Reagent for blood vessel imaging

Abstract

The present invention addresses the problem of developing a novel reagent for blood vessel imaging that is easy to produce, highly sensitive and highly accurate. The present invention provides a reagent for blood vessel imaging that contains a compound comprising an oligoarginine represented by formula and a phosphor or fluorophore group bound to the C-terminal or N-terminal side of the oligoarginine.

Description

Vascular imaging reagents

[0001] The present invention relates to a vascular imaging reagent, and a compound that can be used for the reagent.

[0002] Biological tissue (organ) cells obtain necessary nutrients and oxygen from the blood and release synthesized metabolic products and carbon dioxide into the blood. Therefore, the vascular system plays an important role in maintaining life. If, for some reason, the vascular system becomes abnormal, surrounding tissue cells are unable to function properly. In particular, arteriosclerosis, in which lipid components such as cholesterol accumulate in the arterial lining and form atherosclerotic lesions called atherosclerotic lesions, and liver cirrhosis, in which fibrosis occurs between blood vessels and liver cells, are deeply related to the breakdown of the vascular system. Furthermore, in cancer, angiogenesis cannot keep up with abnormal cell proliferation, resulting in the formation of many immature and abnormally shaped blood vessels. Therefore, visualizing and imaging the shape and course of blood vessels in living individuals is important for the diagnosis and treatment of these pathologies.

[0003] Known clinical methods for vascular imaging include administering a contrast agent into the bloodstream and imaging with MRI, and using photoacoustic imaging technology. While these methods provide insight into the vascular network of the human body, they require large-scale equipment and are unsuitable for use in small animals. Furthermore, they lack spatial resolution at the single-cell level. Optical imaging techniques, on the other hand, offer high sensitivity, simplicity, and real-time measurement, and can easily achieve spatial resolution at the single-cell level when combined with a microscope. The key element here is a probe molecule that remains stable in a contaminated environment such as blood and emits highly bright light.

[0004] Vascular imaging reagents are classified into blood-retention type and endothelial-adsorbed type. The former are reagents that remain in the blood, and fluorescent dextran and fluorescent nanoparticles have been developed. In particular, fluorescent dextran is a reagent in which a fluorescent molecule is covalently bonded to dextran, and various reagents with different molecular weights of dextran are commercially available.

[0005] Meanwhile, fluorescent lectins are commercially available as vascular endothelial-adsorbed imaging reagents. Lectin is a general term for proteins that exhibit specific binding to specific sugar chains, and many are derived from animals, plants, and fungi. Among these, plant-derived lectins recognize and bind to sugar chains present on the surface of vascular endothelial cells in mammals, including humans. To date, fluorescent lectins in which fluorescein or Texas Red is covalently bound to tomato lectin have been commercially available (Vector Laboratories). Synthesizing fluorescent lectins requires the tedious process of extracting tomato lectin from natural sources, and the number of fluorescent molecules bound to the lectin cannot be controlled.

[0006] Fluorescent lectins have the following problems: Because they recognize glycans present on the surface of vascular endothelial cells, only plant-derived lectins can be used; the tedious process of extracting lectins from natural sources is required; because lectins are glycoproteins, the labeling of luminophores can be non-uniform, resulting in differences between lots; and because they utilize fluorescence, they can sometimes be difficult to distinguish from tissue autofluorescence.

[0007] On the other hand, methods have been proposed for quantifying oxygen concentrations in biological tissues or cells that use iridium(III) complexes having cyclometallated ligands such as btq (2-(2'-benzothienyl)-quinolinato-N,C3'), or compounds containing such iridium complexes and fluorescent compounds (e.g., Patent Documents 1 and 2). However, Patent Documents 1 and 2 do not discuss the use of these methods for visualizing blood vessels.

[0008] JP 2018-65768 A JP 2019-7961 A

[0009] The present invention has been made in view of the above problems, and an object of the present invention is to develop a new vascular imaging reagent that is easy to produce and has high sensitivity and accuracy.

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have discovered that clear imaging of blood vessels in an individual can be achieved using a compound containing oligoarginine and a luminophore that is a phosphorescent or fluorescent luminophore. Furthermore, they have developed a compound containing oligoarginine and an iridium complex that can be used as such a luminophore. Based on this discovery, the present invention has been completed. That is, the gist of the present invention relates to the following:

[0011] [1] A vascular imaging reagent comprising a compound containing an oligoarginine represented by the following formula: and a phosphorophore or fluorophore bound to the C-terminal or N-terminal side of the oligoarginine:

[0012]

[0013] In the formula, n is an integer of 4 to 20. [2] The reagent according to [1], wherein n in the oligoarginine is 8 or more. [3] The reagent according to [1] or [2], wherein the phosphorophore is a compound containing an iridium complex. [4] The reagent according to [3], wherein the iridium complex is a compound represented by the following formula (I) or (II):

[0014]

[0015] In formula (I), ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, and ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring; L １ represents a bidentate ligand having a β-diketonate structure;

[0016]

[0017] In formula (II), ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, and ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring; L ２ indicates a bidentate ligand having a phenanthroline skeleton.

[0018] [5] The reagent according to [3] or [4], wherein the iridium complex is a compound represented by the following formula:

[0019]

[0020] In the formula, n is an integer of 4 to 20. [6] A compound represented by the following formula:

[0021]

[0022] In the formula, n is an integer from 4 to 20.

[0023] The present invention provides a vascular imaging reagent comprising a compound containing oligoarginine and a luminophore. Because the compound for the imaging reagent can be chemically synthesized, this reagent can be easily produced without the need for extraction procedures from natural products. Furthermore, the luminophore labeling can be uniform, resulting in a uniform reagent with minimal lot-to-lot variation, enabling high-precision imaging. When a phosphorescent compound is used as the luminophore, autofluorescence can be eliminated by time-resolved measurement, enabling high-sensitivity, high-precision imaging. In particular, compounds with eight or more arginines bonded to the luminophore exhibit excellent vascular endothelial binding, enabling imaging of the vascular endothelium with higher sensitivity and precision. Even when a fluorescent compound is used, compounds that exhibit fluorescence in the near-infrared region can be used to distinguish from autofluorescence and further enable imaging of deep vascular structures. The present invention also provides a compound containing oligoarginine and an iridium complex that can be used as a luminophore for a vascular imaging reagent. The compound exhibits excellent phosphorescence and enables clear imaging of blood vessels. The present invention also provides a compound containing oligoarginine and a fluorescent compound, which can be used as a luminophore for a blood vessel imaging reagent. The compound has excellent fluorescence emission properties and can clearly image blood vessels.

[0024] FIG. 1 shows a conceptual diagram of the compound of the present invention (vascular imaging reagent). FIG. 2 shows the synthesized compound of the present invention (BTQ-R n Figure 3 shows the structural formulas of the FITC-tomato lectin (n = 4, 8, 12, 16). Figure 3 shows images (photographs used as drawing) of kidney capillaries. A is FITC-tomato lectin, B is BTQ, C, D, and E are BTQ-R, respectively.n Figure 4 shows the results of using FITC-lectin (objective lens ×40) and BTQ-R (objective lens ×40) in the kidney. 12 (Objective lens × 40), C is FITC- (Objective lens × 100), D is BTQ-R 12 Figure 5 shows images (photographs in place of drawings) of a kidney section (glomerulus). A shows the results using a 20x objective lens, and B shows the results using a 10x objective lens. Figure 6 shows images (photographs in place of drawings) of liver sinusoidal blood vessels. A and B were taken at different locations within the liver. Figure 7 shows images (photographs in place of drawings) of blood vessels in various organs. A shows the results of the pancreas, B the small intestine, C the subcutaneous tissue, and D the heart. Figure 8 shows images (photographs in place of drawings) of tumor blood vessels in a tumor-bearing mouse. A to C were taken at different locations within the tumor. Figure 9 shows the results of PC6S and BTQ-R in the liver. 12 The multicolor imaging images (photographs used as drawing substitutes) are shown. Column A shows the results for healthy mice, and column B shows the results for fatty liver model mice. The left column shows PC6S, and the center column shows BTQ-R. 12 The right side shows the result of superimposing them. 12 -R 12 Figure 11 shows vascular imaging images (photos in place of drawings) of various organs using NBD-PEG4-R. A shows the liver, B and C show the kidneys (C is an enlarged image of a part of B), D shows the spleen, E shows the pancreas, F shows the testis, G shows the muscle tissue, and H shows the subcutaneous tissue. 12 Figure 12 shows vascular imaging images (photos in place of drawings) of various organs using C6-PEG4-R. A shows the results for the liver, B shows the kidney, C shows the spleen, D shows the subcutaneous tissue, and E shows the results for the adipose tissue. 12 Figure 13 shows vascular imaging images (photos in place of drawings) of various organs using C6-PEG4-R. A shows the results for kidney, B shows the results for spleen, C shows the results for pancreas, D shows the results for testis, E shows the results for adipose tissue, and F shows the results for muscle tissue. 12 Images (photographs substituted for drawings) of cerebral blood vessels are shown. A, B, and C are images of different locations in the brain. D is an enlarged image of part of C.

[0025] The present invention will be described below.

[0026] <Blood vessel imaging reagent> One aspect of the present invention relates to a blood vessel imaging reagent (hereinafter, sometimes referred to as the "blood vessel imaging reagent of the present invention") comprising a compound containing an oligoarginine represented by the following formula: and a phosphorophore or fluorophore bound to the C-terminal or N-terminal side of the oligoarginine:

[0027]

[0028] In the formula, n is an integer from 4 to 20.

[0029] The vascular imaging reagent developed in this invention contains a compound with a structure in which a luminophore is bound to either the C- or N-terminus of an oligoarginine peptide (Figure 1). The luminophore may be bound to either the C- or N-terminus of the oligoarginine peptide, but is preferably bound to the C-terminus. The luminophore can be either a fluorophore (fluorescent compound) or a phosphorophore (phosphorescent compound). When a phosphorescent compound (e.g., an iridium complex) is used, autofluorescence can be eliminated by time-resolved measurement. Even when a fluorescent compound is used, if a compound that fluoresces in the near-infrared region is used, it can be distinguished from autofluorescence and further enables imaging of deep vascular structures.

[0030] <Oligoarginine> The compound used in the vascular imaging reagent of the present invention contains an oligoarginine represented by the following formula.

[0031]

[0032] Here, n is an integer of 4 to 20, preferably an integer of 6 to 16, and more preferably an integer of 8 to 12.

[0033] The compound used in the vascular imaging reagent of the present invention achieves improved adsorption to vascular endothelium by containing oligoarginine. When the oligoarginine has n≧8 or more, it exhibits particularly excellent adsorption to vascular endothelium, and therefore, such an embodiment can be preferably used when the purpose is to image the vascular endothelium.

[0034] The compound used in the vascular imaging reagent of the present invention may contain a group other than oligoarginine, provided that the effects of the present invention are not impaired. The group other than oligoarginine may be one type or two or more types. The group other than oligoarginine may be attached to the terminus of the oligoarginine. That is, the group other than oligoarginine may be located between the oligoarginine and the luminophore (this aspect may be referred to as a "linker"), or may be located on the opposite side of the oligoarginine from the bond to the luminophore. In general, groups containing steroids, peptides, polyethylene glycol, etc. can be relatively easily attached to luminophores and are therefore suitable as the group other than oligoarginine. When the compound contains oligoarginine and a group other than oligoarginine consisting of a peptide, the polypeptide as a whole preferably has 4 to 20 amino acid residues, more preferably 6 to 16 amino acid residues, and even more preferably 8 to 12 amino acid residues. The peptide other than oligoarginine is not limited, but proline is preferred from the standpoint of ease of synthesis. Aspartic acid and lysine are also preferred because they can make the compound water-soluble. When a group other than oligoarginine consisting of oligoarginine and a group containing polyethylene glycol is contained, the degree of polymerization of polyethylene glycol can be adjusted depending on the type of luminophore, etc., but is, for example, 2 to 20, preferably 3 to 16, and more preferably 4 to 12. Compounds used in the vascular imaging reagent of the present invention can be suitably used in both embodiments containing and not containing a group other than oligoarginine. In the case of compounds having a fluorophore, embodiments containing a group other than oligoarginine as a linker can be more preferably used.

[0035] <Fluorophores> The fluorophores contained in the compounds used in the vascular imaging reagents of the present invention can be fluorescent compounds conventionally used in imaging blood vessels, etc., without particular limitation. Furthermore, the compounds of the present invention described below can be used as compounds used in vascular imaging reagents containing fluorophores. Fluorescent compounds can be used singly or in combination of two or more. Examples of fluorescent compounds include, but are not limited to, 4-nitrobenzo-2-oxa-1,3-diazole (NBD), dimethylaminosulfonylbenzoxadiazole (DBD), dimethylaminosulfonylbenzothiadiazole (DBThD), dimethylaminosulfonylbenzoselenadiazole (DBSeD), fluorescein isothiocyanate (FITC), coumarin dyes, rhodamines, boron dipyrromethene (BODIPY), cyanine dyes, and the like. Examples of fluorescent compounds include, but are not limited to, NBD, coumarin dyes such as coumarin 6, and rhodamines such as rhodamine B.

[0036] <Phosphores> The phosphorophores contained in the compounds used in the vascular imaging reagent of the present invention can be any phosphorescent compounds conventionally used in imaging blood vessels, etc., without any particular limitations. Furthermore, the compounds of the present invention described below can be used as compounds used in vascular imaging reagents having phosphorophores. One or more phosphorescent compounds can be used in combination. Examples of phosphorescent compounds include, but are not limited to, compounds containing iridium complexes. Examples of iridium complexes include, but are not limited to, compounds represented by the following formula (I) or (II):

[0037]

[0038] In formula (I), ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, and ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring; L １ represents a bidentate ligand having a β-diketonate structure;

[0039]

[0040] In formula (II), ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, and ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring; L ２ indicates a bidentate ligand having a phenanthroline skeleton.

[0041] <Compound represented by formula (I)> The compound represented by formula (I) (hereinafter, sometimes referred to as "complex (I)") will be described below. The vascular imaging reagent of the present invention may contain one type of complex (I) or two or more types of complex (I).

[0042] Ring R １ Examples of the nitrogen-containing aromatic ring include, but are not limited to, the nitrogen-containing aromatic rings having the structures shown in the following formulas (1-1), (1-2), (1-3), and (1-4).

[0043]

[0044] Here, X １ Among the carbon atoms constituting the ring skeleton, the bond extending from the carbon atom next to N is the ring R ２ N is coordinated to Ir.

[0045] Ring R １ As the ring R ２ In terms of emitting light approaching near-infrared and having good transmittance in a living body when combined with the above, a polycyclic nitrogen-containing aromatic ring is preferred, and a nitrogen-containing aromatic ring having a structure represented by the above formula (1-2), (1-3), or (1-4) is more preferred.

[0046] Ring R ２ Examples of the sulfur-containing aromatic ring include those having the structure represented by the following formula (2-1), (2-2), or (2-3).

[0047]

[0048] Here, X ２ Among the carbon atoms constituting the ring skeleton, the bond extending from the carbon atom next to S is the ring R １ and the carbon atom next to this carbon atom is coordinated to Ir.

[0049] Ring R ２ As the aromatic ring, a sulfur-containing aromatic ring having a structure represented by the above formula (2-1) is preferred.

[0050] Ring R １ and ring R ２ The ligand composed of is a cyclometallated ligand and contributes to the luminescent performance of complex (I).

[0051] L １ represents a bidentate ligand having a β-diketonate structure. １ does not affect the light-emitting performance, but L １ By having L, bioaffinity is increased, and complex (I) is easily taken up into biological tissues. １ Examples of the bidentate ligand include a bidentate ligand having a structure represented by the following formula (L-1): In this case, the complex (I) is represented by the following formula (I-1).

[0052]

[0053] Here, R ３ represents a substituted or unsubstituted alkyl group. The two oxygen atoms in the structure are each coordinated to an Ir atom.

[0054]

[0055] R ３ Examples of the alkyl group include alkyl groups having 1 to 5 carbon atoms. When the alkyl group has a substituent, examples of the substituent include halogen, a hydroxy group, a mercapto group, a carboxy group, a substituted or unsubstituted amino group, a substituted or unsubstituted amido group, a substituted or unsubstituted acyl group, an amino acid residue, a peptide residue, and the like.

[0056] Examples of the structure represented by formula (L-1) include structures represented by the following formulas (L-11), (L-12), (L-13), (L-14), and (L-15). These are the same as those of formula (L-1) except that R ３ Ga-CH ２ CH ２ COCH ２ NH (CH ２ ) ｍ CH ２ NR ４ R５ , -CH ２ CH ２ CONHCH ２ CH ２ N (CH ３ ) ２ , -CH ２ CH ２ COOH, -(CH ２ ) ｎ COR ６ , , or -CH ３ This is the structure.

[0057]

[0058] Here, m represents an integer of 1 to 5, and R ４ represents hydrogen, halogen, a hydroxy group, an amino group, a mercapto group, or a hydrocarbon group having 1 to 20 carbon atoms; R ５ represents hydrogen or a hydrocarbon group having 1 to 6 carbon atoms, n represents an integer of 1 to 5, and R ６ indicates an amino acid residue or a peptide residue.

[0059] In the formula (L-11), m is preferably an integer of 1 to 3, and particularly preferably 2. ４ The halogen in is preferably Cl, Br, or F. The amino group may be -NH2 or an alkylamino group. The hydrocarbon group having 1 to 20 carbon atoms may be linear, branched, or cyclic. It may be saturated or contain an unsaturated bond. One or more hydrogen atoms may be substituted with a substituent such as a halogen, a hydroxy group, an amino group, or a mercapto group. The number of carbon atoms is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. R ５ The hydrocarbon group having 1 to 6 carbon atoms may be linear, branched, or cyclic. It may be saturated or contain an unsaturated bond. One or more hydrogen atoms may be substituted with a substituent such as a halogen, a hydroxy group, an amino group, or a mercapto group. The number of carbon atoms is preferably 1 to 3. In the formula (L-11), R ４ and R ５ are each preferably a methyl group and n is preferably 2.

[0060] In the formula (L-14), n is preferably an integer of 1 to 3, and particularly preferably 2. The term "amino acid residue and peptide residue" refers to a residue of an amino acid or a peptide formed through an amide bond via its amino group. ６ is preferably a residue of an amino acid having a hydroxyl group, a carboxyl group, or an amino group in the side chain, or a residue of a peptide consisting of such an amino acid. Examples of amino acids having a hydroxyl group in the side chain include tyrosine, serine, threonine, etc., with tyrosine being more preferred. Examples of amino acids having a carboxyl group in the side chain include aspartic acid and glutamic acid. Examples of amino acids having an amino group in the side chain include lysine and arginine. The amino acid may be in the L- or D-form, or may be an unnatural amino acid. Examples of peptide residues include peptide residues consisting of one or more of the above amino acids, and the length is preferably 2 to 10, more preferably 2 to 5. R ６ As an example, the aspartic acid residue -NH-CH(COOH) ２ or an aspartic acid dipeptide residue, -NH-CH(COOH)-CO-NH-CH(COOH) ２ is preferred.

[0061] Among the structures represented by formula (L-1), the structures represented by formula (L-11), (L-12), (L-13), or (L-14) are preferred in terms of biocompatibility. These structures are structures in which a functional group has been introduced into the structure represented by formula (L-15) (acetylacetonate (acac)). The introduction of a functional group results in better biocompatibility.

[0062] The complex (I) is preferably a complex having a structure represented by the following formula (Ia), (Ib), (Ic), or (Id).

[0063]

[0064] Here, X １ and X ２ represents hydrogen, and L １１ ~L １４respectively represent bidentate ligands having the structure represented by the formula (L-11), (L-12), (L-13), (L-14), or (L-15). １１ ~L １４ is preferably a bidentate ligand having a structure represented by the formula (L-11), (L-12), (L-13), or (L-14).

[0065] <Compound represented by formula (II)> The compound represented by formula (II) (hereinafter, sometimes referred to as "complex (II)") will be described below. The vascular imaging reagent of the present invention may contain one type of complex (II) or two or more types of complex (II).

[0066]

[0067] Complex (II) is L １ L ２ That is, the ring R in the complex (II) is the same as the complex (I). １ and ring R ２ represents the ring R in complex (I). １ and ring R ２ and contributes to the luminescence performance of the complex (II). １ and ring R ２ A preferred embodiment of the ring R １ , and ring R ２ is the same as:

[0068] L ２ represents a bidentate ligand having a phenanthroline skeleton. ２ does not affect the light-emitting performance, but L ２ By having １ When the complex (II) has L, the bioaffinity is further increased, and the complex (II) is more easily taken up into living tissues. ２ Examples of the bidentate ligand include a bidentate ligand having a structure represented by the following formula (L-2): In this case, the complex (II) is represented by the following formula (II-1):

[0069]

[0070] Here, R ７represents a substituent having a heteroatom such as a nitrogen atom, an oxygen atom, or a sulfur atom, and X ３ represents hydrogen. The two nitrogen atoms (N) constituting the phenanthroline skeleton are each coordinated to an Ir atom.

[0071]

[0072] R ７ Specific examples of the group include an amino group, a dimethylamino group, a diethylamino group, a cyano group, an acetyl group, a carboxyl group, a piperidyl group, and a piperazyl group.

[0073] Complex (II) is preferably a complex having a structure represented by the following formula (IIa), (IIb), (IIc), or (IId).

[0074]

[0075] Here, X １ and X ２ represents hydrogen, and L ２ represents a bidentate ligand having the structure represented by the formula (L-2).

[0076] Complexes (I) and (II) may be cationic iridium complexes or neutral iridium complexes, preferably cationic iridium complexes. In the present invention, it is believed that the binding of oligoarginine to complexes (I) or (II) increases the number of cationic sites and significantly increases the vascular endothelial binding ability. Here, the charge of oligoarginine, for example, becomes +8 when octaarginine is used, and becomes +9 when a cationic iridium complex is attached, while remaining at +8 when neutral. In other words, when the number of arginines is large, the number of charges does not change significantly whether the complex is cationic or neutral, so not only cationic iridium complexes but also neutral iridium complexes can be used. The cationic iridium complex may form a salt with an anion. The anion is not limited, but for example, hexafluorophosphate ion (PF ６ － ), chloride ions (Cl － ), bromide ion (Br － ), trifluoroacetate ion (CF ３ COO－ ) and the like, and preferably PF ６ － is.

[0077] The iridium complex is preferably a compound represented by the formula (IIc), and in formula (L-2), R ７ is a piperazyl group.

[0078] Oligoarginine, complexes (I) and (II), and compounds containing oligoarginine and a luminophore can be synthesized using conventional organic synthesis methods. For example, they can be synthesized according to the methods described in the Examples below. Commercially available raw materials may be used, or those synthesized by known methods may be used.

[0079] <<Method of Use>> The vascular imaging reagent of the present invention may further contain a solvent, an additive, a compound conventionally used in vascular imaging reagents, etc., as long as the effects of the present invention are not impaired. When a solvent is contained, the vascular imaging reagent can be directly added to tissue to perform vascular imaging. The solvent may be any solvent capable of dissolving a compound containing an oligoarginine and a luminophore, and may be appropriately selected from organic solvents such as tetrahydrofuran, acetonitrile, and dimethyl sulfoxide; aqueous solvents such as water and physiological saline (e.g., 0.9% (w / v) physiological saline); and mixtures thereof. When added to a living body, a mixture of dimethyl sulfoxide and water is preferred.

[0080] When the vascular imaging reagent of the present invention is a liquid composition containing a compound containing oligoarginine and a luminophore and a solvent, the concentration of the compound containing oligoarginine and a luminophore may be 0.01 to 500 mM, or 0.1 to 100 mM, depending on the type of compound, etc.

[0081] The vascular imaging reagent of the present invention is used for visualizing blood vessels in biological tissue. The vascular imaging reagent of the present invention accumulates in the vascular endothelium of biological tissue. Therefore, the vascular imaging reagent of the present invention is useful for imaging blood vessels, particularly for imaging the vascular endothelium. The type of biological tissue to be measured is not particularly limited, and examples include organs such as skin, muscle, fat, liver, heart, pancreas, kidney, spleen, intestines, reproductive organs, and brain. The tissue may be either normal tissue or pathological tissue. The biological individual to be administered is not particularly limited, and examples include vertebrates and invertebrates, including mammals (mouse, human, pig, dog, rabbit, human, etc.).

[0082] Imaging of blood vessels in tissue can be performed, for example, as follows. The vascular imaging reagent of the present invention is added to an individual to be measured, and then the compound containing oligoarginine and a luminophore in the vascular imaging reagent incorporated into the specimen is excited and luminescence is observed. The compound can be excited by irradiating the specimen with visible light. The luminescence can be observed using known devices such as a fluorescence microscope, a fluorescence measuring device, or a fluorescence imaging device. The amount of the vascular imaging reagent of the present invention added to an individual can be appropriately adjusted depending on the individual used, vascular density, etc., but can be administered to the individual in the range of, for example, 0.01 to 1,000 μmol / kg body weight, preferably 0.1 to 100 μmol / kg body weight. The vascular imaging reagent of the present invention can be administered, for example, intravenously, subcutaneously, or intramuscularly.

[0083] <Compound of the Present Invention> Another aspect of the present invention relates to a compound represented by the following formula (hereinafter, may be referred to as "compound of the present invention").

[0084]

[0085] In the formula, n is an integer from 4 to 20.

[0086] As shown in the formula above, the compound of the present invention is a compound containing an oligoarginine and an iridium complex.

[0087] Here, n is an integer of 4 to 20, preferably an integer of 6 to 16, and more preferably an integer of 8 to 12. The iridium complex may form a salt with an anion. The anion is not limited, but examples thereof include hexafluorophosphate ion (PF ６ － ), chloride ions (Cl － ), bromide ion (Br － ), trifluoroacetate ion (CF ３ COO － ) and the like, and preferably PF ６ － is.

[0088] The compound of the present invention has excellent phosphorescence emission properties and excellent binding to vascular endothelium, and therefore can be used as a vascular imaging reagent.

[0089] Yet another aspect of the present invention relates to a compound in which oligoarginine is linked to NBD, coumarin 6, or rhodamine B via a linker. Such a compound is also a "compound of the present invention." The linker is preferably, but not limited to, a group containing polyethylene glycol. Polyethylene glycol (PEG ｐ The degree of polymerization p of oligoarginine (R) is not limited, but is, for example, 2 to 20, preferably 3 to 16, and more preferably 4 to 12, from the viewpoint of the ease of retention in the vascular endothelium and the ease of material availability. ｎ The degree of polymerization n of the formula (I) is not limited, but is, for example, 4 to 20, preferably 6 to 16, and more preferably 8 to 12. Such compounds of the present invention have excellent fluorescence emission properties and excellent binding to vascular endothelium, and therefore can be used as vascular imaging reagents.

[0090] The compounds of the present invention can be synthesized using conventional organic synthesis methods. For example, they can be synthesized according to the methods described in the Examples below. The raw materials used may be commercially available products or those synthesized by known methods.

[0091] The present invention will be specifically described below with reference to examples, but these are merely examples of the present invention and the scope of the present invention is not limited to these examples.

[0092] The abbreviations have the following meanings: btq: [2-(2'-benzothienyl)-quinolinate-N,C3'] phen-pipe: 5-piperazinyl-1,10-phenanthroline BTQ: (btq)2Ir(phen-pipe) RhB: Rhodamine B NBD: Nitrobenzofurazan C6: Coumarin 6

[0093] <Synthesis Example> The compounds of the present invention were synthesized according to the following synthesis scheme.

[0094]

[0095] (Boc-[R(Pbf)]4-OH) Boc-[R(Pbf)]4-OH was synthesized by Fmoc solid-phase synthesis using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityl resin (1.7 g, 0.30 mmol / g) containing R(Pbf). The condensation reaction was carried out at room temperature for 1 hour using 0.5 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH as amino acids, 0.6 M DMF solution of HBTU as a condensing agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base. The amounts of the respective components added were 3, 3, 3, and 6 equivalents relative to the molar amount of the amino acids. The Fmoc protecting group was removed using a 2% DBU solution in DMF, with 3.5 equivalents per mole of amino acid introduced, and the mixture was incubated at room temperature for 5 minutes. To ensure complete deprotection, the mixture was incubated again for 10 minutes under the same conditions. To cleave the peptide from the resin, 10 mL of a 1% TFA solution in dichloromethane was added to the vial containing the peptide and resin, and the mixture was incubated at room temperature for 90 minutes. The cleaved peptide solution was evaporated to dryness under reduced pressure and redissolved in a small amount of acetonitrile. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a white solid (734 mg, 0.42 mmol, crude product yield: 84%).

[0096] (BTQ-[R(Pbf)]4-Boc) Boc-[R(Pbf)]4-OH (116 mg, 0.065 mmol), (btq)2Ir(phen-pipe) PF6 CF3COO (63 mg, 0.051 mmol), and HATU (41 mg, 0.10 mmol) were added to a 50 mL eggplant-shaped flask and dissolved in 1 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a red solid (390 mg, 0.14 mmol, crude product yield: 270%).

[0097] (BTQ-R4) BTQ-[R(Pbf)]4-Boc (279 mg, 0.10 mmol) was weighed into a 50 mL centrifuge tube, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether was removed from the vial, and the mixture was dried in a desiccator to obtain a red solid (82 mg, 0.04 mmol, crude product yield: 40%). The red solid was purified by reverse-phase column chromatography to obtain BTQ-R4. Identification was performed using MALDI-TOF-MS, detecting a singly charged ion peak (m / z): calcd.: 1601.9 [M-PF6 - ] + , found: 1601.2

[0098] (Boc-[R(Pbf)]8-OH) Boc-[R(Pbf)]8-OH was synthesized by Fmoc solid-phase synthesis using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityl resin (1.67 g, 0.30 mmol / g) containing R(Pbf). The condensation reaction was carried out at room temperature for 1 hour using 0.6 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH as amino acids, 0.5 M DMF solution of HBTU as a condensing agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base. The condensation reaction was carried out using 3, 3, 3, and 6 equivalents of each of these amino acids relative to the molar amount of the amino acids. The Fmoc protecting group was deprotected using a 2% DBU solution in DMF, with 3.5 equivalents per mole of amino acid introduced, and the mixture was incubated at room temperature for 5 minutes. To ensure complete deprotection, the mixture was incubated again for 10 minutes under the same conditions. To cleave the peptide from the resin, 10 mL of a 1% TFA solution in dichloromethane was added to the vial containing the peptide and resin, and the mixture was incubated at room temperature for 90 minutes. The cleaved peptide solution was evaporated to dryness under reduced pressure and redissolved in a small amount of acetonitrile. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a white solid (1.26 g, 0.37 mmol, crude product yield: 74%).

[0099] (BTQ-[R(Pbf)]8-Boc) Boc-[R(Pbf)]8-OH (209 mg, 0.062 mmol), (btq)2Ir(phen-pipe) PF6 CF3COO (62 mg, 0.050 mmol), and HATU (40 mg, 0.11 mmol) were added to a 50 mL eggplant-shaped flask and dissolved in 1 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a red solid (235 mg, 0.055 mmol, crude product yield: 110%).

[0100] (BTQ-R8) BTQ-[R(Pbf)]8-Boc (110 mg, 0.026 mmol) was weighed into a 50 mL centrifuge tube, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether was removed from the vial, and the mixture was dried in a desiccator to obtain a red solid (72 mg, 0.022 mmol, crude product yield: 85%). The red solid was purified by reverse-phase column chromatography to obtain BTQ-R8. Identification was performed using MALDI-TOF-MS, detecting a singly charged ion peak (m / z): calcd.: 2226.6 [M-PF6 - ] + , found: 2224.8

[0101] (Boc-[R(Pbf)] 12 -OH) Boc-[R(Pbf)] 12-OH was synthesized by Fmoc solid-phase synthesis using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityl resin (1.0 g, 0.30 mmol / g) with R(Pbf) incorporated. The condensation reaction was carried out using 0.5 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH as amino acids, 0.6 M DMF solution of HBTU as a condensing agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base. The reaction was carried out at room temperature for 1 hour, with 3, 3, 3, and 6 equivalents of each, based on the number of moles of amino acid introduced. The Fmoc protecting group was deprotected using 2% DBU in DMF, with 3.5 equivalents based on the number of moles of amino acid introduced, and the reaction was carried out at room temperature for 5 minutes. To ensure complete deprotection, the reaction was carried out again for 10 minutes under the same conditions. To cleave the peptide from the resin, 10 mL of a 1% TFA solution in dichloromethane was added to the vial containing the peptide resin and allowed to react at room temperature for 90 minutes. The cleaved peptide solution was evaporated to dryness under reduced pressure and redissolved in a small amount of acetonitrile. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a white solid (999 mg, 0.20 mmol, crude product yield: 67%).

[0102] (BTQ-[R(Pbf)] 12 -Boc) Boc-[R(Pbf)] in a 50 mL eggplant flask 12-OH (321 mg, 0.064 mmol), (btq)2Ir(phen-pipe) PF6 CF3COO (62 mg, 0.050 mmol), and HATU (0.040 mg, 0.10 mmol) were dissolved in 1 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under nitrogen. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a red solid (320 mg, 0.055 mmol, crude product yield: 110%).

[0103] (BTQ-R 12 ) BTQ-[R(Pbf)] in a 50 mL centrifuge tube 12 1 mL of TFA:water:TIPS (95:2.5:2.5) was added to the solution, and the mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed, and the mixture was dried in a desiccator to obtain a red solid (93 mg, 0.021 mmol, crude product yield: 81%). The red solid was purified by reverse-phase column chromatography to obtain BTQ-R 12 Identification was performed using MALDI-TOF-MS, and a singly charged ion peak (m / z) was detected. Calcd.: 2851.4 [M-PF6 - ] + , found: 2846.8

[0104] (Boc-[R(Pbf)] 16 -OH) Boc-[R(Pbf)] 16-OH was synthesized by Fmoc solid-phase synthesis using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityl resin (0.34 g, 0.30 mmol / g) with R(Pbf) incorporated. The condensation reaction was carried out using 0.3 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH as amino acids, 0.6 M DMF solution of HBTU as a condensing agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base. The reaction was carried out at room temperature for 1 hour using 3, 3, 3, and 6 equivalents of each of these components relative to the number of moles of amino acid introduced. The Fmoc protecting group was deprotected using 2% DBU in DMF, 3.5 equivalents relative to the number of moles of amino acid introduced, and the reaction was carried out at room temperature for 5 minutes. To ensure complete deprotection, the reaction was continued for another 10 minutes under the same conditions. To cleave the peptide from the resin, 10 mL of a 1% TFA solution in dichloromethane was added to the vial containing the peptide resin and the reaction was continued at room temperature for 90 minutes. The cleaved peptide solution was evaporated to dryness under reduced pressure and redissolved in a small amount of acetonitrile. The solution was transferred to a 50 mL centrifuge tube and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a white solid (418 mg, 0.063 mmol, crude product yield: 63%).

[0105] (BTQ-[R(Pbf)] 16 -Boc) Boc-[R(Pbf)] in a 50 mL eggplant flask 16-OH (183 mg, 0.028 mmol), (btq)2Ir(phen-pipe) PF6 CF3COO (37 mg, 0.030 mmol), and HATU (35 mg, 0.09 mmol) were dissolved in 1 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under nitrogen. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a red solid (186 mg, 0.024 mmol, crude product yield: 87%).

[0106] (BTQ-R 16 ) BTQ-[R(Pbf)] in a 50 mL centrifuge tube 16 1 mL of TFA:water:TIPS (95:2.5:2.5) was added to the solution, and the mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed, and the mixture was dried in a desiccator to obtain a red solid (73 mg, 0.013 mmol, crude product yield: 87%). The red solid was purified by reverse-phase column chromatography to obtain BTQ-R 16 Identification was performed using MALDI-TOF-MS, and a singly charged ion peak (m / z) was detected. Calcd.: 3474.8 [M-PF6 - ] + , found: 3473.7

[0107] Example 1 Figure 2 shows the structural formulas of the compounds of the present invention synthesized in the examples (BTQ-R4, BTQ-R8, BTQ-R 12 , BTQ-R 16) are shown. The number of arginine residues is 4, 8, 12, or 16. To demonstrate the effect of arginine, a compound without arginine (BTQ) was used as a reference compound. Measurement of the absorption and phosphorescence spectra of these compounds revealed an absorption maximum near 500 nm and a phosphorescence maximum near 660 nm. Because phosphorescence was observed above 600 nm, multicolor imaging using green-emitting molecules is possible. Furthermore, the phosphorescence quantum yield and phosphorescence lifetime were measured to be 0.32 (0.015 μs under air saturation) and 5.7 μs (0.28 μs under air saturation), respectively. Even though the number of arginine residues differs, the compounds have nearly identical spectral and photophysical properties because the same phosphorophore is attached to the oligoarginine peptide.

[0108] The synthesized compound and FITC-tomato lectin, a commercially available green fluorescent vascular endothelial staining reagent, were administered to mice, and imaging of kidney capillaries was performed. Figure 3 shows the results of FITC-tomato lectin (1 mg / mL in saline, 50 μL administered), BTQ (100 nmol: 1 mmol / L in saline: dimethyl sulfoxide (9:1, v / v) mixed solvent, 100 μL administered), and BTQ-R. n (n: 4, 8, 12, 100 nmol: 1 mmol / L (in physiological saline) 100 μL) was administered through the tail vein of anesthetized mice, and the ventral region was incised to expose the kidney. Images of the kidney surface were taken using a confocal laser microscope. Compared to the image of FITC-tomato lectin, BTQ and BTQ-R4 emitted light from a different region (renal tubular cells) than FITC-tomato lectin, while BTQ-R8 and BTQ-R 12 It can be seen that the kidney capillaries are imaged in the same way as FITC-tomato lectin. 12 In this study, cavities were observed in the blood vessels, and therefore BTQ-R 12 These results suggest that a protein with eight or more arginine residues is preferable for imaging the vascular endothelium.

[0109] Since the kidney is an organ with strong autofluorescence, there is a concern that the autofluorescence of the kidney as well as the vascular endothelium may be detected when FITC-tomato lectin is used. Therefore, we used FITC-tomato lectin (1 mg / mL in saline, 50 μL administered) and BTQ-R. 12 (100 nmol: 100 μL of 1 mmol / L (in physiological saline) was administered) was administered to mice, and imaging was performed at the same location. As shown in Figure 4A and C, when imaging with FITC-tomato lectin fluorescence, fluorescence was observed from areas other than the vascular endothelium. It is impossible to distinguish whether this fluorescence is from FITC-tomato lectin or from autofluorescence. On the other hand, BTQ-R 12 When imaging with phosphorescence from the vascular endothelium (Figure 4B and D), eliminating the fluorescent component allows for the observation of only the phosphorescence from the vascular endothelium, enabling clearer and selective imaging of the vascular endothelium. The kidney filters blood through the glomeruli to excrete waste products and salt from the blood as urine. Many glomeruli are located more than approximately 200 μm below the kidney surface, making them difficult to image with a single-photon microscope. Here, a mouse was euthanized, and the kidney was removed and cut into two pieces, and the cross-sections were observed. Round, glowing areas can be seen as shown in Figure 5A. Magnification reveals a collection of linear structures, allowing for the imaging of the vascular network converging on the glomerulus (Figure 5B).

[0110] Next, we performed vascular imaging of the liver. The liver is a collection of hexagonal functional units called hepatic lobules, with arteries and portal veins near the apexes and a central vein in the center, from which sinusoidal vessels radiate. Figure 6 shows the BTQ-R 12 This shows a phosphorescence microscope image obtained after administration of 100 μL of 100 nmol (1 mmol / L in physiological saline) to a mouse. Sinusoidal blood vessels are clearly imaged. Similar experiments were performed in other organs (pancreas, small intestine, subcutaneous tissue, heart), demonstrating that imaging of capillaries in many organs is possible (Figure 7).

[0111] It has been pointed out that while the vascular structure in normal tissues is robust, cancer tumors have many underdeveloped and fragile new blood vessels. Therefore, anticancer drugs administered into the bloodstream do not reach all cancer cells, and the dosage must be increased. This also leads to an increase in side effects, placing a heavy burden on patients. Here, we will discuss the effects of BTQ-R 12 We attempted to image tumor blood vessels using BTQ-R. We transplanted human colon adenocarcinoma-derived HCT116 cells into nude mice to generate tumor-bearing mice. Figure 8 shows the results of tumor imaging using BTQ-R. 12 This shows a phosphorescence microscope image obtained after administration of 100 μL of 100 nmol (1 mmol / L in saline) to a tumor-bearing mouse. The image shows that many thin blood vessels branch off from large blood vessels, demonstrating successful imaging of the vascular network within the tumor.

[0112] In recent years, attention has been focused on early detection of lipid accumulation in the liver, as fatty liver can progress to cirrhosis and liver cancer. In particular, non-alcoholic fatty liver disease, unlike obesity, is not visible from the outside, so there is a risk that the disease may be discovered late. In a fatty liver, lipids accumulate abnormally within hepatocytes, large lipid droplets form, and hepatocytes become enlarged. This causes the sinusoidal blood vessels to narrow, resulting in hypoxia throughout the liver. Here, we used a green fluorescent lipid droplet reagent (3-(benzo[d]thiazol-2-yl)-8-(diethylamino)-2H-benzo[g]chromen-2-one (PC6S)) and BTQ-R. 12 100 μL of PC6S: 50 nmol: 0.5 mmol / L (physiological saline: dimethyl sulfoxide (9:1, v / v), mixed solvent containing 10 wt% BSA) was administered simultaneously to healthy mice and fatty liver model mice. 12 Multicolor imaging was performed by measuring green fluorescence and deep red phosphorescence after administration of 100 μL of 100 nmol / 1 mmol / L (in physiological saline). In healthy mice, PC6S fluorescence was detected from lipid droplets in hepatocytes, and BTQ-R fluorescence was detected from the sinusoidal endothelium. 12Phosphorescence from the erythrocytes was observed (Figure 9A). The image shows that the sinusoidal vessels run in a straight line between the hepatocytes. On the other hand, in the fatty liver model mice, the hepatocytes become enlarged due to the formation of large lipid droplets, and the sinusoidal vessels become highly tortuous (Figure 9B). This is thought to impede the flow of red blood cells, resulting in a lack of oxygen supply to the liver and a state of hypoxia.

[0113] Synthesis Example 2 The compound of the present invention was synthesized as follows.

[0114] (RhB-pipe-PEG 12 Boc-piperazine (35.6 mg, 0.19 mmol), rhodamine B (73.4 mg, 0.15 mmol), and HATU (58.3 mg, 0.15 mmol) were added to a 100 mL eggplant-shaped flask and dissolved in anhydrous CHCl (5 mL) and DIEA (170 μL). The mixture was then reacted at room temperature under a nitrogen atmosphere for 24 hours. The precipitated crystals were washed with chloroform and dried. The Boc group was then deprotected by reacting with 4N HCl / dioxane (10 mL) at room temperature for 2 hours. The product was washed with anhydrous THF and hexane and dried to obtain a reddish-purple solid (0.224 g, 0.41 mmol, crude yield: 268%).

[0115] Next, t-Boc-N-amido-dPEG was added to a 100 mL eggplant-shaped flask. 12 A mixture of RhB-acid (100 mg, 0.14 mmol), RhB-pipe (79.5 mg, 0.15 mmol), and HATU (105 mg, 0.28 mmol) was added and dissolved in anhydrous CHCl (5 mL) and DIEA (170 μL). The mixture was then incubated at room temperature under a nitrogen atmosphere for 24 hours. The precipitated crystals were washed with chloroform and dried to give a reddish-purple solid (0.399 g, 0.320 mmol, crude yield: 166%). The Boc group was then deprotected by incubation with 4N HCl / dioxane (10 mL) at room temperature for 3 hours. The mixture was washed with anhydrous THF and methanol and dried to give a reddish-purple solid (99.0 mg, 0.086 mmol, product yield: 62%).

[0116] (RhB-pipe-PEG 12 -[R(Pbf)] 12 -Boc) In 100 mL of eggplant flask, add Boc-[R(Pbf)] 12 -OH (286 mg, 0.057 mmol), RhB-pipe-PEG 12 -NH2 (78 mg, 0.068 mmol) and HATU (44.5 mg, 0.117 mmol) were added and dissolved in 10 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a reddish-purple solid (157 mg, 0.026 mmol, crude product yield: 45%).

[0117] (RhB-pipe-PEG 12 -R 12 ) Place RhB-pipe-PEG in a 50 mL centrifuge tube. 12 -[R(Pbf)] 12 A weighed amount of -Boc (21.5 mg, 0.0035 mmol) was added to 1 mL of TFA:water:TIPS (95:2.5:2.5), and the mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed, and the mixture was dried in a desiccator to obtain a red solid (21 mg, 0.0042 mmol, crude product yield: 119%). The reddish-purple solid was purified by reverse-phase column chromatography to obtain RhB-pipe-PEG. 12 -R 12 Identification was performed using ESI-MS, and a triply charged ion peak (m / z) was detected. Calcd.: 995.48 [M;3H] 3+ , found:996.6

[0118]

[0119] (NBD-PEG4-NH2) Boc-NH-PEG4-amine (521 mg, 1.54 mmol), 4-Fluoro-7-nitrobenzofurazan (210 mg, 1.14 mmol), anhydrous CHCl (5 mL), and DIEA (0.17 mL) were dissolved in a 100 mL eggplant-shaped flask and stirred at room temperature for 24 hours under nitrogen. After washing with chloroform and hexane, a yellow liquid was obtained. The Boc group was then deprotected by reaction with 4N HCl / dioxane (10 mL) at room temperature for 3 hours. After washing with anhydrous THF and methanol and drying, a viscous yellow liquid (0.614 g, 1.53 mmol, crude yield: 134%) was obtained.

[0120] (NBD-PEG4-[R(Pbf)] 12 -Boc) In 100 mL of eggplant flask, add Boc-[R(Pbf)] 12 -OH (429 mg, 0.085 mmol), NBD-PEG4-NH2 (0.10 mmol), and HATU (65.9 mg, 0.17 mmol) were added and dissolved in 8 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a yellow solid (313 mg, 0.058 mmol, crude product yield: 68%).

[0121] (NBD-PEG4-R 12 ) NBD-PEG4-[R(Pbf)] in a 50 mL centrifuge tube 12NBD-PEG4-R (30.2 mg, 0.0060 mmol) was weighed out, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was allowed to react at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed, and the mixture was dried in a desiccator to obtain a yellow solid (13 mg, 0.0057 mmol, crude product yield: 95%). The yellow solid was purified by reverse-phase column chromatography to obtain NBD-PEG4-R 12 The compound was identified using ESI-MS, and a quadruply charged ion peak (m / z) was detected. Calcd.: 569.1 [M; 4H] 4+ , found:596.4

[0122]

[0123] (C6-PEG4-NH2) Boc-NH-PEG4-amine (79.0 mg, 0.24 mmol), Coumarin 6-COOH (79.3 mg, 0.20 mmol), and HATU (154.8 mg, 0.41 mmol) were added to a 100 mL eggplant-shaped flask, dissolved in anhydrous CHCl (11 mL) and DIEA (170 μL), and incubated at room temperature under a nitrogen atmosphere for 24 hours. After washing with chloroform, a yellow solid was obtained. The Boc group was then deprotected by incubation with 4N HCl / dioxane (10 mL) at room temperature for 2 hours. After washing with anhydrous THF and methanol and drying, a viscous, deep-red solid was obtained (0.184 g, 0.300 mmol, crude yield: 150%).

[0124] (C6-PEG4-[R(Pbf)] 12 -Boc) In 100 mL of eggplant flask, add Boc-[R(Pbf)] 12-OH (413 mg, 0.082 mmol), C6-PEG4-NH2 (63.0 mg, 0.10 mmol), and HATU (62.3 mg, 0.17 mmol) were added and dissolved in 10 mL of dehydrated DMF. 0.17 mL of DIEA was added to this solution, and the mixture was stirred at room temperature for 24 hours under a nitrogen atmosphere. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This procedure was repeated twice. The water in the vial was removed by lyophilization, yielding a yellow solid (326 mg, 0.058 mmol, crude product yield: 71%).

[0125] (C6-PEG4-R 12 ) C6-PEG4-[R(Pbf)] in a 50 mL centrifuge tube 12 46.0 mg (0.0082 mmol) of -Boc was weighed out, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added, followed by reaction at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed, and the mixture was dried in a desiccator to obtain a yellow solid (34 mg, 0.0014 mmol, crude product yield: 166%). The yellow solid was purified by reverse-phase column chromatography to obtain C6-PEG4-R 12 Identification was performed using ESI-MS, and a quadruply charged ion peak (m / z) was detected. Calcd.: 622.4 [M; 4H] 4+ , found:622.8

[0126]

[0127] Example 2: RhB-pipe-PEG 12 -R 12 RhB-pipe-PEG was administered to mice, and blood vessel imaging of various tissues was performed. 12 -R 12(50μL of 100 nmol:2 mmmol / L (physiological saline) was administered) was administered via the tail vein of an anesthetized mouse, and the ventral region was incised to expose various organs (liver, kidney, spleen, pancreas, testes) and tissues (muscle tissue, subcutaneous tissue). Images of their surfaces were then captured using a confocal laser microscope. The excitation wavelength was 550 nm, and the observation wavelength was >590 nm. Capillaries were successfully imaged in all organs and tissues observed.

[0128] NBD-PEG-R 12 NBD-PEG-R was administered to mice and blood vessel imaging of various tissues was performed. 12 (50 μL of 100 nmol: 2 mmmol / L (physiological saline) was administered) was administered via the tail vein of an anesthetized mouse, and the ventral region was incised to expose various organs (liver, kidney, spleen) and tissues (subcutaneous tissue, adipose tissue). Images of their surfaces were then captured using a confocal laser microscope. The excitation wavelength was 488 nm, and the observation wavelength was 510-550 nm. Capillaries were successfully imaged in all organs and tissues observed.

[0129] C6-PEG-R 12 C6-PEG-R was administered to mice and blood vessel imaging of various tissues was performed. 12 (50 μL of 100 nmol: 2 mmmol / L (physiological saline) was administered) was administered via the tail vein of an anesthetized mouse, and the ventral region was incised to expose various organs (kidney, spleen, pancreas, testis) and tissues (adipose tissue, muscle tissue). Images of their surfaces were then imaged using a confocal laser microscope. The excitation wavelength was 488 nm, and the observation wavelength was 510-550 nm. Capillaries were successfully imaged in all organs and tissues observed. After euthanasia of the mouse, imaging of cerebral blood vessels was also performed. As shown in Figure 13, it was demonstrated that imaging of cerebral blood vessels is also possible.

[0130] From the above results, it was found that the compound containing oligoarginine and a luminophore can be used to clearly image blood vessels in an individual, and can be used as a blood vessel imaging reagent.n can be used as a vascular imaging agent. 12 is a new reagent that can image the vascular course of normal and pathological tissues within an individual. Furthermore, distribution to the vascular endothelium is the action of oligoarginine peptides, and by changing the luminophore (BTQ), it is possible to develop vascular endothelial imaging reagents with various luminescent colors. Furthermore, the RhB-pipe-PEG developed by this invention p -R n , NBD-PEG p -R n , C6-PEG p -R n can also be preferably used as a blood vessel imaging reagent.

[0131] The present invention can be used in fields such as medical diagnosis, pharmaceutical development, and basic medicine.

Claims

1. An oligoarginine represented by the following formula: A phosphorophore or fluorophore bound to the C-terminus or N-terminus of the oligoarginine; and The phosphorophore is a compound comprising an iridium complex; Vascular imaging reagents: 【Chemistry 1】 During the ceremony, n is an integer of 8 or greater.

2. The reagent according to claim 1, wherein the iridium complex is a compound represented by the following formula (I) or (II): 【Chemistry 2】 In formula (I), Ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, Ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring, L １ represents a bidentate ligand having a β-diketonate structure; 【Chemistry 3】 In formula (II), Ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, Ring R ２ represents a monocyclic or polycyclic sulfur-containing aromatic ring, L ２ represents a bidentate ligand having a phenanthroline skeleton.

3. The reagent according to claim 1 or 2, wherein the iridium complex is a compound represented by the following formula: 【Chemistry 4】 During the ceremony, n is an integer of 8 or greater.

4. A compound represented by the formula: 【Chemistry 5】 During the ceremony, n is an integer of 8 or greater.

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