Vascular imaging reagents

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

[Technical Field]

[0001] This invention relates to a vascular imaging reagent and compounds that can be used in the same reagent. [Background technology]

[0002] Cells in living tissues (organs) obtain necessary nutrients and oxygen from the blood and release synthesized metabolites and carbon dioxide into the bloodstream. Therefore, the vascular system plays a crucial role in maintaining life. If an abnormality occurs in the vascular system for any reason, the surrounding tissue cells will not be able to function properly. In particular, arteriosclerosis, in which lipid components such as cholesterol accumulate in the intima of the arteries and form atherosclerotic plaques called atheroma, and cirrhosis of the liver, in which fibrosis occurs between blood vessels and liver cells, are deeply involved in the breakdown of the vascular system. Furthermore, in cancer, the formation of new angiogenesis cannot keep up with the abnormal proliferation of cells, resulting in the observation of numerous 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 the above-mentioned disease conditions.

[0003] In clinical vascular imaging, methods such as administering contrast agents into the bloodstream and imaging with MRI, and photoacoustic imaging techniques are known. While these methods provide insights into the human vascular network, the equipment is large and unsuitable for small animals. Furthermore, they lack single-cell level spatial resolution. On the other hand, optical imaging techniques offer high sensitivity, simplicity, and real-time measurement, and when combined with a microscope, can easily achieve single-cell level spatial resolution. A crucial element here is a probe molecule that exists stably in a congested environment such as blood and exhibits high-luminosity emission.

[0004] Vascular imaging reagents are classified into blood-retention type and vascular endothelial adsorption type. The former are reagents that remain in the bloodstream, and fluorescent dextran and fluorescent nanoparticles have been developed for this purpose. Fluorescent dextran, in particular, 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] On the other hand, fluorescent lectins are commercially available as vascular endothelial adsorption imaging reagents. Lectins are a general term for proteins that exhibit specific binding to particular 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 bonded to tomato lectin have been commercially available (Vector Laboratories). However, the synthesis of fluorescent lectins requires the complicated process of extracting tomato lectin from nature, and the number of fluorescent molecules bound to the lectin cannot be controlled.

[0006] In other words, fluorescent lectins have the following challenges: Because they recognize sugar chains present on the surface of vascular endothelial cells, only plant-derived lectins can be used; the extraction of lectins from natural sources is a complicated process; because lectins are glycoproteins, the labeling of the luminescent phore is non-uniform, resulting in differences between batches; and because they utilize fluorescence, they can sometimes be indistinguishable from the autofluorescence of tissue.

[0007] On the other hand, methods have been proposed for quantifying oxygen concentration in biological tissues and cells using iridium(III) complexes having cyclometallated ligands such as btq(2-(2'-benzothienyl)-quinolinate-N,C3'), or compounds containing the same iridium complex and a fluorescent compound (for example, Patent Documents 1 and 2). However, Patent Documents 1 and 2 do not consider using this method for visualizing blood vessels. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2018-65768 [Patent Document 2] Japanese Patent Publication No. 2019-7961 [Overview of the project] [Problems that the invention aims to solve]

[0009] This invention has been made in view of the above-mentioned problems, and aims to develop a new vascular imaging reagent that is easy to manufacture, highly sensitive, and highly accurate. [Means for solving the problem]

[0010] The inventors of this invention conducted diligent research to solve the above problems and found that blood vessels in an organism can be clearly imaged using a compound containing oligoarginine and a phosphorescent or fluorescent luminescent group. Furthermore, they developed a compound containing oligoarginine and an iridium complex that can be used as such a luminescent group. Based on these findings, the present invention was completed. In other words, the gist of this invention is as follows:

[0011] [1] Oligoarginine represented by the following formula, A phosphorus or fluorophore bound to the C-terminal or N-terminal side of the oligoarginine, Vascular imaging reagents containing compounds including:

[0012] [ka]

[0013] During the ceremony, n is an integer between 4 and 20. [2] The reagent according to [1], wherein the oligoarginine is n = 8 or greater. [3] The reagent according to [1] or [2], wherein the phosphorescent group is a compound containing an iridium complex. [4] The iridium complex is a compound represented by the following formula (I) or (II), as described in the reagent in [3]:

[0014] [ka]

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

[0016] [ka]

[0017] In formula (II), Ring R 1 This represents a monocyclic or polycyclic nitrogen-containing aromatic ring. Ring R 2 This represents a monocyclic or polycyclic sulfur-containing aromatic ring. L 2 This exhibits a bidentate ligand with a phenanthroline skeleton.

[0018] [5] The iridium complex is a compound represented by the following formula, the reagent as described in [3] or [4]:

[0019] [ka]

[0020] During the ceremony, n is an integer between 4 and 20. [6] Compounds represented by the following formula:

[0021] [ka]

[0022] During the ceremony, n is an integer between 4 and 20. [Effects of the Invention]

[0023] The present invention provides a vascular imaging reagent comprising a compound containing oligoarginine and a luminescent phosphatid. Since the compound for imaging reagents can be chemically synthesized, extraction from natural products is unnecessary, and the reagent can be easily manufactured. Furthermore, the labeling of the luminescent phosphatid can be made uniform, resulting in a consistent reagent with minimal lot-to-lot variation, enabling highly accurate imaging. When phosphorescent compounds are used as the luminescent phosphonate, autofluorescence can be eliminated by time-resolved measurements, enabling highly sensitive and accurate imaging. In particular, compounds with eight or more arginine molecules bound to them exhibit excellent binding to vascular endothelium, allowing for even more sensitive and accurate imaging of the vascular endothelium. Even when using fluorescent compounds, if the compounds exhibit fluorescence in the near-infrared region, it becomes possible to distinguish them from autofluorescence, and furthermore, it becomes possible to image the structure of deep blood vessels. Furthermore, the present invention provides a compound containing oligoarginine and an iridium complex that can be used as a luminescent phosphodiester in vascular imaging reagents. This compound has excellent phosphorescent performance and can clearly image blood vessels. Furthermore, the present invention provides a compound containing oligoarginine and a fluorescent compound that can be used as a luminescent phosphodiester in vascular imaging reagents. This compound has excellent fluorescent performance and can clearly image blood vessels. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows a conceptual diagram of the compound of the present invention (a vascular imaging reagent). [Figure 2] Figure 2 shows the structural formula of the synthesized compound of the present invention (BTQ-Rn (n = 4, 8, 12, 16)). [Figure 3] Figure 3 shows imaging images of renal capillaries (photograph used as a substitute for diagram). A shows results using FITC-tomato lectin, B shows results using BTQ, and C, D, and E show results using BTQ-Rn (n = 4, 8, 12), respectively. [Figure 4]Figure 4 shows renal capillary imaging images (photograph used as a substitute for the diagram). A shows the results for FITC-lectin (objective lens ×40), B shows the results for BTQ-R12 (objective lens ×40), C shows the results for FITC- (objective lens ×100), and D shows the results for BTQ-R12 (objective lens ×100). [Figure 5] Figure 5 shows images (photographs used as a substitute for diagrams) of a kidney section (glomerulus). A shows the results with a 20x objective lens, and B shows the results with a 10x objective lens. [Figure 6] Figure 6 shows imaging images (photographs used as a substitute for diagrams) of the sinusoidal vessels of the liver. Images A and B are images of different locations within the liver. [Figure 7] Figure 7 shows vascular imaging images (photographs used as a substitute for diagrams) of various organs. A shows the pancreas, B shows the small intestine, C shows the subcutaneous tissue, and D shows the heart. [Figure 8] Figure 8 shows tumor vascular imaging images (photographs used as a substitute for diagrams) of tumor-bearing mice. Images A-C show different locations within the tumor. [Figure 9] Figure 9 shows multicolor imaging images (photograph used as a substitute for diagram) of PC6S and BTQ-R12 in the liver. Column A shows results from healthy mice, and column B shows results from fatty liver model mice. The left side of the column shows PC6S, the center shows BTQ-R12, and the right side shows the superimposed results of these two. [Figure 10] Figure 10 shows vascular imaging images of various organs using RhB-pipe-PEG12-R12 (photographs used as a substitute for illustrations). A is the liver, B and C are the kidneys (C is a magnified view of a part of B), D is the spleen, E is the pancreas, F is the testes, G is muscle tissue, and H is subcutaneous tissue. [Figure 11] Figure 11 shows vascular imaging images of various organs using NBD-PEG4-R12 (photographs used as a substitute for illustrations). A shows the liver, B shows the kidney, C shows the spleen, D shows the subcutaneous tissue, and E shows the adipose tissue results. [Figure 12] Figure 12 shows vascular imaging images of various organs using C6-PEG4-R12 (photographs used as a substitute for illustrations). A shows the results for the kidney, B for the spleen, C for the pancreas, D for the testes, E for adipose tissue, and F for muscle tissue. [Figure 13]Figure 13 shows imaging images of cerebral blood vessels using C6-PEG4-R12 (photograph used as a substitute for the diagram). A, B, and C are images of different locations in the brain. D is a magnified view of a part of C. [Modes for carrying out the invention]

[0025] The present invention will be described below.

[0026] <Vascular imaging reagents> One aspect of the present invention relates to an oligoarginine represented by the following formula, A phosphorus or fluorophore bound to the C-terminal or N-terminal side of the oligoarginine, Regarding a vascular imaging reagent (hereinafter sometimes referred to as "the vascular imaging reagent of the present invention") containing a compound including:

[0027] [ka]

[0028] During the ceremony, n is an integer between 4 and 20.

[0029] The vascular imaging reagent developed in this invention contains a compound having a structure in which a luminescent phose is bound to either the C-terminus or N-terminus of an oligoarginine peptide (Figure 1). The luminescent phose may be bound to either the C-terminus or N-terminus of the oligoarginine peptide, but is preferably the C-terminus. Both fluorophores (fluorescent compounds) and phosphorescent compounds (phosphorescent compounds) can be used as the luminescent phose. 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 the structure of deep blood vessels can also be imaged.

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

[0031] [ka]

[0032] Here, n is an integer between 4 and 20, preferably an integer between 6 and 16, and more preferably an integer between 8 and 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 amount of oligoarginine is n ≥ 8 or greater, it exhibits particularly excellent adsorption to vascular endothelium, and therefore this embodiment can be preferably used when the purpose is vascular endothelial imaging.

[0034] The compounds used in the vascular imaging reagents of the present invention may contain groups other than oligoarginine, as long as they do not impair the effects of the present invention. The molecule may contain one or more groups other than oligoarginine. The groups other than oligoarginine may be bound to the end of the oligoarginine molecule. That is, the groups other than oligoarginine may be located between the oligoarginine molecule and the luminescent foci (this configuration is sometimes referred to as a "linker"), or they may be located on the opposite side of the oligoarginine molecule from its bond to the luminescent foci. In general, groups containing steroids, peptides, and polyethylene glycol can be relatively easily bound to luminescent phosphodienes, and therefore can be suitably used as groups other than oligoarginine. When oligoarginine and groups other than oligoarginine, consisting of peptides, are included, 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. Other peptides besides oligoarginine are not limited, but proline is preferred from the viewpoint of ease of synthesis. Aspartic acid and lysine are also preferred because they can give the compound water solubility. When the material contains oligoarginine and groups other than oligoarginine, which consist of groups including polyethylene glycol, the degree of polymerization of polyethylene glycol can be adjusted according to the type of luminescent phosphodiol, for example, 2 to 20, preferably 3 to 16, and more preferably 4 to 12. In the compounds used in the vascular imaging reagents of the present invention, both embodiments containing groups other than oligoarginine and embodiments without such groups can be suitably used. In the case of compounds having a fluorophore, embodiments containing groups other than oligoarginine as linkers can be more preferably used.

[0035] ≪Fluorescent Group≫ The fluorophore contained in the compound used in the vascular imaging reagent of the present invention can be any fluorescent compound conventionally used for imaging blood vessels, etc., without particular limitation. Furthermore, the compound of the present invention described below can be used as a compound used in a vascular imaging reagent having a fluorophore. One or more fluorescent compounds can be used. Examples of fluorescent compounds, though not limited to them, include 4-nitrobenzo-2-oxa-1,3-diazole (NBD), dimethylaminosulfonylbenzoxadiazole (DBD), dimethylaminosulfonylbenzothiadiazole (DBThD), dimethylaminosulfonylbenzoselenadiazole (DBSeD), fluorescein isothiocyanate (FITC), or coumarin dyes, rhodamines, borondipyrometen (BODIPY), cyanine dyes, etc. Preferably, fluorescent compounds include NBD, coumarin dyes such as coumarin 6, and rhodamines such as rhodamine B.

[0036] ≪Rinkodan≫ As the phosphor group contained in the compound used in the imaging reagent for blood vessels of the present invention, a phosphorescent compound conventionally used for imaging blood vessels or the like can be used without particular limitation. Further, the compound of the present invention described later can be used as a compound used in an imaging reagent for blood vessels having a phosphor group. The phosphorescent compounds can be used alone or in combination of two or more. Examples of the phosphorescent compound include, but are not limited to, compounds containing an iridium complex. Examples of the iridium complex include, but are not limited to, compounds represented by the following formula (I) or (II).

[0037]

Chemical formula

[0038] 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;

[0039]

Chemical formula

[0040] 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.

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

[0042] Ring R 1 Examples include, but are not limited to, nitrogen-containing aromatic rings with structures represented by the following formulas (1-1), (1-2), (1-3), or (1-4).

[0043] [ka]

[0044] Here, X 1 This represents hydrogen. Among the carbon atoms that make up the ring skeleton, the bond extending from the carbon atom adjacent to N is the ring R 2 It is bound to [Ir]. N is coordinated to Ir.

[0045] Ring R 1 For example, Ring R 2 In combination with the above, the emission approaches the near-infrared region and has good permeability in living organisms, so a polycyclic nitrogen-containing aromatic ring is preferred, and a nitrogen-containing aromatic ring with the structure shown in formulas (1-2), (1-3), or (1-4) is more preferred.

[0046] Ring R 2 Examples include sulfur-containing aromatic rings with structures represented by the following formulas (2-1), (2-2), or (2-3).

[0047] [ka]

[0048] Here, X 2 This represents hydrogen. Of the carbon atoms that make up the ring skeleton, the bond extending from the carbon atom adjacent to S is the ring R 1 It is bonded to this carbon atom, and the carbon atom next to this carbon atom is coordinated to Ir.

[0049] Ring R 2 As such, a sulfur-containing aromatic ring with the structure shown in formula (2-1) is preferred.

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

[0051] L 1 This exhibits a bidentate ligand having a β-diketonate structure. 1 This does not affect the luminescence performance, but L 1 Having this feature increases biocompatibility, making it easier for complex (I) to be taken up into biological tissues. L 1 For example, a bidentate ligand with the structure shown in the following formula (L-1) can be cited. In this case, complex (I) is represented by the following formula (I-1).

[0052] [ka]

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

[0054] [ka]

[0055] R 3 Examples of alkyl groups include alkyl groups having 1 to 5 carbon atoms. When an alkyl group has substituents, examples of substituents include halogens, hydroxyl groups, mercapto groups, carboxyl groups, substituted or unsubstituted amino groups, substituted or unsubstituted amide groups, substituted or unsubstituted acyl groups, amino acid residues, peptide residues, and the like.

[0056] Examples of structures represented by formula (L-1) include those represented by the following formulas (L-11), (L-12), (L-13), (L-14), or (L-15). These are, in each case, R in formula (L-1). 3 -CH2CH2COCH2NH(CH2) m CH2NR 4 R 5 , -CH2CH2CONHCH2CH2N(CH3)2, -CH2CH2COOH, -(CH2) n COR 6 It is a structure that is , or -CH3.

[0057] [ka]

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

[0059] In the above formula (L-11), m is preferably an integer between 1 and 3, and particularly preferably 2. R 4 The halogen 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 also be saturated or contain unsaturated bonds. One or more hydrogen atoms may be substituted with substituents such as halogens, hydroxyl groups, amino groups, or mercapto groups. The number of carbon atoms is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. R 5The hydrocarbon group having 1 to 6 carbon atoms may be linear, branched, or cyclic. It may also be saturated or contain unsaturated bonds. One or more hydrogen atoms may be substituted with substituents such as halogens, hydroxyl groups, amino groups, or mercapto groups. The number of carbon atoms is preferably 1 to 3. In the above formula (L-11), R 4 and R 5 It is preferable that all of these are methyl groups and that n is 2.

[0060] In the above formula (L-14), n is preferably an integer between 1 and 3, and particularly preferably 2. "Amino acid residues and peptide residues" refer to residues formed when an amino acid or peptide is linked by an amide bond via its amino group. R 6 Preferably, the side chain is a residue of an amino acid having a hydroxyl group, a carboxyl group, or an amino group, or a residue of a peptide composed of such an amino acid. Examples of amino acids having a hydroxyl group in the side chain include tyrosine, serine, and threonine, with tyrosine being more preferred. Examples of amino acids having a carboxyl group in their side chain include aspartic acid and glutamic acid. Examples of amino acids having an amino group in their side chain include lysine and arginine. Note that the amino acids may be L-forms or D-forms, and may be unnatural amino acids. Examples of peptide residues include peptide residues consisting of one or more of the above-mentioned amino acids, and their length is preferably 2 to 10, more preferably 2 to 5. R 6 Preferably, the residue is an aspartic acid residue -NH-CH(COOH)2, or an aspartic acid dipeptide residue -NH-CH(COOH)-CO-NH-CH(COOH)2.

[0061] Among the structures represented by formula (L-1), those represented by formulas (L-11), (L-12), (L-13), or (L-14) are preferred in terms of biocompatibility. These structures are formed by introducing functional groups into the structure represented by formula (L-15) (acetylacetonate (acac)). The introduction of functional groups results in superior biocompatibility.

[0062] As complex (I), a complex having the structure shown by the following formulas (Ia), (Ib), (Ic), or (Id) is preferred.

[0063] [ka]

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

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

[0066] [ka]

[0067] Complex (II) is L 1 is L 2 Except for the point that, it is the same as complex (I). That is, the ring R in complex (II) 1and ring R 2 This is the ring R in complex (I). 1 and ring R 2 This is similar to the above and contributes to the luminescence performance of complex (II). Ring R in complex (II) 1 and ring R 2 A preferred embodiment is the ring R in complex (I). 1 , and ring R 2 It is similar to that.

[0068] L 2 This exhibits a bidentate ligand with a phenanthroline skeleton. 2 This does not affect the luminescence performance, but L 2 By having L 1 This further increases biocompatibility compared to when the compound (II) is present, making it easier for the compound (II) to be taken up into living tissues. L 2 For example, a bidentate ligand with the structure shown in formula (L-2) below can be cited. In this case, complex (II) is shown in formula (II-1) below.

[0069] [ka]

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

[0071] [ka]

[0072] R 7 Specific examples include amino groups, dimethylamino groups, diethylamino groups, cyano groups, acetyl groups, carboxyl groups, piperidyl groups, and piperadyl groups.

[0073] As complex (II), a complex having the structure represented by the following formulas (IIa), (IIb), (IIc), or (IId) is preferred.

[0074] [ka]

[0075] Here, X 1 and X 2 represents hydrogen, L 2 This represents a bidentate ligand with the structure shown in equation (L-2) above.

[0076] Complexes (I) and (II) may be cationic iridium complexes or neutral iridium complexes, and are preferably cationic iridium complexes. In this invention, it is believed that the binding of oligoarginine to complex (I) or (II) increases the cationic sites and significantly increases vascular endothelial connectivity. Here, for example, octaarginine has a charge of +8, when a cationic iridium complex is attached it becomes +9, and when neutral it remains +8. In other words, when there are many arginine molecules, the number of charges does not change much whether it is cationic or neutral, so not only cationic iridium complexes but also neutral iridium complexes can be used. Furthermore, cationic iridium complexes may form salts with anions. Examples of anions include, but are not limited to, hexafluorophosphate ions (PF6). - ), chloride ions (Cl - ), bromide ions (Br - ), trifluoroacetate ion (CF3COO - Examples include ) and preferably PF6 - That is the case.

[0077] Preferably, the iridium complex is the compound represented by formula (IIc), where R is... 7 It is a compound in which the group is a piperadyl group.

[0078] Oligoarginine, complexes (I) and (II), and compounds containing oligoarginine and a luminescent phose can be synthesized using conventional organic synthesis methods. For example, they can be synthesized according to the methods described in the examples below. The starting materials may be commercially available or synthesized by known methods.

[0079] ≪How to use≫ The vascular imaging reagent of the present invention may further contain solvents, additives, and compounds conventionally used as vascular imaging reagents, as long as they do not interfere with the effects of the present invention. When a solvent is included, the vascular imaging reagent can be added directly to the tissue to perform vascular imaging. Any solvent capable of dissolving the compound containing oligoarginine and the luminescent phosphatidyl is acceptable. For example, 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 mixed solvents thereof can be appropriately selected. When adding to a living organism, a mixed solvent of dimethyl sulfoxide and water is preferred.

[0080] If the vascular imaging reagent of the present invention is a liquid composition comprising a compound containing oligoarginine and a luminescent phose and a solvent, the concentration of the compound containing oligoarginine and the luminescent phose 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 the vascular endothelium. There are no particular restrictions on the type of biological tissue to be measured, but examples include skin, muscle, fat, liver, heart, pancreas, kidneys, spleen, intestines, reproductive organs, brain, and other organs. Furthermore, the tissue may be either normal tissue or pathological tissue. The organisms to which the drug can be administered are not particularly limited and include, for example, vertebrates and invertebrates, including mammals (mice, humans, pigs, dogs, rabbits, etc.).

[0082] Imaging of intra-tissue blood vessels can be performed, for example, as follows: The vascular imaging reagent of the present invention is added to the individual to be measured, and then a compound containing oligoarginine and a luminescent phose in the vascular imaging reagent that has been taken up by the sample is excited and the luminescence is observed. The compound can be excited by irradiating the sample with visible light. The luminescence can be observed using known devices such as a fluorescence microscope, fluorescence measuring device, or fluorescence imaging device. The amount of the vascular imaging reagent of the present invention added to an individual can be appropriately changed depending on the individual used, vascular density, etc., but for example, it can be administered to an individual in the range of 0.01 to 1,000 μmol / kg body weight, preferably 0.1 to 100 μmol / kg body weight. Examples of administration methods for the vascular imaging reagent of the present invention include intravenous administration, subcutaneous administration, and intramuscular administration.

[0083] ≪The compound of the present invention≫ Another aspect of the present invention relates to a compound represented by the following formula (hereinafter sometimes referred to as "the compound of the present invention").

[0084] [ka]

[0085] During the ceremony, n is an integer between 4 and 20.

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

[0087] Here, n is an integer between 4 and 20, preferably an integer between 6 and 16, and more preferably an integer between 8 and 12. Furthermore, the iridium complex may form a salt with anions. The anions are not limited to iridium complexes, but examples include hexafluorophosphate ions (PF6). - ), chloride ions (Cl - ), bromide ions (Br - ), trifluoroacetate ion (CF3COO - Examples include ) and preferably PF6 - That is the case.

[0088] The compounds of the present invention have excellent phosphorescent properties and excellent binding to vascular endothelium, and therefore can be used as imaging reagents for blood vessels.

[0089] Another aspect of the present invention relates to a compound in which oligoarginine and NBD, coumarin 6, or rhodamine B are linked via a linker. Such a compound is also a "compound of the present invention." The linker is a group that is not limited, but preferably polyethylene glycol. p The degree of polymerization p of ) is not limited, but from the viewpoint of ease of retention in the vascular endothelium and ease of obtaining the material, for example it is 2 to 20, preferably 3 to 16, and more preferably 4 to 12. Also, oligoarginine (R n The degree of polymerization n of ) is not limited, but for example, is 4 to 20, preferably 6 to 16, and more preferably 8 to 12. The compounds of the present invention possess excellent fluorescence emission performance and excellent binding to vascular endothelium, and therefore can be used as imaging reagents for blood vessels.

[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 may be commercially available or synthesized by known methods. [Examples]

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

[0092] The following are the meanings of each abbreviation. 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] <Example of synthesis> The compound of the present invention was synthesized according to the following synthesis scheme.

[0094] [ka]

[0095] (Boc-[R(Pbf)]4-OH) Boc-[R(Pbf)]4-OH was synthesized using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage) via the Fmoc solid-phase synthesis method. The resin used was chlorotrityllase resin (1.7 g, number of moles of amino acid introduced: 0.30 mmol / g) into which R(Pbf) had been introduced. For the condensation reaction, 0.5 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH were used as amino acids, 0.6 M DMF solution of HBTU as a coupling agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base were added in amounts of 3, 3, 3, and 6 equivalents, respectively, relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 1 hour. For deprotection of the Fmoc protecting group, 3.5 equivalents of 2% DBU DMF solution were added relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 5 minutes. To ensure complete deprotection, the reaction was repeated under the same conditions for 10 minutes. To cleave the peptide from the resin, 10 mL of 1% TFA dichloromethane solution was added to the vial containing the peptide resin, and the reaction was allowed to proceed at room temperature for 90 minutes. The cleaved peptide solution was allowed to dry under reduced pressure and redissolved in a small amount of acetonitrile. This solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again for centrifugation. This procedure was repeated twice. The water in the vial was removed by lyophilization to obtain 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 round-bottom flask and dissolved in 1 mL of anhydrous DMF. 0.17 mL of DIEA was added to this solution and the mixture was stirred under nitrogen purging at room temperature for 24 hours. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The obtained 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 to obtain 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 reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes) to remove the diethyl ether, and then 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 (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, and the monovalent ion peak (m / z) was detected. calcd.:1601.9 [M-PF6 - ] + ,found:1601.2

[0098] (Boc-[R(Pbf)]8-OH) Boc-[R(Pbf)]8-OH was synthesized using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage) via the Fmoc solid-phase synthesis method. The resin used was chlorotrityllase resin (1.67 g, number of amino acid moles: 0.30 mmol / g) with R(Pbf) introduced into it. For the condensation reaction, 0.6 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH were used as amino acids, 0.5 M DMF solution of HBTU as a coupling agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base were added in amounts of 3, 3, 3, and 6 equivalents relative to the number of amino acid moles, respectively, and the reaction was carried out at room temperature for 1 hour. For deprotection of the Fmoc protecting group, 3.5 equivalents of 2% DBU DMF solution were added relative to the number of amino acid moles, and the reaction was carried out at room temperature for 5 minutes. To ensure complete deprotection, the reaction was repeated under the same conditions for 10 minutes. To cleave the peptide from the resin, 10 mL of 1% TFA dichloromethane solution was added to the vial containing the peptide resin, and the reaction was allowed to proceed at room temperature for 90 minutes. The cleaved peptide solution was allowed to dry under reduced pressure and redissolved in a small amount of acetonitrile. This solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again for centrifugation. This procedure was repeated twice. The water in the vial was removed by lyophilization to obtain 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 nitrogen substitution. 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 followed by centrifugation. This operation was repeated twice. The water in the vial was removed by lyophilization to obtain a red solid (235 mg, 0.055 mmol, crude product yield: 110%).

[0100] (BTQ-R8) Weighed BTQ-[R(Pbf)]8-Boc (110 mg, 0.026 mmol) into a 50 mL centrifuge tube, added 1 mL of TFA:water:TIPS (95:2.5:2.�), and reacted 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 followed by centrifugation. This operation was repeated twice. The diethyl ether in the vial was removed and 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, and the monovalent ion peak (m / z) was detected. calcd.: 2226.6 [M-PF6 - + , found: 2224.8

[0101] (Boc-[R(Pbf)] 12 -OH) Boc-[R(Pbf)] 12 ​-OH was synthesized by solid-phase synthesis of Fmoc using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityllase resin (1.0 g, number of moles of amino acid introduced: 0.30 mmol / g) with R(Pbf) introduced. For the condensation reaction, 0.5 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH were used as amino acids, 0.6 M DMF solution of HBTU as a coupling agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base were added in amounts of 3, 3, 3, and 6 equivalents, respectively, relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 1 hour. For deprotection of the Fmoc protecting group, 3.5 equivalents of 2% DBU DMF solution were added relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 5 minutes. To completely deprotect, the reaction was carried out again under the same conditions for 10 minutes. To cleave the peptide from the resin, 10 mL of a 1% TFA dichloromethane solution was added to the vial containing the peptide resin, and the mixture was reacted at room temperature for 90 minutes. The cleaved peptide solution was allowed to dry under reduced pressure and then redissolved in a small amount of acetonitrile. This solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again for centrifugation. This procedure was repeated twice. The water in the vial was removed by lyophilization to obtain 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 pear-shaped 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 added 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 nitrogen substitution. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again and centrifuged. This operation was repeated twice. The water in the vial was removed by lyophilization to obtain a red solid (320 mg, 0.055 mmol, crude product yield: 110%).

[0103] (BTQ-R 12 ) Weighed BTQ-[R(Pbf)] 12 -Boc (151 mg, 0.026 mmol) into a 50 mL centrifuge tube, added 1 mL of TFA:water:TIPS (95:2.5:2.5), and reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes), the diethyl ether was removed, and diethyl ether was added again and centrifuged. This operation was repeated twice. The diethyl ether in the vial was removed and 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 monovalent 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 solid-phase synthesis of Fmoc using a fully automated microwave peptide synthesizer (Initiator + Alstra, Biotage). The resin used was chlorotrityllase resin (0.34 g, number of moles of amino acid introduced: 0.30 mmol / g) with R(Pbf) introduced. For the condensation reaction, 0.3 M DMF solutions of Fmoc-R(Pbf)-OH and Boc-R(Pbf)-OH were used as amino acids, 0.6 M DMF solution of HBTU as a coupling agent, 0.5 M DMF solution of HOBt as an additive, and 2.0 M NMP solution of DIEA as a base were added in amounts of 3, 3, 3, and 6 equivalents relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 1 hour. For deprotection of the Fmoc protecting group, 3.5 equivalents of 2% DBU DMF solution were added relative to the number of moles of amino acids introduced, and the reaction was carried out at room temperature for 5 minutes. To ensure complete deprotection, the reaction was repeated under the same conditions for 10 minutes. To cleave the peptide from the resin, 10 mL of 1% TFA dichloromethane solution was added to the vial containing the peptide resin, and the reaction was allowed to proceed at room temperature for 90 minutes. The cleaved peptide solution was allowed to dry under reduced pressure and redissolved in a small amount of acetonitrile. This solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The resulting solid was centrifuged (3500 rpm, 5 minutes), the water was removed, and water was added again for centrifugation. This procedure was repeated twice. The water in the vial was removed by lyophilization to obtain 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 pear-shaped 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 added and dissolved in 1 mL of anhydrous DMF. 0.17 mL of DIEA was added to this solution and the mixture was stirred at room temperature under nitrogen purging for 24 hours. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The obtained 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 to obtain 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 -Boc (113 mg, 0.015 mmol) was weighed out, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes) to remove the diethyl ether, and then diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed and 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 The following was obtained. Identification was performed using MALDI-TOF-MS, and the monovalent 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 16The following results were obtained. The number of arginine residues was 4, 8, 12, and 16. In addition, to demonstrate the effect of arginine, a compound without arginine (BTQ) was used as a reference compound. When the absorption and phosphorescence spectra of these compounds were measured, an absorption maximum wavelength was observed around 500 nm and a phosphorescence maximum wavelength was observed around 660 nm. Since phosphorescence was observed above 600 nm, multicolor imaging using molecules that emit green light is possible. Furthermore, the phosphorescence quantum yield and phosphorescence lifetime were measured to be 0.32 (under 0.015 air saturation) and 5.7 μs (under 0.28 μs air saturation), respectively. Even though the compounds had different numbers of arginine residues, the spectra and photophysical properties were almost the same because the same phosphorescent group was bound to the oligoarginine peptide.

[0108] The synthesized compound and the commercially available green fluorescent endothelial staining reagent FITC-tomato lectin were administered to mice to image the renal capillaries. Figure 3 shows the results of FITC-tomato lectin (50 μL of 1 mg / mL in physiological saline), BTQ (100 μL of 100 nmol:1 mmol / L in physiological saline:dimethyl sulfoxide (9:1,v / v) mixed solvent), and BTQ-R n Images of the kidney surface, obtained by administering (n:4, 8, 12, 100 nmol: 1 mmol / L (in physiological saline) in 100 μL) via the tail vein of anesthetized mice, then incising the ventral side to expose the kidney and imaging the kidney surface with a confocal laser microscope, are shown. Compared with the images of FITC-tomato lectin, luminescence is observed from a different region (tubular cells) than that of FITC-tomato lectin for BTQ and BTQ-R4, whereas for BTQ-R8 and BTQ-R 12 This shows that, similar to FITC-tomato lectin, renal capillaries are being imaged. In particular, BTQ-R 12 Therefore, since cavities are observed within the blood vessels, BTQ-R 12 It is distributed in the vascular endothelium, not in the blood. Therefore, it has become clear that a number of arginine residues of 8 or more is preferable for imaging the vascular endothelium.

[0109] Since the kidney is an organ with strong autofluorescence, there is a concern that when FITC-tomato lectin is used, the autofluorescence of the kidney may be detected in addition to that of the vascular endothelium. Therefore, FITC-tomato lectin (50 μL of 1 mg / mL (in physiological saline) administered) and BTQ-R 12 (100 nmol:1 mmol / L (in physiological saline) was administered to mice in 100 μL doses, and imaging of the same area was performed. As shown in Figures 4A and 4C, when imaging was performed using FITC-tomato lectin fluorescence, fluorescence was observed from areas other than the vascular endothelium. It is not possible to distinguish whether this fluorescence is from FITC-tomato lectin or from autofluorescence. On the other hand, BTQ-R 12 When imaging is performed using phosphorescence (Figure 4B, D), by eliminating the fluorescent component, phosphorescence from the vascular endothelium can be observed, allowing for clearer and more selective imaging of the vascular endothelium. The kidneys filter blood in the glomeruli to excrete waste products and salts from the blood as urine. Many glomeruli are located deeper than approximately 200 μm from the kidney surface, making imaging with a one-photon microscope difficult. Here, after euthanizing mice, the kidneys were removed and cut in half, and the cross-sections were observed. As shown in Figure 5A, round, glowing areas can be seen. Upon magnification, it can be seen that linear structures are aggregated, and the vascular network converging in the glomeruli has been successfully imaged (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 vertices, a central vein in the center, and sinusoidal vessels radiating from the center. Figure 6 shows BTQ-R 12 The image shows a phosphorescent micrograph obtained by administering 100 μL of (100 nmol:1 mmol / L in physiological saline) to mice. Sinusoidal vessels are clearly imaged. Similar experiments were performed on other organs (pancreas, small intestine, subcutaneous tissue, heart), revealing that it is possible to image capillaries in many organs (Figure 7).

[0111] In normal tissue, the vascular structure is robust, whereas in cancerous tumors, there are numerous underdeveloped and fragile neovascularizations. Therefore, because the anticancer drug administered into the bloodstream does not reach all cancer cells, it is necessary to increase the dosage. This also leads to an increase in side effects and places a significant burden on the patient. Here, BTQ-R 12 We attempted tumor vascular imaging using BTQ-R. Human colon adenocarcinoma-derived HCT116 cells were transplanted into nude mice to create tumor-bearing mice. Figure 8 shows BTQ-R 12 The image shows phosphorescent microscopic images obtained after administering (100 μL of 100 nmol:1 mmol / L (in physiological saline)) to tumor-bearing mice. The image reveals that numerous smaller blood vessels branch off from larger vessels, demonstrating successful imaging of the vascular network within the tumor.

[0112] In recent years, because fatty liver disease progresses to cirrhosis and liver cancer, early detection of lipid accumulation in the liver has become a focus of attention. Non-alcoholic fatty liver disease, in particular, unlike obesity, is not outwardly visible, which can lead to delayed diagnosis. In fatty liver disease, lipids accumulate abnormally within hepatocytes, forming large lipid droplets and causing hepatocyte hypertrophy. This leads to narrowing of sinusoidal vessels and a hypoxic state throughout the liver. Here, a green fluorescent lipid droplet reagent (3-(benzo[d]thiazol-2-yl)-8-(diethylamino)-2H-benzo[g]chromen-2-one (PC6S)) and BTQ-R are used. 12 Co-administration of PC6S to healthy mice and fatty liver model mice (100 μL of PC6S: 50 nmol: 0.5 mmol / L in a mixed solvent containing physiological saline, dimethyl sulfoxide (9:1, v / v), and 10 wt% BSA, followed by BTQ-R) 12 Multicolor imaging was performed by administering 100 μL of 100 nmol (1 mmol / L in physiological saline) and measuring green fluorescence and deep red phosphorescence. In healthy mice, PC6S fluorescence was detected from lipid droplets in hepatocytes, and BTQ-R was detected from sinusoidal vascular endothelium. 12Phosphorescence has been observed (Figure 9A). The image shows that sinusoidal vessels run in a straight line between hepatocytes. On the other hand, in fatty liver model mice, hepatocytes enlarge due to the formation of large lipid droplets, and sinusoidal vessels become highly tortuous (Figure 9B). This is thought to inhibit the flow of red blood cells, leading to insufficient oxygen supply to the liver and a hypoxic state.

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

[0114] (RhB-pipe-PEG 12 -NH2) In a 100 mL round-bottom flask, 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 and dissolved with anhydrous CH2Cl2 (5 mL) and DIEA (170 μL). The mixture was then reacted at room temperature under a nitrogen atmosphere for 24 hours. The mixture was washed with chloroform, and the precipitated crystals were dried. Subsequently, the Boc group was deprotected by reacting with 4N-HCl / dioxane (10 mL) at room temperature for 2 hours. The mixture was washed with anhydrous THF and hexane, and dried to obtain a reddish-purple solid. (0.224 g, 0.41 mmol, crude product yield: 268%)

[0115] Next, add t-Boc-N-amido-dPEG to a 100 mL pear-shaped flask. 12-acid (100 mg, 0.14 mmol), RhB-pipe (79.5 mg, 0.15 mmol), and HATU (105 mg, 0.28 mmol) were added, dissolved in anhydrous CH2Cl2 (5 mL) and DIEA (170 μL), and reacted under a nitrogen atmosphere at room temperature for 24 hours. Washed with chloroform, the precipitated crystals were dried to obtain a reddish-purple solid (0.399 g, 0.320 mmol, crude product yield: 166%). Subsequently, the Boc group was deprotected by reacting with 4N-HCl / dioxane (10 mL) at room temperature for 3 hours. Washed with anhydrous THF and methanol and dried to obtain a reddish-purple solid (99.0 mg, 0.086 mmol, product yield: 62%).

[0116] (RhB-pipe-PEG 12 -[R(Pbf)] 12 -Boc) Add Boc-[R(Pbf)] to 100 mL of eggplant frass. 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 anhydrous DMF. 0.17 mL of DIEA was added to this solution and the mixture was stirred at room temperature under nitrogen purging for 24 hours. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The obtained 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 to obtain a reddish-purple solid (157 mg, 0.026 mmol, crude product yield: 45%).

[0117] (RhB-pipe-PEG 12 -R 12 ) RhB-pipe-PEG in a 50 mL centrifuge tube 12 -[R(Pbf)] 12-Boc (21.5 mg, 0.0035 mmol) was weighed out, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes) to remove the diethyl ether, and then 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 The following was obtained. Identification was performed using ESI-MS, and the trivalent ion peak (m / z) was detected. calcd.:995.48 [M;3H] 3+ , found:996.6

[0118] [ka]

[0119] (NBD-PEG4-NH2) In a 100 mL round-bottom flask, Boc-NH-PEG4-amine (521 mg, 1.54 mmol), 4-Fluoro-7-nitrobenzofurazan (210 mg, 1.14 mmol), anhydrous CH2Cl2 (5 mL), and DIEA (0.17 mL) were dissolved, and the mixture was stirred at room temperature for 24 hours under nitrogen purging. Washing with chloroform and hexane yielded a yellow liquid. Subsequently, the Boc group was deprotected by reacting with 4N-HCl / dioxane (10 mL) at room temperature for 3 hours. Washing with anhydrous THF and methanol and drying yielded a viscous yellow liquid (0.614 g, 1.53 mmol, crude product yield: 134%).

[0120] (NBD-PEG4-[R(Pbf)] 12 -Boc) Add Boc-[R(Pbf)] to 100 mL of eggplant frass. 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 anhydrous DMF. 0.17 mL of DIEA was added to this solution and the mixture was stirred at room temperature under nitrogen purging for 24 hours. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The obtained 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 to obtain 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 12 -Boc (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 reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes) to remove the diethyl ether, and then diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed and 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 following was obtained. Identification was performed using ESI-MS, and the tetravalent ion peak (m / z) was detected. calcd.:569.1 [M;4H] 4+ , found:596.4

[0122] [ka]

[0123] (C6-PEG4-NH2) In a 100 mL round-bottom flask, 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 and dissolved with anhydrous CH2Cl2 (11 mL) and DIEA (170 μL). The mixture was then reacted at room temperature under a nitrogen atmosphere for 24 hours. Washing with chloroform yielded a yellow solid. Subsequently, the mixture was reacted with 4N-HCl / dioxane (10 mL) at room temperature for 2 hours to deprotect the Boc group. Washing with anhydrous THF and methanol and drying yielded a viscous deep red solid (0.184 g, 0.300 mmol, crude product yield: 150%).

[0124] (C6-PEG4-[R(Pbf)] 12 -Boc) Add Boc-[R(Pbf)] to 100 mL of eggplant frass. 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 anhydrous DMF. 0.17 mL of DIEA was added to this solution and the mixture was stirred at room temperature under nitrogen purging for 24 hours. The solution was transferred to a 50 mL centrifuge tube, and water was added to precipitate the solid. The obtained 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 to obtain 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-Boc (46.0 mg, 0.0082 mmol) was weighed out, and 1 mL of TFA:water:TIPS (95:2.5:2.5) was added. The mixture was reacted at room temperature for 2 hours. Cold diethyl ether was added to the solution to precipitate a solid. The obtained solid was centrifuged (3500 rpm, 5 minutes) to remove the diethyl ether, and then diethyl ether was added again and centrifuged. This procedure was repeated twice. The diethyl ether in the vial was removed and 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 The following was obtained. Identification was performed using ESI-MS, and the tetravalent ion peak (m / z) was detected. calcd.:622.4 [M;4H] 4+ , found:622.8

[0126] [ka]

[0127] <Example 2> RhB-pipe-PEG 12 -R 12 The drug was administered to mice, and vascular imaging of various tissues was performed. Figure 10 shows RhB-pipe-PEG. 12 -R 12 The following images show images of the surfaces of various organs (liver, kidney, spleen, pancreas, testes) and tissues (muscle tissue, subcutaneous tissue) obtained by administering 50 μL of (100 nmol:2 mmol / L (in physiological saline)) via the tail vein of anesthetized mice. The ventral side of the mice was then incised to expose these organs and tissues, and images were obtained by imaging them with a confocal laser microscope. The excitation wavelength was 550 nm, and the observation wavelength was >590 nm. Capillaries were imaged in all observed organs and tissues.

[0128] NBD-PEG-R 12 The drug was administered to mice, and vascular imaging of various tissues was performed. Figure 11 shows NBD-PEG-R 12The following images show images of the surfaces of various organs (liver, kidney, spleen) and tissues (subcutaneous tissue, adipose tissue) obtained by administering 50 μL of 100 nmol:2 mmol / L (in physiological saline) via the tail vein of anesthetized mice. The ventral side of the mice was then incised to expose these organs (liver, kidney, spleen) and tissues (subcutaneous tissue, adipose tissue), and these surfaces were imaged using a confocal laser microscope. The excitation wavelength was 488 nm, and the observation wavelength was 510-550 nm. Capillaries were imaged in all observed organs and tissues.

[0129] C6-PEG-R 12 The drug was administered to mice, and vascular imaging of various tissues was performed. Figure 12 shows C6-PEG-R 12 50 μL of (100 nmol:2 mmol / L (in physiological saline)) was administered via the tail vein of anesthetized mice. The ventral side was then incised to expose various organs (kidneys, spleen, pancreas, testes) and tissues (adipose tissue, muscle tissue), and images of their surfaces were obtained 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 observed organs and tissues. Furthermore, after euthanasia of the mice, imaging of cerebral blood vessels was performed. As shown in Figure 13, imaging of cerebral blood vessels was also demonstrated.

[0130] Based on these results, it was found that compounds containing oligoarginine and a luminescent phosi can be used to clearly image blood vessels within an organism, and that they can be used as imaging reagents for blood vessels. Furthermore, BTQ-R developed by the present invention n It can be used as an imaging reagent for blood vessels. In particular, BTQ-R 12 This is a novel reagent capable of imaging the vascular pathways of normal and pathological tissues within an individual. Furthermore, its distribution to the vascular endothelium is due to the action of oligoarginine peptides, and by changing the luminescent phosphate (BTQ), it is possible to develop vascular endothelial imaging reagents with various emission colors. In addition, the RhB-pipe-PEG developed by this invention... p -R n NBD-PEG p -R n, C6-PEG p -R n It can also be preferably used as an imaging reagent for blood vessels. [Industrial applicability]

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

Claims

1. Oligoarginine, represented by the following formula, A phosphorus or fluorophore bound to the C-terminal or N-terminal side of the oligoarginine, Contains compounds including The phosphorescent group is a compound containing an iridium complex, The fluorophore is a compound selected from 4-nitrobenzo-2-oxa-1,3-diazole (NBD), dimethylaminosulfonylbenzoxadiazole (DBD), dimethylaminosulfonylbenzothiadiazole (DBThD), dimethylaminosulfonylbenzoselenadiazole (DBSeD), fluorescein isothiocyanate (FITC), coumarin dyes, rhodamines, borondipyrrometene (BODIPY), and cyanine dyes, wherein the oligoarginine and the fluorophore are linked via a linker. Vascular endothelial imaging reagents: 【Chemistry 1】 During the ceremony, n is an integer between 8 and 20.

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 This represents a monocyclic or polycyclic nitrogen-containing aromatic ring. Ring R ２ This represents a monocyclic or polycyclic sulfur-containing aromatic ring. L １ This exhibits a bidentate ligand having a β-diketonate structure; 【Transformation 3】 In formula (II), Ring R １ This represents a monocyclic or polycyclic nitrogen-containing aromatic ring. Ring R ２ This represents a monocyclic or polycyclic sulfur-containing aromatic ring. L ２ This exhibits a bidentate ligand with 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 between 8 and 20.

4. The compound represented by the following formula: 【Transformation 5】 During the ceremony, n is an integer between 8 and 20.

Images