Diaromatic phosphonium cross-linking compound and mitochondrion stain using the same

Abstract

The present application provides a compound represented by general formula (1), wherein the compound represented by general formula (1) is a small molecule and selectively emits fluorescence to mitochondria (particularly, inner membrane of mitochondria).[In general formula (1), Ar 1 and Ar 2 represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. R 1 and R 2 represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group. Among them, R 1 and R 2 optionally form a ring together with the adjacent nitrogen atom. R 1 and / or R 2 optionally form a ring together with the adjacent nitrogen atom and Ar 2 . R 3 and R 4 represent a substituted or unsubstituted (hetero)aryl group. R 5 represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

Description

Technical Field

[0001] This invention relates to diarylphosphonium cross-linked compounds and mitochondrial staining agents using such diarylphosphonium cross-linked compounds. Background Technology

[0002] Mitochondria are oval organelles found in all eukaryotes, separated by an outer membrane and an inner membrane. An intermembrane space exists between the outer and inner membranes; the folded structures of the inner membrane are called cristae, and the inner side of the inner membrane is called the matrix. Within the cristae, oxygen-based oxidative phosphorylation reactions occur using pyruvate, amino acids, and fatty acids from glycolysis to synthesize ATP, the energy currency. Mitochondria produce more than 90% of the ATP required for cellular metabolism, playing a crucial role in cell survival and death. In addition to ATP synthesis, mitochondria participate in various cellular functions, including intracellular ion homeostasis, cell growth, regulation of cell signaling, and control of apoptosis. Therefore, mitochondrial dysfunction is not only closely related to neurodegenerative diseases, cancer, diabetes, and many other diseases, but also has been the subject of numerous research reports in recent years regarding mitochondrial function and aging. In fact, the dramatic increase in the number of mitochondrial-related papers searched on PubMed reflects the high level of attention this field has received.

[0003] Of particular interest is mitochondrial fusion and division. For example, respiratory failure and mitochondrial DNA loss are known to occur in mutant strains of mitochondrial fusion factor. Furthermore, mitochondrial DNA loss has also been observed in HeLa cells under fusion inhibition conditions, indicating that mitochondrial fusion is indispensable for cell survival. Regarding mitochondrial division, reports indicate that apoptosis is delayed under division inhibition conditions, and abnormal synapse formation is associated with a reduction in mitochondria within neurites, suggesting that mitochondrial division plays a crucial biological role. However, the mechanisms of mitochondrial DNA loss due to fusion inhibition and the function of extraneural division remain unclear, and further research into mitochondrial fusion and division is strongly encouraged.

[0004] Besides the fusion and division of mitochondrial monomers, their interactions with other organelles and the dynamics of lipids serving as sites of these interactions have also attracted considerable attention. Mitochondrial lipids include those synthesized within the mitochondria and those synthesized externally. It is known that lipids synthesized internally are transported to other organelles, while those synthesized externally are primarily transported by the endoplasmic reticulum. Membrane contact sites between organelles have been reported to be crucial during these transports. In studies involving mitochondrial interactions and organelle contact, the split-GFP system, reported in 2018, has been used as a powerful tool. In the split-GFP system, GFP remodels and emits strong fluorescence when mitochondria interact with and come into physical proximity with other organelles. However, the possibility that split-GFP itself creates organelle contact sites cannot be ruled out from the reaction mechanism; therefore, there is still a strong desire to develop a probe capable of detecting mitochondrial interactions with other organelles without direct involvement.

[0005] In summary, research on mitochondrial fusion, division, and interactions with different organelles is a highly interested area. To advance this research, efforts are being made to develop fluorescent probes capable of selectively staining the inner mitochondrial membrane and tracking membrane dynamics in real time. Fluorescent probes require several properties, among which the following are particularly important: 1) they should not interfere with organelle interactions; 2) they should be molecularly sized to not affect membrane dynamics; 3) they should exhibit environmental responsiveness, showing changes in fluorescence properties due to organelle interactions; and 4) they should possess photostability.

[0006] In response, the inventors of this application have developed MitoPBYellow, a super-photostable fluorescent imaging probe capable of selectively staining the inner mitochondrial membrane of living cells.

[0007] [Chemical Formula 1] Compared to commercially available MitoTracker series, MitoPBYellow exhibits overwhelming lightfastness, remaining virtually unaffected by fading even in super-resolution fluorescence imaging using STED microscopes that require strong laser illumination, enabling long-duration time-lapse imaging of mitochondrial inner membrane dynamics. Existing technical documents Non-patent literature

[0008] Non-licensed reference 1: Proc. Natl. Acad. Sci., 2019, 116, 15817-15822. Summary of the Invention (a) Technical problems to be solved

[0009] However, in MitoPBYellow, since the phosphorus site responsible for mitochondrial localization is introduced separately from the pigment backbone, there is a technical problem that the molecular weight will increase.

[0010] The present invention is made to solve the prior art problems as described above, and its purpose is to provide a compound that is a small molecule and can selectively fluoresce to mitochondria (especially the inner mitochondrial membrane). (II) Technical Solution

[0011] The inventors of this application conducted repeated and in-depth research in view of the aforementioned technical problems, and discovered that by having a specific structure with a diarylphosphonium cross-linking structure between the aromatic ring and the thiophene ring, even small molecules can introduce the phosphonium site responsible for mitochondrial localization into the pigment backbone. Furthermore, extended functional groups can be introduced onto the pigment backbone, thus imparting selectivity to the mitochondrial inner membrane and enabling selective fluorescence of mitochondria (especially the mitochondrial inner membrane). Based on the above insights, the inventors of this application further conducted repeated research, thereby completing this invention. That is, this invention includes the following solutions.

[0012] Item 1. A compound represented by general formula (1),

[0013] [Chemical Formula 2] In general formula (1), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings. R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl. Among them, R 1 and R 2 They can be chosen to form a ring with adjacent nitrogen atoms. R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring. R 3 and R 4 Same or different indicates (hetero)aryl groups that have been substituted or not substituted. R 5 This indicates a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0014] Item 2. The compound according to item 1, wherein the Ar 1It refers to the substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0015] Item 3. The compound according to item 1 or 2, wherein the Ar 2 It refers to the substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0016] Item 4. The compound according to any one of items 1 to 3, wherein R 1 and R 2 The same or different, and consisting of hydrogen atoms, or substituted or unsubstituted alkyl groups.

[0017] Item 5. The compound according to any one of items 1 to 4, wherein R 3 and R 4 The same or different, and whether the aryl group is substituted or not.

[0018] Item 6. The compound according to any one of items 1 to 5, wherein R 5 It can be a substituted or unsubstituted alkyl group.

[0019] Item 7. A fluorescent dye comprising any one of items 1 to 6 or a solvate thereof.

[0020] Item 8. A mitochondrial staining agent comprising any one of items 1 to 6 or a solvate thereof.

[0021] Item 9. The mitochondrial staining agent according to Item 8, which is a mitochondrial inner membrane staining agent.

[0022] Item 10. A method for preparing the compound described in any one of items 1 to 6, comprising: a step of causing the compound represented by general formula (2) to undergo a ring-closing reaction in the presence of a copper compound.

[0023] [Chemical Formula 3] In general formula (2), Ar 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 Ar in item 1 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 same.]

[0024] Item 11. A compound represented by general formula (2).

[0025] [Chemical Formula 4] In general formula (2), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings. R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl. Among them, R 1 and R 2 They can be chosen to form a ring with adjacent nitrogen atoms. R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring. R 3 and R 4 Same or different indicates (hetero)aryl groups that have been substituted or not substituted. R 5 This refers to a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0026] Item 12. A method for preparing the compound described in Item 11, comprising the steps of reacting a compound represented by general formula (3) with a lithium compound, and then reacting the compound with a compound represented by general formula (4).

[0027] [Chemical Formula 5] In general formula (3), Ar 1 Ar 2 R 1 R 2 and R 5 Ar in item 11 above 1 Ar 2 R 1 R 2 and R 5 Same. X 1 This represents a halogen atom. PR 3 R 4 X 2 (4) [In the formula, R] 3 and R 4 R in item 11 above 3 and R4 Same. X 2 This represents a halogen atom.

[0028] Item 13. A compound represented by general formula (3).

[0029] [Chemical Formula 6] In general formula (3), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings. R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl. Among them, R 1 and R 2 They can be chosen to form a ring with adjacent nitrogen atoms. R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring. R 5 It represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group. X 1 This represents a halogen atom.

[0030] Item 14. A method for preparing the compound described in Item 13, comprising: a step of reacting the compound represented by general formula (5) with the compound represented by general formula (6) in the presence of a palladium catalyst.

[0031] [Chemical Formula 7] In general formula (5), Ar 1 and R 5 Ar in item 13 above 1 and R 5 Same. Y 1 This indicates boric acid (or boronic acid) or its ester group.

[0032] [Chemical Formula 8] In general formula (6), Ar 2 R 1 R 2 and X 1 Ar in item 13 above 2 R1 R 2 and X 1 Same. X 3 Indicates with X 1 Different halogen atoms. (III) Beneficial Effects

[0033] According to the present invention, a compound is provided that is a small molecule but can selectively fluoresce to mitochondria (especially the inner mitochondrial membrane). Attached Figure Description

[0034] Figure 1 The results of X-ray crystallography of compound 8 from Experimental Example 1 are shown. (a) The morphology of two neighboring molecules is shown. (b) The morphology of compound 8 is shown. Hydrogen atoms have been omitted for clarity. Figure 2 The results of the photophysical property measurements of compound 8 in Experimental Example 2 are shown. Solid lines represent the UV-Vis absorption spectrum, and dashed lines represent the fluorescence spectrum (λ). ex =460nm). Figure 3 The results of photophysical property measurements for compound 8 (Example 1; phenyl) and compound 10 (Comparative Example 1; cyclohexyl) from Test Example 2 are shown. Solid lines represent UV-Vis absorption spectra, and dashed lines represent fluorescence spectra. Figure 4 The results of fluorescence imaging of HeLa cells using compound 8 in Experimental Example 3 are shown. (a) shows a confocal image of HeLa cells stained with 500 nM compound 8. Before washing (left), after washing (right). DMEM (+) (top), DMEM (-) (bottom). (b) shows a confocal image of HeLa cells stained with 50 nM compound 8 (unwashed). ex =473nm, λ em =550-650nm. Figure 5 The results of co-staining HeLa cells with Mito Tracker (registered trademark) DeepRed in Experiment Example 3 are shown. Figure 6 The results of mitochondrial staining using compound 8 in Experimental Example 3 are shown using confocal microscopy and STED microscopy. (a) Stained image of mitochondria using confocal microscopy. (b) Stained image of mitochondria using STED microscopy. Detailed Implementation

[0035] In this specification, "comprise" also includes the concepts of "consistes essentially of" and "consist of".

[0036] In this specification, when the range is indicated by "A~B", unless otherwise specified, it means above A and below B.

[0037] 1. Compound The compounds of the present invention are represented by general formula (1).

[0038] [Chemical Formula 9] In general formula (1), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings. R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl. Among them, R 1 and R 2 They can be chosen to form a ring with adjacent nitrogen atoms. R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring. R 3 and R 4 Same or different indicates (hetero)aryl groups that have been substituted or not substituted. R 5 This refers to a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0039] In this invention, the compound represented by general formula (1) is a novel compound. On the other hand, the compound represented by general formula (1) can recognize mitochondria (especially the inner mitochondrial membrane) and can therefore be used as a mitochondrial staining agent (especially the inner mitochondrial membrane staining agent) that can selectively stain mitochondria (especially the inner mitochondrial membrane).

[0040] Furthermore, the compounds represented by the above general formula (1) are environmentally responsive by having an amino group or a substituted amino group as an electron-donating group, and can also achieve longer absorption peak wavelengths, thereby reducing phototoxicity to mitochondria.

[0041] Furthermore, the compound represented by the above general formula (1) has a specific structure with a diarylphosphonium cross-linking structure between the aromatic ring and the thiophene ring. This backbone is a donor-π-acceptor type molecule with a ladder structure that introduces a cationic phosphonium group as an electron-withdrawing group. By directly introducing the cationic site required for mitochondrial localization into the pigment backbone, the molecular weight can be suppressed to a small size, enabling staining of mitochondria. In addition, the compound represented by the above general formula (1) can introduce extended functional groups onto the pigment backbone, thus imparting selectivity to the outer mitochondrial membrane. In summary, the compound represented by general formula (1) is suitable for repeated observation in vivo using super-resolution microscopy such as stimulated emission depletion (STED) imaging.

[0042] Furthermore, in general formula (1), by substituting R on P... 3 and R 4 By setting it to (hetero)aryl, the absorption peak wavelength can be lengthened, and selective staining of mitochondria (especially the inner mitochondrial membrane) can be achieved. For example, when R... 3 and R 4 When the alkyl or cycloalkyl group is used, it is not easy to increase the wavelength of the absorption peak, and it will also stain tissues other than mitochondria, making it impossible to selectively stain mitochondria (especially the inner mitochondrial membrane).

[0043] In general formula (1), as Ar 1 and Ar 2 The aromatic hydrocarbon ring shown can be either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring. For example, benzene ring can be listed as a monocyclic aromatic hydrocarbon ring, and naphthalene ring, anthracene ring, phenanthrene ring, fluorene ring, pyrene ring, benzo[a]phenanthrene ring, etc. can be listed as polycyclic aromatic hydrocarbon rings.

[0044] Ar 1 and Ar 2 The aromatic hydrocarbon ring shown may have substituents. Examples of substituents include alkyl, cycloalkyl, aryl, heteroaryl, alkenyl (vinyl, propenyl, etc.), ynyl (ethynyl, 1-propynyl, etc.), carbonyl, carboxyl, sulfonyl, amide (amide, N-alkylamide, etc.; as alkyl in N-alkylamide, the above groups can be listed), cyano, nitro, etc. The number of substituents is preferably 1 to 5, more preferably 1 to 3.

[0045] In general formula (1), as Ar 1 and Ar 2 The heteroaromatic rings shown can be categorized as monocyclic heteroaromatic rings, such as pyridine rings and pyrazine rings; and as polycyclic heteroaromatic rings, such as indole rings, isoindole rings, benzimidazole rings, quinoline rings, isoquinoline rings, and quinoxaline rings.

[0046] Ar 1 and Ar 2 The heteroaryl ring shown may have substituents. Examples of substituents include alkyl, cycloalkyl, aryl, heteroaryl, alkenyl (vinyl, propenyl, etc.), alkynyl (ethynyl, 1-propynyl, etc.), carbonyl, carboxyl, sulfonyl, amide (amide, N-alkylamide, etc.; the above groups can be listed as alkyl groups in N-alkylamide), cyano, nitro, halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.). The number of substituents is preferably 1 to 5, more preferably 1 to 3.

[0047] As Ar 1 and Ar 2 From the perspectives of mitochondrial (especially mitochondrial inner membrane) staining, structural stability, absorption peak wavelength, phototoxicity to mitochondria, and light resistance, substituted or unsubstituted aromatic hydrocarbon rings are preferred, and substituted or unsubstituted monocyclic aromatic hydrocarbon rings are even more preferred.

[0048] In general formula (1), as R 1 and R 2 The alkyl group shown can be any of straight-chain alkyl or branched alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, etc., which are C1-15 alkyl groups (especially C1-10 alkyl groups).

[0049] R 1 and R 2 The alkyl group shown may have substituents. Examples of substituents include halogen atoms (fluorine, chlorine, bromine, iodine, etc.), cycloalkyl groups (described later), aryl groups (described later), heteroaryl groups (described later), alkenyl groups (vinyl, propenyl, etc.), alkynyl groups (ethynyl, 1-propynyl, etc.), carbonyl groups, carboxyl groups, sulfonyl groups, amide groups (amide groups, N-alkylamide groups, etc.; the above groups can be listed as alkyl groups in N-alkylamide groups), cyano groups, nitro groups, etc. The number of substituents when substituents are present is preferably 1 to 5, more preferably 1 to 3.

[0050] In general formula (1), as R 1 and R 2 The cycloalkyl groups shown include, for example, C3-10 cycloalkyl groups (especially C4-8 cycloalkyl groups) such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0051] R 1 and R 2The cycloalkyl group shown may have substituents. Examples of substituents include halogen atoms (fluorine, chlorine, bromine, iodine, etc.), the aforementioned cycloalkyl groups, the aforementioned alkyl groups, aryl groups (described later), heteroaryl groups (described later), alkenyl groups (vinyl, propenyl, etc.), alkynyl groups (ethynyl, 1-propynyl, etc.), carbonyl groups, carboxyl groups, sulfonyl groups, amide groups (amide groups, N-alkylamide groups, etc.; examples of alkyl groups in N-alkylamide groups include the aforementioned groups), cyano groups, nitro groups, etc. The number of substituents when substituents are present is preferably 1 to 6, more preferably 1 to 3.

[0052] In general formula (1), as R 1 and R 2 The aryl group shown can be any of monocyclic aryl, fused-ring aryl, and polycyclic aryl. For example, phenyl can be listed as a monocyclic aryl, naphthyl, anthracene, phenanthryl, fluorenyl, pyrene, triphenylene, etc. can be listed as fused-ring aryl, and biphenyl, terphenyl, etc., which are C6-18 aryl groups (especially C6-14 aryl groups).

[0053] R 1 and R 2 The aryl group shown may have substituents. Examples of substituents include halogen atoms (fluorine, chlorine, bromine, iodine, etc.), the aforementioned alkyl groups, the aforementioned cycloalkyl groups, the aforementioned aryl groups, the following heteroaryl groups, alkenyl groups (vinyl, propenyl, etc.), alkynyl groups (ethynyl, 1-propynyl, etc.), carbonyl groups, carboxyl groups, sulfonyl groups, amide groups (amide groups, N-alkylamide groups, etc.; examples of alkyl groups in N-alkylamide groups include the aforementioned groups), cyano groups, nitro groups, etc. When substituents are present, the number of substituents is preferably 1 to 6, more preferably 1 to 3.

[0054] In general formula (1), R is used as 1 and R 2 The heteroaryl group shown can be either a monocyclic heteroaryl or a fused-ring heteroaryl. Examples of monocyclic heteroaryl groups include pyrroleyl, thiophenyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, piperidinyl, pyridinyl, and pyrazinyl. Examples of fused-ring heteroaryl groups include indolyl, isoindolyl, benzimidazolyl, quinolinyl, isoquinolinyl, and quinoxalinyl.

[0055] R 1 and R 2The heteroaryl group shown may have substituents. Examples of substituents include, for example, the halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the alkyl group, the cycloalkyl group, the aryl group, the heteroaryl group, the alkenyl group (vinyl group, propenyl group, etc.), the alkynyl group (ethynyl group, 1-propynyl group, etc.), the carbonyl group, the carboxyl group, the sulfonyl group, the amide group (amide group, N-alkylamide group, etc.; examples of alkyl groups in N-alkylamide groups include the above-mentioned groups), the cyano group, the nitro group, etc. When substituents are present, the number of substituents is preferably 1 to 6, more preferably 1 to 3.

[0056] Among them, as R 1 and R 2 From the perspective of easily increasing the wavelength of the absorption peak, easily identifying mitochondria, and easily staining the inner mitochondrial membrane, hydrogen atoms, substituted or unsubstituted alkyl groups, substituted or unsubstituted cycloalkyl groups, etc. are preferred, hydrogen atoms, substituted or unsubstituted alkyl groups, etc. are more preferred, and substituted or unsubstituted alkyl groups are even more preferred.

[0057] Additionally, R 1 and R 2 They can optionally form a ring with adjacent nitrogen atoms. That is, -NR 1 R 2 The group represented can be any of the groups represented by the following groups,

[0058] [Chemical Formula 10] .

[0059] In addition, R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring. That is, Ar 2 -NR 1 R 2 The structure represented can be, for example, the following structures.

[0060] [Chemical Formula 11] .

[0061] At this time, in Ar 2 In the middle, -NR 1 R 2 There are no particular restrictions on the substitution positions to which the indicated groups are bonded.

[0062] In general formula (1), as R 3 and R 4 The aryl and heteroaryl groups shown can be those described above. The types of substituents are also the same. Additionally, to R... 3 and R 4Introducing substituents into the aryl and heteroaryl groups shown, and making them larger groups, can further improve lightfastness.

[0063] In general formula (1), as R 5 The alkyl and cycloalkyl groups shown can be those described above. The types of substituents are also the same, and functional groups can be introduced to consider high selectivity for the mitochondrial inner membrane, etc.

[0064] As compounds represented by general formula (1) that satisfy the conditions described above, examples include the following compounds, etc.

[0065] [Chemical Formula 12] .

[0066] The compounds of the present invention may contain not only the above-described structure, but also counter anions. There are no particular limitations on the counter anions; examples include acetate ions, trifluoroacetate ions, halide ions (fluoride ions, chloride ions, bromide ions, iodide ions, etc.), perchlorate ions, and PF6. - BF4 - Sulfate ions, nitrate ions, phosphate ions, bis(trifluoroimide) ions, etc.

[0067] 2. Fluorescent pigments and mitochondrial staining agents The fluorescent dye of the present invention contains the compounds of the present invention described above.

[0068] The fluorescent dye of the present invention employs a diarylphosphonium cross-linked structure between the aromatic ring and the thiophene ring, thus enabling the absorption peak wavelength to be extended.

[0069] Furthermore, the fluorescent dye of the present invention can selectively stain mitochondria (especially the inner mitochondrial membrane). Moreover, the compounds of the present invention can cause mitochondria (especially the inner mitochondrial membrane) to emit strong fluorescence, suppressing the fluorescence of other tissues. By suppressing the fluorescence of tissues other than mitochondria (especially the inner mitochondrial membrane), the compounds of the present invention enable the fluorescence intensity of mitochondria (especially the inner mitochondrial membrane) to be significantly greater than that of other tissues. Therefore, even at small sizes, mitochondria (especially the inner mitochondrial membrane) can be detected with high sensitivity, thereby suppressing the fluorescence staining of other tissues. Therefore, the fluorescent dye of the present invention, using the compounds of the present invention, is useful as a mitochondrial staining agent (especially a mitochondrial inner membrane staining agent). Furthermore, by using this fluorescent dye of the present invention, the membrane structure (membrane composition) of the inner mitochondrial membrane can be deduced.

[0070] The fluorescent dye (mitochondrial staining agent) of the present invention contains the compound of the present invention described above or a solvate thereof. Its use is not particularly limited; for example, it can be dissolved in an organic solvent to prepare a solution. In this case, even small mitochondria (especially the inner mitochondrial membrane) can be easily detected (stained) with high sensitivity. From the perspective of easily suppressing fluorescence in tissues other than mitochondria (especially the inner mitochondrial membrane), the content of the compound of the present invention described above is preferably 10 nmol / L to 500 nmol / L, more preferably 30 nmol / L to 200 nmol / L. In particular, even when the content is reduced to 30 to 80 nmol / L, mitochondria (especially the inner mitochondrial membrane) can still be detected with high sensitivity. Thus, in the present invention, the content of the compound of the present invention can be suppressed to a low level, thereby easily suppressing damage to living cells.

[0071] When preparing a solution containing the above-described compound of the present invention using the fluorescent dye (mitochondrial staining agent) of the present invention, there are no particular limitations on the organic solvents that can be used. For the compound of the present invention, as a donor-π-acceptor molecule, due to its low solvent dependence in both absorption and fluorescence spectra, various organic solvents can be used, such as aromatic solvents like benzene, toluene, xylene, and mesitylene; aliphatic halogenated hydrocarbons like dichloromethane and dichloroethane; nitrile solvents like acetonitrile; and sulfoxide solvents like dimethyl sulfoxide. Furthermore, water can also be used as a solvent; the fluorescent dye (mitochondrial staining agent) of the present invention can be used even in an aqueous solution environment, and is therefore suitable for use in vivo.

[0072] 3. Preparation method The compound represented by general formula (1) of the present invention can be obtained, for example, by the following preparation method, which includes a step of causing the compound represented by general formula (2) to undergo a ring-closing reaction in the presence of a copper compound.

[0073] [Chemical Formula 13] In general formula (2), Ar 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 Ar in the above general formula (1) 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 same.]

[0074] Furthermore, the compound represented by general formula (2) can be obtained, for example, by the following preparation method, which includes the steps of reacting the compound represented by general formula (3) with a lithium compound, and then reacting it with the compound represented by general formula (4).

[0075] [Chemical Formula 14] In general formula (3), Ar 1 Ar 2 R 1 R 2 and R 5 Ar in the above general formula (2) 1 Ar 2 R 1 R 2 and R 5 Same. X 1 This represents a halogen atom. PR 3 R 4 X 2 (4) In general formula (4), R 3 and R 4 R in the above general formula (2) 3 and R 4 Same. X 2 This represents a halogen atom.

[0076] Furthermore, the compound represented by general formula (3) can be obtained, for example, by the following preparation method, which includes a step of reacting the compound represented by general formula (5) with the compound represented by general formula (6) in the presence of a palladium catalyst.

[0077] [Chemical Formula 15] In general formula (5), Ar 1 and R 5 Ar in the above general formula (3) 1 and R 5 Same. Y 1 This indicates boric acid or its ester group.

[0078] [Chemical Formula 16] In general formula (6), Ar 2 R 1 R 2 and X 1 Ar in the above general formula (3) 2 R 1 R2 and X 1 Same. X 3 Indicates with X 1 Different halogen atoms.

[0079] (3-1) Compound (2) → Compound (1) The preparation method of the compound represented by general formula (2) will be described later.

[0080] Examples of copper compounds include metallic copper and other copper compounds known as catalysts for the synthesis of organic compounds (including polymers). Examples of copper compounds include metallic copper; copper halides (I) such as copper iodide (I), copper bromide (I), and copper chloride (I); and copper trifluoromethanesulfonate (II). In this process, copper trifluoromethanesulfonate (II) is preferred. One of these copper compounds may be used alone, or two or more may be used in combination. Furthermore, these copper compounds may include either substances added as reagents or substances generated in the system.

[0081] From the perspectives of conversion rate, selectivity, and yield, the amount of copper compound used is generally preferably 1.0 to 5.0 moles, more preferably 1.5 to 3.0 moles, relative to 1 mole of the compound represented by general formula (2) as a raw material. Furthermore, when using multiple copper compounds, it is preferable to adjust the total amount to be within the above-mentioned range.

[0082] This process can be carried out in the presence of a solvent. Examples of usable solvents include aliphatic hydrocarbons (hexane, cyclohexane, heptane, etc.), aliphatic halogenated hydrocarbons (dichloromethane, chloroform, carbon tetrachloride, dichloroethane, etc.), aromatic hydrocarbons (benzene, toluene, xylene, chlorobenzene, etc.), nitriles (acetonitrile, propionitrile, etc.), and sulfoxides (dimethyl sulfoxide (DMSO), etc.). One of these solvents can be used alone, or two or more can be used in combination.

[0083] The reaction temperature in this process can typically be 0~50℃, and in particular, 10~40℃. Furthermore, the reaction time can be set to allow the reaction to proceed to its full extent.

[0084] In addition, the reaction atmosphere is not particularly limited and can be set to an inactive gas atmosphere such as argon or nitrogen.

[0085] After the reaction, the compound can be used for routine post-processing such as solvent removal, washing, and chromatographic separation as needed to obtain the compound represented by general formula (1).

[0086] The compound represented by general formula (1) obtained in this way is a novel compound not recorded in the literature.

[0087] (3-2) Compound (3) → Compound (2) The preparation method of the compound represented by general formula (3) will be described later.

[0088] There are no particular limitations on the lithium compounds used; examples include alkyl lithiums such as ethyl lithium, n-propyl lithium, isopropyl lithium, n-butyl lithium, sec-butyl lithium, tert-butyl lithium, n-pentyl lithium, and n-hexyl lithium; cycloalkyl lithiums such as cyclohexyl lithium; and aryl lithiums such as phenyl lithium. In this process, considering conversion, selectivity, and yield, alkyl lithiums are preferred, and n-butyl lithium is more preferred. These lithium compounds can be used alone or in combination of two or more.

[0089] From the perspectives of conversion rate, selectivity, and yield, the amount of lithium compound used is generally preferably 0.7 to 2.0 moles, more preferably 1.0 to 1.5 moles, relative to 1 mole of the compound represented by general formula (3) used as a raw material. In addition, when using multiple lithium compounds, it is preferable to adjust the total amount to be within the above range.

[0090] Then, considering factors such as conversion rate, selectivity, and yield, the amount of the compound represented by general formula (4) is usually preferably 0.7 to 3.0 moles, more preferably 0.7 to 1.5 moles, relative to 1 mole of the compound represented by general formula (3) as a raw material.

[0091] This process can be carried out in the presence of a solvent. Examples of usable solvents include aliphatic hydrocarbons (hexane, cyclohexane, heptane, etc.), aliphatic halogenated hydrocarbons (dichloromethane, chloroform, carbon tetrachloride, dichloroethane, etc.), aromatic hydrocarbons (benzene, toluene, xylene, chlorobenzene, etc.), cyclic ethers (tetrahydrofuran, dioxane, etc.), nitriles (acetonitrile, propionitrile, etc.), and sulfoxides (dimethyl sulfoxide (DMSO), etc.). One of these solvents can be used alone, or two or more can be used in combination.

[0092] The reaction temperature in this process can typically be 0~50℃, and in particular, 10~40℃. Furthermore, the reaction time can be set to allow the reaction to proceed to its full extent.

[0093] In addition, the reaction atmosphere is not particularly limited and can be set to an inactive gas atmosphere such as argon or nitrogen.

[0094] After the reaction, the compound can be subjected to routine post-processing such as solvent removal, cleaning, and chromatographic separation as needed to obtain the compound represented by general formula (2).

[0095] The compound represented by general formula (2) obtained in this way is a novel compound not recorded in the literature.

[0096] (3-3) Compound (5) → Compound (3) The preparation method of the compound represented by general formula (5) will be described later. In addition, the compound represented by general formula (6) can be synthesized according to the examples described later.

[0097] From the perspectives of conversion rate, selectivity, and yield, the amount of compound represented by general formula (6) is usually preferably 0.7 to 3.0 moles, more preferably 0.7 to 1.5 moles, relative to 1 mole of compound represented by general formula (3) as a raw material.

[0098] As palladium compounds, palladium catalysts known for their use in the synthesis of organic compounds can be cited. Specifically, examples include palladium acetate, tetra(triphenylphosphine)palladium (0), palladium trifluoroacetate, palladium chloride, palladium bromide, palladium iodide, tris(dibenzylacetone)palladium (0), bis(dibenzylacetone)palladium (0), dichloro(1,5-cyclooctadiene)palladium (II), and 2,5-norbornadiene palladium dichloride. These palladium compounds can also be used as solvates. Furthermore, these palladium compounds can be used alone or in combination of two or more. In this process, tetra(triphenylphosphine)palladium (0) (or its solvate) is preferred from the perspectives of conversion, selectivity, and yield.

[0099] From the perspectives of conversion rate, selectivity, and yield, the amount of palladium compound used is preferably 0.05 to 0.50 moles, more preferably 0.10 to 0.30 moles, relative to 1 mole of the compound represented by general formula (3) used as a raw material.

[0100] An alkali may also be used in this process as needed. Examples of alkalis include ammonium chloride, potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium tert-butoxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, and calcium acetate. In this process, from the perspectives of conversion rate, selectivity, and yield, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, and cesium carbonate are preferred, with potassium carbonate being more preferred.

[0101] From the perspectives of conversion rate, selectivity, and yield, the amount of alkali used is preferably 2.0 to 15.0 moles, more preferably 3.0 to 10.0 moles, relative to 1 mole of compound (3).

[0102] This process can be carried out in the presence of a solvent. Examples of usable solvents include aliphatic hydrocarbons (hexane, cyclohexane, heptane, etc.), aliphatic halogenated hydrocarbons (dichloromethane, chloroform, carbon tetrachloride, dichloroethane, etc.), aromatic hydrocarbons (benzene, toluene, xylene, chlorobenzene, etc.), cyclic ethers (tetrahydrofuran, dioxane, etc.), alcohols (ethanol, methanol, etc.), nitriles (acetonitrile, propionitrile, etc.), and sulfoxides (dimethyl sulfoxide (DMSO), etc.). Water can also be used as a solvent in this process. These solvents can be used alone or in combination of two or more.

[0103] The reaction temperature in this process can typically be 80~200℃, and in particular 100~150℃. Furthermore, the reaction time can be set to allow the reaction to proceed to its full extent.

[0104] In addition, the reaction atmosphere is not particularly limited and can be set to an inactive gas atmosphere such as argon or nitrogen.

[0105] After the reaction, the compound can be used for routine post-processing such as solvent removal, cleaning, and chromatographic separation as needed to obtain the compound represented by general formula (3).

[0106] The compound represented by general formula (3) obtained in this way is a novel compound not recorded in the literature.

[0107] (3-4) Compound (6) → Compound (5) Furthermore, the compound represented by general formula (5) can be obtained, for example, by the following preparation method, which includes the steps of reacting the compound represented by general formula (6) with a lithium compound, then reacting it with a boric acid compound, and then reacting it with a base.

[0108] [Chemical Formula 17] In general formula (6), Ar 1 and R 5 Ar in the above general formula (5) 1 and R 5 same.]

[0109] There are no particular limitations on the lithium compounds used; examples include alkyl lithiums such as ethyl lithium, n-propyl lithium, isopropyl lithium, n-butyl lithium, sec-butyl lithium, tert-butyl lithium, n-pentyl lithium, and n-hexyl lithium; cycloalkyl lithiums such as cyclohexyl lithium; and aryl lithiums such as phenyl lithium. In this process, considering conversion, selectivity, and yield, alkyl lithiums are preferred, and n-butyl lithium is more preferred. These lithium compounds can be used alone or in combination of two or more.

[0110] From the perspectives of conversion rate, selectivity, and yield, the amount of lithium compound used is generally preferably 0.5 to 2.0 mol, more preferably 0.7 to 1.5 mol, relative to 1 mol of the compound represented by general formula (6) used as a raw material. Furthermore, when using multiple lithium compounds, it is preferable to adjust the total amount to be within the above-mentioned range.

[0111] There are no particular limitations on the types of borate compounds; examples include boric acid, trimethyl borate, and triethyl borate. These borate compounds can be used alone or in combination of two or more.

[0112] From the perspectives of conversion rate, selectivity, and yield, the amount of boric acid compound used is generally preferably 1.0 to 5.0 moles, more preferably 1.5 to 3.0 moles, relative to 1 mole of the compound represented by general formula (6) used as a raw material. Furthermore, when using multiple boric acid compounds, it is preferable to adjust the total amount to be within the above-mentioned range.

[0113] There are no particular limitations on the alkali used, and examples include ammonium chloride, potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium tert-butoxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, and calcium acetate. In this process, considering factors such as conversion rate, selectivity, and yield, ammonium chloride, potassium fluoride, and cesium fluoride are preferred, with ammonium chloride being more preferred.

[0114] From the perspectives of conversion rate, selectivity, and yield, the amount of alkali used is preferably 2.0 to 15.0 moles, more preferably 3.0 to 10.0 moles, relative to 1 mole of compound (5).

[0115] This process can be carried out in the presence of a solvent. Examples of usable solvents include aliphatic hydrocarbons (hexane, cyclohexane, heptane, etc.), aliphatic halogenated hydrocarbons (dichloromethane, chloroform, carbon tetrachloride, dichloroethane, etc.), aromatic hydrocarbons (benzene, toluene, xylene, chlorobenzene, etc.), cyclic ethers (tetrahydrofuran, dioxane, etc.), nitriles (acetonitrile, propionitrile, etc.), and sulfoxides (dimethyl sulfoxide (DMSO), etc.). One of these solvents can be used alone, or two or more can be used in combination.

[0116] For the reaction temperature in this process, the reaction with lithium compounds can typically be set to -100 to -50°C, and subsequent reactions can typically be set to 10 to 50°C. Furthermore, the reaction time can be set to allow the reaction to proceed to completion.

[0117] In addition, the reaction atmosphere is not particularly limited and can be set to an inactive gas atmosphere such as argon or nitrogen.

[0118] After the reaction, the compound can be used for routine post-processing such as solvent removal, cleaning, and chromatographic separation as needed to obtain the compound represented by general formula (5). Example

[0119] The present invention will be specifically described based on the embodiments, but the present invention is not limited thereto.

[0120] 1 H, 13 C{ 1 H} and 31 P{ 1 The ¹H NMR spectra are in CDCl₃ and acetone-d₆, using a JEOL ECZ 400 ( 1H: 400MHz, 13 C{1H}: 100MHz, 31 P{ 1 H}:162MHz), JEOL ECA 500 II spectrometer ( 1 H: 500MHz, 13 C{ 1 H}:125MHz, 31 P{ 1 H:202MHz), or UltraCOOL probe equipped with JEOL ECA 600II spectrometer ( 1 H: 600MHz, 13 C{ 1 Recorded at H:150MHz. 1 Chemical shifts in H NMR spectra were reported in δppm using residual protons in the solvent as an internal standard (CD3OD δ3.31). 13 The chemical shifts in the C NMR spectra were reported in δppm using the solvent signal as an internal standard (CD3OD δ49.00). 31 Chemical shifts in the pNMR spectra were reported using H3PO4 (δ 0.00) as an external standard. High-resolution mass spectrometry was performed using Thermo Fisher Scientific Exactive with ESI ionization. Thin-layer chromatography (TLC) was performed on silica gel 60F coated with a thickness of 0.25 mm. 254 The chromatography was performed on glass plates (manufactured by Merck). Column chromatography was performed using silica gel PSQ100B (manufactured by FUJI SILYSIA CHEMICAL LTD.). Unless otherwise specified, the chemical reagents and solvents were commercially available products from Tokyo Chemical Industry Co., Ltd. (TCI), Sigma-Aldrich, KANTO CHEMICAL CO., INC., and FUJIFILM Wako Pure Chemical Corporation (Wako), and were used directly without purification. Anhydrous THF, CH2Cl2, CH3CN, and DMF were purchased from KANTO CHEMICAL CO., INC. and purified using Glass Contour Solvent Systems. 3-Bromo-N,N-diethylaniline (2) and 5-methoxybenzo[b]thiophene (4) were synthesized according to prior reports.

[0121] [Example 1: Synthesis of Compound 8] The following shows a series of synthetic routes for compound 8. Compound 5, however, was only subjected to liquid-liquid extraction due to its instability in silica gel. Compounds with trivalent phosphorus or phosphonium sites are generally unstable, but compounds 7 and 8 exist as stable solids even in air.

[0122] [Chemical Formula 18]

[0123] Synthesis of 3-bromo-4-iodo-N,N-dimethylaniline (compound 3)

[0124] [Chemical Formula 19] At 0°C, a solution containing 3-bromo-N,N-diethylaniline (compound 2) (2.85 mL, 16.1 mmol) and CaCO3 (2.09 g, 20.9 mmol) dissolved in anhydrous CH2Cl2 (200 mL) and CH3OH (80 mL) was added dropwise to a solution containing benzyltrimethylammonium dichloroiodate (5.60 g, 16.1 mmol) dissolved in anhydrous CH2Cl2 (100 mL) and CH3OH (40 mL). After stirring at room temperature for 2 hours, the resulting black mixture was filtered through diatomaceous earth (Celite) to obtain a yellow solution. The yellow solution was washed with saturated aqueous NaHSO4 and saturated Na2SO3 solutions. Water (300 mL) was added to the combined organic layers, and extraction was performed with CH2Cl2 (50 mL × 4). The combined organic layers were then washed with water (30 mL) and saturated brine (100 mL), and concentrated under reduced pressure. Using hexane / CH2Cl2 as the eluent, the mixture was purified by silica gel column chromatography while gradually increasing the amount of CH2Cl2 (1 / 0 to 20 / 1, Rf=0.19 in hexane) to obtain 3-bromo-4-iodo-N,N-dimethylaniline (compound 3) (4.99 g, 14.1 mmol, 88%) as a colorless solid. 1 H NMR(CDCl3,500MHz)δ7.52(d,J=9.2Hz,1H),6.93(d,J=3.1Hz,1H),6.33(dd,J=9.2,3.1Hz,1H),3.30(q,J=6.9Hz,4H),1.14(t,J=6.9Hz,6H). 13 C{ 1H}NMR(CDCl3,500MHz)δ148.6(C),140.0(CH),130.3(CH),115.7(CH),112.7(CH),81.3(C),44.5(CH2),12.4(CH3).HRMS(ESI)m / z calcd.For C 10 H 13 BrINNa + [M+Na] + :375.9169;found:375.9192.

[0125] Synthesis of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound 6)

[0126] [Chemical Formula 20] To a solution of 5-methoxybenzo[b]thiophene (compound 4) (2.41 g, 14.9 mmol) dissolved in anhydrous tetrahydrofuran (THF) (50 mL), n-butyllithium (n-BuLi) (1.60 M, 10.1 mL, 16.1 mmol in hexane) was added dropwise at -78 °C. After stirring at -78 °C for 1 hour, trimethyl borate (3.30 mL, 29.3 mmol) was added to the mixture at the same temperature. The reaction mixture was then warmed to room temperature over 3 hours. A saturated aqueous solution of NH4Cl (50 mL) was added to the solution, and the mixture was stirred for 30 minutes. Water (100 mL) was added to the resulting white mixture, and the mixture was extracted with diethyl ether (Et2O) (50 mL × 3). The combined organic layers were washed with water (50 mL) and saturated brine (150 mL), and concentrated under reduced pressure to obtain (5-methoxybenzo[b]thiophene-2-yl)boronic acid (compound 5) (2.74 g) as a colorless solid with a small amount of residue.

[0127] Next, 3-bromo-4-iodo-N,N-dimethylaniline (compound 3) (4.56 g, 12.9 mmol) was added to a solution containing (5-methoxybenzo[b]thiophene-2-yl)boronic acid (compound 5) (2.68 g), tetrakis(triphenylphosphine)palladium (0) (2.38 g, 2.06 mmol), and K2CO3 (10.7 g, 77.4 mmol) in xylene (40 mL), ethanol (EtOH) (20 mL), and H2O (10 mL). The mixture was stirred at 130 °C for 22 hours. After cooling to room temperature, the resulting mixture was filtered through diatomaceous earth to obtain a yellow solution. Water (50 mL) was added to the yellow solution, and the solution was extracted with CH2Cl2 (60 mL × 3). The combined organic layers were washed with water (60 mL) and then with saturated brine (70 mL), and concentrated under reduced pressure. Using hexane / CH2Cl2 as the eluent, the mixture was purified by silica gel column chromatography while gradually increasing the amount of CH2Cl2 (from 5 / 1 to 1 / 1, with Rf=0.12 in hexane / CH2Cl2=5 / 1) to obtain 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 6) as a pale yellow solid (3.08 g, 7.88 mmol, 56% (two-step yield)). 1 ¹H NMR (acetone-d6, 400MHz) δ 7.75 (d, J = 8.6 Hz, 1H), 7.40–7.36 (m, 3H), 7.02–6.97 (m, 2H), 6.78 (d, J = 8.6 Hz, 1H), 3.86 (s, 1H), 3.44 (q, J = 7.3 Hz, 4H), 1.18 (t, J = 7.3 Hz, 6H). 13 C{ 1 H}NMR(CDCl3,500MHz)δ157.6(C),148.3(C),144.5(C),141.2(C),132.6(CH),132.4(C),124.0(C),123.0(CH),122 .7(CH),121.5(C),115.6(CH),114.3(CH),110.6(CH),105.6(CH),55.6(CH3),44.5(CH2),12.6(CH3).HRMS(ESI)m / z calcd. for C 19 H 21 BrNOS + [M+H] + :390.0522;found:390.0520.

[0128] 3-(diphenylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound 7) synthesis

[0129] [Chemical Formula 21] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 6) (0.997 g, 2.55 mmol) dissolved in anhydrous tetrahydrofuran (THF) (25 mL), n-butyllithium (n-BuLi) (1.60 M hexane, 1.75 mL, 2.82 mmol) was added dropwise at -78 °C. After stirring at -78 °C for 1 hour, diphenylphosphine chloride (0.471 mL, 2.56 mmol) was added at the same temperature. The reaction mixture was heated to room temperature and stirred for 2 hours. CH3OH (6 mL) was added to the solution. Water (40 mL) was added to the resulting black mixture, and extraction was performed with CH2Cl2 (20 mL × 3). The combined organic layers were washed with water (60 mL) and saturated brine (100 mL), and concentrated under reduced pressure. Using hexane / CH2Cl2 as the eluent, the mixture was purified by silica gel column chromatography while gradually increasing the amount of CH2Cl2 (from 4 / 1 to 2 / 1 to 9 / 4 to 0 / 1, where Rf = 0.15 in hexane / CH2Cl2 = 9 / 4), yielding 3-(diphenylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 7) as a gray solid (0.132 g, 0.266 mmol, 10%). 1 H NMR(CDCl3,400MHz)δ7.63(d,J=8.6Hz,1H),7.48(dd,J=13.5,4.9,1H),7.39-7.36(m,10H),7.04(d,J=2.5Hz,1H),6.97 (s,1H),6.93(dd,J=11.0,2.5,1H),6.26(dd,J=7.3,2.5,1H),3.85(s,3H),3.18(q,J=6.7Hz,4H),0.97(t,J=6.7Hz,6H). 13 C{ 1 H}NMR(acetone-d6,600MHz)δ158.4(C),147.4(C),147.3(C),144.4(C),142.3(C),135.1(C),134.4(CH),134.3(CH ),133.0(C),132.3(CH),132.2(CH),131.8(CH),128.93(CH),128.85(CH),126.9(CH),122.8(CH),118.7(d,J C-P=14.5Hz,CH),114.8(CH),114.7(CH),106.3(CH),55.7(CH3),45.0(CH2),12.6(CH3). 31 P{ 1 H}NMR(CDCl3,400MHz)δ-11.2.HRMS(ESI)m / z calcd.forC 31 H 30 NNaOPS + [M+Na] + :518.1678;found:518.1682.

[0130] 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphanindol[2,3-d]thiophene- Synthesis of 10-onium (compound 8)

[0131] [Chemical Formula 22] Copper(II) trifluoromethanesulfonate (0.1927 g, 0.533 mmol) was added to a solution containing 3-(diphenylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 7) (0.132 g, 0.266 mmol) dissolved in anhydrous CH3CN (7 mL) and CH2Cl2 (4 mL). After stirring at room temperature for 1 hour, water (30 mL) was added to the resulting amber mixture, and the mixture was extracted with CH2Cl2 (10 mL × 3) and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CH2Cl2 / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf=0.17 in CH2Cl2 / MeOH / TFA=94 / 5 / 1) as the eluent to give 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphanindol[2,3-d]thiophene-10-onium (compound 8) (0.0421 g, 0.0693 mmol, 26%) as a yellow solid. 1 H NMR (acetone-d6, 400MHz) δ8.20-8.14(m,4H),8.08(d,J=8.6Hz,1H),8.00-7.97(m,2H),7.90-7.81(m,5H),7.76(dd, J=11.6,1.2Hz,1H),7.25(s,1H),7.18-7.14(m,2H),3.81(s,3H),3.58(q,J=7.3Hz,4H),1.19(t,J=7.3Hz,6H). 13 C{ 1¹H NMR (acetone-d6, 500MHz) δ 164.5(C), 164.3(C), 160.6(C), 160.0(C), 159.7(C), 159.4(C), 151.0(C), 150.8(C), 136.8(CH), 134.7(CH), 134.6(CH), 131.7(CH), 131.6(CH), 126.8(CH), 126.7(CH), 126.4(CH), 118.5(C), 117.8(C), 117.3(C), 115.3(d,J) C-P =15.7Hz,CH),105.3(CH),56.2(CH3),45.5(CH2),12.5(CH3). 31 P{ 1 H}NMR(CD3OD,162MHz)δ16.2.HRMS(ESI) m / z calcd. for C 31 H 29 NOPS + [M+Na] + :494.1702;found:494.1710.

[0132] [Example 2] 3-(xylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound) Synthesis of 11)

[0133] [Chemical Formula 23] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 6) (0.300 g, 0.768 mmol) dissolved in anhydrous tetrahydrofuran (THF) (10 mL), n-butyllithium (n-BuLi) (1.50 M hexane, 0.510 mL, 0.845 mmol) was added dropwise at -78 °C. After stirring at -78 °C for 1 hour, chloroxylphosphine (0.192 g, 0.769 mmol) was added at the same temperature. The reaction mixture was heated to room temperature and stirred for 3 hours. CH3OH (3 mL) was added to the solution. Water (60 mL) was added to the resulting yellow mixture, and extraction was performed with CH2Cl2 (30 mL × 3). The combined organic layers were washed with water (20 mL) and saturated brine (30 mL), and concentrated under reduced pressure. Using hexane / CH2Cl2 as the eluent, the amount of CH2Cl2 was gradually increased (9 / 1 to 4 / 1 to 7 / 3 to 1 / 1 to 0 / 1, Rf=0.26 in hexane / CH2Cl2=7 / 3), and the mixture was purified by silica gel column chromatography. Then, it was purified by gel filtration chromatography with CHCl3 as the solvent to obtain 3-(xylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 11) (0.111 g, 0.212 mmol, 28%) as a yellow liquid. 1 H NMR(acetone-d6,400MHz)δ7.66(d,J=8.6Hz,1H),7.45(dd,J=8.6,4.9Hz,1H),7.31-7.24(m,4H),7.17-7.13(m,3H),6.97(s,1H),6.92-6.88 (m,3H),6.80(dd,J=8.6,2.5Hz,1H).6.25(d,J=4.3,2.5Hz,1H),3.80(s,3H),3.17(q,J=6.7Hz,4H),2.35(s,6H),0.90(t,J=6.7Hz,6H). 31 P{H}NMR(CDCl3,500MHz)δ-27.4.HRMS(ESI) m / z calcd. forC 33 H 35 NOPS + [M+H] + :524.2172;found:524.2171.

[0134] 2-(diethylamino)-8-methoxy-10,10-dimethyl-10H-benzo[b]phosphanindol[2,3-d]thiamethoxy Synthesis of phen-10-onthium (compound 12)

[0135] [Chemical Formula 24] Copper trifluoromethanesulfonate (II) (0.0915 g, 0.253 mmol) was added to a solution containing 3-(xylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 11) (0.0662 g, 0.126 mmol) dissolved in anhydrous CH3CN (7 mL) and CH2Cl2 (4 mL). After stirring at room temperature for 2 hours, water (30 mL) was added to the resulting yellow-green mixture, and the mixture was extracted with CH2Cl2 (20 mL × 3) and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CH2Cl2 / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf=0.18 in CH2Cl2 / MeOH / TFA=94 / 5 / 1). It was further purified by reversed-phase HPLC and finally purified by Sephadex LH-20 to obtain 2-(diethylamino)-8-methoxy-10,10-dimethyl-10H-benzo[b]phosphanindol[2,3-d]thiophene-10-onthium (compound 12) (0.0819 g, 0.109 mmol, 87%) as an orange solid. 1 H NMR (acetone-d6, 400MHz) δ8.10(dd,J=9.17,1.83Hz,1H),8.06-7.52(m,10H),7.18(d,J=8.6Hz,1H),7.12(dd,J=8.86 ,1.83Hz,1H),6.84(d,J=2.45Hz),3.71(s,3H),3.59(q,J=7.3Hz,4H),3.10-2.11(m,6H),1.19(t,J=6.7Hz,6H). 31 P{H}NMR (acetone-d6, 400MHz) δ16.1.HRMS (ESI) m / z calcd. for C 32 H 30 NOPS + [M] + :522.2015;found:522.2013.

[0136] [Comparative Example 1] 3-(Dicyclohexylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 9) Synthesis

[0137] [Chemical Formula 25] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 6) (0.301 g, 0.770 mmol) dissolved in anhydrous tetrahydrofuran (THF) (10 mL), n-butyllithium (n-BuLi) (1.50 M hexane, 0.510 mL, 0.845 mmol) was added dropwise at -78 °C. After stirring at -78 °C for 1 hour, dicyclohexylphosphine chloride (0.170 mL, 0.769 mmol) was added at the same temperature. The reaction mixture was heated to room temperature and stirred for 2 hours. CH3OH (3 mL) was added to the solution. Water (30 mL) was added to the resulting yellow mixture, and extraction was performed with CH2Cl2 (10 mL × 3). The combined organic layers were washed with water (20 mL) and saturated brine (20 mL), and concentrated under reduced pressure. Using hexane / CH2Cl2 as the eluent, the mixture was purified by silica gel column chromatography while gradually increasing the amount of CH2Cl2 (from 4 / 1 to 7 / 3 to 3 / 2 to 1 / 4 to 0 / 1, where Rf = 0.33 in hexane / CH2Cl2 = 3 / 2) to obtain 3-(dicyclohexylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 9) (0.146 g, 0.288 mmol, 37%) as a yellow solid. 1 ¹H NMR (acetone-d6, 400MHz) δ 7.71 (d, J = 8.6 Hz, 1H), 7.35–7.32 (m, 2H), 7.22 (s, 1H), 6.95–6.92 (m, 2H), 6.78 (dd, J = 8.6, 2.5, 1H), 5.63 (s, 2H), 3.86 (s, 3H), 3.48 (q, J = 6.7 Hz, 4H), 2.82 (d, J = 14.1 Hz, 2H), 1.94–1.65 (m, 10H), 1.36–1.04 (m, 16H). 31 P{H}NMR(CDCl3,400MHz)δ-12.4.HRMS(ESI)m / z calcd. for C 31 H 43 NOPS + [M+H] + :508.2798;found:508.2803.

[0138] 2-(diethylamino)-8-methoxy-10,10-dicyclohexyl-10H-benzo[b]phosphanindol[2,3-d]thiamethoxy Synthesis of phen-10-onthium (compound 10)

[0139] [Chemical Formula 26] Copper(II) trifluoromethanesulfonate (0.155 g, 0.428 mmol) was added to a solution containing 3-(dicyclohexylphosphino)-N,N-diethyl-4-(5-methoxybenzo[b]thiophene-2-yl)aniline (compound 9) (0.109 g, 0.214 mmol) dissolved in anhydrous CH3CN (7 mL) and CH2Cl2 (4 mL). After stirring at room temperature for 2 hours, water (30 mL) was added to the resulting yellow-green mixture, and the mixture was extracted with CH2Cl2 (25 mL × 3) and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CH2Cl2 / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf=0.11 in CH2Cl2 / MeOH / TFA=94 / 5 / 1), and further purified by reversed-phase HPLC to obtain 2-(diethylamino)-8-methoxy-10,10-dicyclohexyl-10H-benzo[b]phosphanindol[2,3-d]thiophene-10-onthium (compound 10) (0.141 g, 0.193 mmol, 90%) as a yellow solid. 1 H NMR(methanol-d4,400MHz)δ7.91(dd,J=8.9Hz,J=2.5Hz,1H),7.67(dd,J=8.9,3.7Hz, 1H),7.34(d,J=1.8Hz,1H),7.27(dd,J=11.0,2.5Hz,1H),7.12(dd,J=9.2,2.5H z,1H),7.06(dd,J=8.6,2.5Hz,1H),3.94(s,3H),3.57(q,J=7.3Hz,4H),3.39-3 .31(m,2H),2.05-1.94(m,4H),1.84(d,J=11.0Hz,4H),1.74(m,J=13.5Hz,2H). 31 P{H}NMR(CD3OD,400MHz)δ40.8.HRMS(ESI)m / z calcd. for C 31 H 41 NOPS + [M] + :506.2641;found:506.2646.

[0140] [Experimental Example 1: X-ray Crystallography] 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphanindol[2,3-d]thiophene- Structural analysis of 10-onthium (compound 8) In Example 1, a single crystal of compound 8 was obtained, and therefore its crystal structure was determined by X-ray diffraction.

[0141] Single crystals of compound 8 were grown by slow evaporation from CH₂Cl₂ / hexane at room temperature. Intensity data were collected using MoKα radiation at 293 K using a RIGAKU diffractometer equipped with an FR-X generator, a VariMax optical system, and a PILATUS 200K photon counting detector. A total of 16,161 reflectances were measured at a maximum 2θ angle of 55°, of which 7,319 were independent reflectances (R0). int =0.0909). The structure was measured using the direct method (SHELXS-2013), and all reflections were measured at F. 2 The above was refined using full matrix least squares. All hydrogen atoms were configured using AFIX, and other atoms were refined using anisotropic methods.

[0142] For compound 8, each molecule contains two molecules of trifluoroacetic acid (TFA). Figure 1 (a) Two molecules of trifluoroacetic acid (TFA) share one hydrogen ion. Since the charge of two TFA molecules is -1, it can be understood that it is the counter anion of phosphonium with a charge of +1. Furthermore, the CN bond length is 1.38 Å, which is shorter than the 1.41 Å in aniline. This is further supported by the observed bond alternation on the pigment skeleton. Figure 1 (b) thus suggests that diethylamine acts as an electron-donating group to push electrons toward the pigment skeleton.

[0143] Additionally, the crystallization data is shown below. C 35 H 30 F6NO5PS,FW=721.63,monoclinic,P-1,a=15.9169(4)Å,b=7.28580(10)Å,c=17.8933(4)Å,β=102.453(2)o,V=21647.9(2)Å 3 Z=4,D calcd =1.454 gcm⁻³, μ=0.225 mm -1 ,F(000)=744.The refinement converged to R I [I>2σ(I)]=0.0666, wR2(all data)=0.1930, GOF=1.010.

[0144] [Experimental Example 2: Measurement of Photophysical Properties] The photophysical properties of compound 8 obtained in Example 1 were evaluated. First, the absorption spectrum, fluorescence spectrum, and absolute fluorescence quantum yield were measured in five solvents: toluene, dichloromethane (DCM), acetonitrile (MeCN), water (containing 10% DMSO), and dimethyl sulfoxide (DMSO).

[0145] Compound 8 obtained in Example 1 was diluted to a concentration of 20 μM with various solvents to prepare a sample solution. The UV-Vis absorption and fluorescence spectra of the sample solution, placed in a 1 cm square quartz cuvette, were measured using a Duetta fluorescence spectrophotometer (HORIBA). For the fluorescence measurement, compound 8 was diluted to a concentration of 20 μM with various solvents, and the resulting sample solution was excited with light at a wavelength of 460 nm. Furthermore, the absolute fluorescence quantum yield was measured using a Quantaurus-QY C11347 (manufactured by Hamamatsu Photonics KK) with a calibrated integrating sphere system equipped with a multichannel spectrometer. The results are shown in Table 1 and... Figure 2 .

[0146] [Table 1]

[0147] Compound 8 in toluene exhibits absorption and fluorescence maxima at wavelengths of 472 nm and 562 nm, respectively, while in water they are 465 nm and 575 nm, showing almost no change. As a donor-π-acceptor molecule, its solvent dependence is quite low, suggesting a relatively small contribution from intramolecular charge transfer (ICT) structure in the excited state. Furthermore, the Stokes shift ranges from 3422 to 4214 cm⁻¹. -1 The fluorescence quantum yield shows a large value with increasing solvent polarity. It exhibits a maximum value of 0.734 in dichloromethane and a high value of 0.604 in water. Therefore, this indicates that the compound of the present invention is a donor-π-acceptor molecule and can be used in an aqueous environment.

[0148] Next, the photophysical properties of compound 8 obtained in Example 1 and compound 10 obtained in Comparative Example 1 were compared in water containing 10% dimethyl sulfoxide (DMSO). The results are shown below. Figure 3 Compound 8 obtained in Example 1 exhibits a peak wavelength of 465 nm in the UV-Vis absorption spectrum and a peak wavelength of 575 nm in the fluorescence spectrum in water containing 10% dimethyl sulfoxide (DMSO). In contrast, compound 10 obtained in Comparative Example 1 exhibits a peak wavelength of 436 nm in the UV-Vis absorption spectrum and a peak wavelength of 530 nm in the fluorescence spectrum in water containing 10% dimethyl sulfoxide (DMSO). By introducing a phenyl group, the peak wavelengths in both the UV-Vis absorption and fluorescence spectra can be lengthened.

[0149] [Experiment Example 2: Cell Experiment] To evaluate the cell staining performance of compound 8 obtained in Example 1, fluorescence imaging was performed using a confocal laser microscope.

[0150] Cell culture HeLa (RCB0007) cells were purchased from Riken Cell Bank. The culture medium used was Dalberg Modified Eagle Medium (DMEM) with low glucose, L-glutamine, sodium pyruvate (Wako, 041-29775), fetal bovine serum (FBS, Biosera, 554-0215), and an antibiotic-antifungal agent (AA, Wako, 161-23181). Cells were cultured in DMEM containing 10% FBS and 1% AA at 37°C in a humidified 5% CO2 incubator. Two days before imaging, HeLa cells (5 × 10⁶ cells / year) were cultured... 4 (Number) were inoculated into uncoated glass bottom petri dishes (Matsunami Glass Ind., Ltd.).

[0151] Cell staining and confocal imaging Cells were washed with DMEM(-) (DMEM containing 1% AA). Then, the cells were incubated for 30 minutes with DMEM(-) containing 100 nM of Compound 8. After washing with DMEM(-), the cells were imaged in DMEM(-) using a confocal laser scanning microscope. The confocal microscope used was a FLUOVIEW FV10i (Olympus). Cell culture dishes were mounted in a platform incubator (manufactured by TOKAI HIT Co., Ltd.) and maintained at 37°C and 5% CO2 under humidification. The excitation wavelength was 473 nm, and the fluorescence range was 550–650 nm.

[0152] Figure 4 (a) shows the results of staining HeLa cells with 500 nM compound 8. As shown in the figure, a large number of mitochondria were observed inside the cells. In DMEM(+), before washing, structures such as the endoplasmic reticulum or cell membrane were observed as faint shadows, except for mitochondria, but after washing, the mitochondria were stained more clearly. Furthermore, in DMEM(-), mitochondria could be clearly observed even without washing. From these results, it can be seen that for compound 8 obtained in Example 1, staining with DMEM(-) in the culture medium is more preferable, as fluorescence imaging can be performed under non-washing conditions in this environment. In addition, the results of staining HeLa cells with 50 nM compound 8 without washing and direct observation are as follows: Figure 4(b) 50 nM is a very low concentration for a fluorescent imaging probe, yet mitochondria were still clearly observed. This is because compound 8, which exhibits a high fluorescence quantum yield, was efficiently taken up into the mitochondria, demonstrating its usefulness as a mitochondrial staining agent.

[0153] Here, to verify the specificity of compound 8 obtained in Example 1 and compound 10 obtained in Comparative Example 1 for mitochondrial staining, co-staining was performed with the commercially available mitochondrial staining agent Mito Tracker (registered trademark) Deep Red. The excitation wavelengths of compound 8 and Mito Tracker (registered trademark) Deep Red are 473 nm (compound 8) and 635 nm (Mito Tracker (registered trademark) Deep Red), respectively, allowing for independent detection of their respective fluorescence signals. The staining results of compound 8 or compound 10 with Mito Tracker (registered trademark) Deep Red are shown below. Figure 5 Therefore, it can be understood that compound 8 and Mito Tracker (registered trademark) Deep Red stained the same position. Furthermore, the Pearson coefficient, close to 1 (0.92), indicates that compound 8 can specifically stain mitochondria. On the other hand, compound 10 has a Pearson coefficient of 0.68, suggesting that by setting the phosphorus group to an aryl group, it can particularly specifically stain mitochondria.

[0154] STED imaging As described above, it can be understood that mitochondria can be specifically stained using compound 8 obtained in Example 1 via confocal microscopy. However, since the spatial resolution of confocal microscopy is greater than 200 nm, the location of compound 8 within the mitochondria is not yet clear. Therefore, fluorescence imaging using STED microscopy, which has a higher resolution than confocal microscopy, was performed to evaluate the location of compound 8 in more detail.

[0155] STED imaging was performed using a Leica TCS SP8STED 3X system employing two CW lasers at 592nm and 660nm, along with a pulsed 775nm laser. A HyD detector and a STED WHITE objective lens (100x / 1.40 oz) were used. Unless otherwise specified, STED images were acquired with excitation at 470nm (WLL), fluorescence emission at 550–650nm, depletion at 775nm (CW-STED, 270mW), and gated detection at tg = 1ns. Generally, images were recorded at a pixel resolution of 7.6nm × 7.6nm or higher, a scan speed of 400Hz, bidirectional mode, a line average of 3, and a pinhole size of 151.63μm. Images were processed using ImageJ. Depending on the application, the commercially available Huygens package was used to deconvolve the STED images with theoretical PSF.

[0156] Figure 6 (a) is a staining image of mitochondria based on confocal microscopy. Figure 6 (b) shows the observation results of the same site based on an STED microscope. Figure 6 In (b), cristae, a structural feature of the mitochondrial inner membrane, were observed. This result suggests that compound 8 specifically stained the mitochondrial inner membrane.

Claims

1. A compound represented by general formula (1), [Chemical Formula 1] ， In general formula (1), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings; R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl; in, R 1 and R 2 Choose one to form a ring with an adjacent nitrogen atom; R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring; R 3 and R 4 Same or different indicates (hetero)aryl groups that have been substituted or not substituted; R 5 It represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

2. The compound according to claim 1, wherein, The Ar 1 It refers to the substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

3. The compound according to claim 1, wherein, The Ar 2 It refers to the substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

4. The compound according to claim 1, wherein, R 1 and R 2 The same or different, and consisting of hydrogen atoms, or substituted or unsubstituted alkyl groups.

5. The compound according to claim 1, wherein, R 3 and R 4 The same or different, and whether the aryl group is substituted or not.

6. The compound according to claim 1, wherein, R 5 It can be a substituted or unsubstituted alkyl group.

7. A fluorescent dye comprising the compound or a solvate thereof as described in any one of claims 1 to 6.

8. A mitochondrial staining agent comprising the compound or a solvate thereof as described in any one of claims 1 to 6.

9. The mitochondrial staining agent according to claim 8, wherein it is a mitochondrial inner membrane staining agent.

10. A method for preparing the compound according to any one of claims 1 to 6, comprising: a step of subjecting the compound represented by general formula (2) to a ring-closing reaction in the presence of a copper compound. [Chemical Formula 2] ， In general formula (2), Ar 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 Ar in claim 1 1 Ar 2 R 1 R 2 R 3 R 4 and R 5 same.

11. A compound represented by general formula (2), [Chemical Formula 3] ， In general formula (2), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings; R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl; in, R 1 and R 2 Choose one to form a ring with an adjacent nitrogen atom; R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring; R 3 and R 4 Same or different, and indicating whether it is a (hetero)aryl group that has been substituted or not substituted; R 5 It represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

12. A method for preparing the compound of claim 11, comprising the steps of reacting a compound of general formula (3) with a lithium compound, and then reacting the compound with a compound of general formula (4). [Chemical Formula 4] ， In general formula (3), Ar 1 Ar 2 R 1 R 2 and R 5 Ar in claim 11 1 Ar 2 R 1 R 2 and R 5 Same; X 1 Represents halogen atoms, PR 3 R 4 X 2 (4), In general formula (4), R 3 and R 4 With R in claim 11 3 and R 4 Same; X 2 This represents a halogen atom.

13. A compound represented by general formula (3), [Chemical Formula 5] ， In general formula (3), Ar 1 and Ar 2 Same or different indicates substituted or unsubstituted aromatic hydrocarbon rings or substituted or unsubstituted heteroaromatic rings; R 1 and R 2 Same or different, indicating hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted (hetero)aryl; in, R 1 and R 2 They can either form a ring with an adjacent nitrogen atom; R 1 and / or R 2 Optional and Ar 2 Together with the adjacent nitrogen atom, they form a ring; R 5 Represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group; X 1 This represents a halogen atom.

14. A method for preparing the compound of claim 13, comprising the step of reacting the compound represented by general formula (5) with the compound represented by general formula (6) in the presence of a palladium catalyst. [Chemical Formula 6] ， In general formula (5), Ar 1 and R 5 Ar in claim 13 1 and R 5 Same; Y 1 Indicates boric acid or its ester group. [Chemical Formula 7] ， In general formula (6), Ar 2 R 1 R 2 and X 1 Ar in claim 13 2 R 1 R 2 and X 1 Same; X 3 Indicates with X 1 Different halogen atoms.

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