Iridium complex sonosensitizer as well as preparation method and application thereof

Abstract

The invention discloses an iridium complex sonosensitizer and a preparation method and application thereof, and belongs to the technical field of anti-tumor sonosensitizer medicines.The iridium complex sonosensitizer has two structures which are respectively named as Ir-1 and Ir-2, and the preparation method of the iridium complex sonosensitizer with the two structures is provided. The preparation method is simple, purification is convenient and easy to operate, and the prepared iridium complex sonosensitizer has excellent photophysical properties, shows a good treatment effect in mouse mastadenoma, effectively inhibits tumor growth and has an excellent sonodynamic treatment effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor photosensitizer drugs, and particularly relates to an iridium complex photosensitizer, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer is one of the main causes of death, seriously affecting people's quality of life. At present, the primary clinical treatment methods for cancer are still mainly surgery, chemotherapy, and radiotherapy. However, these methods usually cause serious side effects such as tumor recurrence, multi-drug resistance, and immune system damage. So far, due to efforts to address the challenges of the unsatisfactory precision and serious side effects faced by traditional medicine, a variety of light-wave-based treatment methods have emerged, including photodynamic therapy (PDT), photothermal therapy (PTT), and microwave therapy; among them, PDT has been developed as a powerful method to combat tumors minimally invasively in basic research and clinical practice. Due to the limited tissue penetration of light, the development of PDT in vivo is greatly restricted, resulting in limited application in deep tumors.

[0003] Sonodynamic therapy (SDT) is a novel non-invasive cancer treatment method derived from PDT and has attracted extensive attention in basic research and clinical applications in recent years. In SDT, ultrasound (US) is used as an excitation source to induce the generation of highly cytotoxic reactive oxygen species (ROS) by activating photosensitizers, thereby causing damage to cancer cells. The underlying mechanism shows that US irradiation can induce acoustic cavitation, further leading to sonoluminescence and pyrolysis to activate photosensitizers, resulting in the generation of ROS. US provides great prospects for eradicating deep tumor tissues due to its special tissue penetration depth (>10 cm).

[0004] Iridium is a member of the platinum group metals and is recognized for its promising anti-tumor properties, positioning its complexes as potential successors to established platinum-based drugs. The excellent stability, easy synthesis, and adjustable photophysical properties of iridium complexes contribute to further structural modification to enhance anti-tumor activity. In particular, the long excited-state lifetime, good chemical stability, and high phosphorescence quantum yield of iridium complexes provide a theoretical possibility for the generation of ROS under ultrasound. Therefore, the development of iridium-based complexes with simple structures and easy-to-synthesize ligands has a good promoting effect on the development of SDT. Summary of the Invention

[0005] The present invention provides an iridium complex photosensitizer, a preparation method thereof, and an application thereof. The preparation method is simple, the raw materials are easily available, and the prepared iridium complex can inhibit the proliferation of cancer cells and kill cancer cells at low concentrations.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions: Iridium complex photosensitizers, including two structures, named Ir-1 and Ir-2 respectively, with chemical structures as follows: .

[0007] The preparation method of the above iridium complex photosensitizer Ir-1 includes the following steps: (1) Dissolve 2-chloropyridine, benzo[b]thiophene-2-boronic acid and a catalyst in a solvent. Keep the reaction system anaerobic. Under alkaline conditions, perform a Suzuki coupling reaction at 100 °C for 16 h to obtain 2-(2-pyridyl)benzo[b]thiophene; (2) Dissolve the 2-(2-pyridyl)benzo[b]thiophene and iridium(III) chloride trihydrate in a solvent. Keep the reaction system anaerobic. React at 130 °C for 24 h to obtain the corresponding dimer; (3) Dissolve the dimer and 5-hydroxy-2-pyridinecarboxylic acid in a solvent. Keep the reaction system anaerobic. Under alkaline conditions, perform a coordination reaction at 60 °C for 24 h to obtain the target neutral iridium complex photosensitizer Ir-1; In the above steps, the anaerobic reaction system is preferably carried out under an argon atmosphere; In step (1), the molar ratio of 2-chloropyridine to benzo[b]thiophene-2-boronic acid is 1:(1 - 1.2); the molar ratio of 2-chloropyridine to the catalyst is 1:(0.04 - 0.08), and the catalyst is preferably tetrakis(triphenylphosphine)palladium; the solvent is a mixed solution of water and 1,4-dioxane with a volume ratio of 1:3, and the dosage ratio of 2-chloropyridine to water is 0.5 g:10 mL; the base used in the alkaline environment is potassium carbonate; the molar ratio of 2-chloropyridine to potassium carbonate is 1:(5 - 5.2); In step (2), the molar ratio of 2-(2-pyridyl)benzo[b]thiophene to iridium(III) chloride trihydrate is 1:(0.4 - 0.6); the solvent is a mixed solution of water and 2-ethoxyethanol with a volume ratio of 1:3; the dosage ratio of iridium(III) chloride trihydrate to water is 0.417 g:10 mL; In step (3), the molar ratio of the dimer to 5-hydroxy-2-pyridinecarboxylic acid is 1:(2.3 - 2.8); the solvent is a mixed solution of ethanol and dichloromethane with a volume ratio of 1:3; the dosage ratio of the dimer to ethanol is 0.5 g:10 mL; the base used in the alkaline environment is potassium carbonate; the molar ratio of the dimer to potassium carbonate is 1:(10 - 12).

[0008] The preparation method of the above iridium complex photosensitizer Ir-2 includes the following steps: (1) Dissolve 2-chloroquinoline, benzo[b]thiophene-2-boronic acid and a catalyst in a solvent. Keep the reaction system anaerobic. Under an alkaline condition, perform a Suzuki coupling reaction at 100 °C for 16 h to obtain 2-(1-benzo[b]thiophen-2-yl)quinoline; (2) Dissolve the obtained 2-(1-benzo[b]thiophen-2-yl)quinoline and iridium(III) chloride trihydrate in a solvent. Keep the reaction system anaerobic. React at 130 °C for 24 h to obtain the corresponding dimer; (3) Dissolve the dimer and 5-hydroxypyridine-2-carboxylic acid in a mixed solvent. Keep the reaction system anaerobic. Perform a coordination reaction at 60 °C for 24 h to obtain the target neutral iridium complex photosensitizer Ir-2; In the above steps, it is preferred that the reaction system is anaerobic and carried out under an argon atmosphere; In step (1), the molar ratio of 2-chloroquinoline to benzo[b]thiophene-2-boronic acid is 1:(1 - 1.2); the molar ratio of 2-chloroquinoline to the catalyst is 1:(0.04 - 0.08), and the catalyst is preferably tetrakis(triphenylphosphine)palladium; the solvent is a mixed solution of water and 1,4-dioxane with a volume ratio of 1:3, and the dosage ratio of 2-chloroquinoline to water is 0.5 g:10 mL; the base used in the alkaline environment is potassium carbonate; the molar ratio of 2-chloroquinoline to potassium carbonate is 1:(5 - 5.2); In step (2), the molar ratio of 2-(1-benzo[b]thiophen-2-yl)quinoline to iridium(III) chloride trihydrate is 1:(0.4 - 0.6); the reaction solvent is a mixed solution of water and 2-ethoxyethanol with a volume ratio of 1:3; the dosage ratio of iridium(III) chloride trihydrate to water is 0.506 g:15 mL; In step (3), the molar ratio of the dimer to 5-hydroxypyridine-2-carboxylic acid is 1:(2.3 - 2.8); the reaction solvent is a mixed solution of ethanol and dichloromethane with a volume ratio of 1:3; the dosage ratio of the dimer to ethanol is 0.5 g:10 mL; the base used in the alkaline environment is potassium carbonate; the molar ratio of the dimer to potassium carbonate is 1:(10 - 12).

[0009] The above iridium complex photosensitizers can all be used for the preparation of tumor sonodynamic therapy drugs.

[0010] Beneficial effects: The present invention provides an iridium complex photosensitizer, its preparation method and application, and has the following advantages compared with the prior art: The synthesis method of the neutral iridium complex prepared by the present invention is simple, the raw materials are easily available, and the purification is convenient and easy to operate; The triplet excited state energy level of the neutral iridium complex prepared by the present invention can be well matched with the excited state energy level of oxygen, and at the same time has a long triplet excited state lifetime, and excellent photophysical properties; The neutral iridium complex prepared by the present invention has high reactive oxygen generating ability and relatively high sonotoxicity in N,N-dimethylformamide, can inhibit cancer cell proliferation and kill cancer cells at low concentrations; The neutral iridium complex sonosensitizer prepared by the present invention shows good therapeutic effects in mouse mammary tumors, effectively inhibits tumor growth, and has excellent sonodynamic therapy effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the preparation route diagram of iridium complex Ir-1 in the examples of the present invention; Figure 2 is the preparation route diagram of iridium complex Ir-2 in the examples of the present invention; Figure 3 is the degradation test diagram of neutral iridium complexes Ir-1, Ir-2 and reference ruthenium terpyridine on DPBF under ultrasonic irradiation; Figure 4 is the cytotoxicity test result diagram of neutral iridium complexes Ir-1, Ir-2 on 4T1 cells; Figure 5 is the tumor inhibition diagram of neutral iridium complex Ir-1 on mouse breast cancer. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below with reference to the drawings and specific examples: Example 1

[0013] The preparation method of iridium complex Ir-1 specifically includes the following steps: Add 2-chloropyridine (0.5 g, 4.404 mmol), benzo[b]thiophene-2-boronic acid (0.862 g, 4.844 mmol), tetrakis(triphenylphosphine)palladium (0.407 g, 0.352 mmol), potassium carbonate (3.043 g, 22.02 mmol) into a 100 mL two-necked flask. After adding 1,4-dioxane (30 mL) and water (10 mL), protect with argon, keep the reaction solution temperature at 100 °C, and react for 16 h; after the reaction is completed, cool to room temperature, extract the reaction solution with ethyl acetate and saturated brine, dry with anhydrous sodium sulfate, filter, and remove the solvent by vacuum distillation to obtain a crude product, which is purified by column chromatography, and the eluent is V (petroleum ether): V (ethyl acetate) = 15:1 to obtain a white solid of 2-(2-pyridyl)benzo[b]thiophene; Iridium(III) chloride trihydrate (0.417 g, 1.184 mmol) and 2-(2-pyridyl)benzothiophene (0.5 g, 2.367 mmol) were added to a 100 mL round-bottom flask. 2-Ethoxyethanol (30 mL) and water (10 mL) were added, and the reaction was carried out under an argon atmosphere at 130 °C for 24 h. After the reaction was complete and cooled to room temperature, the reaction solution was slowly poured into a beaker containing 100 mL of water. After stirring for 30 minutes, filtration was carried out, and the filter cake was dried to obtain a red solid, which was the dimer; The dimer (0.5 g, 0.386 mmol), 5-hydroxy-2-pyridinecarboxylic acid (0.134 g, 0.965 mmol), and potassium carbonate (0.587 g, 4.246 mmol) were added to a 100 mL round-bottom flask. After adding dichloromethane (30 mL) and ethanol (10 mL), the reaction was carried out under an argon atmosphere at 60 °C for 24 h. After the reaction was completed, filtration was carried out, and the solvent was removed by distillation under reduced pressure to obtain a crude product, which was purified by column chromatography. The eluent was V (dichloromethane): V (methanol) = 20:1, and a red solid, neutral iridium complex Ir-1, was obtained.

[0014] Ir-1: 1 H NMR (400 MHz, DMSO- d 6) δ 11.13 (s, 1H), 8.58 (d, J = 5.8 Hz,1H), 8.02 – 7.83 (m, 7H), 7.63 (d, J = 5.8 Hz, 1H), 7.41 (dd, J = 8.7, 2.6 Hz,1H), 7.34 (ddd, J = 7.4, 5.8, 1.5 Hz, 1H), 7.24 – 7.10 (m, 4H), 6.86 (dt, J =26.6, 7.6 Hz, 2H), 6.13 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 8.1 Hz, 1H). Example 2

[0015] A preparation method of iridium complex Ir-2 specifically includes the following steps: 2-Chloroquinoline (0.5 g, 3.056 mmol), benzo[b]thiophene-2-boronic acid (0.598 g, 3.362 mmol), tetrakis(triphenylphosphine)palladium(0) (0.282 g, 0.244 mmol), and potassium carbonate (2.112 g, 15.28 mmol) were added to a 100 mL round-bottom flask. After adding 1,4-dioxane (30 mL) and water (10 mL), the reaction mixture was protected with argon. The reaction solution was heated to 100 °C and reacted for 16 h. After the reaction was completed, the reaction mixture was cooled to room temperature. The reaction solution was extracted with ethyl acetate and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography, and the eluent was V (petroleum ether): V (ethyl acetate) = 15:1 to obtain a white solid, 2-(1-benzo[b]thiophen-2-yl)quinoline; Iridium(III) chloride trihydrate (0.506 g, 1.435 mmol) and 2-(1-benzo[b]thiophen-2-yl)quinoline (0.75 g, 2.870 mmol) were added to a 100 mL round-bottom flask. 2-Ethoxyethanol (45 mL) and water (15 mL) were added, and the reaction was carried out under an argon atmosphere at 130 °C for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature. The reaction solution was slowly poured into a beaker containing 100 mL of water. After stirring for 30 minutes, the mixture was filtered by suction, and the filter cake was dried to obtain a red solid, the dimer; The dimer (0.5 g, 0.334 mmol), 5-hydroxy-2-pyridinecarboxylic acid (0.116 g, 0.835 mmol), and potassium carbonate (0.508 g, 3.674 mmol) were added to a 100 mL round-bottom flask. After adding dichloromethane (30 mL) and ethanol (10 mL), the reaction was carried out under an argon atmosphere at 60 °C for 24 h. After the reaction was completed, the mixture was filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography, and the eluent was V (dichloromethane): V (methanol) = 20:1 to obtain a red solid, the neutral iridium complex Ir-2.

[0016] Ir-2: 1 H NMR (400 MHz, DMSO- d 6) δ 11.13 (s, 1H), 8.59 (d, J = 8.7 Hz,1H), 8.53 (d, J = 8.7 Hz, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.18 (d, J = 8.6 Hz, 1H),8.09 (d,J = 8.6 Hz, 1H), 8.03 – 7.91 (m, 3H), 7.87 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H), 7.38 (tdd, J = 16.4, 8.0, 1.5 Hz, 4H), 7.23 – 7.12 (m, 2H), 7.05 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.00 – 6.89 (m, 2H), 6.69 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 6.55 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 6.45 (d, J = 8.1 Hz, 1H), 5.89 (d, J = 8.1 Hz, 1H). Test 1 In N,N-dimethylformamide, using 1,3-diphenylisobenzofuran as the ROS indicator and ruthenium terpyridine as the standard, the ROS generation of neutral iridium complexes Ir-1 and Ir-2 under ultrasound was evaluated. The specific experimental steps are as follows: Prepare a mixed solution of 1,3-diphenylisobenzofuran (1x10 -2 M) and neutral iridium complexes Ir-1 and Ir-2 (1x10 -5 M) in the dark, and then irradiate with ultrasound (2.5 W·cm -2 ). Measure the maximum absorbance at 410 nm every 1 min to evaluate the degradation effect of neutral iridium complexes Ir-1 and Ir-2 and the reference ruthenium terpyridine on 1,3-diphenylisobenzofuran under ultrasound. As Figure 3 shown, the oxidation rates of neutral iridium complexes Ir-1 and Ir-2 were calculated to be 0.04081 min -1 , 0.05358 min -1 , higher than the oxidation rate of the reference ruthenium terpyridine under ultrasound irradiation (0.03493 min −1 ); indicating their excellent ability to generate reactive oxygen species.

[0017] Test 2 Detect the sonotoxicity of neutral iridium complexes Ir-1 and Ir-2 to 4T1 cancer cells under ultrasound and non-ultrasound conditions by the CCK-8 method. The specific experimental steps are as follows: 4T1 cancer cells were inoculated into a well plate, and a culture medium containing neutral iridium complexes Ir-1 and Ir-2 at different concentrations was added. After incubating with the neutral iridium complexes Ir-1 and Ir-2 for 24 h, the cells were irradiated with ultrasound (0.7 W·cm -2 ). The control group was not irradiated and continued to be incubated for 3 or 5 min, then CCK-8 was added and incubated for 1.5 h. The absorbance of the absorption peak at 450 nm in each well was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the cancer cell survival rate was calculated.

[0018] The test results by the CCK-8 method are as Figure 4 shown. Under the action of ultrasound (0.7 W·cm -2 ), as the concentrations of the neutral iridium complexes Ir-1 and Ir-2 (0 - 50 μM) increased continuously, when the concentration reached 50 μM, the cell survival rates of complex Ir-1 at 3 min and 5 min were 61.8% and 17.1% respectively, and the cell survival rates of complex Ir-2 at 3 min and 5 min were 53.2% and 15.8% respectively. It shows that when the concentration reaches 50 μM, with the increase of the ultrasound time, under the action of 5 min ultrasound, the cell survival rate decreases more significantly, indicating that the neutral iridium complexes Ir-1 and Ir-2 of the present invention have high sonotoxicity.

[0019] Test 3 The sonodynamic therapy effect of the neutral iridium complex Ir-1 was evaluated through mouse breast cancer. There were 12 mice in total, which were randomly divided into 4 groups. The grouping situation and specific experimental steps are as follows: (1) Inject phosphate buffer solution without any treatment; (2) Inject phosphate buffer solution and irradiate with ultrasound; (3) Inject the neutral iridium complex Ir-1 (2.66 mM) without using ultrasound; (4) Inject the neutral iridium complex Ir-1 (2.66 mM) and perform treatment with ultrasound; After 15 days of treatment, all the mice were sacrificed, and the tumors and main organ tissues were collected to evaluate the treatment effect. The obtained tumor inhibition diagram is as Figure 5 shown. The tumors in groups 1, 2, and 3 grew rapidly, while the tumor growth in group 4 was significantly inhibited. The neutral iridium complex Ir-1 of the present invention has excellent sonodynamic therapy effect.

[0020] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An iridium complex photosensitizer, characterized in that, It includes two structures, named Ir-1 and Ir-2 respectively, and the chemical structures are as follows: 。 2. The preparation method of the iridium complex photosensitizer according to claim 1, characterized in that, It includes the following steps: (1) Dissolve the corresponding raw materials, benzo[b]thiophene-2-boronic acid and a catalyst in a solvent, keep the reaction system anaerobic, and carry out a Suzuki coupling reaction under a basic environment; (2) Dissolve the product obtained in step (1) and iridium(III) chloride trihydrate in a solvent, keep the reaction system anaerobic, and react at 130 °C for 24 h to obtain the corresponding dimer; (3) Dissolve the dimer and 5-hydroxy-2-pyridinecarboxylic acid in a solvent, keep the reaction system anaerobic, and carry out a coordination reaction to obtain the target neutral iridium complex photosensitizer.

3. The preparation method of the iridium complex sonosensitizer according to claim 2, wherein, In step (1), the molar ratio of the corresponding raw materials to benzo[b]thiophene-2-boronic acid is 1:(1~1.2); the molar ratio of the corresponding raw materials to the catalyst is 1:(0.04~0.08); the base used for the basic environment is potassium carbonate, and the molar ratio of the corresponding raw materials to potassium carbonate is 1:(5~5.2); the solvent is a mixed solution of water and 1,4-dioxane with a volume ratio of 1:3, and the dosage ratio of the corresponding raw materials to water is 0.5 g:10 mL.

4. The preparation method of the iridium complex sonosensitizer according to claim 2 or 3, characterized in that, When the target neutral iridium complex photosensitizer is Ir-1, the corresponding raw material is 2-chloropyridine, and 2-(2-pyridyl)benzo[b]thiophene is obtained after the Suzuki coupling reaction in step (1); when the target neutral iridium complex photosensitizer is Ir-2, the corresponding raw material is 2-chloroquinoline, and 2-(benzo[b]thiophen-2-yl)quinoline is obtained after the Suzuki coupling reaction in step (1).

5. The preparation method of the iridium complex photosensitizer according to claim 2 or 3, characterized in that, The catalyst is tetrakis(triphenylphosphine)palladium.

6. The preparation method of the iridium complex photosensitizer according to claim 2, characterized in that, In step (2), the molar ratio of the product obtained in step (1) to iridium(III) chloride trihydrate is 1:(0.4~0.6).

7. The preparation method of the iridium complex sonosensitizer according to claim 4, characterized in that, In step (2), the solvent is a mixed solution of water and 2-ethoxyethanol with a volume ratio of 1:

3. When the product is Ir-1, the dosage ratio of iridium(III) chloride trihydrate to water is 0.417 g:10 mL; when the product is Ir-2, the dosage ratio of iridium(III) chloride trihydrate to water is 0.506 g:15 mL.

8. The preparation method of the iridium complex sonosensitizer according to claim 2, characterized in that, In step (3), the molar ratio of the dimer to 5-hydroxy-2-pyridinecarboxylic acid is 1:(2.3~2.8); the reaction solvent is a mixed solution of ethanol and dichloromethane with a volume ratio of 1:3; the dosage ratio of the dimer to ethanol is 0.5 g:10 mL.

9. The preparation method of the iridium complex sonosensitizer according to claim 2 or 8, characterized in that, In step (3), the base used for the basic environment is potassium carbonate; the molar ratio of the dimer to potassium carbonate is 1:(10~12).

10. Use of the iridium complex photosensitizer according to claim 1 in the preparation of a drug for sonodynamic therapy of tumors.

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