Diarylphosphonium-bridged compound and mitochondria-dyeing agent using same

Abstract

A compound represented by general formula (1) [wherein Ar1 and Ar2 each independently represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring; R1 and R2 each independently represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, wherein R1 and R2 together may form a ring in conjunction with the adjacent nitrogen atom, and R1 and / or R2 and Ar2 together may form a ring in conjunction with the adjacent nitrogen atom; R3 and R4 each independently represent a substituted or unsubstituted (hetero)aryl group; and R5 represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group] can fluorescently dye a mitochondria (in particular, a mitochondrial inner membrane) selectively although the compound is a small molecule.

Description

Diarylphosphonium-bridged compound and mitochondrial stain using the same

[0001] The present invention relates to a diarylphosphonium-bridged compound and a mitochondrial stain using the same.

[0002] Mitochondria are oval-shaped organelles present in all eukaryotes and are separated by two membranes: an outer membrane and an inner membrane. Between the outer and inner membranes is an intermembrane space. The folded structures of the inner membrane are called cristae, and the inner membrane is called the matrix. The cristae perform oxidative phosphorylation reactions using pyruvate, amino acids, and fatty acids derived from glycolysis, synthesizing ATP, the energy currency. Mitochondria produce over 90% of the ATP required for cellular metabolism and play a crucial role in cell survival and death. In addition to ATP synthesis, they are involved in various cellular functions, including intracellular ion homeostasis, cell growth, regulation of cell signaling, and control of apoptosis. Therefore, mitochondrial dysfunction is closely linked to many diseases, including neurodegenerative disorders, cancer, and diabetes. In recent years, numerous studies have been published on mitochondrial function and aging. Indeed, the rapid increase in the number of searches for articles on mitochondria in PubMed demonstrates the high level of interest in this topic.

[0003] One area that has attracted particular attention is mitochondrial fusion and fission. For example, it is known that mitochondrial fusion factor mutants experience respiratory failure and mitochondrial DNA loss. Furthermore, the loss of mitochondrial DNA observed in HeLa cells under fusion-inhibitory conditions demonstrates that mitochondrial fusion is essential for cell survival. Regarding mitochondrial fission, it has been reported that apoptosis is delayed under fission-inhibitory conditions, and abnormalities in synapse formation due to a reduction in mitochondria in neurites occur. Therefore, mitochondrial fission also plays an important biological role. However, the mechanism of mitochondrial DNA loss due to fusion inhibition and the function of fission in organisms other than neurons remain unclear, and further research into mitochondrial fusion and fission is highly desirable.

[0004] In addition to mitochondrial fusion and fission, attention is also being paid to interactions with other organelles and the dynamics of lipids that are the sites of these interactions. Mitochondrial lipids are synthesized both inside and outside the mitochondria. It is known that lipids synthesized inside the mitochondria are transported to other organelles, while lipids synthesized outside the mitochondria are primarily transported from the endoplasmic reticulum. It has been reported that membrane contact sites between organelles are important for the transport of these lipids to other organelles. The split-GFP system, reported in 2018, is a powerful tool for conducting research on organelle contact involving mitochondria. In the split-GFP system, GFP reconstitutes and emits strong fluorescence when mitochondria interact with other organelles and come into physical proximity. However, due to the reaction mechanism, it cannot be ruled out that split-GFP itself may create organelle contact sites. Therefore, there is a strong need to develop probes that can detect interactions between mitochondria and other organelles without direct involvement.

[0005] To summarize, research into mitochondrial fusion, fission, and interactions with other organelles is a field that is attracting attention, and to advance this research, there is a need for the development of fluorescent probes that can selectively stain the inner mitochondrial membrane and track its membrane dynamics in real time. Fluorescent probes are required to have various properties, but the most important are: 1) they must not interfere with interactions between organelles, 2) they must be of a molecular size that does not affect membrane dynamics, 3) they must be environmentally responsive, changing their fluorescent properties in response to interactions between organelles, and 4) they must be photostability.

[0006] In response to this, the present inventors have developed an ultra-light-resistant fluorescent imaging probe, MitoPBYellow, which can selectively stain the inner membrane of living mitochondria.

[0007] MitoPBYellow exhibits overwhelming light resistance compared to commercially available MitoTracker series and other products, and is hardly affected by bleaching even in super-resolution fluorescence imaging using a STED microscope, which requires strong laser light irradiation, enabling long-term time-lapse imaging of mitochondrial inner membrane dynamics.

[0008] Proc. Natl. Acad. Sci., 2019, 116, 15817-15822.

[0009] However, in MitoPB Yellow, the phosphonium moiety responsible for localization to mitochondria is introduced separately from the dye skeleton, which poses a problem of increasing the molecular weight.

[0010] The present invention is intended to solve the above-mentioned conventional problems, and aims to provide a compound that is a small molecule but can selectively fluoresce in mitochondria (particularly the inner mitochondrial membrane).

[0011] In view of the above-mentioned problems, the present inventors have conducted extensive research and found that by adopting a specific structure in which a diarylphosphonium-bridged structure is used between an aromatic ring and a thiophene ring, it is possible to introduce a phosphonium moiety responsible for localization to mitochondria into the dye skeleton, even though the dye is a small molecule. Furthermore, since an expandable functional group can be introduced onto the dye skeleton, it is possible to impart selectivity to the inner mitochondrial membrane and selectively fluoresce mitochondria (particularly the inner mitochondrial membrane). Based on this finding, the present inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following configurations.

[0012] Item 1. General formula (1):

[0013] [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ３ and R ４ are the same or different and represent a substituted or unsubstituted (hetero)aryl group. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0014] Item 2. Said Ar １ Item 2. The compound according to Item 1, wherein is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0015] Item 3. Said Ar ２ Item 3. The compound according to Item 1 or 2, wherein is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

[0016] Item 4. R １ and R ２ and are the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group.

[0017] Item 5. R ３ and R ４ and are the same or different and are a substituted or unsubstituted aryl group.

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

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

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

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

[0022] Item 10. A method for producing the compound according to any one of items 1 to 6, comprising reacting a compound represented by general formula (2): in the presence of a copper compound:

[0023] [In the formula, Ar １ , Ar ２ , R １ , R ２ , R ３ , R ４ and R ５ are the same as above.] and subjecting a compound represented by the following formula (1) to a ring-closing reaction.

[0024] Item 11. General formula (2):

[0025] [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ３ and R ４ are the same or different and represent a substituted or unsubstituted (hetero)aryl group. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0026] Item 12. A method for producing the compound according to Item 11, comprising reacting a compound represented by general formula (3):

[0027] [In the formula, Ar １ , Ar ２ , R １, R ２ and R ５ is the same as above. １ represents a halogen atom.] is reacted with a lithium compound, and then a compound represented by the general formula (4): PR ３ R ４ X ２ (4) [wherein, R ３ and R ４ is the same as above. ２ represents a halogen atom.] with a compound represented by the following formula (I):

[0028] Item 13. General formula (3):

[0029] [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group. １ represents a halogen atom.] A compound represented by the formula:

[0030] Item 14. A method for producing the compound according to Item 13, comprising reacting a compound represented by general formula (5):

[0031] [In the formula, Ar １ and R ５ is the same as above. １ represents a boronic acid or an ester group thereof.] and a compound represented by general formula (6):

[0032] [In the formula, Ar２ , R １ , R ２ and X １ is the same as above. ３ is X １ and wherein the halogen atom is a halogen atom different from the halogen atom represented by the formula (I) in the presence of a palladium catalyst.

[0033] According to the present invention, it is possible to provide a compound that is a small molecule but can selectively fluoresce in mitochondria (particularly the inner mitochondrial membrane).

[0034] The results of X-ray crystal structure analysis of Compound 8 in Test Example 1 are shown. (a) The state of two nearby molecules is shown. (b) The state of Compound 8 is shown. For clarity, hydrogen atoms are omitted. The results of measurement of the optical properties of Compound 8 in Test Example 2 are shown. The solid line indicates the ultraviolet-visible absorption spectrum, and the dashed line indicates the fluorescence spectrum (λ ｅｘ λ = 460 nm). The results of measuring the optical properties of Compound 8 (Example 1; phenyl group) and Compound 10 (Comparative Example 1; cyclohexyl group) in Test Example 2 are shown. The solid line indicates the UV-visible absorption spectrum, and the dashed line indicates the fluorescence spectrum. The results of fluorescent imaging of HeLa cells using Compound 8 in Test Example 3 are shown. (a) Confocal images of HeLa cells stained with 500 nM Compound 8 are shown. Before washing (left), after washing (right). DMEM(+) (top), DMEM(-) (bottom). (b) Confocal images of HeLa cells stained with 50 nM Compound 8 (without washing). λ ｅｘ = 473 nm, λ ｅｍ = 550-650 nm. In Test Example 3, HeLa cells were co-stained with Compound 8 or Compound 10 and Mito Tracker (registered trademark) Deep Red. In Test Example 3, mitochondria were stained using Compound 8 under a confocal microscope and an STED microscope. (a) Image of stained mitochondria under a confocal microscope. (b) Image of stained mitochondria under an STED microscope.

[0035] In this specification, the term "comprise" is a concept that encompasses "consist essentially of" and "consist only of."

[0036] In this specification, when a range is expressed as "A to B," it means A or more and B or less, unless otherwise specified.

[0037] 1. Compound The compound of the present invention is represented by the general formula (1):

[0038] [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ３ and R ４ are the same or different and represent a substituted or unsubstituted (hetero)aryl group. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

[0039] In the present invention, the compound represented by general formula (1) is a novel compound, and the compound represented by general formula (1) can identify mitochondria (particularly, the inner mitochondrial membrane), and therefore can be used as a mitochondrial staining agent (particularly, an inner mitochondrial membrane staining agent) that can selectively stain mitochondria (particularly, the inner mitochondrial membrane).

[0040] Furthermore, the compound represented by the above general formula (1) has an amino group or a substituted amino group, which is an electron-donating group, and thus can be endowed with environmental responsiveness and can achieve a longer absorption peak wavelength, thereby reducing phototoxicity to mitochondria.

[0041] Furthermore, the compound represented by the above-described general formula (1) has a specific structure employing a diarylphosphonium-bridged structure between the aromatic ring and the thiophene ring. This skeleton is a donor-π-acceptor molecule with a ladder structure incorporating a cationic phosphonium group as an electron-withdrawing group. By directly introducing the cationic moiety required for mitochondrial localization into the dye skeleton, the molecular weight can be kept small, enabling mitochondria to be stained. Furthermore, the compound represented by the above-described general formula (1) can have an expandable functional group introduced into the dye skeleton, imparting selectivity to the outer mitochondrial membrane. For these reasons, the compound represented by general formula (1) is suitable for repeated in vivo observation using super-resolution microscopy, such as stimulated emission depletion (STED) imaging.

[0042] In addition, in the general formula (1), R which is a substituent on P ３ and R ４ By using a (hetero)aryl group, the absorption peak wavelength can be shifted to a longer wavelength, and mitochondria (particularly the inner mitochondrial membrane) can be selectively stained. ３ and R ４ When the group is an alkyl group or a cycloalkyl group, it is difficult to shift the absorption peak wavelength to a longer wavelength, and tissues other than mitochondria are also stained, making it impossible to selectively stain mitochondria (particularly the inner mitochondrial membrane).

[0043] In the general formula (1), Ar １ and Ar ２As the aromatic hydrocarbon ring represented by the formula (I), either a monocyclic aromatic hydrocarbon ring or a polycyclic aromatic hydrocarbon ring can be employed. For example, an example of the monocyclic aromatic hydrocarbon ring is a benzene ring, and examples of the polycyclic aromatic hydrocarbon ring are a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyrene ring, a triphenylene ring, and the like.

[0044] Ar １ and Ar ２ The aromatic hydrocarbon ring represented by the formula (I) may have a substituent. Examples of the substituent include alkyl groups described below, cycloalkyl groups described below, aryl groups described below, heteroaryl groups described below, alkenyl groups (vinyl groups, propenyl groups, etc.), alkynyl groups (ethynyl groups, 1-propynyl groups, etc.), carbonyl groups, carboxy groups, sulfo groups, amido groups (amide groups, N-alkylamido groups, etc.; examples of the alkyl groups in N-alkylamido groups include those mentioned above), cyano groups, nitro groups, etc. When the aromatic hydrocarbon ring has a substituent, the number of the substituents is, for example, preferably 1 to 5, and more preferably 1 to 3.

[0045] In the general formula (1), Ar １ and Ar ２ Examples of the heteroaromatic ring represented by the formula (I) include a pyridine ring, a pyrazine ring, etc. as a monocyclic heteroaromatic ring, and an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, etc. as a polycyclic heteroaromatic ring.

[0046] Ar １ and Ar ２ The heteroaromatic ring represented by the formula (I) may have a substituent. Examples of the substituent include an alkyl group described below, a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, an alkenyl group (a vinyl group, a propenyl group, etc.), an alkynyl group (an ethynyl group, a 1-propynyl group, etc.), a carbonyl group, a carboxy group, a sulfo group, an amido group (an amido group, an N-alkylamido group, etc.; examples of the alkyl group in the N-alkylamido group include those described above), a cyano group, a nitro group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), and the like. When the heteroaromatic ring has a substituent, the number of the substituents is, for example, preferably 1 to 5, and more preferably 1 to 3.

[0047] Ar １ and Ar ２ As the aromatic hydrocarbon ring, from the viewpoints of mitochondrial (particularly mitochondrial inner membrane) staining, structural stability, absorption peak wavelength, phototoxicity to mitochondria, light resistance, etc., a substituted or unsubstituted aromatic hydrocarbon ring is preferred, and a substituted or unsubstituted monocyclic aromatic hydrocarbon ring is more preferred.

[0048] In general formula (1), R １ and R ２ As the alkyl group represented by the formula (I), either a linear alkyl group or a branched alkyl group can be employed, and examples thereof include C1-15 alkyl groups (particularly C1-10 alkyl groups) such as a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, and tert-butyl group.

[0049] R １ and R ２ The alkyl group represented by the formula (I) may have a substituent. Examples of the substituent include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), a cycloalkyl group described below, an aryl group described below, a heteroaryl group described below, an alkenyl group (a vinyl group, a propenyl group, etc.), an alkynyl group (an ethynyl group, a 1-propynyl group, etc.), a carbonyl group, a carboxy group, a sulfo group, an amide group (an amide group, an N-alkylamide group, etc.; examples of the alkyl group in the N-alkylamide group include those described above), a cyano group, a nitro group, etc. When the alkyl group has a substituent, the number of the substituents is, for example, preferably 1 to 5, and more preferably 1 to 3.

[0050] In general formula (1), R １ and R ２ Examples of the cycloalkyl group represented by the formula (I) include C3-10 cycloalkyl groups (particularly C4-8 cycloalkyl groups) such as cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group and cycloheptyl group.

[0051] R １ and R ２The cycloalkyl group represented by the following formula (I) may have a substituent. Examples of the substituent include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), the above-mentioned cycloalkyl group, the above-mentioned alkyl group, the aryl group described below, the heteroaryl group described below, an alkenyl group (a vinyl group, a propenyl group, etc.), an alkynyl group (an ethynyl group, a 1-propynyl group, etc.), a carbonyl group, a carboxy group, a sulfo group, an amido group (an amido group, an N-alkylamido group, etc.; examples of the alkyl group in the N-alkylamido group include those described above), a cyano group, a nitro group, etc. When the cycloalkyl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

[0052] In general formula (1), R １ and R ２ Examples of the aryl group represented by the formula (I) can include a monocyclic aryl group, a fused ring aryl group, and a polycyclic aryl group. Examples of the monocyclic aryl group include a phenyl group, examples of the fused ring aryl group include a naphthyl group, an anthracenyl group, a phenanthrenyl group, a fluorenyl group, a pyrenyl group, and a triphenylenyl group, and examples of the polycyclic aryl group include C6-18 aryl groups (particularly C6-14 aryl groups) such as a biphenyl group and a terphenyl group.

[0053] R １ and R ２ The aryl group represented by the formula (I) may have a substituent. Examples of the substituent include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), the alkyl group, the cycloalkyl group, the aryl group, the heteroaryl group described below, an alkenyl group (a vinyl group, a propenyl group, etc.), an alkynyl group (an ethynyl group, a 1-propynyl group, etc.), a carbonyl group, a carboxy group, a sulfo group, an amide group (an amide group, an N-alkylamide group, etc.; examples of the alkyl group in the N-alkylamide group include those described above), a cyano group, a nitro group, etc. When the aryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

[0054] In general formula (1), R １ and R ２As the heteroaryl group represented by the formula (I), either a monocyclic heteroaryl group or a fused-ring heteroaryl group can be employed. Examples of the monocyclic heteroaryl group include a pyrrolyl group, a thienyl group, a furanyl group, an imidazolyl group, a pyrazolyl group, a thiazolyl group, an oxazolyl group, a piperidyl group, a pyridyl group, and a pyrazyl group. Examples of the fused-ring heteroaryl group include an indolyl group, an isoindolyl group, a benzimidazolyl group, a quinolyl group, an isoquinolyl group, and a quinoxalyl group.

[0055] R １ and R ２ The heteroaryl group represented by the following formula may have a substituent. Examples of the substituent include the above-mentioned halogen atoms (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), the above-mentioned alkyl groups, the above-mentioned cycloalkyl groups, the above-mentioned aryl groups, the above-mentioned heteroaryl groups, alkenyl groups (vinyl group, propenyl group, etc.), alkynyl groups (ethynyl group, 1-propynyl group, etc.), carbonyl groups, carboxy groups, sulfo groups, amido groups (amide groups, N-alkylamido groups, etc.; examples of the alkyl groups in the N-alkylamido groups include those mentioned above), cyano groups, nitro groups, etc. When the heteroaryl group has a substituent, the number of the substituents is, for example, preferably 1 to 6, and more preferably 1 to 3.

[0056] Among them, R １ and R ２ As the dye, from the viewpoints of easily shifting the absorption peak wavelength to a longer wavelength, easily identifying mitochondria, and easily staining the mitochondrial inner membrane, a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, etc. are preferred, a hydrogen atom, a substituted or unsubstituted alkyl group, etc. are more preferred, and a substituted or unsubstituted alkyl group is even more preferred.

[0057] In addition, R １ and R ２ may be taken together to form a ring with the adjacent nitrogen atom. １ R ２ The group represented by

[0058] It may also be a group represented by the following formula:

[0059] Also, R１ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ２ -NR １ R ２ The structure represented by

[0060] etc. may also be used.

[0061] In this case, Ar ２ In the formula, -NR １ R ２ The substitution position to which the group represented by the following formula is bonded is not particularly limited.

[0062] In general formula (1), R ３ and R ４ The aryl group and heteroaryl group represented by the formula (I) can be the same as those described above. The types of the substituents are also the same. ３ and R ４ When a substituent is introduced into the aryl group and heteroaryl group represented by the formula (I) to form a bulky group, it is possible to further improve the light resistance.

[0063] In general formula (1), R ５ The alkyl group and cycloalkyl group represented by the formula (I) can be any of those mentioned above. The same applies to the type of substituent, and functional groups can be introduced in consideration of high selectivity for the mitochondrial inner membrane, etc.

[0064] Examples of the compound represented by the general formula (1) that satisfies the above conditions include:

[0065] etc.

[0066] The compound of the present invention may contain not only the above-described structure but also a counter anion. The counter anion is not particularly limited, but examples thereof include acetate ion, trifluoroacetate ion, halide ion (fluoride ion, chloride ion, bromide ion, iodide ion, etc.), perchlorate ion, PF ６ － , B.F. ４ －, sulfate ion, nitrate ion, phosphate ion, bistriflimide ion, etc.

[0067] 2. Fluorescent Dyes and Mitochondrial Staining Agents The fluorescent dyes of the present invention contain the compounds of the present invention described above.

[0068] The fluorescent dye of the present invention employs a diarylphosphonium-bridged structure between an aromatic ring and a thiophene ring, and therefore can shift the absorption peak wavelength to a longer wavelength.

[0069] Furthermore, the fluorescent dye of the present invention can selectively stain mitochondria (particularly, the inner mitochondrial membrane). Furthermore, the compound of the present invention can cause mitochondria (particularly, the inner mitochondrial membrane) to fluoresce strongly while suppressing the fluorescence of other tissues. The compound of the present invention can suppress the fluorescence of tissues other than mitochondria (particularly, the inner mitochondrial membrane), thereby making the fluorescence intensity of mitochondria (particularly, the inner mitochondrial membrane) significantly greater than that of other tissues. Therefore, even small mitochondria (particularly, the inner mitochondrial membrane) can be detected with high sensitivity, and further, fluorescent staining of other tissues can be suppressed. Therefore, the fluorescent dye of the present invention using the compound of the present invention is useful in that it can be used as a mitochondrial stain (particularly, a stain for the inner mitochondrial membrane). Furthermore, the membrane structure (membrane composition) of the inner mitochondrial membrane can be estimated by using such a fluorescent dye of the present invention.

[0070] The fluorescent dye (mitochondrial stain) of the present invention contains the above-described compound of the present invention or a solvate thereof. The form of use is not particularly limited, and for example, the compound can be dissolved in an organic solvent to form a solution. In this case, from the viewpoints of easily detecting (staining) even small-sized mitochondria (particularly the mitochondrial inner membrane) with high sensitivity and easily suppressing fluorescence of tissues other than mitochondria (particularly the mitochondrial inner membrane), the content of the above-described compound of the present invention 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 as low as 30 to 80 nmol, it is possible to detect mitochondria (particularly the mitochondrial inner membrane) with high sensitivity. As such, in the present invention, the content of the compound of the present invention can be kept low, which makes it easy to suppress damage to living cells.

[0071] When the fluorescent dye (mitochondrial stain) of the present invention is prepared as a solution containing the compound of the present invention, there are no particular limitations on the organic solvent that can be used. Because the compound of the present invention, as a donor-π-acceptor molecule, exhibits low solvent dependence in its absorption and fluorescence spectra, various organic solvents can be used, including aromatic solvents such as benzene, toluene, xylene, and mesitylene; aliphatic halogenated hydrocarbons such as dichloromethane and dichloroethane; nitrile solvents such as acetonitrile; and sulfoxide solvents such as dimethyl sulfoxide. Water can also be used as the solvent, and the fluorescent dye (mitochondrial stain) of the present invention can be used in an aqueous solution environment, making it suitable for use in vivo.

[0072] 3. Production Method The compound represented by general formula (1) of the present invention can be produced, for example, by reacting a compound represented by general formula (2):

[0073] [In the formula, Ar １ , Ar ２ , R １ , R ２ , R ３ , R ４ and R ５are the same as above.] and a step of subjecting a compound represented by the following formula (1) to a ring-closing reaction.

[0074] The compound represented by the general formula (2) can be, for example, a compound represented by the general formula (3):

[0075] [In the formula, Ar １ , Ar ２ , R １ , R ２ and R ５ is the same as above. １ represents a halogen atom.] is reacted with a lithium compound, and then a compound represented by the general formula (4): PR ３ R ４ X ２ (4) [wherein, R ３ and R ４ is the same as above. ２ represents a halogen atom.] with a compound represented by the formula (I).

[0076] The compound represented by the general formula (3) can also be, for example, a compound represented by the general formula (5):

[0077] [In the formula, Ar １ and R ５ is the same as above. １ represents a boronic acid or an ester group thereof.] and a compound represented by general formula (6):

[0078] [In the formula, Ar ２ , R １ , R ２ and X １ is the same as above. ３ is X １ and a compound represented by the formula (I): wherein R represents a halogen atom different from the halogen atom represented by the formula (I):

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

[0080] Examples of copper compounds include metallic copper and copper compounds known as catalysts for synthesizing organic compounds (including polymer compounds). Examples of copper compounds include metallic copper; copper(I) halides such as copper(I) iodide, copper(I) bromide, and copper(I) chloride; and copper(II) trifluoromethanesulfonate. In this step, copper(II) trifluoromethanesulfonate is preferred. These copper compounds can be used alone or in combination of two or more. These copper compounds can include both those added as reagents and those generated in the system.

[0081] The amount of the copper compound used is usually preferably 1.0 to 5.0 moles, more preferably 1.5 to 3.0 moles, per mole of the compound represented by general formula (2) as the raw material, from the viewpoints of conversion rate, selectivity, yield, etc. When multiple copper compounds are used, it is preferable to adjust the total amount to be within the above range.

[0082] This step can be carried out in the presence of a solvent. Examples of solvents that can be used 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.), sulfoxides (dimethyl sulfoxide (DMSO), etc.), etc. These can be used alone or in combination of two or more.

[0083] The reaction temperature in this step can be generally 0 to 50° C., particularly 10 to 40° C. The reaction time can be set to a time that allows the reaction to proceed sufficiently.

[0084] The reaction atmosphere is not particularly limited, but may be an inert gas atmosphere such as an argon gas atmosphere or a nitrogen gas atmosphere.

[0085] After the reaction, if necessary, the reaction mixture may be subjected to general post-treatments such as solvent removal, washing, and chromatographic separation, to obtain the compound represented by general formula (1).

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

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

[0088] The lithium compound is not particularly limited, but examples include alkyllithiums such as ethyllithium, n-propyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, n-pentyllithium, and n-hexyllithium; cycloalkyllithiums such as cyclohexyllithium; and aryllithiums such as phenyllithium. Of these, in this step, alkyllithiums are preferred, and n-butyllithium is more preferred, from the viewpoints of conversion rate, selectivity, yield, and the like. These lithium compounds can be used alone or in combination of two or more.

[0089] The amount of the lithium compound used is usually preferably 0.7 to 2.0 moles, more preferably 1.0 to 1.5 moles, per mole of the compound represented by general formula (3) as the raw material, from the viewpoints of conversion rate, selectivity, yield, etc. When multiple lithium compounds are used, it is preferable to adjust the total amount to be within the above range.

[0090] Thereafter, the amount of the compound represented by general formula (4) used is usually preferably 0.7 to 3.0 mol, more preferably 0.7 to 1.5 mol, per 1 mol of the compound represented by general formula (3) as a raw material, from the viewpoints of conversion rate, selectivity, yield, etc.

[0091] This step can be carried out in the presence of a solvent. Examples of solvents that can be used 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.), sulfoxides (dimethyl sulfoxide (DMSO), etc.), etc. These solvents can be used alone or in combination of two or more.

[0092] The reaction temperature in this step can be generally 0 to 50° C., particularly 10 to 40° C. The reaction time can be set to a time that allows the reaction to proceed sufficiently.

[0093] The reaction atmosphere is not particularly limited, but may be an inert gas atmosphere such as an argon gas atmosphere or a nitrogen gas atmosphere.

[0094] After the reaction, if necessary, the reaction mixture may be subjected to general post-treatments such as solvent removal, washing, and chromatographic separation, to obtain the compound represented by general formula (2).

[0095] The compound represented by the general formula (2) thus obtained is a novel compound not described in any literature.

[0096] (3-3) Compound (5) → Compound (3) The method for producing the compound represented by formula (5) will be described later. The compound represented by formula (6) can be synthesized in accordance with the examples described later.

[0097] The amount of the compound represented by general formula (6) used is usually preferably 0.7 to 3.0 mol, more preferably 0.7 to 1.5 mol, per 1 mol of the compound represented by general formula (3) as a raw material, from the viewpoints of conversion rate, selectivity, yield, etc.

[0098] Examples of palladium compounds include palladium catalysts known as catalysts for synthesizing organic compounds and the like. Specific examples include palladium acetate, tetrakis(triphenylphosphine)palladium(0), palladium trifluoroacetate, palladium chloride, palladium bromide, palladium iodide, tris(dibenzylideneacetone)dipalladium(0), bis(dibenzylideneacetone)palladium(0), dichloro(1,5-cyclooctadiene)palladium(II), and 2,5-norbornadienepalladium dichloride. These palladium compounds can also be used as solvates. These palladium compounds can be used alone or in combination of two or more. In this step, tetrakis(triphenylphosphine)palladium(0) (or a solvate thereof) is preferred from the viewpoints of conversion rate, selectivity, yield, and the like.

[0099] The amount of the palladium compound used is usually preferably 0.05 to 0.50 mol, more preferably 0.10 to 0.30 mol, per 1 mol of the compound represented by the general formula (3) as the raw material, from the viewpoints of conversion rate, selectivity, yield, etc.

[0100] In this step, a base can also be used if necessary. Examples of the base 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, calcium acetate, etc. In this step, from the viewpoints of conversion rate, selectivity, yield, etc., sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, cesium carbonate, etc. are preferred, and potassium carbonate is more preferred.

[0101] When a base is used, the amount used is preferably 2.0 to 15.0 mol, more preferably 3.0 to 10.0 mol, per 1 mol of compound (3) from the viewpoints of conversion rate, selectivity, yield, etc.

[0102] This step can be carried out in the presence of a solvent. Examples of solvents that can be used 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.), sulfoxides (dimethyl sulfoxide (DMSO), etc.), etc. In this step, water can also be used as the solvent. These solvents can be used alone or in combination of two or more.

[0103] The reaction temperature in this step can be generally 80 to 200° C., particularly 100 to 150° C. The reaction time can be set to a time that allows the reaction to proceed sufficiently.

[0104] The reaction atmosphere is not particularly limited, but may be an inert gas atmosphere such as an argon gas atmosphere or a nitrogen gas atmosphere.

[0105] After the reaction, if necessary, the reaction mixture may be subjected to general post-treatments such as solvent removal, washing, and chromatographic separation, to obtain the compound represented by general formula (3).

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

[0107] (3-4) Compound (6) → Compound (5) The compound represented by the general formula (5) can be, for example, a compound represented by the general formula (6):

[0108] [In the formula, Ar １ and R ５ are the same as above.] with a lithium compound, then reacting the compound with a boronic acid compound, and then reacting the compound with a base.

[0109] The lithium compound is not particularly limited, but examples include alkyllithiums such as ethyllithium, n-propyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, n-pentyllithium, and n-hexyllithium; cycloalkyllithiums such as cyclohexyllithium; and aryllithiums such as phenyllithium. Of these, in this step, alkyllithiums are preferred, and n-butyllithium is more preferred, from the viewpoints of conversion rate, selectivity, yield, and the like. These lithium compounds can be used alone or in combination of two or more.

[0110] The amount of the lithium compound used is usually preferably 0.5 to 2.0 moles, more preferably 0.7 to 1.5 moles, per mole of the compound represented by general formula (6) as the raw material, from the viewpoints of conversion rate, selectivity, yield, etc. When multiple lithium compounds are used, it is preferable to adjust the total amount to be within the above range.

[0111] The boronic acid compound is not particularly limited, but examples thereof include boric acid, trimethoxyborane, triethoxyborane, etc. These boronic acid compounds can be used alone or in combination of two or more.

[0112] The amount of the boronic acid compound used is usually preferably 1.0 to 5.0 mol, more preferably 1.5 to 3.0 mol, per mol of the compound represented by general formula (6) as the raw material, from the viewpoints of conversion rate, selectivity, yield, etc. When a plurality of boronic acid compounds are used, it is preferable to adjust the total amount to be within the above range.

[0113] The base is not particularly limited, but examples thereof include ammonium chloride, potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium tert-butoxide, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, cesium carbonate, potassium phosphate, sodium acetate, potassium acetate, calcium acetate, and the like. In this step, from the viewpoints of conversion rate, selectivity, yield, and the like, ammonium chloride, potassium fluoride, cesium fluoride, and the like are preferred, and ammonium chloride is more preferred.

[0114] When a base is used, the amount used is preferably 2.0 to 15.0 mol, more preferably 3.0 to 10.0 mol, per 1 mol of compound (5) from the viewpoints of conversion rate, selectivity, yield, etc.

[0115] This step can be carried out in the presence of a solvent. Examples of solvents that can be used 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.), sulfoxides (dimethyl sulfoxide (DMSO), etc.), etc. These solvents can be used alone or in combination of two or more.

[0116] The reaction temperature in this step can usually be −100 to −50° C. for the reaction with the lithium compound, and can usually be 10 to 50° C. for the subsequent reaction. The reaction time can be a time that allows the reaction to proceed sufficiently.

[0117] The reaction atmosphere is not particularly limited, but may be an inert gas atmosphere such as an argon gas atmosphere or a nitrogen gas atmosphere.

[0118] After the reaction, if necessary, the reaction mixture may be subjected to general post-treatments such as solvent removal, washing, and chromatographic separation, to obtain a compound represented by general formula (5).

[0119] The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

[0120] 1 H, 13 C{ 1 H} and 31 P{ 1 H} NMR spectra were obtained on a JEOL ECZ 400 ( 1 H: 400 MHz, 13 C{1H} : 100 MHz, 31 P{ 1 H} : 162 MHz), JEOL ECA 500 II spectrometer ( 1 H: 500 MHz, 13 C{ 1 H} : 125 MHz, 31 P{ 1 H} : 202 MHz), or a JEOL ECA 600II spectrometer equipped with an UltraCOOL probe ( 1 H: 600 MHz, 13 C{ 1 H} : 150 MHz). 1 Chemical shifts for 1 H NMR spectra are reported in δ ppm using the residual protons of the solvent as the internal standard (CD3OD δ 3.31). 13 Chemical shifts for C NMR spectra are reported in δ ppm using the solvent signal as the internal standard (CD3OD δ 49.00). 31 Chemical shifts for P NMR spectra are reported using H3PO4 (δ 0.00) as an external standard. High-resolution mass spectra were obtained using Thermo Fisher Scientific Exactive with ESI ionization. Thin-layer chromatography (TLC) was performed on 0.25 mm thick silica gel 60F. 254 ​The analysis was performed on glass plates coated with silica gel (Merck). Column chromatography was performed using silica gel PSQ100B (Fuji Silysia Chemical). Unless otherwise noted, chemical reagents and solvents were purchased from Tokyo Chemical Industry Co., Ltd. (TCI), Sigma-Aldrich, Kanto Chemical, and Fujifilm Wako Pure Chemical Industries, Ltd. (Wako) and were used without further purification. Anhydrous THF, CHCl, CHCN, and DMF were purchased from Kanto Chemical and purified using Glass Contour Solvent Systems. 3-Bromo-N,N-diethylaniline (2) and 5-methoxybenzo[b]thiophene (4) were synthesized as previously reported.

[0121] Example 1: Synthesis of Compound 8 The synthesis route for Compound 8 is shown below. Note that Compound 5 was only subjected to a separation procedure because it was unstable on silica gel. Although many compounds having a trivalent phosphorus or phosphonium moiety are generally unstable, Compounds 7 and 8 existed as stable solids even in air.

[0122]

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

[0124] To a solution of 3-bromo-N,N-diethylaniline (compound 2) (2.85 mL, 16.1 mmol) and CaCO3 (2.09 g, 20.9 mmol) in anhydrous CHCl2 (200 mL) and CH3OH (80 mL) was added dropwise a solution of benzyltrimethylammonium dichloroiodate (5.60 g, 16.1 mmol) in anhydrous CHCl2 (100 mL) and CH3OH (40 mL) at 0 °C. After stirring at room temperature for 2 h, the resulting black mixture was filtered through Celite to give a yellow solution. The yellow solution was washed with saturated aqueous NaHSO4 and saturated aqueous Na2SO3. Water (300 mL) was added to the combined organic layer, which was then extracted with CHCl2 (50 mL × 4). The combined organic layer was then washed with water (30 mL), brine (100 mL), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using hexane / CHCl as the eluent, increasing the amount of CHCl stepwise (1 / 0 to 20 / 1, Rf = 0.19 in hexane), to give 3-bromo-4-iodo-N,N-dimethylaniline (compound 3) (4.99 g, 14.1 mmol, 88%) as a colorless solid. 1 H NMR (CDCl3, 500 MHz) δ 7.52 (d, J = 9.2 Hz, 1H), 6.93 (d, J = 3.1 Hz, 1H), 6.33 (dd, J = 9.2, 3.1 Hz, 1H), 3.30 (q, J = 6.9 Hz, 4H), 1.14 (t, J = 6.9 Hz, 6H). 13 C{ 1 H} NMR (CDCl3, 500 MHz) δ 148.6 (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] To a solution of 5-methoxybenzo[b]thiophene (compound 4) (2.41 g, 14.9 mmol) in anhydrous tetrahydrofuran (THF) (50 mL), n-butyllithium (n-BuLi) (1.60 M in hexane, 10.1 mL, 16.1 mmol) was added dropwise at −78°C. After stirring at −78°C for 1 hour, trimethoxyborane (3.30 mL, 29.3 mmol) was added to the mixture at the same temperature. The reaction mixture was allowed to warm to room temperature over 3 hours. Saturated aqueous 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, which was then extracted with diethyl ether (Et₂O) (50 mL × 3). The combined organic layers were washed with water (50 mL), washed with brine (150 mL), and concentrated under reduced pressure to give (5-methoxybenzo[b]thiophen-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 of (5-methoxybenzo[b]thiophen-2-yl)boronic acid (compound 5) (2.68 g), tetrakis(triphenylphosphine)palladium(0) (2.38 g, 2.06 mmol), and KCO (10.7 g, 77.4 mmol) in xylene (40 mL), ethanol (EtOH) (20 mL), and HO (10 mL), and the mixture was stirred at 130 °C for 22 h. After cooling to room temperature, the resulting mixture was filtered through Celite to give a yellow solution. Water (50 mL) was added to the yellow solution, and the mixture was extracted with CHCl (60 mL × 3). The combined organic layers were washed with water (60 mL), brine (70 mL), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using hexane / CHCl as the eluent, increasing the amount of CHCl stepwise (5 / 1 to 1 / 1, Rf = 0.12 in hexane / CHCl = 5 / 1) to give 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 6) (3.08 g, 7.88 mmol, 56% (two-step yield)) as a pale yellow solid. 1 H NMR (acetone-d6, 400 MHz) 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{ 1H} NMR (CDCl3, 500 MHz) δ 157.6 (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] Synthesis of 3-(diphenylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 7)

[0129] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound 6) (0.997 g, 2.55 mmol) in anhydrous tetrahydrofuran (THF) (25 mL), n-butyllithium (n-BuLi) (1.60 M in hexane, 1.75 mL, 2.82 mmol) was added dropwise at −78°C. After stirring at −78°C for 1 hour, chlorodiphenylphosphine (0.471 mL, 2.56 mmol) was added at the same temperature. The reaction mixture was warmed to room temperature and stirred for 2 hours. CHOH (6 mL) was added to the solution. Water (40 mL) was added to the resulting black mixture, which was then extracted with CHCl (20 mL × 3). The combined organic layers were washed with water (60 mL), brine (100 mL), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using hexane / CHCl as the eluent, increasing the amount of CHCl stepwise (4 / 1 to 2 / 1 to 9 / 4 to 0 / 1, Rf = 0.15 in hexane / CHCl = 9 / 4) to give 3-(diphenylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 7) (0.132 g, 0.266 mmol, 10%) as a gray solid. 1 H NMR (CDCl3, 400 MHz) δ 7.63 (d, J = 8.6 Hz , 1H), 7.48 (dd, J = 13.5, 4.9, 1H), 7.39-7.36 (m, 10H), 7.04 (d, J = 2.5 Hz, 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 = 6.7 Hz, 4H), 0.97 (t, J = 6.7 Hz, 6H). 13 C{ 1H} NMR (Acetone-d6, 600 MHz) δ 158.4 (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 = 14.5 Hz, 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, 400 MHz) δ -11.2. HRMS (ESI) m / z calcd. for C 31 H 30 NNaOPS + [M+Na] + : 518.1678; found: 518.1682.

[0130] Synthesis of 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (Compound 8)

[0131] To a solution of 3-(diphenylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 7) (0.132 g, 0.266 mmol) in anhydrous CH3CN (7 mL) and CHCl2 (4 mL) was added copper(II) trifluoromethanesulfonate (0.1927 g, 0.533 mmol). After stirring at room temperature for 1 hour, the resulting brown mixture was added with water (30 mL), extracted with CHCl2 (10 mL × 3), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CHCl / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf = 0.17 in CHCl / MeOH / TFA = 94 / 5 / 1) as the eluent to give 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (compound 8) (0.0421 g, 0.0693 mmol, 26%) as a yellow solid. 1 H NMR (acetone-d6, 400 MHz) δ 8.20-8.14(m, 4H), 8.08 (d, J = 8.6 Hz, 1H), 8.00-7.97 (m, 2H), 7.90-7.81 (m, 5H), 7.76 (dd, J = 11.6, 1.2 Hz, 1H), 7.25 (s, 1H), 7.18-7.14 (m, 2H), 3.81 (s, 3H), 3.58 (q, J = 7.3 Hz, 4H), 1.19 (t, J = 7.3 Hz, 6H). 13 C{ 1 H} NMR (acetone-d6, 500 MHz) 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, JC-P = 15.7 Hz, CH), 105.3 (CH), 56.2 (CH3), 45.5 (CH2), 12.5 (CH3). 31 P{ 1 H} NMR (CD3OD, 162 MHz) δ 16.2. HRMS (ESI) m / z calcd. for C 31 H 29 NOPS + [M+Na] + : 494.1702; found: 494.1710.

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

[0133] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound 6) (0.300 g, 0.768 mmol) in anhydrous tetrahydrofuran (THF) (10 mL), n-butyllithium (n-BuLi) (1.50 M in hexane, 0.510 mL, 0.845 mmol) was added dropwise at −78°C. After stirring at −78°C for 1 hour, chloroditolylphosphine (0.192 g, 0.769 mmol) was added at the same temperature. The reaction mixture was warmed to room temperature and stirred for 3 hours. CHOH (3 mL) was added to the solution. Water (60 mL) was added to the resulting yellow mixture, which was then extracted with CHCl (30 mL × 3). The combined organic layers were washed with water (20 mL), brine (30 mL), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using hexane / CHCl as the eluent, with the amount of CHCl increasing stepwise (9 / 1 to 4 / 1 to 7 / 3 to 1 / 1 to 0 / 1, Rf = 0.26 in hexane / CHCl = 7 / 3), and then further purified by gel filtration chromatography using CHCl as the solvent to give 3-(dicitrylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 11) (0.111 g, 0.212 mmol, 28%) as a yellow liquid. 1 H NMR (acetone-d6, 400 MHz) δ 7.66 (d, J = 8.6 Hz , 1H), 7.45 (dd, J = 8.6, 4.9 Hz, 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, 2.5 Hz, 1H). 6.25 (d, J = 4.3, 2.5 Hz, 1H), 3.80 (s, 3H), 3.17 (q, J = 6.7 Hz, 4H), 2.35 (s, 6H), 0.90 (t, J = 6.7 Hz, 6H). 31P{H} NMR (CDCl3, 500 MHz) δ -27.4. HRMS (ESI) m / z calcd. for C 33 H 35 NOPS + [M+H] + : 524.2172; found: 524.2171.

[0134] Synthesis of 2-(diethylamino)-8-methoxy-10,10-ditolyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (Compound 12)

[0135] To a solution of 3-(ditolylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 11) (0.0662 g, 0.126 mmol) in anhydrous CH3CN (7 mL) and CHCl2 (4 mL) was added copper(II) trifluoromethanesulfonate (0.0915 g, 0.253 mmol). After stirring at room temperature for 2 hours, the resulting yellow-green mixture was added with water (30 mL), extracted with CHCl2 (20 mL × 3), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CHCl / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf = 0.18 in CHCl / MeOH / TFA = 94 / 5 / 1) as the eluent, followed by further purification by reverse-phase HPLC and finally by Sephadex LH-20 to give 2-(diethylamino)-8-methoxy-10,10-ditolyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (compound 12) (0.0819 g, 0.109 mmol, 87%) as an orange solid. 1H NMR (acetone-d6, 400 MHz) δ 8.10(dd, J = 9.17, 1.83 Hz, 1H), 8.06-7.52 (m, 10H), 7.18 (d, J = 8.6 Hz, 1H), 7.12 (dd, J = 8.86, 1.83 Hz, 1H), 6.84(d, J = 2.45 Hz), 3.71 (s, 3H), 3.59 (q, J = 7.3 Hz, 4H), 3.10-2.11 (m, 6H), 1.19 (t, J = 6.7 Hz, 6H). 31 P{H} NMR (Acetone-d6, 400 MHz) δ 16.1. HRMS (ESI) m / z calcd. for C 32 H 30 NOPS + [M] + : 522.2015; found: 522.2013.

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

[0137] To a solution of 3-bromo-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (compound 6) (0.301 g, 0.770 mmol) in anhydrous tetrahydrofuran (THF) (10 mL), n-butyllithium (n-BuLi) (1.50 M in hexane, 0.510 mL, 0.845 mmol) was added dropwise at −78°C. After stirring at −78°C for 1 hour, chlorodicyclohexylphosphine (0.170 mL, 0.769 mmol) was added at the same temperature. The reaction mixture was warmed to room temperature and stirred for 2 hours. CHOH (3 mL) was added to the solution. Water (30 mL) was added to the resulting yellow mixture, which was then extracted with CHCl (10 mL × 3). The combined organic layers were washed with water (20 mL), brine (20 mL), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using hexane / CHCl as the eluent, increasing the amount of CHCl stepwise (4 / 1 to 7 / 3 to 3 / 2 to 1 / 4 to 0 / 1, Rf = 0.33 in hexane / CHCl = 3 / 2) to give 3-(dicyclohexylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 9) (0.146 g, 0.288 mmol, 37%) as a yellow solid. 1 H NMR (acetone-d6, 400 MHz) 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, 400 MHz) δ -12.4. HRMS (ESI) m / z calcd. for C 31 H 43 NOPS + [M+H]+ : 508.2798; found: 508.2803.

[0138] Synthesis of 2-(diethylamino)-8-methoxy-10,10-dicyclohexyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (Compound 10)

[0139] To a solution of 3-(dicyclohexylphosphanyl)-N,N-diethyl-4-(5-methoxybenzo[b]thiophen-2-yl)aniline (Compound 9) (0.109 g, 0.214 mmol) in anhydrous CH3CN (7 mL) and CHCl2 (4 mL) was added copper(II) trifluoromethanesulfonate (0.155 g, 0.428 mmol). After stirring at room temperature for 2 hours, the resulting yellow-green mixture was added with water (30 mL), extracted with CHCl2 (25 mL × 3), and concentrated under reduced pressure. The mixture was purified by silica gel column chromatography using CHCl / methanol (MeOH) / trifluoroacetic acid (TFA) (94 / 5 / 1, Rf = 0.11 in CHCl / MeOH / TFA = 94 / 5 / 1) as the eluent, followed by further purification by reverse-phase HPLC to give 2-(diethylamino)-8-methoxy-10,10-dicyclohexyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (compound 10) (0.141 g, 0.193 mmol, 90%) as a yellow solid. 1H NMR (methanol-d4, 400 MHz) δ 7.91 (dd, J = 8.9 Hz, J = 2.5 Hz, 1H), 7.67 (dd, J = 8.9, 3.7 Hz, 1H), 7.34 (d, J = 1.8 Hz, 1H), 7.27 (dd, J = 11.0, 2.5 Hz, 1H), 7.12 (dd, J = 9.2, 2.5 Hz, 1H), 7.06 (dd, J = 8.6, 2.5 Hz, 1H), 3.94 (s, 3H), 3.57 (q, J = 7.3 Hz, 4H), 3.39-3.31 (m, 2H), 2.05-1.94 (m, 4H), 1.84 (d, J = 11.0 Hz, 4H), 1.74 (m, J = 13.5 Hz, 2H). 31 P{H} NMR (CD3OD, 400 MHz) δ 40.8. HRMS (ESI) m / z calcd. for C 31 H 41 NOPS + [M] + : 506.2641; found: 506.2646.

[0140] Test Example 1: X-ray crystallographic analysis Structural analysis of 2-(diethylamino)-8-methoxy-10,10-diphenyl-10H-benzo[b]phosphine chloro[2,3-d]thiophen-10-ium (Compound 8) In Example 1, single crystals of Compound 8 were obtained, and therefore X-ray crystallographic analysis was performed.

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

[0142] Two molecules of trifluoroacetic acid (TFA) were coordinated per molecule of compound 8 (Figure 1(a)). Two molecules of trifluoroacetic acid (TFA) shared one hydrogen ion, and since the charge of two molecules of trifluoroacetic acid (TFA) is -1, it can be understood that they are counter anions of the phosphonium, which has a charge of +1. In addition, the bond length of the C-N bond was 1.38 Å, which is shorter than the 1.41 Å in aniline. Furthermore, bond alternation was observed on the dye skeleton (Figure 1(b)), suggesting that diethylamine acted as an electron donor group, pushing electrons to the dye skeleton.

[0143] The crystal data was as follows: 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 g cm-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] Test Example 2: Measurement of Optical Properties The optical 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 with each solvent to a concentration of 20 μM to prepare a sample solution. The UV-visible absorption and fluorescence spectra of the sample solution placed in a 1 cm square quartz cuvette were recorded using a Duetta fluorometer (HORIBA). For fluorescence measurements, compound 8 was diluted with each solvent to a concentration of 20 μM, and the resulting sample solution was excited with light at a wavelength of 460 nm. The absolute fluorescence quantum yield was measured using a Quantaurus-QY C11347 (Hamamatsu Photonics) calibrated integrating sphere system equipped with a multichannel spectrometer. The results are shown in Table 1 and Figure 2.

[0146]

[0147] The maximum wavelengths of the absorption and fluorescence spectra of compound 8 in toluene are 472 nm and 562 nm, respectively, whereas in water they are 465 nm and 575 nm, showing almost no change. Since the solvent dependence is quite low for a donor-π-acceptor molecule, it is believed that the contribution of the intramolecular charge transfer (ICT) structure in the excited state is small. The Stokes shift is 3422-4214 cm. -1 The fluorescence quantum yield was 0.734 in dichloromethane and 0.604 in water, indicating that the compound of the present invention can be used in aqueous environments despite being a donor-π-acceptor molecule.

[0148] Next, the optical 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 in Figure 3. Compound 8 obtained in Example 1 had a maximum peak wavelength of 465 nm in the UV-visible absorption spectrum and 575 nm in the fluorescence spectrum in water containing 10% dimethyl sulfoxide (DMSO). In contrast, compound 10 obtained in Comparative Example 1 had a maximum peak wavelength of 436 nm in the UV-visible absorption spectrum and 530 nm in the fluorescence spectrum in water containing 10% dimethyl sulfoxide (DMSO). The introduction of a phenyl group enabled the maximum peak wavelengths to be shifted to longer wavelengths in both the UV-visible absorption spectrum and the fluorescence spectrum.

[0149] Test Example 2: Cell experiment To evaluate the cell staining ability of Compound 8 obtained in Example 1, fluorescence imaging was carried out using a confocal laser microscope.

[0150] Cell culture: HeLa (RCB0007) cells were purchased from the RIKEN Cell Bank. Low-glucose Dulbecco's modified Eagle's medium (DMEM), L-glutamine, sodium pyruvate (Wako, 041-29775), fetal bovine serum (FBS, Biosera, 554-0215), and antibiotic-antimycotic (AA, Wako, 161-23181) were used for cell culture. 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 × 10 4 The cells were seeded onto a non-coated glass-bottom dish (Matsunami Glass).

[0151] Cell staining and confocal imaging. Cells were washed with DMEM(-) (DMEM containing 1% AA). Then, the cells were incubated with DMEM(-) containing 100 nM compound 8 for 30 minutes. After washing with DMEM(-), the cells in DMEM(-) were imaged using a confocal laser scanning microscope. A FLUOVIEW FV10i (Olympus) confocal microscope was used. The cell dish was mounted in a stage-top incubator (Tokai Hit Co., Ltd.) and maintained at 37°C in a humidified 5% CO2 environment. The excitation wavelength was 473 nm, and the emission 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, numerous mitochondria were observed within the cells. In DMEM(+), structures resembling endoplasmic reticulum and cell membranes were observed as faint shadows in addition to mitochondria before washing. However, washing resulted in more vivid mitochondria. Furthermore, in DMEM(-), mitochondria were clearly observed without washing. These results suggest that compound 8 obtained in Example 1 is more suitable for staining using DMEM(-) as the medium, and fluorescent imaging without washing is possible under these conditions. Furthermore, Figure 4(b) shows the results of staining HeLa cells with 50 nM compound 8 without washing. Although 50 nM is a very low concentration for a fluorescent imaging probe, mitochondria were clearly observed. This is due to the efficient uptake of compound 8, which exhibits a high fluorescence quantum yield, into mitochondria, demonstrating its usefulness as a mitochondrial stain.

[0153] To verify the specificity of mitochondria staining by compound 8 obtained in Example 1 and compound 10 obtained in Comparative Example 1, co-staining with MitoTracker® Deep Red, a commercially available mitochondrial stain, was performed. Compound 8 and MitoTracker® Deep Red have excitation wavelengths of 473 nm (compound 8) and 635 nm (MitoTracker® Deep Red), respectively, allowing for independent detection of their respective fluorescent signals. Figure 5 shows the staining results for compound 8 or compound 10 with MitoTracker® Deep Red. From this, it can be seen that compound 8 and MitoTracker® Deep Red stain similar areas. Furthermore, the Pearson coefficient was 0.92, a value close to 1, demonstrating that compound 8 specifically stains mitochondria. Meanwhile, the Pearson coefficient for compound 10 was 0.68, demonstrating that the aryl group on the phosphorus atom allows for particularly specific staining of mitochondria.

[0154] As described above, it can be seen that compound 8 obtained in Example 1 can specifically stain mitochondria using a confocal microscope. However, because the spatial resolution of a confocal microscope exceeds 200 nm, it is unclear where in the mitochondria compound 8 is localized. Therefore, fluorescence imaging was performed using an STED microscope, which has higher resolution than a confocal microscope, to evaluate the localization of compound 8 in more detail.

[0155] STED imaging was performed using a Leica TCS SP8 STED 3X system equipped with two continuous wave lasers (592 nm and 660 nm) and a pulsed 775 nm laser. A HyD detector and a STED WHITE objective (100x magnification / 1.40 OIL) were used. Unless otherwise noted, STED images were acquired with excitation at 470 nm (WLL), emission at 550–650 nm, and depletion at 775 nm (CW-STED, 270 mW) using gated detection with a time of tg = 1 ns. Images were typically recorded with a pixel resolution of 7.6 nm × 7.6 nm or better, a scan speed of 400 Hz, bidirectional mode, line averaging of 3, and a pinhole size of 151.63 μm. Images were processed using ImageJ. In some cases, STED images were deconvolved with a theoretical PSF using the commercially available program package Huygens.

[0156] Figure 6(a) shows a confocal microscopic image of stained mitochondria, and Figure 6(b) shows the results of observation of the same area using an STED microscope. Cristae, a structural feature of the inner mitochondrial membrane, were observed in Figure 6(b). These results demonstrate that compound 8 specifically stains the inner mitochondrial membrane.

Claims

1. General formula (1): [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ３ and R ４ are the same or different and represent a substituted or unsubstituted (hetero)aryl group. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

2. The Ar １ The compound according to claim 1 , wherein is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

3. The Ar ２ The compound according to claim 1 , wherein is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring.

4. R １ and R ２ The compound according to claim 1 , wherein are the same or different and are a hydrogen atom or a substituted or unsubstituted alkyl group.

5. R ３ and R ４ The compound of claim 1 , wherein are the same or different and are substituted or unsubstituted aryl groups.

6. R ５ The compound of claim 1 , wherein is a substituted or unsubstituted alkyl group.

7. A fluorescent dye comprising the compound or solvate thereof according to any one of claims 1 to 6.

8. A mitochondrial staining agent comprising the compound or solvate thereof according to any one of claims 1 to 6.

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

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

11. General formula (2): [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ３ and R ４ are the same or different and represent a substituted or unsubstituted (hetero)aryl group. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group.

12. A method for producing the compound according to claim 11, comprising reacting a compound of formula (3): [In the formula, Ar １ , Ar ２ , R １ , R ２ and R ５ is the same as above. １ represents a halogen atom.] is reacted with a lithium compound, and then a compound represented by the general formula (4): PR ３ R ４ X ２ (4) [wherein, R ３ and R ４ is the same as above. ２ represents a halogen atom.] with a compound represented by the following formula (I):

13. General formula (3): [In the formula, Ar １ and Ar ２ are the same or different and represent a substituted or unsubstituted aromatic hydrocarbon ring or a substituted or unsubstituted heteroaromatic ring. １ and R ２ are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero)aryl group, provided that R １ and R ２ may be joined together to form a ring with the adjacent nitrogen atom. １ and / or R ２ is Ar ２ may be taken together with the adjacent nitrogen atom to form a ring. ５ represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted cycloalkyl group. １ represents a halogen atom.] A compound represented by the formula:

14. A method for producing the compound according to claim 13, comprising: [In the formula, Ar １ and R ５ is the same as above. １ represents a boronic acid or an ester group thereof.] and a compound represented by general formula (6): [In the formula, Ar ２ , R １ , R ２ and X １ is the same as above. ３ is X １ and a compound represented by the formula (I) in the presence of a palladium catalyst.

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