An m6a demethylase alkbh1 small molecule inhibitor, a synthetic method and application thereof
By synthesizing and screening small molecule inhibitors of the m6A demethylase ALKBH1 with specific chemical structures, the problem of lacking effective inhibitors in the existing technology has been solved, and specific inhibition of ALKBH1 protein and effective inhibition of lung cancer cells have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HENAN UNIVERSITY
- Filing Date
- 2024-11-04
- Publication Date
- 2026-04-10
AI Technical Summary
Current technologies lack effective small molecule inhibitors of ALKBH1 to regulate the activity of ALKBH1 protein, which affects the occurrence and development of lung cancer.
A small molecule inhibitor of m6A demethylase ALKBH1 with a specific chemical structure was synthesized. Effective small molecule compounds were screened using molecular docking technology, and the inhibitor was prepared using a specific synthetic method.
This small molecule inhibitor can specifically bind to the ALKBH1 protein and significantly inhibit its enzyme activity. In vitro experiments showed that it has a significant anti-proliferative effect on lung cancer cells, providing a new strategy for the treatment of lung cancer.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of pharmacy, more particularly to a small molecule inhibitor of m6A demethylase ALKBH1, a synthesis method and application. BACKGROUND
[0002] ALKBH1 is a member of the ALKB family, and ALKBH1 is mainly responsible for the methylation modification of DNA and RNA. FTO and ALKBH5 of the ALKB family mainly catalyze the demethylation of DNA m6A, and then affect the transcription and translation of downstream genes. It has been found that ALKBH1 also has the function of demethylation of nucleic acids, mainly catalyzing the 3-mec demethylation of ssDNA and RNA. With the deepening of research, it is found that ALKBH1 can regulate the demethylation of tRNA 1mA and affect the translation process of proteins. ALKBH1 regulates the 6mA demethylation of DNA, and then affects the development of astrocytoma. Silencing the ALKBH1 gene can cause DNA and RNA m6A modification, and then affect the occurrence and development of cancer. In recent years, the role of ALKBH1 and m6A in lung cancer has been explored in vivo and in vitro. Experiments in vivo and in vitro prove that ALKBH1 regulates the occurrence and development of lung cancer by regulating RNA m6A methylation modification. In vitro, silencing the ALKBH1 gene can inhibit the proliferation, migration and invasion ability of lung cancer cells A549. After overexpression of the ALKBH1 gene in vitro, the proliferation, migration and invasion ability of lung cancer cells A549 are significantly enhanced. It is proved that ALKBH1 indeed plays an important role in the occurrence and development of lung cancer. However, at present, it is necessary to develop a small molecule inhibitor of ALKBH1 with biological activity. SUMMARY
[0003] To solve the above problems, the present application provides a small molecule inhibitor of m6A demethylase ALKBH1, a synthesis method and application, which plays a role in the treatment of lung cancer.
[0004] The present application is realized by the following technical solutions:
[0005] In one aspect, the present application provides a small molecule inhibitor of m6A demethylase ALKBH1, which has a chemical structure as shown in formula (I):
[0006] .
[0007] In formula (I), the R group is:
[0008] 、 、 、
[0009] 、 、 、
[0010] 、 or .
[0011] The application further provides a synthesis method of the m6A demethylase ALKBH1 small molecule inhibitor, and specifically comprises the following steps:
[0012] The target compound required by the R group is weighed, dissolved in dimethylformamide, and then potassium carbonate and propargyl bromide toluene solution are added respectively, and mixed to obtain a first mixture.
[0013] The ratio of the target compound, dimethylformamide, potassium carbonate and propargyl bromide toluene solution is 366.24 mg-368.24 mg: 8 ml-10 ml: 226 mg-228 mg: 0.36 ml-0.38 ml.
[0014] The first mixture is stirred in argon for 12-13 hours, cooled to room temperature, and the solvent is removed to obtain an intermediate product.
[0015] The intermediate product is weighed, 3'-azido-3'-deoxythymidine, water and n-butanol are added, mixed, and then sodium ascorbate and copper sulfate pentahydrate are added to obtain a second mixture. The second mixture is stirred in argon for 3-4 hours, the liquid is collected, and the solvent is removed to obtain the m6A demethylase ALKBH1 small molecule inhibitor.
[0016] The ratio of the intermediate product, 3'-azido-3'-deoxythymidine, water and n-butanol solution, sodium ascorbate and copper sulfate pentahydrate is 300 mg-302 mg: 640.8 mg-642.8 mg: 4 ml-6 ml: 38 mg-40 mg: 10 mg-12 mg.
[0017] The target compound is: uracil, adenine, 5-fluorouracil, thymine, 6-azauracil, 5-bromo-6-azauracil, 4-amino-2,6-dihydroxy pyrimidine, 5-ethyluracil, 5-methoxy-2,4-dihydroxy pyrimidine.
[0018] Preferably, the volume ratio of propargyl bromide to toluene in the propargyl bromide toluene solution is 4-5:1.
[0019] Preferably, the volume ratio of water to n-butanol in the water and n-butanol solution is 1-2:1.
[0020] Preferably, the stirring temperature is 60-65°C.
[0021] Preferably, the m6A demethylase ALKBH1 small molecule inhibitor is used for preparing a therapeutic drug for lung cancer.
[0022] The m6A demethylase ALKBH1 small molecule inhibitor is used for preparing a drug for reducing the proliferation of an A549 lung cancer cell line.
[0023] Preferably, the m6A demethylase ALKBH1 small molecule inhibitor is used for treating a therapeutic drug caused by overexpression of ALKBH1.
[0024] Compared with the prior art, the present application has the following beneficial effects:
[0025] The present application provides a m6A demethylase ALKBH1 small molecule inhibitor. The m6A demethylase ALKBH1 small molecule inhibitor has biological activity, can dose-dependently bind to ALKBH1 protein in vitro, reduces ALKBH1 enzyme activity, has nanomolar level of inhibitory activity, and the effect of the small molecule inhibitor on lung cancer cells A549 is verified, which provides a broad application prospect for subsequent computer simulation screening, precise synthesis of the m6A demethylase ALKBH1 small molecule inhibitor and application in diseases caused by overexpression of ALKBH1. BRIEF DESCRIPTION OF DRAWINGS
[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.
[0027] Figure 1 is a chemical structure schematic diagram of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A of the present application;
[0028] Figure 2 is a synthesis route schematic diagram of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A of the present application;
[0029] Figure 2 , A is an intermediate product synthesis method; B is a synthesis method of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A;
[0030] Figure 3 is a nuclear magnetic resonance hydrogen spectrum diagram of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A of the present application;
[0031] Figure 4The nuclear magnetic resonance carbon spectrum of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A of the application is as follows:
[0032] Figure 5 The interaction site schematic diagram of the simulated m6A demethylase ALKBH1 small molecule inhibitor MCY-305A and ALKBH1 obtained based on virtual screening of the application is as follows:
[0033] Figure 6 The anti-proliferation effect of MCY-305A on different cell lines of human non-small cell lung cancer cells is detected by the MTT method of the application.
[0034] Figure 6 In the table, a is the cell proliferation result of MCY-305A on lung cancer cell line A549 after 24h treatment; b is the cell proliferation result of MCY-305A on lung cancer cell line A549 after 48h treatment; c is the cell proliferation result of MCY-305A on lung cancer cell line H1299 after 24h treatment; d is the cell proliferation result of MCY-305A on lung cancer cell line H1299 after 48h treatment, * indicates p <0.1, ** indicates p <0.01, *** indicates p <0.001, **** indicates p <0.0001.
[0035] Figure 7 The interaction schematic diagram of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A and ALKBH1 simulated based on AutoDock of the application is as follows. DETAILED DESCRIPTION
[0036] In order to facilitate the understanding of the present application, the present application will be described more fully below, and preferred embodiments of the present application will be given. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.
[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments, and are not intended to limit the present application.
[0038] The inventive concept of the present application is as follows:
[0039] The application carries out molecular docking between the chemical structure of formula (I) and the ALKBH1 protein structure in the PDB database, and for the first time finds that the chemical structure of formula (I) can be used as an m6A demethylase ALKBH1 small molecule inhibitor. The triazole ring in the small molecule structure is split into alkynyl and azide, and the two can obtain the nitrogen-containing five-membered ring structure in the target small molecule through a cycloaddition reaction, the azide structure is 3'-azido-3'-deoxythymidine, and the alkynyl structure can be obtained by reacting uracil with propargyl bromide. At the same time, according to other similar structures, the group connected by the methylene connected by the triazole ring can be used as a replacement group, and the rest can be used as the parent nucleus structure, and other similar small molecule compounds are synthesized, that is, the reactant uracil in the reaction with propargyl bromide is replaced by other target compounds to be synthesized, and then reacted with the azide structure to complete the replacement of the substituent group.
[0040] The technical solutions of the application will be further described below in combination with specific examples.
[0041] The virtual screening of the m6A demethylase ALKBH1 small molecule inhibitor is carried out by adopting the method of molecular docking of the compound and the active site of the protein structure by using Discovery Studio Client 2019 software. Firstly, the ALKBH1 crystal structure is selected from the PDB database, and the ALKBH1 crystal structure with X-RAY DIFFRACTION less than 3.00 angstroms is finally selected by fixed condition screening, PDB: 6MIC; secondly, the recent 2016 compound 2D structure database is selected from the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) in the United States; finally, the molecular docking is carried out by using the software. Firstly, the selection of the protein target is carried out: the obtained ALKBH1 crystal structure PDB format file is imported into the Discovery Studio Client 2019 software, the water molecules in the protein structure are deleted, the selected protein is processed by using the software "Clean Protein" function, the active site of the ALKBH1 crystal structure is automatically obtained by using the software "Define and Edit Binding Site" function, and the ideal site is selected as the target for subsequent docking; secondly, the preparation of the ligand is carried out: the obtained compound 2D structure database from the NCI-DTP database is imported into the Discovery Studio Client, the target compound is selected, and the target compound is prepared into the ligand required for molecular docking with the protein target by using the software "Prepare Ligands" function; finally, the molecular docking is carried out, and the docking result of the small molecule and the target is obtained: the protein file with the selected target is docked with the obtained ligand and the processed protein target by using the software Dock Ligands function, and the compound with better score is selected as the result obtained in the previous screening according to the docking result. It is found for the first time that the chemical structural formula with formula (I) can be used as the m6A demethylase ALKBH1 small molecule inhibitor.
[0042] .
[0043] In the structural formula (I), the R group is:
[0044] 、 、 、
[0045] 、 、 、
[0046] 、 or .
[0047] The preparation method of the m6A demethylase ALKBH1 small molecule inhibitor is as follows:
[0048] The target compound required by the R group is weighed in a Schlenk tube, dimethylformamide is added to dissolve it, then potassium carbonate and propargyl bromide toluene solution are added to the solution, mixed to obtain a first mixture.
[0049] The ratio of the target compound, dimethylformamide, potassium carbonate and propargyl bromide toluene solution is 366.24 mg-368.24 mg: 8 ml-10 ml: 226 mg-228 mg: 0.36 ml-0.38 ml.
[0050] The first mixture is stirred in argon, cooled to room temperature, and the solvent is removed under reduced pressure, and the desired intermediate product is obtained by column chromatography separation and purification.
[0051] The obtained intermediate product is weighed and added to a Schlenk tube, 3'-azido-3'-deoxythymidine, water and n-butanol are mixed, then sodium ascorbate and copper sulfate pentahydrate are added to obtain a second mixture, the second mixture is stirred in argon, after the reaction is completed, the liquid is collected by filtration, and the solvent is removed under reduced pressure. The residue is purified by column chromatography to obtain the desired small molecule compound with different R group substitution, i.e. the m6A demethylase ALKBH1 small molecule inhibitor.
[0052] The ratio of the intermediate product, 3'-azido-3'-deoxythymidine, water and n-butanol solution, sodium ascorbate and copper sulfate pentahydrate is 300 mg-302 mg: 640.8 mg-642.8 mg: 4 ml-6 ml: 38 mg-40 mg: 10 mg-12 mg.
[0053] The target compound is: uracil, adenine, 5-fluorouracil, thymine, 6-azauracil, 5-bromo-6-azauracil, 4-amino-2,6-dihydroxy pyrimidine, 5-ethyluracil, 5-methoxy-2,4-dihydroxy pyrimidine.
[0054] Example 1, synthesis of m6A demethylase ALKBH1 small molecule inhibitor
[0055] At the same time, refer to Figure 1 and Figure 2 , the chemical structure of the R group is , the specific route of synthesis is as follows:
[0056] (1) The electronic balance was used to weigh 3.27 mmol of uracil 366.24 mg, which was placed in a 25 ml Schlenk tube with a magnet, 8 ml of dimethylformamide was added for dissolution, then 226 mg of potassium carbonate and 0.36 ml of 80% propargyl bromide toluene solution were added, and the mixture was obtained. The volume ratio of propargyl bromide to toluene is 4:1.
[0057] (2) The first mixture was stirred at 60°C for 12h under argon atmosphere, and then cooled to room temperature. The solvent was removed under reduced pressure, and the intermediate product was obtained by column chromatography separation and purification with petroleum ether: ethyl acetate = 1:1.
[0058] (3) The electronic balance was used to weigh 2 mmol of the intermediate product obtained by column chromatography separation and purification 300 mg. 2.4 mmol of 3'-azido-3'-deoxythymidine 640.8 mg, water and n-butanol solution were added and mixed, and then 4 ml of mixed solution was weighed, wherein the volume ratio of water to n-butanol is 1:1. 0.02 mmol of 38 mg of sodium ascorbate, English name L-Ascorbic Acid Sodium Salt, and 0.04 mmol of 10 mg of copper sulfate pentahydrate, English name Copper sulfate pentahydrate, were added and mixed to obtain the second mixture. The second mixture was stirred at 60°C for 3h under argon atmosphere, and then cooled to room temperature. The liquid was collected by filtration, the solvent was removed under reduced pressure, and the residue was separated and purified by column chromatography with ethyl acetate: CH3OH = 1:1 to obtain 657 mg of m6A demethylase ALKBH1 small molecule inhibitor, with a yield of 78.8%.
[0059] Example 2, synthesis of m6A demethylase ALKBH1 small molecule inhibitor
[0060] Also refer to Figure 1 and Figure 2 The specific route for the synthesis of m6A demethylase ALKBH1 small molecule inhibitor is as follows:
[0061] (1) The electronic balance was used to weigh 3.27 mmol of uracil 366.24 mg, which was placed in a 25 ml Schlenk tube with a magnet, 8 ml of dimethylformamide was added for dissolution, then 226 mg of potassium carbonate and 0.36 ml of 80% propargyl bromide toluene solution were added, and the mixture was obtained. The volume ratio of propargyl bromide to toluene is 4:1.
[0062] (2) The first mixture was stirred at 65°C for 13h under argon atmosphere. After the reaction was completed, it was cooled to room temperature, and the solvent was removed under reduced pressure. The intermediate product was obtained by column chromatography purification with petroleum ether: ethyl acetate = 1:1.
[0063] (3) 302mg of the intermediate product obtained by column chromatography purification was weighed by an electronic balance with a substance amount of 2mmol. 3'-azido-3'-deoxythymidine 642.8mg, 2.4mmol, a mixture solution of water and n-butyl alcohol was weighed after mixing, and the volume ratio of water and n-butyl alcohol was 2:1. 6ml of the mixture solution was weighed. 0.02mmol, 40mg of sodium ascorbate, English name: L-Ascorbic Acid Sodium Salt and 0.04mmol, 12mg of copper sulfate pentahydrate, English name: Copper sulfate pentahydrate were added and mixed to obtain a second mixture. The second mixture was stirred at 65°C for 3h under argon atmosphere. After the reaction was completed, it was cooled to room temperature, and the liquid was collected by filtration. The solvent was removed under reduced pressure, and the residue was purified by column chromatography with ethyl acetate: CH3OH = 1:1 to obtain 657mg of m6A demethylase ALKBH1 small molecule inhibitor with a yield of 78.6%.
[0064] The structure of the compound synthesized was confirmed by nuclear magnetic resonance hydrogen spectrum and carbon spectrum. The nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the compound were obtained, which indicated the number of H and C in the compound, respectively, so as to determine the structure. Nuclear magnetic resonance hydrogen spectrum: 1 H NMR (400 MHz, DMSO) δ 11.37-11.30 (m, 2H), 8.26 (s, 1H), 7.82-7.73 (m, 2H), 6.40 (t, J = 6.6 Hz, 1H), 5.60 (dd, J = 7.8, 1.9 Hz, 1H), 5.40-5.26 (m, 2H), 4.95 (s, 2H), 4.19 (q, J = 3.8 Hz, 1H), 3.70-3.58 (m, 2H), 2.75-2.59 (m, 2H), 1.80 (s, 3H). As Figure 3 indicated, 13 C NMR (101 MHz, DMSO) δ 164.22, 151.23, 150.91, 146.00, 143.16, 136.72, 123.73, 110.12, 101.77, 84.86, 84.32, 61.20, 59.78, 42.89, 37.56, 12.71, one carbon atom signal overlapped, as Figure 4 indicated.
[0065] The molecular formula of the m6A demethylase ALKBH1 small molecule inhibitor is: C17 H 19 N7O6.
[0066] The chemical structural formula of the m6A demethylase ALKBH1 small molecule inhibitor is as follows: Figure 1 As shown, the synthesis route is as follows Figure 2 As shown, the small molecule inhibitor of m6A demethylase ALKBH1 is named MCY-305A using the initials of the synthesizer's name and the sequence number of the experiment.
[0067] Example 3: Antiproliferative effect of MCY-305A on human non-small cell lung cancer cell lines
[0068] The effect of the small molecule inhibitor MCY-305A, an inhibitor of the m6A demethylase ALKBH1, on the proliferation of lung cancer cells A549 and H1299 was detected using the MTT assay. 100 μl of cell suspension in logarithmic growth phase A549 and H1299 cells were used to detect the proliferation of lung cancer cells A549 and H1299 at 5 × 10⁻⁶ m² / mL. 3 Cells were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator. After cell attachment, different concentrations of the m6A demethylase ALKBH1 small molecule inhibitor MCY-305A were added to each well, with 6 replicates per group. After 24 h and 48 h of treatment, 150 μl of MTT solution with a final concentration of 0.5 mg / ml was added to each well, and incubation continued for 4 h. After incubation, the culture medium in the wells was discarded, and 150 μl of DMSO solution was added to each well. The mixture was shaken until the crystals were completely dissolved, and the absorbance of each well at 490 nm was measured using a microplate reader. The effect of different concentrations of the synthesized ALKBH1 small molecule inhibitor MCY-305A on the proliferation of lung cancer cells A549 and H1299 was calculated based on the absorbance values.
[0069] Experimental results are as follows Figure 6 As shown, Figure 6 In this context, 'a' represents the cell proliferation result of lung cancer cell line A549 after 24 hours of treatment with MCY-305A; Figure 6 In the figure, b represents the cell proliferation result of lung cancer cell line A549 after treatment with MCY-305A for 48 hours; Figure 6 In this context, 'c' represents the cell proliferation result of lung cancer cell line H1299 after 24 hours of treatment with MCY-305A; Figure 6 In the figure, 'd' represents the cell proliferation result of lung cancer cell line H1299 after treatment with MCY-305A for 48 hours. The results showed that MCY-305A had anti-proliferative effects on both A54 and H1299 lung cancer cell lines, with a significant anti-cancer cell proliferation effect on A549 lung cancer cell line, indicating that the ALKBH1 small molecule inhibitor MCY-305A has biological activity.
[0070] Example 4: Molecular docking of MCY-305A
[0071] First, after obtaining the MCY-305A through virtual screening in Discovery Studio, the software's functions were used to gain a preliminary understanding of the integration between the MCY-305A and the ALKBH1. Figure 5 The diagram shows the interaction site between the simulated m6A demethylase ALKBH1 small molecule inhibitor MCY-305A and ALKBH1, obtained through virtual screening. The binding mode of MCY-305A and ALKBH1 was then accurately analyzed using AutoDock. Molecular docking analysis of MCY-305A and m6A demethylase ALKBH1 was performed using AutoDock 1.5.7 software. The optimal binding and interaction modes of MCY-305A and ALKBH1 were visualized and screened using AutoDock 1.5.7 and Pymol. The chemical structure of MCY-305A was processed using AutoDock 1.5.7, and molecular docking was performed with the ALKBH1 protein structure (PDB: 6MIC). The optimal docking mode was selected from the docking results, with a binding energy of 7.45 kcal / mol. The optimal docking mode was visualized using Pymol. Figure 7 As shown, MCY-305A has one binding site with amino acid residue LYS-182 of ALKBH1 (PDB: 6MIC), one binding site with VAL-232, one binding site with ARG-234, and three binding sites with SER-235, forming hydrogen bonds. Molecular docking and Pymol visualization analysis using AutoDock provided evidence for the action of MCY-305A on ALKBH1, indicating that the amino acid residues obtained by Pymol visualization are the key sites for the pharmacological effect of MCY-305A on ALKBH1.
[0072] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0073] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. An application of a small molecule inhibitor of m6A demethylase ALKBH1 in the preparation of a drug for treating a disease caused by overexpression of ALKBH1, characterized in that, The m6A demethylase ALKBH1 small molecule inhibitor has a chemical structural formula as shown in formula (I): ； In formula (I), the R group is: 、 、 、 、 、 、 , or .
2. Use according to claim 1, wherein The synthesis method of the m6A demethylase ALKBH1 small molecule inhibitor specifically comprises the following steps: The target compound required for the R group is weighed, dissolved in dimethylformamide, and then potassium carbonate and propargyl bromide toluene solution are added respectively, mixed to obtain a first mixture; The ratio of the target compound, dimethylformamide, potassium carbonate and propargyl bromide toluene solution is 366.24mg-368.24mg:8ml-10ml:226mg-228mg:0.36ml-0.38ml; The first mixture is stirred in argon for 12h-13h, cooled to room temperature, the solvent is removed, and the intermediate product is obtained by purification; The intermediate product is weighed, 3'-azido-3'-deoxythymidine, water and n-butanol are added, mixed, and then sodium ascorbate and copper sulfate pentahydrate are added to obtain a second mixture, and the second mixture is stirred in argon for 3h-4h, the liquid is collected, and the solvent is removed to obtain the 6mA demethylase ALKBH1 small molecule inhibitor; The ratio of the intermediate product, 3'-azido-3'-deoxythymidine, water and n-butanol solution, sodium ascorbate and copper sulfate pentahydrate is 300mg-302mg:640.8mg-642.8mg:4ml-6ml:38mg-40mg:10mg-12mg; The target compound is: uracil, adenine, 5-fluorouracil, thymine, 6-azauracil, 5-bromo-6-azauracil, 4-amino-2,6-dihydroxy pyrimidine, 5-ethyluracil, and 5-methoxy-2,4-dihydroxy pyrimidine.
3. Use according to claim 2, wherein the compound is ###0002### The volume ratio of propargyl bromide to toluene in the propargyl bromide toluene solution is 4-5:
1.
4. The use according to claim 2, wherein the compound is ###0002### The volume ratio of water to n-butanol in the water and n-butanol solution is 1-2:
1.
5. The use according to claim 2, wherein the compound is ###0002### The stirring temperature is 60°C-65°C.
6. The use according to claim 1, wherein The disease is lung cancer.