An oxidized indole derivative, a preparation method thereof and a pharmaceutical use thereof for treating liver cancer

Abstract

The application belongs to the technical field of medicines, and specifically discloses an oxidation indole derivative, a preparation method thereof and pharmaceutical use for resisting liver cancer. The compound is prepared from 7-bromo indigo as a starting material through N-alkylation, Suzuki-Miyaura coupling, Wittig reaction, deprotection and acylation reaction. In vitro cell experiments show that the compound has a very strong proliferation inhibiting effect on Hep3B and HepG2 liver cancer cells. Mechanism research shows that the compound can damage the structure of mitochondria, reduce the membrane potential of mitochondria and increase the level of mitochondrial ROS, thereby inducing liver cancer cell death. Animal experiments further confirm the significant tumor growth inhibiting effect. The oxidation indole derivative provided by the application has novel structure, strong activity, low toxicity and clear mechanism, is an excellent candidate compound for preparing liver cancer treating drugs, and has very high clinical application development value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an indole oxide derivative, its preparation method, and its pharmaceutical use in treating liver cancer. Background Technology

[0002] Oxyindoles are a research hotspot in the pharmaceutical field. Their core structure is a benzopyrrole nitrogen heterocycle containing highly electronegative oxygen and nitrogen heteroatoms, exhibiting excellent pharmacodynamic skeleton properties. They can form strong intermolecular interactions with various biomolecules, making them an important class of drug-like skeletons in medicinal chemistry research. These compounds not only demonstrate broad-spectrum antitumor activity but also possess antiviral, anti-inflammatory, and antibacterial activities, and their antitumor potential has attracted widespread attention in recent years.

[0003] Hepatocellular carcinoma (HCC) is a rapidly progressing malignant tumor with a high incidence and mortality rate. Studies have reported that due to an early diagnosis rate of less than 20%, the vast majority of patients are diagnosed at an intermediate or advanced stage. For unresectable or advanced HCC, the 5-year survival rate is only 13%. Currently, chemotherapy, targeted therapy, and immunotherapy are the main treatments for advanced HCC. However, chemotherapy alone offers little clinical benefit; even when combined with the small-molecule targeted drug sorafenib, the treatment efficacy is only about 33%, and over 90% of patients develop drug resistance within 6 months. The widespread drug resistance to existing treatment regimens in liver cancer severely restricts clinical efficacy and patient prognosis, necessitating the development of novel second-line treatment strategies.

[0004] Studies have shown that oxidized indole compounds exhibit outstanding anti-tumor effects. Reports indicate that they can inhibit human gastric adenocarcinoma cells by 87.46±0.71%, possess strong inhibitory activity against MDA-MB-231 breast cancer cells, suppress differentiation of human promyelocytic leukemia cells HL-60, block tumor angiogenesis, and demonstrate inhibitory effects on cell proliferation, migration, and apoptosis in liver cancer models. The mechanism is closely related to the mitochondrial pathway mediated by oxidative stress. Given the significant advantages of the oxidized indole skeleton in anti-liver cancer activity, especially the strong anti-proliferative activity of compounds containing the oxidized indole structure in the HepG2 liver cancer model, modifying and functionalizing its core structure to construct a library of novel and highly active small molecule compounds holds promise for providing new drug candidates for liver cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to provide an indole oxidized derivative, its preparation method, and its pharmaceutical use in treating liver cancer. This indole oxidized derivative can effectively inhibit the proliferation of liver cancer cells and induce cell death by regulating the mitochondrial pathway, exhibiting good anti-liver cancer activity both in vivo and in vitro. To this end, this invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides an indole oxidase derivative having the following chemical structural formula:

[0007] .

[0008] Secondly, the present invention provides a method for preparing the oxidized indole derivative, comprising the following steps:

[0009] S1. 7-Bromoindigo reacts with sodium hydride and 1-BOC-4-(2-chloroethyl)piperazine in dimethylformamide to obtain a first intermediate; wherein the molar ratio of 7-bromoindigodigo to sodium hydride is 1:1.5-2.5, the molar ratio of 7-bromoindigodigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.2-1.8, and the reaction temperature is 60-90℃;

[0010] S2. The first intermediate and 4-methylthiophenylboronic acid are reacted under reflux in 1,4-dioxane in the presence of a palladium catalyst and a base to prepare the second intermediate; the reflux reaction is carried out under inert gas protection at 100-120°C, and the molar ratio of the first intermediate to 4-methylthiophenylboronic acid is 1:1.2-1.8.

[0011] S3. The second intermediate is reacted with ethoxyformylmethylenetriphenylphosphine in tetrahydrofuran to prepare the third intermediate; wherein the molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.0-1.3, and the reaction temperature is -10-10℃.

[0012] S4. The third intermediate is deprotected from the BOC protecting group in dichloromethane containing trifluoroacetic acid to obtain the fourth intermediate;

[0013] S5. The fourth intermediate is reacted with 2-thiopheneacetyl chloride in dichloromethane to obtain the indole oxide derivative; wherein the molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.2-1.8.

[0014] In one or more embodiments, in step S1, the molar ratio of 7-bromoindigo to sodium hydride is 1:2, and the molar ratio of 7-bromoindigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.5; in step S2, the molar ratio of the first intermediate to 4-methylthiophenylboronic acid is 1:1.5; in step S3, the molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.1; and in step S5, the molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.5.

[0015] In one or more embodiments, in step S1, the reaction temperature for obtaining the first intermediate is 80°C; in step S2, the reflux reaction temperature is 110°C; and in step S3, the reaction temperature for obtaining the third intermediate is 0°C.

[0016] In one or more embodiments, in step S2, the palladium catalyst is tris(dibenzylacetone)dipalladium, with triphenylphosphine added as a ligand. The amount of tris(dibenzylacetone)dipalladium is 3 mol%-8 mol% of the first intermediate, and the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine is 1:1.5-2.5. The base is anhydrous potassium carbonate, and the molar ratio of the first intermediate to anhydrous potassium carbonate is 1:1.5-2.5.

[0017] In at least one embodiment, in step S2, the amount of tris(dibenzylacetone)dipalladium is 5 mol% of the first intermediate, the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine is 1:2, and the molar ratio of the first intermediate to anhydrous potassium carbonate is 1:2.

[0018] In one or more embodiments, in step S4, the volume fraction of the trifluoroacetic acid is 3%-20%.

[0019] In at least one embodiment, in step S4, the trifluoroacetic acid has a volume fraction of 5%.

[0020] In one or more embodiments, the first intermediate, the second intermediate, the third intermediate, the fourth intermediate, and the indole oxidase derivative are all purified after being generated in the corresponding steps, and the purification is carried out by column chromatography separation and purification method.

[0021] Thirdly, the present invention provides the use of the aforementioned indole oxidase derivative in the preparation of a medicament for treating liver cancer.

[0022] The drug comprises the indole oxyde derivative and pharmaceutically acceptable excipients.

[0023] Compared with existing technologies, this invention achieves at least the following beneficial effects: This invention provides an oxidized indole derivative, its preparation method, and its pharmaceutical use in treating liver cancer. The preparation method utilizes conventional organic synthesis techniques with a clear route, readily available raw materials, and mild reaction conditions, making it suitable for large-scale preparation. This oxidized indole derivative significantly inhibits the proliferation of liver cancer cells in both in vitro and in vivo experiments. Its mechanism of action in inhibiting liver cancer cell growth primarily involves damaging mitochondria, reducing mitochondrial membrane potential, and increasing mitochondrial ROS concentration. This provides a novel antitumor candidate compound with significant anti-liver cancer activity and a clearly defined mechanism of action, laying an important foundation for the development of novel, highly effective, and low-toxicity anti-liver cancer drugs based on the oxidized indole skeleton. Attached Figure Description

[0024] Figure 1 The results of the plate colony assay were used to detect the ability of compound 6e-2 to inhibit the colony formation of Hep3B and HepG2 cells. A: Crystal violet stained images of Hep3B cell plate colonies; B: Crystal violet stained images of HepG2 cell plate colonies; C: Bar chart showing the number of Hep3B cell colonies; D: Bar chart showing the number of HepG2 cell colonies.

[0025] Figure 2 Figure 1 shows the experimental results of JC-1 staining to observe the effect of compound 6e-2 on the mitochondrial membrane potential of Hep3B cells. A: Experimental results of the Control group; B: Experimental results of the 6e-2-1μM group; C: Experimental results of the 6e-2-2μM group.

[0026] Figure 3 Figure 1 shows the experimental results of JC-1 staining to observe the effect of compound 6e-2 on the mitochondrial membrane potential of HepG2 cells. A: Experimental results of the Control group; B: Experimental results of the 6e-2-2μM group; C: Experimental results of the 6e-2-4μM group.

[0027] Figure 4 Fluorescent staining micrographs showing the effect of compound 6e-2 on mitochondrial morphology. A: Fluorescent staining micrograph of the Control group; B: Fluorescent staining micrograph of the 6e-2 -4 μM group.

[0028] Figure 5 Figure 1 shows the experimental results of observing the effect of compound 6e-2 on mitochondrial ROS under fluorescence microscopy (magnification 200×). A: Experimental results of the Control group; B: Experimental results of the 6e-2-1μM group; C: Experimental results of the 6e-2-2μM group; D: Experimental results of the 6e-2-4μM group.

[0029] Figure 6 Effects of compound 6e-2 on the growth of Hep3B liver cancer xenografts in nude mice. A: Photograph of mice bearing the tumor; B: Photograph of the tumor after dissection in mice. Detailed Implementation

[0030] The present invention will now be described in detail with reference to exemplary embodiments or experimental examples shown in the accompanying drawings. However, it should be understood that the present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided herein to make the disclosure of the present invention more complete and to fully convey the inventive concept to those skilled in the art.

[0031] This invention provides a novel oxidized indole derivative (compound 6e-2), the specific chemical structural formula of which is shown below:

[0032] .

[0033] Furthermore, the present invention also provides a method for preparing the indole oxidase derivative, the specific steps of which are as follows:

[0034] S1. 7-Bromoindigo reacts with sodium hydride and 1-BOC-4-(2-chloroethyl)piperazine in dimethylformamide to give the first intermediate;

[0035] Wherein, the molar ratio of 7-bromoindigo to sodium hydride is 1:1.5-2.5, the molar ratio of 7-bromoindigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.2-1.8, and the reaction temperature is 60-90℃;

[0036] S2. The first intermediate is reacted with 4-methylthiophenylboronic acid in 1,4-dioxane and refluxed under the action of palladium catalyst and base to obtain the second intermediate.

[0037] The reflux reaction is carried out under inert gas protection at 100-120°C, with the molar ratio of the first intermediate to 4-methylthiophenylboronic acid being 1:1.2-1.8; the palladium catalyst is tris(dibenzylacetone)dipalladium, with triphenylphosphine added as a ligand, the amount of tris(dibenzylacetone)dipalladium being 3 mol%-8 mol% of the first intermediate, and the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine being 1:1.5-2.5; the base is anhydrous potassium carbonate, with the molar ratio of the first intermediate to anhydrous potassium carbonate being 1:1.5-2.5;

[0038] S3. The second intermediate is reacted with ethoxyformylmethylenetriphenylphosphine in tetrahydrofuran to prepare the third intermediate;

[0039] The molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.0-1.3, and the reaction temperature is -10 to 10°C.

[0040] S4. The third intermediate is deprotected from the BOC protecting group in dichloromethane containing trifluoroacetic acid to obtain the fourth intermediate;

[0041] The trifluoroacetic acid has a volume fraction of 3%-20%.

[0042] S5. The fourth intermediate is reacted with 2-thiopheneacetyl chloride in dichloromethane to obtain the indole oxide derivative;

[0043] The molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.2-1.8.

[0044] Furthermore, the steps of a preferred embodiment of this preparation method are as follows:

[0045] S1. 7-Bromoindigo reacts with sodium hydride and 1-BOC-4-(2-chloroethyl)piperazine in dimethylformamide to obtain a first intermediate; wherein the molar ratio of 7-bromoindigodigo to sodium hydride is 1:2, the molar ratio of 7-bromoindigodigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.5, and the reaction is carried out at a temperature of 80°C.

[0046] S2. The first intermediate is reacted with 4-methylthiophenylboronic acid in 1,4-dioxane under reflux in the presence of a palladium catalyst and a base to prepare the second intermediate; wherein the reflux reaction is carried out under inert gas protection at 110°C, and the molar ratio of the first intermediate to 4-methylthiophenylboronic acid is 1:1.5; the palladium catalyst is tris(dibenzylacetone)dipalladium, with triphenylphosphine added as a ligand, the amount of tris(dibenzylacetone)dipalladium being 5 mol% of the first intermediate, and the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine being 1:2; the base is anhydrous potassium carbonate, and the molar ratio of the first intermediate to anhydrous potassium carbonate is 1:2;

[0047] S3. The second intermediate is reacted with ethoxyformylmethylenetriphenylphosphine in tetrahydrofuran to obtain the third intermediate; wherein the molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.1, and the reaction temperature is 0°C.

[0048] S4. The third intermediate is deprotected from the BOC protecting group in dichloromethane containing trifluoroacetic acid to obtain the fourth intermediate; wherein the volume fraction of the trifluoroacetic acid is 5%;

[0049] S5. The fourth intermediate is reacted with 2-thiopheneacetyl chloride in dichloromethane to obtain the indole oxide derivative; wherein the molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.5.

[0050] This invention presents a series of studies on this indole oxide derivative (compound 6e-2). The results show that compound 6e-2 significantly inhibits the proliferation of liver cancer cells in both in vitro and in vivo experiments. Its mechanism of action in inhibiting liver cancer cell growth mainly involves damaging mitochondria, reducing mitochondrial membrane potential, and increasing mitochondrial ROS concentration. Compound 6e-2 can provide a new direction and candidate compound for the research of highly effective and low-toxicity anti-liver cancer drugs.

[0051] Therefore, the aforementioned indole oxide derivatives or pharmaceutically acceptable salts thereof can be used to prepare drugs for treating liver cancer, for example, in the form of solid, semi-solid, or liquid pharmaceutical formulations. These pharmaceutical formulations contain the compound 6e-2 of the present invention or a pharmaceutically acceptable salt thereof as the active ingredient, and may also be mixed with inorganic or organic excipients and excipients. Possibly, the active ingredient can be compounded with commonly used non-toxic pharmaceutical excipients to form granules, powders, tablets, capsules, pills, oral liquids, or injections, and any other usable dosage form. The excipients or excipients of the present invention may include talc, water, glucose, honey, lactose, gum arabic, gelatin, mannitol, starch, magnesium trisilicate, keratin, colloidal silica, and any carrier capable of preparing solid, semi-solid, or liquid formulations. Additionally, auxiliary, stabilizing, thickening, coloring, and flavoring agents may also be used.

[0052] To illustrate in detail the medicinal use of the indole oxyoxide derivative (compound 6e-2) in the treatment of liver cancer, the present invention is further illustrated by the following experimental examples.

[0053] Example 1: Design, synthesis, and preliminary screening of anti-hepatocellular carcinoma activity of oxidized indole derivatives.

[0054] I. This invention designs compounds with the following general structural formula:

[0055] ;

[0056] Where R1 are groups in equations (1), (2), (3) and (4) respectively:

[0057] .

[0058] That is, design the following four oxidized indole derivatives with the following structural formulas: 6e-1, 6e-2, 6e-3, and 6e-4:

[0059]

[0060] .

[0061] II. Using 7-bromoindigo as the starting material, the above four compounds were prepared by N-alkylation, Suzuki-Miyaura coupling reaction, Wittig reaction, deBoc protection reaction, and acylation reaction, as follows:

[0062] (1) Synthesis of the first intermediate 2e-1-4

[0063] 7-Bromoindigo (3 mmol, 678 mg) was dissolved in 12 mL of DMF (dimethylformamide) in a 50 cm L flask. The solution was transferred to a cryogenic reactor, and 2 equivalents of NaH were added in three portions at 0 °C. The mixture was stirred for half an hour, and after no more bubbles were produced and the liquid turned purple-black, 1.5 equivalents of 1-BOC-4-(2-chloroethyl)piperazine were added in small, repeated batches. After the reactants dissolved, the mixture was transferred to an oil bath at 80 °C and reacted for approximately 4 hours. After the reaction was confirmed to be complete by thin-layer chromatography, saturated ammonium chloride aqueous solution was added, followed by extraction with ethyl acetate. The organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the ethyl acetate was removed by vacuum distillation. The resulting product was then subjected to rapid silica gel column chromatography, eluted with petroleum ether / ethyl acetate, to give the first intermediate, 2e-1-4.

[0064] (2) Synthesis of the second intermediate 3e-1-4

[0065] 1.5 mmol of the first intermediate 2e-1-4 was placed in a 50 mL three-necked round-bottom flask. 1.5 equivalents of 4-methylthiophenylboronic acid, 2 equivalents of anhydrous potassium carbonate, 5 mol% tris(dibenzylacetone)dipalladium, and 10 mol% triphenylphosphine were added. Nitrogen gas was introduced as a protective atmosphere. 12 mL of Diox (1,4-dioxane) was injected into the flask using a syringe. The flask was refluxed in an oil bath at 110 °C overnight (approximately 14 h). After the reaction was confirmed to be complete by TLC, the mixture was filtered. The residue was washed with dichloromethane, and the filtrate was concentrated under reduced pressure to remove dichloromethane. The filtrate was then subjected to rapid silica gel column chromatography, eluted with petroleum ether / ethyl acetate, to obtain the second intermediate 3e-1-4.

[0066] (3) Synthesis of the third intermediate 4e-1-4

[0067] The second intermediate 3e-1-4 (0.5 mmol) was placed in a 50 mL pear-shaped flask, and 1.1 equivalents of ethoxyformylmethylenetriphenylphosphine were added and dissolved in 5 mL of tetrahydrofuran. The mixture was then reacted at 0 °C for about 2 hours. After the reaction was confirmed to be complete by TLC, the product was subjected to rapid silica gel column chromatography, eluted with petroleum ether / ethyl acetate, to give the third intermediate 4e-1-4.

[0068] (4) Synthesis of intermediate 5e-1-4

[0069] The third intermediate, 4e-1-4, was dissolved in dichloromethane (added in three portions) and stirred until completely dissolved. 5% trifluoroacetic acid was added, and the mixture was refluxed for approximately 1 hour. After TLC analysis showed the reaction was nearly complete, the reaction solution was filtered, the residue was washed with dichloromethane, and the filtrate was distilled under reduced pressure to remove the dichloromethane solvent. The filtrate was then subjected to rapid silica gel column chromatography, eluted with petroleum ether / ethyl acetate, to obtain the fourth intermediate, 5e-1-4.

[0070] (5) Synthesis of the target product

[0071] The fourth intermediate, 5e-1-4, was dissolved in dichloromethane and stirred for 3 min. Depending on the different R1 groups, 1.5 equivalents of a haloalkane or other compound corresponding to its structure were added, and the mixture was refluxed for approximately 2 h. After TLC detection of complete reaction, the mixture was filtered, the filter residue was washed with dichloromethane, and the dichloromethane was removed by vacuum distillation. The mixture was then subjected to rapid silica gel column chromatography with a chloroform / methanol gradient elution to obtain the final products 6e-1, 6e-2, 6e-3, and 6e-4.

[0072] III. Evaluation of the inhibitory effects of different oxidized indole derivatives on the proliferation of Hep3B and HepG2 cells using the MTT assay

[0073] HepG2 and Hep3B liver cancer cells in logarithmic growth phase were digested, centrifuged, and counted. Cells were seeded at a density of 4000 cells / well in sterile 96-well plates and cultured for 24 h. For the control group, 100 μL of complete culture medium was added per well. The drug concentration was prepared and diluted halfway, resulting in 5 drug-treated groups. 100 μL of the corresponding solution was added per well according to the drug concentration. Each concentration was treated in triplicate. After 48 h of drug treatment, the liquid in the wells was discarded, and 100 μL of culture medium containing 10% MTT was added to each well. The cells were incubated at 37°C with 5% CO2 for 4 h. The liquid in the wells was then discarded, and 150 μL of DMSO was added. The cells were shaken thoroughly for 10 min, and the absorbance (OD value) was measured using a microplate reader. The cell growth inhibition rate was calculated as follows: Cell growth inhibition rate % = [1 - (OD value of drug-treated group / OD value of blank group)] × 100%. The results are shown in Table 1 below.

[0074] Table 1. IC50 of indole oxyoxide compound 6e-1-4 on Hep3B and HepG2 cells 50

[0075]

[0076] The inhibitory activity of four novel oxidized indole derivatives against the growth of Hep3B and HepG2 cells was detected by MTT assay. Compound 6e-2 showed good inhibitory activity against both hepatocellular carcinoma cell lines. Furthermore, compound 6e-2 exhibited stronger inhibitory activity against Hep3B and HepG2 cells than the similarly structured anticancer compounds AZD5991 (AstraZeneca) and A12 (A-1210477, AbbVie).

[0077] Experiment Example 2: Plate colony formation assay to detect the ability of compound 6e-2 to inhibit the proliferation of Hep3B and HepG2 cells.

[0078] (1) Hep3B and HepG2 cells in the logarithmic growth phase were digested and counted. They were seeded into 6-well plates at a cell density of 5000 cells / mL. 2 mL of complete culture medium was added to each well beforehand, followed by 100 µL of cell suspension. After shaking, the cells were transferred to a cell culture incubator at 37°C and 5% CO2 and cultured overnight.

[0079] (2) Drug administration

[0080] Observe the cell morphology within the wells of the 6-well plate under an inverted optical microscope. Once the seeded cells have adhered and spread, drug administration can proceed. The dosage will be determined based on the cell growth rate and IC50. 50 Different concentrations of compound 6e-2 were administered: Hep3B at 1µM, 2µM, and 3µM, and HepG2 at 2µM, 4µM, and 8µM. A control group was also set up for each. The 6-well plates were removed, the old culture medium was discarded, and 2mL of drug-containing culture medium was added to each well. The medium was changed approximately every 3 days depending on the cell condition.

[0081] (3) Cloning and observation

[0082] After drug administration, culture the cells for 2-3 weeks according to their growth rate, and observe the formation of cell clone clusters daily under an optical microscope. Once the number of individual clones reaches approximately 50 (visible to the naked eye), fix the cells and terminate the clone formation experiment.

[0083] (4) Fixation, staining and imaging

[0084] Upon reaching the experimental endpoint, discard the old culture medium in the wells, wash gently with PBS, fix with 4% paraformaldehyde for 30 minutes, wash 2-3 times with PBS, and stain with 0.1% crystal violet at room temperature for 30 minutes. After staining, discard the crystal violet solution, wash with tap water, then rinse once with pure water. Finally, invert the plate and dry it in a 50°C oven, photographing and recording the distribution of cell colonies (see...). Figure 1 A, B), and calculate the number of clones formed (see ... Figure 1 C, D).

[0085] The ability of 6e-2 to inhibit the proliferation of Hep3B and HepG2 cells was detected by plate colony formation assay. Figure 1 A and B are photographs of Hep3B and HepG2 cell colonies stained with crystal violet, respectively. The purple color in the images represents the colonies formed by cell clones. Figure 1C and D are bar graphs showing the statistical count of clonal colonies in Hep3B and HepG2 cells, respectively. The experimental results showed that 6e-2 significantly inhibited the proliferation of Hep3B and HepG2 cells compared to the control group. After treatment with 1 μM, 2 μM, and 3 μM 6e-2 for 10 days, 6e-2 significantly reduced the number of Hep3B cell colonies compared to the control group. Similarly, after treatment with 2 μM, 4 μM, and 8 μM 6e-2 for 10 days, 6e-2 also significantly reduced the number of HepG2 cell colonies.

[0086] Experiment Example 3: JC-1 staining to observe the changes in mitochondrial membrane potential caused by compound 6e-2.

[0087] Hep3B / HepG2 cells in logarithmic growth phase were digested, centrifuged at 800 rpm for 5 min, counted, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 4 Cells / mL, totaling 2 mL, were cultured in confocal dishes for 24 hours. After incubation for 24 hours, the following groups were added: Hep3B group: Control group, 6e-2-1μM (i.e., 1μM of 6e-2, the meaning of which is the same for subsequent groupings), 6e-2-2μM; HepG2 group: Control group, 6e-2-2μM, 6e-2-4μM. The culture medium was aspirated, the cells were washed once with PBS, and 1 mL of serum-free cell culture medium containing JC-1 was added (JC-1:medium medium = 1:200). After thorough mixing, the cells were incubated at 37°C for 20 minutes. After incubation at 37°C, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer (1×). 1 mL of serum-free cell culture medium was added, and the changes in green and red fluorescence were observed under a laser confocal microscope. The results are shown in [Figure number missing]. Figure 2 and Figure 3 .

[0088] JC-1 exists in cells in two different physical forms: aggregates and monomers, each with a different fluorescence emission peak. High membrane potential (healthy cells): JC-1 aggregates in mitochondria to form J-aggregates, emitting red fluorescence (Ex / Em≈585 / 590 nm); the red / green fluorescence intensity ratio is typically ≥2.0. Low membrane potential (depolarization / early apoptosis): JC-1 exists in the cytoplasm as monomers, emitting green fluorescence (Ex / Em≈514 / 529 nm), and the red / green fluorescence intensity ratio can decrease to 0.2–0.5.

[0089] In the experiment, a series of concentrations of compound 6e-2 were applied to Hep3B and HepG2 cells for 24 hours. Specifically, Figure 2 A, B and C correspond to the cell groups Control group, 6e-2-1μM group and 6e-2-2μM group, respectively; Figure 3Cell groups A, B, and C correspond to the Control group, the 6e-2-2μM group, and the 6e-2-4μM group, respectively. Cells with high mitochondrial membrane potential exhibit red fluorescence, primarily in aggregate form; cells with low mitochondrial membrane potential exhibit green fluorescence, primarily in monomer form. The yellow fluorescence in the figure is a superposition of green and red fluorescence. Observation of the fluorescence signal after JC-1 staining shows that after treating cells with different concentrations of compound 6e-2, red fluorescence gradually decreases with increasing compound concentration, and yellow fluorescence in the superimposed fluorescence channel diagram also decreases, indicating a gradual decrease in the red / green fluorescence intensity ratio. This suggests that compound 6e-2 can effectively reduce mitochondrial membrane potential in a concentration-dependent manner.

[0090] Experiment Example 4: Observation of mitochondrial damage changes using laser scanning confocal microscopy

[0091] HepG2 cells in logarithmic growth phase were digested, centrifuged at 800 rpm for 5 min, counted, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 4 2 mL of medium was added per cell, and cultured in a confocal dish for 24 h. After incubation for 24 h following drug administration (group: Control group, 6e-2-4 μM [i.e., 4 μM of 6e-2]), drug administration was terminated, the original drug-containing medium in the wells was discarded, and 1 mL of working solution loaded with the Mito-TrackerGreen probe (concentration 100 nM) was added and incubated for 25 min, followed by 3 washes with PBS; nuclei were stained with Hoechst 33342 (original solution 500×), 1 mL was added to 2 μL; incubation was continued for 30 min, followed by 3 washes with PBS. 1 mL of serum-free medium was added to each well, and changes in mitochondria and nuclei were observed and photographed under laser scanning confocal microscopy at 405 nm and 488 nm laser excitation.

[0092] To investigate whether the oxidized indole compound 6e-2 induces apoptosis via the mitochondrial pathway, this experiment observed the effect of compound 6e-2 on mitochondrial morphology using laser confocal microscopy. The results are shown below. Figure 4 . Figure 4 Cell groups A and B correspond to the Control group and the 6e-2-4μM group, respectively. Hoechst 33342 was used for nuclear staining; under normal conditions, the cell nucleus exhibits weak blue fluorescence. When chromatin condensation occurs, the intensity of the blue fluorescence in the cell nucleus increases. The mitochondrial probe stained mitochondria with green fluorescence. Mitochondria are normally rod-shaped or rod-shaped, but become punctate after damage. The results showed that in the control group, mitochondria exhibited normal rod-shaped, strip-shaped, and filamentous morphologies and were evenly distributed in the cytoplasm; however, after treatment with 6e-2 for 24 hours, significant changes in mitochondrial morphology were observed, with mitochondria changing from normal long filaments to short punctate shapes, and even showing obvious breakage.

[0093] Experiment Example 5: Observation of changes in mitochondrial reactive oxygen species using MitoSOX Red (red mitochondrial superoxide fluorescent probe) staining.

[0094] Hep3B cells in logarithmic growth phase were digested, centrifuged at 800 rpm for 5 min, counted, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / year. 5 2 mL of MitoSOX Red was cultured in 6-well plates for 24 hours. After incubation for another 24 hours, the cells were treated with the drug (groups: Control, 6e-2-1 μM, 6e-2-2 μM, 6e-2-4 μM). 50 μg of MitoSOX Red was dissolved in 13 μL LDMSO to obtain a 5 mM stock solution. A 5 μM working solution was prepared using serum-free cell culture medium. To terminate the drug reaction, the original culture medium was discarded, and 1 mL of MitoSOX Red working solution was added. The plate was incubated at 37°C for 30 min. After incubation at 37°C, the supernatant was aspirated, and the cells were washed three times with PBS. 1 mL of serum-free cell culture medium was added, and the intensity of the red fluorescence was observed under a 20× fluorescence microscope.

[0095] This experiment used the MitoSOX Red probe to investigate the effect of compound 6e-2 on mitochondrial ROS levels in Hep3B cells. The results are shown below. Figure 5 . Figure 5 Cell groups A, B, C, and D correspond to the Control group, 6e-2-1μM group, 6e-2-2μM group, and 6e-2-4μM group, respectively. Under normal conditions, mitochondrial ROS levels are low, exhibiting weak red fluorescence. After treatment with different concentrations of compound 6e-2 for 24 hours, mitochondrial ROS levels increased with increasing concentration, as evidenced by a gradual increase in the red fluorescence signal. Furthermore, the intensity of the red fluorescence gradually increased with increasing compound concentration. This indicates that compound 6e-2 can effectively increase the release of ROS from mitochondria, and this effect shows a clear concentration-dependent relationship. It is speculated that compound 6e-2 may accelerate the inhibition of cell growth by promoting mitochondrial ROS production.

[0096] Experimental Example 6: In vivo anti-hepatocellular carcinoma activity study of compound 6e-2

[0097] Hep3B human liver cancer cells were digested when they reached the logarithmic growth phase, washed twice with pre-cooled PBS, centrifuged at 800 rpm for 5 min, resuspended, counted, and the cell concentration was adjusted to 8 × 10⁶ cells / mL. 7Cells / mL: Mix 100 μL of Hep3B human liver cancer cell suspension with 100 μL of Matrigel and inoculate into the skin of the right axilla of nude mice, injecting the cell suspension slowly and evenly. While withdrawing the needle, use a cotton swab to press the needle tip to prevent leakage of the cell suspension. Inoculation is complete when blister-like protrusions are observed at the inoculation site and there is no significant leakage at the needle exit point. Inoculation is complete when the tumor volume reaches 60-100 mmHg. 3 Tumors formed in mice, and the nude mice were randomly divided into 4 groups (see grouping of tumor-bearing mice). Figure 6 A): Solvent control group (control group), 6e-2-15mg / kg group (i.e., 15mg / kg of 6e-2, the meaning of the grouping is the same in the following cases), 6e-2-25mg / kg group, and A12-25mg / kg group, 3 mice in each group. The drugs were administered intraperitoneally once a day. The weight and tumor volume of the nude mice were recorded daily at regular intervals, and the observation continued for 15 days. After the experimental period, the mice were euthanized by cervical dislocation, and the tumor tissue was immediately dissected (tumor tissue from each group is shown in the table below). Figure 6 B), weighing.

[0098] This experiment established a Hep3B liver cancer xenograft model in nude mice and used the existing anti-cancer compound A12 (A-1210477) as a positive control. During the 15-day experiment, compound 6e-2 exhibited significant in vivo antitumor activity. The results showed that, compared with the solvent control group, all treatment groups effectively inhibited tumor growth. While the positive control drug A12 (25 mg / kg) showed some tumor-suppressing effect, the tumor size remained relatively small and did not change significantly. The 6e-2-15 mg / kg and 6e-2-25 mg / kg groups showed significantly stronger inhibition of tumor growth in mice than the A12-25 mg / kg group. In particular, the high-dose groups showed extremely small or even nearly disappeared tumors, with a significant decrease in tumor volume and weight. Simultaneously, the mice in all treatment groups maintained good mental condition and stable weight throughout the experimental period, without significant toxic reactions or abnormal emaciation, indicating that compound 6e-2 has good anti-liver cancer activity in vivo and possesses a certain degree of in vivo safety.

[0099] The above experiments demonstrate that compound 6e-2 significantly inhibits the proliferation of liver cancer cells in both in vitro and in vivo experiments. Its mechanism of action primarily involves damaging mitochondria, reducing mitochondrial membrane potential, and increasing mitochondrial ROS concentration. This invention provides a novel class of oxidized indole antitumor candidate compounds with significant anticancer activity and a clearly defined mechanism of action, laying an important foundation for the development of novel, highly effective, and low-toxicity anticancer drugs based on the oxidized indole skeleton.

Claims

1. An indole oxide derivative, characterized in that, It has the following chemical structural formula: 。 2. The method for preparing the indole oxide derivative as described in claim 1, characterized in that, Includes the following steps: S1. 7-Bromoindigo reacts with sodium hydride and 1-BOC-4-(2-chloroethyl)piperazine in dimethylformamide to obtain a first intermediate; wherein the molar ratio of 7-bromoindigodigo to sodium hydride is 1:1.5-2.5, the molar ratio of 7-bromoindigodigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.2-1.8, and the reaction temperature is 60-90℃; S2. The first intermediate is reacted with 4-methylthiophenylboronic acid in 1,4-dioxane under reflux in the presence of a palladium catalyst and a base to prepare the second intermediate; wherein the reflux reaction is carried out under inert gas protection at 100-120°C, and the molar ratio of the first intermediate to 4-methylthiophenylboronic acid is 1:1.2-1.

8. S3. The second intermediate is reacted with ethoxyformylmethylenetriphenylphosphine in tetrahydrofuran to prepare the third intermediate; wherein the molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.0-1.3, and the reaction temperature is -10-10℃. S4. The third intermediate is deprotected from the BOC protecting group in dichloromethane containing trifluoroacetic acid to obtain the fourth intermediate; S5. The fourth intermediate is reacted with 2-thiopheneacetyl chloride in dichloromethane to obtain the indole oxide derivative; wherein the molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.2-1.

8.

3. The preparation method according to claim 2, characterized in that: In step S1, the molar ratio of 7-bromoindigo to sodium hydride is 1:2, and the molar ratio of 7-bromoindigo to 1-BOC-4-(2-chloroethyl)piperazine is 1:1.

5. In step S2, the molar ratio of the first intermediate to 4-methylthiophenylboronic acid is 1:1.5; In step S3, the molar ratio of the second intermediate to ethoxyformylmethylenetriphenylphosphine is 1:1.1; In step S5, the molar ratio of the fourth intermediate to 2-thiopheneacetyl chloride is 1:1.

5.

4. The preparation method according to claim 2, characterized in that: In step S1, the reaction temperature for obtaining the first intermediate is 80°C; In step S2, the temperature condition for the reflux reaction is 110°C; In step S3, the reaction temperature for obtaining the third intermediate is 0°C.

5. The preparation method according to claim 2, characterized in that: In step S2, the palladium catalyst is tris(dibenzylacetone)dipalladium, and triphenylphosphine is added as a ligand. The amount of tris(dibenzylacetone)dipalladium is 3 mol%-8 mol% of the first intermediate, and the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine is 1:1.5-2.

5. The base is anhydrous potassium carbonate, and the molar ratio of the first intermediate to anhydrous potassium carbonate is 1:1.5-2.

5.

6. The preparation method according to claim 5, characterized in that: In step S2, the amount of tris(dibenzylacetone)dipalladium is 5 mol% of the first intermediate, the molar ratio of tris(dibenzylacetone)dipalladium to triphenylphosphine is 1:2, and the molar ratio of the first intermediate to anhydrous potassium carbonate is 1:

2.

7. The preparation method according to claim 2, characterized in that: In step S4, the volume fraction of the trifluoroacetic acid is 3%-20%.

8. The preparation method according to claim 2, characterized in that: In step S4, the volume fraction of the trifluoroacetic acid is 5%.

9. The use of the indole oxide derivative as described in claim 1 in the preparation of a medicament for treating liver cancer.

10. The application according to claim 9, characterized in that, The drug comprises the indole oxyde derivative and pharmaceutically acceptable excipients.

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