Application of compound in treatment of lung cancer

Abstract

The invention discloses an application of a compound in treatment of lung cancer, the compound is 9, 10-bis (chloromethyl) anthracene, the molecular formula of the compound is C16H12Cl2, the compound is combined with a G-quadruplex structure in cell genome DNA to induce a continuous DNA damage reaction, and the effect of treating the lung cancer is achieved by down-regulating an EGFR / PI3K / Akt / Erk signal channel, up-regulating pro-apoptotic protein and down-regulating anti-apoptotic protein. Therefore, proliferation of EGFR mutation non-small cell lung cancer cells is inhibited, and apoptosis of the cells is induced. The invention can overcome the defects in the prior art that the EGFR mutation NSCLC treatment means is easy to generate drug resistance, has large toxic and side effects and lacks new mechanism small molecule drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a compound in the treatment of lung cancer. Background Technology

[0002] Lung cancer is a leading cause of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer accounting for approximately 80%–85% of all lung cancers. Numerous studies have shown that activating mutations in the EGFR gene kinase domain (such as exon 19 deletion, L858R point mutation, and T790M mutation) occur more frequently in patients with lung adenocarcinoma and are one of the important molecular events driving tumor development and progression.

[0003] First-, second-, and even third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been widely used in the clinical treatment of EGFR-mutant NSCLC targeting EGFR activating mutations, significantly prolonging progression-free survival. However, with prolonged use, tumor cells often acquire drug resistance through various mechanisms such as secondary mutations, bypass activation, and epigenetic alterations, leading to a gradual weakening or loss of efficacy. Furthermore, existing EGFR-TKIs still have drawbacks, including poor efficacy in some patients, significant side effects, and high costs associated with long-term use.

[0004] In recent years, the G-quadruplex (G4), a unique higher-order nucleic acid structure found in telomeres and promoter regions of various oncogenes, has been considered a potential target for antitumor drugs. Small molecule ligands targeting the G-quadruplex can stabilize or perturb this structure, thereby affecting telomere maintenance and oncogene transcriptional activity, inhibiting tumor cell growth through another pathway. However, currently available small molecule G-quadruplex ligands still have room for improvement in terms of selectivity, activity intensity, and clarity of mechanism of action. There is still a lack of small molecule candidates with good selectivity and a clearly defined pathway of action for EGFR-mutant NSCLC, along with their application methods.

[0005] Therefore, there is an urgent need to develop a small molecule compound with a well-defined structure, a clear mechanism of action, high inhibitory activity against EGFR-mutant NSCLC, and relatively low toxicity to normal cells, and to establish corresponding in vitro application and evaluation methods to provide a basis for subsequent drug development. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a compound in the treatment of lung cancer, in order to overcome the shortcomings of existing treatments for EGFR-mutant NSCLC, such as the tendency to develop drug resistance, significant toxic side effects, and the lack of novel small molecule drugs with new mechanisms.

[0007] To achieve the above objectives, the following technical solution is adopted: The application of a compound in the treatment of lung cancer, said compound being 9,10-bis(chloromethyl)anthracene, with the molecular formula C 16 H 12 Cl2, chemical formula as follows:

[0008] The compound binds to the G-quadruplex structure in the cellular genomic DNA, inducing a sustained DNA damage response. It inhibits the proliferation of EGFR-mutant non-small cell lung cancer cells and induces apoptosis by downregulating the EGFR / PI3K / Akt / Erk signaling pathway, upregulating pro-apoptotic proteins, and downregulating anti-apoptotic proteins.

[0009] A medicament comprising an active ingredient, a small molecule compound ML-01 or a pharmaceutically acceptable derivative thereof, and a carrier, wherein the small molecule compound ML-01 is 9,10-bis(chloromethyl)anthracene with the molecular formula C 16 H 12 Cl2, chemical formula as follows:

[0010] The drug is used to treat EGFR gene-mutant non-small cell lung cancer, and it exerts its therapeutic effect by inhibiting the proliferation of EGFR gene-mutant non-small cell lung cancer cells and / or inducing apoptosis.

[0011] Furthermore, the EGFR gene-mutated non-small cell lung cancer is lung adenocarcinoma carrying an EGFR kinase domain mutation, wherein the EGFR kinase domain mutation includes at least one of the following: L858R point mutation, exon 19 deletion mutation, and T790M mutation.

[0012] Furthermore, the pharmaceutically acceptable derivative refers to a compound obtained by forming a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, and / or crystal form without altering the anthracene ring core of ML-01 and its chloromethyl substituents at positions 9 and 10.

[0013] Furthermore, the drug is formulated into a dosage form suitable for oral or injectable administration, wherein the dosage form is selected from at least one of tablets, capsules, granules, oral solutions, oral suspensions, injections, and lyophilized powder for injection; when the drug is an injection or lyophilized powder for injection, the pharmaceutically acceptable carrier includes one or more of the following: water for injection, sodium chloride, buffer salts, cosolvents, isotonic adjusters, pH adjusters, and antioxidants.

[0014] Furthermore, the drug induces a sustained DNA damage response by binding ML-01 to the G-quadruplex nucleic acid structure in the genomic DNA of tumor cells, and downregulates the EGFR / PI3K / Akt / Erk signaling pathway, as well as upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins, thereby inhibiting the proliferation of EGFR-mutant non-small cell lung cancer cells and inducing apoptosis.

[0015] By adopting the above solution, the beneficial effects of the present invention are: This invention is the first to apply 9,10-bis(chloromethyl)anthracene (ML-01) as an active ingredient in an in vitro inhibition and apoptosis induction method for EGFR-mutant NSCLC. Through STD-NMR, ESI-MS and molecular docking, it was demonstrated that ML-01 can specifically interact with telomere G-quadruplex sequences and guanylic acid or form adducts, thus enriching the types and structural basis of G-quadruplex-targeted antitumor small molecule ligands at the molecular level. Attached Figure Description

[0016] Figure 1 This is the STD spectrum; Figure 2 Figure 1 shows the ESI-MS experimental results. Figure 3 Figure showing the results of the molecular docking experiment; Figure 4 The results of the MTT experiment are shown in the figure. Figure 5 Apoptosis was determined by flow cytometry using annexin V-FITC and PI staining. Figure 6 The graph shows the levels of intracellular calcium ions and ROS in ML-01 cells as detected by flow cytometry. Figure 7 To detect apoptosis-related proteins using Western blotting; Figure 8 Mapping of proteins related to EGFR and its downstream pathways using Western blotting; Figure 9 This is a transcriptome sequencing diagram; Figure 10 To detect c-Myc and its downstream pathway-related proteins using Western blotting. Detailed Implementation

[0017] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0018] This invention provides the application of a compound in the treatment of lung cancer, said compound being 9,10-bis(chloromethyl)anthracene, with the molecular formula C. 16 H12 Cl2, chemical formula as follows:

[0019] The compound binds to the G-quadruplex structure in the cellular genomic DNA, inducing a sustained DNA damage response. It inhibits the proliferation of EGFR-mutant non-small cell lung cancer cells and induces apoptosis by downregulating the EGFR / PI3K / Akt / Erk signaling pathway, upregulating pro-apoptotic proteins, and downregulating anti-apoptotic proteins.

[0020] The following discusses the in vitro experimental study of ML-01 against NSCLC: I. Nuclear Magnetic Resonance (STD-NMR) Experiment: 1) Sample preparation NMR sample preparation (volume 240 µL): Prepare 126 µL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄). pH 7.4), 60 µL D2O, 20 µL ammonium acetate (150 mM), 32 µL ML-01 (from 10 mM DMSO-d6 solution, final molar concentration (1.34 mM). Prepare 2 µL of Tel23 G-quadruplex sequence (from 400 µM solution, final molar concentration 3.34 µM).

[0021] dsDNA sample preparation (volume 240 µL): Same as step 1 of the NMR sample preparation described above; Prepare 2 µL of dsDNA sequence (from 400 µM solution, final molar concentration: 3.34 µM), wherein, The sequences are shown in the following table:

[0022] Table 1 Sequence List 2) Experimental Procedure STD-NMR experiments were performed on a Bruker Ascend spectrometer (frequency: 400.13 MHz, 1 hour) using the stddiffesgp.3 pulse program with a ligand / target ratio of 400:1. Two experiments were conducted, recording 45,376 scans with saturation times (d20) of 2 seconds and relaxation delays (d1) of 2.5 seconds. The water signal was suppressed for 1 hour by excitation engraving using a pulse sequence with perfect echo (zgesgppe). The STD effect for each proton in each sample was calculated and correlated with a reference spectrum (REF) at a partial resonance saturation of δ = -40 ppm. The STD amplification factor was calculated using the following formula: , Where ISTD and IREF are the signal intensities in the STD and REF spectra, respectively, and [L]tot and [T]tot are the concentrations of the ligand and target, expressed in molar equivalents. Resonance spectra were precisely recorded by irradiation at δ = 11 ppm by saturating the DNA sequence in the imine region.

[0023] II. Electrospray Ionization Mass Spectrometry (ESI-MS) Analysis 1) Sample preparation The stock solution of the compound was prepared in methanol by thermal denaturation and folding in 150 mM ammonium acetate, resulting in a final concentration of 5 μM for the oligonucleotide in 150 mM ammonium acetate, with a compound / oligonucleotide ratio of 5:1. The guanosine monophosphate (GMP) ligand was prepared in methanol at a compound / GMP ratio of 5:1, with a final ligand concentration of 25 μM.

[0024] 2) Experimental Procedure The precise mass of the Tel23 G-quadruplex sequence was 7270.774 Da. The precise mass of the dsDNA was 10987.922 Da. Samples were acquired after a 30-minute equilibration period, and methanol was added to obtain a stable ESI signal. Mass spectra were recorded via direct injection into an ESI LCQ Fleet ion trap mass spectrometer. The instrument was set to negative ionization mode, capillary voltage of 3.4 kV, capillary temperature of 120°C, and flow rate of 5 μL / min. For the interaction study between ESI-MS and guanosine monophosphate (GMP), the instrument was set to positive ionization mode, capillary voltage of 5.5 kV, capillary temperature of 180°C, and flow rate of 5 μL / min. As a reference, samples containing only GMP or only the ligand were obtained as negative controls.

[0025] III. Molecular Docking Experiment 1) Experimental steps Atomic coordinate profiles were retrieved from the RCSB Protein Database (PDB, www.rcsb.org): the selected 3D structures were 2HY9 (telomere G-quadruplex sequence) and 109D (dsDNA). These structures were prepared using the protein preparation wizard included in the Schrödinger suite with default settings, namely, adding hydrogen, removing water from ligands at distances greater than 5 Å, adjusting charge, covering the ends, and optimizing hydrogen bond clusters. The ligands were prepared for docking with the LigPrep tool under an OPLS4 force field. The docking scheme consisted of rigid acceptor / flexible ligand docking, performed using Glide under an OPLS4 force field with the default settings: a scaling factor of 0.80 for ligand van der Waals radii, a scaling factor of 0.15 for partial charge cutoff, and one position per ligand. This grid was prepared using Receptor Grid Generation included in Schrödinger (SchrödingerRelease 2023-3: Maestro, Schrödinger, LLC, New York, NY, 2023).

[0026] IV. Cell Viability Detection 1) Cell lines The human lung cancer NCI-H1975 cell line used in the experiment was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human lung cancer PC-9 cell line was obtained from Hangzhou Normal University, China.

[0027] 2) Preparation of MTT assay solution The final concentration of the MTT solution is 5 mg / ml. Dissolve 1 g of MTT powder in 200 ml of double-deionized water (ddH2O). The specific procedure is as follows: Add 200 ml of ddH2O to a 250 ml beaker. Pipette 1 ml of ddH2O into a small tube containing the MTT powder, stir several times, and then transfer it to the beaker. Place a stir bar in the beaker and mix thoroughly with a magnetic stirrer, ensuring complete darkness throughout the process. After thoroughly mixing the MTT, filter through a 0.22 μm filter membrane to remove bacteria. Aliquot the solution, protect from light, wrap in aluminum foil, and store at -20°C. Preparation of 1xPBS buffer: Dissolve one packet of PBS instant granules in 1000 mL of ddH2O. After complete dissolution, it contains 10 mM phosphate, 137 mM NaCl and 2.7 mM KCl, and the pH value is 7.4 at 25℃. Preparation of RPMI 1640 complete medium: Add 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin (PS) to RPMI 1640 basal medium and mix well before use; Cell cryopreservation solution preparation: sterile dimethyl sulfoxide (DMSO): fetal bovine serum (FBS) at a ratio of 1:9, that is, 1 mL of DMSO is added to 9 mL of FBS and mixed well before use.

[0028] 3) Experimental steps of MTT detection method Remove the supernatant, add 5 mL of 1xPBS buffer for washing, and digest NCI-H1975 or PC-9 cells with trypsin. After terminating digestion, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell concentration was adjusted to 5 × 10⁴ / mL by cell counting. After preparing the cell suspension, mix it gently. Set up 3-6 auxiliary wells for each concentration, add 100ul to each well to make the density of cells to be tested 5000 cells / well. Fill the edge wells with sterile 1xPBS buffer. The inoculated cell culture plates were placed in an incubator with 5% CO2 at 37°C and cultured until the cells adhered on the second day. Then, different concentrations of drugs were prepared, and the experiment was set up with 5 gradient concentrations. The original culture supernatant in the 96-well plate was removed, and 100 μL of culture medium containing different concentrations of drugs was added. Incubate with 5% CO2 at 37°C for 24 hours, and observe the drug's effects under an inverted microscope. Add MTT solution, 10 μL of MTT solution (5 mg / ml) to each well, and continue incubation at 37°C for 4 hours with 5% CO2. After terminating the culture, dissolve the crystals in dimethyl sulfoxide (DMSO); remove the supernatant, taking care not to remove the formazan crystals during this process. Add 100 μL of DMSO to each well and shake on a shaker at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance (OD value) of each well at 490 nm using a microplate reader.

[0029] 4) Cell cryopreservation experimental procedures Remove the supernatant, add 5 mL of 1xPBS buffer for washing, digest H1975 or PC-9 cells with trypsin, and collect the cells by centrifugation at 1500 rpm for 5 minutes after stopping digestion. Add 1 mL of cell freezing solution to each plate of cells, mix well, place in cryovials, freeze at 4°C for 30 minutes, then freeze at -40°C for 1 hour, and finally store in liquid nitrogen.

[0030] 5) Statistical analysis Statistical analysis of data was performed using Graph Pad Prism 8 software for student t-tests or one-way ANOVA. All experiments were replicated in triplicate and expressed as mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant.

[0031] V. Flow Cytometry 1) Cell lines The human lung cancer NCI-H1975 cell line used in the experiment was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human lung cancer PC-9 cell line was obtained from Hangzhou Normal University, China.

[0032] 2) Preparation of experimental solutions 1xPBS buffer: Same as above; RPMI 1640 complete medium: Same as above; Preparation of 1x Binding Buffer: Dilute 0.5 mL of stock binding buffer (Annexin V-FITC kit, Milltenyi Biotec, Germany) with 9.5 mL of dd H2O to obtain 1x binding buffer; Annexin V-FITC and propidium iodide (PI) staining solution preparation: For every 50,000-100,000 cells resuspended, add 5 μL Annexin V-FITC and 10 μL propidium iodide staining solution to 95 μL of 1x binding buffer. The amount can be adjusted according to the number of cells. Preparation of working solution for DCFH-DA probe for reactive oxygen species detection: Dilute the DCFH-DA probe with serum-free basal culture medium at a ratio of 1:2000 according to the recommended concentration in the instruction manual to make a final concentration of 5 μM. Preparation of Fluo-4 calcium ion detection solution: Take an appropriate amount of Fluo-4 AM stock solution (2mM) and dilute it with 1x PBS buffer to a working solution of 5μM.

[0033] 3) Flow cytometry experimental procedures and steps (I) Detection of apoptosis by assay The effects of Annexin V-FITC and propidium iodide (PI) reagents on cell apoptosis levels were detected by flow cytometry, as follows: NCI-H1975 or PC-9 cells were digested with trypsin. After digestion was terminated, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell count was then adjusted to reach 10⁵ cells / mL. Add the prepared cell suspension to a 6-well cell culture plate, adding 2 mL of cell suspension to each well to make the cell count in each well reach 2 x 10⁵. The 6-well cell culture plates were placed in an incubator with 5% CO2 at 37°C and cultured. After the cells adhered on the second day, the supernatant was removed, and the cells were washed with 1 mL of 1xPBS solution per well. Different concentrations of the prepared drug solution were added to each well, and the plates were then placed back into the incubator for another 24 hours. Cell collection: Collect the supernatant from each well, place it into the correct 15 mL centrifuge tube according to the label, wash with 1 mL of 1xPBS buffer per well and collect; Digest each well of cells with trypsin. After digestion is stopped, transfer the cells to a 15 mL centrifuge tube labeled with the same label. Centrifuge at 1500 rpm for 5 minutes to collect the cells. Wash twice with 1 mL of 1xPBS buffer. The collected cells were resuspended in the prepared Annexin V-FITC and propidium iodide (PI) staining solution (5 μL Annexin V-FITC + 10 μL propidium iodide + 95 μL 1x binding buffer) and incubated at room temperature in the dark for 15 minutes. After incubation, add 0.4 mL of 1x binding buffer to the cells, mix well, and filter into a flow cytometry tube to form a single-cell suspension, preventing cell clumps from affecting the flow cytometry. The samples were collected and quantitatively analyzed using a CytoFLEX flow cytometer. Data such as apoptosis percentage and images were obtained using FlowJo software.

[0034] (II) The experimental steps for detecting reactive oxygen species in cells by flow cytometry are as follows: NCI-H1975 or PC-9 cells were digested with trypsin. After digestion was terminated, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell count was then adjusted to reach 10⁵ cells / mL. Add the prepared cell suspension to a 6-well cell culture plate, adding 2 mL of cell suspension to each well to make the cell count in each well reach 2 x 10⁵. The 6-well cell culture plates were placed in an incubator with 5% CO2 at 37°C and cultured. After the cells adhered on the second day, the supernatant was removed, and the cells were washed with 1 mL of 1xPBS buffer per well. Different concentrations of the prepared drug solution were added to each well, and the plates were then placed back into the incubator for another 24 hours. Cell collection: Remove the supernatant and wash with 1 mL / well of 1xPBS buffer. Digest each well of cells with trypsin. After digestion is stopped, transfer the cells to a labeled 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 minutes to collect the cells. Wash twice with 1 mL of 1xPBS buffer. Add 100 μL of the DCFH-DA probe working solution for reactive oxygen species detection (5 μM) diluted with basal medium to each cell tube, mix well and incubate in the dark for 30 minutes. After incubation, add 0.4 mL of RPMI 1640 basal medium, mix well, and filter into a flow cytometry tube to form a single-cell suspension, preventing cell clumps from affecting machine collection. The samples were collected and quantitatively analyzed using a CytoFLEX flow cytometer. The relevant data, such as the percentage of reactive oxygen species and images, were obtained using FlowJo software.

[0035] (III) The experimental steps for detecting cellular calcium ions by flow cytometry are as follows: NCI-H1975 or PC-9 cells were digested with trypsin. After digestion was terminated, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell count was then adjusted to reach 10⁵ cells / mL. Add the prepared cell suspension to a 6-well cell culture plate, adding 2 mL of cell suspension to each well to make the cell count in each well reach 2 x 10⁵. The 6-well cell culture plates were placed in an incubator with 5% CO2 at 37°C and cultured. After the cells adhered on the second day, the supernatant was removed, and the cells were washed with 1 mL of 1xPBS buffer per well. Different concentrations of the prepared drug solution were added to each well, and the plates were then placed back into the incubator for another 24 hours. Cell collection: Remove the supernatant and wash with 1 mL / well of 1xPBS buffer. Digest each well of cells with trypsin. After digestion is stopped, transfer the cells to a labeled 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 minutes to collect the cells. Wash twice with 1 mL of 1xPBS buffer. Add 100 μL of Fluo-4 AM calcium working solution (5 μM) diluted with 1x PBS buffer to each cell tube, mix well, and incubate in the dark for 30 minutes. After incubation, add 0.4 mL of 1xPBS buffer, mix well, and filter into a flow cytometry tube to form a single-cell suspension, preventing cell clusters from aggregating and affecting the flow cytometry. The samples were collected and quantitatively analyzed using a CytoFLEX flow cytometer. The relevant data, such as the percentage of calcium ions in the cells and images, were obtained using FlowJo software.

[0036] 4) Statistical analysis Statistical analysis of data was performed using Graph Pad Prism 8 software for student t-tests or one-way ANOVA. All experiments were replicated in triplicate and expressed as mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant.

[0037] VI. Detection of specific protein expression using Western blot method 1) Cell lines The human lung cancer NCI-H1975 cell line used in the experiment was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human lung cancer PC-9 cell line was obtained from Hangzhou Normal University, China.

[0038] 2) Preparation of experimental solutions 1xPBS buffer: Same as above; RPMI 1640 complete medium: Same as above; Preparation of 10% sodium dodecyl sulfate (SDS) solution: Weigh 10g of sodium dodecyl sulfate powder on an electronic balance, add it to 100mL of ddH2O, and mix it with a magnetic stir bar on a magnetic stirrer. The solution is easy to precipitate at low temperatures. Before use, it needs to be heated in a 37℃ constant temperature water bath until the solution is completely clear. Preparation of 10% ammonium persulfate (APS) solution: Weigh 1g of ammonium persulfate on an electronic balance, add it to 10mL of ddH2O, mix well before use, dispense into containers, and store at 4℃ or low temperature. Preparation of 1M Tris-HCl (pH 6.8) solution: Weigh 60.5g of Tris Base using an electronic balance, add ddH2O and mix well. Adjust the pH value to 6.8 using a laboratory pH meter, and bring the solution to a final volume of 500mL. Preparation of 1.5M Tris-HCl (pH 8.8) solution: Weigh 181.5g of Tris Base using an electronic balance, add ddH2O and mix well. Adjust the pH value to 8.8 using an experimental pH meter, and bring the solution to a final volume of 1000mL. Preparation of protein lysis buffer (RIPA): Pour 10 mL of protein lysis buffer stock solution into a 15 mL centrifuge tube, add 1 protease inhibitor tablet, melt it, and add the appropriate dose according to the cell volume. After aliquoting, store at 4°C or low temperature. BCA protein assay reagent preparation: Prepare the dosage according to the number of measurement wells. The mixing ratio of solution A to solution B is 50:1. The amount of solution A and B used is 175 μL / well. Prepare and use immediately and protect from light. Preparation of 5x running buffer: Weigh 30.2 g of Tris Base, 188 g of glycine, and 10 g of SDS using an electronic balance, add ddH2O and bring the volume to 2 L. Mix well on a magnetic stirrer. Preparation of 1x electrophoresis buffer (Running Buffer): Dilute 5x electrophoresis buffer with ddH2O by 5 times to obtain 1x electrophoresis buffer; Preparation of 10x transfer buffer: Weigh 60.6g of Tris Base and 288g of glycine using an electronic balance, add ddH2O and bring the volume to 2 L, then mix well on a magnetic stirrer; Preparation of 1x transfer buffer: Pour 100mL of 10x transfer buffer and 200mL of methanol into a 1L bottle, and add ddH2O to bring the volume to 1L. Preparation of 10x Tris buffer solution (TBS): Weigh 160g NaCl + 60g Tris Base + 4g KCl using an electronic balance, add ddH2O and mix well. Adjust the pH value to 7.4 using an experimental pH meter, and bring the solution to a final volume of 2L. Preparation of 1xTris buffer solution (TBST): Pour 100mL into a 1L bottle, add ddH2O and bring the volume to 1L, then add 1mL of Tween-20, mix well and use. Chemiluminescent substrate (ECL) working solution preparation: Mix component A and component B in a 1:1 volume ratio. It is recommended to prepare it fresh for use. After mixing, it can be stored stably at 4°C for 7 days.

[0039] 3) SDS-PAGE gel preparation Pair thick and thin glass plates to form a tank, fix it on a gel casting frame, and add ddH2O to the tank to check for leaks. Mix the separating gel and stacking gel preparation materials from the table below in centrifuge tubes of appropriate capacity. First, pour the separating gel into the appropriate position in the tank, then gently add the stacking gel. Insert a 1.5mm 15-hole comb and allow it to solidify for 30-45 minutes before use.

[0040]

[0041] Table 2. Preparation of Resolving Gel (Quantity for 1 Gel Piece)

[0042] Table 3. Preparation of 5% Stacking Gel (dosage for 1 gel) 4) Experimental operation methods and procedures (a) Protein sample preparation NCI-H1975 or PC-9 cells were digested with trypsin. After digestion was terminated, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell count was then adjusted to reach 10⁵ cells / mL. Add the prepared cell suspension to a 6-well cell culture plate, adding 2 mL of cell suspension to each well to make the cell count in each well reach 2 x 10⁵. The 6-well cell culture plates were placed in an incubator with 5% CO2 at 37°C and cultured. After the cells adhered on the second day, the supernatant was removed, and the cells were washed with 1 mL of 1xPBS buffer per well. Different concentrations of the prepared drug solution were added to each well, and the plates were then placed back into the incubator for another 24 hours. Collect the supernatant from each well and place it into the correct 15 mL centrifuge tube according to the label. Wash with 1 mL of 1xPBS buffer per well and collect. Place the precipitate into a labeled 1.5 mL centrifuge tube, wash twice with 1xPBS buffer, add 50 μL of protein lysis buffer, and lyse on ice for 30 minutes. Add 200 μL of protein lysis buffer to a 6-well cell culture plate and incubate on ice for 30 minutes for lysis. Use a cell scraper to scrape off any cells that have not been completely lysed and collected in a 1.5 mL centrifuge tube (containing supernatant) with the same label. Centrifuge at 13,000 rpm for 15 minutes at 4°C. Prepare a clean 1.5mL centrifuge tube and label it. Aspirate all the supernatant from the tube and transfer it into a new 1.5mL centrifuge tube. Discard any remaining precipitate. The concentration of total protein extract for each sample was determined using the BCA protein assay kit. After dilution with SDS-PAGE protein loading buffer (5X), the samples were heated at 100°C for 10 minutes in a dry bath. The denatured protein samples were then stored at -40°C.

[0043] (II) Protein sample loading and SDS-PAGE gel electrophoresis The solidified SDS-PAGE gel-bonded glass plate was mounted on the electrophoresis tank. 1x electrophoresis buffer was added to the tank. After removing the comb, protein samples were loaded. The protein concentration was calculated to ensure that the loaded protein sample reached 20 μg. Markers and protein samples were added to the loading tank in sequence according to the loading order. Electrophoresis was performed at 80 volts for the stacking gel and 120 volts for the separating gel.

[0044] (III) Transfer and sealing Prepare 1x transfer buffer and a suitable-sized NC or PVDF membrane (activated by soaking in methanol before use). Stop electrophoresis when the protein sample is placed on the bottom layer of the SDS-PAGE gel. After prying open the glass plate, remove the SDS-PAGE gel and place it on the transfer clamp using the "sandwich method" (filter cotton-filter paper-gel-membrane-filter paper-filter cotton). Transfer the membrane at a constant current of 300 mA for 2 hours. After transfer, place the membrane in blocking buffer and incubate at room temperature for 1 hour.

[0045] (iv) Immunohistochemical reaction After blocking, the membrane was washed three times with 1xTBST for 5 minutes each time, and incubated overnight at 4°C with the primary antibodies against the target proteins: Total-EGFR, p-EGFR (Tyr1086), Total-PI3K (p110α), p-PI3K, Total-Akt, p-Akt (Ser473), Total-Erk (1 / 2), p-Erk (1 / 2), BAX, Bcl-2, XIAP, PARP, Cleaved Caspase-3, Cleaved Caspase-9, c-Myc, Total-P53, p-P53 (Ser15) (the above antibodies were diluted 1:1000), β-actin, and GAPDH (the above antibodies were diluted 1:500). The next day, the relevant primary antibody was recovered, and the membrane was washed three times. Then, the corresponding secondary antibody (dilution ratio 1:5000) was added and incubated at room temperature for 1 hour. The secondary antibody was recovered, and the membrane was washed three times.

[0046] (v) Development Chemiluminescent substrate (ECL) working solution was dropped onto the membrane, and the intensity of the protein bands was visualized using an Amersham Imager 600 (AI600) scanner.

[0047] VII. Transcriptome Sequencing 1) Cell lines The human lung cancer NCI-H1975 cell line used in the experiment was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the human lung cancer PC-9 cell line was obtained from Hangzhou Normal University, China.

[0048] 2) Preparation of transcriptome sequencing solution 1xPBS buffer: Same as above; RPMI 1640 complete medium: Same as above.

[0049] 3) Transcriptome sequencing related steps NCI-H1975 or PC-9 cells were digested with trypsin. After digestion was terminated, the cells were collected by centrifugation at 1500 rpm for 5 minutes to prepare a cell suspension. The cell count was then adjusted to reach 10⁵ cells / mL. Add the prepared cell suspension to a 10cm cell culture plate, adding 10mL of cell suspension to each well to make the number of cells in each well reach 106. Place 10cm cell culture dishes in an incubator with 5% CO2 at 37℃ and incubate. After the cells adhere to the wall on the second day, remove the supernatant and wash with 5mL / well of 1xPBS buffer. Add different concentrations of the prepared drug solution to each well and continue incubating for 24 hours. Cell collection: Remove the supernatant and wash with 1 mL / well of 1xPBS buffer. Digest each well of cells with trypsin. After digestion is stopped, transfer the cells to a labeled 15 mL centrifuge tube and centrifuge at 1500 rpm for 5 minutes to collect the cells. Wash twice with 1 mL of 1xPBS buffer. Transfer the cells to 1.5 mL centrifuge tubes, add 1 mL of TRIzol reagent to each tube and mix thoroughly. Place the lysate containing cells and TRIzol at -80°C and transport it to Beijing Novogene Technology Co., Ltd. for transcriptome sequencing as soon as possible using dry ice. Library construction: Obtain mRNA, then randomly fragment the obtained mRNA with divalent cations in NEB Fragmentation Buffer, and construct the library according to NEB's general library construction method or strand-specific library construction method; Quantitative quality control: After library construction, preliminary quantification was performed using a Qubit2.0 Fluorometer to dilute the library to 1.5 ng / ul. Subsequently, the insert size of the library was detected using an Agilent 2100 bioanalyzer. Once the insert size met expectations, the effective concentration of the library was accurately quantified by qRT-PCR (effective concentration of the library is higher than 1.5 nM) to ensure library quality. On-machine testing: After the libraries pass the library test, the different libraries are pooled according to the effective concentration and the target amount of data to be sequenced and then subjected to Illumina sequencing.

[0050] VIII. Results of ML-01 in vitro experiments 1) STD NMR experimental results STD NMR is used to study the binding of small molecules to macromolecular targets in solution. Mechanistically, it offers the potential to understand interactions under conditions very similar to the physiological environment. STD relies on nuclear spin polarization transfer between the target and ligand. Because the behavior of bound and unbound ligands differs, it can also be used to target compounds with specific nucleic acid arrangements; ligands targeting G-quadruplexes have been proposed as antiproliferative drugs. In this study, we tested the interaction of this compound with dsDNA and the biologically relevant telomere G-quadruplex structure Tel23. STD spectroscopy showed ( Figure 1ML-01 exhibits weak interactions with structural nucleic acids in solution, but importantly, it demonstrates high binding to guanine-rich Tel23 sequences, with both targets showing very low calculated binding energies (-4.495 and -6.327 kcal / mol, respectively).

[0051] The signals at 8.5 and 7.6 ppm in the resonance spectra indicate a stronger interaction with the G-quadruplex associated with dsDNA (STDamp values ​​of 12.9 and 11.9, respectively). Green: Off-resonance spectrum of ligand-Tel23 sample; Yellow: Resonance spectrum of ligand-Tel23 sample; Red: Off-resonance spectrum of ligand-dsDNA sample; Blue: Resonance spectrum of ligand-dsDNA sample.

[0052] 2) ESI-MS experimental results ESI-MS analysis is another technique that can be used to study ligand-target interactions and can also be applied to assess the binding affinity of nucleic acids (Ribaudo, Scalabrin et al. 2016, Ribaudo, Ongaro et al. 2021). Even in this case, ML-01 did not exhibit strong interactions with dsDNA and G-quadruplexes because no direct binding peaks were observed in the mass spectrometry. This indicates a lack of strong non-covalent binding with structured nucleic acids. Given the chemical structure of the ligand with two reactive chloromethylene moieties, we cannot rule out the possibility that the compound could covalently modify nucleobases upon proximity to nucleic acids or attack by other nucleophiles. In this regard, the alkylation of guanine / guanosine by covalent drugs has been previously studied using mass spectrometry (Wickramanayake, Arbogast et al. 1985, Andrievsky, Sukhodubet et al. 1991, Ribaudo, Ongaro et al. 2020). Therefore, GMP was used as a nucleobase model in this study to assess its reaction with the compound. ESI-MS analysis revealed the formation of several covalent adducts, demonstrating that the compound can target such nucleophiles. Figure 2 This aspect may become crucial in guanine-rich sequences.

[0053] 3) Results of molecular docking experiments To support these observations, docking studies were conducted on different nucleic acid topologies, including telomere G-quadruplexes and dsDNA (Ongaro, Desiderati et al. 2022). Even though docking provides insights into binding modes, computational models suggest that ligands do not interact through complete nucleobase stacking (…). Figure 3 Furthermore, poor calculated binding energy values ​​(-4.495 and -6.327 kcal / mol) were retrieved for both targets.

[0054] 4) MTT Experiment Results To investigate the toxicity of ML-01 to human lung cancer H1975 and PC-9 cell lines, ML-01 was applied to both cell lines at concentration gradients (0, 0.25, 0.5, 1, 1.5, 2 μM) for 24 hours. The toxicity of ML-01 to the two cell lines was detected by the MTT assay. The IC50 of H1975 cells was 0.84 ± 0.16 μM, and the IC50 of PC-9 cells was 1.48 ± 0.34 μM. In addition, we also examined the cytotoxicity of ML-01 against the normal human lung epithelial cell line BEAS-2B. After treatment with the same drug concentration gradient for 24 hours, the IC50 of ML-01 against the BEAS-2B cell line was 2.23 ± 0.19 μM. The results showed that ML-01 dose-dependently inhibited the proliferation of EGFR mutant NSCLC cells in H1975 and PC-9 cell lines, with a more significant reduction in the activity of the H1975 cell line, and exhibited low cytotoxicity against normal lung epithelial cells. Figure 4 The results in the figure show that ML-01 has a concentration-dependent inhibitory effect on H1975 and PC-9 cells.

[0055]

[0056] Table 4 IC50 values ​​of ML-01 against EGFR-mutant H1975, PC-9 and normal lung epithelial cells BEAS-2B (24 hours) 5) ML-01 induces apoptosis in H1975 and PC-9 cells. To investigate whether ML-01 can induce apoptosis in NSCLC cells, thereby inhibiting the growth of H1975 and PC-9 cells, we treated H1975 and PC-9 cells with different concentrations of ML-01 (0, 0.25, 0.5, 1, 1.5, 2 μM) for 24 hours. The proportion of apoptotic cells was then analyzed by flow cytometry using annexin V-FITC / PI staining. Figure 5The results showed that ML-01 induced apoptosis in both H1975 and PC-9 cell lines, with the proportion of apoptotic cells increasing in a concentration-dependent manner after ML-01 treatment. ML-01 treatment significantly increased the percentage of apoptotic cells: in the H1975 cell line, only 1.05% of cells in the control group were apoptotic; after ML-01 treatment, the percentage of apoptotic cells increased significantly to 2.95%, 4.85%, 26.37%, 31.24%, and 48.94%, respectively. In the PC-9 cell line, the percentage of apoptotic cells was 1.1%, 6.86%, 7.36%, 11.53%, 14.77%, and 33.83% in the control group, respectively.

[0057] Figure 5 The results showed that the percentage of EGFR-mutant H1975 and PC-9 apoptotic cells was significantly increased after 24 hours of treatment with ML-01 drug concentrations (0, 0.25, 0.5, 1, 1.5, 2 μM) (p<0.05, p<0.01, p<0.001).

[0058] We further investigated how ML-01 induces apoptosis and also examined the expression of intracellular calcium ions and ROS. In the experiment, different concentrations of the drug (0µM, 0.5µM, and 1µM) were applied for 24 hours, and quantitative data were collected using flow cytometry. Figure 6 A and Figure 6 The results from B show that the expression of intracellular calcium ions and ROS in H1975 and PC-9 cell lines increases with dosage.

[0059] Figure 6 In the figure, A represents the intracellular calcium ion expression in EGFR-mutant H1975 and PC-9 cells after 24 hours of treatment with ML-01 at concentrations (0, 0.5, 1 μM); Figure 6 B in the table represents the ROS expression in EGFR-mutant H1975 and PC-9 cells after treatment with ML-01 at concentrations (0, 0.5, 1 μM) for 24 hours. The results showed that ML-01 treatment promoted the increase of calcium ion and ROS expression in H1975 and PC-9 cells (p<0.05, p<0.01, p<0.001).

[0060] Flow cytometry analysis of apoptosis demonstrated that ML-01 promotes apoptosis. To further clarify the mechanisms and pathways of apoptosis, this study used Western blotting to detect apoptosis-related proteins, investigating the changes and expression of apoptosis proteins in H1975 and PC-9 cell lines exposed to ML-01. Bcl-2 family proteins play a crucial role in apoptosis. In H1975 and PC-9 cell lines treated with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 µM) of ML-01, after 24 hours, the expression of the anti-apoptotic protein Bcl-2 and the apoptosis-inhibiting protein XIAP decreased with increasing dosage, while the expression of the pro-apoptotic protein BAX was upregulated in a concentration-dependent manner. Figure 7 Furthermore, caspase is an enzyme related to the regulation of apoptosis. To investigate whether ML-01 has a potential impact on the caspase cascade, the promoter caspase 9 and its cleavage form were analyzed. In the experiment, an increase in the protein level of activated caspase 9 was observed in a concentration-dependent manner. Figure 7 The results showed that ML-01 upregulates the expression of the pro-apoptotic protein BAX and downregulates the expression of the anti-apoptotic protein Bcl-2 and the apoptosis inhibitor protein XIAP in H1975 and PC-9 cells through the intrinsic apoptosis pathway, thereby inducing caspase-dependent apoptosis in cells.

[0061] The expression of apoptosis-related proteins XIAP, Bcl-2, BAX, and Caspase 9 cleaved was investigated in H1975 and PC-9 cells after treatment with ML-01 at different concentrations (0, 0.25, 0.5, 1, 1.5, 2 µM) for 24 hours. The results showed that the expression of pro-apoptotic proteins BAX and Caspase 9 cleaved increased in H1975 and PC-9 cells, while the expression of anti-apoptotic proteins Bcl-2 and XIAP decreased, indicating that ML-01 promotes cell death through the intrinsic apoptosis pathway.

[0062] 6) ML-01 inhibits the activation of EGFR and its downstream pathways in NSCLC cells. EGFR mutations abnormally activate its downstream protein PI3K / AKT pathway and Erk pathway, playing an important role in the progression of NSCLC. We examined the effects on EGFR, Erk(1 / 2), Caspase3 cleaved, PI3K and AKT activation in H1975 and PC-9 cells. Figure 8The results showed that 24 hours after treatment with different concentrations of ML-01 (0, 0.25, 0.5, 1, 1.5, 2 µM), the phosphorylation of EGFR and Erk, as well as the phosphorylation of PI3K and AKT, were significantly downregulated, while the Caspase3 cleaved protein was significantly increased. This indicates that ML-01 prevents the activation of EGFR-induced Erk and PI3K / Akt signaling pathways, thereby inhibiting the proliferation of EGFR-dependent NSCLC cells and promoting cell death.

[0063] The expression levels of EGFR and its downstream pathway-related proteins, including Total-EGFR, p-EGFR (Tyr1086), Total-Erk(1 / 2), p-Erk(1 / 2), Caspase3 cleaved, Total-PI3K(p110α), p-PI3K, Total-Akt, and p-Akt (Ser473), were detected in H1975 and PC-9 cells exposed to different concentrations (0, 0.25, 0.5, 1, 1.5, 2 µM) of ML-01 for 24 hours. The results showed that the expression of Caspase3 cleaved increased in both H1975 and PC-9 cells, while the expression of p-EGFR(Tyr1086), p-Erk(1 / 2), p-PI3K, and p-Akt (Ser473) decreased. This indicates that ML-01 inhibits the proliferation of EGFR-dependent NSCLC cells and promotes cell death.

[0064] 7) Transcriptomics sequencing revealed that ML-01 is associated with histone and DNA loss. To investigate the specific molecular mechanism of ML-01's killing effect on NSCLC cells, we performed transcriptome sequencing on both the control and treatment groups. Because ML-01 showed better therapeutic effects on H1975 cells, we selected H1975 cells for transcriptome sequencing to analyze changes in transcriptional levels. We compared samples treated with different concentrations (1 µM and 2 µM) with the control group. Figure 9 The obtained data were analyzed, and the intersection of the two groups' differences was found: in the control group and the 1µM group, the number of upregulated genes was 666 and the number of downregulated genes was 74; in the control group and the 2µM group, the number of upregulated genes was 1233 and the number of downregulated genes was 1020. Figure 9 (A) We performed drug concentration trend cluster analysis on the data ( Figure 9 In the B and C groups, differentially expressed genes were subjected to GO / KEGG enrichment analysis. Figure 9 (D, G) Figure 9 In the table, D and E represent GO enrichment analysis, and F and G represent KEGG enrichment analysis. D and F represent... Figure 9GO / KEGG enrichment analysis of B in E and G, where E and G are... Figure 9 GO / KEGG enrichment analysis of C in C. Figure 9 In the diagram, H represents the differentially intersecting gene-protein interaction network (PPI). Analysis of the PPI suggests that ML-01's regulation of cells may be related to the composition of nuclear histones. Genomic DNA is condensed into chromatin in the cell nucleus. Chromatin is composed of monomeric structural units called nucleosomes, which are DNA molecules surrounding an octamer core of histones (two copies each of histones H2A, H2B, H3, and H4) (Ren, Greenberg et al. 2022). Histones actively participate in DNA damage and repair processes in various ways. The results show that ML-01 can influence histone modifications, regulate the cell's response to DNA damage, cause gene instability, and thus promote cell death.

[0065] Figure 9 In the figure, A represents the number of differentially regulated protein genes, with upregulated genes shown in red and downregulated genes shown in blue. Figure 9 B and C in the table represent cluster analysis of drug concentration trends. Figure 9 Bubble charts of D and E values. GO enrichment analysis. Figure 9 F and G in the figure represent KEGG enrichment analysis. Figure 9 The horizontal axis represents -log10 (P value) for the GO / KEGG Term, and the vertical axis represents the name of the GO / KEGG Term. Each bubble represents the corresponding Term, and the size of the bubble represents the number of genes / pathways annotated to the GO / KEGG Term. The larger the bubble, the more enriched genes / pathways the Term has. Bubbles further to the right indicate a more significant Term, with colors from red to purple representing decreasing significance of enrichment. H. Differential Intersection Protein-Gene Interaction (PPI) Network Diagram: PPI results from the String database were input into Cytoscape software. The CytoHubba plugin was used to obtain node scores based on the Degree method. Finally, visualization was achieved using Gephi software. Bubble size represents decreasing significance of enrichment.

[0066] 8) ML-01 concentration-dependent downregulation of c-Myc protein levels promotes apoptosis. c-Myc is a gene encoding a transcription factor that plays a role in cell cycle progression, apoptosis, and cell transformation (Lin, Loven et al. 2012). The protein encoded by this gene is phosphorylated in the cell nucleus. To investigate whether ML-01 has any related effects on c-Myc and its downstream components, we examined the expression of c-Myc, Total-P53, p-P53 (Ser15), and PARP proteins. The results showed that after 24 hours of treatment with different concentrations (0, 0.25, 0.5, 1, 1.5, 2 µM) of ML-01 in H1975 and PC-9 cells, we found that ML-01 significantly reduced c-Myc expression while increasing the expression of p-P53 (Ser15) and PARP proteins. These results indicate that ML-01 promotes apoptosis induced after DNA damage by inhibiting c-Myc protein expression and upregulating p-P53 (Ser15) protein expression.

[0067] Figure 10 ML-01 was applied at concentrations (0, 0.25, 0.5, 1, 1.5, 2 µM) to H1975 and PC-9 cells for 24 hours. The expression of c-Myc and its downstream pathway-related proteins was then detected. ML-01 significantly downregulated the expression of c-Myc protein and significantly upregulated the expression of p-P53 (Ser15) and PARP proteins. The results indicate that ML-01 can inhibit the growth-inhibiting activity of c-Myc protein expression in NSCLC cells and promote cell death.

[0068] In addition, a medicament is provided, comprising an active ingredient, a small molecule compound ML-01 or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier, wherein the small molecule compound ML-01 is 9,10-bis(chloromethyl)anthracene with the molecular formula C 16 H 12 Cl2, chemical formula as follows:

[0069] The drug is used to treat EGFR gene-mutant non-small cell lung cancer. It exerts its therapeutic effect by inhibiting the proliferation of EGFR gene-mutant non-small cell lung cancer cells and / or inducing apoptosis. The drug induces a sustained DNA damage response by binding ML-01 to the G-quadruplex nucleic acid structure in the genomic DNA of tumor cells, and downregulates the EGFR / PI3K / Akt / Erk signaling pathway, as well as upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins, thereby inhibiting the proliferation of EGFR-mutant non-small cell lung cancer cells and inducing apoptosis.

[0070] The EGFR gene-mutated non-small cell lung cancer is lung adenocarcinoma carrying an EGFR kinase domain mutation, wherein the EGFR kinase domain mutation includes at least one of the L858R point mutation, exon 19 deletion mutation, and T790M mutation; the pharmaceutically acceptable derivative refers to a compound obtained by forming a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, and / or crystal form without altering the anthracene ring core of ML-01 and its chloromethyl substituents at positions 9 and 10.

[0071] Preferably, the drug is formulated into a dosage form suitable for oral or injectable administration, wherein the dosage form is selected from at least one of tablets, capsules, granules, oral solutions, oral suspensions, injections, and lyophilized powder for injection; when the drug is an injection or lyophilized powder for injection, the pharmaceutically acceptable carrier includes one or more of the following: water for injection, sodium chloride, buffer salts, cosolvents, isotonic adjusters, pH adjusters, and antioxidants.

[0072] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a compound in the treatment of lung cancer, characterized in that, The compound is 9,10-bis(chloromethyl)anthracene, with the molecular formula C2. 16 H 12 Cl2, chemical formula as follows: The compound binds to the G-quadruplex structure in the cellular genomic DNA, inducing a sustained DNA damage response. It inhibits the proliferation of EGFR-mutant non-small cell lung cancer cells and induces apoptosis by downregulating the EGFR / PI3K / Akt / Erk signaling pathway, upregulating pro-apoptotic proteins, and downregulating anti-apoptotic proteins.

2. A drug, characterized in that, The active ingredient includes a small molecule compound ML-01 or a pharmaceutically acceptable derivative thereof, and a carrier, wherein the small molecule compound ML-01 is 9,10-bis(chloromethyl)anthracene with the molecular formula C. 16 H 12 Cl2, chemical formula as follows: The drug is used to treat EGFR gene-mutant non-small cell lung cancer, and it exerts its therapeutic effect by inhibiting the proliferation of EGFR gene-mutant non-small cell lung cancer cells and / or inducing apoptosis.

3. The drug according to claim 2, characterized in that, The EGFR gene-mutated non-small cell lung cancer is lung adenocarcinoma carrying EGFR kinase domain mutations, wherein the EGFR kinase domain mutations include at least one of the following: L858R point mutation, exon 19 deletion mutation, and T790M mutation.

4. The drug according to claim 2, characterized in that, The pharmaceutically acceptable derivatives refer to compounds obtained by forming pharmaceutically acceptable salts, hydrates, solvates, prodrugs, stereoisomers and / or crystal forms without altering the anthracene ring core of ML-01 and its chloromethyl substituents at positions 9 and 10.

5. The drug according to claim 2, characterized in that, The drug is formulated into a dosage form suitable for oral or injectable administration, wherein the dosage form is selected from at least one of tablets, capsules, granules, oral solutions, oral suspensions, injections, and lyophilized powder for injection; when the drug is an injection or lyophilized powder for injection, the pharmaceutically acceptable carrier includes one or more of the following: water for injection, sodium chloride, buffer salts, cosolvents, isotonic adjusters, pH adjusters, and antioxidants.

6. The drug according to claim 2, characterized in that, The drug induces a sustained DNA damage response by binding ML-01 to the G-quadruplex nucleic acid structure in the genomic DNA of tumor cells, thereby downregulating the EGFR / PI3K / Akt / Erk signaling pathway, upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins, thereby inhibiting the proliferation of EGFR-mutant non-small cell lung cancer cells and inducing apoptosis.

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