Compound capable of inducing cell ferroptosis as well as preparation method and application thereof

Abstract

The invention discloses a compound capable of inducing cell ferroptosis and a preparation method and application thereof. The compound not only can promote Fe < 2 + > accumulation to trigger iron overload, but also can inhibit GPX4 protein expression to cause accumulation of peroxides in cells and synergistically enhance the ferroptosis effect. The compound has remarkable ferroptosis induction activity on various abnormal hyperplasia cells, shows broad-spectrum anti-tumor potential and has an important clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a compound capable of inducing cell ferroptosis, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant tumors are one of the major diseases that currently pose a serious threat to human life and health. Currently, chemotherapy drugs widely used in clinical practice mainly exert their anti-tumor effects by inducing programmed cell death such as apoptosis, pyroptosis, or ferroptosis. However, due to the high heterogeneity of tumors themselves and the ability of tumor cells to continuously evolve and enhance their adaptability to the external environment under the action of drugs, they eventually develop resistance to multiple chemotherapy drugs, significantly limiting the therapeutic efficacy of traditional chemotherapy regimens.

[0003] In recent years, ferroptosis, a non-apoptotic form of cell death distinct from the classical apoptosis mechanism, has received widespread attention. The main characteristics of ferroptosis include the abnormal accumulation of free iron ions in cells, a significant increase in lipid peroxidation levels, and the massive generation of reactive oxygen species (ROS), which ultimately lead to structural and functional disorders in cells and death. Ferroptosis plays a key role in the occurrence, development, and treatment of tumors. In particular, tumor cells generally have enhanced metabolic activity and disordered iron metabolism, making them significantly more sensitive to ferroptosis than normal cells. This also provides an important therapeutic target and theoretical basis for targeting ferroptosis.

[0004] Currently, a variety of small-molecule ferroptosis inducers have been developed for research purposes. Erastin is a well-studied ferroptosis activator that triggers cellular ferroptosis by inhibiting the Xc-system. However, erastin has significant defects in stability, water solubility, and pharmacokinetic properties, which limit its feasibility for in vivo application. In addition, most existing ferroptosis inducers target only a single regulatory pathway, such as iron metabolism, the Xc-system, glutathione peroxidase 4 (GPX4), or FSP1. These single targets and limited induction strength can easily lead to drug resistance in tumor cells, thereby reducing the therapeutic effect.

[0005] In contrast, novel drugs that can regulate ferroptosis networks at multiple targets have stronger induction abilities and lower risk of drug resistance, thus offering advantages in anti-tumor therapy. Therefore, developing novel ferroptosis-inducing compounds with clear targeting, enhanced stability, and the ability to act on multiple ferroptosis regulatory pathways can not only effectively overcome current treatment bottlenecks but also provide a novel strategy for improving the treatment of malignant tumors, which is of great scientific significance. Summary of the Invention

[0006] In view of the defects in the prior art, the present invention proposes a compound that can induce cell ferroptosis and its preparation method and application. The present invention provides a novel compound that can induce cell ferroptosis and can not only target iron metabolism, but also lead to Fe 2+ The accumulation of iron and subsequent iron overload can also target the GPX4 system, inhibiting GPX4 levels leading to the accumulation of intracellular peroxides and exacerbating ferroptosis.

[0007] The present invention provides a compound that can induce cell ferroptosis, wherein the compound is compound 4, and the structural formula of the compound 4 is as follows:

[0008]

[0009] As used in the description of the embodiments of the present invention and the appended claims, the singular forms "a," "an," "the," and "its" refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, reference to a compound includes one or more than one compound.

[0010] In the present invention, unless otherwise specified, "compound" refers to a chemical substance composed of two or more atoms of different species bound by covalent bonds, which may be of natural origin or synthetically synthesized. Such compounds include neutral molecules, their acceptable salts, esters, prodrugs, crystalline forms (such as amorphous or polymorphic forms), solvates, non-covalent complexes, and optical isomers (including enantiomers and diastereomers), or racemates. Unless otherwise specified, the term "compound" herein implicitly includes all pharmaceutically acceptable forms thereof.

[0011] The present invention also provides a method for preparing the compound, comprising the following steps:

[0012] (1) Compound 1 and Compound 2 were heated to react with sodium acetate in acetic anhydride, and the precipitate was filtered and washed to obtain Compound 3;

[0013] (2) reacting the compound 3 described in step (1) with acetylacetone and triethylamine in dichloromethane under reflux under N2 to obtain a reaction product;

[0014] (3) reacting the reaction product of step (2) with methanol and a saturated NH PF aqueous solution, and purifying to obtain the compound 4;

[0015] Wherein, the structural formula of the compound 1 is as follows:

[0016]

[0017] The structural formula of the compound 2 is shown below:

[0018]

[0019] The structural formula of the compound 3 is shown below:

[0020]

[0021] The present invention also provides use of the compound in preparing a drug for inducing cell ferroptosis.

[0022] In some embodiments, the drug contains the compound as the only active ingredient or one of the active ingredients.

[0023] In the present invention, "active ingredient" refers to an ingredient in a pharmaceutical formulation that has a therapeutic, preventive, diagnostic, or modulatory effect on a physiological function, particularly a chemical substance with biological functions such as inducing ferroptosis, anti-tumor, and anti-proliferative properties. Active ingredients include, but are not limited to, Compound 4 described herein, its pharmaceutically acceptable salts, esters, precursors, optical isomers, racemates, crystalline forms, and solvates. Unless otherwise indicated, the term "active ingredient" shall include all pharmaceutically acceptable forms thereof.

[0024] In some embodiments, the content of Compound 4 in the drug is 0.1 wt% to 99 wt%.

[0025] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient and / or carrier.

[0026] In the present invention, "pharmaceutically acceptable excipients and / or carriers" refer to substances that are used together with the active ingredient in the preparation of pharmaceutical preparations and are non-toxic, non-irritating to humans or animals at the administered dose and do not affect the efficacy and stability of the active ingredient. The excipients and carriers may include, but are not limited to, diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity-increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that certain pharmaceutically acceptable excipients can be used with more than one function and with alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

[0027] In the present invention, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items.

[0028] In some embodiments, the drug can increase the level of malondialdehyde and ROS in cells, while downregulating the expression level of glutathione peroxidase 4.

[0029] In this context, "malondialdehyde level" refers to the intracellular content of malondialdehyde, a lipid peroxidation product, and is an indicator of the extent of lipid peroxidation and cellular oxidative stress. Elevated malondialdehyde levels typically indicate oxidative damage to cell membrane lipids, consistent with the typical biochemical characteristics of ferroptosis.

[0030] In the present invention, "ROS level" refers to the overall concentration or accumulation level of reactive oxygen species in cells. Reactive oxygen species are important effectors in the process of ferroptosis, and their accumulation can lead to intracellular oxidative damage, lipid peroxidation, and cell death.

[0031] In the present invention, "GPX4" refers to a key antioxidant enzyme that reduces lipid peroxides to non-toxic alcohols, preventing oxidative damage to cellular lipid membranes. Decreased expression or inhibition of GPX4 activity is one of the key molecular events that initiate ferroptosis. Therefore, one of the mechanisms of ferroptosis induction described in the present invention is inhibition of GPX4 expression or activity.

[0032] In some embodiments, the cell is a dysplastic cell.

[0033] In the present invention, "abnormally proliferative cells" refer to a cell population formed by the continuous and abnormal proliferation of cells under the influence of pathogenic factors such as genetic factors, inflammatory stimulation, environmental carcinogens, abnormal activation of signal pathways, etc., which usually show characteristics such as cell cycle loss, suppressed apoptosis or differentiation disorders, and are commonly found in various malignant tumors or preneoplastic lesions.

[0034] In some embodiments, the abnormally proliferative cells include any one of breast cancer cells, colon cancer cells, vascular smooth muscle cells, esophageal cancer cells, renal cancer cells, melanoma cells, pancreatic cancer cells, glioma cells, lymphoma cells and non-small cell lung cancer cells.

[0035] The present invention also provides a ferroptosis inducer, which comprises the compound according to claim 1.

[0036] In the present invention, "ferroptosis inducers" refer to substances that can induce or enhance cellular ferroptosis by regulating iron homeostasis, promoting lipid peroxidation, increasing ROS levels, or inhibiting antioxidant defense systems. They are generally used to treat tumors resistant to traditional chemotherapy. The ferroptosis inducers of the present invention include compound 4 as described in claim 1, and pharmaceutically acceptable salts, esters, optical isomers, precursors, or solvates thereof.

[0037] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0038] 1. The compound IR797-acac provided by the present invention is a novel metal complex that can significantly induce typical ferroptosis in tumor cells, including increased malondialdehyde (MDA) levels, accumulation of reactive oxygen species (ROS), and downregulation of glutathione peroxidase 4 (GPX4) expression, thereby effectively inducing cancer cell death; the compound shows good ferroptosis-inducing activity against a variety of abnormally proliferative cells (such as breast cancer, colorectal cancer, etc.) and has broad-spectrum anti-tumor potential.

[0039] 2. Compared with traditional anti-tumor drugs that work through cell apoptosis, necrosis or autophagy mechanisms, the compounds of the present invention achieve anti-tumor effects through the ferroptosis mechanism, opening up a new strategy for tumor treatment. They are particularly suitable for the treatment of malignant tumors that are resistant to conventional chemotherapy drugs and have significant clinical application prospects.

[0040] 3. The synthetic route of the compound IR797-acac of the present invention is clear, the steps are simple, the reaction conditions are mild, the raw materials used are easily available, and it has good scalability, which facilitates large-scale preparation and is suitable for further pharmaceutical research and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 is the structural formula of compound 1 of the present invention;

[0043] Figure 2 is the structural formula of compound 2 of the present invention;

[0044] Figure 3 is the structural formula of compound 3 of the present invention;

[0045] Figure 4 is the structural formula of compound 4 of the present invention;

[0046] Figure 5 is the H NMR spectrum of compound 4 of the present invention;

[0047] Figure 6 is the C NMR spectrum of compound 4 of the present invention;

[0048] Figure 7 This is the CCK8 result of Example 2IR797-acac of the present invention;

[0049] Figure 8This is a confocal imaging image of cells after IR797-acac treatment in Example 3 of the present invention;

[0050] Figure 9 This is the statistical result of MDA content in cells after IR797-acac treatment in Example 4 of the present invention;

[0051] Figure 10 This is the statistical result of intracellular ROS level after IR797-acac treatment in Example 5 of the present invention;

[0052] Figure 11 This is the intracellular GPX4 level after IR797-acac treatment in Example 6 of the present invention. DETAILED DESCRIPTION

[0053] To help those skilled in the art better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0054] The terminology used in this description of the present invention is intended only to describe specific embodiments and is not intended to limit the invention. The nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and biology described herein are well known and commonly used in the art. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available unless otherwise specified.

[0056] Example 1 Synthesis of IR797-acac

[0057] (1) Synthesis process:

[0058] A mixture of compound 1 (7.3 g, 0.023 mol), compound 1 (2.0 g, 0.011 mol) and sodium acetate (1.9 g, 0.023 mol) was added to a round-bottom flask containing 30 mL of acetic anhydride. The mixture was then heated to 130°C for 1 h. The green precipitate was filtered and washed with ether (3×50 mL). 4.4 g of a yellow-green solid was obtained, which was compound 3 (IR797, yield 55.3%). The structural formulas of compounds 1 to 3 are as follows: Figures 1 to 3 As shown, the synthetic route is as follows:

[0059]

[0060] A mixture of compound 3 (1.0 g, 1.6 mmol), 2 mL of acetylacetone, and 8 mL of triethylamine (Et3N) was added to a round-bottomed culture flask containing 40 mL of DCM. The solution was refluxed under N2 for 4 h. The solvent was then removed, and the solid was dissolved in 30 mL of methanol and 1 mL of saturated aqueous NH4PF6 for 1 h. The solvent was removed by rotary evaporation in vacuo. The crude product was purified on silica gel using ethyl acetate:acetonitrile (50:1 to 10:1) as the eluent to obtain a green powder (0.8 g, 61.5%) of pure IR797-acac, whose chemical formula is C 38 H 45 F6N2O2P, the structural formula is Figure 4 As shown, the synthetic route is as follows:

[0061]

[0062] (2) Structural characterization:

[0063] The purified IR797-acac was characterized. Figure 5 As shown, the NMR carbon spectrum is as follows Figure 6 As shown, the results prove that the synthesized IR797-acac has a clear structure and good purity, and can serve as a reliable basis for subsequent functional studies.

[0064] Example 2 Evaluation of Cellular Anti-tumor Effect

[0065] Experimental cells: breast cancer cells (4T1 cells).

[0066] Reagents: IR797-acac solution (2 mM), CCK8 reagent.

[0067] Procedure: 4T1 cells were plated in a 96-well plate (7000 cells / well) and 100 μL of DMEM complete medium was added. After 24 hours of culture, medium containing different concentrations of IR797-acac was added to achieve final concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 μM IR797-acac. Culture was continued for 24 hours. The IR797-acac-containing medium was removed, and the cells were washed three times with PBS. 100 μL of CCK8 working solution prepared in serum-free medium was added to each well. After 30 minutes of culture in a 37°C incubator, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0068] Results: As Figure 7As shown, IR797 had no significant effect on 4T1 cell proliferation within a certain concentration range, while IR797-acac significantly inhibited the proliferation of 4T1 breast cancer cells within a certain concentration range, demonstrating good dose-dependent anti-tumor activity. As the concentration of IR797-acac increased, cell survival gradually decreased, suggesting that IR797-acac has a significant killing effect on breast cancer cells.

[0069] Example 3 Effect of IR797-acac on intracellular Fe 2+ The impact of level

[0070] Experimental cells: 4T1 cells.

[0071] Reagents: IR797-acac solution (2 mM stock solution), ferrous ion probe.

[0072] Procedure: 4T1 cells were plated in 8-well chambers (10,000 cells / well) and 200 μL of DMEM complete medium was added. After culturing for 24 hours, medium containing 2 μM IR797-acac was added and cultured for another 24 hours. The medium containing IR797-acac was removed, and the cells were washed three times with PBS. 200 μL of ferrous ion probe prepared in 2 μM serum-free medium was added to each well. After incubation in a 37°C incubator for 30 minutes, the cells were washed three times with PBS and imaged using a confocal microscope (λex = 560 nm, λem = 570-620 nm).

[0073] Results: As Figure 8 As shown, compared with the untreated group, the red fluorescence signal in the IR797-acac treated group was significantly enhanced, indicating that the intracellular Fe 2+ The levels were significantly increased, suggesting that IR797-acac can effectively induce iron accumulation, providing important mechanistic support for its induction of ferroptosis.

[0074] Example 4 Effect of IR797-acac on intracellular malondialdehyde (MDA) levels

[0075] In this example, the malondialdehyde (MDA) level of the end product of lipid peroxidation was detected by a malondialdehyde (MDA) kit.

[0076] Experimental cells: 4T1 cells.

[0077] Reagents: IR797-acac solution (2 mM), lysis buffer, MDA kit, BCA protein concentration determination kit.

[0078] Procedure: 4T1 cells were plated in a 6-well plate (100,000 cells / well), 2 mL of DMEM complete medium was added, and culture was continued for 24 hours. Then, culture medium containing 2 μM IR797-acac was added and cultured for another 24 hours. The culture medium containing IR797-acac was removed, the cells were washed three times with PBS, and lysed with lysis buffer. After lysis, the supernatant was centrifuged and the sample protein concentration was determined using a BCA protein concentration kit. The MDA content was determined according to the MDA kit instructions. The MDA content per unit protein in the sample was calculated.

[0079] Results: As Figure 9 As shown in the figure, compared with the control group, the MDA level in the IR797-acac-treated group was significantly increased, suggesting that it can effectively induce lipid peroxidation, which is one of the typical signs of ferroptosis.

[0080] Example 5 Effect of IR797-acac on ROS levels in tumor cells

[0081] In this example, the cellular ROS level was detected by flow cytometry.

[0082] Experimental cells: 4T1 cells.

[0083] Reagents: IR797-acac solution (2 mM), ROS probe, trypsin.

[0084] Procedure: 4T1 cells were plated in a 6-well plate (100,000 cells / well) and 2 mL of DMEM complete medium was added. After 24 hours of culture, medium containing 2 μM IR797-acac was added and cultured for another 24 hours. The IR797-acac medium was removed, the cells were washed three times with PBS, ROS probe working solution was added, incubated for 20 minutes, and washed three times with PBS. The cells were digested with trypsin and harvested, and ROS levels were measured using flow cytometry (λex = 480 nm, λem = 500-540 nm).

[0085] Results: As Figure 10 As shown in the figure, the fluorescence intensity of the cells in the IR797-acac-treated group was significantly higher than that in the control group. It can be seen that IR797-acac can significantly increase the ROS level of tumor cells, which is another typical manifestation of inducing ferroptosis.

[0086] Example 6 Effect of IR797-acac on GPX4 protein expression level

[0087] This example verifies whether the compound IR797-acac of the present invention can downregulate the protein expression level of GPX4, a key regulatory factor of ferroptosis, thereby further supporting its mechanism of action of inducing ferroptosis.

[0088] The cellular GPX4 level was detected by Western Blot (WB).

[0089] Reagents: IR797-acac solution (2 mM), lysate, rabbit GPX4 primary antibody, anti-rabbit secondary antibody.

[0090] Procedure: 4T1 cells were plated in a 6-well plate (100,000 cells / well) and incubated with 2 mL of complete DMEM medium for 24 hours. Culture was then added with medium containing 2 μM IR797-acac and continued for another 24 hours. The IR797-acac-containing medium was removed, and the cells were washed three times with PBS. Lysis was performed with lysis buffer. The supernatant was collected and loaded with loading buffer. The sample was boiled, electrophoresed, and transferred to a membrane. Rabbit GPX4 primary antibody (1:2000) was added, and the cells were incubated overnight at 4°C. After washing the membrane, the secondary antibody was added and incubated for 1 hour, followed by development.

[0091] Results: As Figure 11 As shown in the figure, compared with the control group, the expression level of GPX4 protein in the IR797-acac-treated group was significantly downregulated, indicating that IR797-acac can effectively inhibit GPX4 expression, further supporting its mechanism of action of inducing ferroptosis.

[0092] In summary, the present invention systematically evaluated the anti-tumor activity and ferroptosis-inducing effect of compound IR797-acac through a series of cell experiments. First, the CCK-8 cell viability assay was used to verify the dose-dependent killing effect of IR797-acac on 4T1 breast cancer cells. Subsequently, the ferrous ion probe confocal imaging technique was used to demonstrate that IR797-acac significantly promoted the intracellular Fe 2 + levels. Further MDA content assays revealed that the compound significantly increased lipid peroxidation in tumor cells. Flow cytometry confirmed a significant increase in ROS levels in cells treated with IR797-acac. Finally, Western blot analysis revealed a significant downregulation of GPX4 protein expression, suggesting that the compound mediates ferroptosis by inhibiting GPX4. Taken together, these results suggest that IR797-acac can regulate ferroptosis-related markers through multiple pathways, effectively inducing ferroptosis in tumor cells and demonstrating promising anti-tumor potential.

[0093] 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 compound capable of inducing cell ferroptosis, characterized in that: The compound is compound 4, and the structural formula of compound 4 is shown below:

2. The method for preparing the compound according to claim 1, characterized in that The steps include: (1) Compound 1 and Compound 2 were heated to react with sodium acetate in acetic anhydride, and the precipitate was filtered and washed to obtain Compound 3; (2) reacting the compound 3 described in step (1) with acetylacetone and triethylamine in dichloromethane under reflux under N2 to obtain a reaction product; (3) reacting the reaction product of step (2) with methanol and a saturated NH PF aqueous solution, and purifying the resultant to obtain the compound 4; Wherein, the structural formula of the compound 1 is as follows: The structural formula of the compound 2 is shown below: The structural formula of the compound 3 is shown below:

3. Use of compound 4 according to claim 1 in the preparation of a drug for inducing cell ferroptosis.

4. The use according to claim 3, characterized in that The drug contains the compound 4 described in claim 1 as the only active ingredient or one of the active ingredients.

5. The use according to claim 3, characterized in that The content of compound 4 in the medicine is 0.1 wt% to 99 wt%.

6. The use according to claim 3, characterized in that The drug further includes pharmaceutically acceptable excipients and / or carriers.

7. The use according to claim 3, characterized in that The drug can increase the malondialdehyde level and ROS level in cells and simultaneously downregulate the expression level of glutathione peroxidase 4.

8. The use according to any one of claims 3 to 7, characterized in that: The cells are abnormally proliferative cells.

9. The use according to claim 8, characterized in that The abnormally proliferative cells include any one of breast cancer cells, colon cancer cells, vascular smooth muscle cells, esophageal cancer cells, renal cancer cells, melanoma cells, pancreatic cancer cells, glioma cells, lymphoma cells and non-small cell lung cancer cells.

10. A ferroptosis inducing agent, characterized in that The ferroptosis inducer comprises the compound according to claim 1.

Images