JP6468879B2 - Compound that can be used in two-photon absorption material, fluorescent dye using the same, and two-photon absorption material - Google Patents

Description

  The present invention relates to a compound that can be used in a two-photon absorbing material, a fluorescent dye using the compound, and a two-photon absorbing material.

  The two-photon excitation laser microscope uses a two-photon excitable molecule (a molecule with two-photon absorption properties) as a fluorescent dye and excites the fluorescent dye with light in the near-infrared region. It can be observed as it is. In response to this, life technologies sells fluorescent dyes corresponding to various excitation wavelengths.

  Two-photon absorption (TPA) is an excitation process that simultaneously absorbs two photons. Unlike one-photon absorption (OPA), the transition probability is proportional to the square of the excitation light intensity. Therefore, when a condensing laser is used as excitation light, a region near the focal point where the light intensity is high can be spatially excited. In addition, since the energy of photons absorbed in the two-photon absorption process is half the transition energy, it is possible to excite molecules that can be excited only by ultraviolet light or visible light with near infrared light. .

  Utilizing this feature, molecules capable of two-photon excitation are expected to be applied to various fields such as three-dimensional fine stereolithography, cancer treatment by photodynamic therapy, and three-dimensional fluorescence imaging. Especially in the field of fluorescence imaging, two-photon excitation is possible because molecular excitation is possible in the near-infrared region (650-1300 nm), which is high in biological tissue, the so-called “biological window” region. Fluorescent molecules are considered to be effective materials for in vivo visualization, and the development of probe molecules having a large two-photon absorption cross section (TPACS) is desired.

  The existence of two-photon absorption was originally proposed by Mayer in 1929, but the development of two-photon excited molecules originated from the recent development of lasers and microscopes. With the development of measurement technology, molecular design guidelines showing a large two-photon absorption cross-section have been established in the active application of two-photon absorption molecules. Among them, the most widely accepted method is the introduction of an electron donating group (D) and an electron accepting group (A) into the end portion of the π electron system. Albota et al. Found that by introducing an amino group that is an electron donating group at both ends of E-stilbene, the two-photon absorption cross section shows a value that is 10 times or more that of the non-substituted product (for example, Non-Patent Document 1).

  Zhang et al. Synthesized a fluorescent probe molecule having a large two-photon absorption cross section by introducing pyridinium groups, which are strong electron accepting groups, at both ends of the carbazole skeleton (for example, Non-Patent Document 2). This molecule selectively stains DNA and succeeds in imaging nuclei in living plant cells.

Science, 1998, 281, 1653-1656. Org. Biomaol. Chem. 2010, 8, 4582-4588.

  Thus, although the design guideline has already been established and applied, the two-photon absorption dye is still improved. The femtosecond titanium sapphire (Ti: sapphire) laser normally used in a two-photon excitation laser microscope has an oscillation band from 650 nm to 1100 nm. Many molecules excited by this wavelength band have a fluorescence maximum wavelength in a shorter wavelength (about 500 to 600 nm) than in the near infrared region. For more efficient imaging, a fluorescent molecule whose absorption maximum wavelength is in the oscillation band of the Ti: sapphire laser and whose fluorescence maximum wavelength is in the near-infrared region is preferred. Design is required.

  Therefore, an object of the present invention is to provide a compound capable of two-photon absorption in which the fluorescence maximum wavelength is shifted by a longer wavelength while maintaining the fluorescence quantum yield and the fluorescence lifetime.

  As a result of intensive studies in view of the above object, the present inventors introduce an electron-accepting group at the terminal while extending the π-conjugated system to the two thiophene rings of the intramolecularly crosslinked dithienylpyrrole derivative. Thus, the present inventors have found that the above problems can be solved and a compound capable of two-photon absorption in which the fluorescence maximum wavelength is shifted by a longer wavelength while maintaining the fluorescence quantum yield can be provided. This compound can have a fluorescence maximum wavelength that has not existed so far, such as a fluorescence maximum wavelength of 700 nm or more, if a solvent to be used is selected. 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):

[Wherein, ^{R 1} and ^{R 2} are the same or different and are each an electron acceptor group; or different ^{R 3} and ^{R 4} the same, respectively a single bond or a divalent linking group; ^{R 5} represents an organic group; ^{L 1} And L ² are the same or different, and each is a π-conjugated group; Z ¹ and Z ² are the same or different, and each is a heterocyclic group; the dotted line connecting the nitrogen atom and the hydrogen atom is an intramolecular hydrogen bond. ]
A compound represented by

  Item 2. General formula (1A):

[Wherein, R ^{1 to} R ⁵ and L ^{1 to} L ² are the same as described above; a dotted line connecting a nitrogen atom and a hydrogen atom is an intramolecular hydrogen bond. ]
Item 6. The compound according to Item 1, which is a compound represented by:

Item 3. In the general formula (1), L ¹ and L ² are the general formulas (2A) to (2G):

Item 3. The compound according to Item 1 or 2, which is a group obtained by combining one or more groups represented by the formula:

Item 4. In the general formula (1), R ¹ and R ² are a boryl group, a cyano group, a nitro group, a sulfo group, an alkylsulfonyl group, an alkoxysulfonyl group, a phosphate group, a formyl group, a carboxy group, or a pyridinium group. The compound according to any one of Items 1 to 3.

Item 5. Item ^5. The compound according to any one of Items 1 to 4, wherein in General Formula (1), R ⁵ is an alkyl group which may have a substituent.

Item 6. Item 6. The compound according to any one of Items 1 to 5, wherein, in the general formula (1), R ³ and R ⁴ are linear or branched alkylene groups.

  Item 7. Item 7. A fluorescent dye comprising the compound according to any one of Items 1 to 6.

  Item 8. Item 8. A two-photon absorption material containing the fluorescent dye according to Item 7.

  Item 9. Item 9. The two-photon absorption material according to Item 8, further comprising a polar solvent.

  Since the compound of the present invention has a specific intramolecular crosslinking skeleton, the fluorescence maximum wavelength can be shifted by a long wavelength, and an electron accepting group is introduced while extending π conjugation to both ends. Therefore, it is a compound capable of two-photon absorption. For this reason, the compound of the present invention can shift the fluorescence maximum wavelength by a longer wavelength while maintaining the fluorescence quantum yield. In particular, if a solvent is selected, it is possible to have a fluorescence maximum wavelength that has not existed before, such as a fluorescence maximum wavelength of 700 nm or more.

2 is a fluorescence spectrum of each test solution prepared in Test Example 1 (a test solution using the compound obtained in Example 1). In order from the data on the left of the peak position, the solvents used are cyclohexane, benzene, tetrahydrofuran, and acetone. It is the one-photon absorption spectrum and the two-photon absorption spectrum of the test solution prepared in Test Example 2 (the test solution using the compound obtained in Example 1). The solid line is the one-photon absorption spectrum and the point is the two-photon absorption spectrum.

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

[Wherein, ^{R 1} and ^{R 2} are the same or different and are each an electron acceptor group; or different ^{R 3} and ^{R 4} the same, respectively a single bond or a divalent linking group; ^{R 5} represents an organic group; ^{L 1} And L ² are the same or different, and each is a π-conjugated group; Z ¹ and Z ² are the same or different, and each is a heterocyclic group; the dotted line connecting the nitrogen atom and the hydrogen atom is an intramolecular hydrogen bond. ]
It is a compound shown by these. The compound represented by the general formula (1) or a salt thereof is a novel compound not described in any literature.

In the general formula (1), the electron-accepting group represented by R ¹ and R ² is not particularly limited, and examples thereof include a boryl group, a cyano group, a nitro group, a sulfo group, an alkylsulfonyl group, an alkoxysulfonyl group, A phosphoric acid group, a formyl group, a carboxy group, a pyridinium group, etc. are mentioned.

The boryl group as an electron-accepting group represented by R ¹ and R ^2, is not particularly limited, include dimesitylboryl group.

There is no restriction | limiting in particular as an alkylsulfonyl group as an electron-accepting group shown by R < ¹ > and R < ² >, A methylsulfonyl group, an ethylsulfonyl group, etc. are mentioned.

There is no restriction | limiting in particular as an alkoxysulfonyl group as an electron-accepting group shown by R < ¹ > and R < ² >, A methoxysulfonyl group, an ethoxysulfonyl group, etc. are mentioned.

Among these, R ¹ and R ² are preferably a boryl group and more preferably a dimesitylboryl group from the viewpoints of ease of synthesis, ease of two-photon absorption, fluorescence quantum yield, and the like.

In the general formula (1), R ³ and R ⁴ are a single bond or a divalent linking group. Examples of the divalent linking group represented by R ³ and R ^4, is not particularly limited, and examples thereof include straight-chain or branched-chain alkylene group having 1 to 10 carbon atoms (especially 2-6). Examples of such a linking group include an ethylene group, a trimethylene group, and a tetramethylene group.

In the general formula (1), the organic group represented by R ⁵ is not particularly limited, and examples thereof include an alkyl group which may have a substituent and an aryl group which may have a substituent. Can be mentioned.

In the general formula (1), as the alkyl group as the organic group represented by R ⁵ , both a linear alkyl group and a branched alkyl group can be employed.

  As a linear alkyl group, a C1-C6 (especially 1-4) linear alkyl group is preferable, and specifically, a methyl group, an ethyl group, n-propyl group, n-butyl group, n-pentyl. Group, n-hexyl group and the like.

  As the branched alkyl group, a branched alkyl group having 3 to 6 carbon atoms (particularly 3 to 5 carbon atoms) is preferable. Specifically, an isopropyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, a neopentyl group, An isohexyl group, 3-methylpentyl group, etc. are mentioned.

The substituent which the alkyl group as the organic group represented by R ⁵ may have is not particularly limited, and is a hydroxyl group, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), a carboxyl group, Examples include ester groups, amide groups (amide groups, methylamide groups, dimethylamide groups, etc.), azide groups (azide phenyl groups, etc.), alkynyl groups (ethynyl groups, propargyl groups, etc.), and the like. The number of such substituents is not particularly limited, but is preferably 0 to 6, more preferably 0 to 3.

In the general formula (1), as the aryl group as the organic group represented by R ⁵ , any of a monocyclic aryl group and a polycyclic aryl group can be employed. For example, a phenyl group, an oligoaryl group (naphthyl) Group, anthryl group, etc.), biphenyl group, terphenyl group, pyrenyl group, phenanthrenyl group, fluorenyl group and the like.

The substituent which the aryl group as the organic group represented by R ⁵ may have is not particularly limited, and may be a hydroxyl group, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), alkyl group ( Methyl group, ethyl group, n-propyl group, etc.), carboxyl group, ester group, amide group (amide group, methylamide group, dimethylamide group etc.), azido group (azide phenyl group etc.), alkynyl group (ethynyl group, propargyl) Group) and the like. The number of such substituents is not particularly limited, but is preferably 0 to 6, more preferably 0 to 3.

Among these, as R ⁵ , an alkyl group which may have a substituent is preferable from the viewpoints of ease of synthesis, fluorescence quantum yield, long wavelength shift of the fluorescence maximum wavelength, and the like. An optionally branched alkyl group is more preferable, and an unsubstituted alkyl group is more preferable.

In the general formula (1), the π-conjugated group represented by L ¹ and L ² is not particularly limited as long as it is a group capable of extending π-conjugation, but the general formulas (2A) to (2G):

And a group in which one or more of the groups represented by the above are combined. As such a π-conjugated group, for example,

Etc.

In the general formula (1), the heterocyclic ring constituting the heterocyclic group represented by Z ¹ and Z ² is not particularly limited, and examples thereof include a furan ring, a thiophene ring, a pyrrole ring, an imidazole ring, a thiazole ring, and a thiadiazole. Ring, silacyclopentadiene ring, oxazole ring, oxadiazole ring, pyridine ring, pyrimidine ring and the like. In addition, the heterocyclic group may have a substituent on the heterocyclic ring. Examples of the substituent include a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), and the above alkyl. Group, the above aryl group, alkoxyl group, carboxyl group, ester group, amide group and the like. The number of this substituent is preferably 0-3.

  As the compound of the present invention satisfying such conditions, the general formula (1A):

[Wherein, R ^{1 to} R ⁵ and L ^{1 to} L ² are the same as described above; a dotted line connecting a nitrogen atom and a hydrogen atom is an intramolecular hydrogen bond. ]
A compound represented by formula (1A1):

[Wherein, R ^{3 to} R ⁵ are the same as above; Mes is a mesityl group (the same applies hereinafter); a dotted line connecting a nitrogen atom and a hydrogen atom is an intramolecular hydrogen bond. ]
The compound shown by is more preferable. As such a compound, specifically,

Etc.

2. Method for Producing Compound The compound of the present invention is not particularly limited, but when the compound of the present invention is a compound represented by the general formula (1A1), for example, the following reaction formula 1:

[Wherein R ^{3 to} R ⁵ are the same as above; R ⁶ and R ⁷ are the same or different and each is a protecting group; ¹ and X ² are the same or different and each is a halogen atom or a triflate group; TMS is a trimethylsilyl group ; Mes is a mesityl group. ]
Can be synthesized according to

In Reaction Scheme 1, the protecting group represented by R ⁶ and R ⁷ is, for example, a silyl group (trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, etc.), an optionally substituted aralkyl group (benzyl group, p-methoxybenzyl group, p-nitrobenzyl group, etc.), alkoxycarbonyl group (methoxycarbonyl group, tert-butoxycarbonyl group, etc.), alkanoyl group (formyl group, acetyl group, propionyl, etc.) ) And protecting groups such as alkoxyalkyl groups (methoxymethyl group and the like).

  In addition, when a compound represented by the general formula (1) other than the compound represented by the general formula (1A1) is to be synthesized, the compound can be synthesized by the same synthesis method according to the above synthesis method.

In the reaction formula 1, as the halogen atom represented by X ¹ and X ² , any of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom can be adopted, but from the viewpoint of easy progress of the reaction, a chlorine atom, bromine atom An atom etc. are preferable and a bromine atom is more preferable.

(2-1) Halogenation 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 halogenated succinimide compound is added. The halogenation reaction can proceed.

  In addition, the compound (2) in the said Reaction formula 1 may use a well-known or commercially available compound, and may synthesize | combine it.

  There is no restriction | limiting in particular as an organic lithium compound, A well-known thing can be employ | adopted, for example, ethyl lithium, n-propyl lithium, isopropyl lithium, n-butyl lithium, sec-butyl lithium, tert-butyl lithium, pentyl lithium, hexyl Alkyl lithium such as lithium; cycloalkyl lithium such as cyclohexyl lithium; aryl lithium such as phenyl lithium; lithium amide such as lithium diisopropylamide, lithium tetramethylpiperidide and lithium hexamethyldisilazide. Among these, in this step, alkyl lithium is preferable and n-butyl lithium is more preferable from the viewpoint of yield.

  There is no restriction | limiting in particular as a halogenated succinimide compound, A well-known thing can be employ | adopted, for example, N-bromosuccinimide, N-chlorosuccinimide, etc. are mentioned. Among these, N-bromosuccinimide is preferable in this step from the viewpoint of yield.

  Although the usage-amount of the said organolithium compound and a halogenated succinimide compound does not have a restriction | limiting in particular, From viewpoints, such as a yield, it is 1-10 mol (especially 1.5 to 1.5 mol) with respect to 1 mol of compounds (2). ˜5 mol), preferably 1 to 10 mol (particularly 1.5 to 5 mol) of the halogenated succinimide compound.

  The organic solvent that can be used in this step may be a known one, and in this step, for example, ethers such as tetrahydrofuran, dioxane, diethyl ether, t-butyl methyl ether are preferable. The reaction conditions may be such that the reaction proceeds sufficiently, and may be, for example, −150 to 0 ° C., particularly −100 to −50 ° C., 5 minutes to 24 hours, particularly 2 to 12 hours.

(2-2) Sonogashira coupling In this step, the reaction can proceed according to conventionally known Sonogashira coupling. For example, in an organic solvent, the compound (3) in the above reaction scheme 1 can be reacted. In the presence of a palladium catalyst, a copper catalyst and a base, a compound represented by R ⁷ —C≡C—H can be added to cause a Sonogashira coupling reaction.

Examples of the palladium catalyst include metal palladium and palladium compounds known as synthesis catalysts for organic compounds (including polymer compounds). Specifically, as such a palladium compound, for example, Pd (PPh ₃ ) ₄ (Ph is a phenyl group; the same applies hereinafter), PdCl ₂ (PPh ₃ ) ₂ , Pd (OAc) ₂ (Ac is an acetyl group; The same shall apply hereinafter), tris (dibenzylideneacetone) dipalladium (0) (Pd ₂ (dba) ₃ ), tris (dibenzylideneacetone) dipalladium (0) chloroform complex, bis (dibenzylideneacetone) palladium (0), bis (Tri-t-butylphosphino) palladium (0) and the like. In this step, Pd (PPh ₃ ) ₄ or the like is preferable.

  As the copper catalyst, copper halide is preferable, and in this step, copper iodide is more preferable from the viewpoint of yield.

  Examples of the base include amine compounds such as diethylamine, triethylamine, diisopropylamine, and cyclohexyldimethylamine, and these also have a role as a solvent.

The amount of the palladium catalyst, the copper catalyst, the base and the compound represented by R ⁷ —C≡C—H is not particularly limited, but from the viewpoint of yield and the like, palladium is used relative to 1 mol of compound (3). 0.01-0.2 mol (especially 0.02-0.1 mol) of catalyst, 0.02-0.5 mol (especially 0.05-0.2 mol) of copper catalyst, R ⁷ -C≡ It is preferable to use 1 to 10 mol (particularly 1.5 to 5 mol) of the compound represented by C—H. The amount of the base used is not particularly limited. However, when the above liquid amine compound is used, it is preferably an excess amount.

  The organic solvent that can be used in this step may be a known one, and in this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. Moreover, you may use said base as a solvent. In addition, the same solvent as the said halogenation process can be used. The reaction conditions may be such that the reaction proceeds sufficiently, and may be, for example, 10 minutes to 12 hours, particularly 30 minutes to 6 hours at 0 to 150 ° C., particularly 50 to 100 ° C.

(2-3) Deprotection In this step, the protecting groups (R ⁶ and R ⁷ ) can be deprotected by a known method. For example, a base can be added to the compound (4) in the above reaction scheme 1 in an organic solvent for deprotection.

  Examples of the base include alkyllithium such as methyllithium, ethyllithium, n-butyllithium, s-butyllithium and t-butyllithium; aryllithium such as phenyllithium; sodium methoxide, sodium ethoxide, potassium-t- Examples include alkali metal alkoxides such as butoxide; amines such as diisopropylethylamine, tributylamine, morpholine, and N-methylmorpholine. From the viewpoint of yield, an alkali metal alkoxide is preferable, and sodium methoxide is more preferable. Note that it is preferable to use a liquid base as the base because it can be used as a solvent.

  Although the usage-amount of the said base does not have a restriction | limiting in particular, From viewpoints, such as a yield, it is preferable to set it as an excess with respect to a compound (4), and 5-50 mol (1 mol of compound (4) ( It is particularly preferred to use 10 to 30 mol).

  The organic solvent that can be used in this step may be a known one. In this step, for example, cyclic ethers such as tetrahydrofuran and dioxane; alcohols such as methanol and ethanol are preferable. In addition, the same solvent as the said halogenation process can be used. The reaction conditions may be such that the reaction proceeds sufficiently, and may be, for example, 0 to 150 ° C., particularly 50 to 100 ° C., 30 minutes to 24 hours, particularly 1 to 12 hours.

(2-4) Hydroboration In this step, the hydroboration reaction can be caused using dimesitylborane to the compound (5) in the above reaction formula 1 in an organic solvent, for example.

  Although there is no restriction | limiting in particular in the usage-amount of the said dimesityl borane, From viewpoints, such as a yield, it is preferable to use 1-10 mol (especially 1.5-5 mol) with respect to 1 mol of compounds (5).

  The organic solvent that can be used in this step may be a known one, and in this step, for example, cyclic ethers such as tetrahydrofuran and dioxane are preferable. In addition, the same solvent as the said halogenation process can be used. The reaction conditions may be such that the reaction proceeds sufficiently. For example, it may be 1 to 48 hours, particularly 2 to 24 hours at -50 to 100 ° C, particularly 0 to 50 ° C.

  In this way, the compound represented by the general formula (1A1) can be obtained, and can be used through ordinary isolation and purification steps as necessary.

  Note that although an example of a synthesis method of one embodiment of the compound of the present invention has been described above, the synthesis method is not limited to this production method, and the compound can be synthesized by various synthesis methods. Other compounds of the present invention can be synthesized by the same method.

3. Fluorescent dye and two-photon absorbing material The fluorescent dye of the present invention comprises the above-described compound of the present invention.

  In the fluorescent dye of the present invention, since it has a specific intramolecular crosslinking skeleton, the fluorescence maximum wavelength can be shifted by a long wavelength, and an electron accepting group is introduced while extending π conjugation to both ends. Therefore, it is a compound that can also absorb two-photons. For this reason, in addition to the effect of the long wavelength shift of the fluorescence maximum wavelength due to the intramolecular crosslinking skeleton, the effect of two-photon absorption by introducing the electron accepting group while extending the π conjugation to both ends, The fluorescence maximum wavelength can be set to a particularly long wavelength region.

Such fluorescent dyes can be used usually dispersed in an organic solvent. At this time, the concentration of the compound of the present invention is from the viewpoint of introducing into the cell while having a fluorescence maximum wavelength of 650 to 800 nm and a sufficiently high fluorescence quantum yield even under physiological conditions. × 10 ⁻⁸ to 1 × 10 ⁻⁴ mol / L is preferable, and 1 × 10 ⁻⁷ to 1 × 10 ⁻⁵ mol / L is more preferable.

  As the organic solvent, any of a polar solvent and a nonpolar solvent can be used, but if a polar solvent is used, the fluorescence maximum wavelength can be particularly increased. Examples of such polar solvents include ether compounds (tetrahydrofuran, anisole, 1,4-dioxane, cyclopentylmethyl ether, etc.), alcohols (methanol, ethanol, allyl alcohol, etc.), ester compounds (ethyl acetate, etc.), ketones ( Acetone), halogenated hydrocarbons (dichloromethane, chloroform), dimethyl sulfoxide, amide solvents (N, N-dimethylformamide, dimethylacetamide, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, etc.), etc. Can be used. In particular, when the compound of the present invention is dispersed in an ether compound or a ketone, particularly a ketone, especially acetone, it can be obtained with a conventional fluorescent dye having a fluorescence maximum wavelength of 650 to 800 nm, particularly 700 to 750 nm. The fluorescence quantum yield can be increased while having the fluorescence maximum wavelength that has not been obtained.

  On the other hand, if a nonpolar solvent is used as the organic solvent, the fluorescence quantum yield can be particularly increased. As such a nonpolar solvent, for example, an aliphatic organic solvent (pentane, hexane, cyclohexane, heptane, etc.), an aromatic organic solvent (benzene, toluene, xylene, mesitylene, etc.) and the like can be used. Of these, when the compound of the present invention is dispersed in cyclohexane or benzene, the fluorescence quantum yield can be particularly increased.

  For this reason, it is possible to facilitate bioimaging of desired cells (for example, cancer cells such as Hela cells), and the fluorescent dye concentration required for bioimaging can be kept low, greatly reducing damage to the living body. can do. That is, the compound of the present invention is expected to be applied to various fields such as three-dimensional fine stereolithography, cancer treatment by photodynamic therapy, and three-dimensional fluorescence imaging.

  As described above, the two-photon absorption material of the present invention is preferably in the form of a solution, while having a fluorescence maximum wavelength at 650 to 800 nm, while at a sufficiently high fluorescence quantum yield even under physiological conditions, From the viewpoint of introducing into cells, the pH is preferably about 5 to 11, more preferably about 6.5 to 7.5. In order to adjust the pH of the cell detection agent of the present invention, a buffer (Hepes buffer, Tris buffer, tricine-sodium hydroxide buffer, phosphate buffer, phosphate buffered saline, etc.) is used. May be.

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

  [Example 1]

[Wherein Boc is a tert-butoxycarbonyl group; n-BuLi is n-butyllithium; NBS is N-bromosuccinimide; THF is tetrahydrofuran; TMS is a tetramethylsilyl group; iPr is an isopropyl group; Me is a methyl group; Mes Is a mesityl group; the same shall apply hereinafter. ]

Compound 23
To a dehydrated THF (1.5 mL) solution of Compound 13 (370 mg, 0.785 mmol), a hexane solution of n-butyllithium (1.62 M, 1.00 mL, 1.62 mmol) was added at -78 ° C, and the temperature was maintained at -78 ° C. The mixture was stirred for 1.5 hours under an argon atmosphere. Subsequently, a solution of N-bromosuccinimide (290 mg, 1.63 mmol) in dehydrated THF (2.5 mL) was added at −78 ° C., and the mixture was stirred at that temperature for 6.5 hours. The reaction mixture was allowed to warm to room temperature, water was added, and the mixture was extracted 3 times with ethyl acetate (5.0 mL). The organic layer was washed once with saturated brine (2.0 mL). The organic layer was dehydrated with anhydrous sodium sulfate and filtered, and then the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel chromatography (PSQ100B, hexane / ethyl acetate = 1/1, _Rf = 0.20) to obtain Compound 23 as a colorless solid (290 mg, 0.462 mmol, yield). Rate 60%).
¹ H NMR (400 MHz, C ₆ D ₆ ) δ 6.67 (s, 2H), 6.06 (s, 2H), 2.61 (sept, J = 6.4 Hz, 1H), 2.32-2.48 (m, 4H), 2.11- 2.14 (m, 4H), 1.28-1.42 (m, 4H), 1.04 (s, 9H), 0.84 (d, J = 6.4 Hz, 6H); ¹³ C { ¹ H} NMR (100 MHz, C ₆ D ₆ ) δ 148.4, 143.6, 132.4, 131.1, 127.7, 116.4, 110.6, 83.6, 53.0, 51.6, 30.4, 28.1, 27.2, 18.4; HRMS (APCI) Calcd. for C ₂₆ H ₃₃ Br ₂ N ₂ O ₂ S ₂ ⁺ : 629.0325 ([M + H] ⁺ ). Obsd. 629.0305.

Compound 24
Trimethylsilyl was added to a solution of compound 23 (428 mg, 0.682 mmol), Pd (PPh ₃ ) ₄ (40.0 mg, 34.6 μmol) and copper (I) iodide (16.4 mg, 86.1 μmol) in dehydrated diisopropylamine (2.0 mL). Acetylene (200 μL, 1.44 mmol) was added at room temperature, and the mixture was stirred at 70 ° C. for 1.5 hours under an argon atmosphere. The reaction mixture was allowed to cool to room temperature, water and ethyl acetate (5.0 mL) were added, and the mixture was extracted 3 times with ethyl acetate (5.0 mL). The organic layer was washed with saturated brine (5.0 mL). The organic layer was dehydrated with anhydrous sodium sulfate and filtered, and then the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel chromatography (PSQ100B, hexane / ethyl acetate = 1/1, _Rf = 0.43) to obtain Compound 24 as a pale yellow solid (426 mg, 0.642 mmol, Yield 94%).
¹ H NMR (400 MHz, C ₆ D ₆ ) δ 7.07 (s, 2H), 6.03 (s, 2H), 2.51-2.60 (m, 3H), 2.31-2.39 (m, 2H), 2.07-2.11 (m , 4H), 1.28-1.43 (m, 4H), 1.00 (s, 9H), 0.81 (d, J = 6.4 Hz, 6H), 0.21 (s, 9H); ¹³ C { ¹ H} NMR (100 MHz, C ₆ D ₆ ) δ 148.4, 142.8, 135.3, 131.8, 128.0, 121.8, 116.5, 99.0, 98.9, 83.6, 53.0, 51.6, 30.5, 28.1, 27.2, 18.4, 0.01; HRMS (APCI) Calcd. For C ₃₆ H ₅₁ N ₂ O ₂ S ₂ Si ₂ ⁺ : 663.2945 ([M + H] ⁺ ). Obsd. 663.2913.

Compound 25
To a THF (1.0 mL) solution of compound 24 (197 mg, 0.297 mmol), a methanol (1.0 mL) suspension of sodium methoxide (340 mg, 6.30 mmol) was added at room temperature, and the mixture was stirred at 80 ° C. for 3 hours. . The reaction mixture was allowed to cool to room temperature, water and ethyl acetate (4.0 mL) were added, and the mixture was extracted 3 times with ethyl acetate (5.0 mL). The organic layer was washed with saturated brine (5.0 mL). The organic layer was dehydrated with anhydrous sodium sulfate and filtered, and then the solvent was distilled off under reduced pressure. The obtained crude product was purified by silica gel chromatography (PSQ100B, ethyl acetate, R _f = 0.83) to obtain Compound 25 as a yellow solid (104 mg, 0.247 mmol, yield 83%).
¹ H NMR (600 MHz, C ₆ D ₆ ) δ13.0 (br s, 1H), 6.91 (s, 2H), 6.47 (d, J = 1.8 Hz, 2H), 2.99 (s, 2H), 2.55 ( sept, J = 6.6 Hz, 1H), 2.47 (m, 4H), 1.94 (t, J = 5.4 Hz, 4H), 1.43 (m, 4H), 0.31 (d, J = 6.6 Hz); ¹³ C { ¹ H} NMR (100 MHz, C ₆ D ₆ ) δ136.0, 135.4, 133.9, 127.1, 119.6, 111.4, 82.4, 77.6, 47.9, 45.9, 26.9, 25.5, 15.2; HRMS (APCI) Calcd. For C ₂₅ H ₂₇ N ₂ S ₂ ⁺ : 419.1610 ([M + H] ⁺ ). Obsd. 419.1616.

Compound 12
To a dehydrated THF (4.0 mL) solution of compound 25 (119 mg, 0.285 mmol), a dehydrated THF (8.5 mL) suspension of dimesitylborane (143 mg, 0.570 mmol) is added at room temperature, under a nitrogen atmosphere under light shielding conditions. And stirred at room temperature for 13 hours. After evaporating the solvent under reduced pressure, the crude product was washed with diethyl ether to obtain Compound 12 as a red solid (155 mg, 0.169 mmol, 59%).
¹ H NMR (400 MHz, C ₆ D ₆ ) δ 13.2 (br, 1H), 7.43 (d, J = 17 Hz, 2H), 7.29 (d, J = 17 Hz, 2H), 6.85 (s, 8H) , 6.66 (d, J = 1.6 Hz, 2H), 6.31 (s, 1H), 2.62 (sept, J = 6.4 Hz, 1H), 2.53 (m, 4H), 2.35 (s, 24H), 2.22 (s, 12H), 2.02 (m, 4H), 1.51 (m, 4H), 0.29 (d, J = 6.4 Hz, 6H); ¹³ C { ¹ H} NMR (100 MHz, THF-d ₈ ) δ 146.4, 143.1, 141.8, 141.1, 138.8, 137.7, 137.0, 135.4, 135.2, 129.0, 129.0, 112.0, 48.8, 46.6, 27.8, 26.3, 23.6, 21.3, 15.7; ¹¹ B { ¹ H} NMR (100 MHz, THF-d ₈ ) δ 30.4; HRMS (APCI) Calcd. for C ₆₁ H ₇₂ B ₂ N ₂ S ₂ : 918.5336 ([M] ⁺ ). Obsd. 918.5300.

[Test Example 1: Photophysical characteristics (1)]
As the solvent, 1 × 10 ⁻⁶ to 1 × 10 ⁻⁵ mol / L of the compound obtained in Example 1 was added to cyclohexane, benzene, tetrahydrofuran or acetone (pH: 7).

  Using these four types of test solutions using different solvents, the absorption spectrum of the compound obtained in Example 1 was UV-UV / NIR spectrophotometer UV-3150 (Shimadzu Corporation), and the fluorescence spectrum was NIR. -The measurement was performed using a fluorescence spectrometer SPEX Fluorolog 3 (HORIBA Scientific) with a PMT detector (Hamamatsu Photonics). The results are shown in Table 1 and FIG. As a result, the compound obtained in Example 1 showed yellow fluorescence having an emission maximum at 549 nm in cyclohexane, and showed double emission characteristics in a polar solvent. Also, in polar solvents, it shows a relatively strong red emission that spreads widely in the near-infrared region. In particular, when acetone is used, it is a value that cannot be reached by conventional fluorescent dyes of 700 nm or more. It was.

[Test Example 2: Photophysical characteristics (2)]
As a solvent, 1 × 10 ⁻⁶ to 1 × 10 ⁻⁵ mol / L of the compound obtained in Example 1 was added to tetrahydrofuran (pH: 7).

  Using this test solution, a two-photon absorption cross section was measured by a two-photon induced fluorescence method (TPIF method) using a rhodamine 640 / methanol solution as a reference. The results are shown in FIG.

  As a result, the compound obtained in Example 1 had a large two-photon absorption cross section near 800 nm, and a value of 1448 GM was obtained at 830 nm. This value is sufficiently large even when compared with a known compound group having the same extent of π-conjugated system. These known compound groups are as follows.

Further, the compound obtained in Example 1 has a high two-photon excitation brightness (Two-photon brightness = δ × φ _F ). This value is a parameter that represents the brightness when actually observing two-photon excitation fluorescence, and the two-photon excitation luminance of the compound obtained in Example 1 greatly exceeds the value of 9E-BMVC used in live imaging. ing. In addition to this high luminance, considering the near-infrared emission and a large Stokes shift (Δν = 6570 cm ^{−1 in} acetone), it can be said that the compound of the present invention has excellent properties as a two-photon excited luminescent material.

Claims (7)

General formula (1A) :
[Wherein, R ¹ and R ² are the same or different and are each a boryl group, a cyano group, a nitro group, a sulfo group, an alkylsulfonyl group, an alkoxysulfonyl group, a phosphate group, a formyl group, a carboxy group, or a pyridinium group ; R ³ and R ⁴ are the same or different and are each a linear or branched alkylene group ; R ⁵ is an alkyl group that may have a substituent, or an aryl group that may have a substituent ; L ¹ And L ² is any of the following groups:
; Dotted line connecting the nitrogen atom and hydrogen atom are intramolecular hydrogen bonds. ]
A compound represented by In the general formula (1A), R ¹ And R ² The compound according to claim 1, wherein is a boryl group. In the general formula (1A), R ³ And R ⁴ The compound according to claim 1 or 2, wherein is an ethylene group, a trimethylene group, or a tetramethylene group. In the general formula (1A), R ⁵ The compound according to any one of claims 1 to 3, wherein is an unsubstituted alkyl group. Fluorescent dye comprising a compound according to any one of claims 1-4. A two-photon absorption material containing the fluorescent dye according to claim 5 . Furthermore, the two-photon absorption material of Claim 6 containing a polar solvent.