dd, J = 8.3 Hz, J_H-P = 6.3 Hz, d), 7.00 (1H, dd, J = 8.8, 2.4 Hz, h), 7.20 (1H, dd, J = 8.3, 2.7 Hz, e), 3.91-3.65 (2H, m, o), 3.57 (4H, t, J = 5.1 Hz, m), 3.41-3.33 (4H, m, j), 3.20-3.15 (4H, m, l), 1.48 (9H, s, n), 1.13 (6H, t, J = 7.1 Hz, k).
¹³C NMR (100 MHz, CDCl₃): δ 154.4, 149.7 (d, J_C-P = 12.4 Hz), 146.5 (d, J_C-P = 12.4 Hz), 134.3 (d, J_C-P = 104 Hz), 132.9 (d, J_C-P = 8.3 Hz), 130.9, 130.4 (d, J_C-P = 10.8 Hz), 129.5 (d, J_C-P = 16.6 Hz), 129.0 (d, J_C-P = 12.4 Hz), 128.8 (d, J_C-P = 11.6 Hz), 128.6, 127.3 (d, J_C-P = 8.3 Hz), 119.3, 117.6 (d, J_C-P = 7.4 Hz), 114.9, 113.0 (d, J_C-P = 8.3 Hz), 79.6, 48.9, 44.1, 43.2, 35.1, 28.2, 12.2.
HRMS: Calcd for [M+H]⁺, 546.2886, Found, 546.2889 (+0.3 mmu). (3) Synthesis of leuco Boppipe-diEt-PPhR ( 10 )

tert-Butyl 4 (3-bromo-4- (2-bromo-4- (diethylamino) benzyl) phenyl) piperazine-1-carboxylate (580 mg, 1.0 mmol) was dissolved in THF (15 mL) under Ar substitution. S-BuLi (1M hexane solution) (2.0 mL, 2.0 mmol) was gradually added at −78 ^° C., and the mixture was stirred for 1 hour. A solution of dichlorophosphine (162 μL, 1.2 mmol) in THF (3 mL) was added and stirred for 2.5 hours. ^Warmed to 0 ^° C., 30% aqueous hydrogen peroxide (2 mL, 18 mmol) was added and stirred for 2 hours. Water was added thereto, extraction was performed with AcOEt, and the organic phase was dried over anhydrous Na ₂ SO _{4. Then} , the solvent was removed under reduced pressure, and the residue was separated with medium pressure (NH silica gel, 1/2 hexane / DCM → DCM). ) To obtain leuco Boppipe-diEt-PPhR (163 mg, 30% yield).
¹ H NMR (400 MHz, CDCl ₃ ): δ 7.71 (1H, dd, J = 2.4 Hz, J _HP = 13.2 Hz, i), 7.49-7.41 (3H, m, c, f), 7.35 (1H, t , J = 6.8 Hz, a), 7.29 (2H, ddd, J = 7.3, 7.3 Hz, J _HP = 2.1 Hz, b), 7.23 (1H, dd, J = 8.3 Hz, J _HP = 5.9 Hz, g) , 7.18 (1H, dd, J = 8.3 Hz, J _HP = 6.3 Hz, d), 7.00 (1H, dd, J = 8.8, 2.4 Hz, h), 7.20 (1H, dd, J = 8.3, 2.7 Hz, e), 3.91-3.65 (2H, m, o), 3.57 (4H, t, J = 5.1 Hz, m), 3.41-3.33 (4H, m, j), 3.20-3.15 (4H, m, l), 1.48 (9H, s, n), 1.13 (6H, t, J = 7.1 Hz, k).
¹³ C NMR (100 MHz, CDCl ₃ ): δ 154.4, 149.7 (d, J _CP = 12.4 Hz), 146.5 (d, J _CP = 12.4 Hz), 134.3 (d, J _CP = 104 Hz), 132.9 (d , J _CP = 8.3 Hz), 130.9, 130.4 (d, J _CP = 10.8 Hz), 129.5 (d, J _CP = 16.6 Hz), 129.0 (d, J _CP = 12.4 Hz), 128.8 (d, J _CP = 11.6 Hz), 128.6, 127.3 (d, J _CP = 8.3 Hz), 119.3, 117.6 (d, J _CP = 7.4 Hz), 114.9, 113.0 (d, J _CP = 8.3 Hz), 79.6, 48.9, 44.1, 43.2 , 35.1, 28.2, 12.2.
HRMS: Calcd for [M + H] ⁺ , 546.2886, Found, 546.2889 (+0.3 mmu).

(4) Synthesis of Boppipe-diEt-PPhX ( 11 )

Leuco Boppipe-diEt-PPhR (163 mg, 0.30 mmol) and chloranil (220 mg, 0.89 mmol) were dissolved in DCM (50 mL) and stirred at room temperature for 9 hours. The solvent was removed in vacuo and the residue was purified by medium pressure prep (NH silica gel, DCM → 99/1 DCM / MeOH) to give Boppipe-diEt-PPhX (70 mg, 49% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 8.33 (1H, dd, J = 8.8 Hz, J _HP = 5.9 Hz, g), 8.29 (1H, dd, J = 8.8 Hz, J _HP = 5.9 Hz, d ), 7.61 (1H, d, J _HP = 12.5 Hz, i), 7.59 (1H, dd, J = 1.5 Hz, J _HP = 12.5 Hz, f), 7.40-7.30 (4H, m, b, c), 7.13-7.02 (2H, m, a, h), 6.83 (1H, dd, J = 9.2 Hz, J _HP = 2.6 Hz, e), 3.56 (4H, t, J = 5.1 Hz, m), 3.47-3.36 (8H, m, j, l), 1.47 (9H, s, n), 1.15 (6H, t, J = 7.0 Hz, k).
¹³ C NMR (75 MHz, CDCl ₃ ): δ 179.9 (d, J _CP = 8.1 Hz), 154.4, 152.8 (d, J _CP = 12.4 Hz), 150.5 (d, J _CP = 12.4 Hz), 134.8 (d , J _CP = 97.0 Hz), 134.5 (d, J _CP = 106 Hz), 134.4 (d, J _CP = 97.0 Hz), 131.6 (d, J _CP = 10.6 Hz), 131.4 (d, J _CP = 2.5 Hz ), 131.0 (d, J _CP = 9.9 Hz), 130.2 (d, J _CP = 9.9 Hz), 128.5 (d, J _CP = 12.4 Hz), 126.8 (d, J _CP = 6.8 Hz), 123.2 (d, J _CP = 6.8 Hz), 117.1 (d, J _CP = 2.5 Hz), 114.4 (d, J _CP = 7.5 Hz), 114.1 (d, J _CP = 1.9 Hz), 111.3 (d, J _CP = 7.5 Hz) , 80.1, 46.7, 44.4, 42.8, 28.3, 12.3.
HRMS: Calcd for [M + H] ⁺ , 582.2498, Found, 582.2496 (-0.2 mmu).

(5) Synthesis of pEPPR ( 12 ).

Boppipe-diEt-PPhX (20 mg, 0.035 mmol) was dissolved in THF (5 mL) under Ar substitution, and (2,6-dimethylphenyl) magnesium bromide (1.0 M in THF) (2.1 mL, 2.1 mmol) ) Was added in three portions and stirred for 8 hours. To this was added 2N hydrochloric acid (2 mL), and the organic solvent was removed under reduced pressure. The remaining solution was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to obtain pEPPR (6.2 mg, 23% yield).
¹ H NMR (300 MHz, CD ₃ OD): δ 7.74-7.68 (2H, m, i, f), 7.63-7.52 (3H, m, a, c), 7.45 (2H, ddd, J = 7.6, 7.6 Hz, J _HP = 3.3 Hz, b), 7.33 (1H, t, J = 7.6 Hz, n), 7.23-7.17 (2H, m, o), 7.13-7.06 (3H, m, d, g, h) , 6.98 (1H, dd, J = 9.8, 2.4 Hz, e), 3.89 (4H, t, J = 5.4 Hz, j), 3.86-3.69 (4H, m, l), 3.31 (4H, t, J = 5.1 Hz, m), 1.92 (3H, s, p), 1.91 (3H, s, q), 1.29-1.21 (6H, m, k).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

[Synthesis Examples 3 to 5]
According to Scheme 3 below, compound 3 (N- (10- (2,6-dimethylphenyl) -7- (4-methylpiperazin-1-yl) -5-oxide-5-phenylacylphosphine-3 (5H) -Ilidene) -N-ethylethananium (Me-pEPPR)), Compound 4 (N- (10- (2,6-dimethylphenyl) -7- (4-ethylpiperazin-1-yl) -5-oxide- 5-phenylacylphosphine-3 (5H) -ylidene) -N-ethylethanaminium (Et-pEPPR)) and compound 5 (N- (10- (2,6-dimethylphenyl) -7- (4- Isopropylpiperazin-1-yl) -5-oxide-5-phenylacylphosphine-3 (5H) -ylidene) -N-ethylethanaminium (iPr-pEPPR)) It was.

Scheme 3: Synthesis scheme of Me-pEPPR ( 13 ), Et-pEPPR ( 14 ), and iPr-pEPPR ( 15 )

(1) Synthesis of Me-pEPPR ( 13 )

pEPPR (3.1 mg, 4.0 μmol), 37% aqueous HCHO solution (30 μL, 400 μmol), NaBH ₃ CN (2.2 mg, 36 μmol) and AcOH (20 μL) were dissolved in MeOH (10 mL) and stirred at room temperature for 1 hour. did. The solvent was removed under reduced pressure, and the residue was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to give Me-pEPPR (2.1 mg, 67% yield).
¹ H NMR (300 MHz, CD ₃ OD): δ 7.85-7.75 (2H, m, f, i), 7.72-7.60 (3H, m, a, b), 7.58-7.51 (2H, m, c), 7.42 (1H, t, J = 7.3 Hz, n), 7.32-7.25 (2H, m, o), 7.21-7.15 (3H, m, d, g, h), 7.08 (1H, dd, J = 10.3, 2.2 Hz, e), 4.18-3.79 (8H, m, l, m), 3.45 (4H, s, j), 2.95 (3H, s, r), 2.01 (3H, s, p), 2.00 (3H, s, q), 1.36-1.30 (6H, m, k).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

(2) Synthesis of Et-pEPPR ( 14 )

pEPPR (2.8 mg, 3.6 μmol), CH ₃ CHO (22.4 μL, 361 μmol), NaBH ₃ CN (1.0 mg, 16 μmol), and AcOH (20 μL) were dissolved in MeOH (10 mL) for 30 minutes at room temperature. Stir. The solvent was removed under reduced pressure, and the residue was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to give Et-pEPPR (1.7 mg, 59% yield).
¹ H NMR (300 MHz, CD ₂ Cl ₂ ): δ 7.72-7.52 (7H, m, a, b, c, f, i), 7.41 (1H, t, J = 7.7 Hz, n), 7.32-7.17 (4H, m, d, g, o), 6.90 (1H, d, J = 8.8 Hz, h), 6.76 (1H, d, J = 9.5 Hz, e), 3.90-3.43 (12H, m, l, m, j), 3.32 (2H, t, J = 7.3 Hz, r), 2.02 (3H, s, p), 2.00 (3H, s, q), 1.42-1.30 (9H, m, k, s).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

(3) Synthesis of iPr-pEPPR ( 15 )

pEPPR (3.7 mg, 4.8 μmol), acetone (35 μL, 480 μmol), NaBH ₃ CN (1.3 mg, 2.1 μmol) and AcOH (20 μL) were dissolved in MeOH (5 mL) and stirred at room temperature for 4 hours. . The solvent was removed under reduced pressure, and the residue was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to obtain iPr-pEPPR (1.5 mg, 38% yield).
¹ H NMR (400 MHz, CD ₃ OD): δ 7.82 (1H, d, J _HP = 16.1 Hz, i), 7.76 (1H, d, J _HP = 16.2 Hz, f), 7.71-7.62 (3H, m , a, c), 7.54 (1H, ddd, J = 7.7, 7.7 Hz, J _HP = 3.1 Hz, b), 7.42 (1H, t, J = 7.6 Hz, n), 7.31-7.26 (2H, m, o), 7.20-7.14 (3H, m, d, g, h), 7.05 (1H, d, J = 8.8 Hz, e), 3.96-3.76 (5H, m, m, r), 3.21 (4H, q , J = 7.3 Hz, j), 2.90-2.56 (4H, m, l), 2.02 (3H, s, p), 2.01 (3H, s, q), 1.37-1.27 (12H, m, k, s) .
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

[Synthesis Example 6]
According to Scheme 4 below, compound 6 (N- (10- (4-(((2,5-dioxopyrrolidin-1-yl) oxy) carbonyl) -2,6-dimethylphenyl) -7- (4-methyl Piperazin-1-yl) -5-oxide-5-phenylacylphosphine-3 (5H) -ylidene) -N-ethylethanaminium (Me-pEPPR-SE) was synthesized.

Scheme 4: Synthesis of Me-pEPPR-SE ( 20 )

(1) Synthesis of 2,5-dibromo-m-xylene ( 16 ).

4-Bromo-2,6-dimethylaniline (6.00 g, 30 mmol) was suspended in 48% aqueous HBr (20 mL), NaNO ₂ (2.28 g, 33 mmol) dissolved in water at −10 ^° C. was added, Stir for 1 hour and add sulfamic acid (0.58 g, 6 mmol). _{This, FeSO 4 · 7H 2 O (} 4.17g, 15mmol) was slowly added to those which had been heated to dissolve 80 ^o C in aqueous 48% HBr (20 mL), and stirred for 1 hour at 80 ^o C. Water was added thereto, extraction was performed with hexane, and the organic phase was dried over anhydrous Na ₂ SO ₄ , and then the solvent was removed under reduced pressure. Purification by medium pressure fractionation (silica gel, hexane) gave 2,5-dibromo-m-xylene (6.19 g, 72% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.19 (2H, s, a), 2.37 (6H, s, b).

(2) Synthesis of tert-butyl 4-bromo-3,5-dimethylbenzoate ( 17 ).

2,5-Dibromo-m-xylene (4.69 g, 18 mmol) was dissolved in THF (50 mL) under Ar substitution, and n-BuLi (1.6 M hexane solution) (11.1 mL, 18 mmol) at −78 ^° C. ) Was added and stirred for 2 hours. To this was added Boc ₂ O (4.08 mL, 18 mmol) dissolved in THF (5 mL), and the mixture was stirred at room temperature for 2 hours. Water was added thereto, extracted with DCM, and the organic phase was dried over anhydrous Na ₂ SO ₄ , and then the solvent was removed under reduced pressure. The residue was purified by medium pressure prep (silica gel, hexane) to give tert-butyl 4-bromo-3,5-dimethylbenzoate (3.53 g, 69% yield).
¹ H NMR (300 MHz, CDCl ₃ ): δ 7.65 (2H, s, a), 2.43 (6H, s, b), 1.58 (9H, s, c).

(3) Synthesis of pEPPR-COOH ( 18 )

tert-Butyl 4-bromo-3,5-dimethylbenzoate (903 mg, 3.2 mmol) was dissolved in THF (10 mL) under Ar substitution, and s-BuLi (1.0 M hexane solution) was added at −78 ^° C. (3 2 mL, 3.2 mmol) was added and stirred for 30 minutes. To this was added Boppipe-diEt-PPhX (173 mg, 0.31 mmol) dissolved in THF (5 mL), and the mixture was stirred at −78 ^° C. for 1.5 hours. To this was added 2N hydrochloric acid (5 mL), and the organic solvent was removed under reduced pressure. The remaining solution was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to obtain pEPPR-COOH (22.4 mg, 8.8% yield).
¹ H NMR (300 MHz, CD ₃ OD): δ 7.95-7.92 (2H, m, n), 7.84-7.78 (2H, m, i, f), 7.71-7.62 (3H, m, a, c), 7.54 (2H, ddd, J = 7.6, 7.6 Hz, J _HP = 3.3 Hz, b), 7.21-7.05 (4H, m, d, e, g, h), 4.05-3.78 (8H, m, l, m ), 3.41 (4H, t, J = 5.4 Hz, j), 2.08 (3H, s, o), 2.06 (3H, s, p), 1.38-1.30 (6H, m, k).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

(4) Synthesis of Me-pEPPR-COOH ( 19 )

pEPPR-COOH (4.7 mg, 5.7 μmol), 37% aqueous HCHO solution (42.6 μL, 573 μmol), NaBH ₃ CN (1.6 mg, 26 μmol) and AcOH (20 μL) were dissolved in MeOH (10 mL) at room temperature. For 1 hour. The solvent was removed under reduced pressure, and the residue was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to give Me-pEPPR-COOH (4.9 mg, quant. Yield).
¹ H NMR (400 MHz, CD ₃ OD): δ 7.87-7.83 (2H, m, n), 7.76-7.69 (2H, m, i, f), 7.62-7.54 (3H, m, a, c), 7.45 (2H, ddd, J = 7.4, 7.4 Hz, J _HP = 3.4 Hz, b), 7.18-6.95 (4H, m, d, e, g, h), 4.07-3.69 (8H, m, l, m ), 3.38-3.35 (4H, m, j), 1.99 (3H, s, o), 1.97 (3H, s, p), 1.26-1.23 (6H, m, k).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

(5) Synthesis of Me-pEPPR-SE ( 20 )

Me-pEPPR-COOH (4.3 mg, 5.1 μmol), WSCD · HCl (3.0 mg, 15 μmol), NHS (1.8 mg, 15 μmol) and DIEA (9.0 μL, 51 μmol) are dissolved in DCM (5 mL). And stirred at room temperature for 8 hours. The solvent was removed under reduced pressure and the residue was purified by HPLC (ODS silica gel, water / MeCN, 0.1% TFA) to give Me-pEPPR-SE (1.6 mg, 34% yield).
HPLC (A / B = 70/30 → 0/100, 35 min; A: H ₂ O, 0.1 M TFA, B: MeCN / H ₂ O = 80/20, 0.1 M TFA)

[Example 1]
The optical properties of compound 1 (pipeindo-PPhR) of the present invention were evaluated. The absorption spectrum, fluorescence spectrum, and excitation spectrum were measured using Shimadzu UV-1650PC absorptiometer and Hitachi F-4500 fluorometer. The results are shown in FIGS. 1b, 1d-e.

  FIG. 1a shows the chemical structure and photophysical properties of pipeindo-PPhR. FIG. 1b, FIG. 1d, and FIG. 1e represent absorption, fluorescence, and excitation spectra in 0.1M sodium phosphate buffer (containing 0.1% DMSO) with different pH of pipenodo-PPhR, respectively. FIG. 1c represents a plot of the ratio of absorption intensity to pH and the pKa value of the protonation reaction calculated thereby. FIG. 1f shows a plot of the fluorescence intensity ratio against pH and the pKa value of the protonation reaction calculated thereby.

Pipepeindo-PPhR, like the pH probe of Si rhodamine (SiR), was acidic and protonated, and its absorption wavelength was greatly shifted from 734 nm to 645 nm (FIGS. 1a and b). On the other hand, the absorption wavelength was 60-70 nm longer than that of SiR, and both the acidic type and the basic type had absorption in the near infrared region.
pK _a is calculated as 7.4 from the change in the absorption spectrum was shifted to about 1 more acidic than the SiR (Figure 1c). This is thought to be because the P = O group is an electron-attracting group, thereby reducing the electron density of piperazine and reducing the basicity. In addition, all types showed weak fluorescence, and had a fluorescence wavelength in the 700 nm range (FIG. 1d). By taking the ratio of the fluorescence intensity when excited at 640 nm and 730 nm, it is possible to measure the ratio of the fluorescence, and it was calculated as pK _a 7.6 from the change in the ratio value of the fluorescence (FIGS. 1e and f). However, the fluorescence quantum yield was a small value of about 1% at the maximum.

[Example 2]
The reason why the fluorescence quantum yield of compound 1 (pipeindo-PPhR) is low is thought to be due to the large bias of the electron density on the left and right of the xanthene ring. 2 (pipediEt-PPhR (pEPPR)) was synthesized and its optical properties were evaluated.
As a result, the fluorescence quantum yield was 9.4% to 6.7% from acidic to neutral, and it was revealed that the fluorescence quantum yield of pipenedo-PPhR was greatly improved from 1% or less (Fig. 2a). Moreover, the absorption / fluorescence wavelength of pEPPR still had the absorption / fluorescence wavelength in the near infrared region (FIGS. 2b and 2d). When the ratio of the fluorescence intensity at 730 nm when excited at 650 nm and 710 nm was plotted, a change in the fluorescence ratio value was observed, which proved to function as a pH probe (FIGS. 2e and f). However, it is calculated as pK _a 7.4 and 7.9, and it is considered desirable to reduce pK _a to about 7 in order to perform weakly acidic to neutral imaging (FIGS. 2c and f).

[Examples 3 to 5]
Three derivatives Me-pEPPR the optimization of pK _a was synthesized for the purpose, Et-pEPPR, for iPr-pEPPR (Compound 3-5) was examined optical properties. Both absorption and fluorescence spectra compared to PEPPR, while not give significant change in fluorescence quantum yield, the pK _a change was observed (Fig. 3,4,5). In particular, Et-pEPPR, pK _a of the Me-pEPPR indicated a value of around 7.0 which is suitable for observing the acidic environment in the body. Therefore, Me-pEPPR was used for the subsequent investigation of pH imaging.

[Example 6]
Ratio imaging using Me-pEPPR It was examined whether ratio imaging of pH was actually possible using Me-pEPPR. Excitation was performed with two wavelengths of 661 nm and 704 nm with an in vivo imaging apparatus Maestro (PerkinElmer), and a ratio image of each fluorescence image was constructed to examine whether it was applicable to fluorescence ratio imaging of pH.

FIGS. 6a and 6c show fluorescence ratio images of 2 μM Me-pEPPR in 0.1 M sodium phosphate buffer. Figures 6b and 6d represent the ratio of fluorescence intensity to pH and pKa. Error bars indicate standard error (n = 3).
Excitation filter: 661 nm: 641-681 nm, 704 nm: 684-729 nm, emission: 740-850 nm with 740 nm LP filter

FIGS. 6a and 6b show that Me-pEPPR is applicable to pH fluorescence ratio imaging. In particular, in the weakly acidic to neutral pH 6.0 to 7.4 in the living body, the sensitivity was high enough to detect the difference of pH 0.2, and the calibration curve was linearly approximated ( 6c, 6d).
In the following imaging, fluorescence ratio imaging was performed under these conditions.

[Example 7]
The application to in vivo imaging was examined by labeling the probe labeled on the polymer carrier with dextran, which is a water-soluble polymer carrier. Therefore, changes in probe characteristics due to labeling with dextran were investigated. An active ester form of Me-pEPPR was synthesized and labeled according to the following protocol.

Labeling Protocol (1) 40 kDa Dextran, Amino (registered trademark) was dissolved in 0.1 M NaHCO ₃ aqueous solution (0.25 mM). -(A)
(2) Me-pEPPR-SE was dissolved in 0.1M NaHCO ₃ aqueous solution / 40% DMSO. -(B)
(3) (a) and (b) were quickly mixed 1: 1 and vortexed at room temperature for 1 hour.
(4) The dextran-labeled product was separated with PD-10 using PBS (pH 7.4) as an elution solvent. -(C)
(5) (c) was desalted with PD-10 using miliQ as an elution solvent.
(6) Freeze-dried and re-dissolved in miliQ to prepare a 1 mM solution, which was used for measurement and imaging.

FIG. 7a shows the photophysical properties of a probe (Dex-Me-pEPPR) labeled with various equivalents of Me-pEPPR on dextran. From the figure, it was found that Dex-Me-pEPPR functions as a pH probe with no significant change in pK _a and fluorescence quantum yield before dextran labeling, regardless of the number of labels (DOL) (FIG. 7a). ).
Moreover, FIG. 7b shows the confocal fluorescence microscope image of A549 cell which carry | supported Me-pEPPR or Dex-Me-pEPPR (DOL5.7). The cell surface is indicated by a white dotted line. FIG. 7b shows that the probe can be retained extracellularly by binding to dextran (FIG. 7b).
In the following in vivo imaging, DOL5.7 or 1.4 Dex-Me-pEPPR was used.

[Example 8]
In vivo imaging of the gastrointestinal tract The pH of the gastrointestinal tract begins with the acidity of the stomach and is neutralized by pancreatic juice in the upper small intestine. In addition, it is known that from the lower small intestine to the large intestine, it becomes weakly acidic at about pH 5-6 by lactic acid produced by enteric bacteria. If the pH of the intestine can be quantified, great progress will be made in research on the function of intestinal bacteria. Therefore, the following protocol was used to examine whether the intestinal pH could be observed.

Fluorescent probe Dex-Me-pEPPR (DOL = 5.7) was orally administered to protocol mice, and laparotomy was performed after 2 hours, and fluorescence ratio imaging was performed.

FIG. 8 shows a fluorescence image of gastrointestinal pH of mice 2 hours after oral administration of 20 μM Dex-Me-pEPPR (DOL = 5.7) (20 μL). FIG. 8A is a white light image, and the digestive tract is indicated by a white dotted line. B in FIG. 8 is a fluorescence image at Ex / Em = 661 (641-681) nm, and c in FIG. 8 is a fluorescence image at Ex / Em = 704 (684-729) nm (740− 830 nm with 740 nm LP filter). FIG. 8D is a ratio image of b and c (Ex661 / Ex704).
As shown in FIG. 8d, the inventors succeeded in observing a state in which the small intestine (SI) started to be neutral and was acidified as it approached the large intestine (LI).

[Example 9]
In vivo imaging of the stomach Although the pH of the stomach is kept acidic, acid reflux may cause reflux esophagitis. Therefore, it is necessary to administer an antacid that raises the stomach pH to neutrality, but a technique capable of monitoring the stomach pH is important in the development of an antacid. Therefore, the following protocol was used to examine whether the change in gastric pH caused by the administration of existing antacids could be observed.
The fluorescence probe Dex-Me-pEPPR (DOL = 5.7) was orally administered to mice 2 hours before and 5 minutes before protocol fluorescence observation, and fluorescence ratio imaging was performed after laparotomy.

FIG. 9 shows mice after oral administration of 20 μM Dex-Me-pEPPR (DOL = 5.7) (20 μL × 2) (upper row) and after administration of an aqueous NaHCO ₃ solution (stomach antacid) (lower row). 2 shows fluorescent images of gastrointestinal pH. 9a and 9e are white light images, and the digestive tract is indicated by a white dotted line. B and f in FIG. 8 are fluorescence images at Ex / Em = 661 (641-681) nm, and c and g in FIG. 8 are fluorescence images at Ex / Em = 704 (684-729) nm. Yes (740-830 nm with 740 nm LP filter). In FIG. 8, d and h are ratio images of b and c and f and g, respectively (Ex661 / Ex704).
As a result, as shown in FIG. 9 h, the gastric pH was successfully neutralized by administration of about antacid (FIG. 9).

[Example 10]
In vivo pH imaging of tumors by blood administration In cancer cells, the glycolysis is enhanced, and the product H ⁺ derived from lactic acid is excessively produced in the cells. Therefore, in order to keep the inside of the cell neutral, H ⁺ transporter for discharging this H ⁺ out of the cell and CA9 for metabolizing CO ₂ as an acid are overexpressed, and the cancer cell periphery It has been reported that the extracellular pH of these drops to about 6.5-7.0. In addition, previous studies have suggested that the invasion ability of cancer cells is increased by lowering the pH around the cancer cells, which is considered to be one of the causes of cancer metastasis. Thus, if imaging of the acidic environment around cancer cells is achieved, it can contribute to basic research on cancer and evaluation of the efficacy of anticancer agents. Therefore, in vivo pH imaging of tumors by blood administration was performed using the following protocol.
One hour before the protocol fluorescence observation, a fluorescent probe Dex-Me-pEPPR (DOL = 1.4) was intravenously administered to the mice, and fluorescence ratio imaging was performed after incision of the skin.

10a and 10b show fluorescence ratio images at 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to Colon 26 tumor model mice. FIG. 10a shows a fluorescence image in a mouse fed with water, and FIG. 10b shows a fluorescence image in a mouse fed with 200 mM NaHCO ₃ aqueous solution. In the figure, K represents the kidney and T represents the tumor. (Ex / Em = 661 (641-681) nm or 704 (684-729) nm. (740-830 nm with 740 nm LP filter))
FIG. 10c shows pH histograms of mice fed with water (mock) and mice fed with 200 mM NaHCO ₃ (NaHCO ₃ ). FIG. 10d represents the mean pH of mice fed with water (mock) and mice fed with 200 mM NaHCO ₃ (NaHCO ₃ ) (error bar: SE (n = 2)).
The normal site showed neutrality at pH 7.3, but the vicinity of the tumor showed pH 6.9, and it was succeeded in imaging that acidified around pH 6.5-7.0 as reported in the past. (FIGS. 10a, 10c and 10d). In addition, neutralization and neutralization by administration of a drug (NaHCO ₃ aqueous solution) could be observed (FIGS. 10b, 10c, and 10d).

[Example 11]
In vivo pH imaging of the kidney Impaired transporter function in renal tubules causes changes in renal and systemic pH, leading to acidosis and alkalosis. Drug treatment is one of the effective treatments for such renal dysfunction, but it is important to evaluate renal function by drug administration in drug development. Therefore, the following professional sites were used to examine whether changes in renal pH due to drug administration could be detected.
One hour before the protocol fluorescence observation, a fluorescent probe Dex-Me-pEPPR (DOL = 1.4) was intravenously administered to mice, and fluorescence ratio imaging was performed after incision of the skin.

FIGS. 11a and 11b show fluorescence ratio images 1 hour after intravenous administration of Dex-Me-pEPPR (DOL = 1.4) (140 μM, 100 μL) to mice. FIG. 11a shows a fluorescence image in a mouse orally administered with physiological saline for 1 week, and FIG. 11b shows a fluorescence image in a mouse orally administered with acetazolamide for 1 week. In the figure, the kidney is indicated by a white arrow. (Ex / Em = 661 (641-681) nm or 704 (684-729) nm. (740-830 nm with 740 nm LP filter))
FIG. 11 c shows the average pH of mice (mock) orally administered with saline for 1 week and mice (AZM) orally administered with acetazolamide for 1 week (error bar: SE (n = 3)).
As shown in FIG. 11c, an increase in the pH of the kidney (urine) was observed by administration of the drug acetazolamide (FIG. 11c).
ADDIN EN.REFLIST

Claims (15)

The following general formula (I):

(Where
R ¹ represents a hydrogen atom or 1 to 3 identical or different monovalent substituents present on the benzene ring, and R ¹ may be the same or different;
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a fluorinated alkyl group having 1 to 6 carbon atoms. )
Or a salt thereof. The compound or a salt thereof according to claim 1, wherein R ^2a and R ^2b are each independently an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom. ^R2a and ^R2b are the compounds or its salt of Claim 2 which is a C1-C6 alkyl group. The compound or a salt thereof according to any one of claims 1 to 3, wherein R ^2a and R ^2b are each independently a methyl group or an ethyl group. R ¹⁰ is a hydrogen atom, a methyl group, an ethyl group, an isopropyl group, a trifluoromethyl group or a trifluoroethyl group, a compound or a salt thereof according to any one of claims 1 to 4. The compound or a salt thereof according to any one of claims 1 to 5, wherein R ⁸ and R ⁹ are each independently a hydrogen atom, a methyl group or an ethyl group. The compound or a salt thereof according to any one of claims 1 to 6, wherein R ¹ is a hydrogen atom. At least one of R ¹ is a carboxy group, an alkyl group having a carboxy group, an ester group, an alkyl ester group, an amino group, an amide group, an alkylamino group, an isothiocyanate group, a sulfonyl chloride group, a haloalkyl group, a haloacetamide group, The compound or salt thereof according to any one of claims 1 to 6, which is selected from an azide group or an alkynyl group. The compound or a salt thereof according to claim 8, wherein at least one of R ¹ is a carboxy group, an alkyl group having a carboxyl group, an amino group, or an amide group. The following general formula (II):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T;
T represents a bridging group, and the bridging group can be bonded to a functional group, a label site or a target accumulation site capable of introducing a label site or a target accumulation site at one or both ends thereof. May have a functional group;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer of 1 to 2 and n is an integer of 1 to 2, provided that m + n = 3;
Or a salt thereof. The following general formula (III):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a label site or a target accumulation site is bonded to T;
T ′, if present, is the structure after the bridging group is attached to S;
S. Indicates the label site or target accumulation site;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer of 1 to 2 and n is an integer of 1 to 2, provided that m + n = 3;
Or a salt thereof. -X- (T ')-S has the following structure, The compound of Claim 11 or its salt.

The following general formula (IV):

(Where
R ^2a and R ^2b each independently represent a monovalent substituent;
R ³ and R ⁴ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R ⁷ represents an optionally substituted phenyl group;
R ⁸ and R ⁹ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
R ⁸ and R ⁹ together may form a 4-7 membered heterocyclyl containing the nitrogen atom to which R ⁸ and R ⁹ are attached;
R ⁸ or R ⁹ , or both R ⁸ and R ⁹ together with R ³ , R ⁵ , respectively, are 5- to 7-membered heterocyclyls containing the nitrogen atom to which R ⁸ , R ⁹ is attached. Or a heteroaryl may be formed, and may contain 1 to 3 further heteroatoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom as a ring member, and the heterocyclyl. Alternatively, heteroaryl is alkyl having 1 to 6 carbons, alkenyl having 2 to 6 carbons, or alkynyl having 2 to 6 carbons, aralkyl group having 6 to 10 carbons, 6 to 10 carbons Optionally substituted with an alkyl-substituted alkenyl group;
R ¹⁰ represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a fluorinated alkyl group having 1 to 6 carbon atoms;
X represents a structure after a functional group capable of introducing a biopolymer labeling site is bonded to T;
T ′, if present, is the structure after the bridging group is attached to Dx;
Dx represents dextran;
R ¹ ′ is hydrogen or the same or different monovalent substituent;
m is an integer from 1 to 2 and n is an integer from 1 to 2, provided that m + n = 3;
p represents the binding equivalent to dextran;
Or a salt thereof.   The fluorescent probe containing the compound or its salt of any one of Claims 1-13. A method for measuring an acidic region in a cell,
A method comprising the steps of (a) introducing the compound or salt thereof according to claims 1 to 14 into a cell, and (b) measuring fluorescence emitted from the compound or salt thereof in the cell.

Images