A semi-sandwich iridium (III)-quinoline Schiff base metal complex and its preparation method and application

Abstract

The present invention belongs to the field of chemical pharmaceuticals and specifically relates to a half-sandwich iridium (III)-quinoline Schiff base metal complex, its preparation method, and application. The metal complex is complex Ir1, complex Ir2, or complex Ir3, and has the specific structural formula: The metal iridium complex prepared by the present invention exhibits excellent anticancer activity, significantly superior to that of platinum-based anticancer drugs. The metal iridium complex prepared by the present invention has excellent fluorescence properties, facilitating the study of anticancer mechanisms such as drug targeting. The preparation method provided by the present invention has high substrate utilization, low difficulty coefficient for product separation and purification, and broad application prospects. The prepared target complex can accumulate in the mitochondria within cells, thereby causing apoptosis of cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of chemical pharmacy, and specifically relates to a semi-sandwich iridium (III)-quinoline Schiff base metal complex and a preparation method and application thereof. Background Art

[0002] Cancer, also known as malignant tumors, is caused by malignant cell proliferation and is invasive and metastatic. Cancer is a highly devastating disease that can have widespread effects and damage across all human systems. It is one of the leading causes of death, making cancer treatment urgent. Annual medical expenditures in my country exceed 220 billion yuan due to malignant tumors. Surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy are essential approaches to combating cancer. Simultaneously, numerous researchers at home and abroad are dedicated to developing anticancer drugs, which has driven the development and clinical application of platinum-based anticancer drugs. However, with the widespread clinical use of platinum-based drugs, drug resistance is increasing. Due to the inherent weakness of platinum-based drugs (severe toxic side effects), new transition metal drugs, particularly iridium (III) complexes with fewer side effects, have garnered increasing attention. This has prompted the search for alternative metal-based compounds with lower toxicity, higher selectivity, and a broader spectrum of activity. Iridium (III) complexes with fewer side effects have garnered increasing attention due to their distinct anticancer mechanisms from cisplatin.

[0003] As research deepens, the oxidative stability of iridium (I) compounds, as potential therapeutic agents, remains a challenge in both in vitro and in vivo studies. Consequently, researchers have begun extensively studying iridium (III) anticancer complexes. Iridium (III) anticancer complexes can be divided into two broad categories based on their structural characteristics: cyclic iridium (III) structures with excellent luminescence properties and semi-sandwich iridium (III) structures with high activity. The cytotoxicity and selectivity exhibited by metallic iridium (III) complexes have made them a key focus in the research and development of metal-based anticancer drugs in recent years. Therefore, organometallic iridium anticancer drugs are expected to become new anticancer drugs for clinical treatment in the future. The development of iridium complexes with highly effective anticancer activity is of great significance. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a semi-sandwich iridium (III)-quinoline Schiff base metal complex.

[0005] The present invention also provides a method for preparing the semi-sandwich iridium (III)-quinoline Schiff base metal complex.

[0006] The present invention also provides the use of the semi-sandwich iridium (III)-quinoline Schiff base metal complex in the preparation of anti-cancer targeted drugs.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows:

[0008] The present invention provides a half-sandwich iridium (III)-quinoline Schiff base metal complex, wherein the metal complex is complex Ir1, complex Ir2 or complex Ir3, and the specific structural formula is:

[0009] .

[0010] The present invention also provides a method for preparing the above-mentioned half-sandwich iridium (III)-quinoline Schiff base metal complex, comprising the following steps:

[0011] (1) Weigh the iridium dimer and ligand, add sodium acetate solution, evacuate the mixture, fill with nitrogen, and then inject anhydrous methanol as solvent. Mix the solution and stir at room temperature to react. Heat to reflux and detect the reaction by thin layer chromatography.

[0012] (2) After the reaction is completed, heat and reduce pressure to dryness to obtain an orange-red solid powder. Dichloromethane is added dropwise, filtered, and then the precipitate is slowly filled with n-hexane and sealed until crystals appear in the bottle.

[0013] The structural formula of the ligand used in the preparation of the target complex of the present invention is:

[0014] .

[0015] The structural formula of the metal iridium dimer used in the preparation of the target complex of the present invention is:

[0016] .

[0017] Furthermore, in step (1), the mass ratio of the metal iridium dimer to the ligand is 0.45-0.47:1; and the ratio of the metal iridium dimer to the sodium acetate solution is 0.032 g:15-20 mL.

[0018] Furthermore, in step (1), the ligand is pre-dissolved in benzene, and the concentration in benzene is 1.7 mg / mL; the concentration of the sodium acetate solution is 0.4 mol / L.

[0019] Furthermore, in step (1), the stirring reaction time is 6 hours; and the heating reflux is reflux at 65° C. for 4 hours.

[0020] The present invention also provides use of the semi-sandwich iridium (III)-quinoline Schiff base metal complex in the preparation of anticancer drugs.

[0021] The present invention also provides use of the half-sandwich iridium (III)-quinoline Schiff base metal complex in detecting the number of cells at different stages.

[0022] The specific reaction scheme of the present invention is as follows:

[0023]

[0024] This study synthesized three half-sandwich iridium(III)-quinoline Schiff base metal complexes, Ir1-Ir3, with antiproliferative activity against A549 and A549-DDP cells. Complex Ir3 exhibited excellent selectivity and superior antiproliferative activity to cisplatin. Further studies revealed that complex Ir3 entered A549 cells via an energy-dependent pathway. Once inside, it targeted the mitochondria, causing a decrease in mitochondrial membrane potential and arresting the cell cycle in the S phase, thereby promoting early apoptosis. Complex Ir3 also effectively inhibited A549 cell migration. Although complex Ir3 exhibited limited antiproliferative activity against cisplatin-resistant strains, these studies provide insights into the design and research of half-sandwich iridium(III)-quinoline Schiff base anticancer complexes.

[0025] The beneficial effects of the present invention are:

[0026] (1) The metal iridium complex prepared by the present invention exhibits excellent anticancer activity, which is significantly better than that of platinum-based anticancer drugs;

[0027] (2) The compounds of the present invention have good fluorescence properties, which are convenient for studying anti-cancer mechanisms and provide a new idea for the subsequent research of anti-cancer drugs;

[0028] (3) The preparation method provided by the present invention has high substrate utilization rate, low difficulty coefficient of product separation and purification, and broad application prospects. The prepared target complex can accumulate in the mitochondria in cells, thereby causing apoptosis of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the H NMR spectrum of the complex Ir1 of the present invention;

[0030] Figure 2 is the mass spectrum of the complex Ir1 in the present invention;

[0031] Figure 3 is the H NMR spectrum of the complex Ir2 in the present invention;

[0032] Figure 4 is the mass spectrum of the complex Ir2 in the present invention;

[0033] Figure 5 is the H NMR spectrum of the complex Ir3 in the present invention;

[0034] Figure 6 is the mass spectrum of the complex Ir3 in the present invention;

[0035] Figure 7 This is a cell tissue targeting test diagram of the complex of the present invention;

[0036] Figure 8 This is a study of the cell cycle arrest of the complex Ir3; (a) Detection of the cell cycle of A549 cells after treatment with different concentrations of the complex Ir3 for 24 hours; (b) Histogram of cell cycle arrest induced by the complex Ir3;

[0037] Figure 9 This is a study on the effect of the complex Ir3 in the present invention on reducing mitochondrial membrane potential; wherein, (a) a graph showing changes in mitochondrial membrane potential after 24 hours of treatment with complex Ir3 at different concentrations; (b) a histogram showing changes in mitochondrial membrane potential after 24 hours of treatment with complex Ir3 at different concentrations in A549 cells.

[0038] Figure 10 This is a study on the apoptosis induced by the complex Ir3 of the present invention; wherein, (a) a cell apoptosis diagram of A549 cells after 24 hours of treatment with different concentrations of complex Ir3; (b) a histogram of apoptosis of A549 cells induced by different concentrations of complex Ir3;

[0039] Figure 11 This is a study on the inhibition of cell migration by the complex Ir3 of the present invention; wherein, (a) scratch healing image of A549 cells after treatment with different concentrations of complex Ir3 for 24 hours; (b) histogram of scratch healing after treatment with complex Ir3 for 24 hours on A549 cells. DETAILED DESCRIPTION

[0040] The present invention is further illustrated with reference to the following examples of some representative compounds, but these illustrations are not intended to limit the present invention.

[0041] The starting compounds used in the synthesis of the compounds are commercial products or can be prepared by known synthetic methods. The preparation methods of all organic compounds are available from the literature.

[0042] Example 1

[0043] (1) The structural formula of the ligand used in the present invention is:

[0044]

[0045] The synthesis process is as follows:

[0046] Weigh 62.7 mg of toluidine or p-fluoroaniline and 41.7 mg of 8-hydroxyquinoline-2-carboxaldehyde into an eggplant-shaped flask, add 30 mL of methanol to dissolve, add two drops of formic acid, and stir at 65°C for 4 h. Remove the solvent under reduced pressure, and vacuum filter and dry to obtain 67.3 mg of a brown-black solid powder, which is the quinoline Schiff base ligand.

[0047] The synthesis process of quinoline Schiff base ligands L1 and L2 is as follows:

[0048]

[0049] (2) The structural formula of the metal iridium dimer used in the present invention is:

[0050]

[0051] The synthesis process is as follows:

[0052] (1) Synthesis of half-sandwich metal iridium dimer (Dimer 1)

[0053] ① Weigh 0.5 g of hydrated iridium trichloride into a clean, dry microwave digestion vessel. Add 0.75 mL of 1,2,3,4,5-pentamethylcyclopentadiene and 20 mL of anhydrous methanol as the reaction solvent. Mix the above ingredients thoroughly, then fill the microwave digestion vessel with nitrogen and place it in the microwave digester.

[0054] ② After digestion, pour the supernatant into a clean beaker. Rinse the digestion tank with ice-cold methanol and pour the solution into the beaker. Add 10 mL of dichloromethane to the digestion tank to dissolve the remaining crystals. Then filter to remove the remaining iridium trichloride. Spin the solution to dryness to obtain the product.

[0055] ③ The solution in the beaker was spin-dried and washed with ice methanol. Dichloromethane was added to dissolve it, and then the remaining iridium trichloride was filtered out. 16 mL of a 1:1 mixture of n-hexane and anhydrous ether was added to the filtrate. The mixture was placed in a refrigerator for crystallization to obtain a pure half-sandwich metal iridium dimer (Dimer 1). The product was 0.41 g (yield: 66.7%). 1 H NMR (500 MHz, CDCl3): 1.60 ppm (s, J = 1.4 Hz, 15H). Details are shown in Table 1.

[0056] Table 1

[0057]

[0058] Synthesis process of half-sandwich metal iridium dimer (Dimer 1):

[0059]

[0060] (2) Synthesis of half-sandwich metal iridium dimer (Dimer 2)

[0061] 0.5 g (1.7 mmol) of hydrated iridium trichloride and 0.88 g (5.0 mmol) of CpxphH were added to a microwave digestion vessel. Following the method for Dimer 1, 0.396 g (51.40%) of Dimer 2 was synthesized. 1H NMR (500 MHz, CDCl3): 7.58 (m, 2H), 7.35 (m, 3H), 1.72 (s, 6H), 1.63 ppm (s, 6H).

[0062] The synthesis process of half-sandwich metal iridium dimer (Dimer 2) is as follows:

[0063]

[0064] Example 2 Complex Ir1

[0065] (1) Weigh 0.038 g of iridium dimer L1 and 0.083 g of ligand Dimer 1 (pre-added in benzene, the concentration in benzene is 1.7 mg / mL) and add them into a clean and dry Shrek bottle. Add 20 mL of 0.4 mol / L sodium acetate solution into the bottle. After vacuuming and filling with N2, inject 20 mL of anhydrous methanol as solvent. Mix the solution and stir the reaction at room temperature for 6 h. Reflux at 65 °C for 4 h. Detect the reaction by thin layer chromatography.

[0066] (2) After the reaction is completed, connect the evaporator to a rotary evaporator, heat and reduce pressure to dry, and obtain an orange-red solid powder. Add dichloromethane to dissolve the solid, filter it into a diffusion bottle, slowly fill it with n-hexane, and seal it until crystals appear in the bottle.

[0067] The H NMR spectrum and mass spectrum of the complex Ir1 are as follows Figure 1 and Figure 2 shown.

[0068] Example 3 Complex Ir2

[0069] (1) Weigh 0.035 g of iridium dimer L2 and 0.076 g of ligand Dimer 1 (pre-added in benzene, the concentration in benzene is 1.7 mg / mL) and add them into a clean and dry Shrek bottle. Add 20 mL of 0.4 mol / L sodium acetate solution into the bottle. After vacuuming and filling with N2, inject 20 mL of anhydrous methanol as solvent. Mix the solution and stir the reaction at room temperature for 6 h. Reflux at 65 °C for 4 h. Detect the reaction by thin layer chromatography.

[0070] (2) After the reaction is completed, connect the evaporator to a rotary evaporator, heat and reduce pressure to dry, and obtain an orange-red solid powder. Add dichloromethane to dissolve the solid, filter it into a diffusion bottle, slowly fill it with n-hexane, and seal it until crystals appear in the bottle.

[0071] The H NMR spectrum and mass spectrum of the complex Ir1 are as follows Figure 3 and Figure 4 shown.

[0072] Example 4 Complex Ir3

[0073] (1) Weigh 0.083 g of iridium dimer L2 and 0.1814 g of ligand Dimer2 (pre-added in benzene, the concentration in benzene is 1.7 mg / mL) and add them into a clean and dry Shrek bottle. Add 45 mL of 0.4 mmol sodium acetate solution into the bottle, evacuate the bottle and fill it with N2, then inject 45 mL of anhydrous methanol as solvent. Mix the solution and stir at room temperature for 6 h, reflux at 65 °C for 4 h, and detect the reaction by thin layer chromatography.

[0074] (2) After the reaction is completed, connect the evaporator to a rotary evaporator, heat and reduce pressure to dry, and obtain an orange-red solid powder. Add dichloromethane to dissolve the solid, filter it into a diffusion bottle, slowly fill it with n-hexane, and seal it until crystals appear in the bottle.

[0075] The H NMR spectrum and mass spectrum of the complex Ir1 are as follows Figure 5 and Figure 6 shown.

[0076] Effect embodiment

[0077] (I) Experiment on the inhibition of cancer cell proliferation by organometallic iridium complexes:

[0078] (1) Preparation of the complexes to be tested: Weigh the complexes Ir1, Ir2, and Ir3 and dissolve them in 600 μL DMSO to a drug concentration of 10 mM as a stock solution. The stock solution is then diluted to different concentrations using DMEM cell culture medium.

[0079] (2) Cell growth inhibition assay (MTT assay):

[0080] 1) Add different concentrations of drugs to the culture medium of cells in the logarithmic growth phase and incubate them. Add 5µL to each well. Set up 4 parallel experiments for each concentration.

[0081] 2) After 24 hours of incubation, add 15 µL of MTT solution (5 mg / mL) to each well. After 4 hours of incubation in the dark, discard the culture medium and add 100 µL of DMSO solution to each well. Vortex for 2 minutes and measure the absorbance at 570 nm using a microplate reader.

[0082] 3) The cell survival rate curves at different concentrations were fitted using Origin software (V 2021) to obtain the half-lethal concentration (IC 50).

[0083] The proliferation of A549 cells, A549-DDP cells and BEAS-2B cells in vitro after 24 h of treatment with these two complexes was detected by MTT assay, and the IC values ​​of the complex Ir1-Ir3 were calculated. 50 The results are shown in Table 2.

[0084] Table 2 IC values ​​of complexes in different cells after 24h treatment 50

[0085]

[0086] The complexes Ir1-Ir3 showed certain antiproliferative activity against A549 and A549-DDP cells in vitro, especially complexes Ir1 and Ir3. It is worth noting that the IC of complex Ir3 on BEAS-2B cells 50 The value is 1.6 times that of A549 cells, showing good selectivity, which ensures that it kills cancer cells while having less toxic side effects on normal cells. Therefore, the complex Ir3 was selected for subsequent anti-cancer mechanism research.

[0087] (II) Use two-photon laser confocal microscopy to visually detect the targeting of target complexes after they enter cells

[0088] like Figure 7 As shown, laser confocal microscopy was used to investigate the entry mechanism of the complex Ir3 into A549 cells. Compared with the control group, the fluorescence intensity in A549 cells pretreated with CQ was not significantly different from that in the control group. However, the fluorescence intensity in A549 cells pretreated with CCCP or at 4°C was significantly reduced, indicating that the complex Ir3 enters A549 cells through an energy-dependent pathway. Two-photon laser confocal microscopy was used to investigate the targeting of the complex Ir3 after entry into A549 cells. The colocalization coefficient of the complex Ir3 with the cell nucleus was 0, indicating that the complex Ir3 essentially did not enter the cell nucleus. The colocalization coefficient with mitochondria was 0.80, and the colocalization coefficient with lysosomes was 0.09, indicating that the complex Ir3 primarily targeted mitochondria after entry into A549 cells.

[0089] (III) Cell cycle testing of complexes

[0090] A549 cells were seeded in 6-well plates and cultured to the logarithmic growth phase. 50 , 0.75×IC 50 and 1.00×IC 50) for 24 hours, then collect the cells. Add 1 mL of ice-cold 70% ethanol to a new centrifuge tube, resuspend the cell pellet in 50 µL of PBS, then slowly pipette into the ethanol, vortex to mix, and fix overnight at -20°C. After fixation, centrifuge and discard the supernatant. Wash twice with PBS, then resuspend in 500 µL of PBS. Add RNase (2.5 µL), mix thoroughly, and then add PI (25 µL) and mix again. Incubate in a 37°C water bath in the dark for 30 minutes. After incubation, place the tube on ice in the dark until ready for flow cytometry analysis. Results were analyzed using Mod-Fit software (V 5.0).

[0091] pass Figure 8 It can be seen that the cell cycle of A549 cells after 24 h of treatment with different concentrations of complex Ir3 showed that the proportion of cells in the S phase of the control group was only 24.11%. With the increase of drug concentration, more and more cells were in the S phase. When the concentration of complex Ir3 was 2.0×IC 50 When the cells were treated with Ir3, 47.04% of them were arrested in the S phase. Therefore, it can be concluded that the complex Ir3 arrests the A549 cell cycle in the S phase in a concentration-dependent manner. In summary, after the complex Ir3 enters the mitochondria, it induces early apoptosis in A549 cells by causing a decrease in mitochondrial membrane potential and cell cycle arrest.

[0092] (IV) Mitochondrial membrane potential level detection

[0093] A549 cells were seeded in 6-well plates and cultured to the logarithmic growth phase. 50 , 1.0×IC 50 , 1.5×IC 50 and 2.0×IC 50 ) for 24 hours, and then the cells were collected. The positive control group was pretreated with CCCP (2 µL, 5 µM) 2 hours before cell collection. 1 mL of JC-1 working solution was added to each group, mixed, and incubated at 37°C in a dark-protected water bath for 20 minutes. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells were washed twice with 1 mL of JC-1 staining buffer. The cells were then resuspended in 0.5 mL of JC-1 staining buffer and incubated on ice in the dark until analysis by flow cytometry. Results were analyzed using FlowJo software (V10).

[0094] pass Figure 9 It can be seen that as the drug concentration increases, the green fluorescence in the mitochondria gradually increases, that is, the mitochondrial membrane potential gradually decreases, thereby affecting the normal metabolism of the cells.

[0095] (V) Study on the apoptosis induced by the complex TPA-Ir1

[0096] A549 cells were seeded into 6-well plates and cultured in a CO2 incubator until the logarithmic growth phase. 50 , 1.0×IC 50 and 1.5×IC 50 After incubation for 24 hours with 100 µL of the drug, cells were trypsinized and harvested. Resuspend the cells in 195 µL of Annexin V-FITC buffer, add 5 µL of Annexin V-FITC and 10 µL of propidium iodide (PI), mix well, and incubate at 37°C in the dark for 10–20 minutes. Place on ice in the dark until flow cytometry is performed and analysis is performed using FlowJo software (V10).

[0097] pass Figure 10 As can be seen, the complex Ir3 induces apoptosis in A549 cells in a dose-dependent manner, particularly in the early stages of apoptosis (quadrant IV). In particular, when the concentration of the complex Ir3 is 2.0× IC50, the apoptosis rate of A549 cells exceeds 80%, of which 54.9% is in the early stages of apoptosis. Therefore, the complex Ir3 can effectively induce apoptosis in A549 cells.

[0098] (VI) Cell scratch test

[0099] A549 cells seeded in a 6-well plate were allowed to adhere for 4-6 hours. After the culture medium was discarded and the cells were washed once with 1 mL of PBS, 2 mL of PBS was added to each well. A 10 µL pipette tip was used to make a fine, straight scratch perpendicular to the bottom of the 6-well plate (using the 6-well plate lid for assistance). Floating cells were washed with PBS, fresh PBS was added, and the cells were photographed. The position and scratch spacing were recorded for each image. After photography, the PBS was discarded and 2 mL of fresh culture medium containing a specific drug concentration was added. After returning the cells to the incubator and continuing to culture for 12 hours, the culture medium was discarded and the cells were washed once with 1 mL of PBS. After adding 2 mL of PBS to each well, images were acquired at the same location, and the scratch spacing was recorded for each image.

[0100] pass Figure 11 It can be seen that the concentration is 0.5×IC 50 The cell migration rate of the complex Ir3 was 20.52% at a concentration of 1.0×IC 50 The cell migration rate of the complex Ir3 was 3.76%. Compared with the migration rate of the control group of 35.21%, the complex Ir3 could effectively inhibit the migration of A549 cells.

Claims

1. A half-sandwich iridium (III)-quinoline Schiff base metal complex, characterized in that: The metal complex is complex Ir1, complex Ir2 or complex Ir3, and the specific structural formula is: 。 2. A method for preparing the half-sandwich iridium (III)-quinoline Schiff base metal complex according to claim 1, characterized in that: The following steps are involved: (1) Weigh the iridium dimer and ligand, add sodium acetate solution, evacuate the mixture, fill with nitrogen, and then inject anhydrous methanol as solvent. Mix the solution and stir at room temperature to react. Heat to reflux and detect the reaction by thin layer chromatography. (2) After the reaction is completed, heat and reduce pressure to dryness to obtain an orange-red solid powder, add dichloromethane dropwise, filter, and then slowly fill the precipitate with n-hexane and seal it until crystals appear in the bottle; In step (1), the structural formula of the ligand is: ； In step (1), the structural formula of the metal iridium dimer is: 。 3. The preparation method according to claim 2, characterized in that In step (1), the mass ratio of the metal iridium dimer to the ligand is 0.45-0.47:1; the ratio of the metal iridium dimer to the sodium acetate solution is 0.032 g:15-20 mL.

4. The preparation method according to claim 3, characterized in that In step (1), the ligand is pre-dissolved in benzene at a concentration of 1.7 mg / mL; the concentration of the sodium acetate solution is 0.4 mol / L.

5. The preparation method according to claim 2 or 3, characterized in that In step (1), the stirring reaction time is 6 hours; the heating reflux is reflux at 65°C for 4 hours.

6. Use of the half-sandwich iridium (III)-quinoline Schiff base metal complex according to claim 1 in the preparation of anticancer drugs.

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