Oxygen Imaging Reagents

Abstract

To provide an oxygen imaging reagent that shows phosphorescence in a near-infrared light region, has a longer phosphorescent life, and allows for imaging to a deeper area.SOLUTION: A compound of the following formula (I) is used. [R: a monocyclic or polycyclic nitrogen-containing aromatic ring; A1: a heteroatom; Z: H or an optionally substituted C1-20 hydrocarbon group; L: a bidentate ligand].SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an oxygen imaging reagent, a compound that can be used for the reagent, and the like. [Background technology]

[0002] Oxygen is an essential substance for maintaining the vital activities of aerobic organisms. Hypoxia in vivo is commonly observed in cancer, ischemic disease, chronic kidney disease, fatty liver, and other conditions. Therefore, imaging oxygen tension within cells and tissues is important not only for understanding the metabolic processes of normal organs, but also for diagnosing hypoxic pathologies and developing therapeutic drugs. Electrochemical measurement using a needle electrode is currently used to measure oxygen tension within tissues, but this method is invasive and can only measure the area near the needle electrode. On the other hand, imaging techniques using molecular luminescence (phosphorescence) offer high sensitivity, ease of use, and real-time measurement. When combined with a microscope, they can easily achieve spatial resolution at the single-cell level. Therefore, oxygen imaging reagents using metal complexes have been developed and are commercially available (Patent Documents 1 and 2).

[0003] However, the oxygen imaging reagents used to date have the following problems, for example: (1) Because it exhibits absorption and phosphorescence in the visible light region, imaging is only possible up to about 50 μm from the organ surface. (2) Near-infrared phosphorescent metal complexes (BTPHSA in Non-Patent Document 1: Figure 12) exist, but they have low oxygen sensitivity due to their short phosphorescence lifetime. Near-infrared phosphorescent metal complexes (PPY-MD((OC-6-13)-[2-[Phenyl(2H-pyrrol-2-ylidene-κN)methyl]-1H-pyrrolato-κN]bis[2-(1H-pyrazol-1-yl-κN] in Non-Patent Document 2) 2 )phenyl-κC]iridium): Figure 12) has an improved phosphorescence lifetime compared to BTPHSA, but its absorption and emission are more toward shorter wavelengths and its phosphorescence brightness is low, making imaging deep into tissue difficult. (3) Dendrimer-type near-infrared oxygen imaging reagents are also available, but they are limited to measuring blood oxygen partial pressure.

[0004] Therefore, there is a demand for oxygen imaging reagents for cells, biological tissues, etc. that exhibit phosphorescence in the near-infrared region, have a longer phosphorescence lifetime, and are capable of imaging at greater depths. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2010-44059 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-101567 [Non-patent literature]

[0006] [Non-Patent Document 1] S. Zhang et al., Cancer Res., 70, 4490-4498, 2010. [Non-patent document 2] Hanson, K. et al., Inog. Chem. 2010, 49, 6077-6084. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above-mentioned problems, and aims to develop an oxygen imaging reagent that exhibits phosphorescence in the near-infrared light region, has a longer phosphorescence lifetime, and is capable of imaging at deeper locations. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have developed a novel compound that exhibits phosphorescence in the near-infrared region and has a longer phosphorescence lifetime. Furthermore, they have discovered that oxygen imaging at deeper depths is possible using such a compound. Based on these findings, the present invention has been completed.

[0009] That is, the gist of the present invention relates to the following. [1] A compound represented by the following formula (I):

[0010] [ka]

[0011] During the ceremony, Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring; A 1 indicates a heteroatom, Z represents hydrogen or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent; L represents a bidentate ligand. [2] The compound according to [1], wherein the ring R is represented by the following formula (R-1), (R-2), or (R-3):

[0012] [ka]

[0013] During the ceremony, A 2 indicates a heteroatom, X represents hydrogen. [3] A. 2 is sulfur or oxygen. [4] The compound according to [2] or [3], wherein the ring R is represented by formula (R-3). [5] A. 1 is sulfur or oxygen. [6] The compound according to any one of [1] to [5], wherein Z is an alkyl group having 1 to 20 carbon atoms which may have a substituent, or an aryl group having 6 to 20 carbon atoms which may have a substituent. [7] The compound according to any one of [1] to [6], wherein L is represented by the following formula (II):

[0014] [ka]

[0015] During the ceremony, Ring R 1 represents a monocyclic or polycyclic nitrogen-containing aromatic ring, Ring R 2 represents a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring, Ring R 1 and ring R 2 may have a substituent. [8] The compound according to [7], wherein the substituent is represented by the following formula (III):

[0016] [ka]

[0017] During the ceremony, n represents an integer of 1 to 5, X 3 each independently represents hydrogen or a hydrocarbon group having 1 to 6 carbon atoms. [9] An oxygen imaging reagent comprising the compound according to any one of [1] to [8]. [Effects of the Invention]

[0018] The present invention provides a novel compound that exhibits phosphorescence in the near-infrared region and has a longer phosphorescence lifetime. A coupling reagent is provided. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the structural formula of the synthesized oxygen imaging reagent compound of the present invention. [Figure 2] Figure 2 shows the absorption and phosphorescence spectra of PPY-MD, PPY-BMD, PPY-BBMD, and PPYDM-BBMD. [Figure 3] Figure 3 shows the absorption and phosphorescence spectra of BTP-MD, BTP-BMD, BTP-BBMD, and BTPDM-BBMD. [Figure 4] FIG. 4 shows phosphorescence images (photographs) of AML12 cells stained with PPY-MD and PPYDM-BBMD. [Figure 5] FIG. 5 shows phosphorescence images (photographs) of AML12 cells stained with BTP-MD and BTPDM-BBMD. [Figure 6] FIG. 6 shows phosphorescence images (photographs substituting drawings) of AML12 cells stained with BTPDM1 (comparative example), BTPHSA (comparative example), and PPYDM-BBMD. [Figure 7] FIG. 7 shows luminescence imaging images of PPYDM-BBMD and AML12 cells stained with various dyes (intracellular localization of PPYDM-BBMD) (photographs as drawing substitutes). [Figure 8] FIG. 8 shows the results of evaluating the cytotoxicity of PPYDM-BBMD. [Figure 9] Figure 9 shows the results of phosphorescence imaging of cells treated with PPYDM-BBMD and cultured under various oxygen tensions (0 mmHg, 38 mmHg, 76 mmHg, 114 mmHg, and 160 mmHg). (A) Phosphorescence lifetime imaging images (photographs) of AML12 cells and (B) HK-2 cells are shown, and (C) Stern-Vomer plots are shown. [Figure 10] Figure 10 shows the results of imaging the liver of a mouse administered with PPYDM-BBMD. (A) Phosphorescence lifetime imaging image (photograph) of the liver of a mouse administered with PPYDM-BBMD, and (B) oxygen partial pressure in the CV and PV regions. [Figure 11] Figure 11 shows the results of imaging at various depths in the liver of a mouse administered with PPYDM-BBMD. (A) Phosphorescence lifetime imaging image (photograph) of the liver of a mouse administered with PPYDM-BBMD, and (B) oxygen partial pressure in the CV and PV regions. [Figure 12] FIG. 12 shows the structural formula of a conventional oxygen imaging reagent compound. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be described below. <Compound represented by formula (I)> One aspect of the present invention is a compound represented by the following formula (I) (hereinafter referred to as "the compound of the present invention"). Regarding:

[0021] [ka]

[0022] During the ceremony, Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring; A 1 indicates a heteroatom, Z represents hydrogen or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent; L represents a bidentate ligand.

[0023] The compound of the present invention is a complex having a central metal Ir, a bidentate ligand (represented by the following formula) having a dipyrromethene derivative structure containing a ring R coordinated to Ir, and two bidentate ligands L as a structure.

[0024] [ka]

[0025] The compound represented by formula (I) (hereinafter sometimes referred to as "complex (I)") will be described below.

[0026] A 1 The heteroatom represented by the formula (I) includes, for example, sulfur or oxygen, and is preferably sulfur.

[0027] Ring R is not limited to, but examples thereof include nitrogen-containing aromatic rings having structures represented by the following formulas (R-1), (R-2), and (R-3).

[0028] [ka]

[0029] Here, X represents hydrogen. A 2 indicates a heteroatom, and A 2 The heteroatom represented by the formula (I) includes, for example, sulfur or oxygen, and is preferably sulfur. Among the carbon atoms that make up the ring skeleton, the bond extending from the carbon atom next to N is bonded to the carbon atom connecting to the other ring that forms the dipyrromethene derivative structure. N is coordinated to Ir.

[0030] As the ring R, a polycyclic nitrogen-containing aromatic ring is preferred, and a nitrogen-containing aromatic ring having a structure represented by the above formula (R-2) or (R-3) is more preferred, and a nitrogen-containing aromatic ring having a structure represented by the above formula (R-3) is even more preferred, in terms of emitting light approaching near-infrared and having good transmittance in vivo in combination with the other ring forming the dipyrromethene derivative structure and Z.

[0031] Z represents hydrogen or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent. The hydrocarbon group having 1 to 20 carbon atoms may be linear, branched, or cyclic. It may be saturated or contain an unsaturated bond. The number of carbon atoms is preferably 1 to 12, more preferably 1 to 9, and even more preferably 9. The hydrocarbon group having 1 to 20 carbon atoms is, for example, an alkyl group, alkenyl group, or alkynyl group having 1 to 20 carbon atoms, or an aryl group having 6 to 20 carbon atoms, preferably an aryl group. Examples of the substituent include halogen, haloalkyl group, hydroxy group, carboxy group, amino group, alkylamino group, alkyl group, alkoxy group, acyl group, and mercapto group. Preferably, it is an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group. Z is preferably a trimethylphenyl group.

[0032] The bidentate ligand having a dipyrromethene derivative structure containing the ring R is a cyclometallated ligand and contributes to the luminescent performance of complex (I).

[0033] Ring R 1 and R 2 The bidentate ligand L having the formula (I) is a cyclometallated ligand and does not affect the luminescence performance except for the phosphorescence lifetime. Furthermore, the presence of L increases the biocompatibility of the complex (I), making it easier for the complex (I) to be taken up into the body.

[0034] The bidentate ligand L is a ligand that constitutes complex (I) and exhibits photophysical activity as complex (I). There are no limitations as long as L's are each independently selected and may have the same or different structures, and preferably L's have the same structure. L is not limited, but examples thereof include those having a structure represented by the following formula (II).

[0035] [ka]

[0036] Ring R 1 represents a monocyclic or polycyclic nitrogen-containing aromatic ring. 1Examples 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).

[0037] [ka]

[0038] where X 1 indicates hydrogen. Among the carbon atoms that make up the ring skeleton, the bond extending from the carbon atom next to N is the ring R 2 N is coordinated to Ir.

[0039] Ring R 1 As a ring R 2 In combination with the compound (I), the preferable luminescence performance of the compound (I) is maintained. In terms of the supportability, the nitrogen-containing aromatic ring having the structure represented by the formula (1-1) or (1-3) is preferred, and the nitrogen-containing aromatic ring having the structure represented by the formula (1-1) is more preferred.

[0040] Ring R 2 represents a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring. 2 Examples of the aromatic ring include sulfur-containing aromatic rings or aromatic rings having the structure represented by the following formula (2-1), (2-2), (2-3), or (2-4).

[0041] [ka]

[0042] where X 2 indicates hydrogen. Among the carbon atoms constituting the ring skeleton, the bond extending from the carbon atom next to S in the formulas (2-1), (2-2), and (2-3) is a ring R 1 The carbon atom next to this carbon atom is coordinated to Ir. In addition, one of the bonds in the formula (2-4) is a ring R 1The carbon atom next to this carbon atom is coordinated to Ir.

[0043] Ring R 2 As a ring R 1 In combination with the compound (I), the preferable luminescence performance of the compound (I) is maintained. In terms of the supportability, the sulfur-containing aromatic ring having the structure represented by the formula (2-1) or the aromatic ring having the structure represented by the formula (2-4) is preferred.

[0044] In addition, X may be used as long as it does not affect the performance of the complex (I). 1 and X 2 are independently non-hydrogen substitutions The substituent may be, but is not limited to, a halogen, a haloalkyl group, a hydroxy group, a carboxy group, an amino group, an alkylamino group, an alkoxy group, an acyl group, a mercapto group, or a biocompatible group (ring R 1 and / or ring R 2 Here, the term "biocompatible group" refers to a group that has high affinity with biological materials and has the property of facilitating the introduction of Complex (I) into cells or biological tissues. There are no particular limitations on the biocompatible group as long as it is a group capable of binding to the complex (I), and examples thereof include an amide group.

[0045] From the viewpoint of improving affinity with living organisms, X 1 and X 2 It is preferable to introduce a biocompatible group as a substituent of the above. Examples of the biocompatible group include, but are not limited to, a hydrocarbon group having 1 to 20 carbon atoms and having a substituent such as a hydroxy group, a carboxy group, an amino group, an alkylamino group, or a mercapto group, or a biocompatible polymer.

[0046] The hydrocarbon group having 1 to 20 carbon atoms may be linear, branched, or cyclic. It may be saturated or contain an unsaturated bond. The number of carbon atoms is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 2. The hydrocarbon group having 1 to 20 carbon atoms is, for example, an alkyl group, an alkenyl group, an alkynyl group, or an aryl group, and is preferably an alkyl group. Examples of such biocompatible groups include a hydrocarbon group substituted with an alkylamino group and connected to a ring R by an amide bond. 1 or ring R 2 Examples of the group include a group represented by the following formula (III) bonded to the

[0047] [ka]

[0048] n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 1. X 3 each independently represents hydrogen or an alkyl group having 1 to 5 carbon atoms, preferably having 1 to 3 carbon atoms, and more preferably having 1 carbon atom (methyl group).

[0049] Examples of biocompatible polymers include, but are not limited to, residues of oligopeptides, polypeptides, polyamides, polyalkylene glycols, polyglycerin, polysaccharides, polylactic acid, polyvinyl alcohol, polyacrylic acid, and polyacrylamide.

[0050] Examples of the bidentate ligand L include bidentate ligands having a structure represented by the following formula (IIa) or (IIb).

[0051] [ka]

[0052] The complex (I) is preferably a complex having a structure represented by the following formula (Ia) or (Ib): stomach.

[0053] [ka]

[0054] L represents a bidentate ligand having the structure represented by the formula (IIa) or (IIb).

[0055] The complex (I) of the present invention may be, for example, a complex (1) represented by the formula (Ia) and a complex (II) represented by the formula (IIa). a) (BTP-BMD), (2) a compound (PPY-BMD) in which the complex is of the formula (Ia) and L is of the formula (IIb), (3) a compound (PPY-BBMD) in which the complex is of the formula (Ib) and L is of the formula (IIb), (4) a compound (PPY-BBMD) in which the complex is of the formula (Ib) and L is of the formula (IIb) and II) a biocompatible group (n=1, X 3 = methyl group) (PPYDM-BBMD), and the like, and (4) is a preferred example from the viewpoint of luminescence properties, biocompatibility, etc.

[0056] The complex (I) of the present invention can be synthesized by a conventional organic synthesis method. For example, it can be synthesized according to the method described in the Examples below. The raw materials used may be commercially available products or those synthesized by known methods.

[0057] The photophysical properties of the phosphorescence of complex (I), such as the absorption and phosphorescence maximum wavelengths, molar extinction coefficient (ε ), phosphorescence quantum yield (Φ p ) and phosphorescence lifetime (τ p ) can be measured by known measurement methods. For example, the absorption / phosphorescence maximum wavelength and molar absorption coefficient can be measured using a spectrophotometer or the like, and the phosphorescence quantum yield can be measured using a luminescence quantum yield measurement device or the like, using a sample prepared by dissolving complex (I) in a solvent or the like. The phosphorescence lifetime can be measured by measuring the phosphorescence lifetime (τ p ) can be measured. Phosphorescence brightness (εφ p) (under deoxygenated (nitrogen saturated) conditions) depends on the structure of the compound, the type of solvent, etc. It can be changed by, but is not particularly limited to, for example, 3,300 or more, 3,500 or more, 5,000 or more or above 6,000. Phosphorescence lifetime (τ p ) (under deoxygenated (nitrogen saturated) conditions) depends on the compound structure, type of solvent, etc. The time can be varied and is not particularly limited, but may be, for example, 5.0 μs (microseconds) or more, 10.0 μs or more, 15.0 μs or more, or 18.0 μs or more.

[0058] The maximum excitation wavelength of complex (I) in a solvent can be changed depending on the compound structure, the type of solvent, etc. The wavelength is not particularly limited, but is, for example, 500 nm to 580 nm. The phosphorescent wavelength can also be set appropriately, for example, to 700 nm to 780 nm.

[0059] <Oxygen imaging reagent> One aspect of the present invention relates to an oxygen imaging reagent comprising the compound of the present invention (hereinafter, sometimes referred to as "the oxygen imaging reagent of the present invention").

[0060] The oxygen imaging reagent of the present invention contains the compound of the present invention, Complex (I). This structure gives the complex (I) excellent photophysical properties (maximum phosphorescence wavelength, phosphorescence lifetime, molar absorption coefficient, phosphorescence brightness, etc.). Complex (I) exhibits phosphorescence in the near-infrared region. Therefore, the oxygen imaging reagent of the present invention can be distinguished from autofluorescence and can also image oxygen in deeper tissues. Since the fluorescence intensity is longer and the phosphorescence brightness is high, the oxygen imaging reagent of the present invention allows for highly sensitive imaging. Furthermore, since it has the above-mentioned excellent photophysical properties in non-aqueous solvents, it is useful as an oxygen imaging reagent not only in cells but also in tissues and living individuals.

[0061] The oxygen imaging reagent of the present invention is composed solely of the compound of the present invention, Complex (I). The compounds may be used alone or in combination of two or more. Furthermore, as long as the effects of the present invention are not impaired, the composition may further contain solvents, additives, and compounds other than the compound of the present invention used as an oxygen imaging reagent.

[0062] When placed in an environment such as a cell or tissue, the complex (I) exhibits a low oxygen partial pressure. It emits strong phosphorescence. Therefore, it is possible to measure the oxygen partial pressure based on the phosphorescence intensity. That is, it is possible to determine that the oxygen partial pressure is low when the phosphorescence is strong. Furthermore, by determining the relationship between the oxygen partial pressure and the phosphorescence intensity in advance, it is also possible to quantitatively measure the oxygen partial pressure.

[0063] The oxygen imaging reagent of the present invention can be used, for example, as a reagent for imaging oxygen partial pressure in a biological sample. The biological sample can be, but is not limited to, cells or isolated tissue. The oxygen imaging reagent of the present invention can also be applied to living organisms and can be used as an oxygen imaging reagent for detecting oxygen in cells, tissues, etc. in a living organism.

[0064] The oxygen imaging reagent of the present invention is capable of imaging oxygen within cells. Intracellular oxygen imaging can be performed, for example, as follows. The oxygen imaging reagent of the present invention is added to the cells to be measured. The phosphorescence signal of the oxygen imaging reagent of the present invention can then be observed and visualized using an in vitro / in vivo imaging device or a microplate reader equipped with a luminescence lifetime measurement mode, thereby imaging the intracellular oxygen partial pressure. The amount of the oxygen imaging reagent of the present invention added to cells can be varied as appropriate depending on the cells used, oxygen partial pressure, etc., but it can be added to cells at a final concentration of, for example, 0.01 to 1000 μM, preferably 0.1 to 100 μM. The culture time can be changed as appropriate depending on the cells and compounds used, but can be, for example, 0.5 to 10 hours, preferably 1 to 2 hours. When the oxygen imaging reagent of the present invention is dissolved in a solvent and then added to cells, the solvent may be, but is not limited to, an organic solvent such as n-hexane, dibutyl ether, ethyl acetate, acetonitrile, or dimethyl sulfoxide. The cells to which the oxygen imaging reagent of the present invention is added are not particularly limited as long as they are cells that are the subject of measurement of oxygen partial pressure, and examples thereof include established cultured cells and primary cultured cells.

[0065] The oxygen imaging reagent of the present invention can also image oxygen in tissues of living organisms (living organisms). Interstitial oxygen imaging can be performed, for example, as follows. The oxygen imaging reagent of the present invention is added to an individual to be measured. The phosphorescence signal of the oxygen imaging reagent of the present invention is then detected using an in vivo imaging device. By observing and imaging using a device or the like, it is possible to image the oxygen partial pressure in the tissue. The amount of the oxygen imaging reagent of the present invention to be added to an individual can be varied as appropriate depending on the individual used, the oxygen partial pressure, etc., but can be administered to an individual in the range of, for example, 0.01 to 1000 μmol / kg body weight, preferably 0.1 to 100 μmol / kg body weight. The oxygen imaging reagent of the present invention can be administered, for example, intravenously, subcutaneously, or intramuscularly. When the oxygen imaging reagent of the present invention is dissolved in a solvent and then added to tissue, the solvent may be, but is not limited to, an organic solvent such as n-hexane, dibutyl ether, ethyl acetate, acetonitrile, dimethyl sulfoxide, etc. Furthermore, the reagent may be administered in combination with a biocompatible liquid. Tissues that can be detected by the oxygen imaging reagent of the present invention include, but are not limited to, organs such as skin, muscle, liver, heart, pancreas, and kidney. The individual organisms to be administered are not particularly limited, and examples include vertebrates including mammals (mouse, human, pig, dog, rabbit, human, etc.) and invertebrates.

[0066] <Cancer diagnostic drugs> As described above, when the compound of the present invention, Complex (I), is placed in the environment of cells, tissues, etc., Therefore, when the complex (I) of the present invention is administered to experimental animals such as mice and rats, or to humans, the complex (I) emits more intense phosphorescence when the oxygen partial pressure in the environment is low. Since cancer tissue has a shortage of oxygen, detecting areas where the partial pressure of oxygen is low allows specific staining of cancer tissue, which can be used as a cancer diagnostic agent. For example, the complex (I) of the present invention can be administered to a subject, and the subject can be irradiated with visible light from outside the body. Phosphorescence can be observed by fluorophores. This allows for non-invasive, highly sensitive, and selective visualization of cancer tissue. Furthermore, because phosphorescence can be visualized, it can also be used as an imaging reagent for cancer detection. In addition, the complex (I) of the present invention can be used in cancer research using laboratory animals and in the evaluation of cancer therapeutic drugs. It can be used. [Example]

[0067] 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.

[0068] <Synthesis Example> The compounds used in the examples were synthesized as follows.

[0069] The BODIPY derivative was synthesized with some modifications based on the literature (Sun, Z.; Guo, M.; Zhao, C. Synthesis and properties of benzothieno[b]-fused BODIPY dyes, J. Org. Chem. 2016, 81, 229-237.). with some modifications. PPY-MD was synthesized based on the literature (Hanson, K.; Tamayo, A.; Diev, V. V.; Whited, M. T.; Djurovich, P. I.; Thompson, M. E. Efficient dipyrrin-centered phosphorescence at room temperature from bis-cyclometalated iridium(III) dipyrrinato complexes. Inog. Chem. 2010, 49, 6077-6084.).

[0070] <Synthesis of BT-DIPY-MES>

[0071] [Chemical formula]

[0072] (SPh-BODIPY-MES) 2-Bromo BODIPY-MES (89 mg, 0.23 mmol), 2-(methylthio)phenylboronic acid (48 mg, 0.29 mmol), and Pd(PPh3)4 (61 mg, 0.05 mmol) were dissolved in ultra-dehydrated toluene (10 mL) and aqueous K2CO3 solution (4 mL, 2.0 M) and refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was poured into distilled water and extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and the solution was evaporated to dryness under reduced pressure. The resulting crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane:chloroform = 6:4, v / v). ) to give SPh-BODIPY-MES as a dark red solid (93 mg, 0.22 mmol, 94%). .

[0073] 1 H NMR (400 MHz, CDCl3): δ8.23 (s, 1H), 7.91 (s, 1H), 7.26-7.20 (m, 3H), 7.14-7.10 (m, 1H), 6.95 (d, J = 0.5 Hz, 2H), 6.85 (s, 1H), 6.67 (d, J = 4.1 Hz, 1H), 6.47 (dd, J = 4.1, 1.4 Hz, 1H), 2.40 (s, 3H), 2.35 (s, 3H), 2.14 (s, 6H). ESI-MS (positive): calcd. for C 25 H 23 BF2N2S [M] + : 432.16; found: 431.1.

[0074] (SOPh-BODIPY-MES) SPh-BODIPY-MES (93 mg, 0.22 mmol) was dissolved in a mixture of glacial acetic acid and chloroform (6 mL, 2:1). v / v) and cooled in an ice bath until the solvent was almost frozen. H2O2 (30%, 40 μL, 0.5 mmol) was slowly added to the frozen solvent. The solution was removed from the ice bath and stirred at room temperature overnight. Acetic acid was removed under reduced pressure. The organic layer was dried over Na2SO4, filtered, and the solution was evaporated to dryness under reduced pressure. The product was purified by gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane:chloroform = 6:4, v / v) to obtain SOPh-BODIPY-MES as a red solid. (73 mg, 0.16 mmol, 73%).

[0075] 1 H NMR (400 MHz, CDCl3): δ8.05 (dd, J = 7.9, 1.3 Hz, 1H), 8.01 (s, 1H), 7.96 (s, 1H), 7.56-7.45 (m, 2H), 7.35 (dd, J = 7.6, 1.4 Hz, 1H), 6.98 (s, 1H), 6.94 (s, 1H), 6.79 (d, J = 4.4 Hz, 1H), 6.75 (s, 1H), 6.55 (d, J= 3.9 Hz, 1H), 2.49 (s, 3H), 2.36 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H).

[0076] (BT-DIPY-MES) SOPh-BODIPY-MES (64 mg, 0.14 mmol) was added in small portions to concentrated sulfuric acid (0.5 mL) cooled in an ice bath. Added. The reaction solution was stirred at room temperature for 5 minutes, poured into ice water, and then adjusted to pH 8 using an aqueous potassium carbonate solution. Dichloromethane was added to the aqueous solution for extraction. Sodium sulfate was added to the organic layer for drying, and after filtration, the solution was dried under reduced pressure. The obtained crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane: chloroform = 5:5, v / v) to obtain BT-DIPY-MES as a red-violet solid (17 mg, 46 μmol, 32%).

[0077] 1 H NMR (400 MHz, CDCl3): δ12.72 (br, 1H), 7.55 - 7.52 (m, 2H), 7.35 (s, 1H), 7.24 - 7.18 (m, 2H), 6.96 (s, 2H), 6.68 (s, 1H), 6.26 - 6.23 (m, 2H), 2.38 (s, 3H), 2.12 (s, 6H). ESI-MS (positive): calcd. for C 24 H 21 N2S [M + H] + : 369.13; found: 369.2.

[0078] <Synthesis of BBT-DIPY-MES>

[0079]

Chemical Structure

[0080] (BSPh-BODIPY-MES) 2,6-dibromo BODIPY-MES (566 mg, 1.21 mmol), 2-(methylthio)phenylboronic acid Pd(PPh3)4 (487 mg, 2.90 mmol) and Pd(PPh3)4 (289 mg, 0.25 mmol) were dissolved in ultra-dehydrated toluene (40 mL) and aqueous K2CO3 solution (8 mL, 2.0 M) and refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, distilled water was poured into the reaction solution, which was then extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and the solution was evaporated to dryness under reduced pressure. The resulting crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane:chloroform = 3:7, v / v) to give BSPh-BODIPY-MES (455 mg, 0.82 mmol) as a purple solid. 68%).

[0081] 1 H NMR (400 MHz, DMSO-d6): δ 8.42 (s, 2H), 7.41 (d, J = 7.3 Hz, 2H), 7.29-7.28 (m, 4H), 7.16-7.10 (m, 2H), 7.04 (s, 2H), 6.85 (s, 2H), 2.40 (s, 6H), 2.30 (s, 3H), 2.10 (s, 6H). ESI-MS (positive): calcd. for C 32 H 29 BF2N2S2[M] + : 554.18; found: 553.1.

[0082] (BSOPh-BODIPY-MES) BSPh-BODIPY-MES (445 mg, 0.80 mmol) was dissolved in a mixture of glacial acetic acid and chloroform (21 mL, 2:1, v / v) and cooled in an ice bath until the solvent was almost frozen. H2O2 (30%, 204 μL, 2.00 mmol) was slowly added to the frozen solution. After removing from the ice bath, the mixture was stirred at room temperature overnight. The acetic acid was evaporated under reduced pressure, and saturated aqueous sodium bicarbonate was poured into the solution, followed by extraction with dichloromethane. The organic layer was dried over Na2SO4, filtered, and the solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, eluent: ethyl acetate) to give BSOPh-BODIPY-MES (426 mg, 0.73 mol, 91%) as a red solid.

[0083] 1 H NMR (400 MHz, CDCl3): δ8.08-8.06 (m, 4H), 7.57 (td, J= 1.4, 7.7 Hz, 2H), 7.52-7.48 (m, 2H), 7.36 (dd, J = 7.6, 1.4 Hz, 2H), 6.98 (t, J = 16.5 Hz, 2H), 6.86 (s, 2H), 2.51 (s, 6H), 2.36 (s, 3H), 2.22-2.11 (m, 6H).

[0084] (SOPhBT-BODIPY-MES) Add BSOPh-BODIPY-MES (189 mg, 0.32 mmol) in small amounts to concentrated sulfuric acid (0.5 mL) cooled in an ice bath. The reaction solution was stirred at room temperature for 5 minutes, poured into ice water, and then washed with an aqueous potassium carbonate solution. The pH was adjusted to 8. Dichloromethane was added to the aqueous solution and extracted. The mixture was dried over Na2SO4, filtered, and then evaporated to dryness under reduced pressure. The crude product was dissolved in dehydrated dichloromethane (15 mL), and triethylamine (5 mL) was added. After stirring at room temperature for 15 minutes, BF3·OEt2 (7 mL) was added and the mixture was stirred overnight at room temperature. Distilled water was poured into the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was dried over Na2SO4, filtered, and the solution was evaporated to dryness under reduced pressure. The resulting crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n-hexane:ethyl acetate = 2.5:7.5, v / v) to give SOPhBT-BODIPY-MES as a purple solid (118 mg, 0.21 mol, 66%).

[0085] 1 H NMR (400 MHz, CDCl3): δ8.05 (dd, J = 8.0, 1.6 Hz, 1H), 7.89 (s, 1H), 7.71-7.63 (m, 2H), 7.55-7.51 (m, 1H), 7.47 (td, J = 7.4, 1.5 Hz, 1H), 7.38-7.31 (m, 3H), 7.02 (s, 1H), 6.98 (s, 1H), 6.91 (s, 1H), 6.67 (s,1H), 2.50 (s, 3H), 2.39 (s, 3H), 2.21 (s, 3H), 2.16 (s, 3H).

[0086] (BBT-DIPY-MES) SOPhBT-BODIPY-MES (243 mg, 0.44 mmol) was added in small portions to concentrated sulfuric acid (1.0 mL) cooled in an ice bath. The reaction solution was stirred at room temperature for 5 minutes, poured into ice water, and then washed with an aqueous potassium carbonate solution. The pH was adjusted to 8. Dichloromethane was added to the aqueous solution and extracted. The resulting crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane). The resulting product was purified using a solvent mixture of acetic acid and chloroform (5:5, v / v) to give BBT-DIPY-MES as a purple solid. (93 mg, 0.20 mol, 45%).

[0087] 1 1H NMR (400 MHz, CDCl3): δ 14.16 - 11.73 (br, 1H), 7.71 - 7.51 (m, 5H), 7.24 - 7.21 (m, 3H), 7.01 (s, 2H), 6.57 (s, 2H), 2.42 (s, 3H), 2.19 (s, 6H). ESI-MS (positive): calcd. for C 30 H 23 N2S2[M + H] + : 475.12; found: 475.1.

[0088] <Synthesis of PPY-MD>

[0089]

Chem.

[0090] (PPY-MD) 5-mesityl-dipyrromethane (57 mg, 0.22 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 48 mg, 0.21 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (10 mL), and stirred at room temperature for 1 hour. K2CO3 (415 mg, 3.00 mmol) was added to the reaction solution and stirred at room temperature for 15 minutes. Then, PPY chlorine-bridged dinuclear complex (110 mg, 0.10 mmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was dried under reduced pressure. The obtained crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane: chloroform = 5:5, v / v) to obtain red solid PPY-MD (108 mg, 0.14 mmol, 66%).

[0091] 11H NMR (400 MHz, CDCl3): δ 7.89 (d, J = 6.0 Hz, 2H), 7.81 (d, J = 48.2 Hz, 2H), 7.61 - 7.57 (m, 4H), 6.92 (d, J = 7.1 Hz, 2H), 6.89 (s, 2H), 6.86 (d, J = 7.1 Hz, 2H), 6.84 - 6.79 (m, 2H), 6.71 (d, J = 0.9 Hz, 2H), 6.42 (d, J = 7.6 Hz, 2H), 6.37 (d, J = 4.4 Hz, 2H), 6.14 (d, J = 4.1 Hz, 2H), 2.34 (s, 3H), 2.03 (s, 6H). ESI-MS (positive): calcd. for C 40 H 33 IrN4[M] + : 762.23; found: 762.2.

[0092] <Synthesis of PPY - BMD>

[0093]

Chem.

[0094] (PPY - BMD) BT - DIPY - MES (17 mg, 46 μmol) and K2CO3 (97 mg, 0.70 mmol) were dissolved in ultra - dehydrated tetrahydrof ran (10 mL) and stirred at room temperature for 15 minutes. The PPY chlorine bridging dinuclear complex (35 mg, 32 μmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was dried under reduced pressure to dryness. The obtained crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n - hexane: chloroform = 6:4, v / v) to give dark red solid PPY - BMD (20 mg, 23 μmol, 50%).

[0095] 1 1H NMR (400 MHz, CDCl3): δ 8.11 (dd, J = 5.7, 0.9 Hz, 1H), 7.90 (dd, J = 5.8, 0.8 Hz, 1H), 7.81 (m, 2H), 7.63 - 7.55 (m, 5H), 7.46 - 7.44 (m, 1H), 7.08 - 6.98 (m, 3H), 6.95 - 6.90 (m, 3H), 6.87 - 6.78 (m, 4H), 6.66 (t, J = 1.4 Hz, 1H), 6.64 (s, 1H), 6.46 (dd, J = 7.6, 0.7 Hz, 1H), 6.36 - 6.32 (m, 2H), 6.15 (dd, J = 4.4, 1.4 Hz, 1H), 2.39 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H). ESI-MS (positive): calcd. for C 46 H 35 IrN4S [M] + : 868.22; found: 868.1.

[0096] <Synthesis of PPY - BBMD>

[0097]

Chem.

[0098] (PPY - BBMD) BBT - DIPY - MES (30 mg, 63 μmol) and K2CO3 (108 mg, 0.78 mmol) were dissolved in ultra - dehydrated tetrahydro furan (10 mL) and stirred at room temperature for 15 minutes. PPY chlorine - bridged dinuclear complex (47 mg, 44 μmol) was added and refluxed overnight under a nitrogen atmosphere. After the reaction solution was cooled to room temperature, the solution was concentrated under reduced pressure [[ID=三十一]]to dryness. The obtained crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n - hexane: chloroform = 5:5, v / v) to give a purple The colored solid PPY-BBMD was obtained (50 mg, 51 μmol, 81%).

[0099] 1 H NMR (400 MHz, CDCl3): δ 8.12 (d, J = 5.8 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.8 Hz, 2H), 7.46 (d, J = 7.6 Hz, 2H), 7.09 - 6.99 (m, 10H), 6.83 - 6.79 (m, 4H), 6.62 (s, 2H), 6.39 (d, J = 7.6 Hz, 2H), 2.44 (s, 3H), 2.15 (s, 6H). ESI-MS (positive): calcd. for C 52 H 37 IrN4S [M] + : 974.21; found: 974.0.

[0100] <Synthesis of PPYDM-BBMD>

[0101]

Chemical formula

[0102] (PPYCOOH-BBMD) BBT-DIPY-MES (70 mg, 0.15 mmol) and K2CO3 (250 mg, 1.81 mmol) were dissolved in 2-methoxyethanol (20 mL) and stirred at room temperature for 15 minutes. The PPYCOOEt chlorine-bridged dinuclear complex (115 mg, 81 μmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was concentrated The crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, eluent: chloroform:methanol = 9:1, v / v) to give PPYCOOH-BBMD as a purple solid (41 mg, 38 μmol, 25%).

[0103] 1 H NMR (400 MHz, DMSO-d6): δ 13.03-11.72 (br, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.88 (s, 2H), 7.76-7.68 (m, 4H), 7.57 (d, J = 7.0 Hz, 2H), 7.21-7.18 (m, 2H), 7.08-6.96 (m, 10H), 6.74 (t, J = 6.9 Hz, 2H), 6.56 (s, 2H), 6.22 (d, J = 6.7 Hz, 2H), 3.46 (s, 2H), 2.36 (s, 3H), 2.06 (s, 6H). ESI-MS (positive): calcd. for C 56 H 41 IrN4O4S2[M] + : 1090.22; found: 1090.3.

[0104] (PPYDM-BBMD) PPYCOOH-BBMD(54 mg, 50 μmol), N, N-dimethylenediamine(22 μL, 0.20 mmol), o-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 83 mg, 0.22 mmol) and triethylamine (69 μL) were dissolved in ultra-dehydrated dichloromethane. Se was stirred at room temperature for 24 hours under a nitrogen atmosphere. The reaction solution was dried under reduced pressure, and the obtained crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: chloroform: methanol = 9:1, v / v) to obtain purple solid PPYDM-BBMD (20 mg, 16 μmol, 33%).

[0105] 1 1H NMR (400 MHz, CDCl3): δ 7.94 (d, J = 1.4 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.71 - 7.66 (m, 3H), 7.61 (d, J = 7.3 Hz, 2H), 7.45 (d, J = 6.9 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H), 7.11 - 6.98 (m, 9H), 6.82 - 6.78 (m, 2H), 6.62 (s, 2H), 6.38 (d, J = 6.9 Hz, 2H), 3.63 (s, 4H), 3.26 (d, J = 14.2 Hz, 4H), 3.12 (q, J = 5.6 Hz, 4H), 2.45 (s, 3H), 2.17 (s, 6H), 2.06 (s, 12H). ESI-MS (positive): calcd. for C 64 1H 61 IrN8O2S2[M] + : 1230.4; found: 1231.3.

[0106] <Synthesis of BTP-BMD>

[0107]

Chemical formula

[0108] (BTP-MD) 5-Mesityl-dipyrromethane (132 mg, 0.50 mmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 115 mg, 0.51 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (20 mL) and stirred at room temperature for 1 hour. K2CO3 (1.00 g, 7.24 mmol) was added to the reaction solution and stirred at room temperature for 15 minutes, then the BTP chlorine-bridged dinuclear complex (326 mg, 0.25 mmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was dried under reduced pressure. After removing the starting materials from the obtained crude product by silica gel column chromatography (developing solvent: chloroform), it was purified by recycling preparative HPLC (LC-9225 NEXT, Japan Analysis Industry) to obtain red solid BTP-MD (137 mg, 0.16 mmol, 31%).

[0109] 1 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 5.7 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.65 - 7.56 (m, 4H), 7.13 - 7.09 (m, 2H), 6.90 (s, 2H), 6.83 (t, J = 7.7 Hz, 2H), 6.78 - 6.74 (m, 2H), 6.61 (s, 2H), 6.38 (dd, J = 4.2, 1.3 Hz, 2H), 6.23 (d, J = 8.2 Hz, 2H), 6.12 (dd, J = 4.2, 1.3 Hz, 2H), 2.34 (s, 3H), 2.03 (s, 6H). ESI-MS (positive): calcd. for C 44 H 33 IrN4S2[M] + : 874.18; found: 874.2.

[0110] <Synthesis scheme of BTP-BMD>

[0111] [ka]

[0112] (BTP-BMD) BT-DIPY-MES (16 mg, 43 μmol) and K2CO3 (69 mg, 0.50 mmol) were dissolved in superhydrogenated tetrahydrofuran. The reaction mixture was dissolved in 10 mL of ethanol and stirred at room temperature for 15 minutes. BTP chlorine-bridged dinuclear complex (31 mg, 24 μmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling the reaction mixture to room temperature, the solution was evaporated to dryness under reduced pressure. The obtained crude product was purified by silica gel flash chromatography (Isolera Spektra, The resulting solid was purified using Biotage (developing solvent: n-hexane:chloroform = 5:5, v / v). Total BTP-BMD was obtained (25 mg, 26 μmol, 59%).

[0113] 1 H NMR (400 MHz, CDCl3): δ 8.05 (d, J = 5.7Hz, 1H), 7.86 (d, J= 5.5 Hz, 1H), 7.73 (d, J = 18.7, 7.9 Hz, 2H), 7.64-7.53 (m, 4H), 7.43 (d, J = 7.3 Hz, 1H), 7.15-6.96 (m, 7H), 6.84-6.72 (m, 4H), 6.67 (s, 1H), 6.56 (s, 1H), 6.36 (dd, J= 4.2, 1.3 Hz, 1H), 6.18 (dd, J= 7.9, 2.4 Hz, 2H), 6.12 (dd, J= 4.2, 1.3 Hz, 1H), 2.40 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H). ESI-MS (positive): calcd. for C 50 H 35 IrN4S3[M] + : 980.17; found: 980.2.

[0114] <Synthesis of BTP-BBMD>

[0115] [Chemical formula]

[0116] (BTP-BBMD) BBT-DIPY-MES (21 mg, 44 μmol) and K2CO₃ (74 mg, 0.54 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (10 mL) and stirred at room temperature for 15 minutes. The BTP chlorine-bridged dinuclear complex (41 mg, 32 μmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was dried under reduced pressure to a solid. The obtained crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, developing solvent: n-hexane: chloroform = 7:3, v / v), and purple solid BTP-BBMD was obtained (40 mg, 37 μmol, 83%).

[0117] 1 ¹H NMR (400 MHz, CDCl₃): δ 8.13 - 8.11 (m, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.63 - 7.51 (m, 4H), 7.44 (dd, J = 7.6, 0.7 Hz, 2H), 7.13 - 6.95 (m, 10H), 6.82 - 6.73 (m, 4H), 6.64 (s, 2H), 6.17 (d, J = 7.8 Hz, 2H), 2.45 (s, 3H), 2.17 (s, 6H). ESI-MS (positive): calcd. for C 56 H 37 IrN₄S₄[M] + : 1086.15; found: 1086.2.

[0118] <Synthesis of BTPDM-BBMD>

[0119] [Chemical formula]

[0120] (BTPDM-BBMD) BBT-DIPY-MES (14 mg, 29 μmol) and K2CO3 (50 mg, 0.36 mmol) were dissolved in 2-methoxyethanol (10 mL) and stirred at room temperature for 15 minutes. BTPDM chlorine-bridged dinuclear complex (41 mg, 23 μmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling the reaction solution to room temperature, the solution was evaporated to dryness under reduced pressure. The obtained crude product was purified by silica gel flash chromatography (Isolera Spektra, Purification was performed using Biotage (developing solvent: chloroform:methanol = 9:1, v / v) to obtain BTPDM-BBMD as a purple solid (19 mg, 14 μmol, 49%).

[0121] 1 H NMR (400 MHz, CDCl3): δ 7.97 (s, 2H), 7.71 (d, J = 7.6 Hz, 4H), 7.56-7.51 (m, 2H), 7.44 (d, J = 7.6 Hz, 2H), 7.11-6.95 (m, 12H), 6.84 (t, J= 7.0 Hz, 2H), 6.66 (s, 2H), 6.14 (d, J= 7.8 Hz, 2H), 3.32-3.24 (m, 4H), 3.10 (s, 4H), 2.45 (s, 4H), 2.20 (s, 12H), 2.03 (s, 9H). ESI-MS (positive): calcd. for C 70 H 61 IrN6O2S4[M] + : 1342.34; found: 1343.5.

[0122] <Measurement method> The photophysical properties of the compounds, luminescence microscopic images using the compounds, luminescence intensity, and luminescence lifetime were measured using the following equipment. Absorption spectrum, molar absorption coefficient: UV-visible spectrophotometer, Ubest-550; JASCO Corporation Phosphorescence spectrum: Multichannel spectrometer, PMA-12; Hamamatsu Photonics Phosphorescence quantum yield: Luminescence quantum yield measurement device, C9920-01; Hamamatsu Photonics Phosphorescence lifetime: Multichannel scaler, MSA-300; Becker & Hickl Luminescence microscopy image: Research inverted microscope, IX-71; Olympus Phosphorescence lifetime imaging: Research inverted microscope (IX-71; Olympus) equipped with a confocal scanner (DCS-120; Becker & Hickl)

[0123] Example 1 The iridium complex synthesized as the compound of the present invention is shown in FIG. Figure 2 shows the absorption and phosphorescence spectra of PPY-MD (Comparative Example), PPY-BMD, PPY-BBMD, and PPYDM-BBMD in acetonitrile at room temperature. Figure 3 shows the absorption and phosphorescence spectra of BTP-MD (Comparative Example), BTP-BMD, BTP-BBMD, and BTPDM-BBMD in acetonitrile at room temperature. Table 1 also shows the absorption and phosphorescence spectra of BTPHSA (Comparative Example), PPY-MD (Comparative Example), PPY-BMD, BTP-BMD, PPY-BBMD, and PPYDM-BBMD. Photophysical properties at room temperature in acetonitrile.

[0124] [Table 1]

[0125] The compounds of the present invention have an absorption maximum wavelength (λ abs ) is observed at wavelengths above 500 nm, and the maximum emission wavelength (λ phos ) was observed above 700 nm, which is larger than that of previously reported iridium complexes. The emission intensity significantly decreased under air-saturated conditions compared to deoxygenated (nitrogen-saturated) conditions, indicating that the observed emission was phosphorescence. p 0) is 0.049 or more. abs The molar extinction coefficient (ε) at 1000 s was significantly increased. This indicates that the phosphorescence brightness (εφ) under deoxygenation p 0 ) has a higher phosphorescence brightness than the iridium complexes reported so far. p 0 ) significantly increased, indicating that oxygen sensitivity (τ p 0 / τ p ) has also increased significantly.

[0126] <Example 2> The intracellular oxygen responsiveness of PPY-MD (comparison example), PPYDM-BBMD (Figure 4), and BTP-MD and BTPDM-BBMD (Figure 5) was evaluated. Each iridium complex was added to a final concentration of 500 nM, and after 2 hours of incubation, phosphorescence images (λ) of AML12 cells were measured under 21% oxygen partial pressure and 2.5% oxygen partial pressure. ex :545-580 nm λ em :>610 nm). PPYDM-BBMD and BTPDM-BBMD showed a significant increase in phosphorescence intensity under 2.5% oxygen partial pressure, demonstrating their intracellular oxygen responsiveness. Furthermore, the oxygen imaging performance of conventional reagents (BTPDM1, BTPHSA) and the reagent of the present invention, PPYDM-BBMD, was evaluated (Figure 6). In the case of staining with PPYDM-BBMD, It was found that a clearer image could be obtained.

[0127] Example 3 Figure 7 shows inverted microscope images of AML12 cells stained with PPYDM-BBMD and various dyes at 21% oxygen tension. The merged images show that PPYDM-BBMD is mainly distributed in the endoplasmic reticulum. It was.

[0128] Example 4 Next, cytotoxicity was evaluated. The cell viability after 2 hours of addition of PPYDM-BBMD was 4 μM. It was found that high values ​​were observed at or below 500 nM (FIG. 8). The cell experiments for oxygen imaging in this example were carried out at 500 nM, and it was found that cytotoxicity was almost negligible.

[0129] <Example 5> Changes in intracellular oxygen concentration were tracked using phosphorescence lifetime imaging microscopy. AML12 cells (A) and HK-2 cells (B) were seeded in 35 mm Petri dishes and cultured for 48 hours. PPYDM-BBMD was added to a final concentration of 500 nM and cultured for 2 hours. The oxygen partial pressure in the incubator was varied to 160, 114, 76, 38, and 0 mmHg, and phosphorescence lifetime imaging images were acquired at each oxygen partial pressure. The average lifetime of the images was calculated, and the phosphorescence lifetime ratio (phosphorescence lifetime at 0 mmHg / phosphorescence lifetime at each oxygen partial pressure) was plotted against the oxygen partial pressure (C). A calibration curve was created to obtain oxygen partial pressure from the phosphorescence lifetime (Figure 9).

[0130] Example 6 Next, we evaluated the in vivo dynamics. In this experiment, we measured phosphorescence lifetime in addition to phosphorescence intensity images. A PLIM (phosphorescence lifetime imaging microscope) was used to capture images. PPYDM-BBMD (200 nmol) was administered to the tail vein of anesthetized mice, and a phosphorescence lifetime imaging experiment was performed (excitation wavelength: 580 nm, observation wavelength: > 647 nm). Figure 10A shows a lifetime imaging image of the liver of a mouse administered with PPYDM-BBMD. The hepatic lobule, which is the functional unit of the liver, was clearly imaged using this method, and the increase in phosphorescence lifetime from the portal vein area (PV) to the central vein area (CV) indicates the existence of an oxygen partial pressure gradient. A significant difference in the oxygen partial pressure between the PV and CV was observed (Fig. 10B). Similar images were also obtained 180 μm from the surface (Fig. 11A). The oxygen partial pressures of the PV (two locations) and CV showed almost the same tendency from the surface to the interior of 180 μm (Fig. 11B). Therefore, by using PPYDM-BBMD as an oxygen probe, it is possible to obtain information on the oxygen partial pressure deeper than 150 μm. It has been shown that this is possible.

[0131] From the above results, the iridium complex compound represented by the general formula (I) of the present invention has the following properties: It was found to be a new reagent that can measure oxygen concentrations in individuals and perform imaging based on oxygen concentrations. [Industrial Applicability]

[0132] The present invention can be used in fields such as medical diagnosis, pharmaceutical development, and basic medicine.

Claims

1. A compound represented by the following formula (I): 【Chemical 1】 During the ceremony, Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring; A １ indicates a heteroatom, Z represents hydrogen or a hydrocarbon group having 1 to 20 carbon atoms which may have a substituent; L represents a bidentate ligand.

2. The compound according to claim 1, wherein the ring R is represented by the following formula (R-1), (R-2), or (R-3): 【Chemistry 2】 During the ceremony, A ２ indicates a heteroatom, X represents hydrogen.

3. The above A ２ The compound of claim 2, wherein is sulfur or oxygen.

4. The compound according to claim 2 or 3, wherein the ring R is represented by formula (R-3).

5. The above A １ The compound according to any one of claims 1 to 4, wherein is sulfur or oxygen.

6. The compound according to any one of claims 1 to 5, wherein Z is an alkyl group having 1 to 20 carbon atoms which may have a substituent, or an aryl group having 6 to 20 carbon atoms which may have a substituent. 。

7. The compound according to any one of claims 1 to 6, wherein L is represented by the following formula (II): Compound: 【Chemistry 3】 During the ceremony, Ring R １ represents a monocyclic or polycyclic nitrogen-containing aromatic ring, Ring R ２ represents a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring, Ring R １ and ring R ２ may have a substituent.

8. The compound according to claim 7, wherein the substituents bonded to ring R 1 and ring R 2 are represented by the following formula (III): 【Chemistry 4】 During the ceremony, The carbon atom marked with * is the bonding position to the ring R 1 and the ring R 2 ; n represents an integer of 1 to 5, X ３ each independently represents hydrogen or a hydrocarbon group having 1 to 6 carbon atoms.

9. An oxygen imaging reagent comprising the compound according to any one of claims 1 to 8.

Images