Organic compounds modified with lonidamine, cyclometallated complexes based on organic compounds, and preparation methods and applications thereof

Abstract

The present invention discloses a lonidamine-modified organic compound, and also discloses a cyclometallic complex based on the organic compound, and a preparation method and application thereof. The organic compound of the present invention is obtained by chemically modifying the carboxylic acid group of lonidamine, and has good anti-tumor, antibacterial and anti-inflammatory activities. The cyclometallic complex obtained based on the organic compound can solve the problems of poor lipid solubility and large dosage of lonidamine when it is used as an anti-tumor drug or an anti-tumor drug component, and can also solve the problems of cisplatin tolerance and adverse side effects produced by tumor cells when the simple cyclometallic complex is used as an anti-tumor drug or an anti-tumor drug component. In addition, the cyclometallic complex of the present invention can also be used as a glycolysis inhibitor or an energy metabolism inhibitor to stimulate immune function.

Description

Technical Field

[0001] The invention relates to a lonidamine-modified organic compound, and also relates to a cyclometallic complex based on the organic compound and a preparation method and application of the cyclometallic complex. Technical Background

[0002] In recent years, the potential of metal complexes represented by cisplatin in medicinal chemistry has been explored and expanded throughout the periodic table. However, the clinical application and development of platinum anticancer drugs are limited by their drug resistance and adverse side effects. In this case, designing complexes containing other transition metals to make up for the shortcomings of platinum drugs has become the direction of scientific workers' continuous efforts. Among them, Professor Peter Sadler of the University of Warwick in the UK, Professor Paul Dyson of the Swiss Federal Institute of Technology in Lausanne, and Professor Bernhard Keppler of the University of Vienna in Austria successfully connected targeted molecules to transition metals to obtain metal complexes with high efficiency and selectivity.

[0003] Cancer cells mainly obtain energy and substances through glycolysis, glutamine hydrolysis and fatty acid synthesis. Lonidamine, also known as lonidamic acid and trade name Doridamina, was developed by Angelopharm of Germany and launched in Italy in 1986 as an anti-tumor drug. Lonidamine is a tumor thermosensitive drug different from traditional anti-tumor drugs. It does not affect cell proliferation and mainly acts on cell energy metabolism. It changes the mitochondrial ultrastructure of tumor cells, inhibits the activity of hexokinase bound to mitochondria, thereby reducing the glycolysis of tumor cells, and affects the expression of apoptosis-related proteins, inhibiting the oxygen consumption of malignant cells to achieve the purpose of inhibiting and killing tumor cells. It can be used to treat various tumors such as breast cancer, prostate cancer, lung cancer and brain tumors. It is being studied and applied more and more due to its wide anti-cancer spectrum and small side effects. However, lonidamine has defects such as poor lipid solubility, low bioavailability and large dosage, which limit the application of lonidamine as a candidate anti-cancer drug or anti-cancer drug component.

[0004] Breast cancer is the second most common malignant tumor, and triple-negative breast cancer (TNBC) is a highly heterogeneous subtype of breast cancer with the highest recurrence and distant metastasis rates. Due to the lack of expression of hormone receptors and HER2 proteins, current treatment options are still very limited. In recent years, immunotherapy has been shown to be a promising approach for the treatment of TNBC. In 2020, Chinese scientists invented a multi-specific platinum (IV) complex linked to naproxen to inhibit breast cancer proliferation as a PD-L1 inhibitor. Inhibiting glycolysis to block the energy source of tumor cells is an effective strategy to interfere with cell activity and further induce cancer cell death. In 2022, Professor Lu Zhimin and his team studied the increased expression level of PD-L1 in tumor cells under conditions of high levels of glycolysis. Blocking the energy metabolism of triple-negative breast cancer cells and stimulating the immune system to attack are of great significance for cancer treatment. So far, there have been no reports on the study of cyclometallated complexes that induce cancer cell death by inhibiting glycolysis while exerting immune functions. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an organic compound modified with lonidamine, and also to provide a cyclometallic complex based on the above organic compound; the organic compound is obtained by chemically modifying the carboxylic acid group of lonidamine, and itself has good anti-tumor, antibacterial and anti-inflammatory activities. The cyclometallic complex obtained based on the organic compound can solve the problems of poor lipid solubility and large dosage when lonidamine is used as an anti-tumor drug or an anti-tumor drug component, and can also solve the problems of cisplatin tolerance and adverse side effects produced by tumor cells when the simple cyclometallic complex is used as an anti-tumor drug or an anti-tumor drug component; and the cyclometallic complex of the present invention can also be used as a glycolysis inhibitor or an energy metabolism inhibitor to stimulate immune function; another purpose of the present invention is to provide a preparation method of the above cyclometallic complex and its application in preparing anti-tumor drugs or anti-tumor drug components.

[0006] Technical solution: The lonidamine-modified organic compound (LND-NH2) of the present invention has the following structural formula:

[0007]

[0008] The preparation method of the above-mentioned lonidamine-modified organic compound is specifically as follows: under an inert atmosphere, lonidamine, EDCI or DCC, HOBt, TEA and a bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine are dissolved in an organic solvent for reaction, and the crude product generated by the reaction is separated and purified by column chromatography to obtain the lonidamine-modified organic compound.

[0009] The molar ratio of lonidamine to 4-methyl-4'-aminomethyl-2,2'-bipyridine is 1:1 to 1.68.

[0010] Wherein, the structural formula of the bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine is: It was prepared using the synthesis method reported in the literature Bioorthogonal "Labeling after Recognition" Affording an FRET-Based Luminescent Probe for Detecting and Imaging Caspase-3 via Photoluminescence Lifetime Imaging.

[0011] The reaction temperature is -20 to 0°C and the reaction time is 2 to 48 hours.

[0012] Among them, the organic solvent is N,N-dimethylformamide or other formamide solvents, acetamide solvents; EDCI (carbodiimide hydrochloride) or DCC (dicyclohexylcarbodiimide) is used as a carboxyl activation reagent, HOBt (1-hydroxybenzotriazole) is used as a catalyst for the condensation reaction, and TEA (triethylamine) acts as a basic catalyst in the system.

[0013] The above-mentioned lonidamine-modified organic compounds are used for preparing anticancer drugs, anticancer drug components, immune escape inhibition drugs, immune escape inhibition drug components, cell metabolism inhibition drugs, cell metabolism inhibition drug components, immunogenic death inducing drugs, immunogenic death inducing drug components, autophagy inducer drugs and autophagy inducer drug components.

[0014] The cyclometallic complex of the organic compound modified by lonidamine has the following general structural formula:

[0015]

[0016] Wherein, when M is Ir or Rh, X=C, Y=N; when M is Ru or Os, X=N, Y=N.

[0017] Cyclometallic complexes have high lipid solubility, and combining them with lonidamine can effectively increase the uptake of lonidamine by tumor cells. At the same time, cyclometallic complexes have photochemical and photophysical properties and can be localized in cells.

[0018] The synthesis method of the above-mentioned cyclometallic complex tailed with lonidamine is specifically as follows: under an inert atmosphere, the organic ligand modified with lonidamine and the metal precursor are heated to reflux in a mixed solvent of dichloromethane and methanol, the solvent is removed by reduced pressure distillation, and a crude product is obtained by substitution with NH4PF6, and the crude product is separated and purified by column chromatography to obtain the cyclometallic complex tailed with lonidamine.

[0019] The molar ratio of the lonidamine-modified organic ligand to the metal precursor is 1:100.

[0020] Wherein, the temperature of the reflux reaction is 60-80°C and the time is 18h-20h.

[0021] When M is Ir, Rh, Ru or Os, the corresponding structural formulas of the metal precursors are:

[0022] Metallic iridium precursor:

[0023]

[0024] It was prepared using the synthesis method reported in the literature A New Strategy to Fight Metallodrug Resistance: Mitochondria-Relevant Treatment through Mitophagy to Inhibit Metabolic Adaptations of Cancer Cells.

[0025] Metal rhodium precursor:

[0026]

[0027] It was prepared using the synthesis method reported in the literature Mitochondrial targeted rhodium (III) complexes: Synthesis, characterized and antitumor mechanism investigation.

[0028] Ruthenium metal precursor:

[0029]

[0030] The compound is prepared by the synthesis method reported in the literature A lysosome-targeted ruthenium (II) polypyridyl complex as photodynamic anticancer agent.

[0031] Osmium metal precursor:

[0032]

[0033] It was prepared using the synthetic method reported in the literature Physical, spectroscopic, and biological properties of ruthenium and osmium photosensitizers bearing diversely substituted 4,4′-di(styryl)-2,2′-bipyridine ligands.

[0034] The application of the cyclometallic complex tailed with lonidamine in preparing antitumor drugs or antitumor drug components.

[0035] The present invention modifies the active ingredient lonidamine with anti-tumor efficacy with specific groups and coordinates it with a cyclometalated dimer, thereby obtaining a complex with good anticancer activity, excellent immune escape inhibition, cell metabolism inhibition, autophagy induction, and good antibacterial and anti-inflammatory activities. The complex of the present invention can induce tumor cell autophagy, block the energy metabolism of tumor cells, and stimulate the immune system to carry out immune attack.

[0036] Beneficial effects: The cyclometallic complex of the present invention has good lipid solubility, can effectively inhibit tumor cell proliferation, induce ROS production, and induce autophagy in tumor cells; at the same time, it can also inhibit glycolysis and significantly inhibit the immune escape of tumor cells; at the same time, it can also induce immunogenic death in tumor cells, triggering effective anti-tumor immunity, so as to further enhance checkpoint blockade immunotherapy; the cyclometallic complex of the present invention has outstanding effects in anti-cancer, inhibiting cell metabolism, inhibiting cell immune escape, inducing cell immunogenic death, inducing cell autophagy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The localization map of the cyclometalated iridium complex LND-Ir in MDA-MB-231 cells;

[0038] Figure 2 Laser confocal fluorescence imaging and flow cytometry images of the cyclometallated iridium complex LND-Ir promoting the generation of intracellular reactive oxygen species in MDA-MB-231 cells: Figure 2 (a) is a confocal fluorescence imaging image. Figure 2 (b) is the flow cytometry graph of cells at different concentrations;

[0039] Figure 3This is the confocal fluorescence imaging of the cyclometallated iridium complex LND-Ir inducing the production of autophagosomes in MDA-MB-231 cells;

[0040] Figure 4 The immunoblot protein image shows that the cyclometallated iridium complex LND-Ir induces HK II protein and Akt protein in MDA-MB-231 cells;

[0041] Figure 5 This is the confocal fluorescence imaging of PD-L1 of the cyclometallated iridium complex LND-Ir in MDA-MB-231 cells;

[0042] Figure 6 ICD diagram induced by cyclometallated iridium complex LND-Ir in MDA-MB-231 cells: Figure 6 (a) is CRT confocal fluorescence imaging. Figure 6 (b) is the immunoblot protein image of induced HMGB1 protein in cells. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0044] Example 1

[0045] The preparation method of the cyclometalated iridium (III) complex (LND-Ir) of the present invention is specifically as follows:

[0046]

[0047] (1) Preparation of lonidamine-modified bipyridine ligand: Under argon atmosphere, lonidamine (160.58 mg, 0.5 mmol) and EDCI (9.59 g, 50 mmol) were weighed in a two-necked flask, and the mixture was evacuated and introduced with argon. Anhydrous DMF (2 mL) and TEA (100 μL) were added to the mixture under ice bath conditions and stirred for 60 min. A DMF solution (50 μL) containing HOBt (113.50 mg, 0.84 mmol) was then added and stirred for 60 min. min later, the ice bath was removed and the temperature was restored to room temperature; a DMF solution (50 μL) containing 4-methyl-4'-aminomethyl-2,2'-bipyridine (167.10 mg, 0.84 mmol) was added thereto, and the reaction was carried out for 48 h; after the reaction was completed, 20 mL of distilled water was added, and the mixture was extracted with dichloromethane (3×30 ml), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation, and the crude product was further purified by flash chromatography to obtain a beige solid with a yield of 70%. 1H NMR(400MHz, DMSO-d6)δ9.16(t,J=6.3Hz,1H),8.60(d,J=5.0Hz,1H),8.48(d,J= 4.9Hz,1H),8.37(d,J=1.5Hz,1H),8.22(d,J=8.0Hz,2H),7.78(d,J=8.6Hz,1H), 7.71(d,J=2.1Hz,1H),7.52–7.46(m,1H),7.41–7.29(m,3H),7.26(dd,J=4.9,1. 6Hz, 1H), 6.83 (d, J = 8.4Hz, 1H), 5.86 (s, 2H), 4.59 (d, J = 6.2Hz, 2H), 2.40 (s, 3H).

[0048] (2) Under an argon protective atmosphere, the lonidamine-modified bipyridine ligand (25.1 mg, 0.05 mmol) synthesized in step (1) and the cyclometallated iridium dimer (5.36 g, 5 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (in the mixed solvent, the volume ratio of anhydrous dichloromethane to anhydrous methanol was 1:2), and heated under reflux and stirred at 80° C. for 18 h; after the reaction was completed, the solvent was distilled off under reduced pressure, a saturated NH4PF6 methanol solution was added, and the solid was collected by centrifugation after stirring for 5 h. The crude product was further purified by column chromatography to obtain a yellow solid LND-Ir with a yield of 75%. 1H NMR (400MHz, DMSO-d6) δ9.08(t,J=6.1Hz,1H),8.83(d,J=1.6Hz,1H),8.72(d,J=1.6Hz,1H),8.25(t,J=8.3Hz,2H),8.19(d,J=8.2Hz,1H),7.97–7 .86(m,5H),7.78(dd,J=7.1,4.4Hz,2H),7.72(d,J=2.2Hz,1H),7.69(d,J=5.7Hz,1H),7.63(d,J=5.8Hz,2H),7.58(dd,J=5.7,1.8Hz,1H),7.53(dd ,J=5.7,1.6Hz,1H),7.49(ddd,J=8.4,6.8,1.2Hz,1H),7.36–7.33(m,1H),7.30(d,J=7.4Hz,1H),7.16(dddd,J=7.3,5.7,4.3,1.4Hz,2H),7.00(d td,J=9.2,7.5,1.3Hz,2H),6.88(qd,J=7.6,1.4Hz,2H),6.79(d,J=8.4Hz,1H),6.18(d,J=7.6Hz,2H),5.84(s,2H),2.53(s,3H).ESI-MS(positive mode,m / z):calcd.1002.21,found 1002.3.

[0049] Example 2

[0050] The preparation method of the cyclometalated rhodium (III) complex (LND-Rh) of the present invention is specifically as follows:

[0051]

[0052] Under an argon protective atmosphere, the lonidamine-modified bipyridine ligand (25.1 mg, 0.05 mmol) synthesized in step (1) of Example 1 and the cyclometallated rhodium dimer (4.5 g, 5 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (in the mixed solvent, the volume ratio of anhydrous dichloromethane to anhydrous methanol was 1:2), and heated under reflux and stirred at 80°C for 18 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure, a saturated methanol solution of NH4PF6 was added, and the solid was collected by centrifugation after stirring for 8 h. The crude product was further purified by column chromatography to obtain a yellow solid LND-Rh with a yield of 80%. 1H NMR (400MHz, DMSO-d6) δ10.08(t,J=6.1Hz,1H),9.83(d,J=1.6Hz,1H),9.72(d,J=1.6Hz,1H),9.25(t,J=8.3Hz,2H),9.19(d,J=8.2Hz,1H),8.97– 7.86(m,5H),8.78(dd,J=7.1,4.4Hz,2H),8.72(d,J=2.2Hz,1H),8.69(d,J=5.7Hz,1H),8.63(d,J=5.8Hz,2H),8.58(dd,J=5.7,1.8Hz,1H),8.53(d d,J=5.7,1.6Hz,1H),8.49(ddd,J=8.4,6.8,1.2Hz,1H),8.36–8.33(m,1H),8.30(d,J=7.4Hz,1H),8.16(dddd,J=7.3,5.7,4.3,1.4Hz,2H),8.00(d td,J=9.2,7.5,1.3Hz,2H),7.88(qd,J=7.6,1.4Hz,2H),7.79(d,J=8.4Hz,1H),7.18(d,J=7.6Hz,2H),6.84(s,2H),2.53(s,3H).ESI-MS(positive mode,m / z):calcd.912.21,found 912.3.

[0053] Example 3

[0054] The preparation method of the cyclometallated ruthenium (II) complex (LND-Ru) of the present invention is specifically as follows:

[0055]

[0056] Under an argon protective atmosphere, the lonidamine-modified bipyridine ligand (25.1 mg, 0.05 mmol) synthesized in step (1) of Example 1 and a metal ruthenium precursor (2.42 g, 5 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (in the mixed solvent, the volume ratio of anhydrous dichloromethane to anhydrous methanol was 1:2), and heated under reflux and stirred at 80°C for 18 h. After the reaction was completed, the solvent was distilled off under reduced pressure, a saturated methanol solution of NH4PF6 was added, and the solid was collected by centrifugation after stirring for 8 h. The crude product was further purified by column chromatography to obtain a yellow solid LND-Ru with a yield of 78%. 1H NMR (400MHz, DMSO-d6) δ9.18(t,J=6.1Hz,1H),8.93(d,J=1.6Hz,1H),8.82(d,J=1.6Hz,1H),8.35(t,J=8.3Hz,2H),8.29(d,J=8.2Hz,1H),8.07–7 .96(m,5H),7.88(dd,J=7.1,4.4Hz,2H),7.82(d,J=2.2Hz,1H),7.79(d,J=5.7Hz,1H),7.73(d,J=5.8Hz,2H),7.68(dd,J=5.7,1.8Hz,1H),7.63(dd ,J=5.7,1.6Hz,1H),7.59(ddd,J=8.4,6.8,1.2Hz,1H),7.46–7.43(m,1H),7.40(d,J=7.4Hz,1H),7.26(dddd,J=7.3,5.7,4.3,1.4Hz,2H),7.10(d td,J=9.2,7.5,1.3Hz,2H),6.98(qd,J=7.6,1.4Hz,2H),6.89(d,J=8.4Hz,1H),6.28(d,J=7.6Hz,2H),5.94(s,2H),2.63(s,3H).ESI-MS(positive mode,m / z):calcd.457.5,found457.7.

[0057] Example 4

[0058] The preparation method of the cyclometallated osmium (II) complex (LND-Os) of the present invention is specifically as follows:

[0059]

[0060] Under an argon protective atmosphere, the lonidamine-modified bipyridine ligand (25.1 mg, 0.05 mmol) synthesized in step (1) of Example 1 and a metal osmium precursor (2.87 g, 5 mmol) were dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous methanol (in the mixed solvent, the volume ratio of anhydrous dichloromethane to anhydrous methanol was 1:2), and heated under reflux and stirred at 80°C for 18 h. After the reaction was completed, the solvent was distilled off under reduced pressure, a saturated methanol solution of NH4PF6 was added, and the solid was collected by centrifugation after stirring for 8 h. The crude product was further purified by column chromatography to obtain a yellow solid LND-Ir with a yield of 60%. 1H NMR (400MHz, DMSO-d6) δ9.08(t,J=8.1Hz,1H),10.83(d,J=1.6Hz,1H),10.72(d,J=1.6Hz,1H),10.25(t,J=8.3Hz,2H),10.19(d,J=8.2Hz,1H),9.9 7–9.86(m,5H),9.78(dd,J=7.1,4.4Hz,2H),9.72(d,J=2.2Hz,1H),9.69( d,J=5.7Hz,1H),9.63(d,J=5.8Hz,2H),9.58(dd,J=5.7,1.8Hz,1H),9.53( dd,J=5.7,1.6Hz,1H),9.49(ddd,J=8.4,6.8,1.2Hz,1H),9.36–9.33(m,1H),9.30(d,J=7.4Hz,1H),9.16(dddd,J=7.3,5.7,4.3,1.4Hz,2H),9.00( dtd,J=9.2,7.5,1.3Hz,2H),8.88(qd,J=7.6,1.4Hz,2H),8.79(d,J=8.4Hz,1H),8.18(d,J=7.6Hz,2H),7.84(s,2H),4.53(s,3H).ESI-MS(positive mode,m / z):calcd.502.5,found 502.5.

[0061] Comparative Example 1

[0062] The structural formula of the metal complex not modified with lonidamine is:

[0063]

[0064] Wherein, M=Ir or Rh, X=C, Y=N; M=Ru or Os, X=N, Y=N.

[0065] MTT colorimetry was used to analyze the anti-tumor cell proliferation effects of cyclometallated complexes LND-M, LND, cyclometallated complexes M-NH2 without lonidamine, and cisplatin CDDP. 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 570nm, so it can be used to analyze the anti-tumor cell proliferation effects of cyclometallated complexes LND-M, LND, cyclometallated complexes M-NH2 without lonidamine, and cisplatin CDDP. 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 570 nm To analyze cell proliferation.

[0066] The specific experimental steps are as follows:

[0067] (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;

[0068] (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;

[0069] (3) After the cells adhered to the wall, 100 μL of fresh culture medium containing different concentration gradients of LND-Ir, LND, Ir-NH2 and cisplatin CDDP was added to each well, and then placed in a constant temperature box for further incubation;

[0070] (4) After incubation for 48 hours, 2 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). 570nm , calculate the cell proliferation inhibition rate and find the IC 50 Value (drug concentration corresponding to 50% inhibition rate).

[0071] The cytotoxicity of the complex LND-Ir prepared in Example 1 to human lung cancer cells A549, human breast cancer cells MCF-7, human triple-negative breast cancer cells MDA-MB-231 and normal liver cells LO2:

[0072] Table 1 shows the MTT test results of compounds LND (lonidamine), Ir-NH2 (metal Ir complex without lonidamine tail), LND-NH2 (organic ligand modified with lonidamine), LND-Ir and cisplatin CDDP

[0073]

[0074] The results showed that lonidamine itself had little cytotoxicity to cancer cells and normal cell lines. 50 The values ​​were all above 100 μM. The organic compound LND-NH2 modified with the specific group of lonidamine had certain activity against cancer cells. The cyclometallic complex Ir-NH2 without lonidamine had certain toxicity to cancer cells and normal cell lines, but the toxicity was relatively small. The cytotoxicity of the cyclometallic complex LND-Ir after connecting with lonidamine was significantly improved, especially for triple-negative breast cancer cells. Compared with the activity of cisplatin against triple-negative breast cancer (~32.4 μM), the activity of LND-Ir (1.9 μM) increased by nearly 17 times.

[0075] The cytotoxicity of the complex LND-Rh prepared in Example 2 to human lung cancer cells A549, human breast cancer cells MCF-7, human triple-negative breast cancer cells MDA-MB-231 and normal liver cells LO2:

[0076] Table 2 shows the MTT test results of compounds LND, Rh-NH2, LND-Rh and cisplatin CDDP

[0077]

[0078] The results showed that the cyclometal complex Rh-NH2 without lonidamine had a certain toxicity to cancer cells and normal cell lines, but the toxicity was relatively low. The cyclometal complex LND-Rh with lonidamine had a significantly improved cytotoxicity, especially against triple-negative breast cancer cells. Compared with the activity of cisplatin against triple-negative breast cancer (~32.4μM), the activity of LND-Rh (4.9μM) increased by nearly 7 times.

[0079] The cytotoxicity of the complex LND-Ru prepared in Example 3 to human lung cancer cells A549, human breast cancer cells MCF-7, human triple-negative breast cancer cells MDA-MB-231 and normal liver cells LO2:

[0080] Table 3 shows the MTT test results of compounds LND, Ru-NH2, LND-Ru and cisplatin CDDP

[0081]

[0082]

[0083] The results showed that the cyclometallic complex Ru-NH2 without lonidamine had a certain toxicity to cancer cells and normal cell lines, but the toxicity was relatively low. The cyclometallic complex LND-Ru after lonidamine was significantly improved in cytotoxicity, especially for triple-negative breast cancer cells. Compared with the activity of cisplatin against triple-negative breast cancer (~32.4μM), the activity of LND-Ru (2.9μM) increased by nearly 11 times.

[0084] Cytotoxicity of the complex LND-Os prepared in Example 4 to human lung cancer cells A549, human breast cancer cells MCF-7, human triple-negative breast cancer cells MDA-MB-231 and normal liver cells LO2:

[0085] Table 4 shows the MTT test results of compounds LND, Os-NH2, LND-Os and cisplatin CDDP

[0086]

[0087] The results showed that the cyclometallic complex Os-NH2 without lonidamine had a certain toxicity to cancer cells and normal cell lines, but the toxicity was relatively low. The cyclometallic complex LND-Os, which was linked to lonidamine, had a significantly improved cytotoxicity, especially against triple-negative breast cancer cells. Compared with the activity of cisplatin against triple-negative breast cancer (~32.4μM), the activity of LND-Os (3.9μM) increased by nearly 8 times.

[0088] Example 5

[0089] Localization of the cyclometallated iridium complex LND-Ir prepared in Example 1 in cells.

[0090] Methods: MDA-MB-231 cells were seeded in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, 2 μM LND-Ir was added for 8 h, the culture medium was aspirated, the cells were washed twice with PBS, 500 μL of the prepared mitochondrial green fluorescent probe was added, and the cells were incubated in a 37°C incubator for 30 min. The probe was aspirated, the cells were washed twice with PBS, and then fresh preheated serum-free culture medium was replaced. The cells were then observed immediately under a confocal microscope.

[0091] Example 1 Intracellular localization of cyclometallated iridium complex LND-Ir Figure 1 As shown, the results showed that the cyclometalated iridium complex LND-Ir could be taken up by MDA-MB-231 cells through the cell membrane in a large amount in a short time, and was mainly distributed in mitochondria, with a colocalization coefficient of 0.85.

[0092] Example 6

[0093] Application of the cyclometallated iridium complex LND-Ir prepared in Example 1 to inducing the generation of intracellular reactive oxygen species:

[0094] Method 1: Confocal microscopy to detect ROS in cancer cells. MDA-MB-231 cells were seeded in a 35 mm Corning laser confocal culture dish. When the cell density grew to 70%, 4 μM LND-Ir and Ir-NH2 were added for 24 h, and then the cells were stained with serum-free medium containing 10 mM H2DCFH-DA at 37 ° C for 30 min in the dark, and then immediately observed with a confocal microscope, with an excitation wavelength of 488 nm and an emission wavelength of 530 ± 20 nm.

[0095] Method 2: Flow cytometry was used to detect ROS in tumor cells. MDA-MB-231 cells were treated with 2, 4, and 6 μM LND-Ir and 6 μM Ir-NH2 for 24 h, and then stained with serum-free medium containing 10 μm H2DCFH-DA at 37 °C for 30 min in the dark. The cells were centrifuged and the supernatant was discarded. The cells were washed three times with serum-free medium to remove H2DCFH-DA that did not enter the cells. The green fluorescence intensity was measured by flow cytometry within half an hour after the cells were collected. The excitation wavelength was 488 nm and the emission wavelength was 530 ± 20 nm. The average fluorescence intensity of green light was analyzed using FlowJo 7.6 (Tree Star, OR, USA) software.

[0096] Example 1 The results of the cyclometalated iridium complex LND-Ir on inducing the generation of intracellular reactive oxygen species are shown in Figure 2 As shown, the results showed that compared with the control group, both flow cytometry and confocal microscopy results showed that the green fluorescence was significantly enhanced after LND-Ir treatment, indicating that the complex LND-Ir effectively induced an increase in the content of intracellular reactive oxygen species.

[0097] Example 7

[0098] Application of the cyclometallated iridium complex LND-Ir prepared in Example 1 to inducing autophagy in MDA-MB-231 cells:

[0099] Methods: MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density grew to 70%, LND-Ir and Ir-NH2 were added at a concentration of 4 μM for 24 h, and then washed twice with PBS. The cells were fixed with 4% paraformaldehyde (PFA), the fixative was removed, and the cells were washed three times with PBS. After permeabilization with 0.2% TritonX-100 and blocking with 1.5% BSA, LC3 primary antibody dilution was added and incubated for 1 hour; the primary antibody was removed, the cells were washed three times with PBS, and incubated with FITC-conjugated secondary antibody in the dark for 1 hour; the secondary antibody was removed, the cells were 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 488 nm and an emission wavelength of 530 ± 20 nm.

[0100] Example 1: Laser confocal images of cells induced to autophagy by the cyclometallated iridium complex LND-Ir Figure 3 As shown, the results showed that after MDA-MB-231 cells were incubated with 4 μM LND-Ir for 24 h, autophagosomes were produced, indicating that LND-Ir can induce MDA-MB-231 cells to die by autophagy.

[0101] Example 8

[0102] Application of the cyclometallated iridium complex LND-Ir prepared in Example 1 in inhibiting glycolysis in MDA-MB-231 cells:

[0103] Methods: Western blotting (WB) was used to detect changes in autophagy protein content. Pre-prepared cell culture medium containing complexes LND-Ir (2, 4, 6 μM), lonidamine (6 μM) and Ir-NH2 (6 μM) was added to the 100 mm culture dish of MDA-MB-231 cells that had grown on the wall. After 24 hours of drug treatment, the cells were collected by centrifugation, washed with PBS to remove the serum in the residual culture medium, and added with Biyuntian RIPA strong lysis buffer containing PMSF for 20 minutes of whole cell lysis. During this process, a low temperature environment of 4°C was maintained to ensure that the protein was not denatured. After the end, the cells were centrifuged at 13400 rpm for 20 minutes under low temperature conditions. The supernatant obtained by centrifugation was the cell whole protein sample required for the experiment; the protein concentration in the above protein samples was determined using the BCA protein content detection kit; the expression content of different proteins in the samples was detected by SDS-PAGE gel electrophoresis. After the gel is prepared, add the same volume of protein sample to each well for gel electrophoresis experiment, and stop electrophoresis immediately after proper separation; transfer the target protein to the PVDF membrane using the wet method. After completion, place the membrane in a solution containing 5% skim milk powder for blocking for 2 hours. Dilute the primary antibody with skim milk powder according to the instructions for use of the antibody in the corresponding proportion, and place the blocked membrane in the primary antibody incubation solution for a period of time at room temperature to allow it to specifically bind to the target protein. After completion, wash with PBST (5×6min / time). Place the washed membrane in the pre-prepared secondary antibody incubation solution for a period of time to allow it to bind to the primary antibody, and wash it with PBST as well. Prepare an equal volume of ECL developer, cover it on the PVDF membrane, and use the chemiluminescence imaging system to shoot after 2 minutes of treatment.

[0104] Example 1 The experimental results of the complex LND-Ir inducing the expression of glycolysis-related proteins HK II and Akt in MDA-MB-231 cells are as follows Figure 4 As shown, the results showed that compared with the control group without drug treatment, the expression of glycolysis-related proteins HK II and Akt was significantly downregulated after the cells were treated with the complex LND-Ir, indicating that the complex LND-Ir can inhibit the expression of cell HK II and Akt proteins to inhibit their glycolysis ability.

[0105] Example 9

[0106] Application of the cyclometallated iridium complex LND-Ir prepared in Example 1 in inhibiting immune escape in MDA-MB-231 cells:

[0107] Methods: MDA-MB-231 cells were seeded in 35 mm Corning laser confocal culture dishes. When the cell density grew to 70%, LND-Ir and Ir-NH2 at a concentration of 4 μM were added for 24 h, respectively. After treatment, the cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA), the fixative was removed, and the cells were washed 3 times with PBS. After permeabilization with 0.2% TritonX-100 and blocking with 1.5% BSA, PD-L1 primary antibody dilution was added and incubated for 1 hour. The primary antibody was removed, washed 3 times with PBS, and incubated with FITC-conjugated secondary antibody in the dark for 1 hour. The secondary antibody was removed, washed 3 times with PBS, and the cell nucleus was stained with DAPI for 5 minutes. The dye was removed and washed 3 times with PBS, and the cells were imaged under a confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 530 ± 20 nm.

[0108] Example 1: Laser confocal image of the cyclometallated iridium complex LND-Ir inhibiting cellular immune escape Figure 5 The results showed that after MDA-MB-231 cells were incubated with 4 μM LND-Ir for 24 h, the expression of programmed death ligand PD-L1 was inhibited, indicating that LND-Ir can inhibit the immune escape of MDA-MB-231 cells.

[0109] Example 10

[0110] Application of the cyclometallated iridium complex LND-Ir prepared in Example 1 to induce ICD in MDA-MB-231 cells:

[0111] Method 1: MDA-MB-231 cells were seeded in 35mm Corning laser confocal culture dishes. When the cell density grew to 70%, LND-Ir and Ir-NH2 at a concentration of 4μM were added for treatment for 24h. After treatment, the cells were washed twice with PBS, fixed with 4% paraformaldehyde (PFA), the fixative was removed, and the cells were washed 3 times with PBS. After permeabilization with 0.2% TritonX-100 and blocking with 1.5% BSA, CRT primary antibody dilution was added and incubated for 1 hour. The primary antibody was removed, washed 3 times with PBS, and incubated with FITC-conjugated secondary antibody in the dark for 1 hour. The secondary antibody was removed, washed 3 times with PBS, and the cell nucleus was stained with DAPI for 5 minutes. The dye was removed and washed 3 times with PBS, and the cells were imaged under a confocal microscope with an excitation wavelength of 488nm and an emission wavelength of 530±20nm.

[0112] Method 2: Western blotting (WB) was used to detect the changes in the content of ICD-related protein HMGB1. Pre-prepared cell culture medium containing the complex LND-Ir (2, 4, 6 μM), lonidamine (6 μM) and Ir-NH2 (6 μM) was added to the 100 mm culture dish of MDA-MB-231 cells that had grown on the wall. After 24 hours of drug treatment, the cells were collected by centrifugation, washed with PBS to remove the serum in the residual culture medium, and added with Biyuntian RIPA strong lysis buffer containing PMSF for 20 minutes of whole cell lysis. During this whole process, a low temperature environment of 4°C was maintained to ensure that the protein was not denatured. After the end, the cells were centrifuged at 13400 rpm for 20 minutes under low temperature conditions. The supernatant obtained by centrifugation was the cell whole protein sample required for the experiment; the protein concentration in the above protein samples was determined using the BCA protein content detection kit; and the expression content of different proteins in the samples was detected by SDS-PAGE gel electrophoresis. After the gel is prepared, add the same volume of protein sample to each well for gel electrophoresis experiment, and stop electrophoresis immediately after proper separation; transfer the target protein to the PVDF membrane using the wet method. After completion, place the membrane in a solution containing 5% skim milk powder for blocking for 2 hours. Dilute the primary antibody with skim milk powder according to the instructions for use of the antibody in the corresponding proportion, and place the blocked membrane in the primary antibody incubation solution for a period of time at room temperature to allow it to specifically bind to the target protein. After completion, wash with PBST (5×6min / time). Place the washed membrane in the pre-prepared secondary antibody incubation solution for a period of time to allow it to bind to the primary antibody, and wash it with PBST as well. Prepare an equal volume of ECL developer, cover it on the PVDF membrane, and use the chemiluminescence imaging system to shoot after 2 minutes of treatment.

[0113] Example 1 The results of the cyclometallated iridium complex LND-Ir on inducing immunogenic cell death ICD are as follows Figure 6 The results showed that compared with the control group, the CRT of cells was exposed and the expression of HMGB1 protein was downregulated after LND-Ir treatment, indicating that LND-Ir can induce immunogenic death of MDA-MB-231 cells.

Claims

1. A lonidamine-modified organic compound, characterized in that: Its structural formula is as follows:

2. The method for preparing a lonidamine-modified organic compound according to claim 1, characterized in that: Specifically, under an inert atmosphere, lonidamine, EDCI or DCC, HOBt, TEA and a bridging ligand 4-methyl-4'-aminomethyl-2,2'-bipyridine are dissolved in an organic solvent for reaction, and the crude product generated by the reaction is separated and purified by column chromatography to obtain an organic compound modified with lonidamine.

3. The method for preparing a lonidamine-modified organic compound according to claim 2, characterized in that: The molar ratio of lonidamine to 4-methyl-4'-aminomethyl-2,2'-bipyridine is 1:1 to 1.

68.

4. The method for preparing a lonidamine-modified organic compound according to claim 2, characterized in that: The reaction temperature is -20 to 0°C; the reaction time is 2 to 48 hours.

5. Use of the lonidamine-modified organic compound according to claim 1 in the preparation of anticancer drugs, immune escape inhibitory drugs, cell metabolism inhibitory drug components, immunogenic death inducing drug components and autophagy inducer drug components.

6. A cyclometallic complex based on an organic compound according to any one of claims 1 to 5, characterized in that: Its structure is as follows:

7. The method for preparing the cyclometallated complex according to claim 6, characterized in that: Specifically, the organic ligand modified with lonidamine and the metal precursor are heated to reflux in a mixed solvent of dichloromethane and methanol under an inert atmosphere, the solvent is removed by distillation under reduced pressure, and the crude product is replaced with NH4PF6 to obtain a crude product, and the crude product is separated and purified by column chromatography to obtain a cyclometallic complex tailed with lonidamine; Among them, the structural formulas of the metal precursors are: Metallic iridium precursor: Metal rhodium precursor: Ruthenium metal precursor: Osmium metal precursor:

8. The method for preparing a cyclometallated complex according to claim 7, characterized in that: The molar ratio of lonidamine-modified organic ligand to metal precursor was 1:

100.

9. The method for preparing a cyclometallated complex according to claim 7, characterized in that: The temperature of the heating reflux reaction is 60-80°C and the time is 18h-20h.

10. Use of the cyclometallated complex according to claim 7 in preparing anti-tumor drugs.

Images