JP2020050610A - Bioimaging agent - Google Patents

Abstract

To provide a bioimaging agent that is usable in imaging technology using light in the near-infrared region, shows little toxicity to living organisms and emits fluorescence with high intensity.SOLUTION: The bioimaging agent comprises a compound represented by the general formula 1 in the figure or the like. (In the formula, Rrepresents a part of a polymethine chain conjugated to the aromatic rings on both ends and interposed therebetween; Rto Rrepresent H or an arbitrary organic group; and Xrepresents an arbitrary counter anion; provided that a halogen atom is not directly bonded to the meso position of the polymethine chain.)SELECTED DRAWING: None

Description

  The present invention relates to a bioimaging agent.

  As a method of noninvasively imaging a living body, X-ray CT (Computed Tomography), MRI (Magnetic Resonance Imaging), and PET (Positron Emission Tomography) are generally used. 2. Description of the Related Art In recent years, fluorescent imaging technology has attracted attention as a technology for imaging a living body simply and with high sensitivity.

  In the fluorescence imaging technique, first, a dye probe is administered in a living body. Next, the living body is irradiated with light, and the light causes the dye probe to emit fluorescence. Finally, a living body (for example, a specific organ in the living body) is imaged by sensing the fluorescence.

  Conventionally, visible light has been used as light applied to a living body. However, since it is difficult to irradiate the deep part of the living body with the visible light, the fluorescent imaging technique using the visible light is difficult to (i) image the deep part of the living body, and (ii) obtained. There is a problem that an image contains a lot of noise.

  In recent years, attention has been focused on a fluorescent imaging technique using light in the near-infrared region in order to solve the problem of the fluorescent imaging technique using visible light (for example, see Non-Patent Document 1). Since light in the near-infrared region can be applied to a deep part of a living body, it is expected that the above-mentioned problems (i) and (ii) can be solved.

Yuyan Jiang et al., “Molecular Fluorescence and PhotoacousticImaging in the Second Near-Infrared Optical Window Using Organic Contrast Agents”, ADVANCED BIOSYSTEMS, 2018, 2, 1700262

  However, in an imaging technique using light in the near-infrared region, development of a dye probe having low toxicity to a living body and high intensity of emitted fluorescence has not been advanced.

  An object of one embodiment of the present invention is to provide a bioimaging agent that can be used for an imaging technique using light in a near-infrared region, has low toxicity to a living body, and has a high intensity of emitted fluorescence. .

  [1] In order to solve the above problems, a bioimaging agent according to one embodiment of the present invention is characterized by containing a compound represented by the following general formula 1 or general formula 2:

(In the general formula 1, R ₁ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{2 to} R ₅ represent H or any organic group, and X ⁻ represents Any counter anion is shown, provided that a halogen atom is not directly bonded to the meso position (specifically, the central carbon) of the polymethine chain.

(In the general formula 2, R ₁₀ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{11 to} R ₁₄ represent H or an arbitrary organic group, and X ⁻ represents The site indicated by the dotted line represents a condensed ring, H or any independent organic group bonded to the 5- and 6-positions of the thiopyran ring, provided that the meso group of the polymethine chain (Specifically, the central carbon is not directly bonded to a halogen atom).

[2] In the bioimaging agent according to one embodiment of the present invention, it is preferable that R ₁ and R ₁₀ each independently include a structure represented by the following general formula 3:

(In the general formula 3, n represents an arbitrary integer of 1 or more, A ₁ and A ₂ represent H or an arbitrary organic group, provided that the above R ₁ and R ₁₀ represent the aromatic ring at both ends. A part of a polymethine chain sandwiched between conjugates is shown, and a halogen atom is not directly bonded to the meso position (specifically, the central carbon) of the polymethine chain.)

  [3] The bioimaging agent according to one embodiment of the present invention may contain a complex of the above compound and a tag that binds to a biological substance.

  [4] In the bioimaging agent according to one aspect of the present invention, the dosage form is preferably a parenteral agent.

  [5] The bioimaging agent according to one embodiment of the present invention preferably further contains a calixarene derivative.

  According to one embodiment of the present invention, a bioimaging agent having high fluorescence intensity and low toxicity not only in an organic solvent system but also in an aqueous solution system can be provided. In the case of a bioimaging agent having a high fluorescence intensity, not only an image with less noise can be obtained, but also an image of a deep part of a living body can be obtained.

4 shows fluorescent images of compounds according to Examples and Comparative Examples. 4 shows fluorescent images of compounds according to Examples and Comparative Examples. a shows an absorption spectrum of the compound according to the example, and b shows an absorption spectrum of the compound according to the comparative example. a shows the peak of the fluorescence spectrum of the compound which concerns on an Example and a comparative example, and b shows the relative value of the fluorescence intensity of the compound which concerns on an Example and a comparative example. 4 shows a fluorescent image of a cerebral blood vessel of a mouse in an example. 4 shows a fluorescent image of a lymph node of a mouse in an example. 4 shows a fluorescent image of a tumor neovascularization in a mouse in an example. 3 shows the results of cytotoxicity tests of compounds according to Examples and Comparative Examples.

  One embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention can be variously modified within the scope shown in the claims, and the present invention is also applied to embodiments and examples obtained by appropriately combining technical means disclosed in different embodiments and examples. Included in the technical scope. In addition, all of the documents described in this specification are incorporated herein by reference. In the present specification, when the numerical range is described as “A to B”, the description intends “A or more and B or less”.

[1. Bioimaging agent)
The bioimaging agent of the present embodiment contains a compound represented by the following general formula 1 or 2. The compounds represented by the general formulas 1 and 2 may be all stereoisomers and / or all optical isomers. The bioimaging agent may contain either one of the compound represented by the general formula 1 or the compound represented by the general formula 2, or may include both.

(In the general formula 1, R ₁ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{2 to} R ₅ represent H or any organic group, and X ⁻ represents Any counter anion is shown, provided that no halogen atom is directly bonded to the meso position (specifically, the central carbon) of the polymethine chain.

(In the general formula 2, R ₁₀ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{11 to} R ₁₄ represent H or an arbitrary organic group, and X ⁻ represents The site indicated by the dotted line represents a condensed ring, H or any independent organic group bonded to the 5- and 6-positions of the thiopyran ring, provided that the meso group of the polymethine chain (Specifically, the central carbon is not directly bonded to a halogen atom).

  As used herein, the term "bioimaging agent" refers to a substance that emits fluorescence by light of a specific wavelength (eg, light in the near-infrared region) after administration into a living body, and the distribution and / or intensity of the fluorescence. For example, a composition for obtaining an image of a cell and / or a tissue of interest based on the composition is intended.

In the above general formulas 1 and 2, R ₁ and R ₁₀ are a part of a polymethine chain sandwiched conjugated to aromatic rings at both ends, and the meso position of the polymethine chain (specifically, the central carbon ) May be any organic group in which a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, and an astatine atom) is not directly bonded, and the specific structure is not limited. As shown in the examples described later, R ₁ and R ₁₀ are a part of a polymethine chain sandwiched and conjugated to aromatic rings at both ends, and a halogen atom is directly bonded to the meso position of the polymethine chain. In the absence, the intensity of the fluorescence emitted from the compound having the organic group is high, and as a result, an excellent effect that an image with less noise can be obtained and an image of a deep part of a living body can be obtained is obtained.

R ₁ and R ₁₀ may each independently include a structure represented by the following general formula 3, or may have a structure represented by the following general formula 3. Good:

(In the general formula 3, n represents any integer of 1 or more, A ₁ and A ₂ represent H or any organic group. The above R ₁ and R ₁₀ are conjugated to aromatic rings at both ends. Indicates a part of the polymethine chain sandwiched between the polymethine chains, and no halogen atom is directly bonded to the meso position (specifically, the central carbon) of the polymethine chain.)

  In the above general formula 3, n is any integer of 1 or more. Although the upper limit of n is not particularly limited, it may be specifically 1, 2, 3, 4, or 5.

The structure shown when n = 1 in the general formula 3 is considered as one unit. Within a single unit, A ₁ and A ₂ may have the same structure or different structures. Between a plurality of units, A ₁ there are a _plurality, each other, may be the same structure or may have different structures. Further, among a plurality of units, A ₂ existing in plural _numbers, each other, may be the same structure or may have different structures.

In the general formula 3, in a single unit, A ₁ and A ₂ may combine to form a ring structure. In General Formula 3, a ring structure may be formed by combining A ₁ and A ₁ between a plurality of units, or a ring structure may be formed by combining A ₂ and A _2. Or a ring structure may be formed by bonding of A ₁ and A ₂ .

The structure represented by the general formula 3 corresponding to R ₁ in the general formula ₁ and R ₁₀ in the general formula 2 may be more specifically the following structure, for example. Note that R ₂₁ , R ₂₂ and R _{23 in the following} general formula are part of a polymethine chain sandwiched and conjugated to aromatic rings at both ends, and a halogen atom is not directly bonded to the meso position of the polymethine chain. . R ₂₁ , R ₂₂ and R ₂₃ are, for example, H or any organic group.

R ₂₁ , R ₂₂ and R ₂₃ in the above general formula are any organic group other than a halogen atom, and R ₂₁ , R ₂₂ and R ₂₃ are each independently H, OR, SR, Ar (R ) Or NRR ′. However, R and R ′ are H or any organic group, and are not particularly limited. R ′ may be the same as R, and Ar (R) represents H or an aromatic ring having an organic group. .

  The site indicated by the dotted line in the general formula 2 may be a condensed ring or H bonded to the 5- and 6-positions of the thiopyran ring or any independent organic group. The compound represented by the general formula 2 showing the site shown by the dotted line more specifically can be, for example, the following.

R _{2 to} R ₅ of the compound represented by the general formula 1 and R _{11 to} R ₁₄ of the compound represented by the general formula 2 may be each independently H or any organic group. Examples of the organic group include an alkyl group, an alkenyl group, an alkynyl group, a phenyl group, an alkoxy group, a cycloalkyl group, and a sulfonyl group. Examples of the alkyl group include a linear or branched alkyl group having 1 to 12 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group; tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, 1-ethylpropyl group, hexyl group, isohexyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3 -Dimethylbutyl group, 2-ethylbutyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group and the like. Examples of the alkenyl group include a vinyl group, a 1-propenyl group, a 2-propenyl group (allyl group), an isopropenyl group, a 2-butenyl group, a 1,3-butadienyl group, and a 2-pentenyl group. Examples of the alkoxy group include an alkoxy group such as a methoxy group, a trifluoromethoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, and a tert-butoxy group. And cycloalkyl groups such as propyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptanyl group, cyclooctanyl group, cyclononanyl group, and cyclodecanyl group.

The compound represented by the general formula 1 or the general formula 2 of the present invention comprises R _{2 to} R _{5 of} the general formula 1, R _{11 to} R ₁₄ of the general formula 2, and A ₁ and A ₂ of the general formula 3. Multimers such as dimers or trimers can also be formed via at least one organic group selected from the group.

Binding the compound represented by the general formula 1 or the general formula 2 to a tag described later via at least one selected from the group consisting of R _{2 to} R ₅ and R _{11 to} R _14. Can be. In this case, at least one selected from the group consisting of R _{2 to} R ₅ and R _{11 to} R ₁₄ is a hydroxyl group, a carboxyl group, a succinimide group, or a sulfo group capable of forming a chemical bond with a tag. Succinimide group, pentafluorophenyl group, tetrafluorophenyl group, sulfotetrafluorophenyl group, maleimide group, bromoacetamide group, iodoacetamide group, pyridyldisulfide group, hydrazide group, alkoxyamino group, diazirino group, arylazide group, isocyanate group , An azido group, an alkynyl group, a cycloalkynyl group, or a phosphoramidite group, or an alkyl group substituted with the substituent.

In formulas 1 and 2, X ⁻ is an arbitrary counter anion. X ^- is, for _{^{example, I -, Br -, ClO}} 4 -, BF 4 -, PF 6 -, TsO -, CF 3 SO 3 -, (FSO 2) 2 N -, and _{(CF 3} SO ₂₎ 2 N ^- .

  The compound represented by the above general formula 1 or general formula 2 can be synthesized based on a general method. For example, it can be synthesized by the method described in "Photosensitive Dye" Sangyo Tosho Co., Ltd. (published October 17, 1997), supervised by Masaaki Hayami, Japanese Patent No. 5941844. In addition, as the compound represented by the above-described general formula 1 or 2, a commercially available compound can be used. For example, a compound manufactured by Hayashibara can be used.

  Examples of the compounds represented by the general formulas 1 and 2 of the present invention include the following compounds.

  The bioimaging agent of the present embodiment may contain a complex of the compound represented by the general formula 1 or 2 and a tag that binds to a biological substance. Note that the compound represented by the general formula 1 or 2 and the tag can be covalently bonded via an arbitrary linker. As the linker, a hydroxyl group, a carboxyl group, a succinimide group, a sulfosuccinimide group, a pentafluorophenyl group, a tetrafluorophenyl group, a sulfotetrafluorophenyl group, a maleimide group, a bromoacetamide group, an iodoacetamide group, a pyridyl disulfide group, a hydrazide group, Examples include a linker having an alkoxyamino group, a diazirino group, an arylazide group, an isocyanate group, an azido group, an alkynyl group, a cycloalkynyl group, or a phosphoramidite group, or an alkyl group substituted with the substituent. Many of the biological materials are cell-specific and / or tissue-specific. Therefore, with this configuration, the compound represented by the general formula 1 or the general formula 2 binds to the biological substance via the tag, whereby the target cell and / or the target tissue are specifically bound. Can be imaged.

  The biological material is not particularly limited, and examples thereof include proteins, nucleic acids (eg, DNA or RNA), carbohydrates, proteoglycans, lipids, and small molecules (eg, hormones, vitamins).

  The tag that binds to the biological substance is not particularly limited, and is selected from, for example, the group consisting of (i) an oligonucleotide, (ii) an amino group, a sulfhydryl group, a carbonyl group, a hydroxyl group, a carboxyl group, and a thiophosphate group. Oligonucleotides, (iii) antibodies, (iv) lectins, (v) peptides, (vi) aptamers, (vii) drugs, (viii) polymer particles, containing or derivatized to contain one or more. Or (ix) glass beads.

  For example, the antibody is preferably a humanized antibody or a human antibody. Examples of the humanized antibody include anti-HER2 antibody (for example, Herceptin (registered trademark)), anti-VEGF antibody (bevacizumab), anti-IGF1R antibody (AMG479), anti-CD22 antibody (epratuzumab), anti-EGFR antibody (matsuzumab), and the like. No. Examples of the human antibody include an anti-HGF antibody (AMG102), an anti-IGF1R antibody (ixiximab), an anti-IGF1R antibody (darotuzumab), an anti-RANKL antibody (denosumab), an anti-EGFR antibody (manituzumab) and the like. The above antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the viewpoint of obtaining a stable image.

  The compound represented by the general formula 1 or the general formula 2 bonded to a tag can be produced by a known method, for example, the method described in Bioconjugatechem., 2009, 20 (11). P. 2177. A known linker may be used for binding the compound represented by the general formula 1 or 2 to the tag.

  The bioimaging agent of the present embodiment may further contain a calixarene derivative in addition to the compound represented by the general formula 1 or 2. The calixarene derivative has low toxicity to the living body and has an effect of increasing the solubility of the compound represented by the general formula 1 or 2 in water. Therefore, with this configuration, the fluorescence intensity can be increased and the fluorescence intensity can be stabilized. Hereinafter, a specific configuration of the calixarene derivative will be described.

  The calixarene derivative may be a compound represented by the following general formula 4:

(In the above general formula 4, n represents an integer of 4 to 8, n R ¹ each independently represents a C _{4 to 12} alkyl group, and M ⁺ represents a monovalent cation. .).

  The calixarene derivative represented by the above general formula 4 is produced by alkylating a phenolic hydroxyl group of a sulfonated calix [n] arene (n is an integer of 4 to 8) using an alkyl halide. be able to. Alkylation using an alkyl halide is a well-known synthesis method, and can be easily performed by those skilled in the art by appropriately setting synthesis conditions. Sulfonated calix [n] arenes are commercially available and can be easily obtained. As described above, the calixarene derivative can be easily synthesized in large quantities and can be produced at low cost. Further, the calixarene derivative has an advantage of low cytotoxicity.

  When a sodium salt of sulfonated calix [n] arene, which is a starting material of the calixarene derivative represented by the above general formula 4, is added to the bioimaging agent, the luminescence intensity is rather reduced. However, surprisingly, when a calixarene derivative obtained by alkylating a sulfonated calix [n] arene is added to a bioimaging agent, the active ingredient of the bioimaging agent in water (specifically, the general formulas 1 and 2) 2) is remarkably improved in stability and luminescence intensity.

The calixarene derivative represented by the above general formula 4 forms micelles in which a hydrophilic SO ₃ ⁻ group is on the outside and a hydrophobic group R ¹ (that is, a C _{4 to 12} alkyl group) is on the inside. It is thought that. The inside of the micelle of the calixarene derivative becomes a hydrophobic environment, and the active ingredient of the bioimaging agent (specifically, the compound represented by the general formula 1 and the general formula 2) is taken into the hydrophobic environment, It is estimated that the stability and luminescence intensity of the bioimaging agent are improved. However, the present invention is not limited to such estimation.

  In the general formula 4, n represents an integer of 4 to 8, preferably 4, 6 or 8, more preferably 4 or 6, and still more preferably 4.

At the general formula 4, n pieces of ^{R 1} _represents an alkyl group of _{C 4 to 12.} The n R ^{1 s} may be the same or different, and are preferably the same. The alkyl group may be linear or branched, and is preferably linear. Examples of the C _4-12 alkyl group include butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2- Examples include dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like.

In the above general formula 4, M ⁺ represents a monovalent cation such as an alkali metal ion and an ammonium ion. M ⁺ is preferably an alkali metal ion, more preferably a sodium ion.

In the calixarene derivative represented by the general formula 4, when n is 4, four R ^{1 s} are each independently preferably a C _4-11 alkyl group, more preferably a linear C _4-11 An alkyl group, more preferably a linear _C4-9 alkyl group. Specific examples of the C _4-11 alkyl group and the C _4-9 alkyl group include those having 4 to 11 carbon atoms and those having 4 carbon atoms in the specific examples of the C _4-12 alkyl group, respectively. -9. In the calixarene derivative, it is preferable that the four R ¹ are the same. In the calixarene derivative, M ⁺ is preferably an alkali metal ion, more preferably a sodium ion.

In the calixarene derivative represented by the above general formula 4, when n is 6, six R ^{1 s} are each independently preferably a C _4-12 alkyl group, more preferably a linear C _4-12 An alkyl group, more preferably a linear C _6-11 alkyl group. Specific examples of the C _4-11 alkyl group and the C _4-9 alkyl group include those having 4 to 11 carbon atoms and those having 4 carbon atoms in the specific examples of the C _4-12 alkyl group, respectively. -9. In the calixarene derivative, it is preferable that six R ^{1 be} the same. In the calixarene derivative, M ⁺ is preferably an alkali metal ion, more preferably a sodium ion.

When n is 8 in the calixarene derivative represented by the above general formula 4, the eight R ^{1 s} are each independently preferably a C ₅ alkyl group. In the calixarene derivative, eight R ¹ are more preferably n-pentyl. In the calixarene derivative, M ⁺ is preferably an alkali metal ion, more preferably a sodium ion.

  From the viewpoint of stabilization of the bioimaging agent in water and improvement of luminescence intensity, the amount of the calixarene derivative is determined by an effective component of the bioimaging agent (specifically, a compound represented by the general formula 1 or 2). The amount is preferably from 10 to 10,000 mol, more preferably from 100 to 10,000 mol, still more preferably from 500 to 5,000 mol, particularly preferably from 1,000 to 3,000 mol, per mol.

  As the calixarene derivative, those described in WO2016 / 152954A1 can be used. This reference is incorporated herein by reference.

  The bioimaging agent contains a component different from the compounds represented by the general formulas 1 and 2 in addition to the calixarene derivative as long as the component does not inhibit the fluorescence activity of the bioimaging agent. May be.

  As the different components, buffers, pH adjusters, preservatives, antioxidants, isotonic agents, high molecular weight polymers, excipients, simplex, diluents, solvents, solubilizers, stabilizers, fillers, Examples include binders and surfactants.

  Examples of such buffers include phosphoric acid, phosphate, boric acid, borate, citric acid, citrate, acetic acid, acetate, carbonate, carbonate, tartaric acid, tartrate, ε-aminocaproic acid, and Trometamol and the like. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the like. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium hydrogen carbonate. Examples of the tartrate include sodium tartrate and potassium tartrate.

  Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide and the like.

  Examples of the tonicity agent include ionic tonicity agents (such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride) and nonionic tonicity agents (glycerin, propylene glycol, sorbitol, And mannitol).

  Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl paraoxybenzoate, and chlorobutanol.

  Examples of the antioxidant include ascorbic acid, tocophenol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erysorbate, propyl gallate, and sodium sulfite.

  Examples of the high molecular weight polymer include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate , Carboxymethylethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, atelocollagen, and the like.

  The content of the active ingredient (specifically, the compound represented by the general formula 1 and the compound represented by the general formula 2) contained in the bioimaging agent is not particularly limited.

  The content of components other than the active components contained in the bioimaging agent is not particularly limited.

  The target from which an image can be obtained by the bioimaging agent is not particularly limited, and includes, for example, lymph nodes, cerebral blood vessels, tumor new blood vessels, and living tissues.

  Cells in which the bioimaging agent of the present embodiment can be used are not particularly limited, for example, chondrocytes, osteoblasts, fibroblasts, epidermal cells, epithelial cells, adipocytes, hepatocytes, pancreatic cells, muscle cells, and the like. Is mentioned. The animal species from which the cells are derived is not particularly limited, and the animal species may be a non-human mammal or a human. Examples of non-human mammals include primates (monkeys, apes, etc.), artiodactyls (cows, wild boars, pigs, sheep, goats, etc.), ungulates (horses, etc.), rodents (mouse, rat, hamster, etc.) , Squirrels, etc.), lagomorphs (rabbits, etc.), meats (dogs, cats, ferrets, etc.) and the like. The non-human mammal described above may be a domestic animal or a companion animal, or may be a wild animal.

  The dosage form of the bioimaging agent is not particularly limited, but is preferably a parenteral agent, and more preferably an injection. The compound may be blended with another material such as a diluting solvent at any stage in the bioimaging agent manufacturing process. Examples of the method include mixing, kneading, dissolving, melting, dispersing, suspending, and emulsifying. One or more methods are selected from, for example, reverse micelle formation, infiltration, crystallization, spraying, application, adhesion, spraying, coating (coating), injection, dipping, solidification, and supporting. As the diluting solvent, a known solvent can be used, and for example, water and the like can be used.

  In order to detect the bioimaging agent introduced into the living body, the wavelength of the light irradiated to the living body is not particularly limited, but from the viewpoint of increasing the absorbance, light in the near infrared region is preferable. , Preferably near-infrared light of 700 nm to 2000 nm, more preferably near-infrared light of 800 nm to 1500 nm, more preferably near-infrared light of 900 nm to 1500 nm, and near-infrared light of 1000 nm to 1500 nm. More preferably, it is infrared light.

[2. Others)
One embodiment of the present invention can also be configured as follows.
<1> A bioimaging agent containing a compound represented by the general formula 1 or 2 is used as a subject (for example, mammals, non-human mammals, primates, artiodactyla, oddotes, rodents, lagomorphs) Or a meat).
<2> The bioimaging method according to <1>, wherein in the compound, R ₁ and R ₁₀ each independently include a structure represented by General Formula 3.
<3> The bioimaging method according to <1> or <2>, wherein the bioimaging agent contains a complex of the compound and a tag that binds to a biological substance.
<4> The bioimaging method according to any one of <1> to <3>, wherein the dosage form of the bioimaging agent is parenteral.
<5> The bioimaging method according to any one of <1> to <4>, wherein the bioimaging agent further contains a calixarene derivative.

One embodiment of the present invention can also be configured as follows.
<6> Use of a compound represented by General Formula 1 or General Formula 2 for production of a bioimaging agent.
<7> The use according to <6>, wherein R ₁ and R ₁₀ each independently include a structure represented by the general formula 3.
<8> The use according to <6> or <7>, wherein the bioimaging agent contains a complex of the compound and a tag that binds to a biological substance.
<9> The use according to any one of <6> to <8>, wherein the bioimaging agent is administered in a parenteral dosage form.
<10> The use according to any one of <6> to <9>, wherein the bioimaging agent further contains a calixarene derivative.

[Example 1, bioimaging agent]
The structural formula of the compound used in this example is shown below.

  The structural formulas of the compounds to be compared are shown below.

  (I) 1 mg of the above compound was weighed and dissolved in 1 mL of dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries) to obtain a dye solution. Further, 10 μL of the dye solution was diluted with 1 mL of dimethyl sulfoxide (concentration: 0.01 mg / mL) to obtain a DMSO solution.

  (II) 1 mg of the above compound was weighed and dissolved in 1 mL of dimethyl sulfoxide to obtain a dye solution. 10 mg of a hexyl derivative of calix [4] arene represented by the following structural formula was weighed and dispersed in 1 mL of phosphate buffered saline (PBS) (concentration: 1 mg / mL). 10 μL of the above dye solution was added to this aqueous solution, and sonication (Yamato 2510, Branson) was performed for 5 minutes to obtain an S4 aqueous solution.

[Example 2, observation of fluorescence intensity]
Approximately 20 μL of the DMSO solution and the S4 aqueous solution were respectively injected into a glass capillary (Model GD1, NARISHIGE) having a diameter of 1 mm to prepare a sample.

In order to observe the fluorescence intensity of the sample, the sample was irradiated with excitation light having a wavelength of 975 nm using semiconductor laser light (BW TEK INK.). The irradiation power of the laser beam at this time was 40 mW / cm ² . The fluorescence image was taken with a near-infrared camera (C10034-34, Hamamatsu Photonics) using a long-pass filter that transmits only light of 1000 nm or more to remove the excitation light. The obtained fluorescent images were compared after normalizing the fluorescent intensity. FIGS. 1 and 2 show the obtained fluorescent images.

  FIG. 1 shows fluorescent images of the compounds (NK-10053, NK-10054, NK-10057, and NK-10475). Here, compounds (NK-08472, NK-10472) were used as comparison targets. The exposure time was between 0.2 seconds and 2.5 seconds. As a result, all compounds showed fluorescence in the DMSO solution and the S4 aqueous solution, and in particular, strong fluorescence was observed in NK-10053 and NK-10475.

  FIG. 2 shows fluorescent images of the compounds (NK-10053, NK-10475) and the compounds to be compared (IR-26, IR-1048, IR-1061). The exposure time was between 0.15 seconds and 1.5 seconds. As a result, it was revealed that the compounds (NK-10053, NK-10475) showed stronger fluorescence in the DMSO solution and the S4 aqueous solution than the compounds (IR-26, IR-1048, IR-1061).

[Example 3, absorption spectrum measurement]
A DMSO solution of the compound (NK-10053, NK-10054, NK-10057, NK-10475, NK-08472, NK-10472) prepared by the method (I) was filled in a quartz cell having an optical path length of 1 cm, and the spectrophotometer was used. The absorption spectrum in the wavelength region of 500 nm to 1500 nm was measured with a meter (JASCO v-670, JASCO Corporation). The results are shown in FIG. 3A (Example) and FIG. 3B (Comparative Example). The peak of the maximum absorption wavelength was observed at around 1000 nm in all of the compounds.

[Example 4, fluorescence spectrum measurement]
DMSO solution of compounds (NK-10053, NK-10054, NK-10057, NK-10475, NK-08472, NK-10472, IR-26, IR-1048, IR-1061) prepared by the method (II) above Was measured with a near-infrared fluorescence spectrometer (HORIBA Nanolog) in an excitation wavelength of 975 nm and in a total reflection mode (FF mode) in order to reduce absorption of fluorescence by a solution.

  The results are shown in FIGS. 4a and 4b. In FIG. 4a, NK-10053, NK-10054, NK-10057, NK-10475, NK-08472, NK-10472, IR-1061, IR-1061, and IR-26 have peaks between 1000 nm and 1200 nm. Was observed. The peak of NK-10053 was the highest, followed by the peak of NK-10475. FIG. 4B shows a relative value of the fluorescence intensity. It was revealed that NK-10053 and NK-10475 had high fluorescence intensity. Specifically, relative values of the fluorescence intensity are NK-10053 (relative value: 193), NK-10475 (relative value: 179), NK-10057 (relative value: 54), and NK-10054 (relative value: 29), respectively. ), NK-10472 (relative value: 21), NK-08472 (relative value: 17), IR-1061 (relative value: 32), IR-1048 (relative value: 10), IR-26 (relative value: 4) )Met.

[Example 5, near-infrared fluorescence imaging of mouse brain blood vessels]
300 μL of an NK-10053 S4 aqueous solution prepared by the above method (II) was injected into the tail vein of a 5-week-old nude mouse (BALB / c nu / nu, Japan SLC) under isoflurane anesthesia, and cerebrovascular Near infrared fluorescence imaging was performed.

  A 975 nm semiconductor laser beam was irradiated to the mouse head, and a fluorescence image was taken using a near-infrared camera (C10034-34, Hamamatsu Photonics) through bandpass filters of 1100 nm and 1300 nm. The exposure time was 2.5 seconds and 10 seconds, respectively.

  FIG. 5 shows a fluorescence image of a cerebral blood vessel with the scalp and a cerebral blood vessel with the scalp removed. Regardless of the presence or absence of skin on the head, when NK-10053 was used, blood vessels were observed in detail.

[Example 6, near-infrared fluorescence imaging of mouse lymph nodes]
300 μL of an NK-10053 S4 aqueous solution prepared by the above method (II) was injected with a syringe into the left foot surface of a 5-week-old nude mouse under isoflurane anesthesia, and near-infrared fluorescence imaging of lymph nodes of lower limbs was performed. .

The fluorescence image of the lymph node uses a long-pass filter that transmits only light of 1000 nm or more to remove the excitation light, and irradiates the lower limb lymph node with excitation light having a wavelength of 975 nm using a semiconductor laser light, Images were taken with a near-infrared camera (C10034-34, Hamamatsu Photonics). At this time, the irradiation power of the laser beam was 40 mW / cm ² , and the photographing time was 0.25 seconds. Fluorescence images were taken at 4, 6, and 8 minutes after the injection of the S4 aqueous solution.

  FIG. 6 shows near-infrared fluorescence imaging of a mouse lymph node, which clearly shows that fluorescence can be observed even in a mouse lymph node, and that the fluorescence increases as the exposure time increases.

[Example 7, near-infrared fluorescence imaging of mouse tumor neovascularization]
300 μL of an NK-10053 S4 aqueous solution prepared by the above method (II) was injected into the tail vein of a nude mouse carrying a breast cancer tumor (KPL-4, manufactured by Kawasaki Medical University), and near-infrared fluorescence imaging of tumor neovascularization Was done.

A 975 nm semiconductor laser beam was irradiated to the breast cancer tumor, and a fluorescence image was taken with a near-infrared camera (C10034-34, Hamamatsu Photonics) using a 1100 nm bandpass filter. This example was performed in two repetitions. At this time, the irradiation power of the laser beam was 40 mW / cm ² , and the imaging time was 2 seconds.

  FIG. 7 shows near-infrared fluorescence imaging of mouse tumor neovasculature. Fluorescence of tumor neovascularization was observed in all mice.

[Example 8, cytotoxicity test]
HeLa cells were uniformly seeded on a 96-well microplate and cultured at 37 ° C. for 24 hours. In each well, an aqueous S4 solution of a compound (NK-10053, NK-10054, NK-10057, or NK-10475) prepared by the method of (II) above, a lead sulfide quantum dot (PbS-QDs, reference: Y. Nakane, Y. Tsukasaki, S. Takao, H. Yasuda and Takashi Jin, “Aqueous synthesis of glutathione-coated PbS quantum dots with tunable emission for non-invasive fluorescence imaging in the second near-infrared biological window (1000-1400 nm)”, Chem. Commun., 49, 7584-7586 (2013)) and carbon nanotubes (SWNT, NanoIntegris, References: Setsuko Tsuboi, Sayumi Yamada, Yuko Nakane, Takao Sakata, Hidehiro Yasuda, and Takashi Jin, “Water-soluble near-infrared fluorophores emitting over 1000 nm and their application to in vivo imaging in the second optical window (1000-1400 nm) ”, ECS J. Solid State Sci. Technol. 7, R3093-3101 (2018)). .

As a control, a well to which the above compound was not added was also prepared. 48 hours after the addition of the S4 aqueous solution of each of the above compounds, 10 µL of an MTT solution was added to each well, and the cells were cultured in a CO ₂ incubator at 37 ° C for 2 hours.

Next, 100 μL of a formazan solubilization solution was added to each well, mixed by pipetting, each well was sealed with a plate seal, and placed in a CO ₂ incubator at 37 ° C. overnight to completely dissolve the formazan.

  The absorbance of formazan at a wavelength of 570 nm was measured using a Multiskan GO microplate spectrophotometer (ThermoFisher), and the absorbance was used as the cell survival number. More specifically, the background absorbance at 650 nm was subtracted from the absorbance of formazan at a wavelength of 570 nm, and the absorbance after subtraction was taken as the cell viability. At this time, the cell viability of the well to which the above compound was added was calculated assuming that the cell viability of the well to which the above compound was not added was 100%. For the MTT test, a cell number measurement kit (Nacalai Tesque) was used.

  FIG. 8 shows the results. As a result, the cell viability of the lead sulfide quantum dot was about 70%, and the cell viability of the carbon nanotube was about 30%. In the compound of this example, the cell viability was about 100% in all cases. , Indicating that the compound had low toxicity.

  The present invention can be widely used in the medical field (for example, near-infrared fluorescence imaging).

Claims (5)

A bioimaging agent containing a compound represented by the following general formula 1 or 2:
(In the general formula 1, R ₁ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{2 to} R ₅ represent H or any organic group, and X ⁻ represents An arbitrary counter anion is shown, provided that a halogen atom is not directly bonded to the meso position of the polymethine chain.)
(In the general formula 2, R ₁₀ represents a part of a polymethine chain sandwiched between conjugated aromatic rings at both ends, R _{11 to} R ₁₄ represent H or an arbitrary organic group, and X ⁻ represents The site indicated by the dotted line represents a condensed ring, H or any independent organic group bonded to the 5- and 6-positions of the thiopyran ring, provided that the meso group of the polymethine chain No halogen atom is directly bonded to the position.). The bioimaging agent according to claim 1, wherein R ₁ and R ₁₀ each independently include a structure represented by the following general formula 3:
(In the general formula 3, n represents an arbitrary integer of 1 or more, A ₁ and A ₂ represent H or an arbitrary organic group, provided that the above R ₁ and R ₁₀ represent the aromatic ring at both ends. It shows a part of a polymethine chain sandwiched in a conjugate manner, and no halogen atom is directly bonded to the meso position of the polymethine chain.)   The bioimaging agent according to claim 1, comprising a complex of the compound and a tag that binds to a biological substance.   The bioimaging agent according to any one of claims 1 to 3, wherein the dosage form is a parenteral agent.   The bioimaging agent according to any one of claims 1 to 4, further comprising a calixarene derivative.