CN118852124A - A preparation method and application of indole alkaloid compound - Google Patents

Abstract

The invention discloses a preparation method and application of an indole alkaloid compound, wherein the improvement of the preparation method comprises the steps of pre-purifying raw materials, carrying out post-treatment on an intermediate to avoid water washing, and adopting a prepared silica gel plate to replace liquid chromatography in a part of purification process. The preparation method of indole alkaloid provided by the invention improves the purification of raw materials and products, and can effectively improve the preparation efficiency and the product yield. The invention also provides application of the indole alkaloid in preparing an iron death inducer, wherein the indole alkaloid can reduce the oxidation resistance of cells, increase accumulation of cell lipid peroxide and Fe ²⁺, and increase of intracellular iron ion content and peroxide is an important mark of iron death, so that the iron death of cells is induced. The indole alkaloid provided by the invention has the activity of inducing iron death on non-small cell lung cancer cells, and lays a foundation for developing safer and more effective medicines for various diseases related to iron death.

Description

Preparation method and application of indole alkaloid compound

Technical Field

The invention belongs to the technical field of medicine application, and particularly relates to a preparation method and application of an indole alkaloid compound.

Background

Lung cancer is one of the most common malignant tumors in the world, and has high morbidity and mortality, while early lung cancer can usually be treated by surgery, most cases are diagnosed in mid-or late-stages, requiring conservative treatment. Resistance remains a major challenge in lung cancer treatment, and most patients with advanced non-small cell lung cancer (NSCLC) develop resistance to current treatments, resulting in exacerbations. Searching for new therapeutic targets, methods of combating drug resistance, and methods of improving patient quality and longevity are key to lung cancer research. Compounds and therapeutic agents have been shown to treat lung cancer through iron death.

The basic mechanism of iron death is peroxidation of polyunsaturated fatty acids (PUFAs) on cell membranes, mainly catalyzed by lipoxygenase through enzymatic pathways, or non-enzymatic autoxidation of ferrous ions driven by the Fenton reaction. Morphologically, iron death is characterized primarily by mitochondria, including mitochondrial contraction, elevated mitochondrial outer membrane density, and mitochondrial cristae reduction or loss. Lipid peroxidation is accompanied by the formation of Malondialdehyde (MDA), reactive Oxygen Species (ROS) and 4-hydroxynonenal (4-HNE).

APLICYANIN B (r1=ac, r2=r3=h), a class of marine compounds in the indole alkaloid family isolated from ascidians (Aplidium cyaneeum), all of which have antitumor activity. However, the use of APLICYANIN B in iron death induction has not been reported in the literature. In addition, a synthetic method of APLICYANIN B is also disclosed in the related art, but the synthetic process has the problems of low reaction efficiency and incapability of separating effective products, so that a APLICYANIN B preparation method needs to be developed.

Disclosure of Invention

In order to overcome the problems of the prior art, one of the purposes of the present invention is to provide a method for preparing an indole alkaloid compound. The second object of the present invention is to provide an application of an indole alkaloid compound or a pharmaceutically acceptable salt thereof in preparing an iron death inducer or inducing iron death. The invention also provides an application of the indole alkaloid compound or the pharmaceutically acceptable salt thereof in preparing an active oxygen inducer or inducing active oxygen increase. The fourth object of the present invention is to provide an application of an indole alkaloid compound or a pharmaceutically acceptable salt thereof in preparing an iron accumulation inducer or inducing iron accumulation. The fifth object of the present invention is to provide an iron death inducer. The invention also provides application of the iron death inducer in inducing iron death of lung cancer cells.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

The first aspect of the present invention provides a process for the preparation of an indole alkaloid compound comprising the steps of:

s1, adding activated carbon into the compound 1 In the organic solution of (2) to obtain the compound 1

S2, the compound 1 and the compound 2The reaction was carried out to obtain compound 3

S3, reacting the compound 3 with guanidine carbonate to obtain a compound 4

S4, reacting the compound 4 with BH ₃. THF to obtain a compound 5

S5, the compound 5 and the compound 6The reaction was carried out to obtain compound 7

Preferably, in step S1, the activated carbon is porous amorphous carbon of 200-325 mesh.

Preferably, the mass ratio of the activated carbon to the compound 1 is 1: (5-15). More preferably, the mass ratio of the activated carbon to the compound 1 is 1: (8-12).

Preferably, the solvent of the organic solution is an alcohol solvent.

Preferably, the step S3 specifically includes the following steps: and (3) reacting the compound 3 with guanidine carbonate in ethylene glycol monomethyl ether, cooling and separating out after the reaction, filtering to obtain a product, and finally washing with isopropanol and drying to obtain the compound 4.

Preferably, the step S4 specifically includes the following steps: the compound 4 reacts with BH ₃. THF to obtain a crude product, and the crude product is purified by a silica gel plate to obtain a compound 5.

Preferably, step S5 specifically includes the following steps: the compound 5 and the compound 6 react to obtain a crude product, and the crude product is purified by a silica gel plate to obtain the compound 7.

Preferably, in step S1, the purification time is 0.5-3h.

Preferably, in step S2, the reaction time of the reaction is 20 to 50 hours; the reaction temperature is 15-40 ℃.

Preferably, in step S2, the molar ratio of compound 1 to compound 2 is 1: (1-2).

Preferably, in step S3, the reaction time of the reaction is 3 to 5 hours; the reaction temperature is 120-140 ℃.

Preferably, in step S3, the molar ratio of the compound 3 to guanidine carbonate is 1: (0.8-2).

Preferably, in step S4, the reaction time of the reaction is 4 to 6 hours; the reaction temperature is 60-80 ℃.

Preferably, in step S4, the molar ratio of the compound 4 to BH ₃ ·thf is 1: (4-6).

Preferably, in step S5, the reaction time of the reaction is 10 to 20 hours; the reaction temperature is 15-40 ℃.

Preferably, in step S5, the molar ratio of compound 5 to compound 6 is 1: (1-2).

The invention also provides an application of the indole alkaloid compound or pharmaceutically acceptable salt thereof in preparing an iron death inducer or inducing iron death, wherein the structural formula of the indole alkaloid compound is shown as the formula I:

The pharmaceutically acceptable salts include salts with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids; the inorganic acid comprises at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid and hydrobromic acid; the organic acid comprises at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid and tannic acid; the alkali metal comprises at least one of lithium, sodium and potassium; the alkali metal comprises at least one of lithium, sodium and potassium; the basic amino acid includes lysine.

The invention also provides an application of the indole alkaloid compound or the pharmaceutically acceptable salt thereof in preparing an active oxygen inducer or inducing active oxygen increase, wherein the structural formula of the indole alkaloid compound is shown as formula I:

preferably, the active oxygen inducer is an inducer that promotes the production and accumulation of active oxygen.

Preferably, the active oxygen is intracellular active oxygen.

More preferably, the cell is a non-small cell lung cancer cell.

More preferably, the intracellular active oxygen comprises lipid active oxygen and/or mitochondrial active oxygen.

Preferably, the indole alkaloid compound or pharmaceutically acceptable salt thereof is used in the preparation of a peroxide inducer or inducing peroxide increase.

The fourth aspect of the invention also provides an application of an indole alkaloid compound or pharmaceutically acceptable salt thereof in preparing an iron accumulation inducer or inducing iron accumulation, wherein the structural formula of the indole alkaloid compound is shown as formula I:

preferably, the iron accumulation inducer is a ferrous ion accumulation inducer.

Preferably, the iron accumulation inducer is used to induce an increase in intracellular ferrous ion concentration.

Preferably, the induced iron accumulation is an increase in the concentration of ferrous ions in the induced cell.

More preferably, the cell is a non-small cell lung cancer cell.

In a fifth aspect the present invention also provides an iron death inducer, the active substance of which comprises an indole alkaloid compound as described in the second aspect.

Preferably, the only active substance of the iron death inducer is the indole alkaloid compound.

Preferably, the concentration of the indole alkaloid compound is in the range of 0.5 to 50. Mu. Mol/L.

It is a sixth object of the present invention to provide the use of an iron death inducer according to the fifth aspect for inducing iron death in lung cancer cells.

Preferably, the use of an iron death inducer according to the fifth aspect for inducing iron death in non-small cell lung cancer cells

The beneficial effects of the invention are as follows:

(1) The invention provides an application of indole alkaloid in preparing an iron death inducer, which can reduce the oxidation resistance of cells, increase the accumulation of cell lipid peroxide and Fe ²⁺, and increase the content of iron ions and peroxide in cells, which are important marks of iron death, so as to induce the death of cell-induced iron. The indole alkaloid provided by the invention has the activity of inducing iron death on non-small cell lung cancer cells, and lays a foundation for developing safer and more effective medicines for various diseases related to iron death.

(2) The preparation method of indole alkaloid provided by the invention improves the purification of raw materials, and the active carbon is utilized to adsorb the raw materials, so that the preparation efficiency can be effectively improved; and the silica gel plate is used for replacing high performance liquid phase preparation chromatography, so that the preparation cost can be greatly reduced.

Drawings

FIG. 1 is a nuclear magnetic resonance spectrum of crude intermediate 2;

FIG. 2 is a nuclear magnetic hydrogen spectrum of intermediate 3;

FIG. 3 is a mass spectrum of intermediate 3;

FIG. 4 is a nuclear magnetic hydrogen spectrum of intermediate 4;

FIG. 5 is a mass spectrum of intermediate 4;

FIG. 6 is a nuclear magnetic hydrogen spectrum of intermediate 5;

FIG. 7 is a mass spectrum of intermediate 5;

FIG. 8 shows the effect of different concentrations APLICYANIN B on the activity of H1975, H358 cells;

FIG. 9 is a graph showing the status effect (A) and activity effect (B) of APLICYANIN B on H1975 cells;

FIG. 10 is a graph showing the status effect (A) and activity effect (B) of APLICYANIN B on H358 cells;

FIG. 11 is a graph showing the effect of various concentrations APLICYANIN B on the reactive oxygen species level of H1975 cells, wherein (A) is the effect flow cytometry; (B) a result statistical graph;

FIG. 12 is a graph showing the effect of various concentrations APLICYANIN B on the concentration of iron ions in H1975 cells;

FIG. 13 is a graph showing the effect of different concentrations APLICYANIN B on the reactive oxygen species level of H358 cells, wherein (A) is an effect flow cytometry; (B) a result statistical graph;

FIG. 14 is a graph showing the effect of various concentrations APLICYANIN B on the concentration of iron ions in H358 cells;

FIG. 15 shows changes in levels of antioxidant indicators of H1975 cells at various concentrations APLICYANIN B, wherein (A) is the change in Glutathione (GSH) content; (B) is the change of the content of Malondialdehyde (MDA);

FIG. 16 shows changes in levels of antioxidant indicators of H358 cells at various concentrations APLICYANIN B, wherein (A) is the change in Glutathione (GSH) content; (B) is the change of the content of Malondialdehyde (MDA);

FIG. 17 is a graph showing Western blotting results of APLICYANIN B on H1975 cells;

FIG. 18 shows APLICYANIN B versus the expression levels of different proteins in H1975 cells; wherein (A) is ASCL4; (B) is COX-2; (C) is SLC7A11; (D) is PD-L1; (E) is GPX4; (F) is FTH-1;

FIG. 19 is a graph showing Western blotting results of APLICYANIN B on H358 cells;

FIG. 20 shows APLICYANIN B expression levels of different proteins in H358 cells; wherein (A) is ASCL4; (B) is COX-2; (C) is SLC7A11; (D) is PD-L1; (E) is GPX4; (F) is FTH-1.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The starting materials used in the examples below, unless otherwise specified, were all available from conventional commercial sources or were isolated by simple synthetic preparation; the processes used, unless otherwise specified, are all conventional in the art.

Example 1

This example provides indole alkaloids APLICYANIN B, their preparation and their reaction formulas are as follows:

s1, dissolving 3-aldehyde-5-bromo-indole (22.2 g) in 500ml of methanol, adsorbing with medical grade active carbon (2 g, CAS: 64165-11-3, purchased from Pichia pastoris) for 1 hour, filtering, collecting filtrate, and spin-drying the methanol to obtain purified 3-aldehyde-5-bromo-indole.

Trifluoroacetic acid (560 mg,5.0 mmol) and morpholine (435 mg,5.0 mmol) were added to toluene (400 mL) and stirred for 20 minutes, followed by the addition of purified 3-aldehyde-5-bromo-indole (starting material 1, 22.2g,0.1 mol), isopropyl malonate (14.4 g,0.11 mol) and stirring at room temperature for 2 days, and thin layer chromatography detected that the majority of starting material 1 had been consumed. Stirring was stopped, left to stand for 20 minutes, the supernatant was poured out, ethyl acetate (200 mL) was added and stirred for 10 minutes, left to stand for 10 minutes, the supernatant was poured out, and repeated 2 times. Ethanol (200 mL) was then added and stirred for 10 minutes, and left to stand for 10 minutes, the supernatant was decanted and repeated 2 times. Filtering, washing the solid with methanol (100 mLx 2) for 2 times, naturally airing to obtain an intermediate 2 (29.6 g, yield 85% of yellow solid) obtained by condensing the raw material 1 with Mie acid, wherein the nuclear magnetic information of the product is ：¹HNMR(400MHz,CDCl₃)δ9.50(s,1H),8.87(s,1H),8.15(s,1H),7.49(s,1H),7.44(s,1H),1.83(s,6H)., and the nuclear magnetic resonance spectrum is shown in figure 1.

S2, intermediate 2 (6.0 g,17.19 mmol) and guanidine carbonate (1.54 g,17.10 mmol) were added to ethylene glycol monomethyl ether (150 mL), warmed to 130℃and refluxed for 4 hours. Then, the reaction mixture was cooled to room temperature, a large amount of white solid was precipitated, filtered, washed with isopropyl alcohol (5 mL) and dried under vacuum to obtain 2.3g of intermediate 3 in a yield of 38%. The nuclear magnetic hydrogen spectrogram and the mass spectrogram of the product are shown in fig. 2 and 3 respectively.

S3, under the protection of nitrogen, intermediate 3 (306 mg,1.0 mmol) and BH ₃. THF (5 mL,5.0 mmol) were added to dry THF (10 mL), heated to 70℃and refluxed for 5 hours. Then cooled to room temperature, ammonium chloride solution (1.0 mL) was slowly added dropwise, stirred for 30 minutes, then filtered, the solid was washed with THF (20 mL), the mother liquors were combined, and concentrated by rotary evaporation to give crude intermediate 4 (250 mg). Purification of crude intermediate 4 using preparative silica gel plates (CH ₂Cl₂: meoh=10:1) afforded 150mg of intermediate 4 in 45.5% yield, as shown in fig. 4 and 5, respectively, as nuclear magnetic resonance and mass spectra of the product with nuclear magnetic resonance and mass spectrum information of ：¹H NMR(400MHz,MeOD)δ7.75(s,1H),7.33(d,J＝11.7Hz,2H),7.25(d,J＝8.1Hz,1H),4.94(m,1H),3.42(m,2H),2.25(d,J＝4.5Hz,2H).[M+H]⁺＝293.0,295.0..

S4, intermediate 4 (400 mg,1.21 mmol) and acetoxy-succinimide (200 mg,1.27 mmol) were added to dry pyridine (3 mL) and stirred at room temperature for 15 hours. Then concentrated by rotary evaporation under reduced pressure to give crude compound 5 (200 mg). Purification of crude compound 5 using preparative silica gel plates (CH ₂Cl₂: meoh=7:1) gave 100mg of product 5 in 24.7% yield, designated APLICYANIN B, as shown in fig. 6 and 7, respectively, as nuclear magnetic resonance and mass spectrometry for ：¹H NMR(400MHz,MeOD)δ7.79(s,1H),7.35(d,J＝8.0Hz,2H),7.27(d,J＝7.8Hz,1H),5.11(m,1H),3.54(m,2H),2.32(d,J＝4.5Hz,2H),2.20(s,3H).[M+H]⁺＝335.0,337.0..

Comparative example 1

This comparative example provides a process for the preparation of intermediate 2 of example 1, which is as follows:

Trifluoroacetic acid (560 mg,5.0 mmol) and morpholine (435 mg,5.0 mmol) were added to toluene (400 mL) and stirred for 20 minutes, followed by the addition of unpurified 3-aldehyde-5-bromo-indole (starting material 1, 22.2g,0.1 mol), isopropyl malonate (14.4 g,0.11 mol), stirred at room temperature for 5-7 days, and thin layer chromatography detected that the majority of starting material 1 had been consumed. Stirring was stopped, left to stand for 20 minutes, the supernatant was poured out, ethyl acetate (200 mL) was added and stirred for 10 minutes, left to stand for 10 minutes, the supernatant was poured out, and repeated 2 times. Ethanol (200 mL) was then added and stirred for 10 minutes, and left to stand for 10 minutes, the supernatant was decanted and repeated 2 times. Filtering, washing the solid with methanol (100 mLx 2) for 2 times, naturally airing to obtain an intermediate 2 after condensation of the raw material 1 and Mi's acid, wherein the yield is lower than 50%, and the nuclear magnetic information of the intermediate 2 is the same as that of example 1.

Comparative example 1 and example 1 were subjected to comparative analysis, and the pre-purification of raw material 1 in example 1 effectively improved the production efficiency and the product yield.

Comparative example 2

This comparative example provides a process for the preparation of intermediate 3 of example 1, which is as follows:

Intermediate 2 (6.0 g,17.19 mmol) and guanidine carbonate (1.54 g,17.10 mmol) were added to ethylene glycol monomethyl ether (150 mL), warmed to 130℃and refluxed for 4 hours. Then, the mixture was cooled to room temperature, a large amount of white solid was precipitated, filtered, washed with water (5 mL), and the solid was dissolved.

Comparative example 2 was analyzed in comparison with example 1, and the work-up of intermediate 3 in example 1 avoids washing with water, allowing efficient isolation of the intermediate.

Experimental analysis

1. APLICYANIN B Effect on the Activity of H1975, H358 cells

H1975 and H358 cells were seeded at a density of 5X 10 ³ cells/well in 96-well plates, respectively, and cultured overnight. Incubated with APLICYANIN B (0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20. Mu.M) at different concentration gradients for 24 hours. Subsequently, a cell counting kit-8 (CCK-8) solution (NCM Biotech, suzhou, china) was added to each well and incubated for 3 hours. Absorbance (Abs) was measured for each group at 450nm (n=3). A blank group containing only medium and untreated cells was used as a control group. In the formula, cell viability= (experimental group-blank group)/(control group-blank group) ×100%.

As a result, as shown in FIG. 8, it was found that the activities of H1975 and H358 cells gradually decreased with an increase in APLICYANIN B concentration, the IC ₅₀ value of APLICYANIN B acting on H1975 cells was 1.94. Mu. Mol/L, and the IC ₅₀ value acting on H358 cells was 3.63. Mu. Mol/L.

2. APLICYANIN B Effect on proliferation of H1975, H358 cells

H1975 and H358 cells with good growth state are inoculated into 12-well plates, 10 multiplied by 10 ⁴ cells are added into each well, 1000 mu L of 1640 culture medium is added into each well, and the mixture is cultured for 24 hours in a constant temperature box with 5 percent CO ₂ at 37 ℃; cells were divided into a control group (without any treatment, normal culture), APLICYANIN B treatment group (H1975: APLICYANIN B at a final concentration of 2. Mu.M; H358: APLICYANIN B at a final concentration of 4. Mu.M) and APLICYANIN B co-treatment group with iron death inhibitor (Deferoxamine, DFO) (H1975: APLICYANIN B at a final concentration of 2. Mu.M; H358: APLICYANIN B at a final concentration of 4. Mu.M; DFO at a final concentration of 10. Mu.M), 3 treatments each in parallel; after 24h incubation at 37 ℃, the 12-well plate was placed under an electron microscope to observe the state of each group of cells; the activity of H1975 and H358 cells of each treatment group was also examined using CCK-8 kit (Beyotime, cat# C0037).

FIG. 9 shows the status effect (A) and activity effect (B) of APLICYANIN B on H1975 cells, which was observed by electron microscopy (10X) for 2. Mu.M APLICYANIN B to cause H1975 cell death compared to the control group, whereas DFO was able to reverse APLICYANIN B-induced cell death. Further by examining the activity of each group of H1975 cells, the results showed that the activity of H1975 cells in APLICYANIN B-treated group was significantly lower than that of the control group, the group treated with APLICYANIN B together with the iron death inhibitor (p < 0.0001), and that DFO was able to inhibit the effect of APLICYANIN B on H1975 cells (A in FIG. 2).

FIG. 10 is a graph showing the status effect (A) and activity effect (B) of APLICYANIN B on H358 cells; also, APLICYANIN B at 4 μm resulted in H358 cell death, and DFO reversed APLICYANIN B-induced cell death, as compared to the control. By examining the activity of each group of H358 cells, the statistical result shows that the activity of APLICYANIN B treated H358 cells is significantly lower than that of the control group, the H358 cells of APLICYANIN B and iron death inhibitor co-treated group (p < 0.0001), and the DFO can also inhibit the effect of APLICYANIN B on H358 cells.

The above results indicate that APLICYANIN B can inhibit proliferation of H1975, H358 cells by way of iron-dead cell death.

3. APLICYANIN B Effect on reactive oxygen species levels in H1975 and H358 cells

1) The testing method comprises the following steps:

Flow cytometry detects lipid peroxide production levels: h1975 and H358 cells were treated in groups and incubated with 10. Mu. M C11-BODIPY dye (abclonal, wuhan, china) at 37℃for 1H. After washing twice with PBS, the cells were resuspended and then analyzed by flow cytometry (DxP Athena 1L-3L,Cytek Biosciences,USA). The results were analyzed using FlowJo and GRAPHPAD PRISM software.

Fluorescence detection Fe ²⁺ level: h1975 and H358 cells were plated 24H before being treated in groups. FerroOrange working fluid (1. Mu. Mol/L; λex:543 nm; λem:580nm; dojindo, japan) was prepared using RPMI-1640 medium without serum. After half an hour incubation at 37 ℃, real-time observation and image acquisition was performed using a multi-functional microplate detection system (CY-TATION, BIOTEK, USA).

Micro-reduced Glutathione (GSH) assay: h1975 and H358 cells were plated 24H before being treated in groups. After collecting cells, washing the cells for 1 to 2 times by using PBS, centrifuging at a low speed to collect precipitated cells, adding PBS to suspend the cells, and carrying out ultrasonic or manual grinding to break the cells to be detected. Taking 0.1mL of crushed cell suspension, adding 0.1mL of reagent I, uniformly mixing, centrifuging for 10min at 3500g/min, and taking supernatant to be measured. The absorbance of each well was measured by a microplate reader at 405nm after standing for 5min, and mixing well according to the specification of a kit for measuring reduced Glutathione (GSH) (Nanjing institute of biological engineering, cat# A006-2-1). The results were analyzed using EXCEL. The cell calculation formula is:

Lipid oxidation (MDA) detection: h1975 and H358 cells were plated 24H before being treated in groups. Cells were collected and lysed using Western and IP cell lysates. After cell lysis, the supernatant was centrifuged at 10,000g-12,000g for 10 min and used for subsequent assays. After sample preparation, protein concentration was determined using BCA protein concentration assay kit for subsequent calculation of MDA content per protein weight in cells. The preparation of the kit was carried out according to the lipid oxidation (MDA) detection kit (Beyotime, cat# S0131M), the detection reaction system was set according to the kit instructions, and after mixing, the mixture was heated at 100℃or in a boiling water bath for 15 minutes. Cooled to room temperature in a water bath and centrifuged at 1000g for 10 minutes at room temperature. 200. Mu.L of the supernatant was added to a 96-well plate, and absorbance was measured at 532nm using a microplate reader. Calculation of cellular MDA content: after calculating the MDA content in the sample solution, the MDA content in the original sample was expressed by the protein content per unit weight.

2) Test results

FIG. 11 is a graph showing the effect of APLICYANIN B on the reactive oxygen species level of H1975 cells; FIG. 12 is a graph showing the effect of APLICYANIN B on the concentration of iron ions in H1975 cells; FIG. 13 is a graph showing the effect of APLICYANIN B on the reactive oxygen species level of H358 cells; FIG. 14 is a graph showing the effect of APLICYANIN B on the concentration of iron ions in H358 cells; from FIGS. 11-14, APLICYANIN B was found to increase accumulation of lipid peroxides and Fe ²⁺ levels in H1975, H358 cells.

FIG. 15 shows changes in antioxidant index levels for H1975 cells at various concentrations APLICYANIN B; FIG. 16 shows changes in antioxidant index levels of H358 cells at various concentrations APLICYANIN B; the change in lipid oxidation level was observed by measurement of micro-reduced Glutathione (GSH) and detection of Malondialdehyde (MDA), and as a result APLICYANIN B was found to decrease the antioxidant capacity of both H1975 and H358 cells, leading to accumulation of peroxide, and thus iron death.

4. APLICYANIN B induces iron death in H1975, H358 cells

H1975 and H358 cells were plated 24H before being treated in groups. The protein supernatant was collected by lysis using a lysate. Proteins were electrophoresed on 10% SDS-PAGE gels and blotted onto NC membrane by electroblotting. After blocking the membrane, primary antibodies against GAPDH (Sangon Biotech, shanghai, china) were used as an internal reference, SLC7a11, FTH1, GPX4, ACSL4, PD-L1, COX-2 were incubated overnight at 4 ℃. The membranes were then washed with TBST, and then incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (CST, shanghai, china) for 1 hour at room temperature. Protein development was performed using BeyoECL Moon (Beyotime biotechnology, shanghai, china). Finally, the results were analyzed by ImageJ and GRAPHPAD PRISM.

FIG. 17 is a graph showing Western blotting results of APLICYANIN B on H1975 cells; FIG. 18 shows APLICYANIN B versus the expression levels of different proteins in H1975 cells; FIG. 19 is a graph showing Western blotting results of APLICYANIN B on H358 cells; FIG. 20 shows APLICYANIN B expression levels of different proteins in H358 cells; protein expression levels are detected through Western Blot experiments, and statistical analysis proves that APLICYANIN B can induce H1975 and H358 cells to generate iron death, wherein ASCL4, COX-2 and PD-L1 are all expressed up, and FTH-1, GPX4 and SLC7A11 are all expressed down.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. A method for preparing an indole alkaloid compound, characterized in that it comprises the following steps: S1. Add activated carbon to the mixture containing compound 1 In an organic solution, the compound was purified to obtain S2, the compound 1 and the compound 2 Reaction was performed to obtain compound 3 S3, the compound 3 reacts with guanidine carbonate to obtain compound 4 S4, the compound 4 reacts with BH ₃ ·THF to obtain compound 5 S5, the compound 5 and the compound 6 Reaction is carried out to obtain a compound as shown in formula I 2. The method for preparing an indole alkaloid compound according to claim 1, characterized in that in step S1, the activated carbon is a porous amorphous carbon of 200-325 mesh; And/or, the mass ratio of the activated carbon to compound 1 is 1:(5-15); And/or, the solvent of the organic solution is an alcohol solvent. 3. The method for preparing an indole alkaloid compound according to claim 1, characterized in that step S3 specifically comprises the following steps: reacting the compound 3 with guanidine carbonate in ethylene glycol monomethyl ether, cooling and precipitating after the reaction, filtering to obtain a product, and finally washing with isopropanol and drying to obtain compound 4. 4. The method for preparing an indole alkaloid compound according to claim 1, characterized in that step S4 specifically comprises the following steps: reacting the compound 4 with BH ₃ ·THF to obtain a crude product, and purifying the crude product on a silica gel plate to obtain a compound 5; And/or, step S5 specifically comprises the following steps: the compound 5 is reacted with the compound 6 to obtain a crude product, and the crude product is purified by silica gel plate to obtain the compound shown in formula I. 5. The method for preparing an indole alkaloid compound according to any one of claims 1 to 4, characterized in that the reaction conditions of the preparation method are selected from one or more of the following: A) in step S1, the purification time is 0.5-3h; B) In step S2, the reaction time is 20-50h; the reaction temperature is 15-40°C; C) In step S3, the reaction time is 3-5h; the reaction temperature is 120-140°C; D) In step S4, the reaction time is 4-6h; the reaction temperature is 60-80°C; E) In step S5, the reaction time is 10-20 hours and the reaction temperature is 15-40°C. 6. Use of an indole alkaloid compound or a pharmaceutically acceptable salt thereof in preparing a ferroptosis inducer or inducing ferroptosis, characterized in that the structural formula of the indole alkaloid compound is as shown in Formula I: Or it is an indole alkaloid compound prepared by the preparation method according to any one of claims 1 to 5. 7. Use of an indole alkaloid compound or a pharmaceutically acceptable salt thereof in preparing an active oxygen inducer or inducing an increase in active oxygen, characterized in that the structural formula of the indole alkaloid compound is as shown in Formula I: Or it is an indole alkaloid compound prepared by the preparation method according to any one of claims 1 to 5. 8. Use of an indole alkaloid compound or a pharmaceutically acceptable salt thereof in preparing an iron accumulation inducer or inducing iron accumulation, characterized in that the structural formula of the indole alkaloid compound is as shown in Formula I: Or it is an indole alkaloid compound prepared by the preparation method according to any one of claims 1 to 5. 9. A ferroptosis inducer, characterized in that the active substance of the ferroptosis inducer includes the indole alkaloid compound according to claim 6. 10. Use of the ferroptosis inducer according to claim 9 in inducing ferroptosis of lung cancer cells.