An iridium complex ligand, an iridium complex and applications thereof

Abstract

The present invention provides an iridium complex with an ovarian cancer immunotherapeutic effect, having the structure shown in Formula II. The ligand of the iridium complex provided by the present invention contains the structure of the traditional Chinese medicine monomer cinnamic acid, which has good liposolubility, the metal atom iridium has excellent fluorescence properties, and the complex has a variety of coordination configurations and is easy to modify in structure, thus realizing rich biological activities. The experimental results show that the iridium(III) complexes provided by the present invention all have anti-tumor activities to varying degrees, and among them, the toxicity of Complex 1 to ovarian cancer cells is 3.3 times that of the positive drug cisplatin. Further studies were conducted on the effects of Complex 1 at different concentrations on extracellular ATP and the content of HMGB-1 protein, and it was found that Complex 1 can significantly induce the release of ATP and HMGB-1 protein in ovarian cancer cells and has a concentration dependence. Thus, it is shown that the above iridium complex has potential anti-tumor immunotherapeutic activity.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and particularly relates to an iridium complex ligand, an iridium complex and applications thereof. Background Art

[0002] Ovarian cancer is a gynecological malignant tumor with a high incidence rate. It is difficult to diagnose in the early stage, has a high mortality rate and poor prognosis. Its pathogenesis is complex and it is extremely easy to relapse, seriously endangering women's health. In recent years, the combination of anti-tumor drugs and immunotherapy has become a research boom, but its molecular mechanism is still unclear. With the increasing in-depth research on tumor immunity by scientists, more and more targeted therapy drugs and immunotherapy means have been approved for the treatment of ovarian cancer. Existing research has shown that the presence of certain tumor cells locally in tumors, such as NK cells, T lymphocytes, etc., is the main reason for the poor treatment effect of ovarian tumors. Therefore, it is necessary to explore the molecular mechanism of regulating the function of immune cells in the ovarian cancer microenvironment at the cellular and molecular levels, in order to discover new therapeutic targets and lay a foundation for improving the clinical efficacy.

[0003] Research has found that there is a complex relationship between traditional chemotherapy drugs and the body's immune system, not just the simple role of chemotherapy drugs in killing tumor cells. Immunogenic cell death (ICD), as a bridge connecting anti-tumor drugs and anti-tumor immunotherapy, is an acquired immune response mediated by cellular T lymphocytes and forms a lasting immune memory, which is an inflammatory response generated by the body under stress. Some drugs can also induce immunogenic death of tumor cells during the process of killing tumor cells, thereby triggering a tumor immune response. However, there are few drugs that can induce ICD clinically at present, such as doxorubicin, oxaliplatin, mitoxantrone and cyclophosphamide, etc. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an iridium complex ligand, an iridium complex and applications thereof, and the prepared iridium complex can be used for the preparation of anti-tumor immunotherapy drugs.

[0005] The present invention provides an iridium complex ligand having the structure shown in Formula I:

[0006]

[0007] Formula I.

[0008] The present invention provides a preparation method of the above ligand, comprising the following steps:

[0009] Cinnamic acid and 5-amino-1,10-phenanthroline are subjected to a first reaction in a first organic solvent, and then a second solvent is added. After the second reaction, N-(1,10-phenanthrolin-5-yl)cinnamide is obtained.

[0010] The first solvent is preferably dichloromethane.

[0011] Preferably, the first reaction is carried out under the action of a catalyst 4-dimethylaminopyridine and a dehydrating agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0012] The above first reaction is a substitution reaction.

[0013] The temperature of the first reaction is preferably room temperature.

[0014] The time of the above first reaction is preferably 10 - 15 min.

[0015] The above second solvent is preferably N,N-diisopropylethylamine.

[0016] The above second reaction is a substitution reaction.

[0017] After the second reaction is completed, the system is rotary evaporated to dryness to obtain a powder, which is the above ligand.

[0018] The above ligand coordinates with an iridium-containing metal compound and can be used to prepare an iridium complex with the property of inducing ICD.

[0019] Based on this, the present invention provides an iridium complex having the structure shown in Formula II:

[0020] X -

[0021] Formula II;

[0022] C–N is selected from any of the following structures:

[0023] ;

[0024] X - is an anion;

[0025] * represents the connection position.

[0026] The present invention has no special limitation on the anion of the above iridium complex, and any anion that can precipitate the complex can be used. Preferably, the X - is PF6 - .

[0027] Specifically, the cation part of the above iridium complex has any of the following structures:

[0028] .

[0029] The present invention provides a method for preparing the above iridium complex, comprising the following steps:

[0030] Reacting an iridium compound with N-(1,10-phenanthrolin-5-yl)cinnamamide in an organic solvent to obtain an iridium complex;

[0031] The iridium compound is selected from bridged bis(7,8-benzoquinoline)-monochloro-iridium(III), bridged bis(2-(2-thienyl)pyridine)-monochloro-iridium(III) or bridged 2-(2,4-difluorophenyl)pyridine-monochloro-iridium(III).

[0032] The above N-(1,10-phenanthrolin-5-yl)cinnamamide is prepared according to the above method, which will not be elaborated here.

[0033] The temperature of the above reaction is preferably 120-140 °C; the time is preferably 4-6 h.

[0034] The solvent of the above reaction is preferably DMF.

[0035] The above reaction is preferably carried out under light-shielded conditions.

[0036] After the reaction is completed, cool overnight, add NH4PF6, precipitate, add water, filter, and the solid is the iridium complex.

[0037] The obtained iridium complex is a powder with a faint sweet taste.

[0038] The present invention provides a pharmaceutical composition comprising the above iridium complex and an adjuvant.

[0039] The present invention explored the anti-cancer activity and immunotherapeutic activity of the above iridium complex, verified by detecting cytotoxicity through the MTS method, detecting the expression change of CRT protein on the cell membrane surface through the fluorescence immunoassay and detecting the content of HMGB-1 protein through the ELISA method. The experimental results show that the above iridium complex has excellent anti-proliferation activity, can induce immunogenic death of ovarian cancer cells, and generate a series of anti-tumor immune responses.

[0040] Based on this, the present invention provides the application of the above iridium complex or the above pharmaceutical composition in the preparation of anti-tumor immunotherapy drugs.

[0041] Preferably, the tumor is a solid tumor, more preferably ovarian cancer.

[0042] Specifically, it is SKOV-3 ovarian cancer cells.

[0043] Compared with the prior art, the present invention provides an iridium complex with ovarian cancer immunotherapeutic effect, having the structure shown in Formula II.

[0044] The ligand of the iridium complex provided by the present invention contains the structure of the traditional Chinese medicine monomer cinnamic acid, which has good liposolubility, the metal atom iridium has excellent fluorescence properties, and the complex has a variety of coordination configurations and is easy to modify in structure, thus achieving rich biological activities.

[0045] The experimental results show that the iridium (III) complexes provided by the present invention all have anti-tumor activities to varying degrees, and the toxicity of Complex 1 to ovarian cancer cells is 3.3 times that of the positive drug cisplatin. Further study on the effects of Complex 1 on the content of extracellular ATP and HMGB-1 protein at different concentrations found that Complex 1 can significantly induce the release of ATP and HMGB-1 protein in ovarian cancer cells, and has a concentration dependence. This indicates that the above iridium complex has potential anti-tumor immunotherapeutic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a concentration-dependent effect diagram of Complex 1 on the release amount of extracellular ATP in Example 2;

[0047] Figure 2 It is a concentration-dependent effect diagram of Complex 1 on the release amount of extracellular HMGB-1 protein in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0048] In order to further illustrate the present invention, the following describes the present invention in detail in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0049] There is no particular limitation on the sources of all raw materials of the present invention, and they can be purchased on the market or prepared according to the conventional methods well-known to those skilled in the art.

[0050] Example 1

[0051] (1) Preparation of ligand:

[0052] Weigh 148.16 mg (1 mmol) of cinnamic acid, 383.2 mg (2 mmol) of EDC, 244.32 mg (2 mmol) of DMAP, and 196 mg (1 mmol) of 1,10-phenanthroline-5-amine, place them in 40 ml of dichloromethane, stir at room temperature for about 10 min, then add 600 μl of DIPEA, and continue to stir and react for 5 h. After the reaction is completed, the reaction solution is rotary evaporated to obtain a crude product with a faint sweet smell, that is, the ligand, and the yield is 35.7%.

[0053] (2)Elemental analysis C 21 H 15 N3O (molecular weight 325.12), theoretical values: C 77.52%, H 4.65%, N 12.91%; experimental values: C 77.38%, H 4.712%; N 12.98%. ESI-MS: [(L+H) + theoretical value: m / z = 325.12; experimental value: m / z = 362.1. 1 H NMR (400 MHz, Chloroform-d) δ 9.15 (dd, J = 4.3, 1.6 Hz, 1H), 8.89 (dd, J = 4.3, 1.7 Hz, 1H), 8.33 (dd, J = 8.3, 1.7 Hz, 1H), 7.95 (dd, J = 8.1, 1.7 Hz, 1H), 7.71 (d, J = 15.3 Hz, 1H), 7.62 (dd, J = 8.3, 4.3 Hz, 1H), 7.57–7.53 (m, 2H), 7.46 (dd, J = 8.1, 4.3 Hz, 1H), 7.38–7.36 (m, 2H), 7.36–7.34 (m, 1H), 7.15 (d, J = 15.4 Hz, 1H), 6.92 (s, 1H). 13 C NMR (100 MHz, CDCl3) δ 169.39 (C-16), 155.90 (C-13), 155.50 (C-8), 150.06 (C-2), 146.89 (C-1), 140.38 (C-19), 134.85 (C-10, 11, 20), 133.57 (C-4), 130.33 (C-23), 129.87 (C-6), 128.95 (C-22, 24), 128.38 (C-21, 25), 123.37 (C-5), 122.38 (C-9, 12), 122.34 (C-3), 119.40 (C-18).

[0054] (3)Preparation of Complex 1:

[0055] Bis(7,8-benzoquinoline)-iridium(III) monochloride (0.143 mg, 0.125 mmol) and the ligand (0.815 g, 0.25 mmol) were placed in 24 ml of DMF, and stirred under dark at 130 °C for about 4 h. After the reaction, it was cooled, 0.75 mmol of NH4PF6 (0.12 g) was added to precipitate, about 100 ml of water was added, filtered, and the filter residue was dried to obtain the crude product. Silica gel column chromatography was carried out using dichloromethane:methanol (volume ratio 120:1) as the eluent, and dried to obtain Complex 1 with a yield of 61.5%.

[0056] Elemental analysis for C 47 H 31 F6IrN5PO (molecular weight: 1018.98), theoretical values: C 64.59%, H 3.58%, N 8.01%; experimental values: C 64.90%, H 3.89%, N 8.83%. ESI-MS: [(M–PF6) + theoretical value: m / z = 874.0, experimental value: m / z = 873.8.

[0057] (4) Preparation of Complex 2:

[0058] Bis(2-(2-thienyl)pyridine)-iridium(III) monochloride (0.143 mg, 0.125 mmol) and the ligand (0.815 g, 0.25 mmol) were placed in 24 ml of DMF, and stirred under dark at 130 °C for about 4 h. After the reaction, it was cooled, 0.75 mmol of NH4PF6 (0.12 g) was added to precipitate, about 100 ml of water was added, filtered, and the filter residue was dried to obtain the crude product. Silica gel column chromatography was carried out using dichloromethane:methanol (volume ratio 120:1) as the eluent, and dried to obtain Complex 2 with a yield of 66.9%.

[0059] Elemental analysis for C 39 H 27 F6IrN5POS2 (molecular weight: 982.98), theoretical values: C 55.90%, H 3.25%, N 8.36%; experimental values: C 56.34%, H 3.76%, N 8.03%. ESI-MS: [(M–PF6) + theoretical value: m / z = 838.0, experimental value: m / z = 837.7.

[0060] (5) Preparation of Complex 3:

[0061] Bridged 2-(2,4-difluorophenyl)pyridine-iridium(III) monochloride (0.143 mg, 0.125 mmol) and the ligand (0.815 g, 0.25 mmol) were placed in 24 ml of DMF and stirred under dark at 130 °C for about 4 h. After the reaction, it was cooled, 0.75 mmol of NH4PF6 (0.12 g) was added to precipitate, about 100 ml of water was added, filtered, and the filter residue was dried to obtain the crude product. Silica gel column chromatography was carried out using dichloromethane:methanol (volume ratio 120:1) as the eluent, and after drying, the complex 3 was obtained with a yield of 58.1%.

[0062] Elemental analysis C 43 H 27 F 10 IrN5PO (molecular weight 1042.89), theoretical values: C 57.52, H 3.03%, N 7.80%; experimental values: C 57.95%, H 3.83%, N 7.26%. ESI-MS: [(M–PF6) + Theoretical value: m / z = 897.9, experimental value: m / z = 895.9.

[0063] Example 2 In vitro anti-tumor and induction of immunogenic cell death tests of Ir(III) complexes

[0064] Anti-tumor activity test (MTS method): The toxicity of the iridium complex to ovarian cancer was determined by the MTS method: As one of the classical methods for detecting drug toxicity, the experimental steps are as follows. Cells in the logarithmic phase were digested with trypsin into a cell suspension, the cells were counted, 4,500 cells per well, a total of 160 µL of suspension, and inoculated into a 96-well plate. The remaining empty wells at the edges were filled with PBS. After overnight incubation in a CO2 constant temperature incubator, 40 µL of the complex with gradient concentrations containing medium was added to each well, and then incubated in the incubator for 48 h. Then, 20 µL / well of MTS reagent was added respectively. Incubation was continued for 3 hours, and the OD value was detected by machine. Calculate the cell survival rate according to the formula to obtain the half-lethal concentration (IC 50 value).

[0065] Survival rate % = average OD value of the drug-added well / average OD value of the control well × 100%

[0066] The test results are shown in Table 1 below

[0067] Table 1 IC of iridium complex 50 value

[0068]

[0069] a IC50 It is the concentration of the complex corresponding to 50% inhibition of tumor Hela. The experimental data are the averages obtained after three parallel experiments.

[0070] It can be seen from the experimental results that all three iridium complexes have varying degrees of activity in inhibiting the growth of ovarian cancer cells. Notably, their toxicity to ovarian cancer cells is 3.3 times that of the positive drug cisplatin.

[0071] Measurement of extracellular ATP release: Trypsinize SKOV-3 cells and collect the suspension. Adjust the cell density to 1.5×10 5 / dish and seed in 6-well plates, with a volume of 2 mL of cell suspension per well. After incubating for 24 h, add complexes 1 at concentrations of 10 µM, 20 µM, and 40 µM and incubate for another 24 h. Separate the cell supernatant, centrifuge at 1000 rpm / min for 5 min, and transfer the supernatant to a new centrifuge tube. According to the steps in the kit instructions, dissolve each reagent on ice, dilute the ATP standard to 0.01 µM, with gradient concentrations of 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, and 10 µM. Add 100 µL of ATP detection solution to the 96-well detection wells and let stand at room temperature for 5 min. Move the detection plate to the ice surface, add 100 µL of the sample, mix with a pipette, and measure the fluorescence intensity with a multifunctional microplate reader. ATP is the main energy source in the body. A large number of studies have shown that in the late stage of ICD, cells activate, recruit APCs, and the inflammatory signaling pathway through ATP, enhancing the body's immunity. The test results show that as the induction concentration increases, the release level of ATP also increases, showing a significant difference compared with the Control group.

[0072] The results are as Figure 1 shown.

[0073] Detection of extracellular release of HMGB-1 protein: Wash with PBS and then treat SKOV-3 cells with trypsin, and collect the cell suspension. Adjust the suspension density to 1.5×10 per dish 5, inoculated into 6-well plates, with the volume of cell suspension in each well being 2 mL, cultured in an incubator for 24 h. When the cell density reached about 70%, complexes 1 with concentrations of 10 µM, 20 µM, and 40 µM were added and co-incubated for 24 h. Then, the supernatant was taken, centrifuged at 1000 rpm for 5 min, the supernatant was recovered and placed into a new centrifuge tube. It was processed according to the method of the ELISA kit, and finally the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay reader. HMGB-1 is a nuclear protein that ubiquitously exists in various animals and has functions such as gene transcription regulation, nucleosome stabilization, and DNA repair. In the late stage of ICD, cells secrete HMGB-1, which promotes the maturation of dendritic cells and transports the tumor antigens carried by them to T cells. The results are as Figure 2 shown. As can be seen from Figure 2 , after the cells were co-incubated with complex 1, the extracellular HMGB-1 release amount increased significantly.

[0074] The experimental results of the HMGB-1 protein corresponded to the detection results of ATP, jointly indicating that complex 1 can induce immunogenic cell death in SKOV-3 cells and has the potential for anti-ovarian cancer immunotherapy.

[0075] The results of complex 2 and complex 3 were similar, and they could also induce immunogenic cell death in SKOV-3 cells and have the potential for anti-ovarian cancer immunotherapy.

[0076] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. 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 to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An iridium complex ligand having the structure shown in Formula I:

2. An iridium complex having the structure shown in Formula II: C–N is selected from any of the following structures: X - is an anion; * represents the connection position.

3. The iridium complex according to claim 2, characterized in that, The said X - is PF6 - .

4. The preparation method of the iridium complex according to any one of claims 2 to 3, comprising the following steps: Reacting an iridium compound and N-(1,10-phenanthrolin-5-yl)cinnamamide in an organic solvent to obtain an iridium complex; The iridium compound is selected from bridged bis(7,8-benzoquinoline)-monochloro-iridium(III), bridged bis(2-(2-thienyl)pyridine)-monochloro-iridium(III), or bridged 2-(2,4-difluorophenyl)pyridine-monochloro-iridium(III).

5. The preparation method according to claim 4, characterized in that, The N-(1,10-phenanthrolin-5-yl)cinnamamide is prepared according to the following method: Performing a first reaction on cinnamic acid and 5-amino-1,10-phenanthroline in a first organic solvent, then adding a second solvent, and performing a second reaction to obtain N-(1,10-phenanthrolin-5-yl)cinnamamide; The first solvent is selected from dichloromethane; The second solvent is selected from N,N-diisopropylethylamine.

6. The preparation method according to claim 5, characterized in that, The first reaction is carried out under the action of a catalyst 4-dimethylaminopyridine and a dehydrating agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

7. The preparation method according to claim 4, characterized in that, The temperature of the reaction is 120 - 140 °C; the time is 4 - 6 h; The organic solvent is DMF; The reaction is carried out under light-shielded conditions.

8. A pharmaceutical composition comprising the iridium complex according to any one of claims 2 to 3 and an adjuvant.

9. The application of the iridium complex according to any one of claims 2 to 3 or the pharmaceutical composition according to claim 8 in the preparation of an anti-tumor immunotherapy drug.

10. The application according to claim 9, characterized in that, The tumor is ovarian cancer.

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