JP2015101567A - High-sensitivity near-infrared phosphorescent iridium complex for measuring oxygen concentration in cell/tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound with a long phosphorescence lifetime which can efficiently measure the oxygen concentration in a cell, tissue or the like.SOLUTION: The oxygen concentration is measured using a compound represented by the general formula (I). [Rrepresents hydrogen, halogen or the like; Reach independently represent a hydrogen atom or a C1-6 hydrocarbon group; and n represents an integer between 1 and 5.]

Description

  The present invention relates to a novel iridium complex, an oxygen concentration measuring reagent using the same, and a cancer diagnostic agent.

The hypoxic environment in the living body is commonly observed in cancer, stroke, myocardial infarction, etc., which are the three major causes of death in Japan. Therefore, the development of a method for measuring the oxygen concentration in cells and tissues non-invasively in real time is an important issue in the fields of cell biology and medicine.
As a method for quantifying oxygen concentration in living tissue, (1) a method of measuring by inserting a microelectrode into a tissue, (2) a method using an ESR signal of a paramagnetic probe molecule, and (3) of a nitroimidazole probe molecule A method using a reduction reaction and (4) a method using light emission of a water-soluble porphyrin or ruthenium complex are known. The method (1) using a microelectrode can measure only the oxygen partial pressure at one point in the vicinity of the electrode, and has a drawback of being invasive. The method based on the ESR signal of (2) cannot measure oxygen concentration in real time. The method of (3) using a nitroimidazole drug reduces nitroimidazole in hypoxic cells and binds to intracellular proteins and is trapped. However, since this method requires time for the metabolism of the drug, there is a drawback in that data cannot be obtained until several hours have passed after the drug administration. On the other hand, the method (4) is a method for quantifying the oxygen concentration by utilizing the fact that the phosphorescence lifetime of the water-soluble porphyrin derivative or ruthenium complex changes (subjects to quenching) depending on the blood oxygen concentration. This method has a great advantage that the oxygen partial pressure in the tissue can be visualized non-invasively, but since the reagent is water-soluble, the obtained data is limited to the blood oxygen concentration (Non-patent Document 1).

Therefore, the group of the present inventors has developed a method for measuring oxygen concentration in living tissue using room temperature phosphorescence (intensity, lifetime) of an iridium complex (BTP) (Patent Document 1). From the measurement of phosphorescence intensity and lifetime of BTP, we succeeded in quantifying the oxygen concentration in the liposome membrane, phosphorescent imaging using cancer cells, and visualization of tumors in tumor-bearing mice (Non-patent Document 2). Furthermore, we developed an iridium complex BTPHSA that exhibits phosphorescence in the near-infrared light region, and succeeded in visualizing a tumor about 6-7 mm from the skin (Non-patent Document 2 and Patent Document 2).
However, since BTPHSA has a short phosphorescence lifetime of 2.0 μs and low oxygen responsiveness, it is difficult to distinguish normal tissue from hypoxic tissue. In general, the emission lifetime of a compound that emits light (fluorescence, phosphorescence) in the near-infrared region tends to be shortened according to the energy gap law, and there is almost no iridium complex that gives an emission lifetime of μs or more at room temperature ( Non-patent document 3).

Patent No. 4930943 Patent No. 5353509 specification

T. V. Esipova et al., Anal. Chem., 83, 8756-8765, 2011. S. Zhang et al., Cancer Res., 70, 4490-4498, 2010. K. Hanson et al., Inorg. Chem., 49, 6077-6084, 2010.

  An object of the present invention is to provide a near-infrared phosphorescent iridium complex having a long phosphorescence lifetime (5 μs or more) for imaging hypoxic cells / tissues or determining their oxygen concentration.

  As a result of intensive studies to solve the above problems, the present inventors have found that the compound represented by the general formula (I) (hereinafter sometimes referred to as the compound of the present invention or the iridium complex of the present invention). It has been found that it has a long phosphorescence lifetime even in the near-infrared light region and is effective for imaging of hypoxic cells and tissues, and has completed the present invention.

That is, the present invention is as follows.
[1] A compound represented by the following general formula (I).
R ¹ is independently hydrogen, halogen, hydroxyl group, amino group, mercapto group, or hydrocarbon group having 1 to 20 carbon atoms, and R ² is independently hydrogen atom or hydrocarbon group having 1 to 6 carbon atoms. , N represents an integer of 1-5.
[2] The compound according to [1], which is any of the following compounds.
[3] An oxygen concentration measuring reagent containing the compound of [1] or [2].
[4] A cancer diagnostic agent comprising the compound of [1] or [2].

  Since the compound of the present invention has a phosphorescence lifetime of 5 μs or more in the near-infrared light region, it is possible to efficiently perform imaging of hypoxic cells / tissues and quantification of oxygen concentration. The compound of the present invention is useful as a hypoxic tissue imaging reagent, a cell and tissue oxygen concentration measuring reagent, an oxygen sensor, a biosensor, a cancer diagnostic agent and the like.

Absorption and phosphorescence spectra of PPZMD, PPZ4DMMD, PPZ3DMMD and BTPHSA. The figure (photograph) which shows the phosphorescence microscopic image measured after adding 2 mM PPZ3DMMD and PPZ4DMMD to the culture solution of HeLa cells, and culturing for 2 hours. A figure (photograph) showing a phosphorescence microscopic image measured after 2 hours of culturing under normal oxygen (20%) and hypoxia (2.5%) culture with 2 mM BTPHSA, PPZ4DMMD, PPZ3DMM added to the HeLa cell culture medium. The figure which shows the phosphorescence lifetime measuring apparatus of a cultured cell. The figure which shows phosphorescence attenuation | damping of the HeLa cell which took in BTPHSA and PPZ3DMMD obtained using the apparatus of FIG. The figure which shows the schematic of the lifetime measurement system for in-vivo oxygen measurement. The figure (with a photograph) which shows the phosphorescence decay curve of the normal tissue of the tumor bearing mouse | mouth measured using the system of FIG. The figure which shows the phosphorescence decay curve of the tumor tissue of the tumor bearing mouse | mouth measured using the system of FIG. 6 (with a photograph). The figure which shows the phosphorescence decay curve of a normal tissue and a tumor part. The figure (with a photograph) which shows the result of having performed the lifetime imaging of the tumor part of a cancer bearing mouse | mouth using the ICCD camera with a gate.

  Hereinafter, the iridium complex of the present invention will be described with specific examples. However, the present invention is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.

The compound of the present invention is a compound represented by the following general formula (I).

R ¹ independently represents hydrogen, halogen, hydroxyl group, amino group, mercapto group, or hydrocarbon group having 1 to 20 carbon atoms. Here, Cl, Br or F is preferable as the halogen. The amino group may be —NH ₂ or an alkylamino group. The hydrocarbon group having 1 to 20 carbon atoms may be a straight chain alkyl group, a branched chain alkyl group, or a cyclic alkyl group. Further, it may contain an unsaturated bond, and one or more hydrogen atoms may be substituted with a halogen, a hydroxyl group, an amino group, a mercapto group, or the like. Preferably carbon number is 1-10, More preferably, it is 1-5, More preferably, it is 1-3.

Each R ² independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, but may be a linear alkyl group, a branched alkyl group, or a cyclic alkyl group. Further, it may contain an unsaturated bond, and one or more hydrogen atoms may be substituted with a halogen, a hydroxyl group, an amino group, a mercapto group, or the like. Preferably carbon number is 1-3.

  Although n shows the integer of 1-5, Preferably it is an integer of 1-3, More preferably, it is 2.

More specifically, the following compounds wherein R ¹ and R ² are both methyl and n is 2 are exemplified.

  The iridium complex of this invention is compoundable according to the method as described in the below-mentioned Example.

  When the iridium complex as described above is placed in an environment such as a cell or tissue, it emits stronger phosphorescence when the oxygen concentration in the environment is low. Therefore, the oxygen concentration can be measured based on the intensity of phosphorescence. That is, it can be determined that the oxygen concentration is low when phosphorescence is strong. It is also possible to quantitatively measure the oxygen concentration by obtaining the relationship between the oxygen concentration and the phosphorescence intensity in advance.

  In addition, an iridium complex can be administered to a laboratory animal such as a mouse or rat or a human to detect a site where the oxygen concentration is lowered. Since the cancer tissue lacks oxygen supply, it can be used as a diagnostic agent for cancer by specifically staining the cancer tissue by detecting the site where the oxygen concentration is lowered.

  When detecting intracellular oxygen concentration, add an iridium complex to the living body, incubate, and then use a fluorescence microscope, fluorescence measurement device, fluorescence imaging device, etc. that excites the iridium complex to observe phosphorescence. Phosphorescence can be observed.

  Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but can be appropriately changed without departing from the gist of the present invention. Accordingly, the scope of the present invention should not be construed as being limited by the specific examples shown below.

  An iridium complex PPZMD with mesityl dipyrinate (MD) and phenylpyrazole (PPZ) as ligands was synthesized. Furthermore, PPZ4DMMD and PPZ3DMMD with dimethylamino group introduced into PPZ were designed and synthesized to increase the cell affinity of PPZMD.

The synthesis procedure is shown below.

Synthesis of PPZ4DMMD
PPZ4COOH (1054 mg, 5.61 mmol) (obtained from Wako Pure Chemical Industries, Ltd.) was dissolved in EtOH (60 ml), 1 ml of 0.5MH ₂ SO ₄ was added and refluxed at 100 ° C. overnight to obtain PPZ4COOEt (956.8 mg, 4.42 mmol). PPZ4COOEt (956.8 mg, 4.42 mmol) and iridium (III) chloride trihydrate (IrCl ₃ 3H ₂ O) (790 mg, 2.24 mmol) in 2-ethoxyethanol (2 EtOEtOH, 60 ml) and deionized water (20 ml) In addition, the mixture was refluxed at 140 ° C. overnight and filtered to obtain PPZ4COOEt chlorine-bridged binuclear complex (1.4 g, 1.06 mmol). 5-mesityldipyrromethane (290 mg, 1.10 mmol), 2,3-dichloro-5,6-dicyano-1,4-benzoquinoline (DDQ) (250 mg, 1.10 mmol) added to tetrahydrofuran and stirred at room temperature for 1 hour Potassium carbonate (K ₂ CO ₃ ) (1 g, 7.23 mmol) was added and stirred again for 15 minutes, and further PPZ4COOEt chlorine-bridged binuclear complex (730 mg, 0.055 mmol)) was added at 100 ° C. overnight under a nitrogen gas atmosphere. After refluxing, the resulting product was washed with chloroform, filtered, and the raw material was removed by column chromatography (developing solvent: chloroform: ethyl acetate = 9: 1), followed by recycle preparative HPLC (Nippon Analytical Industry, LC9225NEXT To obtain PPZ4COOEtMD. After that, the THF: H ₂ O: EtOH = 3: 1: 3 solution was refluxed overnight at 85 ° C. with a large excess of lithium hydroxide monohydrate (LiOH · H ₂ O). 0.1M HCl was used until it was, and then liquid separation was performed with chloroform to obtain PPZ4COOHMD (173 mg, 0.21 mmol). PPZ4COOHMD (83 mg, 0.10 mmol) to N-hydroxysuccinimide (NHS: 34.5 mg, 0.30 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride (EDCHCl: 57.5 mg, 0.30 mmol) N, N-dimethylformamide (DMF) was added, and the mixture was stirred overnight under a nitrogen atmosphere. Thereafter, the product obtained by adding a large excess of trimethylamine and stirring overnight was purified using column chromatography (developing solvent: chloroform: methanol = 97: 3, filler: alumina), and the target product PPZ4DMMD (60 mg, 0.062). mmol).

¹ H NMR (400 MHz CDCl ₃ ) σ: 8.026 (d, 2H), 7.39-7.37 (s, 1H), 7.21-7.20 (d, 1H), 7.052 (s, 2H), 6.89-6.75 (d, 4H) , 6.78 (s, 1H), 6.58-6.49 (m, 3H), 6.38-6.37 (d, 2H), 6.15-6.14 (d, 2H), 3.40 (d, 2H), 3.23-3.16 (m, 2H) , 2.88 (s, 1H), 2.44-2.40 (m, 4H), 2.34 (s, 2H), 2.251-2.192 (m, 12H), 2.207 (s, 6H), 1.063 (q, 1H), 1.24 (s , 1H), 1.20 (t, 2H)
ESI-MS (positive): calcd.for C ₄₆ H ₅₁ IrN ₁₀ O ₂ : 968.38, found: m / z = 969.5 ([M] ⁺ )

Synthesis of PPZ3DMMD
Microwave synthesis by adding 3-hydrazinobenzoic acid (1 g, 6.57 mmol) and 1,1,3,3-tetramethoxypropane (0.6 ml, 3.66 mg) to a mixed solution of acetic acid (3.0 ml) and ionic water (1.0 ml) Using the apparatus, it was heated at 150 ° C. for 5 minutes to obtain PPZ3COOH (850 mg, 4.52 mmol). PPZ3COOH (850 mg, 4.52 mmol) was dissolved in EtOH (60 ml), 1 ml of 0.5MH ₂ SO ₄ was added, and the mixture was refluxed at 100 ° C. overnight to obtain PPZ3COOEt (750 mg, 3.47 mmol). PPZ3COOEt (750 mg, 3.47 mmol) and iridium (III) chloride trihydrate (IrCl ₃ 3H ₂ O) (630 mg, 1.79 mmol) in 2-ethoxyethanol (2 EtOEtOH, 60 ml) and deionized water (20 ml) In addition, after refluxing at 140 ° C. overnight, filtration was performed to obtain a PPZCOOEt chlorine-bridged binuclear complex.
Add 5-mesityldipyrromethane (423mg, 1.60mmol), 2,3-dichloro-5,6-dicyano-1,4-benzoquinoline (DDQ,) (363mg, 1.86mmol) tetrahydrofuran and stir at room temperature for 1 hour After that, potassium carbonate (K ₂ CO ₃ ) (1 g, 7.23 mmol) was added and stirred again for 15 minutes, and PPZ3COOEt chlorine-bridged binuclear complex was added and refluxed at 100 ° C. overnight under a nitrogen gas atmosphere. After washing with chloroform, filtering and removing the raw material by column chromatography (developing solvent: chloroform: methanol = 9: 1), it was purified by recycle preparative HPLC (Nippon Analytical Industry, LC9225NEXT) and PPZ3COOEtMD (484 mg, 0.55 mg). Then reflux to PPZ3COOEtMD at 85 ° C overnight using THF: H ₂ O: EtOH = 3: 1: 3 solution and a large excess of lithium hydroxide monohydrate (LiOH · H ₂ O), then pH is about 1 Then, 0.1M HCl was used until separation was performed, followed by liquid separation with chloroform to obtain PPZ3COOHMD (369 mg, 0.446 mg). PPZ3CCOHMD (110 mg, 0.133 mmol) and N-hydroxysuccinimide (NHS) (46 mg, 0.400 mol), 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride (EDCHCl) (77 mg, 0.402) mmol), N, N-dimethylformamide (DMF) was added, and the mixture was stirred overnight under a nitrogen atmosphere. Thereafter, the product obtained by adding a large excess of trimethylamine was purified using column chromatography (developing solvent: chloroform: methanol = 97: 3, filler: alumina) to obtain the target product PPZ3DMMD (80 mg, 0.083 mmol). It was.

¹ H NMR (400 MHz CDCl ₃ ) σ: 8.12 (d, 2H), 7.80 (s, 2H), 7.07-6.98 (m, 4H), 6.88-6.79 (m, 5H), 6.48-6.39 (m, 6H) , 6.14-6.13 (d, 2H), 2.48 (m, 3H), 2.34 (s, 3H), 2.25-2.23 (s, 12H), 2.03 (s, 6H), 1.68 (m, 3H), 1.24 (s , 2H), 0.86 (s, 1H)
ESI-MS (positive) ： calcd.for C ₄₆ H ₅₁ IrN ₁₀ O ₂
ESI-MS (positive): calcd.for C ₄₆ H ₅₁ IrN ₁₀ O ₂ : 968.38, found: m / z = 969.4 ([M] ⁺ )

<Measurement of absorption and phosphorescence spectrum>
PPZMD, PPZ4DMMD, and PPZ3DMMD were dissolved in tetrahydrofuran (THF), and absorption / phosphorescence spectra were measured at room temperature. BTPHSA was measured similarly. The results are shown in FIG. In addition, these iridium complexes have a light maximum wavelength λabs, a phosphorescence maximum wavelength λ _phos , a phosphorescence quantum yield (Φ _p , Φ _p ⁰ ), and a phosphorescence lifetime (τ _p , τ _p ⁰ ) in air and under deaeration. Table 1 shows the oxygen responsiveness τp0 / τp. Although the wavelength of the absorption spectrum is shifted by a shorter wavelength than BTPHSA, it can be seen that the phosphorescence spectrum extends to the near infrared region (FIG. 1).

PPZ, PPZ4DMMD and PPZ3DMMD have molar extinction coefficients at 483 nm of 3.59 × 10 ⁴ M ⁻¹ cm ⁻¹ , 3.60 × 10 ⁴ M ⁻¹ cm ⁻¹ and 3.45 × 10 ⁴ M ⁻¹ cm ⁻¹ . The absorption efficiency increased compared with BTPHSA. Furthermore, in PPZ4DMMD and PPZ3DMMD, the phosphorescence lifetime τ _p ⁰ was significantly increased to 10-20 μs, and the oxygen responsiveness could be increased to 40 or more (Table 1).

In Table 1, the phosphorescence lifetime was calculated as follows.
Phosphorescence quenching by oxygen is thought to follow the following Stern-Volmer equation.
Here, τ _p ⁰ is the phosphorescence lifetime when oxygen is not present, that is, when the oxygen partial pressure is pO ₂ = 0 mmHg, and τ _p is the phosphorescence lifetime when the oxygen partial pressure is pO ₂ . k _q is an extinction rate constant, which is an amount related to the magnitude of the interaction between the excited state of the probe and oxygen. Therefore, the oxygen partial pressure pO ₂ can be obtained in principle by measuring the phosphorescence lifetime τ _p of light emission from cells and tissues based on the equation (2).

<Evaluation with cells>
FIG. 2 shows a phosphorescence microscopic image measured after adding 2 mM of PPZ3DMMD and PPZ4DMMD to the culture medium of HeLa cells and culturing for 2 hours. As a result, PPZMD was hardly taken into cells due to its high lipid solubility (not shown), but PPZ3DMMD and PPZ4DMMD introduced with cationic amino groups were taken into cells, and PPZ3DMMD showed higher uptake efficiency. (FIG. 2).

In addition, Fig. 3 shows a phosphorescence microscopic image measured after 2 hours of culturing in normal oxygen (20%) and hypoxia (2.5%) culture with 2 mM BTPHSA, PPZ4DMMD, PPZ3DMM added to the HeLa cell culture medium. . As a result, PPZ3DMMD and PPZ4DMMD showed higher oxygen responsiveness in cells than BTPHSA.

  FIG. 4 shows an apparatus for measuring the phosphorescence lifetime of cultured cells. The second harmonic of the Nd: YAG laser (532 nm, pulse width: 1 ns, repetition rate 20 kHz) or semiconductor laser (488 nm, pulse width: 25 ns, repetition rate 15 kHz) or time correlation is used as the excitation light. The emission decay curve was measured based on the single photon counting method.

FIG. 5 shows phosphorescence decay of HeLa cells incorporating BTPHSA and PPZ3DMMD obtained using this apparatus. All of the phosphorescence decays in FIG. 5 can fit into an exponential decay consisting of two components. The average phosphorescence lifetimes of BTPHSA under 20% O ₂ and 2.5% O ₂ conditions were 0.797 μs and 1.45 μs, respectively. With PPZ3DMMD, the lifetime was increased to 2.71 μs and 9.62 μs, respectively. That is, it can be seen that the oxygen responsiveness increased as τ _p ⁰ increased.

<Evaluation in vivo>
FIG. 6 shows a schematic diagram of a lifetime measurement system for in vivo oxygen measurement. The sensitivity of the detection system has been improved by using six fibers on the light-receiving side for one fiber for excitation. The phosphorescence decay curves of normal tissues and tumor tissues of cancer-bearing mice measured using the system of FIG. 6 are shown in FIGS. 7 and 8, respectively. PPZ4MDDM was used as a probe. PPZ4MDDM 50 nmol was administered from the tail vein of tumor-bearing mice (approximately 2 weeks after transplantation of mouse squamous cell carcinoma SCC7 cells), and LD (488
Phosphorescence decay was observed using nm) as excitation light. Whereas the phosphorescence decay rate of PPZ4MDDM in four normal tissues (1-4 in the left figure in Fig. 7) is almost the same, the phosphorescence decay of the tumor is position-dependent and close to the boundary with the normal tissue In FIG. 8, the lower solid line shows an increase in attenuation rate, that is, an increase in oxygen concentration (FIG. 8).

  FIG. 9 shows a comparison of phosphorescence decay curves of normal tissue and tumor. It can be seen that the phosphorescent lifetime of PPZ4MDDM is greatly increased in tumor tissues compared to normal tissues. That is, it was possible to clearly show that the tumor was in a hypoxic state.

  FIG. 10 shows the results of lifetime imaging of the tumor part of the same tumor-bearing mouse as in FIG. 8 using a gated ICCD camera. 50 nmol of PPZ4DMMD was administered from the tail vein of tumor-bearing mice, the tumor (diameter 0.5-1.0 cm) was irradiated with LD (488 nm, 25 ns) pulsed light, and 1.5 g after excitation using a gated ICCD camera. A time-resolved image of ˜30 μs was measured. The tumor was red in the center and yellow in the periphery (black in the black-and-white photo and white in the periphery), and the center was found to have a longer life span, that is, hypoxia.

Claims (4)

The compound represented by the following general formula (I).
R ¹ is independently hydrogen, halogen, hydroxyl group, amino group, mercapto group, or hydrocarbon group having 1 to 20 carbon atoms, and R ² is independently hydrogen atom or hydrocarbon group having 1 to 6 carbon atoms. , N represents an integer of 1-5. The compound according to claim 1, which is any of the following compounds.
An oxygen concentration measurement reagent comprising the compound according to claim 1 or 2. A cancer diagnostic agent comprising the compound according to claim 1 or 2.