Iridium complex ligand, iridium complex and application

Abstract

The invention provides an iridium complex with an ovarian cancer immunotherapy effect. The iridium complex has a structure as shown in a formula II. The iridium complex ligand provided by the invention contains a traditional Chinese medicine monomer cinnamic acid structure, the lipid solubility is better, the fluorescence property of metal atom iridium is excellent, and the complex has multiple coordination configurations and is easier in structural modification, so that rich biological activity is realized. Experimental results show that the iridium (III) complexes provided by the invention have different degrees of antitumor activity, wherein the toxicity of the complex 1 to ovarian cancer cells is 3.3 times that of a positive drug cis-platinum. The influence of the complex 1 on extracellular ATP and HMGB-1 protein content under different concentrations is further studied, and it is found that the complex 1 can significantly induce release of the ATP and HMGB-1 protein of ovarian cancer cells and has concentration dependence. Therefore, the iridium complex has potential anti-tumor immunotherapy activity.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemistry, in particular to an iridium complex ligand and an iridium complex and application thereof. Background Art

[0002] Ovarian cancer is a gynecological malignancy with a high incidence rate. It is difficult to diagnose early, has a high mortality rate and a poor prognosis. Its pathogenesis is complex, it is very easy to relapse, and it seriously endangers 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. As scientists' research on tumor immunity becomes more and more in-depth, more and more targeted therapeutic drugs and immunotherapy methods have been approved for the treatment of ovarian cancer. Studies have shown that the presence of certain tumor cells in the local tumor, such as NK cells, T lymphocytes, etc., is the main reason for the poor treatment effect of ovarian tumors. Therefore, the molecular mechanism of the regulation of immune cell function by the ovarian cancer microenvironment is explored at the cellular and molecular levels, in order to discover new therapeutic targets and lay the foundation for improving clinical efficacy.

[0003] Studies have found that there is a complex connection between traditional chemotherapy drugs and the body's immune system, which is not just a simple effect of chemotherapy drugs killing tumor cells. As a bridge connecting anti-tumor drugs and anti-tumor immunotherapy, cell immunogenic death (ICD) is an acquired immune response mediated by cell T lymphocytes and forms a lasting immune memory. It is an inflammatory response produced by the body under stress. In the process of killing tumor cells, some drugs can also induce immunogenic death of tumor cells, thereby triggering a tumor immune response. However, there are currently few drugs that can induce ICD in clinical practice, such as doxorubicin, oxaliplatin, mitoxantrone and cyclophosphamide. 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 and an iridium complex and application. The prepared iridium complex can be used to prepare anti-tumor immunotherapy drugs.

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

[0006]

[0007] Formula Ⅰ.

[0008] The present invention provides a method for preparing 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 to carry out a second reaction to obtain N-(1,10-phenanthroline-5-yl)cinnamic amide.

[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 first reaction is preferably 10 to 15 minutes.

[0015] The 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-mentioned ligand.

[0018] The above ligand is coordinated with a metal compound containing iridium and can be used to prepare an iridium complex with induced ICD properties.

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

[0020] X -

[0021] Formula II;

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

[0023] ;

[0024] X - It is an anion;

[0025] *Indicates the connection location.

[0026] The present invention has no particular limitation on the anion of the iridium complex, and any anion that can precipitate the complex is acceptable. Preferably, the X - 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] The iridium compound and N-(1,10-phenanthroline-5-yl)cinnamamide are reacted 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-mentioned N-(1,10-phenanthroline-5-yl)cinnamamide was prepared according to the above method, which will not be described in detail here.

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

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

[0035] The above reaction is preferably carried out under light-shielding 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 light sweet taste.

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

[0039] The present invention explores the anti-cancer activity and immunotherapeutic activity of the iridium complex, detects cytotoxicity by MTS method, detects expression changes of CRT protein on the cell membrane surface by fluorescence immunoassay, and verifies HMGB-1 protein content by ELISA method. The experimental results show that the iridium complex has excellent anti-proliferative activity, can induce immunogenic death of ovarian cancer cells, and produce a series of anti-tumor immune responses.

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

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

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

[0043] Compared with the prior art, the present invention provides an iridium complex having an ovarian cancer immunotherapy effect, which has a structure shown in formula II.

[0044] The iridium complex ligand provided by the invention contains a traditional Chinese medicine monomer cinnamic acid structure, has good fat solubility, and the metal atom iridium has excellent fluorescence properties. In addition, the complex has a variety of coordination configurations and is easy to modify the structure, thereby achieving rich biological activity.

[0045] The experimental results show that the iridium (III) complexes provided by the present invention have different degrees of anti-tumor activity, among which the toxicity of complex 1 to ovarian cancer cells is 3.3 times that of the positive drug cisplatin. The effects of complex 1 on the extracellular ATP and HMGB-1 protein content at different concentrations were further studied, and it was found that complex 1 can significantly induce the release of ATP and HMGB-1 protein in ovarian cancer cells, and is concentration-dependent. This shows that the above-mentioned iridium complex has potential anti-tumor immunotherapy activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a graph showing the concentration-dependent effect of complex 1 on the amount of extracellular ATP released in Example 2;

[0047] Figure 2 This is a graph showing the concentration-dependent effect of complex 1 in Example 2 on the amount of extracellular HMGB-1 protein released. DETAILED DESCRIPTION

[0048] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with the embodiments. 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] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods 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-amino, place in 40 ml of dichloromethane, stir at room temperature for about 10 min, add 600 μl of DIPEA, and continue stirring for 5 h. After the reaction is completed, spin dry the reaction solution to obtain a crude product with a light sweet taste, which is the ligand, with a yield of 35.7%.

[0053] (2) Elemental analysis C 21 H 15 N3O (molecular weight 325.12), theoretical value: C 77.52%, H 4.65%, N 12.91%; experimental value: 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, 122.34 (C-3), 119.40 (C-18).

[0054] (3) Preparation of complex 1:

[0055] Bridged di(7,8-benzoquinoline)-monochloro-iridium(III) (0.143 mg, 0.125 mmol) and ligand (0.815 g, 0.25 mmol) were placed in 24 ml DMF and stirred at 130 °C in the dark for about 4 h. After the reaction was completed, the mixture was cooled and 0.75 mmol NH4PF6 (0.12 g) was added to precipitate. About 100 ml of water was added, filtered, and the residue was dried to obtain a crude product. The crude product was separated by silica gel column chromatography 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 C 47 H 31 F6IrN5PO (molecular weight 1018.98), theoretical value: C 64.59%, H 3.58%, N8.01%; experimental value: 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] Bridged di(2-(2-thienyl)pyridine)-monochloro-iridium(III) (0.143 mg, 0.125 mmol) and ligand (0.815 g, 0.25 mmol) were placed in 24 ml DMF and stirred at 130 °C in the dark for about 4 h. After the reaction was completed, the mixture was cooled and 0.75 mmol NH4PF6 (0.12 g) was added to precipitate. About 100 ml of water was added, filtered, and the residue was dried to obtain a crude product. The crude product was separated by silica gel column chromatography 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 C 39 H 27 F6IrN5POS2 (molecular weight 982.98), theoretical value: C 55.90%, H 3.25%, N 8.36%; experimental value: 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-monochloro-iridium(III) (0.143 mg, 0.125 mmol) and ligand (0.815 g, 0.25 mmol) were placed in 24 ml DMF and stirred at 130 °C in the dark for about 4 h. After the reaction was completed, the mixture was cooled and 0.75 mmol NH4PF6 (0.12 g) was added to precipitate. About 100 ml of water was added, filtered, and the residue was dried to obtain a crude product. The crude product was separated by silica gel column chromatography using dichloromethane:methanol (volume ratio 120:1) as the eluent. The complex 3 was obtained by drying with a yield of 58.1%.

[0062] Elemental Analysis C 43 H 27 F 10 IrN5PO (molecular weight 1042.89), theoretical value: C 57.52, H 3.03%, N7.80%; experimental value: 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 immunogenic death induction test of Ir(III) complexes on ovarian cancer cells

[0064] Anti-tumor activity test (MTS method): The toxicity of iridium complexes to ovarian cancer was determined by the MTS method: The MTS method is one of the classic methods for detecting drug toxicity. The experimental steps are as follows: digest the logarithmic phase cells with trypsin to form a cell suspension, count the cells, and inoculate 4,500 cells per well, a total of 160 µL of suspension, inoculate in a 96-well plate, and fill the remaining empty wells at the edge with PBS. After culturing in a CO2 constant temperature incubator overnight, 40 µL of the complex containing gradient concentrations of culture medium was added to each well, followed by incubation in the incubator for 48 h, and then 20 µL / well of MTS reagent was added. Continue incubation for 3 hours, and detect the OD value on the machine. Calculate the cell survival rate according to the formula to obtain the median lethal concentration (IC 50 value).

[0065] Survival rate % = average OD value of drug-treated wells / average OD value of control wells × 100%

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

[0067] Table 1 IC of iridium complexes 50 value

[0068]

[0069] a IC50 The concentration of the complex corresponding to the inhibition of tumor Hela by 50%. The experimental data are the average values ​​obtained after three parallel experiments.

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

[0071] Determination of extracellular ATP release: SKOV-3 cells were digested with trypsin and the suspension was collected. The cell density was adjusted to 1.5×10 5 / dish, inoculated in a 6-well plate, the volume of cell suspension in each well was 2 mL, incubated for 24 h, and then complex 1 with a concentration of 10 µM, 20 µM, and 40 µM was added. The cells were incubated for 24 h, and the cell supernatant was separated. The cells were centrifuged at 1000 rpm / min for 5 min, and the supernatant was aspirated into a new centrifuge tube. According to the steps in the kit instructions, each reagent was dissolved on ice, and the ATP standard was diluted to 0.01 µM. The gradient concentrations were 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, and 10 µM, respectively. 100 µL of ATP detection solution was added to the 96-well detection well and allowed to 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 most important energy substance in the body. A large number of studies have shown that late ICD cells enhance the body's immunity through ATP activation, recruitment of APCs and inflammatory signaling pathways. The test results showed that as the induction concentration increased, the release level of ATP also increased, which was significantly different from the Control group.

[0072] The results are as follows Figure 1 shown.

[0073] Detection of extracellular release of HMGB-1 protein: After washing with PBS, SKOV-3 cells were treated with trypsin and the cell suspension was collected. The suspension density was adjusted to 1.5×10 5, inoculated in a 6-well plate, with a cell suspension volume of 2 mL in each well, and cultured in an incubator for 24 h. When the cell density reached about 70%, complex 1 was added at concentrations of 10 µM, 20 µM, and 40 µM. The cells were incubated for 24 h, the supernatant was taken, centrifuged at 1000 rpm for 5 min, the supernatant was recovered, and placed back into a new centrifuge tube. The cells were processed according to the ELISA kit method, and the absorbance at 450 nm was finally measured using an enzyme marker. HMGB-1 is a nuclear protein that is ubiquitous in many 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 delivers the tumor antigens they carry to T cells. The results are shown in Figure 2 As shown by Figure 2 It can be seen that after the cells were co-incubated with complex 1, the amount of extracellular HMGB-1 released increased significantly.

[0074] The results of the HMGB-1 protein experiment corresponded to the results of the ATP detection, which together showed that complex 1 could induce immunogenic death of SKOV-3 cells and had the potential for anti-ovarian cancer immunotherapy.

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

[0076] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

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

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

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

4. The method for preparing the iridium complex according to any one of claims 2 to 3, comprising the following steps: The iridium compound and N-(1,10-phenanthroline-5-yl)cinnamamide are reacted 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-phenanthroline-5-yl)cinnamic acid amide is prepared according to the following method: Carrying out a first reaction between cinnamic acid and 5-amino-1,10-phenanthroline in a first organic solvent, and then adding a second solvent to carry out a second reaction to obtain N-(1,10-phenanthroline-5-yl)cinnamic amide; 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 reaction temperature is 120-140°C and the reaction time is 4-6 hours; The organic solvent is DMF; The reaction was carried out under light protection.

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

9. Use 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 anti-tumor immunotherapy drugs.

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

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