A topoisomerase II inhibitor and its preparation method and application

Abstract

The present invention provides a topoisomerase II inhibitor, its preparation method, and application, belonging to the field of medicine. The present invention uses 6-bromopiperonal as a raw material to synthesize a compound having a structural formula as shown in Formula I. This compound is a novel topoisomerase II inhibitor that can be used to treat diseases such as small cell lung cancer, advanced metastatic ovarian cancer, colorectal cancer, and bladder cancer, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a topoisomerase II inhibitor and a preparation method and application thereof. Background Art

[0002] Cancer is the third leading cause of death after cardiovascular and cerebrovascular diseases, becoming a major obstacle to extending human lifespan. Furthermore, the incidence of cancer is increasing at a younger age, placing significant economic pressure on society. Small cell lung cancer (SCLC) is a malignant tumor with neuroendocrine characteristics, characterized by rapid proliferation, early metastasis, high malignancy, and rapid development of drug resistance. Due to its high cell proliferation rate, current drugs used for SCLC directly or indirectly target DNA synthesis, replication, and repair. The standard first-line treatment for SCLC is a combination of a platinum-based chemotherapy agent and the topoisomerase II inhibitor etoposide, established in 1985. The 2019 FDA approval of atezolizumab as a standard treatment marked a major milestone in SCLC treatment. SCLC responds to chemotherapy in up to 80%, but 90% of patients quickly develop drug resistance. For patients who develop resistance within three months, the topoisomerase I inhibitor topotecan is used as a second-line treatment. However, the response rate to topotecan is only approximately 20%. Consequently, in 2020, the FDA granted emergency approval to the RNA polymerase II inhibitor lurbinectedin for second-line treatment of SCLC. In 2021, the CDK4 / 6 inhibitor trilaciclib was approved to reduce the need for chemotherapy-induced bone marrow transplantation. Patients who develop drug resistance after three months still need to resume standard first-line treatment. Therefore, the search for new and effective anti-SCLC drugs is urgent.

[0003] Patent CN114456184B discloses a compound that has the inhibitory effect on both topoisomerase I and topoisomerase II, with the following structure:

[0004]

[0005] However, the compound showed IC 50 =15.24 μM, IC on NCI-H1048 50 =15.41μM, the inhibitory activity needs to be improved. Summary of the Invention

[0006] The purpose of the present invention is to provide a topoisomerase II inhibitor and its preparation method and application.

[0007] The present invention provides a compound represented by formula I, the structural formula of the compound is:

[0008]

[0009] Among them, R1 Selected from C 3～6 alkyl.

[0010] Furthermore, R 1 Selected from C 3～4 alkyl.

[0011] Furthermore, the compound is the following compound:

[0012] The present invention also provides a method for preparing the above compound, which comprises the following steps:

[0013]

[0014] Where R is R 1 As mentioned above;

[0015] (1) reacting 6-bromopiperonal, trimethylsilylacetylene, a catalyst, and a base to prepare compound L2;

[0016] (2) reacting compound L2 with a reducing agent to prepare compound L3;

[0017] (3) reacting compound L1 with a base to prepare compound L4;

[0018] (4) reacting compound L3, compound L4, a catalyst, and a base to prepare compound L5;

[0019] (5) reacting compound L5, CBr4, and PPh3 to obtain an intermediate compound, and then reacting the intermediate compound, an amine compound, and a base to obtain compound L6;

[0020] (6) Compound L6, a nitrogen source compound, and a catalyst are reacted to obtain a 3-arylisoquinoline derivative.

[0021] Furthermore, in step (1), the catalyst is cuprous iodide and tetrakis(triphenylphosphine)palladium; the base is triethylamine; the molar ratio of 6-bromopiperonal, trimethylsilyl acetylene, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:1.5-2.5:0.1-0.2:0.01-0.03; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, the reaction is carried out at 40-60°C for 5-7 hours;

[0022] In step (2), the reducing agent is sodium borohydride; the molar ratio of the compound L2 to the reducing agent is 1:1-3; the solvent of the reaction is an organic solvent; the reaction conditions are: reaction at -5-10°C for 1-15 minutes;

[0023] In step (3), the base is lithium diisopropylamide; the molar ratio of the compound L1 to the base is 1:1-1.5: the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, first react at -85--70°C for 1-2.5 hours, then react at 10-40°C for 5-20 hours;

[0024] In step (4), the catalyst is cuprous iodide and tetrakis(triphenylphosphine)palladium; the base is triethylamine; the molar ratio of compound L3, compound L4, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:1-1.2:0.1-0.3:0.15-0.25; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, the reaction is carried out at 70-90°C for 5-9 hours;

[0025] In step (5), the molar ratio of the compound L5, CBr4 and PPh3 is 1:1.5-2:1.5-2; the solvent of the reaction is an organic solvent; the reaction conditions are: reaction at -5-10°C for 0.1-1 hour; the amine compound is The base is triethylamine; the molar ratio of the intermediate compound, the amine compound and the base is 1:1-3:8-12; the reaction conditions are: reaction at 10-40°C for 5-20 hours;

[0026] In step (6), the nitrogen source compound is ammonium acetate; the catalyst is silver nitrate; the molar ratio of the compound L6, the nitrogen source compound and the catalyst is 1:2-4:0.1-0.5; the solvent of the reaction is an organic solvent; and the reaction conditions are: under argon protection, the reaction is carried out at 10-40°C for 4-15 hours.

[0027] Furthermore, in step (1), the molar ratio of 6-bromopiperonal, trimethylsilyl acetylene, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:2:0.12:0.021; the solvent of the reaction is tetrahydrofuran and triethylamine; and the reaction conditions are: under argon protection, the reaction is carried out at 50°C for 6 hours;

[0028] In step (2), the molar ratio of the compound L2 to the reducing agent is 1:2; the solvent of the reaction is methanol; the reaction conditions are: reaction at 0-4°C for 5-10 minutes;

[0029] In step (3), the molar ratio of the compound L1 to the base is 1:1.25: the solvent of the reaction is tetrahydrofuran; the reaction conditions are: under argon protection, first react at -78°C for 1.5 to 2 hours, then react at 15 to 35°C for 7 to 17 hours;

[0030] In step (4), the molar ratio of compound L3, compound L4, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:1.02:0.23:0.16; the solvent of the reaction is a mixture of tetrahydrofuran, acetonitrile and triethylamine in a volume ratio of 1:2:1; the reaction conditions are: under argon protection, the reaction is carried out at 80°C for 6 to 8 hours;

[0031] In step (5), the molar ratio of the compound L5, CBr4 and PPh3 is 1:1.7:1.7; the solvent of the reaction is dichloromethane; the reaction conditions are: reaction at -5 to 10°C for 0.1 to 1 hour; the molar ratio of the intermediate compound, amine compound and base is 1:2:10; the reaction conditions are: reaction at 15 to 35°C for 7 to 17 hours;

[0032] In step (6), the molar ratio of the compound L6, the nitrogen source compound and the catalyst is 1:3:0.3; the solvent of the reaction is a mixture of tetrahydrofuran and tert-butyl hydroperoxide in a volume ratio of 4:1; the reaction conditions are: under argon protection, the reaction is carried out at 20-35°C for 6-12 hours.

[0033] The present invention also provides use of the above compound in preparing topoisomerase inhibitors.

[0034] Furthermore, the topoisomerase is topoisomerase II.

[0035] Furthermore, the topoisomerase inhibitor is a drug for preventing and / or treating cancer and inhibiting cancer metastasis.

[0036] Furthermore, the cancer is selected from small cell lung cancer, advanced metastatic ovarian cancer, colorectal cancer, and bladder cancer.

[0037] The present invention also provides a pharmaceutical composition, which is a preparation prepared with the above compound as an active ingredient and pharmaceutically acceptable excipients.

[0038] The present invention has achieved the following beneficial effects:

[0039] Compound 52 synthesized in the present invention is a topoisomerase II inhibitor and can be used to treat diseases related to topoisomerase II activity (such as small cell lung cancer, advanced metastatic ovarian cancer, colorectal cancer, bladder cancer, etc.).

[0040] The present invention also found through experiments that compound 52 of the present invention has excellent inhibitory activity on SCLC cells, and IC 50 =0.6μM, IC on NCI-H1048 50 =0.1μM, compared with the compound in patent CN114456184B The activity in NCI-H446 and NCI-H1048 cells was increased by 30 and 150 times, respectively.

[0041] Experimental data showed that compound 52 can intercalate between DNA base pairs, significantly inducing DNA damage and arresting the NCI-H446 cell cycle at the S phase. Compound 52 not only significantly inhibited the proliferation of NCI-H446 and NCI-H1048 cells, but also significantly inhibited the invasion and migration of SCLC cells. Therefore, the compound of the present invention can be used to prepare drugs for treating small cell lung cancer and inhibiting its metastasis.

[0042] Experimental data also showed that compound 52 significantly induced cell apoptosis, significantly outperforming the active agent etoposide at its highest concentration. Compound 52 also demonstrated significantly enhanced SCLC inhibition activity both in vitro and in vivo compared to the active agent etoposide.

[0043] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0044] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 (A) Compound 52 inhibits the relaxation activity of topoisomerase II at a concentration of 100 μM; (B) Compound 52 inhibits the relaxation activity of topoisomerase II at a concentration of 50 μM; (C) Compound 52 and etoposide (vp-16) inhibit the relaxation activity of topoisomerase II at different concentration gradients.

[0046] Figure 2 Schematic diagram of the molecular docking binding mode of compound 52 and topoisomerase II (PBD: 5GWK).

[0047] Figure 3 Comet assay images of compound 52 and vp-16 (magnification 20×, scale bar represents 100 μm).

[0048] Figure 4Effects of compound 52 on SCLC cell proliferation: (A) Effects of compound 52 at the indicated concentrations on the clone formation of NCI-H446 and NCI-H1048 cells; (B) The inhibitory effect of compound 52 on cell proliferation was assessed by EdU incorporation assay, with an incubation time of 24 h (magnification 4×, scale bar represents 500 μm); (C) Cell proliferation ratios in NCI-H446 and (D) NCI-H1048, statistical analysis is expressed as mean ± SD (n = 3): (***) P < 0.001, (**) P < 0.01, (*) P < 0.05.

[0049] Figure 5 Compound 52 inhibits the migration and invasion of NCI-H446 and NCI-H1048 cells: (A) Transwell migration assay of NCI-H446 and NCI-H1048 cells after treatment with compound 52 for 24 hours; (B) the number of migrated cells in each experimental group and the percentage of migrated cells in the blank group; (C) Transwell invasion assay of NCI-H446 and NCI-H1048 cells after treatment with compound 52 for 24 hours (magnification: 10x, scale bar: 200 μm); (D) the number of invasive cells in each experimental group and the percentage of invasive cells in the blank group. Statistical analysis is expressed as mean ± standard deviation (n=3). Each treatment group was compared with the control group, and the significant differences were: (***) P<0.001, (**) P<0.01, (*) P<0.05.

[0050] Figure 6 (A) Representative micrographs of NCI-H446 and NCI-H1048 cells treated with compound 52 for 0 and 24 hours in a wound healing assay (magnification 4x, scale bar represents 500 μm); (B) Percentage of wound closure area in each experimental group of NCI-H446 and NCI-H1048 compared with that in the blank group.

[0051] Figure 7 Compound 52 blocks the SCLC cell cycle at the S phase: (A) Flow cytometric analysis of the cell cycle distribution of NCI-H446 and NCI-H1048 cells after treatment with compound 52 for 48 hours; (B) Quantitative analysis of cell cycle distribution at each stage.

[0052] Figure 8 Compound 52 significantly induced apoptosis of SCLC cells: (A) Effect of the compound on cell morphology was observed by Giemsa staining (magnification 10×, scale bar = 200 μm); (B) Flow cytometric analysis of cell apoptosis in NCI-H446 and NCI-H1048 cells after treatment with compound 52 for 48 hours.

[0053] Figure 9 The trend graph of ROS fluorescence intensity detected by flow cytometry is a graph showing the mitochondrial dysfunction caused by compound 52 in NCI-H446 and NCI-H1048 cells (A: single ROS fluorescence intensity of compound 52 at different ratios in NCI-H446 cells and NCI-H1048 cells; B: ROS fluorescence intensity of compound 52 at different ratios in NCI-H446 cells and NCI-H1048 cells were sorted separately).

[0054] Figure 10 Effects of compound 52 on the expression of apoptotic proteins in NCI-H446 and NCI-H1048 cells: (A) and (B) Immunoblotting was used to detect the expression of mitochondrial-related apoptotic proteins Bcl-2, Bax, cytochrome C, cleaved-caspase-9, and cleaved-caspase-3, with β-actin as an internal reference; (C) Statistical graph of the effect of compound 52 on apoptotic proteins in NCI-H446 cells (n=3); (D) Statistical graph of the effect of compound 52 on apoptotic proteins in NCI-H1048 cells (n=3).

[0055] Figure 11 NCI-H446 and NCI-H1048 cells were treated with different concentrations of compound 52 for 48 hours, and the expression of proteins related to the PI3K / Akt / mTOR signaling pathway was detected by immunoblotting: (A) and (B) The expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR was determined by Western blotting using β-actin as an internal reference; (C) and (D) Statistical analysis of the mean ± SD of three parallel experiments. Significant differences were observed between the drug-treated and blank groups: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05.

[0056] Figure 12 (A) Body weight trend of mice (n=4); (B) Effects of compound 52 administration on various organs of mice.

[0057] Figure 13 Figure 5 shows the inhibition of NCI-H446 xenograft tumor growth by compound 52 in vivo: (A) Changes in tumor volume over 21 days, expressed as mean ± SEM; (B) Trends in mouse body weight over 21 days, expressed as mean ± SD; (C) Histogram of tumor weight on the last day, expressed as mean ± SD; (D) Comparison of tumor volume of compound 52 and vp-16 with that of the control group on the last day, measured using a vernier caliper. DETAILED DESCRIPTION

[0058] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0059] The "room temperature" referred to in the present invention is 25±10°C, and "overnight" is 12±5 hours.

[0060] The preparation of compound 52 of the present invention was carried out according to the following synthetic route:

[0061]

[0062] Example 1. Synthesis of Compound 52 of the Invention

[0063]

[0064] (1) Synthesis of Compound L2: 6-Bromopiperonal (L0, 10.0 g, 40.82 mmol, 1.0 eq), cuprous iodide (CuI, 922 mg, 4.9 mmol, 0.12 eq), and tetrakis(triphenylphosphine)palladium ((PPh3)4Pd, 1.0 g, 0.87 mmol, 0.021 eq) were dissolved in triethylamine (Et3N, 40 mL, 1.0 M) and tetrahydrofuran (60 mL, 1.5 M). The mixture was placed in an anhydrous and oxygen-free argon (Ar2) atmosphere. After stirring at room temperature for 10 minutes, trimethylsilylacetylene (11.5 mL, 81.62 mmol, 2.0 eq) was added dropwise using a needle. The reaction system was heated to 50°C and stirred for 6 hours. Thin-layer chromatography (TLC) was used to monitor the reaction. After the reaction was completed, the solvent was partially removed by rotary evaporation. The residue was filtered through a Buchner funnel lined with filter paper and celite and rinsed several times with dichloromethane (DCM). The solvent was removed by rotary evaporation and then purified by column chromatography on silica gel using petroleum ether / ethyl acetate (20:1) as the eluent to obtain compound L2 (9.5 g, 38.78 mmol). Yield: 95% as a white solid. 1 H NMR (400MHz, CDCl3) δ10.36(s,1H),7.30(s,1H),6.93(s,1H),6.05(s,2H),0.25(s,9H); 13 C NMR (101MHz, CDCl3) δ190.19,152.24,148.81,132.64,123.44,112.32,105.90,102.40,101.06,99.93,-0.21.

[0065] (2) Synthesis of Compound L3: Compound L2 (2.0 g, 8.12 mmol, 1.0 eq) was dissolved in MeOH (20 mL, 0.4 M) and the mixture was placed in an ice bath (0-4°C). Sodium borohydride (NaBH4, 616 mg, 16.24 mmol, 2.0 eq) was then slowly added. The reaction was monitored by TLC until the reaction of the starting material was complete (5-10 min). Water was slowly added to quench the mixture in an ice bath and the mixture was brought to room temperature and stirred for 1-2 hours to obtain Compound L3 after removal of the trimethylsilyl (TMS) protective agent. The mixture was extracted with water and dichloromethane, and the organic phase was collected and the solvent removed. The mixture was purified by silica gel column chromatography using petroleum ether / ethyl acetate (20:1) as the eluent to obtain Compound L3 (1.7 g, 6.9 mmol). Yield: 85%; white solid. 1 HNMR (400MHz, CDCl3) δ7.09–6.76(m,2H),5.97(s,2H),4.72(s,2H),3.24(s,1H); 13 C NMR (101MHz, CDCl3) δ148.64,146.67,138.80,113.22,112.20,108.24,101.56,81.29,80.56,63.56.

[0066] (3) Synthesis of Compound L4: 5-Bromo-2H-1,3-benzodioxazole (Compound L1) (5.33 g, 3.20 mL, 26.40 mmol, 1.0 eq) was dissolved in THF (20 mL). The system was placed in an anhydrous and oxygen-free argon atmosphere at -78°C. After the system cooled, lithium diisopropylamide (LDA, 16.5 mL, 2.0 M in THF, 33 mmol, 1.25 eq) was added dropwise with a needle. After reacting for 1 hour, DMF (4.63 g, 4.9 mL, 63.36 mmol, 2.4 eq) was added dropwise and stirring was continued for 0.5-1 hour. The reaction was then brought to room temperature and stirred overnight. TLC was used to monitor the reaction until the reaction was complete. The system was placed in an ice bath and saturated NH4Cl was slowly added to quench the reaction. The mixture was then extracted with EtOAc. The organic phase was collected and purified by silica gel column chromatography using petroleum ether / ethyl acetate (10:1) as eluent to obtain compound L4 (5.49 g, 25.08 mmol) in a 95% yield as a white solid. 1 H NMR (400MHz, CDCl3) δ10.28 (s, 1H), 7.10 (d, J = 8.2Hz, 1H), 6.84 (d, J = 8.2Hz, 1H), 6.16 (s, 2H); 13C NMR (101MHz, CDCl3) δ190.98,150.09,149.33,126.77,118.00,116.18,114.16,103.97.

[0067] (4) Synthesis of Compound L5: Compound L3 (5.68 mmol, 1.0 g, 1.0 eq), L4 (1268 mg, 5.79 mmol, 1.02 eq), CuI (249 mg, 1.31 mmol, 0.23 eq), and Pd(PPh3)4 (1.0 g, 0.91 mmol, 0.16 eq) obtained above were placed in a round-bottom flask. The system was sealed with a three-way seal in an argon atmosphere and purged three times with an oil pump. THF (5 mL, 1.0 M), MeCN (10 mL, 0.5 M), and triethylamine (5 mL, 1.0 M) were then added to the system with a needle. The reaction was heated to 80°C for 6-8 hours. After completion of the reaction by TLC, the solvent was removed and the product was purified by silica gel column chromatography using PE / EtOAc (3:1) as the eluent to obtain Compound L5 (1.46 g, 4.54 mmol). Yield: 80%; yellow solid. 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),7.23(d,J=8.0Hz,1H),7.19(d,J=8.0Hz,1H),7.12(s ,1H),7.04(s,1H),6.25(s,2H),6.07(s,2H),5.30(t,J=5.7Hz,1H),4.63(d,J=5.6Hz,2H); 13 C NMR(101MHz,DMSO-d6)δ188.65,148.88,148.27,148.16,145.86,139.89,127.39,11 8.15,116.93,113.01,112.15,110.93,107.28,103.37,101.52,91.28,88.79,61.13.

[0068] (5) Synthesis of compound L6:

[0069] 1) Dissolve compound L5 (500 mg, 1.55 mmol, 1.0 eq) obtained above in DCM (16 mL, 1.0 M) and place the mixture in an ice bath. Add CBr4 (875 mg, 2.63 mmol, 1.7 eq) and PPh3 (690 mg, 2.63 mmol, 1.7 eq). After 0.5 hour of reaction, monitor by TLC until complete reaction. Filter through a Buchner funnel lined with filter paper or column chromatography using PE / EtOAc (5:1) to obtain the intermediate benzyl bromide (466.5 mg, 1.08 mmol). Yield: 70% as a yellow solid.

[0070] 2) Add the chain amine to the above intermediate bromide compound (65 mg, 0.15 mmol, 1.0 eq) (0.3 mmol, 2.0 eq), followed by the addition of Et3N (151 mg, 1.5 mmol, 10.0 eq), and stirring at room temperature overnight. After the reaction of the starting materials was completed, monitored by TLC, column chromatography using DCM / MeOH (40:1, 20:1) was performed to obtain compound L6 as a yellow solid.

[0071] (6) Synthesis of compound 52: The intermediate L6 (0.14 mmol, 1.0 eq) obtained above was dissolved in THF (6 mL, 0.025 M) with silver nitrate (AgNO3, 7 mg, 0.042 mmol, 0.3 eq) and ammonium acetate (NH4OAc, 32 mg, 0.42 mmol, 3.0 eq). Tert-butyl hydroperoxide ( t BuOH (1.5 mL, 0.1 M) was used as a co-solvent, and the system was placed under argon. After 6-12 hours at 20-35°C, TLC confirmed complete reaction. NaHCO₃ (35 mg, 0.42 mmol, 3.0 eq) was added and stirred for another 0.5 hour to quench the reaction. The solvent was then removed, and column chromatography was performed using DCM / MeOH (60:1 to 20:1) to afford a yellow solid, Compound 52, in a 42.39% yield with a melting point of 128.4-132.4°C. 1 H NMR (400MHz, CDCl3) δ9.31(s,1H),7.80(s,1H),7.75(s,1H),7.48(s,2H),7.01(s,1H),6.31(s,2H),6. 08(s,2H),4.53(s,2H),3.22–2.99(m,4H),1.71–1.50(m,4H),1.38–1.16(m,4H),0.84(t,J=7.3Hz,6H); 13CNMR (101 MHz, CDCl3) δ149.61,148.52,145.47,144.71,141.99,135.72,132.21,121.86,121.38,120.79,116.07,113.92,113.27,110.53,102.90,102.06,54.46,51.63,25.67,20.14,13.54; LRMS (ESI, m / z) theoretical value is C 26 H 31 N2O4 + [M+H] + 435.2278, actual value 435.0. Purity after LC-MS analysis was 95.22%.

[0072] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0073] Experimental Example 1: Activity test of compounds

[0074] 1. Experimental methods

[0075] (1) Cell culture

[0076] Seven human small cell lung cancer cell lines (NCI-H446, NCI-H196, NCI-H146, NCI-H82, NCI-H1048, NCI-H209, and SHP-77), human normal hepatocytes (LO2), and human bronchial epithelial cells (BEAS-2B) were obtained from CCTCC (China). NCI-H1048 was cultured in DMEM / F12 (1:1), LO2 and BEAS-2B in DMEM, and the remaining SCLC cells in RPMI1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (antibody). Cells were cultured in a constant temperature (37°C) and humidity incubator with 5% CO2, with the culture medium replaced every 1-2 days. When cells reach 80% confluency, for adherent cells, wash away any remaining culture medium with 2 mL of 1× PBS. Add 2 mL of trypsin and digest at room temperature for 0.5-2 minutes. Aspirate the trypsin and quickly add complete culture medium to terminate digestion. Gently dissipate the cells with a pipette tip to form a cell suspension. Passage at a density of 1 to 2 or 1 to 3. For suspended cells, gently dissipate any aggregated or settled cells with a pipette tip to form a cell suspension. Centrifuge at 800 rpm to pellet the cells, discard the original culture medium, and passage at a density of 1 to 2 or 1 to 3.

[0077] (2) MTT and CCK8 experiments

[0078] The cells to be tested were digested or collected according to the above method, and the cell concentration was adjusted to 5-8×10 3Cells / well are seeded into 96-well plates and placed in an incubator for overnight incubation. When the cells have grown to the logarithmic phase after attachment or stabilization, different concentrations of the test compound are added. Generally, 5 concentration gradients are set, 3 replicates are made, and the total volume of each well is 200 μL. The culture is continued for 72 hours. 10 μL of MTT solution or CCK8 solution with a concentration of 5 mg / mL is added, and the adherent cells are initially screened with MTT test. The CCK8 method is greener, simpler, and more accurate, but the cost is high. Therefore, compounds with good activity are screened by MTT or suspended cells are tested with CCK8. The CCK8 method generates water-soluble formazan. It only needs to continue incubation for 2 hours to measure the absorbance of the solution at a wavelength of 490 nm. Inhibition rate % = 1-(OD490 加药孔 –OD490 背景孔 ) / (OD570 对照孔 -OD570 背景孔 )×100%. The MTT method generates water-insoluble formazan and requires incubation for 4 hours. Then, the original culture medium is removed, 100 μL DMSO is added to dissolve it, and the absorbance is measured at a wavelength of 570 nm using a microplate reader. Inhibition rate % = 1-(OD570 加药孔 -OD570 背景孔 ) / (OD570 对照孔 -OD570 背景孔 ) × 100%. IC was calculated by IBM SPSS 50 Value, use express.

[0079] (3) Topoisomerase II inhibitory activity

[0080] To a 50 μL PV tube, add 20 μL of ultrapure water, 2 μL of buffer, 2 μL of 30 mM ATP solution (prepared with ultrapure water), 1 μL of 1 U topoisomerase II (Inspiralis, Norwich, 100 U), 0.2 μL of the test compound (not added for DNA and Topo II groups), and 0.2 μL of 0.5 μg / μL pBR322 plasmid DNA (Takara, 0.5 μg / μL) per well (due to the partial inactivation of Topo II due to long-distance transportation). The total volume was brought to 30 μL with ultrapure water. The remaining procedures were the same as for Topo I.

[0081] (4) Molecular docking

[0082] Molecular docking studies of compound 52 with Topo II were performed using Schrodinger software. First, the X-ray crystal structure of Topo II (PDB code: 5GWK) was obtained from the Protein Data Bank (PDB). Energy and hydrogen bond minimization were performed on the protein, and the docking box was generated using the Protein Preparation Wizard model. The molecular structure of the compound was constructed using Schrodinger software and minimized using the MMFFs force field to generate three-dimensional coordinates. Molecular docking was performed using XP, and all other docking parameters were kept at the default values.

[0083] (5) Comet Experiment

[0084] Comet assay was performed using the Comet Assay Alkaline Kit (Puhe Biopharmaceuticals, China). NCI-H1048 and NCI-H446 cells (5 × 10 4 Cells were seeded in 6-well plates (10 cells / mL). After the cells were allowed to adhere overnight in an incubator, they were treated with different concentrations of compound 52. After 48 hours, the cells were digested and collected, centrifuged at 1200 rpm for 5 minutes at 4°C, and resuspended in 1× PBS. 30 μL of high-melting-point agarose was quickly added to the slide and allowed to stand at room temperature for 10 minutes. The cell concentration was then adjusted to 1×10 5 For each cell / mL, 10 μL of cells was mixed with 60 μL of low-melting-point agarose and dropped onto the solidified high-melting-point agarose (a double layer of agarose allows for better adhesion and prevents slide slippage, but may affect imaging). The slides were then incubated at 4°C in the dark for 30 minutes. Subsequently, the slides were lysed with pre-chilled lysis buffer at 4°C in the dark for 30 minutes to lyse the cell and nuclear membranes. The slides were then washed twice with H2O, placed flat, for 5 minutes each time, and the DNA was melted with an alkaline solution for 20 minutes. Electrophoresis was performed in an electrophoresis tank filled with 1× TAE buffer at 110 V for 25 minutes. After washing, the slides were stained with 10 μL of PI solution for 20–30 minutes in the dark and then washed. Comet images were captured using an inverted fluorescence microscope (Nikon, Japan).

[0085] (6) Plate cloning experiment

[0086] NCI-H1048 and NCI-H446 cells were seeded into 6-well plates at a density of 1000 cells / mL and incubated overnight in a volume of 3 mL per well. Test compounds were then added at varying concentrations. After 2-3 weeks of incubation, the blank control group was observed to have formed more than 50 colonies. The culture medium was then discarded, the cells were washed with 1× PBS, and fixed with 4% paraformaldehyde for 20 minutes. The cells were then stained with 1% crystal violet for 30 minutes, washed with water to remove any residual stain, and the colonies were photographed and recorded.

[0087] (7) EdU test

[0088] NCI-H1048 and NCI-H446 cells were seeded into 96-well plates at a density of 5,000 cells / well. After overnight incubation, the cells were allowed to adhere and then tested compounds were added at varying concentrations, with triplicate wells per group. After 24 hours of incubation with the drug, the old culture medium was discarded and 100 μL of complete culture medium containing 10 μM EdU was added. The cells were incubated in an incubator for 2-4 hours. The culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 15 minutes. The cells were then washed three times with 1× BSA for 5 minutes each. After 100 μL of 0.5% Triton X-100 was added for 15 minutes, and then stained with Apollo stain for 30 minutes in the dark. After three washes, the nuclei were stained with Hoechst 33342 for 30 minutes in the dark. After washing with 1× BSA, the cells were observed and photographed under an inverted fluorescence microscope (Nikon, Japan).

[0089] (8) Cell invasion and migration assay

[0090] 1) Transwell migration assay

[0091] Logarithmic phase NCI-H1048 and NCI-H446 cells were obtained, trypsinized and collected, centrifuged at 800 rpm for 5 minutes at 4°C to remove the complete medium, and serum-free medium was used to make the concentration of 1-2×10 5 Cell suspension of cells / mL. Add 700 μL of culture medium containing 20% ​​serum to a 24-well plate, and place a transwell chamber (Corning 3422) in it. Access the cell suspension prepared above to the upper chamber, 200 μL per chamber, and then add different concentrations of the test compound. Place in a cell culture incubator and incubate for 24-36 hours, remove the chamber, wash with 1×PBS, add 600 μL of 4% paraformaldehyde to the lower chamber to fix the cells for 20 minutes, wash with 1×PBS, add 600 μL of 0.1% crystal violet stain to the lower chamber of each well, and stain at room temperature for 30 minutes. After removing the chamber and cleaning it, gently wipe off the non-migrated cells in the upper chamber with a cotton swab, observe under a microscope, and take pictures and record.

[0092] 2) Transwell invasion assay

[0093] Thaw Matrigel stored at -20°C overnight at 4°C. Mix the thawed Matrigel with serum-free medium at a volume ratio of 1:8. Add 50 μL to each upper chamber of a transwell insert. Spread evenly with a pipette tip and allow to solidify in an incubator for 3-5 hours until a gel forms. Rehydrate the basement membrane. The remaining steps are the same as for the migration assay.

[0094] 3) Scratch test

[0095] Logarithmically phase NCI-H1048 and NCI-H446 cells were trypsinized and seeded into 6-well plates. The cells were incubated until confluent. The culture medium was discarded, and a line of cells was scratched down the middle using a 10μL pipette tip. Cells and cell debris were washed with 1× PBS. Culture medium supplemented with 3% serum and various concentrations of the test compound were added. A reference point was selected and photographed under a microscope to record the scratch wound at 0 hours. After the cells were incubated for an additional 24 hours, the scratch wound was photographed under a microscope (Nikon, Japan). Images were analyzed using Image J.

[0096] (9) Cell cycle distribution

[0097] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate, with 2 mL per well. After cell attachment, the test compounds were added at various concentrations and treated for 48 hours. The cells were then trypsinized, harvested, centrifuged at 1200 rpm for 5 minutes at 4°C, and resuspended in 1× PBS. The cells were then pipetted into pre-chilled 70% ethanol, pipetted evenly, and incubated at 4°C overnight. The cells were centrifuged, the ethanol carefully removed, and washed three times by centrifugation with 1× PBS. RNase A and PI staining solutions were added, and the cells were stained in the dark at 37°C for 30 minutes. The cell cycle was then measured using a flow cytometer (Agilent, USA), and the percentages of cells in the G0 / G1, S, and G2 / M phases were calculated using Graph Pad.

[0098] (10) Giemsa staining

[0099] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5 Cells were seeded at a density of 10 cells / well in a 6-well plate, with 2 mL per well. After cell attachment, the test compounds were added at various concentrations and treated for 48 hours. The culture medium was aspirated and washed with 1× PBS. The cells were fixed with 1 mL of 4% paraformaldehyde at room temperature for 20 minutes. The fixative was removed and the cells were washed with 1× PBS. The cells were then stained with 10 μg / mL Giemsa stain for 30 minutes. The residual stain was washed with PBS, and 600 μL of 1× PBS was added. The stain was observed under a microscope and photographed (Nikon, Japan).

[0100] (11) Cell apoptosis experiment

[0101] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5Cells were seeded at a density of 100 cells / well in a 6-well plate, with 2 mL per well. After cell attachment, the cells were treated with various concentrations of the test compound for 48 hours. The cells were then trypsinized, harvested, centrifuged at 1200 rpm for 5 minutes at 4°C, and resuspended in 1× PBS. After staining with Annexin-V FITC / PI apoptosis reagent at room temperature for 15 minutes, apoptosis was analyzed by flow cytometry (Agilent, USA).

[0102] (12) Reactive oxygen species (ROS) detection

[0103] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5 Cells were seeded at a density of 10 cells / well in a 6-well plate, with 2 mL per well. After cell attachment, the test compounds were added at varying concentrations and treated for 48 hours. The cells were then trypsinized, harvested, centrifuged at 1200 rpm for 5 minutes at 4°C, and resuspended in 1× PBS. 1 mL of 10 mM DCFH-DA reactive oxygen species probe was then added, mixed thoroughly, and incubated at 37°C for 30 minutes. The cells were then centrifuged at 1200 rpm for 5 minutes and washed twice with 1× PBS to remove any residual reactive oxygen species. After resuspending the cells in 500 μL of 1× PBS, fluorescence intensity was measured using a flow cytometer (Agilent, USA).

[0104] (13) Detection of apoptosis-related proteins cleaved-casepase-3, cleaved-casepase-9, Bcl-2, Bax, and cytochrome C

[0105] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5Cells were seeded at a density of 100 cells / well in a 6-well plate, with 2 mL per well. After cells adhered, test compounds were added at varying concentrations and treated for 48 hours. The culture medium was then removed by washing with 1× PBS. 200 μL of Solebol High-Performance RIPA Lysis Buffer was then added to each well and lysed on ice for 15-30 minutes. After lysis, samples were collected and centrifuged at 12,000 rpm for 12 minutes at 4°C. A protein quantification standard curve working buffer was prepared, and 10 μL of the supernatant was aspirated and quantified using a BCA protein concentration kit. The remaining supernatant was aspirated and added to 4× SDS-PAGE protein loading buffer. Mix thoroughly, denature the mixture in a 95°C water bath for 15 minutes, cool, and freeze at -80°C until assayed. Prepare the SDS-PAGE separating and stacking gels based on protein molecular weight, insert a comb, and remove the comb after solidification. The prepared gel plate was placed in the electrophoresis tank, and the appropriate volume of protein sample and marker was added to each well. After concentration at 80V for 15 minutes, the samples were separated at 120V for approximately 1 hour. After electrophoresis, the gel was carefully stripped and a 0.22μM PVDF membrane was pre-activated in methanol for 10 minutes. The separated protein samples were transferred to the activated PVDF membrane using wet blotting at 200-300mA for 1-2 hours. After blotting, the PVDF membrane was blocked in protein rapid blocking buffer (NcmBlot Blocking Buffer, China) for 10 minutes. The membrane was washed twice with 1× TBST buffer (5 minutes each). The PVDF membrane was cut to the molecular weight of the protein marker and incubated in a diluted primary antibody (1:5000) at 4°C on a shaker overnight. The membrane was then washed three times with 1× TBST buffer (5 minutes each). The corresponding secondary antibody was added (1:10,000) and incubated on a shaker at room temperature for 2 hours. After incubation, the membrane was washed three times with 1× TBST buffer (5 minutes each). The strips were added with ECL chemiluminescent solution, chemiluminescent imaging was performed using the bio-rad system, and the grayscale value was measured using Image J software.

[0106] (14) Expression of proteins related to the PI3K / Akt / mTOR signaling pathway

[0107] The Western blot experimental procedure was the same as above.

[0108] (15) Acute toxicity test

[0109] Experimental mice were purchased from Huafukang Biotechnology Co., Ltd. Male Kunming mice weighing between 30 and 35 grams were divided into seven groups (four mice per group): 3g (300 mg / kg and 400 mg / kg), 3x (300 mg / kg and 400 mg / kg), and 52 (200 mg / kg and 300 mg / kg). The experimental groups received an intraperitoneal injection of 200 μL of saline containing varying concentrations of the drug, 5% DMSO, and 2.5% castor oil. The control group received an intraperitoneal injection of an equal volume of blank solution. Mouse body weight was monitored and recorded daily for 10 days.

[0110] (16) The inhibitory effect of the compound on small cell lung cancer tumors in the nude mouse subcutaneous transplanted tumor NCI-H446 model

[0111] Nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. They were first housed in an SPF animal room for about a week to acclimate to the environment. Healthy male nude mice (4-5 weeks old) were selected. NCI-H446 cells in good growth condition and in the logarithmic growth phase were taken and the cell density was adjusted to 8×10 cells using serum-free and double-antibody-free culture medium and Matrigel (mixed in a 1:1 ratio). 6 The cell suspension should be injected subcutaneously into the right armpit of nude mice as soon as possible, 125 μL per mouse. When the average tumor volume reaches 100 mm 3 Afterwards, the mice were randomly divided into 8 groups: 2 control groups, 3g 10mg / kg and 20mg / kg groups, 3x 10mg / kg and 20mg / kg groups, 52 2.5mg / kg and 5mg / kg groups, and positive drug etoposide 5mg / kg and 10mg / kg groups. The mice were intraperitoneally injected once a day for three weeks, and the tumor volume and weight of the nude mice were measured and recorded every other day. The tumor volume formula is: length * width 2 / 2. The tumor inhibition rate formula is:

[0112] [1-(T-T0) / (C–C0)]×100%. T and C represent the average tumor volumes of the drug-treated group and the model group on the last day, respectively. T0 and C0 represent the average tumor volumes of the drug-treated group and the model group on the first day, respectively.

[0113] (17) Data Analysis

[0114] Statistical analyses were performed using IBM SPSS Statistics and GraphPad Prism 9.5. Images were analyzed using ImageJ software, and grouped data are presented as mean ± standard deviation. The following symbols represent the level of statistical significance within each analysis group: * (p-value range: 0.01–0.05), ** (p-value range: 0.001–0.01), *** (p-value < 0.001).

[0115] 2. Experimental results

[0116] (1) In vitro inhibition of cell proliferation activity of the compounds

[0117] Table 1 IC values ​​of compounds on 7 SCLC cell lines (NCI-H446, NCI-H196, NCI-H146, NCI-H82, NCI-H1048, NCI-H209, SHP-77), human bronchial epithelial cells BEAS-2B and human normal liver cells LO2 50 (μM) value

[0118]

[0119] In summary, compound 52 with the longest aliphatic chain showed the strongest anti-SCLC activity (IC 50 = 0.6 μM, IC in NCI-H1048 cells 50 =0.1 μM), and its activity was better than that of the positive drug etoposide.

[0120] (2) Compound 52 inhibits topoisomerase II

[0121] The present invention carried out topoisomerase relaxation test on the synthesized compound, such as Figure 1 As shown, compound 52 has the best in vitro cytotoxicity, compound 52 has topoisomerase II inhibitory activity, and compound 52 and the positive drug etoposide still have inhibitory activity against topoisomerase II at 3.12 μM.

[0122] like Figure 2 Molecular docking of compound 52 with Topo II showed that compound 52 could also insert between DNA base pairs.

[0123] The comet assay (also known as single-cell gel electrophoresis) is a method for detecting DNA damage in cells. By analyzing the tail length of the comet image, the degree of DNA damage in the cell can be assessed. After the cell and its nuclear membrane are ruptured, the DNA unwinds, causing the damaged fragments to migrate toward the positive electrode under the action of the electric field, and then PI staining is used to show a comet-shaped outline. In contrast, intact DNA migrates a shorter distance or does not migrate in the electric field and remains in the cell nucleus, forming a slightly elongated or round image. Depending on the pH value of the electrophoresis buffer, the comet assay can be divided into a neutral comet assay (pH = 8.4) and an alkaline comet assay (pH>13). The neutral comet assay is mainly used to detect double-strand breaks in DNA, while the alkaline comet assay has higher sensitivity and can detect even trace amounts of single-strand and double-strand breaks. As DNA damage intensifies, the breaks and fragments increase, and the tail lengthens. As Figure 3As shown, the alkaline comet assay confirmed that compound 52 induced concentration-dependent DNA damage in NCI-H446 and NCI-H1048 cells. DNA damage was observed in both cell lines at all concentrations, with more pronounced damage in NCI-H1048 cells. In summary, DNA damage can have cytotoxic effects on cancer cells, ultimately leading to apoptosis through a series of cascade reactions.

[0124] (3) Compound 52 inhibits the proliferation of NCI-H446 and NCI-H1048 cells

[0125] In order to evaluate the effect of compound 52 in inhibiting the proliferation of SCLC cells, the present invention conducted a clone formation experiment. Figure 4 As shown in BD, compound 52 exhibited a significant inhibitory effect on the proliferation of SCLC cells, showing a concentration-dependent inhibitory effect. At the same concentration, the inhibitory effect of compound 52 was stronger than that of the positive drug etoposide. In addition, Figure 4 A shows that compound 52 inhibits cell colony formation in a concentration-dependent gradient. In summary, compound 52 significantly inhibits the proliferation of SCLC cells and exhibits a stronger inhibitory effect than etoposide, and can be used to prepare drugs for the treatment of small cell lung cancer.

[0126] (4) Compound 52 inhibits the invasion and migration of LM9 and HuH7 cells

[0127] like Figure 5 As shown, compound 52 significantly inhibited the migration and invasion of NCI-H446 and NCI-H1048 cells in a concentration-dependent manner. In addition, the experimental results showed that compound 52 was significantly more effective than the positive drug etoposide in inhibiting the invasion and migration of SCLC cells.

[0128] Since the number of NCI-H1048 cells that invaded and migrated in the Transwell assay was small, the present invention conducted a scratch test to better confirm the inhibitory effect of the compound on SCLC migration. Figure 6 As shown, compound 52 inhibited the migration of SCLC cells in a concentration-dependent manner, and because the positive drug etoposide at the same concentration. In summary, compound 52 can significantly inhibit the invasion and migration of SCLC cells and can be used to prepare drugs to inhibit the metastasis of small cell lung cancer.

[0129] (5) Compounds block the cell cycle at S phase

[0130] To evaluate the effect of compound 52 on the cell cycle progression of NCI-H446 and NCI-H1048 cells, the present invention performed propidium iodide (PI) staining and then flow cytometry analysis. Figure 7As shown, compound 52 effectively arrests the cell cycle of NCI-H446 cells at the S phase. In contrast, it has no significant effect on the cell cycle progression of NCI-H1048 cells at the selected test concentrations. The positive drug etoposide arrests the cell cycle at the G2 / M phase.

[0131] (6) Compound 52 induces cell apoptosis

[0132] In order to determine the effect of the compounds on SCLC cell apoptosis, Giemsa staining and flow cytometry apoptosis assays were performed. Figure 8 A, Compound 52 reduced the number of NCI-H446 and NCI-H1048 cells in a dose-dependent manner, and NCI-H446 cell membrane blebbing (apoptotic bodies) and chromatin shrinkage were observed. The formation of obvious apoptotic bodies was observed in the etoposide group. Figure 8 B. Compound 52 induces premature apoptosis in SCLC cells in a dose-dependent manner, with a highly significant effect, with almost all cells undergoing apoptosis at the highest dose. Flow cytometry analysis of etoposide at the indicated concentrations revealed little effect on apoptosis in SCLC cells.

[0133] (7) Compound 52 causes mitochondrial dysfunction

[0134] like Figure 9 As shown, as the concentration of compound 52 increased, the ROS level increased, indicating that the degree of mitochondrial dysfunction induced by compound 52 increased.

[0135] like Figure 9 and 10 As the concentration of compound 52 increases, Bcl-2 expression decreases, Bax expression increases, ROS accumulation increases, and mitochondrial membrane permeability increases, leading to mitochondrial dysfunction. The effect is more active than etoposide. This promotes the release of cytochrome C and increases the expression of cleaved-caspase-3 and cleaved-caspase-9, leading to cell apoptosis.

[0136] (8) Compound 52 inhibits the activation of the PI3K / Akt / mTOR signaling pathway

[0137] like Figure 11 The present invention uses immunoblotting experiments to evaluate that compound 52 can indeed inhibit the phosphorylation of the PI3K / Akt / mTOR signaling pathway, thereby inhibiting cell growth processes such as proliferation and apoptosis of SCLC cells.

[0138] (9) Acute toxicity test

[0139] Twelve male Kunming mice weighing 30-35 grams were randomly divided into three groups: a control group and an experimental group (200 mg / kg and 300 mg / kg groups of compound 52, respectively). The experimental group was intraperitoneally injected with 200 μL of saline solution containing different concentrations of the drug, 5% DMSO, and 2.5% castor oil. The control group was intraperitoneally injected with an equal amount of blank solution. No mice in the 200 mg / kg dose group of compound 52 died 10 days after administration, while mice in the 300 mg / kg dose group died. The median lethal dose (LD50) of compound 52 was 200 μL. 50 ) is between 200-300 mg / kg. Monitor the weight changes of mice daily, and the weight change trend is as follows Figure 12 As shown, the results showed that one day after administration, the body weight of the mice changed significantly, and then the body weight of the mice increased steadily.

[0140] (10) NCI-H446 tumor xenografts

[0141] Subsequently, tumor xenografts were performed using NCI-H446 cancer cells, with etoposide as the positive drug. 3 Then, the male nude mice were randomly divided into 4 groups, including: blank group, etoposide group (5 mg / kg / d, ip), low-dose group (2.5 mg / kg / d, ip) and high-dose group (5 mg / kg / d, ip) of compound 52 (5 mice in each group). Figure 13 As shown in A, the tumor volume of the drug-treated group grew very slowly over the 21st day, and the tumor inhibition effect of compound 52 in both the low-dose and high-dose groups was significantly better than that in the etoposide group. Figure 13 As shown in B, there was no significant decrease in the body weight of mice. Figure 13 C, 13D and Table 2, the tumor inhibition rate of the low-dose group of compound 52 was 69.81%, and the tumor inhibition rate of the high-dose group was 83.39%, both of which were significantly better than that of the etoposide group (tumor inhibition rate was 41.77%).

[0142] Table 2. Summary of tumor growth inhibition by compound 52 and vp-16

[0143]

[0144] The above results show that compound 52 synthesized in the present invention has excellent inhibitory activity on SCLC cells, and its IC 50 =0.6μM, IC on NCI-H1048 50=0.1 μM, compound 52 intercalates between DNA base pairs, significantly inducing DNA damage and arresting the NCI-H446 cell cycle at S phase. Compound 52 significantly inhibits the proliferation of NCI-H446 and NCI-H1048 cells, significantly suppresses the invasion and migration of SCLC cells, and significantly induces apoptosis, with activity significantly superior to that of the active agent etoposide at the highest concentration. Compound 52 exhibits significantly enhanced anti-SCLC activity both in vitro and in vivo compared to the active agent etoposide.

[0145] In summary, the present invention provides a topoisomerase II inhibitor, its preparation method, and its application. This compound, synthesized using 6-bromopiperonal as a raw material, is a novel topoisomerase II inhibitor with promising application prospects for the treatment of diseases such as small cell lung cancer, advanced metastatic ovarian cancer, colorectal cancer, and bladder cancer.

Claims

1. A compound, characterized in that The compound is the following compound: .

2. A method for preparing the compound according to claim 1, characterized in that: The method comprises the following steps: Where R is , R 1 As shown in the compound of claim 1; (1) 6-bromopiperonal, trimethylsilylacetylene, a catalyst and a base are reacted to prepare compound L2; (2) reacting compound L2 with a reducing agent to prepare compound L3; (3) Compound L1 is reacted with a base, and then reacted with DMF to prepare compound L4; (4) reacting compound L3, compound L4, a catalyst, and a base to prepare compound L5; (5) Compound L5, CBr4 and PPh3 are reacted to obtain an intermediate compound, and the intermediate compound, an amine compound and a base are reacted to obtain compound L6; (6) Compound L6, a nitrogen source compound, and a catalyst are reacted to obtain the compound according to claim 1.

3. The method according to claim 2, characterized in that In step (1), the catalyst is cuprous iodide and tetrakis(triphenylphosphine)palladium; the base is triethylamine; the molar ratio of 6-bromopiperonal, trimethylsilyl acetylene, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:1.5-2.5:0.1-0.2:0.01-0.03; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, the reaction is carried out at 40-60 ºC for 5-7 hours; In step (2), the reducing agent is sodium borohydride; the molar ratio of the compound L2 to the reducing agent is 1:1-3; the solvent of the reaction is an organic solvent; the reaction conditions are: reaction at -5-10 ºC for 1-15 minutes; In step (3), the base is lithium diisopropylamide; the molar ratio of the compound L1 to the base is 1:1-1.5: the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, first react at -85-70°C for 1-2.5 hours, and then react at 10-40°C for 5-20 hours; In step (4), the catalyst is cuprous iodide and tetrakis(triphenylphosphine)palladium; the base is triethylamine; the molar ratio of compound L3, compound L4, cuprous iodide and tetrakis(triphenylphosphine)palladium is 1:1-1.2:0.1-0.3:0.15-0.25; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, the reaction is carried out at 70-90°C for 5-9 hours; In step (5), the molar ratio of the compound L5, CBr4 and PPh3 is 1:1.5~2:1.5~2; the solvent of the reaction is an organic solvent; the reaction conditions are: reaction at -5~10 ºC for 0.1~1 hour; the amine compound is The base is triethylamine; the molar ratio of the intermediate compound, the amine compound and the base is 1:1~3:8~12; the reaction conditions are: 10~40 ºC for 5~20 hours; In step (6), the nitrogen source compound is ammonium acetate; the catalyst is silver nitrate; the molar ratio of the compound L6, the nitrogen source compound, and the catalyst is 1:2-4:0.1-0.5; the solvent of the reaction is an organic solvent; and the reaction conditions are: under argon protection, at 10-40°C for 4-15 hours.

4. Use of the compound according to claim 1 in the preparation of topoisomerase inhibitors.

5. The use according to claim 4, characterized in that The topoisomerase is topoisomerase II.

6. The use according to claim 5, characterized in that The topoisomerase inhibitor is a drug for preventing and / or treating cancer and inhibiting cancer metastasis.

7. The use according to claim 6, characterized in that The cancer is selected from small cell lung cancer, advanced metastatic ovarian cancer, colorectal cancer, and bladder cancer.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition is a preparation prepared by using the compound according to claim 1 as an active ingredient and adding pharmaceutically acceptable excipients.

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