Double-pharmacophore N-heterocyclic carbene gold compound and application thereof

Abstract

The invention belongs to the technical field of N-heterocyclic carbene gold compounds, relates to a double-pharmacophore N-heterocyclic carbene gold compound 1-14 and application thereof, and discloses a specific structure of the double-pharmacophore N-heterocyclic carbene gold compound 1-14, and the double-pharmacophore N-heterocyclic carbene gold compound 1-14 is prepared by replacing a triethyl phosphine group in gold nofen with NHC, and then adding a compound 1-14 and a compound 2-14, so that the double-pharmacophore N-heterocyclic carbene gold compound 1-14 is obtained. A traditional Chinese medicine micromolecule ferulic acid Fa is introduced for synthesis; the invention discloses application of double-pharmacophore N-heterocyclic carbene gold compounds 1-14 in preparation of anti-hepatoma drugs. The double-pharmacophore N-heterocyclic carbene gold compound Fa-Au has anti-proliferative activity on liver cancer cells HepG2 and Hepa 1-6, obviously inhibits the growth of tumors in a mouse, and improves the tumor immune microenvironment of the mouse at the same time; the N-heterocyclic carbene gold-ferulic acid compound has the advantages of reliable curative effect, no toxic or side effect, simple preparation, low price and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of nitrogen heterocyclic carbene gold compounds, and in particular relates to a dual-pharmacophore nitrogen heterocyclic carbene gold compound and application thereof in the preparation of anti-liver cancer drugs. Background Art

[0002] As the third most common cancer mortality rate in the world, primary liver cancer, with hepatocellular carcinoma accounting for the majority, has an incidence rate of 70-90%. According to the World Health Organization (WHO) 2020 Global Liver Cancer Prevalence Estimation Model Report, the incidence of liver cancer has been increasing year by year, and this trend is likely to continue until 2040. Analysis of clinical liver cancer incidence data shows that the incidence of liver cancer in developing countries is higher than in developed countries; further analysis of data from the Asia-Pacific region shows that the incidence of liver cancer in my country is significantly higher than in other countries in the same region. It is estimated that by 2040, the number of people diagnosed with liver cancer in my country will be 1.45 times that of 2020. The comprehensive clinical treatment plan for liver cancer provided in the "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer 2022 Edition" issued by the National Health Commission was analyzed. We have found that, in the early and middle stages of liver cancer, surgical resection and liver transplantation are the most ideal treatment options. However, most liver cancer patients are diagnosed in the advanced stage, making surgical intervention a suboptimal option. Furthermore, they require systemic medications after surgery to prevent the migration and spread of liver cancer cells. Therefore, compared with surgery, these patients are more suitable for systemic, personalized treatment options such as radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Metal compounds play a crucial role in cancer diagnosis and treatment. Platinum compounds such as cisplatin, carboplatin, and oxaliplatin account for over 50% of all cancer chemotherapy drugs. Cisplatin was the first platinum drug used to treat malignant tumors and remains the preferred chemotherapy drug in clinical practice due to its low cost and proven anticancer activity. However, platinum drug resistance and adverse reactions, such as neurotoxicity, ototoxicity, and nephrotoxicity, are becoming increasingly prominent. The development of new metal-based anticancer drugs to overcome these limitations of existing platinum drugs is urgently needed.

[0003] Nitrogen heterocyclic carbene ligands are highly stable and easily modified. Furthermore, nitrogen heterocyclic carbene ligands are bioisosteres of organophosphine ligands. Therefore, nitrogen heterocyclic carbene gold compounds can be considered derivatives of the gold drug auranofin, possessing potential as druggable agents. Auranofin is an inhibitor of TrxR. The gold ion in its structure binds to the amino acid residue Sec498 at the C-terminus of the TrxR protein, exerting an irreversible inhibitory effect. Ferulic acid, an active ingredient in traditional Chinese medicine, contains an α,β-unsaturated carbonyl functional group. This covalent bond forms an irreversible Michael addition reaction with the Cys497 / Sec498 residues at the C-terminus of TrxR, inhibiting TrxR activity.

[0004] There is currently no report on the anti-hepatocarcinogenic effects of the above compounds. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a dual-pharmacophore nitrogen heterocyclic carbene gold compound and its use in the preparation of anti-liver cancer drugs.

[0006] In order to achieve the purpose of the present invention, the present invention is implemented by adopting the following technical solutions.

[0007] The structures of the bipharmacophore nitrogen heterocyclic carbene gold compounds 1-14 are as follows:

[0008]

[0009] The dual-pharmacophore nitrogen heterocyclic carbene gold compound 1-14 is synthesized by replacing the triethylphosphine group in auranofin with NHC and introducing the traditional Chinese medicine small molecule ferulic acid Fa.

[0010] As a preferred embodiment of the present invention, the structure of the bipharmacophore nitrogen heterocyclic carbene gold compound 8 is as follows:

[0011]

[0012] Application of bipharmacophore nitrogen heterocyclic carbene gold compounds 1-14 in the preparation of anti-liver cancer drugs.

[0013] As a preferred embodiment of the present invention, the dual-pharmacophore nitrogen heterocyclic carbene gold compound 8 has anti-proliferative activity against liver cancer cells HepG2 and Hepa1-6, can inhibit tumor growth, and induce dendritic cell maturation, macrophage M1 polarization and T cell activation in the tumor immune microenvironment.

[0014] Beneficial effects

[0015] The bipharmacophore nitrogen heterocyclic carbene gold compound Fa-Au exhibited strong antiproliferative activity against two liver cancer cell lines, HepG2 and Hepa 1-6, with IC50 values of 0.69±0.15μM and 0.48±0.21μM, respectively. In a C57BL / 6 mouse tumor-bearing model, administration of 2.5mg / kg of Fa-Au significantly inhibited tumor growth and improved the tumor immune microenvironment.

[0016] The nitrogen heterocyclic carbene gold-ferulic acid compound has the advantages of reliable efficacy, no toxic side effects, simple preparation and low price. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the synthesis steps of compounds 1-14 of the present invention;

[0018] Figure 2 The following are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 1, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0019] Figure 3 These are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 2, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0020] Figure 4 These are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 3, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0021] Figure 5 The following are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 4, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0022] Figure 6 These are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 5, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0023] Figure 7 These are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 6, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0024] Figure 8 The hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 7 are shown in Figure a. The hydrogen spectrum and the carbon spectrum are shown in Figure b.

[0025] Figure 9 These are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 8, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0026] Figure 10 The hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 9 are shown in Figure a. The hydrogen spectrum and the carbon spectrum are shown in Figure b.

[0027] Figure 11 The hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 10 are shown in Figure a. The hydrogen spectrum and the carbon spectrum are shown in Figure b.

[0028] Figure 12 The following are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 11, where: Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0029] Figure 13 The following are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 12; Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0030] Figure 14 The hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 13 are shown in Figure a. The hydrogen spectrum and the carbon spectrum are shown in Figure b.

[0031] Figure 15The following are the hydrogen and carbon spectra of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 14; Figure a is the hydrogen spectrum; Figure b is the carbon spectrum;

[0032] Figure 16 Compound Fa-Au inhibits pure TrxR enzyme and TrxR activity in HepG2 liver cancer cells; Figure A shows the structure of the compound; Figures B and C show the activity of compound Fa-Au on TrxR and TrxR(U498) enzymes; Figure D shows the inhibition curve of compound Fa-Au on HepG2 cells; Figure E shows the inhibition of compound Fa-Au on TrxR activity in HepG2 cells;

[0033] Figure 17 The compound Fa-Au causes an increase in ROS and oxidative stress in liver cancer cells HepG2. Figure A shows analysis of ROS levels in HepG2 cells using CDFH-DA probes; Figures B and C show flow cytometry analysis of ROS levels in HepG2 cells; Figures D and E show GSH and GSSG levels in HepG2 cells.

[0034] Figure 18 The compound Fa-Au induces ferroptosis in HepG2 liver cancer cells; Figure A shows transmission electron microscopy analysis of HepG2 organelles; Figure B shows compound Fa-Au-induced ferroptosis in HepG2 cells; Figures C and D show analysis of lipid oxide accumulation in HepG2 cells using a C11 BODIPY probe; Figures E and F show changes in ferroptosis-related proteins in HepG2 cells;

[0035] Figure 19 The compound Fa-Au inhibits the proliferation and migration of HepG2 cells in zebrafish. Figure A shows immunofluorescence analysis of HepG2 migration in zebrafish; Figures B and C show analysis of HepG2 cell migration rate and fluorescence intensity in zebrafish.

[0036] Figure 20 Compound Fa-Au significantly inhibits tumor growth in C57 / BL6 mice; Figure A shows a photo of tumor tissue; Figures B and C show mouse weight and tumor tissue volume; Figure D shows a tumor volume growth curve; Figure E shows HE and Tunel analysis of tumor tissue; Figures F and G show changes in TrxR and ROS in tumor tissue; Figures H-J show statistical analysis of Tunel, TrxR, and ROS;

[0037] Figure 21 This is an analysis of immune cell infiltration in tumor tissues of C57 / BL6 mice after administration of the compound Fa-Au;

[0038] Figure 22 HE staining analysis of five major organs of C57 / BL6 mice after administration of compound Fa-Au. DETAILED DESCRIPTION

[0039] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0040] Unless otherwise specified, the reagents in the examples of the present invention can be purchased from regular channels.

[0041] Unless otherwise specified, HepG2 and Hepa 1-6 cells in the examples of the present invention were purchased from the Cell Bank of the Chinese Academy of Sciences. 50 The 50% inhibition concentration is the concentration corresponding to B / B0=50%. The half inhibition is used to measure the sensitivity of the antibody. The lower the half inhibition, the higher the sensitivity of the antibody.

[0042] Example 1 Synthesis and Structure of Bipharmacophore Nitrogen Heterocyclic Carbene Gold Compounds 1-14

[0043] 1. The structures of the bipharmacophore nitrogen heterocyclic carbene gold compounds 1-14 are as follows:

[0044]

[0045] A series of dual-pharmacophore nitrogen heterocyclic carbene gold compounds 1-14 were synthesized by replacing the triethylphosphine group in auranofin with the more stable NHC and introducing ferulic acid, a small molecule of traditional Chinese medicine with anti-cancer potential. Their structures were determined by NMR analysis.

[0046] The structure of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 8 is as follows:

[0047]

[0048] As an embodiment of the present invention, Figure 1 The synthesis steps for bipharmacophoric nitrogen heterocyclic carbene gold compounds 1-14 are shown in the figure. MeONHC intermediates (a1-a3) or FNHC intermediates (b1-b3) containing bromoaliphatic hydrocarbon substituents of varying lengths are used as starting materials. These intermediates undergo esterification with COOH-containing traditional Chinese medicine molecules, cinnamic acid (Ca) and ferulic acid (Fa), in the presence of potassium carbonate to yield ligand AG. Subsequently, bipharmacophoric nitrogen heterocyclic carbene gold compounds 1-14 are synthesized using a silver transfer method with varying feed ratios.

[0049] Preparation of Ligand AG: Dissolve intermediate (a1, a2, a3, b1, b2, or b3) (1.00 mmol) and ferulic acid (X-COOH: Fa) or cinnamic acid (X-COOH: Ca) (3.00 mmol) in 10 mL of anhydrous dimethylformamide. Then add potassium carbonate (2.00 mmol) and stir at room temperature for 3 days. Monitor the reaction progress by TLC. After completion, quench the reaction by adding 10 mL of water. The solution is then extracted three times with ethyl acetate or dichloromethane. The organic phase is then washed three times with saturated brine, dried over anhydrous sodium sulfate, and the solvent removed in vacuo. Finally, dry-clean the column using a silica gel column (dichloromethane / methanol gradient elution) to obtain Ligand AG.

[0050] Preparation method of compound 1-7: The ligand (A, B, C, D, E, F or G) (0.10 mmol) was dissolved in 5 mL of anhydrous acetonitrile, and then silver oxide (0.06 mmol) was added and stirred overnight at room temperature, nitrogen and dark conditions. Subsequently, dimethyl sulfide gold (0.10 mmol) and potassium hexafluorophosphate (0.98 mmol) were added to the reaction solution and stirring was continued for 24 hours. After the reaction was confirmed to be complete by TLC, the reaction solution was filtered with diatomaceous earth, and finally dry-filtered through a silica gel column (dichloromethane / methanol gradient elution) to obtain compound 1-7. Preparation method of compound 8-14: The ligand (A, B, C, D, E, F or G) (0.15 mmol) was dissolved in 5 mL of anhydrous acetonitrile, and then silver oxide (0.09 mmol) was added and stirred overnight at room temperature, nitrogen and dark conditions. Subsequently, gold dimethyl sulfide (0.08 mmol) and potassium hexafluorophosphate (1.50 mmol) were added to the reaction solution, and stirring was continued for 24 hours. After the reaction was confirmed to be complete by TLC, the reaction solution was filtered through celite and finally passed through a silica gel column (dichloromethane / methanol gradient elution) by dry method to obtain compound 8-14.

[0051] Bipharmacophore nitrogen heterocyclic carbene gold compound 1: yellow powder (24.5 mg, 31%). 1HNMR(500MHz,Chloroform-d)δ7.54(d,J=15.9Hz,1H,CH=CHCOO),7.20(d,J=1.9Hz,1H,ArH),7.08(d d,J=21.5,8.4Hz,5H,ArH),6.91–6.82(m,5H,ArH),6.32(d,J=15.8Hz,1H,CH=CHCOO),4.34(t,J=7.3 Hz,2H,COOCH2),4.17(q,J=7.0Hz,2H,CH2CH3),4.04(t,J=5.6Hz,2H,CH2CH2N),3.97(s,3H,OCH3),3 .79(s,3H,OCH3),3.67(s,3H,OCH3),2.08(q,J=6.7Hz,2H,CH2CH2N),1.29(t,J=7.2Hz,3H,CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ173.09(1C,C-Au-Br),167.40(1C,CO),160.31,160.31(2C,CH3OArC), 147.09,145.36(1C,CH=CHCOO),131.93,131.89,131.08,131.00,123.72,119.77,119.67,115.19,11 4.69,114.53,114.46,109.69,60.55(1C,COOCH2),56.47(1C,OCH3),55.41(1C,OCH3),55.26(1C,OCH 3),46.09(1C,NCH2CH3),44.37(1C,CH2N),30.35,29.84,17.06(1C,CH2CH3).ESI-MSm / z:819.54[M+H] + ,like Figure 2 As shown in Figures a and b.

[0052] Bipharmacophore nitrogen heterocyclic carbene gold compound 2: yellow powder (25.0 mg, 30%). 1HNMR(500MHz,Chloroform-d)δ7.58(d,J=15.9Hz,1H,CH=CHCOO),7.11–7.06(m,5H,ArH),6.9 1(d,J=8.1Hz,1H,ArH),6.89–6.78(m,5H,ArH),6.30(d,J=15.9Hz,1H,CH=CHCOO),5.90(s,1H ,OH),4.17(p,J=7.4Hz,4H),4.05(t,J=6.2Hz,2H,COOCH2),3.95(s,3H,OCH3),3.79(s,3H,OC H3),3.75(s,3H,OCH3),1.88–1.80(m,2H),1.68–1.55(m,2H),1.29(t,J=7.1Hz,3H,CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ172.71(1C,C-Au-Br),167.38(1C,CO),160.29,148.12,146.9 5,145.11(1C,CH=CHCOO),131.93,131.86,130.98,130.93,127.14,123.37,119.84,119.80,1 15.49,114.74,114.49,114.44,109.58,63.46(1C,COOCH2),56.30(1C,OCH3),55.40(1C,OCH3 ),55.35(1C,OCH3),48.80,44.32,27.99,25.89,17.05(1C,CH2CH3).ESI-MSm / z:833.44[M+H] + ,like Figure 3 As shown in Figures a and b.

[0053] Bipharmacophore nitrogen heterocyclic carbene gold compound 3: yellow powder (26.2 mg, 30%). 1HNMR(500MHz,Chloroform-d)δ7.59(d,J=15.9Hz,1H,CH=CHCOO),7.12–7.06(m,6H,ArH),6. 91(d,J=8.6Hz,1H,ArH),6.85(dd,J=8.3,5.1Hz,4H,ArH),6.29(d,J=15.9Hz,1H,CH=CHCOO) ,5.89(s,1H,OH),4.15(p,J=7.2Hz,4H),4.09(t,J=6.5Hz,2H,COOCH2),3.94(s,3H,OCH3),3 .77(d,J=11.1Hz,6H,OCH3),1.74(q,J=7.4Hz,2H),1.59(q,J=7.1Hz,2H),1.34–1.28(m,5H). 13 CNMR(126MHz,Chloroform-d)δ172.64(1C,C-Au-Br),167.46(1C,CO),160.26,148.08, 146.92,144.98(1C,CH=CHCOO),131.92,130.99,130.87,127.15,123.28,119.91,119. 88,115.61,114.78,114.46,114.42,109.52,63.93(1C,COOCH2),56.21(1C,OCH3),55. 39(1C,OCH3),55.37(1C,OCH3),48.97,44.29,30.90,28.24,23.09,17.05(1C,CH2CH3). ESI-MSm / z:847.42[M+H] + ,like Figure 4 As shown in Figures a and b.

[0054] Bipharmacophore nitrogen heterocyclic carbene gold compound 4: yellow powder (43.1 mg, 58%). 1HNMR(500MHz,Chloroform-d)δ7.56(d,J=15.9Hz,1H,CH=CHCOO),7.21(s,1H,ArH),7.20–7. 15(m,4H,ArH),7.10–7.01(m,5H,ArH),6.91(d,J=8.1Hz,1H,ArH),6.31(d,J=15.9Hz,1H,CH =CHCOO),5.91(s,1H,OH),4.34(t,J=7.4Hz,2H,OCH2),4.18(q,J=7.2Hz,2H,CH2CH3),4.05( t,J=5.7Hz,2H,NCH2),3.98(s,3H,OCH3),2.13–2.04(m,2H),1.30(t,J=7.1Hz,3H,CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ170.74(1C,C-Au-Br),167.28(1C,CO),164.37,162.69(2C,Ar CF),148.26(1C,ArCOH),147.02(1C,ArCOCH3),145.61(1C,C=CCOO),132.57,132.51,130.66 ,127.08,123.70,123.40,116.67,116.59,116.50,116.41,115.04,114.65,109.61,60.37(1 C,OCH2),56.38(1C,OCH3),46.36(1C,NCH2),44.66(1C,CH2CH3),30.34,17.02(1C,CH2CH3). ESI-MS m / z:715.51[M–Br] + ,like Figure 5 As shown in Figures a and b.

[0055] Bipharmacophore nitrogen heterocyclic carbene gold compound 5: yellow powder (43.0 mg, 54%). 1HNMR(500MHz,Chloroform-d)δ7.58(d,J=15.9Hz,1H,CH=CHCOO),7.17(td,J=8.8,5.3Hz, 4H,ArH),7.05(dt,J=11.0,8.4Hz,6H,ArH),6.92(d,J=8.2Hz,1H,ArH),6.29(d,J=15.9Hz ,1H,CH=CHCOO),5.88(s,1H,OH),4.18(dt,J=14.5,7.3Hz,4H),4.06(t,J=6.2Hz,2H,NCH2 ), 3.96 (s, 3H, OCH3), 1.87–1.80 (m, 2H), 1.64 (q, J = 6.9Hz, 2H), 1.33–1.30 (m, 3H, CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ173.87(1C,C-Au-Br),167.34(1C,CO),164.35,164.31(1C,ArCF),1 62.35,162.31(1C,ArCF),148.18(1C,ArCOH),146.96(1C,ArCOCH3),145.26(1C,C=CCOO),132.58,1 32.51,132.45,130.54,127.07,123.49,123.40,116.62,116.56,116.45,116.39,115.34,114.77, 109.56,63.28(1C,OCH2),56.29(1C,OCH3),48.99,44.52,29.84,27.97,25.92,17.03(1C,CH2CH3). ESI-MSm / z:809.59[M+H] + ,like Figure 6 As shown in Figures a and b.

[0056] Bipharmacophore nitrogen heterocyclic carbene gold compound 6: yellow powder (25.6 mg, 30%). 1HNMR(500MHz,Chloroform-d)δ7.59(d,J=16.0Hz,1H,CH=CHCOO),7.19–7.15(m,4H ,ArH),7.08–7.03(m,6H,ArH),6.91(d,J=8.6Hz,1H,ArH),6.28(d,J=16.0Hz,1H,C H=CHCOO),5.98(s,1H,OH),4.19–4.12(m,4H),4.10(t,J=6.4Hz,2H,NCH2),3.94(s ,3H,OCH3),1.73(dq,J=15.6,7.7Hz,2H),1.59(t,J=7.6Hz,2H),1.30–1.26(m,5H). 13 CNMR(126MHz,Chloroform-d)δ173.77(1C,C-Au-Br),167.45(1C,CO),164.32,164.31(1C,ArCF),1 62.32,162.31(1C,ArCF),148.13(1C,ArCOH),146.94(1C,ArCOCH3),145.09(1C,C=CCOO),132.57,1 32.51,130.58,130.47,127.09,123.60,123.29,116.58,116.53,116.41,116.35,115.50,114.81, 109.53,63.79(1C,OCH2),56.20(1C,OCH3),49.14,44.47,30.90,28.24,23.08,17.02(1C,CH2CH3). ESI-MSm / z:823.57[M+H] + ,like Figure 7 As shown in Figures a and b.

[0057] Bipharmacophore nitrogen heterocyclic carbene gold compound 7: yellow powder (43.5 mg, 59%). 1HNMR(500MHz,Chloroform-d)δ7.62(d,J=16.1Hz,1H,CH=CHCOO),7.58(ddd,J=6.9,4.7,2.6 Hz,2H,ArH),7.43(p,J=3.9,3.4Hz,3H,ArH),7.23–7.17(m,4H,ArH),7.07(td,J=8.4,5.4Hz, 4H, ArH), 6.34 (dd, J=16.0, 8.7Hz, 1H, CH=CHCOO), 4.34 (td, J=7.4, 4.0Hz, 2H), 4.21 (q, J=7. 3Hz, 2H), 4.11 (t, J = 5.8Hz, 2H), 2.17 (dt, J = 12.0, 6.2Hz, 2H), 0.90 (t, J = 6.9Hz, 3H, CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ174.03,166.68(1C,COO),164.23,164.20,162.23,162 .20(2C,F-ArC),145.31(1C,C=CCOO),134.22,132.52,132.45,132.39,130.61,130.47 ,130.43,128.93,128.28,128.25,117.51(1C,C=CCOO),116.53,116.44,116.35,116. 26(4C,ArC),60.70,46.05,44.45(2C,NCH2),22.71(1C,NCH2CH2),16.91(1C,CH2CH3). ESI-MSm / z:670.27[M–Br] + ,like Figure 8 As shown in Figures a and b.

[0058] Bipharmacophore nitrogen heterocyclic carbene gold compound 8: yellow powder (117.9 mg, 55%). 1HNMR(500MHz,Chloroform-d)δ7.50(d,J=15.9Hz,2H,CH=CHCOO),7.11–6.98(m,12H,ArH),6.90(d ,J=8.1Hz,2H,ArH),6.86–6.76(m,8H,ArH),6.11(d,J=16.0Hz,2H,CH=CHCOO),5.96(s,2H,OH),4. 42(t,J=6.7Hz,4H,OCH2),4.19(q,J=7.0Hz,4H,CH2CH3),4.11(t,J=5.7Hz,4H,NCH2),3.92(s,6H, OCH3),3.79(s,6H,OCH3),3.66(s,6H,OCH3),2.10(t,J=6.3Hz,4H),1.36(t,J=7.1Hz,6H,CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ182.75(2C,C-Au-C),167.19(2C,CO),160.30,160.26,148.26(2C,Ar COH),147.10(2C,ArCOCH3),145.16(2C,C=CCOO),131.99,131.96,131.83,131.65,127.00,122.80,1 19.56,119.42,115.09,114.80,114.50,114.44,110.03,60.90(2C,OCH2),56.30,55.41,55.24(6C, OCH3),46.12(2C,NCH2),44.27(2C,CH2CH3),30.45,17.51(2C,CH2CH3).ESI-MSm / z:1282.08[M–PF6] + ,like Figure 9 As shown in Figures a and b.

[0059] Bipharmacophore nitrogen heterocyclic carbene gold compound 9: yellow powder (43.9 mg, 20%). 1HNMR(500MHz,Chloroform-d)δ7.54(d,J=15.9Hz,2H,CH=CHCOO),7.13(d,J=8.2Hz,6H,ArH),7.04–6 .99(m,4H,ArH),6.92–6.80(m,12H,ArH),6.21(d,J=15.8Hz,2H,CH=CHCOO),5.91(s,2H,OH),4.32–4. 27(m,4H,OCH2),4.21(d,J=7.1Hz,4H,CH2CH3),4.06(t,J=6.1Hz,4H,NCH2),3.91(s,6H,OCH3),3.79( s,6H,OCH3),3.76(s,6H,OCH3),1.86–1.80(m,4H),1.69–1.65(m,4H),1.36(t,J=6.7Hz,6H,CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ182.52(2C,C-Au-C),167.35(2C,CO),160.32,160.31,148 .20(2C,ArCOH),147.00(2C,ArCOCH3),145.18(2C,C=CCOO),132.06,131.99,131.79,131 .75,127.00,123.09,119.62,119.60,115.28,114.76,114.54,114.49,109.65,63.63(2C ,OCH2),56.22,55.42,55.37(6C,OCH3),48.98,44.32,28.21,26.00,17.52(2C,CH2CH3). ESI-MSm / z:1310.10[M–PF6] + ,like Figure 10 As shown in Figures a and b.

[0060] Bipharmacophore nitrogen heterocyclic carbene gold compound 10: yellow powder (41.0 mg, 19%). 1HNMR(500MHz,Chloroform-d)δ7.56(d,J=15.8Hz,2H,CH=CHCOO),7.15–7.11(m,6H,Ar H),7.06–6.98(m,4H,ArH),6.98–6.74(m,12H,ArH),6.24(d,J=15.9Hz,2H,CH=CHCOO) ,5.94(s,2H,OH),4.27–4.16(m,8H),4.11–4.05(m,4H),3.90(s,6H,OCH3),3.78(s,6H ,OCH3),3.76(s,6H,OCH3),1.83–1.75(m,4H),1.61–1.53(m,4H),1.38–1.32(m,10H). 13 CNMR(126MHz,Chloroform-d)δ182.45(2C,C-Au-C),167.44(2C,CO),160.32,148.18(2 C,ArCOH),146.99(2C,ArCOCH3),145.08(2C,C=CCOO),132.04,131.84,131.67,127.03 ,123.07,119.65,119.58,115.41,114.80,114.52,114.48,109.61,63.91(2C,OCH2),5 6.17,55.40,55.39(6C,OCH3),49.05,44.27,31.37,28.27,23.12,17.52(2C,CH2CH3). ESI-MSm / z:1338.14[M–PF6] + ,like Figure 11 As shown in Figures a and b.

[0061] Bipharmacophore nitrogen heterocyclic carbene gold compound 11: yellow powder (112.5 mg, 55%). 1HNMR(500MHz,Chloroform-d)δ7.50(d,J=15.8Hz,2H,CH=CHCOO),7.22–7.14(m,10 H,ArH),7.06–7.00(m,10H,ArH),6.97–6.93(m,2H,ArH),6.10(d,J=15.9Hz,2H,CH= CHCOO),4.53(t,J=7.1Hz,4H,OCH2),4.30(q,J=7.3Hz,4H,CH2CH3),4.15(t,J=5.8 Hz, 4H, NCH2), 3.92 (s, 6H, OCH3), 2.22–2.15 (m, 4H), 1.39 (t, J = 7.3Hz, 6H, CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ183.27(2C,C-Au-C),167.04(2C,CO),164.37,164.31,162.35,162.3 2(4C,ArCF),148.54(2C,ArCOH),147.12(2C,ArCOCH3),145.45(2C,C=CCOO),132.85,132.78,132.72 ,132.65,131.26,126.74,123.29,123.16,116.56,116.50,116.39,116.33,115.00,114.79,109.81, 61.07(2C,OCH2),56.30(2C,OCH3),46.67(2C,NCH2),44.71(2C,CH2CH3),30.82,17.61(2C,CH2CH3). ESI-MSm / z:1233.95[M–PF6] + ,like Figure 12 As shown in Figures a and b.

[0062] Bipharmacophore nitrogen heterocyclic carbene gold compound 12: yellow powder (78.1 mg, 37%). 1HNMR(500MHz,Chloroform-d)δ7.53(d,J=15.9Hz,2H,CH=CHCOO),7.22(ddd,J=8.8,5.2,1.9Hz,8H ,ArH),7.07–7.00(m,12H,ArH),6.89(d,J=8.1Hz,2H,ArH),6.20(d,J=15.9Hz,2H,CH=CHCOO),4.3 0(t,J=7.4Hz,4H,COOCH2),4.22(q,J=7.2Hz,4H,NCH2CH3),4.08(t,J=6.3Hz,4H,CH2CH2N),3.92( s, 6H, OCH3), 1.82 (dt, J = 14.4, 6.7Hz, 4H), 1.66 (p, J = 6.3Hz, 4H), 1.36 (t, J = 7.1Hz, 6H, NCH2CH3). 13 CNMR(126MHz,Chloroform-d)δ183.22(2C,C-Au-C),167.34(2C,CO),164.37,164.33,162.37,162.33(4C, F-ArC),148.27,147.02(2C,ArC),145.29(2C,CH=CHCOO),132.78,132.72,132.65,131.35,131.28,126.9 2,123.44,123.42,123.37,123.35,123.11,116.57,116.50,116.40,116.33,115.16,114.79,109.62,63. 53(2C,COOCH2),56.20(2C,OCH3),49.13(2C,CH2N),44.50(2C,CH2CH3),28.18,25.97,17.41(2C,CH2CH3). ESI-MSm / z:1261.94[M–PF6] + ,like Figure 13 As shown in Figures a and b.

[0063] Bipharmacophore nitrogen heterocyclic carbene gold compound 13: yellow powder (57.0 mg, 27%). 1HNMR(500MHz,Chloroform-d)δ7.56(dd,J=15.8,4.9Hz,2H,CH=CHCOO),7.22(td,J=8.8,5.0 Hz,10H,ArH),7.04(dd,J=15.6,7.3Hz,10H,ArH),6.90(d,J=8.6Hz,2H,ArH),6.23(d,J=15. 8Hz,2H,CH=CHCOO),5.94(s,2H,OH),4.22(q,J=7.2Hz,8H),4.08(t,J=6.5Hz,4H,NCH2),3.9 1(s,6H,OCH3),1.78(p,J=7.8,7.2Hz,4H),1.61–1.56(m,4H),1.34(dt,J=13.0,7.0Hz,10H). 13 CNMR(126MHz,Chloroform-d)δ183.14(2C,C-Au-C),167.44(2C,CO),164.33(2C,ArCF),162.34(2C,ArCF), 148.23(2C,ArCOH),147.01(2C,ArCOCH3),145.14(2C,C=CCOO),132.78,132.75,132.71,132.68,131.37,1 31.19,126.96,123.46,123.44,123.37,123.35,123.09,116.54,116.48,116.36,116.30,115.32,114.83, 109.58,63.81(2C,OCH2),56.15(2C,OCH3),49.19,44.44,31.32,29.83,28.26,23.10,17.40(2C,CH2CH3). ESI-MSm / z:1290.05[M–PF6] + ,like Figure 14 As shown in Figures a and b.

[0064] Bipharmacophore nitrogen heterocyclic carbene gold compound 14: yellow powder (79.3 mg, 41%). 1HNMR(500MHz,Chloroform-d)δ7.56(d,J=16.0Hz,2H,CH=CHCOO),7.52–7.46(m,4H,ArH),7.4 4–7.36(m,6H,ArH),7.18(dd,J=8.5,5.1Hz,4H,ArH),7.12(dd,J=8.5,5.2Hz,4H,ArH),7.02( q,J=8.2Hz,8H,ArH),6.23(d,J=16.1Hz,2H,CH=CHCOO),4.42(t,J=7.0Hz,4H,COOCH2),4.20( dt, J=13.2, 6.6Hz, 8H, NCH2), 2.14 (p, J=6.3Hz, 4H, NCH2CH2), 1.36 (t, J=7.2Hz, 6H, CH2CH3). 13 CNMR(126MHz,Chloroform-d)δ183.42(2C,C-Au-C),166.68(2C,COO),164.34,162. 35(4C,F-ArC),145.25(2C,CH=CHCOO),134.20,132.83,132.76,132.71,132.65,13 1.40,131.28,130.77,129.21,128.23,123.30,123.27,123.24,117.56,116.59,11 6.48,116.41,116.31,61.37(2C,COOCH2),46.45,44.54,30.48,17.41(2C,CH2CH3). ESI-MSm / z:1141.94[M–PF6] + ,like Figure 15 As shown in Figures a and b.

[0065] As an example of the present invention, a dual-pharmacophore nitrogen heterocyclic carbene gold compound has in vitro anti-proliferative activity against liver cancer cells (HepG2 and Hepa1-6).

[0066] Take HepG2, Hepa 1-6 and cells in the logarithmic growth phase. After washing the cells with sterile PBS, digest them with trypsin and terminate the digestion with DMEM complete medium. After centrifugation to remove the supernatant, count the cells and resuspend them for later use. According to the experimental requirements, cells of different densities are plated in 96-well plates, 100 μL per well, and the cells are cultured in an incubator overnight until they adhere to the wall. The concentration of the test compound is set according to the experimental requirements and diluted with DMEM complete medium. Take 100 μL of the diluted compound and add it to the 96-well plate containing adherent cells. Keep 3 replicates for each concentration. After culturing to a fixed time point, use CCK8 method or MTT method to determine the cell proliferation activity. Prepare and detect the reagents according to the instructions of the two detection methods. Calculate the cell viability according to the following formula: Cell viability (%) = (OD 给药组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%. The experimental data were collected and analyzed graphically. The results are shown in Table 1. Compared with the original gold complex, positive controls Auranofin and Oxaliplatin, compound 8 (Fa-Au) showed better anti-proliferation activity against liver cancer cells.

[0067] Table 1. IC values of compounds against HepG2 and Hepa 1-6 cells 50

[0068]

[0069] As an embodiment of the present invention, the anti-tumor mechanism of the compound Fa-Au synthesized by the present invention is studied.

[0070] 1. Compound Fa-Au inhibits TrxR activity.

[0071] Different concentrations of compound Fa-Au were co-incubated with pure TrxR enzyme activated by NADPH, and the inhibitory effect of compound Fa-Au on pure TrxR enzyme was analyzed by DTNB colorimetric principle; HepG2 cells in the logarithmic growth phase were plated in 6 cm cell culture dishes, and after the cells adhered, different concentrations of compound Fa-Au were added for 24 hours, and the inhibition of compound Fa-Au on intracellular TrxR activity was analyzed by TrxR activity kit.

[0072] like Figure 16 As shown in Figure B, the complex Fa-Au can inhibit the pure enzyme activity of TrxR in a dose-dependent manner (IC 50 =3.95μM); the compound Fa-Au was co-incubated with the variant form of TrxR (Usec498), and the results were as follows Figure 16Figure C shows that compound Fa-Au has no significant inhibition on TrxR (Usec498) within the unit monitoring time, indicating that compound Fa-Au binds to the Sec498 site of TrxR to exert pure TrxR enzyme inhibitory activity. The purpose of this study is to screen out the dominant active molecules that can inhibit intracellular TrxR activity and thus play an anti-cancer cell proliferation role. Therefore, we further analyzed the changes in TrxR enzyme activity in HepG2. Before detecting the intracellular TrxR enzyme activity, we first performed a 24h proliferation inhibition test on HepG2 cells, as shown in Figure 4. Figure 16 As shown in Figure D, the IC of the compound Fa-Au 50 is 2.24±0.13μM. According to the experimental results, Figure 16 As shown in Figure E, we found that compound Fa-Au could dose-dependently inhibit intracellular TrxR activity after 24 hours of administration to HepG2 cells.

[0073] 2. Compound Fa-Au induces increased intracellular ROS levels.

[0074] Cells (HepG2 and Hepa 1-6) in the logarithmic growth phase were taken. The two types of cells were plated in 12-well plates (5×10 4 / well) and 6-well plates (2×10 5 After adding different concentrations of drugs and continuing to culture for 3 hours, the cells in the 12-well plate were used for immunofluorescence imaging with a ROS probe (DCFH-DA): the working solution was prepared and incubated according to the DCFH-DA probe instructions, and the changes in ROS levels were analyzed using a fluorescence microscope (Leica DMi8, Germany) and flow cytometer. Figure 17 As shown in Figures A to C, the green fluorescence intensity of HepG2 after administration of Fa-Au and AF was significantly higher than that of the blank control group, indicating that the administration of Fa-Au and AF can significantly increase the level of ROS in HepG2 cells; after pre-protection incubation of HepG2 cells with the antioxidant NAC, it can significantly reverse the stimulation of Fa-Au on the level of intracellular ROS. In order to further analyze the specific mechanism of action of Fa-Au in causing increased ROS levels, we also used reduced glutathione (GSH) and oxidized glutathione (GSSG) detection kits to detect the changes in GSH and GSSG in HepG. As the main source of intracellular thiol groups, GSH participates in the regulation of intracellular redox homeostasis and is considered to be one of the key intracellular antioxidants. Figure 17As shown in Figures D to E, after Fa-Au administration, intracellular GSH levels decreased significantly, GSSG levels increased significantly, and the GSH / GSSG ratio decreased significantly, demonstrating a surge in intracellular ROS levels. This indicates that a large amount of GSH undergoes ROS reduction and is oxidized to GSSG. Pre-incubation with NAC reversed these Fa-Au-induced effects. In summary, Fa-Au significantly stimulates ROS levels in HepG2 cells, disrupting intracellular redox homeostasis.

[0075] 3.Fa-Au induces HepG2 ferroptosis.

[0076] HepG2 cells were plated in 96-well plates (8 × 10 3 / well) and 12-well plates (5×10 4 / well) and cultured overnight until adherence. Ferrostatin-1 (Fer-1) was pre-incubated (37°C, 1.5h) in a 96-well plate, and then the drug intervention continued for 24h. After 24h, cell proliferation was detected using the CCK8 detection kit, and the experimental data were collected for analysis and plotted. For HepG2 cells in 12-well plates, after 24h of drug action, a portion of the cells were washed twice with sterile PBS and set aside. C11 BODIPY probe working solution (2μM) was added to each group, incubated at 37°C for 30min, and then washed again with sterile PBS. The cells were imaged and photographed using a fluorescence microscope, and the imaging data were collected and organized for plotting; after another portion of the cells were collected, the morphological changes of organelles in the cells were observed using a transmission electron microscope. Ferroptosis-related proteins were analyzed by Western Blot experiments.

[0077] According to the TEM experimental results of HepG2, Figure 18 As shown in Figure A, the mitochondria in HepG2 treated with Fa-Au showed a significant increase in double membrane density, overall mitochondrial shrinkage, and reduced or even disappeared cristae. Figure 18 As shown in Figure B, pre-incubation with the ferroptosis inhibitor Ferrostatin-1 (Fer-1) (4 μM, 2 h) significantly inhibited the proliferation inhibitory activity of Fa-Au on HepG2 cells at the final concentration of Fa-Au (0.3125–10 μM). Figure 18 As shown in Figures C to D, the green fluorescence intensity of the Fa-Au-treated group increased significantly, indicating an increase in the accumulation of C11 BODIPY oxidation state, indicating that a large amount of lipid peroxides accumulated in HepG2 cells after Fa-Au administration. Western blot experiments were used to further analyze the changes in ferroptosis regulatory factors in HepG2 after Fa-Au administration. Compared with the blank group, the expression levels of GPX4 and SLC7A11 in the Fa-Au-treated group were significantly reduced. Figure 18 As shown in Figures E to F in Figure 3. Based on the above experimental results, we found that Fa-Au can cause the accumulation of lipid peroxides in HepG2 cells, reduce GSH levels, affect mitochondrial function, downregulate the expression of key factors GPX4 and SLC7A11, and induce ferroptosis.

[0078] As an embodiment of the present invention, the in vivo anti-tumor effect of the compound Fa-Au synthesized by the present invention was studied.

[0079] 1. The zebrafish model was used to evaluate the anti-proliferation and migration effects of the compound Fa-Au on HepG2 cells.

[0080] The implantable zebrafish tumor model can visualize the proliferation and migration of tumor cells and is a fast and efficient model for anticancer drug screening. We used (fli-1:EGFP) zebrafish embryos to implant red-labeled HepG2 cells into the yolk sac of the zebrafish and continuously monitor the proliferation and migration of HepG2 cells in the zebrafish. According to the experimental results, Figure 19 As shown in Figures A to C, HepG2 in the model group continued to proliferate and migrate along the vascular plexus within three days, as shown by the gradual increase and diffusion of red fluorescence; in the AF and Fa-Au treatment groups, the growth rate and diffusion of red fluorescence intensity were significantly inhibited with the increase in treatment time, and the effect of Fa-Au was more significant, indicating that Fa-Au can inhibit the proliferation and migration of HepG2 cells in zebrafish.

[0081] 2. Compound Fa-Au inhibits tumor growth in C57 / BL6 mice.

[0082] The in vivo immunogenic cell death (ICD) effect and tumor growth inhibition effect of Fa-Au were further analyzed using a C57 / BL6 mouse tumor-bearing model. Figure 20 As shown in Figures A and D, we found that during the monitoring period, the tumor growth rate in the model group was significantly faster than that in the Oxa and Fa-Au administration groups; both the Fa-Au and Oxa administration groups could inhibit tumor growth to varying degrees, and the tumor growth inhibition effect of the Fa-Au administration group was better than that of the Oxa administration group, as shown in Figure 4. Figure 20 As shown in Figure C, there was statistical significance compared with the model group; there was no significant change in the body weight of mice in each group. Figure 20 As shown in Figure B, the two drug groups had no obvious toxic side effects on mice under this dosage condition. Figure 20 As shown in Figures E and H, compared with the model group, the tumor tissues of the Fa-Au group and the Oxa group showed different degrees of apoptosis. TrxR in tumor tissues was analyzed by immunofluorescence detection. According to the experimental results, Figure 20 As shown in Figures F and I, there was no significant change in TrxR in the three groups of tumor tissues; further analysis of ROS generation in tumor tissues showed that Figure 20 As shown in Figures G and J, the green fluorescence intensity in the Fa-Au group was higher than that in the Oxa group and the model group, indicating that Fa-Au administration can cause an increase in the level of ROS in tumor tissue.

[0083] According to Figure 21 The experimental results shown in the figure were used for preliminary analysis of immune infiltration, and it was found that the immune infiltration levels of the Fa-Au and Oxa administration groups were higher than those of the model group. After the in vivo pharmacodynamics experiment, HE staining was used to analyze the damage of the main organs of C57 / BL6 mice to preliminarily evaluate the biosafety of the experimental drug dose. Figure 22 As shown, no obvious inflammation and damage were observed in the two drug-treated groups compared with the model group, indicating that the two drug-treated groups had good biosafety at the experimental dosage.

[0084] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the scope and essence of the present invention should be within the scope of the present invention.

Claims

1. A bipharmacophore nitrogen heterocyclic carbene gold compound 1-14, characterized in that: The structure of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 1-14 is as follows: The dual-pharmacophore nitrogen heterocyclic carbene gold compounds 1-14 are synthesized by replacing the triethylphosphine group in auranofin with NHC and introducing the traditional Chinese medicine small molecule ferulic acid Fa.

2. The dual-pharmacophore nitrogen heterocyclic carbene gold compound 1-14 according to claim 1, characterized in that: The structure of the bipharmacophore nitrogen heterocyclic carbene gold compound 8 is shown below:

3. Use of a bipharmacophore nitrogen heterocyclic carbene gold compound 1-14 in the preparation of an anti-liver cancer drug, characterized in that: The structure of the dual-pharmacophore nitrogen heterocyclic carbene gold compound 1-14 is shown below:

4. Use of the bipharmacophore nitrogen heterocyclic carbene gold compound 1-4 according to claim 3 in the preparation of an anti-liver cancer drug, characterized in that: The structure of the bipharmacophore nitrogen heterocyclic carbene gold compound 8 is shown below: The dual-pharmacophore nitrogen heterocyclic carbene gold compound 8 has anti-proliferative activity against liver cancer cells HepG2 and Hepa1-6, can inhibit tumor growth, and induce dendritic cell maturation, macrophage M1 polarization and T cell activation in the tumor immune microenvironment.

Images