Preparation method for ferroptosis inducer, and use thereof

Abstract

Provided are a preparation method for a ferroptosis inducer, and a use thereof. The preparation method for the ferroptosis inducer comprises: mixing a compound A and aldehyde thiophene boric acid under the catalysis of an inorganic base and a palladium catalyst, then adding a mixed solution of 1,4-dioxane and water, heating and stirring at 78-82℃, and purifying to obtain a compound B; and mixing the compound B and a rhodamine derivative under the catalysis of an acid solution, heating and stirring at 110-120℃, and purifying to obtain the ferroptosis inducer. The preparation method is used for tumor suppression, immune response activation, and imaging, can improve the ability of inhibiting tumor cells by using the advantages of ferroptosis and immune activation, and provides a pathway for further research and translation of a ferroptosis-based immunotherapy strategy.

Description

Preparation method and application of ferroptosis inducer Technical Field

[0001] The present invention relates to the technical field of ferroptosis inducers, and in particular to a preparation method and application of a ferroptosis inducer. Background Art

[0002] Ferroptosis is a type of cell death characterized by iron dependence, lipid peroxide accumulation, and regulatable properties. It can be induced by specific inducers, and various ferroptosis inducers play an important role in clinical anticancer and antitumor therapy. Since its initial discovery in 2012, a wide range of new compounds and biomolecules that induce ferroptosis have been discovered. Existing ferroptosis inducers are primarily classified into the following categories: 1. Small molecule drugs, such as VDACs, Erastin, TMZ, and SQS; 2. Nanoparticles; and 3. Nucleotide proteins.

[0003] Despite some progress in inorganic hybrid inducers, current organic ferroptosis inducers have limited performance, require high dosages, and require combination therapy. Furthermore, existing small-molecule ferroptosis inducers are in limited supply and have poor efficacy, making significant efficacy achieved when used alone extremely rare.

[0004] Triphenylamine derivatives (TPA) are a class of small organic compounds known for their excellent electron-donating properties, based on their donor / acceptor properties. Domestic reports have examined their application in hybrid solar cells. By introducing cyanoacetate, malondicyanide, and thiazolidinone (rhodanine), TPA-based small molecules C-TPA, D-TPA, and R-TPA were prepared, respectively, and applied to bulk heterojunction solar cells (Cheng Yuanyuan, "Research on Bulk Heterojunction Energy Level Control and Its Application in Organic / Inorganic Hybrid Solar Cells," June 2016, pp. 16-25). However, no reports have been found regarding their use as ferroptosis inducers or anti-tumor therapies.

[0005] Therefore, new small molecule ferroptosis inducers still need to be developed and improved.

[0006] Summary of the Invention

[0007] In view of this, the present invention proposes a preparation method and application of a ferroptosis inducer, which solves the technical problems of limited supply and poor efficacy of existing small molecule ferroptosis inducers.

[0008] The technical solution of the present invention is achieved as follows:

[0009] In one aspect, the present invention provides a method for preparing a ferroptosis inducer, comprising the following steps:

[0010] S1, Compound A is mixed with aldehyde thiophene boronic acid in the presence of an inorganic base and a palladium catalyst, and then a mixed solution of 1,4-dioxane and water is added. The mixture is heated and stirred at 78-82° C. for 20-24 hours, filtered, extracted, and purified to obtain Compound B;

[0011] S2, under gas protection, the compound B is mixed with a rhodanine derivative under acid catalysis, heated and stirred at 110-120° C. for 8-10 hours, cooled, quenched with water, and the organic phase is washed with a saturated sodium bicarbonate solution, dried, and purified to obtain the ferroptosis inducer.

[0012] The structural formula of the compound A is The structural formula of the rhodanine derivative is The structural formula of the ferroptosis inducer is Wherein R1 is MeO and R2 is CH2COOH.

[0013] Based on this solution, it is further preferred that the molar ratio of the compound formaldehyde, aldehyde thiophene boronic acid, inorganic base and palladium catalyst is 1: (1.8-2): (3.7-4): (7.4-8).

[0014] On the basis of this solution, it is further preferred that the volume ratio of the mixed solution of 1,4-dioxane and water is (1-1.2):1.

[0015] Based on this solution, further preferably, the molar ratio of compound B to the rhodanine derivative is 1:(1.5-1.8).

[0016] On the basis of this scheme, further preferably, the purification method includes silica gel column chromatography, and the eluent is petroleum ether and dichloromethane, and the volume ratio of petroleum ether / dichloromethane is (1 / 10-1 / 5).

[0017] On the basis of this scheme, further preferably, the palladium catalyst includes Pd(PPh3)4.

[0018] On the basis of this solution, it is further preferred that the inorganic base is one of potassium carbonate, potassium hydroxide or sodium carbonate.

[0019] On the basis of this solution, it is further preferred that the gas shielding gas is an inert gas, including one of helium, neon or argon.

[0020] On the other hand, the present invention also provides the application of the preparation method of the ferroptosis inducer in inhibiting tumors and activating immune responses.

[0021] Based on this scheme, it is further preferred that the tumor is a breast tumor.

[0022] The preparation method and application of the ferroptosis inducing agent of the present invention have the following beneficial effects compared with the prior art:

[0023] The preparation method of the ferroptosis inducer provided by the present invention requires, during the synthesis process, to utilize a one-step classic Suzuki-coupling reaction to connect a triphenylamine derivative with thiophene, and then utilize a condensation reaction to connect a rhodanine derivative, thereby obtaining the triphenylamine thiophene-rhodanine molecule. The reaction conditions are mild and the steps are relatively simple. At the same time, a new organic small molecule ferroptosis inducer (MeOTPAV-RHA) based on the rhodanine structure is synthesized, which can effectively induce ferroptosis and apoptosis effects.

[0024] On the other hand, the ferroptosis inducer prepared by the present invention can trigger immunogenic cell death (ICD) and activate immune response, filling a key gap in this field; this innovative method has the potential to improve the effect of inhibiting tumor cells by utilizing the advantages of ferroptosis and immune activation, providing a way for further research and transformation of ferroptosis-based immunotherapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] FIG1 is a synthetic route of the ferroptosis inducer of the present invention;

[0027] FIG2 shows three ferroptosis inducers according to the present invention;

[0028] FIG3 is a hydrogen nuclear magnetic resonance spectrum of the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0029] FIG4 is a carbon NMR spectrum of the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0030] FIG5 is a mass spectrum of the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0031] FIG6 is an absorption and emission spectrum of the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0032] FIG7 shows the tumor inhibition of 4T1 tumor mice by the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0033] FIG8 is a tumor tissue staining image of 4T1 breast tumor-bearing mice after treatment with the ferroptosis inducer MeOTPAV-RHA of the present invention and different controls;

[0034] FIG9 is a flow cytometric graph of immune cells T cells in 4T1 breast tumor-bearing mice after treatment with the ferroptosis inducer MeOTPAV-RHA of the present invention and different controls;

[0035] FIG10 is a graph showing the cell activity of the ferroptosis inducer MeOTPAV-RHA and other ferroptosis inducers on 4T1 cells;

[0036] FIG11 is a schematic diagram of total reactive oxygen species (ROS) detection using different ferroptosis inducers;

[0037] FIG12 is a graph showing the toxicity test of different ferroptosis inducers on 4T1 cells under light conditions;

[0038] Figure 13 is a schematic diagram showing changes in ROS levels in 4T1 cells after treatment with the ferroptosis inducer MeOTPAV-RHA;

[0039] FIG14 is a schematic diagram of the cellular localization of the ferroptosis inducer MeOTPAV-RHA;

[0040] Figures 15 and 16 are schematic diagrams showing ICD of 4T1 cells induced by the ferroptosis inducer MeOTPAV-RHA;

[0041] FIG17 is a staining image of tissue cells of healthy mice using the ferroptosis inducer MeOTPAV-RHA of the present invention;

[0042] Figures 18 and 19 are schematic diagrams showing the results of serum biochemistry and blood routine examinations of 4T1 breast tumor-bearing mice according to the present invention. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] The present invention provides a preparation method and application of a ferroptosis inducer, wherein the synthesis route of the ferroptosis inducer is shown in Figure 1. By designing a triphenylamine thiophene-rhodanine molecule as a ferroptosis inducer, a heteroatom π system, thiophene, is introduced between the electron donor and electron acceptor structures. Such molecules include MeOTPAV-RHA (R1 = MeO, R2 = CH2COOH), TPAV-RHA and TPA-RHA, as shown in Figure 2; a triphenylamine derivative is connected to thiophene by a one-step classic Suzuki coupling reaction, and then a rhodanine derivative is connected by a condensation reaction to obtain the triphenylamine thiophene-rhodanine molecule, which can be used to enhance anti-tumor therapy and activate immune response.

[0045] Example 1

[0046] This embodiment provides a method for preparing a ferroptosis inducing agent, comprising the following steps:

[0047] S1, preparation of aldehyde triphenylamine thiophene compounds; Compound A (15.6 mmol), aldehyde thiophene boronic acid (31.2 mmol), K2CO3 (8.0 g, 58.0 mmol) and palladium catalyst Pd(PPh3)4 (278.4 mg, 116 mmol) were added to a 500 mL reaction flask, and then 30 mL of a mixed solution of 1,4-dioxane and water (volume ratio 1:1) was poured into a 200 mL container, heated and stirred at 78 ° C for 20 h, and after the reaction was completed, the K2CO3 was removed by coarse filtration. The insoluble matter was removed and the filtrate was collected; the filtrate was subjected to rotary evaporation to remove the solvent as much as possible, and then a white solid was obtained in the container, which was filtered again and rinsed with methanol to collect the white solid; 4 mL of a saturated aqueous solution of sodium bicarbonate was added to the container and stirred for 30 minutes, then filtered, and the filtrate was extracted with ethyl acetate (3×30 mL), and the organic phase was washed with a saturated sodium chloride solution and then dried over anhydrous sodium sulfate; the solvent was then removed by rotary evaporation, and the crude product was further purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 10 / 1, volume ratio) to obtain an aldehyde triphenylamine thiophene compound, i.e., compound B, as a bright yellow solid.

[0048] S2, preparation of triphenylamine thiophene-rhodanine molecules; compound B (3 mmol) and rhodanine derivative (4.5 mmol) obtained in step S1 were added to a 50 mL two-necked flask, 10 mL of glacial acetic acid was added under argon gas protection, and heated with stirring at 120 ° C for 10 h; after the reaction was completed, it was cooled to 25 ° C, and the reaction solution was washed 3 times with 1 mL of deionized water; then, while stirring the organic phase, saturated sodium bicarbonate solution was added for neutralization, and after the organic phase was washed with saturated sodium chloride solution, anhydrous sodium sulfate was added for drying and concentrated; the crude product was purified by silica gel column chromatography, and the eluent was petroleum ether / dichloromethane (5 / 1, volume ratio) to obtain a triphenylamine-rhodanine molecular compound as a red-purple solid, that is, the ferroptosis inducer.

[0049] Results: The triphenylamine-rhodanine molecular compound synthesized in Example 1 is MeOTPAV-RHA, and its H NMR spectrum is shown in Figure 3 (MeOTPAV-RHA). 1 H NMR (400MHz, DMSO-d6): δ8.07(s,1H),7.76(d,J=4.1Hz,1H),7.64–7.53(m,3H),7.13 –7.06(m,4H),6.95(d,J=8.9Hz,4H),6.75(d,J=8.7Hz,2H),4.54(s,2H),3.75(s,6H).

[0050] FIG4 proves that the triphenylamine-rhodanine molecular compound synthesized in Example 1 is MeOTPAV-RHA, and its C NMR spectrum is characterized as follows: (MeOTPAV-RHA) 13 C NMR (101MHz, DMSO-d6): δ192.25,167.93,166.58,156.83,153.59,149.92,139.59,138.88 ,135.18,127.88,127.44,127.17,124.45,123.88,118.64,117.96,115.57,55.74,46.42.

[0051] Example 2

[0052] This embodiment provides a method for preparing a ferroptosis inducing agent, comprising the following steps:

[0053] S1, preparation of aldehyde triphenylamine thiophene compounds; Compound A (15.6 mmol), aldehyde thiophene boronic acid (28.1 mmol), K2CO3 (8.6 g, 62.4 mmol) and palladium catalyst Pd(PPh3)4 (124.8 mmol) were added to a 500 mL reaction flask, and then 33 mL of a mixed solution of 1,4-dioxane and water (volume ratio 1.2:1) was poured into a 200 mL container, heated at 82 ° C and stirred for 24 h. After the reaction was completed, coarse filtration was performed to remove K2CO3 insoluble matter, and the filtrate was collected; the filtrate was vortexed The solvent was removed as much as possible by rotary evaporation, and a white solid was obtained in the container. The product was filtered again and rinsed with methanol to collect the white solid. 3 mL of saturated aqueous sodium bicarbonate solution was added to the container and stirred for 30 min, then filtered, and the filtrate was extracted with ethyl acetate (3×30 mL). The organic phase was washed with saturated sodium chloride solution and then dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and the crude product was further purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 10 / 1, volume ratio) to obtain an aldehyde triphenylamine thiophene compound, i.e., compound B, as a bright yellow solid.

[0054] S2, preparation of triphenylaminethiophene-rhodanine molecules; compound B (3 mmol) and rhodanine derivative (5.4 mmol) obtained in Example 1 were added to a 50 mL two-necked flask, 12 mL of glacial acetic acid was added under argon gas protection, and heated with stirring at 120 ° C for 10 h; after the reaction was completed, it was cooled to 25 ° C, and the reaction solution was washed 3 times with 1.5 mL of deionized water; then, while stirring the organic phase, saturated sodium bicarbonate solution was added for neutralization, and after the organic phase was washed with saturated sodium chloride solution, anhydrous sodium sulfate was added for drying and concentrated; the crude product was purified by silica gel column chromatography, and the eluent was petroleum ether / dichloromethane (5 / 1, volume ratio) to obtain a triphenylamine-rhodanine molecular compound as a red-purple solid, i.e., the ferroptosis inducer.

[0055] Results: Figure 5 is the mass spectrum of the triphenylamine-rhodanine molecular compound obtained in Example 2, HRMS (ESI): Calcd for: C 30 H 25 N2O5S3 + ([M+H] + ):589.09201, Found:589.09077, indicating that the compound obtained by purification is the target product.

[0056] Example 3

[0057] This embodiment provides a method for preparing a ferroptosis inducing agent, comprising the following steps:

[0058] S1, preparation of aldehyde triphenylamine thiophene compounds; Compound A (15.6 mmol), aldehyde thiophene boronic acid (28.8 mmol), K2CO3 (8.3 g, 60.1 mmol) and palladium catalyst Pd(PPh3)4 (117.8 mmol) were added to a 500 mL reaction flask, and then 35 mL of a mixed solution of 1,4-dioxane and water (volume ratio 1.1:1) was poured into a 200 mL container, heated at 80 ° C and stirred for 22 h. After the reaction was completed, coarse filtration was performed to remove K2CO3 insoluble matter, and the filtrate was collected; the filtrate was rotated The solvent was removed as much as possible by evaporation, and a white solid was obtained in the container. The product was filtered again and rinsed with methanol to collect the white solid. 3.5 mL of a saturated aqueous solution of sodium bicarbonate was added to the container and stirred for 30 min, then filtered, and the filtrate was extracted with ethyl acetate (3×30 mL). The organic phase was washed with a saturated sodium chloride solution and then dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and the crude product was further purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 8 / 1, volume ratio) to obtain an aldehyde triphenylamine thiophene compound, i.e., compound B, as a bright yellow solid.

[0059] S2, preparation of triphenylaminethiophene-rhodanine molecules; compound B (3 mmol) and rhodanine derivative (5.1 mmol) obtained in Example 1 were added to a 50 mL two-necked flask, 11 mL of glacial acetic acid was added under argon gas protection, and heated with stirring at 120 ° C for 10 h; after the reaction was completed, it was cooled to 25 ° C, and the reaction solution was washed 3 times with 1.5 mL of deionized water; then, while stirring the organic phase, saturated sodium bicarbonate solution was added for neutralization, and after the organic phase was washed with saturated sodium chloride solution, anhydrous sodium sulfate was added for drying and concentrated; the crude product was purified by silica gel column chromatography, and the eluent was petroleum ether / dichloromethane (8 / 1, volume ratio) to obtain a triphenylamine-rhodanine molecular compound as a red-purple solid, i.e., the ferroptosis inducer.

[0060] Results: Figure 6 is the absorption and emission spectra of the ferroptosis inducer MeOTPAV-RHA described in this example. It can be seen that the wavelength of the ferroptosis inducer MeOTPAV-RHA in this example is in the range of 520-700 nm, and the normalized intensity and absorbance are better than TPAV-RHA and TPA-RHA.

[0061] In the preparation method of the ferroptosis inducer described in this embodiment, the inorganic base is explained by taking K2CO3 as an example, but the inorganic base used in this embodiment can also be other inorganic bases such as KOH and Na2CO3; the examples given are only specific examples of the application of the present invention; the present invention is not limited to the embodiments disclosed herein, but the appropriate form is selected according to actual needs to implement the present invention.

[0062] Example 4

[0063] This embodiment provides an application of a ferroptosis inducer in inhibiting tumors. The ferroptosis inducer prepared in Example 1 is applied to anti-breast tumor activity detection, specifically comprising the following steps:

[0064] The 4T1 breast tumor-bearing mouse model was established by subcutaneously inoculating 2×10 6 4T1 breast tumor cells, when the tumor volume reaches 40mm 3 At the time of , 16 mice were randomly divided into 4 groups: group i, group ii, group iii and group iv;

[0065] 1×PBS (200 μL) (group i and group ii) and MeOTPAV-RHA (100 μM, 200 μL) (group iii and group iv) were intravenously injected, and 24 h later, the tumors in group ii and group iv were placed under 180 mW / cm 2 The mice were irradiated under white light for 10 min, and the tumor size was measured every 3 days. At the end of the experiment, the tumor tissues of all mice were collected and photographed.

[0066] To analyze the phenotype of macrophages in tumor tissues after different treatments, samples were collected 24 h after treatment, frozen sections were prepared, and tumor tissues were stained with hematoxylin and eosin (H&E).

[0067] Results: Figure 7 shows the tumor inhibition of 4T1 tumor mice by the ferroptosis inducer MeOTPAV-RHA described in this example; compared with the control group, the tumor in the iv group was almost completely cleared and there was no recurrence, which indicates that the ferroptosis inducer of this example has a synergistically enhanced anti-tumor effect under light irradiation.

[0068] Figure 8 is a tumor tissue staining image of 4T1 breast tumor-bearing mice after treatment with the ferroptosis inducer MeOTPAV-RHA described in the present invention and different controls; it can be seen that the hematoxylin-eosin (HE) staining results show that the tumor tissue in group iv has obvious damage; under light irradiation, the anti-tumor effect of the ferroptosis inducer is synergistically enhanced, thereby stimulating its photodynamic ability.

[0069] Figure 9 is a flow cytometric graph of immune cell T cells in 4T1 breast tumor-bearing mice after treatment with the ferroptosis inducer MeOTPAV-RHA described in the present invention and different controls; the in vivo anti-tumor immunity results can be seen, and the ability of MeOTPAV-RHA to activate immunity to overcome the immunosuppressive tumor microenvironment (TME) was evaluated by flow cytometry analysis; considering the important role of T cells in anti-tumor immunity, the number of T cells in spleen tissue was analyzed; the results showed that the number of CD4+T cells and CD8+T cells in the spleen of the iv group was 2.5 times and 3 times that of the control group, respectively, and the infiltration of CD4+T cells and CD8+T cells increased significantly after tumor treatment.

[0070] Example 5

[0071] This example provides an application of a ferroptosis inducer in inhibiting tumors. The ferroptosis inducer obtained in Example 3 is used in an immune response activation test, specifically comprising the following steps:

[0072] Cell culture:

[0073] 4T1 breast tumor cells were maintained in RPMI-1640 medium (Biological Industries, BI) supplemented with 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% fetal bovine serum (FBS), and cultured at 37° C. and 5% CO 2 .

[0074] Cell viability assay:

[0075] 4T1 breast tumor cells were cultured at 5 × 10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate for 24 h. Different concentrations (0, 3.125, 6.25, 12.5, 25, and 50 μM) of TPA-RHA, TPAV-RHA, and MeOTPAV-RHA were diluted into RPMI 1640 medium. The cells were incubated with the above molecules for 24 h, and then white light (11 MW / cm 2 , 5min), and then treated with the same gradient concentration of iron inhibitor-1 (Fer1) as mentioned above; 100μL of culture medium was added to each sample well, and after treatment, the culture medium was removed and 10μL of CCK-8 was added; 2h later, the absorbance at 450nm of each well was measured using a microplate reader, and cell viability was determined by the ratio of the absorbance of cells cultured with TPA-RHA, TPAV-RHA or MeOTPAV-RHA to that of cells cultured with normal culture medium.

[0076] Total reactive oxygen species (ROS) detection:

[0077] TPAV-RHA, TPA-RHA and MeOTPAV-RHA (100 μM, 200 μL) were prepared into 1 mL solution with 1× PBS, and ROS indicator (DCFH) was used as the indicator for total ROS detection. 25 μL of 40 μM DCFH was added to each solution at 40 mW / cm 2 Under the conditions of white light irradiation and 480nm laser excitation, the time-dependent fluorescence spectrum of DCFH was measured, and then the curve of relative fluorescence intensity changing with illumination time was plotted.

[0078] Detection of ROS in 4T1 cells:

[0079] 5×10 4 4T1 cells were then treated with different concentrations (0, 6.25 μM, and 25 μM) of MeOTPAV-RHA. DCFH was diluted with serum-free medium and incubated at 37°C for 30 min. The medium was then removed and the cells were washed three times with 1x PBS buffer. Finally, the images were imaged under an LSM980 confocal fluorescence microscope using a 488 nm laser for excitation and a 505-550 nm bandpass filter to collect the fluorescence signals of the oxidized and reduced probes, respectively.

[0080] Detection of lipid peroxide (LPO) in 4T1 cells:

[0081] The LPO sensor uses the BODIPY 581 / 591 C11 probe. 4T1 cells were treated with different methods and then incubated with the BODIPY 581 / 591 C11 probe (5 μM) for 1 hour. The culture medium was removed, and the cells were washed three times with 1x PBS. The cells were then imaged under an LSM980 confocal fluorescence microscope. The fluorescence signals of the oxidized and reduced probes were collected using 488 nm and 543 nm laser excitation, respectively, and bandpass filters of 505-550 nm and 580-600 nm were used to collect the fluorescence signals.

[0082] ICD immunofluorescence analysis:

[0083] 4T1 cells were treated with different concentrations of MeOTPAV-RHA (0, 6.25 μM, and 25 μM) with or without light irradiation, fixed in 4% paraformaldehyde (PFA) for 15 min, and incubated with antibodies against glutathione peroxidase 4 (GPX4) (1:400), long-chain acyl-CoA synthetase 4 (ACSL4) (1:200), calreticulin (CRT) (1:400), and high-mobility group protein 1 (HMGB1) (1:400) at 4°C for 12 h. The treated cells were washed three times with 1x TBST buffer and incubated with secondary antibodies against Alexa Fluor 488 or Alexa Fluor 555 (1:1000) at room temperature for 1 h. The nuclei were stained with DAPI at room temperature for 5 min, and the locations of the corresponding labeled proteins were detected using an LSM980 confocal fluorescence microscope.

[0084] Results: Figure 10 is a graph showing the cell activity of 4T1 cells by the ferroptosis inducer MeOTPAV-RHA described in this example and other ferroptosis inducers; it can be seen that the tumor in the iv group was almost completely eliminated without recurrence, indicating that the ferroptosis inducer MeOTPAV-RHA described in this example has a synergistically enhanced anti-tumor effect under light irradiation.

[0085] Figure 11 is a schematic diagram of the total reactive oxygen species (ROS) detection of different ferroptosis inducers; it can be seen that MeOTPAV-RHA has the ability to produce more reactive oxygen species when the drug concentration is 10 μM.

[0086] Figure 12 shows the toxicity test of different ferroptosis inducers on 4T1 cells under light conditions. Subsequently, the phototoxicity of three ferroptosis inducers on 4T1 cancer cells was measured under light conditions. MeOTPAV-RHA also exhibited more pronounced phototoxicity. The addition of the ferroptosis inhibitor Fer1 significantly inhibited the cytotoxic effect.

[0087] Figure 13 is a schematic diagram of the changes in ROS levels in 4T1 cells after treatment with the ferroptosis inducer MeOTPAV-RHA; using DCFH as an indicator of ROS, the changes in the overall intracellular ROS levels of 4T1 cells after MeOTPAV-RHA treatment were evaluated, as shown in Figures 13a and b; at the same time, significant green fluorescence was observed in cells treated with MeOTPAV-RHA, indicating that cell death directly induced by MeOTPAV-RHA is associated with the accumulation of intracellular ROS; the fluorescence intensity can be significantly inhibited by the presence of the ferroptosis inhibitor Fer1.

[0088] To assess the expression levels of ferroptosis-related markers, imaging studies were performed using BODIPY 581 / 591 C11, a fluorescent sensor for detecting intracellular LPO. This probe emits red fluorescence due to its inherent reduced state, and when reacting with peroxide, its emission peak shifts from 590 nm to 510 nm, emitting green fluorescence. As shown in Figures 13c and 13d, after MeOTPAV-RHA treatment, the green fluorescence in the cells increased and the red fluorescence decreased, while this trend was reversed to a certain extent in the presence of ferroptosis inhibitors.

[0089] The results in Figures 13e and 13f indicate that MeOTPAV-RHA is an organic small molecule that can induce ferroptosis in 4T1 cancer cells, and this ferroptosis can be inhibited by Fer1. Immunofluorescence imaging, treatment, and the presence of MeOTPAV-RHA all lead to a significant decrease in the expression of GPX4 in 4T1 cells, while ACSL4 plays a regulatory role, as shown in Figures 13g and 13h. Similar to the detection of intracellular LPO and ROS, the addition of Fer1 inhibited this trend. Therefore, the ability to induce cell ferroptosis should be one of the main reasons for the dark toxicity of MeOTPAV-RHA.

[0090] Figure 14 is a schematic diagram of the cellular localization of the ferroptosis inducer MeOTPAV-RHA; the intracellular localization of MeOTPAV-RHA can be seen, and it is found that the ferroptosis inducer exhibits a strong fluorescence signal that is relatively consistent with ER-Tracker Green.

[0091] Figures 15 and 16 are schematic diagrams of the induction of ICD in 4T1 cells by the ferroptosis inducer MeOTPAV-RHA. It can be seen that after co-incubation with MeOTPAV-RHA, CRT is transferred and exposed on the cell membrane surface, while HMGB1 is released from the nucleus to the extracellular space. This result clearly demonstrates that MeOTPAV-RHA can induce ICD in 4T1 cancer cells.

[0092] Example 6

[0093] This example provides an application of a ferroptosis inducer in inhibiting tumors. The ferroptosis inducer prepared in Example 1 was subjected to an in vitro biosafety evaluation test, which specifically includes the following steps:

[0094] Healthy Balb / c mice (6 in total) were randomly divided into two groups and injected intravenously with 1×PBS (200 μL) and MeOTPAV-RHA (10 mg / kg), respectively. Blood was collected on the 14th day for analysis of serum biochemistry and blood routine. Organs were collected for H&E staining on the 14th day after injection.

[0095] Results: Figure 17 is a staining image of the tissue cells of healthy mice using the ferroptosis inducer MeOTPAV-RHA described in the present invention. To evaluate the biocompatibility of the ferroptosis inducer in healthy mice, mice were sacrificed 14 days after intravenous injection with PBS or MeOTPAV-RHA. The main organs of the mice (heart, liver, spleen, lung, and kidney) were stained, and no abnormal cell morphology or tissue lesions were observed in either group, indicating that MeOTPAV-RHA has good in vivo biocompatibility.

[0096] Figures 18 and 19 are schematic diagrams showing the results of serum biochemistry and blood routine examinations in 4T1 breast tumor-bearing mice described herein. Serum biochemistry and blood routine examinations showed no significant differences in all measured parameters. These results demonstrate that MeOTPAV-RHA has good in vivo biocompatibility at the tested doses, supporting its potential for biological applications.

[0097] In summary, the present invention designed and synthesized a class of organic small molecule ferroptosis inducers (MeOTPAV-RHA) based on the rhodanine structure. As a new type of organic small molecule ferroptosis inducer, it can simultaneously induce ferroptosis, apoptosis and immunogenic death, and has outstanding effects in anti-tumor, activation of immune response and imaging.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A preparation method of an iron death inducer, characterized in that, it comprises the following steps: Compound A is mixed with formylthiopheneboronic acid under the catalysis of an inorganic base and a palladium catalyst, then a mixed solution of 1,4-dioxane and water is added, and the mixture is heated and stirred at 78 - 82 °C for 20 - 24 h, filtered, extracted, and purified to obtain Compound B; Under gas protection, Compound B is mixed with a rhodanine derivative under the catalysis of an acid solution, heated and stirred at 110 - 120 °C for 8 - 10 h, cooled, quenched with water, the organic phase is washed with saturated sodium bicarbonate solution, dried, and purified to obtain the iron death inducer; The structural formula of Compound A is The structural formula of the rhodanine derivative is The structural formula of the ferroptosis inducer is wherein R1 is MeO and R2 is CH 2 COOH.

2. The preparation method of an iron death inducer according to claim 1, characterized in that, the molar ratio of Compound A, formylthiopheneboronic acid, inorganic base and palladium catalyst is 1:(1.8 - 2):(3.7 - 4):(7.4 - 8).

3. The preparation method of an iron death inducer according to claim 1, characterized in that, in the mixed solution of 1,4-dioxane and water, the volume ratio of 1,4-dioxane to water is (1 - 1.2):

1.

4. The preparation method of an iron death inducer according to claim 1, characterized in that, the molar ratio of Compound B and rhodanine derivative is 1:(1.5 - 1.8).

5. The preparation method of an iron death inducer according to claim 1, characterized in that, the purification method includes silica gel column chromatography, and the eluent is petroleum ether and dichloromethane, and the volume ratio of petroleum ether / dichloromethane is (1 / 10 - 1 / 5).

6. The preparation method of an iron death inducer according to claim 1, characterized in that, The palladium catalyst includes Pd(PPh 3 ) 4 .

7. The preparation method of an iron death inducer according to claim 1, characterized in that, the gas in the gas protection includes an inert gas.

8. The preparation method of an iron death inducer according to claim 1, characterized in that, the inorganic base is one of potassium carbonate, potassium hydroxide or sodium carbonate.

9. Application of the preparation method of an iron death inducer according to any one of claims 1 - 8 in inhibiting tumors and activating immune responses.

10. The application of the preparation method of an iron death inducer according to claim 9, characterized in that, the tumor is a breast tumor.

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