JP6529054B2 - Fused thiophene compound and oil droplet stain using the same - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a fused thiophene compound and an oil droplet stain using the same.

  Intracellular lipid droplets (fatty spheres) are a type of organelle that is universally present in all cells, and have a structure in which triglycerides such as triacylglycerols, diacylglycerols and cholesterol esters are covered with a phospholipid monolayer. ing. Recent studies have revealed that many proteins are present on the surface of lipid droplets, and lipid droplets are involved in various biological phenomena such as vesicle transport, signaling, etc., including energy supply by hydrolysis of lipid esters. Reports have been made to suggest that It is also known that the behavior of fat droplets is associated with various diseases such as obesity, cardiovascular disease, diabetes, fatty liver and the like. From these findings, the elucidation of the behavior of fat droplets is an important research topic because it is necessary to understand life phenomena such as lipid metabolism and is useful for early detection and prevention of diseases. However, little is known about the behavior of fat droplets, and it is not possible to directly observe the behavior of various proteins present on the surface of fat droplets, the formation and decomposition process of fat droplets, and the like. If it is possible to analyze intracellular lipid droplet dynamics spatio-temporally with high accuracy, it is considered that research on lipid droplets will be dramatically developed. In order to realize this, it is necessary to keep observing fat droplets non-invasively and for a long time in real time, and means for achieving this are currently required.

  Generally, a method of staining intracellular lipid droplets is employed for observation of these lipid droplets. As such an intracellular fat globule staining agent, BODIPY 493/503 is commercially available.

  Since intracellular lipid droplets are considered to be extremely small in the early developmental stage, high spatial resolution is required to observe intracellular lipid droplets in real time. For this purpose, it is considered to adopt super resolution microscopic techniques such as STED microscope. By using a super-resolution microscope, it is also expected to capture the formation process of extremely small fat droplets, but for that purpose, it is essential to use a fluorescent probe to visualize fat droplets themselves, not proteins on the surface of fat droplets. is there. However, since a super resolution microscope requires the irradiation of a strong laser beam, the fluorescent dye may be strongly discolored if it is not a fluorescent dye extremely excellent in light resistance, and images may be continuously obtained for a long time. Have difficulty. Therefore, in order to observe fat droplets using a super resolution microscope for a long time, in addition to highly selective fat droplet staining ability, a fluorescent probe that has super-light resistance that can suppress fading even under light irradiation It will be necessary.

  Although the commercially available BODIPY 493/503 is easily collected in fat droplets because of its high affinity to neutral fat, it can not selectively stain fat droplets because the cell wall and cell membrane are similarly strongly fluorescent. In addition, since the light resistance is poor, it is not suitable for long-term observation or observation using a strong laser beam.

  On the other hand, BTO in which benzothiophene oxide and a diphenylamino group are linked is capable of highly selective staining of lipid droplets since it hardly fluoresces in polar solvents. However, there is still room for improvement in light stability.

  As described above, since the fluorescence intensity of the conventionally known fluorescent dye decreases with the passage of time, significant fluorescence fading occurs when repeated imaging of fat droplets is performed. It can not be observed for a long time. In addition, when analyzing the lipid metabolism mechanism by fluorescence imaging, it can not be determined whether the fluorescence intensity is reduced due to the decomposition (metabolism) of the lipid droplet or whether the fluorescence of the fluorescent dye itself is faded. Moreover, in order to visualize the formation process and decomposition (metabolism) process of fat droplets in real time, it is necessary to observe with a microscope that emits a strong laser such as a super resolution microscope in order to observe a very small tissue. As described above, when a fluorescent dye having insufficient light resistance is used, the strong laser irradiation rapidly fades the fluorescent dye, and it is impossible at present to visualize the formation process and decomposition process of fat droplets in real time It is.

  The present invention has been made to solve such problems, and it is an object of the present invention to provide a compound which can stain fat droplets highly selectively and which is excellent in light resistance.

  As a result of intensive studies in view of the above problems, the inventors of the present invention have been able to stain fat droplets highly selectively in a fused-ring thiophene compound having a specific structure or a solvate thereof, and light resistance I found it to be excellent. Based on such findings, the present inventors further researched and completed the present invention. That is, the present invention includes the following configurations.

  Item 1. General formula (1-1):

[Wherein, Ar ¹ and Ar ² are the same or different and each represents a substituted or unsubstituted (hetero) aromatic ring. R ^1-1 and R ^2-1 are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. However, the case where R ^1-1 and R ^2-1 are both hydrogen atoms is excluded. R ^1-1 and R ^2-1 may combine to form a ring with the adjacent nitrogen atom. R ^1-1 and / or R ^2-1 may combine with Ar ² to form a ring with an adjacent nitrogen atom. Y ¹ and Y ² one is -SO ₂ - is a group represented by the other is a sulfur atom, -SO ₂ - or a group represented by -CR ³ ₂ in - a group represented by (R ³ are the same Or a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. ]
The fused-ring thiophene compound or its solvate represented by these.

Item 2. The condensed-ring thiophene compound or a solvate thereof according to item 1, wherein ^one of Y ¹ and Y ² is a group represented by —SO ₂ — and the other is a sulfur atom.

Item 3. The fused-ring thiophene compound or a solvate thereof according to Item 1 or 2, wherein Ar ¹ and Ar ² are a substituted or unsubstituted aromatic ring.

Item 4. The thiophene compound or a solvate thereof according to any one of Items 1 to 3, wherein each of R ^1-1 and R ^2-1 is a substituted or unsubstituted aryl group.

  Item 5. An oil droplet stain comprising the fused thiophene compound according to any one of Items 1 to 4 or a solvate thereof.

  Item 6. General formula (1):

[Wherein, Ar ¹ and Ar ² are the same or different and each represents a substituted or unsubstituted (hetero) aromatic ring. R ¹ and R ² are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. R ¹ and R ² may combine to form a ring with the adjacent nitrogen atom. R ¹ and / or R ² may combine with Ar ² to form a ring with the adjacent nitrogen atom. Y ¹ and Y ² one is -SO ₂ - is a group represented by the other is a sulfur atom, -SO ₂ - or a group represented by -CR ³ ₂ in - a group represented by (R ³ are the same Or a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. ]
An oil droplet stain comprising the condensed thiophene compound represented by or a solvate thereof.

  Item 7. 7. The oil droplet stain according to item 5 or 6, wherein the fused thiophene compound or a solvate thereof is dissolved in a nonpolar solvent.

  Item 8. The oil drop stain according to any one of Items 5 to 7, which is a fat stain.

  Item 9. The oil droplet stain according to any one of Items 5 to 8, which is an intracellular fat globule stain.

  Item 10. Item 10. A method for bioimaging a fat, using the oil droplet stain according to any one of Items 5 to 9.

  According to the present invention, certain fused-ring thiophene compounds or solvates thereof are capable of highly sensitively staining oil droplets (particularly intracellular fat spheres) even if they are small in size. Can also suppress the fluorescent staining of

  In addition, this particular fused-ring thiophene compound or a solvate thereof hardly loses its fluorescence intensity upon light irradiation, and is effective for long-term observation or observation using a strong laser beam.

  In addition, since this specific fused-ring thiophene compound or a solvate thereof can be used to stain oil droplets (particularly fat spheres) using a visible light laser light source, the damage to the living body is greatly reduced. It can be used in vivo.

It is an ultraviolet visible absorption spectrum and fluorescence spectrum of DEBSO1 of Example 1, and DEBSO2 of Example 2. a) UV-visible absorption spectrum of DEBSO1 in each solvent. b) Fluorescence spectra of DEBSO1 in each solvent. c) UV-visible absorption spectrum of DEBSO2 in each solvent. d) Fluorescence spectra of DEBSO2 in each solvent. It is a result of the photostability of DEBSO1 of Example 1, DEBSO2 of Example 2, BTO of Comparative Example 1, and BODIPY 493/503 of Comparative Example 2 in toluene. The results of staining of the adipocytes differentiated from 3T3-L1 with 1 μM of DEBSO1, DEBSO2 and BTO for 12 hours are shown. a) Imaging image of adipocytes stained with 1 μM DEBSO1. b) Fluorescence intensity cross section along the white line of a. c) Imaging images of adipocytes stained with 1 μM DEBSO2. d) Fluorescence intensity cross section along the white line of c. e) Imaging image of adipocytes stained with 1 μM BTO. f) Fluorescence intensity cross section along the white line of e. The excitation wavelength is 405 nm in all cases. It shows the results of staining for 12 hours with 100 nM each of DEBSO1, DEBSO2 and BTO on adipocytes differentiated from 3T3-L1. a) Imaging image of adipocytes stained with 100 nM DEBSO1. b) Fluorescence intensity cross section along the white line of a. c) Imaging image of adipocytes stained with 100 nM DEBSO2. d) Fluorescence intensity cross section along the white line of c. e) Imaging images of adipocytes stained with 100 nM BTO. f) Fluorescence intensity cross section along the white line of e. The excitation wavelength is 405 nm in all cases. The results of staining of the adipocytes differentiated from 3T3-L1 with 1 μM of DEBSO1, DEBSO2 and BTO for 12 hours are shown. a) Imaging image of adipocytes stained with 1 μM DEBSO1. b) Fluorescence intensity cross section along the white line of a. c) Imaging images of adipocytes stained with 1 μM DEBSO2. d) Fluorescence intensity cross section along the white line of c. e) Imaging image of adipocytes stained with 1 μM BTO. f) Fluorescence intensity cross section along the white line of e. The excitation wavelength is 473 nm in all cases. An imaging image at the time of continuing irradiating a confocal laser after staining for 1 hour with 1 μM of DEBSO 1, DEBSO 2 and BTO for adipocytes differentiated from 3T3-L1 respectively is shown. The excitation wavelength is 473 nm in all cases. It is a graph which shows the change of the fluorescence intensity at the time of continuing irradiating a confocal laser, after having stained with 1 micromol DEBSO1, DEBSO2, and BTO for 12 hours with respect to the fat cell which differentiation-induced from 3T3-L1, respectively. The excitation wavelength is 473 nm in all cases. It is a schematic diagram of lipolysis. It is the appearance of the contraction | shrinkage of a lipid droplet when the culture | cultivation by the culture medium of 10 micromol forskolin, 0.1 mM IBMX, 2% BSA, and 1 micromol DEBSO1 of the adipocytes stained with 1 micromol DEBSO1 for 12 hours is given. It is a graph which shows the change of the brightness | luminance per area in the area | region (blue circle on the lower side) enclosed with the blue circle of the upper side of FIG. 9, and the area circled on the lower side of FIG.

  As used herein, "(hetero) aromatic ring" means an aromatic ring or a heteroaromatic ring. Moreover, in the present specification, the “(hetero) aryl group” means an aryl group or a heteroaryl group. Furthermore, in the present specification, the term "containing" is a concept including any of "comprise", "consist essentially of", and "consist of". is there.

  In the present specification, as a halogen atom, for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like can be mentioned.

  In the present specification, as the halogenated alkyl group, a trifluoromethyl group, a pentafluoroethyl group and the like can be mentioned.

1. Fused Thiophene Compound The fused thiophene compound of the present invention has the general formula (1):

[Wherein, Ar ¹ and Ar ² are the same or different and each represents a substituted or unsubstituted (hetero) aromatic ring. R ¹ and R ² are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. R ¹ and R ² may combine to form a ring with the adjacent nitrogen atom. R ¹ and / or R ² may combine with Ar ² to form a ring with the adjacent nitrogen atom. Y ¹ and Y ² one is -SO ₂ - is a group represented by the other is a sulfur atom, -SO ₂ - or a group represented by -CR ³ ₂ in - a group represented by (R ³ are the same Or a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. ]
It is a compound represented by

  Among these, general formula (1-1):

[Wherein, Ar ¹ , Ar ² , Y ¹ and Y ² are as defined above. R ^1-1 and R ^2-1 are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. However, the case where R ^1-1 and R ^2-1 are both hydrogen atoms is excluded. R ^1-1 and R ^2-1 may combine to form a ring with the adjacent nitrogen atom. R ^1-1 and / or R ^2-1 may combine with Ar ² to form a ring with an adjacent nitrogen atom. ]
The fused thiophene compound represented by is a novel compound which has not been described in the literature.

In the general formulas (1) and (1-1), as the (hetero) aromatic ring represented by Ar ¹ and Ar ² , any of an aromatic ring and a heteroaromatic ring can be adopted.

Examples of the aromatic ring represented by Ar ¹ and Ar ² include a benzene ring as a monocyclic aromatic hydrocarbon ring, and a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, and a pyrene as a polycyclic aromatic hydrocarbon ring. Rings, triphenylene rings and the like.

The aromatic ring represented by Ar ¹ and Ar ² may have a substituent. As a substituent, a halogen atom, the below-mentioned alkyl group, the below-mentioned cycloalkyl group, a halogenated alkyl group, the below-mentioned aryl group, the below-mentioned heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable.

The heteroaromatic ring represented by Ar ¹ and Ar ² includes, for example, a pyrrolidine ring, a pyrrole ring, a tetrahydrothiophene ring, a thiophene ring, an oxolane ring, a furan ring, an imidazole ring, an imidazole ring, a pyrazole ring, a thiazole ring as a single ring heteroaromatic ring. An oxazole ring, a piperidine ring, a pyridine ring, a pyrazine ring and the like can be mentioned, and as a polycyclic heteroaromatic ring, an indole ring, an isoindole ring, a benzimidazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring and the like can be mentioned.

The heteroaromatic ring represented by Ar ¹ and Ar ² may have a substituent. As a substituent, a halogen atom, the below-mentioned alkyl group, the below-mentioned cycloalkyl group, a halogenated alkyl group, the below-mentioned aryl group, the below-mentioned heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable.

Among them, as Ar ¹ and Ar ² , the absorption maximum wavelength and the fluorescence maximum wavelength are made larger, the light resistance is further improved, the cytotoxicity is lower, and oil droplets (fat droplets etc.) can be stained more clearly From the viewpoint, a substituted or unsubstituted aromatic ring is preferable, and a substituted or unsubstituted monocyclic aromatic hydrocarbon ring is more preferable. On the other hand, when Ar ¹ is a substituted or unsubstituted polycyclic aromatic hydrocarbon ring, when a lipid-soluble functional group is introduced to Ar ¹ , in particular, the localization ability to oil droplets (fat droplets etc.) is enhanced. Is possible.

In the general formula (1), as the alkyl group represented by R ¹ and R ² , either a linear alkyl group or a branched alkyl group can be employed, and examples thereof include a methyl group, an ethyl group, an n-propyl group and an isopropyl group And C1-10 alkyl groups such as n-butyl group, isobutyl group, sec-butyl group, tert-butyl group (especially C1-6 alkyl group). The alkyl group represented by R ^1-1 and R ^2-1 in the general formula (1-1) can also be the same.

The alkyl group represented by R ¹ and R ² may have a substituent. As a substituent, a halogen atom, the below-mentioned cycloalkyl group, the below-mentioned aryl group, the below-mentioned heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable. Substituents which can be possessed by the alkyl group represented by R ^1-1 and R ^2-1 in the general formula (1-1) can be the same.

As the cycloalkyl group represented by R ¹ and R ² in the general formula (1), for example, a C 3-10 cycloalkyl group such as cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group (especially C 4 -8 cycloalkyl group). The cycloalkyl groups represented by R ^1-1 and R ^2-1 in the general formula (1-1) can also be the same.

The cycloalkyl group represented by R ¹ and R ² may have a substituent. As a substituent, a halogen atom, the said alkyl group, the below-mentioned aryl group, the below-mentioned heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable. Substituents which the cycloalkyl group represented by R ^1-1 and R ^2-1 in the general formula (1-1) may have may be the same.

In the general formula (1), as the aryl group represented by R ¹ and R ² , for example, any of a monocyclic aryl group, a fused aryl group and a polycyclic aryl group can be adopted, and for example, phenyl as a monocyclic aryl group Groups include a fused aryl group such as naphthyl, anthracenyl, phenanthrenyl, fluorenyl, pyrenyl and triphenylenyl groups, and a polycyclic aryl group such as a biphenyl group or a terphenyl group. (In particular, a C 6-14 aryl group). The same applies to the aryl group represented by R ^1-1 and R ^2-1 in the general formula (1-1).

The aryl group represented by R ¹ and R ² may have a substituent. As a substituent, a halogen atom, the said alkyl group, the said aryl group, the below-mentioned heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable. Substituents which can be possessed by the aryl group represented by R ^1-1 and R ^2-1 in the general formula (1-1) can be the same.

In the general formula (1), as a heteroaryl group represented by R ¹ and R ² , any of a monocyclic heteroaryl group and a fused heteroaryl group can be adopted, and for example, a pyrrolidinyl group, a pyrrolyl group as a monocyclic heteroaryl group Group, tetrahydrothienyl group, thienyl group, oxoranyl group, furanyl group, imidazolyl group, pyrazolyl group, thiazolyl group, oxazolyl group, piperidyl group, pyridyl group, pyrazyl group, etc., and as a fused heteroaryl group, indolyl group, isoindolyl group Groups, benzimidazolyl groups, quinolyl groups, isoquinolyl groups, quinoxalyl groups and the like. The heteroaryl groups represented by R ^1-1 and R ^2-1 in the general formula (1-1) can also be the same.

The heteroaryl group represented by R ¹ and R ² may have a substituent. As a substituent, the said halogen atom, the said alkyl group, the said aryl group, the said heteroaryl group, a cyano group, a nitro group etc. are mentioned, for example. As for the number of substituents in the case of having a substituent, for example, 1 to 6 is preferable, and 1 to 3 is more preferable. Substituents which may be possessed by the heteroaryl group represented by R ^1-1 and R ^2-1 in the general formula (1-1) may be the same.

Among them, as R ¹ and R ² , a substituted or unsubstituted (hetero) aryl group is preferable, a substituted or unsubstituted aryl group is more preferable, and an unsubstituted aryl group is more preferable. The heteroaryl groups represented by R ^1-1 and R ^2-1 in the general formula (1-1) can also be the same. Incidentally, R ^1-1 and R ^2-1 in the general formula (1-1) can not both be hydrogen atoms.

R ¹ and R ² may combine to form a ring with the adjacent nitrogen atom. That is, the group represented by -NR ¹ R ² is

It may be a group represented by, for example. Similarly, R ^1-1 and R ^2-1 in the general formula (1-1) may form a ring as described above together with the nitrogen atom.

Also, R ¹ and / or R ² may combine with Ar ² to form a ring with the adjacent nitrogen atom. That is, the structure represented by Ar ² -NR ¹ R ² is

Or the like. Similarly, R ^1-1 and / or R ^2-1 in the general formula (1-1) may combine with Ar ² to form a ring as described above with a nitrogen atom.

Incidentally, in the compound represented by the general formula (1), in Ar ^2, the substitution position of a group represented by -NR ¹ R ² bonded are not particularly limited. In the compound represented by the above general formula (1-1), the substitution position to which the group represented by -NR ^1-1 R ^2-1 in Ar ² is also not particularly limited. For example, when Ar ² is a benzene ring, a compound represented by the general formula (1 ′):

[Wherein, Ar ¹ , Y ¹ and Y ² are as defined above. R ¹ and R ² are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. R ¹ and R ² may combine to form a ring with the adjacent nitrogen atom. R ¹ and / or R ² may combine with the adjacent benzene ring to form a ring with the adjacent nitrogen atom. ]
Or a compound represented by the general formula (1-1 ′):

[Wherein, Ar ¹ , Y ¹ and Y ² are as defined above. R ^1-1 and R ^2-1 are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. However, the case where R ^1-1 and R ^2-1 are both hydrogen atoms is excluded. R ^1-1 and R ^2-1 may combine to form a ring with the adjacent nitrogen atom. R ^1-1 and / or R ^2-1 may be combined with the adjacent benzene ring to form a ring with the adjacent nitrogen atom. ]
Are easily formed.

In the general formulas (1) and (1-1), ^one of Y ¹ and Y ² is a group represented by -SO _2- , and the other is a sulfur atom, a group represented by -SO _2- or -CR ³ _2- a group represented by Both Y ¹ and Y ² -SO ₂ - is a group represented by, in order to improve the lipophilic, or a substituted or unsubstituted polycyclic aromatic hydrocarbon rings Ar ^1, in Ar ¹ It is preferred to introduce a lipophilic functional group.

-CR ³ ₂ - In the group represented by the formula, the alkyl group represented by R ^3, cycloalkyl group, aryl group and heteroaryl group may be employed those described above. The types and numbers of substituents are also the same.

Among them, as Y ¹ and Y ² , it is preferable that one of them is a group represented by —SO ₂ — and the other is a sulfur atom, from the viewpoint of easily staining the lipid droplet without excessively increasing the crystallinity. . As Y ¹ and Y ^2, one is -SO ₂ - is a group represented by the other is -CR ³ ₂ - if a group represented by as much as possible the crystallinity by adjusting the R ³ It is preferable to keep it low.

  As the fused-ring thiophene compound of the present invention satisfying the above conditions, it is easier to dye with a visible light laser light source while improving light resistance, and oil droplets (in particular intracellular lipid droplets) can be made higher even with a small size. From the viewpoint of being able to detect sensitivity and further suppressing fluorescent staining of other tissues, general formula (1A) or (1B):

[In the Formula, Y ^1a and Y ^2a represent a sulfur atom or a group represented by —CR ³ ₂ —. Ar ¹ , Ar ² , R ¹ , R ² and R ³ are as defined above. ]
Or a compound represented by the general formula (1A-1) or (1B-1):

[Wherein, Y ^1a , Y ^2a , Ar ¹ , Ar ² , R ¹ , R ² and R ³ are as defined above. ]
Are preferable, and in the general formula (1A1) or (1B1):

[Wherein, Ar ¹ , Ar ² , R ¹ and R ² are as defined above. ]
Or a compound represented by the general formula (1A1-1) or (1B1-1):

[Wherein, Ar ¹ , Ar ² , R ¹ and R ² are as defined above. ]
And more preferably a compound represented by the general formula (1A1A) or (1B1A):

[Wherein, Ar ¹ and Ar ² are as defined above. R ^1a and R ^2a are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. ]
Or a compound represented by the general formula (1A1A-1) or (1B1A-1):

[Wherein, Ar ¹ and Ar ² are as defined above. R ^1a-1 and R ^2a-1 are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted (hetero) aryl group. However, the case where R ^1a-1 and R ^2a-1 are both hydrogen atoms is excluded. ]
Is more preferable,

Etc. are particularly preferred.

  In addition, the fused-ring thiophene compound of the present invention may be a fused thiophene compound represented by the above general formula (1) or (1-1) as a solvate, all of which are included in the scope of the present invention Be done. The solvate is not particularly limited as long as it is a solvate of the fused thiophene compound represented by the above general formula (1) or (1-1) and a solvent. Examples of the solvent for forming a solvate include dichloromethane, chloroform, acetonitrile, diethyl ether, ethyl acetate, methanol, ethanol, cyclohexane, toluene, acetone, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran and the like.

2. Method for Producing Fused Thiophene Compound The method for producing the fused thiophene compound represented by the general formula (1) of the present invention is not particularly limited. For example, when ^one of Y ¹ and Y ² is a group represented by —SO ₂ — and the other is a sulfur atom, the following reaction formula 1:

[Wherein, Ar ¹ , Ar ² , R ¹ and R ² are as defined above. X represents a halogen atom. ]
Can be synthesized according to When a compound represented by the general formula (1-1) is synthesized, synthesis can be performed in the same manner except that R ¹ is R ^1-1 and R ² is R ^2-1 .

  In outline, compound (2) is subjected to an oxidation reaction to obtain compounds (3A) and (3B), and then isolated and subjected to a Backward-Heartwig coupling reaction, or compound (2) is subjected to a backward reaction. An oxidation reaction can be carried out after subjecting the compound to a wig coupling reaction to obtain a compound (4).

The above synthesis is shown only in the case where one of Y ¹ and Y ² is a group represented by —SO ₂ — and the other is a sulfur atom, but ^one of Y ¹ and Y ² is — sO ₂ - in a group represented, the other is -SO ₂ - or a group represented by -CR ³ ₂ in - similarly be a group represented by the oxidation reaction and back against suitable substrate The fused thiophene compound of the present invention can be obtained by combining with the Wald-Heartwig coupling reaction.

(2-1) Oxidation reaction (compound (2) → compound (3A) and compound (3B); compound (4) → compound (1B1))
In this step, for example, an oxidation reaction can be caused to the compound (2) or the compound (4) in the above reaction formula 1 in an organic solvent.

  An oxidizing agent can be used for the oxidation reaction, for example, hydrogen chloride (hydrochloric acid), sulfuric acid, formic acid, trifluoroacetic acid (TFA), trifluoroacetic anhydride, boron trifluoride diethyl ether complex, trifluoromethane sulfonic acid, etc. Perchloric acid such as peracetic acid, perbenzoic acid, metachloroperbenzoic acid (m-CPBA); peroxide such as tert-butyl peroxide; perhalogenate such as sodium metaperiodate Can be mentioned. These can be used alone or in combination of two or more. Among these, in this step, from the viewpoint of yield and easiness of synthesis, peracid is preferable, and metachloroperbenzoic acid (m-CPBA) is more preferable. The amount of the oxidizing agent used is preferably 1 to 5 moles, more preferably 1.5 to 3 moles, per 1 mole of the compound (2) or the compound (4), from the viewpoint of yield and easiness of synthesis.

  As the organic solvent which can be used in this step, known ones can be adopted. For example, halogenated hydrocarbons such as dichloromethane, chloroform and 1,2-dichloroethane; cyclic ether compounds such as tetrahydrofuran and dioxane; dimethyl sulfoxide (DMSO) and the like. These organic solvents can be used alone or in combination of two or more. In this step, halogenated hydrocarbons are preferable, and 1,2-dichloroethane is more preferable, from the viewpoint of yield and easiness of synthesis.

  The reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction atmosphere is preferably an inert gas atmosphere (nitrogen gas atmosphere, argon gas atmosphere, etc.), an air atmosphere, etc., and the reaction temperature is -50 to 100 ° C. The reaction time is preferably 5 minutes to 24 hours, and more preferably 10 minutes to 12 hours.

  When compound (3A) and compound (3B) are obtained by this reaction, it is preferable to carry out purification treatment according to a conventional method as needed, and further to isolate according to a conventional method and use in the next step. In addition, when compound (1B1) is obtained, it can be subjected to purification treatment according to a conventional method as necessary, and can be used as a condensed ring thiophene compound of the present invention.

(2-2) Boldwald-Heartwig coupling reaction (compound (2) → compound (4); compound (3A) → compound (1A1); compound (3B) → compound (1B1))
In this step, for example, a Backward-Heartwig coupling reaction can be caused to the compound (2), the compound (3A), or the compound (3B) in the above Reaction formula 1 in an organic solvent. Specifically, for example, compound (2), compound (3A) or compound (3B) and R ¹ R ² NH (R ¹ and R ² are the same as above in the presence of a palladium catalyst in an organic solvent, for example) Can be reacted with a compound represented by When the compound represented by the general formula (1-1) is to be synthesized, the compound represented by R ^1-1 R ^2-1 NH is substituted for the compound represented by R ¹ R ² NH. use.

The amount of the compound represented by R ¹ R ² NH is usually 1 to 1 mole of Compound (2), Compound (3A) or Compound (3B) from the viewpoint of yield and easiness of synthesis. 10 mol is preferable, and 2 to 5 mol is more preferable.

Examples of the palladium catalyst include palladium catalysts known as catalysts for synthesis of organic compounds and the like. In the present invention, the palladium catalyst used in the Backward-Heartwig coupling reaction can be used. Specifically, palladium acetate (Pd (OAc) ₂ ), tetrakis (triphenylphosphine) palladium (0) (Pd (PPh ₃ ) ₄ ), trifluoroacetate palladium, palladium chloride, palladium bromide, palladium iodide, Tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ), bis (dibenzylideneacetone) palladium (0), dichloro (1,5-cyclooctadiene) palladium (II), 2,5- And norbornadiene palladium dichloride and the like. These palladium catalysts can also use solvates. Moreover, these palladium catalysts can also be used independently and can also be used in combination of 2 or more types. In this step, tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ) (or a solvate thereof) is preferable from the viewpoint of easiness of synthesis, yield and the like. The amount of the palladium catalyst used is preferably 0.001 to 0.1 mol, usually 0.005 to 0.1 mol, per 1 mol of compound (2), compound (3A) or compound (3B), from the viewpoint of easiness of synthesis, yield, etc. 0.05 mol is more preferable.

A ligand compound can also be used at this process as needed. The ligand compound which can be used is not particularly limited. For example, triphenylphosphine, trimethoxyphosphine, triethylphosphine, triisopropylphosphine, tri (t-butyl) phosphine, tri (n-butyl) phosphine, triisopropoxy Phosphine, tricyclopentyl phosphine, tricyclohexyl phosphine, trimesityl phosphine, triphenoxy phosphine, di (t-butyl) methyl phosphine, methyl diphenyl phosphine, dimethyl phenyl phosphine, triethylamine, pyridine, 2,2'-bipyridyl, 4,4 ' -(t-butyl) bipyridyl, 1,1'-bis (diphenylphosphino) ferrocene, 1,1'-bis (t-butyl) ferrocene, diphenylphosphinomethane, 1,2-bis (diphenylphosphino) ethane , 1,3-bis (diphenylphosphino) propane, 1 1,5-bis (diphenylphosphino) pentane, 1,2-bis (dipentafluorophenylphosphino) ethane, 1,2-bis (dicyclohexylphosphino) ethane, 1,3- (dicyclohexylphosphino) propane, 1 , 2-bis (di-t-butylphosphino) ethane, 1,3-bis (di-t-butylphosphino) propane, 1,2-bis (diphenylphosphino) benzene, 1,5-cyclooctadiene , 2- (dicyclohexylphosphino-2 ', 6'-dimethoxy-1,1'-biphenyl (S-Phos), 2- (dicyclohexylphosphino-2', 4 ', 6'-tri-isopropyl-1, 1'-biphenyl (X-Phos), bis (2-diphenylphosphinophenyl) ether (DPEPhos), tetramethylphosphonium chloride, tetraethylphosphonium bromide, tetraphenylphosphonium bromide, tri-tert-butylphosphonium tetrafluoroborate (tert- Bu ₃ PHBF ₄ ) These ligand compounds may also be solvates, and may be used alone or in combination of two or more. From the viewpoint of yield and easiness of synthesis, tri-tert-butylphosphonium tetrafluoroborate (tert-Bu ₃ PHBF ₄ ) is preferable The amount of the ligand compound used is the viewpoint of easiness of synthesis, yield, etc. From this, 1.5 to 10 moles are preferable, and 2 to 5 moles are more preferable, with respect to 1 mole of the palladium catalyst.

  In this step, a base can also be used, if necessary. As a base, ammonium chloride, potassium fluoride, cesium fluoride, sodium hydroxide, potassium hydroxide, sodium methoxide, sodium tert-butoxide, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, phosphorus Potassium acetate, sodium acetate, potassium acetate, calcium acetate etc., and in this step, sodium methoxide, sodium tert-butoxide etc. are preferred from the viewpoint of yield and easiness of synthesis, sodium tert-butoxide is more preferred. . The amount of the base to be used is preferably 1.5 to 10 moles, relative to 1 mole of the compound (2), the compound (3A) or the compound (3B), from the viewpoint of easiness of synthesis, yield, etc. -5 mol is more preferable. As the organic solvent that can be used in this step, known ones can be adopted, and examples thereof include diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentyl methyl Ethers such as ether (CPME) and tert-butyl methyl ether (TBME); aromatic hydrocarbons such as benzene, toluene and xylene; aliphatic halogenated hydrocarbons such as dichloromethane, dichloroethane, chloroform and carbon tetrachloride; N, N -Amide such as dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone etc. may be mentioned. These organic solvents can be used alone or in combination of two or more. In this step, aromatic hydrocarbons are preferable, and xylene (particularly o-xylene) is more preferable, from the viewpoint of yield and easiness of synthesis.

  The reaction conditions are preferably such that the reaction proceeds sufficiently. For example, the reaction atmosphere is preferably an inert gas atmosphere (nitrogen gas atmosphere, argon gas atmosphere etc.) and the like, and the reaction temperature is 50 to 250 ° C., particularly 100 to 200 ° C. is preferred, and the reaction time is preferably 5 minutes to 48 hours, particularly 10 minutes to 36 hours.

  When compound (4) is obtained by this reaction, it is preferable to perform purification treatment according to a conventional method as needed and use it in the next step. When compound (1A1) or compound (1B1) is obtained, the compound can be used as a fused-ring thiophene compound of the present invention, if necessary, after being subjected to purification treatment according to a conventional method.

3. Oil Drop Staining Agent The fused thiophene compound of the present invention has the specific structure described above, so the absorption maximum wavelength is in the visible light region (for example, about 380 to 500 nm), and the fluorescence maximum wavelength is also in the visible light region (For example, about 420 to 650 nm). In particular, since the fused thiophene compound of the present invention has a small absorption maximum wavelength, damage when used in living cells can be extremely reduced. In addition, the fused thiophene compound of the present invention has an extremely high excitation efficiency as a fluorescent dye because the fluorescence maximum wavelength is close to the excitation wavelength of 633 nm of a HeNe laser usually equipped with a laser microscope. For this reason, the fused-ring thiophene compounds of the present invention are very suitable for use as fluorescent dyes in living cells.

  In addition, the fused-ring thiophene compound of the present invention can strongly fluoresce the fat globules (fat droplets) in cells and can suppress the fluorescence of tissues other than fat globules (fat droplets). In the fused thiophene compound of the present invention, the fluorescence intensity of fat globules (fat droplets) is significantly larger than that of other tissues (S / N) by suppressing the fluorescence of tissues other than fat globules (fat droplets). More than twice BTO). Therefore, the fused thiophene compound of the present invention can detect oil droplets (particularly intracellular lipid droplets) with high sensitivity even with a small size, and can further suppress fluorescent staining of other tissues.

  Moreover, although the fluorescent dye used in the conventional oil drop stain has a significant decrease in fluorescence intensity with the passage of light irradiation time, the fused ring thiophene compound of the present invention exhibits fluorescence even when the light irradiation time has elapsed. The strength hardly decreases. Therefore, even fat globules (fat droplets) in the process of fat formation and decomposition can be detected with high sensitivity, and the fluorescence intensity does not decrease even when using a high resolution microscope using a powerful laser beam Since it is possible to visualize fat formation and degradation processes in real time, it is useful for clarifying the behavior of fat cell formation and degradation.

  From the above, the fused-ring thiophene compound of the present invention or a solvate thereof is very useful as an oil droplet stain (particularly as a fat stain, and further as an intracellular fat globule stain (intracellular fat droplet stain)). By using the oil droplet staining agent of the present invention, it is possible to bio-image only fat (particularly intracellular fat globule) with higher sensitivity, and as described above, the fluorescence intensity is almost reduced even when the light irradiation time passes. Because it does not, it can withstand bioimaging using a super resolution microscope using long-time bioimaging or a powerful laser beam.

  In addition, the fused thiophene compound of the present invention can be synthesized in only two steps, as shown in the above-mentioned production method. For this reason, an oil drop stain can be obtained more simply.

Such an oil droplet stain of the present invention (in particular, an intracellular fat globule stain) contains the fused thiophene compound of the present invention or a solvate thereof. The form of use is not particularly limited, and for example, it is preferable to dissolve in an organic solvent to form a solution. At this time, the content of the fused-ring thiophene compound of the present invention is 1 × 10 ⁻¹⁰ from the viewpoint of detecting (staining) even a small size fat sphere with higher sensitivity (staining) and further suppressing the fluorescence of tissues other than fat spheres. 1 * 10 < ^-4 > mol / L is preferable and 1 * 10 < ^-8 > -1 * 10 < ^-5 > mol / L is more preferable. In particular, it is possible to detect oil droplets (especially intracellular fat globules) with high sensitivity even if the content is reduced to 1 × 10 ^{-10 to} 5 × 10 ^-5 mol / L or the like. Thus, in the present invention, since the content of the fused thiophene compound can also be suppressed to a low level, damage to living cells can be further suppressed.

  When the oil droplet stain of the present invention is used as a solution containing the fused thiophene compound of the present invention, an organic solvent which can be used is from the viewpoint of facilitating fluorescence of oil droplets (particularly fat spheres) with higher sensitivity. Nonpolar solvents are preferred. The oil droplet stain of the present invention has a significantly low fluorescence quantum yield in polar solvents (dimethyl sulfoxide, acetonitrile, tetrahydrofuran, methanol, ethanol, ethyl acetate, N-methylpyrrolidone, water, etc.). This is one of the reasons why in response to the surrounding environment, only intracellular fat globules can be highly selectively fluorescent.

  Examples of nonpolar solvents include aliphatic organic solvents such as pentane, hexane, cyclohexane and heptane; aromatic solvents such as benzene, toluene, xylene and mesitylene; and aliphatic halogenated hydrocarbons such as dichloromethane and dichloroethane. .

  When the oil droplet stain of the present invention is in the form of a solution, it is easy to use in living cells, and oil droplets (particularly intracellular fat globules) can be detected more sensitively even in small sizes, and fluorescent staining of other tissues About 5-11 are preferable, and, as for pH from a viewpoint which can suppress more, 6.5-7.5 are more preferable. In order to adjust the pH of the oil droplet stain of the present invention, buffers (Hepes buffer, Tris buffer, Tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) It can also be used.

  Although the present invention will be specifically described based on examples, the present invention is not limited to these.

¹ H NMR spectra and ¹³ C NMR spectra were measured using a nuclear magnetic resonance apparatus ECZ400 (JEOL) equipped with a SuperCOOL probe as a cryogenic probe. Some 13 C NMR spectra were measured using a nuclear magnetic resonance apparatus ECA600II (JEOL) equipped with a cryogenic probe, an UltraCOOL probe. Chemical shift values are expressed in ppm, and deuterated chloroform was used as a solvent. ^{In the 1} H NMR spectrum, signals from residual protons of the solvent were used, and chloroform was set to δ 7.26 ppm as an internal standard, or acetone was set to δ 2.05 ppm. ^{In the 13} C NMR spectrum, heavy solvent was used as an internal standard and deuterated chloroform was set to δ 77.16 ppm. The diphenylamine used was purchased from Tokyo Chemical Industry Co., Ltd. Tri-tert-butylphosphonium tetrafluoroborate (tert-Bu ₃ PHBF ₄ ) and 1,2-dichloroethane used were purchased from Wako Pure Chemical Industries, Ltd. Metachloroperbenzoic acid (m-CPBA) used was purchased from Sigma Aldrich. Thin layer chromatography (TLC) was performed using a glass plate coated with silica gel 60 F-254 (Merck) 0.25 mm. Silica gel column chromatography was performed using silica gel PSQ-100B (Fuji Silysia Chemical Ltd.) as a packing material. For recycling preparative gel chromatography (GPC), LC-Forte / R (YMC) equipped with polystyrene gel columns YMC-GPC T2000 and YMC-GPC T40000 was used.

Synthesis Example 1: N, N-Diphenylbenzo [b] benzo [4,5] thieno [2,3-d] thiophene-2-amine (Compound 2)

  Compound 1 was introduced with a diphenylamino group using a Backward-Heartwig coupling reaction to give Compound 2 in a 66% yield. Specifically, the synthesis was performed as follows.

₂ -bromobenzo [b] benzo [4,5] thieno [2,3-d] thiophene (compound 1; 509 mg, 1.59 mmol) under N ₂ gas atmosphere, tris (dibenzylideneacetone) dipalladium (0) chloroform complex (Pd ₂ (dba) ₃ · CHCl ₃ ; 17.8 mg, 0.0172 mmol), tri-tert-butylphosphonium tetrafluoroborate (tert-Bu ₃ PHBF ₄ ; 19.0 mg, 0.0655 mmol), diphenylamine (Ph ₂ NH; 813 mg, 4.80 mmol) sodium tert-butoxide (t-BuONa; 509 mg, 5.30 mmol) was dissolved in degassed o-xylene (5 mL). The mixture was heated to 160 ° C. and stirred for 9 hours. After cold water was added, it was extracted three times with ethyl acetate. Silica gel column chromatography was performed using hexane as a developing solvent (R _f = 0.46). The resulting solid was dissolved in CHCl ₃ and purified using recycle preparative GPC to give compound 2 as a white solid in 66% yield (434 mg, 1.06 mmol).
^{1 H NMR (400 MHz, acetone} -d 6) δ 8.07 (d, J HH = 7.6 Hz, 1H), 7.94 (d, J HH = 6.4 Hz, 1H), 7.89 (d, J HH = 8.8 Hz, 1H _{), 7.65 (d, J HH} = 2.4 Hz, 1H), 7.52 (td, J HH = 7.6 Hz, 0.8 Hz, 1H), 7.46 (td, J HH = 7.6 Hz, 1.2 Hz, 1H), 7.31-7.38 (m, 4 H), 7.23 (dd, J _HH = 8.8 Hz, 2.4 Hz, 1 H), 7.08-7.16 (m, 6 H); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 147.9, 145.8, 143.8, 142.1, HRMS (APCI): Obsd. 407.0782 ([M] ⁺ ); Calcd. For C ₂₆ H ₁₇ NS ₂ : 407.0797 133.4, 131.5, 129.5, 124.5, 124.3, 122.4, 121.4, 118.8; .

Synthesis Example 2: 7-bromobenzo [b] benzo [2,3-d] thiophene 5,5-dioxide (compound 3) and 2-bromobenzo [b] benzo [4,5] thieno [2,3-d] thiophene 5, 5-dioxide (compound 4)

  Compound 1 was oxidized using mCPBA to give compounds 3 and 4 in 17% and 12% yield, respectively. Specifically, the synthesis was performed as follows.

2-Bromo [b] benzo [4,5] thieno [2,3-d] thiophene (compound 1; 509 mg, 1.59 mmol) was dissolved in 1,2-dichloroethane (150 mL). Thereto, metachloroperbenzoic acid (m-CPBA; 849 mg, 3.44 mmol) was slowly added at 0 ° C. It was returned to room temperature and stirred for 66 hours. Aqueous Na ₂ SO ₃ solution was added, extracted three times with CH ₂ Cl ₂ , dried over anhydrous MgSO ₄ , and the solid was removed by suction filtration and then evaporated under reduced pressure. With respect to the obtained crude product, a mixed solvent of CH ₂ Cl ₂ / hexane = 3/1 was separated by PTLC as a developing solvent (R _{f (3)} = 0.53, R _{f (4)} = 0.64). As a result, Compound 3 was obtained as a white solid in 17% yield (98.1 mg, 0.280 mmol) and Compound 4 as a white solid in a yield of 12%.
Compound ^{3: 1 H NMR (400 MHz} , CDCl 3) δ 8.05 (d, J HH = 1.2 Hz, 1H), 7.90 (d, J HH = 6.8 Hz, 1H), 7.79 (d, J HH = 6.0 Hz, 1H), 7.65 (dd, JHH = 7.2 Hz, 1.6 Hz, 1 H), 7.60-7.63 (m, 1 H), 7.56 (dd, J _HH = 5.6 Hz, 0.8 Hz, 1 H), 7.51-7.54 (m, 1 H) ¹³ C NMR (125 MHz, CDCl ₃ ) δ 155.7, 144.1, 142.6, 133.9, 130.7, 130.5, 129.3, 128.1, 126.5, 123.4, 122.5, 122.2, 120.6; HRMS (APCI): Obsd. ^{. [M] +); Calcd} For C 14 H 7 BrO 2 S 2: 349.9065;
Compound 4: ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (d, J _HH = 6.4 Hz, 1 H), 7.89-7.91 (m, 2 H), 7.73 (dd, J _HH = 1.2 Hz, 6.4 Hz, 1 H , 7.55 (td, J _HH = 0.8 Hz, 6.4 Hz, 1H), 7.48 (td, J _HH = 0.8 Hz, 6.4 Hz, 1 H), 7.39 (d, J _HH = 6.4 Hz, 1 H); ¹³ C NMR HRMS (APCI): Obsd. 350.9127 ([M + H] (125 MHz, CDCl ₃ ) δ 144.0, 143.1, 136.7, 130.4, 127.4, 127.1, 126.9, 125.9, 124.4, 124.0, 123.5, 122.6; HRMS (APCI): ⁺ ); Calcd. For C ₁₄ H ₈ BrO ₂ S ₂ : 350.9144.

Example 1: DEBSO1

  The compound 2 obtained in Synthesis Example 1 was oxidized using mCPBA to synthesize DEBSO1 in 36% yield. In this reaction, DEBSO2 obtained in Example 2 described later was not obtained, but only DEBSO1 was selectively obtained.

N, N-Diphenylbenzo [b] benzo [4,5] thieno [2,3-d] thiophene-2-amine (Compound 2; 80.6 mg, 0.198 mmol) obtained in Synthesis Example 1 with 1,2-dichloroethane Dissolve in (2 mL). Metachloroperbenzoic acid (m-CPBA; 108 mg, 0.437 mmol) was slowly added thereto at 0 ° C. It returned to room temperature and stirred for 5 hours. Aqueous Na ₂ SO ₃ solution was added, extracted three times with CH ₂ Cl ₂ , dried over anhydrous MgSO ₄ , and the solid was removed by suction filtration and then evaporated under reduced pressure. The obtained crude product was purified by silica gel column chromatography using a mixed solvent of CH ₂ Cl ₂ / hexane = 1/1 as a developing solvent (R _f = 0.25). As a result, DEBSO1 was obtained as a yellow solid in a yield of 36% (31.9 mg, 0.0726 mmol).
^{1 H NMR (400 MHz, CDCl} 3) δ 7.98 (d, J HH = 8.4 Hz, 1H), 7.85 (d, J HH = 8.4 Hz, 1H), 7.47-7.52 (m, 1H), 7.41 (d, _{J HH = 7.2 Hz, 1H)} , 7.33-7.38 (m, 6H), 7.15-7.20 (m, 6H), 7.11 (dd, J HH = 8.4 Hz, 2.0 Hz, 1H); 13 C NMR (125 MHz, CDCl ₃ ) δ 151.5, 150.6, 146.2, 142.2, 132.1, 130.1, 126.8, 126.0, 125.9, 124.0, 123.8, 123.0, 122.0, 119.9, 114.0; HRMS (APCI): Obsd. 440.0768 ([ M + H] ⁺ ); Calcd. For C ₂₆ H ₁₈ NO ₂ S ₂ : 440.0773.

Example 2: DEBSO2

  DEBSO2 was obtained with a yield of 16% by introducing a diphenylamino group into Compound 4 obtained in Synthesis Example 2 by a Backward-Heartwig coupling reaction. Specifically, the synthesis was performed as follows.

₂ -bromobenzo [b] benzo [4,5] thieno [2,3-d] thiophene 5,5-dioxide (compound 4; 100 mg, 0.285 mmol) obtained in Synthesis Example 2 under N ₂ gas atmosphere, tris (Dibenzylideneacetone) dipalladium (0) chloroform complex (Pd ₂ (dba) ₃ · CHCl ₃ ; 3.2 mg, 0.00309 mmol), tri-tert-butylphosphonium tetrafluoroborate (tert-Bu ₃ PHBF ₄ ; 3.4 mg, 0.0117 mmol), diphenylamine (Ph ₂ NH; 142.7 mg, 0.843 mmol), sodium tert-butoxide (t-BuONa; 89.4 mg, 0.930 mmol) were dissolved in degassed o-xylene (1 mL). The mixture was heated to 160 ° C. and stirred for 21 hours. After addition of cold water, extraction was carried out three times with ethyl acetate, dried over anhydrous MgSO ₄ and the solid was filtered off with suction and then evaporated under reduced pressure. With respect to the obtained crude product, the mixed solvent of CH ₂ Cl ₂ / hexane = 5/1 was separated by PTLC as a developing solvent (R _f = 0.64). The obtained solid was dissolved in CHCl ₃ and purified by recycle preparative GPC to obtain DEBSO 2 as a yellow solid in a yield of 16% (20.0 mg, 0.0455 mmol).
¹ H NMR (400 MHz, acetone-d ₆ ) δ 7.84-7.87 (m, 2H), 7.74-7.75 (m, 2H), 7.64-7.67 (m, 2H), 7.35-7.39 (m, 4H), 7.29 (dd, J _HH = 8.8 Hz, 2.0 Hz, 1 H), 7.12-7.19 (m, 6 H); ¹³ C NMR (150 MHz, CDCl ₃ ) δ 147.3, 144.7, 142.3, 141.2, 133.8, 129.8, 129.7, 128.9 , 125.2, 125.1, 124.1, 123.3 , 122.7, 122.1, 121.9, 116.7; HRMS (APCI):.. Obsd 440.0792 ([M + H] +); Calcd For C 26 H 18 NO 2 S 2: 440.0773.

Comparative Example 1: 2- [4- (N, N-diphenylamino) phenyl] benzo [b] thiophene-S, S-dioxide (BTO)

  According to the previous report (Angew. Chem. Int. Ed. 2015, 54, 4539-4543), 2- [4- (N, N-diphenylamino) phenyl] benzo [b] thiophene-S, S-dioxide (BTO) I got The obtained BTO was used as the oil droplet stain of Comparative Example 1.

Comparative example 2
Commercially available BODIP Y 493/503:

The oil droplet stain of Comparative Example 2 was used.

Test Example 1 Photophysical Properties With respect to DEBSO1 and DEBSO2 obtained in Examples 1 and 2, measurements of UV-visible absorption spectrum and fluorescence spectrum in cyclohexane, dichloromethane, DMSO, toluene and acetonitrile were performed. The results are shown in Table 1 and FIG. Table 1 also includes data of BTO of Comparative Example 1 for comparison.

As a result of the above, it was found that both the DEBSO1 of Example 1 and the DEBSO2 of Example 2 have an absorption maximum wavelength at around 415 nm in the UV-visible absorption spectrum and are substantially independent of the polarity of the solvent. This result suggests that DEBSO1 and DEBSO2 can be excited using the same visible laser light even in the presence of different environments such as intracellular. On the other hand, in the fluorescence spectrum, it was found that as the polarity of the solvent is increased in both DEBSO1 and DEBSO2, the fluorescence maximum is largely shifted to a long wavelength. The absolute fluorescence quantum yield _{F F} showed high values of 0.85 and 0.82, respectively, in cyclohexane with low polarity for DEBSO1 and DEBSO2, respectively, while low values of 0.18 and 0.21 in DMSO with high polarity, respectively. And showed strong fluorescence only in nonpolar solvents. It was found that the photophysical properties of DEBSO1 and DEBSO2 as described above retain the photophysical properties of BTO. Therefore, when DEBSO1 and DEBSO2 are introduced into cells, it is suggested that a high S / N ratio can be obtained by causing strong fluorescence to be emitted only by those incorporated into lipid droplets with low polarity, like BTO. .

  With respect to DEBSO1 of Example 1, DEBSO2 of Example 2, BTO of Comparative Example 1, and BODIPY 493/503 of Comparative Example 2, the light stability in toluene, which is close in polarity to the environment in the lipid droplet, was evaluated. The change in absorbance every 30 minutes was observed when the xenon lamp (> 325 nm) was continued to be irradiated under the same conditions to a toluene solution of the same concentration of DEBSO1, DEBSO2, BTO and BODIPY 493/503. The results are shown in FIG. As a result, DEBSO1 and DEBSO2 are far superior not only to BODIPY 493/503 but also to BTO, which is highly light resistant, and even when the laser beam is continued for 180 minutes, the absorbance is attenuated by only about 2%. I did not. From these results, it was found that DEBSO1 and DEBSO2 acquired super light resistance by introducing a crosslinked structure in BTO.

  From the measurement results of photophysical properties and photostability of the above-described DEBSO1 and DEBSO2, it can be said that DEBSO1 and DEBSO2 are compounds having more excellent light resistance while retaining the characteristics of BTO. Therefore, it has been suggested that DEBSO1 and DEBSO2 can both be a super-light-resistant lipid droplet staining fluorescent probe having both high sensitivity and super-light resistance.

[Cell experiment]
Cell culture
DMEM (Dulbecco modified) containing 3T3-L1 preadipocytes (RIKEN Cell Bank), 10% FBS (fetal bovine serum; Biosera), 1% penicillin-streptomycin-amphotericin B suspension (Wako Pure Chemical Industries, Ltd.) Eagle's medium; Wako Pure Chemical Industries, Ltd. was used as a medium and cultured in an incubator at 37 ° C., 5% CO ₂ , 95% air.

Cell staining (for determination of dye sensitivity and light stability in fat droplets)
3T3-L1 preadipocytes are seeded in a glass bottom dish, and 2 days after the day when 100% confluence (defined as day 0), the medium is changed to differentiation medium to convert 3T3-L1 into adipocytes It induced differentiation. In the differentiation medium, insulin was added at a final concentration of 10 μg / mL, dexamethasone at a final concentration of 2.5 μM, and 3-isobutyl-1-methylxanthine (IBMX) at a final concentration of 0.5 mM in the culture medium for normal culture. I used one. The insulin, dexamethasone and IBMX used were those contained in Takara Bio Inc.'s Adipoinducer Reagent (for animal cell). On the fourth day, the medium was switched to maintenance medium. As the maintenance medium, one in which insulin was added to a final culture medium to a final concentration of 10 μg / mL was used. The maintenance medium is removed on the 9th day, and a mixture of DMEM and 1 nM or 100 nM DEBSO1, DEBSO2 or BTO (containing 0.1% DMSO) is switched as a staining medium, and maintained at 37 ° C., 5% CO _{2 for} 12 hours. The culture was performed under the condition of 95% air. It was washed three times with DMEM (hereinafter sometimes referred to as “DMEM colorless”; not containing L-glucose and phenol red; gibco) and filled with 2 mL of DMEM colorless.

Cell staining (at the time of observation of fat drop contraction)
The same conditions as described above were used before washing. Washed 3 times with HBSS (+) (phenol red free; Wako Pure Chemical Industries, Ltd.) containing 2% BSA (albumin, bovine serum origin; fatty acid, IgG and protease free; Wako Pure Chemical Industries, Ltd. free) did. 10 μM forskolin (Wako Pure Chemical Industries, Ltd.), 0.1 mM IBMX (Wako Pure Chemical Industries, Ltd.), 2% BSA, DMEM colorless (0.1% DMSO) containing the same concentration of dye as for staining, just before imaging I switched.

Fluorescence imaging
When excited at a wavelength of 405 nm using an FV 10i confocal laser scanning microscope (Olympus Corp.), spectral information of 470 to 570 nm was obtained. When excited at a wavelength of 473 nm, spectral information of 490 to 590 nm was obtained.

Test Example 2 Adipocyte Staining Test From the results of Test Example 1, since both DEBSO1 of Example 1 and DEBSO2 of Example 2 showed utility in fat drop staining, a fat cell staining test was actually performed. Adipocytes differentiated from 3T3-L1 were stained with 1 μM each of DEBSO1, DEBSO2 and BTO for 12 hours, and observed using a confocal laser microscope. The results are shown in FIG. As a result, the S / N ratio was 13 times for DEBSO1, 12 times for DEBSO2, and 12 times for BTO.

  Next, in order to compare the sensitivities of DEBSO1, DEBSO2 and BTO in fat drop staining, when adipocytes are similarly stained for 12 hours when the concentration is 100 nM, respectively, the S / N ratio is 12 times for DEBSO1, DEBSO2 was 18-fold, BTO 4-fold, and DEBSO1 and DEBSO2 could be stained at a signal-to-noise ratio more than twice that of BTO. The results are shown in FIG. Therefore, DEBSO1 and DEBSO2 are superior to BTO in that low concentration and high sensitivity fat drop staining can be performed.

  Although imaging was performed at an excitation wavelength of 405 nm in FIGS. 3 and 4, the sensitivity may be different depending on the excitation wavelength, so the excitation wavelength is changed to 473 nm under the same conditions as in FIG. An imaging image was acquired. The results are shown in FIG. The S / N ratio was 11 times for DEBSO1, 14 times for DEBSO2, and 11 times for BTO, and it was found that the difference in excitation wavelength hardly affected the sensitivity of these dyes.

  According to the results in FIGS. 3 to 5, it is possible to dye fat droplets with the same degree of sensitivity regardless of the excitation wavelength if the concentration is 1 μM for all of DEBSO1, DEBSO2 and BTO, regardless of the excitation wavelength When 100 nM, BTO was desensitized, whereas DEBSO1 and DEBSO2 could be stained without the influence of concentration reduction. For this reason, it can be said that DEBSO1 and DEBSO2 are superior to BTO with respect to the sensitivity of fat drop staining.

Further, from the results shown in FIG. 2, since DEBSO1 of Example 1 and DEBSO2 of Example 2 exhibited higher photostability compared with BTO of Comparative Example 1 in toluene exhibiting a polarity similar to that in the lipid droplet, It is expected to show high light fastness even in drops. Therefore, after staining the adipocytes with each dye, the change of the fluorescence intensity in the fat drop when the confocal laser was continuously irradiated was followed. The conditions were measured at a concentration of 1 μM, λ _ex = 473 nm.

  The results are shown in FIGS. When stained with BTO, the fluorescence intensity was attenuated, but when stained with DEBSO1 and DEBSO2, the fluorescence intensity was almost the same as before irradiation even when the confocal laser irradiation was continued for 50 minutes . The results show that it is possible to continue observing the adipocytes stained with DEBSO1 and DEBSO2 for a long time by imaging using a 473 nm confocal laser.

  Finally, experiments were conducted to follow the process of lipolysis by forskolin using DEBSO1 of Example 1. Lipolysis has a multistage process as shown in FIG. First, by binding the hormone to the beta adrenergic receptor present on the surface of fat cells, adenylate cyclase is activated to increase the concentration of intracellular cyclic adenosine monophosphate (cAMP). Protein kinase (PKA) has the action of phosphorylating proteins in the active state, but since cAMP has the action of activating PKA, the PKA in the active state increases with the increase of cAMP concentration. This active state of PKA phosphorylates perilipin on the surface of lipid droplets and hormone-sensitive lipase (HSL) present in the cytoplasm. Phosphorylated perilipine promotes the transfer of HSL to the surface of the lipid droplet, and binding of HSL to the lipid droplet initiates the decomposition of triacylglycerol in the lipid droplet into fatty acid and diacylglycerol. Since forskolin acts to increase the activity of adenylate cyclase and to elevate the cAMP concentration, administration of forskolin to fat cells promotes lipolysis and shrinks lipid droplets. However, there are also reports that the action of forskolin is inhibited by insulin. In the above cell staining, since insulin was added to the medium for differentiation-inducing 3T3-L1 and for the maintenance medium for adipocytes after differentiation, even after washing and forskolin administration, a slight amount of remaining insulin was used. There was a risk that the function of forskolin would be inhibited. So I decided to add not only forskolin but also IBMX to follow the fat drop contraction. Since IBMX is an inhibitor of phosphodiesterase (PDE), an enzyme that opens cAMP, the addition of IBMX leads to an increase in cAMP concentration. In addition, the fatty acid produced by the decomposition of triacylglycerol may be recycled to the formation of triacyl glyceride, and as a result, the lipid droplet may not shrink. Therefore, BSA, which can trap lipids by hydrophobic interaction, was added to the cell culture medium to prevent the produced fatty acids from being absorbed extracellularly and recycled to the lipid droplet synthesis pathway. From such a concept, fat cells stained with 1 μM DEBSO1 for 12 hours were stimulated with a medium of 10 μM forskolin, 0.1 mM IBMX, 2% BSA, and 1 μM DEBSO1, and the appearance of lipid droplets was observed. The results are shown in FIGS.

  With regard to the area encircled by blue circles in FIG. 9 (the encircled area on the upper side), it was also confirmed by following the change in luminance per area in FIG. Was able to catch On the other hand, for the red circled area in FIG. 9 (lower circled area), the blue circled area (upper circled area) until 1.5 hours after adding forskolin and IBM X The luminance per area decreased in the same manner as in the case of), but then the luminance per area increased. From FIG. The fat droplets that originally existed shrank as time passed, but it can be seen from the reference after 4 hours that the minute bright spots that did not exist at the start of observation increased. This bright spot is considered to be a minute fat droplet. In the present invention, BSA was added to trap fatty acids produced by lipolysis, but in some fat cells, BSA did not function as expected, and fatty acids were recycled to generate new lipid droplets. It is considered a thing. Unexpectedly, DEBSO1 of Example 1 can not only be used to capture the change of the already matured lipid droplet but also capture the generation of a minute lipid droplet, because the new lipid droplet was observed to be generated. It turned out that it can do. This result suggests that DEBSO1 is useful for tracing the process of fat drop formation and degradation through maturation.

Conclusion As described above, when the photophysical properties of DEBSO1 and DEBSO2 were measured, the UV-visible absorption spectra did not substantially change regardless of the polarity of the solvent, but with regard to the fluorescence spectra, high solvent dependence was observed, and in low polar solvents It was found that the property of BTO is maintained, such as strong fluorescence only in Furthermore, when the light stability under xenon lamp irradiation in toluene was measured, it turned out that it has much higher light stability compared with BTO.

  It was found that when fat cells were actually stained using DEBSO1 and DEBSO2, fat droplets could be stained selectively and at high sensitivity. In particular, it was found that fat drops could be sufficiently stained even at a very low concentration of 100 nM, and non-invasive imaging could be achieved. In addition, when the light stability of DEBSO1 and DEBSO2 in the lipid droplet under confocal laser irradiation was examined, it was found that DEBSO1 and DEBSO2 are much more stable than BTO when using 473 nm laser light all right. We also succeeded in tracking the fat drop decomposition by forskolin using DEBSO1. From these results, it was found that DEBSO1 and DEBSO2 are useful dye molecules for selective and sensitive observation of fat droplets for a long time.

Claims (10)

General formula (1-1):
Wherein, Ar ¹ and Ar ² are the same or different and represent a substituted or non-location 換芳 incense ring. R ^1-1 and R ^2-1 are the same or different and represent a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or non-location 換A aryl group. However, the case where R ^1-1 and R ^2-1 are both hydrogen atoms is excluded. R ^1-1 and R ^2-1 may combine to form a ring with the adjacent nitrogen atom. R ^1-1 and / or R ^2-1 may combine with Ar ² to form a ring with an adjacent nitrogen atom. Y ¹ and Y ² one is -SO ₂ - is a group represented by the other is a sulfur atom or -SO ₂ - is a group represented by. ]
The fused-ring thiophene compound or its solvate represented by these. The condensed-ring thiophene compound or a solvate thereof according to claim 1, wherein ^one of Y ¹ and Y ² is a group represented by -SO _2- , and the other is a sulfur atom. The fused thiophene compound or the solvent thereof according to claim 1 or 2, wherein Ar ¹ and Ar ² are substituted or unsubstituted benzene ring, naphthalene ring, anthracene ring, phenanthrene ring, fluorene ring, pyrene ring or triphenylene ring Hydrate. The thiophene compound or a solvate thereof according to any one of claims 1 to 3, wherein each of R ^1-1 and R ^2-1 is a substituted or unsubstituted aryl group. An oil droplet stain comprising the fused thiophene compound according to any one of claims 1 to 4 or a solvate thereof. General formula (1):
Wherein, Ar ¹ and Ar ² are the same or different and represent a substituted or non-location 換芳 incense ring. R 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, a substituted or non-location 換A aryl group. R ¹ and R ² may combine to form a ring with the adjacent nitrogen atom. R ¹ and / or R ² may combine with Ar ² to form a ring with the adjacent nitrogen atom. Y ¹ and Y ² one is -SO ₂ - is a group represented by the other is a sulfur atom or -SO ₂ - is a group represented by. ]
An oil droplet stain comprising the condensed thiophene compound represented by or a solvate thereof. The oil droplet stain according to claim 5 or 6, wherein the fused thiophene compound or a solvate thereof is dissolved in a nonpolar solvent. The oil drop stain according to any one of claims 5 to 7, which is a fat stain. The oil droplet stain according to any one of claims 5 to 8, which is an intracellular fat globule stain. A method of bioimaging a fat, using the oil droplet stain according to any one of claims 5 to 9.