Topoisomerase I / II double-target inhibitor as well as preparation method and application thereof

Abstract

The invention provides a topoisomerase I / II double-target inhibitor as well as a preparation method and application thereof, and belongs to the field of medicines. 6-bromoheliotropin is used as a raw material to synthesize the compound with the structural formula shown in the formula I. The compound is a novel topoisomerase I / II double-target inhibitor, can be used for treating small cell lung cancer, advanced metastatic ovarian cancer, advanced breast cancer, colorectal cancer, bladder cancer and other diseases, and has a good application prospect. # imgabs0 #

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. It has become an important obstacle to extending human lifespan, and cancer patients are getting younger and younger, which brings huge economic pressure to society. Small cell lung cancer (SCLC) is a malignant tumor with neuroendocrine characteristics. Its basic characteristics are rapid proliferation, early metastasis, high malignancy and rapid development of drug resistance. Due to its high cell proliferation rate, the drugs currently used for SCLC are all directly or indirectly targeting DNA synthesis, replication and repair. The standard first-line treatment for SCLC is the combination of platinum and topoisomerase II inhibitor etoposide, which was determined in 1985. In 2019, the FDA approved atezolizumab to join the standard treatment, which became a major milestone in the treatment of SCLC. The response rate of SCLC to chemotherapy can reach 80%, but 90% of patients quickly develop drug resistance. For those who develop drug resistance within 3 months, the second-line drug topoisomerase I inhibitor topotecan is used, but the response rate to topotecan is only about 20%. Therefore, in 2020, the FDA urgently approved the RNA polymerase II inhibitor Lurbinectedin for the second-line treatment of SCLC. In 2021, the CDK4 / 6 inhibitor Trilaciclib was approved to reduce the risk of bone marrow transplantation caused by chemotherapy. For patients who develop drug resistance for more than 3 months, they still receive standard first-line treatment again. Therefore, it is urgent to find new and effective anti-SCLC drugs.

[0003] Patent CN114456184B discloses a compound that has both topoisomerase I and topoisomerase II inhibitory effects, and the structure is as follows:

[0004]

[0005] However, the compound has an IC of 0. 50 =15.24μM, 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 a preparation method and application thereof.

[0007] The present invention provides a compound shown in 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, the method comprising the following steps:

[0013]

[0014] Among them, 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 a 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] Further, 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, react 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: reacting 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, and 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, react 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: reacting 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: reacting 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 compound L6, nitrogen source compound and catalyst is 1:2-4:0.1-0.5; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, react 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; the reaction conditions are: reacting at 50°C for 6 hours under argon protection;

[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: reacting 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, and 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, react 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: reacting at -5 to 10°C for 0.1 to 1 hour; the molar ratio of the intermediate compound, the amine compound and the base is 1:2:10; the reaction conditions are: reacting 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, react at 20-35°C for 6-12 hours.

[0033] The present invention also provides the 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 by using the above compound as an active ingredient and adding pharmaceutically acceptable excipients.

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

[0039] The 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 =1.3μM, IC on NCI-H1048 50 =1.42 μM, compound 3x IC in NCI-H446 cells 50 =1.32μM, IC on NCI-H1048 50 =2.45μM; compared with the compound in patent CN114456184B In NCI-H446 (IC 50 =15.24 μM) and NCI-H1048 (IC 50 =15.41

[0041] μM) cells showed a significant increase in activity.

[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] The experimental data also showed that compounds 3g and 3x can insert into DNA, significantly induce DNA damage, and block the NCI-H446 cell cycle at the S phase. By inducing mitochondrial dysfunction, SCLC cell apoptosis is significantly induced. In addition, compounds 3g and 3x can inhibit the phosphorylation of the PI3K / Akt / mTOR signaling pathway, thereby affecting various SCLC cell activities. The inhibitory activity of compounds 3g and 3x in vivo and in vitro for SCLC is significantly higher than that of the positive drug etoposide.

[0044] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0045] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to 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) The inhibitory effect 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 effect 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 topoisomerase 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 the action of compounds 3g and 3x for 24 hours; (B) and (C) are the percentage of the number of migrated cells in each experimental group and the migrated cells in the blank group; (D) Transwell invasion assay of NCI-H446 and NCI-H1048 cells after the action of compounds 3g and 3x for 24 hours; (magnification is 10 times, scale bar represents 200 μm); (E) and (F) are the percentage of the number of invasive cells in each experimental group and the invasive cells in the blank group, statistical analysis is expressed as mean ± standard deviation (n = 3), each treatment group is compared with the control group, and the significant differences are: (***) 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 of ​​each experimental group compared with the blank group.

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

[0054] Fig. 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 treated with compounds 3g and 3x for 48 hours.

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

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

[0058] Fig.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) β-actin was used as an internal reference, and the expression of PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR was determined by Western blotting experiments; (C) and (D) Statistical analysis of the mean ± SD of three parallel experiments, and significant differences were obtained by comparing the drug-treated group with the blank group: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05; (E) is a schematic diagram of the PI3K / Akt / mTOR signaling pathway.

[0059] Fig.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) β-actin was used as an internal reference to determine the expression of PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR by Western blotting experiments; (C) and (D) Statistical analysis of the mean ± SD of three parallel experiments, and significant differences were found between the drug-treated group and the blank group: (***) P < 0.001, (**) P < 0.01, (*) P < 0.05.

[0060] Fig.15 The graphs are weight trend graphs 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] Fig.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) bar graph 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, which 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 preparation of compounds 3g and 3x of the present invention was carried out 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), and the system was placed in an anhydrous and oxygen-free argon (Ar2) environment. After stirring at room temperature for 10 minutes, trimethylsilyl acetylene (11.5 ml, 81.62 mmol, 2.0 eq) was added dropwise with a needle. The reaction system was heated to 50°C and stirred for 6 hours. After the reaction was completed, the solvent was removed by rotary evaporation. The residue was filtered through a Buchner funnel covered with filter paper and diatomaceous earth and rinsed with dichloromethane (DCM) several times. After the solvent was removed by rotary evaporation, silica gel was used for column chromatography with petroleum ether / ethyl acetate (20:1) as the eluent to obtain compound L2 (9.5 g, 38.78 mmol). Yield: 95%; 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 system was placed in an ice bath (0-4 °C), and then sodium borohydride (NaBH4, 616 mg, 16.24 mmol, 2.0 eq) was slowly added. The reaction was monitored by TLC until the reaction of the raw materials was completed (5-10 min), and water was slowly added to quench the system in an ice bath, and then the mixture was moved to room temperature and stirred for 1-2 hours to obtain Compound L3 after the trimethylsilane (TMS) protective agent was removed. The mixture was extracted with water and dichloromethane, and the organic phase was collected and the solvent was removed. The mixture was purified by silica gel column chromatography with 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), and the system was placed in an anhydrous and oxygen-free argon environment at -78°C. After the system was 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 continued to stir for 0.5-1 hour. The reaction was moved to room temperature and continued to stir overnight. TLC was used to monitor until the reaction of the raw materials was complete, and the system was placed in an ice bath and saturated NH4Cl was slowly added to quench, followed by extraction with EtOAc. The organic phase was collected and subjected to silica gel column chromatography using petroleum ether / ethyl acetate (10:1) as eluent to purify compound L4 (5.49 g, 25.08 mmol). Yield: 95%; 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), 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 environment and ventilated three times with an oil pump. Then THF (5 mL, 1.0 M), MeCN (10 mL, 0.5 M) and triethylamine (5 mL, 1.0 M) were added to the system with a needle. The reaction was heated to 80°C and reacted for 6-8 hours. After the reaction was completed by TLC, the system solvent was removed and the system was purified by silica gel column chromatography and eluted with PE / EtOAc (3:1) 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 Compound 3g of the Invention

[0073]

[0074] (1) Synthesis of compound L6:

[0075] 1) The compound L5 obtained above (500 mg, 1.55 mmol, 1.0 eq) was dissolved in DCM (16 mL, 1.0 M) and the system was placed in an ice bath. CBr4 (875 mg, 2.63 mmol, 1.7 eq) and PPh3 (690 mg, 2.63 mmol, 1.7 eq) were then added. After reacting for 0.5 hours, TLC was monitored until the reaction of the raw materials was complete. The intermediate benzyl bromide compound (466.5 mg, 1.08 mmol) was obtained by filtering through a Buchner funnel covered with filter paper or by PE / EtOAc (5:1) column chromatography. Yield: 70%; yellow solid.

[0076] 2) Add aliphatic cyclic amine to the above intermediate bromide compound (65 mg, 0.15 mmol, 1.0 eq) (0.3mmol, 2.0eq), then Et3N (151mg, 1.5mmol, 10.0eq) was added and stirred at room temperature overnight. After the reaction of the raw material was completed by TLC monitoring, DCM / MeOH (40:1, 20:1) column chromatography was used to obtain compound L6. Yield: 93%; 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, 1.5mL, 0.1M) was used as a co-solvent, and the system was placed in an argon environment. After reacting at 20-35°C for 6-12 hours, TLC was used to detect that the raw materials were completely reacted, and NaHCO3 (35mg, 0.42mmol, 3.0eq) was added and stirred for 0.5 hours to quench the reaction. The solvent was then removed and column chromatography was performed with DCM / MeOH (60:1 to 20:1) to obtain a yellow solid product, namely compound 3g, with a yield of 41.50% and 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 (101MHz,CDCl3)δ149.52,149.16,148.50,145.52,144.65,141.99,135.93,132.20,121.56,120.84,116.11,113.91,113.17,110.62,102.91,102.08,57.65,57.28,39.70,29.36,18.73; LRMS (ESI,m / z)theoretical value is C 25 H 27 N2O4 + [M+H] + 419.1965, actual value is 419.2. Purity after LC-MS analysis is 99.18%.

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

[0079]

[0080] (1) Synthesis of compound L6:

[0081] 1) The compound L5 obtained above (500 mg, 1.55 mmol, 1.0 eq) was dissolved in DCM (16 mL, 1.0 M) and the system was placed in an ice bath. CBr4 (875 mg, 2.63 mmol, 1.7 eq) and PPh3 (690 mg, 2.63 mmol, 1.7 eq) were then added. After reacting for 0.5 hours, TLC was monitored until the reaction of the raw materials was complete. The intermediate benzyl bromide compound (466.5 mg, 1.08 mmol) was obtained by filtering through a Buchner funnel covered with filter paper or by PE / EtOAc (5:1) column chromatography. Yield: 70%; yellow solid.

[0082] 2) Add aliphatic cyclic amine to the above intermediate bromide compound (65 mg, 0.15 mmol, 1.0 eq) (0.3mmol, 2.0eq), then Et3N (151mg, 1.5mmol, 10.0eq) was added and stirred at room temperature overnight. After the reaction of the raw material was completed by TLC monitoring, DCM / MeOH (40:1, 20:1) column chromatography was used to obtain compound L6. Yield: 93%; 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, 1.5mL, 0.1M) was used as a co-solvent, and the system was placed in an argon environment. After reacting at 20-35°C for 6-12 hours, TLC was used to detect that the raw materials were completely reacted, and NaHCO3 (35mg, 0.42mmol, 3.0eq) was added and stirred for 0.5 hours to quench the reaction. The solvent was then removed and column chromatography was performed with DCM / MeOH (60:1 to 20:1) to obtain a yellow solid product, namely compound 3x, with a yield of 38.90% and 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) theoretical value is C 26 H 29 N2O4 + [M+H] + 433.2122, actual value is 433.3. Purity after LC-MS analysis is 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, SHP-77, human normal liver cells LO2 and human bronchial epithelial cells BEAS-2B were all from CCTCC (China). NCI-H1048 was cultured with DMEM / F12 (1:1), LO2 and BEAS-2B were cultured with DMEM, and the remaining SCLC cells were cultured with RPMI1640, and the complete culture medium also needed to be added with 10% embryonic bovine serum and 1% penicillin-streptomycin (double antibody). The cells were cultured in a constant temperature (37°C) and constant humidity incubator containing 5% CO2, and the old culture medium was replaced every 1-2 days. When the cells reach 80% confluence, for adherent cells, wash away the residual medium with 2 mL of 1×PBS, add 2 mL of trypsin and digest for 0.5-2 minutes at room temperature, aspirate the trypsin, quickly add complete medium to terminate digestion, gently blow down the cells with a pipette tip to form a cell suspension, and perform passaging at a density of 1 to 2 or 1 to 3. For suspended cells, gently blow away the aggregated or bottom-settled cells with a pipette tip to form a cell suspension, centrifuge the precipitated cells at 800 rpm, discard the original medium, and perform passaging 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 inoculated into 96-well plates and incubated in an incubator overnight. When the cells are attached or stabilized and grow to the logarithmic phase, different concentrations of the test compound are added. Generally, 5 concentration gradients, 3 replicates, and a total volume of 200 μL per well are set, and the culture is continued for 72 hours. 10 μL of MTT solution or CCK8 solution at a concentration of 5 mg / mL is added, and the MTT test is added for the initial screening of adherent cells. The CCK8 method is greener, simpler, and more accurate, but the cost is high. Therefore, compounds with good activity are screened out 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, which needs to be incubated for 4 hours, and then the original culture medium is removed, 100 μL DMSO is added to dissolve it, and then the absorbance is measured at a wavelength of 570 nm using an enzyme marker. 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] In a 50μL PV tube, add 10μL of ultrapure water, 2μL of Topo I buffer, 2μL of BSA, 1μL of 0.5U topoisomerase I (Takara, 20U / μL), 0.2μL of test compound (no addition for DNA and Topo I groups), add 1μL of 0.25μg / μL pBR322 plasmid DNA (Takara, 0.5μg / μL) to each well, and make up the total volume to 20μL with ultrapure water. After incubation at 37℃ for 0.5 hours, add 2μL of 6× DNA loading buffer, load the sample in 0.8% agarose gel, and electrophoresed at 110V for 45 minutes. Stain with nucleic acid dye for more than 0.5 hours, observe under UV light with bio-rad system and take pictures and record.

[0093] (4) Topoisomerase II inhibitory activity

[0094] In 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 1U topoisomerase II (Inspiralis, Norwich, 100 U), 0.2 μL of test compound (not added to DNA and Topo II groups), 0.2 μL of 0.5 μg / μL pBR322 plasmid DNA (Takara, 0.5 μg / μL) to each well (due to the influence of long-distance transportation, Topo II has been partially inactivated), and fill the total volume to 30 μL with ultrapure water. The rest of the process is consistent with Topo I.

[0095] (5) Molecular docking

[0096] The molecular docking study of compounds 3g and 3x with Topo I and Topo II was analyzed 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), and the energy and hydrogen bond minimization of the proteins were performed, and the Protein Preparation Wizard model was used for preprocessing to generate docking boxes. 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 other docking parameters were all 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 inoculated into 6-well plates (10 cells / mL). After the cells were attached overnight in an incubator, they were treated with different concentrations of compounds 3g and 3x. After 48 hours, the cells were digested and collected and 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 left at room temperature for 10 minutes. The cell concentration was then adjusted to 1×10 5 Cells / mL, 10 μL of cells were mixed with 60 μL of low-melting point agarose, and dropped onto the solidified high-melting point agarose (double-layer agarose allows for better adhesion and prevents slipping, but imaging may be affected), and placed in the dark at 4°C for 30 minutes. Subsequently, the slides were lysed with pre-cooled lysis buffer at 4°C for 30 minutes in the dark to lyse the cell membrane and nuclear membrane. After that, the slides were washed twice with H2O, laid flat for 5 minutes each time, and the DNA was melted with an alkaline solution for 20 minutes. 1×TAE buffer was added to the electrophoresis tank and electrophoresis was performed at 110V for 25 minutes. After washing the slides, they were stained with 10 μL PI solution in the dark for 20 to 30 minutes and washed. The comet image was taken with 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, with a volume of 3 mL per well. Then, different concentrations of the test compound were added, and after incubation for 2-3 weeks, the cells in the blank group were observed to form more than 50 cell colonies with the naked eye, the culture medium was discarded, the cells were washed with 1×PBS, and then fixed with 4% paraformaldehyde for 20 minutes, and then stained with 1% crystal violet for 30 minutes, washed with water to remove the residual dye, and photographed to record the cell colonies.

[0101] (8) EdU test

[0102] NCI-H1048 and NCI-H446 cells were inoculated into 96-well plates at a density of 5000 cells / well. After the cells were incubated overnight and adhered, different concentrations of the test compounds were added, with 3 replicates per group. After 24 hours of drug culture, the old culture medium was discarded, 100 μL of complete culture medium containing 10 μM EdU was added, and incubated in the incubator for 2-4 hours. The culture medium was removed, and then 4% paraformaldehyde was added to fix the cells for 15 minutes, and washed three times with 1×BSA for 5 minutes each time. After adding 100 μL of 0.5% Triton X-100 for 15 minutes, it was stained with Apollo stain for 30 minutes in the dark. After washing three times, the cell nucleus was stained with Hoechst33342 stain for 30 minutes in the dark, and then washed with 1×BSA and observed under an inverted fluorescence microscope and photographed and recorded (Nikon, Japan).

[0103] (9) Cell invasion and migration assay

[0104] 1) Transwell migration assay

[0105] NCI-H1048 and NCI-H446 cells in the logarithmic phase were digested with trypsin and collected. The complete medium was removed by centrifugation at 800 rpm for 5 min at 4°C and the cells were diluted with serum-free medium to a concentration of 1-2 × 10 5 Cell suspension of 1 cell / mL. Add 700 μL of culture medium containing 20% ​​serum to a 24-well plate, and place a transwell chamber (Corning 3422) in it. Connect 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 each lower chamber, 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] Take out the matrigel placed at -20℃ and melt it at 4℃ overnight. Mix the melted matrigel with serum-free medium at a volume ratio of 1:8, add it to the upper chamber of the transwell chamber, 50μL per chamber, spread it evenly with a pipette tip and place it in an incubator to solidify for 3-5 hours until it forms a gel. Hydrate the basement membrane, and the rest of the process is the same as the migration experiment.

[0108] 3) Scratch test

[0109] Take logarithmic phase NCI-H1048 and NCI-H446 cells, digest with trypsin and inoculate in 6-well plates, incubate until cells grow all over the 6-well plates, discard the culture medium, scratch the cells from the middle with a 10μL pipette tip, wash the scratched cells and cell fragments with 1×PBS, add culture medium containing 3% serum and different concentrations of the test compound, select reference points under a microscope to take pictures and record the scratch conditions at 0 hours. After continuing to put it in the incubator for 24 hours, take pictures under a microscope to record the scratch conditions at 24 hours (Nikon, Japan). Image analysis was performed using Image J.

[0110] (10) Cell cycle distribution

[0111] NCI-H1048 and NCI-H446 cells were collected at 1×10 5 The cells were inoculated into 6-well plates at a density of 10 cells / well, 2 mL per well. After the cells adhered to the wall, different concentrations of the test compounds were added and treated for 48 hours. The cells were digested with trypsin and collected, centrifuged at 1200 rpm for 5 minutes at 4°C and resuspended in 1×PBS. The cells were placed in pre-cooled 70% ethanol, blown evenly with a pipette tip, and placed at 4°C overnight. Centrifuge, carefully remove the ethanol and wash three times with 1×PBS by centrifugation. RNase A and PI staining solution were added, and after staining at 37°C in the dark for 30 minutes, the cell cycle was determined by flow cytometry (Agilent, USA), and the percentage of each group in G0 / G1 phase, S phase, and G2 / M phase was counted using Graph Pad.

[0112] (11) Giemsa staining

[0113] NCI-H1048 and NCI-H446 cells were collected at 1×10 5The cells were seeded into 6-well plates at a density of 10 cells / well, 2 mL per well. After the cells adhered to the wall, different concentrations of the test compounds were added for 48 hours, the culture medium was aspirated and washed with 1×PBS, 1 mL of 4% paraformaldehyde was added to fix the cells at room temperature for 20 minutes, the fixative was removed and the cells were washed with 1×PBS, 10 μg / mL of Giemsa dye was added for 30 minutes, the residual dye was washed with PBS, 600 μL of 1×PBS was added, the dye was observed under a microscope and photographed (Nikon, Japan).

[0114] (12) Cell apoptosis experiment

[0115] NCI-H1048 and NCI-H446 cells were collected at 1×10 5 The cells were inoculated into 6-well plates at a density of 10 cells / well, 2 mL per well. After the cells adhered to the wall, different concentrations of the test compounds were added and treated for 48 hours. The cells were then trypsinized and collected, 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, cell 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 1×10 5 The cells were seeded into 6-well plates at a density of 10 cells / well, 2 mL per well. After the cells adhered to the wall, different concentrations of the test compounds were added and treated for 48 hours. The cells were then trypsinized and collected, centrifuged at 1200 rpm for 5 minutes at 4°C, and resuspended in 1×PBS. Then 1 mL of 10 mM DCFH-DA reactive oxygen species probe was added, mixed well, incubated at 37°C for 30 minutes, centrifuged at 1200 rpm / min for 5 minutes, and washed twice with 1×PBS to remove residual reactive oxygen species probes. After adding 500 μL of 1×PBS to suspend the cells, the fluorescence intensity was detected by flow cytometry (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 1×10 5The density of cells / well was inoculated into a 6-well plate, 2mL per well. After the cells adhered to the wall, different concentrations of test compounds were added and treated for 48 hours, and then washed with 1×PBS to remove the culture medium. Then 200μL of Solebao high-efficiency RIPA lysis buffer was added to each well and lysed on ice for 15-30 minutes. After lysis, the samples were collected and centrifuged at 12000 rpm for 12 minutes at 4°C. The protein quantitative standard curve working solution was prepared, and 10μL of supernatant was aspirated and quantified using the BCA protein concentration kit. The remaining supernatant was aspirated and 4×SDS-PAGE protein loading buffer was added, mixed, and the mixture was denatured in a 95°C water bath for 15 minutes. After cooling, it was placed in a -80°C refrigerator for testing. According to the molecular weight of the protein, the SDS-PAGE separation gel and the concentrated gel were selected and prepared, and the comb was inserted. After solidification, the comb was removed. The prepared gel plate was placed in the electrophoresis tank, and the corresponding volume of protein sample and marker were added to each loading well in turn. After concentration at 80V for 15 minutes, the samples were separated at 120V for about 1 hour. After electrophoresis, the gel was carefully peeled off, and the 0.22μM PVDF membrane was activated in methanol for 10 minutes in advance. The separated protein samples were transferred to the activated PVDF membrane by wet transfer at 200-300mA for 1-2 hours. After the transfer, the PVDF membrane was blocked in protein fast blocking solution for 10 minutes (NcmBlot Blocking Buffer, China). The membrane was washed twice with 1×TBST buffer for 5 minutes each time. The PVDF membrane corresponding to the molecular weight of the protein was cut according to the protein marker and placed in the diluted corresponding primary antibody (1:5000), and incubated on a shaker at 4°C overnight. The membrane was washed 3 times with 1×TBST buffer for 5 minutes each time. The diluted corresponding secondary antibody (1:10000) was added and incubated on a shaker at room temperature for 2 hours. After the incubation, the membrane was washed 3 times with 1×TBST buffer for 5 minutes each time. The strips were added with ECL chemiluminescent solution, chemiluminescent imaging was performed using the bio-rad system, and the gray 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 process was the same as above.

[0122] (16) Acute toxicity test

[0123] Experimental mice were purchased from Huafukang Biotechnology Co., Ltd. Kunming male mice weighing between 30 and 35 grams were divided into 7 groups (4 mice per group), namely 3g 300mg / kg group and 400mg / kg group, 3x 300mg / kg group and 400mg / kg group, 52 200mg / kg group and 300mg / kg group. The experimental group was intraperitoneally injected with 200μL of saline containing different concentrations of drugs and 5% DMSO and 2.5% castor oil. The control group was intraperitoneally injected with an equal amount of blank solution. The weight of the mice was monitored and recorded daily for a total of 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 kept in an SPF animal room for about a week to adapt to the environment. Healthy male nude mice (4-5 weeks old) were selected. NCI-H446 cells in good growth state and logarithmic growth phase were taken and the cell density was adjusted to 8×10 using serum-free double antibody-free culture medium and matrix gel (mixed in a ratio of 1:1). 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, they were randomly divided into 8 groups: 2 control groups, 3g 10mg / kg group and 20mg / kg group, 3x 10mg / kg group and 20mg / kg group, 52 2.5mg / kg group and 5mg / kg group, and positive drug etoposide 5mg / kg group and 10mg / kg group. The drugs were intraperitoneally injected once a day for three weeks, and the tumor volume and nude mouse weight 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%. Wherein, T and C represent the average tumor volume of the drug group and the model group on the last day, respectively, and T0 and C0 represent the average tumor volume of the drug group and the model group on the first day, respectively.

[0126] (18) Data Analysis

[0127] Statistical analysis was performed using IBM SPSS Statistics software and GraphPad Prism-9.5 software. Images were analyzed using ImageJ software, and grouped data were expressed as mean ± standard deviation. The following symbols represent the statistical significance level in 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 inhibitory cell proliferation activity of compounds 3g and 3x

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

[0131]

[0132] In summary, the activities of compounds 3g and 3x against NCI-H446 and NCI-H1048 cell lines were comparable to those of the positive drug etoposide, and were more active against other SCLC cell lines. Compounds 3g and 3x were less toxic than the positive drug 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 conducted a topoisomerase relaxation test on the synthesized compounds, such as Figure 1 , 2 As shown, the inhibitory effect of compounds 3g and 3x on Topo I disappeared at a concentration of 50 μM, while the positive control topotecan still retained activity at this concentration. In contrast, compounds 3g and 3x still had inhibitory effects on Topo II at a concentration of 6.25 μM, and were comparable to the positive drug etoposide. Therefore, compounds 3g and 3x have dual inhibitory effects on Topo I and Topo II.

[0135] Molecular docking studies were performed on compounds 3g and 3x with Topo I and II, respectively. Figure 3 As shown. Compounds 3g and 3x both showed the ability to insert into DNA. These findings suggest that compounds 3g and 3x can act 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 a higher sensitivity and can detect even trace amounts of single-strand and double-strand breaks. As DNA damage intensifies, the number of breaks and fragments increases, and the tail becomes longer. As Figure 4 As shown, the results show that the DNA of NCI-H446 and NCI-H1048 cells showed different degrees of damage under the action of different concentrations of 3g and 3x, and showed concentration dependence. At all concentrations, the DNA of both cell lines was damaged, and the damage to NCI-H1048 cells was more obvious. In short, DNA damage can have a cytotoxic effect on cancer cells, and may eventually lead to cell 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 inhibitory effect of compounds 3g and 3x on SCLC cell proliferation, the present invention conducted a clone formation experiment. Figure 5 As shown in Figure A, compounds 3g and 3x inhibited cell clone 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 experiment, 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 about 40%. Compounds 3g and 3x both inhibited cell proliferation in a concentration-dependent manner, and their inhibitory effects on NCI-H446 were comparable to those of etoposide at the same concentration, while their inhibitory effects on NCI-H1048 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 inhibit 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 DF, after adding different concentrations of compounds 3g, 3x and etoposide treatment, the invasion ability of cells 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.

[0141] Since the number of NCI-H1048 cells that invaded and migrated in the Transwell assay was relatively small, the present invention conducted a scratch assay to better confirm the inhibitory effect of the compound on SCLC migration. Figure 7 As shown, compounds 3g and 3x inhibit the migration of SCLC cells in a concentration-dependent manner. It can be seen that 3g has a better inhibitory effect than the positive drug etoposide at the same concentration, and it is not difficult to see that compound 3g has a more obvious inhibitory effect on NCI-H1048. In summary, compounds 3g and 3x can significantly inhibit the invasion and migration of SCLC cells and can be used to prepare drugs for inhibiting the metastasis of small cell lung cancer.

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

[0143] To evaluate the effects of compounds 3g and 3x on the cell cycle progression of NCI-H446 and NCI-H1048, 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 were performed to detect apoptosis. Fig. 9 A, the compound reduced the number of NCI-H446 and NCI-H1048 in a dose-dependent manner. In addition, NCI-H446 cell membrane blebbing, i.e., the generation of apoptotic bodies and chromatin shrinkage were observed. Fig. 9 B and 9C, compound 3g induced premature apoptosis of SCLC cells in a dose-dependent manner, with extremely significant effects and stronger than etoposide in both cell lines. Compound 3x induced the formation of cell fragments in NCI-H446, and etoposide had little effect on the apoptosis of NCI-H446 cells at the selected concentration. Compound 3x can also induce apoptosis of NCI-H1048 cells to a certain extent, but the effect is not as good as etoposide.

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

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

[0148] like Fig.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. Fig.12 With the increase of 3g and 3x concentrations, the expression of Bcl-2 decreased, and 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 activation of the PI3K / Akt / mTOR signaling pathway

[0150] like Fig.13 , 14 The present invention uses WB experiments to confirm that compounds 3g and 3x can inhibit the phosphorylation of 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 between 30-35 grams were randomly divided into five groups, control group and experimental group (300 mg / kg group and 400 mg / kg group of compound 3g, 300 mg / kg group and 400 mg / kg group of compound 3x, respectively). The experimental group was intraperitoneally injected with 200 μL of saline solution containing different concentrations of drugs and 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 1.38 mmol / l. 50 ) is between 300-400 mg / kg. Monitor the weight changes of mice daily, and the weight change trend is as follows Fig.15 As shown, the results showed that one day after administration, the body weight of mice changed significantly, and then the body weight of 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 Afterwards, the 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 were set as low dose (10 mg / kg / d, ip) and high dose group (20 mg / kg / d, ip), respectively. Fig.16 As shown in A and 16B, the tumor volume of the drug-treated group grew very slowly since day 21. Fig.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. Fig.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 =1.3μM, IC on NCI-H1048 50 =1.42 μM, compound 3x IC in NCI-H446 cells 50 =1.32μM, IC on NCI-H1048 50 =2.45μM, compounds 3g and 3x can inhibit the proliferation, invasion and migration of SCLC cells, compounds 3g and 3x can insert into DNA, significantly induce DNA damage, and block the NCI-H446 cell cycle at the S phase. By inducing mitochondrial dysfunction, SCLC cell apoptosis is significantly induced. In addition, compounds 3g and 3x can also inhibit the phosphorylation of the PI3K / Akt / mTOR signaling pathway, thereby affecting various SCLC cell activities. The inhibitory activity of compounds 3g and 3x in vivo and in vitro for SCLC is significantly higher than that of the positive drug etoposide.

[0158] In summary, the present invention provides a topoisomerase I / II dual-target inhibitor and its preparation method and application. The present invention uses 6-bromopiperonal as a raw material to synthesize the compound, which is a new type of topoisomerase I / II dual-target inhibitor, which can be used to treat small cell lung cancer, advanced metastatic ovarian cancer, advanced breast cancer, colorectal cancer, bladder cancer and other diseases, and has good application prospects.

Claims

1. A compound of formula I, characterized in that: The structural formula of the compound is: 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.

2. The compound according to claim 1, characterized in that R 3 is hydrogen, R 1 , R 2 , R 4 and R 5 Each independently selected from C 1～2 alkyl.

3. The compound according to claim 1, characterized in that The compound is selected from one of the following compounds:

4. A method for preparing the compound according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: Among them, R is R 1 , R 2 , R 3 , R 4 and R 5 As claimed in any one of claims 1 to 3; (1) reacting 6-bromopiperonal, trimethylsilylacetylene, a catalyst and a base to prepare a compound L2; (2) reacting compound L2 with a reducing agent to prepare compound L3; (3) reacting compound L1 with a base to prepare compound L4; (4) reacting compound L3, compound L4, a catalyst and a base to prepare compound L5; (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; (6) Compound L6, a nitrogen source compound and a catalyst are reacted to obtain a 3-arylisoquinoline derivative.

5. The method according to claim 4, 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: reacting at 40-60°C for 5-7 hours under argon protection; 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: reacting at -5-10°C 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, react 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: reacting 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: reacting at 10-40°C for 5-20 hours; In step (6), the nitrogen source compound is ammonium acetate; the catalyst is silver nitrate; the molar ratio of compound L6, nitrogen source compound and catalyst is 1:2-4:0.1-0.5; the solvent of the reaction is an organic solvent; the reaction conditions are: under argon protection, react at 10-40°C for 4-15 hours.

6. Use of the compound according to any one of claims 1 to 3 in the preparation of a topoisomerase inhibitor.

7. The use according to claim 6, characterized in that The topoisomerase is topoisomerase I and / or topoisomerase II.

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

9. The use according to claim 8, characterized in that The cancer is selected from small cell lung cancer, advanced metastatic ovarian cancer, advanced breast cancer, colorectal cancer, and bladder cancer.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition is a preparation prepared by using the compound according to any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable excipients.

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