A topoisomerase I / II dual-target inhibitor and its preparation method and application

Abstract

The present invention provides a topoisomerase I / II dual-target 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 I / II dual-target inhibitor that can be used to treat diseases such as small cell lung cancer, advanced metastatic ovarian cancer, advanced breast 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 I / II dual-target 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 has an 80% response rate to chemotherapy, 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 I / II dual-target 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, R 1 、R 2 、R 3 、R 4 and R 5 Each independently selected from hydrogen, C 1～6 Alkyl, and R 1 、R 2 、R 3 、R 4 and R 5 Not hydrogen at the same time.

[0010] Furthermore, R 3 is hydrogen, R 1 、R 2 、R 4 and R 5 Each independently selected from C 1～2 alkyl.

[0011] Furthermore, the compound is selected from one of the following compounds:

[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 、R 2 、R 3 、R 4 and R 5 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 R 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 I and / or 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, advanced breast 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] Compounds 3g and 3x synthesized in the present invention are dual-target inhibitors of topoisomerase I / II and can be used to treat diseases related to topoisomerase I / II activity (such as small cell lung cancer, ovarian cancer, colorectal cancer, bladder cancer, etc.).

[0040] The present invention also found through experiments that compounds 3g and 3x of the present invention have excellent inhibitory activity on SCLC cells. Compound 3g has an IC of 50 =1.3 μM, IC on NCI-H1048 50 =1.42 μM, compound 3x IC on NCI-H446 cells 50 =1.32μM, IC on NCI-H1048 50 =2.45μM; compared to the compound in patent CN114456184B In NCI-H446 (IC 50 =15.24 μM) and NCI-H1048 (IC 50 =15.41

[0041] The activity on the cells was significantly improved.

[0042] Experimental data show that compounds 3g and 3x can not only inhibit the proliferation of SCLC cells, but also inhibit the invasion and migration of SCLC cells. Therefore, the compounds of the present invention can be used to prepare drugs for treating small cell lung cancer and inhibiting the metastasis of small cell lung cancer.

[0043] Experimental data also demonstrated that compounds 3g and 3x can intercalate into DNA, significantly inducing DNA damage and arresting the NCI-H446 cell cycle at the S phase. They also significantly induced apoptosis in SCLC cells by inducing mitochondrial dysfunction. Furthermore, compounds 3g and 3x inhibited phosphorylation of the PI3K / Akt / mTOR signaling pathway, thereby impacting various SCLC cellular activities. Both compounds 3g and 3x demonstrated significantly enhanced SCLC inhibitory activity in vitro and in vivo compared to the active agent etoposide.

[0044] 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.

[0045] 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

[0046] Figure 1 (A) Compounds 3g and 3x inhibit the relaxation activity of Topo I at 100 μM; (B) Compounds inhibit the relaxation activity of Topo I at 50 μM; (C) Compounds inhibit the relaxation activity of Topo I at 25 μM; (D) Inhibitory effects of compounds 3g, 3x and topotecan (TPT) on the relaxation activity of Topo I at different concentration gradients.

[0047] Figure 2 (A) Compounds 3g and 3x inhibit Topo II relaxation activity at 100 μM; (B) Compounds inhibit Topo II relaxation activity at 50 μM; (C) Compounds inhibit Topo II relaxation activity at 25 μM; (D) Inhibitory effects of compounds 3g, 3x and etoposide (vp-16) on Topo II relaxation activity at different concentration gradients.

[0048] Figure 3 (A) Schematic diagram of the molecular docking binding mode of compounds 3g (B) and 3x (C) with topoisomerase I (PBD: 1T8I); (D) Schematic diagram of the molecular docking binding mode of compounds 3g (E) and 3x (F) with topoisomerase II (PBD: 5GWK); (G) Schematic diagram of the molecular docking binding mode of compounds 3g and 3x with topoisomerases I and II.

[0049] Figure 4 (A) Comet assay images of compound 3g and vp-16; (B) Comet assay images of compound 3x and vp-16 (magnification 20×, scale bar represents 100 μm).

[0050] Figure 5 Compounds 3g and 3x inhibit the proliferation of NCI-H446 and NCI-H1048 cells at selected concentrations: (A) Effects of compounds 3g and 3x on SCLC cell clone formation; (B) The inhibitory effect of compounds 3g and 3x on cell proliferation was evaluated by EdU incorporation assay, with an incubation time of 24 hours (magnification 4×, scale bar represents 500 μm); (C) Percentage of EdU-positive cells in NCI-H446 and (D) NCI-H1048 cells. Statistical analysis is expressed as mean ± SD (n = 3), and each experimental group was compared with the control group: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05.

[0051] Figure 6 Compounds 3g and 3x inhibit 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 compounds 3g and 3x for 24 hours; (B) and (C) the number of migrated cells in each experimental group and the percentage of migrated cells in the blank group; (D) Transwell invasion assay of NCI-H446 and NCI-H1048 cells after treatment with compounds 3g and 3x for 24 hours; (magnification: 10x, scale bar: 200 μm); (E) and (F) 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.

[0052] Figure 7 (A) Representative micrographs of NCI-H446 and NCI-H1048 cells treated with compounds 3g and 3x for 0 and 24 hours in a wound healing assay (magnification 4x, scale bar represents 500 μm); (B) and (C) are the percentages of wound closure area in each experimental group compared with the blank group.

[0053] Figure 8 Compounds 3g and 3x block the SCLC cell cycle at S phase: (A) and (B) flow cytometric analysis of cell cycle distribution in NCI-H446 and NCI-H1048 cells after treatment with compounds 3g and 3x for 48 hours; (C) quantitative analysis of cell cycle distribution in each phase.

[0054] Figure 9 Compounds 3g and 3x significantly induced apoptosis of SCLC cells: (A) Effects of the compounds on cell morphology were observed by Giemsa staining (magnification 10×, scale bar = 200 μm); (B) and (C) Flow cytometric analysis of cell apoptosis in NCI-H446 and NCI-H1048 cells after treatment with compounds 3g and 3x for 48 hours.

[0055] Figure 10 The graph shows the trend of ROS fluorescence intensity detected by flow cytometry in NCI-H446 and NCI-H1048 cells induced by compounds 3g and 3x in mitochondrial dysfunction.

[0056] Figure 11Effects of compound 3g 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 3g on apoptotic proteins in NCI-H446 cells (n=3); (D) Statistical graph of the effect of compound 3g on apoptotic proteins in NCI-H1048 cells (n=3).

[0057] Figure 12 Effects of compound 3x 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 effects of compound 3x on apoptotic proteins in NCI-H446 cells (n=3); (D) Statistical graph of the effects of compound 3x on apoptotic proteins in NCI-H1048 cells (n=3).

[0058] Figure 13 NCI-H446 and NCI-H1048 cells were treated with different concentrations of compound 3g 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; (E) Schematic diagram of the PI3K / Akt / mTOR signaling pathway.

[0059] Figure 14 NCI-H446 and NCI-H1048 cells were treated with different concentrations of compound 3x 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.

[0060] Figure 15 The figures are weight trend diagrams of mice (n=4): (A) Effect of compound 3g on weight trend of mice; (B) Effect of compound 3x on weight trend of mice; (C) Effects of compounds 3g and 3x on mouse organs (heart, liver, spleen, lung, and kidney).

[0061] Figure 16 In vivo inhibition of NCI-H446 xenograft tumor growth by compounds 3g and 3x: (A) and (B) changes in tumor volume over 21 days, expressed as mean ± SEM; (C) histogram of tumor volume of each dosing group (3g, 3x, and vp-16) compared with the control group on the last day; (D) and (E) trends in mouse body weight changes over 21 days, expressed as mean ± SD; (F) comparison of tumor volume of 3g, 3x, and vp-16 and the control group on the last day, measured using a vernier caliper. DETAILED DESCRIPTION

[0062] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0063] The "room temperature" referred to in the present invention is 25±10°C, and "overnight" is 12±5 hours.

[0064] The compounds 3g and 3x of the present invention were prepared according to the following synthetic route:

[0065]

[0066] The following is a method for synthesizing intermediate compounds L2-L5:

[0067] (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.

[0068] (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 H NMR (400MHz, CDCl3) δ7.09–6.76(m,2H),5.97(s,2H),4.72(s,2H),3.24(s,1H); 13C NMR (101MHz, CDCl3) δ148.64,146.67,138.80,113.22,112.20,108.24,101.56,81.29,80.56,63.56.

[0069] (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); 13 C NMR (101MHz, CDCl3) δ190.98,150.09,149.33,126.77,118.00,116.18,114.16,103.97.

[0070] (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. 1H 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.

[0071] The following is the method for synthesizing target compounds 3g and 3x:

[0072] Example 1. Synthesis of 3g of the compound of the present invention

[0073]

[0074] (1) Synthesis of compound L6:

[0075] 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.

[0076] 2) Add aliphatic cyclic 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 was complete, monitored by TLC, compound L6 was obtained by column chromatography using DCM / MeOH (40:1, 20:1). Yield: 93% as a yellow solid.

[0077] (2) Synthesis of compound 3g: 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, the reaction was complete as determined by TLC. 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 the product, compound 3g, as a yellow solid in a 41.50% yield with a melting point of 148.5-150.2°C. 1 H NMR(400MHz, CDCl3)δ9.26(s,1H),7.73(s,1H),7.62(s,1H),7.46(s,2H),7.00(s,1H),6.30(s,2H),6.07(s,2H),4. 43(s,2H),3.16(d,J=10.7Hz,2H),2.49(t,J=12.1Hz,2H),2.19(s,2H),1.87–1.74(m,2H),0.91(s,3H),0.89(s,3H); 13 C NMR (101 MHz, CDCl3) δ 149.52, 149.16, 148.50, 145.52, 144.65, 141.99, 135.93, 132.20, 121.56, 120.84, 116.11, 113.91, 113.17, 110.62, 102.91, 102.08, 57.65, 57.28, 39.70, 29.36, 18.73; LRMS (ESI, m / z) theoretical value is C 25 H 27 N2O4 + [M+H] + 419.1965, actual value 419.2. Purity after LC-MS analysis was 99.18%.

[0078] Example 2: Synthesis of Compound 3x of the Invention

[0079]

[0080] (1) Synthesis of compound L6:

[0081] 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.

[0082] 2) Add aliphatic cyclic 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 was complete, monitored by TLC, compound L6 was obtained by column chromatography using DCM / MeOH (40:1, 20:1). Yield: 93% as a yellow solid.

[0083] (2) Synthesis of compound 3x: 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, the reaction was complete as determined by TLC. 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 3x, in a 38.90% yield with a melting point of 148.5-1149.7°C. 1H NMR(400MHz, CDCl3)δ9.26(s,1H),7.97–7.62(m,2H),7.45(s,2H),6.97(s,1H),6.28(s,2H),6.05(s,2H),4.70–4.12(m,2H),3.29–2.79(m,2H) ,2.78–2.44(m,1H),2.28–2.03(m,1H),2.05–1.78(m,2H),1.67(t,J=15 .3Hz,1H),1.60–1.40(m,3H),1.28–1.00(m,3H),0.77(t,J=7.5Hz,3H); 13 C NMR (101 MHz, CDCl3) δ 149.77, 148.90, 148.43, 145.40, 144.50, 141.92, 135.94, 132.19, 121.45, 120.79, 115.97, 113.90, 113.38, 110.52, 110.47, 102.85, 101.98, 61.56, 55.01, 53.79, 35.41, 32.17, 28.54, 26.37, 17.90, 10.52; LRMS (ESI, m / z) calculated value is C 26 H 29 N2O4 + [M+H] + 433.2122, actual value 433.3. Purity after LC-MS analysis was 96.64%.

[0084] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0085] Experimental Example 1: Activity test of compounds 3g and 3x

[0086] 1. Experimental methods

[0087] (1) Cell culture

[0088] 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.

[0089] (2) MTT and CCK8 experiments

[0090] The cells to be tested were digested or collected according to the above method and the cell concentration was adjusted to 5-8×10 3 Cells / 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.

[0091] (3) Topoisomerase I inhibitory activity

[0092] To a 50 μL PV tube, add 10 μL of ultrapure water, 2 μL of Topo I buffer, 2 μL of BSA, 1 μL of 0.5 U topoisomerase I (Takara, 20 U / μL), 0.2 μL of the test compound (not added in the DNA and Topo I groups), and 1 μL of 0.25 μg / μL pBR322 plasmid DNA (Takara, 0.5 μg / μL) to each well. Make up to a total volume of 20 μL with ultrapure water. After incubation at 37°C for 0.5 hour, add 2 μL of 6× DNA loading buffer, load the sample onto a 0.8% agarose gel, and electrophorese at 110 V for 45 minutes. Stain with a nucleic acid dye for at least 0.5 hour, observe under UV light using a Bio-Rad system, and photograph and record.

[0093] (4) Topoisomerase II inhibitory activity

[0094] 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.

[0095] (5) Molecular docking

[0096] Molecular docking studies of compounds 3g and 3x with Topo I and Topo II were performed using Schrodinger software. First, the X-ray crystal structures of Topo I (PDB code: 1T8I) and Topo II (PDB code: 5GWK) were obtained from the Protein Data Bank (PDB). Energy and hydrogen bond minimization was performed on the proteins, and the docking boxes were generated using the Protein Preparation Wizard model. The molecular structures of the compounds were 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 default values.

[0097] (6) Comet Experiment

[0098] 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 into 6-well plates (cells / mL). After the cells were allowed to adhere overnight in an incubator, they were treated with different concentrations of compound 3g and 3x. 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).

[0099] (7) Plate cloning experiment

[0100] 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.

[0101] (8) EdU test

[0102] 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).

[0103] (9) Cell invasion and migration assay

[0104] 1) Transwell migration assay

[0105] 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.

[0106] 2) Transwell invasion assay

[0107] 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.

[0108] 3) Scratch test

[0109] 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.

[0110] (10) Cell cycle distribution

[0111] 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.

[0112] (11) Giemsa staining

[0113] NCI-H1048 and NCI-H446 cells were collected at a rate of 1×10 5Cells 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).

[0114] (12) Cell apoptosis experiment

[0115] 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 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).

[0116] (13) Reactive oxygen species (ROS) detection

[0117] 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).

[0118] (14) Detection of apoptosis-related proteins cleaved-casepase-3, cleaved-casepase-9, Bcl-2, Bax, and cytochrome C

[0119] 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.

[0120] (15) Expression of proteins related to the PI3K / Akt / mTOR signaling pathway

[0121] The Western blot experimental procedure was the same as above.

[0122] (16) Acute toxicity test

[0123] 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.

[0124] (17) The inhibitory effect of the compound on small cell lung cancer tumors in the nude mouse subcutaneous transplanted tumor NCI-H446 model

[0125] 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: [1-(T-T0) / (C-C0)]×100%. Where T and C represent the average tumor volume of the drug-treated group and the model group on the last day, respectively, and T0 and C0 represent the average tumor volume of the drug-treated group and the model group on the first day, respectively.

[0126] (18) Data Analysis

[0127] 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).

[0128] 2. Experimental results

[0129] (1) In vitro inhibition of cell proliferation activity of compounds 3g and 3x

[0130] Table 1 IC values ​​of compounds 3g and 3x on seven 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.

[0131]

[0132] In summary, compounds 3g and 3x showed comparable activity to the active agent etoposide against NCI-H446 and NCI-H1048 cell lines and showed higher activity against other SCLC cell lines. Compounds 3g and 3x were less toxic than the active agent in human bronchial epithelial cells (BEAS-2B) and human normal hepatocytes (LO2).

[0133] (2) Compounds 3g and 3x inhibit topoisomerase I and topoisomerase II

[0134] The present invention carried out topoisomerase relaxation test on the synthesized compound, such as Figure 1 、 2 As shown, the inhibitory effects of compounds 3g and 3x on Topo I disappeared at a concentration of 50 μM, while the positive control topotecan remained active at this concentration. In contrast, compounds 3g and 3x still had inhibitory effects on Topo II at a concentration of 6.25 μM, comparable to the positive control etoposide. Therefore, compounds 3g and 3x exhibit dual inhibitory effects on both Topo I and Topo II.

[0135] Molecular docking studies were performed on compounds 3g and 3x with Topo I and II, respectively. Figure 3 Compounds 3g and 3x both showed the ability to intercalate into DNA. These findings suggest that compounds 3g and 3x can function as dual inhibitors of Topo I and II.

[0136] 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 4 As shown in the figure, the results show that DNA damage in NCI-H446 and NCI-H1048 cells varies in concentration-dependent manner when exposed to 3g and 3x concentrations. 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, potentially leading to apoptosis through a series of cascade reactions.

[0137] (3) Compounds 3g and 3x inhibit the proliferation of NCI-H446 and NCI-H1048 cells

[0138] In order to evaluate the effect of compounds 3g and 3x in inhibiting the proliferation of SCLC cells, the present invention conducted a clone formation experiment. Figure 5 As shown in A, compounds 3g and 3x inhibited cell colony formation in a concentration-dependent manner, and compound 3g had a particularly significant inhibitory effect on NCI-H1048, which was comparable to the effect of the positive drug etoposide at the same concentration. The cell proliferation rate was evaluated by EdU incorporation assay, and the results were shown in Figure 2. Figure 5 As shown in Figures C and 5D, the proliferation rate of the control group was approximately 40%. Compounds 3g and 3x both inhibited cell proliferation in a concentration-dependent manner. Their inhibitory effects on NCI-H446 cells were comparable to those of etoposide at the same concentration, while their inhibitory effects on NCI-H1048 cells exceeded those of the positive control. Therefore, compounds 3g and 3x inhibited the proliferation of NCI-H446 and NCI-H1048 cells and can be used to prepare drugs for the treatment of small cell lung cancer.

[0139] (4) Compounds 3g and 3x inhibited the invasion and migration of LM9 and HuH7 cells

[0140] like Figure 6 As shown in AC, 3g and 3x showed significant anti-migration ability in a concentration-dependent manner. Figure 6As shown in Figures DF, after treatment with different concentrations of compounds 3g, 3x, and etoposide, the cell invasion ability was significantly reduced in a concentration-dependent manner. Compounds 3g and 3x had significant inhibitory effects on the migration and invasion of NCI-H446 and NCI-H1048 cells.

[0141] 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 7 As shown, compounds 3g and 3x inhibited SCLC cell migration in a concentration-dependent manner. At equivalent concentrations, 3g demonstrated superior inhibitory efficacy compared to the active agent etoposide. Furthermore, compound 3g exhibited a more pronounced inhibitory effect against NCI-H1048. In summary, compounds 3g and 3x significantly inhibited SCLC cell invasion and migration and could be used to develop drugs to inhibit small cell lung cancer metastasis.

[0142] (5) Compounds 3g and 3x block the cell cycle at S phase

[0143] To evaluate the effects of compounds 3g and 3x 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 8 As shown, compounds 3g and 3x both induce arrest in the S phase of the cell cycle, which is different from the G2 / M phase arrest of the positive drug etoposide.

[0144] (6) Compounds 3g and 3x induce cell apoptosis

[0145] In order to determine the effect of the compounds on SCLC cell apoptosis, Giemsa staining and flow cytometry apoptosis assays were performed. Figure 9 A, The compound reduced the number of NCI-H446 and NCI-H1048 cells in a dose-dependent manner. In addition, NCI-H446 cell membrane blebbing, i.e., the generation of apoptotic bodies and chromatin shrinkage, were observed. Figure 9 Compound 3g induced premature apoptosis in SCLC cells in a dose-dependent manner, with a highly significant effect and greater efficacy than etoposide in both cell lines. Compound 3x induced cell fragmentation in NCI-H446 cells, while etoposide had minimal effect on apoptosis in NCI-H446 cells at the selected concentration. Compound 3x also induced apoptosis in NCI-H1048 cells to a certain extent, but the effect was not as strong as etoposide.

[0146] (7) Compounds 3g and 3x cause mitochondrial dysfunction

[0147] like Figure 10As shown, with increasing concentrations of compounds 3g and 3x, ROS levels increased, and the enhanced fluorescence reflected greater mitochondrial membrane permeability and more obvious mitochondrial dysfunction.

[0148] like Figure 11 Compounds 3g and 3x promoted the accumulation of mitochondrial ROS and the disturbance of mitochondrial membrane potential. The effect of compound 3g was stronger than that of etoposide, while the activity of compound 3x was comparable to that of etoposide, which was consistent with the apoptosis results. Figure 12 As the concentrations of 3g and 3x increased, the expression of Bcl-2 decreased, while the expression of Bax, cleaved-caspase-3, cleaved-caspase-9, and cytochrome C increased. It is speculated that compounds 3g and 3x activate the mitochondrial apoptosis pathway, modify Bcl-2 family proteins, change mitochondrial membrane permeability, accumulate ROS, release cytochrome C, activate the caspase pathway, and ultimately lead to cell apoptosis.

[0149] (8) Compounds 3g and 3x inhibit the activation of the PI3K / Akt / mTOR signaling pathway

[0150] like Figure 13 、 14 The present invention used WB experiments to confirm that compounds 3g and 3x can inhibit the phosphorylation of the PI3K / Akt / mTOR signaling pathway in a concentration-dependent manner, thereby affecting the proliferation, invasion, migration and apoptosis of SCLC cells.

[0151] (9) Acute toxicity test

[0152] Twenty male Kunming mice weighing 30-35 grams were randomly divided into five groups, control groups and experimental groups (300 mg / kg and 400 mg / kg groups of compound 3g, and 300 mg / kg and 400 mg / kg groups of compound 3x, respectively). The experimental groups were intraperitoneally injected with 200 μL of saline solution containing different concentrations of drugs, 5% DMSO and 2.5% castor oil. The control group was intraperitoneally injected with an equal amount of blank solution. No mice in the 300 mg / kg dose group of compound 3g and 3g died 10 days after administration, while mice in the 400 mg / kg dose group died. The median lethal dose (LD50) of compounds 3g and 3x was 2.5%. 50 ) is between 300-400 mg / kg. Monitor the weight changes of mice daily, and the weight change trend is as follows Figure 15 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.

[0153] (10) NCI-H446 tumor xenografts

[0154] Subsequently, tumor xenografts were performed using NCI-H446 cancer cells, with etoposide as the positive drug. 3 Then, male nude mice were randomly divided into 6 groups (5 mice in each group), including: blank group, etoposide group (10 mg / kg / d, ip), 3g and 3x set low dose (10 mg / kg / d, ip) and high dose group (20 mg / kg / d, ip). Figure 16 As shown in A and 16B, the tumor volume of the drug-treated group grew very slowly since day 21. Figure 16 As shown in D and 16E, the body weight of mice in the 3g and 3x groups was not significantly affected, while the body weight of mice in the positive drug etoposide group was significantly reduced, indicating that 3g and 3x were less toxic than the positive drug. Figure 16 C, 16F and Table 2, the tumor inhibition rates of the low-dose groups of compounds 3g and 3x were 75.19% and 69.16%, respectively, which were comparable to the etoposide group (tumor inhibition rate was 68.73%), while the tumor inhibition rates of the high-dose groups of 3g and 3x were both >80%.

[0155] Table 2. Summary of tumor growth inhibition by compounds 3g, 3x, and vp-16

[0156]

[0157] The above results show that the compounds 3g and 3x synthesized in the present invention have excellent inhibitory activity on SCLC cells. The IC value of compound 3g on NCI-H446 cells is 50 =1.3 μM, IC on NCI-H1048 50 =1.42 μM, compound 3x IC on NCI-H446 cells 50 =1.32μM, IC on NCI-H1048 50 =2.45μM. Compounds 3g and 3x inhibited the proliferation, invasion, and migration of SCLC cells. Compounds 3g and 3x intercalated into DNA, significantly inducing DNA damage and arresting the NCI-H446 cell cycle at the S phase. They significantly induced apoptosis in SCLC cells by inducing mitochondrial dysfunction. Furthermore, compounds 3g and 3x inhibited phosphorylation of the PI3K / Akt / mTOR signaling pathway, thereby affecting various SCLC cell activities. Compounds 3g and 3x demonstrated significantly enhanced anti-SCLC activity in vitro and in vivo compared to the active agent etoposide.

[0158] In summary, the present invention provides a dual-target topoisomerase I / II inhibitor, its preparation method, and application. This compound, synthesized using 6-bromopiperonal as a raw material, is a novel dual-target topoisomerase I / II inhibitor with promising application prospects for the treatment of diseases such as small cell lung cancer, advanced metastatic ovarian cancer, advanced breast cancer, colorectal cancer, and bladder cancer.

Claims

1. A compound, characterized in that The compound is selected from one of the following compounds: 、 .

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 、R 2 、R 3 、R 4 and R 5 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 I and / or 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, advanced breast 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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