An acid-sensitive iridium complex based on isoquinoline benzothiophene and its application

Abstract

The present invention discloses a probe based on an iridium complex of isoquinoline benzothiophene, having the following structure: The iridium complex nanoprobe has acid sensitivity, exhibits phosphorescence emission under hypoxic conditions in the near-infrared region, has good water solubility, has good biocompatibility, and can be used for hypoxic imaging.

Description

Technical Field

[0001] The present invention belongs to the field of phosphorescent probes for bio-optical imaging, and particularly relates to an acid-sensitive probe based on an iridium complex of isoquinoline benzothiophene and its application. Background Art

[0002] Precision imaging of tumors can accurately detect the location of tumors and monitor the development of tumors, which is of great significance for accurate tumor diagnosis and targeted therapy. In recent years, due to its high sensitivity, real-time monitoring, and economy, optical imaging technology has developed rapidly in the field of tumor imaging. Most optical imaging probes for accurate tumor diagnosis usually target biomarkers or respond to the unique microenvironment of tumors. Due to the crazy growth of tumors, a hypoxic and acidified microenvironment is created in tumors, with an oxygen partial pressure <1% and pH <6 in tumors. There are already a wide variety of drug delivery systems for the hypoxic and acidified tumor microenvironment, but the development of optical imaging probes for such tumor microenvironments is still lacking. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides an iridium complex based on isoquinoline benzothiophene. The iridium complex macromolecular nanoprobe has acid sensitivity, has phosphorescent emission under hypoxic conditions in the near-infrared region, has good water solubility, has good biocompatibility, and can be used for hypoxic imaging.

[0004] The specific technical solution of the present invention is as follows:

[0005] An iridium complex of isoquinoline benzothiophene has the following structure:

[0006]

[0007] The present invention also discloses a preparation method of the above iridium complex, including the following steps:

[0008] (1)

[0009] 1-chloroisoquinoline and benzo[b]-2-thiopheneboronic acid are subjected to a catalytic reaction by a palladium catalyst in the presence of a base to obtain the main ligand 1-(benzo[b]thiophen-2-yl)isoquinoline;

[0010] (2)

[0011] p-tolylboronic acid and 6-bromopyridine-3-carbaldehyde are subjected to a catalytic reaction by a palladium catalyst in the presence of a base to obtain the auxiliary ligand 6-(4-methylphenyl)-pyridine-3-carbaldehyde;

[0012] (3)

[0013] React iridium chloride and 1-(benzo[b]thiophen-2-yl)isoquinoline under the protection of an inert gas to obtain a chloro-bridged intermediate;

[0014] (4)

[0015] React the chloro-bridged intermediate, co-ligand 6-(4-methylphenyl)-pyridine-3-carbaldehyde and silver trifluoromethanesulfonate under the protection of argon by heating to obtain an iridium complex of isoquinoline benzothiophene.

[0016] Preferably, the solvent for steps (1) and (2) is tetrahydrofuran; the solvent for step (3) is a mixed solution of 2-ethoxyethanol and water; the solvent for step (4) is diethylene glycol dimethyl ether.

[0017] Preferably, the palladium catalyst in steps (1) and (2) is selected from one or more of tetrakis(triphenylphosphine)palladium and palladium acetate.

[0018] Preferably, the reaction conditions for steps (1) and (2) are heating under reflux, the reaction in step (3) is at 110 °C, and the reaction in step (4) is at 150 °C.

[0019] The present invention also discloses a macromolecular probe of an iridium complex of isoquinoline benzothiophene grafted with PEG, having the following structure:

[0020] Wherein n is a positive integer greater than 40, preferably n = 44 - 227.

[0021] The present invention also discloses a preparation method of the above-mentioned macromolecular probe of an iridium complex of isoquinoline benzothiophene grafted with PEG, which is obtained by reflux reaction of an iridium complex of isoquinoline benzothiophene and p-aminophenyl ether polyethylene glycol under the protection of argon,

[0022]

[0023] Wherein n is a positive integer greater than 40, preferably n = 44 - 227.

[0024] The present invention also discloses the application of the iridium complex of isoquinoline benzothiophene or the macromolecular probe of the iridium complex of isoquinoline benzothiophene grafted with PEG in the preparation of a phosphorescent probe for near-infrared imaging. The phosphorescent probe is an acid-sensitive hypoxia-responsive phosphorescent probe. A specific application is a tumor imaging probe.

[0025] Advantages of the present invention:

[0026] Studies of the present invention have shown that an iridium complex of isoquinoline benzothiophene grafted with PEG can self-assemble into a nanomicelle probe and respond to tumor acidity and hypoxia. Due to the quenching effect of oxygen on phosphorescence, the iridium complex is very sensitive to oxygen and can reversibly transform between different oxygen partial pressures, making it an ideal luminescent material for hypoxic response. The present invention combines water-soluble polyethylene glycol and a strongly hydrophobic iridium complex molecule with near-infrared emission through acid-sensitive imine bonds, and self-assembles into a nanomicelle probe in neutral water or neutral PBS solution. The nanomicelle probe can be better enriched and penetrate into the tumor site through the EPR effect. Subsequently, under the action of tumor acidity, the imine bond breaks, and the hydrophobic iridium complex aggregates accumulate in the tumor site, and can bind to proteins and enhance luminescence under the action of hypoxia, thereby enabling accurate tumor imaging and monitoring the change of tumors with oxygen concentration. Description of the Drawings

[0027] Figure 1 UV absorption spectra of the iridium complex of isoquinoline benzothiophene and the nanoprobes.

[0028] Figure 2 Phosphorescence emission spectra of the nanoprobe before (pH 7.4) and after acid degradation (pH 5.8).

[0029] Figure 3 Emission spectra of the nanoprobe solution after acidolysis at different oxygen concentrations.

[0030] Figure 4 TEM images of the nanoprobe before (pH 7.4) and after acid degradation (pH 5.8).

[0031] Figure 5 Dynamic light scattering characterization of the nanoprobe particle size before and after acid degradation.

[0032] Figure 6 In vivo imaging results of the nanoprobe in tumor-bearing mice. Detailed Description of the Invention

[0033] The following examples illustrate the specific steps of the present invention, but are not limited by the examples.

[0034] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0035] The present invention will be further described in detail below with reference to specific examples and data. It should be understood that these examples are only for illustrating the present invention and not limiting the scope of the present invention in any way.

[0036] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0037] Example 1 Preparation of Grafted PEG Isoquinoline Benzothiophene Iridium Complex of the Present Invention

[0038] 1. Synthesis of the main ligand (Hiqbt):

[0039]

[0040] 1-Chloroisoquinoline (273 mg), benzo[b]-2-thiopheneboronic acid (274 mg), 5 ml of tetrahydrofuran solution and 10 ml of 1M Na2CO3 solution were added to a degassed reaction flask. After one freeze-pump cycle, tetrakis(triphenylphosphine)palladium (2.3 g, 2 mmol) was added, and the freeze-pump cycle was continued twice. Under argon protection, the reaction was refluxed at 150 °C overnight. After the reaction was completed, it was cooled to room temperature, and the organic phase was separated. The aqueous phase was extracted three times with 50 ml of dichloromethane, and the organic phases were combined; the combined organic phase was washed three times with sodium chloride and water, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and then separated by silica gel column chromatography. The mobile phase was petroleum ether:ethyl acetate = 1:1, and 384 mg of the product was collected.

[0041] Hiqbt: 1 1H NMR (400 MHz, Chloroform-d) δ 8.68–8.59 (m, 2H), 7.97–7.85 (m, 4H), 7.76 (ddd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.72–7.64 (m, 2H), 7.46–7.37 (m, 2H).

[0042] 2. Synthesis of the auxiliary ligand:

[0043]

[0044] p-Tolylboronic acid (263 mg), 6-bromopyridine-3-carbaldehyde (772 mg), 5 ml of tetrahydrofuran solution and 15 ml of 2M aqueous Na2CO3 solution were added to a degassed reaction flask. After one freeze-pump cycle, (20 mg) of tetrakis(triphenylphosphine)palladium was added, and the freeze-pump cycle was continued twice. Under argon protection, the reaction was heated to reflux at 130 °C overnight. After the reaction was completed, it was cooled to room temperature, and the organic phase was separated. The aqueous phase was extracted twice with 100 ml of dichloromethane, and the organic phases were combined; the combined organic phase was washed three times with sodium chloride and water, dried over anhydrous sodium sulfate, concentrated by distillation under reduced pressure, and then separated by silica gel column chromatography. The mobile phase was petroleum ether:dichloromethane = 30:1, and 312 mg of the product was collected.

[0045] 6-(4-Methylphenyl)-pyridine-3-carbaldehyde: 11H NMR (400 MHz, Chloroform-d) δ 10.12 (s, 1H), 9.11 (dd, J = 2.2, 0.9 Hz, 1H), 8.22 (dd, J = 8.3, 2.2 Hz, 1H), 8.03–7.95 (m, 2H), 7.89 (d, J = 8.2 Hz, 1H), 7.37–7.29 (m, 2H), 2.43 (s, 3H).

[0046] Synthesis of 3-chloro-bridged intermediate:

[0047]

[0048] Iridium chloride (200 mg) and the main ligand 1-(benzo[b]thiophen-2-yl)isoquinoline (Hiqbt) (475 mg) were added to a reaction flask, and then added to a mixed solution of 2-ethoxyethanol (21 ml) and water (7 ml). Under argon protection, the reaction was carried out at 110 °C overnight. After the reaction was completed, the reaction solution was cooled to room temperature, 20 ml of water was added, and the precipitate was collected by filtration. The precipitate was washed with ethanol and ether and dried in vacuo to obtain 480 mg of the chloro-bridged intermediate.

[0049] Synthesis of isoquinoline benzothiophene iridium complex (Iqbt-Ir)

[0050]

[0051] The chloro-bridged intermediate (300 mg), the auxiliary ligand (197 mg) and silver trifluoromethanesulfonate (100 mg) were placed in a degassed reaction flask, 20 ml of ultra-dry diethylene glycol dimethyl ether was added as the solvent, and the reaction was heated at 150 °C for 24 h under argon protection. After the reaction was completed, the precipitate was collected by precipitation in ethanol; then the collected precipitate was dissolved in dichloromethane, and column chromatography separation was carried out using a silica gel column. The mobile phase was petroleum ether:dichloromethane = 30:1, and finally 10 mg of the product was obtained.

[0052] iqbt-Ir: 11H NMR (400 MHz, Chloroform-d) δ 9.51 (s, 1H), 9.20–9.13 (m, 1H), 9.13–9.06 (m, 1H), 7.93 (dd, J = 8.5, 1.9 Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.77 (t, J = 1.8 Hz, 2H), 7.75–7.69 (m, 4H), 7.68–7.50 (m, 8H), 7.36 (t, J = 2.3 Hz, 1H), 7.24–7.17 (m, 2H), 7.14 (dd, J = 8.6, 2.5 Hz, 1H), 7.11–7.05 (m, 2H), 6.99 (dd, J = 7.5, 5.7 Hz, 2H), 6.86 (d, J = 3.9 Hz, 2H), 6.81–6.76 (m, 2H), 6.74–6.67 (m, 2H), 4.29–4.17 (m, 2H), 2.05 (s, 3H).

[0053] 5. Synthesis of Functionalized Polyethylene Glycol Segment

[0054]

[0055] First, methoxypolyethylene glycol (2 g, 0.4 mmol, Mn = 5 k) and triethylamine (0.4 mL) were dissolved in CH2Cl2 (20 mL) and cooled in an ice bath. Then, p-toluenesulfonyl chloride (Ts-Cl, 0.1 g) was slowly added, and the reaction was carried out at room temperature for 2 h. The solvent was removed under vacuum. It was dissolved in an appropriate amount of CHCl3, washed three times with deionized water, and then the CHCl3 layer was dried with anhydrous Na2SO4, precipitated in ether, and dried under vacuum to obtain a white powder. Then, PEG-Ts (2 g), p-aminobenzoic acid (1 g), and K2CO3 (1 g) were added to DMF (15 mL), stirred at 100 °C for 4 h, cooled to room temperature, and precipitated in ether. The precipitate was filtered out, dissolved in CHCl3 (20 mL), washed three times with deionized water, the CHCl3 layer was dried with anhydrous Na2SO4, precipitated in ether, and dried under vacuum to obtain a white powder.

[0056] 6. Synthesis of Isoquinoline Benzothiophene Iridium Complex Grafted with PEG (iqbt-Ir-PEG)

[0057]

[0058] Dissolve the isoquinoline benzothiophene iridium complex (100 mg) and the functionalized PEG segment (200 mg) in 20 ml of toluene, and reflux the reaction overnight under argon protection. After the reaction, distill off the solvent under reduced pressure, separate the product by column chromatography, with the eluent CH2Cl2:CH3OH = 20:1. After distillation under reduced pressure, dissolve it in a small amount of chloroform, precipitate it in ether, and dry it under vacuum to obtain an orange-red powder product.

[0059] Characterize the product by nuclear magnetic resonance and mass spectrometry:

[0060] Iqbt-Ir-PEG: 1 H NMR (400 MHz, Chloroform-d) δ 9.14–8.98 (m, 2H), 8.22–8.10 (m, 1H), 7.93–7.87 (m, 3H), 7.87–7.82 (m, 1H), 7.80–7.74 (m, 4H), 7.72–7.50 (m, 10H), 7.25–7.20 (m, 2H), 7.16–7.09 (m, 1H), 7.08–7.04 (m, 1H), 7.03–6.97 (m, 1H), 6.95–6.89 (m, 3H), 6.84–6.76 (m, 2H), 6.74–6.67 (m, 3H), 6.66–6.59 (m, 2H), 6.59–6.53 (m, 4H), 3.36 (s, 459H), 3.31 (s, 3H), 1.89 (s, 3H).

[0061] Dissolve the synthesized isoquinoline benzothiophene iridium complex grafted with PEG (Iqbt-Ir-PEG) in neutral water under ultrasound to self-assemble into nanomicelles to obtain a nanoprobe. The ultraviolet absorption spectra of the isoquinoline benzothiophene iridium complex (Iqbt-Ir) and its nanoprobe are as Figure 1 shown. The results show that the ultraviolet absorption spectra of the iridium complex small molecule luminophore and the macromolecular probe grafted with the functionalized PEG segment are similar, with no significant displacement changes. There are obvious absorptions at 400–600 nm, and the relatively obvious absorption peaks are at 475 nm and 525 nm.

[0062] 7. Phosphorescence emission spectra of the nanoprobe before and after acid degradation

[0063] Investigate the phosphorescence emission spectra of the isoquinoline benzothiophene iridium complex (Iqbt-Ir) and its nanoprobe before acid degradation (pH 7.4) and after acid degradation (pH 5.8).

[0064] Before acid degradation (pH 7.4): The iridium complex of isoquinoline benzothiophene (Iqbt-Ir) was dissolved in DMSO solution to measure the phosphorescence emission spectrum, 1 mg / ml; the nanoprobe (Iqbt-Ir-PEG) was dissolved in 0.01 M PBS solution with pH = 7.4, 5 mg / ml, and the phosphorescence emission spectrum was measured after 2 h. Another portion was added with 10% mouse serum to simulate the in vivo protein environment for testing.

[0065] Acid degradation: The nanoprobe (Iqbt-Ir-PEG) was dissolved in 0.01 M PBS solution with pH = 5.8, 5 mg / ml, and the phosphorescence emission spectrum was measured after 2 h. Another portion was added with 10% mouse serum to simulate the in vivo protein environment for testing.

[0066] The results are as Figure 2 shown. The results show that under acidic conditions, the emission intensity of the nanoprobe decreased slightly. This is because the nanoprobe lost the hydrophilic segment due to acid degradation, the hydrophilic-hydrophobic property changed, and the iridium complex could not be water-soluble. Comparing the phosphorescence emission in the PBS solution with pH = 7.4 added with protein, when 10% mouse serum was added to the PBS probe solution with pH = 5.8, the emission peak was significantly enhanced. This is because the small molecule aggregates of the iridium complex after acid degradation combined with the protein in the serum to enhance the phosphorescence.

[0067] Example 2 examines the emission spectrum of the nanoprobe after acidolysis under different oxygen concentrations

[0068] Nitrogen with different oxygen concentrations (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) was prepared. The gas flow with the above different oxygen-nitrogen ratios was bubbled into the aqueous solution of the nanoprobe after acid degradation for 10 min, and the phosphorescence emission spectra of the nanoprobe after acidolysis under different oxygen concentrations were measured respectively.

[0069] The test results of the phosphorescence emission spectrum of the nanoprobe in response to hypoxia after acidolysis are as Figure 3 shown. The results show that when the pH value is constant (pH 5.8), as the oxygen partial pressure in the solution decreases, the intensity of the emission peak at 703 nm gradually increases. The test results of this phosphorescence emission spectrum indicate that the nanoprobe can continue to respond to hypoxia after acidolysis.

[0070] The transmission electron microscope images of the nanoprobe before and after acid degradation are as Figure 4 shown. The results show that the change in the properties of the nanoprobe before and after acid degradation is very obvious. Under neutral conditions, the nanoprobe is in the state of nanomicelles, and the transmission electron microscope shows a spherical structure with an average particle size of 78 nm. Under acidic conditions, the nanoprobe degrades, and the outer hydrophilic segment separates from the iridium complex core, and the spherical structure disintegrates.

[0071] Dynamic light scattering characterization of the particle size of the nanoprobe before and after acid degradation is as follows Figure 5 shown. The results show that under neutral conditions, the hydrodynamic diameter of the nanoprobe in the buffer solution is 98 nm. After acid degradation, the hydrodynamic diameter changes greatly. Some of the diameters are as small as a dozen nanometers, while others are several hundred nanometers. These are due to the aggregates of iridium complexes with different scales generated after acid degradation.

[0072] Example 3 examines the in vivo imaging of the nanoprobe after acid degradation in tumor-bearing mice

[0073] The prepared nanoprobe with enhanced acid degradation and hypoxia response was used for in vivo imaging experiments in tumor-bearing mice. The mouse tumor model was established by subcutaneous injection of mouse hepatoma cell line H22 (10 6 H22 cells per mouse) into the right thigh of ICR mice. When the tumor volume reached about 100 mm 3 3, the nanoprobe was dissolved in PBS (0.01 M, pH = 7.4) to prepare a 12.5 mg / mL solution, and 100 μL of the solution was injected into the mice via the tail vein. The tumor-bearing mice were imaged using a near-infrared in vivo imaging system. The results of the in vivo imaging experiment are as follows Figure 6 shown. The results indicate that from 1 hour to 120 hours after the injection of the probe, the optical signal of the probe detected at the tumor site is much stronger than the background signal. The results of the in vivo imaging test show that the nanoprobe of the present invention can be effectively accumulated in tumors and amplify the signals of the tumor microenvironment after acid degradation and enhanced hypoxia response, achieving tumor detection with a high signal-to-noise ratio.

Claims

1. An iridium complex based on isoquinoline benzothiophene, characterized in that It has the following structure: 。 2. The preparation method of the complex according to claim 1, characterized in that It includes the following steps: （1） ， 1-chloroisoquinoline and benzo[b]-2-thiopheneboronic acid are subjected to a catalytic reaction with a palladium catalyst in the presence of a base to obtain the main ligand 1-(benzo[b]thiophen-2-yl)isoquinoline; （2） ， p-tolylboronic acid and 6-bromopyridine-3-carbaldehyde are subjected to a catalytic reaction with a palladium catalyst in the presence of a base to obtain the auxiliary ligand 6-(4-methylphenyl)pyridine-3-carbaldehyde; （3） ， iridium chloride and 1-(benzo[b]thiophen-2-yl)isoquinoline are reacted under the protection of an inert gas to obtain a chloro-bridged intermediate; （4） ， the chloro-bridged intermediate, the auxiliary ligand 6-(4-methylphenyl)pyridine-3-carbaldehyde and silver trifluoromethanesulfonate are heated and reacted under the protection of argon to obtain an iridium isoquinoline benzothiophene complex; 3. The method according to claim 2, characterized in that The solvent for steps (1) and (2) is tetrahydrofuran; the solvent for step (3) is a mixed solution of 2-ethoxyethanol and water; the solvent for step (4) is diethylene glycol dimethyl ether.

4. The method according to claim 2, wherein The palladium catalyst in steps (1) and (2) is selected from one or more of tetrakis(triphenylphosphine)palladium and palladium acetate.

5. The method according to claim 2, wherein The reaction conditions for steps (1) and (2) are heating under reflux, the reaction in step (3) is carried out at 100-115 °C, and the reaction in step (4) is carried out at 140-160 °C.

6. A grafted PEG isoquinoline benzothiophene iridium complex, characterized in that It has the following structure: , where n is a positive integer greater than 40.

7. The preparation method of the grafted PEG isoquinoline benzothiophene iridium complex according to claim 6, characterized in that It is obtained by refluxing an iridium isoquinoline benzothiophene complex and p-aminophenyl ether polyethylene glycol under the protection of argon, ， where n is a positive integer greater than 40.

8. Use of the iridium complex of isoquinoline benzothiophene according to claim 1 or the grafted PEG iridium isoquinoline benzothiophene complex according to claim 6 in the preparation of a phosphorescent probe for near-infrared imaging.

9. The application according to claim 8, wherein The phosphorescent probe is an acid-sensitive hypoxia-responsive phosphorescent probe.

10. The application according to claim 9, wherein The phosphorescent probe is a tumor imaging probe.

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