Schiff base gold (III) compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation of anti-cancer drugs, in particular to a Schiff base gold (III) compound and a preparation method and application thereof.The series of symmetric or asymmetric Schiff base gold (III) compounds are synthesized on the basis of the bioisostere principle in combination with a Schiff base ligand, the cyclohexanediamine structure and the NNOO coordination mode of oxaliplatin are reserved, platinum (II) is replaced with gold (III), and the Schiff base gold (III) compound is synthesized. Meanwhile, the invention relates to application of the Schiff base gold (III) compound in preparation of anti-hepatoma drugs, the defects that oxaliplatin is strong in side effect and the like are overcome, in-vivo and in-vitro experiment results of the Schiff base gold (III) compound provided by the invention show that the compounds Au3 and Au7 are remarkable in anti-hepatoma activity and can target thioredoxin reductase (TrxR) and mitochondrial DNA (mtDNA) at the same time, and the Schiff base gold (III) compound can be used for preparing anti-hepatoma drugs. The further mechanism research shows that the compound can promote the generation of reactive oxygen species (ROS) to cause endoplasmic reticulum stress (ERS) and mitochondrial injury and finally induce pyroptosis and immunogenic cell death, and has great application potential.

Description

Technical Field

[0001] This invention relates to the field of compound preparation technology, specifically to the preparation of a Schiff base gold (III) compound targeting mtDNA and TrxR and its application in the preparation of anti-liver cancer drugs. Background Technology

[0002] Platinum-based drugs are widely used to treat various cancers. Oxaliplatin is the only metalloimmunogenic cell death inducer approved by the U.S. Food and Drug Administration (FDA). However, platinum-based drugs have drawbacks such as poor selectivity, significant side effects, and marked drug resistance. The main cause of cisplatin resistance is targeting nuclear DNA (nDNA). Developing drugs targeting non-nuclear DNA (such as mtDNA) holds promise for overcoming cisplatin resistance. Mitochondrial mtDNA encodes an important subunit of the mitochondrial respiratory chain complex, which is crucial for ATP secretion. Pyroptosis is considered highly immunogenic, promoting and stimulating endogenous and adaptive immune responses; inducing pyroptosis may be a novel approach for developing anti-tumor drugs.

[0003] Gold compounds have a long history of application in medicine. Unlike platinum-based drugs, the primary target of gold compounds is thioredoxin reductase (TrxR). Numerous studies have shown that TrxR is involved in various pathological processes, including tumorigenesis and development, and is overexpressed in these processes. Furthermore, in various cancers, including primary liver cancer, high expression levels of the TrxR / Trx system are directly associated with poor prognosis and reduced survival. Therefore, TrxR is considered a promising target for anticancer therapy. Our previous studies have demonstrated that gold compounds can effectively inhibit TrxR activity and promote the generation of reactive oxygen species (ROS). This prevents cells from maintaining endoplasmic reticulum homeostasis, leading to the accumulation of misfolded proteins and ultimately endoplasmic reticulum stress (ERS). The generation of ERS and ROS has been shown to be key factors regulating immunogenic cell death pathways.

[0004] Platinum(II) and gold(III) are bioisosteres, and therefore may possess similar properties, including damage to DNA. Early studies have shown that gold(III) compounds are the best alternatives to platinum(II) drugs. Schiff bases are a class of excellent ligands with diverse activities; when bound to metal ions, they can enhance their stability and increase their cell membrane permeability. Numerous Schiff base metal compounds with antitumor activity have been reported. Therefore, researching new Schiff base gold(III) compounds with great development potential is crucial. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Schiff base gold (III) compound, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The Schiff base gold (III) compound involved in this invention is any one of the following compounds:

[0008]

[0009] A further embodiment is that the Schiff base gold (III) compound is compound Au3 or compound Au7, and the structural formulas of compound Au3 and compound Au7 are as follows:

[0010]

[0011] The present invention also provides a method for preparing Schiff base gold(III) compounds, which is synthesized by combining Schiff base ligands, retaining the cyclohexanediamine structure and NNOO coordination mode of oxaliplatin, and using gold(III) instead of platinum(II).

[0012] This invention relates to the use of any of the above-mentioned Schiff base gold (III) compounds in the preparation of anti-liver cancer drugs.

[0013] Among them, Schiff base gold (III) compounds simultaneously target mtDNA and TrxR. Further mechanistic studies have shown that these compounds can promote ROS generation, leading to ERS and mitochondrial damage, ultimately inducing pyroptosis and immunogenic cell death. In addition, Schiff base gold (III) compounds have anti-proliferative activity against liver cancer cells Hepa 1-6, Huh7, and Hepa3B, and can inhibit tumor growth.

[0014] The Schiff base gold (III) compounds, their preparation methods, and applications provided in the above technical solutions aim to overcome the defects of oxaliplatin, such as its strong side effects. Based on the principle of bioisosteres, this invention combines Schiff base ligands, retains the cyclohexanediamine structure and NNOO coordination mode of oxaliplatin, and uses gold (III) to replace platinum (II) to synthesize a series of symmetrical or asymmetrical Schiff base gold (III) compounds. The synthesized Schiff base gold (III) compounds have the advantages of simple synthesis, low price, significant activity, and strong stability.

[0015] Among them, Schiff base gold (III) compounds Au1-Au12 all showed good anti-hepatocellular carcinoma activity. After comprehensive evaluation, the symmetrical compound Au3 and the asymmetric compound Au7 were screened for further research. In vitro and in vivo experimental results showed that compounds Au3 and Au7 had significant anti-hepatocellular carcinoma activity and could simultaneously target mtDNA and TrxR. By promoting ROS generation, they caused ERS and mitochondrial damage, ultimately inducing pyroptosis and immunogenic cell death, showing great application potential. Attached Figure Description

[0016] Figure 1 This is the basic synthetic route diagram for Schiff base gold (III) compounds Au1-Au12;

[0017] Figure 2 and Figure 3 These are the 1H and 1C spectra of Au1, a Schiff base gold (III) compound.

[0018] Figure 4 and Figure 5 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au2;

[0019] Figure 6 and Figure 7 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au3;

[0020] Figure 8 and Figure 9 These are the 1H and 1C spectra of Au4, a Schiff base gold (III) compound.

[0021] Figure 10 and Figure 11 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au5;

[0022] Figure 12 and Figure 13 These are the 1H and 1C spectra of Au6, a Schiff base gold (III) compound.

[0023] Figure 14 and Figure 15 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au7;

[0024] Figure 16 and Figure 17 These are the proton and carbon spectra of the Schiff base gold (III) compound Au8;

[0025] Figure 18 and Figure 19 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au9;

[0026] Figure 20 and Figure 21 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au10;

[0027] Figure 22 and Figure 23 These are the 1H and 1C spectra of Au11, a Schiff base gold (III) compound.

[0028] Figure 24 and Figure 25 These are the 1H and 1C spectra of the Schiff base gold (III) compound Au12;

[0029] Figure 26 The following are the results of the Schiff base gold (III) compound Au3: (A) the pure enzyme inhibitory activity of TrxR; (B) the intracellular inhibitory activity of TrxR; (C) the inhibitory ability of mtDNA; (D) pyroptosis electron microscopy image; (E) CRT efflux immunofluorescence image; (F) WB images of CRT and HMGB1; (G) changes in extracellular ATP.

[0030] Figure 27 The images show: (A) Fluorescence pattern promoting ROS expression changes (Schiff base gold (III) compound Au3; (B) Fluorescence pattern promoting calcium ion changes; (C) Fluorescence pattern causing mitochondrial damage; (DF) Tumor image, volume, and weight after treatment; (G) Changes in mouse body weight during treatment.

[0031] Figure 28 The images show the following: (A) the pure enzyme inhibitory activity of the Schiff base gold (III) compound Au7; (B) the intracellular inhibitory activity of TrxR; (C) pyroptosis electron microscopy image; (D) CRT efflux immunofluorescence image; and (E) the change in extracellular ATP.

[0032] Figure 29 The images show: (A) Fluorescence pattern of Schiff base gold (III) compound Au7 promoting changes in ROS expression; (B) Fluorescence pattern of ERS; (C) Fluorescence pattern of mitochondrial damage; (DF) Tumor image, volume, and weight after treatment; (G) Changes in mouse body weight during treatment. Detailed Implementation

[0033] To make the objectives and advantages of this invention clearer, the invention will be specifically described below with reference to embodiments. It should be understood that the following text is merely used to describe one or more specific embodiments of the invention and does not strictly limit the scope of protection specifically claimed by the invention.

[0034] This invention investigates the preparation of a series of Schiff base gold (III) compounds Au1-Au12 targeting mtDNA and TrxR, and their in vitro and in vivo anti-hepatocellular carcinoma mechanisms.

[0035] Example 1:

[0036] A series of Schiff base gold(III) compounds retaining the cyclohexanediamine structure and NNOO coordination mode of oxaliplatin were synthesized via one-pot or stepwise synthesis methods. Utilizing the bioisosteric principle, Schiff base ligands were bound to gold(III) compounds, preserving the cyclohexanediamine and NNOO coordination mode of oxaliplatin, and synthesized through chemical methods (such as... Figure 1 The Schiff base gold (III) compound Au1-Au12 was obtained; and its structure was determined using 1H and 1C NMR spectroscopy (as shown). Figures 2-25(As shown).

[0037] The synthesis of Schiff gold (III) compounds Au3 and Au7 is introduced using the synthesis method of Schiff gold (III) compounds as an example:

[0038] The synthesis steps of Au3 are as follows: Accurately weigh 50.00 mg (0.13 mmol) of NaAuCl4·2H2O and dissolve it in 2 mL of ethanol. Then, accurately weigh 67.20 mg (0.13 mmol) of L3 and completely dissolve it in 1 mL of dichloromethane. Add the dichloromethane solution dropwise to the ethanol solution, stir well, add ammonium hexafluorophosphate (102.40 mg, 0.65 mmol), and react under argon protection, protected from light, at room temperature for 24 h. After the reaction is complete, a clear, brownish-red substance is obtained. Filter to remove ammonium hexafluorophosphate, and evaporate the filtrate to dryness to obtain the crude product. Add 1 mL of methanol, 1 mL of toluene, and 2 mL of n-hexane, and let stand at -20 °C for at least 6 h. An orange-red solid precipitates, which is filtered, washed with a small amount of dichloromethane and methanol, and dried to obtain the product.

[0039] The synthesis steps of Au7 are as follows: Accurately weigh NaAuCl4·2H2O (50.00 mg, 0.13 mmol) and dissolve it completely in 1 mL of ethanol. Accurately weigh L7 (63.90 mg, 0.13 mmol) and add 2 mL of dichloromethane to dissolve it completely. Then, add the dichloromethane solution dropwise to the ethanol solution, stir well, add ammonium hexafluorophosphate (102.40 mg, 0.65 mmol), and stir at room temperature for 24 h under argon protection and in the dark. After the reaction is complete, an orange-red solid precipitates. Filter the solid, wash it with dichloromethane and ethanol, dissolve it in acetonitrile, and filter to obtain the crude product. Add methanol to the crude product, stir at 55 °C for 10 min, then add toluene, let it stand at -20 °C for more than 6 h, filter, wash with a small amount of dichloromethane and methanol, and dry to obtain the product.

[0040] The structures of compounds Au1-Au12 are as follows:

[0041]

[0042] The specific chemical data are as follows:

[0043] Au1: Brown solid, yield 15.2%; 11H NMR (500 MHz, DMSO-d6) δ 8.64 (s, 2H, 2×N=CH), 7.77 (d, J = 8.9 Hz, 2H, ArH), 6.77 (d, J = 2.4 Hz, 2H, ArH), 6.68 (dd, J = 8.9, 2.4 Hz, 2H, ArH), 4.39 (s, 2H, 2×N-CH), 3.89 (s, 6H, 2×OCH3), 2.37 (s, 2H), 1.96 (d, J = 11.8 Hz, 2H), 1.53 - 1.43 (m, 4H). 13 13C NMR (126 MHz, DMSO-d6) δ 168.38, 162.11, 158.09, 137.44, 113.39, 109.13, 100.29, 71.25, 56.06, 25.3, 20.42. <​​​​​​​​​​​C NMR (126MHz, DMSO-d6)δ

[0046] 167.28,161.98,158.10,137.45,136.15,128.58,128.15,127.86,113.49,109.79,101.27,71.27,69.85,25.99,20.44.

[0047] Au4: Orange-red solid, yield 33.0%; 1 H NMR (500MHz, DMSO-d6) δ9.38(s,1H,N=CH),9.30(s,1H,N=CH),8.25(d,J=9.2Hz,1H,ArH),8.15(d,J=9.1Hz, 1H,ArH),7.98(d,J=7.9Hz,1H,ArH),7.90(d,J=8.8Hz,1H,ArH),7.76-7.71(m,ArH),7.54-7.46(m,2H,ArH) ,7.29(d,J=9.1Hz,1H,ArH),7.17(dd,J=8.8,2.2Hz,1H,ArH),4.72(d,J=5.2Hz,1H,N-CH),4.64(dt,J=9.3, 4.5Hz,1H,N-CH),3.98(s,3H,OCH3),2.79(s,1H),2.52(s,1H),2.16-2.01(m,2H),1.68(s,1H),1.63(s,3H). 13 C NMR (126MHz, DMSO-d6) δ162.39,161.88,160.07,153.76,153.51,139.45,139.41,135.10,133.00,130.99,129.22,129.12,127. 74,124.51,122.71,121.90,120.37,117.41,114.48,109.99,109.44,103.81,72.58,72.20,55.69,25.19,21.27,21.17,19.73.

[0048] Au5: Brownish-yellow solid, yield 21.5%; 1HNMR (500MHz, DMSO-d6) δ10.25(s,1H,-OH),9.41(s,1H,N=CH),9.07(s,1H,N=CH),8.14(d,J=9.1Hz,1H,ArH),8.08(d,J=9. 1Hz,1H,ArH),7.89(d,J=8.8Hz,1H,ArH),7.79(d,J=8.6Hz,1H,ArH),7.74(d,J=2.4Hz,1H,ArH),7.56(d,J=2.2Hz,1H,ArH) ,7.28(d,J=9.0Hz,1H,ArH),7.20(d,J=10.0Hz,1H,ArH),7.16(dd,J=2.4Hz,8.8Hz,1H,ArH),7.03(dd,J=8.6,2.0Hz,1H,Ar H),4.76-4.70(m,1H,N-CH),4.68-4.62(m,1H,N-CH),3.99(s,3H,OCH3),2.76(s,1H),2.18-2.03(m,2H),176-1.49(m,5H). 13 C NMR (126MHz, DMSO-d6) δ162.47,160.08,158.60,153.77,153.09,139.57,139.33,135.59,135.16,131.07,130.98,122.71, 121.88,117.54,116.45,115.42,114.48,109.46,109.11,105.46,103.82,72.57,71.85,55.71,31.84,27.20,26.49,19.51.

[0049] Au6: Orange-red solid, yield 15.4%; 11H NMR (500 MHz, DMSO-d6) δ 9.41 (s, 1H, N=CH), 9.25 (s, 1H, N=CH), 8.62 (d, J = 1.8 Hz, 1H, ArH), 8.26 (d, J = 9.2 Hz, 1H, ArH), 8.16 (d, J = 9.2 Hz, 1H, ArH), 7.92 (dd, J = 13.3, 8.6 Hz, 2H, ArH), 7.71 - 7.63 (m, 2H, ArH), 7.52 (d, J = 9.1 Hz, 1H, ArH), 7.30 (d, J = 9.0 Hz, 1H, ArH), 7.18 (dd, J = 8.8, 2.2 Hz, 1H, ArH), 4.73 - 4.65 (m, 2H, N-CH), 3.99 (s, 3H, OCH3), 2.11 (d, J = 12.1 Hz, 2H), 1.60 (s, 4H), 1.05 (t, J = 7.0 Hz, 2H). 13 13C NMR (126 MHz, DMSO-d6) δ 162.42, 162.16, 160.05, 154.03, 153.48, 139.48, 139.05, 135.04, 134.49, 131.5, 127.55, 126.45, 124.27, 123.31, 122.70, 121.07, 117.33, 114.42, 109.44, 103.91, 72.63, 72.33, 56.03, 55.63, 54.93, 31.96, 25.95, 20.14, 18.57.

[0050] Au7: Orange-red solid, 20.10%; 1H NMR (500MHz, DMSO-d6) δ9.47(s,1H,N=CH),8.55(s,1H,N=CH),8.11(dq,J=13.3,8.1,5.6Hz,1H,ArH),7.85(ddt,J= 22.9,8.5,4.2Hz,2H,ArH),7.73(s,1H,ArH),7.51-7.34(m,5H,ArH),7.26-7.09(m,2H,ArH),6.86(s,1H,ArH),6.68 (q,J=8.5Hz,1H,ArH),5.27(d,J=14.2Hz,2H,OCH2),4.65(s,1H,N-CH),4.51(d,J=10.7Hz,1H,N-CH),3.98(s,3H,O CH3),2.78(d,J=15.5Hz,1H),2.19-1.94(m,3H),1.79(d,J=12.8Hz,1H),1.58-1.44(m,2H),1.34(d,J=12.8Hz,1H). 13 C NMR (126MHz, DMSO-d6) δ167.50,162.41,162.10,160.24,157.93,154.20,139.43,137.84,136.26,135.26,131.06,128.71,128.27, 127.82,122.82,117.46,114.76,113.93,109.78,109.48,103.80,101.35,73.25,70.57,69.98,55.88,29.04,23.35,22.71,18.38.

[0051] Au8: Orange-yellow solid, yield 24.7%; 1HNMR(500MHz, DMSO-d6) δ 9.49 (s, 1H, N=CH), 8.57 (s, 1H, N=CH), 8.13 (d, J=9.1Hz, 1H, ArH), 7.87 (dd, J=16.2, 8.9Hz, 2H, ArH), 7.76 (s, 1H, ArH), 7.36 (d, J=7.7Hz, 2H, ArH), 7.23 (d, J=7.2Hz, 3H, ArH), 7.16 (dd, J=8.7, 2.2Hz, 1H, ArH), 6.87 (d, J=2.5Hz, 1H, ArH), 6.71 (dd, J=9.0, 2.4Hz, 1H, ArH), 5.23 (s, 2H, OCH2), 4.63 (s, 1H, N-CH), 4.50 (dt, J=10.8, 4.9Hz, 1H, N-CH), 3.98 (s, 3H, OCH3), 2.78 (d, J=14.3Hz, 1H), 2.31 (s, 3H,), 2.19 - 1.94 (m, 3H), 1.77 (d, J=12.6Hz, 1H), 1.50 (dd, J=22.8, 12.4Hz, 2H), 1.40 - 1.27 (m, 1H). 13 C NMR(126MHz, DMSO-d6) δ 167.43, 162.35, 162.03, 160.13, 157.82, 154.15, 139.29, 137.68, 137.45, 135.18, 133.14, 130.97, 129.12, 127.85, 122.72, 117.44, 114.63, 113.81, 109.77, 109.39, 103.73, 101.27, 73.10, 70.38, 69.80, 55.78, 23.23, 22.66, 20.80, 18.31, 17.82.

[0052] Au9: Orange solid, yield 15.1%; 1H NMR (500MHz, DMSO-d6) δ9.50(s,1H,N=CH),8.58(s,1H,N=CH),8.13(d,J=9.2Hz,1H,ArH),7.88(dd,J=11.0,8.9Hz,2H,ArH),7.76 (d,J=2.3Hz,1H,ArH),7.54(dd,J=8.4,5.5Hz,2H,ArH),7.30-7.20(m,3H,ArH),7.16(dd,J=8.8,2.2Hz,1H,ArH),6.91(d,J=2.4H z,1H,ArH),6.72(dd,J=8.9,2.4Hz,1H,ArH),5.27(s,2H,OCH2),4.64(d,J=4.7Hz,1H,N-CH),4.51(dt,J=10.4,4.8Hz,1H,N-CH), 3.98(s,3H,OCH3),2.78(d,J=15.4Hz,1H),2.20-1.96(m,3H),1.78(d,J=11.9Hz,1H),1.57-1.43(m,2H),1.35(t,J=13.0Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ167.25,162.90,162.29,162.01,160.95,160.12,15 7.84,154.14,139.29,137.71,135.17,132.42(d,J=3.0Hz),130.96,130.12( d, J=8.4Hz),122.71,117.39,115.51,115.34,114.61,113.89,109.65,109. 38,103.72,101.22,73.10,70.41,69.13,55.78,28.92,23.18,22.69,18.28.

[0053] Au10: Orange-yellow solid, yield 25.7%; 1H NMR (500MHz, DMSO-d6) δ9.50(s,1H,N=CH),8.57(s,1H,N=CH),8.13(d,J=9.2Hz,1H,ArH),7.87(dd,J=12.6,8.9Hz,2H,ArH),7.76(d, J=2.3Hz,1H,ArH),7.42(dd,J=8.2Hz,25.0Hz,4H,ArH),7.23(d,J=9.1Hz,1H,ArH),7.15(dd,J=8.8,2.2Hz,1H,ArH),6.89(d,J=2.4H z,1H,ArH),6.71(dd,J=9.0,2.4Hz,1H,ArH),5.25(s,2H,OCH2),4.64(d,J=4.5Hz,1H,N-CH),4.51(dt,J=10.4,5.0Hz,1H,N-CH),3.9 8(s,3H,OCH3),2.78(d,J=15.3Hz,1H),2.19-1.96(m,3H),1.78(d,J=11.7Hz,1H),1.57-1.42(m,2H),1.38-1.31(m,1H),1.29(s,9H). 13 C NMR (126MHz, DMSO-d6) δ167.44,162.32,162.01,160.10,157.80,150.64,139.26,137.69,135.16,133.16,130.94,127.66,125.29,12 2.69,117.40,114.60,113.80,109.70,109.37,103.72,101.19,73.07,70.36,69.66,55.76,34.33,31.11,23.16,22.67,21.27,18.26.

[0054] Au11: Orange-red solid, yield 32.0%; 1H NMR (500MHz, DMSO-d6) δ9.49(s,1H,N=CH),8.57(s,1H,N=CH),8.13(d,J=9.2Hz,1H,ArH),7.89(d,J=9.0Hz,1H,ArH),7.85(d,J=9.0Hz,1H, ArH),7.76(d,J=2.4Hz,1H,ArH),7.39-7.30(m,4H,ArH),7.28–7.23(m,1H,ArH),7.22(d,J=9.1Hz,1H,ArH),7.19-7.13(m,1H,ArH),6.85(d ,J=2.4Hz,1H,ArH),6.65(dd,J=8.9,2.4Hz,1H,ArH),4.64(s,1H,N-CH),4.52(dt,J=9.9,4.5Hz,1H,N-CH),4.38(t,J=6.8Hz,2H,OCH2),3. 99(s,3H,OCH3),3.09(t,J=6.8Hz,2H),2.78(d,J=14.7Hz,1H),2.21-1.94(m,3H),1.82-1.74(m,1H),1.57-1.46(m,2H),1.41-1.29(m,1H). 13 C NMR(126MHz,DMSO-d6)δ168.09,162.82,162.56,160.60,158.27,154.61,139.75,138.48,138.21,135.65,131.42,129.44,128.82,128.67,126 .88,125.78,123.18,117.85,115.07,114.21,109.86,109.84,104.23, 101.31,73.57,70.87,69.34,56.24,35.03,29,34,23.72,23.08,18.78.

[0055] Au12: Orange-red solid, yield 24.5%; 1H NMR (500MHz, DMSO-d6) δ9.50(s,1H,N=CH),8.58(s,1H,N=CH),8.14(d,J=9.2Hz,1H,ArH),7.88(dd,J=16.7,8.9Hz,2H,A rH),7.77(s,2H,ArH),7.31(t,J=7.5Hz,2H,ArH),7.28-7.14(m,5H,ArH),6.81(d,J=2.4Hz,1H,ArH),6.67(dd,J=8.9,2. 4Hz,1H,ArH),4.65(s,1H,N-CH),4.52(dt,J=9.9,4.6Hz,1H,N-CH),4.13(t,J=6.5Hz,2H,OCH2),3.99(s,3H,OCH3),2.8 3-2.73(m,3H),2.22-2.12(m,1H),2.11-1.97(m,4H),1.79(d,J=11.0Hz,1H),1.58-1.44(m,2H),1.37(t,J=12.1Hz,1H). 13 CNMR(126MHz,DMSO-d6)δ167.81,162.35,162.10,160.10,157.76,154.12,141.14,139.25,137.68,135.15,130.92,128.35,128.33,125.8 9,122.68,117.38,114.56,113.66,109.42,109.36,103.74,100.66,7 3.07,70.35,67.64,55.74,31.28,30.00,28.91,23.23,22.58,18.28.

[0056] Example 2: In vitro inhibitory effect of Schiff base gold (III) compounds Au3 and Au7 on liver cancer cells.

[0057] The inhibitory effects of compounds Au1-Au12 on Hepa 1-6, Huh7, and Hepa3B cells were determined using the MTT assay. In vitro screening results for anti-hepatocellular carcinoma activity showed that compounds Au1-Au12 all exhibited good anti-hepatocellular carcinoma activity (Table 1). Among them, the symmetrical compound Au3 and the asymmetric compound Au7 showed significantly higher anti-hepatocellular carcinoma activity than the positive control drugs aurinophene and oxaliplatin.

[0058] Table 1. Inhibitory effect of Schiff base gold (III) compounds Au1-Au12 on liver cancer cells (IC50, 100%) 50 Value [μM], Mean ± SD, 72h)

[0059]

[0060] Example 3: TrxR Pure Enzyme Activity Detection

[0061] First, take 80 μL of the TrxR enzyme stock solution and dilute it with 720 μL of ddH2O. Then, add 25 μL of TrxR enzyme to each well of a 96-well plate. Next, add 25 μL of the compound diluted with DMF (0.01 μM, 0.1 μM, 1 μM, and 10 μM) and DMF in sequence, setting up three replicates for each concentration. At the same time, prepare the reaction solution. Then, immediately add 225 μL of the reaction solution to each well, and then add 25 μL of the colorimetric reagent (DTNB) to each well. Mix well by pipetting and repositioning. After confirming that there are no air bubbles, use a microplate reader to detect the change in absorbance at 405 nm within 10 minutes. Figure 26 A and Figure 28 Studies on the inhibitory activity of purified TrxR enzyme A in the study showed that Au3 and Au7 have significant inhibitory activity against TrxR, with inhibitory capacity reaching the nanomolar level, and corresponding IC50 values ​​of [missing information]. 50 The values ​​are 954 nM and 970 nM, respectively.

[0062] Example 4: Detection of TrxR activity at the cellular level

[0063] Huh7 cells in logarithmic growth phase were treated with compounds (2.5 μM, 5 μM, 10 μM, 20 μM). Proteins were extracted after 24 hours, and TrxR activity in the cells was detected using a TrxR activity assay kit. Protein concentrations for each group were determined using a BCA protein quantification kit. Figure 26 B and Figure 28 As shown in B, with increasing compound concentration, Au3 and Au7 significantly enhanced their inhibitory ability on intracellular TrxR, indicating that Au3 and Au7 can significantly inhibit TrxR activity at the cellular level.

[0064] Example 5: RNA extraction and qPCR experiment

[0065] 1×10⁶ Huh7 cells were seeded into 6-well plates. 6 / well / 2mL, add compound Au3 for 24h treatment, then extract RNA, detect RNA concentration, synthesize cDNA and perform qPCR. GAPDH was used as a normalized control, and the relative difference in gene expression was calculated using the ΔΔCt method. The results showed that treatment with compound Au3 for 24h significantly reduced the level of intracellular mtDNA (mtDNA). Figure 26 (C in the middle).

[0066] Example 6: Scanning Electron Microscopy Detection

[0067] Huh7 cells were seeded into 12-well plates. After cell adhesion, the cells were treated with a compound for 12 hours, followed by washing with PBS and fixation with 3% glutaraldehyde. After further sample processing, images were acquired using a JSM-IT700HR scanning electron microscope. Figure 26 D and Figure 28 As shown in Figure C, treatment with Au3 and Au7 disrupted the microvilli on the surface of Huh7 cells, resulting in the formation of prominently protruding vesicles and obvious pores on the cell membrane. This phenomenon exhibits clear characteristics of pyroptosis, demonstrating that Au3 and Au7 can significantly induce pyroptosis in Huh7 cells.

[0068] Example 7: Immunofluorescence Experiment

[0069] After sterilization, 20mm cell slides were placed in 12-well plates. Huh7 cells were seeded into the wells, and after cell adhesion, drugs were administered for 24 hours. The supernatant was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. After washing with PBS, 0.1% Triton X-100 was added for permeabilization for 10 minutes. Then, 1% BSA was added for blocking for 60 minutes. Primary antibody was added, and the cells were incubated overnight at 4°C, followed by washing three times with PBS. Subsequently, under light-protected conditions, fluorescently labeled rabbit secondary antibody was added, and the cells were incubated at room temperature for 2 hours, followed by washing three times with PBS. After nuclear staining with DAPI dye, the cells were washed three times with PBS. The slides were removed, anti-fluorescence quencher was added, and the slides were inverted onto a glass slide, mounted, and observed under a fluorescence microscope. Pyroptosis is a specific type of immunogenic cell death, and the significant markers of immunogenic cell death are CRT exposure, HMGB1 efflux, and ATP release. Figure 26 E and Figure 28 As shown in D, the intensity of green fluorescence on the surface of Huh7 cell membranes was significantly enhanced after treatment with Au3 and Au7, indicating that the two compounds can promote the efflux of CRT, which is consistent with the basic characteristics of immunogenic cell death, demonstrating that Au3 and Au7 have the potential to induce immunogenic cell death.

[0070] Example 8: Immunoblot Analysis

[0071] To further investigate whether Au3 can induce immunogenic cell death, the changes in CRT and HMGB1 expression levels in Huh7 cells after Au3 treatment were detected by Western blotting. The results showed that Au3 treatment decreased intracellular HMGB1 expression (…). Figure 26 The phenomenon described in the F (referring to the expression level of HMGB1) is consistent with the characteristics of immunogenic cell death, as HMGB1 is released from the nucleus to the extracellular space during immunogenic cell death, leading to a decrease in its intracellular content. However, the expression level of CRT did not change significantly after Au3 treatment. Figure 26The presence of F in the figure further confirms that CRT is merely a membrane translocation, and combined with previous immunofluorescence experiments, it demonstrates that Au3 can significantly induce immunogenic cell death.

[0072] Example 9: ATP Release

[0073] Huh7 cells were treated with Au3 and Au7 for 24 h. All supernatant was collected from the wells, centrifuged, and the supernatant was used. 100 μL of ATP detection working solution was added to an opaque 96-well plate. The plate was incubated at room temperature for 3 min to remove background ATP. 100 μL of sample was added to the corresponding well and mixed immediately. After approximately 2 seconds, the RLU value was measured using a chemiluminescence analyzer. Specific detection methods are described in the Beyotime Enhanced ATP Detection Kit. ATP release is also a significant marker of compound-induced immunogenic cell death; the extracellular ATP content of Huh7 cells after Au3 and Au7 treatment was measured. Figure 26 G and Figure 28 As shown in E, compared with DMF, Au3 and Au7 can significantly promote the release of ATP, and the release capacity is positively correlated with concentration.

[0074] Example 10: ROS Level Detection

[0075] Huh7 cells were seeded in 12-well plates. After cell attachment, the cells were drug-treated for 6 hours and washed with PBS. 1 mL of 10 μM DHE staining solution was added to each well, and the cells were incubated at 37°C for 30 min, followed by washing with PBS. Finally, 1 mL of PBS was added, and the cells were observed and photographed under a fluorescence microscope. ROS plays an important role in the treatment of cancer cells; inhibition of TrxR is often accompanied by ROS generation, and ROS is closely related to pyroptosis and immunogenic cell death. Therefore, we investigated the ability of Au3 and Au7 to induce ROS generation in Huh7 cells. Figure 27 A and Figure 29 As shown in Figure A, the red fluorescence intensity was significantly enhanced after treatment with Au3 and Au7, indicating that the ROS level in Huh7 cells was significantly increased and positively correlated with the concentration of the compounds.

[0076] Example 11: ERS detection

[0077] Huh7 cells were seeded in 12-well plates. After attachment, Huh7 cells were treated with different concentrations of compounds for 24 h. After washing twice with PBS, 1 mL of Fluo-4AM probe was added, and the cells were incubated at 37°C for 30 min, followed by two more washes with PBS. Finally, 1 mL of PBS was added, and images were taken under a fluorescence microscope. The endoplasmic reticulum (ER) is one of the main reservoirs of intracellular calcium ions. ERS interferes with the folding process of oxidative proteins in the ER, leading to the release of calcium ions from the ER into the cytoplasm. Numerous studies have shown that ROS can cause cancer cells to produce ERS; therefore, we used the calcium ion fluorescent probe Fluo-4AM and detected whether Au3 can induce ERS. Figure 27 As shown in Figure B, the green fluorescence intensity of Huh7 cells was significantly enhanced after Au3 treatment, and this enhancement was positively correlated with the concentration. This result indicates that Au3 can release calcium ions stored in the endoplasmic reticulum into the cytoplasm, leading to an increase in intracellular calcium ion concentration. Furthermore, immunofluorescence results showed ( Figure 29 In part B), Au7 treatment leads to enhanced fluorescence of Calnexin, a protein associated with ERS activation. In conclusion, Au3 and Au7 can induce ROS generation, which in turn generates ERS.

[0078] Example 12: Mitochondrial membrane potential detection

[0079] Huh7 cells were seeded in 12-well plates. After attachment, the cells were drug-treated for 24 hours and washed twice with PBS. 1 mL of JC-1 dye (10 μM) was added to each well, and the plates were incubated at 37°C for 30 min, followed by two washes with PBS. Finally, 1 mL of PBS was added, and images were taken under a fluorescence microscope. Previous studies have shown that Au3 and Au7 can promote ROS generation. Excessive ROS can also lead to mitochondrial membrane lipid peroxidation, protein oxidation, and DNA damage, thereby affecting normal mitochondrial function and causing mitochondrial dysfunction. Changes in mitochondrial membrane potential after Au3 and Au7 treatment were detected using JC-1. Figure 27 C and Figure 29 As shown in Figure C, the green fluorescence of JC-1 monomer is enhanced after treatment with Au3 and Au7, while the red fluorescence of JC-1 polymer is weakened, indicating that Au3 and Au7 can reduce the mitochondrial membrane potential.

[0080] Example 13: In vivo inhibitory activity assay

[0081] Male C57BL / 6 mice (18-22 g) were purchased from Hangzhou Medical College, China. Mice were randomly divided into three groups: a saline group (n=6), an OXA group (n=6), and an Au3 / Au7 group (n=6). Hepa1-6 cells (1×10⁻⁶) were inoculated into the left axilla of each mouse. 6 / 100μL / animal). After tumor formation, administer the drug intraperitoneally (once every 2 days). Record body weight and tumor volume daily. The formula for calculating tumor volume is: V (cm²). 3 ) = a 2 ×b / 2, (a: short diameter; b: long diameter; V: tumor volume). Mice were sacrificed 14 days later, and tumors, blood, and internal organs (heart, liver, spleen, lungs, and kidneys) were collected for subsequent experiments. Figure 27 DF and Figure 29 As shown in the EG diagram, after 14 days of treatment with Au3 and Au7, the tumor volume and weight were significantly smaller compared to the saline and OXA groups, indicating that compounds Au3 and Au7 also exhibit significant anti-hepatocellular carcinoma activity in vivo. Monitoring of mouse body weight during treatment with compounds Au3 and Au7 revealed that, compared to the saline group, compounds Au3 and Au7 did not cause a decrease in mouse body weight. Figure 27 G and Figure 29 (G in the text). The above results indicate that compounds Au3 and Au7 exhibit good anti-hepatocellular carcinoma activity in vivo, with no obvious toxicity and good biocompatibility.

[0082] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. For those skilled in the art, after learning the contents described in the present invention, several equivalent changes and substitutions can be made without departing from the principle of the present invention. These equivalent changes and substitutions should also be considered to fall within the protection scope of the present invention.

Claims

1. A Schiff base gold (III) compound, characterized in that, The Schiff base gold (III) compound is any one of the following compounds:

2. The Schiff base gold (III) compound according to claim 1, characterized in that, The Schiff base gold (III) compound is either the symmetrical compound Au3 or the asymmetrical compound Au7, and the structural formulas of the compounds Au3 and Au7 are as follows:

3. The method for preparing the Schiff base gold(III) compound as described in claim 1, characterized in that, It is synthesized by combining Schiff base ligands, retaining the cyclohexanediamine structure and NNOO coordination mode of oxaliplatin, and replacing platinum (II) with gold (III).

4. The method for preparing the Schiff base gold(III) compound as described in claim 3, characterized in that, The general reaction formula for the preparation method is as follows:

5. A class of drugs with potential for treating liver cancer, characterized in that: It comprises any one of the Schiff base gold (III) compounds as described in claim 1.

6. The use of the Schiff base gold (III) compound as described in claim 1 in the preparation of an anti-hepatocellular carcinoma drug.

7. The use of the Schiff base gold (III) compound as described in claim 2 in the preparation of an anti-hepatocellular carcinoma drug.

8. The application according to claim 7, characterized in that: The Schiff base gold (III) compound has anti-proliferative activity against liver cancer cells Hepa1-6, Huh7 and Hep3B, and can inhibit tumor growth.

9. The application according to claim 7, characterized in that: The Schiff base gold (III) compound simultaneously targets mtDNA and TrxR, promotes ROS generation, leads to ERS and mitochondrial damage, and ultimately induces pyroptosis and immunogenic cell death in liver cancer cells.

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