A metal complex tailed with a TDO inhibitor and its preparation method and application

Abstract

The present invention discloses a metal complex tailed with a TDO inhibitor, and its preparation method and application. The preparation method of the metal complex tailed with a TDO inhibitor of the present invention is specifically as follows: in a light-proof and inert gas environment, a metal dimer precursor and a dechlorinating agent are added to anhydrous methanol, refluxed and stirred at 60°C for 0.2 to 72 hours, and filtered after the reaction; an immune checkpoint TDO inhibitor ligand is added to the filtrate, and the reaction is carried out in an anaerobic environment. After the reaction is complete, an anion salt is added thereto, the solution is filtered and concentrated, and then frozen. After freezing, centrifugation is performed to obtain a crude product, which is purified by column chromatography to obtain a metal complex. The aryl / cyclopentadienyl metal complex of the present invention can be well positioned in the nucleus of cancer cells, induce cell necrosis, lead to immunogenic cell death, and inhibit the expression of immune checkpoint TDO, thereby achieving good anticancer activity, ICD induction activity and immune checkpoint TDO inhibition activity.

Description

Technical Field

[0001] The present invention relates to a metal complex tailed with a TDO inhibitor, and also relates to a preparation method and application of the metal complex. Technical Background

[0002] The development of platinum-based anticancer drugs, including cisplatin, carboplatin, and oxaliplatin, is one of the great successes in the field of metal preparations. However, the serious side effects and drug resistance of platinum-based anticancer drugs have greatly limited their clinical application in cancer treatment. In recent years, aryl metal complexes have attracted great interest as the basis for new anticancer drugs due to their specificity in treatment, low drug resistance, and low side effects. Designing drugs that target tumor cells has become a research hotspot in modern medicine. In this regard, Professor Peter Sadler of the University of Warwick, UK, Professor Paul Dyson of the Swiss Federal Institute of Technology in Lausanne, Switzerland, and Professor Bernhard Keppler of the University of Vienna, Austria, and others have successfully attached targeted molecules to metals and obtained drugs with high selectivity.

[0003] A potential target of arylruthenium / iridium complexes in cells may be DNA, which induces apoptosis and necrosis. Necrosis is usually considered to be random and unregulated, but recently it has been recognized that there is a programmed form of necrosis called necroptosis. Necroptosis can destroy the integrity of the plasma membrane, leading to the leakage of intracellular damage-associated molecular patterns (DMAPs), and participate in tumor-specific immune responses by triggering immunogenic cell death (ICD), so that dying cancer cells can initiate a powerful immune response. The arylruthenium complex KP1339 reported by Kepler and the complex Ir1 published by Professor Chao Hui's research group can both stimulate immune responses by inducing immunogenic cell death (ICD) through chemotherapy and promoting the maturation of immune cells.

[0004] Tryptophan 2,3-dioxygenase (TDO) is a typical immunosuppressive checkpoint that is overexpressed in most tumor cells, which may catalyze the metabolism of the essential amino acid tryptophan to kynurenine. TDO-derived kynurenine activates the aryl hydrocarbon receptor (AHR), contributing to the immunosuppression of the tumor microenvironment (TME) and supporting tumor cell immune escape. Small molecule inhibitors of indole derivatives (e.g., 680C91) can interfere with downstream AHR metabolism by inhibiting TDO activity in tumor cells, leading to a reversal of the tumor microenvironment. However, the current small molecule inhibitors have a relatively short circulation half-life, which seriously restricts their further application in enhancing tumor immunotherapy. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a metal complex tailed with an immune checkpoint TDO inhibitor, and also provide a preparation method and application of the above-mentioned metal complex. By coordinating an aryl metal dimer or a cyclopentadienyl metal dimer with an immune checkpoint TDO inhibitor to synthesize a metal complex, taking into account the chemotherapeutic effect and immune checkpoint inhibitory effect of the metal, the effect of the metal complex in anti-cancer, anti-inflammatory, inhibiting immune checkpoint TDO expression and inducing ICD effect can be effectively improved.

[0006] Technical solution: The metal complex tailed with a TDO inhibitor of the present invention has a general structural formula as shown in formula (I) or formula (II):

[0007]

[0008] Wherein, in formula (I), R1 is In formula (I) and formula (II), Cp x for M is Ru or Ir; R is H or F.

[0009] The preparation method of the metal complex tailed with a TDO inhibitor is specifically as follows: in a light-proof and inert gas environment, a metal dimer precursor and a dechlorinating agent are added to anhydrous methanol, refluxed and stirred at 60° C. for 0.2 to 72 hours, and filtered after the reaction; an immune checkpoint TDO inhibitor ligand is added to the filtrate, and the reaction is carried out in an anaerobic environment (dry argon is bubbled into the reaction solution to drive away the air), and after the reaction is complete at room temperature, an anion salt is added thereto, and the solution is filtered and concentrated after stirring, and then frozen. After freezing, the crude product is centrifuged to obtain a crude product, which is purified by column chromatography to obtain a metal complex.

[0010] Wherein, the dechlorinating agent is silver nitrate or silver trifluoromethanesulfonate.

[0011] Among them, the added molar ratio of the metal dimer precursor, the dechlorinating agent and the immune checkpoint TDO inhibitor ligand is 1:10:30 to 1:100:300.

[0012] Among them, the immune checkpoint TDO inhibitor ligand is an indole ligand.

[0013] Wherein, the anion salt is ammonium hexafluorophosphate salt.

[0014] The stirring temperature for adding the anionic salt is 10 to 50° C., and the stirring time is 2 to 72 hours.

[0015] Among them, the freezing temperature is -20~4℃, and the freezing time is 0.2~5h.

[0016] Application of the above metal complex in the preparation of anticancer drugs, anticancer drug components, anti-inflammatory drugs, anti-inflammatory drug components, immune checkpoint TDO inhibitor drugs, immune checkpoint TDO inhibitor drug components, ICD inducer drugs and ICD inducer drug components.

[0017] The chemical reaction formula for the synthesis of the metal complex of the present invention is:

[0018]

[0019] Beneficial effects: The present invention synthesizes a metal complex with targeted anti-cancer, anti-inflammatory, immune checkpoint TDO expression inhibition and ICD effect induction by tailing an immune checkpoint TDO inhibitor with an aryl metal dimer or a cyclopentadienyl metal dimer; the aryl / cyclopentadienyl metal complex of the present invention can be well localized in the nucleus of cancer cells, induce cell necrosis, lead to immunogenic cell death, and can inhibit the expression of the immune checkpoint TDO, thereby achieving good anti-cancer activity, ICD induction activity and immune checkpoint TDO inhibition activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The uptake and localization diagram of the iridium complex IndoF-Ir and the ruthenium complex IndoF-Ru of Example 1 and Example 3 in HepG2 cells;

[0021] Figure 2 The diagrams are of cell necrosis induced by the iridium complex IndoF-Ir and the ruthenium complex IndoF-Ru of Examples 1 and 3;

[0022] Figure 3 The flow cytometry data of the iridium complex IndoF-Ir and the ruthenium complex IndoF-Ru inducing CRT exposure and HMGB1 extravasation in Example 1 and Example 3;

[0023] Figure 4 Western blots showing the inhibition of TDO protein expression by the iridium complex IndoF-Ir and the ruthenium complex IndoF-Ru of Examples 1 and 3;

[0024] Figure 5 The graphs show the anti-proliferation and permeability effects of the iridium complex IndoF-Ir and the ruthenium complex IndoF-Ru of Examples 1 and 3 on HepG2 tumor spheroid cells. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0026] Example 1

[0027]

[0028] Under light-proof and argon protection, 1.5 mmol pyridine-3-acetic acid hydrochloride and 2.5 mmol triethylamine were dissolved in anhydrous 1,4-dioxane, stirred at room temperature for 0.2 to 6 hours, and then 3.5 mmol 6-fluoroindole-3-carboxaldehyde and 4.2 mmol piperidine were added thereto. After the reaction was completed, a small amount of water was used to quench the reaction, the product was extracted with ethyl acetate, and the organic layer was washed with a saturated NaHCO3 aqueous solution and dried with Na2SO4, and the solvent was removed under reduced pressure. Purification was performed by column chromatography on silica gel (petroleum ether / ethyl acetate = 10:1 to 0.5:1) to obtain the ligand compound IndoF, which was a yellow powder with a yield of 64%. 1 H NMR (400MHz, DMSO-d6) δ11.45(s,1H),8.76(d,J=2.2Hz,1H),8.38(dd,J=4.7,1.6Hz,1H),8.07-7.98(m,2H),7.69(d,J=2.4Hz,1 H), 7.54 (d, J = 16.6Hz, 1H), 7.36 (dd, J = 8.0, 4.7Hz, 1H), 7.22 (dd, J = 9.9, 2.4Hz, 1H), 7.11 (d, J = 16.7Hz, 1H), 7.02-6.96 (m, 1H). ESI-MS[IndoF] + :Theoretical value: 239.08, experimental value: 239.1.

[0029] Under light-proof and argon protection, 0.2 mmol of dichloro(p-methylisopropylphenyl)ruthenium(II) dimer, 1 mmol of 2-phenylpyridine and 5 mmol of sodium acetate were dissolved in a dichloromethane solution. After stirring at room temperature until the reaction was complete, the dichloromethane was removed under reduced pressure and recrystallized with chloroform / ether to precipitate a relatively pure yellow powder product of ppy-Ru metal precursor with a yield of 73%. 1H NMR (400MHz, DMSO-d6) δ9.36(ddd,J=5.7,1.6,0.7Hz,1H),8.13(dd,J=7.5,1.2Hz,1H),7.95(ddd,J=8.3,1.4,0.7H z,1H),7.83(ddd,J=8.1,7.3,1.5Hz,1H),7.73(dd,J=7.7,1.4Hz,1H),7.21(ddd,J=7.2,5.7,1.4Hz,1H),7.06(td, J=7.3,1.4Hz,1H),6.95(td,J=7.4,1.2Hz,1H),5.77-5.74(m,1H),5.69(dd,J=6.0,1.3Hz,1H),5.42(dd,J=6.0,1. 3Hz,1H),5.16(dd,J=6.0,1.3Hz,1H),2.31-2.24(m,1H),1.93(s,3H),0.85(d,J=6.9Hz,3H),0.77(d,J=6.9Hz,3H).

[0030] In the light-proof and argon-protected environment, 0.1 mmol of metal ruthenium precursor (ppy-Ru) was dissolved in 6 mL of methanol, 0.4 mmol of silver nitrate was added thereto, and the mixture was heated under reflux and stirred at 60°C for 0.5 to 8 hours until the silver nitrate was completely reacted, and the generated AgCl was filtered; 0.6 mmol of indole fluoride ligand (IndoF) was added to the filtrate, and the air was driven out by bubbling under dry argon. After the reaction, the mixture was stirred at room temperature and the reaction was monitored by TCL; after the reaction was completed, the mixture was cooled to room temperature, the solution was concentrated, and a saturated methanol solution of ammonium hexafluorophosphate was added. The formed precipitate was collected by centrifugation, washed with ether for multiple times, recrystallized with chloroform, and dried in vacuo to obtain a yellow solid ruthenium metal complex with a yield of 44.5%. 1H NMR (400MHz, DMSO-d6) δ9.71(d,J=5.8Hz,1H),8.57(d,J=1.9Hz,1H),8.51(d,J=7.5Hz,1H),8.13(d,J=5.4H z,1H),8.03-7.93(m,4H),7.76(d,J=7.8Hz,1H),7.68(d,J=2.5Hz,1H),7.49(t,J=6.1Hz,1H),7.36-7.22(m, 4H),7.12-7.02(m,2H),6.89(d,J=16.6Hz,1H),6.23(d,J=6.2Hz,1H),6.15(d,J=6.1Hz,1H),5.96(d,J=6.2 Hz, 1H), 5.54 (d, J = 5.8Hz, 1H), 2.21-2.15 (m, 1H), 1.64 (s, 3H), 0.78 (d, J = 6.8Hz, 3H), 0.72 (d, J = 6.9Hz, 3H). ESI-MS[IndoF-Ru-PF6] + Theoretical value: 628.13, experimental value: 628.2.

[0031] Example 2

[0032]

[0033] Under light-proof and argon protection, 1.5 mmol pyridine-3-acetic acid hydrochloride and 2.5 mmol triethylamine were dissolved in anhydrous 1,4-dioxane, stirred at room temperature for 0.2 to 6 hours, and then 3.5 mmol 1-hydrogen-indole-3-carboxaldehyde and 4.2 mmol piperidine were added thereto. After the reaction was completed, a small amount of water was used to quench the reaction, and the product was extracted with ethyl acetate, and the organic layer was washed with a saturated NaHCO3 aqueous solution and dried with Na2SO4, and the solvent was removed under reduced pressure. Column chromatography was used for purification with a developing solvent of petroleum ether / ethyl acetate = 10:1 to 0.5:1 to obtain a ligand compound Indo, which was a yellow powder with a yield of 71.9%. 1 H NMR (400MHz, DMSO-d6) δ11.47-11.35(m,1H),8.76(d,J=2.2Hz,1H),8.37(dd,J=4.7,1.6Hz,1H),8.06-7.99(m,2H),7 .69(d,J=2.7Hz,1H),7.56(d,J=16.6Hz,1H),7.46-7.42(m,1H),7.36(ddd,J=8.0,4.8,0.8Hz,1H),7.20-7.08(m,3H). ESI-MS[Indo] + Theoretical value: 221.08, experimental value: 221.2.

[0034] Under light-proof and argon protection, 0.1 mmol of the metal ruthenium precursor (ppy-Ru) prepared in Example 1 was dissolved in 6 mL of methanol, 0.4 mmol of silver nitrate was added thereto, and the mixture was heated under reflux and stirred at 60° C. for 0.5 to 8 hours until the silver nitrate was completely reacted, and the generated AgCl was filtered; 0.6 mmol of indole ligand (Indo) was added to the filtrate, and the air was driven out by bubbling under dry argon. After the reaction, the mixture was stirred at room temperature and the reaction was monitored by TCL; after the reaction was completed, the mixture was cooled to room temperature, the solution was concentrated, and a saturated methanol solution of ammonium hexafluorophosphate was added. The formed precipitate was collected by centrifugation, washed with ether for multiple times, recrystallized from chloroform, and dried in vacuo to obtain a yellow solid ruthenium metal complex with a yield of 56.3%. 1 H NMR (400MHz, DMSO-d6) δ11.59(s,1H),9.73-9.70(m,1H),8.57(d,J=2.0Hz,1H),8.52(dd,J=7.7,1.1Hz,1H),8.12(dd,J=5.6, 1.2Hz,1H),8.03-7.95(m,4H),7.76(dd,J=7.9,1.3Hz,1H),7.68(s,1H),7.52-7.44(m,2H),7.33(d,J=16.5Hz,2H),7.24-7.17 (m,3H),7.10(td,J=7.5,1.1Hz,1H),6.88(d,J=16.6Hz,1H),6.24(dd,J=6.2,1.3Hz,1H),6.15(dd,J=6.0,1.3Hz,1H),5.97(dd ,J=6.2,1.3Hz,1H),5.55(dd,J=6.0,1.3Hz,1H),2.22-2.16(m,1H),1.65(s,3H),0.78(d,J=6.9Hz,3H),0.73(d,J=6.9Hz,3H). ESI-MS[Indo-Ru-PF6] + Theoretical value: 610.14, experimental value: 610.2.

[0035] Example 3

[0036]

[0037] Under light-proof and argon protection, 0.2 mmol of dichloro(pentamethylcyclopentadiene)iridium(III) dimer, 1 mmol of 2-phenylpyridine and 5 mmol of sodium acetate were dissolved in a dichloromethane solution. After stirring at room temperature until the reaction was complete, the dichloromethane was removed under reduced pressure and recrystallized with chloroform / ether to precipitate a relatively pure yellow powder product ppy-Ir metal precursor with a yield of 74.3%.1 H NMR(400MHz, DMSO-d6)δ8.70(ddd,J=5.7,1.6,0.7Hz,1H),8.08(dt,J=8.1,1.1Hz,1H),7.89-7.84(m,1H),7.81(dd,J=7.7,1.4Hz,1H), 7.71(dd,J=7.6,1.2Hz,1H), 7.30(ddd,J=7.3,5.7,1.4Hz,1H), 7.12(td,J=7.3,1.4Hz,1H), 6.99(td,J=7.4,1.2Hz,1H), 1.60(s,15H).

[0038] Under light-proof and argon protection, 0.1 mmol of metal iridium precursor (ppy-Ir) was dissolved in 6 mL of methanol, 0.4 mmol of silver nitrate was added thereto, and the mixture was heated under reflux and stirred at 60° C. for 0.5 to 8 hours until the silver nitrate was completely reacted, and the generated AgCl was filtered; 0.6 mmol of indole fluoride ligand (IndoF) prepared in Example 1 was added to the filtrate, and the air was driven out by bubbling under dry argon. After the reaction, the mixture was stirred at room temperature and the reaction was monitored by TCL; after the reaction was completed, the mixture was cooled to room temperature, the solution was concentrated, and a saturated methanol solution of ammonium hexafluorophosphate was added. The formed precipitate was collected by centrifugation, washed with ether for multiple times, recrystallized with chloroform, and dried in vacuo to obtain a yellow solid iridium metal complex with a yield of 73.9%. 1 H NMR (400MHz, DMSO-d6) δ11.59(s,1H),9.13-9.09(m,1H),8.36(d,J=2.0Hz,1H),8.21-8.19(m,1H),8.1 7(d,J=8.2Hz,1H),8.09(dt,J=8.1,1.6Hz,1H),8.06-8.01(m,2H),7.95(dd,J=8.7,5.3Hz,1H),7.86(d d,J=7.9,1.3Hz,1H),7.71(s,1H),7.61-7.56(m,1H),7.45-7.41(m,1H),7.36-7.30(m,2H),7.23(dd,J =9.8, 2.4Hz, 1H), 7.18 (dd, J = 7.5, 1.1Hz, 1H), 7.06-7.01 (m, 1H), 6.93 (d, J = 16.6Hz, 1H), 1.60 (s, 15H). ESI-MS[IndoF-Ir-PF6] + Theoretical value: 720.25, experimental value: 720.3.

[0039] Example 4

[0040]

[0041] Under light-proof and argon protection, 0.1 mmol of the metal iridium precursor (ppy-Ir) prepared in Example 3 was dissolved in 6 mL of methanol, 0.4 mmol of silver nitrate was added thereto, and the mixture was heated under reflux and stirred at 60° C. for 0.5 to 8 hours until the silver nitrate was completely reacted, and the generated AgCl was filtered; 0.6 mmol of the indole ligand (Indo) prepared in Example 2 was added to the filtrate, and the air was driven away by bubbling under dry argon. After the reaction, the mixture was stirred at room temperature and the reaction was monitored by TCL; after the reaction was completed, the mixture was cooled to room temperature, the solution was concentrated, and a saturated methanol solution of ammonium hexafluorophosphate was added. The formed precipitate was collected by centrifugation, washed with ether for multiple times, recrystallized from chloroform, and dried in vacuo to obtain a yellow solid iridium metal complex with a yield of 49.7%. 1 H NMR (400MHz, DMSO-d6) δ11.56(s,1H),9.12(d,J=5.7Hz,1H),8.36(d,J=1.9Hz,1H),8.18(dd,J=9.5, 6.8Hz,2H),8.08(d,J=8.2Hz,1H),8.04(d,J=7.6Hz,2H),7.95(dd,J=6.8,2.1Hz,1H),7.89-7.85(m,1 H),7.71(s,1H),7.59(ddd,J=7.2,5.6,1.4Hz,1H),7.46-7.41(m,2H),7.36(d,J=16.5Hz,1H),7.30(d d,J=8.1,5.6Hz,1H),7.21-7.15(m,3H),6.92(d,J=16.5Hz,1H),1.61(s,15H).ESI-MS[Indo-Ir-PF6] + Theoretical value: 702.25, experimental value: 702.3.

[0042] Application of the metal complexes prepared in Examples 1 to 4 of the present invention in tumor cell therapy:

[0043] Example 5

[0044] The anticancer activities of the ligands, metal ruthenium / iridium precursors and ruthenium / iridium complexes prepared in Examples 1, 2, 3 and 4 were determined by MTT colorimetry.

[0045] The in vitro cytotoxic activity of the complexes against three cancer cell lines expressing TDO: human cervical epithelioid carcinoma (HeLa), human lung cancer (A549) and human hepatocellular carcinoma (HepG2) was evaluated by MTT colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with human normal liver cell line LO2 as control. The cytotoxicity of the ligands Indo and IndoF and the precursors ppy-Ru and ppy-Ir were compared under the same biological conditions. HeLa, A549, HepG2 and LO2 cells were cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C in a cell culture incubator with 5% CO2. The cells were seeded into 96-well cell culture plates at an initial density of 5000 cells / well. The medium was removed after 24 hours of culture, and three parallel comparison experiments were set up, with five concentrations of the tested samples, and the incubation continued for 48 hours. Then, 20 μL of 5 mg / mL MTT was added to each well, and the mixture was incubated in a 37°C incubator for 4 hours before adding 150 μL of DMSO. The mixture was shaken until the purple crystals were completely dissolved, and the absorbance at 490 nm was read on a microplate reader.

[0046] Table 1 shows the IC values ​​of the complexes Indo-Ir, Indo-Ru, Indo-Ir, IndoF-Ru and the precursors phenylpyridine ruthenium (ppy-Ru), phenylpyridine iridium (ppy-Ir) and the ligands indole (Indo), indole fluoride (IndoF) and cisplatin prepared outside the cell. 50 (μM) value.

[0047] Table 1

[0048]

[0049] The results showed that metal complexes tailed with immune checkpoint TDO inhibitors (IndoF-Ir, Indo-Ir, Indo-Ru, IndoF-Ru) all showed high antitumor activity against tumor cells, and the activity of some complexes was about 4 times that of cisplatin.

[0050] Example 6

[0051] Example 1: Uptake and localization of ruthenium precursor ppy-Ru and ruthenium complex IndoF-Ru; Example 3: Uptake and localization of iridium precursor ppy-Ir and metal iridium complex IndoF-Ir in HepG2 cells.

[0052] Methods: HepG2 cells were seeded in a 100 mm culture dish. After confluence, 8 μmol of the compounds in Example 1 and Example 3 were added and cultured. After 6 hours, the cells were collected and washed twice with 1×PBS. The nuclei, mitochondria and cytoplasm were removed using a mitochondrial / nucleus preparation kit, and then digested with concentrated nitric acid for 2 hours, hydrogen peroxide for 1.5 hours, and concentrated hydrochloric acid for 2 hours. After cooling, it was diluted to 2 mL with ultrapure water, and the content of metal ruthenium / iridium in the samples in different organelles was determined by inductively coupled plasma mass spectrometry ICP-MS.

[0053] The uptake and localization of the ruthenium / iridium complexes in HepG2 cells in Examples 1 and 3 are shown in FIG. Figure 1 As shown, Figure 1 The data in the Figure 3 indicate that the metal dimer precursors and metal complexes are mainly localized in the nucleus after being taken up by HepG2 cells.

[0054] Example 7

[0055] Flow cytometry was used to detect the ways in which the ligand IndoF, the ruthenium precursor ppy-Ru, the ruthenium complex IndoF-Ru in Example 1 and the iridium complex IndoF-Ir in Example 3 induced cell apoptosis.

[0056] Methods: HepG2 cells were seeded in a 6-well plate. After confluence, they were incubated with the iridium complex IndoF-Ir (8 μmol) and the ruthenium complex IndoF-Ru (4 μmol, 8 μmol) in Examples 1 and 3, respectively, at 37°C for 24 hours. The cells were collected and washed twice with PBS, and then 5 μL of Annexin V-FITC apoptosis detection reagent was added and mixed. After 5 minutes, 5 μL of Propidium Iodide was added and mixed. After incubation in the dark, the samples were analyzed by flow cytometry, and the data were analyzed using FlowJo7.6 software.

[0057] The ruthenium complex IndoF-Ru of Example 1 and the iridium complex IndoF-Ir of Example 3 induced cell necrosis as shown in Figure 2 As shown in the figure, it can be seen from the data that the ruthenium complex IndoF-Ru induces cell necrosis.

[0058] Example 8

[0059] Flow cytometry was used to detect the exposure of cell CRT and the extravasation of HMGB1 induced by the ligand IndoF in Example 1, the ruthenium precursor ppy-Ru, the ruthenium complex IndoF-Ru and the metal iridium complex IndoF-Ir in Example 3.

[0060] Methods: HepG2 cells were seeded in 6-well plates. After confluence, they were incubated with the above compounds at 37°C for 24 hours. The cells were collected and washed twice with PBS. After being fixed with 4% formaldehyde at room temperature for 15 minutes, 0.2% Triton X-100 solution was added and incubated for 15 minutes. They were blocked with 5% bovine serum for 30 minutes, and primary antibodies such as anti-CRT and anti-HMGB1 were incubated overnight at 4°C. After being washed three times with PBST, secondary antibodies were incubated with peroxidase-labeled goat anti-rabbit (HRP, 1:50) for 1 hour in the dark at room temperature. Samples were analyzed by flow cytometry, and data were analyzed by FlowJo.

[0061] Figure 3 The results of the data processing by FlowJo after the ruthenium complex IndoF-Ru and iridium complex IndoF-Ir of Examples 1 and 3 were co-incubated with HepG2 cells for 24 hours, and the CRT and HMGB1 immunofluorescence were processed by flow cytometry. The results showed that the flow CRT immunofluorescence gradually shifted to the right, and the flow HMGB1 immunofluorescence gradually shifted to the left. This indicates that CRT is gradually exposed on the cell membrane and high mobility group protein 1 (HMGB1) is released into the extracellular space in necrotic cells, causing cell immunogenic death.

[0062] Example 9

[0063] Western blotting was used to detect the inhibitory effects of the ruthenium complex IndoF-Ru in Example 1 and the iridium complex IndoF-Ir in Example 3 on the expression of TDO protein in cells.

[0064] Method: HepG2 cells were inoculated in a 100 mm culture dish. After confluence, they were incubated with the chemically synthesized indole fluoride IndoF (8 μmol), ruthenium precursor ppy-Ru (8 μmol), iridium complex IndoF-Ir (8 μmol) and ruthenium complex IndoF-Ru (4 μmol, 8 μmol) in Examples 1 and 3 respectively at room temperature for 24 hours, and then the cells were collected, treated with RIPA lysis buffer on ice for 30 minutes, centrifuged for 20 minutes, and the supernatant was aliquoted. The protein concentration of the supernatant was determined by BCA protein quantification. 6×SDS-PAGE sample loading buffer (China Baitai Biotechnology Co., Ltd.) was added, heated at 95°C for 5 minutes, and stored in a freezer. 30 μg of protein was analyzed by Western blotting, PVDF membrane was used to transfer protein, skim milk blocked the PVDF membrane, and then anti-TDO primary antibody was incubated at 4°C overnight. The membrane was washed three times with PBST in a shaker. Then it was incubated with secondary antibody for 1 hour and washed three times in a shaker. Add chemiluminescent reagent, react for 1 minute, and expose in a dark room. Finally, use ImageJ software to quantitatively analyze protein expression.

[0065] In view of the TDO inhibition mechanism of the above ligands, protein immunoblotting was used to explore the expression of TDO by the ruthenium complexes IndoF-Ru and the iridium complex IndoF-Ir in Examples 1 and 3. Figure 4 The expression of TDO in HepG2 cells was analyzed by western blot, and the data showed that the complex had a higher activity in inhibiting the expression of TDO protein, which helped to inhibit the immune escape of cancer cells.

[0066] Example 10

[0067] Three-dimensional multicellular tumor spheroids (MCTs) were used to verify the anti-proliferation and penetration abilities of the ruthenium complex IndoF-Ru in Example 1 and the iridium complex IndoF-Ir in Example 3 on HepG2 tumor spheroids.

[0068] Methods: In solid tumor models, tumor cells are generally less sensitive to chemotherapy than cultured monolayer cells. Three-dimensional multicellular tumor spheroids (MCTs) are usually used to simulate the microenvironment of solid tumors. Two days after the formation of three-dimensional multicellular tumor spheroids of HepG2 cells, the above compounds (drug concentration of 30 μmol) were added and co-incubated, and the anti-proliferative effect of the drugs on HepG2 tumor cell spheroids was monitored for eight consecutive days. Calcein AM / PI live and dead cell staining was performed on the tumor cell spheroids on the eighth day to analyze the anti-tumor effect.

[0069] Figure 5 The results are as follows: HepG2 cells were co-incubated with the ruthenium complexes IndoF-Ru and iridium complex IndoF-Ir in Examples 1 and 3 for 8 days after forming spheres. The results showed that no drug addition and single precursor or single ligand did not inhibit the proliferation of tumor cell spheres, and the volume of the cell spheres gradually increased, while the complex IndoF-Ru significantly inhibited the growth of tumor cells and destroyed the cell sphere structure, and had stronger anti-proliferation activity and tumor penetration ability on HepG2 tumor cell spheres.

[0070] The metal complex of the present invention has good ability to inhibit the activity of immune checkpoint TDO, thereby inhibiting the metabolism of AHR, and can also induce immunogenic cell death, and is a good immune checkpoint TDO inhibitor and ICD inducer.

Claims

1. A metal complex tailed with a TDO inhibitor, characterized in that: The structural formula of the metal complex is shown below: or .

2. The method for preparing the metal complex tailed with a TDO inhibitor according to claim 1, characterized in that: Specifically, in a light-proof, inert gas environment, a metal precursor and a dechlorinating agent are added to anhydrous methanol, refluxed and stirred at 60°C for 0.2 to 72 hours, and filtered after the reaction; an immune checkpoint TDO inhibitor ligand is added to the filtrate to allow the reaction to proceed in an anaerobic environment, and an anion salt is added after the reaction is complete, the solution is filtered and concentrated, and then frozen. After freezing, the crude product is centrifuged to obtain a crude product, which is purified by column chromatography to obtain a metal complex.

3. The method for preparing a metal complex tailed with a TDO inhibitor according to claim 2, characterized in that: The dechlorinating agent is silver nitrate or silver trifluoromethanesulfonate.

4. The method for preparing a metal complex tailed with a TDO inhibitor according to claim 2, characterized in that: The metal precursor, dechlorinating agent, and immune checkpoint TDO inhibitor ligand were added in a molar ratio of 1:10:30 to 1:100:

300.

5. The method for preparing a metal complex tailed with a TDO inhibitor according to claim 2, characterized in that: The anion salt is ammonium hexafluorophosphate.

6. The method for preparing a metal complex tailed with a TDO inhibitor according to claim 2, characterized in that: The reaction temperature after adding the anionic salt is 10-50°C, and the reaction time is 2-72h.

7. The method for preparing a metal complex tailed with a TDO inhibitor according to claim 2, characterized in that: The freezing temperature is -20~4℃ and the freezing time is 0.2~5h.

8. Use of the metal complex tailed with a TDO inhibitor according to claim 1 in the preparation of anti-tumor drugs, immune checkpoint TDO inhibitor drugs and ICD inducer drugs; wherein, The tumors were human cervical epithelioid carcinoma, human lung cancer, and human hepatocellular carcinoma.

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