Metal complex with anti-tumor immunocompetence as well as preparation method and application thereof

Abstract

The invention discloses a metal complex with anti-tumor immunocompetence. The invention further discloses a preparation method of the metal complex and application of the metal complex in preparation of anti-tumor drugs or anti-tumor cell metastasis drugs. The metal complex is obtained by chemically modifying hydroxyl groups of natural products such as emodin and 4-methylumbelliferone and then carrying out a copper catalytic reaction with cyclometalated iridium azide or cyclometalated rhodium azide. The metal complex disclosed by the invention can solve the problem that the dosage is large when natural products such as emodin are used as antitumor drugs, and the fat solubility of the metal complex is remarkably improved; the metal complex can effectively polarize tumor-promoting M2 macrophages into tumor-inhibiting M1 phenotypes or inhibit expression of tumor cell surface signals CD47 by triggering immunogenic death, so that cancer cells are effectively killed by promoting phagocytosis of the macrophages, and the metal complex has good anti-tumor immunocompetence.

Description

Technical Field

[0001] The present invention relates to a metal complex with anti-tumor immune activity, and also relates to a preparation method of the metal complex and its application in the preparation of anti-tumor drugs or anti-tumor cell metastasis drugs. Technical Background

[0002] In the tumor environment, different factors promote the differentiation of tumor-associated macrophages into the M2 phenotype. The dominant M2 macrophages secrete pro-angiogenic and immunosuppressive cytokines in the tumor microenvironment to promote tumor growth and metastasis. In contrast, M1 polarized macrophages produce a large number of pro-inflammatory cytokines and promote the phagocytosis of tumor cells. Therefore, the functional transformation of tumor-associated macrophages from M2 to M1 phenotype can be achieved to promote the dynamic transition of the tumor immune microenvironment from an immunosuppressive state to an anti-tumor state, thereby achieving the inhibition of tumor growth and metastasis.

[0003] Metal drugs have attracted extensive attention due to their unique pharmacological properties and mechanisms of action. Due to their multifunctional structure, redox activity, and metal-ligand interactions, they provide a huge space for the design of anticancer candidates. The discovery and clinical application of cisplatin marked the beginning of metal drugs in cancer treatment and greatly promoted the research on the antitumor activity of metal compounds and their derivatives. In addition to platinum drugs, other metal anticancer drug substitutes that have been studied and developed include metal drugs based on ruthenium, gold, copper, iridium, and osmium, which are effective against a variety of cancers, including cancers resistant to traditional chemotherapy drugs. However, metal complexes currently have disadvantages such as low solubility in water, insufficient tumor targeting, and large toxic side effects. Although natural products themselves have good anticancer effects, they have poor structural stability and low bioavailability in the body. Using natural products alone as anticancer drugs has the disadvantages of large doses and low efficacy. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a metal complex with anti-tumor immune activity, which can solve the problem of large dosage when natural products such as rhein are used as anti-tumor drugs, and can also solve the problems of low solubility in water, insufficient tumor targeting and large toxic side effects of existing metal complexes; the metal complex shows good anti-proliferative activity against a series of tumor (including lung cancer, breast cancer, etc.) cell lines, and can overcome cisplatin tolerance and adverse side effects, and at the same time can significantly improve the fat solubility of the metal complex; another purpose of the present invention is to provide a preparation method of the above-mentioned metal complex and its application in the preparation of anti-tumor drugs or anti-tumor cell metastasis drugs.

[0005] Technical solution: The metal complex with anti-tumor immune activity described in the present invention has a general structural formula as shown below:

[0006]

[0007] Among them, R1 is M is Ir or Rh; R2 is

[0008] The preparation method of the above metal complex comprises the following steps:

[0009] (1) dissolving rhein or 4-methylumbelliferyl acetone and K2CO3 in an organic solvent, and then adding propyne bromide to react, and after the reaction, performing rotary evaporation and column purification to obtain rhein alkyne Ed-Alkyne or 4-methylumbelliferyl alkyne Mu-Alkyne;

[0010] Wherein, the structural formula of the rhubarb acetylene is:

[0011]

[0012] Wherein, the structural formula of the 4-methylumbelliferyl acetylene is:

[0013]

[0014] (2) Preparing a metal azide complex precursor: dissolving a cyclometallated iridium dimer or a cyclometallated rhodium dimer and 4-azidomethyl-4'-methyl-2,2'-bipyridine in an organic solvent under inert gas protection and dark conditions, stirring and refluxing overnight, and removing the solvent by rotary evaporation; dissolving with methanol, and then adding ammonium hexafluorophosphate thereto until a large amount of precipitate is precipitated; and purifying by column to obtain a metal azide complex precursor;

[0015] (3) Under the protection of inert gas and in the dark, Ed-Alkyne or Mu-Alkyne, a copper catalyst, and sodium ascorbate (to reduce divalent copper to monovalent copper) are dissolved in an organic solvent, and then a metal azide complex is added thereto and stirred for reaction; the reaction is monitored by TLC until the reaction is completed, and then the solvent is removed by rotary evaporation, and the metal complex is purified by silica gel column to obtain the metal complex.

[0016] Wherein, in step (1), the molar ratio of propargyl bromide, rhamnosine or 4-methylumbelliferone, and K2CO3 added is 1-2:1:2.

[0017] Wherein, in step (2), the molar ratio of the cyclometallated iridium dimer or cyclometallated rhodium dimer to 4-azidomethyl-4'-methyl-2,2'-bipyridine is 1:2-4.

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

[0019] Wherein, in step (3), the molar ratio of rhubarb acetylene or 4-methylumbelliferyl acetylene, metal azide complex precursor, copper catalyst and sodium ascorbate is 1:1:0.4-0.5:0.4-0.5.

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

[0021] Use of the above metal complex in the preparation of anti-tumor drugs or anti-tumor 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 complex of the present invention is obtained by chemically modifying the hydroxyl group of the natural product rhamnosine and 4-methylumbelliferone, and then reacting with cyclometallated iridium azide or cyclometallated rhodium azide by copper catalysis. Such metal complex can effectively polarize tumor-promoting M2 macrophages to tumor-suppressing M1 phenotypes or inhibit the expression of tumor cell surface signal CD47 by inducing immunogenic death, thereby effectively killing cancer cells by promoting macrophage phagocytosis, and has good anti-tumor immune activity.

[0023] The reaction mechanism of the metal complex synthesis process of the present invention is:

[0024]

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention combines a natural product with anticancer activity with a metal complex, and the obtained metal complex has good lipid solubility, which helps to penetrate the cell membrane and produce good anticancer activity and inhibit cancer cell metastasis; on the one hand, the metal complex can induce the production of intracellular reactive oxygen species, target mitochondria and reduce mitochondrial membrane potential, thereby inducing mitochondrial function damage and causing cell autophagy and necroptosis; on the other hand, the metal complex can effectively induce the immunogenic death of tumor cells, release a series of damage-associated molecular patterns (DAMPs), such as calcium These molecules can activate pattern recognition receptors (PRRs) on the surface of macrophages, promote the polarization of macrophages from the tumor-promoting M2 phenotype to the tumor-suppressing M1 phenotype, thereby upregulating the expression of M1 macrophage markers IL1B, CD86, and IL12, and downregulating the expression of M2 macrophage marker IL10, so as to effectively kill cancer cells by the phagocytosis of macrophages; in addition, Ir-Mu can promote the phagocytosis of tumor cells by macrophages by inhibiting the expression of tumor cell surface signal CD47, so the metal complex in the present invention can induce effective anti-tumor immune activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The uptake of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 into cells and their distribution in subcellular organelles;

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

[0028] Figure 3 The scratch test diagram of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 inhibiting cell metastasis;

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

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

[0031] Figure 6 This is a flow cytometric graph of the complex Ir-Mu synthesized in Example 3 inhibiting CD47 in MDA-MB-231 cells;

[0032] Figure 7 The results of using the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 to promote macrophages to phagocytize tumor cells;

[0033] Figure 8 The application result of the complex Ir-Mu synthesized in Example 3 in promoting macrophages to phagocytize tumor cells;

[0034] Fig. 9 The proximal tumor (left) and distal tumor (right) volumes of mice after treatment with cisplatin, Ir-Ed, and Rh-Ed;

[0035] Fig.10 The flow cytometric graphs of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 in activating anti-tumor immunity in mice. DETAILED DESCRIPTION

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

[0037] Example 1

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

[0039]

[0040] (1) Rhein (270 mg, 1 mmol) and K2CO3 (276.4 mg, 2 mmol) were dissolved in 4 mL of dichloromethane, propargyl bromide (118.0 mg, 1 mmol) was added and stirred, and the reaction was monitored by TLC; after reacting for 12 hours, the mixture was evaporated and purified by column to obtain 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 inert atmosphere and dark conditions, cyclometallated iridium 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 obtained solid was dissolved with a small amount of methanol, and then a saturated ammonium hexafluorophosphate solution was slowly added dropwise thereto until a large amount of precipitate was precipitated to obtain 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 dark conditions, Ed-Alkyne (21.5 mg, 0.07 mmol), CuSO4 . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL N,N-dimethylformamide (DMF), and then Ir-N3 (60.9 mg, 0.07 mmol) was added and stirred. The reaction was monitored by a plate. 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. 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 metal rhodium (III) complex (Rh-Ed) of the present invention is specifically as follows:

[0045]

[0046] (1) Under inert atmosphere and dark conditions, cyclometallated 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 obtained solid was dissolved with a trace amount of methanol, and then a saturated ammonium hexafluorophosphate solution was slowly added dropwise thereto until a large amount of precipitate was precipitated to obtain a light 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 dark conditions, Ed-Alkyne (21.5 mg, 0.07 mmol), CuSO4 . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL DMF, and then Rh-N3 (54.6 mg, 0.07 mmol) was added and stirred for reaction. The reaction was monitored by a plate. 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. 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 metal iridium (III) complex (Ir-Mu) of the present invention is specifically 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 propargyl bromide (118.0 mg, 1 mmol) was added and stirred. After reacting for 12 hours, the mixture was evaporated and purified by column to obtain 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) In an inert atmosphere and dark conditions, Mu-Alkyne (15.0 mg, 0.07 mmol), CuSO4 . 5H2O (7.0 mg, 0.028 mmol) and sodium ascorbate (5.5 mg, 0.028 mmol) were dissolved in 5 mL DMF, and then Ir-N3 (60.9 mg, 0.07 mmol) was added and stirred. The reaction was monitored by a plate. After 24 hours of reaction, the solvent was removed by rotary evaporation and the product Ir-Mu (40 mg, 53%) was obtained by purification by silica gel column. 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 (rhubarb), Ed-Alkyne, Ir-N3, Rh-N3, Ir-Ed, Rh-Ed, Mu (4-methylumbelliferone), Mu-Alkyne, 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 by MTT method, and cisplatin (CDDP) was selected as a control. MTT (thiazolyl blue) is a tetrazolium salt that can be reduced by succinate dehydrogenase in the mitochondria of living cells to generate a blue-purple product formazan (the product is soluble in DMSO), and the product has an absorption peak at 490nm, so it can be used A 490 nm To analyze cell proliferation.

[0054] The specific experimental steps are as follows:

[0055] (1) First, thaw a tube of tumor cells, culture them with fresh culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin and streptomycin), and use them after two passages;

[0056] (2) When the cells reached the logarithmic growth phase, they were seeded into a 96-well plate at a density of 5,000 cells / well (100 μL of culture medium per well), and then placed 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 drugs at different concentration gradients was added to each well, and then the cells were placed in an incubator for further incubation;

[0058] (4) After incubation for 48 hours, 20 μL of MTT (5 mg / mL) was added to each well and incubated for another 4 hours in a 37°C incubator. The supernatant was removed and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. A was detected using an enzyme-linked immunosorbent assay (ELISA). 490nm , calculate the cell proliferation inhibition rate and find the IC 50 Value (drug concentration corresponding to 50% inhibition rate).

[0059] Example 1 The toxicity of the synthesized complex Ir-Ed 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 mammary epithelial (MCF-10A) cells:

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

[0061]

[0062] The results showed that Ed-Alkyne itself had little cytotoxicity to cancer cells and normal cell lines. 50 The values ​​were all above 50μM. The metal azide complex Ir-N3 was not only highly toxic to cancer cells, but also to normal cell lines, so it did not have selectivity for cancer cells. The metal complex Ir-Ed after connecting with emodin had good selectivity for cancer cells, and its activity against human triple-negative breast cancer cells was the best. Compared with the activity of cisplatin against triple-negative breast cancer (~29.1μM), the activity of Ir-Ed (4.3μM) was increased by nearly 7 times.

[0063] Example 2 The toxicity of the complex Rh-Ed synthesized in Example 2 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 of compounds Ed-Alkyne, Rh-N3, Rh-Ed and CDDP

[0065]

[0066] The results showed that compared with Rh-N3, the cytotoxicity of the metal complex Rh-Ed after connecting with rhein was improved, and it had good selectivity for cancer cells, among which the activity against human triple-negative breast cancer cells was the best. Compared with the activity of cisplatin against triple-negative breast cancer (~29.1μM), the activity of Rh-Ed (3.9μM) 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 of compounds Mu-Alkyne, Ir-N3, Ir-Mu and CDDP

[0069]

[0070] The results showed that the toxicity of the cyclometallated iridium complex connected with Mu-Alkyne was not increased, and the toxicity of Ir-Mu to the three cancer cells was equivalent (IC 50 :6~8μM), compared with Ir-N3, the toxicity of Ir-Mu to normal MCF-10A cells was significantly reduced (IC 50 :34.9μM), with excellent selectivity for cancer cells.

[0071] Example 4

[0072] Cellular uptake and distribution of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 in subcellular organelles:

[0073] Methods: MDA-MB-231 cells were inoculated in a 10 cm culture dish. After the cells adhered to the wall, medium containing Ir-N3, Ir-Ed, Rh-N3 and Rh-Ed were added respectively and incubated for 8 hours. After the incubation, the cells were collected and centrifuged. The collected cells were fractionated to obtain mitochondrial precipitate, nuclear precipitate and supernatant cytoplasm. Concentrated nitric acid, H2O2 and concentrated HCl were added to digest each organelle component in a 95℃ sand bath. The contents of Ir and Rh in each component were determined by ICP-MS.

[0074] The uptake of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 into cells and their distribution in subcellular organelles are shown in FIG. Figure 1 The results showed that since Ir-N3 and Rh-N3 are mainly distributed in mitochondria, the cyclometallated complex modified by emodin is also mainly distributed in mitochondria, and the uptake of the cyclometallated complex is significantly increased after being modified by emodin (the uptake of Ir-Ed is increased from 363.6 ng / 10 6 cells increased to 457.6ng / 10 6 cells, Rh-Ed intake 810.9ng / 10 6 cells increased to 1546.3ng / 10 6cells), indicating that the lipid solubility of the cyclometallated complex was significantly improved after being modified by emodin, making it easier to pass through the cell membrane and selectively enrich in mitochondria (the distribution content of Ir-Ed in mitochondria was 10.6 ng / 10 6 cells, the distribution content of Rh-Ed in mitochondria was 146.3ng / 10 6 cells), further confirming the synergistic enhancement effect of combining natural products and metal ligands.

[0075] Example 5

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

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

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

[0079] Example 6

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

[0081] Methods: MDA-MB-231 cells were inoculated in six-well plates. After the cells adhered to the wall, the cell surface was scraped with the end of a 200μL pipette to make cell gaps appear, and the non-adherent cells were washed three times with PBS to remove them. The culture medium containing Ed (5μM), Ed-Alkyne (5μM), Ir-N3 (5μM), CDDP (5μM), Ir-Ed (5μM), Rh-N3 (5μM), and Rh-Ed (5μM) was added for 12h, among which the control group was a cell culture medium containing 1% DMSO. Immediately after the addition, the cell scratches in each well were photographed, and the scratch images at 0h, 24, and 48h were taken respectively (the part where the scratch crossed the marked line was selected for photography), and the scratch area changes before and after were compared. The scratch area was calculated by Image J software.

[0082] The scratch test of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 to inhibit cell metastasis is shown in Figure 3 The results showed 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 almost healed after 48 hours, indicating that Ir-Ed and Rh-Ed treatment had significant anti-cancer cell metastasis ability.

[0083] Example 7

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

[0085] Methods: MDA-MB-231 cells were plated in 6-well plates. After the cells adhered to the wall, medium 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) was added and incubated for 24 hours. After incubation, the cells and supernatant were collected, the medium was washed with PBS, and the cells were resuspended in blocking buffer and incubated at room temperature for 30 minutes. The primary antibody was added and incubated at room temperature for 45 minutes, the primary antibody was washed away, and the diluted secondary antibody coupled to the fluorescent probe was added and incubated at room temperature for 45 minutes in the dark, and the secondary antibody was washed away. Flow cytometry was used for detection, and FlowJov10 was used for data processing. For cell supernatant: add ATP detection working solution to a light-proof 96-well black board, leave it at room temperature for 3-5 minutes, add cell supernatant and mix well, measure the absorbance value with a chemiluminometer, and calculate the ATP concentration based on the standard curve.

[0086] The results of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 on inducing immunogenic cell death ICD are as follows: Figure 4 The results showed 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, indicating that both Ir-Ed and Rh-Ed can induce CRT exposure, HMGB1 release, and reduce cellular ATP levels, thereby inducing immunogenic death of MDA-MB-231 cells.

[0087] Example 8

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

[0089] Method 1: RAW 264.7 cells were plated into 6-well plates. After the cells adhered to the wall, IL-4 (25 ng / mL) was added for stimulation for 24 hours, and then the culture 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) was added for 24 hours. The cells were collected by centrifugation and washed twice with PBS. 500 μL of cell staining buffer was added, and PE-CD86 Antibody and APC-CD206 were added in sequence. The cells were incubated on ice in the dark for 15-20 minutes, and the probes were removed by centrifugation. The detection was performed by flow cytometry, and the 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 inoculated in a 10 cm culture dish. When the cell density grew to 70%, a pre-prepared cell culture medium containing compounds Ed-Alkyne (5 μM), Ir-N3 (5 μM), CDDP (5 μM), Ir-Ed (5 μM, 10 μM), Rh-N3 (5 μM), and Rh-Ed (5 μM, 10 μM) was added. After 24 hours of drug treatment, the cells were collected by centrifugation, washed twice with PBS, and lysed on ice for 20-30 minutes by adding RIPA strong lysis buffer containing PMSF. Then, the supernatant was taken by centrifugation at 4°C and 13400rpm for 20 minutes. The concentration of the supernatant was determined according to the instructions of the BCA protein content detection kit. All protein samples were diluted to a uniform concentration with PBS. 6×SDS-PAGE protein loading buffer was added and heated at 100°C for 5 minutes. After cooling to room temperature, place in a -80℃ refrigerator for later use. Prepare PAGE gel of appropriate concentration according to the instructions of the SDS-PAGE gel preparation kit. Load the sample at a standard of 20-40μg / well and perform 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 in a 4℃ refrigerator. Rinse four times with rinse solution. Add the corresponding secondary antibody and incubate at room temperature for 1 hour, rinse four times with PBST. Rinse the transfer membrane in distilled water, add developer to develop color, and use a chemiluminescence instrument for on-machine detection.

[0091] The complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 induce macrophage polarization. Figure 5As shown. The results showed that with the increase of the incubation concentration of Ir-Ed and Rh-Ed, RAW264.7 cells were significantly polarized to the M1 phenotype. After Ir-Ed treatment, the proportion of M1 expression marker CD86 increased from 12.07% to 46.9%, and after Rh-Ed treatment, the proportion of M1 expression marker CD86 increased from 66.8% to 93.8%, which was significantly higher than the control group (1.3%). WB results showed that after Ir-Ed and Rh-Ed treatment, the expression levels of macrophage M1 markers iNOS, IL1B, and TLR-2 increased, and the expression level of M2 marker Arg1 protein was significantly reduced, indicating that Ir-Ed can induce macrophages to polarize from M2 to M1, and has the potential to phagocytize tumor cells and induce immunity.

[0092] Example 9

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

[0094] Methods: MDA-MB-231 cells were seeded in confocal culture dishes. When the cell density grew to 70%, 7 μM Mu-Alkyne, Ir-N3, CDDP and Ir-Mu (7 μM, 14 μM) were added and treated for 24 h, then washed twice with PBS, fixed with 4% paraformaldehyde, and the fixative was removed and washed three times with PBS. After permeabilization with 0.2% TritonX-100 and blocking with 1.5% BSA, CD47 primary antibody dilution was added and incubated for 1 hour; the primary antibody was removed, washed three times with PBS, and incubated with Tritc-coupled secondary antibody in the dark for 1 hour; the secondary antibody was removed, washed three times with PBS, and the cell nucleus was stained with DAPI for 5 minutes; the dye was removed and washed three times with PBS, and the cells were imaged under a confocal microscope with an excitation wavelength of 550 nm and an emission wavelength of 570 nm.

[0095] The results of the inhibition of CD47 by the complex Ir-Mu synthesized in Example 3 are as follows Figure 6 The results showed that compared with the control group, the fluorescence intensity of the CD47 expression on the cell surface decreased with the increase of drug concentration after Ir-Mu treatment, indicating that the content of CD47 on the cell surface decreased, indicating that Ir-Mu can inhibit the expression of tumor cell signal CD47.

[0096] Example 10

[0097] Application of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 in promoting macrophages to phagocytize tumor cells:

[0098] Method 1: RAW 264.7 cells were polarized to M2 phenotype with IL-4, PE anti-mouse / human CD11bAntibody was co-incubated with RAW 264.7 cells for 15 minutes, CFSE Cell Division Tracker Kit was co-incubated with 4T1 cells for 20 minutes, washed with PBS, and the cells were collected. RAW 264.7 and 4T1 cells were mixed 1:1 and plated in 6-well plates, and medium containing Ed (5μM), Ed-Alkyne (5μM), Ir-N3 (5μM), Rh-N3 (5μM), CDDP (5μM), Ir-Ed (5μM, 10μM, 15μM), and Rh-Ed (5μM, 10μM, 15μM) was added, respectively, and incubated for 12 hours. The cells were collected by centrifugation, washed with PBS, detected by flow cytometry, and data were processed using FlowJo v10.

[0099] Method 2: RAW 264.7 cells were polarized to M2 phenotype with IL-4, PE anti-mouse / human CD11bAntibody was co-incubated with RAW 264.7 cells for 15 minutes, CFSE Cell Division Tracker Kit was co-incubated with 4T1 cells for 20 minutes, washed with PBS, and cells were collected. RAW 264.7 and 4T1 cells were mixed 1:1 and plated in a confocal dish, and medium containing Ed-Alkyne (5μM), Ir-N3 (5μM), Rh-N3 (5μM), CDDP (5μM), Ir-Ed (5μM), and Rh-Ed (5μM) was added, respectively, and cells were imaged under a confocal microscope after incubation for 12 hours.

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

[0101] Embodiment 11

[0102] Example 3 Application of the synthesized complex Ir-Mu in promoting macrophages to phagocytize tumor cells:

[0103] Methods: RAW 264.7 cells were polarized to M1 phenotype by LPS, PE anti-mouse / human CD11bAntibody was co-incubated with RAW 264.7 cells for 15 minutes, CFSE Cell Division Tracker Kit was co-incubated with 4T1 cells for 20 minutes, washed with PBS, and the cells were collected. RAW 264.7 and 4T1 cells were mixed 1:1 and plated in 6-well plates, and culture medium containing Mu-Alkyne (7μM), Ir-N3 (7μM), CDDP (7μM), and Ir-Mu (7μM, 14μM, 21μM) was added for 12 hours, and the cells were collected by centrifugation, washed with PBS, and detected by flow cytometry. FlowJo v10 was used for data processing.

[0104] The results of the complex Ir-Mu synthesized in Example 3 promoting macrophages to phagocytize tumor cells are shown in Figure 8 The results showed that after Ir-Mu treatment, the ratio of macrophages engulfing tumor cells 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 engulfment of tumor cells by macrophages by inhibiting the expression of CD47.

[0105] Example 12

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

[0107] Methods: BALB / c female mice (4-6 weeks old) were randomly divided into 4 groups, with 5 mice in each group. 4T1 cells (2×10 6 4 days later, 4T1 cells were injected into the right back to establish distal tumors (1×10 6 ). Subsequently, different drugs (100 μL (5 mg / Kg) were injected intratumorally on the first, third, and fifth days as follows: (1) control group (injected with normal saline); (2) CDDP group; (3) Ir-Ed group; (4) Rh-Ed group. After 21 days, the mice were dissected, and one mouse tumor was randomly taken from each group and immersed in 2 mL of cell digestion solution at 37°C for 1-2 hours. The cells were collected using a 70 μM cell sieve, centrifuged, and washed with PBS. Cell staining buffer was added and incubated for 20 minutes, and CD8 Plus 647Anti-Mouse / CD3FITCAnti-Mouse, CD4 APC Anti-Mouse / CD3FITC Anti-Mouse, Foxp3 PE Anti-Mouse / CD4 APCAnti-Mouse, CD80 FITC Anti-Mouse / CD86 CoraLiteR Plus 647Anti-Mouse, CD86CoraLiteR Plus 647Anti-Mouse / CD206 Anti-Mouse, F4 / 80Anti-Mouse / CD86 CoraLiteRPlus 647Anti-Mouse was incubated in the dark for 30 minutes. The cells were collected and washed with PBS, detected by flow cytometry, and data were processed using FlowJo v10.

[0108] The results of the complexes Ir-Ed and Rh-Ed synthesized in Example 1 and Example 2 in activating anti-tumor immunity in mice are as follows: Fig. 9 and Fig.10 As shown, in mice injected with Ir-Ed and Rh-Ed, the proximal tumor changes were comparable to those with cisplatin, but the distal tumor volume was significantly smaller than that in the CDDP group. The maturation of dendritic cells (DCs) within the tumor tissue was improved, and the percentage of CD80 / CD86 increased significantly 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 evaluate the adaptive immune response. The proportions of CD4 and CD8 T cells mediated by Ir-Ed and Rh-Ed were approximately 20.3% / 13.3% and 21.7% / 17.9%, respectively, which were higher than those in the control group and CDDP group. Ir-Ed and Rh-Ed improved DC maturation and initiated adaptive antitumor immunity within the tumor. In addition, the M1 macrophage marker CD86 increased from 4.36% to 10.6%, and the M2 macrophage marker CD206 increased from 4.49% to 2.02%.

Claims

1. A metal complex having anti-tumor immune activity, characterized in that: Its general structure is shown below: Among them, R1 is M is Ir or Rh; R2 is 2. The method for preparing the metal complex according to claim 1, characterized in that: The steps include: (1) dissolving rhein or 4-methylumbelliferone and a catalyst in an organic solvent, adding propyne bromide thereto for reaction, and after the reaction, rotary evaporation and column purification are performed to obtain rhein acetylene or 4-methylumbelliferone acetylene; Wherein, the structural formula of the rhubarb acetylene is: Wherein, the structural formula of the 4-methylumbelliferyl acetylene is: (2) preparing a metal azide complex precursor: dissolving a cyclometallated iridium dimer or a cyclometallated rhodium dimer and 4-azidomethyl-4'-methyl-2,2'-bipyridine in an organic solvent under inert gas protection and dark conditions, stirring and refluxing overnight, and removing the solvent by rotary evaporation; after recrystallization, purifying by column to obtain a metal azide complex precursor; (3) Under the protection of inert gas and in the dark, Ed-Alkyne or Mu-Alkyne, a copper catalyst and a reducing agent are dissolved in an organic solvent, and then a metal azide complex is added thereto and stirred for reaction; after the reaction is completed, the solvent is removed by rotary evaporation, and the metal complex is purified by silica gel column to obtain the metal complex.

3. The method for preparing the metal complex according to claim 2, characterized in that: In step (1), the molar ratio of propargyl bromide, rhamnosine or 4-methylumbelliferone and the 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 cyclometallated iridium dimer or cyclometallated rhodium 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: dissolving with methanol, and then adding ammonium hexafluorophosphate thereto until a large amount of precipitate is precipitated.

7. The method for preparing a metal complex according to claim 2, characterized in that: In step (3), the molar ratio of rhubarb acetylene or 4-methylumbelliferyl acetylene, metal azide complex precursor, copper catalyst and reducing agent added 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 a metal complex according to claim 2, characterized in that: In step (1) and step (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. Use of the metal complex according to claim 1 in the preparation of anti-tumor drugs or anti-tumor 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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