Ru (II) complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of antitumor drugs, in particular to a Ru (II) complex and a preparation method and application thereof, and the Ru (II) complex has a structure as shown in a formula I. The ruthenium (II) complex has excellent anti-tumor activity, can induce tumor cells to generate ICD death, can be quickly positioned in an endoplasmic reticulum, causes the intracellular active oxygen level to rise, blocks the cell cycle in the S phase, and finally induces the tumor cells to generate pyroptosis. More importantly, MCF-7 cells can be caused to generate endoplasmic reticulum stress, so that an ICD effect is triggered, DC in tumor tissues is increased, chemotaxis of effector T cells in the tumor tissues is improved, and meanwhile, the number of Treg cells with an immunosuppression effect can be reduced, so that tumor immunity of an organism is activated. In addition, the Ru (II) complex can activate a cGAS-STING pathway, the proportion of CD8 + T cells is increased, and the anti-tumor immunocompetence is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and in particular to a Ru(II) complex and a preparation method and application thereof. Background Art

[0002] There are four common methods for treating cancer, namely surgical resection, radiotherapy, chemotherapy and immunotherapy. Currently, chemotherapy, which is the most widely used, can improve the survival rate of patients, but the remaining tumor cells in the patient's body will increase the risk of tumor metastasis and cause the disease to recur. In recent years, cancer immunology research has made significant progress, and immunotherapy has become a research hotspot that is expected to conquer cancer. Tumor immunotherapy is essentially different from traditional anti-cancer treatment methods in that it can mobilize the body's own immune system to attack and eliminate cancer cells, thereby improving the anti-tumor effect. It has the characteristics of specificity, high efficiency, safety, effectiveness, and few side effects.

[0003] At present, there are five main methods of cancer-based immunotherapy: molecular targeted therapy, immune checkpoint inhibitors, adoptive immune cell therapy, cytokine therapy, and tumor vaccines. Molecular targeted therapy targets the marker molecules on tumor cells and achieves anti-tumor effects by inhibiting tumor cell proliferation, inhibiting metastasis, and inducing tumor cell apoptosis. It is characterized by high efficiency, low toxicity, and little effect on normal tissue cells. In 1997, the FDA approved the world's first molecular targeted drug, rituximab, for the treatment of CD20-positive malignant lymphoma. Immune checkpoint molecules refer to immune-suppressing regulatory molecules expressed on immune cells, which prevent autoimmune reactions and maintain self-tolerance. At the same time, they inhibit the function of immune cells, thereby causing tumors to form immune escape. Immune checkpoint inhibitors are some monoclonal antibody drugs developed for tumor-related immune checkpoint molecules (such as PD1 and CTLA4) in order to block the inhibitory effect of tumor cells on immune cells. The immune checkpoint inhibitors currently on the market are mainly CTLA-4 inhibitors and PD-1 inhibitors. However, some tumors have too few immune cells, which limits the effect of PD-1 / L1 inhibitors. Adoptive immune cell therapy (ACT) refers to the process of culturing and modifying the patient's own immune cells in vitro, enhancing their targeted killing function, and then returning them to the patient's body to stimulate the body's anti-tumor immune response. However, adoptive immune cell therapy has certain limitations in clinical practice. After the modified immune cells are returned to the patient's body, they survive for a short time in the body, making the treatment time insufficient. In addition, due to the large heterogeneity of solid tumors, the expression of few tumor-specific antigens, and the inhibition of immune suppression checkpoints in the tumor, the use of ACT therapy in solid tumors has great limitations. In addition, before ACT treatment, in order to prolong the survival time of adoptive immune cells, patients need to receive high-dose chemotherapy to eliminate immunosuppressive cells in the body, and high-dose chemotherapy often brings significant side effects, which greatly reduces patient compliance. In recent years, tumor vaccines have been a research hotspot in immunotherapy. It is an active immunotherapy method to introduce tumor cells or fragments containing tumor-specific antigens into the human body to activate the body's own immunity, overcome the immunosuppressive state, and improve resistance to specific tumors. The tumor vaccines currently being developed and used are divided into whole cell vaccines, tumor peptide vaccines, genetic engineering vaccines and antibody tumor vaccines. According to different uses, they can be divided into two categories: one is preventive tumor vaccines, such as HPV vaccines; the other is therapeutic tumor vaccines, which are mainly used for adjuvant therapy after traditional treatment, such as the first truly therapeutic tumor vaccine approved by the FDA, Provenge / sipuleucel-T, which is used to treat advanced prostate cancer.

[0004] Some chemotherapy drugs can stimulate tumor cell death and cause immune responses in dead tumor cells. We call this phenomenon immunogenic death (ICD). Under the promotion of certain treatment methods, ICD can induce dendritic cell maturation, activate tumor-specific T cell immune responses and eliminate residual tumor cells in the body, further improving the anti-tumor efficacy. Endoplasmic reticulum stress has been proven to be a prerequisite for inducing ICD in cells. Therefore, it is of great significance to design novel chemotherapy drugs that can cause endoplasmic reticulum stress. The gold standard for determining whether ICD can be induced in tumor cells is whether the in vivo vaccination experiment is effective. That is, in the absence of adjuvants, tumor cells treated with ICD inducers are injected into mice with immune function, which can activate the anti-tumor immune response of mice and protect them from attacks by specific tumors.

[0005] At present, there are very few metal complexes that can induce ICD, and only a few metal complexes have been reported to induce ICD in breast cancer cells and have anti-tumor immune activity. The rapid growth of malignant tumor cells has seriously affected human survival, and it is necessary to develop some new metal complexes with anti-tumor immune activity to provide a feasible reference for cancer treatment. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a Ru(II) complex and a preparation method and application thereof, wherein the Ru(II) complex can cause immunogenic death of tumor cells.

[0007] To achieve the above object, the present invention provides a Ru(II) complex having a structure shown in Formula I:

[0008]

[0009] Formula Ⅰ.

[0010] The anion of the above complex is preferably PF 6 - .

[0011] The above complex can be referred to as [Ru(NN)(CN)(L)]PF 6 , wherein ligand L = 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazine-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide, NN = bipyridine, CN = 2-(2,4-difluorophenyl)pyridine.

[0012] The above-mentioned Ru(II) complex has anti-tumor immune effect. The present invention studies the anti-tumor activity of the above-mentioned Ru(II) complex and finds that the complex Ru1 has high cytotoxicity to a variety of human tumor cells, especially has excellent anti-tumor activity to MCF-7 cells, and can specifically target the endoplasmic reticulum to induce cell pyroptosis. Importantly, further studies have found that Ru1 can induce ICD in MCF-7 cells and 4T1 cells.

[0013] When the Ru(II) complex acts on human breast cancer (MCF-7) cells, it can cause the cells to produce a large amount of reactive oxygen species and induce cell pyroptosis. Most importantly, in the verification of in vitro and in vivo experiments, it can cause CRT shift, HMGB1 migration and extracellular release of ATP in MCF-7 cells, thereby inducing immunogenic cell death. The dendritic cells and CD4 + T cells and CD8 + T cells increased significantly, and the immunosuppressive T reg In addition, the above-mentioned Ru(II) complex can activate the cGAS-STING pathway, thereby increasing the CD8 + T cell ratio, further improving the body's anti-tumor immune activity.

[0014] The present invention provides a method for preparing the above-mentioned Ru(II) complex, comprising the following steps:

[0015] In an organic solvent, 6 -C 6 H 6 )Ru(bpy)Cl]Cl, 2-(2,4-difluorobenzazole)pyridine and ligand L are reacted to obtain a Ru(II) complex shown in formula I;

[0016] The [(η 6 -C 6 H 6 )Ru(bpy)Cl]Cl has the following structure:

[0017] ;

[0018] The ligand L is 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazine-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide, and the structural formula is as follows:

[0019]

[0020] Preferably, the organic solvent is selected from N,N-dimethylformamide.

[0021] Preferably, the reaction is specifically:

[0022] Reflux reaction in N,N-dimethylformamide, after the reaction is completed, add excess saturated NH 4 PF 6 The solution is filtered and the solvent is dried by spin drying to obtain a solid which is the Ru(II) complex shown in formula I.

[0023] The equation for the above reaction is as follows:

[0024]

[0025] The present invention provides the use of the Ru(II) complex or the Ru(II) complex prepared by the above preparation method in the preparation of anti-tumor drugs.

[0026] Specifically, the present invention provides the use of the above-mentioned Ru(II) complex or the Ru(II) complex prepared by the above-mentioned preparation method in the preparation of anti-tumor immune active drugs.

[0027] Specifically, the Ru(II) complex provided by the present invention or the Ru(II) complex prepared by the above preparation method can be used to prepare type II ICD inducers.

[0028] Preferably, the tumor is a solid tumor, more preferably breast cancer.

[0029] In some specific embodiments of the present invention, the tumor is human MCF-7 cells and / or mouse 4T1 tumor cells.

[0030] The Ru(II) complex provided by the present invention can simultaneously induce immunogenic cell death (ICD) of MCF-7 and 4T1 tumor cells.

[0031] Preferably, the mice used in the experiment are female BALB / c mice. The experimental results show that the Ru(II) complex provided by the present invention can enable female BALB / c mice inoculated with 4T1 cells treated with the Ru(II) complex to acquire immunity to 4T1 tumors, specifically, it can activate the cGAS-STING pathway and thus enhance the immunity of female BALB / c mice.

[0032] Compared with the prior art, the present invention provides a Ru(II) complex having a structure shown in formula I.

[0033] The present invention has the following beneficial effects:

[0034] (1) The present invention provides a ruthenium (II) complex with a phenanthroline derivative as the main ligand, which has excellent anti-tumor immune activity. Since it contains metal ions, it has an electric charge, which enhances the in vivo penetration and retention effect compared with traditional organic small molecules. In addition, the metal complex has a multi-coordinated configuration and can be modified with different ligands to achieve its excellent biological activity.

[0035] (2) The experimental results show that the ruthenium (II) complex provided by the present invention not only has excellent anti-tumor activity, but also can induce ICD death of tumor cells. The anti-tumor mechanism of the complex Ru1 was further studied. The study found that the complex Ru1 can quickly localize in the endoplasmic reticulum, causing an increase in the level of intracellular reactive oxygen species, arresting the cell cycle in the S phase, and ultimately inducing pyroptosis of tumor cells. More importantly, Ru1 can cause endoplasmic reticulum stress in MCF-7 cells, thereby inducing the ICD effect, increasing the number of DCs in tumor tissues, improving the chemotaxis of effector T cells in tumor tissues, and at the same time reducing the immunosuppressive T cells. reg In addition, Ru1 can activate the cGAS-STING pathway and increase the number of CD8 + T cell ratio, further improving anti-tumor immune activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the effect of the complex Ru1 in Example 2 on the expression of cGAS-STING pathway-related proteins in MCF-7 cells;

[0037] Figure 2 This is an effect diagram of the complex Ru1 in Example 2 on the expression of cGAS-STING pathway-related proteins in 4T1 cells;

[0038] Figure 3 This is a diagram showing the immune effect of female BALB / c mice inoculated with 4T1 cells treated with the complex Ru1 on subcutaneously implanted tumors in Example 2. DETAILED DESCRIPTION

[0039] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than for limiting the claims of the invention.

[0040] 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.

[0041] Example 1

[0042] Preparation of complex Ru1

[0043] Take [(η 6 -C 6 H 6 )Ru(bpy)Cl]Cl (0.17 g, 0.4 mmol) and 2-(2,4-difluorophenyl)pyridine (0.076 g, 0.4 mmol) were dissolved in 20 mL of N,N-dimethylformamide (DMF). Under argon protection, heat to 140°C and reflux for 18 h in the dark. Weigh L (0.22 g, 0.4 mmol) and add it to the reaction solution. Continue the reaction for 1 h. After the reaction is completed, cool to room temperature and add an appropriate amount of deionized water. Add NH 4 PF 6 Stir at room temperature until a precipitate is formed. The mixture is filtered, and the precipitate is washed several times with water and ether. The precipitate is filtered and dried to obtain a crude product. The crude product is dissolved in a small amount of acetonitrile and separated by silica gel column chromatography using acetonitrile: water: saturated sodium nitrate solution (50:6:0.1, v / v / v) as the eluent to obtain 0.14 g of purple-red powder Ru1 with a yield of 35.0%.

[0044] Elemental Analysis C 54 H 46 CIF 8 N 8 O 2 PRu (molecular weight 1158.21), theoretical value: C, 55.99%; H, 4.00%; N, 9.67%; experimental value: C, 55.93%; H, 3.94%; N, 9.75%. ESI-MS: [(M-PF 6 )] + Theoretical value: m / z = 1013.24, experimental value: m / z = 1013.0.

[0045] Example 2

[0046] In vitro antitumor activity experiments of Ru(II) complexes

[0047] Anti-tumor activity test (MTT method): The anti-tumor ability of ruthenium complexes is mainly determined by the MTT method: The MTT method is a classic method for determining the anti-tumor activity of drugs. The operating steps are as follows:

[0048] Cells in the logarithmic growth phase were digested with 0.25% trypsin to form a single-cell suspension. 5,000 cells (MCF-7 or 4T1 cells) (100 μL) were seeded into each well of a 96-well plate and incubated in an incubator containing 5% CO. 2Incubate in a cell culture incubator for 24 h. Dilute Ru1 with culture medium, add 100 μL / well of diluted Ru1, and then incubate in an incubator for 44 h. Subsequently, add 10 μL of MTT solution (5 mg / mL) to each well and incubate in an incubator for 4 h. Then aspirate the culture medium, add 100 μL of DMSO to each well, and detect the OD value at 595 nm with an enzyme marker. Calculate the cell survival rate of different concentration groups using the following formula, and plot the half lethal concentration (IC 50 ). The cell viability of the wells without drug treatment is taken as 100% as the reference object to evaluate the cytotoxicity of the drug. This analysis method provides a reliable screening method based on the MTT classic experiment, which can be used to directly determine whether the compound has the ability to have anti-tumor activity.

[0049] Survival rate % = average OD value of drug-treated wells / average OD value of control wells × 100%

[0050] The test results are shown in Table 1 below:

[0051] Table 1 In vitro antitumor activity of Ru(II) complexes

[0052]

[0053] a IC 50 The concentration of the complex corresponding to 50% inhibition of cell proliferation. The experimental data are the average values ​​obtained after three parallel experiments.

[0054] Western blot analysis of proteins related to the cGAS-STING pathway: MCF-7 cells or 4T1 cells were inoculated in a 60 mm cell culture dish. When the cell density reached 70%, different concentrations of Ru1 were added for co-culture for 24 h, and then the cell protein samples were extracted. The extracted protein samples were denatured at 95 °C, and then SDS-PAGE gel electrophoresis was used to separate the desired target proteins, transfer to the membrane, block, incubate with primary antibodies (primary antibodies were human and mouse cGAS, Sting, p-Sting and p-NF-κB, respectively), incubate with secondary antibodies, and detect proteins.

[0055] The cGAS-STING signaling pathway plays an important role in anti-tumor immune response. When cyclic guanosine-adenylate synthase (cGAS) is activated, it catalyzes ATP and guanosine triphosphate (GTP) to form 2'-3'-cyclic GMP-AMP (cGAMP), which then binds to STING in the endoplasmic reticulum, induces STING transport and mediates the expression of type I interferon and various inflammatory factors. At the same time, STING is ubiquitinated and recruits TBK1, further activating nuclear factor kappa-B (NF-κB), which can induce the expression of other immune-related factors, thereby activating dendritic cells and increasing CD8 + The activity of T cells can improve the body's anti-tumor immunity. Figure 1 and Figure 2 As shown, after co-culture of MCF-7 cells or 4T1 cells with different concentrations of Ru1, the expression of cGAS and p-Sting was upregulated, which further led to increased expression of p-NF-κB, indicating that Ru1 can effectively activate the cGAS-STING pathway and induce anti-tumor immune response.

[0056] Vaccine experiments to determine anti-tumor immunity in mice:

[0057] (1) Preparation of tumor cell vaccine: Mouse breast cancer 4T1 cells in the logarithmic growth phase were inoculated into a cell culture dish and the cell concentration was adjusted to 1×10 6 / dish, and cultured in a cell culture incubator overnight. Different drugs were given according to different groups: 1) 4T1 cells were co-cultured with CDDP (20 μM) for 24 hours; 2) 4T1 cells were co-cultured with Ru1 (6 μM) for 24 hours. After drug treatment, the cell suspension was collected into a centrifuge tube, washed twice with PBS, centrifuged to collect the cell precipitate, and resuspended in PBS to obtain a single cell suspension, which is the tumor cell vaccine.

[0058] (2) Mouse tumor vaccine injection and tumor cell inoculation

[0059] Thirty healthy 4-week-old SPF female BALB / c mice were selected and randomly divided into three groups: control group, Ru1 group and CDDP group. The mice were kept at a constant temperature of (22 ± 1) ℃, with light from 7:00 to 19:00 every day, and sufficient water and food. The right groin to the midpoint of the dorsal thigh of the mice was disinfected, and the mice were injected subcutaneously in this area with a syringe: Control group: only 100 μL PBS was injected; CDDP group: injection of cell suspension treated with CDDP; Ru1 group: injection of cell suspension treated with Ru1. This treatment time was recorded as -7 days of the experiment; 7 days later, the right groin to the midpoint of the dorsal thigh of 30 mice was disinfected, and active 4T1 cells were injected subcutaneously in this area of ​​the mice. The status of the mice was observed every day, and the weight of the mice and the changes in the long and short diameters of the tumors were recorded. On the 16th day after tumor inoculation, three mice in each group were killed, and tumor and spleen tissues were collected for flow cytometry detection of immune-related cells. On the 30th day after tumor inoculation, the remaining mice were sacrificed and the tumor masses and major organs were removed.

[0060] The formula for calculating tumor size is V=0.5*L*W 2 (W is the tumor width and L is the tumor length).

[0061] Figure 3 In the figure, A shows the tumor mass in the mouse body on the 30th day after injection of 4T1 cells, with back view (left) and side view (right); B shows the statistical graph of the weight change of the mouse; C shows the statistical graph of the tumor volume change of the mouse; D shows the solid graph of the tumor size of the mouse on the 30th day after injection of 4T1 cells. Note: Cisplatin was used as the experimental control drug.

[0062] like Figure 3 As shown, the weight of mice in each group increased slowly. The tumor growth rate of mice in the Ru1 group was significantly inhibited, much smaller than that of the control group and CDDP group. When the tumors of mice in each group were observed 30 days after tumor inoculation, it was found that the tumor size of mice in the Ru1 group was significantly smaller than that of the control group and CDDP group, indicating that Ru1 has anti-tumor immune activity.

[0063] The above experiments show that the Ru(II) complex provided by the present invention can cause immunogenic death of tumor cells.

[0064] 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. A Ru(II) complex having the structure shown in Formula I: Formula Ⅰ.

2. The Ru(II) complex according to claim 1, characterized in that The anion of the Ru(II) complex is PF6 - .

3. The method for preparing the Ru(II) complex according to claim 1, comprising the following steps: In an organic solvent, 6 -C6H6)Ru(bpy)Cl]Cl, 2-(2,4-difluorobenzazole)pyridine and ligand L are reacted to obtain the Ru(II) complex shown in formula I; The [(η 6 -C6H6)Ru(bpy)Cl]Cl has the following structure: ； The ligand L is 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)ethoxy)-N-(1,10-phenanthroline-5-yl)acetamide.

4. The preparation method according to claim 3, characterized in that: The organic solvent is selected from N,N-dimethylformamide.

5. The preparation method according to claim 3, characterized in that: The reaction is specifically: Reflux reaction in N,N-dimethylformamide, add excess saturated NH4PF6 solution, filter and spin-dry the solvent, and the obtained solid is the Ru(II) complex shown in formula I.

6. Use of the Ru(II) complex according to any one of claims 1 to 2 or the Ru(II) complex prepared by the preparation method according to any one of claims 3 to 5 in the preparation of antitumor drugs.

7. The use according to claim 6, characterized in that: The anti-tumor drug is an anti-tumor immune active drug.

8. The use according to claim 6, characterized in that: The anti-tumor drug is a type II ICD inducer.

9. The use according to claim 6, characterized in that: The tumor is a solid tumor.

10. The use according to claim 6, characterized in that: The tumor is breast cancer.

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