Metal complex with antitumor immune activity, preparation method and application thereof

Abstract

This invention discloses a metal complex with antitumor immunomodulatory activity, and also discloses a method for preparing the aforementioned metal complex and its application in the preparation of antitumor drugs or antitumor cell metastasis drugs. The metal complex of this invention is obtained by chemically modifying the hydroxyl groups of the natural products emodin and 4-methylumbelliferone, followed by a copper-catalyzed reaction with cycloiridium or cyclorhodium azides. This metal complex solves the problem of high dosage when using natural products such as emodin as antitumor drugs, and significantly improves the lipophilicity of the metal complex. This type of metal complex can effectively polarize pro-tumor M2 macrophages to a tumor-suppressive M1 phenotype or inhibit the expression of the tumor cell surface signal CD47 by inducing immunogenic death, thereby effectively killing cancer cells by promoting macrophage phagocytosis and exhibiting good antitumor immunomodulatory activity.

Description

Technical Field

[0001] This invention relates to a metal complex with antitumor immunomodulatory activity, and also to a method for preparing the aforementioned metal complex and its application in the preparation of antitumor drugs or antitumor cell metastasis drugs. Technical Background

[0002] In the tumor environment, various factors promote the differentiation of tumor-associated macrophages into the M2 phenotype. Dominant M2 macrophages secrete pro-angiogenic and immunosuppressive cytokines within the tumor microenvironment to promote tumor growth and metastasis. Conversely, M1-polarized macrophages produce a large number of pro-inflammatory cytokines and promote phagocytosis by tumor cells. Therefore, the functional shift of tumor-associated macrophages from the M2 to the M1 phenotype can be achieved to promote a dynamic transition of the tumor immune microenvironment from an immunosuppressive to an anti-tumor state, thereby inhibiting tumor growth and metastasis.

[0003] Metal-based drugs have attracted widespread attention due to their unique pharmacological properties and mechanisms of action. Their multifunctional structures, redox activities, and metal-ligand interactions provide a vast space for the design of anticancer candidates. The discovery and clinical application of cisplatin marked the beginning of metal-based drugs in cancer treatment and greatly promoted research on the antitumor activity of metal compounds and their derivatives. Besides platinum-based drugs, other researched and developed alternatives to metal-based anticancer drugs include those based on ruthenium, gold, copper, iridium, and osmium. These metal-based drugs are effective against various cancers, including those resistant to traditional chemotherapy drugs. However, current metal complexes suffer from drawbacks such as low water solubility, insufficient tumor targeting, and significant toxic side effects. While natural products themselves possess good anticancer effects, their poor structural stability and low bioavailability in vivo, coupled with the drawbacks of high dosage and low efficacy when used alone as anticancer drugs, contribute to their limited effectiveness. Summary of the Invention

[0004] Objective of this invention: The objective of this invention is to provide a metal complex with antitumor immunomodulatory activity. This metal complex can solve the problem of high dosage when natural products such as emodin are used as antitumor drugs, and also solve the problems of low water solubility, insufficient tumor targeting, and high toxicity of existing metal complexes. This metal complex exhibits good antiproliferative activity against a range of tumor cell lines (including lung cancer and breast cancer), overcomes cisplatin tolerance and adverse side effects, and significantly improves the lipophilicity of the metal complex. Another objective of this invention is to provide a method for preparing the above-mentioned metal complex and its application in the preparation of antitumor drugs or antitumor cell metastasis drugs.

[0005] Technical solution: The metal complex with antitumor immunomodulatory activity described in this invention has the following general structural formula:

[0006]

[0007] Where R1 is M is Ir or Rh; R2 is

[0008] The preparation method of the above-mentioned metal complex includes the following steps:

[0009] (1) Dissolve emodin or 4-methylumbelliferone with K2CO3 in an organic solvent, then add bromopropyne to react, and after the reaction, rotary evaporate and column purify to obtain emodin alkynyl Ed-Alkyne or 4-methylumbelliferone alkynyl Mu-Alkyne.

[0010] The structural formula of the emodinyne is as follows:

[0011]

[0012] The structural formula of the 4-methylumbelliferyl ketyne is as follows:

[0013]

[0014] (2) Preparation of metal azide complex precursor: Under inert gas protection and dark conditions, cycloiridium or cyclorhodium dimer and 4-azidomethyl-4'-methyl-2,2'-bipyridine were dissolved in an organic solvent, stirred and refluxed overnight, and the solvent was removed by rotary evaporation; dissolved in methanol, and then ammonium hexafluorophosphate was added until a large amount of precipitate was formed; purified by column chromatography to obtain the metal azide complex precursor;

[0015] (3) Under inert gas protection and dark conditions, Ed-Alkyne or Mu-Alkyne, copper catalyst, and sodium ascorbate (which reduces divalent copper to monovalent copper) were dissolved in an organic solvent, and then a metal azide complex was added and stirred to react. The reaction was monitored by TLC until the reaction was completed, and the solvent was removed by rotary evaporation. The metal complex was obtained by silica gel column purification.

[0016] In step (1), the molar ratio of bromopropyne, emodin or 4-methylumbelliferone, and K2CO3 is 1 to 2:1:2.

[0017] In step (2), the molar ratio of the cycloiridium dimer or cyclorhodium dimer to 4-azidomethyl-4'-methyl-2,2'-bipyridine is 1:2 to 4.

[0018] In step (2), the organic solvent is a mixture of dichloromethane and methanol in a volume ratio of 10 to 1:1.

[0019] In step (3), the molar ratio of emodin yne or 4-methyl umbelliferone yne, metal azide complex precursor, copper catalyst and sodium ascorbate is 1:1:0.4-0.5:0.4-0.5.

[0020] In steps (1) and (3), the organic solvent is one of acetonitrile, N,N-dimethylformamide or dichloromethane.

[0021] The above-mentioned metal complexes are used in the preparation of antitumor drugs or antitumor cell metastasis drugs, wherein the tumor is one of human non-small cell lung cancer, breast cancer, or triple-negative breast cancer.

[0022] The metal complexes of this invention are obtained by chemically modifying the hydroxyl groups of the natural products emodin and 4-methylumbelliferone, followed by a copper-catalyzed reaction with cycloiridium or cyclorhodium azides. These metal complexes can effectively polarize pro-tumor M2 macrophages to a tumor-suppressive M1 phenotype or inhibit the expression of the tumor cell surface signaling CD47 by inducing immunogenic cell death, thereby effectively killing cancer cells through macrophage phagocytosis and exhibiting good anti-tumor immunomodulatory activity.

[0023] The reaction mechanism of the metal complex synthesis process of this invention is as follows:

[0024]

[0025] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention combines natural products with anticancer activity with metal complexes. The resulting metal complexes have good lipid solubility, which helps them penetrate cell membranes and produce good anticancer activity and inhibit cancer cell metastasis. On the one hand, the metal complexes can induce the production of intracellular reactive oxygen species, target mitochondria, and reduce mitochondrial membrane potential, thereby inducing mitochondrial dysfunction and causing autophagy and necrotizing apoptosis. On the other hand, the metal complexes can effectively induce immunogenic death of tumor cells, releasing a series of damage-associated molecular patterns (DAMPs), such as calcium... Molecules such as reticulin and HMGB1 can activate pattern recognition receptors (PRRs) on the surface of macrophages, promoting macrophage polarization from a pro-tumor M2 phenotype to a tumor suppressor M1 phenotype. This upregulates the expression of M1 macrophage markers IL1B, CD86, and IL12, and downregulates the expression of M2 macrophage marker IL10, thereby enabling the effective killing of cancer cells through macrophage phagocytosis. In addition, Ir-Mu can promote macrophage phagocytosis of tumor cells by inhibiting the expression of the tumor cell surface signal CD47. Therefore, the metal complex in this invention can induce effective anti-tumor immune activity. Attached Figure Description

[0026] Figure 1The uptake of Ir-Ed and Rh-Ed by cells and their distribution in subcellular organelles are shown in Examples 1 and 2.

[0027] Figure 2 Distribution of the Ir-Mu complex synthesized in Example 3 in subcellular organelles;

[0028] Figure 3 The diagram shows the scratch assay results of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2, which inhibit cell metastasis.

[0029] Figure 4 The Ir-Ed and Rh-Ed complexes synthesized in Examples 1 and 2 induced ICD in MDA-MB-231 cells. Figure 4 A is a flow cytometry diagram from a CRT camera; Figure 4 B is a flow cytometry diagram of HMGB1; Figure 4 C is a quantitative graph showing the changes in ATP content in the extracellular supernatant;

[0030] Figure 5 Flow cytometry and protein immunoblotting analysis of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 on the induction of polarization in RAW264.7 cells;

[0031] Figure 6 Flow cytometry image of the Ir-Mu complex synthesized in Example 3 inhibiting CD47 in MDA-MB-231 cells;

[0032] Figure 7 The results of the application of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 in promoting the phagocytosis of tumor cells by macrophages;

[0033] Figure 8 The results of the application of the Ir-Mu complex synthesized in Example 3 to promote the phagocytosis of tumor cells by macrophages;

[0034] Figure 9 The volumes of proximal (left) and distal (right) tumors in mice treated with cisplatin, Ir-Ed, and Rh-Ed are shown.

[0035] Figure 10 Flow cytometry images of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 activating anti-tumor immunity in mice. Detailed Implementation

[0036] The cycloiridium dimer (CAS: 92220-65-0) of this invention was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. The cyclorhodium dimer was synthesized using the method reported in the literature "AMetal-Based Inhibitor of Tumor Necrosis Factor-α". 4-Azidemethyl-4'-methyl-2,2'-bipyridine was prepared using the method reported in the literature "Artificial photosynthesis dendrimers integrating light-harvesting, electron delivery and hydrogen production".

[0037] Example 1

[0038] The preparation method of the metallic iridium(III) complex (Ir-Ed) of the present invention is as follows:

[0039]

[0040] (1) Emodin (270 mg, 1 mmol) and K2CO3 (276.4 mg, 2 mmol) were dissolved in 4 mL of dichloromethane, and bromopropyne (118.0 mg, 1 mmol) was added and stirred. The reaction was monitored by TLC. After reacting for 12 hours, the mixture was rotary evaporated and purified by column chromatography to obtain a yellow solid Ed-Alkyne (287 mg, yield: 93%). 1 H NMR(400MHz,Chloroform-d)δ12.32(s,1H),12.10(s,1H),7.65(d,J=1.7Hz,1H),7.44(d,J=2.6Hz,1H ),7.14–7.08(m,1H),6.81(d,J=2.6Hz,1H),4.85(d,J=2.4Hz,2H),2.63(t,J=2.4Hz,1H),2.48(s,3H).

[0041] (2) Under an inert atmosphere and in the dark, cycloiridium dimer (160.8 mg, 0.15 mmol) and 4-azidomethyl-4'-methyl-2,2'-bipyridine (67.6 mg, 0.3 mmol) were dissolved in 10 mL of a mixed solvent of dichloromethane and methanol (v dichloromethane / v methanol = 3:1). After stirring and refluxing for 12 hours, the solvent was removed by rotary evaporation. The resulting solid was dissolved in a trace amount of methanol, and then a saturated ammonium hexafluorophosphate solution was slowly added dropwise until a large amount of precipitate was formed, yielding a yellow solid Ir-N3 (198 mg, yield: 76%). 1H NMR (400MHz, DMSO-d6) δ8.81(s,2H),8.26(d,J=8.1Hz,2H),7.92(s,4H),7.82(s,1H),7.65(d,J=17. 2Hz,4H),7.53(s,1H),7.15(s,2H),7.01(s,2H),6.89(s,2H),6.18(s,2H),4.83(s,2H),2.53(s,3H).

[0042] (3) In an inert atmosphere and under dark conditions, Ed-Alkyne (21.5 mg, 0.07 mmol) and CuSO4 were added. . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL of N,N-dimethylformamide (DMF), followed by the addition of Ir-N3 (60.9 mg, 0.07 mmol) and stirring. The reaction was monitored by TLC. After 24 hours of reaction, the solvent was removed by rotary evaporation, and the product Ir-Ed (48 mg, 58%) was purified by silica gel column chromatography. 1 H NMR(400MHz,Chloroform-d)δ12.30(s,1H),8.64–8.58(m,1H),8.48(s,1H),8.22(s,1H),7.97–7.8 6(m,3H),7.81–7.74(m,3H),7.68(ddd,J=7.8,3.3,1.3Hz,2H),7.63–7.60(m,1H),7.56(dt,J=5.6,1 .2Hz,1H),7.51–7.47(m,1H),7.40(d,J=2.5Hz,1H),7.23–7.20(m,1H),7.13–6.99(m,5H),6.92(tdd ,J=7.4,2.3,1.3Hz,2H),6.80(d,J=2.6Hz,1H),5.86(s,2H),3.73(s,2H),2.60(s,3H),2.46(s,3H).

[0043] Example 2

[0044] The preparation method of the rhodium(III) complex (Rh-Ed) of the present invention is as follows:

[0045]

[0046] (1) Under an inert atmosphere and in the dark, cyclic rhodium dimer (100.0 mg, 0.11 mmol) and 4-azidomethyl-4'-methyl-2,2'-bipyridine (49.5 mg, 0.22 mmol) were dissolved in 10 mL of a mixed solvent of dichloromethane and methanol (v / v = 3:1), stirred and refluxed for 12 hours, and the solvent was removed by rotary evaporation. The resulting solid was dissolved in a trace amount of methanol, and then a saturated ammonium hexafluorophosphate solution was slowly added dropwise until a large amount of precipitate was formed, yielding a pale yellow solid Rh-N3 (146 mg, yield: 78%). 1 H NMR(500MHz,Chloroform-d)δ8.75(dd,J=7.6,1.5Hz,1H),8.63–8.53(m,4H),7.94(dd,J=7.4,1.6Hz,2H),7.79(dd,J=7.4,1.3Hz,2H),7.73(d,J=1.5Hz ,1H),7.53–7.34(m,7H),7.24(td,J=7.4,1.5Hz,2H),7.12(dd,J=7.6,1.5Hz ,1H),7.00(td,J=7.6,1.5Hz,1H),6.86(td,J=7.4,1.5Hz,2H),3.40(s,2H).

[0047] (2) In an inert atmosphere and under dark conditions, Ed-Alkyne (21.5 mg, 0.07 mmol) and CuSO4 were added. . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL of DMF, followed by the addition of Rh-N3 (54.6 mg, 0.07 mmol) and stirring. The reaction was monitored by TLC. After 24 hours of reaction, the solvent was removed by rotary evaporation, and the product Rh-Ed (42 mg, 55%) was purified by silica gel column chromatography. 1H NMR (400MHz, Chloroform-d) δ8.57(s,1H),8.45(s,1H),8.22(s,1H),7.86(ddd,J=38.1,11. 4,6.6Hz,6H),7.71(dd,J=8.0,1.9Hz,2H),7.60(d,J=1.7Hz,1H),7.51(d,J=5.2Hz,1H),7.46 (d,J=5.5Hz,1H),7.39(d,J=2.4Hz,1H),7.17–6.98(m,6H),6.95(t,J=7.5Hz,2H),6.79(d,J= 2.4Hz,1H),6.28(dd,J=7.6,4.6Hz,2H),5.83(s,2H),5.25(s,2H),2.56(s,3H),2.44(s,3H).

[0048] Example 3

[0049] The preparation method of the metallic iridium(III) complex (Ir-Mu) of the present invention is as follows:

[0050]

[0051] (1) 4-methylumbelliferone (176 mg, 1 mmol) and K2CO3 (276.4 mg, 2 mmol) were dissolved in 4 mL of dichloromethane, and bromopropyne (118.0 mg, 1 mmol) was added and stirred. After reacting for 12 hours, the mixture was rotary evaporated and purified by column chromatography to obtain a white solid Mu-Alkyne (204 mg, yield: 95%). 1 H NMR (400MHz, Chloroform-d) δ7.56–7.49(m,1H),6.94(s,2H),6.17(d,J=1.5Hz,1H),4.76(d,J=2.3Hz,2H),2.57(t,J=2.4Hz,1H),2.41(d,J=1.1Hz,3H).

[0052] (2) Under an inert atmosphere and in the dark, Mu-Alkyne (15.0 mg, 0.07 mmol) and CuSO4 were added. . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL of DMF, and then Ir-N3 (60.9 mg, 0.07 mmol) was added and stirred. The reaction was monitored by TLC. After 24 hours of reaction, the solvent was removed by rotary evaporation, and the product Ir-Mu (40 mg, 53%) was purified by silica gel column chromatography. 1H NMR(400MHz,Chloroform-d)δ8.59–8.55(m,1H),8.44(s,1H),8.19(s,1H),7.94–7.85(m,3H ),7.79–7.72(m,3H),7.69–7.64(m,2H),7.56–7.50(m,2H),7.46(dt,J=5.7,1.3Hz,1H),7.21 –7.17(m,1H),7.10–6.96(m,5H),6.90(td,J=7.1,6.6,2.8Hz,3H),6.27(ddd,J=7.7,3.9,1.2 Hz,2H),6.13(d,J=1.3Hz,1H),5.81(s,2H),5.21(s,2H),2.57(s,3H),2.39(d,J=1.2Hz,3H).

[0053] The toxicity of Ed (emodin), Ed-Alkyne, Ir-N3, Rh-N3, Ir-Ed, Rh-Ed, Mu (4-methylumbelliferone), Mu-Alkyne, and Ir-Mu to human non-small cell lung cancer cells (A549), human breast cancer cells (MCF-7), human triple-negative breast cancer cells (MDA-MB-231), and human normal breast epithelial cells (MCF-10A) was determined using the MTT assay, with cisplatin (CDDP) used as a control. MTT (thiazolyl blue) is a tetrazolium salt that can be reduced by succinate dehydrogenase in the mitochondria of living cells to produce a blue-violet product, formazan (the product is soluble in DMSO), which has an absorption peak at 490 nm. Therefore, it can be used as a reference for the toxicity of 4-methylumbelliferone. 490 nm To analyze cell proliferation.

[0054] The specific experimental steps are as follows:

[0055] (1) First, revive a tube of tumor cells, culture them in fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use them after passage twice;

[0056] (2) When the cells reach the logarithmic growth phase, seed them into 96-well plates at a density of 5000 cells / well (100 μL of culture medium per well) and then place them in an incubator (37°C, 5% CO2) for culture.

[0057] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing different concentration gradients of drugs was added to each well, and then the cells were placed in an incubator for further incubation.

[0058] (4) After incubation for 48 hours, add 20 μL of MTT (5 mg / mL) to each well and continue incubation at 37°C for another 4 hours. After removing the supernatant, add 150 μL of dimethyl sulfoxide (DMSO) to each well and detect A using an ELISA reader. 490nm Calculate the cell proliferation inhibition rate and determine the IC50. 50 Value (the drug concentration corresponding to an inhibition rate of 50%).

[0059] The toxicity of the synthesized complex Ir-Ed in Example 1 to human non-small cell lung cancer (A549) cells, human breast cancer (MCF-7) cells, human triple-negative breast cancer (MDA-MB-231) cells, and human normal breast epithelial (MCF-10A) cells:

[0060] Table 1 shows the MTT test results for compounds Ed-Alkyne, Ir-N3, Ir-Ed, and CDDP.

[0061]

[0062] The results showed that ed-alkyne itself had almost no cytotoxicity to cancer cells and normal cell lines, with an IC50 value of 1.5%. 50 The concentrations were all above 50 μM. The metal azide complex Ir-N3 exhibited significant toxicity not only to cancer cells but also to normal cell lines, thus lacking selectivity for cancer cells. In contrast, the metal complex Ir-Ed, linked to emodin, showed good selectivity for cancer cells, exhibiting the best activity against human triple-negative breast cancer cells. Compared to cisplatin's activity against triple-negative breast cancer (~29.1 μM), Ir-Ed's activity (4.3 μM) was nearly 7-fold higher.

[0063] Example 2: The toxicity of the synthesized complex Rh-Ed to human non-small cell lung cancer cells (A549), human breast cancer cells (MCF-7), human triple-negative breast cancer cells (MDA-MB-231), and human normal breast epithelial cells (MCF-10A):

[0064] Table 2 shows the MTT test results for compounds Ed-Alkyne, Rh-N3, Rh-Ed, and CDDP.

[0065]

[0066] The results showed that, compared with Rh-N3, the metal complex Rh-Ed, which is linked to emodin, exhibited enhanced cytotoxicity and good selectivity for cancer cells. Among them, the activity against human triple-negative breast cancer cells was the best. Compared with the activity of cisplatin against triple-negative breast cancer (~29.1 μM), the activity of Rh-Ed (3.9 μM) was increased by nearly 7 times.

[0067] Example 3: The toxicity of the synthesized complex Ir-Mu to human non-small cell lung cancer cells (A549), human breast cancer cells (MCF-7), human triple-negative breast cancer cells (MDA-MB-231), and human normal breast epithelial cells (MCF-10A):

[0068] Table 3 shows the MTT test results for compounds Mu-Alkyne, Ir-N3, Ir-Mu, and CDDP.

[0069]

[0070] The results showed that the toxicity of the cyclic iridium complex with Mu-Alkyne was not increased, and Ir-Mu exhibited comparable toxicity to the three cancer cell lines (IC50). 50 Compared to Ir-N3, Ir-Mu showed significantly reduced toxicity to MCF-10A in normal cells (IC50, 6–8 μM). 50 (34.9 μM), exhibiting excellent selectivity for cancer cells.

[0071] Example 4

[0072] The uptake of Ir-Ed and Rh-Ed by cells and their distribution in subcellular organelles by the complexes synthesized in Examples 1 and 2:

[0073] Methods: MDA-MB-231 cells were seeded in 10 cm culture dishes. After cell attachment, culture media containing Ir-N3, Ir-Ed, Rh-N3, and Rh-Ed were added and incubated for 8 hours. After incubation, cells were collected and centrifuged. The collected cells were fractionated to obtain mitochondrial precipitate, nuclear precipitate, and supernatant. Each organelle component was digested in a 95°C sand bath by adding concentrated nitric acid, H2O2, and concentrated HCl sequentially. The content of Ir and Rh in each component was determined by ICP-MS.

[0074] The uptake of Ir-Ed and Rh-Ed by cells and their distribution in subcellular organelles were as follows in Examples 1 and 2. Figure 1 As shown, the results indicate that since both Ir-N3 and Rh-N3 are mainly distributed in mitochondria, the cyclic metal complexes modified with emodin are also mainly distributed in mitochondria. Furthermore, the uptake of these cyclic metal complexes is significantly increased after modification with emodin (Ir-Ed uptake increased from 363.6 ng / 10⁻⁶ ng / L). 6 The cell count increased to 457.6 ng / 10. 6 Cells, Rh-Ed uptake 810.9 ng / 10 6 The cell count increased to 1546.3 ng / 10. 6The results showed that the lipid solubility of the cyclic metal complex was significantly enhanced after modification with emodin, making it more conducive to permeation of the cell membrane and selective accumulation in mitochondria (the distribution of Ir-Ed in mitochondria was 10.6 ng / 10^6). 6 The concentration of Rh-Ed in mitochondria was 146.3 ng / 10 cells. 6 The study further confirmed that combining natural products and metal ligands has a synergistic enhancement effect.

[0075] Example 5

[0076] Distribution of the synthesized complex Ir-Mu in subcellular organelles in Example 3:

[0077] Methods: MDA-MB-231 cells were seeded in confocal culture dishes. After cell adhesion, medium containing Ir-Mu was added and incubated for 8 hours. After incubation, the medium was removed, and the cells were washed with PBS. Serum-free medium containing 0.2 μM MitoDeep Red, 2 μM Lyso Red, and 2 μM ER Red were added and incubated for 20-30 minutes. The medium was removed, the cells were washed with PBS, and laser confocal microscopy was performed immediately.

[0078] The distribution of the synthesized complex Ir-Mu in subcellular organelles in Example 3 is as follows: Figure 2 As shown, the results indicate that the colocalization coefficients of the complex Ir-Mu with mitochondria, lysosomes, and endoplasmic reticulum are 0.641, 0.819, and 0.725, respectively, indicating that Ir-Mu has the highest binding degree with mitochondria and is mainly located in lysosomes.

[0079] Example 6

[0080] Application of the synthesized complexes Ir-Ed and Rh-Ed in Example 1 and Example 2 in inhibiting cancer cell metastasis:

[0081] Methods: MDA-MB-231 cells were seeded in six-well plates. After cell attachment, the cell surface was scraped with the tip of a 200 μL pipette to create cell notches, and the cells were washed three times with PBS to remove unattached cells. Cells were treated with culture medium containing Ed (5 μM), Ed-Alkyne (5 μM), Ir-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM), Rh-N3 (5 μM), and Rh-Ed (5 μM) for 12 h. The control group received cell culture medium containing 1% DMSO. Immediately after addition, cell scratches were photographed in each well at 0 h, 24 h, and 48 h (images were taken of the areas where the scratches intersect with the marker lines). The changes in scratch area before and after treatment were compared, and the scratch area was calculated using ImageJ software.

[0082] The scratch assays of the synthesized complexes Ir-Ed and Rh-Ed in Examples 1 and 2, which inhibit cell metastasis, are as follows: Figure 3 As shown in the figure. The results indicate that the healing rate of MDA-MB-231 cells treated with Ir-Ed and Rh-Ed was significantly lower than that of the control group. In the control group, the wound was nearly healed after 48 hours, indicating that Ir-Ed and Rh-Ed treatment has significant anti-cancer cell metastasis ability.

[0083] Example 7

[0084] Application of the synthesized complexes Ir-Ed and Rh-Ed in inducing ICD in MDA-MB-231 cells: Examples 1 and 2

[0085] Methods: MDA-MB-231 cells were seeded into 6-well plates. After cell attachment, culture media containing Ed (5 μM), Ed-Alkyne (5 μM), Ir-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM, 10 μM, 15 μM), Rh-N3 (5 μM), and Rh-Ed (5 μM, 10 μM, 15 μM) were added and incubated for 24 hours. After incubation, cells and supernatant were collected. After washing away the culture medium with PBS, the cells were resuspended in blocking buffer and incubated at room temperature for 30 minutes. Primary antibody was added and incubated at room temperature for 45 minutes. After washing away the primary antibody, secondary antibody with diluted fluorescent probe was added and incubated at room temperature in the dark for 45 minutes. After washing away the secondary antibody, the cells were analyzed by flow cytometry, and data were processed using FlowJov10. For cell supernatant: Add ATP detection working solution to an opaque 96-well black plate, let stand at room temperature for 3-5 minutes, then add cell supernatant and mix well. Measure the absorbance value using a chemiluminescence analyzer and calculate the ATP concentration based on the standard curve.

[0086] The ICD results of the synthesized complexes Ir-Ed and Rh-Ed in Examples 1 and 2 on inducing cellular immunogenic death are as follows: Figure 4 As shown in the figure. The results indicated that, according to flow cytometry analysis, compared with the control group, the expression levels of CRT and HMGB1 in cells treated with Ir-Ed and Rh-Ed were significantly increased. Compared with the control group, the in vitro ATP content of Ir-Ed increased by 1.3 times, and the in vitro ATP content of Rh-Ed increased by 5.9 times. This indicates that both Ir-Ed and Rh-Ed can induce CRT exposure and HMGB1 release, and reduce cellular ATP levels, thereby inducing immunogenic cell death in MDA-MB-231 cells.

[0087] Example 8

[0088] Application of the synthesized complexes Ir-Ed and Rh-Ed in Examples 1 and 2 in inducing polarization in RAW264.7 cells:

[0089] Method 1: RAW 264.7 cells were seeded into 6-well plates. After cell adhesion, IL-4 (25 ng / mL) was added for stimulation for 24 hours, followed by incubation for 24 hours in a medium containing Ed-Alkyne (5 μM), Ir-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM, 10 μM, 15 μM), Rh-N3 (5 μM), and Rh-Ed (5 μM, 10 μM, 15 μM). Cells were collected by centrifugation and washed twice with PBS. 500 μL of cell staining buffer was added, followed by PE-CD86 Antibody and APC-CD206, and incubated on ice in the dark for 15-20 minutes. The probes were removed by centrifugation. Flow cytometry was used for analysis, and data were processed using FlowJo v10.

[0090] Method 2: Western blot (WB) was used to detect changes in macrophage markers. MDA-MB-231 cells were seeded in 10cm culture dishes. When the cell density reached 70%, pre-prepared cell culture medium containing Ed-Alkyne (5μM), Ir-N3 (5μM), CDDP (5μM), Ir-Ed (5μM, 10μM), Rh-N3 (5μM), and Rh-Ed (5μM, 10μM) was added. After 24 hours of treatment, cells were collected by centrifugation, washed twice with PBS, and lysed on ice for 20-30 minutes with RIPA strong lysis buffer containing PMSF. The supernatant was then collected by centrifugation at 13400 rpm for 20 minutes at 4°C. The concentration of the supernatant was determined according to the BCA protein assay kit instructions. All protein samples were diluted to a uniform concentration with PBS. 6×SDS-PAGE protein loading buffer was added, and the mixture was heated at 100°C for 5 minutes. After cooling to room temperature, store at -80°C for later use. Prepare an appropriate concentration of PAGE gel according to the SDS-PAGE gel preparation kit instructions. Load the standard sample (20-40 μg / well) for electrophoresis and transfer. Rinse the transfer membrane with distilled water and block it in 5% skim milk powder for 2 hours. Add the diluted primary antibody and incubate overnight at 4°C. Rinse four times with washing buffer. Add the corresponding secondary antibody and incubate at room temperature for 1 hour, then rinse four times with PBST. Rinse the transfer membrane with distilled water, add developing solution for color development, and perform chemiluminescence analysis.

[0091] The complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 induce macrophage polarization as follows: Figure 5As shown in the figure, the results indicated that with increasing Ir-Ed and Rh-Ed incubation concentrations, RAW264.7 cells exhibited significant M1 phenotype polarization. After Ir-Ed treatment, the proportion of CD86, a marker of M1 expression, increased from 12.07% to 46.9%, and after Rh-Ed treatment, it increased from 66.8% to 93.8%, significantly higher than the control group (1.3%). Western blot results showed that after Ir-Ed and Rh-Ed treatment, the expression levels of macrophage M1 markers iNOS, IL1B, and TLR-2 increased, while the expression level of M2 marker Arg1 protein significantly decreased, indicating that Ir-Ed can induce macrophage polarization from M2 to M1, possessing the potential to phagocytose tumor cells and trigger immunity.

[0092] Example 9

[0093] Application of the synthesized complex Ir-Mu in inhibiting CD47 in MDA-MB-231 cells (Example 3):

[0094] Methods: MDA-MB-231 cells were seeded in confocal culture dishes. When the cell density reached 70%, they were treated with 7 μM Mu-Alkyne, Ir-N3, CDDP, and Ir-Mu (7 μM, 14 μM) for 24 h, respectively. Cells were then washed twice with PBS, fixed with 4% paraformaldehyde, and after removing the fixative, washed three times with PBS. Cells were then infiltrated with 0.2% Triton X-100 and blocked with 1.5% BSA, followed by incubation with CD47 primary antibody dilution for 1 h. The primary antibody was removed, and the cells were washed three times with PBS and incubated with Tritc-conjugated secondary antibody in the dark for 1 h. The secondary antibody was removed, and the cells were washed three times with PBS. Cell nuclei were stained with DAPI for 5 min. The stain was removed, and the cells were washed three times with PBS. Cell imaging was performed under a confocal microscope with an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0095] The results of the synthesized Ir-Mu complex in Example 3 inhibiting CD47 are as follows: Figure 6 As shown in the figure. The results indicate that, compared with the control group, the fluorescence intensity of CD47 expression on the cell surface decreased with increasing drug concentration after Ir-Mu treatment, indicating that the content of CD47 on the cell surface was reduced, suggesting that Ir-Mu can inhibit the expression of CD47 signaling in tumor cells.

[0096] Example 10

[0097] Application of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 in promoting macrophage phagocytosis of tumor cells:

[0098] Method 1: RAW 264.7 cells were polarized to the M2 phenotype with IL-4. PE anti-mouse / human CD11b antibody was co-incubated with RAW 264.7 cells for 15 minutes, and CFSE Cell Division Tracker Kit was used to co-incubate with 4T1 cells for 20 minutes. Cells were washed with PBS and collected. RAW 264.7 and 4T1 cells were mixed 1:1 and seeded into 6-well plates. Culture media containing Ed (5 μM), Ed-Alkyne (5 μM), Ir-N3 (5 μM), Rh-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM, 10 μM, 15 μM), and Rh-Ed (5 μM, 10 μM, 15 μM) were added and incubated for 12 hours. Cells were collected by centrifugation, washed with PBS, and analyzed by flow cytometry. Data were processed using FlowJo v10.

[0099] Method 2: RAW 264.7 cells were polarized to the M2 phenotype using IL-4. PE anti-mouse / human CD11b antibody was co-incubated with RAW 264.7 cells for 15 minutes, and 4T1 cells were co-incubated with a CFSE Cell Division Tracker Kit for 20 minutes. Cells were washed with PBS and collected. RAW 264.7 and 4T1 cells were mixed 1:1 and seeded into confocal microscopy dishes. Culture media containing Ed-Alkyne (5 μM), Ir-N3 (5 μM), Rh-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM), and Rh-Ed (5 μM) were added and incubated for 12 hours. Cell imaging was then performed under a confocal microscope.

[0100] The results of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 promoting the phagocytosis of tumor cells by macrophages are as follows: Figure 7 As shown in the figure. The results indicated that, compared with the control group, the green fluorescence of RAW 264.7 and the red fluorescence of 4T1 overlapped after treatment with Ir-Ed and Rh-Ed, indicating that Ir-Ed and Rh-Ed could promote the phagocytosis of tumor cells by macrophages. Flow cytometry data showed that, with increasing drug concentration, the uptake rate of cancer cells by Ir-Ed-induced macrophages increased from 8.35% to 40.1%, and the uptake rate of cancer cells by Rh-Ed-induced macrophages increased from 66.8% to 93.8%. This was positively correlated with the M1 polarization rate of macrophages induced by Ir-Ed and Rh-Ed, indicating that Ir-Ed and Rh-Ed phagocytose tumor cells by inducing macrophage M1 polarization.

[0101] Example 11

[0102] Application of the synthesized complex Ir-Mu in Example 3 to promote macrophage phagocytosis of tumor cells:

[0103] Methods: RAW 264.7 cells were polarized to the M1 phenotype using LPS. PE anti-mouse / human CD11b antibody was co-incubated with RAW 264.7 cells for 15 minutes, and CFSE Cell Division Tracker Kit was used to co-incubate with 4T1 cells for 20 minutes. Cells were washed with PBS and collected. RAW 264.7 and 4T1 cells were mixed 1:1 and seeded into 6-well plates. Culture media containing Mu-Alkyne (7 μM), Ir-N3 (7 μM), CDDP (7 μM), and Ir-Mu (7 μM, 14 μM, 21 μM) were added and incubated for 12 hours. Cells were collected by centrifugation, washed with PBS, and analyzed by flow cytometry. Data were processed using FlowJo v10.

[0104] Example 3: The synthesized complex Ir-Mu promoted the phagocytosis of tumor cells by macrophages. Figure 8 As shown in the figure. The results indicated that after Ir-Mu treatment, the phagocytosis rate of tumor cells by macrophages increased from 11.9% to 45%, which was significantly higher than that of the control group (1.86%), indicating that Ir-Mu can effectively promote the phagocytosis of tumor cells by macrophages by inhibiting CD47 expression.

[0105] Example 12

[0106] Application of the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 in activating anti-tumor immunity in mice:

[0107] Methods: BALB / c female mice (4-6 weeks old) were randomly divided into 4 groups of 5 mice each. 4T1 cells (2×10⁻⁶) were subcutaneously injected into the left back. 6 Four days later, 4T1 cells were injected into the right back to establish a distal tumor (1×10⁻⁶ cells). 6 (1). Subsequently, on days 1, 3, and 5, 100 μL (5 mg / Kg) of different drugs were injected into the tumor as follows: (1) control group (injected with physiological saline); (2) CDDP group; (3) Ir-Ed group; (4) Rh-Ed group. After 21 days, the mice were dissected, and one mouse tumor from each group was randomly selected and immersed in 2 mL of cell digestion solution for 1-2 hours at 37°C. Cells were collected using a 70 μM cell sieve, centrifuged to collect cells, and washed with PBS. Cell staining buffer was added and incubated for 20 minutes, and CD8+ was added respectively. Cells were incubated with the following formulations for 30 minutes in the dark: Plus 647 Anti-Mouse / CD3FITC Anti-Mouse, CD4 APC Anti-Mouse / CD3FITC Anti-Mouse, Foxp3 PE Anti-Mouse / CD4 APC Anti-Mouse, CD80 FITC Anti-Mouse / CD86 CoraLiteR Plus 647 Anti-Mouse, CD86 CoraLiteR Plus 647 Anti-Mouse / CD206 Anti-Mouse, F4 / 80 Anti-Mouse / CD86 CoraLiteR Plus 647 Anti-Mouse. Cells were collected, washed with PBS, and analyzed by flow cytometry. Data were processed using FlowJo v10.

[0108] The results of activating anti-tumor immunity in mice by the complexes Ir-Ed and Rh-Ed synthesized in Examples 1 and 2 are as follows. Figure 9 and Figure 10 As shown, in mice injected with Ir-Ed and Rh-Ed, the proximal tumor changes were comparable to those in the cisplatin group, but the distal tumor volume was significantly smaller than that in the CDDP group. Dendritic cell (DC) maturation within the tumor tissue was improved, with the CD80 / CD86 percentage significantly increasing from 15.1% (control group) to 19.5% (Ir-Ed group) and 25.5% (Rh-Ed group). Tumor-infiltrating helper T cells (CD4) and cytotoxic T cells (CD8) were quantified to assess adaptive immune responses. The proportions of Ir-Ed and Rh-Ed-mediated CD4 and CD8 T cells were approximately 20.3% / 13.3% and 21.7% / 17.9%, respectively, both higher than in the control and CDDP groups. Ir-Ed and Rh-Ed improved DC maturation and simultaneously initiated adaptive anti-tumor immunity within the tumor. In addition, the percentages of CD86, a marker for M1 macrophages, increased from 4.36% to 10.6%, and CD206, a marker for M2 macrophages, increased from 4.49% to 2.02%.

Claims

1. A metal complex having antitumor immunological activity, characterized by comprising a metal atom, a ligand having a nitrogen atom, and a ligand having a sulfur atom. Its structural formula is: ;or ;or .

2. The method for preparing the metal complex according to claim 1, characterized in that, Includes the following steps: (1) Dissolve emodin or 4-methylumbelliferone with a catalyst in an organic solvent, then add bromopropyne to react, and after the reaction, rotary evaporate and purify by column to obtain emodin acetylin or 4-methylumbelliferone acetylin. The structural formula of the emodinyne is as follows: ； The structural formula of the 4-methylumbelliferyl ketyne is as follows: ； (2) Preparation of metal azide complex precursor: Under inert gas protection and dark conditions, cycloiridium dimer or cyclorhodium dimer and 4-azidomethyl-4'-methyl-2,2'-bipyridine were dissolved in an organic solvent, stirred and refluxed overnight, and the solvent was removed by rotary evaporation; after recrystallization, the metal azide complex precursor was obtained by column purification. (3) Under inert gas protection and dark conditions, Ed-Alkyne or Mu-Alkyne, copper catalyst and reducing agent are dissolved in organic solvent, and then metal azide complex is added to it and stirred to react; after the reaction is completed, the solvent is removed by rotary evaporation and the metal complex is obtained by silica gel column purification.

3. The method for preparing the metal complex according to claim 2, characterized in that: In step (1), the molar ratio of bromopropyne, emodin or 4-methylumbelliferone, and catalyst is 1~2:1:

2.

4. The method for preparing the metal complex according to claim 3, characterized in that: The catalyst is potassium carbonate or sodium carbonate.

5. The method for preparing the metal complex according to claim 2, characterized in that: In step (2), the molar ratio of the cycloiridium dimer or cyclorhodium dimer to 4-azidomethyl-4'-methyl-2,2'-bipyridine is 1:2~4.

6. The method for preparing the metal complex according to claim 2, characterized in that: In step (2), the recrystallization process is as follows: dissolve in methanol, then add ammonium hexafluorophosphate until a large amount of precipitate is formed.

7. The method for preparing the metal complex according to claim 2, characterized in that: In step (3), the molar ratio of emodin yne or 4-methyl umbelliferone yne, metal azide complex precursor, copper catalyst and reducing agent is 1:1:0.4~0.5:0.4~0.

5.

8. The method for preparing the metal complex according to claim 7, characterized in that: The reducing agent is sodium ascorbate.

9. The method for preparing the metal complex according to claim 2, characterized in that: In steps (1) and (3), the organic solvent is one of acetonitrile, N,N-dimethylformamide or dichloromethane; in step (2), the organic solvent is a mixture of dichloromethane and methanol in a volume ratio of 1 to 10:

1.

10. The use of the metal complex of claim 1 in the preparation of antitumor drugs or antitumor cell metastasis drugs, wherein, The tumor is one of human non-small cell lung cancer, breast cancer, or triple-negative breast cancer.

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