JP2018145246A - Sila dithieno rhodamine compound and fluorescent dye using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent dye which is easy to chemically modify for adding various functions, has a maximum fluorescence wavelength in a near-infrared wavelength region or a region closer thereto (wavelength of 720 nm or more) without expanding a π conjugation system, and is high in a fluorescence quantum yield.SOLUTION: There is provided a sila dithieno rhodamine compound represented by following 1a, or a hydrate or a solvation thereof.SELECTED DRAWING: None

Description

  The present invention relates to a siladithienorhodamine compound and a fluorescent dye using the same.

  In the near-infrared wavelength region, organic molecules (pigments) that have absorption and fluorescence maximum wavelengths in this region are attracting attention in deep observation of biological tissues from the viewpoint of high tissue permeability and suppression of autofluorescence. . Organic molecules that exhibit fluorescence in the near-infrared wavelength region have high importance beyond the framework of applications such as bioimaging fluorescent probes and fluorescent materials for organic EL devices. In many cases, a fluorescent probe for bioimaging is required to be water-soluble. In addition, it can be labeled on specific small molecules with biological activity, proteins such as antibodies, and can be given functions according to the purpose, such as the ability to detect specific substances and environments and emit fluorescence. This is important for application to fluorescent probes, and near-infrared fluorescent dyes that satisfy these requirements are demanded. Various types of fluorescent dyes have been known so far, but cyanine dyes, BODIPY dyes, etc. that are widely used at present are difficult to modify the structure to give various functions, and have high fat solubility. These fluorescent dyes are not effective.

  On the other hand, compounds having a xanthene skeleton typified by rhodamine compounds, fluorescein compounds, etc. are highly water-soluble and excellent in terms of imparting functions according to the purpose, and are widely used as representatives of fluorescent dyes for fluorescent probes. ing. However, the problem is that the absorption maximum wavelength and the fluorescence maximum wavelength are in the visible light region. Although it is possible to lengthen the absorption maximum wavelength and the fluorescence maximum wavelength by extending the π-conjugated system of these compounds having a xanthene skeleton, the water solubility decreases.

  In recent years, analogs (silicon rhodamine compounds) in which the oxygen atom in the skeleton ring of the xanthene skeleton is replaced with a silicon atom have been reported, and the absorption and fluorescence maximum wavelengths shift long wavelengths while maintaining high water solubility (absorption). It has been reported that the maximum wavelength is 646 nm and the maximum fluorescence wavelength is 660 nm (for example, see Non-Patent Document 1). Although this dye plays a central role in fluorescence imaging that requires long-wavelength emission, further increase in the wavelength of absorption maximum wavelength and fluorescence maximum wavelength is required. Therefore, as described above, it is also known to extend the π-conjugated system of the silicon rhodamine compound. For example, the absorption maximum wavelength can be set to 721 nm, and the fluorescence maximum wavelength can be set to 740 nm (for example, see Non-Patent Document 2). As the water solubility decreases, the fluorescence quantum yield is only 0.05, and the fluorescence intensity is not sufficient.

ACS Chem. Biol., 2011, 6, 600. J. Am. Chem. Soc. 2012, 134, 5029.

  As described above, the near-infrared fluorescent dye having a fluorescence maximum wavelength in the near-infrared wavelength region (wavelength of 720 nm or more) has a fluorescence quantum yield of less than 0.10. In consideration of water solubility, it is preferable to lengthen the absorption maximum wavelength and the fluorescence maximum wavelength without extending the π-conjugated system of the xanthene skeleton.

  For this reason, the present invention is easy to modify the structure to provide various functions, and has a fluorescence maximum wavelength in the near infrared wavelength region or a region close to it (wavelength of 720 nm or more) without extending the π-conjugated system. However, an object is to provide a fluorescent dye having a high fluorescence quantum yield.

  As a result of diligent investigations in view of the above object, the present inventors have found that siladithienorhodamine compounds in which the benzene ring of a silicon rhodamine compound is substituted with a thiophene ring can be easily modified to give various functions. The present inventors have found that the fluorescence quantum yield is high while having the fluorescence maximum wavelength in the near-infrared wavelength region (wavelength of 720 nm or more) without extending the π-conjugated system. The present invention has been completed by further research based on such knowledge. That is, the present invention includes the following configurations.

  Item 1. General formula (1):

[In the formula, R ¹ represents the general formula (2A) or (2B):

(In the formula, R ¹⁰ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted (hetero) aryl group. Ar represents substituted or unsubstituted. (Shows a (hetero) aromatic ring.)
The group represented by these is shown. R ² and R ³ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted (hetero) aryl group. R ⁴ and R ⁵ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonyl group, or a substituted or unsubstituted alkyl group. R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group. R ⁶ and R ⁷ , and R ⁸ and R ⁹ may combine to form a ring with the adjacent nitrogen atom. X represents an anion. ]
Or a hydrate or solvate thereof.

Item 2. Item 2. The siladithienorhodamine compound according to item 1, or a hydrate or solvate thereof, wherein in the general formula (1), R ¹ is a group represented by the general formula (2A).

Item 3. The siladithienorhodamine compound according to claim 2, or a hydrate or solvate thereof, wherein in the general formula (1), R ¹⁰ is a substituted or unsubstituted aryl group.

Item 4. Item 4. The siladithienorhodamine compound according to any one of Items 1 to 3, or a hydrate or solvate thereof, wherein in General Formula (1), R ² and R ³ are both substituted or unsubstituted alkyl groups. .

Item 5. Item ^{5. The} siladithienorhodamine compound according to any one of Items 1 to 4, or a hydrate or solvate thereof, wherein in the general formula (1), R ⁴ and R ⁵ are both hydrogen atoms.

Item 6. In the general formula (1), R ⁶ , R ⁷ , R ⁸ and R ⁹ are all substituted or unsubstituted alkyl groups, or R ⁶ and R ⁷ , and R ⁸ and R ⁹ are bonded to each other adjacent nitrogen. Item 6. The siladithienorhodamine compound according to any one of Items 1 to 5, which forms a ring with atoms, or a hydrate or solvate thereof.

  Item 7. A siladithienorhodamine compound or a hydrate or solvate thereof according to any one of Items 1 to 6, which has a fluorescence maximum wavelength at 720 to 850 nm and a fluorescence quantum yield of 0.10 or more. .

  Item 8. Item 8. A fluorescent dye containing the siladithienorhodamine compound according to any one of Items 1 to 7, or a hydrate or solvate thereof.

  The siladithienorhodamine compound of the present invention has an absorption maximum and a fluorescence maximum in the near-infrared wavelength region or a region near it by replacing the benzene ring portion of the silicon rhodamine compound with a thiophene skeleton, and has a high fluorescence quantum yield. Have a rate. Moreover, it is easy to modify the structure to give various functions according to the required characteristics.

2 shows an ultraviolet-visible near-infrared absorption spectrum and a fluorescence spectrum of Compound 1a of Example 1 and Compound 1b of Example 2. (A) Fluorescence microscope image, (b) Bright field image, and (c) Fluorescence microscope image and bright field image of HeLa cells stained with compound 1b of Example 2. It is the fluorescence-microscope image of the HeLa cell co-stained using MitoTracker (trademark) Green FM and the compound 1b. (a) Fluorescence microscope image of compound 1b. (b) Fluorescent microscope image of MitoTracker (registered trademark) Green FM. (c) Superposition of both.

  In the present specification, the “(hetero) aryl group” means an aryl group or a heteroaryl group. “(Hetero) aromatic ring” means an aromatic ring or a heteroaromatic ring. “Contains” is a concept encompassing any of “comprise”, “consist essentially of”, and “consist of”.

  In the present specification, examples of the amide group include a dimethylamide group, a diethylamide group, and an acetamide group. Examples of the ester group include a methoxycarbonyl group and an ethoxycarbonyl group.

1． Siladithienorhodamine Compound The siladithienorhodamine compound of the present invention has the general formula (1):

[In the formula, R ¹ represents the general formula (2A) or (2B):

(In the formula, R ¹⁰ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted (hetero) aryl group. Ar represents substituted or unsubstituted. (Indicates a (hetero) aromatic ring.)
The group represented by these is shown. R ² and R ³ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted (hetero) aryl group. R ⁴ and R ⁵ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonyl group, or a substituted or unsubstituted alkyl group. R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group. R ⁶ and R ⁷ , and R ⁸ and R ⁹ may combine to form a ring with the adjacent nitrogen atom. X represents an anion. ]
It is a compound represented by these. The siladithienorhodamine compound represented by the general formula (1) is a novel compound not described in any literature.

In the general formula (1), R ¹ represents the general formula (2A) or (2B):

[Wherein, R ¹⁰ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted (hetero) aryl group. Ar represents a substituted or unsubstituted (hetero) aromatic ring. ]
It is group represented by these.

As the alkyl group represented by R ¹⁰ , either a linear alkyl group or a branched alkyl group can be employed. As the linear alkyl group, a linear alkyl group having 1 to 6 (particularly 1 to 4) carbon atoms is preferable. For example, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, n -A hexyl group etc. are mentioned. As the branched alkyl group, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5) is preferable. For example, isopropyl group, isobutyl group, tert-butyl group, sec-butyl group, neopentyl group, isohexyl group, Examples include 3-methylpentyl group.

Examples of the substituent which the alkyl group represented by R ¹⁰ may have include, for example, a hydroxyl group, a halogen atom described below, an alkenyl group described below, an alkynyl group described below, an aryl group described below, a heteroaryl group described below, and a carboxy group. Group, amide group, ester group and the like. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

The alkenyl group represented by R ¹⁰ is preferably an alkenyl group having 2 to 6 carbon atoms (particularly 2 to 4), and examples thereof include a vinyl group, an allyl group, a 1-butenyl group, and a 2-butenyl group.

Examples of the substituent that the alkenyl group represented by R ¹⁰ may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, an alkynyl group described later, an aryl group described later, and a heteroaryl described later. Group, carboxy group, amide group, ester group and the like. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

The alkynyl group represented by R ¹⁰ is preferably an alkynyl group having 2 to 6 (particularly 2 to 4) carbon atoms, such as ethynyl group, 1-propynyl group, propargyl group, 1-butynyl group, 2-butynyl group and the like. Is mentioned.

Examples of the substituent that the alkynyl group represented by R ¹⁰ may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, the alkynyl group, an aryl group described later, and a heteroaryl group described later. , Carboxy group, amide group, ester group and the like. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

As the aryl group represented by R ¹⁰ , any of a monocyclic aryl (phenyl group) group and a polycyclic aryl group can be employed. For example, a phenyl group, an oligoaryl group (naphthyl group, anthryl group, etc.), phenanthrenyl Group, fluorenyl group, pyrenyl group, triphenylenyl group, biphenyl group and the like.

Examples of the substituent that the aryl group represented by R ¹⁰ may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, the alkynyl group, the aryl group, a heteroaryl group described later, A carboxy group, an amide group, an ester group, etc. are mentioned. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

Examples of the heteroaryl group represented by R ¹⁰ include a thienyl group, a furyl group, a pyridyl group, and the like.

Examples of the substituent that the heteroaryl group represented by R ¹⁰ may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the heteroaryl group, A carboxy group, an amide group, an ester group, etc. are mentioned. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

Among these, as R ¹⁰ , a substituted or unsubstituted aryl group is preferable from the viewpoint of having a higher fluorescence quantum yield and facilitating recognition during bioimaging, and a substituted or unsubstituted monocyclic aryl group (substituted or substituted). Unsubstituted phenyl group), monocyclic aryl groups having a substituent at the ortho position (phenyl groups having a substituent at the ortho position) are more preferable, and o-tolyl group, 2,6-dimethoxyphenyl group and the like are particularly preferable preferable. In addition, from a viewpoint of the stability with respect to water in a neutral area | region, it is preferable to set it as a bulkier group.

  As the aromatic ring represented by Ar, any of a monocyclic aromatic hydrocarbon ring (benzene ring) and a polycyclic aromatic hydrocarbon ring can be adopted. For example, a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring , Pyrene ring, triphenylene ring and the like.

  Examples of the substituent that the aromatic ring represented by Ar may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the heteroaryl group, and a carboxy group. Amide group, ester group and the like. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

  Examples of the heteroaromatic ring represented by Ar include a thiophene ring, a furan ring, and a pyridine ring.

  Examples of the substituent that the heteroaromatic ring represented by Ar may have include, for example, a hydroxyl group, a halogen atom described later, the alkyl group, the alkenyl group, the alkynyl group, the aryl group, the heteroaryl group, a carboxy group, and the like. Group, amide group, ester group and the like. The number in the case of having a substituent is not particularly limited and is preferably 1 to 6, more preferably 1 to 3.

  Among them, Ar is preferably a substituted or unsubstituted aromatic hydrocarbon ring from the viewpoint of having a higher fluorescence quantum yield and facilitating recognition during bioimaging, and is preferably a substituted or unsubstituted monocyclic aromatic carbonization. A hydrogen ring (substituted or unsubstituted benzene ring) is more preferable.

Among these, R ¹ is preferably a group represented by the general formula (2A) from the viewpoint of easier synthesis. In addition, when R ¹ is a group represented by the general formula (2B), it is expected that a function of switching fluorescence properties (on / off) by ring opening and ring closing will appear.

As the alkyl group, aryl group and heteroaryl group represented by R ² and R ³ , those described above can be adopted. The kind and number of substituents are the same.

Depending on the type of R ² and R ³ , the electronic structure can be finely adjusted and the physical properties (solubility, cell permeability, etc.) can be adjusted. In particular, from the viewpoint of synthesis, a substituted or unsubstituted alkyl group is preferable, a substituted or unsubstituted methyl group is more preferable, and a methyl group is more preferable.

In the general formula (1), examples of the halogen atom represented by R ⁴ and R ⁵ include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom is preferable from the viewpoint of further improving light resistance.

In the general formula (1), the sulfonyl group represented by R ⁴ and R ⁵ includes not only a sulfo group (a group represented by —SO ₃ H), but also a methanesulfonyl group, an ethanesulfonyl group, a phenylsulfonyl group, p -Toluenesulfonyl group, trifluoromethanesulfonyl group, etc. are also mentioned.

In the general formula (1), as the alkyl group represented by R ⁴ and R ⁵ , those described above can be adopted. The kind and number of substituents are the same.

Among these, as R ⁴ and R ⁵ , a hydrogen atom is preferable from the viewpoint of easier synthesis, a halogen atom is preferable and a fluorine atom is preferable from the viewpoint of light resistance.

In the general formula (1), as the alkyl group, alkenyl group and aryl group represented by R ⁶ , R ⁷ , R ⁸ and R ⁹ , those described above can be adopted. The kind and number of substituents are the same.

R ⁶ and R ⁷ , R ⁸ and R ⁹ may be bonded to form a ring with the adjacent nitrogen atom. In this case, as a ring composed of NR ⁶ R ⁷ and NR ⁸ R ⁹ ,

Etc.

Among these, R ⁶ , R ⁷ , R ⁸ and R ⁹ are easy to synthesize, easy to cationize, make the absorption maximum wavelength and fluorescence maximum wavelength longer, and improve the fluorescence quantum yield. From the viewpoint of easy formation, it is preferably a substituted or unsubstituted alkyl group, or R ⁶ and R ⁷ , or R ⁸ and R ⁹ are bonded to form a ring with an adjacent nitrogen atom. In addition, when R ⁶ and R ⁷ and R ⁸ and R ⁹ are alkyl groups, the absorption maximum wavelength and the fluorescence maximum wavelength are higher than when R ⁶ and R ⁷ and R ⁸ and R ⁹ are hydrogen atoms. Further, the wavelength can be increased. Further, in the silicon rhodamine compound, it is known that the fluorescence quantum yield is improved when the amino groups at both ends are cyclic amino groups (Nat. Methods, 2015, 12, 244.). However, from the viewpoint of particularly improving the fluorescence quantum yield, it is more preferable that R ⁶ and R ⁷ , R ⁸ and R ⁹ are bonded to form a ring with the adjacent nitrogen atom.

  In the general formula (1), examples of the anion represented by X include halogen ions (fluoride ions, chloride ions, bromide ions, iodide ions, etc.), cyanide ions, acetate ions, trifluoroacetate ions, and the like. Can be mentioned.

  The siladithienorhodamine compound of the present invention that satisfies such conditions is easier to synthesize, has higher light resistance and water solubility, has a higher fluorescence quantum yield, has a maximum absorption wavelength and a maximum fluorescence. From the viewpoint of making the wavelength higher and making it easier to recognize during bioimaging, the general formula (1A):

[Wherein R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and X are the same as defined above. ]
A siladithienorhodamine compound represented by the formula is preferred.

  As such a siladithienorhodamine compound of the present invention, for example,

Etc.

  The siladithienorhodamine compound of the present invention can have an absorption maximum wavelength of preferably 650 to 800 nm, more preferably 680 to 750 nm, and a fluorescence maximum wavelength of 720 to 850 nm, more preferably 730 to 800 nm. In addition, the fluorescence quantum yield can be 0.10 or more (particularly 0.10 to 0.20, and further 0.12 to 0.18). For this reason, with conventional fluorescent dyes, when the fluorescence maximum wavelength is set to 720 nm or more, the fluorescence intensity in the long wavelength region can be dramatically improved compared to the case where only the fluorescent quantum yield is less than 0.10. it can. For this reason, it is useful for uses such as fluorescent probes for bioimaging and fluorescent materials for organic EL elements.

  The siladithienorhodamine compound of the present invention may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention.

2． Method for Producing Siladithienorhodamine Compound The siladithienorhodamine compound of the present invention is not particularly limited and can be synthesized by various methods. For example, the siladithienorhodamine compound (1A) of the present invention in which R ¹ is a group represented by the general formula (2A) is represented by the following reaction formula 1:

[Wherein R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are the same as defined above. X ¹ and X ² are the same or different and each represents a halogen atom. ]
Can be synthesized according to

In the reaction formula 1, examples of the halogen atom represented by X ¹ and X ² include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. From the viewpoint of yield, a chlorine atom, a bromine atom, an iodine atom, and the like A bromine atom is preferable.

(2-1) Cyclization reaction (compound (2) → compound (3))
In this step, for example, the compound (2) in the above reaction formula 1 is reacted with the organolithium compound in an organic solvent, and then the desired cyclization reaction is advanced by adding the organosilicon compound. Can do.

  The compound (2) in the above reaction formula 1 can be a known or commercially available compound, or can be synthesized. In the case of synthesis, it can be synthesized according to a report (Angew. Chem. Int. Ed., 2013, 52, 8990.).

  The organolithium compound is not particularly limited, and known compounds can be used. For example, ethyllithium, n-propyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, n-pentyllithium And alkyl lithium such as n-hexyl lithium; cycloalkyl lithium such as cyclohexyl lithium; aryl lithium such as phenyl lithium; Among these, in this step, alkyl lithium is preferable and n-butyl lithium is more preferable from the viewpoint of yield and ease of synthesis. These organolithium compounds can be used alone or in combination of two or more. The amount of the organolithium compound used is not particularly limited, and is preferably 1.0 to 10.0 mol, and more preferably 1.5 to 5.0 mol, per 1 mol of compound (2), from the viewpoints of yield, ease of synthesis, and the like.

The organosilicon compound is not particularly limited as long as the compound (3) can be obtained in this step, and a known compound can be adopted. However, since a group represented by —SiR ² R ³ — is introduced, it is preferable to use a dihalogenated silicon compound having R ² and R ^3. For example, dichlorosilane, dibromosilane, dichlorodimethylsilane, dibromo Examples include dimethylsilane, dichlorodiphenylsilane, and dibromodiphenylsilane. These organosilicon compounds can be used alone or in combination of two or more. The amount of the organosilicon compound used is not particularly limited, and from the viewpoints of yield, ease of synthesis, etc., 0.2 to 3.0 moles of the organosilicon compound is preferably used relative to 1 mole of the compound (2), and 0.5 to 2.0 Mole is more preferred.

  As the organic solvent that can be used in this step, a known one can be employed. In this step, for example, cyclic ether compounds such as tetrahydrofuran and dioxane are preferable. 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.). The reaction temperature can be any of heating, room temperature and cooling. Usually, the reaction with the organolithium compound is carried out at -150 to 0 ° C (particularly -100 to -50 ° C). It is preferable to carry out the reaction at −50 to 100 ° C. (especially 0 to 50 ° C.). The reaction time is preferably a time for which the reaction proceeds sufficiently, preferably 5 minutes to 36 hours, particularly preferably 10 minutes to 24 hours.

  After completion of the reaction, purification may be performed according to a conventional method as necessary. Moreover, the following process can also be performed without performing a refinement | purification process.

(2-2) Compound (3) → Compound (4)
In this step, compound (4) can be obtained by reacting compound (3) with an organolithium compound, then reacting with an electrophilic halogenating agent, and then adding an acid.

  Examples of the organic lithium compound include alkyllithium such as ethyllithium, n-propyllithium, isopropyllithium, n-butyllithium, sec-butyllithium, tert-butyllithium, pentyllithium, and hexyllithium; and cycloalkyl such as cyclohexyllithium. Lithium; aryl lithium such as phenyl lithium; lithium diethylamide, lithium n-propylamide, lithium di n-propylamide, lithium isopropylamide, lithium diisopropylamide (LDA), lithium n-butylamide, lithium di n-butylamide, lithium bistrimethylsilylamide 1 type, or 2 or more types, such as lithium alkylamides, etc. are mentioned. Among these, in this step, lithium alkylamide is preferable and lithium diisopropylamide is more preferable from the viewpoint of ease of synthesis and yield. The amount of the organic lithium compound used is usually preferably 1 to 10 mol, more preferably 2 to 5 mol, per 1 mol of compound (3).

Examples of the electrophilic halogenating agent include iodine (I ₂ ), bromine (Br ₂ ), iodine monochloride (ICl), N-iodosuccinimide (NIS), N-bromosuccinimide (NBS), 1,2- One such as diiodoethane (ICH ₂ CH ₂ I), 1,2-dibromoethane (BrCH ₂ CH ₂ Br), 1,2-dibromo-1,1,2,2-tetrachloroethane (BrCl ₂ CCCl ₂ Br) Or 2 or more types are mentioned. The amount of the electrophilic halogenating agent used is usually preferably 1 to 10 mol, more preferably 2 to 5 mol, per 1 mol of compound (3).

  Examples of the acid include hydrogen chloride (hydrochloric acid), sulfuric acid, hydrogen peroxide, formic acid, acetic acid, trifluoroacetic acid (TFA), trifluoroacetic anhydride, boron trifluoride diethyl ether complex, and trifluoromethanesulfonic acid. . The amount of the acid used is preferably 0.2 to 3.0 mol, more preferably 0.5 to 1.5 mol with respect to 1 mol of the compound (3), from the viewpoint of ease of synthesis, yield and the like. When the acid is a liquid, an excess amount, for example, a solvent amount can be used.

  The reaction can usually be performed in the presence of a reaction solvent. Examples of the reaction solvent that can be used include diethyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentyl methyl ether (CPME), tert-butyl methyl ether ( TBME), ethers such as anisole; aromatic hydrocarbons such as benzene, toluene, and xylene, and the like. Ethers are preferable and tetrahydrofuran is more preferable from the viewpoint of ease of synthesis and yield. In addition, the above-mentioned acid can also be used as a reaction solvent. These reaction solvents can be used alone or in combination of two or more.

  As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, room temperature and cooling. Usually, the reaction with the organolithium compound is carried out at -150 to 0 ° C (particularly -100 to -50 ° C). The reaction with the agent is preferably performed at -50 to 100 ° C (particularly 0 to 50 ° C). The reaction time is preferably a time for which the reaction proceeds sufficiently, preferably 5 minutes to 36 hours, particularly preferably 10 minutes to 24 hours.

  After completion of the reaction, purification may be performed according to a conventional method as necessary. Moreover, the following process can also be performed without performing a refinement | purification process.

(2-3) Compound (4) → Compound (6)
In this step, compound (4) and general formula (5):

[Wherein, R ⁶ and R ⁷ are the same as defined above. ]
The compound (6) can be obtained by reacting the compound represented by the above in the presence of a palladium catalyst.

  The amount of compound (5) to be used is generally preferably 1.5 to 5 mol, particularly preferably 2 to 3 mol, per 1 mol of compound (4).

Examples of the palladium catalyst include palladium acetate (Pd (OAc) ₂ ), tetrakis (triphenylphosphine) palladium (0) (Pd (PPh ₃ ) ₄ ), palladium trifluoroacetate, palladium chloride, palladium bromide, and iodide. Palladium, tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ), and the like are listed. In this step, tris (dibenzylideneacetone) dipalladium is used from the viewpoint of ease of synthesis, yield, and the like. (0) (Pd ₂ (dba) ₃ ) is preferred. A solvate may be used as the palladium catalyst. Further, the palladium catalyst can be used alone or in combination of two or more. The amount of palladium catalyst used is usually preferably 0.02 to 0.5 mol, more preferably 0.05 to 0.2 mol, per 1 mol of compound (4).

  In this step, a ligand compound can be used as necessary. Examples of ligand compounds that can be used include triphenylphosphine, trimethoxyphosphine, triethylphosphine, triisopropylphosphine, tri (tert-butyl) phosphine, tri (n-butyl) phosphine, triisopropoxyphosphine, and tricyclopentylphosphine. , Tricyclohexylphosphine, 2,2'-bipyridyl, diphenylphosphinomethane, 1,2-bis (diphenylphosphino) ethane, 1,3-bis (diphenylphosphino) propane, 1,5-bis (diphenylphosphino) ) Pentane, 1,5-cyclooctadiene, 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl (BINAP), 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene 1 type or 2 types or more, such as (XantPhos). Among these, 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (XantPhos) is preferable in this step from the viewpoint of yield and ease of synthesis. The amount of the ligand compound used is usually preferably 0.5 to 5.0 mol, more preferably 1.0 to 3.0 mol, per 1 mol of the palladium catalyst.

  In this step, a base can be used as necessary. Examples of the base include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate, potassium carbonate and cesium carbonate; alkali metal phosphates such as sodium phosphate and potassium phosphate; sodium Examples thereof include alkali metal alkoxides such as methoxide, potassium methoxide, sodium tert-butoxide, potassium tert-butoxide, etc. In this step, alkali metal alkoxides are preferable from the viewpoint of yield and ease of synthesis, and sodium tert- Butoxide is preferred. In the case of using a base, the amount used is preferably from 1.0 to 5.0 mol, more preferably from 1.5 to 3.0 mol, based on 1 mol of the compound (4), from the viewpoint of ease of synthesis, yield and the like. The reaction can usually be performed in the presence of a reaction solvent. Examples of the reaction solvent that can be used include diethyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentyl methyl ether (CPME), tert-butyl methyl ether ( TBME), ethers such as anisole; aromatic hydrocarbons such as benzene, toluene and xylene; nitrile solvents such as acetonitrile; amide solvents such as dimethylformamide, etc., from the viewpoint of ease of synthesis, yield, etc. Aromatic hydrocarbons are preferred, and toluene is more preferred. These reaction solvents can be used alone or in combination of two or more.

  As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, room temperature, and cooling, and it is usually preferably 0 to 150 ° C. (particularly 50 to 100 ° C.). The reaction time is preferably a time for which the reaction proceeds sufficiently, preferably 5 minutes to 36 hours, particularly preferably 10 minutes to 24 hours.

  After completion of the reaction, purification may be performed according to a conventional method as necessary. Moreover, the following process can also be performed without performing a refinement | purification process.

(2-4) Compound (6) → Compound (1A)
In this step, the siladithienorhodamine compound (1A) of the present invention can be obtained by reacting the compound (6) with an organometallic nucleophile (Grignard reagent, organolithium compound, etc.) and an acid reagent. At this time, the anion contained in the acid reagent constitutes the anion X of the siladithienorhodamine compound (1A) of the present invention.

As the Grignard reagent, those capable of introducing the desired R ¹⁰ in the dithienophospholine compound (1A) of the present invention are preferred, and R ¹⁰ MgX ³ (R ¹⁰ is the same as described above. X ³ represents a halogen atom. ) Is preferred.

As the halogen atom represented by X ³ , those described above can be adopted. The same applies to preferred embodiments.

  As a Grignard reagent satisfying such conditions, for example,

Etc.

As the organolithium compound, those capable of introducing the desired R ¹⁰ in the siladithienorhodamine compound (1A) of the present invention are preferable, and the organolithium compound represented by R ¹⁰ Li (R ¹⁰ is as defined above) Is preferred.

  As an organic lithium compound that satisfies such conditions, for example,

Etc.

  The amount of the organometallic nucleophile used is preferably 1 to 30 mol (particularly 2 to 15 mol) with respect to 1 mol of compound (6) from the viewpoint of yield and the like.

  Examples of the acid reagent that can be used include hydrogen chloride (hydrochloric acid), sulfuric acid, formic acid, acetic acid, trifluoroacetic acid (TFA), trifluoroacetic anhydride, boron trifluoride diethyl ether complex, trifluoromethanesulfonic acid, and the like. . The amount of the acid reagent used is preferably from 0.2 to 3.0 mol, more preferably from 0.5 to 1.5 mol, based on 1 mol of the compound (6), from the viewpoint of ease of synthesis, yield, and the like. When the acid reagent is a liquid, it may be in an excess amount, for example, a solvent amount.

  The reaction can usually be performed in the presence of a reaction solvent. Examples of usable reaction solvents include diethyl ether, dibutyl ether, diisopropyl ether, tetrahydrofuran (THF), 1,4-dioxane, dimethoxyethane (DME), diglyme, cyclopentyl methyl ether (CPME), tert-butyl methyl ether ( TBME), ethers such as anisole; aromatic hydrocarbons such as benzene, toluene, and xylene, and the like. Ethers are preferable and cyclopentylmethyl ether (CPME) is more preferable from the viewpoint of ease of synthesis and yield. The acid reagent described above can also be used as a reaction solvent. These reaction solvents can be used alone or in combination of two or more.

  As the reaction atmosphere, an inert gas atmosphere (argon gas atmosphere, nitrogen gas atmosphere, etc.) can be usually adopted. The reaction temperature can be any of heating, room temperature and cooling, and it is usually preferable to carry out at -50 to 150 ° C (particularly 0 to 100 ° C). The reaction time is preferably a time for which the reaction proceeds sufficiently, preferably 5 minutes to 36 hours, particularly preferably 10 minutes to 24 hours.

  After completion of the reaction, purification can be carried out according to a conventional method as necessary to obtain the siladithienorhodamine compound (1A) of the present invention.

3． Fluorescent dye The fluorescent dye of the present invention contains the above-mentioned siladithienorhodamine compound of the present invention.

In the fluorescent dye of the present invention, the benzene ring portion of the rhodamine dye is replaced with a thiophene skeleton, and the oxygen atom in the skeleton is replaced with a silicon-containing group, so that the absorption maximum wavelength is preferably 650 to 800 nm, more preferably 680 to 750 nm. It can have a fluorescence maximum wavelength of 720 to 850 nm, more preferably 730 to 800 nm, and the fluorescence quantum yield is 0.10 or more (especially 0.10 to 0.20, more preferably 0.12 to 0.18), while maintaining light resistance. Can also be superior. For this reason, with conventional fluorescent dyes, when the fluorescence maximum wavelength is set to 720 nm or more, the fluorescence intensity in the long wavelength region can be dramatically improved compared to the case where only the fluorescent quantum yield is less than 0.10. it can. Moreover, it is possible to introduce a desired group into R ¹ according to the required characteristics. For this reason, the fluorescent dye of the present invention is particularly useful for uses such as a fluorescent probe for bioimaging and a fluorescent material for organic EL elements. In particular, the siladithienorhodamine compound of the present invention has a high absorption wavelength and a fluorescence maximum wavelength at a high wavelength, but is not high in conventional fluorescent dyes having an absorption maximum wavelength and a fluorescence maximum wavelength in a high wavelength region. Because it can have a fluorescent quantum yield and has excellent light resistance, it is easy to perform bioimaging, the fluorescent dye concentration required for bioimaging can be kept low, and damage to the living body can be greatly reduced. It is possible to observe the deep part for a long time.

When the fluorescent dye of the present invention is used for bioimaging applications, it is preferably dissolved in an organic solvent to form a solution, and the content of the siladithienorhodamine compound of the present invention is 1 × 10 ⁻⁸ to 1 × 10 ⁻⁴ mol / L is preferable, and 1 × 10 ⁻⁷ to 1 × 10 ⁻⁵ mol / L is more preferable. Thus, in the present invention, the content of the siladithienorhodamine compound can be kept low as compared with the conventional fluorescent dye.

  When the fluorescent dye (siladithienorhodamine compound) of the present invention is used as a solution, the organic solvent that can be used is not particularly limited, and any of a polar solvent and a nonpolar solvent can be used.

  Examples of polar solvents include ether compounds (tetrahydrofuran, anisole, 1,4-dioxane, cyclopentyl methyl ether, etc.), alcohols (methanol, ethanol, allyl alcohol, etc.), ester compounds (ethyl acetate, etc.), ketones (acetone, etc.) , Halogenated hydrocarbons (dichloromethane, chloroform), dimethyl sulfoxide, amide solvents (N, N-dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, etc.) .

  Examples of the nonpolar solvent include aliphatic organic solvents such as pentane, hexane, cyclohexane and heptane; aromatic solvents such as benzene, toluene, xylene and mesitylene.

  In the case of a solution, while increasing the absorption maximum wavelength and the fluorescence maximum wavelength, the fluorescence quantum yield is sufficiently high even under physiological conditions, and the light resistance is excellent. About 3 to 12 is preferable. Buffers (Hepes buffer, Tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) are used to adjust the pH of the cell detection agent of the present invention. You can also

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

General operation
¹ H NMR, ¹³ C { ¹ H} NMR and ²⁹ Si { ¹ H} NMR spectra were measured using a nuclear magnetic resonance apparatus JEOL AL-400 spectrometer. Chemical shift values are expressed in ppm, and deuterated chloroform, deuterated benzene, deuterated dichloromethane and deuterated methanol were used as solvents. ^{In 1} H NMR, signals derived from residual protons of the solvent were used as internal standards, and deuterated chloroform was set to δ7.26 ppm, deuterated benzene to δ7.16 ppm, deuterated dichloromethane to δ5.32 ppm, and deuterated methanol to δ3.31 ppm. ^{In 13} C NMR, a heavy solvent was used as an internal standard, deuterated chloroform was set to δ77.16 ppm, deuterated dichloromethane was set to δ5.32 ppm, and deuterated methanol was set to δ3.31 ppm. ^{In 29} Si NMR, Si (CH ₃ ) ₄ (δ0.00 ppm) was used as an external standard. The high resolution mass spectrometry spectrum was measured using Exactive (Thermo Fisher Scientific), and the ESI method was used for the ionization method. The thin-layer chromatography using TLC plate coated with a thickness of 0.25mm to silica gel 60 F ₂₅₄ a (Merck). For column chromatography, Fuji Silysia Chemical Silica gel or Aluminum oxide 90 active basic (Merck) was used. LC-918 (Nippon Analytical Industry) equipped with JAIGEL 1H and 2H was used for preparative recycling GPC. LC-force / R (YMC) equipped with YMC-Actus Triart C18 was used for high performance liquid chromatography (HPLC). Unless otherwise specified, a dehydrated solvent obtained by purifying a commercially available anhydrous solvent (Kanto Chemical) using an organic solvent purifier (Nikko Hansen) was used. 2,2-bis (3-bromo-2-thienyl) -1,3-dioxolane is described in the literature (X. He, JB Lin, WH Kan, T. Baumgartner Angew. Chem. Int. Ed. 2013, 52, 8990. ). Reactions were performed under a nitrogen atmosphere unless otherwise stated.

The UV-visible absorption spectrum was measured using UV-3600 (Shimadzu Corporation). A 1 cm square quartz cell was used for the measurement, and a sample solution prepared to about 10 ⁻⁵ M was used. Fluorolog FL3-11 (Horiba Seisakusho) was used for the measurement of the fluorescence spectrum. Quantaurus-QY C10027 (Hamamatsu Photonics) was used for measurement of absolute fluorescence quantum yield.

[Example]
A siladithienorhodamine compound was synthesized according to the following synthesis method.

[Wherein n-BuLi represents n-butyllithium. Me represents a methyl group. LDA represents lithium diisopropylamide. Pd ₂ (dba) ₃ · CHCl ₃ represents tris (dibenzylideneacetone) dipalladium (0) chloroform adduct. XantPhos represents 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene. NaO ^t Bu represents sodium tert-butoxide. Et represents an ethyl group. TFA indicates trifluoroacetic acid. The same applies hereinafter. ]

Synthesis Example 1: Compound 9

2,2-bis (3-bromo-2-thienyl) -1,3-dioxolane (1.00 g, 2.53 mmol) in diethyl ether / THF mixed solution (3/1, 84.0 mL) was added to n-butyllithium ( n-BuLi; 1.60M, 3.17 mL, 5.06 mmol) was added dropwise at −78 ° C. over 10 minutes. Subsequently, a THF (3.40 mL) solution of dichlorodimethylsilane (Me ₂ SiCl ₂ ; 0.365 mL, 3.03 mmol) was added dropwise over 3 hours, and the temperature was gradually raised to room temperature overnight. After the solvent was distilled off under reduced pressure, the solid was dissolved in diethyl ether (Et ₂ O; 10 mL) and H ₂ O (10 mL). This was extracted three times with diethyl ether (Et ₂ O; 20 mL), and then the organic layer was washed with saturated brine. The obtained organic layer was dehydrated with anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The resulting crude product is separated by column chromatography (PSQ100B, hexane / ethyl acetate = 10/1) (R _f = 0.39), and the resulting fraction is purified using preparative recycling GPC (CHCl ₃ ) 0.434 g of compound 9 was obtained as a white solid (1.47 mmol, 58% yield).
¹ H NMR (CDCl ₃ , 400 MHz): δ 0.46 (s, 6H), 4.35 (s, 4H), 7.15 (d, J = 4.8 Hz, 2H), 7.40 (d, J = 4.8 Hz, 2H). ¹³ C { ¹ H} NMR (CDCl ₃ , 100 MHz): δ -1.15, 66.31, 104.97, 126.15, 130.64, 137.56, 153.76. ²⁹ Si NMR (CDCl ₃ , 79 MHz): δ -22.29. HRMS (ESI) : Calcd for C ₁₃ H ₁₅ O ₂ S ₂ Si: 295.0277 ([M + H] ⁺ ). Obsd. 295.0277 ([M + H] ⁺ ).

Synthesis Example 2: Compound 10

To a THF (1.21 mL) solution of diisopropylamine (0.70 mL, 5.00 mmol), n-butyllithium (n-BuLi; 1.63M, 3.08 mL, 5.02 mmol) was added dropwise at −78 ° C. over 5 minutes. The reaction solution was stirred at -78 ° C for 30 minutes, then brought to 0 ° C for 15 minutes, and then returned to -78 ° C. The lithium diisopropylamide (LDA) thus prepared was added dropwise to a solution of compound 9 (0.500 g, 1.70 mmol) in THF (15 mL) at −78 ° C. over 10 minutes. The mixture was stirred for 2 hours while maintaining at -78 ° C., and then dropwise added to a THF (2.0 mL) solution of C ₂ Cl ₄ Br ₂ (1.22 g, 3.74 mmol) over 5 minutes. After gradually warming to room temperature overnight, 5% aqueous HCl was added and stirred for 5 hours. The reaction solution was extracted 3 times with 10 mL of CH ₂ Cl ₂ , and then the organic layer was washed with saturated brine. The obtained organic layer was dehydrated with anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The obtained crude product was separated by adsorption column chromatography (PSQ100B, hexane / CHCl ₃ = 3/1) (R _f = 0.30) to obtain 0.611 g of compound 10 as a pale yellow solid (1.50 mmol, 88 % yield).
¹ H NMR (CDCl ₃ , 400 MHz): δ 0.47 (s, 6H), 7.24 (s, 2H). ¹³ C { ¹ H} NMR (CDCl ₃ , 100 MHz): δ -1.93, 124.19, 135.09, 147.39 , 150.48, 174.31. ²⁹ Si NMR (CDCl ₃ , 79 MHz): δ -22.14. HRMS (ESI): Calcd for C ₁₁ H ₉ Br ₂ OS ₂ Si: 406.8225 ([M + H] ⁺ ). Obsd. 406.8192 ([M + H] ⁺ ).

Synthesis Example 3: Compound 5a

Tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ; 0.06 equivalent, 0.0735-0.0739 mmol) and 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (Xantphos; 0.1 equivalent) , 0.122-0.124 mmol) was added 1 mL of dehydrated and degassed toluene, and the mixture was stirred for 10 minutes under a nitrogen atmosphere. This suspension was added to a toluene (1 mL) solution of Compound 10 (500 mg, 1.22 mmol), sodium tert-butoxide (NaO ^t Bu; 2.8 equivalents, 3.43-3.44 mmol), and amine (4.0 equivalents, 4.90-5.03 mmol). And washed with 8 mL of toluene. Thereafter, the oil bath was set to 75 ° C. and heating was started. After 18 hours of heating, the temperature was lowered to room temperature, the reaction solution was extracted 3 times with 15 mL of CH ₂ Cl ₂ , and the organic layer was washed with saturated brine. The obtained organic layer was dehydrated with anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The resulting crude product was separated by column chromatography (Aluminium oxide 90 active basic, hexane / CH ₂ Cl ₂ = 2/3, 1% triethylamine added) (R _f = 0.28), and the resulting fraction was fractionated. Compound 5a was obtained as an orange powder of 118 mg (0.301 mmol, 25% yield) by purification using Toray Recycle GPC (with CHCl ₃ , 1% triethylamine added).
¹ H NMR (C ₆ D ₆ , 400 MHz): δ 0.80 (d, J = 7.0 Hz, 12H), 2.85 (q, J = 7.1 Hz, 8H), 5.91 (s, 2H). ¹³ C { ¹ H _{} NMR (CD 2 Cl 2,} 100 MHz):. δ -1.50, 12.42, 47.98, 105.62, 131.71, 147.96, 164.31, 173.91 29 Si NMR (C 6 D 6, 79 MHz):. δ -24.24 HRMS (ESI ): Calcd for C ₁₉ H ₂₉ N ₂ OS ₂ Si: 393.1485 ([M + H] ⁺ ). Obsd. 393.1479 ([M + H] ⁺ ).

Synthesis Example 4: Compound 5b

Tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ; 0.06 equivalent, 0.0735-0.0739 mmol) and 4,5-bis (diphenylphosphino) -9,9-dimethylxanthene (Xantphos; 0.1 equivalent) , 0.122-0.124 mmol) was added 1 mL of dehydrated and degassed toluene, and the mixture was stirred for 10 minutes under a nitrogen atmosphere. This suspension was added to a toluene (1 mL) solution of Compound 10 (500 mg, 1.22 mmol), sodium tert-butoxide (NaO ^t Bu; 2.8 equivalents, 3.43-3.44 mmol), and amine (4.0 equivalents, 4.90-5.03 mmol). And washed with 8 mL of toluene. Thereafter, the oil bath was set to 75 ° C. and heating was started. After 18 hours of heating, the temperature was lowered to room temperature, the reaction solution was extracted 3 times with 15 mL of CH ₂ Cl ₂ , and the organic layer was washed with saturated brine. The obtained organic layer was dehydrated with anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The resulting crude product was separated by column chromatography (Aluminium oxide 90 active basic, hexane / CH ₂ Cl ₂ = 2/3, 1% triethylamine added) (R _f = 0.28), and the resulting fraction was fractionated. Compound 5b was obtained as an orange powder of 225 mg (0.625 mmol, 51% yield) by purification using Toray Recycle GPC (with CHCl ₃ , 1% triethylamine added).
¹ H NMR (CDCl ₃ , 400 MHz): δ 0.37 (s, 6H), 2.45 (quint., J = 7.3 Hz, 4H), 4.03 (t, J = 7.2 Hz, 8H), 5.86 (s, 2H) ¹³ C { ¹ H} NMR (CD ₂ Cl ₂ , 100 MHz): δ -1.62, 17.70, 55.76, 61.99, 103.62, 107.20, 147.87, 181.58. ²⁹ Si NMR (CD ₂ Cl ₂ , 79 MHz): δ -23.11. HRMS (ESI): Calcd for C ₁₇ H ₂₁ N ₂ OS ₂ Si: 361.0859 ([M + H] ⁺ ). Obsd. 361.0857 ([M + H] ⁺ ).

Example 1: Compound 1a

To a solution of 2-bromotoluene (0.10 mL, 0.830 mmol) in cyclopentyl methyl ether (CPME; 5.0 mL), add sec-butyllithium (sec-BuLi; 1.00M, 0.830 mL, 0.830 mmol) at -78 ° C. The solution was added dropwise over a period of minutes to obtain a solution of compound 11 (2-methylphenyllithium). Subsequently, a CPME (5.0 mL) solution of Compound 11 (50.0 mg, 0.127-0.140 mmol) was added dropwise at −78 ° C. over 5 minutes. After that, heating started in an oil bath set at 70 ° C. After 14 hours, heating was terminated, 0.1% trifluoroacetic acid (TFA) aqueous solution was added, and extraction was performed 5 times with ₂ mL of CH ₂ Cl ₂ . The solvent was distilled off from the obtained organic layer under reduced pressure, and the resulting crude product was purified using reverse-phase high performance liquid chromatography (HPLC) to obtain Compound 1a as 6.4 mg of a blue solid. (0.0110mmol, 8.6% yield).
¹ H NMR (CD ₃ OD, 400 MHz): δ 0.59 (s, 3H), 0.60 (s, 3H), 1.26 (t, J = 7.2 Hz, 12H), 2.28 (s, 3H), 3.58 (q, J = 7.2 Hz, 8H), 7.03 (s, 2H), 7.34-7.49 (m, 4H). ¹³ C { ¹ H} NMR (CD ₃ OD, 100 MHz): δ -1.70, -1.45, 12.46, 19.46 , 50.28, 120.25, 127.19, 129.91, 131.37, 131.76, 131.87, 137.16, 139.17, 149.26, 156.65, 173.80. ²⁹ Si NMR (CD ₃ OD, 79 MHz): δ -18.83. HRMS (ESI): Calcd for C ₂₆ H ₃₅ N ₂ S ₂ Si: 467.2005 (M ⁺ ). Obsd. 467.2003 (M ⁺ ).

Example 2: Compound 1b

To a solution of 2-bromotoluene (0.10 mL, 0.830 mmol) in cyclopentyl methyl ether (CPME; 5.0 mL), add sec-butyllithium (sec-BuLi; 1.00M, 0.830 mL, 0.830 mmol) at -78 ° C. The solution was added dropwise over a period of minutes to obtain a solution of compound 11 (2-methylphenyllithium). Subsequently, a CPME (5.0 mL) solution of Compound 11 (50.0 mg, 0.127-0.140 mmol) was added dropwise at −78 ° C. over 5 minutes. After that, heating started in an oil bath set at 70 ° C. After 14 hours, heating was terminated, 0.1% trifluoroacetic acid (TFA) aqueous solution was added, and extraction was performed 5 times with ₂ mL of CH ₂ Cl ₂ . The solvent was distilled off from the obtained organic layer under reduced pressure, and the obtained crude product was purified using reverse phase high performance liquid chromatography (HPLC) to obtain Compound 1b as 19.6 mg of a blue solid. (0.0357mmol, 25% yield).
¹ H NMR (CD ₃ OD, 400 MHz): δ 0.59 (s, 3H), 0.60 (s, 3H), 1.26 (t, J = 7.2 Hz, 12H), 2.28 (s, 3H), 3.58 (q, J = 7.2 Hz, 8H), 7.03 (s, 2H), 7.34-7.49 (m, 4H). ¹³ C { ¹ H} NMR (CD ₃ OD, 100 MHz): δ -1.71, -1.44, 17.81, 19.37 , 56.54, 119.75, 127.15, 129.82, 131.38, 131.73, 133.28, 137.12, 139.39, 149.03, 155.89, 173.17. ²⁹ Si NMR (CD ₃ OD, 79 MHz): δ -18.48.HRMS (ESI): Calcd for C ₂₄ H ₂₇ N ₂ S ₂ Si: 435.1379 (M ⁺ ). Obsd. 435.1391 (M ⁺ ).

[Test Example 1: Optical properties]
Measurement of UV-Vis near-infrared absorption spectrum and fluorescence spectrum of Compound 1a and Compound 1b of Example 2 using a sample solution dissolved in PBS buffer aqueous solution (pH 7.4) at a concentration of 1.26 × 10 ⁻⁵ M Went. As a result, in the UV-Vis near-infrared absorption spectrum of Compound 1a, the absorption maximum wavelength (λ _abs ) is 710 nm and the molar absorption coefficient is ε44800M ⁻¹ cm ⁻¹ , and in the fluorescence spectrum, the fluorescence maximum wavelength (λ _em ) 743 nm, fluorescence The quantum yield was Φ _F 0.13. Compound 1b has an absorption maximum wavelength (λ _abs ) of 706 nm and a molar extinction coefficient of ε32800 M ⁻¹ cm ⁻¹ in the ultraviolet-visible near-infrared absorption spectrum, and has a fluorescence maximum wavelength (λ _em ) of 740 nm and a fluorescence quantum in the fluorescence spectrum. The yield was Φ _F 0.13. The results are shown in FIG. As a result, considering that the conventional silicon rhodamine compound SiR650 has an absorption maximum wavelength (λ _abs ) 646 nm and a fluorescence maximum wavelength (λ _em ) 660 nm (J. Am. Chem. Soc., 2012, 134, 5029.) It can be understood that both the absorption maximum wavelength and the fluorescence maximum wavelength can be increased by using a siladithienorhodamine skeleton. Among the compounds in which the π conjugation of the conventional silicon rhodamine compound is expanded, SiR720 has an absorption maximum wavelength (λ _abs ) of 721 nm and a fluorescence maximum wavelength (λ _em ) of 740 nm, which is inferior to the siladithienorhodamine skeleton of the present invention. Although both the absorption maximum wavelength and the fluorescence maximum wavelength can be lengthened to such an extent that there is no loss, the fluorescence quantum yield Φ _F is 0.05 (J. Am. Chem. Soc., 2012, 134, 5029.) The siladithienorhodamine skeleton can be improved by about 2.5 times the conventional fluorescent dye having a fluorescence maximum wavelength of 720 nm or more.

[Test Example 2: Fluorescence imaging]
HeLa cells (RIKEN Cell Bank, Japan) were used as the cells. HeLa cells are cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic (AA, Sigma) at 37 ° C in a 5% CO ₂ incubator. The day before imaging, it was transferred to a 35 mm glass-bottom dish. In the staining experiment, 500 nM compound 1b was cultured for 2 minutes at 37 ° C. in DMEM medium containing HEPES buffer, and then the medium was replaced. After another 60 minutes, the medium was again replaced with a new medium, and then observed with an excitation wavelength of 635 nm using a confocal laser scanning microscope Olympus FV10i. The results are shown in FIG.

  In addition, as a result of co-staining experiments with MitoTracker (registered trademark) Green FM, the fluorescence sites showed good agreement. Compound 1b was confirmed to localize to mitochondria as well as general rhodamine dyes. This result suggests that it is possible to apply an imaging technique that has been used in conventional rhodamine dyes.

Claims (8)

General formula (1):
[In the formula, R ¹ represents the general formula (2A) or (2B):
(In the formula, R ¹⁰ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, or a substituted or unsubstituted (hetero) aryl group. Ar represents substituted or unsubstituted. (Indicates a (hetero) aromatic ring.)
The group represented by these is shown. R ² and R ³ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted (hetero) aryl group. R ⁴ and R ⁵ are the same or different and each represents a hydrogen atom, a halogen atom, a sulfonyl group, or a substituted or unsubstituted alkyl group. R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, or a substituted or unsubstituted aryl group. R ⁶ and R ⁷ , and R ⁸ and R ⁹ may combine to form a ring with the adjacent nitrogen atom. X represents an anion. ]
Or a hydrate or solvate thereof. The siladithienorhodamine compound according to claim 1, or a hydrate or solvate thereof, wherein, in the general formula (1), R ¹ is a group represented by the general formula (2A). The siladithienorhodamine compound according to claim 2, or a hydrate or solvate thereof, wherein in the general formula (1), R ¹⁰ is a substituted or unsubstituted aryl group. In Formula (1), both R ² and R ³ is a substituted or unsubstituted alkyl group, shea Raj thieno rhodamine compound, or a hydrate or solvate thereof according to any of claims 1 to 3 object. The siladithienorhodamine compound according to any one of claims 1 to 4, or a hydrate or solvate thereof, wherein in the general formula (1), R ⁴ and R ⁵ are both hydrogen atoms. In the general formula (1), R ⁶ , R ⁷ , R ⁸ and R ⁹ are all substituted or unsubstituted alkyl groups, or R ⁶ and R ⁷ , and R ⁸ and R ⁹ are bonded to each other adjacent nitrogen. The siladithienorhodamine compound according to any one of claims 1 to 5, or a hydrate or solvate thereof, which forms a ring together with atoms. The siladithienorhodamine compound according to any one of claims 1 to 6, or a hydrate or solvate thereof, having a fluorescence maximum wavelength at 720 to 850 nm and a fluorescence quantum yield of 0.10 or more. A fluorescent dye comprising the siladithienorhodamine compound according to any one of claims 1 to 7, or a hydrate or solvate thereof.