Compounds and labeled biological substances using the same

Abstract

This invention provides a compound of formula (1) below, and a labeled biological substance having the compound. (Z-ring) 1 and ring Z 2 This indicates a 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms. R 1 ~R 4 R 11 ~R 13 L 1 L 2 These represent specific functional groups, and n, α1, and α2 represent specific numbers. R 11 ~R 13 At least one of the groups contains a carboxyl group or a substituent capable of bonding with biological material. Compounds represented by formula (1) have at least one substituent consisting of -(CH2-CH2-O). m -R 21 The structure represented. R 21 The radical represents a specific group, and m represents a specific number. The compound represented by formula (1) is a neutral compound.

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 immunoblotting (hereinafter also referred to as WB) for detecting 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 is bonded 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] As a fluorescent pigment used in the aforementioned fluorescent labeling, anthocyanin is known, for example, as described in Patent Document 1. However, when anthocyanin is 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, for example, Patent Document 2 describes anthocyanins incorporating water-soluble PEG (polyethylene glycol) groups. According to Patent Document 2, the anthocyanins described in each patent document suppress self-association between labeled pigments through the PEG groups present in the pigment, exhibiting higher fluorescence intensity than conventional anthocyanins.

[0007] Previous technical documents

[0008] Patent documents

[0009] Patent Document 1: U.S. Patent Application Publication No. 2021 / 0093737

[0010] Patent Document 2: International Publication No. 2009 / 078970 Summary of the Invention

[0011] The technical problem to be solved by the invention

[0012] However, through the inventors' research, it was found that the fluorescence intensity obtained by fluorescent labeling using the anthocyanin described in Patent Document 1 was low and could not be obtained sufficiently. Furthermore, it was found that in fluorescent labeling using the anthocyanin described in Patent Document 2, the binding affinity of the anthocyanin to living molecules such as antibodies was inherently low, resulting in low fluorescence intensity and the inability to obtain sufficient fluorescence intensity.

[0013] The objective of this invention is to provide a compound that represents excellent fluorescence intensity of the obtained labeled biological substance. Furthermore, the objective of this invention is to provide a labeled biological substance formed by bonding this compound to a biological substance.

[0014] means for solving technical problems

[0015] That is, the above-mentioned problems of the present invention are solved by the following method.

[0016] [1]

[0017] A compound represented by the following general formula (1).

[0018] [Chemical Formula 1]

[0019]

[0020] In the above formula, R 1 ~R 4 Indicates alkyl, aryl, heteroaryl, or -(CH2-CH2-O) m -R 21 m is 1 to 10, R 21 Indicates alkyl group.

[0021] R 11 ~R 13 This represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, amide, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring. Among them, R... 11 ~R 13 At least one of them contains a carboxyl group or a substituent capable of bonding with biological material.

[0022] n is an integer from 1 to 3.

[0023] L 1 and L 2 Indicates alkyl or -(CH2-CH2-O) m -R 21 R 21 and m are respectively related to the above R 21 The meaning of "and m" is the same. L 1 and L 2 They can bond together to form a ring.

[0024] α1 and α2 are 0 or 1.

[0025] Ring Z 1 and ring Z 2 This indicates a 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms. It can have substituents and can form a fused ring. When ring Z... 1 and ring Z 2 When a 6-membered ring with a cyclic nitrogen atom is formed, the cyclic nitrogen atom can be substituted by a substituent.

[0026] The compound represented by formula (1) has at least one -(CH2-CH2-O) group. m -R 21 The structure represented. R 21 and m are respectively related to the above R 21 The meanings of "and m" are the same.

[0027] Among them, the compound represented by formula (1) is a neutral compound.

[0028] [2]

[0029] The compound according to [1] is represented by any one of the following general formulas (2-1) to (2-4).

[0030] [Chemical Formula 2]

[0031]

[0032] In the above formula, R 45 Indicates alkyl or -(CH2-CH2-O) m -R 21 .

[0033] R 41 ~R 44 and R 81 ~R 86 It represents a hydrogen atom, alkyl group, alkoxy group, aryl group, sulfonyl group, sulfonamide group, nitro group, carboxyl group, amide group, or halogen atom. Adjacent groups can bond with each other to form a fused ring.

[0034] R 1 ~R 4 R 11 ~R 13 L 1 L 2 and m are respectively related to the above R 1 ~R 4 R 11 ~R 13 L 1 L 2 The meanings of "and m" are the same.

[0035] R 1 ~R 4 L 1 L 2 or R 45 At least one of them is -(CH2-CH2-O). m -R 21 R 21 and m are respectively related to the above R 21 The meanings of "and m" are the same.

[0036] l represents an integer of 2 or 3.

[0037] Among them, the compound represented by any one of formulas (2-1) to (2-4) is a neutral compound.

[0038] [3]

[0039] According to the compound described in [1] or [2], wherein,

[0040] The above R 1 and R 2 At least one of the above R 3 and R 4 At least one of them contains -(CH2-CH2-O) m - indicates a structure where m is 1 to 10.

[0041] [4]

[0042] According to the compound described in [3], wherein,

[0043] The compound contains four groups consisting of -(CH2-CH2-O). m - indicates a structure where m is 1 to 10.

[0044] [5]

[0045] The compound according to any one of [1] to [4], wherein,

[0046] The above L 1 and L 2 At least one of them is an alkyl group having at least one of alkoxy, carboxyl, sulfonyl and phosphonoyl groups as substituents.

[0047] [6]

[0048] According to the compound described in [5], wherein,

[0049] The above L 1 and L 2 It is an alkyl group having at least one of the following as a substituent: alkoxy, carboxyl, sulfonyl, and phosphonoyl.

[0050] [7]

[0051] The compound according to any one of [1] to [6], wherein,

[0052] The above R 11 ~R 13 The substituents having a carboxyl group or a group having a substituent capable of bonding with biological material are substituents represented by any one of the following general formulas (I) to (V).

[0053] [Chemical Formula 3]

[0054]

[0055] In the above formula, Y represents an oxygen atom or =NR 38 Q ring 1 This indicates a 5- or 6-membered heterocyclic or hydrocarbon ring.

[0056] R 31 ~R 44 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, amide, halogen atom, sulfonyl, nitro, cyano, carboxyl, or a substituent that can bond with biological substances.

[0057] The group represented by any one of the general formulas (I) to (V) has at least one carboxyl group or a substituent capable of bonding with biological material.

[0058] * indicates a bond.

[0059] [8]

[0060] According to the compound described in [7], wherein,

[0061] The substituent represented by the above general formula (II) is a substituent represented by any one of the following formulas (E-1) to (E-8).

[0062] [Chemical Formula 4]

[0063]

[0064] In the above formula, R 51 ~R 72 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, halogen atom, sulfonyl, nitro, cyano, carboxyl, or a substituent that can bond with biological substances.

[0065] The substituent represented by any one of the general formulas (E-1) to (E-8) has at least one carboxyl group or a substituent capable of bonding with biological material.

[0066] * indicates a bond.

[0067] [9]

[0068] According to the compound described in [7], wherein,

[0069] The above R 11 ~R 13 Substituents containing carboxyl groups or groups containing substituents capable of bonding with biological substances are substituents represented by the above general formula (IV).

[0070]

[10]

[0071] According to the compound described in [9], wherein,

[0072] The above R 39 ~R 43 At least one of them is a carboxyl group, an alkoxy group having a carboxyl group, an alkyl carbamoyl group having a carboxyl group, or a poly(alkylene oxide) carbamoyl group having a carboxyl group.

[0073]

[11]

[0074] A biolabeled substance formed by bonding a biolabel with any one of the compounds described in [1] to

[10] .

[0075]

[12]

[0076] According to the labeled biological material described in

[11] , wherein,

[0077] The aforementioned biological substances are any one of proteins, amino acids, nucleic acids, sugar chains, and phospholipids.

[0078] Invention Effects

[0079] The compounds of the present invention can produce labeled biological substances exhibiting excellent fluorescence intensity. Furthermore, the labeled biological substances of the present invention display excellent fluorescence intensity. Detailed Implementation

[0080] 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, unless otherwise specified, each substituent, etc., may be identical or different from each other (regardless of whether it is expressed as "separately and independently"), each substituent, etc., may be identical or different from each other. The same applies to the number of substituents, etc. Furthermore, when multiple substituents, etc., are close together (especially adjacent), unless otherwise specified, they may connect to each other to form a ring, and the formed ring may or may not have unsaturated bonds. Furthermore, unless otherwise specified, rings, such as alicyclic rings, aromatic rings, and heterocyclic rings, may further fuse to form fused rings.

[0081] 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.

[0082] 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.

[0083] 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 a mixture of salt-type and free acid-type compounds.

[0084] 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), L 2 The formal charge of the bonded nitrogen atom is +1 except when α2 = 0. In relation to this formal charge, the ionizable groups such as the sulfonyl group in the compound have ionic structures such as sulfonate ions, thus the compound of the present invention, as a whole, is a compound with a charge of 0. When α2 = 0, L 2 The formal charge of the bonded nitrogen atom is 0.

[0085] Furthermore, in compounds represented by general formula (1), L 1 The formal charge of the bonded nitrogen atoms is 0 when α1 = 1. When α1 = 0, the L... 1The bonded nitrogen atom has a formal charge of 0, replacing the L-containing atoms in general formula (1). 1 The bonded nitrogen atom's 5-membered ring and ring Z 1 The double bonds in the fused portion, and having L 1 The bonded nitrogen atom and ring Z 1 The bonding portion becomes a conjugated structure of double bonds.

[0086] 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.

[0087] 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 not explicitly described as substituted or unsubstituted, 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.), linking groups (e.g., groups expressed as "alkylene", "methylene", "methylene"), and rings (e.g., rings expressed as "heterocyclic", "hydrocarbon ring", etc.). Among such arbitrary substituents, in the present invention, substituents preferably selected from the substituent group T described later.

[0088] 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 of carbon atoms 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.

[0089] Furthermore, in this invention, the numerical range represented by “~” refers to the range of values ​​recorded before and after “~”, which are both the lower and upper limits.

[0090] 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 yield labeled biological substances with excellent fluorescence intensity are not yet clear, but are thought to be as follows.

[0091] As shown in general formula (1), the compounds of the present invention contain nitrogen atoms as cyclic atoms and have ring Z at both ends. 1 or ring Z 2Fused heterocyclic polymethimide chains (in this invention, a polymethimide chain refers to a hydrocarbon chain with multiple double bonds within the chain, which is an integrally conjugated chain, and the hydrocarbon chain has substituents R) 11 ~R 13 Furthermore, the number of carbon atoms constituting this hydrocarbon chain is 2n+3, which is composed of () n The carbon atoms of the enclosed hydrocarbon chain, enclosed by R 11 The substituted carbon atoms and the rings forming the heterocycles on either side of them constitute the carbon atom composition. (The same applies below.) Furthermore, the ring Z... 1 The nitrogen atom of the fused heterocycle has a tertiary amine structure, and the ring Z... 2 The nitrogen atoms of the fused heterocycles have a quaternary ammonium structure, thereby generating absorption through charge transfer via the polymethyl backbone. Thus, the compounds of the present invention are classified as compounds called polymethyl pigments (broadly defined as anthocyanins).

[0092] The compounds of the present invention, having the above-described structure, have substituent R on the methylene chain with repeating number 2n+3. 11 ~R 13 At least one of them further comprises a carboxyl group or a substituent capable of bonding with biological material, and in the compound, has at least one structure comprising a PEG (polyethylene glycol) group, said PEG (polyethylene glycol) group being -(CH2-CH2-O). m -R 21 This indicates that m is 1 to 10. Thus, through R in the compound... 11 ~R 13 Having a carboxyl group or a substituent capable of bonding with biological matter at at least any position, it can be more easily discovered by means of -(CH2-CH2-O). m -R 21 The compound with the PEG group exhibits a chemical structure that eliminates the volumetric effect, and as a result of effectively suppressing the interaction between compounds, it is believed that the decrease in fluorescence intensity due to the self-association of the compounds can be suppressed. Furthermore, by setting the repeating number m in the PEG group to 10 or less, the decrease in binding affinity to biological substances caused by the large volumetric effect resulting from PEG groups with m exceeding 10 can be suppressed, demonstrating a practically problem-free level of binding affinity to biological substances.

[0093] The compounds of the present invention have an excitation absorption wavelength in the wavelength region of 520–600 nm (around 585 nm) depending on the length of the methylene chain with repeating number 2n+3. When n = 1, they have an excitation absorption wavelength in the wavelength region of 620–700 nm (around 685 nm); when n = 2, they have an excitation absorption wavelength in the wavelength region of 740–830 nm (around 785 nm); and when n = 3, they have an excitation absorption wavelength in the wavelength region of 740–830 nm (around 785 nm). Therefore, the compounds represented by these general formulas (1) can be used as fluorescent labels for biomarkers that exhibit excellent fluorescence intensity when using a light source of any wavelength (e.g., around 600 nm, 700 nm, or 800 nm) in the wavelength region of approximately 500–800 nm corresponding to the excitation wavelength of the compound.

[0094] 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.

[0095] 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.

[0096] 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, the compounds of the present invention with n=2 or 3 can be used as compounds that can be used to label biological substances and exhibit excellent fluorescence intensity. In particular, for the requirement of higher sensitivity observation and detection of proteins, it can exhibit superior fluorescence intensity compared to conventional anthocyanin fluorescent labels containing anthocyanins described in Patent Documents 1 and 2.

[0097] Hereinafter, the compounds represented by general formula (1) of the present invention will be described in detail.

[0098] <Compounds represented by general formula (1)>

[0099] The compounds of the present invention represented by general formula (1) are described below.

[0100] [Chemical Formula 5]

[0101]

[0102] In the formula, R 1 ~R 4 Indicates alkyl, aryl, heteroaryl, or -(CH2-CH2-O) m -R 21 m is 1 to 10, R 21 Indicates alkyl group.

[0103] R 11 ~R 13 This represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, amide, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring. Among them, R... 11 ~R 13 At least one of them contains a carboxyl group or a substituent capable of bonding with biological material.

[0104] n is an integer from 1 to 3.

[0105] L 1 and L 2 Indicates alkyl or -(CH2-CH2-O) m -R 21 R 21 and m are respectively related to the above R 21 The meaning of "and m" is the same. L 1 and L 2 They can bond together to form a ring.

[0106] α1 and α2 are 0 or 1.

[0107] Ring Z 1 and ring Z 2 This indicates a 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms. It can have substituents and can form a fused ring. When ring Z... 1 and ring Z 2 When a 6-membered ring with a cyclic nitrogen atom is formed, the cyclic nitrogen atom can be substituted by a substituent.

[0108] The compound represented by formula (1) has at least one -(CH2-CH2-O) group. m -R 21 The structure represented. R 21 and m are respectively related to the above R 21 The meanings of "and m" are the same.

[0109] Among them, the compound represented by formula (1) is a neutral compound.

[0110] The substituents in general formula (1) will be explained in detail below.

[0111] (1)R 1 ~R 4

[0112] R 1 ~R 4 Indicates alkyl, aryl, heteroaryl, or -(CH2-CH2-O) m -R 21 R 1 With R 2 They can be connected to form a ring, R 3 With R 4 They can also be connected to form a ring.

[0113] Can be used as R 1 ~R 4 The alkyl, aryl, and heteroaryl groups have the same meanings as the alkyl, aryl, and heteroaryl groups in the substituent group T described later.

[0114] 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.

[0115] 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 even more preferably 3 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 30, and even more preferably 3 to 25.

[0116] 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.

[0117] 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 that do 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.

[0118] As can be used as R 1 ~R 4 Alkyl groups can have various substituents, including alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, amide, sulfonyl, phosphonoyl, and -(CH2-CH2-O). m -R 21 -(C3H6-O) m -R21 And groups formed by 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 formed by combinations of these substituents, may have substituents capable of bonding with biological substances as described later.

[0119] As can be used as R 1 ~R 4 The alkyl group having substituents is not particularly limited, but from the viewpoint of suppressing intermolecular interactions, it is preferred to have -(CH2-CH2-O). m -R 21 Alkyl groups as substituents. In this case, -(CH2-CH2-O) m -R 21 It can directly replace alkyl groups, or it can be composed of carbamoyl group and -(CH2-CH2-O). m -R 21 The combination of groups constitutes substitution.

[0120] (-(CH2-CH2-O) m -R 21 )

[0121] In the ability to be used as R 1 ~R 4 -(CH2-CH2-O) m -R 21 In the middle, m is 1 to 10, R 21 Indicates alkyl group.

[0122] m refers to the average number of repetitions (also simply called the number of repetitions), which is preferably 4 to 10, and more preferably 4 to 8.

[0123] 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.

[0124] R 21 The alkyl group in the above-mentioned R is suitable for use as R. 1 ~R 4 The record of alkyl groups.

[0125] As can be used as R 1 ~R 4 -(CH2-CH2-O) m -R 21 and R 1 ~R4 Contains -(CH2-CH2-O) m -R 21 Preferably -(CH2-CH2-O) m - Unsubstituted alkyl group.

[0126] (-(C3H6-O) m -R 21 )

[0127] Can be used as R 1 ~R 4 -(C3H6-O) m -R 21 m and R in 21 With -(CH2-CH2-O) m -R 21 m and R in 21 They have the same meaning.

[0128] Can be used as R 1 ~R 4 -(C3H6-O) m -R 21 The -C3H6- in it can be any of the structures -CH2-CH2-CH2-, -CH2-CH(CH3)-, and -CH(CH3)-CH2-.

[0129] R 1 ~R 4 At least one preferred component comprises -(CH2-CH2-O) m - Represents the structure, from the viewpoint of further improving fluorescence intensity, R 1 and R 2 At least one of them and R 3 and R 4 At least one more preferably comprises -(CH2-CH2-O) m - represents the structure, R 1 and R 2 At least one of them and R 3 and R 4 At least one of them is further preferably composed of -(CH2-CH2-O). m - indicates a structure that contains a total of 4 -(CH2-CH2-O) groups in the compound. m - represents the structure. Where R 1 and R 2 At least one of them, R 3 and R 4 At least one of them, L 1 and L 2 Especially preferred are those containing -(CH2-CH2-O) mThe structure indicated by - is as follows. The above is derived from -(CH2-CH2-O). m The structure represented by - is preferably -(CH2-CH2-O). m -R 21 It is directly bonded to the cyclizing atom of the heterocycle that is directly bonded to the aforementioned methine chain.

[0130] The above -(CH2-CH2-O) m The 'm' in - is related to the -(CH2-CH2-O) above. m -R 21 The meaning of 'm' in the text is the same.

[0131] R 1 ~R 4 The substituents are present in R groups having at least one carboxyl group or substituents capable of bonding with biological material. 11 ~R 13 The separated positions, and the projections perpendicular to the anthocyanin skeleton (plane), suggest that this is achieved by including -(CH2-CH2-O). m The structure represented by - as the substituent is more effectively expressed due to -(CH2-CH2-O). m The structure indicated by - produces a volume exclusion effect, making it difficult for π-π interactions to occur in the fused ring region (enhancing the effect of inhibiting association), which can further suppress the decrease in fluorescence intensity caused by association.

[0132] (2)R 11 ~R 13

[0133] 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, amide group, or halogen atom. Adjacent groups can bond to each other to form a 5-membered or 6-membered ring. As described below, R... 11 ~R 13 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material.

[0134] 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.

[0135] Furthermore, as a form that can be used as R 11 ~R 13The amide group, in addition to the amide group in substituent group T described below, can also include amide or carbamoyl groups as monovalent substituents derived from carboxylic amides, or sulfonyl or aminosulfonyl groups as monovalent substituents derived from sulfonic amides. These groups have the same meaning as amide, carbamoyl, sulfonyl, and aminosulfonyl groups in substituent group T described below, and the preferred range is also the same. As a group capable of being used as R 11 ~R 13 The amide group, wherein, preferably, is an amide group.

[0136] Can be used as R 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, and amide groups may be unsubstituted or have substituents.

[0137] As R 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, and amide groups in the group can have substituents, such as those in substituent group T described later. Preferred substituents include alkyl, alkoxy, aryloxy, alkylthio, aryl, heteroaryl, acyl, alkoxycarbonyl, alkylsulfonyl, amino, amide, nitro, cyano, sulfonyl, or halogen atoms. Furthermore, oxo groups (=O) or imino groups (=NH, where the hydrogen atom can be substituted) can also be included. Carboxyl groups or other substituents capable of bonding with biological substances described later are also preferred. 11 ~R 13 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, or amide groups may have substituents that can be groups formed by combining two or more of the groups listed above.

[0138] In R 11 ~R 13 When adjacent groups in the compound bond to each other to form a 5-membered ring or a 6-membered ring, the formed 5-membered ring or 6-membered ring can be either aromatic or aliphatic, preferably aliphatic. The number of the aforementioned 5-membered ring or 6-membered ring in the compound is not particularly limited, but is preferably one or two, more preferably one.

[0139] 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 does not form a ring structure. 11 ~R 13 The hydrogen atom is the ring structure, which is recorded as having no substituents, but as mentioned above, R 11 ~R 13At least one of them is a group having a carboxyl group or a substituent capable of bonding with biological material, for other R 11 ~R 13 It is not limited to the following structures.

[0140] [Chemical Formula 6]

[0141]

[0142] R 11 ~R 13 The substituent having a carboxyl group or a substituent having a substituent capable of bonding with biological material is preferably a substituent represented by any one of the following general formulas (I) to (V), and more preferably a substituent represented by the following general formula (IV).

[0143] [Chemical Formula 7]

[0144]

[0145] In the above formula, Y represents an oxygen atom or =NR 38 Q ring 1 This indicates a 5- or 6-membered heterocyclic or hydrocarbon ring.

[0146] R 31 ~R 44 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, amide, halogen atom, sulfonyl, nitro, cyano, carboxyl, or a substituent that can bond with biological substances.

[0147] Additionally, R 36 With R 37 They do not bond with each other to form 5 or 6-membered rings, and the substituents represented by general formula (II) are different from those represented by (III).

[0148] The group represented by any one of the general formulas (I) to (V) has at least one carboxyl group or a substituent capable of bonding with biological material.

[0149] * indicates a bond.

[0150] R 31 ~R 35 Adjacent groups in R can connect with each other to form a ring. 39 ~R 43 Adjacent groups in the compound can also connect with each other to form a ring.

[0151] Can be used as R 31 ~R 44The 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.

[0152] Can be used as R 31 ~R 44 The alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, and halogen atoms may have substituents, for example, preferably sulfonyl or halogen atoms (preferably fluorine atoms).

[0153] Can be used as R 31 ~R 44 The amide group can be used as R 11 ~R 13 The amide group is described. As it can be used as R 31 ~R 44 The amide group, wherein carbamoyl and aminosulfonyl groups are preferably examples.

[0154] Can be used as R 31 ~R 44 The substituents that can bond with biological substances have the same meaning as the substituents that can bond with biological substances described later, and the preferred range is also the same.

[0155] In the substituents represented by the above general formula (I), as R 31 ~R 35 Among the groups listed above, the preferred groups are hydrogen atoms, alkyl groups, alkoxy groups, alkylthio groups, amino groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, cyano groups, carboxyl groups, or substituents that can bond with biological substances, and more preferably hydrogen atoms, alkyl groups, alkoxy groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, carboxyl groups, or substituents that can bond with biological substances.

[0156] R is preferred as the substituent represented by the above general formula (I). 32 ~R 34 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, more preferably R. 32 ~R 34 At least one of them (preferably any one) has a carboxyl group or a substituent capable of bonding with biological material. As R 31 ~R 35 R is a form in which any of the substituents has a carboxyl group or a substituent capable of bonding with biological material. 31 ~R 35 It can be a carboxyl group or a substituent capable of bonding with biological substances, or it can be used as an R group. 31 ~R 35The substituents are replaced by carboxyl groups or substituents that can bond with biological substances.

[0157] In the substituents represented by the above general formula (II), Y is preferably an oxygen atom.

[0158] Q Ring 1 This indicates a 5- or 6-membered heterocyclic or hydrocarbon ring.

[0159] Q Ring 1 It can be an aromatic ring or an aliphatic ring, but an aliphatic ring is preferred.

[0160] Furthermore, as a component of ring Q 1 In addition to the carbon atom bonded to Y and the carbon atom with bonding bond*, the atoms in the ring-forming atoms can be carbon atoms, nitrogen atoms, oxygen atoms or sulfur atoms. There can be one or more types of ring-forming atoms, but it is preferred to have one or two types.

[0161] Q Ring 1 It can have substituents and can also form fused rings.

[0162] The substituent represented by the above general formula (II) is preferably a substituent represented by any one of the following formulas (E-1) to (E-8).

[0163] [Chemical Formula 8]

[0164]

[0165] In the above formula, R 51 ~R 72 It represents a hydrogen atom, alkyl, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, amino, halogen atom, sulfonyl, nitro, cyano, carboxyl, or a substituent that can bond with biological substances.

[0166] The substituent represented by any one of the general formulas (E-1) to (E-8) has at least one carboxyl group or a substituent capable of bonding with biological material.

[0167] * indicates a bond.

[0168] Can be used as R 51 ~R 72 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.

[0169] Can be used as R 51 ~R 72The substituents that can bond with biological substances have the same meaning as the substituents that can bond with biological substances described later, and the preferred range is also the same.

[0170] As R 51 ~R 72 Among the groups listed above, hydrogen atoms, alkyl groups, cyano groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred.

[0171] As a substituent represented by any one of the general formulas (E-1) to (E-8), as long as it has R in each substituent... 51 ~R 72 At least one carboxyl group or a substituent capable of bonding with biological material is required, wherein, preferably, in general formula (E-1), R 52 Having a carboxyl group or a substituent capable of bonding with biological matter, in the general formula (E-2), R 54 Having a carboxyl group or a substituent capable of bonding with biological matter, in general formula (E-3), R 57 ~R 59 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, in general formula (E-4), R 61 and R 62 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, in general formula (E-5), R 63 and R 64 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, in general formula (E-6), R 65 and R 66 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, in general formula (E-7), R 67 and R 68 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, in the general formula (E-8), R 69 ~R 72 At least one of them has a carboxyl group or a substituent capable of bonding with biological material. As R 51 ~R 72 R is a form in which any of the substituents has a carboxyl group or a substituent capable of bonding with biological material. 51 ~R 72 It can be a carboxyl group or a substituent capable of bonding with biological substances, or it can be used as an R group. 51 ~R 72 The substituents are replaced by carboxyl groups or substituents that can bond with biological substances.

[0172] Among the substituents represented by the above general formula (III), as R 36 or R 37Among the groups listed above, hydrogen atoms, alkyl groups, amino groups, cyano groups, carboxyl groups, or substituents that can bond with biological substances are preferred.

[0173] As a substituent represented by the above general formula (III), as long as R 36 or R 37 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, preferably R. 36 It has a carboxyl group or a substituent capable of bonding with biological substances. As R 36 or R 37 R is a form in which any of the substituents has a carboxyl group or a substituent capable of bonding with biological material. 36 or R 37 It can be a carboxyl group or a substituent capable of bonding with biological substances, or it can be used as an R group. 36 or R 37 The substituents are in the form of carboxyl groups or substituents that can bond with biological substances.

[0174] In this invention, R is preferred. 11 And besides the Z ring 2 The R-type carbon atoms of the fused heterocyclic bonded structure 13 Other than R 12 or R 13 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, more preferably in the linking ring Z. 1 Fused heterocyclic rings and ring Z 2 The central portion of the bond in the fused heterocycle has a carboxyl group or a substituent that can bond with biological material.

[0175] R 11 and with ring Z 2 The R-type carbon atoms of the fused heterocyclic bonded structure 13 Hydrogen atoms are preferred.

[0176] As R 12 and other R besides those mentioned above 13 The group does not contain a carboxyl group or a substituent that can bond with biological substances, preferably a hydrogen atom, alkyl group, aryloxy group or arylthio group, more preferably a hydrogen atom or alkyl group, and even more preferably a hydrogen atom.

[0177] Furthermore, R 11 ~R 13 In the middle, R 11 and with ring Z 2 In addition to R, the carbon atoms of the fused heterocyclic bonds possess other properties. 13 Other than R 12 ~R 13Adjacent groups in the ring are preferably bonded to each other to form a 5-membered ring or a 6-membered ring, more preferably a 6-membered ring. Furthermore, it is preferable that the connecting ring Z... 1 Fused heterocyclic rings and ring Z 2 The central portion of the bond in the fused heterocyclic ring forms the aforementioned 5-membered or 6-membered ring. In the connecting ring Z... 1 Fused heterocyclic rings and ring Z 2 The ring formed by the central portion of the bond in a fused heterocycle represents a ring containing an equal number of carbon atoms from the two heterocycles as cyclic atoms.

[0178] Additionally, when the above R 11 ~R 13 When adjacent groups in a ring bond to each other to form a 5- or 6-membered ring, the carboxyl group or a substituent that can bond with biological material is preferably located at a site outside the ring.

[0179] In the substituents represented by the above general formula (IV), as R 39 ~R 43 Among the groups listed above, hydrogen atoms, alkyl, alkoxy, alkylthio, amino, amide, halogen, sulfonyl, nitro, cyano, carboxyl or substituents capable of bonding with biological substances are preferred, hydrogen atoms, alkyl, alkoxy, amide, halogen, sulfonyl, nitro, cyano, carboxyl or substituents capable of bonding with biological substances are more preferred, and hydrogen atoms, alkyl, alkoxy, amide, halogen, sulfonyl, nitro, carboxyl or substituents capable of bonding with biological substances are even more preferred.

[0180] As a substituent represented by the above general formula (IV), R is preferred from the viewpoint of further improving fluorescence intensity or from the viewpoint of obtaining excellent lightfastness. 39 and R 43 It is an alkyl, alkoxy, or halogen atom. Preferably, it is R. 40 ~R 42 At least one of them is a carboxyl group, an alkoxy group having a carboxyl group, an alkylcarbamoyl group having a carboxyl group, or a poly(alkylene oxide)carbamoyl group having a carboxyl group, or has a substituent capable of bonding with biological material, more preferably R. 40 ~R 42 At least one of them is a carboxyl group, an alkoxy group having a carboxyl group, an alkyl carbamoyl group having a carboxyl group, or a poly(alkylene oxide) carbamoyl group having a carboxyl group, more preferably R 41 The group is a carboxyl group, an alkoxy group having a carboxyl group, an alkyl carbamoyl group having a carboxyl group, or a poly(alkylene oxide) carbamoyl group having a carboxyl group, especially R. 41 It is a carboxyl group.

[0181] As R 39 ~R 43R is a form in which any of the substituents has a carboxyl group or a substituent capable of bonding with biological material. 39 ~R 43 It can be a carboxyl group or a substituent capable of bonding with biological substances, or it can be used as an R group. 39 ~R 43 The substituents are replaced by carboxyl groups or substituents that can bond with biological substances.

[0182] In the substituents represented by the above general formula (V), as R 44 Among the groups listed above, aryl groups having a carboxyl group or a substituent capable of bonding with biological material are preferred. The aryl group may have substituents other than a carboxyl group or a substituent capable of bonding with biological material; for example, alkyl, alkoxy, or halogen atoms are preferably included.

[0183] (3)L 1 and L 2

[0184] L 1 and L 2 Indicates alkyl or -(CH2-CH2-O) m -R 21 R 21 and m are respectively related to the above R 21 The meaning of "and m" is the same. L 1 and L 2 They can bond together to form a ring.

[0185] As L 1 and L 2 The alkyl group can have substituents, including alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, amide, sulfonyl, and phosphonoyl groups, as well as combinations of these substituents. Furthermore, it can have -(CH2-CH2-O). m -R 21 (R 21 and m are respectively related to the above R 21 The meanings of 'm' and 'm' are the same. ) and the substituents described below that can bond with biological substances. Furthermore, the alkyl portion of the above-mentioned alkoxy, carboxyl, alkoxycarbonyl, acyloxy, carbamoyl, amide, sulfonyl, and phosphonoyl groups, as well as combinations of these substituents, may have -(CH2-CH2-O). m -R 21 Or, as described later, substituents that can bond with biological substances.

[0186] Can be used as L 1 and L 2 The alkyl group has the same meaning as the alkyl group in the substituent group T described later.

[0187] 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 3.

[0188] The alkyl portion of the alkyl group having a substituent preferably has 1 to 10 carbon atoms, more preferably 1 to 8, even more preferably 1 to 7, even more preferably 1 to 6, and even more preferably 1 to 5. Furthermore, the longest chain constituting the alkyl group having a substituent preferably has 3 to 14 atoms, more preferably 3 to 12, and even more preferably 3 to 10.

[0189] As having the ability to be used as L 1 and L 2 From the viewpoint of further improving water solubility, the alkyl group with substituents is preferably an alkyl group having at least one of the substituents selected from alkoxy, carboxyl, sulfonyl, and phosphonoyl groups; more preferably, it is an alkyl group having at least one of the substituents selected from carboxyl, sulfonyl, and phosphonoyl groups; and even more preferably, it is an alkyl group having at least one of the substituents selected from carboxyl and sulfonyl groups. Alternatively, it may be an alkyl group having a combination of substituents consisting of the above-mentioned preferred substituents (alkoxy, carboxyl, sulfonyl, and phosphonoyl groups) and groups other than these substituents.

[0190] Furthermore, the aforementioned R can also be preferentially applied. 1 ~R 4 Alkyl groups with substituents can be used.

[0191] Can be used as L 1 and L 2 -(CH2-CH2-O) m -R 21 The above-mentioned R can be preferred 1 ~R 4 -(CH2-CH2-O) m -R 21 The records.

[0192] As can be used as L 1 and L 2 -(CH2-CH2-O) m -R 21 R is preferred. 21 The alkyl group that does not have a carboxyl group or a substituent at the end that can bond with biological material is more preferably an unsubstituted alkyl group.

[0193] From the perspective of inhibiting the association between pigments caused by the highly lipophilic structure of the compound, L 1 and L 2 Preferably L 1 and L 2 At least one of them has an alkyl group selected from alkoxy, carboxyl, sulfonyl and phosphonoyl as a substituent, more preferably L1 and L 2 Both of these have an alkyl group that is a substituent of at least one of alkoxy, carboxyl, sulfonyl, and phosphonoyl groups.

[0194] As R 1 ~R 4 With L 1 and L 2 The combination of R can be preferably cited as an example. 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains -(CH2-CH2-O) m - represents the structure, and L 1 and L 2 At least one of them is a combination of alkyl groups having at least one substituent of an alkoxy group, a carboxyl group, a sulfonyl group, and a phosphonoyl group, more preferably R. 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains -(CH2-CH2-O) m - represents the structure, L 1 and L 2 It is a combination of alkyl groups having at least one substituent of an alkoxy group, a carboxyl group, a sulfonyl group, and a phosphonoyl group, and is more preferably R. 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains -(CH2-CH2-O) m - represents the structure, L 1 and L 2 It is an alkyl group having a sulfonyl substituent.

[0195] In addition, the above-mentioned features are capable of being used as L 1 and L 2 The alkyl group having at least one of the following as a substituents (alkoxy, carboxyl, sulfonyl, and phosphonoyl) is preferably an alkyl group having at least one of the following as a substituents (carboxyl, sulfonyl, and phosphonoyl), more preferably an alkyl group having at least one of the following as a substituents (carboxyl and sulfonyl), and even more preferably an alkyl group having a sulfonyl as a substituent.

[0196] (4) Ring Z 1 and ring Z 2

[0197] Ring Z 1 and ring Z 2 It represents a 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms. It can have substituents and can also form a fused ring.

[0198] In the Z-ring 1 and ring Z 2 In the case of a fused ring consisting of a 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms, the fused ring as a whole is understood as ring Z. 1 and ring Z 2 .

[0199] When ring Z 1 and ring Z 2 When a 6-membered ring with a cyclic nitrogen atom is formed, this cyclic nitrogen atom can be substituted with a substituent. From the viewpoint of further improving fluorescence intensity, cyclic Z... 1 and ring Z 2 It is preferably a single ring.

[0200] As can be used as a ring Z 1 and ring Z 2 The 6-membered ring formed by cyclic atoms selected from carbon and nitrogen atoms can be an aliphatic ring or an aromatic ring, but is preferably an aromatic ring.

[0201] Examples of the aforementioned 6-membered ring include hydrocarbon rings and nitrogen-containing heterocycles. As a nitrogen-containing heterocycle, it is preferable to have a ring relative to L... 1 or L 2 The cyclic atom adjacent to the bonded nitrogen atom is a nitrogen-containing heterocycle, more preferably a nitrogen-containing heterocycle relative to L. 1 or L 2 The nitrogen atom bonded to the cyclic atom located in the adjacent position is a pyridine ring of nitrogen.

[0202] Examples of the aforementioned 6-membered rings include benzene rings, pyridine rings, pyrimidine rings, pyridazine rings, and 1,2,3 or 1,2,4-triazine rings, with benzene rings or pyridine rings being preferred. Furthermore, examples can be made by describing the conjugated structure so that these aromatic rings have the same type and position as the cyclic atoms in aliphatic rings.

[0203] Substituents that may be present in the above-mentioned 6-membered ring or the fused ring of the above-mentioned 6-membered ring include those in the substituent group T described below, preferably alkyl, alkoxy, aryl, carboxyl, sulfonyl, phosphonyl, sulfonamide, nitro or halogen atom, more preferably alkyl, sulfonyl, sulfonamide, nitro or halogen atom.

[0204] From the perspective of improving water solubility and inhibiting association, cyclo-Z 1 and ring Z 2 Preferably, each has one or more hydrophilic groups independently, more preferably forming the ring Z. 1 or ring Z 2 Each of the rings has at least one hydrophilic group. For example, it is more preferable to have a hydrophilic group in ring Z. 1 and ring Z 2When all rings are naphthalene rings, ring Z is formed. 1 and ring Z 2 The number of rings is 2, and ring Z 1 and ring Z 2 All have at least two hydrophilic groups. There is no particular upper limit on the number of possible structures, and it can be appropriately adjusted according to the number of hydrophilic groups as a whole in the compound, as described later.

[0205] There are no particular limitations on the hydrophilic group; for example, alkoxy, carboxyl, sulfonyl, and phosphonyl groups with substituents can be included, with sulfonyl being preferred. These hydrophilic groups can be directly bonded to the ring Z. 1 or ring Z 2 The two bonds can also be bonded via linking groups, preferably by direct bonding.

[0206] (5) n, α1 and α2

[0207] n is an integer from 1 to 3, preferably an integer of 2 or 3.

[0208] α1 and α2 are either 0 or 1. α1 being 0 indicates that L is not present. 1 α1 being 1 indicates having L 1 Similarly, α2 being 0 indicates the absence of L. 2 α2 being 1 indicates having L 2 .

[0209] When ring Z 1 For relative to L 1 or L 2 When the bonded nitrogen atom is located in a nitrogen-containing heterocycle with a nitrogen atom in its adjacent position, α1 can be 0. Furthermore, when ring Z... 2 For relative to L 1 or L 2 When the bonded nitrogen atom is located in a nitrogen-containing heterocycle with a nitrogen atom in the adjacent position, α2 can be 0.

[0210] R 11 ~R 13 At least one of them contains a carboxyl group or a substituent capable of bonding with biological material.

[0211] 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.

[0212] In this invention, "substituents capable of bonding with biological material" include substituents capable of bonding with biological material derived from carboxyl groups.

[0213] Thus, the compound represented by the above general formula (1) is obtained by the presence of substituents at specific positions in the anthocyanin skeleton structure (specifically, R as substituents on the polymethimide chain with repeating number n+1). 11 ~R 13 At least one of the markers is bound to the biomaterial, and therefore the obtained labeled biomaterial is considered to exhibit excellent fluorescence intensity as described above.

[0214] R 11 ~R 13 The total number of groups having a carboxyl group or a substituent that can bond with biological substances 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.

[0215] 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.

[0216] As a hydrophilic group, it can be used in the aforementioned cyclo-Z 1 and ring Z 2 A list of the hydrophilic groups that can be used.

[0217] The position of the hydrophilic group is not particularly limited. For example, R can be preferably cited as a group having the above-mentioned hydrophilic group. 11 ~R 13 , Ring Z 1 , Ring Z 2 L 1 and L 2 .

[0218] Furthermore, when the compound represented by the above general formula (1) has the above-mentioned hydrophilic group as the carboxyl group or the substituent capable of bonding with biological material, it actually has one or more hydrophilic groups in addition to the above-mentioned carboxyl group or the substituent capable of bonding with biological material, which is therefore preferred. As specific examples, carboxyl groups, sulfonyl groups, etc. can be used, and the substituents can be alkyl groups, alkoxy groups, etc., having carboxyl groups, sulfonyl groups, etc. as substituents. For example, sulfonylalkyl groups, sulfonylalkoxy groups, etc., can be used.

[0219] <Compounds represented by any one of the general formulas (2-1) to (2-4)>

[0220] The compound of the present invention represented by general formula (1) is preferably represented by any one of the following general formulas (2-1) to (2-4).

[0221] [Chemical Formula 9]

[0222]

[0223] In the formula, R 45 Indicates alkyl or -(CH2-CH2-O) m -R 21 .

[0224] R 41 ~R 44 and R 81 ~R 86 It represents a hydrogen atom, alkyl group, alkoxy group, aryl group, sulfonyl group, sulfonamide group, nitro group, carboxyl group, amide group, or halogen atom. Adjacent groups can bond with each other to form a fused ring.

[0225] Furthermore, for compounds represented by either general formula (2-1) or (2-4), the classification as compounds represented by general formula (2-4) is preferred. That is, R in general formula (2-1) 43 With R 44 It bonds without forming a benzene ring.

[0226] R 1 ~R 4 R 11 ~R 13 L 1 L 2 and m are respectively related to R in the above general formula (1) 1 ~R 4 R 11 ~R 13 L 1 L 2 The meanings of 'm' and 'm' are the same, and the preferred ranges are also the same.

[0227] R 1 ~R 4 L 1 L 2 or R 45 At least one of them is -(CH2-CH2-O). m -R 21 That is, it refers to R in general formulas (2-1), (2-2), and (2-4). 1 ~R 4 L 1 and L 2 At least one of them is -(CH2-CH2-O). m -R 21 In general formula (2-3), R 1 ~R 4 L 1 and R 45 At least one of them is -(CH2-CH2-O). m -R 21 R21 and m are respectively related to R in the above general formula (1) 21 The meanings of "and m" are the same.

[0228] l represents an integer of 2 or 3.

[0229] Similarly, in compounds represented by any one of the above general formulas (1), R 11 ~R 13 At least one of them contains a carboxyl group or a substituent capable of bonding with biological material.

[0230] Among them, the compound represented by any one of formulas (2-1) to (2-4) is a neutral compound.

[0231] Can be used as R 45 Alkyl groups and -(CH2-CH2-O) m -R 21 With the ability to be used as L 1 or L 2 Alkyl groups and -(CH2-CH2-O) m -R 21 They have the same meaning.

[0232] Can be used as R 41 ~R 44 and R 81 ~R 86 The alkyl, alkoxy, aryl, sulfonyl, sulfonamide, nitro, carboxyl, and halogen atoms in the substituent group T described below have the same meanings as the alkyl, alkoxy, aryl, sulfonyl, sulfonamide, nitro, carboxyl, and halogen atoms in the substituent group T described below.

[0233] Can be used as R 41 ~R 44 and R 81 ~R 86 The amide group can be used as R in the above general formula (1). 11 ~R 13 The amide group is recorded.

[0234] As R 41 ~R 44 Preferably, it is a hydrogen atom, alkyl group, alkoxy group, sulfonyl group, or carboxyl group.

[0235] R 41 ~R 44 Adjacent groups in the ring can connect to each other to form a ring. The formed ring can be a monocyclic ring or a fused ring. The ring is preferably 5 or 6-membered, and can be an aliphatic ring or an aromatic ring. When it is a 6-membered ring, it is preferably an aromatic ring.

[0236] In the case of a fused ring, containing R 41 ~R 44 The bonded benzene ring is preferably a fused ring comprising 2 to 5 rings, more preferably a fused ring comprising 2 to 4 rings, and even more preferably a fused ring comprising 2 or 3 rings.

[0237] As a preferred method for forming the ring, it is in R 41 ~R 44 Adjacent groups in the group are linked together to form at least a benzene ring, i.e., with R 41 ~R 44 The bonded benzene ring corresponds to at least a naphthalene ring structure.

[0238] Among the compounds represented by any one of general formulas (2-1) to (2-3), R is preferred. 41 With R 42 Forming a ring, or R 42 With R 43 Forming a ring, or R 43 With R 44 Forming a ring, more preferably R 41 With R 42 Forming a ring, or R 42 With R 43 A ring is formed.

[0239] By R 41 ~R 44 The bonded benzene ring and R 41 ~R 44 The ring formed by the interconnection of adjacent groups in R can have substituents. Examples of substituents that can be present include those described above that can be used as R. 41 ~R 44 The alkyl, alkoxy, aryl, sulfonyl, sulfonamide, nitro, carboxyl, and halogen atoms are preferred, with alkyl, alkoxy, sulfonyl, or carboxyl groups being preferred.

[0240] Among the compounds represented by any one of general formulas (2-1) to (2-3), from the viewpoint of improving water solubility and inhibiting association, the compound represented by general formula (1) with ring Z is preferred. 1 and ring Z 2 Each of the corresponding rings independently has one or more hydrophilic groups, more preferably in the configuration of ring Z. 1 or ring Z 2 Each of the corresponding rings has at least one hydrophilic group. There is no particular upper limit on the number of possible structures, and it can be appropriately adjusted according to the number of hydrophilic groups as a whole in the compound, as described later.

[0241] There are no particular limitations on the hydrophilic group; examples include alkoxy, carboxyl, sulfonyl, and phosphonyl groups with substituents, with sulfonyl being preferred. These hydrophilic groups can react with cyclo[Z]... 1 or ring Z 2 The corresponding rings can be directly bonded, or they can be bonded via linking groups, but direct bonding is preferred.

[0242] With ring Z 1 or ring Z 2 The position of the hydrophilic group in the corresponding ring is not particularly limited; for example, it is preferable to have at least one hydrophilic group as R. 43 More preferably, R has a hydrophilic group. 43 .

[0243] As R 81 ~R 86 Preferably, the atom is a hydrogen atom, alkyl group, alkoxy group, aryl group, sulfonyl group, or carboxyl group.

[0244] Can be used as R 81 ~R 86 The alkyl, alkoxy, and aryl groups may have substituents. Examples of substituents that may be present include alkyl, alkoxy, sulfonyl, sulfonamide, nitro, carboxyl, and halogen atoms, with alkyl, alkoxy, sulfonyl, or carboxyl being preferred.

[0245] Among the compounds represented by general formulas (2-4), from the viewpoint of improving water solubility and inhibiting association, the compounds represented by general formula (1) with ring Z are preferred. 1 and ring Z 2 Each of the corresponding rings independently has one or more hydrophilic groups, more preferably in the configuration of ring Z. 1 or ring Z 2 Each of the corresponding rings has at least one hydrophilic group. There is no particular upper limit on the number of possible structures, and it can be appropriately adjusted according to the number of hydrophilic groups as a whole in the compound, as described later.

[0246] There are no particular limitations on the hydrophilic group; examples include alkoxy, carboxyl, sulfonyl, and phosphonyl groups with substituents, with sulfonyl being preferred. These hydrophilic groups can react with cyclo[Z]... 1 or ring Z 2 The corresponding rings can be directly bonded, or they can be bonded via linking groups, but direct bonding is preferred.

[0247] With ring Z 1 or ring Z 2 The position of the hydrophilic group in the corresponding ring is not particularly limited; for example, it is preferred to have it at least in R. 83 ~R 85 One of them has a hydrophilic group, more preferably at least as R83 and R 85 Having a hydrophilic group or at least as R 84 Having a hydrophilic group, it is further preferred as R 83 and R 85 Having hydrophilic groups or as R 84 Having hydrophilic groups, especially preferred as R 83 and R 85 It has hydrophilic groups.

[0248] The following are specific examples of compounds of the present invention represented by general formula (1), but the present invention is not limited to these compounds. In the following specific examples, X in -SO3X + Represents hydrogen ions (H) + Sodium ions, potassium ions, ammonium (NH4+) + ) or triethylamine (NH(CH2CH3)3 + ).

[0249] [Chemical Formula 10]

[0250]

[0251] [Chemical Formula 11]

[0252]

[0253] [Chemical Formula 12]

[0254]

[0255] [Chemical Formula 13]

[0256]

[0257] [Chemical Formula 14]

[0258]

[0259] [Chemical Formula 15]

[0260]

[0261] [Chemical Formula 16]

[0262]

[0263] [Chemical Formula 17]

[0264]

[0265] [Chemical Formula 18]

[0266]

[0267] [Chemical Formula 19]

[0268]

[0269] As a first preferred embodiment of the present invention, one can exemplify this by having an excitation absorption wavelength of 740–830 nm in compounds represented by the above general formula (1). In the above R… 11 ~R 13 In this context, the substituent having a carboxyl group or the group having a substituent capable of bonding with biological material is a substituent represented by any one of the above general formulas (I) to (V), preferably a substituent represented by any one of the above general formulas (I), (II) and (IV), and more preferably a substituent represented by the above general formulas (I) or (IV).

[0270] As a first preferred embodiment of the present invention, for example, compounds represented by any one of the above general formulas (2-1) to (2-4) (where 1 = an integer of 3) can be cited. Among the compounds represented by the above general formula (2-1), compounds represented by the above general formula (2-1) can be cited more preferably, compounds in which each substituent in general formula (2-1) satisfies any one of the following embodiments I or II.

[0271] (Method I)

[0272] The above R 11 ~R 13 In this context, the substituents containing carboxyl groups or polysubstituents capable of bonding with biological substances are substituents represented by the above general formulas (I) or (II), L 1 and L 2 For the above -(CH2-CH2-O) m -R 21 Or, it is an alkyl group having at least one of alkoxy, carboxyl, sulfonyl, and phosphonoyl groups as substituents, R 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains the above-mentioned -(CH2-CH2-O) m - represents the structure, R 41 ~R 44 At least one of them (preferably R) 43 () is in the form of sulfonyl.

[0273] In this manner, R, as a substituent in the above general formula (I), 31 ~R 35Among the above-mentioned groups, hydrogen atoms, alkyl groups, alkoxy groups, alkylthio groups, amino groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, cyano groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred; more preferably, hydrogen atoms, alkyl groups, alkoxy groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred. Among the substituents represented by the above general formula (I), from the viewpoint of further improving fluorescence intensity or from the viewpoint of obtaining excellent lightfastness, R is preferred. 31 and R 35 It is an alkyl, alkoxy, or halogen atom. Furthermore, R is particularly preferred. 32 ~R 34 At least one of them has a carboxyl group or a substituent that can bond with biological material.

[0274] In this manner, the substituent represented by the above general formula (II) is preferably any of the substituents represented by formulas (E-4) to (E-8), and more preferably the substituent represented by formula (E-6). The above description applies to the substituents in formulas (E-4) to (E-8).

[0275] (Method II)

[0276] The above R 11 ~R 13 In this context, the substituents containing carboxyl groups or polysubstituents capable of bonding with biological substances are substituents represented by the above general formula (IV), L 1 and L 2 For the above -(CH2-CH2-O) m -R 21 Or it may be an alkyl group having at least one of the following as substituents: alkoxy, carboxyl, sulfonyl, and phosphonoyl, R 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains the above-mentioned -(CH2-CH2-O) m - represents the structure, R 41 ~R 44 At least one of them (preferably R) 43 () is in the form of sulfonyl.

[0277] In this manner, R, as a substituent in the above general formula (IV), 39 ~R 43In the above-mentioned groups, hydrogen atoms, alkyl groups, alkoxy groups, alkylthio groups, amino groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, cyano groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred; more preferably, hydrogen atoms, alkyl groups, alkoxy groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred. Among the substituents represented by the above general formula (IV), from the viewpoint of further improving fluorescence intensity or from the viewpoint of obtaining excellent lightfastness, R is preferred. 39 and R 43 It is an alkyl, alkoxy, or halogen atom. Furthermore, R is particularly preferred. 40 ~R 42 At least one of them has a carboxyl group or a substituent that can bond with biological material.

[0278] As a second preferred embodiment of the present invention, examples include compounds having an excitation absorption wavelength of 620–700 nm, and preferably those represented by any one of the above general formulas (2-1) to (2-4) (where 1 = an integer of 2). In the above R... 11 ~R 13 In this context, the substituent having a carboxyl group or the group having a substituent capable of bonding with biological material is a substituent represented by any one of the above general formulas (I) to (V), preferably a substituent represented by any one of the above general formulas (I), (IV) and (V), and more preferably a substituent represented by the above general formula (IV).

[0279] As the substituent R in the above general formula (IV) 39 ~R 43 In the above-mentioned groups, hydrogen atoms, alkyl groups, alkoxy groups, alkylthio groups, amino groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, cyano groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred; more preferably, hydrogen atoms, alkyl groups, alkoxy groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred. Among the substituents represented by the above general formula (IV), from the viewpoint of further improving fluorescence intensity or from the viewpoint of obtaining excellent lightfastness, R is preferred. 39 and R 43 It is an alkyl, alkoxy, or halogen atom. Furthermore, R is particularly preferred. 40 ~R 42 At least one of them has a carboxyl group or a substituent that can bond with biological material.

[0280] In the second preferred embodiment of the invention, the compound represented by the above general formula (2-1) (where l = an integer of 2) preferably includes L as a substituent other than the substituent. 1 and L 2R is an alkyl group having at least one substituent of an alkoxy group, a carboxyl group, a sulfonyl group, and a phosphonoyl group. 1 and R 2 At least one of them and R 3 and R 4 At least one of them contains the above-mentioned -(CH2-CH2-O) m - represents the structure, R 41 ~R 44 At least one of them (preferably R) 43 () is in the form of sulfonyl.

[0281] As a third preferred embodiment of the present invention, a compound having an excitation absorption wavelength of 620–720 nm, represented by any one of the above general formulas (2-1) to (2-4) (where 1 = an integer of 2), is preferably represented by a compound represented by the above general formula (2-4). 11 ~R 13 In this context, the substituent having a carboxyl group or the group having a substituent capable of bonding with biological material is a substituent represented by any one of the above general formulas (I) to (V), preferably a substituent represented by any one of the above general formulas (I), (IV) and (V), and more preferably a substituent represented by the above general formula (IV).

[0282] As the substituent R in the above general formula (IV) 39 ~R 43 Among the above-mentioned groups, hydrogen atoms, alkyl groups, alkoxy groups, alkylthio groups, amino groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, cyano groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred; more preferably, hydrogen atoms, alkyl groups, alkoxy groups, amide groups, halogen atoms, sulfonyl groups, nitro groups, carboxyl groups, or substituents capable of bonding with biological substances are preferred. From the viewpoint of further improving fluorescence intensity and obtaining excellent lightfastness, R is preferred among the substituents represented by the above general formula (IV). 39 or R 43 At least one of them is an alkyl, alkoxy, or halogen atom, more preferably R. 39 and R 43 It is an alkyl, alkoxy, or halogen atom. Preferably, it is R. 40 ~R 42 At least one of the components has a carboxyl group or a substituent capable of bonding with biological material, particularly preferably R. 41 It has a carboxyl group or a substituent that can bond with biological substances.

[0283] In the compounds represented by the above general formulas (2-4) in the third preferred embodiment of the present invention (where l = an integer of 2), L can preferably be cited as a substituent other than the substituents. 1 and L2 For example, -(CH2-CH2-O) m -R 21 Or, it is an alkyl group having at least one of alkoxy, carboxyl, sulfonyl, and phosphonoyl groups as substituents, R 1 and R 2 At least one of them is related to R 3 and R 4 At least one of them contains the above-mentioned -(CH2-CH2-O) m - represents the structure, R 83 ~R 85 At least one of them (preferably R) 83 and R 85 or R 84 R is preferred. 83 and R 85 () is in the form of sulfonyl.

[0284] The compounds of the present invention represented by general formula (1) can be obtained through R 11 ~R 13 At least one of the substituents has the ability to bond with biological substances, such as proteins, peptides, amino acids, nucleic acids, sugar chains, and lipids, and can be used as a labeling agent for biological substances.

[0285] 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 ester), 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.

[0286] In the compounds of the present invention represented by general formula (1), as in R 11 ~R 13 Specific examples of compounds having at least one substituent capable of bonding with biological material include, for example, the exemplary compounds of labeled biological material described later, in addition to the compounds represented by the general formula (1) of the present invention described above. Furthermore, among the exemplary compounds of the present invention represented by the general formula (1) described above, examples may be given of compounds having substituents capable of bonding with biological material, as shown in the exemplary compounds of labeled biological material described later. 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, may be used with a salt structure by dissociating the hydrogen atoms.

[0287] Regarding the compound represented by general formula (1) of the present invention, the compound structure can be set as specified in general formula (1), and it can also be synthesized using known methods. For example, methods described in Patent Document 1, Patent Document 2, etc. can be cited.

[0288] 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 using known methods. For example, one can refer to Bioconjugate Techniques (Third Edition, by Greg T. Hermanson).

[0289] <<Tagged Biological Material>>

[0290] 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.

[0291] As the aforementioned biological substances, proteins (including 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.

[0292] 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.

[0293] Furthermore, examples include bacteria such as diphtheria bacteria, botulinum toxin, mycoplasma, and spirochetes and their antibodies; protozoa such as Toxoplasma gondii, Trichomonas vaginalis, Leishmaniasis, Trypanosomiasis, 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.

[0294] 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.

[0295] Examples include:

[0296] 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;

[0297] ii) Van der Waals forces between long-chain alkyl groups in compound (1) and lipid double membranes and lipids in biological substances;

[0298] iii) Based on the reaction of NHS ester (N-hydroxysuccinimide ester) in compound (1) with amino groups in biological substances, amide bonds;

[0299] iv) Thioether bonds based on the reaction of the maleimide group in compound (1) with thioalkyl (-SH) groups in biological material; and

[0300] 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.

[0301] 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.

[0302] The following illustrates the compounds of the present invention represented by general formula (1), wherein at least in R 11 ~R 13 The present invention includes specific examples of compounds having substituents capable of bonding with biological material and the labeled biological material obtained by bonding with biological material through interaction therewith, but the present invention is not limited to these compounds, etc. In the following specific examples, regarding groups such as sulfonyl groups having dissociable hydrogen atoms, the hydrogen atoms can be dissociated to adopt a salt structure.

[0303] [Chemical Formula 20]

[0304]

[0305] [Chemical Formula 21]

[0306]

[0307] <Reagents containing labeled biological substances>

[0308] 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.

[0309] 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.

[0310] <Uses of Labeled Biological Substances>

[0311] The labeled biomaterials of the present invention, obtained from compounds represented by general formula (1), exhibit excellent fluorescence intensity and can stably detect fluorescence emitted from 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 for use as fluorescent labeling reagents in multicolor Western blotting or as in vivo fluorescence imaging reagents.

[0312] 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 a compound of the present invention, while fluorescence detection including steps (iv) to (vii) corresponds to the indirect method using a secondary antibody fluorescently labeled with a compound of the present invention.

[0313] (i) Prepare the procedures described in (a) and (b) below respectively.

[0314] (a) A sample containing the target biological substance (hereinafter also referred to as "target biological substance").

[0315] (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").

[0316] (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.

[0317] (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.

[0318] (iv) Prepare the procedures (c) to (e) below respectively.

[0319] (c) Samples containing the target biological material

[0320] (d) Biomaterials capable of binding to the target biomaterials in (c) above (hereinafter also referred to as "primary biomaterials").

[0321] (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.

[0322] (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.

[0323] (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.

[0324] (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.

[0325] Examples of the biomaterials in the labeled biomaterials of the present invention include biomaterials capable of binding to the target biomaterials (primary biomaterials) and biomaterials capable of binding to primary biomaterials (secondary biomaterials). Depending on whether the target biomaterial (biomaterial in the test subject) or the primary biomaterial is appropriately selected, biomaterials capable of specifically binding to the biomaterial in the test subject or the primary biomaterial can be selected.

[0326] As proteins among the aforementioned target biological substances, disease markers can be cited. 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), BCA225 (breast carcinoma-associated antigen), 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.

[0327] The target biological substance mentioned above 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 pose a public health problem.

[0328] 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.

[0329] The target biological substance mentioned above can be a cell, and this cell is not limited to living cells; it can also be a dead cell.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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 (specifically, in the wavelength region of 560–600 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.

[0337] 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 (specifically, in the wavelength region of 620–700 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 in the near-infrared region such as multicolor WB around 700 nm.

[0338] 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 (specifically, in the wavelength region of 740–830 nm). Therefore, the wavelength region of the irradiated light is preferably 700–850 nm, more preferably 730–830 nm. The labeled biomaterial using this compound is suitable for use as a labeled biomaterial that exhibits excellent fluorescence intensity to excitation light sources in the near-infrared region such as multicolor WB around 800 nm.

[0339] As the fluorescence excitation light source used in this invention, there are no particular limitations as long as it emits a light source capable of exciting the labeled biological material of this invention at a wavelength (excitation wavelength). For example, various laser light sources can be used. Furthermore, various filters can be used to obtain a preferred excitation wavelength or to detect fluorescence only.

[0340] 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.

[0341] Furthermore, for the steps other than (i) to (vii) above, the commonly used methods, reagents, apparatus and other conditions can be appropriately selected according to the various methods of using fluorescent labeling.

[0342] For example, using the multicolor WB of the labeled biological material of the present invention, a blot membrane is prepared by methods commonly used for target biological materials (separation of proteins by electrophoresis, blotting of membranes, and membrane agglomeration), and the labeled biological material of the present invention is used as a labeled antibody (preferably a secondary antibody), enabling the detection of target biological materials with excellent fluorescence intensity.

[0343] -Substituted basis set T-

[0344] In this invention, preferred substituents can be selected from the following group of substituents T.

[0345] Furthermore, in this invention, when only substituents are described, referring to the substituent group T, when only individual groups such as alkyl groups are described, it is preferable to apply the corresponding group of the substituent group T.

[0346] 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 applies to groups (alkoxy, alkathio, alkenyl, etc.) containing groups capable of cyclic structures (alkyl, alkenyl, alkynyl, etc.) and compounds containing groups capable of cyclic structures. 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 capable of adopting the structure specifically described below, and is 3 or more, preferably 5 or more.

[0347] 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 to distinguish them.

[0348] The groups included in the substituent group T include the following groups.

[0349] Examples of suitable carbon 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).

[0350] 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 replace the unsubstituted amino group are the same as those corresponding to 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),

[0351] Amino group (preferably with 1 to 20 carbon atoms, for example, acetamido, cyclohexylcarbonylamino, benzoylamino, etc.), amide group (aminocarbonyl), sulfonamide group (which can be any of sulfonamide or aminosulfonamide, preferably with 0 to 20 carbon atoms, preferably alkyl, cycloalkyl, or aryl sulfonamide group), alkylthio group (preferably with 1 to 20 carbon atoms, more preferably with 1 to 12 carbon atoms), cycloalkylthio group (preferably with 3 to 20 carbon atoms), arylthio group (preferably with 6 to 40 carbon atoms, more preferably with 6 to 26 carbon atoms, and even more preferably with 6 to 14 carbon atoms), heterocyclic thio group (preferably with 2 to 20 carbon atoms), alkyl, cycloalkyl, or arylsulfonamide group (preferably with 1 to 20 carbon atoms).

[0352] 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.

[0353] Furthermore, examples can be given that contain carboxyl, phosphono, sulfonyl, ononyl, amino acid residues, polyamino acid residues, or -(CH2-CH2-O). m-alkyl(m with R) 1 ~R 6 The 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.

[0354] 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.

[0355] 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.

[0356] Example

[0357] 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.

[0358] Compounds (1) to (20) represented by the above general formula (1), comparative compounds (1) to (5) and AlexaFluor647 are shown below.

[0359] Compounds (1) to (4) and (17) to (20) are compounds of the first preferred embodiment of the present invention having an excitation absorption wavelength of 740 to 830 nm, compounds (5) to (7) are compounds of the second preferred embodiment of the present invention having an excitation absorption wavelength of 620 to 700 nm, and compounds (8) to (16) are compounds of the third preferred embodiment of the present invention having an excitation absorption wavelength of 620 to 700 nm.

[0360] Furthermore, in the example and comparative compounds, the sulfonyl group represented by -SO3H can be incorporated into a structure containing a portion of a salt structure (e.g., potassium salt, sodium salt, TEA (triethylamine) salt, or DIPEA (N,N-diisopropylethylamine) salt) during purification processes, etc. Also, in the example and comparative compounds, the number added in parentheses enclosing the ethyleneoxy structure refers to the average repetition count. All compounds were synthesized using a compound with an average repetition count whose first decimal place was 0 as a starting material.

[0361] [Chemical Formula 22]

[0362]

[0363] [Chemical Formula 23]

[0364]

[0365] [Chemical Formula 24]

[0366]

[0367] [Chemical Formula 25]

[0368]

[0369] Comparative compound (1) is the compound described in U.S. Patent Application Publication No. 2021 / 0093737.

[0370] Comparative compound (2) is an NHS ester derivative of the compound disclosed in International Publication No. 2009 / 078970.

[0371] Comparative compound (5) is an NHS ester derivative of AlexaFluor647 (product name, product number A33084, manufactured by Thermo Fisher Scientific, Inc.) which is presumed to have the above chemical structure.

[0372] The following describes in detail the synthesis methods of compounds (1) to (16) and comparative compounds (1) to (4) used in each embodiment, but the starting materials, pigment intermediates and synthesis routes are not limited to these.

[0373] In the following synthetic routes, room temperature refers to 25°C.

[0374] Unless otherwise specified, the carrier used in reversed-phase column chromatography was SNAP Ultra C18 (product name, manufactured by Biotage) or Sfar C18 (product name, manufactured by Biotage), and the carrier used in normal-phase column chromatography was Hi-Flash Column (product name, manufactured by YAMAZEN CORPORATION).

[0375] 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".

[0376] The separation and purification HPLC (High Performance Liquid Chromatography) used 2767 (product name, manufactured by Waters Corporation).

[0377] MS spectra were determined using an ACQUITY SQD LC / MS System (manufactured by Waters Corporation, ionization method: ESI (ElectroSpray Ionization)) or an LCMS-2010EV (manufactured by Shimadzu Corporation, ionization method: ESI and APCI (Atmospheric Pressure Chemical Ionization)).

[0378] <Synthesis of Compound (1)>

[0379] Compound (1) was synthesized according to the following scheme.

[0380] [Chemical Formula 26]

[0381]

[0382] 1) Synthesis of compound (1-C)

[0383] 1.9 g of compound (1-A), 20 ml of acetic acid (AcOH), 1.3 g of compound (1-B), and 0.98 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-C) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 35 / 65) to obtain 0.99 g of compound (1-C).

[0384] 2) Synthesis of compound (1-D)

[0385] Compound (1-C) 478 mg, mPEG4-Br 1.08 g, sulfolane 2 mL, and triethylamine (Et3N) 0.132 mL were added to a 50 mL volumetric flask, and the mixture was heated and stirred at 120 °C for 6 hours. Ethyl acetate / hexane (1 / 1) 20 mL was added to the reaction mixture to induce precipitation. The precipitate was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 20 / 100) to obtain compound (1-D) 369 mg.

[0386] 3) Synthesis of compound (1-F)

[0387] 214 mg of compound (1-D), 43 mg of compound (1-E), 25 mg of potassium acetate (AcOK), and 1 mL of acetic anhydride (Ac2O) were added to a test tube, and 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 buffer: acetonitrile / water = 0 / 100 → 25 / 75) to obtain 170 mg of compound (1-F).

[0388] 4) Synthesis of compound (1-H)

[0389] 10 mg of compound (1-F) and 300 μL of distilled water were added to a test tube and stirred at 95 °C. A solution containing 3 mg of compound (1-G) and 5 mg of sodium hydroxide in 200 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 6.0 mg of compound (1-H). The MS determination results of compound (1-H) are as follows.

[0390] MS(ESI m / z): (M+H + ) + =1097, (MH) + ) - =1095

[0391] 5) Synthesis of compound (1)

[0392] 0.30 mL of N,N-dimethylformamide (DMF) and 1 mg of compound (1-I) were dissolved in 3.0 mg of compound (1-H), and the mixture was stirred for 1 hour. Then, the solvent was removed by vacuum distillation, 1.5 mL of ethyl acetate was added to remove the supernatant, and the mixture was dried under vacuum to obtain compound (1).

[0393] <Synthesis of Compound (2)>

[0394] Compound (2) was synthesized in the same manner as compound (1) according to the following scheme. The MS determination results of compound (2-G) are as follows.

[0395] MS(ESI m / z): (M+H + ) + =1313

[0396] [Chemical Formula 27]

[0397]

[0398] In addition, compound (2-C) was synthesized as follows.

[0399] 200 ml of tert-butanol (tBuOH) and 12 g of potassium tert-butoxide (tBuOK) were added to a 500 ml three-necked flask purged with nitrogen. While stirring, 14.4 g of compound (2-A) was added dropwise, and the mixture was stirred briefly. Next, 32.5 g of triethylene glycol 2-bromoethyl methyl ether (Br-mPEG4) was added dropwise, and the mixture was heated and stirred. After stirring at 80 °C for 1 hour, the solvent was removed by vacuum distillation. The crude product was extracted using 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. Then, the solvent was removed by vacuum distillation, and the product was purified by normal-phase column chromatography (eluent: hexane / ethyl acetate = 50 / 50 → 30 / 70) to obtain 11.0 g of compound (2-C).

[0400] <Synthesis of Compound (3)>

[0401] Compound (3) was synthesized in the same manner as compound (1) according to the following scheme. The results of MS determination of compound (3-C) are as follows.

[0402] MS(ESI m / z): (M+H + ) + =1450, (MH) + ) - =1448

[0403] [Chemical Formula 28]

[0404]

[0405] <Synthesis of Compound (4)>

[0406] Compound (4) was synthesized in the same manner as compound (1) according to the following scheme. The results of MS determination of compound (4-F) are as follows.

[0407] MS(ESI m / z): (M+H + ) +=1484, (MH + ) - =1482

[0408] [Chemical Formula 29]

[0409]

[0410] In addition, compound (4-C) was synthesized in the following manner.

[0411] To a 50 mL flask purged with nitrogen, 500 mg of compound (4-A), 1.28 mL of compound (4-B), 0.8 g of sodium methoxide, and 10 mL of methanol were added, and the mixture was heated under reflux for 3 hours. After cooling to room temperature, 0.52 g of compound (4-C) was obtained by filtration.

[0412] <Synthesis of Compound (5)>

[0413] Compound (5) was synthesized according to the following scheme.

[0414] [Chemical Formula 30]

[0415]

[0416] 1) Synthesis of compound (5-B)

[0417] 94 mg of compound (2-E), 34 mg of compound (5-A), 80 mg of potassium acetate (AcOK), and 4.5 mL of acetic anhydride (Ac2O) were added to a 5 mL flask. The mixture was stirred at 60 °C for 2.5 hours under a nitrogen atmosphere. The reaction mixture was then added to ethyl acetate to induce precipitation. The precipitate was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 10 / 90) to obtain 46 mg of compound (5-B). The MS determination results of compound (5-B) are as follows.

[0418] MS(ESI m / z): (M+H + ) + =1190, 1192

[0419] 2) Synthesis of compound (5-D)

[0420] Compound (5-B) 30 mg, compound (5-C) 28 mg, [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct 21 mg, potassium carbonate 35 mg, ethanol 1.26 ml, and distilled water 0.126 ml were added to a test tube. The mixture was heated and stirred at 55 °C for 30 minutes under a nitrogen atmosphere. The solvent was removed by vacuum distillation. Compound (5-D) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 20 / 100) and HPLC, and then freeze-dried to obtain 2.6 mg of compound (5-D). The MS determination results of compound (5-D) are as follows.

[0421] MS(ESI m / z): (M+H + ) + =1291

[0422] 3) Synthesis of compound (5)

[0423] 0.10 ml of N,N-dimethylformamide (DMF), 0.001 ml of triethylamine, and 1 mg of compound (1-I) were dissolved in 0.9 mg of compound (5-D), and the mixture was stirred for 1 hour. Then, the solvent was removed by vacuum distillation, and 1 mL of ethyl acetate / hexane (1 / 1) was added to remove the supernatant. The mixture was then dried under vacuum to obtain compound (5).

[0424] In addition, compound (5-A) was synthesized in the following manner.

[0425] 2.89 g of compound (5-E) and 15 ml of ethanol were added to a 100 ml three-necked flask. After adding 2.05 ml of aniline and 15 ml of ethanol dropwise under ice-cold conditions, the mixture was heated and stirred at 60 °C for 4 hours. After the reaction was complete, the solvent was removed by vacuum distillation until the volume was halved, and then the mixture was transferred to an ice-salt bath and stirred. The resulting solid was filtered and dried to obtain 2.55 g of compound (5-A). The MS analysis results of compound (5-A) are as follows.

[0426] MS(ESI m / z): (M+H + ) + =301, 303

[0427] In addition, compound (5-C) was synthesized as follows.

[0428] To a 50 mL three-necked flask, 316 mg of compound (5-F), 141 mg of tetrahydroxydiborane, 1.5 mg of dichlorobis[di-tert-butyl-(p-dimethylaminophenyl)phosphine]palladium(II), 3.2 mL of tetrahydrofuran, 1.6 mL of methanol, and 0.348 mL of N,N-diisopropylethylamine were added. After heating and stirring at 55 °C for 2 hours, the solvent was removed by vacuum distillation. 3.2 mL of trifluoroacetic acid was added to the residue, and the mixture was stirred at room temperature for 15 minutes. After the reaction was complete, the solvent was removed by vacuum distillation, and the compound (5-C) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 10 / 90) and freeze-dried to obtain 93 mg of compound (5-C). The MS determination results of compound (5-C) are as follows.

[0429] MS(ESI m / z): (M+H + ) + =227, (MH) + ) - =225

[0430] <Synthesis of Compound (6)>

[0431] Compound (6) was synthesized in the same manner as compound (5) according to the following scheme. The MS determination results of compound (6-B) are as follows.

[0432] MS(ESI m / z): (M+H + ) + =1259

[0433] [Chemical Formula 31]

[0434]

[0435] <Synthesis of Compound (7)>

[0436] Compound (7) was synthesized in the same manner as compound (5) according to the following scheme.

[0437] [Chemical Formula 32]

[0438]

[0439] In addition, compound (7-D) was synthesized as follows.

[0440] 500 mg of compound (7-C), 1.20 g of potassium carbonate (K₂CO₃), 0.95 ml of 1,3-propanesulfonyl lactone, 20 ml of N,N-dimethylformamide (DMF), and 10 ml of distilled water were added to a 200 ml g-faced flask. After heating and stirring at 80 °C for 1 hour, 0.57 ml of 1,3-propanesulfonyl lactone was added. After heating and stirring at 80 °C for 1 hour, the solvent was removed by vacuum distillation. 30 ml of ethyl acetate was added to the residue, and the mixture was heated and stirred at 60 °C for 15 minutes, then air-cooled to 0 °C. The solid was filtered and dried under reduced pressure to obtain a white solid. The obtained solid was dissolved in 5 ml of distilled water, and 400 mg of sodium hydroxide (NaOH) was added. After heating and stirring at 80 °C for 1 hour, the mixture was cooled to room temperature, and 1.2 ml of a 30% aqueous hydrogen chloride solution was added. The reaction solution was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 10 / 90) and then freeze-dried to obtain 333 mg of compound (7-D). The MS determination results of compound (7-D) are as follows.

[0441] MS(ESI m / z): (MH + ) - =475, 477

[0442] In addition, compound (7-A) was synthesized as follows.

[0443] In the synthesis of compound (5-C), compound (7-D) was used instead of compound (5-F). Otherwise, a colorless solid compound (7-A) (118 mg, 30% yield) was obtained in the same manner.

[0444] [M+H + ] + :443

[0445] <Synthesis of Compound (8)>

[0446] Compound (8) was synthesized in the same manner as compound (5) according to the following scheme. The MS determination results of compound (8-B) are as follows.

[0447] MS(ESI m / z): (M+H + ) + =1231

[0448] [Chemical Formula 33]

[0449]

[0450] <Synthesis of Compound (9)>

[0451] Compound (9) was synthesized according to the following scheme.

[0452] [Chemical Formula 34]

[0453]

[0454] Synthesis of compound (9-B)

[0455] 2.9 g of compound (9-A) and 15 mL of ethanol were added to a 100 mL flask. Then, 15 mL of ethanol containing 2.1 mL of aniline was added dropwise at 0 °C. The mixture was stirred at 60 °C for 4 hours under a nitrogen atmosphere. The solvent was removed by vacuum distillation to a volume of 15 mL, and the flask was cooled to 0 °C. The precipitated yellow crystals were filtered to obtain 2.6 g of compound (9-B).

[0456] Synthesis of compound (9-E)

[0457] 200 ml of tert-butanol (tBuOH) and 12 g of potassium tert-butoxide (tBuOK) were added to a 500 ml three-necked flask purged with nitrogen. While stirring, 14.4 g of compound (9-C) was added dropwise, and the mixture was stirred briefly. Next, 32.5 g of triethylene glycol 2-bromoethyl methyl ether (Br-mPEG4) was added dropwise, and the mixture was heated and stirred. After stirring at 80 °C for 1 hour, the solvent was removed by vacuum distillation. The crude product was extracted using 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. Then, the solvent was removed by vacuum distillation, and the product was purified by normal-phase column chromatography (elution: hexane / ethyl acetate = 50 / 50 → 30 / 70) to obtain 11.0 g of compound (9-E).

[0458] Synthesis of compound (9-G)

[0459] 100 mg of compound (9-F), 0.3 ml of acetic acid (AcOH), 124 mg of compound (9-E), and 31 mg of potassium acetate (AcOK) were added to a 100 ml three-necked flask. The mixture was stirred at 130 °C for 1 hour under a nitrogen atmosphere. The solvent was removed by vacuum distillation, and the compound (9-G) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 20 / 80) to obtain 58 mg of compound (9-G).

[0460] Synthesis of compound (9-H)

[0461] In a 50 mL flask, 470 mg of compound (9-G), 0.15 mL of 1,3-propanesulfonyl lactone, 2 mL of sulfolane, and 254 mg of potassium acetate (AcOK) were added, and the mixture was heated and stirred at 110 °C for 1 hour. 20 mL of ethyl acetate was added to the reaction mixture to induce precipitation. The precipitate was purified by reversed-phase column chromatography (elution: acetonitrile / water = 0 / 100 → 30 / 70) to obtain 475 mg of compound (9-H).

[0462] Synthesis of compound (9-I)

[0463] 171 mg of compound (9-H), 48 mg of compound (9-B), 71 μL of triethylamine (Et3N), 5 μL of methanol (MeOH), and 0.48 mL of acetic anhydride (Ac2O) were added to a test tube, and the mixture was stirred at 50 °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 150 mg of compound (9-I).

[0464] Synthesis of compound (9-K)

[0465] 10 mg of compound (9-I), 10 mg of potassium carbonate (K₂CO₃), 300 μL of ethanol (EtOH), and 100 μL of distilled water were added to a test tube and stirred at 50 °C. A solution prepared by mixing 6 mg of compound (9-J) and [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (Pd(dppf)Cl₂) in 200 μL of distilled water was added dropwise, and the mixture was stirred at 50 °C for 30 minutes. The reaction solution was cooled to room temperature, purified by HPLC, and freeze-dried to obtain 3.6 mg of compound (9-K). The MS determination results of compound (9-K) are as follows.

[0466] MS(ESI m / z): (M+H + ) + =1491, (MH + ) - =1489

[0467] Synthesis of compound (9)

[0468] 0.30 mL of N,N-dimethylformamide (DMF) and 1 mg of compound (9-L) were dissolved in 3.6 mg of compound (9-K), and the mixture was stirred for 1 hour. Then, the solvent was removed by vacuum distillation, 1.5 mL of ethyl acetate was added to remove the supernatant, and the mixture was dried under vacuum to obtain compound (9).

[0469] <Synthesis of Compound (10)>

[0470] Compound (10) was synthesized in the same manner as compound (9) according to the following scheme. The results of MS determination of compound (10-C) are as follows.

[0471] MS(ESI m / z): (M+H + ) + =1505, (MH) + ) - =1503

[0472] In addition, compound (10-B) was synthesized as follows: 500 mg of compound (10-A), 309 mg of tetrahydroxydiborane, 3 mg of dichlorobis[di-tert-butyl-(4-dimethylaminophenyl)phosphine]palladium(II), 760 μL of N,N-diisopropylethylamine (DIPEA), 5 mL of tetrahydrofuran (THF), and 2.5 mL of methanol (MeOH) were added to a 100 mL three-necked flask, and the mixture was stirred at 55 °C for 3 hours under a nitrogen atmosphere. The solvent was removed by vacuum distillation, and the compound (10-B) was purified by reversed-phase column chromatography (eluent: acetonitrile / water = 0 / 100 → 30 / 70) to obtain 220 mg of compound (10-B).

[0473] [Chemical Formula 35]

[0474]

[0475] <Synthesis of Compound (11)>

[0476] Compound (11) was synthesized in the same manner as compound (10) according to the following scheme. The results of MS determination of compound (11-C) are as follows.

[0477] MS(ESI m / z): (M+H + ) + =1551, (MH + ) - =1549

[0478] [Chemical Formula 36]

[0479]

[0480] <Synthesis of Compound (12)>

[0481] Compound (12) was synthesized in the same manner as compound (10) according to the following scheme. The results of MS determination of compound (12-C) are as follows.

[0482] MS(ESI m / z): (M+H + ) + =1519, (MH + )- =1517

[0483] [Chemical Formula 37]

[0484]

[0485] <Synthesis of Compound (13)>

[0486] Compound (13) was synthesized in the same manner as compound (10) according to the following scheme. The results of MS determination of compound (13-C) are as follows.

[0487] MS(ESI m / z): (M+H + ) + =1767, (MH) + ) - =1765

[0488] [Chemical Formula 38]

[0489]

[0490] <Synthesis of Compound (14)>

[0491] Compound (14) was synthesized in the same manner as compound (10) according to the following scheme. The results of MS determination of compound (14-C) are as follows.

[0492] MS(ESI m / z): (M+H + ) + =1521, (MH) + ) - =1519

[0493] [Chemical Formula 39]

[0494]

[0495] <Synthesis of Compound (15)>

[0496] Compound (15) was synthesized in the same manner as compound (9) according to the following scheme. The results of MS determination of compound (15-K) are as follows.

[0497] MS(ESI m / z): (M+H + ) + =1331, (MH + ) - =1329

[0498] [Chemical Formula 40]

[0499]

[0500] <Synthesis of Compound (16)>

[0501] Compound (16) was synthesized in the same manner as compound (13) according to the following scheme. The MS determination results of compound (16-A) are as follows.

[0502] MS(ESI m / z): (M+H + ) + =1608, (MH) + ) - =1606

[0503] [Chemical Formula 41]

[0504]

[0505] <Synthesis of Compound (17)>

[0506] Synthesis of compound (17)

[0507] 5 mg of compound (2-F), 8 μL of triethylamine (TEA), and 100 μL of distilled water were added to a test tube and stirred at 80 °C. A solution prepared by mixing 2 mg of compound (9-J) and tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4) in 100 μL of distilled water was added dropwise to this solution, and the mixture was stirred at 80 °C for 1 hour. The reaction solution was cooled to room temperature, purified by HPLC, and freeze-dried to obtain 2.8 mg of compound (17). The results of MS determination and absorption fluorescence determination of compound (17) are as follows. Additionally, λ ex This refers to the excitation wavelength, λ em This refers to the fluorescence wavelength, and the following meanings are the same.

[0508] MS(ESI m / z): (M+H + ) + =1297, (MH + ) - =1295

[0509] λ ex =768nm, λ em =787nm

[0510] [Chemical Formula 42]

[0511]

[0512] <Synthesis of Compound (18)>

[0513] Compound (18) was synthesized in the same manner as compound (17) according to the following scheme. The results of MS and absorption fluorescence measurements of compound (18) are described below.

[0514] MS(ESI m / z): (M+H + ) + =1327, (MH) + ) - =1325

[0515] λ ex =765nm, λ em =784nm

[0516] [Chemical Formula 43]

[0517]

[0518] <Synthesis of Compound (19)>

[0519] Compound (19) was synthesized in the same manner as compound (17) according to the following scheme. The results of MS and absorption fluorescence measurements of compound (19) are described below.

[0520] MS(ESI m / z): (M+H + ) + =1325, (MH) + ) - =1323

[0521] λ ex =770nm, λ em =789nm

[0522] [Chemical Formula 44]

[0523]

[0524] <Synthesis of Compound (20)>

[0525] Compound (20) was synthesized in the same manner as compound (17) according to the following scheme. The results of MS and absorption fluorescence measurements of compound (20) are described below.

[0526] MS(ESI m / z): (M+H + ) + =1573, (MH) + ) - =1571

[0527] λ ex =777nm, λ em =794nm

[0528] [Chemical Formula 45]

[0529]

[0530] <Comparative Synthesis of Compound (1)>

[0531] Comparative compound (1) was synthesized in the same manner as compound (1) according to the following scheme. The results of MS determination of compound (c1-C) are as follows.

[0532] MS(ESI m / z): (M+H + ) + =989, (MH + ) - =987

[0533] [Chemical Formula 46]

[0534]

[0535] <Comparative Synthesis of Compound (2)>

[0536] Comparative compound (2) was synthesized in the same manner as compound (1) according to the following scheme. The results of MS determination of compound (c2-C) are as follows.

[0537] MS(ESI m / z): (M+H + ) + =2858, (MH) + ) - =2856

[0538] [Chemical Formula 47]

[0539]

[0540] <Comparative Synthesis of Compound (3)>

[0541] Comparative compound (3) was synthesized in the same manner as compound (1) according to the following scheme. The results of MS determination of compound (c3-D) are as follows.

[0542] MS(ESI m / z): (M+H + ) + =1111, (MH) + ) - =1109

[0543] [Chemical Formula 48]

[0544]

[0545] <Comparative Synthesis of Compound (4)>

[0546] Comparative compound (4) was synthesized in the same manner as compound (8) according to the following protocol. The MS determination results of comparative compound (4-G) are as follows.

[0547] MS(ESI m / z): (M+H + ) + =1565, (MH) + ) - =1563

[0548] [Chemical Formula 49]

[0549]

[0550] <Example 1: Evaluation at an excitation wavelength of 785nm>

[0551] The fluorescence labeling rate, solution fluorescence intensity, and membrane fluorescence intensity of the above compounds (1) to (4) and comparative compounds (1) to (3) were evaluated.

[0552] [1] Evaluation of fluorescent labeling rate

[0553] 217 μL of anti-rabbit IgG antibody (2.3 mg / ml) and 21.7 μL of carbonate buffer were added to a microtube and shaken. Then, a dimethyl sulfoxide solution of compound (1) was added in the same molar ratio as shown in Table 1, and the mixture was shaken again. After standing at room temperature for 1 hour, the reaction solution was purified using gel filtration column chromatography (PD10, manufactured by GE Healthcare Life Sciences) and PBS (phosphate-buffered saline) to obtain labeled anti-rabbit IgG antibodies using compound (1). Labeled anti-rabbit IgG antibodies using each compound or a comparative compound were obtained in the same manner. The fluorescence labeling rate (DOL) of the obtained labeled antibodies was calculated using the method shown below. The results are summarized in Table 1.

[0554] The fluorescent labeling rate was calculated using the standard method shown below. [] indicates units, and [-] indicates no units. In this experiment, protein refers to anti-rabbit IgG antibody.

[0555] Fluorescent labeling rate = Concentration of fluorescent dye / Concentration of protein

[0556] The concentration of fluorescent dye refers to the total molar concentration [M] of the labeled fluorescent dye, and the concentration of protein refers to the molar concentration [M] of the fluorescently labeled protein, which are calculated using the following formulas respectively.

[0557] Fluorescent dye concentration = Dye max / ε dye

[0558] Protein concentration = (IgG) 280 -(Dye max ×CF)) / εprotein

[0559] The symbols in the above formula are described below.

[0560] Dye max Absorption of fluorescent dyes at their maximum absorption wavelength [-]

[0561] ε dye Molar absorptivity of fluorescent dyes [M] -1 cm -1 ]

[0562] IgG 280 Absorption of fluorescently labeled proteins at 280 nm [-]

[0563] Dye 280 Absorption of fluorescent dye at 280 nm [-]

[0564] ε protein ; Molar absorptivity of protein [M] -1 cm -1 ]

[0565] CF (Correction Factor); Dye 280 / Dye max [-]

[0566] [Table 1]

[0567] No. Labeled antibodies 3 equivalent 5 equivalent 7 equivalent 10 equivalent 101 Compound (1)-IgG 2.3 3.6 4.5 6.2 102 Compound (2)-IgG 2.2 3.5 4.5 6.1 103 Compound (3)-IgG 2.3 3.7 4.4 6.5 104 Compound (4)-IgG 2.2 3.4 4.3 6.0 c11 Comparison of compound (1)-IgG 2.2 3.2 4.2 5.9 c12 Comparison of compound (2)-IgG 1.2 1.8 2.4 3.3 c13 Comparison of compound (3)-IgG 2.3 3.3 4.2 6.1

[0568] (Table Notes)

[0569] In the labeled antibody column, the descriptions of Compound (Z)-IgG or Comparative Compound (Z)-IgG refer to labeled anti-rabbit IgG antibodies labeled with Compound (Z) or labeled anti-rabbit IgG antibodies labeled with Comparative Compound (Z), respectively. Z refers to the compound number. The same meaning applies in subsequent tables.

[0570] The following information can be obtained from the results in Table 1 above.

[0571] When compounds (1) to (4), which are compounds of the present invention, are added in any molar equivalent of 3, 5, 7, and 10 equivalents relative to 1 equivalent of antibody, they also show the same level of fluorescence labeling as when using the comparative compound (1) which is the labeling compound described in prior art document 1, and exceed the fluorescence labeling rate when using the comparative compound (2) which is the labeling compound described in prior art document 2. As for the binding to the antibody, they show a sufficient level of practically no problem. This can be seen from the comparison between No. c11 or c12 and No. 101 to 104.

[0572] On the other hand, L in the general formula (1) of the present invention 1 Considering the presence of substituents capable of binding to biological substances, comparative compound (3) is not a compound specified in this invention. The compounds of this invention, namely compounds (1) to (4) and comparative compound (3), showed the same level of fluorescence labeling rate, and no difference in fluorescence labeling rate was found.

[0573] [2] Evaluation of solution fluorescence intensity

[0574] The solutions of each labeled antibody prepared in the evaluation of the fluorescent labeling rate in [1] were adjusted to a protein concentration of 0.005 mg / mL. Using a spectrophotometer (product name: RF-5300 or RF-6000, manufactured by Shimadzu Corporation), under uniform exposure conditions with excitation light of 785 nm, the integral value of fluorescence intensity in the range of 810–840 nm was calculated. Using the integral value of fluorescence intensity in the range of 810–840 nm of DOL2.2 (with 3 equivalents of dye) of compound (1)-IgG as a benchmark, the ratio (integral value of fluorescence intensity in the range of 810–840 nm of labeled antibody / benchmark value) was calculated, and the evaluation was carried out according to the following evaluation criteria. The results are summarized in Table 2.

[0575] Evaluation Criteria for Fluorescence Intensity (Integral Value)-

[0576] A: The ratio of fluorescence intensity to the baseline value is 2.0 times or more.

[0577] B: The ratio of fluorescence intensity to the reference value is greater than 1.7 and less than 2.0.

[0578] C: The ratio of fluorescence intensity to the reference value is greater than 1.4 and less than 1.7.

[0579] D: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 1.4.

[0580] E: The ratio of fluorescence intensity to the reference value is greater than 1.1 and less than 1.2.

[0581] F: The ratio of fluorescence intensity to the reference value is greater than 0.9 and less than 1.1.

[0582] G: The ratio of fluorescence intensity to the reference value is less than 0.9 times.

[0583] [Table 2]

[0584]

[0585] The following information can be obtained from the results in Table 2 above.

[0586] From the perspective of not having a structure containing a PEG group, comparative compound (1) is not the structure specified in this invention. The fluorescence intensity of the labeled antibody labeled with this comparative compound (1) in solution is low in any DOL (No. c11).

[0587] Considering that the number of PEG repeats (m as specified in this invention) is 24, the number of PEG repeats in comparative compound (2) is longer than the number of PEG repeats specified in this invention. The fluorescence intensity of the labeled antibody using this comparative compound (2) in solution is lower than that of the labeled antibodies (No. c12) labeled with compounds (1) to (4) with the same DOL.

[0588] From general formula (1) not R 11 ~R 13 L 1 Considering the presence of a carboxyl group or substituents capable of binding to biological substances, the comparative compound (3) is not the structure specified in this invention. The fluorescence intensity of the labeled antibody labeled with the comparative compound (3) in solution was low in any DOL (No. c13).

[0589] In contrast, the labeled antibodies using compounds (1) to (4) which are compounds of the present invention all showed significantly higher fluorescence intensity (relative to No. 101 to 104 of No. c11) than the labeled antibodies using the aforementioned comparative compound (1).

[0590] Furthermore, in the labeled antibodies using compounds (1) to (4) which are compounds of the present invention, it was also found that setting the position of the structure containing the PEG group as R... 1 and R 2 At least one of them and R 3 and R 4 At least one of the labeled antibodies using compounds (2) to (4) of the present invention showed superior fluorescence intensity, and the labeled antibodies using compounds (3) and (4) of the present invention, having a structure that also contains two PEG groups in the compound, showed even superior fluorescence intensity.

[0591] Furthermore, comparing No. 101 of compound (1) using 4 repeats of PEG, as shown in Tables 1 and 2, with No. c12 of comparative compound (2) using only a structurally different compound in that the repeat number of PEG is 24, it was found that by setting the repeat number of PEG within the range specified in this invention, antibodies can be adequately labeled with a smaller amount of fluorescent compound used (Table 1). Moreover, by comparing solutions of labeled antibodies showing the same degree of DOL, it was also found that by setting the repeat number of PEG within the range specified in this invention, superior fluorescence intensity can be achieved (Table 2).

[0592] [3] Fluorescence intensity on the membrane

[0593] Rabbit IgG solution was adjusted 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 (Tris Buffered Saline with Tween 20) with 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 (the labeled antibody solution prepared above [1], with a concentration of 1 mg·mL before dilution) in PBS was diluted 20,000 times with TBS buffer. The membrane was immersed in the diluted solution and incubated with stirring for 1 hour. The membrane was washed 3 times with TBS-T buffer for 10 minutes each time, and finally washed with TBS buffer 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 (manufactured by GEHC) with uniform exposure conditions of 785 nm excitation light, and the 6.48 mm diameter was measured. 2 The fluorescence intensity of the component in the fluorescence wavelength range of 810–840 nm was used as the signal fluorescence intensity. The integral value of the signal fluorescence intensity of compound (1)-IgG DOL2.2 in the fluorescence wavelength range of 810–840 nm was used as a benchmark. The ratio to this benchmark value (integral value of the signal 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 3.

[0594] Evaluation Criteria for Fluorescence Intensity (Integral Value)-

[0595] A: The ratio of the signal fluorescence intensity to the reference value is greater than 2.0 times.

[0596] B: The ratio of the signal fluorescence intensity to the reference value is greater than 1.7 and less than 2.0.

[0597] C: The ratio of the signal fluorescence intensity to the reference value is greater than 1.4 and less than 1.7.

[0598] D: The ratio of the signal fluorescence intensity to the reference value is greater than 1.2 and less than 1.4.

[0599] E: The ratio of the signal fluorescence intensity to the reference value is greater than 1.1 and less than 1.2.

[0600] F: The ratio of the signal fluorescence intensity to the reference value is greater than 0.9 and less than 1.1.

[0601] G: The ratio of the signal fluorescence intensity to the reference value is less than 0.9 times.

[0602] [Table 3]

[0603]

[0604] The fluorescence intensity of the labeled antibody on the membrane shown in Table 3 above was obtained in the same way as the fluorescence intensity of the labeled antibody in the solution shown in Table 2 above.

[0605] <Example 2: Evaluation at an excitation wavelength of 633nm>

[0606] The fluorescence intensity in stained cells was evaluated for each of the compounds (5) to (8) and the comparative compound (5). Furthermore, the fluorescence quantum yield and photostability of the compounds and labeled antibodies were evaluated.

[0607] [4] Evaluation of fluorescence intensity of labeled antibodies in stained cells

[0608] In the preparation of each labeled antibody solution in the fluorescence labeling rate evaluation above [1], the compounds used in the labeling of the antibody and the molar equivalent ratio of the compounds relative to 1 antibody equivalent were changed as shown in Table 4 below. Otherwise, each labeled antibody solution was prepared in the same manner.

[0609] Cell staining was performed using the labeled antibody synthesized above and a comparative labeled antibody, as follows. The characteristics of the stained cells were evaluated. The results are summarized in Table 4.

[0610] [Preparation of stained cell samples]

[0611] HeLa cells (European Collection of Authenticated Cell Cultures) were seeded into 96-well plates (Thermo Fisher Scientific, Inc.) and cultured for 20 hours in D-MEM medium (all from FUJIFILM WakoPure Chemical Corporation) containing 10% fetal bovine serum, 1% penicilline / streptomycin, and 1% MEM non-essential amino acids.

[0612] Next, the culture medium was removed, and the cells were immobilized by treating with methanol at -20°C for 5 minutes. After immobilization, the cells were washed with PBS (Thermo Fisher Scientific, Inc.), and a PBS solution containing 0.2% Triton X-100 (polyethylene glycol monooctylphenyl ether) (Aldrich) and 2% BSA (bovine serum albumin) (Biological Industries) was added to prevent clumping and membrane permeability, and the solution was incubated for 1 hour. After removing clumping and membrane permeability, anti-α-tubulin antibody (rabbit polyclonal antibody, GeneTex) dilution was added as the primary antibody, and the solution was incubated at 4°C for 15 hours at a final antibody concentration of 1 μg / mL. After washing with PBST, 2 μg / mL of the above-prepared labeled antibody aqueous solution was added as the secondary antibody, and the solution was incubated at room temperature for 1 hour while protected from light, followed by washing with PBST again. After further washing with PBS, one drop of Prolong Gold (Thermo Fisher Scientific, Inc.) was added to each well using a syringe as an anti-fading agent to obtain stained cell samples. In addition, the fluorescence intensity evaluation described below used freshly prepared stained cell samples.

[0613] [Evaluation of fluorescence intensity in stained cells]

[0614] The fluorescence intensity of the stained cells was measured using the EnSight microplate reader manufactured by PerkinElmer under the following conditions.

[0615] (Measurement conditions) Well scanning mode, excitation wavelength 633nm, fluorescence wavelength 670-720nm (data interval 5nm), bandwidth 8nm, 100 cumulative measurements, 3×3 points, measurement height 3mm, point spacing 1mm)

[0616] The sum (integral value) of fluorescence intensities in the fluorescence wavelength range of 670–720 nm was set as the fluorescence intensity of the sample. Using the integral value of the fluorescence intensity in the fluorescence wavelength range of 670–720 nm of compound (5)-IgG DOL3.7 as a benchmark, the ratio to this benchmark (integral value of fluorescence intensity in the fluorescence wavelength range of 670–720 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 4.

[0617] Evaluation Criteria for Fluorescence Intensity (Integral Value)-

[0618] A: The ratio of fluorescence intensity to the baseline value is more than 2.5 times.

[0619] B: The ratio of fluorescence intensity to the reference value is greater than 2.0 and less than 2.5.

[0620] C: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 2.0.

[0621] D: The ratio of fluorescence intensity to the reference value is greater than 1.0 and less than 1.2.

[0622] E: The ratio of fluorescence intensity to the reference value is less than 1.0 times.

[0623] [Table 4]

[0624]

[0625] The following information can be obtained from the results in Table 4 above.

[0626] Compounds (5) to (8), which are compounds of the present invention, exhibit a fluorescence labeling rate (DOL) of 4.7 to 8.2, and show a sufficient level of practically no problem in terms of binding to antibodies.

[0627] From general formula (1) not R 11 ~R 13 R 3 It has a carboxyl group or a substituent that can bond with biological substances, but does not have a -(CH2-CH2-O) group. m -R 21 Considering the structural aspects, the comparative compound (5) does not have the structure specified in this invention.

[0628] The labeled antibodies labeled with compounds (5) to (8), which are compounds of the present invention, all showed significantly higher fluorescence intensity (relative to No. 105 to 108 of No. c15) than the labeled antibody using the above-mentioned comparative compound (5).

[0629] [5] Evaluation of fluorescence quantum yield

[0630] The PBS solution (pH 7.4) containing the above-mentioned compounds or labeled antibodies was evaluated using the methods described in the following references.

[0631] "A Guide to Recording Fluorescence Quantum Yields" (from HORIBA Scientific, available at https: / / www.horiba.com / fileadmin / uploads / Scientific / Documents / Fluorescence / quantumyieldstrad.pdf)

[0632] For fluorescence quantum yield, the higher the evaluation level, the better the fluorescence quantum yield, and therefore the more desirable it is.

[0633] - Evaluation Criteria for Fluorescence Quantum Yield -

[0634] A: 0.5 or more

[0635] B: 0.4 or higher and less than 0.5

[0636] C: 0.3 or higher and less than 0.4

[0637] D: 0.2 or higher and less than 0.3

[0638] E: Less than 0.2

[0639] [6] Evaluation of lightfastness

[0640] The synthesized compounds or labeled antibodies described above were dissolved in PBS solution (pH 7.4) with absorbance at the absorption wavelength peak of 0.095–0.105. A carousel light irradiation machine (Ushio Inc.) with a xenon lamp UXL-500D-O, HA-50 filter, Y44 filter, and an exposure intensity of 22 mW / cm² was used. 2 [500nm conversion] While the solution was exposed, the absorbance of the absorption wavelength peaks of each compound or labeled antibody was measured over time using a spectrometer (manufactured by Agilent Technologies, Inc., Agilent 8453). The absorbance of the absorption wavelength peak before exposure was set to 100%, and the exposure time until the absorbance of that absorption wavelength peak first decreased by 50% (the absorbance of the absorption wavelength peak reached 50%) was calculated. Evaluation was conducted based on the following criteria.

[0641] Since a higher evaluation level results in a longer period of stability, it is preferred.

[0642] - Evaluation Criteria for Lightfastness -

[0643] A: More than 40 hours

[0644] B: More than 30 hours but less than 40 hours

[0645] C: More than 20 hours but less than 30 hours

[0646] D: 10 hours or more but less than 20 hours

[0647] E: Less than 10 hours

[0648] [Table 5]

[0649]

[0650] (Table Notes)

[0651] As mentioned above, the comparative compound (5)-IgG refers to the labeled antibody made using the NHS ester of AlexaFluor647.

[0652] From the results in Tables 4 and 5 above, it was found that the compounds of the second preferred embodiment of the present invention and their labeled antibodies, which have an excitation absorption wavelength of 620-700 nm, not only exhibit excellent fluorescence intensity of the obtained labeled biomaterials, but also show excellent fluorescence quantum yield, and have better photostability compared with commercially available fluorescent compounds such as AlexaFluor647 and its labeled antibodies.

[0653] <Example 3: Evaluation at an excitation wavelength of 685nm>

[0654] The fluorescence intensity in solution and on the membrane of the above compounds (9) to (13) and comparative compound (4) were evaluated.

[0655] [7] Evaluation of solution fluorescence intensity

[0656] In the preparation of each labeled antibody solution in the fluorescence labeling rate evaluation above [1], the compounds used in the antibody labeling and the molar equivalent ratio of the compounds relative to 1 antibody equivalent were changed as shown in Table 6 below. Otherwise, each labeled antibody solution was prepared in the same manner. The obtained labeled antibody solution was adjusted to a protein concentration of 0.005 mg / mL, and the integral value of fluorescence intensity in the fluorescence wavelength range of 710–730 nm was calculated using a spectrophotometer (product name: RF-6000, manufactured by Shimadzu Corporation) under uniform exposure conditions with excitation light of 685 nm. The integral value of fluorescence intensity in the fluorescence wavelength range of 710–730 nm of the comparative compound (4)-IgG DOL3.4 (with 10 equivalents of pigment) was used as a reference value. The ratio of the integral value of fluorescence intensity in the fluorescence wavelength range of 710–730 nm of the labeled antibody to the reference value was calculated, and the evaluation was carried out according to the following evaluation criteria. The results are summarized in Table 6.

[0657] Evaluation Criteria for Fluorescence Intensity (Integral Value)-

[0658] A: The ratio of fluorescence intensity to the baseline value is 2.0 times or more.

[0659] B: The ratio of fluorescence intensity to the reference value is greater than 1.7 and less than 2.0.

[0660] C: The ratio of fluorescence intensity to the reference value is greater than 1.4 and less than 1.7.

[0661] D: The ratio of fluorescence intensity to the reference value is greater than 1.2 and less than 1.4.

[0662] E: The ratio of fluorescence intensity to the reference value is greater than 1.1 and less than 1.2.

[0663] F: The ratio of fluorescence intensity to the reference value is greater than 0.9 and less than 1.1.

[0664] G: The ratio of fluorescence intensity to the reference value is less than 0.9 times.

[0665] [Table 6]

[0666]

[0667] The following information can be obtained from the results in Table 6 above.

[0668] Compounds (9) to (13), which are compounds of the present invention, exhibit a fluorescence labeling rate (DOL) of 2.1 to 7.8, and show a sufficient level of practically no problem in terms of binding to antibodies.

[0669] From general formula (1) not R 11~R 13 L 1 Considering the presence of a carboxyl group or substituents capable of binding to biological substances, the comparative compound (4) is not the structure specified in this invention. The fluorescence intensity of the labeled antibody using the comparative compound (4) in solution was low in any DOL (No. c14).

[0670] In contrast, the labeled antibodies of compounds (9) to (13) of the present invention all showed significantly higher fluorescence intensity than the labeled antibody using the above-mentioned comparative compound (4) (compared to No. 109 to 113 of No. c14).

[0671] Furthermore, in R with substituents capable of binding with biological substances 13 In the labeled antibodies of the compounds (9) to (13) of the present invention, which are groups represented by general formula (IV), it was also found that R in general formula (IV) is present. 39 or R 43 Compounds (10) to (13) of the present invention having at least one substituent exhibit superior fluorescence intensity in R 39 and R 43 The compounds of the present invention (11) to (13) having substituents on both of these exhibit further superior fluorescence intensity.

[0672] [8] Evaluation of immunoblotting fluorescence intensity

[0673] Transferrin (manufactured by Merck KGaA) was diluted with Fluorescent Compatible Sample Buffer (4X, non-reducing) (manufactured by Thermo Fisher Scientific, Inc.) to 1 ng / μL, 0.3 ng / μL, and 0.1 ng / μL, and heated at 95°C for 5 minutes. The aforementioned transferrin sample was loaded onto Novex 4-20% Tris-Glycine Mini Gels (manufactured by Thermo Fisher Scientific, Inc.) and PageRuler Prestained NIR Protein Ladder (manufactured by Thermo Fisher Scientific, Inc.), and electrophoresis was performed at a constant voltage of 225V for 45 minutes. The electrophoresed gel was then stacked on a nitrocellulose membrane (manufactured by Cytiva), and protein transfer was performed at a constant voltage of 20V for 1 hour. The membrane was then immersed in Western Blot Blocking Buffer (FishGelatin) (manufactured by Takara Bio Inc.) and incubated at 4°C for 12 hours. After washing the membrane with TBS-T buffer, it was immersed in a liquid containing the primary antibody for Transferrin (manufactured by Dako) (5000-fold dilution), and agitated for 1 hour. The membrane was then washed with TBS-T buffer again. A liquid containing comparative compound (4)-IgG (25000-fold dilution) was prepared, and the membrane was immersed in it and agitated for 1 hour under light-protected conditions. After agitation, it was washed with TBS-T buffer. The membrane was dried in a 40°C incubator for 1 hour in the dark. Imaging was performed using an Amersham Typhoon scanner (manufactured by Cytiva), and measurements were taken at 3.24 mm under uniform conditions with an excitation light of 685 nm. 2 The fluorescence intensity of the component in the fluorescence wavelength range of 710–730 nm was used as the signal fluorescence intensity. The fluorescence intensity of the labeled antibodies for other compounds was determined in the same manner as described above.

[0674] Using the integral value of the fluorescence intensity of the labeled antibody in the fluorescence wavelength range of 710–730 nm as a baseline, the ratio to this baseline (integral value of the fluorescence intensity of the labeled antibody in the fluorescence wavelength range of 710–730 nm / baseline value) was calculated, and the results were evaluated according to the following evaluation criteria. The results are summarized in Table 7.

[0675] Evaluation Criteria for Fluorescence Intensity (Integral Value)-

[0676] A: The ratio of the signal fluorescence intensity to the reference value is 3.0 times or more.

[0677] B: The ratio of the signal fluorescence intensity to the reference value is greater than 2.5 and less than 3.0.

[0678] C: The ratio of the signal fluorescence intensity to the reference value is greater than 2.0 and less than 2.5.

[0679] D: The ratio of the signal fluorescence intensity to the reference value is greater than 1.5 and less than 2.0.

[0680] E: The ratio of the signal fluorescence intensity to the reference value is greater than 1.1 and less than 1.5.

[0681] F: The ratio of the signal fluorescence intensity to the reference value is greater than 0.9 and less than 1.1.

[0682] G: The ratio of the signal fluorescence intensity to the reference value is less than 0.9 times.

[0683] [Table 7]

[0684]

[0685] The fluorescence intensity in the immunoblots of the labeled antibodies shown in Table 7 above was obtained in the same way as the fluorescence intensity in the solution of the labeled antibodies shown in Table 7 above.

[0686] <Example 4>

[0687] The lightfastness of the above compounds (19) and (20) as well as the commercially available product (Alexa Fluor 750) was evaluated.

[0688] [9] Evaluation of lightfastness

[0689] The compounds synthesized above or commercially available products were dissolved in PBS solution (pH 7.4) with an absorbance of 0.095–0.105 at the absorption wavelength peak. A carousel light irradiation machine (Ushio Inc.) with a xenon lamp UXL-500D-O, HA-50 filter, Y44 filter, and an exposure intensity of 22 mW / cm² was used. 2 [500nm conversion] While the solution was exposed, the absorbance of the absorption wavelength peaks of each compound or commercially available product was measured over time using a spectrometer (manufactured by Agilent Technologies, Inc., Agilent 8453). The absorbance of the absorption wavelength peak before exposure was set to 100%, and the exposure time until the absorbance of that absorption wavelength peak first decreased by 50% (the absorbance of the absorption wavelength peak reached 50%) was calculated. Evaluation was conducted based on the following criteria.

[0690] Since a higher evaluation level results in a longer period of stability, it is preferred.

[0691] - Evaluation Criteria for Lightfastness -

[0692] A: More than 9 hours

[0693] B: 7 hours or more but less than 9 hours

[0694] C: More than 5 hours but less than 7 hours

[0695] D: More than 3 hours but less than 5 hours

[0696] E: Less than 3 hours

[0697] [Table 8]

[0698] No. Fluorescent compounds Lightfastness 119 Compound (19) A 120 Compound (20) A c15 Alexa Fluor 750 E

[0699] (Table Notes)

[0700] Alexa Fluor 750: Alexa Fluor 750 (product name, manufactured by Thermo Fisher Scientific, Inc.). It is a type of λ... ex =749nm, λ em Fluorescent pigments with a wavelength of 775 nm are known for their excellent lightfastness.

[0701] From the results in Table 8 above, it was found that the compound of the first preferred embodiment of the present invention, which has an excitation absorption wavelength of 740–830 nm, exhibits superior lightfastness compared to Alexa Fluor 750, a commercially available fluorescent compound.

[0702] Thus, the labeled biological material using the compounds of the present invention exhibits excellent fluorescence intensity in at least one of the following modes: solution, membrane, stained cells, and immunoblotting.

[0703] 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.

[0704] This application claims priority based on Japanese Patent Application No. 2021-101305 filed in Japan on June 18, 2021 and Japanese Patent Application No. 2022-044427 filed in Japan on March 18, 2022, the contents of which are incorporated herein by reference as a part of this specification.

Claims

1. A compound represented by the following general formula (2-1) or (2-4), In the above formula, R 1 With R 2 One of them represents an alkyl group with 1 carbon atom, and the other represents -(CH2-CH2-O). m -R 21 R 3 With R 4 One of them represents an alkyl group with 1 carbon atom, and the other represents -(CH2-CH2-O). m -R 21 m is 4, R 21 Indicates an alkyl group with 1 carbon atom. R 11 R represents a hydrogen atom. 12 Represents a hydrogen atom, general formula (I), general formula (IV), or general formula (E-6), R 13 Represents a hydrogen atom, general formula (I) or general formula (IV), or adjacent R. 13 They bond together to form 5-membered or 6-membered rings, where... R 12 ~R 13 At least one of them contains a carboxyl group or a substituent capable of bonding with biological material. R 43 R represents sulfonyl. 83 R 85 and R 84 Indicates a hydrogen atom or a sulfonyl group. R 41 R 42 R 44 R 81 R 82 and R 86 Represents a hydrogen atom. L 1 and L 2 This indicates an alkyl group with 3 carbon atoms and a sulfonyl group as a substituent, or -(CH2-CH2-O). m -R 21 R 21 and m are respectively related to the above R 21 The meaning of 'm' is the same as 'm'. l represents an integer of 2 or 3. Among them, the compounds represented by formula (2-1) or (2-4) are neutral compounds. The R 12 ~R 13 The substituents containing a carboxyl group or substituents capable of bonding with biological substances are substituents represented by any one of the following general formulas (I), (IV), and (E-6). In the above general formula (I), R 31 R 32 R 34 and R 35 R represents a hydrogen atom. 33 A carboxyl group or a substituent that can bond with biological substances. In the above general formula (IV), R 39 and R 43 R represents a hydrogen atom, an alkyl group with one carbon atom, or an alkoxy group with three carbon atoms having a sulfonyl substituent. 40 ~R 42 Substituents that represent hydrogen atoms, carboxyl groups, or substances capable of bonding with biological matter. In the above general formula (E-6), R 65 With R 66 One of them represents a carboxyl group or a substituent that can bond with biological substances, and the other represents a hydrogen atom. * indicates a bond. Wherein, the group represented by any one of general formulas (I), (IV), and (E-6) has at least one carboxyl group or a substituent capable of bonding with biological material. The substituents that can bond with biological substances are selected from the group consisting of NHS ester structure, succinimide structure, maleimide structure, azide group, ethynyl group, peptide structure, long-chain alkyl group, and quaternary ammonium group.

2. The compound according to claim 1, wherein, The compound contains four groups consisting of -(CH2-CH2-O). m - indicates a structure where m has the same meaning as the m mentioned above.

3. The compound according to claim 1, wherein, The R 12 ~R 13 Substituents having a carboxyl group or substituents capable of bonding with biological material are substituents represented by the general formula (IV).

4. The compound according to claim 1, wherein, The compounds are selected from the group consisting of the following compounds (2) to (13), (19), and (20): 。 5. A biolabeled substance, which is formed by bonding a biolabeled substance with a compound according to any one of claims 1 to 4.

6. The labeled biological material according to claim 5, wherein, The biological substance is any one of protein, amino acid, nucleic acid, sugar chain and phospholipid.

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