Iridium complex photosensitizer and its preparation method and application

Abstract

The present invention discloses an iridium complex photosensitizer and a preparation method and application thereof, belonging to the technical field of anti-tumor drugs. The primary purpose of the present invention is to provide an iridium complex photosensitizer with ultra-high phototoxicity and a preparation method thereof, and another purpose is to provide the application of the above-mentioned iridium complex photosensitizer in tumor photodynamic therapy. The iridium complex photosensitizer prepared by the present invention has an ultra-high relative reactive oxygen yield, which is close to 3 times that of the reference substance terpyridine ruthenium, and exhibits ultra-high phototoxicity in mouse breast cancer cells 4T1 (half inhibition concentration IC50 = 91.10 nM). The phototoxicity is improved by one order of magnitude compared with the reported iridium complex photosensitizers, and can achieve excellent photodynamic therapy effects at extremely low concentrations, and has excellent biological safety.

Description

Technical Field

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

[0002] Cancer, also known as malignant tumor, is one of the major diseases that seriously endangers human health and life. Traditional tumor treatment methods mainly include surgical resection, chemotherapy and radiotherapy, but they have disadvantages such as easy recurrence after surgery, large side effects on normal tissues, and easy to develop drug resistance. In recent years, with the rapid development of medical technology, a non-invasive and light-controlled treatment method - photodynamic therapy, has been considered an ideal and potential treatment method in the field of clinical treatment.

[0003] Cyclometallated iridium (Ir) complexes have been widely used in biological detection, imaging and cancer treatment as biological imaging probes and drugs in the past decade due to their high quantum yield, large Stokes shift, long luminescence lifetime, good photostability and easy structural modification. Iridium (III) complexes have long-lived triplet excited states, and their suitable energy levels allow effective energy transfer to oxygen or electron transfer with various receptor molecules to produce phototoxic reactive oxygen species, which makes iridium (III) complexes have great potential as photosensitizers in the field of photodynamic therapy. Most of the iridium complex photosensitizers reported so far are rigid structures containing complex ligands, and the synthesis steps are relatively complicated. Compared with the reference substance terpyridine ruthenium, their relative reactive oxygen yield is lower (about 0.1 to 2.0 times that of terpyridine), and the phototoxicity IC50 values ​​are mostly maintained at the micromolar level. Summary of the invention

[0004] The invention provides an iridium complex photosensitizer and a preparation method and application thereof. The prepared iridium complex photosensitizer has ultra-high relative active oxygen yield and phototoxicity, and has excellent subcutaneous tumor treatment effect and biological safety.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions:

[0006] An iridium complex photosensitizer, the chemical structure is:

[0007]

[0008] A method for preparing an iridium complex photosensitizer comprises the following steps:

[0009] (1) placing 4-trifluoromethylquinolin-2(H)-one and phosphorus oxychloride in a reaction flask, keeping the reaction system oxygen-free, and carrying out a halogenation reaction at 100-110° C. for 2-4 hours to obtain 2-chloro-4-(trifluoromethyl)quinoline;

[0010] (2) dissolving the 2-chloro-4-(trifluoromethyl)quinoline, benzothiophene-2-boric acid and a catalyst in a solvent, keeping the reaction system oxygen-free, and carrying out a Suzuki coupling reaction at 90 to 116° C. in an alkaline environment for 14 to 19 hours to obtain 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline;

[0011] (3) dissolving the 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline and iridium trichloride trihydrate in a solvent, keeping the reaction system oxygen-free, reacting at 115 to 127° C. for 20 to 25 hours to obtain the corresponding dimer;

[0012] (4) dissolving the dimer and 5-hydroxypicolinic acid in a mixed solvent, keeping the reaction system oxygen-free, and carrying out coordination reaction at 53-65° C. in an alkaline environment for 20-25 hours to obtain the target iridium complex photosensitizer HIr-NO;

[0013] In the above steps, the reaction system is oxygen-free and preferably carried out under an argon atmosphere;

[0014] The molar ratio of 4-trifluoromethylquinoline-2(H)-one to phosphorus oxychloride in step (1) is 1:(20-25); the mixing method of 4-trifluoromethylquinoline-2(H)-one and phosphorus oxychloride is specifically as follows: firstly, 4-trifluoromethylquinoline-2(H)-one is placed in a round-bottom flask, and the reaction flask is kept in an argon atmosphere, and chlorine oxychloride is added dropwise to the reaction flask under continuous stirring;

[0015] The molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to benzothiophene-2-boric acid in step (2) is 1:(0.8-1.2); the catalyst is tetrakis(triphenylphosphine)palladium or [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride; the molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to the catalyst is 1:(0.06-0.08); the solvent for the Suzuki coupling reaction is a mixed solvent of water and 1,4-dioxane, the volume ratio of water to 1,4-dioxane is 1:(2.7-3.3), the amount ratio of 2-chloro-4-(trifluoromethyl)quinoline to water is 1g:15mL; the base used in the alkaline environment is potassium carbonate, potassium acetate or potassium phosphate; the molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to the base is 1:(4.3-5.2);

[0016] In step (3), the molar ratio of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline to iridium trichloride trihydrate is 1:(0.3-0.6); the reaction solvent is a mixed solvent of water and 2-ethoxyethanol, the volume ratio of which is 1:(2.7-3.3), and the amount ratio of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline to water is 1 g:(17-25) mL;

[0017] In step (4), the molar ratio of the dimer to 5-hydroxypicolinic acid is 1:(2.3-3.1); the mixed solvent is a mixed solvent of ethanol and dichloromethane, and the volume ratio is 1:(2.7-3.4); the dosage ratio of the dimer to ethanol is 1 g:(16-23) mL; the base used in the alkaline environment is potassium carbonate, potassium acetate or potassium phosphate; the molar ratio of the dimer to the base is 1:(8.3-11.2);

[0018] The iridium complex photosensitizer can be used to prepare tumor photodynamic therapy drugs. In specific applications, the tumor is a subcutaneous tumor.

[0019] Beneficial effects: The present invention provides an iridium complex photosensitizer and a preparation method and application thereof, which have the following advantages over the prior art:

[0020] 1. The iridium complex photosensitizer of the present invention has a simple structure, readily available raw materials, and simple synthesis and purification operations;

[0021] 2. The iridium complex photosensitizer of the present invention has an ultra-high relative active oxygen yield, which is close to 3 times that of the reference material terpyridine ruthenium, and is much higher than the currently reported iridium complex photosensitizers that also use terpyridine ruthenium as a reference material, and has excellent core performance;

[0022] 3. The iridium complex photosensitizer of the present invention is effective under laser (535nm, 100mW / cm -2 ) irradiation, it has ultra-high phototoxicity to cancer cells, with a half-inhibitory concentration IC50 as low as 91.10nM; compared with the traditional reported iridium complex photosensitizer (half-inhibitory concentration is at the micromolar level), the phototoxicity is increased by an order of magnitude, and excellent therapeutic effects can be achieved at extremely low concentrations, which can effectively reduce the dosage and cost of medication;

[0023] 4. When the iridium complex photosensitizer of the present invention is applied to a mouse breast cancer subcutaneous tumor model, it can effectively alleviate the splenomegaly symptoms (leukemoid reaction characteristics) caused by the mouse breast cancer model;

[0024] 5. When the iridium complex photosensitizer of the present invention is applied to a mouse breast cancer subcutaneous tumor model, it has no malignant effect on the mouse organs and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a test diagram of the degradation of diphenylisobenzofuran by the iridium complex HIr-NO and the reference terpyridine ruthenium under the action of laser in the embodiment of the present invention;

[0026] Figure 2 This is a photodynamic therapy test result diagram of iridium complex HIr-NO on 4T1 cells in an embodiment of the present invention;

[0027] Figure 3 This is a graph showing the tumor coefficient of the iridium complex HIr-NO after photodynamic therapy of a mouse breast cancer subcutaneous tumor model;

[0028] Figure 4 This is a diagram of organ coefficients after photodynamic therapy of a mouse breast cancer subcutaneous tumor model by the iridium complex HIr-NO in an embodiment of the present invention;

[0029] Figure 5 The synthetic route of the iridium complex photosensitizer HIr-NO in the present invention is shown in FIG. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments:

[0031] like Figure 5 As shown, a method for preparing an iridium complex photosensitizer comprises the following steps:

[0032] The preparation method of 2-chloro-4-(trifluoromethyl)quinoline is:

[0033] Place 4-trifluoromethylquinolin-2(H)-one (3g) in a round-bottom flask, maintain an argon atmosphere, and add trichloroethylene (30mL) to the reaction flask under continuous stirring. The reaction system reacts at 106°C for 3h. When the reaction liquid temperature drops to room temperature, slowly add the reaction liquid to a beaker containing distilled water (300mL) and continue stirring. A white solid precipitates. No additional purification is required. The white solid obtained by suction filtration is 2-chloro-4-(trifluoromethyl)quinoline.

[0034] The preparation method of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline is:

[0035] 2-Chloro-4-(trifluoromethyl)quinoline (2g), benzothiophene-2-boric acid (1.69g), tetrakis(triphenylphosphine)palladium (0.79g), potassium carbonate (5.98g) were added to a 250mL round-bottom flask, and water (30mL) and 1,4-dioxane (90mL) were added. The reaction system was protected by argon gas to remove oxygen, and the temperature of the reaction solution was raised to 100°C for 16h. After the reaction was completed, the mixture was cooled to room temperature, washed with distilled water 3 times, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by distillation under reduced pressure to obtain a crude product, which was purified by column chromatography with an eluent of V (petroleum ether): V (ethyl acetate) = 10:1 to obtain a white solid of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline.

[0036] The preparation method of the dimer is:

[0037] Add iridium trichloride trihydrate (0.96 g), 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline (1.8 g), 2-ethoxyethanol (120 mL) and water (40 mL) to a 100 mL round-bottomed double-necked flask. Remove oxygen from the reaction system under argon atmosphere, raise the temperature of the reaction solution to 120 ° C, and react for 24 hours. After the reaction is completely cooled to room temperature, slowly pour the reaction solution into a beaker containing 100 mL of water. After stirring for 15 minutes, filter with suction, and dry the filter residue to obtain a black-red solid as the corresponding dimer.

[0038] The preparation method of iridium complex HIr-NO is as follows:

[0039] The corresponding dimer (0.38 g), 5-hydroxypicolinic acid (0.078 g), potassium carbonate (0.33 g), dichloromethane (30 mL), and ethanol (10 mL) were added to a 100 mL two-necked flask and reacted at 60° C. for 24 h under an argon atmosphere. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain a crude product, which was purified by column chromatography with an eluent of V (dichloromethane): V (methanol) = 50:1 to obtain a black solid as the iridium complex HIr-NO.

[0040] The NMR data of the iridium complex HIr-NO obtained are:

[0041] 1 H NMR (400MHz, DMSO-d6) δ8.45 (s, 1H), 8.39 (d, J = 10.6Hz, 2H), 8.09-7.95 (m, 3H), 7 .93(d,J=8.0Hz,1H),7.60(t,J=7.7Hz,1H),7.56-7.48(m,3H),7.37(d,J=8.6Hz,1 H),7.25(t,J=7.6Hz,1H),7.20(dd,J=8.6,2.3Hz,1H),7.12(t,J=7.6Hz,1H),7.07 (t,J=7.2Hz,2H),6.80(t,J=7.7Hz,1H),6.72-6.52(m,3H),5.95(d,J=8.2Hz,1H).

[0042] 19 F NMR(376MHz,DMSO-d6)δ-60.16(s),-60.27(s).

[0043] Test 1:

[0044] The relative active oxygen yield of the iridium complex HIr-NO was tested by a chemical method using diphenylisobenzofuran as an active oxygen indicator and terpyridine ruthenium as a reference. The specific experimental steps are as follows:

[0045] The iridium complex HIr-NO in acetonitrile (10 μM) was mixed with diphenylisobenzofuran (10 μM) solution and then illuminated with a laser (535 nm, 10 mW / cm -2 ) was irradiated, and the absorption spectrum of the mixed solution was detected. The absorption spectrum and the absorbance at 415 nm were recorded every 30 seconds. The same experiment was carried out using terpyridine ruthenium as a reference.

[0046] The degradation effects of iridium complex HIr-NO and reference compound terpyridine ruthenium on diphenylisobenzofuran under laser irradiation are shown in Figure 2. Figure 1 As shown, after laser irradiation, the iridium complex HIr-NO causes diphenylisobenzofuran to degrade rapidly, and its degradation rate is significantly faster than that of the reference substance terpyridine ruthenium. The relative active oxygen yield of the iridium complex HIr-NO is calculated to be close to 3 times that of the reference substance terpyridine ruthenium, indicating that the iridium complex HIr-NO prepared above has excellent active oxygen generation ability.

[0047] Test 2:

[0048] The CCK-8 method was used to detect the toxicity and photodynamic therapy effect of the iridium complex photosensitizer HIr-NO on 4T1 cancer cells under laser action and non-laser action conditions. The specific experimental steps are as follows:

[0049] Cells in the logarithmic growth phase were cultured at 1×10 4 The cells were inoculated at a density of 100 cells / well in a 96-well plate and incubated in an incubator (37°C, 5% CO2). When the cell density reached 70%, culture medium containing different concentrations of iridium complex HIr-NO was added. For the laser irradiation group, after incubation with iridium complex HIr-NO for 16 h, laser (535 nm, 100 mW / cm -2 , 5min) irradiate cells; for the laser non-irradiation group, the cells were always kept in the dark, among which 0nM was set as the control group. After incubation for 24h, 15μL of CCK-8 was added to each well and incubated for 75min. The absorbance of the absorption peak of each well at 450nm was tested by an ELISA instrument, and the average and standard deviation of the absorbance of 6 wells in each group were calculated to calculate the survival rate of cancer cells.

[0050] The specific preparation method of the above-mentioned culture medium containing different concentrations of iridium complex HIr-NO is: use biochemical grade dimethyl sulfoxide to prepare a mother solution of iridium complex HIr-NO with a concentration of 1 mM, and use complete culture medium (RPMI 1640, containing 10% fetal bovine serum) to dilute the mother solution to 20nM, 40nM, 60nM, 80nM, 100nM, 120nM, 140nM, 160nM.

[0051] The above CCK-8 test results are as follows Figure 2As shown, under the condition of no laser action, the cell survival rate at all test concentrations can be maintained above 95%, indicating that the iridium complex photosensitizer HIr-NO prepared above has excellent biocompatibility; under the condition of laser action, with the continuous increase of the test concentration, the cell survival rate continues to decrease, and its half inhibition concentration IC50 is calculated to be 91.10nM, indicating that the iridium complex photosensitizer HIr-NO prepared above has excellent photodynamic therapy effect.

[0052] Test 3:

[0053] The photodynamic therapy effect of iridium complex HIr-NO was evaluated by using a mouse breast cancer subcutaneous tumor model. The specific experimental steps are as follows:

[0054] 4T1 cells (2×10 6 The tumor model was established with 100 μL of cells and the tumor volume reached 100 mm 3 The group treatment was started and the tumor-bearing mice were divided into 4 groups (5 mice in each group):

[0055] Group 1: no treatment;

[0056] Group 2: Normal saline was injected into the tail vein. Two hours after injection, laser (535 nm, 100 mW / cm -2 , 5min) for treatment;

[0057] Group 3: Tail vein injection of iridium complex HIr-NO (2 μM);

[0058] Group 4: Injection of iridium complex HIr-NO (2 μM) into the tail vein. Two hours after injection, laser (535 nm, 100 mW / cm -2 , 5min) for treatment;

[0059] At the same time, in order to verify the death mechanism of cancer cells induced by iridium complex HIr-NO, GPX4 gene knockout 4T1 tumor-bearing mice were divided into two groups (5 mice in each group).

[0060] Group 5: no treatment;

[0061] Group 6: Injection of iridium complex HIr-NO (2 μM) into the tail vein. Two hours after injection, laser (535 nm, 100 mW / cm -2 , 5min) for treatment;

[0062] After 10 days of treatment, all mice were sacrificed, and tumors and major organ tissues (heart, liver, spleen, lung, and kidney) were collected, photographed, weighed, and recorded.

[0063] Effect of iridium complex HIr-NO on photodynamic therapy of subcutaneous breast cancer tumor model in mice Figure 3 As shown, the tumors of mice in the groups without drug administration and only light irradiation and the group with drug administration and no light irradiation grew rapidly, while the tumors of mice in the group with drug administration and light irradiation were effectively inhibited, indicating that the iridium complex photosensitizer HIr-NO prepared above has excellent photodynamic therapy effect.

[0064] Effect of iridium complex HIr-NO on organ function of mouse breast cancer subcutaneous tumor model after photodynamic therapy Figure 4 As shown, the organ coefficients of heart, liver, lung and kidney of all groups remained normal, indicating that the iridium complex photosensitizer HIr-NO prepared above has excellent biocompatibility; the mice in the no-drug and light-only groups and the drug-only and light-untreated groups showed symptoms of splenomegaly, which is one of the pathological characteristics of the 4T1 tumor model, while the splenomegaly of the mice in the drug-treated and light-treated groups was effectively alleviated, which indirectly indicates that the iridium complex photosensitizer HIr-NO prepared above has excellent photodynamic therapy effect.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An iridium complex photosensitizer, characterized in that: Its chemical structure is: 。 2. A method for preparing the iridium complex photosensitizer according to claim 1, characterized in that: The following steps are involved: (1) 4-trifluoromethylquinoline-2( H )-ketone is mixed with phosphorus oxychloride, the reaction system is kept oxygen-free, and a halogenation reaction is carried out at 100-110 °C for 2-4 h to obtain 2-chloro-4-(trifluoromethyl)quinoline; (2) dissolving the 2-chloro-4-(trifluoromethyl)quinoline, benzothiophene-2-boric acid and a catalyst in a solvent, keeping the reaction system oxygen-free, and carrying out a Suzuki coupling reaction at 90-116° C. in an alkaline environment for 14-19 h to obtain 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline; (3) dissolving the 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline and iridium trichloride trihydrate in a solvent, keeping the reaction system oxygen-free, reacting at 115-127° C. for 20-25 hours to obtain the corresponding dimer; (4) dissolving the dimer and 5-hydroxypicolinic acid in a mixed solvent, keeping the reaction system oxygen-free, and carrying out coordination reaction at 53-65° C. in an alkaline environment for 20-25 h to obtain an iridium complex photosensitizer.

3. The method for preparing an iridium complex photosensitizer according to claim 2, characterized in that: The 4-trifluoromethylquinoline-2( H The molar ratio of )-ketone to phosphorus oxychloride is 1:(20~25).

4. The method for preparing the iridium complex photosensitizer according to claim 3, characterized in that: The 4-trifluoromethylquinoline-2( H )-ketone and phosphorus oxychloride are mixed in the following manner: firstly, 4-trifluoromethylquinoline-2( H )-ketone is placed in a reaction bottle, and the reaction bottle is kept in an argon atmosphere. Under continuous stirring, chlorine oxychloride is added dropwise into the reaction bottle.

5. The method for preparing an iridium complex photosensitizer according to claim 2, characterized in that: In step (2), the molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to benzothiophene-2-boric acid is 1:(0.8-1.2); the molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to the catalyst is 1:(0.06-0.08), and the molar ratio of 2-chloro-4-(trifluoromethyl)quinoline to the base is 1:(4.3-5.2).

6. The method for preparing the iridium complex photosensitizer according to claim 5, characterized in that: The solvent for the Suzuki coupling reaction in step (2) is a mixed solvent of water and 1,4-dioxane, the volume ratio of water to 1,4-dioxane is 1:(2.7-3.3), and the amount ratio of 2-chloro-4-(trifluoromethyl)quinoline to water is 1 g:15 mL.

7. The method for preparing the iridium complex photosensitizer according to claim 2 or 5, characterized in that: The catalyst in step (2) is tetrakis(triphenylphosphine)palladium or [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride.

8. The method for preparing an iridium complex photosensitizer according to claim 2, characterized in that: In step (3), the molar ratio of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline to iridium trichloride trihydrate is 1:(0.3-0.6); the solvent is a mixed solvent of water and 2-ethoxyethanol, the volume ratio is 1:(2.7-3.3), and the amount ratio of 2-(benzothiophene-2-yl)-4-(trifluoromethyl)quinoline to water is 1 g:(17-25) mL.

9. The method for preparing an iridium complex photosensitizer according to claim 2, characterized in that: In step (4), the molar ratio of the dimer to 5-hydroxypicolinic acid is 1:(2.3-3.1); the mixed solvent is a mixed solvent of ethanol and dichloromethane, and the volume ratio is 1:(2.7-3.4); the dosage ratio of the dimer to ethanol is 1 g:(16-23) mL; and the molar ratio of the dimer to the base is 1:(8.3-11.2).

10. Use of the iridium complex photosensitizer according to claim 1 in the preparation of drugs for photodynamic therapy of breast cancer.

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