Squarylium pigment derivative

Abstract

To provide a stable squarylium pigment derivative which exhibits low cytotoxicity and safety, and which enables selective and efficient staining of mitochondria contained in eukaryotic cells, particularly live eukaryotic cells, a mitochondrial staining reagent comprising the squarylium pigment derivative, and a method for staining mitochondria using the squarylium pigment derivative.SOLUTION: The present invention provides a squarylium pigment derivative or a salt thereof, represented by the following formula (I). [In the formula, R1 and R2 independently represent a C1-6 alkyl group, a C6-12 aryl group, or a C6-12 arylmethyl group; R3 and R4 independently represent a single bond or a linker group; and Z1 and Z2 independently represent H or a cationic group, with at least one of Z1 and Z2 representing a cationic group].SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a stable squarylium dye derivative that is safe with low cytotoxicity and capable of selectively and effectively staining mitochondria contained in eukaryotic cells, particularly living eukaryotic cells, a mitochondrial staining reagent containing the squarylium dye derivative, and a method for staining mitochondria using the squarylium dye derivative. [Background technology]

[0002] Mitochondria, organelles found in almost all eukaryotic cells, produce the high-energy substance adenosine triphosphate (ATP) mainly through the TCA cycle and electron transport chain, so changes in their activity and dysfunction are closely related to cancer, aging, neurodegenerative diseases, etc. Therefore, evaluation of mitochondrial activity is very important as it helps evaluate cell activity.

[0003] Highly active mitochondria have a high membrane potential, while low activity mitochondria have a low membrane potential. In normal mitochondria where the membrane potential difference is maintained, JC-1 dye aggregates and emits red fluorescence, and when the membrane potential decreases, it exists as a monomer and emits green fluorescence. Therefore, the state of mitochondria can be evaluated based on the change in the red and green fluorescence intensity of JC-1 dye (Non-Patent Document 1).

[0004] Tetramethylrhodamine methyl ester (TMRM) is a cell-permeable fluorescent dye that accumulates in active mitochondria with normal membrane potential, and its signal is bright when cells are healthy and have normally functioning mitochondria. On the other hand, when mitochondrial activity decreases and the membrane potential decreases, TMRM accumulation stops and the signal becomes dim or disappears (Non-Patent Document 2).

[0005] However, the fluorescence intensity of JC-1 and TMRE decreases about 10 minutes after staining. Therefore, when observing cells over a long period of time using JC-1 or TMRE, the cells must be stained each time they are observed, which places a heavy burden on the cells. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dojin Chemical Research Institute Co., Ltd., “JC-1 MitoMP Detection Kit”, [online], [Searched on December 22, 2020], Internet<URL:https: / / www.dojindo.co.jp / products / MT09 / > [Non-Patent Document 2] Thermo Fisher Scientific, "Fluorescent labeling of mitochondria", [online], [Retrieved December 22, 2022], Internet <URL:https: / / www.thermofisher.com / jp / ja / home / life-science / cell-analysis / cell-analysis-learning-center / molecular-probes-school-of-fluorescence / imaging-basics / labeling-your-samples / mitochondrial-labeling.html> Summary of the Invention [Problem to be solved by the invention]

[0007] The ability to stain mitochondria, the main energy-producing organelles, for extended periods of time without harming them has major advantages. Therefore, an object of the present invention is to provide a stable squarylium dye derivative that is safe with low cytotoxicity and capable of selectively and effectively staining mitochondria contained in eukaryotic cells, particularly living eukaryotic cells, a mitochondrial staining reagent containing the squarylium dye derivative, and a method for staining mitochondria using the squarylium dye derivative. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems, and as a result have found that a squarylium dye derivative developed by the present inventors can be taken up into living eukaryotic cells to specifically stain mitochondria, has no or very low toxicity to cells, and can stain mitochondria continuously for a long period of time, thereby completing the present invention. The present invention will now be described.

[0009] [1] A squarylium dye derivative or a salt thereof, characterized by being represented by the following formula (I): [ka] [In the formula, R 1 and R 2 is independently 1-6 Alkyl group, C 6-12 Aryl group, or C 6-12 represents an aryl-methyl group, R 3 and R 4 each independently represents a single bond or a linker group, Z 1 and Z 2 each independently represents H or a cationic group; Z 1 and Z 2 At least one of represents a cationic group.] [2] Z 1 and Z 2 At least one of them is -P + Ph 35. The squarylium dye derivative or salt thereof according to the above [1], wherein Ph represents a phenyl group which may have a substituent, and a plurality of Phs may be the same or different. [3] Z 1 indicates H, and Z 2 represents a cationic group, and R 1 and R 2 But independently, C 1-6 R represents an alkyl group. 3 C 1-6 The squarylium dye derivative or a salt thereof according to the above [1], which represents an alkanediyl group. [4] The squarylium dye derivative or a salt thereof according to [2] above, wherein Ph is a phenyl group. [5] The squarylium dye derivative or a salt thereof according to [1] above, which contains a halide ion.

[0010] [6] A mitochondrial staining reagent comprising the squarylium dye derivative or a salt thereof according to any one of [1] to [5] above. [7] The mitochondrial staining reagent according to [6], further comprising an aqueous solvent and a betaine compound.

[0011] [8] adding the mitochondrial staining reagent according to [6] to a eukaryotic cell; and A method for staining mitochondria, comprising the step of irradiating the eukaryotic cells with light and observing the fluorescence.

[0012] In the present invention, "C 1-6 The term "alkyl group" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, and n-hexyl. 1-5 is an alkyl group, more preferably C 2-5 is an alkyl group, and even more preferably C 3-4 It is an alkyl group.

[0013] "C6-12 The term "aryl group" refers to a monovalent aromatic hydrocarbon group having 6 to 12 carbon atoms. Examples include a phenyl group, a naphthyl group, an indenyl group, a biphenyl group, etc., and preferably a phenyl group.

[0014] "C 6-12 The "aryl-methyl group" is 6-12 It refers to a methyl group substituted with an aryl group, and is preferably a benzyl group.

[0015] The linker group has the effect of facilitating the synthesis of the squarylium dye derivative (I) and increasing the degree of freedom of the triphenylphosphonium group. The linker group is not particularly limited, but examples thereof include C 1-6 Alkanediyl group, C 6-12 Divalent aromatic group, amino group (-NH-), imino group (>C=N- or -N=C<), ether group (-O-), thioether group (-S-), carbonyl group (-C(=O)-), thionyl group (-C(=S)-), ester group (-C(=O)-O- or -OC(=O)-), amide group (-C(=O)-NH- or -NH-C(=O)-), sulfoxide group (-S(=O)-), sulfonyl group (-S(=O) 2 -), sulfonylamido group (-NH-S(=O) 2 - and -S(=O) 2 In the case where two or more of these groups are bonded to form the linker group, the number of bonds is preferably 10 or less or 5 or less, and more preferably 3 or less. In addition, examples of the linker group include C 1-6 Alkanediyl groups are preferred.

[0016] "C 1-6 The term "alkanediyl group" refers to a linear or branched divalent saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms. Examples include methylene, ethylene, methylmethylene, n-propylene, methylethylene, n-butylene, methylpropylene, dimethylethylene, n-pentylene, and n-hexylene. 1-4 alkylene, more preferably C2-4 It is alkylene.

[0017] The cationic group Z is not particularly limited as long as it is a substituent containing a cation. For example, a triphenylphosphino group: -P + Ph 3 group (wherein Ph represents a phenyl group which may have a substituent, and a plurality of Ph may be the same or different), a quaternary ammonium group: -N + R 5 3 (In the formula, R 5 is C 1-6 Indicates an alkyl group, and multiple R 5 may be the same or different from each other.), pyridinium group: -N + C 5 H 5 or -C 5 H 4 N + R 6 (In the formula, R 6 is H or C 1-6 It represents an alkyl group.

[0018] The substituent that the phenyl group Ph may have is not particularly limited, and examples thereof include, for example, C 1-6 Alkyl groups, halogenated C 1-6 Alkyl group, C 1-6 Alkoxy groups, halogenated C 1-6 The substituents may be one or more selected from an alkoxy group and a halogeno group. 1-6 Alkyl and halogenated C 1-6 The halogeno group of the alkoxy group includes fluoro, chloro, bromo and iodo, preferably fluoro or chloro, more preferably fluoro. The number of substituents is not particularly limited as long as it is substitutable, but is preferably 5 or less, 4 or less or 3 or less, more preferably 1 or 2, and even more preferably 1.

[0019] The counter anion that forms a salt with the squarylium dye derivative according to the present invention is not particularly limited, and examples thereof include halide ions such as fluoride ion, chloride ion, bromide ion, and iodide ion; inorganic acid ions such as nitrate ion, sulfate ion, and phosphate ion; and organic acid ions such as methanesulfonate ion, p-toluenesulfonate ion, acetate ion, trifluoroacetate ion, citrate ion, tartrate ion, maleate ion, fumarate ion, malate ion, and lactate ion. Effect of the Invention

[0020] The squarylium dye derivative according to the present invention can be incorporated into living eukaryotic cells and even into mitochondria, and can specifically stain mitochondria. In addition, it has no or very low toxicity to cells, is relatively stable against light, and is unlikely to fade due to decomposition. Therefore, by using the squarylium dye derivative according to the present invention, it is possible to continuously observe mitochondria for a long period of time. As a result, it is also possible to evaluate stress, etc., suffered by living eukaryotic cells by continuously observing the morphology and movement of mitochondria. For example, it is considered that it may be used to confirm the quality of cultured cells. Therefore, the present invention is very useful industrially because it may be widely applicable to biochemical research using cells. [Brief description of the drawings]

[0021] [Figure 1] Figure 1(1) shows the results of staining with squarylium dye derivatives, Figure 1(2) shows the results of staining with MitoTracker, and Figure 1(3) shows the results of co-staining with squarylium dye derivatives and MitoTracker. [Diagram 2] FIG. 2 is a micrograph showing that the squarylium dye derivative according to the present invention enables continuous fluorescence observation of cells over a long period of time. [Diagram 3] FIG. 3 is a graph showing the results of a toxicity test of the squarylium dye derivative according to the present invention. [Figure 4]FIG. 4 is a micrograph showing that the squarylium dye derivative according to the present invention enables continuous fluorescence observation of cells over a long period of time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The squarylium dye derivative (I) according to the present invention can be synthesized, for example, according to the following scheme, although it is not particularly limited thereto.

[0023] 1-1. Synthesis of squaric acid unit [ka]

[0024] Compound 1 can be synthesized by those skilled in the art by alkylating the amino group of m-aminophenol. Compound 2 is a known compound, but can also be synthesized by halogenating squaric acid as described in Example 1(1) below.

[0025] X 1 represents a halogeno group selected from chloro, bromo and iodo, and since squaric acid itself has high reactivity, chloro or bromo is preferred, and chloro is more preferred.

[0026] The Friedel-Crafts reaction produces a halogenated compound, which is a precursor of the squaric acid unit 3, from compound 1 and compound 2, and the halogenated compound is then hydrolyzed to produce the squaric acid unit 3. In compound 1, the o-position of the dialkylamino group and the o-position of the phenolic hydroxyl group are activated, but due to the steric hindrance of the dialkylamino group, compound 2 reacts preferentially with the p-position of the dialkylamino group of compound 1.

[0027] 1-2. Synthesis of cationic units [ka]

[0028] X 2 represents a halogeno group selected from chloro, bromo, and iodo, and is preferably bromo or iodo, which are more reactive, and more preferably iodo. X 3- is a counter anion that forms a salt with the cationic group Z.

[0029] Compound 4 can be synthesized by alkylation of the amino group of m-aminophenol by one skilled in the art. Compound 5 can be synthesized, for example, by the reaction of X 2 -R 4 -X 3 and triphenylphosphine compounds (PPh 3 ) can be easily synthesized by reacting X 2 and X 3 It is preferable that X is the same. 3- can later be exchanged for the desired ion.

[0030] The above reaction can be carried out, for example, by reacting compound 4 with compound 5 in a solvent in the presence of a base. Since the above reaction is a modification reaction of an amino group, it proceeds easily to give cationic unit 6.

[0031] 1-3. Condensation reaction of each unit [ka]

[0032] In this reaction, the squaric acid unit 3 is condensed with the cationic unit 6. The reaction between the squaric acid unit 3 and the cationic unit 6 can be easily carried out by simply dissolving the squaric acid unit 3 and the cationic unit 6 in a solvent and heating them due to a resonance structure or the like.

[0033] After each of the above reactions, each target compound may be purified by a purification method that may be appropriately selected depending on the target compound and the state of the reaction solution, and examples of the purification method include chromatography and recrystallization.

[0034] The squarylium dye derivative (I) according to the present invention can be incorporated into cells, particularly living cells, and selectively incorporated into mitochondria in the cytoplasm, and can therefore serve as a specific staining reagent for mitochondria. The method for staining mitochondria according to the present invention will be described below, but the present invention is not limited to the following specific examples.

[0035] 2-1. Addition of mitochondrial staining reagent In this step, the mitochondrial staining reagent according to the present invention is added to eukaryotic cells. Mitochondria are organelles present in almost all eukaryotic cells and are involved in energy production, specifically, producing ATP, an energy source necessary for life activities. Since mitochondria exhibit the lowest membrane potential among all organelles, it is believed that the squarylium dye derivative (I) according to the present invention is selectively taken up into mitochondria, probably due to the cationic group, and accumulates in the mitochondria.

[0036] The squarylium dye derivative (I) or a salt thereof according to the present invention may be added as it is to a culture solution containing eukaryotic cells to be observed as a mitochondrial staining reagent. However, since it may be difficult to dissolve in the culture solution, it is preferable to add a solution of the squarylium dye derivative (I) or a salt thereof as a mitochondrial staining reagent.

[0037] Aqueous solvents can be used as the solvent for the solution of the squarylium dye derivative (I) or its salt. The aqueous solvent refers to water or a mixed solvent of water and a water-miscible organic solvent. The water-miscible organic solvent refers to an organic solvent that is miscible with water without restrictions, and examples thereof include lower alcohol solvents such as methanol, ethanol, and 2-propanol; amide solvents such as dimethylformamide and dimethylacetamide; and sulfoxide solvents such as dimethylsulfoxide. The water-miscible organic solvent has a role of assisting the dissolution of the squarylium dye derivative (I) or its salt, but since it may have a negative effect on cells, it is preferable not to use it or to reduce the amount of use. The ratio of the water-miscible organic solvent in the mixed solvent is, for example, preferably 10% by mass or less or 5% by mass or less, more preferably 2% by mass or less or 1% by mass or less. The lower limit of the water-miscible organic solvent in the solvent is preferably 0% by mass, and when a water-miscible organic solvent is used, it is preferably 0.1% by mass.

[0038] The concentration of the squarylium dye derivative (I) or its salt in the mitochondrial staining reagent may be adjusted to a level sufficient to sufficiently stain the mitochondria of the target eukaryotic cells when the mitochondrial staining reagent is added to a liquid culture medium of the target eukaryotic cells, and may be adjusted to, for example, 1 nM or more and 10 mM or less.

[0039] A solubilizing agent may be added to the solution of the squarylium dye derivative (I) or its salt in addition to or instead of the water-miscible organic solvent. A surfactant is generally used as the solubilizing agent, but the surfactant may have a negative effect on cells. The present inventors have found that betaine assists in the dissolution of the squarylium dye derivative (I) or its salt and has little effect on cells.

[0040] Betaine is a compound that has a positive charge and a negative charge in non-adjacent positions in the same molecule, and the positively charged atom has no dissociable hydrogen bonded to it, so the molecule as a whole has no charge. In the narrow sense, it is a compound in which the amino group of an amino acid is converted into a quaternary ammonium. For example, tri(C1-6 alkyl)glycine.

[0041] The concentration of betaine in the mitochondrial staining reagent may be appropriately adjusted within a range in which the squarylium dye derivative (I) can be sufficiently dissolved, and may be, for example, about 100 mM or more and 10 M or less. When the mitochondrial staining reagent containing a solvent is added to a liquid medium, the concentration of the squarylium dye derivative (I) in the liquid medium will be less than the concentration of the squarylium dye derivative (I) in the mitochondrial staining reagent. Therefore, if the squarylium dye derivative (I) can be sufficiently dissolved in the mitochondrial staining reagent, precipitation of the squarylium dye derivative (I) in the liquid medium can be sufficiently suppressed.

[0042] The amount of mitochondrial staining reagent added to the liquid medium of the target eukaryotic cells may be appropriately adjusted within a range that sufficiently stains the mitochondria of the target eukaryotic cells. For example, when the mitochondrial staining reagent is in liquid form, the volume ratio before addition to the liquid medium is 1 × 10 -4 More than twice, 1×10 -2 Alternatively, the liquid medium may be replaced with a solution of the squarylium dye derivative (I). The concentration of the solution of the squarylium dye derivative (I) may be, for example, 0.1 M or more and 10 M or less.

[0043] The squarylium dye derivative (I) according to the present invention is taken up by eukaryotic cells and further taken up by mitochondria in the cytoplasm. Therefore, by adjusting the amount of squarylium dye derivative (I) relative to the target eukaryotic cells so that substantially all of it is taken up by the target eukaryotic cells, it may be possible to observe the fluorescence of mitochondria without washing the target eukaryotic cells. For example, when 1×10 target eukaryotic cells are dissolved in a liquid medium, 3 cell / mL or more, 1×10 7 After culturing to below 1×10 cells / mL, the ratio of squarylium dye derivative (I) to the target eukaryotic cells was 1×10 -15 mol / cell or more, 1×10 -9 Simply add a mitochondrial staining reagent so that the concentration is less than mol / cell.

[0044] After adding the mitochondrial staining reagent according to the present invention to the target eukaryotic cells, the target eukaryotic cells are incubated until the squarylium dye derivative (I) is sufficiently incorporated into the mitochondria of the target eukaryotic cells. The temperature and atmosphere of the incubation may be adjusted to the optimal temperature and optimal atmosphere of the target eukaryotic cells. The incubation time may be until fluorescence in the liquid medium becomes unobservable, or may be appropriately determined in a preliminary experiment, and may be, for example, 10 minutes or more and 5 hours or less. The incubation time is preferably 4 hours or less or 3 hours or less, and more preferably 2 hours or less. The above incubation time is the time from the addition of the mitochondrial staining reagent to the time when mitochondria can be observed, and the culture may be continued thereafter for continuous observation.

[0045] When observing the target eukaryotic cells for a long period of time, the culture may be continued with the mitochondrial staining reagent added to the medium. When observing the cells with a general cell staining reagent, the cells need to be washed one by one, which causes stress to the cells. In contrast, the mitochondrial staining reagent of the present invention may be added to the medium and then continuously observed. However, when the cells are cultured for a long period of time, certain nutrients are consumed and become insufficient, or waste products are excreted and accumulated in the medium, so that the medium needs to be replaced. In addition, the cells may grow confluently in the medium. In such a case, the mitochondrial staining reagent may be newly added together with the replacement of the medium. The time from the start of culture to the replacement of the medium may be appropriately adjusted depending on the culture conditions, and may be, for example, 40 hours or more and 200 hours or less.

[0046] 2-2. Cleaning process In this step, the target eukaryotic cells are washed to reduce background fluorescence for the purpose of observing mitochondria. This step is optional, and if the squarylium dye derivative (I) is sufficiently incorporated into the target eukaryotic cells and further into mitochondria, and is not present in the liquid medium, or is present to the extent that it does not inhibit observation of mitochondria, this step may not be performed.

[0047] The washing operation may be performed according to a conventional method. For example, the liquid medium may be separated from the cells by decantation, filtration, centrifugation, or the like, and new liquid medium or a solution for fluorescence observation such as a phosphate buffer solution may be added. The washing operation may be performed multiple times.

[0048] When the target eukaryotic cells are to be observed for a long period of time, this step may be performed on only a portion of the target eukaryotic cells. Specifically, a portion of the culture medium may be collected as a sample, the collected sample may be washed, and the remaining portion may be allowed to continue culturing.

[0049] 2-3. Fluorescence observation process In this step, the culture medium of the target eukaryotic cells or the culture medium washed in the washing step 2 is irradiated with light and the fluorescence is observed. The irradiated light is preferably light having a peak excitation wavelength of the squarylium dye derivative (I). Furthermore, the fluorescence observed is within a range including the peak fluorescence wavelength of the squarylium dye derivative (I).

[0050] The squarylium dye derivative (I) according to the present invention is selectively incorporated into eukaryotic cells, and further into mitochondria in the cytoplasm, and is therefore effective for observing the mitochondria of target eukaryotic cells. In addition, the squarylium dye derivative (I) is stable, and therefore can be observed for a longer period of time than conventional fluorescent dyes. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the present invention by making appropriate modifications within the scope of the above and below-mentioned aims, and all of these modifications are included in the technical scope of the present invention.

[0052] Example 1: Synthesis of squarylium dye derivatives (1) Synthesis of 3,4-dichlorocyclo-3-butene-1,2-dione [ka] Under an argon atmosphere, squaric acid (1.0 g, 8.77 mM) was dissolved in dichloromethane (5 mL) in a three-neck flask, and one drop of DMF was added. Oxalyl chloride (2.2 g, 17.5 mM) was added to this solution, and the mixture was stirred for about 5 hours at an internal temperature of 40°C using a water bath. The solid generated in the reaction solution was filtered off, and the filtrate was concentrated to obtain a yellow solid containing the target compound (yield: 0.92 g (6.1 mM), yield: 70%). The obtained compound was used in the next reaction without purification.

[0053] (2) Synthesis of 3-(4-(dipropylamino)-2-hydroxyphenyl)-4-hydroxycyclo-3-butene-1,2-dione [ka] Under an argon atmosphere, aluminum chloride (0.81 g, 6.1 mM) was added to dichloromethane (5 mL) in a three-neck flask and stirred overnight at room temperature. The resulting white suspension was cooled in a water bath, and a solution of 3-(dipropylamino)phenol (0.96 g, 5.0 mM) dissolved in dichloromethane (5 mL) and a solution of 3,4-dichlorocyclo-3-butene-1,2-dione (0.92 g, 6.1 mM) dissolved in dichloromethane (5 mL) were simultaneously added dropwise over 15 minutes. Then, the mixture was stirred for about 2 hours at an internal temperature of 40°C using a hot water bath. The temperature of the reaction solution was returned to room temperature, and the reaction solution was added to water (50 mL) and stirred for about 10 minutes. The organic phase was collected, and the aqueous phase was further extracted twice with methylene chloride (10 mL). The organic phase and the extract were combined, washed twice with water (30 mL), and then dried over anhydrous magnesium sulfate. The organic phase was concentrated to obtain a dark green paste crude product (1.4 g). The crude product was purified by column chromatography (silica gel amount: 25 g, elution solvent: heptane / ethyl acetate = 100 / 0 → 30 / 70) to obtain the target product as a yellow solid (yield: 0.10 g (0.35 mM), yield: 7%). 1 H NMR (400 MHz, CDCl 3 )δ=0.97(6H,t,J=7.2Hz),1.66(4H,m),3.34(4H,t,J=8.0Hz),6.15(1H,d,J=2.0Hz),6.39(1H,t,J=2.4Hz),7.86(1H,d,J=9.2Hz),9.75(1H,br,s) MS(m / z+1): 290

[0054] (3) Synthesis of (3-((3-hydroxyphenyl)(propyl)amino)propyl)triphenylphosphonium iodide [ka] 3-(Propylamino)phenol (100 mg, 0.66 mM), (3-iodopropyl)triphenylphosphonium iodide (384 mg, 0.66 mM), methylene chloride (5.0 mL), and triethylamine (90.2 mg, 0.89 mM) were added to a three-neck flask and stirred overnight at room temperature. Triethylamine (230 mg, 2.27 mM) was further added and stirred at room temperature for about 48 hours. The reaction solution was concentrated, and the resulting crude product was purified by column chromatography (SiO 2 The target compound, which was an amorphous substance, was purified using a column of 20×60 mm column and elution solvent: methylene chloride / methanol=100 / 0→90 / 10 (yield: 335 mg (0.58 mM), yield: 87%). MS(m / z+1): 455

[0055] (4) Synthesis of (3-((4-(3-(4-(diisopropylamino)-2-hydroxyphenyl)-2,4-dihydroxycyclobuta-1,3-dien-1-yl)-3-hydroxyphenyl)(propyl)amino)propyl)triphenylphosphonium iodide [ka] Under an argon atmosphere, in a three-neck flask, (3-((3-hydroxyphenyl)(propyl)amino)propyl)triphenylphosphonium iodide (100 mg, 0.172 mM) and 3-(4-(dipropylamino)-2-hydroxyphenyl)-4-hydroxycyclo-3-butene-1,2-dione (49.8 mg, 0.172 mM) were dissolved in 1-butanol (5 mL) and toluene (4 mL). The mixture was then stirred at an external temperature of 120° C. for about 17 hours. The resulting reaction solution was concentrated to obtain a crude product (150 mg). The target product, which was a blue-green solid, was purified from the resulting crude product by column chromatography (yield: 68 mg (80 mM), yield: 47%). 1 H NMR (400MHz, DMSO-d 6)δ=0.80-0.89(9H,m),1.44-1.60(6H,m),1.72-1.84(2H,m),3.30-3.35(2H,m ),3.37-3.45(4H,m),3.50-3.60(4H,m),6.13-6.16(2H,m),6.48(1H,dd,J=2.0 and 9.2Hz),6.61(1H,dd,J=2.4 and 9.6Hz),7.61-7.82(12H,m),7.81-7.91(3H,m),11.70-12.10(2H,br) MS(m / z):727

[0056] Example 2: Synthesis of squarylium dye derivatives (1) Synthesis of N,N,N-triethyl-3-iodopropane-1-ammonium iodide [ka] Acetonitrile (5.0 mL), triethylamine (1.0 g, 9.91 mM), and diiodopropane (5.86 g, 19.8 mM) were added to a three-neck flask and stirred at a bath temperature of 60° C. for about 3 hours. The reaction solution was then concentrated to obtain a pale yellow solid. Hexane (5 mL) was further added to the obtained solid and stirred, then filtered to recover the filtrate. The recovered filtrate was concentrated to obtain the target product as a pale yellow solid. The obtained compound was used as it was in the next reaction.

[0057] (2) Synthesis of N,N,N-triethyl-3-((3-hydroxyphenyl)(propyl)amino)propane-1-ammonium iodide [ka] 3-(propylamino)phenol (118 mg, 0.78 mM), N,N,N-triethyl-3-iodopropane-1-ammonium iodide (310 mg, 0.78 mM), and methylene chloride (5 mL) were added to a test tube, and triethylamine (300 mg, 3.0 mM) was further added. The resulting solution was heated and stirred at an external temperature of 40° C. for about 23 hours. Then, N,N,N-trimethyl-3-iodopropane-1-ammonium iodide (310 mg, 0.78 mM) was further added, and the mixture was stirred at an external temperature of 40° C. for 4 days. Next, the reaction solution was directly subjected to silica gel column chromatography (elution solvent: methylene chloride / methanol = 100 / 0 → 50 / 50) to purify the target product (yield: 389 mg).

[0058] (3) Synthesis of (3-((4-(3-(4-(dipropylamino)-2-hydroxyphenyl)-2,4-dihydroxycyclobuta-1,3-dien-1-yl)-3-hydroxyphenyl)(propyl)amino)propyl)triethylammonium iodide [ka] In a test tube, 3-(4-(dipropylamino)-2-hydroxyphenyl)-4-hydroxycyclo-3-butene-1,2-dione (10 mg, 0.035 mM) and N,N,N-triethyl-3-((3-hydroxyphenyl)(propyl)amino)propane-1-ammonium iodide (14 mg, 0.033 mM) were added to a mixture of toluene (2.5 mL), 1-butanol (2.5 mL), and water (1 drop), and the mixture was stirred at 120°C for 7 hours, and the resulting dark green solution was concentrated. The resulting crude product was subjected to silica gel column chromatography (eluent: methylene chloride / methanol = 8 / 2), and the blue-green fraction was collected and concentrated to obtain the target product. MS(m / z):564.6

[0059] Example 3: Synthesis of squarylium dye derivatives (1) Synthesis of 1-(3-iodopropyl)-1-pyridinium iodide [ka] Acetonitrile (5.0 mL), pyridine (1.0 g, 12.3 mM), and diiodopropane (7.45 g, 25.2 mM) were added to a three-neck flask and stirred at a bath temperature of 60° C. for about 15 hours. The reaction solution was then concentrated to obtain a yellow solid. Hexane (5 mL) was further added to the obtained solid and stirred, and then filtered to recover the filtrate. The recovered filtrate was concentrated to obtain the target product as a pale yellow solid. The obtained compound was used as it was in the next reaction.

[0060] (2) Synthesis of 1-(3-((3-hydroxyphenyl)(propyl)amino)propyl)-1-pyridinium iodide [ka] 3-(Propylamino)phenol (102 mg, 0.68 mM), 1-(3-iodopropyl)-1-pyridinium iodide (250 mg, 0.67 mg), and methylene chloride (5 mL) were added to a test tube and stirred for about 23 hours at an external temperature of 40° C. Then, 1-(3-iodopropyl)-1-pyridinium iodide (250 mg, 0.67 mg) was further added and stirred for 4 days at an external temperature of 40° C. Next, the reaction solution was directly subjected to silica gel column chromatography (elution solvent: methylene chloride / methanol=100 / 0→50 / 50) to purify the target product (yield: 340 mg).

[0061] (3) Synthesis of (3-((4-(3-(4-(dipropylamino)-2-hydroxyphenyl)-2,4-dihydroxycyclobuta-1,3-dien-1-yl)-3-hydroxyphenyl)(propyl)amino)propyl)pyridinium iodide [ka] In a test tube, 3-(4-(dipropylamino)-2-hydroxyphenyl)-4-hydroxycyclo-3-butene-1,2-dione (10 mg, 0.035 mM) and 1-(3-((3-hydroxyphenyl)(propyl)amino)propyl)-1-pyridinium iodide (14 mg, 0.035 mM) were added to a mixed solution of toluene (2.5 mL), 1-butanol (2.5 mL), and water (1 drop), and the mixture was stirred at 120° C. for 7 hours. The resulting dark green solution was concentrated. The resulting crude product was subjected to silica gel column chromatography (eluent: methylene chloride / methanol=8 / 2), and the blue-green fraction was collected and concentrated to obtain the target product. MS(m / z):542.6

[0062] Example 4: Synthesis of squarylium dye derivatives [ka] Under an argon atmosphere, (3-((3-hydroxyphenyl)(propyl)amino)propyl)triphenylphosphonium iodide (100 mg, 0.172 mM), squaric acid (9.0 mg, 0.078 mM), 1-butanol (5.0 mL), and toluene (4.0 mL) were mixed in a three-necked flask equipped with a Dean-Stark distillation apparatus and heated under reflux for 1.5 hours to obtain a green suspension. The resulting solid was collected by filtration to obtain the target product (yield: 40 mg, 38%). MS(m / 2z):493.7.

[0063] Example 5 (1) Preparation of squarylium dye derivative solution Betaine ("Betaine 5" manufactured by Nard Laboratory, 1.71 g) was dissolved in purified water to prepare a 2000 mM betaine aqueous solution. The squarylium dye derivative (2.4 mg, 2.8 μM) synthesized in Example 1 was dissolved in a 2000 mM aqueous betaine solution (2.8 mL) to prepare a blue, transparent 1 mM squarylium dye derivative-2000 mM betaine solution.

[0064] (2) Dyeing test A 1 mM squarylium dye derivative-2000 mM betaine solution (10 μL) was mixed with MEM medium (10 mL) to prepare a 500 nM squarylium dye derivative MEM solution. In addition, an existing mitochondrial staining reagent ("MitoTracker Green (registered trademark)" manufactured by Thermo Fisher Scientific) was dissolved in MEM medium at a concentration of 200 nM according to the attached instructions.

[0065] 1 x 10 in a 35 mm glass bottom dish 5 Rat heart-derived H9c2 cells were seeded on the cells and incubated at 37°C, 5% CO 2 The medium was removed from the culture solution and replaced with the 500 nM squarylium dye derivative MEM solution (1 mL) prepared above, and the cells were incubated at 37°C, 5% CO 2 The cells were then stained by incubating for 30 minutes under reduced pressure. The 200 nM MitoTracker MEM solution prepared above was then added and incubated at 37°C, 5% CO 2 The cells were stained by incubating under 5% CO for 30 minutes. The medium was then removed and the cells were washed three times with PBS. Next, 1 mL of 1 μg / mL Hoechst solution (Hoechst 33342 product number (catalog number): H1399, Thermo Fisher Scientific) was added and the cells were incubated at 37°C, 5% CO 2 The sections were then incubated under reduced pressure for 30 minutes for staining, and then washed three times with Hank's balanced salt solution (HBSS, calcium, magnesium, no phenol red, product number (catalog number): 14025076, Thermo Fisher Scientific Co.), and finally replaced with 1 mL of Hank's balanced salt solution. Fluorescence micrographs were taken using a fluorescence microscope ("BZ-700" manufactured by Keyence Corporation) and a fluorescence filter with an excitation wavelength of 620±60 nm ("BZ-X filter Cy5" manufactured by Keyence Corporation), a fluorescence filter with an excitation wavelength of 470 / 40 nm ("BZ-X filter GFP" manufactured by Keyence Corporation), or no fluorescence filter. The squarylium dye derivative had an excitation wavelength peak of 655 nm and a fluorescence wavelength peak of 676 nm, allowing selective observation with a BZ-X filter Cy5. MitoTracker had an excitation wavelength peak of 490 nm and a fluorescence wavelength peak of 513 nm, allowing selective observation with a BZ-X filter GFP. The results of staining with squarylium dye derivatives are shown in Figure 1(1), the results of staining with MitoTracker in Figure 1(2), and the results of co-staining with squarylium dye derivatives and MitoTracker in Figure 1(3). As shown in FIG. 1, the sites stained by the squarylium dye derivative of the present invention are consistent with the sites stained by existing mitochondrial staining reagents, demonstrating that the squarylium dye derivative of the present invention accumulates in mitochondria within cells and can specifically stain mitochondria.

[0066] Example 6: Long-term observation The 1 mM squarylium dye derivative-2000 mM betaine solution (5 μL) prepared in Example 5(1) was mixed with MEM medium. The resulting 500 nM squarylium dye derivative MEM solution (0.2 mL) was diluted with MEM medium to adjust the total volume to 10 mL, thereby obtaining a 10 nM squarylium dye derivative MEM solution. 3 x 10 cells in a 35 mm glass bottom dish. 4 H9c2 cells derived from rat heart were seeded in 2 mL of MEM medium. After one day, the medium was removed from the dish and replaced with the 10 nM squarylium dye derivative MEM solution (2 mL) prepared above. The cells were incubated at 37°C, 5% CO 2 The cells were incubated for 4 days under low temperature. After 48 hours, the medium was replaced with a fresh 10 nM squarylium dye derivative MEM solution to add nutrients for the culture and remove waste products. Fluorescence observation was performed using a fluorescence microscope ("BZ-700" manufactured by Keyence Corporation) and a fluorescence filter ("BZ-X filter Cy5" manufactured by Keyence Corporation) 24 hours, 48 ​​hours, 72 hours, and 96 hours after the start of the culture. The results are shown in Figure 2.

[0067] As shown in FIG. 2, cells can be cultured even in a medium containing the squarylium dye derivative according to the present invention, and fluorescence can be observed continuously for a long period of time.

[0068] Example 7: Toxicity Testing Rat heart-derived H9c2 cells were seeded in each well of a 96-well microplate and incubated at 37°C, 5% CO 2 The medium was removed from the pre-incubated microplate, and the medium in each well was replaced with medium alone or a MEM solution (100 μL) containing 0 nM, 50 nM, 100 nM, 250 nM, 500 nM, or 1000 nM of the squarylium dye derivative (Example 1) and 2 mM betaine, and incubated at 37° C., 5% CO 2 The cells were incubated for 24 hours under reduced pressure. Next, 10 μL of WST-8 reagent was added and cultured for 1 to 4 hours. After that, the absorbance at 450 nm, which is the maximum absorption wavelength of WST-8 formazan generated by reduction of WST-8 reagent by live cells, was measured using a microreader. The results are shown in Figure 3. As shown in the results in FIG. 3, the squarylium dye derivative according to the present invention caused almost no change in cell number even at a concentration of 1000 μM, and is harmless to cells, demonstrating that its toxicity is extremely low.

[0069] Example 8: Long-term observation Rat heart-derived H9c2 cells were seeded on a 35 mm glass-bottom dish and incubated at 37°C, 5% CO 2 After one day, the medium was removed from the dish and replaced with MEM solution (2 mL) containing 500 nM of the squarylium dye derivative (Example 1), and the cells were incubated at 37° C., 5% CO 2 Incubated under Time-lapse photography was performed using a fluorescence microscope (Keyence BZ-700) and a fluorescence filter with an excitation wavelength of 620±60 nm (Keyence BZ-X filter Cy5). The results are shown in Figure 4. In Figure 4, time progresses from the upper left to the lower right. As shown in FIG. 4, the squarylium dye derivative according to the present invention was demonstrated to be capable of staining live cells for a long period of time, both before and after cell division.

Claims

1. 1. A squarylium dye derivative or a salt thereof, characterized by being represented by the following formula (I): 【Chemistry 1】 [In the formula, R 1 and R 2 is independently 1-6 Alkyl group, C 6-12 Aryl group, or C 6-12 represents an aryl-methyl group, R 3 and R 4 each independently represents a single bond or a linker group, Z 1 and Z 2 each independently represents H or a cationic group; Z 1 and Z 2 At least one of the groups represents a cationic group.

2. Z 1 and Z 2 At least one of the following is -P + Ph 3 2. The squarylium dye derivative or salt thereof according to claim 1 , wherein the formula (I) represents a phenyl group which may have a substituent, and a plurality of Phs may be the same or different.

3. Z 1 indicates H, and Z 2 represents a cationic group, R 1 and R 2 But independently, C 1-6 represents an alkyl group, R 3 C 1-6 The squarylium dye derivative or a salt thereof according to claim 1, which represents an alkanediyl group.

4. 3. The squarylium dye derivative or a salt thereof according to claim 2, wherein Ph is a phenyl group.

5. 2. The squarylium dye derivative or a salt thereof according to claim 1, which contains a halide ion.

6. A mitochondrial staining reagent comprising the squarylium dye derivative or a salt thereof according to any one of claims 1 to 5.

7. The mitochondrial staining reagent according to claim 6 , further comprising an aqueous solvent and a betaine compound.

8. adding the mitochondrial staining reagent according to claim 6 to a eukaryotic cell; and A method for staining mitochondria, comprising the step of irradiating the eukaryotic cells with light and observing the fluorescence.

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