Diaminobenzene compounds containing sulfinyl or sulfonyl groups or salts thereof, and organic fluorescent materials containing them

Abstract

To develop a material suitable for fluorescent labeling agents that can absorb long-wavelength light and emit fluorescence with small molecular size.SOLUTION: The invention provides compounds having a sulfonyl aniline skeleton represented by the general formula (1), or salts thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Detailed Description of the Invention] The present invention relates to a compound having a sulfonylaniline skeleton and an organic fluorescent material using the same. [Background technology]

[0002] Fluorescence imaging, which uses fluorescent dyes to visualize target proteins and molecules and capture the space and time of biological phenomena, is an important technology in life sciences, including not only biochemistry and biology but also medicine. It is expected to lead to elucidation of biological functions and disease mechanisms, providing clues to medical technologies such as prevention, diagnosis, and treatment. In particular, in recent years, customer needs have diversified for fluorescently labeled glycans and peptides, which serve as important core tools supporting life science research, such as the development of fluorescent probes and fluorescent imaging.

[0003] However, fluorescent proteins such as GFP (green fluorescent protein), as well as small molecule fluorescent dyes such as fluorescein and rhodamine, are often large in size and can interfere with the function of complex targets, making them difficult to introduce into targets. Furthermore, these molecules have a small Stokes shift (the difference between excitation light and emission light), making them unsuitable for multicolor imaging, which visualizes multiple emission signals simultaneously.

[0004] Labeling agents with a large Stokes shift can reduce the overlap of the excitation light with the detection wavelength range compared to many conventional fluorescent labeling agents with a small Stokes shift, and have the great advantage of improving sensitivity and precision. Therefore, fluorescent dyes with a small molecular structure and a large Stokes shift are required.

[0005] Because extended π-conjugated systems are advantageous for exhibiting excellent fluorescence properties, molecular designs with extended aromatic backbones have been widely used. However, many of the materials produced by such molecular designs have high planarity and rigid backbones, resulting in problems such as large molecular size, low stability, poor water solubility, small Stokes shift, and strong intermolecular interactions that cause concentration quenching and reduced fluorescence intensity.

[0006] The present inventors have reported that sulfonylaniline dyes, which have multiple amino groups and sulfonyl groups on one benzene ring, exhibit superior properties to conventional organic fluorescent materials, such as: (1) extremely small molecular size; (2) longer absorption wavelength due to the push-pull effect; (3) high solubility due to the bent structure of the sulfonyl group, which allows the molecules to associate and exhibit solid-state fluorescence, emitting light in a solid or aggregated state; and (4) hydrogen bonds between the amino group and sulfonyl group suppress the free rotation of the amino group, improving fluorescence quantum efficiency and stability (Non-Patent Document 1, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6249210 [Patent Document 2] Patent Publication No. 2021-127305 [Non-patent literature]

[0008] [Non-Patent Document 1] Single Benzene Green Fluorophore: Solid-State Emissive, Water-Soluble, and Solvent- and pH-Independent Fluorescence with Large Stokes Shifts., Teruo Beppu, Kosuke Tomiguchi, AkitoMasuhara, Yong-Jin Pu, and Hiroshi Katagiri., Angewandte Chemie InternationalEdition, 54 (2015) 7332-7335. Summary of the Invention [Problem to be solved by the invention]

[0009] The general mechanism of fluorescence is that a compound absorbs light in a certain wavelength range, causing excited electrons to return to their ground state, resulting in the emission of fluorescence in a specific wavelength range. Ultraviolet light is typically absorbed, but ultraviolet light can be damaging to living organisms, especially when administered to living organisms. For this reason, low-energy, long-wavelength light is desirable. However, in order to absorb light with a longer wavelength, the molecular conjugation system must generally be expanded, resulting in a problem of large molecular size. While various organic fluorescent materials have been investigated and developed as fluorescent imaging agents, there is a continuing need for compounds with small molecular sizes that can absorb long-wavelength light and emit fluorescence. The present invention aims to further improve the sulfonylaniline dyes and develop materials suitable for fluorescent labeling agents. [Means for solving the problem]

[0010] The present inventors discovered that it is possible to introduce four sulfides into a benzene ring with high reproducibility, and that a compound in which at least a portion of the sulfides is oxidized can solve the above-mentioned problems, and thus arrived at the present invention.

[0011] That is, the present invention comprises the following items. The present invention relates to a compound represented by the following general formula (1): [ka] (In the formula, R 1 is independently in each occurrence an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom or an amino group or an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom; R 2 is independently in each occurrence a hydrogen atom, a group —RH or one of the following: [ka] (In each of the above formulas, R is a linear or branched alkylene group having 1 to 18 carbon atoms, which may contain one or more heteroatoms in the chain, or a cyclic alkylene group having 3 to 18 carbon atoms, and * represents a bond to the nitrogen atom), m, n, p, and q are each independently 0, 1, or 2, provided that any one of m, n, p, and q is 1 or 2. The compound has a sulfonylaniline skeleton and is represented by the following formula:

[0012] Another aspect of the present invention is an organic fluorescent material containing the compound having a sulfonylaniline skeleton or a salt thereof. [Effects of the Invention]

[0013] The compound having a sulfonylaniline skeleton or a salt thereof of the present invention has a molecular size equivalent to that of BODIPY (registered trademark, boron dipyrromethene), one of the smallest and most versatile dyes, and can emit fluorescence even when excited by visible light, which has a longer wavelength than conventional organic fluorescent materials. Furthermore, this group of compounds having a sulfonylaniline skeleton can emit fluorescence over a wide range of wavelengths, making it possible to control the fluorescence wavelength, i.e., the emission color, by selecting the compound. They also have a large Stokes shift, high stability, high quantum efficiency, and water solubility. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below. [Compounds having a sulfonylaniline skeleton or salts thereof] The present invention relates to a compound represented by the following general formula (1): [ka] (In the formula, R 1 is independently in each occurrence an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom or an amino group or an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom; R 2 is independently in each occurrence a hydrogen atom, a group —RH or one of the following: [ka] (In each of the above formulae, R is a linear or branched alkylene group having 1 to 18 carbon atoms, which may contain one or more heteroatoms in the chain, or a cyclic alkylene group having 3 to 18 carbon atoms, and * represents a bond to the nitrogen atom), m, n, p, and q are each independently 0, 1, or 2, provided that any one of m, n, p, and q is 1 or 2. or a salt thereof.

[0015] The alkyl group constituting the alkyl group and fluoroalkyl group is specifically an alkyl group having 1 to 18 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms, which may be linear or branched. Specific examples of the alkyl group include methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and octadecyl. The fluoroalkyl group refers to a group in which at least a portion of the hydrogen atoms of the alkyl group are substituted with fluorine. The substitution sites are not particularly limited, and any site can be substituted in any number. Specific examples of the fluoroalkyl group include a fluoromethyl group, a trifluoromethyl group, a pentafluoroethyl group, and a 3,3,3-trifluoropropyl group.

[0016] The cyclic alkyl group is a hydrocarbon skeleton group having at least one cyclic structure and having 3 to 18 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 8 carbon atoms. The cyclic structure may be formed by condensing two or more rings. The cyclic alkyl group may have at least one cyclic structure. Bonds do not necessarily have to be present in the cyclic structure, and a linear or branched alkyl group may be present in addition to the cyclic structure. Examples of such cyclic alkyl groups include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclohexylmethyl group, and a norbornyl group.

[0017] A phenyl group substituted with a halogen atom refers to a phenyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine, or iodine). Examples of such groups include a pentafluorophenyl group. The phenyl group substituted with an amino group refers to a group in which some or all of the hydrogen atoms of the phenyl group have been substituted with a primary amino group or a secondary amino group. The alkoxy group constituting the alkoxy-substituted phenyl group is specifically an alkoxy group which may be linear or branched and has 1 to 18 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Here, the alkoxy group refers to a group in which an oxygen atom is introduced into the bonding site of the alkyl group. A thienyl group substituted with a halogen atom refers to a thienyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine or iodine). The thiazolyl group substituted with a halogen atom refers to a thiazolyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine, iodine). The pyridyl group substituted with a halogen atom refers to a pyridyl group in which some or all of the hydrogen atoms have been substituted with a halogen atom (fluorine, chlorine, bromine, iodine).

[0018] R 1 is independently in each occurrence an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom, an amino group, or an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom. 1 are preferably the same group. From the viewpoint of controlling the molecular size to be small and increasing the water solubility of the molecule, it is preferable that R 1 is preferably an alkyl group, more preferably an alkyl group having 1 to 6 carbon atoms which may be linear or branched, and even more preferably a methyl group.

[0019] R 2 is independently in each occurrence a hydrogen atom, a group —RH or one of the following: [ka] is a substituent selected from the group consisting of: In each of the above formulas, R represents a linear, branched, or cyclic alkylene group having 1 to 18 carbon atoms, which may contain one or more heteroatoms in the chain, including both ends. Here, the term "alkylene group" refers to a divalent group in which any one of the hydrogen atoms in the linear, branched, or cyclic alkyl group is replaced with a single bond. R may contain one or more heteroatoms in the chain, including both ends. Examples of heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and halogen atoms. Oxygen and sulfur atoms may be present as a (thio)ether bond or an ester bond, or as an atom having a double bond, such as a (thio)carbonyl group or a phosphate group. Nitrogen atoms may be present as an -NH- group or an -NR'- group (where R' represents an alkyl group having 1 to 6 carbon atoms). These may be adjacent to a carbonyl group to form an amide bond. The valence of the phosphorus atom may be any value. The position of the heteroatom is not limited as long as it can be synthesized as a stable compound, and any number of halogen atoms may be substituted at any position in the alkylene group, and if it can be a divalent or higher group such as an oxygen atom, it may be included as part of the ring structure. Examples of such alkylene groups include divalent groups derived from oxyethylene groups (-OCH2CH2- or -CH2CHO-), linear ethers such as ethoxyethylene and PEG oligomers, or cyclic ethers such as epoxy and oxetanyl groups.

[0020] The substituents represented by the above formulas are reactive groups for fluorescent labeling. Examples of reactive groups include succinidyl esters, maleimides, isothiocyanates, tetrafluorophenyl (TFP) esters, and iodoacetamides, as shown in the following structural formulas, as well as reactive groups that utilize a click reaction, which has an alkyne moiety and binds an azide group incorporated into a biomolecule to an alkyne to form a carbon-heteroatom bond. Of these, succinidyl esters and maleimides are preferred in terms of compound stability and ease of operation.

[0021] R 2 From the viewpoint of reducing the molecular size, R is preferably a hydrogen atom. 2 and R are each a hydrogen atom. On the other hand, when the compound is to be administered to a living body, it is preferable that at least one of the groups is a reactive group for fluorescent labeling selected from the group consisting of the above formulas. Therefore, another aspect of the present invention is a compound in which R 2 is a substituent selected from the group consisting of the above formulas, i.e., R 2 is a compound in which one of the atoms is not a hydrogen atom.

[0022] In general formula (1), m, n, p, and q are each independently 0, 1, or 2, with the exception that any one of m, n, p, and q is 1 or 2. Therefore, the compounds of the present invention are compounds in which at least one of the four sulfides is oxidized. The values ​​of m, n, p, and q are not particularly limited, except that any one of them is 1 or 2, and the total value of m, n, p, and q can be freely designed within the range of 1 to 8. In one embodiment, when the total of m, n, p, and q is 3 to 5, the electron-withdrawing ability of the sulfur functional group is increased, the basicity of the amino group is appropriately reduced, and the acidity of the hydrogen atoms on the amino group is appropriately increased. This improves the stability, fluorescence properties, water solubility, and other properties of the compound. Hereinafter, the total value of m, n, p, and q may be referred to as the "oxidation degree."

[0023] The compound of the present invention can be described by the above general formula (1). When the value of m, n, p, or q in the compound of the present invention is 1, the corresponding sulfur atom has an unpaired electron in addition to an S=O bond, and therefore the sulfur atom becomes an asymmetric center. Therefore, when the value of at least two of m, n, p, and q is 1, the compound of the present invention may exist as the following set of stereoisomers: [ka] In the above example, the difference is whether both methyl groups on the left side of the molecule are pointing outward from the paper (left) or whether one of the methyl groups is pointing outward from the paper (right). 1 or R 2 In addition to the case where the compound itself has an asymmetric center, stereoisomers such as enantiomers and diastereomers may exist depending on the oxidation state of the sulfide, and the compound of the present invention also includes such stereoisomers.

[0024] The ionic hydrophilic group (eg, amino group) of the compound of the present invention may form a salt such as a hydrochloride or sulfate.

[0025] Specific examples of the compound of the present invention, taking stereoisomers into consideration, include compounds selected from the following group or salts thereof. [ka]

[0026] [Method for synthesizing the compound of the present invention] The compound having a sulfonylaniline skeleton or a salt thereof of the present invention can be synthesized by a method including the following steps. 1) reacting 1,4-diaminobenzene with thiosulfate in the presence of an oxidizing agent; 2) a step of treating the compound obtained in the step 1) with a strong acid and then with a strong base to form a crosslinked sulfide; 3) The crosslinked sulfide is reacted with a base and R 1I (where R 1 is as defined above) to form a tetra-sulfide of 1,4-diaminobenzene; and 4) a step of oxidizing the tetrasubstituted sulfide in the presence of an oxidizing agent. The present invention also relates to a method for producing a compound represented by the general formula (1), which comprises:

[0027] The compound of the present invention can be obtained by oxidizing a tetrasubstituted sulfide such as the following compound (2) in the step 4). [ka] Although the existence of compound (2) has been known (Lakshmikantham, MV, Raasch, MS, Cava, MP, Bott, SG, & Atwood, JL (1987). Thioquinones. A reinvestigation of Perkin and Green's diaminodithioquinone. The Journal of Organic Chemistry, 52(9), 1874-1877), the present inventors have found that the selection of reagents and recrystallization solvents according to the method described in the publication may result in poor reproducibility in some cases. The present invention is based in part on the discovery that the above method can synthesize not only compound (2) but also the compounds of the present invention with high reproducibility. Therefore, one aspect of the present invention relates to compound (2) and a method for synthesizing compound (2) for synthesizing a compound represented by general formula (1).

[0028] In step 1, 1,4-diaminobenzene is reacted with thiosulfate in the presence of an oxidizing agent to introduce thiosulfate groups into the 2, 3, 5, and 6 positions of 1,4-diaminobenzene. Examples of oxidizing agents that can be used include sodium dichromate (Na2Cr2O7), potassium dichromate (K2Cr2O7), potassium permanganate (KMnO4), sodium peroxodisulfate (Na2S2O8), and potassium peroxodisulfate (K2S2O8).

[0029] In step 2), the compound obtained in step 1) is treated with a strong acid and then with a strong base to form a crosslinked sulfide. The crosslinked sulfide has the following formula: [ka] and forming a polymer, the sulfides at the 2- and 3-positions may each be bridged with the same benzene ring or with different benzene rings.

[0030] In step 3), the crosslinked sulfide is reacted with a base and R 1 I (where R 1 is as defined above) to form a tetra-substituted sulfide of 1,4-diaminobenzene. 1 The compound represented by I can be prepared by a known method, for example, by R 1 It can be obtained by reacting a compound represented by —OH with phosphorus triiodide. 1 The reaction for introducing the group can be carried out by known means. For example, in the case of iodomethane (CH3I), a methyl group can be introduced by reaction in the presence of a base.

[0031] In step 4), the compound of the present invention can be obtained by oxidizing the tetrasubstituted sulfide in the presence of an oxidizing agent. The oxidation reaction can be carried out under known conditions, but the degree of oxidation, i.e., the values ​​of m, n, p, and q, can be controlled by changing the strength of the oxidizing agent, the oxidation reaction time, etc. In the method including these steps 1) to 4), each step may be carried out in one pot, or a step of isolating and purifying the product may be introduced at any stage. Furthermore, as long as a compound represented by general formula (1) is ultimately obtained, reaction steps other than the above steps 1) to 4) may be inserted midway. For example, prior to the oxidation reaction in step 4), the amino group may be converted to a protecting group such as carbamate, amide, imide, or sulfonamide, and after the oxidation reaction, each protecting group can be deprotected by a known method.

[0032] Furthermore, oxidation may produce multiple compounds with different oxidation degrees or different positions of the oxidized sulfur atom. In such cases, each compound can be separated and purified by known purification methods such as column chromatography. After separation into compounds with individual oxidation states, they can be further purified by procedures such as recrystallization. The raw material compounds, reagents, etc. used in the above steps may be commercially available products of commercial grade or may be purified before use.

[0033] As an example, the synthesis scheme for the final product is shown below, assuming that m, n, p, and q are all 2. [ka] In the above formula, compounds with different oxidation degrees can be produced by controlling the conditions of the final oxidation reaction.

[0034] The compound having the sulfonylaniline skeleton represented by general formula (1) can further react with two amino groups in the compound. By further modifying the amino group portion with an organic group, further properties can be introduced into the compound, such as effectiveness as a biological probe. As the organic group, the above-mentioned R 2 Examples of the substituents include those other than hydrogen atoms. Preferred substituents include an N-hydroxysuccinimide (NHS) group. These groups are known as biomolecule binding groups, and the introduction of these groups can provide compounds that are more useful as fluorescent labeling agents. Compounds with an additional organic group introduced into the amino group moiety can be obtained using known organic synthesis methods. For example, they can be synthesized by using a ring-opening reaction of a lactone via an intermediate product having a carboxylic acid. In this method, by selecting dicyclohexylcarbodiimide (DCC) as a condensing agent, the lactone can be reacted with the amino group at the 4-position with high selectivity. See Patent Document 2 for a method of suppressing by-products and increasing yields by optimizing the reaction temperature and concentration.

[0035] One aspect of the present invention is an organic fluorescent material containing a compound having a sulfonylaniline skeleton represented by general formula (1) or a salt thereof. Measurement of the excitation / fluorescence spectrum of the compound having a sulfonylaniline skeleton or a salt thereof reveals that it exhibits a large Stokes shift. The emission energy is lower than the absorption energy, and the emission spectrum has longer wavelengths than the absorption spectrum. The Stokes shift refers to the difference in maximum wavelengths between the absorption and wavelength spectra. In the compound having a sulfonylaniline skeleton or a salt thereof, the large Stokes shift is thought to be caused by the large difference between the structure of the ground state and the structure of the excited state due to photoexcitation. The compound having a sulfonylaniline skeleton or a salt thereof of the present invention exhibits a large Stokes shift, and is therefore expected to be applicable to fluorescent probes and the like that can simultaneously monitor multiple target molecules.

[0036] The compounds or salts thereof having the sulfonylaniline skeleton can emit fluorescence with lower energy light than conventional organic fluorescent materials. Conventional organic fluorescent materials often require high-energy ultraviolet light, making them particularly unsuitable for biological applications. In contrast, the compounds of the present invention have absorption peaks at wavelengths longer than the near-ultraviolet range. Molecular orbital calculations show that some compounds have absorption peaks at wavelengths as long as 500 nm, making them useful as organic fluorescent materials that can prevent damage to biological tissues when administered to living organisms. Furthermore, the group of compounds of the present invention exhibits a high correlation between the oxidation state of the sulfide and the fluorescence wavelength. Specifically, the more the sulfur atom is oxidized, the longer the fluorescence wavelength. In other words, the greater the sum of the values ​​of m, n, p, and q in formula (1), the longer the fluorescence wavelength. Therefore, it is possible to create organic fluorescent materials with different emission colors by varying the molecular design.

[0037] When the optimized structure of the compound having the sulfonylaniline skeleton or its salt is determined by quantum chemical calculation, the molecular size is 250 to 300 Å. 3 This molecular size is equivalent to that of BODIPY (registered trademark), which is considered to have the smallest molecular size. Therefore, the compound having a sulfonylaniline skeleton of the present invention or a salt thereof has the advantages of causing little steric hindrance when labeling biopolymers such as sugar chains, peptides, or proteins having complex structures, and not inhibiting the function of the target molecule.

[0038] Furthermore, most conventional general-purpose dyes have a rigid extended π-conjugated system in their structure, which causes a concentration quenching problem, in which the emission intensity decreases when the dye accumulates to a certain concentration. In contrast, the compounds or salts thereof having a sulfonylaniline skeleton of the present invention exhibit no concentration quenching at all due to the bent structure of the sulfonyl group. Furthermore, because the compounds of the present invention do not exhibit concentration quenching, they emit fluorescence in the solid state that is similar to that in the solution state. Furthermore, while it is well known that fluorescent materials can have different emission wavelengths when dissolved in a solvent due to interference from the solution, the compounds of the present invention emit fluorescence with similar wavelengths regardless of the type of solution. Furthermore, contrary to predictions based on their molecular structure, the compounds of the present invention are surprisingly water-soluble.

[0039] Furthermore, the absorption wavelength and fluorescence wavelength of the compound of the present invention can be predicted with good accuracy by molecular orbital calculation.

[0040] Among the compounds having a sulfonylaniline skeleton or salts thereof, R 2 Compounds in which at least one of the groups is not a hydrogen atom form an NHS ester in their structure, which serves as a binding group for biomolecules. NHS esters are activated esters of organic carboxylic acids that react quickly and selectively with amino groups on biomolecules to form stable amide bonds. For this reason, they are widely used to label glycans and peptides.

[0041] Among the compounds having a sulfonylaniline skeleton, R 2 The compounds in which at least one of the groups is not a hydrogen atom have an NHS ester bond, which makes them highly polar and water-soluble. Furthermore, because they do not have ionic functional groups, their fluorescence intensity and wavelength do not change depending on the environment (pH, polarity, ultraviolet light, etc.), and they exhibit stable luminescence properties. [Example]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0043] [Synthesis of compounds with sulfonylaniline skeleton] [Manufacturing Example 1] [Synthesis of 1,4-diamino-2,3,5,6-tetra(methylmercapto)benzene] Synthesis of Compound 2 [ka] The reagents and their amounts used in the synthesis of Compound 2 are as follows.

[0044] [Table 1]

[0045] p-Phenylenediamine, acetic acid, and ice were added to a 500 mL three-neck flask and stirred at room temperature for 1.5 minutes. To this, aqueous sodium thiosulfate solution (110 mL) and aqueous sodium dichromate solution (110 mL) were simultaneously added so that the mixture filled up completely within 7 seconds. The mixture was then stirred at room temperature for 2.5 minutes, potassium chloride was added, and the mixture was stirred for 5 minutes. The reaction solution was then filtered. The filter cake was transferred to a 1 L beaker and dissolved in water (300 mL). Saturated aqueous potassium chloride solution (300 mL) was added, and the mixture was allowed to stand at room temperature for 2.5 hours. The precipitated solid was filtered under suction to obtain compound 2 (13.9 g, 42%) as a yellow solid.

[0046] Synthesis of Compound 3 [ka] The reagents and their amounts used in the synthesis of compound 3 are as follows.

[0047] [Table 2]

[0048] Compound 2, hot water, and concentrated hydrochloric acid were placed in a 1 L two-neck flask and refluxed with stirring for 30 minutes. The mixture was then cooled to 0°C and filtered. The filter cake was transferred to a 200 mL beaker, water was added, and the mixture was stirred at room temperature for approximately 30 minutes. A 3 M aqueous solution of sodium hydroxide was then added and the mixture was stirred at room temperature for approximately 30 minutes. The mixture was filtered and washed with water, acetone, and methanol to obtain compound 3 (7.88 g, 92%) as a black powder.

[0049] Synthesis of Compound 4 [ka] The reagents and their amounts used in the synthesis of Compound 4 are as follows.

[0050] [Table 3]

[0051] Compound 3, sodium borohydride, 3 M aqueous sodium hydroxide, and tetrahydrofuran (THF) were added to a nitrogen-purged 1 L three-neck flask and stirred at 45 °C for 2 days. The mixture was then cooled to 0 °C, and methyl iodide was added dropwise over 1.5 hours, followed by stirring for 1.5 hours. After completion of the reaction, the THF was evaporated, and the reaction solution was transferred to a separatory funnel and separated into an organic layer and an aqueous layer by adding dichloromethane (150 mL). The aqueous layer was extracted with dichloromethane (150 mL x 3) and combined with the organic layer. The resulting organic layer was dried over anhydrous sodium sulfate and concentrated. The reaction mixture was then purified by neutral silica gel column chromatography (CHCl) to give compound 4 (804 mg, 16%) as a tan solid.

[0052] [Example 1] [Synthesis of monoxide] [ka] The reagents and their amounts used in the synthesis of 1oxide are as follows:

[0053] [Table 4]

[0054] Compound 4 and ethyl acetate were added to a 100 mL recovery flask and stirred at room temperature for 10 minutes. Water and NaOCl·5H2O were then added and stirred at room temperature for 18 minutes. Saturated aqueous sodium sulfite was then added and stirred to confirm the disappearance of the oxidizing agent. The reaction solution was transferred to a separatory funnel and toluene (50 mL) was added to separate the organic and aqueous layers. The aqueous layer was extracted with toluene (50 mL x 3) and combined with the organic layer. The resulting organic layer was dried over anhydrous sodium sulfate and concentrated. The reaction product was then purified by silica gel column chromatography (NH silica gel, ethyl acetate:hexane = 2:3) to obtain 1oxide (21.7 mg, 10%) as a brown solid. 1oxide: 1 H NMR (500 MHz, CDCl3):δ = 6.15 (s, 2H, -NH2), 5.00 (s, 2H, -NH2), 3.01 (s, 3H, -CH3), 2.39 (s, 3H, -CH3), 2.35 (s, 3H, -CH3), 2.33 (s, 3H, -CH3). HRMS(FD + ): m / z calcd for C 10 H 16 N2O1S4[M + ]: 308.01455; found 308.01492.

[0055] [Example 2] [Synthesis of dioxide] [ka] The reagents and their amounts used in the synthesis of dioxide are as follows:

[0056] [Table 5]

[0057] Sodium tungstate dihydrate, hydrogen peroxide, and acetic acid were added to a 100 mL two-neck flask and stirred at room temperature for 10 minutes. Toluene and compound 4 were added and stirred at -50°C for 3 hours. Methyl sulfide was then added and stirred to confirm the disappearance of the oxidant. The reaction solution was concentrated, and the resulting gray solid was separated and purified by neutral silica gel column chromatography (dichloromethane:acetone = 5:1) to give 1oxide (19.8 mg, 9.4%) as a brown solid and 2oxide (4.4 mg, 1.9%) as a yellow solid. 2oxide: 1 H NMR (500 MHz, CDCl3):δ = 6.09 (s, 4H, -NH2), 3.02 (s, 6H, -CH3), 2.32 (s, 6H, -CH3).

[0058] [Example 3] [Synthesis of tetraoxide-a / tetraoxide-b / pentoxide] [ka] The reagents and their amounts used in Example 3 are as follows:

[0059] [Table 6]

[0060] Sodium tungstate dihydrate, hydrogen peroxide, and acetic acid were added to a 100 mL two-neck flask and stirred at room temperature for 10 minutes. Toluene and compound 4 were added and stirred at 0°C for 1 hour. Methyl sulfide was then added and stirred to confirm the disappearance of the oxidizing agent. The reaction solution was concentrated, and the resulting orange oil was separated and purified by neutral silica gel column chromatography (ethyl acetate:methanol = 20:1) to give 4oxide-a (6 mg, 2.5%), 4oxide-b (4.1 mg, 1.7%), and 5oxide (6.5 mg, 2.4%) as yellow solids. 4oxide-a: 1 H NMR (500 MHz, CDCl3):δ = 6.75 (s, 4H, -NH2), 3.17 (s, 12H, -CH3); 13 C NMR (125 MHz, CDCl3):δ =141.02, 125.47, 36.99 ppm. 5oxide: 1 H NMR (500 MHz, CDCl3):δ = 7.04 (s, 2H, -NH2), 6.89 (s, 2H, -NH2), 3.32 (s, 3H, -CH3), 3.23 (s, 3H, -CH3), 3.21 (s, 3H, -CH3) , 3.20 (s, 3H, -CH3); 13 C NMR (125 MHz, CDCl3): δ=141.81, 139.62, 129.66, 128.10, 126.36, 123.25, 44.42, 37.09, 36.31, 29.71 ppm.

[0061] [Comparative Example 1] Fluorescein-NHS-ester A commercially available product from Tokyo Chemical Industry Co., Ltd. was used. [ka]

[0062] [Comparative Example 2] BODIPY-NHS-ester A commercially available product from Tokyo Chemical Industry Co., Ltd. was used. [ka]

[0063] <Evaluation as an organic fluorescent material> [Optical properties] To investigate the optical properties of the sulfonylaniline dyes of Examples 1 to 5, absorption spectra (double beam spectrophotometer U-2810, manufactured by Hitachi High-Tech Science Corporation) and excitation / fluorescence spectra (spectrofluorophotometer RF-6000, manufactured by Shimadzu Corporation) were measured using water, taking into consideration cell imaging. The excitation / emission wavelengths were measured using a fluorescence spectrophotometer. The quantum efficiency is the absolute luminous quantum efficiency measured by placing a sample in an integrating sphere. The Stokes shift was determined from the difference in peak positions of the excitation and fluorescence spectra.

[0064] [Molecular size] Molecular volumes (Å) were optimized by quantum chemical calculations using Gaussian 09 (B3LYP / 6-31g(d)). 3 ) was sought.

[0065] (1) Excitation / emission wavelength The sulfonylaniline dyes of the examples exhibited blue to yellow fluorescence despite having a single benzene ring in their cyclic structure. This is thought to be due to intramolecular hydrogen bonding and a push-pull structure. Another factor is that, unlike conventional dyes, the molecular size does not change because the emission wavelength varies depending on the arrangement of the substituents. Furthermore, the compounds of the examples tended to exhibit a fluorescent color that shifted toward longer wavelengths depending on the degree of oxidation of each compound, demonstrating that they possess fluorescent wavelengths independent of the type of solvent. No significant differences were observed in the fluorescent wavelengths of the various solvents used for measuring the fluorescent wavelength. The fluorescent wavelength peaks (nm) of compound 4 and each of the examples in dichloromethane (DCM), acetonitrile (MeCN), methanol (MeOH), and water are summarized in the table below.

[0066] [Table 7]

[0067] (2) Molecular size The sulfonylaniline dyes of the examples have smaller molecular sizes than conventional small-molecule fluorescent dyes, and their volumes are comparable to those of BODIPY in Comparative Example 2, which has the smallest molecular size. Therefore, the sulfonylaniline dyes of the present invention have the advantage of less steric hindrance when labeling biopolymers such as sugar chains, peptides, or proteins with complex structures. Furthermore, their small molecular size is also advantageous in that they do not inhibit the function of target molecules.

[0068] (3) Stokes shift The sulfonylaniline dyes of the examples exhibited extremely large Stokes shifts of at least 87 nm, comparable to those of quantum dots despite being organic low-molecular-weight compounds. The Stokes shift values ​​can be determined from Table 1 above. This is because the sulfonylaniline dyes are less rigid than conventional dyes such as the general-purpose dyes of Comparative Examples 1 and 2, and therefore have different structures in the excited and ground states. Clear superiority was observed compared to the fluorescein-NHS-ester (27 nm) of Comparative Example 1 and the BODIPY-NHS-ester (6 nm) of Comparative Example 2.

[0069] (4) Quantum efficiency Although the compounds of the present invention are somewhat insufficient compared to the general-purpose dyes of Comparative Examples 1 and 2, they exhibit stable quantum efficiency and can be said to have a sufficiently practical quantum efficiency. Furthermore, since they exhibit fluorescence even in the solid state, it can be said that the effect of concentration quenching is extremely small. Table 8 shows the quantum efficiencies of Compound 4 and the compounds of the Examples for each solvent.

[0070] [Table 8]

[0071] (5) Concentration quenching Most general-purpose dyes, including those in Comparative Examples 1 and 2, have rigid conjugated systems and therefore exhibit concentration quenching. On the other hand, the sulfonylaniline dyes in the Examples have a unique molecular structure that is completely different from conventional extended π-conjugated systems, and therefore not only do they not exhibit concentration quenching at all, but also exhibit solid-state fluorescence. This property is due to the bent structure of the sulfonyl group. The peak fluorescence wavelengths and quantum efficiencies in the solid state are summarized in Table 9 below.

[0072] [Table 9]

[0073] [Optical properties calculated by quantum chemical calculations] The theoretical values ​​of absorption and fluorescence wavelengths can be determined from the molecular structure of each compound using quantum chemical calculations. Therefore, for some compounds represented by general formula (1), the absorption and fluorescence wavelengths of each compound were calculated using quantum chemical calculations. The Gaussian16 program was used for the calculations. The polarizable continuum model (PCM) was used to account for the solvent effect (water). Absorption wavelengths were calculated using the linear response (LR) model with B3LYP / 6-31+G(d,p). Fluorescence wavelengths were calculated using the state-specific (SS) model with wB97XD / 6-31+G(d,p). These calculation methods are commonly used to calculate absorption or fluorescence wavelengths. The theoretical values ​​of the absorption wavelength and the fluorescence wavelength calculated by the above calculation are shown below.

[0074] [ka]

[0075] [Calculation of optical properties of NHS bodies by quantum chemical calculations] A compound incorporating an N-hydroxysuccinimide (NHS) structure, useful as a fluorescent probe, was synthesized from 5oxide according to the method described in Patent Document 2, and the absorption and fluorescence wavelengths were calculated by quantum chemical calculations. The absorption and fluorescence wavelengths of the resulting compound (5oxide-NHS) were calculated to be 411 nm / 607 nm. [ka] The emission wavelength of the NHS-modified pentoxide calculated in the previous section is slightly longer than that of the non-NHS-modified pentoxide, but this supports the idea that NHS modification does not sacrifice optical properties. [Industrial Applicability]

[0076] The compounds of the present invention are not only useful as organic fluorescent materials themselves, but can also serve as suitable fluorescent probes for labeling complex structures such as sugar chains and peptide chains. Therefore, they enable multicolor imaging and high-precision imaging, and are expected to contribute to a wide range of fields, including new drug development, clinical testing, and life science research.

Claims

1. The following general formula (1): 【Chemical 1】 (In the formula, R １ is independently in each occurrence an alkyl group, a fluoroalkyl group, a phenyl group, a phenyl group substituted with a halogen atom or an amino group or an alkoxy group, a naphthyl group, a thienyl group, a thienyl group substituted with a halogen atom, a thiazolyl group, a thiazolyl group substituted with a halogen atom, a pyridyl group, or a pyridyl group substituted with a halogen atom; R ２ is independently in each occurrence a hydrogen atom, a group —RH or one of the following: 【Chemistry 2】 (In each of the above formulas, R is a linear or branched alkylene group having 1 to 18 carbon atoms, which may contain one or more heteroatoms in the chain, or a cyclic alkylene group having 3 to 18 carbon atoms, and * represents a bond to the nitrogen atom), m, n, p, and q each independently represent 0, 1, or 2, provided that any one of m, n, p, and q is 1 or 2. A compound or a salt thereof represented by the formula:

2. R １ and each represent an alkyl group having 1 to 6 carbon atoms, or a salt thereof, according to claim 1.

3. R １ and each represent a methyl group, or a salt thereof, according to claim 1 or 2.

4. R ２ The compound or salt thereof according to any one of claims 1 to 3, wherein each of

5. R ２ One of them is: 【Chemistry 3】 (wherein R and * are as defined in claim 1)

6. The compound or salt thereof according to any one of claims 1 to 5, wherein the sum of m, n, p, and q is 3 to 5.

7. The compound or salt thereof according to any one of claims 1 to 4, which is a compound selected from the group consisting of: 【Chemistry 4】

8. 1) reacting 1,4-diaminobenzene with thiosulfate in the presence of an oxidizing agent; 2) a step of treating the compound obtained in the step 1) with a strong acid and then with a strong base to form a crosslinked sulfide; 3) The crosslinked product of the sulfide is reacted with a base and R １ I (where R １ is as defined in claim 1) to form a tetra-substituted sulfide of 1,4-diaminobenzene; and 4) Oxidizing the tetrasubstituted sulfide in the presence of an oxidizing agent. A method for producing the compound or salt thereof according to any one of claims 1 to 7, comprising:

9. An organic fluorescent material comprising the compound or salt thereof according to any one of claims 1 to 7.

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