JP2018025399A - Hydro-polysulfide detection fluorescence probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new fluorescence probe for detecting and visualizing intracellular hydro-polysulfide selectively and reversibly.SOLUTION: There is provided a fluorescence probe containing a compound expressed by the chemical formula (I) or a salt thereof (in which X denotes Si(R)(R), and Rand Rdenote H or an alkyl group).SELECTED DRAWING: None

Description

The present invention relates to a novel fluorescent probe for detecting hydropolysulfide, which is a compound having a —S _n SH group. More specifically, the present invention relates to a fluorescent probe for reversibly detecting hydropolysulfide in real time, a detection method using the fluorescent probe, and a detection kit including the probe.

Low molecular weight compounds containing elemental sulfur play an important role in maintaining the homeostasis of the intracellular redox environment. In recent years, biochemical and medical research on reactive sulfur species (RSS) has attracted attention. Has been. For example, zero-valent sulfur as represented by polysulfide (Sulfane Sulfur), and hydropersulfide (Cys-SSH, GSSH, etc.) The important role of hydropolysulfides (GSSSH, GSSSSH, etc.) in cells and in vivo has become clear. In addition, reports that polysulfidation of cysteine that constitutes protein plays an important role in the redox of active sulfur species, and that hydrogen sulfide (H ₂ S ₃ etc.) is produced during metabolic processes in neurons. Has been made in recent years.

Therefore, in order to investigate the causal relationship between such active sulfur species, especially hydropolysulfide and oxidative stress, and the relationship with diseases, development of a method capable of detecting and analyzing intracellular hydropolysulfide is required. . However,
At present, there is no choice but to rely on a destructive analysis technique that crushes cells and extracts components, and there are few means for visualizing the cells alive.

  On the other hand, although development of fluorescent probes for detecting hydropolysulfide has been attempted in recent years (for example, Non-Patent Documents 1 to 3), these existing probes have poor selectivity for hydropolysulfide and have a high response. Since it has no reversibility, there is a problem that it is impossible to visualize changes in concentration over time in cells.

W. Chen et al., Chem. Sci., 2013, 4, 2892-2896 C. Liu et al., J. Am. Chem. Soc., 2014, 136, 7257-7260 W. Chen et al., Angew. Chem. Int. Ed., 2015, 54, 13961-13965

International Publication WO2015 / 129705

  Accordingly, an object of the present invention is to provide a novel fluorescent probe for selectively and reversibly detecting and visualizing intracellular hydropolysulfide.

  The present inventors have already developed a fluorescent probe having a rhodamine skeleton capable of reversibly detecting a compound containing an —SH group such as glutathione (Patent Document 1). Here, glutathione is present in cells in an amount of about 0.5 to 10 mM, whereas hydropolysulfide is in a very small amount of about 1 to 10 μM in cells and about several tens of μM in organs. Therefore, a fluorescent probe for quantitatively detecting a hydropolysulfide having one or more —S—S— bonds in the molecule in an environment where a compound containing an —SH group such as glutathione exists is at least 1000 Double or more hydropolysulfide selectivity is required. On the other hand, as a result of intensive studies to solve the above problems, hydropolysulfide can be detected as a fluorescence response in a reversible and concentration-dependent manner by devising the structure of the rhodamine skeleton fluorophore. It was found that hydropolysulfide can be selectively detected even in the presence of a —SH group-containing compound such as a large excess of glutathione, and the present invention has been completed. Furthermore, the present inventors have developed a fluorescence resonance energy transfer (FRET) type fluorescent probe using the fluorophore as an acceptor, and found that real-time imaging of hydropolysulfide concentration change in living cells can be achieved.

〔式中、
Ｘは、Ｓｉ（Ｒ^ａ）（Ｒ^ｂ）を表し（ここで、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素原子、又はアルキル基を表す）；
Ｒ^１は、水素原子、シアノ基、又はそれぞれ置換されていてもよいアルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、及びアジド基よりなる群から独立に選択される１〜４個の同一又は異なる置換基を表し；
Ｒ^２は、ハロゲン原子、それぞれ置換されていてもよいアルキル基、又はアルコキシ基を表し；
Ｒ^３及びＲ^４は、それぞれ独立に、水素原子、又はヒドロキシル基、ハロゲン原子、それぞれ置換されていてもよいアルキル基、スルホ基、カルボキシル基、エステル基、アミド基及びアジド基よりなる群から独立に選択される１〜３個の同一又は異なる置換基を表し；
Ｒ^５、Ｒ^６、Ｒ^７及びＲ^８は、いずれもアルキル基を表し、
ここで、Ｒ^５又はＲ^６は、それぞれＲ^３と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよく、
Ｒ^７又はＲ^８は、それぞれＲ^４と一緒になって、それらが結合する窒素原子を含む環構造を形成してもよい。〕；
＜２＞Ｒ^５、Ｒ^６、Ｒ^７及びＲ^８が、いずれもメチル基である、上記＜１＞に記載の蛍光プローブ；
＜３＞Ｒ^２が、塩素原子、メチル基、又はメトキシ基である、上記＜１＞に記載の蛍光プローブ；
＜４＞Ｒ^５又はＲ^６が、それぞれＲ^３と一緒になって、それらが結合する窒素原子を含む５員環を形成し；及び／又は、Ｒ^７又はＲ^８が、それぞれＲ^４と一緒になって、それらが結合する窒素原子を含む５員環を形成している、上記＜１＞に記載の蛍光プローブ；
＜５＞Ｒ^１が、蛍光共鳴エネルギー移動（ＦＲＥＴ）のドナーとなる蛍光団を有する、上記＜１＞〜＜４＞のいずれか１に記載の蛍光プローブ；及び
＜６＞前記蛍光団が、キサンテン骨格を有する化合物である、上記＜５＞に記載の蛍光プローブ
を提供するものである。 That is, the present invention in one aspect,
<1> A fluorescent probe for detecting hydropolysulfide, comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
X represents Si (R ^a ) (R ^b ) (where R ^a and R ^b each independently represents a hydrogen atom or an alkyl group);
R ¹ is independently selected from the group consisting of a hydrogen atom, a cyano group, or an optionally substituted alkyl group, carboxyl group, ester group, alkoxy group, amide group, and azide group. Represents the same or different substituents;
R ² represents a halogen atom, an optionally substituted alkyl group, or an alkoxy group;
R ³ and R ⁴ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents selected from
R ⁵ , R ⁶ , R ⁷ and R ⁸ all represent an alkyl group,
Here, R ⁵ or R ⁶ together with R ³ may form a ring structure containing a nitrogen atom to which they are bonded,
R ⁷ or R ⁸ may be combined with R ⁴ to form a ring structure containing a nitrogen atom to which they are bonded. ];
<2> The fluorescent probe according to <1>, wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ are all methyl groups;
<3> The fluorescent probe according to <1>, wherein R ² is a chlorine atom, a methyl group, or a methoxy group;
<4> R ⁵ or R ⁶ together with R ³ form a 5-membered ring containing the nitrogen atom to which they are attached; and / or R ⁷ or R ⁸ together with R ⁴ respectively And forming a 5-membered ring containing a nitrogen atom to which they are bonded; the fluorescent probe according to <1>above;
<5> The fluorescent probe according to any one of <1> to <4>, wherein R ¹ has a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET); and <6> the fluorophore is The fluorescent probe according to <5>, which is a compound having a xanthene skeleton, is provided.

In another aspect, the present invention provides:
<7> A method for detecting hydropolysulfide using the fluorescent probe according to any one of <1> to <6>above;
<8> The detection method according to <7>, wherein the presence of the hydropolysulfide is detected by observing a fluorescence response or a change in absorbance due to a reaction between the hydropolysulfide and the fluorescent probe;
<9> The detection method according to <8>, wherein the fluorescence response is a fluorescence change caused by fluorescence resonance energy transfer (FRET);
<10> The detection method according to <8> above, wherein the fluorescence response is visualized using a fluorescence imaging means; and <11> any one of <1> to <6> above A kit for detecting a hydropolysulfide including a fluorescent probe is provided.

According to the present invention, hydropolysulfide can be detected reversibly and in a concentration-dependent manner by a fluorescence response, and the detection and visualization of hydropolysulfide can be performed selectively and reversibly in living cells. In particular, the fluorescent probe of the present invention can select a hydropolysulfide having one or more —SS—bonds in the molecule 1000 times or more even in the presence of a large excess of an —SH group-containing compound such as glutathione. Since the dissociation constant (K _d ) is within the range of intracellular hydropolysulfide concentration, it is suitable for live cell imaging.

  The detection method of the present invention can be performed with a microscope capable of normal cell imaging, and does not require any special equipment. In addition, since quantification over time is possible, it is possible to grasp the decrease in hydropolysulfide and the recovery from oxidative stress exposure when various stimuli are given or repeatedly added to the same cell. From the basic research of the present invention, it can be said that the industrial utility value and the economic effect are extremely large.

FIG. 1 is a diagram showing a chemical structural formula of Compound 1 (2Me SiR650), which is a fluorescent probe of the present invention, and a change in absorption spectrum associated with the addition of sodium disulfide (Na ₂ S ₂ ). FIG. 2 is a graph showing changes in the fluorescence spectrum associated with the addition of sodium disulfide (Na ₂ S ₂ ) to compound 1 (2Me SiR650), which is the fluorescent probe of the present invention. FIG. 3 shows the time dependence of the change in absorption spectrum intensity when sodium disulfide (Na ₂ S ₂ ) is added to the fluorescent probe compound 1 of the present invention and then N-ethylmaleimide (NEM) is added. It is a graph. FIG. 4a is a schematic diagram of the FRET type luminescence mechanism in Compound 5 (TMR-SiR650) which is a fluorescent probe of the present invention; FIG. 4b is a sodium disulfide (5) of Compound 5 (TMR-SiR650) which is a fluorescent probe of the present invention. Na ₂ S ₂₎ Figure showing the absorption spectrum changes due to the addition; Figure 4c, disodium sulfide (Na ₂ S ₂₎ fluorescent spectra change with the addition of a fluorescent probe that compounds of the present invention 5 (TMR-SiR650) FIG. 4d is a graph plotting the change in the fluorescence intensity ratio between the short wavelength side peak and the isofluorescent point in FIG. 4c. FIG. 5 shows A549 cells obtained by administering sodium disulfide (Na ₂ S ₂ ) and thiol group scavenger N-ethylmaleimide (NEM) to compound 5 (TMR-SiR650) which is a fluorescent probe of the present invention. It is the figure which showed the time change (FIG. 5a) of the fluorescence imaging, and the time change (FIG. 5b) of the fluorescence intensity in that case. FIG. 6 shows temporal changes in fluorescence imaging of A549 cells when BnSSH and N-ethylmaleimide (NEM) were administered to Compound 5 (TMR-SiR650), which is the fluorescent probe of the present invention (FIG. 6a), and the fluorescence at that time. The figure which showed the time change of an intensity | strength (FIG. 6b); It is the figure which showed the time change (FIG. 6c) of the fluorescence imaging at the time of washing | cleaning after BnSSH administration (FIG. 6c) and the fluorescence intensity at that time (FIG. 6d). FIG. 7 shows the fluorescence of A549 cells when membrane-permeable glutathione (GSH (Et)), sodium disulfide (Na ₂ S ₂ ), and BnSSH are added to compound 5 (TMR-SiR650), which is the fluorescent probe of the present invention. It is a figure (Drawing 7b) which shows a change of an imaging picture (Drawing 7a) and a signal ratio value. FIG. 8 is a diagram showing a change (FIG. 8a) in the fluorescence imaging image of Compound 5 (TMR-SiR650), which is a fluorescent probe of the present invention in A549 cells, (FIG. 8a) and a change in signal ratio value at that time (FIG. 8b). ). FIG. 9 is a diagram showing changes in the fluorescence imaging image of the compound 5 (TMR-SiR650), which is the fluorescent probe of the present invention in A549 cells (FIG. 8a), and changes in the signal ratio at that time (FIG. 8b). It is.

  Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Definitions In this specification, “hydropolysulfide” (sometimes abbreviated simply as polysulfide) has one or more —S—S— bonds in the molecule and has the general formula R—S _n SH. It is a sulfur compound represented by these. When n = 1, it is called hydropersulfide (sometimes abbreviated simply as persulfide), and when R is hydrogen, it is called hydrogen polysulfide (Hydrogen polysulfide). In this specification, hydro polysulfide is intended to include both of these hydro Perth sulfide and polysulfide hydrogen, collectively may be referred to as R-S n _SH. Although the typical compound example is shown below, it is not limited to these. Detailed definitions and other specific examples of hydropolysulfides can be found in, for example, TV Mishanina et al., Nat. Chem. Biol., 2015, 11, 457-464; C.-M. Park et al., Mol. BioSyst., 2015, 11, 1775-1785; alternatively, TS Bailey et al., Free Radical Biol. Med., 2015, 89, 662-667.

  In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present specification, “alkyl” may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited, for example, 1-20 carbon atoms _{(C 1-20),} 1 to 15 carbon atoms _{(C 1 to 15),} 1 to 10 carbon atoms _{(C 1 to 10} ). When the number of carbons is specified, it means “alkyl” having the number of carbons within the range. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of such a substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl part of other substituents containing an alkyl part (for example, an alkoxy group, an arylalkyl group, etc.).

  In the present specification, when a functional group is defined as “may be substituted”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different. Examples of the substituent group include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.

  In the present specification, “alkenyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon double bond. For example, as non-limiting examples, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl 4-pentenyl, 1,3-pentanedienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1,4-hexanedienyl). The double bond may be either cis or trans conformation.

  In the present specification, “alkynyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond. For example, non-limiting examples include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl.

  In the present specification, “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic ring system composed of the above alkyl. The cycloalkyl can be unsubstituted or substituted by one or more substituents, which can be the same or different, and non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl Non-limiting examples of polycyclic cycloalkyl include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl and the like. In addition, the cycloalkyl may be a heterocycloalkyl containing one or more hetero atoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as ring-constituting atoms. Any —NH in the heterocycloalkyl ring may be protected, for example as a —N (Boc) group, —N (CBz) group and —N (Tos) group, a nitrogen atom in the ring or The sulfur atom may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. For example, non-limiting examples of monocyclic heterocycloalkyl include diazapanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, lactam and Examples include lactones.

  As used herein, “cycloalkenyl” refers to a monocyclic or polycyclic non-aromatic ring system containing at least one carbon-carbon double bond. The cycloalkenyl may be unsubstituted or substituted by one or more substituents, which may be the same or different, and non-limiting examples of monocyclic cycloalkenyl include cyclopentenyl, cyclohexenyl And cyclohepta-1,3-dienyl, and non-limiting examples of polycyclic cycloalkenyl include norbornylenyl and the like. In addition, the cycloalkyl may be a heterocycloalkenyl which may be a heterocycloalkenyl containing one or more heteroatoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. Atoms may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.

  In the present specification, “aryl” may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom Etc.) may be an aromatic heterocyclic ring. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether aryl is a single ring or a fused ring, it can be attached at all possible positions. Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl Group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazole-3 -An yl group etc. are mentioned. Non-limiting examples of fused polycyclic aryl include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3 -Dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, Isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 , 3-Dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, thioxanthenyl group, etc. And the like. In the present specification, an aryl group may have one or more arbitrary substituents on the ring. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and acyl. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing the aryl moiety (for example, an aryloxy group and an arylalkyl group).

  In the present specification, “arylalkyl” represents an alkyl substituted with the above aryl. The arylalkyl may have one or more arbitrary substituents. Examples of the substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, and an acyl group. When the acyl group has two or more substituents, they may be the same or different. Non-limiting examples of arylalkyl include benzyl group, 2-thienylmethyl group, 3-thienylmethyl group, 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group, 2-furylmethyl group, 3-furylmethyl group, 2-thiazolylmethyl group, 4-thiazolylmethyl group, 5-thiazolylmethyl group, 2-oxazolylmethyl group, 4-oxazolylmethyl group, 5-oxazolylmethyl group, 1-pyrazolylmethyl group 3-pyrazolylmethyl group, 4-pyrazolylmethyl group, 2-pyrazinylmethyl group, 2-pyrimidinylmethyl group, 4-pyrimidinylmethyl group, 5-pyrimidinylmethyl group, 1-pyrrolylmethyl group, 2-pyrrolylmethyl group, 3-pyrrolylmethyl group 1-imidazolylmethyl group, 2-imidazolylmethyl group, 4-imidazolylmethyl 3-pyridazinylmethyl group, 4-pyridazinylmethyl group, 3-isothiazolylmethyl group, 3-isoxazolylmethyl group, 1,2,4-oxadiazol-5-ylmethyl group Or a 1,2,4-oxadiazol-3-ylmethyl group etc. are mentioned.

  Similarly, in the present specification, “arylalkenyl” represents alkenyl substituted with aryl.

  In the present specification, the “alkoxy group” is a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group that is linear, branched, cyclic, or a combination thereof. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n- Pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy or cyclopentylmethyloxy are preferred Take as an example.

  In the present specification, “alkylene” is a divalent group consisting of a linear or branched saturated hydrocarbon, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1 -Methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1 -Diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethyl , Tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyl Trimethylene and the like.

  In the present specification, “alkenylene” is a divalent group consisting of an unsaturated hydrocarbon having at least one linear or branched carbon-carbon double bond, such as ethenylene. 1-methylethenylene, 1-ethylethenylene, 1,2-dimethylethenylene, 1,2-diethylethenylene, 1-ethyl-2-methylethenylene, propenylene, 1-methyl-2-propenylene, 2-methyl -2-propenylene, 1,1-dimethyl-2-propenylene, 1,2-dimethyl-2-propenylene, 1-ethyl-2-propenylene, 2-ethyl-2-propenylene, 1,1-diethyl-2-propenylene 1,2-diethyl-2-propenylene, 1-butenylene, 2-butenylene, 1-methyl-2-butenylene, 2-methyl-2-butenylene, , 1-dimethyl-2-butenylene, 1,2-dimethyl-2-butenylene, and the like.

  In the present specification, “arylene” and “arylalkylene” mean a divalent group based on the above “aryl” and “arylalkyl”, respectively. Similarly, “oxyalkylene” and “aryleneoxy” mean a divalent group based on the above “alkoxy” and “aryloxy”, respectively.

  As used herein, “amide” includes both RNR′CO— (when R = alkyl, alkaminocarbonyl-) and RCONR′- (where R = alkyl, alkylcarbonylamino-).

  As used herein, “ester” includes both ROCO— (in the case of R = alkyl, alkoxycarbonyl-) and RCOO— (in the case of R = alkyl, alkylcarbonyloxy-).

  As used herein, the term “ring structure”, when formed by a combination of two substituents, means a heterocyclic or carbocyclic group, such group being saturated, unsaturated, or aromatic. Can be. Accordingly, it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperidinyl, imidazolyl, pyrrolidinyl, pyridyl and the like. In the present specification, a substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will recognize a specific substitution, such as bonding to hydrogen. Can be understood. Therefore, when it is described that specific substituents together form a ring structure, those skilled in the art can understand that the ring structure can be formed by an ordinary chemical reaction and can be easily generated. Both such ring structures and their process of formation are within the purview of those skilled in the art.

2. Fluorescent probe for detecting hydropolysulfide according to the present invention In one aspect, the fluorescent probe according to the present invention includes a compound having a structure represented by the following formula (I).

In the above formula (I), X represents Si (R ^a ) (R ^b ). Here, R ^a and R ^b each independently represent a hydrogen atom or an alkyl group. When R ^a and R ^b are alkyl groups, they can have one or more substituents, and examples of such substituents include alkyl groups, alkoxy groups, halogen atoms, hydroxyl groups, carboxyl groups, You may have 1 or 2 or more amino groups, sulfo groups, etc. R ^a and R ^b are preferably both methyl groups. In some cases, R ^a and R ^b may be bonded to each other to form a ring structure. For example, when R ^a and R ^b are both alkyl groups, R ^a and R ^b can be bonded to each other to form a spirocarbocycle. The ring formed is preferably about 5 to 8 membered ring, for example. X is preferably Si (CH ₃ ) ₂ .

R ¹ represents a hydrogen atom, a cyano group, or an optionally substituted alkyl group (for example, the “optionally substituted alkyl group” may include haloalkyl such as fluoroalkyl), a carboxyl group, 1 to 4 identical or different substituents independently selected from the group consisting of an ester group, an alkoxy group, an amide group, and an azide group. When R ¹ is other than a hydrogen atom, the position in the benzene ring is not particularly limited, but is preferably a meta position with respect to the substituent having R ² . Moreover, when it has two or more substituents on the benzene ring, they may be the same or different. From the viewpoint of suppressing localization of the probe molecule in the cell, R ¹ is preferably, for example, iminodiacetic ester.

Further, as will be described later, R ¹ may have a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET). In this case, typically, R ¹ is preferably an amide group, and a fluorophore serving as a donor can be linked via the amide group. As such a fluorophore, a fluorophore known in the technical field related to FRET can be used as long as it is a fluorophore that can serve as a donor for the rhodamine-like skeleton site in formula (I). Non-limiting examples include pyrene, perylene, coumarin, fluorescein, rhodamine, cyanine, boron dipyrromethene (BODIPY), oxazine, and the like. Preferably, such a fluorophore is a compound having a xanthene skeleton, such as tetramethylrhodamine.

R ² represents a halogen atom, an optionally substituted alkyl group, or an alkoxy group. Preferably, they are a chlorine atom, a methyl group, or a methoxy group.

R ³ and R ⁴ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents. Preferably, R ³ and R ⁴ are both hydrogen atoms. Similarly to R ¹ , R ³ or R ⁴ may have a fluorophore that serves as a donor for fluorescence resonance energy transfer (FRET).

R ⁵ , R ⁶ , R ⁷ and R ⁸ all represent an alkyl group. Here, R ⁵ or R ⁶ may be combined with R ³ to form a ring structure containing a nitrogen atom to which they are bonded. In that case, only one of R ⁵ and R ³ or a combination of R ⁶ and R ³ may form a ring structure, or both may form a ring structure. The ring structure may contain further hetero atoms other than the nitrogen atom.

Similarly, when either R ⁷ or R ⁸ is an alkyl group, it may be combined with R ⁴ to form a ring structure containing the nitrogen atom to which they are attached. In that case, only one of R ⁷ and R ⁴ , or a combination of R ⁸ and R ⁴ may form a ring structure, or both may form a ring structure. The ring structure may contain further hetero atoms other than the nitrogen atom.

Preferably, R ⁵ , R ⁶ , R ⁷ and R ⁸ are all methyl groups.

が挙げられる。ただし、これらに限定されるものではない。 Specific examples of compounds of formula (I) that are particularly suitable as fluorescent probes for detecting hydropolysulfides of the present invention include:

Is mentioned. However, it is not limited to these.

Since the compound represented by the above formula (I) has a monovalent positive charge at the N atom to which R ⁷ and R ⁸ are linked, it usually exists as a salt. Examples of such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. As, for example, mineral acid salts such as hydrochloride, sulfate, and nitrate, carboxylate, trifluoroacetate, methanesulfonate, paratoluenesulfonate, citrate, oxalate, and other organic acid salts Can be mentioned. Examples of amino acid salts include glycine salts. However, it is not limited to these salts.

  The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

  The compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.

  The above-described fluorescent probe may be used as a composition by blending additives usually used in the preparation of reagents as required. For example, additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for use in a physiological environment, and the amount of these can be appropriately selected by those skilled in the art. is there. These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, or a liquid.

  In the examples of the present specification, production methods for representative compounds included in the compounds of the present invention represented by the formula (I) are specifically shown. Any compound included in the formula (I) can be easily produced by referring to, and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.

3. Method for Detecting Hydropolysulfide Using Fluorescent Probe of the Present Invention The fluorescent probe of the present invention has a hydropolysulfide S-position as shown in the following equilibrium formula at the 9-position of the xanthene-like condensed ring represented by the formula (I). A mechanism is used in which absorption and fluorescence in the visible light region disappear when a concentration-dependent nucleophilic attack is caused by the -SR group (herein, hydropolysulfide is indicated as "R-SSH"). The reaction is reversible. Thereby, the hydropolysulfide having one or more -SS-bonds can be detected reversibly, with time, and quantitatively. As described above, examples of the “hydropolysulfide” include both hydropersulfide and hydrogen polysulfide, and can broadly include compounds having a structure of R—S _n SH, and the following reaction proceeds. As long as this is the case, the fluorescent probe of the present invention can be applied to these compounds and the like.

Here, hydropolysulfide is very small amount of about 1 to 10 μM in cells and about several tens of μM in organs, whereas glutathione is present in cells at a concentration of about 0.5 to 10 mM. A fluorescent probe for quantitatively detecting a hydropolysulfide having one or more -SS-bonds in a molecule in an environment where a compound containing an -SH group exists includes at least a 1000-fold or more hydropolysulfide Selectivity is required. The present invention has surprisingly found a fluorescent probe molecule capable of specifically detecting hydropolysulfide despite having a molecular skeleton relatively similar to the fluorescent probe molecule for glutathione. More specifically, although not necessarily bound by theory, the present invention uses a Si atom as X in formula (I), R ² is a specific substituent, and R ⁵ , R ⁶ , R By using a molecule having a structure in which both ⁷ and R ⁸ are alkyl groups, glutathione (approximately 0.5 to 10 mM) in a large excess does not show a response and exhibits high selectivity for hydropolysulfide, and It is characterized in that it has found a fluorescent probe molecule that provides a low dissociation constant K _d (around 10 μM) with respect to the intracellular hydropolysulfide concentration.

Further, by providing a further fluorophore that can serve as a donor of fluorescence resonance energy transfer (FRET) in the molecule of formula (I), the fluorescence resonance energy transfer (FRET) in which the rhodamine-like skeleton of formula (I) serves as an acceptor. It is also possible to detect the reaction with hydropolysulfide as a change in emission wavelength. That is, in an environment where no hydropolysulfide exists, light emission at a fluorescence wavelength unique to the rhodamine-like skeleton is observed, but the reaction from the compound of formula (I) with hydropolysulfide quenches the fluorescence derived from the rhodamine-like skeleton. By increasing the emission at the fluorescence wavelength inherent to the donor fluorophore, the detection of hydropolysulfide can be detected as the fluorescence emission. Therefore, the fluorescence spectrum of the donor fluorophore overlaps with the absorption spectrum of the rhodamine-like skeleton, and the fluorescence wavelength of the donor fluorophore is preferably in a wavelength region different from the fluorescence wavelength of the rhodamine-like skeleton. As described above, the donor fluorophore may be a fluorophore known in the technical field related to FRET, such as pyrene, perylene, coumarin, fluorescein, rhodamine, cyanine, boron dipyrromethene (BODIPY), oxazine, and the like. Can be mentioned. Tetramethylrhodamine is preferable. Typically, such fluorophores can be introduced into compounds of formula (I) via the amide group of R ¹ .

  According to the above light emission mechanism, the hydropolysulfide detection method of the present invention is characterized in that the presence of hydropolysulfide is detected by observing a fluorescence response or a change in absorbance due to a reaction between the hydropolysulfide and the fluorescent probe. In this specification, the term “detection” should be interpreted in the broadest sense including measurement for various purposes such as quantification and qualitative. As described above, the fluorescence response is preferably a fluorescence change caused by fluorescence resonance energy transfer (FRET).

  As a means for observing the fluorescence response, a fluorometer having a wide measurement wavelength can be used, but it is also possible to visualize the fluorescence response using a fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image. By using the fluorescence imaging means, the fluorescence response can be visualized in two dimensions, so that the hydropolysulfide can be viewed instantaneously. As the fluorescence imaging apparatus, an apparatus known in the technical field can be used.

  The means for bringing the hydropolysulfide sample to be measured into contact with the fluorescent probe typically includes adding, applying, or spraying a solution containing the fluorescent probe, but the sample form, measurement environment, etc. It is possible to select appropriately according to the situation.

  The application concentration of the fluorescent probe of the present invention is not particularly limited, but for example, a solution having a concentration of about 0.1 to 10 μM can be applied.

  As the fluorescent probe of the present invention, the compound represented by the above formula (I) or a salt thereof may be used as it is, but if necessary, an additive usually used for the preparation of a reagent is blended as a composition. It may be used. For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible. These compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It is sufficient to dissolve and apply to.

4). Kit for detecting hydropolysulfide using the fluorescent probe of the present invention In the detection method of the present invention, it is preferable to use a kit for detecting hydropolysulfide containing the fluorescent probe. In the kit, the fluorescent probe of the present invention is usually prepared as a solution. However, for example, it is provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid. It can also be applied by dissolving in distilled water for injection or an appropriate buffer.

  In addition, the kit may appropriately contain other reagents as necessary. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives, and the amount of these additives can be appropriately selected by those skilled in the art.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.

[Reagents, equipment, etc.]
The organic solvents used in the reactions shown below were all dehydrated grades. Commercial raw materials were purchased from reagent manufacturers (Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., Kanto Chemical Co., Ltd., Sigma-Aldrich Co. Ltd., Acros Co. Ltd.).

The following apparatus and column were used for purification by high performance liquid chromatography.
・ Pump: PU-2080 and PU-2087 (JASCO Corporation)
・ Detector: MD-2010 (JASCO Corporation)
Column: Inertsil ODS-3 (10 x 250 mm or 20 x 250 mm, GL Science Inc.)

In the separation and purification by HPLC, the following solvents A, B and C were used and purified by mixing them in an arbitrary composition unless otherwise specified.
A: Purified water (containing 1% acetonitrile, 0.1% trifluoroacetic acid)
B: Acetonitrile (including 1% purified water)
C: Purified water (including 0.2M acetic acid-triethylamine)
For HPLC separation, the flow rate is 25 ml / min (pump: PU-2087, column: 20 x 250 mm), 5 ml / min (pump: PU-2080, column: 10 x 250 mm), 1 ml / min, respectively. (Pump: PU-2080, column: 4.6 x 200 mm). Purification by medium pressure column chromatography was performed using YFLC-Al580 (Yamazen Co., Ltd.).

  NMR measurement was performed using AVANCE III 400 Nanobay (Bruker, Co. Ltd.) (400 MHz for 1H NMR, 101 MHz for 13C NMR). Mass spectrometric measurements were performed using MicrOTOF (ESI-TOF, Bruker, Co. Ltd.). For high-resolution MS (HRMS) measurement, sodium formate was used as an external standard.

Abbreviations used in the following examples are as follows.
THF: tetrahydrofuran
DMF: N, N-dimethylformamide
TFA: trifluoroacetic acid

これらのうち、化合物１と４は、公知の文献（J. Am. Chem. Soc., 2012, 134 (11), pp 5029‐5031）に記載の合成例に基づいて合成した。また、以下の合成例で用いた化合物のうち、化合物Ａ１はJ. Am. Chem. Soc., 2012, 134 (11), pp 5029‐5031、化合物Ｃ１はBioorg. Med. Chem. Lett. 21, 2869-2872 (2011)、化合物Ｃ３はWO2015/129705の実施例の記載の手法に基づいてそれぞれ合成した。 1. Synthesis of fluorescent probe The following compounds 1 to 5, which are compounds of the formula (I) of the present invention, were synthesized.

Of these, compounds 1 and 4 were synthesized based on synthesis examples described in known literature (J. Am. Chem. Soc., 2012, 134 (11), pp 5029-5031). Of the compounds used in the following synthesis examples, compound A1 is J. Am. Chem. Soc., 2012, 134 (11), pp 5029-5031, and compound C1 is Bioorg. Med. Chem. Lett. 21, 2869-2872 (2011), Compound C3 was synthesized based on the procedure described in the examples of WO2015 / 129705.

Synthesis of Compound 2 Compound 2, which is a fluorescent probe of the present invention, was synthesized according to the following scheme.

2-Bromoanisole (50 mg, 0.293 mmol, 8.4 eq.) Was dissolved in THF (2 ml) and stirred at −78 ^° C. for 10 minutes under an argon atmosphere. 1 M sec-butyllithium cyclohexane / n-hexane solution (250 μl, 0.250 mmol, 7.1 eq.) Was slowly added and stirred for 10 minutes. Compound A1 (10 mg, 0.035 mmol, 1 eq.) Dissolved in THF (2 ml) was added, and the mixture was stirred at −78 ^° C. for 1 hour and at room temperature for 6 hours. Add 1 N hydrochloric acid to acidify, then add saturated aqueous sodium hydrogen carbonate solution, extract twice with dichloromethane, wash the resulting organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure . The obtained residue was purified by HPLC (A / B = 90/10 to 10/90, 25 min) to obtain the target compound (2) (15.3 mg, 94%) as a blue solid.
¹ H NMR (CD ₃ OD): δ 7.56 (dt, J = 7.4 Hz, 1.8 Hz, 1H), 7.32 (d, J = 2.8 Hz, 2H), 7.22-7.16 (m, 3H) 7.15 (d, J = 7.4 Hz, 1H), 7.08 (dd, J = 7.4 Hz, 1.8 Hz, 1H), 6.75 (dd, J = 9.6 Hz, 2.8 Hz, 2H), 3.71 (s, 3H), 3.34 (s, 12H) , 0.61 (s, 3H), 0.58 (s, 3H) .; ¹³ C NMR (CD ₃ OD) δ 169.5, 158.1, 155.7, 149.4, 142.7, 131.8, 131.4, 129.2, 129.1, 121.8, 121.4, 114.9, 112.3 , 56.2, 40.8, -1.0, -1.4 ppm.

Synthesis of compound 3
According to the following scheme, Compound 3, which is a fluorescent probe of the present invention, was synthesized.

2-Chlorophenylboronic acid was dried under reduced pressure at 110 ° C. for 6 hours to obtain Compound B1 as a white solid. Compound B1 was used in the next reaction without separation and purification.
Compound A1 (30 mg, 0.092 mmol, 1 eq.) Was dissolved in deoxygenated acetonitrile (10 ml) and stirred at room temperature under an argon atmosphere. Slowly add trifluoromethanesulfonic anhydride (20 μl, 0.112 mmol, 1.2 eq.) And stir for another 15 minutes at room temperature, then compound B1 (61.1 mg, 0.144 mmol, 1.6 eq.), Bis (triphenylphosphine) ) Palladium (II) dichloride (13.1 mg, 0.018 mmol, 0.2 eq.) And sodium carbonate (62.3 mg, 0.587 mmol, 6.2 eq.) Were added, and the mixture was stirred at 70 ° C. overnight. After returning to room temperature, water was added to the reaction solution, and the mixture was extracted twice with dichloromethane. The obtained organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 100/0 to 90/10) and HPLC (A / B = 90/10 to 10/90, 25 min) to obtain the target compound 3. (3.0 mg, 6%) was obtained as a blue solid.
¹ H NMR (CD ₃ OD): δ 7.65 (dd, J = 8.0 Hz, 1.4 Hz, 1H), 7.59 (ddd, J = 8.0 Hz, 7.3 Hz, 1.8 Hz, 1H), 7.53 (dt, J = 1.4 Hz.7.3 Hz, 1H), 7.37 (d, J = 2.8 Hz, 2H), 7.32 (dd, J = 7.3 Hz, 1.8 Hz, 1H), 7.04 (d, J = 9.6 Hz, 2H), 6.80 (dd , J = 9.6 Hz, 2.8 Hz, 2H), 3.36 (s, 12H), 0.613 (s, 3H), 0.598 (s, 3H) .; ¹³ C NMR (CD ₃ OD): δ 167.0 (C), 155.8 (C), 149.5 (C), 142.0 (CH), 139.3 (C), 134.1 (C), 132.2 (CH), 131.7 (CH), 130.9 (CH), 128.4 (C), 128.1 (CH), 122.3 (CH), 115.3 (CH), 40.9 (CH ₃ ), -1.1 (CH ₃ ), -1.4 (CH ₃ ) .; HRMS-ESI (m / z): [M] ⁺ calcd for C ₂₅ H ₂₈ ClN ₂ Si: 419.17048; found: 419.16962 (0.9 mDa, 2.0 ppm).

Synthesis of compound 5
According to the following scheme, Compound 5, which is a fluorescent probe of the present invention, was synthesized.

[Synthesis of Compound C2]
Compound C1 (323 mg, 1.20 mmol, 3.2 eq.) Was dissolved in THF (10 ml) and stirred at −78 ^° C. for 10 minutes under an argon atmosphere. 1.1 M sec-butyllithium cyclohexane / normal hexane solution (550 μl, 0.61 mmol, 2.0 eq.) Was slowly added and stirred for 10 minutes. Compound A1 (122 mg, 0.376 mmol, 1 eq.) Was added by dissolving in THF (5 ml), and the mixture was stirred at −78 ^° C. for 1 hour and at room temperature for 2 hours. Add 1 N hydrochloric acid to acidify, then add saturated aqueous sodium hydrogen carbonate solution, extract twice with dichloromethane, wash the resulting organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure . The obtained compound was dissolved in trifluoroacetic acid (2 ml) and stirred at room temperature for 30 minutes. The solvent was distilled off under reduced pressure, and the resulting residue was purified by HPLC (A / B = 90/10 to 10/90, 25 min) to obtain the target compound C2 (126 mg, 60%) as a blue-violet solid. .
¹ H NMR (CD ₃ OD): δ 7.97 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 2.8 Hz, 2H), 7.12 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 9.6 Hz, 2H), 6.77 (dd, J = 9.6 Hz, 2.8 Hz, 2H), 3.34 (s, 12H), 2.05 (s, 3H), 0.62 (s, 3H) , 0.60 (s, 3H) .; ¹³ C NMR (CD ₃ OD): δ 173.5 (C), 170.6 (C), 155.8 (C), 149.4 (C), 142.34 (C), 142.24 (CH), 138.7 (C), 136.7 (C), 132.1 (CH), 129.8 (CH), 128.3 (CH), 127.7 (C), 122.2 (CH), 115.3 (CH), 40.9 (CH ₃ ), 19.5 (CH ₃ ) , -1.1 (CH ₃ ), -1.3 (CH ₃ ) .; HRMS-ESI (m / z): [M] ⁺ calcd for C ₂₇ H ₃₁ N ₂ O ₂ Si: 443.21493; found: 443.21524 (-0.3 mDa , -0.7 ppm).

[Synthesis of Compound 5]
Compound C2 (5.3 mg, 9 μmol, 1 eq.), Compound C3 (7.0 mg, 9 μmol, 1 eq.) And hexafluorophosphate (benzotriazol-1-yloxy) tripyrrolidinophosphonium (9.1 mg, 17 μmol) , 1.9 eq.) Was dissolved in DMF (500 μl) and stirred at room temperature. N, N-Diisopropylethylamine (15 μl, 85 μmol, 9.4 eq.) Was added, and the mixture was further stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, and the resulting residue was separated and purified by HPLC (first time; A / B = 90/10 to 10/90, 25 min: second time; C / B = 90/10 to 10 / 90, 40 min), and the target compound 5 (6.4 mg, 59%) was obtained as a dark purple solid.
¹ H NMR (CD ₃ OD): δ 8.77 (d, J = 1.7 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H, -CONH-), 8.49 (d, J = 8.0 Hz, 1H,- CONH-), 8.31 (dd, J = 8.0 Hz, 1.7 Hz, 1H), 7.91 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.38 (d, J = 2.8 Hz, 2H), 7.26 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 9.5 Hz, 2H), 7.07 (dd, J = 9.5 Hz, 2.4 Hz, 2H) , 7.05 (d, J = 9.6 Hz, 2H), 7.02 (d, J = 2.3 Hz, 2H), 6.79 (dd, J = 9.7 Hz, 2.8 Hz, 2H), 4.07-4.01 (m, 2H), 3.66 (s, 3H), 3.36 (s, 12H), 3.33 (s, 12H), 2.18-2.16 (m, 4H), 2.12 (s, 3H), 1.69-1.64 (m, 4H), 0.627 (s, 3H .), 0.612 (s, 3H ) 13 C NMR (CD 3 OD): δ 169.3 (C), 168.9 (C), 167.5 (C), 166.4 (C), 159.9 (C), 159.08 (C), 159.05 (C), 155.9 (C), 149.5 (C), 143.4 (C), 142.0 (CH), 138.0 (C), 137.9 (C), 137.6 (C), 136.5 (C), 132.7 (CH), 132.4 (CH), 132.08 (CH), 131.94 (C), 131.83 (CH), 131.1 (CH), 130.5 (CH), 130.2 (CH), 128.1 (C), 125.8 (CH), 122.3 (CH), 115.7 (CH), 115.3 (CH), 114.7 (C), 53.1 (CH ₃ ), 50.2 (CH), 49.9 (CH), 40.97 (CH ₃ ), 40.94 (CH ₃ ), 3 2.34 (CH ₂ ), 32.32 (CH ₂ ), 19.5 (CH ₃ ), -1.1 (CH ₃ ), -1.3 (CH ₃ ) .; HRMS-ESI (m / z): [M] ²⁺ / 2 calcd for C ₅₉ H ₆₆ N ₆ O ₅ Si: 483.24265; found: 483.24277 (-0.1 mDa, -0.3 ppm).

Fluorescence assay for hydropolysulfide Compound 1 (2Me SiR650), which is the fluorescent probe compound synthesized in Example 1, was reacted with sodium disulfide (Na ₂ S ₂ ), which is a commercial product, as a hydropolysulfide, and the fluorescence assay was performed. . Ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy were performed using Shimadzu UV-2450 (Shimadzu Corporation) and Hitachi F-7000 (Hitachi Corporation). As the solution conditions, a fluorescent probe concentration was 1 μM, 0.2 M sodium phosphate buffer (pH 7.4), and a solution containing 0.1% DMSO was used. An absorption spectrum and a fluorescence spectrum were measured when the concentration of sodium disulfide to be added was changed in the range of 0 to 200 μM. The excitation wavelength was 620 nm. The obtained results are shown in FIGS.

As shown in FIGS. 1 and 2, a significant decrease in absorption and fluorescence intensity was observed with the addition of sodium disulfide. As a result of plotting the sodium disulfide concentration dependence of the intensity change of the absorption peak wavelength (646 nm) and the fluorescence peak wavelength (667 nm) and calculating the dissociation constant K _{d, H2S2} (μM), 10 μM was obtained.

In addition, in order to compare the selectivity for glutathione (GSH) having -SH bond, compound 1 was allowed to act on glutathione and the same fluorescence assay was performed. As a result, the dissociation constant K _{d, GSH} (mM) was> 100 mM. It was.

Similarly, other probes, Compound 2 (2OMe SiR650), Compound 3 (2Cl SiR650), Compound 4 (2Me SiR700), and Compound 6 (2F SiR650), are also shown in the table. It is shown in 1. Further, the structure of the fluorophore is different, that is, the substituent R at the 2′-position of phenyl (corresponding to R ² of the formula (I)) and the substituent R ′ in the amino group (R ⁵ and R ⁶ of the formula (I)) Table 1 shows the results of assaying under the same conditions for Comparative Compounds 1 to 6 having different R, R ⁷ and R ⁸ equivalents).

From the results shown in Table 1, it was found that the compounds 1 to 4 showed a selectivity of 1000 times or more with respect to the selectivity ( _{Kd, GSH} / _{Kd, H2S2} ) of _{hydropolysulfide} (sodium disulfide) with respect to glutathione. Particularly in Compound 1, the selectivity of hydropolysulfide to glutathione is 10,000 times or more, and the dissociation constant K _{d, H2S2 for hydropolysulfide} is 10 μM, and the concentration of hydropolysulfide in the cell (about 1 to 10 μM) The result was detectable. That is, it was demonstrated that these fluorescent probes are useful for detection of hydropolysulfides in cells.

Fluorescence assay for other hydropolysulfides Instead of sodium disulfide used above, commercially available sodium trisulfide (Na ₂ S ₃ ), sodium tetrasulfide (Na ₂ S ₄ ), and synthetic compound 4-trimethylsilyl The responsiveness to benzylpersulfide (BnSSH, below) was evaluated. Compound 1 was used as a fluorescent probe.

As a result, concentration-dependent decrease in absorbance / fluorescence was confirmed for Na ₂ S ₃ , Na ₂ S ₄ and BnSSH, and the dissociation constants K _d were calculated as 5 μM, 5 μM and 11 μM, respectively. These results, the fluorescent probe of the present invention, it was confirmed other hydro polysulfide represented by R-S n _SH can also be detected.

Evaluation of reversibility of fluorescence response Observed change in absorbance upon addition of BnSSH (5 μM) and addition of thiol group scavenger (0.1 or 1 mM) to confirm that the reaction between the fluorescent probe and hydropolysulfide is reversible. The evaluation was performed. The measurement was performed using Compound 1. As the solution conditions, a fluorescent probe concentration was 1 μM, a solution containing 0.2 M sodium phosphate buffer (pH 7.4), 0.1% DMSO and 0.1% Triton-X was used. The results are shown in FIG.

  From the results in FIG. 3, it was found that these fluorescent probes responded quickly to BnSSH, which is a hydropolysulfide, and a change in absorbance occurred depending on the amount of BnSSH. Furthermore, by adding N-ethylmaleimide (NEM), which is a thiol scavenger, the absorbance returned to the same value as when BnSSH was not added, so it was confirmed that the responsiveness to hydropolysulfide was reversible.

In evaluation cell imaging as a FRET sensor , a technique (ratio imaging) in which imaging is performed by taking a ratio of excitation or fluorescence of two wavelengths is a very frequently used technique. Therefore, the compound 5 (TMR-SiR650) synthesized in Example 1, which is a probe having an intramolecular donor fluorophore, functions as a FRET-type fluorescent probe exhibiting one-wavelength excitation and two-wavelength fluorescence based on the FRET principle. I verified. Compound 5 is obtained by introducing tetramethylrhodamine (TMR, TAMRA) into compound 1 as a fluorophore (donor) on the short wavelength side.

Using compound 5, the absorption and fluorescence spectrum changes accompanying the addition of Na ₂ S ₂ were measured in the same manner as in Example 2. As the solution conditions, a solution containing a fluorescent probe concentration of 1 μM, 0.2 M sodium phosphate buffer (pH 7.4), 0.1% DMSO, and 0.1% Triton-X was used. The concentration of Na ₂ S _{2 to be} added was changed in the range of 0 to 100 μM. The excitation wavelength was 550 nm. The obtained results are shown in FIG.

As shown in FIGS. 4c and 4d, the fluorescence intensity of the long wavelength peak decreased with the addition of Na ₂ S ₂ and the fluorescence intensity of the short wavelength peak increased. The _{Kd, H2S2} was also about 11 μM, which was found to be almost the same as in the case of Compound 1 alone. This result shows that the fluorescent probe (compound 5) can achieve detection of a physiological concentration of hydropolysulfide by the luminescence type (wavelength change type) by the FRET mechanism, not by the quenching type detection as in Example 2. Is.

Cell Imaging with Fluorescent Probe Compound Next, various fluorescence imaging in cells was performed using Compound 5 as a fluorescent probe. The measurement was performed using a confocal microscope.

A) Reversibility evaluation 1: Evaluation by Na ₂ S ₂ / NEM A perfusion device was installed for signal reversibility by sodium disulfide (Na ₂ S ₂ ) and N-ethylmaleimide (NEM), a thiol group scavenger. Was evaluated with a confocal microscope. The results are shown in FIG.

The measurement conditions are as follows.
[Experimental conditions]
・ Cells used: A549
・ Dish used: 35 mm glass bottom dish, observation surface: 14 mm
Staining conditions: 1 μM TMR-SiR650 in HBSS (including 0.01% Pluronic F-127 and 0.1% DMSO), 10 min, 37 ^o C → washing x3 → imaging
・ Liquid feeding conditions Solution A: HBSS
Solution B: 10 μM Na ₂ S ₂ in HBSS
Solution C: 50 μM NEM (0.1% DMSO) in HBSS
・ Flow rate: 4 ml / min
・ Imaging interval: 5 seconds [microscope setting conditions]
・ Excitation wavelength: 550 nm, 20%
・ Detection wavelength: Ch1: 560-640 nm (Gain: 700 V), Ch2: 650-750 nm (Gain: 950 V)
・ Pinhole: 2 airy

  As shown in FIG. 5a, a fluorescence image was observed immediately after administration of sodium disulfide, and as shown in FIG. 5b, an increase in fluorescence intensity was observed under the condition of glutathione depletion by adding N-ethylmaleimide (NEM). This demonstrates that reversible imaging in real time can be achieved.

B) Reversibility evaluation 2: Evaluation by BnSSH / NEM Next, an experiment was conducted using 4-trimethylsilyl benzylpersulfide (BnSSH) instead of sodium disulfide.
[Experimental conditions]
Same as above except for the conditions for liquid delivery. Solution A: HBSS
Solution B: 10 μM BnSSH in HBSS (0.1% DMSO)
Solution C: 50 μM NEM (0.1% DMSO) in HBSS

  When reversibility was evaluated using NEM, it was confirmed that rapid changes in intracellular BnSSH concentration could be monitored in real time (FIGS. 6a and b). Furthermore, when BnSSH was removed in the form of washing with cell culture medium instead of NEM, the point at which the ratio value reached a plateau increased with each reversible cycle while showing a reversible cycle (FIGS. 6c, d). ). This result seems to suggest that the sulfur element contained in BnSSH is retained in endogenous glutathione and cysteine and accumulated in the form of GSSH and Cys-SSH. The demonstration of reversible imaging of hydropolysulfides on the second scale shown above has not been achieved so far.

C) Selectivity evaluation of hydropolysulfide in cells It was verified by using membrane-permeable glutathione (glutathione monoethyl ether: GSH (Et)) that the cells have hydropolysulfide selectivity for glutathione. The experimental conditions are as follows. The results are shown in FIG.
[Experiment protocol]
The staining conditions are the same as above. Ibidi 8 well is used.
Before: Imaging of A549 in HBSS (the upper part of Fig. 7a)
↓
NEM: Replacement to HBSS in 100 μM NEM (0.1% DMSO), imaged in 3 min.
↓
Stimulation: Washing with HBSS x2, stimulation with the following reagents, then imaging in 5 min. (Bottom of Fig. 7a)
・ HBSS (control)
・ 10 mM GSH (Et) in HBSS
・ 10 μM Na ₂ S ₂ in HBSS
・ 10 μM BnSSH in HBSS

As shown in FIG. 7b, compound 5 can be obtained by removing intracellular thiol components (including R-SSH, GSH, Cys, etc.) once with NEM and adding 10 mM (equivalent to intracellular concentration) of GSH (Et). Showed little signal change. On the other hand, when Na ₂ S ₂ and BnSSH (10 μM: equivalent to intracellular concentration) of 1,000 times thinner concentration were added, a significant increase in the ratio value was recognized. From this result, it was demonstrated that Compound 5 selectively detected and visualized a trace amount of hydropolysulfide without being affected by GSH present in a large amount in the cell.

D) Detection of change in concentration of endogenous hydropolysulfide component In the above c), it was demonstrated that a dramatic ratio change was observed with respect to external addition of hydropolysulfide. Next, it was verified whether or not the concentration change of hydropolysulfide actually existing in cells can be visualized using Compound 5.

  In this experiment, a model experiment proposed by T. Ida et al., Proc. Natl. Acad. Sci. U. S. A, 2014, 111, 7606-7611 was adopted. According to this non-patent document, it is stated that proteins such as xCT, SNAT2, CBS, and CSE are involved in intracellular Cys-SSH synthesis.

Therefore, by inhibiting these proteins, if a change in the amount of CysSSH is seen, visualization of changes in the endogenous CysSSH concentration can be achieved. Therefore, the inhibition experiments with inhibitors and siRNA against these proteins were verified. The inhibitor is as follows. siRNA was purchased from Invitrogen or Santa Cruz.
XCT inhibitor: Sulfasalazine (SSZ)
・ CBS / CSE inhibitor: Propargylglycine (PAG) or β-cyanoalanine (BCA)

[Protocol: Inhibitor]
A549 cells seeded on a ibidi 8-well plate were treated with the following inhibitors for 4 hr at 37 ^o C.
Control: no treatment
Inhibition of xCT: 10 μM SSZ
Inhibition of CBS / CSE: 1 mM PAG or BCA
・ Cells were washed with FBS-free HBSS, then probe (1 μM, in HBSS including 0.1% DMSO and 0.01% Pluronic F-127) was loaded for 10 min.
・ Cells were washed with HBSS x2 and replaced to HBSS, then imaged.

[Protocol: siRNA]
・ A549 cells (~ 20% confl.) Seeded on a ibidi 8-well plate and alternatively overnight.
・ SiRNA (for xCT, SNAT2, CBS, CSE) was treated under the following condition.
siRNA for xCT and SNAT2: 10 nM for 72 hrs
siRNA for CBS and CSE: 30 or 60 nM for 24 hrs
・ Cells were washed with FBS-free HBSS, then probe (1 μM, in HBSS including 0.1% DMSO and 0.01% Pluronic F-127) was loaded for 10 min.
・ Cells were washed with HBSS x2 and replaced to HBSS, then imaged.

The results of inhibition experiments with inhibitors and siRNA are shown in FIGS. 8 and 9, respectively. From the results shown in FIGS. 8 and 9, there was a significant difference in the ratio reduction in cells treated with various inhibitors or siRNA compared to untreated cells. This is clearly considered to reflect a decrease in the amount of Cys-SSH due to inhibition of Cys-SSH synthesis, so that compound 5 has a response sufficient to visualize changes in the amount of endogenous Cys-SSH. It was proved to be a fluorescent probe having the property.

  The above results demonstrate that the fluorescent probe of the present invention is a highly practical fluorescent probe that selectively and reversibly visualizes hydropolysulfide.

Claims (11)

A fluorescent probe for detecting hydropolysulfide comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
X represents Si (R ^a ) (R ^b ) (where R ^a and R ^b each independently represents a hydrogen atom or an alkyl group);
R ¹ is independently selected from the group consisting of a hydrogen atom, a cyano group, or an optionally substituted alkyl group, carboxyl group, ester group, alkoxy group, amide group, and azide group. Represents the same or different substituents;
R ² represents a halogen atom, an optionally substituted alkyl group, or an alkoxy group;
R ³ and R ⁴ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, an independently substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group. Represents 1 to 3 identical or different substituents selected from
R ⁵ , R ⁶ , R ⁷ and R ⁸ all represent an alkyl group,
Here, R ⁵ or R ⁶ together with R ³ may form a ring structure containing a nitrogen atom to which they are bonded,
R ⁷ or R ⁸ may be combined with R ⁴ to form a ring structure containing a nitrogen atom to which they are bonded. ] The fluorescent probe according to claim 1, wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ are all methyl groups. The fluorescent probe according to claim 1, wherein R ² is a chlorine atom, a methyl group, or a methoxy group. R ⁵ or R ⁶ together with R ³ each form a 5-membered ring containing the nitrogen atom to which they are attached; and / or R ⁷ or R ⁸ together with R ⁴ respectively The fluorescent probe according to claim 1, which forms a 5-membered ring containing a nitrogen atom to which they are bonded. The fluorescent probe according to any one of claims 1 to 4, wherein R ¹ has a fluorophore serving as a donor of fluorescence resonance energy transfer (FRET).   The fluorescent probe according to claim 5, wherein the fluorophore is a compound having a xanthene skeleton.   A method for detecting hydropolysulfide using the fluorescent probe according to claim 1.   The detection method according to claim 7, wherein the presence of the hydropolysulfide is detected by observing a fluorescence response or a change in absorbance due to a reaction between the hydropolysulfide and the fluorescent probe.   The detection method according to claim 8, wherein the fluorescence response is a fluorescence change caused by fluorescence resonance energy transfer (FRET).   The detection method according to claim 8, wherein the fluorescence response is visualized using a fluorescence imaging means.   A kit for detecting hydropolysulfide, comprising the fluorescent probe according to any one of claims 1 to 6.

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