Schiff base trivalent osmium complex as well as synthesis method and application thereof

Abstract

The invention relates to a Schiff base trivalent osmium complex and a synthesis method and application thereof, the chemical formula of the Schiff base trivalent osmium complex is trans-[OsIII (NHPPh3) (L) (p-Me2Npy)] (PF6), and the structural formula is as follows: # imgabs0 #. The preparation method comprises the following steps: stirring a hexavalent osmium complex [OsVI (N) (L) (OH2)] (PF6), 4-dimethylaminopyridine and triphenylphosphine (PPh3) in dichloromethane at room temperature for 1 hour, concentrating, passing through a column to obtain a brown product, and diffusing with diethyl ether to obtain the product. The reduction potential value of the Schiff base trivalent osmium complex is-0.18 V, and the Schiff base trivalent osmium complex has good solubility and stability; under the tumor redox microenvironment, in-situ valence state conversion (OsIII and OsIV) and redox reaction circulation can be achieved, the redox steady state in cells can be bidirectionally broken, hydrogen peroxide is catalyzed to generate hydroxyl free radicals, active oxygen is generated, glutathione is consumed, the killing power on tumor cells is improved by 5 times, and the toxicity on normal cells is low; the compound has a better anti-tumor effect than cis-platinum (Pt (NH3) 2Cl2), and can be used for tumor treatment, especially treatment of lung cancer, colon cancer, liver cancer, ovarian cancer and breast cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical compounds, and in particular relates to a Schiff base trivalent osmium complex and a synthesis method thereof and application in tumor treatment. Background Art

[0002] According to the 2022 China Malignant Tumor Disease Burden published by the National Cancer Center in the Journal of the National Cancer Center (JNCC), there will be 4.8247 million new cancer patients and 2.5742 million deaths in China in 2022. The global cancer burden is also increasing rapidly, and it is expected that the global cancer burden will increase by as much as 47% in 20 years, reaching 28.4 million cases in 2040. Therefore, people hope to find effective treatments to curb this deadly disease.

[0003] Redox homeostasis in cancer cells is a carefully regulated balance between the generation and elimination of reactive oxygen species (ROS), which is essential for maintaining cellular function and cancer progression. This balance is dynamic and involves a complex interplay between intracellular oxidation and reduction systems. Cancer cells have evolved to exploit certain concentrations of ROS to stimulate tumorigenesis and support their uncontrolled proliferation and invasion. However, if ROS levels exceed a critical threshold, they overwhelm cellular antioxidant defense mechanisms, leading to oxidative stress and cell death.

[0004] The concentrations of ROS and glutathione (GSH) are key indicators of cellular redox homeostasis, representing the balance between pro-oxidant species and antioxidant defenses. Cancer cells often have higher ROS levels than normal cells due to increased metabolic activity and mitochondrial dysfunction. For example, the intracellular concentration of hydrogen peroxide (H2O2) in healthy cells is usually maintained in the nanomolar to low micromolar range, however, the concentration of H2O2 in tumors can be as high as 100 μM. The oxidation potential of hydrogen peroxide is 1.77 V. GSH is a major antioxidant that is present at higher concentrations in cancer cells to offset the increased ROS production. The intracellular GSH concentration is approximately 2-10 mM, which is significantly higher than the GSH concentration in the extracellular environment (2-10 μM). Although normal cells also need GSH for antioxidant defense, they usually show lower GSH concentrations, which reflects their lower ROS production and oxidative stress levels. The reduction potential of GSH is -0.3V.

[0005] Given the redox properties of cancer cells, strategic approaches to cancer therapy involve disrupting redox homeostasis by increasing the production of ROS (especially hydroxyl radicals) and depleting GSH. This approach aims to enhance oxidative stress in cancer cells, thereby improving the efficacy of cancer therapy, while selectively inducing cancer cell death without causing significant toxicity to normal cells. If this hypothesis can be successfully implemented in clinical practice, it may confer tremendous therapeutic advantages in the field of oncology. However, it is worth noting that single molecules, especially metal complexes, that can simultaneously achieve these dual goals are scarce.

[0006] Platinum drugs are one of the most widely used clinical drugs. However, due to the indiscriminate killing effect of platinum anti-tumor drugs and the obvious drug resistance after long-term use, the search for low-toxic and high-efficiency non-platinum drugs has become an urgent problem to be solved. Osmium and ruthenium have similar properties and belong to the platinum group metals, but there is a lack of relevant research on osmium complexes.

[0007] Preliminary studies have shown that metal osmium complexes have broad application prospects as potential anti-tumor drugs. However, it is still difficult to achieve an optimal result between efficacy and toxic side effects. Therefore, how to design the structure of osmium complexes so that they can target and regulate the redox homeostasis of tumors, while exerting the best anti-tumor effect of the drug and reducing the toxic side effects on normal tissues is the most pressing problem to be solved by existing technologies. Summary of the invention

[0008] The purpose of the present invention is to provide a Schiff base trivalent osmium complex and a synthesis method and application thereof, so as to solve the problem that it is difficult to achieve an optimal result between the efficacy and toxic side effects of the current metal osmium complex.

[0009] A Schiff base trivalent osmium complex having the chemical formula trans -[Os III (NHPPh3)(L)( p -Me2Npy)](PF6), the structural formula is as follows: .

[0010] Schiff base trivalent osmium complex of the present invention trans -[Os III The cation of [(NHPPh3)(L)(p-Me2Npy)](PF6) has a distorted octahedral geometry, with the osmium center coordinated by a tetradentate salen ligand (L), a nitrogen-triphenylphosphine ligand, and a 4-dimethylaminopyridine.

[0011] The Schiff base trivalent osmium complex of the present invention has a reduction potential value of -0.18V, good solubility and stability, strong ability to kill tumor cells and low toxicity to normal cells, and can bidirectionally break the redox homeostasis in cells, catalyze hydrogen peroxide to produce hydroxyl free radicals, generate active oxygen and consume glutathione, thereby increasing the killing power of tumor cells by about 5 times.

[0012] The synthesis method of the above-mentioned Schiff base trivalent osmium complex comprises the following steps: S1. Potassium osmate dihydrate K2[OsO2(OH)4] is reacted with tetrabutylammonium chloride [Bu4N]Cl in sodium azide solution to generate hexavalent osmium salt [N n Bu4][Os Ⅵ (N)Cl4]; S2. The Schiff base H2L (salen) and the hexavalent osmium salt obtained in step S1 [N n Bu4][Os Ⅵ (N)Cl4], and then react with ammonium hexafluorophosphate to obtain the hexavalent osmium nitrogen complex [Os Ⅵ (N)(L)(OH2)](PF6); the reaction formula is as follows:

[0013] S3. The hexavalent osmium ammine complex obtained in step S2 [Os Ⅵ (N)(L)(OH2)](PF6) was dissolved in dichloromethane, 4-dimethylaminopyridine was added and reacted for ten minutes, and then triphenylphosphine was added and stirred at room temperature for 1-3 hours under alkaline conditions, concentrated and column-filtered, and ether was diffused to obtain the Schiff base trivalent osmium complex. trans -[Os III (NHPPh3)(L)( p -Me2Npy)] (PF6), the reaction formula is as follows: .

[0014] Step S3 needs to react under alkaline conditions, wherein 4-dimethylaminopyridine is alkaline.

[0015] Preferably, in step (1), the mass ratio of potassium osmate dihydrate and tetrabutylammonium chloride added is (1.5-2): 1. More preferably, the mass ratio of K2[OsO2(OH)4] to [Bu4N]Cl is 1.6:1.

[0016] Preferably, in step (2), the molar ratio of Schiff base H2L, the hexavalent osmium salt obtained in step (1), and ammonium hexafluorophosphate is 1: (1-2): (3-6). More preferably, H2L (salen), [N n Bu4][OsⅥ The molar ratio of (N)Cl4] and ammonium hexafluorophosphate added is 1:1:6.

[0017] Preferably, in step (3), the molar ratio of triphenylphosphine, the hexavalent osmium nitrogen complex obtained in step (2), and 4-dimethylaminopyridine is 1:(1-2):(6-10). More preferably, triphenylphosphine, [Os Ⅵ The molar ratio of (N)(L)(OH2)](PF6) and 4-dimethylaminopyridine added is 1:1:8.8.

[0018] Preferably, the method for synthesizing the above-mentioned Schiff base trivalent osmium complex comprises the following steps: S1. Potassium osmate dihydrate K2[OsO2(OH)4] (3 g) was dissolved in concentrated hydrochloric acid (40 ml), and sodium azide solution (1.125 g dissolved in 20 ml water) was added. After stirring for 20 minutes, tetrabutylammonium chloride [Bu4N]Cl (1.875 g) was added to obtain a purple precipitate [N n Bu4][Os Ⅵ (N)Cl4].

[0019] S2. Add H2L (salen) (322 mg, 1.01 mmol) to [N n Bu4][Os Ⅵ (N)Cl4] (588 mg, 1.01 mmol) in methanol (150 ml) was added and the mixture was stirred for 1 hour. Then ammonium hexafluorophosphate (1 g, 6.13 mmol) was added to the brown solution and stirred for another 3 hours at room temperature. The orange crystalline solid was filtered and washed with methanol (5 ml) and then with ether (3×10 ml) to obtain the osmium nitrogen complex [Os Ⅵ (N)(L)(OH2)](PF6).

[0020] S3. Add triphenylphosphine (90 mg, 0.34 mmol) to the hexavalent osmium complex [Os Ⅵ A mixture of (N)(L)(OH2)](PF6) (230 mg, 0.34 mmol) and 4-dimethylaminopyridine (366 mg, 3.00 mmol) in dichloromethane (30 ml) was stirred at room temperature for 1 hour. The brown solution was concentrated and then loaded onto a silica gel column (20×2 cm). The silica gel column was first eluted with dichloromethane and then with dichloromethane / acetone (30:1, v / v). The brown band was collected and concentrated to give a brown solid ( trans - [Os III (NHPPh3)(L)( p-Me2Npy)](PF6)).

[0021] Application of the above-mentioned Schiff base trivalent osmium complex in the preparation of anti-tumor drugs.

[0022] Preferably, the tumor includes lung cancer, colon cancer, liver cancer, ovarian cancer and breast cancer.

[0023] An antitumor preparation comprises the above-mentioned Schiff base trivalent osmium complex.

[0024] The Schiff base trivalent osmium complex of the present invention has a half-maximal inhibition concentration of 1 μM for lung cancer cells NCI-H460, and a half-maximal inhibition concentration of between 2.5 and 5 μM for normal human embryonic lung cells MRC5; it can catalyze hydrogen peroxide to produce hydroxyl free radicals, significantly increase the level of reactive oxygen species (ROS) in lung cancer cells NCI-H460, and the increase multiple is as high as 23 times; it can induce apoptosis of NCI-H460 cells and activate immunogenic cell death by disturbing the redox homeostasis in the cells, and can be used for tumor treatment.

[0025] Compared with the prior art, the present invention has the following technical advantages: (1) The present invention synthesized a Schiff base trivalent osmium complex ( trans -[Os III (NHPPh3)(L)( p -Me2Npy)](PF6)), the redox potential is -0.18V, and it has good redox properties. In the redox microenvironment of the tumor, such as in the presence of high concentrations of hydrogen peroxide (H2O2) and antioxidant glutathione (GSH), the osmium complex of the present invention can achieve in situ valence state conversion (Os III and Os IV ) and redox reaction cycles.

[0026] (2) The osmium complex of the present invention bidirectionally breaks the redox homeostasis in tumor cells by increasing oxidative stress and reducing antioxidant defense, inducing cell apoptosis and activating immunogenic cell death. Hydrogen peroxide is catalyzed to produce hydroxyl radicals, which increases the reactive oxygen species in NCI-H460 cells by about 23 times, enhances oxidative stress, induces cell apoptosis, and activates damage-associated molecular patterns, further causing immunogenic cell death. The toxicity of the osmium complex of the present invention to tumor cells NCI-H460 is about 5 times higher than that to normal cells MRC5, and selectively kills tumor cells by targeting the redox homeostasis of tumors.

[0027] (3) The osmium complex of the present invention can increase the reactive oxygen and lipid peroxides in tumor cells, and at the same time reduce the level of antioxidant glutathione, bidirectionally breaking the redox homeostasis in cells, showing a stronger anti-tumor effect than the classic metal complex cisplatin (Pt(NH3)2Cl2), showing a higher killing effect on tumor cells, but lower toxicity to normal cells. It can be used for lung cancer (NCI-H460 and A549), colon cancer (HCT116 and CT26), liver cancer (HepG2), breast cancer, ovarian cancer (A2780) and other tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The single crystal structure and structural formula of the osmium complex of the present invention; Figure 2 It is a synthetic route diagram of the osmium complex of the present invention; Figure 3 is a high-resolution mass spectrum of the osmium complex of the present invention; Figure 4 The UV-visible spectrum change of the osmium complex of the present invention in the organic solvent dimethyl sulfoxide (DMSO) for 48 hours; Figure 5 It is the UV-visible spectrum change of the osmium complex of the present invention in cell culture medium (1% DMSO-RPMI) for 24 hours; Figure 6 The EPR method is used to detect the hydroxyl radical produced by hydrogen peroxide catalyzed by the osmium complex of the present invention; Figure 7 The absorbance method is used to detect the hydroxyl free radicals produced by hydrogen peroxide catalyzed by the osmium complex of the present invention; Figure 8 The present invention compares the antiproliferative activity of the osmium complex on tumor cells and normal cells; Fig. 9 The osmium complex of the present invention has the ability to induce death in lung cancer cells NCI-H460; Fig.10 The effect of the osmium complex of the present invention on the level of active oxygen in lung cancer cells NCI-H460; Fig.11 The effect of the osmium complex of the present invention on the glutathione level in lung cancer cells NCI-H460; Fig.12 The effect of the osmium complex of the present invention on the level of lipid peroxides in lung cancer cells NCI-H460; Fig.13 The effect of the osmium complex of the present invention on apoptosis of lung cancer cells NCI-H460; Fig.14 The effect of the osmium complex of the present invention on the lung cancer cell NCI-H460 calreticulin CRT; Fig.15 The effect of the osmium complex of the present invention on adenosine triphosphate ATP in lung cancer cells NCI-H460; Fig.16 The invention relates to the influence of the osmium complex of the present invention on the high mobility group protein HMGB1 of lung cancer cells NCI-H460. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] Example 1 A method for preparing an antitumor Schiff base trivalent osmium complex comprises the following steps: S1. K2[OsO2(OH)4] reacts with [Bu4N]Cl in a sodium azide solution to generate [N n Bu4][Os Ⅵ (N)Cl4]; K2[OsO2(OH)4] (3 g) was dissolved in concentrated hydrochloric acid (40 ml), and sodium azide solution (1.125 g dissolved in 20 ml water) was added. After stirring for 20 minutes, [Bu4N]Cl (1.875 g) was added to obtain a purple precipitate [N n Bu4][Os Ⅵ (N)Cl4].

[0031] S2. First, H2L (salen) and [N obtained in step S1 n Bu4][Os Ⅵ (N)Cl4] and then react with ammonium hexafluorophosphate to obtain [Os Ⅵ (N)(L)(OH2)](PF6) H2L (salen) (322 mg, 1.01 mmol) was added to [N n Bu4][Os Ⅵ (N)Cl4] (588 mg, 1.01 mmol) in methanol (150 ml) was added and the mixture was stirred for 1 hour. Then, ammonium hexafluorophosphate (1 g, 6.13 mmol) was added to the brown solution and stirred for another 3 hours at room temperature. The orange crystalline solid was filtered and washed with methanol (5 ml) and then with ether (3×10 ml) to obtain [Os Ⅵ (N)(L)(OH2)](PF6); the chemical reaction equation is as follows Figure 2 shown.

[0032] S3. Triphenylphosphine, 4-dimethylaminopyridine and [Os Ⅵ (N)(L)(OH2)](PF6) trans -[Os III (NHPPh3)(L)( p -Me2Npy)] (PF6) Triphenylphosphine (90 mg, 0.34 mmol) was added to [Os Ⅵ A mixture of (N)(L)(OH2)](PF6) (230 mg, 0.34 mmol) and 4-dimethylaminopyridine (366 mg, 3.00 mmol) in dichloromethane (30 ml) was stirred at room temperature for 1 hour. The brown solution was concentrated and then loaded onto a silica gel column (20×2 cm). The silica gel column was first eluted with dichloromethane and then with dichloromethane / acetone (30:1, v / v). The brown band was collected and concentrated to give a brown solid ( trans -[Os III (NHPPh3)(L)( p -Me2Npy)](PF6)); the chemical reaction equation is as follows Figure 2 shown.

[0033] Performance Testing 1. Characterization of metal osmium complexes (1) Single crystal structure detection: The osmium complex obtained in Example 1 was dissolved in acetonitrile and diffused with ether to obtain a single crystal of the target osmium complex. The single crystal X-ray diffraction detection was then performed. The crystal structure is shown in FIG. Figure 1 , Osmium complex trans -[Os III The cation of [(NHPPh3)(L)(p-Me2Npy)](PF6) has a distorted octahedral geometry, with the osmium center coordinated by a tetradentate salen ligand (L), a nitrogen-triphenylphosphine ligand, and a 4-dimethylaminopyridine.

[0034] (2) The osmium complex obtained in Example 1 was characterized by high-resolution mass spectrometry, and the result was consistent with the molecular formula. Figure 3 It can be seen that the molecular weight of the osmium complex obtained in Example 1 is trans -[Os III The molecular weight of the complex is consistent with that of (NHPPh3)(L)(p-Me2Npy)](PF6). The molecular ion peak of the complex is m / z=911.3003. trans -[Os III (NHPPh3)(L)(p-Me2Npy)] + , is the target product trans -[OsIII (NHPPh3)(L)(p-Me2Npy)](PF6) loses a hexafluorophosphate: HR-ESI-MS: m / z Calcd. for C 45 H 46 F6N5O2OsP2([M-PF6] + ): 911.2998, found:911.3003. These two values ​​are very close, and the error is within the acceptable range. Therefore, it can be considered that in this mass spectrometry test, the compound trans -[Os III The molecular mass of (NHPPh3)(L)(p-Me2Npy)](PF6) was effectively confirmed.

[0035] (3) Element analysis: Theoretical value C 45 H 46 F6N5O2OsP2: C, 51.23; H, 4.39; N, 6.64; Observation C, 51.30; H, 4.40; N, 6.45. 2. Solubility and stability of osmium complexes UV-visible spectroscopy was used to detect the stability of the osmium complex obtained in Example 1. A metal osmium complex was prepared at a concentration of 25 μM. trans -[Os III (NHPPh3)(L)( p -Me2Npy)](PF6) solution and scan its UV spectrum from 200 to 800 nm.

[0036] (1) Stability in the organic solvent DMSO: The results are as follows Figure 4 As shown, after the osmium complex (25 μM) was dissolved in DMSO solution for 48 hours, the absorption peak of the osmium complex remained basically unchanged.

[0037] (2) Stability in cell culture medium (1% DMSO-RPMI): The results are as follows Figure 5 As shown, when the osmium complex (25 μM) was dissolved in 1% DMSO-RPMI solution for 24 hours, the absorption peak of the osmium complex was relatively stable.

[0038] 3. The osmium complex obtained in Example 1 catalyzes hydrogen peroxide (H2O2) to generate hydroxyl radicals (•OH) (1) Electron paramagnetic resonance method: 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as a spin trap for •OH in vitro. Different solution samples were prepared: (1) 200 μM osmium complex was dissolved in acetonitrile and reacted with hydrogen peroxide (H2O2) (100 mM) for 6 h; (2) 200 μM osmium complex was dissolved in acetonitrile for 6 h; (3) H2O2 (100 mM) was dissolved in acetonitrile for 6 h. Subsequently, the trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (100 mM) was added and quickly transferred to a quartz capillary for detection using a Bruker EMX-plus instrument. Figure 6 As shown, the characteristic peaks of hydroxyl radicals (•OH) of 1:2:2:1 can be detected when the osmium complex obtained in Example 1 reacts with hydrogen peroxide, indicating that the osmium complex obtained in Example 1 can catalyze hydrogen peroxide to generate hydroxyl radicals.

[0039] (2) The generation of •OH was then detected by absorbance method: 3,3',5,5'-tetramethylbenzidine (TMB) was used as an indicator. The experiment was divided into 5 groups: (1) TMB + Os III (Osmium complex obtained in Example 1, 200 μM) + H2O2 (100 mM), (2) TMB (5 mM) + H2O2 (100 mM), (3) TMB (5 mM) + Os III (Osmium complex obtained in Example 1, 200 μM), (4) TMB (5 mM) and (5) H2O2 (100 mM) + Os III (Osmium complex obtained in Example 1, 200 μM), the osmium complex was dissolved in acetonitrile, TMB and hydrogen peroxide were dissolved and diluted in water, and after each group reacted for 6 hours, the absorbance at the characteristic peak of 652 nm was measured by an ELISA instrument to determine the generation of •OH. Figure 7 As shown, the solution appears green only when all three substances are present, indicating that the osmium complex obtained in Example 1 reacts with hydrogen peroxide to generate hydroxyl radicals, and the oxidation indicator TMB presents a green result.

[0040] 4. Antitumor activity of metal osmium complexes The naphthol blue black (NBB) method was used to detect the in vitro antiproliferative ability of the osmium complex obtained in Example 1 on different cell lines, including lung cancer (NCI-H460 and A549), colon cancer (HCT116 and CT26), liver cancer (HepG2), breast cancer (MDA-MB-231), ovarian cancer (A2780) cells and normal human embryonic lung cells MRC5. The main steps are as follows: the osmium complex obtained in Example 1 is ultrasonically dissolved in dimethyl sulfoxide to prepare a stock solution, and cisplatin is dissolved in physiological saline. The logarithmic phase cells are inoculated and cultured for 12-24 hours, and different concentrations of osmium complexes (<1% DMSO) are incubated with the cells for 48 hours. Then, the culture medium is removed and the cells are fixed with 50 μl of 3% formaldehyde in PBS for 15 minutes. The cells are then stained overnight with a staining solution containing 0.05% NBB, 9% acetic acid solution and 0.1 M sodium acetate (50 μl / well). After removing the staining reagent, the stained cells were gently washed with deionized water and dissolved in 50 mM NaOH solution (100 μl / well). The absorbance of each well was read at 590 nm on a microplate reader. The antiproliferative activity was expressed as the half inhibitory concentration (IC) compared with the untreated control. 50 ), and IC was calculated from the concentration-effect curves by logarithmic interpolation using Origin 50 The evaluation was based on the mean of three independent experiments, each of which included three replicates at each concentration level. The half-maximal inhibitory concentration (IC) values ​​of the osmium complexes for 48 h 50 ) and the normal cell viability curves are shown in Table 1 and Figure 8 After the cells were treated with the osmium complex obtained in Example 1 for 48 h, the IC 50 The values ​​ranged from 0.3 to 3.9 μM, showing better antiproliferative activity than cisplatin, but less toxic to normal human embryonic lung cells MRC5.

[0041] Table 1. In vitro antitumor activity of osmium complexes (48h, μM)

[0042] 5. Ability of metal osmium complexes to induce cell death The live cell probe Calcein AM and the dead cell probe Propidium Iodide (PI) were used for staining and imaging. The main steps are as follows: the osmium complex obtained in Example 1 was dissolved in DMSO solution, ultrasonically dissolved, and a 10 mM stock solution was prepared for use; logarithmic phase NCI-H460 (1×10 5Cells were seeded on 12-well plates for 24 hours, and then treated with different concentrations of osmium complexes. After 24 hours, the cells were stained with a dye solution consisting of Calcein AM and PI. After co-staining at 37°C for 30 minutes, images were taken using an inverted fluorescence microscope. Fig. 9 As shown, compared with the control group, with the increase of the concentration of osmium complex, the green fluorescence of NCI-H460 cells gradually decreased and the red fluorescence increased significantly, indicating that the osmium complex not only inhibited the proliferation of tumor cells NCI-H460, but also induced cell death.

[0043] 6. Effects of metal osmium complexes on intracellular redox homeostasis (1) The intracellular reactive oxygen species (ROS) level was determined using 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. The main steps were as follows: the osmium complex obtained in Example 1 was dissolved in dimethyl sulfoxide solution, ultrasonically dissolved, and a 10 mM stock solution was prepared for use; logarithmic phase NCI-H460 (1×10 5 Cells were seeded on 12-well plates for 24 hours, and then different concentrations of osmium complexes were prepared to treat the cells. After 6 hours, the culture medium was removed and the cells were washed with buffer PBS. Subsequently, the DCFH-DA probe was diluted in serum-free medium to a final concentration of 10 μM and stained for 20 minutes at 37°C. The staining solution was removed and the cells were washed three times with FBS-free medium. The cells were collected and analyzed by flow cytometry. The test results are shown in Fig.10 Compared with the control group, the levels of reactive oxygen species increased by 2.5 times (2 μM) and 22.9 times (4 μM) after osmium complex treatment, respectively.

[0044] (2) Quantify the intracellular glutathione (GSH) level using a kit. The main steps are as follows: dissolve the osmium complex obtained in Example 1 in DMSO solution, dissolve it by ultrasonication, and prepare a 10 mM stock solution for later use; 5 ) cells were seeded on 12-well plates for 24 hours, and then different concentrations of osmium complexes were prepared to treat the cells. After 6 hours, the cells were lysed by freezing and thawing cycles, which were repeated at least three times to ensure complete cell disruption. Subsequently, the lysate was centrifuged and the resulting supernatant was harvested for GSH analysis according to the standard protocol of the glutathione assay kit. The absorbance at 412 nm was measured, and the concentration of reduced GSH in each sample was determined and normalized to the total protein content with reference to the standard curve. The test results are shown in Fig.11 Compared with the control group (92 μg / mg protein), the levels of osmium complexes were reduced to 74.1 (2 μM) and 65.3 (4 μM) μg / mg protein, respectively.

[0045] (3) Use a kit to quantify intracellular lipid peroxide (LPO). The main steps are as follows: dissolve the osmium complex obtained in Example 1 in dimethyl sulfoxide solution, dissolve it by ultrasonication, and prepare a 10 mM stock solution for use; 6 ) cells were inoculated on culture dishes for 24 hours, and then different concentrations of osmium complexes were prepared to treat the cells. After 24 hours, the cells were treated according to the standard protocol of the LPO assay kit. The concentration of LPO in each sample was determined by reference to the standard curve and normalized to the total protein content. The test results are shown in Fig.12 Compared with the control group (0.2 nmol / mg protein), lipid peroxide levels increased to 0.6 (2 μM) and 1.0 (4 μM) nmol / mg protein after osmium complex treatment, respectively.

[0046] The above experimental results collectively indicate that the osmium complex of the present invention can increase the reactive oxygen species and lipid peroxides in tumor cells and simultaneously reduce the level of the antioxidant glutathione, thus bidirectionally disrupting the redox homeostasis in cells.

[0047] 7. Flow cytometric analysis of cell apoptosis The main steps are as follows: dissolve the osmium complex obtained in Example 1 in DMSO solution, dissolve it by ultrasonication, and prepare a 10 mM stock solution for use; add logarithmic phase NCI-H460 (1×10 5 Cells were seeded on 12-well plates for 24 hours, and then treated with different concentrations of osmium complexes. After 24 hours, cells were collected and washed twice with PBS. Annexin-V FITC and PI dyes were used for staining. After incubation at room temperature for 15 minutes, the cells were analyzed by flow cytometry. Fig.13 As shown, the total proportion of apoptotic cells increased to 41.7% (Q2+Q3) after treatment with osmium complexes (4 μM) compared with the control group (6.43%, Q2+Q3).

[0048] 8. Damage-associated molecular patterns (DAMPs) of osmium complexes that induce immunogenic cell death (ICD) (1) Flow cytometric analysis of cell surface calreticulin (CRT). The main steps are as follows: the osmium complex obtained in Example 1 was dissolved in DMSO solution, dissolved by ultrasonication, and a 10 mM stock solution was prepared for use; logarithmic phase NCI-H460 (1×10 5Cells were seeded on 12-well plates for 24 hours, and then treated with different concentrations of osmium complexes. After 24 hours, the culture medium was removed, and the cells were collected and washed twice with PBS. The cells were then incubated with CRT primary antibody diluted in 3% BSA / PBS on ice for 2 hours, then washed and incubated with goat anti-rabbit IgG Alexa Fluor 488 antibody for 1 hour at room temperature. They were stained with PI for 10 minutes at room temperature, then washed with PBS and detected by flow cytometry. Fig.14 As shown, compared with the control group, with the increase of osmium complex concentration, the proportion of calreticulin CRT on the surface of NCI-H460 cells increased to 11.2% (1 μM), 19.2% (2 μM) and 21.1% (4 μM), respectively.

[0049] (2) Detection of extracellular ATP content. The main steps are as follows: dissolve the osmium complex obtained in Example 1 in DMSO solution, dissolve it by ultrasonication, and prepare a 10 mM stock solution for use; 4 ) cells were inoculated on a 96-well plate for 24 hours, and then different concentrations of osmium complexes were prepared to treat the cells. After 24 hours, the supernatant was transferred to a white-walled opaque-bottomed 96-well plate. After completion, an equal volume of ATP detection reagent (Cell Titer Luminescent Cell Viability Assay-ATP, Promega) was added to each well and vigorous shaking was used to mix. The extracellular ATP content of these cells was detected by the GloMax Navigator microplate luminescence detector. Fig.15 As shown, the extracellular ATP content of NCI-H460 cells increased by 1.3-fold (2 μM) and 1.6-fold (4 μM) compared with the control group, respectively.

[0050] (3) Detection of extracellular HMGB1 content. The release of HMGB1 was detected by ELISA kit. The main steps are as follows: dissolve the osmium complex obtained in Example 1 in DMSO solution, dissolve it by ultrasonication, and prepare a 10 mM stock solution for use; 5 Cells were seeded in 12-well plates for 24 hours, and then treated with different concentrations of osmium complexes. The supernatant was collected and centrifuged to remove cell debris. According to the instructions of the kit (Chondrex, #6010), the detection reagent was gradually added to determine the HMGB1 concentration. The absorbance was measured using an Infinite200PRO M Nano microplate reader. Fig.16 As shown, the proportion of HMGB1 outside NCI-H460 cells increased to 8 ng / mL in the high concentration osmium complex treatment group (4 μM) compared with the control group.

Claims

1. A Schiff base trivalent osmium complex, characterized in that: The structural formula is as follows: 。 2. The method for synthesizing the Schiff base trivalent osmium complex according to claim 1, characterized in that: The following steps are involved: (1) adding potassium osmate dihydrate to a sodium azide solution, and then reacting with tetrabutylammonium chloride to generate a hexavalent osmium salt; (2) reacting the Schiff base H2L with the hexavalent osmium salt obtained in step (1), and then reacting with ammonium hexafluorophosphate to obtain a hexavalent osmium nitrogen complex; (3) Add triphenylphosphine and 4-dimethylaminopyridine to the hexavalent osmium nitrogen complex obtained in step (2), stir in dichloromethane at room temperature for 1-3 hours, concentrate and pass through a column, and diffuse with ether to obtain the Schiff base trivalent osmium complex.

3. The synthesis method according to claim 2, characterized in that: In step (1), the mass ratio of potassium osmate dihydrate and tetrabutylammonium chloride added is (1.5-2):

1.

4. The synthesis method according to claim 2, characterized in that: In step (2), the molar ratio of the Schiff base H2L, the hexavalent osmium salt obtained in step (1) and ammonium hexafluorophosphate added is 1:(1-2):(3-6).

5. The synthesis method according to claim 2, characterized in that: In step (3), the molar ratio of triphenylphosphine, the hexavalent osmium nitrogen complex obtained in step (2), and 4-dimethylaminopyridine added is 1:(1-2):(6-10).

6. Use of the Schiff base trivalent osmium complex according to claim 1 in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that: Such tumors include lung cancer, colon cancer, liver cancer, ovarian cancer and breast cancer.

8. An anti-tumor preparation, characterized in that: The invention comprises the Schiff base trivalent osmium complex according to claim 1.

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