An Ir(III) complex with anti-tumor immune activity, its preparation method and application

Abstract

The present invention provides an Ir(III) complex with anti-tumor immune activity, having the structure shown in Formula I. The Ir(III) complex provided by the present invention has a certain selectivity for normal cells, can target and kill HepG2 cells through the paraptosis pathway, and can simultaneously cause immunogenic death of tumor cells. The test results show that the Ir(III) complex can induce ICD in human-derived HepG2 and mouse-derived Hepa1-6 cells and can be applied to the field of anti-tumor immunity.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor and antibacterial drugs, and particularly relates to an Ir(III) complex with anti-tumor immune activity, a preparation method thereof, and applications thereof. Background Art

[0002] Tumor immunotherapy refers to the induction and stimulation of the human immune system, thereby enhancing the ability of anti-tumor immune response and fighting tumors through the body's own immune function. Unlike traditional treatment methods, its target is the body's own immune system, not cancer tissues and cells. Immunotherapy is mainly divided into the following categories: The first category is to use the body's own immune cells to fight tumors, such as adoptive T cell therapy (ACT), that is, to genetically engineer T lymphocytes in the body in vitro, screen out specific anti-tumor T cells, and then amplify them in large quantities and re-transfer them back into the body to enhance the immune cells' attack on tumor cells, such as chimeric antigen receptor T cell immunotherapy (CAR-T). However, this method also has its limitations: First, CAR-T cells can recognize and act on normal cells expressing target antigens even at low levels. When the infused CAR-T cells are activated by normal cells expressing low levels of target antigens in the heart, liver or lungs, this off-target tumor toxicity may lead to death. Second, even with specific tumor targeting, serious side effects may occur after large-dose CAR-T cell infusion. What is more noteworthy is that due to the characteristics of tumors promoting connective tissue hyperplasia and the immunosuppressive tumor microenvironment, CAR-T cells usually have low infiltration efficiency in tumors, so that CAR-T cell therapy is not ideal for the treatment of solid tumors. The second category is to relieve the tolerance or shielding effect of tumor cells on immune cells to restore the immune system's recognition, killing and clearance functions of tumor cells, that is, immune surveillance function, which is represented by immune checkpoint therapy (ICB). This method can significantly prolong the survival of patients with various solid tumors, but it is ineffective for tumors with poor immunogenicity. It is well known that advanced tumors can escape immune surveillance by dysregulating signal pathways, hijacking immunosuppressive cells, and exhausting effector cells and cytokines to avoid being eliminated by immune cells. Cycle disorders caused by inhibitory receptor signal transduction often lead to immune escape. In addition, there is tumor vaccine therapy, which mainly introduces tumor antigens into patients to enhance the immune system's recognition function of tumors, stimulate specific cellular immunity, and improve the tumor microenvironment. It mainly includes: ① whole cell vaccine (WTCV), which can be divided into tumor cell vaccine and dendritic cell (DC) vaccine according to the cell source; ② polypeptide vaccine; ③ genetic engineering vaccine; ④ antibody tumor vaccine, etc. Therapeutic WTCV has the advantages of carrying relatively complete tumor antigens and not being restricted by the histocompatibility complex. In the case where tumor-specific antigens have not yet been identified, tumor cell vaccines have their unique advantages. However, compared with the first two types of immunotherapy, tumor vaccine therapy has developed relatively slowly, and there are fewer drugs on the market.

[0003] It can induce tumor cell death, activate the body's immune system to fight against dead cell antigens, and endow the dead tumor cells with immunogenicity. This process is called immunogenic cell death (ICD). Chemotherapeutic drugs can promote the expression and release of immunogenic substances in tumor cells by causing a series of cellular reactions, enhancing their immunogenicity. Whether a metal complex has the property of inducing ICD in tumors can be judged by some molecular markers. Among them, the occurrence of endoplasmic reticulum stress is one of the important markers for inducing ICD. The gold standard for judging whether a molecule / drug can induce ICD in tumor cells is an in vivo vaccination experiment, that is, in the absence of adjuvants, tumor cells treated with an ICD inducer are injected into immunocompetent mice, which can cause an antigen-adaptive immune response in the mice and can protect the mice from the attack of the same-type tumor.

[0004] So far, only a few metal complexes can induce ICD, and only one iridium complex has been found to have anti-tumor activity while also being able to induce ICD. The mortality and morbidity caused by malignant tumors are increasing year by year, seriously threatening human survival. Therefore, designing and synthesizing novel iridium complexes with both anti-cancer and immune activities undoubtedly provides a feasible strategy for future cancer treatment. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an Ir(III) complex with anti-tumor immune activity, its preparation method and application, which can induce ICD while having anti-tumor activity.

[0006] The present invention provides an Ir(III) complex with anti-tumor immune activity, having the structure shown in Formula I:

[0007]

[0008] Optionally, the anion of the Ir(III) complex is hexafluorophosphate.

[0009] The Ir(III) complex provided by the present invention can be denoted as [Ir(C-N)2L]PF6, where the ligand L = 2-(6-quinoxalinyl)-1H-imidazo[4,5-f][1,10]phenanthroline, and C-N = 1-phenylisoquinoline (piq).

[0010] The present invention conducts research on the anti-tumor immune activity of the Ir(III) complex and finds that Ir1 has excellent anti-tumor immune activity. The IC 50 of Ir1 against human hepatocellular carcinoma cells (HepG2) is 4.2 ± 0.4 μM, and the IC 50 against normal cells LO2 is 24.4 ± 0.2 μM, and the IC against murine hepatocellular carcinoma cells (Hepa1-6)50 = 10.5 ± 0.1 μM. It can be seen that this complex can selectively kill tumors, and mechanism studies have shown that this complex mainly exerts its effect through the paraptosis pathway. It is worth noting that the complex also has very good immune induction activity. Cell experiments and animal experiments have proved that while inducing apoptosis in HepG2 and Hepa1-6 cells, the complex can further induce the ICD effect, promote the maturation of DC cells, weaken the immunosuppression in tumor tissues by downregulating the number of regulatory Treg cells in tissues, thereby activating the body's tumor immunity. Eventually, mice inoculated with tumor immune cells can be spared from the invasion of liver cancer tumor tissues. Thus, Ir1 has anti-tumor immune activity.

[0011] The present invention provides a preparation method of the above-mentioned Ir(III) complex, comprising the following steps:

[0012] S1. Preparing the ligand: Dissolve 1,10-phenanthroline-5,6-dione and ammonium acetate in an organic solvent, then add quinoxaline-6-carboxaldehyde, and reflux the reaction. After the reaction solution is cooled, add ammonia water to adjust the pH value of the solution, filter, and wash to obtain the ligand;

[0013] S2. Preparing the complex: React bridged bis(1-phenylisoquinoline)-iridium(III) chloride and the ligand prepared in S1 in an organic solvent to obtain the Ir(III) complex shown in Formula I.

[0014] The reaction equation is as follows:

[0015]

[0016] Optionally, the organic solvent in step S1 is glacial acetic acid.

[0017] Optionally, the temperature of the reflux reaction is 90 - 110 °C, more preferably 100 °C.

[0018] Optionally, the reflux time is 80 - 120 min, more preferably 90 min.

[0019] Optionally, in step S1, the pH value of the solution is adjusted to neutral.

[0020] Optionally, the organic solvent in step S2 is a mixed solvent of dichloromethane and methanol. The volume ratio of dichloromethane to methanol is preferably 1 - 4:1, more preferably 2:1.

[0021] Optionally, after the reaction in step S2 is completed, an excess of saturated NH4PF6 solution is added, and the precipitated solid is the iridium(III) complex.

[0022] The iridium(III) complex provided by the present invention can be used to prepare drugs with anti-tumor immune activity.

[0023] Based on this, the present invention provides the application of the above-mentioned Ir(III) complex in the preparation of anti-tumor drugs.

[0024] Optionally, the tumor is a solid tumor.

[0025] Optionally, the anti-tumor is to inhibit the proliferation of human HepG2 liver cancer cells and / or murine Hepa1-6 liver cancer cells.

[0026] The present invention provides the application of the above-mentioned Ir(III) complex in the preparation of drugs for inhibiting the proliferation of human HepG2 liver cancer cells and / or murine Hepa1-6 liver cancer cells.

[0027] More preferably, the mice used in the experiment are male C57BL / 6 mice.

[0028] The iridium(III) complex can simultaneously induce immunogenic cell death (ICD) in HepG2 and Hepa1-6 tumor cells, and can endow male C57BL / 6 mice inoculated with Hepa1-6 cells treated with Ir1 with immune ability against Hepa1-6 tumors.

[0029] Based on this, the present invention provides a tumor vaccine obtained by extracting tumor cells treated with the above-mentioned Ir(III) complex.

[0030] Optionally, the tumor cells are HepG2 cells or Hepa1-6 cells.

[0031] The present invention provides an Ir(III) complex with anti-tumor immune activity, which has the structure shown in Formula I.

[0032] Compared with the prior art, the present invention has the following excellent effects:

[0033] (1) The compound with anti-tumor immune activity described in the present invention is an iridium(III) complex containing a phenanthroline derivative as a ligand, which contains metal ions and is charged itself. Compared with traditional organic small molecules, it enhances the in vivo penetration and retention effects, and the metal complex has a multi-coordination configuration, which can be modified with different ligands to achieve its excellent biological activity.

[0034] (2) Experimental results show that the iridium(III) complex provided by the present invention not only has excellent anti-tumor activity, but also can induce ICD death in tumor cells. Further in-depth study on the anti-tumor mechanism of complex Ir1: Complex Ir1 mainly enters cells in an energy-dependent manner and localizes in lysosomes, has no effect on the cell cycle, and mainly induces tumor cell death through paraptosis. At the same time, it is found that Ir1 can induce endoplasmic reticulum stress response in HepG2 cells, further induce ICD effect, promote the maturation of DC, improve the chemotaxis of effector T cells in tumor tissues, reduce immune suppression in tumor tissues by down-regulating the number of regulatory Treg cells in tumor tissues, activate the body's tumor immunity, and finally enable the mice inoculated with tumor vaccines to be immune from the invasion of liver cancer tumor tissues. Brief Description of the Drawings

[0035] Figure 1 It is the effect diagram of the influence of complex Ir1 in Example 2 on the expression of endoplasmic reticulum stress-related proteins in HepG2 cells;

[0036] Figure 2 It is the effect diagram of the influence of complex Ir1 in Example 2 on the expression of endoplasmic reticulum stress-related proteins in Hepa1-6 cells;

[0037] Figure 3 It is the immune effect diagram of transplanted tumors after inoculating Hepa1-6 cells treated with complex Ir1 in male C57BL / 6 mice in Example 2. Detailed Embodiments

[0038] To further illustrate the present invention, the Ir(III) complex with anti-tumor immune activity provided by the present invention, its preparation method and application will be described in detail below in conjunction with embodiments.

[0039] Example 1

[0040] (1) Preparation of ligand L:

[0041] Dissolve 0.53 g of 1,10-phenanthroline-5,6-dione (2.5 mmol) and 3 g of ammonium acetate (39 mmol) in 80 mL of glacial acetic acid by stirring at 60 °C, then add 0.4 g of quinoxaline-6-carbaldehyde (2.5 mmol), adjust the temperature to 100 °C and reflux for 90 minutes. At this time, the color of the solution changes from yellow to orange-red. After the reaction is completed, cool the reaction solution to room temperature, dropwise add ammonia water to adjust the pH of the solution to neutral, a large amount of yellow precipitate will precipitate. Filter, wash several times with pure water and anhydrous ethanol respectively, and dry to obtain 0.60 g (yield: 69.0%) of yellow compound, which is ligand L1.

[0042] Elemental analysis C 21 H 12N6 (molecular weight 348.11), theoretical values: C 72.40%, H 3.47%, N 24.12%; experimental values: C 72.25%, H 3.43%, N 24.10%. ESI-MS: [(M+H)] + Theoretical value: m / z = 349.37, experimental value: m / z = 349.0.

[0043] (2) Preparation of complex Ir1

[0044] 0.32 g of [Ir(piq)2Cl]2 (0.25 mmol) and 0.18 g of ligand L1 (0.50 mmol) were placed in a three-necked flask, 80 mL of dichloromethane:methanol mixed solution (2:1, v / v) was added, and the mixture was refluxed for 4 h at 45 °C under argon protection in the dark. After cooling to room temperature, 0.22 g of NH4PF6 (1.50 mmol) was added, and the mixture was stirred at room temperature in the dark for 1 h. The reaction mixture was filtered, and the solvent was removed by a rotary evaporator to obtain a crude solid product. The crude product was dissolved in a small amount of dichloromethane, and then silica gel column chromatography was carried out using dichloromethane:methanol (60:1, v / v) as the eluent to obtain 0.16 g of orange-yellow Ir1 (yield: 29.3%).

[0045] Elemental analysis C 51 H 32 F6IrN8P (molecular weight 1094.03), theoretical values: C 55.99%, H 2.95%, N 10.24%; experimental values: C 56.10%, H 2.31%, N 10.10%. ESI-MS: [(M–PF6)] + Theoretical value: m / z = 951.82, experimental value: m / z = 951.7; [(M–PF6)] + Theoretical value: m / z = 949.07, experimental value: m / z = 949.0.

[0046] Example 2 In vitro anti-tumor activity test of Ir(III) complexes

[0047] Anti-tumor activity test (MTT method): The anti-tumor ability of iridium complexes was mainly determined by the MTT method: The MTT method is a classic method for determining the anti-tumor activity of drugs. The operation steps are roughly as follows: 4,000 HepG2 or Hepa1-6 cells were added to a 96-well culture plate. After culturing in a CO2 incubator for 24 h, different concentrations of drugs diluted with medium were added respectively. Then, after incubating in the incubator for 44 h, MTT was added. After 4 h, the medium was removed, and DMSO was added. After shaking well, the OD value at 595 nm was measured.

[0048] The test results are shown in Table 1 below:

[0049] Table 1

[0050]

[0051] a IC 50 It is the concentration of the complex corresponding to 50% cell inhibition. The experimental data are the averages obtained after three parallel experiments.

[0052] It can be seen from the experimental results that the complex Ir1 not only has excellent anti-tumor activity, with an IC 50 of only 4.2 ± 0.4 μM, but also has a certain selectivity.

[0053] Western Blot analysis: HepG2 and Hepa1-6 cells were cultured in 60-mm tissue culture dishes. When the cell density reached 70%, the complex Ir1 at a specified concentration was added. After incubation for 24 h, protein samples were collected. The prepared protein samples were denatured at 95 °C, and then subjected to SDS-PAGE gel electrophoresis to separate the required proteins, followed by membrane transfer, blocking, incubation with primary antibodies (the primary antibodies were human and mouse Alix, eIF2α, p-eIF2α, and Chop respectively), incubation with secondary antibodies, and protein detection. Endoplasmic reticulum stress is an important indicator of ICD in cells. For example, Figure 1 and Figure 2 in HepG2 and Hepa1-6 cells treated with different concentrations of Ir1, two endoplasmic reticulum stress-related proteins: C / EBP homologous protein (CHOP) and eukaryotic initiation factor 2α (eIF2α) were detected. Among them, Chop was upregulated and phosphorylation of eIF2α (p-eIF2α) occurred, which are typical phenomena of endoplasmic reticulum stress. At the same time, there was an obvious downward trend in the exosome protein Alix that helps cancer cells escape the immune system. This indicates that the complex Ir1 can simultaneously induce ICD in human and mouse liver cancer cells and enhance the immune ability of the human or mouse body against tumors.

[0054] Vaccine experiment to determine the anti-tumor immune ability of mice:

[0055] 1) Mouse tumorigenesis experiment: Ten healthy 4-week-old C57BL / 6 mice were selected. The hair on the left groin to the midpoint of the dorsal thigh of the mice was removed and disinfected, and 1 × 10 7 mouse tumor cells in the logarithmic growth phase were injected with a syringe. After 20 - 30 days, the tumor grew into a tumor with a major axis of about 1.5 cm, and the tumor was removed and cut into small tumor pieces with a major axis of 0.5 cm.

[0056] 2) Preparation of tumor cell vaccine: Tumor cells in the logarithmic growth phase were washed with PBS and then digested with trypsin to collect the cell suspension, which was inoculated into cell culture dishes. The cell concentration was adjusted to 1×10 6 / dish and cultured overnight in an incubator; Different drug treatments were given according to different groups: CDDP (cisplatin) group: The cells were incubated with CDDP solution for 24 hours; Drug group: The cells were incubated with drug solution for 24 hours. After the drug treatment, the supernatant was discarded, and the cells were washed with PBS. After digestion with trypsin containing EDTA, the cell suspension was collected into a centrifuge tube; After centrifugation, the supernatant was discarded, and the cells were washed with PBS. Then, PBS was added to each tube to resuspend the cells, which was the tumor cell vaccine.

[0057] 3) Mouse tumor vaccine injection and transplanted tumor experiment: Thirty healthy 4-week-old C57BL / 6 male mice were selected and divided into three groups (drug group, cisplatin group, control group). They were raised under the conditions of constant temperature (22±1)°C, with light from 7:00 to 19:00 every day, and sufficient water and food. The hair on the left groin to the midpoint of the dorsal thigh of the mice was removed and disinfected, and then injected subcutaneously in this area of the mice with a syringe: Control group: Only 100 μL of PBS was injected; CDDP group: The cell suspension treated with CDDP was injected; Drug group: The cell suspension treated with the drug was injected. This treatment time was recorded as -7 days of the experiment; Seven days later, the hair on the right groin to the midpoint of the dorsal thigh of the 30 mice was removed and disinfected, and the tumor pieces from the previous step were transplanted into the mice and sutured. The status of the mice was observed every day, and the body weight, long diameter, and short diameter changes of the tumors of the mice were recorded. The tumor size calculation formula was V = 0.5 * L * W 2 (W is the tumor width and L is the tumor length).

[0058] Figure 3 It is the immunization effect diagram of transplanted tumors after inoculating Hepa1-6 cells treated with complex Ir1 in male C57BL / 6 mice (A: Curve of mouse body weight change; B: Curve of mouse tumor volume change; C: Solid tumor diagrams of each group 30 days after tumor transplantation; D: In vivo situation 30 days after mouse tumor mass transplantation, back view (left) and side view (right). Note: Cisplatin was used as the experimental control drug).

[0059] As Figure 3 shown: The body weights of the mice in each group showed a slow upward trend. The tumor mass inhibited in the Ir1 group did not grow or even decreased in volume, while the tumor mass of the mice in the Control group grew rapidly along the groin side of the mice. The tumor mass of the mice in the CDDP group was slightly smaller than that of the Control group but significantly larger than that of the Ir1 group, indicating that Ir1 has anti-tumor immune activity.

[0060] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to 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 Ir(III) complex with anti-tumor immune activity, having the structure shown in Formula I: The anion of the Ir(III) complex is hexafluorophosphate.

2. A method for preparing the Ir(III) complex according to claim 1, comprising the following steps: S1. Prepare the ligand: Dissolve 1,10-phenanthroline-5,6-dione and ammonium acetate in an organic solvent, then add quinoxaline-6-carbaldehyde, and reflux the reaction. After the reaction solution is cooled, add ammonia water to adjust the pH value of the solution, filter, and wash to obtain the ligand; S2. Prepare the complex: React bridged bis(1-phenylisoquinoline)-iridium(III) chloride and the ligand obtained in S1 in an organic solvent to obtain the Ir(III) complex shown in Formula I.

3. The preparation method according to claim 2, wherein, The organic solvent in step S1 is glacial acetic acid.

4. The preparation method according to claim 2, wherein, In step S1, the pH value of the solution is adjusted to neutral.

5. The preparation method according to claim 2, wherein The organic solvent in step S2 is a mixed solvent of dichloromethane and methanol.

6. Use of the Ir(III) complex according to claim 1 in the preparation of anti-tumor drugs; The anti-tumor effect is to inhibit the proliferation of human HepG2 liver cancer cells and / or murine Hepa1-6 liver cancer cells.

7. A tumor vaccine obtained by treating tumor cells with the Ir(III) complex according to claim 1 and then extracting; The tumor cells are HepG2 cells or Hepa1-6 cells.

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