JP7738889B2 - Oxygen Imaging Reagents - Google Patents

Description

The present invention relates to an oxygen imaging reagent and compounds that can be used in the reagent.

Oxygen is an essential substance for maintaining the vital activities of aerobic organisms. Hypoxia in the body is commonly observed in cancer, ischemic disease, chronic kidney disease, fatty liver, and other conditions. Therefore, imaging oxygen partial pressure 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 partial pressure within tissues, but this is invasive and can only measure the area near the needle electrode. On the other hand, imaging technology using molecular luminescence (phosphorescence) offers high sensitivity, simplicity, and real-time measurement. When combined with a microscope, it can easily achieve spatial resolution at the single-cell level. For this reason, oxygen imaging reagents using metal complexes have been developed and are commercially available (Patent Documents 1 and 2).

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 ² )phenyl-κC]iridium in Non-Patent Document 2: Figure 12) have an improved phosphorescence lifetime compared to BTPHSA, but their absorption and emission wavelengths are shorter, and their phosphorescence brightness is low, making deep imaging difficult.
(3) Dendrimer-type near-infrared oxygen imaging reagents are also available, but they are limited to measuring blood oxygen partial pressure.

Therefore, there is a need for oxygen imaging reagents for cells, biological tissues, etc. that phosphoresce in the near-infrared region, have a longer phosphorescence lifetime, and enable imaging at deeper depths.

Japanese Patent Application Laid-Open No. 2010-44059 Japanese Patent Application Laid-Open No. 2015-101567

S. Zhang et al., Cancer Res., 70, 4490-4498, 2010. Hanson, K. et al., Inog. Chem. 2010, 49, 6077-6084.

The present invention was developed in consideration 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 enables imaging at deeper locations.

As a result of intensive research aimed at solving the above-mentioned problems, the inventors have developed a novel compound that exhibits phosphorescence in the near-infrared region and has a longer phosphorescence lifetime. They have also discovered that using such a compound, oxygen imaging can be performed at greater depths. Based on these findings, they have completed the present invention.

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

During the ceremony,
Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring;
A ¹ represents 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):

During the ceremony,
^A2 represents a heteroatom;
X represents hydrogen.
[3] The compound according to [2], wherein 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] The compound according to any one of [1] to [4], wherein 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):

During the ceremony,
Ring ^R1 represents a monocyclic or polycyclic nitrogen-containing aromatic ring;
Ring ^R2 represents a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring;
The ring R ¹ and the ring R ² may have a substituent.
[8] The compound according to [7], wherein the substituent is represented by the following formula (III):

During the ceremony,
n represents an integer of 1 to 5,
^X3 's each independently represent 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].

The present invention provides a novel compound that exhibits phosphorescence in the near-infrared region and has a longer phosphorescence lifetime, and also provides an oxygen imaging reagent using the compound that enables imaging at deeper depths.

FIG. 1 shows the structural formula of the synthesized oxygen imaging reagent compound of the present invention. Figure 2 shows the absorption and phosphorescence spectra of PPY-MD, PPY-BMD, PPY-BBMD, and PPYDM-BBMD. Figure 3 shows the absorption and phosphorescence spectra of BTP-MD, BTP-BMD, BTP-BBMD, and BTPDM-BBMD. FIG. 4 shows phosphorescence images (photographs) of AML12 cells stained with PPY-MD and PPYDM-BBMD. FIG. 5 shows phosphorescence images (photographs) of AML12 cells stained with BTP-MD and BTPDM-BBMD. FIG. 6 shows phosphorescence images (photographs substituting drawings) of AML12 cells stained with BTPDM1 (comparative example), BTPHSA (comparative example), and PPYDM-BBMD. 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). FIG. 8 shows the results of evaluating the cytotoxicity of PPYDM-BBMD. 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 shows the results of imaging the liver of a mouse administered with PPYDM-BBMD. (A) Phosphorescence lifetime imaging image (photograph substitute for drawing) of the liver of a mouse administered with PPYDM-BBMD, and (B) oxygen partial pressure in the CV and PV regions. 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 substitute for drawing) of the liver of a mouse administered with PPYDM-BBMD, and (B) oxygen partial pressure in the CV and PV regions. FIG. 12 shows the structural formula of a conventional oxygen imaging reagent compound.

The present invention will be described below.
<Compound represented by formula (I)>
One aspect of the present invention relates to a compound represented by the following formula (I) (hereinafter, sometimes referred to as the "compound of the present invention"):

During the ceremony,
Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring;
A ¹ represents 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.

The compound of the present invention is a complex having a central metal Ir, a bidentate ligand (shown in the formula below) with a dipyrromethene derivative structure including a ring R coordinated to Ir, and two bidentate ligands L.

The compound represented by formula (I) (hereinafter sometimes referred to as "Complex (I)") is described below.

Examples of the heteroatom represented by A ¹ include sulfur and oxygen, and sulfur is preferred.

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

Here, X represents hydrogen.
^A2 represents a heteroatom, and examples of the heteroatom represented by ^A2 include sulfur and oxygen, and preferably sulfur.
Among the carbon atoms constituting the ring skeleton, the bond extending from the carbon atom next to N is bonded to the carbon atom connecting to the other ring forming the dipyrromethene derivative structure. N is coordinated to Ir.

Ring R is preferably a polycyclic nitrogen-containing aromatic ring, in that, in combination with the other ring forming the dipyrromethene derivative structure and Z, it emits light approaching the near-infrared range and has good transmittance in vivo. Nitrogen-containing aromatic rings having a structure represented by formula (R-2) or (R-3) are more preferred, and nitrogen-containing aromatic rings having a structure represented by formula (R-3) are even more preferred.

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.

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).

The bidentate ligand L having rings ^R1 and ^R2 is a cyclometallated ligand and does not affect the luminescence performance except for the phosphorescence lifetime. Furthermore, the presence of L increases the bioaffinity of the complex (I), making it easier for the complex (I) to be taken up into the body.

The bidentate ligand L is not limited as long as it constitutes the complex (I) and exhibits the photophysical activity of the complex (I). L may be independent and may have the same or different structures, and preferably, L have the same structure.
L is not limited, but examples thereof include those having a structure represented by the following formula (II):

Ring ^R1 represents a monocyclic or polycyclic nitrogen-containing aromatic ring. Ring ^R1 is not limited to, but examples thereof include nitrogen-containing aromatic rings having a structure represented by the following formula (1-1), (1-2), (1-3), or (1-4).

Here, ^X1 represents hydrogen.
Among the carbon atoms constituting the ring skeleton, the bond extending from the carbon atom next to N is bonded to the ring ^R2 . N is coordinated to Ir.

As the ring R ¹ , from the viewpoint of maintaining the preferable light-emitting performance of the complex (I) in combination with the ring R ² , a nitrogen-containing aromatic ring having a structure represented by the formula (1-1) or (1-3) is preferred, and a nitrogen-containing aromatic ring having a structure represented by the formula (1-1) is more preferred.

Ring ^R2 is a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring. Examples of ring ^R2 include sulfur-containing aromatic rings or aromatic rings having the structures shown in the following formulas (2-1), (2-2), (2-3), and (2-4).

Here, ^X2 represents hydrogen.
Among the carbon atoms constituting the ring skeleton, a bond extending from the carbon atom adjacent to S in the formulae (2-1), (2-2), and (2-3) is bonded to the ring ^R1 , and the carbon atom adjacent to this carbon atom is coordinated to Ir. Also, one bond in the formula (2-4) is bonded to the ring ^R1 , and the carbon atom adjacent to this carbon atom is coordinated to Ir.

As the ring ^R2 , a sulfur-containing aromatic ring having a structure represented by ^the formula (2-1) or an aromatic ring having a structure represented by the formula (2-4) is preferred in terms of maintaining the preferable luminescence performance of the complex (I) in combination with the ring R1.

In addition, ^X1 and ^X2 may independently be a substituent other than hydrogen, as long as it does not affect the performance of complex (I). Examples of the substituent include, but are not limited to, halogen, haloalkyl group, hydroxy group, carboxy group, amino group, alkylamino group, alkoxy group, acyl group, mercapto group, or bioaffinity group (which may include a bonding group to ring ^R1 and/or ring ^R2 ). Here, the term "bioaffinity group" refers to a group that has high affinity with biological materials and has the property of promoting the introduction of complex (I) into cells or biological tissues. The bonding group is not limited as long as it is a group that can bond the bioaffinity group to complex (I), and examples thereof include an amide group.

From the viewpoint of improving affinity with living organisms, it is preferable to introduce a biocompatible group as the substituent of ^X1 and ^X2 . 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 polymer having biocompatibility.

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. Examples of the hydrocarbon group having 1 to 20 carbon atoms include an alkyl group, an alkenyl group, an alkynyl group, and an aryl group, and preferably an alkyl group. Examples of such biocompatible groups include groups represented by the following formula (III), in which a hydrocarbon group substituted with an alkylamino group is bonded to ring ^R1 or ring ^R2 via an amide bond:

n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 1.
^X3 's each independently represent hydrogen or an alkyl group having 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms, and more preferably 1 carbon atom (methyl group).

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

Examples of the bidentate ligand L include bidentate ligands having the structure shown in formula (IIa) or (IIb) below.

Complex (I) is preferably a complex having a structure represented by the following formula (Ia) or (Ib):

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

As the complex (I) of the present invention, for example, (1) the complex is represented by the formula (Ia) and L is represented by the formula (II)
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) (PPYDM-BBMD) having a biocompatible group (n=1, X ³ =methyl group), and the like. From the viewpoints of luminescence properties, biocompatible properties, etc., the compound (4) is preferred.

Complex (I) of the present invention can be synthesized using conventional organic synthesis methods. For example, it can be synthesized according to the methods described in the Examples below. Commercially available raw materials may be used, or those synthesized by known methods may be used.

The photophysical properties of the phosphorescence of complex (I), such as the absorption and phosphorescence maximum wavelengths, the molar extinction coefficient (ε
The absorption/phosphorescence maximum wavelength and molar extinction coefficient can be measured using a spectrophotometer or the _like , and the phosphorescence quantum yield can be measured using an emission quantum yield measurement device or the like, with the complex ( _I ) being used as a sample dissolved in a solvent or the like. The phosphorescence lifetime (τ _p ) of the compound can be measured in each solvent using a phosphorescence lifetime measurement device.
The phosphorescence brightness (εφ _p ) (under deoxygenated (nitrogen saturated) conditions) can be changed depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 3,300 or more, 3,500 or more, 5,000 or more, or 6,000 or more.
The phosphorescence lifetime (τ _p ) (under deoxygenated (nitrogen saturated) conditions) can be varied depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 5.0 μs (microseconds) or more, 10.0 μs or more, 15.0 μs or more, or 18.0 μs or more.

The maximum excitation wavelength of Complex (I) in a solvent can be varied depending on the structure of the compound, the type of solvent, etc., and is not particularly limited, but is, for example, 500 nm to 580 nm. The maximum phosphorescence wavelength in the solvent can also be appropriately set, but is, for example, 700 nm to 780 nm.

<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").

The oxygen imaging reagent of the present invention contains the compound complex (I) of the present invention. This structure provides excellent photophysical properties in phosphorescence (e.g., phosphorescence maximum wavelength, phosphorescence lifetime, molar extinction coefficient, phosphorescence brightness, etc.). Because complex (I) exhibits phosphorescence in the near-infrared region, the oxygen imaging reagent of the present invention can be distinguished from autofluorescence and can also image oxygen in deeper tissues. Furthermore, because complex (I) has a longer phosphorescence lifetime and higher phosphorescence brightness, the oxygen imaging reagent of the present invention enables 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.

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

When placed in a cell or tissue environment, complex (I) emits stronger phosphorescence when the oxygen partial pressure in the environment is low. Therefore, the oxygen partial pressure can be measured based on the phosphorescence intensity. That is, when the phosphorescence is strong, it can be determined that the oxygen partial pressure is low. Furthermore, by determining the relationship between the oxygen partial pressure and the phosphorescence intensity in advance, it is possible to quantitatively measure the oxygen partial pressure.

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. Biological samples include, but are not limited to, cells or isolated tissues. 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. within a living organism.

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 incubation time can be varied 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.

The oxygen imaging reagent of the present invention can also image oxygen in tissues of 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.
Thereafter, the phosphorescent signal of the oxygen imaging reagent of the present invention can be observed and imaged using an in vivo imaging device or the like, thereby imaging 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.

<Cancer diagnostic drugs>
As described above, when placed in a cell or tissue environment, Complex (I) of the present invention emits stronger phosphorescence when the oxygen partial pressure in the environment is low. Therefore, Complex (I) of the present invention can be administered to experimental animals such as mice and rats, or to humans, to detect sites with reduced oxygen partial pressure. Since cancer tissues suffer from a shortage of oxygen, detecting sites with reduced oxygen partial pressure allows specific staining of cancer tissues, which can be used as a cancer diagnostic agent.
For example, phosphorescence can be observed by administering the complex (I) of the present invention to a specimen and irradiating the specimen with visible light ex vivo. This allows non-invasive, highly sensitive, and selective visualization of cancer tissue. Furthermore, since phosphorescence can be visualized, it can also be used as an imaging reagent for cancer detection.
Furthermore, the complex (I) of the present invention can also be used in cancer research using laboratory animals and in the evaluation of cancer therapeutic drugs.

The present invention will be explained in detail below using examples, but these are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

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

The BODIPY derivatives were 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.).
PPY-MD was synthesized with reference to the literature (Hanson, K.; Tamayo, A.; Diev, VV; Whited, MT; Djurovich, PI; Thompson, ME. Efficient dipyrrin-centered phosphorescence at room temperature from bis-cyclometalated iridium(III) dipyrrinato complexes. Inog. Chem. 2010, 49, 6077-6084.).

<Synthesis of BT-DIPY-MES>

(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%).
.

¹ H NMR (400 MHz, CDCl ₃ ): 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 BF ₂ N ₂ S [M] ⁺ : 432.16; found: 431.1.

(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).
The resulting solution was dissolved in a 200 ml solution of 30% ethanol (v/v) and cooled in an ice bath until the solvent was almost frozen. H ₂ O ₂ (30%, 40 μL, 0.5 mmol) was slowly added to the frozen solution. The mixture was removed from the ice bath and stirred overnight at room temperature. The acetic acid was removed under reduced pressure, and saturated aqueous sodium bicarbonate was poured into the mixture, followed by extraction with dichloromethane. The organic layer was dried over Na ₂ SO ₄ , 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 = 6:4, v/v) to give SOPh-BODIPY-MES as a red solid (73 mg, 0.16 mmol, 73%).

¹ H NMR (400 MHz, CDCl ₃ ): 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).

(BT-DIPY-MES)
SOPh-BODIPY-MES (64 mg, 0.14 mmol) was added portionwise 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 adjusted to pH 8 with aqueous _potassium carbonate. The resulting solution was extracted with dichloromethane. The organic layer was dried over _Na2SO4 , filtered, and then evaporated to dryness under reduced pressure. The resulting crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n-hexane:chloroform = 5:5, v/v) to give BT-DIPY-MES as a reddish-purple solid (17 mg, 46 μmol, 32%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 N ₂ S [M+H] ⁺ : 369.13; found: 369.2.

<Synthesis of BBT-DIPY-MES>

(BSPh-BODIPY-MES)
2,6-dibromo BODIPY-MES (566 mg, 1.21 mmol), 2-(methylthio)phenylboronic acid
(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, 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 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%).

¹ H NMR (400 MHz, DMSO-d ₆ ): 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 BF ₂ N ₂ S ₂ [M] ⁺ : 554.18; found: 553.1.

(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 overnight at room temperature. 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.

¹ H NMR (400 MHz, CDCl ₃ ): 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).

(SOPhBT-BODIPY-MES)
BSOPh-BODIPY-MES (189 mg, 0.32 mmol) was added portionwise 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 adjusted to pH 8 with aqueous potassium carbonate. Dichloromethane was added to the aqueous solution and extracted. The organic layer was dried over Na ₂ SO ₄ , filtered, and the solution was evaporated to dryness under reduced pressure. The crude product was dissolved in anhydrous 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 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:ethyl acetate = 2.5:7.5, v/v) to give SOPhBT-BODIPY-MES as a purple solid (118 mg, 0.21 mol, 66%).

¹ H NMR (400 MHz, CDCl ₃ ): 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).

(BBT-DIPY-MES)
SOPhBT-BODIPY-MES (243 mg, 0.44 mmol) was added portionwise 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 adjusted to pH 8 with aqueous _potassium carbonate. The resulting solution was extracted with dichloromethane. The organic layer was dried over _Na2SO4 , filtered, and then evaporated to dryness under reduced pressure. The resulting crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n-hexane:chloroform = 5:5, v/v) to give BBT-DIPY-MES as a purple solid (93 mg, 0.20 mol, 45%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 N ₂ S ₂ [M+H] ⁺ : 475.12; found: 475.1.

<Synthesis of PPY-MD>

(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).
The _mixture was stirred at room temperature for 1 hour. _K2CO3 (415 mg, 3.00 mmol) was added to the reaction solution, which was then stirred at room temperature for 15 minutes. Then, PPY chlorine-bridged binuclear complex (110 mg, 0.10 mmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure. The crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: n-hexane:chloroform = 5:5, v/v) to give PPY-MD as a red solid (108 mg, 0.14 mmol, 66%).

¹ H NMR (400 MHz, CDCl ₃ ): δ7.89 (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 IrN ₄ [M] ⁺ : 762.23; found: 762.2.

<Synthesis of PPY-BMD>

(PPY-BMD)
BT-DIPY-MES (17 mg, 46 μmol) and K ₂ CO ₃ (97 mg, 0.70 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (10 mL) and stirred at room temperature for 15 minutes. PPY chlorine-bridged binuclear complex (35 mg, 32 μmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was evaporated to dryness under reduced pressure. The resulting crude product was purified by silica gel flash chromatography (Isolera Spektra,
The product was purified using Biotage (developing solvent: n-hexane:chloroform = 6:4, v/v) to obtain PPY-BMD as a dark red solid (20 mg, 23 μmol, 50%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 IrN ₄ S [M] ⁺ : 868.22; found: 868.1.

<Synthesis of PPY-BBMD>

(PPY-BBMD)
BBT-DIPY-MES (30 mg, 63 μmol) and K ₂ CO ₃ (108 mg, 0.78 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (10 mL) and stirred at room temperature for 15 minutes. PPY chlorine-bridged dinuclear complex (47 mg, 44 μmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction 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:chloroform = 5:5, v/v) to give PPY-BBMD as a purple solid (50 mg, 51 μmol, 81%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 IrN ₄ S [M] ⁺ : 974.21; found: 974.0.

<Synthesis of PPYDM-BBMD>

(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 min. PPYCOOEt chlorine-bridged dinuclear complex (115 mg,
The reaction mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure. The crude product was purified using silica gel flash chromatography (Isolera Spektra, Biotage, eluent: chloroform:methanol = 9:1, v/v) to give PPYCOOH-BBMD (41 mg, 38 μmol, 25%) as a purple solid.

¹ H NMR (400 MHz, DMSO-d ₆ ): 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 IrN ₄ O ₄ S ₂ [M] ⁺ : 1090.22; found: 1090.3.

(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 and stirred at room temperature under a nitrogen atmosphere for 24 hours. The reaction solution was evaporated to dryness under reduced pressure, and the resulting crude product was purified by silica gel flash chromatography (Isolera Spektra, Biotage, eluent: chloroform:methanol = 9:1, v/v) to give PPYDM-BBMD as a purple solid (20 mg, 16 μmol, 33%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 H ₆₁ IrN ₈ O ₂ S ₂ [M] ⁺ : 1230.4; found: 1231.3.

<Synthesis of BTP-BMD>

(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 h. _K2CO3 (1.00 g, 7.24 mmol) was added to the reaction solution, _which was then stirred at room temperature for 15 min. Then, BTP chlorine-bridged dinuclear complex (326 mg, 0.25 mmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: chloroform) to remove starting materials, followed by purification by recycling preparative HPLC (LC-9225 NEXT, Japan Analysis Industry) to yield BTP-MD as a red solid (137 mg, 0.16 mmol, 31%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 IrN ₄ S ₂ [M] ⁺ : 874.18; found: 874.2.

<BTP-BMD synthesis scheme>

(BTP-BMD)
BT-DIPY-MES (16 mg, 43 μmol) and K ₂ CO ₃ (69 mg, 0.50 mmol) were dissolved in ultra-dehydrated tetrahydrofuran (10 mL) and stirred at room temperature for 15 minutes. BTP chlorine-bridged dinuclear complex (31 mg, 24 μmol) was added and refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was evaporated to dryness under reduced pressure. The resulting crude product was purified by silica gel flash chromatography (Isolera Spektra,
The product was purified using Biotage (developing solvent: n-hexane:chloroform = 5:5, v/v) to obtain BTP-BMD as a red solid (25 mg, 26 μmol, 59%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 IrN ₄ S ₃ [M] ⁺ : 980.17; found: 980.2.

<Synthesis of BTP-BBMD>

(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 min _. BTP chlorine-bridged dinuclear complex (41 mg, 32 μmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction 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:chloroform = 7:3, v/v) to give BTP-BBMD as a purple solid (40 mg, 37 μmol, 83%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 ESI-MS (positive): calcd. for C ₅₆ H ₃₇ IrN ₄ S ₄ [M] ⁺ : 1086.15; found: 1086.2.

<Synthesis of BTPDM-BBMD>

(BTPDM-BBMD)
BBT-DIPY-MES (14 mg, 29 μmol) and K ₂ CO ₃ (50 mg, 0.36 mmol) were dissolved in 2-methoxyethanol (10 mL) and stirred at room temperature for 15 minutes. BTPDM chlorine-bridged binuclear complex (41 mg, 23 μmol) was added and the mixture was refluxed overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction solution was evaporated to dryness under reduced pressure. The resulting crude product was purified by silica gel flash chromatography (Isolera Spektra,
Purification using Biotage (developing solvent: chloroform:methanol = 9:1, v/v) gave BTPDM-BBMD as a purple solid (19 mg, 14 μmol, 49%).

¹ H NMR (400 MHz, CDCl ₃ ): 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 IrN ₆ O ₂ S ₄ [M] ⁺ : 1342.34; found: 1343.5.

<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 extinction 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. Emission microscopy image: Research inverted microscope, IX-71; Olympus Corporation. Phosphorescence lifetime imaging image: Research inverted microscope (IX-71; Olympus) equipped with a confocal scanner (DCS-120; Becker & Hickl).

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 shows the photophysical properties of BTPHSA (comparative example), PPY-MD (comparative example), PPY-BMD, BTP-BMD, PPY-BBMD, and PPYDM-BBMD in acetonitrile at room temperature.

The compounds of the present invention exhibited absorption maxima (λ _abs ) at wavelengths greater than 500 nm and emission maxima (λ _phos ) at wavelengths greater than 700 nm, significantly longer than 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. The phosphorescence quantum yield (φ _p ⁰ ) under deoxygenated conditions was greater than 0.049. Furthermore, the molar extinction coefficient (ε) at λ _abs significantly increased. Therefore, the phosphorescence brightness under deoxygenated conditions (εφ _p ⁰ ) was higher than that of previously reported iridium complexes. Furthermore, the phosphorescence lifetime (τ _p ⁰ ) under deoxygenated conditions significantly increased, resulting in a significant increase in oxygen sensitivity (τ _p ⁰ /τ _p ).

Example 2
The intracellular oxygen responsiveness of PPY-MD (comparison), 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. After 2 hours of incubation, phosphorescence images (λ _ex : 545-580 nm, λ _em : >610 nm) of AML12 cells were measured under 21% and 2.5% oxygen tension. PPYDM-BBMD and BTPDM-BBMD exhibited a significant increase in phosphorescence intensity under 2.5% oxygen tension, demonstrating their intracellular oxygen responsiveness. Furthermore, the oxygen imaging performance of conventional reagents (BTPDM1, BTPHSA) and the present reagent, PPYDM-BBMD, was evaluated (Figure 6). It was found that cells stained with PPYDM-BBMD exhibited clearer images.

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 revealed that PPYDM-BBMD was primarily distributed in the endoplasmic reticulum.

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 (Figure 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.

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 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 derive oxygen partial pressure from the phosphorescence lifetime (Figure 9).

Example 6
Next, we evaluated in vivo dynamics. In this experiment, we used a PLIM (phosphorescence lifetime imaging microscope) capable of acquiring phosphorescence lifetime images in addition to phosphorescence intensity images. PPYDM-BBMD (200 nmol) was administered via the tail vein of anesthetized mice, and phosphorescence lifetime imaging experiments were performed (excitation wavelength: 580 nm, observation wavelength: >647 nm). Figure 10A shows a lifetime imaging image of the liver of a PPYDM-BBMD-administered mouse. The hepatic lobule, the functional unit of the liver, was clearly imaged within 20 μm from the surface. The increase in phosphorescence lifetime from the portal venous area (PV) to the central venous area (CV) indicates an oxygen partial pressure gradient. A significant difference in oxygen partial pressure between the PV and CV was observed (Figure 10B). Similar images were also obtained within 180 μm from the surface (Figure 11A). The oxygen partial pressures in the PV (two locations) and CV showed similar trends from the surface to a depth of 180 μm (Fig. 11B). This indicates that using PPYDM-BBMD as an oxygen probe can provide insight into the oxygen partial pressure at depths greater than 150 μm.

From the above results, it was found that the iridium complex compound represented by general formula (I) of the present invention is a new reagent that can measure oxygen concentrations in cells and individuals and perform imaging based on oxygen concentrations.

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

Claims (9)

A compound represented by the following formula (I):
During the ceremony,
Ring R represents a monocyclic or polycyclic nitrogen-containing aromatic ring;
A ¹ represents 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. The compound according to claim 1, wherein the ring R is represented by the following formula (R-1), (R-2), or (R-3):
During the ceremony,
^A2 represents a heteroatom;
X represents hydrogen. The compound of claim 2, wherein ^A2 is sulfur or oxygen. The compound according to claim 2 or 3, wherein the ring R is represented by formula (R-3). The compound according to any one of claims 1 to 4, wherein A ¹ is sulfur or oxygen. 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. The compound according to any one of claims 1 to 6, wherein L is represented by the following formula (II):
During the ceremony,
Ring ^R1 represents a monocyclic or polycyclic nitrogen-containing aromatic ring;
Ring ^R2 represents a monocyclic or polycyclic aromatic ring, or a monocyclic or polycyclic sulfur-containing aromatic ring;
The ring R ¹ and the ring R ² may have a substituent. 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):
During the ceremony,
The carbons marked with * are the bonding positions to the rings ^R1 and R2 ^,
n represents an integer of 1 to 5,
^X3 's each independently represent hydrogen or a hydrocarbon group having 1 to 6 carbon atoms. An oxygen imaging reagent comprising the compound of any one of claims 1 to 8.