Application of small molecular compound Necrathione in preparation of antitumor drugs

Abstract

The invention relates to an application of a small molecule compound Necrathione in preparation of antitumor drugs, and belongs to the field of biological medicines, the small molecule compound is a TXNRD1 inhibitor, has an inhibition effect in occurrence and development of non-small cell lung cancer, is applied to preparation of drugs for treating solid tumors, and has a molecular formula of C18H14N2O6. The Necrathione is used as the thioredoxin reductase inhibitor and is targeted to the thioredoxin reductase 1 in a thioredoxin system of a tumor cell. The anti-tumor drug is used for causing oxidative stress of non-small cell lung cancer, inhibiting proliferation and clone formation of non-small cell lung cancer cells and regulating and controlling a non-classical mechanism of necrotic apoptosis of the non-small cell lung cancer. The Necrathione disclosed by the invention can be used for inhibiting proliferation, clone formation and migration of lung cancer cells in a concentration-dependent manner and inducing non-classical necroptosis, and the Necrathione reaches the antitumor biological activity at a relatively low concentration.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a small molecule compound, Necrathionone, in the preparation of antitumor drugs. Currently, no literature or patent discloses or suggests that the compound Necrathionone possesses activity against non-small cell lung cancer. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is the most prevalent histological subtype of lung cancer, accounting for approximately 85% of all lung cancer cases. Its global incidence and mortality rates consistently rank first among malignant tumors. According to GLOBOCAN 2022 data, it is estimated that over 2.4 million new lung cancer cases and approximately 1.8 million deaths occurred globally that year, with NSCLC being a major contributing factor to this heavy disease burden. In China, lung cancer also has the highest number of new cases and deaths, and NSCLC constitutes a major challenge in clinical practice.

[0003] Pathologically, NSCLC mainly includes subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. With the development of molecular biology, NSCLC has been confirmed as a highly heterogeneous disease, and the discovery of multiple driver gene mutations (such as EGFR, ALK, ROS1, KRAS, MET, etc.) is the basis for its precision treatment.

[0004] While targeted therapies against specific driver genes and the use of immune checkpoint inhibitors have significantly improved the prognosis of patients with advanced NSCLC, current clinical treatment still faces significant challenges. These challenges include, but are not limited to, the emergence of primary or secondary drug resistance, limited response to immunotherapy in some patient groups, and tumor recurrence and metastasis. Therefore, there remains a significant unmet clinical need to develop novel anti-tumor drugs with novel mechanisms of action, high efficacy, and low toxicity for NSCLC, especially for patients who are insensitive to existing therapies or have developed resistance. Summary of the Invention

[0005] This invention provides a small molecule compound, Necrathionone, and its application in the preparation of antitumor drugs. The purpose of this invention is to provide new pharmaceutical uses for the compound Necrathionone. In-depth experiments have revealed that the compound Necrathionone exhibits significant inhibitory activity against non-small cell lung cancer cells. Therefore, this invention claims protection for the use of the compound Necrathionone in the preparation of drugs for treating non-small cell lung cancer. To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of a small molecule compound, Necrathionone, in the preparation of antitumor drugs. The chemical name of Necrathionone is 3,4-bis(4-methoxybenzoyl)-1,2,5-oxadiazole 2-oxide, and its molecular formula is C2. 18 H 14 N2O6, with a molecular weight of 354.31, is used in the preparation of drugs for treating solid tumors. The C... 18 H 14 The structural formula of N2O6 is shown in formula (I): (I) The solid tumor is non-small cell lung cancer.

[0006] The application involves Necrathionone as a thioredoxin reductase inhibitor targeting thioredoxin reductase 1 (TXNRD1) in the thioredoxin system of tumor cells.

[0007] The aforementioned antitumor drug is used to induce oxidative stress and ROS accumulation in non-small cell lung cancer. The aforementioned antitumor drug is used to inhibit the proliferation and colony formation of non-small cell lung cancer cells; The antitumor drug described herein uses a non-classical mechanism to regulate necrotizing apoptosis in non-small cell lung cancer.

[0008] The present invention also provides the application of the small molecule compound Necrathionone in the preparation of antitumor drugs.

[0009] The beneficial effects of this invention are as follows: This invention, through the study of the pharmacological effects of the small molecule compound Necrathionone, has discovered that Necrathionone possesses significant antitumor activity. Specifically, this small molecule compound can be used to control or treat non-small cell lung cancer. Further research shows that Necrathionone can effectively inhibit the activity of thioredoxin reductase 1 (TXNRD1), leading to increased intracellular oxidative stress levels and significant accumulation of reactive oxygen species (ROS), thereby irreversibly regulating and activating the necrosis-apoptosis signaling pathway in non-small cell lung cancer cells. Based on this mechanism, Necrathionone can significantly inhibit the proliferation and colony formation ability of non-small cell lung cancer cells, thereby exerting an antitumor effect. Attached Figure Description

[0010] Figure 1 The graph shows the inhibitory effect of Necrathionone on the in vitro recombinant TXNRD1 enzyme activity. Figure 2The graph shows the inhibitory effect of Necrathionone on the intracellular TXNRD1 enzyme activity of H1299 cells; Figure 3 The results of the DCFH-DA fluorescent probe staining experiment in Example 3; Figure 3 (a) in the figure is the bright field result diagram of the control group; Figure 3 (b) in the figure is the bright field result of the experimental group treated with 5 μM ecrathionone; Figure 3 (c) in the figure is the bright field result of the experimental group treated with 10 μM ecrathionone; Figure 3 (d) in the figure represents the dark field results for the control group; Figure 3 (e) in the figure is the dark field result of the experimental group treated with 5 μM N ecrathionone; Figure 3 (f) in the figure represents the dark-field results of the experimental group treated with 10 μM N ecrathionone; Figure 4 The results are from the MTT assay of the Necrathionone compound in Example 4; Figure 5 The results of the cloning test of the Necrathionone compound in Example 5; Figure 5 (a) in the figure shows the control group results for A549 cells; Figure 5 (b) in the figure shows the results of the A549 cell experimental group treated with 1 μM N ecrathionone; Figure 5 (c) in the figure shows the results of the A549 cell experimental group treated with 5 μM ecrathionone; Figure 5 (d) in the figure represents the control group results for H1299 cells; Figure 5 (e) in the figure represents the results of the H1299 cell experimental group treated with 1 μM N ecrathionone; Figure 5 (f) in the figure shows the results of the H1299 cell experimental group treated with 5 μM N ecrathionone; Figure 6 The results are from the lactate dehydrogenase release assay of the Necrathionone compound in Example 6. Detailed Implementation

[0011] The present invention will be further described below with reference to specific implementation examples.

[0012] The compound Necrathionone used in this invention is a commercially available product, purchased from Bidex Pharmaceuticals, catalog number BD628778, CAS number 40499-78-3. The following experiments will be used to demonstrate the technical effects claimed in this invention.

[0013] Example 1: Inhibition of recombinant TXNRD1 enzyme activity and cellular TXNRD1 enzyme activity assay This invention analyzes the inhibitory effect of Necrathionone on TXNRD1 using recombinant enzyme activity inhibition assays and cellular enzyme activity inhibition assays. After pre-incubating recombinant TXNRD1 protein in vitro for 10 min, the protein was treated with gradient concentrations of Necrathionone (0, 1, 2, 3, 5, 10 μM) for 1 h. TXNRD1 activity was then measured using the endpoint method TXN1-insulin conjugation assay. In the TXN1 conjugation assay, the final reaction mixture contained 50 nM wild-type TXNRD1, 160 μM insulin, 200 μM NADPH, and 10 μM TXN1, dissolved in 50 mM TE buffer (pH 7.5). The enzyme activity for TXN reduction was calculated by monitoring the oxidation rate of NADPH at 340 nm, and the half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism. The experimental results are as follows: Figure 1 As shown, the results indicate that Necrathionone has a significant dose-dependent inhibitory effect on recombinant TXNRD1.

[0014] To further investigate the inhibitory effect of Necrathionone on TXNRD1 under physiological conditions, an enzyme activity inhibition assay of TXNRD1 in cells was performed using Necrathionone. Similarly, the TXNRD1 activity in cells was measured using the endpoint method of TXN1-insulin conjugation. In short, H1299 cells in logarithmic growth phase were prepared into a cell suspension using standard digestion and passage methods, and the cells were evenly seeded into six-well plates at a rate of 20 weeks / well. After cell attachment, cells were treated with gradient concentrations of Necrathionone (0, 1, 2, 3, 5, 10 μM) for 4 hours. After drug treatment, cell lysis buffer was added to lyse the cells. After thorough lysis, approximately 5 μg of the cell lysis buffer was added to a premix containing 15 μM TXN1, 300 μM insulin, 660 μM NADPH, and 3 mM EDTA, dissolved in 200 mM Hepes buffer (pH 7.5). A reaction mixture without TXN1 was used as a blank control. Samples were incubated at 37 °C for 30 minutes. Subsequently, a 6 M guanidine hydrochloride solution containing 1 mM MDTNB and 10 mM EDTA was added to each well, and the endpoint absorbance at 412 nm was measured to calculate TXN1 reductase activity. Thioredoxin reductase activities in cell lysates were normalized to protein concentration to ensure accurate comparisons. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism. Experimental results are shown below. Figure 2As shown, the results indicate that Necrathionone has a significant dose-dependent inhibitory effect on intracellular TXNRD1.

[0015] Example 2: Necrathionone induces oxidative stress and reactive oxygen species (ROS) accumulation in tumor cells. DCFH-DA itself is an uncharged, non-fluorescent probe that can freely cross the cell membrane and enter the cell. Inside the cell, esterases hydrolyze it to form DCFH. DCFH still cannot freely permeate the cell membrane and is thus retained inside the cell. When reactive oxygen species (such as O2) are present in the cell... - When exposed to ROS such as H2O2, ·OH, etc., DCFH is oxidized by these ROS, generating DCF, a product with strong green fluorescence. The overall level of intracellular ROS can be indirectly reflected by detecting the fluorescence intensity of DCF (excitation light approximately 485 nm, emission light approximately 525 nm). This invention determines the degree of intracellular ROS accumulation by detecting the fluorescence intensity of ROS. H1299 cells in logarithmic growth phase were prepared into a cell suspension using conventional digestion and passage methods. Cells were evenly seeded into six-well plates at a rate of 20 mg / well. After cell adhesion, the cells were treated with gradient concentrations of Necrathionone (0, 5, 10 μM) for 4 hours. The six-well plates were then removed from the incubator, observed under a fluorescence microscope, and photographed. The experimental results are as follows: Figure 3 As shown, the results indicate that Necrathionone significantly and in a concentration-dependent manner causes ROS accumulation.

[0016] Example 3: MTT test results of the Necrathionone compound; This invention analyzes the effect of Necrathionone on the proliferation of non-small cell lung cancer (NSCLC) cells using MTT assays and cloning experiments. Two NSCLC cell lines, H1299 and A549, were selected. Logarithmic-phase cells were collected and seeded at 3000 cells / well in 96-well plates. After the cells adhered and formed a monolayer covering the bottom of the wells, a gradient concentration of Necrathionone (0, 1, 2, 3, 5, 10 μM) was added, and the treatment time was 24 h. The MTT solution was diluted with serum-free basal medium to a final concentration of 0.5 mg / mL. The drug-containing medium was aspirated, and 100 μL of the diluted medium containing 0.5 mg / mL MTT was added to each well. The plates were incubated at 37°C for 4 h. After discarding the supernatant, 150 μL of DMSO was added per well to dissolve the crystals. The absorbance was measured at 570 nm and 630 nm using a microplate reader, and the absorbance difference (OD570-OD630) was calculated. Using the average absorbance of the DMSO-treated group as 100%, data from each drug group were compared to obtain the relative cell viability and assess the cytotoxic effect of the drugs. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism. Experimental results are as follows: Figure 6 As shown, the results indicate that Necrathionone can dose-dependently inhibit the proliferation of non-small cell lung cancer cells after 24 hours of treatment.

[0017] Example 4: Cloning test results of Necrathionone compound Tumor cells can proliferate indefinitely to form cell colonies. Through clonogenic assays, this invention discovered that Necrathionone can inhibit the clonogenic formation of lung cancer cells. H1299 cells in logarithmic growth phase were prepared into a cell suspension using standard digestion and passage methods, and then evenly seeded into six-well plates at a density of 1000 cells / well. The culture dishes were placed in a 5% CO2 incubator overnight. The next day, a low concentration of Necrathionone was added for 6 hours. The supernatant was then discarded, and fresh culture medium was added for continued culturing. The culture medium was changed every 3 days, and culture was terminated after one week. The culture medium was discarded, and each well was washed twice with 1 mL of PBS, followed by fixation with 1 mL of 4% paraformaldehyde for 1 hour. Afterward, the cells were washed twice with 1 mL of PBS, and then stained with crystal violet solution for 10 minutes. Finally, the cells were washed several times with PBS until the crystal violet dye was removed, and then observed and photographed. The results showed that Necrathionone could limit the clonogenic formation of non-small cell lung cancer cells in a concentration-dependent manner.

[0018] Example 5: Induction of lactate dehydrogenase release from tumor cells This invention uses lactate dehydrogenase release levels as an indicator of necrosis and apoptosis. H1299 cells in logarithmic growth phase were prepared into a cell suspension using standard digestion and passage methods. H1299 cells were evenly seeded into 96-well plates at a density of 3000 cells per well and incubated overnight at 37°C to allow complete cell adhesion before subsequent drug treatment. The original culture medium was discarded, and the cells were gently washed once with PBS and replaced with serum-free medium. The wells were divided into the following groups: cell-free culture medium wells, untreated control cell wells, untreated cell wells used for subsequent lysis, and wells treated with Necrathiononee, and clearly labeled. Gradual concentrations of Necrathiononee were used for treatment for 3h, 6h, and 9h, while an equal volume of DMSO was added to the control wells. After the treatment time was reached, the 96-well plates were removed, and LDH release reagent was added to the "maximum enzyme activity control wells," mixed thoroughly, and incubated for another hour. After reaching the processing endpoint, centrifuge at 400×g for 5 minutes using a plate centrifuge. Transfer 120 μL of the supernatant from each well to the corresponding well of a new 96-well plate. Add 60 μL of LDH detection working solution to each well, mix well, wrap with aluminum foil at room temperature, and incubate slowly on a shaker in the dark for 30 minutes. Measure the absorbance at 490 nm using a microplate reader, with a reference wavelength of 600 nm for dual-wavelength calibration. Subtract the absorbance values ​​from the background blank control wells to calculate the LDH release rate. Experimental results are as follows: Figure 4 As shown, the results indicate that Necrathiononee can induce the release of lactate dehydrogenase from tumor cells in a concentration- and time-dependent manner.

[0019] Example 6: Necrathionone treatment of tumor cells followed by Nec-1 salvage assay This invention utilizes Nec-1, a necrosis-rescue agent, to rescue Necrathionone-induced cell death as an indicator of necrosis-induced apoptosis. H1299 and A549 cells in logarithmic growth phase were prepared into cell suspensions using standard digestion and passage methods. Cells were evenly seeded at a density of 3000 cells per well in 96-well plates and incubated overnight at 37°C to ensure complete cell adhesion before subsequent drug treatment. While treating tumor cells with gradient concentrations of Necrathionone, 10 μM of the necrosis-induced apoptosis inhibitor Nec-1 was added for rescue. Cell viability was assessed using an MTT assay 24 hours after drug treatment. Experimental results are as follows: Figure 5 As shown, the results indicate that the cytotoxicity of Necrathionone on tumor cells can be significantly reversed by Nec-1.

[0020] The above specific embodiments further illustrate the purpose, technical solution and beneficial effects of this application. It should be understood that the above are only specific embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made on the basis of the technical solution of this application should be included within the scope of protection of this application.

Claims

1. The use of a small molecule compound, Necrathionone, in the preparation of an antitumor drug, wherein the chemical name of Necrathionone is 3,4-bis(4-methoxybenzoyl)-1,2,5-oxadiazole 2-oxide, and the molecular formula is C2. 18 H 14 N₂O₆, with a molecular weight of 354.31, is characterized by: The small molecule compound Necrathionone was used in the preparation of drugs for treating solid tumors.

2. The application of the small molecule compound Necrathionone according to claim 1 in the preparation of antitumor drugs, characterized in that, The solid tumor is non-small cell lung cancer.

3. The application of the small molecule compound Necrathionone according to claim 1 in the preparation of antitumor drugs, characterized in that, The application involves Necrathionone as a thioredoxin reductase inhibitor targeting thioredoxin reductase 1 in the thioredoxin system of tumor cells.

4. The application of the small molecule compound Necrathionone according to claim 1 in the preparation of antitumor drugs, characterized in that, The aforementioned antitumor drug is used to induce oxidative stress and ROS accumulation in non-small cell lung cancer.

5. The application of the small molecule compound Necrathionone according to claim 1 in the preparation of antitumor drugs, characterized in that, The aforementioned antitumor drug is used to inhibit the proliferation and clonal formation of non-small cell lung cancer cells.

6. The application of the small molecule compound Necrathionone according to claim 1 in the preparation of antitumor drugs, characterized in that, The antitumor drug described herein uses a non-classical mechanism to regulate necrotizing apoptosis in non-small cell lung cancer.

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