Quinolone derivative for treating esophageal cancer through targeting ACSL4 as well as preparation method and application of quinolone derivative

Abstract

The invention discloses a quinolone derivative for treating esophageal cancer by targeting ACSL4 as well as a preparation method and application of the quinolone derivative. The structural formula of the quinolone derivative is # imgabs0 #. The invention also specifically discloses a preparation method of the quinolone derivative for treating esophageal cancer by targeting ACSL4 and application of the quinolone derivative in preparation of drugs for treating or / and preventing esophageal cancer. The molecular structure of the quinolone compound is innovatively modified, a specific substituent group is introduced to improve the selectivity and treatment effect of the quinolone compound on esophageal cancer cells, and the prepared quinolone derivative not only shows an efficient inhibition effect on the esophageal cancer cells, but also remarkably reduces the toxicity to normal cells, and has a good application prospect. The medicine is expected to become a novel medicine for treating esophageal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of quinolone derivatives and their anti-tumor applications, and specifically relates to a quinolone derivative for treating esophageal cancer by targeting ACSL4, and a preparation method and application thereof. Background Art

[0002] Esophageal cancer is one of the most common malignant tumors of the esophageal mucosal epithelium, and its morbidity and mortality are among the highest among cancers. Since the early symptoms of esophageal cancer are not obvious, most patients have already progressed to the late stage when diagnosed, which greatly limits the therapeutic effect of traditional treatments (such as surgery, radiotherapy and chemotherapy). In addition, traditional treatments have significant toxic side effects, which seriously affect the quality of life of patients.

[0003] Ferroptosis is a new type of non-apoptotic regulated cell death process, accompanied by the accumulation of iron ions and lipid peroxides. It plays an important regulatory role in the occurrence and development of many diseases, such as tumors, nervous system diseases, and acute kidney injury. Activating or blocking the ferroptosis pathway to alleviate the progression of the disease provides a promising therapeutic strategy for many diseases.

[0004] As an important component of nitrogen heterocyclic compounds, quinolone derivatives have multiple biological activities such as antibacterial, anti-inflammatory, anti-cancer, anti-tuberculosis, anti-malaria, anti-AIDS and anti-Alzheimer's disease. They are an important class of biologically active heterocyclic compounds in the medical field and are widely used in drug research and development. Quinolone derivatives are widely present in a large number of bioactive molecules, natural products and organic functional materials. Their structures can connect different functional groups to produce different pharmacological activities, and they play a very important role in the design and synthesis of drug molecules. In summary, quinolone derivatives have the advantages of good safety, wide range of action and high bioavailability. Therefore, quinolone derivatives always have the potential for first-line drug development. Their structural innovation and related activity research are continuous topics in drug research and development, and are also one of the important ways to develop new drugs.

[0005] The most striking thing is the potential of quinolone derivatives in the field of anti-tumor. Previous studies have shown that quinolone derivatives exert anti-tumor activity by inhibiting DNA topoisomerase (TopoII) to induce DNA damage. DNA topoisomerase includes TopoI and TopoII, which can catalyze the breakage and combination of DNA double strands, thereby controlling the topological state of DNA. Related studies have shown that a series of new quinolone derivatives synthesized by substituent substitution of quinolone derivatives have significantly improved anti-tumor activity, stronger selectivity, and good high efficiency and low toxicity. The present invention uses structure-activity relationship to introduce different electrophilic groups on the basis of quinolone mother nucleus. The results show that R 3 OCH 3 With R5 The -OH group can significantly enhance the antitumor activity of quinolone derivatives.

[0006] Recent studies have found that quinolone derivatives can effectively regulate a variety of cancer-related cell death signaling pathways. For example, some quinolone derivatives induce cell ferroptosis by targeting the NRF2 / SLC7A11 / HMOX1 pathway. In addition, relevant literature reports that quinolone derivatives can act as inducers of ferroptosis to induce ferroptosis in tumor cells, thereby exerting anti-tumor effects. However, there are currently no reports on new quinolone derivatives targeting ACSL4 to treat esophageal cancer.

[0007] Based on this background, the novel quinolone derivative prepared by the present invention, Qu-5, is prepared by innovatively modifying the molecular structure of quinolone compounds and introducing specific substituent groups to improve their selectivity and therapeutic effect on esophageal cancer cells. This novel quinolone derivative not only shows a highly effective inhibitory effect on esophageal cancer cells, but also significantly reduces its toxicity to normal cells, and is expected to become a new drug for the treatment of esophageal cancer. Summary of the invention

[0008] The technical problem solved by the present invention is to provide a quinolone derivative for treating esophageal cancer by targeting ACSL4 and a preparation method thereof. The method innovatively modifies the molecular structure of quinolone compounds and introduces specific substituent groups to improve their selectivity and therapeutic effect on esophageal cancer cells. The prepared quinolone derivative not only shows a highly effective inhibitory effect on esophageal cancer cells, but also significantly reduces the toxicity to normal cells, and is expected to become a new drug for treating esophageal cancer.

[0009] The present invention adopts the following technical solution to solve the above technical problems: a quinolone derivative for treating esophageal cancer by targeting ACSL4, characterized in that: the structural formula of the quinolone derivative is:

[0010]

[0011] Where R 1 For H, R 2 For H, R 3 OCH 3 , R 4 For H, R 5 It is -OH.

[0012] The method for preparing a quinolone derivative for treating esophageal cancer by targeting ACSL4 of the present invention is characterized by the specific steps of: dissolving a substituted aromatic aldehyde and 2-aminobenzamide in an ethanol solution, adding a catalyst p-toluenesulfonic acid (p-TsOH), and reacting under a nitrogen atmosphere under reflux, cooling to room temperature after the reaction is completed, and then adding 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ), stirring and reacting at room temperature to obtain a target product, a quinolone derivative for treating esophageal cancer by targeting ACSL4, and the reaction equation in the preparation process is:

[0013]

[0014] Where R 1 For H, R 2 For H, R 3 OCH 3 , R 4 For H, R 5 It is -OH.

[0015] It is further defined that the molar ratio of the substituted aromatic aldehyde, 2-aminobenzamide and 2,3-dicyano-5,6-dichlorobenzoquinone is 1:1-2:0.8-1.5; the ratio of the substituted aromatic aldehyde to the catalyst p-toluenesulfonic acid is 1 mmol:4-6 mg.

[0016] The present invention discloses an application of a quinolone derivative for treating esophageal cancer by targeting ACSL4 in the preparation of a drug for treating or / and preventing esophageal cancer. The quinolone derivative exerts an anti-esophageal cancer effect by targeting ACSL4. The quinolone derivative directly binds to the targeted ACSL4 to induce ferroptosis, thereby inhibiting esophageal cancer cells.

[0017] Further defined, the esophageal cancer is esophageal squamous cell carcinoma Eca-109, Ec-9706 and KYSE-150 cells.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention synthesizes for the first time a quinolone derivative for treating esophageal cancer by targeting ACSL4 and uses the synthesized quinolone derivative for treating esophageal cancer, especially esophageal squamous cell carcinoma. In addition, the present invention proposes for the first time that quinolone derivatives induce ferroptosis of esophageal cancer cells by directly targeting ACSL4. In vitro tests show that the quinolone derivatives prepared by the present invention show a strong inhibitory effect on esophageal squamous cell carcinoma Eca-109, Ec-9706 and KYSE-150 cells, and the therapeutic effect is dose-dependent and time-dependent. In vivo tests show that the quinolone derivatives prepared by the present invention show a significant effect of inhibiting tumor progression in the KYSE-150 cell tumor-bearing mouse model of esophageal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The IC values ​​of the five quinolone derivatives synthesized in Example 1 are 50 Value determination diagram.

[0020] Figure 2 This is a graph showing the apoptosis level of Eca-109, Ec-9706 and KYSE-150 cells in Example 2.

[0021] Figure 3 This is a graph showing the apoptosis ratio analysis of Eca-109, Ec-9706 and KYSE-150 cells in Example 2.

[0022] Figure 4 This is a graph showing the cell cycle changes of Eca-109, Ec-9706 and KYSE-150 cells in Example 3.

[0023] Figure 5 This is a graph showing the cell cycle ratio analysis of Eca-109, Ec-9706 and KYSE-150 cells in Example 3.

[0024] Figure 6 This is a graph showing the MDA level determination of Ec-9706 and KYSE-150 cells in Example 4.

[0025] Figure 7 This is a graph showing the determination of GSH levels in Ec-9706 and KYSE-150 cells in Example 4.

[0026] Figure 8 This is the in vivo inhibitory effect curve of high and low doses of quinolone derivatives in Example 4 on tumor-bearing mice inoculated with KYSE-150 cells.

[0027] Fig. 9 The weights of the tumors removed from the tumor-bearing mice inoculated with KYSE-150 cells after being treated with high and low doses of the quinolone derivatives in Example 4 were analyzed.

[0028] Fig.10 The toxicity test results of different doses of quinolone derivatives in Example 4 are shown.

[0029] Fig.11 This is the docking result of the quinolone derivative and the ferroptosis-related protein in Example 5.

[0030] Fig.12 The thermal shift test results of quinolone derivatives and ACSL4 in Example 6 are shown.

[0031] Figure 4 , Figures 6 to 12In the table, *, **, and *** indicate that the differences among different treatment groups were statistically significant, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0032] In order to illustrate the technical solution of the present invention, a series of embodiments are given below, which are completely illustrative and are only used to specifically describe the technical solution of the present invention, and should not be understood as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0033] Example 1

[0034] Design, synthesis and screening of quinolone derivatives

[0035]

[0036] Substituted aromatic aldehyde (386 mg, 2.0 mmol) and 2-aminobenzamide (408 mg, 3.0 mmol) were dissolved in 30 mL of ethanol solution, and then a catalyst p-toluenesulfonic acid (p-TsOH) (10 mg) was added. The mixture was refluxed for 6 h under nitrogen protection. After cooling to room temperature, 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ) (454 mg, 2.0 mmol) was added. The mixture was stirred at room temperature overnight and five novel quinolone derivatives were obtained by vacuum filtration (Table 1). The corresponding structural formulas of the five novel quinolone derivatives are shown below, and the product yield is 70% to 90%. The structures of the novel quinolone derivatives Qu-1 to Qu-5 are shown in Table 1. 1 H and 13 C nuclear magnetic resonance (NMR) confirmation.

[0037] Table 1

[0038]

[0039] KYSE-150 esophageal squamous cell carcinoma cells were cultured at 5 × 10 3 The cells were evenly plated in a 96-well cell culture plate and incubated at 37°C with 5% CO 2 Culture in an incubator for 24 hours. Set up 5 different quinolone derivative treatment groups, add a different quinolone derivative to each well, and repeat three times. After drug treatment, add CCK-8 reagent for color development. Detect OD value at 450nm wavelength on iMark microplate reader, and calculate cell growth inhibition rate IC 50 .

[0040] The experimental results are as follows Figure 1 shown. Figure 1 IC of five quinolone derivatives50 Value, the vertical axis in the figure is the IC of different derivatives 50 The horizontal axis is the value, and the horizontal axis is the five new quinolone derivatives synthesized. The experimental results show that the inhibitory effect of the quinolone derivative Qu-5 on the growth of esophageal cancer KYSE-150 cells is significantly stronger than that of the other four new quinolone derivatives.

[0041] Example 2

[0042] Quinolone derivative Qu-5 induces apoptosis in esophageal cancer cells

[0043] Esophageal squamous cell carcinoma Eca-109, Ec-9706 and KYSE-150 cells were cultured at 5×10 6 Each well was evenly plated in a 6-well plate. The cells in each well were treated with the following four treatments: (1) 0.1 wt% DMSO (Control); (2) 20 μM quinolone derivative Qu-5; (3) 40 μM quinolone derivative Qu-5; (4) 80 μM quinolone derivative Qu-5. The cells were cultured at 37°C and 5% CO 2 The cells were incubated in an incubator for 48 hours, then stained with Annexin V and propidium iodide (PI), and the proportion of apoptotic cells was determined by flow cytometry.

[0044] The experimental results are as follows Figure 2 and Figure 3 As shown, Figure 2 This is the measurement diagram of apoptosis level of Eca-109, Ec-9706 and KYSE-150 cells. Figure 3 The figure shows the apoptosis ratio analysis of Eca-109, Ec-9706 and KYSE-150 cells. The ratio of apoptotic cells after treatment with the quinolone derivative Qu-5 was significantly higher than that in the control group. This result further explains the mechanism of action of the novel quinolone derivative Qu-5 in inhibiting the growth of esophageal cancer Eca-109, Ec-9706 and KYSE-150 tumor cells.

[0045] Example 3

[0046] Quinolone derivative Qu-5 promotes cell cycle arrest in esophageal cancer cells at the G2 / M phase

[0047] Eca-109, Ec-9706 and KYSE-150 cells with good growth status were collected and plated at 5×10 6 Each well was evenly plated in a 6-well plate. The cells in each well were treated with the following four treatments: (1) 0.1 wt% DMSO (Control); (2) 20 μM quinolone derivative Qu-5; (3) 40 μM quinolone derivative Qu-5; (4) 80 μM quinolone derivative Qu-5. The cells were cultured at 37°C and 5% CO2 The cells were incubated in an incubator for 48 hours, then stained with Annexin V and propidium iodide (PI), and the cell cycle changes were determined by flow cytometry.

[0048] The experimental results are as follows Figure 4 and Figure 5 As shown, Figure 4 This is the measurement chart of cell cycle changes of Eca-109, Ec-9706 and KYSE-150. Figure 5 The figure shows the cell cycle ratio analysis of Eca-109, Ec-9706 and KYSE-150 cells. After treatment with the quinolone derivative Qu-5, the proportion of cells in the G2 / M phase in the drug-treated group increased in a dose-dependent manner compared with the blank control group. Therefore, the increase in the cell cycle ratio after treatment with the quinolone derivative Qu-5 further explains the mechanism of action of the novel quinolone derivative Qu-5 in inhibiting the growth of esophageal cancer Eca-109, Ec-9706 and KYSE-150 cells.

[0049] Example 4

[0050] Quinolone derivative Qu-5 induces ferroptosis in esophageal cancer cells

[0051] Iron ion accumulation and lipid peroxidation are the main characteristics of ferroptosis. Malondialdehyde (MDA) is a natural product of lipid oxidation in organisms, and its content can represent the occurrence of ferroptosis. The MDA level was determined using a lipid peroxidation MDA assay kit (Beyotime Biotechnology, China). KYSE-150 cells or Ec-9706 cells were lysed with a lysis buffer, centrifuged at 13000g for 15 minutes, and filtered with a 0.2μm pore size filter to obtain a clear supernatant for protein concentration determination. 0.1mL of lysis supernatant was mixed with 0.2mL of malondialdehyde detection working solution and incubated at 100℃ for 15 minutes. It was then cooled to room temperature and centrifuged at 1000g for 10 minutes at room temperature. Finally, 0.2mL of supernatant was added to a 96-well plate, and the absorbance was measured at 532nm using an ELISA reader.

[0052] The experimental results are as follows Figure 6 As shown, Figure 6 The figure shows the MDA level determination of Ec-9706 and KYSE-150 cells. After treatment with the quinolone derivative Qu-5, the MDA level of the drug-treated group was significantly increased compared with the blank control group. This effect was reversed after the use of ferroptosis inhibitors, indicating that the quinolone derivative Qu-5 promoted ferroptosis of esophageal cancer cells.

[0053] Example 5

[0054] Reduced glutathione is the main source of sulfhydryl groups in most living cells. It plays an important role in maintaining the proper redox state of sulfhydryl groups in proteins and is a key antioxidant in cells. The intracellular GSH level was measured using a GSH assay kit (Beyotime Biotechnology, China). The cell suspension of the collected KYSE-150 cells and Ec-9706 cells was centrifuged, and a protein removal reagent M solution (Beyotime Biotechnology, S0053-5) with a volume 3 times that of the cell pellet was added, and the mixture was fully vortexed, and then the samples were quickly frozen and thawed twice using liquid nitrogen and a 37°C water bath. Place at 4°C for 5 minutes, centrifuge at 10,000g for 10 minutes, and take the supernatant for the determination of total glutathione. Take part of the above-prepared samples for the total glutathione content to be measured, add the diluted GSH removal auxiliary solution according to the ratio of 20 μl of diluted GSH removal auxiliary solution per 100 μl of sample, and immediately vortex to mix. Then add GSH scavenging working solution at a ratio of 4 μl of GSH scavenging reagent working solution per 100 μl of sample, vortex mix immediately, and react at 25°C for 60 minutes. Add the supernatant to a 96-well plate and incubate with the corresponding total glutathione assay at room temperature for 25 minutes. Measure the absorbance at 412 nm. Calculate the GSH content based on the total glutathione content and GSSG content obtained by the determination (GSH = Total Glutathione-GSSG × 2).

[0055] The experimental results are as follows Figure 7 As shown, Figure 7 The figure shows the determination of GSH levels in Ec-9706 and KYSE-150 cells. After treatment with the quinolone derivative Qu-5, the GSH level in the drug-treated group was significantly lower than that in the blank control group, and this effect was reversed after the use of ferroptosis inhibitors, indicating that the quinolone derivative Qu-5 promoted ferroptosis in esophageal cancer cells.

[0056] Example 6

[0057] Quinolone derivative Qu-5 is used to inhibit tumor cell growth in vivo

[0058] KYSE-150 esophageal cancer cells were subcutaneously inoculated into BALB / c nude female mice. After the tumor cells were inoculated, the tumor tissue reached 100 mm. 3The mice were randomly divided into 3 groups, with 5 mice in each group: (1) blank control group (Control); (2) low-dose quinolone derivative Qu-5 group (50 mg / kg); (3) high-dose quinolone derivative Qu-5 group (100 mg / kg). The administration of Qu-5 was once every three days for a total of three weeks. During the administration process, the tumor growth was observed every three days, and the tumor size and weight were measured to evaluate the tumor inhibition effect of the drug.

[0059] The experimental results are as follows Figure 8 and Fig. 9 shown. Figure 8 The vertical axis is tumor volume, and the horizontal axis is days. Fig. 9 The vertical axis is the weight of the tumor after the drug administration, and the horizontal axis is the different treatment groups. The experimental results show that in the KYSE-150 esophageal cancer xenograft model, the high and low dose groups of quinolone derivatives showed a more significant effect of inhibiting tumor progression compared with the control group, and the differences were statistically significant.

[0060] Example 7

[0061] Toxicity test of quinolone derivatives

[0062] During the administration of the drug to tumor-bearing mice, the weight changes of the tumor-bearing mice in the three treatment groups were monitored to evaluate the drug toxicity of the quinolone derivative Qu-5, including the following three treatment groups: (1) blank control group (Control); (2) low-dose quinolone derivative Qu-5 group (50 mg / kg); (3) high-dose quinolone derivative Qu-5 group (100 mg / kg). The experimental results are as follows Fig.10 The results showed that the average body weight of mice was not significantly reduced compared with the blank control group, regardless of whether it was treated with low or high doses of Qu-5, which means that the quinolone derivative Qu-5 did not have potential toxicity.

[0063] Example 8

[0064] The potential binding target of the quinolone derivative Qu-5 is ACSL4

[0065] The target protein crystal structure is from the Protein Data Bank (https: / / www.rcsb.org / ), GPX4 (PDBID: 5H5Q), COX2 (PDB ID: 5z62), ACSL4 (simulated protein: AF-O60488-F1-model_v4, UniProt), SLC7A11 (PDB ID: 7epz). After deleting irrelevant small molecules and water molecules in the protein molecule using Pymol 2.6 software, the protein molecule was imported into AutoDock Tools-1.5.7 software, hydrogen atoms were added, and the atom type was set. The compound structure was energy minimized by Chem3D, and the small molecule compound was imported into AutoDock Tools-1.5.7 software, and atomic charges and atomic types were added, and all flexible bonds were rotatable by default. Finally, molecular docking was performed by AutoDock, and the molecular docking results were analyzed. The binding interaction between the compound and the protein was visualized using Pymol2.6 software.

[0066] The results are as follows Fig.11 As shown in the docking results with ferroptosis proteins, the binding energies of the four proteins (ACSL4, COX2, SLC7A11, and GPX) with the quinolone derivative Qu-5 are all lower than -5 kcal / mol, indicating that they all have potential binding effects. Since ACSL4 has the lowest binding energy with the quinolone derivative Qu-5, ACSL4 is considered to be the target of the quinolone derivative Qu-5.

[0067] Example 9

[0068] ACSL4 is the direct target of the novel quinolone derivative Qu-5

[0069] KYSE-150 and EC-9706 cells in good condition were plated in 6-well plates. When the cells were in the logarithmic growth phase, they were treated with quinolone and DMSO for 24 hours respectively. The cells were digested with trypsin for 3 minutes, and the digestion was terminated with complete medium. Subsequently, the mixture was collected in a 1.5mL ep tube and heated in a constant metal bath for 10 minutes at the specified temperature. Centrifuge at 800rpm for 5 minutes, discard the supernatant, resuspend in PBS and repeat centrifugation. The centrifuged precipitate was lysed with lysis buffer for 30 minutes, then centrifuged at 4℃12000rpm for 15 minutes, and the supernatant was retained for WB detection.

[0070] The results are as follows Fig.12 As shown in the results, after treatment with quinolone derivatives, the degradation of ACSL4 was significantly improved compared with the control group. Therefore, ACSL4 is the direct target of the quinolone derivative Qu-5.

[0071] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which all fall within the scope of the present invention to be protected.

Claims

1. A quinolone derivative for treating esophageal cancer by targeting ACSL4, characterized in that: The structural formula of the quinolone derivative is: Wherein R1 is H, R2 is H, R3 is -OCH3, R4 is H, and R5 is -OH.

2. A method for preparing a quinolone derivative for treating esophageal cancer by targeting ACSL4 according to claim 1, characterized in that The specific steps are as follows: dissolving the substituted aromatic aldehyde and 2-aminobenzamide in an ethanol solution, adding a catalyst of p-toluenesulfonic acid, and reacting under reflux in a nitrogen atmosphere. After the reaction is completed, the mixture is cooled to room temperature, and then 2,3-dicyano-5,6-dichlorobenzoquinone is added. The mixture is stirred at room temperature to react to obtain the target product, which is a quinolone derivative for treating esophageal cancer by targeting ACSL4. The reaction equation in the preparation process is: Wherein R1 is H, R2 is H, R3 is -OCH3, R4 is H, and R5 is -OH.

3. The method for preparing a quinolone derivative for treating esophageal cancer by targeting ACSL4 according to claim 2, characterized in that: The molar ratio of the substituted aromatic aldehyde, 2-aminobenzamide and 2,3-dicyano-5,6-dichlorobenzoquinone is 1:1-2:0.8-1.5; the ratio of the substituted aromatic aldehyde and the catalyst p-toluenesulfonic acid is 1mmol:4-6mg.

4. Use of the quinolone derivative for treating esophageal cancer by targeting ACSL4 as claimed in claim 1 in the preparation of a drug for treating or / and preventing esophageal cancer, wherein the quinolone derivative exerts an anti-esophageal cancer effect by targeting ACSL4, and the quinolone derivative directly binds to the targeted ACSL4 to induce ferroptosis, thereby achieving inhibition of esophageal cancer cells.

5. The use according to claim 4, characterized in that: The esophageal cancer cells are esophageal squamous cell carcinoma Eca-109, Ec-9706 and KYSE-150 cells.

Images