Application of cytisine-N-isoflavone derivative in preparation of medicine for resisting non-small cell lung cancer

Abstract

The invention discloses application of a cytisine-N-isoflavone derivative in preparation of a medicine for resisting non-small cell lung cancer, and belongs to the technical field of biological medicine. The derivative is a compound CNI3, and the medicine takes the compound CNI3 or a pharmaceutically acceptable solvate thereof as an active ingredient; the dosage form can be selected from tablets, capsules, granules, dripping pills, suspensions, syrups, enteric preparations, gels, suppositories, ointments, emulsions and injections, and the administration route can be selected from oral administration, injection administration, respiratory tract inhalation administration, local administration, sublingual administration and rectal administration. Experiments in combination with in-vivo cell level and in-vitro animal level prove that the compound CNI3 promotes apoptosis of non-small cell lung cancer, exerts activity of resisting non-small cell lung cancer, has no obvious toxic or side effect on normal tissue cells, is good in safety, and has a good clinical application prospect when being used for preparing the medicine for resisting non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of cytisine-N-isoflavones derivatives in the preparation of drugs for treating non-small cell lung cancer. Background Technology

[0002] Cancer is a major global health problem. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers, is highly metastatic and aggressive, and is often diagnosed at an advanced stage. While its 5-year survival rate has improved, it remains lower than that of other cancers. Despite significant advances in treatment options (including chemotherapy, radiotherapy, immunotherapy, and surgery), the efficacy of existing treatments remains limited due to frequent metastasis and poor prognosis. Therefore, there is an urgent need to develop drugs with novel mechanisms of action to improve the treatment outcomes of NSCLC.

[0003] Natural products play a crucial role in drug therapy. Sophora alopecuroides, an important medicinal herb distributed in Northwest China, contains active ingredients such as alkaloids, flavonoids, and polysaccharides. In recent years, the anti-tumor effects of the active ingredients in Sophora alopecuroides have been effectively verified in various cancer types. Studies have reported the isolation and extraction of Sophora alopecuroides L., a traditional Chinese medicine, and the design and synthesis of a series of cytisine-N-isoflavone derivatives with dual pharmacophore groups of cytisine and isoflavone. Some of these compounds have been found to possess anti-tumor activity. For example, patent document CN109970738A discloses cytisine-N-isoflavone compounds and their application in anti-tumor therapy, indicating that these compounds have certain anti-breast cancer potential, but it does not reveal the application of specific compounds in non-small cell lung cancer, nor does it clarify their mechanism of action. Furthermore, a research paper (Discovery and evaluation of cytisine N-isoflavones as novel EGFR / HER2 dual inhibitors. Bioorganic Chemistry. 2022, 127:105868.) reported that cytisine-N-isoflavones CNI3 and CNI4 can target and inhibit the activity of EGFR and HER2, while also exhibiting cytotoxicity to breast cancer cells. However, this paper did not reveal the specific mechanism by which the compounds exert toxicity on breast cancer cells. That is, it did not clarify whether the specific compounds exert toxicity on breast cancer cells by dual inhibition of EGFR and HER2 or by single-target inhibition of one of the targets, through the loss or gain of dual-target function of EGFR and HER2 or the loss or gain of function of a single target.In addition, the research literature (Molecular Mechanisms of Reversal of Multidrug Resistance in Breast Cancer by Inhibition of P-gp by Cytisine N-Isoflavones Derivatives Explored Through Network Pharmacology, Molecular Docking, and Molecular Dynamics. International Journal of Molecular Sciences. 2025, 26(8):3813.) reported that the cytisine-N-isoflavone compounds CNI1-CNI4 have binding effects on both AKT1 and P-gp and have the activity of reversing multidrug resistance in breast cancer. However, it did not reveal the mechanism by which the specific compounds exert toxicity on breast cancer cells. That is, it did not clarify whether the specific compounds reversed the multidrug resistance of breast cancer cells by dual inhibition of AKT1 and P-gp or single-target inhibition of a single target through binding to the loss or gain of dual target function of AKT1 and P-gp or single-target inhibition of a single target. The above results indicate that the existing technology has not revealed the application and specific mechanism of action of cytisine-N-isoflavone compounds in non-small cell lung cancer.

[0004] Mammalian target of rapamycin (mTOR) is a crucial regulator of cell growth, proliferation, and metabolism. Transduced by the upstream PI3K / AKT signaling pathway, it exhibits overactivation in various malignant tumors, making it an important potential target for cancer therapy. Numerous studies have shown that sustained activation of the mTOR pathway not only promotes tumor cell survival and drug resistance but is also closely related to the development and progression of non-small cell lung cancer (NSCLC), making it a potential therapeutic target for NSCLC. While existing mTOR inhibitors (such as rapamycin and its derivatives) have entered clinical trials, their efficacy in NSCLC is limited, and they suffer from poor pharmacokinetics and significant toxic side effects, hindering their clinical application. Therefore, developing novel inhibitors targeting this pathway is of great significance for lung cancer treatment.

[0005] Although research literature (Cytisine, a Partial Agonist of α4β2 Nicotinic Acetylcholine Receptors, Reduced Unpredictable Chronic Mild Stress-Induced Depression-Like Behaviors. Biomolecules & Therapeutics. 2016, 24(3):291-7.) reported that cytisine, as a partial agonist of α4β2 nicotinic acetylcholine receptors, significantly reversed the decrease in mTOR levels in the hippocampus and amygdala of mice under chronic mild stress and alleviated depressive symptoms, it did not clarify whether cytisine improves depression by targeting and regulating mTOR expression. The literature (Ononin inhibits triple-negative breast cancer lung metastasis by targeting the EGFR-mediated PI3K / Akt / mTOR pathway. ScienceChina-Life Sciences.2024,67(9):1849-1866.) reported that soy isoflavones (Ononin) inhibit lung metastasis of breast cancer by targeting and binding to EGFR, reducing its phosphorylation, and thus inhibiting the PI3K / Akt / mTOR signaling pathway. However, it did not reveal whether Ononin has a direct effect on mTOR, and it did not involve research on non-small cell lung cancer. The literature (Antitumor Effects of Ononin by Modulation of Apoptosis in Non-Small-Cell Lung Cancer through Inhibiting PI3K / Akt / mTOR Pathway. Oxidative Medicine And Cellular Longevity. 2022, 2022: 5122448.) reports that ononin exerts an anti-non-small cell lung cancer effect by increasing cell apoptosis and inhibiting the PI3K / AKT / mTOR signaling pathway, but it does not reveal whether ononin can inhibit the progression of non-small cell lung cancer by targeting and regulating mTOR expression, thereby increasing cell apoptosis.The literature (A preclinical report of acobimetinib-inspired novel anticancer small-molecule scaffold of isoflavones, NSC777213, for targeting PI3K / AKT / mTOR / MEK in multiple cancers. American Journal of Cancer Research. 2021, 11(6): 2590-2617.) reported that a novel isoflavone, NSC777213, exhibited antiproliferative effects in various tumor cells, including non-small cell lung cancer, ovarian cancer, and colon cancer, and targeted the inhibition of mTOR protein expression in human glioma cells U251 and U87MG. However, it did not clarify whether NSC777213 inhibits the proliferation of human glioma cells by inhibiting mTOR through the loss or gain of mTOR target function. Furthermore, this study did not reveal whether the proliferation of NSC777213 against non-small cell lung cancer is directly related to mTOR, i.e., whether NSC777213 can inhibit the progression of non-small cell lung cancer by targeting and inhibiting the expression of mTOR.

[0006] In summary, current research on the cytisine-N-isoflavone compound CNI3 remains limited, particularly regarding its potential to inhibit non-small cell lung cancer (NSCLC) progression through targeted regulation of mTOR, which lacks systematic reports. Therefore, exploring the role and potential mechanisms of compound CNI3 in NSCLC treatment holds significant scientific importance and application prospects. Summary of the Invention

[0007] The purpose of this invention is to provide the application of cytisine-N-isoflavone derivatives in the preparation of drugs for treating non-small cell lung cancer. The cytisine-N-isoflavone derivative is compound CNI3. The invention aims to clarify the efficacy of compound CNI3 in treating non-small cell lung cancer and its mechanism of action in targeting and inhibiting mTOR, thus providing a new drug approach for the treatment of non-small cell lung cancer.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides the use of a cytisine-N-isoflavone derivative in the preparation of a drug for treating non-small cell lung cancer, wherein the cytisine-N-isoflavone derivative is compound CNI3 with the following structural formula:

[0010] Preferably, the non-small cell lung cancer includes human non-small cell lung cancer cell lines A549 and H1299.

[0011] Preferably, the drug uses compound CNI3 or its pharmaceutically acceptable solvate as the active ingredient, with an effective dose of 0.1–100 μM.

[0012] More preferably, the drug uses compound CNI3 or its pharmaceutically acceptable solvate as the active ingredient, with an effective dose of 20–50 μM.

[0013] Preferably, the drug comprises a pharmaceutically acceptable carrier or diluent.

[0014] Preferably, the dosage form of the drug is selected from at least one of the following formulations: tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, gels, suppositories, ointments, emulsions, and injections.

[0015] Preferably, the route of administration of the drug is selected from at least one of oral administration, injection administration, inhalation administration, topical administration, sublingual administration, and rectal administration.

[0016] A second aspect of the present invention provides a pharmaceutical composition for treating non-small cell lung cancer, comprising a compound CNI3 or a pharmaceutically acceptable solvate thereof as an active ingredient, the compound CNI3 having the structural formula described above; or further comprising a pharmaceutically acceptable carrier or diluent.

[0017] Preferably, the effective dose of the compound CNI3 or its pharmaceutically acceptable solvate is 0.1–100 μM.

[0018] A third aspect of the present invention provides the use of a cytisine-N-isoflavone derivative in the preparation of a drug that targets and inhibits mTOR expression, wherein the cytisine-N-isoflavone derivative is compound CNI3, with the following structural formula:

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) Through in vitro cellular studies and in vivo tumor model studies, this invention demonstrates that compound CNI3 targets and binds to mTOR, inhibits mTOR protein expression, and promotes cell apoptosis, thereby exerting an anti-non-small cell lung cancer effect and showing good clinical application prospects.

[0021] (2) The compound CNI3 applied in this invention can effectively inhibit tumor growth in in vivo and in vitro models, while having no obvious toxic side effects on normal tissue cells, and has good safety.

[0022] (3) This invention provides new target strategies and candidate compounds for drug development in non-small cell lung cancer, which have important clinical translation potential. It not only provides new strategies for the treatment of non-small cell lung cancer, but also provides new directions for the development of related drugs. Attached Figure Description

[0023] Figure 1 The effect of compound CNI3 in Example 1 on the viability of non-small cell lung cancer cell lines A549 and H1299 is shown in Figure 1. A represents the viability of A549 cells, and B represents the viability of H1299 cells. Data are expressed as mean ± standard error (Mean ± SEM). Each group had 6 replicates (n = 6). * and ** indicate significant differences (P < 0.05) and highly significant differences (P < 0.01) compared to the control group, respectively.

[0024] Figure 2 The effect of compound CNI3 in Example 1 on the intracellular ROS content of non-small cell lung cancer cell lines A549 and H1299 is shown below. A: Results of ROS fluorescence intensity in A549 and H1299 cells; B: Statistical results of ROS fluorescence intensity in A549 cells; C: Statistical results of ROS fluorescence intensity in H1299 cells. Data are expressed as mean ± standard error (Mean ± SEM). Each group had 3 replicates (n = 3). ** indicates a highly significant difference compared to the control group (P < 0.01).

[0025] Figure 3 The effect of compound CNI3 in Example 1 on the survival rate of the non-small cell lung cancer cell line A549 is shown in Figure 1. A represents the fluorescence intensity of live and dead cells in A549 cells; B represents the statistical results of A549 cell survival rate. Data are expressed as mean ± standard error (Mean ± SEM). Each group had 3 replicates (n = 3). ** indicates a highly significant difference compared to the control group (P < 0.01).

[0026] Figure 4 The effect of compound CNI3 in Example 1 on the survival rate of the non-small cell lung cancer cell line H1299 is shown in Figure 1. A: Results of fluorescence intensity of live and dead cells in H1299 cells; B: Statistical results of H1299 cell survival rate. Data are expressed as mean ± standard error (Mean ± SEM). Each group had 3 replicates (n = 3). ** indicates a highly significant difference compared to the control group (P < 0.01).

[0027] Figure 5The effect of compound CNI3 in Example 1 on the apoptosis rate of non-small cell lung cancer cell lines A549 and H1299 is shown below. A: Distribution of apoptotic cells in A549 and H1299 cells; B: Statistical results of apoptosis rate in A549 cells; C: Statistical results of apoptosis rate in H1299 cells. Data are expressed as mean ± standard error (Mean ± SEM). Each group had 3 replicates (n = 3). ** indicates a highly significant difference compared to the control group (P < 0.01).

[0028] Figure 6 The effects of compound CNI3 in Example 1 on the expression of apoptosis markers Bax, Caspase 3, and Bcl-2 proteins in the non-small cell lung cancer cell line A549 are shown in Figure 1. A: Results of apoptosis marker protein expression in A549 cells; B: Statistical results of apoptosis marker protein expression in A549 cells. Data are presented as mean ± standard error (Mean ± SEM). The number of replicates for each group was 3 (n = 3). * and ** indicate significant differences (P < 0.05) and highly significant differences (P < 0.01) compared with the control group, respectively.

[0029] Figure 7 The effects of compound CNI3 in Example 1 on the expression of apoptosis markers Bax, Caspase 3, and Bcl-2 proteins in the non-small cell lung cancer cell line H1299 are shown in Figure 1. A: Results of apoptosis marker protein expression in H1299 cells; B: Statistical results of apoptosis marker protein expression in H1299 cells. Data are presented as mean ± standard error (Mean ± SEM). The number of replicates for each group was 3 (n = 3). * indicates a significant difference compared with the control group (P < 0.05).

[0030] Figure 8 The effect of compound CNI3 in Example 2 on the size and volume of mouse tumors; A: Appearance of mouse tumors after 2 weeks of treatment with compound CNI3; B: Statistical results of mouse tumor volume during 2 weeks of treatment with compound CNI3; Data are expressed as mean ± standard error (Mean ± SEM), with 6 replicates per group (n = 6), * indicates a significant difference compared with the control group (P < 0.05).

[0031] Figure 9 The effects of compound CNI3 in Example 2 on the final volume and weight of mouse tumors are shown in Figure 2. A: Statistical results of the final volume of mouse tumors after 2 weeks of treatment with compound CNI3; B: Statistical results of the final weight of mouse tumors after 2 weeks of treatment with compound CNI3. Data are expressed as mean ± standard error (Mean ± SEM). The number of replicates in each group is 6 (n = 6). * indicates a significant difference compared with the control group (P < 0.05).

[0032] Figure 10 The effects of compound CNI3 in Example 2 on the expression of apoptosis markers Bax, Caspase 3, and Bcl-2 proteins in mouse tumor tissues; A: Results of apoptosis marker protein expression in mouse tumor tissues; B: Statistical results of apoptosis marker protein expression in mouse tumor tissues; Data are expressed as mean ± standard error (Mean ± SEM), with 6 replicates per group (n = 6), * indicates significant difference compared to the control group (P < 0.05).

[0033] Figure 11 The effect of compound CNI3 on ​​mouse body weight in Example 2 is shown below; A: Statistical results of mouse body weight during 2 weeks of treatment with compound CNI3; B: Statistical results of final mouse body weight after 2 weeks of treatment with compound CNI3; Data are expressed in the form of mean ± standard error (Mean ± SEM), with 6 replicates per group (n = 6).

[0034] Figure 12 The effects of compound CNI3 in Example 2 on mouse blood routine and blood biochemistry; A: Statistical results of mouse blood routine related indicators; B: Statistical results of mouse blood biochemistry related indicators; Data are expressed in the form of mean ± standard error (Mean ± SEM), and the number of replicates in each group is 6 (n=6).

[0035] Figure 13 In Example 2, the effects of compound CNI3 on ​​the tissue structure of mouse heart, liver, spleen, lung, and kidney were detected by HE staining.

[0036] Figure 14 The results show the analysis of the binding sites and binding ability of compound CNI3 and mTOR in Example 3; A: Molecular docking results of the binding sites of compound CNI3 and mTOR; B: Thermal displacement experimental results of the binding ability of compound CNI3 and mTOR.

[0037] Figure 15 The effect of compound CNI3 in Example 3 on mTOR protein expression in non-small cell lung cancer cell lines A549 and H1299 and tumor tissues; A: Results of mTOR protein expression in A549 and H1299 cells and tumor tissues; B: Statistical results of mTOR protein expression in A549 and H1299 cells and tumor tissues; Data are expressed as mean ± standard error (Mean ± SEM). The number of replicates per group at the cellular level was 3 (n=3), and the number of replicates per group at the animal level was 6 (n=6). * and ** indicate significant difference (P<0.05) and extremely significant difference (P<0.01) compared with the control group, respectively.

[0038] Figure 16This section describes the effects of mTOR overexpression on the expression of mTOR and the apoptosis marker Caspase 3 protein in the non-small cell lung cancer cell line A549 treated with compound CNI3 in Example 3. A: Results of mTOR and apoptosis marker Caspase 3 protein expression in A549 cells; B: Statistical results of mTOR and apoptosis marker Caspase 3 protein expression in A549 cells. Data are presented as mean ± standard error (Mean ± SEM). The number of replicates for each group was 3 (n = 3). * and ** indicate significant differences (P < 0.05) and highly significant differences (P < 0.01) compared with the control group, respectively.

[0039] Figure 17 The effects of mTOR overexpression on the expression of mTOR and the apoptosis marker Caspase 3 protein in the non-small cell lung cancer cell line H1299 treated with compound CNI3 in Example 3 are shown in Figure 3. A: Results of mTOR and apoptosis marker Caspase 3 protein expression in H1299 cells; B: Statistical results of mTOR and apoptosis marker Caspase 3 protein expression in H1299 cells. Data are presented as mean ± standard error (Mean ± SEM). The number of replicates for each group was 3 (n = 3). ** indicates that the difference is extremely significant compared with the control group (P < 0.01).

[0040] Figure 18 The effect of mTOR overexpression on the apoptosis rate of non-small cell lung cancer cell lines A549 and H1299 treated with compound CNI3 in Example 3 is shown in Figure 3. A: Distribution of apoptotic cells in A549 and H1299 cells; B: Statistical results of apoptosis rate of A549 cells; C: Statistical results of apoptosis rate of H1299 cells. Data are expressed as mean ± standard error (Mean ± SEM). The number of replicates for each group was 3 (n = 3). ** indicates that the difference is extremely significant compared with the control group (P < 0.01). Detailed Implementation

[0041] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0042] In the following examples, non-small cell lung cancer (NSCLC) cell lines A549 and H1299, as well as an in vivo tumor model, were treated with compound CNI3. The results showed that compound CNI3 significantly reduced the viability of A549 and H1299 cells, induced excessive ROS accumulation in cells, increased the apoptosis rate, and decreased cell survival. Furthermore, compound CNI3 significantly increased the expression of pro-apoptotic proteins Bax and Caspase 3 in A549 and H1299 cells and tumor tissues, inhibited the expression of the anti-apoptotic protein Bcl-2, and promoted apoptosis in NSCLC cells, demonstrating good anti-tumor activity in NSCLC. Mechanistically, compound CNI3 showed good binding properties to mTOR, improving the thermal stability of mTOR. Moreover, after treatment with compound CNI3, the protein expression of mTOR in A549 and H1299 cells and tumor tissues was significantly decreased. Furthermore, when A549 and H1299 cells were treated with compound CNI3 and simultaneously overexpressed mTOR, it was found that compared with the compound CNI3 group, compound CNI3+mTOR overexpression significantly increased mTOR protein expression in cells and decreased the protein expression of the pro-apoptotic marker Caspase 3, thereby reducing the cell apoptosis rate. This confirms that compound CNI3 can serve as a novel drug for treating non-small cell lung cancer, and its mechanism of action is to target and inhibit mTOR expression, promote cell apoptosis, and thus exert its anti-non-small cell lung cancer effect.

[0043] In some embodiments, the non-small cell lung cancer cell lines used in in vitro studies are A549 and H1299 cells, while the animal tumor model used in in vivo studies is tumor formation in BALB / c nude mice subcutaneously transplanted with human non-small cell lung cancer cells H1299. The non-small cell lung cancer cell lines can also be other related cell lines, and the nude mouse tumor formation can be achieved using other strains of nude mice and different tumor-seeding methods.

[0044] In some embodiments, compound CNI3 promotes excessive accumulation of intracellular ROS, leading to increased expression of pro-apoptotic proteins Bax and Caspase 3 and decreased expression of anti-apoptotic protein Bcl-2 in cells and tissues, thereby increasing the apoptosis rate of tumor cells and reducing cell survival.

[0045] In some embodiments, compound CNI3 induces apoptosis by targeting and inhibiting mTOR, thereby promoting apoptosis in non-small cell lung cancer and inhibiting tumor growth.

[0046] In some embodiments, the effective dose of compound CNI3 in human non-small cell lung cancer cell lines A549 and H1299 is 0.1–100 μM, with preferred doses of 50 μM and 20 μM, respectively.

[0047] In some embodiments, the novel application of compound CNI3 in the preparation of drugs for non-small cell lung cancer was demonstrated with an effective dose of 20 mg / kg in mice.

[0048] Example 1: Compound CNI3 promotes apoptosis in non-small cell lung cancer cells and exerts anti-tumor activity.

[0049] Using CCK8 assays to detect the viability of non-small cell lung cancer cell lines A549 and H1299, the results showed that compound CNI3 significantly reduced the viability of non-small cell lung cancer cells compared to the control group. Figure 1 It exhibited excellent antitumor activity. The intracellular ROS content in A549 and H1299 cells was detected using the DCFH-DA probe, and cell viability was assessed using Calcein-AM / PI staining. The results showed that, compared with the control group, compound CNI3 induced excessive accumulation of intracellular ROS. Figure 2 ), significantly reducing the survival rate of non-small cell lung cancer cells ( Figure 3 and 4 Apoptosis rates of A549 and H1299 cells were detected by flow cytometry, and protein expression of apoptosis markers was detected by Western blotting. The results showed that, compared with the control group, compound CNI3 significantly increased the apoptosis rate. Figure 5 It reduced the expression of pro-apoptotic proteins Bax and Caspase 3, and decreased the expression of the anti-apoptotic protein Bcl-2. Figure 6 and 7 The results showed that compound CNI3 exerts its antitumor activity by promoting apoptosis in non-small cell lung cancer cells.

[0050] The specific method is as follows:

[0051] (1) Materials

[0052] Lung cancer human alveolar basal epithelial cells A549 (hereinafter referred to as A549 cells): Manufacturer: Pronosai, catalog number: CL-0016.

[0053] Human non-small cell lung cancer cells H1299 (hereinafter referred to as H1299 cells): Manufacturer: Pronosai, catalog number: CL-0165.

[0054] F12K complete medium: F12K basal medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin-streptomycin mixture; the fetal bovine serum is manufactured by BDBIO, catalog number F801-500; the penicillin-streptomycin mixture is manufactured by Biosharp, catalog number BL505A; the F12K basal medium is manufactured by BDBIO, catalog number L112-500.

[0055] RPMI 1640 complete medium: RPMI 1640 basal medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin-streptomycin mixture; the fetal bovine serum is manufactured by BDBIO, catalog number F801-500; the penicillin-streptomycin mixture is manufactured by Biosharp, catalog number BL505A; the RPMI 1640 basal medium is manufactured by BDBIO, catalog number 46223201.

[0056] 1×PBS buffer: Manufacturer: Biosharp, Catalog No.: BL601A.

[0057] CCK-8 reagent: Manufacturer: GlpBio, Catalog No.: GK10001.

[0058] CNI3 stock solution: Weigh 5 mg of compound CNI3 and dissolve it in 424 μL of DMSO. After thorough mixing, a stock solution with a concentration of 25 mM is obtained and stored in a refrigerator at -20°C.

[0059] CNI3 working solution: The CNI3 stock solution was diluted with complete culture medium at a certain dilution factor according to the final concentration of the cells treated in the experiment.

[0060] 10% SDS: Dissolve 10g SDS (manufacturer: Biosharp, catalog number: BS088) in 80mL ddH2O, and finally bring the volume up to 100mL with ddH2O.

[0061] Electrophoresis buffer: 2.72g Tris (manufacturer: Biosharp, catalog number: BS083), 18.8g Glycine (manufacturer: Biosharp, catalog number: BS082) and 10mL 10% SDS, add ddH2O to bring the volume to 1L.

[0062] Transfer buffer: 3.03g Tris (manufacturer: Biosharp, catalog number: BS083), 14.4g Glycine (manufacturer: Biosharp, catalog number: BS082) and 200mL methanol solution (manufacturer: Titan, catalog number: 67-56-1), add ddH2O to a final volume of 1L, pre-cool before use.

[0063] Tween 20: Manufacturer is Biosharp, product number is BS100.

[0064] (2) Cell viability detection

[0065] ① Cell Culture and Grouping: Healthy non-small cell lung cancer cell lines A549 and H1299 were digested with trypsin (manufacturer: Beyotime, catalog number C0201) to prepare cell suspensions. The cell suspensions were then diluted to 5 × 10⁻⁶ with F12K complete medium and RPMI 1640 complete medium, respectively. 4 Cells / mL were seeded into 96-well plates, with 100 μL of cell suspension added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator (Thermo Scientific BB150-2TCS-L). Once the cells reached approximately 70% confluence, control and experimental groups were established for 48 hours. The control group received 100 μL of complete culture medium, while the experimental groups received 100 μL of CNI3 working solution at different final concentrations (5 μM, 10 μM, 20 μM, 50 μM, and 100 μM). (Final concentration refers to the final concentration of the target substance added to each well of the cell culture plate; complete culture medium refers to F12K complete medium for A549 and PMI1640 complete medium for H1299.)

[0066] ② CCK8 assay for cell viability: Discard the culture medium in the A549 and H1299 cell culture plates after cell culture. Wash each well once with 100 μL of 1×PBS buffer. Add 100 μL of CCK-8 dilution buffer (obtained by diluting CCK-8 reagent 10 times with basal culture medium) to each well. Incubate in a 37℃ incubator (manufacturer: Lepotrex, model: ZQPL-200) for 1 hour in the dark. Detect the absorbance at 450 nm using a multi-functional microplate reader (manufacturer: Tecan, model: SPARK). (The basal culture medium refers to F12K basal medium used for A549 culture and PMI 1640 basal medium used for H1299 culture, respectively.)

[0067] (3) Cell staining

[0068] ① Cell culture and grouping: The A549 and H1299 cell suspensions digested with trypsin (manufacturer: Beyotime, catalog number: C0201) were diluted to 2×10⁻⁶. 5Cells / mL were seeded in 24-well plates, with 500 μL of cell suspension added to each well. The plates were then incubated at 7°C in a 5% CO2 incubator (Thermo Scientific BB150-2TCS-L). Once the cells reached approximately 70% confluence, control and experimental groups were established for each cell type and cultured for 48 hours. The control groups for both cell types received 500 μL of complete culture medium. The experimental groups for A549 cells received 500 μL of CNI3 working solution at a final concentration of 50 μM; the experimental groups for H1299 cells received 500 μL of CNI3 working solution at a final concentration of 20 μM. (Final concentration refers to the final concentration of the target substance added to each well of the culture medium; complete culture medium refers to F12K complete medium for A549 and PMI 1640 complete medium for H1299, respectively.)

[0069] ②Calcein-AM / PI staining: Prepare the Calein-AM / PI staining working solution according to the instructions of the Calein / PI Cell Viability and Cytotoxicity Assay Kit (manufacturer: Beyotime, catalog number: NO.C2015M). Discard the culture medium in the A549 and H1299 cell culture plates after cell culture. Add 300 μL of 1×PBS buffer to each well and wash three times. Add 200 μL of Calein-AM / PI staining working solution to each well. Incubate at 37°C in a constant temperature incubator (manufacturer: Lepotex, model: ZQPL-200) in the dark for 30 min. Then, use a fluorescence microscope (manufacturer: Thermo Scientific, model: EVOSM5000) to take pictures and analyze (Calcein AM is green fluorescence, excitation wavelength: 494 nm, emission wavelength: 517 nm; PI is red fluorescence, excitation wavelength: 535 nm, emission wavelength: 617 nm).

[0070] ③ DCFH-DA staining: Prepare the DCFH-DA staining working solution according to the instructions of the DCFH-DA probe (manufacturer: Glpbio, catalog number: GC30006). Discard the culture medium in the A549 and H1299 cell culture plates after cell culture. Add 300 μL of 1×PBS buffer to each well and wash 3 times. Add 200 μL of DCFH-DA staining working solution to each well and incubate in a 37°C incubator (manufacturer: Lepote, model: ZQPL-200) in the dark for 30 min. After incubation, discard the staining working solution and wash 300 μL of 1×PBS buffer to each well. Then, use a fluorescence microscope (manufacturer: Thermo Scientific, model: EVOSM5000) to take pictures and analyze (excitation wavelength: 488 nm, emission wavelength: 525 nm).

[0071] (4) Apoptosis rate detection

[0072] ① Cell culture and grouping: The A549 and H1299 cell suspensions digested with trypsin (manufacturer: Beyotime, catalog number: C0201) were diluted to 4×10⁻⁶. 5 Cells / mL were seeded in 12-well plates, with 1 mL of cell suspension added to each well. The plates were then incubated at 7°C in a 5% CO2 incubator (Thermo Scientific BB150-2TCS-L). Once the cells reached approximately 70% confluence, control and experimental groups were established for each cell type and cultured for 48 hours. The control groups for both cell types received 1 mL of complete culture medium. The experimental groups for A549 cells received 1 mL of CNI3 working solution at a final concentration of 50 μM; the experimental groups for H1299 cells received 1 mL of CNI3 working solution at a final concentration of 20 μM. (Final concentration refers to the final concentration of the target substance added to each well of the culture medium; complete culture medium refers to F12K complete medium for A549 and PMI 1640 complete medium for H1299, respectively.)

[0073] ② Flow cytometry detection of apoptosis rate: After cell culture, A549 and H1299 cells were processed according to the instructions of the Annexin V-FITC / PI apoptosis detection kit (manufacturer: Yeasen, catalog number: 40302ES60). In short, A549 and H1299 cells were digested with trypsin (manufacturer: Beyotime, catalog number: C0201), and the cell suspension was collected in 1.5 mL centrifuge tubes and centrifuged at 1,000 rpm for 5 min at room temperature. The supernatant was discarded, and the cell pellet was resuspended in 100 μL of the kit's 1× Binding Buffer. Then, 5 μL of Annexin V-FITC and 10 μL of PIStaining Solution were added, gently mixed, and incubated at room temperature in the dark for 30 min. The mixture was then placed on ice and analyzed using a flow cytometer (manufacturer: Agilent, model: Novocyte 3000).

[0074] (5) Western blot (WB) detection of protein expression of apoptosis markers in cells.

[0075] ① Cell culture and grouping: The A549 and H1299 cell suspensions digested with trypsin (manufacturer: Beyotime, catalog number: C0201) were diluted to 5×10⁻⁶. 5Cells / mL were seeded in 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated at 7°C in a 5% CO2 incubator (Thermo Scientific BB150-2TCS-L). Once the cells reached approximately 70% confluence, control and experimental groups were established for each cell type and cultured for 48 hours. The control groups for both cell types received 2 mL of complete culture medium. The experimental groups for A549 cells received 2 mL of CNI3 working solution at a final concentration of 50 μM; the experimental groups for H1299 cells received 2 mL of CNI3 working solution at a final concentration of 20 μM. (Final concentration refers to the final concentration of the target substance added to each well of the culture medium; complete culture medium refers to F12K complete medium for A549 and PMI 1640 complete medium for H1299, respectively.)

[0076] ② Extraction of cellular proteins: Discard the culture medium from A549 and H1299 cell culture plates after cell culture. Wash each well three times with 600 μL of 1×PBS buffer. Add 45 μL of RIPA protein lysis buffer (Biosharp, catalog number BL504A) to each well. Incubate at 4°C on a shaker for 30 min. Scrape the cells from the wells with a cell scraper and collect them into 1.5 mL EP tubes. Perform sonication lysis on an ultrasonic cell disruptor (Sonic, model VCX130) at 130 W and 20 Hz for 3 cycles, 10 s each, with a 10 s interval between cycles, all on ice. Centrifuge the lysate at 4°C, 13,500 rpm for 15 min. Collect the supernatant as the extracted protein stock solution and store at -80°C for later use.

[0077] ③ Protein concentration determination and denaturation: Take 20 μL of the above protein stock solution and add it to each of the 96 wells. Then add 160 μL of the prepared BCA working solution (A solution: B solution = 50:1, manufacturer: GlpBio, catalog number: GK10009). After mixing thoroughly, incubate in a 37℃ constant temperature incubator (manufacturer: Lepote, model: ZQPL-200) for 30 min. Then use a multi-functional microplate reader (manufacturer: Tecan, model: SPARK) to detect the absorbance at a wavelength of 562 nm. A standard curve was plotted based on the absorbance values, and the protein concentration in each well was calculated. After adjusting the protein stock solution in each well to a uniform concentration and volume, 6× loading buffer (manufacturer: TransGen, catalog number: DL101-02, volume: 1 / 5 of the protein stock solution volume) was added, and the mixture was incubated in a metal bath (manufacturer: MIO, model: DTC-100) at 100℃ for 10 min to obtain denatured protein, which was then stored at -80℃ for later use.

[0078] ④SDS-PAGE gel electrophoresis

[0079] Prepare an 8% separating gel as needed. After thorough mixing, quickly and evenly add it to the gel plate. Slowly and evenly add ddH2O to the top of the gel plate to form a water seal. Let it stand until a clear boundary appears between the separating gel and the water seal, then pour off the water from the gel plate. Prepare a 5% stacking gel. After thorough mixing, quickly add it to the gel plate. Insert a comb with gel holes vertically into the stacking gel and let it stand for 40 minutes. The gel preparation method is shown in Table 1.

[0080] Table 1

[0081]

[0082] Fix the solidified gel in the electrophoresis apparatus (Bio-Rad Laboratories, PowerPac HC 1645052), and add fresh electrophoresis buffer to completely immerse the gel. Slowly remove the comb and thoroughly mix the denatured protein. Use a pipette to add the denatured protein (40 μg per lane) to each lane, and add 3 μL of protein marker (Thermo Fisher Scientific, 26619) to each side. Turn on the power. Adjust the voltage to 80V until the sample passes through the stacking gel and forms a straight line. Then, adjust the voltage to 120V to separate proteins of different molecular weights. Stop electrophoresis when the indicator band corresponding to the smallest molecular weight of the marker reaches the bottom of the gel.

[0083] ⑤ Transfer: Prepare a PVDF membrane (Invitrogen, catalog number 88520) and qualitative filter paper according to the width of the target protein gel. Activate the PVDF membrane by immersing it in methanol solution (Titanium, catalog number 67-56-1) for 1 minute. Soak the qualitative filter paper in the transfer solution for 10 minutes beforehand. Open the transfer clamp and lay out the sponge, filter paper, SDS-PAGE strip, PVDF membrane, filter paper, and sponge in that order, removing any air bubbles. After fixing the clamp and determining the positive and negative electrodes, place the membrane in the transfer instrument (Bio-Rayet, catalog number 10104001), pour in the pre-cooled transfer solution, and transfer at a constant voltage of 100V for 90 minutes.

[0084] ⑥ Blocking, antibody hybridization, and imaging:

[0085] After the transfer was completed, the transferred PVDF membrane was placed in 4% skim milk blocking solution and blocked at room temperature for 2 hours. The blocked PVDF membrane was then incubated overnight at 4°C with the diluted first antibody corresponding to the target protein (diluted with 1×PBS buffer according to the volume ratio in Table 2), followed by washing three times with 1×PBST buffer (1×PBST buffer is prepared by mixing 1×PBS buffer and Tween 20 at a ratio of 1000:1), each time for 10 minutes. The PVDF membrane was incubated with diluted secondary antibody CoraLite488-conjugated Goat Anti-Rabbit IgG (H+L) (manufacturer: Proteintech, catalog number: SA00013-2, diluted 1:10000 with 1×PBST buffer) at room temperature in the dark for 2 hours. After washing three times with 1×PBST buffer, images were acquired and protein bands were analyzed using a dual-color infrared laser imaging system (manufacturer: Odyssey, model: DLX-3307). Information on the diluted primary antibody corresponding to the target protein is shown in Table 2.

[0086] Table 2

[0087] Target protein name Antibody categories brand Item number Dilution ratio Bax First Antibody Proteintech 50599-2-AP 1:2000 Caspase-3 First Antibody Proteintech 19677-1-AP 1:2000 Bcl-2 First Antibody Proteintech 26539-1-AP 1:2000 β-actin First Antibody Proteintech 66009-1-lg 1:5000

[0088] Example 2: Compound CNI3 promotes apoptosis in non-small cell lung cancer cells, inhibits tumor growth, and has no obvious toxic side effects on normal tissues and organs.

[0089] An in vivo tumor model was established by subcutaneously transplanting human non-small cell lung cancer cells H1299 into BALB / c nude mice. The mice were then treated with compound CNI3 via tail vein injection every two days for two weeks. Tumor volume was measured every two days during treatment, and the mice were weighed. The tumor tissue was weighed again after treatment. The results showed that, compared with the control group, compound CNI3 significantly reduced tumor volume and weight. Figure 8 and 9 Western blot analysis of apoptosis marker protein expression in tumor tissues showed that, compared with the control group, compound CNI3 significantly increased the expression of pro-apoptotic proteins Bax and Caspase3, and decreased the expression of the anti-apoptotic protein Bcl-2. Figure 10 The above results demonstrate that compound CNI3 inhibits tumor growth by promoting apoptosis in non-small cell lung cancer cells.

[0090] Furthermore, compared with the control group, compound CNI3 had no significant effect on the body weight of tumor-bearing mice. Figure 11 The results of blood biochemistry and routine blood tests in tumor-bearing mice showed that, compared with the control group, compound CNI3 had no significant effect on blood biochemistry and routine blood test results in tumor-bearing mice. Figure 12 HE staining results showed that, compared with the control group, compound CNI3 had no significant effect on the tissue structure of the heart, liver, spleen, lungs, and kidneys in tumor-bearing mice. Figure 13 The above results demonstrate that compound CNI3 has no significant toxic side effects on normal tissues and organs of tumor-bearing mice, and has good safety.

[0091] The specific method is as follows:

[0092] (1) Establishment of tumor model and drug administration

[0093] Preparation of cell culture medium: Healthy non-small cell lung cancer cell line H1299 (producer: Pronosai, catalog number: CL-0165) were digested with trypsin (producer: Beyotime, catalog number: C0201) to prepare a cell suspension. The cells were centrifuged at 1200 rpm for 3 min at room temperature, the supernatant was discarded, and the cells were resuspended in 1×PBS buffer (producer: Biosharp, catalog number: BL601A) to a concentration of 2×10⁶ cells / mL. 7 Cell resuspension at a density of cells / mL was prepared by mixing 75 μL of cell resuspension with 75 μL of Matrigel (manufacturer: Corning, catalog number: 356234) in equal volumes to obtain 1×10⁻⁶ cells / mL. 7 Cells / mL of cell seeding medium, stored on ice for later use.

[0094] Male BALB / c nude mice aged 4-6 weeks (16-18g) were selected. The skin of the right posterior axilla of the mouse was disinfected with a 75% alcohol swab. Cell implantation solution was slowly injected subcutaneously into the right posterior axilla using a 1mL insulin syringe (manufacturer: Yuyang Medical, model A). The needle was slowly withdrawn, and the mouse skin was gently pressed with a cotton swab at the injection site for 30 seconds to prevent extravasation of the cell suspension (the formation of a small bulge at the injection site indicates successful inoculation). 7-10 days after tumor growth, the tumor size was measured with calipers. The tumor was weighed every 3 days, and the longest (longest) and shortest (shortest) diameters were measured. The volume of the transplanted tumor was calculated using the formula: V = (shortest diameter / longest diameter) * (shortest diameter / shortest ... 2 Calculate the tumor volume by multiplying the length by the diameter () / 2. The subcutaneous transplanted tumor should reach a volume of 60-90 mm. 3 A successful xenograft tumor model was established in nude mice. Mice were randomly divided into a control group and an experimental group (n=6 each) according to body weight and tumor size to reduce intergroup differences. The experimental group was injected intraperitoneally with 20 mg / kg of compound CNI3, while the control group was injected with an equal volume of physiological saline. The injections were given every 2 days, and the body weight and tumor size of the mice were recorded for a total of 2 weeks.

[0095] After the experiment, mice were anesthetized with 1% aphthylazine (manufacturer: Biosharp, catalog number: BR4108423) at a dose of 20 uL / g. Blood was collected from the eyeballs, and 0.5 mL of blood was collected in an EDTA-anticoagulated tube for routine blood tests. The remaining blood was collected in a heparin sodium anticoagulated tube, allowed to stand at room temperature for 30 min, centrifuged at 3,000 rpm for 15 min, and the supernatant was collected to obtain serum, which was stored at -80℃ for blood biochemical tests. Tumors were weighed, measured, photographed, and stored in liquid nitrogen for Western blotting experiments. Heart, liver, spleen, lung, and kidney were fixed in 4% paraformaldehyde (manufacturer: Biosharp, catalog number: BL539A) for HE staining.

[0096] (2) Western blot (WB) detection of protein expression of apoptosis markers in tumor tissues

[0097] ① Tissue protein extraction: Weigh 50 mg of tumor tissue into a fragmentation tube containing 3 sterile steel balls, add 700 μL of LRIPA protein lysis buffer (manufacturer: Biosharp, catalog number: BL504A), homogenize using a multichannel tissue homogenizer (manufacturer: Wix-many-GRIND, model: 5,000×g) for 15 s, pause for 30 s, repeat this process 5 times until the tissue homogenizes thoroughly, incubate on ice for 20 min, centrifuge at 12,000×g for 20 min at 4℃, and collect the supernatant into a new 1.5 mL centrifuge tube to obtain the protein stock solution, which is stored at -80℃ for later use.

[0098] ② Protein concentration determination and denaturation: Take 10 μL of protein stock solution and dilute it 20 times. Take 20 μL of the diluted protein sample and add it to each well of a 96-well plate. Then add 160 μL of freshly prepared BCA working solution (A solution: B solution = 50:1, manufacturer: GlpBio, catalog number: GK10009). After thorough mixing, incubate in a 37℃ constant temperature incubator (manufacturer: Lepote, model: ZQPL-200) for 30 min. Then use a multi-functional microplate reader (manufacturer: Tecan, model: SPARK) to detect the absorbance at a wavelength of 562 nm. A standard curve was plotted based on the absorbance values, and the protein concentration in each well was calculated. The original protein concentration was calculated based on the dilution factor. After adjusting the protein stock solution in each well to a uniform concentration and volume, 6× loading buffer (manufacturer: TransGen, catalog number: DL101-02, volume: 1 / 5 of the protein stock solution volume) was added, and the mixture was incubated in a metal bath (manufacturer: MIO, model: DTC-100) at 100℃ for 10 min to obtain denatured protein, which was then stored at -80℃ for later use.

[0099] ③ The denatured protein was subjected to SDS-PAGE gel electrophoresis, transfer blocking, antibody hybridization, and development before analysis. The relevant operating steps and the target protein antibody used for detection were the same as those in Example 1 (5).

[0100] (3) Hematoxylin and eosin (HE) staining

[0101] ① Preparation of sections: The fixed tissue was trimmed smooth at the target site using a scalpel. The trimmed tissue and corresponding labels were placed in an embedding frame and then subjected to a gradient ethanol treatment (75% immersion for 4 hours, 85% immersion for 2 hours, 90% immersion for 2 hours, 95% immersion for 1 hour, anhydrous ethanol I for 30 minutes, and anhydrous ethanol II for 30 minutes). The tissue samples were then immersed in xylene I for 10 minutes and xylene II for 10 minutes to make the tissue transparent. The transparent tissue samples were then immersed in molten paraffin I at 65°C for 1 hour, molten paraffin II at 65°C for 1 hour, and molten paraffin III at 65°C for 1 hour. After the paraffin had completely penetrated the tissue samples, the samples were placed in an embedding cassette containing molten paraffin and cooled to -20°C to allow the paraffin to completely solidify. After solidification, the paraffin block was removed from the embedding frame and trimmed. Paraffin tissue samples were cut into 5μm sections using a Leica HistoCore MULTICUT microtome. After being fully spread on a Leica HI1210 slide spreader, the sections were mounted on glass slides and placed in a 37°C constant temperature incubator (Leica ZQPL-200) to dry completely in preparation for staining.

[0102] ② HE staining: The sample sections were soaked in xylene (I) for 10 min and xylene (II) for 10 min to dewax. Then, they were soaked in anhydrous ethanol 1:1 mixture for 3 min. The sections were then soaked in a gradient of ethanol (100%, 95%, 85%, 75%) for 3 min at each gradient. Finally, they were soaked in distilled water for 5 min to wash away the residual liquid. Then, staining was performed according to the instructions of the hematoxylin and eosin staining kit (manufacturer: Solarbio, catalog number: G1120), namely, staining with hematoxylin solution for 2 min, washing with distilled water for 5 min to remove excess stain; then adding 1% hydrochloric acid ethanol for differentiation for 30 s, rinsing twice with tap water for 5 min each time, then adding eosin staining solution for 1 min to remove excess staining solution, and then quickly performing gradient dehydration, that is, immersing the sections in gradient ethanol (75%, 85%, 95% and 100% ethanol (I)) for 3 s each, then immersing in 100% ethanol (II) for 1 min, then transferring to xylene (I) for 2 min, then xylene (II) for 2 min, and finally using neutral resin glue (manufacturer: Beyotime, catalog number: C0173) to fix the tissue sections, and observing the tissue morphology under a microscope (manufacturer: Olympus, model: CKX53) for imaging and image analysis.

[0103] (4) Complete blood count

[0104] According to the operating procedures provided by the manufacturer, the collected blood samples were gently mixed and then a complete blood count (red blood cell and platelet system) was performed using a fully automated blood cell analyzer (manufacturer: Mindray Medical, model: BC-2800).

[0105] (5) Blood biochemistry test

[0106] After thawing and mixing the collected serum samples on ice, 100 μL of each sample was transferred to a new 1.5 mL centrifuge tube. Following the manufacturer's operating procedures, blood biochemical indicators were measured using an automated biochemical analyzer (HITACHI, model 7020), including alanine aminotransferase (ALT, Maccura, catalog number CH0105201), aspartate aminotransferase (AST, Maccura, catalog number CH0105202), alkaline phosphatase (ALP, Maccura, catalog number CH0105203), gamma-glutamyl transferase (GGT, Maccura, catalog number H115), creatinine (CREA, Maccura, catalog number CH0101053), and urea (UREA, Bio-Rad, catalog number GL1963), to assess whether compound CNI3 caused damage to organs such as the liver and kidneys in tumor-bearing mice.

[0107] Example 3: Compound CNI3 exerts its anti-non-small cell lung cancer effect by targeting and inhibiting mTOR to promote apoptosis.

[0108] The binding affinity between compound CNI3 and mTOR was analyzed using computer-aided molecular docking and thermal displacement experiments. The results showed that compound CNI3 and mTOR have excellent binding affinity and can increase the thermal stability of mTOR. Figure 14 Western blot analysis of mTOR protein expression in non-small cell lung cancer cell lines A549 and H1299, as well as tumor tissues, showed that, compared with the control group, compound CNI3 significantly reduced mTOR protein expression in A549 and H1299 cells and tumor tissues. Figure 15 Simultaneously, mTOR overexpression was induced by treatment with compound CNI3. Western blotting was used to detect the expression of mTOR protein and the pro-apoptotic protein Caspase 3 in non-small cell lung cancer cell lines A549 and H1299, as well as tumor tissues. Flow cytometry was used to detect the apoptosis rate of A549 and H1299 cells. The results showed that, compared with the control group, compound CNI3 significantly reduced mTOR protein expression and increased Caspase 3 protein expression and apoptosis rate. Compared with the compound CNI3 group, CNI3+mTOR overexpression significantly increased mTOR protein expression and decreased Caspase 3 protein expression and apoptosis rate. Figure 16 , 17 (and 18). The results showed that compound CNI3 exerts its anti-non-small cell lung cancer effect by targeting and inhibiting mTOR protein expression and promoting cell apoptosis.

[0109] The specific method is as follows:

[0110] (1) Preparation of materials

[0111] Lung cancer human alveolar basal epithelial cells A549 (hereinafter referred to as A549 cells): Manufacturer: Pronosel, catalog number: CL-0016;

[0112] Human non-small cell lung cancer cells H1299 (hereinafter referred to as H1299 cells): Manufacturer: Pronosai, catalog number: CL-0165;

[0113] F12K complete medium: This is F12K basal medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin-streptomycin mixture; the manufacturer of the fetal bovine serum is BDBIO, catalog number F801-500; the manufacturer of the penicillin-streptomycin mixture is Biosharp, catalog number BL505A; the manufacturer of the F12K basal medium is BDBIO, catalog number L112-500.

[0114] RPMI 1640 complete medium: This is RPMI 1640 basal medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin-streptomycin mixture; the fetal bovine serum is manufactured by BDBIO, catalog number F801-500; the penicillin-streptomycin mixture is manufactured by Biosharp, catalog number BL505A; the RPMI 1640 basal medium is manufactured by BDBIO, catalog number 46223201.

[0115] CNI3 stock solution: Weigh 5 mg of compound CNI3 and dissolve it in 424 μL of DMSO. After thorough mixing, a stock solution with a concentration of 25 mM is obtained and stored in a refrigerator at -20°C.

[0116] CNI3 working solution: The CNI3 stock solution was diluted with complete culture medium at a certain dilution factor according to the final concentration of the cells treated in the experiment.

[0117] mTOR empty vector plasmid (pCMV) and overexpression plasmid (pCMV-mTOR): both were constructed by Shanghai Jiying Biotechnology Co., Ltd.

[0118] (2) Molecular docking

[0119] The 3D-SDF file of compound CNI3 was downloaded from the PubChem database and converted to mol2 format in OpenBabel software. The mol2 file was then imported into Autodock 1.5.6, where full hydrogen bonding and charge were added to set it as a ligand, and saved as a PABQT file. The target protein mTOR was searched for in the Uniport database and its PDB file was downloaded. This PDB file was then imported into PyMOL software, where it underwent dehydration, solvent removal, and metal ion removal before being saved as a mol2 file. It was then imported into Autodock 1.5.6, where full hydrogen bonding and charge were added, set as an acceptor, and exported as a PABQT file. Molecular docking calculations for the ligand and acceptor were performed using Autodock 1.5.6, and potential binding modes were screened based on docking energy scores. Finally, PyMOL was used to perform 3D visualization of the molecular docking structure to evaluate the binding of compound CNI3 and the target protein mTOR.

[0120] (3) Cell thermal migration assay (CETSA)

[0121] ① Extraction of cellular proteins: Discard the culture medium from the A549 and H1299 cell culture flasks after cell culture. Add 2 mL of 1×PBS buffer (Biosharp, catalog number BL601A) and wash three times. Then add 150 μL of protein lysis buffer (Biosharp, catalog number BL504A) and incubate at 4°C on a shaker for 30 min. Scrape the cells from the wells of the plate with a cell scraper and collect them in 1.5 mL EP tubes. Perform sonication lysis on an ultrasonic cell disruptor (Sonic, model VCX130) at 130 W and 20 Hz for 3 cycles, 10 s each, with a 10 s interval between cycles, all on ice. Centrifuge the lysate at 4°C, 13,500 rpm for 15 min. Collect the supernatant as the extracted protein stock solution and store at -80°C for later use.

[0122] ② Thermal displacement experiment: 600 μL of protein stock solution from each of the two cell types was transferred to four 1.5 EP tubes. For the A549 cell protein stock solution, an equal volume of 0.1% DMSO (manufacturer: Sigma, catalog number: D8418) and an equal volume of 50 μM CNI3 working solution were added to the two centrifuge tubes, respectively. The tubes were incubated at room temperature in the dark for 3 hours. For the H1299 cell protein stock solution, an equal volume of 0.1% DMSO (manufacturer: Sigma, catalog number: D8418) and an equal volume of 20 μM CNI3 working solution were added to the two centrifuge tubes, respectively. The tubes were incubated at room temperature in the dark for 3 hours. The incubation solutions for the two cell types were divided into 10 equal portions and 50 μL each in 200 μL centrifuge tubes. The tubes were heated for 3 min in a standard PCR instrument (manufacturer: Dongshenglong, catalog number: ETC811) with temperature gradients (45℃, 49℃, 53℃, 57℃, 61℃, 65℃, 69℃, 73℃, 77℃, 81℃) and cooled for 3 min. The tubes were then centrifuged at 4℃ and 13,000 rpm for 25 min. The supernatant was collected, and 6× loading buffer (manufacturer: TransGen, catalog number: DL101-02, volume 1 / 5 of the protein stock solution volume) was added. The tubes were then incubated in a metal bath (manufacturer: MIO, model: DTC-100) at 100℃ for 10 min to obtain denatured protein, which was then stored at -80℃ for later use.

[0123] ③ The denatured protein was subjected to SDS-PAGE gel electrophoresis, transfer blocking, antibody hybridization and development, and the results were analyzed. The relevant operation steps are the same as those in Example 1 (5) of the second antibody, the difference being that there is only one first antibody in the antibody hybridization step, namely mTOR (manufacturer is Proteintech, catalog number is 66888-1-AP, diluted with 1×PBST buffer at a volume ratio of 1:5000).

[0124] (4) Cell transfection

[0125] ① Preparation of the transfection system: Invert and mix (do not shake vigorously). Add 150 μL of 1×OPTI-MEM (Gibco, catalog number 31985-062) diluent to 1.5 mL centrifuge tubes containing 3 μg of mTOR empty vector plasmid (pCMV) and overexpression plasmid (pCMV-mTOR), respectively. Invert and mix well, then let stand for 5 min. Separately, take 6 μL of Celopener (Gibco Biotechnology, catalog number TRAN002D) and add 150 μL of 1×OPTI-MEM (Gibco, catalog number 31985-062) diluent. Invert and mix well, then let stand for 5 min. Two diluted Celopener fractions were added to the mTOR empty vector plasmid (pCMV) and the overexpression plasmid (pCMV-mTOR) dilution buffer, respectively. After mixing by pipetting, the mTOR empty vector plasmid (pCMV) and the overexpression plasmid (pCMV-mTOR) transfection complexes were obtained and allowed to stand for 20 min.

[0126] ② Cell transfection and grouping: The A549 and H1299 cell suspensions digested with trypsin (manufacturer: Beyotime, product number: C0201) were diluted to 5×10⁻⁶. 5 Cells / mL were seeded in 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated at 7°C in a 5% CO2 incubator (Thermo Scientific, model BB150-2TCS-L). When the cells reached approximately 70% confluence, they were washed once with 1×PBS buffer (Biosharp, catalog number BL601A). 1.7 mL of the corresponding basal medium was added to each well. Control and experimental groups were set up for both cell types. In the control group, 300 μL of the mTOR empty vector plasmid (pCMV) transfection complex was added to each well, while in the experimental group, 300 μL of the overexpression plasmid (pCMV-mTOR) transfection complex was added to each well. After mixing, the plates were incubated for 6 hours. The culture medium was then discarded and replaced with fresh medium. In this study, the control groups for both cell types were given 500 μL of complete culture medium. The experimental groups for A549 cells were given 500 μL of CNI3 working solution with a final concentration of 50 μM. The experimental groups for H1299 cells were given 500 μL of CNI3 working solution with a final concentration of 20 μM. After 48 hours of incubation, the results were analyzed. (The basal culture medium refers to F12K basal medium used for A549 and PMI 1640 basal medium used for H1299; the complete culture medium refers to F12K complete medium used for A549 and PMI 1640 complete medium used for H1299.)

[0127] (5) Western blot (WB) detection of mTOR and apoptosis marker protein expression in non-small cell lung cancer and tumor tissues.

[0128] The cell-related procedures are the same as in (5) of Example 1, and the tissue-related procedures and the second antibody are the same as in (2) of Example 2. The information of the diluted first antibody corresponding to the target protein is shown in Table 3.

[0129] Table 3

[0130] Target protein name Antibody categories brand Item number Dilution ratio Caspase-3 First Antibody Proteintech 19677-1-AP 1:2000 mTOR First Antibody Proteintech 66888-1-AP 1:5000 β-actin First Antibody Proteintech 66009-1-lg 1:5000

[0131] (6) Apoptosis rate detection

[0132] The relevant operating steps are the same as those in (4) of Example 1.

[0133] As demonstrated in Examples 1-3, the cytisine-N-isoflavone derivative (compound CNI3) inhibits mTOR protein expression, increases the expression of apoptosis-promoting proteins Bax and Caspase3, and decreases the expression of the anti-apoptotic protein Bcl-2, thereby inducing apoptosis in non-small cell lung cancer (NSCLC) cells and thus resisting the pathological progression of NSCLC. These findings clarify the role and specific mechanism of action of compound CNI3 in the progression of NSCLC, providing new theoretical basis and drug research directions for its application in the treatment of NSCLC.

[0134] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of cytisine-N-isoflavone derivatives in the preparation of drugs for treating non-small cell lung cancer, wherein the cytisine-N-isoflavone derivative is compound CNI3, with the structural formula as follows:

2. The application according to claim 1, characterized in that, The non-small cell lung cancer includes human non-small cell lung cancer cell lines A549 and H1299.

3. The application according to claim 1, characterized in that, The drug uses compound CNI3 or its pharmaceutically acceptable solvate as the active ingredient, with an effective dose of 0.1–100 μM.

4. The application according to claim 1, characterized in that, The drug uses compound CNI3 or its pharmaceutically acceptable solvate as the active ingredient, with an effective dose of 20–50 μM.

5. The application according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier or diluent, and the dosage form of the drug is selected from at least one formulation of tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, gels, suppositories, ointments, emulsions, and injections.

6. The application according to claim 1, characterized in that, The route of administration of the drug is selected from at least one of oral administration, injection administration, inhalation administration, topical administration, sublingual administration, and rectal administration.

7. A pharmaceutical composition for treating non-small cell lung cancer, characterized in that, Includes compound CNI3 as an active ingredient or a pharmaceutically acceptable solvate thereof, wherein the structural formula of compound CNI3 is as follows: It may also include pharmaceutically acceptable carriers or diluents.

8. The application according to claim 7, characterized in that, The effective dose of the compound CNI3 or its pharmaceutically acceptable solvate is 0.1–100 μM.

9. The application of cytisine-N-isoflavone derivatives in the preparation of drugs targeting and inhibiting mTOR expression, wherein the cytisine-N-isoflavone derivative is compound CNI3, with the structural formula [insert structural formula here].

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