N-heterocyclic carbene gold compound as well as preparation method and application thereof

Abstract

The invention belongs to the field of organic synthesis, and particularly relates to an N-heterocyclic carbene gold compound and a preparation method and application thereof.The preparation method comprises the steps that an intermediate II is prepared, and after the intermediate II, a compound X and alkali are mixed and dissolved, an esterification reaction is conducted to generate an intermediate III; and fully mixing the intermediate III with an inorganic silver reagent, then adding an organic gold reagent and potassium salt, carrying out gold ion coordination reaction, and after the reaction is completed, carrying out post-treatment, separation and purification to obtain the product. The N-heterocyclic carbene gold compound disclosed by the invention has an anti-tumor immune effect and an anti-ferroptosis effect, and has a liver cancer specific targeting capability.

Description

Technical Field

[0001] The present invention belongs to the field of organic matter preparation, and in particular relates to a nitrogen heterocyclic carbene gold compound and a preparation method and application thereof. Background Art

[0002] Ferroptosis is an iron-dependent, regulated form of cell death triggered by lipid peroxide overload. During ferroptosis, glutathione (GSH) depletion (such as inhibition of SLC7A11 leading to impaired cystine transport) and inactivation of glutathione peroxidase 4 (GPX4) disrupt cellular redox balance, leading to a surge in lipid peroxidation (LPO) and reactive oxygen species (ROS). By inhibiting the activity of thioredoxin reductase (TrxR) within the Trx system (an important component of tumor antioxidant defense), increased ROS production can be induced, leading to increased oxidative stress in tumor cells and affecting redox balance, ultimately triggering ferroptosis. Importantly, tumor cells undergoing ferroptosis can release damage-associated molecular patterns (DAMPs), various immunogenic signals that promote the maturation of dendritic cells (DCs) and enhance the infiltration of CD8+ cytotoxic T cells into tumors, thereby enhancing anti-tumor immune responses. In addition, activated CD8+ cytotoxic T cells secrete interferon gamma (IFN-γ), activating the JAK1-2 / STAT1 / SLC7A11 pathway, further amplifying LPO and iron accumulation in tumors. It is worth noting that macrophage polarization also plays a role in its participation in ferroptosis in the tumor microenvironment. Reprogramming tumor-associated macrophages (TAMs) from the M2 phenotype to the M1 phenotype helps regulate the immunosuppressive tumor microenvironment and improve tumor immune escape. Therefore, combining ferroptosis with immunotherapy through a "closed-loop" strategy is expected to become a new cancer treatment method and is also a hot topic in the research of metal-based anti-tumor drugs.

[0003] In recent years, a variety of metal compounds have been reported to induce ferroptosis while combining anti-tumor immunity. However, their therapeutic effects often require delivery systems or external stimuli (such as light and heat) to achieve. Currently, no studies have reported that traditional gold compounds can achieve a "closed loop" effect of synergistically enhancing ferroptosis and tumor immunity by themselves (independent of delivery systems or external stimuli) and have tumor-specific targeting.

[0004] In summary, developing a gold compound with anti-tumor immune effects and ferroptosis effects that can achieve tumor targeting is an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the low selectivity, limited efficacy, and susceptibility to drug resistance of traditional metal anticancer drugs, the present invention discloses a liver cancer-targeting nitrogen heterocyclic carbene gold compound with anti-tumor immune and ferroptosis effects, as well as its preparation method and application. The technical solution is as follows:

[0006] A nitrogen heterocyclic carbene gold compound, the structure of which is shown in Formula I:

[0007]

[0008] Wherein, X is one of the bile acid homologues; and Y is MeO or F.

[0009] Furthermore, the bile acid homologues include

[0010] One of them.

[0011] A method for preparing the above-mentioned nitrogen heterocyclic carbene gold compound comprises the following steps: preparing intermediate II

[0012] After the intermediate II is mixed and dissolved with compound X and a base, an esterification reaction is performed to generate intermediate III.

[0013] The intermediate III is fully mixed with an inorganic silver reagent, and then an organic gold reagent and potassium salt are added to carry out a gold ion coordination reaction. After the reaction is complete, the intermediate III is subjected to post-treatment and separation and purification to obtain the product.

[0014] Further, the following steps are included:

[0015] a. Dissolve 1,3-dibromopropane and 1-ethyl-4,5-diaryl-1H-imidazole in an organic solvent, reflux reaction, filter and evaporate the solvent after the reaction is complete, and purify to obtain intermediate II; further, when the 1-ethyl-4,5-diaryl-1H-imidazole is 1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole, intermediate II is: When 1-ethyl-4,5-diaryl-1H-imidazole is 1-ethyl-4,5-bis(4-fluorophenyl)-1H-imidazole, intermediate II is:

[0016] b. The intermediate II is mixed with an excess of compound X and a base in an organic solvent and dissolved, and an esterification reaction is carried out. After the esterification is completed, extraction is performed to obtain an organic phase, and the organic phase is thoroughly washed, dried, and then eluted to obtain the intermediate III;

[0017] c. After dissolving intermediate III, add an inorganic silver reagent and mix thoroughly in a dark and protective atmosphere. After the reaction is complete, add an organic gold reagent and potassium salt to carry out a gold ion coordination reaction. After the reaction is complete, post-process and separate and purify to obtain the product.

[0018] In addition, the valence of gold in the nitrogen heterocyclic carbene gold compound of the present invention is +1.

[0019] A use of the above-mentioned nitrogen heterocyclic carbene gold compound in the preparation of anti-tumor drugs.

[0020] By adopting the above scheme, the method of the present invention has the following advantages:

[0021] 1. The nitrogen heterocyclic carbene gold compound of the present invention introduces the molecular skeleton of cholesteric acid-based traditional Chinese medicine through the application of intermolecular "synergistic" design strategy, achieves targeted effect on liver cancer, overcomes the problem of poor selectivity of traditional metal drugs, and fills the research gap in the field of traditional gold compounds in synergistically enhancing the "closed-loop" effect of ferroptosis and tumor immunity, as well as having tumor targeting effect.

[0022] 2. The nitrogen heterocyclic carbene gold compound of the present invention can induce the massive production of ROS in tumor cells by inhibiting thioredoxin reductase (TrxR), thereby destroying the redox homeostasis in tumor cells, leading to damaged mitochondrial function, cell cycle arrest and cell apoptosis in tumor cells, triggering mitochondrial-related ferroptosis and causing endoplasmic reticulum stress, inducing immunogenic cell death (ICD) in tumor cells, activating anti-tumor immunity, successfully achieving multiple anti-tumor effects, and having good application prospects.

[0023] 3. The nitrogen heterocyclic carbene gold compound of the present invention can reshape the tumor immune microenvironment (TIME) by promoting DCs maturation, T cell infiltration, M1 polarization of TAMs, and reduction of Tregs, activate systemic anti-tumor immune response through IFN-γ / STAT1 / SLC7A11 cascade reaction, and enhance the sensitivity of tumor cells to ferroptosis, thereby achieving a "closed-loop" effect of ferroptosis and anti-tumor immunity, opening up a new path for the development of new metal anti-tumor drugs.

[0024] 4. The present invention adopts the coordination structure of the dinitrogen heterocyclic carbene ligand to form an ionic nitrogen heterocyclic carbene gold compound, which is beneficial to improving the membrane permeability and stability of the nitrogen heterocyclic carbene gold compound, thereby improving the anti-tumor activity.

[0025] 5. The preparation method of the present invention is simple and can be prepared without high temperature, low temperature and special treatment, which is conducive to expanding production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is the nuclear magnetic resonance spectrum of compound Ⅰ-1 of Example 1 of the present invention;

[0027] Figure 2 This is the nuclear magnetic resonance spectrum of compound I-2 of Example 2 of the present invention;

[0028] Figure 3 This is the nuclear magnetic resonance spectrum of compound I-3 of Example 3 of the present invention;

[0029] Figure 4 This is the nuclear magnetic resonance spectrum of compound I-4 of Example 4 of the present invention;

[0030] Figure 5 This is the nuclear magnetic resonance spectrum of compound I-5 of Example 5 of the present invention;

[0031] Figure 6 This is a comparative table of the anti-proliferative activity of carbene gold on tumor cells of the present invention;

[0032] Figure 7 HPLC purity analysis chart of the compounds of Examples 1 to 5 of the present invention;

[0033] Figure 8 Effects of Compound I-2 of Example 2 on TrxR activity and ROS levels: A) Inhibition of purified TrxR activity; B, C) Inhibitory effects on intracellular enzyme activity and protein levels in HepG2 cells after 48 h of treatment; D) Inhibitory effects on TrxR in HepG2 cells; E) Flow cytometry analysis of changes in intracellular ROS levels in HepG2 cells after different treatments; F) DCFH-DA assay for the proportion of ROS-positive cells in fused images of bright and fluorescent areas after treatment; G) MTT assay for the antagonistic effect of NAC; H) Schematic diagram of ROS burst caused by TrxR inhibition;

[0034] Figure 9 Compound I-2 of Example 2 induces mitochondrial-associated ferroptosis: A, B) Flow cytometry analysis of the amount of MMP in HepG2 cells under different treatments, with gates P2 and P3 showing the red (aggregates) and green (monomers) fluorescence of JC-10, respectively; C) Colocalization of ROS and mitochondria; D) Transmission electron microscopy image of HepG2 cells after incubation with 5 μM; E) MTT assay for the counteracting effect of ferrostatin-1; F) Western blot analysis of ferroptosis-related proteins; G) Flow cytometric analysis of LPO in HepG2 cells; H) Effect on GSH / GSSG in HepG2 cells; I) Colocalization of LPO and mitochondria; J) Schematic diagram of induced redox imbalance promoting mitochondrial-associated ferroptosis;

[0035] Figure 10Compound I-2 of Example 2 induces translocation and release of DAMPs and activation of ERS: A, B) Western blot analysis of ERS-related proteins; C) Fluorescence microscopy images for detection of ER-ROS; D, E) Immunofluorescence analysis of CRT exposure and HMGB1 translocation in HepG2 cells; F, G) Flow cytometry analysis of CRT and HMGB1 expression in HepG2 cells; H) Luciferase assay for detection of intracellular ATP concentration in HepG2 cells after treatment; I) Schematic diagram of the enhanced immunogenicity of induced DAMPs translocation and release;

[0036] Figure 11 Compound I-2 of Example 2 enhances cell sensitivity to ferroptosis through an immune-driven mechanism: A) CCK8 assay for the effects of different treatment conditions on HepG2 cell viability; B-D) Flow cytometry and fluorescence image analysis of the effects of different treatment conditions on LPO expression in HepG2 cells; E) Effects on STAT1 luciferase activity in the presence or absence of IFN-γ; F) Cell viability after treatment of WT or STAT1- / - HepG2 cells in the presence or absence of IFN-γ; G) Western blot analysis of the expression of ferroptosis-related proteins in HepG2 cells; H) Schematic diagram of the "closed loop" of ferroptosis and anti-tumor immunity;

[0037] Figure 12 This is the in vivo tumor model experiment of compound I-2 in Example 2: A) Mouse tumor images taken on day 14 after treatment; B) Tumor weight; C) Tumor volume; D) Mouse body weight; E~I) Immunofluorescence analysis of related marker expression in tumor tissue; J) Immunohistochemical analysis of GPX4 expression in tumor tissue.

[0038] Figure 13 This is the immunohistochemical analysis of ATF-4 expression and immune infiltration in tumor tissues of the in vivo tumor-bearing model of compound I-2 of Example 2. DETAILED DESCRIPTION

[0039] Intermediate Example 1: 1-Ethyl-4,5-bis(4-fluorophenyl)-1h-imidazole (6.0 mmol) was placed in a 25 mL round-bottom flask and dissolved by adding 20 mL of anhydrous acetonitrile. Subsequently, 1,3-dibromopropane (26.0 mmol) was added dropwise to the reaction mixture. The reaction mixture was refluxed at 80°C for 72 to 120 hours while monitoring the progress using TLC. After the reaction was complete, the solvent was removed under low pressure. The crude product was purified by silica gel column chromatography using a DCM / MeOH gradient elution method to obtain 2.3 g of the product intermediate IIA with a yield of 80%;

[0040] Intermediate ⅡA.

[0041] Intermediate Example 2: 1-Ethyl-4,5-bis(4-methoxyphenyl)-1h-imidazole (6.0 mmol) was placed in a 25 mL round-bottom flask and dissolved by adding 20 mL of anhydrous acetonitrile. Subsequently, 1,3-dibromopropane (26.0 mmol) was added dropwise to the reaction mixture. The reaction mixture was refluxed at 80°C for 72 to 120 hours while monitoring the progress using TLC. After the reaction was complete, the solvent was removed under low pressure. The crude product was purified by silica gel column chromatography using a DCM / MeOH gradient elution method to obtain 2.4 g of the product, Intermediate IIB, with a yield of 78%.

[0042] Intermediate ⅡB.

[0043] Example 1: (1) Intermediate IIA (1.0 mmol) and 3.0 mmol of cholic acid were placed in a 25 mL round-bottom flask, followed by the addition of 2.0 mmol of potassium carbonate and 10 mL of anhydrous DMF. The reaction mixture was reacted at room temperature for 48 to 96 hours, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, water was added to the solution and stirred thoroughly, and then extracted three times with ethyl acetate or dichloromethane to obtain an organic phase. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and then evaporated under low pressure until dry. Finally, the crude product was eluted on silica gel with a dichloromethane / methanol gradient to obtain 414.6 mg of the product intermediate III-1 with a yield of 51%. The structure is shown below:

[0044] Intermediate III-1.

[0045] (2) Intermediate III-1 (0.125 mmol) was dissolved in 5 mL of anhydrous acetonitrile. Silver oxide (0.075 mmol) was then added to the mixture and stirred overnight at room temperature under nitrogen and in the dark. Me2SAuCl (0.0625 mmol) and potassium hexafluorophosphate (1.25 mmol) were then added to the reaction mixture for a total of 24 hours. The reaction mixture was filtered through celite and then evaporated under low pressure until dry. The crude product was purified by silica gel (gradient elution with DCM / MeOH) to obtain 36.9 mg of compound I-1 with a yield of 33%. The nuclear magnetic resonance spectrum was as shown below. Figure 1 As shown, its structure is as follows:

[0046]

[0047] Example 2: (1) Intermediate IIA (1.0 mmol) and 3.0 mmol of dehydrocholic acid were placed in a 25 mL round-bottom flask, followed by the addition of 2.0 mmol of potassium carbonate and 10 mL of anhydrous DMF. The reaction mixture was reacted at room temperature for 48 to 96 hours, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, water was added to the solution and stirred thoroughly, and then extracted three times with ethyl acetate or dichloromethane to obtain an organic phase. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and then evaporated under low pressure until dry. Finally, the crude product was eluted on silica gel with a dichloromethane / methanol gradient to obtain 427.7 mg of the product intermediate III-2 with a yield of 53%. The structure is as follows:

[0048] Intermediate III-2.

[0049] (2) Using intermediate III-2, the preparation was continued according to step (2) of Example 1 to obtain 42.0 mg of compound I-2 with a yield of 34%. The NMR spectrum is as follows: Figure 2 As shown, its structure is as follows:

[0050] Example 3: (1) Intermediate IIA (1.0 mmol) and 3.0 mmol ursodeoxycholic acid were placed in a 25 mL round-bottom flask, followed by the addition of 2.0 mmol potassium carbonate and 10 mL anhydrous DMF. The reaction mixture was reacted at room temperature for 48 to 96 hours, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, water was added to the solution and stirred thoroughly, and then extracted three times with ethyl acetate or dichloromethane to obtain an organic phase. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and then evaporated under low pressure until dry. Finally, the crude product was eluted on silica gel with a dichloromethane / methanol gradient to obtain 549.9 mg of the product intermediate III-3 with a yield of 69%, and the structure was as follows:

[0051] Intermediate III-3.

[0052] (2) Using intermediate III-3, the preparation was continued according to step (2) of Example 1 to obtain 42.0 mg of compound I-3 with a yield of 38%. The NMR spectrum is as follows: Figure 3 As shown, its structure is as follows:

[0053]

[0054] Example 4: (1) Intermediate IIA (1.0 mmol) and 3.0 mmol chenodeoxycholic acid were placed in a 25 mL round-bottom flask, followed by the addition of 2.0 mmol potassium carbonate and 10 mL anhydrous DMF. The reaction mixture was reacted at room temperature for 48 to 96 hours, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, water was added to the solution and stirred thoroughly, and then extracted three times with ethyl acetate or dichloromethane to obtain an organic phase. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and then evaporated under low pressure until dry. Finally, the crude product was eluted on silica gel with a dichloromethane / methanol gradient to obtain 526.0 mg of the product intermediate III-4 with a yield of 66%, and the structure was as follows:

[0055] Intermediate III-4.

[0056] (2) Using intermediate III-4, the preparation was continued according to step (2) of Example 1 to obtain 56.1 mg of compound I-4 with a yield of 50%. The NMR spectrum is as follows: Figure 4 As shown, its structure is as follows:

[0057] Example 5: (1) Intermediate IIB (1.0 mmol) and 3.0 mmol of dehydrocholic acid were placed in a 25 mL round-bottom flask, followed by the addition of 2.0 mmol of potassium carbonate and 10 mL of anhydrous DMF. The reaction mixture was reacted at room temperature for 48 to 96 hours, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, water was added to the solution and stirred thoroughly, and then extracted three times with ethyl acetate or dichloromethane to obtain an organic phase. The organic phase was washed three times with water and saturated brine, dried over anhydrous sodium sulfate, and then evaporated under low pressure until dry. Finally, the crude product was eluted on silica gel with a dichloromethane / methanol gradient to obtain 332.4 mg of the product intermediate III-5 with a yield of 40%, and the structure was as follows:

[0058] Intermediate III-5.

[0059] (2) Using intermediate III-5, the preparation was continued according to step (2) of Example 1 to obtain 62.8 mg of compound I-5 with a yield of 55%. The NMR spectrum is as follows: Figure 5 As shown, its structure is as follows:

[0060]

[0061] Example sample test:

[0062] The purity of the obtained compounds (Ⅰ-1, Ⅰ-2, Ⅰ-3, Ⅰ-4 and Ⅰ-5) was detected by HPLC. The experimental conditions were: chromatographic column: Inertex C18 (250mm×4.6mm×5μm); mobile phase: acetonitrile / water; temperature: 25℃; detection wavelength: 220nm.

[0063] The results are as follows Figure 7 As shown in Figure AE in the figure, the purity of the obtained compounds (I-5, I-1, I-2, I-3 and I-4) was greater than 95% by HPLC.

[0064] Evaluation of the liver cancer-specific targeting effect and anti-tumor activity of the compounds of the present invention:

[0065] Anti-proliferative activity of the compounds on tumor cells: HepG2, Hep3B, Hepa1-6, Huh7-LR, A2780, IOSE80, Hela and H8 cells in the logarithmic growth phase were digested with trypsin and incubated at 2×10 3 The cells were seeded in 96-well plates at a density of 100 μL per well. The cultures were incubated overnight at 37°C in DMEM containing 10% fetal bovine serum. After adding different concentrations of the complex, positive reagents (auranofin and cisplatin), and negative control (0.1% DMF), the cells were incubated at 37°C for 72 hours. The final volume was 200 μL / well, and the final drug concentration ranged from 0.078125 to 20 μM. The antiproliferative effect of the complex was detected by the MTT assay. 5% MTT (5 mg / mL, PBS) reagent was added and incubated for approximately 4 hours. The supernatant was collected, 200 μL DMSO was added to dissolve the blue-purple methyl nitrogen, and the cells were gently shaken for approximately 10 minutes. The absorbance was measured at 490 nm, and the IC50 value was calculated.

[0066] See the results Figure 6 The effects of the compounds on liver cancer cells HepG2, Hepa1-6, Hep3B, Huh7-LR, human ovarian cancer cells A2780, human cervical cancer cells Hela, human normal ovarian epithelial cells IOSE80 and human normal cervical cells H8 were analyzed. Auranofin and cisplatin were used as positive controls. Figure 6 As shown, I-2 exhibited significant cytotoxicity among this series of complexes. The IC50 values of I-2 against tumor cells ranged from 0.60 to 2.30 μM, demonstrating a stronger antiproliferative effect than auranofin and cisplatin. Furthermore, I-2 exhibited lower anti-proliferative properties against normal cells (IOSE80: 2.08 μM; H8: 3.21 μM) than against tumor cells (A2780: 1.68 μM; HeLa: 2.30 μM), demonstrating the compound's selectivity and biocompatibility.

[0067] Furthermore, I-2 exhibited strong inhibitory activity against various liver tumor cell lines, with IC50 values of 2.23 μM (HepG2), 1.63 μM (Hepa1-6), and 0.60 μM (Hep3B). Notably, I-2 exhibited a stronger inhibitory effect against the lenvatinib-resistant Huh7-LR cell line, with an IC50 value of 1.89 μM, compared to auranofin (IC50: 3.15 μM) and cisplatin (IC50: 3.40 μM). These findings suggest that I-2 contributes to its specific targeting of liver cancer.

[0068] Compound I-2 of Example 2 was used to study the mechanism of inducing TrxR inhibition and triggering ROS burst:

[0069] Enzyme level detection: Detection was performed using the DTNB assay. In a 96-well plate, DMF and I-2 (0.625, 1.25, 2.5, 5, 10, and 20 μM) were added to 0.15 U of purified TrxR enzyme (Sigma-Aldrich). TE buffer (50 mM Tris-HCl, pH 7.5; 1 mM EDTA) was then added to a final volume of 50 μL per well. The reaction was allowed to proceed at room temperature. Immediately after the reaction, 50 μL of DTNB assay solution (2 mM DTNB; 200 μM NAPDH) was aspirated and the absorbance was recorded at 405 nm for 300 s using a microplate reader.

[0070] Intracellular activity assay: HepG2 cells were seeded in 10 cm culture dishes and cultured overnight. DMF, a positive drug group (5 μM auranofin), and I-2 groups (2, 5, and 10 μM) were then established. Cells were harvested 48 hours after drug administration. Total protein was extracted and measured using a BCA assay. Enzyme activity was assessed using a TrxR assay according to the provided instructions.

[0071] Probe method: HepG2 cells were cultured at 5×10 4 Cells were seeded at a density of 100 μg / well in 12-well plates and cultured overnight. After 48 hours of treatment, 10 μM TRFS-green was added to fresh culture medium and stained for 0.5 hours. After rinsing twice with PBS, images were captured under a fluorescence microscope.

[0072] Western blot analysis of TrxR expression levels: Cells were treated under various conditions for 24 hours. Cell extracts were then washed with cold PBS and resuspended in RIPA lysis buffer to prepare cell extracts. Samples were loaded based on the calculated protein concentration. Electrophoresis was performed at 20 mA for 120 minutes. After separation, proteins were blotted onto PVDF membranes and probed with various specific antibodies. They were then washed and incubated with secondary antibodies for 2 hours, and protein bands were detected using chemiluminescence (ECL).

[0073] The results are as follows Figure 8 As shown, Ⅰ-2 exhibited a dose-dependent inhibition of TrxR pure enzyme activity with an IC50 value of 4.66 μM ( Figure 8 A). In addition, compared with the DMF control group, it dose-dependently inhibited the TrxR activity in cells ( Figure 8 B). In addition to these findings, we used TRFS-green as a reported probe to observe the inhibitory effect of TrxR in cells. Ⅰ-2 effectively eliminated TrxR activity in cells, as evidenced by the bright green fluorescence intensity reflecting its cellular activity ( Figure 8 D). This result was further confirmed by Western blot analysis ( Figure 8 C).

[0074] The fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect the ability of Ⅰ-2 to induce intracellular ROS accumulation. Our results showed that cells incubated with Ⅰ-2 showed a strong fluorescence signal, indicating a significant accumulation of intracellular ROS. Notably, after 1 hour of pretreatment with the ROS scavenger acetylcysteine (NAC), the fluorescence intensity decreased significantly ( Figure 8 F). Flow cytometry analysis showed that Ⅰ-2 significantly increased the intracellular ROS level; however, the decrease in fluorescence intensity caused by NAC pretreatment was comparable to that observed in the control group ( Figure 8 E). In addition, our results showed that I-2-induced intracellular ROS accumulation led to cell death. After 72 h of I-2 treatment, pretreatment with 2 mM NAC partially improved cell viability. However, when the NAC concentration reached or exceeded 4 mM, the inhibitory effect was almost completely reversed ( Figure 8 G).

[0075] All these results indicate that the carbene gold of the present invention targets TrxR and induces a surge in intracellular ROS levels by inhibiting TrxR activity, ultimately leading to cell death ( Figure 8 H).

[0076] Compound I-2 of Example 2 was used to study the mechanism of inducing redox imbalance in tumor cells and promoting mitochondrial-related ferroptosis:

[0077] Intracellular ROS determination and colocalization: HepG2 cells were seeded in 12-well plates (5×10 4 / well) and 6-well plates (2×10 5 Cells were then treated with DCFH-DA probes (cells per well) overnight for 6 hours. After 6 hours of treatment, the cells were stained with the DCFH-DA probe according to the manufacturer's instructions. Fluorescence microscopy images were obtained, and flow cytometry was used for quantification. Additionally, mitochondria were stained with Mito-Tracker-Red, and ER was labeled with ER-tracker-red.

[0078] Glutathione level determination: HepG2 cells were seeded in 10 cm culture dishes and cultured overnight. After 48 hours of treatment, cells from each experimental group were harvested and intracellular GSH and GSSG levels were measured according to the kit instructions.

[0079] Determination of MMP: HepG2 cells were seeded in 12-well plates (5×10 4 / well) or 6-well plate (2×10 5 After 48 hours of treatment, the cells were stained with a JC-10 probe and incubated at 37°C for 30 minutes according to the manufacturer's instructions. Fluorescence microscopy was then used to capture images, while flow cytometry was used for quantification.

[0080] Lipid peroxidation analysis: HepG2 cells were seeded in 12-well plates (5 × 10 4 / well) and 6-well plates (2×10 5 Cells were then treated with the different treatments for 6 and 12 hours. After treatment, cell samples were collected from each group and stained with a C11-BODIPY probe (Thermo Fisher Scientific Life Sciences) according to the manufacturer's instructions. Fluorescence microscopy was used for imaging and quantitative analysis by flow cytometry. Mitochondria were stained with Mito-Tracker-Red.

[0081] The results are as follows Figure 9 As shown in Figure 2, Fer-1 pretreatment partially increased the survival rate of cells after I-2 treatment. This effect was more pronounced when the dose was equal to or lower than 5 μM ( Figure 9 E) In addition, we evaluated the effect of I-2 on the ferroptosis marker LPO using a C11-BODIPY assay. Figure 9Results shown in G show that LPO levels were significantly increased 12 h after I-2 treatment compared with the DMF control group and could be reversed by Fer-1 pretreatment. The ratio of GSH to GSSG was significantly decreased after I-2 treatment; however, NAC pretreatment significantly improved this situation ( Figure 9 H). In addition, western blot showed that the intracellular expression levels of SLC7A11 and GPX4 were reduced after Ⅰ-2 treatment ( Figure 9 F) These findings suggest that Ⅰ-2 treatment can induce a decrease in cellular antioxidant defenses, thereby promoting ferroptosis.

[0082] To further elucidate the potential of I-2 to induce mitochondria-associated ferroptosis, we evaluated the fluorescence intensity of LPO and ROS within mitochondria. Figure 9 As shown in Figures C and 9I, cells treated with I-2 exhibited stronger green fluorescence signals compared to the DMF control group, and colocalized with mitochondria labeled with the Mito-Tracker probe. This observation suggests that the accumulation of LPO and ROS in mitochondria is increased, and Fer-1 treatment can partially alleviate this situation. In addition, transmission electron microscopy (TEM) images of HepG2 cells showed that exposure to I-2 caused mitochondrial structural damage compared to the DMF control group ( Figure 9 D). These changes include atrophy or disappearance of mitochondrial cristae, mitochondrial vacuolization, and outer membrane rupture. These results suggest that ferroptosis induced by I-2 may be related to its effects on mitochondria. Alternatively, changes in mitochondrial structure may lead to functional impairment. Mitochondrial membrane potential (MMP or ΔΨm) was measured using JC-10 staining to assess oxidative stress caused by increased ROS levels. Figure 9 As shown in Figures A and 9B, treatment with I-2 significantly reduced MMP, as evidenced by an increase in green fluorescence accompanied by a decrease in red fluorescence signal. These effects were significantly reversed after NAC intervention.

[0083] Compound I-2 of Example 2 was used to conduct an experiment on enhancing immunogenicity by inducing the translocation and release of DAMPs: the relevant methods refer to the above experiment.

[0084] The results are as follows Figure 10 As shown in Figure 1, the accumulation and exposure of calreticulin (CRT) on the cell membrane of HepG2 cells treated with Ⅰ-2 ( Figure 10 D), flow cytometry results ( Figure 10 This was further confirmed by F), which showed that the content of CRT increased significantly after treatment. Similarly, laser confocal microscopy showed that in I-2 treated cells, high mobility group box 1 protein (HMGB1) translocated from the nucleus to the cytoplasm ( Figure 10 E). Flow cytometry results showed that the expression level of HMGB1 increased, e.g. Figure 10 G. In addition, our results show that Figure 10 As observed by H, I-2 treatment led to the release of ATP from cells. These results indicate that I-2 treatment significantly stimulated the activation of common intracellular DAMPs.

[0085] In addition, we further investigated the effect of I-2 on ER stress in HepG2 cells by colocalization analysis of ER-Tracker and DCFH-DA probes. Our results showed that I-2 could induce the coactivation of ROS and ER, while NAC pretreatment partially attenuated the ER-ROS ( Figure 10 C). In addition, Ⅰ-2 significantly upregulated the expression levels of ERS marker proteins CHOP and Calnaxin, as well as the phosphorylated forms of PERK, atf6, eIF2α, and ATF4 ( Figure 10 A and 10B). Similarly, NAC pretreatment led to a decrease in the expression levels of these proteins. All these results indicate that I-2 can induce intracellular ERS and damage ER structure through the surge of ROS.

[0086] Compound I-2 of Example 2 was used to study the enhancement of cell sensitivity to ferroptosis through immune-mediated mechanisms:

[0087] Luciferase activity analysis: HepG2 / STAT1 cells (8×10 4 cells / well) were inoculated into 96-well plates and cultured overnight. 5 Cells were stimulated with 100 IU / ml of IFN-α for 24 h, then treated with DMF or I-2, with or without IFN-γ (10 ng / ml) for 24 h. Luciferase activity was determined using the ONE-Glo kit according to the manufacturer's instructions and quantified using Varioskan Flash.

[0088] Study on ferroptosis-related STAT1 knockdown in HepG2 cells: HepG2 cells were plated and cultured in 96-well plates. After cell adhesion, Fer-1 was given as a pretreatment, and then different concentrations of I-2 or IFN-γ (10 ng / ml) were added for 24 hours. To evaluate the effects of treatment on cell growth and viability, freshly prepared CCK-8 solution was added to the wells. siRNA oligonucleotides were synthesized by GenePharma. Specific siRNA sequences for STAT1 (Sense: 5'-GCGUAAUCUUCAGGAUAAUTT-3'; Antisense: 5'-AUUAUCCUGAAGAUUACGCTT-3') were used and verified by western blot. In addition, STAT1-deficient HepG2 cells were established and used for further studies. Other experimental methods refer to the above experiments.

[0089] The results are as follows Figure 11 As shown in [ 15 ], the sensitivity of immune-driven mechanisms to ferroptosis was investigated using in vitro cell models. Figure 11 As shown in A to 11D, IFN-γ pretreatment increased the susceptibility of tumor cells to I-2 and enhanced its ability to induce lipid ROS production in these cells (assessed using BODIPY-C11). The above effects were significantly weakened after co-administration with Fer-1. Compared with I-2 intervention alone, IFN-γ enhanced the luciferase activity of STAT1 to a certain extent, thereby enhancing the I-2-induced downregulation of SLC7A11 and GPX4 expression, and further promoted LPO in tumor cells ( Figure 11 E and 11G). In addition, in HepG2 cells pretreated with IFN-γ, knockdown of STAT1 (si-STAT1) reversed I-2-induced cell death ( Figure 11 F) Ⅰ-2 enhances the susceptibility of tumor cells to ferroptosis through IFN-γ / STAT1 / slc7a11-mediated immune drive.

[0090] In vivo anti-tumor experiments were conducted using compound I-2 of Example 2: After one week of adaptive feeding, male C57 / BL6 mice were subcutaneously inoculated with Hepa1-6 cells (2×10 6 After 7 days of observation, when the axillary tumor volume of the mice reached about 80 mm 3 The mice were randomly divided into groups. The treatment groups included the complex I-2 (2.5 mg / kg) group, the oxaliplatin (2.5 mg / kg) group, and the model group. The mice were intraperitoneally injected once every two days, and the diet and mental state of the mice were monitored. Tumor volume was measured with a vernier caliper and calculated as V (cm 3) = a² × (b / 2), where a represents the short diameter of the tumor, b represents the long diameter of the tumor, and V represents the tumor volume. In addition, body weight was monitored throughout the experiment. Two weeks later, all mice were sacrificed, and tumors and major organs, including the heart, liver, spleen, lungs, and kidneys, were removed for HE staining and immunofluorescence analysis.

[0091] The in vivo anti-tumor effect of complex I-2 was further evaluated using a C57BL / 6 mouse tumor model. Figure 12 A~12D), treatment with Ⅰ-2 (2.5 mg / kg) significantly inhibited tumor growth, and there was no significant change in the body weight of mice in each group. Figure 12 Immunofluorescence analysis shown in E-12G showed that compared with the model group, TrxR expression in tumor tissues of the I-2 treatment group was significantly reduced and ROS levels were increased. These findings, combined with previous studies, indicate that I-2 can inhibit TrxR expression, enhance ROS accumulation, and thus inhibit tumor growth. Immunohistochemistry was used to examine whether I-2 induces ERS and promotes the release of DAMPs. The results showed that compared with the model group, the expression of the ERS-related activating transcription factor ATF-4 was increased in the I-2 treatment group ( Figure 13 In addition, the distribution patterns of ICD-related markers HMGB1 and CRT in tumor tissues of the I-2 treatment group changed significantly, including the efflux of CRT and the nuclear diffusion of HMGB1 ( Figure 12 E, 12H and 12I). In addition, compared with the tumor tissues of model animals, immunohistochemistry ( Figure 13 ) showed that in the tumor tissues of the I-2 treatment group, CD86, CD4, and CD8 markers were activated, suggesting that I-2 induced an anti-tumor immune response. At the same time, the expression of GPX4, a marker related to ferroptosis, was inhibited in tumor tissues ( Figure 12 J).

[0092] In summary, the present invention has studied the anti-tumor activity and mechanism of the liver cancer-targeting nitrogen heterocyclic carbene gold compound, which has both anti-tumor immune and ferroptosis effects and is prepared by the preparation method of the present invention, through a series of experiments. The results show that the compound can exert effects on both ferroptosis and anti-tumor immunity and has liver cancer-specific targeting capabilities. It enhances the sensitivity of tumor cells to ferroptosis through an immune-driven mechanism mediated by IFN-γ / STAT1 / SLC7A11, forming a "closed-loop" therapeutic effect that combines ferroptosis with anti-tumor immunity, which has important practical application value and good market application prospects.

Claims

1. A nitrogen heterocyclic carbene gold compound, characterized in that Its structure is shown in Formula I Show: Wherein, X is one of the bile acid homologues; and Y is MeO or F.

2. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 1, wherein The bile acid homologues include one of cholic acid, dehydrocholic acid, ursodeoxycholic acid and chenodeoxycholic acid.

3. A method for preparing the nitrogen heterocyclic carbene gold compound according to claim 1, characterized in that: The following steps are included: preparation of intermediate II After the intermediate II is mixed and dissolved with compound X and a base, an esterification reaction is performed to generate intermediate III. The intermediate III is fully mixed with an inorganic silver reagent, and then an organic gold reagent and potassium salt are added to carry out a gold ion coordination reaction. After the reaction is complete, the intermediate III is subjected to post-treatment and separation and purification to obtain the product.

4. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 3, wherein The following steps are involved: a. Dissolve 1,3-dibromopropane and 1-ethyl-4,5-diaryl-1H-imidazole in an organic solvent and reflux. After the reaction is complete, filter and evaporate the solvent to obtain intermediate II after purification. b. The intermediate II is mixed with an excess of compound X and a base in an organic solvent and dissolved, and an esterification reaction is carried out. After the esterification is completed, extraction is performed to obtain an organic phase, and the organic phase is thoroughly washed, dried, and then eluted to obtain the intermediate III; c. After dissolving intermediate III, add an inorganic silver reagent and mix thoroughly in a dark and protective atmosphere. After the reaction is complete, add an organic gold reagent and potassium salt to carry out a gold ion coordination reaction. After the reaction is complete, post-process and separate and purify to obtain the product.

5. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 3 or 4, wherein The temperature of the reflux reaction is 70-90° C.; the temperature of the esterification reaction is 0-50° C. and the time is 48-96 hours; after adding the silver reagent, the mixture is fully mixed for 12-24 hours; the time of the gold ion coordination reaction is 20-32 hours and the temperature is 15-40° C.

6. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 3 or 4, wherein: The base includes any one of potassium carbonate, sodium hydroxide, potassium hydroxide or sodium carbonate; the inorganic silver reagent includes any one of silver oxide, silver chloride, silver sulfide or silver bromide; the organic gold reagent includes any one of Me2SAuCl, CAuClO or (Ph3P)AuCl; the potassium salt includes any one of potassium bromide, potassium chloride, potassium iodide or potassium hexafluorophosphate.

7. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 3 or 4, wherein: The molar ratio of the intermediate II to the compound X is 1:2.2-4; the molar ratio of the intermediate II to the base is 1:1.5-2.5; the molar ratio of the intermediate III to the inorganic silver reagent is 1.5-2:1; the molar ratio of the inorganic silver reagent to the organic gold reagent is 1.1-1.5:1; and the molar ratio of the organic gold reagent to the potassium salt is 1:15-25.

8. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 4, wherein The 1-ethyl-4,5-diaryl-1H-imidazole is 1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazole or 1-ethyl-4,5-bis(4-fluorophenyl)-1H-imidazole; the organic solvent includes one or more of anhydrous acetonitrile, anhydrous DMF, DMSO, methanol, anhydrous dichloromethane or tetrahydrofuran.

9. The method for preparing the nitrogen heterocyclic carbene gold compound according to claim 4, wherein The washing in step b is performed using water and saturated brine; the drying in step b is performed using anhydrous sodium sulfate and then evaporating under low pressure until dry; the extraction in step b is performed using ethyl acetate or dichloromethane; the post-treatment and separation and purification in step c are performed by filtering the reaction mixture through diatomaceous earth, then evaporating under low pressure until dry, and then performing gradient elution through silica gel to purify the crude product.

10. Use of the nitrogen heterocyclic carbene gold compound according to claim 1 or 2 in the preparation of anti-tumor drugs.

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