Fluorescent compounds and fluorescently labeled biomaterials using the same

Abstract

A fluorescent compound of the following formula or a salt thereof, and a labeled biological substance.　Y represents -NR10R11 or -OR12, R1-R12, R21-R23, and Z represent specific groups and satisfy the following provisions.　R21 and / or R22 is an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group. When R21=aryl group and R22=alkyl group or aryl group, R7 and R8 and / or R10 and R11 bond to form a 4-membered ring.　A compound of formula (I) or (II) or salt thereof satisfies condition α, and a compound of formula (III) or (IV) or salt thereof satisfies condition β. (Condition α) When an aryl group, Z has a specific structure. (Condition β) R7 and R8 and / or R10 and R11 bond to form a 4-membered ring.

Description

[Technical Field]

[0001] This invention relates to fluorescent compounds and fluorescently labeled biomaterials using the same. [Background technology]

[0002] In pathological examinations and the identification of infectious diseases, fluorescently labeled biomolecules are used, which are biomolecules that bind to the target substance with a compound (dye). In particular, bioimaging technology, which analyzes the dynamics and functions of biomolecules, cells, and tissues in living organisms, is being utilized in the diagnosis of various diseases. In recent years, biofluorescence imaging, which visualizes and observes specific parts of living organisms using fluorescent dyes, has been attracting attention as a new technology for observing living organisms. In biofluorescence imaging, organic fluorescent dyes are generally used. However, organic fluorescent dyes have poor lightfastness and degrade when exposed to excitation light, which can prevent sufficient observation of the target organism.

[0003] Rhodamine is known as a fluorescent dye with high fluorescence quantum yield and high lightfastness (resistance to photofading), and is used as a dye in fluorescently labeled biomaterials. On the other hand, as described in Patent Documents 1 to 7, research is also underway on fluorescent dyes in which the absorption and fluorescence wavelengths are lengthened by replacing the oxygen atoms, which are ring constituent atoms of the xanthene skeleton of rhodamine, with silicon atoms or phosphorus atoms. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2020 / 033681 [Patent Document 2] International Publication No. 2014 / 106957 [Patent Document 3] International Publication No. 2014 / 144793 [Patent Document 4] U.S. Patent Application Publication No. 2018 / 0284105 [Patent Document 5] U.S. Patent Application Publication No. 2019 / 0100653 [Patent Document 6] U.S. Patent No. 8506655 [Patent Document 7] International Publication No. 2018 / 043579 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, our investigations have revealed that the rhodamine compounds described in the above-mentioned Patent Documents 1 to 7 are not sufficient in terms of achieving both excellent fluorescence quantum yield and excellent light resistance. Furthermore, there is a demand for compounds in which the oxygen atoms in the xanthene skeleton of rhodol are replaced with silicon or phosphorus atoms, resulting in compounds that exhibit both excellent fluorescence quantum yield and excellent photostability.

[0006] The present invention aims to provide a fluorescent compound, which is a rhodamine or rhodol compound in which the oxygen atom, a ring constituent atom of the xanthene ring, is replaced with a silicon atom or a phosphorus atom, and which can achieve both excellent fluorescence quantum yield and excellent photostability. The present invention also aims to provide a fluorescently labeled biomaterial obtained by bonding this fluorescent compound with a biomaterial. [Means for solving the problem]

[0007] In other words, the above-mentioned problems of the present invention were solved by the following means. [1] A fluorescent compound represented by any of the following general formulas (I) to (IV), or a salt thereof. [ka] In the formula, Y is -NR 10 R 11 OR 12 This indicates that Z represents the group shown in formula (A) below. R 1 ~R 6 represents a halogen atom, a cyano group or a group represented by the following formula (A). R 7 ~R 12 represents a group represented by the following formula (A). R 7 and R 8 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring, and R 10 and R 11 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. Further, R 7 ~R 11 each may be bonded to an adjacent R 2 ~R 5 to form a 5- to 7-membered aliphatic heterocyclic ring or aromatic heterocyclic ring. R 21 and R 22 represent a group represented by the following formula (A), and R 21 and R 22 may be bonded to each other to form a 4- to 7-membered aliphatic heterocyclic ring. However, at least one of R 21 and R 22 is an alkenyl group, an alkynyl group, an aryl group or a heteroaryl group. Further, when R 21 is an aryl group and R 22 is an alkyl group or an aryl group, at least one of each combination of R 7 and R 8 , or R 10 and R 11 is bonded to each other to form a 4-membered aliphatic heterocyclic ring. R 23 represents a group represented by the following formula (A).

Chemical formula

[10] The fluorescent compound or salt thereof described above is the fluorescent compound or salt thereof described in [1], which is represented by the general formula (III) or (IV) described above.

[11] The above R 7 ~R 11 The fluorescent compound or salt thereof according to

[10] , wherein at least one of the groups is an electron-withdrawing group.

[12] The above R 23 A fluorescent compound or salt thereof according to

[10] or

[11] , wherein is a hydroxyl group, an alkoxy group, an aryl group, or an alkyl group. However, the above R 23 If the group is a hydroxyl group, the hydrogen atoms in this hydroxyl group may be dissociated.

[13] A fluorescently labeled biomaterial obtained by binding a fluorescent compound or a salt thereof described in any one of [1] to

[12] to a biomaterial.

[14] The fluorescently labeled biomaterial described in

[13] , wherein the above-mentioned biomaterial is one of a protein, amino acid, nucleic acid, glycan, or lipid.

[15] The fluorescently labeled biomaterial according to

[13] or

[14] , wherein the binding of the above-mentioned fluorescent compound or salt thereof to the above-mentioned biomaterial is by any of the following i) to v). i) Non-covalent or covalent bonds between peptides, ii) Van der Waals interactions between long-chain alkyl groups in fluorescent compounds and lipid bilayers or lipids in biomolecules. iii) An amide bond formed by reacting an N-hydroxysuccinimide ester in a fluorescent compound with an amino group in a biomolecule, iv) A thioether bond formed by reacting a maleimide group in a fluorescent compound with a sulfanyl group in a biomaterial, v) A bond involving the formation of a triazole ring, which is formed by subjecting an azide group in a fluorescent compound and an acetylene group in a biological substance, or an acetylene group in a fluorescent compound and an azide group in a biological substance, to a Click reaction

Advantages of the Invention

[0008] The fluorescent compound of the present invention is a compound that can achieve both excellent fluorescence quantum yield and excellent light resistance. Further, the fluorescently labeled biological substance of the present invention is obtained by using a fluorescent compound excellent in fluorescence quantum yield and light resistance.

Modes for Carrying Out the Invention

[0009] In the present invention, when there are a plurality of substituents or linking groups (hereinafter referred to as substituents, etc.) represented by specific symbols or formulas, or when a plurality of substituents, etc. are defined simultaneously, unless otherwise specified, each of the substituents, etc. may be the same as or different from each other. This also applies to the definition of the number of substituents, etc. Further, when a plurality of substituents, etc. are close to each other (particularly when adjacent), unless otherwise specified, they may be linked to each other to form a ring. Further, unless otherwise specified, a ring, such as an alicyclic ring, an aromatic ring, and a heterocyclic ring, may be further condensed to form a condensed ring. In the present invention, unless otherwise specified, for a double bond, when both E-type and Z-type exist in the molecule, either one or a mixture thereof may be present. For example, in the compound represented by any one of General Formulas (II) and (IV), the substituent R on the nitrogen atom 9 and the carbon atom to which R 5 is bonded are shown for convenience as having a structure on the same side with respect to the double bond. However, the substituent R on the nitrogen atom 9 and R 5The structure may have the carbon atom to which it is bonded on opposite sides of the double bond, or it may be a mixture of these structures, and these compounds are included in the compounds represented by either general formula (II) or (IV). Furthermore, unless otherwise specified, if diastereomers and enantiomers exist as a compound, it may be either one of them or a mixture thereof.

[0010] In this invention, the designation of compounds and substituents includes not only the compound itself and the substituent itself, but also its salt and its ion. For example, a carboxyl group, a sulfo group or R 23 Groups having dissociable hydrogen atoms, such as hydroxyl groups, that can be adopted as such may have the hydrogen atoms dissociate to form the corresponding ionic or salt structure. In the case of a salt structure, there may be only one type of salt, or two or more types may be mixed together, and the compound may contain a mixture of salt-type and free acid-type groups, as well as a mixture of salt-type compounds and free acid-type compounds. The term "salt" above includes forms that form a salt within the molecule of the fluorescent compound of the present invention. For example, a carboxyl group, a sulfo group, or R 23 Examples of hydroxyl group salts that can be used include alkali metal salts such as Na, Li, and K, alkaline earth metal salts such as Mg, Ca, and Ba, and organic amine salts such as tetraalkylammonium. Also, for example, carboxyl groups, sulfo groups, or R 23 The hydroxyl group that can be adopted has an ionic structure, R 7 and R 8 One example is a form in which a salt is formed intramolecularly by bonding with a positively charged nitrogen atom. The term "ion" above refers to an atom or group of atoms that possesses a negative or positive charge.

[0011] In the compounds of the present invention, for example, those represented by general formulas (I) or (III), the positive charge of the compound is conveniently identified and shown as the structure possessed by a specific nitrogen atom. However, since the compounds of the present invention have a conjugated system, in reality, other atoms besides the nitrogen atom may also take on a positive charge, and any compound that can take on the structure represented by general formula (I) or (III) as one of its chemical structures is included in the compounds represented by general formulas (I) or (III). The same applies to negative charges. The same also applies to the compounds represented by each of the other general formulas. Furthermore, this includes compounds in which a part of the structure has been altered to the extent that it does not impair the effects of the present invention. Moreover, for compounds in which substitution or unsubstituted is not specified, it means that they may have any substituents to the extent that it does not impair the effects of the present invention. This also applies to substituents (for example, groups expressed as "alkyl group," "methyl group," "methyl," etc.) and linking groups (for example, groups expressed as "alkylene group," "methylene group," "methylene," etc.). Among such arbitrary substituents, the substituents preferred in the present invention are those selected from the substituent group T described later. In the present invention, "rhodamine compound" means a rhodamine compound in which the 3rd and 6th positions of the xanthene ring are substituted with amino groups, and "lodol structure" means a lodol compound in which the 3rd or 6th position of the xanthene ring is substituted with an amino group. Furthermore, in the present invention, Si-rhodamine compound or Si-lodol compound means a rhodamine compound or lodol compound in which the oxygen atom, which is a ring constituent atom of the xanthene skeleton, is replaced with a silicon atom, and P-rhodamine compound or P-lodol compound means a rhodamine compound or lodol compound in which the oxygen atom, which is a ring constituent atom of the xanthene skeleton, is replaced with a phosphorus atom. Furthermore, in the compounds of the present invention, R 1 ~R 9 Adjacent substituents among Y and Z may bond to each other to form a ring, thus forming a fused ring structure. The number of rings formed is not particularly limited as long as it is structurally feasible, and multiple rings may be formed. In this invention, when specifying the number of carbon atoms in a group, this number of carbon atoms refers to the total number of carbon atoms in the group unless otherwise specified in this invention or specification. That is, if the group has further substituents, this refers to the total number of carbon atoms including those substituents.

[0012] Furthermore, in this invention, a numerical range represented using "~" means a range that includes the numerical values ​​written before and after "~" as the lower limit and upper limit, respectively.

[0013] <Fluorescent compounds represented by any of the general formulas (I) to (IV)> The fluorescent compounds represented by any of the general formulas (I) to (IV) of the present invention, or salts thereof, are as follows and emit fluorescence. Hereinafter, the fluorescent compounds represented by any of the general formulas (I) to (IV) of the present invention, or salts thereof, will also be simply referred to as the fluorescent compounds of the present invention.

[0014] [ka]

[0015] In the formula, Y is -NR 10 R 11 OR 12 This indicates that Z represents the group shown in formula (A) below. R 1 ~R 6 represents a halogen atom, a cyano group, or a group represented by the following formula (A). R 7 ~R 12 R represents the group shown in formula (A) below. 7 and R 8 They may be bonded to each other to form a 4-7 membered aliphatic heterocycle, R 10 and R 11 They may be bonded to each other to form a 4- to 7-membered aliphatic heterocycle. Also, R 7 ~R 11 Each of the adjacent R 2 ~R 5 It may bond with other elements to form a 5-7 membered aliphatic or aromatic heterocycle. R21 and R 22 R represents the group shown in formula (A) below, 21 and R 22 These may be bonded to each other to form a 4- to 7-membered aliphatic heterocycle. However, R 21 and R 22 At least one of them is an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group. Also, R 21 is an aryl group, 22 If R is an alkyl group or an aryl group, 7 and R 8 , or R 10 and R 11 At least one of each combination is bonded to each other to form a four-membered aliphatic heterocycle. R 23 This represents the group shown in formula (A) below. However, the compound represented by formula (I) or (II) above, or a salt thereof, satisfies condition α described below, and the compound represented by formula (III) or (IV) above, or a salt thereof, satisfies condition β described below.

[0016] The substituents in formulas (I) to (IV) will be described in detail below.

[0017] (Base represented by formula (A))

[0018] [ka]

[0019] In the formula, L 3 This indicates a single bond or a linking group consisting of one or more of the alkylene group, alkenylene group, alkynylene group, arylene group, heteroarylene group, and any of the groups represented by formulas (1-1) to (1-8) below. R 111 This represents a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. However, R 111If the hydrogen atom in is a dissociable hydrogen atom, this dissociable hydrogen atom may be dissociated. Also, L 3 and R 111 Each group in may have further substituents. * indicates a connection point.

[0020] Furthermore, for the base represented by formula (A), L is defined according to the following rules (i) and (ii). 3 and R 111 This shall be interpreted as follows. (i) When the group represented by formula (A) has a group represented by any of the general formulas (1-1) to (1-8) *From the (bonding part) to the group represented by any of the general formulas (1-1) to (1-8) L 3 It is interpreted as follows. For example, if the group represented by formula (A) is a carboxyphenyl group, then the group represented by formula (A) is L 3 : A linking group formed by combining a phenylene group with a group represented by formula (1-4) and a group represented by formula (1-1), and R 111 : is a group represented by a hydrogen atom. Also, if the group represented by formula (A) is a sulfoalkyl group, the group represented by formula (A) is L 3 : A linking group formed by combining an alkylene group with a group represented by formula (1-7) and a group represented by formula (1-1), and R 111 : A group represented by a hydrogen atom. However, groups represented by any of the general formulas (1-1) to (1-8) do not constitute a ring as ring-forming atoms. Furthermore, if the group represented by formula (A) contains two or more groups represented by general formulas (1-1) to (1-8), then in the group represented by formula (A), the group represented by general formulas (1-1) to (1-8) located at the position where the minimum number of bonded atoms from * (bonding part) to any of the groups represented by general formulas (1-1) to (1-8) is maximized is L 3 It is interpreted as follows. (ii) When the group represented by formula (A) does not have a group represented by any of the general formulas (1-1) to (1-8) The alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, or monovalent aliphatic heterocyclic group located at the terminal structure of the group represented by formula (A) is R 111 It is interpreted as follows. For example, if the group represented by formula (A) is a phenyl group, then the group represented by formula (A) is L 3 : Single bond and R 111 : This is a group represented by a phenyl group. Also, if the group represented by formula (A) is -CH2=CHPh, then the group represented by formula (A) is L 3 :-CH2=CH- and R 111 This is a group represented by :Ph, where Ph represents the phenyl group. However, "terminal structure in the group represented by formula (A)" means the structure located at the very end of the longest bonding chain in the group represented by formula (A), counting from the * (bonding site). In (i) and (ii) above, if the group represented by formula (A) is a group having substituents that can be bonded to biomolecules as described later, we read the group obtained by replacing the substituents that can be bonded to biomolecules as described later in the group represented by formula (A) with hydrogen atoms. That is, for substituents that can be bonded to biomolecules as described later, L 3 or R 111 Each group in is interpreted as an optional substituent that it may have. Furthermore, within the scope equivalent to the provisions of (i) and (ii) above, R 111 or L 3 Each of the groups defined by may have further substituents.

[0021] (L 3 ) L 3 The alkylene group that can be chosen is equivalent to a group obtained by removing one more hydrogen atom from an alkyl group selected from the substituent group T described later, and the preferred group is also the same. L 3 The alkenylene group that can be chosen is equivalent to the group obtained by removing one more hydrogen atom from an alkenyl group selected from the substituent group T described later, and the preferred group is also the same. L 3The alkynylene group that can be chosen is equivalent to the group obtained by removing one more hydrogen atom from the alkynyl group selected from the substituent group T described later, and the preferred group is also the same. L 3 The arylene group that can be adopted is equivalent to an aryl group selected from the substituent group T described later, from which one more hydrogen atom has been removed, and the preferred group is also the same. L 3 The heteroarylene groups that can be adopted are equivalent to the heteroaryl groups selected from the substituent group T described later, from which one more hydrogen atom has been removed, and the preferred ones are also the same. L 3 The alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group that can be selected may be an unsubstituted group or a substituted group. L 3 The substituents that the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may have are not particularly limited, and are preferably selected from the substituent group T described later, and more preferably halogen atoms, alkyl groups, or alkoxy groups. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms, with fluorine atoms or chlorine atoms being preferred. Also, L 3 The number of substituents that the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may have is not particularly limited as long as it can be adopted structurally, and can be at least one or more. There is no particular upper limit, for example, all hydrogen atoms in the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group may be substituted with substituents.

[0022] L 3In linking groups that can be adopted as alkylene groups, alkenylene groups, alkynylene groups, arylene groups, heteroarylene groups, and linking groups that combine one or more of the groups represented by any of the following formulas (1-1) to (1-8), the types of groups to be combined are not particularly limited as long as a reasonable chemical structure is obtained. For example, L 3 In this case, groups consisting of two or more consecutive groups represented by any of the following formulas (1-1) to (1-3) are not included. L 3 In a linking group that can be adopted as such, one or more groups from alkylene groups, alkenylene groups, alkynylene groups, arylene groups, heteroarylene groups, and any of the following formulas (1-1) to (1-8) are combined, and the types of groups to be combined are not particularly limited, but for example, 1 to 6 types are preferred, and 1 to 4 types are more preferred. L 3 In a linking group that can be adopted as such, one or more groups from alkylene groups, alkenylene groups, alkynylene groups, arylene groups, heteroarylene groups, and any of the following formulas (1-1) to (1-8) are combined, and the number of groups to be combined is not particularly limited, but for example, 1 to 10 is preferred, 1 to 6 is more preferred, and 1 to 4 is even more preferred.

[0023] L 3 The bases that can be represented by any of the equations (1-1) to (1-8) are as follows:

[0024] [ka]

[0025] In the formula, R 31 and R 32 represents a hydrogen atom or substituent. * indicates a connection point.

[0026] R 31 The substituents that can be taken are not particularly limited, and are preferably selected from the substituent group T described later. 31Examples thereof include a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, an acyl group or a sulfonyl group, with a hydrogen atom or an alkyl group being more preferred and a hydrogen atom being even more preferred. Note that R 31 The alkyl group, aryl group, heteroaryl group, acyl group and sulfonyl group that can be adopted as R may each be an unsubstituted group or a group having a substituent. R 32 The substituent that can be adopted as R is not particularly limited and is preferably selected from the substituent group T described later. R 32 Examples thereof include a hydrogen atom, a hydroxy group, an alkoxy group, an aryloxy group, an alkyl group, an aryl group or a heteroaryl group, with a hydroxy group, an alkoxy group or an aryloxy group being more preferred and a hydroxy group being even more preferred.

[0027] As the group formed by combining the groups represented by any of the above formulas (1-1) to (1-8), groups represented by any of the following formulas (1A-1) to (1A-9) are preferably mentioned.

[0028]

Chemical formula

[0029] R 31 and R 32 are synonymous with the above R 31 and R 32 respectively. * and ** indicate bonding sites. Note that ** indicates the bonding site with the R 3 side when it is a group that can be adopted as L 111 Formula (1A-2) may be bonded to R 3 on either * side in L 111 respectively.

[0030] The above L 3As a linking group that can be adopted, a group represented by any of the above formulas (1-1) to (1-8) is preferably a group represented by any of the above formulas (1A-1), (1A-2), or (1A-4), more preferably a group represented by the above formulas (1A-1) or (1A-2), and even more preferably a group represented by the above formula (1A-1). For example, the group represented by the above formula (1A-1) and R 111 The group represented by a hydrogen atom corresponds to a carboxyl group, and the group represented by the above formula (1A-4) and R 111 A group represented by a hydrogen atom as a carboxyl group corresponds to a sulfo group. Furthermore, a group formed by the dissociation of a hydrogen atom from this carboxyl group as a dissociable hydrogen atom corresponds to a carboxyl group having an ionic or salt structure, and a group formed by the dissociation of a hydrogen atom from this sulfo group as a dissociable hydrogen atom corresponds to a sulfo group having an ionic or salt structure.

[0031] Also, L 3 As such, a linking group may be a linking group formed by combining any of the groups represented by formulas (1-1) to (1-8) or a combination thereof with at least one of the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group. Alternatively, a linking group may be formed by linking two or more of the groups represented by formulas (1-1) to (1-8) or a combination thereof via a group formed by combining one or more of the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group. L 3 Specific examples of linking groups that combine two or more types that can be taken include, for example, a linking group that combines at least two (preferably 2 to 4) of alkylene groups, alkenylene groups, alkynylene groups, arylene groups, and heteroarylene groups, and a linking group that combines at least one (preferably 1 to 4) of alkylene groups, arylene groups, and heteroarylene groups with at least one (preferably 1 to 4) represented by any of formulas (1-1) to (1-8).

[0032] (R 111 ) R111 If the hydrogen atom that can be taken as is a dissociable hydrogen atom, the group represented by formula (A) may form an ionic structure or a salt structure when the dissociable hydrogen atom dissociates. This will be explained in subsequent R 111 The same applies to the description relating thereto. For a hydrogen atom to be dissociable, for example, its acid dissociation constant (pKa) is 10 or less, preferably 7 or less, and more preferably 5 or less. The above acid dissociation constant refers to the value at 25°C in water.

[0033] R 111 The alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and monovalent aliphatic heterocyclic groups that can be selected are, in each case, equivalent to the corresponding groups in substituent group T, and the preferred ones are also the same. R 111 The alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and monovalent aliphatic heterocyclic groups that can be selected as such may all be unsubstituted groups or substituted groups. R 111 The substituents that each of the above groups that can be adopted as such may have are not particularly limited and include groups selected from the substituent group T described later, preferably halogen atoms. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms, with fluorine atoms being preferred. They may also have substituents that can be bonded to biomolecules, as described later. R 111 The number of substituents that each of the above groups that can be adopted as such may have is not particularly limited as long as it is structurally feasible, and can be at least one. There is no particular upper limit, and for example, all hydrogen atoms in alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, and monovalent aliphatic heterocyclic groups may be substituted with substituents. R 111 Among the alkyl groups that can be selected, halogenoalkyl groups are preferred as alkyl groups having substituents. 111The halogenoalkyl groups that can be chosen are synonymous with the alkyl groups in substituent group T, except that at least one hydrogen atom in the alkyl group in substituent group T is substituted with a halogen atom, and the preferred ones are also the same. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, and iodine atoms, with fluorine atoms being preferred. There are no particular restrictions on the number of halogen atoms that make up a halogen alkyl group; for example, a perhalogenoalkyl group may be used.

[0034] Specifically, preferred forms of the group represented by formula (A) include the following groups (1) or (2). However, in these examples, L 3 and R 111 The substituent in L does not exclude the form in which the substituent has a substituent; it may be unsubstituted or have a substituent. 3 and R 111 The substituents that the substituents in the above L may have are, respectively, 3 and R 111 The description of optional substituents in can be preferably applied. (1) When the group is represented by any of the general formulas (1-1) to (1-8) The group represented by formula (A) is L 3 R is a linking group which is a combination of one or more of the following groups: a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and any of the groups represented by formulas (1-1) to (1-8) above, and 111 This is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. However, it has at least one of the bases represented by any of formulas (1-1) to (1-8). This also applies to the following description of (1). In this case, L 3The linking group is preferably a combination of a single bond, an alkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, and one or more of the groups represented by any of the above formulas (1-1), (1-3), (1-4), and (1-7). A linking group combining these is more preferred, a linking group combining one or more of the alkylene group, arylene group, heteroarylene group, and the groups represented by formulas (1-1), (1-3), (1-4), and (1-7) is even more preferred, and a linking group combining one or more of the alkylene group, arylene group, heteroarylene group, and the groups represented by formulas (1-1), (1-3), and (1-4) is particularly preferred. In these cases, R 111 The group is preferably a hydrogen atom, an alkyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group, more preferably a hydrogen atom or an alkyl group, and even more preferably a hydrogen atom. (2) When there is no group represented by any of the general formulas (1-1) to (1-8) The group represented by formula (A) is L 3 is a single bond, or a linking group formed by combining one or more of the alkylene group, alkenylene group, alkynylene group, arylene group, and heteroarylene group, and R 111 is a hydrogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a monovalent aliphatic heterocyclic group. In this case, L 3 It is a single bond, and R 111 L is a hydrogen atom, alkyl group, alkenyl group, aryl group or heteroaryl group. 3 is an alkenylene group or an alkynylene group, and R 111 It is preferable that L is an alkyl group or an aryl group. 3 It is a single bond, and R 111 It is more preferable that the group is a hydrogen atom, an alkyl group, an alkenyl group, or an aryl group.

[0035] 1) R 1 ~R 6 The above R 1 ~R 6 represents a halogen atom, a cyano group, or a group represented by the above formula (A), with a halogen atom or a group represented by the above formula (A) being preferred, a hydrogen atom, a halogen atom, an alkyl group, or a sulfo group being more preferred, and a hydrogen atom or an alkyl group being even more preferred. R 1 ~R 6 Possible halogen atoms include fluorine, chlorine, bromine, and iodine atoms, with chlorine being preferred. R 1 ~R 6 The alkyl groups that can be adopted are the above R 111 The description of alkyl groups that can be adopted as such can be applied. R 1 ~R 6 A preferred substituent for the alkyl group that can be adopted is a sulfo group. R 1 ~R 6 Specific examples include hydrogen atoms, methyl groups, ethyl groups, chlorine atoms, sulfo groups, or sulfomethyl groups.

[0036] 2) Z The above Z represents a group represented by the above formula (A), and is preferably a group in the preferred form of (1) or (2) above, preferably an alkyl group, alkoxy group, amino group, aryl group or heteroaryl group, more preferably an alkyl group, aryl group or heteroaryl group, and even more preferably an aryl group. The alkyl, alkoxy, amino, aryl, and heteroaryl groups that Z can take can be described as alkyl, alkoxy, amino, aryl, and heteroaryl groups represented by the above formula (A).

[0037] Z preferably has a carboxyl group or a substituent that can be bonded to a biomolecule as described later. In the description regarding each group that can be taken as Z below, a form having a substituent capable of binding to a biomaterial described later instead of a carboxy group is also preferable. The alkyl group, alkoxy group, and amino group that Z can take preferably have a carboxy group, and -(CH2) n COOH, -O(CH2) n COOH or -NH(CH2) n COOH is more preferable. Here, n is an integer of 1 to 15, preferably an integer of 1 to 10, and more preferably an integer of 1 to 5. The heteroaryl group that Z can take preferably has a carboxy group. As the heteroaryl group, a monocyclic group is preferable, a thiophene ring group or an imidazole ring group is more preferable, and an imidazole ring group is even more preferable.

[0038] The aryl group that Z can take is preferably a phenyl group, and a group represented by the following formula (C) is more preferable.

[0039]

Chemical formula

[0040] In the formula, R 41 ~R 45 represents a hydrogen atom or a substituent. * represents a bonding site. However, it satisfies at least any one of the following (C-1) to (C-3). (C-1) At least one of R 42 ~R 44 has a group having either a carboxy group or a substituent capable of binding to a biomaterial. (C-2) R 41 and R 45 are an alkyl group or a halogen atom. (C-3) At least one of R 41 and R 45 is a fluorine atom or an alkoxy group.

[0041] If (C-1) above is satisfied, non-radiative deactivation is suppressed; if (C-2) above is satisfied, thermal deactivation due to free rotation of the aryl group is suppressed due to steric hindrance; and if (C-3) above is satisfied, thermal deactivation due to free rotation of the aryl group is suppressed due to intramolecular hydrogen bonding. By satisfying at least one of these conditions (C-1) to (C-3), the fluorescence quantum yield is further improved. Note, R 41 and R 45 If both are fluorine atoms, it is determined that both (C-2) and (C-3) above are satisfied.

[0042] R 41 ~R 45 Possible substituents include hydrogen atoms, halogen atoms, cyano groups, alkyl groups, alkoxy groups, carboxyl groups, amide groups, sulfo groups, sulfamide groups, amino groups, alkenyl groups, aryl groups, heteroaryl groups, aliphatic heterocyclic groups, or substituents that can be bonded to biomolecules as described later, with halogen atoms, alkyl groups, alkoxy groups, carboxyl groups, amide groups, or substituents that can be bonded to biomolecules as described later being preferred. R 41 ~R 45 The alkyl group, alkoxy group, amide group, sulfamide group, amino group, alkenyl group, aryl group, heteroaryl group, or aliphatic heterocyclic group that can be selected as such may have substituents, for example, the groups in substituent group T described later, and poly(alkylene oxy) groups, sulfo groups, carboxyl groups, or substituents that can be bonded to biomolecules are preferred. R 41 and R 45 From the viewpoint of suppressing rotation of the benzene ring in formula (C), halogen atoms, alkyl groups, or alkoxy groups are preferred as substituents in formula (C). R 42 ~R 44The substituents that can be taken are not particularly limited, but are preferably selected from the substituent group T described later, and it is preferable that they have a carboxyl group or a substituent that can be bonded to a biomolecule. The carboxyl group or the substituent that can be bonded to a biomolecule may be directly bonded to the benzene ring in general formula (C), or it may be bonded via a linking group, but it is preferable that it is directly bonded. Examples of linking groups include alkylene groups, alkylene oxy groups, amide groups, or groups formed in combination thereof. Examples of alkylene groups, alkylene oxy groups, amide groups, or groups formed in combination thereof include -CONH(CH2CH2O) m CH2CH2- is an example. m is an integer from 1 to 30, preferably from 1 to 15, and more preferably from 1 to 10. Among them, R 43 It is preferable from the viewpoint of bonding with biomolecules that the substituent has a carboxyl group or a substituent that can be bonded to a biomolecule. R 42 ~R 44 Preferably, the substituent is a hydrogen atom, or a carboxyl group or a substituent that can be bonded to a biomolecule, and more preferably, it is a hydrogen atom, a carboxyl group or a substituent that can be bonded to a biomolecule. R 41 ~R 45 The halogen atom, alkyl group, and alkoxy group that can be used are the corresponding groups in substituent group T, respectively.

[0043] 3) R 7 ~R 12 The fluorescent compounds of the present invention are characterized by a ring structure in which the carbon atom at position 10 of the anthracene ring is replaced with a silicon atom or a phosphorus atom, as represented by any of the above general formulas (I) to (IV), with =N at position 3. + R 7 R 8 or =NR 9 It has a substituent Y at the 6th position. This substituent Y is -NR 10 R 11 OR 12 That is the case. The above R 7~R 12 represents a group represented by the above formula (A), and a preferred form of the group described in (1) or (2) above can be preferably applied.

[0044] R 7 and R 8 They may be bonded to each other to form a 4-7 membered aliphatic heterocycle, R 10 and R 11 They may be bonded to each other to form a 4- to 7-membered aliphatic heterocycle. Also, R 7 ~R 11 Each of the adjacent R 2 ~R 5 It may bond with other elements to form a 5-7 membered aliphatic heterocycle or a 5-7 membered aromatic heterocycle. The above, R 7 and R 8 A ring may be formed by the bonding of these elements together, R 10 and R 11 A ring which may be formed by the bonding of these elements together, or R 7 ~R 11 Each adjacent R 2 ~R 5 The rings that may be formed by bonding with each other all have R as a ring constituent atom. 7 ~R 11 In addition to the nitrogen atom to which it is bonded, it may also have 1 to 3 heteroatoms selected from oxygen, nitrogen, and sulfur atoms, with sulfur atoms being preferred.

[0045] R 7 and R 8 The preferred elements are hydrogen atoms, alkyl groups, or aryl groups, with hydrogen atoms or alkyl groups being more preferred. R 7 and R 8 The alkyl or aryl group that can be adopted can be described as an alkyl or aryl group represented by the above formula (A). R 7 and R 8 Preferred substituents that the alkyl or aryl group may have include halogen atoms, carbonyl groups, and other electron-withdrawing groups described later. 7and R 8 It is preferably an alkyl group, and more preferably an alkyl group substituted with a fluorine atom. R 7 and R 8 If it is not bonded to any group, R 7 and R 8 They may be the same or they may be different, but it is preferable that they be the same.

[0046] R 7 and R 8 The aliphatic heterocycle, which can be formed by the bonding of these atoms to each other and has 4 to 7 members, is not particularly limited, but is preferably a saturated aliphatic heterocycle, and preferably has 4 to 6 members. This aliphatic heterocycle may be a monocycle, or it may have a fused ring structure such as a structure in which at least one atom in the norbornane structure is replaced by a nitrogen atom. It is also preferable that the ring constituent atoms include >SO2, and that the substituents include electron-withdrawing groups described later, such as halogen atoms (preferably fluorine atoms).

[0047] R 7 R 5 A ring that can be formed by bonding with, and R 8 R 4 The rings that can be formed by bonding with may be aliphatic or aromatic, as long as they are heterocycles with 5 to 7 members. Among these rings, those with 5 or 6 members are preferred, and the ring constituent atoms are R 7 or R 8 It is preferable that the compound does not have heteroatoms other than the substituted nitrogen atom. Furthermore, it may be a monoring or a fused ring, but the ring formed by the bond is preferably a monoring.

[0048] R 10 and R 11 As for the above R 7 and R 8 The description in the above R may be applied. However, the above R 7 and R 8 While the nitrogen atom to which R is bonded has a positive charge, 10 and R 11It differs in that the nitrogen atom to which it is bonded does not have a positive charge. Also, R 7 R 5 The ring that can be formed by bonding with R 10 R 2 A ring that can be formed by bonding with, or R 111 R 3 These can be reinterpreted as rings that can be formed by combining with each other.

[0049] R 9 Alkyl alkyl groups are preferred. R 9 The alkyl groups that can be adopted are the above R 7 and R 8 The description of alkyl groups that can be adopted can be applied. 7 R 5 The ring that can be formed by bonding with R 9 adjacent R 4 or R 5 This can be reinterpreted as a ring that can be formed by combining with it.

[0050] R 12 Preferably, the group is a hydroxyl group, an acyl group, or a sulfonyl group. R 12 The acyl group or sulfonyl group that can be adopted is the above R 7 and R 8 The description of alkyl groups that can be adopted can be applied. 7 R 5 The ring that can be formed by bonding with R 9 adjacent R 4 or R 5 This can be reinterpreted as a ring that can be formed by combining with it.

[0051] 4) Y The above Y is -NR 10 R 11 OR 12 This indicates -NR 10 R 11 It is preferable. R 10 ~R 12 Regarding the above R 10 ~R 12 As stated above.

[0052] 5) R 21 and R 22 The above R 21 and R 22 represents a group represented by the above formula (A), and a preferred form of the group described in (1) or (2) above can be preferably applied. R 21 and R 22 These elements may be bonded to each other to form a 4- to 7-membered aliphatic heterocycle. However, R 21 and R 22 At least one of them is an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group. Also, R 21 is an aryl group, 22 If R is an alkyl group or an aryl group, 7 and R 8 , or R 10 and R 11 At least one of each combination is bonded to each other to form a four-membered aliphatic heterocycle. That is, the fluorescent compound of the present invention is R 10 and R 11 If R 7 and R 8 , or R 10 and R 11 At least one of each combinations bonds with each other to form a four-membered aliphatic heterocycle, and the fluorescent compound of the present invention is R 10 and R 11 If it does not have R 7 and R 8 These elements are bonded to each other to form a four-membered aliphatic heterocycle. These R 21 and R 22 The following description pertains to R 21 and R 22 The same applies to the description relating thereto. R 21 and R 22If at least one of the substituents has an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group, these substituents are sterically bulky and are thought to improve the stability of the silicon atom by suppressing the approach of photodegradation-causing substances such as reactive oxygen species to the silicon atom, thereby exhibiting excellent light resistance. 21 is an aryl group, 22 If R is an alkyl group or an aryl group, it is presumed that the fluorescence quantum yield will decrease due to non-radiative deactivation caused by the free rotation of the aryl group. 7 and R 8 , or R 10 and R 11 It is believed that at least one of each combination is bonded to each other to form a four-membered aliphatic heterocycle, which improves the fluorescence quantum yield through the suppression of TICT (as described later) and allows for the maintenance of high brightness. R 21 and R 22 Preferably, the group is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or a heteroaryl group. R 21 and R 22 The alkyl group, alkenyl group, alkynyl group, aryl group, or heteroaryl group that can be adopted can be described as an alkyl group, alkenyl group, alkynyl group, aryl group, or heteroaryl group represented by the above formula (A). R 21 and R 22 Preferred substituents on alkyl, alkenyl, alkynyl, aryl, or heteroaryl groups that can be formed include carboxyl, alkoxy, or aryl groups. Among these, alkenyl groups substituted with carboxyl or alkoxy groups, and alkenyl or alkynyl groups substituted with aryl groups are particularly preferred. R 21 and R 22Preferred combinations include an alkyl group with an alkenyl group, alkynyl group, aryl group, or heteroaryl group; an alkenyl group with an alkenyl group; an alkynyl group with an alkynyl group; and a heteroaryl group with a heteroaryl group. A combination of an alkyl group with an alkenyl group or aryl group is more preferred.

[0053] 6)R 23 The above R 23 represents a group represented by the above formula (A), and is preferably a hydroxyl group, an alkoxy group, an aryl group, or an alkyl group. R 23 If it is a hydroxyl group, the hydrogen atoms in this hydroxyl group may dissociate to have an ionic structure or a salt structure. This will be explained in subsequent R 23 The same applies to the hydroxyl group that can be adopted.

[0054] (Fluorescent compounds represented by formula (I) or (II) or salts thereof) Among the fluorescent compounds of the present invention, the fluorescent compound represented by formula (I) or (II) above, or a salt thereof (hereinafter referred to as "fluorescent compound represented by formula (I) or (II)"), is a compound having a ring structure in which the carbon atom at the 10th position of the anthracene ring is replaced with a silicon atom, and in addition to the provisions relating to each substituent described above, it satisfies the following condition α. (Condition α) If Z is an aryl group, this aryl group is the group represented by formula (C) above.

[0055] In the fluorescent compound represented by formula (I) or (II) above, if Z is an aryl group, unless this aryl group is a group represented by formula (C) above that satisfies at least one of (C-1) to (C-3) above, the fluorescence quantum yield will decrease due to thermal deactivation caused by the free rotation of the aryl group, and it will not be possible to maintain an excellent fluorescence quantum yield.

[0056] Among the fluorescent compounds represented by the above formula (I) or (II), the following compounds are preferred. R 7 , R 8 , R 10 and R 11 It is preferably a hydrogen atom, an alkyl group, or an aryl group, and is either a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, or R 7 and R 8 , or R 10 and R 11 It is more preferable that these elements are bonded to each other to form a four- or five-membered aliphatic heterocycle. Among them, R 7 , R 8 , R 10 and R 11 However, whether it is a hydrogen atom or a methyl group, R 7 and R 8 , or R 10 and R 11 The elements are bonded to each other to form a four-membered ring, and it is preferable that this four-membered ring is either an unsubstituted azetidine ring or an azetidine ring having a fluorine atom as a substituent, in order to minimize non-radiative deactivation due to the stretching and contracting of various molecular bonds at the substituent, and as a result be able to exhibit a high fluorescence quantum yield.

[0057] Also, the above R 7 and R 8 , or R 10 and R 11 It is preferable, from the viewpoint of further improving the fluorescence quantum yield as a result of suppressing TICT, that at least one of each combination is bonded to each other to form a 4- to 7-membered aliphatic heterocycle. However, the above R 21 is an aryl group, and the above R 22 If R is an alkyl group or an aryl group, then the above R 7 and R 8 , or R 10 and R 11 In each combination, at least one of the elements is bonded to form a ring that is a four-membered aliphatic heterocycle.

[0058] The above R 7 ~R 11 At least one of the groups is an electron-withdrawing group, that is, the fluorescent compound of the present invention is R10 and R 11 If it has R, then in the fluorescent compound represented by general formula (I), 7 , R 8 , R 10 or R 11 At least one of these groups contains an electron-withdrawing group, and in fluorescent compounds represented by general formula (II), R 9 ~R 11 At least one of these groups contains an electron-withdrawing group, and the fluorescent compound of the present invention is R 10 and R 11 If it does not have R 7 or R 8 At least one of these groups contains an electron-withdrawing group, and in fluorescent compounds represented by general formula (II), R 9 It is preferable that the group contains an electron-withdrawing group, as this suppresses TICT and further improves the fluorescence quantum yield. TICT is described below, and it is presumed that by having an electron-withdrawing group, the ionization potential of the nitrogen atom of the amino group located at the 3rd or 6th position of the xanthene skeleton can be increased, thereby suppressing TICT. An electron-withdrawing group is a group that has the characteristic of increasing the ionization potential of an amino group located at the 3rd or 6th position through an inductive effect and / or mesomeric effect. Examples of electron-withdrawing groups include halogen atoms (fluorine, chlorine, bromine, or iodine atoms), nitro groups, cyano groups, sulfonyl groups, phosphoryl groups, azide groups (-N3), carbonyl groups, alkoxycarbonyl groups, aryloxycarbonyl groups, and heterocyclic oxycarbonyl groups, with halogen atoms or sulfonyl groups being preferred. Examples of alkoxycarbonyl groups, aryloxycarbonyl groups, and heterocyclic oxycarbonyl groups include the respective groups in substituent group T described later. Furthermore, the sulfonyl group can refer to the alkyl, cycloalkyl, or arylsulfonyl groups in substituent group T described later. The number of electron-withdrawing groups is not particularly limited; one or more are sufficient, and one to two are preferred.

[0059] As mentioned above, it is preferable that the above Z is a group represented by formula (C) above, from the viewpoint of improving the fluorescence quantum yield by suppressing the free rotation of the aryl group.

[0060] The above Y is -NR 10 R 11 This is preferable from the viewpoint of wavelength compatibility with commonly used fluorescence microscope filters.

[0061] From the viewpoint of improving the fluorescence quantum yield, the fluorescent compound represented by the above formula (I) or (II) is more preferably a compound represented by the following general formula (IA) or (IIA) or a salt thereof.

[0062] [ka]

[0063] In the formula, R 1 ~R 11 and R 41 ~R 45 The above R 1 ~R 11 and R 41 ~R 45 It is synonymous with [the above]. R 21 and R 22 R represents the group represented by the above formula (A), 21 and R 22 These may be bonded to each other to form a 4- to 7-membered aliphatic heterocycle. However, R 21 and R 22 At least one of them is an alkenyl group or an aryl group. Also, R 21 is an aryl group, 22 If R is an alkyl group or an aryl group, then the above R 7 and R 8 , or R 10 and R 11 At least one of each combination is bonded to each other to form a four-membered aliphatic heterocycle. That is, the fluorescent compound of the present invention is R 10 and R 11 If R7 and R 8 , or R 10 and R 11 At least one of each combinations bonds with each other to form a four-membered aliphatic heterocycle, and the fluorescent compound of the present invention is R 10 and R 11 If it does not have R 7 and R 8 These elements are bonded to each other to form a four-membered aliphatic heterocycle.

[0064] One preferred embodiment of the present invention is represented by the above general formula (IA), and the above R 7 , R 8 , R 10 and R 11 is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and the above R 41 ~R 45 However, examples include fluorescent compounds or salts thereof that satisfy conditions (C-1) and (C-2) in the above condition α, or (C-1) and (C-3), or all of (C-1) to (C-3). Comfortably, the above R 7 , R 8 , R 10 and R 11 is an alkyl group having 1 to 3 carbon atoms, and the above R 41 ~R 45 However, it is a fluorescent compound or a salt thereof that satisfies (C-1) and (C-3) in the above condition α, or satisfies all of (C-1) to (C-3).

[0065] Another preferred embodiment of the present invention is represented by the above general formula (IA) and the above R 7 and R 8 , or R 10 and R 11 At least one of each combinations joins with each other to form a 4- to 7-membered aliphatic heterocycle, and the above R 41 ~R 45 However, examples include fluorescent compounds or salts thereof that satisfy conditions (C-1) and (C-2) in the above condition α, or (C-1) and (C-3), or all of (C-1) to (C-3). Comfortably, the above R 7 and R 8 , and R 10 and R 11 Each combination of these elements combines with one another to form a 4-7 membered aliphatic heterocycle, as described above. 41 ~R 45 However, it is a fluorescent compound or a salt thereof that satisfies (C-1) and (C-3) in the above condition α, or satisfies all of (C-1) to (C-3), and more preferably the above R 7 and R 8 , and R 10 and R 11 Each combination of these elements combines with one another to form a 4- or 5-membered aliphatic heterocycle, and the above R 41 ~R 45 However, if (C-1) and (C-3) in the above condition α are satisfied, or if all of (C-1) to (C-3) are satisfied, then R for (C-3) 41 and R 45 It is a fluorescent compound or a salt thereof, in which both are alkoxy groups.

[0066] (Fluorescent compounds represented by formula (III) or (IV) or salts thereof) Among the fluorescent compounds of the present invention, the fluorescent compound represented by formula (III) or (IV) above, or a salt thereof (hereinafter referred to as "fluorescent compound represented by formula (III) or (IV)"), is a p-rhodamine compound or a p-rhodol compound, each having a ring structure in which an oxygen atom, which is a ring constituent atom of the xanthene skeleton, is replaced with a phosphorus atom, and in addition to the provisions relating to each substituent described above, it satisfies the following condition β. (Condition β) R 7 and R 8 , or R 10 and R 11 At least one of each combination is bonded to each other to form a four-membered aliphatic heterocycle. In other words, the fluorescent compound of the present invention is R 10 and R 11 If R 7 and R 8 , or R 10 and R 11At least one of each combinations bonds with each other to form a four-membered aliphatic heterocycle, and the fluorescent compound of the present invention is R 10 and R 11 If it does not have R 7 and R 8 These elements are bonded to each other to form a four-membered aliphatic heterocycle.

[0067] In the fluorescent compound represented by formula (III) or (IV) above, if the above condition β is not satisfied, twisted intramolecular charge transfer (TICT) occurs, resulting in a decrease in the fluorescence quantum yield. TICT refers to the R of the amino group located at the 3rd or 6th position of the xanthene skeleton. 7 and R 8 , or R 10 and R 11 This refers to a transition to a charge-separated state (=torsional state) that exists on a non-planar plane relative to the xanthene skeleton. In this invention, it is hypothesized that by making at least one of the amino groups located at the 3rd or 6th position of the xanthene skeleton a highly strained 4-membered ring, the planar state (=non-torsional state) can be stabilized, thereby suppressing the transition to the TICT state.

[0068] Among the fluorescent compounds represented by the above formula (III) or (IV), the following compounds are preferred. The above R 7 ~R 11 At least one of the groups is an electron-withdrawing group, that is, the fluorescent compound of the present invention is R 10 and R 11 If it has R, then in the fluorescent compound represented by general formula (III), 7 , R 8 , R 10 or R 11 At least one of these groups contains an electron-withdrawing group, and in fluorescent compounds represented by general formula (IV), R 9 ~R 11 At least one of these groups contains an electron-withdrawing group, and the fluorescent compound of the present invention is R 10 and R 11If it does not have R, then in the fluorescent compound represented by general formula (III), 7 or R 8 At least one of these groups contains an electron-withdrawing group, and in fluorescent compounds represented by general formula (IV), R 9 It is preferable that the group contains an electron-withdrawing group, as this suppresses TICT and further improves the fluorescence quantum yield. Regarding electron-withdrawing groups, R is described in the preferred form of the fluorescent compound represented by formula (I) or (II) above. 7 ~R 11 The description of electron-withdrawing groups, which preferably include at least one of the following, can be applied.

[0069] The above R 23 It is preferable that the group is a hydroxyl group, alkoxy group, aryl group, or alkyl group from the viewpoint of wavelength compatibility with commonly used microscope filters. However, the above R 23 If the group is a hydroxyl group, the hydrogen atoms in this hydroxyl group may be dissociated.

[0070] Specific examples of fluorescent compounds represented by any of the general formulas (I) to (IV) of the present invention are shown below, but the present invention is not limited to these compounds or their salts. In the compounds represented by the following formula (1) or (2) or salts thereof, X, Y, Z, R are provided so as to satisfy the requirements of any of the general formulas (I) to (IV) of the present invention. 1 ~R 6 and R 7 ~R 9 It has. * indicates a binding site. In the example compounds below, Me represents a methyl group, Et represents an ethyl group, Ac represents an acetyl group, Tf represents a trifluoromethylsulfonyl group, and Ph represents a phenyl group.

[0071] [ka]

[0072] [ka]

[0073] The fluorescent compounds of the present invention can be synthesized by known methods. For example, see International Publication No. 2020 / 033681, International Publication No. 2014 / 106957, International Publication No. 2014 / 144793, U.S. Patent Application Publication No. 2018 / 0284105, U.S. Patent Application Publication No. 2019 / 0100653, U.S. Patent No. 8506655, or International Publication No. 2018 / 043579.

[0074] The fluorescent compounds of the present invention exhibit excellent fluorescence quantum yield and photoresistance, and can be used as reagents for biofluorescence imaging by binding them to biomolecules such as proteins, amino acids, nucleic acids, glycans, and lipids. In other words, the fluorescent compounds of the present invention also include compounds having groups that interact with biomolecules (such as through physical adsorption and chemical bonding), and such forms are particularly preferred when applying the fluorescent compounds of the present invention to the fluorescent labeling of biomolecules, such as in biofluorescence imaging. The fluorescent compounds of the present invention preferably have at least one substituent capable of binding to biomolecules. There are no particular restrictions on the substituents that can bind to biomolecules, but for example, Z, R 21 ~R 23 Z is preferred, and Z is more preferred. Examples of substituents capable of binding to biomolecules include substituents capable of binding to biomolecules in the fluorescent compounds of the present invention described later in "Specific forms in which the fluorescent compounds of the present invention interact with and bind to biomolecules," and preferably, the following substituents are included. Examples of fluorescent compounds of the present invention having substituents capable of binding to biomolecules include compounds in which the carboxyl group in the above-mentioned fluorescent compounds is replaced with substituents capable of binding to biomolecules.

[0075] [ka]

[0076] Compounds having groups for acting on (including adhering to) or binding to biomolecules can be synthesized by known methods. For example, one can refer to Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).

[0077] <<Fluorescently labeled biomaterials>> The fluorescently labeled biomaterial of the present invention (also simply referred to as a labeled biomaterial) is a substance in which the fluorescent compound of the present invention is bound to a biomaterial. The binding of the fluorescent compound of the present invention to the biomaterial may be in the form of direct bonding between the fluorescent compound and the biomaterial, or in the form of linkage via a linking group.

[0078] Preferred biomaterials include proteins, amino acids, nucleic acids, glycans, and lipids. In this invention, the term "protein" is used to include peptides and refers to a compound formed by the linkage of two or more amino acids by peptide bonds. Preferred proteins include antibodies, and preferred lipids include phospholipids, fatty acids, and sterols. A labeled biomaterial formed by the linkage of the fluorescent compound of this invention to an antibody is called a labeled antibody. Among the above-mentioned biological substances, there are no particular limitations on substances that are clinically and pathologically useful, but examples include immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, and IgD; complement; plasma proteins such as CRP (C-reactive protein), ferritin, α1-microglobulin, and β2-microglobulin and their antibodies; α-fetoprotein; tumor markers such as carcinoembryonic antigen (CEA), prostate acid phosphatase (PAP), CA (carbohydrate antigen) 19-9, and CA-125 and their antibodies; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human ciliary gonadotropin (hCG), estrogen, and insulin and their antibodies; viral infection-related substances such as hepatitis B virus (HBV)-related antigens (HBs, HBe, HBc), human immunodeficiency virus (HIV), and adult T-cell leukemia (ATL), and their antibodies. Furthermore, other examples include bacteria such as *Clostridium diphtheriae*, *Clostridium botulinum*, *Mycoplasma*, and *Treponema pallidum*, and their antibodies; protozoa such as *Toxoplasma toxoplasma*, *Trichomonas*, *Leishmania*, *Trivanozoma*, and *Parasomalis*, and their antibodies; ES cells (Embryonic Stem Cells) such as ELM3, HM1, KH2, v6.5, v17.2, v26.2 (derived from mice 129, 129 / SV, C57BL / 6, BALB / c) and their antibodies; drugs such as antiepileptic drugs like phenytoin and phenobarbital; cardiovascular drugs like quinidine and digoxin; antiasthmatic drugs like theophylline; antibiotics like chloramphenicol and gentamicin, and their antibodies; other enzymes; and extracellular toxins (such as styrelyzine O) and their antibodies. Antibody fragments such as Fab'2, Fab, and Fv can also be used.

[0079] Specific forms in which the fluorescent compound of the present invention interacts with and binds to a biomolecule include, for example, the forms described below. i) Non-covalent bonds (e.g., ionic bonds including hydrogen bonds and chelate formation) or covalent bonds between the peptide in the fluorescent compound of the present invention and the peptide in the biomaterial, ii) Van der Waals forces between the long-chain alkyl group in the fluorescent compound of the present invention and lipid bilayers and lipids in biomaterials, iii) Amide bond formation by reaction between the NHS ester (N-hydroxysuccinimide ester) in the fluorescent compound of the present invention and an amino group in a biomolecule, iv) Thioether bonding by reaction between a maleimide group in the fluorescent compound of the present invention and a sulfanyl group (-SH) in a biomaterial, v) Click reaction between an azide group in the fluorescent compound of the present invention and an acetylene group in a biomaterial, or formation of a triazole ring by a click reaction between an acetylene group in the fluorescent compound of the present invention and an azide group in a biomaterial. These are some examples. In addition to the forms described in i) to v) above, the molecules can also be conjugated in the form described in, for example, Lucas CD de Rezende and Flavio da Silva Emery, A Review of the Synthetic Strategies for the Development of BODIPY Dyes for Conjugation with Proteins, Orbital: The Electronic Journal of Chemistry, 2013, Vol 5, No. 1, pp. 62-83. Furthermore, the methods described in the same document can be appropriately referenced in the preparation of the fluorescently labeled biomolecule of the present invention.

[0080] Among the fluorescent compounds of the present invention, the labeled biomaterial obtained from a compound having substituents capable of binding to biomaterials and a biomaterial that binds to it through interaction includes compounds and their products obtained by replacing the substituents other than those capable of binding to biomaterials in paragraph 0038 of Japanese Patent Application Publication No. 2019-172826 with the dye portion of the fluorescent compound of the present invention. However, the present invention is not limited to these compounds.

[0081] The fluorescently labeled biomaterial of the present invention uses the fluorescent compound of the present invention, which exhibits excellent fluorescence quantum yield, making it easier to identify target biomaterials. It also has excellent lightfastness. Furthermore, because the fluorescently labeled biomaterial of the present invention uses the fluorescent compound of the present invention, which exhibits excellent lightfastness, it can be applied to observations under more severe conditions, such as long-term observation or observation using a strong laser, compared to conventional fluorescently labeled biomaterials containing Si, P-rhodamine, or rhodol compounds. Therefore, the fluorescently labeled biomaterial of the present invention can be applied to a variety of applications in the observation of biomaterials.

[0082] <Reagents containing fluorescently labeled biomaterials> In the reagent containing the fluorescently labeled biomaterial of the present invention, the fluorescently labeled biomaterial of the present invention can be in any form, without particular limitations, such as a solution in which it is dissolved in an aqueous medium such as physiological saline and phosphate buffer, or in a solid form such as fine particulate powder and freeze-dried powder, and its form can be appropriately selected according to the purpose of use. For example, when using the fluorescently labeled biomaterial of the present invention as a biofluorescence imaging reagent as described below, it can also be used as a reagent containing any of the above forms of the fluorescently labeled biomaterial.

[0083] <Applications of fluorescently labeled biomaterials> The fluorescently labeled biomaterial of the present invention, obtained from the fluorescent compound of the present invention, is expected to be able to stably detect the fluorescence emitted from the fluorescent compound excited by light irradiation. For this reason, the fluorescently labeled biomaterial of the present invention is suitable, for example, as a biofluorescence imaging reagent. Cells stained with the fluorescently labeled biomaterial of the present invention as a fluorescent dye exhibit highly suppressed color fading, allowing them to maintain fluorescence intensity for extended periods. Therefore, the fluorescently labeled biomaterial of the present invention is particularly suitable for biofluorescence imaging applications requiring excellent photoresistance, such as long-term observation of biomaterials using time-lapse microscopy, and observation of biomaterials using high-resolution microscopes like confocal laser microscopes or super-resolution microscopes like STED microscopes (stimulated emission suppression microscopes).

[0084] The biofluorescence imaging using the fluorescently labeled biomaterial of the present invention includes the following steps (i) to (iii). (i) A step of preparing a target biomolecule (hereinafter referred to as the "target biomolecule") and a fluorescently labeled biomolecule of the present invention, which is formed by binding a biomolecule capable of binding to the target biomolecule to the fluorescent compound of the present invention. (ii) A step of binding a target biomaterial with the fluorescently labeled biomaterial of the present invention. (iii) A step of irradiating a substance to which the fluorescently labeled biological material of the present invention is bound with a target biological material with light in the wavelength range absorbed by the fluorescently labeled biological material of the present invention, and detecting the fluorescence emitted by the fluorescently labeled biological material of the present invention.

[0085] In the above biofluorescence imaging, the biomolecule capable of binding to the target biomolecule is the biomolecule in the fluorescently labeled biomolecule of the present invention. It can be appropriately selected according to the target biomolecule (subject), and a biomolecule capable of specifically binding to the subject can be selected.

[0086] Among the target biomolecules listed above, proteins include so-called disease markers. While there are no particular restrictions on disease markers, examples include α-fetoprotein (AFP), PIVKA-II (protein induced by vitamin K absence or antagonist II), BCA (breast carcinoma-associated antigen) 225, basic fetoprotein (BFP), CA (carbohydrate antigen) 15-3, CA19-9, CA72-4, CA125, CA130, CA602, CA54 / 61 (CA546), carcinoembryonic antigen (CEA), DUPAN-2, elastase 1, immunosuppressive acid protein (IAP), NCC-ST-439, γ-seminoprotein (γ-Sm), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), nerve-specific enolase (NSE), Iba1, and amyloid. Examples include β, tau, squamous cell carcinoma-associated antigen (SCC antigen), sialyl LeX-i antigen (SLX), SPan-1, tissue polypeptide (TPA), serial Tn antigen (STN), cytokeratin (CYFRA), pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, and ventricular myosin light chain I.

[0087] Among the target biological substances mentioned above, bacteria include those that are the subject of cellular microbiological testing, and are not particularly limited, but examples include Escherichia coli, Salmonella, Legionella, and bacteria that cause public health problems.

[0088] Among the target biological substances mentioned above, there are no particular restrictions on which viruses can be used, but examples include hepatitis virus antigens such as those of hepatitis C and B viruses, the p24 protein antigen of the HIV virus, the pp65 protein antigen of CMV (cytomegalovirus), and the E6 and E7 proteins of HPV (papillomavirus).

[0089] In (i) above, the target biomaterial can be prepared according to conventional methods without any particular restrictions. Furthermore, the fluorescently labeled biomaterial of the present invention is not particularly limited and can be prepared by binding a biomaterial capable of binding to a target biomaterial with the fluorescent compound of the present invention according to a conventional method. Examples of binding forms and reactions that form the binding include binding through interactions described above for the fluorescently labeled biomaterial of the present invention.

[0090] In (ii) above, the fluorescently labeled biomaterial of the present invention and the target biomaterial may be bound directly, or they may be bound via other biomaterials different from the fluorescently labeled biomaterial of the present invention and the target biomaterial. While not particularly limited, the use of the fluorescently labeled biomaterial of the present invention for biofluorescence imaging can be, for example, fluorescent cell staining. Fluorescent cell staining can be performed using a direct method with a fluorescently labeled antibody as the primary antibody, or an indirect method in which a secondary antibody, as a fluorescently labeled antibody, is reacted with the primary antibody. The fluorescently labeled biomaterial of the present invention can be used as a fluorescently labeled antibody in either the direct or indirect method, but it is preferable to use it as a fluorescently labeled antibody in the indirect method. The binding of the fluorescently labeled biomaterial of the present invention to the target biomaterial can be carried out according to conventional methods without particular limitations.

[0091] In (iii) above, the wavelength for exciting the fluorescently labeled biological material of the present invention is not particularly limited as long as it is a wavelength (wavelength light) capable of exciting the fluorescently labeled biological material of the present invention. Typically, 300 to 1000 nm is preferred, and 400 to 800 nm is more preferred.

[0092] The fluorescence excitation light source used in the present invention is not particularly limited as long as it emits wavelengths (wavelength light) capable of exciting the fluorescence-labeled biological material of the present invention, and various laser light sources can be used. Examples include gas lasers such as He-Ne lasers, CO2 lasers, Ar ion lasers, Kr ion lasers, He-Cd lasers, excimer lasers, and nitrogen lasers; solid-state lasers such as ruby ​​lasers, yttrium-aluminum-garnet (YAG) lasers, and glass lasers; dye lasers; and semiconductor lasers. Furthermore, various optical filters can be used to obtain a preferred excitation wavelength or to detect only fluorescence.

[0093] Other matters in (i) to (iii) above are not particularly limited and can be appropriately selected from commonly used methods, reagents, equipment, and other conditions.

[0094] Biofluorescence imaging using the fluorescently labeled biomaterial of the present invention allows for long-term observation of biomaterials while maintaining fluorescence intensity, because the color fading of the substance bound to the target biomaterial by the fluorescently labeled biomaterial of the present invention is highly suppressed. Furthermore, observation with maintained fluorescence intensity is possible even when using high-resolution microscopes and super-resolution microscopes. In addition, since the substance bound to the target biomaterial by the fluorescently labeled biomaterial of the present invention exhibits high brightness, its identification is easy.

[0095] Furthermore, the fluorescently labeled biomaterial of the present invention can be suitably used, in addition to the above, for long-term storage of stained cells, etc., by appropriately adjusting the storage conditions.

[0096] - Substituent group T - In the present invention, preferred substituents include substituents selected from the following substituent group T. Furthermore, in this specification, when a substituent is mentioned only as such, it refers to this substituent group T, and when only an individual group, such as an alkyl group, is mentioned, the corresponding group of this substituent group T is preferably applied. Furthermore, in this specification, when alkyl groups are described separately from cyclic (cyclo) alkyl groups, the term alkyl group is used to encompass both linear alkyl groups and branched alkyl groups. On the other hand, when alkyl groups are not described separately from cyclic alkyl groups, and unless otherwise specified, the term alkyl group is used to encompass both linear alkyl groups, branched alkyl groups, and cycloalkyl groups. This also applies to groups that can form a cyclic structure (alkyl groups, alkenyl groups, alkynyl groups, etc.) and compounds that contain groups that can form a cyclic structure. When a group can form a cyclic skeleton, the lower limit of the number of atoms in the group forming the cyclic skeleton is 3 or more, and preferably 5 or more, regardless of the lower limit of the number of atoms specifically described below for groups that can form this structure. In the description of the substituent group T below, linear or branched groups and cyclic groups are sometimes described separately to clearly distinguish between them, for example, alkyl groups and cycloalkyl groups.

[0097] The substituent group T includes the following groups: Alkyl groups (preferably 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, even more preferably 1 to 12 carbon atoms, even more preferably 1 to 8 carbon atoms, even more preferably 1 to 6 carbon atoms, particularly preferably 1 to 3 carbon atoms), alkenyl groups (preferably 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, even more preferably 2 to 12 carbon atoms, even more preferably 2 to 6 carbon atoms, even more preferably 2 to 4 carbon atoms), alkynyl groups (preferably 2 to 30 carbon atoms, more preferably 2 to 20 carbon atoms, even more preferably C2-C12, more preferably C2-C6, even more preferably C2-C4), cycloalkyl group (preferably C3-C20), cycloalkenyl group (preferably C5-C20), aryl group (may be a monocyclic group or a fused ring group (preferably a fused ring group of 2-6 rings). If it is a fused ring group, it consists of a 5-7 membered ring, etc. The aryl group preferably has C6-C40, more preferably C6-C30, even more preferably C6-C26, particularly preferably C6-C10), A heterocyclic group (having at least one nitrogen, oxygen, sulfur, phosphorus, silicon, or selenium atom as a ring constituent atom, and may be a monocyclic group or a fused ring group (preferably a fused ring group of 2 to 6 rings). If it is a monocyclic group, the number of ring members is preferably 5 to 7, more preferably 5 or 6. The number of carbon atoms in a heterocyclic group is preferably 2 to 40, more preferably 2 to 20. Heterocyclic groups include aromatic heterocyclic groups (heteroaryl groups) and aliphatic heterocyclic groups (aliphatic heterocyclic groups). ), alkoxy group (preferably with 1 to 20 carbon atoms, more preferably with 1 to 12 carbon atoms), alkenyloxy group (preferably with 2 to 20 carbon atoms, more preferably with 2 to 12 carbon atoms), alkynyloxy group (preferably with 2 to 20 carbon atoms, more preferably with 2 to 12 carbon atoms), cycloalkyloxy group (preferably with 3 to 20 carbon atoms), aryloxy group (preferably with 6 to 40 carbon atoms, more preferably with 6 to 26 carbon atoms, even more preferably with 6 to 14 carbon atoms), heterocyclic oxy group (preferably with 2 to 20 carbon atoms),

[0098] Alkoxycarbonyl groups (preferably with 2 to 20 carbon atoms), cycloalkoxycarbonyl groups (preferably with 4 to 20 carbon atoms), aryloxycarbonyl groups (preferably with 6 to 20 carbon atoms), amino groups (preferably with 0 to 20 carbon atoms, including unsubstituted amino groups (-NH2), (mono- or di-)alkylamino groups, (mono- or di-)alkenylamino groups, (mono- or di-)alkynylamino groups, (mono- or di-)cycloalkylamino groups, (mono- or di-)cycloalkenylamino groups, and (mono- or di-)arylamino groups) , (mono- or di-)heterocyclic amino groups. Each of the above groups substituting the unsubstituted amino group is synonymous with the corresponding group of substituent group T.) ), sulfamoyl group (preferably C0-C20, alkyl, cycloalkyl or aryl sulfamoyl groups are preferred), acyl group (preferably C1-C20, more preferably C2-C15), acyloxy group (preferably C1-C20), carbamoyl group (preferably C1-C20, alkyl, cycloalkyl or aryl carbamoyl groups are preferred),

[0099] Acylamino group (preferably C1-C20), alkylthio group (preferably C1-C20, more preferably C1-C12), cycloalkylthio group (preferably C3-C20), arylthio group (preferably C6-C40, more preferably C6-C26, even more preferably C6-C14), heterocyclic thio group (preferably C2-C20), alkyl, cycloalkyl or arylsulfonyl group (preferably C1-C20),

[0100] Silyl groups (preferably silyl groups having 1 to 30 carbon atoms, more preferably silyl groups having 1 to 20 carbon atoms substituted with alkyl, aryl, alkoxy, or aryloxy), silyloxy groups (preferably silyloxy groups having 1 to 20 carbon atoms substituted with alkyl, aryl, alkoxy, or aryloxy), hydroxyl groups, cyano groups, nitro groups, halogen atoms (e.g., fluorine, chlorine, bromine, or iodine), oxygen atoms (specifically, replacing the >CH2 ring with >C=O), carboxyl groups (-CO2H), phosphono groups [-PO(OH)2], phosphonooxy groups [-O-PO(OH)2], sulfo groups (-SO3H), boric acid groups [-B(OH)2], onio groups (ammonio groups including cyclic ammonia), sulfonio groups (-SH2) + ), phosphonio group (-PH3 + Examples include a ) which preferably has 0 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, a sulfanyl group (-SH), an amino acid residue, or a polyamino acid residue. Furthermore, examples include alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, cycloalkenyl groups, aryl groups, heterocyclic groups, alkoxy groups, alkenyloxy groups, alkynyloxy groups, cycloalkyloxy groups, aryloxy groups, heterocyclic oxy groups, alkoxycarbonyl groups, cycloalkoxycarbonyl groups, aryloxycarbonyl groups, amino groups, sulfamoyl groups, acyl groups, acyloxy groups, carbamoyl groups, acylamino groups, alkylthio groups, cycloalkylthio groups, arylthio groups, heterocyclic thio groups, alkyl groups, cycloalkyl or arylsulfonyl groups having carboxyl groups, phosphono groups, sulfo groups, onio groups, amino acid residues, amino acid residues, amino acid residues, carboxyl groups, phosphono groups, sulfo groups, onio groups, amino acid residues, amino acid residues, cycloalkenyl groups, aryloxy groups, heterocyclic thio groups, alkyl groups, cycloalkyl or arylsulfonyl groups as substituents.

[0101] The substituents selected from substituent group T are more preferably alkyl groups, alkenyl groups, cycloalkyl groups, aryl groups, heterocyclic groups, alkoxy groups, cycloalkoxy groups, aryloxy groups, alkoxycarbonyl groups, cycloalkoxycarbonyl groups, amino groups, acylamino groups, cyano groups, or halogen atoms, and are particularly preferably alkyl groups, alkenyl groups, aryl groups, heterocyclic groups, alkoxy groups, alkoxycarbonyl groups, amino groups, acylamino groups, or cyano groups.

[0102] Unless otherwise specified, substituents selected from substituent group T include groups formed by combining multiple of the above groups. For example, when a compound or substituent contains alkyl groups, alkenyl groups, etc., these may be substituted or unsubstituted. Also, when it contains aryl groups, heterocyclic groups, etc., these may be monocyclic or fused rings, and may be substituted or unsubstituted. [Examples]

[0103] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto. In this invention, room temperature means 25°C.

[0104] The compounds used as examples and comparative examples are shown below. In the following structural formulas, Me represents a methyl group. In compounds (10) to (12), it is sufficient if at least one of the carboxyl groups has a negative charge.

[0105] [ka]

[0106] The synthesis methods for the compounds and labeled antibodies are described in detail below, but the starting materials, dye intermediates, and synthesis routes are not limited to these.

[0107] Unless otherwise specified, the silica gel column chromatography used was the SNAP KP-Sil Cartridge (Biotage), with high-flash columns W001, W002, W003, W004, or W005 (Yamazen). For NH silica, the SNAP KP-NH Cartridge (Biotage) was used. The mixing ratio in the eluent is expressed as a volume ratio. For example, "ethyl acetate:n-hexane = 0:100 → 100:0" means that the eluent was changed from "ethyl acetate:n-hexane = 0:100" to "ethyl acetate:n-hexane = 100:0".

[0108] MS spectra were measured using either the ACQUITY SQD LC / MS System (Waters, ionization method: ESI (ElectroSpray Ionization)) or the LCMS-2010EV (Shimadzu Corporation, ionization method: simultaneous ESI and APCI (Atomospheric Pressure Chemical Ionization)).

[0109] Unless otherwise specified, synthesized compounds and labeled antibodies were stored under light-shielding conditions if not used immediately after preparation. Similarly, commercially available compounds and labeled antibodies were stored under light-shielding conditions until they were used after purchase.

[0110] The abbreviations used are listed below. DDQ:2,3-Dichloro-5,6-dicyano-1,4-benzoquinone HSTU:O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate NMO: N-methylmorpholine-N-oxide TBAF: Tetrabutylammonium fluoride TPAP: Tetrapropylammonium perlutenate TFA: Trifluoroacetic acid X-phos:2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl CPME: Cyclopentyl methyl ether diF-azetidine-HCl: 3,3-difluoroazetidine hydrochloride F-azetidine-HCl:3-fluoroazetidine hydrochloride F-pyrrolidine-HCl:3-fluoropyrrolidine hydrochloride DCM: Methylene chloride DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide THF: Hetrahydrofuran Ac: Acetyl group nBu: n-butyl group Et: Ethyl group Me: Methyl group PEG4:-(CH2CH2O)4- Ph: Phenyl group iPr: Isopropyl group TBS: tert-butyldimethylsilyl group Tf: Trifluoromethylsulfonyl group dba: dibenzylideneacetone

[0111] [Synthesis Example 1: Synthesis of Compound (1) and Labeled Antibody (1)] Compound (1) and labeled antibody (1) were synthesized according to the following scheme.

[0112] [ka]

[0113] <Synthesis of compound (1-A)> 3-bromo-N,N-dimethylaniline (1.00 g, Fujifilm Wako Pure Chemical Industries, Ltd.) and tetrahydrofuran (30 mL, super-dehydrated, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 100 mL three-necked flask. After purging with nitrogen, the mixture was cooled to -78 °C, and n-butyllithium (3.17 mL, 1.6 M, n-hexane solution, Kanto Chemical Co., Ltd.) was added dropwise and the mixture was stirred for 30 minutes. Subsequently, dichloromethylvinylsilane (0.309 mL, Tokyo Chemical Co., Ltd.) was added, and the mixture was heated to room temperature and stirred for 5 minutes. The completion of the reaction was confirmed by LC / MS, and under ice cooling, saturated ammonium chloride aqueous solution was added, the mixture was extracted with ethyl acetate, dried with Glauber's salt, and the solvent was removed under reduced pressure. The obtained residue was dissolved in methylene chloride and purified by silica gel column chromatography (ethyl acetate:n-hexane = 0:100 → 10:90). The target product was recovered, and the solvent was removed under reduced pressure to obtain a colorless to yellow oily compound (1-A) (0.62 g, yield 83%). [M+H + ] + :311

[0114] <Synthesis of compound (1)> Compound (1-A) (58 mg), 3,5-difluoro-4-formylbenzoic acid (35 mg, COMBI-BLOCKS), zinc(II) chloride (76 mg, FUJIFILM Wako Pure Chemical Industries, Ltd.), and ethanol (0.64 mL, super-dehydrated, FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to a 5 mL test tube reaction vessel, and the mixture was heated at 140 °C for 30 minutes under microwave irradiation. The completion of the reaction was confirmed by LC / MS, and the solvent was removed under reduced pressure. Methylene chloride (1.85 mL, FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to the resulting residue, followed by the addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (42 mg) under ice cooling, and the mixture was stirred for 5 minutes after being heated to room temperature. The reaction was confirmed to be complete by LC / MS, the solvent was removed under reduced pressure, and the solution to which acetonitrile was added was purified by reverse-phase silica gel column chromatography (C18, Biotage, acetonitrile:distilled water = 0:100 → 60:40). The target product was recovered and the solvent was removed by freeze-drying. The resulting residue was dissolved in DMSO and purified by preparative HPLC (Water, acetonitrile:0.1% ammonium formate aqueous solution = 10:90 → 90:10). The target product was recovered and the solvent was removed by freeze-drying to obtain compound (1) (3 mg, yield 3%), a dark blue powder. [M+H + ] + :477

[0115] <Synthesis of compound (1-NHS)> Compound (1) (1 mg), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2 mg, Tokyo Chemical Industry Co., Ltd.), triethylamine (2 μL, Fujifilm Wako Pure Chemical Industries, Ltd.), and DMF (100 μL, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 2 mL test tube reaction vessel, and the mixture was stirred at room temperature for 2 hours. The completion of the reaction was confirmed by LC / MS, and the reaction solution was purified directly by reversed-phase silica gel column chromatography (C18, Biotage, acetonitrile:distilled water = 0:100 → 60:40). The target product was recovered, and the solvent was removed by freeze-drying to obtain the dark blue powder compound (1-NHS) (0.5 mg, yield 41%). [M + ] + :574

[0116] <Synthesis of labeled antibody (1)> 400 μL of anti-rabbit IgG antibody [host: goat, 2.4 mg / mL, catalog number: 111-005-003, JacksonImmunoResearch] was mixed with 40 μL of carbonate pH standard solution (pH=10.01) (Fujifilm Wako Pure Chemical Industries) and 3.2 μL of DMSO solution with a concentration of 20 mM of compound (1-NHS), and the mixture was stirred. The mixture was then allowed to stand at room temperature for 1 hour. Subsequently, the reaction mixture was directly charged onto a Sephadex G-25 column [catalog number: 17085101, GE Healthcare] and purified with PBS [pH=7.4, Fujifilm Wako Pure Chemical Industries] to obtain labeled antibody (1).

[0117] [Synthesis Example 2: Synthesis of Compound (2) and Labeled Antibody (2)]

[0118] [ka]

[0119] <Synthesis of compound (2)> Compound (2), a dark blue powder, was obtained in the same manner as in the synthesis method of compound (1), except that 3-fluoro-4-formylbenzoic acid (manufactured by ChemExpress) was used instead of 3,5-difluoro-4-formylbenzoic acid. [M+H + ] + :459

[0120] <Synthesis of compound (2-NHS)> In the synthesis method for compound (1-NHS), 0.5 mg of compound (2-NHS) in a dark blue powder was obtained in the same manner, except that compound (2) was used instead of compound (1). [M + ] + :556

[0121] <Synthesis of labeled antibody (2)> Labeled antibody (2) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (2-NHS) was used instead of compound (1-NHS).

[0122] [Synthesis Example 3: Synthesis of Compound (3) and Labeled Antibody (3)]

[0123] [ka]

[0124] <Synthesis of compound (3)> Compound (3), a dark blue powder, was obtained in the same manner as in the synthesis method of compound (1), except that 4-formyl-3,5-dimethylbenzoic acid (manufactured by AOBChem) was used instead of 3,5-difluoro-4-formylbenzoic acid. [M+H + ] + :469

[0125] <Synthesis of compound (3-NHS)> In the synthesis method for compound (1-NHS), 0.2 mg of compound (3-NHS) in a dark blue powder was obtained in the same manner as before, except that compound (3) was used instead of compound (1). [M + ] + :566

[0126] <Synthesis of labeled antibody (3)> Labeled antibody (3) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (3-NHS) was used instead of compound (1-NHS).

[0127] [Synthesis Example 4: Synthesis of Compound (4) and Labeled Antibody (4)]

[0128] [ka]

[0129] <Synthesis of compound (4)> Compound (4), a dark blue powder, was obtained in the same manner as in the synthesis method of compound (1), except that 4-formyl-3,5-dimethoxybenzoic acid (manufactured by COMBI-BLOCKS) was used instead of 3,5-difluoro-4-formylbenzoic acid. [M+H + ] + :501

[0130] <Synthesis of compound (4-NHS)> In the synthesis method for compound (1-NHS), 0.4 mg of compound (4-NHS) in a dark blue powder was obtained in the same manner, except that compound (4) was used instead of compound (1). [M + ] + :598

[0131] <Synthesis of labeled antibody (4)> Labeled antibody (4) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (4-NHS) was used instead of compound (1-NHS).

[0132] [Synthesis Example 5: Synthesis of Compound (5) and Labeled Antibody (5)]

[0133] [ka]

[0134] <Synthesis of compound (5)> Compound (5), a dark blue powder, was obtained in the same manner as in the synthesis method of compound (1), except that 5-formyl-4-methylthiophene-2-carboxylic acid (manufactured by Enamine) was used instead of 3,5-difluoro-4-formylbenzoic acid. [M+H + ] + :461

[0135] <Synthesis of compound (5-NHS)> In the synthesis method for compound (1-NHS), 0.3 mg of compound (5-NHS) in a dark blue powder was obtained by the same procedure, except that compound (5) was used instead of compound (1). [M+ ] + :558

[0136] <Synthesis of labeled antibody (5)> Labeled antibody (5) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (5-NHS) was used instead of compound (1-NHS).

[0137] [Synthesis Example 6: Synthesis of Compound (6) and Labeled Antibody (6)]

[0138] [ka]

[0139] <Synthesis of compound (6-A)> In a 20 mL test tube reaction vessel, 1,3-dibromobenzene (1.89 g, Fujifilm Wako Pure Chemical Industries, Ltd.), thiomorpholine (0.83 g, Fujifilm Wako Pure Chemical Industries, Ltd.), palladium acetate (0.09 g, Fujifilm Wako Pure Chemical Industries, Ltd.), BINAP (0.37 g, racemic mixture, Fujifilm Wako Pure Chemical Industries, Ltd.), sodium t-butoxide (0.92 g, Fujifilm Wako Pure Chemical Industries, Ltd.), and toluene (10 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the mixture was irradiated with microwaves and stirred at 140°C for 45 minutes. The completion of the reaction was confirmed by LC / MS, and the reaction solution was purified directly by silica gel column chromatography (ethyl acetate:n-hexane = 0:100 → 20:80). The target product was recovered, and the solvent was removed under reduced pressure to obtain a colorless oily compound (6-A) (1.22 g, yield 59%). [M+H + ] + :258, 260

[0140] <Synthesis of compound (6-B)> The same method was used for the synthesis of compound (1-A), except that compound (6-A) (2.30 g) was used instead of 3-bromo-N,N-dimethylaniline to obtain the yellow oily compound (6-B) (471 mg, yield 26%). [M+H + ] + :427

[0141] <Synthesis of compound (6-C)> Compound (6-B) (50 mg), 3,5-difluoro-4-formylbenzoic acid (65 mg, COMBI-BLOCKS), zinc(II) chloride (48 mg, FUJIFILM Wako Pure Chemical Industries, Ltd.), and ethanol (0.47 mL, super-dehydrated, FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to a 5 mL test tube reaction vessel, and the mixture was microwaved and heated at 140 °C for 30 minutes. The completion of the reaction was confirmed by LC / MS, and the solvent was removed under reduced pressure. Methylene chloride (1.85 mL, FUJIFILM Wako Pure Chemical Industries, Ltd.) was added to the resulting residue, and the mixture was purified by silica gel column chromatography (ethyl acetate:n-hexane = 0:100 → 50:50). The target product was recovered, and the solvent was removed under reduced pressure to obtain a yellow to pale blue oily compound (6-C) (50 mg, yield 72%). [M+H + ] + :595

[0142] <Synthesis of compound (6)> Compound (6-C) (24 mg), tetrapropylammonium perlutenate (7 mg, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), N-methylmorpholine-N-oxide (118 mg, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and methylene chloride (1.01 mL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 5 mL test tube reaction vessel and stirred at 80°C for 20 minutes. The completion of the reaction was confirmed by LC / MS, and then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (24 mg) was added under ice cooling, and the mixture was stirred for 5 minutes after being raised to room temperature. The reaction was confirmed to be complete by LC / MS, the solvent was removed under reduced pressure, and the solution to which acetonitrile was added was purified by reverse-phase silica gel column chromatography (C18, Biotage, acetonitrile:distilled water = 0:100 → 60:40). The target product was recovered, and the solvent was removed by freeze-drying to obtain compound (6) (0.4 mg, yield 2%), a deep blue powder. [M+H + ] + :657

[0143] <Synthesis of compound (6-NHS)> In the synthesis method for compound (1-NHS), 0.1 mg of compound (6-NHS) in a dark blue powder was obtained by the same procedure, except that compound (6) was used instead of compound (1). [M + ] + :754

[0144] <Synthesis of labeled antibody (6)> Labeled antibody (6) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (6-NHS) was used instead of compound (1-NHS).

[0145] [Synthesis Example 7: Synthesis of Compound (7) and Labeled Antibody (7)]

[0146] [ka]

[0147] <Synthesis of compound (7-A)> 3-bromo-N-methylaniline (5.00 g, Fujifilm Wako Pure Chemical Industries, Ltd.), allyl bromide (2.80 mL, Fujifilm Wako Pure Chemical Industries, Ltd.), potassium carbonate (7.50 g, Fujifilm Wako Pure Chemical Industries, Ltd.), and acetonitrile (25 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 100 mL round-bottom flask and stirred at 90°C for 16 hours. The reaction was confirmed to be complete by LC / MS, insoluble matter was removed by filtration, and the solvent was removed under reduced pressure. The resulting residue was dissolved in n-hexane and purified by silica gel column chromatography (ethyl acetate:n-hexane = 0:100 → 10:90). The target product was recovered, and the solvent was removed under reduced pressure to obtain a yellow oily compound (7-A) (5.57 g, yield 92%). [M+H + ] + :226, 228

[0148] <Synthesis of compound (7-B)> The same method was used for the synthesis of compound (1-A), except that compound (7-A) (2.01 g) was used instead of 3-bromo-N,N-dimethylaniline to obtain the yellow oily compound (7-B) (1.51 g, yield 98%). [M+H + ] + :363

[0149] <Synthesis of compound (7-C)> In the synthesis method for compound (6-C), compound (7-C) (109 mg, yield 25%) was obtained in the same manner as before, except that compound (7-B) (300 mg) was used instead of compound (6-B).

[0150] <Synthesis of compound (7)> Compound (7-C) (109 mg), barbituric acid (481 mg, Fujifilm Wako Pure Chemical Industries, Ltd.), tetrakistriphenylphosphine palladium (48 mg, Fujifilm Wako Pure Chemical Industries, Ltd.), and methylene chloride (11 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 50 mL round-bottom flask and stirred at room temperature for 30 minutes. After confirming completion of the reaction by LC / MS, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (50 mg) was added under ice cooling, and the mixture was heated to room temperature and stirred for 5 minutes. After confirming completion of the reaction by LC / MS, the solvent was removed under reduced pressure, and the solution with added acetonitrile was purified by reverse-phase silica gel column chromatography (C18, Biotage, acetonitrile:distilled water = 0:100 → 60:40). The target product was recovered and the solvent was removed by freeze-drying to obtain compound (7) (5 mg, yield 6%) as a dark blue powder. [M+H + ] + :449

[0151] <Synthesis of compound (7-NHS)> In the synthesis method for compound (1-NHS), 2 mg of compound (7-NHS) was obtained as a dark blue powder, except that compound (7) was used instead of compound (1). [M+H + ] + :546

[0152] <Synthesis of labeled antibody (7)> Labeled antibody (7) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (7-NHS) was used instead of compound (1-NHS).

[0153] [Synthesis Example 8: Synthesis of Compound (8) and Labeled Antibody (8)]

[0154] [ka]

[0155] <Synthesis of compound (8-A)> Compound (8-A) (56 mg, yield 13%) was obtained in the same manner as in the synthesis of compound (7-C), except that 5-formyl-4-methylthiophene-2-carboxylic acid (manufactured by Enamine) was used instead of 3,5-difluoro-5-formylbenzoic acid. [M+H + ] + :515

[0156] <Synthesis of compound (8)> Compound (8) (1 mg, yield 1%) was obtained as a dark blue powder in the same manner as in the synthesis method of compound (7), except that compound (8-A) was used instead of compound (7-C). [M+H + ] + :433

[0157] <Synthesis of compound (8-NHS)> In the synthesis method for compound (7-NHS), 0.1 mg of compound (8-NHS) in a dark blue powder was obtained in the same manner as before, except that compound (8) was used instead of compound (7). [M+H + ] + :530

[0158] <Synthesis of labeled antibody (8)> Labeled antibody (8) was obtained in the same manner as in the synthesis method for labeled antibody (7), except that compound (8-NHS) was used instead of compound (7-NHS).

[0159] [Synthesis Example 9: Synthesis of Compound (9) and Labeled Antibody (9)]

[0160] [ka]

[0161] <Synthesis of compound (9)> Compound (7-NHS) (1 mg), amino-PEG4-t-butyl ester (2 mg, manufactured by Tokyo Chemical Industry Co., Ltd.), triethylamine (1 μL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and DMF (200 μL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 2 mL test tube reaction vessel, and the mixture was stirred at room temperature for 16 hours. After confirming the completion of the reaction by LC / MS, the solvent was removed under reduced pressure, and methylene chloride (400 μL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and trifluoroacetic acid (100 μL, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the resulting residue, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed to be complete by LC / MS, and the solution to which acetonitrile was added was purified by reverse-phase silica gel column chromatography (C18, Biotage, acetonitrile:distilled water = 0:100 → 60:40). The target product was recovered, and the solvent was removed by freeze-drying to obtain compound (9) (1 mg, yield 79%), a dark blue powder. [M+H + ] + :696

[0162] <Synthesis of compound (9-NHS)> In the synthesis method for compound (7-NHS), 0.2 mg of compound (9-NHS) was obtained as a dark blue powder, except that compound (9) was used instead of compound (7). [M+H + ] + :793

[0163] <Synthesis of labeled antibody (9)> Labeled antibody (9) was obtained in the same manner as in the synthesis method of labeled antibody (7), except that compound (9-NHS) was used instead of compound (7-NHS).

[0164] [Synthesis Example 10: Synthesis of Compound (10) and Labeled Antibody (10)]

[0165] [ka]

[0166] <Synthesis of compound (10-A) to compound (10-NHS)> In the synthesis method for compound example 33 (2-(3,7-di(azetidine-1-yl)-5,5-dimethyldibenzo[b,e]silin-10-illium-10(5H)-yl)-4-(((2,5-dioxopyrrolidine-1-yl)oxy)carbonyl)benzoate) described in U.S. Patent Application Publication No. 2018 / 0284105, compound (10) (2 mg) and compound (10-NHS) (0.5 mg) were obtained in the same manner, except that dichloromethylvinylsilane (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of dichlorodimethylsilane. Compound (10) [M+H + ] + :509 Compound (10-NHS) [M+H + ] + :606

[0167] <Synthesis of labeled antibody (10)> Labeled antibody (10) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (10-NHS) was used instead of compound (1-NHS).

[0168] [Synthesis Example 11: Synthesis of Compound (11) and Labeled Antibody (11)]

[0169] [ka]

[0170] <Synthesis of compound (11) and compound (11-NHS)> Compound (11) (1 mg) and compound (11-NHS) (0.3 mg) were obtained in the same manner as in the synthesis of compound (10) and compound (10-A), except that 3-fluoroazetidine hydrochloride (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of azetidine hydrochloride. Compound (11) [M+H + ] + :545 Compound (11-NHS) [M+H + ] + :642

[0171] <Synthesis of labeled antibody (11)> Labeled antibody (11) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (11-NHS) was used instead of compound (1-NHS).

[0172] [Synthesis Example 12: Synthesis of Compound (12) and Labeled Antibody (12)]

[0173] [ka]

[0174] <Synthesis of compound (12) and compound (12-NHS)> Compound (12) (0.6 mg) and compound (12-NHS) (0.1 mg) were obtained in the same manner as in the synthesis of compound (10) and compound (10-A), except that 3,3-difluoroazetidine hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used instead of azetidine hydrochloride. Compound (12) [M+H + ] + :581 Compound (12-NHS) [M+H + ] + :678

[0175] <Synthesis of labeled antibody (12)> Labeled antibody (12) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (12-NHS) was used instead of compound (1-NHS).

[0176] [Synthesis Example 13: Synthesis of Compound (13) and Labeled Antibody (13)]

[0177] [ka]

[0178] <Synthesis of compound (13-A)> Compound (13-A), a white powder, was obtained in the same manner as in the synthesis method of compound (10-F), except that compound (10-C) was used instead of compound (10-E).

[0179] <Synthesis of compound (13-B)> Compound (13-B) (414 mg, yield 62%) was obtained in the same manner as in the synthesis method of compound (10), except that compound (13-A) was used instead of compound (10-F) and 3-fluoroazetidine hydrochloride was used instead of azetidine hydrochloride. [M+H + ] + :397

[0180] <Synthesis of compound (13)> 200 mg of tert-butyl-4-bromo-3,5-dimethoxybenzoic acid (Combi-Blocks) and 2 mL of tetrahydrofuran were added to a 10 mL round-bottom flask. After purging with nitrogen, the mixture was cooled to -78°C, and n-butyllithium (0.405 mL, 1.6 M, n-hexane solution, Kanto Chemical Co., Ltd.) was added dropwise and the mixture was stirred for 15 minutes. Subsequently, 3 mL of tetrahydrofuran solution of 51 mg of compound (13-B) was added, and the mixture was heated to -10°C while stirring. The completion of the reaction was confirmed by LC / MS, and 5 M aqueous hydrochloric acid was added. After removing the solvent under reduced pressure, the resulting residue was purified by reverse-phase silica gel column chromatography (C18, Biotage, acetonitrile: distilled water containing 0.1% trifluoroacetic acid = 5:95 → 50:50). The target product was recovered, and the solvent was removed by freeze-drying to obtain compound (13) (37 mg, yield 44%) as a dark blue powder. [M+H + ] + :561 <Synthesis of compound (13-NHS)> Compound (13-NHS), a dark blue powder, was obtained in the same manner as the synthesis method for compound (1-NHS), except that compound (13) was used instead of compound (1).

[0181] <Synthesis of labeled antibody (13)> Labeled antibody (13) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (13-NHS) was used instead of compound (1-NHS).

[0182] [Synthesis Example 14: Synthesis of Compound (14) and Labeled Antibody (14)]

[0183] [ka]

[0184] <Synthesis of compound (14-A)> Compound (14-A) (213 mg, yield 47%) was obtained in the same manner as in the synthesis of compound (13-B), except that 3-fluoroazetidine hydrochloride was replaced with 3-fluoropyrrolidine hydrochloride (manufactured by Enamine). [M+H + ] + :425

[0185] <Synthesis of compound (14)> Compound (14) (3.5 mg, yield 5.6%) was obtained in the same manner as in the synthesis method of compound (13), except that compound (14-A) was used instead of compound (13-B). [M+H + ] + :589 <Synthesis of compound (14-NHS)> Compound (14-NHS), a dark blue powder, was obtained in the same manner as in the synthesis method of compound (1-NHS), except that compound (14) was used instead of compound (1).

[0186] <Synthesis of labeled antibody (14)> Labeled antibody (14) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (14-NHS) was used instead of compound (1-NHS).

[0187] [Synthesis Example 15: Synthesis of Compound (15) and Labeled Antibody (15)]

[0188] [ka]

[0189] <Synthesis of compound (15-A)> Compound (15-A) (45 mg, yield 34%) was obtained in the same manner as in the synthesis of compound (13-B), except that methyl(2,2,2-trifluoroethyl)amine hydrochloride (manufactured by Matrix Scientific) was used instead of 3-fluoroazetidine hydrochloride. [M+H + ] + :473

[0190] <Synthesis of compound (15)> Compound (15) (0.96 mg, yield 1.6%) was obtained in the same manner as in the synthesis method of compound (13), except that compound (15-A) was used instead of compound (13-B). [M+H + ] + :637 <Synthesis of compound (15-NHS)> Compound (15-NHS), a dark blue powder, was obtained in the same manner as the synthesis method for compound (1-NHS), except that compound (15) was used instead of compound (1).

[0191] <Synthesis of labeled antibody (15)> Labeled antibody (15) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that compound (15-NHS) was used instead of compound (1-NHS).

[0192] [Synthesis Example 16: Synthesis of Comparative Compound (1) and Comparative Labeled Antibody (1)]

[0193] [ka]

[0194] <Synthesis of comparative compound (1-A) to comparative compound (1-NHS)> Comparative compound (1) (5 mg) and comparative compound (1-NHS) (1 mg) were obtained in the same manner as in the synthesis methods of compound (1) and compound (1-NHS), except that dichlorodimethylsilane (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of dichloromethylvinylsilane. Compound (11) [M+H + ] + :465 Compound (11-NHS) [M+H + ] + :563

[0195] <Synthesis of comparative labeled antibody (1)> Comparative labeled antibody (1) was obtained in the same manner as in the synthesis method of labeled antibody (1), except that comparative compound (1-NHS) was used instead of compound (1-NHS).

[0196] [1] Evaluation of water solubility 5 μL of DMSO solution containing the above compound (hereinafter referred to as "sample") at a concentration of 20 mM and 495 μL of PBS at pH 7.4 were added to a 1.5 mL Eppendorf tube and mixed. The mixture was stirred at 2000 rpm for 30 minutes using a Multi Shaker MS 300 (product name, manufactured by AS ONE Corporation). The resulting mixture was allowed to stand in the dark for 60 minutes, and then centrifuged (12000 rpm, 5 minutes). The filtrate, filtered through a 0.20 μm pore size filter, was measured for sample concentration (the sample concentration is 200 μM when the sample is completely dissolved) using a Nexera UHPLC (product name, manufactured by Shimadzu Corporation, column: Shim-pack XR-ODSII), and evaluated based on the following evaluation criteria. Regarding water solubility, a higher evaluation rank is highly desirable because it indicates higher hydrophilicity. In this test, it is preferable for the water solubility to meet an evaluation rank of "C" or higher from the viewpoint of practicality as a fluorescent compound. - Criteria for evaluating water solubility - A: 100μM or more B: 10 μM or more and less than 100 μM C: 1 μM or more and less than 10 μM D: Less than 1 μM, or low water solubility making concentration measurement impossible.

[0197] [2] Evaluation of fluorescence quantum yield The above compounds were evaluated using PBS solutions (pH 7.4) according to the method described in the following reference materials. "A Guide to Recording Fluorescence Quantum Yields" (a document from HORIBA Scientific, available at https: / / www.horiba.com / fileadmin / uploads / Scientific / Documents / Fluorescence / quantumyieldstrad.pdf) Regarding fluorescence quantum yield, a higher evaluation rank indicates superior fluorescence quantum yield and is therefore highly desirable. In this test, a fluorescence quantum yield of evaluation rank "C" or higher is preferable from the viewpoint of practicality as a fluorescent compound. - Criteria for evaluating fluorescence quantum yield - A: 0.5 or higher B: 0.3 or higher, less than 0.5 C: 0.1 or greater, less than 0.3 D: Less than 0.1 E: Evaluation not possible due to low water solubility.

[0198] [3] Evaluation of the maximum molar extinction coefficient The above compounds were evaluated using a PBS solution (pH 7.4) according to the method described in the following reference materials. The unit of the maximum molar extinction coefficient in the evaluation criteria below is mole. -1 Lcm -1 That is the case. “TECH TIP #6 Extinction Coefficients” (Available from Thermo Scientific's document at https: / / assets.thermofisher.com / TFS-Assets / LSG / Application-Notes / TR0006-Extinction-coefficients.pdf) Regarding the maximum molar extinction coefficient, a higher evaluation rank is highly desirable because it indicates superior maximum molar extinction coefficient. In this test, it is preferable from the viewpoint of practicality as a fluorescent compound that the maximum molar extinction coefficient meets an evaluation rank of "C" or higher. - Criteria for evaluating the maximum molar extinction coefficient - A: Over 100,000 B: 50,000 or more, less than 100,000 C: 10,000 or more, less than 50,000 D: Less than 10,000 E: Evaluation not possible due to low water solubility.

[0199] [4] Evaluation of lightfastness The compound synthesized above was dissolved in PBS solution (pH 7.4) so ​​that the absorbance of the absorption wavelength peak was 0.095 to 0.105. This solution was exposed to light using a merry-go-round light irradiation machine [Ushio Inc. xenon lamp UXL-500D-O, HA-50 filter, Y44 filter, exposure intensity 22 mW / cm²]. 2 Under exposure using [500nm equivalent], the absorbance of the absorption wavelength peak of each compound was measured over time using a spectrometer (HP, Agilent 8453). The absorbance of the absorption wavelength peak before exposure was set to 100%, and the exposure time until the absorbance of this absorption wavelength peak decreased by 20% (the absorbance of the absorption wavelength peak reached 80%) was determined and evaluated based on the following evaluation criteria. A higher evaluation rank is highly desirable because it indicates a longer period of stability. In this test, it is preferable for the lightfastness to meet an evaluation rank of "B" or higher from the viewpoint of practicality as a fluorescent compound. - Criteria for evaluating lightfastness - A: Over 100 hours B: 50 hours or more but less than 100 hours C: 25 hours or more but less than 50 hours D: 2 hours or more but less than 25 hours E: Less than 2 hours F: Evaluation not possible due to low water solubility.

[0200] [Table 1]

[0201] Compounds (1) to (15) and comparative compound (1) are the compounds (1) to (15) and comparative compound (1) synthesized above, respectively, and AlexaFluor647 is AlexaFluor647 (trade name, product number A33084) manufactured by Thermo Scientific. In Examples 1-13, 1-14, and 1-15, the evaluation of lightfastness was not always accurate, and is indicated as "-" in the table above. However, it has been confirmed that compounds (13) to (15) tend to exhibit sufficient lightfastness at the same level as compounds (1) to (12).

[0202] From the results in Table 1 above, the following can be seen. The commercially available fluorescent compounds used in Reference Examples 1-2 exhibited poor lightfastness. In contrast, compounds (1) to (15) and comparative compound (1), which are fluorescent compounds of the present invention, exhibit excellent lightfastness, water solubility, fluorescence quantum yield, and maximum molar extinction coefficient, all of which are at a desirable level from a practical standpoint. It is expected that labeled biomaterials exhibiting excellent lightfastness and excellent fluorescence quantum yield can be obtained. Among these compounds, for example, in the fluorescent compound of the present invention, R 7 ~R 11 Compound (6) in which at least one of the groups contains an electron-withdrawing group is R 7 ~R 11 In addition, in the fluorescent compounds of the present invention, R 7 ~R 11 A compound (15) in which at least one of the groups contains an electron-withdrawing group is R 7 ~R 11 However, all of these compounds show better fluorescence quantum yields compared to compound (4) which does not contain electron-withdrawing groups. Furthermore, in the fluorescent compound of the present invention, R 7 and R 8 , or R 10 and R11 Compounds (10) to (14) in which at least one of each combination is bonded to each other to form a 4- to 7-membered aliphatic heterocycle are R 7 and R 8 , or R 10 and R 11 All of these combinations show better fluorescence quantum yields compared to compound (9), which does not form a 4- to 7-membered aliphatic heterocycle. Although compounds (9) and compounds (10) to (14) have different structures of substituent Z in the fluorescent compounds of the present invention, the effect of achieving good fluorescence quantum yields is considered to be due to the formation of a 4- to 7-membered aliphatic heterocycle, considering that there is almost no change in fluorescence quantum yield associated with changes in the structure of substituent Z as described in J. Am. Chem. Soc. 2017, 139, 17397-17404.

[0203] Although we have described the present invention along with its embodiments, we do not intend to limit our invention in any detail of the description unless specifically designated, and we believe that it should be interpreted broadly without contradicting the spirit and scope of the invention as set forth in the appended claims.

[0204] This application claims priority based on Japanese Patent Application No. 2021-036777, filed in Japan on 8 March 2021, the contents of which are incorporated herein by reference as part of this specification.

Claims

1. A fluorescent compound represented by the following general formula (IA) or a salt thereof. 【Chemistry 1】 In the formula, R １ ~R ６ This indicates a hydrogen atom or a halogen atom. R ７ , R ８ , R １０ and R １１ This indicates an alkyl group. However, the above R ７ and R ８ , and R １０ and R １１ are each bonded to each other to form a 4- or 5-membered aliphatic heterocyclic ring, or at least one of the above R ７ and R ８ , and at least one of R １０ and R １１ is an alkyl group substituted with a fluorine atom. R ２１ and R ２２ In this case, one element represents an alkyl group, and the other represents an alkenyl group. R ４１ ~R ４５ This represents a hydrogen atom, halogen atom, alkyl group, alkoxy group, carboxyl group, amide group, or substituent that can be bonded to the following biomolecules. 【Chemistry 2】 However, the fluorescent compound represented by formula (IA) above, or a salt thereof, satisfies at least one of the following (C-1) to (C-3). (C-1)R ４２ ~R ４４ At least one of these groups is a group having either a carboxyl group or a substituent capable of being bonded to the biomolecule. (C-2)R ４１ and R ４５ is an alkyl group or halogen atom. (C-3)R ４１ and R ４５ At least one of them is a fluorine atom or an alkoxy group.

2. The aforementioned R ７ , R ８ , R １０ and R １１ However, it is an alkyl group having 1 to 3 carbon atoms, and the above R ７ and R ８ At least one of the following, and R １０ and R １１ At least one of them is a C1-C3 alkyl group substituted with a fluorine atom, The aforementioned R ４１ ~R ４５ A fluorescent compound or salt thereof according to claim 1, which satisfies (C-1) and (C-2) above, or satisfies (C-1) and (C-3) above, or satisfies all of (C-1) to (C-3) above.

3. The aforementioned R ７ and R ８ , and R １０ and R １１ These elements bond to each other to form a four- or five-membered aliphatic heterocycle. The aforementioned R ４１ ~R ４５ A fluorescent compound or salt thereof according to claim 1, which satisfies (C-1) and (C-2) above, or satisfies (C-1) and (C-3) above, or satisfies all of (C-1) to (C-3) above.

4. A fluorescently labeled biomaterial comprising a fluorescent compound or salt thereof according to any one of claims 1 to 3, bound to a biomaterial.

5. The fluorescently labeled biomaterial according to claim 4, wherein the biomaterial is any of a protein, amino acid, nucleic acid, glycan, and lipid.

6. The fluorescently labeled biomaterial according to claim 4 or 5, wherein the binding of the fluorescent compound or a salt thereof to the biomaterial is by any of the following methods i) to v). i) Non-covalent or covalent bonds between peptides, ii) Van der Waals interactions between long-chain alkyl groups in fluorescent compounds and lipid bilayers or lipids in biomaterials. iii) An amide bond formed by reacting an N-hydroxysuccinimide ester in a fluorescent compound with an amino group in a biomolecule, iv) A thioether bond formed by reacting a maleimide group in a fluorescent compound with a sulfanyl group in a biomaterial, v) A bond involving the formation of a triazole ring, which is formed by a click reaction between an azide group in a fluorescent compound and an acetylene group in a biomaterial, or between an acetylene group in a fluorescent compound and an azide group in a biomaterial.

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