Compounds and labeled biological substances using the same

Abstract

This invention provides the following compounds and labeled biological substances having the compounds. 1 ~R 6 R 11 ~R 13 R 22 ~R 29 This represents a specific group, where n is an integer from 1 to 3. (Selected from R...) 1 R 2 R 5 R 22 ~R 25 One of them and selected from R 3 R 4 R 6 R 26 ~R 29 One of them is bonded via a linking group LL. The linking group LL represents a linking group with 1 to 100 atoms that does not possess any of the following: an aromatic hydrocarbon ring, a sulfonyl group, or a phosphonoyl group. R 1 ~R 6 R 22 ~R 29 At least one of them contains -(CH2-CH2-O) m - indicates a structure where m = 1 to 50. The following compounds are neutral compounds containing at least one carboxyl group or a substituent capable of bonding with biological material.

Description

Technical Field

[0001] This invention relates to a compound and a labeled biological substance using the compound. Background Technology

[0002] To observe changes in response to various stimuli (diseases, environmental changes, etc.) in living organisms, fluorescently labeled biological substances (antibodies, etc.) that have a binding affinity to the target substance are mostly used.

[0003] For example, in Western blotting (hereinafter also referred to as WB), which detects a specific protein from a mixture of proteins, a fluorescence method is also used to detect the presence or amount of the specific protein by using a fluorescently labeled antibody that has a bond to the protein.

[0004] Furthermore, in bioimaging techniques that analyze the dynamics and functions of living molecules, cells, and tissues in living organisms, in vivo fluorescence imaging, which visualizes specific parts of living organisms through fluorescent labeling, is one of the techniques for in vivo observation.

[0005] Anthocyanins are known as fluorescent pigments for the aforementioned fluorescent labeling. However, when anthocyanins are used for fluorescent labeling, self-association and other interactions easily occur between the labeled pigments, tending to reduce the fluorescence quantum yield.

[0006] As a technology to address this problem, patent documents 1-3 disclose, for example, a macrocyclic anthocyanin with a large-membered ring structure introduced into a compound. According to patent document 1, the macrocyclic anthocyanin compound, formed by linking heterocycles at both ends of the polymethyl chain, exhibits improved stability against exopeptidases, inhibited aggregation, and easier excretion from the liver when used as an imaging agent in clinical settings. According to patent document 2, by forming a ring with one of the heterocycles at both ends of the polymethyl chain... 3 In macrocyclic anthocyanin compounds formed by carbon atoms linked to cyclic nitrogen atoms of another heterocycle, self-association is suppressed, fluorescence intensity is increased, and stability is improved. Furthermore, in Patent Document 3, by preparing macrocyclic anthocyanin compounds formed by crosslinking of at least one water-soluble group having a sulfonate (sulfonic acid or its salt) group and a phosphonate (phosphonic acid or its salt) group, quantum yield and water solubility are improved, thereby suppressing self-association and increasing brightness (fluorescence intensity).

[0007] Previous technical documents

[0008] Patent documents

[0009] Patent Document 1: International Publication No. 2005 / 000218

[0010] Patent Document 2: International Publication No. 2006 / 047452

[0011] Patent Document 3: International Publication No. 2012 / 012595 Summary of the Invention

[0012] The technical problem to be solved by the invention

[0013] Pigments used for fluorescent labeling are required to exhibit excellent fluorescence intensity in various states, such as solution, film, or spot imprint. However, the inventors conducted further research on fluorescence intensity and found that, in fluorescent labeling using anthocyanins specifically disclosed in Patent Documents 1 to 3, a sufficient level of fluorescence intensity could not be obtained in any state, including solution, film, and spot imprint.

[0014] The objective of this invention is to provide a compound that exhibits excellent fluorescence intensity as a labeled biological substance in any of the following states: solution, membrane, and dot blot. Furthermore, the objective of this invention is to provide a labeled biological substance formed by bonding this compound to a biological substance.

[0015] means for solving technical problems

[0016] That is, the above-mentioned problems of the present invention are solved by the following method.

[0017] [1]

[0018] A compound represented by the following general formula (1),

[0019] [Chemical Formula 1]

[0020]

[0021] In the formula, R 1 ~R 6 This indicates that the alkyl group or -(CH2-CH2-O) can have substituents. m -R 21 m is 1–50, R 21 This indicates an alkyl group that can have substituents.

[0022] R 11 ~R 13 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring.

[0023] R 22 ~R 29It represents a hydrogen atom, alkyl, alkoxy, aryl, sulfonyl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, amide, nitro, or halogen atom. Adjacent groups can bond to each other to form a fused ring.

[0024] n is an integer from 1 to 3.

[0025] Among them, selected from R 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R 6 and R 26 ~R 29 One of them is bonded via a linking group LL. The linking group LL represents a divalent linking group with 1 to 100 atoms. The linking group LL does not contain any groups selected from aromatic hydrocarbon rings, sulfonyl groups, and phosphonyl groups.

[0026] R 1 ~R 6 and R 22 ~R 29 At least one of them contains -(CH2-CH2-O) m - indicates the structure. m has the same meaning as the m mentioned above.

[0027] The compound represented by the above general formula (1) is a neutral compound containing at least one carboxyl group or a substituent capable of bonding with biological material.

[0028] [2]

[0029] The compound according to [1] is represented by any one of the following general formulas (1-1) to (1-3).

[0030] [Chemical Formula 2]

[0031]

[0032] In the formula, L 1 ~L 6 Indicates alkylene or -(CH2-CH2-O) m -alkylene-*. * indicates the bonding position with the linking group LL.

[0033] R 7 and R 8 This indicates that the alkyl group or -(CH2-CH2-O) can have substituents. m -R 21 .

[0034] R 1 ~R6 R 11 ~R 13 R 21 ~R 29 ,LL, m and n and the above R 1 ~R 6 R 11 ~R 13 R 21 ~R 29 , LL, m and n have the same meaning.

[0035] [3]

[0036] According to the compound described in [2], wherein the above-mentioned L 1 ~L 6 Each contains -(CH2-CH2-O) m - indicates the structure, where m = 1 to 50.

[0037] [4]

[0038] The compound according to any one of [1] to [3], wherein the linking group LL is a divalent linking group having a carboxyl group or a substituent capable of bonding with biological material.

[0039] [5]

[0040] The compound according to any one of [1] to [4], wherein both heterocycles in the above general formula satisfy the following condition I.

[0041] (Condition I)

[0042] sp as a cyclic atom of a heterocycle 3 At least one substituent on the carbon atom and the substituent on the cyclic nitrogen atom of the heterocycle include -(CH2-CH2-O). m - indicates the structure, where m = 1 to 50.

[0043] [6]

[0044] The compound according to any one of [1] to [5], wherein the linking group LL of the above-mentioned linking group R 1 ~R 6 R 22 ~R 29 or L 1 ~L 6 The connecting parts are -O-base, -S-base, and -NR. 50 -base, -COO-base or -CONR 50 -Base. Wherein, R 50 It is a hydrogen atom or an alkyl group.

[0045] [7]

[0046] The compound according to any one of [1] to [6], wherein the above R 11 ~R 13 At least one of them is an aryloxy group.

[0047] [8]

[0048] A biolabeled substance, which is formed by bonding a compound, any one of [1] to [7], to a biolabel.

[0049] [9]

[0050] According to the labeled biological material described in [8], wherein,

[0051] The aforementioned biological substances are any one of proteins, amino acids, nucleic acids, sugar chains, and phospholipids.

[0052] Invention Effects

[0053] The compounds of the present invention can produce labeled biological substances exhibiting excellent fluorescence intensity in any state, including solution, membrane, and dot-printed samples. Furthermore, the labeled biological substances of the present invention exhibit excellent fluorescence intensity. Detailed Implementation

[0054] In this invention, when multiple substituents or linking groups (hereinafter referred to as substituents, etc.) represented by specific symbols or formulas are present, or when multiple substituents, etc. are specified simultaneously, each substituent, etc., may be the same or different from each other unless otherwise specified. This also applies to the specification of the number of substituents, etc. Furthermore, when multiple substituents, etc., are close together (especially adjacent), they may connect to each other to form a ring unless otherwise specified. Furthermore, unless otherwise specified, rings, such as alicyclic rings, aromatic rings, and heterocyclic rings, may further fuse to form fused rings.

[0055] In this specification, unless otherwise specified, double bonds, when present in a molecule, can be either E-type or Z-type, or a mixture thereof. Furthermore, unless otherwise specified, when present as a compound, diastereomers and enantiomers can be either either, or a mixture thereof.

[0056] In this invention, the designation of compounds and substituents includes not only the compound itself and the substituent itself, but also its salts and ions. For example, carboxyl, sulfonyl, and phosphonoyl (-P(=O)(OH)2) can be used to adopt an ionic structure by dissociating hydrogen atoms, or a salt structure. That is, in this invention, "carboxyl" is used to mean that it includes a carboxylate ion or its salt, "sulfonyl" is used to mean that it includes a sulfonate ion or its salt, and "phosphonoyl" is used to mean that it includes a phosphonate ion or its salt. There are no particular limitations on the monovalent or polyvalent cations that constitute the above-mentioned salt structures; inorganic cations, organic cations, etc., can be cited. Specifically, Na can be cited as an example. + Li + and K + Alkali metal cations, Mg 2+ Ca 2+ And Ba 2+ Alkaline earth metal cations, as well as organic ammonium cations such as trialkylammonium cations and tetraalkylammonium cations.

[0057] In the case of a salt structure, there can be one type of salt, or two or more types of salt can coexist. There can also be a mixture of salt-type and free acid-type groups in the compound, and there can also be a mixture of salt-type and free acid-type compounds.

[0058] All compounds of this invention are neutral compounds. In this invention, neutrality means electroneutrality. Specifically, the overall charge of the compound is adjusted to 0 by means of charged groups or counterions within the compound. For example, in compounds represented by general formula (1), R 6 The bonded nitrogen atom has a formal charge of +1, and in a manner that pairs with this formal charge, the ionizable groups such as sulfonyl groups in the compound have ionic structures such as sulfonate ions, thereby making the compound of the present invention a compound with a charge of 0 as a whole.

[0059] In the general formulas specified in this invention, for convenience, the positive charge of the compound is defined as the structure possessed by a specific nitrogen atom. Since the compounds of this invention have conjugated systems, in practice, atoms other than the nitrogen atom can sometimes also carry a positive charge. Compounds whose chemical structures can be represented by the general formulas are included in the compounds represented by the general formulas. This also applies to negative charges.

[0060] Furthermore, this indicates that the compound includes compounds whose structure has been modified to a degree that does not impair the effects of the present invention. Moreover, regarding compounds where substitution or non-substitution is not explicitly stated, this indicates that any substituent may be present to a degree that does not impair the effects of the present invention. This also applies to substituents (e.g., groups expressed as "alkyl", "methyl", "methyl", etc.) and linking groups (e.g., groups expressed as "alkylene", "methylene", "methylene", etc.). Among such arbitrary substituents, in the present invention, substituents preferably selected from the substituent group T described later.

[0061] In this invention, when the number of carbon atoms of a certain group is specified, unless otherwise specified in this invention or specification, that number represents the total number of carbon atoms of the group. That is, when the group is further substituented, it represents the total number of carbon atoms including the substituent.

[0062] Furthermore, in this invention, the numerical range represented by “~” indicates the range of values ​​recorded before and after “~”, which are included as the lower limit and upper limit values.

[0063] The compounds of the present invention are represented by the following general formula (1). The detailed reasons why the compounds of the present invention can produce labeled biological substances exhibiting excellent fluorescence intensity in any state, including solution, membrane, and dot-printed samples, are not yet certain, but are believed to be as follows.

[0064] As shown in general formula (1), the compounds of the present invention have a polymethyl chain with an indoline ring and a pseudoindoline ring at both ends, and R 5 The bonded indoline cyclic nitrogen atom has a tertiary amine structure, R 6 The bonded pseudoindole cyclic nitrogen atom has a quaternary ammonium structure, thereby generating absorption through charge movement via the polymethyl backbone.

[0065] The aforementioned polymethine chain refers to a methine chain linked by conjugated double bonds in this invention, and the methine chain having 2n+3 carbon atoms. The hydrogen atom of the methine can be R. 11 ~R 13 Substituents that can be used are excluded from the number of carbon atoms constituting the aforementioned methine chain. Thus, the compounds of the present invention are classified as compounds called polymethine pigments (broadly defined as anthocyanins).

[0066] The compounds of the present invention, in addition to having the above-described structure, also possess the following characteristics: the substituent R in the above-described indoline ring and pseudoindoline ring. 1 ~R 6 and R 22~R 29 At least one of them contains an ethoxy group repeating 1 to 50 times, and is selected from the substituent R of the indoline ring. 1 R 2 R 5 and R 22 ~R 25 One of them is selected from the substituent R of the pseudoindole ring. 3 R 4 R 6 and R 26 ~R 29 One of them is bonded to a specific linking group LL that does not contain any groups selected from aromatic hydrocarbon rings, sulfonyl and phosphonyl groups to form a large member ring.

[0067] That is, by including ethoxy groups with a repeat number of 1 to 50 that have a volume exclusion effect, and by not including highly hydrophobic aromatic hydrocarbon rings in the linking group LL, the interaction between compounds is suppressed, and the decrease in fluorescence intensity caused by self-association of compounds can be suppressed. Furthermore, by including ethoxy groups with a repeat number of 1 to 50 that have a volume exclusion effect, and by not including any of the highly hydrophilic sulfonyl or phosphonyl groups in the linking group LL, the decrease in bonding with biological substances (e.g., antibodies, hereinafter the same) is suppressed, and the decrease in the activity of biological substances based on the charge of sulfonyl or phosphonyl groups (e.g., antibody activity, hereinafter the same) is suppressed, and the obtained labeled biological substances are considered to exhibit excellent fluorescence intensity. In other words, in the labeled biological substances obtained from the compounds of the present invention, the linking group LL is considered to help suppress the self-association of compounds, but it is considered that the presence of sulfonyl or phosphonyl groups on the linking group LL affects the charge balance on the surface of the biological substance, resulting in reduced activity of the obtained labeled biological substances (labeled antibodies, etc.) and reduced fluorescence intensity in target detection. Especially when the aforementioned linking group LL has a carboxyl group or a substituent that can bond with biological material, since sulfonyl and phosphonyl groups are present near the substituent that bonds with biological material, it is believed that the bonding with biological material is further reduced due to charge repulsion.

[0068] In contrast, as specifically described in Patent Document 1 or 2 above, even if a large-membered ring structure is formed by connecting heterocycles at both ends of the polymethyl chain, the compound will have poor water solubility and its fluorescence intensity will decrease due to association if the heterocycle (including fused ring) does not contain ethoxy groups with a repeating number of 1 to 50. Moreover, as described in Patent Document 3 above, by having at least any sulfonyl or phosphonoyl groups in the linking group forming the large-membered ring structure, the binding affinity with biological material is reduced, or the activity of the biological material is reduced, and the resulting labeled biological material is unlikely to exhibit excellent fluorescence intensity.

[0069] The compounds of the present invention have an excitation absorption wavelength near 585 nm when n = 1, near 685 nm when n = 2, and near 785 nm when n = 3, depending on the length of the methylene chain with repeating number 2n+3. Therefore, the compounds represented by these general formulas (1) can be used as compounds exhibiting excellent fluorescence intensity in fluorescent labels using excitation light sources near 600 nm, 700 nm, and 800 nm, respectively.

[0070] In multicolor Western blotting (WB), multiple emission colors are detected across a range from the visible to the near-infrared region. Therefore, it is necessary to select the excitation sources in a way that ensures an appropriate wavelength relationship between the absorption and emission waveforms of the multiple pigments, preventing crosstalk caused by interference when the pigments are excited to emit light. Ideally, the excitation light should be adjusted so that only one pigment emits light in a given excitation light, while the others do not. From this perspective, for example, in the near-infrared region of multicolor WB, two excitation sources with somewhat separated wavelengths, such as those around 700 nm and 800 nm, can be used.

[0071] Compared to visible light-excited detection, near-infrared fluorescence detection suppresses membrane autofluorescence, i.e., background fluorescence, thus easily improving the signal-to-noise ratio (S / N ratio) and enabling highly sensitive detection of target proteins. Therefore, in recent years, the necessity of near-infrared fluorescence detection (WB) has increased in the analysis of trace proteins.

[0072] However, in the near-infrared region, fluorescent dyes typically exhibit low fluorescence quantum yields, making it difficult to obtain high signal quantities. Even in multicolor WB with the two compounds mentioned above, one near 700 nm and the other near 800 nm, compounds of the present invention with n=2 or 3 can be used as compounds exhibiting excellent fluorescence intensity. In particular, for the requirement of higher sensitivity observation and detection of proteins, they can exhibit superior fluorescence intensity compared to conventional anthocyanin fluorescent labels containing anthocyanins described in Patent Documents 1 to 3.

[0073] Hereinafter, the compounds represented by general formula (1) of the present invention will be described in detail.

[0074] <Compounds represented by general formula (1)>

[0075] The compounds of the present invention represented by general formula (1) are described below.

[0076] [Chemical Formula 3]

[0077]

[0078] In the formula, R 1 ~R6 This indicates that the alkyl group or -(CH2-CH2-O) can have substituents. m -R 21 m is 1–50, R 21 This indicates an alkyl group that can have substituents.

[0079] R 11 ~R 13 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring.

[0080] R 22 ~R 29 It represents a hydrogen atom, alkyl, alkoxy, aryl, sulfonyl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, amide, nitro, or halogen atom. Adjacent groups can bond to each other to form a fused ring.

[0081] n is an integer from 1 to 3.

[0082] Among them, selected from R 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R 6 and R 26 ~R 29 One of them is bonded via a linking group LL. This is represented in the above chemical structural formula by connecting the structure surrounded by two [] with the linking group LL.

[0083] The linking group LL mentioned above represents a divalent linking group with 1 to 100 atoms. The linking group LL does not contain any groups selected from aromatic hydrocarbon rings, sulfonyl groups, and phosphonyl groups.

[0084] R 1 ~R 6 and R 22 ~R 29 At least one of them contains -(CH2-CH2-O) m - indicates the structure. m has the same meaning as the m mentioned above.

[0085] The compound represented by the above general formula (1) is a neutral compound containing at least one carboxyl group or a substituent capable of bonding with biological material.

[0086] The substituents in general formula (1) will be explained in detail below.

[0087] (1)R 1 ~R6

[0088] R 1 ~R 6 This indicates that the alkyl group or -(CH2-CH2-O) can have substituents. m -R 21 .

[0089] Can be used as R 1 ~R 6 The alkyl group has the same meaning as the alkyl group in the substituent group T described later.

[0090] The number of carbon atoms in the unsubstituted alkyl group is preferably 1 to 6, more preferably 1 to 4, and even more preferably 1 to 2.

[0091] The number of carbon atoms in the alkyl portion of the alkyl group having the substituent is preferably 1 to 10, more preferably 1 to 8, even more preferably 2 to 6, and particularly preferably 2 to 5. Furthermore, the number of atoms in the longest chain constituting the alkyl group having the substituent is preferably 3 to 35, more preferably 3 to 25, even more preferably 3 to 15, and particularly preferably 3 to 11.

[0092] In the ability to be used as R 1 ~R 6 When an alkyl group is bonded to a linking group LL, an alkylene group obtained by removing one hydrogen atom or a substituent from an alkyl group that may have substituents is bonded to the linking group LL. In this case, all atoms constituting the shortest molecular chain of the linking heterocycle and the linking group LL are sp. 3 Carbon atom.

[0093] The number of carbon atoms in the alkylene moiety of the alkylene group bonded to the linking group LL is preferably the same as the number of carbon atoms in the alkyl moiety of the substituted alkyl group described above. Furthermore, in the alkylene moiety of the alkylene group bonded to the linking group LL, the number of atoms constituting the bond connecting the indoline ring or pseudoindoline ring to the linking group LL is preferably 1 to 10, more preferably 1 to 8, further preferably 2 to 6, and particularly preferably 3 to 5.

[0094] In this invention, "the number of carbon atoms of the alkyl portion of the alkyl group having a substituent" refers to the number of carbon atoms other than the substituent portion of the alkyl group.

[0095] In this invention, "the number of atoms constituting the longest chain of the alkyl group having substituents" refers to the number of atoms including the substituent portion (i.e., the number of atoms obtained by subtracting the number of atoms in the molecular chain that does not constitute the longest chain from the total number of atoms). Furthermore, when a substituent such as a sulfonyl or carboxyl group, which has dissociable hydrogen atoms, constitutes the longest chain, hydrogen atoms are included in the calculation regardless of whether dissociation occurs. Also, the number of atoms in the substituent portion capable of bonding with biological substances, as described later, is not included.

[0096] In this invention, "the number of atoms constituting the bond between the indoline ring or pseudoindoline ring and the linking group LL in the alkylene moiety when bonded to the linking group LL" refers to the number of atoms including the substituent moiety (i.e., from the atoms constituting R...). 1 ~R 6 The total number of atoms is obtained by subtracting the number of atoms in the molecular chain and branches that do not form the chain connecting the indoline ring or pseudoindoline ring to the linking group LL from the total number of atoms.

[0097] As can be used as R 1 ~R 6 Alkyl groups can have various substituents, including alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, amide, sulfonyl, phosphonoyl, and -(CH2-CH2-O). m -R 21 And groups consisting of combinations of these substituents. Furthermore, examples of substituents capable of bonding with biological substances can be given later. Additionally, the alkyl portion of the above-mentioned alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, amide, sulfonyl, and phosphonoyl groups, and groups consisting of combinations of these substituents, may have substituents capable of bonding with biological substances as described later.

[0098] As can be used as R 1 ~R 6 The alkyl group having substituents is not particularly limited, but is preferably an alkyl group having a carboxyl group or a substituent capable of bonding with biological material at the end. In this case, the carboxyl group or the substituent capable of bonding with biological material can directly replace the alkyl group, or can be replaced by a group consisting of a combination of an alkoxy group and a carboxyl group or a substituent capable of bonding with biological material.

[0099] Additionally, in R 1 ~R 6 When R is bonded to the linking group LL, 1 ~R 6 Preferably, it is an alkyl group that does not have either sulfonyl or phosphonoyl as a substituent.

[0100] (-(CH2-CH2-O) m -R 21 )

[0101] In the ability to be used as R 1 ~R 6 -(CH2-CH2-O) m -R 21 In the middle, m is 1 to 50, R 21 This indicates an alkyl group that can have substituents.

[0102] m represents the average number of replicates (also simply the number of replicates). From the viewpoint of suppressing the reduction in binding with the antibody through a moderate exclusion volume effect and obtaining superior fluorescence intensity, m is preferably 1 to 24, more preferably 1 to 12, even more preferably 1 to 10, particularly preferably 1 to 6, and most preferably 1 to 4.

[0103] Regarding the above average number of replicates, it is possible to perform [analysis / analysis] on the compound. 1 H-NMR measurements are performed, calculated from the average integral value. The average number of repetitions, as defined in this invention, is the number obtained by rounding the first decimal place of the average number of repetitions calculated using the above method.

[0104] R 21 The alkyl groups that can have substituents are suitable for use as R. 1 ~R 6 The alkyl group may have a substituent.

[0105] In the ability to be used as R 1 ~R 6 -(CH2-CH2-O) m -R 21 When bonded to the linking group LL, -(CH2-CH2-O) m -R 21 In, from alkyl groups that can have substituents, i.e., R 21 -(CH2-CH2-O) is obtained by removing one hydrogen atom or a substituent. m -The alkylene group is bonded to the linking group LL. The shortest molecular chain forming the linking heterocycle and the linking group LL is -(CH2-CH2-O). m In -alkylene groups, all the atoms constituting the alkylene moiety are sp. 3 Carbon atom.

[0106] -(CH2-CH2-O) when bonded to the linking group LL m The number of carbon atoms in the alkylene moiety of the -alkylene group is preferably 1 to 10, more preferably 1 to 8, even more preferably 2 to 6, and particularly preferably 2 to 4. Furthermore, the -(CH2-CH2-O) moiety is preferred when bonded to the linking group LL. m -The alkylene moiety of the alkylene group forms a linker -(CH2-CH2-O). m - The number of atoms in the bond with the linking group LL is preferably 1 to 10, more preferably 1 to 8, even more preferably 2 to 6, and especially preferably 2 to 4.

[0107] In this invention, "-(CH2-CH2-O) when bonded to the linking group LL" m -The alkylene moiety of the alkylene group forms a linker -(CH2-CH2-O).m "Number of atoms in the bond with the linking group LL" refers to the number of atoms including the substituent portion (i.e., subtracting the number of atoms that do not form the linking group -(CH2-CH2-O) from the total number of atoms). m - The number of atoms obtained by considering the number of atoms in the molecular chain and branches of the chain with the linking group LL).

[0108] As can be used as R 1 ~R 6 -(CH2-CH2-O) m -R 21 and as R 1 ~R 6 Alkyl groups can function as substituents with the -(CH2-CH2-O) group. m -R 21 Preferably -(CH2-CH2-O) m - Unsubstituted alkyl group.

[0109] R 1 ~R 6 At least one preferred component comprises -(CH2-CH2-O) m From the viewpoint of further improving the fluorescence intensity in solution, film and spot imprint states, it is more preferable that both heterocycles in general formula (1) satisfy the following condition I.

[0110] (Condition I)

[0111] sp as a cyclic atom of a heterocycle 3 At least one substituent on the carbon atom and the substituent on the cyclic nitrogen atom of the heterocycle include -(CH2-CH2-O). m - indicates the structure, where m = 1 to 50.

[0112] "Both heterocycles in general formula (1) satisfy the following condition I" means that R 1 and R 2 At least one of them, R 3 and R 4 At least one of them, and R 5 and R 6 Contains -(CH2-CH2-O) m - indicates the structure, where m = 1 to 50.

[0113] The above is composed of -(CH2-CH2-O) m The structure represented by - is preferably -(CH2-CH2-O). m -R 21 It is directly bonded to the heterocycle that is directly bonded to the aforementioned methine chain.

[0114] The above -(CH2-CH2-O)m The 'm' in - is related to the above -(CH2-CH2-O) m -R 21 The meaning of 'm' in the text is the same.

[0115] R 1 ~R 6 The substituents protrude vertically relative to the anthocyanin skeleton (planar), thus it is speculated that this is achieved by including -(CH2-CH2-O). m The structure indicated by -, when used as the substituent, makes the fused ring portion less susceptible to π-π interactions (enhancing the effect of inhibiting association), thus suppressing the decrease in fluorescence intensity caused by association.

[0116] (2)R 11 ~R 13

[0117] R 11 ~R 13 This represents a hydrogen atom, alkyl group, aryl group, heteroaryl group, alkoxy group, aryloxy group, alkylthio group, arylthio group, amino group, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring.

[0118] Can be used as R 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, and halogen atoms in the substituent group T described below have the same meaning and preferred range as the alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, and halogen atoms in the substituent group T described below.

[0119] Can be used as R 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, and amino groups can be unsubstituted or have substituents.

[0120] As R 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, and amino groups may have substituents, such as those in substituent group T described later, for example, preferably alkoxy or sulfonyl. Furthermore, the aforementioned -(CH2-CH2-O) group is also preferably mentioned. m -R 21 Furthermore, it can also be a group composed of a combination of these substituents, such as -O-(CH2-CH2-O). m -R 21 .

[0121] R 11 ~R 13In this compound, the 5-membered or 6-membered rings formed by the bonding of adjacent groups can be either aromatic or aliphatic, preferably aliphatic. Furthermore, a 6-membered ring is preferred. The number of the aforementioned 5-membered or 6-membered rings in the compound is not particularly limited, but is preferably one or two, more preferably one.

[0122] For example, taking the case of n=3 as an example, as having R 11 ~R 13 The structure formed by adjacent groups bonding together to form a ring is preferably exemplified by the following structures. Additionally, in the following examples, R without a ring structure is described. 11 ~R 13 The structures are hydrogen atoms, and the ring structures do not have substituents, but are not limited to these. Furthermore, structures at the ends of the wavy lines are omitted in the following descriptions.

[0123] [Chemical Formula 4]

[0124]

[0125] R 11 and the R of the carbon atom bonded to the pseudoindole ring 13 Hydrogen atoms are preferred.

[0126] R 12 and other Rs mentioned above 13 Preferably, it is a hydrogen atom, alkyl group, aryloxy group, or arylthio group, and more preferably, it is a hydrogen atom, alkyl group, or aryloxy group.

[0127] R 11 ~R 13 At least one of them is preferably an aryloxy group, R 12 and the R of the carbon atom bonded to the pseudoindole ring mentioned above. 13 Other than R 13 At least one of them is more preferably an aryloxy group.

[0128] R 11 ~R 13 In the middle, R 11 and the R of the carbon atom bonded to the pseudoindole ring 13 Other than R 12 ~R 13 Adjacent groups in the ring (i.e., the R groups on the carbon atoms bonded to the pseudoindole ring) 13 Other than R 13 and R 12Adjacent groups in the ring preferably bond to each other to form a 5- or 6-membered ring, more preferably a 6-membered ring. Furthermore, it is preferable that the aforementioned 5- or 6-membered ring is formed at the central portion of the bond connecting the indoline ring and the pseudoindoline ring. The ring formed at the central portion of the bond connecting the indoline ring and the pseudoindoline ring refers to a ring containing an equal number of carbon atoms from both the indoline ring and the pseudoindoline ring as cyclic atoms.

[0129] (3)R 22 ~R 29

[0130] R 22 ~R 29 It represents a hydrogen atom, alkyl, alkoxy, aryl, sulfonyl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, amide, nitro, or halogen atom. Adjacent groups can bond to each other to form a fused ring.

[0131] In R 22 ~R 29 When bonded to the linking group LL, from which it can be used as R 22 ~R 29 The alkyl, alkoxy, aryl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, or acylamino group with substituents can be obtained by removing one hydrogen atom from an alkylene, alkyleneoxy, arylene, alkylenesulfonamide, cycloalkylenesulfonamide, alkyleneoxycarbonyl, alkylenecarbonyloxy, alkylenecarbamoyl, cycloalkylenecarbamoyl, or alkylenecarbonylamino group with substituents, and then bonded to a linking group LL. The linking group R constitutes the linking group R. 22 ~R 29 All atoms of the shortest molecular chain of the bonded benzene ring and linking group LL are derived from those that can be used as R. 22 ~R 29 The group formed by removing one hydrogen atom from an unsubstituted alkyl, alkoxy, aryl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, or amide group constitutes R. 22 ~R 29 The bonding atoms of the linking group LL are sp 3 Carbon atom.

[0132] Can be used as R 22 ~R 29 The alkyl, alkoxy, aryl, sulfonyl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, amide, nitro, and halogen atoms in this group have the same meaning as the alkyl, alkoxy, aryl, sulfonyl, sulfonamide, alkoxycarbonyl, acyloxy, carbamoyl, amide, nitro, and halogen atoms in substituent group T described later. Furthermore, these groups may have substituents, such as those in substituent group T described later or the aforementioned -(CH2-CH2-O). m -R21 .

[0133] Among them, in R 22 ~R 29 When bonded to the linker group LL, from the viewpoint of obtaining superior fluorescence intensity, R 22 ~R 29 Preferably, the substituent does not have an aromatic hydrocarbon ring and does not have either a sulfonyl or phosphonoyl group. The absence of an aromatic hydrocarbon ring refers to the absence of 1- to 6-valent aromatic hydrocarbon groups such as aryl or arylene. Examples include alkylene-carbamoyl and alkylene groups.

[0134] As R 22 ~R 29 There are no particular limitations on the fused rings formed by the bonding of adjacent groups; for example, naphthalene rings can be cited. Furthermore, from the viewpoint of inhibiting association, R is preferred. 22 ~R 29 Adjacent groups do not bond to each other and do not form fused rings.

[0135] From the perspective of improving water solubility and inhibiting association, R is preferred. 22 ~R 25 At least one of them and R 26 ~R 29 At least one of them has a hydrophilic group, more preferably R 22 ~R 25 The bonded ring and R 26 ~R 29 Each of the bonded rings has at least one hydrophilic group. For example, R 22 ~R 25 and R 26 ~R 29 When adjacent groups in the fused ring form naphthalene rings by bonding with each other, it represents R. 22 ~R 25 The number of bonded rings is 2, R 26 ~R 29 The number of bonded rings is 2, preferably R. 22 ~R 25 At least two and R 26 ~R 29 It must have at least two hydrophilic groups. There is no particular upper limit, as long as it is a possible structure, and the number of hydrophilic groups that can be combined with the overall hydrophilic groups of the compound as described later can be adjusted appropriately.

[0136] As a hydrophilic group, there are no particular limitations as long as it can impart hydrophilicity to the compound. Examples include alkoxy, carboxyl, sulfonyl, and phosphonyl groups with substituents, with sulfonyl being preferred.

[0137] R22 ~R 29 Preferably, it is a hydrogen atom, alkyl group, sulfonyl group, sulfonamide group, alkoxycarbonyl group, acyloxy group, carbamoyl group, amide group, nitro group, or halogen atom.

[0138] In R 22 ~R 29 When R is bonded to the linking group LL, 22 ~R 29 More preferably, it is alkyl, alkoxycarbonyl, acyloxy, carbamoyl or amide, and even more preferably alkyl, carbamoyl or amide.

[0139] In R 22 ~R 29 If not bonded to the linking group LL, it is more preferably a hydrogen atom, sulfonyl, nitro or halogen atom, and even more preferably a hydrogen atom, sulfonyl or halogen atom.

[0140] (4) Linking group LL

[0141] Selected from R 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R 6 and R 26 ~R 29 One of them is bonded via a linker group LL.

[0142] The linking group LL represents a divalent linking group with 1 to 100 atoms. The linking group LL does not contain any group selected from aromatic hydrocarbon rings, sulfonyl groups, or phosphonyl groups. Through bonding via this linking group LL, the rotation of the indoline ring and pseudoindoline ring is suppressed, thereby increasing fluorescence intensity. On the other hand, if the linking group LL contains any group selected from aromatic hydrocarbon rings, sulfonyl groups, or phosphonyl groups, it is believed that the hydrophobicity of the aromatic hydrocarbon ring promotes association, or the charge repulsion exhibited by the sulfonyl or phosphonyl groups reduces the bonding affinity with biological material or decreases the activity of the biological material, thus reducing fluorescence intensity. The phrase "not containing an aromatic hydrocarbon ring" means not containing any 1- to 6-valent aromatic hydrocarbon group such as aryl or arylene.

[0143] In this invention, "the number of atoms of the linking group LL" refers to the number of atoms selected from R that constitute the linking group. 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R6 and R 26 ~R 29 The number of connecting atoms in the shortest molecular chain of one of the constituent molecules. That is, it represents the total number of atoms forming the connecting group LL minus the number of atoms not forming the connecting group selected from R. 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R 6 and R 26 ~R 29 The number of atoms is determined by the number of atoms in the molecular chain and branches (including -H and =O) of one of the chains. For example, when the linking group LL is -CONH-CH2CH(CH3)-NHCO-, the number of atoms in the linking group LL is 6.

[0144] In addition, the linking group LL and R 1 ~R 6 Or R 22 ~R 29 The bonded atoms become, except for sp 3 The linking group LL is determined by atoms other than carbon atoms. Furthermore, according to the aforementioned R... 1 ~R 6 Records of bonding with linking group L and R 22 ~R 29 The record of bonding with the linking group L determines the equivalent of R when bonding with the linking group LL. 1 ~R 6 Or R 22 ~R 29 The groups and linking groups LL.

[0145] The number of atoms in the linking group LL is preferably 5 to 70, more preferably 5 to 50, even more preferably 5 to 30, and especially preferably 5 to 20.

[0146] The linking group LL is preferably selected from alkylene, -O-, -S-, and -NR. 50 -、-COO-、-CONR 50 - and -(CH2-CH2-O) p - A divalent linker formed by the bonding of one or more groups in -. 50 It represents a hydrogen atom or an alkyl group.

[0147] The number of carbon atoms in the alkylene moiety that can be used as the linking group LL is preferably 1 to 10, more preferably 1 to 8, further preferably 1 to 7, especially preferably 1 to 6, and most preferably 1 to 5.

[0148] In this invention, "the number of carbon atoms of the alkylene portion of the alkylene group" refers to the number of carbon atoms other than the substituent portion of the alkylene group.

[0149] Can be used as R 50 The alkyl group is preferably suitable for the above-mentioned R 1 ~R 6 The alkyl group in the text is recorded.

[0150] As R 50 Preferably, it contains hydrogen atoms.

[0151] p represents the average number of repetitions (also simply the number of repetitions), preferably 1 to 50, more preferably 1 to 30, even more preferably 1 to 24, particularly preferably 1 to 20, and most preferably 1 to 12, and most preferably 1 to 4.

[0152] The method and definition for determining the average number of replicates are the same as those described above -(CH2-CH2-O) m - The meaning of the average number of repetitions is the same as that in the text.

[0153] The above-mentioned alkylene groups, -O-, -S-, and -NR that constitute the linking group LL 50 -、-COO-、-CONR 50 - and -(CH2-CH2-O) p The quantity of - is preferably 3 to 11, more preferably 3 to 9, further preferably 3 to 7, especially preferably 3 to 5, and most preferably 3.

[0154] The linking group LL is more preferably selected from alkylene groups, -CONR 50 - and -(CH2-CH2-O) p - is composed of groups, and more preferably is selected from alkylene and -CONR. 50 - is composed of groups.

[0155] In the linking group LL, it is selected from R 1 R 2 R 5 and R 22 ~R 25 One of the connecting parts and the part selected from R 3 R 4 R 6 and R 26 ~R 29 One of the connecting portions is preferably -O-, -S-, or -NR. 50 -、-COO- or -CONR 50 That is, the linking group LL is preferably connected via -O-, -S-, or -NR that constitute the linking group LL. 50 -、-COO- or -CONR50 -and selected from R 1 R 2 R 5 and R 22 ~R 25 One of them and one selected from R 3 R 4 R 6 and R 26 ~R 29 One bond in the linker group LL. The linker in the linker group LL is preferably -O- or -CONR. 50 -, more preferably -CONR 50 -

[0156] Furthermore, in compounds represented by any of the general formulas (1-1) to (1-3), the linking group LL is related to L 1 ~L 6 The connecting parts are preferably -O-, -S-, or -NR. 50 -、-COO- or -CONR 50 That is, the linking group LL is preferably connected via -O-, -S-, or -NR that constitute the linking group LL. 50 -、-COO- or -CONR 50 -with L 1 ~L 6 Bonding. The linker in the linking group LL is preferably -O- or -CONR. 50 -, more preferably -CONR 50 -

[0157] The linking group LL is preferably a divalent linking group having a carboxyl group or a substituent capable of bonding with biological material. In the linking group LL, examples of substituents having a carboxyl group or capable of bonding with biological material include alkylene groups or substituents such as R. 50 Alkyl groups, preferably alkylene groups.

[0158] In the linking group LL, the carboxyl group or a substituent capable of bonding with biological material can be associated with an alkylene group or act as R. 50 Alkyl groups can be directly bonded or bonded via a ZZZ linker.

[0159] Examples of ZZZ linkages include alkylene groups, -O-, -S-, and -NR. 60 -、-COO-、-CONR 60 - and -(CH2-CH2-O) p - and groups consisting of combinations of these substituents. The number of combinations is preferably 2 to 7, more preferably 2 to 5, and even more preferably 2 or 3.

[0160] The number of carbon atoms in the alkylene moiety that can be used as the linking group ZZZ is preferably the number of carbon atoms in the alkylene moiety that can be used as the linking group LL.

[0161] The linking group ZZZ is preferably selected from alkylene groups, -CONR 60 - and -(CH2-CH2-O) p - is composed of groups, more preferably -CONR 60 -alkylene or -CONR 60 -(CH2-CH2-O) p -alkylene groups, more preferably those represented by -CONR 60 -(CH2-CH2-O) p -alkylene groups.

[0162] R 60 It is a hydrogen atom or an alkyl group, preferably a hydrogen atom. As a component capable of being used as R... 60 Alkyl groups, preferably suitable for the above-mentioned R 50 The alkyl group in the text is recorded, but it can be used as R. 60 The alkyl group will not have a carboxyl group or a substituent that can bond with biological substances.

[0163] p has the same meaning as the p mentioned above.

[0164] (5)n

[0165] n is an integer from 1 to 3, preferably an integer of 2 or 3.

[0166] The compound represented by the above general formula (1) contains at least one carboxyl group or a substituent that can bond with biological substances.

[0167] The compound represented by the above general formula (1) can be bonded to the target labeled biological substance by the carboxyl group or a substituent capable of bonding with the biological substance. In addition, the carboxyl group can be readily derived into substituents capable of bonding with the biological substance by conventional methods.

[0168] In this invention, "substituents capable of bonding with biological material" include substituents capable of bonding with biological material derived from carboxyl groups.

[0169] In compounds represented by general formula (1), there are no particular restrictions on the position of the carboxyl group or the substituent capable of bonding with biological substances, but it is preferred that the carboxyl group is present in R. 1 ~R 6 The linking group LL may have at least one linking group at any position, more preferably at least one linking group LL.

[0170] The total number of groups having a carboxyl group or a substituent capable of bonding with biological substances in the compound represented by general formula (1) is at least one. From the viewpoint of quantitative detection of the target substance, it is preferred to have 1 to 3 groups, more preferably 1 or 2 groups, and even more preferably 1 group.

[0171] In the compounds represented by the above general formula (1), R 1 ~R 6 and R 22 ~R 29 At least one of them contains -(CH2-CH2-O) m - indicates the structure. m has the same meaning as the m above. Therefore, it is believed that the compounds of the present invention can have moderate hydrophilicity and moderate volume exclusion effect, and the obtained labeled biomaterials can exhibit excellent fluorescence intensity.

[0172] Furthermore, from the viewpoint of imparting sufficient hydrophilicity to the compound, the compound represented by the above general formula (1) preferably has two or more hydrophilic groups as a whole, more preferably two to eight, even more preferably two to six, and especially preferably three to six.

[0173] As a hydrophilic group, it can be used in the aforementioned R 22 ~R 29 A list of the hydrophilic groups that can be used.

[0174] Unless otherwise specified, the position of the hydrophilic group is not particularly limited. For example, R is a preferred example of a group having the aforementioned hydrophilic group. 11 ~R 13 Or R 22 ~R 29 The linking group LL does not contain sulfonyl or phosphonoyl groups.

[0175] Furthermore, when the compound represented by the above general formula (1) has the above-mentioned hydrophilic group as the carboxyl group or the substituent that can bond with biological substances, it actually has one or more hydrophilic groups in addition to the above-mentioned carboxyl group or the substituent that can bond with biological substances, which is therefore preferred. As a specific example, the carboxyl group can be cited.

[0176] <Compounds represented by any one of the general formulas (1-1) to (1-3)>

[0177] The compound of the present invention represented by general formula (1) is preferably represented by any one of the following general formulas (1-1) to (1-3).

[0178] [Chemical Formula 5]

[0179]

[0180] In the formula, L 1 ~L 6 Indicates alkylene or -(CH2-CH2-O) m -alkylene-*. * indicates the bonding position with the linking group LL.

[0181] R 7 and R 8 R in the above general formula (1) 1 ~R 4 The meaning is the same; it indicates that an alkyl group or -(CH2-CH2-O) can have substituents. m -R 21 .

[0182] R 1 ~R 6 R 11 ~R 13 R 21 ~R 29 ,LL,m andn and R in the above general formula (1) 1 ~R 6 R 11 ~R 13 R 21 ~R 29 , LL, m, and n have the same meaning. Among them, R 1 ~R 6 and R 21 ~R 29 It does not bond with the linking group LL.

[0183] R in the compound 1 ~R 8 R 22 ~R 29 and L 1 ~L 6 At least one of them contains -(CH2-CH2-O) m The structure indicated. m has the same meaning as the m mentioned above.

[0184] Similar to the compounds represented by the above general formula (1), the compounds represented by any one of the general formulas (1-1) to (1-3) are neutral compounds containing at least one carboxyl group or a substituent capable of bonding with biological material.

[0185] Can be used as L 1 ~L 3 The alkylene group is equivalent to the R group that can be used as described above. 5 and R 6 The alkylene group obtained by removing one hydrogen atom or a substituent from a substituted alkyl group is preferably suitable for use as described above. 4 ~L 6The alkylene group is equivalent to the one that can be used as R 1 ~R 4 The alkylene group obtained by removing one hydrogen atom or a substituent from an alkyl group having a substituent is preferably applicable to this description.

[0186] Can be used as L 1 ~L 3 -(CH2-CH2-O) m -alkylene-* is equivalent to the R that can be used as described above. 5 and R 6 -(CH2-CH2-O) m -R 21 The alkyl group that can have substituents, i.e., R 21 -(CH2-CH2-O) is obtained by removing one hydrogen atom or a substituent. m -alkylene groups are preferably suitable for use in this study.

[0187] Can be used as L 4 ~L 6 -(CH2-CH2-O) m -alkylene-* is equivalent to the derivative that can be used as R 1 ~R 4 -(CH2-CH2-O) m -R 21 The alkyl group that can have substituents, i.e., R 21 -(CH2-CH2-O) is obtained by removing one hydrogen atom or a substituent. m -alkylene groups are preferably suitable for use in this study.

[0188] From the perspective of further improving fluorescence intensity, the preferred compound contains L. 1 ~L 6 Each contains -(CH2-CH2-O) m - indicates the structure. m has the same meaning as the m mentioned above.

[0189] From the viewpoint of further improving the fluorescence intensity in solution, membrane and spot imprint states, it is more preferable that the two heterocycles in each of the general formulas (1-1) to (1-3) satisfy the following condition I.

[0190] (Condition I)

[0191] sp as a cyclic atom of a heterocycle 3 At least one substituent on the carbon atom and the substituent on the cyclic nitrogen atom of the heterocycle include -(CH2-CH2-O). m - indicates the structure.

[0192] "The two heterocycles in each of the general formulas (1-1) to (1-3) satisfy the following condition I" means that, in general formula (1-1), R 1 and R 2 At least one of them, R 3 and R 4 At least one of them, and L 1 and L 2 Contains -(CH2-CH2-O) m - represents the structure. In general formula (1-2), it represents R. 1 and R 2 At least one of them, L 4 and R 7 At least one of them, and R 6 and L 3 Contains -(CH2-CH2-O) m - represents the structure. In general formula (1-3), it represents R. 7 and L 6 At least one of them, R 8 and L 5 At least one of them, and R 5 and R 6 Contains -(CH2-CH2-O) m - indicates the structure.

[0193] From the viewpoint of obtaining superior fluorescence intensity, compounds represented by any one of the above general formulas (1-1) to (1-3) are preferred, especially those represented by general formula (1-1). This is because compounds represented by general formula (1-1) tend to maintain the planarity of the compound, thus minimizing thermal deactivation caused by structural strain.

[0194] From the perspective of obtaining superior fluorescence intensity, the structure (composed of L) 1 and L 2 L 3 and L 4 、 or L 5 and L 6 Linking chains formed with linking groups LL (i.e., chains formed by -L) 1 -LL-L 2 -、-L 3 -LL-L 4 -or-L 5 -LL-L 6 The upper limit of the number of connecting atoms in the shortest molecular chain (represented by the connecting chain) is preferably 37 or less, more preferably 31 or less, and even more preferably 25 or less.

[0195] In this invention, from the viewpoint of obtaining superior fluorescence intensity, compounds represented by the above general formula (1-1) are preferred, and R 11 ~R 13 At least one of them is an aryloxy group, and the two heterocycles in formula (1-1) satisfy the above condition I, and R 1 and R 2 At least one of them, R 3 and R 4 At least one of them, and L 1 and L 2 Compounds containing ethoxide groups with a repeating number of 1 to 50, more preferably R 1 ~R 4 The number of repetitions of all ethoxide groups is 1 to 6 (preferably 1 to 4), and the composition is further preferred (composed of L...). 1 and L 2 Linking chains formed with linking groups LL (i.e., chains formed by -L) 1 -LL-L 2 - The upper limit of the number of connecting atoms in the shortest molecular chain (represented by the connecting chain) is below the above-mentioned preferred range.

[0196] The following are specific examples of compounds represented by general formula (1) according to the present invention, but the present invention is not limited to these compounds. In the following specific examples, the sulfonyl group can dissociate hydrogen atoms to adopt a salt structure. In the following specific examples, EO m The following structure is represented, where Me represents a methyl group. Where EO... m It bonds to the cyclic atom of the indoline ring or pseudoindoline ring, or to the nitrogen atom or oxygen atom on the carbon atom side.

[0197] [Chemical Formula 6]

[0198]

[0199] [Chemical Formula 7]

[0200]

[0201] [Chemical Formula 8]

[0202]

[0203] The compounds of the present invention represented by general formula (1) can be used to label biological substances such as proteins, peptides, amino acids, nucleic acids, sugar chains and lipids through at least one substituent in the compound that can bond with biological substances.

[0204] As a substituent capable of bonding with biological substances, any group that is used for interaction (including attachment) or bonding with biological substances can be used without particular restriction, and examples include those described in International Publication No. 2002 / 026891. Among these, preferred examples include NHS ester structures (N-hydroxysuccinimide esters), succinimide structures, maleimide structures, azide groups, ethynyl groups, peptide structures (polyamino acid structures), long-chain alkyl groups (preferably with 12 to 30 carbon atoms), and quaternary ammonium groups.

[0205] Among the compounds of the present invention represented by general formula (1), specific examples of compounds having at least one substituent capable of bonding with biological material include the exemplary compounds of labeled biological material described later. Furthermore, among the exemplary compounds of the present invention represented by general formula (1), those having a substituent capable of bonding with biological material, as shown in the exemplary compounds of labeled biological material described later, can also be specifically exemplified. However, the present invention is not limited to these compounds. For example, in these specific examples, groups with dissociable hydrogen atoms, such as carboxyl and sulfonyl groups, can be dissociated to adopt a salt structure.

[0206] Regarding the compounds of the present invention represented by general formula (1), the compound structure can be set as specified in general formula (1), and otherwise synthesized by known methods. For example, methods described in Patent Documents 1 to 3, etc., can be cited.

[0207] For compounds having substituents capable of bonding with biological substances, the compound structure is set as specified in general formula (1), and otherwise it can be synthesized by known methods. For example, one can refer to Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).

[0208] <<Tagged Biological Material>>

[0209] The labeled biomaterial of the present invention is a substance formed by bonding a compound of the present invention represented by general formula (1) with a biomaterial. The compound of the present invention represented by general formula (1) is fluorescent, exhibiting an absorption wavelength peak suitable for color development in the near-infrared region and excellent fluorescence intensity, and is therefore preferably used for labeling biomaterials. The bonding between the compound represented by general formula (1) and the biomaterial can be by direct bonding between the compound represented by general formula (1) and the biomaterial, or by linkage via a linking group.

[0210] As the aforementioned biological substances, proteins, peptides, amino acids, nucleic acids, sugar chains, and lipids are preferred examples. As proteins, antibodies are preferred examples, and as lipids, phospholipids, fatty acids, and sterols are preferred examples, with phospholipids being more preferred.

[0211] Among the aforementioned biological substances, there are no particular limitations on substances that are useful in clinical pathology. Examples include immunoglobulins such as Ig (Immunoglobulin) G, IgM, IgE, IgA, and IgD; complement, C-reactive protein (CRP), ferritin, α1-microglobulin, β2-microglobulin, and their antibodies; alpha-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA (carbohydrate antigen) 19-9, CA-125, and their antibodies; hormones such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), human chorionic 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.

[0212] Furthermore, examples include bacteria such as diphtheria bacteria, botulinum toxin, mycoplasma, and Treponema pallidum, and their antibodies; protozoa such as Toxoplasma gondii, Trichomonas vaginalis, Leishmaniasis, Trypanosoma cruzi, and Plasmodium, and their antibodies; ES cells (embryonic stem cells) such as ELM3, HM1, KH2, v6.5, v17.2, and v26.2 (derived from mouse 129, 129 / SV, C57BL / 6, and BALB / c) and their antibodies; antiepileptic drugs such as phenytoin and phenobarbital; cardiovascular drugs such as quinidine and digoxin; anti-asthmatic drugs such as theophylline; antibiotics such as chloramphenicol and gentamicin, and their antibodies; other enzymes; exotoxins (such as styrelidine O) and their antibodies; and antibody fragments such as Fab'2, Fab, and Fv.

[0213] As a specific way in which the compound of the present invention represented by general formula (1) (hereinafter also simply referred to as compound (1)) interacts with biological substances and bonds, the following description can be cited as an example.

[0214] Examples include:

[0215] i) The peptide in compound (1) is bonded to a peptide in biological material by a non-covalent bond (e.g., hydrogen bond, ionic bond including chelation) or a covalent bond;

[0216] ii) Van der Waals forces between long-chain alkyl groups in compound (1) and lipid bilayer membranes and lipids in biological substances;

[0217] iii) Based on the reaction of NHS ester (N-hydroxysuccinimide ester) in compound (1) with amino groups in biological substances, amide bonds;

[0218] iv) Thioether bonds based on the reaction of the maleimide group in compound (1) with thioalkyl (-SH) groups in biological material; and

[0219] v) Formation of a triazole ring based on a click reaction between the azido group in compound (1) and the acetylene group in the biomaterial or based on a click reaction between the acetylene group in compound (1) and the azido group in the biomaterial.

[0220] In addition to the methods described in i) to v) above, bonding can also be achieved, for example, through the methods described in Lucas CDde 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 that literature can be appropriately referenced in the preparation of the labeled biomaterials of this invention.

[0221] The following are specific examples of the labeled biomaterials of the present invention obtained from compounds represented by general formula (1) having substituents capable of bonding with biological substances and biological substances bonded thereto through interaction, but the present invention is not limited to these labeled biomaterials, etc. In the following specific examples, regarding groups with dissociable hydrogen atoms such as sulfonyl groups, the hydrogen atoms can be dissociated to adopt a salt structure. EO1 and EO4 have the same meaning as EO1 and EO4 in the specific examples of compounds represented by the aforementioned general formula (1).

[0222] [Chemical Formula 9]

[0223]

[0224] [Chemical Formula 10]

[0225]

[0226] <Reagents containing labeled biological substances>

[0227] In reagents containing the labeled biological material of the present invention, the labeled biological material of the present invention is not particularly limited, and its form can be appropriately selected according to the purpose of use, such as a solution form dissolved in aqueous media such as physiological saline and phosphate buffer, or a solid form such as microparticle powder and freeze-dried powder.

[0228] For example, when using the labeled biological material of the present invention as a fluorescent labeling reagent, it can also be used as a reagent containing any of the above-described forms of labeled biological material.

[0229] <Uses of Labeled Biological Substances>

[0230] The labeled biomaterials of the present invention, obtained from compounds represented by general formula (1), exhibit excellent fluorescence intensity and can be stably detected from the fluorescence emitted by the labeled biomaterials excited by light irradiation. Therefore, the labeled biomaterials of the present invention are applicable to various techniques using fluorescent labeling, for example, suitable as fluorescent labeling reagents in multicolor WB or dot blot hybridization or as in vivo fluorescence imaging reagents.

[0231] Fluorescence detection using the labeled biological material of the present invention typically includes the following steps (i) to (iii) or (iv) to (vii). Fluorescence detection including steps (i) to (iii) corresponds to the direct method using a primary antibody fluorescently labeled with the compound of the present invention, and fluorescence detection including steps (iv) to (vii) corresponds to the indirect method using a secondary antibody fluorescently labeled with the compound of the present invention.

[0232] (i) Prepare the procedures described in (a) and (b) below respectively.

[0233] (a) A sample containing the target biological substance (hereinafter also referred to as "target biological substance").

[0234] (b) The labeled biomaterial of the present invention (hereinafter also referred to as "labeled biomaterial A of the present invention") is formed by bonding a biomaterial capable of binding to the target biomaterial in (a) above (hereinafter also referred to as "primary biomaterial").

[0235] (ii) The process of preparing a conjugate (hereinafter also referred to as "fluorescently labeled conjugate A") formed by bonding the target biomaterial in (a) above with the primary biomaterial in the labeled biomaterial A of the present invention in (b) above.

[0236] (iii) The step of irradiating the fluorescently labeled binder A with light in the wavelength region absorbed by the labeled biological substance A of the present invention, and detecting the fluorescence emitted by the labeled biological substance A of the present invention.

[0237] (iv) Prepare the procedures (c) to (e) below respectively.

[0238] (c) Samples containing the target biological material

[0239] (d) Biomaterials capable of binding to the target biomaterials in (c) above (hereinafter also referred to as "primary biomaterials").

[0240] (e) The labeled biomaterial of the present invention (hereinafter also referred to as "labeled biomaterial B of the present invention") formed by bonding a biomaterial capable of bonding with the primary biomaterial of (d) above (hereinafter also referred to as "secondary biomaterial") to the compound of the present invention.

[0241] (v) The process of preparing a conjugate (hereinafter also referred to as "conjugate b") formed by bonding the target biomaterial in (c) above with the primary biomaterial in (d) above.

[0242] (vi) The step of preparing a bond (hereinafter also referred to as "fluorescently labeled bond B2") formed by bonding the primary biological substance in the above-mentioned bonded body b with the secondary biological substance in the labeled biological substance B of the present invention.

[0243] (vii) Irradiating the fluorescently labeled binder B2 with light in the wavelength region absorbed by the labeled biological substance B of the present invention, and detecting the fluorescence emitted by the labeled biological substance B of the present invention.

[0244] Examples of the biomaterials in the labeled biomaterials of the present invention include those capable of binding to the target biomaterial (primary biomaterial) and those capable of binding to the primary biomaterial (secondary biomaterial). Appropriate selection can be made for the target biomaterial (biomaterial in the test subject) or the primary biomaterial, and biomaterials capable of specifically binding to the biomaterial in the test subject or the primary biomaterial can be selected.

[0245] Proteins among the aforementioned target biological substances can be cited as so-called disease markers. There are no particular limitations on what constitutes a disease marker; examples include alpha-fetoprotein (AFP), PIVKA-II (protein induced by vitamin Kabsence or antagonist II: protein with missing antigen K or reversed antigen II), BCA (breast carcinoma-associated antigen) 225, basic alpha-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 acidic protein (IAP), NCC-ST-439, γ-protamine (γ-Sm), prostate-specific antigen (PSA), prostate acid phosphatase (PAP), neuron-specific enolase (NSE), Iba1, and amyloidosis protein. White β, Tau, flotillin, squamous cell carcinoma-associated antigen (SCC antigen), sialic acid LeX-i antigen (SLX), SPan-1, tissue polypeptide antigen (TPA), sialic acid Tn antigen (STN), CYFRA (cytokeratin), pepsinogen (PG), C-reactive protein (CRP), serum amyloid A protein (SAA), myoglobin, creatine kinase (CK), troponin T, ventricular myosin light chain I, etc.

[0246] The aforementioned target biological substance can be bacteria. Examples of bacteria that can be used as subjects for cellular microbiological examinations include Escherichia coli, Salmonella, respiratory pathogens, and bacteria that cause public health problems.

[0247] The target biological substance mentioned above can be a virus, serving as an antigen of the virus, without particular limitation. Examples include hepatitis virus antigens such as hepatitis C and hepatitis B virus antigens, HIV p24 protein antigen, CMV (cytomegalovirus) pp65 protein antigen, and HPV (human papillomavirus) E6 and E7 proteins.

[0248] In (i) or (iv) above, there are no particular restrictions on the sample containing the target biological substance, and it can be prepared according to conventional methods.

[0249] Furthermore, the labeled biomaterials of the present invention are not particularly limited, and can be prepared by bonding biomaterials capable of bonding with the target biomaterials to the compounds of the present invention using conventional methods. The morphology of the bonds and the reactions that form the bonds are as described in the labeled biomaterials of the present invention above.

[0250] In (v) above, the target biological substance and the primary biological substance can be directly bonded, or they can be bonded via other biological substances different from the target biological substance and the primary biological substance. Furthermore, in (vi) above, the primary biological substance in the bonded body b can be directly bonded to the secondary biological substance in the labeled biological substance B of the present invention, or they can be bonded via other biological substances different from the primary biological substance and the secondary biological substance.

[0251] The labeled biological material of the present invention can also be used as a fluorescent labeling antibody in either the direct or indirect method, and is preferably used as a fluorescent labeling antibody in the indirect method.

[0252] In (ii) or (v) and (vi) above, there are no particular limitations on the bonding of the labeled biological material of the present invention to the target biological material, and it can be carried out by conventional methods.

[0253] In (iii) or (vii) above, there is no particular limitation on the wavelength used to excite the labeled biological material of the present invention, as long as it is a light emission wavelength (excitation wavelength) capable of exciting the labeled biological material of the present invention.

[0254] In compound (1) of the present invention, the labeled biomaterial using a compound with n=1 has a wavelength of maximum absorption around 585 nm (560–620 nm), therefore the wavelength region of the irradiated light is preferably 530–650 nm, more preferably 550–630 nm. The labeled biomaterial using this compound is suitable for use as a labeled biomaterial that exhibits excellent fluorescence intensity to an excitation light source around 600 nm in the visible region.

[0255] In compound (1) of the present invention, the labeled biomaterial using a compound with n=2 has a wavelength of maximum absorption around 685 nm (660–720 nm), therefore the wavelength region of the irradiated light is preferably 630–750 nm, more preferably 650–730 nm. The labeled biomaterial using this compound is suitable for use as a labeled biomaterial that exhibits excellent fluorescence intensity to excitation light sources around 700 nm in the near-infrared region such as multicolor WB.

[0256] In compound (1) of the present invention, the labeled biomaterial using a compound with n=3 has a wavelength of maximum absorption around 785 nm (760–820 nm), therefore the wavelength region of the irradiated light is preferably 730–850 nm, more preferably 750–830 nm. The labeled biomaterial using this compound is suitable for use as a labeled biomaterial that exhibits excellent fluorescence intensity to excitation light sources around 800 nm in the near-infrared region such as multicolor WB.

[0257] As the fluorescence excitation light source used in this invention, there are no particular limitations as long as it emits a light source that emits a light emission wavelength (excitation wavelength) capable of exciting the labeled biological material of this invention; for example, various laser light sources can be used. Furthermore, various filters can be used to obtain a preferred excitation wavelength or to detect only fluorescence.

[0258] There are no particular restrictions on the other matters mentioned in (i) to (vii) above, and the methods, reagents, devices, and other conditions commonly used in fluorescence detection using fluorescent labels can be appropriately selected.

[0259] Furthermore, for the steps other than (i) to (vii) mentioned above, it is also possible to appropriately select the commonly used methods, reagents, apparatus, and other conditions in conjunction with various fluorescent labeling methods.

[0260] For example, multicolor Western blotting using the labeled biological material of the present invention involves fabricating an imprinted membrane using methods commonly used for target biological materials (protein separation by electrophoresis, membrane blotting, membrane agglomeration), and using the labeled biological material of the present invention as a labeling antibody (preferably a secondary antibody), enabling the detection of the target biological material with excellent fluorescence intensity. Similarly, dot blot hybridization using the labeled biological material of the present invention involves fabricating an imprinted nitrocellulose membrane or an imprinted PVDF (polyvinylidene fluoride) membrane using methods commonly used for target biological materials, and using the labeled biological material of the present invention as a labeling antibody (preferably a secondary antibody), enabling the detection of the target biological material with excellent fluorescence intensity.

[0261] -Substituted basis set T-

[0262] In this invention, preferred substituents can be selected from the following group of substituents T.

[0263] Furthermore, in this invention, when only substituents are described, referring to the substituent group T, when only individual groups such as alkyl are described, it is preferable to apply the corresponding group of the substituent group T.

[0264] Furthermore, in this specification, when alkyl and cyclic (cyclo)alkyl are described separately, alkyl is used to mean that it includes both straight-chain alkyl and branched alkyl. On the other hand, when alkyl and cyclic alkyl are not described separately and unless otherwise specified, alkyl is used to mean that it includes straight-chain alkyl, branched alkyl, and cycloalkyl. This also applies to compounds containing groups (alkoxy, alkathio, alkenyl, etc.) that can adopt a cyclic structure, and to compounds containing groups that can adopt a cyclic structure. When a group can form a cyclic skeleton, the lower limit of the number of atoms of the group forming the cyclic skeleton is not related to the lower limit of the number of atoms of the group that can adopt the structure specifically described below, and is 3 or more, preferably 5 or more.

[0265] In the following description of substituent group T, for example, alkyl and cycloalkyl, groups with straight or branched structures and groups with cyclic structures are sometimes described separately in order to distinguish them.

[0266] The groups included in the substituent group T include the following groups.

[0267] Examples of suitable groups include alkyl groups (preferably with 1 to 30 carbon atoms, more preferably with 1 to 20 carbon atoms, even more preferably with 1 to 12 carbon atoms, even more preferably with 1 to 8 carbon atoms, even more preferably with 1 to 6 carbon atoms, and particularly preferably with 1 to 3 carbon atoms), alkenyl groups (preferably with 2 to 30 carbon atoms, more preferably with 2 to 20 carbon atoms, even more preferably with 2 to 12 carbon atoms, even more preferably with 2 to 6 carbon atoms, and even more preferably with 2 to 4 carbon atoms), and alkynyl groups (preferably with 2 to 30 carbon atoms, more preferably with 2 to 20 carbon atoms). Further preferably, the carbon number is 2 to 12, further preferably 2 to 6, further preferably 2 to 4; cycloalkyl (preferably 3 to 20 carbon atoms); cycloalkenyl (preferably 5 to 20 carbon atoms); and aryl (which can be a monocyclic group or a fused-ring group (preferably a fused-ring group with 2 to 6 rings). When it is a fused-ring group, it is composed of 5 to 7 membered rings, etc. The aryl group preferably has 6 to 40 carbon atoms, more preferably 6 to 30 carbon atoms, further preferably 6 to 26 carbon atoms, and especially preferably 6 to 40 carbon atoms. 6-10) Heterocyclic groups (having at least one nitrogen, oxygen, sulfur, phosphorus, silicon, or selenium atom as the cyclic atom, and can be monocyclic or fused-ring groups (preferably fused-ring groups with 2-6 rings). When it is a monocyclic group, the number of ring members is preferably 5-7, more preferably 5 or 6. The number of carbon atoms in the heterocyclic group is preferably 2-40, more preferably 2-20. Heterocyclic groups include aromatic heterocyclic groups (heteroaryl) and aliphatic heterocyclic groups (aliphatic heterocyclic groups).) Alkoxy groups (preferably with 1-20 carbon atoms). More preferably, the carbon atoms are 1 to 12, alkenyloxy (preferably 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms), alkynyloxy (preferably 2 to 20 carbon atoms, more preferably 2 to 12 carbon atoms), cycloalkoxy (preferably 3 to 20 carbon atoms), aryloxy (preferably 6 to 40 carbon atoms, more preferably 6 to 26 carbon atoms, even more preferably 6 to 14 carbon atoms), heterocyclic oxy (preferably 2 to 20 carbon atoms), and polyalkylene oxy (preferably 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms).

[0268] Alkoxycarbonyl (preferably 2-20 carbon atoms), cycloalkoxycarbonyl (preferably 4-20 carbon atoms), aryloxycarbonyl (preferably 6-20 carbon atoms), amino (preferably 0-20 carbon atoms, including unsubstituted amino (-NH2), (mono- or di-)alkylamino, (mono- or di-)enylamino, (mono- or di-)alkynylamino, (mono- or di-)cycloalkylamino, (mono- or di-)cycloenylamino, (mono- or di-)arylamino, (mono- or di-)arylamino, (mono- or di-)cycloalkylamino, (mono- or di-)cycloalkenyl ... - or di-)heterocyclic amino. The meanings of the groups above that substituted the unsubstituted amino group are the same as those corresponding to the substituent group T. ), aminosulfonyl (preferably 0-20 carbon atoms, preferably alkyl, cycloalkyl, or aryl aminosulfonyl), acyl (preferably 1-20 carbon atoms, more preferably 2-15 carbon atoms), acyloxy (preferably 1-20 carbon atoms), carbamoyl (preferably 1-20 carbon atoms, preferably alkyl, cycloalkyl, or aryl carbamoyl),

[0269] Acylamino (preferably 1-20 carbon atoms), sulfonamide (preferably 0-20 carbon atoms, preferably alkyl, cycloalkyl, or aryl sulfonamide), alkylthio (preferably 1-20 carbon atoms, more preferably 1-12 carbon atoms), cycloalkylthio (preferably 3-20 carbon atoms), arylthio (preferably 6-40 carbon atoms, more preferably 6-26 carbon atoms, even more preferably 6-14 carbon atoms), heterocyclic thio (preferably 2-20 carbon atoms), alkyl, cycloalkyl, or arylsulfonyl (preferably 1-20 carbon atoms).

[0270] Silyyl groups (preferably having 1 to 30 carbon atoms, more preferably 1 to 20 carbon atoms, and preferably silyl groups substituted with alkyl, aryl, alkoxy, or aryloxy groups.), silanoxy groups (preferably having 1 to 20 carbon atoms, and preferably silanoxy groups substituted with alkyl, aryl, alkoxy, or aryloxy groups.), hydroxyl groups, cyano groups, nitro groups, halogen atoms (e.g., fluorine, chlorine, bromine, or iodine atoms), oxygen atoms (specifically, replacing >CH2 constituting the ring with >C=O), carboxyl groups (-CO2H), phosphonyl groups [-PO(OH)2], phosphoryl groups [-O-PO(OH)2], sulfonyl groups (-SO3H), borate groups [-B(OH)2], ononyl groups (including ammonium groups containing cyclic ammonium groups, sulfonyl groups, phosphonyl groups, preferably having 0 to 30 carbon atoms, more preferably 1 to 20 carbon atoms), thioalkyl groups (-SH), amino acid residues, or polyamino acid residues.

[0271] Furthermore, examples include those with carboxyl, phosphono, sulfonyl, ononyl, amino acid residues, polyamino acid residues, or -(CH2-CH2-O). m -alkyl(m with R) 1 ~R 6The meaning of 'm' in the above-mentioned alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclic, alkoxy, alkenyloxy, cycloalkoxy, aryloxy, heterocyclic, alkoxycarbonyl, cycloalkoxycarbonyl, aryloxycarbonyl, amino, aminosulfonyl, acyl, acyloxy, carbamoyl, amide, sulfonamide, alkylthio, cycloalkylthio, arylthio, heterocyclic thio, alkyl, cycloalkyl, or arylsulfonyl.

[0272] The substituents selected from the substituent group T are more preferably alkyl, alkenyl, cycloalkyl, aryl, heterocyclic, alkoxy, cycloalkoxy, aryloxy, alkoxycarbonyl, cycloalkoxycarbonyl, amino, amide, cyano or halogen atoms, and are particularly preferred to be alkyl, alkenyl, aryl, heterocyclic, alkoxy, alkoxycarbonyl, amino, amide or cyano.

[0273] The substituents selected from substituent group T include groups formed by combining multiple of the above groups, unless otherwise specified. For example, when the compound or substituent contains alkyl, alkenyl, etc., they may be substituted or unsubstituted. Furthermore, when it contains aryl, heterocyclic, etc., they may be monocyclic or fused-ring, and may be substituted or unsubstituted.

[0274] Example

[0275] The present invention will be further described in detail below with reference to embodiments, but the present invention is not limited thereto. Additionally, room temperature refers to 25°C.

[0276] Compounds (1) to (7) used in the examples and comparative compounds (1) to (6) are shown below.

[0277] Additionally, in the compound examples, even if not specifically stated otherwise, the sulfonyl group may contain a salt structure (e.g., potassium salt, sodium salt, TEA (triethylamine) salt, or DIPEA (N,N-diisopropylethylamine) salt). EO1 and EO4 have the same meaning as in the specific examples of compounds represented by the aforementioned general formula (1). m represents the average number of repetitions. Each compound was synthesized using a compound with an average number of repetitions whose first decimal place was 0 as the starting material.

[0278] [Chemical Formula 11]

[0279]

[0280] [Chemical Formula 12]

[0281]

[0282] [Chemical Formula 13]

[0283]

[0284] The following provides a detailed description of the synthetic methods for each compound, but the starting materials, pigment intermediates, and synthetic routes are not limited to these.

[0285] In the following synthetic routes, room temperature means 25°C.

[0286] Unless otherwise specified, the carrier used in reversed-phase column chromatography was SNAP Ultra C18 (trade name, manufactured by Biotage) or Sfar C18 (trade name, manufactured by Biotage), and the carrier used in normal-phase column chromatography was Hi-Flash Column (trade name, manufactured by YAMAZEN CORPORATION).

[0287] In reversed-phase column chromatography or normal-phase column chromatography, the mixing ratio of the eluent is a volume ratio. For example, "acetonitrile:water = 0:100 → 20:80" means changing the eluent from "acetonitrile:water = 0:100" to "acetonitrile:water = 20:80".

[0288] The HPLC (High Performance Liquid Chromatography) used was 2767 (trade name, manufactured by Waters Corporation).

[0289] MS spectra were determined using an ACQUITY SQD LC / MS System (trade name, manufactured by Waters Corporation, ionization method: ESI (ElectroSpray Ionization)) or an LCMS-2010EV (trade name, manufactured by Shimadzu Corporation, ionization method: ESI and APCI (Atmospheric Pressure Chemical Ionization)).

[0290] <Synthesis of compound (1-NHS)>

[0291] The compound (1-NHS) was synthesized according to the following scheme.

[0292] [Chemical Formula 14]

[0293]

[0294] [Chemical Formula 15]

[0295]

[0296] 1) Synthesis of compound (1-C)

[0297] 200 mL of tert-butanol (tBuOH) and 12 g of potassium tert-butoxide (tBuOK) were added to a nitrogen-purged 50 mL three-necked flask. While stirring, 14.4 g of compound (1-A) was added dropwise, and the mixture was stirred briefly. Next, 36.3 g of polyethylene glycol methyl ether toluenesulfonate (mean repeat number of ethylene glycol units = 4.0, TsO-EO4-Me) was added dropwise, and the mixture was stirred at 80 °C for 1 hour. The solvent was then removed by vacuum distillation, and the crude product was extracted with ethyl acetate and distilled water. 30 mL of 30% hydrochloric acid aqueous solution was added to the obtained crude product, and the mixture was stirred at 100 °C for 3 hours. The solvent was then removed by vacuum distillation, and the product was purified by normal-phase column chromatography (eluent: hexane / ethyl acetate = 50 / 50 → 30 / 70) to obtain 10.7 g of compound (1-C).

[0298] 2) Synthesis of compound (1-E)

[0299] 20 g of compound (1-D) and 120 mL of distilled water were added to a 1 L three-necked flask, and 40 mL of 30% hydrochloric acid aqueous solution was added dropwise while stirring. The flask was cooled in a salt-ice bath, maintained below 3 °C, while a solution of 4.22 g of sodium nitrite dissolved in 80 mL of distilled water was slowly added dropwise, followed by stirring at 0–3 °C for 45 minutes. Next, a solution of 21 g of stannous chloride (II) dissolved in 60 mL of distilled water and 20 mL of 30% HCl was slowly added dropwise, followed by stirring below 7 °C for 40 minutes. The solvent was concentrated, and the residue was washed with isopropanol to obtain 15 g of compound (1-E).

[0300] 3) Synthesis of compound (1-F)

[0301] 2.0 g of compound (1-E), 30 mL of acetic acid (AcOH), 2.5 g of compound (1-C), and 1.24 g of potassium acetate (AcOK) were added to a 200 mL flask. The mixture was stirred at 140 °C for 1 hour under a nitrogen atmosphere. The solvent was removed by vacuum distillation, and the compound (1-F) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 35 / 65) to obtain 1.0 g of compound (1-F).

[0302] 4) Synthesis of compound (1-G)

[0303] 500 mg of compound (1-F), 2 mL of sulfolane, 365 mg of 6-bromohexanoic acid, and 0.169 mL of triethylamine (Et3N) were added to a 50 mL volumetric flask, and the mixture was heated and stirred at 120 °C for 6 hours. Ethyl acetate was added to the reaction solution to induce precipitation. The precipitate was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 20 / 100) to obtain 105 mg of compound (1-G).

[0304] 5) Synthesis of compound (1-H)

[0305] 50 mg of compound (1-G) and 1 mL of methanol (MeOH) were added to a 25 mL flask. While stirring, 9 mg of glutaraldehyde dimethyl hydrochloride, 8 μl of acetic anhydride (Ac₂O), and 4 μl of triethylamine (Et₃N) were added, and the mixture was stirred briefly under a nitrogen atmosphere. After the reaction converged, distilled water was added, and the mixture was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 30 / 70) to obtain 28 mg of compound (1-H).

[0306] 6) Synthesis of compound (1-K)

[0307] 700 mg of compound (1-I), 20 mL of N,N-dimethylformamide (DMF), 1.2 g of N,N'-dicyclohexylcarbodiimide (DCC), and 1 mL of triethylamine (TEA) were added to a 50 mL flask. The mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. 1.2 g of compound (1-J) was then added, and the mixture was stirred further at room temperature for 1 hour. The solvent was then concentrated, and the mixture was extracted with 100 mL of ethyl acetate. Insoluble matter was removed by diatomaceous earth filtration. After further solvent concentration, 20 mL of formic acid was added, and the mixture was heated and stirred at 80 °C for 3 hours. The solvent was removed by vacuum distillation, and the mixture was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 20 / 80) to obtain 527 mg of compound (1-K).

[0308] 7) Synthesis of compound (1-L)

[0309] 20 mg of compound (1-H), 2 mL of DMF, 80 mg of N,N,N',N'-tetramethyl-O-(N-succinimide)urea hexafluorophosphate (HSTU), and 77 μL of triethylamine (Et3N) were added to a 10 mL flask and stirred for 3 hours. Then, the above reaction solution was added dropwise to a solution in which 10 mg of compound (1-K) and 3 mg of sodium carbonate were dissolved in 40 mL of water, and the mixture was stirred for 3 hours. After concentrating the reaction solution, it was purified by HPLC and freeze-dried to obtain 15.1 mg of compound (1-L).

[0310] 8) Synthesis of compound (1)

[0311] 10.0 mg of compound (1-L), 2 mL of DMF, 2 mg of N,N,N',N'-tetramethyl-O-(N-succinimide)urea hexafluorophosphate (HSTU), and 5 μL of triethylamine (Et3N) were added to a 10 mL flask and allowed to react for 3 hours. Then, 1 mg of aminocaproic acid was added to the reaction solution, and the mixture was stirred for 3 hours. After concentrating the reaction solution, it was purified by HPLC and freeze-dried to obtain 6.1 mg of compound (1). The MS determination results of compound (1) are as follows.

[0312] MS(ESI m / z): (M+H + ) + =1662, (MH + ) - =1660

[0313] 9) Synthesis of compound (1-NHS)

[0314] 2.6 mg of compound (1) was added to 0.28 mL of N,N-dimethylformamide (DMF), a solution of N,N,N',N'-tetramethyl-O-(N-succinimide)urea hexafluorophosphate (N,N-dimethylformamide) containing 1 mg of DMF, and 1.3 μL of triethylamine (Et3N), and stirred for 1 hour. The solvent was then removed by vacuum distillation, the supernatant was removed by adding ethyl acetate, and the mixture was dried under vacuum to obtain compound (1-NHS).

[0315] <Synthesis of compound (2-NHS)>

[0316] Compound (2-NHS) was synthesized in the same manner as compound (1-NHS) according to the following scheme. The MS determination results of compound (2) are as follows.

[0317] MS(ESI m / z): (M+H + ) + =1614, (MH + ) - =1612

[0318] In addition, compounds (2-F) and (2-G) were synthesized as follows.

[0319] 282 mg of compound (2-D), 45.7 mg of compound (2-E), 54.7 mg of potassium acetate (AcOK), and 2.0 mL of acetic anhydride (Ac2O) were added to a 25 mL flask. The mixture was stirred at 60 °C for 2 hours under a nitrogen atmosphere. After the reaction converged, distilled water was added, and the mixture was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 25 / 75) to obtain 174.2 mg of compound (2-F).

[0320] 10 mg of compound (2-F) and 500 μL of distilled water were added to a test tube and stirred at 95 °C. A solution containing 20 mg of sodium 4-hydroxybenzenesulfonate and 6 mg of sodium hydroxide in 500 μL of distilled water was then added dropwise, and the mixture was stirred at 95 °C for 30 minutes. The reaction solution was cooled to room temperature, purified by HPLC, and freeze-dried to obtain 5.5 mg of compound (2-G).

[0321] [Chemical Formula 16]

[0322]

[0323] [Chemical Formula 17]

[0324]

[0325] <Synthesis of Compound (3-NHS)>

[0326] Compound (3-NHS) was synthesized in the same manner as compound (2-NHS) according to the following scheme. The MS determination results of compound (3) are as follows.

[0327] MS(ESI m / z): (M+H + ) + =1855, (MH + ) - =1853

[0328] [Chemical Formula 18]

[0329]

[0330] [Chemical Formula 19]

[0331]

[0332] <Synthesis of compound (4-NHS)>

[0333] Compound (4-NHS) was synthesized in the same manner as compound (2-NHS) according to the following scheme. The MS determination results of compound (4) are as follows.

[0334] MS(ESI m / z): (M+H + ) + =1695, (MH) + ) - =1693

[0335] [Chemical Formula 20]

[0336]

[0337] [Chemical Formula 21]

[0338]

[0339] <Synthesis of Compound (5-NHS)>

[0340] Compound (5-NHS) was synthesized in the same manner as compound (2-NHS) according to the following scheme. The MS determination results of compound (5) are as follows.

[0341] MS(ESI m / z): (M+H + ) + =1723, (MH) + ) - =1721

[0342] [Chemical Formula 22]

[0343]

[0344] <Synthesis of Compound (6-NHS)>

[0345] Compound (6-NHS) was synthesized in the same manner as compound (2-NHS) according to the following scheme. The MS determination results of compound (6) are as follows.

[0346] MS(ESI m / z): (M+H + ) + =1547, (MH) + ) - =1545

[0347] [Chemical Formula 23]

[0348]

[0349] <Synthesis of Compound (7-NHS)>

[0350] In the synthesis of compound (4-NHS), compound (7-B), synthesized according to the following scheme, was used instead of compound (3-C). Otherwise, compound (7-NHS) was synthesized in the same manner as compound (4-NHS). The MS determination results of compound (7) are as follows.

[0351] MS(ESI m / z): (M+H + ) + =2253, (MH + ) - =2251

[0352] [Chemical Formula 24]

[0353]

[0354] [Chemical Formula 25]

[0355]

[0356] <Comparative Synthesis of Compound (1-NHS)>

[0357] According to the following scheme, the comparative compound (1-NHS) was synthesized in the same manner as compound (2-NHS). The MS determination results of the comparative compound (1) are as follows.

[0358] MS(ESI m / z): (M+H + ) + =1279, (MH + ) - =1277

[0359] [Chemical Formula 26]

[0360]

[0361] <Comparative Synthesis of Compound (2-NHS)>

[0362] The comparative compound (2-NHS) was synthesized in the same manner as compound (2-NHS) according to the following scheme. The MS determination results of the comparative compound (2) are as follows.

[0363] MS(ESI m / z): (M+H + ) + =1204, (MH) + ) - =1202

[0364] [Chemical Formula 27]

[0365]

[0366] [Chemical Formula 28]

[0367]

[0368] <Comparative Synthesis of Compound (3-NHS)>

[0369] According to the following scheme, the comparative compound (3-NHS) was synthesized in the same manner as compound (2-NHS). The MS determination results of the comparative compound (3) are as follows.

[0370] MS(ESI m / z): (M+H + ) + =1708, (MH) + ) - =1706

[0371] Additionally, Boc is the abbreviation for tert-butyloxycarbonyl.

[0372] [Chemical Formula 29]

[0373]

[0374] [Chemical Formula 30]

[0375]

[0376] Comparative compound (4) is compound 11 as described in Patent Document 1, and was synthesized according to the method described in the document. The MS determination results of comparative compound (4) are as follows.

[0377] MS(ESI m / z): (M+H + ) + =864, (MH + ) - =862

[0378] Comparative compound (5) is compound 22 as described in Patent Document 2, and was synthesized according to the method described in the document. The MS determination results of comparative compound (5) are as follows.

[0379] MS(ESI m / z): (M+H + ) + =1100, (MH) + ) - =1098

[0380] Comparative compound (6) is compound 17 as described in International Publication No. 2001 / 002374, and was synthesized according to the method described in the literature. The MS determination results of comparative compound (6) are as follows.

[0381] MS(ESI m / z): (M+H+ ) + =885, (MH + ) - =883

[0382] The NHS esters (N-hydroxysuccinimide esters) of the comparative compounds (4), (5) and (6) were synthesized in the same manner as the synthesis of compound (1-NHS) from compound (1).

[0383] <Example 1>

[0384] For each of the above compounds, the fluorescence labeling rate, solution fluorescence intensity, and on-film fluorescence intensity were evaluated.

[0385] [0] Preparation of fluorescently labeled antibodies

[0386] 217 μL of anti-rabbit IgG antibody (2.3 mg / mL) and 21.7 μL of carbonate buffer were added to a microtube and vortexed. Then, a dimethyl sulfoxide solution of compound (1-NHS) was added at a ratio of 10 equivalents to 1 antibody equivalent, followed by further vortexing. After standing at room temperature for 1 hour, the reaction solution was purified using gel filtration chromatography on a PD10 column (manufactured by GE HealthcareLife Sciences) and PBS (Phosphate Buffered Saline) to obtain the labeled antibody (1).

[0387] Labeled antibodies for each compound and the comparative compounds were obtained in the same manner.

[0388] [1] Evaluation of solution fluorescence intensity

[0389] The labeled antibody solution prepared above was prepared to a protein concentration of 0.1 mg / mL. Using a spectrophotometer (trade name: RF-5300, manufactured by Shimadzu Corporation) under uniform exposure conditions with excitation light of 785 nm, the integral value of fluorescence intensity in the fluorescence wavelength range of 810–840 nm was calculated. Using the integral value of fluorescence intensity in the fluorescence wavelength range of 810–840 nm of compound (1)-IgG as a benchmark, the ratio to this benchmark (integral value of fluorescence intensity in the fluorescence wavelength range of 810–840 nm of the labeled antibody / benchmark value) was calculated, and the results were evaluated according to the following evaluation criteria. The results are summarized in Table 1.

[0390] In this test, a rating of "D" or above is considered acceptable for fluorescence intensity.

[0391] - Evaluation Criteria for Fluorescence Intensity -

[0392] A: The ratio of fluorescence intensity to the baseline value is more than 1.5 times.

[0393] B: The ratio of fluorescence intensity to the reference value is greater than 1.4 and less than 1.5.

[0394] C: The ratio of fluorescence intensity to the reference value is greater than 1.3 and less than 1.4.

[0395] D: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 1.3.

[0396] E: The ratio of fluorescence intensity to the reference value is greater than 1.1 and less than 1.2.

[0397] F: The ratio of fluorescence intensity to the reference value is greater than 0.9 and less than 1.1.

[0398] G: The ratio of fluorescence intensity to the reference value is less than 0.9 times.

[0399] [Table 1]

[0400] No. Labeled antibodies Fluorescence intensity (solution) 101 Compound (1)-IgG C 102 Compound (2)-IgG C 103 Compound (3)-IgG B 104 Compound (4)-IgG A 105 Compound (5)-IgG A 106 Compound (6)-IgG A 107 Compound (7)-IgG B c11 Comparison of compound (1)-IgG 1.0 (Base value) c12 Comparison of compound (2)-IgG F c13 Comparison of compound (3)-IgG E c14 Comparison of compound (4)-IgG - c15 Comparison of compound (5)-IgG F c16 Comparison of compound (6)-IgG F

[0401] (Table Notes)

[0402] In the labeled antibody column, the descriptions of compound (Z)-IgG or comparative compound (Z)-IgG indicate the IgG-labeled antibody of compound (Z-NHS) or the IgG-labeled antibody of comparative compound (Z-NHS), respectively. Z represents the compound number. The same meaning applies in subsequent tables.

[0403] For the IgG-labeled antibody of the comparative compound (4-NHS), since labeling was not possible using the methods described above, it is indicated by "-" in the table above. This is presumably because of its low water solubility, causing precipitation in the reaction system with the antibody. The same "-" will be used for the evaluation of fluorescence intensity related to the comparative compound (4)-IgG in Tables 2 and 3.

[0404] [2] Evaluation of fluorescence intensity on membrane

[0405] The labeled antibody solution (anti-rabbit IgG solution) prepared above was prepared to a protein concentration of 5.0 ng / mL, and 2 μL was carefully spotted onto a nitrocellulose membrane. After drying the membrane, it was then blocked in TBS-T using Fish Gelatin clumping buffer. The membrane was incubated at room temperature with stirring for 1 hour. The clumping solution was removed, and the labeled antibody PBS solution was diluted 20,000 times with TBS (Tris Buffered Saline: Tris buffer). The membrane was immersed in the diluted solution and incubated for 1 hour with stirring. The membrane was washed three times with TBS-T (Tris Buffered Saline with Tween 20: a mixture of Tris buffer and polysorbate 20), 10 minutes each time, and finally washed with TBS for 10 minutes. The obtained membrane was dried on a hot plate at 40°C for 1 hour and imaged using an Amersham Typhoon scanner (GEHC). Under uniform exposure conditions with excitation light of 785 nm, the fluorescence intensity in the range of 810–840 nm was calculated. Using the integral value of the fluorescence intensity of the labeled antibody in the fluorescence wavelength range of 810–840 nm as a benchmark, the ratio to this benchmark (integral value of fluorescence intensity of the labeled antibody in the fluorescence wavelength range of 810–840 nm / benchmark value) was calculated, and the results were evaluated according to the following evaluation criteria. The results are summarized in Table 2.

[0406] In this test, a rating of "D" or above is considered acceptable for fluorescence intensity.

[0407] - Evaluation Criteria for Fluorescence Intensity -

[0408] A: The ratio of fluorescence intensity to the baseline value is 2.0 times or more.

[0409] B: The ratio of fluorescence intensity to the reference value is greater than 1.8 and less than 2.0.

[0410] C: The ratio of fluorescence intensity to the reference value is greater than 1.6 and less than 1.8.

[0411] D: The ratio of fluorescence intensity to the reference value is greater than 1.4 and less than 1.6.

[0412] E: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 1.4.

[0413] F: The ratio of fluorescence intensity to the reference value is greater than 0.9 and less than 1.2.

[0414] G: The ratio of fluorescence intensity to the reference value is less than 0.9 times.

[0415] [Table 2]

[0416] No. Labeled antibodies Fluorescence intensity (membrane) 201 Compound (1)-IgG D 202 Compound (2)-IgG C 203 Compound (3)-IgG B 204 Compound (4)-IgG A 205 Compound (5)-IgG A 206 Compound (6)-IgG A 207 Compound (7)-IgG B c21 Comparison of compound (1)-IgG 1.0 (Base value) c22 Comparison of compound (2)-IgG G c23 Comparison of compound (3)-IgG E c24 Comparison of compound (4)-IgG - c25 Comparison of compound (5)-IgG G c26 Comparison of compound (6)-IgG G

[0417] [3] Evaluation of spotted imprints

[0418] Transferrin (20 mg / mL) was prepared to a concentration of 50 ng / mL using TBS-T, and 2 μL was carefully spotted onto a nitrocellulose membrane. After drying the membrane, it was then blocked in TBS-T using Fish Gelatin clump buffer. Next, 6 μL of rabbit anti-human transferrin polyclonal antibody was added to 30 mL of PBS-T (Phosphate Buffered Saline with Tween 20), the membrane was immersed, and shaken for 1 hour. Then, the membrane was removed and washed 4 times with TBS-T. Next, 15 μL of the labeled antibody (anti-rabbit IgG) prepared above was added to 30 mL of TBS-T, the membrane was immersed, and incubated at room temperature for 1 hour with stirring. The membrane was then washed 3 times with TBS-T at 10 minutes per wash, and finally washed with TBS for 10 minutes. The obtained membrane was dried on a hot plate at 40°C for 1 hour and imaged using an Amersham Typhoonscanner (GEHC). Under uniform exposure conditions with excitation light at 785 nm, the fluorescence intensity in the range of 810–840 nm was calculated. Using the integral value of the fluorescence intensity in the range of 810–840 nm of compound (1)-IgG as a benchmark, the ratio to this benchmark (integral value of the fluorescence intensity in the range of 810–840 nm of the labeled antibody / benchmark value) was calculated, and the results were evaluated according to the following evaluation criteria. The results are summarized in Table 3.

[0419] In this test, a rating of "D" or above is considered acceptable for fluorescence intensity.

[0420] - Evaluation Criteria for Fluorescence Intensity -

[0421] A: The ratio of fluorescence intensity to the baseline value is 2.0 times or more.

[0422] B: The ratio of fluorescence intensity to the reference value is greater than 1.8 and less than 2.0.

[0423] C: The ratio of fluorescence intensity to the reference value is greater than 1.6 and less than 1.8.

[0424] D: The ratio of fluorescence intensity to the reference value is greater than 1.4 and less than 1.6.

[0425] E: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 1.4.

[0426] F: The ratio of fluorescence intensity to the reference value is greater than 0.9 and less than 1.2.

[0427] G: The ratio of fluorescence intensity to the reference value is less than 0.9 times.

[0428] [Table 3]

[0429] No. Labeled antibodies Fluorescence intensity (spotted imprint) 301 Compound (1)-IgG D 302 Compound (2)-IgG C 303 Compound (3)-IgG B 304 Compound (4)-IgG A 305 Compound (5)-IgG A 306 Compound (6)-IgG A 307 Compound (7)-IgG C c31 Comparison of compound (1)-IgG 1.0 (Base value) c32 Comparison of compound (2)-IgG G c33 Comparison of compound (3)-IgG G c33 Comparison of compound (4)-IgG - c34 Comparison of compound (5)-IgG G c35 Comparison of compound (6)-IgG G

[0430] The following information can be obtained from the results in Tables 1-3 above.

[0431] From general formula (1), selected from R 1 R 2 R 5 and R 22 ~R 25 One of them is not selected from R 3 R 4 R 6 and R 26 ~R 29 Considering the aspect of forming a large ring through one bond in the formula (1), compound (1) is not a compound specified in this invention. From the R in general formula (1) 1 ~R 6 and R 22 ~R 29 Considering that at least one of the ethoxy groups does not have a repeating number of 1 to 50, comparative compound (2) is not a compound specified in this invention. Considering that the linking group LL forming the large member ring in general formula (1) has a sulfonyl group, comparative compound (3) is not a compound specified in this invention. The labeled antibodies using these comparative compounds (1) to (3) exhibit low fluorescence intensity in solution, on membrane, and in dot blots (No. c11 to c13, c21 to c23, c31 to c33). Comparative compound (4) is a compound disclosed in International Publication No. 2005 / 000218, and antibodies cannot be labeled using comparative compound (4) in the first place. Comparative compound (5) is a compound disclosed in International Publication No. 2006 / 047452, and comparative compound (6) is a compound disclosed in International Publication No. 2001 / 002374. The labeled antibodies using these comparative compounds (5) or (6) have low fluorescence intensity in solution, on membrane, and in dot blot (No. c14-c16, c24-c26, c34-c36).

[0432] In contrast, the labeled antibodies of compounds (1) to (7) specified in this invention exhibit fluorescence intensity that is more than 1.3 times that of the comparative labeled antibody (1) in solution and more than 1.4 times that in either membrane or spot blot, showing excellent fluorescence intensity (No. 101 to 107 relative to No. c11, No. 201 to 207 relative to No. c21, and No. 301 to 307 relative to No. c31).

[0433] Among them, R in general formula (1) is used. 11 ~R 13 Antibodies labeled with compounds (2) to (7) in which at least one of the compounds is an aryloxy group showed superior fluorescence intensity in membrane and dot blot conditions (compared to No. 202 to 207 of No. 201, and No. 302 to 307 of No. 301). Furthermore, both heterocycles in general formula (1) satisfy condition I, and R... 1 and R 2 At least one of them, R 3 and R 4 At least one of them, and R 5 and R 6 Labeled antibodies containing ethoxide groups with repeat numbers of 1 to 50 (3) to (7) exhibited superior fluorescence intensity (No. 103 to 107, 203 to 207) in both solution and membrane states. Furthermore, using R... 1 ~R 4 Labeled antibodies of compounds (3) to (6) having 1 to 4 repeats of ethoxide groups and using R 1 ~R 4 Compared to labeled antibodies containing 10 repeats of ethoxy groups (7), it showed superior fluorescence intensity even in the dot blot state (No. 303-306).

[0434] Thus, the compounds of the present invention represented by general formula (1) are selected from R 1 R 2 R 5 and R 22 ~R 25 One of them and selected from R 3 R 4 R 6 and R 26 ~R 29 One of them is a large-membered ring compound formed by bonding via a linking group LL, wherein the linking group LL does not contain any group selected from aromatic hydrocarbon rings, sulfonyl and phosphonyl groups, R 1 ~R 6 and R 22 ~R 29At least one of them contains ethoxy groups with a repeat number of 1 to 50, thereby imparting excellent fluorescence intensity to the obtained labeled biological material in any state, such as in solution, membrane, or spot imprint.

[0435] The present invention has been described together with its embodiments, but the inventors believe that unless otherwise specified, the invention will not be limited in any detail of the description, and should be interpreted broadly without departing from the spirit and scope of the invention shown in the appended technical solutions.

[0436] This application claims priority based on Japanese Patent Application No. 2020-129754, filed on July 30, 2020, the contents of which are incorporated herein by reference and are incorporated herein by reference as part of the description.

Claims

1. A compound represented by the following general formula (1-1), ###0001### (1-1) wherein n is 3, and the substituent capable of bonding to a biological substance is selected from the group consisting of an NHS ester structure, a succinimide structure, a maleimide structure, an azido group, an ethynyl group, a peptide structure, a long-chain alkyl group having 12 to 30 carbon atoms, and a quaternary ammonium group. wherein R 1 represents an alkyl group having 1 to 6 carbon atoms or -(CH2-CH2-O) 4 m -R 21 , m is 1 to 10, R 21 represents an alkyl group having 1 to 3 carbon atoms,​ R 12 represents a hydrogen atom or an aryloxy group having a sulfo group as a substituent, R 11 and R 13 represents a hydrogen atom, or the adjacent groups can be bonded to each other to form an aliphatic 6-membered ring, R 22 ~R 29 represents a hydrogen atom, an alkyl group having 1 to 3 carbon atoms or a sulfo group, and adjacent groups can be bonded to each other to form a naphthalene ring, R 22 ~R 25 The bonded ring and R 26 ~R 29 Each of the bonded rings has at least one sulfonyl group. The compound represented by the general formula (1-1) is a neutral compound containing at least one carboxyl group or a substituent capable of bonding to a biological substance. L 1 ~L 2 represents an alkylene group having 3 to 5 carbon atoms or -(CH2-CH2-O) m - an alkylene group having 2 to 4 carbon atoms, * represents a bonding position to the linking group LL, and m is 1 to 4, The linking group LL represents a divalent linking group having 5 to 20 atoms, which is composed of an alkylene group having 1 to 5 carbon atoms and -CONR 50 The linking group LL represents a divalent linking group having 5 to 20 atoms, which is composed of an alkylene group having 1 to 5 carbon atoms and -CONR 50 represents a hydrogen atom, and the linking group LL has a carboxyl group or a substituent capable of bonding to a biological substance, which is directly bonded to the alkylene group or is bonded to the alkylene group via a linking group ZZZ, the linking group ZZZ being a group represented by -CONH-(CH2-CH2-O) p a group represented by an alkylene group having 1 to 7 carbon atoms, the p is 1 to 4, 2. The compound according to claim 1, wherein R 1 ~R 4 At least one of them contains -(CH2-CH2-O) m - indicates a structure, and m has the same meaning as the m mentioned above.

3. The compound according to claim 1 or 2, wherein Both of the two heterocyclic rings in the general formula satisfy the following condition I. The L 1 ~L 2 respectively comprising a structure represented by -(CH2-CH2-O) m - wherein m = 1 to 4. Condition I:

4. The compound according to claim 1 or 2, wherein 5. A labeled biological substance obtained by bonding a compound according to any one of claims 1 to 4 to a biological substance. sp 3 at least one substituent on the carbon atom and the substituent on the ring-forming nitrogen atom of the heterocycle comprise -(CH2-CH2-O) m a structure represented by -CH2-CH(OH)-CH2- wherein m = 1 to 4.

6. The labeled biological substance according to claim 5, wherein The connecting part of L 1 ~L 2 in the connecting group LL is -CONR 50 - group, wherein R 50 is a hydrogen atom. The biological substance is any one of a protein, an amino acid, a nucleic acid, a sugar chain, and a phospholipid. ​ ​

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