Preparation method of glycyrrhetinic acid-gold (I) compound and application of glycyrrhetinic acid-gold (I) compound in liver cancer

Abstract

The invention discloses a preparation method of a glycyrrhetinic acid-gold (I) compound and application of the glycyrrhetinic acid-gold (I) compound in liver cancer. Belongs to the field of medicines. The glycyrrhetinic acid-gold (I) compound comprises an active ingredient of a traditional Chinese medicine liquorice, glycyrrhetinic acid with a liver targeting function, an N-heterocyclic carbene gold (I) part and a BMS-1 skeleton with PD-L1 inhibitory activity. The compounds are useful in the immunotherapy of hepatocellular carcinoma (HCC). Pharmacodynamic evaluation on HepG2 cells proves that the compound can inhibit expression of tumor cells PD-L1 while causing an effect of immunogen cell death (ICD) of liver cancer cells, the anti-tumor immune effect is enhanced from the positive and negative aspects, and a high-efficiency and low-toxicity potential compound is provided for clinical treatment of hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to a preparation method of glycyrrhetinic acid-gold (I) compound and its application in liver cancer; specifically, it relates to the synthesis of glycyrrhetinic acid-gold derivative with dual immune functions of ICD and PD-L1 inhibition and its application in liver cancer immunotherapy. Background Art

[0002] At present, hepatocellular carcinoma (HCC) has ranked the 4th among all newly added cancer cases; with the gradual becoming of immunotherapy represented by immune checkpoint inhibitors as the first-line treatment method, its disadvantages are gradually emerging, such as insufficient treatment targeting, sequelae left over, large individual differences in the treatment process, etc. Moreover, since hepatocellular carcinoma belongs to "cold tumor", problems such as immune cell deficiency and antigen presentation process disorder appear, resulting in a decline in the effect of immunotherapy; therefore, it is urgent to find a lead compound that can enhance the immunogenicity of liver cancer cells and specifically target liver cancer cells, in order to solve the disadvantages of insufficient targeting and excessive toxicity of single use of immune checkpoint inhibitors.

[0003] According to clinical manifestations, targeted therapy is a treatment method mainly based on sorafenib and lenvatinib for specific tumor targets or signaling pathways, which can accurately attack cells and reduce the damage to normal cells; immunotherapy uses PD-1 inhibitors such as camrelizumab and pembrolizumab to block the interaction between PD-L1 and PD-L2 and the PD-1 receptor, activate the patient's own immune system, and help it attack and kill tumor cells, and has relatively low toxic and side effects; but at the same time, it is also found that single use is prone to problems such as drug resistance, insufficient targeting, sequelae left over, and large individual differences in the treatment process. Therefore, glycyrrhetinic acid-gold (I) compound and PD-L1 inhibitor are combined to obtain a more prominent treatment effect. Summary of the Invention

[0004] In view of the above problems, the present invention provides a glycyrrhetinic acid-gold (I) compound for treating hepatocellular carcinoma, which can induce the ICD effect of liver cancer cells while inhibiting the expression of tumor cell PD-L1. The present invention also provides its preparation method and application.

[0005] The technical solution of the present invention is: the present invention discloses a glycyrrhetinic acid-gold (I) compound; the compound includes glycyrrhetinic acid, PD-L1 immune checkpoint inhibitory group BMS-1 and diazabicyclic carbene gold (I) compound;

[0006] The molar ratio is 1:1:1.

[0007] Furthermore, the chemical structural formula of the compound is shown in formula (I):

[0008]

[0009] Further, its synthetic route is as follows:

[0010]

[0011] Further, a pharmaceutical composition contains a therapeutically effective amount of one or more of the glycyrrhetinic acid-gold(I) compounds as described above and a pharmaceutically acceptable carrier.

[0012] Further, the use of the glycyrrhetinic acid-gold(I) compound in the preparation of a drug for treating liver cancer.

[0013] Further, the glycyrrhetinic acid-gold(I) compound for treating liver cancer achieves immune killing of liver cancer cells by targeting and activating the immunogenic cell death (ICD) effect of liver cancer cells and inhibiting the expression of PD-L1.

[0014] Specifically, the preparation of a class of glycyrrhetinic acid-gold(I) compounds for treating hepatocellular carcinoma and their inhibitory effect on liver cancer cells; based on the basic skeleton of diazabicycloalkylidene gold(I), introducing a glycyrrhetinic acid ligand with liver cancer cell targeting function and a BMS-1 fragment with PD-L1 inhibitory activity can inhibit the expression of PD-L1 while inducing the ICD effect of liver cancer cells, achieving the purpose of enhancing the effect of immunotherapy.

[0015] Licorice has the effects of coordinating various herbs and detoxifying; 18β-glycyrrhetinic acid (GA), as its active ingredient, has better curative effects and lower toxicity, and GA has an affinity for protein kinase Cα on the surface of liver cells, thus having the property of liver targeting; at the same time, according to the potential of gold compounds to induce the ICD effect and the PD-L1 inhibitor BMS-1 to inhibit the escape of tumor cells from the immune system, we chose to introduce glycyrrhetinic acid and the PD-L1 inhibitor BMS-1 on the basis of gold compounds to design a novel structure of a compound that can both activate the ICD effect of tumor cells and inhibit the expression of PD-L1 - the glycyrrhetinic acid-gold(I) compound, in order to form an immune cycle of ICD effect and PD-L1 immune checkpoint inhibition and enhance the immune therapy effect of liver cancer.

[0016] The beneficial effects of the invention are: the glycyrrhetinic acid-gold(I) compound 7c has a good inhibitory effect on hepatocellular carcinoma, can inhibit the expression of PD-L1 while inducing the ICD effect of liver cancer cells; and 7c has the advantages of strong targeting, good therapeutic effect, high stability, etc. Description of the Drawings

[0017] Figure 1 is the hydrogen spectrum of the glycyrrhetinic acid-gold compound 7a in the embodiment of the present invention;

[0018] Figure 2 It is the carbon spectrum of glycyrrhetinic acid-gold compound 7a in the embodiment of the present invention;

[0019] Figure 3 It is the hydrogen spectrum of glycyrrhetinic acid-gold compound 7b in the embodiment of the present invention;

[0020] Figure 4 It is the carbon spectrum of glycyrrhetinic acid-gold compound 7b in the embodiment of the present invention;

[0021] Figure 5 It is the hydrogen spectrum of glycyrrhetinic acid-gold compound 7c in the embodiment of the present invention;

[0022] Figure 6 It is the carbon spectrum of glycyrrhetinic acid-gold compound 7c in the embodiment of the present invention;

[0023] Figure 7 It is the fluorescence schematic diagram of the inhibition of the expression of PD-L1 on the surface of HepG2 cells by glycyrrhetinic acid-gold compounds 7a, 7b and 7c in the embodiment of the present invention;

[0024] Figure 8 It is the in vivo anti-tumor effect diagram of glycyrrhetinic acid-gold compound 7c in the C57BL / 6 mouse tumor model established by Hepa 1-6 cells in the embodiment of the present invention. Detailed implementation manners

[0025] To more clearly illustrate the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below: Definitions:

[0026] IC 50 is the 50% inhibitory concentration, that is, the concentration corresponding to B / B0 = 50%. Half inhibition is used to measure the anti-tumor proliferation activity of a compound. The lower the half inhibition, the stronger the anti-proliferation activity.

[0027] Western Blot colors a cell or biological tissue sample treated by gel electrophoresis with a specific antibody, and obtains information on the expression of a specific protein in the analyzed cell or tissue by analyzing the position and depth of the coloring.

[0028] 1 The 1H-NMR spectrum is the response of hydrogen atomic nuclei in a magnetic field; using nuclear magnetic resonance hydrogen spectrum, the position and number of hydrogen atoms in an organic molecule can be determined on the carbon skeleton, and then the carbon skeleton structure of the organic compound can be inferred.

[0029] The CCK-8 method is a method for cell proliferation and cytotoxicity assays; the principle of this method is based on the reduction reaction of WST-8 (water-soluble tetrazolium salt); in the presence of an electron-coupling reagent, WST-8 is reduced by mitochondrial dehydrogenase in cells to a water-soluble orange product, namely formazan dye; the amount of this product generated is proportional to the number of living cells, so the number of cells can be indirectly reflected by measuring the absorbance of the product.

[0030] Example 1

[0031] Synthesis method of glycyrrhetinic acid-gold(I) compound:

[0032] Preparation of intermediate 2a: In a 50 mL single-neck flask, successively add compound 1 (BMS-1) (1.00 g, 2.10 mmol), K 2 CO 3 (0.87 g, 6.29 mmol), 1,3-dibromopropane (0.63 g, 3.15 mmol) and anhydrous DMF (20 mL), stir at room temperature for 1 - 24 h, monitor by TLC, after the reaction is complete, add ethyl acetate (100 mL) for dilution, wash successively with 1N HCl (10 mL) and saturated brine (10 mL × 3), dry over anhydrous sodium sulfate, remove the solvent, purify by column chromatography (SiO 2 , EA / PE = 1:10 (v:v)), concentrate, dry to obtain 1.04 g of a pale yellow oil, yield: 84%.

[0033] The preparation method of intermediate 2b is the same as that of 2a, where 1,3-dibromopropane is replaced with an equimolar amount of 1,4-dibromobutane, yield: 96%.

[0034] The preparation method of intermediate 2c is the same as that of 2a, where 1,3-dibromopropane is replaced with an equimolar amount of 1,5-dibromopentane, yield: 88%. 11H NMR (500 MHz, Chloroform-d) δ 7.41 (dt, J = 34.0, 7.2 Hz, 5H), 7.35–7.33 (m, 2H), 7.31 (d, J = 7.6 Hz, 1H), 6.26 (s, 2H), 5.11 (s, 2H), 4.32–4.25 (m, 1H), 4.16 (dd, J = 11.8, 5.3 Hz, 1H), 3.94–3.85 (m, 2H), 3.78 (s, 6H), 3.44 (dd, J = 11.4, 4.8 Hz, 2H), 3.17 (s, 1H), 2.98 (s, 1H), 2.91 (s, 1H), 2.30 (s, 3H), 2.29–2.21 (m, 1H), 1.92 (dd, J = 14.6, 7.1 Hz, 2H), 1.80–1.70 (m, 4H), 1.68 (s, 2H), 1.57 (dt, J = 15.0, 7.5 Hz, 4H).

[0035] Preparation of Intermediate 3a: Add Intermediate 2a (0.98 g, 1.65 mmol), 1-ethylimidazole (1.58 g, 16.5 mmol) and anhydrous acetonitrile (100 mL) into a 100 mL single-necked round-bottom flask. Install a reflux condenser and heat to reflux for 1 - 5 days; after monitoring the reaction to completion by TLC, remove the oil bath, cool naturally to room temperature, rotary evaporate the solvent under reduced pressure, and purify by column chromatography (SiO 2 , eluent polarity is increased from MeOH / DCM = 1:100 (v:v) to MeOH / DCM = 1:10 (v:v) for gradient elution), concentrate, dry, to obtain a white solid, yield: 67.1%. 11H NMR (500 MHz, Chloroform-d) δ 10.64 (d, J = 1.8 Hz, 1H), 8.56 (s, 1H), 7.48–7.40 (m, 3H), 7.40–7.35 (m, 1H), 7.33 (d, J = 1.7 Hz, 1H), 7.32 (d, J = 1.9 Hz, 1H), 7.31–7.29 (m, 2H), 7.25 (d, J = 3.8 Hz, 1H), 6.28 (s, 2H), 5.14 (s, 2H), 5.04–4.90 (m, 2H), 4.56 (s, 1H), 4.49 (q, J = 13.8 Hz, 2H), 4.39 (ddd, J = 11.3, 7.9, 3.1 Hz, 1H), 4.34 (q, J = 7.4 Hz, 2H), 3.81 (s, 6H), 3.64 (d, J = 72.4 Hz, 2H), 2.68–2.42 (m, 4H), 2.29 (s, 3H), 2.24 (d, J = 13.6 Hz, 1H), 2.12 (tt, J = 13.0, 6.4 Hz, 1H), 1.92 (d, J = 13.6 Hz, 1H), 1.80 (d, J = 13.8 Hz, 1H), 1.63 (t, J = 7.4 Hz, 3H), 1.39 (s, 1H). 13 13C NMR (126 MHz, Chloroform-d) δ 162.88, 160.85, 143.24, 141.73, 136.82, 134.53, 134.34, 130.62, 129.33, 128.35, 128.15, 127.00, 125.75, 124.45, 120.39, 91.37, 69.58, 55.75, 46.81, 45.19, 30.11, 21.77, 16.28, 15.43。

[0036] The preparation of intermediate 3b is the same as that of 3a, except that raw material 2a is replaced with 2b, obtaining a pale yellow solid, yield: 65.2%. 1HNMR(500 MHz, Chloroform-d) δ 10.43 (d, J = 1.7 Hz, 1H), 7.98 (s, 1H), 7.45–7.40 (m, 3H), 7.37 (td, J = 3.4, 1.7 Hz, 2H), 7.33–7.30 (m, 2H), 7.27 (d, J = 1.8 Hz, 2H), 6.26 (s, 2H), 5.11 (s, 2H), 4.53 (td, J = 7.9, 2.1 Hz, 2H), 4.39 (q, J = 7.4 Hz, 2H), 4.31 (dq, J = 11.1, 6.0, 5.5 Hz, 2H), 4.22 (d, J = 3.6 Hz, 2H), 3.78 (s, 6H), 3.48–3.40 (m, 2H), 2.46 (t, J = 11.7 Hz, 1H), 2.28 (s, 5H), 2.13–2.04 (m, 2H), 1.89 (d, J = 12.9 Hz, 2H), 1.79–1.66 (m, 2H), 1.61 (t, J = 7.4 Hz, 3H), 1.40–1.21 (m, 2H). 13 C NMR(126 MHz, Chloroform-d) δ 161.75, 160.59, 143.15, 141.80, 136.82, 134.64, 134.51, 130.47, 129.34, 128.32, 128.12, 126.94, 125.69, 123.29, 121.15, 91.34, 69.47, 64.74, 63.20, 55.75, 51.12, 49.40, 45.24, 27.41, 25.33, 16.26, 15.51.

[0037] The preparation of intermediate 3c is the same as that of 3a, except that raw material 2a is replaced with 2c, obtaining a yellowish-green solid, yield: 63.4%. 1HNMR(500MHz, Chloroform-d) δ 10.47(s, 1H), 7.90(s, 1H), 7.44(d, J = 5.5Hz, 3H), 7.42(s, 1H), 7.36(t, J = 7.3Hz, 1H), 7.31(d, J = 7.2Hz, 3H), 7.27(d, J = 5.6Hz, 1H), 6.27(s, 2H), 5.13(s, 2H), 4.50(t, J = 7.3Hz, 2H), 4.45–4.40(m, 3H), 4.39–4.35(m, 1H), 4.31(dt, J = 11.1, 5.7Hz, 1H), 3.80(s, 6H), 3.63(d, J = 12.1Hz, 1H), 3.56(s, 1H), 2.58(s, 1H), 2.35(s, 1H), 2.28(s, 3H), 2.25–2.18(m, 1H), 2.10(dd, J = 15.3, 7.6Hz, 2H), 2.05–1.98(m, 1H), 1.82(ddd, J = 28.4, 17.9, 10.7Hz, 4H), 1.66(dd, J = 15.3, 7.5Hz, 2H), 1.61(t, J = 7.4Hz, 3H), 1.35(dd, J = 27.3, 8.5Hz, 2H). 13 C NMR(126MHz, Chloroform-d) δ 160.26, 143.03, 141.94, 135.40, 135.11, 134.55, 130.32, 129.40, 128.37, 128.11, 126.89, 125.65, 122.18, 121.77, 91.24, 69.29, 63.95, 63.29, 55.69, 50.16, 50.01, 46.33, 45.34, 30.23, 29.78, 28.36, 25.64, 25.19, 25.12, 16.25, 15.06.

[0038] The preparation method of intermediates 6a - 6c is the same as that of intermediates 3a - 3c, except that the raw material BMS - 1 is replaced with GA;

[0039] Intermediate 6a: White solid, yield: 84.1%. 11H NMR (500 MHz, Chloroform-d) δ 10.61 (s, 1H), 7.61 (t, J = 1.8 Hz, 1H), 7.47 (t, J = 1.7 Hz, 1H), 5.58 (s, 1H), 4.60–4.54 (m, 1H), 4.46 (dq, J = 18.5, 7.3 Hz, 3H), 4.24 (dp, J = 23.8, 5.9 Hz, 2H), 3.24 (dd, J = 11.0, 5.3 Hz, 1H), 2.77 (dt, J = 13.5, 3.6 Hz, 1H), 2.40 (p, J = 7.0 Hz, 2H), 2.36 (s, 1H), 2.03 (ddt, J = 16.9, 12.9, 3.6 Hz, 3H), 1.93–1.87 (m, 2H), 1.82 (dd, J = 13.6, 4.6 Hz, 1H), 1.70–1.62 (m, 7H), 1.61 (d, J = 3.5 Hz, 1H), 1.46 (t, J = 2.7 Hz, 1H), 1.44 (d, J = 2.5 Hz, 1H), 1.42 (d, J = 3.3 Hz, 1H), 1.39 (s, 3H), 1.34 (dd, J = 13.3, 3.0 Hz, 1H), 1.31 (d, J = 3.0 Hz, 1H), 1.22 (s, 1H), 1.19 (s, 3H), 1.13 (s, 6H), 1.07–1.03 (m, 1H), 1.02 (s, 3H), 0.98 (dd, J = 13.0, 4.3 Hz, 1H), 0.82 (d, J = 4.4 Hz, 6H), 0.72 (d, J = 12.4 Hz, 1H). 13 13C NMR (126 MHz, Chloroform-d) δ 200.32, 176.21, 169.69, 137.30, 128.25, 122.56, 121.58, 78.64, 61.93, 60.61, 54.94, 48.73, 47.28, 45.52, 45.45, 44.14, 43.30, 41.26, 39.19, 39.15, 37.72, 37.15, 32.75, 31.93, 31.05, 29.99, 28.62, 28.41, 28.11, 27.28, 26.49, 26.35, 23.37, 18.72, 17.47, 16.39, 15.60, 15.49.

[0040] Intermediate 6b: White solid, yield: 90.5%. 11H NMR (500 MHz, Chloroform-d) δ 10.56 (d, J = 1.7 Hz, 1H), 7.59 (t, J = 1.8 Hz, 1H), 7.46 (t, J = 1.8 Hz, 1H), 5.49 (s, 1H), 4.51 (t, J = 7.6 Hz, 2H), 4.44 (q, J = 7.3 Hz, 2H), 4.23 (dt, J = 12.0, 6.2 Hz, 1H), 4.10 (dt, J = 11.5, 6.0 Hz, 1H), 3.25 (dd, J = 11.0, 5.2 Hz, 1H), 2.75 (dt, J = 13.4, 3.5 Hz, 1H), 2.35 (s, 1H), 2.08 (ddt, J = 16.0, 8.0, 4.8 Hz, 2H), 2.01 (dq, J = 11.9, 3.5, 2.5 Hz, 3H), 1.90 (dd, J = 4.3, 2.6 Hz, 1H), 1.89–1.85 (m, 2H), 1.85–1.81 (m, 1H), 1.79 (dd, J = 9.0, 5.6 Hz, 2H), 1.67 (d, J = 13.3 Hz, 2H), 1.65–1.61 (m, 5H), 1.50–1.45 (m, 1H), 1.43 (s, 1H), 1.41 (s, 1H), 1.38 (s, 3H), 1.32 (d, J = 9.7 Hz, 2H), 1.23–1.18 (m, 1H), 1.16 (s, 3H), 1.13 (d, J = 3.8 Hz, 6H), 1.08–1.03 (m, 1H), 1.02 (s, 3H), 0.98 (dd, J = 12.9, 4.3 Hz, 1H), 0.82 (d, J = 1.6 Hz, 6H), 0.74–0.68 (m, 1H). 13 13C NMR (126 MHz, Chloroform-d) δ 200.40, 176.32, 170.10, 137.15, 128.11, 122.24, 121.76, 78.59, 63.39, 61.92, 54.91, 49.55, 48.84, 45.47, 45.40, 44.09, 43.34, 41.22, 39.20, 39.15, 37.62, 37.15, 32.73, 31.90, 31.05, 28.63, 28.43, 28.11, 27.66, 27.27, 26.49, 26.37, 25.75, 23.37, 18.72, 17.46, 16.40, 15.61, 15.49.

[0041] Intermediate 6c: White solid, Yield: 93.9%. 11H NMR (500 MHz, Chloroform-d) δ 10.55 (s, 1H), 7.63 (s, 1H), 7.47 (s, 1H), 5.56 (s, 1H), 4.50–4.43 (m, 3H), 4.42–4.35 (m, 1H), 4.24–4.16 (m, 1H), 4.06–3.98 (m, 1H), 3.24 (dd, J=10.9, 5.3 Hz, 1H), 2.73 (dd, J=10.2, 3.3 Hz, 1H), 2.36 (s, 1H), 2.07 (d, J=7.6 Hz, 1H), 2.05 (d, J=8.3 Hz, 2H), 2.02–1.97 (m, 2H), 1.91 (d, J=13.3 Hz, 2H), 1.87–1.80 (m, 2H), 1.75 (s, 1H), 1.72 (s, 1H), 1.70 (s, 1H), 1.67 (s, 1H), 1.63 (t, J=7.4 Hz, 5H), 1.54 (d, J=8.0 Hz, 1H), 1.51 (d, J=7.4 Hz, 1H), 1.45 (s, 1H), 1.42 (d, J=3.3 Hz, 1H), 1.41 (s, 1H), 1.39 (d, J=5.1 Hz, 3H), 1.34 (dd, J=8.8, 5.0 Hz, 1H), 1.31–1.29 (m, 1H), 1.27 (s, 1H), 1.21 (d, J=13.7 Hz, 2H), 1.15 (s, 3H), 1.13 (s, 6H), 1.06 (s, 1H), 1.01 (s, 3H), 0.98 (dd, J=13.0, 4.3 Hz, 1H), 0.81 (d, J=2.8 Hz, 6H), 0.72 (d, J=11.5 Hz, 1H). 13 13C NMR (126 MHz, Chloroform-d) δ 200.60, 176.26, 170.34, 137.00, 128.08, 122.37, 121.49, 78.56, 63.85, 61.92, 54.90, 49.95, 48.89, 45.51, 45.34, 44.06, 43.35, 41.25, 39.21, 39.14, 37.58, 37.14, 32.74, 31.91, 31.06, 30.28, 28.62, 28.40, 28.11, 27.24, 26.52, 26.36, 23.35, 23.30, 18.73, 17.46, 16.41, 15.62, 15.54.

[0042] Preparation of the final product 7a: Under nitrogen protection, intermediate 6a (69.3 mg, 0.10 mmol) and Ag were successively added to a 50 mL single-necked round-bottom flask2 O (13.9 mg, 0.06 mmol) and anhydrous DCM (10 mL), under aluminum foil protection from light, react at room temperature for 12 h, then add Me 2 SAuCl (29.46 mg, 0.10 mmol) and solid NaBr (10.3 mg, 1.00 mmol), continue the reaction for 12 h; monitor by TLC, after the reaction is complete, filter through celite to remove the solid, wash the solid with a small amount of anhydrous DCM (about 3 mL), remove the solvent by rotary evaporation under reduced pressure, dissolve in a small amount of DCM (about 0.5 mL) and dropwise add to an appropriate amount of n-hexane (about 10 mL), white solid precipitates, filter by suction, dry, dissolve the white solid in anhydrous acetone (10 mL), under nitrogen protection, add intermediate 3a (70.53 mg, 0.10 mmol) and K 2 CO 3 (41.46 mg, 0.30 mmol), under aluminum foil protection from light, stir at room temperature for 12 h, finally add KPF 6 (184 mg, 1.00 mmol), continue to stir at room temperature for 24 - 48 h; monitor by TLC, after the reaction is complete, directly remove the solvent by rotary evaporation under reduced pressure, purify by column chromatography (SiO 2 , elute with a gradient from MeOH / DCM = 1:100 (v:v) to MeOH / DCM = 1:100 (v:v)), concentrate, dissolve the product in an appropriate amount of anhydrous dichloromethane (about 0.5 mL), slowly dropwise add to an appropriate amount of anhydrous n-hexane (about 10 mL), solid precipitates, remove the solvent, dry to obtain a white solid, yield: 42%. 11H NMR (500 MHz, Chloroform-d) δ 7.48–7.42 (m, 3H), 7.38 (d, J = 8.3 Hz, 2H), 7.35–7.29 (m, 3H), 7.21–7.12 (m, 3H), 7.10 (s, 1H), 6.29 (s, 2H), 5.64 (s, 1H), 5.14 (s, 2H), 4.47–4.39 (m, 4H), 4.34–4.29 (m, 4H), 4.25 (dd, J = 14.1, 7.0 Hz, 4H), 4.20 (t, J = 6.6 Hz, 2H), 4.02 (s, 1H), 3.87 (s, 6H), 3.45 (s, 1H), 3.25 (dd, J = 10.9, 5.2 Hz, 1H), 2.94 (s, 1H), 2.76 (dd, J = 8.7, 5.1 Hz, 1H), 2.40 (d, J = 6.8 Hz, 1H), 2.36 (s, 1H), 2.29 (s, 3H), 2.26 (d, J = 7.3 Hz, 1H), 2.08 (dd, J = 13.0, 5.4 Hz, 2H), 2.04–2.00 (m, 2H), 1.96 (d, J = 13.2 Hz, 2H), 1.87–1.80 (m, 4H), 1.63 (d, J = 14.6 Hz, 6H), 1.55 (dt, J = 7.5, 4.0 Hz, 6H), 1.46 (d, J = 3.8 Hz, 1H), 1.44 (s, 1H), 1.41 (s, 1H), 1.38 (s, 3H), 1.35 (s, 1H), 1.32 (s, 1H), 1.31 (s, 1H), 1.20 (s, 2H), 1.15 (d, J = 3.1 Hz, 3H), 1.13 (s, 6H), 1.05 (s, 1H), 1.03 (s, 3H), 0.99 (d, J = 9.8 Hz, 1H), 0.83–0.81 (m, 6H), 0.72 (d, J = 11.6 Hz, 1H). 1313C NMR (126 MHz, Chloroform-d) δ 200.57, 183.12, 182.96, 182.72, 176.29, 169.89–169.87 (m), 162.71, 160.34, 143.09, 141.85, 134.65, 134.59, 130.49, 129.39, 128.47, 128.37, 128.12, 126.92, 125.69, 122.58, 121.88, 121.73, 121.33, 121.26, 120.96, 91.44, 78.65, 69.54, 63.44, 63.02, 61.88, 61.19, 56.03, 54.86, 50.53, 48.43, 48.19, 46.60, 46.48, 45.45, 44.02, 43.29, 41.05, 39.14, 37.80, 37.11, 32.73, 31.88, 30.99, 30.63, 30.44, 29.70, 28.59, 28.31, 28.09, 27.27, 26.44, 26.31, 23.40, 18.71, 17.47, 16.84, 16.80, 16.39, 16.27, 15.59. ESI-MS calcd. for C 75 H 103 AuN 5 O 9 [M–PF 6 + 1414.7416, found 1414.83. HPLC purity: 97.08%.

[0043] The preparation method of the final product 7b is the same as that of 7a, only by replacing the intermediates 6a and 3a with equimolar amounts of 6b and 3b. White solid, yield: 53%. 1 ​1H NMR (500 MHz, Chloroform-d) δ 7.43 (d, J = 7.1 Hz, 3H), 7.40–7.36 (m, 1H), 7.33 (d, J = 7.4 Hz, 2H), 7.30 (s, 1H), 7.28 (s, 2H), 7.16 (d, J = 16.0 Hz, 2H), 7.09 (dd, J = 10.1, 1.8 Hz, 1H), 6.28 (d, J = 2.8 Hz, 2H), 5.58 (d, J = 7.9 Hz, 1H), 5.13 (d, J = 3.9 Hz, 2H), 4.48–4.39 (m, 2H), 4.28 (q, J = 9.2, 8.0 Hz, 8H), 4.15 (ddd, J = 20.7, 9.8, 5.6 Hz, 2H), 3.86 (d, J = 3.4 Hz, 6H), 3.45 (d, J = 11.8 Hz, 1H), 3.25 (dd, J = 11.2, 5.3 Hz, 1H), 2.91 (s, 1H), 2.77 (ddd, J = 12.6, 7.6, 3.8 Hz, 1H), 2.36 (s, 1H), 2.29 (d, J = 2.9 Hz, 3H), 2.19 (d, J = 14.6 Hz, 1H), 2.08–2.02 (m, 5H), 2.01–1.96 (m, 4H), 1.88 (d, J = 14.6 Hz, 2H), 1.82 (dd, J = 10.4, 5.3 Hz, 3H), 1.72 (t, J = 7.6 Hz, 3H), 1.69–1.66 (m, 2H), 1.65–1.62 (m, 3H), 1.61 (d, J = 3.9 Hz, 1H), 1.56–1.52 (m, 6H), 1.46 (d, J = 3.1 Hz, 1H), 1.44 (s, 1H), 1.43–1.41 (m, 1H), 1.39 (s, 1H), 1.38 (s, 3H), 1.35 (s, 1H), 1.34 (s, 1H), 1.31 (s, 2H), 1.21 (d, J = 14.1 Hz, 2H), 1.14 (t, J = 5.2 Hz, 9H), 1.05 (s, 1H), 1.02 (s, 3H), 0.98 (dd, J = 13.4, 4.0 Hz, 1H), 0.82 (d, J = 2.5 Hz, 3H), 0.80 (s, 3H), 0.72 (d, J = 11.6 Hz, 1H). 1313C NMR(126MHz,Chloroform-d)δ200.38,183.10,182.91,182.72,176.41,160.37,143.10,141.86,134.65,134.60,130.49,129.39,128.48,128.32,128.12,126.92,125.69,122.02,121.54,121.44,121.32,121.09,91.43,78.68,69.55,63.36,61.87,56.00,54.89,50.88,48.53,46.53,46.37,45.44,44.03,43.29,41.11,39.14,37.74,37.11,32.74,31.87,31.03,30.15,29.70,28.61,28.38,28.09,27.95,27.76,27.27,26.46,26.34,25.94,25.43,23.40,18.70,17.48,16.84,16.39,16.27,15.59.ESI-MS calcd.for C 77 H 107 AuN 5 O 9 [M–PF 6 + 1442.7729,found1442.84.HPLC purity:95.08%.

[0044] The preparation method of the final product 7c is the same as that of 7a, only by replacing the intermediates 6a and 3a with equimolar amounts of 6c and 3c. White solid, yield: 39%. 1 ​1H NMR (500 MHz, Chloroform-d) δ 7.43 (s, 3H), 7.37 (d, J = 4.9 Hz, 1H), 7.32 (d, J = 6.4 Hz, 2H), 7.25 (s, 2H), 7.20 (d, J = 10.2 Hz, 1H), 7.15 (d, J = 10.6 Hz, 2H), 7.09 (s, 1H), 6.29 (s, 2H), 5.60 (s, 1H), 5.14 (s, 2H), 4.50 (d, J = 15.2 Hz, 2H), 4.27–4.21 (m, 8H), 4.09 (d, J = 20.6 Hz, 2H), 3.87 (s, 6H), 3.53 (s, 1H), 3.25 (d, J = 6.5 Hz, 1H), 2.95 (s, 1H), 2.76 (s, 1H), 2.36 (s, 1H), 2.28 (s, 3H), 2.25–2.20 (m, 1H), 2.06 (s, 1H), 2.03 (s, 2H), 1.97 (s, 6H), 1.86 (s, 2H), 1.82 (s, 1H), 1.80 (s, 2H), 1.70 (s, 2H), 1.69 (s, 1H), 1.67 (s, 1H), 1.64 (s, 4H), 1.55 (s, 1H), 1.54 (s, 2H), 1.53 (s, 2H), 1.52–1.51 (m, 1H), 1.46 (s, 2H), 1.44 (s, 2H), 1.41 (s, 1H), 1.39 (s, 3H), 1.35 (s, 1H), 1.30 (s, 5H), 1.23–1.20 (m, 3H), 1.13 (s, 9H), 1.04 (s, 1H), 1.02 (s, 3H), 0.99–0.95 (m, 1H), 0.85 (d, J = 5.9 Hz, 1H), 0.81 (d, J = 5.6 Hz, 6H), 0.72 (d, J = 10.7 Hz, 1H). 13CNMR(126MHz,Chloroform-d)δ200.33,182.97,182.81,182.64,176.39,169.74,160.48,143.07,141.84,134.60(d,J=11.5Hz),130.47,129.36,128.38(d,J=22.7Hz),128.10,126.90,125.67,122.24,121.75(d,J=20.2Hz),121.13(d,J=7.0Hz),120.84,91.43,78.65,69.56,66.60,63.85(d,J=6.2Hz),61.87,56.00,54.87,51.23(dd,J=10.3,4.2Hz),50.97,48.59(d,J=4.0Hz),46.42(d,J=16.2Hz),45.45,44.02,43.30,41.12,39.13,37.71,37.11,32.74,31.93,31.51,31.44,31.37,31.23(d,J=7.8Hz),31.04,30.53,30.15,29.70,29.36,28.61,28.35,28.09,27.27,26.41(d,J=15.9Hz),23.41,23.19(d,J=4.4Hz),22.82,18.71,17.48,16.92(dd,J=7.6,3.4Hz),16.39,16.27,15.60.ESI-MS calcd.for C 79 H 17a uN 5 O 9 [M–PF 6 + 1470.8042,found1470.88.HPLC purity:97.69%.

[0045] Example 2

[0046] Selection of Glycyrrhetinic Acid-Gold(I) Dominant Compounds:

[0047] (1) Evaluation of in vitro anti-proliferation ability:

[0048] All cell lines involved in the present invention were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; all cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin double antibody at 37°C in a 5% CO 2 incubator.

[0049] ​The specific experimental operations are as follows: Hepa1-6, HepG2, and Hep3B cells in the logarithmic growth phase are digested with trypsin, centrifuged, and the supernatant is discarded. They are then resuspended evenly with complete medium and counted, and the cell concentration is adjusted to 2×10 4 / mL and seeded in 96-well plates (100 μL per well, 2×10 3 wells) and cultured overnight. The test compound is dissolved in DMF (20 mM) and serially diluted with complete medium to make the compound concentrations: 1.25, 2.5, 5, 10, 20, and 40 μM. Then 100 μL is added to the original 96-well plates to make the final compound concentrations 0.625, 1.25, 2.5, 5, 10, and 20 μM. The 96-well plates are then returned to the incubator and cultured for another 72 h. After that, 20 μL of MTT solution (5 mg / mL, dissolved in PBS, pH = 7.4) is added to each well of the 96-well plates. After incubation for 4 h, the supernatant is discarded, 200 μL of DMSO is added, and the plates are shaken on a shaker at a constant speed for 10 min. The absorbance at 490 nm is measured using a microplate reader. The cells without drug addition are used as a control, and three replicates are set for each concentration. The cell viability (%) is calculated and the result data is analyzed using GraphPad Prism 8.0 software;

[0050] Cell viability (%) = (OD d - OD 0 ) / (OD C – OD 0 ) × 100%

[0051] OD d is the absorbance value of the drug-treated group, OD 0 is the absorbance value of the blanking well, and OD C is the absorbance value of the blank group;

[0052] Results: The anti-proliferative activities (IC 50 ) of 7a, 7b, and 7c on HepG2 cells are 1.19 ± 0.15 μM, 1.70 ± 0.20 μM, and 2.64 ± 0.36 μM, respectively. The IC 50 values decrease with the elongation of the carbon chain of the Linker (the number of carbon atoms in 7a, 7b, and 7c are 3, 4, and 5, respectively). And the anti-proliferative activity of 7c on HepG2 cells at 24 h is slightly better than that of auranofin (the IC 50 values of auranofin at 72 h and 24 h are 1.77 ± 0.29 μM and 3.79 ± 0.49 μM, respectively), and there is only a slight decrease compared to the IC 50 value at 72 h (the IC 50 values of 7c at 72 h and 24 h are 2.64 ± 0.36 μM and 3.22 ± 0.31 μM, respectively). This indicates that 7c takes effect faster than the positive drug auranofin and can maintain the drug effect for a long time;

[0053] (2) Evaluation of the ability to inhibit PD-L1 protein expression:

[0054] First, evaluate the expression of PD-L1 protein in HepG2 cells by Western Blot at the protein level, and use confocal technology to more intuitively observe the PD-L1 expression in HepG2 cells treated with the compound. Combining with the anti-proliferative activity results, select the dominant compound for subsequent stability and other pharmacodynamic evaluations;

[0055] Results: Evaluate the expression of PD-L1 protein in HepG2 cells by Western Blot. 7b and 7c can significantly reduce the expression of PD-L1, while 7a has a poor effect; the statistical chart of the relative expression level of PD-L1 protein also verifies this conclusion. In addition, to more intuitively observe the expression of PD-L1 protein, use confocal technology to evaluate the PD-L1 expression in HepG2 cells treated with 7a, 7b, and 7c. It can be seen that compared with DMF, all three compounds can inhibit the expression of PD-L1 protein on the surface of HepG2 cells, and this inhibitory intensity is consistent with the results at the protein level. Combining with the anti-proliferative activity results in (1), select 7c as the dominant compound in this series for subsequent stability and other pharmacodynamic evaluations.

[0056] Example 3

[0057] Stability evaluation of glycyrrhetinic acid-gold(I) compound:

[0058] Nuclear magnetic resonance method (proton NMR): React compound 7c (5 mM) with GSH (5 mM) in a mixed deuterated reagent of 10% D 2 O / 90% DMSO-d6 at room temperature, and collect the 1 H-NMR spectra at different time points and analyze the 1 changes in the H-NMR spectra;

[0059] Results: Incubate 7c in aqueous DMSO-d6 at room temperature for 7 days. No obvious new peaks or chemical shift changes are observed in the proton NMR spectrum, indicating that 4c has good stability in aqueous solution. Mix 7c and GSH in a molar ratio of 1:1 and incubate them in a mixed DMSO-d6 solution containing 10% D 2 O at room temperature for 72 h. At 6 h, a triplet appears at a chemical shift of 3.5 ppm and a quartet appears at 4.5 ppm. It is more obvious at 12 h, but it is speculated that only about 10% of 7c has reacted at 24 h, indicating that compound 7c can react with GSH slowly. In summary, 7c has good stability.

[0060] Example 4

[0061] Pharmacodynamic evaluation of glycyrrhetinic acid-gold(I) compound in HepG2:

[0062] (1) In vitro inhibitor anti-proliferation experiment of the dominant compound:

[0063] To preliminarily explore the specific mechanism of the dominant compound's anti-proliferation, four inhibitors (NAC, Sal, Fer-1, 3-MA) were co-cultured with the dominant compound in HepG2 cells respectively, and the CCK-8 method was used to detect cell viability;

[0064] Results: After co-culturing NAC and 7c for 24 h, it could dose-dependently reverse the cell proliferation inhibition caused by 7c; similarly, the ERS inhibitor Sal (Salubrinal) could achieve a similar reversal effect; while the ferroptosis inhibitor Fer-1 (Ferrostatin-1) and the autophagy inhibitor 3-MA (3-Methyladenine) did not show an obvious reversal effect; in summary, the anti-proliferation ability of 7c in HepG2 cells may be related to oxidative stress and endoplasmic reticulum stress;

[0065] (2) Evaluate the inhibitory ability of the dominant compound on HepG2 TrxR:

[0066] ① Detection of TrxR pure enzyme activity:

[0067] Prepare TrxR pure enzyme, dilute the dominant compound to a suitable concentration, add 25 μL of enzyme solution and 25 μL of compound dilution in a 96-well plate, and incubate at room temperature; after mixing, immediately add 225 μL of reaction solution to each well in the 96-well plate, then add 25 μL of DTNB (5 mg / 200 μL, dissolved in 95% ethanol) to each well, and immediately continuously detect the absorbance change at 405 nm for 300 seconds with an enzyme-labeling instrument;

[0068] ② Detection of intracellular TrxR activity:

[0069] Inoculate HepG2 cells in the logarithmic growth phase into 6 cm, and when the cell confluence reaches 70%, administer drugs. Set the groups as the DMF solvent control group and the drug administration groups with different concentrations. After 24 h, collect the cells and operate according to the kit instructions, and detect the protein concentration;

[0070] ③ Detection of intracellular TrxR probe:

[0071] Inoculate HepG2 cells in the logarithmic growth phase into a 6-well plate. After the cells adhere to the wall, administer drugs. After 24 h, add the TRFS-green staining working solution, stain at 37 °C for 30 min, discard the supernatant, wash twice with PBS, and observe under a fluorescence microscope;

[0072] ④ Immunofluorescence (confocal) detection:

[0073] The sterilized cell slides were placed in a 24-well plate. HepG2 cells in the logarithmic growth phase were inoculated into the wells and cultured overnight. After the cells adhered to the wall, the drug was administered. After 24 h, the 24-well plate was taken out, the culture medium was discarded, and after washing 3 times with PBS, 4% paraformaldehyde was added and fixed at 37 °C for 30 min. Then, it was taken out, washed 3 times with PBS, 0.1% Triton was added for permeabilization for 10 min, washed 3 times with PBS, 1% BSA prepared with PBS was added for blocking for 1 h, washed three times with PBS, and the primary antibody working solutions TrxR, GRP78, CHOP, CRT, HMGB1, and PD-L1 were added, and incubated overnight at 4 °C. The 24-well plate was taken out, washed 3 times with PBS, wrapped with tin foil, and slowly shaken on a shaker with a rabbit secondary antibody labeled with fluorescence (FITC) for 2 h (room temperature), washed 3 times with PBS, incubated with the DAPI staining working solution in the dark at room temperature for 10 min, washed 3 times with PBS, and after mounting the slides, photographed with a Leica DMi8 fluorescence microscope or a TCSSP8 laser confocal microscope;

[0074] Results: The results of immunofluorescence analysis showed that the green fluorescence of TrxR was significantly reduced after treatment with 7c (4 μM), indicating that 7c significantly inhibited the expression of TrxR in HepG2 cells; in addition, 7c could dose-dependently inhibit the expression of TrxR protein in HepG2 cells; compared with the DMF group, the relative expression levels of TrxR decreased by 24%, 55%, and 75% after treatment with 1 μM, 2 μM, and 4 μM of 7c, respectively. This was very different from the experimental data in Example 3 where only about 10% of 7c reacted with GSH. The results showed that 7c mainly did not react with the sulfhydryl groups on TrxR to inhibit its activity, but mostly coordinated with the strongly nucleophilic selenolate anions in its active center to inhibit its activity; in addition, after treating HepG2 cells with different concentrations of 7c for 24 h, the TrxR enzyme activity in HepG2 cells was detected using a TrxR enzyme activity kit. The results showed that 7c also had a dose-dependent inhibitory effect on the TrxR activity in cells; in summary, the results of the activity of 7c on TrxR indicated that while improving the overall stability of the compound, it maintained the inhibitory effect on TrxR enzyme activity;

[0075] (3) Evaluate the ability of the superior compound to induce apoptosis and cell cycle arrest in HepG2 cells:

[0076] Take HepG2 cells in the logarithmic growth phase and inoculate them into a 6-well plate (2×10 5Cultivate overnight in (number / hole), after different administration groups were treated for 24 h, collect the cells; Use the Annexin V-FITC apoptosis kit or cell cycle detection kit to detect apoptosis or cell cycle according to the kit instructions; Analyze and measure using a flow cytometer, and use FlowJo software for data processing;

[0077] Results: The ability of 7c to induce apoptosis in HepG2 cells at 0.5 μM was comparable to that of auranofin (4 μM), and the ability of 7c to induce apoptosis showed a certain dose-dependence. However, after 2 μM, it entered a plateau phase, and the apoptotic cells did not increase further. On the other hand, after treatment with 7c, the cell cycle of HepG2 cells was mainly arrested in the G0 / G1 phase. This cell cycle arrest pattern was consistent with that of auranofin at the same concentration (4 μM). The difference was that 7c also arrested the cell cycle in the G2 / M phase, while auranofin was mainly in the S phase; Therefore, 7c can induce apoptosis and cell cycle arrest in HepG2 cells;

[0078] (4) Evaluate the ability of the lead compound to damage HepG2 mitochondria:

[0079] After inoculating and administering drugs to HepG2 cells in the logarithmic growth phase for 24 h, discard the culture medium. After washing with PBS, add the JC-1 probe working solution and stain at 37 °C for 30 min. Discard the working solution, wash with PBS, and observe under a fluorescence microscope; The cell inoculation and drug administration operations were as described above. After 24 h, discard the culture medium, wash with PBS, digest and collect the cells, add the JC-1 staining working solution, and the staining conditions and time were the same as above. Centrifuge (800 rpm, 5 min), discard the working solution, wash with PBS (800 rpm, 3 min), resuspend the cells with the cell staining buffer, and analyze using a flow cytometer. Use FlowJo software to process the data;

[0080] Results: After treating HepG2 cells with 7c, compared with the DMF control group, the red fluorescence decreased and the green fluorescence increased significantly, indicating a decrease in mitochondrial membrane potential. The results of flow cytometry analysis also verified this conclusion; It shows that 7c can induce mitochondrial dysfunction, but the ability of 7c to reduce MMP is not as strong as that of auranofin;

[0081] (5) Evaluate the ability of the lead compound to induce oxidative stress, endoplasmic reticulum stress and ICD effect in HepG2:

[0082] ① Evaluate the ability of the lead compound to induce oxidative stress in HepG2:

[0083] After inoculating and administering drugs to HepG2 cells in the logarithmic growth phase for 6 h, discard the culture medium, wash with PBS, add the DCFH-DA staining working solution, and incubate at 37 °C for 30 min. Discard the working solution, wash with PBS, and observe and take pictures under a fluorescence microscope;

[0084] Results: DHE (Dihydroethidium) staining and flow cytometry results showed that the amount of reactive oxygen species in HepG2 cells caused by 7c was dose-dependent. The ability of 7c to increase reactive oxygen species at a concentration of 1μM or even 0.5μM was similar to that of auranofin (4μM). Compared with the results that the IC50 values ​​of 7c and auranofin at 24h were similar, it was shown that auranofin might not be a specific TrxR inhibitor to a certain extent. The similar IC50 values ​​of 7c at 24h and 72h indicated that 7c could quickly enter cells and take effect, and its main target was TrxR, that is, 7c was a more specific TrxR inhibitor than auranofin.

[0085] ②Evaluate the ability of the superior compounds to induce endoplasmic reticulum stress in HepG2:

[0086] HepG2 cells in logarithmic growth phase were seeded in 12-well plates (5×10 4 After 24 h of treatment with different drug groups, the cells were stained with Fluo 4-AM probe and photographed under a fluorescence microscope. The probe was prepared and incubated according to the instructions.

[0087] Results: The ability of 7c and oxaliplatin to promote the increase of calnexin and CHOP expression at the same concentration (4 μM) was evaluated. The results showed that both 7c and oxaliplatin could greatly increase the expression of these two proteins, but 7c was more effective than oxaliplatin (after 7c treatment, the expression of Calnexin and CHOP increased by 3.45 and 3.42 times, respectively, compared with the DMF group, while oxaliplatin only increased by 2.34 and 2.56 times, respectively); the confocal experiment results also verified the above conclusions again; the effects of 7c and oxaliplatin on the phosphorylation of these two proteins were tested, and the results showed that 7c could increase the expression of p-PERK and p-eif2α, and this ability was stronger than oxaliplatin at the same dose; in summary, 7c can cause ERS, and its effect is stronger than oxaliplatin;

[0088] The intracellular Ca was detected by the calcium fluorescent probe Fluo4-AM. 2+ The results showed that after 24h of treatment with 7c (4μM) and oxaliplatin (4μM), compared with the DMF group, obvious dot-like or block-like green fluorescence appeared in the cytoplasm of HepG2 cells, indicating a large amount of Ca2+ release; and the intensity analysis by flow cytometry showed that 7c caused Ca2+ to increase. 2+ The release capacity is stronger than that of oxaliplatin at the same concentration;

[0089] ③Evaluate the ability of the superior compounds to induce the ICD effect of HepG2:

[0090] The ability of the advantageous compound to trigger the biochemical characteristic markers (DAMPs) of ICD was evaluated by immunofluorescence as in (4), and the efflux of HMGB1 from HepG2 cells was then detected. The steps are as follows:

[0091] HepG2 cells in the logarithmic growth phase were seeded in six-well plates. After 24 h of drug administration, the culture medium was discarded, and the cells were washed twice with PBS. 200 μL of lysis buffer was added to each well, and the cells were collected after thorough pipetting. The cells were centrifuged at 12,000 g for 5 min at 4 °C, and the supernatant was taken and placed in a 96-well plate. The HMGB1 level was determined according to the standard operation of the ELISA manufacturer;

[0092] Results: In HepG2 cells treated with 7c (4 μM) or oxaliplatin (4 μM), characteristic green surface patches could be observed in the confocal images, indicating that CRT was transferred from the lumen of endoplasmic reticulum vesicles to the surface of tumor cells, and the green surface patches of 7c were more obvious than those in the oxaliplatin group, suggesting that 7c had a stronger ability to cause the efflux of CRT from HepG2 cells than oxaliplatin. In addition, the results of flow cytometry showed that after treatment with 7c and oxaliplatin, the curve shifted significantly to the right, and the shift in the 7c group was more obvious than that in the oxaliplatin group. Finally, the results of Western blot analysis were consistent with the confocal and flow cytometry results. The expression level of CRT protein after treatment with 7c was 2.91 times that of the DMF group, while that in the oxaliplatin group was only 2.21 times;

[0093] The efflux of HMGB1 from HepG2 cells after treatment with 7c and oxaliplatin was evaluated. The expression levels of HMGB1 in the culture medium (S-HMGB1) and in the cells (C-HMGB1) increased after treatment with 7c or oxaliplatin, while the expression level of HMGB1 in the cells (C-HMGB1) decreased, and 7c had a stronger ability to cause the efflux of HMGB1 protein than oxaliplatin. In addition, the release of HMGB1 from the cell nucleus was detected using an HMGB1 ELISA kit. The results showed that the amount of HMGB1 released from HepG2 cells after treatment with 7c was nearly 8 times that of the DMF group, while that in the oxaliplatin group was only 4 times. Therefore, 7c could promote the release of HMGB1 from the cell nucleus of HepG2 cells, and this ability was stronger than that of oxaliplatin;

[0094] The effect of 7c (4 μM)-treated Hepa1-6 cells on DCs of C57BL / 6 mice was investigated by flow cytometry; the expressions of both CD80 and CD86, two markers of DC maturation, increased; compared with the DMF control group, the proportion of DCs in the 7c group doubled (31.6% vs 15.6%);

[0095] In summary, 7c could promote the release of DMAPs from HepG2 cells and promote the maturation of murine DCs cells; therefore, 7c could trigger the ICD effect.

[0096] Example 5

[0097] Evaluating the anti-angiogenic ability of glycyrrhetinic acid-gold(I) compounds using a zebrafish model:

[0098] Zebrafish embryos were purchased from a biotechnology company; the embryos were placed in a standard zebrafish culture system with a day-night lighting cycle and the temperature maintained at 28.0 ± 1.0 °C; all experiments were carried out on the 2nd day after successful embryo transplantation; the control group was treated with 0.1% DMF, and the treatment groups were 7c (2 μM) and auranofin (2 μM) respectively; the embryos of each group were observed continuously for three days, and fluorescence images of the embryos were taken; the embryos were anesthetized with 0.03 mg / mL tricaine during photography;

[0099] Results: Angiogenesis in zebrafish embryos treated with compound 7c was significantly inhibited; compared with the control group (Control), after treatment with 7c (4 μM), the area of the subintestinal vessels in zebrafish was significantly reduced, and the intersegmental vessels were thinner (and there were multiple breaks, indicating that 7c inhibited angiogenesis in zebrafish embryos; compared with auranofin, 7c had a stronger inhibitory effect on angiogenesis; Example 6

[0100] Studying the anti-tumor immune mechanism of glycyrrhetinic acid-gold(I) compounds in animals:

[0101] Male C57BL / 6 mice (18 - 22 g) were purchased from a medical college; all animal experiments were carried out in accordance with the guidelines approved by the institutional and local ethics committees of a university (the experimental animal use license number is SYXK (Su) 2023 - 0077); the animals were housed under standard environmental conditions (temperature 22 - 24 °C, light / dark cycle 12 hours, humidity 50 ± 10%), and had free access to food and water; the mice started the experiment after one week of adaptive feeding; the Hepa1-6 cells inoculated in a 10 cm dish were digested and collected, resuspended by pipetting with serum-free cell medium and counted, and the cell density was adjusted to 1×10 6 cells / 100 μL / animal, inoculated in the left axilla of the mice, and administered intraperitoneally once every 2 days. The body weight of the tumor-bearing mice was recorded every day, and the tumor volume was measured with a vernier caliper. The tumor volume calculation formula was; V (cm 3 ) = a 2 × b / 2, (a: short diameter; b: long diameter; V: tumor volume); the mice were sacrificed after two weeks, and the tumors and main organs were removed for subsequent experiments; the tumor tissues were photographed, and after the tumor tissues were digested, the activation of immune cells was analyzed using a flow cytometer;

[0102] Results: The tumor volume and weight of mice treated with the compound were significantly reduced compared to the control group; flow cytometry analysis results showed that the proportions of mature DC cells and activated T cells were significantly increased, indicating enhanced antigen presentation and lymphocyte killing after compound treatment; the proportions of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) were decreased, suggesting weakened immunosuppressive ability of tumors; the increase in M1 macrophages was decreased, accompanied by a decrease in M2 macrophages, indicating a transformation of the tumor immune microenvironment from an immunosuppressive type to an immune-enhanced type.

[0103] Therefore, compound treatment not only reduced tumor growth and development through the chemotherapy pathway but also activated the immune response in mice and reversed tumor immune escape.

Claims

1. A glycyrrhetinic acid-gold (I) compound, characterized in that: The compounds include glycyrrhizic acid, PD-L1 immune checkpoint inhibitory group BMS-1 ​​and diazaheterocyclic carbene gold (I) compound.

2. The glycyrrhetinic acid-gold (I) compound according to claim 1, characterized in that The compound uses diazaheterocyclic carbene gold (I) as a basic skeleton, and introduces a glycyrrhetinic acid ligand with liver cancer cell targeting function and a BMS-1 ​​fragment with PD-L1 activity inhibition.

3. The glycyrrhetinic acid-gold (I) compound according to claim 1, characterized in that The molar ratio of the glycyrrhetinic acid, the PD-L1 immune checkpoint inhibitory group BMS-1 ​​and the diazaheterocyclic carbene gold (I) compound is 1:1:

1.

4. The glycyrrhetinic acid-gold (I) compound according to claim 1, characterized in that: The chemical structural formula of the compound is shown in formula (I):

5. A method for synthesizing the glycyrrhetinic acid-gold (I) compound according to claim 1.

6. The method for synthesizing the glycyrrhetinic acid-gold (I) compound according to claim 5, characterized in that: Its synthetic route is as follows:

7. A pharmaceutical composition comprising a therapeutically effective amount of one or more glycyrrhetinic acid-gold (I) compounds according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. Use of the glycyrrhetinic acid-gold (I) compound according to any one of claims 1 to 6.

9. The use according to claim 8, characterized in that The use is specifically the use of glycyrrhizic acid-gold (I) compound in the preparation of drugs for treating liver cancer.

10. The use according to claim 9, characterized in that The glycyrrhizic acid-gold (I) compound for treating liver cancer achieves immune killing of liver cancer cells by targeted activation of the immunogenic cell death ICD effect of liver cancer cells and inhibition of PD-L1 expression.

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